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

4.2.3 Do not bake reusable glassware in an oven as a routine part of cleaning. Baking may be warranted after particularly dirty samples are encountered but should be minimized, as repeated baking of glassware may cause active sites on the glass surface that will irreversibly adsorb CDDs/CDFs.

4.2.4 Immediately prior to use, the Soxhlet apparatus should be pre-extracted with toluene for approximately three hours (see Sections 12.3.1 through 12.3.3). Separatory funnels should be shaken with methylene chloride/toluene (80/20 mixture) for two minutes, drained, and then shaken with pure methylene chloride for two minutes.

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

4.3.1 The reference matrix must simulate, as closely as possible, the sample matrix under test. Ideally, the reference matrix should not contain the CDDs/CDFs in detectable amounts, but should contain potential interferents in the concentrations expected to be found in the samples to be analyzed. For example, a reference sample of human adipose tissue containing pentachloronaphthalene can be used to exercise the cleanup systems when samples containing pentachloronaphthalene are expected.

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

4.4 Interferences coextracted from samples will vary considerably from source to source, depending on the diversity of the site being sampled. Interfering compounds may be present at concentrations several orders of magnitude higher than the CDDs/CDFs. The most frequently encountered interferences are chlorinated biphenyls, methoxy biphenyls, hydroxydiphenyl ethers, benzylphenyl ethers, polynuclear aromatics, and pesticides. Because very low levels of CDDs/CDFs are measured by this method, the elimination of interferences is essential. The cleanup steps given in Section 13 can be used to reduce or eliminate these interferences and thereby permit reliable determination of the CDDs/CDFs at the levels shown in Table 2.

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

4.6 Cleanup of tissue—The natural lipid content of tissue can interfere in the analysis of tissue samples for the CDDs/CDFs. The lipid contents of different species and portions of tissue can vary widely. Lipids are soluble to varying degrees in various organic solvents and may be present in sufficient quantity to overwhelm the column chromatographic cleanup procedures used for cleanup of sample extracts. Lipids must be removed by the lipid removal procedures in Section 13.7, followed by alumina (Section 13.4) or Florisil (Section 13.8), and carbon (Section 13.5) as minimum additional cleanup steps. If chlorodiphenyl ethers are detected, as indicated by the presence of peaks at the exact m/z's monitored for these interferents, alumina and/or Florisil cleanup must be employed to eliminate these interferences.

5.0 Safety

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

5.1.1 The 2,3,7,8-TCDD isomer has been found to be acnegenic, carcinogenic, and teratogenic in laboratory animal studies. It is soluble in water to approximately 200 ppt and in organic solvents to 0.14%. On the basis of the available toxicological and physical properties of 2,3,7,8-TCDD, all of the CDDs/CDFs should be handled only by highly trained personnel thoroughly familiar with handling and cautionary procedures and the associated risks.

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

5.2 The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material safety data sheets (MSDSs) should also be made available to all personnel involved in these analyses. It is also suggested that the laboratory perform personal hygiene monitoring of each analyst who uses this method and that the results of this monitoring be made available to the analyst. Additional information on laboratory safety can be found in References 10–13. The references and bibliography at the end of Reference 13 are particularly comprehensive in dealing with the general subject of laboratory safety.

5.3 The CDDs/CDFs and samples suspected to contain these compounds are handled using essentially the same techniques employed in handling radioactive or infectious materials. Well-ventilated, controlled access laboratories are required. Assistance in evaluating the health hazards of particular laboratory conditions may be obtained from certain consulting laboratories and from State Departments of Health or Labor, many of which have an industrial health service. The CDDs/CDFs are extremely toxic to laboratory animals. Each laboratory must develop a strict safety program for handling these compounds. The practices in References 2 and 14 are highly recommended.

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

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

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

5.3.4 Personal hygiene—Hands and forearms should be washed thoroughly after each manipulation and before breaks (coffee, lunch, and shift).

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

5.3.6 Effluent vapors—The effluents of sample splitters from the gas chromatograph (GC) and from roughing pumps on the mass spectrometer (MS) should pass through either a column of activated charcoal or be bubbled through a trap containing oil or high-boiling alcohols to condense CDD/CDF vapors.

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

5.3.8 Decontamination

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

5.3.8.2 Glassware, tools, and surfaces—Chlorothene NU Solvent is the least toxic solvent shown to be effective. Satisfactory cleaning may be accomplished by rinsing with Chlorothene, then washing with any detergent and water. If glassware is first rinsed with solvent, then the dish water may be disposed of in the sewer. Given the cost of disposal, it is prudent to minimize solvent wastes.

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

5.3.10 Wipe tests—A useful method of determining cleanliness of work surfaces and tools is to wipe the surface with a piece of filter paper. Extraction and analysis by GC with an electron capture detector (ECD) can achieve a limit of detection of 0.1 µg per wipe; analysis using this method can achieve an even lower detection limit. Less than 0.1 µg per wipe indicates acceptable cleanliness; anything higher warrants further cleaning. More than 10 µg on a wipe constitutes an acute hazard and requires prompt cleaning before further use of the equipment or work space, and indicates that unacceptable work practices have been employed.

5.3.11 Table or wrist-action shaker—The use of a table or wrist-action shaker for extraction of tissues presents the possibility of breakage of the extraction bottle and spillage of acid and flammable organic solvent. A secondary containment system around the shaker is suggested to prevent the spread of acid and solvents in the event of such a breakage. The speed and intensity of shaking action should also be adjusted to minimize the possibility of breakage.

6.0 Apparatus and Materials

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

6.1 Sampling Equipment for Discrete or Composite Sampling

6.1.1 Sample bottles and caps

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

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

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

6.1.1.4 Bottle caps—Threaded to fit sample bottles. Caps shall be lined with fluoropolymer.

6.1.1.5 Cleaning

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

6.1.1.5.2 Liners are detergent water washed, rinsed with reagent water (Section 7.6.1) followed by solvent, and baked at approximately 200 °C for a minimum of 1 hour prior to use.

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

6.2 Equipment for Glassware Cleaning—Laboratory sink with overhead fume hood.

6.3 Equipment for Sample Preparation

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

6.3.2 Glove box (optional).

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

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

6.3.5 Equipment for determining percent moisture

6.3.5.1 Oven—Capable of maintaining a temperature of 110 ±5 °C.

6.3.5.2 Dessicator.

6.3.6 Balances

6.3.6.1 Analytical—Capable of weighing 0.1 mg.

6.3.6.2 Top loading—Capable of weighing 10 mg.

6.4 Extraction Apparatus

6.4.1 Water samples

6.4.1.1 pH meter, with combination glass electrode.

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

6.4.1.3 Graduated cylinder, 1 L capacity.

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

6.4.1.5 Solid-phase extraction

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

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

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

6.4.1.5.4 Solid-phase extraction disk containing octadecyl (C18) bonded silica uniformly enmeshed in an inert matrix—Fisher Scientific 14–378F (or equivalent), to fit filtration apparatus in Section 6.4.1.5.1.

6.4.2 Soxhlet/Dean-Stark (SDS) extractor (Figure 5)—For filters and solid/sludge samples.

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

6.4.2.2 Thimble—43 × 123 to fit Soxhlet (Cal-Glass LG–6901–122, or equivalent).

6.4.2.3 Moisture trap—Dean Stark or Barret with fluoropolymer stopcock, to fit Soxhlet.

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

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

6.4.3 Apparatus for extraction of tissue.

6.4.3.1 Bottle for extraction (if digestion/extraction using HCl is used)” 500–600 mL wide-mouth clear glass, with fluoropolymer-lined cap.

6.4.3.2 Bottle for back-extraction—100–200 mL narrow-mouth clear glass with fluoropolymer-lined cap.

6.4.3.3 Mechanical shaker—Wrist-action or platform-type rotary shaker that produces vigorous agitation (Sybron Thermolyne Model LE “Big Bill” rotator/shaker, or equivalent).

6.4.3.4 Rack attached to shaker table to permit agitation of four to nine samples simultaneously.

6.4.4 Beakers—400–500 mL.

6.4.5 Spatulas—Stainless steel.

6.5 Filtration Apparatus.

6.5.1 Pyrex glass wool—Solvent-extracted by SDS for three hours minimum.

Note: Baking glass wool may cause active sites that will irreversibly adsorb CDDs/CDFs.

6.5.2 Glass funnel—125–250 mL.

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

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

6.5.5 Buchner funnel—15 cm.

6.5.6 Glass-fiber filter paper—to fit Buchner funnel in Section 6.5.5.

6.5.7 Filtration flasks—1.5–2.0 L, with side arm.

6.5.8 Pressure filtration apparatus—Millipore YT30 142 HW, or equivalent.

6.6 Centrifuge Apparatus.

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

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

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

6.7 Cleanup Apparatus.

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

6.7.1.1 Column—600–700 mm long × 25 mm ID, packed with 70 g of

SX–3 Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).

6.7.1.2 Syringe—10 mL, with Luer fitting.

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

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

6.7.2 Reverse-phase high-performance liquid chromatograph.

6.7.2.1 Column oven and detector—Perkin-Elmer Model LC–65T (or equivalent) operated at 0.02 AUFS at 235 nm.

6.7.2.2 Injector—Rheodyne 7120 (or equivalent) with 50 µL sample loop.

6.7.2.3 Column—Two 6.2 mm × 250 mm Zorbax-ODS columns in series (DuPont Instruments Division, Wilmington, DE, or equivalent), operated at 50 °C with 2.0 mL/min methanol isocratic effluent.

6.7.2.4 Pump—Altex 110A (or equivalent).

6.7.3 Pipets.

6.7.3.1 Disposable, pasteur—150 mm long × 5-mm ID (Fisher Scientific 13–678–6A, or equivalent).

6.7.3.2 Disposable, serological—10 mL (6 mm ID).

6.7.4 Glass chromatographic columns.

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

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

6.7.4.3 300 mm long × 25 mm ID, with 300 mL reservoir and glass or fluoropolymer stopcock.

6.7.5 Stirring apparatus for batch silica cleanup of tissue extracts.

6.7.5.1 Mechanical stirrer—Corning Model 320, or equivalent.

6.7.5.2 Bottle—500–600 mL wide-mouth clear glass.

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

6.8 Concentration Apparatus.

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

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

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

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

6.8.2 Kuderna-Danish (K-D) Concentrator.

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

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

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

6.8.2.4 Boiling chips.

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

6.8.2.4.2 Fluoropolymer (optional)—Extracted with methylene chloride.

6.8.2.5 Water bath—Heated, with concentric ring cover, capable of maintaining a temperature within ±2 °C, installed in a fume hood.

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

6.8.4 Sample vials.

6.8.4.1 Amber glass—2–5 mL with fluoropolymer-lined screw-cap.

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

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

6.9.1 GC column for CDDs/CDFs and for isomer specificity for 2,3,7,8-TCDD—60 ±5 m long × 0.32 ±0.02 mm ID; 0.25 µm 5% phenyl, 94% methyl, 1% vinyl silicone bonded-phase fused-silica capillary column (J&W DB–5, or equivalent).

6.9.2 GC column for isomer specificity for 2,3,7,8-TCDF—30 ±5 m long × 0.32 ±0.02 mm ID; 0.25 µm bonded-phase fused-silica capillary column (J&W DB–225, or equivalent).

6.10 Mass Spectrometer—28–40 eV electron impact ionization, shall be capable of repetitively selectively monitoring 12 exact m/z's minimum at high resolution (=10,000) during a period of approximately one second, and shall meet all of the performance specifications in Section 10.

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

6.12 Data System—Capable of collecting, recording, and storing MS data.

7.0 Reagents and Standards

7.1 pH Adjustment and Back-Extraction.

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

7.1.2 Sulfuric acid—Reagent grade (specific gravity 1.84).

7.1.3 Hydrochloric acid—Reagent grade, 6N.

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

7.2 Solution Drying and Evaporation.

7.2.1 Solution drying—Sodium sulfate, reagent grade, granular, anhydrous (Baker 3375, or equivalent), rinsed with methylene chloride (20 mL/g), baked at 400 °C for one hour minimum, cooled in a dessicator, and stored in a pre-cleaned glass bottle with screw-cap that prevents moisture from entering. If, after heating, the sodium sulfate develops a noticeable grayish cast (due to the presence of carbon in the crystal matrix), that batch of reagent is not suitable for use and should be discarded. Extraction with methylene chloride (as opposed to simple rinsing) and baking at a lower temperature may produce sodium sulfate that is suitable for use.

7.2.2 Tissue drying—Sodium sulfate, reagent grade, powdered, treated and stored as above.

7.2.3 Prepurified nitrogen.

7.3 Extraction.

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

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

7.4 GPC Calibration Solution—Prepare a solution containing 300 mg/mL corn oil, 15 mg/mL bis(2-ethylhexyl) phthalate, 1.4 mg/mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.

7.5 Adsorbents for Sample Cleanup.

7.5.1 Silica gel.

7.5.1.1 Activated silica gel—100–200 mesh, Supelco 1–3651 (or equivalent), rinsed with methylene chloride, baked at 180 °C for a minimum of one hour, cooled in a dessicator, and stored in a precleaned glass bottle with screw-cap that prevents moisture from entering.

7.5.1.2 Acid silica gel (30% w/w)—Thoroughly mix 44.0 g of concentrated sulfuric acid with 100.0 g of activated silica gel in a clean container. Break up aggregates with a stirring rod until a uniform mixture is obtained. Store in a bottle with a fluoropolymer-lined screw-cap.

7.5.1.3 Basic silica gel—Thoroughly mix 30 g of 1N sodium hydroxide with 100 g of activated silica gel in a clean container. Break up aggregates with a stirring rod until a uniform mixture is obtained. Store in a bottle with a fluoropolymer-lined screw-cap.

7.5.1.4 Potassium silicate.

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

7.5.1.4.2 Add 100 g of silica gel and a stirring bar, and stir on a hot plate at 60–70 °C for one to two hours.

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

7.5.1.4.4 Spread the potassium silicate on solvent-rinsed aluminum foil and dry for two to four hours in a hood.

7.5.1.4.5 Activate overnight at 200–250 °C.

7.5.2 Alumina—Either one of two types of alumina, acid or basic, may be used in the cleanup of sample extracts, provided that the laboratory can meet the performance specifications for the recovery of labeled compounds described in Section 9.3. The same type of alumina must be used for all samples, including those used to demonstrate initial precision and recovery (Section 9.2) and ongoing precision and recovery (Section 15.5).

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

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

7.5.3 Carbon.

7.5.3.1 Carbopak C—(Supelco 1–0258, or equivalent).

7.5.3.2 Celite 545—(Supelco 2–0199, or equivalent).

7.5.3.3 Thoroughly mix 9.0 g Carbopak C and 41.0 g Celite 545 to produce an 18% w/w mixture. Activate the mixture at 130 °C for a minimum of six hours. Store in a dessicator.

7.5.4 Anthropogenic isolation column—Pack the column in Section 6.7.4.3 from bottom to top with the following:

7.5.4.1 2 g silica gel (Section 7.5.1.1).

7.5.4.2 2 g potassium silicate (Section 7.5.1.4).

7.5.4.3 2 g granular anhydrous sodium sulfate (Section 7.2.1).

7.5.4.4 10 g acid silica gel (Section 7.5.1.2).

7.5.4.5 2 g granular anhydrous sodium sulfate.

7.5.5 Florisil column.

7.5.5.1 Florisil—60–100 mesh, Floridin Corp (or equivalent). Soxhlet extract in 500 g portions for 24 hours.

7.5.5.2 Insert a glass wool plug into the tapered end of a graduated serological pipet (Section 6.7.3.2). Pack with 1.5 g (approx 2 mL) of Florisil topped with approx 1 mL of sodium sulfate (Section 7.2.1) and a glass wool plug.

7.5.5.3 Activate in an oven at 130–150 °C for a minimum of 24 hours and cool for 30 minutes. Use within 90 minutes of cooling.

7.6 Reference Matrices—Matrices in which the CDDs/CDFs and interfering compounds are not detected by this method.

7.6.1 Reagent water—Bottled water purchased locally, or prepared by passage through activated carbon.

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

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

7.6.4 Tissue reference matrix—Corn or other vegetable oil. May be prepared by extraction with methylene chloride.

7.6.5 Other matrices—This method may be verified on any reference matrix by performing the tests given in Section 9.2. Ideally, the matrix should be free of the CDDs/CDFs, but in no case shall the background level of the CDDs/CDFs in the reference matrix exceed three times the minimum levels in Table 2. If low background levels of the CDDs/CDFs are present in the reference matrix, the spike level of the analytes used in Section 9.2 should be increased to provide a spike-to-background ratio in the range of 1:1 to 5:1 (Reference 15).

7.7 Standard Solutions—Purchased as solutions or mixtures with certification to their purity, concentration, and authenticity, or prepared from materials of known purity and composition. If the chemical purity is 98% or greater, the weight may be used without correction to compute the concentration of the standard. When not being used, standards are stored in the dark at room temperature in screw-capped vials with fluoropolymer-lined caps. A mark is placed on the vial at the level of the solution so that solvent loss by evaporation can be detected. If solvent loss has occurred, the solution should be replaced.

7.8 Stock Solutions.

7.8.1 Preparation—Prepare in nonane per the steps below or purchase as dilute solutions (Cambridge Isotope Laboratories (CIL), Woburn, MA, or equivalent). Observe the safety precautions in Section 5, and the recommendation in Section 5.1.2.

7.8.2 Dissolve an appropriate amount of assayed reference material in solvent. For example, weigh 1–2 mg of 2,3,7,8-TCDD to three significant figures in a 10 mL ground-glass-stoppered volumetric flask and fill to the mark with nonane. After the TCDD is completely dissolved, transfer the solution to a clean 15 mL vial with fluoropolymer-lined cap.

7.8.3 Stock standard solutions should be checked for signs of degradation prior to the preparation of calibration or performance test standards. Reference standards that can be used to determine the accuracy of calibration standards are available from CIL and may be available from other vendors.

7.9 PAR Stock Solution

7.9.1 All CDDs/CDFs—Using the solutions in Section 7.8, prepare the PAR stock solution to contain the CDDs/CDFs at the concentrations shown in Table 3. When diluted, the solution will become the PAR (Section 7.14).

7.9.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the PAR stock solution to contain these compounds only.

7.10 Labeled-Compound Spiking Solution.

7.10.1 All CDDs/CDFs—From stock solutions, or from purchased mixtures, prepare this solution to contain the labeled compounds in nonane at the concentrations shown in Table 3. This solution is diluted with acetone prior to use (Section 7.10.3).

7.10.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the labeled-compound solution to contain these compounds only. This solution is diluted with acetone prior to use (Section 7.10.3).

7.10.3 Dilute a sufficient volume of the labeled compound solution (Section 7.10.1 or 7.10.2) by a factor of 50 with acetone to prepare a diluted spiking solution. Each sample requires 1.0 mL of the diluted solution, but no more solution should be prepared than can be used in one day.

7.11 Cleanup Standard—Prepare 37Cl4-2,3,7,8-TCDD in nonane at the concentration shown in Table 3. The cleanup standard is added to all extracts prior to cleanup to measure the efficiency of the cleanup process.

7.12 Internal Standard(s).

7.12.1 All CDDs/CDFs—Prepare the internal standard solution to contain 13C12-1,2,3,4-TCDD and 13C2-1,2,3,7,8,9-HxCDD in nonane at the concentration shown in Table 3.

7.12.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the internal standard solution to contain 13C12–1,2,3,4-TCDD only.

7.13 Calibration Standards (CS1 through CS5)—Combine the solutions in Sections 7.9 through 7.12 to produce the five calibration solutions shown in Table 4 in nonane. These solutions permit the relative response (labeled to native) and response factor to be measured as a function of concentration. The CS3 standard is used for calibration verification (VER). If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, combine the solutions appropriate to these compounds.

7.14 Precision and Recovery (PAR) Standard—Used for determination of initial (Section 9.2) and ongoing (Section 15.5) precision and recovery. Dilute 10 µL of the precision and recovery standard (Section 7.9.1 or 7.9.2) to 2.0 mL with acetone for each sample matrix for each sample batch. One mL each are required for the blank and OPR with each matrix in each batch.

7.15 GC Retention Time Window Defining Solution and Isomer Specificity Test Standard—Used to define the beginning and ending retention times for the dioxin and furan isomers and to demonstrate isomer specificity of the GC columns employed for determination of 2,3,7,8-TCDD and 2,3,7,8-TCDF. The standard must contain the compounds listed in Table 5 (CIL EDF—4006, or equivalent), at a minimum. It is not necessary to monitor the window-defining compounds if only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined. In this case, an isomer-specificity test standard containing the most closely eluted isomers listed in Table 5 (CIL EDF-4033, or equivalent) may be used.

7.16 QC Check Sample—A QC Check Sample should be obtained from a source independent of the calibration standards. Ideally, this check sample would be a certified reference material containing the CDDs/CDFs in known concentrations in a sample matrix similar to the matrix under test.

7.17 Stability of Solutions—Standard solutions used for quantitative purposes (Sections 7.9 through 7.15) should be analyzed periodically, and should be assayed against reference standards (Section 7.8.3) before further use.

8.0 Sample Collection, Preservation, Storage, and Holding Times

8.1 Collect samples in amber glass containers following conventional sampling practices (Reference 16). Aqueous samples that flow freely are collected in refrigerated bottles using automatic sampling equipment. Solid samples are collected as grab samples using wide-mouth jars.

8.2 Maintain aqueous samples in the dark at 0–4 °C from the time of collection until receipt at the laboratory. If residual chlorine is present in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 17). If sample pH is greater than 9, adjust to pH 7–9 with sulfuric acid.

Maintain solid, semi-solid, oily, and mixed-phase samples in the dark at <4 °C from the time of collection until receipt at the laboratory.

Store aqueous samples in the dark at 0–4 °C. Store solid, semi-solid, oily, mixed-phase, and tissue samples in the dark at <-10 °C.

8.3 Fish and Tissue Samples.

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

8.3.2 Fish collected in the field should be wrapped in aluminum foil, and must be maintained at a temperature less than 4 °C from the time of collection until receipt at the laboratory.

8.3.3 Samples must be frozen upon receipt at the laboratory and maintained in the dark at <-10 °C until prepared. Maintain unused sample in the dark at <-10 °C.

8.4 Holding Times.

8.4.1 There are no demonstrated maximum holding times associated with CDDs/CDFs in aqueous, solid, semi-solid, tissues, or other sample matrices. If stored in the dark at 0–4 °C and preserved as given above (if required), aqueous samples may be stored for up to one year. Similarly, if stored in the dark at <-10 °C, solid, semi-solid, multi-phase, and tissue samples may be stored for up to one year.

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

9.0 Quality Assurance/Quality Control

9.1 Each laboratory that uses this method is required to operate a formal quality assurance program (Reference 18). The minimum requirements of this program consist of an initial demonstration of laboratory capability, analysis of samples spiked with labeled compounds to evaluate and document data quality, and analysis of standards and blanks as tests of continued performance. Laboratory performance is compared to established performance criteria to determine if the results of analyses meet the performance characteristics of the method.

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

9.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 9.2.

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

9.1.2.1 Each time a modification is made to this method, the analyst is required to repeat the procedure in Section 9.2. If the detection limit of the method will be affected by the change, the laboratory is required to demonstrate that the MDL (40 CFR Part 136, Appendix B) is lower than one-third the regulatory compliance level or one-third the ML in this method, whichever is higher. If calibration will be affected by the change, the analyst must recalibrate the instrument per Section 10.

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

9.1.2.2.1 The names, titles, addresses, and telephone numbers of the analyst(s) who performed the analyses and modification, and of the quality control officer who witnessed and will verify the analyses and modifications.

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

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

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

(a) Calibration (Section 10.5 through 10.7).

(b) Calibration verification (Section 15.3).

(c) Initial precision and recovery (Section 9.2).

(d) Labeled compound recovery (Section 9.3).

(e) Analysis of blanks (Section 9.5).

(f) Accuracy assessment (Section 9.4).

9.1.2.2.5 Data that will allow an independent reviewer to validate each determination by tracing the instrument output (peak height, area, or other signal) to the final result. These data are to include:

(a) Sample numbers and other identifiers.

(b) Extraction dates.

(c) Analysis dates and times.

(d) Analysis sequence/run chronology.

(e) Sample weight or volume (Section 11).

(f) Extract volume prior to each cleanup step (Section 13).

(g) Extract volume after each cleanup step (Section 13).

(h) Final extract volume prior to injection (Section 14).

(i) Injection volume (Section 14.3).

(j) Dilution data, differentiating between dilution of a sample or extract (Section 17.5).

(k) Instrument and operating conditions.

(l) Column (dimensions, liquid phase, solid support, film thickness, etc).

(m) Operating conditions (temperatures, temperature program, flow rates).

(n) Detector (type, operating conditions, etc).

(o) Chromatograms, printer tapes, and other recordings of raw data.

(p) Quantitation reports, data system outputs, and other data to link the raw data to the results reported.

9.1.3 Analyses of method blanks are required to demonstrate freedom from contamination (Section 4.3). The procedures and criteria for analysis of a method blank are described in Sections 9.5 and 15.6.

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

9.1.5 The laboratory shall, on an ongoing basis, demonstrate through calibration verification and the analysis of the ongoing precision and recovery aliquot that the analytical system is in control. These procedures are described in Sections 15.1 through 15.5.

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

9.2 Initial Precision and Recovery (IPR)—To establish the ability to generate acceptable precision and recovery, the analyst shall perform the following operations.

9.2.1 For low solids (aqueous) samples, extract, concentrate, and analyze four 1 L aliquots of reagent water spiked with the diluted labeled compound spiking solution (Section 7.10.3) and the precision and recovery standard (Section 7.14) according to the procedures in Sections 11 through 18. For an alternative sample matrix, four aliquots of the alternative reference matrix (Section 7.6) are used. All sample processing steps that are to be used for processing samples, including preparation (Section 11), extraction (Section 12), and cleanup (Section 13), shall be included in this test.

9.2.2 Using results of the set of four analyses, compute the average concentration (X) of the extracts in ng/mL and the standard deviation of the concentration (s) in ng/mL for each compound, by isotope dilution for CDDs/CDFs with a labeled analog, and by internal standard for 1,2,3,7,8,9-HxCDD, OCDF, and the labeled compounds.

9.2.3 For each CDD/CDF and labeled compound, compare s and X with the corresponding limits for initial precision and recovery in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare s and X with the corresponding limits for initial precision and recovery in Table 6a. If s and X for all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If, however, any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, system performance is unacceptable for that compound. Correct the problem and repeat the test (Section 9.2).

9.3 The laboratory shall spike all samples with the diluted labeled compound spiking solution (Section 7.10.3) to assess method performance on the sample matrix.

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

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

9.3.3 The recovery of each labeled compound must be within the limits in Table 7 when all 2,3,7,8-substituted CDDs/CDFs are determined, and within the limits in Table 7a when only 2,3,7,8-TCDD and 2,3,7,8-TCDF are determined. If the recovery of any compound falls outside of these limits, method performance is unacceptable for that compound in that sample. To overcome such difficulties, water samples are diluted and smaller amounts of soils, sludges, sediments, and other matrices are reanalyzed per Section 18.4.

9.4 Recovery of labeled compounds from samples should be assessed and records should be maintained.

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

9.4.2 Update the accuracy assessment for each labeled compound in each matrix on a regular basis (e.g., after each 5–10 new measurements).

9.5 Method Blanks—Reference matrix method blanks are analyzed to demonstrate freedom from contamination (Section 4.3).

9.5.1 Prepare, extract, clean up, and concentrate a method blank with each sample batch (samples of the same matrix started through the extraction process on the same 12-hour shift, to a maximum of 20 samples). The matrix for the method blank shall be similar to sample matrix for the batch, e.g., a 1 L reagent water blank (Section 7.6.1), high-solids reference matrix blank (Section 7.6.2), paper matrix blank (Section 7.6.3); tissue blank (Section 7.6.4) or alternative reference matrix blank (Section 7.6.5). Analyze the blank immediately after analysis of the OPR (Section 15.5) to demonstrate freedom from contamination.

9.5.2 If any 2,3,7,8-substituted CDD/CDF (Table 1) is found in the blank at greater than the minimum level (Table 2) or one-third the regulatory compliance level, whichever is greater; or if any potentially interfering compound is found in the blank at the minimum level for each level of chlorination given in Table 2 (assuming a response factor of 1 relative to the 13C12-1,2,3,4-TCDD internal standard for compounds not listed in Table 1), analysis of samples is halted until the blank associated with the sample batch shows no evidence of contamination at this level. All samples must be associated with an uncontaminated method blank before the results for those samples may be reported for regulatory compliance purposes.

9.6 QC Check Sample—Analyze the QC Check Sample (Section 7.16) periodically to assure the accuracy of calibration standards and the overall reliability of the analytical process. It is suggested that the QC Check Sample be analyzed at least quarterly.

9.7 The specifications contained in this method can be met if the apparatus used is calibrated properly and then maintained in a calibrated state. The standards used for calibration (Section 10), calibration verification (Section 15.3), and for initial (Section 9.2) and ongoing (Section 15.5) precision and recovery should be identical, so that the most precise results will be obtained. A GC/MS instrument will provide the most reproducible results if dedicated to the settings and conditions required for the analyses of CDDs/CDFs by this method.

9.8 Depending on specific program requirements, field replicates may be collected to determine the precision of the sampling technique, and spiked samples may be required to determine the accuracy of the analysis when the internal standard method is used.

10.0 Calibration

10.1 Establish the operating conditions necessary to meet the minimum retention times for the internal standards in Section 10.2.4 and the relative retention times for the CDDs/CDFs in Table 2.

10.1.1 Suggested GC operating conditions:

Injector temperature: 270 °C

Interface temperature: 290 °C

Initial temperature: 200 °C

Initial time: Two minutes

Temperature program:

200–220 °C, at 5 °C/minute

220 °C for 16 minutes

220–235 °C, at 5 °C/minute

235 °C for seven minutes

235–330 °C, at 5 °C/minute

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

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

10.1.2 Mass spectrometer (MS) resolution—Obtain a selected ion current profile (SICP) of each analyte in Table 3 at the two exact m/z's specified in Table 8 and at =10,000 resolving power by injecting an authentic standard of the CDDs/CDFs either singly or as part of a mixture in which there is no interference between closely eluted components.

10.1.2.1 The analysis time for CDDs/CDFs may exceed the long-term mass stability of the mass spectrometer. Because the instrument is operated in the high-resolution mode, mass drifts of a few ppm (e.g., 5 ppm in mass) can have serious adverse effects on instrument performance. Therefore, a mass-drift correction is mandatory and a lock-mass m/z from PFK is used for drift correction. The lock-mass m/z is dependent on the exact m/z's monitored within each descriptor, as shown in Table 8. The level of PFK metered into the HRMS during analyses should be adjusted so that the amplitude of the most intense selected lock-mass m/z signal (regardless of the descriptor number) does not exceed 10% of the full-scale deflection for a given set of detector parameters. Under those conditions, sensitivity changes that might occur during the analysis can be more effectively monitored.

Note: Excessive PFK (or any other reference substance) may cause noise problems and contamination of the ion source necessitating increased frequency of source cleaning.

10.1.2.2 If the HRMS has the capability to monitor resolution during the analysis, it is acceptable to terminate the analysis when the resolution falls below 10,000 to save reanalysis time.

10.1.2.3 Using a PFK molecular leak, tune the instrument to meet the minimum required resolving power of 10,000 (10% valley) at m/z 304.9824 (PFK) or any other reference signal close to m/z 304 (from TCDF). For each descriptor (Table 8), monitor and record the resolution and exact m/z's of three to five reference peaks covering the mass range of the descriptor. The resolution must be greater than or equal to 10,000, and the deviation between the exact m/z and the theoretical m/z (Table 8) for each exact m/z monitored must be less than 5 ppm. (continued)