Method 1613: Tetra- through Octa-Chlorinated Dioxins …

1 Revision B. Method 1613. Tetra- through Octa-Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS. October 1994. environmental protection agency Office of Water Engineering and Analysis Division (4303). 401 M Street Washington, 20460. Acknowledgments This Method was prepared under the direction of William A. Telliard of the Engineering and Analysis Division within the EPA Office of Water. This document was prepared under EPA. Contract No. 68-C3-0337 by DynCorp environmental Services Division with assistance from its subcontractor Interface, Inc. Disclaimer This Method has been reviewed by the Engineering and Analysis Division, environmental protection agency , and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Method 1613. Introduction Method 1613 was developed by the united states environmental protection agency 's Office of Science and Technology for isomer-specific determination of the 2,3,7,8-substituted, tetra- through Octa-Chlorinated , dibenzo-p- Dioxins and dibenzofurans in aqueous, solid, and tissue matrices by isotope dilution, high resolution capillary column gas chromatography (HRGC)/high resolution mass spectrometry (HRMS).

2 Questions concerning this Method or its application should be addressed to: Telliard US EPA Office of Water Analytical methods Staff Mail Code 4303. 401 M Street, SW. Washington, 20460. 202/260-7120. Requests for additional copies should be directed to: Water Resource Center Mail Code RC-4100. 401 M Street, SW. Washington, 20460. 202/260-7786 or 202/260-2814. October 1994 iii Method 1613. Method 1613, Revision B. Tetra- through Octa-Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS. Scope and Application This Method is for determination of tetra- through Octa-Chlorinated dibenzo-p- Dioxins (CDDs) and dibenzofurans (CDFs) in water, soil, sediment, sludge, tissue, and other sample matrices by high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). The Method is for use in EPA's data gathering and monitoring programs associated with the Clean Water Act, the Resource Conservation and Recovery Act, the Comprehensive environmental Response, Compensation and Liability Act, and the Safe Drinking Water Act.

3 The Method is based on a compilation of EPA, industry, commercial laboratory, and academic methods (References 1-6). The seventeen 2,3,7,8-substituted CDDs/CDFs listed in Table 1 may be determined by this Method . Specifications are also provided for separate determination of 2,3,7,8-tetrachloro-dibenzo-p- dioxin (2,3,7,8-TCDD) and 2,3,7,8-tetrachloro-dibenzofuran (2,3,7,8-TCDF). The detection limits and quantitation levels in this Method are usually dependent on the level of interferences rather than instrumental limitations. The minimum levels (MLs). in Table 2 are the levels at which the CDDs/CDFs can be determined with no interferences present. The Method Detection Limit (MDL) for 2,3,7,8-TCDD has been determined as pg/L (parts-per-quadrillion) using this Method . The GC/MS portions of this Method are for use only by analysts experienced with HRGC/HRMS or under the close supervision of such qualified persons.

4 Each laboratory that uses this Method must demonstrate the ability to generate acceptable results using the procedure in Section This Method is "performance-based". The analyst is permitted to modify the Method to overcome interferences or lower the cost of measurements, provided that all performance criteria in this Method are met. The requirements for establishing Method equivalency are given in Section Any modification of this Method , beyond those expressly permitted, shall be considered a major modification subject to application and approval of alternate test procedures under 40 CFR and Summary of Method Flow charts that summarize procedures for sample preparation, extraction, and analysis are given in Figure 1 for aqueous and solid samples, Figure 2 for multi-phase samples, and Figure 3 for tissue samples. October 1994 1. Method 1613. Extraction Aqueous samples (samples containing less than 1% solids) Stable isotopically labeled analogs of 15 of the 2,3,7,8-substituted CDDs/CDFs are spiked into a 1 L.

5 Sample, and the sample is extracted by one of three procedures: Samples containing no visible particles are extracted with methylene chloride in a separatory funnel or by the solid-phase extraction technique summarized in Section The extract is concentrated for cleanup. Samples containing visible particles are vacuum filtered through a glass- fiber filter. The filter is extracted in a Soxhlet/Dean-Stark (SDS) extractor (Reference 7), and the filtrate is extracted with methylene chloride in a separatory funnel. The methylene chloride extract is concentrated and combined with the SDS extract prior to cleanup. The sample is vacuum filtered through a glass-fiber filter on top of a solid- phase extraction (SPE) disk. The filter and disk are extracted in an SDS. extractor, and the extract is concentrated for cleanup. Solid, semi-solid, and multi-phase samples (but not tissue) The labeled compounds are spiked into a sample containing 10 g (dry weight) of solids.

6 Samples containing multiple phases are pressure filtered and any aqueous liquid is discarded. Coarse solids are ground or homogenized. Any non-aqueous liquid from multi-phase samples is combined with the solids and extracted in an SDS. extractor. The extract is concentrated for cleanup. Fish and other tissue The sample is extracted by one of two procedures: Soxhlet or SDS extraction A 20 g aliquot of sample is homogenized, and a 10 g aliquot is spiked with the labeled compounds. The sample is mixed with sodium sulfate, allowed to dry for 12-24 hours, and extracted for 18- 24 hours using methylene chloride:hexane (1:1) in a Soxhlet extractor. The extract is evaporated to dryness, and the lipid content is determined. HCl digestion A 20 g aliquot is homogenized, and a 10 g aliquot is placed in a bottle and spiked with the labeled compounds. After equilibration, 200 mL of hydrochloric acid and 200 mL of methylene chloride:hexane (1:1) are added, and the bottle is agitated for 12-24 hours.

7 The extract is evaporated to dryness, and the lipid content is determined. After extraction, 37Cl4-labeled 2,3,7,8-TCDD is added to each extract to measure the efficiency of the cleanup process. Sample cleanups may include back-extraction with acid and/or base, and gel permeation, alumina, silica gel, Florisil and activated carbon chromatography. High-performance liquid chromatography (HPLC) can be used for further isolation of the 2,3,7,8-isomers or other specific isomers or congeners. Prior to the cleanup procedures cited above, tissue extracts are cleaned up using an anthropogenic isolation column, a batch silica gel adsorption, or sulfuric acid and base back-extraction, depending on the tissue extraction procedure used. 2 October 1994. Method 1613. After cleanup, the extract is concentrated to near dryness. Immediately prior to injection, internal standards are added to each extract, and an aliquot of the extract is injected into the gas chromatograph.

8 The analytes are separated by the GC and detected by a high- resolution ( 10,000) mass spectrometer. Two exact m/z's are monitored for each analyte. An individual CDD/CDF is identified by comparing the GC retention time and ion- abundance ratio of two exact m/z's with the corresponding retention time of an authentic standard and the theoretical or acquired ion-abundance ratio of the two exact m/z's. The non-2,3,7,8 substituted isomers and congeners are identified when retention times and ion-abundance ratios agree within predefined limits. Isomer specificity for 2,3,7,8-TCDD. and 2,3,7,8-TCDF is achieved using GC columns that resolve these isomers from the other Tetra- isomers. Quantitative analysis is performed using selected ion current profile (SICP) areas, in one of three ways: For the 15 2,3,7,8-substituted CDDs/CDFs with labeled analogs (see Table 1), the GC/MS system is calibrated, and the concentration of each compound is determined using the isotope dilution technique.

9 For 1,2,3,7,8,9-HxCDD, OCDF, and the labeled compounds, the GC/MS system is calibrated and the concentration of each compound is determined using the internal standard technique. For non-2,3,7,8-substituted isomers and for all isomers at a given level of chlorination ( , total TCDD), concentrations are determined using response factors from calibration of the CDDs/CDFs at the same level of chlorination. The quality of the analysis is assured through reproducible calibration and testing of the extraction, cleanup, and GC/MS systems. Definitions Definitions are given in the glossary at the end of this Method . Contamination and Interferences Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or elevated baselines causing misinterpretation of chromatograms (References 8-9). Specific selection of reagents and purification of solvents by distillation in all-glass systems may be required.

10 Where possible, reagents are cleaned by extraction or solvent rinse. Proper cleaning of glassware is extremely important, because glassware may not only contaminate the samples but may also remove the analytes of interest by adsorption on the glass surface. Glassware should be rinsed with solvent and washed with a detergent solution as soon after use as is practical. Sonication of glassware containing a detergent solution for approximately 30 seconds may aid in cleaning. Glassware with October 1994 3. Method 1613. removable parts, particularly separatory funnels with fluoropolymer stopcocks, must be disassembled prior to detergent washing. After detergent washing, glassware should be rinsed immediately, first with methanol, then with hot tap water. The tap water rinse is followed by another methanol rinse, then acetone, and then methylene chloride. Do not bake reusable glassware in an oven as a routine part of cleaning.