vvEPA
   United States
   Environmental Protection
   Agency
Method 608.3- Organochlorine Pesticides and
             RGBs by GC/HSD
December 2014

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                 U.S. Environmental Protection Agency
                           Office of Water
                   Office of Science and Technology
               Engineering and Analysis Division (4303T)
                   1200 Pennsylvania Avenue, NW
                       Washington, DC 20460
                         EPA-821-R-14-016
Method 608.3                        i                            December 2014

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         METHOD 608.3 - ORGANOCHLORINE PESTICIDES AND PCBS BY GC/HSD
1.    Scope and Application

1.1   This method is for determination of organochlorine pesticides and polychlorinated biphenyls
      (PCBs) in industrial discharges and other environmental samples by gas chromatography (GC)
      combined with a halogen-specific detector (HSD; e.g., electron capture, electrolytic conductivity),
      as provided under 40 CFR 136.1.  This revision is based on a previous protocol (Reference 1), on
      the revision promulgated October 26, 1984 (49 FR 43234), on an inter-laboratory method
      validation study (Reference 2), and on EPA Method 1656 (Reference 16). The analytes that may
      be qualitatively and quantitatively determined using this method and their CAS Registry numbers
      are listed in Table 1.

1.2   This method may be extended to determine the analytes listed in Table 2. However, extraction or
      gas chromatography challenges for some of these analytes may make quantitative determination
      difficult.

1.3   When this method is used to analyze unfamiliar samples for an analyte listed in Table 1 or Table 2,
      analyte identification must be supported by at least one additional qualitative technique. This
      method gives analytical conditions for a second GC column that can be used to confirm and
      quantify measurements.

      Additionally, Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions
      appropriate for the qualitative confirmation of results for the analytes listed in Tables 1 and 2 using
      the extract produced by this method, and Method 1699 (Reference 18) provides high resolution
      GC/MS conditions  for qualitative confirmation of results using the original sample. When such
      methods are used to confirm the identifications of the target analytes, the quantitative results should
      be derived from the procedure with the calibration range and sensitivity that are most appropriate
      for the intended application.

1.4   The large number of analytes in Tables 1 and 2 makes testing difficult if all analytes are determined
      simultaneously. Therefore, it is necessary to determine and perform quality control (QC) tests for
      the "analytes of interest" only. The analytes of interest are those required to be determined by a
      regulatory/control authority or in a permit, or by a client. If a list of analytes is not specified, the
      analytes in Table 1  must be determined, at a minimum, and QC testing must be performed for these
      analytes. The analytes  in Table 1 and some of the analytes in Table  2 have been identified as Toxic
      Pollutants (40 CFR 401.15), expanded to a list of Priority Pollutants (40 CFR 423, Appendix A).

1.5   In this revision to Method 608, Chlordane has been listed as the alpha- and gamma- isomers in
      Table 1. Reporting may be by the individual  isomers, or as the sum of the concentrations of these
      isomers, as requested or required by a regulatory/control authority or in a permit. Technical
      Chlordane is listed in Table 2 and may be used in cases where historical reporting has only been
      the Technical Chlordane. Toxaphene and the PCBs have been moved from Table  1 to Table 2
      (Additional Analytes) to distinguish these analytes from the analytes required in quality control
      tests (Table 1).  QC acceptance criteria for Toxaphene and the PCBs have been retained in Table 4
      and may continue to be applied if desired, or if these analytes are requested or required by a
      regulatory/control authority or in a permit. Method 1668C (Reference 17) may be useful for
      determination of PCBs as individual chlorinated biphenyl congeners, and Method  1699
      (Reference  18) may be useful for determination of the pesticides listed in this method. However,
      at the time of writing of this revision, Methods 1668C and 1699 had not been approved for use at
      40 CFR part 136.

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1.6   Method detection limits (MDLs; Reference 3) for the analytes in Tables 1 and some of the analytes
      in Table 2 are listed in those tables. These MDLs were determined in reagent water (Reference 3).
      Advances in analytical technology, particularly the use of capillary (open-tubular) columns,
      allowed laboratories to  routinely achieve MDLs for the analytes in this method that are 2-10 times
      lower than those in the version promulgated in 1984 (40 FR 43234). The MDL for an analyte in a
      specific wastewater may differ from those listed, depending upon the nature of interferences in the
      sample matrix.

      1.6.1   EPA has promulgated this method at 40 CFR Part 136 for use in wastewater compliance
             monitoring under the National Pollutant Discharge Elimination System (NPDES). The data
             reporting practices described in  Section 15.2 are focused on such monitoring needs and
             may not be relevant to other uses of the method.

      1.6.2   This method includes "reporting limits" based on EPA's "minimum level" (ML) concept
             (see the glossary in Section 23). Tables 1 and 2 contain MDL values and ML values for
             many of the analytes.  The MDL for an analyte in a specific wastewater may differ from
             those listed  in Tables  1 or 2, depending upon the nature of interferences in the sample
             matrix.

1.7   The separatory funnel and continuous liquid-liquid sample extraction and concentration steps in
      this method are essentially the same as those steps in Methods 606, 609, 611, and 612. Thus, a
      single sample may be extracted to measure the analytes included in the scope of each  of these
      methods. Samples may also be extracted using a disk-based solid-phase extraction (SPE)
      procedure developed by the 3M Corporation and approved by EPA as an Alternate Test Procedure
      (ATP) for wastewater analyses in 1995  (Reference 20).

1.8   This method is performance-based. It may be modified to improve performance (e.g., to overcome
      interferences or improve the accuracy of results) provided all performance requirements are met.

      1.8.1   Examples of allowed method modifications  are described at 40 CFR 136.6. Other
             examples of allowed modifications specific to this method are described in Section 8.1.2.

      1.8.2   Any modification beyond those  expressly permitted at 40 CFR 136.6 or in Section 8.1.2 of
             this method shall be considered  a major modification subject to application and approval of
             an alternate test procedure under 40 CFR 136.4 and 136.5.

      1.8.3   For regulatory compliance, any  modification must be demonstrated to produce results
             equivalent or superior to results  produced by this method when applied to relevant
             wastewaters (Section 8.1.2).

1.9   This method is restricted to use by or under the supervision of analysts experienced in the use of
      GC/HSD.  The laboratory must demonstrate the ability to generate acceptable results with this
      method using the procedure in Section 8.2.

1.10  Terms and units of measure used in this method are  given in the glossary at the end of the method.
2.    Summary of Method

2.1   A measured volume of sample, the amount required to meet an MDL or reporting limit (nominally
      1-L), is extracted with methylene chloride using a separatory funnel, a continuous liquid/liquid
      extractor, or disk-based solid-phase extraction equipment.  The extract is dried and concentrated for

Method 608.3                                 2                                       December 2014

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      cleanup, if required. After cleanup, or if cleanup is not required, the extract is exchanged into an
      appropriate solvent and concentrated to the volume necessary to meet the required compliance or
      detection limit, and analyzed by GC/HSD.

2.2   Qualitative identification of an analyte in the extract is performed using the retention times on
      dissimilar GC columns.  Quantitative analysis is performed using the peak areas or peak heights for
      the analyte on the dissimilar columns with either the external or internal standard technique.

2.3   Florisil®, alumina, a CIS solid-phase cleanup, and an elemental sulfur cleanup procedure are
      provided to aid in elimination of interferences that may be encountered. Other cleanup procedures
      may be used if demonstrated to be effective for the analytes in a wastewater matrix.
3.    Contamination and Interferences

3.1   Solvents, reagents, glassware, and other sample processing lab ware may yield artifacts, elevated
      baselines, or matrix interferences causing misinterpretation of chromatograms.  All materials used
      in the analysis must be demonstrated free from contamination and interferences by running blanks
      initially and with each extraction batch (samples started through the extraction process in a given
      24-hour period, to a maximum of 20 samples).  Specific selection of reagents and purification of
      solvents by distillation in all-glass systems may be required.  Where possible, lab ware is cleaned
      by extraction or solvent rinse, or baking in a kiln or oven. All materials used must be routinely
      demonstrated to be free from interferences under the conditions of the analysis by running blanks
      as described in Section 8.5.

3.2   Glassware must be scrupulously cleaned (Reference 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 reagent water. The glassware should then be
      drained dry, and heated at 400 °C for 15-30 minutes.  Some thermally stable materials, such as
      PCBs, may require higher temperatures and longer baking times for removal. Solvent rinses with
      pesticide quality acetone, hexane, or other solvents may be substituted for heating. Volumetric lab
      ware should not be heated excessively or for long periods of time. After drying and cooling,
      glassware should be sealed and stored in a clean environment to prevent accumulation of dust or
      other contaminants. Store inverted or capped with aluminum foil.

3.3   Interferences by phthalate esters can pose a major problem in pesticide analysis when using the
      electron capture detector. The phthalate esters 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 that may be 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 use of non-fluoropolymer plastics
      in the laboratory.  Exhaustive cleanup of reagents and glassware may be required to eliminate
      background phthalate contamination (References 5 and 6). Interferences from phthalate esters can
      be avoided by using a microcoulometric or electrolytic conductivity detector.

3.4   Matrix interferences may be caused by contaminants 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.  Interferences extracted from
      samples high in total organic carbon (TOC) may result in elevated baselines, or by enhancing or
      suppressing a signal at or near the retention time of an analyte of interest. Analyses of the matrix
      spike and duplicate (Section 8.3) may be useful in identifying matrix interferences, and the cleanup

Method 608.3                                  3                                        December 2014

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      procedures in Section 11 may aid in eliminating these interferences. EPA has provided guidance
      that may aid in overcoming matrix interferences (Reference 7); however, unique samples may
      require additional cleanup approaches to achieve the MDLs listed in Table 3.
4.    Safety

4.1   The 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 safety data sheets (SDSs, OSHA, 29 CFR 1910.1200[g]) should also be made available to all
      personnel involved in sample handling and chemical analysis. Additional references to laboratory
      safety are available and have been identified (References  8 and 9) for the information of the
      analyst.

4.2   The following analytes covered by this method have been tentatively classified as known or
      suspected human or mammalian carcinogens: 4,4'-DDT, 4,4'-DDD, the BHCs, and the PCBs.
      Primary standards of these toxic analytes should be prepared in a chemical fume hood, and a
      NIOSFi/MESA approved toxic gas respirator should be worn when high concentrations are
      handled.

4.3   This method allows the use of hydrogen as a carrier gas in place of helium (Section 5.8.2). The
      laboratory should take the necessary precautions in dealing with hydrogen, and should limit
      hydrogen flow at the source to prevent buildup of an explosive mixture of hydrogen in air.
5.    Apparatus and Materials

      Note:   Brand names and suppliers are for illustration purposes only. No endorsement is implied.
             Equivalent performance may be achieved using equipment and materials other than those
             specified here.  Demonstrating that the equipment and supplies used in the laboratory
             achieve the required performance is the responsibility of the laboratory. Suppliers for
             equipment and materials in this method may be found through an on-line search. Please
             do not contact EPA for supplier information.

5.1   Sampling equipment, for discrete or composite sampling

      5.1.1   Grab sample bottle—amber glass bottle large enough to contain the necessary sample
             volume (nominally 1 L), fitted with a fluoropolymer-lined screw cap.  Foil may be
             substituted for fluoropolymer if the sample is not corrosive.  If amber bottles are not
             available, protect samples from light. Unless pre-cleaned, the bottle and cap liner must be
             washed, rinsed with acetone or methylene chloride, and dried before use to minimize
             contamination.

      5.1.2   Automatic sampler (optional)—the sampler must use a glass or fluoropolymer container
             and tubing for sample collection. 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
             rinsing with reagent water to minimize the potential for sample contamination. An
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             integrating flow meter is required to collect flow proportional composites. The sample
             container must be kept refrigerated at <6 °C and protected from light during compositing.

5.2.   Lab ware

      5.2.1   Extraction

             5.2.1.1   pH measurement

                      5.2.1.1.1    pH meter, with combination glass electrode

                      5.2.1.1.2    pH paper, wide range (Hydrion Papers, or equivalent)

             5.2.1.2   Separatory funnel—Size appropriate to hold the sample and extraction solvent
                      volumes, equipped with fluoropolymer stopcock.

             5.2.1.3   Continuous liquid-liquid extractor—Equipped with fluoropolymer or glass
                      connecting joints and stopcocks requiring no lubrication. (Hershberg-Wolf
                      Extractor, Ace Glass Company, Vineland, NJ, or equivalent.)

                      5.2.1.3.1    Round-bottom flask, 500-mL, with heating mantle

                      5.2.1.3.2    Condenser, Graham, to fit extractor

             5.2.1.4   Solid-phase extractor—90-mm filter apparatus (Figure 2) or multi-position
                      manifold

                      5.2.1.4.1    Vacuum system—Capable of achieving 0.1 bar (25 inch) Hg (house
                                 vacuum, vacuum pump, or water aspirator), equipped with shutoff
                                 valve and vacuum gauge

                      5.2.1.4.2    Vacuum trap—Made from 500-mL sidearm flask fitted with single-
                                 hole rubber stopper and glass tubing

              Note:  The approved ATP for solid-phase extraction is limited to disk-based extraction
                      media and associated peripheral equipment.

      5.2.2   Filtration

             5.2.2.1   Glass powder funnel, 125- to 250-mL

             5.2.2.2   Filter paper for above, Whatman 41, or equivalent

             5.2.2.3   Prefiltering aids—90-mm 1-um glass fiber filter or Empore® Filter Aid 400

      5.2.3   Drying column

             5.2.3.1   Chromatographic column—approximately 400 mm long x 15 mm ID, with
                      fluoropolymer stopcock and coarse frit filter disc (Kontes or equivalent).

             5.2.3.2   Glass wool—Pyrex, extracted with methylene chloride or baked at 450 °C for 1
                      hour minimum

Method 608.3                                 5                                        December 2014

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      5.2.4   Column for Florisil® or alumina cleanup—approximately 300 mm long x 10 mm ID, with
             fluoropolymer stopcock. (This column is not required if cartridges containing Florisil® are
             used.)

      5.2.5   Concentration/evaporation

             Note:   Use of a solvent recovery system with the K-D or other solvent evaporation
                    apparatus is strongly recommended.

             5.2.5.1   Kuderna-Danish concentrator

                      5.2.5.1.1   Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes or
                                 equivalent). Calibration must be checked at the volumes employed
                                 for extract volume measurement.  A ground-glass stopper is used to
                                 prevent evaporation of extracts.

                      5.2.5.1.2   Evaporative flask, Kuderna-Danish—500-mL (Kontes or
                                 equivalent). Attach to concentrator tube with connectors.

                      5.2.5.1.3   Snyder column, Kuderna/Danish—Three-ball macro (Kontes or
                                 equivalent)

                      5.2.5.1.4   Snyder column—Two-ball micro (Kontes or equivalent)

                      5.2.5.1.5   Water bath—Heated, with concentric ring cover, capable of
                                 temperature control (± 2 °C), installed in a hood using appropriate
                                 engineering controls to limit exposure to solvent vapors.

             5.2.5.2   Nitrogen evaporation device—Equipped with heated bath that can be maintained
                      at an appropriate temperature for the solvent and analytes.
                      (N-Evap, Organomation Associates, Inc., or equivalent)

             5.2.5.3   Rotary evaporator—Buchi/Brinkman-American Scientific or equivalent, equipped
                      with a variable temperature water bath, vacuum source with shutoff valve at the
                      evaporator, and vacuum gauge.

                      5.2.5.2.1   A 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.

                      5.2.5.2.2   Round-bottom flask - 100-mL and 500-mL or larger, with ground-
                                 glass fitting compatible with the rotary evaporator

              Note:  This equipment is used to prepare copper foil or copper powder for removing
                      sulfur from sample extracts (see Section 6.7.4).

             5.2.5.4   Automated concentrator—Equipped with glassware sufficient to concentrate 3-
                      400 mL extract to a final volume of 1-10 mL under controlled conditions of
                      temperature and nitrogen flow (Turbovap, or equivalent). Follow manufacturer's
                      directions and requirements.
Method 608.3                                  6                                       December 2014

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             5.2.5.5   Boiling chips—Glass, silicon carbide, or equivalent, approximately 10/40 mesh.
                      Heat at 400 °C for 30 minutes, or solvent rinse or Soxhlet extract with methylene
                      chloride.

      5.2.5   Solid-phase extraction disks—90-mm extraction disks containing 2 g of 8-um octadecyl
             (CIS) bonded silica uniformly enmeshed in a matrix of inert PTFE fibrils (3M Empore® or
             equivalent).  The disks should not contain any organic compounds, either from the PTFE or
             the bonded silica, which will leach into the methylene chloride eluant.  One liter of reagent
             water should pass through the disks in 2-5 minutes, using a vacuum of at least 25 inches of
             mercury.

             Note: Extraction disks from other manufacturers may be used in this procedure, provided
                   that they use the  same solid phase materials (i.e., octadecyl bonded silica). Disks of
                   other diameters also may be used, but may adversely affect the flow rate of the
                   sample through the disk.

5.3   Vials

      5.3.1   Extract storage—10- to  15-mL, amber glass, with fluoropolymer-lined screw cap

      5.3.2   GC autosampler—1- to  5-mL, amber glass, with fluoropolymer-lined screw- or crimp-cap,
             to fit GC autosampler

5.4   Balances

      5.4.1   Analytical—capable of accurately weighing 0.1 mg

      5.4.2   Top loading—capable of weighing 10 mg

5.5   Sample cleanup

      5.5.1   Oven—For baking and storage of adsorbents, capable of maintaining a constant
             temperature (± 5 °C) in  the range of 105-250 °C.

      5.5.2   Muffle furnace—Capable of cleaning glassware or baking sodium sulfate in the range of
             400-450 °C.

      5.5.3   Vacuum system and cartridges for solid-phase cleanup (see Section 11.2)

             5.5.3.1   Vacuum system—Capable of achieving  0.1 bar (25 in.) Hg (house vacuum,
                      vacuum pump, or water aspirator), equipped with shutoff valve and vacuum
                      gauge

             5.5.3.2   VacElute Manifold (Analytichem International, or equivalent)

             5.5.3.3   Vacuum trap—Made from 500-mL sidearm flask fitted with single-hole rubber
                      stopper and glass tubing

             5.5.3.4   Rack for holding 50-mL volumetric flasks in the manifold
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             5.5.3.5   Cartridge—Mega Bond Elute, Non-polar, CIS Octadecyl, 10 g/60 mL
                      (Analytichem International or equivalent), used for solid-phase cleanup of
                      sample extracts (see Section 11.2)

                      5.5.3.5.1   Cartridge certification—Each cartridge lot must be certified to
                                 ensure recovery of the analytes of interest and removal of 2,4,6-
                                 trichlorophenol. To make the test mixture, add the trichlorophenol
                                 solution (Section 6.7.2.1) to the same standard used to prepare the
                                 Quality Control Check Sample (Section 6.8.3).  Transfer the mixture
                                 to the column and dry the column. Pre-elute with three 10-mL
                                 portions of elution solvent,  drying the column between elutions.
                                 Elute the cartridge with 10 mL each of methanol and water, as in
                                 Section 11.2.3.3.

                      5.5.3.5.2   Concentrate the eluant to per Section 10.3.3, exchange to isooctane
                                 or hexane per Section 10.3.3,  and inject 1.0 joL of the concentrated
                                 eluant into the GC using the procedure in Section 12.  The recovery
                                 of all analytes (including the unresolved GC peaks) shall be within
                                 the ranges for calibration verification (Section 13.6 and Table 4), and
                                 the peak for trichlorophenol shall not be detectable; otherwise the
                                 SPE cartridge is not performing properly and the cartridge lot shall
                                 be rejected.

      5.5.4   Sulfur removal tube—40- to 50-mL bottle, test tube, or Erlenmeyer flask with
             fluoropolymer-lined screw cap

5.6   Centrifuge apparatus

      5.6.1   Centrifuge—Capable of rotating 500-mL centrifuge bottles or 15-mL  centrifuge tubes at
             5,000 rpm minimum

      5.6.2   Centrifuge bottle—500-mL, with screw cap, to fit centrifuge

      5.6.3   Centrifuge tube—15-mL, with screw cap, to fit centrifuge

5.7   Miscellaneous lab ware—graduated cylinders, pipettes, beakers, volumetric flasks, vials, syringes,
      and other lab ware necessary to support the operations in this method

5.8   Gas chromatograph—Dual-column with simultaneous split/splitless, temperature programmable
      split/splitless (PTV), or on-column injection; temperature program with isothermal holds, and all
      required accessories including syringes, analytical columns, gases, and detectors.  An autosampler
      is highly recommended because it injects volumes more reproducibly than manual injection
      techniques. Alternatively, two separate single-column gas chromatographic systems may be
      employed.

      5.8.1   Example columns and operating conditions

             5.8.1.1   DB-608 (or equivalent), 30-m long x 0.53-mm ID fused-silica capillary, 0.83-|o,m
                      film thickness.

             5.8.1.2   DB-1701 (or equivalent), 30-m long x 0.53-mm ID fused-silica capillary, 1.0-|o,m
                      film thickness.

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             5.8.1.3   Suggested operating conditions used to meet the retention times shown in Table 3
                      are:

                      Carrier gas flow rate: approximately 7 mL/min
                      Initial temperature: 150 °C for 0.5 minute,
                      Temperature program: 150-270 °C at 5 °C/min, and
                      Final temperature: 270 °C, until trans-Permethrin elutes

             Note:    Other columns, internal diameters, film thicknesses,  and operating conditions
                      may be used, provided that the performance requirements in this method are met.
                      However, the column pair chosen must have dissimilar phases/chemical
                      properties in order to separate the compounds of interest in different retention
                      time order. Columns that only differ in the length, ID, or film thickness, but use
                      the same stationary phase do not qualify as "dissimilar. "

      5.8.2   Carrier gas—Helium or hydrogen.  Data in the tables in this method were obtained using
             helium carrier gas. If hydrogen is used, analytical conditions may need to be adjusted for
             optimum performance, and calibration and all QC tests must be performed with hydrogen
             carrier gas.  See Section 4.3 for precautions regarding the use of hydrogen as a carrier gas.

             Detector—Halogen-specific detector (electron capture detector (BCD), electrolytic
             conductivity detector (ELCD), or equivalent). The BCD has proven effective in the
             analysis of wastewaters for the analytes listed in Tables 1 and 2, and was used to develop
             the method performance data in Section 17 and Tables 4 and 5.

             Data system—A computer system must be interfaced to the GC that allows continuous
             acquisition and storage of data from the detectors throughout the chromatographic program.
             The computer must have software that allows searching GC data for specific analytes, and
             for plotting responses versus time.  Software must also be available that allows integrating
             peak areas or peak heights in selected retention time windows and calculating
             concentrations of the analytes.
6.    Reagents and Standards

6.1   pH adjustment

      6.1.1   Sodium hydroxide solutions

             6.1.1.1   Concentrated (10 M)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute
                      to 100 mL.

             6.1.1.2   Dilute (1 M)—Dissolve 40 g NaOH in 1 L of reagent water.

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

      6.1.3   Hydrochloric acid—Reagent grade, 6 N

6.2   Sodium thiosulfate—(ACS) granular.
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6.3   Sodium sulfate—Sodium sulfate, reagent grade, granular anhydrous (Baker or equivalent), rinsed
      with methylene chloride (20 mL/g), baked in a shallow tray at 450 °C for 1 hour minimum, cooled
      in a desiccator, and stored in a pre-cleaned glass bottle with screw cap which 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 suitable for use.

6.4   Reagent water—Reagent water is defined as water in which the analytes of interest and interfering
      compounds are not observed at the MDLs of the analytes in this method.

6.5   Solvents—methylene chloride, acetone, methanol, hexane, acetonitrile, and isooctane, high purity
      pesticide quality, or equivalent, demonstrated to be free of the analytes and interferences (Section
      3). Purification of solvents by distillation in all-glass systems may be required.

      Note:  The standards and final sample extracts must be prepared in the same final solvent.

6.6   Ethyl ether—Nanograde, redistilled in glass if necessary

      Ethyl ether must be shown to be free of peroxides before use, as indicated by EM Laboratories
      Quant test strips (available from Scientific Products Co. and other suppliers). Procedures
      recommended for removal of peroxides are provided with the test strips. After removal of
      peroxides, add 20 mL of ethyl alcohol preservative to each liter of ether.

6.7   Materials for sample cleanup

      6.7.1   Florisil®—PR grade (60/100 mesh), activated at 650 - 700 °C, stored in the dark in a glass
             container with fluoropolymer-lined screw cap. Activate each batch immediately prior to
             use for 16 hours  minimum at 130 °C in a foil-covered glass container and allow to cool.
             Alternatively, 500 mg cartridges (J.T. Baker, or equivalent) may be used.

      6.7.2   Solutions for solid-phase cleanup

             6.7.2.1    SPE cartridge calibration solution—2,4,6-trichlorophenol, 0.1 |o,g/mL in acetone.

             6.7.2.2    SPE elution solvent—methylene chloride:acetonitrile:hexane (50:3:47).

      6.7.3   Alumina, neutral, Brockman Activity I, 80-200 mesh (Fisher Scientific certified, or
             equivalent). Heat in a glass bottle for 16 hours at 400 to 450 °C. Seal  and cool to room
             temperature. Add 7% (w/w) reagent water and mix for 10 to 12 hours. Keep bottle tightly
             sealed.

      6.7.4   Sulfur removal

             6.7.4.1    Copper foil or powder—Fisher, Alfa Aesar, or equivalent. Cut copper foil into
                       approximately 1-cm squares.  Copper must be activated on each day it will be
                       used, as described below.

                       6.7.4.1.1    Place the quantity of copper needed for sulfur removal (Section
                                  11.5.1.3) in a ground-glass-stoppered Erlenmeyer flask or bottle.
                                  Cover the foil or powder with methanol.
Method 608.3                                  10                                        December 2014

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                      6.7.4.1.2   Add HC1 dropwise (0.5 - 1.0 mL) while swirling, until the copper
                                 brightens.

                      6.7.4.1.3   Pour off the methanol/HCl and rinse 3 times with reagent water to
                                 remove all traces of acid, then 3 times with acetone, then 3 times
                                 with hexane.
                      6.7.4.1.4   For copper foil, cover with hexane after the final rinse. Store in a
                                 stoppered flask under nitrogen until used.  For the powder, dry on a
                                 rotary evaporator. Store in a stoppered flask under nitrogen until
                                 used.

             6.7.4.2   Tetrabutylammonium sulfite (TEA sulfite)

                      6.7.4.2.1   Tetrabutylammonium hydrogen sulfate, [CH3(CH2)3]4NHSO4

                      6.7.4.2.2   Sodium sulfite, Na2SO3

                      6.7.4.2.3   Dissolve approximately 3 g tetrabutylammonium hydrogen sulfate in
                                 100 mL of reagent water in an amber bottle with fluoropolymer-lined
                                 screw cap. Extract with three 20-mL portions of hexane and discard
                                 the hexane extracts.

                      6.7.4.2.4   Add 25 g sodium sulfite to produce a saturated solution.  Store at
                                 room temperature. Replace after 1 month.

6.8   Standard solutions—Purchase as solutions or mixtures with certification to their purity,
      concentration, and authenticity, or prepare from materials of known purity and composition. If
      compound purity is 96% or greater, the weight may be used without correction to compute the
      concentration of the standard. Store neat standards or single analyte standards in the dark at -20 to
      -10 °C in screw-cap vials with fluoropolymer-lined caps. Store multi-analyte standards at 4°C or
      per manufacturer's recommendations.  Place a mark on the vial at the level of the solution so that
      solvent evaporation loss can be detected. Bring the vial to room temperature prior to use to re-
      dissolve any precipitate.

      6.8.1   Stock standard solutions—Standard solutions may be prepared from pure standard
             materials or purchased as certified solutions.  Traceability must be to a national standard,
             when available. Except as noted below for solutions spiked into samples, prepare stock
             standards in isooctane or hexane. Observe the safety precautions in Section 4. The
             following procedure may be used to prepare standards from neat materials.

             6.8.1.1   Dissolve an appropriate amount of assayed reference material in solvent.  For
                      example, weigh 10 mg of aldrin in a 10-mL ground-glass-stoppered volumetric
                      flask and fill to the mark with isooctane or hexane. Larger volumes may be used
                      at the convenience of the laboratory.  After the aldrin  is completely dissolved,
                      transfer the solution to a 15-mL vial with fluoropolymer-lined cap.

             6.8.1.2   Check for signs of degradation prior to preparation of calibration or performance-
                      test standards.

             6.8.1.3   Replace stock solutions after 12 months, or sooner if comparison with quality
                      control check standards indicates a change in concentration.
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      6.8.2   Calibration solutions—It is necessary to prepare calibration solutions for the analytes of
             interest (Section 1.4) only using an appropriate solvent (isooctane or hexane may be used).
             Whatever solvent is used, both the calibration standards and the final sample extracts must
             use the same solvent.  Other analytes may be included as desired.

             6.8.2.1    Prepare calibration standards for the single-component analytes of interest and
                       surrogates at a minimum of three concentration levels (five are suggested) by
                       adding appropriate volumes of one or more stock standards to volumetric flasks.
                       One of the calibration standards should be at a concentration of the analyte near
                       the ML in Table 1 or 2. The ML value may be rounded to a whole number that is
                       more convenient for preparing the standard, but must not exceed the ML values
                       listed in Tables 1 or 2 for those analytes which list ML values. Alternatively, the
                       laboratory may establish the ML for each analyte based on the concentration of
                       the lowest calibration standard in a series of standards obtained from a
                       commercial vendor, again, provided that the ML values does not exceed the MLs
                       in Table 1 and 2, and provided that the resulting calibration meets the acceptance
                       criteria in Section 7.5.2. based on the RSD, RSE, or R2.

                       The other concentrations should correspond to the expected range of
                       concentrations found in real samples or should define the working range of the
                       GC system. A minimum of six concentration levels is required for a second
                       order, non-linear (e.g., quadratic; ax2 + bx + c) calibration. Calibrations higher
                       than second order are not allowed.

                       Given the number of analytes included in this method, it is highly likely that
                       some will coelute on one or both of the GC columns used for the  analysis.
                       Therefore, divide the analytes two or more groups and prepare separate
                       calibration standards for each group, at multiple concentrations (e.g., a five-point
                       calibration will require ten solutions to cover two groups of analytes).

             Note:    Many commercially available standards are divided into separate mixtures to
                       address this issue.

                       The other concentrations should correspond to the expected range of
                       concentrations found in real samples or should define the working range of the
                       GC system. A separate standard near the MDL may be analyzed  as a check on
                       sensitivity, but should not be included in the linearity assessment. A minimum of
                       six concentration levels is required for a non-linear (e.g., quadratic) calibration
                       (Section 7.5.2 or 7.6.2). The solvent for the standards must match the final
                       solvent for the sample extracts (e.g., isooctane or hexane).

             Note:     The option for non-linear calibration may be necessary to address specific
                       instrumental techniques. However, it is not EPA's intent to allow  non-linear
                       calibration to be used to compensate for detector saturation or to avoid proper
                       instrument maintenance.

             6.8.2.2    Multi-component analytes (e.g., PCBs as Aroclors,  and Toxaphene)

                       6.8.2.2.1    A standard containing a mixture of Aroclor 1016 and Aroclor 1260
                                  will include many of the peaks represented in the other Aroclor
                                  mixtures. As a result, a multi-point initial calibration employing a
                                  mixture of Aroclors 1016 and 1260 at three to five concentrations

Method 608.3                                  12                                       December 2014

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                                  should be sufficient to demonstrate the linearity of the detector
                                  response without the necessity of performing multi-point initial
                                  calibrations for each of the seven Aroclors. In addition, such a
                                  mixture can be used as a standard to demonstrate that a sample does
                                  not contain peaks that represent any  one of the Aroclors. This
                                  standard can also be used to determine the concentrations of either
                                  Aroclor 1016 or Aroclor 1260, should they be present in a sample.

                                  Therefore, prepare a minimum of three calibration standards
                                  containing equal concentrations of both Aroclor 1016 and Aroclor
                                  1260 by dilution of the stock standard with isooctane or hexane.  The
                                  concentrations should correspond to the expected range  of
                                  concentrations found in real samples and should bracket the linear
                                  range of the detector.

                       6.8.2.2.2    Single standards of each of the other five Aroclors are required to aid
                                  the analyst in pattern  recognition.  Assuming that the Aroclor
                                  1016/1260 standards described in Section 6.8.2.2.1 have been used to
                                  demonstrate the linearity of the detector, these single standards of the
                                  remaining five Aroclors also may be used to determine the
                                  calibration factor for each Aroclor. Prepare a standard for each of
                                  the other Aroclors. The concentrations should generally correspond
                                  to the mid-point of the linear range of the detector, but lower
                                  concentrations may be employed at the discretion of the analyst
                                  based on project requirements.

                       6.8.2.2.3    For Toxaphene, prepare a minimum  of three calibration  standards
                                  containing Toxaphene by dilution of the stock standard with
                                  isooctane or hexane.  The concentrations should correspond to the
                                  expected range of concentrations found in real samples and should
                                  bracket the linear range of the detector.

      6.8.3   Quality Control (QC) Check Sample —Also known as the  Laboratory Control Sample
             (LCS).  Prepare a mid-level  standard mixture in acetone (or water miscible solvent) from a
             stock solution from the same source as the calibration standards. This standard will be used
             to generate extracts to evaluate the capability of the laboratory.

      6.8.4   Second Source Standard—Obtain standards from a second source (different manufacturer
             or different certified lot), and prepare a mid-level standard  mixture in isooctane or hexane.
             This standard will be analyzed with the calibration curve to verify the accuracy of the
             calibration.

      6.8.5   Internal standard solution—If the internal standard calibration technique is to be used,
             prepare pentachloronitrobenzene (PCNB) at a concentration of 10 |o,g/mL in ethyl acetate.
             Alternative and multiple internal standards;  e.g., tetrachloro-m-xylene, 4,4'-
             dibromobiphenyl, and/or decachlorobiphenyl may be used  provided that the laboratory
             performs all QC tests and meets all QC acceptance criteria with the alternate or additional
             internal standard(s) as an integral part of this method.

      6.8.6   Surrogate solution—Prepare a solution containing one or more surrogates  at a
             concentration of 2 |o,g/mL in acetone. Potential surrogates include:  dibutyl chlorendate
             (DEC), tetrachloro-m-xylene (TCMX), 4,4'-dibromobiphenyl, or decachlorobiphenyl

Method 608.3                                  13                                        December 2014

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             provided that the laboratory performs all QC tests and meets all QC acceptance criteria
             with the alternative surrogate(s) as an integral part of this method. If the internal standard
             calibration technique is used, do not use the internal standard as a surrogate.

      6.8.7   DDT and endrin decomposition (breakdown) solution—Prepare a solution containing
             endrin at a concentration of 1 |J,g/mL and 4,4'-DDT at a concentration of 2 ng/mL, in
             isooctane or hexane.

      6.8.8   Quality control check sample (laboratory control sample; LCS) concentrate—See Sections
             8.2. land 8.4.

      6.8.9   Stability of solutions—Analyze all standard solutions (Sections 6.8.1 through 6.8.8) within
             48 hours of preparation.  Replace purchased certified stock standard  solutions per the
             expiration date.  Replace stock standard solutions prepared by the laboratory or mixed with
             purchased solutions after one year, or sooner if comparison with QC check samples
             indicates a problem.
7.    Calibration

7.1   Establish gas chromatographic operating conditions equivalent to those in Section 5.8.1 and
      Footnote 2 to Table 3.  Alternative temperature program and flow rate conditions may be used.
      The system may be calibrated using the external standard technique (Section 7.5) or the internal
      standard technique (Section 7.6).  It is necessary to calibrate the system for the analytes of interest
      (Section 1.4) only.

7.2   Separately inject the mid-level calibration standard for each calibration mixture. Store the
      retention time on each GC column.

7.3   Demonstrate that each column/detector system meets the MDLs in Table 3 or demonstrates
      sufficient sensitivity for the intended application and passes the DDT/endrin decomposition test
      (Section 13.5).

7.4   Injection of calibration solutions—Inject a constant volume in the range of 0.5 to 2.0 |oL of each
      calibration solution into the GC column/detector pairs.  Beginning with the lowest level mixture
      and proceeding to the highest level mixture may limit the risk of carryover from one standard to the
      next, but other sequences may be used. A blank sample should be analyzed after the highest
      standard to demonstrate that there is no carry-over within the system for this calibration range. For
      each analyte, compute, record, and store, as a function of the concentration injected, the retention
      time and peak area on each column/detector system. If multi-component analytes are to be
      analyzed,  store the retention time and peak area for the three to five exclusive (unique large) peaks
      for each PCB or technical chlordane. Use four to six peaks for toxaphene.

7.5   External standard calibration

      7.5.1    From the calibration data (Section 7.4), calculate the calibration factor (CF) for each
              analyte at each concentration according to the following equation:

                                                  AS
                                             CF= —
Method 608.3                                  14                                        December 2014

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             where:
             Cs  =   Concentration of the analyte in the standard (ng/mL)
             As  =   Peak height or area

             For multi-component analytes, choose a series of characteristic peaks for each analyte (3 to
             5 for each Aroclor, 4 to 6 for toxaphene) and calculate individual calibration factors for
             each peak. Alternatively, for toxaphene, sum the areas of all of the peaks in the standard
             chromatogram and use the summed area to determine the calibration factor. (If this
             alternative is used, the same approach must be used to quantitate the analyte in the
             samples.)

      7.5.2   Calculate the mean (average) and relative standard deviation (RSD) of the calibration
             factors. If the RSD is less than 20%,  linearity through the origin can be assumed and the
             average CF can be used for calculations. Alternatively, the results can be used to fit a
             linear or quadratic regression of response ratios, AS/A1S, vs.  concentration ratios CS/C1S.  If
             used, the regression must be weighted inversely proportional to concentration.  The
             coefficient of determination (R2) of the weighted regression must be greater than 0.99.
             Alternatively, the relative standard error (Reference 10) may be used as an acceptance
             criterion.  As with the RSD, the RSE  must be less than 20%. If an RSE less than 20%
             cannot be achieved for a quadratic regression, system performance is unacceptable and the
             system must be adjusted and re-calibrated.

             Note:   Regression calculations are not included in this method because the calculations
                     are cumbersome and because many GC/ECD data  systems allow selection of
                     weighted regression for calibration and calculation of analyte concentrations.

7.6   Internal standard calibration

      7.6.1   From the calibration data (Section 7.4), calculate the response factor (RF) for each analyte
             at each concentration according to the following equation:

                                               (As x Cis)
                                          Rp=^J	isJ_
                                               (Ais x Cs)

             where:
             As  =   Response for the analyte to be measured.
             AJS  =   Response for the internal standard.
             Cjs  =   Concentration of the internal  standard (ng/mL)
             Cs  =   Concentration of the analyte to be measured  (ng/mL).

      7.6.2   Calculate the mean (average) and relative standard deviation (RSD) of the response factors.
             If the RSD is less than 15%, linearity through the origin can be assumed and the average
             RF can be used for calculations.  Alternatively, the results can be used to prepare a
             calibration curve of response ratios, AS/A1S, vs. concentration ratios, CS/C1S, for the analyte.
             A minimum of six concentration levels is required for a non-linear (e.g., quadratic)
             regression. If used, the regression must be weighted inversely proportional to
             concentration, and the correlation coefficient of the weighted regression must be greater
             than 0.99. The relative standard error (Reference 11) may also be used as an acceptance
             criterion.  As with the RSD, the RSE  must be less than 15%. If an RSE less than 15%
             cannot be achieved for a quadratic regression, system performance is unacceptable and the
             system must be adjusted and re-calibrated.
Method 608.3                                  15                                       December 2014

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7.7   Second source standard—After the calibration curves are analyzed, analyze a second source
      standard at the mid-level concentration. This standard confirms the accuracy of the calibration
      curve. The concentrations must be within 20% difference of the true value. If the observed
      concentration exceeds this criteria, a third source may be analyzed to determine which standard
      was not accurate, and subsequent corrective actions taken.

7.8   The working calibration curve, CF, or RF must be verified at the beginning and end of each 24-
      hour shift by the analysis  of a mid-level calibration standard or the combined QC standard (Section
      6.8.2.1.3). Requirements  for calibration verification are given in Section 13.6 and Table 4.
      Alternatively, calibration  verification may be performed after a set number of injections (e.g., every
      20 injections), to include injection of extracts of field samples, QC samples, instrument blanks, etc.
      (i.e., it is based on the number of injections performed, not sample extracts).

     Note:  The 24-hour shift  begins after analysis of the combined QC standard (calibration
             verification) and ends 24 hours later. The ending calibration verification standard is run
             immediately after the last sample run during the 24-hour shift, so the beginning and ending
             calibration verifications are outside of the 24-hour shift. If calibration verification is based
             on the number of injections instead of time, then the ending verification standard for  one
             group of 20 injections may be used  as the beginning verification for the next group of 20
             injections.

7.9   Florisil® calibration—The column cleanup  procedure in Section 11.3 utilizes Florisil  column
      chromatography. Florisil® from different batches or sources may vary in adsorptive capacity. To
      standardize the amount of Florisil® which is used, use of the lauric acid value (Reference 11) is
      suggested. The referenced procedure determines the adsorption from a 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.  If cartridges containing Florisil® are used, then
      this step is not necessary.
8.    Quality Control

8.1   Each 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 and ongoing analysis of spiked samples and blanks to evaluate and document data
      quality. The laboratory must maintain records to document the quality of data generated. Ongoing
      data quality checks are compared with established performance criteria to determine if the results
      of analyses meet performance requirements of this method. A quality control check standard
      (LCS, Section 8.4) must be prepared and analyzed with each batch of samples to confirm that the
      measurements were performed in an in-control mode of operation. A laboratory may develop its
      own performance criteria (as QC acceptance criteria), provided such criteria are as or more
      restrictive than the criteria in this method.

      8.1.1   The laboratory must make an initial demonstration of the capability (IDC) to generate
             acceptable precision and recovery with this method. This demonstration is detailed in
             Section 8.2.  On a continuing basis, the laboratory should repeat demonstration of
             capability (DOC) annually.

      8.1.2   In recognition of advances that are occurring in analytical technology, and to overcome
             matrix interferences, the laboratory is permitted certain options (Section 1.8 and 40 CFR
             136.6(b) [Reference  12]) to improve separations or lower the costs of measurements. These
             options may include alternative extraction (e.g., other solid-phase extraction materials and

Method 608.3                                 16                                       December 2014

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             formats), concentration, and cleanup procedures, and changes in GC columns (Reference 12).
             Alternative determinative techniques, such as the substitution of spectroscopic or
             immunoassay 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 greater than the specificity of the techniques in
             this method for the analytes of interest.  The laboratory is also encouraged to participate in
             performance evaluation studies (see Section 8.8).

             8.1.2.1   Each time a modification listed above is made to this method, the laboratory is
                      required to repeat the procedure in Section 8.2.  If the detection limit of the method
                      will be affected by the change, the laboratory is required to demonstrate that the
                      MDLs (40 CFR Part 136, Appendix B) are lower than one-third the regulatory
                      compliance limit or as low as the MDLs in this method, whichever are greater.  If
                      calibration will be affected by the change, the instrument must be recalibrated per
                      Section 7.  Once the modification is demonstrated to produce results equivalent or
                      superior to results produced by this  method as written, that modification may be
                      used routinely thereafter, so long as the other requirements in this method are met
                      (e.g., matrix spike/matrix spike duplicate recovery and relative percent difference).

                      8.1.2.1.1   If an allowed method modification, is to be applied to aspecific
                                 discharge, the laboratory must prepare and analyze matrix
                                 spike/matrix spike duplicate (MS/MSD) samples (Section 8.3) and
                                 LCS samples (Section  8.4). The  laboratory must include surrogates
                                 (Section 8.7) in each of the samples.  The MS/MSD and LCS
                                 samples must be fortified with the analytes of interest (Section 1.4).
                                 If the modification is for nationwide use, MS/MSD samples must be
                                 prepared from a minimum of nine different discharges (See  Section
                                 8.1.2.1.2), and all QC acceptance criteria in this method must be
                                 met.  This evaluation only needs to be performed once other than for
                                 the routine QC required by this method (for example it could be
                                 performed by the vendor of an alternate material) but any laboratory
                                 using that specific material must have the results of the study
                                 available. This includes a full data package with the raw data that
                                 will allow an independent reviewer to verify each  determination and
                                 calculation performed by the laboratory (see Section 8.1.2.2.5, items
                                 a-q).

                      8.1.2.1.2   Sample matrices on which MS/MSD tests must be performed for
                                 nationwide use of an allowed modification:
                                 (a) Effluent from a POTW
                                 (b) ASTMD5905 Standard Specification for Substitute Wastewater
                                 (c) Sewage sludge, if sewage sludge will be in the permit
                                 (d) ASTM Dl 141 Standard Specification for Substitute Ocean
                                      Water, if ocean water will be in the permit
                                 (e) Untreated and treated wastewaters up to a total of nine matrix
                                     types (see http:water.epa.gov/scitech/wastetech
                                     /guide/industry.cfm) for a list of industrial categories with
                                     existing effluent guidelines).

                                     At least one of the above wastewater matrix types must  have at
                                     least one of the following characteristics:
Method 608.3                                  17                                       December 2014

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                                      (i)   Total suspended solids greater than 40 mg/L
                                      (ii)   Total dissolved solids greater than 100 mg/L
                                      (iii)  Oil and grease greater than 20 mg/L
                                      (iv)  NaCl greater than 120 mg/L
                                      (v)   CaCO3 greater than 140 mg/L

                                  The interim acceptance criteria for MS, MSB recoveries that do not
                                  have recovery limits specified in Table 5, and recoveries for
                                  surrogates that do not have recovery limits specified in Table 8, must
                                  be no wider than 60-140 %, and the relative percent difference
                                  (RPD) of the concentrations in the MS and MSB that do not have
                                  RPD limits specified in Table 5 must be less than 30%.
                                  Alternatively, the laboratory may use the laboratory's in-house limits
                                  if they are tighter.

                                  (f)  A proficiency testing (PT) sample from a recognized provider, in
                                      addition to tests of the nine matrices (Section 8.1.2.1.1).

             8.1.2.2    The laboratory must maintain records of modifications made to this method.
                       These records include the following, at a minimum:

                       8.1.2.2.1    The names, titles, street addresses, telephone numbers, and
                                  e-mail addresses of the analyst(s) that performed the analyses and
                                  modification, and of the quality control officer that witnessed and will
                                  verify the analyses and modifications.
                       8.1.2.2.2    A list of analytes, by name and CAS Registry number.

                       8.1.2.2.3    A narrative stating reason(s) for the modifications.

                       8.1.2.2.4    Results from all quality control (QC) tests comparing the modified
                                  method to this  method, including:
                                  a)  Calibration (Section 7).
                                  b)  Calibration verification (Section 13.6).
                                  c)  Initial demonstration of capability (Section 8.2).
                                  d)  Analysis of blanks (Section 8.5).
                                  e)  Matrix spike/matrix spike duplicate analysis (Section 8.3).
                                  f)   Laboratory control sample analysis (Section 8.4).

                       8.1.2.2.5    Data 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 10).
                                  f)   Extract volume prior to each cleanup step (Sections 10 and 11).
                                  g)  Extract volume after each cleanup step (Section 11).
                                  h)  Final extract volume prior to injection (Sections 10 and 12).
                                  i)   Injection volume (Sections 12.3 and 13.2).
                                  j)   Sample or extract dilution (Section 15.4).
                                  k)  Instrument and operating conditions.

Method 608.3                                  18                                        December 2014

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                                  1)  Column (dimensions, material, etc).
                                  m) Operating conditions (temperatures, flow rates, etc).
                                  n)  Detector (type, operating conditions, etc).
                                  o)  Chromatograms and other recordings of raw data.
                                  p)  Quantitation reports, data system outputs, and other data to link
                                     the raw data to the results reported.
                                  q)  A written Standard Operating Procedure (SOP)

                      8.1.2.2.6    Each individual laboratory wishing to use a given modification must
                                  perform the start-up tests in  Section 8.1.2 (e.g., DOC, MDL), with
                                  the modification as an integral part of this method prior to applying
                                  the modification to specific discharges.  Results of the DOC must
                                  meet the QC acceptance criteria in Table 5 for the analytes of interest
                                  (Section 1.4), and the MDLs must be equal to or lower than the
                                  MDLs in Table 3 for the analytes of interest.

      8.1.3   Before analyzing samples, the laboratory must analyze a blank to demonstrate that
             interferences from the analytical system, lab ware,  and reagents, are under control. Each
             time a batch of samples is extracted or reagents are changed, a blank must be extracted and
             analyzed as a safeguard against laboratory contamination.  Requirements for the blank are
             given in Section 8.5.

      8.1.4   The laboratory must, on an ongoing basis, spike and analyze a minimum of 5 % of all
             samples in  a batch (Section 22.2) or from a given site or discharge, in duplicate, to monitor
             and evaluate method and laboratory performance on the sample matrix. This procedure is
             described in Section 8.3.

      8.1.5   The laboratory must, on an ongoing basis, demonstrate through analysis of a quality control
             check sample (laboratory control sample, LCS; on-going precision and recovery sample,
             OPR) that the measurement system is in control. This procedure is described in Section
             8.4.

      8.1.6   The laboratory should maintain performance records to document the quality of data that is
             generated.  This procedure is given in Section 8.7.

      8.1.7   The large number of analytes  tested in performance tests in this method present a
             substantial  probability that one or more will fail acceptance criteria when all  analytes are
             tested simultaneously, and a re-test (reanalysis) is allowed if this situation should occur. If,
             however, continued re-testing results in further repeated failures, the laboratory should
             document the failures and either avoid reporting results for the analytes that failed or report
             the problem and failures with the data. A QC failure does not relieve a discharger or
             permittee of reporting timely results.

8.2   Demonstration of capability (DOC)—To establish the ability to generate acceptable recovery and
      precision, the laboratory must perform the DOC in Sections 8.2.1 through 8.2.6 for the analytes of
      interest initially and in an on-going manner at least annually.  The laboratory must also establish
      MDLs for the analytes of interest using the MDL procedure at 40 CFR 136, Appendix B. The
      laboratory's MDLs must be  equal to or lower than those listed in Table 3 or lower than one-third
      the regulatory compliance limit, whichever is greater. For MDLs not listed in Tables 1 or 2, the
      laboratory must determine the MDLs using the MDL procedure at 40 CFR 136, Appendix B under
      the same conditions used to determine the MDLs for the analytes listed in Tables 1 and 2. All
      procedures used in the analysis, including cleanup procedures, must be included in the DOC.

Method 608.3                                  19                                       December 2014

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      8.2.1   For the DOC, a QC check sample concentrate containing each analyte of interest (Section
             1.4) is prepared in a water-miscible solvent using the solution in Section 6.8.3. The QC
             check sample concentrate must be prepared independently from those used for calibration,
             but should be from the same source and prepared in a water-miscible solvent.  The
             concentrate should produce concentrations of the analytes of interest in water at or below
             the mid-point of the calibration range. Multiple solutions may be required.

             Note:   QC check sample concentrates are no longer available from EPA.

      8.2.2   Using a pipet or syringe, prepare four QC check samples by adding an appropriate volume
             of the concentrate and of the surrogate(s) to each of four 1-L aliquots of reagent water.
             Swirl or stir to mix.

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

      8.2.4   Calculate the average percent recovery (X) and the standard deviation (s) of the percent
             recovery for each analyte using the four results.

      8.2.5   For each analyte,  compare s and  X with the corresponding acceptance criteria for precision
             and recovery in Table 4.  For analytes in Table  2 that are not listed in Table 4, QC
             acceptance criteria must be developed by the  laboratory.  EPA has provided guidance for
             development of QC acceptance criteria (References 12 and 13). If s and X for all analytes
             of interest meet the acceptance criteria, system  performance is acceptable and analysis of
             blanks and samples can begin. If any individual s exceeds the precision limit or any
             individual X falls outside the range for recovery, system performance is unacceptable for
             that analyte.

             Note:   The large number of analytes in Tables 1 and 2 present a substantial probability
                     that one or more  will fail at least one of the acceptance criteria when  many or all
                     analytes are  determined simultaneously.

      8.2.6   When one or more of the  analytes tested fail at  least one of the acceptance criteria, repeat
             the test for only the analytes that failed. If results for these analytes  pass, system
             performance is acceptable and analysis of samples and blanks may proceed. If one or more
             of the analytes again fail, system performance is unacceptable for the analytes that failed
             the acceptance criteria. Correct the problem and repeat the test (Section 8.2).  See Section
             8.1.7 for disposition of repeated failures.

             Note: To maintain the validity of the test and re-test, system maintenance and/or
                   adjustment is  not permitted between this pair of tests.

8.3   Matrix spike and matrix spike duplicate (MS/MSD)—The laboratory must, on an ongoing basis,
      spike at least 5% of the samples in duplicate from each sample site being monitored to assess
      accuracy (recovery and precision).  The data user should identify the sample and the analytes of
      interest (Section 1.4) to be spiked.  If direction cannot be obtained, the laboratory must spike at
      least one sample in duplicate per  extraction batch of up to 20 samples (Section 22.2) with the
      analytes in Table 1.  Spiked sample results should be reported only to the data user whose sample
      was spiked, or as requested or required by a regulatory/control authority.

      8.3.1.  If, as in compliance monitoring, the concentration of a specific analyte will be checked
             against a regulatory concentration limit, the concentration of the spike should be at that
Method 608.3                                  20                                        December 2014

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             limit; otherwise, the concentration of the spike should be one to five times higher than the
             background concentration determined in Section 8.3.2, at or near the midpoint of the
             calibration range, or at the concentration in the LCS (Section 8.4) whichever concentration
             would be larger. When no information is available, the mid-point of the calibration may be
             used, as long as it is the same or less than the regulatory limit.

      8.3.2   Analyze one sample aliquot to determine the background concentration (B) of the each
             analyte of interest.  If necessary to meet the requirement in Section 8.3.1, prepare a new
             check sample concentrate (Section 8.2.1) appropriate for the background concentration.
             Spike and analyze two additional  sample aliquots of the same volume as the original
             sample, and determine the concentrations after spiking (Al and A2) of each analyte.
             Calculate the percent recoveries (Pi and P2) as:

                                                  AX-B
                                              Px= -y- x 100

             where T is the known true value of the spike.

             Also calculate the  relative percent difference (RPD) between the concentrations (A] and
             A2):
                                            Mi- A2\
                                    RPD =  '    i   .    x100
                                             A! + A2
                                                2

      8.3.3   Compare the percent recoveries (Pi and P2) and the RPD for each analyte in the MS/MSD
             aliquots with the corresponding QC acceptance criteria for recovery (P) and RPD in Table
             4.

             If any individual P falls outside the designated range for recovery in either aliquot, or the
             RPD limit is exceeded, the result for the analyte in the unspiked sample is suspect and may
             not be reported or used for permitting or regulatory compliance.  See Section 8.1.7 for
             disposition of failures.

             For analytes in Table 2 not listed in Table 5, QC acceptance criteria must be developed by
             the laboratory. EPA has provided guidance for development of QC acceptance criteria
             (References 12 and 13).

      8.3.4   After analysis of a minimum of 20 MS/MSD samples for each target analyte and surrogate,
             the laboratory must calculate and  apply in-house QC limits for recovery and RPD of future
             MS/MSD samples (Section 8.3).  The QC limits for recovery are calculated as the mean
             observed recovery ± 3  standard deviations, and the upper QC limit  for  RPD is calculated as
             the mean RPD plus 3 standard deviations of the RPDs. The in-house QC limits must be
             updated at least every  two years and re-established after any major change in the analytical
             instrumentation or process. At least 80% of the analytes tested in the MS/MSD must have
             in-house QC acceptance criteria that are tighter than those in Table 4.  If an in-house QC
             limit for the RPD is greater than the limit in Table 4, then the limit  in Table 4 must be used.
             Similarly, if an in-house lower limit for recovery is below the lower limit in Table 4, then
             the lower limit in Table 4 must be used, and if an in-house upper limit  for recovery is above
             the upper limit in Table 4, then the upper limit in Table 4 must be used. The laboratory
             must evaluate surrogate recovery  data in each  sample against its in-house surrogate
             recovery limits.  The laboratory may use 60 -140% as interim acceptance criteria for
             surrogate recoveries until in-house limits are developed.

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8.4   Laboratory control sample (LCS)—A QC check sample (laboratory control sample, LCS; on-going
      precision and recovery sample, OPR) containing each single-component analyte of interest
      (Section 1.4) must be extracted, concentrated, and analyzed with each extraction batch of up to 20
      samples (Section 3.1) to demonstrate acceptable recovery of the analytes of interest from a clean
      sample matrix. If multi-peak analytes are required, extract and prepare at least one as an LCS for
      each batch. Alternatively, the laboratory may set up a program where multi-peak LCS is rotated
      with a single-peak LCS.

      8.4.1   Prepare the LCS by adding QC check sample concentrate (Section 8.2.1) to reagent water.
             Include all analytes of interest (Section 1.4) in the LCS. The volume of reagent water must
             be the same as the nominal volume used for the sample, the DOC (Section 8.2), the blank
             (Section 8.5), and the MS/MSD (Section 8.3).  Also add a volume of the surrogate solution
             (Section 6.8.6).

      8.4.2   Analyze the LCS prior to analysis of samples in the extraction batch (Section 3.1).
             Determine the concentration (A) of each analyte. Calculate the percent recovery as:


                                          Ps=jx 100

             where T is the true value of the concentration in the LCS.

      8.4.3   For each analyte, compare the percent recovery (P) with its corresponding QC acceptance
             criterion in Table 4.  For analytes of interest in Table 2 not listed in Table 4, use the QC
             acceptance criteria developed for the MS/MSD (Section 8.3.3.2).  If the recoveries for all
             analytes of interest fall within the designated ranges, analysis of blanks and field samples
             may proceed. If any individual recovery falls outside the range, proceed according to
             Section 8.4.4.

             Note:  The large number of analytes in Tables 1 and 2 present a substantial probability
                    that one or more will fail the acceptance criteria when all analytes are tested
                    simultaneously.  Because a re-test is allowed in event of failure (Sections 8.1.7 and
                    8.4.4), it may be prudent to extract and analyze two LCSs together and evaluate
                    results of the second analysis against the QC acceptance criteria only if an analyte
                    fails the first test.

      8.4.4   Repeat the test only for those analytes that failed to meet the acceptance criteria (P). If
             these analytes now pass, system performance is acceptable and  analysis of blanks and
             samples may proceed. Repeated failure, however, will confirm a general problem with the
             measurement system.  If this occurs, repeat the test using a fresh LCS (Section 8.2.1) or an
             LCS prepared with a fresh QC check sample concentrate (Section 8.2.1), or perform and
             document system repair. Subsequent to repair, repeat the LCS test (Section 8.4).  See
             Section 8.1.7 for disposition of repeated failures.

      8.4.5   After analysis of 20 LCS samples, the laboratory must calculate and apply in-house QC
             limits for recovery to future LCS samples (Section  8.4). Limits for recovery in the LCS are
             calculated as the mean recovery ±3 standard deviations. A minimum of 80% of the
             analytes tested for in the LCS must have QC acceptance criteria tighter than those in Table
             4.  As noted in Section 8.6, each laboratory must develop QC acceptance criteria for the
             surrogates they employ.  The laboratory should use 60 -140% as interim acceptance criteria
             for recoveries of spiked analytes and surrogates until in-house LCS and surrogate limits are
             developed. If an in-house lower limit for LCS recovery is lower than the lower limit in

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             Table 4, the lower limit in Table 4 must be used, and if an in-house upper limit for recovery
             is higher than the upper limit in Table 4, the upper limit in Table 4 must be used.

8.5   Blank—Extract and analyze a blank with each extraction batch (Section 22.2) to demonstrate that
      the reagents and equipment used for preparation and analysis are free from contamination.

      8.5.1   Prepare the blank from reagent water and spike it with the surrogates. The volume of
             reagent water must be the same as the volume used for samples, the DOC (Section 8.2), the
             LCS (Section 8.4), and the MS/MSD (Section 8.3).  Extract, concentrate, and analyze the
             blank using the same procedures and reagents used for the samples, LCS, and MS/MSD in
             the batch.  Analyze the blank immediately after analysis of the LCS (Section 8.4) and prior
             to analysis of the MS/MSD and samples to demonstrate freedom from contamination.

      8.5.2   If any analyte of interest is found in the blank at a concentration greater than the MDL for
             the analyte, at a concentration greater than one-third the regulatory compliance limit, or at a
             concentration greater than one-tenth the concentration in a sample in the batch (Section
             3.1), whichever is greatest, analysis of samples must be halted and samples in the batch
             must be re-extracted and the extracts reanalyzed.  Samples in a batch must be associated
             with an uncontaminated blank before the results for those samples may be reported or used
             for permitting or regulatory compliance purposes. If re-testing of blanks results in repeated
             failures, the laboratory should document the failures and report the problem and failures
             with the data.

8.6   Surrogate recovery—As a quality control check, the laboratory must spike all samples with the
      surrogate standard spiking solution (Section 6.8.6) per Section 10.2.2 or 10.4.2, analyze the
      samples, and calculate the percent recovery of each surrogate. QC acceptance criteria for
      surrogates must be developed by the laboratory. EPA has provided guidance for development of
      QC acceptance criteria (References 12 and 13). If any recovery fails its criterion, attempt to find
      and correct the cause of the failure, and if sufficient volume is available, re-extract another aliquot
      of the affected sample.  Surrogate recoveries from the blank and LCS may be used as pass/fail
      criteria by the laboratory or as required by a regulatory authority, or may be used to diagnose
      problems with the analytical system.

8.7   As part of the QC program for the laboratory, it is suggested but not required that method accuracy
      for wastewater samples be assessed and records maintained. After analysis of five or more spiked
      wastewater samples as in Section 8.4, calculate the average percent recovery (X) and the standard
      deviation of the percent recovery  (sp). Express the accuracy assessment as a percent interval from
      X -2sp to X +2sp.  For example, if X  = 90% and  sp=  10%, the accuracy interval is expressed as 70
      -  110%. Update the accuracy assessment for each analyte on a regular basis to ensure process
      control (e.g., after each 5-10 new accuracy measurements).

8.8   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
      environmental measurements. When doubt exists over the identification of a peak on the
      chromatogram, confirmatory techniques such as gas chromatography with another 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.
Method 608.3                                 23                                       December 2014

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9.    Sample Collection, Preservation, and Handling

9.1   Collect samples as grab samples in glass bottles, or in refrigerated bottles using automatic sampling
      equipment. Collect 1-L of ambient waters, effluents, and other aqueous samples. If high
      concentrations of the analytes of interest are expected (e.g., for untreated effluents or in-process
      waters), collect a smaller volume (e.g., 250 mL), but not less than 100 mL, in addition to the 1-L
      sample. Follow conventional sampling practices, except do not pre-rinse the bottle with sample
      before collection. Automatic sampling equipment must be as free as possible of polyvinyl chloride
      or other tubing or other potential sources of contamination. If needed, collect additional sample(s)
      for the MS/MSD (Section 8.3).

9.2   Ice or refrigerate the sample at <6°C from the time of collection until extraction, but do not freeze.
      If aldrin is to be determined and residual chlorine  is present, add 80 mg/L of sodium thiosulfate but
      do not add excess. Any method suitable for field use may be employed to test for residual chlorine
      (Reference 14). If sodium thiosulfate interferes in the determination of the analytes, an alternative
      preservative (e.g., ascorbic acid or sodium sulfite) may be used.

9.3   Extract all samples within seven days of collection and completely analyze within 40 days of
      extraction (Reference 1).  If the sample will not be extracted within 72 hours of collection, adjust
      the sample pH to range of 5.0  - 9.0 with sodium hydroxide solution or sulfuric acid.  Record the
      volume of acid or base used.
10.   Sample Extraction

10.1  This section contains procedures for separatory funnel liquid-liquid extraction (SFLLE, Section
      10.2), continuous liquid-liquid extraction (CLLE, Section 10.4), and disk-based solid-phase
      extraction (SPE, Section 10.5). SFLLE is faster, but may not be as effective as CLLE for
      extracting polar analytes. SFLLE is labor intensive and may result in formation of emulsions that
      are difficult to break. CLLE is less labor intensive, avoids emulsion formation, but requires more
      time (18-24 hours), more hood space, and may require more solvent. SPE can be faster, unless the
      particulate load in an aqueous sample is so high that it slows the filtration process. If an alternative
      extraction scheme to those detailed in this method is used, all QC tests must be performed and all
      QC acceptance criteria must be met with that extraction scheme as an integral part of this method.

10.2  Separatory funnel liquid-liquid extraction (SFLLE)

      10.2.1  The SFLLE procedure below assumes a sample volume of 1 L. When a different sample
             volume is extracted, adjust the volume of methylene chloride accordingly.

      10.2.2  Mark the water meniscus on the side of the sample bottle for later determination of sample
             volume.  Pour the entire sample into the separatory funnel.  Pipetthe surrogate standard
             spiking solution (Section 6.8.6) into the separatory funnel. If the sample will be used for
             the LCS or MS or MSB, pipet the appropriate QC check sample concentrate (Section 8.2.1)
             into the separatory funnel.  Mix well.  If the sample arrives in a larger sample bottle, 1 L
             may be measured in a graduated cylinder, then added to the separatory funnel.

             Note:  Instances in which the sample is collected in an oversized bottle should be reported
                    by the laboratory to the data user.  Of particular concern is that fact that this
                    practice precludes rinsing the empty bottle with solvent as described below, which
                    could leave hydrophobic pesticides on the wall of the bottle, and underestimate the
                    actual sample concentrations.

Method 608.3                                 24                                       December 2014

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      10.2.3  Add 60 mL of methylene chloride to the sample bottle, seal, and shake for 30 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 to release excess pressure.
             Allow the organic layer to separate from the water phase for a minimum of 10 minutes. If
             an emulsion forms and the emulsion interface between the layers is more than one-third the
             volume of the solvent layer, 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, freezing, or other physical
             methods.  Collect the methylene chloride extract in a 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 10.4.

      10.2.4  Add 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 flask. Perform a third
             extraction in the same manner.  Proceed to macro-concentration (Section 10.3.1).

      10.2.5  Determine the original sample volume by refilling the sample bottle to the mark and
             transferring the liquid to an appropriately sized graduated cylinder. Record the sample
             volume to the nearest 5 mL. Sample volumes may also be determined by weighing the
             container before and after extraction or filling to the mark with water.

10.3  Concentration

      10.3.1  Macro concentration

             10.3.1.1  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 so long as the
                      requirements of Section 8.2 are met.

             10.3.1.2  Pour the 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 flask and column with 20-30 mL of methylene chloride to complete the
                      quantitative transfer.

             10.3.1.3  If no cleanup is to be  performed on the sample, add 500 (iL (0.5 mL) of isooctane
                      to the extract to act as a keeper during concentration.

               10.3.1.4        Add one  or two clean boiling chips and attach athree-ball Snyder
                              column to the K-D evaporative flask. Pre-wet 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 - 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 - 20 minutes.  At the proper rate of evaporation 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 or other determined amount, remove the K-D apparatus from the
                              water bath and allow  it to drain and cool for at least 10 minutes.
Method 608.3                                 25                                       December 2014

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             10.3.1.5  If the extract is to be cleaned up by a procedure for sulfur removal, 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.  Adjust the final volume to 10 mL in methylene chloride and proceed
                      to sulfur removal (Section 11.5).  If the extract is to cleaned up using one of the
                      other cleanup procedures or is to be injected into the GC, proceed to Kuderna-
                      Danish micro-concentration (Section  10.3.2) or nitrogen evaporation and solvent
                      exchange (Section 10.3.3).

      10.3.2  Kuderna-Danish micro concentration

             10.3.2.1  Add another one or two clean boiling  chips to the concentrator tube and attach a
                      two-ball micro-Snyder column.  Pre-wet 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 - 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 - 10 minutes.  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
                      approximately 1  mL or other required amount, remove the K-D apparatus from
                      the water bath and allow it 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 approximately 0.2 mL of methylene chloride, and proceed to Section 10.3.3
                      for nitrogen evaporation and solvent exchange.

      10.3.3  Nitrogen evaporation and solvent exchange—Extracts to be subjected to solid-phase
             cleanup (SPE)  are exchanged into  1.0 mL of the SPE elution solvent (Section 6.7.2.2).
             Extracts to be subjected to Florisil® or alumina cleanups are exchanged into hexane.
             Extracts that have been cleaned up and are ready for analysis are exchanged into isooctane
             or hexane, to match the solvent used for the calibration standards.

             10.3.3.1  Transfer the vial containing the sample extract to the nitrogen evaporation
                      (blowdown) device (Section  5.2.5.2).  Lower the vial into a 50-55 °C water bath
                      and begin concentrating. During the solvent evaporation process, do not allow
                      the extract to become dry. Adjust the flow of nitrogen so that the surface of the
                      solvent is just visibly disturbed. A large vortex in the solvent may cause analyte
                      loss.

             10.3.3.2  Solvent exchange

                      10.3.3.2.1   When the volume of the liquid is approximately 500  |oL, add 2 to 3
                                  mL of the desired solvent (SPE elution solvent for SPE cleanup,
                                  hexane for Florisil or alumina, or isooctane for final injection into
                                  the GC) and continue concentrating to approximately 500 |oL.
                                  Repeat the addition of solvent and concentrate once more.

                      10.3.3.3.2   Adjust the volume of an extract to be cleaned up by SPE,  Florisil®,
                                  or alumina to 1.0 mL.  Proceed to extract cleanup (Section 11).

             10.3.3.3  Extracts that have been cleaned up and are ready for analysis—Adjust the final
                      extract volume to be consistent with the volume extracted and the sensitivity
Method 608.3                                 26                                       December 2014

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                      desired. The goal is for a full-volume sample (e.g., 1-L) to have a final extract
                      volume of 10 mL, but other volumes may be used.

      10.3.4  Transfer the concentrated extract to a vial with fluoropolymer-lined cap.  Seal the vial and
             label with the sample number. Store in the dark at room temperature until ready for GC
             analysis.  If GC analysis will not be performed on the same day, store the vial in the dark at
             4 °C.  Analyze the extract by GC per the procedure in Section 12.

10.4  Continuous liquid/liquid extraction (CLLE)

      10.4.1  Use CLLE when experience with a sample from a given source indicates an emulsion
             problem, or when an emulsion is encountered using SFLLE.  CLLE may be used for all
             samples, if desired.

      10.4.2  Mark the water meniscus on the side of the sample bottle for later determination of sample
             volume. Transfer the sample to the continuous extractor and, using a pipet, add surrogate
             standard spiking solution. If the sample will be used for the LCS, MS, or MSB, pipet the
             appropriate check sample concentrate (Section 8.2.1 or 8.3.2) into the separatory funnel.
             Mix well. Add 60 mL of methylene chloride to the sample bottle,  seal, and shake for 30
             seconds to rinse the inner surface. Transfer the solvent to the extractor.

      10.4.3  Repeat the sample bottle rinse with two additional  50-100 mL portions of methylene
             chloride and add the rinses to the extractor.

      10.4.4  Add a suitable volume of methylene chloride to the distilling flask (generally
             200 - 500 mL) and sufficient reagent water to ensure proper operation of the extractor, and
             extract the sample for 18-24 hours. A shorter or longer extraction time may be used if all
             QC acceptance criteria are met. Test and, if necessary, adjust the pH of the water during
             the second or third hour of the extraction. After extraction, allow the apparatus to cool,
             then detach the distilling flask. Dry, concentrate, solvent exchange, and transfer the extract
             to a vial with fluoropolymer-lined cap, per Section 10.3.

      10.4.5  Determine the  original sample volume by refilling  the sample bottle to the mark and
             transferring the liquid to an appropriately sized graduated cylinder. Record the sample
             volume to the nearest 5 mL. Sample volumes may also be determined by weighing the
             container before and after extraction or filling to the mark with water.

10.5  Solid-phase extraction of aqueous samples

      The steps in this section address the extraction of aqueous  field samples using disk-based solid-
      phase extraction (SPE) media, based on an ATP approved  by EPA in 1995 (Reference 20). This
      application of SPE is distinct from that used in this method for the cleanup of sample extracts in
      Section 11.2.  Analysts must be careful not to confuse the equipment, supplies, or the procedural
      steps from these two different uses  of SPE.

      Note:   Changes to the extraction conditions described below may be made by the laboratory under
             the allowance for method flexibility described in Section 8.1, provided that the performance
             requirements in Section 8.2 are met. However, changes  in SPE materials, formats, and
             solvents must meet the requirements in Section 8.1.2 and its subsections.
Method 608.3                                 27                                       December 2014

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      10.5.1  Mark the water meniscus on the side of the sample bottle for later determination of sample
             volume.  If the sample contains particulates, let stand to settle out the particulates before
             extraction.

      10.5.2  Extract the sample as follows:

             10.5.2.1  Place a 90-mm standard filter apparatus on a vacuum filtration flask or manifold
                      and attach to a vacuum source. The vacuum gauge should read at least 25 in. of
                      mercury when all valves are closed. Position a 90-mm CIS extraction disk onto
                      the filter screen.  Wet the entire disk with methanol. To aid in filtering samples
                      with particulates, a 1-um glass fiber filter or Empore® Filter Aid 400 can be
                      placed on the top of the disk and wetted with methanol. Install the reservoir and
                      clamp.  Resume vacuum to dry the disk. Interrupt the vacuum. Wash the disk
                      and reservoir with 20 mL of methylene chloride. Resume the vacuum briefly to
                      pull methylene chloride through the disk.  Interrupt the vacuum and allow the
                      disk to soak for about a minute. Resume vacuum and completely dry the disk.

             10.5.2.2  Condition the disk with 20 mL of methanol.  Apply vacuum until nearly all the
                      solvent has passed through the disk, interrupting it while solvent remains on the
                      disk.  Allow the disk to soak for about a minute. Resume vacuum to pull most of
                      the methanol through, but interrupting it to leave a layer of methanol on the
                      surface of the disk. Do not allow disk to dry.

                      For uniform flow and good recovery, it is critical the disk not be allowed to dry
                      from now until the end of the extraction.  Discard waste solvent. Rinse the disk
                      with 20 mL of deionized water. Resume vacuum to pull most of the water
                      through, but interrupt it to leave a layer of water on the surface of the disk.  Do
                      not allow the disk to dry. If disk does dry, recondition with methanol as above.

             10.5.2.3  Add the water sample to the reservoir and immediately apply the vacuum. If
                      particulates have settled in the  sample, gently decant the clear layer into the
                      apparatus until most of the sample has been processed. Then pour the remainder
                      including the particulates into the reservoir. Empty the sample bottle completely.
                      When the filtration is complete, dry the disk for three minutes. Turn off the
                      vacuum.

      10.5.3  Discard sample filtrate. Insert tube to collect the eluant.  The tube should fit around the
             drip tip of the base. Reassemble the apparatus. Add 5.0 mL of acetone to the center of the
             disk, allowing it to spread evenly over the disk.  Turn the vacuum on and quickly off when
             the filter  surface nears dryness but still remains wet.  Allow to soak for 15 seconds. Add  20
             mL of methylene chloride to the sample bottle, seal and shake to rinse the inside of the
             bottle.  Transfer the methylene chloride from the bottle to the filter.  Resume  the vacuum
             slowly so as to avoid splashing.

             Interrupt  the vacuum when the filter surface nears dryness but still remains wet. Allow
             disk to soak in solvent for 20 seconds. Rinse the reservoir glass and disk with 10 mL of
             methylene chloride.  Resume vacuum slowly. Interrupt vacuum when disk is  covered with
             solvent. Allow to soak for  20 seconds. Resume vacuum to dry the disk. Remove the
             sample tube.

      10.5.4  Dry,  concentrate, solvent exchange, and transfer the extract to a vial with fluoropolymer-
             lined cap, per Section 10.3.

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      10.5.5  Determine the original sample volume by refilling the sample bottle to the mark and
             transferring the liquid to an appropriately sized graduated cylinder. Record the sample
             volume to the nearest 5 mL. Sample volumes may also be determined by weighing the
             container before and after extraction or filling to the mark with water.
11.   Extract Cleanup

11.1  Cleanup may not be necessary for a relatively clean sample matrix. If particular circumstances
      require the use of a cleanup procedure, the laboratory may use any or all of the procedures below or
      any other appropriate procedure (e.g., gel permeation chromatography).  However, the laboratory
      must first repeat the tests in Sections 8.2, 8.3, and 8.4 to demonstrate that the requirements of those
      sections can be met using the cleanup procedure(s) as an integral part of this method. This is
      particularly important when the target analytes for the analysis include any of the single component
      pesticides in Table 2, because some cleanups have not been optimized for all of those analytes.

      11.1.1  The solid-phase cartridge (Section 11.2) removes polar organic compounds such as
             phenols.

      11.1.2  The Florisil® column (Section 11.3) allows for selected fractionation of the organochlorine
             analytes and will also eliminate polar interferences.

      11.1.3  Alumina column cleanup (Section 11.4) also removes polar materials.

      11.1.4  Elemental sulfur, which interferes with the electron capture gas chromatography of some of
             the pesticides, may be removed using activated copper, or TEA sulfite. Sulfur removal
             (Section 11.5) is required when sulfur is known or suspected to be present. Some
             chlorinated pesticides which also contain sulfur may be removed by this cleanup.

11.2  Solid-phase extraction (SPE) as a cleanup

      In order to use the C18 SPE cartridge in Section 5.5.3.5 as a cleanup procedure, the sample extract
      must be exchanged from methylene chloride to methylene chloride: acetonitrile:hexane. Follow
      the solvent exchange  steps in Section 10.3.3.2 prior to attempting solid-phase cleanup.

      Note:   This application of SPE is distinct from that used in this method for the extraction of
             aqueous samples in Section 10.5. Analysts must be careful not to confuse the equipment,
             supplies, or procedural steps from these two different uses of SPE.

      11.2.1  Setup

              11.2.1.1  Attach the VacElute Manifold (Section 5.5.3.2) to a water aspirator or vacuum
                      pump with the trap and gauge installed between the manifold and vacuum
                      source.

              11.2.1.2  Place the SPE cartridges in the manifold, turn on the vacuum source, and adjust
                      the vacuum to 5 to 10 psi.

      11.2.2  Cartridge washing—Pre-elute each cartridge prior to use sequentially with 10-mL portions
             each of hexane, methanol, and water using vacuum for 30 seconds after each eluting
             solvent. Follow this pre-elution with 1 mL methylene chloride and three 10-mL portions of
             the elution solvent (Section 6.7.2.2) using vacuum for 5 minutes  after each eluting solvent.

Method 608.3                                  29                                       December 2014

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             Tap the cartridge lightly while under vacuum to dry between solvent rinses. The three
             portions of elution solvent may be collected and used as a cartridge blank, if desired.
             Finally, elute the cartridge with 10 mL each of methanol and water, using the vacuum for
             30 seconds after each eluant.

      11.2.3  Extract cleanup

             11.2.3.1  After cartridge washing (Section 11.2.2), release the vacuum and place the rack
                      containing the 50-mL volumetric flasks (Section 5.5.3.4) in the vacuum
                      manifold. Re-establish the vacuum at 5 to  10 psi.

             11.2.3.2  Using a pipette or a 1-mL syringe, transfer  1.0 mL of extract to the SPE
                      cartridge. Apply vacuum for five minutes to dry the cartridge. Tap gently to aid
                      in drying.

             11.2.3.3  Elute each cartridge into its volumetric flask sequentially with three 10-mL
                      portions of the methylene chloride :acetonitrile:hexane (50:3:47) elution solvent
                      (Section 6.7.2.2), using vacuum for five minutes after each portion. Collect the
                      eluants in the 50-mL volumetric flasks.

             11.2.3.4  Release the vacuum and remove the 50-mL volumetric flasks.

             11.2.3.5  Concentrate the eluted extracts per Section  10.3.

11.3  Florisil®

      In order to use Florisil cleanup, the sample extract must be exchanged from methylene chloride to
      hexane. Follow the  solvent exchange steps in Section 10.3.3.2 prior to attempting Florisil®
      cleanup.

      Note: Alternative formats for  this cleanup may be used by the laboratory,  including cartridges
            containing Florisif.  If an alternative format is used, consult the manufacturer's
            instructions and develop a formal documented procedure to replace the steps in Section 11.3
            of this method and demonstrate that the alternative meets the relevant quality control
            requirements of this method.

      11.3.1  If the chromatographic column does not contain a frit at the bottom, place a small plug of
             pre-cleaned glass wool in the column (Section 5.2.4) to retain the Florisil®. Place the mass
             of Florisil® (nominally 20 g) predetermined by calibration (Section 7.9 and Table 6) in a
             chromatographic column. Tap the column to settle the Florisil® and add 1 to 2 cm of
             granular anhydrous sodium sulfate to the top.

      11.3.2  Add 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 eluant.

      11.3.3  Transfer the  concentrated extract  (Section 10.3.3) onto the column. Complete the transfer
             with two 1-mL hexane rinses, drawing the extract and rinses down to the level of the
             sodium sulfate.

      11.3.4  Place a clean 500-mL K-D flask and  concentrator tube under the column. Elute Fraction 1
             with 200 mL of 6% (v/v) ethyl ether in hexane at a rate of approximately 5 mL/min.

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             Remove the K-D flask and set it aside for later concentration. Elute Fraction 2 with 200
             mL of 15% (v/v) ethyl ether in hexane into a second K-D flask.  Elute Fraction 3 with 200
             mL of 50% (v/v) ethyl ether in hexane into a third K-D flask. The elution patterns for the
             pesticides and PCBs are shown in Table 6.

      11.3.5  Concentrate the fractions as in Section 10.3, except use hexane to prewetthe 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 Fraction 1 to approximately 10 mL for sulfur removal (Section 11.5), if required;
             otherwise, adjust the volume of the fractions to 10 mL, 1.0 mL, or other volume needed for
             the sensitivity desired. Analyze the concentrated extract by gas chromatography (Section
             12).

11.4  Alumina

      The sample extract must be exchanged from methylene chloride to hexane.  Follow the solvent
      exchange steps in Section 10.3.3.2 prior to attempting alumina cleanup.

      11.4.1  If the chromatographic column does not contain a frit at the bottom, place a small plug of
             pre-cleaned glass wool in the chromatographic column (Section 5.2.4) to retain the
             alumina.  Add 10 g of alumina (Section 6.7.3) on top of the plug. Tap the column to settle
             the alumina. Place 1 - 2 g of anhydrous sodium sulfate on top of the alumina.

      11.4.2  Close the stopcock and fill the column to just above the sodium sulfate with hexane.  Add
             25 mL of hexane. Open the stopcock and adjust the flow rate of hexane to approximately 2
             mL/min.  Do not allow the column to go dry throughout the elutions.

      11.4.3  When the level of the hexane is at the top of the column, quantitatively transfer the extract
             to the column. When the level of the extract is at the top of the column, slowly add 25 mL
             of hexane and elute the column to the level of the sodium sulfate.  Discard the hexane.

      11.4.4  Place a K-D flask (Section 5.2.5.1.2) under the column and elute the pesticides with
             approximately 150 mL of hexane:ethyl ether (80:20 v/v). It may be necessary to adjust the
             volume of elution solvent for slightly different alumina activities.

      11.4.5  Concentrate the extract per Section  10.3.

11.5  Sulfur removal—Elemental sulfur will usually elute  in Fraction 1 of the Florisil® column cleanup.
      If Florisil® cleanup is not used, or to remove sulfur from any of the Florisil® fractions, use one of
      the sulfur removal procedures below.  These procedures may be applied to extracts in hexane, ethyl
      ether, or methylene chloride.

      Note:   Separate procedures using copper or TEA sulfite are provided in this section for sulfur
             removal.  They may be used separately or in  combination, if desired.

      11.5.1  Removal with copper (Reference 15)

             Note:  Some of the analytes in Table 2 are not amenable to sulfur removal with copper
                    (e.g.,  atrazine and diazinon).  Therefore, before using copper to remove sulfur from
                    an extract that will be analyzed for any of the non-PCB analytes in Table 2, the
                    laboratory must demonstrate that the analytes can be extracted from an  aqueous
                    sample matrix that contains sulfur and recovered from an extract treated with

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                     copper.  Acceptable performance can be demonstrated through the preparation
                     and analysis of a matrix spike sample that meets the QC requirements for recovery.

             11.5.1.1  Quantitatively transfer the extract to a 40- to 50-mL flask or bottle. If there is
                       evidence of water in the K-D or round-bottom flask after the transfer, rinse the
                       flask with small portions of hexane:acetone (40:60) and add to the flask or bottle.
                       Mark and set aside the concentration flask for future use.

             11.5.1.2  Add 10 - 20 g of granular anhydrous sodium sulfate to the flask. Swirl to dry the
                       extract.

             11.5.1.3  Add activated copper (Section 6.7.4.1.4) and allow to stand for 30-60 minutes,
                       swirling occasionally. If the copper does not remain bright, add more and swirl
                       occasionally for another 30  - 60 minutes.

             11.5.1.4  After drying and sulfur removal, quantitatively transfer the extract to a nitrogen-
                       evaporation vial or tube and proceed to Section 10.3.3 for nitrogen evaporation
                       and solvent exchange, taking care to leave the sodium sulfate and copper foil in
                       the flask.

      11.5.2  Removal with TEA sulfite

             11.5.2.1  Using small volumes of hexane, quantitatively transfer the extract to a40-to 50-
                       mL centrifuge tube with fluoropolymer-lined screw cap.

             11.5.2.2  Add 1 - 2 mL of TEA sulfite reagent (Section 6.7.4.2.4), 2 - 3 mL of 2-propanol,
                       and approximately 0.7 g of sodium sulfite (Section 6.7.4.2.2) crystals to the tube.
                       Cap and shake for 1 - 2 minutes. If the sample is colorless or if the initial color is
                       unchanged, and if clear crystals (precipitated sodium sulfite) are observed,
                       sufficient sodium sulfite is present. If the precipitated sodium sulfite disappears,
                       add more crystalline sodium sulfite in approximately 0.5-g portions until a solid
                       residue remains after repeated shaking.

             11.5.2.3  Add 5-10 mL of reagent water and shake for 1 - 2 minutes. Centrifuge  to settle
                       the solids.

             11.5.2.4  Quantitatively transfer the hexane (top) layer through a small funnel containing a
                       few grams of granular anhydrous sodium sulfate to a nitrogen-evaporation vial or
                       tube and proceed to Section 10.3.3 for micro-concentration and solvent
                       exchange.
12.   Gas Chromatography

12.1  Establish the same operating conditions used in Section 7.1 for instrument calibration.

12.2  If the internal standard calibration procedure is used, add the internal standard solution (Section
      6.9.3) to the extract as close as possible to the time of injection to minimize the possibility of loss
      by evaporation, adsorption, or reaction. For example, add 1 (iL of 10 (ig/mL internal standard
      solution into the extract, assuming no dilutions.  Mix thoroughly.
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12.3  Simultaneously inject an appropriate volume of the sample extract or standard solution onto both
      columns, using split, splitless, solvent purge, large-volume, or on-column injection.  Alternatively,
      if using a single-column GC configuration, inject an appropriate volume of the sample extract or
      standard solution onto each GC column independently. If the sample is injected manually, the
      solvent-flush technique should be used. The injection volume depends upon the technique used
      and the sensitivity needed to meet MDLs or reporting limits for regulatory compliance. Injected
      volumes must be the same for all standards and sample extracts. Record the volume injected to the
      nearest 0.05  (iL.

12.4  Set the data system or GC control to start the temperature program upon sample injection, and
      begin data collection after the solvent peak elutes. Set the data system to stop data collection after
      the last analyte is expected to elute and to return the column to the initial  temperature.

12.5  Perform all qualitative and quantitative measurements as described in Sections 14 and 15.  When
      standards and extracts are not being used for analyses, store them refrigerated at <6°C, protected
      from light, in screw-cap vials equipped with un-pierced fluoropolymer-lined septa.
13.   System and Laboratory Performance

13.1  At the beginning of each shift during which standards or extracts are analyzed, GC system
      performance and calibration must be verified for all analytes and surrogates on both
      column/detector systems.  Adjustment and/or recalibration (per Section 7) are performed until all
      performance criteria are met. Only after all performance criteria are met may samples, blanks and
      other QC samples, and standards be analyzed.

13.2  Inject an aliquot of the combined QC standard (Section 6.8.4) on both columns. Inject an aliquot
      of each of the multi-component standards.

13.3  Retention times—The absolute retention times of the peak maxima shall be within ±2 seconds of
      the retention times in the calibration verification (Section 7.8).

13.4  GC resolution—Resolution is acceptable if the valley height between two peaks (as measured from
      the baseline) is less than 40% of the shorter of the two peaks.

      13.4.1  DB-608 column —DDT and endrin aldehyde

      13.4.2  DB-1701 column—alpha and gamma chlordane

      Note:   If using other GC columns or stationary phases, these resolution criteria apply to these
            four target analytes and any other closely eluting analytes on those other GC columns.

13.5  Decomposition of DDT and  endrin—If DDT, endrin, or their breakdown products are to be
      determined, this test must be performed prior to calibration verification (Section 13.6). DDT
      decomposes to DDE and ODD. Endrin decomposes to endrin aldehyde and endrin ketone.
      13.5.1  Inject 1 |oL  of the DDT and endrin decomposition solution (Section 6.9.5).

      13.5.2  Measure the areas of the peaks for DDT, DDE, ODD, Endrin, Endrin aldehyde, and  Endrin
             ketone in the chromatogram and calculate the percent breakdown as shown in the equations
             below:
Method 608.3                                 33                                       December 2014

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                                     sum of degradation peak areas (ODD + DDE)
               % breakdown of DDT =	—-	^- x 100
                                      sum of all peak areas (DDT+ DDE + ODD)

                             sum of degradation peak areas (Endrin aldehyde + Endrin ketone)
    % breakdown of Endrin =  	-	-
                             sum of all peak areas (Endrin + Endrin aldehyde + Endrin ketone)
xlOO
      13.5.3  Both the % breakdown of DDT and of Endrin must be less than 20%, otherwise the system
             is not performing acceptably for DDT and endrin. In this case, repair the GC column
             system that failed and repeat the performance tests (Sections 13.2 to  13.6) until the
             specification is met.

             Note: DDT and endrin decomposition are usually caused by accumulations ofparticulates
                   in the injector and in the front end of the column. Cleaning and silanizing the
                   injection port liner, and breaking off a short section of the front end of the column
                   will usually eliminate the decomposition problem.  Either of these corrective actions
                   may affect retention times, GC resolution, and calibration linearity.

13.6  Calibration verification

      13.6.1  Compute the percent recovery of each analyte and of the coeluting analytes, based on the
             initial calibration data (Section 7.5 or 7.6).

      13.6.2  For each analyte or for coeluting analytes, compare the concentration with the limits for
             calibration verification in Table 4.  For coeluting analytes, use the coeluting analyte with
             the least restrictive specification (the widest range).  For analytes in Table 2 not listed in
             Table 4, QC acceptance criteria must be developed by the laboratory. EPA has provided
             guidance for development of QC acceptance criteria (References  13 and 14).  If the
             recoveries for all analytes meet the acceptance criteria, system performance is acceptable
             and analysis of blanks and samples may continue. If, however, any recovery falls outside
             the calibration verification range, system performance is unacceptable for that analyte.  If
             this occurs, repair the system and repeat the test (Section 13.6), or prepare a fresh
             calibration standard and repeat the test, or recalibrate (Section 7). See Section 8.1.7 for
             information on repeated test failures.

13.7  Laboratory control sample

      13.7.1  Analyze the extract of the combined QC standard (a.k.a. LCS) (Section 6.8.3) extracted
             with each sample batch (Section 8.4).

      13.7.2  Compute the percent recovery of each analyte and of the coeluting analytes.

      13.7.3  For each analyte or coeluting analytes, compare the percent recovery with the limits for "P"
             in Table 4. For coeluting analytes, use the coeluting analyte with the least restrictive
             specification (widest range). If all analytes  pass, the extraction, concentration, and cleanup
             processes are in control and analysis of blanks and samples may proceed. If, however,  any
             of the analytes fail, these processes are not in control.  In this event, correct the problem, re-
             extract the sample batch,  and repeat the ongoing precision and recovery test.

      13.7.4  It is suggested, but not required, that the laboratory update statements of data quality. Add
             results that pass the specifications in Section 13.7.3 to initial (Section 8.7) and previous
             ongoing data.  Update QC charts to form a graphic representation of continued laboratory
             performance.  Develop a  statement of laboratory data quality for each analyte by
             calculating the average percent recovery (R) and the standard deviation of percent recovery,
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             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%.

13.8  Internal standard response—If internal standard calibration is used, verify that detector sensitivity
      has not changed by comparing the response (area or height) of each internal standard in the sample,
      blank, LCS, MS, and MSB to the response in the combined QC standard (Section 6.8.3). The peak
      area or height of the internal standard should be within 50% to 200% (1/2 to 2x) of its respective
      peak area or height in the verification standard. If the area or height is not within this range,
      compute the concentration of the analytes using the external standard method (Section 7.5).
14.   Qualitative Identification

14.1  Identification is accomplished by comparison of data from analysis of a sample, blank, or other QC
      sample with data from calibration verification (Section 7.7.1 or 13.5), and with data stored in the
      retention-time and calibration libraries (Section 7.7). The retention time window is determined as
      described in Section 14.2. Identification is confirmed when retention time agrees on both GC
      columns, as described below.

14.2  Establishing retention time windows

      14.2.1  Using the data from the multi-point initial calibration (Section 7.4), determine the retention
             time in decimal minutes (not minutes: seconds) of each peak representing a single-
             component target analyte on each column/detector system. For the multi-component
             analytes, use the retention times of the five largest peaks in the chromatograms on each
             column/detector system.

      14.2.2  Calculate the  standard deviation of the retention times for each single-component analyte
             on each column/detector system and for the three to five exclusive (unique large) peaks for
             each multi-component analyte.

      14.2.3  Define the width of the retention time window as three times  that standard deviation.
             Establish the center of the retention time window for each analyte by using the absolute
             retention time for each analyte from the calibration verification standard at the beginning of
             the analytical shift. For samples run during the same  shift as  an initial calibration, use the
             retention time of the mid-point standard of the initial calibration. If the calculated RT
             window is less than 0.02 minutes, then use 0.02 minutes as the window.

      Note:   Procedures for establishing retention time windows from other sources may be employed
             provided that they are clearly  documented and provide acceptable performance.  Such
             performance may be evaluated using the results for the spiked QC samples described in
             this method, such as laboratory control samples and matrix spike samples.

      14.2.4  New retention time windows must be established when a new GC column is installed or if a
             GC column has been shortened during maintenance to a degree that the retention times of
             analytes in the calibration verification standard have shifted close to the lower limits of the
             established retention time windows.

      14.2.5  RT windows should be checked periodically by examining the peaks in spiked samples
             such as the LCS or MS/MSD to confirm that peaks for known analytes are properly
             identified.
Method 608.3                                 35                                       December 2014

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      14.2.6  If the retention time of an analyte in the initial calibration data has been evaluated as
             described in Section 7.4.1 and it varied by more than 5 seconds across the calibration range
             as a function of the concentration of the standard (see Section 7.4.2), then using the
             standard deviation of the retention times to set the width of the retention time window may
             not adequately serve to identify the analyte in question under routine conditions. In such
             cases, data from additional analyses of standards may be required to adequately model the
             chromatographic behavior of the analyte.

14.3  Identifying the analyte in a sample

      14.3.1  In order to identify a single-component analyte from analysis of a sample, blank, or other
             QC sample, the peak representing the analyst must fall within its respective retention time
             windows on both column/detector systems (as defined in Section 14.2). That identification
             is further supported by the comparison of the numerical results on both columns, as
             described in Section 15.7.

      14.3.2  In order to identify a multi-component analyte, pattern matching (fingerprinting) may be
             used, or the three to five exclusive (unique, baseline resolved, and largest) peaks for that
             analyte must fall within their respective retention time windows on both column/detector
             systems (as defined in Section  14.2).  That identification is further supported by the
             comparison of the numerical results on both columns, as described in Section 15.7.

14.4  GC/MS confirmation

      When the concentration of an analyte is sufficient, or if the presence  or identity is suspect, its
      presence should be confirmed by GC/MS.  In  order to match the sensitivity of the GC/ECD,
      confirmation will have to be by SIM-GC/MS, or estimated the concentration would have to be 100
      times higher than the GC/ECD calibration range.

14.5  Additional information that may aid the laboratory in the identification of an analyte

      The occurrence of peaks eluting near the retention time of an analyte of interest increases the
      probability of a false positive for the analyte.  If the concentration is insufficient for confirmation
      by GC/MS, the laboratory may use the  cleanup procedures in this method (Section 11) on a new
      sample aliquot to attempt to remove the interferent.  After attempts at cleanup are exhausted, the
      following steps may be helpful to assure that the substance that appears in the RT windows on both
      columns is the analyte of interest.

      14.5.1  Determine the consistency of the RT data for the analyte on each column. For example, if
             the RT is very stable (i.e., varies by no more than a few seconds) for the calibration,
             calibration verification, blank, LCS, and MS/MSD, the RT for the analyte of interest in the
             sample should be within this variation regardless of the window established in Section 14.2.
             If the analyte is not within this  variation on both columns, it is likely not present.

      14.5.2  The possibility exists that the RT for the analyte in a sample  could shift if extraneous
             materials are present. This possibility may be able to be confirmed or refuted by the
             behavior of the surrogates in the sample. If multiple surrogates are used that span the
             length of the chromatographic run, the RTs for the surrogates on both columns are
             consistent with their RTs in calibration, calibration verification, blank, LCS, and MS/MSD,
             it is unlikely that the RT for the analyte of interest has shifted.
Method 608.3                                  36                                       December 2014

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      14.5.3  If the RT for the analyte is shifted slightly later on one column and earlier on the other, and
             the surrogates have not shifted, it is highly unlikely that the analyte is present, because
             shifts nearly always occur in the same direction on both columns.
15.   Quantitative Determination

15.1  External standard quantitation—Calculate the concentration of the analyte in the extract using the
      calibration curve or average calibration factor determined in calibration (Section 7.5.2) and the
      following equation:
                                                   AS
                                             r   — —
                                             ex ~ CF

       where:
       Cex = Concentration of the analyte in the extract (ng/mL)
       As = Peak height or area for the analyte in the standard or sample
       CF = Calibration factor, as defined in Section 7.5.1

15.2  Internal standard quantitation—Calculate the concentration of the analyte in the extract using the
      calibration curve or average response factor determined in calibration (Section 7.6.2) and the
      following equation:
                                             ^  As x LIS
                                           "    Ais x RF

       where:
       Cex = Concentration of the analyte in the extract (ng/mL)
       AS = Peak height or area for the analyte  in the standard or sample
       C;s = Concentration of the internal standard (ng/mL)
       AJS = Area of the internal standard
       RF = Response factor, as defined in Section 7.6.1

15.3  Calculate the concentration of the analyte in the sample using the concentration in the extract, the
      extract volume, the sample volume,  and the dilution factor, per the following equation:

                                           =  CexxVexxDF
                                          s~   VsxlOOO

       where:
       Cs = Concentration of the analyte in the sample ((ig/L)
       Vex = Final extract volume (mL)
       Cex = Concentration in the extract  (ng/mL)
       Vs = Volume of sample (L)
       DF = Dilution factor
       and the factor of 1,000 in the denominator converts the final units from ng/L to (ig/L

15.4  If the concentration of any target analyte exceeds the calibration range, either extract and analyze a
      smaller sample volume, or dilute and analyze the diluted extract.
Method 608.3                                  37                                        December 2014

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15.5  Quantitation of multi-component analytes

      15.5.1  PCBs as Aroclors

             Quantify an Aroclor by comparing the sample chromatogram to that of the most similar
             Aroclor standard as indicated in Section 14.3.2. Compare the responses of 3 to 5 major
             peaks in the calibration standard for that Aroclor with the peaks observed in the sample
             extract. The amount of Aroclor is calculated using the individual calibration factor for each
             of the 3 to 5 characteristic peaks chosen in Sec. 7.5.1. Determine the concentration of each
             of the characteristic peaks, using the average calibration factor calculated for that peak in
             Sec. 7.5.2, and then those 3 to 5 concentrations  are averaged to determine the concentration
             of that Aroclor.

      15.5.2  Other multi-component analytes

             Quantify any other multi-component analytes (technical chlordane or toxaphene) using the
             same peaks used to develop the average calibration factors in Section 7.5.2.  Determine the
             concentration of each of the characteristic peaks, and then the concentrations represented
             by those characteristic peaks are averaged to determine the concentration of the analyte.
             Alternatively, for toxaphene, the analyst may determine the calibration factor in Section
             7.5.2 by summing the areas of all of the peaks for the analyte and using the summed of the
             peak areas in the sample chromatogram to determine the concentration. However, the
             approach used for toxaphene must be the same for the calibration and the sample analyses.

15.6  Reporting of re suits

      As noted in Section 1.6.1, EPA has promulgated this method at 40 CFR Part 136 for use in
      wastewater  compliance monitoring under the National Pollutant Discharge Elimination System
      (NPDES). The data reporting practices described here are focused on such monitoring needs and
      may not be  relevant to other uses of the method.

      15.6.1  Report results for wastewater samples in (ig/L without correction for recovery.  (Other units
             may be used if required by in a permit.) Report all QC data with the sample results.

      15.6.2  Reporting level

             Unless otherwise specified in by a regulatory authority or in a discharge permit, results for
             analytes that meet the identification criteria are  reported down to the concentration of the
             ML established by the laboratory through calibration of the instrument (see Section 7.5 or
             7.6  and the glossary for the derivation of the ML).  EPA considers the terms "reporting
             limit," "quantitation limit," and "minimum level" to be synonymous.

             15.6.2.1  Report the lower result from the two columns (see Section 15.7below) for each
                      analyte in each sample, blank, or standard at or above the ML to 3 significant
                      figures. Report a result for each analyte found in each sample below the ML as
                      "
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                      sample may be subtracted from the result for that sample^ but only if requested or
                      required by a regulatory authority or in a permit. In this case, both the sample
                      result and the blank results must be reported together.

             15.6.2.3  Report the result for an analyte in a sample or extract that has been diluted at the
                      least dilute level at which the peak area is within the calibration range (i.e., above
                      the ML for the analyte) and the MS/MSD recovery and RPD are within their
                      respective QC acceptance criteria (Table 4). This may require reporting results
                      for some analytes from different analyses.

                      The results for each analyte in the MS/MSD samples should be reported from the
                      same GC column as used to report the results for that analyte in the unspiked
                      sample. If the MS/MSD recoveries and RPDs calculated in this manner do not
                      meet the acceptance criteria in Table 4, then the analyst may use the results from
                      the other GC column to determine if the MS/MSD results meet the acceptance
                      criteria. If such a situation occurs, the results for the sample should be
                      recalculated using the same GC column data as used for the MS/MSD samples,
                      and reported with appropriate annotations that alert the data user of the issue.

             15.6.2.4  Results from tests performed with an analytical system that is not in control (i.e.,
                      that does not meet acceptance criteria for all of QC tests in this method) must not
                      be reported or otherwise used for permitting or regulatory compliance purposes,
                      but do not relieve a discharger or permittee of reporting timely results. If the
                      holding time would be exceeded for a re-analysis of the sample, the
                      regulatory/control authority should be consulted for disposition.

      15.6.3  Analyze the sample  by GC/MS or on athird column when analytes have co-eluted or
             interfere with determination on both columns.

             Note:  Dichlone and kepone do not elute from the DB-1701 column and must be
                    confirmed on a DB-5 column, or by GC/MS.

15.7  Quantitative information that may aid in the confirmation of the presence of an analyte

      15.7.1  As noted in Section  14.3, the relative agreement between the numerical results from the
             two GC columns may be used to support the identification of the target analyte by
             providing evidence that that co-eluting interferences are not present at the retention time of
             the target analyte. Calculate the percent difference (%D) between the results for the analyte
             from both columns, as follows:

                                        Hiqher result — Lower result
                                 %D = —	—	x 100
                                                Higher result

             In general, if the %D of the two results is less than 50% (e.g., a factor of 2), then the
             pesticide is present.  This %D is generous and allows for the pesticide that has the largest
             measurement error.

             Note:  Laboratories may employ metrics less than 50% for this comparison, including
                    those specified in other analytical methods for these pesticides (e.g., CLP or
                    SW-846).
Method 608.3                                  39                                       December 2014

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      15.7.2  If the amounts do not agree, and the RT data indicate the presence of the analyte (per
             Section 14), it is likely that a positive interference is present on the column that yielded the
             higher result.  That interferent may be represented by a separate peak on the other column
             that does not coincide with the retention time of any of the target analytes. If the
             interfering peak is evident on the other column, report the result from that column and
             advise the data user that the interference resulted in a %D value greater than 50%.

             If an interferent is not identifiable on the second column, then the results must be reported
             as "not detected" at the lower concentration. In this event, the pesticide is not confirmed
             and the reporting limit is elevated.

             Note:   The resulting elevation of the reporting limit may not meet the requirements for
                     compliance monitoring and the use of additional cleanup procedures may be
                     required.
16.   Analysis of Complex Samples

16.1  Some samples may contain high levels (greaterthan 1 (ig/L) of the analytes of interest, interfering
      analytes, and/or polymeric materials.  Some samples may not concentrate to 1.0 mL (Section
      10.3.3.3.2); others may overload the GC column and/or detector.

16.2  When an interference is known or suspected to be present, the laboratory should attempt to clean
      up the sample extract using the SPE cartridge (Section 11.2), by Florisil® (Section 11.3), Alumina
      (Section 11.4), sulfur removal (Section 11.5), or another clean up procedure appropriate to the
      analytes of interest.  If these techniques do not remove the interference, the extract is diluted by a
      known factor and reanalyzed (Section 12). Dilution until the extract is lightly colored is preferable.
      Typical dilution factors are 2, 5, and 10.

16.3  Recovery of surrogate(s)—In most samples, surrogate recoveries will be similar to those from
      reagent water.  If surrogate recovery is outside the range developed in Section 8.6, the sample is re-
      extracted and reanalyzed if there is sufficient sample and if it is within the 7-day extraction holding
      time.  If the surrogate recovery is still  outside this range, extract and analyze one-tenth the volume
      of sample to overcome any matrix interference problems.  If a sample is highly colored or
      suspected to be high in concentration,  a 1-L sample aliquot and a 100-mL sample aliquot could be
      extracted simultaneously and still meet the holding time criteria, while providing information about
      a complex matrix.

16.4  Recovery of the matrix spike and matrix spike duplicate (MS/MSD)—In most samples, MS/MSD
      recoveries will be similar to those from reagent water.  If either the MS or MSB recovery is outside
      the range specified in Section 8.3.3, one-tenth the volume of sample is spiked and analyzed. If the
      matrix spike recovery is still outside the range, the result for the unspiked sample may not be
      reported or used for permitting or regulatory compliance purposes. Poor matrix spike recovery
      does not relieve a discharger or permittee of reporting timely results.
17.   Method Performance

17.1  This method was tested for linearity of spike recovery from reagent water and has been
      demonstrated to be applicable over the concentration range from 4x MDL to lOOOx MDL with the
      following exceptions:  Chlordane recovery at 4x MDL was low (60%); Toxaphene recovery was
      demonstrated linear over the range of lOx MDL to lOOOx MDL (Reference 3).

Method 608.3                                  40                                       December 2014

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17.2  The 1984 version of this method was tested by 20 laboratories using reagent water, drinking water,
      surface water, and three industrial wastewaters spiked at six concentrations (Reference 2).
      Concentrations used in the study ranged from 0.5 to 30 |o,g/L for single-component pesticides and
      from 8.5 to 400 |o,g/L for multi-component analytes.  These data are for a subset of analytes
      described in the current version of the method.

17.3  During the development of Method 1656, a similar EPA procedure for the organochlorine
      pesticides, single-operator precision, overall precision, and method accuracy were found to be
      directly related to the concentration of the analyte and essentially independent of the sample
      matrix. Linear equations to describe these relationships are presented in Table 5.
18.   Pollution Prevention

18.1  Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity
      of waste at the point of generation. Many opportunities for pollution prevention exist in laboratory
      operations. EPA has established a preferred hierarchy of environmental management techniques
      that places pollution prevention as the management option of first choice. Whenever feasible, the
      laboratory should use pollution prevention techniques to address waste generation. When wastes
      cannot be reduced at the source, the Agency recommends recycling as the next best option.

18.2  The analytes in this method are used in extremely small amounts and pose little threat to the
      environment when managed properly.  Standards should be prepared in volumes consistent with
      laboratory use to minimize the disposal of excess volumes of expired standards.  This method
      utilizes significant quantities of methylene chloride. Laboratories are encouraged to recover and
      recycle this and other solvents during extract concentration.

18.3  For information about pollution prevention that may be applied to laboratories and research
      institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction
      (Reference 19).
19.   Waste Management

19.1  The laboratory is responsible for complying 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. An overview of requirements can be found in Environmental Management Guide
      for Small Laboratories (EPA 233-B-98-001).

19.2  Samples at pH <2, or pH >12 are hazardous and must be neutralized before being poured down a
      drain, or must be handled as hazardous waste.

19.3  Many analytes in this method decompose above 500 °C. Low-level waste such as absorbent paper,
      tissues, animal remains, and plastic gloves may be burned in an appropriate incinerator.  Gross
      quantities of neat or highly concentrated solutions of toxic or hazardous chemicals should be
      packaged securely and disposed of through commercial or governmental channels that are capable
      of handling toxic wastes.
Method 608.3                                 41                                       December 2014

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20.   References

1.     "Determination of Pesticides and PCBs in Industrial and Municipal Wastewaters," EPA 600/4-82-
      023, National Technical Information Service, PB82-214222, Springfield, Virginia 22161, April
      1982.

2.     "EPA Method Study 18 Method 608-Organochlorine Pesticides and PCBs," EPA 600/4-84-061,
      National Technical Information Service, PB84-211358, Springfield, Virginia 22161, June 1984.

3.     "Method Detection Limit and Analytical Curve Studies, EPA Methods 606, 607, and 608," Special
      letter report for EPA Contract 68-03-2606, U.S. Environmental Protection Agency, Environmental
      Monitoring and Support Laboratory, Cincinnati, Ohio  45268, June 1980.

4.     ASTM Annual Book of Standards, Part 31, D3694-78. "Standard Practice for Preparation of
      Sample Containers and for Preservation of Organic Constituents," American Society for Testing
      and Materials, Philadelphia.

5.     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).

6.     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. 701-708, 1976.

7.     Solutions to Analytical Chemistry Problems with Clean Water Act Methods, EPA 821-R-07-002,
      March 2007.

8.     "Carcinogens-Working With Carcinogens," Department of Health, Education, and Welfare, Public
      Health Service, Center for Disease Control, National Institute for Occupational Safety and Health,
      Publication No. 77-206, August 1977.

9.     "Occupational Exposure to Hazardous Chemicals in Laboratories," (29 CFR Part 1910, Subpart
      1450), Occupational Safety and Health Administration, OSHA .

10.   40 CFR 136.6(b)(4)(j)

11.   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).

12.   40 CFR 136.6(b)(2)(/)

13.   Protocol^or EPA Approval of New Methods for Organic and Inorganic Analytes in Wastewater
      and Drinking Water (EPA-821-B-98-003) March 1999

14.   Methods 4500 Cl F and 4500 Cl G, Standard Methods for the Examination of Water and
      Wastewater, published jointly by the American Public Health Association, American Water Works
      Association, and Water Environment Federation, 1015 Fifteenth St., Washington, DC 20005, 20th
      Edition, 2000.

15.   "Manual of Analytical Methods for the Analysis of Pesticides in Human and Environmental
      Samples," EPA-600/8-80-038, U.S. Environmental Protection Agency, Health Effects Research
      Laboratory, Research Triangle Park, North Carolina.

Method 608.3                                42                                      December 2014

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16.   USEPA, 2000, Method 1656 Organo-Halide Pesticides In Wastewater, Soil, Sludge, Sediment, and
      Tissue by GC/HSD, EPA-821-R-00-017, September 2000.

17.   USEPA, 2010, Method 1668C Chlorinated Biphenyl Congeners in Water, Soil, Sediment,
      Biosolids, and Tissue by HRGC/HRMS, EPA-820-R-10-005, April 2010.

18.   USEPA, 2007, Method 1699: Pesticides in Water, Soil, Sediment, Biosolids, and Tissue by
      HRGC/HRMS, EPA-821-R-08-001, December 2007.

19.   "Less is Better," American Chemical Society on-line publication,
      http://www.acs.org/content/dam/acsorg/about/governance/committees/chemicalsafety/publications/
      less-is-better.pdf

20.   EPA Method 608 ATP 3M0222, An alternative test procedure for the measurement of
      organochlorine pesticides and polychlorinated biphenyls in waste water. Federal Register/Vol. 60,
      No. 148 August 2, 1995
Method 608.3                                43                                      December 2014

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21.   Tables
Table 1 - Pesticides 1
Analyte
Aldrin
alpha-BHC
beta-BHC
delta-BHC
gamma-BHC (Lindane)
alpha-Chlordane
gctmmct-Chlordane
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
CAS Number
309-00-2
319-84-6
319-85-7
319-86-8
58-89-9
5103-71-9
5103-74-2
72-54-8
72-55-9
50-29-3
60-57-1
959-98-8
33213-65-9
1031-07-8
72-20-8
7421-93-4
76-44-8
1024-57-3
MDL2
(ng/L)
8
6
7
5
1
9
8
5
10
12
6
11
8
7
4
11
5
12
ML3
(ng/L
24
18
21
15
33
27
24
15
30
36
18
33
24
21
12
33
15
36
                       All analytes in this table are Priority Pollutants (40 CFR 423, Appendix A)
                     2 40 CFR 136, Appendix B.  MDLs were obtained by a single laboratory
                       with an electrolytic conductivity detector, and are estimates of what can be
                       achieved using an electron capture detector.
                     3 ML = Minimum Level - see Glossary for definition and derivation
Method 608.3
44
December 2014

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Table 2 - Additional Analytes
Analyte
Acephate
Alachlor
Atrazine
Benfluralin (Benefin)
Bromacil
Bromoxynil octanoate
Butachlor
Captafol
Captan
Carbophenothion (Trithion)
Chlorobenzilate
Chloroneb (Terraneb)
Chloropropylate (Acaralate)
Chlorothalonil
Cyanazine
DCPA (Dacthal)
2,4'-DDD
2,4'-DDE
2,4'-DDT
Diallate (Avadex)
1 ,2-Dibromo-3 -chloropropane (DBCP)
Dichlone
Dichloran
Dicofol
Endrin ketone
Ethalfluralin (Sonalan)
Etridiazole
Fenarimol (Rubigan)
Hexachlorobenzene :
Hexachlorocyclopentadiene :
Isodrin
Isopropalin (Paarlan)
Kepone
Methoxychlor
Metolachlor
Metribuzin
Mirex
Nitrofen (TOK)
c/'s-Nonachlor
trans -Nonachlor
Norfluorazon
Octachlorostyrene
Oxychlordane
PCNB (Pentachloronitrobenzene)
Pendamethalin (Prowl)
c/s-Permethrin
fra«5-Permethrin
CAS Number
30560-19-1
15972-60-8
1912-24-9
1861-40-1
314-40-9
1689-99-2
23184-66-9
2425-06-1
133-06-2
786-19-6
510-15-6
2675-77-6
5836-10-2
1897-45-6
21725-46-2
1861-32-1
53-19-0
3424-82-6
789-02-6
2303-16-4
96-12-8
117-80-6
99-30-9
115-32-2
53494-70-5
55283-68-6
2593-15-9
60168-88-9
118-74-1
77-47-4
465-73-6
33820-53-0
143-50-0
72-43-5
51218-45-2
21087-64-9
2385-85-5
1836-75-5
5103-73-1
39765-80-5
27314-13-2
29082-74-4
27304-13-8
82-68-8
40487-42-1
61949-76-6
61949-77-7
MDL3
(ng/L)
2,000
20
500
20
70
30
30
100
100
50
25


15

3



45




8
5

20


13
20
100
30

5
4
13


50


6

200
200
ML4
(ng/L
6,000
60
1,500
60
210
90
90
300
300
150
75


45

9



135




24
15

30


39
60
300
90

15
12
39


150


18

600
600
Method 608.3
45
December 2014

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Table 2 - Additional Analytes
Analyte
Perthane (Ethylan)
Propachlor
Propanil
Propazine
Quintozene
Simazine
Strobane
Technazene
Technical Chlordane2
Terbacil
Terbuthylazine
Toxaphene :
Trifluralin
PCB-10161
PCB-12211
PCB-12321
PCB-12421
PCB-12481
PCB-12541
PCB-12601
CAS Number
72-56-0
1918-16-7
709-98-8
139-40-2
82-68-8
122-34-9
8001-50-1
117-18-0

5902-51-2
5915-41-3
8001-35-2
1582-09-8
12674-11-2
11104-28-2
11141-16-5
53469-21-9
12672-29-6
11097-69-1
11096-82-5
MDL3
(ng/L)





400



200
300
910
50
150
150
150
150
150
150
140
ML4
(ng/L





1,200



600
900
2,730
150
450
450
450
450
450
450
420
         1  Priority Pollutants (40 CFR 423, Appendix A)
         2  Technical Chlordane may be used in cases where historical reporting has only been for this
           form of Chlordane.
         3  40 CFR 136, Appendix B.  MDLs were obtained by a single laboratory with an electrolytic
           conductivity detector, and are estimates of what can be achieved using an electron capture
           detector.
         4  ML = Minimum Level - see Glossary for definition and derivation
Method 608.3
46
December 2014

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Table 3 - Example Retention Times1
Analyte
Acephate
Trifluralin
Ethalfluralin
Benfluralin
Diallate-A
Diallate-B
alpha-BHC
PCNB
Simazine
Atrazine
Terbuthylazine
gamma-BHC (Lindane)
beta-BHC
Heptachlor
Chlorothalonil
Dichlone
Terbacil
delta-BHC
Alachlor
Propanil
Aldrin
DCPA
Metribuzin
Triadimefon
Isopropalin
Isodrin
Heptachlor epoxide
Pendamethalin
Bromacil
a//>/2a-Chlordane
Butachlor
gamma -Chlordane
Endosulfan I
4,4 '-DDE
Dieldrin
Captan
Chlorobenzilate
Endrin
Nitrofen (TOK)
Kepone
4,4 '-ODD
Endosulfan II
Bromoxynil octanoate
Retention time (min)2
DB-608
5.03
5.16
5.28
5.53
7.15
7.42
8.14
9.03
9.06
9.12
9.17
9.52
9.86
10.66
10.66
10.80
11.11
11.20
11.57
11.60
11.84
12.18
12.80
12.99
13.06
13.47
13.97
14.21
14.39
14.63
15.03
15.24
15.25
16.34
16.41
16.83
17.58
17.80
17.86
17.92
18.43
18.45
18.85
DB-1701
3
6.79
6.49
6.87
6.23
6.77
7.44
7.58
9.29
9.12
9.46
9.91
11.90
10.55
10.96
4
12.63
12.98
11.06
14.10
11.46
12.09
11.68
13.57
13.37
11.12
12.56
13.46
3
14.20
15.69
14.36
13.87
14.84
15.25
15.43
17.28
15.86
17.47
3,5
17.77
18.57
18.57
Method 608.3
47
December 2014

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Table 3 - Example Retention Times1
Analyte
4,4 '-DDT
Carbophenothion
Endrin aldehyde
Endosulfan sulfate
Captafol
Norfluorazon
Mirex
Methoxychlor
Endrin ketone
Fenarimol
c/5-Permethrin
frara-Permethrin
PCB-1242
PCB-1232
PCB-1016
PCB-1221
PCB-1248
PCB-1254
PCB-1260 (5 peaks)




Toxaphene (5 peaks)




Retention time (min)2
DB-608
19.48
19.65
19.72
20.21
22.51
20.68
22.75
22.80
23.00
24.53
25.00
25.62






15.44
15.73
16.94
17.28
19.17
16.60
17.37
18.11
19.46
19.69
DB-1701
18.32
18.21
19.18
20.37
21.22
22.01
19.79
20.68
21.79
23.79
23.59
23.92






14.64
15.36
16.53
18.70
19.92
16.60
17.52
17.92
18.73
19.00
                          Data from EPA Method 1656 (Reference 16)
                          Columns: 30-m long x 0.53-mm ID fused-silica capillary;
                          DB-608, 0.83 (am; and DB-1701, 1.0 |am.
                          Conditions suggested to meet retention times shown:
                          150 °C for 0.5 minute, 150-270 °C at 5°C/min, and 270 °C
                          until trans-Permethrin elutes.
                          Carrier gas flow rates approximately 7 mL/min.
                          Does not elute from DB-1701 column at level tested.
                          Not recovered from water at the levels tested.
                          Dichlone and Kepone do not elute from the DB-1701
                          column and should be confirmed on DB-5.
Method 608.3
December 2014

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Table 4 - QC Acceptance Criteria
Analyte
Aldrin
alpha-BHC
beta-BHC
delta-BHC
gamma-BHC
alpha-Chlordane
gamma-Chlordane
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Heptachlor
Heptachlor epoxide
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Calibration
verification
(%)
75 - 125
69 - 125
75 - 125
75 - 125
75 - 125
73 - 125
75 - 125
75 - 125
75 - 125
75 - 125
48 - 125
75 - 125
75 - 125
70 - 125
5-125
75 - 125
75 - 125
68-134
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
Test
concen-
tration
(Hg/L)
2.0
2.0
2.0
2.0
2.0
50.0
50.0
10.0
2.0
10.0
2.0
2.0
10.0
10.0
10.0
2.0
2.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
Limit for
s (% SD)
25
28
38
43
29
24
24
32
30
39
42
25
63
32
42
28
22
30
24
50
32
26
32
34
28
Range for
X (%)
54-130
49-130
39-130
51-130
43 - 130
55-130
55-130
48-130
54 - 130
46-137
58-130
57-141
22-171
38-132
51-130
43 - 130
57-132
56-130
61-103
44-150
28 - 197
50-139
58-140
44-130
37-130
Range
forP
(%)
42 - 140
37 - 140
17-147
19-140
32 - 140
45 - 140
45 - 140
31 -141
30 - 145
25 - 160
36 - 146
45 - 153
D-202
26 - 144
30 - 147
34 - 140
37 - 142
41 - 140
50 - 140
15-178
10-215
39-150
38-158
29 - 140
8-140
Maximum
MS/MSD
RPD (%)
35
36
44
52
39
35
35
39
35
42
49
28
53
38
48
43
26
41
36
48
25
29
35
45
38
S  =  Standard deviation of four recovery measurements (Section 8.2.4).

Note: These criteria were developed from data in Table 5 (Reference 2).  Where necessary, limits for recovery have
      been broadened to assure applicability to concentrations below those in Table 5.
Method 608.3
49
December 2014

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Table 5 - Precision and Recovery as Functions of Concentration
Analyte
Aldrin
alpha-BHC
beta-BHC
delta-BHC
gamma-BHC (Lindane)
Chlordane
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Heptachlor
Heptachlor epoxide
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Recovery, X'
(H8/L)
0.81C + 0.04
0.84C + 0.03
0.81C + 0.07
0.81C + 0.07
0.82C-0.05
0.82C-0.04
0.84C + 0.30
0.85C + 0.14
0.93C-0.13
0.90C + 0.02
0.97C + 0.04
0.93C + 0.34
0.89C-0.37
0.89C-0.04
0.69C + 0.04
0.89C + 0.10
0.80C+ 1.74
0.81C + 0.50
0.96C + 0.65
0.91C+ 10.8
0.93C + 0.70
0.97C+ 1.06
0.76C + 2.07
0.66C + 3.76
Single analyst
precision, s/ ((J,g/L)
0.16(X)-0.04
0.13(X) + 0.04
0.22(X)-0.02
0.18(X) + 0.09
0.12(X) + 0.06
0.13(X) + 0.13
0.20(X)-0.18
0.13(X) + 0.06
0.17(X) + 0.39
0.12(X) + 0.19
0.10(X) + 0.07
0.41(X)-0.65
0.13(X) + 0.33
0.20(X) + 0.25
0.06(X) + 0.13
0.18(X)-0.11
0.09(X) + 3.20
0.13(X) + 0.15
0.29(X)-0.76
0.21(X)-1.93
0.11(X) + 1.40
0.17(X) + 0.41
0.15(X) + 1.66
0.22(X)-2.37
Overall precision,
S'Oig/L)
0.20(X)-0.01
0.23(X)-0.00
0.33(X)-0.05
0.25(X) + 0.03
0.22(X) + 0.04
0.18(X) + 0.18
0.27(X)-0.14
0.28(X)-0.09
0.31(X)-0.21
0.16(X) + 0.16
0.18(X) + 0.08
0.47(X)-0.20
0.24(X) + 0.35
0.24(X) + 0.25
0.16(X) + 0.08
0.25(X)-0.08
0.20(X) + 0.22
0.15(X) + 0.45
0.35(X)-0.62
0.31(X) + 3.50
0.21(X)+ 1.52
0.25(X)-0.37
0.17(X) + 3.62
0.39(X)-4.86
    X' =  Expected recovery for one or more measurements of a sample containing a concentration of C, in u.g/L.
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Table 6 - Distribution of Chlorinated Pesticides and PCBs into Florisil® Column Fractions
Analyte
Aldrin
alpha-BHC
beta-BHC
delta-BHC
gamma-BHC (Lindane)
Chlordane
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Percent Recovery by Fraction1
1
100
100
97
98
100
100
99

100
0
37
0
0
4
0
100
100
96
97
97
95
97
103
90
95
2







98

100
64
7
0
96
68





4




3











91
106

26










      1 Eluant composition:
          Fraction 1 - 6% ethyl ether in hexane
          Fraction 2 -15% ethyl ether in hexane
          Fraction 3 - 50% ethyl ether in hexane
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22.   Figures
    5   6
                                    13   14  15   16   17   18  19  20  21  22  23  24  25
Figure 1    Example Chromatogram of Selected Organochlorine Pesticides
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                            1000




                            750




                            500



                            250
                                 XCD
                                                               GMF150 Filter
                                              1-Liter Suction Flask
Figure 2   Disk-based solid-phase extraction apparatus
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23.     Glossary

        These definitions and purposes are specific to this method but have been conformed to common
        usage to the extent possible.

23.1    Units of weight and measure and their abbreviations

        23.1.1  Symbols

               °C    degrees Celsius
               (ig    microgram
               uL    microliter
               <     less than
               <     less than or equal to
               >     greater than
               %    percent

        23.1.2  Abbreviations (in alphabetical order)

               cm    centimeter
               g     gram
               hr    hour
               ID    inside diameter
               in.    inch
               L     liter
               M    molar solution - one mole or gram molecular weight of solute in one liter of solution
               mg    milligram
               min   minute
               mL   milliliter
               mm   millimeter
               N     Normality - one equivalent of solute in one liter of solution
               ng    nanogram
               psia  pounds-per-square inch absolute
               psig  pounds-per-square inch gauge
               v/v    volume per unit volume
               w/v   weight per unit volume

23.2    Definitions and acronyms (in alphabetical order)

        Analyte - A compound or mixture of compounds (e.g., PCBs) tested for by this method. The
        analytes are listed in Tables 1 and 2.

        Analytical batch - The set  of samples analyzed on a given instrument during a 24-hour period that
        begins and ends with calibration verification (Sections 7.8 and 13). See also "Extraction batch."

        Blank (method blank; laboratory 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 blank is used to determine if analytes or interferences
        are present in the laboratory environment, the reagents, or the apparatus.

        Calibration factor (CF) - See Section 7.5.1.
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        Calibration standard - A solution prepared from stock solutions and/or a secondary standards and
        containing the analytes of interest, surrogates, and internal standards.  This standard is used to
        model the response of the GC instrument against analyte concentration.

        Calibration verification - The process of confirming that the response of the analytical system
        remains within specified limits of the calibration.

        Calibration verification standard - The combined QC standard (Section 7.7) used to verify
        calibration (Section 13.5) and for LCS tests (Section 8.4).

        Extraction Batch - A set of up to 20 field samples (not including QC samples) started through the
        extraction process in a given 24-hour shift. Each extraction batch of 20 or fewer samples must be
        accompanied by a blank (Section 8.5), a laboratory control sample (LCS, Section 8.4), a matrix
        spike and duplicate (MS/MSD; Section 8.3), resulting in a minimum of five  samples (1 field
        sample, 1 blank, 1 LCS, 1 MS, and 1 MSB) and a maximum of 24 samples (20 field samples, 1
        blank, 1 LCS, 1 MS, and 1 MSB) for the batch. If greater than 20 samples are to be extracted in a
        24-hour shift, the samples must be separated into extraction batches of 20 or fewer samples.

        Field Buplicates - Two samples collected at the same time and place under identical conditions, and
        treated identically throughout field and laboratory procedures. Results of analyses the field
        duplicates provide an estimate of the precision associated with sample collection, preservation, and
        storage, as well as with laboratory procedures.

        Field blank - An aliquot of reagent water or other reference matrix that is placed in a sample
        container in 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. See also "Blank."

        GC - Gas chromatograph or gas chromatography

        Gel-permeation chromatography (GPC) - A form of liquid chromatography in which the analytes
        are separated based on exclusion from the solid phase by size.

        Internal standard - A compound added to  an extract or standard solution in a known amount and
        used as a reference for quantitation of the analytes of interest and surrogates. Also see Internal
        standard quantitation.

        Internal standard quantitation - A means of determining the concentration of an analyte of interest
        (Tables 1 and 2) by reference to a compound not expected to be found in a sample.

        IBC - Initial Bemonstration of Capability (Section 8.2); four aliquots of a reference matrix spiked
        with the analytes of interest and analyzed to establish the ability of the laboratory to generate
        acceptable precision and recovery.  An IBC is performed prior to the first time this method is used
        and any time the method or instrumentation is modified.

        Laboratory Control Sample (LCS; laboratory fortified blank; Section 8.4) - An aliquot of reagent
        water spiked with known quantities of the analytes of interest and surrogates. The LCS 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.

        Laboratory Fortified Sample Matrix - See Matrix spike

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       Laboratory reagent blank - See blank

       Matrix spike (MS) and matrix spike duplicate (MSB) (laboratory fortified sample matrix and
       duplicate) - Two aliquots of an environmental sample to which known quantities of the analytes of
       interest and surrogates are  added in the laboratory. The MS/MSD are prepared and analyzed
       exactly like a field sample. Their purpose is to quantify any additional bias and imprecision caused
       by the sample matrix. The background concentrations of the analytes in the sample matrix must be
       determined in a separate aliquot and the measured values in the MS/MSD corrected for background
       concentrations.

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

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

       Method detection limit (MDL) - A detection limit determined by the procedure at 40 CFR 136,
       Appendix B. The MDLs determined by EPA are listed in Tables 1 and 2. As noted in Sec. 1.6, use
       the MDLs in Tables 1 and 2 in conjunction with current MDL data from the laboratory actually
       analyzing samples to assess the sensitivity of this procedure relative to project objectives and
       regulatory requirements (where applicable)

       Minimum level (ML) - The term "minimum level" refers to either the sample concentration
       equivalent to the lowest calibration point in a method or a multiple of the method detection limit
       (MDL), whichever is higher. Minimum levels may be obtained in several ways: They may be
       published in a method; they may be based on the lowest acceptable calibration point used by a
       laboratory; or they may be calculated by multiplying the MDL in a method, or the MDL determined
       by a laboratory, by a factor of 3. For the purposes of NPDES compliance monitoring, EPA
       considers the following terms to be synonymous: "quantitation limit," "reporting limit,"  and
       "minimum level."

       MS - Mass spectrometer or mass spectrometry

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

       Preparation blank - See blank

       Quality control sample (QCS) - A sample containing analytes of interest at known concentrations.
       The QCS is obtained from a source external to the laboratory or is prepared from standards obtained
       from a different source than the calibration standards. The purpose is to check laboratory
       performance using test materials that have been prepared independent of the normal preparation
       process.

       Reagent water - Water demonstrated to be free from the analytes of interest and  potentially
       interfering substances at the MDLs for the analytes in this method.

       Regulatory compliance limit - A limit on the concentration or amount of a pollutant or contaminant
       specified in a nationwide standard, in a permit, or otherwise established by a regulatory/control
       authority.

       Relative standard deviation (RSD) -  The standard deviation times 100 divided by the mean. Also
       termed "coefficient of variation."

       RF - Response factor. See Section 7.6.2

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        RPD - Relative percent difference

        RSD - See relative standard deviation

        Safety Data Sheet (SDS) - Written information on a chemical's toxicity, health hazards, physical
        properties, fire, and reactivity, including storage, spill, and handling precautions that meet the
        requirements of OSHA, 29 CFR 1910.1200(g) and Appendix D. United Nations Globally
        Harmonized System of Classification and Labelling of Chemicals (GHS), third revised edition,
        United Nations, 2009.

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

        SPE - Solid-phase extraction; a sample extraction or extract cleanup technique in which an analyte
        is selectively removed from a sample or extract by passage over or through a material capable of
        reversibly adsorbing the analyte.

        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.

        Surrogate - A compound unlikely to be found in a sample, which is spiked into the sample in a
        known amount before extraction, and which is quantified with the same procedures used to
        quantify other sample components. The purpose of the surrogate is to monitor method performance
        with each sample.
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