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


   May 2010

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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-10-005

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                                    Method 1614A
           Brominated Diphenyl Ethers in Water, Soil, Sediment,
                           and Tissue by HRGC/HRMS

                                        May 2010

Introduction

The Office of Science and Technology (OST) in EPA's Office of Water developed Method 1614A for use
in Clean Water Act (CWA) programs.  EPA is publishing this method for users who wish to measure
polybrominated diphenyl ethers (PBDEs), and in 2010, EPA expects to publish a proposal in the Federal
Register for public comment to add this method to other CWA methods published at 40 CFR Part 136.
EPA Method 1614A was developed to determine polybrominated diphenyl ether (PBDE) congeners in
aqueous, solid, tissue, and multi-phase matrices. These ethers are used as flame retardants.  The method
uses isotope dilution and internal standard high resolution gas chromatography/high resolution mass
spectrometry (HRGC/HRMS). This revision of Method 1614 resulted from comments received in a peer
review conducted in September of 2007 and changes to the GC temperature program for the short column
in Section 10.1.

Acknowledgments

EPA Method 1614A was developed under EPA contract by CSC, Interface, Inc., and by AXYS
Analytical Services, Ltd. Sidney, BC, Canada.

Disclaimer

Mention of trade names or commercial products does not constitute endorsement or recommendation for
use.

Summary of changes between  EPA Method 1614A (May 2010) and 1614 (August
2007) excluding corrections to  references and typographical errors

Section 7.10.2.2.1 has been revised to include the word "evaluation" for clarity.

The following note has been added to Section 10.1: "RTs, RRTs, and RRT limits may differ slightly from
those in Table 2." This statement has also been added to the footnotes to Table 2

Section 10.2.1 has been revised to include "PFK."

Table 8 has been revised to make all lower and upper QC limits 15% of the theoretical ratio.

Section 4.2.6 has been added to specify that all possible sources of contamination (glassware, vials, caps,
etc.) should be as clean as possible.

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

Sections 6.9 and 10.1 have been expanded to allow use of a short (15-m) column and to clarify
differences and requirements among the long (30-m) column, a 2-column system, and the short column.

EPA Method 1614A                               i                                    May 2010

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Section 7.7.2 has been revised to indicate that standard solutions should be stored in the dark at
approximately 4 C in screw-capped vials with fluoropolymer-lined caps.

Section 8.4.1 has been revised to indicate the following, "Lipid content, and hence PBDE content, may
vary depending on which area offish is taken (e.g. dorsal vs. ventral). The sampler should observe the
precautions to preclude contamination in Section 5.  In particular, tissue should be processed in a dust-
free zone because dust may contain high levels of PBDEs."

Section 9.1.2.1 has been revised to require a laboratory to achieve MDLs no greater than five times the
MDLs in this method.

Section 9.5.2 has been revised to require BDE congener concentrations no greater than two times the ML
in blanks.

Operating conditions for the long and short column have been further defined in Sections 10.1, 10.1.1,
and 10.1.3.

The exact injection volume requirement in Section  10.3 has been edited to allow for an injection volume
of 5 (iL into the short column.

In Section 14.2, the volume necessary to bring an extract back to the original 20 (iL volume when
evaporative loss has occurred has been expanded to allow for a 5 (iL injection volume with the short
column.

A sentence was added to Section 11.4.2.1 to require weighing the sample bottle after emptying, and to
determine the volume using the density of water.

Section 13.4.6 has been revised to state the  following, "Elute the BDEs with methylene chloride:hexane
(50:50 v/v) and collect the  eluate. A higher percent methylene chloride solution may be required for more
highly activated alumina."

The following  note has been added to Section 14, "It has been reported that decabromodiphenylethane
elutes approximately 3 minutes after DeBDE, has m/z's in common with 13Ci2-DeBDE, and  may interfere
in a subsequent run. If known or suspected to be present, sufficient time should be allowed for
decabromodiphenylethane  to elute."

Section 17.2.1  has been changed to clarify that concentrations of native compounds other than those in the
Native Toxics/LOC standard, in the Labeled cleanup standard, and in the Labeled injection internal
standard (except for labeled CB  178) should be determined using the response factors from Section  10.5
or Section 15.4.2.2.

Section 17.6.5  has been added to provide information on the use of optional data qualifier flags for
reporting coeluting congeners.

Reference 11 was replaced with, "Standard Methods for the  Examination of Water and Wastewater, 20th
Edition, published jointly by American Public Health Association, American Water Works Association,
and Water Environment Federation, Washington, DC, 2005."

Tables 10-12 have been added to provide additional data for the short column and TPI options.
EPA Method 1614A                                ii                                        May 2010

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Summary of changes between EPA Method 1614 (August 2007) and 1614 (August
2003) excluding corrections to references and typographical errors

Section 6.9.2 and its subsections have been added to provide the specifications and parameters for the gas
chromatograph short column.

Section 10.1.3 has been added to give diameters of the short column, the experimental conditions for the
injector and the GC temperature program.

Section 15.4.3 has been added to specify the use of a DeBDE breakdown test when using the TPI and/or
short column.

Please address your questions or comments to:

Richard Reding, Brian Englert or 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 1614A                              Hi                                    May 2010

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                                     Method 1614A
  Brominated  Diphenyl Ether Congeners  in Water, Soil, Sediment, and
                               Tissue by HRGC/HRMS
                                         May 2010
1.0    Scope and application
1.1    EPA Method 1614A ("Method 1614A"; 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 the analytical techniques in EPA Method 1668A (Reference 4).

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" which means that you may make modifications without
       additional EPA review to improve performance (e.g., overcome interferences, or improve the
       sensitivity, accuracy or precision of the results) provided that you meet all performance criteria in
       this Method. Requirements for establishing equivalency are in Section 9.1.2, and include
       9.1.2.2.3 - explaining the reason for your modifications. For CWA uses, additional flexibility is
       described at 40 CFR 136.6. You must document changes in performance, sensitivity, selectivity,
       precision, recovery, etc., that result from modifications within the scope of Part 136.6, and
       Section 9 of this Method, and how these modifications compare to the specifications in this
       Method. Changes outside the scope of Part 136.6 and Section 9 of this Method may require prior
       review or approval.
EPA Method 1614A                               1                                      May 2010

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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
        (Section 7.5.3), and all extracts are cleaned up using back-extraction with sulfuric acid and/or
        base, and gel permeation, silica gel,  and/or Florisil or alumina chromatography, as required.

2.3     After cleanup, the  extract is concentrated to 20 joL 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  (>5000) mass spectrometer. Two exact
        m/z's 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/z's with the corresponding retention time of an authentic standard and the
        theoretical or acquired ion-abundance ratio of the two exact m/z's.

2.5     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 1614A                                 2                                        May 2010

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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 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.

2.7     For laboratories wishing to use the short column and temperature programmable injector (TPI),
        Tables 10-12 have been added to provide MDL, retention time, retention time windows, and
        accuracy and precision data obtained when using the TPI and short column. When using the short
        column and TPI use the MDLs in Table 10, the retention times, relative retention times, retention
        time windows and ion abundance ratios in Table 11 to replace other data found elsewhere in this
        method. Table 12 should not be used to replace existing  QA/QC requirements.
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


EPA Method 1614A                                3                                       May 2010

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               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.

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

        4.2.6  To aid in elimination of BDE 209 background all possible sources of contamination
               (glassware, vials,  caps, etc.) should be as clean as possible.

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 interferents  in the concentrations expected to be found in the
               samples to be analyzed.

        4.3.2  When a reference matrix that simulates the sample matrix under test is not available,
               reagent water (Section 7.6.1) can be  used to simulate water; 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

EPA Method 1614A                                 4                                        May 2010

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        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.
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 5-8.  The references and
        bibliography at the end of Reference 7 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. We 11-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

EPA Method 1614A                                5                                        May 2010

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               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.

        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 isopropanol 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.
EPA Method 1614A                                 6                                        May 2010

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               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.

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

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

6.1    Sampling equipment for discrete or composite sampling

       6.1.1   Sample bottles and caps

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

               6.1.1.2    Solid samples (soils, sediments, sludges, paper pulps, filter cake, compost,
                          and similar materials that contain more than 5 percent solids) - Sample
                          bottle, wide-mouth, amber glass, 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.
EPA Method 1614A                                7                                       May 2010

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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.

6.3    Equipment for sample preparation

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

       6.3.2  Glove box (optional)

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

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

       6.3.5  Equipment for determining percent moisture

               6.3.5.1     Oven - Capable of maintaining a temperature of 110  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

EPA Method 1614A                                8                                        May 2010

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                                     (Figure 4).  For wastewater samples, the apparatus should accept
                                     90 or 144 mm disks.  For drinking water or other samples
                                     containing low solids, smaller disks may be used.

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

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

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

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

        6.4.2   Soxhlet/Dean-Stark (SDS) extractor (Figure 5 and Reference 9) 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.
EPA Method 1614A                                 9                                        May 2010

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       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 runnel - 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

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
EPA Method 1614A                                10                                       May 2010

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               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

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
                          temperature 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
EPA Method 1614A                                11                                       May 2010

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               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
       Sections 10 and 15.4. A gooseneck liner (Restek or equivalent) was commonly used. The injector
       liner should be deactivated.

       6.9.1   GC long column and 2-column system- A single- or two-column system may be used, as
               follows:

               6.9.1.1    Long column - 30  5-m long x 0.25  0.02-mm ID; O.l-^m film; 95%
                         methyl, 4% phenyl, 1% vinyl silicone (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 7.10.2.2.2) 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 to 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  The column that elutes the mono- through nona- BDEs must
                                    meet the resolution and tailing factor specifications in Section
                                    6.9.1.1.2 and the column that elutes DeBDE must meet the
                                    tailing factor specification (Section 6.9.1.1.3) for DeBDE.
EPA Method 1614A                                12                                      May 2010

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                          6.9.1.2.3  The respective column must be replaced when any of the criteria
                                     in Sections 6.9.1.2.1 to 6.9.1.2.2 are not met.

               6.9.1.3    If an alternate column or column system is used, specifications similar to
                          those for the long column or two-column system (Sections 6.9.1.1 to 6.9.1.2)
                          must be developed and must be functionally equivalent to those
                          specifications.

        6.9.2   Short column - Optionally, to prevent breakdown of the octa-, nona-, and deca-
               congeners, a short column and temperature-programmable injector may be used, provided
               that the system then meets the performance specifications in Section 9 and 10.

               6.9.2.1    Short column - 15  1-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). Retention time data for the short column are given in
                          Table 11.

               6.9.2.2    The retention time for decabromodiphenyl ether (DeBDE) must be greater
                          than 27 minutes.

               6.9.2.3    The column must meet the resolution and tailing factor specifications in
                          Sections 6.9.1.1.2 and 6.9.1.1.3.

               6.9.2.4    The column must be replaced when any of the criteria in Sections 6.9.2.2 to
                          6.9.2.3 are not met.

               6.9.2.5    The absolute retention times must be approximately equal to those  in Table
                          11.

               6.9.2.6    If a column alternate to the short column is  used, specifications similar to
                          those for the short column (Sections 6.9.2.1 to 6.9.2.3) must be developed
                          and must be functionally equivalent to those specifications.

6.10    Mass spectrometer - 28- to 40-eV electron ionization, must be capable of selectively monitoring
        a minimum of 15 exact m/z's in a single function at high resolution (>5000) 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 beam.

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 1614A                                13                                       May 2010

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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, diethyl  ether, 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.  It may be necessary to include DeBDE in the calibration solution.

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.

EPA Method 1614A                                14                                        May 2010

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        7.5.1   Silica gel

               7.5.1.1    Activated silica gel - 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  Place the potassium silicate in an enclosed flow-through
                                     container and purge dry with high-purity nitrogen to prevent
                                     contamination from dust.  Observe the other contamination
                                     precautions  in Section 5.3.1.

                          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
                          mL/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 1614A                                15                                        May 2010

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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 1/2 to 2/3 full with Florisil and place
                                     in an oven at 130 to 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 to 1.0 cm of
                                     warm to hot anhydrous sodium sulfate (Section 7.2.1),  10 to 10.5
                                     cm of warm to hot activated Florisil (Section 7.5.4.1.1), and 1 to
                                     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

EPA Method 1614A                                16                                       May 2010

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               Table 2.  If low background levels of the BDEs are present in the reference matrix, the
               spike level of the analytes used in Section 9.2 should be increased to provide a spike-to-
               background ratio of approximately 5 (Reference  10).

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

        7.7.1   For preparation of stock solutions from neat materials, dissolve an appropriate amount of
               assayed reference material in solvent. For example, weigh 10 to 20 mg  of 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 approximately 4 C 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 column calibration 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.


        7.8.3   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.

        7.8.4   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.

7.9     Labeled compound stock solutions
EPA Method 1614A                                17                                        May 2010

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        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 (CS's) 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 7.8.3) is given in Table
                                     2.

                          7.10.2.1.2 Individually combine an aliquot of each individual mix stock
                                     solution (Section 7.8.3) 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 evaluation of 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.
EPA Method 1614A                                18                                       May 2010

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                          7.1 0.2.2.2 Combine an aliquot of the combined BDE congener solution
                                     (Section 7.8.3) 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).

7.1 1   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 joL, the
       concentrations in the final volume will be 50 and 500 ng/mL (pg/joL).  Prepare only the amount
       necessary for each reference matrix with each sample batch.

7.1 2   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 joL, the concentrations in the final volume will be 100 and  1000 ng/mL
       (pg/|oL). 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 joL,  the concentration in the final volume will be
       100 ng/mL
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 |oL of this solution is spiked into a 20 |oL
        extract, the concentration of each injection internal standard will be nominally 100 ng/mL
Note:   The addition of 2 ^L of the labeled injection internal standard spiking solution to a 20 pL final
extract has the effect of diluting the concentration of the components In the extract by 10%.  Provided that
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.


7.1 5    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. 1 6    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 (if

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       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.

8.2     Aqueous samples (Reference 11)

        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 12).

        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 less than  -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.
               Lipid content, and hence PBDE content, may vary depending on which area of fish is
               taken (e.g. dorsal vs. ventral). The sampler should observe the precautions to preclude
               contamination in Section 5.  In particular, tissue should be processed in a dust-free zone
               because dust may contain high levels of PBDEs.

        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 less than
               -10  C until prepared. Maintain unused sample in the dark at less than -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.

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        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.

9.0     Quality assurance/quality control

9.1     Each laboratory that uses this method is required to operate a formal quality assurance program
        (Reference 13).  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 to 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 lower than five times the MDLs 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:

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                                     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)
                                     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 or 6.9.2.5.

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

        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 (Section
               15.6) 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 two times 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    Suggested operating conditions for the long column (Section 6.9.1.1) and for the long column in
        the 2-column system (Section 6.9.1.2) are given in Section 10.1.1; suggested operating conditions
        for the short column (Section 6.9.2) are given in Section 10.1.3.

Note:   RTs, RRTs, and RRT limits may differ slightly from those in Table 2.
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        10.1.1  Long Column & Two Column System - Suggested GC operating conditions necessary to
               meet the retention times (RTs) and relative retention times (RRTs) in Table 2 for the long
               column and column that elutes the mono- through nona- BDEs in the 2-column system:

               Injector temperature:    300 C
               Interface temperature:   320 C
               Initial temperature:      100 C
               Initial time:            3 minutes
               Temperature program:  100 - 320 C at 5 C per 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.

               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.

               Typical Septum Temperatures were around 200 C.

        10.1.2  Retention time calibration for the BDE congeners (all columns)

               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 30-m
                          DB-5ht column (27 minutes on the short 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 30-m DB-5ht, 15-m or two column system 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 a 30-m alternate column or 27 minutes on a 15-m
                          alternate column.

               10.1.2.3    If BDEs otherthan 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-, two- or short column system in Section
                          6.9.1.1.1 to 6.9.1.1.4 or 6.9.1.2.1  to 6.9.1.2.3 or 6.9.2.2 to 6.9.2.5,
                          respectively, are met.  If an  alternate column or column system is used,
                          adjust the conditions for that column. If column performance is

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                         unacceptable, optimize the analysis conditions or replace the column and
                         repeat the performance tests.  Confirm that the scan descriptor changes at
                         times when BDEs do not elute.

               10.1.2.4   After the column performance tests are passed (Section 10.1.2.2 to 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  15 meter GC Column and Temperature-Programmable 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:
               15m DB-5ht (or equivalent), 0.25 mm x 0.1 (im film
               Constant helium flow at 1.5 mL/min

               Temperature-Programmable Injector (TPI):
               Sample Injection Volume:    5 joL
               Vent Flow:                 100 mL/min
               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:
               75 C for 0.4 min
               40 C/min to 200  C
               10C/minto260C
               5 C/min to 300 C hold for 10 min
               Total Run Time: 27.5 min

10.2   Mass spectrometer (MS) resolution

       10.2.1  Using perfluorokerosene (PFK, Fluka high boiling or equivalent) (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/z's 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

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               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/z's 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/z's 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 >5000 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 joL (5 (iL if using the short column), consistent with the capability of the
        HRGC/HRMS instrument. Inject an 1 or 2 joL (5 (iL if using the short column) aliquot of the  CS-
        1 calibration solution (Table 5) using the GC conditions in Section 10.1.1 or 10.3.1.

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

               10.3.1.1   The exact m/z's 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/z's may be monitored in each descriptor, and the m/z's may be
                         divided among more than the descriptors listed in Table 7, provided that the
                         laboratory is able to monitor the m/z's of all BDEs that elute from the GC in
                         a given level of bromination (LOB) window. The laboratory must also
                         monitor exact m/z's 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/z's. The lock mass for each
                         group of m/z's 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
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               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 and its labeled analog are
        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.

        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/z's specified in Table 7, for
               each calibration standard,  as follows:

                                      RR=(AkA2JCL
                                            (Al1 + A21)Cn
               where:
               Aln and A2n = The measured areas at the primary and secondary m/z's for the BDE
               Ali and A2i = The measured areas at the primary and secondary m/z's for the labeled
                             compound
                      Ci  = 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 or 10.1.3 for the long or short column, respectively.
               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
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        10.5.1  Internal standard calibration is applied to determination of the native BDEs for which a
               labeled compound is not available, determination of the labeled congeners and labeled
               cleanup standard for performance tests and intra-laboratory statistics (Sections 9.4 and
               15.5.4), and 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=(A1S + A2S)CIS
                                           (A1IS + A2IS)CS

               where:
               Als and A2S =  The measured areas at the primary and secondary m/z's for the BDE
               Alis and A21S=  The measured areas at the primary and secondary m/z's for the internal
                              standard
                      C;s  =  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/z's for 99L,
               100L, and 126L are summed and the total area is divided by 3 (because there are 3
               congeners as the quantitation reference).

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

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        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
               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:

                                   _ weight of sample aliquot after drying (g) - weight of filter (g)
                          /O oOllLlo                                                          A.  Iv/VJ
                                                              lOg
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        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:

                          .   ,. ,    weight of sample aliquot after drying (g)
                          % solids =	      x 100
                                     weight of sample 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.

        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.  After extraction (Section 12.2), re-
                          weigh the sample bottle and convert the weight to volume assuming a density
                          of l.OOg/mL.

               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

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        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 a hood. 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.

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.
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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.

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.
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               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.
12.0    Extraction and concentration

12.1    Extraction procedures include solid phase (Section 12.2.1), separatory funnel (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.


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

               12.2.1.2   Sample extraction

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

                          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 eluent 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.

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                          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
                          separatory funnel, and extract the sample by shaking the funnel for 2 minutes
                          with periodic venting. Allow the organic layer to separate from the aqueous
                          phase for a minimum of 10 minutes. If an emulsion forms and is more than
                          one-third the volume of the solvent layer, employ mechanical techniques to
                          complete the phase separation (see note below).  Drain the methylene
                          chloride extract through a solvent-rinsed glass funnel approximately one-half
                          full of granular anhydrous 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.
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               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. 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.

        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
               nonaqueous 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

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               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 to
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 PBDEs 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.

        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.


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               1 2.4.9.2   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.

               1 2.4.9.3   Calculate the lipid content to the nearest three significant figures as follows:
                                     weight of tissue (g)

               1 2.4.9.4   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 12.2.1.3.4 or 12.3.9.

        1 2.5.2 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
               labeled compound recovery and demonstrates acceptable performance using the
               procedure in Section 9.2.

        1 2.5.3 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.

        1 2.5.4 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.

        1 2.5.6 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.


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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.

               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
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               methylene chloride and hexane. Toluene is difficult to concentrate using the K-D
               technique unless a water bath fed by a steam generator is used.

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

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

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

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

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

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

               12.6.3.7   Proceed to 12.6.4 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.

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        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 40-45 C water bath and continue concentrating. Care should be
               taken to avoid solvent bumping.

               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 joL, 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 joL. 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
               approximately 100 |o,L.  Add  20 |oL of nonane to the vial, and evaporate the solvent to the
               level of the nonane. Seal the vial and label with the sample number.  Store in the dark  at
               room temperature until ready for GC/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,
        and 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).
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        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.

               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  |oL aliquot.

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               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.

        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
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               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.
               Elute the BDEs with methylene chloride:hexane (50:50 v/v) and collect the eluate.  A
               higher percent methylene chloride solution may be required for more highly activated
               alumina.

       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  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  14) - Used for removal of lipids from tissue extracts

       13.5.1  Prepare the column as given in Section 7.5.3.

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

       13.5.5  If necessary, exchange the extract to a solvent suitable for the additional cleanups to be
               used (Section 13.2-13.4 and 13.6).
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        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 joL as described in Section 12.7 and
               proceed with the analysis in Section 14.

13.6    Florisil cleanup

        13.6.1 Begin to drain the n-hexane from the column (Section 7.5.4.1.2).  Adjust the flow rate of
               eluantto 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.

        13.6.3 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.
14.0    HRGC/HRMS analysis

14.1    Establish the operating conditions given in Section 10.1.

14.2    Add 2 joL of the labeled injection internal standard spiking solution (Section 7.14) to the 20 |o,L
        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., 21 joL if a 1 |oL injection volume was used; 20 joL if a 2 joL
        injection volume was used; 17 (iL if a 5 (iL injection volume was used).

14.3    Inject 1.0 or 2.0|oL (5 (iL if using the short column) of the the concentrated extract that contains
        the labeled injection internal standards (Section 14.2) 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/z's at each LOB throughout the LOB retention time window. Where
               warranted, monitor m/z's associated with congeners at higher levels of bromination to
               assure that fragments are not interfering with the m/z's for congeners at lower levels of
               bromination.  Also where warranted, monitor m/z's associated with interferents expected
               to be present.

        14.3.3 Stop data collection after 13Ci2-DeBDE has eluted. Return the column to the initial
               temperature for analysis of the next extract or standard.
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Note:  It has been reported that decabromodiphenylethane elutes approximately 3 minutes after DeBDE,
has m/z 's in common with 13C12-DeBDE, and may interfere in a subsequent run. If known or suspected to
be present,  sufficient time should be allowed for decabromodiphenylethane to elute.
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 7.10.2.2.2) 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.

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 (7.10.2.2.2).

       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

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        15.4.1  Retention times

               15.4.1.1    Absolute-The absolute retention times for the labeled congeners (Section
                          7.12 and Table 3) in the verification test (Section 15.3) must be within  15
                          seconds of their respective retention times in the calibration (Section 10).

               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 for the long column (Section 6.9.1.1), for the column that
                          elutes the mono- through nona- BDEs in the 2-column system (Section
                          6.9.1.2) or within their respective RRT limits for the alternate column or
                          column system (Section 6.9.1.3). If a short column (Section 6.9.2) is
                          employed, the RRTs of the BDEs and labeled compounds must be within the
                          limits established from the data in Table 11 (see the footnote to that table for
                          establishing RT and RRT limits for the short column).

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

        15.4.2  GC minimum analysis time and peak tailing

               15.4.2.1    The peak tailing, resolution, and minimum analysis time specifications in
                          Sections 6.9.1 and 6.9.2 must be met for the column or column system
                          employed..  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  DeBDE Breakdown Test - Optionally, a DeBDE breakdown test may be performed -
               Analyze DeBDE by itself and if more than 10% total OcBDE + NoBDE are present,
               further adjust injector 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

EPA Method 1614A                                48                                       May 2010

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               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 (SR). Express the accuracy
               as a recovery interval from R - 2SR to R + 2SR. For example, if R = 95% and SR = 5%,
               the accuracy is 85 to 105%.

15.6    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.
16.0    Qualitative determination

Note:   A BDE or labeled compound is identified in a standard, blank, or sample when all of the criteria
in Sections 16.1-16.4 are met.

16.1    The signals for the two exact m/z's 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/z's 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 one 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
EPA Method 1614A                                49                                       May 2010

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       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.

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

                                               (Aln + A2n)C1
                                Cex (ng/mL) = - -
                                  e            (A11 + A21)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 of the labeled compounds (except labeled PCB 138L) and of
              native BDEs other than those that have a labeled analog using the response factors
              determined from calibration (Section 10.5) or calibration verification (Section 15.4.2.2)
              and the following equation:
                                               (A1S + A2S)C-
                                Cex  (ng/mL) =            *
               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:
                        _,        ,0/.   Concentrai on found (ng/mL)
                        Recovery (%) = -  - - - x 100
                                       Concentrai on spiked (ng/mL)
EPA Method 1614A                                50                                       May 2010

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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:
                                                               C   V
                         Concentration in solid sample (ng/kg) = -
                                                                 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
       11.4.2.1), as follows:

                                                             C  V
                    Concentration in aqueous sample (ng/L) = - x 1000
                                                                's
       where:
       Cex  =  concentration of the BDE in the extract (pg/mL)
       Vex  =  extract volume (mL)
       Vs  =  sample volume (L)

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/|oL
       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
EPA Method 1614A                                51                                       May 2010

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                                     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 a minimum of 10 blanks (Reference 15).
                                     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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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 |oL
        (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
        |oL after all cleanup procedures have been exhausted. If a smaller aliquot of soils or mixed-phase
        samples is analyzed, attempt to assure that the sample is representative.

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

18.4    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).

        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.

EPA Method 1614A                                53                                        May 2010

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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.

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 1614A was developed in a single laboratory (Reference 16). 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.
EPA Method 1614A                                54                                       May 2010

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

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

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

8.  "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.

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

10. Provost, L.P., and Elder, R.S., "Interpretation of Percent Recovery Data," American Laboratory, 15:
    56-83, 1983.

11. Standard Methods  for the Examination of Water and Wastewater, 20th Edition, published jointly by
    American Public Health Association, American Water Works Association, and Water Environment
    Federation, Washington, DC, 2005.

12. "Methods 330.4 and 330.5  for Total Residual Chlorine," USEPA, EMSL, Cincinnati, OH 45268,
    EPA 600/4-70-020, May 1979.

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

14. 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
    Poly chlorinated Biphenyls: Application to Fish and Marine Mammals," Chemosphere 22:9-10,  849-
    858, 1991.

15. Ferrario, J.C., C. Byrne, A.E. Dupuy, Jr., "Background Contamination by Coplanar Fob/chlorinated
    Biphenyls (PCBs) in Trace Level High Resolution Gas Chromatography/High Resolution Mass
    Spectrometry (HRGC/HRMS) Analytical  Procedures" Chemosphere 34:11, 2451-2465, 1997.

16. Initial information for this method was provided to EPA by AXYS Analytical Systems and this
    information is contained in this method. No further report is available.

EPA Method 1614A                                55                                      May 2010

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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-MoBDE 3
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
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
BDE-3 9
Labeled analog 1
Name


13C12-4-MoBDE











13C12-4,4'-DiBDE












13C12-2,4,4'-TrBDE











Number


BDE-3L











BDE-15L












BDE-28L











EPA Method 1614A
56
May 2010

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       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
2,2',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'-TeBDE*'5
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'-TeBDE4
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'-TeBDE 4
3,3',4,5-TeBDE
3,3',4,5'-TeBDE4
3,3',5,5'-TeBDE
Number
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
Labeled analog 1
Name







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





























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



Number







BDE-47L





























BDE-77L



EPA Method 1614A
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       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
3,4,4',5-TeBDE
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
Number
BDE-81
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- 11 6
BDE-117
BDE-118
BDE-119
BDE-120
BDE-121
Labeled analog 1
Name


















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





















Number


















BDE-99L
BDE-100L





















EPA Method 1614A
58
May 2010

-------
       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',3,3',4,5-PeBDE
2',3,4,4',5-PeBDE
2',3,4,5,5'-PeBDE
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'-HxBDE 4 5
2,2',4,4',5',6-HxBDE 4 5
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
Number
BDE-122
BDE-123
BDE-124
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
Labeled analog 1
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 1614A
59
May 2010

-------
       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
2,3,3',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
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-HpBDE 4 5
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-OcBDE 3
Number
BDE-163
BDE-164
BDE-165
BDE- 166
BDE-167
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
Labeled analog 1
Name




















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




















Number




















BDE-183L




















EPA Method 1614A
60
May 2010

-------
       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
2,2',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'-NoBDE 3
DeBDE 4'5
Number
BDE-204
BDE-205
BDE-206
BDE-207
BDE-208
BDE-209
Labeled analog 1
Name





13C12-DeBDE
Number





BDE-209L
        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 1614A
61
May 2010

-------
 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 the long (30-m) DB-5ht Column (Section 6.9.1.1)
Br
No.1
Congener
No. 2'3'4
RT Ref 5
RT
(min:sec) 6
RRT7
RRT limits 8
Window
(sec) 9
Quantitation
reference 10
MDLs and MLs by Matrix "
Water (pg/L)
MDL
ML
Other (ng/kg)
MDL
ML
Extract
(pg/HL)
ML
Compounds using PCB-52L (13Ci2-2,2',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(12)





100





4





10





5



Tribromodiphenyl ethers
3
3
o
J
3
3
3
3
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
4
4
4
4
75
51
49
71
47
79
66
77
47L
47L
47L
47L
47L
47L
47L
77L
26:04
26:12
26:25
26:33
27:05
27:26
27:40
28:34
0.9625
0.9674
0.9754
0.9803
1.0000
1.0123
1.0215
1.0000
0.9563-0.9686
0.9612-0.9735
0.9692-0.9815
0.9742-0.9865
0.9988-1.0018
1.0062-1.0185
1.0154-1.0277
0.9988-1.0018
 10
 10
 10
 10
-2+3
 10
 10
-2+3
47L/77L
47L/77L
47L/77L
47L/77L
47L
47L/77L
47L/77L
77L
30



25 (12)

20

100



100

50

3



2.5

2

10



10

5

5



5

2.5

EPA Method 1614A
62
May 2010

-------
 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 the long (30-m) DB-5ht Column (Section 6.9.1.1)
Br
No.1
Congener
No. 2'3'4
RT Ref 5
RT
(min:sec) 6
RRT7
RRT limits 8
Window
(sec) 9
Quantitation
reference 10
MDLs and MLs by Matrix "
Water (pg/L)
MDL
ML
Other (ng/kg)
MDL
ML
Extract
(pg/HL)
ML
Labeled compounds
1
2
o
5
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
Hexabromodi
6
6
6
6
6
6
155
154
153
140
138/166
128
100L
100L
99L
99L
126L
126L
126L
30:10
30:28
31:04
31:19
32:34
32:51
33:08 12
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


phenyl ethers
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

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
o
J

2
10

5
5

2.5
Octabromodiphenyl ether
8
203
209L
42:4013
1.1282
1.1194-1.1370
20
209L





EPA Method 1614A
63
May 2010

-------
 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 the long (30-m) DB-5ht Column (Section 6.9.1.1)
Br
No.1
Congener
No. 2'3'4
Nonabromodi
9
9
9
208
207
206
RT Ref 5
RT
(min:sec) 6
RRT7
RRT limits 8
Window
(sec) 9
Quantitation
reference 10
MDLs and MLs by Matrix "
Water (pg/L)
MDL
ML
Other (ng/kg)
MDL
ML
Extract
(PS/HL)
ML
phenyl ether
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 02)
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










1. Number of bromines on congener 10. Labeled congeners that form the quantitation reference. Areas from the exact m/z's of the
2. Suffix "L" indicates labeled compound. congeners listed in the quantitation reference are summed, and divided by the number of
3. Two congeners in a cell indicate a coelution congeners in the quantitation reference. For example, for congener 75, the areas at the exact
4. BDEs of primary interest are shown in boldface. m/z's for 47L and 77L are summed and the sum is divided by 2 (because there are 2 congeners
5. Retention time reference that is used to locate the target congener in the quantitation reference).
6. Retention time (RT) of the target congener. 1 1 . MDLs for "Other" matrix calculated per procedure at 40 CFR 1 36, Appendix B; MLs for
7. Relative retention time (RRT) between the RT for the congener and "Other" matrix calculated per procedure at 68 FR 1 1790 (May 12, 2003); MDLs and MLs for
RT for the reference. aqueous matrix calculated as 10 times the MDLs and MLs for the "Other" matrix. MDLs and
8. Limits based on the magnitude of the RRT, adjusted for closely MLs for congeners for which MDLs and MLs are not shown will be similar to the MDLs and
eluting isomers MLs for congeners at the same level of bromination.
9. Window width for congener or congener pair. RTs, RRTs, and RRT 12. This MDL may not be achievable with a laboratory background present. See Table
limits may differ slightly from those in Table 2. 10 for MDL achievable with background present for this congener.
1 3 . Estimated retention time
EPA Method 1614A
64
May 2010

-------
    Table 3.  Concentrations of Native and Labeled Brominated Diphenyl Ethers in Stock
             Solution, Spiking Solution, and Final Extract

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
5.0
5.0
1000
1000
100
100
Diluted combined congener

Standard
Solution concentration (ng/mL)
Native
Labeled
Native congeners
MoBDEthruNoBDE
DeBDE
50
500


Labeled congeners
MoBDEthruNoBDE
DeBDE
Labeled cleanup
Labeled injection internal




100
1000
100
100
EPA Method 1614A
65
May 2010

-------
                           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
Total number of congeners
34
BDE-A2
11
13
25
33
51
71
79
120
116
155
105
166
183
190
207




















15
                           1.   Congeners listed 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 1614A
66
May 2010

-------
 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
13C12-2,2',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
EPA Method 1614A
67
May 2010

-------
 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.   See Section 15.3.
EPA Method 1614A
68
May 2010

-------
 Table 7.  Scan Descript
          Monitored by
ors, Levels of Bromination and Chlorination, m/z's, and BDEs and PCBs
HRGC/HRMS
Function and bromine
or chlorine level
Fn-l;Br-l
Fn-2; Br-2; Cl-4
Fn-3Br-3;Br-4;Cl-6
Fn-4; Br-5; Br-6
Fn-5; Br-7; Br-8
m/z1'2
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
716.9569
721.4406
723.4386
733.4809
735.4788
799.3511
801.3491
811.3914
813.3893
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
lock
M+6
M+8
M+6
M+8
M+6
M+8
M+6
M+8
m/z formula
12C12H916079Br
12C12H916O81Br
13C12H916079Br
13C12H916081Br
12C6Fn
13C12H635C14
13C12H635C1337C1
12C12H816O79Br2
12C12H816079Br81Br
12C7 F13
13C12H816079Br2
13C12H816079Br81Br
13C12H535C1537C1
13C12H635C1437C12
12C12H716079Br281Br
12C12H716079Br81Br2
13C12H716079Br281Br
13C12H716079Br81Br2
CioFn
12C12H616079Br381Br
12C12H616079Br281Br2
13C12H616079Br281Br2
13C12H616079Br81Br3
12C13F21
12C12H516079Br381Br2
12C12H516079Br281Br3
13C12H516079Br381Br2
13C12H516079Br281Br3
12C12H416079Br481Br2
12C12H416079Br381Br3
13C12H416079Br381Br3
13C12H416079Br281Br4
12C17 F27
12C12H316079Br481Br3
12C12H316079Br381Br4
13C12H316079Br481Br3
13C12H316079Br381Br4
12C12H216079Br581Br3
12C12H216079Br481Br4
13C12H216079Br581Br3
13C12H216079Br481Br4
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
PFK
HpBDE
HpBDE
13C12 HpBDE
13C12 HpBDE
OcBDE
OcBDE
13C12 OcBDE
13C12 OcBDE
EPA Method 1614A
                     69
May 2010

-------
 Table 7.  Scan Descriptors, Levels of Bromination and Chlorination, m/z's, and BDEs and PCBs
          Monitored by HRGC/HRMS
Function and bromine
or chlorine level
Fn-6; Br-9; Br-10
m/z1'2
879.2596
881.2575
891.2998
892.9441
893.2978
957.1701
959.1680
971.2083
973.2063
m/z type
M+8
M+10
M+8
lock
M+10
M+8
M+10
M+10
M+12
m/z formula
12C12H16O79Br581Br4
12C12H16079Br481Br5
13C12H16079Br581Br4
Ci9F35
13C12H16079Br481Br5
12C1216079Br681Br4
12C1216079Br581Br5
13C1216079Br581Br5
13C1216079Br481Br6
Substance
NoBDE
NoBDE
13C12 NoBDE
PFK
13C12 NoBDE
DeBDE
DeBDE
13C12 DeBDE
13C12 DeBDE
1. Isotopic masses used for accurate mass calculation
'H
12C
13C
16Q
35C1
37C1
79Br
81Br
19F
1.0078
12.0000
13.0034
15.9949
34.9689
36.9659
78.9813
80.9163
18.9984
2. The masses we have listed here are those which provided the most unambiguous identification in our
work.
EPA Method 1614A
70
May 2010

-------
 Table 8. Theoretical Ion Abundance Ratios and QC Limits
Bromine Atoms
1
2
o
J
4

5
6

7
8
9
10
10
m/z's 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)
(M+10)/(M+12)
Theoretical Ratio
1.03
0.51
1.03
0.70
1.54
1.03
0.77
1.37
1.03
0.82
1.03
0.86
1.23
Lower QC Limit
0.88
0.43
0.88
0.60
1.31
0.88
0.65
1.16
0.88
0.70
0.88
0.73
1.05
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
1.41
Chlorine atoms
4 M/(M+2)
6 (M+2)/(M+4)
0.78
1.25
0.66
1.06
0.90
1.44
EPA Method 1614A
71
May 2010

-------
 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

10 g
10 g
1.  The quantity of sample to be extracted is adjusted to provide 10 g of solids (dry weight). One liter of aqueous
    samples containing one percent solids will contain 10 grams of solids. For aqueous samples containing greater
    than one percent solids, a lesser volume is used so that 10 grams of solids (dry weight) will be extracted.

2.  The sample matrix may be amorphous for some samples. In general, when the 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 1614A
72
May 2010

-------
Table 10. MDLs Obtained Using the Short (15-Meter) Column and
Temperature-Programmable Injector
Analyte
2,4-DiBDE
2,4'-DiBDE/3,3'-DiBDE
2,6-DiBDE
3,4-DiBDE/3,4'-DiBDE
4,4'-DiBDE
2,2',4-TriBDE/2,3 ',4-TriBDE
2,4,4'-TriBDE/2',3,4-TrBDE
2,4,6-TriBDE
2,4',6-TriBDE
3,3',4-TriBDE
3,4,4'-TriBDE
2,2',4,4'-TeBDE
2,2',4,5'-TeBDE
2,2',4,6'-TeBDE
2,3',4,4'-TeBDE
2,3',4',6-TeBDE
2,4,4',6-TeBDE
3,3',4,4'-TeBDE
3,3',4,5'-TeBDE
2,2',3,4,4'-PeBDE
2,2',4,4',5-PeBDE
2,2',4,4',6-PeBDE
2,3,3',4,4'-PeBDE
2,3,4,5,6-PeBDE
2,3',4,4',6-PeBDE/2,3',4,5,5'-
PdBDE
3,3',4,4',5-PeBDE
2,2',3,3',4,4'-HxBDE
2,2',3,4,4',5'-
HxBDE/2,3,4,4',5,6-HxBDE
2,2',3,4,4',6'-HxBDE
2,2',4,4',5,5'-HxBDE
2,2',4,4',5,6'-HxBDE
2,2',4,4',6,6'-HxBDE
2,2',3,4,4',5,6-HpBDE
2,2',3,4,4',5',6-HpBDE
2,3,3',4,4',5,6-HpBDE
2,2',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'-NoBDE
2,2',3,3',4,4',5,5',6,6'-DeBDE
Congener No.
7
8/11
10
12/13
15
17/25
28/33
30
32
35
37
47
49
51
66
71
75
77
79
85
99
100
105
116
119/120
126
128
138/166
140
153
154
155
181
183
190
203
206
207
208
209
MDL (pg/L)
27
79
9
60
29
29
9
10
5
13
16
48
8
10
11
10
12
9
5
9
33
7
8
13
12
9
15
34
22
11
5
6
13
18
20
95
296
151
95
1499
EPA Method 1614A
73
May 2010

-------
Table 11. Retention Times Using the Short (15-m Column) and Temperature
Programmable Injector
Analyte
2,4-DiBDE
2,4'-DiBDE/3,3'-DiBDE
2,6-DiBDE
3,4-DiBDE/3,4'-DiBDE
4,4'-DiBDE
2,2',4-TriBDE/2,3 ',4-TriBDE
2,4,4'-TriBDE/2',3,4-TrBDE
2,4,6-TriBDE
2,4',6-TriBDE
3, 3 ',4-TriBDE
3,4,4'-TriBDE
2,2',4,4'-TeBDE
2,2',4,5'-TeBDE
2,2',4,6'-TeBDE
2,3',4,4'-TeBDE
2,3',4',6-TeBDE
2,4,4',6-TeBDE
3,3',4,4'-TeBDE
3,3',4,5'-TeBDE
2,2',3,4,4'-PeBDE
2,2',4,4',5-PeBDE
2,2',4,4',6-PeBDE
2,3,3',4,4'-PeBDE
2,3,4,5,6-PeBDE
2,3',4,4',6-PeBDE/2,3',4,5,5'-
PdBDE
3,3',4,4',5-PeBDE
2,2',3,3',4,4'-HxBDE
2,2',3,4,4',5'-HxBDE/2,3,4,4',5,6-
HxBDE
2,2',3,4,4',6'-HxBDE
2,2',4,4',5,5'-HxBDE
2,2',4,4',5,6'-HxBDE
2,2',4,4',6,6'-HxBDE
2,2',3,4,4',5,6-HpBDE
2,2',3,4,4',5',6-HpBDE
2,3,3',4,4',5,6-HpBDE
2,2',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
lUPACCongener
No.
7
8/11
10
12/13
15
17/25
28/33
30
32
35
37
47
49
51
66
71
75
77
79
85
99
100
105
116
119/120
126
128
138/166
140
153
154
155
181
183
190
203
206
207
RT (min:sec)*
6:36
6:45
6:18
6:51
6:58
8:18
8:33
7:39
8:08
8:43
8:55
10:38
10:15
10:09
10:57
10:20
10:04
11:31
10:49
14:12
13:08
12:32
14:35
13:19
12:44
14:24
18:08
16:55
16:17
15:44
14:51
14:24
19:49
18:22
20:01
22:07
26:19
25:33
EPA Method 1614A
74
May 2010

-------
Table 11. Retention Times Using the Short (15-m Column) and Temperature
Programmable Injector
Analyte
2,2',3,3',4,5,5',6,6'-NoBDE
2 2' 3 3' 4 4' 5 5' 6 6'-DeBDE

Labels
13C12-4,4'-DiBDE
13C12-2,4,4'-TriBDE
13C12-2,2',4,4'-TeBDE
13C12-3,3',4,4'-TeBDE
13C12-2,2',4,4',5-PeBDE
13C12-2,2',4,4',6-PeBDE
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
13C12-2,2',3,4,4',5',6-HpBDE
lUPACCongener
No.
208
209

15L
28L
47L
77L
99L
100L
126L
139L
153L
154L
183L
RT (min:sec)*
25:12
31:19

6:58
8:32
10:38
11:30
13:07
12:32
14:24
16:03
15:43
14:50
18:22
          * See Table 2 for retention time (RT) and quantitation references, and for RT windows.
            RRT limits may be constructed from the RTs in this table and the RT windows in Table 2
            if desired.
EPA Method 1614A
75
May 2010

-------
Table 12. Precision and Recovery Data Obtained in Reagent Water Using the
Short (15-m) Column and Temperature-Programmable Injector*
Analyte
BDE-28/BDE-33
BDE-47
BDE-100
BDE-99
BDE-154
BDE-153
BDE-183
BDE-209
13C-BDE-28
13C-BDE-47
13C-BDE-100
13C-BDE-99
13C-BDE-154
13C-BDE-153
13C-BDE-183
13C-BDE-209
13C-BDE-139
IPR1
% Rec
97
107
94
98
96
94
95
121
108
89
98
100
100
97
96
51
109
IPR2
% Rec
97
108
97
96
94
94
95
122
100
88
92
100
103
96
96
50
104
IPR3
% Rec
96
113
94
98
93
94
90
115
105
90
104
110
111
108
107
57
118
IPR4
% Rec
96
114
95
100
93
94
96
126
111
96
112
113
117
110
109
54
124
Avg %
Rec
97
110
95
98
94
94
94
121
106
91
102
106
108
103
102
53
114
SD
0.6
3.6
1.4
1.6
1.5
0.4
2.6
4.7
4.6
3.8
8.7
6.7
7.6
7.3
7.1
3
8.8
         *Recoveries and standard deviation expressed as percent
EPA Method 1614A
76
May 2010

-------
                                      Determine % solids
                                            11.2
                                     Determine particle size
                                            11.3
             Prep per 11. 5
        Spike Labeled Toxics/LOC
          window-definers per
               11.5.2.2
                 Prep per 11. 4
             Spike Labeled Toxics/LOC
               window-definers per
                   11.4.2.2
                                         Spike Cleanup standard per
                                                12.5.1
                                             Back extract per
                                                 12.5
                                             Transfer through
                                            Na2S04 per 12.5.6
                                                                  Extract per 12.2.1,
                                                                  12.2.2, or 12.2.3
                                                                                    1
                                                                               Concentrate per
                                                                                12.6 -12.7
                                                                                 Clean up per
                                                                            13.2 -13.5, or 13.7
                                                                               Concentrate per
                                                                                12.6 -12.7
                                                                                    I
                                                                             Spike injection internal
                                                                              standard per 14.2
                                                                                 Analyze per
                                                                                 14-18
       Figure  1   Flow Chart for Analysis of Aqueous and Solid Samples
EPA Method 1614A
11
May 2010

-------
                          Aqueous
            Discard
                                             Determine %solids
                                                 per 11.2
                                           Determine particle size
                                                per 11.3
                                          Spike LabeledToxics/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 C lean up sta ndard per
                                                  12.5.1
                                                Back extract
                                                 per 12.5
                                               Transfer thro ugh
                                              Na2S04 per 12.5.6
    Figure 2  Flow Chart forAnalysis 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 hjection internal
                                                                                 standard per  14.2
                               Analyze per
                                14-18
                                                                                             SCC-9 9-01 8
EPA Method 1614A
78
May 2010

-------
                                                 Homogenize tissue
                                                   per 11.8.1
                                                   Remove 10 g
                                                   per 11.8.1.4
                                              Spike Labeled Toxics/LOC
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,18
                           Figure  3 Flow Chart for Analysis of Tissue Samples
EPA Method 1614A
79
May 2010

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                                                      90-mm 6MF150 Filter
                                             1-liter Suction Flask
                           Figure 4.  Solid-phase Extraction Apparatus
EPA Method 1614A
80
May 2010

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                           Figure 5.  Soxhlet/Dean-Stark Extractor
EPA Method 1614A
81
May 2010

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  Figure 6. DB-5HT column resolution test. Separation of BDE-49 and BDE-71 with a valley
           less than 40% (i.e., 100 x/y < 40%)
EPA Method 1614A
82
May 2010

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24.0   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
               |oL     microliter
               jam    micrometer
               <      less than
               >      greater than
               o/
               7o
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

24.2   Definitions and acronyms (in alphabetical order)

Analyte - A BDE tested for by this method.  The analytes are listed in Table  1.

BDE - See brominated diphenyl ether

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.

EPA Method 1614A                              84                                      May 2010

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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


EPA Method 1614A                              85                                      May 2010

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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

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; May 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.

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.

EPA Method 1614A                              86                                      May 2010

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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 un-retained 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

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 standard
EPA Method 1614A                               87                                      May 2010

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