Method 8276 8276 - 1 Revision 1 October 2012 METHOD 8276

TOXAPHENE AND TOXAPHENE CONGENERS BY GAS CHROMATOGR APHY/NEGATIVE ION

CHEMICAL IONIZATION MASS SPECTROMETRY (GC-NICI/MS)

SW-846 is not intended to be an analytical training manual. Therefore, method procedures are written based on the assumption that they will be performed by analysts formally trained in the basic principles of chemical analysis and in the us e of the subject technology.

In addition, SW-846 methods, with the exception of required use for the analysis of method-defined parameters, are intended to be guida nce methods which contain general information on how to perform an analytical procedu re or technique, which a laboratory can use as a basic starting point for generating its own de tailed standard operating procedure (SOP), either for its own general use or for a specific pr oject application. Performance data included in this method are for guidance purposes only, and mus t not be used as absolute quality control (QC) acceptance criteria for the purposes of labora tory QC or accreditation.

1.0 SCOPE AND APPLICATION

Please see Appendix A at the back of this document for a summary of revisions to Method
8276 (From March 2010).

1.1 This method is used to determine the concentrat ions of various toxaphene congeners (see table below) and technical toxaphene (along with the possible addition of other toxaphene congeners and compounds from Method 8081) in extracts from solid and liquid matrices, using fused-silica, open-tubular capillar y columns with negative ion chemical ionization mass spectrometry (NICI/MS). The approach emphasize s the analytical conditions recommended for technical toxaphene and for toxaphene congeners as compared to weathered toxaphene. Technical toxaphene can be definitively quantitated by NICI/MS while weathered toxaphene may only appear to be present based on the detection of ions found in toxaphene or the presence of known degradation products of toxaphene (e.g., Hx-S ed and Hp-Sed). For this reason, the quantitation of weathered toxaphene may be consider ed subjective and qualitative with the success highly dependent on matching the calibratio n standards to the weathered peak pattern. For clarification, any notation of toxaphene in the method hereafter will refer to technical toxaphene.

Compound CAS Registry No. a

Toxaphene 8001-35-2 Toxaphene Congeners:

2-exo,3-endo,6-exo,8,9,10-Hexachlorobornane (Hx-Se d) 57981-29-0 2-endo,3-exo,5-endo,6-exo,8,9,10-Heptachlorobornane (Hp-Sed) 70649-42-2 2-endo,3-exo,5-endo,6-exo,8,8,10,10-Octachloroborna ne (P26) 142534-71-2 2-endo,3-exo,5-endo,6-exo,8,9,10,10-Octachloroborna ne (P40) 166021-27-8 2-exo,3-endo,5-exo,8,9,9,10,10-Octachlorobornane (P 41) 165820-16-6 2-exo,5,5,8,9,9,10,10-Octachlorobornane (P44) 16582 0-17-7 2-endo,3-exo,5-endo,6-exo,8,8,9,10,10-Nonachlorobor nane (P50) 6680-80-8 2,2,5,5,8,9,9,10,10-Nonachlorobornane (P62) 154159- 06-5

a Chemical Abstract Service Registry NumberMethod 8276 8276 - 2 Revision 1 October 2012 1.2 The analyst should select gas chromatography (G C) columns, detectors, and calibration procedures most appropriate for the spe cific analytes of interest in a particular project application. Matrix-specific performance da ta should be generated, and the stability of the analytical system and instrument calibration mu st be established for each analytical matrix (e.g., hexane solutions from various sample matrix extractions). Example chromatograms and GC-NICI/MS conditions are provided as guidance.

1.3 Although performance data are presented only fo r toxaphene and toxaphene congeners, in future method revisions additional ta rget analytes (e.g., from Method 8081) may be added if acceptable performance can be demonstra ted. When more analytes are included, it may become likely that not all of them can be deter mined in a single analysis. The chemical and chromatographic behaviors of these additional chemi cals may result in coelution of some target analytes. Several cleanup/fractionation schemes are provided in this method for additional

details refer to Method 3600.

1.4 Multi-component mixtures (e.g., chlordane and t oxaphene) present additional difficulties that include the need to separate cong eners (Sec. 2.5 and 3.1) of the mixture. When samples contain more than one multi-component analy te, a higher level of expertise is necessary to attain acceptable levels of qualitativ e and quantitative analysis. The same is true of multi-component analytes that have been subjecte d to degradation by the environment or treatment technologies. These processes result in “ weathered” multi-component mixtures that may have significant differences in peak patterns c ompared to those of the standards.

1.5 Unless present in relatively high concentration s, 4,4’-DDD, 4,4’-DDE, 4,4’-DDT and related compounds will not pose significant int erferences to toxaphene analysis by NICI/MS because they do not respond with similar sensitivit y.

1.6 This method has primarily been validated for th e analysis of target analytes listed in Sec. 1.1. Extracts suitable for analysis by this method may also be analyzed for other organochlorine and organophosphorus pesticides (Met hods 8081 and 8141) provided acceptable performance data can be generated. Addit ionally, some extracts may also be suitable for triazine herbicide analysis. However, low recoveries of triazine herbicides may result from lack of sample preservation. In addition, shou ld users generate acceptable performance data for organochlorine pesticide compounds as note d in Method 8081, this method may be considered as an appropriate alternative to Method 8081.

1.7 Prior to employing this method, analysts are ad vised to consult the base method for each type of procedure that may be employed in the overall analysis (e.g., Methods 3500, 3600, and 8000) for additional information on QC pr ocedures, development of QC acceptance criteria, calculations, and general guidance. Analy sts also should consult the disclaimer

statement at the front of the SW-846 manual and the information in Chapter Two for 1) guidance on the intended flexibility in the choice of method s, apparatus, materials, reagents, and supplies; and 2) the responsibilities of the analys t for demonstrating that the techniques employed are appropriate for the target analytes in the matrix of interest, and at the level(s) of

concern.

In addition, analysts and data users are advised th at, except where explicitly specified in a regulation, the use of SW-846 methods is not mand atory in response to Federal testing requirements. The information contained in this met hod is provided by the Environmental Protection Agency (EPA) as guidance to be used by t he analyst and the regulatory community in making judgments necessary to generate results t hat meet the data quality objectives (DQOs)Method 8276 8276 - 3 Revision 1 October 2012 for the intended application.

1.8 Application of this method is restricted to use by, or under the supervision of, personnel appropriately experienced and trained in the use of GC-NICI/MS and skilled in the interpretation of applicable chromatograms. Each an alyst must demonstrate the ability to generate acceptable results with this method.

1.9 This method depends on resonance electron captu re (electron attachment) or dissociative electron capture in the ion source whe n the instrument is operated under methane chemical ionization (CI) conditions. Thus, the tech nique produces a transient ionic species that can be detected as a molecular anion of a compound or as a dissociated ion of that species produced by dissociative electron capture. Other mo derating gases besides methane may be used provided acceptable project-specific performan ce data can be generated. Within this description, the technique does not strictly result from a CI event (i.e., the result of an ionmolecule reaction) and refers to the production of negative ions by any of these processes as

NICI/MS. See Ref. 1 for a more detailed discussion of NICI/MS theory.

2.0 SUMMARY OF METHOD

2.1 The samples are prepared for analysis by GC-NIC I/MS using the appropriate sample preparation (refer to Method 350 0) and, if necessary, sample cleanup procedures (refer to Method 3600).

2.2 The extract is analyzed by injection of a sampl e aliquot into a gas chromatograph with a narrow-bore fused-silica capillary column in terfaced to a mass spectrometer capable of performing NIMS under CI conditions with methane as the introduced gas.

2.3 Analysis of toxaphene involves monitoring a ser ies of ions representing various congener groups found in the mixture and integratin g all of these signals for a total toxaphene response. In the case of the toxaphene congeners, i ndividual compounds are quantitated separately and reported separately.

2.4 This method does not address all the congeners that comprise toxaphene. Toxaphene congeners can be added to the method by i dentifying retention windows with the additional congeners, assuming their responses are resolved from other congeners. Any added analytes must meet performance-based QC acceptance criteria.

3.0 DEFINITIONS

3.1 Toxaphene - A complex mixture of polychlorinate d monoterpenes (primarily bornanes and camphenes) produced commercially from 1947 to 1982 and purported to contain 600-plus separate congeners. Also known as camphechlor, Melipax, chlorocamphene, polychlorocamphene, and chlorinated camphene.

3.2 GC-NICI/MS - All techniques producing negativel y charged ions used for confirmation and quantitation of analytes. See Sec. 1.9 for additional details.

3.3 Congener - One of many variants or configuratio ns of a common starting

material. Most often the term is used to describe a chlorinated or brominated species of aMethod 8276 8276 - 4 Revision 1 October 2012 given starting material that gives rise to multiple levels of halogenation to fully halogenated compounds.

3.4 Parlar number - Individual toxaphene congeners based on GC elution order are also known as "Parlars". For ease of reference, spe cific congeners have been given a Parlar number (e.g., P26, P50). More information on Parla r nomenclature can be found in reference

28.

3.5 Weathered toxaphene - In the environment, techn ical toxaphene degrades over time via both biotic and abiotic processes. The maj or processes of this degradation seem to be dechlorination and dehydrochlorination that lead to a pronounced shift toward lower chlorinated homologs. Weathered toxaphene may have a different chromatographic profile and NICI/MS detector response when compared to “virgin” toxaphe ne; this is a result of the composition changes which occurs from environmental degradation

.

3.6 Total Ion Current (TIC) - The sum of the separa te ion currents carried by the different ions contributing to the spectrum (so metimes called the reconstructed ion current).

3.7 Refer to Chapter One and the instrument manufac turer's instructions for additional definitions that may be relevant to this procedure.

4.0 INTERFERENCES

4.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or interferences during sample analys is. All of these materials must be

demonstrated to be free from interferences under co nditions of analysis by method blanks. Specific selection of reagents may be necessary. Re fer to each method used for specific guidance on QC procedures and to Chapter Four for g eneral guidance on glassware cleaning. Also refer to Methods 3500, 3600, and 8000 for a di scussion of interferences.

4.2 Interferences co-extracted from the samples wil l vary considerably from matrix to matrix. While general cleanup techniques are refere nced or provided as part of this method, unique samples may require additional cleanup appro aches to achieve desired degrees of discrimination and quantitation. Sources of interfe rence in this method can be grouped into three broad categories as follows.

4.2.1 Contaminated solvents, reagents, or sample pr ocessing hardware

4.2.2 Contaminated GC carrier gas, parts, column su rfaces, or detector

surfaces

4.2.3 Compounds extracted from the sample matrix to which the detector will respond

4.3 Interferences by phthalate esters introduced du ring sample preparation can pose a major problem in some pesticide determinations bu t not for toxaphene analysis provided the scan window or ion monitoring signal is greater tha n m/z 300. However, extremely high levels of phthalate contamination could adversely affect quan titative responses of coeluting target components by reducing the availability of thermal electrons or reagent ions needed to ionizeMethod 8276 8276 - 5 Revision 1 October 2012 target analytes. Interferences from phthalate ester s can best be minimized by avoiding contact with any plastic materials and checking all solvent s and reagents for phthalate contamination.

4.3.1 Common flexible plastics contain varying amou nts of phthalate esters that are easily extracted or leached from such mate rials during laboratory operations.

4.3.2 Exhaustive cleanup of solvents, reagents, and glassware may be necessary to eliminate background phthalate ester c ontamination.

4.3.3 Phthalate esters may be removed prior to anal ysis using Method 3640 (GPC) or Method 3630 (silica gel cleanup).

4.3.3 Cross-contamination of clean glassware routin ely occurs when plastics are handled during extraction steps, especially whe n solvent-wetted surfaces are manipulated.

4.4 The presence of sulfur will result in broad pea ks that interfere with the detection of early-eluting compounds only if the monitored io ns occur near m/z 256, 224, 192, 160, 128, and 96 (as well as the M+2 isotope due to 34 S and the corresponding M+1 peaks from 33 S). Therefore, sulfur should not interfere with monitor ed ions from toxaphene but should

nevertheless be removed because of deleterious effe cts on chromatography and on the response of the NICI/MS ion source during coelution . Sulfur contamination should be expected with sediment samples.

Method 3660 is suggested for removal of sulfur. Bec ause the recovery of endrin aldehyde is drastically reduced when using the tetr abutylammonium (TBA) procedure in Method 3660, it must be determined prior to sulfur cleanup if it is an analyte of interest. Endrin aldehyde is not affected by copper powder, so it can be dete rmined after the removal of sulfur by using the copper powder technique in Method 3660. However , as indicated in Method 3660, copper powder may adversely affect the recoveries of other potential analytes of interest, including some organochlorine compounds and many organophosph orus compounds.

4.5 Waxes, lipids, and other high molecular weight materials can be removed by Method 3640 (GPC cleanup) but are generally transpa rent to NICI/MS. Naturally occurring compounds such as flavonoids (e.g., coumarins) that contain conjugated carbonyl groups may respond sensitively under GC-NICI/MS conditions as will other kinds of compounds containing functional groups that facilitate ionization under these conditions.

4.6 Other halogenated pesticides or industrial chem icals may interfere with the analysis of pesticides. Certain coeluting organopho sphorus pesticides may be eliminated using Method 3640 (pesticide option). Coeluting chlorophe nols may be removed with Methods 3630 (silica gel), Method 3620 (Florisil®), or Method 36 10 (alumina).

4.7 PCB Interference

4.7.1 Polychlorinated biphenyls (PCBs) also may int erfere with the analysis of organochlorine pesticides. The problem may be most severe for the analysis of multicomponent analytes such as chlordane, toxaphen e, and Strobane. If PCBs are known or expected to occur in samples, the analyst should consult Methods 3620 and 3630 for techniques that may be used to separate th e pesticides from the PCBs. Under NICI/MS conditions in the absence of oxygen (oxygen reacts with PCBs), PCBs shouldMethod 8276 8276 - 6 Revision 1 October 2012 not constitute an interference concern for toxaphen e ions. The presence of the oxygen reaction does not preclude analysis, but must lead to reproducible results and would require removal of PCBs from extracts prior to toxa phene analysis. Overall, the success of analysis is strongly dependent on adequate clean up, separations of congeners/analytes, and quantitative recoveries.

4.7.2 Based on limited studies to date, the oxygen reaction observed with PCBs that gives rise to ions potentially interferin g with toxaphene determination (i.e.,

ions at the same nominal mass but not the same elem ental composition) is completely eliminated in modern instruments under appropriate conditions. The use of a PCB congener (e.g., PCB #204) serves to monitor this si tuation by the absence of (M - Cl + O) ─ ions (e.g., m/z

411) at its retention time (less than 0.5% possibl e attributable response). Thus the success of this method depends heavily on the proper extraction, cleanup, concentration, separations, and mass spect rometric conditions to achieve the quantitation and confirm the target analytes.

4.8 Coelution among the target analytes in this met hod can cause interference problems. Toxaphene congener coelution may be a pro blem for all possible GC columns regardless of the proposed analysis scheme.

4.9 Chlordane interference

4.9.1 Under GC-NICI/MS conditions, the determinatio n of toxaphene and individual congeners of toxaphene may be affected b y the presence of chlordane. In addition, chlordane congeners may coelute with cert ain toxaphene congeners and the 13 C isotope of the chlordane ion may add intensity to t he corresponding isobaric ion of toxaphene.

4.9.2 As shown in Table 3, ions for monitoring the presence of chlordane

indicate that their 13 C isotopes can contribute signals to monitored toxa phene ions. Therefore, use Table 3 to confirm whether chlordane is present at levels which significantly affect toxaphene quantitation.

4.10 Table 4 gives ions that can be used to confirm the presence of organochlorine pesticides in the sample. None of these compounds s hould result in interference with toxaphene

determination.

5.0 SAFETY

This method does not address all safety issues asso ciated with its use. The laboratory is responsible for maintaining a safe work environment and a current awareness file of OSHA regulations regarding the safe handling of the chem icals specified in this method. A reference file of material safety data sheets (MSDSs) should be available to all personnel involved in these analyses.Method 8276 8276 - 7 Revision 1 October 2012 6.0 EQUIPMENT AND SUPPLIES

The mention of trade names or commercial products i n this manual is for illustrative purposes only, and does not constitute an EPA endor sement or exclusive recommendation for use. The products and instrument settings cited in SW-846 methods represent those used during method development or subsequently evaluated by the Agency. Glassware, reagents, supplies, equipment, and settings other than those listed in this manual may be employed provided that method performance appropriate for th e intended application has been

demonstrated and documented.

6.1 Gas chromatograph (GC) - An analytical system e quipped with a temperature programmable oven suitable for any applicable injec tion technique and all required accessories including syringes, analytical columns, and gases. The capillary column should elute directly into the ion source of the mass spectrometer.

6.2 GC columns - The columns listed in this section were used in developing the

method and are not intended to exclude others that are available or may be developed. Laboratories may use these columns or other columns provided that they document method performance data (e.g., chromatographic resolution, analyte breakdown, and sensitivity) appropriate for the intended application.

Given the performance data available to date, the D B-XLB stationary phase columns may provide the maximum practical resolving power f or congener separations.

6.2.1 30-m x 0.25-mm ID fused-silica capillary colu mn DB-XLB MSD (J&W Scientific), 0.25-μm film thickness

6.2.2 30-m x 0.25-mm ID fused silica capillary colu mn ZB-MultiResidue-1 (Phenomenex), 0.25-μm film thickness. This column a ppears similar to DB-XLB

MSD.

6.2.3 40-m x 0.18-mm ID fused silica capillary col umn DB-5MS (J&W Scientific), 0.18- μm film thickness

NOTE : DB-5 columns have been problematic, however, in separating some of the Parlars, which thus results in summed values.

6.3 Column rinsing kit - Bonded-phase column rinse kit (J&W Scientific, Catalog No. 430-3000 or equivalent).

6.4 Mass spectrometer

6.4.1 The mass spectrometer must be capable of prod ucing a mass spectrum in the negative ion mode under chemical io nization conditions. Before acquiring spectra in this mode the instrument must be tuned u nder NICI conditions according to the guidance outlined in Sec. 11.4.1 and using a cu stomized NICI tune table.

6.4.2 An ion trap mass spectrometer may be used if capable of axial

modulation to reduce ion-molecule reactions and the production of NCI spectra. The mass spectrometer must be capable of producing a sp ectrum using a NCI-customized tuning file according to the guidance outlined in S ec. 11.4.Method 8276 8276 - 8 Revision 1 October 2012 6.4.3 GC/MS interface - Any GC-to-MS interface may be used that gives acceptable calibration points for each compound of interest and achieves acceptable tuning performance criteria. For a narrow-bore capi llary column, the interface is usually capillary-direct into the mass spectrometer source.

6.5 Data system - A computer system should be inter faced to the mass spectrometer. The system must allow the continuous acquisition and storage on machinereadable media of all mass spectra obtained through out the duration of the chromatographic program. The computer should have software that can search any GC/MS data file for ions of a specific mass and can plot such ion abundances vers us time or scan number. This type of plot is defined as an extracted ion current profile (EIC P). Software should also be available that allows integrating the abundances in any EICP betwe en specified time or scan-number limits.

6.6 Analytical balance, capable of weighing to 0.00 01 g

7.0 REAGENTS AND STANDARDS

7.1 Reagent-grade or pesticide-grade chemicals, at a minimum, should be used in all tests. Unless otherwise indicated, all reagents should conform to specifications (where available) of the Committee on Analytical Reagents of the American Chemical Society. Other grades may be used provided the reagent is of suffi ciently high purity to permit its use without lessening the accuracy of the determination. Reagen ts should be stored in glass to prevent leaching of contaminants from plastic containers.

NOTE: Store all standard solutions (stock, composite, c alibration, internal, and surrogate) at ≤6°C in polytetrafluoroethylene (PTFE) sealed gl ass containers in the dark. When a standard lot is prepared, aliquots should be stored in indiv idual small vials. All stock standards must be replaced after one year or sooner if routine QC (Se c. 9.0) indicates a problem. All other standard solutions must be replaced after six month s or sooner if routine QC (Sec. 9.0)

deteriorates.

7.2 Solvents used in the extraction and cleanup pro cedures (see appropriate 3500 and 3600 series methods) may include n -hexane, diethyl ether, methylene chloride (dichloromethane), acetone, ethyl acetate, and isoo ctane (2,2,4-trimethylpentane). All solvents should be pesticide grade quality or equivalent, an d each lot of solvent should be demonstrated to have negligible contamination of both target and non-target compounds (e.g., phthalates) at a minimum below the typical laboratory reporting limi t.

7.3 Organic-free reagent water - Reagent water must be interference free. All references to water in this method refer to org anic-free reagent water unless otherwise specified.

7.4 Stock standard solutions (1000 mg/L) - May be p repared from pure standard materials or can be purchased as certified solution s. Other concentrations may be used as appropriate for the intended application. If suffic ient neat material exists, prepare stock standard solutions by accurately weighing 0.0100 g of pure compound. Dissolve the compound

in isooctane or hexane and dilute to volume in a 10 -mL volumetric flask. If compound purity is ≥96%, the weight may be used without correction to calculate the concentration of the stockMethod 8276 8276 - 9 Revision 1 October 2012 standard solution. Commercially prepared stock stan dard solutions may be used at any concentration if they are certified by the manufact urer or by an independent source.

7.5 Composite stock standard - May be prepared from individual stock solutions.

7.5.1 As an example, for composite stock standards containing <25 components, add solvent to 1 mL of each individual stock solution at a concentration of 1000 mg/L (or other concentrations as appropriat e) and mix the solutions in a 25- mL volumetric flask. The resulting concentration of each component in the mixture, after the volume is adjusted to 25 mL, will be 1 mg /25 mL or 40 mg/L. This composite

solution can be further diluted to obtain the desir ed concentrations.

7.5.2 For composite stock standards containing > 25 components, use volumetric flasks of the appropriate volume (e.g., 50-mL, 100-mL), and follow the procedure described above.

7.6 Calibration standards - Should be prepared at a minimum of five different concentrations by dilution of the composite stock s tandard with isooctane or hexane and should match the sample extract solvent. Recommende d standard concentrations for establishing a calibration curve for toxaphene are 50 - 400 pg/μL and 1.0 - 500 pg/μL for all toxaphene congeners, except Parlar 62. The recommen ded standard concentrations for establishing a calibration curve for Parlar 62 are 5 - 500 pg/μL. Ranges may be extended provided that the linear response can be adequately verified through satisfaction of all calibration criteria and QC requirements. The low s tandard must be equivalent to or below the lowest result to be reported. All reported results must be within the calibration range. Concentrations should correspond to the expected am ounts found in real samples and should bracket the linear range of the detector. See Metho d 8000 for additional information on the preparation of calibration standards.

Analysts should evaluate the specific toxaphene sta ndard carefully. Some toxaphene components, particularly the more heavily chlorinat ed, are subject to dechlorination reactions. Consequently, standards from different v endors may exhibit marked differences which could lead to possible false negatives or to large differences in quantitative results.

7.7 Internal standard - PCB congener #204 at a conc entration range of 50 - 100 pg/μL in the final extract is suggested for use as an internal standard for the quantitation of toxaphene and toxaphene congeners. For example, spi ke 2 - 4 μL of a 25-ng/μL solution into each 1 mL of sample extract for a final concentrati on of 50 - 100 ng/mL (pg/μL). Other concentrations and volumes are acceptable.

7.8 Surrogate standards - The performance of the me thod should be monitored using surrogate compounds. Surrogate standards are added to all samples, method blanks, matrix spikes, and calibration standards. The follo wing compounds are recommended as possible surrogates. Other surrogates may be used p rovided that the analyst can demonstrate and document performance appropriate for the data q uality needs of the particular application. Method 3500 describes the procedures for preparing these surrogates.

7.8.1 є-hexachlorocyclohexane (є-HCH) and decachlor obiphenyl (PCB congener #209) have been found to be a useful pair of surrogates. Decachlorobiphenyl

should have a minimum retention time of 45 minutes to ensure adequate resolution ofMethod 8276 8276 - 10 Revision 1 October 2012 target compounds.

7.8.2 Alternatively, if an adequate response can be achieved for the particular application, tetrachloro- m -xylene or possibly isotopically labeled toxaphene congeners may be used.

8.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE

Sample collection, preservation, and storage requi rements may vary by EPA program and may be specified in a regulation or project pla nning document that requires compliance monitoring for a given contaminant. Where such req uirements are specified in the regulation, follow those requirements. In the absence of speci fic regulatory requirements, use the following information as guidance in determining the sample c ollection, preservation, and storage requirements.

8.1 See Chapter Four, "Organic Analytes" for sample collection and preservation

instructions.

8.2 Store the sample extracts at ≤6 °C, protected f rom light, in sealed vials (e.g., screw- cap or crimp- capped vials) equipped with in tact PTFE-lined septa. Extracts should be analyzed within 40 days of extraction.

9.0 QUALITY CONTROL

9.1 Refer to SW-846 Chapter One for guidance on qua lity assurance (QA) and QC protocols. When inconsistencies exist among QC guid elines, method-specific criteria take precedence over both technique-specific and Chapter One criteria. Technique-specific QC criteria take precedence over Chapter One criteria. Any effort involving the collection of analytical data should include development of a str uctured and systematic planning document, such as a Quality Assurance Project Plan (QAPP), Standard Operating Procedure (SOP) or a Sampling and Analysis Plan (SAP); these documents translate project objectives and specifications into directions for implementing the project and assessing the results.

Each laboratory should maintain a formal QC program and maintain records to document the quality of the data generated. Develop ment of in-house QC limits for each method is encouraged. Use of instrument specific Q C limits is encouraged provided such limits will generate data appropriate for use in the inten ded application. All data sheets and QC data should be saved for reference or inspection.

9.2 Refer to Method 8000 for specific determinative method QC procedures and to Method 3500 and 5000 for QC procedures to ensure th e proper operation of the various sample preparation techniques. If an extract cleanup proce dure is performed, refer to Method 3600 for the appropriate QC procedures. Any more specific QC procedures provided in this method will supersede those noted in Methods 8000, 5000, 3500, or 3600.

9.3 QC procedures necessary to evaluate the GC syst em operation are found in

Method 8000 and include evaluation of retention tim e windows, calibration verification, and chromatographic analysis of samples. In addition, d iscussions regarding the instrument QC requirements listed below can be found in the refer enced sections of this method.Method 8276 8276 - 11 Revision 1 October 2012 9.3.1 The GC/MS is tuned and calibrated using a NIC I-customized tune table. The analytical system should be tuned prior to the initial calibration and for each occurrence when a new initial calibration may be necessary. See Sec. 11.4.1

for further details.

9.3.2 There must be an initial calibration of the G C/MS system and subsequent second source verification as described in Sec.11.4.

9.3.3 The GC/MS system must meet the calibration ve rification acceptance criteria in Sec. 11.5 every 12 hours.

9.3.4 The absolute or relative retention time (RRT) of each sample component must fall within the specified window of its corresponding standard component provided in Sec. 11.4.4.

9.4 Initial demonstration of proficiency - The init ial demonstration of method proficiency (IDP) must be performed by the laborato ry prior to independently running an analytical method, and should be repeated if other changes occur (e.g., instrument repair, significant change in procedure, and change in anal yst). Refer to Method 8000 Sec. 9.0 for additional information regarding instrument, proced ure, and analyst IDPs. An IDP must consist of replicate reference samples from each sample pre paration and determinative method combination it utilizes by generating data of accep table accuracy and precision for target analytes in a clean reference matrix taken through the entire preparation and analysis. If an autosampler is used to perform sample dilutions, pr ior to use, the laboratory should satisfy itself that those dilutions are of equivalent or be tter accuracy than is achieved by an experienced analyst performing manual dilutions.

9.5 Method blank - Before processing any samples, t he analyst should demonstrate that all parts of the equipment in contact with the sample and reagents are interference-free through analysis of a method blank. The blank shoul d be carried through all stages of sample preparation and analysis. As a continuing check, ea ch time samples are extracted, cleaned up, and analyzed, a method blank must be prepared and a nalyzed for the compounds of interest as a safeguard against laboratory contamination. If a peak is observed within the retention time window of an analyte that would prevent its determi nation, the source must be found and eliminated, if possible, before samples are process ed. Method blank re-extraction may be necessary if the source of contamination cannot be determined.

When new reagents or chemicals are received, the la b should monitor the preparation and/or analysis blanks associated with samples for any signs of contamination. It is not necessary to test every new batch of reagents or ch emicals prior to sample preparation if the source shows no prior problems. However, if reagent s are changed during a preparation batch, separate blanks need to be prepared for each set of reagents.

9.6 Sample QC for preparation and analysis - The l aboratory must also have procedures for documenting matrix effect on method performance (precision, accuracy, method sensitivity). Minimally, this should include analysis of QC samples including a method blank, a matrix spike/matrix spike duplicate (MS/MSD) or a sample duplicate (if a matrix spike duplicate cannot be prepared and suffi cient sample exists), and a laboratory control sample (LCS) in each analytical batch. Addi tion of surrogates to each field sampleMethod 8276 8276 - 12 Revision 1 October 2012 and QC sample is also recommended. Method blanks, m atrix spike samples, and replicate samples should be subjected to the same analytical procedures (Sec. 11.0) as those used on actual samples.

9.6.1 Documenting the effect of the matrix should i nclude analysis of at least one matrix spike and one duplicate unspiked sample or one MS/MSD pair. The decision on whether to prepare and analyze duplicate samples or a MS/MSD must be based on knowledge of the samples in the sample batch. If sa mples are expected to contain target analytes, laboratories may use a matrix spike and a duplicate analysis of an unspiked field sample. If samples are not expected to contai n target analytes, then laboratories
should use a MS and MSD.

9.6.2 An LCS should be included with each analytica l batch and consists of an aliquot of a clean (control) matrix similar to t he sample matrix and of the same weight or volume. The LCS is spiked in identical ma nner as the matrix spike, when appropriate. When results of the matrix spike analy sis indicate a potential problem due to the sample matrix itself, the LCS results are us ed to verify that the laboratory can perform the analysis in a clean matrix.

9.6.3 Also see Method 8000 for the details on carry ing out sample QC procedures for preparation and analysis. In-house method performance criteria for evaluating method performance should be developed u sing the guidance found in

Method 8000.

9.7 Lower limit of quantitation (LLOQ) check standa rd

The laboratory shall establish the LLOQ as the lowe st point of quantitation, which in most cases, is the lowest concentration in the cali bration curve. The LLOQ verification is recommended for each project application to validat e quantitation capability at low analyte concentration levels. This verification may be acco mplished with either clean control material (e.g., reagent water, solvent blank, Ottawa sand, d iatomaceous earth, etc.) or a representative sample matrix, free of target compounds. Optimally, the LLOQ should be less than the desired regulatory action levels based on the stated DQOs.

9.7.1 Determination of LLOQs using spiked clean co ntrol material represents a best-case scenario, and does not evalu ate potential matrix effects of real-world samples. For the application of LLOQs on a project-specific basis with established DQOs, a representative matrix-specific LLOQ verification may provide a more reliable estimate of the lower quantitation limit capabilities.

9.7.1.1 A LLOQ check standard (not part of an initi al calibration) is prepared by spiking a clean control material with t he analyte(s) of interest at the predicted LLOQ concentration level(s). Alternat ively, a representative sample matrix may be spiked with the analytes of in terest at the predicted
LLOQ concentration levels. The LLOQ check is carrie d through the same preparation procedures as the environmental samples and other QC samples.

9.7.1.2 Recovery of target analytes in the LLOQ che ck standard should be within established in-house limits, or ot her such project-specific acceptance limits, to demonstrate acceptable method performance at the LLOQ. Until the laboratory has sufficient data to determi ne acceptance limits, the LCSMethod 8276 8276 - 13 Revision 1 October 2012 criteria ± 20% may be used for the LLOQ acceptance criteria. This acknowledges the poorer overall response at the low end of the c alibration curve. Historicallybased LLOQ acceptance criteria should be determined as soon as practical once sufficient data points have been acquired.

9.7.2 In-house acceptance criteria for recovery of the LLOQ check standard for a sample matrix can be calculated when sufficie nt data points exist. The laboratory should have a documented procedure for establishing in-house acceptance ranges.

9.8 Surrogate recoveries - The laboratory should ev aluate recovery data from individual samples against historically developed s urrogate control limits. See Method 8000 for information on evaluating surrogate data a nd developing and updating surrogate limits. Procedures for evaluating recoveries of mul tiple surrogates and associated corrective actions when they are deemed unacceptabl e should be defined in a QAPP or

SOP.

9.9 The experience of the analyst performing GC-NIC I/MS is invaluable to the success of the method. Each day that analysis is pe rformed, the calibration verification

standard should be evaluated to determine if the ch romatographic system is operating properly. Do the peaks look normal? Is the response obtained comparable to the response from previous

calibrations?

Careful examination of the standard chromatogram ca n indicate if the column is still performing acceptably, the injector is leaking, the injector septum needs replacing, etc. When any changes are made to the system (e.g., the colum n or septum is changed), see guidance in Method 8000 regarding whether recalibration of t he system must take place.

9.10 It is recommended that the laboratory adopt ad ditional QA practices for use with this method. Specific practices that are most productive depend upon the needs of the laboratory, the nature of the samples, and project- specific requirements. Field duplicates may be analyzed to assess precision of the environm ental measurements. When doubt exists over identification of a peak on the chromat ogram, confirmatory techniques such as GC with a dissimilar column, element-specific detec tor, or mass spectrometer (selected ion monitoring or full scan) must be used. Whenever po ssible, the laboratory should analyze standard reference materials and participate in rel evant performance evaluation studies.

10.0 CALIBRATION AND STANDARDIZATION

See Sec. 11.4 and 11.5 for information on calibrati on and standardization.

11.0 PROCEDURE

11.1 Sample preparationMethod 8276 8276 - 14 Revision 1 October 2012 Samples are normally prepared by one of the followi ng methods prior to GC-NICI/MS analysis.

Matrix Methods

Water 3510, 3520 Soil/sediment 3540, 3541, 3545, 3546, 3550, 356 2 Tissue 3540, 3541, 3545, 3550, 3562

11.2 Extract cleanup - Cleanup procedures may not b e necessary for a relatively clean sample matrix, but most extracts from environ mental and waste samples will require additional preparation before analysis. The specifi c cleanup procedure used will depend on the nature of the sample to be analyzed, expected inter ferences coextracted from the sample, and the DQOs. General guidance on the selection of clea nup methods is provided in Sec. 11.2

and in Method 3600. Refer to Table 13 for a data co mparison of real-world samples extracted with and without cleanup procedures.

11.2.1 Method 3610 (alumina) may be used to remove phthalate esters.

11.2.2 Method 3620 (Florisil ® ) may be used to separate organochlorine pesticides from aliphatic compounds, aromatics, and nitrogen-containing compounds.

11.2.3 Method 3630 (silica gel) may be used to sepa rate single- and multi- component organochlorine compounds from inte rferences.

11.2.4 Method 3640 (GPC - pesticide option) is a si ze exclusion procedure that may be used to remove high molecular weight materials.

11.2.5 Method 3660 (sulfur cleanup) should be used to remove possible sulfur interfering compounds which may be present i n certain sediments and industrial wastes. (These compounds should not interfere with monitored ions from toxaphene

but should nevertheless be removed because of delet erious effects on chromatography and on the response of the NICI/MS i on source during coelution.)

11.2.6 Method 3665 (sulfuric acid/permanganate) may be used prior to toxaphene analysis to remove more fragile organic c ontaminants.

11.3 GC conditions - This method uses a single colu mn in conjunction with an electron capture NICI/MS detector. If an autosample r is not used, the use of an internal standard is required. It is highly recommended to u se an internal standard in all samples even if an autosampler is used. The list in Sec. 6.0 ind icates the columns used to develop the method performance data in the tables at the end of this method and is not intended to exclude other columns that are available or may be develope d. Laboratories may use these or other capillary columns (including columns of other dimen sions), provided that they document method performance data (e.g., chromatographic reso lution, analyte breakdown, and sensitivity) appropriate for the intended applicati on.

11.4 Tuning, initial calibration, and second-source calibration accuracy check - Establish the GC-NICI/MS operating conditions accor ding to the recommendations in TableMethod 8276 8276 - 15 Revision 1 October 2012 1. Alternative operating conditions may be acceptab le provided that appropriate compound sensitivity and selectivity can be achieved.

11.4.1 Prior to analysis of samples or calibration standards, the instrument must be tuned for NCI conditions. If possible, use the CI autotune procedure provided by the instrument manufacturer. Without sp ecific manufacturer guidance, the following generic recommendations should be fol lowed.

11.4.1.1 The source is operated in the CI mode usin g methane as the reagent gas.

11.4.1.2 When the source pressure has stabilized at a value known to produce satisfactory results, the instrument is manually tuned and calibrated using a NCI-customized tuning file. Pressure stabil ization usually takes about

45 minutes.

NOTE: The internal standard noted in Sec. 7.7 should pe rmit most of the components of interest in a chromatogram to have re tention times of 0.80- 1.20 relative to it. Use the base peak ion from the internal standard as the primary ion for quantitation (Table 2).

11.4.2 Analyze 1 μ L of each calibration standard (containing the quant itation compounds, as well as, the appropriate surrogates a nd internal standard) and calculate the response factor (RF) as shown in Sec. 11.4.3. A set of at least five calibration standards is necessary (Sec. 7.6 and Method 8000). Alternate injection volumes may be used if applicable QC requirements are met, but must be the same for all standards and sample extracts. Figure 1 is a representative c hromatogram of select congeners.

NOTE: Toxaphene will require a separate calibration cu rve from the congeners due to possible congener interferences present in the t oxaphene calibration standard chromatograms.

11.4.3 Initial calibration calculations

11.4.3.1 Calculate a RF for each target analyte rel ative to one of

the internal standards as follows:



###  
 

###   



###   

###  



where:

A

s = Peak response of the analyte or surrogate.

A
is = Peak response of the internal standard.

C

s = Concentration or mass of the analyte or surrogate in the sample aliquot.

C
is = Concentration or mass of the internal standard in the sample aliquot.

11.4.3.2 Calculate the average RF and the percent r elative standard deviation (%RSD) of the RFs for each targe t analyte using theMethod 8276 8276 - 16 Revision 1 October 2012 equations below. The %RSD should be ≤20% for each t arget analyte. It is also

recommended that a minimum RF for the most common t arget analytes be

demonstrated for each individual calibration level to ensure that these compounds are behaving as expected. In addition, me eting the minimum RF

criteria for the lowest calibration standard is cri tical in establishing and demonstrating the desired sensitivity.

Due to the large number of compounds that may poten tially be analyzed by this method, some compounds will fail t o meet these criteria. For these occasions, it is acknowledged that the failin g compounds may not be critical to the specific project and therefore they may be used as qualified data or estimated values for screening purposes. The analyst should strive to place more emphasis on meeting the calibration criteria f or compounds that are critical to the project.



### 

        ∑  

 

    ∑        





###   

     100

where:

= RF for each of the calibration standards.

     = Average RF for each compound from the initial cal ibration.

n = Number of calibration standards (e.g., 5).

11.4.3.3 If more than 10% of the compounds included in the initial
calibration exceed the 20% RSD limit or do not meet the minimum coefficient of determination (r 2 = 0.99) for alternate curve fits, the chromatograph ic system may be considered too reactive for analysis to begi n. Clean or replace the injector liner, capillary column, and/or ion source , and then repeat the calibration procedure beginning with Sec. 11.4.

Either of the two procedures described in Sec. 11.4 .4.1 or 11.4.4.2 may be used to determine calibration function accep tability. These include refitting the calibration data back to the model or the determination of the relative standard error (RSE) for the curve when co mparing the actual response with the predicted response.

11.4.4.1 Refitting the calibration data back to the model or calculating the % Error is determined by using t he following equation:



### % #$  % &' % ( &

% & 

###  100Method 8276 8276 - 17 Revision 1 October 2012 where:



### x’ i = Measured amount of analyte at calibration level i, in mass or

concentration units.



### x i = True amount of analyte at calibration level i, i n mass or

concentration units.

Percent error between the calculated and expected a mounts of an analyte should be ≤30% for all standards. For so me data uses, ≤50% may be acceptable for the lowest calibration p oint.

11.4.4.2 Relative Standard Error is calculated by u sing the following equation:



### #  100 , -

. /  0 

### 1 

 2 3 

4 

### 56 1 78

9

where:



### x i = True amount of analyte in calibration level i , in mass or

concentration units.



### x ́ i = Measured amount of analyte in calibration level i , in mass or

concentration units.



### p = Number of terms in the fitting equation.
(average = 1, linear = 2, quadratic = 3, cubic = 4 )



### n = Number of calibration points.

RSE acceptance limit criterion for the calibration model is 

### ≤20%

.

11.4.5 Evaluation of retention times -Either the ab solute or relative retention time (RRT) used in conjunction with spect ral matching is allowed for use by the laboratory to establish peak identity. The abso lute or RRT of each target analyte in each calibration standard should agree within es tablished limits set by the laboratory in their SOP. These limits should be app ropriate for reliable identification of the target analyte. If this criterion is not met and there are no other indicators of an analyte's identification (such as a very unique and high probability mass spectral match), that analyte may not be considered as ident ified using either RRT or absolute retention times. The RRT equation follows units.



### =  >6>?$6 >?@ $A >B 6
CD>

>6>?$6 >?@ $A >B E6>6
C >
6F
FMethod 8276 8276 - 18 Revision 1 October 2012 11.4.6 Linearity of target analytes - If the RSD of any target analyte is



### ≤ 20%, then the relative response is assumed to be co nstant over the calibration range, and the average RF may be used for quantitat ion (Sec. 11.8.2).

11.4.6.1 If the RSD of any target analyte is >20%, refer to Method 8000 for additional calibration options. One of the options must be applied to the GC/MS calibration in this situation, or a new initial cal ibration must be performed. The average RF should not be used for compounds that ha ve an RSD >20% unless the concentration is reported as estimated.

11.4.6.2 When the RSD is >20%, plotting and visual inspection of a calibration curve can be a useful diagnostic tool. The inspection may indicate analytical problems, including errors in standard p reparation, presence of active sites in the chromatographic system, analytes that exhibit poor chromatographic behavior, etc.

11.4.6.3 Due to the large number of compoun ds that may be analyzed by this method, some compounds may fail to meet either the 20% RSD, minimum coefficient of determination (r 2 = 0.99), or the acceptance criteria for alternative calibration procedures in Method 8000. Any calibrat ion method described in Method 8000 may be used for a compound, but it shou ld be used consistently until the next calibration is performed. If compoun ds fail to meet these criteria, the associated concentrations may still be determin ed but they must be reported as estimated. In order to report non-detects, it mu st be demonstrated that there is adequate sensitivity to detect the failed compou nds at the applicable lower quantitation limit.

11.4.6.4 The method of linear regression analysis h as the potential for a significant bias at the lower portion of a calibrat ion curve, while the average response factor and quadratic methods of calibratio n do not typically have this problem. Also, when a least squares linear regressi on calibration model is used, inverse weighting of the concentrations used in the initial calibration has been shown to emphasize precision at the low end of the calibration range (see Method 8000 for all calibration model options).

When evaluating the calibration curves with the lin ear regression model, the analyst should perform a minimum quantit ation check on the viability of the lowest calibration point by refitting the re sponse from the low

concentration calibration standard back into the cu rve (see Method 8000 for additional details). It is not necessary to reanaly ze a low concentration standard; rather the data system can recalculate this low con centration as if it were an unknown sample. The recalculated concentration of t he low calibration point should be ±50% (or in-house historically based acce ptance criteria) of the

standard’s true concentration.

Other recovery criteria may be applicable dependin g on the project’s DQOs. For those situations, the minimum quantitatio n check criteria should be outlined in a laboratory SOP, or a project-specific QAPP. Analytes which do not meet the minimum quantitation calibration refitting criteria should be considered "out-of-control." Corrective actions may be appropr iate such as redefining theMethod 8276 8276 - 19 Revision 1 October 2012 LLOQ and/or reporting "out-of-control" target analy tes as estimated when the

concentration is at or near the lowest calibration point.

11.4.7 The initial calibration function for each ta rget analyte should be checked by injecting the second-source standard immediately a fter the standards

used for initial calibration. The second-source sta ndard should be prepared at a

concentration near the middle of the calibration ra nge with a standard from a source different (if available) from that used for the ini tial calibration. The measured values of the parameters in the second-source check standa rd should fall within 70 - 130% of the expected value(s). Due to the variability of technical mixtures (e.g., technical toxaphene), this criterion for the second source ch eck standard should be applied with caution. An alternative recovery limit may be appropriate based on the desired project-specific DQOs. Quantitative sample analyses should not proceed for those analytes that fail the second-source standard initi al calibration verification. However, analyses may continue for those analytes t hat fail the criteria with an understanding these results could be used for scree ning purposes and would be

considered estimated values.

11.5 Calibration verification consists of several s teps that are performed at the beginning of each 12-hour analytical shift.

11.5.1 Calibration verification - Initial calibrati on (Sec. 11.4) for each compound of interest should be verified once every 12 hours prior to sample analysis, with the introduction technique and conditions used for samples. This is accomplished by analyzing a calibration verification standard (c ontaining all the compounds for quantitation) at a concentration either near the mi dpoint concentration for the calibration range of the GC-NICI/MS or near the action level fo r the project. The results must be compared against the most recent initial calibratio n curve and should meet the verification acceptance criteria provided in Sec. 1 1.5.1.1 through 11.5.1.5.

11.5.1.1 Each of the target analytes in the calibra tion verification standard should meet the appropriate m inimum RFs. These criteria are particularly important when the target analytes are also critical project-required compounds. This is the same check that is applied during

the initial calibration.

11.5.1.2 If minimum RFs are not met, the system sho uld be evaluated, and corrective action taken before sampl e analysis begins. Possible problems include standard mixture degradat ion; contamination of the injection port inlet, source and/or front end of th e column; active sites in the column or chromatographic system; etc.

11.5.1.3 All target compounds of interest must be e valuated using a 20% criterion: percent difference when performing t he average RF model calibration and percent drift when calibrating with a regression fit model. Refer to Method 8000 for guidance on calculating percent dif ference and drift.

11.5.1.4 If the percent difference or percent drift for a compound is ≤20%, the initial calibration for that compound is assumed to be valid. Due to large numbers of compounds that may potentially be analyzed by this method, itMethod 8276 8276 - 20 Revision 1 October 2012 is expected that some compounds will fail to meet t he criterion. If the criterion is not met for more than 20% of the compounds included in the initial calibration, then corrective action must be taken prior to the a nalysis of samples. When compounds fail, they may still be reported as non-d etects if it can be demonstrated that there was adequate sensitivity to detect the compound at the applicable quantitation limit. For situations when the failed compound is present, the concentrations in the affected samples must be reported as estimated.

11.5.1.5 Problems similar to those listed under ini tial calibration could affect the ability to pass the calibration ve rification standard analysis. If the problem cannot be corrected by other measures, a new initi al calibration must be generated. Calibration verification criteri a must be met before sample analysis may begin.

11.5.2 Method blank - A method blank should be anal yzed prior to sample analysis to demonstrate that the total system (intr oduction device, transfer lines and GC-NICI/MS system) is free of contaminants. If the method blank indicates contamination, it may be appropriate to analyze a s olvent blank to demonstrate that the contamination is not a result of carryover from standards or samples. See Method 8000 for information regarding method blank perform ance criteria.

11.5.3 Internal standard retention time - Retention times of the internal

standard in the calibration verification standard m ust be evaluated immediately during or after data acquisition. If the retention time for any internal standard changes by >30 seconds from that in the midpoint st andard level of the most recent initial calibration sequence, the chromatographic s ystem must be inspected for malfunctions and corrections must be made, as requi red. When corrections are made, reanalysis of samples analyzed while the syst em was malfunctioning is required.

11.5.4 Internal standard response - If the EICP are a for the internal standard in the calibration verification standard changes by greater than a factor of two (-50% to +100%) from that in the midpoint standard level of the most recent initial calibration sequence, the mass spectrometer must be inspected f or malfunctions and corrections must be made, as appropriate. When corrections are made, reanalysis of samples analyzed while the system was malfunctioning is req uired.

NOTE : A decrease in the EICP area of the internal stan dard may be due to the use of a hydrocarbon reagent gas (e.g., methane ) in the negative ion chemical ionization mode and a decreased source temperature (160 °C, versus 250 °C for EI). These conditions make the ion source susceptible to a buildup of a hydrocarbon film on its surface. This in turn may lead to a lower s ignal. If this decrease of signal is gradual, it should not signify anything worse than this hydrocarbon buildup. If the signal drops drastically over one or two runs, some thing else is happening and measures should be taken immediately to remedy this

.

11.6 GC-NICI/MS analysis of samples

11.6.1 It is highly recommended that sample extract s be screened on a GC with an electron capture detector (ECD) using the s ame type of capillary column employed in the GC-NICI/MS system. Pre-screening ca n detect the possible presenceMethod 8276 8276 - 21 Revision 1 October 2012 of interferences (e.g., sulfur, PCBs) to determine if extract cleanups are needed. Screening will minimize contamination of the GC-NIC I/MS system from unexpectedly high concentrations of organic compounds. The QAPP often contains historical contaminant information of the sampling site which may indicate if screening is

necessary.

11.6.2 Allow the sample extract to warm to room tem perature. Just prior to analysis, add an aliquot of the internal standard s olution to the concentrated sample

extract. The internal standard concentration in the injected sample extract must be

the same as that in the calibration standards.

11.6.3 Inject an aliquot of the sample extract usin g the same operating conditions employed for the calibration (Sec. 11.4) . The volume to be injected should include an appropriate concentration that is within the calibration range of the standards as noted in Sec. 7.7. The injection volum e must be the same volume used

for the calibration standards.

11.6.4 If any quantitation ion response exceeds the GC-NICI/MS system initial calibration range, the sample extract must be diluted and reanalyzed. Additional

internal standard solution must be added to the dil uted extract to maintain the same concentration as in the calibration standards (usua lly 50 - 100 pg/ μ L or other concentrations as appropriate).

NOTE: It may be a useful diagnostic tool to monitor in ternal standard retention times in all samples, spikes, blanks, and standards to ch eck drifting, method performance, poor injection execution, and the need for system i nspection and/or maintenance. Internal standard responses (area counts) must be m onitored in all samples, spikes,

blanks for similar reasons. If the EICP area for th e internal standard in samples, spikes and blanks changes by a factor of two (-50% to +100%) from the areas

determined in the calibration verification standard analyzed that day, corrective action must be taken. The samples, spikes, or blanks shoul d be reanalyzed or the data qualified.

11.6.4.1 When ions from a compound in the sample sa turate the detector, this analysis should be followed by a n instrument blank consisting of clean solvent. If the blank analysis is not free of interferences, then the system must be decontaminated. Sample anal ysis may not resume until the blank analysis is demonstrated to be free of interferences. Contamination from one sample to the next on the in strument usually takes place in the syringe. If adequate syringe washes ar e employed, carryover from high concentration samples can usually be avoi ded.

11.6.4.2 All dilutions should keep the response of the major constituents (previously saturated peaks) within th e linear range of the

curve.

11.6.5 The use of selected ion monitoring (SIM) is preferred. Using the primary ion for quantitation and the secondary ions for confirmation sets up the collection groups based on their retention times. M ost compounds have a small mass defect in their spectra, usually <0.2 amu. These ma ss defects should be accounted forMethod 8276 8276 - 22 Revision 1 October 2012 during data acquisition. The total scan time should be <1 second and produce at least 5 -10 scans per chromatographic peak. The start and stop times for the SIM groups are determined from a full scan analysis.

11.7 Analyte identification

11.7.1 The qualitative identification of compounds determined by this method is based on retention time and on comparison of the sample mass spectrum, after background correction, with characteristic ions in a reference mass spectrum. The reference mass spectrum must be generated by the la boratory using the conditions of

this method. The characteristic ions from the refer ence mass spectrum are defined as the three ions of greatest relative intensity, or a ny ions over 30% relative intensity, if

fewer than three such ions occur in the reference s pectrum. Compounds are identified when the following criteria are met.

11.7.1.1 The intensities of the characteristic ions of a compound

must maximize in the same scan or within one scan o f each other. Selection of a peak by a data system target compound search rout ine where the search is based on the presence of a target chromatographic p eak containing ions specific for the target compound at a compound-spec ific retention time will be accepted as meeting this criterion.

11.7.1.2 Either absolute or RRT, used in conjunctio n with spectral matching is allowed for use by the laboratory to es tablish peak identity. The absolute or RRT of each target analyte, when compar ed against the daily calibration verification standard, should agree wit hin established limits set by the laboratory in their SOP. These limits should be appropriate for reliable identification of the target analyte. If this crite rion is not met and there are no other indicators of an analyte's identification (su ch as a very unique and high probability mass spectral match), that analyte may not be considered as identified using either RRT or absolute retention t imes.

11.7.1.3 The relative intensities of the character istic ions agree
within 30% of the relative intensities of these ion s in the reference spectrum. (e.g., for an ion with an abundance of 50% in the r eference spectrum, the corresponding abundance in a sample spectrum should be 20 - 80%.) Use professional judgment when interferences are observ ed.

11.7.1.4 Structural isomers that produce very simil ar mass spectra should be identified as individual isomers if they have sufficiently different GC

retention times. Sufficient GC resolution is achiev ed if the height of the valley between two isomer peaks is less than 50% of the av erage of the two peak heights. Otherwise, structural isomers are identified as i someric pairs. The resolution should be verified on the midpoint conce ntration of the initial calibration as well as the laboratory-designated ca libration verification standard level if closely eluting isomers are to be reported

.

CAUTION: Because of the complexity of toxaphene, decreasi ng run time will likely cause additional isomers to be reported as t he target congeners, thus inflating concentrationsMethod 8276 8276 - 23 Revision 1 October 2012 11.7.1.5 Identification is hampered when sample com ponents are not resolved chromatographically and produce mass s pectra containing ions contributed by more than one analyte. When GC peaks obviously represent more than one sample component (i.e., a broadened p eak with shoulder(s) or a valley between two or more maxima), appropriate sel ection of analyte spectra and background spectra is important.

11.7.1.6 Examination of EICPs of appropriate ions c an aid in the selection of spectra and in qualitative identificat ion of compounds. When analytes coelute (i.e., only one chromatographic pe ak is apparent), the identification criteria may be met, but each analyt e spectrum will contain extraneous ions contributed by the coeluting compou nd.

11.7.2 The identification of mixtures such as toxap hene is not based on a single peak, but rather on the characteristic peaks that comprise the "fingerprint" of the mixture, using both the retention times and shapes of the indicator peaks. See Method

8000 for information on confirmation of tentative i dentifications.

11.8 Quantitation

11.8.1 Multi-component analytes such as toxaphene - Complex analytes present problems in measurement. Quantitation is ba sed on the areas of the characteristic peaks as compared to the areas of th e corresponding peaks at the same retention times in the calibration standard, using either internal or external calibration procedures. Suggestions are offered in the followin g sections for handling toxaphene.

11.8.1.1 Calibrate the instrument for toxaphene usi ng the guidance noted in Sec. 11.4

11.8.1.2 Quantitate toxaphene by summing the area o f all peaks originating from this compound using the tota l area of the toxaphene pattern or using the 4 to 6 major peaks that closel y match the corresponding peaks in the most recent calibration verification s tandard. Whichever approach is employed should be documented and avail able to the data user, if necessary.

11.8.1.3 While toxaphene contains a large number of compounds that will produce well-resolved peaks in a GC-NICI/ MS chromatogram, it also contains many other components that are not chromat ographically resolved. This unresolved complex mixture results in the "hum p" in the chromatogram that is characteristic of this compound. Although t he resolved peaks are important for identification, the area of the unres olved complex mixture contributes a significant portion of the area of th e total response.

11.8.1.4 To measure total area , construct the baseline of toxaphene in the sample chromatogram between the re tention times of the first and last eluting toxaphene components in the standa rd. In order to use the total area approach, the pattern in the sample chromatogr am must be compared to that of the standard to ensure that all of the majo r components in the standard are present in the sample. Otherwise, the sample co ncentration may be significantly underestimated. As an example, either manually or by using theMethod 8276 8276 - 24 Revision 1 October 2012 data system’s control settings, integrate the entir e area under the total ion chromatogram for the toxaphene response and subtrac t out the internal standard response ( m/z 429.8) and ions associated with surrogate or other compounds if they are present.

11.8.1.5 When toxaphene is deter mined using the 4 to 6 peaks approach, the analyst must take care to evaluate th e relative areas of the peaks chosen in the sample and standard chromatograms. It is highly unlikely that the peaks will match exactly, but the analyst should no t employ peaks from the sample chromatogram whose relative sizes or areas a ppear to be disproportionally larger or smaller in the sample c ompared to the standard.

11.8.1.6 The heights or areas of the selected 4 - 6 peaks should be summed together and used to determine the toxaphene concentration. Alternatively, utilize each peak in the standard to calculate a calibration factor for that peak, using the total mass of toxaphene in the standard. These calibration

factors are then used to calculate the concentratio n of each corresponding peak in the sample chromatogram and the 4 - 6 resulting concentrations are averaged to provide the final result for the sample.

11.8.2 Once a target compound has been identified, the quantitation of that compound will be based on the integrated abund ance of the primary

characteristic ion from the EICP.

11.8.2.1 Use the integration produced by the softwa re if the integration is correct because the software should produce more consistent integrations. However, manual integrations may be n ecessary when the software does not produce proper integrations becau se baseline selection is improper, the correct peak is missed, a coelution i s integrated, the peak is partially integrated, etc. The analyst is responsib le for ensuring that the integration is correct whether performed by the sof tware or done manually.

11.8.2.2 Manual integrations should not be substitu ted for proper maintenance of the instrument or setup of th e method (e.g., retention time updates, integration parameter files, etc). Th e analyst should seek to minimize manual integration by properly maintaining the instrument, updating retention times, and configuring peak integration p arameters.

11.8.3 If the RSD of a compound's response factor i s ≤ 20%, the concentration in the extract may be determined usin g the average RF from initial calibration data (Sec. 11.4.4). See Method 8000 for the equations describing internal

standard calibration and either linear or non-linea r calibrations.

11.8.4 Table 5 lists example retention times for th e target analytes. The retention times listed in this table are provided f or illustrative purposes only. Each laboratory must determine retention times and reten tion time windows for their specific application of the method.Method 8276 8276 - 25 Revision 1 October 2012 12.0 DATA ANALYSIS AND CALCULATIONS

See Secs. 11.4 through 11.8 and Method 8000 for inf ormation on data analysis

and calculations.

13.0 METHOD PERFORMANCE

13.1 Performance data and related information are p rovided in SW-846 methods only

as examples and guidance. The data do not represent r equired performance criteria for users of the methods. Instead, performance criteria should be developed on a project-specific basis, and the laboratory should establish in-house QC per formance criteria for the application of this

method. Performance data must not be used as absolu te QC acceptance criteria for the purposes of laboratory QC or accreditation . All performance data included resulted from both single and multi-laboratory analyses.

13.2 The chromatographic separations in this method were tested in multiple laboratories by using clean hexane and liquid/solid extracts spiked with the test compounds at various concentrations. Single-operator and multi-l aboratory precision and method accuracy

were found to be related to the concentration of th e compound and the type of matrix.

13.3 The levels of accuracy and precision that can be achieved with this method depend on the sample matrix, sample preparat ion, optional cleanup techniques, and calibration procedures used.

13.4 Tables 2 through 4 represent SIM for method an alytes using GC-NICI/MS.

13.5 Table 5 contains representative retention time s.

NOTE: Decachlorobiphenyl should have a minimum RT of 4 5 minutes to ensure adequate resolution of target compounds.

13.6 Table 6 contains single-laboratory performance data on spiked soil for toxaphene and chlordane. Data are provided for guid ance purposes only.

13.7 Table 7 contains single-laboratory performance data on spiked soil for toxaphene congeners. Data are provided for guidance purposes only.

13.8 Table 8 contains single-laboratory performance data on spiked soil for organochlorine pesticides. Data are provided fo r guidance purposes only.

13.9 Table 9 contains multi-laboratory phase I vali dation data using unknown
standard concentrations.

13.10 Table 10 contains multi-laboratory phase II v alidation data using spiked extracts of uncontaminated real-world sample matrices.

13.11 Table 11 contains multi-laboratory phase II v alidation data using realworld samples from Terry Creek in Brunswick, Georgi a.

13.12 Table 12 contains multi-laboratory phase II v alidation data using real worldMethod 8276 8276 - 26 Revision 1 October 2012 samples from Terry Creek in Brunswick, Georgia. Ext racts from this study were further subjected to copper, acid, and silica gel cleanup m ethods.

13.13 Table 13 includes a Terry Creek real-world sa mple data comparison between extraction only to those extracts subjected to copper, acid, and silica gel cleanup
methods.

13.14 Table 14 contains multiple-laboratory validat ion data using multiple technical toxaphene spiking protocols of uncontamin ated real world sample matrices.

13.15 Table 15 contains multi-laboratory phase III validation data using spiked extracts of uncontaminated real world sample matric es.

13.16 Table 16 contains multi-laboratory phase III validation data using real world samples from Terry Creek in Brunswick, Georgia.

13.17 Figure 1 is an example chromatogram of a mixe d toxaphene congeners standard.

13.18 Figure 2 is an example chromatogram of a fish tissue extract containing weathered toxaphene and spiked with decachlorobiphe nyl.

13.19 Figure 3 is an example chromatogram of a toxa phene congener
validation standard.

13.20 Figure 4 is an example chromatogram of an EPA Region 4 sediment extract spiked with decachlorobiphenyl.

NOTE: Not all components shown in the figures may be t arget analytes.

14.0 POLLUTION PREVENTION

14.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity and/or toxicity of waste at the point of g eneration. Numerous opportunities for pollution prevention exist in laboratory operations. The EPA has established a preferred hierarchy of environmental management techniques that places pol lution prevention as the management option of first choice. Whenever feasible, laborato ry personnel should use pollution prevention techniques to address their waste generation. When wastes cannot be feasibly reduced at the source, the Agency recommends recycling as the next best option.

14.2 For information about pollution prevention tha t may be applicable to laboratories

and research institutions consult Less is Better: Laboratory Chemical management for Waste

Reduction , a free publication available from the American Ch emical Society (ACS), Committee on Chemical Safety, http://portal.acs.org/portal/fileFetch/C/WPCP_01229 0/pdf/WPCP_012290.pdf

.Method 8276 8276 - 27 Revision 1 October 2012 15.0 WASTE MANAGEMENT

The EPA requires that laboratory waste management practices be conducted consistent with all applicable rules and regulations. Laborato ries are urged to protect air, water, and land by minimizing and controlling all releases from hoo ds and bench operations, complying with the letter and spirit of any sewer discharge permits an d regulations, and by complying with all solid and hazardous waste regulations, particularly the h azardous waste identification rules and land disposal restrictions. For further information on w aste management, consult The Waste Management Manual for Laboratory Personnel available from the American Chemical Society at

the address listed in Sec. 14.2.Method 8276 8276 - 28 Revision 1 October 2012 16.0 REFERENCES

1. A.G. Harrison (2 nd ed.), Chemical Ionization Mass Spectrometry, CRC Pr ess, Boca Raton, FL, 1992.

2. W. C. Brumley, C. M. Brownrigg, and A. H. Grange , "Determination of Toxaphene in Soil by Electron-capture Negative Ion Mass Spectrometry after Fractionation by Highperformance Gel Permeation Chromatography," J. Chro matogr., 633, 177-183, 1993.

3. Brumley, W. C. “Analytical Protocol (GC/NIMS) Fo r OSW's Response to OIG Report (2005-P-00022) on Toxaphene Analysis.” EPA/600/R-08 /048. (TIP # 08-069, Published Report) U.S. Environmental Protection Agency, Washi ngton, DC, 2008.

4. V. Lopez-Avila, E. Baldin, J. Benedicto, J. Mila nes, W. F. Beckert, "Application of OpenTubular Columns to SW-846 GC Methods," Report for t he U.S. Environmental Protection Agency, Contract 68-03-3511, Mid-Pacific Environmen tal Laboratory, Mountain View, CA,

1990.

5. "Development and Application of Test Procedures for Specific Organic Toxic Substances in Wastewaters," Category 10, Pesticides and PCB Re port for the U.S. Environmental Protection Agency, Contract 68-03-2606.

6. D. F. Goerlitz, L. M. Law, "Removal of Elemental Sulfur Interferences from Sediment Extracts for Pesticide Analysis," Bull. Environ. Co ntam. Toxicol., 6, 9, 1971.

7. S. Jensen, L. Renberg, L. Reutergardth, "Residue Analysis of Sediment and Sewage Sludge for Organochlorines in the Presence of Eleme ntal Sulfur," Anal. Chem., 49, 316- 318, 1977.

8. R. H. Wise, D. F. Bishop, R. T. Williams, B. M. Austern, "Gel Permeation Chromatography in the GC/MS Analysis of Organics in Sludges," U.S. Environmental Protection Agency, Cincinnati, OH.

9. H. B. Pionke, G. Chesters, D.E. Armstrong, "Extr action of Chlorinated Hydrocarbon Insecticides from Soil," Agron. J., 60, 289, 1968.

10. J. A. Burke, P. A. Mills, D.C. Bostwick, "Exper iments with Evaporation of Solutions of Chlorinated Pesticides," J. Assoc. Off. Anal. Chem. , 49, 999, 1966.

11. J. A. Glazer, et al., "Trace Analyses for Waste waters," Environ. Sci. and Technol., 15, 1426, 1981.

12. P. J. Marsden, "Performance Data for SW-846 Met hods 8270, 8081, and 8141," U.S. Environmental Protection Agency, EMSL-Las Vegas, E PA/600/4-90/015.

13. V. Lopez-Avila (Beckert, W., Project Officer), "Development of a Soxtec Extraction Procedure for Extracting Organic Compounds from Soi ls and Sediments," EPA 600/X91/140, US Environmental Protection Agency, Environ mental Monitoring Systems Laboratory, Las Vegas, NV, October 1991.Method 8276 8276 - 29 Revision 1 October 2012 14. C. Markell, "Solid-Phase Extraction of TCLP Lea chates," Proceedings of the Tenth Annual Waste Testing and Quality Assurance Symposium, Arli ngton, VA, July, 1994.

15. D. Bennett, B. Lesnik, S. M. Lee, "Supercritica l Fluid Extraction of Organochlorine Pesticide Residues from Soils," Proceedings of the Tenth Annual Waste Testing and Quality Assurance Symposium, Arlington, VA, July, 1 994.

16. C. Markell, "3M Data Submission to EPA," letter to B. Lesnik, June 27, 1995.

17. B. Richter, J. Ezzell, and D. Felix, "Single La boratory Method Validation Report - Extraction of Organophosphorus Pesticides, Herbicid es and Polychlorinated Biphenyls Using Accelerated Solvent Extraction (ASE) with Ana lytical Validation by GC/NPD and GC/ECD," Dionex, Salt Lake City, UT, Document 10112 4, December 2, 1994.

18. K. Li, J. M. R. Bélanger, M. P. Llompart, R. D. Turpin, R. Singhvi, and J. R. J. Paré. Evaluation of rapid solid sample extraction using t he microwave-assisted process (MAP TM ) under closed-vessel conditions. Spectros. Int. J. 1 3 (1), 1-14, 1997.

19. L.D. Betowski, H.M. Webb, and A.D. Sauter. Puls ed Positive Ion Negative Ion Chemical Ionization Mass Spectrometric Applications to Envir onmental and Hazardous Waste Analysis. Biomed. Mass Spectrom. 10, 369, 1983.

20. Science Applications International Corporation, "Interlaboratory Study Plan for Validation of Method 8276," Submitted to U.S. Environmental Pr otection Agency, November 2008.

21. USEPA Region 4 Modified Method 3545A Standard O perating Procedure (SOP) for Pressurized Fluid Extraction (PFE), Revision 4.1, N ovember 5, 2010.

22. USEPA Region 4 Modified Method 3620C, SOP for F lorisil® Cleanup, Revision 3.0, August 23, 2010.

23. USEPA Region 4 Modified 3630C SOP for Silica Ge l Cleanup, Revision 3.1, August 10,

2011.

24. TechLaw Incorporated, “Inter-laboratory Validat ion for Fish Tissue Study Plan,” Submitted to U.S. Environmental Protection Agency, September 2011.

25. TechLaw Incorporated, “Inter-laboratory Validat ion for Fish Tissue Analytical Data Report,” Submitted to U.S. Environmental Protection Agency, September 2012.

26. TechLaw Incorporated, “Inter-laboratory Validat ion for Fish Tissue Data Validation Report,” Submitted to U.S. Environmental Protection Agency, September 2012.

27. TechLaw Incorporated, “Inter-laboratory Validat ion for Fish Tissue Statistical Analysis Report,” Submitted to U.S. Environmental Protection Agency, September 2012.

28. Parlar H, Angerhöfer D, Coelhan M, Kimmel L, "H RGC and HRGC-ECNI Determination of Toxaphene Residues in Fish with a New 22 Components Standard," Organohalogen Compd., 26, 357, 1995Method 8276 8276 - 30 Revision 1 October 2012 17.0 TABLES, VALIDATION DATA, FIGURES

The following pages contain the tables and figures referenced by this method.Method 8276 8276 - 31 Revision 1 October 2012 

### Table 1

RECOMMENDED GC-NICI/MS OPERATING CONDITIONS FOR TOX APHENE AND

TOXAPHENE CONGENERS USING A NARROW-BORE CAPILLARY COLUMN 1

Column - 30-m x 0.25-mm ID fused silica capillary c olumn J&W DB-XLB, 0.25- μm film thickness.

Carrier gas Helium

Carrier gas pressure 10.4 psi Carrier gas flow 1.2 mL/min

EPC Constant flow Injector temperature 205°C Purge flow 60 mL/min Purge time 1 min Injector mode Splitless Injector liner 4-mm I.D. splitless Transfer line temperature 280°C Initial oven temperature 60°C, hold 1 min Temperature program 60 °C to 150 ° C at 10 °C/min, followed by

150 ° C to 260 ° C at 3 °C/min, followed by 260 °C to 320 °C at 20 °C/min, hold 0.33 min followed by 320 °C to 330 °C at 50 °C/min,

hold 3 min Final oven temperature 330 °C

MS mode Negative CI CI gas Methane CI gas flow 40% Source pressure N/A MS quad temperature 150 °C MS Source temperature 160 °C Solvent delay 15 min Acquisition mode SIM SIM parameters

Resolution Low

Dwell time 35 Group 1 Ions Time, initial 306.9, 308.9, 310.9, 340.9, 342.9, 34 4.9, 376.9, 378.9, 380.9, 410.9, 412.9, 414.9, 444.8, 446.8, 448.8

429.8 (IS), 410.8 (IS O
2 rxn) 254.9 (Surr), 497.7 (Surr)

Scan parameters (optional) 300-500, 0.5 sec/scan

1 Data provided courtesy of Scott Sivertsen from U S EPA Region 4 Laboratory.Method 8276 8276 - 32 Revision 1 October 2012 

### Table 2

SELECTED ION MONITORING FOR ANALYTES BY GC-NICI/MS

Compound Quantitation

Ion Other Ions Comments

Toxaphene T IC 1 306.9, 308.9, 310.9, 340.9, 342.9,
344.9, 376.9, 378.9, 380.9, 410.8,
412.8, 414.8, 444.8, 446.8, 448.8 Manual Integration

Parlar 26 378.9 376.9, 380.9

Parlar 40, 41 378.9 376.9, 380.9

Parlar 50, 62 412.8 410.8, 414.8

H x-Sed 308.9 306.9, 310.9

H p-Sed 342.9 340.9, 344.9

IS, PC B 204 429.8 410.8 Monitor oxygen reaction

Surr, PC B 209 497.7

Surr, ε-HCH 254.9

1 Total Ion CurrentMethod 8276 8276 - 33 Revision 1 October 2012 

### Table 3

SELECTED ION MONITORING FOR CHLORDANE BY GC-NICI/MS

Compound Quantitation

Ion Other Ions Comments

Chlordane 1 T IC 2 303.9, 305.9, 337.9, 339.9, 341.9,
371.8, 373.8, 375.8, 407.8, 409.8,
411.8, 441.8, 443.8, 445.8 Manual integration

IS, PC B#204 429.8 Q C check for response

1 Ions are responses observed for technical chlordane ; they may be used to confirm the presence of chlordane in the sample. 13 C -isotope contributions from the higher members of the chlordane congeners
contribute to monitored ions for technical toxaphe ne and some toxaphene congener ions.

2 Total Ion CurrentMethod 8276 8276 - 34 Revision 1 October 2012 2 Table 4

SELECTED ION MONITORING FOR ANALYTES BY GC-NICI/MS

Compound Quantitation

Ion 1 Other Ions Comments

BH C 254.9 252.9, 256.9 M -C l

Heptachlor 2 299.9 297.9, 301.9 M -C l

Aldrin 2

Dieldrin 2 , endrin 2 , endrin aldehyde 2

Heptachlor epoxide 2 329.9 327.9, 331.9 M -C l+H

379.8 377.9, 381.9 M

387.8 385.9, 389.9 M

Endosulfan I, II 403.7 405.7, 407.7 M

Endosulfan sulfate 421.7 419.7, 423.7 M

IS, PC B 204 429.8 Q C

1 Ions are responses observed for organochlorine pest icides; they may be used to confirm the presence of pesticides
in the sample. These com pounds do not pose major i nterferences to toxaphene determination under NICI/ M S

conditions.

2 These compounds may exhibit fragmentation resulting in a response at m /z 234.9, 236.9, and 238.9 that aids in

confirmation.Method 8276 8276 - 35 Revision 1 October 2012 Table 5

EXAMPLE GAS CHROMATOGRAPHIC RETENTION TIMES FOR TOXAPHENE CONGENERS USING A NARROW-BORE CAPILLARY COLUMN (DB- XLB MSD)

Compound Retention Time (min)

Hx-Sed 31.31

Hp-Sed 31.95

Parlar 26 32.38

Parlar 41 36.69

Parlar 40 37.03

Parlar 44 37.35

Parlar 50 37.89

Parlar 62 41.91

Decachlorobiphenyl (DCB) 48.30Method 8276 8276 - 36 Revision 1 October 2012 Table 6

QUANTITATIONS OF SPIKED SOIL FOR TOXAPHENE (25 ppb, 5-g sample) AND CHLORDANE (50 ppb, 5-g sample) 1

Replicate No. Toxaphene (ppb) Chlordane (ppb)

1 34.2 31.4

2 46.4 35.2

3 42.7 29.4

4 35.0 41.6

5 27.0 27.0

6 42.0 27.8

7 43.9 28.0

Avg 38.7 31.5

% RSD 17.8 16.7

1 Data provided courtesy of Dr. Bill Brumley from US EPA ORD, 2008. Results were obtained using a 40-m Agilent J&W 0.18- mm ID, 0.18-μm film thickness column.Method 8276 8276 - 37 Revision 1 October 2012 Table 7

QUANTITATIONS OF SPIKED SOIL FOR TOXAPHENE CONGENER S (500 ppt, 5-g sample) 1

Replicate No. Hx-Sed Hp-Sed Parlar 26

1 468 532 552

2 690 722 788

3 768 746 778

4 688 724 808

5 780 752 804

6 778 786 822

7 672 784 706

Avg 692 720 752

% RSD 15.8 12.1 12.7

Replicate No. Parlar 38, 40, 41 Parlar 44 Parlar 50, 62

1 508 696 544

2 792 930 812

3 808 876 702

4 856 1,016 844

5 860 986 790

6 826 942 750

7 866 1,270 772

Avg 788 958 744

% RSD 16.1 17.8 13.4

1 Data provided courtesy of Dr. Bill Brumley from US EPA ORD, 2008. Results were obtained using a 40-m Agilent J&W 0.18- mm ID, 0.18-μm film thickness column.Method 8276 8276 - 38 Revision 1 October 2012 

### Table 8

QUANTITATIONS OF SPIKED SOIL FOR ORGANOCHLORINE PESTICIDES (50 ppb, 5-g sample) 1

Replicate

No. α-BHC β-BHC δ-BHC Heptachlor Aldrin Heptachlor Epoxide

1 28.0 52.0 39.8 34.8 40.8 51.2

2 40.6 57.6 50.0 44.6 53.4 53.0

3 33.0 46.4 37.8 35.4 42.0 46.2

4 27.0 71.6 40.4 28.2 34.4 51.8

5 42.4 71.4 53.4 49.2 56.6 59.6

6 43.2 64.4 51.2 53.0 59.8 62.4

7 37.6 43.2 32.2 49.0 53.0 54.2

Avg 36.0 58.0 43.6 42.0 48.6 54.0

% RSD 18.7 19.9 18.3 22.0 19.5 10.0

Replicate

No. Endosulfan I Dieldrin Endrin Endosulfan II Endrin Aldehyde Endosulfan

Sulfate

1 16.8 73.8 73.6 17.7 26.0 36.6

2 21.4 70.4 72.6 29.4 44.6 47.4

3 37.8 61.2 63.6 38.4 19.7 37.6

4 51.0 69.6 68.6 43.0 25.6 42.8

5 25.2 74.4 72.8 37.8 44.2 51.0

6 20.4 74.2 70.6 29.8 42.8 52.4

7 13.0 62.8 66.6 11.1 37.8 46.2

Avg 26.6 69.4 69.8 29.6 34.4 44.8

% RSD 50.1 7.9 5.3 39.2 30.1 13.8

1 Data provided courtesy of Dr. Bill Brumley from U S EPA O R D, 2008. Results were obtained using a 40 -m Agilent J&W 0.18-mm ID, 0.18-μm film thickness column and are i ncluded for informational purposes only to indicate that quantitation and acceptable recoveries of other organochlorine pesti cide com pounds using GC-NICI/MS are possible.Method 8276 8276 - 39 Revision 1 October 2012 

### Table 9

PHASE I MULTI-LABORATORY VALIDATION DATA FOR UNKNOW N SPIKED STANDARDS 1

T ox a ph e n e

ID No. of L abs True V a lue Grand M e an Std. Reprodu c ibility (pg/μL) (pg/μL) Deviation (RSD)

Unknown 1 9 10.0 13.0 5.7 44%

Unknown 2 10 125 130 17.4 13%

QC 9 150 148 10.3 7%

H x -SED

ID No. of L abs True V a lue Grand M e an Std. Reprodu c ibility (pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.3 2.1 23%

P-Unknown 2 10 125 126 10.9 9%

H p -SED

ID No. of L abs True V a lue Grand M e an Std. Reprodu c ibility (pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.1 2.1 23%

P-Unknown 2 10 125 124 9.3 7%

P26

ID No. of L abs True V a lue Grand M e an Std. Reprodu c ibility (pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.7 2.7 28%

P-Unknown 2 10 125 123 7.8 6%

P-QC 9 200 200 22.5 11%

P40

ID No. of L abs True V a lue Grand M e an Std. Reprodu c ibility (pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 9 10.0 9.4 2.6 28%

P-Unknown 2 9 125 121 9.0 7%

P-QC 8 200 182 22.5 12%Method 8276 8276 - 40 Revision 1 October 2012 

### Table 9 (cont.)

PHASE I MULTI-LABORATORY VALIDATION DATA FOR UNKNOW N SPIKED STANDARDS

P41

ID No. of L abs True V a lue Grand M e an Std. Reprodu c ibility (pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 9 10.0 9.6 3.8 40%

P-Unknown 2 9 125 121 9.8 8%

P-QC 8 200 176 22.0 13%

P44

ID No. of L abs True V a lue Grand M e an Std. Reprodu c ibility (pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.2 3.0 33%

P-Unknown 2 10 125 121 15.0 12%

P-QC 9 200 269 41.9 16%

P50

ID No. of L abs True V a lue Grand M e an Std. Reprodu c ibility (pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.1 2.9 32%

P-Unknown 2 10 125 122 9.2 8%

P-QC 9 200 182 22.0 12%

P62

ID No. of La b s True Val u e Grand Mean Std. Repro d uc i bi l ity (pg/μ L ) (pg/μ L ) Deviati o n (RSD)

P-Unknown 1 10 10.0 7.1 2.7 38%

P-Unknown 2 10 125 127 19.4 15%

P-QC 9 200 320 62.0 19%

1 The values of the quality control standards were un known to the analysts.Method 8276 8276 - 41 Revision 1 October 2012 

### Table 10

PHASE II MULTI-LABORATORY VALIDATION DATA USING SPI KED EXTRACTS OF

UNCONTAMINATED REAL WORLD SAMPLE MATRICES 1

Toxaphene

ID No. of L abs True V a lue Grand M e an Std. Reprodu c ibility (pg/μL) (pg/μL) Deviation (RSD)

GW 2 9 600 694 58.6 8.4%

Sed 2 9 700 881 159 18.0%

Soil 2 9 400 458 71.0 15.5%

Hx-SED

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) Std.

Deviation Reproduc ibility (RSD)

GW 1 9 1.0 0.98 0.11 11.2%

GW 2 9 150 146 9.2 6.3%

Sed 1 9 4.0 3.57 0.36 10.1%

Sed 2 9 200 198 22.0 11.1%

Soil 1 9 2.0 1.92 0.26 13.5%

Soil 2 9 250 253 25.7 10.2%

Hp-SED

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) Std.

Deviation Reproduc ibility (RSD)

GW 1 9 1.0 1.00 0.11 11.0%

GW 2 9 150 148 7.9 5.3%

Sed 1 9 4.0 3.61 0.44 12.2%

Sed 2 9 200 198 18.9 9.5%

Soil 1 9 2.0 1.99 0.26 13.1%

Soil 2 9 250 255 24.1 9.5%

1 Liquid samples were solvent extracted with methylen e chloride according to Method 3510 using a sample volume of one
L and concentrated to a final extract volume of 5 m L in isooctane. Solid samples were solvent extracte d using methylene chloride according to Method 3540 with a sample mass of 30-g and concentrated to a final ext ract volume of

5 mL in isooctane.Method 8276 8276 - 42 Revision 1 October 2012 

### Table 10 (cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING SPI KED EXTRACTS OF

UNCONTAMINATED REAL WORLD SAMPLE MATRICES 1

P26

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) Std.

Deviation Reproduc ibility (RSD)

GW 1 9 1.0 0.96 0.12 12.5%

GW 2 9 150 145 6.8 4.7%

Sed 1 9 4.0 3.56 0.34 9.6%

Sed 2 9 200 196 17.9 9.1%

Soil 1 9 2.0 1.95 0.27 13.8%

Soil 2 9 250 257 23.0 8.9%

P40

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) Std.

Deviation Reproduc ibility (RSD)

GW 1 9 1.0 1.03 0.15 14.6%

GW 2 9 150 149 7.1 4.8%

Sed 1 9 4.0 3.72 0.47 12.6%

Sed 2 9 200 203 20.7 10.2%

Soil 1 9 2.0 2.11 0.25 11.8%

Soil 2 9 250 267 26.6 10.0%

P41

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) Std.

Deviation Reproduc ibility (RSD)

GW 1 9 1.0 0.97 0.12 12.4%

GW 2 9 150 146 9.0 6.2%

Sed 1 9 4.0 3.68 0.46 12.5%

Sed 2 9 200 196 23.8 12.1%

Soil 1 9 2.0 2.04 0.28 13.7%

Soil 2 9 250 263 27.3 10.4%

1 Liquid samples were solvent extracted with methylen e chloride according to Method 3510 using a sample volume of one

L and concentrated to a final extract volume of 5 m L in isooctane. Solid samples were solvent extracte d using methylene chloride according to Method 3540 with a sample mas s of 30-g and concentrated to a final extract volum e of 5 mL in

isooctane.Method 8276 8276 - 43 Revision 1 October 2012 Table 10 (cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING SPI KED EXTRACTS OF

UNCONTAMINATED REAL WORLD SAMPLE MATRICES 1

P44

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) Std.

Deviation Reproduc ibility (RSD)

GW 1 9 1.0 0.93 0.18 19.4%

GW 2 9 150 149 13.1 8.8%

Sed 1 9 4.0 3.91 1.05 26.9%

Sed 2 9 200 208 34.4 16.5%

Soil 1 9 2.0 2.01 0.30 14.9%

Soil 2 9 250 282 32.1 11.4%

P50

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) Std.

Deviation Reproduc ibility (RSD)

GW 1 9 1.0 0.97 0.18 18.6%

GW 2 9 150 148 8.0 5.4%

Sed 1 9 4.0 3.68 0.44 12.0%

Sed 2 9 200 199 21.2 10.7%

Soil 1 9 2.0 2.00 0.27 13.5%

Soil 2 9 250 265 22.7 8.6%

P62

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) Std.

Deviation Reproduc ibility (RSD)

GW 1 9 1.0 0.96 0.33 34.4%

GW 2 9 150 143 18.6 13.0%

Sed 1 9 4.0 4.09 1.08 26.4%

Sed 2 9 200 198 44.5 22.5%

Soil 1 9 2.0 2.02 0.29 14.4%

Soil 2 9 250 273 57.9 21.2%

1 Liquid samples were solvent extracted with methylen e chloride according to Method 3510 using a sample volume of one

L and concentrated to a final extract volume of 5 m L in isooctane. Solid samples were solvent extracte d using methylene chloride according to Method 3540 with a sample mas s of 30-g and concentrated to a final extract volum e of 5 mL in

isooctane.Method 8276 8276 - 44 Revision 1 October 2012 

### Table 11

PHASE II MULTI-LABORATORY VALIDATION DATA USING REA L WORLD SAMPLES FROM TERRY CREEK IN BRUNSWICK, GEORGIA 1

SEDIMENT

ID N o. of L a b s G rand Mea n ( pg /μ L ) G rand Mea n

( m g / k g ) Std. De v i a t i on

( pg /μ L ) Re p r odu c i b ili t y (R SD )

Toxaphene 9 173,000 36,300 91,000 53%

Hx-Sed 9 3,600 754 366 10%

Hp-Sed 9 3,700 776 526 14%

P26 8 1,250 261 163 13%

P40 9 2,660 557 951 36%

P41 9 1,310 273 1530 117%

P44 8 902 189 172 19%

P50 9 1,780 372 231 13%

P62 8 3,840 803 1,410 37%

SO I L

ID N o. of L a b s G rand Mea n ( pg /μ L ) G rand Mea n

( m g / k g ) Std. De v i a t i on

( pg /μ L ) Re p r odu c i b ili t y (R SD )

Toxaphene 9 2,980 624 1,730 58%

Hx-Sed 9 153 32 37 24%

Hp-Sed 9 129 27 30 23%

P26 8 6.74 1.4 2.08 31%

P40 9 15.0 3.1 7.4 49%

P41 9 6.0 1.3 8.5 142%

P44 8 2.53 0.5 1.30 51%

P50 9 4.0 0.8 1.2 30%

P62 8 20.7 4.3 45.8 221%

1 Samples were solvent extracted using methylene chlo ride according to Method 3540 with a sample mass of 30 g

and concentrated to a final extract volume of 5 mL in isooctane.Method 8276 8276 - 45 Revision 1 October 2012 

### Table 12

PHASE II MULTI-LABORATORY VALIDATION DATA USING REA L WORLD SAMPLES FROM TERRY CREEK IN BRUNSWICK, GEORGIA WITH EXTRACTS SUB JECTED TO CLEANUP 1

Technical Tox aphene

ID No. of Labs True Va lue Grand Me an Grand Me an % Rec. Std. Dev. Reproduc ibility (pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD)

TC Sed 4

--- 30 5,000 63, 900

--- 47, 2 00 15%

TC Soil 4 --- 15,600 3,260 --- 4,570 29%

PCB 209 Sed Surr 4 100 106 --- 106% 4.0 4%

ε -HCH Sed Surr 4 250 231 --- 92% 21.0 9%

PCB 209 Soil Surr 4 100 106

--- 10 6 % 1.0 1%

ε

- HCH Soil Surr 4 250 240

--- 96% 11.8 5%

L C S 4 200 228

--- 11 4 % 27.5 12

Hx

- SED

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

TC Sed 4

--- 3,6 8 0 772

--- 183 5%

TC Soil 4 --- 1,380 290 --- 91 7%

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4%

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5%

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8%

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2%

LCS 4 200 211 --- 105% 12.8 6%

Hx-SED

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

TC Sed 4

--- 4,2 20 883

--- 993 24%

TC Soil 4 --- 1,100 231 --- 29 3%

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4%

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5%

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8%

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2%

LCS 4 200 212 --- 106% 14.2 7%

1 Samples were solvent extracted using methylene chlo ride according to Method 3540 with a sample mass of 30 g

and concentrated to a volume of 10 mL. These extrac ts were further subjected to clean-up using a coppe r, acid, and silica gel approach according to Methods 3660, 3665 , and 3630, respectively, to a final extract volume of 5 mL in

isooctane.Method 8276 8276 - 46 Revision 1 October 2012 

### Table 12 (cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING REA L WORLD SAMPLES FROM TERRY CREEK IN BRUNSWICK, GEORGIA WITH EXTRACTS SUB JECTED TO CLEANUP 1

P26

ID No. of Labs True Va lue Grand Me an Grand Me an % Rec. Std. Dev. Reproduc ibility (pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD)

TC Sed 4

--- 1,8 7 0 392

--- 156 8%

TC Soil 4 --- 60 13 --- 6 11%

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4%

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5%

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8%

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2%

LCS 4 200 216 --- 108% 22.8 11%

P40

ID No. of Labs True Va lue Grand Me an Grand Me an % Rec. Std. Dev. Reproduc ibility (pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD)

TC Sed 4

--- 4, 2 0 0 880

--- 1,8 9 0 45%

TC Soil 4 --- 143 30 --- 60 42%

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4%

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5%

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8%

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2%

LCS 4 200 219 --- 110% 24.3 11%

P41

ID No. of Labs True Va lue Grand Me an Grand Me an % Rec. Std. Dev. Reproduc ibility (pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD)

TC Sed 4

--- 1, 2 3 0 258

--- 175 14%

TC Soil 4 --- 33 7 --- 17 51%

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4%

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5%

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8%

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2%

LCS 4 200 220 --- 110% 28.1 13%

1 Samples were solvent extracted using methylene chlo ride according to Method 3540 with a sample mass of 30 g

and concentrated to a volume of 10 mL. These extrac ts were further subjected to clean-up using a coppe r, acid, and silica gel approach according to Methods 3660, 3665 , and 3630, respectively, to a final extract volume of 5 mL in

isooctane.Method 8276 8276 - 47 Revision 1 October 2012 

### Table 12 (cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING REA L WORLD SAMPLES FROM TERRY CREEK IN BRUNSWICK, GEORGIA WITH EXTRACTS SUB JECTED TO CLEANUP 1

P44

ID No. of Labs True Va lue Grand Mean Grand Me an % Rec. Std. Dev. Reproduc ibility (pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD)

TC Sed 3

--- 1, 3 9 0 292

--- 194 14%

TC Soil 3 --- 30 6 --- 5 18%

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4%

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5%

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8%

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2%

LCS 4 200 209 --- 104% 29.8 14%

P50

ID No. of Labs True Va lue Grand Mean Grand Me an % Rec. Std. Dev. Reproduc ibility (pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD)

TC Sed 4

--- 2, 9 2 0 612

--- 342 12%

TC Soil 4 --- 69 14 --- 7 11%

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4%

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5%

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8%

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2%

LCS 4 200 221 --- 110% 23.8 11%

P62

ID No. of Labs True Va lue Grand Mean Grand Me an % Rec. Std. Dev. Reproduc ibility (pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD)

TC Sed 3

--- 8, 8 40 1, 8 5 0

--- 1, 5 50 18%

TC Soil 3 --- 219 46 --- 54 24%

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4%

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5%

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8%

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2%

LCS 4 200 226 --- 113% 27.6 12%

1 Samples were solvent extracted using methylene chlo ride according to Method 3540 with a sample mass of 30 g and

concentrated to a volume of 10 mL. These extracts were further subjected to clean-up using a copper, acid, and silica gel approach according to Methods 3660, 3665 , and 3630, respectively, to a final extract volume of 5 mL in

isooctane.Method 8276 8276 - 48 Revision 1 October 2012 

### Table 13

PHASE II MULTI-LABORATORY VALIDATION DATA COMPARISO N USING REAL WORLD SAMPLES FROM TERRY CREEK IN BRUNSWICK, GEORGIA (EXT RACTION ONLY COMPARED

TO EXTRACTS SUBJECTED TO CLEANUP)

SEDIMENT

ID N o. of L a b s 1 N o. of Labs 2 G rand Mean G rand Mea n G rand Mea n G rand Mea n

Extraction only Cleanup ( pg /μ L ) 1

Extraction only ( pg /μ L ) 2

Cleanup ( m g / k g ) 1

Extraction only ( m g / k g ) 2

Cleanup

T oxaphene 9 4 173,000 305,000 36,300 63,900

H x-Sed 9 4 3,600 3,680 754 772

H p-Sed 9 4 3,700 4,220 776 883

P26 8 4 1,250 1,870 261 392

P40 9 4 2,660 4,200 557 880

P41 9 4 1,310 1,230 273 258

P44 8 3 902 1,390 189 292

P50 9 4 1,780 2,920 372 612

P62 8 3 3,840 8,840 803 1,850

SO I L

ID N o. of L a b s 1 N o. of L a b s 2 G rand Mea n ( pg / μ L ) 1 G rand Mea n

( pg / μ L ) 2 G rand Mea n

( m g / k g ) 1 G rand Mea n

( m g / k g ) 2

Extrac t ion o n ly Cleanup Extrac t ion o n ly Cleanup Extrac t ion o n ly Cleanup

Toxaphene 9 4 2,980 15,600 624 3,260

Hx-Sed 9 4 153 1,380 32 290

Hp-Sed 9 4 129 1,100 27 231

P26 8 4 6.74 60 1.4 13

P40 9 4 15.0 143 3.1 30

P41 9 4 6.0 33 1.3 7

P44 8 3 2.53 30 0.5 6

P50 9 4 4.0 69 0.8 14

P62 8 3 20.7 219 4.3 46

1 Samples were solvent extracted using methylene chlo ride according to Method 3540 with a sample mass of 30 g and

concentrated to a final extract volume of 5 mL in i sooctane.

2 Samples were solvent extracted using methylene chlo ride according to Method 3540 with a sample mass of 30 g and

concentrated to a volume of 10 mL. These extracts w ere further subjected to clean-up using a copper, a cid, and silica gel approach according to Methods 3660, 3665, and 3 630, respectively, to a final extract volume of 5 m L in isooctane.Method 8276 8276 - 49 Revision 1 October 2012 

### Table 14

PHASE II MULTI-LABORATORY VALIDATION DATA USING MUL TIPLE TECHNICAL

TOXAPHENE SPIKING PROTOCOLS FOR UNCONTAMINATED REAL WORLD SAMPLE

MATRICES 1

Technical Tox aphene Spiked Prior to Extraction and Cleanu p

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) % Recovery Std.

Deviation (pg/μL) Reproduc ibility

RSD

Sed 1L 4 75 78 105% 6.9 9%

PCB 209 Surr 4 100 109 109% 2 2%

ε -HCH Surr 4 250 275 110% 10 4%

Sed 1M 4 150 148 99% 16 11%

PCB 209 Surr 4 100 97 97% 5 5%

ε -HCH Surr 4 250 238 95% 17 7%

LCS L 4 75 71 95% 9 12%

LCS M 4 200 185 93% 27 15%

Low-level Check 4 40 49 121% 7 14%

Technical Tox aphene Spiked After Extraction and Prior to Clea n-up

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) % Recovery Std.

Deviation (pg/μL) Reproduc ibility

RSD

Sed 2L 4 75 73 97% 9.1 12%

PCB 209 Surr 4 100 100 100% 4 4%

ε -HCH Surr 4 250 258 103% 14 5%

Sed 2M 4 150 153 102% 18 12%

PCB 209 Surr 4 100 99 99% 6 6%

ε -HCH Surr 4 250 255 102% 19 7%

LCS L 4 75 71 95% 9 12%

LCS M 4 200 185 93% 27 15%

Low-level Check 4 40 49 121% 7 14%

1 Samples were solvent extracted using methylene chlo ride according to Method 3540 with a sample mass of 30 g

and concentrated to a volume of 10 mL. These extrac ts were further subjected to clean-up using a coppe r, acid, and silica gel approach according to Methods 3660, 3665 , and 3630, respectively, to a final extract volume of 5 mL in

isooctane.Method 8276 8276 - 50 Revision 1 October 2012 

### Table 14 (cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING MUL TIPLE SPIKING

PROTOCOLS FOR UNCONTAMINATED REAL WORLD SAMPLE MATRICES 1

Technical

Tox aphene Spiked in the Final

Ex tract

ID No. of L abs True Va lue (pg/μL) Grand Me an

(pg/μL) % Recovery Std. Deviation (pg/μL) Reproduc ibility

RSD

Sed 3L 4 75 86 114% 10 12%

PCB 209 Surr 4 100 105 105% 5 5%

ε -HCH Surr 4 250 276 111% 21 7%

Sed 3M 4 150 157 105% 21 13%

PCB 209 Surr 4 100 105 105% 5 5%

ε-HCH Surr 4 250 273 109% 18 7%

LCS L 4 75 71 95% 9 12%

LCS M 4 200 185 93% 27 15%

Low-level Check 4 40 49 121% 7 14%

1 Samples were solvent extracted using methylene chlo ride according to Method 3540 with a sample mass of 30 g
and concentrated to a volume of 10 mL. These extrac ts were further subjected to clean-up using a coppe r, acid, and silica gel approach according to Methods 3660, 3665, and 3630, respectively, to a final extract vo lume of 5

mL in isooctane.Method 8276 8276 - 51 Revision 1 October 2012 

### Table 15

PHASE III MULTI-LABORATORY VALIDATION DATA USING SP IKED LABORATORY QUALITY

CONTROL SAMPLES CARRIED THROUGH EXTRACTION AND CLEA NUP 1

Toxaphene

ID No. of Participants True

Value (pg/ μ L) Grand

Mean (pg/ μ L) %

Recovery Std.

Deviation Reproducibility

(% RSD)

Second Source

Standard 8 150 159 106 9.9 6.2

LCS 1 8 50 57.3 115 9.7 16.9

LCS 2 8 200 207 104 13.4 6.5

PCB 209 LCS1 8 100 95.0 95.0 1.9 2.0

ε-HCH LCS 1 8 250 231 92.2 21.0 9.1

PCB 209 LCS2 8 100 90.2 90.2 5.4 6.0

ε-HCH LCS2 8 250 240 95.8 15.4 6.4

Hx-Sed

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(% RSD)

LCS1 8 100 98.4 98.4 5.84 5.94

LCS2 8 2 1.95 97.4 0.16 8.00

Hp-Sed

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(% RSD)

LCS1 8 100 98.9 98.9 5.77 5.84

LCS2 8 2 1.99 99.3 0.13 6.30

P26

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(% RSD)

LCS1 8 100 97.9 97.9 5.27 5.39

LCS2 8 2 1.83 91.4 0.36 19.7Method 8276 8276 - 52 Revision 1 October 2012 Table 15 (cont.)

P40

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(% RSD)

LCS1 8 100 98.1 98.1 6.33 6.45

LCS2 8 2 1.94 97.2 0.15 7.46

P41

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(% RSD)

LCS1 8 100 98.6 98.6 6.31 6.40

LCS2 8 2 2.03 102 0.17 8.32

P44

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(RSD)

LCS1 8 100 99.4 99.4 8.85 8.91

LCS2 8 2 1.99 99.7 0.16 8.09

P50

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(% RSD)

LCS1 8 100 96.9 96.9 7.47 7.71

LCS2 8 2 2.01 101 0.07 3.55

P62

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(% RSD)

LCS1 8 226 237.0 105 26.0 11.0

LCS2 8 22.6 21.0 92.9 4.51 21.5Method 8276 8276 - 53 Revision 1 October 2012 

### Table 15 (cont.)

Congeners - PCB 209

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(% RSD)

LCS1 8 100 95.1 95.1 5.13 5.39

LCS2 8 100 94.9 94.9 5.55 5.85

Congeners – ε-HCH

ID No. of

Participants True Value

(pg/μL) Grand Mean

(pg/μL) %

Recovery Std.

Deviation Reproducibility

(% RSD)

LCS1 8 250 248.0 99.0 11.7 4.72

LCS2 8 250 247.0 98.9 7.84 3.17

1 Samples were extracted in hexane in an Accelerated Solvent Extractor (ASE) using a modified version of Method 3545A with a sample mass of 10 g of solid ma terial (Ottawa sand). In-cell cleanup was done in t he ASE extraction cell using activated, acidified (30% H 2 SO 4 ) silica gel per Method 3665A and Method 3630C. These extracts were further subjected to cleanup us ing a Florisil® cartridge (1% deactivated with DI w ater) approach according to Method 3620C. The sample was in hexane throughout the process and was concentrat ed
to a final extract volume of 1 mL.Method 8276 8276 - 54 Revision 1 October 2012 

### Table 16

PHASE III MULTI-LABORATORY VALIDATION DATA USING FI SH SAMPLES COLLECTED FROM
TERRY CREEK IN BRUNSWICK, GEORGIA WITH EXTRACTS SUB JECTED TO CLEANUP 1

Toxaphene

ID No. of

Participants Grand Mean (pg/μL) Std.

Deviation Reproducibility

% RSD

TC 1 8 351 71.9 20.5

TC 2 8 317 79.3 25.0

Hp-Sed

ID No. of Participants Grand Mean (pg/μL) Std.

Deviation Reproducibility

% RSD

TC1 8 91.8 5.66 6.17

TC2 8 75.6 5.80 7.67

P26

ID No. of Participants Grand Mean (pg/μL) Std.

Deviation Reproducibility

% RSD

TC1 8 8.05 0.40 4.93

TC2 8 11.6 0.52 4.46

P40

ID No. of Participants Grand Mean (pg/μL) Std.

Deviation Reproducibility

% RSD

TC1 8 11.1 1.02 9.19

TC2 8 12.6 0.71 5.66 Hx-Sed

ID No. of Participants Grand Mean (pg/μL) Std.

Deviation Reproducibility

% RSD

TC1 8 80.3 5.59 6.97

TC2 8 54.2 4.49 8.27Method 8276 8276 - 55 Revision 1 October 2012 

### Table 16 (cont.)

P41

ID No. of Participants Grand Mean (pg/μL) Std.

Deviation Reproducibility

% RSD

TC1 8 4.49 0.19 4.31

TC2 8 5.29 0.21 3.90

P44

ID No. of Participants Grand Mean (pg/μL) Std.

Deviation Reproducibility

% RSD

TC1 8 4.76 0.33 6.86

TC2 8 6.60 0.55 8.33

P50

ID No. of Participants Grand Mean (pg/μL) Std.

Deviation Reproducibility

% RSD

TC1 8 8.34 0.73 8.76

TC2 8 12.9 1.06 8.18

P62

ID No. of Participants Grand Mean (pg/μL) Std.

Deviation Reproducibility

% RSD

TC1 8 10.5 2.81 26.9

TC2 8 14.8 4.13 27.8

1 Samples were extracted in hexane in an Accelerated Solvent Extractor (ASE) using a
modified version of Method 3545A with a sample mass of 10 g of solid material (Ottawa
sand). In-cell cleanup was done in the ASE extracti on cell using activated, acidified (30%

H 2 SO 4 ) silica gel per Method 3665A and Method 3630C. The se extracts were further
subjected to cleanup using a Florisil® cartridge (1 % deactivated with DI water) approach
according to Method 3620C. The sample was in hexane throughout the process and was

concentrated to a final extract volume of 1 mL.Method 8276 8276 - 56 Revision 1 October 2012 

### FIGURE 1

EXAMPLE GAS CHROMATOGRAM OF A MIXED CONGENER STANDA RDMethod 8276 8276 - 57 Revision 1 October 2012 

### FIGURE 2

EXAMPLE GAS CHROMATOGRAM OF A FISH TISSUE EXTRACTMethod 8276 8276 - 58 Revision 1 October 2012 

### FIGURE 3

EXAMPLE GAS CHROMATOGRAM OF A 100 pg/μL CONGENER CA LIBRATION VERIFICATION

STANDARDMethod 8276 8276 - 59 Revision 1 October 2012 

### FIGURE 4

EXAMPLE GAS CHROMATOGRAM OF AN EPA REGION 4 SEDIMEN T EXTRACT SPIKED USING

DECACHLOROBIPHENYLMethod 8276 8276 - 60 Revision 1 October 2012 

### Appendix A: Changes in Revision 1 from the Revision 0, 2010 Version.

1. The acronym NIMS (negative ion mass spectroscop y) has been replaced with NICI/MS (negative ion chemical ionization/mass spectrometry).

2. Section 2.0 now points to Method 3500 for sample preparation guidance, rather than listing all methods specifically.

3. References to the method quantitation limit (MQL ) have been replaced with the lower limit of quantitation (LLOQ).

4. Section 6.4.1 no longer specifies exact mass spe ctrometer conditions. Instead, it states that the detector must tuned per Section 11.4.1 and Table 1 conditions.

5. Sections 9.4 (IDP) and 9.7 (LLOQ) were expanded to reflect current guidance.

6. Section 11.1 was re-organized into tabular form to increase clarity and ease of preparatory method

selection.

7. Improved overall method formatting for consisten cy with new SW-846 methods style guidance. The format was updated to Microsoft Word 2007.docx. Many minor editorial and technical revisions were made throughout to improve method clarity.

8. The revision number was changed to 1 and the dat e published was changed to October 2012.

9. This appendix was added showing changes from the previous revision.