&EPA  Method 1699: Pesticides in Water,
        Soil, Sediment, Biosolids, and
        Tissue by HRGC/HRMS

        December 2007
                           Cl
    i.e.

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Method 1699                                           December 2007
                 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-08-001
                          December 2007

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Method 1699                                                          December 2007
                                      Introduction

EPA Method 1699 determines organochlorine, organophosphorus, triazine, and pyrethroid pesticides in
environmental samples by high resolution gas chromatography/high resolution mass spectrometry
(HRGC/HRMS) using isotope dilution and internal standard quantitation techniques. This method has
been developed for use with aqueous, solid, tissue and biosolids matrices.
                                       Disclaimer

This method has been reviewed by the Engineering and Analytical Support Branch of the Engineering
and Analysis Division (EAD) in OST. The method is available for general use, but has not been
published in 40 CFR Part 136. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.

                                        Contacts

Questions concerning this method or its application should be addressed to:

Brian Englert, Ph.D.
Environmental Scientist
Engineering & Analytical Support Branch
Engineering and Analysis Division (43 OST)
Office of Science and Technology, Office of Water
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue NW
Washington, DC 20460
http://www.epa.gov/waterscience
ostcwamethods@epa.gov

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Method 1699                                               December 2007





Table of Contents





INTRODUCTION	3




DISCLAIMER	3




1.0    SCOPE AND APPLICATION	5




2.0    SUMMARY OF METHOD	6




3.0    DEFINITIONS AND UNITS OF MEASURE	7




4.0    INTERFERENCES	7




5.0    SAFETY	9




6.0    APPARATUS AND MATERIALS	12




7.0    REAGENTS AND STANDARDS	19




8.0    SAMPLE COLLECTION, PRESERVATION, STORAGE, AND HOLDING TIMES	25




9.0    QUALITY ASSURANCE/QUALITY CONTROL	26




10.0   CALIBRATION	31




11.0   SAMPLE PREPARATION	36




12.0   EXTRACTION AND CONCENTRATION	42




13.0   EXTRACT CLEANUP	51




14.0   HRGC/HRMS ANALYSIS	57




15.0   SYSTEM AND LABORATORY PERFORMANCE	58




16.0   QUALITATIVE DETERMINATION	61




17.0   QUANTITATIVE DETERMINATION	61




18.0   ANALYSIS OF COMPLEX SAMPLES	64




19.0   POLLUTION PREVENTION	65




20.0   WASTE MANAGEMENT	65




21.0   METHOD PERFORMANCE	66




22.0   REFERENCES	66

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Method 1699                                                     December 2007
Method 1699: Pesticides in Water, Soil,

Sediment,  Biosolids, and  Tissue by HRGC/HRMS



1.0   Scope and Application

       1.1   Method  1699 is for determination of selected organochlorine, organo-phosphorus, triazine,
            and pyrethroid pesticides in multi-media environmental samples by high resolution gas
            chromatography/high resolution mass spectrometry (HRGC/HRMS).

       1.2   This Method was developed for use in EPA's Clean Water Act (CWA) programs; other
            applications are possible. It is based on existing EPA methods (Reference 1) and
            procedures developed at Axys Analytical Services (Reference 2).

       1.3   The analytes that may be measured by this method and their corresponding Chemical
            Abstracts Service Registry Numbers (CASRNs) and ambient water quality criteria are listed
            in Table 1.

       1.4   The detection limits and quantitation levels in this Method are usually dependent on the
            level of interferences rather than instrumental limitations.  The method detection limits
            (MDLs;  40 CFR 136, appendix B) and minimum levels of quantitation (MLs; 68 FR
            11790) in Table 1 are the levels at which pesticides can be determined in the absence of
            interferences.

       1.5   This Method is restricted for use by analysts experienced in HRGC/HRMS or under the
            close supervision of such qualified persons. Each laboratory that uses this Method must
            demonstrate the ability to generate acceptable results using the procedure in Section 9.2.

       1.6 This method is performance-based which means that you may modify the method to improve
          performance (e.g., to overcome interferences or improve the accuracy or precision of the
          results) provided that you meet all performance requirements in this method. These
          requirements for establishing equivalency of a modification are in Section 9.1.2. For Clean
          Water Act (CWA) uses, additional flexibility is described at 40 CFR 136.6. Modifications
          not in the scope of Part 136.6 or in Section 9 of this method may require prior review and
          approval.

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Method 1699                                                             December 2007

2.0    Summary of Method

        Flow charts that summarize procedures for sample preparation, extraction, and analysis are given
        in Figure 1 for aqueous and solid samples, Figure 2 for multi-phase samples, and Figure 3 for
        tissue samples.

        2.1   Extraction (Section 12)

             2.1.1  Aqueous samples (samples containing less than one percent solids) - Stable
                    isotopically labeled analogs of the pesticides are spiked into a 1-L sample. The
                    sample is extracted at neutral pH with methylene chloride using separatory funnel
                    extraction (SFE) or continuous liquid/liquid extraction (CLLE).

             2.1.2  Solid, semi-solid, and multi-phase samples (excluding municipal sludge and tissue)
                    - The labeled compounds are spiked into a sample containing 10 g (dry weight) of
                    solids. Samples containing multiple phases are pressure filtered and any aqueous
                    liquid is discarded. Coarse solids are ground or homogenized.  Any non-aqueous
                    liquid from multi-phase samples is combined with the solids and extracted with
                    methylene chloride, methylene chloride :hexane (1:1) or acetone :hexane (1:1) in a
                    Soxhlet extractor or with toluene in a Soxhlet/Dean-Stark (SDS) extractor
                    (Reference 3).

             2.1.3  Municipal sludges are homogenized, spiked with labeled compounds, and Soxhlet
                    extracted with dichloromethane.

             2.1.4  Fish and other tissue - A 20-g aliquot of sample is homogenized, and a 10-g
                    aliquot is spiked with the labeled compounds. The sample is mixed with
                    anhydrous sodium sulfate, allowed to dry for 30 minutes minimum, and extracted
                    for 18 - 24 hours using methylene chloride in a Soxhlet extractor.  The extract is
                    evaporated to dryness, and the lipid content is determined.

        2.2   Concentration (Section 12)

             2.2.1  Extracts are macro-concentrated using rotary evaporation, a heating mantle, or a
                    Kuderna-Danish evaporator.

             2.2.2  Extracts to be injected into the HRGC/HRMS are concentrated to a final volume of
                    20 |oL using nitrogen evaporation (blowdown).

        2.3   Cleanup (Section 13)

             2.3.1  Extracts of aqueous, solid or mixed phase samples are cleaned up using an
                    aminopropyl SPE column followed by a microsilica column.

             2.3.2  Extracts may be further cleaned up using gel permeation chromatography (GPC) or
                    solid-phase cartridge techniques.

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Method 1699                                                             December 2007

             2.3.3  Extracts in which the organo-chlorine pesticides only are to be determined may be
                    further cleaned up using silica gel, Florisil, or alumina chromatography.

        2.4  Determination by GC/HRMS - Immediately prior to injection, a labeled injection internal
             standard is added to each extract and an aliquot of the extract is injected into the gas
             chromatograph (GC).  The analytes are separated by the GC and detected by a high-resolu-
             tion (>8,000) mass spectrometer.  Two  exact m/z's for each pesticide are monitored
             throughout a pre-determined retention time window.

        2.5  An individual pesticide is identified by comparing the GC retention time and ion-
             abundance ratio of two exact m/z's with the corresponding retention time of an authentic
             standard and the theoretical or acquired ion-abundance ratio of the two exact m/z's.

        2.6  Quantitative analysis is performed in one of two ways using selected ion current profile
             (SICP) areas:

             2.6.1  For pesticides for which a labeled analog is available, the GC/HRMS is multi-point
                    calibrated and the concentration is determined using the isotope dilution technique.

             2.6.2  Pesticides for which a labeled analog is not available are determined using the
                    internal standard technique.  The labeled compounds are used as internal standards,
                    affording recovery correction for all pesticides.

        2.7  The quality of the analysis is assured through reproducible calibration and testing of the
             extraction, cleanup, and GC/MS systems.
3.0    Definitions and units of measure

        Definitions and units of measure are given in the glossary at the end of this Method.


4.0    Interferences

        4.1   Solvents, reagents, glassware, and other sample processing hardware may yield artifacts,
             elevated baselines, and/or lock-mass suppression causing misinterpretation of
             chromatograms.  Specific selection of reagents and purification of solvents by distillation in
             all-glass systems may be required. Where possible, reagents are cleaned by extraction or
             solvent rinse.

        4.2   Proper cleaning of glassware is extremely important, because glassware may not only
             contaminate the samples but may also remove the analytes of interest by adsorption on the
             glass surface.

             4.2.1   Glassware should be rinsed with solvent and washed with a detergent solution as
                    soon after use as is practical.  Sonication of glassware containing a detergent

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Method 1699                                                              December 2007

                     solution for approximately 30 seconds may aid in cleaning. Glassware with
                     removable parts, particularly separatory runnels with fluoropolymer stopcocks,
                     must be disassembled prior to detergent washing.

             4.2.2   After detergent washing, glassware should be rinsed immediately, first with
                     methanol, then with hot tap water. The tap water rinse is followed by another
                     methanol rinse, then acetone, and then methylene chloride.

             4.2.3   Baking of glassware in a kiln or other high temperature furnace (300 - 500C) may
                     be warranted after particularly dirty samples are encountered. The kiln or furnace
                     should be vented to prevent laboratory contamination by pesticide vapors.  Baking
                     should be minimized, as repeated baking of glassware may cause active sites on the
                     glass surface that may irreversibly adsorb pesticides. Volumetric ware should not
                     be baked at high temperature.

             4.2.4   After drying and cooling, glassware should be sealed and stored in a clean
                     environment to prevent any accumulation of dust or other contaminants.  Store
                     inverted or capped with aluminum foil.

             4.2.5   Immediately prior to use, the Soxhlet apparatus should be pre-extracted for
                     approximately 3 hours and the extraction apparatus should be rinsed with the
                     extraction solvent.

        4.3  All materials used in the analysis must be demonstrated to be free from interferences by
             running reference matrix method blanks (Section 9.5) initially and with  each sample batch
             (samples started through the extraction process on a given 12-hour shift, to a maximum of
             20 samples).

             4.3.1   The reference matrix must simulate, as closely as possible, the sample matrix under
                     test. Ideally, the reference matrix should not contain the pesticides in detectable
                     amounts, but should contain potential interferents in the concentrations expected to
                     be found in the samples to be analyzed.

             4.3.2   When a reference matrix that simulates the sample matrix under test is not
                     available, reagent water (Section 7.6.1) can be used to simulate water samples;
                     playground sand (Section  7.6.2) or white quartz sand (Section 7.3.2) can be used to
                     simulate soils; filter paper (Section 7.6.3) can be used to simulate papers and
                     similar materials; and corn oil (Section 7.6.4) can be used to simulate tissues.

        4.4  Interferences co-extracted from samples will vary considerably from source to source,
             depending on the diversity of the site being sampled. Interfering compounds may be
             present at concentrations several orders of magnitude higher than the pesticides.  The most
             frequently encountered interferences are chlorinated biphenyls, chlorinated and brominated
             dibenzodioxins and dibenzofurans, methoxy biphenyls, hydroxydiphenyl ethers,
             benzylphenyl ethers, brominated diphenyl ethers, polynuclear aromatics, and
             polychlorinated naphthalenes.  Because very low levels of pesticides are measured  by this
             Method, elimination of interferences is essential. The cleanup steps given in Section 13

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Method 1699                                                              December 2007

             can be used to reduce or eliminate these interferences and thereby permit reliable
             determination of the pesticides at the levels shown in Table 1.

        4.5  Each piece of reusable glassware should be numbered to associate that glassware with the
             processing of a particular sample. This will assist the laboratory in tracking possible
             sources of contamination for individual samples, identifying glassware associated with
             highly contaminated samples that may require extra cleaning, and determining when
             glassware should be discarded.

        4.6  Organic acids and other substances make it difficult to extract and clean up biosolids
             (sewage sludge) samples.  The exact procedures to be used are dependent on the analytes to
             be determined. If all analytes in this Method are to be determined, gel permeation
             chromatography (GPC), the amino-propyl SPE column, and the layered alumina/Florisil
             column have been found effective. For the organo-chlorine pesticides, sequential
             extraction with acetonitrile and methylene chloride followed by back extraction with
             sodium sulfate-saturated water has been found effective. An anthropogenic isolation
             column (Section 13.6; see Section 7.5.2 for column details), GPC (Section 13.2), high
             performance liquid chromatography (HPLC; Section 13.5), Florisil (Section 13.7), and
             alumina (Section 13.8) are additional steps that may be employed to minimize interferences
             in the sludge matrix.

        4.7  The natural lipid content of tissue can interfere in the analysis of tissue samples for
             measurement of pesticides.  The lipid  contents of different species and portions of tissue
             can vary widely.  Lipids are soluble to varying degrees in various organic solvents and may
             be present in sufficient quantity to overwhelm the column chromatographic cleanup
             procedures used for sample extracts. Lipids must be removed by the anthropogenic
             isolation column procedure in Section 13.6, followed by GPC (Section  13.2).
5.0    Safety

        5.1   The toxicity or carcinogenicity of each chemical used in this Method has not been precisely
             determined; however, each compound should be treated as a potential health hazard.
             Exposure to these compounds should be reduced to the lowest possible level.

             5.1.1   Some pesticides, most notably 4,4'-DDT and 4,4'-DDD, have been tentatively
                     classified as known or suspected human or mammalian carcinogens. Pure
                     standards of the pesticides should be handled only by highly trained personnel
                     thoroughly familiar with handling and cautionary procedures and the associated
                     risks.

             5.1.2   It is recommended that the laboratory purchase dilute standard solutions of the
                     analytes in this Method. However, if primary solutions are prepared, they must be
                     prepared in a hood,  and a NIOSH/MESA approved toxic gas respirator must be
                     worn when high concentrations are handled.

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Method 1699                                                              December 2007

        5.2  This Method does not address all safety issues associated with its use. The laboratory is
             responsible for maintaining a current awareness file of OSHA regulations regarding the
             safe handling of the chemicals specified in this Method.  A reference file of material safety
             data sheets (MSDSs) should also be made available to all personnel involved in these
             analyses.  It is also suggested that the laboratory perform personal hygiene monitoring of
             each analyst who uses this Method and that the results of this monitoring be made available
             to the analyst.  Additional information on laboratory safety can be found in References 4-7.
              The references and bibliography at the end of Reference 6 are particularly comprehensive
             in dealing with the general subject of laboratory safety.

        5.3  The pure pesticides and samples suspected to contain high concentrations of these
             compounds are handled using essentially the same techniques employed in handling
             radioactive or infectious materials. Well-ventilated, controlled access laboratories are
             required.  Assistance in evaluating the health hazards of particular laboratory conditions
             may be obtained from certain consulting laboratories and from State Departments of Health
             or Labor, many of which have an industrial health service. Each laboratory must develop a
             strict safety program for handling these compounds.  The practices in Reference 8 for
             handling chlorinated dibenzo-p-dioxins and dibenzofurans (CDDs/CDFs) are also
             recommended for handling pesticides.

             5.3.1  Facility - When finely divided samples (dusts, soils, dry chemicals) are handled, all
                    operations (including removal of samples from sample containers, weighing,
                    transferring, and mixing) should be performed in a glove box demonstrated to be
                    leak tight or in a fume hood demonstrated to have adequate air flow. Gross losses
                    to the laboratory ventilation system must not be  allowed. Handling  of the dilute
                    solutions normally used in analytical and  animal work presents no inhalation
                    hazards except in the case of an accident.

             5.3.2  Protective equipment - Disposable plastic gloves, apron or lab coat, safety glasses
                    or mask, and a glove box or fume hood adequate for radioactive work should be
                    used.  During analytical operations that may give rise to aerosols or dusts,
                    personnel should wear respirators equipped with activated carbon filters. Eye
                    protection (preferably full face shields) must be  worn while working with exposed
                    samples or pure  analytical standards. Latex gloves are commonly used to reduce
                    exposure of the hands. When handling samples  suspected or known to contain
                    high concentrations of the pesticides, an additional set of gloves can also be worn
                    beneath the latex gloves.

             5.3.3  Training - Workers must be trained in the proper method of removing
                    contaminated gloves and clothing without contacting the exterior surfaces.

             5.3.4  Personal hygiene - Hands and forearms should be washed thoroughly after each
                    operation involving high concentrations of the pesticides, and before breaks
                    (coffee, lunch, and shift).
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Method 1699                                                              December 2007

             5.3.5   Confinement - Isolated work areas posted with signs, segregated glassware and
                     tools, and plastic absorbent paper on bench tops will aid in confining
                     contamination.

             5.3.6   Effluent vapors - The effluent of the sample splitter from the gas chromatograph
                     (GC) and from roughing pumps on the mass spectrometer (MS) should pass
                     through either a column of activated charcoal or be bubbled through a trap contain-
                     ing oil or high-boiling alcohols to condense pesticide vapors.

             5.3.7   Waste handling - Good technique includes minimizing contaminated waste.
                     Plastic bag liners should be used in waste cans. Janitors and other personnel should
                     be trained in the safe handling of waste.

             5.3.8   Decontamination

                     5.3.8.1   Decontamination of personnel - Use any mild soap with plenty of
                              scrubbing action.

                     5.3.8.2   Glassware, tools, and surfaces - Chlorothene NU Solvent is a less toxic
                              solvent that should be effective in removing pesticides.  Satisfactory
                              cleaning may be accomplished by rinsing with Chlorothene, then
                              washing with any detergent and water.  If glassware is first rinsed with
                              solvent, the wash water may be disposed of in the sewer. Given the cost
                              of disposal, it is prudent to minimize solvent wastes.

             5.3.9   Laundry - Clothing known to be contaminated should be collected in plastic bags.
                     Persons that convey the bags and launder clothing should be advised of the hazard
                     and trained in proper handling.  Clothing may be put into a washer without contact
                     if the launderer knows of the potential problem. The washer should be run through
                     a cycle before being used again for other clothing.

             5.3.10  Wipe tests - A useful method of determining cleanliness of work surfaces and tools
                     is to perform a wipe test of the surface suspected of being contaminated.

                     5.3.10.1  Using a piece of filter paper moistened with Chlorothene or other
                              solvent, wipe an area approximately 10 x 10 cm.

                     5.3.10.2  Extract and analyze the wipe by GC with an electron capture detector
                              (BCD) or by this Method.

                     5.3.10.3  Using the area wiped (e.g., 10 x 10 cm = 0.01 m2), calculate the
                              concentration in (ig/m2. A concentration less than 1 (ig/m2 indicates
                              acceptable cleanliness; anything higher warrants further cleaning. More
                              than  100 (ig/m2 constitutes an acute hazard and requires prompt cleaning
                              before further use of the equipment or work space, and indicates that
                              unacceptable work practices have been employed.


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Method 1699                                                            December 2007

        5.4     Biosolids samples may contain high concentrations of biohazards, and must be handled
               with gloves and opened in a hood or biological safety cabinet to prevent exposure.
               Laboratory staff should know and observe the safety procedures required in a
               microbiology laboratory that handles pathogenic organisms when handling biosolids
               samples.
6.0   Apparatus and materials


    Note: Brand names, suppliers, and part numbers are for illustration purposes only and no
    endorsement is implied.  Equivalent performance may be achieved using apparatus and materials
    other than those specified here. Meeting the performance requirements of this Method is the
    responsibility of the laboratory.


       6.1   Sampling equipment for discrete or composite sampling

             6.1.1   Sample bottles and caps

                    6.1.1.1  Liquid samples (waters, sludges and similar materials containing 5
                             percent solids or less) - Sample bottle, amber glass, 1.1-L minimum,
                             with screw cap.

                    6.1.1.2  Solid samples (soils, sediments, sludges, paper pulps, filter cake,
                             compost, and similar materials that contain more than 5 percent solids) -
                             Sample bottle, wide mouth, amber glass, 500-mL minimum.

                    6.1.1.3  If amber bottles are not available, samples must be protected from light.

                    6.1.1.4  Bottle caps - Threaded to fit sample bottles. Caps must be lined with
                             fluoropolymer.

                    6.1.1.5  Cleaning

                             6.1.1.5.1   Bottles are detergent water washed, then solvent rinsed
                                        before use.

                             6.1.1.5.2   Liners are detergent water washed and rinsed with reagent
                                        water (Section 7.6.1).

             6.1.2  Compositing equipment - Automatic or manual compositing system incorporating
                    glass containers cleaned per bottle cleaning procedure above.  Only glass or
                    fluoropolymer tubing must be used. If the sampler uses a peristaltic pump, a
                    minimum length of compressible silicone rubber tubing may be used in the pump
                    only.  Before use, the tubing must be thoroughly  rinsed with methanol, followed by
                    repeated rinsing with reagent water to minimize sample contamination. An
                    integrating flow meter is used to collect proportional composite samples.
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Method 1699                                                             December 2007


        6.2   Equipment for glassware cleaning


    Note: If blanks from bottles or other glassware or with fewer cleaning steps than required in this
    Method show no detectable pesticide contamination, unnecessary cleaning steps and equipment may
    be eliminated.

             6.2.1  Laboratory sink with overhead fume hood

             6.2.2  Kiln - Capable of reaching 450C within 2 hours and maintaining 450 - 500C
                    within V10C, with temperature controller and safety switch (Cress Manufacturing
                    Co, Santa Fe Springs, CA, B31H, X3 ITS, or equivalent).  See the precautions in
                    Section 4.2.3.

             6.2.3  Aluminum foil - solvent rinsed or baked in a kiln.  If baked in a kiln, heavy-duty
                    aluminum foil is required, as thinner foil will become brittle and unusable.

        6.3   Equipment for sample preparation

             6.3.1  Laboratory fume hood of sufficient size to contain the sample preparation
                    equipment listed below.

             6.3.2  Glove box (optional)

             6.3.3  Tissue homogenizer - VirTis Model 45 Macro homogenizer (American Scientific
                    Products H-3515, or equivalent) with stainless steel Macro-shaft and Turbo-shear
                    blade.

             6.3.4  Meat grinder - Hobart, or equivalent, with 3- to 5-mm holes in inner plate.

             6.3.5  Equipment for determining percent moisture

                    6.3.5.1    Oven - Capable of maintaining a temperature of 110 V5C

                    6.3.5.2    Desiccator

             6.3.6  Balances

                    6.3.6.1    Analytical - Capable of weighing 0.1 mg

                    6.3.6.2    Top loading - Capable of weighing 10 mg

        6.4   Extraction apparatus

             6.4.1  Water and solid samples


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Method 1699                                                              December 2007

                     6.4.1.1   pH meter, with combination glass electrode

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

                     6.4.1.3   Graduated cylinder, glass, 1-L capacity and Erlenmeyer Flask, glass, 1-L
                              capacity

                     6.4.1.4   Liquid/liquid extraction - Separatory runnels, 250-, 500-, and 2000-mL,
                              with fluoropolymer stopcocks

                     6.4.1.5   Solid-phase extraction

                              6.4.1.5.1   1-L filtration apparatus, including glass funnel, frit support,
                                         clamp, adapter, stopper, filtration flask, and vacuum tubing
                                         (Figure 4).  For wastewater samples, the apparatus should
                                         accept 90 or 144 mm disks.  For drinking water or other
                                         samples containing low solids, smaller disks may be used.

                              6.4.1.5.2  Vacuum source - Capable of maintaining 25 in. Hg,
                                         equipped with shutoff valve and vacuum gauge

                              6.4.1.5.3  Glass-fiber filter - Whatman GMF 150 (or equivalent), 1
                                         micron pore size, to fit filtration apparatus in Section
                                         6.4.1.5.1

                              6.4.1.5.4  Solid-phase extraction disk containing octadecyl (Ci8)
                                         bonded silica uniformly enmeshed in an inert matrix - Fisher
                                         Scientific 14-378F (or equivalent), to fit filtration apparatus
                                         in Section 6.4.1.5.1

                     6.4.1.6   Continuous liquid/liquid extraction (CLLE) - Fluoropolymer or glass
                              connecting joints and stopcocks without lubrication, 1.5-2 L capacity
                              (Hershberg-Wolf Extractor, Cal-Glass, Costa Mesa, California, 1000 mL
                              or 2000 mL, or equivalent)

             6.4.2   Soxhlet/Dean-Stark (SDS) extractor (Figure 5 and Reference 3) for filters and
                     solid/sludge samples

                     6.4.2.1   Soxhlet - 50-mm ID, 200-mL capacity with 500-mL flask (Cal-Glass
                              LG-6900, or equivalent, except substitute 500-mL round-bottom flask for
                              300-mL flat-bottom flask)

                     6.4.2.2   Thimble - 43 H 123 to fit Soxhlet (Cal-Glass LG-6901-122, or
                              equivalent)
                     6.4.2.3   Moisture trap - Dean Stark or Barret with fluoropolymer stopcock, to fit
                              Soxhlet
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Method 1699                                                            December 2007


                    6.4.2.4  Heating mantle - Hemispherical, to fit 500-mL round-bottom flask (Cal-
                             Glass LG-8801-112, or equivalent)

                    6.4.2.5  Variable transformer - Powerstat (or equivalent),  110-volt, 10-amp

             6.4.3  Beakers - 400- to 500-mL

             6.4.4  Spatulas - Stainless steel

        6.5   Filtration apparatus

             6.5.1  Pyrex glass wool - Solvent-extracted using a Soxhlet or SDS extractor for 3 hours
                    minimum

             6.5.2  Glass funnel - 125- to 250-mL

             6.5.3  Glass-fiber filter paper - Whatman GF/D (or equivalent), to fit glass funnel in
                    Section 6.5.2.

             6.5.4  Drying column - 15- to 20-mm ID Pyrex chromatographic column equipped with
                    coarse-glass frit or glass-wool plug

             6.5.5  Buchner funnel -  15-cm

             6.5.6  Glass-fiber filter paper for Buchner funnel above

             6.5.7  Filtration flasks - glass, 1.5- to 2.0-L, with side arm

             6.5.8  Pressure filtration apparatus - Millipore YT30 142 HW, or equivalent

        6.6   Centrifuge apparatus

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

             6.6.2  Centrifuge bottles - 500-mL, with screw-caps, to fit centrifuge

             6.6.3  Centrifuge tubes - 12- to 15-mL, with screw-caps, to fit centrifuge

        6.7   Cleanup apparatus

             6.7.1  Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc,
                    Columbia, MO, Model GPC Autoprep 1002, or equivalent)
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Method 1699                                                             December 2007

                    6.7.1.1  Column - 600-700 mm long H 25 mm ID glass, packed with 70 g of
                             200-400 mesh SX-3 Bio-beads (Bio-Rad Laboratories, Richmond, CA,
                             or equivalent)

                    6.7.1.2  Syringe - 10-mL, with Luer fitting

                    6.7.1.3  Syringe filter holder - stainless steel, and glass-fiber or fluoropolymer
                             filters (Gelman 4310, or equivalent)

                    6.7.1.4  UV detectors - 254-nm, preparative or semi-preparative flow cell (Isco,
                             Inc., Type 6; Schmadzu, 5-mm path length; Beckman-Altex 152W, 8-|oL
                             micro-prep flow cell, 2-mm path; Pharmacia UV-1, 3-mm flow cell;
                             LDC Milton-Roy UV-3, monitor #1203; or equivalent)

             6.7.2  Reverse-phase high-performance liquid chromatograph (Reference 9)

                    6.7.2.1  Pump - Perkin-Elmer Series 410, or equivalent

                    6.7.2.2  Injector - Perkin-Elmer ISS-100 Autosampler, or equivalent

                    6.7.2.3  6-Port switching valve - Valco N60, or equivalent

                    6.7.2.4  Column - Hypercarb, 100 x 4.6 mm, 5 Om particle size, Keystone
                             Scientific, or equivalent

                    6.7.2.5  Detector - Altex 110A (or equivalent) operated at 0.02 AUFS at 235 nm

                    6.7.2.6  Fraction collector - Isco Foxy II, or equivalent

             6.7.3  Pipets, precleaned

                    6.7.3.1  Disposable, Pasteur, 150-mm long x 5-mm ID (Fisher Scientific 13-678-
                             6A, or equivalent)

                    6.7.3.2  Disposable, serological, 50-mL (8- to 10- mm ID)

             6.7.4  Glass chromatographic columns

                    6.7.4.1  150-mm long x 8-mm ID, (Kontes K-420155, or equivalent) with coarse-
                             glass frit or glass-wool plug and  250-mL reservoir

                    6.7.4.2  200-mm long x 15-mm ID, with  coarse-glass frit or glass-wool plug and
                             250-mL reservoir

                    6.7.4.3  300-mm long x 22-mm ID, with  coarse-glass frit, 300-mL reservoir, and
                             glass or fluoropolymer stopcock

16

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Method 1699                                                             December 2007

             6.7.5  Oven - For baking and storage of adsorbents, capable of maintaining a constant
                    temperature (V 5C) in the range of 105-250C

             6.7.6  System for solid-phase extraction

                    6.7.6.1  Vac-Elute Manifold (Analytichem International, or equivalent)

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

                    6.7.6.3  Rack for holding 50-mL volumetric flasks in the manifold

        6.8   Concentration apparatus

             6.8.1  Rotary evaporator - Buchi/Brinkman-American Scientific No. E5045-10 or
                    equivalent, equipped with a variable temperature water bath

                    6.8.1.1  Vacuum source for rotary evaporator equipped with vacuum gauge and
                             with shutoff valve at the evaporator

                    6.8.1.2  A recirculating water pump and chiller are recommended. Use of tap
                             water for cooling the evaporator wastes large volumes of water and can
                             lead to inconsistent performance as water temperatures and pressures
                             vary.

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

             6.8.2  Kuderna-Danish (K-D) concentrator

                    6.8.2.1  Concentrator tube - 10-mL, graduated (Kontes K-570050-1025, or
                             equivalent) with calibration verified. Ground-glass stopper (size 19/22
                             joint) is used to prevent evaporation of extracts.

                    6.8.2.2  Evaporation flask - 500-mL (Kontes K-570001-0500, or equivalent),
                             attached to concentrator tube with springs (Kontes K-662750-0012 or
                             equivalent)

                    6.8.2.3  Snyder column - Three-ball macro (Kontes K-503000-0232, or
                             equivalent)
                                                                                             17

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Method 1699                                                             December 2007
                    6.8.2.4  Boiling chips

                             6.8.2.4.1  Glass or silicon carbide - Approximately 10/40 mesh,
                                        extracted with methylene chloride and baked at 450C for
                                        one hour minimum

                             6.8.2.4.2  Fluoropolymer (optional) - Extracted with methylene
                                        chloride

                    6.8.2.5  Water bath - Heated, with concentric ring cover, capable of maintaining
                             a temperature within V 2C, installed in a fume hood

             6.8.3  Nitrogen evaporation apparatus - Equipped with water bath controlled in the range
                    of 30 - 60C (N-Evap, Organomation Associates, Inc., South Berlin, MA, or
                    equivalent), installed in a fume hood

             6.8.4  Sample vials

                    6.8.4.1  Amber glass, 2- to 5-mL with fluoropolymer-lined screw-cap

                    6.8.4.2  Glass, 0.3-mL, conical, with fluoropolymer-lined screw or crimp cap

        6.9   Gas chromatograph - Must have splitless or on-column injection port for capillary column,
             temperature program with isothermal hold, and must meet all of the performance
             specifications in Section 10.

             6.9.1  GC column - 60 V 5-m long x 0.25 V 0.02-mm ID; 0.1 (Hun film DB-17, or
                    equivalent

                    6.9.1.1  The column must meet the following minimum retention time and
                             resolution criteria, and must be adjusted or replaced when these criteria
                             are not met:

                             6.9.1.1.1  The retention time for methoxychlor must be greater than 39
                                        minutes.

                             6.9.1.1.2  trans-chlordane and trans-nonachlor (or the labeled analogs)
                                        must be uniquely resolved to a valley height less than 10
                                        percent  of the shorter of the two peaks.

                    6.9.1.2  Endrin and DDT breakdown - The column must meet the endrin/DDT
                             breakdown criteria in Section 10.6.2.3.  Some GC injectors may be
                             unable to meet requirements for endrin and DDT breakdown.  This
                             problem can be minimized by operating the injector at 200 - 205  C,
                             using a Pyrex (not quartz) methyl silicone deactivated injector liner, and
                             deactivating the injector with dichlorodimethylsilane. A temperature
18

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Method 1699                                                             December 2007

                             programmed injector has also been shown to minimize decomposition of
                             labile substances such as endrin and DDT (Reference  10).

        6.10 Mass spectrometer - 28- to 40-eV electron impact ionization, must be capable of
             selectively monitoring a minimum of 22 exact m/z's minimum at high resolution (greater
             than 8,000) during a period less than 1.5 seconds, and must meet all of the performance
             specifications in Section 10.

        6.11 GC/MS interface - The mass spectrometer (MS) must be interfaced to the GC such that the
             end of the capillary column terminates within 1 cm of the ion source but does not intercept
             the electron or ion beams.

        6.12 Data system - Capable of collecting, recording, storing, and processing MS data

             6.12.1 Data acquisition - The signal at each exact m/z must be collected repetitively
                    throughout the monitoring period and stored on a mass storage device.

             6.12.2 Response factors and multipoint calibrations - The data system must record and
                    maintain lists of response factors (response ratios for isotope dilution) and
                    multipoint calibrations.  Computations of relative standard deviation (RSD) are be
                    used to test calibration linearity.  Statistics on initial (Section 9.4) and ongoing
                    (Section  15.6.4) performance should be computed and maintained, either on the
                    instrument data system, or on a separate computer system.

7.0    Reagents and standards

        7.1   pH adjustment and back-extraction

             7.1.1   Potassium hydroxide (KOH) - Dissolve 20 g reagent grade KOH in 100 mL
                    reagent water.

             7.1.2  Sulfuric acid (H2SO4) - Reagent grade (specific gravity 1.84)

             7.1.3  Hydrochloric acid - Reagent grade, 6N

             7.1.4  Sodium chloride solution - Prepare at 5% (w/v) solution in reagent water

             7.1.4  Sodium sulfate solution - Prepare at 2% (w/v) in reagent water;  pH adjust to 8.5 -
                    9.0withKOHorH2SO4

        7.2  Solution and tissue drying, municipal sludge extract back-extraction, and solvent
             evaporation (blowdown)

             7.2.1   Solution  drying - Sodium sulfate, reagent grade, granular, anhydrous (Baker 3375,
                    or equivalent), rinsed with methylene chloride (20 mL/g), baked at 400C for 1
                    hour minimum,  cooled in a desiccator, and stored in a pre-cleaned glass bottle with
                    screw-cap that prevents moisture from entering.  If, after heating, the sodium
                                                                                             19

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Method 1699                                                              December 2007

                     sulfate develops a noticeable grayish cast (due to the presence of carbon in the
                     crystal matrix), that batch of reagent is not suitable for use and should be discarded.
                     Extraction with methylene chloride (as opposed to simple rinsing) and baking at a
                     lower temperature may produce sodium sulfate that is suitable for use.

             7.2.2   Tissue drying - Sodium sulfate, reagent grade, powdered, treated and stored as in
                     Section 7.2.1

             7.2.3   Solution for back-extraction of municipal sludge extracts - Sodium sulfate
                     solution: 2% (w/v) in reagent water, pH adjusted to pH 8.5 to 9.0 with KOH or
                     H2SO4

             7.2.4   Prepurified nitrogen

        7.3  Extraction

             7.3.1   Solvents - Acetone, toluene, cyclohexane, hexane, methanol, methylene chloride,
                     isooctane, and nonane; distilled in glass, pesticide quality, lot-certified to be free of
                     interferences

             7.3.2   White quartz sand, 60/70 mesh - For Soxhlet/Dean-Stark extraction (Aldrich
                     Chemical, Cat. No. 27-437-9, or equivalent).  Bake at 450 - 500C for 4 hours
                     minimum.

        7.4  GPC calibration solution - Prepare a solution containing 2.5 mg/mL corn oil, 0.05 mg/mL
             bis(2-ethylhexyl)  phthalate (BEHP), 0.01 mg/mL methoxychlor, 0.002 mg/mL perylene,
             and 0.008 mg/mL sulfur, or at concentrations appropriate to the response of the detector.

        7.5  Adsorbents for sample cleanup

             7.5.1   Silica gel

                     7.5.1.1   Activated silica gel - 100-200 mesh, Supelco 1-3651 (or equivalent),
                              mesh, rinsed with methylene chloride, baked at  1805 C for a minimum
                              of 1 hour, cooled in a desiccator, and stored in a precleaned glass bottle
                              with screw-cap that prevents moisture from entering.

                           7.5.1.1.1  10% deactivated silica - Place 100 g of activated silica gel
                              (Section 7.5.1.1) in a clean glass bottle or jar and add 10 g (ormL) of
                              reagent water.  Cap the  bottle tightly to prevent  moisture from entering
                              or escaping.

                           7.5.1.1.2  Tumble the bottle for 5-10 hours to thoroughly mix the water
                              and silica.  Keep bottle tightly sealed when silica is not being removed
                              for use.
20

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Method 1699                                                               December 2007

                     7.5.1.2  Acid silica gel (30% w/w) - Thoroughly mix 44 g of concentrated
                              sulfuric acid with 100 g of activated silica gel in a clean container.
                              Break up aggregates with a stirring rod until a uniform mixture is
                              obtained.  Store in a screw-capped bottle with fluoropolymer-lined cap.

                     7.5.1.3  Basic silica gel - Thoroughly mix 30 g of IN sodium hydroxide with 100
                              g of activated silica gel in a clean container. Break up  aggregates with a
                              stirring rod until a uniform mixture is obtained.  Store in a screw-capped
                              bottle with fluoropolymer-lined cap.

                     7.5.1.4  Potassium silicate

                              7.5.1.4.1   Dissolve 56 g  of high purity potassium hydroxide (Aldrich,
                                         or equivalent) in 300 mL of methanol in a  750- to 1000-mL
                                         flat-bottom flask.

                              7.5.1.4.2  Add 100 g of activated silica gel (Section 7.5.1.1) and a
                                         stirring bar, and stir on an explosion-proof hot plate at 60-
                                         70C for 1-2 hours.

                              7.5.1.4.3  Decant the liquid and rinse the potassium silicate twice with
                                         100-mL portions of methanol, followed by a single rinse
                                         with 100 mL of methylene chloride.

                              7.5.1.4.4  Spread  the potassium silicate on solvent-rinsed aluminum
                                         foil and dry for 2-4 hours in a hood.  Observe the precaution
                                         in Section 5.3.2.

                              7.5.1.4.5  Activate overnight at 200-250C prior to use.

             7.5.2   Anthropogenic isolation column - Pack the column in Section  6.7.4.3 from bottom
                     to top with the following:

                     7.5.2.1  2 g silica gel  (Section 7.5.1.1)

                     7.5.2.2  2 g potassium silicate (Section 7.5.1.4)

                     7.5.2.3  2 g granular anhydrous sodium sulfate (Section 7.2.1)

                     7.5.2.4  10 g acid silica gel (Section 7.5.1.2)

                     7.5.2.5  2 g granular anhydrous sodium sulfate

             7.5.3   Aminopropyl solid-phase extraction  (SPE) column  - 1 g aminopropyl-bonded
                     silica (Varian NH2, or equivalent).
                                                                                               21

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Method 1699                                                             December 2007

    Note: Other SPE columns (e.g., C18 octadecyl, cyanopropyl) may be used provided the laboratory
    establishes the elution conditions and meets the requirements in Section 9.2 with the SPE column as
    an integral part of the analysis.

             7.5.4  Florisil column

                    7.5.4.1  Florisil - PR grade, 60-100 mesh (U.S. Silica Corp, Berkeley Springs,
                             WV, or equivalent). Alternatively, prepacked Florisil columns may be
                             used. Use the following procedure for Florisil  activation and column
                             packing.

                    7.5.4.2  Fill a clean 1- to 2-L bottle 1/2 to 2/3 full with  Florisil and place in an
                             oven at 130-150C for a minimum of three days to activate the Florisil.

                    7.5.4.3  Immediately prior to use, dry pack a 300-mm x 22-mm ID glass column
                             (Section 6.7.4.3) bottom to top with 0.5-1.0 cm of warm to hot
                             anhydrous sodium sulfate (Section 7.2.1), 10-10.5 cm of warm to hot
                             activated Florisil (Section 7.5.4.2), and 1-2 cm of warm to hot anhydrous
                             sodium sulfate. Allow the column to cool and  pre-elute immediately
                             with 100 mL of n-hexane. Keep column wet with hexane to prevent
                             water from entering.

                    7.5.4.4  Using the procedure in Section 13.7.3, establish the elution pattern for
                             each carton of Florisil or each lot of Florisil columns received.

             7.5.5  Alumina column

                    7.5.5.1  Alumina - Neutral, Brockman Activity I, 80-200 mesh (Fisher Scientific
                             Certified, or equivalent).  Heat for 16 hours at 400 to 450C.  Seal and
                             cool to room temperature. Add 7% (WAV) reagent water and tumble for
                             1 to 2 hours.  Keep bottle tightly sealed.

                    7.5.5.2  Immediately prior to use, partially fill a 150-mm x 8-mm ID glass
                             column (Section 6.7.4.1) with n-hexane.  Pack  the column bottom to top
                             with 0.5 - 1 cm of warm to hot anhydrous sodium sulfate (Section 7.2.1),
                             10 - 10.5 cm alumina (Section 7.5.5.1) and 1 -  1.5 cm of warm to hot
                             anhydrous sodium sulfate. Allow the column to cool and pre-elute
                             immediately with 100 mL of hexane. Keep column wet with hexane to
                             prevent moisture from entering.

        7.6   Reference matrices - Matrices in which the pesticides and interfering compounds are not
             detected by this Method

             7.6.1  Reagent water - Bottled water purchased locally, or prepared by passage through
                    activated carbon
22

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Method 1699                                                              December 2007

             7.6.2   High-solids reference matrix - Playground sand or similar material. Prepared by
                     extraction with methylene chloride and/or baking at 450C for a minimum of 4
                     hours.

             7.6.3   Paper reference matrix - Glass-fiber filter, Gelman type A, or equivalent. Cut
                     paper to simulate the surface area of the paper sample being tested.

             7.6.4   Tissue reference matrix - Corn or other vegetable oil.

             7.6.5   Other matrices - This Method may be verified on any reference matrix by
                     performing the tests given in Section 9.2.  Ideally, the matrix should be free of the
                     pesticides, but in no case must the background level of the pesticides in the
                     reference matrix exceed the minimum levels in Table 1.  If low background levels
                     of the pesticides are present in the reference matrix, the spike level of the analytes
                     used in Section 9.2 should be increased to provide a spike-to-background ratio of
                     approximately 5 (Reference 11).

        7.7  Standard solutions - Prepare from materials of known purity and composition or purchase
             as solutions or mixtures with certification to their purity, concentration, and authenticity. If
             the chemical purity is 98 % or greater, the weight may be used without correction to
             calculate the concentration of the standard.  Observe the safety precautions in Section 5  and
             the recommendation in Section  5.1.2.

             7.7.1   For preparation of stock solutions from neat materials, dissolve an appropriate
                     amount of assayed reference material in solvent. For example, weigh 10 to 20 mg
                     of lindane to three significant figures in a 10-mL ground-glass-stoppered
                     volumetric flask and fill to the mark with nonane.  After the compound is
                     completely dissolved, transfer the solution to a clean 15-mL vial with
                     fluoropolymer-lined cap.

             7.7.2   When not being used, store standard solutions in the dark at room temperature in
                     screw-capped vials with fluoropolymer-lined caps.  Place a mark on the vial at the
                     level of the solution so that solvent loss by evaporation can be detected. Replace
                     the solution if solvent loss has occurred.

        7.8  Native (unlabeled) stock solutions

             7.8.1   Native stock solution -  Prepare to contain the pesticides at the concentrations
                     shown in Table 3, or purchase prepared solutions. If additional pesticides are to be
                     determined, include the additional native compounds in this stock solution.

             7.8.2   Stock solutions should be checked for signs of degradation (e.g., discoloration,
                     precipitation) prior to preparing calibration or performance test standards.
                     Reference standards that can be used to determine the accuracy of standard
                     solutions are available from several vendors.

        7.9  Labeled compound stock solutions (Table 3)

                                                                                              23

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Method 1699                                                              December 2007
             7.9.1   Labeled pesticide stock solution - Prepare the labeled pesticides in isooctane or
                     nonane at the concentrations in Table 3 or purchase prepared standards.  If
                     additional pesticides are to be determined by isotope dilution, include the
                     additional labeled compounds in this stock solution.

             7.9.2   Labeled injection internal standard stock solution - Prepare labeled PCB 52 in
                     nonane or isooctane at the concentration shown in Table 3, or purchase a prepared
                     standard.

        7.10 Calibration standards - Combine and dilute the solutions in Sections 7.8 and  7.9 to produce
             the calibration solutions in Table 4 or purchase prepared standards for the CS-1 to  CS-6 set
             of calibration solutions.  These solutions permit the relative response  (labeled to native) and
             response factor to be measured as a function of concentration.  The CS-4 standard  is used
             for calibration verification (VER).

        7.11 Native IPR/OPR standard spiking solution - Used for determining initial precision and
             recovery (IPR; Section 9.2) and ongoing precision and  recovery (OPR;  Section 15.6).
             Dilute the Native stock solution (Section 7.8.1) with acetone to produce the concentrations
             of the pesticides as shown in Table 3. When 1 mL of this solution is spiked into the IPR
             (Section 9.2.1) or OPR (Section 15.6) and concentrated to a final volume of 20 joL, the
             concentration of the pesticides in the final volume  will  be either 8 or 20 ng/mL (pg/OL), as
             shown in Table 3. Prepare only the amount necessary for each reference matrix with each
             sample batch.

        7.12 Labeled standard spiking solution - This solution is spiked into each sample (Section 9.3)
             and into the IPR (Section 9.2.1), OPR (Section 15.6), and blank (Section 9.5) to measure
             recovery. Dilute the Labeled pesticide stock solution (Section 7.9.1) with acetone  to
             produce the concentrations of the labeled compounds shown in Table 3. When 1 mL of this
             solution is spiked into an IPR, OPR, blank, or sample and concentrated  to a final extract
             volume of 20 |oL, the concentration in the final volume will be as shown in Table  3.
             Prepare only the amount necessary for each reference matrix with each  sample batch.

        7.13 Endrin/4,4'-DDT breakdown solution - Prepare a solution to contain  100 ng/mL (pg/joL) of
             DDT and 50 ng/mL (pg/joL) of endrin in isooctane or nonane.  This solution is to determine
             endrin/4,4'-DDT breakdown in Sections 10.6 and 15.5.

        7.14 Labeled injection internal standard spiking solution - This solution is added to each
             concentrated extract prior to injection into the HRGC/HRMS.  Dilute the Labeled injection
             internal standard stock solution (Section 7.9.2) in nonane to produce a concentration of the
             injection internal standards at 800 ng/mL, as shown in Table 3. When 2 |oL of this solution
             is spiked into a 20 |oL extract, the concentration of each injection internal standard will be
             nominally 80 ng/mL (pg/joL), as shown in Table 3.


    Note: The addition of 2 ^L of the Labeled injection internal standard spiking solution to a 20 ^L
    final extract has the effect of diluting the concentration of the components in the extract by 10%.

24

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Method 1699                                                            December 2007

    Provided all calibration solutions and all extracts undergo this dilution as a result of adding the
    Labeled injection internal standard spiking solution, the effect of the 10% solution is compensated,
    and correction for this dilution should not be made.

        7.15 QC Check Sample - A QC Check Sample should be obtained from a source independent of
             the calibration standards. Ideally, this check sample would be a certified Standard
             Reference Material (SRM) containing the pesticides in known concentrations in a sample
             matrix similar to the matrix under test. The National Institute of Standards and Technology
             (NIST) in Gaithersburg, Maryland has SRMs, and the Institute for National Measurement
             Standards of the National Research Council of Canada in Ottawa has certified reference
             materials (CRMs), for pesticides in various matrices.

        7.16 Stability of solutions - Standard solutions used for quantitative purposes (Sections 7.8 -
             7.14) should be assayed periodically (e.g., every 6 months) against SRMs from NIST (if
             available), or certified reference materials  from a source that will attest to the authenticity
             and concentration, to assure that the composition and concentrations have not changed.

8.0    Sample collection, preservation, storage, and holding times

        8.1   Collect samples in amber glass containers  following conventional sampling practices
             (Reference  12); collect field and trip blanks as necessary to validate the sampling.

        8.2  Aqueous samples

             8.2.1   Samples that flow freely are collected as grab samples or in refrigerated bottles
                    using  automatic sampling equipment. Collect  1-L. If high concentrations of the
                    pesticides are expected,  collect a smaller volume (e.g., 100 mL) in addition to the
                    1-L sample.  Do not rinse the bottle with sample before collection.

             8.2.2   If residual chlorine is present, add 80 mg sodium thiosulfate per liter of water. Any
                    method suitable for field use may be employed to test for residual chlorine (Reference
                    9).

             8.2.3   Maintain aqueous samples in the dark at <6C  from the time of collection until
                    receipt at the laboratory (see 40 CFR 136.6(e), Table II).  If the sample will be
                    frozen, allow room for expansion.

             8.2.4   If the  sample will not be analyzed within 72 hours, adjust the pH to a range of 5.0
                    to 9.0 with sodium hydroxide or sulfuric acid solution. Record the volume of acid
                    or base used.

        8.3  Solid, mixed-phase, semi-solid,  and oily samples, excluding tissue.

             8.3.1   Collect samples as grab  samples using wide-mouth jars.  Collect a sufficient
                    amount of wet material to produce a minimum of 20 g of solids.
                                                                                            25

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Method 1699                                                             December 2007

             8.3.2  Maintain solid, semi-solid, oily, and mixed-phase samples in the dark at <6C from
                    the time of collection until receipt at the laboratory. Store solid, semi-solid, oily,
                    and mixed-phase samples in the dark at less than -10C.

        8.4  Fish and other tissue samples

             8.4.1  Fish may be cleaned, filleted, or processed in other ways in the field, such that the
                    laboratory may expect to receive whole fish, fish fillets, or other tissues for
                    analysis.

             8.4.2  Collect fish, wrap in aluminum foil, and maintain at <6C from the time of
                    collection until receipt at the laboratory, to a maximum time of 24 hours. If a
                    longer transport time is necessary, freeze the sample. Ideally,  fish should be frozen
                    upon collection and shipped to the laboratory under dry ice.

             8.4.3  Freeze tissue samples upon receipt at the laboratory and maintain in the dark at less
                    than -10C until prepared. Maintain unused sample in the dark at less than -10C.

             8.4.4  Store sample extracts in the dark at less than -10C until analyzed.

        8.5  Holding times - See 40 CFR 136.3(e) Table II

             8.5.1  Aqueous samples - Extract within 7 days of collection, and analyze within 40 days
                    of extraction.

             8.5.2  Solid, mixed-phase, semi-solid, tissue, and oily samples - Extract and analyze
                    within 1 year of collection.  If a sample is to be stored for more than 14 days, and
                    results are to be reported in solids units, either hermetically seal the sample
                    container or determine the moisture content upon receipt and immediately prior to
                    analysis. Adjust the final concentration based on the original moisture content.

9.0    Quality assurance/quality control

        9.1   Each laboratory that uses this Method is required to operate a formal quality assurance
             program (Reference 14). The minimum requirements of this program  consist of an initial
             demonstration of laboratory capability, analysis of samples spiked with labeled compounds
             to evaluate and  document data quality, and analysis of standards and blanks as tests of
             continued performance.  Laboratory performance is compared to established performance
             criteria to determine if the results of analyses meet the performance characteristics of the
             Method.

             If the Method is to be applied to a sample matrix other than water (e.g., soils, filter cake,
             compost, tissue) the most appropriate alternate reference  matrix (Sections 7.6.2 - 7.6.5  and
             7.15) is substituted for the reagent water matrix (Section  7.6.1) in all performance tests.
26

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Method 1699                                                              December  2007

             9.1.1   The laboratory must make an initial demonstration of the ability to generate
                     acceptable precision and recovery with this Method. This demonstration is given
                     in Section 9.2.

             9.1.2   In recognition of advances that are occurring in analytical technology, and to
                     overcome matrix interferences, the laboratory is permitted certain options to
                     improve separations or lower the costs of measurements. These options include
                     alternate extraction, concentration, and cleanup procedures, and changes in
                     columns and detectors (see also 40 CFR 136.6). Alternate determinative
                     techniques, such as the substitution of spectroscopic or immuno-assay techniques,
                     and changes that degrade Method performance, are not allowed. If an analytical
                     technique other than the techniques specified in this Method is used, that technique
                     must have a specificity equal to or  greater than the specificity of the techniques in
                     this Method for the analytes of interest.

                     9.1.2.1   Each time a modification is made to this Method, the laboratory is
                              required to repeat the procedure  in Section 9.2.  If the detection limit of
                              the Method will be affected by the change, the laboratory is required to
                              demonstrate that the MDLs (40 CFR Part 136, Appendix B) are lower
                              than one-third the regulatory compliance level or the MDLs in this
                              Method, whichever are greater.  If calibration will be affected by the
                              change, the instrument must be recalibrated per Section 10.  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., labeled compound recovery).

                     9.1.2.2   The laboratory is required to maintain records of modifications made to
                              this Method.  These records include the following, at a minimum:

                              9.1.2.2.1  The names, titles, addresses,  and telephone numbers 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.

                              9.1.2.2.2  A listing of pollutant(s) measured, by name and CAS
                                         Registry number.

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

                              9.1.2.2.4  Results from all quality control (QC) tests comparing the
                                         modified method to  this Method,  including:

                                         a) Calibration (Section 10)
                                         b) Calibration verification (Section 15.3)
                                         c) Initial precision and recovery (Section 9.2)
                                         d) Labeled compound recovery (Section 9.3)

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Method 1699                                                             December 2007

                                         e)  Analysis of blanks (Section 9.5)
                                         f)  Accuracy assessment (Section 9.4)

                              9.1.2.2.5   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 11)
                                         f)  Extract volume prior to each cleanup step  (Section 13)
                                         g)  Extract volume after each cleanup step (Section 13)
                                         h)  Final extract volume prior to injection (Section 14)
                                         i)  Injection volume (Sections  10.3 and 14.3)
                                         j)  Dilution data, differentiating between dilution of a
                                            sample or extract (Section 17.5)
                                         k)  Instrument and operating conditions
                                         1)  Column (dimensions, liquid phase, solid support, film
                                            thickness, etc)
                                         m) Operating conditions (temperatures, temperature
                                            program, flow rates)
                                         n)  Detector (type, operating conditions, etc)
                                         o)  Chromatograms, printer tapes, and other recordings of
                                            raw data
                                         p)  Quantitation reports, data system outputs,  and other data
                                            to link the raw data to  the results reported

                     9.1.2.3   Alternate HRGC columns and column systems - If a column or column
                              system alternate to those specified in this Method is used, that column or
                              column system must meet the requirements in Section 6.9.1.

             9.1.3   Analyses of method blanks are required to demonstrate freedom from
                     contamination (Section 4.3). The procedures and criteria for analysis of a method
                     blank are given in Sections 9.5 and 15.7.

             9.1.4   The laboratory must spike all samples with labeled compounds to monitor Method
                     performance.  This test is described in Section 9.3. When results of these spikes
                     indicate atypical Method performance for samples, the samples are diluted to bring
                     Method performance within  acceptable limits. Procedures for dilution are given in
                     Section 17.5.

             9.1.5   The laboratory must, on an ongoing basis, demonstrate through calibration verifica-
                     tion and the analysis of the ongoing precision and recovery standard  (OPR) and


28

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Method 1699                                                              December 2007

                    blanks that the analytical system is in control. These procedures are given in
                    Sections 15.1 through 15.7.

             9.1.6  The laboratory should maintain records to define the quality of data generated.
                    Development of accuracy statements is described in Sections 9.4 and 15.6.4.

        9.2  Initial precision and recovery (IPR) - To establish the ability to generate acceptable
             precision and recovery, the laboratory must perform the following operations:

             9.2.1  For low solids (aqueous) samples, extract, concentrate, and analyze four 1-L
                    aliquots of reagent water spiked with 1 mL each of the Native spiking solution
                    (Section 7.11) and the Labeled spiking solution (Section 7.12), according to the
                    procedures in Sections 11 through 18.  For an alternate sample matrix, four aliquots
                    of the alternate reference matrix (Sections 7.6.1 - 7.6.5) are used. All sample
                    processing steps that are to be used for processing samples, including preparation
                    (Section 11), extraction (Section 12), and cleanup (Section 13), must be included in
                    this test.

             9.2.2  Using results of the set of four analyses, compute the average percent recovery (X)
                    of the extracts and the relative standard deviation (RSD) of the concentration for
                    each compound, by isotope dilution for pesticides with a labeled analog, and by
                    internal standard for pesticides without a labeled analog and for the labeled
                    compounds.

             9.2.3  For each pesticide and labeled compound, compare RSD and X with the
                    corresponding limits for initial precision and recovery in Table 5.  If RSD and X
                    for all compounds meet the acceptance  criteria, system performance is acceptable
                    and analysis of blanks and samples may begin.  If, however, any individual RSD
                    exceeds the precision limit or any individual X falls outside the range for recovery,
                    system performance is unacceptable for that compound.  Correct the problem and
                    repeat the test (Section 9.2).

        9.3  To assess Method performance on the sample matrix, the laboratory must spike  all samples
             with the Labeled spiking solution (Section 7.12).

             9.3.1  Analyze each sample according to the procedures in Sections 11 through 18.

             9.3.2  Compute the percent recovery of the labeled pesticides using the internal standard
                    method (Section 17.2).

             9.3.3  The recovery of each labeled compound must be within the limits in Table 5. If the
                    recovery of any compound falls outside of these limits, Method performance is
                    unacceptable for that compound in that sample. Additional cleanup procedures
                    must then be employed to attempt to bring the recovery within the normal range. If
                    the recovery cannot be brought within the normal range after all cleanup
                    procedures have been employed, water samples are diluted and smaller amounts of
                    soils, sludges, sediments, and other matrices are analyzed per Section 18.

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Method 1699                                                              December 2007
        9.4  It is suggested but not required that recovery of labeled compounds from samples be
             assessed and records maintained.

             9.4.1   After the analysis of 30 samples of a given matrix type (water, soil, sludge, pulp,
                     etc.) for which the labeled compounds pass the tests in Section 9.3, compute the
                     average percent recovery (R) and the standard deviation of the percent recovery
                     (SR) for the labeled compounds only.  Express the assessment as a percent recovery
                     interval from R ! 2SR to R + 2SR for each matrix.  For example, if R = 90% and SR
                     = 10% for five analyses of pulp, the recovery interval is expressed as 70 to 110%.

             9.4.2   Update the accuracy assessment for each labeled compound in each matrix on a
                     regular basis (e.g., after each five to ten new measurements).

        9.5  Method blanks - A reference matrix Method blank is analyzed with each sample batch
             (Section 4.3) to demonstrate freedom from contamination. The matrix for the Method
             blank must be similar to the sample matrix for the batch, e.g., a 1-L reagent water blank
             (Section 7.6.1), high-solids reference matrix blank (Section 7.6.2), paper matrix blank
             (Section 7.6.3); tissue  blank (Section 7.6.4), or alternate reference matrix blank (Section
             7.6.5).

             9.5.1   Spike 1.0 mL  each of the Labeled spiking solution (Section 7.12) into the Method
                     blank, according to the procedures in Sections 11  through  18. Prepare, extract,
                     clean up, and concentrate the Method blank.  Analyze the blank immediately after
                     analysis of the OPR (Section 15.6) to demonstrate freedom from contamination.

             9.5.2   If any pesticide (Table 1) is found in the  blank at greater than the minimum level
                     (Table 1) or one-third the regulatory compliance limit, whichever is greater; or if
                     any potentially interfering compound is found in the blank at the minimum level
                     for each pesticide in Table 1 (assuming a response factor of 1 relative to the
                     quantitation reference in Table 2 for a potentially interfering compound; i.e.,  a
                     compound not listed in this Method), analysis of samples must be halted until the
                     sample batch is re-extracted and the extracts re-analyzed, and the blank associated
                     with the sample batch shows no evidence of contamination at these levels.  All
                     samples must  be associated with an uncontaminated Method blank before the
                     results for those samples may be reported or used for permitting or regulatory
                     compliance purposes.

        9.6  QC Check Sample - Analyze the QC Check Sample (Section 7.15) periodically to assure
             the accuracy of calibration standards and the overall reliability of the analytical process. It
             is suggested that the QC Check Sample be analyzed at least quarterly.

        9.7  The specifications contained in this Method can be met if the apparatus used is calibrated
             properly and then maintained in a calibrated state.  The standards used for calibration
             (Section 10), calibration verification (Section 15.3), and for initial  (Section 9.2) and
             ongoing (Section 15.6) precision and recovery should be identical, so that the most precise
             results will be obtained. A GC/HRMS instrument will provide the most reproducible

30

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Method 1699
                           December  2007
             results if dedicated to the settings and conditions required for determination of pesticides by
             this Method.
       9.8   Depending on specific program requirements, field replicates may be collected to determine
             the precision of the sampling technique, and spiked samples may be required to determine
             the accuracy of the analysis when the internal standard method is used.
10.0  Calibration
       10.1  Establish the operating conditions necessary to meet the retention times (RTs) and relative
             retention times (RRTs) for the pesticides in Table 2.
             10.1.1  Suggested operating conditions:
              GC conditions
              Injector
              Carrier gas
              Injector temperature
              Maximum column temperature
              GC Temperature program
              Initial temperature and hold
              Initial ramp
              Second hold
              Second ramp
              Third hold
              Third ramp
              Final hold
              Interface temperature

              Mass spectrometer conditions
              Source temperature
              Electron energy
              Trap current
              Mass resolution
              Detector potential
Split/splitless, 2 min
Helium @ 200 kPa
180 - 220C or temperature programmed
300C

50C for 1 minute
50 -  180C @ 10C per minute
180C for 0 minute
180-200C@1.5C per minute
200C for 2 minutes
200 - 295C @ 6C per minute
295C for 1 minutes or until methoxychlor elutes
290C


250C
35 eV
500 - 900 OA
8000
340 - 400 V

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Method 1699                                                             December 2007

                    10.1.1.1 All portions of the column that connect the GC to the ion source should
                             remain at or above the interface temperature during analysis to preclude
                             condensation of less volatile compounds.

                    10.1.1.2 The GC conditions may be optimized for compound separation and
                             sensitivity. Once optimized, the same GC conditions must be used for
                             the analysis of all standards, blanks, IPR and OPR standards, and
                             samples.

             10.1.2 Retention time calibration for the native and labeled pesticides

                    10.1.2.1 Inject the CS-4 calibration standard (Section 7.10 and Table 4).
                             Establish the beginning and ending retention times for the scan
                             descriptors in Table 6. Scan descriptors other than those listed in Table 6
                             may be used provided the MLs in Table 1 are met. Store the retention
                             time (RT) and relative retention time (RRT) for each compound in the
                             data system.

                    10.1.2.2 The absolute retention time of methoxychlor must exceed 39 minutes on
                             the DB-17 column; otherwise, the GC temperature program must be
                             adjusted and this test repeated until the minimum retention time criterion
                             is met. If a GC column or column system alternate to the DB-17 column
                             is used,  a similar minimum retention time specification must be
                             established for the alternate column or column systems so that
                             interferences that may be encountered in environmental samples will be
                             resolved from the analytes of interest. This specification is deemed to be
                             met if the retention time of methoxychlor is greater than 39 minutes on
                             such alternate column.

        10.2 Mass spectrometer (MS) resolution

             10.2.1 Using PFK (or other reference substance) and a molecular leak, tune the instrument
                    to meet the minimum required resolving power of 8,000 (10% valley) at m/z
                    280.9825 or other significant PFK fragment in the range of 250 -  300. For each
                    descriptor (Table 6), monitor and record the resolution and exact m/z's of three to
                    five reference peaks covering the mass range of the descriptor.  The level of PFK
                    (or other reference substance) metered into the HRMS during analyses should be
                    adjusted so that the amplitude of the most intense selected lock-mass m/z signal
                    (regardless of the descriptor number) does not exceed 10% of the full-scale
                    deflection for a given set of detector parameters. Under those conditions,
                    sensitivity changes that might occur during the analysis can be more effectively
                    monitored.

    Note: Different lots and types of PFK can contain varying levels of contamination,  and excessive
    PFK (or other reference substance) may cause noise problems and contamination of the ion source
    necessitating increased frequency of source cleaning.


32

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Method 1699                                                              December 2007

             10.2.2 The analysis time for the pesticides may exceed the long-term mass stability of the
                    mass spectrometer.  Because the instrument is operated in the high-resolution
                    mode, mass drifts of a few ppm (e.g., 5 ppm in mass) can have serious adverse
                    effects on instrument performance. Therefore, mass-drift correction is mandatory
                    and a lock-mass m/z from perfluorokerosene (PFK) or other reference substance is
                    used for drift correction. The lock-mass m/z is dependent on the exact m/z's
                    monitored within each descriptor, as shown in Table 6. The deviation between
                    each monitored exact m/z and the theoretical m/z (Table 6) must be less than 5
                    ppm.

             10.2.3 Obtain a selected ion current profile (SICP) at the two exact m/z's specified in
                    Table 6 and at 38,000 resolving power for each native and labeled pesticide.
                    Because of the extensive mass range covered in each function, it may not be
                    possible to maintain 8,000 resolution throughout the mass range during the
                    function.  Therefore, resolution must be 36,000 throughout the mass range and
                    must be 38,000 in the center of the mass range for each function.

             10.2.4 If the HRMS has the capability to monitor resolution during the analysis, it is
                    acceptable to terminate the analysis when the resolution falls below the minimum
                    (Section 10.2.1 and 10.2.3) to save re-analysis time.

        10.3 Ion abundance ratios, minimum levels, and signal-to-noise ratios during calibration.
             Choose an injection volume of either 1 or 2 (iL, consistent with the capability of the
             HRGC/HRMS instrument. Inject a 1 or 2 (iL aliquot of the CS-1 calibration solution
             (Table 4) using the GC conditions in Section 10.1.1.

             10.3.1 Measure the SICP areas for each  pesticide, and compute the ion abundance ratios at
                    the exact m/z's specified in Table 6.  Compare the computed ratio to the theoretical
                    ratio given in Table 6.

                    10.3.1.1  The exact m/z's to be monitored in each descriptor are shown  in Table 6.
                              Each group or descriptor must be monitored in succession as  a function
                              of GC retention time to ensure that the pesticides are detected.
                              Additional m/z's may be monitored in each descriptor, and the m/z's  may
                              be divided among more than the descriptors listed in Table 6,  provided
                              that the laboratory is able to monitor the m/z's of all pesticides that may
                              elute from the GC in a  given RT window.

                    10.3.1.2  The mass spectrometer must be operated in a mass-drift correction mode,
                              using PFK (or other reference substance) to provide lock m/z's.  The lock
                              mass for each group of m/z's is shown in Table 6. Each lock mass must
                              be monitored and must not vary by more than V  20% throughout its
                              respective retention time window. Variations of lock mass by more than
                              20% indicate the presence of co-eluting interferences that raise the
                              source pressure and may significantly reduce the sensitivity of the mass
                              spectrometer. Re-injection of another aliquot of the sample extract may
                              not resolve the problem and additional cleanup of the extract may be
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Method 1699                                                              December 2007

                              required to remove the interference. A lock mass interference or
                              suppression in a retention time region in which pesticides and labeled
                              compounds do not elute may be ignored.

             1 0.3.2  All pesticides and labeled compounds in the CS-1 standard must be within the QC
                     limits in Table 6 for their respective ion abundance  ratios; otherwise, the mass
                     spectrometer must be adjusted and this test repeated until the m/z ratios fall within
                     the limits specified.  If the adjustment alters the resolution of the mass
                     spectrometer, resolution must be verified (Section 10.2.1) prior to repeat of the test.

             1 0.3.3  Verify that the HRGC/HRMS instrument meets the minimum levels (MLs) in
                     Table 1. The peaks representing the pesticides and  labeled compounds in the CS-1
                     calibration standard must have signal-to-noise ratios (S/N) 3 3; otherwise, the mass
                     spectrometer must be adjusted and this test repeated until the minimum levels in
                     Table 1 are met.

        10.4 Calibration by isotope dilution - Isotope dilution is used for calibration of the native
             pesticides for which a labeled analog is available. The reference compound for each  native
             compound is its labeled analog, as listed in Table 2. A 6-point calibration encompassing
             the concentration range is prepared for each native compound.

             1 0.4.1  For the pesticides determined by isotope dilution, the  relative response (RR)
                     (labeled to native) vs. concentration in the calibration solutions (Table 4) is
                     computed over the calibration range according to the procedures described below.
                     Five calibration points are employed for less-sensitive HRMS instruments (e.g.,
                     VG 70); five or six points may be employed for more -sensitive instruments (e.g.,
                     Micromass Autospec Ultima).

             1 0.4.2  Determine the response  of each pesticide relative to its labeled analog using the
                     area responses of both the primary and secondary exact m/z's specified in Table 6,
                     for each calibration standard. Use the labeled compounds listed in Table 2 as the
                     quantitation reference and the two exact m/z's listed in Table 6 for quantitation.
                     The areas at the two exact m/z's for the compound is summed and divided by the
                     summed area of the two exact m/z's for the quantitation reference.

    Note: Both exact m/z's are used as reference to reduce the effect of an interference at a single  m/z.
    Other quantitation references and procedures may be used provided that the results produced are as
    accurate as results produced by the quantitation references and procedures described in this Section.

             1 0.4.3  Calibrate the native compounds with a labeled analog using the following equation:

                                             RR =  (A
                     Where:
                             A \n and A2n  =    The areas of the primary and secondary m/z's for the
                                               pesticide
34

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Method 1699                                                              December 2007

                             A\i and A2t   =   The areas of the primary and secondary m/z's for the
                                              labeled compound.
                                     Ci   =   The concentration of the labeled compound in the
                                              calibration standard (Table 4).
                                     Cn   =   The concentration of the native compound in the
                                              calibration standard (Table 4).

             10.4.4  To calibrate the analytical system by isotope dilution, inject calibration standards
                     CS-2 through CS-6 (Section 7.10 and Table 4) for a less sensitive instrument (e.g.
                     VG 70) or CS-1 through CS-6 for a more sensitive instrument (e.g., Micromass
                     Autospec Ultima).  Use a volume identical to the volume chosen in Section 10.3,
                     the procedure in Section 14, and the conditions in Section  10.1.1.  Compute and
                     store the relative response (RR) for each pesticide at each concentration.  Compute
                     the average  (mean) RR and the RSD of the 6 RRs.

             10.4.5  Linearity - If the RRs for any pesticide are constant (less than 20% RSD), the
                     average RRmay be used for that pesticide; otherwise, the complete calibration
                     curve for that pesticide must be used over the calibration range.

        10.5 Calibration by internal standard - Internal standard calibration is applied to determination
             of the native pesticides for which a labeled compound is not available, and to determination
             of the labeled compounds for performance tests and intra-laboratory statistics (Sections 9.4
             and 15.6.4). The reference compound for each compound is listed in Table 2. For the
             labeled compounds, calibration is performed at a single concentration using data from the 6
             points in the calibration (Section 10.4).

             10.5.1  Response factors - Internal standard calibration requires the determination  of
                     response factors (RF) defined by the following equation:

                                             RF=  (A
                                                    (Alls+A2ls)Cs

                    Where:
                             A \s and A2S   =   The areas of the primary and secondary m/z's for the
                                              pesticide.
                             A\is and A2is  =   The areas of the primary and secondary m/z's for the
                                              internal standard.
                                      Cis  =   The concentration of the internal standard (Table 4).
                                      Cs  =   The concentration of the compound in the calibration
                                              standard (Table 4).

             10.5.2 To calibrate the analytical system for pesticides that do not have a labeled analog,
                    and for the labeled compounds, use the data from the 6-point calibration (Section
                     10.4.4 and Table 4).

             10.5.3 Compute and store the  response factor (RF) for all native pesticides that do not
                    have a labeled analog and for the labeled compounds. Use the labeled compounds

                                                                                              35

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Method 1699                                                             December 2007

                    listed in Table 2 as the quantitation reference and the two exact m/z's listed in
                    Table 6 for quantitation. For example, the areas at the two exact m/z's for the
                    compound is summed and divided by the summed area of the two exact m/z's for
                    the quantitation reference.

             10.5.4 Compute and store the response factor (RF) for the labeled compounds using the
                    Labeled injection internal standard as the quantitation reference, as given in Table
                    2.

             10.5.5 Linearity - If the RFs for any pesticide are constant (less than 35% RSD), the
                    average RF may be used for that pesticide; otherwise, the complete calibration
                    curve for that pesticide must be used over the calibration range.

        10.6 Endrin/4,4'-DDT breakdown - This test is run after calibration (Section  10.4 and 10.5) or
             calibration verification (Section 15.3) to assure that the labile pesticides  do not decompose
             in the GC.

             10.6.1 Inject the endrin/4,4'-DDT breakdown solution (Section 7.13) using the same
                    volume chosen in Section 10.3.

             10.6.2 Measure and sum the peak areas for both exact m/z's separately for 4,4'-DDD, 4,4'-
                    DDE, 4,4'-DDT, endrin, endrin aldehyde, and endrin ketone using the calibration
                    data from Section 10.4.

                    10.6.2.1  Add the summed peak areas for endrin aldehyde and endrin ketone and
                              separately add the peak areas for 4,4'-DDD and 4,4'-DDE.

                    10.6.2.2  Calculate the endrin and 4,4'-DDT breakdown as follows:

                    Endrin breakdown  (percent) = (areas for endrin aldehyde + endrin ketone) xlOO
                                                       areas for endrin

                    4,4'-DDTbreakdown (percent) = (areas for 4.4'-DDD + 4,4'-DDE) xWO
                                                           areas for 4,4'-DDT

                    10.6.2.3  If the breakdown of endrin or 4,4'-DDT exceeds 20 percent, endrin or
                              4,4'-DDT  is decomposing.  If decomposition greater than 20 percent of
                              either endrin or 4,4'-DDT occurs, clean and recondition the injector,
                              break off a short section of the inlet end of the column, or alter the GC
                              conditions to reduce the decomposition to where the 20 percent  criterion
                              is met (see Section 6.9.1.2).

11.0  Sample preparation

        11.1 Sample preparation involves modifying the physical form of the sample  so that the
             pesticides can be extracted  efficiently. In general, the samples must be in a liquid form or

36

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Method 1699                                                              December 2007

             in the form of finely divided solids in order for efficient extraction to take place. Table 7
             lists the phases and suggested quantities for extraction of various sample matrices.

             For samples known or expected to contain high levels of the pesticides, the smallest sample
             size representative of the entire sample should be used (see Section 18).  For all samples,
             the blank and IPR/OPR aliquots must be processed through the same steps as the sample to
             check for contamination and losses in the preparation processes.

             11.1.1  For samples that contain particles, percent solids and particle size are determined
                     using the procedures in  Sections 11.2 and 11.3, respectively.

             11.1.2  Aqueous samples - Because the pesticides may be bound to suspended particles,
                     the preparation of aqueous samples is dependent on the solids content of the
                     sample.

                     11.1.2.1  Aqueous samples containing one percent solids or less are prepared per
                               Section 11.4 and extracted directly using one  of the extraction
                               techniques in Section 12.2.

                     11.1.2.2  For aqueous samples containing greater than one percent solids, a
                               sample aliquot sufficient to provide 10 g of dry solids is  used, as
                               described in Section 11.5.

             11.1.3  Solid Samples - Solid samples are prepared using the procedure described in
                     Section  11.5 followed by extraction using the SDS  procedure in Section 12.3.

             11.1.4  Multi-phase samples - The phase(s) containing the pesticides is separated from the
                     non-pesticide phase using pressure filtration and centrifugation, as described in
                     Section  11.6. The pesticides will be in the organic phase in a multi-phase sample
                     in which an organic phase exists.

             11.1.5  Procedures for grinding, homogenization, and blending of various sample phases
                     are given in Section  11.7.

             11.1.6  Tissue samples - Preparation procedures for fish and other tissues are given in
                     Section  11.8.

        11.2 Determination of percent suspended solids

    Note: 777/5 aliquot is used for determining the solids content of the sample, not for pesticide
    determination.

             11.2.1  Aqueous liquids and multi-phase samples consisting of mainly an aqueous phase.

                     11.2.1.1  Desiccate and weigh a GF/D filter (Section 6.5.3) to three significant
                               figures.


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Method 1699                                                              December 2007

                     11.2.1.2  Filter 10.0 V0.02 mL of well-mixed sample through the filter.

                     11.2.1.3  Dry the filter a minimum of 12 hours at 110  5C and cool in a
                               desiccator.

                     11.2.1.4  Calculate percent solids as follows:

                     % Solids = Weight of sample aliquot after drying (g) - weight of filter (g) xlOO
                                                    Wg

             11.2.2  Non-aqueous liquids, solids, semi-solid samples, and multi-phase samples in which
                     the main phase is not aqueous; but not tissues.

                     11.2.2.1  Weigh 5 to  10 g of sample to three significant figures in a tared beaker.

                     11.2.2.2  Dry a minimum of 12 hours at 110 V5C, and cool in a desiccator.

                     11.2.2.3  Calculate percent solids as follows:

                     % Solids = Weight of sample aliquot after drying   x 100
                               Weight of sample aliquot before drying

        11.3 Estimation of particle size

             11.3.1  Spread the dried sample from Section 11.2.1.3 or 11.2.2.2 on apiece of filter paper
                     or aluminum foil in a fume hood or glove box.

             11.3.2  Estimate the size of the particles in the sample.  If the size of the largest particles is
                     greater than 1 mm, the particle size must be reduced to 1 mm or less prior to
                     extraction using the procedures in Section 11.7.

        11.4 Preparation of aqueous samples containing one percent suspended solids or less.

             11.4.1  Aqueous samples containing one percent suspended solids or less are prepared
                     using the procedure below and extracted using the one of the extraction techniques
                     in Section  12.2.

             11.4.2  Preparation of sample and QC aliquots

                     11.4.2.1  Mark the original level of the sample on the sample bottle for reference.
                               Weigh the sample plus bottle to V 1 g.

                     11.4.2.2  Spike 1.0 mL of the  Labeled pesticide spiking solution (Section 7.12)
                               into the sample bottle.  Cap the bottle and mix the sample by shaking.
                               Allow the sample to equilibrate for 1 to 2 hours, with occasional
                               shaking.

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Method 1699                                                              December 2007
                     11.4.2.3  For each sample or sample batch (to a maximum of 20 samples) to be
                               extracted during the same 12-hour shift, place two 1.0-L aliquots of
                               reagent water in clean sample bottles or flasks.

                     11.4.2.4  Spike 1.0 mL of the Labeled pesticide spiking solution (Section 7.12)
                               into both reagent water aliquots.  One of these aliquots will serve as the
                               Method blank.

                     11.4.2.5  Spike 1.0 mL of the Native pesticide spiking solution (Section 7.11)
                               into the remaining reagent water aliquot. This aliquot will serve as the
                               OPR (Section 15.6).

                     11.4.2.6  For extraction using SPE, add 5 mL of methanol to the sample and  QC
                               aliquots. Cap and shake the sample and QC aliquots to mix thoroughly,
                               and proceed to  Section 12.2 for extraction.

        11.5 Preparation of samples containing  greater than one percent solids.

             11.5.1  Weigh a well-mixed aliquot of each  sample  (of the same matrix type) sufficient to
                     provide 10 g of dry solids  (based on the solids determination in Section 11.2) into a
                     clean beaker or glass jar, to a maximum of 1 L of sample.

             11.5.2  Spike 1.0 mL of the Labeled pesticide spiking solution (Section 7.12) into the
                     sample.

             11.5.3  Prepare the blank and OPR aliquots per Sections 11.4.2.3 - 11.4.2.5.

             11.5.4  Stir or tumble and equilibrate the aliquots for 1 to 2 hours.

             11.5.5  Decant excess water.  If necessary to remove water, filter the sample through a
                     glass-fiber filter and discard the aqueous liquid.

             11.5.6  If particles >1 mm are present in the sample (as determined in Section 11.3.2),
                     spread the sample on clean aluminum foil in a hood.  After the sample is dry, grind
                     to reduce the particle size (Section 11.7).

             11.5.7  Extract the sample and QC aliquots using the SDS procedure in Section 12.3.1.

        11.6 Multi-phase samples, including high solids municipal sludge samples

             11.6.1  Using the percent solids determined  in Section  11.2.1.4 or 11.2.2.3, determine  the
                     volume of sample that will provide 10 g of solids, up to 1 L of sample.

             11.6.2  Spike 1.0 mL of the Labeled pesticide spiking solution (Section 7.12) into the
                     amount of sample determined in Section 11.6.1, and into the OPR and blank.

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Method 1699                                                             December 2007

             11.6.3 Prepare the blank and OPRaliquots per Sections 11.4.2.3 - 11.4.2.5.

             11.6.4 Pressure filter the sample, blank, and OPR through Whatman GF/D glass-fiber
                    filter paper (Section 6.5.3).  If necessary to separate the phases and/or settle the
                    solids, centrifuge these aliquots prior to filtration. Discard any aqueous phase (if
                    present). Remove any non-aqueous liquid present and reserve the maximum
                    amount filtered from the sample (Section 11.5.5) or 10 g, whichever is less, for
                    combination with the solid phase (Section 12.3.1.5).

             11.6.5 If particles >1 mm are present in the sample (as determined in Section 11.3.2) and
                    the sample is capable of being dried, spread the sample and QC aliquots on clean
                    aluminum foil in a hood.  Observe the precaution in Section 5.3.1.

             11.6.6 After the aliquots are dry or if the sample cannot be dried, reduce the particle size
                    using the procedures in Section 11.7 and extract the reduced-size particles using the
                    SDS procedure in Section 12.3. If particles >1 mm are not present, extract the
                    particles and filter in the sample and QC aliquots directly using the SDS procedure
                    in Section 12.3.

        11.7 Sample grinding, homogenization, or blending - Samples with particle sizes greater than 1
             mm (as determined in Section 11.3.2) are subjected to grinding, homogenization,  or
             blending. The method of reducing particle size to less than 1 mm is matrix-dependent. In
             general, hard particles can be reduced by grinding with a mortar and pestle. Softer particles
             can be reduced by grinding in a Wiley mill or meat grinder, by homogenization, or in a
             blender.

             11.7.1 Each size-reducing preparation procedure on each matrix must be verified by
                    running the tests in Section 9.2 before the procedure is employed routinely.

             11.7.2 The grinding, homogenization, or blending procedures must be carried out in a
                    glove box or fume hood to prevent particles from contaminating the work
                    environment.

             11.7.3 Grinding - Certain papers and pulps, slurries, and amorphous solids can be ground
                    in a Wiley mill  or heavy duty meat grinder.  In some cases, reducing the
                    temperature of the sample to freezing or to dry ice or liquid nitrogen temperatures
                    can aid in the grinding process.  Grind the sample aliquots from  Sections  11.5.7 or
                    11.6.6 in a clean grinder.  Do not allow the sample temperature to exceed 50C.
                    Grind the blank and reference matrix aliquots using a clean grinder.

             11.7.4 Homogenization or blending - Particles that are not ground effectively, or particles
                    greater than 1 mm in size after grinding, can often be reduced in size by high speed
                    homogenization or blending.  Homogenize and/or blend the particles or filter from
                    Sections 11.5.7 or 11.6.6  for the sample, blank, and OPRaliquots.

             11.7.5 Extract the aliquots using the SDS procedure in Section 12.3.1.


40

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Method 1699                                                              December 2007

        11.8 Fish and other tissues - Prior to processing tissue samples, the laboratory must determine
             the exact tissue to be analyzed. Common requests for analysis offish tissue include whole
             fish-skin on, whole fish-skin removed, edible fish fillets (filleted in the field or by the
             laboratory), specific organs, and other portions. Once the appropriate tissue has been
             determined, the sample must be homogenized.

             11.8.1  Tissue homogenization

                     11.8.1.1   Samples are homogenized while still frozen, where practical.  If the
                               laboratory must dissect the whole fish to obtain the appropriate tissue
                               for analysis, the unused tissues may be rapidly refrozen and stored in a
                               clean glass jar for subsequent use.

                     11.8.1.2   Each analysis requires  10 g of tissue (wet weight). Therefore, the
                               laboratory should homogenize at least 20 g of tissue to allow for re-
                               extraction of a second aliquot of the same homogenized sample, if re-
                               analysis is required.  When whole fish analysis  is necessary, the entire
                               fish is homogenized.

                     11.8.1.3   Homogenize the sample in a tissue homogenizer (Section 6.3.3) or grind
                               in a meat grinder (Section 6.3.4). Cut tissue too large to feed into the
                               grinder into smaller pieces. To assure homogeneity, grind three times.

                     11.8.1.4   Transfer approximately 10 g (wet weight) of homogenized tissue  to a
                               clean, tared, 400- to 500-mL beaker.

                     11.8.1.5   Transfer the remaining homogenized tissue to a clean jar with a
                               fluoropolymer-lined lid. Seal the jar and store the tissue at less than
                               -10C. Return any tissue that was not homogenized to its original
                               container and store at less than -10C.

             11.8.2  Tissue QC aliquots

                     11.8.2.1   Prepare a Method blank by adding approximately 1-2 g of the oily
                               liquid reference matrix (Section 7.6.4) to a 400- to 500-mL beaker.
                               Record the weight to the nearest 10 mg.

                     11.8.2.2   Prepare an ongoing precision and recovery aliquot by adding 1-2  g of
                               the oily liquid reference matrix (Section  7.6.4) to a separate 400-  to
                               500-mL beaker. Record the weight to the nearest 10 mg.

             11.8.3  Spiking

                     11.8.3.1   Spike 1.0 mL of the Labeled pesticide spiking solution (Section 7.12)
                               into the sample, blank, and  OPR aliquot.
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Method 1699                                                              December 2007

                     11.8.3.2  Spike 1.0 mL of the Native spiking solution (Section 7.11) into the OPR
                               aliquot.

             11.8.4  Extract the aliquots using the Soxhlet procedure in Section 12.4.

12.0  Extraction  and  concentration

        12.1 Extraction procedures include: solid phase (Section 12.2.1), separatory runnel (Section
             12.2.2), or continuous liquid/liquid (Section 12.2.3) for aqueous liquids; Soxhlet/Dean-
             Stark (Section 12.3.1) for sludge, solids and filters; and Soxhlet extraction (Section 12.4)
             for tissues.

             Macro-concentration procedures include:  rotary evaporation (Section 12.6.1), heating
             mantle (Section 12.6.2), and Kuderna-Danish (K-D) evaporation (Section 12.6.3). Micro-
             concentration uses nitrogen evaporation (Section 12.7).

        12.2 Extraction of aqueous liquids - separatory or continuous liquid/liquid extraction.


             12.2.1  Solid-phase extraction of samples containing less than one percent solids

                     12.2.1.1   Disk preparation

                               12.2.1.1.1     Remove the test tube from the suction flask (Figure 4).
                                             Place an SPE disk on the base of the filter holder and
                                             wet with methylene chloride. While holding a GMF 150
                                             filter above the SPE disk with tweezers, wet the filter
                                             with methylene chloride and lay the  filter on the SPE
                                             disk, making sure that air is not trapped between the
                                             filter and disk.  Clamp the filter and  SPE disk between
                                             the 1-L glass reservoir and the vacuum filtration flask.

                               12.2.1.1.2    Rinse the sides of the reservoir with approx 15 mL of
                                             methylene chloride using a squeeze bottle or pipet.
                                             Apply vacuum momentarily until a few drops appear at
                                             the drip tip. Release the vacuum and allow the filter/disk
                                             to soak for approx one minute.  Apply vacuum and draw
                                             all of the methylene chloride through the filter/disk.
                                             Repeat the wash step with approx  15 mL of acetone and
                                             allow the filter/disk to air dry.

                     12.2.1.2  Sample extraction

                               12.2.1.2.1     Pre-wetthe disk by adding approx 20 mL of methanol to
                                             the reservoir. Pull most of the methanol through the
                                             filter/disk, retaining a layer of methanol approx 2 mm

42

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Method 1699                                                               December 2007

                                             thick on the filter.  Do not allow the filter/disk to go dry
                                             from this point until the extraction is completed.

                               12.2.1.2.2    Add approx 20 mL of reagent water to the reservoir and
                                             pull most through, leaving a layer approx 2 mm thick on
                                             the filter/disk.

                               12.2.1.2.3    Allow the sample (Section 11.4.2.6) to stand for 1-2
                                             hours, if necessary, to settle the suspended particles.
                                             Decant the clear layer of the sample, the blank (Section
                                             11.4.2.4), or IPR/OPR aliquot (Section  11.4.2.5) into its
                                             respective reservoir and turn on the vacuum to begin the
                                             extraction.  Adjust the vacuum to complete the
                                             extraction in no less than 10 minutes. For samples
                                             containing  a high concentration of particles (suspended
                                             solids), the extraction time may be an hour or longer.

                               12.2.1.2.4    Before all of the sample has been pulled through the
                                             filter/disk, add approx 50 mL of reagent water to the
                                             sample bottle, swirl to suspend the solids (if present),
                                             and pour into the reservoir. Pull through the filter/disk.
                                             Use additional reagent water rinses until all solids are
                                             removed.

                               12.2.1.2.5    Before all of the sample and rinses have been pulled
                                             through the filter/disk, rinse the sides of the reservoir
                                             with small portions of reagent water.

                               12.2.1.2.6    Partially dry the filter/disk under vacuum for approx 3
                                             minutes.
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Method 1699                                                              December  2007

                     12.2.1.3  Elution of the filter/disk

                               12.2.1.3.1     Release the vacuum, remove the entire
                                             filter/disk/reservoir assembly from the vacuum flask, and
                                             empty the flask. Insert a test tube for eluant collection
                                             into the flask. The test tube should have sufficient
                                             capacity to contain the total volume of the elution
                                             solvent (approx 50 mL) and should fit around the drip
                                             tip.  The drip tip should protrude into the test tube to
                                             preclude loss of sample from  spattering when vacuum is
                                             applied (see Figure 4). Re-assemble the
                                             filter/disk/reservoir assembly on the vacuum flask.

                               12.2.1.3.2    Wet the filter/disk with 4-5 mL of acetone.  Allow the
                                             acetone to spread evenly across the disk and soak for 15-
                                             20 seconds. Pull the acetone through the disk, releasing
                                             the vacuum when approx 1 mm thickness remains on the
                                             filter.

                               12.2.1.3.3    Rinse the sample bottle with approx 20 mL of methylene
                                             chloride and transfer to the reservoir. Pull approx half of
                                             the solvent through the filter/disk and release the
                                             vacuum.  Allow the filter/disk to soak for approx 1
                                             minute.  Pull all of the solvent through the disk. Repeat
                                             the bottle rinsing and elution step with another 20 mL of
                                             methylene chloride.  Pull all of the solvent through the
                                             disk.

                               12.2.1.3.4    Release the vacuum, remove the filter/disk/reservoir
                                             assembly, and remove the test tube containing the
                                             sample solution. Quantitatively transfer the solution to  a
                                             250-mL separatory funnel and proceed to Section 12.5
                                             for back-extraction.

             12.2.2  Separatory funnel extraction

                     12.2.2.1  Pour the spiked sample (Section 11.4.2.2) into a 2-L separatory funnel.
                               Rinse the bottle or flask twice with 5 mL of reagent water and add these
                               rinses to the separatory funnel.

                     12.2.2.2  Add 100 mL methylene chloride to the empty sample bottle. Cap the
                               bottle and shake 60 seconds to rinse the inner surface.  Transfer the
                               solvent to the separatory funnel, and extract the sample by  shaking the
                               funnel for 2 minutes with periodic venting. Allow the organic layer to
                               separate from the aqueous phase for a minimum of 10 minutes. If an
                               emulsion forms and is more than one-third the volume of the solvent
                               layer, employ mechanical techniques to complete the phase separation

44

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Method 1699                                                              December 2007

                               (see note below). Drain the methylene chloride extract through a
                               solvent-rinsed glass funnel and dry over anhydrous sodium sulfate
                               (Section 7.2.1) into an Erlenmeyer flask (1 L).

    Note: If an emulsion forms, the laboratory must employ mechanical techniques to complete the
    phase separation. The optimum technique depends upon the sample, but may include stirring,
    filtration through glass wool, use of phase separation paper, centrifugation,  use of an ultrasonic bath
    with ice, addition ofNaCl, or other physical methods. Alternatively, solid-phase (Section 12.2.1),
    CLLE (Section 12.2.3), or other extraction techniques may be used to prevent emulsion formation.
    Any alternative technique is acceptable so long as the requirements in Section 9.2 are met.	

                     12.2.2.3   Extract the water sample two more times with 100-mL portions of
                               methylene chloride.  Dry each portion over anhydrous sodium sulfate .
                               After the third extraction, rinse the separatory funnel with at least 20
                               mL of methylene chloride, and add to the three 100-mL portions of
                               methylene chloride.  Repeat this rinse at least twice. Allow the
                               methylene chloride extract to dry for 30 min. Transfer to a solvent-
                               rinsed concentration device (Section 12.6).

                     12.2.2.4   Add 1 mL of a toluene "keeper" to the extract and concentrate using one
                               of the macro-concentration procedures in Section 12.6, then proceed to
                               back extraction in Section 12.5.

             12.2.3 Continuous liquid/liquid extraction

                     12.2.3.1   Place 100-150 mL methylene chloride in each continuous extractor and
                               200-300 mL in each  distilling flask.

                     12.2.3.2   Pour the sample(s), blank, and QC aliquots into the extractors. Rinse
                               the sample containers with 50-100 mL methylene chloride and add to
                               the respective extractors. Include all solids in the extraction process.

                     12.2.3.3   Begin the extraction by heating the flask until the methylene chloride is
                               boiling. When properly adjusted, 1-2 drops of methylene chloride per
                               second will fall from the condenser tip into the water. Extract for 16-24
                               hours.

                     12.2.3.4   Remove the distilling flask, estimate and record the volume of extract
                               (to the nearest 100 mL), and pour the contents through a drying column
                               containing 7 to 10 cm of granular anhydrous sodium sulfate into the
                               concentration flask.  Rinse the distilling flask with 30-50 mL  of
                               methylene chloride and pour through the drying column.

                     12.2.3.5   Add 1 mL of a toluene "keeper" to the extract and concentrate using one
                               of the macro-concentration procedures in Section 12.6, then proceed to
                               back extraction in Section 12.5.


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Method 1699                                                               December 2007

        12.3 Extraction of solids - Solid or sludge samples_are extracted using a Soxhlet/Dean-Stark
             extractor (Section 12.3.1).

             12.3.1  Soxhlet/Dean-Stark extraction

                     12.3.1.1  Charge a clean extraction thimble (Section 6.4.2.2) with 5.0 g of
                               100/200 mesh silica (Section 7.5.1.1) topped with 100 g of quartz sand
                               (Section 7.3.2). Do not disturb the silica layer throughout the extraction
                               process.

                     12.3.1.2  Place the thimble in a clean extractor. Place 30 to 40 mL of toluene in
                               the receiver and 200 to 250 mL of toluene in the flask.

                     12.3.1.3  Pre-extract the glassware by heating the flask until the toluene is
                               boiling. When properly adjusted, 1 to 2 drops of toluene will fall per
                               second from the condenser tip into the receiver.  Extract the apparatus
                               for a minimum of 3 hours.

                     12.3.1.4  After pre-extraction, cool and disassemble the apparatus. Rinse the
                               thimble with toluene and allow to air dry.

                     12.3.1.5  Load the wet sample and/or filter from Sections 11.5.7, 11.6.6, or 11.7.5
                               and any non-aqueous liquid from Section 11.6.4 into the thimble and
                               manually mix into the sand layer with a clean metal spatula, carefully
                               breaking up any large lumps of sample.

                     12.3.1.6  Reassemble the pre-extracted SDS apparatus, and add a fresh charge of
                               300 mL 80:20 toluene:acetone to the receiver and reflux flask. Apply
                               power to the heating mantle to begin re-fluxing. Adjust the reflux rate
                               to match the rate of percolation through the sand and silica beds until
                               water removal lessens the restriction to toluene flow. Frequently check
                               the apparatus for foaming during the first 2 hours of extraction.  If
                               foaming occurs, reduce the reflux rate until foaming subsides. Soxhlet
                               extract for 12-24 hours.

                     12.3.1.7  Drain the water from the receiver at  1-2 hours and 8-9 hours, or sooner
                               if the receiver fills with water. After 12-24 hours cool and disassemble
                               the apparatus. Record the total volume of water collected.

                     12.3.1.8  Remove the distilling flask. Drain the water from the receiver and add
                               any toluene in the receiver to the extract in the flask.

                     12.3.1.9  Concentrate the extracts from particles to approximately 10 mL using
                               the rotary evaporator (Section 12.6.1) or heating mantle (Section
                               12.6.2), transfer to a 250-mL separatory funnel, and proceed with back-
                               extraction (Section 12.5).


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Method 1699                                                              December 2007

        12.4 Soxhlet extraction of tissue

    Note: This procedure includes determination of the lipid content of the sample (Section 12.4.9),
    using the same sample extract that is analyzed by GC/HRMS.  Alternatively, a separate sample
    aliquot may be used for the lipid determination. If a separate aliquot is used for GC/HRMS
    determination, use nitrogen to evaporate the main portion of the sample extract only to the extent
    necessary to effect the solvent exchange to n-hexane, so that loss of low molecular weight pesticides
    is avoided, i.e., it is not necessary to dry the main portion of the sample to constant weight (Section
    12.4.8).

             12.4.1  Add 30 to 40 g of powdered anhydrous sodium sulfate (Section 7.2.2) to each of
                     the beakers (Section 11.8.4) and mix thoroughly. Cover the beakers with
                     aluminum foil and dry until the mixture becomes a free-flowing powder (30
                     minutes minimum).  Remix prior to extraction to prevent clumping.

             12.4.2 Assemble and pre-extract the Soxhlet apparatus per Sections 12.3.1-12.3.1.4,
                     except use methylene chloride for the pre-extraction and rinsing and omit the
                     quartz sand.

             12.4.3 Re-assemble the pre-extracted Soxhlet apparatus and add a fresh charge of
                     methylene chloride to the reflux flask.

             12.4.4 Transfer the sample/sodium sulfate mixture (Section  12.4.1) to the Soxhlet thimble,
                     and install the thimble in the Soxhlet apparatus.

             12.4.5 Rinse the beaker with several portions of solvent and add to the thimble.  Fill the
                     thimble/receiver with solvent. Extract for 18-24 hours.

             12.4.6 After extraction, cool and disassemble the apparatus.

             12.4.7 Quantitatively transfer the extract to a macro-concentration device (Section 12.6)
                     and concentrate to near dryness. Set aside the concentration apparatus for re-use.

             12.4.8 Complete the removal of the solvent using the nitrogen blowdown procedure
                     (Section 12.7) and a water bath temperature of 60C.  Weigh the receiver, record
                     the weight, and return the receiver to the blowdown apparatus, concentrating the
                     residue until a constant weight is obtained.

             12.4.9 Percent lipid determination

                     12.4.9.1  Re-dissolve the  residue in the  receiver in hexane.

                     12.4.9.2  Transfer the residue/hexane to the anthropogenic isolation column
                               (Section  13.6); retaining the boiling chips in the concentration
                               apparatus.  Use several rinses to assure that all material is transferred. If
                               necessary, sonicate or heat the receiver slightly to assure that all


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Method 1699                                                              December 2007

                               material is re-dissolved.  Allow the receiver to dry. Weigh the receiver
                               and boiling chips.

                     12.4.9.3  Calculate the lipid content to the nearest three significant figures as
                               follows:

                                     Percent lipid = Weight of residue (g)  xlOO
                                                    Weight of tissue (g)

                     12.4.9.4  The laboratory should determine the lipid content of the blank, IPR, and
                               OPRto assure that the extraction system is working effectively.

        12.5 Back-extraction with base and acid

    Note: Some pesticides may be decomposed by acid or base.  If acid or base back-extraction is
    employed, the laboratory must evaluate the strengths of the acid and base solutions, and the exposure
    times, to preclude decomposition.

             12.5.1  Back-extraction may not be necessary for some samples, and back-extraction with
                     strong acid and/or base with long contact times may destroy some pesticides. For
                     some samples, the presence of color in the extract may indicate that back-extraction
                     is necessary. If back-extraction is not necessary, concentrate the extract for
                     cleanup or analysis (Section 12.6 and/or 12.7). If back-extraction is necessary,
                     back-extract the extracts from Section 12.2.3.5 or 12.3.1.9 as follows:

             12.5.2  Back-extract each extract three times sequentially with 500 mL of the aqueous
                     sodium sulfate solution (Section 7.1.5), returning the bottom (organic) layer to the
                     separatory funnel the first two times while discarding the top (aqueous) layer. On
                     the final back-extraction, filter each pesticide  extract through a prerinsed drying
                     column containing 7 to  10 cm anhydrous sodium sulfate into a 500- to 1000-mL
                     graduated cylinder. Record the final extract volume ._Re-concentrate the sample and
                     QC aliquots per Sections 12.6-12.7, and clean up the samples and QC aliquots per
                     Section 13.

        12.6 Macro-concentration - Extracts in toluene are concentrated using a rotary evaporator or a
             heating mantle; extracts in methylene chloride or hexane are concentrated using a rotary
             evaporator, heating mantle, or Kuderna-Danish apparatus.

    Note: In the concentration procedures below, the extract must not be allowed to concentrate  to
    dryness because low molecular weight pesticides may be totally or partially lost. It may be
    advantageous to add 1 mL of toluene as a "keeper" to prevent loss of the low molecular weight
    pesticides.

             12.6.1  Rotary evaporation - Concentrate the extracts in separate round-bottom flasks.

                     12.6.1.1   Assemble the rotary evaporator according to manufacturer's
                               instructions, and warm the water bath to 45C. On a daily basis, pre-

48

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Method 1699                                                              December 2007

                               clean the rotary evaporator by concentrating 100 mL of clean extraction
                               solvent through the system. Archive both the concentrated solvent and
                               the solvent in the catch flask for a contamination check if necessary.
                               Between samples, three 2- to 3- mL aliquots of solvent should be rinsed
                               down the feed tube into a waste beaker.

                     12.6.1.2   Attach the round-bottom flask containing the sample extract to the
                               rotary evaporator. Slowly apply vacuum to the system, and begin
                               rotating the sample flask.

                     12.6.1.3   Lower the flask into the water bath, and adjust the speed of rotation and
                               the temperature as required to complete concentration in 15 to 20
                               minutes. At the proper rate of concentration, the flow of solvent into the
                               receiving flask will be steady, but no bumping or visible boiling of the
                               extract will occur.

    Note: If the rate of concentration is too fast, analyte loss may occur.

                     12.6.1.4   When the liquid in the concentration flask has reached an apparent
                               volume of approximately 2 mL, remove the flask from the water bath
                               and stop the rotation.  Slowly and carefully admit air into the system.
                               Be sure not to open the valve so quickly that the sample is blown out of
                               the flask. Rinse the feed tube with approximately 2 mL of solvent.

                     12.6.1.5   Proceed to Section  12.5 for back-extraction or Section 12.7 for micro-
                               concentration and solvent exchange.

             12.6.2  Heating mantle - Concentrate the extracts in separate round-bottom flasks.

                     12.6.2.1   Add one or two clean boiling chips to the round-bottom flask, and
                               attach a three-ball macro Snyder column. Prewet the column by adding
                               approximately 1 mL of solvent through the top. Place the round-bottom
                               flask in a heating mantle, and apply heat as required to complete the
                               concentration in 15 to 20 minutes.  At the proper rate of distillation, the
                               balls of the column will actively chatter, but the chambers will not
                               flood.

                     12.6.2.2   When the liquid has reached an apparent volume of approximately 10
                               mL, remove the round-bottom flask from the heating mantle and allow
                               the solvent to drain and cool for at least 10 minutes.  Remove the
                               Snyder column and rinse the glass joint into the receiver with small
                               portions of solvent.

                     12.6.2.3   Proceed to Section  12.6 for preparation for back-extraction or Section
                               12.7 for micro-concentration and solvent exchange.
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Method 1699                                                             December 2007

             12.6.3 Kuderna-Danish (K-D) - Concentrate the extracts in separate 500-mL K-D flasks
                    equipped with 10-mL concentrator tubes. The K-D technique is used for solvents
                    such as methylene chloride and hexane.  Toluene is difficult to concentrate using
                    the K-D technique unless a water bath fed by a steam generator is used.

                    12.6.3.1   Add 1 to 2 clean boiling chips to the receiver.  Attach a three-ball macro
                               Snyder column. Prewet the column by adding approximately 1 mL of
                               solvent through the top. Place the K-D apparatus in a hot water bath so
                               that the entire lower rounded surface of the flask is bathed with steam.

                    12.6.3.2  Adjust the vertical position of the apparatus and the water temperature
                               as required to complete the concentration in 15 to 20 minutes. At the
                               proper rate of distillation, the  balls of the column will actively chatter
                               but the chambers will not flood.

                    12.6.3.3  When the liquid has reached an apparent volume of 1 mL, remove the
                               K-D apparatus from the bath and allow the solvent to drain and cool for
                               at least 10 minutes. Remove the Snyder column and rinse the flask and
                               its lower joint into the concentrator tube with 1 to 2 mL of solvent. A 5-
                               mL syringe is recommended for this operation.

                    12.6.3.4  Remove the three-ball Snyder column, add a fresh boiling chip, and
                               attach a two ball micro Snyder column to the concentrator tube. Prewet
                               the column by adding approximately 0.5 mL of solvent through the top.
                               Place the apparatus in the hot water bath.

                    12.6.3.5  Adjust the vertical position and the water temperature as required to
                               complete the concentration in 5 to 10 minutes. At the proper rate of
                               distillation, the balls of the column will actively chatter but the
                               chambers will not flood.

                    12.6.3.6  When the liquid reaches an apparent volume of 0.5 mL, remove the
                               apparatus from the water bath and allow to drain and cool for at least 10
                               minutes.

                    12.6.3.7  Proceed to 12.6 for preparation for back-extraction or Section 12.7 for
                               micro-concentration and solvent exchange.

        12.7 Micro-concentration and solvent exchange

             12.7.1 Extracts to be subjected to GPC cleanup are exchanged into methylene chloride.
                    Extracts to be cleaned up using silica gel, Florisil, the SPE cartridge, and/or HPLC
                    are exchanged into hexane.

             12.7.2 Transfer the vial containing the sample extract to a nitrogen evaporation device.
                    Adjust the  flow of nitrogen so that the surface of the solvent is just visibly
                    disturbed.

50

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Method 1699                                                             December 2007
    Note: A large vortex in the solvent may cause analyte loss.

             12.7.3 Lower the vial into a 30C water bath and continue concentrating.

                    12.7.3.1  If the extract or an aliquot of the extract is to be concentrated to dryness
                              for weight determination (Sections 12.4.8 and 13.6.4), blow dry until a
                              constant weight is obtained.

                    12.7.3.2  If the extract is to be concentrated for injection into the GC/HRMS or
                              the solvent is to be exchanged for extract cleanup, proceed as follows:

             12.7.4 When the volume of the liquid is approximately 100 joL, add 2 to 3 mL of the
                    desired solvent (methylene chloride for GPC and HPLC, or hexane for the other
                    cleanups) and continue concentration to approximately 100 (iL. Repeat the
                    addition of solvent and concentrate once more.

             12.7.5 If the extract is to be cleaned up by GPC, adjust the volume of the extract to 5.0
                    mL with methylene chloride. If the extract is to be cleaned up by HPLC,
                    concentrate the extract to  1.0 mL.  Proceed with GPC or HPLC cleanup (Section
                    13.2 or 13.5, respectively).

             12.7.6 If the extract is to be cleaned up by column chromatography or the SPE cartridge,
                    bring the final volume to  1.0 mL with hexane.  Proceed with column cleanup
                    (Sections 13.3, 13.4,  13.7, or 13.8).

             12.7.7 If the extract is to be concentrated  for injection into the GC/HRMS (Section 14),
                    quantitatively transfer the extract to a 0.3-mL conical vial for final concentration,
                    rinsing the larger vial with hexane  and adding the rinse to the conical vial. Reduce
                    the volume to approximately 100 |oL. Add 20 |oL of nonane to the vial, and
                    evaporate the solvent to the level of the nonane. Seal the vial and label with the
                    sample number. Store in the dark at room temperature until ready for GC/HRMS
                    analysis. If GC/HRMS analysis will not be performed on the same day, store the
                    vial at less than -10C.
13.0  Extract cleanup

        13.1  Cleanup may not be necessary for relatively clean samples (e.g., treated effluents,
             groundwater, drinking water). If particular circumstances require the use of a cleanup
             procedure, the laboratory may use any or all of the procedures below or any other
             appropriate procedure. Before using a cleanup procedure, the laboratory must demonstrate
             that the requirements  of Section 9.2 can be met using the cleanup procedure. The following
             table suggests cleanups that may be used for the various analyte groups.
Analyte group
All
Suggested cleanups
GPC (13.2); SPE (13.3); Micro-silica (13.4)
                                                                                             51

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Method 1699                                                             December 2007
Organo-chlorine
Specific
compounds
GPC,
GPC,
SPE,
SPE,
Micro-silica
Micro-silica
plus
plus
Florisil (13.7)
HPLC(13.5)
or alumina (13

8)

             13.1.1 Gel permeation chromatography (Section 13.2) removes high molecular weight
                    interferences that cause GC column performance to degrade.  It should be used for
                    all soil and sediment extracts. It may be used for water extracts that are expected to
                    contain high molecular weight organic compounds (e.g., polymeric materials,
                    humic acids). It should also be used for tissue extracts after initial cleanup on the
                    anthropogenic isolation column (Section 13.6).

             13.1.2 Micro-silica (Section 13.4), the SPE cartridge (Section 13.3), Florisil (Section
                    13.7), and alumina (Section 13.8) may be used to remove non-polar and polar
                    interferences.

             13.1.3 HPLC (Section 13.5) is used to provide specificity for certain pesticides.

             13.1.4 The anthropogenic isolation column (Section 13.6) is used for removal of lipids
                    from tissue samples.

        13.2 Gel permeation chromatography (GPC)

             13.2.1 Column packing

                    13.2.1.1  Place 70 to 75 g of SX-3 Bio-beads (Section 6.7.1.1) in a 400-to 500-
                              mL beaker.

                    13.2.1.2  Cover the beads with methylene chloride and allow to swell overnight
                              (a minimum of 12 hours).

                    13.2.1.3  Transfer the swelled beads to the column (Section 6.7.1.1) and pump
                              solvent through the column, from bottom to top, at 4.5 to 5.5 mL/minute
                              prior to connecting the column to the detector.

                    13.2.1.4  After purging the column with solvent  for 1 to 2 hours, adjust the
                              column head pressure to 7 to 10 psig and purge for 4 to 5 hours to
                              remove air.  Maintain a head pressure of 7 to 10 psig. Connect the
                              column to the detector (Section 6.7.1.4).

             13.2.2 Column calibration

                    13.2.2.1  Load 5 mL of the GPC calibration solution (Section 7.4) into the sample
                              loop.

                    13.2.2.2  Inject the GPC calibration solution and record the signal from the
                              detector. The elution pattern will be corn oil, bis(2-ethylhexyl) phthalate
                              (BEHP), methoxychlor, perylene, and sulfur.
52

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Method 1699                                                             December 2007
                     13.2.2.3   Set the "dump time" to allow >85% removal of BEHP and >85%
                               collection of methoxychlor.

                     13.2.2.4   Set the "collect time" to the time of the sulfur peak maximum.

                     13.2.2.5   Verify calibration with the GPC calibration solution after every 20
                               extracts. Calibration is verified if the recovery of methoxychlor is
                               greater than 85%. If calibration is not verified, the system must be
                               recalibrated using the GPC calibration solution, and the previous sample
                               batch must be re-extracted and cleaned up using a calibrated GPC
                               system.

             13.2.3  Extract cleanup - GPC requires that the column not be overloaded.  The column
                     specified in this Method is designed to handle a maximum of 0.5 g of material from
                     an aqueous, soil, or mixed-phase sample in a 5-mL extract, and has been shown to
                     handle 1.5  g of lipid from a tissue sample in a 5-mL extract. If the extract is known
                     or expected to contain more than these amounts, the extract is split into aliquots for
                     GPC, and the aliquots are combined after elution from the column. The residue
                     content of the extract may be obtained gravimetrically by evaporating the solvent
                     from a 50-|oL aliquot.

                     13.2.3.1   Filter the extract or load through the filter holder (Section 6.7.1.3) to
                               remove particles. Load the 5.0-mL extract onto the column.

                     13.2.3.2   Elute the extract using the calibration data determined in Section 13.2.2.
                               Collect the eluate in a clean 400- to  500-mL beaker. Allow the  system
                               to rinse for additional 10 minutes before injecting the next sample.

                     13.2.3.3   Rinse the sample loading tube thoroughly with methylene chloride
                               between extracts to prepare for the next sample.

                     13.2.3.4   If an extract is encountered that could overload the GPC column to the
                               extent that carry-over could occur, a 5.0-mL methylene chloride blank
                               must be run through the  system to check for carry-over.

                     13.2.3.5   Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or
                               injection into the GC/MS.

        13.3 Solid-phase extraction (SPE) cartridge

             13.3.1  Setup

                     13.3.1.1   Attach the Vac-elute manifold (6.7.6.1) to a water aspirator or vacuum
                               pump with the trap and gauge installed between the manifold and
                               vacuum source.

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Method 1699                                                              December 2007

                     13.3.1.2  Place the SPE cartridge(s) in the manifold, turn on the vacuum source,
                               and adjust the vacuum to 5 to 10 psig.

    Note: Do not allow the SPE cartridge to go dry during the following steps.

             13.3.2  Cartridge washing - Pre-elute the cartridge sequentially with two 6-mL volumes of
                     1:2:1 ethyl acetate:acetonitrile:toluene.

             13.3.3  Using a pipette or a 1-mL syringe, transfer 1.0 mL of the extract in 1:2:1 ethyl
                     acetate:acetonitrile:toluene (Section_12.2.3.5, 12.3.1.9, 12.4.8 or 12.5.2) onto the
                     SPE cartridge followed by a rinse of 1 mL 1:2:1 ethyl acetate:acetonitrile:toluene.

             13.3.4  As soon as the sample is loaded, begin to collect the eluate in a round bottom flask
                     or centrifuge tube (if using a manifold). Elute the SPE cartridge with 11 mL of
                     1:2:1 ethyl acetate:acetonitrile: toluene.

             13.3.5  Concentrate the eluted extract per Sections 12.6 and 12.7 and proceed to other
                     cleanups or determination by HRGC/HRMS.

        13.4 Micro-silica column

             13.4.1  Place a small glass-wool plug in a clean Pasteur pipette. Rinse the pipette and glass
                     wool twice with small (e.g., 2-5 mL) volumes of toluene, followed by two
                     rinsings  with small volumes of hexane.  Allow the pipette to drain.  Dry pack the
                     column bottom to top with 0.75 gram of 10% deactivated silica (Section 7.5.1.1).
                     Tap the column to settle the silica.

             13.4.2  Rinse the column with hexane until the column is completely wetted (typically 5-
                     10 mL).  Allow the hexane to drain to the  top of the silica.

             13.4.3  Adjust the extract volume to 1.0 mL and apply to the column. Allow the extract to
                     drain to the top of the silica. Rinse the extract onto the column with 500 |oL of
                     hexane.

             13.4.4  Rinse the centrifuge tube that contained the extract with 300-|oL of 10% methanol
                     in dichloromethane and apply to the column.  Collect the eluate in a round-bottom
                     flask. Repeat this rinse and collect the eluate in the flask.

             13.4.5  Elute the column  with 5 mL of 10% methanol in dichloromethane. Collect the
                     eluate in the round bottom flask.

             13.4.6  Add 5 mL of acetone and 1 mL of iso-octane to the round bottom flask and
                     concentrate the eluate per Section 12.6 and 12.7 for further cleanup or injection
                     intotheHPLCorGC/MS.
54

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Method 1699                                                              December  2007

             13.4.7  For extracts of samples known to contain large quantities of other organic
                     compounds, it may be advisable to increase the capacity of the silica gel column.
                     This may be accomplished by increasing the strength of the acid silica and
                     including basic silica gel.  The acid silica gel (Section 7.5.1.2) may be increased in
                     strength to as much as 40% w/w (6.7 g sulfuric acid added to 10 g silica gel).  The
                     basic silica gel (Section 7.5.1.3) may be  increased in strength to as much as 33%
                     w/w (50 mL IN NaOH added to 100 g silica gel), or the potassium silicate (Section
                     7.5.1.4) may be used. Larger columns may also be used if needed.

    Note: The use of stronger acid and basic silica gel (44% w/w) may lead to charring of organic
    compounds in some extracts. The charred material may retain some of the analytes and lead to lower
    recoveries of the pesticides.  Increasing the strengths of the acid and basic silica gel may also require
    different volumes ofeluants than those specified above to elute the analytes from the column.  The
    performance of the Method after such modifications must be verified by the procedure in Section 9.2.

        13.5 HPLC (Reference 9)

             13.5.1  Column calibration

                     13.5.1.1   Prepare a calibration standard containing the pesticides at the
                               concentrations of the stock solution in Table 3, or at a concentration
                               appropriate to the response of the detector.

                     13.5.1.2   Inject the calibration standard into the HPLC and record the signal from
                               the detector. Collect the eluant for reuse.

                     13.5.1.3   Establish the collection time for the pesticides of interest. Following
                               calibration, flush the injection system with solvent to ensure that
                               residual pesticides are removed from the system.

                     13.5.1.4   Verify the calibration with the calibration solution after every 20
                               extracts. Calibration is  verified if the recovery of the pesticides is 75 to
                               125% compared to the calibration (Section 13.5.1.1). If calibration is
                               not verified, the system must be recalibrated using the calibration
                               solution, and the batch of samples run on the uncalibrated system must
                               be re-extracted and cleaned up using a calibrated system.

             13.5.2  Extract cleanup - HPLC requires that the column not be overloaded. The column
                     specified in this  Method is designed to handle a maximum of 50 Og of a given
                     pesticide, depending on the particular compound.  If the amount of material in the
                     extract will overload the column, split the extract into fractions and combine the
                     fractions after elution from the column.

                     13.5.2.1   Rinse the sides of the vial containing the sample and adjust to the
                               volume required  for the sample loop for injection.

                     13.5.2.2   Inject the sample extract into the HPLC.

                                                                                              55

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Method 1699                                                              December 2007
                     13.5.2.3  Elute the extract using the calibration data determined in Section 13.5.1.
                               Collect the fraction(s) in clean 20-mL concentrator tubes.

                     13.5.2.4  If an extract containing greater than 500 (ig of total material is encoun-
                               tered, a blank must be run through the system to check for carry-over.

                     13.5.2.5  Concentrate the eluate per Section 12.7 for injection into the
GC/HRMS.

        13.6 Anthropogenic isolation column (Reference 15) - Used for removal of lipids from tissue
             extracts

             13.6.1  Prepare the column as given in Section 7.5.2.

             13.6.2  Pre-elute the column with 100 mL of hexane. Drain the hexane layer to the top of
                     the column, but do not expose the sodium sulfate.

             13.6.3  Load the sample and rinses (Section 12.4.9.2) onto the  column by draining each
                     portion to the top of the bed.  Elute the pesticides from the column into the
                     apparatus used for concentration (Section 12.4.7) using 200 mL of hexane.

             13.6.4  Remove a small portion (e.g., 50 joL) of the extract for determination of residue
                     content.  Estimate the percent of the total that this portion represents.  Concentrate
                     the small portion to constant weight per Section 12.7.3.1. Calculate the total
                     amount of residue in the extract. If more than 500 mg of material  remains, repeat
                     the cleanup using a fresh anthropogenic isolation column.

             13.6.5  If necessary, exchange the extract to a solvent suitable  for the additional cleanups
                     to be used (Section 13.2-13.8).

             13.6.6  Clean up the extract using the procedures in Sections 13.2 - 13.8.  GPC (Section
                     13.2) and Florisil (Section 13.7) are recommended as minimum additional cleanup
                     steps.

             13.6.7  Following cleanup, concentrate the extract to 20 OL per Section 12.7 and proceed
                     with the  analysis in Section 14.

        13.7 Florisil

             13.7.1  Begin to drain the n-hexane from the column (Section 7.5.4.3). Adjust the flow
                     rate of eluantto 4.5-5.0 mL/min.

             13.7.2  When the n-hexane is within 1 mm of the sodium sulfate, apply the sample extract
                     (in hexane) to the column. Rinse the sample container twice with  1-mL portions of
                     hexane and apply to the column, allowing the hexane to drain to the top of the
                     sodium sulfate  layer.
56

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Method 1699                                                              December 2007
             13.7.3  Elute Fraction 1 with 200 mL of 6% ethyl ether in n-hexane and collect the eluate.
                     Elute Fraction 2 with 200 mL of 15% ethyl ether in hexane and collect the eluate.
                     Elute Fraction 3 with 50% ethyl ether in hexane and collect the eluate. The exact
                     volumes of solvents will need to be determined for each batch of Florisil. If the
                     pesticides are not to be collected in separate fractions, elute all pesticides with 50%
                     ethyl ether in hexane.

             13.7.4  Concentrate the eluate(s) per Sections 12.6 - 12.7 for further cleanup or for
                     injection into the HPLC or GC/HRMS.

        13.8 Alumina

             13.8.1  Begin to drain the hexane from the column (Section 7.5.5.2).  Adjust the flow rate
                     of eluantto 4.5 - 5.0 mL/min.

             13.8.2  When the n-hexane is within 1 mm of the sodium sulfate, apply the sample extract
                     (in hexane) to the column. Rinse the sample container twice with 1-mL portions of
                     hexane and apply to the column, allowing the hexane to drain to the top of the
                     sodium sulfate layer.

             13.8.3  Elute the pesticides with 150 mL of n-hexane. If all pesticides are not eluted, elute
                     the remaining pesticides with 50 mL of 15% methylene chloride in n-hexane.

             13.8.4  Concentrate the eluate(s) per Sections 12.6 - 12.7 for further cleanup or for
                     injection into the HPLC or GC/HRMS.
14.0  HRGC/HRMS analysis

        14.1 Establish the operating conditions given in Section 10.1.

        14.2 Add 2 |oL of the labeled injection internal standard spiking solution (Section 7.14) to the 20
             (iL sample extract immediately prior to injection to minimize the possibility of loss by
             evaporation, adsorption, or reaction. If an extract is to be reanalyzed and evaporation has
             occurred, do not add more labeled injection internal standard spiking solution. Rather,
             bring the extract back to its previous volume (e.g., 19 joL) with pure nonane (18 |oL if 2 |oL
             injections are used).

        14.3 Inject 1.0 or 2.0 joL of the concentrated extract containing the Labeled injection internal
             standards using on-column or splitless injection. The volume injected must be identical to
             the volume used for calibration (Section 10.3).

             14.3.1  Start the GC column initial isothermal hold upon injection. Start MS data
                     collection after the solvent peak elutes.
                                                                                              57

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Method 1699                                                             December 2007

             14.3.2 Monitor the exact m/z's for each pesticide throughout its retention time window.
                    Where warranted, monitor m/z's associated with pesticides at higher levels of
                    chlorination to assure that fragments are not interfering with the m/z's for
                    pesticides at lower levels of chlorination. Also where warranted, monitor m/z's
                    associated with interferents expected to be present.

             14.3.3 Stop data collection after permethrin and cypermethrin have eluted.  Return the
                    column to the initial temperature for analysis of the next sample extract or standard.
15.0  System and laboratory performance

        15.1  At the beginning of each 12-hour shift during which analyses are performed, GC/MS
             system performance and calibration are verified for all the pesticides and labeled
             compounds.  For these tests, analysis of the CS-4 calibration verification (VER) standard
             (Section 7.10 and Table 4) must be used to verify all performance criteria. Adjustment
             and/or recalibration (Section 10) must be performed until all performance criteria are met.
             Only after all performance criteria are met may samples, blanks, IPRs, and OPRs be
             analyzed.

        15.2 MS resolution - Static resolving power checks must be performed at the beginning and at
             the end of each shift per Sections 10.2.1.  If analyses are performed on successive shifts,
             only the beginning  of shift static resolving power check is required. If the requirement in
             Section 10.2.1 cannot be met, the problem must be corrected before analyses can proceed.
             If any of the samples in the previous shift may be affected by poor resolution, those
             samples must be re-analyzed.

        15.3 Calibration verification

             15.3.1 Inject the VER (CS-4) calibration standard using the procedure in Section 14.

             15.3.2 The m/z abundance ratios for all pesticides must be within the limits in Table 6;
                    otherwise, the mass spectrometer  must be adjusted until the m/z abundance ratios
                    fall within the limits specified when the verification test is be repeated.  If the
                    adjustment alters the resolution of the mass spectrometer, resolution must be
                    verified (Section 10.2.1) prior to repeat of the verification test.

             15.3.3 The GC peak representing each native pesticide and labeled compound in the VER
                    standard must be present with a S/N of at least  10; otherwise, the mass
                    spectrometer must be adjusted and the verification test repeated.

             15.3.4 Compute the concentration of the pesticides that have labeled analogs by isotope
                    dilution and the concentration of the pesticides that do not have labeled analogs by
                    the internal standard technique. These concentrations are computed based on the
                    calibration  data in Section 10.
58

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Method 1699                                                              December 2007

             15.3.5  For each compound, compare the concentration with the calibration verification
                     limit in Table 5.  If all compounds meet the acceptance criteria, calibration has
                     been verified and analysis of standards and sample extracts may proceed.  If,
                     however, any compound fails its respective limit, the measurement system is not
                     performing properly. In this event, prepare a fresh calibration standard or correct
                     the problem and repeat the resolution (Section  15.2) and verification (Section 15.3)
                     tests, or recalibrate (Section 10).

        15.4 Retention times and GC resolution

             15.4.1  Retention times.

                     15.4.1.1  Absolute - The absolute retention times of the Labeled compounds in
                               the verification test (Section 15.3) must be within V 15 seconds of the
                               respective retention times in the calibration (Section 10.1)

                     15.4.1.2  Relative - The relative retention times of native pesticides and the
                               labeled compounds in the verification test (Section 15.3) must be within
                               their respective RRT limits in Table 2 or, if an alternate column or
                               column system is employed, within their respective RRT limits for the
                               alternate column or column system  (Sections 9.1.2.3 and 6.9.1).

                     15.4.1.3  If the absolute or relative retention time of any compound is not within
                               the limits specified, the GC is not performing properly. In this event,
                               adjust the GC and repeat the verification  test (Section 15.3) or
                               recalibrate (Section 10), or replace the GC column and either verify
                               calibration or recalibrate.

             15.4.2  GC resolution and minimum analysis time

                     15.4.2.1  The resolution and minimum analysis time  specifications in Sections
                               6.9.1.1.2  and 6.9.1.1.1, respectively, must be met for the DB-17 column
                               or, if an alternate column or column system is employed, must be met as
                               specified for the alternate column or column system (Sections 9.1.2.3
                               and 6.9.1).  If these specifications are not met, the GC analysis
                               conditions must be adjusted until the specifications are met, or the
                               column must be replaced and the calibration verification tests  repeated
                               Sections  15.3 - 15.4), or the system must be recalibrated (Section 10).

                     15.4.2.2  After the resolution and minimum analysis  time specifications are met,
                               update the retention times and relative retention times, but not the
                               relative responses and response factors. For the relative responses and
                               response  factors, the multi-point calibration data (Sections 10.4 and
                               10.5) must be used.
                                                                                               59

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Method 1699                                                             December 2007

        15.5 Endrin/4,4'-DDT breakdown - Perform the endrin/4,4'-DDT breakdown test (Section 10.6).
             The breakdown specification (Section 10.6.2.3) must be met before an OPR, sample, or
             blank may be analyzed.

        15.6 Ongoing precision and recovery

             15.6.1 Analyze the extract of the ongoing precision and recovery (OPR) aliquot (Section
                     11.4.2.5, 11.5.3, 11.6.3, or 11.8.3.2) prior to analysis of samples from the same
                    batch.

             15.6.2 Compute the percent recovery of the pesticides with labeled analogs by isotope
                    dilution (Section 10.4). Compute the percent recovery of each labeled compound
                    by the internal standard method (Section 10.5).

             15.6.3 For the pesticides and labeled compounds, compare the recovery to the OPR limits
                    given in Table 5.  If all  compounds meet the acceptance criteria, system
                    performance is acceptable and analysis of blanks and samples may proceed. If,
                    however, any individual concentration falls outside of the range given, the
                    extraction/concentration processes are not being performed properly for that
                    compound.  In this event, correct the problem, re-prepare, extract, and clean up the
                    sample batch and repeat the ongoing precision and recovery test (Section 15.6).

             15.6.4 If desired, add results that pass the specifications in Section 15.6.3 to initial
                    (Section 9.4) and previous ongoing data for each compound in each matrix.
                    Update QC charts to form a graphic representation of continued laboratory
                    performance. Develop  a statement of laboratory accuracy for each pesticide in
                    each matrix type by calculating the average percent recovery (R) and the standard
                    deviation of percent recovery (SR). Express the accuracy as a recovery interval
                    from R ! 2SR to R + 2SR. For example, if R = 95% and SR = 5%, the accuracy is 85
                    to 105%.

        15.7 Blank - Analyze the Method blank extracted with each sample batch immediately
             following analysis of the OPR aliquot to demonstrate freedom from contamination and
             freedom from carryover from the OPR analysis. If pesticides will be carried from the OPR
             into the Method blank, analyze  one or more aliquots of solvent between the OPR and the
             Method blank. The  results of the analysis of the blank must meet the specifications in
             Section 9.5.2 before sample analyses may proceed.
60

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Method 1699                                                             December  2007

16.0  Qualitative determination

        A pesticide or labeled compound is identified in a standard, blank, or sample when all of the
        criteria in Sections 16.1 through 16.4 are met.

        16.1 The signals for the two exact m/z's in Table 6 must be present and must maximize within
             the same two scans.

        16.2 The signal-to-noise ratio (S/N) for the GC peak at each exact m/z must be greater than or
             equal to 2.5 for each pesticide detected in a sample extract, and greater than or equal to 10
             for all pesticides in the calibration and verification standards (Sections 10.3.3 and 15.6.3).

        16.3 The ratio of the integrated areas of the two exact m/z's specified in Table 6 must be within
             the limit in Table 6, or within V 15 percent of the ratio in the midpoint (CS-4) calibration or
             calibration verification (VER), whichever is most recent.

        16.4 The relative retention time of the peak for a pesticide must be within the RRT QC limits
             specified in Table 2 or within similar limits developed from calibration data (Section
             10.1.2).  If an alternate column (Section 9.1.2.3) is employed, the RRT for the pesticide
             must be within its respective RRT QC limits for the alternate column or column system
             (Section 6.9.1).

    Note: For native pesticides determined by internal standard quantitation, a pesticide with the same
    exact m/z's as other pesticides may fall within more than one RT window and be mis-identified unless
    the RRT windows are made very narrow, as in Table 2. Therefore, consistency of the RT and RRT
    with other pesticides and the labeled compounds may be required for rigorous pesticide
    identification.  Retention time regression may aid in this identification.

        16.5 Because of pesticide RT overlap and the potential for interfering substances, it is possible
             that all of the identification criteria (Sections 16.1 - 16.4) may not be met. It is also
             possible that loss of one or more chlorines from a highly chlorinated pesticide or interferent
             may inflate or produce a false concentration for a less-chlorinated pesticide that elutes at
             the same retention time (see Section 18).  If identification is ambiguous, an experienced
             spectrometrist (Section 1.5) must determine the presence or absence of the pesticide.

        16.6 If the criteria for identification in Sections 16.1 - 16.5 are not met, the pesticide has not
             been identified and the result for that pesticide may not be reported or used for permitting
             or regulatory compliance purposes. If interferences preclude identification, a new aliquot
             of sample must be extracted, further cleaned up, and analyzed.

17.0  Quantitative determination

        17.1 Isotope dilution quantitation

             17.1.1  By adding a known amount of the labeled pesticides to every sample prior to
                     extraction, correction for recovery of each pesticide can be made because the native

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Method 1699                                                              December 2007

                    compound and its labeled analog exhibit similar effects upon extraction,
                    concentration, and gas chromatography. Relative responses (RRs) are used in
                    conjunction with the calibration data in Section 10.4 to determine concentrations in
                    the final extract, so long as labeled compound spiking levels are constant.

             17.1 .2 Compute the concentrations of the pesticides in the extract using the RRs from the
                    calibration data (Section 10.4) and following equation:

                                 Cex (ng/mL)  =


                    Where:
                           Cex  =  The concentration of the pesticide in the extract, and the other
                                   terms are as defined in Section 10.4.3

        17.2 Internal standard quantitation and labeled compound recovery

             1 7.2.1 Compute the concentrations in the extract of the native compounds that do not have
                    labeled analogs using the response factors determined from the calibration data
                    (Section 10.5) and the following equation:

                                 Cex (ng/mL)  =
                                              (Alls+A2ls}RF

                    Where:
                           Cex =  The concentration of the labeled compound in the extract, and the
                                   other terms are as defined in Section 10.5.1

             1 7.2.2 Using the concentration in the extract determined above, compute the percent
                    recovery of the labeled pesticides other labeled cleanup standard using the
                    following equation:

                           Recovery (%) = Concentration found (ng/mL)  xlOO
                                          Concentration spiked (ng/mL)

        17.3 The concentration of a native compound in the solid phase of the sample is computed using
             the concentration of the compound in the extract and the weight of the solids (Section
             11.2.2.3), as follows:

                           Concentration in solid (ng/kg) = (Qr x )
                                                            Ws

                    Where:
                           Cex =  The concentration of the compound in the extract.
                           Vex =  The extract volume in mL.
                           Ws  =  The sample weight (dry weight) in kg.
62

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Method 1699                                                              December 2007

        17.4 The concentration of a native pesticide in the aqueous phase of the sample is computed
             using the concentration of the compound in the extract and the volume of water extracted
             (Section 11.4), as follows:

             Concentration in aqueous phase (pg/L) = IQOOx (QrX )
                                                              Vs

                     Where:
                           Cex  =  The concentration of the compound in the extract.
                           V^  =  The extract volume in mL.
                           Vs   =  The sample volume in liters.

        17.5 If the SICP area at either quantitation m/z for any pesticide exceeds the calibration range of
             the system, dilute the sample extract by the factor necessary to bring the concentration
             within the calibration range, adjust the concentration of the Labeled injection internal
             standard to 100 pg/^L in the extract, and analyze an aliquot of this diluted extract. If the
             pesticides cannot be measured reliably by isotope dilution, dilute and analyze an aqueous
             sample or analyze a smaller portion of a soil, tissue, or mixed-phase sample. Adjust the
             pesticide concentrations, detection limits, and minimum levels to account for the dilution.

        17.6 Reporting of re suits

             17.6.1  Reporting units and levels

                     17.6.1.1  Aqueous samples - Report results in pg/L (parts-per-quadrillion).

                     17.6.1.2  Samples containing greater than 1% solids (soils, sediments, filter cake,
                               compost) - Report results in ng/kg based on the dry weight of the
                               sample.  Report the percent solids so that the result may be converted to
                               aqueous units.

                     17.6.1.3  Tissues - Report results in ng/kg of wet tissue, not on the basis of the
                               lipid content of the tissue. Report the percent lipid content, so  that the
                               data user can calculate the concentration on a lipid basis if desired.

             17.6.2  Reporting level

                     17.6.2.1  Report the result for each pesticide in each sample, blank, or standard
                               (VER, IPR, OPR) at or above the minimum level of quantitation (ML;
                               Table  1) to 3 significant figures. Report the result below the ML in
                               each sample as 
-------
Method 1699                                                             December 2007

                              the samples and blank(s) separately, the concentration of each pesticide
                              in a method blank or field blank associated with the sample may be
                              subtracted from the results for that sample, or must be subtracted if
                              requested or required by a regulatory authority or in a permit.

                    17.6.2.3  Results for a pesticide in a sample that has been diluted are reported at
                              the least dilute level at which the area at the quantitation m/z is within
                              the calibration range (Section 17.5).

                    17.6.2.4  For a pesticide having a labeled analog, report results at the least dilute
                              level at which the area at the quantitation m/z is within the calibration
                              range (Section 17.5) and the labeled compound recovery is within the
                              normal range for the Method (Section 9.3 and Table 5).

                    17.6.2.5  Results from tests performed with an analytical system that is not in
                              control 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.

18.0  Analysis of complex samples

        18.1  Some samples may contain high levels (>10 ng/L; >1000 ng/kg) of the compounds of
             interest, interfering compounds, and/or polymeric materials. Some extracts may not
             concentrate to 20 (iL (Section  12.7); others may overload the GC column and/or mass
             spectrometer. A fragment ion  from a pesticide  at a higher level of chlorination may
             interfere with determination  of a pesticide at a lower level of chlorination.

        18.2 Analyze a smaller aliquot of the sample (Section 17.5) when the extract will not
             concentrate to 20 OL after all cleanup procedures have been exhausted.  If a smaller aliquot
             of soils or mixed-phase samples is analyzed, attempt to assure that the sample is
             representative.

        18.3 Perform integration of peak areas and calculate concentrations manually when interferences
             preclude computerized calculations.

        18.4 Recovery of labeled compounds - In most samples, recoveries of the labeled compounds
             will be similar to those from reagent water or from the alternate matrix (Section 7.6).

             18.4.1 If the recovery of any  of the labeled compounds is outside of the normal range
                    (Table 5), a diluted sample must be analyzed (Section 17.5).

             18.4.2 If the recovery of any  of the labeled compounds in the diluted sample is outside of
                    normal range, the calibration verification standard (Section 7.10 and Table 5) must
                    be analyzed and calibration verified (Section 15.3).

             18.4.3 If the calibration cannot be verified, a new calibration must be performed and the
                    original sample extract reanalyzed.
64

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Method 1699                                                            December 2007
             18.4.4 If calibration is verified and the diluted sample does not meet the limits for labeled
                    compound recovery, the Method does not apply to the sample being analyzed and
                    the result may not be reported or used for permitting or regulatory compliance
                    purposes. In this case, alternate extraction and cleanup procedures in this Method
                    or an alternate GC column must be employed to resolve the interference. If all
                    cleanup procedures in this Method and an alternate GC column have been
                    employed and labeled compound recovery remains outside of the normal range,
                    extraction and/or cleanup procedures that are beyond this scope of this Method will
                    be required to analyze the sample.

19.0   Pollution prevention

        19.1  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 operation. EPA has established a preferred hierarchy of environmental
             management techniques that places pollution prevention as the management option of first
             choice. Whenever feasible, laboratory personnel 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.

        19.2  The pesticides 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.

        19.3  For information about pollution prevention that may be applied to  laboratories and research
             institutions, consult Less is Better: Laboratory Chemical Management for Waste
             Reduction, available from the American Chemical Society's Department of Governmental
             Relations and Science Policy, 1155 16th Street NW, Washington DC 20036, 202/872-4477
             (http://membership.acs.0rg/c/ccs/pubs/less is  better.pdf).

20.0   Waste  management

        20.1  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).

        20.2  Samples containing HC1 or H2SO4 to pH <2, or KOH or NaOH to pH >12 must be handled
             as hazardous waste, or must be neutralized before being poured down a drain.

        20.3  The pesticides decompose above 800C. Low-level waste such as absorbent paper, tissues,
             animal remains, and plastic gloves may be  burned in an appropriate incinerator. Gross

                                                                                            65

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Method 1699                                                          December 2007

             quantities (milligrams) should be packaged securely and disposed of through commercial or
             governmental channels that are capable of handling extremely toxic wastes.

       20.4 For further information on waste management, consult The Waste Management Manual for
             Laboratory Personnel and Less is Better-Laboratory Chemical Management for Waste
             Reduction, available from the American Chemical Society's Department of Government
             Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.

21.0  Method performance

       Method 1699 was validated and preliminary data were collected in a single laboratory (Reference
       2). Single laboratory performance data are included in Table 8.

22.0  References

       1     EPA Methods  608, 1656, 1613, and 1668A.

       2     "Analytical Method for the Analysis of Multi-residue Pesticides in Aqueous and XAD
             Column Samples by HRGC/HRMS," Axys Analytical Services  (proprietary).

       3     Lamparski, L.L., and Nestrick, T.J., "Novel Extraction Device for the Determination of
             Chlorinated Dibenzo-p-dioxins (PCDDs) and Dibenzofurans (PCDFs) in Matrices
             Containing Water," Chemosphere, 19:27-31, 1989.

       4     "Working with Carcinogens," Department of Health, Education, & Welfare, Public Health
             Service, Centers for Disease Control, NIOSH, Publication 77-206, August 1977, NTIS PB-
             277256.

       5     "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910.

       6     "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety, 1979.

       7     "Standard Methods for the Examination of Water and Wastewater,"  18th edition and later
             revisions, American Public Health Association, 1015 15th St, N.W., Washington, DC
             20005, 1-35: Section 1090 (Safety), 1992.

       8     "Method 613 - 2,3,7,8-Tetrachlorodibenzo-/?-dioxin," 40 CFR 136 (49 FR 43234),
             December 26,  1984, Section 4.1.

       9     Echols, Kathy, Robert Gale, Donald E. Tillitt, Ted Schwartz, and Jerome O'Laughlin,
             Environmental Toxicology and Chemistry 16:8 1590-1597 (1997)

       10    U.S. EPA Office of Superfund Remediation and Technology Innovation, Contract
             Laboratory Program Summary of Requirements; Reporting and Deliverables Requirements;
             Target Compound List and Contract Required Quantitation Limits; and Analytical Methods
             (http://www.epa.gov/superfund/programs/clp/olm4.htm).

66

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Method 1699                                                            December 2007
        11    Provost, L.P., and Elder, R.S., "Interpretation of Percent Recovery Data," American
             Laboratory, 15: 56-83, 1983.

        12    "Standard Practice for Sampling Water," ASTM Annual Book of Standards, ASTM, 1916
             Race Street, Philadelphia, PA 19103-1187, 1980.

        13    e.g., "Standard Methods for the Examination of Water and Wastewater," 18th edition and
             later revisions, American Public Health Association, 1015 15th St, N.W., Washington, DC
             20005, Methods 4500-C1 adapted for field use.

        14    "Handbook of Analytical Quality Control in Water and Wastewater Laboratories," USEPA
             EMSL, Cincinnati, OH 45268, EPA-600/4-79-019, March 1979.

        15    "Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF
             in Fish", U.S. Environmental Protection Agency, Environmental Research Laboratory,
             Duluth MN  55804, EPA/600/3-90/022, March 1990.
                                                                                           67

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Method 1699
December 2007
23.0     Tables and Figures

Table 1. Names, CAS Registry numbers, and ambient water quality criteria for pesticides determined by
isotope dilution and internal standard HRGC/HRMS.
Pesticide
Organochlorine
Aldrin
BHC, alpha
BHC, beta
BHC, delta
BHC, gamma (lindane)
Captan
Chlordane, alpha (cis)
Chlordane, gamma (trans)
Chlorothalonil
Dacthal
ODD, o,p-
DDD, p,p-
DDE, o,p-
DDE, p,p-
DDT, o,p-
DDT, p,p-
Dieldrin
Endosulfan-alpha
Endosulfan-beta
Endosulfan-sulfate
Endrin
Endrin-ketone
Heptachlor
Heptachlor-epoxide
Hexachlorobenzene
Methoxychlor
Mi rex
Nonachlor, cis-
Nonachlor, trans-
Octachlorostyrene
Oxychlordane
Perthane
Quintozene
Tecnazene
CAS
Number

309-00-2
319-84-6
319-85-7
319-86-8
58-89-9
133-06-2
5103-71-9
5103-74-2
1897-45-6
1861-32-1
53-19-0
72-54-8
3424-82-6
72-55-9
789-02-6
50-29-3
60-57-1
959-98-8
33213-65-9
1031-07-8
72-20-8
53494-70-5
76-44-8
1024-57-3
118-74-1
72-43-5
2385-85-5
5103-73-1
39765-80-5
29082-74-4
27304-13-8
72-56-0
82-68-8
117-18-0
Labeled analog

13C12-Aldrin
13C6-BHC, alpha
13C6-BHC, beta
13C6-BHC, delta
13C6-BHC, gamma


13Cio-Chlordane, gamma





13C12-p,p-DDE
13C12-o,p-DDT
13C12-p,p-DDT
13C12-Dieldrin
13C9-alpha-Endosulfan
13C9-beta-Endosulfan

13C12-Endrin

13C4-Heptachlor
13C10-Heptachlor-epoxide
13C6-Hexachlorobenzene
13C12-Methoxychlor
13C8-Mirex
13C10-Nonachlor, cis-
13C10-Nonachlor, trans-

13C10-Oxychlordane



Lowest
Ambient
Criterion
(pg/L)(i)

49
2600
9100

160000

800
800



11

11

11
52
8700
8700
62000000
2300

79
40

30000
1000







MDLs and MLs, matrix and
concentration (2)
Water (pg/L)
MDL
6
7
6
5
9
182
7
6
35
4
3
5
3
6
2
1
5
24
30
13
3
12
7
12
4
7
35
4
11
12
7
36
18
22
ML
90
60
60
60
60
500
30
50
100
20
30
30
30
30
30
30
30
100
100
40
30
40
30
40
40
30
100
30
40
40
60
100
80
80
Solid
(ng/kg)
MD
L
0.6
1.3
0.6
2.0
0.7
35
0.6
0.8
1.9
0.9
0.8
1.5
0.5
0.7
0.3
0.3
0.5
-
-
11
0.4
1.6
-
0.3
1.9
0.3
-
0.5
0.8
1.1
0.5
-
4.7
3.2
ML
10
10
10
10
10
100
5
5
10
2
5
5
5
5
5
5
5
-
-
50
5
5
-
5
5
5
-
5
5
5
10
-
20
10
Extract
(pg/ML)
ML
3
3
3
3
3
25
1.5
2
5
1
1.5
1.5
1.5
1.5
1.5
1.5
1.5
5
5
2
1.5
2
1.5
2
2
1.5
5
1.5
2
2
3
5
4
4
68

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Method 1699
December 2007
Organophosphate
Azinphos-methyl
Chlorpyriphos
Chlorpyriphos-methyl
Chlorpyriphos-oxon
Diazinon
Diazinon-oxon
Disulfoton
Disulfoton sulfone
Fenitrothion
Fonofos
Malathion
Methamidophos
Parathion-ethyl
Parathion-methyl
Phorate
Phosmet
Pirimiphos-methyl
Triazine
Ametryn
Atrazine
Cyanazine
Desethyl atrazine
Hexazinone
Metribuzin
Simazine
Pyrethroid
Cypermethrin
Permethrins-peak 1
Permethrins-peak2

86-50-0
2921-88-2
5598-13-0
5598-15-2
333-41-5
962-58-3
298-04-4
2497 05 06
122-14-5
944-22-9
121-75-5
10265-92-6
56-38-2
298-00-0
298-02-2
732-11-6
29232-93-7

834-12-8
1912-24-9
21725-46-2
6190-65-4
51235-04-2
21087-64-9
122-34-9

52315-07-8
52645-53-1
52645-53-1

Azinphos-methyl-d6



Diazinon-d10




13C6-Fonofos









13C3-Atrazine







13C6-cis/trans-Permethrin3
13C6-cis/trans-Permethrin3





170000





100000

13000

















57
20
19
24
27
22
64
9
24
11
296
269
15
39
49
63
14

11
14
38
5
20
14
12

66
59
44

200
80
100
80
80
80
400
30
80
80
1000
1000
80
200
200
200
80

80
80
80
40
100
60
80

200
200
100

1.4
2.0
3.0
3.5
24
-
7.1
1.6
4.6
0.8
41
-
3.5
6.1
3.5
12
7.3

13
-
-
1.3
1.0
-
1.4

2.4
230
340

20
10
10
10
100
-
100
5
20
8
200
-
10
20
20
50
20

50
-
-
5
10
-
10

20
1000
1000

10
4
5
4
4
4
20
1.5
4
4
50
50
4
10
10
10
4

4
4
4
2
5
3
4

10
10
5
1.  National Recommended Water Quality Criteria, 2004, http://epa.gov/waterscience/criteria/wqcriteria.html. and
    Great Lakes Criteria (40 CFR 132.6), whichever is lower.  A blank cell means there is no ambient criterion.
2.  Method detection limits (MDLs) and minimum levels of quantitation (MLs) with no interferences present.
3.  Elution order of cis/trans permethrin unknown
                                                                                                  69

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Method 1699
December  2007
Table 2.  Retention times (RTs); relative retention times (RRTs); and retention time and
quantitation references for the pesticides
Pesticide

Methamidophos
Tecnazene
njC6-Hexachlorobenzene
Hexachlorobenzene
Phorate
BHC-alpha
Desethylatrazine
Diazinon-d10
Quintozene
Diazinon
Diazinon-oxon
1JC3-Atrazine
Atrazine
njC6-gamma-BHC
gamma-BHC
Simazine
Fonofos
1JC6-Fonofos
Disulfoton
1JC6-beta-BHC
beta-BHC
1JC4-Heptachlor
Heptachlor
1JC6-delta-BHC
delta-BCH
Chlorothalonil.
lJC12-Aldrin
Aldrin
Chlorpyriphos-methyl
lJC12-PCB-52
Parathion-methyl
Ametryn
Pirimiphos-methyl
Metribuzin
Octachlorostyrene
Dacthal
Chlorpyriphos
Fenitrothion
njCio-Oxychlordane
Oxychlordane
Malathion
Heptachlor-epoxide
njC-Permethrins-Peak 2
Parathion-ethyl
Chlorpyriphos-oxon
njC6-Permethrins-Peak 1
Azinphos-ethyl-d6
njC12-Heptachlor-epoxide
RT(1)

09:01
14:44
15:54
15:55
16:11
16:35
16:50
17:32
17:39
17:44
17:55
18:00
18:01
18:15
18:16
18:21
18:25
18:25
18:34
19:26
19:27
19:36
19:37
21:00
21:01
21:08
21:15
21:17
21:26
21:51
22:28
22:41
22:42
23:04
23:18
23:18
23:33
24:07
24:09
24:11
24:12
25:14
42:21
24:26
24:30
42:04
24:33
25:11
RRT
(2)

0.413
0.927
0.728
1.001
0.741
0.909
0.935
0.802
1.110
1.011
1.022
0.824
1.001
0.835
1.001
1.019
1.000
0.843
0.850
0.889
1.001
0.897
1.001
0.961
1.001
0.967
0.973
1.002
0.981
N/A
1.028
1.038
1.039
1.056
1.096
1.066
1.078
1.104
1.105
1.001
1.108
0.962
1.114
1.118
1.121
1.124
1.124
1.153
RRT Limits
(3)

0.397-0.428
0.906-0.948
0.712-0.743
0.991 -1.012
0.725-0.756
0.890-0.927
0.917-0.954
0.787-0.818
1.089-1.131
1.002-1.021
1.003-1.041
0.809-0.839
0.992-1.010
0.820-0.850
0.992-1.010
1.001 -1.038
0.991 -1.009
0.828-0.858
0.834-0.865
0.874-0.905
0.992-1.009
0.882-0.912
0.992-1.009
0.946-0.976
0.993-1.009
0.952-0.982
0.957-0.988
0.994-1.009
0.966-0.996
N/A
1.013-1.043
1.023-1.053
1.024-1.054
1.040-1.071
1.081 -1.112
1.051 -1.082
1.063-1.093
1.088-1.119
1.090-1.121
0.994-1.008
1.092-1.123
0.956-0.969
1.099-1.130
1.103-1.133
1.106-1.137
1.108-1.139
1.108-1.139
1.137-1.168
Retention time and
quantitation reference (4)

lJC12-PCB-52
13C6-HCB
lJC12-PCB-52
1JC6-HCB
lJC12-PCB-52
1JC6-gamma-BHC
njC3-Atrazine
lJC12-PCB-52
1JC6-HCB
Diazinon-d10
Diazinon-d10
lJC12-PCB-52
1JC3-Atrazine
lJC12-PCB-52
njC6-gamma-BHC
njC3-Atrazine
1JC6-Fonofos
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
1JC6-beta-BHC
lJC12-PCB-52
1JC4-Heptachlor
lJC12-PCB-52
1JC6-delta-BHC
lJC12-PCB-52
lJC12-PCB-52
lJC12-Aldrin
lJC12-PCB-52
N/A
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
1JCi2-Aldrin
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
lJC10-Oxychlordane
lJC12-PCB-52
lJC12-Heptachlor-epoxide
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
Quant Ref RT

21:51
15:54
21:51
15:54
21:51
18:15
18:00
21:51
15:54
17:32
17:32
21:51
18:00
21:51
18:15
18:00
18:25
21:51
21:51
21:51
19:26
21:51
19:36
21:51
21:00
21:51
21:51
21:15
21:51
N/A
21:51
21:51
21:51
21:51
21:15
21:51
21:51
21:51
21:51
24:09
21:51
25:11
21:51
21:51
21:51
21:51
21:51
21:51
70

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Method 1699
December 2007
lJC10-t-Chlordane
t-Chlordane
lJC10-t-Nonachlor
t-Nonachlor
c-Chlordane
njC9-alpha-Endosulfan
Alpha-Endosulfan
o.p-DDE
Cyanazine
njC12-Dieldrin
Dieldrin
p.p-DDE
Captan
o.p-DDD
njC12-p,p-DDE
Disulfoton-Sulfone.
njC12-Endrin
Endrin
Perthane
lJC10-c-Nonachlor
c-Nonachlor
lJC12-o,p-DDT
o.p-DDT
1JC9-beta-Endosulfan
p.p-DDD
beta-Endosulfan
p.p-DDT
Endosulfan-sulfate
njC8-Mirex
Mi rex
Hexazinone
njC12-Methoxychlor
Methoxychlor
Endrin-Ketone
lJC12-p,p-DDT
Phosmet
Permethrins-Peak 1
Permethrins-Peak 2
Azinphos-methyl
Cypermethrins-Peak 1
Cypermethrins-Peak 2
Cypermethrins-Peak 3
26:39
26:41
26:48
26:50
27:44
27:51
27:53
28:07
28:13
30:31
30:34
30:38
31:26
32:21
30:36
32:49
32:53
32:56
32:58
33:17
33:19
33:58
33:59
34:30
34:31
34:32
35:54
36:54
39:29
39:30
39:38
39:43
39:44
39:47
35:53
40:55
42:04
42:21
42:39
43:52
44:03
44:11
1.220
1.001
1.227
1.001
1.041
1.275
1.001
0.862
1.291
1.397
1.002
0.940
1.439
0.952
1.492
1.502
1.505
1.002
1.509
1.523
1.001
1.555
1.000
1.579
0.865
1.001
0.900
1.070
1.807
1.000
1.814
1.818
1.000
1.210
1.825
1.873
1.714
1.739
1.737
N/A
N/A
N/A
1.204-1.235
0.995-1.008
1.211 -1.242
0.995-1.007
1.028-1.053
1.259-1.290
0.995-1.007
0.852-0.873
1.276-1.307
1.381 -1.412
0.996-1.007
0.935-0.945
1.423-1.454
0.943-0.962
1.477-1.507
1.487-1.517
1.490-1.520
0.996-1.007
1.494-1.524
1.508-1.539
0.996-1.006
1.539-1.570
0.996-1.005
1.564-1.594
0.857-0.874
0.996-1.006
0.896-0.904
1.060-1.079
1.792-1.822
0.996-1.005
1.799-1.829
1.802-1.833
0.996-1.005
1.200-1.220
1.810-1.841
1.857-1.888
1.707-1.72
1.732-1.746
1.730-1.744
N/A
N/A
N/A
lJC12-PCB-52
lJC10-t-Chlordane
lJC12-PCB-52
lJC10-t-Nonachlor
lJC10-t-Chlordane
lJC12-PCB-52
1JCg-alpha-Endosulfan
lJC12-p,p-DDE
lJC12-PCB-52
lJC12-PCB-52
lJC12-Dieldrin
lJC12-p,p-DDE
lJC12-PCB-52
lJC12-o,p-DDT
lJC12-PCB-52
lJC12-PCB-52
lJC12-PCB-52
lJC12-Endrin
lJC12-PCB-52
lJC12-PCB-52
lJC10-c-Nonachlor
lJC12-PCB-52
1JC-o,p-DDT
lJC12-PCB-52
"C12-p,p-DDT
njCg-beta-Endosulfan
"C12-p,p-DDT
njCg-beta-Endosulfan
lJC12-PCB-52
1JC8-Mirex
lJC12-PCB-52
lJC12-PCB-52
lJC12-Methoxychlor
lJC12-Endrin
lJC12-PCB-52
lJC12-PCB-52
1JC6-Permethrins-Peak 1
njC6-Permethrins-Peak 2
Azinphos-methyl-d6
njC6-Permethrins-Peak 1+2
njC6-Permethrins-Peak 1+2
njC6-Permethrins-Peak 1+2
21:51
26:39
21:51
26:48
26:39
21:51
27:51
30:36
21:51
21:51
30:31
30:36
21:51
33:58
21:51
21:51
21:51
32:53
21:51
21:51
33:17
21:51
33:58
21:51
35:53
34:30
35:53
34:30
21:51
39:29
21:51
21:51
39:43
32:53
21:51
21:51
42:04
42:21
42:33



1 . Retention time of pesticide or labeled compound.
2. Relative retention time (RRT) between the target and reference compounds.
3 . RRT limits based on estimated RRT variability.
4. Labeled compounds that form both the retention time and quantitation reference.
5. Method detection limits (MDLs) and minimum levels of quantitation (MLs) with no interferences present.
                                                                                71

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Method 1699
December 2007
Table 3.  Concentrations of native and labeled pesticides in stock solutions, spiking solutions, and final
extracts
Pesticide
Tecnazene
Hexachlorobenzene
Quintozene
Heptachlor
Alpha-BHC
gamma-BHC (Lindane)
beta-BHC
delta-BHC
Aldrin
Dacthal
Octachlorostyrene
Oxychlordane
Heptachlor epoxide B
Trans-Chlordane
cis-Chlordane
Trans-Nonachlor
cis-Nonachlor
Endosulfan 1 (alpha)
Endosulfan II (beta)
Dieldrin
2,4'-DDD
4,4'-DDD
2,4'-DDE
4,4'-DDE
2,4'-DDT
4,4'-DDT
Perthane
Endrin
Endosulfan sulfate
Mi rex
Methoxychlor
Endrin ketone
Desethylatrazine
Simazine
Atrazine
Ametryn
Metribuzin
Cyanazine
Hexazinone
Stock
(ng/mL)
800
800
1600
600
1200
1200
1200
1200
1200
400
600
1200
600
600
600
800
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
800
1600
1600
1600
400
1600
2000
Spiking
solution
(pg/mL)
800
800
1600
600
1200
1200
1200
1200
1200
400
600
1200
600
600
600
800
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
800
1600
1600
1600
400
1600
2000
In 20 (jL extract
(ng/mL; pg/HL)
40
40
80
30
60
60
60
60
60
20
30
60
30
30
30
40
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
40
80
80
80
20
80
100
72

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Method 1699
December 2007
Permethrin
Cypermethrin
Chlorothalonil
Diazinon
Disulfoton
Phorate
Methamidophos
Diazinon-oxon
Fonofos
Chlorpyriphos-methyl
Parathion-methyl
Pirimphos-methyl
Chlorpyriphos
Fenitrothion
Malathion
Parathion-ethyl
Chlorpyriphos-oxon
Disulfoton sulfone
Azinphos-methyl
Captan
Phosmet (Imidan)
13C6-HCB
13C6-gamma-BHC
13C4-Heptachlor
13C6-beta-BHC
13C6-delta-BHC
13C12-Aldrin
13C10-Oxychlordane
13C10-Heptachlor-epoxide
13C9-alpha-Endosulfan
13C12-Dieldrin
13C10-t-Chlordane
13C10-t-Nonachlor
13C12-p,p-DDE
13C12-Endrin
13C9-beta-Endosulfan
13C10-c-Nonachlor
13C12-o,p-DDT
13C12-p,p-DDT
13C8-Mirex
13C12-Methoxychlor
Azinphos-methyl-d6
Diazinon-d10
13C6-Fonofos
800
4000
800
1600
8000
1600
1600
1600
1600
2000
4000
1600
1600
1600
20000
1600
1600
400
2000
4000
4000
1800
2600
1400
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
800
4000
800
1600
8000
1600
1600
1600
1600
2000
4000
1600
1600
1600
20000
1600
1600
400
2000
4000
4000
1800
2600
1400
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
1600
40
200
40
80
400
80
80
80
80
100
200
80
80
80
1000
80
80
20
100
200
200
90
130
70
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
                                                                                 73

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Method 1699
December 2007
13C3-Atrazine
13C6-Permethrins
13C12PCB52
1600
1600
1600
1600
1600
1600
80
80
80
74

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Method 1699
December 2007
Table 4.  Concentration of pesticides in calibration and calibration verification standards (ng/mL)
Solution concentration (ng/mL)
Pesticide
Tecnazene
Hexachlorobenzene
Quintozene
Heptachlor
alpha-BHC
gamma-BHC (Lindane)
beta-BHC
delta-BHC
Aldrin
Dacthal
Octachlorostyrene
Oxychlordane
Heptachlor epoxide
trans-Chlordane
cis-Chlordane
trans-Nonachlor
cis-Nonachlor
Endosulfan 1 (alpha)
Endosulfan II (beta)
Dieldrin
2,4'-DDD
4,4'-DDD
2,4-DDE
4,4-DDE
2,4-DDT
4,4-DDT
Perthane
Endrin
Endosulfan sulfate
Mi rex
Methoxychlor
Endrin ketone
Desethylatrazine
Simazine
Atrazine
Ametryn
Metribuzin
Cyanazine
Hexazinone
Permethrin
CS-1 (Hi
sens) (1)
2.0
2.0
4.0
1.5
3.0
3.0
3.0
3.0
3.0
1.0
1.5
3.0
1.5
1.5
1.5
2.0
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
2.0
4.0
4.0
4.0
1.0
4.0
5.0
2.0
CS-2
8.0
8.0
16.0
6.0
12.0
12.0
12.0
12.0
12.0
4.0
6.0
12.0
6.0
6.0
6.0
8.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
8.0
16.0
16.0
16.0
4.0
16.0
20.0
8.0
CS-3
16.0
16.0
32.0
12.0
24.0
24.0
24.0
24.0
24.0
8.0
12.0
24.0
12.0
12.0
12.0
16.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
12.0
16.0
32.0
32.0
32.0
8.0
32.0
40.0
16.0
CS-4
(VER)
40.0
40.0
80.0
30.0
60.0
60.0
60.0
60.0
60.0
20.0
30.0
60.0
30.0
30.0
30.0
40.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
30.0
40.0
80.0
80.0
80.0
20.0
80.0
100.0
40.0
CS-5
100.0
100.0
200.0
75.0
150.0
150.0
150.0
150.0
150.0
50.0
75.0
150.0
75.0
75.0
75.0
100.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
75.0
100.0
200.0
200.0
200.0
50.0
200.0
250.0
100.0
CS-6
200.0
200.0
400.0
150.0
300.0
300.0
300.0
300.0
300.0
100.0
150.0
300.0
150.0
150.0
150.0
200.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
150.0
200.0
400.0
400.0
400.0
100.0
400.0
500.0
200.0
                                                                                             75

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Method 1699
December 2007
Cypermethrin
Chlorothalonil
Diazinon
Disulfoton
Phorate
Methamidophos (Monitor)
Diazinon-oxon
Fonofos (Dyfonate)
Chlorpyriphos-methyl
Parathion-methyl
Pirimphos-methyl
Chlorpyriphos (Dursban)
Fenitrothion
Malathion
Parathion-ethyl (Parathion)
Chlorpyriphos-oxon
Disulfoton sulfone
Azinphos-methyl
Captan
Phosmet (Imidan)
13C6-HCB
13C6-gamma-BHC
13C4-Heptachlor
13C6-beta-BHC
13C6-delta-BHC
13C12-Aldrin
13C10-Oxychlordane
13C10-Heptachlor-epoxide
13C9-alpha-Endosulfan
13C12-Dieldrin
13C10-t-Chlordane
13C10-t-Nonachlor
13C12-p,p-DDE
13C12-Endrin
13C9-beta-Endosulfan
13C10-c-Nonachlor
13C12-o,p-DDT
13C12-p,p-DDT
13C8-Mirex
13C12-Methoxychlor
Azinphos-methyl-d6
Diazinon-d10
13C6-Fonofos
13C3-Atrazine
10.0
2.0
4.0
20.0
4.0
4.0
4.0
4.0
5.0
10.0
4.0
4.0
4.0
50.0
4.0
4.0
1.0
5.0
10.0
10.0
90.0
130.0
70.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
40.0
8.0
16.0
80.0
16.0
16.0
16.0
16.0
20.0
40.0
16.0
16.0
16.0
200.0
16.0
16.0
4.0
20.0
40.0
40.0
90.0
130.0
70.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
16.0
32.0
160.0
32.0
32.0
32.0
32.0
40.0
80.0
32.0
32.0
32.0
400.0
32.0
32.0
8.0
40.0
80.0
80.0
90.0
130.0
70.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
200.0
40.0
80.0
400.0
80.0
80.0
80.0
80.0
100.0
200.0
80.0
80.0
80.0
1000.0
80.0
80.0
20.0
100.0
200.0
200.0
90.0
130.0
70.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
500.0
100.0
200.0
1000.0
200.0
200.0
200.0
200.0
250.0
500.0
200.0
200.0
200.0
2500.0
200.0
200.0
50.0
250.0
500.0
500.0
90.0
130.0
70.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
1000.0
200.0
400.0
2000.0
400.0
400.0
400.0
400.0
500.0
1000.0
400.0
400.0
400.0
5000.0
400.0
400.0
100.0
500.0
1000.0
1000.0
100.0
150.0
100.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
76

-------
Method 1699
December 2007
13C6-Permethrins
13C12PCB52
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
1. Additional concentration used for calibration of high sensitivity HRGC/HRMS systems
                                                                                           77

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Method 1699
December 2007
Table 5.  QC acceptance criteria for pesticides in VER, IPR, OPR, and samples1
Pesticide (1) (2)
13C12-Aldrin
13C3-Atrazine
13C6-beta-BHC
13C10-c-Nonachlor
13C6-delta-BHC
13C12-Dieldrin
13C6-Fonofos
13C6-gamma-BHC
13C6-Hexachlorobenzene
13C4-Heptachlor
13C10-Heptachlor-epoxide
13C8-Mirex
13C12-o,p-DDT
13C10-Oxychlordane
13C12-p,p-DDE
13C12-p,p-DDT
13C6-Permethrin-Peak 1
13C6-Permethrin-Peak 2
13C10-T-Chlordane
13C10-T-Nonachlor
13C12-Endrin
13C12-Methoxychlor
13C9-alpha-Endosulfan
13C9-beta-Endosulfan
Diazinon-d10
Azinphos-methyl-d6
o,p'-DDD
o,p'-DDE
o,p'-DDT
p,p'-DDD
p,p'-DDE
p,p'-DDT
Aldrin
Alpha-Endosulfan
beta-Endosulfan
Disulfoton
alpha-BHC
Ametryn
Atrazine
Azinphos-methyl
VER (%)
(3)
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
75-125
70-130
75-125
75-125
75 - 125
75-125
75-125
75 - 125
75 - 125
75 - 125
75-125
75-125
75 - 125
75-125
75-125
75-125
IPR Rec.
Limits %
(4)
6-113
20-133
19-127
18-139
18-135
21 -145
6-108
6-112
6-108
6-115
9-131
6-125
16-180
6-129
29-152
15-180
35-180
35-180
17-130
15-134
22-141
8-180
6-130
6-108
6-130
6-180
55-108
26 - 1 1 1
55-108
47-108
55-108
55-108
55-108
55-108
5-200
5-200
55-108
6-160
55-108
55-108
IPR RSD
Max
75
45
46
47
47
46
63
62
70
67
52
56
51
54
43
52
43
43
47
49
45
54
63
59
54
57
30
30
30
30
30
30
30
30
50
50
30
52
30
30
OPR Rec.
limits (%)
(5)
5-126
18-147
17-141
17-154
16-150
19-161
5-120
5-124
5-120
5-128
8-146
5-138
14-200
5-144
26-169
13-200
32 - 200
31 -200
15-144
13-149
20-157
8-200
5-144
5-120
5-145
5-200
50-120
24-123
50-120
42-120
50-120
50-120
50-120
50-120
5-200
5-200
50-120
5-178
50-120
50-120
Recovery in
samples
(%) (6)
5-120
36-132
32-130
36-139
36-137
40-151
5-132
11 -120
5-120
5-120
27-137
5-120
5-199
23-135
47-160
5-120
35-189
31 -192
21 -132
14-136
35-155
5-120
15-148
5-122
21 -141
20-179














78

-------
Method 1699
December 2007
beta-BHC
c-Chlordane
c-Nonachlor
Captan
Chlorothalonil
Chlorpyrifos
Chlorpyrifos-methyl
Chlorpyrifos-oxon
Octachlorostyrene
Cyanazine
Dacthal
delta-BHC
Desethylatrazine
Diazinon
Diazinon-oxon
Dieldrin
Disulfoton sulfone
Endosulfan-sulfate
Endrin
Endrin-ketone
Fenitrothion
Fonofos
Gamma-BHC
Hexachlorobenzene
Heptachlor
Heptachlor-epoxide
Hexazinone
Malathion
Methamidophos
Methoxychlor
Metribuzin
Mi rex
Oxychlordane
Parathion-ethyl
Parathion-methyl
Perthane
Phorate
Phosmet
Pirimiphos-methyl
Quintozene
Simazine
t-Chlordane
t-Nonachlor
Technazene
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75-125
75 - 125
70-130
75-125
75 - 125
75-125
75-125
75 - 125
75-125
75 - 125
75-125
75-125
75 - 125
75-125
75-125
75 - 125
75-125
75-125
75 - 125
75 - 125
75-125
75 - 125
75-125
75 - 125
75-125
75 - 125
75-125
75-125
75-125
75 - 125
75-125
75 - 125
75-125
75 - 125
75 - 125
75 - 125
75-125
75 - 125
55-108
55-108
55-108
6-108
6-108
21 -147
10-130
6-143
55-158
10-176
18-129
55-108
55-108
55-108
55-144
55-108
6-180
55-180
55-108
55-120
15-168
55-108
55-108
55-108
55-108
55-108
6-154
15-136
6-108
55-108
6-134
55-108
55-108
13-147
7-136
26-180
6-108
14-138
6-151
55-180
55-108
55-108
55-108
55-154
30
30
30
39
47
46
51
43
30
53
46
30
30
30
30
30
79
30
30
30
50
30
30
30
30
30
74
48
68
30
58
30
30
50
53
46
291
49
64
30
30
30
30
30
50-120
50-120
50-120
5-120
5-120
19-163
9-145
5-158
50-175
9-195
16-143
50-120
50-120
50-120
50-160
50-120
5-200
50 - 200
50-120
50-134
14-186
50-120
50-120
50-120
50-120
50-120
5-171
14-151
5-120
50-120
5-149
50-120
50-120
12-164
7-151
24 - 200
5-120
13-153
5-168
50 - 200
50-120
50-120
50-120
50-171












































                                                                                79

-------
Method 1699
December 2007
Total-Cypermethrins
Total-Permethrins
75-125
75-125
55-108
55-180
30
30
50-120
50 - 200


1.      QC acceptance criteria for IPR, OPR, and samples based on a 20 uL extract final volume
2.      For concentrations see Table 3 spike solutions.
3.      Section 15.3.
4.      Section 9.2.
5.      Section 15.6.
6.      Section 9.3: Recovery of labeled compounds from samples.
80

-------
Method 1699
December 2007
Table 6.  Scan functions; exact m/z's (ml and m2), ratios and tolerances; retention times (RTs); and
quantitation references.
Func
-tion
1
2
2
2
2
3
3
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
7
7
7
7
Pesticide
Methamidophos
HCB
Tecnazene
13C6-HCB
Phorate
Desethylatrazine
Alpha-HCH
Atrazine
Simazine
Fonofos
gamma-HCH
Quintozene
13C3-Atrazine
13C6-Fonofos
13C6-gamma-BHC
Diazinon-d10
Disulfoton
Diazinon
Diazinon-oxon
Aldrin
Beta-BHC
Delta-BHC
Heptachlor
13C12-Aldrin
13C6-beta-BHC
13C6-delta-BHC
13C4-Heptachlor
Chlorothalonil.
Chlorpyriphos-methyl
13C12-PCB-52
Octachlorostyrene
Ametryn
Dacthal
Metribuzin
Parathion-methyl
Pirimiphos-methyl
Oxychlordane
13C10-Oxychlordane
Chlorpyriphos
Chlorpyriphos-oxon
m1(1)
93.9642
283.8102
258.8761
289.8303
260.0128
172.0390
218.9116
215.0938
201.0781
246.0302
218.9116
236.8413
218.1038
252.0503
222.9346
282.1074
274.0285
276.0698
273.1004
262.8569
218.9116
218.9116
271.8102
269.8804
222.9346
222.9346
276.8269
263.8816
285.9261
301.9626
270.8443
227.1205
298.8836
198.0701
263.0017
276.0572
262.8569
269.8804
313.9574
269.9490
m2(1)
94.9721
285.8072
260.8732
291.8273
262.0086
174.0360
220.9086
217.0908
203.0752
247.0336
220.9086
238.8384
220.1009
253.0537
224.9317
314.1638
275.0318
304.1011
288.1239
264.854
220.9086
220.9086
273.8072
271.8775
224.9317
224.9317
278.824
265.8786
287.9232
303.9597
272.8413
228.1238
300.8807
199.0735
264.0051
290.0728
264.8540
271.8775
315.9545
271.9462
m1/m2
Ratio

1.25
0.78
1.25
6.92
3.11
2.08
3.08
3.1

2.08
1.56
3.08
1000
0.77
1000



1.56
2.08
2.08
1.25
1.56
0.77
0.77
1.24
0.78
1.44
0.78
0.63

0.78



1.56
1.56
1.44
1.54
Tolerance (+/-)
0.35
0.25
0.35
0.25
0.35
0.35
0.25
0.35
0.35
0.35
0.25
0.35
0.35
0.35
0.25
0.35
0.35
0.35
0.35
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.35
0.35
0.25
0.25
0.35
0.35
0.35
0.35
0.35
0.25
0.25
0.35
0.35
RT
(min)
09:01
15:55
14:44
15:54
16:11
16:50
16:35
18:01
18:21
18:25
18:16
17:39
18:00
18:25
18:15
17:32
18:34
17:44
17:55
21:17
19:27
21:01
19:37
21:15
19:26
21:00
19:36
21:08
21:26
21:51
23:18
22:41
23:18
23:04
22:28
22:42
24:11
24:09
23:33
24:30
Quantified against
labeled standard
13C12-PCB-52
13C6-HCB
13C6-HCB
13C12-PCB-52
13C12-PCB-52
13C3-Atrazine
13C6-gamma-BHC
13C3-Atrazine
13C3-Atrazine
13C6-Fonofos
13C6-gamma-BHC
13C6-HCB
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
Diazinon-d10
Diazinon-d10
13C12-Aldrin
13C6-beta-BHC
13C6-delta-BHC
13C4-Heptachlor
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52

13C12-Aldrin
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C10-Oxychlordane
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
                                                                                            81

-------
Method 1699
December 2007
1
1
1
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
10
10
10
10
10
10
10
10
10
11
11
11
11
11
11
11
11
11
11
Fenitrothion
Malathion
Parathion-ethyl
Heptachlor-epoxide
alpha-Endosulfan
Dieldrin
o.p-DDE
p.p-DDE
13C12-Heptachlor-epoxide
13C9-alpha-Endosulfan
13C12-Dieldrin
13C12-p,p-DDE
13C10-t-Chlordane
13C10-t-Nonachlor
Cyanazine
c-Chlordane
t-Chlordane
t-Nonachlor
Endrin
c-Nonachlor
o.p-DDD
13C12-Endrin
Captan
Disulfoton-Sulfone.
Perthane
beta-Endosulfan
Endosulfan-sulfate
o.p-DDT
p.p-DDD
p.p-DDT
13C9-beta-Endosulfan
13C10-c-Nonachlor
13C12-o,p-DDT
13C12-p,p-DDT
Endrin-ketone
Methoxychlor
Mi rex
13C12-Methoxychlor
13C8-Mirex
13C6-Permethrins-Peak 1
13C6-Permethrins-Peak_2
Azinphos-methyl-d6
Hexazinone
Phosmet
260.0146
283.9942
291.0330
262.8569
262.8569
262.8569
246.0003
246.0003
269.8804
269.8804
269.8804
258.0406
269.8804
269.8804
240.0890
262.8569
262.8569
262.8569
262.8569
262.8569
235.0081
269.8804
263.9653
213.0173
224.1520
264.8540
264.8540
235.0081
235.0081
235.0081
269.8804
269.8804
247.0484
247.0484
247.8521
227.1072
236.8413
239.1475
241.8581
189.1011
189.1011
160.0511
171.0882
160.0399
277.0174
285.0020
292.0364
264.8540
264.8540
264.8540
247.9974
247.9974
271.8775
271.8775
271.8775
260.0376
271.8775
271.8775
242.0861
264.854
264.854
264.854
264.854
264.854
237.0052
271.8775
265.9623
214.0251
223.1487
262.8569
262.8569
237.0052
237.0052
237.0052
271.8775
271.8775
249.0454
249.0454
249.8491
228.1106
238.8384
240.1508
243.8551
190.1045
190.1045
161.0544
172.0916
161.0432



1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
3.06
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.44


0.64
0.64
1.56
1.56
1.56
1.56
1.56
1.56
1.56
0.63

1.56

1.56





0.35
0.35
0.35
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.35
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.35
0.35
0.35
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.35
0.25
0.35
0.25
0.35
0.35
0.35
0.35
0.35
24:07
24:12
24:26
24:14
27:53
30:34
28:07
30:38
25:11
27:51
30:31
32:36
26:39
26:48
28:13
27:44
26:41
26:50
32:56
33:19
32:21
32:53
31:26
32:49
32:58
34:32
36:54
33:59
34:31
35:54
34:30
33:17
33:58
39:53
39:47
39:44
39:30
39:43
39:29
24:33
24:21
24:33
39:38
40:55
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-Heptachlor-epoxide
1 3C9-alpha-Endosulfan
13C12-Dieldrin
13C12-p,p-DDE
13C12-p,p-DDE
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C10-t-Chlordane
13C10-t-Chlordane
13C10-t-Nonachlor
13C12-Endrin
13C10-c-Nonachlor
13C12-o,p-DDT
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C9-beta-Endosulfan
13C9-beta-Endosulfan
13C12-o,p-DDT
13C12-p,p-DDT
13C12-p,p-DDT
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-Endrin
13C12-Methoxychlor
13C8-Mirex
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
13C12-PCB-52
82

-------
Method 1699
                                                                 December  2007
11
11
11
11
11
11
11
11
Permethrins-Peak_1
Cypermethrins-Peak_1
Cypermethrins-Peak_2
Cypermethrins-Peak_3
Permethrins-Peak_2
Azinphos-methyl
Total-Cypermethrins
Total-Permethrins
183.0081
163.0081
163.0081
163.0081
183.0081
160.0511
163.0081
183.0081
184.0843
165.0052
165.0052
165.0052
184.0843
161.0544
165.0052
184.0843

1.56
1.56
1.56


1.56

0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
42:04
43:52
44:03
44:11
42:21
42:39


1 3C6-Permethrins-Peak_1
1 3C6-Permethrins-Peak_1 +2
1 3C6-Permethrins-Peak_1 +2
1 3C6-Permethrins-Peak_1 +2
1 3C6-Permethrins-Peak_2
Azinphos-methyl-d6


1.   Isotopic masses used for accurate mass calculation
     'H
    12C
    13C
    35C1
    37C1
    19F
    14N
    16Q
 1.0078
12.0000
13.0034
34.9689
36.9659
18.9984
14.0031
15.9949
                                                                                                 83

-------
Method 1699
December 2007
Table 7.  Suggested sample quantities to be extracted for various matrices'
Sample matrix2
Example
Percent
solids
Phase
Quantity
extracted
Single-phase
Aqueous
Solid
Organic
Tissue
Drinking water
Groundwater
Treated wastewater
Dry soil
Compost
Ash
Waste solvent
Waste oil
Organic polymer
Fish
Human adipose
<1
>20
<1
	
3
Solid
Organic
Organic
1000 mL
10 g
10 g
10 g
Multi-phase
Liquid/Solid
Aqueous/Solid
Organic/solid
Wet soil
Untreated effluent
Digested municipal sludge
Filter cake
Paper pulp
Industrial sludge
Oily waste
1-30
1-100
Solid
Both
10 g
10 g
Liquid/Liquid
Aqueous/organic
Aqueous/organic/solid
In-process effluent
Untreated effluent
Drum waste
Untreated effluent
Drum waste
<1
>1
Organic
Organic & solid
10 g
10 g
1 .   The quantity of sample to be extracted is adjusted to provide 10 g of solids (dry weight). One liter of aqueous
    samples containing one percent solids will contain 10 grams of solids. For aqueous samples containing greater
    than one percent solids, a lesser volume is used so that 10 grams of solids (dry weight) will be extracted.

2.   The sample matrix may be amorphous for some samples. In general, when the pesticides are in contact with a
    multi-phase system in which one of the phases is water, they will be preferentially dispersed in or adsorbed on
    the alternate phase because of their low water solubility.
84

-------
Method 1699
December 2007
3.  Aqueous samples are filtered after spiking with the labeled compounds. The filtrate and the materials trapped
    on the filter are extracted separately, and the extracts are combined for cleanup and analysis.

Table 8. Performance data from single laboratory validation.



13C12-ENDRIN
13C12-METHOXYCHLOR
13C9-ALPHA-
ENDOSULPHAN
13C9-BETA-
ENDOSULPHAN
13C-ALDRIN
13C-ATRAZINE
13C-BETA-HCH
13C-C-NONACHLOR
13C-DELTA-HCH
13C-DIELDRIN
13C-FONOFOS
13C-GAMMA-HCH
13C-HCB
13C-HEPTACHLOR
13C-HEPTACHLOR-
EPOXIDE
13C-MIREX
13C-O.P-DDT
13C-OXYCHLORDANE
13C-P.P-DDE
13C-P.P-DDT
13C-PERMETHRINS-
PEAK 1
13C-PERMETHRINS-
PEAK 2
13C-T-CHLORDANE
13C-T-NONACHLOR
2,4'-DDD
2,4'-DDE
2,4'-DDT
4,4'-DDD
4,4'-DDE
4,4'-DDT
ALDRIN
ALPHA-ENDOSULPHAN
Solid-Based
on 5 samples
Solids
Average
Recovery
90.43
128.41
78.70
41.74
63.24
79.29
82.51
90.04
88.30
93.89
50.73
68.29
52.33
68.85
83.80
79.07
123.18
83.09
99.83
140.36
116.88
118.48
86.23
87.23
81.14
84.86
97.46
45.89
99.45
95.81
97.30
94.02
Solids
Standard
Deviation
31.77
32.12
35.89
33.54
40.59
33.93
31.58
31.72
31.10
31.51
40.18
32.52
31.93
33.42
31.90
31.94
30.97
30.78
30.31
30.94
32.00
32.32
29.38
31.29
1.09
15.50
0.50
3.04
0.67
0.94
1.88
4.50
Solids
Relative
Standard
Deviation
28.73
41.25
28.25
14.00
25.67
26.90
26.05
28.56
27.46
29.59
20.38
22.21
16.71
23.01
26.73
25.25
38.16
25.58
30.25
43.42
37.40
38.30
25.34
27.30
0.89
13.15
0.48
1.40
0.66
0.90
1.83
4.23
Reagent Water-Based
on 4 samples
Water
Average
Recovery
86.81
100.11
58.13
29.95
44.20
87.33
74.98
79.79
76.82
85.15
49.40
48.74
29.07
42.58
68.38
59.67
99.18
62.67
94.99
110.00
128.45
130.18
71.73
74.09
102.00
58.65
98.77
46.43
92.40
97.66
99.82
91.74
Water
Standard
Deviation
8.60
7.61
8.21
7.71
23.24
6.03
6.06
5.75
6.14
5.83
27.30
23.03
37.79
27.19
10.13
9.65
7.07
10.04
6.26
4.43
6.05
6.11
7.67
7.72
1.33
7.59
0.77
1.70
1.23
0.66
5.14
4.03
Water
Relative
Standard
Deviation
7.46
7.62
4.77
2.31
10.27
5.27
4.55
4.59
4.72
4.96
13.49
11.22
10.98
11.58
6.93
5.76
7.02
6.29
5.95
4.87
7.77
7.95
5.50
5.72
1.36
4.45
0.76
0.79
1.14
0.64
5.13
3.70
Biosolids-Based on 8 (native)
or 6 (label) samples
Biosolids
Average
Recovery
104.57
20.64
99.81
84.12
55.15
85.34
84.55
89.72
91.15
103.06
90.65
71.55
54.51
55.32
90.61
46.84
48.25
87.54
111.80
33.09
97.01
94.06
72.27
66.33
270.95
101.19
98.26
430.55
102.43
97.19
97.94
90.06
Biosolids
Standard
Deviation
13.16
53.78
7.05
10.42
13.54
7.72
11.92
8.54
6.01
5.37
8.48
17.26
17.21
34.65
7.70
24.47
43.86
11.61
5.68
50.74
11.02
11.15
21.41
26.20
81.98
2.88
9.75
97.71
7.38
7.90
15.24
13.49
Biosolids
Relative
Standard
Deviation
13.76
11.10
7.04
8.76
7.47
6.59
10.08
7.66
5.48
5.53
7.69
12.35
9.38
19.17
6.98
11.46
21.16
10.16
6.34
16.79
10.69
10.49
15.47
17.38
222.13
2.91
9.58
420.69
7.56
7.68
14.93
12.15
                                                                                                   85

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Method 1699
December 2007
ALPHA-HCH
AMETRYN
ATRAZINE
AZINPHOS-METHYL
BETA-ENDOSULPHAN
BETA-HCH
CAPTAN
C-CHLORDANE
CHLOROTHALONIL
CHLORPYRIPHOS
CHLORPYRIPHOS-METHYL
CHLORPYRIPHOS-OXON
CL8-STYRENE
C-NONACHLOR
CYANAZINE
D10-DIAZINON
D6-AZINPHOS-METHYL
DACTHAL
DELTA-HCH
DESETHYLATRAZINE
DIAZINON
DIAZINON-OXON
DIELDRIN
DIMETHOATE
DISULFOTON
DISULFOTONSULFONE
ENDOSULPHAN-
SULPHATE
ENDRIN
ENDRIN-KETONE
ETHION
FENITROTHION
FONOFOS
GAMMA-HCH
HCB
HEPTACHLOR
HEPTACHLOR-EPOXIDE
HEXAZINONE
MALATHION
METHAMIDOPHOS
METHOXYCHLOR
METRIBUZIN
MIREX
OXYCHLORDANE
PARATHION-ETHYL
86.44
38.46
99.47
95.50
*
101.26
2.03
97.89
18.32
95.97
82.40
0.64
122.49
99.49
104.03
77.75
93.60
81.66
102.16
99.84
98.58
*
101.70
75.95
*
477.45
231 .97
101.22
104.25
90.12
101.29
106.68
95.92
102.21
100.14
101.40
27.26
83.84
29.71
101.14
80.54
103.40
97.11
91.86
2.34
57.63
1.51
1.32
*
1.23

1.77
43.37
30.61
31.23
42.62
16.49
1.65
38.26
36.37
42.99
31.90
1.56
2.63
1.85
*
0.95
38.51
*
41.55
1.94
3.00
10.11
61.34
35.89
2.93
1.44
0.10
1.30
1.84
90.24
34.77
37.83
2.51
37.63
2.45
0.88
34.54
2.03
22.17
1.50
1.27
*
1.24

1.73
7.94
29.38
25.73
0.27
20.20
1.64
39.80
28.28
40.24
26.05
1.59
2.63
1.83
*
0.97
29.25
*
198.40
4.50
3.04
10.54
55.28
36.36
3.13
1.39
0.10
1.30
1.87
24.60
29.15
11.24
2.54
30.31
2.53
0.86
31.72
80.00
105.31
98.95
91.43
*
103.95
39.76
95.29
46.46
85.71
70.07
84.10
109.15
99.95
99.88
71.13
118.19
77.16
99.31
86.50
98.08
131.28
103.87
85.52
*
651.23
271 .05
103.49
95.59
49.68
98.41
98.46
94.86
102.89
99.58
101.50
89.16
81.02
*
98.17
67.41
94.01
103.17
82.74
12.67
6.94
0.76
2.10
*
0.62
15.77
2.47
12.52
9.45
17.14
14.59
12.37
0.99
5.26
15.58
4.83
12.06
3.08
4.41
1.36
5.15
0.71
10.34
*
39.89
3.40
1.60
8.87
4.65
9.50
2.43
0.82
2.58
3.27
2.31
19.32
7.61
*
1.44
5.70
3.69
2.14
7.33
10.14
7.31
0.75
1.92
*
0.65
6.27
2.36
5.82
8.10
12.01
12.27
13.50
0.99
5.25
11.08
5.71
9.31
3.06
3.81
1.33
6.76
0.74
8.84
*
259.79
9.22
1.66
8.48
2.31
9.35
2.40
0.78
2.65
3.26
2.35
17.23
6.17
*
1.41
3.84
3.46
2.21
6.06
94.43
124.18
108.25
92.45
97.41
96.72
*
109.10
5.43
112.15
95.67
59.89
132.75
99.96
121.57
88.11
90.12
100.50
97.90
111.16
93.14
82.64
95.23
114.22
60.23
139.98
88.54
98.44
71.24
167.53
151.55
98.06
95.09
100.09
91.71
102.08
130.31
95.88
32.62
105.54
130.08
103.95
93.78
146.11
11.18
15.18
25.55
12.98
12.31
10.14
*
98.97
124.49
9.12
16.32
47.09
7.10
7.50
31.95
16.37
18.63
25.52
9.92
20.68
7.25
36.03
16.77
20.38
46.33
57.09
20.18
9.29
41.83
32.83
17.43
7.60
10.82
7.31
9.12
7.48
21.84
37.53
40.38
9.42
13.38
10.84
12.65
22.28
10.56
18.85
27.66
12.00
11.99
9.81
*
107.98
6.76
10.23
15.61
28.20
9.42
7.50
38.84
14.43
16.79
25.65
9.71
22.99
6.75
29.78
15.97
23.28
27.91
79.91
17.87
9.15
29.80
54.99
26.42
7.45
10.28
7.32
8.37
7.64
28.45
35.98
13.17
9.95
17.41
11.27
11.86
32.55
86

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Method 1699
December 2007
PARATHION-METHYL
PERTHANE
PHORATE
PHOSMET
PIRIMIPHOS-METHYL
QUINTOZENE
SIMAZINE
T-CHLORDANE
TECNAZENE
TERBUFOS
T-NONACHLOR
TOTAL-CYPERMETHRINS
TOTAL-PERMETHRINS
82.54
120.82
5.72
80.19
73.75
129.60
106.25
101.65
120.17
7.50
100.91
93.50
145.58
35.71
31.57
167.39
36.61
48.99
3.06
2.05
5.24
3.76
167.78
1.81
3.29
16.20
29.48
38.14
9.57
29.36
36.13
3.96
2.18
5.33
4.52
12.58
1.83
3.07
23.58
74.72
108.82
14.53
86.54
87.90
163.36
1 04.43
104.93
107.67
20.86
102.74
93.57
1 44.77
13.29
6.85
137.43
8.34
8.09
15.06
3.02
1.01
23.63
127.25
4.02
4.36
2.53
9.93
7.45
19.96
7.22
7.11
24.60
3.15
1.06
25.44
26.54
4.13
4.08
3.66
139.95
144.00
81.78
86.09
112.14
142.76
110.07
104.36
132.60
84.92
94.64
71.64
655.40
16.25
23.38
21.37
75.25
33.53
25.37
11.58
26.02
36.97
26.80
35.56
7.06
124.74
22.74
33.67
17.48
64.78
37.60
36.22
12.75
27.16
49.02
22.75
33.65
5.06
817.53
                                                                                 87

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Method 1699
                     December 2007
      Prep per  11.5
  Spike Labeled Pesticides
         11.5.2.2
                                    Determine % solids
                                         11.2
                                  Determine particle size
                                         11.3
                           -Yes
         Particle
       size > 1mm?
       (from 11.3)
                                     Back extract per
                                         12.5
    Prep per 11.4
Spike Labeled Pesticides
       11.4.2.2
                                                                  Extract per  12.2.1,
                                                                   12.2.2, or  12.2.3
                                 Transfer through Na2SO4
                                       per 12.5.6
    Concentrate per
    12.6-12.7
                                                                     Clean up per
                                                                  13.2- 13.5;  13.7-
                                                                	13.8
                                                                    Concentrate per
                                                                     12.6-12.7
                                                                 Spike injection internal
                                                                   standard per  14.2
                                                                      Analyze per
                                                                      14-18
          Figure 1  Flow Chart for Analysis of Aqueous and Solid Samples
88

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Method 1699
                December  2007
                                                Determine % solids
                                                      11.2
                                                       I.
                                               Determine particle size
                                                      11.3
                                              Spike Labeled Pesticides
                                                     11.6.2.2
                                Aqueous
                  Discard
                                              Pressure filter aliquot per
                                                     11.6.5
Non-aqueous (organic)
            Reserve 10g or amount up
             to 1 L, whichever is less
                                              Transfer through Na2SO4
                                                   per 12.5.6
                 Concentrate per
                  12.6-12.7
                                                                                       Clean up per
                                                                                    13.2- 13.5;  13.7
                                                                                  	13.8
                                                                                      Concentrate per
                                                                                      12.6-12.7
                                                                                   Spike injection internal
                                                                                     standard per 14.2
                                                                                        Analyze per
                                                                                        14-18
                           Figure 2 Flow Chart for Analysis of Multi-Phase Samples
                                                                                                           89

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Method 1699
December 2007
90
                                                   Homogenize tissue
                                                      per 11.8.1
                                                      Remove 10g
                                                     per 11.8.1.4
                                                Spike Labeled Pesticides
                                                      per 11.8.3
                                                     Soxhlet extract
                                                       per 12.4
                                                 Concentrate to dryness
                                                 per 12.4.7- 12.4.8
                                                   Determine % lipids
                                                      per 12.4.9
                                                   Re-dissolve in n-Ce
                                                     per 12.4.9.1
                                                         I.
                                                   Remove lipids per
                                                        13.6
                                                    Concentrate per
                                                     12.6-12.7
                                                         I.
                                                      Clean up per
                                                  13.2- 13.5;  13.7
                                                	 13.8
                                                    Concentrate per
                                                      12.6- 12.7
                                                  Spike injection internal
                                                   standard per  14.2
                                                      Analyze per
                                                      14-18
                                Figure 3  Flow Chart for Analysis of Tissue Samples

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Method 1699
December 2007
                         1000
                         7SO
                         SCO
                         250
            r
                                          l-LltB/SucttooFte*
                            Figure 4  Solid-phase Extraction Apparatus
                                                                                         91

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Method 1699
December 2007
                          Figure 5  Soxhlet/Dean-Stark Extractor
92

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Method 1699
                                                                 December 2007
24.0
            Glossary

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

24.1  Units of weight and measure

      24.1.1  Symbols

             EC   degrees Celsius
             OL   microliter
             m  micrometer
             <    less than
             >    greater than
             %    percent

      24.1.2  Alphabetical abbreviations

             cm   centimeter
             g    gram
                  hour
                  inside diameter
                  inch
                  liter
                  Molecular ion
                  mass or meter
                  milligram
                  minute
                  milliliter
                  millimeter
                  mass-to-charge ratio
                  normal; gram molecular weight of solute divided by hydrogen equivalent of
                  solute, per liter of solution
                  outside diameter
                    h
                    ID
                    in.
                    L
                    M
                    m
                    mg
                    min
                    mL
                    mm
                    m/z
                    N

                    OD
                    Pg
                    ppb
                    ppm
                    ppq
                    ppt
                    psig
                    v/v
                    w/v
                  picogram
                  part-per-billion
                  part-per-million
                  part-per-quadrillion
                  part-per-trillion
                  pounds-per-square inch gauge
                  volume per unit volume
                  weight per unit volume
25.0    Definitions and acronyms (in alphabetical order)

        Analyte - A pesticide tested for by this Method.  The analytes are listed in Table 1.
                                                                                            93

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Method 1699                                                             December 2007

        Calibration standard (CAL) - A solution prepared from a secondary standard and/or stock
        solution and used to calibrate the response of the HRGC/HRMS instrument.

        Calibration verification standard (VER) - The mid-point calibration standard (CS-4) that is
        used to verify calibration.  See Table 4.

        CS-1, CS-2, CS-3, CS-4, CS-5, CS-6 - See Calibration standards and Table 4.

        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.

        GC - Gas chromatograph or gas chromatography

        GPC - Gel permeation chromatograph or gel permeation chromatography

        HPLC - High performance liquid chromatograph or high performance liquid chromatography

        HRGC - High resolution GC

        HRMS - High resolution MS

        Labeled injection internal standard - Labeled PCB52 is spiked into the concentrated extract
        immediately prior to injection of an aliquot of the extract into the HRGC/HRMS.

        Internal standard - a labeled compound used as a reference  for quantitation of other labeled
        compounds and for quantitation of native pesticides other than the pesticide of which it is a
        labeled analog.  See Internal standard quantitation.

        Internal standard quantitation - A means of determining the concentration of (1) a naturally
        occurring (native) compound by reference to a compound other than its labeled analog and (2) a
        labeled compound by reference to another labeled compound.

        IPR - Initial precision and recovery; four aliquots of a reference matrix spiked with the analytes
        of interest and labeled compounds and analyzed to establish the ability of the laboratory to
        generate acceptable precision and recovery. An IPR is performed prior to the first time this
        Method is used and any time the Method or instrumentation is modified.

        Isotope dilution quantitation - A means of determining a naturally occurring (native)
        compound by reference to the same compound in which one or more atoms has been isotopically
        enriched. In this Method, labeled are enriched with carbon-13 to produce 13C-labeled  analogs.
        The 13C-labeled pesticides are spiked into each sample to allow identification and correction of
        the concentration of the native compounds in the analytical process.

        K-D - Kuderna-Danish concentrator; a device used to concentrate the  analytes in  a solvent

94

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Method 1699                                                             December 2007


        Laboratory blank - See Method blank

        Laboratory control sample (LCS) - See Ongoing precision and recovery standard (OPR)

        Laboratory reagent blank - See Method blank

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

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

        Method blank - An aliquot of reagent water that is treated exactly as a sample including
        exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that
        are used with samples. The Method blank is used to determine if analytes or interferences are
        present in the laboratory environment, the reagents, or the apparatus.

        Method detection limit (MDL) - The lowest concentration at which a pesticide can be detected
        under routine operating conditions (see 40 CFR 136, appendix B). MDLs are listed in Table 1.

        Minimum level (ML) - The greater of a multiple of the MDL or the lowest calibration point (see
        68 FR 11790, March 12, 2003.)

        MS - Mass spectrometer or mass spectrometry

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

        OPR - Ongoing precision and recovery standard (OPR); a method blank spiked with known
        quantities of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that
        the results produced by the laboratory remain within the limits specified in this Method for
        precision and recovery.

        Perfluorokerosene (PFK) - A mixture of compounds used to calibrate the exact m/z  scale in the
        HRMS.

        Preparation blank - See Method blank

        Quality control check sample (QCS) - A sample containing all or a subset of the analytes at
        known concentrations. The QCS is obtained from a source external to the laboratory or is
        prepared from a source of standards different from the source of calibration standards.  It is used
        to check laboratory performance with test materials prepared external to the normal preparation
        process.

        Reagent water - water demonstrated to be free from the analytes of interest and potentially
        interfering substances at the method detection limit for the analyte.
        Relative standard deviation (RSD) - The standard deviation times 100 divided by the mean.
        Also termed "coefficient of variation."
                                                                                             95

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Method 1699                                                              December  2007


        RF - Response factor. See Section 10.5

        RR - Relative response.  See Section 10.4

        RSD - See relative standard deviation

        SDS - Soxhlet/Dean-Stark extractor; an extraction device applied to the extraction of solid and
        semi-solid materials (Reference 3 and Figure 5).

        Signal-to-noise ratio (S/N) - The height of the signal as measured from the mean (average) of
        the noise to the peak maximum divided by the width of the noise.

        Should - Although this action, activity, or procedural step is suggested and not required, you
        may be asked why you changed or omitted this action, activity, or procedural step.

        SICP - Selected ion current profile; the line described by the signal at an exact m/z.

        SPE - Solid-phase extraction; an extraction technique in which an analyte is extracted from  an
        aqueous sample by passage over or through a material capable of reversibly adsorbing the
        analyte.  Also termed liquid-solid extraction.

        Stock 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.

        Unique GC resolution or uniquely resolved - Two adjacent chromatographic peaks in which
        the height of the valley is less than 10 percent of the height of the shorter peak (see Section
        6.9.1.1.2).

        VER - See Calibration verification.
96

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