N normal; gram molecular weight of solute

divided by hydrogen equivalent of solute,

per liter of solution OD outside diameter ppb part-per-billion ppm part-per-million ppt part-per-trillion psig pounds-per-square inch gauge V/v volume per unit volume W/v weight per unit volume

18.2 Definitions and acronyms (in alphabetical order).

Analyte: AOX tested for by this method.

Calibration standard (CAL): A solution prepared from a secondary standard and/or stock solution which is 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 to verify calibration.

Field blank: An 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.

IPR: Initial 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.

Laboratory blank: See Method blank.

Laboratory control sample (LCS): See Ongoing precision and recovery sample (OPR).

Laboratory reagent blank: See Method blank.

May: This action, activity, or procedural step is neither required nor prohibited.

May not: This action, activity, or procedural step is prohibited.

Method blank: An 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.

Must: This action, activity, or procedural step is required.

OPR: Ongoing precision and recovery standard; a laboratory blank spiked with a known quantity of analyte. 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.

PAR: Precision and recovery standard; secondary standard that is diluted and spiked to form the IPR and OPR.

Preparation blank: See Method blank.

Primary dilution standard: A 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 i 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 water: Water demonstrated to be free from the analyte of interest and potentially interfering substances at the method detection limit for the analyte.

Relative standard deviation (RSD): The standard deviation multiplied by 100, divided by the mean.

RSD: See Relative standard deviation.

Should: This action, activity, or procedural step is suggested but not required.

Stock solution: A 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.

VER: See Calibration verification standard.

Method 1653—Chlorinated Phenolics in Wastewater by In Situ Acetylation and GCMS

1.0 Scope and Application 1.1 This method is for determination of chlorinated phenolics (chlorinated phenols, guaiacols, catechols,

vanillins, syringaldehydes) and other compounds associated with the Clean Water Act; the Resource Conservation and Recovery Act; and the Comprehensive Environmental Response. Compensation, and Liability Act; and that are amenable to in situ acetylation, extraction, and analysis by capillary column gas chromatography/mass spectrometry (GCMS). This method is based on existing methods for determination of chlorophenolics in pulp and paper industry wastewaters (References 1 and 2).

1.2 The chemical compounds listed in Table 1 may be determined in waters and, specifically, in in-process streams and

3.0 Definitions 3.1 Chlorinated phenolics are the chlorinated phenols, guaiacols, catechols, vanillins, syringaldehydes and other compounds amenable to in situ acetylation, extraction, and determination by GCMS using this method.

3.2 Definitions for other terms used in this method are given in the glossary at the end of the method (Section 20.0).

wastewaters associated with the pulp and paper industry. The method is designed to meet the survey and monitoring requirements of the Environmental Protection Agency (EPA).

1.3 The detection limit of this method is usually dependent on the level of interferences rather than instrumental limitations. The method detection limits (MDLS) in Table 2 typify the minimum quantity that can be detected with no interferences present.

1.4 The GCMS portions of this method are for use only by persons experienced with GCMS or under the close supervision of such qualified persons. Laboratories unfamiliar with analyses of environmental samples by GCMS should run the performance tests in Reference 3 before beginning.

1.5 Any modification of the method beyond those expressly permitted is subject to the application and approval of alternative test procedures under 40 CFR Parts 136.4 and 136.5.

4.0 Interferences 4.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or elevated baselines, causing misinterpretation of chromatograms and spectra. All materials used in the analysis shall be demonstrated to be free from interferences under the conditions of analysis by running method blanks initially and with each sample batch (samples started through the extraction process on a given eight-hour shift, to a maximum of 20). Specific selection of reagents and purification of solvents by distillation in all-glass systems may be required. Glassware and, where possible, reagents are cleaned by using solvent rinse and baking at 450 °C for a minimum of one hour.

4.2 Interferences co-extracted from samples will vary considerably from source to source, depending on the diversity of the site being sampled. Industry experience suggests that high levels of non-chlorinated phenols may cause poor recovery of the compounds of interest, particularly in samples collected in the vicinity of a source of creosote, such as a wood-preserving plant (Reference 1).

4.3 The internal standard, 3,4,5trichlorophenol, has been reported to be an anaerobic degradation product of 2,3,4,5tetrachlorophenol and/or pentachlorophenol (Reference 1). When an interference with this

another compound occurs, labeled pentachlorophenol or another labeled compound may be used as an alternative internal standard; otherwise, the internal standards and reference compounds must be used as specified in this method.

4.4 Blank contamination by pentachlorophenol has been reported (Reference 1) to be traceable to potassium carbonate; it has also been reported that this contamination may be removed by baking overnight at 400 to 500 °C.

4.5 Catechols are susceptible to degradation by active sites on injection port liners and columns, and are subject to oxidation to the corresponding chloro-o-benzoquinones (Reference 2). A small amount of ascorbic acid may be added to samples to prevent auto-oxidation (Reference 2; also see Section 11.1.6). For pulp and paper industry samples, ascorbic acid may be added to treated effluent samples only.

2.0 Summary of Method 2.1 A 1000-ml aliquot of water is spiked with stable isotopically labeled analogs of the compounds of interest and an internal standard. The solution is adjusted to neutral PH, potassium carbonate buffer is added, and the

pH is raised to 9-11.5. The chlorophenolics are converted in situ to acetates by the addition of acetic anhydride. After acetylation, the solution is extracted with hexane. The hexane is concentrated to a final volume of 0.5 mL, an instrument internal standard is added, and an aliquot of the concentrated extract is injected into the gas chromatograph (GC). The compounds are separated by GC and detected by a mass spectrometer (MS). The labeled compounds and internal standard serve to correct the variability of the analytical technique.

2.2 Identification of a pollutant (qualitative analysis) is performed by comparing the relative retention time and mass spectrum to that of an authentic standard. A compound is identified when its relative retention time and mass spectrum agree.

2.3 Quantitative analysis is performed in one of two ways by GCMS using extracted ion-current profile (EICP) areas: (1) For those compounds listed in Table 1 for which standards and labeled analogs are available, the GCMS system is calibrated and the compound concentration is determined using an isotope dilution technique; (2) for those compounds listed in Table 1 for which authentic standards but no labeled compounds are available, the GCMS system is calibrated and the compound concentration is determined using an internal standard technique.

2.4 Quality is assured through reproducible calibration and testing of the extraction and GCMS systems.

or

5.0 Safety 5.1 The 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 materials safety data sheets (MSDSs) should be made available to all personnel involved in these analyses. Additional information on laboratory safety can be found in References 4 through 6.

5.2 Samples may contain high concentrations of toxic compounds, and should be handled with gloves and a hood opened to prevent exposure.

6.0 Equipment and Supplies NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only. No endorsement is implied. Equivalent performance may be achieved using apparatus and materials other than those specified here, but demonstration of equivalent performance that meets the requirements of this method is the responsibility of the laboratory.

6.1 Sampling equipment for discrete or composite sampling.

6.1.1 Sample bottles and caps.

6.1.1.1 Sample bottle: Amber glass, 1000mL minimum, with screw-cap. If amber bottles are not available, samples shall be protected from light.

6.1.1.2 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with PTFE.

6.1.1.3 Cleaning bottles: Detergent water wash, cap with aluminum foil, and bake at 450 °C for a minimum of one hour before use.

6.1.1.4 Cleaning liners: Detergent water wash, reagent water (Section 7.4) and solvent rinse, and bake at approximately 200 °C for a minimum of 1 hour prior to use.

6.1.1.5 Bottles and liners must be lot-certified to be free of chlorophenolics by running blanks according to this method. If blanks from bottles and/or liners without cleaning or with fewer cleaning steps show no detectable chlorophenolics, the bottle and liner cleaning steps that do not eliminate chlorophenolics may be omitted.

6.1.2 Compositing equipment: Automatic or manual compositing system incorporating glass containers cleaned per bottle cleaning procedure above. Sample containers are kept at 0 to 4 °C during sampling. Glass or PTFE tubing only 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 meth

anol, followed by repeated rinsing with reagent water (Section 7.4) to minimize sample contamination. An integrating flow meter is used to collect proportional composite samples.

6.2 Extraction apparatus.

6.2.1 Bottle or beaker: 1500-to 2000-mL capacity.

6.2.2 Separatory funnel: 500-to 2000-ml, glass, with PTFE stopcock.

6.2.3 Magnetic stirrer: Corning Model 320, or equivalent, with stirring bar.

6.3 Polyethylene gloves: For handling samples and extraction equipment (Fisher 11-394-110-B, or equivalent).

6.4 Graduated cylinders: 1000-mL, 100-ml, and 10-mL nominal.

6.5 Centrifuge: Capable of accepting 50mL centrifuge tubes and achieving 3000 RPM.

6.5.1 Centrifuge tubes.

6.5.1.1 35-ml nominal, with PTFE-lined screw-cap.

6.5.1.2 15-mL nominal, conical graduated, with ground-glass stopper.

6.6 Concentration apparatus.

6.6.1 Kuderna-Danish (K-D) concentrator tube: 10-ml, graduated (Kontes K570050–1025, or equivalent) with calibration verified. Ground-glass stopper (size 19:22 joint) is used to prevent evaporation of extracts.

6.6.2 Kuderna-Danish (K-D) evaporation flask: 1000-ml (Kontes K-570001-1000, or equivalent), attached to concentrator tube with springs (Kontes K-662750-0012).

6.6.3 Snyder column: Three-ball macro (Kontes K-503000-0232, or equivalent).

6.6.4 Snyder column: Two-ball micro (Kontes K-469002–0219, or equivalent).

6.6.5 Boiling chips: Approximately 10-40 mesh, extracted with methylene chloride and baked at 450 °C for a minimum of one hour.

6.6.6 Nitrogen evaporation apparatus: Equipped with a water bath controlled at 35 to 40 °C (N-Evap, Organomation Associates, Inc., South Berlin, MA, or equivalent), installed in a fume hood. This device may be used in place of the micro-Snyder column concentrator in Section 6.6.4 above.

6.7 Water bath: Heated, with concentric ring cover, capable of temperature control (12 °C), installed in a fume hood.

6.8 Sample vials: Amber glass, 1- to 3-mL, with PTFE-lined screw-cap.

6.9 Balances.

6.9.1 Analytical: Capable of weighing 0.1 mg.

6.9.2 Top loading: Capable of weighing 10 mg

6.10 pH meter.

6.11 Gas chromatograph: Shall have splitless or on-column injection port for capillary column, temperature program with 50 °C hold, and shall meet all of the performance specifications in Section 9.

6.12 Gas chromatographic column: 30 m (5 m) x 0.25 mm (10.02 mm) I.D. X 0.25 micron, 5% phenyl, 94% methyl, 1% vinyl silicone bonded-phase fused-silica capillary column (J & W DB-5, or equivalent).

6.13 Mass spectrometer: 70 eV electron impact ionization, shall repetitively scan from 42 to 450 amu in 0.95 to 1.00 second, and shall produce a unit resolution (valleys between miz 441-442 less than 10% of the height of the 441 peak), background-corrected mass spectrum from

50

ng decafluorotriphenylphosphine (DFTPP) introduced through the GC inlet. The spectrum shall meet the mass-intensity criteria in Table 3 (Reference 7). The mass spectrometer 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. All portions of the column which connect the GC to the ion source shall remain at or above the column temperature during analysis to preclude condensation of less volatile compounds.

6.14 Data system: Shall collect and record MS data, store mass-intensity data in spectral libraries, process GCMS data, generate reports, and compute and record response factors.

6.14.1 Data acquisition: Mass spectra shall be collected continuously throughout the analysis and stored on mass storage device.

6.14.2 Mass spectral libraries: User-created libraries containing mass spectra obtained from analysis of authentic standards shall be employed to reverse search GCMS runs for the compounds of interest (Section 10.2).

6.14.3 Data processing: The data system shall be used to search, locate, identify, and quantify the compounds of interest in each GCMS analysis. Software routines shall be employed to compute retention times, and to compute peak areas at the m/z's specified (Table 4). Displays of spectra, mass chromatograms, and library comparisons are required to verify results.

6.14.4 Response factors and multi-point calibrations: The data system shall be used to record and maintain lists of response factors (response ratios for isotope dilution) and multi-point calibration curves (Section 10). Computations of relative standard deviation (coefficient of variation) are used for testing calibration linearity. Statistics on initial (Section 9.3.2) and ongoing (Section 9.6) performance shall be computed and maintained.

7.0 Reagents and Standards 7.1 Reagents for adjusting sample pH. 7.1.1 Sodium hydroxide: Reagent grade, 6 N in reagent water.

7.1.2 Sulfuric acid: Reagent grade, 6 N in reagent water. 7.2 Reagents for sample preservation. 7.2.1 Sodium thiosulfate (Na2S203) solution (1 N): Weigh 79 g Na2S2O3 in a 1-L volu

metric flask and dilute to the mark with reagent water.

7.2.2 Ascorbic acid solution: Prepare a solution of ascorbic acid in reagent water at a concentration of 0.1 g/mL. This solution must be prepared fresh on each day when derivatizations will be performed. Therefore, do not prepare more than will be used that day. (A 50-ml volume is sufficient for ten analyses).

7.3 Solvents: Hexane, acetone, and methanol. Distilled in glass (Burdick and Jackson, or equivalent).

7.4 Reagent water: Water in which the compounds of interest and interfering compounds are not detected by this method.

7.5 Reagents for derivatization. 7.5.1 Potassium carbonate (K2CO3).

7.5.1.1 Purification: Spread in a shallow baking dish, heat overnight at 400 to 500 °C.

7.5.1.2 Solution: Dissolve 150 g purified K2CO3 in 250 mL reagent water.

7.5.2 Acetic anhydride: Redistilled reagent grade.

7.6 Analytical standards.

7.6.1 Derivatization: Because the chlorinated phenolics are determined as their acetate derivatives after in situ acetylation, the method requires that the calibration standards be prepared by spiking the underivatized materials into reagent water and carrying the spiked reagent water aliquot through the entire derivatization and extraction procedure that is applied to the field samples.

7.6.2 Standard solutions: Purchased as solutions or mixtures with certification to their purity, concentration, and authenticity, or prepared from materials of known purity and composition. If chemical purity of a compound 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 - 20 to -10 °C in screw-capped vials with PTFE-lined lids. A mark is placed on the vial at the level of the solution so that solvent evaporation loss can be detected. The vials are brought to room temperature prior to use.

7.6.3 If the chemical purity of any standard does not meet the 98% requirement above, the laboratory must correct all calculations, calibrations, etc., for the difference in purity.

7.7 Preparation of stock solutions: Prepare chlorovanillins

and chlorosyringaldehydes in acetone, as these compounds are subject to degradation in methanol Prepare the remaining chlorophenolics in methanol. Prepare all standards per the steps below. Observe the safety precautions in Section 5.

7.7.1 Dissolve an appropriate amount of assayed reference material in a suitable solvent. For example, weigh 50 mg (10.1 mg) of pentachlorophenol in a 10-mL ground-glass