vvEPA
   United States
   Environmental Protection
   Agency
  Method 1614
  Brominated Diphenyl Ethers in Water Soil,
  Sediment and Tissue by HRGC/HRMS


  August 2007

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  U.S. Environmental Protection Agency
            Office of Water
    Office of Science and Technology
Engineering and Analysis Division (4303T)
     1200 Pennsylvania Avenue, NW
        Washington, DC 20460
          EPA-821-R-07-005

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Method 1614


Brominated Diphenyl  Ethers in Water, Soil, Sediment, and Tissue by
HRGC/HRMS

August 2007

Introduction

   EPA Method 1614 was developed by the Office of Water's Office of Science and Technology (OST)
to determine polybrominated diphenyl ether (PBDE) congeners in aqueous, solid, tissue, and multi-phase
matrices.  These ethers are used in brominated flame retardants. The method uses isotope dilution and
internal standard high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS).

   This version revises the August 2003 version to include use of a temperature programmed
injector/vaporizer and a short column to improve recoveries of the octa-, nona- and decabrominated
diphenyl ethers.

Acknowledgments

   EPA Method 1614 was developed under EPA contract by DynCorp Environmental and Interface, Inc.
Data were provided by Axys Analytical Services, Ltd., Sidney, BC, Canada.

Disclaimer

   This PBDE-congener method is patterned after the EPA PCB-congener Method 1668A.  The method
specifications are based on data from Axys Analytical. Method 1614 has been reviewed by the
Engineering and Analytical Support Branch in the Engineering and Analysis Division (EAD) of 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.
Please address your questions or comments to:

CWA Methods Team
Engineering and Analytical Support Branch/EAD  (4303T)
Office of Science and Technology
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue
Washington, DC 20460

E-mail: OSTCWAMethods@,epa.gov
                                EPA Method 1614, August 2007

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Method 1614
Brominated Diphenyl Ether Congeners in Water, Soil, Sediment, Biosolids and
Tissue by HRGC/HRMS
August 2007


1.0 Scope and application

    1.1  EPA Method 1614 ("Method 1614"; the "Method") is for determination of brominated diphenyl
       ether (BDE) congeners in water, soil, sediment, biosolids, tissue, and other sample matrices by
       high resolution gas chromatography combined with high resolution mass spectrometry
       (HRGC/HRMS).

       1.1.1    The 209 BDE congeners are listed in Table 1. The 8 congeners of primary interest
               ("BDEs of primary interest"; Reference 1) are shown in boldface type. Congeners that
               have been reported as being found in environmental samples (References 2 and 3) are
               shown in italics.  Congeners that allow determination of BDEs at those levels of
               bromination not found in environmental samples (References 2 and 3) are shown by
               footnote in Table 1.  Other congeners in Table 1 may be determined as standards become
               available and as needs arise. Data are provided in Tables 2-6 for congeners that have
               been tested.

       1.1.2   This Method can also be used to test for other brominated flame retardants (BFR) and
               brominated organic compounds in the event that new products come on the market.

    1.2 This method is based on EPA Method 1668A (Reference 4) and a method developed by a
       commercial testing laboratory (Reference 5).

    1.3 The detection and quantitation limits in this Method are usually dependent on the level of
       interferences and laboratory backgrounds rather than instrumental limitations.  The method
       detection limits and (MDLs) and minimum levels of quantitation (MLs) in Table 2 are the levels
       at which the BDEs can be determined with laboratory contamination present. The estimated
       MDL for BDE 99 in water is 5 pg/L (picograms-per-liter (pg/L); parts-per-quadrillion (ppq)) with
       no interferences present.

    1.4 The GC/MS portions of this Method are for use only by analysts experienced with HRGC/HRMS
       or under the close supervision of such qualified persons. Each laboratory that uses this Method
       must demonstrate the ability to generate acceptable results using the procedure in Section 9.2.

    1.5 This method is performance-based.  The laboratory is permitted to omit any step or modify any
       procedure (e.g., to overcome interferences or lower the cost of measurements), provided that all
       performance requirements in this method are met.  Requirements for establishing equivalency are
       given in Section 9.1.2, and additionally for Clean Water Act (CWA) uses, at 40 CFR 136.6. For
       use in regulatory programs, such as the CWA, any modification beyond the modifications
       expressly permitted in this method, or at 40 CFR 136.6 is a modification that may require prior
       review and approval.
                                 EPA Method 1614, August 2007

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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, concentration, and cleanup

       2.1.1   Aqueous samples (samples containing less than one percent solids)—Stable isotopically
               labeled analogs of the BDEs are spiked into a 1-L sample.  The sample is extracted using
               solid-phase extraction (SPE), separatory funnel extraction (SFE), or continuous
               liquid/liquid extraction (CLLE).

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

       2.1.3   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,
               dried for a minimum of 30 minutes, 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 After extraction, a labeled cleanup standard is spiked into the extract and the extract is
       concentrated.  Tissue extracts are first cleaned up using an anthropogenic isolation column, and
       all extracts are cleaned up using back-extraction with sulfuric acid and/or base, and gel perme-
       ation, silica gel, and/or Florisil or alumina chromatography, as required.

    2.3 After cleanup, the extract is concentrated to 20 (iL and labeled injection internal standards are
       added. An aliquot of the  extract is injected into the gas chromatograph  (GC).  The analytes are
       separated by the GC and  detected by a high-resolution (>5,000) mass spectrometer.  Two exact
       m/zs are monitored at each level of bromination (LOB) throughout a pre-determined retention
       time window.

    2.4 An individual  BDE congener is identified by comparing the GC retention time and ion-abundance
       ratio of two exact m/zs with the corresponding retention time of an authentic standard and the
       theoretical or acquired ion-abundance ratio of the two exact m/zs.

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

       2.5.1   For a BDE of primary interest and for other congeners of interest for which  a labeled
               analog is available, the GC/MS is multi-point calibrated and the concentration is
               determined using the  isotope dilution technique.

       2.5.2   For a BDE of primary interest for which a labeled analog is not available and for a
               congener of interest for which a multi-point calibration is desired, the GC/MS is multi-
               point calibrated and the concentration is determined using the internal standard technique.
                                   EPA Method 1614, August 2007

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       2.5.3   For other congeners for which a multi-point calibration is not necessary, the GC/MS is
               calibrated at a single concentration and the concentration is determined using the internal
               standard technique.

       2.5.4   For the labeled analogs of the BDEs quantitated by isotope dilution (Section 2.5.1) and
               for the cleanup standards, the GC/MS is calibrated at a single concentration and the
               concentrations of these labeled compounds in samples are determined using the internal
               standard technique.

    2.6 The quality of the analysis is assured through reproducible calibration and testing of the
       extraction, cleanup, and HRGC/HRMS systems.
3.0 Definitions

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


4.0 Contamination and 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.  Environmentally
       abundant BDEs have shown to be difficult to completely eliminate from the laboratory at levels
       lower than the MDLs in this Method (Table 2).

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

       4.2.2   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 - 500 °C) may be
               warranted after particularly dirty samples are encountered. The kiln or furnace should be
               vented to prevent laboratory contamination by BDE vapors.  Baking should be
               minimized, as repeated baking of glassware may cause active sites on the glass surface
               that may irreversibly adsorb BDEs.

       4.2.4   Immediately prior to use, the Soxhlet apparatus  should be pre-extracted for
               approximately 3 hours with the solvent to be used for the extraction (see Sections 12.3.1-
               12.3.3).  Other extraction apparatus  (Section 6.4) should also be rinsed prior to use.
                                   EPA Method 1614, August 2007

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    4.2.5  A separate set of glassware may be necessary to effectively preclude contamination when
           low-level samples are analyzed.

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 BDEs in detectable amounts, but
           should contain potential interferants  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; playground sand (Section
           7.6.2) or white quartz sand (Section  7.3.2) can be used to simulate soil; filter paper
           (Section 7.6.3) can be used to simulate paper and similar materials; and corn oil (Section
           7.6.4) can be  used to simulate tissue.

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 BDEs.  Because low levels of BDEs
    are measured by this Method, the elimination of interferences is essential. The cleanup steps
    given in Section  13 can be used to reduce or eliminate these interferences and thereby permit
    reliable determination of the BDEs at the levels shown in Table 2.

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 Contamination of calibration solutions—The MDLs and MLs in Table 2 are the levels that can be
    achieved with normal laboratory backgrounds present. Many of the MLs are greater than the
    equivalent concentrations of the  calibration solutions.  In order to prevent contamination of the
    calibration solutions with the backgrounds allowed by the MLs, the calibration solutions must be
    prepared in an area free from BDE contamination using glassware free from contamination. If
    these requirements cannot be met or are difficult to meet in the laboratory, the laboratory should
    prepare the calibration solutions  in a contamination-free facility or have a vendor prepare the
    calibration standards and guarantee freedom  from contamination.

4.7 Cleanup of tissue—The natural lipid content of tissue can interfere in the analysis  of tissue
    samples for the BDEs. The lipid contents of different species and portions of tissue can vary
    widely. Lipids are soluble to varying degrees in various organic solvents and may be present in
    sufficient quantity to  overwhelm the  column chromatographic cleanup procedures used for
    cleanup of sample extracts. Lipids must be removed by the anthropogenic isolation column
    procedure in Section  13.5, followed by the gel permeation chromatography procedure in Section
    13.2. Florisil (Section 13.6) and/or alumina  (Section 13.4) are recommended as additional
    cleanup steps.
                               EPA Method 1614, August 2007

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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   BDEs are under investigation as suspected human or mammalian carcinogens. On the
               basis of the available toxicological and physical properties of the BDEs, pure standards
               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.

    5.2 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.  The laboratory should perform personal hygiene monitoring of each analyst who uses
       this Method and the results of this monitoring be made available to the analyst. Additional
       information on laboratory safety can be found in References 6-9. The references and
       bibliography at the end of Reference 8 are particularly comprehensive in dealing with the general
       subject of laboratory safety.

    5.3 The pure BDEs and samples suspected to contain these compounds are handled using essentially
       the same techniques employed  in handling radioactive or infectious materials. Well-ventilated,
       controlled access laboratories are required.  Assistance in  evaluating the health hazards of
       particular laboratory conditions may be obtained from certain consulting laboratories and from
       State Departments of Health or Labor, many of which have an industrial health service. Each
       laboratory must develop a strict safety program for handling these compounds. The practices in
       EPA Method 613 (40 CFR 136, Appendix A, Section 4.1) for handling chlorinated dibenzo-p-
       dioxins  and dibenzofurans (CDDs/CDFs) are also recommended for handling the BDEs.

       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 BDEs, an
               additional set of gloves can also be worn beneath  the latex gloves.
                                  EPA Method 1614, August 2007

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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
       manipulation and before breaks (coffee, lunch, and shift).

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 containing oil or high-boiling
       alcohols to condense BDE 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—An ethanol solution is a less toxic solvent that
           should be effective in removing BDEs. Satisfactory cleaning may be accomplished
           by rinsing with ethanol, then washing with 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 the clothing should be advised of the hazard
       and trained in proper handling. The clothing may be put into a washing machine without
       contact if the launderer knows of the potential problem.  The machine 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 ethanol or other solvent, wipe  an
               area approximately 10x10 cm.

    5.3.10.2    Extract and analyze the wipe by GC with an electron capture detector (ECD) 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.
                           EPA Method 1614, August 2007

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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, 100-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 the 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.

   6.2 Equipment for glassware cleaning

   Note:   If blanks from bottles or other glassware or with fewer cleaning steps than required above
   show no detectable BDE 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 450 °C within 2 hours and maintaining 450 - 500 °C within ±
               10 °C, 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.
                                  EPA Method 1614, August 2007

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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 atemperature of 110 ± 5 °C

       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 samples

       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,  1-L capacity

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

       6.4.1.5 Solid-phase extraction

           6.4.1.5.1   Filtration apparatus, 1-L, 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
                               EPA Method 1614, August 2007

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           6.4.1.5.4  Solid-phase extraction disk containing octadecyl (C18) 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, or equivalent).

    6.4.2  Soxhlet/Dean-Stark (SDS) extractor (Figure 5 and Reference 10) 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 x 123 to fit Soxhlet (Cal-Glass LG-6901-122, or equivalent)

       6.4.2.3 Moisture trap—Dean-Stark or Barrett with fluoropolymer stopcock, to fit Soxhlet

       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—1.5- to 2.0-L, with side arm

    6.5.8  Pressure filtration apparatus—Millipore YT30 142 HW, or equivalent
                               EPA Method 1614, August 2007

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   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)

           6.7.1.1 Column—600-700 mm long x 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-(iL 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  Pipets

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

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

       6.7.3  Glass chromatographic columns

           6.7.3.1 150-mm long  x 8-mm ID, (Kontes K-420155, or equivalent) with coarse-glass frit or
                  glass-wool plug and 250-mL reservoir

           6.7.3.2 200-mm long  x 15-mm ID, with coarse-glass frit or glass-wool plug and 250-mL
                  reservoir

           6.7.3.3 300-mm long x 22-mm ID, with coarse-glass frit, 300-mL reservoir, and glass or
                  fluoropolymer stopcock

       6.7.4  Oven—For baking and storage of absorbents, capable  of maintaining a constant
              temperature (±5 °C) in the range of 105-250 °C
10                                EPA Method 1614, August 2007

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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 shutoff valve at the evaporator
              and vacuum gauge

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

       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)

       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 450 °C 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 tempera-
              ture in the range of 70-100 °C within ±2 °C, installed in a fume hood

    6.8.3   Nitrogen  evaporation apparatus—Equipped with water bath controlled in the range of 30-
           60 °C (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.
                               EPA Method 1614, August 2007                                11

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       6.9.1   GC column—A single- or two-column system may be used, as follows:

           6.9.1.1 Single-column—30±5-m long x 0.25±0.02-mm ID; O.l-^im film; 95% methyl, 4%
                  phenyl, 1% vinyl silicone for high temperature use (J&W DB-5HT, or equivalent).
                  The column must meet the following specifications:

              6.9.1.1.1   The absolute and relative retention times must be approximately equal to
                         those in Table 2.

              6.9.1.1.2   If the additional congeners listed in Tables 2 and 4 are determined, the
                         column must uniquely resolve congener 49 from 71. Unique resolution
                         means a valley height less than 40 percent of the shorter of the two peaks that
                         result when the diluted combined congener solution (Section 7.10.2.2) is
                         analyzed  (see Figure 6). If the BDEs of primary interest only are determined,
                         there is no column resolution test because these congeners are adequately
                         resolved on the DB-5HT column.

              6.9.1.1.3   The tailing factor (see Figure 13 in EPA Method 625; 40 CFR 136, Appendix
                         A) for congener 99L in the CS-3 standard must be less than 3.00.

              6.9.1.1.4   The retention time for decabromodiphenyl ether (DeBDE) must be greater
                         than 48 minutes.

              6.9.1.1.5   The column must be replaced when any of the criteria in Sections 6.9.1.1.1 -
                         6.9.1.1.4 are not met.

           6.9.1.2 Two-column system—If a two-column system is used,  one column to elute the
                  mono- through nona- BDEs and another column to elute the DeBDE separately, the
                  two-column system must meet the following specifications:

              6.9.1.2.1   The retention time for the latest-eluted BDE on the column that elutes the
                         mono- through nona- BDEs must be greater than the retention time for this
                         compound on the DB-5HT column, as shown in Table 2, and the retention
                         time for DeBDE must be greater than  10 minutes on the column used for
                         determination of DeBDE.

              6.9.1.2.2   Congener 49 must be uniquely resolved from congener 71 per Section
                         6.9.1.1.2 on the column that elutes the mono- through nona- BDEs if
                         congeners in addition to the BDEs of primary interest are determined
                         (Section 6.9.1.1.2).

              6.9.1.2.3   The tailing factor specification in Section 6.9.1.1.3 must be met for congener
                         99L on the column that elutes the mono- through nona- BDEs and for
                         DeBDE on its column.

              6.9.1.2.4   The respective column must be replaced when any of the criteria in Sections
                         6.9.1.2.1 -6.9.1.2.3 are not met.

           6.9.1.3 If a column or column system alternate to the column or two-column system  above is
                  used, specifications similar to those for the column or two-column system (Sections
12                                EPA Method 1614, August 2007

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               6.9.1.1 -6.9.1.2) must be developed and must be functionally equivalent to those
               specifications.

    6.9.2 Gas chromatograph short column - Optionally to prevent breakdown of octa, nona and deca
    congeners and improve recovery - temperature-programmed injector TPI (splitless) or on-column
    injection port for capillary column, temperature program for injector and column, with isothermal
    hold, and must meet all of the performance specifications in Section 9 and 10.

       6.9.2.1 GC column-15±l-m long x 0.25±0.02-mm ID; 0.1-|im film; 95% methyl, 4%
               phenyl, 1% vinyl silicone for high temperature use (J&W DB-5HT, or
               equivalent). The column must meet the specifications in sections 6.9.2-6.

       6.9.2.2 The absolute and relative retention times must be approximately
               equal to those in Table 2.

       6.9.2.3 The column must uniquely resolve congener 49 from 71. Unique
               resolution means a valley height less than 40 percent of the shorter of the
               two peaks that result when the diluted combined congener solution (Section
               7.10.2.2) is analyzed (see Figure 6). If the BDEs of primary interest only
               are determined, there is no column resolution test because these congeners
               are adequately resolved on the DB-5HT column.

       6.9.2.4 The tailing factor (see Figure 13 in EPA Method 625; 40 CFR 136,
               Appendix A) for congener 99L in the CS-3 standard must be less than 3.00.

       6.9.2.5 The retention time for decabromodiphenyl ether (DeBDE) must be
               greater than 45 minutes.

       6.9.2.6 The column must be replaced when any of the criteria in Sections
               6.9.2.2 - 6.9.2.5 are not met.

6.10   Mass spectrometer—28- to 40-eV electron impact ionization, must be capable of selectively
       monitoring a minimum of 15 exact m/zs minimum in a single function at high resolution
       (> 5,000) 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 used to test calibration linearity.
           Statistics on initial (Section 9.4) and ongoing (Section 15.5.4) performance should be
           computed and maintained, either on the instrument data system, or on a separate
           computer system.


                               EPA Method 1614, August 2007                                13

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7.0 Reagents and standards

    7.1 pH adjustment and back-extraction

       7.1.1  Potassium hydroxide—Dissolve 20 g reagent grade KOH in 100 mL reagent water.

       7.1.2  Sulfuric acid—Reagent grade (specific gravity 1.84)

       7.1.3  Hydrochloric acid—Reagent grade, 6N

       7.1.4  Sodium chloride—Reagent grade, prepare at 5% (w/v) solution in reagent water

    7.2 Solution drying and evaporation

       7.2.1  Solution drying—Sodium sulfate, reagent grade, granular, anhydrous (Baker 3375, or
              equivalent), rinsed with methylene chloride (20 mL/g), baked at 400 °C 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 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  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

    Note:   Some solvents; e.g., isooctane and nonane, may need to be re-distilled to eliminate BDE
    backgrounds.	

       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 °C for 4 hour 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.

    Note:   At the time  of writing of this method the calibration solution and the dump and collect
    times for BDEs had not been optimized. Because DeBDE has a molecular weight much higher
    than BEHP and methoxychlor, it may be necessary to include DeBDE in the calibration solution.
14                                EPA Method 1614, August 2007

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7.5 Adsorbents for sample cleanup—Any of the adsorbents listed below may be used for sample
    cleanup. Regardless of the cleanup used, the same quantity and type of adsorbent and the same
    procedure must be used for cleanup of all standards and samples.

    7.5.1   Silica gel

       7.5.1.1 Activated silica gel—100-200 mesh, Supelco 1-3651 (or equivalent), 100-200 mesh,
              rinsed with methylene chloride, baked at 180 °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.

       7.5.1.2 Acid silica gel  (30% w/w)—Thoroughly mix 44 g of concentrated sulfuric acid
              (Section 7.1.2) with 100 g of activated silica gel (Section 7.5.1.1) 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 (Section 7.5.1.1) 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 60-70 °C 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 4.8.

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

    7.5.2   Alumina—Either one of two types of alumina, acid or basic, may be used in the cleanup
           of sample extracts, provided that the laboratory can meet the performance specifications
           for the recovery of labeled compounds in Section 9.3.

       7.5.2.1 Acid alumina—Supelco 19996-6C (or equivalent). Activate by heating to 130 °C for
              a minimum of 12 hours.

       7.5.2.2 Basic alumina—Supelco 19944-6C  (or equivalent). Activate by heating to 600 °C for
              a minimum of 24 hours.  Alternatively, activate by heating in a tube furnace at 650 to
              700 °C under an air flow rate of approximately 400 cc/minute. Do not heat  over 700
              °C, as this can lead to reduced capacity for retaining the analytes. Store at 130 °C in
              a covered flask. Use within five days of baking.
                               EPA Method 1614, August 2007                                 15

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       7.5.3  Anthropogenic isolation column—Pack the column in Section 6.7.3.3 from bottom to top
              with the following:

           7.5.3.1 2 g silica gel (Section 7.5.1.1)

           7.5.3.2 2 g potassium silicate (Section 7.5.1.4)

           7.5.3.3 2 g granular anhydrous sodium sulfate (Section 7.2.1)

           7.5.3.4 10 g acid silica gel (Section 7.5.1.2)

           7.5.3.5 2 g granular anhydrous sodium sulfate

       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.

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

              7.5.4.1.2  Immediately prior to use, dry pack a 300-mm x 22-mm ID glass column
                         (Section 6.7.3.3) 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 7.5.4.1.1), and 1-2 cm of warm to hot anhydrous sodium sulfate.
                         Allow the column to cool and wet immediately with 100 mL of n-hexane to
                         prevent water from entering.

           7.5.4.2 Using the procedure in Section 13.6.3, establish the elution pattern for each carton of
                  Florisil or each lot of Florisil columns received.

    7.6 Reference matrices—Matrices in which BDEs 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

       7.6.2  High-solids reference matrix—Playground sand or similar material. Prepared by
              extraction with methylene chloride and/or baking at 450 °C 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 BDEs, but in no case
              must the background level of BDEs in the reference matrix exceed the minimum levels in
              Table 2. If low background levels of the BDEs are present in the reference matrix, the
16                                EPA Method 1614, August 2007

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           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 1 to 2 mg of BDE 99 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 native BDEs of primary interest (see Table
           1, footnote 5) at the concentrations shown in Table 3. If additional BDEs are to be
           determined by multi-point calibration, include the additional native compounds in this
           stock solution.

    7.8.2   Native BDE congener stock solutions—Solutions containing the native congeners to
           calibrate  the DB-5HT  column. If BDE congeners other than the BDEs of primary
           interest are to be determined, and co-elution of these congeners will occur, prepare stock
           solutions that will allow separation of the congeners on the DB-5HT column. For
           example, if it is desired to test for the congeners found in EPA's 2001 literature search
           and the congeners that will cover all levels of bromination (shown in italics and the
           footnotes in Table 1), prepare stocks of the 2 congener solutions in Table 4 at
           concentrations of 20 (ig/mL each for all BDEs except DeBDE and at 200 (ig/mL for
           DeBDE.

Note:  If a column other than the  DB-5HT column is used, solutions that allow separation of the
congeners of interest  on that column must be prepared.	

       7.8.2.1 Combined congener stock solution—Combine equal volumes of the solutions in
               Section 7.8.2 to form a stock solution containing all BDE congeners. Because 2
               solutions  are used, this solution will be at 1A the concentration of the 2 individual
               solutions.

    7.8.3   Stock solutions should be checked for signs of degradation prior to the preparation of
           calibration or performance test standards.  Reference standards that can be used to
           determine the accuracy of standard solutions are available from several vendors.
                               EPA Method 1614, August 2007                                 17

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    7.9 Labeled compound stock solutions

       7.9.1  Labeled compound stock solution—Prepare in isooctane or nonane at the concentrations
              in Table 3. If additional BDEs are to be determined by isotope dilution, include the
              additional labeled compounds in this stock solution.

       7.9.2  Labeled cleanup standard stock solution—Prepare labeled BDE 139L in iso-octane or
              nonane at the concentration shown in Table 3.

       7.9.3  Labeled injection internal standard stock solution—Prepare labeled polychlorinated
              biphenyl (PCB) PCB-52L and PCB-138L in nonane or isooctane at the concentrations
              shown in Table 3.

    7.10   Calibration standards

       7.10.1 Calibration standards—Combine and dilute the solutions in Sections 7.8.1 and 7.9 to
              produce the calibration solutions in Table 5 for the CS-1 to CS-5 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-3 standard is used for
              calibration verification (VER).

       7.10.2 Solutions of congener mixes

           7.10.2.1   Diluted individual solutions—Required if congeners other than the BDEs of
                      primary interest are to be determined.

              7.10.2.1.1 The two individual solutions listed in Table 4, when analyzed individually,
                         allow resolution of all of the BDE congeners on the DB-5HT column, and
                         are used for establishing retention time and other data for each congener.
                         The elution order of the congeners present in each  of the solutions (Section
                         7.8.2.1) is given in Table 2.

              7.10.2.1.2 Individually combine an aliquot of each individual mix stock solution
                         (Section 7.8.2.1) with an aliquot of the labeled stock solution (Section 7.9.1),
                         the labeled cleanup standard stock solution (Section 7.9.2), and the labeled
                         injection internal standard stock solution (7.9.3) to produce concentrations of
                         50 and 500 ng/mL for MoBDE - NoBDE and DeBDE, respectively, for the
                         native compounds; and 100 and 1000 ng/mL for MoBDE - NoBDE and
                         DeBDE, respectively, for the labeled compounds, as shown in the "Extract"
                         column in Table 3.  The congeners will be at the same concentration as in the
                         CS-3 (VER) calibration solution in Table 5.

           7.10.2.2   Diluted combined congener solution—Required if BDEs other than the BDEs of
                      primary interest are to be determined.

              7.10.2.2.1 This solution combines the two individual mixes with the labeled compounds
                         to allow single-point calibration of the congeners not included in the multi-
                         point calibration, and establishes an average response  factor for the co-
                         eluting isomeric congeners.
18                                EPA Method 1614, August 2007

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           7.10.2.2.2 Combine an aliquot of the combined BDE congener solution (Section
                      7.8.2.1) with an aliquot of the labeled stock solution (Section 7.9.1), the
                      labeled cleanup standard stock solution (Section 7.9.2), and the labeled
                      injection internal standard stock solution (7.9.3) to produce the same
                      concentrations as in the diluted individual mix solutions (Section 7.10.2.1.2,
                      the "Extract" column Table 3 and the CS-3 (VER) solution in Table 5).

7.11   Native standard spiking solution—Used for determining initial precision and recovery (IPR;
       Section 9.2) and ongoing precision and recovery (OPR; Section 15.5). Dilute the native stock
       solution (Section 7.8.1) with acetone to produce concentrations of MoBDE - NoBDE and
       DeBDE at 1.0 and 2.0 ng/mL, respectively, as shown in Table 3. When 1 mL of this solution
       spiked into the IPR (Section 9.2.1) or OPR (Section 15.5) and concentrated to a final volume
       of 20 (iL, the concentrations in the final volume will be 50 and 500 ng/mL (pg/(iL).  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 15.5), and blank (Section 9.5) to measure
       recovery. Dilute the labeled stock solution (Section 7.9.1) with acetone to produce
       concentrations of labeled Mo-BDE - NoBDE and DeBDE, respectively, at 2.0 and 20 ng/mL,
       as 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 (iL, the concentrations in the final
       volume will be 100 and 1000 ng/mL (pg/(iL). Prepare  only the amount necessary for each
       reference matrix with each sample batch.

7.13   Labeled cleanup standard spiking solution—This solution is spiked into each extract prior to
       cleanup to measure the efficiency of the cleanup process. Dilute the labeled cleanup standard
       stock solution (Section 7.9.2) in methylene chloride to  produce a concentration of the cleanup
       standard at 2.0 ng/mL, as shown in Table 3.  When 1.0 mL of this solution is spiked into  a
       sample extract and concentrated to a final volume of 20 (iL, the concentration in the final
       volume will be 100 ng/mL (pg/(iL).

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 7.9.3) in nonane to produce a concentration of the
       injection internal standards at 1000 ng/mL, as shown in Table 3. When 2 (iL of this solution
       is spiked into a 20 (iL extract, the concentration of each injection internal standard will be
       nominally 100 ng/mL (pg/(iL).

Note:  The addition of 2 (iL of the labeled injection internal standard spiking solution to a 20 (iL
final extract has the effect of diluting the concentration of the components in the extract by 10%.
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 Standard Reference Material
       (SRM) containing the BDEs in known concentrations in a sample matrix similar to the matrix
       under test.

7.16   Stability of solutions—Standard solutions used for quantitative purposes (Sections 7.9
       through 7.14) should be assayed periodically (e.g., every 6 months) against SRMs from NIST

                               EPA Method 1614, August 2007                                 19

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           (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).

    8.2 Aqueous samples

       8.2.1   Collect samples that flow freely as grab samples or in refrigerated bottles using automatic
               sampling equipment.

       8.2.2   If residual chlorine is present, add 80 mg sodium thiosulfate per liter of water. EPA
               Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 13).

       8.2.3   Maintain aqueous samples in the dark at <6 °C from the time of collection until receipt at
               the laboratory. If the sample will be frozen, allow room for expansion. Store in the dark
               at <6 °C.

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

       8.3.1   Collect samples as grab samples using wide-mouth jars.

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

    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 <6 °C from the time of collection
               until receipt at the laboratory, to a maximum 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 <  -10 °C
               until prepared. Maintain unused sample in the dark at < -10 °C.

    8.5 Holding times

       8.5.1   There are no demonstrated maximum holding  times associated with the BDEs in aqueous,
               solid, semi-solid, tissue, or other sample matrices.  If stored in the dark at <6 °C, aqueous
               samples may be stored for up to one year.  Similarly, if stored in the dark at less than -10
               °C,  solid, semi-solid, multi-phase, and tissue samples may be stored for up to one year.

       8.5.2   Store sample extracts in the dark at less than -10 °C until analyzed. If stored in the dark
               at less than -10 °C, sample extracts may be stored for up to one year.

20                                EPA Method 1614, August 2007

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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 this Method.  If this Method is to be
       applied to a sample  matrix other than water (e.g., soil, 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.

       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.  Alternate
               determinative techniques, such  as substitution of spectroscopic or immunoassay
               techniques,  and changes that  degrade Method performance, are not allowed. If an
               analytical technique other than the techniques specified in this Method is used, that
               technique must have a specificity equal to or greater than the specificity of the techniques
               in this Method for the analytes of interest.

           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 136,
                  Appendix B) are lower than one-third the regulatory compliance limit or one-third
                  the EMDLs in this Method, whichever are greater. If calibration will be affected by
                  the change, the instrument must be recalibrated per Section 10.

           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 modification.

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

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

               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).
                  e)      Analysis of blanks  (Section 9.5).


                                   EPA Method 1614, August 2007                                 21

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                   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 (Section 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 GC column or column system—See Section 6.9.1.3.

       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 15.6.

       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 verification
               and analysis of the ongoing precision and recovery standard (OPR) and blanks that the
               analytical system is in control.  These procedures are given in Sections 15.1 through 15.6.

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

    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 standard spiking solution (Section 7.12), and spike the extracts with the labeled
               cleanup standard spiking solution (Section 7.13), according to the procedures in Sections
               11 through 18. For an alternate sample matrix, four aliquots of the alternate reference
               matrix (Section 7.6) are used.  All sample processing steps that are to be used for


22                                 EPA Method 1614, August 2007

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           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 percent recovery for each BDE in
           each analysis, the average percent recovery (X) for each BDE in the four analyses, and
           the standard deviation of the percent recoveries (relative standard deviation; RSD) for
           each compound, by isotope dilution for BDEs with a labeled analog, and by internal
           standard for BDEs without a labeled analog and for the labeled compounds.

    9.2.3   For each BDE and labeled compound, compare RSD and X with the corresponding limits
           for initial precision and recovery in Table 6.  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 standard spiking solution (Section 7.12) and all sample extracts with the labeled
    cleanup standard spiking solution (Section 7.13).

    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 congeners and the labeled cleanup congener
           using the internal standard method (Section 17.2).

    9.3.3   The recovery of each labeled  compound must be within the limits in Table 6.  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.

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 (SjJ 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 alternative reference matrix blank (Section 7.6.5).


                              EPA Method 1614, August 2007                                23

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       9.5.1   Spike 1.0 mL of the labeled standard spiking solution (Section 7.12) into the method
               blank and 1 mL the labeled cleanup standard spiking solution (Section 7.13) into the
               extract of 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 15.5) to demonstrate freedom from
               contamination.

       9.5.2   If any BDE (Table  1) is found in the blank at greater than the minimum level (Table 2) or
               one-third the regulatory compliance limit, whichever is greater; or if any potentially
               interfering compound is found in the blank at the ML for each BDE given in Table 2
               (assuming a response factor of 1 relative to the quantitation reference in Table 2 at that
               level of bromination 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—The laboratory should analyze a 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 maintained in  a calibrated state.  The standards used for calibration (Section 10),
       calibration verification (Section 15.3), and for initial (Section 9.2) and ongoing (Section 15.5)
       precision and recovery should be identical, so that the most precise results will be obtained. The
       HRGC/HRMS instrument will provide the most reproducible results if dedicated to the settings
       and conditions required for determination of BDEs 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 BDEs in Table 2.

       10.1.1  Suggested GC operating conditions:
           Inj ector temperature:    3 00 °C
           Interface temperature:  320 °C
           Initial temperature:     100 °C
           Initial time:            3 minutes
           Temperature program:  100 - 320 °C @ 5 °C/minute
           Final time:            5 minutes

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


24                                 EPA Method 1614, August 2007

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

10.1.2 Retention time calibration for the BDE congeners

   10.1.2.1   If only the eight BDEs of primary interest are to be analyzed, inject the CS-3
              calibration solution (Section 7.10.1 and Table 5). If congeners in addition to the
              BDEs of primary interest are to be analyzed, separately inject each of the diluted
              individual congener solutions (Section 7.10.2.1.2). Establish the beginning and
              ending retention times at each level of bromination for the scan descriptors in
              Table 7. Scan descriptors other than those listed in Table 7 may be used
              provided the MDLs in Table 2 are met. Store the retention time (RT) and relative
              retention time (RRT) for each congener in the data system.

   10.1.2.2   The absolute retention time of BDE 209 must exceed 48 minutes on the DB-5HT
              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-5HT 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 BDE 209 is greater than 48 minutes on
              such alternate column.

   10.1.2.3   If BDEs other than the eight BDEs of primary interest are to be determined,
              inject the Diluted combined congener solution (Section 7.10.2.2); otherwise, use
              the data from injection of the CS-3 calibration solution (Section 10.1.2.1).
              Adjust the chromatographic conditions and scan descriptors until the
              specifications for the one- or two-column system in Section 6.9.1.1.1 -6.9.1.1.4
              or 6.9.1.2.1 - 6.9.1.2.3, respectively, are met. If an alternate column or column
              system is used, adjust the conditions for that column. If column performance is
              unacceptable, optimize the analysis conditions or replace the column and repeat
              the performance tests.  Confirm that the scan descriptor changes at times when
              BDEs donotelute.

   10.1.2.4   After the column performance tests are passed (Section 10.1.2.2 - 10.1.2.3), store
              the RTs and RRTs for the resolved congeners and the RTs and RRTs for the
              isomeric congeners that co-elute.

10.1.3 GC and Temperature-Programmed Injector Conditions - Optional to prevent breakdown
       of octa, nona and deca congeners and improve recovery. These conditions are optimized
       to target minimization of BDE-209 breakdown, while attempting to resolve and
       quantitate as many of the other target BDE congeners as possible.

       GC Column:
       15 m DB-5ms, 0.25 mm x 0.1  ?m film
       Constant Helium flow at 1.5 mL/min
       TPI Injector:
       Sample Injection Volume: 5 ?L
       Vent Flow: 100 mL/min
                           EPA Method 1614, August 2007                                25

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               Vent Pressure: 2 psi until 0.75 min
               Pure Flow to Spit Vent: 500 mL/min at 5 min
               Temperature Program: 45 °C for 0.8 min, 600 °C/min to 330 °C, hold for 20 min
               GC Temperature Program:
               40 ° for 1.5 min
               45 "C/min to 140 °C
               5  "C/min to 310 °C, hold for 8.0 min
               Total Run Time: 45.72 min
    10.2   Mass spectrometer (MS) resolution

       10.2.1  Using perfluorokerosene (or other reference substance) and a molecular leak, tune the
               instrument to meet a resolving power of 5,000 (10% valley) at m/z 554.9665 or any other
               significant fragment in the range of 540 to 580. For each descriptor (Table 7), monitor
               and record the resolution and exact m/zs of three to five reference peaks covering the
               mass range of the descriptor.  The level of 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 reference substance can contain varying levels of
    contamination, and an excessive amount of reference substance may cause noise problems and
    contamination of the ion source necessitating increased frequency of source cleaning.	

       10.2.2  The analysis time for BDEs may exceed the long-term mass stability of the mass
               spectrometer.  Because the instrument is operated in the high-resolution mode, a mass
               drift of a few ppm (e.g.,  5 ppm in mass) can have a serious adverse effect on instrument
               performance. Therefore, mass-drift correction is mandatory and a lock-mass m/z from
               PFK or other reference substance is used for drift correction. The lock-mass m/z is
               dependent  on the exact m/zs monitored within each descriptor, as shown in Table 7. The
               deviation between each exact m/z monitored and the theoretical m/z (Table 7) must be
               less than 5  ppm.

       10.2.3  Obtain a selected ion current profile (SICP)  at the two exact m/zs specified in Table 7 and
               at >5,000 resolving power at each level of bromination (LOB) for the native congeners
               and congener groups and for the labeled congeners.  Because of the extensive mass range
               covered  in  each function and the requirement for >5,000 resolution throughout the mass
               range during the function, the resolution may need to be greater than 5,000 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 10.2.3) to
               save analysis time.

    10.3   Ion abundance  ratios, minimum levels, and signal-to-noise (S/N) ratios. 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 5) using the GC conditions
           in Section 10.1.1.


26                                EPA Method 1614, August 2007

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    10.3.1 Measure the SICP areas for each congener or congener group, and compute the ion
           abundance ratios at the exact m/zs specified in Table 7. Compare the computed ratio to
           the theoretical ratio given in Table 8.

        10.3.1.1    The exact m/zs to be monitored in each descriptor are shown in Table 7.  Each
                   group or descriptor must be monitored in succession as a function of GC
                   retention time to ensure that the BDEs of interest are detected.  Additional m/zs
                   may be monitored in each descriptor, and the m/zs may be divided among more
                   than the descriptors listed in Table 7, provided that the laboratory is able to
                   monitor the m/zs of all BDEs that elute from the GC in a given level of
                   bromination (LOB) window. The laboratory must also monitor exact m/zs for
                   congeners at higher LOBs to determine if fragments will compromise
                   measurement of congeners at lower LOBs.

        10.3.1.2    The mass spectrometer must be operated in a mass-drift correction mode, using a
                   reference substance to provide lock  m/zs. The lock mass for each group  of m/zs
                   is shown in Table 7. Each lock mass must be monitored and must not vary by
                   more than ±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 required to
                   remove the interference. A lock mass interference or suppression in a retention
                   time region in which BDEs and labeled compounds do not elute may be ignored.

    10.3.2 All BDEs and labeled compounds in the CS-1 standard must be within the QC limits in
           Table 8 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.

    10.3.3 Verify that the HRGC/HRMS instrument meets the minimum levels  (MLs) in Table 2.
           The peaks representing the  BDEs and labeled compounds in the CS-1 calibration
           standard must have S/Ns >  10; otherwise, the mass spectrometer must be adjusted and
           this test repeated until the minimum levels in Table 2 are met.

Note:   The MDLs and MLs in Table 2 are based on the levels of contamination  normally found
in laboratories. Lower levels may be readily achievable  if segregation and extensive cleaning of
glassware  are employed. If lower levels are achievable,  these levels must be established as
described in Section 17.6.1.4.1.	

10.4   Calibration by isotope dilution—Isotope dilution is used for calibration of the BDEs that have
        a labeled analog. The reference compound for each native compound its labeled analog, as
        listed in Table 2.  A 5-point calibration encompassing the concentration range is prepared for
        each native congener.

    10.4.1 For the native BDEs determined by  isotope dilution, the relative response (RR) (labeled
           to native) vs. concentration in the calibration solutions (Table 5) is computed over the
           calibration range according to the procedures described below.
                               EPA Method 1614, August 2007                                27

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       10.4.2  The response of each native BDE relative to its labeled analog is determined using the
               area responses of both the primary and secondary exact m/zs specified in Table 7, for
               each calibration standard, as follows:

                      RR = (Aln+A2J Cl/(All+A2l) Cn

           where:
               Aln and A2n = The measured areas at the primary and secondary m/zs for the BDE
               Alj and A2{ = The measured areas at the primary and secondary m/zs for the labeled
                  compound
               Q  = The concentration of the labeled compound in the calibration standard (Table 5)
               Cn = The concentration of the native compound in the calibration standard (Table 5)

       10.4.3  To calibrate the instrument for the BDEs of primary interest by isotope dilution, inject
               calibration standards CS-1 through CS-5 (Section 7.10.1 and Table 5). 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
               native BDE at each concentration. Compute the average (mean) RR and the RSD of the 5
               RRs.

       10.4.4  Linearity—If the RR for any BDE is constant (less than 20% RSD), the average RR may
               be used for that congener; otherwise, the complete calibration curve for that congener
               must be used over the calibration range.

    10.5   Calibration by internal standard

       10.5.1  Internal standard calibration is applied to determination of the native BDEs for which a
               labeled compound is not available, to determination of the labeled congeners and labeled
               cleanup standard for performance tests and intra-laboratory statistics (Sections 9.4 and
               15.5.4), and to determination of the labeled injection internal standards except for PCB
               138L.  The reference for each congener is listed in Table 2.

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

                  RF = (Als  + A2J C1S / (A11S + A2J Cs

           where:
               Als and A2S = The measured areas at the primary and secondary m/zs for the BDE
               A11S and A21S =  The measured areas at the primary and secondary m/zs for the internal
                  standard
               C1S = The concentration of the internal standard (Table 5)
               Cs = The concentration of the compound in the calibration standard (Table 5)

       10.5.3  Compute the response factor (RF) for all native BDEs except those that have a labeled
               analog. Use the average (mean) response of the labeled compounds at each level of
               bromination (LOB) as the quantitation reference, as shown in Table 2.  For the
               combinations of isomeric congeners that co-elute, compute a combined RF for the co-
               eluted group. For example, for congener 116, the areas at the two exact m/zs for 99L,
               100L,  and 126L are  summed and the total area is divided by 3 (because there are 3
               congeners as the quantitation reference).


28                                EPA Method 1614, August 2007

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   Note:   All labeled congeners at each LOB are used as reference to reduce the effect of
   interferences. 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 method.	

       10.5.4 For the labeled compounds and the labeled cleanup standards, use the nearest eluted
              labeled injection internal standard as the quantitation reference, as given in Table 2.
              Labeled  injection internal standard PCB  52L is referenced to PCB 138L, and PCB  138L
              is referenced to itself, as shown in Table 2.

       10.5.5 Multi-point calibration—Calibrate the instrument for compounds to be multi-point
              calibrated (other than those calibrated by isotope dilution) using data from the injections
              performed for isotope dilution calibration (Section 10.4.3).

       10.5.6 Linearity—If the RF for any compound is constant (less than 35% RSD for all congeners
              other than congener 209L; less than 100% for congener 209L), an average RF may be
              used for that compound; otherwise, a calibration curve must be used for that compound.

       10.5.7 Single-point calibration—Calibrate the instrument for any additional native BDEs by
              injecting the diluted combined congener solution (Section 7.10.2.2).
11.0   Sample preparation

    11.1    Sample preparation involves modifying the physical form of the sample so that the BDEs can
           be extracted efficiently. In general, the samples must be in a liquid form or in the form of
           finely divided solids in order for efficient extraction to take place. Table 9 lists the phases
           and suggested quantities for extraction of various sample matrices.
               For samples known or expected to contain high levels of the BDEs, 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 BDEs 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 are prepared using the procedure 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 BDEs is separated from the non-BDE
               phase using pressure filtration and centrifugation, as described in Section  11.6.  The

                                   EPA Method 1614, August 2007                                29

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           BDEs 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.  This aliquot is used for determining the solids
       content of the sample, not for determination of BDEs.

    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.

       11.2.1.2   Filter 10.0 ± 0.02 mL of well-mixed sample through the filter.

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

       11.2.1.4   Calculate percent solids as follows:

           % solids  =100 (weight of sample aliquot after drying (g) - weight of filter (g) ) /10 g

    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 a 5- to 10-g aliquot of sample to three significant figures in a tared beaker.

       11.2.2.2   Dry a minimum of 12 hours at 110 ± 5 °C, and cool in a desiccator.

       11.2.2.3   Calculate percent solids as follows:

           % solids  =100 weight of aliquot after drying (g) /weight of aliquot before drying (g)

11.3   Estimation of particle size

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

    11.3.2 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 Prepare using the procedure below and extract using the one of the extraction techniques
           in Section 12.2.
                               EPA Method 1614, August 2007

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    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 ±1 g.

       11.4.2.2   Spike 1.0 mL of the labeled standard 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.

       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 standard spiking solution (Section 7.12) into both
                  reagent water aliquots. Spike 1.0 mL of the native standard spiking solution
                  (Section 7.11) into one reagent water aliquot. This aliquot will serve as the OPR
                  (Section 15.5).  The other aliquot will serve as the method blank.

       11.4.2.5   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.

    11.5.2 Spike 1.0 mL of the labeled standard spiking solution (Section 7.12)  into the sample.

    11.5.3 For each sample or sample batch (to a maximum of 20 samples) to be extracted during
           the same  12 hour shift, weigh two 10-g aliquots of the appropriate reference matrix
           (Section 7.6) into clean beakers or glass jars.

    11.5.4 Spike 1.0 mL of the labeled standard spiking solution (Section 7.12)  into both reference
           matrix aliquots. Spike 1.0 mL of the native standard spiking  solution (Section 7.11) into
           one reference matrix aliquot. This aliquot will serve as the OPR (Section 15.5). The
           other aliquot will serve as the method blank.

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

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

    11.5.7 If particles >1 mm are present in the sample (as  determined in Section 11.3.2), spread the
           sample on clean aluminum foil in ahood. Observe the precaution in  Section 5.3.1. After
           the sample is dry, grind to reduce the particle size (Section 11.7).

    11.5.8 Extract the sample and QC aliquots using the SDS procedure  in Section  12.3.
                               EPA Method 1614, August 2007                                 31

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11.6   Multi-phase samples

    11.6.1  Using the percent solids determined in Section 11.2.1 or 11.2.2, determine the volume of
           sample that will provide 10 g of solids, up to 1 L of sample.

    11.6.2  Spike 1.0 mL of the labeled standard spiking solution (Section 7.12) into the amount of
           sample determined in Section 11.6.1, and into the OPR and blank.  Spike  1.0 mL of the
           native standard spiking solution (Section 7.11) into the OPR. 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.

    11.6.3  Discard any aqueous phase (if present).  Remove any non-aqueous  liquid present and
           reserve the maximum amount filtered from the sample (Section 11.6.1)  or 10 g,
           whichever is less, for combination with the solid phase (Section 12.3.5).

    11.6.4  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.

    11.6.5  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 applied to samples and 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 11.6.5 in a clean grinder. Do
           not allow the sample temperature to exceed 50 °C.  Grind the blank and reference matrix
           aliquots using a clean grinder.

    11.7.4  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 11.6.5 for the sample, blank, and OPR aliquots.

    11.7.5  Extract the aliquots using  the SDS procedure in Section 12.3.


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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  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 a minimum of 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 -10 °C. Return any tissue
                  that was not homogenized to its original container and store at less than -10 °C.

    11.8.2 QCaliquots

       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.

       11.8.2.2  Prepare a 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. If the initial precision and recovery test is to be
                  performed, use four aliquots; if the ongoing precision and recovery test is to be
                  performed, use a single aliquot.

    11.8.3 Spiking

       11.8.3.1   Spike 1.0 mL of the labeled standard spiking solution (Section 7.12) into the
                  sample, blank, and OPR aliquot.

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

    11.8.4 Extract the aliquots using the procedures  in Section 12.4.
                               EPA Method 1614, August 2007                                 33

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12.0   Extraction and concentration

    12.1    Extraction procedures include solid phase (Section 12.2.1), separatory runnel (Section
           12.2.2), and continuous liquid/liquid (Section 12.2.3) for aqueous liquids; Soxhlet/Dean-
           Stark (Section 12.3) for solids and filters; and Soxhlet extraction (Section 12.4) for tissues.
           Acid/base back-extraction (Section 12.5) is used for initial cleanup of extracts.

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

    12.2   Extraction of aqueous liquids

       12.2.1  SPE 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-wet the 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 thick on the filter. Do not allow the filter/disk to go dry 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 11.4.2.5) 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  11.4.2.4) 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.
                                   EPA Method 1614, August 2007

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       12.2.1.2.4 Before all of the sample has been pulled through the filter/disk, add
                  approximately 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 approximately 3 minutes.

    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 (approximately 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.
                  Reassemble 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 approximately 1 mm thickness remains
                  on the filter.

       12.2.1.3.3 Rinse the sample bottle with approximately 20 mL of methylene chloride and
                  transfer to the reservoir. Pull approximately half of the solvent through the
                  filter/disk and release the vacuum.  Allow the filter/disk to soak for
                  approximately 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 60 mL methylene chloride to the empty sample bottle. Seal the bottle and
               shake  60 seconds to  rinse the inner surface. Transfer the solvent to the separa-
               tory 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 (see note  below). Drain the methylene chloride extract through a
               solvent-rinsed glass  funnel approximately one-half full of granular anhydrous


                           EPA Method 1614, August 2007                                 35

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                   sodium sulfate (Section 7.2.1) supported on clean glass-fiber paper into a
                   solvent-rinsed concentration device (Section 12.6).

Note:   If an emulsion forms, the 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 of NaCl, or other physical methods.  Alternatively, solid-phase (Section
12.2.1), continuous liquid/liquid (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 60-mL portions of methylene
                   chloride.  Drain each portion through the sodium sulfate into the concentrator.
                   After the  third extraction, rinse the separatory funnel with at least 20 mL of
                   methylene chloride, and drain this rinse through the sodium sulfate into the
                   concentrator.  Repeat this rinse at least twice. Set aside the funnel with sodium
                   sulfate if the extract is to be combined with the extract from the particles.

        12.2.2.4   Concentrate the extract using one of the macro-concentration procedures in
                   Section 12.6 and 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. Test and adjust the pH after 1-2 hours.
                   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 a 500-mL K-D evaporator flask
                   equipped with a 10-mL concentrator tube. Rinse the distilling flask with 30-50
                   mL of methylene chloride and pour through the drying column.  Concentrate and
                   exchange to hexane per Section 12.6 and back extract per Section  12.5.

12.3   SDS extraction of samples containing particles

    12.3.1 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.

    12.3.2 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.
                               EPA Method 1614, August 2007

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    12.3.3 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.

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

    12.3.5 Load the wet sample and/or filter from Sections 11.5.8, 11.6.5, or 11.7.5 and any non-
           aqueous liquid from Section 11.6.3 into the thimble and manually mix into the sand layer
           with a clean metal spatula, carefully breaking up any large lumps of sample.

    12.3.6 Reassemble the pre-extracted  SDS apparatus, and add a fresh charge of toluene to the
           receiver and reflux flask. Apply power to the heating mantle to begin refluxing. Adjust
           the reflux rate to match the rate of percolation through the sand and silica beds until water
           removal lessens the restriction to toluene flow. Frequently check the apparatus for
           foaming during the first 2 hours of extraction.  If foaming occurs, reduce the reflux rate
           until foaming subsides.

    12.3.7 Drain the water from the receiver at 1-2 hours and 8-9 hours, or sooner if the receiver fills
           with water.  Save the water. Reflux the sample for a total of 16-24 hours.

    12.3.8 Cool and disassemble the apparatus. Remove the distilling flask. Drain the water from
           the Dean-Stark receiver and add any toluene in the receiver to the extract in the flask.
           Record the total volume of water collected.

    12.3.9 Quantitatively transfer the extract to a macro-concentration device (Section 12.6), and
           concentrate to near dryness. Dilute to approximately 100 mL with methylene chloride,
           quantitatively transfer to a 250-mL separatory funnel, and proceed with back-extraction
           (Section 12.5).

12.4   Soxhlet extraction of tissue

Note:   This procedure includes determination of the lipid content of the sample (Sections 12.4.8
- 12.4.9), using the same sample extract that is analyzed by GC/MS. Alternatively, a separate
sample aliquot may be used for the lipid determination. If a separate aliquot is used, 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 CBs 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
           allow to equilibrate 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 12.3.1-12.3.4, substituting
           methylene chloride for the pre-extraction and rinsing, and omitting the quartz sand.

    12.4.3 Reassemble the pre-extracted  Soxhlet apparatus and add a fresh charge of methylene
           chloride to the reflux flask.
                               EPA Method 1614, August 2007                                 37

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    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 mixture 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 evaporation procedure (Section
           12.7) and  a water bath temperature of 60 °C. Weigh the receiver, record the weight, and
           return the receiver to the evaporation apparatus, concentrating the residue until a constant
           weight is obtained.

    12.4.9 Percent lipid determination

        12.4.9.1    Redissolve the residue in the receiver in hexane and spike 1.0 mL of the labeled
                   cleanup standard spiking solution  (Section 7.13) into the solution.

        12.4.9.2   Transfer the residue/hexane to the anthropogenic isolation column (Section 13.5),
                   retaining the boiling chips in the concentration apparatus. Use several rinses to
                   assure that all material is transferred. If necessary, sonicate or heat the receiver
                   slightly to assure that all material is re-dissolved. Allow the receiver to dry.
                   Weigh the receiver and boiling chips.

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

               % lipid = 100 x weight of residue (g) /weight of tissue (g)

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

12.5    Back-extraction with base and acid

    12.5.1 Back-extraction may not be necessary for some samples. For some samples, the presence
           of color in the extract may indicate that back-extraction is necessary.  If back-extraction
           is not performed, spike 1.0 mL of the labeled cleanup standard spiking solution (Section
           7.13) into the extract and concentrate the extract for cleanup or analysis (Section 12.7).
           If back-extraction is necessary,  spike 1.0 mL of the labeled cleanup standard spiking
           solution (Section 7.13) into the  separatory funnels containing the sample and QC extracts
           from Section 12.2.3.4 or  12.3.9.

    12.5.2 Partition the extract against 50 mL of potassium hydroxide solution (Section 7.1.1).
           Shake for 2 minutes with periodic venting into a hood.  Remove and discard the aqueous
           layer. Repeat the base washing until no color is visible in the aqueous layer, to a
           maximum of four washings. Minimize contact time between the extract and the base to
           prevent degradation of the BDEs. Stronger potassium hydroxide solutions may be
           employed for back-extraction, provided that the laboratory meets the specifications for
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           labeled compound recovery and demonstrates acceptable performance using the
           procedure in Section 9.2.

    12.5.3 Partition the extract against 50 mL of sodium chloride solution (Section 7.1.4) in the
           same way as with base.  Discard the aqueous layer.

    12.5.4 Partition the extract against 50 mL of sulfuric acid (Section 7.1.2) in the same way as
           with base.  Repeat the acid washing until no color is visible in the aqueous layer, to a
           maximum of four washings.

    12.5.5 Repeat the partitioning against sodium chloride solution and discard the aqueous layer.

    12.5.6 Pour each extract through a drying column containing 7 to 10 cm of granular anhydrous
           sodium sulfate (Section 7.2.1). Rinse the separatory funnel with 30 to 50 mL of solvent,
           and pour through the drying column.  Collect each extract in a round-bottom flask. Re-
           concentrate the sample and  QC aliquots per Sections 12.6-12.7, 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 the mono- through di- BDEs may be totally or partially lost.	

    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 45 °C. On a daily basis, pre-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, rinse three 2- to 3-mL
                  aliquots of solvent down the feed tube into a waste beaker to preclude sample
                  cross-contamination.

       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.

                               EPA Method 1614, August 2007                                  39

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           12.6.1.5   Proceed to Section 12.6.4 for preparation for back-extraction or 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 12.6.4 for preparation for back-extraction or micro-
                      concentration and solvent exchange.

       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.
40                                 EPA Method 1614, August 2007

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       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 12.6.4 for preparation for back-extraction or micro-concentration and
                  solvent exchange.

    12.6.4 Preparation for back-extraction or micro-concentration and solvent exchange

       12.6.4.1   For back-extraction (Section 12.5), transfer the extract to a 250-mL separatory
                  funnel. Rinse the concentration vessel with small portions of hexane, adjust the
                  hexane volume in the separatory funnel to 10 to 20 mL, and proceed to back-
                  extraction (Section 12.5).

       12.6.4.2   For determination  of the weight of residue in the  extract, or for clean-up
                  procedures other than back-extraction, transfer the extract to a evaporation vial
                  using 2-3 rinses of solvent. Proceed with micro-concentration and solvent
                  exchange (Section 12.7).

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, and/or alumina  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.  A large
           vortex in the solvent may cause analyte loss.

    12.7.3 Lower the vial into a 45 °C 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 -12.4.9 and 13.5.4), blow dry until a
                  constant weight is  obtained.

       12.7.3.2   If the extract is to be concentrated for injection into the GC/MS 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  (iL,  add 2 to 3 mL of the desired
           solvent (methylene chloride for GPC or hexane for the other cleanups) and continue
           concentrating 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 and proceed with GPC cleanup (Section 13.2).

    12.7.6 If the extract is to be cleaned up by column chromatography (silica gel, Florisil, or
           alumina), bring the final volume to 1.0 mL with hexane.  Proceed with column cleanup
           (Sections  13.3, 13.4, or 13.6).

    12.7.7 If the extract is to be concentrated for injection into the GC/MS (Section 14),
           quantitatively transfer the extract to a 0.3-mL conical vial for final concentration, rinsing
           the larger vial with hexane and adding the rinse to the conical vial. Reduce the volume to

                               EPA  Method 1614, August 2007                                 41

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               approximately 100 (iL. Add 20 (iL of nonane to the vial, and evaporate the solvent to the
               level of the nonane. Seal the vial and label with the sample number.  Store in the dark at
               room temperature until ready for GC/MS analysis.  If GC/MS analysis will not be
               performed on the same day, store the vial at less than -10 °C.
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.

       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 13.5).

       13.1.2  Acid, neutral, and basic silica gel (Section 13.3), alumina (Section  13.4), and Florisil
               (Section 13.6) are used to remove non-polar and polar interferences.

       13.1.3  The anthropogenic isolation column (Section 13.5) 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.
42                                EPA Method 1614, August 2007

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       13.2.2.2   Inject the GPC calibration solution and record the signal from the detector.  The
                  elution pattern will be corn oil, DeBDE, BEHP, methoxychlor, perylene, and
                  sulfur.

       13.2.2.3   Set the "dump time" to allow >85% removal the corn oil and >85% collection of
                  DeBDE (see the note in Section 7.4).

       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 the DeBDE 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 the 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-(iL 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   Silica gel cleanup

    13.3.1 Place a glass-wool plug in a 15-mm ID chromatography column (Section 6.7.3.2).  Pack
           the column bottom to top with: 1 g silica gel (Section 7.5.1.1), 4 g basic silica gel
           (Section 7.5.1.3), 1 g silica gel, 8 g acid silica gel (Section 7.5.1.2), 2 g silica gel, and 4 g
           granular anhydrous sodium sulfate (Section 7.2.1). Tap the column to settle the
           adsorbents.

    13.3.2 Pre-elute the column with 50 to 100 mL of hexane. Close the stopcock when the hexane
           is within  1 mm of the sodium sulfate. Discard the eluate.  Check the column for
           channeling.  If channeling is present, discard the column and prepare another.

                               EPA Method 1614, August 2007                                 43

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       13.3.3  Apply the concentrated extract to the column. Open the stopcock until the extract is
               within 1 mm of the sodium sulfate.

       13.3.4  Rinse the receiver twice with 1-mL portions of hexane, and apply separately to the
               column. Elute the BDEs with 25 mL of n-hexane and collect the eluate.

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

       13.3.6  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 strengths of the acid and basic silica gels.  The acid silica
               gel (Section 7.5.1.2) may be increased in strength to as much as 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.

               The use of stronger acid silica gel (44% w/w) may lead to charring of organic compounds
               in some extracts.  The charred material may retain some of the analytes and lead to lower
               recoveries of the BDEs. Increasing the strengths of the acid and basic silica gel may also
               require different volumes of solvent 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.

    13.4   Alumina

       13.4.1  Place a glass-wool plug in a 15-mm ID chromatography column (Section 6.7.3.2).

       13.4.2  If using acid alumina, pack the column by adding 6 g acid alumina (Section 7.5.2.1); if
               using basic alumina, substitute 6 g basic alumina (Section 7.5.2.2).  Tap the column to
               settle the adsorbent.

       13.4.3  Pre-elute the column with 50 to 100 mL of hexane.  Close the stopcock when the hexane
               is within 1 mm of the  alumina.

       13.4.4  Discard the eluate.  Check the column  for channeling. If channeling is present, discard
               the column and prepare another.

       13.4.5  Apply the concentrated extract to the column. Open the stopcock until the extract is
               within 1 mm of the alumina.

       13.4.6  Rinse the receiver twice with 1-mL portions of hexane and apply separately to the
               column. Elute the interfering compounds with 15 mL hexane and discard the eluate.

       13.4.7  The volume of eluting solvents will depend on the choice (acid or basic) and activity of
               the alumina.  Determine the volume by test.

       13.4.8  Elute interferences with n-hexane and  discard the elute.  Elute the BDEs with methylene
               chloride:hexane (50:50 v/v) and collect the eluate.
44                                 EPA Method 1614, August 2007

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

13.5   Anthropogenic isolation column (Reference 15) — Used for removal of lipids from tissue
       extracts.

    13.5.1 Prepare the column as given in Section 7.5.3.

    1 3.5.2 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.

    13.5.3 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 BDEs from the column into the apparatus used for
           concentration (Section 12.4.7) using 200 mL of hexane.
    13.5.4 Remove a small portion (e.g, 50 (iL) 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.

    13.5.5 If necessary, exchange the extract to a solvent suitable for the additional cleanups to be
           used (Section 13.2-13.4 and 13.6).

    13.5.6 Clean up the extract using the procedures in Sections 13.2-13.4 and 13.6. GPC (Section
           13.2) and Florisil (Section 13.6) are recommended as minimum additional cleanup steps.

    13.5.7 Following cleanup, concentrate the extract to 20 (iL as described in Section 12.7 and
           proceed with the analysis in Section 14.

13.6   Florisil cleanup (Reference 16)

    13.6.1 Begin to drain the n-hexane from the column (Section 7.5.4.1.2). Adjust the flow rate of
           eluant to 4.5-5.0 mL/min.

    13.6.2 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.

    1 3.6.3 Elute the BDEs with n-hexane and/or ether in n-hexane and collect the eluate. The exact
           volumes of solvents will need to be  determined for each batch of Florisil. If the BDEs
           are not to be separated according to  polarity, elute all BDEs with ether in n-hexane.

    13.6.4 Concentrate the eluate(s) per Sections 12.6-12.7 for further cleanup or for injection into
           the GC/MS.
                               EPA Method 1614, August 2007                                 45

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14.0   HRGC/HRMS analysis

    14.1    Establish the operating conditions given in Section 10.1.

    14.2   Add 2 (iL 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 with nonane (e.g., 19 (iL if a l-(iL injection is used;
           18 (iL if a 2-(iL injection is used).

    14.3   Inject 1.0 or 2.0 (iL 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.

       14.3.2 Monitor the exact m/zs at each LOB throughout the LOB retention time window. Where
               warranted, monitor m/zs associated with congeners at higher levels of bromination to
               assure that fragments are not interfering with the m/zs for congeners at lower levels of
               bromination. Also where warranted, monitor m/zs associated with interferants expected
               to be present.

       14.3.3 Stop data collection after 13C12-DeBDE has eluted. Return the column to the initial
               temperature for analysis of the next 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 native BDEs and labeled compounds.  For
           these tests, the CS-3 calibration verification (VER) standard (Section 7.10.1 and Table 5) is
           analyzed.  If BDEs in addition to the BDEs of primary interest are to be determined, the
           Diluted combined congener solution (Section 7.10.2.2) is used in place of the VER standard
           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.
46                                 EPA Method 1614, August 2007

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15.3   Calibration verification

    15.3.1  Inject the VER(CS-3) standard using the procedure in Section 14. If BDEs in addition to
           the BDEs of primary interest are to be determined, inject the diluted combined congener
           solution (7.10.2.2).

    15.3.2  The ion abundance ratios for all BDEs must be within the limits in Table 8; 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 BDE 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 BDEs that have a labeled analog by isotope dilution
           (Section 17.1). These concentrations are computed based on the calibration data in
           Section 10.

    15.3.5  For each compound, compare the concentration with the calibration verification limit in
           Table 6. 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). If
           recalibration is required, recalibration for all congeners (Section  10.5) must also be
           performed.

15.4   GC performance

    15.4.1  Retention times

       15.4.1.1   Absolute—The absolute retention times for the congeners in the labeled standard
                  (Section 7.12) in the verification test (Section 15.3) must be within ±15  seconds
                  of the respective retention times in the calibration.

       15.4.1.2   Relative—The relative retention times of native BDEs and labeled compounds in
                  the verification test (Section  15.3) must be within their respective RRT limits in
                  Table 2 (Section 6.9.1.1)  or,  if an alternate column or column system is
                  employed, within their respective RRT limits for the alternate column or column
                  system (Section 6.9.1.3).

       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.
                               EPA Method 1614, August 2007                                 47

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15.4.2 GC minimum analysis time and peak tailing

           15.4.2.1   The peak tailing, resolution, and minimum analysis time specifications in
                      Sections 6.9.1.1.2 - 6.9.1.1.4 must be met for the DB-5HT column or, if an
                      alternate column or column system is employed, must be met as specified for the
                      alternate column or column system (Section 6.9.1.2.1 - 6.9.1.2.3). 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 15.4.1 through 15.4.2), or the system must be
                      recalibrated (Section 10).

           15.4.2.2   After the peak tailing and minimum analysis time specifications are met, update
                      the retention times, relative retention times, and response factors for all
                      congeners except the response factors for the compounds that are multi-point
                      calibrated. For these compounds, the multi-point calibration data must be used
                      (see Section  10.4 and 15.3).

       15.4.3  When using either the TPI in section 6.9.2 or the short column in section 10.1.3 a DeBDE
               breakdown test should be performed - Analyze DeBDE by itself and if more than 10%
               total OcBDE + NoBDE are present, adjust chromatographic conditions to eliminate the
               breakdown.

    15.5   Ongoing precision and recovery

       15.5.1  Analyze the extract of the ongoing precision and recovery (OPR) aliquot (Section
               11.4.2.4, 11.5.4, 11.6.2, or 11.8.3.2) prior to analysis of samples from the same batch.

       15.5.2  Compute the percent recovery of the native compounds that have a labeled analog by
               isotope dilution (Section  10.4). Compute the percent recovery of each labeled compound
               by the internal standard method (Section 10.5).

       15.5.3  For the native BDEs  and labeled compounds, compare the recovery to the OPR limits
               given in Table 6.  If all compounds meet the acceptance criteria, system  performance is
               acceptable and analysis of blanks and samples may proceed.  If, however, any individual
               concentration falls outside of the range given, the extraction/concentration processes are
               not being performed  properly for that compound. In this event, correct the problem, re-
               prepare, extract, and  clean up the sample batch and repeat the ongoing precision and
               recovery test (Section 15.5).

       15.5.4  If desired, add results that pass the specifications in Section 15.5.3 to initial and previous
               ongoing data for each compound in each matrix. Update QC charts to form a graphic
               representation of continued laboratory performance. Develop a statement of laboratory
               accuracy for each congener in each matrix type by calculating the average percent
               recovery (R) and the standard deviation of percent recovery (Sg). 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%.

    15.6   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. The results of the analysis of the blank must meet the
           specifications in Section  9.5.2 before sample analyses may proceed.


48                                EPA Method 1614, August 2007

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16.0   Qualitative determination

   A BDE 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/zs in Table 7 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 BDE detected in a sample extract, and greater than  or equal to 10 for all
           BDEs in the calibration and verification standards (Sections 10.3.3 and 15.3.3).

   16.3   The ratio of the integrated areas of the two exact m/zs specified in Table 7 must be within the
           limit in Table 8, or within ±15 percent of the ratio in the midpoint (CS-3) calibration or
           calibration verification (VER), whichever is most recent.

   16.4   The relative retention time of the peak for a BDE must be within the RRT QC limits specified
           in Table 2 or developed from calibration data or, if an alternate column or column system is
           employed, within its respective RRT QC limits for the alternate column or column system
           (Section 6.9.1.1-6.9.1.3).

           For native BDEs determined by internal standard quantitation, a given BDE congener may
           fall within more than RT window and be misidentified unless the RRT windows are made
           very narrow, as in Table 2. Therefore, consistency of the RT and RRT with other congeners
           and the labeled compounds may be required for rigorous congener identification. Retention
           time regression analysis may aid in this identification.

   16.5   Because of congener 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 bromines from a highly brominated congener may inflate or produce a false
           concentration for a less-brominated congener that elutes at the same retention time.  If
           identification is ambiguous, an experienced spectrometrist (Section 1.4)  must determine the
           presence or absence of the congener.

   16.6   If the criteria for identification in Sections 16.1-16.5 are not met, the congener has not been
           identified and the result for that congener 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 compounds to every sample prior to extraction,
              correction for recovery of the BDE can be made because the native 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.


                                  EPA Method 1614, August 2007                                 49

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       17.1.2 Compute the concentrations in the extract of the BDEs that have a labeled analog using
              the RRs from the calibration data (Section 10.4) and following equation:

              Cex (ng/mL)  = (Aln + A2J  Cl/(All + A2J RR

              where:
              Cex =  concentration of the BDE in the extract (ng/mL) and the other terms are as
                     defined in Section 10.4.2

    17.2   Internal standard quantitation and labeled compound recovery

       17.2.1 Compute the concentrations in the extract of the BDEs other than those that have  a
              labeled analog using the response factors determined from the calibration data (Section
              10.5) and the following equation:

              Cex (ng/mL)  = (Als + A2J Cls/(Alls + A2J RF

              where:
              Cex =  concentration of the BDE in the extract (ng/mL) and the other terms are as
                     defined in Section 10.5.2

       17.2.2 Using the concentration in the extract determined above, compute the percent recovery of
              the labeled standard BDEs and the labeled cleanup standard BDEs using the following
              equation:

              Recovery (%) = 100 x Concentration found (ng/mL) /Concentration spiked (ng/mL)

    17.3   The concentration of a native BDE 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) = 1000 (Cex x Vey.) / Ws

           where:
           Cex = concentration of the BDE in the extract (ng/mL)
           Vex = extract volume (mL)
           Ws = sample dry weight (g)

    17.4   The concentration of a native BDE 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)  = 1000 ( Cex x Vex) /Vs

           where:
           Cex = concentration of the BDE in the extract (ng/mL)
           Vex = extract volume (mL)
           Vs = sample volume (L)
50                                EPA Method 1614, August 2007

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17.5   If the SICP area at either quantitation m/z for any congener 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 BDEs 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 BDE congener
       concentrations, detection limits, and minimum levels to account for the dilution.

17.6   Reporting of results—Results are reported to three significant figures for the BDEs and
       labeled compounds found in all standards, blanks, and samples.

    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.

       17.6.1.4   Reporting level

           17.6.1.4.1 Report results above the minimum level of quantitation (ML) for analyses of
                      blanks, standards, and samples. The MLs in Table 2 are the levels that can
                      be achieved in the presence of common laboratory contamination. A
                      laboratory may establish an ML for a BDE lower than the MLs in Table 2.
                      MLs may be established as low as the lowest calibration point (Table 5)
                      provided that the concentration of the congener in a minimum of 10 blanks
                      for a sample medium (e.g., water, soil, sludge, tissue) is significantly below
                      the ML in Table 2. Significant means that the ML for the congener is no less
                      than 2 standard deviations above the mean  (average) level in the minimum of
                      10 blanks (Reference 17). The blanks must be analyzed during the same
                      period that samples are analyzed, ideally over an approximately 1-month
                      period.

           17.6.1.4.2 Standards (VER, IPR, OPR) and samples—Report the result for each
                      congener at or above the ML to 3 significant figures. Report results  below
                      the ML as 
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                         (Reference 17) is that a result is significantly above the blank level, and the
                         level in the blank may be subtracted, if the result is greater than 2 standard
                         deviations above the mean (average) of results of analyses of 10 or more
                         blanks for a sample medium.

       17.6.2 Results for a BDE 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).

       17.6.3 For a BDE 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 6).

       17.6.4 If requested, the total concentration of all congeners at a given level of bromination
              (homolog; i.e., total TrBDE, total PeBDE, total HxBDE, etc) may be reported by
              summing the concentrations of all congeners identified at that LOB, including the BDEs
              of primary interest and other BDEs.  Also if requested, total BDE may be reported by
              summing all congeners identified at all LOBs.
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. Fragment ions from congeners at higher levels of bromination may interfere
           with determination of congeners at lower levels of bromination.

    18.2   Analyze a smaller aliquot of the sample (Section 17.5) when the extract will not concentrate
           to 20 (iL 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   Several laboratories have reported that backgrounds of many of the BDE congeners are
           difficult to eliminate, and that these backgrounds can interfere with the determination of the
           BDEs in environmental samples. Backgrounds of the BDEs of primary interest (Section
           1.1.1) are common. The effects of contamination on results for these congeners should be
           understood in order to make a reliable determination.

    18.5   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).

       18.5.1  If the recovery of any of the labeled compounds is outside of the normal range (Table 6),
               a diluted sample must be analyzed (Section 17.5).

       18.5.2  If the recovery of any of the labeled compounds in the diluted sample is outside of
               normal range, the calibration verification standard (Section 7.10.1 and Table 5) must  be
               analyzed and calibration verified (Section 15.3).
52                                EPA Method 1614, August 2007

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

       18.5.4 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 operations.  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 feasibly at the source, the
           Agency recommends recycling as the next best option.

    19.2   The BDEs 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.
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 are hazardous and must be neutralized before
           being poured down a drain or must be handled as hazardous waste.

    20.3   The BDEs decompose above 800 °C. Low-level waste such as absorbent paper, tissues,
           animal remains, and plastic gloves may be burned in an appropriate incinerator.  Gross
           quantities (milligrams) should be packaged securely and disposed of through commercial or
           governmental channels that are capable of handling extremely toxic wastes.
                                  EPA Method 1614, August 2007                                53

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    20.4   Liquid or soluble waste should be dissolved in methanol or ethanol and irradiated with
           ultraviolet light with a wavelength shorter than 290 nm for several days. Use F40 BL or
           equivalent lamps. Analyze liquid wastes, and dispose of the solutions when the BDEs can no
           longer be detected.

    20.5   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 NW, Washington, DC 20036.

21.0   Method performance

    Method 1614 was developed in a single laboratory (Reference 18). Figure 8 is a chromatogram
showing method performance at each level of bromination.
22.0   References

1.   Telliard, W.A. and D.R. Rushneck, 2002, Consensus obtained at the "4th Annual Workshop on
    Brominated Flame Retardants in the Environment," June 17-18, 2002, Canada Centre for Inland
    Waters, 867 Lakeshore Road, Burlington, Ontario, Canada
2.   "Results of the Literature Search on Polybrominated Diphenyl Ethers," December 7, 2001, Prepared
    for the Statistics and Analytical Support Branch; Engineering and Analysis Division (4303T); U.S.
    Environmental Protection Agency; 1200 Pennsylvania Ave., NW; Washington, DC 20460
3.   Congeners reported in "Proceedings of the 22nd International Symposium on Halogenated
    Environmental Pollutants and Persistent Organic Pollutants" (Dioxin 2002), Barcelona, Spain, August
    11-16, 2002; Organohalogen Compounds 58 161-249.
4.   "Method 1668, Revision A: Chlorinated Biphenyl Congeners in Water,  Soil, Sediment, and Tissue by
    HRGC/HRMS," December 1999, (EPA-821-R-00-002), Statistics and Analytical Support Branch;
    Engineering and Analysis Division (4303T); U.S. Environmental Protection Agency; 1200
    Pennsylvania Ave., NW; Washington, DC 20460
5.   "Analytical Method for the Determination of Polybrominated Diphenylethers by High Resolution
    GC/MS," July 9, 2002, (MLA-025, Rev 02), Axys Analytical Services,  PO Box 2219, Sidney, BC,
    Canada V8L 3S8.  The Axys method is proprietary and is not available.
6.   "Working with Carcinogens," Department of Health, Education, & Welfare, Public Health Service,
    Centers for Disease Control, NIOSH, Publication 77-206, August 1977, NTIS PB-277256.
7.   "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910.
8.   "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety, 1979.
9.   "Standard Methods for the Examination of Water and Wastewater,"  18th and later editions, American
    Public Health Association, 1015 15th St, NW, Washington, DC 20005,  1-35: Section 1090 (Safety),
    1992.
10. Lamparski, L.L., andNestrick, 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.
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. "Methods 330.4 and 330.5 for Total Residual Chlorine," USEPA, EMSL, Cincinnati, OH 45268, EPA
    600/4-70-020, March 1979.
54                                EPA Method 1614, August 2007

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14. "Handbook of Analytical Quality Control in Water and Wastewater Laboratories," USEPA EMSL,
   Cincinnati, OH 45268, EPA-600/4-79-019, March 1979.
15. Kuehl, D.W., B.C. Butterworth, J. Libal, and P. Marquis, "An Isotope Dilution High Resolution Gas
   Chromatography-High Resolution Mass Spectrometric Method for the Determination of Coplanar
   Polychlorinated Biphenyls: Application to Fish and Marine Mammals," Chemosphere 22:9-10, 849-
   858, 1991.
16. Tessari, J.D., Personal communication with Dale Rushneck, available from the EPA Sample Control
   Center, operated by DynCorp Environmental, 6101 Stevenson Avenue, Alexandria, VA 22304, 703-
   461-2100.
17. Ferrario, J.C., C. Byrne, A.E. Dupuy, Jr., "Background Contamination by Coplanar Polychlorinated
   Biphenyls (PCBs) in Trace Level High Resolution Gas Chromatography/High Resolution Mass
   Spectrometry (HRGC/HRMS) Analytical Procedures" Chemosphere 34:11, 2451-2465, 1997.
18. Initial information for this method was generously provided to EPA by Axys Analytical Systems and
   is contained in this method. No further report is available.
                                 EPA Method 1614, August 2007                               55

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23.0   Tables and Figures
Table 1.  Names and congener numbers for native and labeled bromodiphenyl ether (BDE) congeners
determined by isotope dilution and internal standard HRGC/HRMS
Native BDE congener
Name2
2-MoBDE
3-MoBDE3
4-MoBDE
2,2'-DiBDE
2,3-DiBDE
2,3'-DiBDE
2,4-DiBDE4
2,4'-DiBDE4
2,5-DiBDE
2,6-DiBDE
3,3'-DiBDE
3,4-DiBDE4
3,4'-DiBDE4
3,5-DiBDE
4,4'-DiBDE4
2,2',3-TrBDE
2,2',4-TrBDE4
2,2',5-TrBDE
2,2',6-TrBDE
2,3,3'-TrBDE
2,3,4-TrBDE
2,3,4'-TrBDE
2,3,5-TrBDE
2,3,6-TrBDE
2,3',4-TrBDE4
2,3',5-TrBDE
2,3',6-TrBDE
2,4,4'-TrBDE45
2,4,5-TrBDE
2,4,6-TrBDE4
2,4',5-TrBDE
2,4',6-TrBDE4
2',3,4-TrBDE4
2',3,5-TrBDE
3,3',4-TrBDE4
3,3',5-TrBDE
3,4,4'-TrBDE4
3,4,5-TrBDE
Number
BDE-1
BDE-2
BDE-3
BDE-4
BDE-5
BDE-6
BDE- 7
BDE-8
BDE-9
BDE- 10
BDE- 11
BDE- 12
BDE- 13
BDE- 14
BDE- 15
BDE- 16
BDE-1 7
BDE- 18
BDE- 19
BDE-20
BDE-2 1
BDE-22
BDE-23
BDE-24
BDE-25
BDE-26
BDE-27
BDE-28
BDE-29
BDE-30
BDE-3 1
BDE-32
BDE-33
BDE-34
BDE-35
BDE-36
BDE-3 7
BDE-3 8
Labeled analog1
Name


13C12-4-MoBDE











13C12-4,4'-DiBDE












13C12-2,4,4'-TriBDE










Number


BDE-3L











BDE-15L












BDE-28L










56
EPA Method 1614, August 2007

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Native BDE congener
Name2
3,4',5-TrBDE
2,2',3,3'-TeBDE
2,2',3,4-TeBDE
2,2',3,4'-TeBDE
2,2',3,5-TeBDE
2,2',3,5'-TeBDE
2,2',3,6-TeBDE
2,2',3,6'-TeBDE
2,2',4,4'-TeBDE45
2,2',4,5-TeBDE
2,2',4,5'-TeBDE4
2,2',4,6-TeBDE
2,2',4,6'-TeBDE4
2,2',5,5'-TeBDE
2,2',5,6'-TeBDE
2,2',6,6'-TeBDE
2,3,3',4'-TeBDE
2,3,3',4'-TeBDE
2,3,3',5-TeBDE
2,3,3',5'-TeBDE
2,3,3',6-TeBDE
2,3,4,4'-TeBDE
2,3,4,5-TeBDE
2,3,4,6-TeBDE
2,3,4',5-TeBDE
2,3,4',6-TeBDE
2,3,5,6-TeBDE
2,3',4,4'-TeBDE*
2,3',4,5-TeBDE
2,3',4,5'-TeBDE
2,3',4,6-TeBDE
2,3',4',5-TeBDE
2,3',4',6-TeBDE4
2,3',5,5'-TeBDE
2,3',5',6-TeBDE
2,4,4',5-TeBDE
2,4,4',6-TeBDE4
2',3,4,5-TeBDE
3,3',4,4'-TeBDE4
3,3',4,5-TeBDE
3,3',4,5'-TeBDE4
3,3',5,5'-TeBDE
3,4,4',5-TeBDE
Number
BDE-39
BDE-40
BDE-41
BDE-42
BDE-43
BDE-44
BDE-45
BDE-46
BDE-47
BDE-48
BDE-49
BDE-50
BDE-51
BDE-52
BDE-53
BDE-54
BDE-55
BDE-56
BDE-57
BDE-58
BDE-59
BDE-60
BDE-61
BDE-62
BDE-63
BDE-64
BDE-65
BDE-66
BDE-67
BDE-68
BDE-69
BDE-70
BDE-71
BDE-72
BDE-73
BDE-74
BDE- 7 5
BDE-76
BDE-77
BDE-78
BDE- 7 9
BDE-80
BDE-81
Labeled analog1
Name








13C12-2,2',4,4'-TeBDE





























13C12-3,3',4,4'-TeBDE




Number








BDE-47L





























BDE-77L




EPA Method 1614, August 2007
57

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Native BDE congener
Name2
2,2',3,3',4-PeBDE
2,2',3,3',5-PeBDE4
2,2',3,3',6-PeBDE
2,2',3,4,4'-PeBDE4
2,2',3,4,5-PeBDE
2,2',3,4,5'-PeBDE
2,2',3,4,6-PeBDE
2,2',3,4,6'-PeBDE
2,2',3,4',5-PeBDE
2,2',3,4',6-PeBDE
2,2',3,5,5'-PeBDE
2,2',3,5,6-PeBDE
2,2',3,5,6'-PeBDE
2,2',3,5',6-PeBDE
2,2',3,6,6'-PeBDE
2,2',3',4,5-PeBDE
2,2',3',4,6-PeBDE
2,2',4,4',5-PeBDE45
2,2',4,4',6-PeBDE45
2,2',4,5,5'-PeBDE
2,2',4,5,6'-PeBDE
2,2',4,5,'6-PeBDE
2,2',4,6,6'-PeBDE
2,3,3',4,4'-PeBDE4
2,3,3',4,5-PeBDE
2,3,3',4',5-PeBDE
2,3,3',4,5'-PeBDE
2,3,3',4,6-PeBDE
2,3,3',4',6-PeBDE
2,3,3',5,5'-PeBDE
2,3,3',5,6-PeBDE
2,3,3',5',6-PeBDE
2,3,4,4',5-PeBDE
2,3,4,4',6-PeBDE
2,3,4,5,6-PeBDE4
2,3,4',5,6-PeBDE
2,3',4,4',5-PeBDE
2,3',4,4',6-PeBDE4
2,3',4,5,5'-PeBDE4
2,3',4,5,'6-PeBDE
2',3,3',4,5-PeBDE
2',3,4,4',5-PeBDE
2',3,4,5,5'-PeBDE
Number
BDE-82
BDE-83
BDE-84
BDE-85
BDE-86
BDE-87
BDE-88
BDE-89
BDE-90
BDE-91
BDE-92
BDE-93
BDE-94
BDE-95
BDE-96
BDE-97
BDE-98
BDE-99
BDE-100
BDE-101
BDE- 102
BDE- 103
BDE- 104
BDE- 105
BDE- 106
BDE- 107
BDE- 108
BDE- 109
BDE-110
BDE-111
BDE-112
BDE-113
BDE-114
BDE-115
BDE-116
BDE-117
BDE-118
BDE- 119
BDE- 120
BDE- 121
BDE- 122
BDE-123
BDE- 124
Labeled analog1
Name

















13C12-2,2',4,4',5-PeBDE
13C12-2,2',4,4',6-PeBDE
























Number

















BDE-99L
BDE-100L
























58
EPA Method 1614, August 2007

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Native BDE congener
Name2
2',3,4,5,6'-PeBDE
3,3',4,4',5-PeBDE4
3,3',4,5,5'-PeBDE
2,2',3,3',4,4'-HxBDE4
2,2',3,3',4,5-HxBDE
2,2',3,3',4,5'-HxBDE
2,2',3,3',4,6-HxBDE
2,2',3,3',4,6'-HxBDE
2,2',3,3',5,5'-HxBDE
2,2',3,3',5,6-HxBDE
2,2',3,3',5,6'-HxBDE
2,2',3,3',6,6'-HxBDE
2,2',3,4,4',5-HxBDE
2,2',3,4,4',5'-HxBDE4
2,2',3,4,4',6-HxBDE
2,2',3,4,4',6'-HxBDE4
2,2',3,4,5,5'-HxBDE
2,2',3,4,5,6-HxBDE
2,2',3,4,5,6'-HxBDE
2,2',3,4,5',6-HxBDE
2,2',3,4,6,6'-HxBDE
2,2',3,4',5,5'-HxBDE
2,2',3,4',5,6-HxBDE
2,2',3,4',5,6'-HxBDE
2,2',3,4',5',6-HxBDE
2,2',3,4',6,6'-HxBDE
2,2',3,5,5',6-HxBDE
2,2',3,5,6,6'-HxBDE
2,2',4,4',5,5'-HxBDE45
2,2',4,4',5',6-HxBDE45
2,2',4,4',6,6'-HxBDE4
2,3,3',4,4',5-HxBDE
2,3,3',4,4',5'-HxBDE
2,3,3',4,4',6-HxBDE
2,3,3',4,5,5'-HxBDE
2,3,3',4,5,6-HxBDE
2,3,3',4,5',6-HxBDE
2,3,3',4',5,5'-HxBDE
2,3,3',4',5,6-HxBDE
2,3,3',4',5',6-HxBDE
2,3,3',5,5',6-HxBDE
2,3,4,4',5,6-HxBDE4
2,3',4,4',5,5'-HxBDE
Number
BDE-125
BDE- 126
BDE-127
BDE- 128
BDE-129
BDE-130
BDE-131
BDE-132
BDE-133
BDE- 134
BDE-135
BDE-136
BDE- 137
BDE- 138
BDE- 139
BDE- 140
BDE-141
BDE-142
BDE-143
BDE- 144
BDE-145
BDE-146
BDE-147
BDE-148
BDE-149
BDE-150
BDE-151
BDE-152
BDE-153
BDE-154
BDE-155
BDE- 156
BDE-157
BDE-158
BDE-159
BDE- 160
BDE-161
BDE- 162
BDE- 163
BDE- 164
BDE- 165
BDE- 166
BDE- 167
Labeled analog1
Name

13C12-3,3',4,4',5-PeBDE












13C12-2,2',3,4,4',6-HxBDE













13C12-2,2',4,4',5,5'-HxBDE
13C12-2,2',4,4',5',6-HxBDE













Number

BDE-126L












BDE-139L













BDE-153L
BDE-154L













EPA Method 1614, August 2007
59

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Native BDE congener
Name2
2,3',4,4',5',6-HxBDE
3,3',4,4',5,5'-HxBDE
2,2',3,3',4,4',5-HpBDE
2,2'3,3',4,4',6-HpBDE
2,2',3,3',4,5,5'-HpBDE
2,2',3,3',4,5,6-HpBDE
2,2',3,3',4,5,6'-HpBDE
2,2',3,3',4,5',6-HpBDE
2,2',3,3',4,6,6'-HpBDE
2,2',3,3',4',5,6-HpBDE
2,2',3,3',5,5',6-HpBDE
2,2',3,3',5,6,6'-HpBDE
2,2',3,4,4',5,5'-HpBDE
2,2',3,4,4',5,6-HpBDE4
2,2',3,4,4',5,6'-HpBDE
2,2',3,4,4',5',6-HpBDE45
2,2',3,4,4',6,6'-HpBDE
2,2',3,4,5,5',6-HpBDE
2,2',3,4,5,6,6'-HpBDE
2,2',3,4',5,5',6-HpBDE
2,2',3,4',5,6,6'-HpBDE
2,3,3',4,4',5,5'-HpBDE
2,3,3',4,4',5,6-HpBDE4
2,3,3',4,4',5',6-HpBDE
2,3,3',4,5,5',6-HpBDE
2,3,3',4',5,5',6-HpBDE
2,2',3,3',4,4',5,5'-OcBDE
2,2',3,3',4,4',5,6-OcBDE
2,2',3,3',4,4',5,6'-OcBDE
2,2',3,3',4,4',6,6'-OcBDE
2,2',3,3',4,5,5',6-OcBDE
2,2',3,3',4,5,5',6'-OcBDE
2,2',3,3',4,5,6,6'-OcBDE
2,2',3,3',4,5',6,6'-OcBDE
2,2',3,3',5,5',6,6'-OcBDE
2,2',3,4,4',5,5',6-OcBDE3
2,2',3,4,4',5,6,6'-OcBDE
2,3,3',4,4',5,5',6-OcBDE
2,2',3,3',4,4',5,5',6-NoBDE
2,2',3,3',4,4',5,6,6'-NoBDE
2,2',3,3',4,5,5',6,6'-NoBDE3
DeBDE45
Number
BDE-168
BDE- 169
BDE- 170
BDE-171
BDE- 172
BDE- 173
BDE- 174
BDE- 175
BDE- 176
BDE- 177
BDE-178
BDE- 179
BDE-180
BDE-181
BDE- 182
BDE-183
BDE- 184
BDE-185
BDE-186
BDE- 187
BDE- 188
BDE- 189
BDE- 190
BDE-191
BDE- 192
BDE- 193
BDE- 194
BDE- 195
BDE- 196
BDE- 197
BDE-198
BDE- 199
BDE-200
BDE-201
BDE-202
BDE-203
BDE-204
BDE-205
BDE-206
BDE-207
BDE-208
BDE-209
Labeled analog1
Name















13C12-2,2',3,4,4',5',6-HpBDE

























13C19-DeBDE
Number















BDE-183L

























BDE-209L
60
EPA Method 1614, August 2007

-------
1.   Labeled compound in standard solution

2.   Abbreviations for levels of bromination
    MoBDE    =   monobromodiphenyl ether
    DiBDE     =   dibromodiphenyl ether
    TrBDE     =   tribromodiphenyl ether
    TeBDE     =   tetrabromodiphenyl ether
    PeBDE     =   pentabromodiphenyl ether
    HxBDE     =   hexabromodiphenyl ether
    HpBDE     =   heptabromodiphenyl ether
    OcBDE     =   octabromodiphenyl ether
    NoBDE     =   nonabromodiphenyl ether
    DeBDE     =   decabromodiphenyl ether

3.   Congener suggested for coverage of this level of bromination

4.   Congener (shown in italics) reported in the technical literature in EPA's 2001 literature survey

5.   BDEs of primary interest (shown in bold) as determined at the "4th Annual Workshop on Brominated Flame
    Retardants in the Environment,"  June 17-18, 2002, Canada Centre for Inland Waters, Burlington, Ontario,
    Canada
                                   EPA Method 1614, August 2007
61

-------
Table 2. Retention times (RT), RT references, relative retention times (RRTs), method detection limits (MDLs), and minimum levels (MLs) for
the selected BDE congeners on DB-5HT
Br
No.1
Congener No. 2'3'4
RTRef
RT
(min:sec)6
RRT7
RRT limits8
Window
(sec)9
Quantitation
reference10
Detection limits and minimum levels -
Matrix and concentration11
Water
(pg/L)
MDL
ML
Other
(ng/kg)
MDL
ML
Extract
(pg/nL)
ML
Compounds using PCB-52L (13C12-2,2',5,5'-TeCB) as Labeled injection internal standard
Monobromodiphenyl ether
1
1
1
1
2
3
3L
3L
3L
11:26
11:45
12:04
0.9488
0.9751
1.0014
0.9350-0.9627
0.9613-0.9889
0.9986-1.0055
±10
±10
-2+3
3L
3L
3L

50


200


5


20


10

Dibromodiphenyl ethers
2
2
2
2
2
2
10
7
8/11
12
13
15
15L
15L
15L
15L
15L
15L
15:48
16:59
17:32
17:50
17:54
18:18
0.8642
0.9289
0.9590
0.9754
0.9790
1.0009
0.8459-0.8824
0.9152-0.9426
0.9499-0.9681
0.9663-0.9845
0.9699-0.9881
0.9991-1.0036
±20
±15
±10
±10
±10
-2+3
15L
15L
15L
15L
15L
15L


40





100





4





10





5



Tribromodiphenyl ethers
3
o
J
3
o
J
o
J
3
o
J
30
32
17
25
28/33
35
37
28L
28L
28L
28L
28L
28L
28L
20:26
21:45
22:11
22:17
22:49
23:14
23:41
0.8955
0.9533
0.9722
0.9766
1.0000
1.0183
1.0380
0.8809-0.9102
0.9459-0.9606
0.9649-0.9795
0.9693-0.9839
0.9985-1.0022
1.0110-1.0256
1.0307-1.0453
±20
±10
±10
±10
-2+3
±10
±10
28L
28L
28L
28L
28L
28L
28L

50

20



200

50



5

2



20

5



10

2.5


Tetrabromodiphenyl ethers
4
4
4
4
75
51
49
71
47L
47L
47L
47L
26:04
26:12
26:25
26:33
0.9625
0.9674
0.9754
0.9803
0.9563-0.9686
0.9612-0.9735
0.9692-0.9815
0.9742-0.9865
±10
±10
±10
±10
47L/77L
47L/77L
47L/77L
47L/77L
30



100



3



10



5



62
EPA Method 1614, August 2007

-------
Br
No.1
4
4
4
4
Congener No. 2'3'4
47
79
66
77
RTRef5
47L
47L
47L
77L
RT
(min:sec)6
27:05
27:26
27:40
28:34
RRT7
1.0000
1.0123
1.0215
1.0000
RRT limits8
0.9988-1.0018
1.0062-1.0185
1.0154-1.0277
0.9988-1.0018
Window
(sec)9
-2+3
±10
±10
-2+3
Quantitation
reference10
47L
47L/77L
47L/77L
77L
Detection limits and minimum levels -
Matrix and concentration11
Water
(pg/L)
MDL
25

20

ML
100

50

Other
(ng/kg)
MDL
2.5

2

ML
10

5

Extract
(pg/nL)
ML
5

2.5

Labeled compounds
1
2
o
J
4
4
3L
15L
28L
47L
77L
PCB-52L
PCB-52L
PCB-52L
PCB-52L
PCB-52L
12:03
18:17
22:49
27:05
28:34
0.6992
1.0609
1.3240
1.5716
1.6576
0.6702-0.7282
1.0319-1.0899
1.2950-1.3530
1.5426-1.6006
1.6286-1.6867
±30
±30
±30
±30
±30
PCB-52L
PCB-52L
PCB-52L
PCB-52L
PCB-52L

























Compounds using PCB-138L (13C122,2',3,4,4',5'-HxCB) as Labeled injection internal standard
Pentabromodiphenyl ethers
5
5
5
5
5
5
5
100
119/120
99
116
85
126
105
100L
100L
99L
99L
126L
126L
126L
30:10
30:28
31:04
31:19
32:34
32:51
33:0812
1.0000
1.0099
1.0005
1.0086
0.9914
1.0000
1.0086
0.9989-1.0017
1.0044-1.0155
0.9995-1.0021
1.0032-1.0140
0.9863-0.9964
0.9990-1.0015
1.0036-1.0137
-2+3
±10
-2+3
±10
±10
-2+3
±10
100L
99L/100L/126L
99L
99L/100L/126L
99L/100L/126L
126L
99L/100L/126L
20

40

40


50

100

100


2

4

4


5

10

10


2.5

5

5


Hexabromodiphenyl ethers
6
6
6
6
6
6
155
154
153
140
138/166
128
154L
154L
153L
153L
153L
153L
32:50
33:28
34:38
35:2012
36:09
37:43
0.9816
1.0005
1.0005
1.0207
1.0443
1.0896
0.9766-0.9865
0.9995-1.0020
0.9995-1.0019
1.0159-1.0255
1.0395-1.0491
1.0823-1.0968
±10
-2+3
-2+3
±10
±10
±15
153L/154L
154L
153L
153L/154L
153L/154L
153L/154L

20
20

40


50
50

100


2
2

4


5
5

10


2.5
2.5

5

EPA Method 1614, August 2007
63

-------
Br
No.1
Congener No. 2'3'4
RTRef5
RT
(min:sec)6
RRT7
RRT limits8
Window
(sec)9
Quantitation
reference10
Detection limits and minimum levels -
Matrix and concentration11
Water
(pg/L)
MDL
ML
Other
(ng/kg)
MDL
ML
Extract
(pg/nL)
ML
Heptabromodiphenyl ethers
7
7
7
183
181
190
183L
183L
183L
37:58
39:40
39:54
1.0000
1.0448
1.0509
0.9991-1.0013
1.0404-1.0492
1.0465-1.0553
-2+3
±10
±10
183L
183L
183L
30

20
100

50
3

2
10

5
5

2.5
Octabromodiphenyl ether
8
203
209L
42:4012
1.1282
1.1194-1.1370
±20
209L





Nonabromodiphenyl ether
9
9
9
208
207
206
209L
209L
209L
45:33
45:52
46:31
0.9050
0.9113
0.9242
0.9000-0.9099
0.9063-0.9162
0.9192-0.9291
±15
±15
±15
209L
209L
209L















Decabromodiphenyl ether
10
209
209L
50:20
1.0000
0.9993-0.1010
-2+3
209L
700
2000
70
200
100
Labeled compounds
5
5
5
6
6
7
10
100L
99L
126L
154L
153L
183L
209L
PCB-138L
PCB-138L
PCB-138L
PCB-138L
PCB-138L
PCB-138L
PCB-138L
30:10
31:03
32:50
33:27
34:37
37:58
50:20
1.2230
1.2588
1.3318
1.3561
1.4034
1.5392
2.0405
1.2095-1.2365
1.2453-1.2723
1.3115-1.3520
1.3358-1.3764
1.3831-1.4236
1.4986-1.5797
2.0000-2.0811
±20
±20
±30
±30
±30
±60
±60
PCB-138L
PCB-138L
PCB-138L
PCB-138L
PCB-138L
PCB-138L
PCB-138L



































Labeled clean-up standard
6
139L
153L
35:03
1.0125
1.0077-1.0173
±10
PCB-138L





Labeled injection internal standards
4
6
PCB-52L
PCB-138L
PCB-138L
PCB-138L
17:14
24:40
0.6986
1.0000
0.6581-0.7392
1.0000-1.0000
±60
±100
PCB-138L
PCB-138L










64
EPA Method 1614, August 2007

-------
1.   Number of bromines on congener.

2.   Suffix "L" indicates labeled compound.

3.   Two congeners in a cell indicate a coelution.

4.   BDEs of primary interest are shown in boldface.

5.   Retention time reference that is used to locate the target congener.

6.   Retention time (RT) of the target congener.

7.   Relative retention time (RRT) between the RT for the congener and RT for the reference.

8.   Limits based on the magnitude of the RRT, adjusted for closely eluted isomers.

9.   Window width for congener or congener pair.

10. Labeled congeners that form the quantitation reference. Areas from the exact m/zs of the congeners listed in the quantitation reference are summed, and
    divided by the number of congeners in the quantitation reference. For example, for congener 75, the areas at the exact m/zs for 47L and 77L are summed and
    the sum is  divided by 2 (because there are 2 congeners in the quantitation reference).

11. MDLs for  "Other" matrix calculated per procedure at 40 CFR 136, appendix B; MLs for "Other" matrix calculated per procedure at 68 FR 11790 (March 12,
    2003); MDLs and MLs for aqueous matrix calculated as 10 times the MDLs and MLs for the "Other" matrix. MDLs and MLs for congeners for which
    MDLs and MLs are not shown will be similar to the MDLs and MLs for congeners at the same level of bromination.

12. Estimated
                                     EPA Method 1614, August 2007                                                                           65

-------
Table 3.  Concentrations of native and labeled brominated diphenyl ethers in stock solutions, spiking
solutions, and final extracts

BDE congener
Solution concentrations
Stock (jig/mL)
Spiking (ng/mL)
Extract (ng/mL)
Native congener by isotope dilution
28
47
99
100
153
154
183
209
20
20
20
20
20
20
20
200
1.0
1.0
1.0
1.0
1.0
1.0
1.0
10
50
50
50
50
50
50
50
500
Labeled BDE Congener
28L
47L
99L
100L
153L
154L
183L
209L
1.0
1.0
1.0
1.0
1.0
1.0
1.0
10
2.0
2.0
2.0
2.0
2.0
2.0
2.0
20
100
100
100
100
100
100
100
1000
Labeled clean-up
139L
1.0
2.0
100
Labeled injection internal
PCB-52L
PCB-138L
5
5
1000
1000
100
100
Diluted combined congener

Standard
Solution concentration (ng/mL)
Native
Labeled
Native congeners
MoBDE thru NoBDE
DeBDE
50
500


Labeled congeners
MoBDE thru NoBDE
DeBDE
Labeled cleanup
Labeled injection internal




100
1000
100
100
66
EPA Method 1614, August 2007

-------
Table 4. Composition of individual native BDE congener solutions1
Solution Identifier
BDE-A1
1
2
3
10
7
8
12
15
30
32
17
28
35
37
75
49
47
66
77
100
119
99
85
126
154
153
140
138
128
181
203
208
206
209
Totals
34
BDE-A2
11
13
25
33
51
71
79
120
116
155
105
166
183
190
207




















15
1.   Congeners listed by congener number in retention time order in each solution.  See Table 3 for concentrations of
    congeners in stock solutions and Table 5 for concentrations in calibration standards.
                                     EPA Method 1614, August 2007
67

-------
Table 5.  Concentration of BDE congeners in calibration and calibration verification standards1

BDE congener
Native
2,4,4'-TrBDE
2,2',4,4'-TeBDE
2,2',4,4',5-PeBDE
2,2',4,4',6-PeBDE
2,2',4,4',5,5'-HxBDE
2,2',4,4',5',6-HxBDE
2,2',3,4,4',5',6-HpBDE
DeBDE

Congener
number2

28
47
99
100
153
154
183
209
Solution concentration (ng/mL)
CS-1

1.0
1.0
1.0
1.0
1.0
1.0
1.0
10
CS-2

5.0
5.0
5.0
5.0
5.0
5.0
5.0
50
CS-3
(VER)

50
50
50
50
50
50
50
500
CS-4

500
500
500
500
500
500
500
5000
CS-5

2500
2500
2500
2500
2500
2500
2500
25000
Labeled
13C12-2,4,4'-TrBDE
13C12-2,2',4,4'-TeBDE
13C12-2,2',4,4',5-PeBDE
13C12-2,2',4,4',6-PeBDE
13C12-2,2',4,4',5,5'-HxBDE
13C12-2,2',4,4',5',6-HxBDE
13C12-2,2',3,4,4',5',6-HpBDE
13C12-DeBDE
28L
47L
99L
100L
153L
154L
183L
209L
100
100
100
100
100
100
100
1000
100
100
100
100
100
100
100
1000
100
100
100
100
100
100
100
1000
100
100
100
100
100
100
100
1000
100
100
100
100
100
100
100
1000
Labeled clean-up
13C12-2,2',3,4,4',6-HxBDE
139L
100
100
100
100
100
Labeled injection internal
13C12-2,2',5,5'-TeCB
13C19-2,2',3,4,4',5'-HxCB
PCB-52L
PCB-138L
100
100
100
100
100
100
100
100
100
100
1.   Other congeners may be included in calibration solutions if desired.




2.   Suffix "L" indicates labeled compound
68
EPA Method 1614, August 2007

-------
Table 6.  QC acceptance criteria for bromodiphenyl ethers in VER, IPR, OPR, and samples1

Congener2
Native congeners
2,4,4'-TrBDE
2,2'4,4'TeBDE
2,2',4,4',5-PeBDE
2,2',4,4',6-PeBDE
2,2',4,4',5,5'-HxBDE
2,2',4,4',5',6-HxBDE
2,2',3,4,4',5',6-HpBDE
DeBDE
Labeled congeners
13C12-2,4,4'-TrBDE
13C12-2,2',4,4'-TeBDE
13C12-2,2'4,4',5-PeBDE
13C12-2,2',4,4',6-PeBDE
13C12-2,2',4,4',5,5'-HxBDE
13C12-2,2',4,4',5',6-HxBDE
13C12-2,2',3,4,4',5',6-HpBDE
13C12-DeBDE
Cleanup standard
13C12-2,2',3,4,4',6-HxBDE
Congener
number3

28
47
99
100
153
154
183
209

28L
47L
99L
100L
153L
154L
183L
209L

139L
Test cone
(ng/mL)4

50
50
50
50
50
50
50
500

100
100
100
100
100
100
100
1000

100
VER5
(%)

70-130
70-130
70-130
70-130
70-130
70-130
70-130
50-200

50-150
50-150
50-150
50-150
50-150
50-150
50-150
25-200

60-130
IP
RSD (%)

40
40
40
40
40
40
40
40

50
50
50
50
50
50
50
50

45
R
X(%)

60-140
60-140
60-140
60-140
60-140
60-140
60-140
50-200

35-135
35-135
35-135
35-135
35-135
35-135
35-135
25-200

45-120
OPR
(%)

50-150
50-150
50-150
50-150
50-150
50-150
50-150
40-200

30-140
30-140
30-140
30-140
30-140
30-140
30-140
20-200

40-125
Labeled compound
recovery in samples (%)










25-150
25-150
25-150
25-150
25-150
25-150
25-150
20-200

30-135
1.   QC acceptance criteria for IPR, OPR, and samples based on a 20 uL extract final volume




2.   Other congeners may be included in test solutions if desired.




3.   Suffix "L" indicates labeled compound.




4.   See Table 5.




5.   Section 15.3.
                                   EPA Method 1614, August 2007
69

-------
Table 7. Scan descriptors, levels of bromination and chlorination, m/zs, and BDEs and PCBs monitored
by HRGC/HRMS
Function and
bromine or chlorine
level
Fn-l;Br-l




Fn-2; Br-2; Cl-4






Fn-3 Br-3; Br-4; Cl-6










Fn-4; Br-5; Br-6








m/z1
247.9837
249.9816
260.0239
262.0219
280.9824
301.9626
303.9597
325.8942
327.8921
330.9792
337.9344
339.9324
371.8817
373.8788
405.8027
407.8002
417.8429
419.8409
442.9728
483.7132
485.7111
497.7514
499.7493
554.9665
563.6216
565.6196
575.6619
577.6598
641.5322
643.5302
655.5704
657.5683
m/z type
M
M+2
M
M+2
lock
M
M+2
M
M+2
lock
M
M+2
M+2
M+4
M+2
M+4
M+2
M+4
lock
M+2
M+4
M+4
M+6
lock
M+4
M+6
M+4
M+6
M+4
M+6
M+6
M+8
m/z formula
12C12H916079Br
12C12H916081Br
13C12H916079Br
13C12H916081Br
12r F
*-6 -Ml
M2 H6 C14
13C12 H6 35C13 37C1
12C12H816079Br2
12C12H816079Br81Br
12C7F13
13C12H816079Br2
13C12H816079Br81Br
13C12 H5 35C15 37C1
13C12 H6 35C14 37C12
12C12H716079Br281Br
12C12H716079Br81Br2
13C12H716079Br281Br
13C12H716079Br81Br2
12c F
Mo riv
12C12H616079Br381Br
12C12H616079Br281Br2
13C12H616079Br281Br2
13C12H616079Br81Br3
12C13 F21
12C12H516079Br381Br2
12C12H516079Br281Br3
13C12H516079Br381Br2
13C12H516079Br281Br3
12C12H416079Br481Br2
12C12H416079Br381Br3
13C12H416079Br381Br3
13C12H416079Br281Br4
Substance
MoBDE
MoBDE
13C12 MoBDE
13C12 MoBDE
PFK
13C12 TeCB
13C12 TeCB
DiBDE
DiBDE
PFK
13C12 DiBDE
13C12 DiBDE
13C12 HxCB
13C12 HxCB
TrBDE
TrBDE
13C12 TrBDE
13C12 TrBDE
PFK
TeBDE
TeBDE
13C12 TeBDE
13C12 TeBDE
PFK
PeBDE
PeBDE
13C12 PeBDE
13C12 PeBDE
HxBDE
HxBDE
13C12 HxBDE
13C12 HxBDE
70
EPA Method 1614, August 2007

-------
Function and
bromine or chlorine
level
Fn-5; Br-7; Br-8








Fn-6;Br-9;Br-10








m/z1
716.9569
721.4406
723.4386
733.4809
735.4788
799.3511
801.3491
811.3914
813.3893
879.2596
881.2575
891.2998
892.9441
893.2978
957.1701
959.1680
971.2083
973.2063
m/z type
lock
M+6
M+8
M+6
M+8
M+6
M+8
M+6
M+8
M+8
M+10
M+8
lock
M+10
M+8
M+10
M+10
M+12
m/z formula
12Q7F27
12C12H316079Br481Br3
12C12H316079Br381Br4
13C12H316079Br481Br3
13C12H316079Br381Br4
12C12H216079Br581Br3
12C12H216079Br481Br4
13C12H216079Br581Br3
13C12H216079Br481Br4
12C12H16079Br581Br4
12C12H16079Br481Br5
13C12H16079Br581Br4
12p p
M9 r35
13C12H16079Br481Br5
12C12 160 79Br6 81Br4
12C12 160 79Br5 81Br5
13C12 160 79Br5 81Br5
13C12 160 79Br4 81Br6
Substance
PFK
HpBDE
HpBDE
13C12 HpBDE
13C12 HpBDE
OcBDE
OcBDE
13C12 OcBDE
13C12 OcBDE
NoBDE
NoBDE
13C12 NoBDE
PFK
13C12 NoBDE
DeBDE
DeBDE
13C12 DeBDE
13C12 DeBDE
1. Isotopic masses used for accurate mass calculation
                    1H                  1.0078
                    12C                12.0000
                    13C                13.0034
                    16O                15.9949
                    35C1               34.9689
                    37C1               36.9659
                    79Br               78.9813
                    81Br               80.9163
                    19F                18.9984
                                  EPA Method 1614, August 2007
71

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Table 8. Theoretical ion abundance ratios and QC limits
Bromine atoms
1
2
3
4

5
6

7
8
9
10
m/zs forming ratio
m/m+2
m/(m+2)
(m+2)/(m+4)
(m+2)/(m+4)
(m+4)/(m+6)
(m+4)/(m+6)
(m+4)/(m+6)
(m+6)/(m+8)
(m+6)/(m+8)
(m+6)/(m+8)
(m+8)/(m+10)
(m+8)/(m+10)
Theoretical ratio
1.03
0.43
1.03
0.70
1.54
1.03
0.77
1.37
1.03
0.82
1.03
0.73
Lower QC limit
0.88
0.47
0.88
0.60
1.31
0.88
0.65
1.16
0.88
0.70
0.88
0.86
Upper QC limit
1.18
0.59
1.18
0.81
1.77
1.18
0.89
1.58
1.18
0.94
1.18
0.99

Chlorine atoms
4
6

m/(m+2)
(m+2)/(m+4)

0.78
1.25

0.66
1.06

0.90
1.44
72
EPA Method 1614, August 2007

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Table 9.  Suggested Sample Quantities to be Extracted for Various Matrices1
Sample matrix2
Example
Percent
solids
Phase
Quantity
extracted
Single-phase
Aqueous


Solid




Tissue
Drinking water
Groundwater
Treated wastewater
Dry soil
Compost
Ash
Waste oil
Organic polymer
Fish
Human adipose
<1


>20




—
3


Solid




Organic
1000 mL


10 g




10 g
Multi-phase
Liquid/Solid
Aqueous/Solid




Organic/solid

Liquid/Liquid
Aqueous/organic


Aqueous/organic/solid


Wet soil
Untreated effluent
Digested municipal sludge
Filter cake
Paper pulp
Industrial sludge
Oily waste

In-process effluent
Untreated effluent
Drum waste
Untreated effluent
Drum waste

1-30




1-100


<1


>1


Solid




Both


Organic


Organic & solid


10 g




10 g


lOg


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 BDEs 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 solubility in water.

3.  Aqueous samples are filtered after spiking with the labeled compounds. The filtrate and the materials
trapped on the filter are extracted separately, and the extracts are combined for cleanup and analysis.
                                   EPA Method 1614, August 2007
73

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                                      Determine % solids
                                           §11.2
                                     Determine particle size
                                           §11.3
           Prep per § 11.5
       Spike Labeled Toxics/LOC
         window-definers per
             §11.5.2.2
           SDS extraction
             per §12.3
              Particle
            size > 1 mm?
            (from §11.3)
                               Prep per § 11.4
                          Spike Labeled Toxics/LOC
                            window-definers per
                                 §11.4.2.2
                                                                  Extract per §12.2.1,
                                                                  §12.2.2, or§ 12.2.3
   Spike Cleanup standard per
           §12.5.1
                                             Back extract per
                                                §12.5
                                            Transfer through
                                           Na2S04 per §12.5.6
                                                                               Concentrate per
                                                                                §12.6-§12.7
                                                                                 Clean up per
                                                                            §13.2-§13.5, or §13.7
                                                                               Concentrate per
                                                                                §12.6-§12.7
                                                                             Spike injection internal
                                                                              standard per § 14.2
                                                                                 Analyze per
                                                                                  §14-§18
                                                                                          SCC-99-020
      Figure 1   Flow Chart for Analysis of Aqueous and Solid Samples
74
EPA Method 1614, August 2007

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                      Aqueous
        Discard
                                         Determine % solids
                                             per §11.2
                                        Determine particle size
                                             per §11.3
                                      Spike Labeled Toxics/LOC
                                        window-definers per §
                                              11.6.2
                                       Pressure filter aliquot per
                                              §11.6.2
                                                            No
                                                  Yes
                                           Grind per §11.7
                                           SDS extract per
                                               §12.3
                                       Spike Cleanup standard per
                                              §12.5.1
                                             Back extract
                                              per §12.5
                                           Transfer through
                                          Na2S04 per 12.5.6
Figure 2  Flow Chart for Analysis of Multi-Phase Samples
Non-aqueous (organic)
      Concentrate per
      §12.6-§12.7
       Clean up per
   §13.2-§13.5, §13.7
      Concentrate per
       §12.6-§12.7
                                                                            Spike injection internal
                                                                              standard per§ 14.2
       Analyze per
        §14-§18
                                   EPA Method 1614, August 2007
                             75

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                                                 Homogenize tissue
                                                    per §11.8.1
                                                    Remove 10 g
                                                    per§ 11.8.1.4
                                              Spike Labeled Toxics/LOC
                                              window-definers 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
                                              Redissolve in n-C6 and spike
                                                  cleanup standard
                                                    per §12.4.9.1
                                                  Remove lipids per
                                                       §13.6
                                                   Concentrate per
                                                   §12.6-§12.7
                                                     Clean up per
                                                  §13.2-§13.5, §13.7
                                                   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
76
EPA Method 1614, August 2007

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                                                              GMF150 Filter
                                             - Test Tube, 25-mm x 200-mm
                                               1-Liter Suction Flask
Figure 4        Solid-phase Extraction Apparatus
                                  EPA Method 1614, August 2007
77

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                                                                                       $2-027-02
Figure 5       Soxhlet/Dean-Stark Extractor
78
EPA Method 1614, August 2007

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       103 Text .•HtH=BDOjA-£*L,,/6J     ixfS :BI-DBSWT-l_
Figure 6  DB-5HT column resolution test:  Separation of Br-4 congeners 49
and 71 with valley less than 40% (i.e.  100 x/y < 40%)
                        EPA Method 1614, August 2007
19

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80
EPA Method 1614, August 2007

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24  Glossary of Definitions and Purposes

    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 and their abbreviations

       24.1.1  Symbols
           °C     degrees Celsius
           (iL     microliter
           (im    micrometer
           <      less than
           >      greater than
           %     percent

       24.1.2  Alphabetical abbreviations
           cm     centimeter
           g      gram
           h      hour
           ID     inside diameter
           in.     inch
           L      liter
           M     Molecular ion
           m     meter
           mg     milligram
           min   minute
           mL    milliliter
           mm   millimeter
           m/z   mass-to-charge ratio
           N     normal; gram molecular weight of solute divided by hydrogen equivalent of solute,
                  per liter of solution
           OD    outside diameter
           pg     picogram
           ppb   part-per-billion
           ppm   part-per-million
           ppq   part-per-quadrillion
           ppt    part-per-trillion
           psig   pounds-per-square inch gauge
           v/v    volume per unit volume
           w/v   weight per unit volume
                                  EPA Method 1614, August 2007
81

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    24.2   Definitions and acronyms (in alphabetical order).

Analyte—A BDE tested for by this Method. The analytes are listed in Table 1.
Brominated diphenyl ether (BDE)—Any of the 209 congeners tested for by this Method and listed in
    Table 1.
Calibration standard (CAL)—A solution prepared from a secondary standard and/or stock solutions and
    used to calibrate the response of the HRGC/HRMS instrument.
Calibration verification standard (VER)—The mid-point calibration standard (CS-3) that is used to verify
    calibration. See Table 5.
CS-0.2, CS-1, CS-2, CS-3, CS-4, CS-5—See Calibration  standards and Table 5.
DeBDE—decabromodiphenyl ether (BDE 209)
DiBDE—dibromodiphenyl ether
Field blank—An aliquot of reagent water or other reference matrix that is placed in a sample container in
    the laboratory or the field, and treated as a sample in all respects, including exposure to sampling site
    conditions, storage, preservation, and all analytical procedures. The purpose of the field blank is to
    determine if the field or sample transporting procedures and environments have contaminated the
    sample.
GC—Gas chromatograph or gas  chromatography
GPC—Gel  permeation chromatograph or gel permeation chromatography
HpBDE—heptabromodiphenyl ether
HRGC—High resolution GC
HRMS—High resolution MS
HxBDE—hexabromodiphenyl ether
Labeled injection internal standard—The 13C12-labeled PCB congeners spiked into the concentrated
    extract  immediately prior to injection of an aliquot of the extract into the HRGC/HRMS.  The labeled
    injection internal standards in this Method are PCBs with congener numbers 52L and 138L.
Internal standard—a labeled compound used as a reference for quantitation of other labeled compounds
    and for quantitation of native BDE congeners other than the congener for 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, all 12 carbon atoms in the BDE are enriched with carbon-13 to produce 13C12-labeled analogs
    of the brominated diphenyl ethers. The 13C12-labeled  BDEs are spiked into each sample and 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
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.
MDL—See Method Detection Limit

82                                EPA Method 1614, August 2007

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Method blank—An aliquot of reagent water or other reference matrix 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—A detection limit determined by the procedure at 40 CFR 136, appendix B.
Minimum level of quantitation (ML)—The level at which the entire analytical system must give a
    recognizable signal and acceptable calibration point for the analyte. It is equivalent to the
    concentration of the lowest calibration standard, assuming that all Method-specified sample weights,
    volumes, and cleanup procedures have been employed (see 68 FR 11790; March 12, 2003).
MoBDE—monobromodiphenyl ether
MS—Mass spectrometer or mass spectrometry
Must—This action, activity, or procedural step is required.
NoBDE—nonabromodiphenyl ether
OcBDE—octabromodiphenyl ether
OPR—Ongoing precision and recovery standard (OPR; also laboratory control sample, LCS); an aliquot
    of reagent water or other reference matrix spiked with known quantities of the 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.
BDE—See brominated diphenyl ether.
Perfluorokerosene (PFK)—A mixture of compounds used to calibrate the exact m/z scale in the HRMS.
Polybrominated diphenyl ether (PBDE)—See Brominated diphenyl ether
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.
PeBDE—pentabromodiphenyl ether
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 retention time (RRT)—The retention time of the component of interest divided by the retention
    time of its reference. The RRT references for the BDEs and labeled compounds are listed in Table 2.
Relative standard deviation (RSD)—The standard deviation times  100 divided by the mean. Also termed
    "coefficient of variation."
Retention time (RT)—The time between the time that an unretained component elutes and a component
    of interest elutes from a chromatographic column. Usually the time from the air peak or solvent front
    to the time at which the component of interest elutes, but also sometimes the time from the time of
    injection until the component of interest elutes. RTs for the BDEs on the DB-5HT column are listed
    in Table 2.
RF—Response  factor.  See Section 10.5
RR—Relative response.  See Section 10.4
RRT—See Relative retention time
RSD—See Relative standard deviation
RT—See Retention time
SDS—Soxhlet/Dean-Stark extractor; an extraction device applied to the extraction of solid and semi-solid
    materials (Reference 10 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—This action, activity, or procedural step  is suggested but not required.
SICP—Selected ion current profile; the line described by the signal at an exact m/z.

                                  EPA Method 1614, August 2007                                 83

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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.
Specificity—the capability of an analytical system to identify and quantify an analyte when other analytes
    and interferences are present in the sample.
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.
TeBDE—tetrabromodiphenyl ether
TrBDE—tribromodiphenyl ether
Unique GC resolution or uniquely resolved—Two adjacent chromatographic peaks in which the height of
    the valley is less than 40 percent of the height of the shorter peak (See Section 6.9.1.1.2 and Figure 6
    for unique resolution specific to the DB-5HT column).
VER—See Calibration verification.
84                                 EPA Method 1614, August 2007

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