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8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none by (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.
8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. In necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (A-B)%/T, where T is the known true value of the spike.
8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1. 7 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X') using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S') using the equation in Table 3, substituting X' for X ; (3) calculate the range for recovery at the spike concentration as (100 X'/T) ±2.44 (100 S'/T)%. 7
8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.
8.4. If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.
Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.
8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Sections 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.
8.4.2 Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.
8.4.3 Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.
8.5 As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P ) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P-2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70–110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevent performance evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional sampling practices8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.
9.2 All samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2–L separatory funnel.
10.2 Add 60 mL of methylele chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 to 2 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.
Note: The dichloribenzenes have a sufficiently high volatility that significant losses may occur in concentration steps if care is not exercised. It is important to maintain a constant gentle evaporation rate and not to allow the liquid volume to fall below 1 to 2 mL before removing the K-D apparatus from the hot water bath.
10.7 Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the tempeature of the water bath to 85 to 90 °C. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.
10.8 Romove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.
11.2 Florisil column cleanup for chlorinated hydrocarbons:
11.2.1 Adjust the sample extract to 10 mL with hexane.
11.2.2 Place 12 g of Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.
11.2.3 Preelute the column with 100 mL of petroleum ether. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column by decantation and subsequent petroleum ether washings. Discard the eluate. Just prior to exposure of the sodium sulfate layer to the air, begin eluting the column with 200 mL of petroleum ether and collect the eluate in a 500-mL K-D flask equipped with a 10-mL concentrator tube. This fraction should contain all of the chlorinated hydrocarbons.
11.2.4 Concentrate the fraction as in Section 10.6, except use hexane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Analyze by gas chromatography (Section 12).
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Columl 2 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed throughly immediately before injection into the gas chromatograph.
12.4 Inject 2 to 5 µL of the sample extract or standard into the gas chromatograph using the solvent-flush techlique.9 Smaller (1.0 µL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 µL, the total extract volume, and the resulting peak size in area or peak height units.
12.5 Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.
12.6 If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.
Equation 2
where:
A=Amount of material injected (ng).
Vi=Volume of extract injected (µL).
Vt=Volume of total extract (µL).
Vs=Volume of water extracted (mL).
13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.
Equation 3
where:
As=Response for the parameter to be measured.
Ais=Response for the internal standard.
Is=Amount of internal standard added to each extract (µg).
Vo=Volume of water extracted (L).
13.2 Report results in µg/L without correction for recovery data. All QC data obtained should be reported with the sample results.
14. Method Performance
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.
14.2 This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4×MDL to 1000×MDL.10
14.3 This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 356 µg/L.11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.
References
1. 40 CFR part 136, appendix B.
2. “Determination of Chlorinated Hydrocarbons In Industrial and Municipal Wastewaters, “EPA 6090/4–84–ABC, National Technical Information Service, PBXYZ, Springfield, Virginia, 22161 November 1984.
3. ASTM Annual Book of Standards, Part 31, D3694–78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.
4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77–206, August 1977.
5. “OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).
6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.
7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,”American Laboratory, 15, 58–63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)
8. ASTM Annual Book of Standards, Part 31, D3370–76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.
9. Burke, J.A. “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
10. “Development of Detection Limits, EPA Method 612, Chlorinated Hydrocarbons,” Special letter report for EPA Contract 68–03–2625, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
11. “EPA Method Study Method 612—Chlorinated Hydrocarbons,” EPA 600/4–84–039, National Technical Information Service, PB84–187772, Springfield, Virginia 22161, May 1984.
12. “Method Performance for Hexachlorocyclopentadiene by Method 612,” Memorandum from R. Slater, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, December 7, 1983.
Table 1_Chromatographic Conditions and Method Detection Limits
------------------------------------------------------------------------
Retention time (min) Method
-------------------------- detection
Parameter limit
Column 1 Column 2 (µg/
L)
------------------------------------------------------------------------
1,3-Dichlorobenzene.............. 4.5 6.8 1.19
Hexachloroethane................. 4.9 8.3 0.03
1,4-Dichlorobenzene.............. 5.2 7.6 1.34
1,2-Dichlorobenzene.............. 6.6 9.3 1.14
Hexachlorobutadiene.............. 7.7 20.0 0.34
1,2,4-Trichlorobenzene........... 15.5 22.3 0.05
Hexachlorocyclopentadiene........ nd c 16.5 0.40
2-Chloronaphthalene.............. a 2.7 b 3.6 0.94
Hexachlorobenzene................ a 5.6 b 10.1 0.05
------------------------------------------------------------------------
Column 1 conditions: Supelcoport (100/120 mesh) coated with 1% SP-1000
packed in a 1.8 m x 2 mm ID glass column with 5% methane/95% argon
carrier gas at 25 mL/min. flow rate. Column temperature held
isothermal at 65 °C, except where otherwise indicated.
Column 2 conditions: Supelcoport (80/100 mesh) coated with 1.5% OV-1/
2.4% OV-225 packed in a 1.8 m x 2 mm ID glass column with 5% methane/
95% argon carrier gas at 25 mL/min. flow rate. Column temperature held
isothermal at 75 °C, except where otherwise indicated.
nd=Not determined.
a 150 °C column temperature.
b 165 °C column temperature.
c 100 °C column temperature.
Table 2_QC Acceptance Criteria_Method 612
----------------------------------------------------------------------------------------------------------------
Limit for Range for X
Test conc. s (µg/ Range for
Parameter (µg/ (µg/ L) P, Ps
L) L) (percent)
----------------------------------------------------------------------------------------------------------------
2-Chloronaphthalene............................................. 100 37.3 29.5-126.9 9-148
1,2-Dichlorobenzene............................................. 100 28.3 23.5-145.1 9-160
1,3-Dichlorobenzene............................................. 100 26.4 7.2-138.6 D-150
1,4-Dichlorobenzene............................................. 100 20.8 22.7-126.9 13-137
Hexachlorobenzene............................................... 10 2.4 2.6-14.8 15-159
Hexachlorobutadiene............................................. 10 2.2 D-12.7 D-139
Hexachlorocyclopentadiene....................................... 10 2.5 D-10.4 D-111
Hexachloroethane................................................ 10 3.3 2.4-12.3 8-139
1,2,4-Trichlorobenzene.......................................... 100 31.6 20.2-133.7 5-149
----------------------------------------------------------------------------------------------------------------
s=Standard deviation of four recovery measurements, in µg/L (Section 8.2.4).
X=Average recovery for four recovery measurements, in µg/L (Section 8.2.4).
P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).
D=Detected; result must be greater than zero.
Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits
for recovery have been broadened to assure applicability of the limits to concentrations below those used to
develop Table 3.
Table 3_Method Accuracy and Precision as Functions of Concentration_Method 612
----------------------------------------------------------------------------------------------------------------
Single analyst
Parameter Acccuracy, as recovery, precision, sr[prime] Overall precision,
X[prime] (µg/L) (µg/L) S[prime] (µg/L)
----------------------------------------------------------------------------------------------------------------
2-Chloronaphthalene................... 0.75C+3.21 0.28X-1.17 0.38X-1.39
1,2-Dichlorobenzene................... 0.85C-0.70 0.22X-2.95 0.41X-3.92
1,3-Dichlorobenzene................... 0.72C+0.87 0.21X-1.03 0.49X-3.98
1,4-Dichlorobenzene................... 0.72C+2.80 0.16X-0.48 0.35X-0.57
Hexachlorobenzene..................... 0.87C-0.02 0.14X+0.07 0.36X-0.19
Hexachlorobutadiene................... 0.61C+0.03 0.18X+0.08 0.53X-0.12
Hexachlorocyclopentadiene a........... 0.47C 0.24X 0.50X
Hexachloroethane...................... 0.74C-0.02 0.23X+0.07 0.36X-0.00
1,2,4-Trichlorobenzene................ 0.76C+0.98 0.23X-0.44 0.40X-1.37
----------------------------------------------------------------------------------------------------------------
X[prime]=Expected recovery for one or more measurements of a sample containing a concentration of C, in µg/
L.
sr[prime]=Expected single analyst standard deviation of measurements at an average concentration found of X, in
µg/L.
S[prime]=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in
µg/L.
C=True value for the concentration, in µg/L.
X=Average recovery found for measurements of samples containing a concentration of C, in µg/L.
a Estimates based upon the performance in a single laboratory. \12\
View or download PDF
View or download PDF
Method 613—2,3,7,8-Tetrachlorodibenzo-p-Dioxin
1. Scope and Application
1.1 This method covers the determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The following parameter may be determined by this method:
------------------------------------------------------------------------
STORET
Parameter No. GAS No.
------------------------------------------------------------------------
2,3,7,8-TCDD..................................... 34675 1746-01-6
------------------------------------------------------------------------
1.2 This is a gas chromatographic/mass spectrometer (GC/MS) method applicable to the determination of 2,3,7,8–TCDD in municipal and industrial discharges as provided under 40 CFR 136.1. Method 625 may be used to screen samples for 2,3,7,8–TCDD. When the screening test is positive, the final qualitative confirmation and quantification must be made using Method 613.
1.3 The method detection limit (MDL, defined in Section 14.1) 1 for 2,3,7,8–TCDD is listed in Table 1. The MDL for a specific wastewater may be different from that listed, depending upon the nature of interferences in the sample matrix.
1.4 Because of the extreme toxicity of this compound, the analyst must prevent exposure to himself, of to others, by materials knows or believed to contain 2,3,7,8–TCDD. Section 4 of this method contains guidelines and protocols that serve as minimum safe-handling standards in a limited-access laboratory.
1.5 Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.
1.6 This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.
2. Summary of Method
2.1 A measured volume of sample, approximately 1–L, is spiked with an internal standard of labeled 2,3,7,8–TCDD and extracted with methylene chloride using a separatory funnel. The methylene chloride extract is exchanged to hexane during concentration to a volume of 1.0 mL or less. The extract is then analyzed by capillary column GC/MS to separate and measure 2,3,7,8–TCDD. 2,3
2.2 The method provides selected column chromatographic cleanup proceudres to aid in the elimination of interferences that may be encountered.
3. Interferences
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated backgrounds at the masses (m/z) monitored. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.
3.1.1 Glassware must be scrupulously cleaned. 4 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by the treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.
3.1.2 The use of high purity reagents and solvents helps to mininmize interference problems. Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. 2,3,7,8–TCDD is often associated with other interfering chlorinated compounds which are at concentrations several magnitudes higher than that of 2,3,7,8–TCDD. The cleanup producers in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches1,5–7 to eliminate false positives and achieve the MDL listed in Table 1.
3.3 The primary column, SP–2330 or equivalent, resolves 2,3,7,8–TCDD from the other 21 TCDD insomers. Positive results using any other gas chromatographic column must be confirmed using the primary column.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. 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 data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified8–10 for the information of the analyst. Benzene and 2,3,7,8–TCDD have been identified as suspected human or mammalian carcinogens.
4.2 Each laboratory must develop a strict safety program for handling 2,3,7,8–TCDD. The following laboratory practices are recommended:
4.2.1 Contamination of the laboratory will be minimized by conducting all manipulations in a hood.
4.2.2 The effluents of sample splitters for the gas chromatograph and roughing pumps on the GC/MS should pass through either a column of activated charcoal or be bubbled through a trap containing oil or high-boiling alcohols.
4.2.3 Liquid waste should be dissolved in methanol or ethanol and irradiated with ultraviolet light with a wavelength greater than 290 nm for several days. (Use F 40 BL lamps or equivalent). Analyze liquid wastes and dispose of the solutions when 2,3,7,8–TCDD can no longer be detected.
4.3 Dow Chemical U.S.A. has issued the following precautimns (revised November 1978) for safe handling of 2,3,7,8–TCDD in the laboratory:
4.3.1 The following statements on safe handling are as complete as possible on the basis of available toxicological information. The precautions for safe handling and use are necessarily general in nature since detailed, specific recommendations can be made only for the particular exposure and circumstances of each individual use. Inquiries about specific operations or uses may be addressed to the Dow Chemical Company. Assistance in evaluating the health hazards of particular plant conditions may be obtained from certain consulting laboratories and from State Departments of Health or of Labor, many of which have an industrial health service. 2,3,7,8–TCDD is extremely toxic to laboratory animals. However, it has been handled for years without injury in analytical and biological laboratories. Techniques used in handling radioactive and infectious materials are applicable to 2,3,7,8,–TCDD.
4.3.1.1 Protective equipment—Throw-away plastic gloves, apron or lab coat, safety glasses, and a lab hood adequate for radioactive work.
4.3.1.2 Training—Workers must be trained in the proper method of removing contaminated gloves and clothing without contacting the exterior surfaces.
4.3.1.3 Personal hygiene—Thorough washing of hands and forearms after each manipulation and before breaks (coffee, lunch, and shift).
4.3.1.4 Confinement—Isolated work area, posted with signs, segregated glassware and tools, plastic-backed absorbent paper on benchtops.
4.3.1.5 Waste—Good technique includes minimizing contaminated waste. Plastic bag liners should be used in waste cans. Janitors must be trained in the safe handling of waste.
4.3.1.6 Disposal of wastes—2,3,7,8–TCDD decomposes above 800 °C. Low-level waste such as absorbent paper, tissues, animal remains, and plastic gloves may be burned in a good incinerator. Gross quantities (milligrams) should be packaged securely and disposed through commercial or governmental channels which are capable of handling high-level radioactive wastes or extremely toxic wastes. Liquids should be allowed to evaporate in a good hood and in a disposable container. Residues may then be handled as above.
4.3.1.7 Decontamination—For personal decontamination, use any mild soap with plenty of scrubbing action. For decontamination of glassware, tools, and surfaces, Chlorothene NU Solvent (Trademark of the Dow Chemical Company) 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. Dishwater may be disposed to the sewer. It is prudent to minimize solvent wastes because they may require special disposal through commercial sources which are expensive.
4.3.1.8 Laundry—Clothing known to be contaminated should be disposed with the precautions described under Section 4.3.1.6. Lab coats or other clothing worn in 2,3,7,8–TCDD work areas may be laundered.
Clothing 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 the problem. The washer should be run through a cycle before being used again for other clothing.
4.3.1.9 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 gas chromatography can achieve a limit of sensitivity of 0.1 µg per wipe. Less than 1 µg of 2,3,7,8–TCDD per sample indicates acceptable cleanliness; anything higher warrants further cleaning. More than 10 µg on a wipe sample constitutes an acute hazard and requires prompt cleaning before further use of the equipment or work space. A high (>10 µg) 2,3,7,8–TCDD level indicates that unacceptable work practices have been employed in the past.
4.3.1.10 Inhalation—Any procedure that may produce airborne contamination must be done with good ventilation. Gross losses to a 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.
4.3.1.11 Accidents—Remove contaminated clothing immediately, taking precautions not to contaminate skin or other articles. Wash exposed skin vigorously and repeatedly until medical attention is obtained.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle—1–L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.
5.1.3 Clearly label all samples as “POISON” and ship according to U.S. Department of Transportation regulations.
5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):
5.2.1 Separatory funnels—2–L and 125-mL, with Teflon stopcock.
5.2.2 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K–570050–1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.
5.2.3 Evaporative flask, Kuderna-Danish—500–mL (Kontes K–570001–0500 or equivalent). Attach to concentrator tube with springs.
5.2.4 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K–503000–0121 or equivalent).
5.2.5 Snyder column, Kuderna-Danish—Two-ball micro (Kontes K–569001–0219 or equivalent).
5.2.6 Vials—10 to 15–mL, amber glass, with Teflon-lined screw cap.
5.2.7 Chromatographic column—300 mm long × 10 mm ID, with Teflon stopcock and coarse frit filter disc at bottom.
5.2.8 Chromatographic column—400 mm long × 11 mm ID, with Teflon stopcock and coarse frit filter disc at bottom.
5.3 Boiling chips—Approximately 10/40 mesh. Heat to 400 °C for 30 min or Soxhlet extract with methylene chloride.
5.4 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.
5.5 GC/MS system:
5.5.1 Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph and all required accessories including syringes, analytical columns, and gases. The injection port must be designed for capillary columns. Either split, splitless, or on-column injection techniques may be employed, as long as the requirements of Section 7.1.1 are achieved.
5.5.2 Column—60 m long × 0.25 mm ID glass or fused silica, coated with SP–2330 (or equivalent) with a film thickness of 0.2 µm. Any equivalent column must resolve 2, 3, 7, 8–TCDD from the other 21 TCDD isomers.16
5.5.3 Mass spectrometer—Either a low resolution mass spectrometer (LRMS) or a high resolution mass spectrometer (HRMS) may be used. The mass spectrometer must be equipped with a 70 V (nominal) ion source and be capable of aquiring m/z abundance data in real time selected ion monitoring (SIM) for groups of four or more masses.
5.5.4 GC/MS interface—Any GC to MS interface can be used that achieves the requirements of Section 7.1.1. GC to MS interfaces constructed of all glass or glass-lined materials are recommended. Glass surfaces can be deactivated by silanizing with dichlorodimethylsilane. To achieve maximum sensitivity, the exit end of the capillary column should be placed in the ion source. A short piece of fused silica capillary can be used as the interface to overcome problems associated with straightening the exit end of glass capillary columns.
5.5.5 The SIM data acquired during the chromatographic program is defined as the Selected Ion Current Profile (SICP). The SICP can be acquired under computer control or as a real time analog output. If computer control is used, there must be software available to plot the SICP and report peak height or area data for any m/z in the SICP between specified time or scan number limits.
5.6 Balance—Analytical, capable of accurately weighing 0.0001 g.
6. Reagents
6.1 Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of 2, 3, 7, 8–TCDD.
6.2 Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL. Wash the solution with methylene chloride and hexane before use.
6.3 Sodium thiosulfate—(ACS) Granular.
6.4 Sulfuric acid—Concentrated (ACS, sp. gr. 1.84).
6.5 Acetone, methylene chloride, hexane, benzene, ortho-xylene, tetradecane—Pesticide quality or equivalent.
6.6 Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400 °C for 4 h in a shallow tray.
6.7 Alumina—Neutral, 80/200 mesh (Fisher Scientific Co., No. A–540 or equivalent). Before use, activate for 24 h at 130 °C in a foil-covered glass container.
6.8 Silica gel—High purity grade, 100/120 mesh (Fisher Scientific Co., No. S–679 or equivalent).
6.9 Stock standard solutions (1.00 µg/µL)—Stock standard solutimns can be prepared from pure standard materials or purchased as certified solutions. Acetone should be used as the solvent for spiking solutions; ortho-xylene is recommended for calibration standards for split injectors; and tetradecane is recommended for splitless or on-colum injectors. Analyze stock internal standards to verify the absence of native 2,3,7,8–TCDD.
6.9.1 Prepare stock standard solutions of 2,3,7,8–TCDD (mol wt 320) and either 37C14 2,3,7,8–TCDD (mol wt 328) or 13C112 2,3,7,8–TCDD (mol wt 332) in an isolated area by accurately weighing about 0.0100 g of pure material. Dissolve the material in pesticide quality solvent and dilute to volume in a 10-mL volumetric flask. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.
6.9.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store in an isolated refrigerator protected from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards or spiking solutions from them.
6.9.3 Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.
6.10 Internal standard spiking solution (25 ng/mL)—Using stock standard solution, prepare a spiking solution in acetone of either 13Cl12 or 37Cl4 2,3,7,8–TCDD at a concentration of 25 ng/mL. (See Section 10.2)
6.11 Quality control check sample concentrate—See Section 8.2.1.
7. Calibration
7.1 Establish gas chromatograhic operating conditions equivalent to those given in Table 1 and SIM conditions for the mass spectrometer as described in Section 12.2 The GC/MS system must be calibrated using the internal standard technique.
7.1.1 Using stock standards, prepare calibration standards that will allow measurement of relative response factors of at least three concentration ratios of 2,3,7,8–TCDD to internal standard. Each calibration standard must be prepared to contain the internal standard at a concentration of 25 ng/mL. If any interferences are contributed by the internal standard at m/z 320 and 322, its concentration may be reduced in the calibration standards and in the internal standard spiking solution (Section 6.10). One of the calibration standards should contain 2,3,7,8–TCDD at a concentration near, but above, the MDL and the other 2,3,7,8–TCDD concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the GC/MS system.
7.1.2 Using injections of 2 to 5 µL, analyze each calibration standardaccording to Section 12 and tabulate peak height or area response against the concentration of 2,3,7,8–TCDD and internal standard. Calculate response factors (RF) for 2,3,7,8–TCDD using Equation 1.
Equation 1
where:
As=SIM response for 2,3,7,8–TCDD m/z 320.
Ais=SIM response for the internal standard, m/z 332 for 13 C12 2,3,7,8–TCDD m/z 328 for 37Cl4 2,3,7,8–TCDD.
Cis=Concentration of the internal standard (µg/L).
Cs=Concentration of 2,3,7,8–TCDD (µg/L).
If the RF value over the working range is a constant (<10% relative standard deviation, RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.
7.1.3 The working calibration curve or RF must be verified on each working day by the measurement of one or more 2,3,7,8–TCDD calibration standards. If the response for 2,3,7,8–TCDD varies from the predicted response by more than ±15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared.
7.2 Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.
8. Quality Control
8.1 Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.
8.1.1 The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.
8.1.2 In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.5, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2
8.1.3 Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.
8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples with native 2,3,7,8–TCDD to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.
8.1.5 The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.
8.1.6 The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.
8.2.1 A quality control (QC) check sample concentrate is required containing 2,3,7,8–TCDD at a concentration of 0.100 µg/mL in acetone. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.
8.2.2 Using a pipet, prepare QC check samples at a concentration of 0.100 µg/L (100 ng/L) by adding 1.00 mL of QC check sample concentrate to each of four 1–L aliquots of reagent water.
8.2.3 Analyze the well-mixed QC check samples according to the method beginning in Section 10.
8.2.4 Calculate the average recovery (X ) in µg/L, and the standard deviation of the recovery (s) in µg/L, for 2,3,7,8–TCDD using the four results.
8.2.5 Compare s and (X ) with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If s exceeds the precision limit or X falls outside the range for accuracy, the system performance is unacceptable for 2,3,7,8–TCDD. Locate and correct the source of the problem and repeat the test beginning with Section 8.2.2.
8.3 The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.
8.3.1 The concentration of the spike in the sample should be determined as follows:
8.3.1.1 If, as in compliance monitoring, the concentration of 2,3,7,8–TCDD in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.
8.3.1.2 If the concentration of 2,3,7,8–TCDD in the sample is not being checked against a limit specific to that parameter, the spike should be at 0.100 µg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.
8.3.1.3 If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 0.100 µg/L.
8.3.2 Analyze one sample aliquot to determine the background concentration (B) of 2,3,7,8–TCDD. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentration in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of 2,3,7,8–TCDD. Calculate percent recovery (P) as 100(A-B)%T, where T is the known true value of the spike. (continued)