stoppered volumetric flask and fill to the mark with methanol. After the pentachlorophenol is completely dissolved, transfer the solution to a 15-mL vial with PTFE-lined cap.

7.7.2 Stock solutions should be checked for signs of degradation prior to the preparation of calibration or performance test standards and shall be replaced after six months, or sooner if comparison with quality control check standards indicates a change in concentration.

7.8 Labeled compound spiking solution: From stock solutions prepared as above, or from mixtures, prepare one spiking solution to contain the labeled chlorovanillin in acetone and a second spiking solution to contain the remaining chlorophenolics, including the 3,4,5-trichlorophenol sample matrix internal standard (SMIS), in methanol. The labeled compounds and SMIS are each at a concentration of 12.5 ug/mL.

7.9 Secondary standards for calibration: Using stock solutions (Section 7.7), prepare one secondary standard containing the chlorovanillins and chlorsyringaldehydes listed in Table 1 in acetone and a second secondary standard containing the remaining chlorophenolics in methanol. The monochlorinated phenol, guaiacol, and catechol are included at a concentration of 25 ug/mL; the trichlorinated catechols, tetrachlorinated guaiacol and catechol, pentachlorophenol, 5,6-dichlorovanillin, and 2,6-dichlorosyringaldehyde are included at a concentration of 100 ug/mL; and the remaining compounds are included at a concentration of 50 ug/mL, each in their respective solutions.

7.10 Instrument internal standard (IIS): Prepare a solution of 2,2-difluorobiphenyl (DFB) at a concentration of 2.5 mg/mL in hexane.

7.11 DFTPP solution: Prepare a solution of DFTPP at 50 ug/mL in acetone.

7.12 Solutions for obtaining authentic mass spectra (Section 10.2): Prepare mixtures of compounds at concentrations which will assure authentic spectra are obtained for storage in libraries.

7.13 Preparation of calibration solutions.

7.13.1 Into five 1000-ml aliquots of reagent water, spike 50, 100, 200, 500 and 1000 ML of each of the two solutions in Section 7.9. Spike 1.00 mL of each of the two labeled compound spiking solutions (Section 7.8) into each of the five aliquots.

7.13.2 Using the procedure in Section 11, derivatize and extract each solution, and concentrate the extract to a final volume of 0.50 mL. This will produce calibration solutions of nominal 5, 10, 20, 50, and 100 ug/mL of the native chlorophenolics and a constant concentration of 25 ug/mL of each labeled compound and the SMIS (assuming 100% derivatization and recovery). As noted in Section 11.1.6, ascorbic acid is added to all

samples of final effluents to stabilize chlorocatechols, but is not added to samples of pulp and paper in-process wastewaters. Therefore, it is necessary to prepare separate sets of five initial calibration standards with and without the addition of ascorbic acid. Also, in the event that the laboratory is extracting final effluent samples by both the stir-bar and separatory funnel procedures (see Section 11.3), initial calibration standards should be prepared by both methods.

7.13.3 These solutions permit the relative response (labeled to unlabeled) and the response factor to be measured as a function of concentration (Sections 10.4 and 10.5).

7.13.4 The nominal 50 ug/mL standard may also be used as a calibration verification standard (see Section 9.6).

7.14 Ongoing precision and recovery (OPR) standard: Used for determination of initial (Section 9.3.2) and ongoing (Section 9.6) precision and recovery. This solution is prepared by spiking 500 uL of each the two solutions of the secondary calibration standards (Section 7.9) and 1 mL of each of the two labeled compound spiking solutions (Section 7.8) into 1000 mL of reagent water.

7.15 Stability of solutions: All standard solutions (Sections 7.7 through 7.14) shall be analyzed within 48 hours of preparation and on a monthly basis thereafter for signs of degradation. Standards will remain acceptable if the peak area at the quantitation m z relative to the DFB internal standard remains within +15% of the area obtained in the initial analysis of the standard.

8.0 Sample Collection, Preservation, and

Storage 8.1 Collect samples in glass containers (Section 6.1) following conventional sampling practices (Reference 9). Aqueous samples are collected in refrigerated bottles using automatic sampling equipment.

8.2 Sample preservation.

8.2.1 Residual chlorine: If the sample contains residual chlorine, the chlorine must be reduced to eliminate positive interference resulting from continued chlorination reactions. Immediately after sampling, test for residual chlorine using the following method or an alternative EPA method (Reference 10).

8.2.1.1 Dissolve a few crystals of potassium iodide in the sample and add three to five drops of a 1% starch solution. A blue color indicates the presence of residual chlorine.

8.2.1.2 If residual chlorine is found, add 1 mL of sodium thiosulfate solution (Section 7.2.1) for each 2.5 ppm of free chlorine or until the blue color disappears.

8.2.2 Acidification: Adjust pH of all aqueous samples to <2 with sulfuric acid (Section 7.1.2). Failure to acidify samples may result in positive interferences from continued chlorination reactions.

8.2.3 Refrigeration: Maintain sample temperature at 0 to 4 °C from time of collection until extraction, and maintain extracts at a temperature of 0 to 4 °C from time of extraction until analysis.

8.3 Collect a minimum of 2000 mL of sample. This will provide a sufficient amount for all testing. Smaller amounts may be collected if the stream is known to contain high levels of chlorophenolics.

8.4 All samples must be acetylated and extracted within 30 days of collection, and must be analyzed within 30 days of acetylation. If labeled compound recoveries for a sample do not meet the acceptance criteria in Table 5 and the 30-day holding time is not met, a new sample must be collected.

9.0 Quality Control 9.1 Each laboratory that uses this method is required to operate a formal quality assurance program (Reference 8). 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.

9.1.1 DFTPP spectrum validity shall be checked at the beginning of each eight-hour shift during which analyses are performed. This test is described in Section 9.2.

9.1.2 The laboratory shall make an initial demonstration of the ability to generate acceptable results with this method. This ability is established as described in Section 9.3.

9.1.3 The laboratory 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 laboratory is required to repeat the procedures in Sections 10.3 and 9.3.2 to demonstrate method performance. If the detection limits for the analytes in this method will be affected by the modification, the laboratory should demonstrate that each MDL (40 CFR 136, Appendix B) is less than or equal to the MDL in this method or one-third the regulatory compliance level, whichever is higher.

9.1.4 The laboratory shall spike all samples with labeled compounds and the sample matrix internal standard (SMIS) to monitor method performance. This test is described in Section 9.4. When results of these spikes indicate atypical method performance for samples, the samples are diluted to bring method performance within acceptable limits (Section 13).

9.1.5 Analyses of blanks are required to demonstrate freedom from contamination.

The procedures and criteria for analysis of a blank are described in Section 9.5.

9.1.6 The laboratory shall, on an ongoing basis, demonstrate through analysis of the ongoing precision and recovery standard (Section 7.14) that the analysis system is in control. These procedures are described in Section 9.6.

9.1.7 The laboratory shall maintain records to define the quality of data that is generated. Development of accuracy statements is described in Section 9.4.4 and 9.6.3.

9.2 DFTPP spectrum validity: Inject 1 uL of the DFTPP solution (Section 7.11) either separately or within a few seconds of injection of the OPR standard (Section 9.6) analyzed at the beginning of each shift. The criteria in Table 3 shall be met.

9.3 Initial demonstration of laboratory capability.

9.3.1 Method Detection Limit (MDL): To establish the ability to detect the analytes in this method, the laboratory should determine the MDL per the procedure in 40 CFR 136, Appendix B using the apparatus, reagents, and standards that will be used in the practice of this method. MDLs less than or equal to the MDLs in Table 2 should be achieved prior to the practice of this method.

9.3.2 Initial precision and recovery (IPR): To establish the ability to demonstrate control over the analysis system and to generate acceptable precision and accuracy, the laboratory shall perform the following operations:

9.3.2.1 Derivatize, extract, concentrate, and analyze four 1000-mL aliquots of the ongoing precision and recovery standard (OPR; Section 7.14), according to the procedure in Section 11. Separate sets of IPR aliquots must be prepared with the addition of ascorbic acid and without.

9.3.2.2 Using results of the four analyses, compute the average percent recovery (X) and the relative standard deviation of the recovery (s) for each compound, by isotope dilution for pollutants with a labeled analog, and by internal standard for pollutants with no labeled analog and for the labeled compounds and the SMIS.

9.3.2.3 For each compound, compare s and X with the corresponding limits for initial precision and recovery 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, however, any individual s exceeds the precision limit or any individual X falls outside the range for recovery, system performance is unacceptable for that compound. In this event, correct the problem and repeat the test (Section 9.3.2).

9.4 Labeled compound recovery: The laboratory shall spike all samples with labeled compounds and the sample matrix internal standard (SMIS) to assess method performance on the sample matrix.

9.4.1 Analyze each sample according to the method beginning in Section 11.

9.4.2 Compute the percent recovery (P) of the labeled compounds and the SMIS using the internal standard method (Section 14.3) with 2,2-difluorobiphenyl as the reference compound.

9.4.3 Compare the labeled compound and SMIS recovery for each compound with the corresponding limits 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 is complex. The sample is diluted and reanalyzed per Section 13.

9.4.4 As part of the QA program for the laboratory, it is suggested, but not required, that method accuracy for samples be assessed and records maintained. After the analysis of five samples for which the labeled compounds pass the tests in Section 9.4.3, 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 P-2sp to P = 2sp for each matrix. For example, if P = 90% and sp = 10%, the accuracy interval is expressed as 70 to 110%. Update the accuracy assessment for each compound on a regular basis (e.g., after each 20 to 30 new accuracy measurements).

9.5 Blanks: Reagent water blanks are analyzed to demonstrate freedom from contamination.

9.5.1 Extract and concentrate a 1000-ml reagent water blank with each sample batch (samples started through the extraction process on the same eight-hour shift, to a maximum of 20 samples). Blanks associated with samples to which ascorbic acid is added must be prepared with ascorbic acid, and blanks associated with samples to which ascorbic acid is not added must be prepared without ascorbic acid. Analyze the blank immediately after analysis of the OPR (Section 7.14) to demonstrate freedom from contamination.

9.5.2 If any of the compounds of interest (Table 1) or any potentially interfering compound is found in an aqueous blank at greater than 5 ug/L (assuming a response factor of one relative to the sample matrix 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.

9.6 Calibration verification and ongoing precision and recovery: At the beginning of each eight-hour shift during which analyses are performed, analytical system performance is verified for all compounds. Analysis of DFTPP (Section 9.2) and the nominal 50 ug/mL OPR (Section 11.1.5) is used to verify

all performance criteria. Adjustment and/or recalibration, per Section 10, shall be performed until all performance criteria are met. Only after all performance criteria are met may samples and blanks be analyzed.

9.6.1 Analyze the extract of the OPR (Section 11.1.5) at the beginning of each eighthour shift and prior to analysis of samples from the same batch. Alternatively, a separate calibration verification may be performed using an aliquot of the midpoint calibration standard from Section 7.13 (with a nominal concentration of 50 ug/mL). This alternative may be used to check instrument performance on failure of an OPR, or when samples extracted with an OPR aliquot are not analyzed within the same eight-hour analysis shift.

9.6.1.1 Retention times: The absolute retention time of 2,2-difluorobiphenyl shall be within the range of 765 to 885 seconds, and the relative retention times of all pollutants and labeled compounds shall fall within the limits given in Table 2.

9.6.1.2 GC resolution: The valley height between 4,6-dichloroguaiacol and 3,4dichloroguaiacol at m/z 192 shall not exceed 10% of the height of the taller of the two peaks.

9.6.1.3 Multiple peaks: Each compound injected shall give a single, distinct GC peak.

9.6.2 Compute the percent recovery of each pollutant (Table 1) by isotope dilution (Section 10.4) for those compounds that have labeled analogs. Compute the percent recovery of each pollutant that has no labeled analog by the internal standard method (Section 10.5), using the 3,4,5-trichlorophenol (SMIS) as the internal standard. Compute the percent recovery of the labeled compounds and the SMIS by the internal standard method, using the 2,2-difluorobiphenyl as the internal standard.

9.6.2.1 For each compound, compare the recovery with the limits for ongoing precision and recovery in Table 5. If all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may proceed. If, however, any individual recovery falls outside of the range given, system performance is unacceptable for that compound. In this event, there may be a problem with the GCMS or with the derivatization/extraction concentration systems.

9.6.2.2 GCMS system: To determine if the failure of the OPR test (Section 9.6.2.1) is due to instrument drift, analyze the current calibration verification extract (Section 7.13.4), calculate the percent recoveries of all compounds, and compare with the OPR recovery limits in Table 5. If all compounds meet these criteria, GCMS performance/stability is verified, and the failure of the OPR analysis is attributed to problems in the derivatization/extraction concentration of the OPR. In this case, analysis of the sample extracts may proceed. However, failure of any of the recovery criteria in the analysis of a sample extract requires rederivatization of that sample (Sections 13.3.1 and 13.3.2). If, however, the performance/stability of the GCMS is not verified by analysis of the calibration verification extract, the GCMS requires recalibration and all extracts associated with the failed OPR must be reanalyzed.

9.6.3 Add results that pass the specifications in Section 9.6.2.1 to initial and previous ongoing data for each compound. Update QC charts to form a graphic representation of continued laboratory performance. Develop a statement of laboratory accuracy for each pollutant and labeled compound in each matrix type (reagent water, C-stage filtrate, Estage filtrate, final effluent, etc.) by calculating the average percent recovery (R) and the standard deviation of percent recovery (sr). Express the accuracy as a recovery interval from R- 2sr to R = 2sr. For example, if R = 95% and sr = 5%, the accuracy is 85 to 105%.

9.7 The 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 10) and for initial (Section 9.3.2) and ongoing (Section 9.6) precision and recovery should be identical, so that the most precise results will be obtained. The GCMS instrument in particular will provide the most reproducible results if dedicated to the settings and conditions required for the analyses of chlorophenolics by this method.

9.8 Depending 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.

study indicate the presence of an interfering compound.

10.2.2 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 mz's or introduce other distortion.

10.2.3 The authentic reference spectrum is obtained under DFTPP tuning conditions (Section 10.1 and Table 3) to normalize it to spectra from other instruments.

10.2.4 The spectrum is edited by removing all peaks in the m/z 42 to 45 range, and saving the five most intense mass spectral peaks and all other mass spectral peaks greater than 10% of the base peak (excluding the peaks in the m/z 42 to 45 range). The spectrum may be further edited to remove common interfering m/z's. The spectrum obtained is stored for reverse search and for compound confirmation. 10.3 Minimum level: Demonstrate that the chlorophenolics are detectable at the minimum level (per all criteria in Section 14). The nominal 5 ug/mL calibration standard (Section 7.13) can be used to demonstrate this performance.

10.4 Calibration with isotope dilution: Isotope dilution is used when (1) labeled compounds are available, (2) interferences do not preclude its use, and (3) the quantitation m z (Table 4) extracted ion-current profile (EICP) area for the compound is in the calibration range. Alternative labeled compounds and quantitation m/z's may be used based on availability. If any of the above conditions preclude isotope dilution, the internal standard calibration method (Section 10.5) is used.

10.4.1 A calibration curve encompassing the concentration range is prepared for each compound to be determined. The relative response (pollutant to labeled) vs. concentration in standard solutions is plotted or computed using a linear regression. The example in Figure 1 shows a calibration curve for phenol using phenol-d5 as the isotopic diluent. Also shown are the +10% error limits (dotted lines). Relative response (RR) is determined according to the procedures described below. A minimum of five data points are employed for calibration.

10.4.2 The relative response of a pollutant to its labeled analog is determined from isotope ratio values computed from acquired data. Three isotope ratios are used in this process: Rx = the isotope ratio measured for the pure

pollutant. Ry = the isotope ratio measured for the la

beled compound. Rm = the isotope ratio of an analytical mix

ture of pollutant and labeled compounds.

10.0 Calibration and Standardization 10.1 Assemble the GCMS and establish the operating conditions in Section 12. Analyze standards per the procedure in Section 12 to demonstrate that the analytical system meets the minimum levels in Table 2, and the mass-intensity criteria in Table 3 for 50 ng DFTPP,

10.2 Mass-spectral libraries: Detection and identification of compounds of interest are dependent upon spectra stored in user-created libraries.

10.2.1 Obtain a mass spectrum of the acetyl derivative of each chlorophenolic compound (pollutant, labeled compound, and the sample matrix internal standard) by derivatizing and analyzing an authentic standard either singly or as part of a mixture in which there is no interference between closely eluting components. That only a single compound is present is determined by examination of the spectrum. Fragments not attributable to the compound under

Rm

10.4.4 When the pollutant-labeled pair is not separated (as occurs with carbon-13-labeled compounds), or when another labeled compound with interfering spectral masses overlaps the pollutant (a case which can occur with isomeric compounds), it is necessary to determine the contributions of the pollutant and labeled compound to the respective EICP areas. If the peaks are separated well enough to permit the data system or operator to remove the contributions of the compounds to each other, the equations in Section 10.4.3 apply. This usually occurs when the height of the valley between the two GC peaks at the same m/z is less than 70 to 90% of the height of the shorter of the two peaks. If significant GC and spectral overlap occur, RR is computed using the following equation:

compound shall be used over the five-point calibration range.

10.5 Calibration by internal standard: The method contains two types of internal standards, the sample matrix internal standard (SMIS) and the instrument internal standard (IIS), and they are used for different quantitative purposes. The 3,4,5-trichlorophenol sample matrix internal standard (SMIS) is used for measurement of all pollutants with no labeled analog and when the criteria for isotope dilution (Section 10.4) cannot be met. The 2,2-difluorobiphenyl instrument internal standard (IIS) is used for determination of the labeled compounds and the SMIS. The results are used for intralaboratory statistics (Sections 9.4.4 and 9.6.3).

10.5.1 Response factors: Calibration requires the determination of response factors (RF) for both the pollutants with no labeled analog and for the labeled compounds and the SMIS. The response factor is defined by the following equation:

Where:
Rx is measured as shown in figure 3A,
Ry is measured as shown in figure 3B,
Rm is measured as shown in figure 3C.

For example, Rx = 46100/4780 = 9.644; Ry = 2650/43600 = 0.0608; Rm = 49200/48300 = 1.1019; thus, RR = 1.114. 10.4.5 To calibrate the analytical system by isotope dilution, analyze a 1-4L aliquot of each of the calibration standards (Section 7.13) using the procedure in Section 12. Compute the RR at each concentration.

10.4.6 Linearity: If the ratio of relative response to concentration for any compound is constant (less than 20% coefficient of variation) over the five-point calibration range, an averaged relative response concentration ratio may be used for that compound; otherwise, the complete calibration curve for that

Where:
Ar=the area of the characteristic mass for

the compound in the daily standard. Ais=the area of the characteristic mass for

the internal standard. Cis=the concentration of the internal stand

ard (ug/mL). Cs=is the concentration of the compound in the calibration standard (ug/

mL). When this equation is used to determine the response factors for pollutant compounds without labeled analogs, use the area (Ais) and concentration

of 3,4,5trichlorophenol (SMIS) the internal standard. When this equation is used to determine the response factors for the labeled

(Cis)

as