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13.2.3.1 Filter the extract or load through the filter holder (Section 6.7.1.3) to remove the particles. Load the 5.0 mL extract onto the column.

13.2.3.2 Elute the extract using the calibration data determined in Section 13.2.2. Collect the eluate in a clean 400–500 mL beaker.

13.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between extracts to prepare for the next sample.

13.2.3.4 If a particularly dirty extract is encountered, a 5.0 mL methylene chloride blank shall be run through the system to check for carry-over.

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

13.3 Silica Gel Cleanup.

13.3.1 Place a glass-wool plug in a 15 mm ID chromatography column (Section 6.7.4.2). Pack the column bottom to top with: 1 g silica gel (Section 7.5.1.1), 4 g basic silica gel (Section 7.5.1.3), 1 g silica gel, 8 g acid silica gel (Section 7.5.1.2), 2 g silica gel, and 4 g granular anhydrous sodium sulfate (Section 7.2.1). Tap the column to settle the adsorbents.

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

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

13.3.4 Rinse the receiver twice with 1 mL portions of hexane, and apply separately to the column. Elute the CDDs/CDFs with 100 mL hexane, and collect the eluate.

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

13.3.6 For extracts of samples known to contain large quantities of other organic compounds (such as paper mill effluents), it may be advisable to increase the capacity of the silica gel column. This may be accomplished by increasing the strengths of the acid and basic silica gels. The acid silica gel (Section 7.5.1.2) may be increased in strength to as much as 44% w/w (7.9 g sulfuric acid added to 10 g silica gel). The basic silica gel (Section 7.5.1.3) may be increased in strength to as much as 33% w/w (50 mL 1N NaOH added to 100 g silica gel), or the potassium silicate (Section 7.5.1.4) may be used.

Note: The use of stronger acid silica gel (44% w/w) may lead to charring of organic compounds in some extracts. The charred material may retain some of the analytes and lead to lower recoveries of CDDs/CDFs. Increasing the strengths of the acid and basic silica gel may also require different volumes of hexane than those specified above to elute the analytes off the column. Therefore, the performance of the method after such modifications must be verified by the procedure in Section 9.2.

13.4 Alumina Cleanup.

13.4.1 Place a glass-wool plug in a 15 mm ID chromatography column (Section 6.7.4.2).

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

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

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

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

13.4.6 Rinse the receiver twice with 1 mL portions of hexane and apply separately to the column. Elute the interfering compounds with 100 mL hexane and discard the eluate.

13.4.7 The choice of eluting solvents will depend on the choice of alumina (acid or basic) made in Section 13.4.2.

13.4.7.1 If using acid alumina, elute the CDDs/CDFs from the column with 20 mL methylene chloride:hexane (20:80 v/v). Collect the eluate.

13.4.7.2 If using basic alumina, elute the CDDs/CDFs from the column with 20 mL methylene chloride:hexane (50:50 v/v). Collect the eluate.

13.4.8 Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the HPLC or GC/MS.

13.5 Carbon Column.

13.5.1 Cut both ends from a 10 mL disposable serological pipet (Section 6.7.3.2) to produce a 10 cm column. Fire-polish both ends and flare both ends if desired. Insert a glass-wool plug at one end, and pack the column with 0.55 g of Carbopak/Celite (Section 7.5.3.3) to form an adsorbent bed approximately 2 cm long. Insert a glass-wool plug on top of the bed to hold the adsorbent in place.

13.5.2 Pre-elute the column with 5 mL of toluene followed by 2 mL of methylene chloride: methanol:toluene (15:4:1 v/v), 1 mL of methylene chloride:cyclohexane (1:1 v/v), and 5 mL of hexane. If the flow rate of eluate exceeds 0.5 mL/minute, discard the column.

13.5.3 When the solvent is within 1 mm of the column packing, apply the sample extract to the column. Rinse the sample container twice with 1 mL portions of hexane and apply separately to the column. Apply 2 mL of hexane to complete the transfer.

13.5.4 Elute the interfering compounds with two 3 mL portions of hexane, 2 mL of methylene chloride:cyclohexane (1:1 v/v), and 2 mL of methylene chloride:methanol:toluene (15:4:1 v/v). Discard the eluate.

13.5.5 Invert the column, and elute the CDDs/CDFs with 20 mL of toluene. If carbon particles are present in the eluate, filter through glass-fiber filter paper.

13.5.6 Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or injection into the HPLC or GC/MS.

13.6 HPLC (Reference 6).

13.6.1 Column calibration.

13.6.1.1 Prepare a calibration standard containing the 2,3,7,8-substituted isomers and/or other isomers of interest at a concentration of approximately 500 pg/µL in methylene chloride.

13.6.1.2 Inject 30 µL of the calibration solution into the HPLC and record the signal from the detector. Collect the eluant for reuse. The elution order will be the tetra- through octa-isomers.

13.6.1.3 Establish the collection time for the tetra-isomers and for the other isomers of interest. Following calibration, flush the injection system with copious quantities of methylene chloride, including a minimum of five 50 µL injections while the detector is monitored, to ensure that residual CDDs/CDFs are removed from the system.

13.6.1.4 Verify the calibration with the calibration solution after every 20 extracts. Calibration is verified if the recovery of the CDDs/CDFs from the calibration standard (Section 13.6.1.1) is 75–125% compared to the calibration (Section 13.6.1.2). If calibration is not verified, the system shall be recalibrated using the calibration solution, and the previous 20 samples shall be re-extracted and cleaned up using the calibrated system.

13.6.2 Extract cleanup—HPLC requires that the column not be overloaded. The column specified in this method is designed to handle a maximum of 30 µL of extract. If the extract cannot be concentrated to less than 30 µL, it is split into fractions and the fractions are combined after elution from the column.

13.6.2.1 Rinse the sides of the vial twice with 30 µL of methylene chloride and reduce to 30 µL with the evaporation apparatus (Section 12.7).

13.6.2.2 Inject the 30 µL extract into the HPLC.

13.6.2.3 Elute the extract using the calibration data determined in Section 13.6.1. Collect the fraction(s) in a clean 20 mL concentrator tube containing 5 mL of hexane:acetone (1:1 v/v).

13.6.2.4 If an extract containing greater than 100 ng/mL of total CDD or CDF is encountered, a 30 µL methylene chloride blank shall be run through the system to check for carry-over.

13.6.2.5 Concentrate the eluate per Section 12.7 for injection into the GC/MS.

13.7 Cleanup of Tissue Lipids—Lipids are removed from the Soxhlet extract using either the anthropogenic isolation column (Section 13.7.1) or acidified silica gel (Section 13.7.2), or are removed from the HCl digested extract using sulfuric acid and base back-extraction (Section 13.7.3).

13.7.1 Anthropogenic isolation column (References 22 and 27)—Used for removal of lipids from the Soxhlet/SDS extraction (Section 12.4.1).

13.7.1.1 Prepare the column as given in Section 7.5.4.

13.7.1.2 Pre-elute the column with 100 mL of hexane. Drain the hexane layer to the top of the column, but do not expose the sodium sulfate.

13.7.1.3 Load the sample and rinses (Section 12.4.1.9.2) onto the column by draining each portion to the top of the bed. Elute the CDDs/CDFs from the column into the apparatus used for concentration (Section 12.4.1.7) using 200 mL of hexane.

13.7.1.4 Concentrate the cleaned up extract (Sections 12.6 through 12.7) to constant weight per Section 12.7.3.1. If more than 500 mg of material remains, repeat the cleanup using a fresh anthropogenic isolation column.

13.7.1.5 Redissolve the extract in a solvent suitable for the additional cleanups to be used (Sections 13.2 through 13.6 and 13.8).

13.7.1.6 Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent.

13.7.1.7 Clean up the extract using the procedures in Sections 13.2 through 13.6 and 13.8. Alumina (Section 13.4) or Florisil (Section 13.8) and carbon (Section 13.5) are recommended as minimum additional cleanup steps.

13.7.1.8 Following cleanup, concentrate the extract to 10 µL as described in Section 12.7 and proceed with the analysis in Section 14.

13.7.2 Acidified silica gel (Reference 28)—Procedure alternate to the anthropogenic isolation column (Section 13.7.1) that is used for removal of lipids from the Soxhlet/SDS extraction (Section 12.4.1).

13.7.2.1 Adjust the volume of hexane in the bottle (Section 12.4.1.9.2) to approximately 200 mL.

13.7.2.2 Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent.

13.7.2.3 Drop the stirring bar into the bottle, place the bottle on the stirring plate, and begin stirring.

13.7.2.4 Add 30–100 g of acid silica gel (Section 7.5.1.2) to the bottle while stirring, keeping the silica gel in motion. Stir for two to three hours.

Note: 30 grams of silica gel should be adequate for most samples and will minimize contamination from this source.

13.7.2.5 After stirring, pour the extract through approximately 10 g of granular anhydrous sodium sulfate (Section 7.2.1) contained in a funnel with glass-fiber filter into a macro contration device (Section 12.6). Rinse the bottle and sodium sulfate with hexane to complete the transfer.

13.7.2.6 Concentrate the extract per Sections 12.6 through 12.7 and clean up the extract using the procedures in Sections 13.2 through 13.6 and 13.8. Alumina (Section 13.4) or Florisil (Section 13.8) and carbon (Section 13.5) are recommended as minimum additional cleanup steps.

13.7.3 Sulfuric acid and base back-extraction. Used with HCl digested extracts (Section 12.4.2).

13.7.3.1 Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent (Section 12.4.2.8.2).

13.7.3.2 Add 10 mL of concentrated sulfuric acid to the bottle. Immediately cap and shake one to three times. Loosen cap in a hood to vent excess pressure. Cap and shake the bottle so that the residue/solvent is exposed to the acid for a total time of approximately 45 seconds.

13.7.3.3 Decant the hexane into a 250 mL separatory funnel making sure that no acid is transferred. Complete the quantitative transfer with several hexane rinses.

13.7.3.4 Back extract the solvent/residue with 50 mL of potassium hydroxide solution per Section 12.5.2, followed by two reagent water rinses.

13.7.3.5 Drain the extract through a filter funnel containing approximately 10 g of granular anhydrous sodium sulfate in a glass-fiber filter into a macro concentration device (Section 12.6).

13.7.3.6 Concentrate the cleaned up extract to a volume suitable for the additional cleanups given in Sections 13.2 through 13.6 and 13.8. Gel permeation chromatography (Section 13.2), alumina (Section 13.4) or Florisil (Section 13.8), and Carbopak/Celite (Section 13.5) are recommended as minimum additional cleanup steps.

13.7.3.7 Following cleanup, concentrate the extract to 10 L as described in Section 12.7 and proceed with analysis per Section 14.

13.8 Florisil Cleanup (Reference 29).

13.8.1 Pre-elute the activated Florisil column (Section 7.5.3) with 10 mL of methylene chloride followed by 10 mL of hexane:methylene chloride (98:2 v/v) and discard the solvents.

13.8.2 When the solvent is within 1 mm of the packing, apply the sample extract (in hexane) to the column. Rinse the sample container twice with 1 mL portions of hexane and apply to the column.

13.8.3 Elute the interfering compounds with 20 mL of hexane:methylene chloride (98:2) and discard the eluate.

13.8.4 Elute the CDDs/CDFs with 35 mL of methylene chloride and collect the eluate. Concentrate the eluate per Sections 12.6 through 12.7 for further cleanup or for injection into the HPLC or GC/MS.

14.0 HRGC/HRMS Analysis

14.1 Establish the operating conditions given in Section 10.1.

14.2 Add 10 uL of the appropriate internal standard solution (Section 7.12) to the sample extract immediately prior to injection to minimize the possibility of loss by evaporation, adsorption, or reaction. If an extract is to be reanalyzed and evaporation has occurred, do not add more instrument internal standard solution. Rather, bring the extract back to its previous volume (e.g., 19 L) with pure nonane only (18 L if 2 L injections are used).

14.3 Inject 1.0 µL or 2.0 µL of the concentrated extract containing the internal standard solution, using on-column or splitless injection. The volume injected must be identical to the volume used for calibration (Section 10). Start the GC column initial isothermal hold upon injection. Start MS data collection after the solvent peak elutes. Stop data collection after the OCDD and OCDF have eluted. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, stop data collection after elution of these compounds. Return the column to the initial temperature for analysis of the next extract or standard.

15.0 System and Laboratory Performance

15.1 At the beginning of each 12-hour shift during which analyses are performed, GC/MS system performance and calibration are verified for all CDDs/CDFs and labeled compounds. For these tests, analysis of the CS3 calibration verification (VER) standard (Section 7.13 and Table 4) and the isomer specificity test standards (Section 7.15 and Table 5) shall be used to verify all performance criteria. Adjustment and/or recalibration (Section 10) shall be performed until all performance criteria are met. Only after all performance criteria are met may samples, blanks, IPRs, and OPRs be analyzed.

15.2 MS Resolution—A static resolving power of at least 10,000 (10% valley definition) must be demonstrated at the appropriate m/z before any analysis is performed. Static resolving power checks must be performed at the beginning and at the end of each 12-hour shift according to procedures in Section 10.1.2. Corrective actions must be implemented whenever the resolving power does not meet the requirement.

15.3 Calibration Verification.

15.3.1 Inject the VER standard using the procedure in Section 14.

15.3.2 The m/z abundance ratios for all CDDs/CDFs shall be within the limits in Table 9; otherwise, the mass spectrometer shall be adjusted until the m/z abundance ratios fall within the limits specified, and the verification test shall be repeated. If the adjustment alters the resolution of the mass spectrometer, resolution shall be verified (Section 10.1.2) prior to repeat of the verification test.

15.3.3 The peaks representing each CDD/CDF and labeled compound in the VER standard must be present with S/N of at least 10; otherwise, the mass spectrometer shall be adjusted and the verification test repeated.

15.3.4 Compute the concentration of each CDD/CDF compound by isotope dilution (Section 10.5) for those compounds that have labeled analogs (Table 1). Compute the concentration of the labeled compounds by the internal standard method (Section 10.6). These concentrations are computed based on the calibration data in Section 10.

15.3.5 For each compound, compare the concentration with the calibration verification limit in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare the concentration to the limit in Table 6a. If all compounds meet the acceptance criteria, calibration has been verified and analysis of standards and sample extracts may proceed. If, however, any compound fails its respective limit, the measurement system is not performing properly for that compound. In this event, prepare a fresh calibration standard or correct the problem causing the failure and repeat the resolution (Section 15.2) and verification (Section 15.3) tests, or recalibrate (Section 10).

15.4 Retention Times and GC Resolution.

15.4.1 Retention times.

15.4.1.1 Absolute—The absolute retention times of the 13C12-1,2,3,4–TCDD and 13C12-1,2,3,7,8,9-HxCDD GCMS internal standards in the verification test (Section 15.3) shall be within ±15 seconds of the retention times obtained during calibration (Sections 10.2.1 and 10.2.4).

15.4.1.2 Relative—The relative retention times of CDDs/CDFs and labeled compounds in the verification test (Section 15.3) shall be within the limits given in Table 2.

15.4.2 GC resolution.

15.4.2.1 Inject the isomer specificity standards (Section 7.15) on their respective columns.

15.4.2.2 The valley height between 2,3,7,8-TCDD and the other tetra-dioxin isomers at m/z 319.8965, and between 2,3,7,8-TCDF and the other tetra-furan isomers at m/z 303.9016 shall not exceed 25% on their respective columns (Figures 6 and 7).

15.4.3 If the absolute retention time of any compound is not within the limits specified or if the 2,3,7,8-isomers are not resolved, the GC is not performing properly. In this event, adjust the GC and repeat the verification test (Section 15.3) or recalibrate (Section 10), or replace the GC column and either verify calibration or recalibrate.

15.5 Ongoing Precision and Recovery.

15.5.1 Analyze the extract of the ongoing precision and recovery (OPR) aliquot (Section 11.4.2.5, 11.5.4, 11.6.2, 11.7.4, or 11.8.3.2) prior to analysis of samples from the same batch.

15.5.2 Compute the concentration of each CDD/CDF by isotope dilution for those compounds that have labeled analogs (Section 10.5). Compute the concentration of 1,2,3,7,8,9-HxCDD, OCDF, and each labeled compound by the internal standard method (Section 10.6).

15.5.3 For each CDD/CDF and labeled compound, compare the concentration to the OPR limits given in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare the concentration to the limits in Table 6a. If all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may proceed. If, however, any individual concentration falls outside of the range given, the extraction/concentration processes are not being performed properly for that compound. In this event, correct the problem, re-prepare, extract, and clean up the sample batch and repeat the ongoing precision and recovery test (Section 15.5).

15.5.4 Add results that pass the specifications in Section 15.5.3 to initial and previous ongoing data for each compound in each matrix. Update QC charts to form a graphic representation of continued laboratory performance. Develop a statement of laboratory accuracy for each CDD/CDF in each matrix type by calculating the average percent recovery (R) and the standard deviation of percent recovery (SR). Express the accuracy as a recovery interval from R-2SR to R=2SR. For example, if R=95% and SR=5%, the accuracy is 85–105%.

15.6 Blank—Analyze the method blank extracted with each sample batch immediately following analysis of the OPR aliquot to demonstrate freedom from contamination and freedom from carryover from the OPR analysis. The results of the analysis of the blank must meet the specifications in Section 9.5.2 before sample analyses may proceed.

16.0 Qualitative Determination

A CDD, CDF, or labeled compound is identified in a standard, blank, or sample when all of the criteria in Sections 16.1 through 16.4 are met.

16.1 The signals for the two exact m/z's in Table 8 must be present and must maximize within the same two seconds.

16.2 The signal-to-noise ratio (S/N) for the GC peak at each exact m/z must be greater than or equal to 2.5 for each CDD or CDF detected in a sample extract, and greater than or equal to 10 for all CDDs/CDFs in the calibration standard (Sections 10.2.3 and 15.3.3).

16.3 The ratio of the integrated areas of the two exact m/z's specified in Table 8 must be within the limit in Table 9, or within ±10% of the ratio in the midpoint (CS3) calibration or calibration verification (VER), whichever is most recent.

16.4 The relative retention time of the peak for a 2,3,7,8-substituted CDD or CDF must be within the limit in Table 2. The retention time of peaks representing non-2,3,7,8-substituted CDDs/CDFs must be within the retention time windows established in Section 10.3.

16.5 Confirmatory Analysis—Isomer specificity for 2,3,7,8-TCDF cannot be achieved on the DB–5 column. Therefore, any sample in which 2,3,7,8-TCDF is identified by analysis on a DB–5 column must have a confirmatory analysis performed on a DB–225, SP–2330, or equivalent GC column. The operating conditions in Section 10.1.1 may be adjusted to optimize the analysis on the second GC column, but the GC/MS must meet the mass resolution and calibration specifications in Section 10.

16.6 If the criteria for identification in Sections 16.1 through 16.5 are not met, the CDD or CDF has not been identified and the results may not be reported for regulatory compliance purposes. If interferences preclude identification, a new aliquot of sample must be extracted, further cleaned up, and analyzed.

17.0 Quantitative Determination

17.1 Isotope Dilution Quantitation—By adding a known amount of a labeled compound to every sample prior to extraction, correction for recovery of the CDD/CDF can be made because the CDD/CDF and its labeled analog exhibit similar effects upon extraction, concentration, and gas chromatography. Relative response (RR) values are used in conjunction with the initial calibration data described in Section 10.5 to determine concentrations directly, so long as labeled compound spiking levels are constant, using the following equation:


where:

Cex = The concentration of the CDD/CDF in the extract, and the other terms are as defined in Section 10.5.2.

17.1.1 Because of a potential interference, the labeled analog of OCDF is not added to the sample. Therefore, OCDF is quantitated against labeled OCDD. As a result, the concentration of OCDF is corrected for the recovery of the labeled OCDD. In instances where OCDD and OCDF behave differently during sample extraction, concentration, and cleanup procedures, this may decrease the accuracy of the OCDF results. However, given the low toxicity of this compound relative to the other dioxins and furans, the potential decrease in accuracy is not considered significant.

17.1.2 Because 13C12-1,2,3,7,8,9-HxCDD is used as an instrument internal standard (i.e., not added before extraction of the sample), it cannot be used to quantitate the 1,2,3,7,8,9-HxCDD by strict isotope dilution procedures. Therefore, 1,2,3,7,8,9-HxCDD is quantitated using the averaged response of the labeled analogs of the other two 2,3,7,8-substituted HxCDD's: 1,2,3,4,7,8-HxCDD and 1,2,3,6,7,8-HxCDD. As a result, the concentration of 1,2,3,7,8,9-HxCDD is corrected for the average recovery of the other two HxCDD's.

17.1.3 Any peaks representing non-2,3,7,8-substituted CDDs/CDFs are quantitated using an average of the response factors from all of the labeled 2,3,7,8-isomers at the same level of chlorination.

17.2 Internal Standard Quantitation and Labeled Compound Recovery.

17.2.1 Compute the concentrations of 1,2,3,7,8,9-HxCDD, OCDF, the 13C-labeled analogs and the 37C-labeled cleanup standard in the extract using the response factors determined from the initial calibration data (Section 10.6) and the following equation:


where:

Cex = The concentration of the CDD/CDF in the extract, and the other terms are as defined in Section 10.6.1.

Note: There is only one m/z for the 37Cl-labeled standard.

17.2.2 Using the concentration in the extract determined above, compute the percent recovery of the 13C-labeled compounds and the 37C-labeled cleanup standard using the following equation:


17.3 The concentration of a CDD/CDF in the solid phase of the sample is computed using the concentration of the compound in the extract and the weight of the solids (Section 11.5.1), as follows:


where:

Cex = The concentration of the compound in the extract.

Vex = The extract volume in mL.

Ws = The sample weight (dry weight) in kg.

17.4 The concentration of a CDD/CDF in the aqueous phase of the sample is computed using the concentration of the compound in the extract and the volume of water extracted (Section 11.4 or 11.5), as follows:


where:

Cex = The concentration of the compound in the extract.

Vex = The extract volume in mL.

Vs = The sample volume in liters.

17.5 If the SICP area at either quantitation m/z for any compound exceeds the calibration range of the system, a smaller sample aliquot is extracted.

17.5.1 For aqueous samples containing 1% solids or less, dilute 100 mL, 10 mL, etc., of sample to 1 L with reagent water and re-prepare, extract, clean up, and analyze per Sections 11 through 14.

17.5.2 For samples containing greater than 1% solids, extract an amount of sample equal to 1/10, 1/100, etc., of the amount used in Section 11.5.1. Re-prepare, extract, clean up, and analyze per Sections 11 through 14.

17.5.3 If a smaller sample size will not be representative of the entire sample, dilute the sample extract by a factor of 10, adjust the concentration of the instrument internal standard to 100 pg/µL in the extract, and analyze an aliquot of this diluted extract by the internal standard method.

17.6 Results are reported to three significant figures for the CDDs/CDFs and labeled compounds found in all standards, blanks, and samples.

17.6.1 Reporting units and levels.

17.6.1.1 Aqueous samples—Report results in pg/L (parts-per-quadrillion).

17.6.1.2 Samples containing greater than 1% solids (soils, sediments, filter cake, compost)—Report results in ng/kg based on the dry weight of the sample. Report the percent solids so that the result may be corrected.

17.6.1.3 Tissues—Report results in ng/kg of wet tissue, not on the basis of the lipid content of the sample. Report the percent lipid content, so that the data user can calculate the concentration on a lipid basis if desired.

17.6.1.4 Reporting level.

17.6.1.4.1 Standards (VER, IPR, OPR) and samples—Report results at or above the minimum level (Table 2). Report results below the minimum level as not detected or as required by the regulatory authority.

17.6.1.4.2 Blanks—Report results above one-third the ML.

17.6.2 Results for CDDs/CDFs in samples that have been diluted are reported at the least dilute level at which the areas at the quantitation m/z's are within the calibration range (Section 17.5).

17.6.3 For CDDs/CDFs having a labeled analog, results are reported at the least dilute level at which the area at the quantitation m/z is within the calibration range (Section 17.5) and the labeled compound recovery is within the normal range for the method (Section 9.3 and Tables 6, 6a, 7, and 7a).

17.6.4 Additionally, if requested, the total concentration of all isomers in an individual level of chlorination (i.e., total TCDD, total TCDF, total Paced, etc.) may be reported by summing the concentrations of all isomers identified in that level of chlorination, including both 2,3,7,8-substituted and non-2,3,7,8-substituted isomers.

18.0 Analysis of Complex Samples

18.1 Some samples may contain high levels (>10 ng/L; >1000 ng/kg) of the compounds of interest, interfering compounds, and/or polymeric materials. Some extracts will not concentrate to 10 µL (Section 12.7); others may overload the GC column and/or mass spectrometer.

18.2 Analyze a smaller aliquot of the sample (Section 17.5) when the extract will not concentrate to 10 µL after all cleanup procedures have been exhausted.

18.3 Chlorodiphenyl Ethers—If chromatographic peaks are detected at the retention time of any CDDs/CDFs in any of the m/z channels being monitored for the chlorodiphenyl ethers (Table 8), cleanup procedures must be employed until these interferences are removed. Alumina (Section 13.4) and Florisil (Section 13.8) are recommended for removal of chlorodiphenyl ethers.

18.4 Recovery of Labeled Compounds—In most samples, recoveries of the labeled compounds will be similar to those from reagent water or from the alternate matrix (Section 7.6).

18.4.1 If the recovery of any of the labeled compounds is outside of the normal range (Table 7), a diluted sample shall be analyzed (Section 17.5).

18.4.2 If the recovery of any of the labeled compounds in the diluted sample is outside of normal range, the calibration verification standard (Section 7.13) shall be analyzed and calibration verified (Section 15.3).

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

18.4.4 If the calibration is verified and the diluted sample does not meet the limits for labeled compound recovery, the method does not apply to the sample being analyzed and the result may not be reported for regulatory compliance purposes. In this case, alternate extraction and cleanup procedures in this method must be employed to resolve the interference. If all cleanup procedures in this method have been employed and labeled compound recovery remains outside of the normal range, extraction and/or cleanup procedures that are beyond this scope of this method will be required to analyze these samples.

19.0 Pollution Prevention

19.1 The solvents used in this method pose little threat to the environment when managed properly. The solvent evaporation techniques used in this method are amenable to solvent recovery, and it is recommended that the laboratory recover solvents wherever feasible.

19.2 Standards should be prepared in volumes consistent with laboratory use to minimize disposal of standards.

20.0 Waste Management

20.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance is also required with any sewage discharge permits and regulations.

20.2 Samples containing HCl to pH <2 are hazardous and must be neutralized before being poured down a drain or must be handled as hazardous waste.

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

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

20.5 For further information on waste management, consult “The Waste Management Manual for Laboratory Personnel” and “Less is Better—Laboratory Chemical Management for Waste Reduction,” available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.

21.0 Method Performance

Method performance was validated and performance specifications were developed using data from EPA's international interlaboratory validation study (References 30–31) and the EPA/paper industry Long-Term Variability Study of discharges from the pulp and paper industry (58 FR 66078).

22.0 References

1. Tondeur, Yves. “Method 8290: Analytical Procedures and Quality Assurance for Multimedia Analysis of Polychlorinated Dibenzo-p-dioxins and Dibenzofurans by High Resolution Gas Chromatography/High Resolution Mass Spectrometry,” USEPA EMSL, Las Vegas, Nevada, June 1987.

2. “Measurement of 2,3,7,8-Tetrachlorinated Dibenzo-p-dioxin (TCDD) and 2,3,7,8-Tetrachlorinated Dibenzofuran (TCDF) in Pulp, Sludges, Process Samples and Wastewaters from Pulp and Paper Mills,” Wright State University, Dayton, OH 45435, June 1988.

3. “NCASI Procedures for the Preparation and Isomer Specific Analysis of Pulp and Paper Industry Samples for 2,3,7,8-TCDD and 2,3,7,8-TCDF,” National Council of the Paper Industry for Air and Stream Improvement Inc., 260 Madison Avenue, New York, NY 10016, Technical Bulletin No. 551, Pre-Release Copy, July 1988.

4. “Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in Fish,” USEPA, Environmental Research Laboratory, 6201 Congdon Boulevard, Duluth, MN 55804, April 1988.

5. Tondeur, Yves. “Proposed GC/MS Methodology for the Analysis of PCDDs and PCDFs in Special Analytical Services Samples,” Triangle Laboratories, Inc., 801–10 Capitola Dr, Research Triangle Park, NC 27713, January 1988; updated by personal communication September 1988.

6. Lamparski, L.L. and Nestrick, T.J. “Determinationof Tetra-, Hexa-, Hepta-, and Octachlorodibenzo-p-dioxin Isomers in Particulate Samples at Parts per Trillion Levels,” Analytical Chemistry, 52: 2045–2054, 1980.

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

8. Patterson, D.G., et. al. “Control of Interferences in the Analysis of Human Adipose Tissue for 2,3,7,8-Tetrachlorodibenzo-p-dioxin,” Environmental Toxicological Chemistry, 5:355–360, 1986.

9. Stanley, John S. and Sack, Thomas M. “Protocol for the Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin by High Resolution Gas Chromatography/High Resolution Mass Spectrometry,” USEPA EMSL, Las Vegas, Nevada 89114, EPA 600/4–86–004, January 1986.

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

11. “OSHA Safety and Health Standards, General Industry,” OSHA 2206, 29 CFR 1910.

12. “Safety in Academic Chemistry Laboratories,” ACS Committee on Chemical Safety, 1979.

13. “Standard Methods for the Examination of Water and Wastewater,” 18th edition and later revisions, American Public Health Association, 1015 15th St, N.W., Washington, DC 20005, 1–35: Section 1090 (Safety), 1992.

14. “Method 613—2,3,7,8-Tetrachlorodibenzo-p-dioxin,” 40 CFR 136 (49 FR 43234), October 26, 1984, Section 4.1.

15. Provost, L.P. and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15: 56–83, 1983.

16. “Standard Practice for Sampling Water,” ASTM Annual Book of Standards, ASTM, 1916 Race Street, Philadelphia, PA 19103–1187, 1980.

17. “Methods 330.4 and 330.5 for Total Residual Chlorine,” USEPA, EMSL, Cincinnati, OH 45268, EPA 600/4–79–020, March 1979.

18. “Handbook of Analytical Quality Control in Water and Wastewater Laboratories,” USEPA EMSL, Cincinnati, OH 45268, EPA–600/4–79–019, March 1979.

19. Williams, Rick. Letter to Bill Telliard, June 4, 1993, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 703–519–1140.

20. Barkowski, Sarah. Fax to Sue Price, August 6, 1992, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria VA 22314, 703–519–1140.

21. “Analysis of Multi-media, Multi-concentration Samples for Dioxins and Furans, PCDD/PCDF Analyses Data Package”, Narrative for Episode 4419, MRI Project No. 3091-A, op.cit. February 12, 1993, Available from the EPA Sample Control Center operated by DynCorp Viar Inc, 300 N Lee St, Alexandria, VA 22314 (703–519–1140).

22. “Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in Fish”, U.S. Environmental Protection Agency, Environmental Research Laboratory, Duluth, MN 55804, EPA/600/3–90/022, March 1990.

23. Afghan, B.K., Carron, J., Goulden, P.D., Lawrence, J., Leger, D., Onuska, F., Sherry, J., and Wilkenson, R.J., “Recent Advances in Ultratrace Analysis of Dioxins and Related Halogenated Hydrocarbons”, Can J. Chem., 65: 1086–1097, 1987.

24. Sherry, J.P. and Tse, H. “A Procedure for the Determination of Polychlorinated Dibenzo-p-dioxins in Fish”, Chemosphere, 20: 865–872, 1990.

25. “Preliminary Fish Tissue Study”, Results of Episode 4419, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 703–519–1140.

26. Nestrick, Terry L. DOW Chemical Co., personal communication with D.R. Rushneck, April 8, 1993. Details available from the U.S. Environmental Protection Agency Sample Control Center operated by DynCorp Viar Inc, 300 N Lee St, Alexandria, VA 22314, 703–519–1140.

27. Barnstadt, Michael. “Big Fish Column”, Triangle Laboratories of RTP, Inc., SOP 129–90, 27 March 27, 1992.

28. “Determination of Polychlorinated Dibenzo-p-Dioxins (PCDD) and Dibenzofurans (PCDF) in Environmental Samples Using EPA Method 1613”, Chemical Sciences Department, Midwest Research Institute, 425 Volker Boulevard, Kansas City, MO 44110–2299, Standard Operating Procedure No. CS–153, January 15, 1992.

29. Ryan, John J. Raymonde Lizotte and William H. Newsome, J. Chromatog. 303 (1984) 351-360.

30. Telliard, William A., McCarty, Harry B., and Riddick, Lynn S. “Results of the Interlaboratory Validation Study of USEPA Method 1613 for the Analysis of Tetra-through Octachlorinated Dioxins and Furans by Isotope Dilution GC/MS,” Chemosphere, 27, 41–46 (1993).

31. “Results of the International Interlaboratory Validation Study of USEPA Method 1613”, October 1994, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 703–519–1140.

23.0 Tables and Figures


Table 1_Chlorinated Dibenzo-p-Dioxins and Furans Determined by Isotope Dilution and Internal Standard High
Resolution Gas Chromatography (HRGC)/High Resolution Mass Spectrometry (HRMS)
----------------------------------------------------------------------------------------------------------------
CDDs/CDFs \1\ CAS registry Labeled analog CAS registry
----------------------------------------------------------------------------------------------------------------
2,3,7,8-TCDD.................................. 1746-01-6 13C12-2,3,7,8-TCDD.............. 76523-40-5
37Cl4-2,3,7,8-TCDD.............. 85508-50-5
Total TCDD.................................... 41903-57-5
2,3,7,8-TCDF.................................. 51207-31-9 13C12-2,3,7,8-TCDF.............. 89059-46-1
Total-TCDF.................................... 55722-27-5
1,2,3,7,8-PeCDD............................... 40321-76-4 13C12-1,2,3,7,8-PeCDD........... 109719-79-1
Total-PeCDD................................... 36088-22-9
1,2,3,7,8-PeCDF............................... 57117-41-6 13C12-1,2,3,7,8-PeCDF........... 109719-77-9
2,3,4,7,8-PeCDF............................... 57117-31-4 13C12-2,3,4,7,8-PeCDF........... 116843-02-8
Total-PeCDF................................... 30402-15-4
1,2,3,4,7,8-HxCDD............................. 39227-28-6 13C12-1,2,3,4,7,8-HxCDD......... 109719-80-4
1,2,3,6,7,8-HxCDD............................. 57653-85-7 13C12-1,2,3,6,7,8-HxCDD......... 109719-81-5
1,2,3,7,8,9-HxCDD............................. 19408-74-3 13C12-1,2,3,7,8,9-HxCDD......... 109719-82-6
Total-HxCDD................................... 34465-46-8
1,2,3,4,7,8-HxCDF............................. 70648-26-9 13C12-1,2,3,4,7,8-HxCDF......... 114423-98-2
1,2,3,6,7,8-HxCDF............................. 57117-44-9 13C12-1,2,3,6,7,8-HxCDF......... 116843-03-9
1,2,3,7,8,9-HxCDF............................. 72918-21-9 13C12-1,2,3,7,8,9-HxCDF......... 116843-04-0
2,3,4,6,7,8-HxCDF............................. 60851-34-5 13C12-2,3,4,6,7,8-HxCDF......... 116843-05-1
Total-HxCDF................................... 55684-94-1
1,2,3,4,6,7,8-HpCDD........................... 35822-46-9 13C12-1,2,3,4,6,7,8-HpCDD....... 109719-83-7
Total-HpCDD................................... 37871-00-4
1,2,3,4,6,7,8-HpCDF........................... 67562-39-4 13C12-1,2,3,4,6,7,8-HpCDF....... 109719-84-8
1,2,3,4,7,8,9-HpCDF........................... 55673-89-7 13C12-1,2,3,4,7,8,9-HpCDF....... 109719-94-0
Total-HpCDF................................... 38998-75-3
OCDD.......................................... 3268-87-9 13C12-OCDD...................... 114423-97-1
OCDF.......................................... 39001-02-0 Not used........................
----------------------------------------------------------------------------------------------------------------
\1\ Chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans.
TCDD = Tetrachlorodibenzo-p-dioxin.
TCDF = Tetrachlorodibenzofuran.
PeCDD = Pentachlorodibenzo-p-dioxin.
PeCDF = Pentachlorodibenzofuran.
HxCDD = Hexachlorodibenzo-p-dioxin.
HxCDF = Hexachlorodibenzofuran.
HpCDD = Heptachlorodibenzo-p-dioxin.
HpCDF = Heptachlorodibenzofuran.
OCDD = Octachlorodibenzo-p-dioxin.
OCDF = Octachlorodibenzofuran.




Table 2_Retention Time References, Quantitation References, Relative Retention Times, and Minimum Levels for
CDDS and DCFS
----------------------------------------------------------------------------------------------------------------
Minimum level \1\
--------------------------------
Retention time and Relative Extract
CDD/CDF quantitation reference retention time Water (pg/ Solid (ng/ (pg/
L; ppq) kg; ppt) µL;
ppb)
----------------------------------------------------------------------------------------------------------------
Compounds using 13 C12-1,2,3,4-TCDD as the Injection Internal Standard
----------------------------------------------------------------------------------------------------------------
2,3,7,8-TCDF......................... 13 C12-2,3,7,8-TCDF..... 0.999-1.003 10 1 0.5
2,3,7,8-TCDD......................... 13 C12-2,3,7,8-TCDD..... 0.999-1.002 10 1 0.5
1,2,3,7,8-Pe......................... 13 C12-1,2,3,7,8-PeCDF.. 0.999-1.002 50 5 2.5
2,3,4,7,8-PeCDF...................... 13 C12-2,3,4,7,8-PeCDF.. 0.999-1.002 50 5 2.5
1,2,3,7,8-PeCDD...................... 13 C12-1,2,3,7,8-PeCDD.. 0.999-1.002 50 5 2.5
13 C12-2,3,7,8-TCDF.................. 13 C12-1,2,3,4-TCDD..... 0.923-1.103 ......... ......... .........
13 C12-2,3,7,8-TCDD.................. 13 C12-1,2,3,4-TCDD..... 0.976-1.043 ......... ......... .........
13 C12-2,3,7,8-TCDD.................. 13 C12-1,2,3,4-TCDD..... 0.989-1.052 ......... ......... .........
13 C12-1,2,3,7,8-PeCDF............... 13 C12-1,2,3,4-TCDD..... 1.000-1.425 ......... ......... .........
13 C12-2,3,4,7,8-PeCDF............... 13 C12-1,2,3,4-TCDD..... 1.001-1.526 ......... ......... .........
13 C12-1,2,3,7,8-PeCDF............... 13 C12-1,2,3,4-TCDD..... 1.000-1.567 ......... ......... .........
----------------------------------------------------------------------------------------------------------------
Compounds using 13 C12-1,2,3,7,8,9-HxCDD as the Injection Internal Standard
----------------------------------------------------------------------------------------------------------------
1,2,3,4,7,8-HxCDF.................... 13 C12-1,2,3,4,7,8-HxCDF 0.999-1.001 50 5 2.5
1,2,3,6,7,8-HxCDF.................... 13 C12-1,2,3,6,7,8-HxCDF 0.997-1.005 50 5 2.5
1,2,3,7,8,9-HxCDF.................... 13 C12-1,2,3,7,8,9-HxCDF 0.999-1.001 50 5 2.5
2,3,4,6,7,8-HxCDF.................... 13 C12-2,3,4,6,7,8-HxCDF 0.999-1.001 50 5 2.5
1,2,3,4,7,8-HxCDD.................... 13 C12-1,2,3,4,7,8-HxCDD 0.999-1.001 50 5 2.5
1,2,3,6,7,8-HxCDD.................... 13 C12-1,2,3,6,7,8-HxCDD 0.998-1.004 50 5 2.5
1,2,3,7,8,9-HxCDD.................... (\2\)................... 1.000-1.019 50 5 2.5
1,2,3,4,6,7,8-HpCDF.................. 13 C12-1,2,3,4,6,7,8- 0.999-1.001 50 5 2.5
HpCDF.
1,2,3,4,7,8,9-HpCDF.................. 13 C12-1,2,3,4,7,8,9- 0.999-1.001 50 5 2.5
HpCDF.
1,2,3,4,6,7,8-HpCDD.................. 13 C12-1,2,3,4,6,7,8- 0.999-1.001 50 5 2.5
HpCDD.
OCDF................................. 13 C12-OCDD............. 0.999-1.001 100 10 5.0
OCDD................................. 13 C12-OCDD............. 0.999-1.001 100 10 5.0
1,2,3,4,6,7,8,-HxCDF................. 13 C12-1,2,3,7,8,9-HpCDD 0.949-0.975 ......... ......... .........
13 C121,2,3,7,8,9-HxCDF.............. 13 C12-1,2,3,7,8,9-HpCDD 0.977-1.047 ......... ......... .........
13 C122,3,4,6,7,8,-HxCDF............. 13 C12-1,2,3,7,8,9-HpCDD 0.959-1.021 ......... ......... .........
13 C121,2,3,4,7,8,-HxCDF............. 13 C12-1,2,3,7,8,9-HpCDD 0.977-1.000 ......... ......... .........
13 C121,2,3,6,7,8,-HxCDF............. 13 C12-1,2,3,7,8,9-HpCDD 0.981-1.003 ......... ......... .........
13 C121,2,3,4,6,7,8-HxCDF............ 13 C12-1,2,3,7,8,9-HpCDD 1.043-1.085 ......... ......... .........
13 C121,2,3,4,7,8,9-HxCDF............ 13 C12-1,2,3,7,8,9-HpCDD 1.057-1.151 ......... ......... .........
13 C121,2,3,4,6,7,8-HxCDF............ 13 C12-1,2,3,7,8,9-HpCDD 1.086-1.110 ......... ......... .........
13 C12OCDD........................... 13 C12-1,2,3,7,8,9-HpCDD 1.032-1.311 ......... ......... .........
----------------------------------------------------------------------------------------------------------------
\1\ The Minimum Level (ML) for each analyte is defined as the level at which the entire analytical system must
give a recognizable signal and acceptable calibration point. It is equivalent to the concentration of the
lowest calibration standard, assuming that all method-specified sample weights, volumes, and cleanup
procedures have been employed.
\2\ The retention time reference for 1,2,3,7,8,9-HxCDD is 13C12-1,2,3,6,7,8-HxCDD, and 1,2,3,7,8,9-HxCDD is
quantified using the averaged responses for 13C12-1,2,3,4,7,8-HxCDD and 13C12-1,2,3,6,7,8-HxCDD.




Table 3_Concentration of Stock and Spiking Solutions Containing CDDS/CDFS and Labeled Compounds
----------------------------------------------------------------------------------------------------------------
Labeled Labeled
compound compound PAR stock PAR spiking
CDD/CDF stock spiking solution solution \4\
solution \1\ solution \3\ (ng/mL) (ng/mL)
(ng/mL) \2\ (ng/mL)
----------------------------------------------------------------------------------------------------------------
2,3,7,8-TCDD.............................................. ............ ........... 40 0.8
2,3,7,8-TCDF.............................................. ............ ........... 40 0.8
1,2,3,7,8-PeCDD........................................... ............ ........... 200 4
1,2,3,7,8-PeCDF........................................... ............ ........... 200 4
2,3,4,7,8-PeCDF........................................... ............ ........... 200 4
1,2,3,4,7,8-HxCDD......................................... ............ ........... 200 4 (continued)