40 CFR Appendix A to Part 136 - Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater - Regulations - VLEX 19813273

40 CFR Appendix A to Part 136 - Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater

Actualizado a:December 2005
CONTENT

TITLE 40 - PROTECTION OF ENVIRONMENT

CHAPTER I - ENVIRONMENTAL PROTECTION AGENCY

SUBCHAPTER D - WATER PROGRAMS

PART 136 - GUIDELINES ESTABLISHING TEST PROCEDURES FOR THE ANALYSIS OF POLLUTANTS

Appendix A to Part 136 - Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater

Method 601Purgeable Halocarbons 1. Scope and Application 1.1This method covers the determination of 29 purgeable halocarbons.

The following parameters may be determined by this method: ------------------------------------------------------------------------ STORET Parameter No. CAS No.

------------------------------------------------------------------------ Bromodichloromethane........................... 32101 75-27-4 Bromoform...................................... 32104 75-25-2 Bromomethane................................... 34413 74-83-9 Carbon tetrachloride........................... 32102 56-23-5 Chlorobenzene.................................. 34301 108-90-7 Chloroethane................................... 34311 75-00-3 2-Chloroethylvinyl ether....................... 34576 100-75-8 Chloroform..................................... 32106 67-66-3 Chloromethane.................................. 34418 74-87-3 Dibromochloromethane........................... 32105 124-48-1 1,2-Dichlorobenzene............................ 34536 95-50-1 1,3-Dichlorobenzene............................ 34566 541-73-1 1,4-Dichlorobenzene............................ 34571 106-46-7 Dichlorodifluoromethane........................ 34668 75-71-8 1,1-Dichloroethane............................. 34496 75-34-3 1,2-Dichloroethane............................. 34531 107-06-2 1,1-Dichloroethane............................. 34501 75-35-4 trans-1,2-Dichloroethene....................... 34546 156-60-5 1,2-Dichloropropane............................ 34541 78-87-5 cis-1,3-Dichloropropene........................ 34704 10061-01-5 trans-1,3-Dichloropropene...................... 34699 10061-02-6 Methylene chloride............................. 34423 75-09-2 1,1,2,2-Tetrachloroethane...................... 34516 79-34-5 Tetrachloroethene.............................. 34475 127-18-4 1,1,1-Trichloroethane.......................... 34506 71-55-6 1,1,2-Trichloroethane.......................... 34511 79-00-5 Tetrachloroethene.............................. 39180 79-01-6 Trichlorofluoromethane......................... 34488 75-69-4 Vinyl chloride................................. 39715 75-01-4 ------------------------------------------------------------------------ 1.2This is a purge and trap gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 624 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for most of the parameters listed above.

1.3The method detection limit (MDL, defined in Section 12.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1An inert gas is bubbled through a 5-mL water sample contained in a specially-designed purging chamber at ambient temperature. The halocarbons are efficiently transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the halocarbons are trapped. After purging is completed, the trap is heated and backflushed with the inert gas to desorb the halocarbons onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the halocarbons which are then detected with a halide-specific detector.2,3 2.2The method provides an optional gas chromatographic column that may be helpful in resolving the compounds of interest from interferences that may occur.

3. Interferences 3.1Impurities in the purge gas and organic compounds outgassing from the plumbing ahead of the trap account for the majority of contamination problems. The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2Samples can be contaminated by diffusion of volatile organics (particularly fluorocarbons and methylene chloride) through the septum seal ilto the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed with reagent water between sample analyses. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high organohalide levels, it may be necessary to wash out the purging device with a detergent solution, rinse it with distilled water, and then dry it in a 105C oven between analyses. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified46 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: carbon tetrachloride, chloroform, 1,4-dichlorobenzene, and vinyl chloride.

Primary standards of these toxic compounds should be prepared in a hood.

A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials 5.1Sampling equipment, for discrete sampling.

5.1.1Vial25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 C before use.

5.1.2SeptumTeflon-faced silicone (Pierce #12722 or equivalent).

Detergent wash, rinse with tap and distilled water, and dry at 105 C for 1 h before use.

5.2Purge and trap systemThe purge and trap system consists of three separate pieces of equipment: a purging device, trap, and desorber.

Several complete systems are now commercially available.

5.2.1The purging device must be designed to accept 5-mL samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL.

The purge gas must pass through the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column.

The purging device illustrated in Figure 1 meets these design criteria.

5.2.2The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in. The trap must be packed to contain the following minimum lengths of adsorbents: 1.0 cm of methyl silicone coated packing (Section 6.3.3), 7.7 cm of 2,6-diphenylene oxide polymer (Section 6.3.2), 7.7 cm of silica gel (Section 6.3.4), 7.7 cm of coconut charcoal (Section 6.3.1). If it is not necessary to analyze for dichlorodifluoromethane, the charcoal can be eliminated, and the polymer section lengthened to 15 cm. The minimum specifications for the trap are illustrated in Figure 2.

5.2.3The desorber must be capable of rapidly heating the trap to 180 C.

The polymer section of the trap should not be heated higher than 180 C and the remaining sections should not exceed 200 C. The desorber illustrated in Figure 2 meets these design criteria.

5.2.4The purge and trap system may be assembled as a separate unit or be coupled to a gas chromatograph as illustrated in Figures 3 and 4.

5.3Gas chromatographAn analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.3.1Column 18 ft long 0.1 in. ID stainless steel or glass, packed with 1% SP1000 on Carbopack B (60/80 mesh) or equivalent. This column was used to develop the method performance statements in Section 12.

Guidelines for the use of alternate column packings are provided in Section 10.1.

5.3.2Column 26 ft long 0.1 in. ID stainless steel or glass, packed with chemically bonded n-octane on Porasil-C (100/120 mesh) or equivalent.

5.3.3DetectorElectrolytic conductivity or microcoulometric detector.

These types of detectors have proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1). The electrolytic conductivity detector was used to develop the method performance statements in Section 12. Guidelines for the use of alternate detectors are provided in Section 10.1.

5.4Syringes5-mL glass hypodermic with Luerlok tip (two each), if applicable to the purging device.

5.5Micro syringes25-L, 0.006 in. ID needle.

5.6Syringe valve2-way, with Luer ends (three each).

5.7Syringe5-mL, gas-tight with shut-off valve.

5.8Bottle15-mL, screw-cap, with Teflon cap liner.

5.9BalanceAnalytical, capable of accurately weighing 0.0001 g.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3Reagent water may also be prepared by boiling water for 15 min.

Subsequently, while maintaining the temperature at 90 C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.2Sodium thiosulfate(ACS) Granular.

6.3Trap Materials: 6.3.1Coconut charcoal6/10 mesh sieved to 26 mesh, Barnabey Cheney, CA58026 lot # M2649 or equivalent.

6.3.22,6-Diphenylene oxide polymerTenax, (60/80 mesh), chromatographic grade or equivalent.

6.3.3Methyl silicone packing3% OV1 on Chromosorb-W (60/80 mesh) or equivalent.

6.3.4Silica gel35/60 mesh, Davison, grade-15 or equivalent.

6.4MethanolPesticide quality or equivalent.

6.5Stock standard solutionsStock standard solutions may be prepared from pure standard materials or purchased as certified solutions. Prepare stock standard solutions in methanol using assayed liquids or gases as appropriate. Because of the toxicity of some of the organohalides, primary dilutions of these materials should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.5.1Place about 9.8 mL of methanol into a 10-mL ground glass stoppered volumetric flask. Allow the flask to stand, unstoppered, for about 10 min or until all alcohol wetted surfaces have dried. Weigh the flask to the learest 0.1 mg.

6.5.2Add the assayed reference material: 6.5.2.1LiquidUsing a 100 L syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the alcohol without contacting the neck of the flask.

6.5.2.2GasesTo prepare standards for any of the six halocarbons that boil below 30C (bromomethane, chloroethane, chloromethane, dichlorodifluoromethane, trichlorofluoromethane, vinyl chloride), fill a 5-mL valved gas-tight syringe with the reference standard to the 5.0-mL mark. Lower the needle to 5 mm above the methanol meniscus. Slowly introduce the reference standard above the surface of the liquid (the heavy gas will rapidly dissolve into the methanol).

6.5.3Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in g/L from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the malufacturer or by an independent source.

6.5.4Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store, with minimal headspace, at 10 to 20 C and protect from light.

6.5.5Prepare fresh standards weekly for the six gases and 2-chloroethylvinyl ether. All other standards must be replaced after one month, or sooner if comparison with check standards indicates a problem.

6.6Secondary dilution standardsUsing stock standard solutions, prepare secondary dilution standards in methanol that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3.1 or 7.4.1 will bracket the working range of the analytical system. Secondary dilution standards should be stored with minimal headspace and should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1. Calibrate the purge and trap-gas chromatographic system using either the external standard technique (Section 7.3) or the internal standard technique (Section 7.4).

7.3External standard calibration procedure: 7.3.1Prepare calibration standards at a miminum of three concentration levels for each parameter by carefully adding 20.0 L of one or more secondary dilution standards to 100, 500, or 1000 L of reagent water. A 25-L syringe with a 0.006 in. ID needle should be used for this operation. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector. These aqueous standards can be stored up to 24 h, if held in sealed vials with zero headspace as described in Section 9.2. If not so stored, they must be discarded after 1 h.

7.3.2Analyze each calibration standard according to Section 10, and tabulate peak height or area responses versus the concentration in the standard. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to concentration (calibration factor) is a constant over the working range (7.4Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples. The compounds recommended for use as surrogate spikes in Section 8.7 have been used successfully as internal standards, because of their generally unique retention times.

7.4.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest as described in Section 7.3.1.

7.4.2Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.5 and 6.6. It is recommended that the secondary dilution standard be prepared at a concentration of 15 g/mL of each internal standard compound. The addition of 10 L of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 g/L.

7.4.3Analyze each calibration standard according to Section 10, adding 10 L of internal standard spiking solution directly to the syringe (Section 10.4). Tabulate peak height or area responses against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1. (image) Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard.

Cs=Concentration of the parameter to be measured.

If the RF value over the working range is a constant (7.5The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of a QC check sample.

7.5.1Prepare the QC check sample as described in Section 8.2.2.

7.5.2Analyze the QC check sample according to Section 10.

7.5.3For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 2. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, proceed according to Section 7.5.4.

Note: The large number of parameters in Table 2 present a substantial probability that one or more will not meet the calibration acceptance criteria when all parameters are analyzed.

7.5.4Repeat the test only for those parameters that failed to meet the calibration acceptance criteria. If the response for a parameter does not fall within the range in this second test, a new calibration curve, calibration factor, or RF must be prepared for that parameter according to Section 7.3 or 7.4.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 10.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 10 g/mL in methanol. The QC check sample concentrate must be obtained from the U.S.

Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Prepare a QC check sample to contain 20 g/L of each parameter by adding 200 L of QC check sample concentrate to 100 mL of reagent water.

8.2.3Analyze four 5-mL aliquots of the well-mixed QC check sample according to Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter of interest using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, then the system performance is unacceptable for that parameter.

Note: The large number of parameters in Table 2 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.3.

8.2.6.2Beginning with Section 8.2.3, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.3.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 L of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 20 g/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 3, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T)2.44(100 S/T)%.7 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 2 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spiked sample.

8.4.1Prepare the QC check standard by adding 10 L of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If p =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

8.7The analyst should monitor both the performance of the analytical system and the effectiveness of the method in dealing with each sample matrix by spiking each sample, standard, and reagent water blank with surrogate halocarbons. A combination of bromochloromethane, 2-bromo-1-chloropropane, and 1,4-dichlorobutane is recommended to encompass the range of the temperature program used in this method. From stock standard solutions prepared as in Section 6.5, add a volume to give 750 g of each surrogate to 45 mL of reagent water contained in a 50-mL volumetric flask, mix and dilute to volume for a concentration of 15 ng/L. Add 10 L of this surrogate spiking solution directly into the 5-mL syringe with every sample and reference standard analyzed. Prepare a fresh surrogate spiking solution on a weekly basis. If the internal standard calibration procedure is being used, the surrogate compounds may be added directly to the internal standard spiking solution (Section 7.4.2).

9. Sample Collection, Preservation, and Handling 9.1All samples must be iced or refrigerated from the time of collection until analysis. If the sample contains free or combined chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2Grab samples must be collected in glass containers having a total volume of at least 25 mL. Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. If preservative has been added, shake vigorously for 1 min. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3All samples must be analyzed within 14 days of collection.3 10. Procedure 10.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are estimated retention times and MDL that can be achieved under these conditions. An example of the separations achieved by Column 1 is shown in Figure 5. Other packed columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

10.2Calibrate the system daily as described in Section 7.

10.3Adjust the purge gas (nitrogen or helium) flow rate to 40 mL/min.

Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

10.4Allow the sample to come to ambient temperature prior to introducing it to the syringe. Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing.

Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 L of the surrogate spiking solution (Section 8.7) and 10.0 L of the internal standard spiking solution (Section 7.4.2), if applicable, through the valve bore, then close the valve.

10.5Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

10.6Close both valves and purge the sample for 11.0 0.1 min at ambient temperature.

10.7After the 11-min purge time, attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 4), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 C while backflushing the trap with an inert gas between 20 and 60 mL/min for 4 min. If rapid heating of the trap cannot be achieved, the GC column must be used as a secondary trap by cooling it to 30 C (subambient temperature, if poor peak geometry or random retention time problems persist) instead of the initial program temperature of 45 C 10.8While the trap is being desorbed into the gas chromatograph, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

10.9After desorbing the sample for 4 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at 180 C After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

10.10Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

10.11If the response for a peak exceeds the working range of the system, prepare a dilution of the sample with reagent water from the aliquot in the second syringe and reanalyze.

11. Calculations 11.1Determine the concentration of individual compounds in the sample.

11.1.1If the external standard calibration procedure is used, calculate the concentration of the parameter being measured from the peak response using the calibration curve or calibration factor determined in Section 7.3.2.

11.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.4.3 and Equation 2. Equation 2 (image) where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard.

11.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

12. Method Performance 12.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero. 1 The MDL concentration listed in Table 1 were obtained using reagent water.11. Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

12.2This method is recommended for use in the concentration range from the MDL to 1000MDL. Direct aqueous injection techniques should be used to measure concentration levels above 1000MDL.

12.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 8.0 to 500 g/L.9 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References 1. 40 CFR part 136, appendix B.

2. Bellar, T.A., and Lichtenberg, J.J. Determining Volatile Organics at Microgram-per-Litre-Levels by Gas Chromatography, Journal of the American Water Works Association, 66, 739 (1974).

3. Bellar, T.A., and Lichtenberg, J.J. Semi-Automated Headspace Analysis of Drinking Waters and Industrial Waters for Purgeable Volatile Organic Compounds, Proceedings from Symposium on Measurement of Organic Pollutants in Water and Wastewater, American Society for Testing and Materials, STP 686, C.E. Van Hall, editor, 1978.

4. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 8. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA 600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

9. EPA Method Study 24, Method 601Purgeable Halocarbons by the Purge and Trap Method, EPA 600/484064, National Technical Information Service, PB84212448, Springfield, Virginia 22161, July 1984.

10. Method Validation Data for EPA Method 601, Memorandum from B.

Potter, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, November 10, 1983.

11. Bellar, T. A., Unpublished data, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, 1981.

Table 1_Chromatographic Conditions and Method Detection Limits ---------------------------------------------------------------------------------------------------------------- Retention time (min) Method detection Parameter ------------------------------------ limit (g/ Column 1 Column 2 L) ---------------------------------------------------------------------------------------------------------------- Chloromethane............................................. 1.50 5.28 0.08 Bromomethane.............................................. 2.17 7.05 1.18 Dichlorodifluoromethane................................... 2.62 nd 1.81 Vinyl chloride............................................ 2.67 5.28 0.18 Chloroethane.............................................. 3.33 8.68 0.52 Methylene chloride........................................ 5.25 10.1 0.25 Trichlorofluoromethane.................................... 7.18 nd nd 1,1-Dichloroethene........................................ 7.93 7.72 0.13 1,1-Dichloroethane........................................ 9.30 12.6 0.07 trans-1,2-Dichloroethene.................................. 10.1 9.38 0.10 Chloroform................................................ 10.7 12.1 0.05 1,2-Dichloroethane........................................ 11.4 15.4 0.03 1,1,1-Trichloroethane..................................... 12.6 13.1 0.03 Carbon tetrachloride...................................... 13.0 14.4 0.12 Bromodichloromethane...................................... 13.7 14.6 0.10 1,2-Dichloropropane....................................... 14.9 16.6 0.04 cis-1,3-Dichloropropene................................... 15.2 16.6 0.34 Trichloroethene........................................... 15.8 13.1 0.12 Dibromochloromethane...................................... 16.5 16.6 0.09 1,1,2-Trichloroethane..................................... 16.5 18.1 0.02 trans-1,3-Dichloropropene................................. 16.5 18.0 0.20 2-Chloroethylvinyl ether.................................. 18.0 nd 0.13 Bromoform................................................. 19.2 19.2 0.20 1,1,2,2-Tetrachloroethane................................. 21.6 nd 0.03 Tetrachloroethene......................................... 21.7 15.0 0.03 Chlorobenzene............................................. 24.2 18.8 0.25 1,3-Dichlorobenzene....................................... 34.0 22.4 0.32 1,2-Dichlorobenzene....................................... 34.9 23.5 0.15 1,4-Dichlorobenzene....................................... 35.4 22.3 0.24 ---------------------------------------------------------------------------------------------------------------- Column 1 conditions: Carbopack B (60/80 mesh) coated with 1% SP-1000 packed in an 8 ft x 0.1 in. ID stainless steel or glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held at 45 C for 3 min then programmed at 8 C/min to 220 C and held for 15 min.

Column 2 conditions: Porisil-C (100/120 mesh) coated with n-octane packed in a 6 ft x 0.1 in. ID stainless steel or glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held at 50 C for 3 min then programmed at 6 C/min to 170 C and held for 4 min.

nd=not determined.

Table 2_Calibration and QC Acceptance Criteria_Method 601 a ---------------------------------------------------------------------------------------------------------------- Limit for Range for Q s Range for X Range P, Parameter (g/L) (g/ (g/L) Ps (%) L) ---------------------------------------------------------------------------------------------------------------- Bromodichloromethane.................................... 15.2-24.8 4.3 10.7-32.0 42-172 Bromoform............................................... 14.7-25.3 4.7 5.0-29.3 13-159 Bromomethane............................................ 11.7-28.3 7.6 3.4-24.5 D-144 Carbon tetrachloride.................................... 13.7-26.3 5.6 11.8-25.3 43-143 Chlorobenzene........................................... 14.4-25.6 5.0 10.2-27.4 38-150 Chloroethane............................................ 15.4-24.6 4.4 11.3-25.2 46-137 2-Chloroethylvinyl ether................................ 12.0-28.0 8.3 4.5-35.5 14-186 Chloroform.............................................. 15.0-25.0 4.5 12.4-24.0 49-133 Chloromethane........................................... 11.9-28.1 7.4 D-34.9 D-193 Dibromochloromethane.................................... 13.1-26.9 6.3 7.9-35.1 24-191 1,2-Dichlorobenzene..................................... 14.0-26.0 5.5 1.7-38.9 D-208 1,3-Dichlorobenzene..................................... 9.9-30.1 9.1 6.2-32.6 7-187 1,4-Dichlorobenzene..................................... 13.9-26.1 5.5 11.5-25.5 42-143 1,1-Dichloroethane...................................... 16.8-23.2 3.2 11.2-24.6 47-132 1,2-Dichloroethane...................................... 14.3-25.7 5.2 13.0-26.5 51-147 1,1-Dichloroethene...................................... 12.6-27.4 6.6 10.2-27.3 28-167 trans-1,2-Dichloroethene................................ 12.8-27.2 6.4 11.4-27.1 38-155 1,2-Dichloropropane..................................... 14.8-25.2 5.2 10.1-29.9 44-156 cis-1,3-Dichloropropene................................. 12.8-27.2 7.3 6.2-33.8 22-178 trans-1,3-Dichloropropene............................... 12.8-27.2 7.3 6.2-33.8 22-178 Methylene chloride...................................... 15.5-24.5 4.0 7.0-27.6 25-162 1,1,2,2-Tetrachloroethane............................... 9.8-30.2 9.2 6.6-31.8 8-184 Tetrachloroethene....................................... 14.0-26.0 5.4 8.1-29.6 26-162 1,1,1-Trichloroethane................................... 14.2-25.8 4.9 10.8-24.8 41-138 1,1,2-Trichloroethane................................... 15.7-24.3 3.9 9.6-25.4 39-136 Trichloroethene......................................... 15.4-24.6 4.2 9.2-26.6 35-146 Trichlorofluoromethane.................................. 13.3-26.7 6.0 7.4-28.1 21-156 Vinyl chloride.......................................... 13.7-26.3 5.7 8.2-29.9 28-163 ---------------------------------------------------------------------------------------------------------------- a Criteria were calculated assuming a QC check sample concentration of 20 g/L.

Q=Concentration measured in QC check sample, in g/L (Section 7.5.3).

s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3_Method Accuracy and Precision as Functions of Concentration_Method 601 ---------------------------------------------------------------------------------------------------------------- Single analyst Parameter Accuracy, as recovery, precision, sr[prime] Overall precision, X[prime] (g/L) (g/L) S[prime] (g/L) ---------------------------------------------------------------------------------------------------------------- Bromodichloromethane................ 1.12C-1.02 0.11X+0.04 0.20X+1.00 Bromoform........................... 0.96C-2.05 0.12X+0.58 0.21X+2.41 Bromomethane........................ 0.76C-1.27 0.28X+0.27 0.36X+0.94 Carbon tetrachloride................ 0.98C-1.04 0.15X+0.38 0.20X+0.39 Chlorobenzene....................... 1.00C-1.23 0.15X-0.02 0.18X+1.21 Choroethane......................... 0.99C-1.53 0.14X-0.13 0.17X+0.63 2-Chloroethylvinyl ether a.......... 1.00C 0.20X 0.35X Chloroform.......................... 0.93C-0.39 0.13X+0.15 0.19X-0.02 Chloromethane....................... 0.77C+0.18 0.28X-0.31 0.52X+1.31 Dibromochloromethane................ 0.94C+2.72 0.11X+1.10 0.24X+1.68 1,2-Dichlorobenzene................. 0.93C+1.70 0.20X+0.97 0.13X+6.13 1,3-Dichlorobenzene................. 0.95C+0.43 0.14X+2.33 0.26X+2.34 1,4-Dichlorobenzene................. 0.93C-0.09 0.15X+0.29 0.20X+0.41 1,1-Dichloroethane.................. 0.95C-1.08 0.09X+0.17 0.14X+0.94 1,2-Dichloroethane.................. 1.04C-1.06 0.11X+0.70 0.15X+0.94 1,1-Dichloroethene.................. 0.98C-0.87 0.21X-0.23 0.29X-0.40 trans-1,2-Dichloroethene............ 0.97C-0.16 0.11X+1.46 0.17X+1.46 1,2-Dichloropropane a............... 1.00C 0.13X 0.23X cis-1,3-Dichloropropene a........... 1.00C 0.18X 0.32X trans-1,3-Dichloropropene a......... 1.00C 0.18X 0.32X Methylene chloride.................. 0.91C-0.93 0.11X+0.33 0.21X+1.43 1,1,2,2-Tetrachloroethene........... 0.95C+0.19 0.14X+2.41 0.23X+2.79 Tetrachloroethene................... 0.94C+0.06 0.14X+0.38 0.18X+2.21 1,1,1-Trichloroethane............... 0.90C-0.16 0.15X+0.04 0.20X+0.37 1,1,2-Trichloroethane............... 0.86C+0.30 0.13X-0.14 0.19X+0.67 Trichloroethene..................... 0.87C+0.48 0.13X-0.03 0.23X+0.30 Trichlorofluoromethane.............. 0.89C-0.07 0.15X+0.67 0.26X+0.91 Vinyl chloride...................... 0.97C-0.36 0.13X+0.65 0.27X+0.40 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sn[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S\1\=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

a Estimates based upon the performance in a single laboratory.\10\ (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF Method 602Purgeable Aromatics 1. Scope and Application 1.1This method covers the determination of various purgeable aromatics.

The following parameters may be determined by this method: ------------------------------------------------------------------------ STORET Parameter No. CAS No.

------------------------------------------------------------------------ Benzene.......................................... 34030 71-43-2 Chlorobenzene.................................... 34301 108-90-7 1,2-Dichlorobenzene.............................. 34536 95-50-1 1,3-Dichlorobenzene.............................. 34566 541-73-1 1,4-Dichlorobenzene.............................. 34571 106-46-7 Ethylbenzene..................................... 34371 100-41-4 Toluene.......................................... 34010 108-88-3 ------------------------------------------------------------------------ 1.2This is a purge and trap gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 624 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above.

1.3The method detection limit (MDL, defined in Section 12.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1An inert gas is bubbled through a 5-mL water sample contained in a specially-designed purging chamber at ambient temperature. The aromatics are efficiently transferred from the aqueous phase to the vapor phase.

The vapor is swept through a sorbent trap where the aromatics are trapped. After purging is completed, the trap is heated and backflushed with the inert gas to desorb the aromatics onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the aromatics which are then detected with a photoionization detector.2,3 2.2The method provides an optional gas chromatographic column that may be helpful in resolving the compounds of interest from interferences that may occur.

3. Interferences 3.1Impurities in the purge gas and organic compounds outgassing from the plumbing ahead of the trap account for the majority of contamination problems. The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2Samples can be contaminated by diffusion of volatile organics through the septum seal into the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed with reagent water between sample analyses. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high aromatic levels, it may be necessary to wash the purging device with a detergent solution, rinse it with distilled water, and then dry it in an oven at 105C between analyses. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified46 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzene and 1,4-dichlorobenzene. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials 5.1Sampling equipment, for discrete sampling.

5.1.1Vial]25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 C before use.

5.1.2SeptumTeflon-faced silicone (Pierce #12722 or equivalent).

Detergent wash, rinse with tap and distilled water, and dry at 105 C for 1 h before use.

5.2Purge and trap systemThe purge and trap system consists of three separate pieces of equipment: A purging device, trap, and desorber.

Several complete systems are now commercially available.

5.2.1The purging device must be designed to accept 5-mL samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL.

The purge gas must pass through the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column.

The purging device illustrated in Figure 1 meets these design criteria.

5.2.2The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in.

5.2.2.1The trap is packed with 1 cm of methyl silicone coated packing (Section 6.4.2) and 23 cm of 2,6-diphenylene oxide polymer (Section 6.4.1) as shown in Figure 2. This trap was used to develop the method performance statements in Section 12.

5.2.2.2Alternatively, either of the two traps described in Method 601 may be used, although water vapor will preclude the measurement of low concentrations of benzene.

5.2.3The desorber must be capable of rapidly heating the trap to 180 C.

The polymer section of the trap should not be heated higher than 180 C and the remaining sections should not exceed 200 C. The desorber illustrated in Figure 2 meets these design criteria.

5.2.4The purge and trap system may be assembled as a separate unit or be coupled to a gas chromatograph as illustrated in Figures 3, 4, and 5.

5.3Gas chromatographAn analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.3.1Column 16 ft long 0.082 in. ID stainless steel or glass, packed with 5% SP1200 and 1.75% Bentone-34 on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 12. Guidelines for the use of alternate column packings are provided in Section 10.1.

5.3.2Column 28 ft long 0.1 in ID stainless steel or glass, packed with 5% 1,2,3-Tris(2-cyanoethoxy)propane on Chromosorb W-AW (60/80 mesh) or equivalent.

5.3.3DetectorPhotoionization detector (h-Nu Systems, Inc. Model PI5102 or equivalent). This type of detector has been proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 12. Guidelines for the use of alternate detectors are provided in Section 10.1.

5.4Syringes5-mL glass hypodermic with Luerlok tip (two each), if applicable to the purging device.

5.5Micro syringes25-L, 0.006 in. ID needle.

5.6Syringe valve2-way, with Luer ends (three each).

5.7Bottle15-mL, screw-cap, with Teflon cap liner.

5.8BalanceAnalytical, capable of accurately weighing 0.0001 g.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3Reagent water may also be prepared by boiling water for 15 min.

Subsequently, while maintaining the temperature at 90 C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.2Sodium thiosulfate(ACS) Granular.

6.3Hydrochloric acid (1+1)Add 50 mL of concentrated HCl (ACS) to 50 mL of reagent water.

6.4Trap Materials: 6.4.12,6-Diphenylene oxide polymerTenax, (60/80 mesh), chromatographic grade or equivalent.

6.4.2Methyl silicone packing3% OV1 on Chromosorb-W (60/80 mesh) or equivalent.

6.5MethanolPesticide quality or equivalent.

6.6Stock standard solutionsStock standard solutions may be prepared from pure standard materials or purchased as certified solutions. Prepare stock standard solutions in methanol using assayed liquids. Because of the toxicity of benzene and 1,4-dichlorobenzene, primary dilutions of these materials should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.6.1Place about 9.8 mL of methanol into a 10mL ground glass stoppered volumetric flask. Allow the flask to stand, unstoppered, for about 10 min or until all alcohol wetted surfaces have dried. Weigh the flask to the nearest 0.1 mg.

6.6.2Using a 100L syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the alcohol without contacting the neck of the flask.

6.6.3Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in g/L from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.6.4Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store at 4 C and protect from light.

6.6.5All standards must be replaced after one month, or sooner if comparison with check standards indicates a problem.

6.7Secondary dilution standardsUsing stock standard solutions, prepare secondary dilution standards in methanol that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3.1 or 7.4.1 will bracket the working range of the analytical system. Secondary solution standards must be stored with zero headspace and should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.8Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1. Calibrate the purge and trap-gas chromatographic system using either the external standard technique (Section 7.3) or the internal standard technique (Section 7.4).

7.3External standard calibration procedure: 7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter by carefully adding 20.0 L of one or more secondary dilution standards to 100, 500, or 1000 mL of reagent water. A 25-L syringe with a 0.006 in. ID needle should be used for this operation. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector. These aqueous standards must be prepared fresh daily.

7.3.2Analyze each calibration standard according to Section 10, and tabulate peak height or area responses versus the concentration in the standard. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to concentration (calibration factor) is a constant over the working range (7.4Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples. The compound, ,,,-trifluorotoluene, recommended as a surrogate spiking compound in Section 8.7 has been used successfully as an internal standard.

7.4.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest as described in Section 7.3.1.

7.4.2Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.6 and 6.7. It is recommended that the secondary dilution standard be prepared at a concentration of 15 g/mL of each internal standard compound. The addition of 10 l of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 g/L.

7.4.3Analyze each calibration standard according to Section 10, adding 10 L of internal standard spiking solution directly to the syringe (Section 10.4). Tabulate peak height or area responses against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

RF= (As)(Cis) (Ais)(Cs) ---------------------------------------------------------------------------------------------------------------- Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard Cs=Concentration of the parameter to be measured.

If the RF value over the working range is a constant (7.5The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of a QC check sample.

7.5.1Prepare the QC check sample as described in Section 8.2.2.

7.5.2Analyze the QC check sample according to Section 10.

7.5.3For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 2. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, a new calibration curve, calibration factor, or RF must be prepared for that parameter according to Section 7.3 or 7.4.

8. Quality Control 8.1 Each laboratory that uses this method is required to operate a formal quality control program. The mimimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 10.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 10 g/mL in methanol. The QC check sample concentrate must be obtained from the U.S.

Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Prepare a QC check sample to contain 20 g/L of each parameter by adding 200 L of QC check sample concentrate to 100 mL of reagant water.

8.2.3Analyze four 5-mL aliquots of the well-mixed QC check sample according to Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter of interest using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note: The large number of parameters in Table 2 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.3.

8.2.6.2Beginning with Section 8.2.3, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.3.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 L of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.

7 If spiking was performed at a concentration lower than 20 g/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 3, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T) 2.44(100 S/T)%. 7 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 10 L of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

8.7The analyst should monitor both the performance of the analytical system and the effectiveness of the method in dealing with each sample matrix by spiking each sample, standard, and reagent water blank with surrogate compounds (e.g. , , ,-trifluorotoluene) that encompass the range of the temperature program used in this method. From stock standard solutions prepared as in Section 6.6, add a volume to give 750 g of each surrogate to 45 mL of reagent water contained in a 50-mL volumetric flask, mix and dilute to volume for a concentration of 15 mg/L.

Add 10 L of this surrogate spiking solution directly into the 5-mL syringe with every sample and reference standard analyzed. Prepare a fresh surrogate spiking solution on a weekly basis. If the internal standard calibration procedure is being used, the surrogate compounds may be added directly to the internal standard spiking solution (Section 7.4.2).

9. Sample Collection, Preservation, and Handling 9.1The samples must be iced or refrigerated from the time of collection until analysis. If the sample contains free or combined chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Method 330.4 or 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2Collect about 500 mL of sample in a clean container. Adjust the pH of the sample to about 2 by adding 1+1 HCl while stirring. Fill the sample bottle in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3All samples must be analyzed within 14 days of collection.3 10. Procedure 10.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are estimated retention times and MDL that can be achieved under these conditions. An example of the separations achieved by Column 1 is shown in Figure 6. Other packed columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

10.2Calibrate the system daily as described in Section 7.

10.3Adjust the purge gas (nitrogen or helium) flow rate to 40 mL/min.

Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

10.4Allow the sample to come to ambient temperature prior to introducing it to the syringe. Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing.

Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 L of the surrogate spiking solution (Section 8.7) and 10.0 L of the internal standard spiking solution (Section 7.4.2), if applicable, through the valve bore, then close the valve.

10.5Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

10.6Close both valves and purge the sample for 12.0 0.1 min at ambient temperature.

10.7After the 12-min purge time, disconnect the purging device from the trap. Dry the trap by maintaining a flow of 40 mL/min of dry purge gas through it for 6 min (Figure 4). If the purging device has no provision for bypassing the purger for this step, a dry purger should be inserted into the device to minimize moisture in the gas. Attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 5), and begin to temperature program the gas chromatograph.

Introduce the trapped materials to the GC column by rapidly heating the trap to 180 C while backflushing the trap with an inert gas between 20 and 60 mL/min for 4 min. If rapid heating of the trap cannot be achieved, the GC column must be used as a secondary trap by cooling it to 30 C (subambient temperature, if poor peak geometry and random retention time problems persist) instead of the initial program temperature of 50 C.

10.8While the trap is being desorbed into the gas chromatograph column, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

10.9After desorbing the sample for 4 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s, then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at 180 C. After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

10.10Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

10.11If the response for a peak exceeds the working range of the system, prepare a dilution of the sample with reagent water from the aliquot in the second syringe and reanalyze.

11. Calculations 11.1Determine the concentration of individual compounds in the sample.

11.1.1If the external standard calibration procedure is used, calculate the concentration of the parameter being measured from the peak response using the calibration curve or calibration factor determined in Section 7.3.2.

11.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.4.3 and Equation 2. (image) Equation 2 where: As = Response for the parameter to be measured.

Ais = Response for the internal standard.

Cis = Concentration of the internal standard.

11.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

12. Method Performance 12.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.9 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

12.2This method has been demonstrated to be applicable for the concentration range from the MDL to 100 MDL.9 Direct aqueous injection techniques should be used to measure concentration levels above 1000 MDL.

12.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 2.1 to 550 g/L.9 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References 1. 40 CFR part 136, appendix B.

2. Lichtenberg, J.J. Determining Volatile Organics at Microgram-per-Litre-Levels by Gas Chromatography, Journal American Water Works Association, 66, 739 (1974).

3. Bellar, T.A., and Lichtenberg, J.J. Semi-Automated Headspace Analysis of Drinking Waters and Industrial Waters for Purgeable Volatile Organic Compounds, Proceedings of Symposium on Measurement of Organic Pollutants in Water and Wastewater. American Society for Testing and Materials, STP 686, C.E. Van Hall, editor, 1978.

4. CarcinogensWorking with Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health. Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3. is two times the value 1.22 derived in this report.) 8.Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Office of Research and Development, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268. March 1979.

9. EPA Method Study 25, Method 602, Purgeable Aromatics, EPA 600/484042, National Technical Information Service, PB84196682, Springfield, Virginia 22161, May 1984.

Table 1_Chromatographic Conditions and Method Detection Limits ------------------------------------------------------------------------ Retention time (min) Method ---------------------- detection Parameter limit Column 1 Column 2 (g/ L) ------------------------------------------------------------------------ Benzene............................... 3.33 2.75 0.2 Toluene............................... 5.75 4.25 0.2 Ethylbenzene.......................... 8.25 6.25 0.2 Chlorobenzene......................... 9.17 8.02 0.2 1,4-Dichlorobenzene................... 16.8 16.2 0.3 1,3-Dichlorobenzene................... 18.2 15.0 0.4 1,2-Dichlorobenzene................... 25.9 19.4 0.4 ------------------------------------------------------------------------ Column 1 conditions: Supelcoport (100/120 mesh) coated with 5% SP-1200/ 1.75% Bentone-34 packed in a 6 ft x 0.085 in. ID stainless steel column with helium carrier gas at 36 mL/min flow rate. Column temperature held at 50 C for 2 min then programmed at 6 C/ min to 90 C for a final hold.

Column 2 conditions: Chromosorb W-AW (60/80 mesh) coated with 5% 1,2,3- Tris(2-cyanoethyoxy)propane packed in a 6 ft x 0.085 in. ID stainless steel column with helium carrier gas at 30 mL/min flow rate. Column temperature held at 40 C for 2 min then programmed at 2 C/ min to 100 C for a final hold.

Table 2_Calibration and QC Acceptance Criteria_Method 602 a ---------------------------------------------------------------------------------------------------------------- Limit for Range for X Range for Q s (g/ Range for Parameter (g/ (g/ L) P, Ps(%) L) L) ---------------------------------------------------------------------------------------------------------------- Benzene........................................................ 15.4-24.6 4.1 10.0-27.9 39-150 Chlorobenzene.................................................. 16.1-23.9 3.5 12.7-25.4 55-135 1,2-Dichlorobenzene............................................ 13.6-26.4 5.8 10.6-27.6 37-154 1,3-Dichlorobenzene............................................ 14.5-25.5 5.0 12.8-25.5 50-141 1,4-Dichlorobenzene............................................ 13.9-26.1 5.5 11.6-25.5 42-143 Ethylbenzene................................................... 12.6-27.4 6.7 10.0-28.2 32-160 Toluene........................................................ 15.5-24.5 4.0 11.2-27.7 46-148 ---------------------------------------------------------------------------------------------------------------- Q=Concentration measured in QC check sample, in g/L (Section 7.5.3).

s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

Ps, P=Percent recovery measured (Section 8.3.2, Section 8.4.2).

a Criteria were calculated assuming a QC check sample concentration of 20 g/L.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3_Method Accuracy and Precision as Functions of Concentration_Method 602 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Single analyst Overall recovery, precision, precision, Parameter X[prime] s[prime] S[prime] (g/L) (g/L) (g/L) ---------------------------------------------------------------------------------------------------------------- Benzene......................................................... 0.92C+0.57 0.09X+0.59 0.21X+0.56 Chlorobenzene................................................... 0.95C+0.02 0.09X+0.23 0.17X+0.10 1,2-Dichlorobenzene............................................. 0.93C+0.52 0.17X-0.04 0.22X+0.53 1,3-Dichlorobenzene............................................. 0.96C-0.05 0.15X-0.10 0.19X+0.09 1,4-Dichlorobenzene............................................. 0.93C-0.09 0.15X+0.28 0.20X+0.41 Ethylbenzene.................................................... 0.94C+0.31 0.17X+0.46 0.26X+0.23 Toluene......................................................... 0.94C+0.65 0.09X+0.48 0.18X+0.71 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

S[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in X g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the Concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

(image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF Method 603Acrolein and Acrylonitrile 1. Scope and Application 1.1This method covers the determination of acrolein and acrylonitrile.

The following parameters may be determined by this method: ------------------------------------------------------------------------ STORET Parameter No. CAS No.

------------------------------------------------------------------------ Acrolein......................................... 34210 107-02-8 Acrylonitrile.................................... 34215 107-13-1 ------------------------------------------------------------------------ 1.2This is a purge and trap gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for either or both of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 624 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for the parameters listed above, if used with the purge and trap conditions described in this method.

1.3The method detection limit (MDL, defined in Section 12.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1An inert gas is bubbled through a 5-mL water sample contained in a heated purging chamber. Acrolein and acrylonitrile are transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the analytes are trapped. After the purge is completed, the trap is heated and backflushed with the inert gas to desorb the compound onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the analytes which are then detected with a flame ionization detector.2,3 2.2The method provides an optional gas chromatographic column that may be helpful in resolving the compounds of interest from the interferences that may occur.

3. Interferences 3.1Impurities in the purge gas and organic compound outgassing from the plumbing of the trap account for the majority of contamination problems.

The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2Samples can be contaminated by diffusion of volatile organics through the septum seal into the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed between samples with reagent water. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high analyte levels, it may be necessary to wash the purging device with a detergent solution, rinse it with distilled water, and then dry it in an oven at 105C between analyses. The trap and other parts of the system are also subject to contamination, therefore, frequent bakeout and purging of the entire system may be required.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this view point, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4,6 for the information of the analyst.

5. Apparatus and Materials 5.1Sampling equipment, for discrete sampling.

5.1.1Vial25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 C before use.

5.1.2SeptumTeflon-faced silicone (Pierce #12722 or equivalent).

Detergent wash, rinse with tap and distilled water and dry at 105 C for 1 h before use.

5.2Purge and trap systemThe purge and trap system consists of three separate pieces of equipment: a purging device, trap, and desorber.

Several complete systems are now commercially available.

5.2.1The purging device must be designed to accept 5-mL, samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL.

The purge gas must pass through the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column.

The purging device must be capable of being heated to 85 C within 3.0 min after transfer of the sample to the purging device and being held at 85 2 C during the purge cycle. The entire water column in the purging device must be heated. Design of this modification to the standard purging device is optional, however, use of a water bath is suggested.

5.2.1.1Heating mantleTo be used to heat water bath.

5.2.1.2Temperature controllerEquipped with thermocouple/sensor to accurately control water bath temperature to 2 C. The purging device illustrated in Figure 1 meets these design criteria.

5.2.2The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in. The trap must be packed to contain 1.0 cm of methyl silicone coated packing (Section 6.5.2) and 23 cm of 2,6-diphenylene oxide polymer (Section 6.5.1). The minimum specifications for the trap are illustrated in Figure 2.

5.2.3The desorber must be capable of rapidly heating the trap to 180 C, The desorber illustrated in Figure 2 meets these design criteria.

5.2.4The purge and trap system may be assembled as a separate unit as illustrated in Figure 3 or be coupled to a gas chromatograph.

5.3pH paperNarrow pH range, about 3.5 to 5.5 (Fisher Scientific Short Range Alkacid No. 2, #148372 or equivalent).

5.4Gas chromatographAn analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.4.1Column 110 ft long 2 mm ID glass or stainless steel, packed with Porapak-QS (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 12. Guidelines for the use of alternate column packings are provided in Section 10.1.

5.4.2Column 26 ft long 0.1 in. ID glass or stainless steel, packed with Chromosorb 101 (60/80 mesh) or equivalent.

5.4.3DetectorFlame ionization detector. This type of detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 12. Guidelines for the use of alternate detectors are provided in Section 10.1.

5.5Syringes5-mL, glass hypodermic with Luerlok tip (two each).

5.6Micro syringes25-L, 0.006 in. ID needle.

5.7Syringe valve2-way, with Luer ends (three each).

5.8Bottle15-mL, screw-cap, with Teflon cap liner.

5.9BalanceAnalytical, capable of accurately weighing 0.0001 g.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3Regent water may also be prepared by boiling water for 15 min.

Subsequently, while maintaining the temperature at 90 C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.2Sodium thiosulfate(ACS) Granular.

6.3Sodium hydroxide solution (10 N)Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.4Hydrochloric acid (1+1)Slowly, add 50 mL of concentrated HCl (ACS) to 50 mL of reagent water.

6.5Trap Materials: 6.5.12,6-Diphenylene oxide polymerTenax (60/80 mesh), chromatographic grade or equivalent.

6.5.2Methyl silicone packing3% OV1 on Chromosorb-W (60/80 mesh) or equivalent.

6.6Stock standard solutionsStock standard solutions may be prepared from pure standard materials or purchased as certified solutions. Prepare stock standard solutions in reagent water using assayed liquids. Since acrolein and acrylonitrile are lachrymators, primary dilutions of these compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.6.1Place about 9.8 mL of reagent water into a 10-mL ground glass stoppered volumetric flask. For acrolein standards the reagent water must be adjusted to pH 4 to 5. Weight the flask to the nearest 0.1 mg.

6.6.2Using a 100-L syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the water without contacting the neck of the flask.

6.6.3Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in g/L from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock staldard. Optionally, stock standard solutions may be prepared using the pure standard material by volumetrically measuring the appropriate amounts and determining the weight of the material using the density of the material. Commercially prepared stock standards may be used at any concentration if they are certified by the manufactaurer or by an independent source.

6.6.4Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store at 4 C and protect from light.

6.6.5Prepare fresh standards daily.

6.7Secondary dilution standardsUsing stock standard solutions, prepare secondary dilution standards in reagent water that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3.1 or 7.4.1 will bracket the working range of the analytical system. Secondary dilution standards should be prepared daily and stored at 4 C.

6.8Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1. Calibrate the purge and trap-gas chromatographic system using either the external standard technique (Section 7.3) or the internal standard technique (Section 7.4).

7.3External standard calibration procedure: 7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter by carefully adding 20.0 L of one or more secondary dilution standards to 100, 500, or 1000 mL of reagent water. A 25-L syringe with a 0.006 in. ID needle should be used for this operation. One of the external standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector. These standards must be prepared fresh daily.

7.3.2Analyze each calibration standard according to Section 10, and tabulate peak height or area responses versus the concentration of the standard. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to concentration (calibration factor) is a constant over the working range (7.4Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.4.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest as described in Section 7.3.1.

7.4.2Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.6 and 6.7. It is recommended that the secondary dilution standard be prepared at a concentration of 15 g/mL of each internal standard compound. The addition of 10 L of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 g/L.

7.4.3Analyze each calibration standard according to Section 10, adding 10 L of internal standard spiking solution directly to the syringe (Section 10.4). Tabulate peak height or area responses against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

RF= (As)(Cis) (Ais)(Cs) ---------------------------------------------------------------------------------------------------------------- Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard.

Cs=Concentration of the parameter to be measured.

If the RF value over the working range is a constant (7.5The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of a QC check sample.

7.5.1Prepare the QC check sample as described in Section 8.2.2.

7.5.2Analyze the QC check sample according to Section 10.

7.5.3For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 2. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, a new calibration curve, calibration factor, or RF must be prepared for that parameter according to Section 7.3 or 7.4.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 10.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 25 g/mL in reagent water. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Prepare a QC check sample to contain 50 g/L of each parameter by adding 200 L of QC check sample concentrate to 100 mL of reagent water.

8.2.3Analyze four 5-mL aliquots of the well-mixed QC check sample according to Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If either s exceeds the precision limit or X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for each compound of interest.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 50 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 L of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 10 L of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1All samples must be iced or refrigerated from the time of collection until analysis. If the sample contains free or combined chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2If acrolein is to be analyzed, collect about 500 mL of sample in a clean glass container. Adjust the pH of the sample to 4 to 5 using acid or base, measuring with narrow range pH paper. Samples for acrolein analysis receiving no pH adjustment must be analyzed within 3 days of sampling.

9.3Grab samples must be collected in glass containers having a total volume of at least 25 mL. Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. If preservative has been added, shake vigorously for 1 min. Maintain the hermetic seal on the sample bottle until time of analysis.

9.4All samples must be analyzed within 14 days of collection.3 10. Procedure 10.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are estimated retention times and MDL that can be achieved under these conditions. An example of the separations achieved by Column 1 is shown in Figure 5. Other packed columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

10.2Calibrate the system daily as described in Section 7.

10.3Adjust the purge gas (nitrogen or helium) flow rate to 20 mL-min.

Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

10.4Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing. Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 L of the internal standard spiking solution (Section 7.4.2), if applicable, through the valve bore then close the valve.

10.5Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

10.6Close both valves and purge the sample for 15.0 0.1 min while heating at 85 2 C.

10.7After the 15-min purge time, attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 4), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 C while backflushing the trap with an inert gas between 20 and 60 mL/min for 1.5 min.

10.8While the trap is being desorbed into the gas chromatograph, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

10.9After desorbing the sample for 1.5 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at 210 C. After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

10.10Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

11. Calculations 11.1Determine the concentration of individual compounds in the sample.

11.1.1If the external standard calibration procedure is used, calculate the concentration of the parameter being measured from the peak response using the calibration curve or calibration factor determined in Section 7.3.2.

11.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.4.3 and Equation 2. (image) Equation 2 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard.

11.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

12. Method Performance 12.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.9 The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

12.2This method is recommended for the concentration range from the MDL to 1,000MDL. Direct aqueous injection techniques should be used to measure concentration levels above 1,000MDL.

12.3In a single laboratory (Battelle-Columbus), the average recoveries and standard deviations presented in Table 2 were obtained.9 Seven replicate samples were analyzed at each spike level.

References 1. 40 CFR part 136, appendix B.

2. Bellar, T.A., and Lichtenberg, J.J. Determining Volatile Organics at Microgram-per-Litre-Levels by Gas Chromatography, Journal American Water Works Association, 66, 739 (1974).

3. Evaluate Test Procedures for Acrolein and Acrylonitrile, Special letter report for EPA Project 4719A, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, 27 June 1979.

4. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983).

8. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

9. Evaluation of Method 603 (Modified), EPA600/484ABC, National Technical Information Service, PB84, Springfield, Virginia 22161, Nov.

1984.

Table 1_Chromatographic Conditions and Method Detection Limits ------------------------------------------------------------------------ Retention time (min) Method ------------------------ detection Parameter limit Column 1 Column 2 (g/ L) ------------------------------------------------------------------------ Acrolein............................ 10.6 8.2 0.7 Acrylonitrile....................... 12.7 9.8 0.5 ------------------------------------------------------------------------ Column 1 conditions: Porapak-QS (80/100 mesh) packed in a 10 ft x 2 mm ID glass or stainless steel column with helium carrier gas at 30 mL/ min flow rate. Column temperature held isothermal at 110 C for 1.5 min (during desorption), then heated as rapidly as possible to 150 C and held for 20 min; column bakeout at 190 C for 10 min.\9\ Column 2 conditions: Chromosorb 101 (60/80 mesh) packed in a 6 ft. x 0.1 in. ID glass or stainless steel column with helium carrier gas at 40 mL/min flow rate. Column temperature held isothermal at 80 C for 4 min, then programmed at 50 C/min to 120 C and held for 12 min.

Table 2_Single Laboratory Accuracy and Precision_Method 603 ---------------------------------------------------------------------------------------------------------------- Spike Average Standard Sample conc. recovery deviation Average Parameter matrix (g/ (g/ (g/ percent L) L) L) recovery ---------------------------------------------------------------------------------------------------------------- Acrolein............................................... RW 5.0 5.2 0.2 104 RW 50.0 51.4 0.7 103 POTW 5.0 4.0 0.2 80 POTW 50.0 44.4 0.8 89 IW 5.0 0.1 0.1 2 IW 100.0 9.3 1.1 9 Acrylonitrile.......................................... RW 5.0 4.2 0.2 84 RW 50.0 51.4 1.5 103 POTW 20.0 20.1 0.8 100 POTW 100.0 101.3 1.5 101 IW 10.0 9.1 0.8 91 IW 100.0 104.0 3.2 104 ---------------------------------------------------------------------------------------------------------------- ARW=Reagent water.

APOTW=Prechlorination secondary effluent from a municipal sewage treatment plant.

AIW=Industrial wastewater containing an unidentified acrolein reactant.

Table 3_Calibration and QC Acceptance Criteria_Method 603 \a\ ---------------------------------------------------------------------------------------------------------------- Limit for Range for Q S Range for X Range for Parameter (g/ (g/ (g/ P, Ps (%) L) L) L) ---------------------------------------------------------------------------------------------------------------- Acrolein..................................................... 45.9-54.1 4.6 42.9-60.1 88-118 Acrylonitrile................................................ 41.2-58.8 9.9 33.1-69.9 71-135 ---------------------------------------------------------------------------------------------------------------- a=Criteria were calculated assuming a QC check sample concentration of 50 g/L.9 Q=Concentration measured in QC check sample, in g/L (Section 7.5.3).

s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

(image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF Method 604Phenols 1. Scope and Application 1.1 This method covers the determination of phenol and certain substituted phenols. The following parameters may be determined by this method: ------------------------------------------------------------------------ STORET Parameter No. CAS No.

------------------------------------------------------------------------ 4-Chloro-3-methylphenol.......................... 34452 59-50-7 2--Chlorophenol.................................. 34586 95-57-8 2,4-Dichlorophenol............................... 34601 120-83-2 2,4-Dimethylphenol............................... 34606 105-67-9 2,4-Dinitrophenol................................ 34616 51-28-5 2-Methyl-4,6-dinitrophenol....................... 34657 534-52-1 2-Nitrophenol.................................... 34591 88-75-5 4-Nitrophenol.................................... 34646 100-02-7 Pentachlorophenol................................ 39032 87-86-5 Phenol........................................... 34694 108-95-2 2,4,6-Trichlorophenol............................ 34621 88-06-2 ------------------------------------------------------------------------ 1.2This is a flame ionization detector gas chromatographic (FIDGC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for derivatization, cleanup, and electron capture detector gas chromatography (ECDGC) that can be used to confirm measurements made by FIDGC. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3 The method detection limit (MDL, defined in Section 14.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix. The MDL listed in Table 1 for each parameter was achieved with a flame ionization detector (FID). The MDLs that were achieved when the derivatization cleanup and electron capture detector (ECD) were employed are presented in Table 2.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1-L, is acidified and extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to 2-propanol during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the phenols are then measured with an FID. 2 2.2A preliminary sample wash under basic conditions can be employed for samples having high general organic and organic base interferences.

2.3The method also provides for a derivatization and column chromatography cleanup procedure to aid in the elimination of interferences.2,3 The derivatives are analyzed by ECDGC.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned. 4 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating.

Thorough rinsing with such solvents usually eliminates PCB interference.

Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants.

Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are coextracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The derivatization cleanup procedure in Section 12 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Tables 1 and 2.

3.3The basic sample wash (Section 10.2) may cause significantly reduced recovery of phenol and 2,4-dimethylphenol. The analyst must recognize that results obtained under these conditions are minimum concentrations.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this mothod has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified57 for the information of analyst.

4.2Special care should be taken in handling pentafluorobenzyl bromide, which is a lachrymator, and 18-crown-6-ether, which is highly toxic.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.): 5.2.1Separatory funnel2L, with Teflon stopcock.

5.2.2Drying columnChromatographic column, 400 mm long 19 mm ID, with coarse frit filter disc.

5.2.3Chromatographic column100 mm long 10 mm ID, with Teflon stopcock.

5.2.4Concentrator tube, Kuderna-Danish10-mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5Evaporative flask, Kuderna-Danish500-mL (Kontes K5700010500 or equivalent). Attach to concentrator tube with springs.

5.2.6Snyder column, Kuderna-DanishThree-ball macro (Kontes K5030000121 or equivalent).

5.2.7Snyder column, Kuderna-DanishTwo-ball micro (Kontes K5690010219 or equivalent).

5.2.8Vials10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.2.9Reaction flask15 to 25-mL round bottom flask, with standard tapered joint, fitted with a water-cooled condenser and U-shaped drying tube containing granular calcium chloride.

5.3Boiling chipsApproximately 10/40 mesh. Heat to 400 C for 30 min or Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2C). The bath should be used in a hood.

5.5BalanceAnalytical, capable of accurately weighting 0.0001 g.

5.6Gas chromatographAn analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column for underivatized phenols1.8 m long 2 mm ID glass, packed with 1% SP1240DA on Supelcoport (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 14.

Guidelines for the use of alternate column packings are provided in Section 11.1.

5.6.2Column for derivatized phenols1.8 m long 2 mm ID glass, packed with 5% OV17 on Chromosorb W-AW-DMCS (80/100 mesh) or equivalent. This column has proven effective in the analysis of wastewaters for derivatization products of the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14.

Guidelines for the use of alternate column packings are provided in Section 11.1.

5.6.3DetectorsFlame ionization and electron capture detectors. The FID is used when determining the parent phenols. The ECD is used when determining the derivatized phenols. Guidelines for the use of alternatve detectors are provided in Section 11.1.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Sodium hydroxide solution (10 N)Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3Sodium hydroxide solution (1 N)Dissolve 4 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.4Sodium sulfate(ACS) Granular, anhydrous. Purify by heating at 400C for 4 h in a shallow tray.

6.5Sodium thiosulfate(ACS) Granular.

6.6Sulfuric acid (1+1)Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.7Sulfuric acid (1 N)Slowly, add 58 mL of H2SO4 (ACS, sp. gr. 1.84) to reagent water and dilute to 1 L.

6.8Potassium carbonate(ACS) Powdered.

6.9Pentafluorobenzyl bromide (-Bromopentafluorotoluene)97% minimum purity.

Note: This chemical is a lachrymator. (See Section 4.2.) 6.1018-crown-6-ether (1,4,7,10,13,16-Hexaoxacyclooctadecane)98% minimum purity.

Note: This chemical is highly toxic.

6.11Derivatization reagentAdd 1 mL of pentafluorobenzyl bromide and 1 g of 18-crown-6-ether to a 50-mL volumetric flask and dilute to volume with 2-propanol. Prepare fresh weekly. This operation should be carried out in a hood. Store at 4 C and protect from light.

6.12Acetone, hexane, methanol, methylene chloride, 2-propanol, toluenePesticide quality or equivalent.

6.13Silica gel100/200 mesh, Davison, grade-923 or equivalent. Activate at 130 C overnight and store in a desiccator.

6.14Stock standard solutions (1.00 g/L)Stock standard solutions may be prepared from pure standard materials or purchased as certified solutions.

6.14.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in 2-propanol and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.14.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.14.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.15Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1To calibrate the FIDGC for the anaylsis of underivatized phenols, establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure for FIDGC: 7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with 2-propanol. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 l, analyze each calibration standard according to Section 11 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (7.3Internal standard calibration procedure for FIDGCTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with 2-propanol. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 11 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF= (As)(Cis) (Ais)(Cs) ---------------------------------------------------------------------------------------------------------------- Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 15%, a new calibration curve must be prepared for that compound.

7.5To calibrate the ECDGC for the analysis of phenol derivatives, establish gas chromatographic operating conditions equivalent to those given in Table 2.

7.5.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with 2-propanol. One of the external standards should be at a concentration near, but above, the MDL (Table 2) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.5.2Each time samples are to be derivatized, simultaneously treat a 1-mL aliquot of each calibration standard as described in Section 12.

7.5.3After derivatization, analyze 2 to 5 L of each column eluate collected according to the method beginning in Section 12.8 and tabulate peak height or area responses against the calculated equivalent mass of underivatized phenol injected. The results can be used to prepare a calibration curve for each compound.

7.6Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.6 and 11.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 100 g/mL in 2-propanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 100 g/L by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note: The large number of parameters in Talbe 3 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 100 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any, or, if none, (2) the larger of either 5 times higher than the expected background concentration or 100 g/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.8 If spiking was performed at a concentration lower than 100 g/L, the analyst must use either the QC acceptance criteria in Table 3, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 4, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 4, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T)2.44(100 S/T)%.8 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6.It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices9 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.10 Field test kits are available for this purpose.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2 10. Sample Extraction 10.1Mark the water meniscus on the side of sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2For samples high in organic content, the analyst may solvent wash the sample at basic pH as prescribed in Sections 10.2.1 and 10.2.2 to remove potential method interferences. Prolonged or exhaustive contact with solvent during the wash may result in low recovery of some of the phenols, notably phenol and 2,4-dimethylphenol. For relatively clean samples, the wash should be omitted and the extraction, beginning with Section 10.3, should be followed.

10.2.1Adjust the pH of the sample to 12.0 or greater with sodium hydroxide solution.

10.2.2Add 60 mL of methylene chloride to the sample by shaking the funnel for 1 min with periodic venting to release excess pressure.

Discard the solvent layer. The wash can be repeated up to two additional times if significant color is being removed.

10.3Adjust the sample to a pH of 1 to 2 with sulfuric acid.

10.4Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.5Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.6Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.7Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.8Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.9Increase the temperature of the hot water bath to 95 to 100 C.

Remove the Synder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of 2-propanol. A 5-mL syringe is recommended for this operation. Attach a two-ball micro-Snyder column to the concentrator tube and prewet the column by adding about 0.5 mL of 2-propanol to the top. Place the micro-K-D apparatus on the water bath so that the concentrator tube is partially immersed in the hot water.

Adjust the vertical position of the apparatus and the water temperature as required to complete concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 2.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Add an additional 2 mL of 2-propanol through the top of the micro-Snyder column and resume concentrating as before. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.10Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of 2-propanol. Adjust the extract volume to 1.0 mL. Stopper the concentrator tube and store refrigerated at 4 C if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with FIDGC analysis (Section 11). If the sample requires further cleanup, proceed to Section 12.

10.11Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder.

Record the sample volume to the nearest 5 mL.

11. Flame Ionization Detector Gas Chromatography 11.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. An example of the separations achieved by this column is shown in Figure 1. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

11.2Calibrate the system daily as described in Section 7.

11.3If the internal standard calibration procedure is used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

11.4Inject 2 to 5 L of the sample extract or standard into the gas chromatograph using the solvent-flush technique.11 Smaller (1.0 L) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L, and the resulting peak size in area or peak height units.

11.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound may be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

11.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

11.7If the measurement of the peak response is prevented by the presence of interferences, an alternative gas chromatographic procedure is required. Section 12 describes a derivatization and column chromatographic procedure which has been tested and found to be a practical means of analyzing phenols in complex extracts.

12. Derivatization and Electron Capture Detector Gas Chromatography 12.1Pipet a 1.0-mL aliquot of the 2-propanol solution of standard or sample extract into a glass reaction vial. Add 1.0 mL of derivatizing reagent (Section 6.11). This amount of reagent is sufficient to derivatize a solution whose total phenolic content does not exceed 0.3 mg/mL.

12.2Add about 3 mg of potassium carbonate to the solution and shake gently.

12.3Cap the mixture and heat it for 4 h at 80 C in a hot water bath.

12.4Remove the solution from the hot water bath and allow it to cool.

12.5Add 10 mL of hexane to the reaction flask and shake vigorously for 1 min. Add 3.0 mL of distilled, deionized water to the reaction flask and shake for 2 min. Decant a portion of the organic layer into a concentrator tube and cap with a glass stopper.

12.6Place 4.0 g of silica gel into a chromatographic column. Tap the column to settle the silica gel and add about 2 g of anhydrous sodium sulfate to the top.

12.7Preelute the column with 6 mL of hexane. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, pipet onto the column 2.0 mL of the hexane solution (Section 12.5) that contains the derivatized sample or standard. Elute the column with 10.0 mL of hexane and discard the eluate. Elute the column, in order, with: 10.0 mL of 15% toluene in hexane (Fraction 1); 10.0 mL of 40% toluene in hexane (Fraction 2); 10.0 mL of 75% toluene in hexane (Fraction 3); and 10.0 mL of 15% 2-propanol in toluene (Fraction 4). All elution mixtures are prepared on a volume: volume basis. Elution patterns for the phenolic derivatives are shown in Table 2. Fractions may be combined as desired, depending upon the specific phenols of interest or level of interferences.

12.8Analyze the fractions by ECDGC. Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions.

An example of the separations achieved by this column is shown in Figure 2.

12.9Calibrate the system daily with a minimum of three aliquots of calibration standards, containing each of the phenols of interest that are derivatized according to Section 7.5.

12.10Inject 2 to 5 L of the column fractions into the gas chromatograph using the solvent-flush technique. Smaller (1.0 L) volumes can be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L, and the resulting peak size in area or peak height units. If the peak response exceeds the linear range of the system, dilute the extract and reanalyze.

13. Calculations 13.1Determine the concentration of individual compounds in the sample analyzed by FIDGC (without derivatization) as indicated below.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2. (image) Equation 2 where: A=Amount of material injected (ng).

Vi=Volume of extract injected (L).

Vt=Volume of total extract (L).

Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3. (image) Equation 3 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

13.2Determine the concentration of individual compounds in the sample analyzed by derivatization and ECDGC according to Equation 4. (image) Equation 4 where: A=Mass of underivatized phenol represented by area of peak in sample chromatogram, determined from calibration curve in Section 7.5.3 (ng).

Vi=Volume of eluate injected (L).

Vt=Total volume of column eluate or combined fractions from which Vi was taken (L).

Vs=Volume of water extracted in Section 10.10 (mL).

B=Total volume of hexane added in Section 12.5 (mL).

C=Volume of hexane sample solution added to cleanup column in Section 12.7 (mL).

D=Total volume of 2-propanol extract prior to derivatization (mL).

E=Volume of 2-propanol extract carried through derivatization in Section 12.1 (mL).

13.3Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Tables 1 and 2 were obtained using reagent water.12 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked as six concentrations over the range 12 to 450 g/L. 13 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships for a flame ionization detector are presented in Table 4.

References 1. 40 CFR part 136, appendix B.

2. Determination of Phenols in Industrial and Municipal Wastewaters, EPA 600/484ABC, National Technical Information Service, PBXYZ, Springfield, Virginia 22161, November 1984.

3. Kawahara, F. K. Microdetermination of Derivatives of Phenols and Mercaptans by Means of Electron Capture Gas Chromatography, Analytical Chemistry, 40, 1009 (1968).

4. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

5. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

6. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

7. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

8. Provost, L. P., and Elder, R. S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 9. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

10. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methmds for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

11. Burke, J. A. Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects, Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

12. Development of Detection Limits, EPA Method 604, Phenols, Special letter report for EPA Contract 68032625, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.

13. EPA Method Study 14 Method 604-Phenols, EPA 600/484044, National Technical Information Service, PB84196211, Springfield, Virginia 22161, May 1984.

Table 1_Chromatographic Conditions and Method Detection Limits ------------------------------------------------------------------------ Method Retention detection Parameter time (min) limit (g/L) ------------------------------------------------------------------------ 2-Chlorophenol................................ 1.70 0.31 2-Nitrophenol................................. 2.00 0.45 Phenol........................................ 3.01 0.14 2,4-Dimethylphenol............................ 4.03 0.32 2,4-Dichlorophenol............................ 4.30 0.39 2,4,6-Trichlorophenol......................... 6.05 0.64 4-Chloro-3-methylphenol....................... 7.50 0.36 2,4-Dinitrophenol............................. 10.00 13.0 2-Methyl-4,6-dinitrophenol.................... 10.24 16.0 Pentachlorophenol............................. 12.42 7.4 4-Nitrophenol................................. 24.25 2.8 ------------------------------------------------------------------------ Column conditions: Supelcoport (80/100 mesh) coated with 1% SP-1240DA packed in a 1.8 m long x 2 mm ID glass column with nitrogen carrier gas at 30 mL/min flow rate. Column temperature was 80 C at injection, programmed immediately at 8 C/min to 150 C final temperature. MDL were determined with an FID.

Table 2_Silica Gel Fractionation and Electron Capture Gas Chromatography of PFBB Derivatives ---------------------------------------------------------------------------------------------------------------- Percent recovery by Method fraction a Retention detection Parent compound ---------------------------- time limit (min) (g/ 1 2 3 4 L) ---------------------------------------------------------------------------------------------------------------- 2-Chlorophenol............................................... ..... 90 1 ..... 3.3 0.58 2-Nitrophenol................................................ ..... ..... 9 90 9.1 0.77 Phenol....................................................... ..... 90 10 ..... 1.8 2.2 2,4-Dimethylphenol........................................... ..... 95 7 ..... 2.9 0.63 2,4-Dichlorophenol........................................... ..... 95 1 ..... 5.8 0.68 2,4,6-Trichlorophenol........................................ 50 50 ..... ..... 7.0 0.58 4-Chloro-3-methylphenol...................................... ..... 84 14 ..... 4.8 1.8 Pentachlorophenol............................................ 75 20 ..... ..... 28.8 0.59 4-Nitrophenol................................................ ..... ..... 1 90 14.0 0.70 ---------------------------------------------------------------------------------------------------------------- Column conditions: Chromosorb W-AW-DMCS (80/100 mesh) coated with 5% OV-17 packed in a 1.8 m long x 2.0 mm ID glass column with 5% methane/95% argon carrier gas at 30 mL/min flow rate. Column temperature held isothermal at 200 C. MDL were determined with an ECD.

a Eluant composition: Fraction 1_15% toluene in hexane.

Fraction 2_40% toluene in hexane.

Fraction 3_75% toluene in hexane.

Fraction 4_15% 2-propanol in toluene.

Table 3_QC Acceptance Criteria_Method 604 ---------------------------------------------------------------------------------------------------------------- Limit for Range for X Test conc. s (g/ Range for Parameter (g/ (g/ L) P, Ps L) L) (percent) ---------------------------------------------------------------------------------------------------------------- 4-Chloro-3-methylphenol....................................... 100 16.6 56.7-113.4 49-122 2-Chlorophenol................................................ 100 27.0 54.1-110.2 38-126 2,4-Dichlorophenol............................................ 100 25.1 59.7-103.3 44-119 2,4-Dimethylphenol............................................ 100 33.3 50.4-100.0 24-118 4,6-Dinitro-2-methylphenol.................................... 100 25.0 42.4-123.6 30-136 2,4-Dinitrophenol............................................. 100 36.0 31.7-125.1 12-145 2-Nitrophenol................................................. 100 22.5 56.6-103.8 43-117 4-Nitrophenol................................................. 100 19.0 22.7-100.0 13-110 Pentachlorophenol............................................. 100 32.4 56.7-113.5 36-134 Phenol........................................................ 100 14.1 32.4-100.0 23-108 2,4,6-Trichlorophenol......................................... 100 16.6 60.8-110.4 53-119 ---------------------------------------------------------------------------------------------------------------- s_Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X_Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps_Percent recovery measured (Section 8.3.2, Section 8.4.2).

Note: These criteria are based directly upon the method performance data in Table 4. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 4.

Table 4_Method Accuracy and Precision as Functions of Concentration_Method 604 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Single Analyst Overall recovery, precision, precision, Parameter X[prime] sr[prime] S[prime] (g/L) (g/L) (g/L) ---------------------------------------------------------------------------------------------------------------- 4-Chloro-3-methylphenol................................ 0.87C-1.97 0.11X-0.21 0.16X+1.41 2-Chlorophenol......................................... 0.83C-0.84 0.18X+0.20 0.21X+0.75 2,4-Dichlorophenol..................................... 0.81C+0.48 0.17X-0.02 0.18X+0.62 2,4-Dimethylphenol..................................... 0.62C-1.64 0.30X-0.89 0.25X+0.48 4,6-Dinitro-2-methylphenol............................. 0.84C-1.01 0.15X+1.25 0.19X+5.85 2,4-Dinitrophenol...................................... 0.80C-1.58 0.27X-1.15 0.29X+4.51 2-Nitrophenol.......................................... 0.81C-0.76 0.15X+0.44 0.14X+3.84 4-Nitrophenol.......................................... 0.46C+0.18 0.17X+2.43 0.19X+4.79 Pentachlorophenol...................................... 0.83C+2.07 0.22X-0.58 0.23X+0.57 Phenol................................................. 0.43C+0.11 0.20X-0.88 0.17X+0.77 2,4,6-Trichlorophenol.................................. 0.86C-0.40 0.10X+0.53 0.13X+2.40 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

(image) View or download PDF (image) View or download PDF Method 605Benzidines 1. Scope and Application 1.1This method covers the determination of certain benzidines. The following parameters can be determined by this method: ------------------------------------------------------------------------ Parameter Storet No CAS No.

------------------------------------------------------------------------ Benzidine..................................... 39120 92-87-5 3,3[prime]-Dichlorobenzidine.................. 34631 91-94-1 ------------------------------------------------------------------------ 1.2This is a high performance liquid chromatography (HPLC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for the compounds above, identifications should be supported by at least one additional qualitative technique. This method describes electrochemical conditions at a second potential which can be used to confirm measurements made with this method. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of the interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the use of HPLC instrumentation and in the interpretation of liquid chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1L, is extracted with chloroform using liquid-liquid extractions in a separatory funnel. The chloroform extract is extracted with acid. The acid extract is then neutralized and extracted with chloroform. The final chloroform extract is exchanged to methanol while being concentrated using a rotary evaporator. The extract is mixed with buffer and separated by HPLC. The benzidine compounds are measured with an electrochemical detector.2 2.2The acid back-extraction acts as a general purpose cleanup to aid in the elimination of interferences.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures that are inherent in the extraction step are used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3Some dye plant effluents contain large amounts of components with retention times closed to benzidine. In these cases, it has been found useful to reduce the electrode potential in order to eliminate interferences and still detect benzidine. (See Section 12.7.) 4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health harzard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 46 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzidine and 3,3-dichlorobenzidine. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

4.3Exposure to chloroform should be minimized by performing all extractions and extract concentrations in a hood or other well-ventiliated area.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested): 5.2.1Separatory funnels2000, 1000, and 250-mL, with Teflon stopcock.

5.2.2Vials10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.2.3Rotary evaporator.

5.2.4FlasksRound bottom, 100mL, with 24/40 joints.

5.2.5Centrifuge tubesConical, graduated, with Teflon-lined screw caps.

5.2.6PipettesPasteur, with bulbs.

5.3BalanceAnalytical, capable of accurately weighing 0.0001 g.

5.4High performance liquid chromatograph (HPLC)An analytical system complete with column supplies, high pressure syringes, detector, and compatible recorder. A data system is recommended for measuring peak areas and retention times.

5.4.1Solvent delivery systemWith pulse damper, Altex 110A or equivalent.

5.4.2Injection valve (optional)Waters U6K or equivalent.

5.4.3Electrochemical detectorBioanalytical Systems LC2A with glassy carbon electrode, or equivalent. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

5.4.4Electrode polishing kitPrinceton Applied Research Model 9320 or equivalent.

5.4.5ColumnLichrosorb RP2, 5 micron particle diameter, in a 25 cm 4.6 mm ID stainless steel column. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Sodium hydroxide solution (5 N)Dissolve 20 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3Sodium hydroxide solution (1 M)Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 1 L.

6.4Sodium thiosulfate(ACS) Granular.

6.5Sodium tribasic phosphate (0.4 M)Dissolve 160 g of trisodium phosphate decahydrate (ACS) in reagent water and dilute to 1 L.

6.6Sulfuric acid (1+1)Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.7Sulfuric acid (1 M)Slowly, add 58 mL of H2SO4 (ACS, sp. gr. 1.84) to reagent water and dilute to 1 L.

6.8Acetate buffer (0.1 M, pH 4.7)Dissolve 5.8 mL of glacial acetic acid (ACS) and 13.6 g of sodium acetate trihydrate (ACS) in reagent water which has been purified by filtration through a RO4 Millipore System or equivalent and dilute to 1 L.

6.9Acetonitrile, chloroform (preserved with 1% ethanol), methanolPesticide quality or equivalent.

6.10Mobile phasePlace equal volumes of filtered acetonitrile (Millipore type FH filter or equivalent) and filtered acetate buffer (Millipore type GS filter or equivalent) in a narrow-mouth, glass container and mix thoroughly. Prepare fresh weekly. Degas daily by sonicating under vacuum, by heating and stirring, or by purging with helium.

6.11Stock standard solutions (1.00 g/L)Stock standard solutions may be prepared from pure standard materials or purchased as certified solutions.

6.11.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in methanol and dilute to volume in a 10mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.11.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.11.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.12Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Establish chromatographic operating conditions equivalent to those given in Table 1. The HPLC system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure: 7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with mobile phase. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using syringe injections of 5 to 25 L or a constant volume injection loop, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected.

The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (7.3Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with mobile phase. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using syringe injections of 5 to 25 L or a constant volume injection loop, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF= (As)(Cis) (Ais)(Cs) ---------------------------------------------------------------------------------------------------------------- Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 15%, a new calibration curve must be prepared for that compound. If serious loss of response occurs, polish the electrode and recalibrate.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.9, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing benzidine and/or 3,3-dichlorobenzidine at a concentration of 50 g/mL each in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 50 g/L by adding 1.00 mL of QC check sample concentrate to each of four 1L-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 50 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 50 g/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.

7 If spiking was performed at a concentration lower than 50 g/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 3, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T)2.44(100 S/T)%. 7 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Sections 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as HPLC with a dissimilar column, gas chromatography, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices 8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4C and stored in the dark from the time of collection until extraction. Both benzidine and 3,3-dichlorobenzidine are easily oxidized. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine. 9 Field test kits are available for this purpose.

After mixing, adjust the pH of the sample to a range of 2 to 7 with sulfuric acid.

9.3If 1,2-diphenylhydrazine is likely to be present, adjust the pH of the sample to 4.0 0.2 to prevent rearrangement to benzidine.

9.4All samples must be extracted within 7 days of collection. Extracts may be held up to 7 days before analysis, if stored under an inert (oxidant free) atmosphere. 2 The extract should be protected from light.

10. Sample Extraction 10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2L separatory funnel. Check the pH of the sample with wide-range pH paper and adjust to within the range of 6.5 to 7.5 with sodium hydroxide solution or sulfuric acid.

10.2Add 100 mL of chloroform to the sample bottle, seal, and shake 30 s to rinse the inner surface. (Caution: Handle chloroform in a well ventilated area.) Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the chloroform extract in a 250-mL separatory funnel.

10.3Add a 50-mL volume of chloroform to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the separatory funnel. Perform a third extraction in the same manner.

10.4Separate and discard any aqueous layer remaining in the 250-mL separatory funnel after combining the organic extracts. Add 25 mL of 1 M sulfuric acid and extract the sample by shaking the funnel for 2 min.

Transfer the aqueous layer to a 250-mL beaker. Extract with two additional 25-mL portions of 1 M sulfuric acid and combine the acid extracts in the beaker.

10.5Place a stirbar in the 250-mL beaker and stir the acid extract while carefully adding 5 mL of 0.4 M sodium tribasic phosphate. While monitoring with a pH meter, neutralize the extract to a pH between 6 and 7 by dropwise addition of 5 N sodium hydroxide solution while stirring the solution vigorously. Approximately 25 to 30 mL of 5 N sodium hydroxide solution will be required and it should be added over at least a 2-min period. Do not allow the sample pH to exceed 8.

10.6Transfer the neutralized extract into a 250-mL separatory funnel.

Add 30 mL of chloroform and shake the funnel for 2 min. Allow the phases to separate, and transfer the organic layer to a second 250-mL separatory funnel.

10.7Extract the aqueous layer with two additional 20-mL aliquots of chloroform as before. Combine the extracts in the 250-mL separatory funnel.

10.8Add 20 mL of reagent water to the combined organic layers and shake for 30 s.

10.9Transfer the organic extract into a 100-mL round bottom flask. Add 20 mL of methanol and concentrate to 5 mL with a rotary evaporator at reduced pressure and 35 C. An aspirator is recommended for use as the source of vacuum. Chill the receiver with ice. This operation requires approximately 10 min. Other concentration techniques may be used if the requirements of Section 8.2 are met.

10.10Using a 9-in. Pasteur pipette, transfer the extract to a 15-mL, conical, screw-cap centrifuge tube. Rinse the flask, including the entire side wall, with 2-mL portions of methanol and combine with the original extract.

10.11Carefully concentrate the extract to 0.5 mL using a gentle stream of nitrogen while heating in a 30 C water bath. Dilute to 2 mL with methanol, reconcentrate to 1 mL, and dilute to 5 mL with acetate buffer.

Mix the extract thoroughly. Cap the centrifuge tube and store refrigerated and protected from light if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with HPLC analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.12Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1,000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation 11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

12. High Performance Liquid Chromatography 12.1Table 1 summarizes the recommended operating conditions for the HPLC. Included in this table are retention times, capacity factors, and MDL that can be achieved under these conditions. An example of the separations achieved by this HPLC column is shown in Figure 1. Other HPLC columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met. When the HPLC is idle, it is advisable to maintain a 0.1 mL/min flow through the column to prolong column life.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the instrument.

12.4Inject 5 to 25 L of the sample extract or standard into the HPLC. If constant volume injection loops are not used, record the volume injected to the nearest 0.05 L, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract with mobile phase and reanalyze.

12.7If the measurement of the peak response for benzidine is prevented by the presence of interferences, reduce the electrode potential to +0.6 V and reanalyze. If the benzidine peak is still obscured by interferences, further cleanup is required.

13. Calculations 13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2. (image) Equation 2 where: A=Amount of material injected (ng).

Vi=Volume of extract injected (L).

Vt=Volume of total extract (L).

Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3. (image) Equation 3 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

13.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero. 1 The MDL concentrations listed in Table 1 were obtained using reagent water. 10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 7MDL to 3000MDL. 10 14.3This method was tested by 17 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 70 g/L. 11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References 1. 40 CFR part 136, appendix B.

2. Determination of Benzidines in Industrial and Muncipal Wastewaters, EPA 600/482022, National Technical Information Service, PB82196320, Springfield, Virginia 22161, April 1982.

3. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

4. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 8. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

9. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

10. EPA Method Study 15, Method 605 (Benzidines), EPA 600/484062, National Technical Information Service, PB84211176, Springfield, Virginia 22161, June 1984.

11. EPA Method Validation Study 15, Method 605 (Benzidines), Report for EPA Contract 68032624 (In preparation).

Table 1_Chromatographic Conditions and Method Detection Limits ------------------------------------------------------------------------ Method Column detection Parameter Retention capacity limit time (min) factor (g/ (k[prime]) L) ------------------------------------------------------------------------ Benzidine........................ 6.1 1.44 0.08 3,3[prime]-Dichlorobenzidine..... 12.1 3.84 0.13 ------------------------------------------------------------------------ HPLC Column conditions: Lichrosorb RP-2, 5 micron particle size, in a 25 cmx4.6 mm ID stainless steel column. Mobile Phase: 0.8 mL/min of 50% acetonitrile/50% 0.1M pH 4.7 acetate buffer. The MDL were determined using an electrochemical detector operated at +0.8 V.

Table 2_QC Acceptance Criteria_Method 605 ---------------------------------------------------------------------------------------------------------------- Limit for Range for Test conc. s X Range for Parameter (g/ (g/ (g/ P, Ps L) L) L) (percent) ---------------------------------------------------------------------------------------------------------------- Benzidine........................................................ 50 18.7 9.1-61.0 D-140 3.3[prime]-Dichlorobenzidine..................................... 50 23.6 18.7-50.0 5-128 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3_Method Accuracy and Precision as Functions of Concentration_Method 605 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Single analyst Overall recovery, precision, precision, Parameter X[prime](g/ sr[prime] S[prime] L) (g/L) (g/L) ---------------------------------------------------------------------------------------------------------------- Benzidine................................................... 0.70C+0.06 0.28X+0.19 0.40X+0.18 3,3[prime]-Dichlorobenzidine................................ 0.66C+0.23 0.39X-0.05 0.38X+0.02 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

(image) View or download PDF Method 606Phthalate Ester 1. Scope and Application 1.1This method covers the determination of certain phthalate esters. The following parameters can be determined by this method: ------------------------------------------------------------------------ STORET Parameter No. CAS No.

------------------------------------------------------------------------ Bis(2-ethylhexyl) phthalate........................ 39100 117-81-7 Butyl benzyl phthalate............................. 34292 85-68-7 Di-n-butyl phthalate............................... 39110 84-74-2 Diethyl phthalate.................................. 34336 84-66-2 Dimethyl phthalate................................. 34341 131-11-3 Di-n-octyl phthalate............................... 34596 117-84-0 ------------------------------------------------------------------------ 1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 608, 609, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the phthalate esters are then measured with an electron capture detector. 2 2.2Analysis for phthalates is especially complicated by their ubiquitous occurrence in the environment. The method provides Florisil and alumina column cleanup procedures to aid in the elimination of interferences that may be encountered.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned. 3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating.

Thorough rinsing with such solvents usually eliminates PCB interference.

Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants.

Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Phthalate esters are contaminants in many products commonly found in the laboratory. It is particularly important to avoid the use of plastics because phthalates are commonly used as plasticizers and are easily extracted from plastic materials. Serious phthalate contamination can result at any time, if consistent quality control is not practiced.

Great care must be experienced to prevent such contamination. Exhaustive cleanup of reagents and glassware may be required to eliminate background phthalate contamination.4,5 3.3Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 68 for the information of the analyst.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1L or 1qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4 C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only).

5.2.1Separatory funnel2L, with Teflon stopcock.

5.2.2Drying columnChromatographic column, approximately 400 mm long 19 mm ID, with coarse frit filter disc.

5.2.3Chromatographic column300 mm long 10 mm ID, with Teflon stopcock and coarse frit filter disc at bottom (Kontes K4205400213 or equivalent).

5.2.4Concentrator tube, Kuderna-Danish10mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5Evaporative flask, Kuderna-Danish500mL (Kontes K5700010500 or equivalent). Attach to concentrator tube with springs.

5.2.6Snyder column, Kuderna-DanishThree-ball macro (Kontes K5030000121 or equivalent).

5.2.7Snyder column, Kuderna-DanishTwo-ball micro (Kontes K5690010219 or equivalent).

5.2.8Vials10 to 15mL, amber glass, with Teflon-lined screw cap.

5.3Boiling chipsApproximately 10/40 mesh. Heat to 400 C for 30 min or Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2 C). The bath should be used in a hood.

5.5BalanceAnalytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatographAn analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder.

A data system is recommended for measuring peak areas.

5.6.1Column 11.8 m long 4 mm ID glass, packed with 1.5% SP2250/1.95% SP2401 Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statemelts in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.2Column 21.8 m long 4 mm ID glass, packed with 3% OV1 on Supelcoport (100/120 mesh) or equivalent.

5.6.3DetectorElectron capture detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Acetone, hexane, isooctane, methylene chloride, methanolPesticide quality or equivalent.

6.3Ethyl ethernanograde, redistilled in glass if necessary.

6.3.1Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat. No. P11268, and other suppliers.) 6.3.2Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.4Sodium sulfate(ACS) Granular, anhydrous. Several levels of purification may be required in order to reduce background phthalate levels to an acceptable level: 1) Heat 4 h at 400 C in a shallow tray, 2) Heat 16 h at 450 to 500 C in a shallow tray, 3) Soxhlet extract with methylene chloride for 48 h.

6.5FlorisilPR grade (60/100 mesh). Purchase activated at 1250 F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. To prepare for use, place 100 g of Florisil into a 500-mL beaker and heat for approximately 16 h at 40 C. After heating transfer to a 500-mL reagent bottle. Tightly seal and cool to room temperature. When cool add 3 mL of reagent water. Mix thoroughly by shaking or rolling for 10 min and let it stand for at least 2 h. Keep the bottle sealed tightly.

6.6AluminaNeutral activity Super I, W200 series (ICN Life Sciences Group, No. 404583). To prepare for use, place 100 g of alumina into a 500-mL beaker and heat for approximately 16 h at 400 C. After heating transfer to a 500-mL reagent bottle. Tightly seal and cool to room temperature. When cool add 3 mL of reagent water. Mix thoroughly by shaking or rolling for 10 min and let it stand for at least 2 h. Keep the bottle sealed tightly.

6.7Stock standard solutions (1.00 g/L)Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in isooctane and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Establish gas chromatograph operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure: 7.2.1Prepared calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with isooctane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (7.3Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flash. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with isooctane. One of the standards should be at a concentraton near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF= (As)(Cis) (Ais)(Cs) ---------------------------------------------------------------------------------------------------------------- Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 15%, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality contrml (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetone: butyl benzyl phthalate, 10 g/mL; bis(2-ethylhexyl) phthalate, 50 g/mL; di-n-octyl phthalate, 50 g/mL; any other phthlate, 25 g/mL. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agancy, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.

9 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 3, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T)2.44(100 S/T)%. 9 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices 10 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 C from the time of collection until extraction.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction. 2 10. Sample Extraction 10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2L separatory funnel.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phrase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentrator devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7Increase the temperature of the hot water bath to about 80 C.

Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Adjust the extract volume to 10 mL.

Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.9Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder.

Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation 11.Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2If the entire extract is to be cleaned up by one of the following procedures, it must be concentrated to 2.0 mL. To the concentrator tube in Section 10.8, add a clean boiling chip and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of hexane to the top. Place the micro-K-D apparatus on a hot water bath (80 C) so that the concentrator tube is partially immersed in the hot water.

Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of hexane. Adjust the final volume to 2.0 mL and proceed with one of the following cleanup procedures.

11.3Florisil column cleanup for phthalate esters: 11.3.1Place 10 g of Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 cm of anhydrous sodium sulfate to the top.

11.3.2Preelute the column with 40 mL of hexane. The rate for all elutions should be about 2 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2-mL sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 40 mL of hexane and continue the elution of the column. Discard this hexane eluate.

11.3.3Next, elute the column with 100 mL of 20% ethyl ether in hexane (V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube.

Concentrate the collected fraction as in Section 10.6. No solvent exchange is necessary. Adjust the volume of the cleaned up extract to 10 mL in the concentrator tube and analyze by gas chromatography (Section 12).

11.4Alumina column cleanup for phthalate esters: 11.4.1Place 10 g of alumina into a chromatographic column. Tap the column to settle the alumina and add 1 cm of anhydrous sodium sulfate to the top.

11.4.2Preelute the column with 40 mL of hexane. The rate for all elutions should be about 2 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2-mL sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 35 mL of hexane and continue the elution of the column. Discard this hexane eluate.

11.4.3Next, elute the column with 140 mL of 20% ethyl ether in hexane (V/V) into a 500-mL K-D flask equipped with a 10mL concentrator type.

Concentrate the collected fraction as in Section 10.6. No solvent exchange is necessary. Adjust the volume of the cleaned up extract to 10 mL in the concentrator tube and analyze by gas chromatography (Section 12).

12. Gas Chromatography 12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal staldard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.4Inject 2 to 5 L of the sample extract or standard into the gas-chromatograph using the solvent-flush technique. 11 Smaller (1.0 L) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations 13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2. (image) Equation 2 where: A=Amount of material injected (ng).

Vi=Volume of extract injected (L).

Vt=Volume of total extract (L).

Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3. (image) Equation 3 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

13.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero. 1 The MDL concentrations listed in Table 1 were obtained using reagent water. 12 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 5 MDL to 1000 MDL with the following exceptions: dimethyl and diethyl phthalate recoveries at 1000 MDL were low (70%); bis-2-ethylhexyl and di-n-octyl phthalate recoveries at 5 MDL were low (60%). 12 14.3This method was tested by 16 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.7 to 106 g/L. 13 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References 1. 40 CFR part 136, appendix B.

2. Determination of Phthalates in Industrial and Muncipal Wastewaters, EPA 600/481063, National Technical Information Service, PB81232167, Springfield, Virginia 22161, July 1981.

3. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

4. Giam, C.S., Chan, H.S., and Nef, G.S. Sensitive Method for Determination of Phthalate Ester Plasticizers in Open-Ocean Biota Samples, Analytical Chemistry, 47, 2225 (1975).

5. Giam, C.S., and Chan, H.S. Control of Blanks in the Analysis of Phthalates in Air and Ocean Biota Samples, U.S. National Bureau of Standards, Special Publication 442, pp. 701708, 1976.

6. CarcinogensWorking with Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

7. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

8. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

9. Provost L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 10. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

11. Burke, J.A. Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects, Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

12. Method Detection Limit and Analytical Curve Studies, EPA Methods 606, 607, and 608, Special letter report for EPA Contract 68032606, U.S.

Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

13. EPA Method Study 16 Method 606 (Phthalate Esters), EPA 600/484056, National Technical Information Service, PB84211275, Springfield, Virginia 22161, June 1984.

Table 1_Chromatographic Conditions and Method Detection Limits ------------------------------------------------------------------------ Retention time (min) Method ---------------------------- detection Parameter limit Column 1 Column 2 (g/L) ------------------------------------------------------------------------ Dimethyl phthalate............ 2.03 0.95 0.29 Diethyl phthalate............. 2.82 1.27 0.49 Di-n-butyl phthalate.......... 8.65 3.50 0.36 Butyl benzyl phthalate........ a 6.94 a 5.11 0.34 Bis(2-ethylhexyl) phthalate... a 8.92 a 10.5 2.0 Di-n-octyl phthalate.......... a 16.2 a 18.0 3.0 ------------------------------------------------------------------------ Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5% SP-2250/ 1.95% SP-2401 packed in a 1.8 m long x 4 mm ID glass column with 5% methane/95% argon carrier gas at 60 mL/min flow rate. Column temperature held isothermal at 180C, except where otherwise indicated.

Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% OV-1 packed in a 1.8 m long x 4 mm ID glass column with 5% methane/95% argon carrier gas at 60 mL/min flow rate. Column temperature held isothermal at 200 C, except where otherwise indicated.

a 220 C column temperature.

Table 2_QC Acceptance Criteria_Method 606 ---------------------------------------------------------------------------------------------------------------- Limit for Range for Test conc. s X Range for Parameter (g/ (g/ (g/ P, Ps L) L) L) (percent) ---------------------------------------------------------------------------------------------------------------- Bis(2-ethylhexyl) phthalate...................................... 50 38.4 1.2-55.9 D-158 Butyl benzyl phthalate........................................... 10 4.2 5.7-11.0 30-136 Di-n-butyl phthalate............................................. 25 8.9 10.3-29.6 23-136 Diethyl phthalate................................................ 25 9.0 1.9-33.4 D-149 Dimethyl phathalate.............................................. 25 9.5 1.3-35.5 D-156 Di-n-octyl phthalate............................................. 50 13.4 D-50.0 D-114 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3_Method Accuracy and Precision as Functions of Concentration_Method 606 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Single analyst Overall recovery, precision, precision, Parameter X[prime] sr[prime] S[prime] (g/L) (g/L) (g/L) ---------------------------------------------------------------------------------------------------------------- Bis(2-ethylhexyl) phthalate..................................... 0.53C+2.02 0.80X-2.54 0.73X-0.17 Butyl benzyl phthalate.......................................... 0.82C+0.13 0.26X+0.04 0.25X+0.07 Di-n-butyl phthalate............................................ 0.79C+0.17 0.23X+0.20 0.29X+0.06 Diethyl phthalate............................................... 0.70C+0.13 0.27X+0.05 0.45X+0.11 Dimethyl phthalate.............................................. 0.73C+0.17 0.26X+0.14 0.44X+0.31 Di-n-octyl phthalate............................................ 0.35C-0.71 0.38X+0.71 0.62X+0.34 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

(image) View or download PDF (image) View or download PDF Method 607Nitrosamines 1. Scope and Application 1.1This method covers the determination of certain nitrosamines. The following parameters can be determined by this method: ------------------------------------------------------------------------ Parameter Storet No. CAS No.

------------------------------------------------------------------------ N-Nitrosodimethylamine........................ 34438 62-75-9 N-Nitrosodiphenylamine........................ 34433 86-30-6 N-Nitrosodi-n-propylamine..................... 34428 621-64-7 ------------------------------------------------------------------------ 1.2This is a gas chromatographic (GC) method applicable to the determination of the parameters listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compmunds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditimns for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for N-nitrosodi-n-propylamine. In order to confirm the presence of N-nitrosodiphenylamine, the cleanup procedure specified in Section 11.3 or 11.4 must be used. In order to confirm the presence of N-nitrosodimethylamine by GC/MS, Column 1 of this method must be substituted for the column recommended in Method 625. Confirmation of these parameters using GC-high resolution mass spectrometry or a Thermal Energy Analyzer is also recommended. 1,2 1.3The method detection limit (MDL, defined in Section 14.1)3 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is washed with dilute hydrochloric acid to remove free amines, dried, and concentrated to a volume of 10 mL or less. After the extract has been exchanged to methanol, it is separated by gas chromatography and the parameters are then measured with a nitrogen-phosphorus detector.4 2.2The method provides Florisil and alumina column cleanup procedures to separate diphenylamine from the nitrosamines and to aid in the elimination of interferences that may be encountered.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.5 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3N-Nitrosodiphenylamine is reported69 to undergo transnitrosation reactions. Care must be exercised in the heating or concentrating of solutions containing this compound in the presence of reactive amines.

3.4The sensitive and selective Thermal Energy Analyzer and the reductive Hall detector may be used in place of the nitrogen-phosphorus detector when interferences are encountered. The Thermal Energy Analyzer offers the highest selectivity of the non-MS detectors.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified1012 for the information of the analyst.

4.2These nitrosamines are known carcinogens1317, therefore, utmost care must be exercised in the handling of these materials. Nitrosamine reference standards and standard solutions should be handled and prepared in a ventilated glove box within a properly ventilated room.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4 C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flowmeter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.): 5.2.1Separatory funnels2L and 250mL, with Teflon stopcock.

5.2.2Drying columnChromatographic column, approximately 400 mm long 19 mm ID, with coarse frit filter disc.

5.2.3Concentrator tube, Kuderna-Danish10-mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.4Evaporative flask, Kuderna-Danish500-mL (Kontes K5700010500 or equivalent). Attach to concentrator tube with springs.

5.2.5Snyder column, Kuderna-DanishThree-ball macro (Kontes K5030000121 or equivalent).

5.2.6Snyder column, Kuderna-DanishTwo-ball micro (Kontes K5690010219 or equivalent).

5.2.7Vials10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.2.8Chromatographic columnApproximately 400 mm long 22 mm ID, with Teflon stopcock and coarse frit filter disc at bottom (Kontes K4205400234 or equivalent), for use in Florisil column cleanup procedure.

5.2.9Chromatographic columnApproximately 300 mm long 10 mm ID, with Teflon stopcock and coarse frit filter disc at bottom (Kontes K4205400213 or equivalent), for use in alumina column cleanup procedure.

5.3Boiling chipsApproximately 10/40 mesh. Heat to 400 C for 30 min or Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2 C). The bath should be used in a hood.

5.5BalanceAnalytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatographAn analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder.

A data system is recommended for measuring peak areas.

5.6.1Column 11.8 m long 4 mm ID glass, packed with 10% Carbowax 20 M/2% KOH on Chromosorb W-AW (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.2.

5.6.2Column 21.8 m long 4 mm ID glass, packed with 10% SP2250 on Supel- coport (100/120 mesh) or equivalent.

5.6.3DetectorNitrogen-phosphorus, reductive Hall, or Thermal Energy Analyzer detector.1,2 These detectors have proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1). A nitrogen-phosphorus detector was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.2.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Sodium hydroxide solution (10 N)Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 ml.

6.3Sodium thiosulfate(ACS) Granular.

6.4Sulfuric acid (1+1)Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.5Sodium sulfate(ACS) Granular, anhydrous. Purify by heating at 400 C for 4 h in a shallow tray.

6.6Hydrochloric acid (1+9)Add one volume of concentrated HCl (ACS) to nine volumes of reagent water.

6.7Acetone, methanol, methylene chloride, pentanePesticide quality or equivalent.

6.8Ethyl etherNanograde, redistilled in glass if necessary.

6.8.1Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat No. P11268, and other suppliers.) 6.8.2Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.9FlorisilPR grade (60/100 mesh). Purchase activated at 1250 F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 C in a foil-covered glass container and allow to cool.

6.10AluminaBasic activity Super I, W200 series (ICN Life Sciences Group, No. 404571, or equivalent). To prepare for use, place 100 g of alumina into a 500-mL reagent bottle and add 2 mL of reagent water. Mix the alumina preparation thoroughly by shaking or rolling for 10 min and let it stand for at least 2 h. The preparation should be homogeneous before use. Keep the bottle sealed tightly to ensure proper activity.

6.11Stock standard solutions (1.00 g/L)Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.11.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in methanol and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.11.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.11.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.12Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure: 7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with methanol. One of the external standards should be at a concentraton near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (7.3Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with methanol. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF= (As)(Cis) (Ais)(Cs) ---------------------------------------------------------------------------------------------------------------- Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 15%, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.2) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 20 g/mL in methanol. The QC check sample concentrate must be obtained from the U.S.

Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 20 g/L by adding 1.00 mL of QC check sample concentrate to each of four 1L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 20 g/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were caluclated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.

18 If spiking was performed at a concentration lower than 20 g/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria caluclated for the specific spike concentration. To calculate optional acceptance crtieria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 3, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T) 2.44(100 S/T)%. 18 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices 19 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine. 20 Field test kits are available for this purpose. If N-nitrosodiphenylamine is to be determined, adjust the sample pH to 7 to 10 with sodium hydroxide solution or sulfuric acid.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction. 4 9.4Nitrosamines are known to be light sensitive. 7 Samples should be stored in amber or foil-wrapped bottles in order to minimize photolytic decomposition.

10. Sample Extraction 10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2L separatory funnel. Check the pH of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with sodium hydroxide solution or sulfuric acid.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Add 10 mL of hydrochloric acid to the combined extracts and shake for 2 min. Allow the layers to separate. Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If N-nitrosodiphenylamine is to be measured by gas chromatography, the analyst must first use a cleanup column to eliminate diphenylamine interference (Section 11). If N-nitrosodiphenylamine is of no interest, the analyst may proceed directly with gas chromatographic analysis (Section 12).

10.8Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000- mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation 11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure. Diphenylamine, if present in the original sample extract, must be separated from the nitrosamines if N-nitrosodiphenylamine is to be determined by this method.

11.2If the entire extract is to be cleaned up by one of the following procedures, it must be concentrated to 2.0 mL. To the concentrator tube in Section 10.7, add a clean boiling chip and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of methylene chloride to the top. Place the micr-K-D apparatus on a hot water bath (60 to 65C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of methylene chloride. Adjust the final volume to 2.0 mL and proceed with one of the following cleanup procedures.

11.3Florisil column cleanup for nitrosamines: 11.3.1Place 22 g of activated Florisil into a 22-mm ID chromatographic column. Tap the column to settle the Florisil and add about 5 mm of anhydrous sodium sulfate to the top.

11.3.2Preelute the column with 40 mL of ethyl ether/pentane (15+85)(V/V). Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2-mL sample extract onto the column using an additional 2 mL of pentane to complete the transfer.

11.3.3Elute the column with 90 mL of ethyl ether/pentane (15+85)(V/V) and discard the eluate. This fraction will contain the diphenylamine, if it is present in the extract.

11.3.4Next, elute the column with 100 mL of acetone/ethyl ether (5+95)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. This fraction will contain all of the nitrosamines listed in the scope of the method.

11.3.5Add 15 mL of methanol to the collected fraction and concentrate as in Section 10.6, except use pentane to prewet the column and set the water bath at 70 to 75C. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of pentane. Analyze by gas chromatography (Section 12).

11.4Alumina column cleanup for nitrosamines: 11.4.1Place 12 g of the alumina preparation (Section 6.10) into a 10-mm ID chromatographic column. Tap the column to settle the alumina and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.4.2Preelute the column with 10 mL of ethyl ether/pentane (3+7)(V/V).

Discard the eluate (about 2 mL) and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2 mL sample extract onto the column using an additional 2 mL of pentane to complete the transfer.

11.4.3Just prior to exposure of the sodium sulfate layer to the air, add 70 mL of ethyl ether/pentane (3+7)(V/V). Discard the first 10 mL of eluate. Collect the remainder of the eluate in a 500mL K-D flask equipped with a 10 mL concentrator tube. This fraction contains N-nitrosodiphenylamine and probably a small amount of N-nitrosodi-n-propylamine.

11.4.4Next, elute the column with 60 mL of ethyl ether/pentane (1+1)(V/V), collecting the eluate in a second K-D flask equipped with a 10mL concentrator tube. Add 15 mL of methanol to the K-D flask. This fraction will contain N-nitrosodimethylamine, most of the N-nitrosodi-n-propylamine and any diphenylamine that is present.

11.4.5Concentrate both fractions as in Section 10.6, except use pentane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of pentane. Analyze the fractions by gas chromatography (Section 12).

12. Gas Chromatography 12.1N-nitrosodiphenylamine completely reacts to form diphenylamine at the normal operating temperatures of a GC injection port (200 to 250C).

Thus, N-nitrosodiphenylamine is chromatographed and detected as diphenylamine. Accurate determination depends on removal of diphenylamine that may be present in the original extract prior to GC analysis (See Section 11).

12.2Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.3Calibrate the system daily as described in Section 7.

12.4If the extract has not been subjected to one of the cleanup procedures in Section 11, it is necessary to exchange the solvent from methylene chloride to methanol before the thermionic detector can be used. To a 1 to 10-mL volume of methylene chloride extract in a concentrator tube, add 2 mL of methanol and a clean boiling chip. Attach a two-ball micro-Snyder column to the concentrator tube. Prewet the column by adding about 0.5 mL of methylene chloride to the top. Place the micro-K-D apparatus on a boiling (100C) water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of methanol. Adjust the final volume to 2.0 mL.

12.5If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.6Inject 2 to 5 L of the sample extract or standard into the gas chromatograph using the solvent-flush technique. 21 Smaller (1.0 L) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L, and the resulting peak size in area or peak height units.

12.7Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.8If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.9If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations 13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2. (image) Equation 2 where: A=Amount of material injected (ng).

Vi=Volume of extract injected (L).

Vt=Volume of total extract (L).

Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3. (image) Equation 3 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

13.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero. 3 The MDL concentrations listed in Table 1 were obtained using reagent water. 22 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4 MDL to 1000 MDL. 22 14.3This method was tested by 17 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.8 to 55 g/L. 23 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References 1. Fine, D.H., Lieb, D., and Rufeh, R. Principle of Operation of the Thermal Energy Analyzer for the Trace Analysis of Volatile and Non-volatile N-nitroso Compounds, Journal of Chromatography, 107, 351 (1975).

2. Fine, D.H., Hoffman, F., Rounbehler, D.P., and Belcher, N.M. Analysis of N-nitroso Compounds by Combined High Performance Liquid Chromatography and Thermal Energy Analysis, Walker, E.A., Bogovski, P.

and Griciute, L., Editors, N-nitroso CompoundsAnalysis and Formation, Lyon, International Agency for Research on Cancer (IARC Scientific Publications No. 14), pp. 4350 (1976).

3. 40 CFR part 136, appendix B.

4. Determination of Nitrosamines in Industrial and Municipal Wastewaters, EPA 600/482016, National Technical Information Service, PB82199621, Springfield, Virginia 22161, April 1982.

5. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

6. Buglass, A.J., Challis, B.C., and Osborn, M.R. Transnitrosation and Decomposition of Nitrosamines, Bogovski, P. and Walker, E.A., Editors, N-nitroso Compounds in the Environment, Lyon, International Agency for Research on Cancer (IARC Scientific Publication No. 9), pp. 94100 (1974).

7. Burgess, E.M., and Lavanish, J.M. Photochemical Decomposition of N-nitrosamines, Tetrahedon Letters, 1221 (1964) 8. Druckrey, H., Preussmann, R., Ivankovic, S., and Schmahl, D.

Organotrope Carcinogene Wirkungen bei 65 Verschiedenen N-NitrosoVerbindungen an BD-Ratten, Z. Krebsforsch., 69, 103 (1967).

9. Fiddler, W. The Occurrence and Determination of N-nitroso Compounds, Toxicol. Appl. Pharmacol., 31, 352 (1975).

10. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

11. OSHA Safety and Health Standards, General Industry, (29 CFR Part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

12. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

13. Lijinsky, W. How Nitrosamines Cause Cancer, New Scientist, 73, 216 (1977).

14. Mirvish, S.S. N-Nitroso compounds: Their Chemical and in vivo Formation and Possible Importance as Environmental Carcinogens, J.

Toxicol. Environ. Health, 3, 1267 (1977).

15. Reconnaissance of Environmental Levels of Nitrosamines in the Central United States, EPA330/177001, National Enforcement Investigations Center, U.S. Environmental Protection Agency (1977).

16. Atmospheric Nitrosamine Assessment Report, Office of Air Quality Planning and Standards, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina (1976).

17. Scientific and Technical Assessment Report on Nitrosamines, EPA660/67001, Office of Research and Development, U.S. Environmental Protection Agency (1976).

18. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value of 1.22 derived in this report.) 19. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

20. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

21. Burke, J. A. Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects, Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

22. Method Detection Limit and Analytical Curve Studies EPA Methods 606, 607, and 608, Special letter report for EPA Contract 68032606, U.S.

Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

23. EPA Method Study 17 Method 607Nitrosamines, EPA 600/484051, National Technical Information Service, PB84207646, Springfield, Virginia 22161, June 1984.

Table 1_Chromatographic Conditions and Method Detection Limits ------------------------------------------------------------------------ Retention time (min) Method -------------------------- detection Parameter limit Column 1 Column 2 (g/ L) ------------------------------------------------------------------------ N-Nitrosodimethylamine........... 4.1 0.88 0.15 N-Nitrosodi-n-propylamine........ 12.1 4.2 .46 N-Nitrosodiphenylamine \a\....... \b\ 12.8 \c\ 6.4 .81 ------------------------------------------------------------------------ Column 1 conditions: Chromosorb W-AW (80/100 mesh) coated with 10% Carbowax 20 M/2% KOH packed in a 1.8 m long x 4mm ID glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held isothermal at 110 C, except where otherwise indicated.

Column 2 conditions: Supelcoport (100/120 mesh) coated with 10% SP-2250 packed in a 1.8 m long x 4 mm ID glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held isothermal at 120 C, except where otherwise indicated.

\a\ Measured as diphenylamine.

\b\ 220 C column temperature.

\c\ 210 C column temperature.

Table 2_QC Acceptance Criteria_Method 607 ---------------------------------------------------------------------------------------------------------------- Range for X Test conc. Limit for s (g/ Range for Parameter (g/ (g/ L) P, Ps L) L) (percent) ---------------------------------------------------------------------------------------------------------------- N-Nitrosodimethylamine...................................... 20 3.4 4.6-20.0 13-109 N-Nitrosodiphenyl........................................... 20 6.1 2.1-24.5 D-139 N-Nitrosodi-n-propylamine................................... 20 5.7 11.5-26.8 45-146 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation for four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3_Method Accuracy and Precision as Functions of Concentration_Method 607 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Single analyst Overall recovery, precision, precision, Parameter X[prime] sr[prime] S[prime] (g/L) (g/L) (g/L) ---------------------------------------------------------------------------------------------------------------- N-Nitrosodimethylamine.......................................... 0.37C+0.06 0.25X-0.04 0.25X+0.11 N-Nitrosodiphenylamine.......................................... 0.64C+0.52 0.36X-1.53 0.46X-0.47 N-Nitrosodi-n-propylamine....................................... 0.96C-0.07 0.15X+0.13 0.21X+0.15 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

(image) View or download PDF (image) View or download PDF Method 608Organochlorine Pesticides and PCBs 1. Scope and Application 1.1This method covers the determination of certain organochlorine pesticides and PCBs. The following parameters can be determined by this method: ------------------------------------------------------------------------ Parameter STORET No. CAS No.

------------------------------------------------------------------------ Aldrin...................................... 39330 309-00-2 -BHC................................. 39337 319-84-6 -BHC.................................. 39338 319-85-7 -BHC................................. 34259 319-86-8 -BHC................................. 39340 58-89-9 Chlordane................................... 39350 57-74-9 4,4[prime]-DDD.............................. 39310 72-54-8 4,4[prime]-DDE.............................. 39320 72-55-9 4,4[prime]-DDT.............................. 39300 50-29-3 Dieldrin.................................... 39380 60-57-1 Endosulfan I................................ 34361 959-98-8 Endosulfan II............................... 34356 33212-65-9 Endosulfan sulfate.......................... 34351 1031-07-8 Eldrin...................................... 39390 72-20-8 Endrin aldehyde............................. 34366 7421-93-4 Heptachlor.................................. 39410 76-44-8 Heptachlor epoxide.......................... 39420 1024-57-3 Toxaphene................................... 39400 8001-35-2 PCB-1016.................................... 34671 12674-11-2 PCB-1221.................................... 39488 1104-28-2 PCB-1232.................................... 39492 11141-16-5 PCB-1242.................................... 39496 53469-21-9 PCB-1248.................................... 39500 12672-29-6 PCB-1254.................................... 39504 11097-69-1 PCB-1260.................................... 39508 11096-82-5 ------------------------------------------------------------------------ 1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 609, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the parameters are then measured with an electron capture detector.

2 2.2The method provides a Florisil column cleanup procedure and an elemental sulfur removal procedure to aid in the elimination of interferences that may be encountered.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned. 3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating.

Thorough rinsing with such solvents usually eliminates PCB interference.

Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants.

Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Interferences by phthalate esters can pose a major problem in pesticide analysis when using the electron capture detector. These compounds generally appear in the chromatogram as large late eluting peaks, especially in the 15 and 50% fractions from Florisil. Common flexible plastics contain varying amounts of phthalates. These phthalates are easily extracted or leached from such materials during laboratory operations. Cross contamination of clean glassware routinely occurs when plastics are handled during extraction steps, especially when solvent-wetted surfaces are handled. Interferences from phthalates can best be minimized by avoiding the use of plastics in the laboratory.

Exhaustive cleanup of reagents and glassware may be required to eliminate background phthalate contamination.4,5 The interferences from phthalate esters can be avoided by using a microcoulometric or electrolytic conductivity detector.

3.3Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified68 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: 4,4-DDT, 4,4DDD, the BHCs, and the PCBs. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4 C and protected from light during composting. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2.Glassware (All specifications are suggested. Catalog numbers are included for illustration only.): 5.2.1Separatory funnel2L, with Teflon stopcock.

5.2.2Drying columnChromatographic column, approximately 400 mm long 19 mm ID, with coarse frit filter disc.

5.2.3Chromatographic column400 mm long 22 mm ID, with Teflon stopcock and coarse frit filter disc (Kontes K42054 or equivalent).

5.2.4Concentrator tube, Kuderna-Danish10mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5Evaporative flask, Kuderna-Danish500mL (Kontes K5700010500 or equivalent). Attach to concentrator tube with springs.

5.2.6Snyder column, Kuderna/DanishThree-ball macro (Kontes K5030000121 or equivalent).

5.2.7Vials10 to 15mL, amber glass, with Teflon-lined screw cap.

5.3Boiling chipsApproximately 10/40 mesh. Heat to 400 C for 30 min or Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2 C). The bath should be used in a hood.

5.5BalanceAnalytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatographAn analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip- chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column 11.8 m long 4 mm ID glass, packed with 1.5% SP2250/1.95% SP2401 on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.2Column 21.8 m long 4 mm ID glass, packed with 3% OV1 on Supelcoport (100/120 mesh) or equivalent.

5.6.3DetectorElectron capture detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Sodium hydroxide solution (10 N)Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3Sodium thiosulfate(ACS) Granular.

6.4Sulfuric acid (1+1)Slowly, add 50 mL to H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.5Acetone, hexane, isooctane, methylene chloridePesticide quality or equivalent.

6.6Ethyl etherNanograde, redistilled in glass if necessary.

6.6.1Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat. No. P11268, and other suppliers.) 6.6.2Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.7Sodium sulfate(ACS) Granular, anhydrous. Purify by heating at 400 C for 4 h in a shallow tray.

6.8FlorisilPR grade (60/100 mesh). Purchase activated at 1250 F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 C in a foil-covered glass container and allow to cool.

6.9MercuryTriple distilled.

6.10Copper powderActivated.

6.11Stock standard solutions (1.00 g/L)Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.11.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in isooctane and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.11.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.11.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.12Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure: 7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with isooctane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (7.3Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with isooctane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1. (image) Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentraton of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared for that compound.

7.5The cleanup procedure in Section 11 utilizes Florisil column chromatography. Florisil from different batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which is used, the use of lauric acid value 9 is suggested. The referenced procedure determines the adsorption from hexane solution of lauric acid (mg) per g of Florisil. The amount of Florisil to be used for each column is calculated by dividing 110 by this ratio and multiplying by 20 g.

7.6Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each single-component parameter of interest at the following concentrations in acetone: 4,4DDD, 10 g/mL; 4,4DDT, 10 g/mL; endosulfan II, 10 g/mL; endosulfan sulfate, 10 g/mL; endrin, 10 g/mL; any other single-component pesticide, 2 g/mL. If this method is only to be used to analyze for PCBs, chlordane, or toxaphene, the QC check sample concentrate should contain the most representative multicomponent parameter at a concentration of 50 g/mL in acetone. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 3 by adding 1.00 mL of QC check sample concentrate to each of four 1L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/mL; and the standard deviation of the recovery (s) in g/mL, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note: The large number of parameters in Table 3 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compmunds of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.

10 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 3, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 4, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 4, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T)2.44(100 S/T)%. 10 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 3 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spike sample.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standards to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2 sp to P +2 sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices 11 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 C from the time of collection until extraction. If the samples will not be extracted within 72 h of collection, the sample should be adjusted to a pH range of 5.0 to 9.0 with sodium hydroxide solution or sulfuric acid. Record the volume of acid or base used. If aldrin is to be determined, add sodium thiosulfate when residual chlorine is present. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine. 12 Field test kits are available for this purpose.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction. 2 10. Sample Extraction 10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2L separatory funnel.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optium technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7Increase the temperature of the hot water bath to about 80 C.

Momeltarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.9Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder.

Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation 11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure. The Florisil column allows for a select fractionation of the compounds and will eliminate polar interferences. Elemental sulfur, which interferes with the electron capture gas chromatography of certain pesticides, can be removed by the technique described in Section 11.3.

11.2Florisil column cleanup: 11.2.1Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5), into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.2.2Add 60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to exposure of the sodium sulfate layer to the air, stop the elution of the hexane by closing the stopcock on the chromatographic column. Discard the eluate.

11.2.3Adjust the sample extract volume to 10 mL with hexane and transfer it from the K-D concentrator tube onto the column. Rinse the tube twice with 1 to 2 mL of hexane, adding each rinse to the column.

11.2.4Place a 500-mL K-D flask and clean concentrator tube under the chromatographic column. Drain the column into the flask until the sodium sulfate layer is nearly exposed. Elute the column with 200 mL of 6% ethyl ether in hexane (V/V) (Fraction 1) at a rate of about 5 mL/min.

Remove the K-D flask and set it aside for later concentration. Elute the column again, using 200 mL of 15% ethyl ether in hexane (V/V) (Fraction 2), into a second K-D flask. Perform the third elution using 200 mL of 50% ethyl ether in hexane (V/V) (Fraction 3). The elution patterns for the pesticides and PCBs are shown in Table 2.

11.2.5Concentrate the fractions as in Section 10.6, except use hexane to prewet the column and set the water bath at about 85 C. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Adjust the volume of each fraction to 10 mL with hexane and analyze by gas chromatography (Section 12).

11.3Elemental sulfur will usually elute entirely in Fraction 1 of the Florisil column cleanup. To remove sulfur interference from this fraction or the original extract, pipet 1.00 mL of the concentrated extract into a clean concentrator tube or Teflon-sealed vial. Add one to three drops of mercury and seal. 13 Agitate the contents of the vial for 15 to 30 s. Prolonged shaking (2 h) may be required. If so, this may be accomplished with a reciprocal shaker. Alternatively, activated copper powder may be used for sulfur removal. 14 Analyze by gas chromatography.

12. Gas Chromatography 12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 to 10. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.4Inject 2 to 5 L of the sample extract or standard into the gas chromatograph using the solvent-flush technique. 15 Smaller (1.0 uL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L, the total extract volume, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations 13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2. (image) Equation 2 where: A=Amount of material injected (ng).

Vi=Volume of extract injected (L).

Vt=Volume of total extract (L).

Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3. (image) Equation 3 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

13.2When it is apparent that two or more PCB (Aroclor) mixtures are present, the Webb and McCall procedure 16 may be used to identify and quantify the Aroclors.

13.3For multicomponent mixtures (chlordane, toxaphene, and PCBs) match retention times of peaks in the standards with peaks in the sample.

Quantitate every identifiable peak unless interference with individual peaks persist after cleanup. Add peak height or peak area of each identified peak in the chromatogram. Calculate as total response in the sample versus total response in the standard.

13.4Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero. 1 The MDL concentrations listed in Table 1 were obtained using reagent water. 17 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4MDL to 1000MDL with the following exceptions: Chlordane recovery at 4MDL was low (60%); Toxaphene recovery was demonstrated linear over the range of 10MDL to 1000MDL. 17 14.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations. 18 Concentrations used in the study ranged from 0.5 to 30 g/L for single-component pesticides and from 8.5 to 400 g/L for multicomponent parameters. Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 4.

References 1. 40 CFR part 136, appendix B.

2. Determination of Pesticides and PCBs in Industrial and Municipal Wastewaters, EPA 600/482023, National Technical Information Service, PB82214222, Springfield, Virginia 22161, April 1982.

3. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

4. Giam, C.S., Chan, H.S., and Nef, G.S., Sensitive Method for Determination of Phthalate Ester Plasticizers in Open-Ocean Biota Samples, Analytical Chemistry, 47, 2225 (1975).

5. Giam, C.S., Chan, H.S. Control of Blanks in the Analysis of Phthalates in Air and Ocean Biota Samples, U.S. National Bureau of Standards, Special Publication 442, pp. 701708, 1976.

6. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

7. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

8. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

9. Mills, P.A. Variation of Florisil Activity: Simple Method for Measuring Absorbent Capacity and Its Use in Standardizing Florisil Columns, Journal of the Association of Official Analytical Chemists, 51, 29, (1968).

10. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 11. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

12. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

13. Goerlitz, D.F., and Law, L.M. Bulletin for Environmental Contamination and Toxicology, 6, 9 (1971).

14. Manual of Analytical Methods for the Analysis of Pesticides in Human and Environmental Samples, EPA600/880038, U.S. Environmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, North Carolina.

15. Burke, J.A. Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects, Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

16. Webb, R.G., and McCall, A.C. Quantitative PCB Standards for Election Capture Gas Chromatography, Journal of Chromatographic Science, 11, 366 (1973).

17. Method Detection Limit and Analytical Curve Studies, EPA Methods 606, 607, and 608, Special letter report for EPA Contract 68032606, U.S.

Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

18. EPA Method Study 18 Method 608Organochlorine Pesticides and PCBs, EPA 600/484061, National Technical Information Service, PB84211358, Springfield, Virginia 22161, June 1984.

Table 1_Chromatographic Conditions and Method Detection Limits ------------------------------------------------------------------------ Retention time (min) Method -------------------------- detection Parameter limit Col. 1 Col. 2 (g/L) ------------------------------------------------------------------------ -BHC.................... 1.35 1.82 0.003 -BHC.................... 1.70 2.13 0.004 -BHC..................... 1.90 1.97 0.006 Heptachlor..................... 2.00 3.35 0.003 -BHC.................... 2.15 2.20 0.009 Aldrin......................... 2.40 4.10 0.004 Heptachlor epoxide............. 3.50 5.00 0.083 Endosulfan I................... 4.50 6.20 0.014 4,4[prime]-DDE................. 5.13 7.15 0.004 Dieldrin....................... 5.45 7.23 0.002 Endrin......................... 6.55 8.10 0.006 4,4[prime]-DDD................. 7.83 9.08 0.011 Endosulfan II.................. 8.00 8.28 0.004 4,4[prime]-DDT................. 9.40 11.75 0.012 Endrin aldehyde................ 11.82 9.30 0.023 Endosulfan sulfate............. 14.22 10.70 0.066 Chlordane...................... mr mr 0.014 Toxaphene...................... mr mr 0.24 PCB-1016....................... mr mr nd PCB-1221....................... mr mr nd PCB-1232....................... mt mr nd PCB-1242....................... mr mr 0.065 PCB-1248....................... mr mr nd PCB-1254....................... mr mr nd PCB-1260....................... mr mr nd ------------------------------------------------------------------------ AColumn 1 conditions: Supelcoport (100/120 mesh) coated with 1.5% SP- 2250/1.95% SP-2401 packed in a 1.8 m long x 4 mm ID glass column with 5% methane/95% argon carrier gas at 60 mL/min flow rate. Column temperature held isothermal at 200 C, except for PCB-1016 through PCB-1248, should be measured at 160 C.

AColumn 2 conditions: Supelcoport (100/120 mesh) coated with 3% OV-1 packed in a 1.8 m long x 4 mm ID glass column with 5% methane/95% argon carrier gas at 60 mL/min flow rate. Column temperature held isothermal at 200 C for the pesticides; at 140 C for PCB- 1221 and 1232; and at 170 C for PCB-1016 and 1242 to 1268.

Amr=Multiple peak response. See Figures 2 thru 10.

And=Not determined.

Table 2_Distribution of Chlorinated Pesticides and PCBs into Florisil Column Fractions 2 ------------------------------------------------------------------------ Percent recovery by fraction \a\ Parameter -------------------------------------- 1 2 3 ------------------------------------------------------------------------ Aldrin........................... 100 ........... ...........

-BHC...................... 100 ........... ...........

-BHC....................... 97 ........... ...........

-BHC...................... 98 ........... ...........

-BHC...................... 100 ........... ...........

Chlordane........................ 100 ........... ...........

4,4[prime]-DDD................... 99 ........... ...........

4,4[prime]-DDE................... 98 ........... ...........

4,4[prime]-DDT................... 100 ........... ...........

Dieldrin......................... 0 100 ...........

Endosulfan I..................... 37 64 ...........

Endosulfan II.................... 0 7 91 Endosulfan sulfate............... 0 0 106 Endrin........................... 4 96 ...........

Endrin aldehyde.................. 0 68 26 Heptachlor....................... 100 ........... ...........

Heptachlor epoxide............... 100 ........... ...........

Toxaphene........................ 96 ........... ...........

PCB-1016......................... 97 ........... ...........

PCB-1221......................... 97 ........... ...........

PCB-1232......................... 95 4 ...........

PCB-1242......................... 97 ........... ...........

PCB-1248......................... 103 ........... ...........

PCB-1254......................... 90 ........... ...........

PCB-1260......................... 95 ........... ...........

------------------------------------------------------------------------ \a\ Eluant composition: Fraction 1-6% ethyl ether in hexane.

Fraction 2-15% ethyl ether in hexane.

Fraction 3-50% ethyl ether in hexane.

Table 3_QC Acceptance Criteria_Method 608 ---------------------------------------------------------------------------------------------------------------- Range for Test conc. Limit for s X Range for Parameter (g/ (g/L) (g/ P, Ps(%) L) L) ---------------------------------------------------------------------------------------------------------------- Aldrin...................................................... 2.0 0.42 1.08-2.24 42-122 -BHC................................................. 2.0 0.48 0.98-2.44 37-134 -BHC.................................................. 2.0 0.64 0.78-2.60 17-147 -BHC................................................. 2.0 0.72 1.01-2.37 19-140 -BHC................................................. 2.0 0.46 0.86-2.32 32-127 Chlordane................................................... 50 10.0 27.6-54.3 45-119 4,4 [prime]-DDD............................................. 10 2.8 4.8-12.6 31-141 4,4 [prime]-DDE............................................. 2.0 0.55 1.08-2.60 30-145 4,4[prime]-DDT.............................................. 10 3.6 4.6-13.7 25-160 Dieldrin.................................................... 2.0 0.76 1.15-2.49 36-146 Endosulfan I................................................ 2.0 0.49 1.14-2.82 45-153 Endosulfan II............................................... 10 6.1 2.2-17.1 D-202 Endosulfan Sulfate.......................................... 10 2.7 3.8-13.2 26-144 Endrin...................................................... 10 3.7 5.1-12.6 30-147 Heptachlor.................................................. 2.0 0.40 0.86-2.00 34-111 Heptachlor epoxide.......................................... 2.0 0.41 1.13-2.63 37-142 Toxaphene................................................... 50.0 12.7 27.8-55.6 41-126 PCB-1016.................................................... 50 10.0 30.5-51.5 50-114 PCB-1221.................................................... 50 24.4 22.1-75.2 15-178 PCB-1232.................................................... 50 17.9 14.0-98.5 10-215 PCB-1242.................................................... 50 12.2 24.8-69.6 39-150 PCB-1248.................................................... 50 15.9 29.0-70.2 38-158 PCB-1254.................................................... 50 13.8 22.2-57.9 29-131 PCB-1260.................................................... 50 10.4 18.7-54.9 8-127 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 4. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 4.

Table 4_Method Accuracy and Precision as Functions of Concentration_Method 608 ---------------------------------------------------------------------------------------------------------------- Single analyst Accuracy, as precision, Overall precision, Parameter recovery, X[prime] sr[prime] S[prime] (g/ (g/L) (g/L) L) ---------------------------------------------------------------------------------------------------------------- Aldrin.............................................. 0.81C+0.04 0.16X-0.04 0.20X-0.01 -BHC......................................... 0.84C+0.03 0.13X+0.04 0.23X-0.00 -BHC.......................................... 0.81C+0.07 0.22X-0.02 0.33X-0.05 -BHC......................................... 0.81C+0.07 0.18X+0.09 0.25X+0.03 -BHC......................................... 0.82C-0.05 0.12X+0.06 0.22X+0.04 Chlordane........................................... 0.82C-0.04 0.13X+0.13 0.18X+0.18 4,4[prime]-DDD...................................... 0.84C+0.30 0.20X-0.18 0.27X-0.14 4,4[prime]-DDE...................................... 0.85C+0.14 0.13X+0.06 0.28X-0.09 4,4[prime]-DDT...................................... 0.93C-0.13 0.17X+0.39 0.31X-0.21 Dieldrin............................................ 0.90C+0.02 0.12X+0.19 0.16X+0.16 Endosulfan I........................................ 0.97C+0.04 0.10X+0.07 0.18X+0.08 Endosulfan II....................................... 0.93C+0.34 0.41X_0.65 0.47X-0.20 Endosulfan Sulfate.................................. 0.89C-0.37 0.13X+0.33 0.24X+0.35 Endrin.............................................. 0.89C-0.04 0.20X+0.25 0.24X+0.25 Heptachlor.......................................... 0.69C+0.04 0.06X+0.13 0.16X+0.08 Heptachlor epoxide.................................. 0.89C+0.10 0.18X-0.11 0.25X-0.08 Toxaphene........................................... 0.80C+1.74 0.09X+3.20 0.20X+0.22 PCB-1016............................................ 0.81C+0.50 0.13X+0.15 0.15X+0.45 PCB-1221............................................ 0.96C+0.65 0.29X-0.76 0.35X-0.62 PCB-1232............................................ 0.91C+10.79 0.21X-1.93 0.31X+3.50 PCB-1242............................................ 0.93C+0.70 0.11X+1.40 0.21X+1.52 PCB-1248............................................ 0.97C+1.06 0.17X+0.41 0.25X-0.37 PCB-1254............................................ 0.76C+2.07 0.15X+1.66 0.17X+3.62 PCB-1260............................................ 0.66C+3.76 0.22X-2.37 0.39X-4.86 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

(image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF Method 609Nitroaromatics and Isophorone 1. Scope and Application 1.1This method covers the determination of certain nitroaromatics and isophorone. The following parameters may be determined by this method: ------------------------------------------------------------------------ Parameter STORET No. CAS No.

------------------------------------------------------------------------ 2,4-Dinitrotoluene............................ 34611 121-14-2 2,6-Dinitrotoluene............................ 34626 606-20-2 Isophorone.................................... 34408 78-59-1 Nitrobenzene.................................. 34447 98-95-3 ------------------------------------------------------------------------ 1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. Isophorone and nitrobenzene are measured by flame ionization detector gas chromatography (FIDGC). The dinitrotoluenes are measured by electron capture detector gas chromatography (ECDGC). 2 2.2The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baseliles in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned. 3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating.

Thorough rinsing with such solvents usually eliminates PCB interference.

Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants.

Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified46 for the information of the analyst.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.): 5.2.1Separatory funnel2L, with Teflon stopcock.

5.2.2Drying columnChromatographic column, approximately 400 mm long 19 mm ID, with coarse frit filter disc.

5.2.3Chromatographic column100 mm long 10 mm ID, with Teflon stopcock.

5.2.4Concentrator tube, Kuderna-Danish10-mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5Evaporative flask, Kuderna-Danish500-mL (Kontes K5700010500 or equivalent). Attach to concentrator tube with springs.

5.2.6Snyder column, Kuderna-DanishThree-ball macro (Kontes K5030000121 or equivalent).

5.2.7Snyder column, Kuderna-DanishTwo-ball micro (Kontes K5690010219 or equivalent).

5.2.8Vials10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.3Boiling chipsApproximately 10/40 mesh. Heat to 400C for 30 min or Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2C). The bath should be used in a hood.

5.5BalanceAnalytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatographAn analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder.

A data system is recommended for measuring peak areas.

5.6.1Column 11.2 m long 2 or 4 mm ID glass, packed with 1.95% QF1/1.5% OV17 on Gas-Chrom Q (80/100 mesh) or equivalent. This column was used to develop the method performance statements given in Section 14.

Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.2Column 23.0 m long 2 or 4 mm ID glass, packed with 3% OV101 on Gas-Chrom Q (80/100 mesh) or equivalent.

5.6.3DetectorsFlame ionization and electron capture detectors. The flame ionization detector (FID) is used when determining isophorone and nitrobenzene. The electron capture detector (ECD) is used when determining the dinitrotoluenes. Both detectors have proven effective in the analysis of wastewaters and were used in develop the method performance statements in Section 14. Guidelines for the use to alternate detectors are provided in Section 12.1.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Sodium hydroxide solution (10 N)Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3Sulfuric acid (1+1)Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.4Acetone, hexane, methanol, methylene chloridePesticide quality or equivalent.

6.5Sodium sulfate(ACS) Granular, anhydrous. Purify by heating at 400 C for 4 h in a shallow tray.

6.6FlorisilPR grade (60/100 mesh). Purchase activated at 1250 F and store in dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 200 C in a foil-covered glass container and allow to cool.

6.7Stock standard solutions (1.00 g/L)Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in hexane and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure: 7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with hexane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (7.3Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flash. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with hexane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

Equation 1. (image) where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 15%, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1,5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest in acetone at a concentration of 20 g/mL for each dinitrotoluene and 100 g/mL for isophorone and nitrobenzene. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determile background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.

7 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 3, substituting X for X 8; (3) calculate the range for recovery at the spike concentration as (100 X/T) 2.44 (100 S/T)%. 7 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4.If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp = 10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices 8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 C from the time of collection until extraction.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction. 2 10. Sample Extraction 10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2L separatory funnel. Check the pH of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with sodium hydroxide solution or sulfuric acid.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Sections 10.7 and 10.8 describe a procedure for exchanging the methylene chloride solvent to hexane while concentrating the extract volume to 1.0 mL. When it is not necessary to achieve the MDL in Table 2, the solvent exchange may be made by the addition of 50 mL of hexane and concentration to 10 mL as described in Method 606, Sections 10.7 and 10.8.

10.7Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.8Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Add 1 to 2 mL of hexane and a clean boiling chip to the concentrator tube and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of hexane to the top. Place the micro-K-D apparatus on a hot water bath (60 to 65C) so that the concentrator tube is partially immersed in the hot water.

Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.9Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of hexane. Adjust the extract volume to 1.0 mL. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.10Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder.

Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation 11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2Florisil column cleanup: 11.2.1Prepare a slurry of 10 g of activated Florisil in methylene chloride/hexane (1+9)(V/V) and place the Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 cm of anhydrous sodium sulfate to the top. Adjust the elution rate to about 2 mL/min.

11.2.2Just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 30 mL of methylene chloride/hexane (1 + 9)(V/V) and continue the elution of the column.

Discard the eluate.

11.2.3Next, elute the column with 30 mL of acetone/methylene chloride (1 + 9)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction as in Sections 10.6, 10.7, 10.8, and 10.9 including the solvent exchange to 1 mL of hexane. This fraction should contain the nitroaromatics and isophorone. Analyze by gas chromatography (Section 12).

12. Gas Chromatography 12.1Isophorone and nitrobenzene are analyzed by injection of a portion of the extract into an FIDGC. The dinitrotoluenes are analyzed by a separate injection into an ECDGC. Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions.

Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the same extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.4Inject 2 to 5 L of the sample extract or standard into the gas chromatograph using the solvent-flush technique. 9 Smaller (1.0 L) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L, the total extract volume, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations 13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2. (image) Equation 2 where: A=Amount of material injected (ng).

Vi=Volume of extract injected (L).

Vt=Volume of total extract (L).

Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3. (image) Equation 3 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

13.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero. 1 The MDL concentrations listed in Table 1 were obtained using reagent water. 10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 7MDL to 1000MDL. 10 14.3This method was tested by 18 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 515 g/L. 11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References 1. 40 CFR part 136, appendix B.

2. Determination of Nitroaromatic Compounds and Isophorone in Industrial and Municipal Wastewaters, EPA 600/ 482024, National Technical Information Service, PB82208398, Springfield, Virginia 22161, May 1982.

3. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

4. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 8. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

9. Burke, J.A. Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects, Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

10. Determination of Method Detection Limit and Analytical Curve for EPA Method 609Nitroaromatics and Isophorone, Special letter report for EPA Contract 68032624, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

11. EPA Method Study 19, Method 609 (Nitroaromatics and Isophorone), EPA 600/484018, National Technical Information Service, PB84176908, Springfield, Virginia 22161, March 1984.

Table 1_Chromatographic Conditions and Method Detection Limits ---------------------------------------------------------------------------------------------------------------- Retention time (min) Method detection limit ---------------------------- (g/L) Parameter --------------------------- Col. 1 Col. 2 ECDGC FIDGC ---------------------------------------------------------------------------------------------------------------- Nitrobenzene............................................ 3.31 4.31 13.7 3.6 2,6-Dinitrotoluene...................................... 3.52 4.75 0.01 - Isophorone.............................................. 4.49 5.72 15.7 5.7 2,4-Dinitrotoluene...................................... 5.35 6.54 0.02 - ---------------------------------------------------------------------------------------------------------------- AAColumn 1 conditions: Gas-Chrom Q (80/100 mesh) coated with 1.95% QF-1/1.5% OV-17 packed in a 1.2 m long x 2 mm or 4 mm ID glass column. A 2 mm ID column and nitrogen carrier gas at 44 mL/min flow rate were used when determining isophorone and nitrobenzene by FIDGC. The column temperature was held isothermal at 85 C. A 4 mm ID column and 10% methane/90% argon carrier gas at 44 mL/min flow rate were used when determining the dinitrotoluenes by ECDGC. The column temperature was held isothermal at 145 C.

AAColumn 2 conditions: Gas-Chrom Q (80/100 mesh) coated with 3% OV-101 packed in a 3.0 m long x 2 mm or 4 mm ID glass column. A 2 mm ID column and nitrogen carrier gas at 44 mL/min flow rate were used when determining isophorone and nitrobenzene by FIDGC. The column temperature was held isothermal at 100 C. A 4 mm ID column and 10% methane/90% argon carrier gas at 44 mL/min flow rate were used when determining the dinitrotoluenes by ECDGC. The column temperature was held isothermal at 150 C.

Table 2_QC Acceptance Criteria_Method 609 ---------------------------------------------------------------------------------------------------------------- Test Conc. Range for X Parameter (g/ Limit for s (g/L) Range for L) (g/L) P, Ps (%) ---------------------------------------------------------------------------------------------------------------- 2,4-Dinitrotoluene........................................ 20 5.1 3.6-22.8 6-125 2,6-Dinitrotoluene........................................ 20 4.8 3.8-23.0 8-126 Isophorone................................................ 100 32.3 8.0-100.0 D-117 Nitrobenzene.............................................. 100 33.3 25.7-100.0 6-118 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3_Method Accuracy and Precision as Functions of Concentration_Method 609 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Single analyst Overall recovery, precision, precision, Parameter X[prime] sr[prime] S[prime] (g/L) (g/L) (g/L) ---------------------------------------------------------------------------------------------------------------- 2,4-Dinitro- toluene............................................... 0.65C+0.22 0.20X+0.08 0.37X-0.07 2,6-Dinitro- toluene............................................... 0.66C+0.20 0.19X+0.06 0.36X-0.00 Isophorone............................................. 0.49C+2.93 0.28X+2.77 0.46X+0.31 Nitrobenzene........................................... 0.60C+2.00 0.25X+2.53 0.37X-0.78 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

(image) View or download PDF (image) View or download PDF Method 610Polynuclear Aromatic Hydrocarbons 1. Scope and Application 1.1This method covers the determination of certain polynuclear aromatic hydrocarbons (PAH). The following parameters can be determined by this method: ------------------------------------------------------------------------ Parameter STORET No. CAS No.

------------------------------------------------------------------------ Acenaphthene................................ 34205 83-32-9 Acenaphthylene.............................. 34200 208-96-8 Anthracene.................................. 34220 120-12-7 Benzo(a)anthracene.......................... 34526 56-55-3 Benzo(a)pyrene.............................. 34247 50-32-8 Benzo(b)fluoranthene........................ 34230 205-99-2 Benzo(ghi)perylene.......................... 34521 191-24-2 Benzo(k)fluoranthene........................ 34242 207-08-9 Chrysene.................................... 34320 218-01-9 Dibenzo(a,h)anthracene...................... 34556 53-70-3 Fluoranthene................................ 34376 206-44-0 Fluorene.................................... 34381 86-73-7 Indeno(1,2,3-cd)pyrene...................... 34403 193-39-5 Naphthalene................................. 34696 91-20-3 Phenanthrene................................ 34461 85-01-8 Pyrene...................................... 34469 129-00-0 ------------------------------------------------------------------------ 1.2This is a chromatographic method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for many of the parameters listed above, using the extract produced by this method.

1.3This method provides for both high performance liquid chromatographic (HPLC) and gas chromatographic (GC) approaches for the determination of PAHs. The gas chromatographic procedure does not adequately resolve the following four pairs of compounds: Anthracene and phenanthrene; chrysene and benzo(a)anthracene; benzo(b)fluoranthene and benzo(k)fluoranthene; and dibenzo(a,h) anthracene and indeno (1,2,3-cd)pyrene. Unless the purpose for the analysis can be served by reporting the sum of an unresolved pair, the liquid chromatographic approach must be used for these compounds. The liquid chromatographic method does resolve all 16 of the PAHs listed.

1.4The method detection limit (MDL, defined in Section 15.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.5The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. Selection of the aliquots must be made prior to the solvent exchange steps of this method. The analyst is allowed the latitude, under Sections 12 and 13, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.6Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.7This method is restricted to use by or under the supervision of analysts experienced in the use of HPLC and GC systems and in the interpretation of liquid and gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and concentrated to a volume of 10 mL or less. The extract is then separated by HPLC or GC. Ultraviolet (UV) and fluorescence detectors are used with HPLC to identify and measure the PAHs. A flame ionization detector is used with GC. 2 2.2The method provides a silica gel column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardward that lead to discrete artifacts and/or elevated baselines in the chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned. 3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400C for 15 to 30 min.

Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3The extent of interferences that may be encountered using liquid chromatographic techniques has not been fully assessed. Although the HPLC conditions described allow for a unique resolution of the specific PAH compounds covered by this method, other PAH compounds may interfere.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method have not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified46 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzo(a)anthracene, benzo(a)pyrene, and dibenzo(a,h)-anthracene. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.): 5.2.1Separatory funnel2L, with Teflon stopcock.

5.2.2Drying columnChromatographic column, approximately 400 mm long 19 mm ID, with coarse frit filter disc.

5.2.3Concentrator tube, Kuderna-Danish10-mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.4Evaporative flask, Kuderna-Danish500-mL (Kontes K5700010500 or equivalent). Attach to concentrator tube with springs.

5.2.5Snyder column, Kuderna-DanishThree-ball macro (Kontes K5030000121 or equivalent).

5.2.6Snyder column, Kuderna-DanishTwo-ball micro (Kontes K5690010219 or equivalent).

5.2.7Vials10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.2.8Chromatographic column250 mm long 10 mm ID, with coarse frit filter disc at bottom and Teflon stopcock.

5.3Boiling chipsApproximately 10/40 mesh. Heat to 400 C for 30 min or Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2 C). The bath should be used in a hood.

5.5BalanceAnalytical, capable of accurately weighing 0.0001 g.

5.6High performance liquid chromatograph (HPLC)An analytical system complete with column supplies, high pressure syringes, detectors, and compatible strip-chart recorder. A data system is recommended for measuring peak areas and retention times.

5.6.1Gradient pumping systemConstant flow.

5.6.2Reverse phase columnHC-ODS Sil-X, 5 micron particle diameter, in a 25 cm 2.6 mm ID stainless steel column (Perkin Elmer No. 0890716 or equivalent). This column was used to develop the method performance statements in Section 15. Guidelines for the use of alternate column packings are provided in Section 12.2.

5.6.3DetectorsFluorescence and/or UV detectors. The fluorescence detector is used for excitation at 280 nm and emission greater than 389 nm cutoff (Corning 375 or equivalent). Fluorometers should have dispersive optics for excitation and can utilize either filter or dispersive optics at the emission detector. The UV detector is used at 254 nm and should be coupled to the fluorescence detector. These detectors were used to develop the method performance statements in Section 15. Guidelines for the use of alternate detectors are provided in Section 12.2.

5.7Gas chromatographAn analytical system complete with temperature programmable gas chromatograph suitable for on-column or splitless injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.7.1Column1.8 m long 2 mm ID glass, packed with 3% OV17 on Chromosorb W-AW-DCMS (100/120 mesh) or equivalent. This column was used to develop the retention time data in Table 2. Guidelines for the use of alternate column packings are provided in Section 13.3.

5.7.2DetectorFlame ionization detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), excluding the four pairs of unresolved compounds listed in Section 1.3. Guidelines for the use of alternate detectors are provided in Section 13.3.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Sodium thiosulfate(ACS) Granular.

6.3Cyclohexane, methanol, acetone, methylene chloride, pentanePesticide quality or equivalent.

6.4AcetonitrileHPLC quality, distilled in glass.

6.5Sodium sulfate(ACS) Granular, anhydrous. Purify by heating at 400C for 4 h in a shallow tray.

6.6Silica gel100/200 mesh, desiccant, Davison, grade-923 or equivalent.

Before use, activate for at least 16 h at 130 C in a shallow glass tray, loosely covered with foil.

6.7Stock standard solutions (1.00 g/L)Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in acetonitrile and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Establish liquid or gas chromatographic operating conditions equivalent to those given in Table 1 or 2. The chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure: 7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with acetonitrile. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 5 to 25 L for HPLC and 2 to 5 L for GC, analyze each calibration standard according to Section 12 or 13, as appropriate.

Tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound.

Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (7.3Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with acetonitrile. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 5 to 25 L for HPLC and 2 to 5 L for GC, analyze each calibration standard according to Section 12 or 13, as appropriate.

Tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1. (image) Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, 12.2, and 13.3) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetonitrile: 100 g/mL of any of the six early-eluting PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, and anthracene); 5 g/mL of benzo(k)fluoranthene; and 10 g/mL of any of the other PAHs. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 3 by adding 1.00 mL of QC check sample concentrate to each of four 1L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note: The large number of parameters in Table 3 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none, (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.

7 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 3, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 4, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 4, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T)2.44(100 S/T)%. 7 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the critiera must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 3 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spike sample.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices 8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4C from the time of collection until extraction. PAHs are known to be light sensitive; therefore, samples, extracts, and standards should be stored in amber or foil-wrapped bottles in order to minimize photolytic decomposition. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine. 9 Field test kits are available for this purpose.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction. 2 10. Sample Extraction 10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2L separatory funnel.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial and protected from light. If the sample extract requires no further cleanup, proceed with gas or liquid chromatographic analysis (Section 12 or 13). If the sample requires further cleanup, proceed to Section 11.

10.8Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder.

Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation 11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the methods as revised to incorporate the cleanup procedure.

11.2Before the silica gel cleanup technique can be utilized, the extract solvent must be exchanged to cyclohexane. Add 1 to 10 mL of the sample extract (in methylene chloride) and a boiling chip to a clean K-D concentrator tube. Add 4 mL of cyclohexane and attach a two-ball micro-Snyder column. Prewet the column by adding 0.5 mL of methylene chloride to the top. Place the micro-K-D apparatus on a boiling (100 C) water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of the liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of cyclohexane. Adjust the extract volume to about 2 mL.

11.3Silica gel column cleanup for PAHs: 11.3.1Prepare a slurry of 10 g of activiated silica gel in methylene chloride and place this into a 10-mm ID chromatographic column. Tap the column to settle the silica gel and elute the methylene chloride. Add 1 to 2 cm of anhydrous sodium sulfate to the top of the silica gel.

11.3.2Preelute the column with 40 mL of pentane. The rate for all elutions should be about 2 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, transfer the 2-mL cyclohexane sample extract onto the column using an additional 2 mL cyclohexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 25 mL of pentane and continue the elution of the column. Discard this pentane eluate.

11.3.3Next, elute the column with 25 mL of methylene chloride/pentane (4+6)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction to less than 10 mL as in Section 10.6. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint with pentane. Proceed with HPLC or GC analysis.

12. High Performance Liquid Chromatography 12.1To the extract in the concentrator tube, add 4 mL of acetonitrile and a new boiling chip, then attach a two-ball micro-Snyder column.

Concentrate the solvent as in Section 10.6, except set the water bath at 95 to 100 C. When the apparatus is cool, remove the micro-Snyder column and rinse its lower joint into the concentrator tube with about 0.2 mL of acetonitrile. Adjust the extract volume to 1.0 mL.

12.2Table 1 summarizes the recommended operating conditions for the HPLC. Included in this table are retention times, capacity factors, and MDL that can be achieved under these conditions. The UV detector is recommended for the determination of naphthalene, acenaphthylene, acenapthene, and fluorene and the fluorescence detector is recommended for the remaining PAHs. Examples of the separations achieved by this HPLC column are shown in Figures 1 and 2. Other HPLC columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.3Calibrate the system daily as described in Section 7.

12.4If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the instrument.

12.5Inject 5 to 25 L of the sample extract or standard into the HPLC using a high pressure syringe or a constant volume sample injection loop. Record the volume injected to the nearest 0.1 L, and the resulting peak size in area or peak height units. Re-equilibrate the HPLC column at the initial gradient conditions for at least 10 min between injections.

12.6Identify the parameters in the sample by comparing the retention time of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.7If the response for a peak exceeds the working range of the system, dilute the extract with acetonitrile and reanalyze.

12.8If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Gas Chromatography 13.1The packed column GC procedure will not resolve certain isomeric pairs as indicated in Section 1.3 and Table 2. The liquid chromatographic procedure (Section 12) must be used for these parameters.

13.2To achieve maximum sensitivity with this method, the extract must be concentrated to 1.0 mL. Add a clean boiling chip to the methylene chloride extract in the concentrator tube. Attach a two-ball micro-Snyder column. Prewet the micro-Snyder column by adding about 0.5 mL of methylene chloride to the top. Place the micro-K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of methylene chloride. Adjust the final volume to 1.0 mL and stopper the concentrator tube.

13.3Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times that were obtained under these conditions. An example of the separations achieved by this column is shown in Figure 3. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

13.4Calibrate the gas chromatographic system daily as described in Section 7.

13.5If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

13.6Inject 2 to 5 L of the sample extract or standard into the gas chromatograph using the solvent-flush technique. 10 Smaller (1.0 L) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L, and the resulting peak size in area or peak height units.

13.7Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

13.8If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

13.9If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

14. Calculations 14.1Determine the concentration of individual compounds in the sample.

14.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2. (image) Equation 2 where: A=Amount of material injected (ng).

Vi=Volume of extract injected (L).

Vt=Volume of total extract (L).

Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3. (image) Equation 3 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

14.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

15. Method Performance 15.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero. 1 The MDL concentrations listed in Table 1 were obtained using reagent water. 11 Similar results were achieved using representative wastewaters. MDL for the GC approach were not determined. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

15.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 8 MDL to 800 MDL 11 with the following exception: benzo(ghi)perylene recovery at 80 and 800 MDL were low (35% and 45%, respectively).

15.3This method was tested by 16 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.1 to 425 g/L. 12 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 4.

References 1. 40 CFR part 136, appendix B.

2. Determination of Polynuclear Aromatic Hydrocarbons in Industrial and Municipal Wastewaters, EPA 600/482025, National Technical Information Service, PB82258799, Springfield, Virginia 22161, June 1982.

3. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

4. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 8. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

9. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

10. Burke, J.A. Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects, Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

11. Cole, T., Riggin, R., and Glaser, J. Evaluation of Method Detection Limits and Analytical Curve for EPA Method 610PNAs, International Symposium on Polynuclear Aromatic Hydrocarbons, 5th, Battelle's Columbus Laboratories, Columbus, Ohio (1980).

12. EPA Method Study 20, Method 610 (PNA's), EPA 600/484063, National Technical Information Service, PB84211614, Springfield, Virginia 22161, June 1984.

Table 1_High Performance Liquid Chromatography Conditions and Method Detection Limits ------------------------------------------------------------------------ Method Retention Column detection Parameter time capacity limit (min) factor (g/ (k[prime]) L) a ------------------------------------------------------------------------ Naphthalene.......................... 16.6 12.2 1.8 Acenaphthylene....................... 18.5 13.7 2.3 Acenaphthene......................... 20.5 15.2 1.8 Fluorene............................. 21.2 15.8 0.21 Phenanthrene......................... 22.1 16.6 0.64 Anthracene........................... 23.4 17.6 0.66 Fluoranthene......................... 24.5 18.5 0.21 Pyrene............................... 25.4 19.1 0.27 Benzo(a)anthracene................... 28.5 21.6 0.013 Chrysene............................. 29.3 22.2 0.15 Benzo(b)fluoranthene................. 31.6 24.0 0.018 Benzo(k)fluoranthene................. 32.9 25.1 0.017 Benzo(a)pyrene....................... 33.9 25.9 0.023 Dibenzo(a,h)anthracene............... 35.7 27.4 0.030 Benzo(ghi)perylene................... 36.3 27.8 0.076 Indeno(1,2,3-cd)pyrene............... 37.4 28.7 0.043 ------------------------------------------------------------------------ AAAHPLC column conditions: Reverse phase HC-ODS Sil-X, 5 micron particle size, in a 25 cm x 2.6 mm ID stainless steel column.

Isocratic elution for 5 min. using acetonitrile/water (4+6), then linear gradient elution to 100% acetonitrile over 25 min. at 0.5 mL/ min flow rate. If columns having other internal diameters are used, the flow rate should be adjusted to maintain a linear velocity of 2 mm/ sec.

a The MDL for naphthalene, acenaphthylene, acenaphthene, and fluorene were determined using a UV detector. All others were determined using a fluorescence detector.

Table 2_Gas Chromatographic Conditions and Retention Times ------------------------------------------------------------------------ Retention Parameter time (min) ------------------------------------------------------------------------ Naphthalene................................................. 4.5 Acenaphthylene.............................................. 10.4 Acenaphthene................................................ 10.8 Fluorene.................................................... 12.6 Phenanthrene................................................ 15.9 Anthracene.................................................. 15.9 Fluoranthene................................................ 19.8 Pyrene...................................................... 20.6 Benzo(a)anthracene.......................................... 24.7 Chrysene.................................................... 24.7 Benzo(b)fluoranthene........................................ 28.0 Benzo(k)fluoranthene........................................ 28.0 Benzo(a)pyrene.............................................. 29.4 Dibenzo(a,h)anthracene...................................... 36.2 Indeno(1,2,3-cd)pyrene...................................... 36.2 Benzo(ghi)perylene.......................................... 38.6 ------------------------------------------------------------------------ GC Column conditions: Chromosorb W-AW-DCMS (100/120 mesh) coated with 3% OV-17 packed in a 1.8 x 2 mm ID glass column with nitrogen carrier gas at 40 mL/min. flow rate. Column temperature was held at 100 C for 4 min., then programmed at 8 C/min. to a final hold at 280 C.

Table 3_QC Acceptance Criteria_Method 610 ---------------------------------------------------------------------------------------------------------------- Range for X Test conc. Limit for s (g/ Range for Parameter (g/ (g/ L) P, Ps (%) L) L) ---------------------------------------------------------------------------------------------------------------- Acenaphthene................................................ 100 40.3 D-105.7 D-124 Acenaphthylene.............................................. 100 45.1 22.1-112.1 D-139 Anthracene.................................................. 100 28.7 11.2-112.3 D-126 Benzo(a)anthracene.......................................... 10 4.0 3.1-11.6 12-135 Benzo(a)pyrene.............................................. 10 4.0 0.2-11.0 D-128 Benzo(b)fluor-anthene....................................... 10 3.1 1.8-13.8 6-150 Benzo(ghi)perylene.......................................... 10 2.3 D-10.7 D-116 Benzo(k)fluo-ranthene....................................... 5 2.5 D-7.0 D-159 Chrysene.................................................... 10 4.2 D-17.5 D-199 Dibenzo(a,h)an-thracene..................................... 10 2.0 0.3-10.0 D-110 Fluoranthene................................................ 10 3.0 2.7-11.1 14-123 Fluorene.................................................... 100 43.0 D-119 D-142 Indeno(1,2,3-cd)pyrene...................................... 10 3.0 1.2-10.0 D-116 Naphthalene................................................. 100 40.7 21.5-100.0 D-122 Phenanthrene................................................ 100 37.7 8.4-133.7 D-155 Pyrene...................................................... 10 3.4 1.4-12.1 D-140 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 4. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 4.

Table 4_Method Accuracy and Precision as Functions of Concentration_Method 610 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Single analyst Overall recovery, precision, precision, Parameter X[prime] sr[prime] S[prime] (g/L) (g/L) (g/L) ---------------------------------------------------------------------------------------------------------------- Acenaphthene.................................................... 0.52C + 0.54 0.39X + 0.76 0.53X + 1.32 Acenaphthylene.................................................. 0.69C - 1.89 0.36X + 0.29 0.42X + 0.52 Anthracene...................................................... 0.63C - 1.26 0.23X + 1.16 0.41X + 0.45 Benzo(a)anthracene.............................................. 0.73C + 0.05 0.28X + 0.04 0.34X + 0.02 Benzo(a)pyrene.................................................. 0.56C + 0.01 0.38X - 0.01 0.53X - 0.01 Benzo(b)fluoranthene............................................ 0.78C + 0.01 0.21X + 0.01 0.38X - 0.00 Benzo(ghi)perylene.............................................. 0.44C + 0.30 0.25X + 0.04 0.58X + 0.10 Benzo(k)fluoranthene............................................ 0.59C + 0.00 0.44X - 0.00 0.69X + 0.01 Chrysene........................................................ 0.77C - 0.18 0.32X - 0.18 0.66X - 0.22 Dibenzo(a,h)anthracene.......................................... 0.41C + 0.11 0.24X + 0.02 0.45X + 0.03 Fluoranthene.................................................... 0.68C + 0.07 0.22X + 0.06 0.32X + 0.03 Fluorene........................................................ 0.56C - 0.52 0.44X - 1.12 0.63X - 0.65 Indeno(1,2,3-cd)pyrene.......................................... 0.54C + 0.06 0.29X + 0.02 0.42X + 0.01 Naphthalene..................................................... 0.57C - 0.70 0.39X - 0.18 0.41X + 0.74 Phenanthrene.................................................... 0.72C - 0.95 0.29X + 0.05 0.47X - 0.25 Pyrene.......................................................... 0.69C - 0.12 0.25X + 0.14 0.42X - 0.00 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

(image) View or download PDF (image) View or download PDF (image) View or download PDF Method 611Haloethers 1. Scope and Application 1.1This method covers the determination of certain haloethers. The following parameters can be determined by this method: ------------------------------------------------------------------------ Parameter STORET No. CAS No.

------------------------------------------------------------------------ Bis(2-chloroethyl) ether...................... 34273 111-44-4 Bis(2-chloroethoxy) methane................... 34278 111-91-1 Bis(2-chloroisopropyl) ether.................. 34283 108-60-1 4-Bromophenyl phenyl ether.................... 34636 101-55-3 4-Chlorophenyl phenyl either.................. 34641 7005-72-3 ------------------------------------------------------------------------ 1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the parameters are then measured with a halide specific detector.2 2.2The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed be detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials, such a PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference.

Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants.

Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3Dichlorobenzenes are known to coelute with haloethers under some gas chromatographic conditions. If these materials are present together in a sample, it may be necessary to analyze the extract with two different column packings to completely resolve all of the compounds.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified46 for the information of the analyst.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4 C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.): 5.2.1Separatory funnel2-L, with Teflon stopcock.

5.2.2Drying columnChromatographic column, approximately 400 mm long 19 mm ID, with coarse frit filter disc.

5.2.3Chromatographic column400 mm long 19 mm ID, with Teflon stopcock and coarse frit filter disc at bottom (Kontes K4205400224 or equivalent).

5.2.4Concentrator tube, Kuderna-Danish10-mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5Evaporative flask, Kuderna-Danish500-mL (Kontes K5700010500 or equivalent). Attach to concentrator tube with springs.

5.2.6Snyder column, Kuderna-DanishThree-ball macro (Kontes K5030000121 or equivalent).

5.2.7Vials10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.3Boiling chipsApproximately 10/40 mesh. Heat to 400 C for 30 min or Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2C). The bath should be used in a hood.

5.5BalanceAnalytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatographAn analytical system complete with temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column 11.8 m long 2 mm ID glass, packed with 3% SP1000 on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.2Column 21.8 m long 2 mm ID glass, packed with 2,6-diphenylene oxide polymer (60/80 mesh), Tenax, or equivalent.

5.6.3DetectorHalide specific detector: electrolytic conductivity or microcoulometric. These detectors have proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1). The Hall conductivity detector was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1. Although less selective, an electron capture detector is an acceptable alternative.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Sodium thiosulfate(ACS) Granular.

6.3Acetone, hexane, methanol, methylene chloride, petroleum ether (boiling range 3060 C)Pesticide quality or equivalent.

6.4Sodium sulfate(ACS) Granular, anhydrous. Purify by heating at 400 C for 4 h in a shallow tray.

6.5FlorisilPR Grade (60/100 mesh). Purchase activated at 1250 F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 C in a foil-covered glass container and allow to cool.

6.6Ethyl etherNanograde, redistilled in glass if necessary.

6.6.1Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat. No. P11268, and other suppliers.) 6.6.2Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.7Stock standard solutions (1.00 g/L)Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in acetone and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure: 7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with hexane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (7.3Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with hexane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1. (image) Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 15%, a new calibration curve must be prepared for that compound.

7.5The cleanup procedure in Section 11 utilizes Florisil column chromatography. Florisil from different batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which is used, the use of lauric acid value 7 is suggested. The referenced procedure determines the adsorption from hexane solution of lauric acid (mg) per g of Florisil. The amount of Florisil to be used for each column is calculated by dividing 110 by this ratio and multiplying by 20 g.

7.6Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 100 g/mL in acetone. The QC check sample concentrate must be obtained from the U.S.

Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 100 g/L by adding 1.00 mL of QC check sample concentrate to each of four 1L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1.The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 100 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 100 g/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.

8 If spiking was performed at a concentration lower than 100 g/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 3, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T)2.44(100 S/T)%. 8 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 m/L of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices9 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.10 Field test kits are available for this purpose.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2 10. Sample Extraction 10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

Note: Some of the haloethers are very volatile and significant losses will occur in concentration steps if care is not exercised. It is important to maintain a constant gentle evaporation rate and not to allow the liquid volume to fall below 1 to 2 mL before removing the K-D apparatus from the hot water bath.

10.7Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the temperature of the water bath to 85 to 90 C. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.9Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder.

Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation 11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2Florisil column cleanup for haloethers: 11.2.1Adjust the sample extract volume to 10 mL.

11.2.2Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5), into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.2.3Preelute the column with 50 to 60 mL of petroleum ether. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column by decantation and subsequent petroleum ether washings. Discard the eluate.

Just prior to exposure of the sodium sulfate layer to the air, begin eluting the column with 300 mL of ethyl ether/petroleum ether (6+94) (V/V). Adjust the elution rate to approximately 5 mL/min and collect the eluate in a 500-mL K-D flask equipped with a 10-mL concentrator tube.

This fraction should contain all of the haloethers.

11.2.4Concentrate the fraction as in Section 10.6, except use hexane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Adjust the volume of the cleaned up extract to 10 mL with hexane and analyze by gas chromatography (Section 12).

12. Gas Chromatography 12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Columns 1 and 2 are shown in Figures 1 and 2, respectively.

Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatrograph.

12.4Inject 2 to 5 L of the sample extract or standard into the gas chromatograph using the solvent-flush technique.11 Smaller (1.0 L) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L, the total extract volume, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weight heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations 13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2. (image) Equation 2 where: A=Amount of material injected (ng).

Vi=Volume of extract injected (L).

Vt=Volume of total extract (L).

Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3. (image) Equation 3 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

13.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.12 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4 MDL to 1000 MDL.12 14.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 626 /L.12 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References 1. 40 CFR part 136, appendix B.

2. Determination of Haloethers in Industrial and Municipal Wastewaters, EPA 600/481062, National Technical Information Service, PB81232290, Springfield, Virginia 22161, July 1981.

3. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constitutents, American Society for Testing and Materials, Philadelphia.

4. CarcinogensWorking Carcinogens, Department of Health, Education, and Welfare, Public Health Services, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Mills., P.A. Variation of Florisil Activity: Simple Method for Measuring Absorbent Capacity and Its Use in Standardizing Florisil Columns, Journal of the Association of Official Analytical Chemists, 51, 29 (1968).

8. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 9. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

10. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

11. Burke, J.A. Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects, Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

12. EPA Method Study 21, Method 611, Haloethers, EPA 600/484052, National Technical Information Service, PB84205939, Springfield, Virginia 22161, June 1984.

Table 1_Chromatographic Conditions and Methods Detection Limits ------------------------------------------------------------------------ Retention time (min) Method ---------------------- detection Parameters limit Column 1 Column 2 (/ L) ------------------------------------------------------------------------ Bis(2-chloroisopropyl) ether........... 8.4 9.7 0.8 Bis(2-chloroethyl) ether............... 9.3 9.1 0.3 Bis(2-chloroethoxy) methane............ 13.1 10.0 0.5 4-Chlorophenyl ether................... 19.4 15.0 3.9 4-Bromophenyl phenyl ether............. 21.2 16.2 2.3 ------------------------------------------------------------------------ AColumn 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-1000 packed in a 1.8 m long x 2 mm ID glass column with helium carrier gas at 40 mL/min. flow rate. Column temperature held at 60 C for 2 min. after injection then programmed at 8 C/min. to 230 C and held for 4 min. Under these conditions the retention time for Aldrin is 22.6 min.

AColumn 2 conditions: Tenax-GC (60/80 mesh) packed in a 1.8 m long x 2mm ID glass column with helium carrier gas at 40 mL/min. flow rate.

Column temperature held at 150 C for 4 min. after injection then programmed at 16 C/min. to 310 C. Under these conditions the retention time for Aldrin is 18.4 min.

Table 2_QC Acceptance Criteria_Method 611 ---------------------------------------------------------------------------------------------------------------- Range for X Test conc. Limit for s (g/ Range for Parameter (g/ (g/L) L) P, Ps L) percent ---------------------------------------------------------------------------------------------------------------- Bis (2-chloroethyl)ether................................... 100 26.3 26.3-136.8 11-152 Bis (2-chloroethoxy)methane................................ 100 25.7 27.3-115.0 12-128 Bis (2-chloroisopropyl)ether............................... 100 32.7 26.4-147.0 9-165 4-Bromophenyl phenyl ether................................. 100 39.3 7.6 -167.5 D-189 4-Chlorophenyl phenyl ether................................ 100 30.7 15.4-152.5 D-170 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3_Method Accuracy and Precision as Functions of Concentration_Method 611 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Single analyst Overall recovery, precision, precision, Parameter X[prime] sr[prime] S[prime] (g/L) (g/L) (g/L) ---------------------------------------------------------------------------------------------------------------- Bis(2-chloroethyl) ether........................................ 0.81C+0.54 0.19X+0.28 0.35X+0,36 Bis(2-chloroethoxy) methane..................................... 0.71C+0.13 0.20X+0.15 0.33X+0.11 Bis(2-chloroisopropyl) ether.................................... 0.85C+1.67 0.20X+1.05 0.36X+0.79 4-Bromophenyl phenyl ether...................................... 0.85C+2.55 0.25X+0.21 0.47X+0.37 4-Chlorophenyl phenyl ether..................................... 0.82C+1.97 0.18X+2.13 0.41X+0.55 ---------------------------------------------------------------------------------------------------------------- X[prime] = Expected recovery for one or more measuremelts of a sample containing a concentration of C, in g/L.

sr[prime] = Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime] = Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C =True value for the concentration, in g/L.

X = Average recovery found for measurements of samples containing a concentration of C, in g/L.

(image) View or download PDF (image) View or download PDF Method 612Chlorinated Hydrocarbons 1. Scope and Application 1.1This method covers the determination of certain chlorinated hydrocarbons. The following parameters can be determined by this method: ------------------------------------------------------------------------ STORET Parameter No. CAS No.

------------------------------------------------------------------------ 2-Chloronaphthalene.............................. 34581 91-58-7 1,2-Dichlorobenzene.............................. 34536 95-50-1 1,3-Dichlorobenzene.............................. 34566 541-73-1 1,4-Dichlorobenzene.............................. 34571 106-46-7 Hexachlorobenzene................................ 39700 118-74-1 Hexachlorobutadiene.............................. 34391 87-68-3 Hexachlorocyclopentadiene........................ 34386 77-47-4 Hexachloroethane................................. 34396 67-72-1 1,2,4-Trichlorobenzene........................... 34551 120-82-1 ------------------------------------------------------------------------ 1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, and 611. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the parameters are then measured with an electron capture detector.

2 2.2The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned. 3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating.

Thorough rinsing with such solvents usually eliminates PCB interference.

Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants.

Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified46 for the information of the analyst.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1cL or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4 C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.): 5.2.1Separatory funnel2L, with Teflon stopcock.

5.2.2Drying columnChromatographic column, approximately 400 mm long 19 mm ID, with coarse frit filter disc.

5.2.3Chromatographic column300 long 10 mm ID, with Teflon stopcock and coarse frit filter disc at bottom.

5.2.4Concentrator tube, Kuderna-Danish10-mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.5Evaporative flask, Kuderna-Danish500-mL (Kontes K5700010500 or equivalent). Attach to concentrator tube with springs.

5.2.6Snyder column, Kuderna-DanishThree-ball macro (Kontes K5030000121 or equivalent).

5.2.7Vials10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.3Boiling chipsApproximately 10/40 mesh. Heat to 400 C for 30 min or Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2 C). The bath should be used in a hood.

5.5BalanceAnalytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatographAn analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder.

A data system is recommended for measuring peak areas.

5.6.1Column 11.8 m long 2 mm ID glass, packed with 1% SP1000 on Supelcoport (100/120 mesh) or equivalent. Guidelines for the use of alternate column packings are provide in Section 12.1.

5.6.2Column 21.8 m long 2 mm ID glass, packed with 1.5% OV1/2.4% OV225 on Supelcoport (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 14.

5.6.3DetectorElectron capture detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Acetone, hexane, isooctane, methanol, methylene chloride, petroleum ether (boiling range 30 to 60 C)Pesticide quality or equivalent.

6.3Sodium sulfate(ACS) Granular, anhydrous. Purify heating at 400 C for 4 h in a shallow tray.

6.4FlorisilPR grade (60/100 mesh). Purchase activated at 1250 F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 C in a foil-covered glass container and allow to cool.

6.5Stock standard solution (1.00 g/L)Stock standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.5.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in isooctane and dilute to volume in a 120-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.5.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.5.3Stock standard solutions must be replaced after six months, or sooner if comparision with check standards indicates a problem.

6.6Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure: 7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with isooctane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (7.3Internal standard calibration procedureTo use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with isooctane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1. (image) Equation 1 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 15%, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When the results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetone: Hexachloro-substituted parameters, 10 g/mL; any other chlorinated hydrocarbon, 100 g/mL. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note: The large number of parameters in Table 2 presents a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spike sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none by (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. In necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (AB)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.

7 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 3, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T) 2.44 (100 S/T)%. 7 8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4. If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Sections 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevent performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4C from the time of collection until extraction.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2 10. Sample Extraction 10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2L separatory funnel.

10.2Add 60 mL of methylele chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 to 2 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

Note: The dichloribenzenes have a sufficiently high volatility that significant losses may occur in concentration steps if care is not exercised. It is important to maintain a constant gentle evaporation rate and not to allow the liquid volume to fall below 1 to 2 mL before removing the K-D apparatus from the hot water bath.

10.7Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the tempeature of the water bath to 85 to 90 C. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8Romove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.9Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder.

Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation 11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2Florisil column cleanup for chlorinated hydrocarbons: 11.2.1Adjust the sample extract to 10 mL with hexane.

11.2.2Place 12 g of Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.2.3Preelute the column with 100 mL of petroleum ether. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column by decantation and subsequent petroleum ether washings. Discard the eluate.

Just prior to exposure of the sodium sulfate layer to the air, begin eluting the column with 200 mL of petroleum ether and collect the eluate in a 500-mL K-D flask equipped with a 10-mL concentrator tube. This fraction should contain all of the chlorinated hydrocarbons.

11.2.4Concentrate the fraction as in Section 10.6, except use hexane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Analyze by gas chromatography (Section 12).

12. Gas Chromatography 12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Columl 2 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed throughly immediately before injection into the gas chromatograph.

12.4Inject 2 to 5 L of the sample extract or standard into the gas chromatograph using the solvent-flush techlique.9 Smaller (1.0 L) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L, the total extract volume, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations 13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2. (image) Equation 2 where: A=Amount of material injected (ng).

Vi=Volume of extract injected (L).

Vt=Volume of total extract (L).

Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3. (image) Equation 3 where: As=Response for the parameter to be measured.

Ais=Response for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

13.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4MDL to 1000MDL.10 14.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 356 g/L.11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References 1. 40 CFR part 136, appendix B.

2. Determination of Chlorinated Hydrocarbons In Industrial and Municipal Wastewaters, EPA 6090/484ABC, National Technical Information Service, PBXYZ, Springfield, Virginia, 22161 November 1984.

3. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

4. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data,American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 8. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

9. Burke, J.A. Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects, Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

10. Development of Detection Limits, EPA Method 612, Chlorinated Hydrocarbons, Special letter report for EPA Contract 68032625, U.S.

Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.

11. EPA Method Study Method 612Chlorinated Hydrocarbons, EPA 600/484039, National Technical Information Service, PB84187772, Springfield, Virginia 22161, May 1984.

12. Method Performance for Hexachlorocyclopentadiene by Method 612, Memorandum from R. Slater, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, December 7, 1983.

Table 1_Chromatographic Conditions and Method Detection Limits ------------------------------------------------------------------------ Retention time (min) Method -------------------------- detection Parameter limit Column 1 Column 2 (g/ L) ------------------------------------------------------------------------ 1,3-Dichlorobenzene.............. 4.5 6.8 1.19 Hexachloroethane................. 4.9 8.3 0.03 1,4-Dichlorobenzene.............. 5.2 7.6 1.34 1,2-Dichlorobenzene.............. 6.6 9.3 1.14 Hexachlorobutadiene.............. 7.7 20.0 0.34 1,2,4-Trichlorobenzene........... 15.5 22.3 0.05 Hexachlorocyclopentadiene........ nd c 16.5 0.40 2-Chloronaphthalene.............. a 2.7 b 3.6 0.94 Hexachlorobenzene................ a 5.6 b 10.1 0.05 ------------------------------------------------------------------------ Column 1 conditions: Supelcoport (100/120 mesh) coated with 1% SP-1000 packed in a 1.8 m x 2 mm ID glass column with 5% methane/95% argon carrier gas at 25 mL/min. flow rate. Column temperature held isothermal at 65 C, except where otherwise indicated.

Column 2 conditions: Supelcoport (80/100 mesh) coated with 1.5% OV-1/ 2.4% OV-225 packed in a 1.8 m x 2 mm ID glass column with 5% methane/ 95% argon carrier gas at 25 mL/min. flow rate. Column temperature held isothermal at 75 C, except where otherwise indicated.

nd=Not determined.

a 150 C column temperature.

b 165 C column temperature.

c 100 C column temperature.

Table 2_QC Acceptance Criteria_Method 612 ---------------------------------------------------------------------------------------------------------------- Limit for Range for X Test conc. s (g/ Range for Parameter (g/ (g/ L) P, Ps L) L) (percent) ---------------------------------------------------------------------------------------------------------------- 2-Chloronaphthalene............................................. 100 37.3 29.5-126.9 9-148 1,2-Dichlorobenzene............................................. 100 28.3 23.5-145.1 9-160 1,3-Dichlorobenzene............................................. 100 26.4 7.2-138.6 D-150 1,4-Dichlorobenzene............................................. 100 20.8 22.7-126.9 13-137 Hexachlorobenzene............................................... 10 2.4 2.6-14.8 15-159 Hexachlorobutadiene............................................. 10 2.2 D-12.7 D-139 Hexachlorocyclopentadiene....................................... 10 2.5 D-10.4 D-111 Hexachloroethane................................................ 10 3.3 2.4-12.3 8-139 1,2,4-Trichlorobenzene.......................................... 100 31.6 20.2-133.7 5-149 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3_Method Accuracy and Precision as Functions of Concentration_Method 612 ---------------------------------------------------------------------------------------------------------------- Single analyst Parameter Acccuracy, as recovery, precision, sr[prime] Overall precision, X[prime] (g/L) (g/L) S[prime] (g/L) ---------------------------------------------------------------------------------------------------------------- 2-Chloronaphthalene................... 0.75C+3.21 0.28X-1.17 0.38X-1.39 1,2-Dichlorobenzene................... 0.85C-0.70 0.22X-2.95 0.41X-3.92 1,3-Dichlorobenzene................... 0.72C+0.87 0.21X-1.03 0.49X-3.98 1,4-Dichlorobenzene................... 0.72C+2.80 0.16X-0.48 0.35X-0.57 Hexachlorobenzene..................... 0.87C-0.02 0.14X+0.07 0.36X-0.19 Hexachlorobutadiene................... 0.61C+0.03 0.18X+0.08 0.53X-0.12 Hexachlorocyclopentadiene a........... 0.47C 0.24X 0.50X Hexachloroethane...................... 0.74C-0.02 0.23X+0.07 0.36X-0.00 1,2,4-Trichlorobenzene................ 0.76C+0.98 0.23X-0.44 0.40X-1.37 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

a Estimates based upon the performance in a single laboratory.\12\ (image) View or download PDF (image) View or download PDF Method 6132,3,7,8-Tetrachlorodibenzo-p-Dioxin 1. Scope and Application 1.1This method covers the determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The following parameter may be determined by this method: ------------------------------------------------------------------------ STORET Parameter No. GAS No.

------------------------------------------------------------------------ 2,3,7,8-TCDD..................................... 34675 1746-01-6 ------------------------------------------------------------------------ 1.2This is a gas chromatographic/mass spectrometer (GC/MS) method applicable to the determination of 2,3,7,8TCDD in municipal and industrial discharges as provided under 40 CFR 136.1. Method 625 may be used to screen samples for 2,3,7,8TCDD. When the screening test is positive, the final qualitative confirmation and quantification must be made using Method 613.

1.3The method detection limit (MDL, defined in Section 14.1) 1 for 2,3,7,8TCDD is listed in Table 1. The MDL for a specific wastewater may be different from that listed, depending upon the nature of interferences in the sample matrix.

1.4Because of the extreme toxicity of this compound, the analyst must prevent exposure to himself, of to others, by materials knows or believed to contain 2,3,7,8TCDD. Section 4 of this method contains guidelines and protocols that serve as minimum safe-handling standards in a limited-access laboratory.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1A measured volume of sample, approximately 1L, is spiked with an internal standard of labeled 2,3,7,8TCDD and extracted with methylene chloride using a separatory funnel. The methylene chloride extract is exchanged to hexane during concentration to a volume of 1.0 mL or less.

The extract is then analyzed by capillary column GC/MS to separate and measure 2,3,7,8TCDD. 2,3 2.2The method provides selected column chromatographic cleanup proceudres to aid in the elimination of interferences that may be encountered.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated backgrounds at the masses (m/z) monitored. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned. 4 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by the treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating.

Thorough rinsing with such solvents usually eliminates PCB interference.

Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants.

Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to mininmize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are coextracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled.

2,3,7,8TCDD is often associated with other interfering chlorinated compounds which are at concentrations several magnitudes higher than that of 2,3,7,8TCDD. The cleanup producers in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches1,57 to eliminate false positives and achieve the MDL listed in Table 1.

3.3The primary column, SP2330 or equivalent, resolves 2,3,7,8TCDD from the other 21 TCDD insomers. Positive results using any other gas chromatographic column must be confirmed using the primary column.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified810 for the information of the analyst. Benzene and 2,3,7,8TCDD have been identified as suspected human or mammalian carcinogens.

4.2Each laboratory must develop a strict safety program for handling 2,3,7,8TCDD. The following laboratory practices are recommended: 4.2.1Contamination of the laboratory will be minimized by conducting all manipulations in a hood.

4.2.2The effluents of sample splitters for the gas chromatograph and roughing pumps on the GC/MS should pass through either a column of activated charcoal or be bubbled through a trap containing oil or high-boiling alcohols.

4.2.3Liquid waste should be dissolved in methanol or ethanol and irradiated with ultraviolet light with a wavelength greater than 290 nm for several days. (Use F 40 BL lamps or equivalent). Analyze liquid wastes and dispose of the solutions when 2,3,7,8TCDD can no longer be detected.

4.3Dow Chemical U.S.A. has issued the following precautimns (revised November 1978) for safe handling of 2,3,7,8TCDD in the laboratory: 4.3.1The following statements on safe handling are as complete as possible on the basis of available toxicological information. The precautions for safe handling and use are necessarily general in nature since detailed, specific recommendations can be made only for the particular exposure and circumstances of each individual use. Inquiries about specific operations or uses may be addressed to the Dow Chemical Company. Assistance in evaluating the health hazards of particular plant conditions may be obtained from certain consulting laboratories and from State Departments of Health or of Labor, many of which have an industrial health service. 2,3,7,8TCDD is extremely toxic to laboratory animals. However, it has been handled for years without injury in analytical and biological laboratories. Techniques used in handling radioactive and infectious materials are applicable to 2,3,7,8,TCDD.

4.3.1.1Protective equipmentThrow-away plastic gloves, apron or lab coat, safety glasses, and a lab hood adequate for radioactive work.

4.3.1.2TrainingWorkers must be trained in the proper method of removing contaminated gloves and clothing without contacting the exterior surfaces.

4.3.1.3Personal hygieneThorough washing of hands and forearms after each manipulation and before breaks (coffee, lunch, and shift).

4.3.1.4ConfinementIsolated work area, posted with signs, segregated glassware and tools, plastic-backed absorbent paper on benchtops.

4.3.1.5WasteGood technique includes minimizing contaminated waste.

Plastic bag liners should be used in waste cans. Janitors must be trained in the safe handling of waste.

4.3.1.6Disposal of wastes2,3,7,8TCDD decomposes above 800 C. Low-level waste such as absorbent paper, tissues, animal remains, and plastic gloves may be burned in a good incinerator. Gross quantities (milligrams) should be packaged securely and disposed through commercial or governmental channels which are capable of handling high-level radioactive wastes or extremely toxic wastes. Liquids should be allowed to evaporate in a good hood and in a disposable container. Residues may then be handled as above.

4.3.1.7DecontaminationFor personal decontamination, use any mild soap with plenty of scrubbing action. For decontamination of glassware, tools, and surfaces, Chlorothene NU Solvent (Trademark of the Dow Chemical Company) is the least toxic solvent shown to be effective.

Satisfactory cleaning may be accomplished by rinsing with Chlorothene, then washing with any detergent and water. Dishwater may be disposed to the sewer. It is prudent to minimize solvent wastes because they may require special disposal through commercial sources which are expensive.

4.3.1.8LaundryClothing known to be contaminated should be disposed with the precautions described under Section 4.3.1.6. Lab coats or other clothing worn in 2,3,7,8TCDD work areas may be laundered.

Clothing should be collected in plastic bags. Persons who convey the bags and launder the clothing should be advised of the hazard and trained in proper handling. The clothing may be put into a washer without contact if the launderer knows the problem. The washer should be run through a cycle before being used again for other clothing.

4.3.1.9Wipe testsA useful method of determining cleanliness of work surfaces and tools is to wipe the surface with a piece of filter paper.

Extraction and analysis by gas chromatography can achieve a limit of sensitivity of 0.1 g per wipe. Less than 1 g of 2,3,7,8TCDD per sample indicates acceptable cleanliness; anything higher warrants further cleaning. More than 10 g on a wipe sample constitutes an acute hazard and requires prompt cleaning before further use of the equipment or work space. A high (>10 g) 2,3,7,8TCDD level indicates that unacceptable work practices have been employed in the past.

4.3.1.10InhalationAny procedure that may produce airborne contamination must be done with good ventilation. Gross losses to a ventilation system must not be allowed. Handling of the dilute solutions normally used in analytical and animal work presents no inhalation hazards except in the case of an accident.

4.3.1.11AccidentsRemove contaminated clothing immediately, taking precautions not to contaminate skin or other articles. Wash exposed skin vigorously and repeatedly until medical attention is obtained.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle1L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4 C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.1.3Clearly label all samples as POISON and ship according to U.S.

Department of Transportation regulations.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.): 5.2.1Separatory funnels2L and 125-mL, with Teflon stopcock.

5.2.2Concentrator tube, Kuderna-Danish10-mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.3Evaporative flask, Kuderna-Danish500mL (Kontes K5700010500 or equivalent). Attach to concentrator tube with springs.

5.2.4Snyder column, Kuderna-DanishThree-ball macro (Kontes K5030000121 or equivalent).

5.2.5Snyder column, Kuderna-DanishTwo-ball micro (Kontes K5690010219 or equivalent).

5.2.6Vials10 to 15mL, amber glass, with Teflon-lined screw cap.

5.2.7Chromatographic column300 mm long 10 mm ID, with Teflon stopcock and coarse frit filter disc at bottom.

5.2.8Chromatographic column400 mm long 11 mm ID, with Teflon stopcock and coarse frit filter disc at bottom.

5.3Boiling chipsApproximately 10/40 mesh. Heat to 400 C for 30 min or Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2 C). The bath should be used in a hood.

5.5GC/MS system: 5.5.1Gas chromatographAn analytical system complete with a temperature programmable gas chromatograph and all required accessories including syringes, analytical columns, and gases. The injection port must be designed for capillary columns. Either split, splitless, or on-column injection techniques may be employed, as long as the requirements of Section 7.1.1 are achieved.

5.5.2Column60 m long 0.25 mm ID glass or fused silica, coated with SP2330 (or equivalent) with a film thickness of 0.2 m. Any equivalent column must resolve 2, 3, 7, 8TCDD from the other 21 TCDD isomers.16 5.5.3Mass spectrometerEither a low resolution mass spectrometer (LRMS) or a high resolution mass spectrometer (HRMS) may be used. The mass spectrometer must be equipped with a 70 V (nominal) ion source and be capable of aquiring m/z abundance data in real time selected ion monitoring (SIM) for groups of four or more masses.

5.5.4GC/MS interfaceAny GC to MS interface can be used that achieves the requirements of Section 7.1.1. GC to MS interfaces constructed of all glass or glass-lined materials are recommended. Glass surfaces can be deactivated by silanizing with dichlorodimethylsilane. To achieve maximum sensitivity, the exit end of the capillary column should be placed in the ion source. A short piece of fused silica capillary can be used as the interface to overcome problems associated with straightening the exit end of glass capillary columns.

5.5.5The SIM data acquired during the chromatographic program is defined as the Selected Ion Current Profile (SICP). The SICP can be acquired under computer control or as a real time analog output. If computer control is used, there must be software available to plot the SICP and report peak height or area data for any m/z in the SICP between specified time or scan number limits.

5.6BalanceAnalytical, capable of accurately weighing 0.0001 g.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of 2, 3, 7, 8TCDD.

6.2Sodium hydroxide solution (10 N)Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL. Wash the solution with methylene chloride and hexane before use.

6.3Sodium thiosulfate(ACS) Granular.

6.4Sulfuric acidConcentrated (ACS, sp. gr. 1.84).

6.5Acetone, methylene chloride, hexane, benzene, ortho-xylene, tetradecanePesticide quality or equivalent.

6.6Sodium sulfate(ACS) Granular, anhydrous. Purify by heating at 400 C for 4 h in a shallow tray.

6.7AluminaNeutral, 80/200 mesh (Fisher Scientific Co., No. A540 or equivalent). Before use, activate for 24 h at 130 C in a foil-covered glass container.

6.8Silica gelHigh purity grade, 100/120 mesh (Fisher Scientific Co., No.

S679 or equivalent).

6.9Stock standard solutions (1.00 g/L)Stock standard solutimns can be prepared from pure standard materials or purchased as certified solutions. Acetone should be used as the solvent for spiking solutions; ortho-xylene is recommended for calibration standards for split injectors; and tetradecane is recommended for splitless or on-colum injectors. Analyze stock internal standards to verify the absence of native 2,3,7,8TCDD.

6.9.1Prepare stock standard solutions of 2,3,7,8TCDD (mol wt 320) and either 37C14 2,3,7,8TCDD (mol wt 328) or 13C112 2,3,7,8TCDD (mol wt 332) in an isolated area by accurately weighing about 0.0100 g of pure material. Dissolve the material in pesticide quality solvent and dilute to volume in a 10-mL volumetric flask. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.9.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store in an isolated refrigerator protected from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards or spiking solutions from them.

6.9.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.10Internal standard spiking solution (25 ng/mL)Using stock standard solution, prepare a spiking solution in acetone of either 13Cl12 or 37Cl4 2,3,7,8TCDD at a concentration of 25 ng/mL. (See Section 10.2) 6.11Quality control check sample concentrateSee Section 8.2.1.

7.Calibration 7.1Establish gas chromatograhic operating conditions equivalent to those given in Table 1 and SIM conditions for the mass spectrometer as described in Section 12.2 The GC/MS system must be calibrated using the internal standard technique.

7.1.1Using stock standards, prepare calibration standards that will allow measurement of relative response factors of at least three concentration ratios of 2,3,7,8TCDD to internal standard. Each calibration standard must be prepared to contain the internal standard at a concentration of 25 ng/mL. If any interferences are contributed by the internal standard at m/z 320 and 322, its concentration may be reduced in the calibration standards and in the internal standard spiking solution (Section 6.10). One of the calibration standards should contain 2,3,7,8TCDD at a concentration near, but above, the MDL and the other 2,3,7,8TCDD concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the GC/MS system.

7.1.2Using injections of 2 to 5 L, analyze each calibration standardaccording to Section 12 and tabulate peak height or area response against the concentration of 2,3,7,8TCDD and internal standard.

Calculate response factors (RF) for 2,3,7,8TCDD using Equation 1.

(image) Equation 1 where: As=SIM response for 2,3,7,8TCDD m/z 320.

Ais=SIM response for the internal standard, m/z 332 for 13 C12 2,3,7,8TCDD m/z 328 for 37Cl4 2,3,7,8TCDD.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of 2,3,7,8TCDD (g/L).

If the RF value over the working range is a constant (7.1.3The working calibration curve or RF must be verified on each working day by the measurement of one or more 2,3,7,8TCDD calibration standards. If the response for 2,3,7,8TCDD varies from the predicted response by more than 15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared.

7.2Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.5, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2 8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples with native 2,3,7,8TCDD to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing 2,3,7,8TCDD at a concentration of 0.100 g/mL in acetone. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 0.100 g/L (100 ng/L) by adding 1.00 mL of QC check sample concentrate to each of four 1L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for 2,3,7,8TCDD using the four results.

8.2.5Compare s and (X ) with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If s exceeds the precision limit or X falls outside the range for accuracy, the system performance is unacceptable for 2,3,7,8TCDD. Locate and correct the source of the problem and repeat the test beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of 2,3,7,8TCDD in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of 2,3,7,8TCDD in the sample is not being checked against a limit specific to that parameter, the spike should be at 0.100 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 0.100 g/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of 2,3,7,8TCDD. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentration in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of 2,3,7,8TCDD. Calculate percent recovery (P) as 100(AB)%T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for 2,3,7,8TCDD with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.11 If spiking was performed at a concentration lower than 0.100 g/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of 2,3,7,8TCDD: (1) Calculate accuracy (X) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 3, substituting X for X; (3) calculate the range for recovery at the spike concentration as (100 X/T)2.44(100 S/T)%.11 8.3.4If the recovery of 2,3,7,8TCDD falls outside the designated range for recovery, a check standard must be analyzed as described in Section 8.4.

8.4If the recovery of 2,3,7,8TCDD fails the acceptance criteria for recovery in Section 8.3, a QC check standard must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the complexity of the sample matrix and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of 2,3,7,8TCDD. Calculate the percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) with the corresponding QC acceptance criteria found in Table 2. If the recovery of 2,3,7,8TCDD falls outside the designated range, the laboratory performance is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for 2,3,7,8TCDD in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the spandard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices12 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 C and protected from light from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.13 Field test kits are available for this purpose.

9.3Label all samples and containers POISON and ship according to applicable U.S. Department of Transportation regulations.

9.4All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2 10. Sample Extraction Caution: When using this method to analyze for 2,3,7,8TCDD, all of the following operations must be performed in a limited-access laboratory with the analyst wearing full protective covering for all exposed skin surfaces. See Section 4.2.

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2L separatory funnel.

10.2Add 1.00 mL of internal standard spiking solution to the sample in the separatory funnel. If the final extract will be concentrated to a fixed volume below 1.00 mL (Section 12.3), only that volume of spiking solution should be added to the sample so that the final extract will contain 25 ng/mL of internal standard at the time of analysis.

10.3Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the vmlume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.4Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.5Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.6Pour the combined extract into the K-D concentrator. Rinse the Erlenmeyer flask with 20 to 30 mL of methylele chloride to complete the quantitative transfer.

10.7Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.8Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the temperature of the water bath to 85 to 90C. Concentrate the extract as in Section 10.7, except use hexane to prewet the column. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Set aside the K-D glassware for reuse in Section 10.14.

10.9Pour the hexane extract from the concentrator tube into a 125-mL separatory funnel. Rinse the concentrator tube four times with 10-mL aliquots of hexane. Combine all rinses in the 125-mL separatory funnel.

10.10Add 50 mL of sodium hydroxide solution to the funnel and shake for 30 to 60 s. Discard the aqueous phase.

10.11Perform a second wash of the organic layer with 50 mL of reagent water. Discard the aqueous phase.

10.12Wash the hexane layer with a least two 50-mL aliquots of concentrated sulfuric acid. Continue washing the hexane layer with 50-mL aliquots of concentrated sulfuric acid until the acid layer remains colorless. Discard all acid fractions.

10.13Wash the hexane layer with two 50-mL aliquots of reagent water.

Discard the aqueous phases.

10.14Transfer the hexane extract into a 125-mL Erlenmeyer flask containing 1 to 2 g of anhydrous sodium sulfate. Swirl the flask for 30 s and decant the hexane extract into the reassembled K-D apparatus.

Complete the quantitative transfer with two 10-mL hexane rinses of the Erlenmeyer flask.

10.15Replace the one or two clean boiling chips and concentrate the extract to 6 to 10 mL as in Section 10.8.

10.16Add a clean boiling chip to the concentrator tube and attach a two-ball micro-Snyder column. Prewet the column by adding about 1 mL of hexane to the top. Place the micro-K-D apparatus on the water bath so that the concentrator tube is partially immersed in the hot water.

Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of hexane.

Adjust the extract volume to 1.0 mL with hexane. Stopper the concentrator tube and store refrigerated and protected from light if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with GC/MS analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.17Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder.

Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation 11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure.1,57 However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure. Two cleanup column options are offered to the analyst in this section. The alumina column should be used first to overcome interferences. If background problems are still encountered, the silica gel column may be helpful.

11.2Alumina column cleanup for 2,3,7,8TCDD: 11.2.1Fill a 300 mm long 10 mm ID chromatographic column with activated alumina to the 150 mm level. Tap the column gently to settle the alumina and add 10 mm of anhydrous sodium sulfate to the top.

11.2.2Preelute the column with 50 mL of hexane. Adjust the elution rate to 1 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 1.0-mL sample extract onto the column using two 2-mL portions of hexane to complete the transfer.

11.2.3Just prior to exposure of the sodium sulfate layer to the air, add 50 mL of 3% methylene chloride/95% hexane (V/V) and continue the elution of the column. Discard the eluate.

11.2.4Next, elute the column with 50 mL of 20% methylene chloride/80% hexane (V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction to 1.0 mL as in Section 10.16 and analyze by GC/MS (Section 12).

11.3Silica gel column cleanup for 2,3,7,8TCDD: 11.3.1Fill a 400 mm long 11 mm ID chromatmgraphic column with silica gel to the 300 mm level. Tap the column gently to settle the silica gel and add 10 mm of anhydrous sodium sulfate to the top.

11.3.2Preelute the column with 50 mL of 20% benzene/80% hexane (V/V).

Adjust the elution rate to 1 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 1.0-mL sample extract onto the column using two 2-mL portions of 20% benzene/80% hexane to complete the transfer.

11.3.3Just prior to exposure of the sodium sulfate layer to the air, add 40 mL of 20% benzene/80% hexane to the column. Collect the eluate in a clean 500-mL K-D flask equipped with a 10-mL concentrator tube.

Concentrate the collected fraction to 1.0 mL as in Section 10.16 and analyze by GC/MS.

12. GC/MS Analysis 12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Other capillary columns or chromatographic conditions may be used if the requirements of Sections 5.5.2 and 8.2 are met.

12.2Analyze standards and samples with the mass spectrometer operating in the selected ion monitoring (SIM) mode using a dwell time to give at least seven points per peak. For LRMS, use masses at m/z 320, 322, and 257 for 2,3,7,8TCDD and either m/z 328 for 37Cl4 2,3,7,8TCDD or m/z 332 for 13C12 2,3,7,8TCDD. For HRMS, use masses at m/z 319.8965 and 321.8936 for 2,3,7,8TCDD and either m/z 327.8847 for 37Cl4 2,3,7,8TCDD or m/z 331.9367 for 13C12 2,3,7,8TCDD.

12.3If lower detection limits are required, the extract may be carefully evaporated to dryness under a gentle stream of nitrogen with the concentrator tube in a water bath at about 40 C. Conduct this operation immediately before GC/MS analysis. Redissolve the extract in the desired final volume of ortho-xylene or tetradecane.

12.4Calibrate the system daily as described in Section 7.

12.5Inject 2 to 5 L of the sample extract into the gas chromatograph.

The volume of calibration standard injected must be measured, or be the same as all sample injection volumes.

12.6The presence of 2,3,7,8TCDD is qualitatively confirmed if all of the following criteria are achieved: 12.6.1The gas chromatographic column must resolve 2,3,7,8TCDD from the other 21 TCDD isomers.

12.6.2The masses for native 2,3,7,8TCDD (LRMS-m/z 320, 322, and 257 and HRMS-m/z 320 and 322) and labeled 2,3,7,8TCDD (m/z 328 or 332) must exhibit a simultaneous maximum at a retention time that matches that of native 2,3,7,8TCDD in the calibration standard, with the performance specifications of the analytical system.

12.6.3The chlorine isotope ratio at m/z 320 and m/z 322 must agree to within10% of that in the calibration standard.

12.6.4The signal of all peaks must be greater than 2.5 times the noise level.

12.7For quantitation, measure the response of the m/z 320 peak for 2,3,7,8TCDD and the m/z 332 peak for 13 C12 2,3,7,8TCDD or the m/z 328 peak for 37Cl4 2,3,7,8TCDD.

12.8Co-eluting impurities are suspected if all criteria are achieved except those in Section 12.6.3. In this case, another SIM analysis using masses at m/z 257, 259, 320 and either m/a 328 or m/z 322 can be performed. The masses at m/z 257 and m/z 259 are indicative of the loss of one chlorine and one carbonyl group from 2,3,7,8TCDD. If masses m/z 257 and m/z 259 give a chlorine isotope ratio that agrees to within 10% of the same cluster in the calibration standards, then the presence of TCDD can be confirmed. Co-eluting DDD, DDE, and PCB residues can be confirmed, but will require another injection using the appropriate SIM masses or full repetitive mass scans. If the response for 37Cl4 2,3,7,8TCDD at m/z 328 is too large, PCB contamination is suspected and can be confirmed by examining the response at both m/z 326 and m/z 328. The 37Cl4 2,3,7,8TCDD internal standard gives negligible response at m/z 326.

These pesticide residues can be removed using the alumina column cleanup procedure.

12.9If broad background interference restricts the sensitivity of the GC/MS analysis, the analyst should employ additional cleanup procedures and reanalyze by GC/MS.

12.10In those circumstances where these procedures do not yield a definitive conclusion, the use of high resolution mass spectrometry is suggested.5 13. Calculations 13.1Calculate the concentration of 2,3,7,8TCDD in the sample using the response factor (RF) determined in Section 7.1.2 and Equation 2. (image) Equation 2 where: As=SIM response for 2,3,7,8TCDD at m/z 320.

Ais=SIM response for the internal standard at m/z 328 or 332.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

13.2For each sample, calculate the percent recovery of the internal standard by comparing the area of the m/z peak measured in the sample to the area of the same peak in the calibration standard. If the recovery is below 50%, the analyst should review all aspects of his analytical technique.

13.3Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentration listed in Table 1 was obtained using reagent water.14 The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method was tested by 11 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.02 to 0.20 g/L.15 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References 1. 40 CFR part 136, appendix B.

2. Determination of TCDD in Industrial and Municipal Wastewaters, EPA 600/482028, National Technical Information Service, PB82196882, Springfield, Virginia 22161, April 1982.

3. Buser, H.R., and Rappe, C. High Resolution Gas Chromatography of the 22 Tetrachlorodibenzo-p-dioxin Isomers, Analytical Chemistry, 52, 2257 (1980).

4. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

5. Harless, R. L., Oswald, E. O., and Wilkinson, M. K. Sample Preparation and Gas Chromatography/Mass Spectrometry Determination of 2,3,7,8-Tetrachlorodibenzo-p-dioxin, Analytical Chemistry, 52, 1239 (1980).

6. Lamparski, L. L., and Nestrick, T. J. Determination of Tetra-, Hepta-, and Octachlorodibenzo-p-dioxin Isomers in Particulate Samples at Parts per Trillion Levels, Analytical Chemistry, 52, 2045 (1980).

7. Longhorst, M. L., and Shadoff, L. A. Determination of Parts-per-Trillion Concentrations of Tetra-, Hexa-, and Octachlorodibenzo-p-dioxins in Human Milk, Analytical Chemistry, 52, 2037 (1980).

8. CarcinogensWorking with Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

9. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occuptional Safety and Health Administration, OSHA 2206 (Revised, January 1976).

10. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

11. Provost, L. P., and Elder, R. S., Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 12. ASTM Annual Book of Standards, Part 31, D337076, Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

13. Methods, 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

14. Wong, A.S. et al. The Determination of 2,3,7,8TCDD in Industrial and Municipal Wastewaters, Method 613, Part 1Development and Detection Limits, G. Choudhay, L. Keith, and C. Ruppe, ed., Butterworth Inc., (1983).

15. EPA Method Study 26, Method 613: 2,3,7,8Tetrachlorodibenzo-p-dioxin, EPA 600/484037, National Technical Information Service, PB84188879, Springfield, Virginia 22161, May 1984.

Table 1_Chromatographic Conditions and Method Detection Limit ------------------------------------------------------------------------ Method Retention detection Parameter time limit (min) (g/ L) ------------------------------------------------------------------------ 2,3,7,8-TCDD..................................... 13.1 0.002 ------------------------------------------------------------------------ Column conditions: SP-2330 coated on a 60 m long x 0.25 mm ID glass column with hydrogen carrier gas at 40 cm/sec linear velocity, splitless injection using tetradecane. Column temperature held isothermal at 200C for 1 min, then programmed at 8C/min to 250 C and held. Use of helium carrier gas will approximately double the retention time.

Table 2_QC Acceptance Criteria_Method 613 ---------------------------------------------------------------------------------------------------------------- Limit for Test conc. s Range for X Range Parameter (g/ (g/ (g/L) for P, L) L) Ps (%) ---------------------------------------------------------------------------------------------------------------- 2,3,7,8-TCDD................................................... 0.100 0.0276 0.0523-0.1226 45-129 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.

Table 3_Method Accuracy and Precision as Functions of Concentration_Method 613 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Single analyst, recovery, X precision, sr Overall precision, Parameter [prime] (g/ [prime] (/ S [prime] (/ L) L) g/L) ---------------------------------------------------------------------------------------------------------------- 2,3,7,8-TCDD........................................ 0.86C+0.00145 0.13X+0.00129 0.19X+0.00028 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements. of a sample containing a concentration of C, in g/L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

Method 624Purgeables 1. Scope and Application 1.1This method covers the determination of a number of purgeable organics. The following parameters may be determined by this method: ------------------------------------------------------------------------ STORET Parameter No. CAS No.

------------------------------------------------------------------------ Benzene.......................................... 34030 71-43-2 Bromodichloromethane............................. 32101 75-27-4 Bromoform........................................ 32104 75-25-2 Bromomethane..................................... 34413 74-83-9 Carbon tetrachloride............................. 32102 56-23-5 Chlorobenzene.................................... 34301 108-90-7 Chloroethane..................................... 34311 75-00-3 2-Chloroethylvinyl ether......................... 34576 110-75-8 Chloroform....................................... 32106 67-66-3 Chloromethane.................................... 34418 74-87-3 Dibromochloromethane............................. 32105 124-48-1 1,2-Dichlorobenzene.............................. 34536 95-50-1 1,3-Dichlorobenzene.............................. 34566 541-73-1 1,4-Dichlorobenzene.............................. 34571 106-46-7 1,1-Dichloroethane............................... 34496 75-34-3 1,2-Dichloroethane............................... 34531 107-06-2 1,1-Dichloroethane............................... 34501 75-35-4 trans-1,2-Dichloroethene......................... 34546 156-60-5 1,2-Dichloropropane.............................. 34541 78-87-5 cis-1,3-Dichloropropene.......................... 34704 10061-01-5 trans-1,3-Dichloropropene........................ 34699 10061-02-6 Ethyl benzene.................................... 34371 100-41-4 Methylene chloride............................... 34423 75-09-2 1,1,2,2-Tetrachloroethane........................ 34516 79-34-5 Tetrachloroethene................................ 34475 127-18-4 Toluene.......................................... 34010 108-88-3 1,1,1-Trichloroethene............................ 34506 71-55-6 1,1,2-Trichloroethene............................ 34511 79-00-5 Trichloroethane.................................. 39180 79-01-6 Trichlorofluoromethane........................... 34488 75-69-4 Vinyl chloride................................... 39175 75-01-4 ------------------------------------------------------------------------ 1.2The method may be extended to screen samples for acrolein (STORET No.

34210, CAS No. 107028) and acrylonitrile (STORET No. 34215, CAS No. 107131), however, the preferred method for these two compounds in Method 603.

1.3This is a purge and trap gas chromatographic/mass spectrometer (GC/MS) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1.

1.4The method detection limit (MDL, defined in Section 14.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.5Any modification to this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

Depending upon the nature of the modification and the extent of intended use, the applicant may be required to demonstrate that the modifications will produce equivalent results when applied to relevant wastewaters.

1.6This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1An inert gas is bubbled through a 5-mL water sample contained in a specially-designed purging chamber at ambient temperature. The purgeables are efficiently transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the purgeables are trapped. After purging is completed, the trap is heated and backflushed with the inert gas to desorb the purgeables onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the purgeables which are then detected with a mass spectrometer.2,3 3. Interferences 3.1Impurities in the purge gas, organic compounds outgassing from the plumbing ahead of the trap, and solvent vapors in the laboratory account for the majority of contamination problems. The analytical system must be demonstated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2Samples can be contaminated by diffusion of volatile organics (particularly fluorocarbons and methylene chloride) through the septum seal into the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed with reagent water between sample analyses. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high pureeable levels, it may be necessary to wash the purging device with a detergent solution, rinse it with distilled water, and then dry it in a 105 C oven between analyses. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this methmd. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified46 for the information of the analyst.

4.2.The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzene, carbon tetrachloride, chloroform, 1,4-dichlorobenzene, and vinyl chloride. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials 5.1Sampling equipment, for discrete sampling.

5.1.1Vial25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 C before use.

5.1.2SeptumTeflon-faced silicane (Pierce #12722 or equivalent).

Detergent wash, rinse with tap and distilled water, and dry at 105 C for 1 h before use.

5.2Purge and trap systemThe purge and trap system consists of three separate pieces of equipment: A purging device, trap, and desorber.

Several complete systems are now commercially available.

5.2.1The purging device must be designed to accept 5-mL samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL.

The purge gas must pass though the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column.

The purging device illustrated in Figure 1 meets these design criteria.

5.2.2The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in. The trap must be packed to contain the following minimum lengths of adsorbents: 1.0 cm of methyl silicone coated packing (Section 6.3.2), 15 cm of 2,6-dyphenylene oxide polymer (Section 6.3.1), and 8 cm of silica gel (Section 6.3.3). The minimum specifications for the trap are illustrated in Figure 2.

5.2.3The desorber should be capable of rapidly heating the trap to 180 C. The polymer section of the trap should not be heated higher than 180 C and the remaining sections should not exceed 200 C. The desorber illustrated in Figure 2 meets these design criteria.

5.2.4The purge and trap system may be assembled as a separate unit or be coupled to a gas chromatograph as illustrated in Figures 3 and 4.

5.3GC/MS system: 5.3.1Gas chromatographAn analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, and gases.

5.3.2Column6 ft long 0.1 in ID stainless steel or glass, packed with 1% SP1000 on Carbopack B (60/80 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 11.1.

5.3.3Mass spectrometerCapable of scanning from 20 to 260 amu every 7 s or less, utilizing 70 V (nominal) electron energy in the electron impact ionization mode, and producing a mass spectrum which meets all the criteria in Table 2 when 50ng of 4-bromofluorobenzene (BFB) is injected through the GC inlet.

5.3.4GC/MS interfaceAny GC to MS interface that gives acceptable calibration points at 50 ng or less per injection for each of the parameters of interest and achieves all acceptable performance criteria (Section 10) may be used. GC to MS interfaces constructed of all glass or glass-lined materials are recommended. Glass can be deactivated by silanizing with dichlorodimethylsilane.

5.3.5Data systemA computer system must be interfaced to the mass spectrometer that allows the continuous acquisition and storage on machine-readable media of all mass spectra obtained throughout the duration of the chromatographic program. The computer must have software that allows searching any GC/MS data file for specific m/z (masses) and plotting such m/z abundances versus time or scan number. This type of plot is defined as an Extracted Ion Current Profile (EICP). Software must also be available that allows integrating the abundance in any EICP between specified time or scan number limits.

5.4Syringes5-mL, glass hypodermic with Luerlok tip (two each), if applicable to the purging device.

5.5Micro syringes25-L, 0.006 in. ID needle.

5.6Syringe valve2-way, with Luer ends (three each).

5.7Syringe5-mL, gas-tight with shut-off valve.

5.8Bottle15-mL, screw-cap, with Teflon cap liner.

5.9BalanceAnalytical, capable of accurately weighing 0.0001 g.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb300, Calgon Corp., or equivalent).

6.1.2A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3Reagent water may also be prepared by boiling water for 15 min.

Subsequently, while maintaining the temperature at 90 C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.2Sodium thiosulfate(ACS) Granular.

6.3Trap materials: 6.3.12,6-Diphenylene oxide polymerTenax, (60/80 mesh), chromatographic grade or equivalent.

6.3.2Methyl silicone packing3% OV1 on Chromosorb-W (60/80 mesh) or equivalent.

6.3.3Silica gel35/60 mesh, Davison, grade-15 or equivalent.

6.4MethanolPesticide quality or equivalent.

6.5Stock standard solutionsStock standard solutions may be prepared from pure standard materials or purchased as certified solutions. Prepare stock standard solutions in methanol using assayed liquids or gases as appropriate. Because of the toxicity of some of the compounds, primary dilutions of these materials should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.5.1Place about 9.8 mL of methanol into a 10-mL ground glass stoppered volumetric flask. Allow the flask to stand, unstoppered, for about 10 min or until all alcohol wetted surfaces have dried. Weigh the flask to the nearest 0.1 mg.

6.5.2Add the assayed reference material: 6.5.2.1LiquidsUsing a 100-L syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the alcohol without contacting the neck of the flask.

6.5.2.2GasesTo prepare standards for any of the four halocarbons that boil below 30 C (bromomethane, chloroethane, chloromethane, and vinyl chloride), fill a 5-mL valved gas-tight syringe with the reference standard to the 5.0-mL mark. Lower the needle to 5 mm above the methanol meniscus. Slowly introduce the reference standard above the surface of the liquid (the heavy gas will rapidly dissolve in the methanol).

6.5.3Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in g/L from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight may be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards may be used at any concentration if they are certified by the manufacturer or by an independent source.

6.5.4Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store, with minimal headspace, at 10 to 20 C and protect from light.

6.5.5Prepare fresh standards weekly for the four gases and 2-chloroethylvinyl ether. All other standards must be replaced after one month, or sooner if comparison with check standards indicates a problem.

6.6Secondary dilution standardsUsing stock solutions, prepare secondary dilution standards in methanol that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3 will bracket the working range of the analytical system. Secondary dilution standards should be stored with minimal headspace and should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7Surrogate standard spiking solutionSelect a minimum of three surrogate compounds from Table 3. Prepare stock standard solutions for each surrogate standard in methanol as described in Section 6.5. Prepare a surrogate standard spiking solution from these stock standards at a concentration of 15 g/mL in water. Store the solutions at 4 C in Teflon-sealed glass containers with a minimum of headspace. The solutions should be checked frequently for stability. The addition of 10 L of this solution of 5 mL of sample or standard is equivalent to a concentration of 30 g/L of each surrogate standard.

6.8BFB StandardPrepare a 25 g/mL solution of BFB in methanol.

6.9Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1.

7.3Internal standard calibration procedureTo use this approach, the analyst must select three or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Some recommended internal standards are listed in Table 3.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter by carefully adding 20.0 L of one or more secondary dilution standards to 50, 250, or 500 mL of reagent water. A 25-L syringe with a 0.006 in. ID needle should be used for this operation. One of the calibration standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the GC/MS system. These aqueous standards can be stored up to 24 h, if held in sealed vials with zero headspace as described in Section 9.2. If not so stored, they must be discarded after 1 h.

7.3.2Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.5 and 6.6. It is recommended that the secondary dilution standard be prepared at a concentration of 15 g/mL of each internal standard compound. The addition of 10 L of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 g/L.

7.3.3Analyze each calibration standard according to Section 11, adding 10 L of internal standard spiking solution directly to the syringe (Section 11.4). Tabulate the area response of the characteristic m/z against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

(image) Equation 1 where: As=Area of the characteristic m/z for the parameter to be measured.

Ais=Area of the characteristic m/z for the inernal standard.

Cis=Concentration of the internal standard.

Cs=Concentration of the parameter to be measured.

If the RF value over the working range is a constant (7.4The working calibration curve or RF must be verified on each working day by the measurement of a QC check sample.

7.4.1Prepare the QC check sample as described in Section 8.2.2.

7.4.2Analyze the QC check sample according to the method beginning in Section 10.

7.4.3For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 5. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, proceed according to Section 7.4.4.

Note: The large number of parameters in Table 5 present a substantial probability that one or more will not meet the calibration acceptance criteria when all parameters are analyzed.

7.4.4Repeat the test only for those parameters that failed to meet the calibration acceptance criteria. If the response for a parameter does not fall within the range in this second test, a new calibration curve or RF must be prepared for that parameter according to Section 7.3.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 11.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 5% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 5% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must spike all samples with surrogate standards to monitor continuing laboratory performance. This procedure is described in Section 8.5.

8.1.7The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.6.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 10 g/mL in methanol. The QC check sample concentrate must be obtained from the U.S.

Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Prepare a QC check sample to contain 20 g/L of each parameter by adding 200 L of QC check sample concentrate to 100 mL of reagent water.

8.2.3Analyze four 5-mL aliquots of the well-mixed QC check sample according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X ) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter of interest using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 5. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note: The large number of parameters in Table 5 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.3.

8.2.6.2Beginning with Section 8.2.3, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.3.

8.3The laboratory must, on an ongoing basis, spike at least 5% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing 1 to 20 samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows: 8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 L of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 5. These acceptance criteria wer calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 20 g/L, the analyst must use either the QC acceptance criteria in Table 5, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recoveryof a parameter: (1) Calculate accuracy (X) using the equation in Table 6, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 6, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T) (2.44(100 S/T)%.7 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required anlaysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 5 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spiked sample.

8.4.1Prepare the QC check standard by adding 10 L of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (PS) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (PS) for each parameter with the corresponding QC acceptance criteria found in Table 5. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As a quality control check, the laboratory must spike all samples with the surrogate standard spiking solutions as described in Section 11.4, and calculate the percent recovery of each surrogate compound.

8.6As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P 2sp to P + 2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter a regular basis (e.g. after each five to ten new accuracy measurements).

8.7It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1All samples must be iced or refrigerated from the time of collection until analysis. If the sample contains residual chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site.

EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine. 8 Field test kits are available for this purpose.

9.2Grab samples must be collected in glass containers having a total volume of at least 25 mL. Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. If preservative has been added, shake vigorously for 1 min. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3Experimental evidence indicates that some aromatic compounds, notably benzene, toluene, and ethyl benzene are susceptible to rapid biological degradation under certain environmental conditions. 3 Refrigeration alone may not be adequate to preserve these compounds in wastewaters for more than seven days. For this reason, a separate sample should be collected, acidified, and analyzed when these aromatics are to be determined. Collect about 500 mL of sample in a clean container. Adjust the pH of the sample to about 2 by adding 1+1 HCl while stirring vigorously, Check pH with narrow range (1.4 to 2.8) pH paper. Fill a sample container as described in Section 9.2.

9.4All samples must be analyzed within 14 days of collection. 3 10. Daily GC/MS Performance Tests 10.1At the beginning of each day that analyses are to be performed, the GC/MS system must be checked to see if acceptable performance criteria are achieved for BFB. 9 The performance test must be passed before any samples, blanks, or standards are analyzed, unless the instrument has met the DFTPP test described in Method 625 earlier in the day. 10 10.2These performance tests require the following instrumental parameters: Electron Energy: 70 V (nominal) Mass Range: 20 to 260 amu Scan Time: To give at least 5 scans per peak but not to exceed 7 s per scan.

10.3At the beginning of each day, inject 2 L of BFB solution directly on the column. Alternatively, add 2 L of BFB solution to 5.0 mL of reagent water or standard solution and analyze the solution according to section 11. Obtain a background-corrected mass spectrum of BFB and confirm that all the key m/z criteria in Table 2 are achieved. If all the criteria are not achieved, the analyst must retune the mass spectrometer and repeat the test until all criteria are achieved.

11. Sample Purging and Gas Chromatography 11.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. An example of the separations achieved by this column is shown in Figure 5. Other packed columns or chromatographic conditions may be used if the requirements of Section 8.2 are met.

11.2After achieving the key m/z abundance criteria in Section 10, calibrate the system daiy as described in Section 7.

11.3Adjust the purge gas (helium) flow rate to 40 mL/min. Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

11.4Allow the sample to come to ambient temperature prior to introducing it into the syringe. Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing.

Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 L of the surrogate spiking solution (Section 6.7) and 10.0 L of the internal standard spiking solution (Section 7.3.2) through the valve bore, then close the valve. The surrogate and internal standards may be mixed and added as a single spiking solution.

11.5Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

11.6Close both valves and purge the sample for 11.0 0.1 min at ambient temperature.

11.7After the 11-min purge time, attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 4), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 C while backflushing the trap with an inert gas between 20 and 60 mL/min for 4 min. If rapid heating of the trap cannot be achieved, the GC cloumn must be used as a secondary trap by cooling it to 30 C (subambient temperature, if problems persist) instead of the initial program temperature of 45 C.

11.8While the trap is being desorbed into the gas chromatograph, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

11.9After desorbing the sample for 4 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at 180 C. After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

11.10If the response for any m/z exceeds the working range of the system, prepare a dilution of the sample with reagent water from the aliquot in the second syringe and reanalyze.

12. Qualitative Identification 12.1Obtain EICPs for the primary m/z (Table 4) and at least two secondary masses for each parameter of interest. The following criteria must be met to make a qualitative identification: 12.1.1The characteristic masses of each parameter of interest must maximize in the same or within one scan of each other.

12.1.2The retention time must fall within 30 s of the retention time of the authentic compound.

12.1.3The relative peak heights of the three characteristic masses in the EICPs must fall within 20% of the relative intensities of these masses in a reference mass spectrum. The reference mass spectrum can be obtained from a standard analyzed in the GC/MS system or from a reference library.

12.2Structural isomers that have very similar mass spectra and less than 30 s difference in retention time, can be explicitly identified only if the resolution between authentic isomers in a standard mix is acceptable. Acceptable resolution is achieved if the baseline to valley height between the isomers is less than 25% of the sum of the two peak heights. Otherwise, structural isomers are identified as isomeric pairs.

13. Calculations 13.1When a parameter has been identified, the quantitation of that parameter should be based on the integrated abundance from the EICP of the primary characteristic m/z given in Table 4. If the sample produces an interference for the primary m/z, use a secondary characteristic m/z to quantitate.

Calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.3 and Equation 2. (image) Equation 2 where: AS=Area of the characteristic m/z for the parameter or surrogate standard to be measured.

Ais=Area of the characteristic m/z for the internal standard.

Cis=Concentration of the internal standard.

13.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance 14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero. 1 The MDL concentrations listed in Table 1 were obtained using reagent water. 11 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method was tested by 15 laboratories using reagent water, drinking water, surface water, and industrial wastewaters spiked at six concentrations over the range 5600 g/L.12Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 5.

References 1. 40 CFR part 136, appendix B.

2. Bellar, T.A., and Lichtenberg, J.J. Determining Volatile Organics at Microgram-per-Litre Levels by Gas Chromatography, Journal American Water Works Association, 66, 739 (1974).

3. Bellar, T.A., and Lichtenberg, J.J. Semi-Automated Headspace Analysis of Drinking Waters and Industrial Waters for Purgeable Volatile Organic Compounds, Measurement of Organic Pollutants in Water and Wastewater, C.E. Van Hall, editor, American Society for Testing and Materials, Philadelphia, PA. Special Technical Publication 686, 1978.

4. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.2.3 is two times the value 1.22 derived in this report.) 8. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

9. Budde, W.L., and Eichelberger, J.W. Performance Tests for the Evaluation of Computerized Eas Chromatography/Mass Spectrometry Equipment and Laboratories, EPA600/480025, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, April 1980.

10. Eichelberger, J.W., Harris, L.E., and Budde, W.L. Reference Compound to Calibrate Ion Abundance Measurement in Gas ChromatographyMass Spectrometry Systems, Analytical Chemistry, 47, 9951000 (1975).

11. Method Detection Limit for Methods 624 and 625, Olynyk, P., Budde, W.L., and Eichelberger, J.W. Unpublished report, May 14, 1980.

12. EPA Method Study 29 EPA Method 624Purgeables, EPA 600/484054, National Technical Information Service, PB84209915, Springfield, Virginia 22161, June 1984.

13.Method Performance Data for Method 624, Memorandum from R. Slater and T. Pressley, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, January 17, 1984.

Table 1_Chromatographic Conditions and Method Detection Limits ------------------------------------------------------------------------ Method detection Parameter Retention limit time (min) (g/ L) ------------------------------------------------------------------------ Chloromethane................................... 2.3 nd Bromomethane.................................... 3.1 nd Vinyl chloride.................................. 3.8 nd Chloroethane.................................... 4.6 nd Methylene chloride.............................. 6.4 2.8 Trichlorofluoromethane.......................... 8.3 nd 1,1-Dichloroethene.............................. 9.0 2.8 1,1-Dichloroethane.............................. 10.1 4.7 trans-1,2-Dichloroethene........................ 10.8 1.6 Chloroform...................................... 11.4 1.6 1,2-Dichloroethane.............................. 12.1 2.8 1,1,1-Trichloroethane........................... 13.4 3.8 Carbon tetrachloride............................ 13.7 2.8 Bromodichloromethane............................ 14.3 2.2 1,2-Dichloroproane.............................. 15.7 6.0 cis-1,3-Dichloropropene......................... 15.9 5.0 Trichloroethene................................. 16.5 1.9 Benzene......................................... 17.0 4.4 Dibromochloromethane............................ 17.1 3.1 1,1,2-Trichloroethane........................... 17.2 5.0 trans-1,3-Dichloropropene....................... 17.2 nd 2-Chloroethylvinlyl ether....................... 18.6 nd Bromoform....................................... 19.8 4.7 1,1,2,2-Tetrachloroethane....................... 22.1 6.9 Tetrachloroethene............................... 22.2 4.1 Toluene......................................... 23.5 6.0 Chlorobenzene................................... 24.6 6.0 Ethyl benzene................................... 26.4 7.2 1,3-Dichlorobenzene............................. 33.9 nd 1,2-Dichlorobenzene............................. 35.0 nd 1,4-Dichlorobenzene............................. 35.4 nd ------------------------------------------------------------------------ Column conditions: Carbopak B (60/80 mesh) coated with 1% SP-1000 packed in a 6 ft by 0.1 in. ID glass column with helium carrier gas at 30 mL/ min. flow rate. Column temperature held at 45C for 3 min., then programmed at 8C/min. to 220C and held for 15 min.

nd=not determined.

Table 2_BFB Key m/z Abundance Criteria ------------------------------------------------------------------------ Mass m/z Abundance criteria ------------------------------------------------------------------------ 50........................................ 15 to 40% of mass 95.

75........................................ 30 to 60% of mass 95.

95........................................ Base Peak, 100% Relative Abundance.

96........................................ 5 to 9% of mass 95.

173....................................... 174....................................... >50% of mass 95.

175....................................... 5 to 9% of mass 174.

176....................................... >95% but 177....................................... 5 to 9% of mass 176.

------------------------------------------------------------------------ Table 3_Suggested Surrogate and Internal Standards ------------------------------------------------------------------------ Retention Compound time Primary Secondary (min)a m/z masses ------------------------------------------------------------------------ Benzene d-6............................ 17.0 84 ...........

4-Bromofluorobenzene................... 28.3 95 174, 176 1,2-Dichloroethane d-4................. 12.1 102 ...........

1,4-Difluorobenzene.................... 19.6 114 63, 88 Ethylbenzene d-5....................... 26.4 111 ...........

Ethylbenzene d-10...................... 26.4 98 ...........

Fluorobenzene.......................... 18.4 96 70 Pentafluorobenzene..................... 23.5 168 ...........

Bromochloromethane..................... 9.3 128 49, 130, 51 2-Bromo-1-chloropropane................ 19.2 77 79, 156 1, 4-Dichlorobutane.................... 25.8 55 90, 92 ------------------------------------------------------------------------ a For chromatographic conditions, see Table 1.

Table 4_Characteristic Masses for Purgeable Organics ------------------------------------------------------------------------ Parameter Primary Secondary ------------------------------------------------------------------------ Chloromethane........................ 50 52.

Bromomethane......................... 94 96.

Vinyl chloride....................... 62 64.

Chloroethane......................... 64 66.

Methylene chloride................... 84 49, 51, and 86.

Trichlorofluoromethane............... 101 103.

1,1-Dichloroethene................... 96 61 and 98.

1,1-Dichloroethane................... 63 65, 83, 85, 98, and 100.

trans-1,2-Dichloroethene............. 96 61 and 98.

Chloroform........................... 83 85.

1,2-Dichloroethane................... 98 62, 64, and 100.

1,1,1-Trichloroethane................ 97 99, 117, and 119.

Carbon tetrachloride................. 117 119 and 121.

Bromodichloromethane................. 127 83, 85, and 129.

1,2-Dichloropropane.................. 112 63, 65, and 114.

trans-1,3-Dichloropropene............ 75 77.

Trichloroethene...................... 130 95, 97, and 132.

Benzene.............................. 78 ........................

Dibromochloromethane................. 127 129, 208, and 206.

1,1,2-Trichloroethane................ 97 83, 85, 99, 132, and 134.

cis-1,3-Dichloropropene.............. 75 77.

2-Chloroethylvinyl ether............. 106 63 and 65.

Bromoform............................ 173 171, 175, 250, 252, 254, and 256.

1,1,2,2-Tetrachloroethane............ 168 83, 85, 131, 133, and 166.

Tetrachloroethene.................... 164 129, 131, and 166.

Toluene.............................. 92 91.

Chlorobenzene........................ 112 114.

Ethyl benzene........................ 106 91.

1,3-Dichlorobenzene.................. 146 148 and 113.

1,2-Dichlorobenzene.................. 146 148 and 113.

1,4-Dichlorobenzene.................. 146 148 and 113.

------------------------------------------------------------------------ Table 5_Calibration and QC Acceptance Criteria_Method 624a ---------------------------------------------------------------------------------------------------------------- Limit for Range for Q s Range for X Range for P, Parameter (/g/L) (/ (/g/L) Ps (%) g/L) ---------------------------------------------------------------------------------------------------------------- Benzene.............................................. 12.8-27.2 6.9 15.2-26.0 37-151 Bromodichloromethane................................. 13.1-26.9 6.4 10.1-28.0 35-155 Bromoform............................................ 14.2-25.8 5.4 11.4-31.1 45-169 Bromomethane......................................... 2.8-37.2 17.9 D-41.2 D-242 Carbon tetrachloride................................. 14.6-25.4 5.2 17.2-23.5 70-140 Chlorobenzene........................................ 13.2-26.8 6.3 16.4-27.4 37-160 Chloroethane......................................... 7.6-32.4 11.4 8.4-40.4 14-230 2-Chloroethylvinyl ether............................. D-44.8 25.9 D-50.4 D-305 Chloroform........................................... 13.5-26.5 6.1 13.7-24.2 51-138 Chloromethane........................................ D-40.8 19.8 D-45.9 D-273 Dibromochloromethane................................. 13.5-26.5 6.1 13.8-26.6 53-149 1,2-Dichlorobenzene.................................. 12.6-27.4 7.1 11.8-34.7 18-190 1,3-Dichlorobenzene.................................. 14.6-25.4 5.5 17.0-28.8 59-156 1,4-Dichlorobenzene.................................. 12.6-27.4 7.1 11.8-34.7 18-190 1,1-Dichloroethane................................... 14.5-25.5 5.1 14.2-28.5 59-155 1,2-Dichloroethane................................... 13.6-26.4 6.0 14.3-27.4 49-155 1,1-Dichlorothene.................................... 10.1-29.9 9.1 3.7-42.3 D-234 trans-1,2-Dichloroethene............................. 13.9-26.1 5.7 13.6-28.5 54-156 1,2-Dichloropropane.................................. 6.8-33.2 13.8 3.8-36.2 D-210 cis-1,3-Dichloropropene.............................. 4.8-35.2 15.8 1.0-39.0 D-227 trans-1,3-Dichloropropene............................ 10.0-30.0 10.4 7.6-32.4 17-183 Ethyl benzene........................................ 11.8-28.2 7.5 17.4-26.7 37-162 Methylene chloride................................... 12.1-27.9 7.4 D-41.0 D-221 1,1,2,2-Tetrachloroethane............................ 12.1-27.9 7.4 13.5-27.2 46-157 Tetrachloroethene.................................... 14.7-25.3 5.0 17.0-26.6 64-148 Toluene.............................................. 14.9-25.1 4.8 16.6-26.7 47-150 1,1,1-Trichloroethane................................ 15.0-25.0 4.6 13.7-30.1 52-162 1,1,2-Trichloroethane................................ 14.2-25.8 5.5 14.3-27.1 52-150 Trichloroethene...................................... 13.3-26.7 6.6 18.6-27.6 71-157 Trichlorofluoromethane............................... 9.6-30.4 10.0 8.9-31.5 17-181 Vinyl chloride....................................... 0.8-39.2 20.0 D-43.5 D-251 ---------------------------------------------------------------------------------------------------------------- Q= Concentration measured in QC check sample, in g/L (Section 7.5.3).

s= Standard deviation of four recovery measurements, in g/L (Section 8.2.4).

X= Average recovery of four recovery measurements, in g/L (Section 8.2.4).

P, Ps= Percent recovery measured, (Section 8.3.2, Section 8.4.2).

D= Detected; result must be greater than zero.

a Criteria were calculated assuming a QC check sample concentration of 20 g/L.

Note: These criteria are based directly upon the method performance data in Table 6. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 6.

Table 6_Method Accuracy and Precision as Functions of Concentration_Method 624 ---------------------------------------------------------------------------------------------------------------- Single analyst Parameter Accuracy, as recovery, precision, sr[prime] Overall precision, X[prime] (g/L) (g/L) S[prime] (g/L) ---------------------------------------------------------------------------------------------------------------- Benzene............................... 0.93C+2.00 0.26X-1.74 0.25X-1.33 Bromodichloromethane.................. 1.03C-1.58 0.15X+0.59 0.20X+1.13 Bromoform............................. 1.18C-2.35 0.12X+0.36 0.17X+1.38 Bromomethane a........................ 1.00C 0.43X 0.58X Carbon tetrachloride.................. 1.10C-1.68 0.12X+0.25 0.11X+0.37 Chlorobenzene......................... 0.98C+2.28 0.16X-0.09 0.26X-1.92 Chloroethane.......................... 1.18C+0.81 0.14X+2.78 0.29X+1.75 2-Chloroethylvinyl ether a............ 1.00C 0.62X 0.84X Chloroform............................ 0.93C+0.33 0.16X+0.22 0.18X+0.16 Chloromethane......................... 1.03C+0.81 0.37X+2.14 0.58X+0.43 Dibromochloromethane.................. 1.01C-0.03 0.17X-0.18 0.17X+0.49 1,2-Dichlorobenzene b................. 0.94C+4.47 0.22X-1.45 0.30X-1.20 1,3-Dichlorobenzene................... 1.06C+1.68 0.14X-0.48 0.18X-0.82 1,4-Dichlorobenzene b................. 0.94C+4.47 0.22X-1.45 0.30X-1.20 1,1-Dichloroethane.................... 1.05C+0.36 0.13X-0.05 0.16X+0.47 1,2-Dichloroethane.................... 1.02C+0.45 0.17X-0.32 0.21X-0.38 1,1-Dichloroethene.................... 1.12C+0.61 0.17X+1.06 0.43X-0.22 trans-1,2,-Dichloroethene............. 1.05C+0.03 0.14X+0.09 0.19X+0.17 1,2-Dichloropropane a................. 1.00C 0.33X 0.45X cis-1,3-Dichloropropene a............. 1.00C 0.38X 0.52X trans-1,3-Dichloropropene a........... 1.00C 0.25X 0.34X Ethyl benzene......................... 0.98C+2.48 0.14X+1.00 0.26X-1.72 Methylene chloride.................... 0.87C+1.88 0.15X+1.07 0.32X+4.00 1,1,2,2-Tetrachloroethane............. 0.93C+1.76 0.16X+0.69 0.20X+0.41 Tetrachloroethene..................... 1.06C+0.60 0.13X-0.18 0.16X-0.45 Toluene............................... 0.98C+2.03 0.15X-0.71 0.22X-1.71 1,1,1-Trichloroethane................. 1.06C+0.73 0.12X-0.15 0.21X-0.39 1,1,2-Trichloroethane................. 0.95C+1.71 0.14X+0.02 0.18X+0.00 Trichloroethene....................... 1.04C+2.27 0.13X+0.36 0.12X+0.59 Trichloroflouromethane................ 0.99C+0.39 0.33X-1.48 0.34X-0.39 Vinyl chloride........................ 1.00C 0.48X 0.65X ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

Sr=Expected single analyst standard deviation of measurements at an average concentration found ofX, in g/ L.

S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found ofX, in g/L.

C=True value for the concentration, in g/L.

X=Average recovery found for measurements of samples containing a concentration of C, in g/L.

a Estimates based upon the performance in a single laboratory.13 b Due to chromatographic resolution problems, performance statements for these isomers are based upon the sums of their concentrations.

(image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF Method 625Base/Neutrals and Acids 1. Scope and Application 1.1This method covers the determination of a number of organic compounds that are partitioned into an organic solvent and are amenable to gas chromatography. The parameters listed in Tables 1 and 2 may be qualitatively and quantitatively determined using this method.

1.2The method may be extended to include the parameters listed in Table 3. Benzidine can be subject to oxidative losses during solvent concentration. Under the alkaline conditions of the extraction step, BHC, BHC, endosulfan I and II, and endrin are subject to decomposition.

Hexachlorocyclopentadiene is subject to thermal decomposition in the inlet of the gas chromatograph, chemical reaction in acetone solution, and photochemical decomposition. N-nitrosodimethylamine is difficult to separate from the solvent under the chromatographic conditions described. N-nitrosodiphenylamine decomposes in the gas chromatographic inlet and cannot be separated from diphenylamine. The preferred method for each of these parameters is listed in Table 3.

1.3This is a gas chromatographic/mass spectrometry (GC/MS) method2,14 applicable to the determination of the compounds listed in Tables 1, 2, and 3 in municipal and industrial discharges as provided under 40 CFR 136.1.

1.4The method detection limit (MDL, defined in Section 16.1) 1 for each parameter is listed in Tables 4 and 5. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.5Any modification to this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

Depending upon the nature of the modification and the extent of intended use, the applicant may be required to demonstrate that the modifications will produce equivalent results when applied to relevant wastewaters.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method 2.1 A measured volume of sample, approximately 1L, is serially extracted with methylene chloride at a pH greater than 11 and again at a pH less than 2 using a separatory funnel or a continuous extractor. 2 The methylene chloride extract is dried, concentrated to a volume of 1 mL, and analyzed by GC/MS. Qualitative identification of the parameters in the extract is performed using the retention time and the relative abundance of three characteristic masses (m/z). Quantitative analysis is performed using internal standard techniques with a single characteristic m/z.

3. Interferences 3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in the total ion current profiles. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it.

Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating.

Thmrough rinsing with such solvents usually eliminates PCB interference.

Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants.

Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled.

3.3The base-neutral extraction may cause significantly reduced recovery of phenol, 2-methylphenol, and 2,4-dimethylphenol. The analyst must recognize that results obtained under these conditions are minimum concentrations.

3.4The packed gas chromatographic columns recommended for the basic fraction may not exhibit sufficient resolution for certain isomeric pairs including the following: anthracene and phenanthrene; chrysene and benzo(a)anthracene; and benzo(b)fluoranthene and benzo(k)fluoranthene.

The gas chromatographic retention time and mass spectra for these pairs of compounds are not sufficiently different to make an unambiguous identification. Alternative techniques should be used to identify and quantify these specific compounds, such as Method 610.

3.5In samples that contain an inordinate number of interferences, the use of chemical ionization (CI) mass spectrometry may make identification easier. Tables 6 and 7 give characteristic CI ions for most of the compounds covered by this method. The use of CI mass spectrometry to support electron ionization (EI) mass spectrometry is encouraged but not required.

4. Safety 4.1The toxicity or carcinogenicity of each reagent used in this method have not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified46 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzo(a)anthracene, benzidine, 3,3-dichlorobenzidine, benzo(a)pyrene, -BHC, -BHC, -BHC, -BHC, dibenzo(a,h)anthracene, N-nitrosodimethylamine, 4,4-DDT, and polychlorinated biphenyls (PCBs). Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials 5.1Sampling equipment, for discrete or composit sampling.

5.1.1Grab sample bottle1-L or 1-gt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample.

Sample containers must be kept refrigerated at 4 C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used.

before use, however, the compressible tubing should be throughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.): 5.2.1Separatory funnel2L, with Teflon stopcock.

5.2.2Drying columnChromatographic column, 19 mm ID, with coarse frit 5.2.3Concentrator tube, Kuderna-Danish10-mL, graduated (Kontes K5700501025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.2.4Evaporative flask, Kuderna-Danish500-mL (Kontes K570010500 or equivalent). Attach to concentrator tube with springs.

5.2.5Snyder column, Kuderna-DanishThree all macro (Kontes K5030000121 or equivalent).

5.2.6Snyder column, Kuderna-DanishTwo-ball macro (Kontes K5690010219 or equivalent).

5.2.7Vials10 to 15-mL, amber glass, with Teflon-lined screw cap.

5.2.8Continuous liquidliquid extractorEquipped with Teflon or glass connecting joints and stopcocks requiring no lubrication.

(Hershberg-Wolf Extractor, Ace Glass Company, Vineland, N.J., P/N 684110 or equivalent.) 5.3Boiling chipsApproximately 10/40 mesh. Heat to 400 C for 30 min of Soxhlet extract with methylene chloride.

5.4Water bathHeated, with concentric ring cover, capable of temperature control (2C). The bath should be used in a hood.

5.5BalanceAnalytical, capable of accurately weighing 0.0001 g.

5.6GC/MS system: 5.6.1Gas ChromatographAn analytical system complete with a temperature programmable gas chromatograph and all required accessores including syringes, analytical columns, and gases. The injection port must be designed for on-column injection when using packed columns and for splitless injection when using capillary columns.

5.6.2Column for base/neutrals1.8 m long 2 mm ID glass, packed with 3% SP2250 on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 16. Guidelines for the use of alternate column packings are provided in Section 13.1.

5.6.3Column for acids1.8 m long 2 mm ID glass, packed with 1% SP1240DA on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 16. Guidelines for the use of alternate column packings are given in Section 13.1.

5.6.4Mass spectrometerCapable of scanning from 35 to 450 amu every 7 s or less, utilizing a 70 V (nominal) electron energy in the electron impact ionization mode, and producing a mass spectrum which meets all the criteria in Table 9 when 50 ng of decafluorotriphenyl phosphine (DFTPP; bis(perfluorophenyl) phenyl phosphine) is injected through the GC inlet.

5.6.5GC/MS interfaceAny GC to MS interface that gives acceptable calibration points at 50 ng per injection for each of the parameters of interest and achieves all acceptable performance criteria (Section 12) may be used. GC to MS interfaces constructed of all glass or glass-lined materials are recommended. Glass can be deactivated by silanizing with dichlorodimethylsilane.

5.6.6Data systemA computer system must be interfaced to the mass spectrometer that allows the contiluous acquisition and storage on machine-readable media of all mass spectra obtained throughout the duration of the chromatographic program. The computer must have software that allows searching any GC/MS data file for specific m/z and plotting such m/z abundances versus time or scan number. This type of plot is defined as an Extracted Ion Current Profile (EICP). Software must also be available that allows integrating the abundance in any EICP between specified time or scan number limits.

6. Reagents 6.1Reagent waterReagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.2Sodium hydroxide solution (10 N)Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL.

6.3Sodium thiosulfate(ACS) Granular.

6.4Sulfuric acid (1+1)Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.

6.5Acetone, methanol, methlylene chloridePesticide quality or equivalent.

6.6Sodium sulfate(ACS) Granular, anhydrous. Purify by heating at 400 C for 4 h in a shallow tray.

6.7Stock standard solutions (1.00 g/L)standard solutions can be prepared from pure standard materials or purchased as certified solutions.

6.7.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in pesticide quality acetone or other suitable solvent and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight may be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards may be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3Stock standard solutions must be replaced after six months, or sooner if comparison with quality control check samples indicate a problem.

6.8Surrogate standard spiking solutionSelect a minimum of three surrogate compounds from Table 8. Prepare a surrogate standard spiking solution containing each selected surrogate compound at a concentration of 100 g/mL in acetone. Addition of 1.00 mL of this solution to 1000 mL of sample is equivalent to a concentration of 100 g/L of each surrogate standard. Store the spiking solution at 4 C in Teflon-sealed glass container. The solution should be checked frequently for stability. The solution must be replaced after six months, or sooner if comparison with quality control check standards indicates a problem.

6.9DFTPP standardPrepare a 25 g/mL solution of DFTPP in acetone.

6.10Quality control check sample concentrateSee Section 8.2.1.

7. Calibration 7.1Establish gas chromatographic operating parameters equivalent to those indicated in Table 4 or 5.

7.2Internal standard calibration procedureTo use this approach, the analyst must select three or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standards is not affected by method or matrix interferences. Some recommended internal standards are listed in Table 8. Use the base peak m/z as the primary m/z for quantification of the standards. If interferences are noted, use one of the next two most intense m/z quantities for quantification.

7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding appropriate volumes of one or more stock standards to a volumetric flask. To each calibration standard or standard mixture, add a known constant amount of one or more internal standards, and dilute to volume with acetone. One of the calibration standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the GC/MS system.

7.2.2Using injections of 2 to 5 L, analyze each calibration standard according to Section 13 and tabulate the area of the primary characteristic m/z (Tables 4 and 5) against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1. (image) Equation 1 where: As=Area of the characteristic m/z for the parameter to be measured.

Ais=Area of the characteristic m/z for the internal standard.

Cis=Concentration of the internal standard (g/L).

Cs=Concentration of the parameter to be measured (g/L).

If the RF value over the working range is a constant (7.3The working calibration curve or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than 20%, the test must be repeated uning a fresh calibration standard.

Alternatively, a new calibration curve must be prepared for that compound.

8. Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method.

This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occuring in chromatography, the analyst is permitted certain options (detailed in Sections 10.6 and 13.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 5% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 5% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 100 g/mL in acetone. Multiple solutions may be required. PCBs and multicomponent pesticides may be omitted from this test. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 100 g/L by adding 1.00 mL of QC check sample concentrate to each of four 1L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10 or 11.

8.2.4Calculate the average recovery (X) in g/L, and the standard deviation of the recovery (s) in g/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 6. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note: The large number of parameters in Table 6 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 5% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing 1 to 20 samples per month, at least one spiked sample per month is required.

8.3.1.The concentration of the spike in the sample should be determined as follows: 8.3.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 100 g/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 100 g/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(AB)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 6. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 100 g/L, the analyst must use either the QC acceptance criteria in Table 6, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X) using the equation in Table 7, substituting the spike concentration (T) for C; (2) calculate overall precision (S) using the equation in Table 7, substituting X for X ; (3) calculate the range for recovery at the spike concentration as (100 X/T)2.44(100 S/T)%7 8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of single-component parameters in Table 6 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spike sample.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (PS) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 6. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained.

After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent interval from P 2sp to P +2sp. If P =90% and sp=10%, for example, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6As a quality control check, the laboratory must spike all samples with the surrogate standard spiking solution as described in Section 10.2, and calculate the percent recovery of each surrogate compound.

8.7It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling 9.1Grab samples must be collected in glass containers. Conventional sampling practices8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All sampling must be iced or refrigerated at 4 C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.9 Field test kits are available for this purpose.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.

10. Separatory Funnel Extraction 10.1Samples are usually extracted using separatory funnel techniques. If emulsions will prevent achieving acceptable solvent recovery with separatory funnel extractions, continuous extraction (Section 11) may be used. The separatory funnel extraction scheme described below assumes a sample volume of 1 L. When sample volumes of 2 L are to be extracted, use 250, 100, and 100-mL volumes of methylene chloride for the serial extraction of the base/neutrals and 200, 100, and 100-mL volumes of methylene chloride for the acids.

10.2Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2L separatory funnel. Pipet 1.00 mL of the surrogate standard spiking solution into the separatory funnel and mix well. Check the pH of the sample with wide-range pH paper and adjust to pH>11 with sodium hydroxide solution.

10.3Add 60 mL of methylene chloride to the sample bottle, seal, and shake for 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask. If the emulsion cannot be broken (recovery of less than 80% of the methylene chloride, corrected for the water solubility of methylene chloride), transfer the sample, solvent, and emulsion into the extraction chamber of a continuous extractor and proceed as described in Section 11.3.

10.4Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner. Label the combined extract as the base/neutral fraction.

10.5Adjust the pH of the aqueous phase to less than 2 using sulfuric acid. Serially extract the acidified aqueous phase three times with 60-mL aliquots of methylene chloride. Collect and combine the extracts in a 250-mL Erlenmeyer flask and label the combined extracts as the acid fraction.

10.6For each fraction, assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.7For each fraction, pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.8Add one or two clean boiling chips and attach a three-ball Snyder column to the evaporative flask for each fraction. Prewet each Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus from the water bath and allow it to drain and cool for at least 10 min. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation.

10.9Add another one or two clean boiling chips to the concentrator tube for each fraction and attach a two-ball micro-Snyder column. Prewet the Snyder column by adding about 0.5 mL of methylene chloride to the top.

Place the K-D apparatus on a hot water bath (60 to 65 C) so that the concentrator tube is partially immersed in hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus from the water bath and allow it to drain and cool for at least 10 min. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with approximately 0.2 mL of acetone or methylene chloride. Adjust the final volume to 1.0 mL with the solvent. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extracts will be stored longer than two days, they should be transferred to Teflon-sealed screw-cap vials and labeled base/neutral or acid fraction as appropriate.

10.10Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder.

Record the sample volume to the nearest 5 mL.

11. Continuous Extraction 11.1When experience with a sample from a given source indicates that a serious emulsion problem will result or an emulsion is encountered using a separatory funnel in Section 10.3, a continuous extractor should be used.

11.2Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Check the pH of the sample with wide-range pH paper and adjust to pH >11 with sodium hydroxide solution.

Transfer the sample to the continuous extractor and using a pipet, add 1.00 mL of surrogate standard spiking solution and mix well. Add 60 mL of methylene chloride to the sample bottle, seal, and shake for 30 s to rinse the inner surface. Transfer the solvent to the extractor.

11.3Repeat the sample bottle rinse with an additional 50 to 100-mL portion of methylene chloride and add the rinse to the extractor.

11.4Add 200 to 500 mL of methylene chloride to the distilling flask, add sufficient reagent water to ensure proper operation, and extract for 24 h. Allow to cool, then detach the distilling flask. Dry, concentrate, and seal the extract as in Sections 10.6 through 10.9.

11.5Charge a clean distilling flask with 500 mL of methylene chloride and attach it to the continuous extractor. Carefully, while stirring, adjust the pH of the aqueous phase to less than 2 using sulfuric acid.

Extract for 24 h. Dry, concentrate, and seal the extract as in Sections 10.6 through 10.9.

12. Daily GC/MS Performance Tests 12.1At the beginning of each day that analyses are to be performed, the GC/MS system must be checked to see if acceptable performance criteria are achieved for DFTPP.10 Each day that benzidine is to be determined, the tailing factor criterion described in Section 12.4 must be achieved.

Each day that the acids are to be determined, the tailing factor criterion in Section 12.5 must be achieved.

12.2These performance tests require the following instrumental parameters: Electron Energy: 70 V (nominal) Mass Range: 35 to 450 amu Scan Time: To give at least 5 scans per peak but not to exceed 7 s per scan.

12.3DFTPP performance testAt the beginning of each day, inject 2 L (50 ng) of DFTPP standard solution. Obtain a background-corrected mass spectra of DFTPP and confirm that all the key m/z criteria in Table 9 are achieved. If all the criteria are not achieved, the analyst must retune the mass spectrometer and repeat the test until all criteria are achieved. The performance criteria must be achieved before any samples, blanks, or standards are analyzed. The taililg factor tests in Sections 12.4 and 12.5 may be performed simultaneously with the DFTPP test.

12.4Column performance test for base/neutralsAt the beginning of each day that the base/neutral fraction is to be analyzed for benzidine, the benzidine tailing factor must be calculated. Inject 100 ng of benzidine either separately or as a part of a standard mixture that may contain DFTPP and calculate the tailing factor. The benzidine tailing factor must be less than 3.0. Calculation of the tailing factor is illustrated in Figure 13. 11 Replace the column packing if the tailing factor criterion cannot be achieved.

12.5Column performance test for acidsAt the beginning of each day that the acids are to be determined, inject 50 ng of pentachlorophenol either separately or as a part of a standard mix that may contain DFTPP. The tailing factor for pentachlorophenol must be less than 5. Calculation of the tailing factor is illustrated in Figure 13. 11 Replace the column packing if the tailing factor criterion cannot be achieved.

13. Gas Chromatography/Mass Spectrometry 13.1Table 4 summarizes the recommended gas chromatographic operating conditions for the base/neutral fraction. Table 5 summarizes the recommended gas chromatographic operating conditions for the acid fraction. Included in these tables are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by these columns are shown in Figures 1 through 12. Other packed or capillary (open-tubular) columns or chromatographic conditions may be used if the requirements of Section 8.2 are met.

13.2After conducting the GC/MS performance tests in Section 12, calibrate the system daily as described in Section 7.

13.3The internal standard must be added to sample extract and mixed thoroughly immediately before it is injected into the instrument. This procedure minimizes losses due to adsorption, chemical reaction or evaporation.

13.4Inject 2 to 5 L of the sample extract or standard into the GC/MS system using the solvent-flush technique. 12 Smaller (1.0 L) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 L.

13.5If the response for any m/z exceeds the working range of the GC/MS system, dilute the extract and reanalyze.

13.6Perform all qualitative and quantitative measurements as described in Sections 14 and 15. When the extracts are not being used for analyses, store them refrigerated at 4C, protected from light in screw-cap vials equipped with unpierced Teflon-lined septa.

14. Qualitative Identification 14.1Obtain EICPs for the primary m/z and the two other masses listed in Tables 4 and 5. See Section 7.3 for masses to be used with internal and surrogate standards. The following criteria must be met to make a qualitative identification: 14.1.1The characteristic masses of each parameter of interest must maximize in the same or within one scan of each other.

14.1.2The retention time must fall within 30 s of the retention time of the authentic compound.

14.1.3The relative peak heights of the three characteristic masses in the EICPs must fall within 20% of the relative intensities of these masses in a reference mass spectrum. The reference mass spectrum can be obtained from a standard analyzed in the GC/MS system or from a reference library.

14.2Structural isomers that have very similar mass spectra and less than 30 s difference in retention time, can be explicitly identified only if the resolution between authentic isomers in a standard mix is acceptable. Acceptable resolution is achieved if the baseline to valley height between the isomers is less than 25% of the sum of the two peak heights. Otherwise, structural isomers are identified as isomeric pairs.

15. Calculations 15.1When a parameter has been identified, the quantitation of that parameter will be based on the integrated abundance from the EICP of the primary characteristic m/z in Tables 4 and 5. Use the base peak m/z for internal and surrogate standards. If the sample produces an interference for the primary m/z, use a secondary characteristic m/z to quantitate.

Calculate the concentration in the sample using the response factor (RF) determined in Section 7.2.2 and Equation 3. (image) Equation 3 where: As=Area of the characteristic m/z for the parameter or surrogate standard to be measured.

Ais=Area of the characteristic m/z for the internal standard.

Is=Amount of internal standard added to each extract (g).

Vo=Volume of water extracted (L).

15.2Report results in g/L without correction for recovery data. All QC data obtained should be reported with the sample results.

16. Method Performance 16.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Tables 4 and 5 were obtained using reagent water.13 The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

16.2This method was tested by 15 laboratories using reagent water, drinking water, surface water, and industrial wastewaters spiked at six concentrations over the range 5 to 1300 g/L.14 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 7.

17. Screening Procedure for 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8TCDD) 17.1If the sample must be screened for the presence of 2,3,7,8TCDD, it is recommended that the reference material not be handled in the laboratory unless extensive safety precautions are employed. It is sufficient to analyze the base/neutral extract by selected ion monitoring (SIM) GC/MS techniques, as follows: 17.1.1Concentrate the base/neutral extract to a final volume of 0.2 ml.

17.1.2Adjust the temperature of the base/neutral column (Section 5.6.2) to 220 C.

17.1.3Operate the mass spectrometer to acquire data in the SIM mode using the ions at m/z 257, 320 and 322 and a dwell time no greater than 333 milliseconds per mass.

17.1.4Inject 5 to 7 L of the base/neutral extract. Collect SIM data for a total of 10 min.

17.1.5The possible presence of 2,3,7,8TCDD is indicated if all three masses exhibit simultaneous peaks at any point in the selected ion current profiles.

17.1.6For each occurrence where the possible presence of 2,3,7,8TCDD is indicated, calculate and retain the relative abundances of each of the three masses.

17.2False positives to this test may be caused by the presence of single or coeluting combinations of compounds whose mass spectra contain all of these masses.

17.3Conclusive results of the presence and concentration level of 2,3,7,8TCDD can be obtained only from a properly equipped laboratory through the use of EPA Method 613 or other approved alternate test procedures.

References 1. 40 CFR part 136, appendix B.

2. Sampling and Analysis Procedures for Screening of Industrial Effluents for Priority Pollutants, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1977, Revised April 1977. Available from Effluent Guidelines Division, Washington, DC 20460.

3. ASTM Annual Book of Standards, Part 31, D369478. Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents, American Society for Testing and Materials, Philadelphia.

4. CarcinogensWorking With Carcinogens, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77206, August 1977.

5. OSHA Safety and Health Standards, General Industry, (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

6. Safety in Academic Chemistry Laboratories,American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

7. Provost, L.P., and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15, 5863 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.) 8. ASTM Annual Book of Standards, Part 31, D337076. Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia.

9. Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, EPA600/479020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

10. Eichelberger, J.W., Harris, L.E., and Budde, W.L. Reference Compound to Calibrate Ion Abundance Measurement in Gas Chromatography-Mass Spectometry, Analytical Chemistry, 47, 995 (1975).

11. McNair, N.M. and Bonelli, E.J. Basic Chromatography, Consolidated Printing, Berkeley, California, p. 52, 1969.

12. Burke, J.A. Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects, Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

13. Olynyk, P., Budde, W.L., and Eichelberger, J.W. Method Detection Limit for Methods 624 and 625, Unpublished report, May 14, 1980.

14. EPA Method Study 30, Method 625, Base/Neutrals, Acids, and Pesticides, EPA 600/484053, National Technical Information Service, PB84206572, Springfield, Virginia 22161, June 1984.

Table 1_Base/Neutral Extractables ------------------------------------------------------------------------ STORET Parameter No. CAS No.

------------------------------------------------------------------------ Acenaphthene..................................... 34205 83-32-9 Acenaphthylene................................... 34200 208-96-8 Anthracene....................................... 34220 120-12-7 Aldrin........................................... 39330 309-00-2 Benzo(a)anthracene............................... 34526 56-55-3 Benzo(b)fluoranthene............................. 34230 205-99-2 Benzo(k)fluoranthene............................. 34242 207-08-9 Benzo(a)pyrene................................... 34247 50-32-8 Benzo(ghi)perylene............................... 34521 191-24-2 Benzyl butyl phthalate........................... 34292 85-68-7 -BHC....................................... 39338 319-85-7 -BHC...................................... 34259 319-86-8 Bis(2-chloroethyl) ether......................... 34273 111-44-4 Bis(2-chloroethoxy)methane....................... 34278 111-91-1 Bis(2-ethylhexyl) phthalate...................... 39100 117-81-7 Bis(2-chloroisopropyl) ether a................... 34283 108-60-1 4-Bromophenyl phenyl ether a..................... 34636 101-55-3 Chlordane........................................ 39350 57-74-9 2-Chloronaphthalele.............................. 34581 91-58-7 4-Chlorophenyl phenyl ether...................... 34641 7005-72-3 Chrysene......................................... 34320 218-01-9 4,4[prime]-DDD................................... 39310 72-54-8 4,4[prime]-DDE................................... 39320 72-55-9 4,4[prime]-DDT................................... 39300 50-29-3 Dibenzo(a,h)anthracene........................... 34556 53-70-3 Di-n-butylphthalate.............................. 39110 84-74-2 1,3-Dichlorobenzene.............................. 34566 541-73-1 1,2-Dichlorobenzene.............................. 34536 95-50-1 1,4-Dichlorobenzene.............................. 34571 106-46-7 3,3[prime]-Dichlorobenzidine..................... 34631 91-94-1 Dieldrin......................................... 39380 60-57-1 Diethyl phthalate................................ 34336 84-66-2 Dimethyl phthalate............................... 34341 131-11-3 2,4-Dinitrotoluene............................... 34611 121-14-2 2,6-Dinitrotoluene............................... 34626 606-20-2 Di-n-octylphthalate.............................. 34596 117-84-0 Endosulfan sulfate............................... 34351 1031-07-8 Endrin aldehyde.................................. 34366 7421-93-4 Fluoranthene..................................... 34376 206-44-0 Fluorene......................................... 34381 86-73-7 Heptachlor....................................... 39410 76-44-8 Heptchlor epoxide................................ 39420 1024-57-3 Hexachlorobenzene................................ 39700 118-74-1 Hexachlorobutadiene.............................. 34391 87-68-3 Hexachloroethane................................. 34396 67-72-1 Indeno(1,2,3-cd)pyrene........................... 34403 193-39-5 Isophorone....................................... 34408 78-59-1 Naphthalene...................................... 34696 91-20-3 Nitrobenzene..................................... 34447 98-95-3 N-Nitrosodi-n-propylamine........................ 34428 621-64-7 PCB-1016......................................... 34671 12674-11-2 PCB-1221......................................... 39488 11104-28-2 PCB-1232......................................... 39492 11141-16-5 PCB-1242......................................... 39496 53469-21-9 PCB-1248......................................... 39500 12672-29-6 PCB-1254......................................... 39504 11097-69-1 PCB-1260......................................... 39508 11096-82-5 Phenanthrene..................................... 34461 85-01-8 Pyrene........................................... 34469 129-00-0 Toxaphene........................................ 39400 8001-35-2 1,2,4-Trichlorobenzene........................... 34551 120-82-1 ------------------------------------------------------------------------ a The proper chemical name is 2,2[prime]-oxybis(1-chloropropane).

Table 2_Acid Extractables ------------------------------------------------------------------------ STORET Parameter No. CAS No.

------------------------------------------------------------------------ 4-Chloro-3-methylphenol.......................... 34452 59-50-7 2-Chlorophenol................................... 34586 95-57-8 2,4-Dichlorophenol............................... 34601 120-83-2 2,4-Dimethylphenol............................... 34606 105-67-9 2,4-Dinitrophenol................................ 34616 51-28-5 2-Methyl-4,6-dinitrophenol....................... 34657 534-52-1 2-Nitrophenol.................................... 34591 88-75-5 4-Nitrophenol.................................... 34646 100-02-7 Pentachlorophenol................................ 39032 87-86-5 Phenol........................................... 34694 108-95-2 2,4,6-Trichlorophenol............................ 34621 88-06-2 ------------------------------------------------------------------------ Table 3_Additional Extractable Parameters a ------------------------------------------------------------------------ STORET Parameter No. CAS No. Method ------------------------------------------------------------------------ Benzidine................................ 39120 92-87-5 605 -BHC............................... 39337 319-84-6 608 -BHC.............................. 39340 58-89-8 608 Endosulfan I............................. 34361 959-98-8 608 Endosulfan II............................ 34356 33213-65-9 608 Endrin................................... 39390 72-20-8 608 Hexachlorocylopentadiene................. 34386 77-47-4 612 N-Nitrosodimethylamine................... 34438 62-75-9 607 N-Nitrosodiphenylamine................... 34433 86-30-6 607 ------------------------------------------------------------------------ a See Section 1.2.

Table 4_Chromatographic Conditions, Method Detection Limits, and Characteristic Masses for Base/Neutral Extractables -------------------------------------------------------------------------------------------------------------------------------------------------------- Method Characteristic masses Retention detection ------------------------------------------------------------- Parameter time limit Electron impact Chemical ionization (min) (g/ ------------------------------------------------------------- L) Primary Secondary Secondary Methane Methane Methane -------------------------------------------------------------------------------------------------------------------------------------------------------- 1,3-Dichlorobenzene................................................ 7.4 1.9 146 148 113 146 148 150 1,4-Dichlorobenzene................................................ 7.8 4.4 146 148 113 146 148 150 Hexachloroethane................................................... 8.4 1.6 117 201 199 199 201 203 Bis(2-chloroethyl) ether a......................................... 8.4 5.7 93 63 95 63 107 109 1,2-Dichlorobenzene................................................ 8.4 1.9 146 148 113 146 148 150 Bis(2-chloroisopropyl) ether a..................................... 9.3 5.7 45 77 79 77 135 137 N-Nitrosodi-n-propylamine.......................................... ......... .......... 130 42 101 ........ ........ ........

Nitrobenzene....................................................... 11.1 1.9 77 123 65 124 152 164 Hexachlorobutadiene................................................ 11.4 0.9 225 223 227 223 225 227 1,2,4-Trichlorobenzene............................................. 11.6 1.9 180 182 145 181 183 209 Isophorone......................................................... 11.9 2.2 82 95 138 139 167 178 Naphthalene........................................................ 12.1 1.6 128 129 127 129 157 169 Bis(2-chloroethoxy) methane........................................ 12.2 5.3 93 95 123 65 107 137 Hexachlorocyclopentadiene a........................................ 13.9 .......... 237 235 272 235 237 239 2-Chloronaphthalene................................................ 15.9 1.9 162 164 127 163 191 203 Acenaphthylene..................................................... 17.4 3.5 152 151 153 152 153 181 Acenaphthene....................................................... 17.8 1.9 154 153 152 154 155 183 Dimethyl phthalate................................................. 18.3 1.6 163 194 164 151 163 164 2,6-Dinitrotoluene................................................. 18.7 1.9 165 89 121 183 211 223 Fluorene........................................................... 19.5 1.9 166 165 167 166 167 195 4-Chlorophenyl phenyl ether........................................ 19.5 4.2 204 206 141 ........ ........ ........

2,4-Dinitrotoluene................................................. 19.8 5.7 165 63 182 183 211 223 Diethyl phthalate.................................................. 20.1 1.9 149 177 150 177 223 251 N-Nitrosodiphenylamine b........................................... 20.5 1.9 169 168 167 169 170 198 Hexachlorobenzene.................................................. 21.0 1.9 284 142 249 284 286 288 -BHC b....................................................... 21.1 .......... 183 181 109 ........ ........ ........

4-Bromophenyl phenyl ether......................................... 21.2 1.9 248 250 141 249 251 277 -BHC b...................................................... 22.4 .......... 183 181 109 ........ ........ ........

Phenanthrene....................................................... 22.8 5.4 178 179 176 178 179 207 Anthracene......................................................... 22.8 1.9 178 179 176 178 179 207 -BHC......................................................... 23.4 4.2 181 183 109 ........ ........ ........

Heptachlor......................................................... 23.4 1.9 100 272 274 ........ ........ ........

-BHC........................................................ 23.7 3.1 183 109 181 ........ ........ ........

Aldrin............................................................. 24.0 1.9 66 263 220 ........ ........ ........

Dibutyl phthalate.................................................. 24.7 2.5 149 150 104 149 205 279 Heptachlor epoxide................................................. 25.6 2.2 353 355 351 ........ ........ ........

Endosulfan I b..................................................... 26.4 .......... 237 339 341 ........ ........ ........

Fluoranthene....................................................... 26.5 2.2 202 101 100 203 231 243 Dieldrin........................................................... 27.2 2.5 79 263 279 ........ ........ ........

4,4[prime]-DDE..................................................... 27.2 5.6 246 248 176 ........ ........ ........

Pyrene............................................................. 27.3 1.9 202 101 100 203 231 243 Endrin b........................................................... 27.9 .......... 81 263 82 ........ ........ ........

Endosulfan II b.................................................... 28.6 .......... 237 339 341 ........ ........ ........

4,4[prime]-DDD..................................................... 28.6 2.8 235 237 165 ........ ........ ........

Benzidine b........................................................ 28.8 44 184 92 185 185 213 225 4,4[prime]-DDT..................................................... 29.3 4.7 235 237 165 ........ ........ ........

Endosulfan sulfate................................................. 29.8 5.6 272 387 422 ........ ........ ........

Endrin aldehyde.................................................... ......... .......... 67 345 250 ........ ........ ........

Butyl benzyl phthalate............................................. 29.9 2.5 149 91 206 149 299 327 Bis(2-ethylhexyl) phthalate........................................ 30.6 2.5 149 167 279 149 ........ ........

Chrysene........................................................... 31.5 2.5 228 226 229 228 229 257 Benzo(a)anthracene................................................. 31.5 7.8 228 229 226 228 229 257 3,3[prime]-Dichlorobenzidine....................................... 32.2 16.5 252 254 126 ........ ........

Di-n-octyl phthalate............................................... 32.5 2.5 149 Benzo(b)fluoranthene............................................... 34.9 4.8 252 253 125 252 253 281 Benzo(k)fluoranthene............................................... 34.9 2.5 252 253 125 252 253 281 Benzo(a)pyrene..................................................... 36.4 2.5 252 253 125 252 253 281 Indeno(1,2,3-cd) pyrene............................................ 42.7 3.7 276 138 277 276 277 305 Dibenzo(a,h)anthracene............................................. 43.2 2.5 278 139 279 278 279 307 Benzo(ghi)perylene................................................. 45.1 4.1 276 138 277 276 277 305 N-Nitrosodimethylamine b........................................... ......... .......... 42 74 44 ........ ........ ........

Chlordane c........................................................ 19-30 .......... 373 375 377 ........ ........ ........

Toxaphene c........................................................ 25-34 .......... 159 231 233 ........ ........ ........

PCB 1016 c......................................................... 18-30 .......... 224 260 294 ........ ........ ........

PCB 1221 c......................................................... 15-30 30 190 224 260 ........ ........ ........

PCB 1232 c......................................................... 15-32 .......... 190 224 260 ........ ........ ........

PCB 1242 c......................................................... 15-32 .......... 224 260 294 ........ ........ ........

PCB 1248 c......................................................... 12-34 .......... 294 330 262 ........ ........ ........

PCB 1254 c......................................................... 22-34 36 294 330 362 ........ ........ ........

PCB 1260 c......................................................... 23-32 .......... 330 362 394 ........ ........ ........

-------------------------------------------------------------------------------------------------------------------------------------------------------- a The proper chemical name is 2,2[prime]-bisoxy(1-chloropropane).

b See Section 1.2.

c These compounds are mixtures of various isomers (See Figures 2 through 12). Column conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a 1.8 m long x 2 mm ID glass column with helium carrier gas at 30 mL/min. flow rate. Column temperature held isothermal at 50 C for 4 min., then programmed at 8 C/min. to 270 C and held for 30 min.

Table 5_Chromatographic Conditions, Method Detection Limits, and Characteristic Masses for Acid Extractables -------------------------------------------------------------------------------------------------------------------------------------------------------- Method Characteristic masses Retention detection ------------------------------------------------------------- Parameter time limit Electron Impact Chemical ionization (min) (g/ ------------------------------------------------------------- L) Primary Secondary Secondary Methane Methane Methane -------------------------------------------------------------------------------------------------------------------------------------------------------- 2-Chlorophenol..................................................... 5.9 3.3 128 64 130 129 131 157 2-Nitrophenol...................................................... 6.5 3.6 139 65 109 140 168 122 Phenol............................................................. 8.0 1.5 94 65 66 95 123 135 2,4-Dimethylphenol................................................. 9.4 2.7 122 107 121 123 151 163 2,4-Dichlorophenol................................................. 9.8 2.7 162 164 98 163 165 167 2,4,6-Trichlorophenol.............................................. 11.8 2.7 196 198 200 197 199 201 4-Chloro-3-methylphenol............................................ 13.2 3.0 142 107 144 143 171 183 2,4-Dinitrophenol.................................................. 15.9 42 184 63 154 185 213 225 2-Methyl-4,6-dinitrophenol......................................... 16.2 24 198 182 77 199 227 239 Pentachlorophenol.................................................. 17.5 3.6 266 264 268 267 265 269 4-Nitrophenol...................................................... 20.3 2.4 65 139 109 140 168 122 -------------------------------------------------------------------------------------------------------------------------------------------------------- Column conditions: Supelcoport (100/120 mesh) coated with 1% SP-1240DA packed in a 1.8 m long x 2mm ID glass column with helium carrier gas at 30 mL/ min. flow rate. Column temperature held isothermal at 70 C for 2 min. then programmed at 8 C/min. to 200 C.

Table 6_QC Acceptance Criteria_Method 625 ---------------------------------------------------------------------------------------------------------------- Test conclusion Limits for Range for Range for Parameter (g/ s (g/ X(g/ P, Ps L) L) L) (Percent) ---------------------------------------------------------------------------------------------------------------- Acenaphthene................................................ 100 27.6 60.1-132.3 47-145 Acenaphthylene.............................................. 100 40.2 53.5-126.0 33-145 Aldrin...................................................... 100 39.0 7.2-152.2 D-166 Anthracene.................................................. 100 32.0 43.4-118.0 27-133 Benzo(a)anthracene.......................................... 100 27.6 41.8-133.0 33-143 Benzo(b)fluoranthene........................................ 100 38.8 42.0-140.4 24-159 Benzo(k)fluoranthene........................................ 100 32.3 25.2-145.7 11-162 Benzo(a)pyrene.............................................. 100 39.0 31.7-148.0 17-163 Benzo(ghi)perylene.......................................... 100 58.9 D-195.0 D-219 Benzyl butyl phthalate...................................... 100 23.4 D-139.9 D-152 -BHC.................................................. 100 31.5 41.5-130.6 24-149 -BHC................................................. 100 21.6 D-100.0 D-110 Bis(2-chloroethyl) ether.................................... 100 55.0 42.9-126.0 12-158 Bis(2-chloroethoxy)methane.................................. 100 34.5 49.2-164.7 33-184 Bis(2-chloroisopropyl) ether a.............................. 100 46.3 62.8-138.6 36-166 Bis(2-ethylhexyl) phthalate................................. 100 41.1 28.9-136.8 8-158 4-Bromophenyl phenyl ether.................................. 100 23.0 64.9-114.4 53-127 2-Chloronaphthalene......................................... 100 13.0 64.5-113.5 60-118 4-Chlorophenyl phenyl ether................................. 100 33.4 38.4-144.7 25-158 Chrysene.................................................... 100 48.3 44.1-139.9 17-168 4,4[prime]-DDD.............................................. 100 31.0 D-134.5 D-145 4,4[prime]-DDE.............................................. 100 32.0 19.2-119.7 4-136 4,4[prime]-DDT.............................................. 100 61.6 D-170.6 D-203 Dibenzo(a,h)anthracene...................................... 100 70.0 D-199.7 D-227 Di-n-butyl phthalate........................................ 100 16.7 8.4-111.0 1-118 1,2-Dichlorobenzene......................................... 100 30.9 48.6-112.0 32-129 1,3-Dichlorobenzene......................................... 100 41.7 16.7-153.9 D-172 1,4,-Dichlorobenzene........................................ 100 32.1 37.3-105.7 20-124 3,3[prime]-Dhlorobenzidine.................................. 100 71.4 8.2-212.5 D-262 Dieldrin.................................................... 100 30.7 44.3-119.3 29-136 Diethyl phthalate........................................... 100 26.5 D-100.0 D-114 Dimethyl phthalate.......................................... 100 23.2 D-100.0 D-112 2,4-Dinitrotoluene.......................................... 100 21.8 47.5-126.9 39-139 2,6-Dinitrotoluene.......................................... 100 29.6 68.1-136.7 50-158 Di-n-octyl phthalate........................................ 100 31.4 18.6-131.8 4-146 Endosulfan sulfate.......................................... 100 16.7 D-103.5 D-107 Endrin aldehyde............................................. 100 32.5 D-188.8 D-209 Fluoranthene................................................ 100 32.8 42.9-121.3 26-137 Fluorene.................................................... 100 20.7 71.6-108.4 59-121 Heptachlor.................................................. 100 37.2 D-172.2 D-192 Heptachlor epoxide.......................................... 100 54.7 70.9-109.4 26-155 Hexachlorobenzene........................................... 100 24.9 7.8-141.5 D-152 Hexachlorobutadiene......................................... 100 26.3 37.8-102.2 24-116 Hexachloroethane............................................ 100 24.5 55.2-100.0 40-113 Indeno(1,2,3-cd)pyrene...................................... 100 44.6 D-150.9 D-171 Isophorone.................................................. 100 63.3 46.6-180.2 21-196 Naphthalene................................................. 100 30.1 35.6-119.6 21-133 Nitrobenzene................................................ 100 39.3 54.3-157.6 35-180 N-Nitrosodi-n-propylamine................................... 100 55.4 13.6-197.9 D-230 PCB-1260.................................................... 100 54.2 19.3-121.0 D-164 Phenanthrene................................................ 100 20.6 65.2-108.7 54-120 Pyrene...................................................... 100 25.2 69.6-100.0 52-115 1,2,4-Trichlorobenzene...................................... 100 28.1 57.3-129.2 44-142 4-Chloro-3-methylphenol..................................... 100 37.2 40.8-127.9 22-147 2-Chlorophenol.............................................. 100 28.7 36.2-120.4 23-134 2,4-Dichlorophenol.......................................... 100 26.4 52.5-121.7 39-135 2,4-Dimethylphenol.......................................... 100 26.1 41.8-109.0 32-119 2,4-Dinitrophenol........................................... 100 49.8 D-172.9 D-191 2-Methyl-4,6-dinitrophenol.................................. 100 93.2 53.0-100.0 D-181 2-Nitrophenol............................................... 100 35.2 45.0-166.7 29-182 4-Nitrophenol............................................... 100 47.2 13.0-106.5 D-132 Pentachlorophenol........................................... 100 48.9 38.1-151.8 14-176 Phenol...................................................... 100 22.6 16.6-100.0 5-112 2,4,6-Trichlorophenol....................................... 100 31.7 52.4-129.2 37-144 ---------------------------------------------------------------------------------------------------------------- s=Standard deviation for four recovery measurements, in g/L (Section 8.2.4).

X=Average recovery for four recovery measurements, in g/L (Section 8.2.4).

P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).

D=Detected; result must be greater than zero.

Note: These criteria are based directly upon the method performance data in Table 7. Where necessary, the limits for recovery have been broadened to assure applicability of the limts to concentrations below those used to develop Table 7.

a The proper chemical name is 2,2[prime]oxybis(1-chloropropane).

Table 7_Method Accuracy and Precision as Functions of Concentration_Method 625 ---------------------------------------------------------------------------------------------------------------- Accuracy, as Overall recovery, Single analyst precision, Parameter X[prime] precision, sr' S[prime] (g/L) (g/L) (g/L) ---------------------------------------------------------------------------------------------------------------- Acenaphthene.................................................... 0.96C=0.19 0.15X-0.12 0.21X-0.67 Acenaphthylene.................................................. 0.89C=0.74 0.24X-1.06 0.26X-0.54 Aldrin.......................................................... 0.78C=1.66 0.27X-1.28 0.43X=1.13 Anthracene...................................................... 0.80C=0.68 0.21X-0.32 0.27X-0.64 Benzo(a)anthracene.............................................. 0.88C-0.60 0.15X=0.93 0.26X-0.28 Benzo(b)fluoranthene............................................ 0.93C-1.80 0.22X=0.43 0.29X=0.96 Benzo(k)fluoranthene............................................ 0.87C-1.56 0.19X=1.03 0.35X=0.40 Benzo(a)pyrene.................................................. 0.90C-0.13 0.22X=0.48 0.32X=1.35 Benzo(ghi)perylene.............................................. 0.98C-0.86 0.29X=2.40 0.51X-0.44 Benzyl butyl phthalate.......................................... 0.66C-1.68 0.18X=0.94 0.53X=0.92 -BHC...................................................... 0.87C-0.94 0.20X-0.58 0.30X-1.94 -BHC..................................................... 0.29C-1.09 0.34X=0.86 0.93X-0.17 Bis(2-chloroethyl) ether........................................ 0.86C-1.54 0.35X-0.99 0.35X=0.10 Bis(2-chloroethoxy)methane...................................... 1.12C-5.04 0.16X=1.34 0.26X=2.01 Bis(2-chloroisopropyl) ether a.................................. 1.03C-2.31 0.24X=0.28 0.25X=1.04 Bis(2-ethylhexyl) phthalate..................................... 0.84C-1.18 0.26X=0.73 0.36X=0.67 4-Bromophenyl phenyl ether...................................... 0.91C-1.34 0.13X=0.66 0.16X=0.66 2-Chloronaphthalene............................................. 0.89C=0.01 0.07X=0.52 0.13X=0.34 4-Chlorophenyl phenyl ether..................................... 0.91C=0.53 0.20X-0.94 0.30X-0.46 Chrysene........................................................ 0.93C-1.00 0.28X=0.13 0.33X-0.09 4,4[prime]-DDD.................................................. 0.56C-0.40 0.29X-0.32 0.66X-0.96 4,4[prime]-DDE.................................................. 0.70C-0.54 0.26X-1.17 0.39X-1.04 4,4[prime]-DDT.................................................. 0.79C-3.28 0.42X=0.19 0.65X-0.58 Dibenzo(a,h)anthracene.......................................... 0.88C=4.72 0.30X=8.51 0.59X=0.25 Di-n-butyl phthalate............................................ 0.59C=0.71 0.13X=1.16 0.39X=0.60 1,2-Dichlorobenzene............................................. 0.80C=0.28 0.20X=0.47 0.24X=0.39 1,3-Dichlorobenzene............................................. 0.86C-0.70 0.25X=0.68 0.41X=0.11 1,4-Dichlorobenzene............................................. 0.73C-1.47 0.24X=0.23 0.29X=0.36 3,3[prime]-Dichlorobenzidine.................................... 1.23C-12.65 0.28X=7.33 0.47X=3.45 Dieldrin........................................................ 0.82C-0.16 0.20X-0.16 0.26X-0.07 Diethyl phthalate............................................... 0.43C=1.00 0.28X=1.44 0.52X=0.22 Dimethyl phthalate.............................................. 0.20C=1.03 0.54X=0.19 1.05X-0.92 2,4-Dinitrotoluene.............................................. 0.92C-4.81 0.12X=1.06 0.21X=1.50 2,6-Dinitrotoluene.............................................. 1.06C-3.60 0.14X=1.26 0.19X=0.35 Di-n-octyl phthalate............................................ 0.76C-0.79 0.21X=1.19 0.37X=1.19 Endosulfan sulfate.............................................. 0.39C=0.41 0.12X=2.47 0.63X-1.03 Endrin aldehyde................................................. 0.76C-3.86 0.18X=3.91 0.73X-0.62 Fluoranthene.................................................... 0.81C=1.10 0.22X-0.73 0.28X-0.60 Fluorene........................................................ 0.90C-0.00 0.12X=0.26 0.13X=0.61 Heptachlor...................................................... 0.87C-2.97 0.24X-0.56 0.50X-0.23 Heptachlor epoxide.............................................. 0.92C-1.87 0.33X-0.46 0.28X=0.64 Hexachlorobenzene............................................... 0.74C=0.66 0.18X-0.10 0.43X-0.52 Hexachlorobutadiene............................................. 0.71C-1.01 0.19X=0.92 0.26X=0.49 Hexachloroethane................................................ 0.73C-0.83 0.17X=0.67 0.17X=0.80 Indeno(1,2,3-cd)pyrene.......................................... 0.78C-3.10 0.29X=1.46 0.50X=0.44 Isophorone...................................................... 1.12C=1.41 0.27X=0.77 0.33X=0.26 Naphthalene..................................................... 0.76C=1.58 0.21X-0.41 0.30X-0.68 Nitrobenzene.................................................... 1.09C-3.05 0.19X=0.92 0.27X=0.21 N-Nitrosodi-n-propylamine....................................... 1.12C-6.22 0.27X=0.68 0.44X=0.47 PCB-1260........................................................ 0.81C-10.86 0.35X=3.61 0.43X=1.82 Phenanthrene.................................................... 0.87C-0.06 0.12X=0.57 0.15X=0.25 Pyrene.......................................................... 0.84C-0.16 0.16X=0.06 0.15X=0.31 1,2,4-Trichlorobenzene.......................................... 0.94C-0.79 0.15X=0.85 0.21X=0.39 4-Chloro-3-methylphenol......................................... 0.84C=0.35 0.23X=0.75 0.29X=1.31 2-Chlorophenol.................................................. 0.78C=0.29 0.18X=1.46 0.28X=0.97 2,4-Dichlorophenol.............................................. 0.87C=0.13 0.15X=1.25 0.21X=1.28 2,4-Dimethylphenol.............................................. 0.71C=4.41 0.16X=1.21 0.22X=1.31 2,4-Dinitrophenol............................................... 0.81C-18.04 0.38X=2.36 0.42X=26.29 2-Methyl-4,6-Dinitrophenol...................................... 1.04C-28.04 0.05X=42.29 0.26X=23.10 2-Nitrophenol................................................... 1.07C-1.15 0.16X=1.94 0.27X=2.60 4-Nitrophenol................................................... 0.61C-1.22 0.38X=2.57 0.44X=3.24 Pentachlorophenol............................................... 0.93C=1.99 0.24X=3.03 0.30X=4.33 Phenol.......................................................... 0.43C=1.26 0.26X=0.73 0.35X=0.58 2,4,6-Trichlorophenol........................................... 0.91C-0.18 0.16X=2.22 0.22X=1.81 ---------------------------------------------------------------------------------------------------------------- X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in g/ L.

sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in g/L.

S[prime]= Expected interlaboratory standard deviation of measurements at an average concentration found of X, in g/L.

C= True value for the concentration, in g/L.

X= Average recovery found for measurements of samples containing a concentration of C, in g/L.

a The proper chemical name is 2,2[prime]oxybis(1-chloropropane).

Table 8_Suggested Internal and Surrogate Standards ------------------------------------------------------------------------ Base/neutral fraction Acid fraction ------------------------------------------------------------------------ Aniline-d5................................ 2-Fluorophenol.

Anthracene-d10............................ Pentafluorophenol.

Benzo(a)anthracene-d12.................... Phenol-d5 4,4[prime]-Dibromobiphenyl................ 2-Perfluoromethyl phenol.

4,4[prime]-Dibromooctafluorobiphenyl......

Decafluorobiphenyl........................

2,2 \1\-Difluorobiphenyl.................. ............................

4-Fluoroaniline........................... ............................

1-Fluoronaphthalene....................... ............................

2-Fluoronaphthalene....................... ............................

Naphthalene-d8............................ ............................

Nitrobenzene-d5........................... ............................

2,3,4,5,6-Pentafluorobiphenyl............. ............................

Phenanthrene-d10.......................... ............................

Pyridine-d5............................... ............................

------------------------------------------------------------------------ Table 9_DFTPP Key Masses and Abundance Criteria ------------------------------------------------------------------------ Mass m/z Abundance criteria ------------------------------------------------------------------------ 51 30-60 percent of mass 198.

68 Less than 2 percent of mass 69.

70 Less than 2 percent of mass 69.

127 40-60 percent of mass 198.

197 Less than 1 percent of mass 198.

198 Base peak, 100 percent relative abundance.

199 5-9 percent of mass 198.

275 10-30 percent of mass 198.

365 Greater than 1 percent of mass 198.

441 Present but less than mass 443.

442 Greater than 40 percent of mass 198.

443 17-23 percent of mass 442.

------------------------------------------------------------------------ (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF Attachment 1 to Method 625 Introduction To support measurement of several semivolatile pollutants, EPA has developed this attachment to EPA Method 625. 1 The modifications listed in this attachment are approved only for monitoring wastestreams from the Centralized Waste Treatment Point Source Category (40 CFR Part 437) and the Landfills Point Source Category (40 CFR Part 445). EPA Method 625 (the Method) involves sample extraction with methylene chloride followed by analysis of the extract using either packed or capillary column gas chromatography/mass spectrometry (GC/MS). This attachment addresses the addition of the semivolatile pollutants listed in Tables 1 and 2, to all applicable standard, stock, and spiking solutions utilized for the determination of semivolatile organic compounds by EPA Method 625.

1 EPA Method 625: Base/Neutrals and Acids, 40 CFR Part 136, Appendix A.

1.0EPA METHOD 625 MODIFICATION SUMMARY The additional semivolatile organic compounds listed in Tables 1 and 2 are added to all applicable calibration, spiking, and other solutions utilized in the determination of base/neutral and acid compounds by EPA Method 625. The instrument is to be calibrated with these compounds, using a capillary column, and all procedures and quality control tests stated in the Method must be performed.

2.0SECTION MODIFICATIONS Note: All section and figure numbers in this Attachment reference section and figure numbers in EPA Method 625 unless noted otherwise.

Sections not listed here remain unchanged.

Section 6.7The stock standard solutions described in this section are modified such that the analytes in Tables 1 and 2 of this attachment are required in addition to those specified in the Method.

Section 7.2The calibration standards described in this section are modified to include the analytes in Tables 1 and 2 of this attachment.

Section 8.2The precision and accuracy requirements are modified to include the analytes listed in Tables 1 and 2 of this attachment.

Additional performance criteria are supplied in Table 5 of this attachment.

Section 8.3The matrix spike is modified to include the analytes listed in Tables 1 and 2 of this attachment.

Section 8.4The QC check standard is modified to include the analytes listed in Tables 1 and 2 of this attachment. Additional performance criteria are supplied in Table 5 of this attachment.

Section 16.0Additional method performance information is supplied with this attachment.

Table 1_Base/Neutral Extractables ------------------------------------------------------------------------ Parameter CAS No.

------------------------------------------------------------------------ acetophenone 1............................................. 98-86-2 alpha-terpineol 3.......................................... 98-55-5 aniline 2.................................................. 62-53-3 carbazole 1................................................ 86-74-8 o-cresol 1................................................. 95-48-7 n-decane 1................................................. 124-18-5 2,3-dichloroaniline 1...................................... 608-27-5 n-octadecane 1............................................. 593-45-3 pyridine 2................................................. 110-86-1 ------------------------------------------------------------------------ CAS = Chemical Abstracts Registry.

1 Analysis of this pollutant is approved only for the Centralized Waste Treatment industry.

2 Analysis of this pollutant is approved only for the Centralized Waste Treatment and Landfills industries.

3 Analysis of this pollutant is approved only for the Landfills industry.

Table 2_Acid Extractables ------------------------------------------------------------------------ Parameter CAS No.

------------------------------------------------------------------------ p-cresol 1................................................. 106-44-5 ------------------------------------------------------------------------ CAS = Chemical Abstracts Registry.

1 Analysis of this pollutant is approved only for the Centralized Waste Treatment and Landfills industries.

Table 3_Chromatographic Conditions,\1\ Method Detection Limits (MDLs), and Characteristic m/z's for Base/Neutral Extractables ---------------------------------------------------------------------------------------------------------------- Characteristic m/z's Retention MDL -------------------------------------- Analyte time (min) (g/ Electron impact \2\ L) -------------------------------------- Primary Secondary Secondary ---------------------------------------------------------------------------------------------------------------- pyridine \3\................................... 4.93 4.6 79 52 51 N-Nitro sodimethylamine........................ 4.95 ........... 42 74 44 aniline \3\.................................... 10.82 3.3 93 66 65 Bis(2-chloroethyl)ether........................ 10.94 ........... 93 63 95 n-decane \4\................................... 11.11 5.0 57 ........... ...........

1,3-Dichlorobenzene............................ 11.47 ........... 146 148 113 1,4-Dichlorobenzene............................ 11.62 ........... 146 148 113 1,2-Dichlorobenzene............................ 12.17 ........... 146 148 113 o-creso \1\.................................... 12.48 4.7 108 107 79 Bis(2-chloro- isopropyl)ether.................. 12.51 ........... 45 77 79 acetophenone \4\............................... 12.88 3.4 105 77 51 N-Nitrosodi-n-propylamine...................... 12.97 ........... 130 42 101 Hexachloroethane............................... 13.08 ........... 117 201 199 Nitrobenzene................................... 13.40 ........... 77 123 65 Isophorone..................................... 14.11 ........... 82 95 138 Bis (2-chloro ethoxy)methane................... 14.82 ........... 93 95 123 1,2,4-Trichlorobenzene......................... 15.37 ........... 180 182 145 alpha-terpineol................................ 15.55 5.0 59 ........... ...........

Naphthalene.................................... 15.56 ........... 128 129 127 Hexachlorobutadiene............................ 16.12 ........... 225 223 227 Hexachlorocyclopentadiene...................... 18.47 ........... 237 235 272 2,3-dichloroaniline \4\........................ 18.82 2.5 161 163 90 2-Chloronaphthalene............................ 19.35 ........... 162 164 127 Dimethyl phthalate............................. 20.48 ........... 163 194 164 Acenaphthylene................................. 20.69 ........... 152 151 153 2,6-Dinitrotoluene............................. 20.73 ........... 165 89 121 Acenaphthene................................... 21.30 ........... 154 153 152 2,4-Dinitrotoluene............................. 22.00 ........... 165 63 182 Diethylphthalate............................... 22.74 ........... 149 177 150 4-Chlorophenyl phenyl ether.................... 22.90 ........... 204 206 141 Fluorene....................................... 22.92 ........... 166 165 167 N-Nitro sodiphenylamine........................ 23.35 ........... 169 168 167 4-Bromophenyl phenyl ether..................... 24.44 ........... 248 250 141 Hexachlorobenzene.............................. 24.93 ........... 284 142 249 n-octadecane \4\............................... 25.39 2.0 57 ........... ...........

Phenanthrene................................... 25.98 ........... 178 179 176 Anthracene..................................... 26.12 ........... 178 179 176 Carbazole \4\.................................. 26.66 4.0 167 ........... ...........

Dibutyl phthalate.............................. 27.84 ........... 149 150 104 Fluoranthene................................... 29.82 ........... 202 101 100 Benzidine...................................... 30.26 ........... 184 92 185 Pyrene......................................... 30.56 ........... 202 101 100 Butyl benzyl phthalate......................... 32.63 ........... 149 91 206 3,3'-Dichlorobenzidine......................... 34.28 ........... 252 254 126 Benzo(a)anthracene............................. 34.33 ........... 228 229 226 Bis(2-ethyl hexyl)phthalate.................... 34.36 ........... 149 167 279 Chrysene....................................... 34.44 ........... 228 226 229 Di-n-octyl-phthalate........................... 36.17 ........... 149 ........... ...........

Benzo(b)fluoranthene........................... 37.90 ........... 252 253 125 Benzo(k)fluoranthene........................... 37.97 ........... 252 253 125 Benzo(a)pyrene................................. 39.17 ........... 252 253 125 Dibenzo(a,h) anthracene........................ 44.91 ........... 278 139 279 Indeno(1,2,3-c,d)pyrene........................ 45.01 ........... 276 138 277 Benzo(ghi)perylene............................. 46.56 ........... 276 138 277 ---------------------------------------------------------------------------------------------------------------- \1\ The data presented in this table were obtained under the following conditions: Column_30 5 meters x 0.25 .02 mm i.d., 94% methyl, 5% phenyl, 1% vinyl, bonded phase fused silica capillary column (DB-5).

Temperature program_Five minutes at 30 C; 30-280 C at 8 C per minute; isothermal at 280 C until benzo(ghi)perylene elutes.

Gas velocity_30 5 cm/sec at 30 C.

\2\ Retention times are from Method 1625, Revision C, using a capillary column, and are intended to be consistent for all analytes in Tables 4 and 5 of this attachment.

\3\ Analysis of this pollutant is approved only for the Centralized Waste Treatment and Landfills industries.

\4\ Analysis of this pollutant is approved only for the Centralized Waste Treatment industry.

Table 4_Chromatographic Conditions,\1\ Method Detection Limits (MDLs), and Characteristic m/z's for Acid Extractables ---------------------------------------------------------------------------------------------------------------- Characteristic m/z's Retention MDL -------------------------------------- Analyte time \2\ (g/ Electron impact (min) L) -------------------------------------- Primary Secondary Secondary ---------------------------------------------------------------------------------------------------------------- Phenol......................................... 10.76 ........... 94 65 66 2-Chlorophenol................................. 11.08 ........... 128 64 130 p-cresol \3\................................... 12.92 7.8 108 107 77 2-Nitrophenol.................................. 14.38 ........... 139 65 109 2,4-Dimethylphenol............................. 14.54 ........... 122 107 121 2,4-Dichlorophenol............................. 15.12 ........... 162 164 98 4-Chloro-3-methylphenol........................ 16.83 ........... 142 107 144 2,4,6-Trichlorophenol.......................... 18.80 ........... 196 198 200 2,4-Dinitrophenol.............................. 21.51 ........... 184 63 154 4-Nitrophenol.................................. 21.77 ........... 65 139 109 2-Methyl-4,6-dinitrophenol..................... 22.83 ........... 198 182 77 Pentachlorophenol.............................. 25.52 ........... 266 264 268 ---------------------------------------------------------------------------------------------------------------- \1\ The data presented in this table were obtained under the following conditions: Column_30 5 meters x 0.25 .02 mm i.d., 94% methyl, 5% phenyl, 1% vinyl silicone bonded phase fused silica capillary column (DB-5).

Temperature program_Five minutes at 30 C; 30-280 C at 8 C per minute; isothermal at 280 C until benzo(ghi)perylene elutes.

Gas velocity_30 5 cm/sec at 30 C \2\ Retention times are from EPA Method 1625, Revision C, using a capillary column, and are intended to be consistent for all analytes in Tables 3 and 4 of this attachment.

\3\ Analysis of this pollutant is approved only for the Centralized Waste Treatment and Landfills industries.

Table 5_QC Acceptance Criteria ---------------------------------------------------------------------------------------------------------------- Test Limits for conclusion s Range for X Range for Analyte (g/ (g/ (g/ P, Ps(%) L) L) L) --------------------------------------------------------------------------------------------------------------- acetophenone \1\.......................................... 100 51 23-254 61-144 alpha-terpineol........................................... 100 47 46-163 58-156 aniline \2\............................................... 100 71 15-278 46-134 carbazole \1\............................................. 100 17 79-111 73-131 o-cresol \1\.............................................. 100 23 30-146 55-126 p-cresol \2\.............................................. 100 22 11-617 76-107 n-decane \1\.............................................. 100 70 D-651 D-ns 2,3-dichloroaniline \1\................................... 100 13 40-160 68-134 n-octadecane \1\.......................................... 100 10 52-147 65-123 pyridine \2\.............................................. 100 ns 7-392 33-158 ---------------------------------------------------------------------------------------------------------------- s = Standard deviation for four recovery measurements, in g/L (Section 8.2) X = Average recovery for four recovery measurements in g/L (Section 8.2) P,Ps = Percent recovery measured (Section 8.3, Section 8.4) D = Detected; result must be greater than zero.

ns = no specification; limit is outside the range that can be measured reliably.

\1\ Analysis of this pollutant is approved only for the Centralized Waste Treatment industry.

\2\ Analysis of this pollutant is approved only for the Centralized Waste Treatment and Landfills industries.

Method 1613, Revision B Tetra- Through Octa-Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS 1.0Scope and Application 1.1This method is for determination of tetra- through octa-chlorinated dibenzo-p-dioxins (CDDs) and dibenzofurans (CDFs) in water, soil, sediment, sludge, tissue, and other sample matrices by high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). The method is for use in EPA's data gathering and monitoring programs associated with the Clean Water Act, the Resource Conservation and Recovery Act, the Comprehensive Environmental Response, Compensation and Liability Act, and the Safe Drinking Water Act. The method is based on a compilation of EPA, industry, commercial laboratory, and academic methods (References 16).

1.2The seventeen 2,3,7,8-substituted CDDs/CDFs listed in Table 1 may be determined by this method. Specifications are also provided for separate determination of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2,3,7,8-TCDD) and 2,3,7,8-tetrachloro-dibenzofuran (2,3,7,8-TCDF).

1.3The detection limits and quantitation levels in this method are usually dependent on the level of interferences rather than instrumental limitations. The minimum levels (MLs) in Table 2 are the levels at which the CDDs/CDFs can be determined with no interferences present. The Method Detection Limit (MDL) for 2,3,7,8-TCDD has been determined as 4.4 pg/L (parts-per-quadrillion) using this method.

1.4The GC/MS portions of this method are for use only by analysts experienced with HRGC/HRMS or under the close supervision of such qualified persons. Each laboratory that uses this method must demonstrate the ability to generate acceptable results using the procedure in Section 9.2.

1.5This method is performance-based. The analyst is permitted to modify the method to overcome interferences or lower the cost of measurements, provided that all performance criteria in this method are met. The requirements for establishing method equivalency are given in Section 9.1.2.

1.6Any modification of this method, beyond those expressly permitted, shall be considered a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

2.0Summary of Method Flow charts that summarize procedures for sample preparation, extraction, and analysis are given in Figure 1 for aqueous and solid samples, Figure 2 for multi-phase samples, and Figure 3 for tissue samples.

2.1Extraction.

2.1.1Aqueous samples (samples containing less than 1% solids)Stable isotopically labeled analogs of 15 of the 2,3,7,8-substituted CDDs/CDFs are spiked into a 1 L sample, and the sample is extracted by one of three procedures: 2.1.1.1Samples containing no visible particles are extracted with methylene chloride in a separatory funnel or by the solid-phase extraction technique summarized in Section 2.1.1.3. The extract is concentrated for cleanup.

2.1.1.2Samples containing visible particles are vacuum filtered through a glass-fiber filter. The filter is extracted in a Soxhlet/Dean-Stark (SDS) extractor (Reference 7), and the filtrate is extracted with methylene chloride in a separatory funnel. The methylene chloride extract is concentrated and combined with the SDS extract prior to cleanup.

2.1.1.3The sample is vacuum filtered through a glass-fiber filter on top of a solid-phase extraction (SPE) disk. The filter and disk are extracted in an SDS extractor, and the extract is concentrated for cleanup.

2.1.2Solid, semi-solid, and multi-phase samples (but not tissue)The labeled compounds are spiked into a sample containing 10 g (dry weight) of solids. Samples containing multiple phases are pressure filtered and any aqueous liquid is discarded. Coarse solids are ground or homogenized. Any non-aqueous liquid from multi-phase samples is combined with the solids and extracted in an SDS extractor. The extract is concentrated for cleanup.

2.1.3Fish and other tissueThe sample is extracted by one of two procedures: 2.1.3.1Soxhlet or SDS extractionA 20 g aliquot of sample is homogenized, and a 10 g aliquot is spiked with the labeled compounds. The sample is mixed with sodium sulfate, allowed to dry for 1224 hours, and extracted for 1824 hours using methylene chloride:hexane (1:1) in a Soxhlet extractor. The extract is evaporated to dryness, and the lipid content is determined.

2.1.3.2HCl digestionA 20 g aliquot is homogenized, and a 10 g aliquot is placed in a bottle and spiked with the labeled compounds. After equilibration, 200 mL of hydrochloric acid and 200 mL of methylene chloride:hexane (1:1) are added, and the bottle is agitated for 1224 hours. The extract is evaporated to dryness, and the lipid content is determined.

2.2After extraction, 37Cl4-labeled 2,3,7,8-TCDD is added to each extract to measure the efficiency of the cleanup process. Sample cleanups may include back-extraction with acid and/or base, and gel permeation, alumina, silica gel, Florisil and activated carbon chromatography.

High-performance liquid chromatography (HPLC) can be used for further isolation of the 2,3,7,8-isomers or other specific isomers or congeners.

Prior to the cleanup procedures cited above, tissue extracts are cleaned up using an anthropogenic isolation column, a batch silica gel adsorption, or sulfuric acid and base back-extraction, depending on the tissue extraction procedure used.

2.3After cleanup, the extract is concentrated to near dryness.

Immediately prior to injection, internal standards are added to each extract, and an aliquot of the extract is injected into the gas chromatograph. The analytes are separated by the GC and detected by a high-resolution (10,000) mass spectrometer. Two exact m/z's are monitored for each analyte.

2.4An individual CDD/CDF is identified by comparing the GC retention time and ion-abundance ratio of two exact m/z's with the corresponding retention time of an authentic standard and the theoretical or acquired ion-abundance ratio of the two exact m/z's. The non-2,3,7,8 substituted isomers and congeners are identified when retention times and ion-abundance ratios agree within predefined limits. Isomer specificity for 2,3,7,8-TCDD and 2,3,7,8-TCDF is achieved using GC columns that resolve these isomers from the other tetra-isomers.

2.5Quantitative analysis is performed using selected ion current profile (SICP) areas, in one of three ways: 2.5.1For the 15 2,3,7,8-substituted CDDs/CDFs with labeled analogs (see Table 1), the GC/MS system is calibrated, and the concentration of each compound is determined using the isotope dilution technique.

2.5.2For 1,2,3,7,8,9-HxCDD, OCDF, and the labeled compounds, the GC/MS system is calibrated and the concentration of each compound is determined using the internal standard technique.

2.5.3For non-2,3,7,8-substituted isomers and for all isomers at a given level of chlorination (i.e., total TCDD), concentrations are determined using response factors from calibration of the CDDs/CDFs at the same level of chlorination.

2.6The quality of the analysis is assured through reproducible calibration and testing of the extraction, cleanup, and GC/MS systems.

3.0Definitions Definitions are given in the glossary at the end of this method.

4.0Contamination and Interferences 4.1Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or elevated baselines causing misinterpretation of chromatograms (References 89). Specific selection of reagents and purification of solvents by distillation in all-glass systems may be required. Where possible, reagents are cleaned by extraction or solvent rinse.

4.2Proper cleaning of glassware is extremely important, because glassware may not only contaminate the samples but may also remove the analytes of interest by adsorption on the glass surface.

4.2.1Glassware should be rinsed with solvent and washed with a detergent solution as soon after use as is practical. Sonication of glassware containing a detergent solution for approximately 30 seconds may aid in cleaning. Glassware with removable parts, particularly separatory funnels with fluoropolymer stopcocks, must be disassembled prior to detergent washing.

4.2.2After detergent washing, glassware should be rinsed immediately, first with methanol, then with hot tap water. The tap water rinse is followed by another methanol rinse, then acetone, and then methylene chloride.

4.2.3Do not bake reusable glassware in an oven as a routine part of cleaning. Baking may be warranted after particularly dirty samples are encountered but should be minimized, as repeated baking of glassware may cause active sites on the glass surface that will irreversibly adsorb CDDs/CDFs.

4.2.4Immediately prior to use, the Soxhlet apparatus should be pre-extracted with toluene for approximately three hours (see Sections 12.3.1 through 12.3.3). Separatory funnels should be shaken with methylene chloride/toluene (80/20 mixture) for two minutes, drained, and then shaken with pure methylene chloride for two minutes.

4.3All materials used in the analysis shall be demonstrated to be free from interferences by running reference matrix method blanks initially and with each sample batch (samples started through the extraction process on a given 12-hour shift, to a maximum of 20 samples).

4.3.1The reference matrix must simulate, as closely as possible, the sample matrix under test. Ideally, the reference matrix should not contain the CDDs/CDFs in detectable amounts, but should contain potential interferents in the concentrations expected to be found in the samples to be analyzed. For example, a reference sample of human adipose tissue containing pentachloronaphthalene can be used to exercise the cleanup systems when samples containing pentachloronaphthalene are expected.

4.3.2When a reference matrix that simulates the sample matrix under test is not available, reagent water (Section 7.6.1) can be used to simulate water samples; playground sand (Section 7.6.2) or white quartz sand (Section 7.3.2) can be used to simulate soils; filter paper (Section 7.6.3) can be used to simulate papers and similar materials; and corn oil (Section 7.6.4) can be used to simulate tissues.

4.4Interferences coextracted from samples will vary considerably from source to source, depending on the diversity of the site being sampled.

Interfering compounds may be present at concentrations several orders of magnitude higher than the CDDs/CDFs. The most frequently encountered interferences are chlorinated biphenyls, methoxy biphenyls, hydroxydiphenyl ethers, benzylphenyl ethers, polynuclear aromatics, and pesticides. Because very low levels of CDDs/CDFs are measured by this method, the elimination of interferences is essential. The cleanup steps given in Section 13 can be used to reduce or eliminate these interferences and thereby permit reliable determination of the CDDs/CDFs at the levels shown in Table 2.

4.5Each piece of reusable glassware should be numbered to associate that glassware with the processing of a particular sample. This will assist the laboratory in tracking possible sources of contamination for individual samples, identifying glassware associated with highly contaminated samples that may require extra cleaning, and determining when glassware should be discarded.

4.6Cleanup of tissueThe natural lipid content of tissue can interfere in the analysis of tissue samples for the CDDs/CDFs. The lipid contents of different species and portions of tissue can vary widely. Lipids are soluble to varying degrees in various organic solvents and may be present in sufficient quantity to overwhelm the column chromatographic cleanup procedures used for cleanup of sample extracts. Lipids must be removed by the lipid removal procedures in Section 13.7, followed by alumina (Section 13.4) or Florisil (Section 13.8), and carbon (Section 13.5) as minimum additional cleanup steps. If chlorodiphenyl ethers are detected, as indicated by the presence of peaks at the exact m/z's monitored for these interferents, alumina and/or Florisil cleanup must be employed to eliminate these interferences.

5.0Safety 5.1The toxicity or carcinogenicity of each compound or reagent used in this method has not been precisely determined; however, each chemical compound should be treated as a potential health hazard. Exposure to these compounds should be reduced to the lowest possible level.

5.1.1The 2,3,7,8-TCDD isomer has been found to be acnegenic, carcinogenic, and teratogenic in laboratory animal studies. It is soluble in water to approximately 200 ppt and in organic solvents to 0.14%. On the basis of the available toxicological and physical properties of 2,3,7,8-TCDD, all of the CDDs/CDFs should be handled only by highly trained personnel thoroughly familiar with handling and cautionary procedures and the associated risks.

5.1.2It is recommended that the laboratory purchase dilute standard solutions of the analytes in this method. However, if primary solutions are prepared, they shall be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator shall be worn when high concentrations are handled.

5.2The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material safety data sheets (MSDSs) should also be made available to all personnel involved in these analyses. It is also suggested that the laboratory perform personal hygiene monitoring of each analyst who uses this method and that the results of this monitoring be made available to the analyst.

Additional information on laboratory safety can be found in References 1013. The references and bibliography at the end of Reference 13 are particularly comprehensive in dealing with the general subject of laboratory safety.

5.3The CDDs/CDFs and samples suspected to contain these compounds are handled using essentially the same techniques employed in handling radioactive or infectious materials. Well-ventilated, controlled access laboratories are required. Assistance in evaluating the health hazards of particular laboratory conditions may be obtained from certain consulting laboratories and from State Departments of Health or Labor, many of which have an industrial health service. The CDDs/CDFs are extremely toxic to laboratory animals. Each laboratory must develop a strict safety program for handling these compounds. The practices in References 2 and 14 are highly recommended.

5.3.1FacilityWhen finely divided samples (dusts, soils, dry chemicals) are handled, all operations (including removal of samples from sample containers, weighing, transferring, and mixing) should be performed in a glove box demonstrated to be leak tight or in a fume hood demonstrated to have adequate air flow. Gross losses to the laboratory ventilation system must not be allowed. Handling of the dilute solutions normally used in analytical and animal work presents no inhalation hazards except in the case of an accident.

5.3.2Protective equipmentDisposable plastic gloves, apron or lab coat, safety glasses or mask, and a glove box or fume hood adequate for radioactive work should be used. During analytical operations that may give rise to aerosols or dusts, personnel should wear respirators equipped with activated carbon filters. Eye protection equipment (preferably full face shields) must be worn while working with exposed samples or pure analytical standards. Latex gloves are commonly used to reduce exposure of the hands. When handling samples suspected or known to contain high concentrations of the CDDs/CDFs, an additional set of gloves can also be worn beneath the latex gloves.

5.3.3TrainingWorkers must be trained in the proper method of removing contaminated gloves and clothing without contacting the exterior surfaces.

5.3.4Personal hygieneHands and forearms should be washed thoroughly after each manipulation and before breaks (coffee, lunch, and shift).

5.3.5ConfinementIsolated work areas posted with signs, segregated glassware and tools, and plastic absorbent paper on bench tops will aid in confining contamination.

5.3.6Effluent vaporsThe effluents of sample splitters from the gas chromatograph (GC) and from roughing pumps on the mass spectrometer (MS) should pass through either a column of activated charcoal or be bubbled through a trap containing oil or high-boiling alcohols to condense CDD/CDF vapors.

5.3.7Waste HandlingGood technique includes minimizing contaminated waste. Plastic bag liners should be used in waste cans. Janitors and other personnel must be trained in the safe handling of waste.

5.3.8Decontamination 5.3.8.1Decontamination of personnelUse any mild soap with plenty of scrubbing action.

5.3.8.2Glassware, tools, and surfacesChlorothene NU Solvent is the least toxic solvent shown to be effective. Satisfactory cleaning may be accomplished by rinsing with Chlorothene, then washing with any detergent and water. If glassware is first rinsed with solvent, then the dish water may be disposed of in the sewer. Given the cost of disposal, it is prudent to minimize solvent wastes.

5.3.9LaundryClothing known to be contaminated should be collected in plastic bags. Persons who convey the bags and launder the clothing should be advised of the hazard and trained in proper handling. The clothing may be put into a washer without contact if the launderer knows of the potential problem. The washer should be run through a cycle before being used again for other clothing.

5.3.10Wipe testsA useful method of determining cleanliness of work surfaces and tools is to wipe the surface with a piece of filter paper.

Extraction and analysis by GC with an electron capture detector (ECD) can achieve a limit of detection of 0.1 g per wipe; analysis using this method can achieve an even lower detection limit. Less than 0.1 g per wipe indicates acceptable cleanliness; anything higher warrants further cleaning. More than 10 g on a wipe constitutes an acute hazard and requires prompt cleaning before further use of the equipment or work space, and indicates that unacceptable work practices have been employed.

5.3.11Table or wrist-action shakerThe use of a table or wrist-action shaker for extraction of tissues presents the possibility of breakage of the extraction bottle and spillage of acid and flammable organic solvent. A secondary containment system around the shaker is suggested to prevent the spread of acid and solvents in the event of such a breakage. The speed and intensity of shaking action should also be adjusted to minimize the possibility of breakage.

6.0Apparatus and Materials Note: Brand names, suppliers, and part numbers are for illustration purposes only and no endorsement is implied. Equivalent performance may be achieved using apparatus and materials other than those specified here. Meeting the performance requirements of this method is the responsibility of the laboratory.

6.1Sampling Equipment for Discrete or Composite Sampling 6.1.1Sample bottles and caps 6.1.1.1Liquid samples (waters, sludges and similar materials containing 5% solids or less)Sample bottle, amber glass, 1.1 L minimum, with screw cap.

6.1.1.2Solid samples (soils, sediments, sludges, paper pulps, filter cake, compost, and similar materials that contain more than 5% solids)Sample bottle, wide mouth, amber glass, 500 mL minimum.

6.1.1.3If amber bottles are not available, samples shall be protected from light.

6.1.1.4Bottle capsThreaded to fit sample bottles. Caps shall be lined with fluoropolymer.

6.1.1.5Cleaning 6.1.1.5.1Bottles are detergent water washed, then solvent rinsed before use.

6.1.1.5.2Liners are detergent water washed, rinsed with reagent water (Section 7.6.1) followed by solvent, and baked at approximately 200 C for a minimum of 1 hour prior to use.

6.1.2Compositing equipmentAutomatic or manual compositing system incorporating glass containers cleaned per bottle cleaning procedure above. Only glass or fluoropolymer tubing shall be used. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used in the pump only. Before use, the tubing shall be thoroughly rinsed with methanol, followed by repeated rinsing with reagent water to minimize sample contamination. An integrating flow meter is used to collect proportional composite samples.

6.2Equipment for Glassware CleaningLaboratory sink with overhead fume hood.

6.3Equipment for Sample Preparation 6.3.1Laboratory fume hood of sufficient size to contain the sample preparation equipment listed below.

6.3.2Glove box (optional).

6.3.3Tissue homogenizerVirTis Model 45 Macro homogenizer (American Scientific Products H3515, or equivalent) with stainless steel Macro-shaft and Turbo-shear blade.

6.3.4Meat grinderHobart, or equivalent, with 35 mm holes in inner plate.

6.3.5Equipment for determining percent moisture 6.3.5.1OvenCapable of maintaining a temperature of 110 5 C.

6.3.5.2Dessicator.

6.3.6Balances 6.3.6.1AnalyticalCapable of weighing 0.1 mg.

6.3.6.2Top loadingCapable of weighing 10 mg.

6.4Extraction Apparatus 6.4.1Water samples 6.4.1.1pH meter, with combination glass electrode.

6.4.1.2pH paper, wide range (Hydrion Papers, or equivalent).

6.4.1.3Graduated cylinder, 1 L capacity.

6.4.1.4Liquid/liquid extractionSeparatory funnels, 250 mL, 500 mL, and 2000 mL, with fluoropolymer stopcocks.

6.4.1.5Solid-phase extraction 6.4.1.5.1One liter filtration apparatus, including glass funnel, glass frit support, clamp, adapter, stopper, filtration flask, and vacuum tubing (Figure 4). For wastewater samples, the apparatus should accept 90 or 144 mm disks. For drinking water or other samples containing low solids, smaller disks may be used.

6.4.1.5.2Vacuum source capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum gauge.

6.4.1.5.3Glass-fiber filterWhatman GMF 150 (or equivalent), 1 micron pore size, to fit filtration apparatus in Section 6.4.1.5.1.

6.4.1.5.4Solid-phase extraction disk containing octadecyl (C18) bonded silica uniformly enmeshed in an inert matrixFisher Scientific 14378F (or equivalent), to fit filtration apparatus in Section 6.4.1.5.1.

6.4.2Soxhlet/Dean-Stark (SDS) extractor (Figure 5)For filters and solid/sludge samples.

6.4.2.1Soxhlet50 mm ID, 200 mL capacity with 500 mL flask (Cal-Glass LG6900, or equivalent, except substitute 500 mL round-bottom flask for 300 mL flat-bottom flask).

6.4.2.2Thimble43 123 to fit Soxhlet (Cal-Glass LG6901122, or equivalent).

6.4.2.3Moisture trapDean Stark or Barret with fluoropolymer stopcock, to fit Soxhlet.

6.4.2.4Heating mantleHemispherical, to fit 500 mL round-bottom flask (Cal-Glass LG8801112, or equivalent).

6.4.2.5Variable transformerPowerstat (or equivalent), 110 volt, 10 amp.

6.4.3Apparatus for extraction of tissue.

6.4.3.1Bottle for extraction (if digestion/extraction using HCl is used) 500600 mL wide-mouth clear glass, with fluoropolymer-lined cap.

6.4.3.2Bottle for back-extraction100200 mL narrow-mouth clear glass with fluoropolymer-lined cap.

6.4.3.3Mechanical shakerWrist-action or platform-type rotary shaker that produces vigorous agitation (Sybron Thermolyne Model LE Big Bill rotator/shaker, or equivalent).

6.4.3.4Rack attached to shaker table to permit agitation of four to nine samples simultaneously.

6.4.4Beakers400500 mL.

6.4.5SpatulasStainless steel.

6.5Filtration Apparatus.

6.5.1Pyrex glass woolSolvent-extracted by SDS for three hours minimum.

Note: Baking glass wool may cause active sites that will irreversibly adsorb CDDs/CDFs.

6.5.2Glass funnel125250 mL.

6.5.3Glass-fiber filter paperWhatman GF/D (or equivalent), to fit glass funnel in Section 6.5.2.

6.5.4Drying column1520 mm ID Pyrex chromatographic column equipped with coarse-glass frit or glass-wool plug.

6.5.5Buchner funnel15 cm.

6.5.6Glass-fiber filter paperto fit Buchner funnel in Section 6.5.5.

6.5.7Filtration flasks1.52.0 L, with side arm.

6.5.8Pressure filtration apparatusMillipore YT30 142 HW, or equivalent.

6.6Centrifuge Apparatus.

6.6.1CentrifugeCapable of rotating 500 mL centrifuge bottles or 15 mL centrifuge tubes at 5,000 rpm minimum.

6.6.2Centrifuge bottles500 mL, with screw-caps, to fit centrifuge.

6.6.3Centrifuge tubes1215 mL, with screw-caps, to fit centrifuge.

6.7Cleanup Apparatus.

6.7.1Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc, Columbia, MO, Model GPC Autoprep 1002, or equivalent).

6.7.1.1Column600700 mm long 25 mm ID, packed with 70 g of SX3 Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).

6.7.1.2Syringe10 mL, with Luer fitting.

6.7.1.3Syringe filter holderstainless steel, and glass-fiber or fluoropolymer filters (Gelman 4310, or equivalent).

6.7.1.4UV detectors254 nm, preparative or semi-preparative flow cell (Isco, Inc., Type 6; Schmadzu, 5 mm path length; Beckman-Altex 152W, 8 L micro-prep flow cell, 2 mm path; Pharmacia UV1, 3 mm flow cell; LDC Milton-Roy UV3, monitor #1203; or equivalent).

6.7.2Reverse-phase high-performance liquid chromatograph.

6.7.2.1Column oven and detectorPerkin-Elmer Model LC65T (or equivalent) operated at 0.02 AUFS at 235 nm.

6.7.2.2InjectorRheodyne 7120 (or equivalent) with 50 L sample loop.

6.7.2.3ColumnTwo 6.2 mm 250 mm Zorbax-ODS columns in series (DuPont Instruments Division, Wilmington, DE, or equivalent), operated at 50 C with 2.0 mL/min methanol isocratic effluent.

6.7.2.4PumpAltex 110A (or equivalent).

6.7.3Pipets.

6.7.3.1Disposable, pasteur150 mm long 5-mm ID (Fisher Scientific 136786A, or equivalent).

6.7.3.2Disposable, serological10 mL (6 mm ID).

6.7.4Glass chromatographic columns.

6.7.4.1150 mm long 8-mm ID, (Kontes K420155, or equivalent) with coarse-glass frit or glass-wool plug and 250 mL reservoir.

6.7.4.2200 mm long 15 mm ID, with coarse-glass frit or glass-wool plug and 250 mL reservoir.

6.7.4.3300 mm long 25 mm ID, with 300 mL reservoir and glass or fluoropolymer stopcock.

6.7.5Stirring apparatus for batch silica cleanup of tissue extracts.

6.7.5.1Mechanical stirrerCorning Model 320, or equivalent.

6.7.5.2Bottle500600 mL wide-mouth clear glass.

6.7.6OvenFor baking and storage of adsorbents, capable of maintaining a constant temperature (5 C) in the range of 105250 C.

6.8Concentration Apparatus.

6.8.1Rotary evaporatorBuchi/Brinkman-American Scientific No. E504510 or equivalent, equipped with a variable temperature water bath.

6.8.1.1Vacuum source for rotary evaporator equipped with shutoff valve at the evaporator and vacuum gauge.

6.8.1.2A recirculating water pump and chiller are recommended, as use of tap water for cooling the evaporator wastes large volumes of water and can lead to inconsistent performance as water temperatures and pressures vary.

6.8.1.3Round-bottom flask100 mL and 500 mL or larger, with ground-glass fitting compatible with the rotary evaporator.

6.8.2Kuderna-Danish (K-D) Concentrator.

6.8.2.1Concentrator tube10 mL, graduated (Kontes K5700501025, or equivalent) with calibration verified. Ground-glass stopper (size 19/22 joint) is used to prevent evaporation of extracts.

6.8.2.2Evaporation flask500 mL (Kontes K5700010500, or equivalent), attached to concentrator tube with springs (Kontes K6627500012 or equivalent).

6.8.2.3Snyder columnThree-ball macro (Kontes K5030000232, or equivalent).

6.8.2.4Boiling chips.

6.8.2.4.1Glass or silicon carbideApproximately 10/40 mesh, extracted with methylene chloride and baked at 450 C for one hour minimum.

6.8.2.4.2Fluoropolymer (optional)Extracted with methylene chloride.

6.8.2.5Water bathHeated, with concentric ring cover, capable of maintaining a temperature within 2 C, installed in a fume hood.

6.8.3Nitrogen blowdown apparatusEquipped with water bath controlled in the range of 3060 C (N-Evap, Organomation Associates, Inc., South Berlin, MA, or equivalent), installed in a fume hood.

6.8.4Sample vials.

6.8.4.1Amber glass25 mL with fluoropolymer-lined screw-cap.

6.8.4.2Glass0.3 mL, conical, with fluoropolymer-lined screw or crimp cap.

6.9Gas ChromatographShall have splitless or on-column injection port for capillary column, temperature program with isothermal hold, and shall meet all of the performance specifications in Section 10.

6.9.1GC column for CDDs/CDFs and for isomer specificity for 2,3,7,8-TCDD60 5 m long 0.32 0.02 mm ID; 0.25 m 5% phenyl, 94% methyl, 1% vinyl silicone bonded-phase fused-silica capillary column (J&W DB5, or equivalent).

6.9.2GC column for isomer specificity for 2,3,7,8-TCDF30 5 m long 0.32 0.02 mm ID; 0.25 m bonded-phase fused-silica capillary column (J&W DB225, or equivalent).

6.10Mass Spectrometer2840 eV electron impact ionization, shall be capable of repetitively selectively monitoring 12 exact m/z's minimum at high resolution (10,000) during a period of approximately one second, and shall meet all of the performance specifications in Section 10.

6.11GC/MS InterfaceThe mass spectrometer (MS) shall be interfaced to the GC such that the end of the capillary column terminates within 1 cm of the ion source but does not intercept the electron or ion beams.

6.12Data SystemCapable of collecting, recording, and storing MS data.

7.0Reagents and Standards 7.1pH Adjustment and Back-Extraction.

7.1.1Potassium hydroxideDissolve 20 g reagent grade KOH in 100 mL reagent water.

7.1.2Sulfuric acidReagent grade (specific gravity 1.84).

7.1.3Hydrochloric acidReagent grade, 6N.

7.1.4Sodium chlorideReagent grade, prepare at 5% (w/v) solution in reagent water.

7.2Solution Drying and Evaporation.

7.2.1Solution dryingSodium sulfate, reagent grade, granular, anhydrous (Baker 3375, or equivalent), rinsed with methylene chloride (20 mL/g), baked at 400 C for one hour minimum, cooled in a dessicator, and stored in a pre-cleaned glass bottle with screw-cap that prevents moisture from entering. If, after heating, the sodium sulfate develops a noticeable grayish cast (due to the presence of carbon in the crystal matrix), that batch of reagent is not suitable for use and should be discarded.

Extraction with methylene chloride (as opposed to simple rinsing) and baking at a lower temperature may produce sodium sulfate that is suitable for use.

7.2.2Tissue dryingSodium sulfate, reagent grade, powdered, treated and stored as above.

7.2.3Prepurified nitrogen.

7.3Extraction.

7.3.1SolventsAcetone, toluene, cyclohexane, hexane, methanol, methylene chloride, and nonane; distilled in glass, pesticide quality, lot-certified to be free of interferences.

7.3.2White quartz sand, 60/70 meshFor Soxhlet/Dean-Stark extraction (Aldrich Chemical, Cat. No. 274379, or equivalent). Bake at 450 C for four hours minimum.

7.4GPC Calibration SolutionPrepare a solution containing 300 mg/mL corn oil, 15 mg/mL bis(2-ethylhexyl) phthalate, 1.4 mg/mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.

7.5Adsorbents for Sample Cleanup.

7.5.1Silica gel.

7.5.1.1Activated silica gel100200 mesh, Supelco 13651 (or equivalent), rinsed with methylene chloride, baked at 180 C for a minimum of one hour, cooled in a dessicator, and stored in a precleaned glass bottle with screw-cap that prevents moisture from entering.

7.5.1.2Acid silica gel (30% w/w)Thoroughly mix 44.0 g of concentrated sulfuric acid with 100.0 g of activated silica gel in a clean container.

Break up aggregates with a stirring rod until a uniform mixture is obtained. Store in a bottle with a fluoropolymer-lined screw-cap.

7.5.1.3Basic silica gelThoroughly mix 30 g of 1N sodium hydroxide with 100 g of activated silica gel in a clean container. Break up aggregates with a stirring rod until a uniform mixture is obtained. Store in a bottle with a fluoropolymer-lined screw-cap.

7.5.1.4Potassium silicate.

7.5.1.4.1Dissolve 56 g of high purity potassium hydroxide (Aldrich, or equivalent) in 300 mL of methanol in a 7501000 mL flat-bottom flask.

7.5.1.4.2Add 100 g of silica gel and a stirring bar, and stir on a hot plate at 6070 C for one to two hours.

7.5.1.4.3Decant the liquid and rinse the potassium silicate twice with 100 mL portions of methanol, followed by a single rinse with 100 mL of methylene chloride.

7.5.1.4.4Spread the potassium silicate on solvent-rinsed aluminum foil and dry for two to four hours in a hood.

7.5.1.4.5Activate overnight at 200250 C.

7.5.2AluminaEither one of two types of alumina, acid or basic, may be used in the cleanup of sample extracts, provided that the laboratory can meet the performance specifications for the recovery of labeled compounds described in Section 9.3. The same type of alumina must be used for all samples, including those used to demonstrate initial precision and recovery (Section 9.2) and ongoing precision and recovery (Section 15.5).

7.5.2.1Acid aluminaSupelco 199966C (or equivalent). Activate by heating to 130 C for a minimum of 12 hours.

7.5.2.2Basic aluminaSupelco 199446C (or equivalent). Activate by heating to 600 C for a minimum of 24 hours. Alternatively, activate by heating in a tube furnace at 650700 C under an air flow rate of approximately 400 cc/minute. Do not heat over 700 C, as this can lead to reduced capacity for retaining the analytes. Store at 130 C in a covered flask.

Use within five days of baking.

7.5.3Carbon.

7.5.3.1Carbopak C(Supelco 10258, or equivalent).

7.5.3.2Celite 545(Supelco 20199, or equivalent).

7.5.3.3Thoroughly mix 9.0 g Carbopak C and 41.0 g Celite 545 to produce an 18% w/w mixture. Activate the mixture at 130 C for a minimum of six hours. Store in a dessicator.

7.5.4Anthropogenic isolation columnPack the column in Section 6.7.4.3 from bottom to top with the following: 7.5.4.12 g silica gel (Section 7.5.1.1).

7.5.4.22 g potassium silicate (Section 7.5.1.4).

7.5.4.32 g granular anhydrous sodium sulfate (Section 7.2.1).

7.5.4.410 g acid silica gel (Section 7.5.1.2).

7.5.4.52 g granular anhydrous sodium sulfate.

7.5.5Florisil column.

7.5.5.1Florisil60100 mesh, Floridin Corp (or equivalent). Soxhlet extract in 500 g portions for 24 hours.

7.5.5.2Insert a glass wool plug into the tapered end of a graduated serological pipet (Section 6.7.3.2). Pack with 1.5 g (approx 2 mL) of Florisil topped with approx 1 mL of sodium sulfate (Section 7.2.1) and a glass wool plug.

7.5.5.3Activate in an oven at 130150 C for a minimum of 24 hours and cool for 30 minutes. Use within 90 minutes of cooling.

7.6Reference MatricesMatrices in which the CDDs/CDFs and interfering compounds are not detected by this method.

7.6.1Reagent waterBottled water purchased locally, or prepared by passage through activated carbon.

7.6.2High-solids reference matrixPlayground sand or similar material.

Prepared by extraction with methylene chloride and/or baking at 450 C for a minimum of four hours.

7.6.3Paper reference matrixGlass-fiber filter, Gelman Type A, or equivalent. Cut paper to simulate the surface area of the paper sample being tested.

7.6.4Tissue reference matrixCorn or other vegetable oil. May be prepared by extraction with methylene chloride.

7.6.5Other matricesThis method may be verified on any reference matrix by performing the tests given in Section 9.2. Ideally, the matrix should be free of the CDDs/CDFs, but in no case shall the background level of the CDDs/CDFs in the reference matrix exceed three times the minimum levels in Table 2. If low background levels of the CDDs/CDFs are present in the reference matrix, the spike level of the analytes used in Section 9.2 should be increased to provide a spike-to-background ratio in the range of 1:1 to 5:1 (Reference 15).

7.7Standard SolutionsPurchased as solutions or mixtures with certification to their purity, concentration, and authenticity, or prepared from materials of known purity and composition. If the chemical purity is 98% or greater, the weight may be used without correction to compute the concentration of the standard. When not being used, standards are stored in the dark at room temperature in screw-capped vials with fluoropolymer-lined caps. A mark is placed on the vial at the level of the solution so that solvent loss by evaporation can be detected. If solvent loss has occurred, the solution should be replaced.

7.8Stock Solutions.

7.8.1PreparationPrepare in nonane per the steps below or purchase as dilute solutions (Cambridge Isotope Laboratories (CIL), Woburn, MA, or equivalent). Observe the safety precautions in Section 5, and the recommendation in Section 5.1.2.

7.8.2Dissolve an appropriate amount of assayed reference material in solvent. For example, weigh 12 mg of 2,3,7,8-TCDD to three significant figures in a 10 mL ground-glass-stoppered volumetric flask and fill to the mark with nonane. After the TCDD is completely dissolved, transfer the solution to a clean 15 mL vial with fluoropolymer-lined cap.

7.8.3Stock standard solutions should be checked for signs of degradation prior to the preparation of calibration or performance test standards.

Reference standards that can be used to determine the accuracy of calibration standards are available from CIL and may be available from other vendors.

7.9PAR Stock Solution 7.9.1All CDDs/CDFsUsing the solutions in Section 7.8, prepare the PAR stock solution to contain the CDDs/CDFs at the concentrations shown in Table 3. When diluted, the solution will become the PAR (Section 7.14).

7.9.2If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the PAR stock solution to contain these compounds only.

7.10Labeled-Compound Spiking Solution.

7.10.1All CDDs/CDFsFrom stock solutions, or from purchased mixtures, prepare this solution to contain the labeled compounds in nonane at the concentrations shown in Table 3. This solution is diluted with acetone prior to use (Section 7.10.3).

7.10.2If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the labeled-compound solution to contain these compounds only.

This solution is diluted with acetone prior to use (Section 7.10.3).

7.10.3Dilute a sufficient volume of the labeled compound solution (Section 7.10.1 or 7.10.2) by a factor of 50 with acetone to prepare a diluted spiking solution. Each sample requires 1.0 mL of the diluted solution, but no more solution should be prepared than can be used in one day.

7.11Cleanup StandardPrepare 37Cl4-2,3,7,8-TCDD in nonane at the concentration shown in Table 3. The cleanup standard is added to all extracts prior to cleanup to measure the efficiency of the cleanup process.

7.12Internal Standard(s).

7.12.1All CDDs/CDFsPrepare the internal standard solution to contain 13C12-1,2,3,4-TCDD and 13C2-1,2,3,7,8,9-HxCDD in nonane at the concentration shown in Table 3.

7.12.2If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the internal standard solution to contain 13C121,2,3,4-TCDD only.

7.13Calibration Standards (CS1 through CS5)Combine the solutions in Sections 7.9 through 7.12 to produce the five calibration solutions shown in Table 4 in nonane. These solutions permit the relative response (labeled to native) and response factor to be measured as a function of concentration. The CS3 standard is used for calibration verification (VER). If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, combine the solutions appropriate to these compounds.

7.14Precision and Recovery (PAR) StandardUsed for determination of initial (Section 9.2) and ongoing (Section 15.5) precision and recovery.

Dilute 10 L of the precision and recovery standard (Section 7.9.1 or 7.9.2) to 2.0 mL with acetone for each sample matrix for each sample batch. One mL each are required for the blank and OPR with each matrix in each batch.

7.15GC Retention Time Window Defining Solution and Isomer Specificity Test StandardUsed to define the beginning and ending retention times for the dioxin and furan isomers and to demonstrate isomer specificity of the GC columns employed for determination of 2,3,7,8-TCDD and 2,3,7,8-TCDF. The standard must contain the compounds listed in Table 5 (CIL EDF4006, or equivalent), at a minimum. It is not necessary to monitor the window-defining compounds if only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined. In this case, an isomer-specificity test standard containing the most closely eluted isomers listed in Table 5 (CIL EDF-4033, or equivalent) may be used.

7.16QC Check SampleA QC Check Sample should be obtained from a source independent of the calibration standards. Ideally, this check sample would be a certified reference material containing the CDDs/CDFs in known concentrations in a sample matrix similar to the matrix under test.

7.17Stability of SolutionsStandard solutions used for quantitative purposes (Sections 7.9 through 7.15) should be analyzed periodically, and should be assayed against reference standards (Section 7.8.3) before further use.

8.0Sample Collection, Preservation, Storage, and Holding Times 8.1Collect samples in amber glass containers following conventional sampling practices (Reference 16). Aqueous samples that flow freely are collected in refrigerated bottles using automatic sampling equipment.

Solid samples are collected as grab samples using wide-mouth jars.

8.2Maintain aqueous samples in the dark at 04 C from the time of collection until receipt at the laboratory. If residual chlorine is present in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 17). If sample pH is greater than 9, adjust to pH 79 with sulfuric acid.

Maintain solid, semi-solid, oily, and mixed-phase samples in the dark at Store aqueous samples in the dark at 04 C. Store solid, semi-solid, oily, mixed-phase, and tissue samples in the dark at 8.3Fish and Tissue Samples.

8.3.1Fish may be cleaned, filleted, or processed in other ways in the field, such that the laboratory may expect to receive whole fish, fish fillets, or other tissues for analysis.

8.3.2Fish collected in the field should be wrapped in aluminum foil, and must be maintained at a temperature less than 4 C from the time of collection until receipt at the laboratory.

8.3.3Samples must be frozen upon receipt at the laboratory and maintained in the dark at 8.4Holding Times.

8.4.1There are no demonstrated maximum holding times associated with CDDs/CDFs in aqueous, solid, semi-solid, tissues, or other sample matrices. If stored in the dark at 04 C and preserved as given above (if required), aqueous samples may be stored for up to one year. Similarly, if stored in the dark at 8.4.2Store sample extracts in the dark at 9.0Quality Assurance/Quality Control 9.1Each laboratory that uses this method is required to operate a formal quality assurance program (Reference 18). The minimum requirements of this program consist of an initial demonstration of laboratory capability, analysis of samples spiked with labeled compounds to evaluate and document data quality, and analysis of standards and blanks as tests of continued performance. Laboratory performance is compared to established performance criteria to determine if the results of analyses meet the performance characteristics of the method.

If the method is to be applied to sample matrix other than water (e.g., soils, filter cake, compost, tissue) the most appropriate alternate matrix (Sections 7.6.2 through 7.6.5) is substituted for the reagent water matrix (Section 7.6.1) in all performance tests.

9.1.1The analyst shall make an initial demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 9.2.

9.1.2In recognition of advances that are occurring in analytical technology, and to allow the analyst to overcome sample matrix interferences, the analyst is permitted certain options to improve separations or lower the costs of measurements. These options include alternate extraction, concentration, cleanup procedures, and changes in columns and detectors. Alternate determinative techniques, such as the substitution of spectroscopic or immuno-assay techniques, and changes that degrade method performance, are not allowed. If an analytical technique other than the techniques specified in this method is used, that technique must have a specificity equal to or better than the specificity of the techniques in this method for the analytes of interest.

9.1.2.1Each time a modification is made to this method, the analyst is required to repeat the procedure in Section 9.2. If the detection limit of the method will be affected by the change, the laboratory is required to demonstrate that the MDL (40 CFR Part 136, Appendix B) is lower than one-third the regulatory compliance level or one-third the ML in this method, whichever is higher. If calibration will be affected by the change, the analyst must recalibrate the instrument per Section 10.

9.1.2.2The laboratory is required to maintain records of modifications made to this method. These records include the following, at a minimum: 9.1.2.2.1The names, titles, addresses, and telephone numbers of the analyst(s) who performed the analyses and modification, and of the quality control officer who witnessed and will verify the analyses and modifications.

9.1.2.2.2A listing of pollutant(s) measured, by name and CAS Registry number.

9.1.2.2.3A narrative stating reason(s) for the modifications.

9.1.2.2.4Results from all quality control (QC) tests comparing the modified method to this method, including: (a) Calibration (Section 10.5 through 10.7).

(b) Calibration verification (Section 15.3).

(c) Initial precision and recovery (Section 9.2).

(d) Labeled compound recovery (Section 9.3).

(e) Analysis of blanks (Section 9.5).

(f) Accuracy assessment (Section 9.4).

9.1.2.2.5Data that will allow an independent reviewer to validate each determination by tracing the instrument output (peak height, area, or other signal) to the final result. These data are to include: (a) Sample numbers and other identifiers.

(b) Extraction dates.

(c) Analysis dates and times.

(d) Analysis sequence/run chronology.

(e) Sample weight or volume (Section 11).

(f) Extract volume prior to each cleanup step (Section 13).

(g) Extract volume after each cleanup step (Section 13).

(h) Final extract volume prior to injection (Section 14).

(i) Injection volume (Section 14.3).

(j) Dilution data, differentiating between dilution of a sample or extract (Section 17.5).

(k) Instrument and operating conditions.

(l) Column (dimensions, liquid phase, solid support, film thickness, etc).

(m) Operating conditions (temperatures, temperature program, flow rates).

(n) Detector (type, operating conditions, etc).

(o) Chromatograms, printer tapes, and other recordings of raw data.

(p) Quantitation reports, data system outputs, and other data to link the raw data to the results reported.

9.1.3Analyses of method blanks are required to demonstrate freedom from contamination (Section 4.3). The procedures and criteria for analysis of a method blank are described in Sections 9.5 and 15.6.

9.1.4The laboratory shall spike all samples with labeled compounds to monitor method performance. This test is described in Section 9.3. When results of these spikes indicate atypical method performance for samples, the samples are diluted to bring method performance within acceptable limits. Procedures for dilution are given in Section 17.5.

9.1.5The laboratory shall, on an ongoing basis, demonstrate through calibration verification and the analysis of the ongoing precision and recovery aliquot that the analytical system is in control. These procedures are described in Sections 15.1 through 15.5.

9.1.6The laboratory shall maintain records to define the quality of data that is generated. Development of accuracy statements is described in Section 9.4.

9.2Initial Precision and Recovery (IPR)To establish the ability to generate acceptable precision and recovery, the analyst shall perform the following operations.

9.2.1For low solids (aqueous) samples, extract, concentrate, and analyze four 1 L aliquots of reagent water spiked with the diluted labeled compound spiking solution (Section 7.10.3) and the precision and recovery standard (Section 7.14) according to the procedures in Sections 11 through 18. For an alternative sample matrix, four aliquots of the alternative reference matrix (Section 7.6) are used. All sample processing steps that are to be used for processing samples, including preparation (Section 11), extraction (Section 12), and cleanup (Section 13), shall be included in this test.

9.2.2Using results of the set of four analyses, compute the average concentration (X) of the extracts in ng/mL and the standard deviation of the concentration (s) in ng/mL for each compound, by isotope dilution for CDDs/CDFs with a labeled analog, and by internal standard for 1,2,3,7,8,9-HxCDD, OCDF, and the labeled compounds.

9.2.3For each CDD/CDF and labeled compound, compare s and X with the corresponding limits for initial precision and recovery in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare s and X with the corresponding limits for initial precision and recovery in Table 6a. If s and X for all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If, however, any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, system performance is unacceptable for that compound. Correct the problem and repeat the test (Section 9.2).

9.3The laboratory shall spike all samples with the diluted labeled compound spiking solution (Section 7.10.3) to assess method performance on the sample matrix.

9.3.1Analyze each sample according to the procedures in Sections 11 through 18.

9.3.2Compute the percent recovery of the labeled compounds and the cleanup standard using the internal standard method (Section 17.2).

9.3.3The recovery of each labeled compound must be within the limits in Table 7 when all 2,3,7,8-substituted CDDs/CDFs are determined, and within the limits in Table 7a when only 2,3,7,8-TCDD and 2,3,7,8-TCDF are determined. If the recovery of any compound falls outside of these limits, method performance is unacceptable for that compound in that sample. To overcome such difficulties, water samples are diluted and smaller amounts of soils, sludges, sediments, and other matrices are reanalyzed per Section 18.4.

9.4Recovery of labeled compounds from samples should be assessed and records should be maintained.

9.4.1After the analysis of five samples of a given matrix type (water, soil, sludge, pulp, etc.) for which the labeled compounds pass the tests in Section 9.3, compute the average percent recovery (R) and the standard deviation of the percent recovery (SR) for the labeled compounds only. Express the assessment as a percent recovery interval from R2SR to R=2SR for each matrix. For example, if R = 90% and SR = 10% for five analyses of pulp, the recovery interval is expressed as 70110%.

9.4.2Update the accuracy assessment for each labeled compound in each matrix on a regular basis (e.g., after each 510 new measurements).

9.5Method BlanksReference matrix method blanks are analyzed to demonstrate freedom from contamination (Section 4.3).

9.5.1Prepare, extract, clean up, and concentrate a method blank with each sample batch (samples of the same matrix started through the extraction process on the same 12-hour shift, to a maximum of 20 samples). The matrix for the method blank shall be similar to sample matrix for the batch, e.g., a 1 L reagent water blank (Section 7.6.1), high-solids reference matrix blank (Section 7.6.2), paper matrix blank (Section 7.6.3); tissue blank (Section 7.6.4) or alternative reference matrix blank (Section 7.6.5). Analyze the blank immediately after analysis of the OPR (Section 15.5) to demonstrate freedom from contamination.

9.5.2If any 2,3,7,8-substituted CDD/CDF (Table 1) is found in the blank at greater than the minimum level (Table 2) or one-third the regulatory compliance level, whichever is greater; or if any potentially interfering compound is found in the blank at the minimum level for each level of chlorination given in Table 2 (assuming a response factor of 1 relative to the 13C12-1,2,3,4-TCDD internal standard for compounds not listed in Table 1), analysis of samples is halted until the blank associated with the sample batch shows no evidence of contamination at this level. All samples must be associated with an uncontaminated method blank before the results for those samples may be reported for regulatory compliance purposes.

9.6QC Check SampleAnalyze the QC Check Sample (Section 7.16) periodically to assure the accuracy of calibration standards and the overall reliability of the analytical process. It is suggested that the QC Check Sample be analyzed at least quarterly.

9.7The specifications contained in this method can be met if the apparatus used is calibrated properly and then maintained in a calibrated state. The standards used for calibration (Section 10), calibration verification (Section 15.3), and for initial (Section 9.2) and ongoing (Section 15.5) precision and recovery should be identical, so that the most precise results will be obtained. A GC/MS instrument will provide the most reproducible results if dedicated to the settings and conditions required for the analyses of CDDs/CDFs by this method.

9.8Depending on specific program requirements, field replicates may be collected to determine the precision of the sampling technique, and spiked samples may be required to determine the accuracy of the analysis when the internal standard method is used.

10.0Calibration 10.1Establish the operating conditions necessary to meet the minimum retention times for the internal standards in Section 10.2.4 and the relative retention times for the CDDs/CDFs in Table 2.

10.1.1Suggested GC operating conditions: Injector temperature: 270 C Interface temperature: 290 C Initial temperature: 200 C Initial time: Two minutes Temperature program: 200220 C, at 5 C/minute 220 C for 16 minutes 220235 C, at 5 C/minute 235 C for seven minutes 235330 C, at 5 C/minute Note: All portions of the column that connect the GC to the ion source shall remain at or above the interface temperature specified above during analysis to preclude condensation of less volatile compounds.

Optimize GC conditions for compound separation and sensitivity. Once optimized, the same GC conditions must be used for the analysis of all standards, blanks, IPR and OPR aliquots, and samples.

10.1.2Mass spectrometer (MS) resolutionObtain a selected ion current profile (SICP) of each analyte in Table 3 at the two exact m/z's specified in Table 8 and at 10,000 resolving power by injecting an authentic standard of the CDDs/CDFs either singly or as part of a mixture in which there is no interference between closely eluted components.

10.1.2.1The analysis time for CDDs/CDFs may exceed the long-term mass stability of the mass spectrometer. Because the instrument is operated in the high-resolution mode, mass drifts of a few ppm (e.g., 5 ppm in mass) can have serious adverse effects on instrument performance.

Therefore, a mass-drift correction is mandatory and a lock-mass m/z from PFK is used for drift correction. The lock-mass m/z is dependent on the exact m/z's monitored within each descriptor, as shown in Table 8. The level of PFK metered into the HRMS during analyses should be adjusted so that the amplitude of the most intense selected lock-mass m/z signal (regardless of the descriptor number) does not exceed 10% of the full-scale deflection for a given set of detector parameters. Under those conditions, sensitivity changes that might occur during the analysis can be more effectively monitored.

Note: Excessive PFK (or any other reference substance) may cause noise problems and contamination of the ion source necessitating increased frequency of source cleaning.

10.1.2.2If the HRMS has the capability to monitor resolution during the analysis, it is acceptable to terminate the analysis when the resolution falls below 10,000 to save reanalysis time.

10.1.2.3Using a PFK molecular leak, tune the instrument to meet the minimum required resolving power of 10,000 (10% valley) at m/z 304.9824 (PFK) or any other reference signal close to m/z 304 (from TCDF). For each descriptor (Table 8), monitor and record the resolution and exact m/z's of three to five reference peaks covering the mass range of the descriptor. The resolution must be greater than or equal to 10,000, and the deviation between the exact m/z and the theoretical m/z (Table 8) for each exact m/z monitored must be less than 5 ppm.

10.2Ion Abundance Ratios, Minimum Levels, Signal-to-Noise Ratios, and Absolute Retention TimesChoose an injection volume of either 1 L or 2 L, consistent with the capability of the HRGC/HRMS instrument. Inject a 1 L or 2 L aliquot of the CS1 calibration solution (Table 4) using the GC conditions from Section 10.1.1. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the operating conditions and specifications below apply to analysis of those compounds only.

10.2.1Measure the SICP areas for each analyte, and compute the ion abundance ratios at the exact m/z's specified in Table 8. Compare the computed ratio to the theoretical ratio given in Table 9.

10.2.1.1The exact m/z's to be monitored in each descriptor are shown in Table 8. Each group or descriptor shall be monitored in succession as a function of GC retention time to ensure that all CDDs/CDFs are detected.

Additional m/z's may be monitored in each descriptor, and the m/z's may be divided among more than the five descriptors listed in Table 8, provided that the laboratory is able to monitor the m/z's of all the CDDs/CDFs that may elute from the GC in a given retention-time window.

If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the descriptors may be modified to include only the exact m/z's for the tetra-and penta-isomers, the diphenyl ethers, and the lock m/z's.

10.2.1.2The mass spectrometer shall be operated in a mass-drift correction mode, using perfluorokerosene (PFK) to provide lock m/z's.

The lock-mass for each group of m/z's is shown in Table 8. Each lock mass shall be monitored and shall not vary by more than 20% throughout its respective retention time window. Variations of the lock mass by more than 20% indicate the presence of coeluting interferences that may significantly reduce the sensitivity of the mass spectrometer.

Reinjection of another aliquot of the sample extract will not resolve the problem. Additional cleanup of the extract may be required to remove the interferences.

10.2.2All CDDs/CDFs and labeled compounds in the CS1 standard shall be within the QC limits in Table 9 for their respective ion abundance ratios; otherwise, the mass spectrometer shall be adjusted and this test repeated until the m/z ratios fall within the limits specified. If the adjustment alters the resolution of the mass spectrometer, resolution shall be verified (Section 10.1.2) prior to repeat of the test.

10.2.3Verify that the HRGC/HRMS instrument meets the minimum levels in Table 2. The peaks representing the CDDs/CDFs and labeled compounds in the CS1 calibration standard must have signal-to-noise ratios (S/N) greater than or equal to 10.0. Otherwise, the mass spectrometer shall be adjusted and this test repeated until the minimum levels in Table 2 are met.

10.2.4The absolute retention time of 13C12-1,2,3,4TCDD (Section 7.12) shall exceed 25.0 minutes on the DB5 column, and the retention time of 13C12-1,2,3,4TCDD shall exceed 15.0 minutes on the DB225 column; otherwise, the GC temperature program shall be adjusted and this test repeated until the above-stated minimum retention time criteria are met.

2010.3Retention-Time WindowsAnalyze the window defining mixtures (Section 7.15) using the optimized temperature program in Section 10.1.

Table 5 gives the elution order (first/last) of the window-defining compounds. If 2,3,7,8-TCDD and 2,3,7,8-TCDF only are to be analyzed, this test is not required.

10.4Isomer Specificity.

10.4.1Analyze the isomer specificity test standards (Section 7.15) using the procedure in Section 14 and the optimized conditions for sample analysis (Section 10.1.1).

10.4.2Compute the percent valley between the GC peaks that elute most closely to the 2,3,7,8-TCDD and TCDF isomers, on their respective columns, per Figures 6 and 7.

10.4.3Verify that the height of the valley between the most closely eluted isomers and the 2,3,7,8-substituted isomers is less than 25% (computed as 100 x/y in Figures 6 and 7). If the valley exceeds 25%, adjust the analytical conditions and repeat the test or replace the GC column and recalibrate (Sections 10.1.2 through 10.7).

10.5Calibration by Isotope DilutionIsotope dilution calibration is used for the 15 2,3,7,8-substituted CDDs/CDFs for which labeled compounds are added to samples prior to extraction. The reference compound for each CDD/CDF compound is shown in Table 2.

10.5.1A calibration curve encompassing the concentration range is prepared for each compound to be determined. The relative response (RR) (labeled to native) vs. concentration in standard solutions is plotted or computed using a linear regression. Relative response is determined according to the procedures described below. Five calibration points are employed.

10.5.2The response of each CDD/CDF relative to its labeled analog is determined using the area responses of both the primary and secondary exact m/z's specified in Table 8, for each calibration standard, as follows: (image) where: A1n and A2n = The areas of the primary and secondary m/z's for the CDD/CDF.

A1l and A2l = The areas of the primary and secondary m/z's for the labeled compound.

Cl = The concentration of the labeled compound in the calibration standard (Table 4).

Cn = The concentration of the native compound in the calibration standard (Table 4).

10.5.3To calibrate the analytical system by isotope dilution, inject a volume of calibration standards CS1 through CS5 (Section 7.13 and Table 4) identical to the volume chosen in Section 10.2, using the procedure in Section 14 and the conditions in Section 10.1.1 and Table 2. Compute the relative response (RR) at each concentration.

10.5.4LinearityIf the relative response for any compound is constant (less than 20% coefficient of variation) over the five-point calibration range, an averaged relative response may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the five-point calibration range.

10.6Calibration by Internal StandardThe internal standard method is applied to determination of 1,2,3,7,8,9-HxCDD (Section 17.1.2), OCDF (Section 17.1.1), the non 2,3,7,8-substituted compounds, and to the determination of labeled compounds for intralaboratory statistics (Sections 9.4 and 15.5.4).

10.6.1Response factorsCalibration requires the determination of response factors (RF) defined by the following equation: (image) where: A1s and A2s = The areas of the primary and secondary m/z's for the CDD/CDF.

A1is and A2is = The areas of the primary and secondary m/z's for the internal standard.

Cis = The concentration of the internal standard (Table 4).

Cs = The concentration of the compound in the calibration standard (Table 4).

Note: There is only one m/z for 37Cl4-2,3,7,8-TCDD. See Table 8.

10.6.2To calibrate the analytical system by internal standard, inject 1.0 L or 2.0 L of calibration standards CS1 through CS5 (Section 7.13 and Table 4) using the procedure in Section 14 and the conditions in Section 10.1.1 and Table 2. Compute the response factor (RF) at each concentration.

10.6.3LinearityIf the response factor (RF) for any compound is constant (less than 35% coefficient of variation) over the five-point calibration range, an averaged response factor may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the five-point range.

10.7Combined CalibrationBy using calibration solutions (Section 7.13 and Table 4) containing the CDDs/CDFs and labeled compounds and the internal standards, a single set of analyses can be used to produce calibration curves for the isotope dilution and internal standard methods. These curves are verified each shift (Section 15.3) by analyzing the calibration verification standard (VER, Table 4). Recalibration is required if any of the calibration verification criteria (Section 15.3) cannot be met.

10.8Data StorageMS data shall be collected, recorded, and stored.

10.8.1Data acquisitionThe signal at each exact m/z shall be collected repetitively throughout the monitoring period and stored on a mass storage device.

10.8.2Response factors and multipoint calibrationsThe data system shall be used to record and maintain lists of response factors (response ratios for isotope dilution) and multipoint calibration curves.

Computations of relative standard deviation (coefficient of variation) shall be used to test calibration linearity. Statistics on initial performance (Section 9.2) and ongoing performance (Section 15.5) should be computed and maintained, either on the instrument data system, or on a separate computer system.

11.0Sample Preparation 11.1Sample preparation involves modifying the physical form of the sample so that the CDDs/CDFs can be extracted efficiently. In general, the samples must be in a liquid form or in the form of finely divided solids in order for efficient extraction to take place. Table 10 lists the phases and suggested quantities for extraction of various sample matrices.

For samples known or expected to contain high levels of the CDDs/CDFs, the smallest sample size representative of the entire sample should be used (see Section 17.5).

For all samples, the blank and IPR/OPR aliquots must be processed through the same steps as the sample to check for contamination and losses in the preparation processes.

11.1.1For samples that contain particles, percent solids and particle size are determined using the procedures in Sections 11.2 and 11.3, respectively.

11.1.2Aqueous samplesBecause CDDs/CDFs may be bound to suspended particles, the preparation of aqueous samples is dependent on the solids content of the sample.

11.1.2.1Aqueous samples visibly absent particles are prepared per Section 11.4 and extracted directly using the separatory funnel or SPE techniques in Sections 12.1 or 12.2, respectively.

11.1.2.2Aqueous samples containing visible particles and containing one percent suspended solids or less are prepared using the procedure in Section 11.4. After preparation, the sample is extracted directly using the SPE technique in 12.2 or filtered per Section 11.4.3. After filtration, the particles and filter are extracted using the SDS procedure in Section 12.3 and the filtrate is extracted using the separatory funnel procedure in Section 12.1.

11.1.2.3For aqueous samples containing greater than one percent solids, a sample aliquot sufficient to provide 10 g of dry solids is used, as described in Section 11.5.

11.1.3Solid samples are prepared using the procedure described in Section 11.5 followed by extraction via the SDS procedure in Section 12.3.

11.1.4Multiphase samplesThe phase(s) containing the CDDs/CDFs is separated from the non-CDD/CDF phase using pressure filtration and centrifugation, as described in Section 11.6. The CDDs/CDFs will be in the organic phase in a multiphase sample in which an organic phase exists.

11.1.5Procedures for grinding, homogenization, and blending of various sample phases are given in Section 11.7.

11.1.6Tissue samplesPreparation procedures for fish and other tissues are given in Section 11.8.

11.2Determination of Percent Suspended Solids.

Note: This aliquot is used for determining the solids content of the sample, not for determination of CDDs/CDFs.

11.2.1Aqueous liquids and multi-phase samples consisting of mainly an aqueous phase.

11.2.1.1Dessicate and weigh a GF/D filter (Section 6.5.3) to three significant figures.

11.2.1.2Filter 10.0 0.02 mL of well-mixed sample through the filter.

11.2.1.3Dry the filter a minimum of 12 hours at 110 5 C and cool in a dessicator.

11.2.1.4Calculate percent solids as follows: (image) 11.2.2Non-aqueous liquids, solids, semi-solid samples, and multi-phase samples in which the main phase is not aqueous; but not tissues.

11.2.2.1Weigh 510 g of sample to three significant figures in a tared beaker.

11.2.2.2Dry a minimum of 12 hours at 110 5 C, and cool in a dessicator.

11.2.2.3Calculate percent solids as follows: (image) 11.3Determination of Particle Size.

11.3.1Spread the dried sample from Section 11.2.2.2 on a piece of filter paper or aluminum foil in a fume hood or glove box.

11.3.2Estimate the size of the particles in the sample. If the size of the largest particles is greater than 1 mm, the particle size must be reduced to 1 mm or less prior to extraction using the procedures in Section 11.7.

11.4Preparation of Aqueous Samples Containing 1% Suspended Solids or Less.

11.4.1Aqueous samples visibly absent particles are prepared per the procedure below and extracted directly using the separatory funnel or SPE techniques in Sections 12.1 or 12.2, respectively. Aqueous samples containing visible particles and one percent suspended solids or less are prepared using the procedure below and extracted using either the SPE technique in Section 12.2 or further prepared using the filtration procedure in Section 11.4.3. The filtration procedure is followed by SDS extraction of the filter and particles (Section 12.3) and separatory funnel extraction of the filtrate (Section 12.1). The SPE procedure is followed by SDS extraction of the filter and disk.

11.4.2Preparation of sample and QC aliquots.

11.4.2.1Mark the original level of the sample on the sample bottle for reference. Weigh the sample plus bottle to 1.

11.4.2.2Spike 1.0 mL of the diluted labeled-compound spiking solution (Section 7.10.3) into the sample bottle. Cap the bottle and mix the sample by careful shaking. Allow the sample to equilibrate for one to two hours, with occasional shaking.

11.4.2.3For each sample or sample batch (to a maximum of 20 samples) to be extracted during the same 12-hour shift, place two 1.0 L aliquots of reagent water in clean sample bottles or flasks.

11.4.2.4Spike 1.0 mL of the diluted labeled-compound spiking solution (Section 7.10.3) into both reagent water aliquots. One of these aliquots will serve as the method blank.

11.4.2.5Spike 1.0 mL of the PAR standard (Section 7.14) into the remaining reagent water aliquot. This aliquot will serve as the OPR (Section 15.5).

11.4.2.6If SPE is to be used, add 5 mL of methanol to the sample, cap and shake the sample to mix thoroughly, and proceed to Section 12.2 for extraction. If SPE is not to be used, and the sample is visibly absent particles, proceed to Section 12.1 for extraction. If SPE is not to be used and the sample contains visible particles, proceed to the following section for filtration of particles.

11.4.3Filtration of particles.

11.4.3.1Assemble a Buchner funnel (Section 6.5.5) on top of a clean filtration flask. Apply vacuum to the flask, and pour the entire contents of the sample bottle through a glass-fiber filter (Section 6.5.6) in the Buchner funnel, swirling the sample remaining in the bottle to suspend any particles.

11.4.3.2Rinse the sample bottle twice with approximately 5 mL portions of reagent water to transfer any remaining particles onto the filter.

11.4.3.3Rinse any particles off the sides of the Buchner funnel with small quantities of reagent water.

11.4.3.4Weigh the empty sample bottle to 1 g. Determine the weight of the sample by difference. Save the bottle for further use.

11.4.3.5Extract the filtrate using the separatory funnel procedure in Section 12.1.

11.4.3.6Extract the filter containing the particles using the SDS procedure in Section 12.3.

11.5Preparation of Samples Containing Greater Than 1% Solids.

11.5.1Weigh a well-mixed aliquot of each sample (of the same matrix type) sufficient to provide 10 g of dry solids (based on the solids determination in Section 11.2) into a clean beaker or glass jar.

11.5.2Spike 1.0 mL of the diluted labeled compound spiking solution (Section 7.10.3) into the sample.

11.5.3For each sample or sample batch (to a maximum of 20 samples) to be extracted during the same 12-hour shift, weigh two 10 g aliquots of the appropriate reference matrix (Section 7.6) into clean beakers or glass jars.

11.5.4Spike 1.0 mL of the diluted labeled compound spiking solution (Section 7.10.3) into each reference matrix aliquot. One aliquot will serve as the method blank. Spike 1.0 mL of the PAR standard (Section 7.14) into the other reference matrix aliquot. This aliquot will serve as the OPR (Section 15.5).

11.5.5Stir or tumble and equilibrate the aliquots for one to two hours.

11.5.6Decant excess water. If necessary to remove water, filter the sample through a glass-fiber filter and discard the aqueous liquid.

11.5.7If particles >1mm are present in the sample (as determined in Section 11.3.2), spread the sample on clean aluminum foil in a hood.

After the sample is dry, grind to reduce the particle size (Section 11.7).

11.5.8Extract the sample and QC aliquots using the SDS procedure in Section 12.3.

11.6Multiphase Samples.

11.6.1Using the percent solids determined in Section 11.2.1 or 11.2.2, determine the volume of sample that will provide 10 g of solids, up to 1 L of sample.

11.6.2Pressure filter the amount of sample determined in Section 11.6.1 through Whatman GF/D glass-fiber filter paper (Section 6.5.3). Pressure filter the blank and OPR aliquots through GF/D papers also. If necessary to separate the phases and/or settle the solids, centrifuge these aliquots prior to filtration.

11.6.3Discard any aqueous phase (if present). Remove any non-aqueous liquid present and reserve the maximum amount filtered from the sample (Section 11.6.1) or 10 g, whichever is less, for combination with the solid phase (Section 12.3.5).

11.6.4If particles >1mm are present in the sample (as determined in Section 11.3.2) and the sample is capable of being dried, spread the sample and QC aliquots on clean aluminum foil in a hood. After the aliquots are dry or if the sample cannot be dried, reduce the particle size using the procedures in Section 11.7 and extract the reduced particles using the SDS procedure in Section 12.3. If particles >1mm are not present, extract the particles and filter in the sample and QC aliquots directly using the SDS procedure in Section 12.3.

11.7Sample grinding, homogenization, or blendingSamples with particle sizes greater than 1 mm (as determined in Section 11.3.2) are subjected to grinding, homogenization, or blending. The method of reducing particle size to less than 1 mm is matrix-dependent. In general, hard particles can be reduced by grinding with a mortar and pestle. Softer particles can be reduced by grinding in a Wiley mill or meat grinder, by homogenization, or in a blender.

11.7.1Each size-reducing preparation procedure on each matrix shall be verified by running the tests in Section 9.2 before the procedure is employed routinely.

11.7.2The grinding, homogenization, or blending procedures shall be carried out in a glove box or fume hood to prevent particles from contaminating the work environment.

11.7.3GrindingCertain papers and pulps, slurries, and amorphous solids can be ground in a Wiley mill or heavy duty meat grinder. In some cases, reducing the temperature of the sample to freezing or to dry ice or liquid nitrogen temperatures can aid in the grinding process. Grind the sample aliquots from Section 11.5.7 or 11.6.4 in a clean grinder. Do not allow the sample temperature to exceed 50 C. Grind the blank and reference matrix aliquots using a clean grinder.

11.7.4Homogenization or blendingParticles that are not ground effectively, or particles greater than 1 mm in size after grinding, can often be reduced in size by high speed homogenization or blending.

Homogenize and/or blend the particles or filter from Section 11.5.7 or 11.6.4 for the sample, blank, and OPR aliquots.

11.7.5Extract the aliquots using the SDS procedure in Section 12.3.

11.8Fish and Other TissuesPrior to processing tissue samples, the laboratory must determine the exact tissue to be analyzed. Common requests for analysis of fish tissue include whole fishskin on, whole fishskin removed, edible fish fillets (filleted in the field or by the laboratory), specific organs, and other portions. Once the appropriate tissue has been determined, the sample must be homogenized.

11.8.1Homogenization.

11.8.1.1Samples are homogenized while still frozen, where practical. If the laboratory must dissect the whole fish to obtain the appropriate tissue for analysis, the unused tissues may be rapidly refrozen and stored in a clean glass jar for subsequent use.

11.8.1.2Each analysis requires 10 g of tissue (wet weight). Therefore, the laboratory should homogenize at least 20 g of tissue to allow for re-extraction of a second aliquot of the same homogenized sample, if re-analysis is required. When whole fish analysis is necessary, the entire fish is homogenized.

11.8.1.3Homogenize the sample in a tissue homogenizer (Section 6.3.3) or grind in a meat grinder (Section 6.3.4). Cut tissue too large to feed into the grinder into smaller pieces. To assure homogeneity, grind three times.

11.8.1.4Transfer approximately 10 g (wet weight) of homogenized tissue to a clean, tared, 400500 mL beaker. For the alternate HCl digestion/extraction, transfer the tissue to a clean, tared 500600 mL wide-mouth bottle. Record the weight to the nearest 10 mg.

11.8.1.5Transfer the remaining homogenized tissue to a clean jar with a fluoropolymer-lined lid. Seal the jar and store the tissue at Return any tissue that was not homogenized to its original container and store at 11.8.2QC aliquots.

11.8.2.1Prepare a method blank by adding approximately 10 g of the oily liquid reference matrix (Section 7.6.4) to a 400500 mL beaker. For the alternate HCl digestion/extraction, add the reference matrix to a 500600 mL wide-mouth bottle. Record the weight to the nearest 10 mg.

11.8.2.2Prepare a precision and recovery aliquot by adding approximately 10 g of the oily liquid reference matrix (Section 7.6.4) to a separate 400500 mL beaker or wide-mouth bottle, depending on the extraction procedure to be used. Record the weight to the nearest 10 mg. If the initial precision and recovery test is to be performed, use four aliquots; if the ongoing precision and recovery test is to be performed, use a single aliquot.

11.8.3Spiking 11.8.3.1Spike 1.0 mL of the labeled compound spiking solution (Section 7.10.3) into the sample, blank, and OPR aliquot.

11.8.3.2Spike 1.0 mL of the PAR standard (Section 7.14) into the OPR aliquot.

11.8.4Extract the aliquots using the procedures in Section 12.4.

12.0Extraction and Concentration Extraction procedures include separatory funnel (Section 12.1) and solid phase (Section 12.2) for aqueous liquids; Soxhlet/Dean-Stark (Section 12.3) for solids, filters, and SPE disks; and Soxhlet extraction (Section 12.4.1) and HCl digestion (Section 12.4.2) for tissues.

Acid/base back-extraction (Section 12.5) is used for initial cleanup of extracts.

Macro-concentration procedures include rotary evaporation (Section 12.6.1), heating mantle (Section 12.6.2), and Kuderna-Danish (K-D) evaporation (Section 12.6.3). Micro-concentration uses nitrogen blowdown (Section 12.7).

12.1Separatory funnel extraction of filtrates and of aqueous samples visibly absent particles.

12.1.1Pour the spiked sample (Section 11.4.2.2) or filtrate (Section 11.4.3.5) into a 2 L separatory funnel. Rinse the bottle or flask twice with 5 mL of reagent water and add these rinses to the separatory funnel.

12.1.2Add 60 mL methylene chloride to the empty sample bottle (Section 12.1.1), seal, and shake 60 seconds to rinse the inner surface. Transfer the solvent to the separatory funnel, and extract the sample by shaking the funnel for two minutes with periodic venting. Allow the organic layer to separate from the aqueous phase for a minimum of 10 minutes. If an emulsion forms and is more than one-third the volume of the solvent layer, employ mechanical techniques to complete the phase separation (see note below). Drain the methylene chloride extract through a solvent-rinsed glass funnel approximately one-half full of granular anhydrous sodium sulfate (Section 7.2.1) supported on clean glass-fiber paper into a solvent-rinsed concentration device (Section 12.6).

Note: If an emulsion forms, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration through glass wool, use of phase separation paper, centrifugation, use of an ultrasonic bath with ice, addition of NaCl, or other physical methods.

Alternatively, solid-phase or other extraction techniques may be used to prevent emulsion formation. Any alternative technique is acceptable so long as the requirements in Section 9 are met.

Experience with aqueous samples high in dissolved organic materials (e.g., paper mill effluents) has shown that acidification of the sample prior to extraction may reduce the formation of emulsions. Paper industry methods suggest that the addition of up to 400 mL of ethanol to a 1 L effluent sample may also reduce emulsion formation. However, studies by EPA suggest that the effect may be a result of sample dilution, and that the addition of reagent water may serve the same function. Mechanical techniques may still be necessary to complete the phase separation. If either acidification or addition of ethanol is utilized, the laboratory must perform the startup tests described in Section 9.2 using the same techniques.

12.1.3Extract the water sample two more times with 60 mL portions of methylene chloride. Drain each portion through the sodium sulfate into the concentrator. After the third extraction, rinse the separatory funnel with at least 20 mL of methylene chloride, and drain this rinse through the sodium sulfate into the concentrator. Repeat this rinse at least twice. Set aside the funnel with sodium sulfate if the extract is to be combined with the extract from the particles.

12.1.4Concentrate the extract using one of the macro-concentration procedures in Section 12.6.

12.1.4.1If the extract is from a sample visibly absent particles (Section 11.1.2.1), adjust the final volume of the concentrated extract to approximately 10 mL with hexane, transfer to a 250 mL separatory funnel, and back-extract using the procedure in Section 12.5.

12.1.4.2If the extract is from the aqueous filtrate (Section 11.4.3.5), set aside the concentration apparatus for addition of the SDS extract from the particles (Section 12.3.9.1.2).

12.2SPE of Samples Containing Less Than 1% Solids (References 1920).

12.2.1Disk preparation.

12.2.1.1Place an SPE disk on the base of the filter holder (Figure 4) and wet with toluene. While holding a GMF 150 filter above the SPE disk with tweezers, wet the filter with toluene and lay the filter on the SPE disk, making sure that air is not trapped between the filter and disk.

Clamp the filter and SPE disk between the 1 L glass reservoir and the vacuum filtration flask.

12.2.1.2Rinse the sides of the filtration flask with approx 15 mL of toluene using a squeeze bottle or syringe. Apply vacuum momentarily until a few drops appear at the drip tip. Release the vacuum and allow the filter/disk to soak for approx one minute. Apply vacuum and draw all of the toluene through the filter/disk. Repeat the wash step with approx 15 mL of acetone and allow the filter/disk to air dry.

12.2.1.3Re-wet the filter/disk with approximately 15 mL of methanol, allowing the filter/disk to soak for approximately one minute. Pull the methanol through the filter/disk using the vacuum, but retain a layer of methanol approximately 1 mm thick on the filter. Do not allow the disk to go dry from this point until the end of the extraction.

12.2.1.4Rinse the filter/disk with two 50-mL portions of reagent water by adding the water to the reservoir and pulling most through, leaving a layer of water on the surface of the filter.

12.2.2Extraction.

12.2.2.1Pour the spiked sample (Section 11.4.2.2), blank (Section 11.4.2.4), or IPR/OPR aliquot (Section 11.4.2.5) into the reservoir and turn on the vacuum to begin the extraction. Adjust the vacuum to complete the extraction in no less than 10 minutes. For samples containing a high concentration of particles (suspended solids), filtration times may be eight hours or longer.

12.2.2.2Before all of the sample has been pulled through the filter/disk, rinse the sample bottle with approximately 50 mL of reagent water to remove any solids, and pour into the reservoir. Pull through the filter/disk. Use additional reagent water rinses until all visible solids are removed.

12.2.2.3Before all of the sample and rinses have been pulled through the filter/disk, rinse the sides of the reservoir with small portions of reagent water.

12.2.2.4Allow the filter/disk to dry, then remove the filter and disk and place in a glass Petri dish. Extract the filter and disk per Section 12.3.

12.3SDS Extraction of Samples Containing Particles, and of Filters and/or Disks.

12.3.1Charge a clean extraction thimble (Section 6.4.2.2) with 5.0 g of 100/200 mesh silica (Section 7.5.1.1) topped with 100 g of quartz sand (Section 7.3.2).

Note: Do not disturb the silica layer throughout the extraction process.

12.3.2Place the thimble in a clean extractor. Place 3040 mL of toluene in the receiver and 200250 mL of toluene in the flask.

12.3.3Pre-extract the glassware by heating the flask until the toluene is boiling. When properly adjusted, one to two drops of toluene will fall per second from the condenser tip into the receiver. Extract the apparatus for a minimum of three hours.

12.3.4After pre-extraction, cool and disassemble the apparatus. Rinse the thimble with toluene and allow to air dry.

12.3.5Load the wet sample, filter, and/or disk from Section 11.4.3.6, 11.5.8, 11.6.4, 11.7.3, 11.7.4, or 12.2.2.4 and any nonaqueous liquid from Section 11.6.3 into the thimble and manually mix into the sand layer with a clean metal spatula, carefully breaking up any large lumps of sample.

12.3.6Reassemble the pre-extracted SDS apparatus, and add a fresh charge of toluene to the receiver and reflux flask. Apply power to the heating mantle to begin refluxing. Adjust the reflux rate to match the rate of percolation through the sand and silica beds until water removal lessens the restriction to toluene flow. Frequently check the apparatus for foaming during the first two hours of extraction. If foaming occurs, reduce the reflux rate until foaming subsides.

12.3.7Drain the water from the receiver at one to two hours and eight to nine hours, or sooner if the receiver fills with water. Reflux the sample for a total of 1624 hours. Cool and disassemble the apparatus.

Record the total volume of water collected.

12.3.8Remove the distilling flask. Drain the water from the Dean-Stark receiver and add any toluene in the receiver to the extract in the flask.

12.3.9Concentrate the extract using one of the macro-concentration procedures in Section 12.6 per the following: 12.3.9.1Extracts from the particles in an aqueous sample containing less than 1% solids (Section 11.4.3.6).

12.3.9.1.1Concentrate the extract to approximately 5 mL using the rotary evaporator or heating mantle procedures in Section 12.6.1 or 12.6.2.

12.3.9.1.2Quantitatively transfer the extract through the sodium sulfate (Section 12.1.3) into the apparatus that was set aside (Section 12.1.4.2) and reconcentrate to the level of the toluene.

12.3.9.1.3Adjust to approximately 10 mL with hexane, transfer to a 250 mL separatory funnel, and proceed with back-extraction (Section 12.5).

12.3.9.2Extracts from particles (Sections 11.5 through 11.6) or from the SPE filter and disk (Section 12.2.2.4)Concentrate to approximately 10 mL using the rotary evaporator or heating mantle (Section 12.6.1 or 12.6.2), transfer to a 250 mL separatory funnel, and proceed with back-extraction (Section 12.5).

12.4Extraction of TissueTwo procedures are provided for tissue extraction.

12.4.1Soxhlet extraction (Reference 21).

12.4.1.1Add 3040 g of powdered anhydrous sodium sulfate to each of the beakers (Section 11.8.4) and mix thoroughly. Cover the beakers with aluminum foil and allow to equilibrate for 1224 hours. Remix prior to extraction to prevent clumping.

12.4.1.2Assemble and pre-extract the Soxhlet apparatus per Sections 12.3.1 through 12.3.4, except use the methylene chloride:hexane (1:1) mixture for the pre-extraction and rinsing and omit the quartz sand. The Dean-Stark moisture trap may also be omitted, if desired.

12.4.1.3Reassemble the pre-extracted Soxhlet apparatus and add a fresh charge of methylene chloride:hexane to the reflux flask.

12.4.1.4Transfer the sample/sodium sulfate mixture (Section 12.4.1.1) to the Soxhlet thimble, and install the thimble in the Soxhlet apparatus.

12.4.1.5Rinse the beaker with several portions of solvent mixture and add to the thimble. Fill the thimble/receiver with solvent. Extract for 1824 hours.

12.4.1.6After extraction, cool and disassemble the apparatus.

12.4.1.7Quantitatively transfer the extract to a macro-concentration device (Section 12.6), and concentrate to near dryness. Set aside the concentration apparatus for re-use.

12.4.1.8Complete the removal of the solvent using the nitrogen blowdown procedure (Section 12.7) and a water bath temperature of 60 C. Weigh the receiver, record the weight, and return the receiver to the blowdown apparatus, concentrating the residue until a constant weight is obtained.

12.4.1.9Percent lipid determinationThe lipid content is determined by extraction of tissue with the same solvent system (methylene chloride:hexane) that was used in EPA's National Dioxin Study (Reference 22) so that lipid contents are consistent with that study.

12.4.1.9.1Redissolve the residue in the receiver in hexane and spike 1.0 mL of the cleanup standard (Section 7.11) into the solution.

12.4.1.9.2Transfer the residue/hexane to the anthropogenic isolation column (Section 13.7.1) or bottle for the acidified silica gel batch cleanup (Section 13.7.2), retaining the boiling chips in the concentration apparatus. Use several rinses to assure that all material is transferred. If necessary, sonicate or heat the receiver slightly to assure that all material is re-dissolved. Allow the receiver to dry.

Weigh the receiver and boiling chips.

12.4.1.9.3Calculate the lipid content to the nearest three significant figures as follows: (image) 12.4.1.9.4It is not necessary to determine the lipid content of the blank, IPR, or OPR aliquots.

12.4.2HCl digestion/extraction and concentration (References 2326).

12.4.2.1Add 200 mL of 6 N HCl and 200 mL of methylene chloride:hexane (1:1) to the sample and QC aliquots (Section 11.8.4).

12.4.2.2Cap and shake each bottle one to three times. Loosen the cap in a hood to vent excess pressure. Shake each bottle for 1030 seconds and vent.

12.4.2.3Tightly cap and place on shaker. Adjust the shaker action and speed so that the acid, solvent, and tissue are in constant motion.

However, take care to avoid such violent action that the bottle may be dislodged from the shaker. Shake for 1224 hours.

12.4.2.4After digestion, remove the bottles from the shaker. Allow the bottles to stand so that the solvent and acid layers separate.

12.4.2.5Decant the solvent through a glass funnel with glass-fiber filter (Sections 6.5.2 through 6.5.3) containing approximately 10 g of granular anhydrous sodium sulfate (Section 7.2.1) into a macro-concentration apparatus (Section 12.6). Rinse the contents of the bottle with two 25 mL portions of hexane and pour through the sodium sulfate into the apparatus.

12.4.2.6Concentrate the solvent to near dryness using a macro-concentration procedure (Section 12.6).

12.4.2.7Complete the removal of the solvent using the nitrogen blowdown apparatus (Section 12.7) and a water bath temperature of 60 C. Weigh the receiver, record the weight, and return the receiver to the blowdown apparatus, concentrating the residue until a constant weight is obtained.

12.4.2.8Percent lipid determinationThe lipid content is determined in the same solvent system [methylene chloride:hexane (1:1)] that was used in EPA's National Dioxin Study (Reference 22) so that lipid contents are consistent with that study.

12.4.2.8.1Redissolve the residue in the receiver in hexane and spike 1.0 mL of the cleanup standard (Section 7.11) into the solution.

12.4.2.8.2Transfer the residue/hexane to the narrow-mouth 100200 mL bottle retaining the boiling chips in the receiver. Use several rinses to assure that all material is transferred, to a maximum hexane volume of approximately 70 mL. Allow the receiver to dry. Weigh the receiver and boiling chips.

12.4.2.8.3Calculate the percent lipid per Section 12.4.1.9.3. It is not necessary to determine the lipid content of the blank, IPR, or OPR aliquots.

12.4.2.9Clean up the extract per Section 13.7.3.

12.5Back-Extraction with Base and Acid.

12.5.1Spike 1.0 mL of the cleanup standard (Section 7.11) into the separatory funnels containing the sample and QC extracts from Section 12.1.4.1, 12.3.9.1.3, or 12.3.9.2.

12.5.2Partition the extract against 50 mL of potassium hydroxide solution (Section 7.1.1). Shake for two minutes with periodic venting into a hood. Remove and discard the aqueous layer. Repeat the base washing until no color is visible in the aqueous layer, to a maximum of four washings. Minimize contact time between the extract and the base to prevent degradation of the CDDs/CDFs. Stronger potassium hydroxide solutions may be employed for back-extraction, provided that the laboratory meets the specifications for labeled compound recovery and demonstrates acceptable performance using the procedure in Section 9.2.

12.5.3Partition the extract against 50 mL of sodium chloride solution (Section 7.1.4) in the same way as with base. Discard the aqueous layer.

12.5.4Partition the extract against 50 mL of sulfuric acid (Section 7.1.2) in the same way as with base. Repeat the acid washing until no color is visible in the aqueous layer, to a maximum of four washings.

12.5.5Repeat the partitioning against sodium chloride solution and discard the aqueous layer.

12.5.6Pour each extract through a drying column containing 710 cm of granular anhydrous sodium sulfate (Section 7.2.1). Rinse the separatory funnel with 3050 mL of solvent, and pour through the drying column.

Collect each extract in a round-bottom flask. Re-concentrate the sample and QC aliquots per Sections 12.6 through 12.7, and clean up the samples and QC aliquots per Section 13.

12.6Macro-ConcentrationExtracts in toluene are concentrated using a rotary evaporator or a heating mantle; extracts in methylene chloride or hexane are concentrated using a rotary evaporator, heating mantle, or Kuderna-Danish apparatus.

12.6.1Rotary evaporationConcentrate the extracts in separate round-bottom flasks.

12.6.1.1Assemble the rotary evaporator according to manufacturer's instructions, and warm the water bath to 45 C. On a daily basis, preclean the rotary evaporator by concentrating 100 mL of clean extraction solvent through the system. Archive both the concentrated solvent and the solvent in the catch flask for a contamination check if necessary. Between samples, three 23 mL aliquots of solvent should be rinsed down the feed tube into a waste beaker.

12.6.1.2Attach the round-bottom flask containing the sample extract to the rotary evaporator. Slowly apply vacuum to the system, and begin rotating the sample flask.

12.6.1.3Lower the flask into the water bath, and adjust the speed of rotation and the temperature as required to complete concentration in 1520 minutes. At the proper rate of concentration, the flow of solvent into the receiving flask will be steady, but no bumping or visible boiling of the extract will occur.

Note: If the rate of concentration is too fast, analyte loss may occur.

12.6.1.4When the liquid in the concentration flask has reached an apparent volume of approximately 2 mL, remove the flask from the water bath and stop the rotation. Slowly and carefully admit air into the system. Be sure not to open the valve so quickly that the sample is blown out of the flask. Rinse the feed tube with approximately 2 mL of solvent.

12.6.1.5Proceed to Section 12.6.4 for preparation for back-extraction or micro-concentration and solvent exchange.

12.6.2Heating mantleConcentrate the extracts in separate round-bottom flasks.

12.6.2.1Add one or two clean boiling chips to the round-bottom flask, and attach a three-ball macro Snyder column. Prewet the column by adding approximately 1 mL of solvent through the top. Place the round-bottom flask in a heating mantle, and apply heat as required to complete the concentration in 1520 minutes. At the proper rate of distillation, the balls of the column will actively chatter, but the chambers will not flood.

12.6.2.2When the liquid has reached an apparent volume of approximately 10 mL, remove the round-bottom flask from the heating mantle and allow the solvent to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the glass joint into the receiver with small portions of solvent.

12.6.2.3Proceed to Section 12.6.4 for preparation for back-extraction or micro-concentration and solvent exchange.

12.6.3Kuderna-Danish (K-D)Concentrate the extracts in separate 500 mL K-D flasks equipped with 10 mL concentrator tubes. The K-D technique is used for solvents such as methylene chloride and hexane. Toluene is difficult to concentrate using the K-D technique unless a water bath fed by a steam generator is used.

12.6.3.1Add one to two clean boiling chips to the receiver. Attach a three-ball macro Snyder column. Prewet the column by adding approximately 1 mL of solvent through the top. Place the K-D apparatus in a hot water bath so that the entire lower rounded surface of the flask is bathed with steam.

12.6.3.2Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 1520 minutes.

At the proper rate of distillation, the balls of the column will actively chatter but the chambers will not flood.

12.6.3.3When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus from the bath and allow the solvent to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 12 mL of solvent. A 5 mL syringe is recommended for this operation.

12.6.3.4Remove the three-ball Snyder column, add a fresh boiling chip, and attach a two-ball micro Snyder column to the concentrator tube.

Prewet the column by adding approximately 0.5 mL of solvent through the top. Place the apparatus in the hot water bath.

12.6.3.5Adjust the vertical position and the water temperature as required to complete the concentration in 510 minutes. At the proper rate of distillation, the balls of the column will actively chatter but the chambers will not flood.

12.6.3.6When the liquid reaches an apparent volume of 0.5 mL, remove the apparatus from the water bath and allow to drain and cool for at least 10 minutes.

12.6.3.7Proceed to 12.6.4 for preparation for back-extraction or micro-concentration and solvent exchange.

12.6.4Preparation for back-extraction or micro-concentration and solvent exchange.

12.6.4.1For back-extraction (Section 12.5), transfer the extract to a 250 mL separatory funnel. Rinse the concentration vessel with small portions of hexane, adjust the hexane volume in the separatory funnel to 1020 mL, and proceed to back-extraction (Section 12.5).

12.6.4.2For determination of the weight of residue in the extract, or for clean-up procedures other than back-extraction, transfer the extract to a blowdown vial using two to three rinses of solvent. Proceed with micro-concentration and solvent exchange (Section 12.7).

12.7Micro-Concentration and Solvent Exchange.

12.7.1Extracts to be subjected to GPC or HPLC cleanup are exchanged into methylene chloride. Extracts to be cleaned up using silica gel, alumina, carbon, and/or Florisil are exchanged into hexane.

12.7.2Transfer the vial containing the sample extract to a nitrogen blowdown device. Adjust the flow of nitrogen so that the surface of the solvent is just visibly disturbed.

Note: A large vortex in the solvent may cause analyte loss.

12.7.3Lower the vial into a 45 C water bath and continue concentrating.

12.7.3.1If the extract is to be concentrated to dryness for weight determination (Sections 12.4.1.8, 12.4.2.7, and 13.7.1.4), blow dry until a constant weight is obtained.

12.7.3.2If the extract is to be concentrated for injection into the GC/MS or the solvent is to be exchanged for extract cleanup, proceed as follows: 12.7.4When the volume of the liquid is approximately 100 L, add 23 mL of the desired solvent (methylene chloride for GPC and HPLC, or hexane for the other cleanups) and continue concentration to approximately 100 L.

Repeat the addition of solvent and concentrate once more.

12.7.5If the extract is to be cleaned up by GPC, adjust the volume of the extract to 5.0 mL with methylene chloride. If the extract is to be cleaned up by HPLC, further concentrate the extract to 30 L. Proceed with GPC or HPLC cleanup (Section 13.2 or 13.6, respectively).

12.7.6If the extract is to be cleaned up by column chromatography (alumina, silica gel, Carbopak/Celite, or Florisil), bring the final volume to 1.0 mL with hexane. Proceed with column cleanups (Sections 13.3 through 13.5 and 13.8).

12.7.7If the extract is to be concentrated for injection into the GC/MS (Section 14), quantitatively transfer the extract to a 0.3 mL conical vial for final concentration, rinsing the larger vial with hexane and adding the rinse to the conical vial. Reduce the volume to approximately 100 L. Add 10 L of nonane to the vial, and evaporate the solvent to the level of the nonane. Seal the vial and label with the sample number.

Store in the dark at room temperature until ready for GC/MS analysis. If GC/MS analysis will not be performed on the same day, store the vial at 13.0Extract Cleanup 13.1Cleanup may not be necessary for relatively clean samples (e.g., treated effluents, groundwater, drinking water). If particular circumstances require the use of a cleanup procedure, the analyst may use any or all of the procedures below or any other appropriate procedure. Before using a cleanup procedure, the analyst must demonstrate that the requirements of Section 9.2 can be met using the cleanup procedure. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the cleanup procedures may be optimized for isolation of these two compounds.

13.1.1Gel permeation chromatography (Section 13.2) removes high molecular weight interferences that cause GC column performance to degrade. It should be used for all soil and sediment extracts and may be used for water extracts that are expected to contain high molecular weight organic compounds (e.g., polymeric materials, humic acids).

13.1.2Acid, neutral, and basic silica gel (Section 13.3), alumina (Section 13.4), and Florisil (Section 13.8) are used to remove nonpolar and polar interferences. Alumina and Florisil are used to remove chlorodiphenyl ethers.

13.1.3Carbopak/Celite (Section 13.5) is used to remove nonpolar interferences.

13.1.4HPLC (Section 13.6) is used to provide specificity for the 2,3,7,8-substituted and other CDD and CDF isomers.

13.1.5The anthropogenic isolation column (Section 13.7.1), acidified silica gel batch adsorption procedure (Section 13.7.2), and sulfuric acid and base back-extraction (Section 13.7.3) are used for removal of lipids from tissue samples.

13.2Gel Permeation Chromatography (GPC).

13.2.1Column packing.

13.2.1.1Place 7075 g of SX3 Bio-beads (Section 6.7.1.1) in a 400500 mL beaker.

13.2.1.2Cover the beads with methylene chloride and allow to swell overnight (a minimum of 12 hours).

13.2.1.3Transfer the swelled beads to the column (Section 6.7.1.1) and pump solvent through the column, from bottom to top, at 4.55.5 mL/minute prior to connecting the column to the detector.

13.2.1.4After purging the column with solvent for one to two hours, adjust the column head pressure to 710 psig and purge for four to five hours to remove air. Maintain a head pressure of 710 psig. Connect the column to the detector (Section 6.7.1.4).

13.2.2Column calibration.

13.2.2.1Load 5 mL of the calibration solution (Section 7.4) into the sample loop.

13.2.2.2Inject the calibration solution and record the signal from the detector. The elution pattern will be corn oil, bis(2-ethyl hexyl)phthalate, pentachlorophenol, perylene, and sulfur.

13.2.2.3Set the dump time to allow >85% removal of the corn oil and >85% collection of the phthalate.

13.2.2.4Set the collect time to the peak minimum between perylene and sulfur.

13.2.2.5Verify the calibration with the calibration solution after every 20 extracts. Calibration is verified if the recovery of the pentachlorophenol is greater than 85%. If calibration is not verified, the system shall be recalibrated using the calibration solution, and the previous 20 samples shall be re-extracted and cleaned up using the calibrated GPC system.

13.2.3Extract cleanupGPC requires that the column not be overloaded. The column specified in this method is designed to handle a maximum of 0.5 g of high molecular weight material in a 5 mL extract. If the extract is known or expected to contain more than 0.5 g, the extract is split into aliquots for GPC, and the aliquots are combined after elution from the column. The residue content of the extract may be obtained gravimetrically by evaporating the solvent from a 50 L aliquot.

13.2.3.1Filter the extract or load through the filter holder (Section 6.7.1.3) to remove the particles. Load the 5.0 mL extract onto the column.

13.2.3.2Elute the extract using the calibration data determined in Section 13.2.2. Collect the eluate in a clean 400500 mL beaker.

13.2.3.3Rinse the sample loading tube thoroughly with methylene chloride between extracts to prepare for the next sample.

13.2.3.4If a particularly dirty extract is encountered, a 5.0 mL methylene chloride blank shall be run through the system to check for carry-over.

13.2.3.5Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the GC/MS.

13.3Silica Gel Cleanup.

13.3.1Place a glass-wool plug in a 15 mm ID chromatography column (Section 6.7.4.2). Pack the column bottom to top with: 1 g silica gel (Section 7.5.1.1), 4 g basic silica gel (Section 7.5.1.3), 1 g silica gel, 8 g acid silica gel (Section 7.5.1.2), 2 g silica gel, and 4 g granular anhydrous sodium sulfate (Section 7.2.1). Tap the column to settle the adsorbents.

13.3.2Pre-elute the column with 50100 mL of hexane. Close the stopcock when the hexane is within 1 mm of the sodium sulfate. Discard the eluate. Check the column for channeling. If channeling is present, discard the column and prepare another.

13.3.3Apply the concentrated extract to the column. Open the stopcock until the extract is within 1 mm of the sodium sulfate.

13.3.4Rinse the receiver twice with 1 mL portions of hexane, and apply separately to the column. Elute the CDDs/CDFs with 100 mL hexane, and collect the eluate.

13.3.5Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the HPLC or GC/MS.

13.3.6For extracts of samples known to contain large quantities of other organic compounds (such as paper mill effluents), it may be advisable to increase the capacity of the silica gel column. This may be accomplished by increasing the strengths of the acid and basic silica gels. The acid silica gel (Section 7.5.1.2) may be increased in strength to as much as 44% w/w (7.9 g sulfuric acid added to 10 g silica gel). The basic silica gel (Section 7.5.1.3) may be increased in strength to as much as 33% w/w (50 mL 1N NaOH added to 100 g silica gel), or the potassium silicate (Section 7.5.1.4) may be used.

Note: The use of stronger acid silica gel (44% w/w) may lead to charring of organic compounds in some extracts. The charred material may retain some of the analytes and lead to lower recoveries of CDDs/CDFs.

Increasing the strengths of the acid and basic silica gel may also require different volumes of hexane than those specified above to elute the analytes off the column. Therefore, the performance of the method after such modifications must be verified by the procedure in Section 9.2.

13.4Alumina Cleanup.

13.4.1Place a glass-wool plug in a 15 mm ID chromatography column (Section 6.7.4.2).

13.4.2If using acid alumina, pack the column by adding 6 g acid alumina (Section 7.5.2.1). If using basic alumina, substitute 6 g basic alumina (Section 7.5.2.2). Tap the column to settle the adsorbents.

13.4.3Pre-elute the column with 50100 mL of hexane. Close the stopcock when the hexane is within 1 mm of the alumina.

13.4.4Discard the eluate. Check the column for channeling. If channeling is present, discard the column and prepare another.

13.4.5Apply the concentrated extract to the column. Open the stopcock until the extract is within 1 mm of the alumina.

13.4.6Rinse the receiver twice with 1 mL portions of hexane and apply separately to the column. Elute the interfering compounds with 100 mL hexane and discard the eluate.

13.4.7The choice of eluting solvents will depend on the choice of alumina (acid or basic) made in Section 13.4.2.

13.4.7.1If using acid alumina, elute the CDDs/CDFs from the column with 20 mL methylene chloride:hexane (20:80 v/v). Collect the eluate.

13.4.7.2If using basic alumina, elute the CDDs/CDFs from the column with 20 mL methylene chloride:hexane (50:50 v/v). Collect the eluate.

13.4.8Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the HPLC or GC/MS.

13.5Carbon Column.

13.5.1Cut both ends from a 10 mL disposable serological pipet (Section 6.7.3.2) to produce a 10 cm column. Fire-polish both ends and flare both ends if desired. Insert a glass-wool plug at one end, and pack the column with 0.55 g of Carbopak/Celite (Section 7.5.3.3) to form an adsorbent bed approximately 2 cm long. Insert a glass-wool plug on top of the bed to hold the adsorbent in place.

13.5.2Pre-elute the column with 5 mL of toluene followed by 2 mL of methylene chloride: methanol:toluene (15:4:1 v/v), 1 mL of methylene chloride:cyclohexane (1:1 v/v), and 5 mL of hexane. If the flow rate of eluate exceeds 0.5 mL/minute, discard the column.

13.5.3When the solvent is within 1 mm of the column packing, apply the sample extract to the column. Rinse the sample container twice with 1 mL portions of hexane and apply separately to the column. Apply 2 mL of hexane to complete the transfer.

13.5.4Elute the interfering compounds with two 3 mL portions of hexane, 2 mL of methylene chloride:cyclohexane (1:1 v/v), and 2 mL of methylene chloride:methanol:toluene (15:4:1 v/v). Discard the eluate.

13.5.5Invert the column, and elute the CDDs/CDFs with 20 mL of toluene.

If carbon particles are present in the eluate, filter through glass-fiber filter paper.

13.5.6Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the HPLC or GC/MS.

13.6HPLC (Reference 6).

13.6.1Column calibration.

13.6.1.1Prepare a calibration standard containing the 2,3,7,8-substituted isomers and/or other isomers of interest at a concentration of approximately 500 pg/L in methylene chloride.

13.6.1.2Inject 30 L of the calibration solution into the HPLC and record the signal from the detector. Collect the eluant for reuse. The elution order will be the tetra- through octa-isomers.

13.6.1.3Establish the collection time for the tetra-isomers and for the other isomers of interest. Following calibration, flush the injection system with copious quantities of methylene chloride, including a minimum of five 50 L injections while the detector is monitored, to ensure that residual CDDs/CDFs are removed from the system.

13.6.1.4Verify the calibration with the calibration solution after every 20 extracts. Calibration is verified if the recovery of the CDDs/CDFs from the calibration standard (Section 13.6.1.1) is 75125% compared to the calibration (Section 13.6.1.2). If calibration is not verified, the system shall be recalibrated using the calibration solution, and the previous 20 samples shall be re-extracted and cleaned up using the calibrated system.

13.6.2Extract cleanupHPLC requires that the column not be overloaded.

The column specified in this method is designed to handle a maximum of 30 L of extract. If the extract cannot be concentrated to less than 30 L, it is split into fractions and the fractions are combined after elution from the column.

13.6.2.1Rinse the sides of the vial twice with 30 L of methylene chloride and reduce to 30 L with the evaporation apparatus (Section 12.7).

13.6.2.2Inject the 30 L extract into the HPLC.

13.6.2.3Elute the extract using the calibration data determined in Section 13.6.1. Collect the fraction(s) in a clean 20 mL concentrator tube containing 5 mL of hexane:acetone (1:1 v/v).

13.6.2.4If an extract containing greater than 100 ng/mL of total CDD or CDF is encountered, a 30 L methylene chloride blank shall be run through the system to check for carry-over.

13.6.2.5Concentrate the eluate per Section 12.7 for injection into the GC/MS.

13.7Cleanup of Tissue LipidsLipids are removed from the Soxhlet extract using either the anthropogenic isolation column (Section 13.7.1) or acidified silica gel (Section 13.7.2), or are removed from the HCl digested extract using sulfuric acid and base back-extraction (Section 13.7.3).

13.7.1Anthropogenic isolation column (References 22 and 27)Used for removal of lipids from the Soxhlet/SDS extraction (Section 12.4.1).

13.7.1.1Prepare the column as given in Section 7.5.4.

13.7.1.2Pre-elute the column with 100 mL of hexane. Drain the hexane layer to the top of the column, but do not expose the sodium sulfate.

13.7.1.3Load the sample and rinses (Section 12.4.1.9.2) onto the column by draining each portion to the top of the bed. Elute the CDDs/CDFs from the column into the apparatus used for concentration (Section 12.4.1.7) using 200 mL of hexane.

13.7.1.4Concentrate the cleaned up extract (Sections 12.6 through 12.7) to constant weight per Section 12.7.3.1. If more than 500 mg of material remains, repeat the cleanup using a fresh anthropogenic isolation column.

13.7.1.5Redissolve the extract in a solvent suitable for the additional cleanups to be used (Sections 13.2 through 13.6 and 13.8).

13.7.1.6Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent.

13.7.1.7Clean up the extract using the procedures in Sections 13.2 through 13.6 and 13.8. Alumina (Section 13.4) or Florisil (Section 13.8) and carbon (Section 13.5) are recommended as minimum additional cleanup steps.

13.7.1.8Following cleanup, concentrate the extract to 10 L as described in Section 12.7 and proceed with the analysis in Section 14.

13.7.2Acidified silica gel (Reference 28)Procedure alternate to the anthropogenic isolation column (Section 13.7.1) that is used for removal of lipids from the Soxhlet/SDS extraction (Section 12.4.1).

13.7.2.1Adjust the volume of hexane in the bottle (Section 12.4.1.9.2) to approximately 200 mL.

13.7.2.2Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent.

13.7.2.3Drop the stirring bar into the bottle, place the bottle on the stirring plate, and begin stirring.

13.7.2.4Add 30100 g of acid silica gel (Section 7.5.1.2) to the bottle while stirring, keeping the silica gel in motion. Stir for two to three hours.

Note: 30 grams of silica gel should be adequate for most samples and will minimize contamination from this source.

13.7.2.5After stirring, pour the extract through approximately 10 g of granular anhydrous sodium sulfate (Section 7.2.1) contained in a funnel with glass-fiber filter into a macro contration device (Section 12.6).

Rinse the bottle and sodium sulfate with hexane to complete the transfer.

13.7.2.6Concentrate the extract per Sections 12.6 through 12.7 and clean up the extract using the procedures in Sections 13.2 through 13.6 and 13.8. Alumina (Section 13.4) or Florisil (Section 13.8) and carbon (Section 13.5) are recommended as minimum additional cleanup steps.

13.7.3Sulfuric acid and base back-extraction. Used with HCl digested extracts (Section 12.4.2).

13.7.3.1Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent (Section 12.4.2.8.2).

13.7.3.2Add 10 mL of concentrated sulfuric acid to the bottle.

Immediately cap and shake one to three times. Loosen cap in a hood to vent excess pressure. Cap and shake the bottle so that the residue/solvent is exposed to the acid for a total time of approximately 45 seconds.

13.7.3.3Decant the hexane into a 250 mL separatory funnel making sure that no acid is transferred. Complete the quantitative transfer with several hexane rinses.

13.7.3.4Back extract the solvent/residue with 50 mL of potassium hydroxide solution per Section 12.5.2, followed by two reagent water rinses.

13.7.3.5Drain the extract through a filter funnel containing approximately 10 g of granular anhydrous sodium sulfate in a glass-fiber filter into a macro concentration device (Section 12.6).

13.7.3.6Concentrate the cleaned up extract to a volume suitable for the additional cleanups given in Sections 13.2 through 13.6 and 13.8. Gel permeation chromatography (Section 13.2), alumina (Section 13.4) or Florisil (Section 13.8), and Carbopak/Celite (Section 13.5) are recommended as minimum additional cleanup steps.

13.7.3.7Following cleanup, concentrate the extract to 10 L as described in Section 12.7 and proceed with analysis per Section 14.

13.8Florisil Cleanup (Reference 29).

13.8.1Pre-elute the activated Florisil column (Section 7.5.3) with 10 mL of methylene chloride followed by 10 mL of hexane:methylene chloride (98:2 v/v) and discard the solvents.

13.8.2When the solvent is within 1 mm of the packing, apply the sample extract (in hexane) to the column. Rinse the sample container twice with 1 mL portions of hexane and apply to the column.

13.8.3Elute the interfering compounds with 20 mL of hexane:methylene chloride (98:2) and discard the eluate.

13.8.4Elute the CDDs/CDFs with 35 mL of methylene chloride and collect the eluate. Concentrate the eluate per Sections 12.6 through 12.7 for further cleanup or for injection into the HPLC or GC/MS.

14.0HRGC/HRMS Analysis 14.1Establish the operating conditions given in Section 10.1.

14.2Add 10 uL of the appropriate internal standard solution (Section 7.12) to the sample extract immediately prior to injection to minimize the possibility of loss by evaporation, adsorption, or reaction. If an extract is to be reanalyzed and evaporation has occurred, do not add more instrument internal standard solution. Rather, bring the extract back to its previous volume (e.g., 19 L) with pure nonane only (18 L if 2 L injections are used).

14.3Inject 1.0 L or 2.0 L of the concentrated extract containing the internal standard solution, using on-column or splitless injection. The volume injected must be identical to the volume used for calibration (Section 10). Start the GC column initial isothermal hold upon injection. Start MS data collection after the solvent peak elutes. Stop data collection after the OCDD and OCDF have eluted. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, stop data collection after elution of these compounds. Return the column to the initial temperature for analysis of the next extract or standard.

15.0System and Laboratory Performance 15.1At the beginning of each 12-hour shift during which analyses are performed, GC/MS system performance and calibration are verified for all CDDs/CDFs and labeled compounds. For these tests, analysis of the CS3 calibration verification (VER) standard (Section 7.13 and Table 4) and the isomer specificity test standards (Section 7.15 and Table 5) shall be used to verify all performance criteria. Adjustment and/or recalibration (Section 10) shall be performed until all performance criteria are met. Only after all performance criteria are met may samples, blanks, IPRs, and OPRs be analyzed.

15.2MS ResolutionA static resolving power of at least 10,000 (10% valley definition) must be demonstrated at the appropriate m/z before any analysis is performed. Static resolving power checks must be performed at the beginning and at the end of each 12-hour shift according to procedures in Section 10.1.2. Corrective actions must be implemented whenever the resolving power does not meet the requirement.

15.3Calibration Verification.

15.3.1Inject the VER standard using the procedure in Section 14.

15.3.2The m/z abundance ratios for all CDDs/CDFs shall be within the limits in Table 9; otherwise, the mass spectrometer shall be adjusted until the m/z abundance ratios fall within the limits specified, and the verification test shall be repeated. If the adjustment alters the resolution of the mass spectrometer, resolution shall be verified (Section 10.1.2) prior to repeat of the verification test.

15.3.3The peaks representing each CDD/CDF and labeled compound in the VER standard must be present with S/N of at least 10; otherwise, the mass spectrometer shall be adjusted and the verification test repeated.

15.3.4Compute the concentration of each CDD/CDF compound by isotope dilution (Section 10.5) for those compounds that have labeled analogs (Table 1). Compute the concentration of the labeled compounds by the internal standard method (Section 10.6). These concentrations are computed based on the calibration data in Section 10.

15.3.5For each compound, compare the concentration with the calibration verification limit in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare the concentration to the limit in Table 6a. If all compounds meet the acceptance criteria, calibration has been verified and analysis of standards and sample extracts may proceed. If, however, any compound fails its respective limit, the measurement system is not performing properly for that compound. In this event, prepare a fresh calibration standard or correct the problem causing the failure and repeat the resolution (Section 15.2) and verification (Section 15.3) tests, or recalibrate (Section 10).

15.4Retention Times and GC Resolution.

15.4.1Retention times.

15.4.1.1AbsoluteThe absolute retention times of the 13C12-1,2,3,4TCDD and 13C12-1,2,3,7,8,9-HxCDD GCMS internal standards in the verification test (Section 15.3) shall be within 15 seconds of the retention times obtained during calibration (Sections 10.2.1 and 10.2.4).

15.4.1.2RelativeThe relative retention times of CDDs/CDFs and labeled compounds in the verification test (Section 15.3) shall be within the limits given in Table 2.

15.4.2GC resolution.

15.4.2.1Inject the isomer specificity standards (Section 7.15) on their respective columns.

15.4.2.2The valley height between 2,3,7,8-TCDD and the other tetra-dioxin isomers at m/z 319.8965, and between 2,3,7,8-TCDF and the other tetra-furan isomers at m/z 303.9016 shall not exceed 25% on their respective columns (Figures 6 and 7).

15.4.3If the absolute retention time of any compound is not within the limits specified or if the 2,3,7,8-isomers are not resolved, the GC is not performing properly. In this event, adjust the GC and repeat the verification test (Section 15.3) or recalibrate (Section 10), or replace the GC column and either verify calibration or recalibrate.

15.5Ongoing Precision and Recovery.

15.5.1Analyze the extract of the ongoing precision and recovery (OPR) aliquot (Section 11.4.2.5, 11.5.4, 11.6.2, 11.7.4, or 11.8.3.2) prior to analysis of samples from the same batch.

15.5.2Compute the concentration of each CDD/CDF by isotope dilution for those compounds that have labeled analogs (Section 10.5). Compute the concentration of 1,2,3,7,8,9-HxCDD, OCDF, and each labeled compound by the internal standard method (Section 10.6).

15.5.3For each CDD/CDF and labeled compound, compare the concentration to the OPR limits given in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare the concentration to the limits in Table 6a. If all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may proceed. If, however, any individual concentration falls outside of the range given, the extraction/concentration processes are not being performed properly for that compound. In this event, correct the problem, re-prepare, extract, and clean up the sample batch and repeat the ongoing precision and recovery test (Section 15.5).

15.5.4Add results that pass the specifications in Section 15.5.3 to initial and previous ongoing data for each compound in each matrix.

Update QC charts to form a graphic representation of continued laboratory performance. Develop a statement of laboratory accuracy for each CDD/CDF in each matrix type by calculating the average percent recovery (R) and the standard deviation of percent recovery (SR).

Express the accuracy as a recovery interval from R2SR to R=2SR. For example, if R=95% and SR=5%, the accuracy is 85105%.

15.6BlankAnalyze the method blank extracted with each sample batch immediately following analysis of the OPR aliquot to demonstrate freedom from contamination and freedom from carryover from the OPR analysis. The results of the analysis of the blank must meet the specifications in Section 9.5.2 before sample analyses may proceed.

16.0Qualitative Determination A CDD, CDF, or labeled compound is identified in a standard, blank, or sample when all of the criteria in Sections 16.1 through 16.4 are met.

16.1The signals for the two exact m/z's in Table 8 must be present and must maximize within the same two seconds.

16.2The signal-to-noise ratio (S/N) for the GC peak at each exact m/z must be greater than or equal to 2.5 for each CDD or CDF detected in a sample extract, and greater than or equal to 10 for all CDDs/CDFs in the calibration standard (Sections 10.2.3 and 15.3.3).

16.3The ratio of the integrated areas of the two exact m/z's specified in Table 8 must be within the limit in Table 9, or within 10% of the ratio in the midpoint (CS3) calibration or calibration verification (VER), whichever is most recent.

16.4The relative retention time of the peak for a 2,3,7,8-substituted CDD or CDF must be within the limit in Table 2. The retention time of peaks representing non-2,3,7,8-substituted CDDs/CDFs must be within the retention time windows established in Section 10.3.

16.5Confirmatory AnalysisIsomer specificity for 2,3,7,8-TCDF cannot be achieved on the DB5 column. Therefore, any sample in which 2,3,7,8-TCDF is identified by analysis on a DB5 column must have a confirmatory analysis performed on a DB225, SP2330, or equivalent GC column. The operating conditions in Section 10.1.1 may be adjusted to optimize the analysis on the second GC column, but the GC/MS must meet the mass resolution and calibration specifications in Section 10.

16.6If the criteria for identification in Sections 16.1 through 16.5 are not met, the CDD or CDF has not been identified and the results may not be reported for regulatory compliance purposes. If interferences preclude identification, a new aliquot of sample must be extracted, further cleaned up, and analyzed.

17.0Quantitative Determination 17.1Isotope Dilution QuantitationBy adding a known amount of a labeled compound to every sample prior to extraction, correction for recovery of the CDD/CDF can be made because the CDD/CDF and its labeled analog exhibit similar effects upon extraction, concentration, and gas chromatography. Relative response (RR) values are used in conjunction with the initial calibration data described in Section 10.5 to determine concentrations directly, so long as labeled compound spiking levels are constant, using the following equation: (image) where: Cex = The concentration of the CDD/CDF in the extract, and the other terms are as defined in Section 10.5.2.

17.1.1Because of a potential interference, the labeled analog of OCDF is not added to the sample. Therefore, OCDF is quantitated against labeled OCDD. As a result, the concentration of OCDF is corrected for the recovery of the labeled OCDD. In instances where OCDD and OCDF behave differently during sample extraction, concentration, and cleanup procedures, this may decrease the accuracy of the OCDF results. However, given the low toxicity of this compound relative to the other dioxins and furans, the potential decrease in accuracy is not considered significant.

17.1.2Because 13C12-1,2,3,7,8,9-HxCDD is used as an instrument internal standard (i.e., not added before extraction of the sample), it cannot be used to quantitate the 1,2,3,7,8,9-HxCDD by strict isotope dilution procedures. Therefore, 1,2,3,7,8,9-HxCDD is quantitated using the averaged response of the labeled analogs of the other two 2,3,7,8-substituted HxCDD's: 1,2,3,4,7,8-HxCDD and 1,2,3,6,7,8-HxCDD. As a result, the concentration of 1,2,3,7,8,9-HxCDD is corrected for the average recovery of the other two HxCDD's.

17.1.3Any peaks representing non-2,3,7,8-substituted CDDs/CDFs are quantitated using an average of the response factors from all of the labeled 2,3,7,8-isomers at the same level of chlorination.

17.2Internal Standard Quantitation and Labeled Compound Recovery.

17.2.1Compute the concentrations of 1,2,3,7,8,9-HxCDD, OCDF, the 13C-labeled analogs and the 37C-labeled cleanup standard in the extract using the response factors determined from the initial calibration data (Section 10.6) and the following equation: (image) where: Cex = The concentration of the CDD/CDF in the extract, and the other terms are as defined in Section 10.6.1.

Note: There is only one m/z for the 37Cl-labeled standard.

17.2.2 Using the concentration in the extract determined above, compute the percent recovery of the 13C-labeled compounds and the 37C-labeled cleanup standard using the following equation: (image) 17.3The concentration of a CDD/CDF in the solid phase of the sample is computed using the concentration of the compound in the extract and the weight of the solids (Section 11.5.1), as follows: (image) where: Cex = The concentration of the compound in the extract.

Vex = The extract volume in mL.

Ws = The sample weight (dry weight) in kg.

17.4The concentration of a CDD/CDF in the aqueous phase of the sample is computed using the concentration of the compound in the extract and the volume of water extracted (Section 11.4 or 11.5), as follows: (image) where: Cex = The concentration of the compound in the extract.

Vex = The extract volume in mL.

Vs = The sample volume in liters.

17.5If the SICP area at either quantitation m/z for any compound exceeds the calibration range of the system, a smaller sample aliquot is extracted.

17.5.1For aqueous samples containing 1% solids or less, dilute 100 mL, 10 mL, etc., of sample to 1 L with reagent water and re-prepare, extract, clean up, and analyze per Sections 11 through 14.

17.5.2For samples containing greater than 1% solids, extract an amount of sample equal to 1/10, 1/100, etc., of the amount used in Section 11.5.1. Re-prepare, extract, clean up, and analyze per Sections 11 through 14.

17.5.3If a smaller sample size will not be representative of the entire sample, dilute the sample extract by a factor of 10, adjust the concentration of the instrument internal standard to 100 pg/L in the extract, and analyze an aliquot of this diluted extract by the internal standard method.

17.6Results are reported to three significant figures for the CDDs/CDFs and labeled compounds found in all standards, blanks, and samples.

17.6.1Reporting units and levels.

17.6.1.1Aqueous samplesReport results in pg/L (parts-per-quadrillion).

17.6.1.2Samples containing greater than 1% solids (soils, sediments, filter cake, compost)Report results in ng/kg based on the dry weight of the sample. Report the percent solids so that the result may be corrected.

17.6.1.3TissuesReport results in ng/kg of wet tissue, not on the basis of the lipid content of the sample. Report the percent lipid content, so that the data user can calculate the concentration on a lipid basis if desired.

17.6.1.4Reporting level.

17.6.1.4.1Standards (VER, IPR, OPR) and samplesReport results at or above the minimum level (Table 2). Report results below the minimum level as not detected or as required by the regulatory authority.

17.6.1.4.2BlanksReport results above one-third the ML.

17.6.2Results for CDDs/CDFs in samples that have been diluted are reported at the least dilute level at which the areas at the quantitation m/z's are within the calibration range (Section 17.5).

17.6.3For CDDs/CDFs having a labeled analog, results are reported at the least dilute level at which the area at the quantitation m/z is within the calibration range (Section 17.5) and the labeled compound recovery is within the normal range for the method (Section 9.3 and Tables 6, 6a, 7, and 7a).

17.6.4Additionally, if requested, the total concentration of all isomers in an individual level of chlorination (i.e., total TCDD, total TCDF, total Paced, etc.) may be reported by summing the concentrations of all isomers identified in that level of chlorination, including both 2,3,7,8-substituted and non-2,3,7,8-substituted isomers.

18.0Analysis of Complex Samples 18.1Some samples may contain high levels (>10 ng/L; >1000 ng/kg) of the compounds of interest, interfering compounds, and/or polymeric materials. Some extracts will not concentrate to 10 L (Section 12.7); others may overload the GC column and/or mass spectrometer.

18.2Analyze a smaller aliquot of the sample (Section 17.5) when the extract will not concentrate to 10 L after all cleanup procedures have been exhausted.

18.3Chlorodiphenyl EthersIf chromatographic peaks are detected at the retention time of any CDDs/CDFs in any of the m/z channels being monitored for the chlorodiphenyl ethers (Table 8), cleanup procedures must be employed until these interferences are removed. Alumina (Section 13.4) and Florisil (Section 13.8) are recommended for removal of chlorodiphenyl ethers.

18.4Recovery of Labeled CompoundsIn most samples, recoveries of the labeled compounds will be similar to those from reagent water or from the alternate matrix (Section 7.6).

18.4.1If the recovery of any of the labeled compounds is outside of the normal range (Table 7), a diluted sample shall be analyzed (Section 17.5).

18.4.2If the recovery of any of the labeled compounds in the diluted sample is outside of normal range, the calibration verification standard (Section 7.13) shall be analyzed and calibration verified (Section 15.3).

18.4.3If the calibration cannot be verified, a new calibration must be performed and the original sample extract reanalyzed.

18.4.4If the calibration is verified and the diluted sample does not meet the limits for labeled compound recovery, the method does not apply to the sample being analyzed and the result may not be reported for regulatory compliance purposes. In this case, alternate extraction and cleanup procedures in this method must be employed to resolve the interference. If all cleanup procedures in this method have been employed and labeled compound recovery remains outside of the normal range, extraction and/or cleanup procedures that are beyond this scope of this method will be required to analyze these samples.

19.0Pollution Prevention 19.1The solvents used in this method pose little threat to the environment when managed properly. The solvent evaporation techniques used in this method are amenable to solvent recovery, and it is recommended that the laboratory recover solvents wherever feasible.

19.2Standards should be prepared in volumes consistent with laboratory use to minimize disposal of standards.

20.0Waste Management 20.1It is the laboratory's responsibility to comply with all federal, state, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance is also required with any sewage discharge permits and regulations.

20.2Samples containing HCl to pH 20.3The CDDs/CDFs decompose above 800 C. Low-level waste such as absorbent paper, tissues, animal remains, and plastic gloves may be burned in an appropriate incinerator. Gross quantities (milligrams) should be packaged securely and disposed of through commercial or governmental channels that are capable of handling extremely toxic wastes.

20.4Liquid or soluble waste should be dissolved in methanol or ethanol and irradiated with ultraviolet light with a wavelength shorter than 290 nm for several days. Use F40 BL or equivalent lamps. Analyze liquid wastes, and dispose of the solutions when the CDDs/CDFs can no longer be detected.

20.5For further information on waste management, consult The Waste Management Manual for Laboratory Personnel and Less is BetterLaboratory Chemical Management for Waste Reduction, available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.

21.0Method Performance Method performance was validated and performance specifications were developed using data from EPA's international interlaboratory validation study (References 3031) and the EPA/paper industry Long-Term Variability Study of discharges from the pulp and paper industry (58 FR 66078).

22.0References 1. Tondeur, Yves. Method 8290: Analytical Procedures and Quality Assurance for Multimedia Analysis of Polychlorinated Dibenzo-p-dioxins and Dibenzofurans by High Resolution Gas Chromatography/High Resolution Mass Spectrometry, USEPA EMSL, Las Vegas, Nevada, June 1987.

2. Measurement of 2,3,7,8-Tetrachlorinated Dibenzo-p-dioxin (TCDD) and 2,3,7,8-Tetrachlorinated Dibenzofuran (TCDF) in Pulp, Sludges, Process Samples and Wastewaters from Pulp and Paper Mills, Wright State University, Dayton, OH 45435, June 1988.

3. NCASI Procedures for the Preparation and Isomer Specific Analysis of Pulp and Paper Industry Samples for 2,3,7,8-TCDD and 2,3,7,8-TCDF, National Council of the Paper Industry for Air and Stream Improvement Inc., 260 Madison Avenue, New York, NY 10016, Technical Bulletin No.

551, Pre-Release Copy, July 1988.

4. Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in Fish, USEPA, Environmental Research Laboratory, 6201 Congdon Boulevard, Duluth, MN 55804, April 1988.

5. Tondeur, Yves. Proposed GC/MS Methodology for the Analysis of PCDDs and PCDFs in Special Analytical Services Samples, Triangle Laboratories, Inc., 80110 Capitola Dr, Research Triangle Park, NC 27713, January 1988; updated by personal communication September 1988.

6. Lamparski, L.L. and Nestrick, T.J. Determinationof Tetra-, Hexa-, Hepta-, and Octachlorodibenzo-p-dioxin Isomers in Particulate Samples at Parts per Trillion Levels, Analytical Chemistry, 52: 20452054, 1980.

7. Lamparski, L.L. and Nestrick, T.J. Novel Extraction Device for the Determination of Chlorinated Dibenzo-p-dioxins (PCDDs) and Dibenzofurans (PCDFs) in Matrices Containing Water, Chemosphere, 19:2731, 1989.

8. Patterson, D.G., et. al. Control of Interferences in the Analysis of Human Adipose Tissue for 2,3,7,8-Tetrachlorodibenzo-p-dioxin, Environmental Toxicological Chemistry, 5:355360, 1986.

9. Stanley, John S. and Sack, Thomas M. Protocol for the Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin by High Resolution Gas Chromatography/High Resolution Mass Spectrometry, USEPA EMSL, Las Vegas, Nevada 89114, EPA 600/486004, January 1986.

10. Working with Carcinogens, Department of Health, Education, & Welfare, Public Health Service, Centers for Disease Control, NIOSH, Publication 77206, August 1977, NTIS PB277256.

11. OSHA Safety and Health Standards, General Industry, OSHA 2206, 29 CFR 1910.

12. Safety in Academic Chemistry Laboratories, ACS Committee on Chemical Safety, 1979.

13. Standard Methods for the Examination of Water and Wastewater, 18th edition and later revisions, American Public Health Association, 1015 15th St, N.W., Washington, DC 20005, 135: Section 1090 (Safety), 1992.

14. Method 6132,3,7,8-Tetrachlorodibenzo-p-dioxin, 40 CFR 136 (49 FR 43234), October 26, 1984, Section 4.1.

15. Provost, L.P. and Elder, R.S. Interpretation of Percent Recovery Data, American Laboratory, 15: 5683, 1983.

16. Standard Practice for Sampling Water, ASTM Annual Book of Standards, ASTM, 1916 Race Street, Philadelphia, PA 191031187, 1980.

17. Methods 330.4 and 330.5 for Total Residual Chlorine, USEPA, EMSL, Cincinnati, OH 45268, EPA 600/479020, March 1979.

18. Handbook of Analytical Quality Control in Water and Wastewater Laboratories, USEPA EMSL, Cincinnati, OH 45268, EPA600/479019, March 1979.

19. Williams, Rick. Letter to Bill Telliard, June 4, 1993, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 7035191140.

20. Barkowski, Sarah. Fax to Sue Price, August 6, 1992, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria VA 22314, 7035191140.

21. Analysis of Multi-media, Multi-concentration Samples for Dioxins and Furans, PCDD/PCDF Analyses Data Package, Narrative for Episode 4419, MRI Project No. 3091-A, op.cit. February 12, 1993, Available from the EPA Sample Control Center operated by DynCorp Viar Inc, 300 N Lee St, Alexandria, VA 22314 (7035191140).

22. Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in Fish, U.S. Environmental Protection Agency, Environmental Research Laboratory, Duluth, MN 55804, EPA/600/390/022, March 1990.

23. Afghan, B.K., Carron, J., Goulden, P.D., Lawrence, J., Leger, D., Onuska, F., Sherry, J., and Wilkenson, R.J., Recent Advances in Ultratrace Analysis of Dioxins and Related Halogenated Hydrocarbons, Can J. Chem., 65: 10861097, 1987.

24. Sherry, J.P. and Tse, H. A Procedure for the Determination of Polychlorinated Dibenzo-p-dioxins in Fish, Chemosphere, 20: 865872, 1990.

25. Preliminary Fish Tissue Study, Results of Episode 4419, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 7035191140.

26. Nestrick, Terry L. DOW Chemical Co., personal communication with D.R. Rushneck, April 8, 1993. Details available from the U.S.

Environmental Protection Agency Sample Control Center operated by DynCorp Viar Inc, 300 N Lee St, Alexandria, VA 22314, 7035191140.

27. Barnstadt, Michael. Big Fish Column, Triangle Laboratories of RTP, Inc., SOP 12990, 27 March 27, 1992.

28. Determination of Polychlorinated Dibenzo-p-Dioxins (PCDD) and Dibenzofurans (PCDF) in Environmental Samples Using EPA Method 1613, Chemical Sciences Department, Midwest Research Institute, 425 Volker Boulevard, Kansas City, MO 441102299, Standard Operating Procedure No.

CS153, January 15, 1992.

29. Ryan, John J. Raymonde Lizotte and William H. Newsome, J. Chromatog.

303 (1984) 351-360.

30. Telliard, William A., McCarty, Harry B., and Riddick, Lynn S.

Results of the Interlaboratory Validation Study of USEPA Method 1613 for the Analysis of Tetra-through Octachlorinated Dioxins and Furans by Isotope Dilution GC/MS, Chemosphere, 27, 4146 (1993).

31. Results of the International Interlaboratory Validation Study of USEPA Method 1613, October 1994, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 7035191140.

23.0Tables and Figures Table 1_Chlorinated Dibenzo-p-Dioxins and Furans Determined by Isotope Dilution and Internal Standard High Resolution Gas Chromatography (HRGC)/High Resolution Mass Spectrometry (HRMS) ---------------------------------------------------------------------------------------------------------------- CDDs/CDFs \1\ CAS registry Labeled analog CAS registry ---------------------------------------------------------------------------------------------------------------- 2,3,7,8-TCDD.................................. 1746-01-6 13C12-2,3,7,8-TCDD.............. 76523-40-5 37Cl4-2,3,7,8-TCDD.............. 85508-50-5 Total TCDD.................................... 41903-57-5 2,3,7,8-TCDF.................................. 51207-31-9 13C12-2,3,7,8-TCDF.............. 89059-46-1 Total-TCDF.................................... 55722-27-5 1,2,3,7,8-PeCDD............................... 40321-76-4 13C12-1,2,3,7,8-PeCDD........... 109719-79-1 Total-PeCDD................................... 36088-22-9 1,2,3,7,8-PeCDF............................... 57117-41-6 13C12-1,2,3,7,8-PeCDF........... 109719-77-9 2,3,4,7,8-PeCDF............................... 57117-31-4 13C12-2,3,4,7,8-PeCDF........... 116843-02-8 Total-PeCDF................................... 30402-15-4 1,2,3,4,7,8-HxCDD............................. 39227-28-6 13C12-1,2,3,4,7,8-HxCDD......... 109719-80-4 1,2,3,6,7,8-HxCDD............................. 57653-85-7 13C12-1,2,3,6,7,8-HxCDD......... 109719-81-5 1,2,3,7,8,9-HxCDD............................. 19408-74-3 13C12-1,2,3,7,8,9-HxCDD......... 109719-82-6 Total-HxCDD................................... 34465-46-8 1,2,3,4,7,8-HxCDF............................. 70648-26-9 13C12-1,2,3,4,7,8-HxCDF......... 114423-98-2 1,2,3,6,7,8-HxCDF............................. 57117-44-9 13C12-1,2,3,6,7,8-HxCDF......... 116843-03-9 1,2,3,7,8,9-HxCDF............................. 72918-21-9 13C12-1,2,3,7,8,9-HxCDF......... 116843-04-0 2,3,4,6,7,8-HxCDF............................. 60851-34-5 13C12-2,3,4,6,7,8-HxCDF......... 116843-05-1 Total-HxCDF................................... 55684-94-1 1,2,3,4,6,7,8-HpCDD........................... 35822-46-9 13C12-1,2,3,4,6,7,8-HpCDD....... 109719-83-7 Total-HpCDD................................... 37871-00-4 1,2,3,4,6,7,8-HpCDF........................... 67562-39-4 13C12-1,2,3,4,6,7,8-HpCDF....... 109719-84-8 1,2,3,4,7,8,9-HpCDF........................... 55673-89-7 13C12-1,2,3,4,7,8,9-HpCDF....... 109719-94-0 Total-HpCDF................................... 38998-75-3 OCDD.......................................... 3268-87-9 13C12-OCDD...................... 114423-97-1 OCDF.......................................... 39001-02-0 Not used........................

---------------------------------------------------------------------------------------------------------------- \1\ Chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans.

TCDD = Tetrachlorodibenzo-p-dioxin.

TCDF = Tetrachlorodibenzofuran.

PeCDD = Pentachlorodibenzo-p-dioxin.

PeCDF = Pentachlorodibenzofuran.

HxCDD = Hexachlorodibenzo-p-dioxin.

HxCDF = Hexachlorodibenzofuran.

HpCDD = Heptachlorodibenzo-p-dioxin.

HpCDF = Heptachlorodibenzofuran.

OCDD = Octachlorodibenzo-p-dioxin.

OCDF = Octachlorodibenzofuran.

Table 2_Retention Time References, Quantitation References, Relative Retention Times, and Minimum Levels for CDDS and DCFS ---------------------------------------------------------------------------------------------------------------- Minimum level \1\ -------------------------------- Retention time and Relative Extract CDD/CDF quantitation reference retention time Water (pg/ Solid (ng/ (pg/ L; ppq) kg; ppt) L; ppb) ---------------------------------------------------------------------------------------------------------------- Compounds using 13 C12-1,2,3,4-TCDD as the Injection Internal Standard ---------------------------------------------------------------------------------------------------------------- 2,3,7,8-TCDF......................... 13 C12-2,3,7,8-TCDF..... 0.999-1.003 10 1 0.5 2,3,7,8-TCDD......................... 13 C12-2,3,7,8-TCDD..... 0.999-1.002 10 1 0.5 1,2,3,7,8-Pe......................... 13 C12-1,2,3,7,8-PeCDF.. 0.999-1.002 50 5 2.5 2,3,4,7,8-PeCDF...................... 13 C12-2,3,4,7,8-PeCDF.. 0.999-1.002 50 5 2.5 1,2,3,7,8-PeCDD...................... 13 C12-1,2,3,7,8-PeCDD.. 0.999-1.002 50 5 2.5 13 C12-2,3,7,8-TCDF.................. 13 C12-1,2,3,4-TCDD..... 0.923-1.103 ......... ......... .........

13 C12-2,3,7,8-TCDD.................. 13 C12-1,2,3,4-TCDD..... 0.976-1.043 ......... ......... .........

13 C12-2,3,7,8-TCDD.................. 13 C12-1,2,3,4-TCDD..... 0.989-1.052 ......... ......... .........

13 C12-1,2,3,7,8-PeCDF............... 13 C12-1,2,3,4-TCDD..... 1.000-1.425 ......... ......... .........

13 C12-2,3,4,7,8-PeCDF............... 13 C12-1,2,3,4-TCDD..... 1.001-1.526 ......... ......... .........

13 C12-1,2,3,7,8-PeCDF............... 13 C12-1,2,3,4-TCDD..... 1.000-1.567 ......... ......... .........

---------------------------------------------------------------------------------------------------------------- Compounds using 13 C12-1,2,3,7,8,9-HxCDD as the Injection Internal Standard ---------------------------------------------------------------------------------------------------------------- 1,2,3,4,7,8-HxCDF.................... 13 C12-1,2,3,4,7,8-HxCDF 0.999-1.001 50 5 2.5 1,2,3,6,7,8-HxCDF.................... 13 C12-1,2,3,6,7,8-HxCDF 0.997-1.005 50 5 2.5 1,2,3,7,8,9-HxCDF.................... 13 C12-1,2,3,7,8,9-HxCDF 0.999-1.001 50 5 2.5 2,3,4,6,7,8-HxCDF.................... 13 C12-2,3,4,6,7,8-HxCDF 0.999-1.001 50 5 2.5 1,2,3,4,7,8-HxCDD.................... 13 C12-1,2,3,4,7,8-HxCDD 0.999-1.001 50 5 2.5 1,2,3,6,7,8-HxCDD.................... 13 C12-1,2,3,6,7,8-HxCDD 0.998-1.004 50 5 2.5 1,2,3,7,8,9-HxCDD.................... (\2\)................... 1.000-1.019 50 5 2.5 1,2,3,4,6,7,8-HpCDF.................. 13 C12-1,2,3,4,6,7,8- 0.999-1.001 50 5 2.5 HpCDF.

1,2,3,4,7,8,9-HpCDF.................. 13 C12-1,2,3,4,7,8,9- 0.999-1.001 50 5 2.5 HpCDF.

1,2,3,4,6,7,8-HpCDD.................. 13 C12-1,2,3,4,6,7,8- 0.999-1.001 50 5 2.5 HpCDD.

OCDF................................. 13 C12-OCDD............. 0.999-1.001 100 10 5.0 OCDD................................. 13 C12-OCDD............. 0.999-1.001 100 10 5.0 1,2,3,4,6,7,8,-HxCDF................. 13 C12-1,2,3,7,8,9-HpCDD 0.949-0.975 ......... ......... .........

13 C121,2,3,7,8,9-HxCDF.............. 13 C12-1,2,3,7,8,9-HpCDD 0.977-1.047 ......... ......... .........

13 C122,3,4,6,7,8,-HxCDF............. 13 C12-1,2,3,7,8,9-HpCDD 0.959-1.021 ......... ......... .........

13 C121,2,3,4,7,8,-HxCDF............. 13 C12-1,2,3,7,8,9-HpCDD 0.977-1.000 ......... ......... .........

13 C121,2,3,6,7,8,-HxCDF............. 13 C12-1,2,3,7,8,9-HpCDD 0.981-1.003 ......... ......... .........

13 C121,2,3,4,6,7,8-HxCDF............ 13 C12-1,2,3,7,8,9-HpCDD 1.043-1.085 ......... ......... .........

13 C121,2,3,4,7,8,9-HxCDF............ 13 C12-1,2,3,7,8,9-HpCDD 1.057-1.151 ......... ......... .........

13 C121,2,3,4,6,7,8-HxCDF............ 13 C12-1,2,3,7,8,9-HpCDD 1.086-1.110 ......... ......... .........

13 C12OCDD........................... 13 C12-1,2,3,7,8,9-HpCDD 1.032-1.311 ......... ......... .........

---------------------------------------------------------------------------------------------------------------- \1\ The Minimum Level (ML) for each analyte is defined as the level at which the entire analytical system must give a recognizable signal and acceptable calibration point. It is equivalent to the concentration of the lowest calibration standard, assuming that all method-specified sample weights, volumes, and cleanup procedures have been employed.

\2\ The retention time reference for 1,2,3,7,8,9-HxCDD is 13C12-1,2,3,6,7,8-HxCDD, and 1,2,3,7,8,9-HxCDD is quantified using the averaged responses for 13C12-1,2,3,4,7,8-HxCDD and 13C12-1,2,3,6,7,8-HxCDD.

Table 3_Concentration of Stock and Spiking Solutions Containing CDDS/CDFS and Labeled Compounds ---------------------------------------------------------------------------------------------------------------- Labeled Labeled compound compound PAR stock PAR spiking CDD/CDF stock spiking solution solution \4\ solution \1\ solution \3\ (ng/mL) (ng/mL) (ng/mL) \2\ (ng/mL) ---------------------------------------------------------------------------------------------------------------- 2,3,7,8-TCDD.............................................. ............ ........... 40 0.8 2,3,7,8-TCDF.............................................. ............ ........... 40 0.8 1,2,3,7,8-PeCDD........................................... ............ ........... 200 4 1,2,3,7,8-PeCDF........................................... ............ ........... 200 4 2,3,4,7,8-PeCDF........................................... ............ ........... 200 4 1,2,3,4,7,8-HxCDD......................................... ............ ........... 200 4 1,2,3,6,7,8-HxCDD......................................... ............ ........... 200 4 1,2,3,7,8,9-HxCDD......................................... ............ ........... 200 4 1,2,3,4,7,8-HxCDF......................................... ............ ........... 200 4 1,2,3,6,7,8-HxCDF......................................... ............ ........... 200 4 1,2,3,7,8,9-HxCDF......................................... ............ ........... 200 4 2,3,4,6,7,8-HxCDF......................................... ............ ........... 200 4 1,2,3,4,6,7,8-HpCDD....................................... ............ ........... 200 4 1,2,3,4,6,7,8-HpCDF....................................... ............ ........... 200 4 1,2,3,4,7,8,9-HpCDF....................................... ............ ........... 200 4 OCDD...................................................... ............ ........... 400 8 OCDF...................................................... ............ ........... 400 8 13C12-2,3,7,8-TCDD........................................ 100 2 ........... ............

13C12-2,3,7,8-TCDF........................................ 100 2 ........... ............

13C12-1,2,3,7,8-PeCDD..................................... 100 2 ........... ............

13C12-1,2,3,7,8-PeCDF..................................... 100 2 ........... ............

13C12-2,3,4,7,8-PeCDF..................................... 100 2 ........... ............

13C12-1,2,3,4,7,8-HxCDD................................... 100 2 ........... ............

13C12-1,2,3,6,7,8-HxCDD................................... 100 2 ........... ............

13C12-1,2,3,4,7,8-HxCDF................................... 100 2 ........... ............

13C12-1,2,3,6,7,8-HxCDF................................... 100 2 ........... ............

13C12-1,2,3,7,8,9-HxCDF................................... 100 2 ........... ............

13C12-2,3,4,6,7,8-HxCDF................................... 100 2 ........... ............

13C12-1,2,3,4,6,7,8-HpCDD................................. 100 2 ........... ............

13C12-1,2,3,4,6,7,8-HpCDF................................. 100 2 ........... ............

13C12-1,2,3,4,7,8,9-HpCDF................................. 100 2 ........... ............

13C12-OCDD................................................ 200 4 ........... ............

Cleanup Standard \5\ 37Cl4-2,3,7,8-TCDD.................................... 0.8 ........... ........... ............

Internal Standards \6\ 13C12-1,2,3,4-TCDD.................................... 200 ........... ........... ............

13C12-1,2,3,7,8,9-HxCDD............................... 200 ........... ........... ............

---------------------------------------------------------------------------------------------------------------- \1\ Section 7.10_prepared in nonane and diluted to prepare spiking solution.

\2\ Section 7.10.3_prepared in acetone from stock solution daily.

\3\ Section 7.9_prepared in nonane and diluted to prepare spiking solution.

\4\ Section 7.14_prepared in acetone from stock solution daily.

\5\ Section 7.11_prepared in nonane and added to extract prior to cleanup.

\6\ Section 7.12_prepared in nonane and added to the concentrated extract immediately prior to injection into the GC (Section 14.2).

Table 4_Concentration of CDDS/CDFS in Calibration and Calibration Verification Solutions \1\ (Section 15.3) ---------------------------------------------------------------------------------------------------------------- CDD/CDF CS2 (ng/mL) CS3 (ng/mL) CS4 (ng/mL) CS5 (ng/mL) ---------------------------------------------------------------------------------------------------------------- 2,3,7,8-TCDD.................................. 0.5 2 10 40 200 2,3,7,8-TCDF.................................. 0.5 2 10 40 200 1,2,3,7,8-PeCDD............................... 2.5 10 50 200 1000 1,2,3,7,8-PeCDF............................... 2.5 10 50 200 1000 2,3,4,7,8-PeCDF............................... 2.5 10 50 200 1000 1,2,3,4,7,8-HxCDD............................. 2.5 10 50 200 1000 1,2,3,6,7,8-HxCDD............................. 2.5 10 50 200 1000 1,2,3,7,8,9-HxCDD............................. 2.5 10 50 200 1000 1,2,3,4,7,8-HxCDF............................. 2.5 10 50 200 1000 1,2,3,6,7,8-HxCDF............................. 2.5 10 50 200 1000 1,2,3,7,8,9-HxCDF............................. 2.5 10 50 200 1000 2,3,4,6,7,8-HxCDF............................. 2.5 10 50 200 1000 1,2,3,4,6,7,8-HpCDD........................... 2.5 10 50 200 1000 1,2,3,4,6,7,8-HpCDF........................... 2.5 10 50 200 1000 1,2,3,4,7,8,9-HpCDF........................... 2.5 10 50 200 1000 OCDD.......................................... 5.0 20 100 400 2000 OCDF.......................................... 5.0 20 100 400 2000 13 C12-2,3,7,8-TCDD........................... 100 100 100 100 100 13 C12-2,3,7,8-TCDF........................... 100 100 100 100 100 13 C12-1,2,3,7,8-PeCDD........................ 100 100 100 100 100 13 C12-PeCDF.................................. 100 100 100 100 100 13 C12-2,3,4,7,8-PeCDF........................ 100 100 100 100 100 13 C12-1,2,3,4,7,8-HxCDD...................... 100 100 100 100 100 13 C12-1,2,3,6,7,8-HxCDD...................... 100 100 100 100 100 13 C12-1,2,3,4,7,8-HxCDF...................... 100 100 100 100 100 13 C12-1,2,3,6,7,8-HxCDF...................... 100 100 100 100 100 13 C12-1,2,3,7,8,9-HxCDF...................... 100 100 100 100 100 13 C12-1,2,3,4,6,7,8-HpCDD.................... 100 100 100 100 100 13 C12-1,2,3,4,6,7,8-HpCDF.................... 100 100 100 100 100 13 C12-1,2,3,4,7,8,9-Hp CDF................... 100 100 100 100 100 13 C12-OCDD................................... 200 200 200 200 200 Cleanup Standard: 37 C14-2,3,7,8-TCDD....................... 0.5 2 10 40 200 Internal Standards: 13 C12-1,2,3,4-TCDD........................... 100 100 100 100 100 13 C12-1,2,3,7,8,9-HxCDD...................... 100 100 100 100 100 ---------------------------------------------------------------------------------------------------------------- Table 5_GC Retention Time Window Defining Solution and Isomer Specificity Test Standard (Section 7.15) ---------------------------------------------------------------------------------------------------------------- DB-5 column GC retention-time window defining solution ----------------------------------------------------------------------------------------------------------------- CDD/CDF First eluted Last eluted ---------------------------------------------------------------------------------------------------------------- TCDF................................. 1,3,6,8-.................................. 1,2,8,9- TCDD................................. 1,3,6,8-.................................. 1,2,8,9- PeCDF................................ 1,3,4,6,8-................................ 1,2,3,8,9- PeCDD................................ 1,2,4,7,9-................................ 1,2,3,8,9- HxCDF................................ 1,2,3,4,6,8-.............................. 1,2,3,4,8,9- HxCDD................................ 1,2,4,6,7,9-.............................. 1,2,3,4,6,7- HpCDF................................ 1,2,3,4,6,7,8-............................ 1,2,3,4,7,8,9- HpCDD................................ 1,2,3,4,6,7,9-............................ 1,2,3,4,6,7,8- ---------------------------------------------------------------------------------------------------------------- DB-5 Column TCDD Specificity Test Standard 1,2,3,7=1,2,3,8-TCDD 2,3,7,8-TCDD 1,2,3,9-TCDD DB-225 Column TCDF Isomer Specificity Test Standard 2,3,4,7-TCDF 2,3,7,8-TCDF 1,2,3,9-TCDF Table 6_Acceptance Criteria for Performance Tests When All CDDS/CDFS Are Tested \1\ ---------------------------------------------------------------------------------------------------------------- IPR 2 3 CDD/CDF Test conc. ---------------------------- OPR (ng/mL) VER (ng/mL) (ng/mL) s (ng/mL) X (ng/mL) ---------------------------------------------------------------------------------------------------------------- 2,3,7,8-TCDD............................... 10 2.8 8.3-12.9 6.7-15.8 7.8-12.9 2,3,7,8-TCDF............................... 10 2.0 8.7-13.7 7.5-15.8 8.4-12.0 1,2,3,7,8-PeCDD............................ 50 7.5 38-66 35-71 39-65 1,2,3,7,8-PeCDF............................ 50 7.5 43-62 40-67 41-60 2,3,4,7,8-PeCDF............................ 50 8.6 36-75 34-80 41-61 1,2,3,4,7,8-HxCDD.......................... 50 9.4 39-76 35-82 39-64 1,2,3,6,7,8-HxCDD.......................... 50 7.7 42-62 38-67 39-64 1,2,3,7,8,9-HxCDD.......................... 50 11.1 37-71 32-81 41-61 1,2,3,4,7,8-HxCDF.......................... 50 8.7 41-59 36-67 45-56 1,2,3,6,7,8-HxCDF.......................... 50 6.7 46-60 42-65 44-57 1,2,3,7,8,9-HxCDF.......................... 50 6.4 42-61 39-65 45-56 2,3,4,6,7,8-HxCDF.......................... 50 7.4 37-74 35-78 44-57 1,2,3,4,6,7,8-HpCDD........................ 50 7.7 38-65 35-70 43-58 1,2,3,4,6,7,8-HpCDF........................ 50 6.3 45-56 41-61 45-55 1,2,3,4,7,8,9-HpCDF........................ 50 8.1 43-63 39-69 43-58 OCDD....................................... 100 19 89-127 78-144 79-126 OCDF....................................... 100 27 74-146 63-170 63-159 13C12-2,3,7,8-TCDD......................... 100 37 28-134 20-175 82-121 13C12-2,3,7,8-TCDF......................... 100 35 31-113 22-152 71-140 13C12-1,2,3,7,8-PeCDD...................... 100 39 27-184 21-227 62-160 13C12-1,2,3,7,8-PeCDF...................... 100 34 27-156 21-192 76-130 13C12-2,3,4,7,8-PeCDF...................... 100 38 16-279 13-328 77-130 13C12-1,2,3,4,7,8-HxCDD.................... 100 41 29-147 21-193 85-117 13C12-1,2,3,6,7,8-HxCDD.................... 100 38 34-122 25-163 85-118 13C12-1,2,3,4,7,8-HxCDF.................... 100 43 27-152 19-202 76-131 13C12-1,2,3,6,7,8-HxCDF.................... 100 35 30-122 21-159 70-143 13C12-1,2,3,7,8,9-HxCDF.................... 100 40 24-157 17-205 74-135 13C12-2,3,4,6,7,8,-HxCDF................... 100 37 29-136 22-176 73-137 13C12-1,2,3,4,6,7,8-HpCDD.................. 100 35 34-129 26-166 72-138 13C12-1,2,3,4,6,7,8-HpCDF.................. 100 41 32-110 21-158 78-129 13C12-1,2,3,4,7,8,9-HpCDF.................. 100 40 28-141 20-186 77-129 13C12-OCDD................................. 200 95 41-276 26-397 96-415 37Cl4-2,3,7,8-TCDD......................... 10 3.6 3.9-15.4 3.1-19.1 7.9-12.7 ---------------------------------------------------------------------------------------------------------------- \1\ All specifications are given as concentration in the final extract, assuming a 20 L volume.

\2\ s = standard deviation of the concentration.

\3\ X = average concentration.

Table 6a_Acceptance Criteria for Performance Tests When Only Tetra Compounds are Tested 1 ---------------------------------------------------------------------------------------------------------------- IPR 2 3 CDD/CDF Test Conc. --------------------------- OPR (ng/mL) VER (ng/mL) (ng/mL) s (ng/mL) X (ng/mL) ---------------------------------------------------------------------------------------------------------------- 2,3,7,8-TCDD................................ 10 2.7 8.7-12.4 7.314.6 8.2-12.3 2,3,7,8-TCDF................................ 10 2.0 9.1-13.1 8.0-14.7 8.6-11.6 13C12-2,3,7,8-TCDD.......................... 100 35 32-115 25-141 85-117 13C12-2,3,7,8-TCDF.......................... 100 34 35-99 26-126 76-131 37Cl4-2,3,7,8-TCDD.......................... 10 3.4 4.5-13.4 3.7-15.8 8.3-12.1 ---------------------------------------------------------------------------------------------------------------- 1 All specifications are given as concentration in the final extract, assuming a 20 L volume.

2 s = standard deviation of the concentration.

3 X = average concentration.

Table 7_Labeled Compounds Recovery in Samples When all CDDS/CDFS are Tested ------------------------------------------------------------------------ Labeled compound recovery Compound Test conc. -------------------------- (ng/mL) (ng/mL) 1 (%) ------------------------------------------------------------------------ 13C12-2,3,7,8-TCDD.............. 100 25-164 25-164 13C12-2,3,7,8-TCDF.............. 100 24-169 24-169 13C12-1,2,3,7,8-PeCDD........... 100 25-181 25-181 13C12-1,2,3,7,8-PeCDF........... 100 24-185 24-185 13C12-2,3,4,7,8-PeCDF........... 100 21-178 21-178 13C12-1,2,3,4,7,8-HxCDD......... 100 32-141 32-141 13C12-1,2,3,6,7,8-HxCDD......... 100 28-130 28-130 13C12-1,2,3,4,7,8-HxCDF......... 100 26-152 26-152 13C12-1,2,3,6,7,8-HxCDF......... 100 26-123 26-123 13C12-1,2,3,7,8,9-HxCDF......... 100 29-147 29-147 13C12-2,3,4,6,7,8-HxCDF......... 100 28-136 28-136 13C12-1,2,3,4,6,7,8-HpCDD....... 100 23-140 23-140 13C12-1,2,3,4,6,7,8-HpCDF....... 100 28-143 28-143 13C12-1,2,3,4,7,8,9-HpCDF....... 100 26-138 26-138 13C12-OCDD...................... 200 34-313 17-157 37Cl4-2,3,7,8-TCDD.............. 10 3.5-19.7 35-197 ------------------------------------------------------------------------ 1 Specification given as concentration in the final extract, assuming a 20-L volume.

Table 7a_Labeled Compound Recovery in Samples When Only Tetra Compounds are Tested ------------------------------------------------------------------------ Labeled compound recovery Compound Test conc. -------------------------- (ng/mL) (ng/mL) \1\ (%) ------------------------------------------------------------------------ 13C12-2,3,7,8-TCDD.............. 100 31-137 31-137 13C12-2,3,7,8-TCDF.............. 100 29-140 29-140 37Cl4-2,3,7,8-TCDD.............. 10 4.2-16.4 42-164 ------------------------------------------------------------------------ \1\ Specification given as concentration in the final extract, assuming a 20 L volume.

Table 8_Descriptors, Exact M/Z's, M/Z Types, and Elemental Compositions of the CDDs and CDFs ---------------------------------------------------------------------------------------------------------------- Exact M/Z Descriptor \1\ M/Z type Elemental composition Substance \2\ ---------------------------------------------------------------------------------------------------------------- 1........................ 292.9825 Lock C7F11.................... PFK 303.9016 M C12H435Cl4O.............. TCDF 305.8987 M=2 C12H435Cl337ClO.......... TCDF 315.9419 M 13C12H435Cl4O............ TCDF \3\ 317.9389 M=2 13C12H435Cl337ClO........ TCDF \3\ 319.8965 M C12H435Cl4O2............. TCDD 321.8936 M=2 C12H435Cl337ClO2......... TCDD 327.8847 M C12H437Cl4O2............. TCDD \4\ 330.9792 QC C7F13.................... PFK 331.9368 M 13C12H435Cl4O2........... TCDD \3\ 333.9339 M=2 13C12H435Cl337ClO2....... TCDD \3\ 375.8364 M=2 C12H435Cl537ClO.......... HxCDPE 2........................ 339.8597 M=2 C12H335Cl437ClO.......... PeCDF 341.8567 M=4 C12H335Cl337Cl2O......... PeCDF 351.9000 M=2 13C12H335Cl437ClO........ PeCDF 353.8970 M=4 13C12H335Cl337Cl2O....... PeCDF \3\ 354.9792 Lock C9F13.................... PFK 355.8546 M=2 C12H335Cl437ClO2......... PeCDD 357.8516 M=4 C12H335Cl337Cl2O2........ PeCDD 367.8949 M=2 13C12H335Cl437ClO2....... PeCDD \3\ 369.8919 M=4 13C12H335Cl337Cl2O2...... PeCDD \3\ 409.7974 M=2 C12H335Cl637ClO.......... HpCDPE 3........................ 373.8208 M=2 C12H235Cl537ClO.......... HxCDF 375.8178 M=4 C12H235Cl437Cl2O......... HxCDF 383.8639 M 13C12H235Cl6O............ HxCDF \3\ 385.8610 M=2 13C12H235Cl537ClO........ HxCDF \3\ 389.8157 M=2 C12H235Cl537ClO2......... HxCDD 391.8127 M=4 C12H235Cl437Cl2O2........ HxCDD 392.9760 Lock C9F15.................... PFK 401.8559 M=2 13C12H235Cl537ClO2....... HxCDD \3\ 403.8529 M=4 13C12H235Cl437Cl2O2...... HxCDD \3\ 430.9729 QC C9F17.................... PFK 445.7555 M=4 C12H235Cl637Cl2O......... OCDPE 4........................ 407.7818 M=2 C12H35Cl637ClO........... HpCDF 409.7789 M=4 C12H35Cl537Cl2O.......... HpCDF 417.8253 M 13C12H35Cl7O............. HpCDF \3\ 419.8220 M=2 13C12H35Cl637ClO......... HpCDF \3\ 423.7766 M=2 C12H35Cl637ClO2.......... HpCDD 425.7737 M=4 C12H35Cl537Cl2O2......... HpCDD 430.9729 Lock C9F17.................... PFK 435.8169 M=2 13C12H35Cl637ClO2........ HpCDD \3\ 437.8140 M=4 13C12H35Cl537Cl2O2....... HpCDD \3\ 479.7165 M=4 C12H35Cl737Cl2O.......... NCDPE 5........................ 441.7428 M=2 C1235Cl737ClO............ OCDF 442.9728 Lock C10F17................... PFK 443.7399 M=4 C1235Cl637Cl2O........... OCDF 457.7377 M=2 C1235Cl737ClO2........... OCDD 459.7348 M=4 C1235Cl637Cl2O2.......... OCDD 469.7779 M=2 13C1235Cl737ClO2......... OCDD\3\ 471.7750 M=4 13C1235Cl637Cl2O2........ OCDD\3\ 513.6775 M=4 C1235Cl837Cl2O........... DCDPE ---------------------------------------------------------------------------------------------------------------- \1\ Nuclidic masses used: H = 1.007825.

O = 15.994915.

C = 12.00000.

35Cl = 34.968853.

13C = 13.003355.

37Cl = 36.965903.

F = 18.9984.

\2\ TCDD = Tetrachlorodibenzo-p-dioxin.

PeCDD = Pentachlorodibenzo-p-dioxin.

HxCDD = Hexachlorodibenzo-p-dioxin.

HpCDD = Heptachlorodibenzo-p-dioxin.

OCDD = Octachlorodibenzo-p-dioxin.

HxCDPE = Hexachlorodiphenyl ether.

OCDPE = Octachlorodiphenyl ether.

DCDPE = Decachlorodiphenyl ether.

TCDF = Tetrachlorodibenzofuran.

PeCDF = Pentachlorodibenzofuran.

HxCDF = Hexachlorodibenzofuran.

HpCDF = Heptachlorodibenzofuran.

OCDF = Octachlorodibenzofuran.

HpCDPE = Heptachlorodiphenyl ether.

NCDPE = Nonachlorodiphenyl ether.

PFK = Perfluorokerosene.

\3\ Labeled compound.

\4\ There is only one m/z for 37Cl4-2,3,7,8,-TCDD (cleanup standard).

Table 9_Theoretical Ion Abundance Ratios and QC Limits ---------------------------------------------------------------------------------------------------------------- QC limit \1\ Number of chlorine atoms M/Z's forming ratio Theoretical ------------------------- ratio Lower Upper ---------------------------------------------------------------------------------------------------------------- 4 \2\................................... M/(M=2)........................ 0.77 0.65 0.89 5....................................... (M=2)/(M=4).................... 1.55 1.32 1.78 6....................................... (M=2)/(M=4).................... 1.24 1.05 1.43 6 \3\................................... M/(M=2)........................ 0.51 0.43 0.59 7....................................... (M=2)/(M=4).................... 1.05 0.88 1.20 7 \4\................................... M/(M=2)........................ 0.44 0.37 0.51 8....................................... (M=2)/(M=4).................... 0.89 0.76 1.02 ---------------------------------------------------------------------------------------------------------------- \1\ QC limits represent 15% windows around the theoretical ion abundance ratios.

\2\ Does not apply to 37Cl4-2,3,7,8-TCDD (cleanup standard).

\3\ Used for 13C12-HxCDF only.

\4\ Used for 13C12-HpCDF only.

Table 10_Suggested Sample Quantities To Be Extracted for Various Matrices \1\ ---------------------------------------------------------------------------------------------------------------- Quantity Sample Matrix \2\ Example Percent solids Phase extracted ---------------------------------------------------------------------------------------------------------------- Single-phase: Aqueous...................... Drinking water...... Groundwater Treated wastewater Solid........................ Dry soil............ >20 Solid............... 10 g.

Compost Ash Organic...................... Waste solvent....... Waste oil Organic polymer Tissue....................... Fish................ .............. Organic............. 10 g.

Human adipose Multi-phase: Liquid/Solid: Aqueous/Solid............ Wet soil............ 1-30 Solid............... 10 g.

Untreated effluent..

Digested municipal sludge.

Filter cake.........

Paper pulp..........

Organic/solid............ Industrial sludge... 1-100 Both................ 10 g.

Oily waste Liquid/Liquid: Aqueous/organic.......... In-process effluent. Untreated effluent Drum waste Aqueous/organic/solid.... Untreated effluent.. >1 Organic and solid... 10 g.

Drum waste ---------------------------------------------------------------------------------------------------------------- \1\ The quantity of sample to be extracted is adjusted to provide 10 g of solids (dry weight). One liter of aqueous samples containing 1% solids will contain 10 g of solids. For aqueous samples containing greater than 1% solids, a lesser volume is used so that 10 g of solids (dry weight) will be extracted.

\2\ The sample matrix may be amorphous for some samples. In general, when the CDDs/CDFs are in contact with a multiphase system in which one of the phases is water, they will be preferentially dispersed in or adsorbed on the alternate phase because of their low solubility in water.

\3\ Aqueous samples are filtered after spiking with the labeled compounds. The filtrate and the materials trapped on the filter are extracted separately, and the extracts are combined for cleanup and analysis.

(image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF (image) View or download PDF 24.0Glossary of Definitions and Purposes These definitions and purposes are specific to this method but have been conformed to common usage as much as possible.

24.1Units of weight and Measure and Their Abbreviations.

24.1.1Symbols: Cdegrees Celsius Lmicroliter mmicrometer greater than %percent 24.1.2Alphabetical abbreviations: ampampere cmcentimeter ggram hhour Dinside diameter in.inch Lliter MMolecular ion mmeter mgmilligram minminute mLmilliliter mmmillimeter m/zmass-to-charge ratio Nnormal; gram molecular weight of solute divided by hydrogen equivalent of solute, per liter of solution ODoutside diameter pgpicogram ppbpart-per-billion ppmpart-per-million ppqpart-per-quadrillion pptpart-per-trillion psigpounds-per-square inch gauge v/vvolume per unit volume w/vweight per unit volume 24.2Definitions and Acronyms (in Alphabetical Order).

AnalyteA CDD or CDF tested for by this method. The analytes are listed in Table 1.

Calibration Standard (CAL)A solution prepared from a secondary standard and/or stock solutions and used to calibrate the response of the instrument with respect to analyte concentration.

Calibration Verification Standard (VER)The mid-point calibration standard (CS3) that is used in to verify calibration. See Table 4.

CDDChlorinated Dibenzo-p-ioxinThe isomers and congeners of tetra-through octa-chlorodibenzo-p-dioxin.

CDFChlorinated DibenzofuranThe isomers and congeners of tetra-through octa-chlorodibenzofuran.

CS1, CS2, CS3, CS4, CS5See Calibration standards and Table 4.

Field BlankAn aliquot of reagent water or other reference matrix that is placed in a sample container in the laboratory or the field, and treated as a sample in all respects, including exposure to sampling site conditions, storage, preservation, and all analytical procedures. The purpose of the field blank is to determine if the field or sample transporting procedures and environments have contaminated the sample.

GCGas chromatograph or gas chromatography.

GPCGel permeation chromatograph or gel permeation chromatography.

HPLCHigh performance liquid chromatograph or high performance liquid chromatography.

HRGCHigh resolution GC.

HRMSHigh resolution MS.

IPRInitial precision and recovery; four aliquots of the diluted PAR standard analyzed to establish the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first time this method is used and any time the method or instrumentation is modified.

K-DKuderna-Danish concentrator; a device used to concentrate the analytes in a solvent.

Laboratory BlankSee method blank.

Laboratory Control sample (LCS)See ongoing precision and recovery standard (OPR).

Laboratory Reagent BlankSee method blank.

MayThis action, activity, or procedural step is neither required nor prohibited.

May NotThis action, activity, or procedural step is prohibited.

Method BlankAn aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples.

The method blank is used to determine if analytes or interferences are present in the laboratory environment, the reagents, or the apparatus.

Minimum Level (ML)The level at which the entire analytical system must give a recognizable signal and acceptable calibration point for the analyte. It is equivalent to the concentration of the lowest calibration standard, assuming that all method-specified sample weights, volumes, and cleanup procedures have been employed.

MSMass spectrometer or mass spectrometry.

MustThis action, activity, or procedural step is required.

OPROngoing precision and recovery standard (OPR); a laboratory blank spiked with known quantities of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results produced by the laboratory remain within the limits specified in this method for precision and recovery.

PARPrecision and recovery standard; secondary standard that is diluted and spiked to form the IPR and OPR.

PFKPerfluorokerosene; the mixture of compounds used to calibrate the exact m/z scale in the HRMS.

Preparation BlankSee method blank.

Primary Dilution StandardA solution containing the specified analytes that is purchased or prepared from stock solutions and diluted as needed to prepare calibration solutions and other solutions.

Quality Control Check Sample (QCS)A sample containing all or a subset of the analytes at known concentrations. The QCS is obtained from a source external to the laboratory or is prepared from a source of standards different from the source of calibration standards. It is used to check laboratory performance with test materials prepared external to the normal preparation process.

Reagent WaterWater demonstrated to be free from the analytes of interest and potentially interfering substances at the method detection limit for the analyte.

Relative Standard Deviation (RSD)The standard deviation times 100 divided by the mean. Also termed coefficient of variation.

RFResponse factor. See Section 10.6.1.

RRRelative response. See Section 10.5.2.

RSDSee relative standard deviation.

SDSSoxhlet/Dean-Stark extractor; an extraction device applied to the extraction of solid and semi-solid materials (Reference 7).

ShouldThis action, activity, or procedural step is suggested but not required.

SICPSelected ion current profile; the line described by the signal at an exact m/z.

SPESolid-phase extraction; an extraction technique in which an analyte is extracted from an aqueous sample by passage over or through a material capable of reversibly adsorbing the analyte. Also termed liquid-solid extraction.

Stock SolutionA solution containing an analyte that is prepared using a reference material traceable to EPA, the National Institute of Science and Technology (NIST), or a source that will attest to the purity and authenticity of the reference material.

TCDDTetrachlorodibenzo-p-dioxin.

TCDFTetrachlorodibenzofuran.

VERSee calibration verification standard.

Method 1624 Revision BVolatile Organic Compounds by Isotope Dilution GC/MS 1.Scope and Application 1.1This method is designed to determine the volatile toxic organic pollutants associated with the 1976 Consent Decree and additional compounds amenable to purge and trap gas chromatography-mass spectrometry (GC/MS).

1.2The chemical compounds listed in table 1 may be determined in municipal and industrial discharges by this method. The methmd is designed to meet the survey requirements of Effluent Guidelines Division (EGD) and the National Pollutants Discharge Elimination System (NPDES) under 40 CFR 136.1 and 136.5. Any modifications of this method, beyond those expressly permitted, shall be considered as major modifications subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.3The detection limit of this method is usually dependent on the level of interferences rather than instrumental limitations. The limits in table 2 represent the minimum quantity that can be detected with no interferences present.

1.4The GC/MS portions of this method are for use only by analysts experienced with GC/MS or under the close supervision of such qualified persons. Laboratories unfamiliar with the analyses of environmental samples by GC/MS should run the performance tests in reference 1 before beginning.

2.Summary of Method 2.1Stable isotopically labeled analogs of the compounds of interest are added to a 5 mL water sample. The sample is purged at 2025 C with an inert gas in a specially designed chamber. The volatile organic compounds are transferred from the aqueous phase into the gaseous phase where they are passed into a sorbent column and trapped. After purging is completed, the trap is backflushed and heated rapidly to desorb the compounds into a gas chromatograph (GC). The compounds are separated by the GC and detected by a mass spectrometer (MS) (references 2 and 3).

The labeled compounds serve to correct the variability of the analytical technique.

2.2Identification of a compound (qualitative analysis) is performed by comparing the GC retention time and the background corrected characteristic spectral masses with those of authentic standards.

2.3Quantitative analysis is performed by GC/MS using extracted ion current profile (EICP) areas. Isotope dilution is used when labeled compounds are available; otherwise, an internal standard method is used.

2.4Quality is assured through reproducible calibration and testing of the purge and trap and GC/MS systems.

3.Contamination and Interferences 3.1Impurities in the purge gas, organic compounds out-gassing from the plumbing upstream of the trap, and solvent vapors in the laboratory account for the majority of contamination problems. The analytical system is demonstrated to be free from interferences under conditions of the analysis by analyzing blanks initially and with each sample lot (samples analyzed on the same 8 hr shift), as described in Section 8.5.

3.2Samples can be contaminated by diffusion of volatile organic compounds (particularly methylene chloride) through the bottle seal during shipment and storage. A field blank prepared from reagent water and carried through the sampling and handling protocol serves as a check on such contamination.

3.3Contamination by carry-over can occur when high level and low level samples are analyzed sequentially. To reduce carry-over, the purging device and sample syringe are rinsed between samples with reagent water.

When an unusually concentrated sample is encountered, it is followed by analysis of a reagent water blank to check for carry-over. For samples containing large amounts of water soluble materials, suspended solids, high boiling compounds, or high levels or purgeable compounds, the purge device is washed with soap solution, rinsed with tap and distilled water, and dried in an oven at 100125 C. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

3.4Interferences resulting from samples will vary considerably from source to source, depending on the diversity of the industrial complex or municipality being sampled.

4.Safety 4.1The toxicity or carcinogenicity of each compound or reagent used in this method has not been precisely determined; however, each chemical compound should be treated as a potential health hazard. Exposure to these compounds should be reduced to the lowest possible level. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of data handling sheets should also be made available to all personnel involved in these analyses. Additional information on laboratory safety can be found in references 46.

4.2The following compounds covered by this method have been tentatively classified as known or suspected human or mammalian carcinogens: benzene, carbon tetrachloride, chloroform, and vinyl chloride. Primary standards of these toxic compounds should be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator should be worn when high concentrations are handled.

5.Apparatus and Materials 5.1Sample bottles for discrete sampling.

5.1.1Bottle25 to 40 mL with screw cap (Pierce 13075, or equivalent).

Detergent wash, rinse with tap and distilled water, and dry at >105 C for one hr minimum before use.

5.1.2SeptumTeflon-faced silicone (Pierce 12722, or equivalent), cleaned as above and baked at 100200 C, for one hour minimum.

5.2Purge and trap deviceconsists of purging device, trap, and desorber.

Complete devices are commercially available.

5.2.1Purging devicedesigned to accept 5 mL samples with water column at least 3 cm deep. The volume of the gaseous head space between the water and trap shall be less than 15 mL. The purge gas shall be introduced less than 5 mm from the base of the water column and shall pass through the water as bubbles with a diameter less than 3 mm. The purging device shown in Figure 1 meets these criteria.

5.2.2Trap25 to 30 cm 2.5 mm i.d. minimum, containing the following: 5.2.2.1Methyl silicone packingone 0.2 cm, 3 percent OV1 on 60/80 mesh Chromosorb W, or equivalent.

5.2.2.2Porous polymer15 1.0 cm, Tenax GC (2,6-diphenylene oxide polymer), 60/80 mesh, chromatographic grade, or equivalent.

5.2.2.3Silica gel8 1.0 cm, Davison Chemical, 35/60 mesh, grade 15, or equivalent. The trap shown in Figure 2 meets these specifications.

5.2.3Desorbershall heat the trap to 175 5 C in 45 seconds or less. The polymer section of the trap shall not exceed 180 C, and the remaining sections shall not exceed 220 C. The desorber shown in Figure 2 meets these specifications.

5.2.4The purge and trap device may be a separate unit or coupled to a GC as shown in Figures 3 and 4.

5.3Gas chromatographshall be linearly temperature programmable with initial and final holds, shall contain a glass jet separator as the MS interface, and shall produce results which meet the calibration (Section 7), quality assurance (Section 8), and performance tests (Section 11) of this method.

5.3.1Column2.8 0.4 m 2 0.5 mm i. d. glass, packekd with one percent SP1000 on Carbopak B, 60/80 mesh, or equivalent.

5.4Mass spectrometer70 eV electron impact ionization; shall repetitively scan from 20 to 250 amu every 23 seconds, and produce a unit resolution (valleys between m/z 174176 less than 10 percent of the height of the m/z 175 peak), background corrected mass spectrum from 50 ng 4-bromo-fluorobenzene (BFB) injected into the GC. The BFB spectrum shall meet the mass-intensity criteria in Table 3. All portions of the GC column, transfer lines, and separator which connect the GC column to the ion source shall remain at or above the column temperature during analysis to preclude condensation of less volatile compounds.

5.5Data systemshall collect and record MS data, store mass intensity data in spectral libraries, process GC/MS data and generate reports, and shall calculate and record response factors.

5.5.1Data acquisitionmass spectra shall be collected continuously throughout the analysis and stored on a mass storage device.

5.5.2Mass spectral librariesuser created libraries containing mass spectra obtained from analysis of authentic standards shall be employed to reverse search GC/MS runs for the compounds of interest (Section 7.2).

5.5.3Data processingthe data system shall be used to search, locate, identify, and quantify the compounds of interest in each GC/MS analysis.

Software routines shall be employed to compute retention times and EICP areas. Displays of spectra, mass chromatograms, and library comparisons are required to verify results.

5.5.4Response factors and multipoint calibrationsthe data system shall be used to record and maintain lists of response factors (response ratios for isotope dilution) and generate multi-point calibration curves (Section 7). Computations of relative standard deviation (coefficient of variation) are useful for testing calibration linearity. Statistics on initial and on-going performance shall be maintained (Sections 8 and 11).

5.6Syringes5 mL glass hypodermic, with Luer-lok tips.

5.7Micro syringes10, 25, and 100 uL.

5.8Syringe valves2-way, with Luer ends (Telfon or Kel-F).

5.9Syringe5 mL, gas-tight, with shut-off valve.

5.10Bottles15 mL., screw-cap with Telfon liner.

5.11Balanceanalytical, capable of weighing 0.1 mg.

6.Reagents and Standards 6.1Reagent waterwater in which the compounds of interest and interfering compounds are not detected by this method (Section 11.7). It may be generated by any of the following methods: 6.1.1Activated carbonpass tap water through a carbon bed (Calgon Filtrasorb-300, or equivalent).

6.1.2Water purifierpass tap water through a purifier (Millipore Super Q, or equivalent).

6.1.3Boil and purgeheat tap water to 90100 C and bubble contaminant free inert gas through it for approx one hour. While still hot, transfer the water to screw-cap bottles and seal with a Teflon-lined cap.

6.2Sodium thiosulfateACS granular.

6.3Methanolpesticide quality or equivalent.

6.4Standard solutionspurchased as solution or mixtures with certification to their purity, concentration, and authenticity, or prepared from materials of known purity and composition. If compound purity is 96 percent or greater, the weight may be used without correction to calculate the concentration of the standard.

6.5Preparation of stock solutionsprepare in methanol using liquid or gaseous standards per the steps below. Observe the safety precautions given in Section 4.

6.5.1Place approx 9.8 mL of methanol in a 10 mL ground glass stoppered volumetric flask. Allow the flask to stand unstoppered for approximately 10 minutes or until all methanol wetted surfaces have dried. In each case, weigh the flask, immediately add the compound, then immediately reweigh to prevent evaporation losses from affecting the measurement.

6.5.1.1Liquidsusing a 100 L syringe, permit 2 drops of liquid to fall into the methanol without contacting the leck of the flask.

Alternatively, inject a known volume of the compound into the methanol in the flask using a micro-syringe.

6.5.1.2Gases (chloromethane, bromomethane, chloroethane, vinyl chloride)fill a valved 5 mL gas-tight syringe with the compound. Lower the needle to approximately 5 mm above the methanol meniscus. Slowly introduce the compound above the surface of the meniscus. The gas will dissolve rapidly in the methanol.

6.5.2Fill the flask to volume, stopper, then mix by inverting several times. Calculate the concentration in mg/mL (g/L ) from the weight gain (or density if a known volume was injected).

6.5.3Transfer the stock solution to a Teflon sealed screw-cap-bottle.

Store, with minimal headspace, in the dark at 10 to 20 C.

6.5.4Prepare fresh standards weekly for the gases and 2-chloroethylvinyl ether. All other standards are replaced after one month, or sooner if comparison with check standards indicate a change in concentration.

Quality control check standards that can be used to determine the accuracy of calibration standards are available from the US Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.

6.6Labeled compound spiking solutionfrom stock standard solutions prepared as above, or from mixtures, prepare the spiking solution to contain a concentration such that a 510 L spike into each 5 mL sample, blank, or aqueous standard analyzed will result in a concentration of 20 g/L of each labeled compound. For the gases and for the water soluble compounds (acrolein, acrylonitrile, acetone, diethyl ether, and MEK), a concentration of 100 g/L may be used. Include the internal standards (Section 7.5) in this solution so that a concentration of 20 g/L in each sample, blank, or aqueous standard will be produced.

6.7Secondary standardsusing stock solutions, prepare a secondary standard in methanol to contain each pollutant at a concentration of 500 g/mL For the gases and water soluble compounds (Section 6.6), a concentration of 2.5 mg/mL may be used.

6.7.1Aqueous calibration standardsusing a 25 L syringe, add 20 L of the secondary standard (Section 6.7) to 50, 100, 200, 500, and 1000 mL of reagent water to produce concentrations of 200, 100, 50, 20, and 10 g/L, respectively. If the higher concentration standard for the gases and water soluble compounds was chosen (Section 6.6), these compounds will be at concentrations of 1000, 500, 250, 100, and 50 g/L in the aqueous calibration standards.

6.7.2Aqueous performance standardan aqueous standard containing all pollutants, internal standards, labeled compounds, and BFB is prepared daily, and analyzed each shift to demonstrate performance (Section 11).

This standard shall contain either 20 or 100 g/L of the labeled and pollutant gases and water soluble compounds, 10 g/L BFB, and 20 g/L of all other pollutants, labeled compounds, and internal standards. It may be the nominal 20 g/L aqueous calibration standard (Section 6.7.1).

6.7.3A methanolic standard containing all pollutants and internal standards is prepared to demonstrate recovery of these compounds when syringe injection and purge and trap analyses are compared. This standard shall contain either 100 g/mL or 500 g/mL of the gases and water soluble compounds, and 100 g/mL of the remaining pollutants and internal standards (consistent with the amounts in the aqueous performance standard in 6.7.2).

6.7.4Other standards which may be needed are those for test of BFB performance (Section 7.1) and for collection of mass spectra for storage in spectral libraries (Section 7.2).

7.Calibration 7.1Assemble the gas chromatographic apparatus and establish operating conditions given in table 2. By injecting standards into the GC, demonstrate that the analytical system meets the detection limits in table 2 and the mass-intensity criteria in table 3 for 50 ng BFB.

7.2Mass spectral librariesdetection and identification of the compound of interest are dependent upon the spectra stored in user created libraries.

7.2.1Obtain a mass spectrum of each pollutant and labeled compound and each internal standard by analyzing an authentic standard either singly or as part of a mixture in which there is no interference between closely eluted components. That only a single compound is present is determined by examination of the spectrum. Fragments not attributable to the compound under study indicate the presence of an interfering compound. Adjust the analytical conditions and scan rate (for this test only) to produce an undistorted spectrum at the GC peak maximum. An undistorted spectrum will usually be obtained if five complete spectra are collected across the upper half of the GC peak. Software algorithms designed to enhance the spectrum may eliminate distortion, but may also eliminate authentic m/z's or introduce other distortion.

7.2.3The authentic reference spectrum is obtained under BFB tuning conditions (Section 7.1 and table 3) to normalize it to spectra from other instruments.

7.2.4The spectrum is edited by saving the 5 most intense mass spectral peaks and all other mass spectral peaks greater than 10 percent of the base peak. This spectrum is stored for reverse search and for compound confirmation.

7.3Assemble the purge and trap device. Pack the trap as shown in Figure 2 and condition overnight at 170180 C by backflushing with an inert gas at a flow rate of 2030 mL/min. Condition traps daily for a minimum of 10 minutes prior to use.

7.3.1Analyze the aqueous performance standard (Section 6.7.2) according to the purge and trap procedure in Section 10. Compute the area at the primary m/z (table 4) for each compound. Compare these areas to those obtained by injecting one L of the methanolic standard (Section 6.7.3) to determine compound recovery. The recovery shall be greater than 20 percent for the water soluble compounds, and 60110 percent for all other compounds. This recovery is demonstrated initially for each purge and trap GC/MS system. The test is repeated only if the purge and trap or GC/MS systems are modified in any way that might result in a change in recovery.

7.3.2Demonstrate that 100 ng toluene (or toluene-d8) produces an area at m/z 91 (or 99) approx one-tenth that required to exceed the linear range of the system. The exact value must be determined by experience for each instrument. It is used to match the calibration range of the instrument to the analytical range and detection limits required.

7.4Calibration by isotope dilutionthe isotope dilution approach is used for the purgeable organic compounds when appropriate labeled compounds are available and when interferences do not preclude the analysis. If labeled compounds are not available, or interferences are present, internal standard methods (Section 7.5 or 7.6) are used. A calibration curve encompassing the concentration range of interest is prepared for each compound determined. The relative response (RR) vs concentration (g/L) is plotted or computed using a linear regression. An example of a calibration curve for toluene using toluene-d8 is given in figure 5.

Also shown are the 10 percent error limits (dotted lines). Relative response is determined according to the procedures described below. A minimum of five data points are required for calibration (Section 7.4.4).

7.4.1The relative response (RR) of pollutant to labeled compound is determined from isotope ratio values calculated from acquired data.

Three isotope ratios are used in this process: RX=the isotope ratio measured in the pure pollutant (figure 6A).

Ry=the isotope ratio of pure labeled compound (figure 6B).

Rm=the isotope ratio measured in the analytical mixture of the pollutant and labeled compounds (figure 6C).

The correct way to calculate RR is: RR=(RyRm) (RX+1)/(RmRX)(Ry+1) If Rm is not between 2Ry and 0.5RX, the method does not apply and the sample is analyzed by internal or external standard methods (Section 7.5 or 7.6).

7.4.2In most cases, the retention times of the pollutant and labeled compound are the same and isotope ratios (R's) can be calculated from the EICP areas, where: R=(area at m1/z)/(area at m2/z) If either of the areas is zero, it is assigned a value of one in the calculations; that is, if: area of m1/z=50721, and area of m2/z=0, then R=50721/1=50720.

The m/z's are always selected such that RX>Ry. When there is a difference in retention times (RT) between the pollutant and labeled compounds, special precautions are required to determine the isotope ratios.

RX, Ry, and Rm are defined as follows: RX=[area m1/z (at RT1)]/1 Ry=1/[area m2/z (at RT2)] Rm=[area m1/z (at RT1)]/[area m2/z (at RT2)] 7.4.3An example of the above calculations can be taken from the data plotted in figure 6 for toluene and toluene-d8. For these data, RX=168920/1=168900, Ry=1/60960=0.00001640, and Rm=96868/82508=1.174. The RR for the above data is then calculated using the equation given in Section 7.4.1. For the example, RR=1.174.

Note: Not all labeled compounds elute before their pollutant analogs.

7.4.4To calibrate the analytical system by isotope dilution, analyze a 5 mL aliquot of each of the aqueous calibration standards (Section 6.7.1) spiked with an appropriate constant amount of the labeled compound spiking solution (Section 6.6), using the purge and trap procedure in section 10. Compute the RR at each concentration.

7.4.5Linearityif the ratio of relative response to concentration for any compound is constant (less than 20 percent coefficient of variation) over the 5 point calibration range, an averaged relative response/concentration ratio may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the 5 point calibration range.

7.5Calibration by internal standardused when criteria for isotope dilution (Section 7.4) cannot be met. The method is applied to pollutants having no labeled analog and to the labeled compounds. The internal standards used for volatiles analyses are bromochloromethane, 2-bromo-1-chloropropane, and 1,4-dichlorobutane. Concentrations of the labeled compounds and pollutants without labeled analogs are computed relative to the nearest eluted internal standard, as shown in table 2.

7.5.1Response factorscalibration requires the determination of response factors (RF) which are defined by the following equation: RF=(AsxCis)/(AisxCs), where As is the EICP area at the characteristic m/z for the compound in the daily standard. Ais is the EICP area at the characteristic m/z for the internal standard.

Cis is the concentration (ug/L) of the internal standard Cs is the concentration of the pollutant in the daily standard.

7.5.2The response factor is determined at 10, 20, 50, 100, and 200 ug/L for the pollutants (optionally at five times these concentrations for gases and water soluble pollutantssee Section 6.7), in a way analogous to that for calibration by isotope dilution (Section 7.4.4). The RF is plotted against concentration for each compound in the standard (Cs) to produce a calibration curve.

7.5.3Linearityif the response factor (RF) for any compound is constant (less than 35 percent coefficient of variation) over the 5 point calibration range, an averaged response factor may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the 5 point range.

7.6Combined calibrationby adding the isotopically labeled compounds and internal standards (Section 6.6) to the aqueous calibration standards (Section 6.7.1), a single set of analyses can be used to produce calibration curves for the isotope dilution and internal standard methods. These curves are verified each shift (Section 11.5) by purging the aqueous performance standard (Section 6.7.2). Recalibration is required only if calibration and on-going performance (Section 11.5) criteria cannot be met.

8.Quality Assurance/Quality Control 8.1Each laboratory that uses this method is required to operate a formal quality assurance program. The minimum requirements of this program consist of an initial demonstration of laboratory capability, analysis of samples spiked with labeled compounds to evaluate and document data quality, and analysis of standards and blanks as tests of continued performance. Laboratory performance is compared to established performance criteria to determine if the results of analyses meet the performance characteristics of the method.

8.1.1The analyst shall make an initial demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2The analyst is permitted to modify this method to improve separations or lower the costs of measurements, provided all performance specifications are met. Each time a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2 to demonstrate method performance.

8.1.3Analyses of blanks are required to demonstrate freedom from contamination and that the compounds of interest and interfering compounds have not been carried over from a previous analysis (Section 3). The procedures and criteria for analysis of a blank are described in Sections 8.5 and 11.7.

8.1.4The laboratory shall spike all samples with labeled compounds to monitor method performance. This test is described in Section 8.3. When results of these spikes indicate atypical method performance for samples, the samples are diluted to bring method performance within acceptable limits (Section 14.2).

8.1.5The laboratory shall, on an on-going basis, demonstrate through the analysis of the aqueous performance standard (Section 6.7.2) that the analysis system is in control. This procedure is described in Sections 11.1 and 11.5.

8.1.6The laboratory shall maintain records to define the quality of data that is generated. Development of accuracy statements is described in Sections 8.4 and 11.5.2.

8.2Initial precision and accuracyto establish the ability to generate acceptable precision and accuracy, the analyst shall perform the following operations: 8.2.1Analyze two sets of four 5mL aliquots (8 aliquots total) of the aqueous performance standard (Section 6.7.2) according to the method beginning in Section 10.

8.2.2Using results of the first set of four analyses in Section 8.2.1, compute the average recovery (X ) in g/L and the standard deviation of the recovery (s) in g/L for each compound, by isotope dilution for polluitants with a labeled analog, and by internal standard for labeled compounds and pollutants with no labeled analog.

8.2.3For each compound, compare s and X with the corresponding limits for initial precision and accuracy found in table 5. If s and X for all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If individual X falls outside the range for accuracy, system performance is unacceptable for that compound.

Note: The large number of compounds in table 5 present a substantial probability that one or more will fail one of the acceptance criteria when all compoulds are analyzed. To determine if the analytical system is out of control, or if the failure can be attributed to probability, proceed as follows: 8.2.4Using the results of the second set of four analyses, compute s and X for only those compounds which failed the test of the first set of four analyses (Section 8.2.3). If these compounds now pass, system performance is acceptable for all compounds and analysis of blanks and samples may begin. If, however, any of the same compounds fail again, the analysis system is not performing properly for the compound(s) in question. In this event, correct the problem and repeat the entire test (Section 8.2.1).

8.3The laboratory shall spike all samples with labeled compounds to assess method performance on the sample matrix.

8.3.1Spike and analyze each sample according to the method beginning in Section 10.

8.3.2Compute the percent recovery (P) of the labeled compounds using the internal standard method (Section 7.5).

8.3.3Compare the percent recovery for each compound with the corresponding labeled compound recovery limit in table 5. If the recovery of any compound falls outside its warning limit, method performance is unacceptable for that compound in that sample. Therefore, the sample matrix is complex and the sample is to be diluted and reanalyzed, per Section 14.2.

8.4As part of the QA program for the laboratory, method accuracy for wastewater samples shall be assessed and records shall be maintained.

After the analysis of five wastewater samples for which the labeled compounds pass the tests in Section 8.3.3, compute the average percent recovery (P) and the standard deviation of the percent recovery (sp) for the labeled compounds only. Express the accuracy assessment as a percent recovery interval from P2sp to P+2sp. For example, if P=90% and sp=10%, the accuracy interval is expressed as 70110%. Update the accuracy assessment for each compound on a regular basis (e.g. after each 510 new accuracy measurements).

8.5Blanksreagent water blanks are analyzed to demonstrate freedom from carry-over (Section 3) and contamination.

8.5.1The level at which the purge and trap system will carry greater than 5 g/L of a pollutant of interest (table 1) into a succeeding blank shall be determined by analyzing successively larger concentrations of these compounds. When a sample contains this concentration or more, a blank shall be analyzed immediately following this sample to demonstrate no carry-over at the 5 g/L level.

8.5.2With each sample lot (samples analyzed on the same 8 hr shift), a blank shall be analyzed immediately after analysis of the aqueous performance standard (Section 11.1) to demonstrate freedom from contamination. If any of the compounds of interest (table 1) or any potentially interfering compound is found in a blank at greater than 10 g/L (assuming a response factor of 1 relative to the nearest eluted internal standard for compounds not listed in table 1), analysis of samples is halted until the source of contamination is eliminated and a blank shows no evidence of contamination at this level.

8.6The specifications contained in this method can be met if the apparatus used is calibrated properly, then maintained in a calibrated state.

The standards used for calibration (Section 7), calibration verification (Section 11.5) and for initial (Section 8.2) and on-going (Section 11.5) precision and accuracy should be identical, so that the most precise results will be obtained. The GC/MS instrument in particular will provide the most reproducible results if dedicated to the settings and conditions required for the analyses of volatiles by this method.

8.7Depending on specific program requirements, field replicates may be collected to determine the precision of the sampling technique, and spiked samples may be required to determine the accuracy of the analysis when internal or external standard methods are used.

9.Sample Collection, Preservation, and Handling 9.1Grab samples are collected in glass containers having a total volume greater than 20 mL. Fill sample bottles so that no air bubbles pass through the sample as the bottle is filled. Seal each bottle so that no air bubbles are entrapped. Maintain the hermetic seal on the sample bottle until time of analysis.

9.2Samples are maintained at 04 C from the time of collection until analysis. If the sample contains residual chlorine, add sodium thiosulfate preservative (10 mg/40 mL) to the empty sample bottles just prior to shipment to the sample site. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine (Reference 8). If preservative has been added, shake bottle vigorously for one minute immediately after filling.

9.3Experimental evidence indicates that some aromatic compounds, notably benzene, toluene, and ethyl benzene are susceptible to rapid biological degradation under certain environmental conditions. Refrigeration alone may not be adequate to preserve these compounds in wastewaters for more than seven days. For this reason, a separate sample should be collected, acidified, and analyzed when these aromatics are to be determined.

Collect about 500 mL of sample in a clean container.

Adjust the pH of the sample to about 2 by adding HCl (1+1) while stirring. Check pH with narrow range (1.4 to 2.8) pH paper. Fill a sample container as described in Section 9.1. If residual chlorine is present, add sodium thiosulfate to a separate sample container and fill as in Section 9.1.

9.4All samples shall be analyzed within 14 days of collection.

10.Purge, Trap, and GC/MS Analysis 10.1Remove standards and samples from cold storage and bring to 2025 .

10.2Adjust the purge gas flow rate to 40 4 mL/min. Attach the trap inlet to the purging device and set the valve to the purge mode (figure 3).

Open the syringe valve located on the purging device sample introduction needle (figure 1).

10.3Remove the plunger from a 5mL syringe and attach a closed syringe valve. Open the sample bottle and carefully pour the sample into the syringe barrel until it overflows. Replace the plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Because this process of taking an aliquot destroys the validity of the sample for future analysis, fill a second syringe at this time to protect against possible loss of data. Add an appropriate amount of the labeled compound spiking solution (Section 6.6) through the valve bore, then close the valve.

10.4Attach the syringe valve assembly to the syringe valve on the purging device. Open both syringe valves and inject the sample into the purging chamber.

10.5Close both valves and purge the sample for 11.0 0.1 minutes at 2025 C.

10.6After the 11 minute purge time, attach the trap to the chromatograph and set the purge and trap apparatus to the desorb mode (figure 4).

Desorb the trapped compounds into the GC column by heating the trap to 170180 C while backflushing with carrier gas at 2060 mL/min for four minutes. Start MS data acquisition upon start of the desorb cycle, and start the GC column temperature program 3 minutes later. Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and detection limits that were achieved under these conditions. Other columns may be used provided the requirements in Section 8 can be met. If the priority pollutant gases produce GC peaks so broad that the precision and recovery specifications (Section 8.2) cannot be met, the column may be cooled to ambient or sub-ambient temperatures to sharpen these peaks.

10.7While analysis of the desorbed compounds proceeds, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL portions of reagent water. After the purging device has been emptied, allow the purge gas to vent through the chamber until the frit is dry, so that it is ready for the next sample.

10.8After desorbing the sample for four minutes, recondition the trap by returning to the purge mode. Wait 15 seconds, then close the syringe valve on the purging device to begin gas flow through the trap. Maintain the trap t