METHOD OF ANALYSIS FOR CHLORINATED
DIBENZOpDIOXINS AND DIBENZOFURANSl,2,3,4
This method is the same as that listed in Appendix X to 40 CFR Part 261.
1. Scope and Application.
1.1 This method measures the concentration of chlorinated dibenzopdioxins and chlorinated dibenzofurans in chemical wastes including still bottoms, filter aids, sludges, spent carbon, and reactor residues, and in soils.
1.2 The sensitivity of this method is dependent upon the level of interferences.
1.3 This method is recommended for use only by analysts experienced with residue analysis and skilled in mass spectral analytical techniques.
1.4 Because of the extreme toxicity of these compounds, the analyst must take necessary precautions to prevent exposure to himself, or to others, of materials known or believed to contain CDDs or CDFs.
1This method is appropriate for the analysis of tetra, penta, and hexa chlorinated dibenzopdioxins and di benzofurans.
2Analytical protocol for determination of TCDDs in phenolic chemical wastes and soil samples obtained from the proximity of chemical dumps. T.O. Tiernan and M. Taylor, Berhm Laboratory, Wright State University, Dayton, Ohio 45435.
3Analytical protocol for determination of chlorinated dibenzop- dioxins and chlorinated dibenzofurans in river water. T.O. Tiernan and M. Taylor, Berhm Laboratory, Wright State University, Dayton, Ohio 45435.
4In general, the techniques that should be used to handle these materials are those which are followed for radioactive or infectious laboratory materials. Assistance in evaluating laboratory practices may be obtained from industrial hygienists and persons specializing in safe laboratory practices. Typical infectious waste incinerators are probably not satisfactory devices for disposal of materials highly contaminated with CDDs and CDFs. Safety instructions are outlined in EPA Test Method 613(4.0)
See also: 1) "Program for monitoring potential contamination in the laboratory following the handling and analyses of chlorinated dibenzop dioxins and dibenzofurans" by F.D. Hileman et al., In: Human and Environmental Risks of Chlorinated Dioxins and Related Compounds, R.E. Tucker, et al., eds., Plenum Publishing Corp., 1983. 2) Safety procedures outlined in EPA Method 613, Federal Register Volume 44, No. 233, December 3, 1979.
2. Summary of the Method.
2.1 This method is an analytical extraction cleanup procedure, and capillary column gas chromatographlow resolution mass spectrometry method, using capillary column GC/MS conditions and internal standard techniques, which allow for the measurement of PCDDs and PCDFs in the extract.
2.2 If interferences are encountered, the method provides selected general purpose cleanup procedures to aid the analyst in their elimination.
3.1 Solvents, reagents, glassware, and other sample processing hardware may yield discrete artifacts and/or elevated baselines causing misinterpretation of gas chromatograms. All of these materials must be demonstrated to be free from interferences under the conditions of the analysis by running method blanks. Specific selection of reagents and purification of solvents by distillation in allglass systems may be required.
3.2 Interferences coextracted from the samples will vary considerably from source to source, depending upon the diversity of the industry being sampled. PCDD is often associated with other interfering chlorinated compounds such as PCB's which may be at concentrations several orders of magnitude higher than that of PCDD. While general cleanup techniques are provided as part of this method, unique samples may require additional cleanup approaches to achieve the sensitivity stated in Table 1.
3.3 The other isomers of tetrachlorodibenzopdioxin may interfere with the measurement of 2,3,7,8TCDD. Capillary column gas chromatography is required to resolve those isomers that yield virtually identical mass fragmentation patterns.
4. Apparatus and Material.
4.1 Sampling equipment for discrete or composite sampling.
4.1.1 Grab sample bottle amber glass, l liter or l quart volume. French or Boston Round design is recommended. The container must be washed and solvent rinsed before use to minimize interferences.
4.1.2 Bottle caps threaded to screw on to the sample bottles. Caps must be lined with Teflon. Solvent washed foil, used with the shiny side towards the sample, may be substituted for the Teflon if sample is not corrosive.
4.1.3 Compositing equipment automatic or manual composing system. No tygon or rubber tubing may be used, and the system must incorporate glass sample containers for the collection of a minimum of 250 ml. Sample containers must be kept refrigerated after sampling.
4.2 Water bath heated, with concentric ring cover, capable of temperature control (+2º C). The bath should be used in a hood.
4.3 Gas chromatograph/mass spectrometer data system.
4.3.1 Gas chromatograph: An analytical system with a temperatureprogrammable gas chromatograph and all required accessories including syringes, analytical columns, and gases.
4.3.2 Column: SP2250 coated on a 30 m long X 0.25 mm I.D. glass column (Supelco No. 23714 or equivalent). Glass capillary column conditions: Helium carrier gas at 30 cm/sec linear velocity run splitless. Column temperature is 210o C.
4.3.3 Mass spectrometer: Capable of scanning from 35 to 450 amu every l sec or less, utilizing 70 volts (nominal) electron energy in the electron impact ionization mode and producing a mass spectrum which meets all the criteria in Table 2 when 40 ng of decafluorotriphenyl phosphine (DFTPP) is injected through the GC inlet. The system must also be capable of selected ion monitoring (SIM) for at least 4 ions simultaneously, with a cycle time of 1 sec or less. Minimum integration time for SIM is l00 ms. Selected ion monitoring is verified by injecting .015 ng of TCDD Cl37 to give a minimum signal to noise ratio of 5 to l at mass 328.
4.3.4 GC/MS interface: Any GCtoMS interface that gives acceptable calibration points at 50 ng per injection for each compound of interest and achieves acceptable tuning performance criteria (see Section 6.16.3) may be used. GCtoMS interfaces constructed of all glass or glasslined materials are recommended. Glass can be deactivated by silanizing with dichlorodi- methylsilane. The interface must be capable of interest from GC to the MS.
4.3.5 Data system: A computer system must be interfaced to the mass spectrometer. The system must allow the continuous acquisition and storage on machinereadable media of all mass spectra obtained throughout the duration of the chromatographic program. The computer must have software that can search any GC/MS data file for ions of a specific mass and that can plot such ion abundance versus time or scan number. This type of plot is defined as an Extracted Ion Current Profile (EICP). Software must also be able to integrate the abundance, in any EICP, between specified time or scan number limits.
4.4 PipettesDisposable, Pasteur, 150 mm long X 5 ID (Fisher Scientific Co., No. 136786A or equivalent).
4.5 Flint glass bottle (Teflonlined screw cap).
4.6 Reactivial (silanized) (Pierce Chemical Co.).
5.1 Potassium hydroxide(ACS), 2% in distilled water.
5.2 Sulfuric acid(ACS), concentrated.
5.3 Methylene chloride, hexane, benzene, petroleum ether, methanol, tetradecanepesticide quality or equivalent
5.4 Prepare stock standard solutions of TCDD and 37ClTCDD (molecular weight 328) in a glove box. The stock solutions are stored in a glovebox, and checked frequently for signs of degradation or evaporation, especially just prior to the preparation of working standards.
5.5 Aluminabasic, Woelm: 80/200 mesh. Before use activate overnight at 600oC, cool to room temperature in a dessicator.
5.6 Prepurified nitrogen gas.
6.1 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 reagents.
6.2 Prepare GC/MS calibration standards for the internal standard technique that will allow for measurement of relative response factors of at least three CDD/37CDD ratios. Thus, for TCDDs at least three TCDD/37clTCDD and TCDF/37ClTCDF must be determined.5
5 37Cllabelled 2,3,7,8TCDD and 2,3,7,8 TCDF are available from K.O.R. Isotopes and Cambridge Isotopes, Inc., Cambridge, MA. Proper standardization requires the use of a specific labelled isomer for each congener to be determined. However, the only labelled isomers readily available are 37Cl2,3,7,8TCDD and Cl2,3,7,8TCDF. This method therefore uses these isomers as surrogates for the CDDs and CDFs. When other labelled CDDs and CDFs are available, their use will be required.
The 37ClTCDD/F concentration in the standard should be fixed and selected to yield a reproducible response at the most sensitive setting of the mass spectrometer. Response factors for PCDD and HxCDD may be determined by measuring the response of the tetrachloro labelled compounds relative to that of the unlabelled 1,2,3,4 or 2,3,7,8TCDD, 1,2,3,4,7 PCDD or 1,2,3,4,7,8HxCDD, which are commercially available.6
6.3 Assemble the necessary GC/MS apparatus and establish operating parameters equivalent to those indicated in Section 11.1 of this method. Calibrate the GC/MS system according to Eichelbarger, et al. (1975) by the use of decafluorotriphenyl phosphine (DFTPP). By injecting calibration standards, establish the response factors for CDDs vs. 37ClTCDD, and for CDFs vs. 37ClTCDF. The detection limit provided in Table 1 should be verified by injecting .015 ng of 37ClTCDD which should give a minimum signal to noise ratio of 5 to 1 at mass 328.
7. Quality Control.
7.1 Before processing any samples, the analyst should demonstrate through the analysis of a distilled water method blank, that all glassware and reagents are interferencefree. Each time a set of samples is extracted, or there is a change in reagents, a method blank should be processed as a safeguard against laboratory contamination.
7.2 Standard quality assurance practices must be used with this method. Field replicates must be collected to measure the precision ofthe sampling technique. Laboratory replicates must be analyzed to establish the precision of the analysis. Fortified samples must be analyzed to establish the accuracy of the analysis.
8. Sample Collection, Preservation, and Handling.
8.1 Grab and composite samples must be collected in glass containers. Conventional sampling practices should be followed, except that the bottle must not be prewashed with sample before collection. Composite samples should be collected in glass containers in accordance with the requirements of the RCRA program. Sampling equipment must be free of tygon and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of collection until extraction. Chemical preservatives should not be used in the field unless more than 24 hours will elapse before delivery to the laboratory. If an aqueous sample is taken and the sample will not be extracted within 48 hours of collection, the sample should be adjusted to a pH range of 6.08.0 with sodium hydroxide or sulfuric acid.
8.3 All samples must be extracted within 7 days and completely analyzed within 30 days of collection.
6This procedure is adopted because standards are not available for most of the CDDs and CDFs and assumes that all the cogeners will show the same response as the unlabelled cogener used as a standard. Although this assumption may not be true in all cases, the error will be small.
9. Extraction and Cleanup Procedures.
9.1 Use an aliquot of 110 g sample of the chemical waste or soil to be analyzed. Soils should be dried using a stream or prepurified nitrogen and pulverized in a ballmill or similar device. Perform this operation in a clear are a with proper hood space. Transfer the sample to a tared 125 ml flint glass bottle (Teflonlined screw cap) and determine the weight of the sample. Add an appropriate quantity of 37Cl labelled 2,3,7,8TCDD (adjust the quantity according to the required minimum detectable concentration), which is employed as an internal standard.
9.2.1 Extract chemical waste samples by adding 10 ml methanol, 40 ml petroleum ether, 50 ml doubly distilled water, and then shaking the mixture for 2 minutes. Tars should be completely dissolved in any of the recommended neat solvents. Activated carbon samples must be extracted with benzene using method 3540 in SW 846 (Test Methods for Evaluating Solid Waste Physical/Chemical Methods, available from G.P.O. Stock #055022810012). Quantitatively transfer the organic extract or dissolved sample toa clean 250 ml flint doubly distilled water and shake for 2 minutes. Discard the aqueous layer and proceed with Step 9.3.
9.2.2 Extract soil samples by adding 40 ml of petroleum ether to the sample, and then shaking for 20 minutes. Quantitatively transfer the organic extract to a clean 250 ml flint glass bottle (Teflonlined screw cap), add 50 ml doubly distilled water and shake for 20 minutes. Discard the aqueous layer and proceed with step 9.3.
9.3 Wash the organic layer with 50 ml of 20% aquoid potassium hydroxide by shaking for 10 minutes and then remove and discard the aqueous layer.
9.4 Wash the organic layer with 50 ml of doubly distilled water by shaking for 2 minutes, and discard the aqueous layer.
9.5 Cautiously add 50 ml concentrated sulfuric acid and shake for 10 minutes. Allow the mixture to stand until layers separate (approximately 10 minutes) and remove and discard the acid layer. Repeat acid washing until no color is visible in the acid layer.
9.6 Add 50 ml doubly distilled water to the organic extract and shake for 2 minutes. Remove and discard the aqueous layer and dry the organic layer by adding 10g of anhydrous sodium sulfate.
9.7 Concentrate the extract to incipient dryness by heating in a 55º C water bath and simultaneously flowing a stream of prepurified nitrogen over the extract. Quantitively transfer the residue to an alumina microcolumn fabricated as follows:
9.7.1 Cut off the top section of a 10 ml disposable Pyrex pipette at the 4.0 ml mark and insert a plug of silanized glass wool into the tip of the lower portion of the pipette.
9.7.2 Add 2.8g of Woelm basic alumina (previously activiated at 600º C overnight and then cooled to room temperature in a dessicator just prior to use).
9.7.3 Transfer sample extract with a small volume of methylene chloride.
9.8 Elute the microcolumn with 10 ml of 3% methylene chlorideinhexane and discard these effluents. Elute the column with 15 ml of 50% methylene chlorideinhexane and concentrate this effluent (55º C water bath, stream of prepurified nitrogen) to about 0.30.5 ml.
9.9 Quantitatively transfer the residue (using methylene chloride to rinse the container) to a silanized ReactiVial (Pierce Chemical Co.). Evaporate, using a stream of prepurified nitrogen, almost to dryness, rinse the walls of the vessel with approximately 0.5 ml methylene chloride, evaporate just to dryness, and tightly cap the vial. Store the vial at 5º C until analysis, at which time the sample is reconstituted by the addition of tridecane.
9.10 Approximately 1 hour before the GC/MA (HRGCLRMS) analysis, dilute the residue in the microreaction vessel with an appropriate quantity of tridecane. Gently swirl the tridecane on the lower portion of the vessel to ensure dissolution of the CDDs and CDFs. Analyze a sample by GC/EC to provide insight into the complexity of the problem, and to determine the manner in which the mass spectrometer should be used. Inject an appropriate aliquot of the sample into the GC/MS instrument, using a syringe.
9.11 If, upon preliminary GC/MS analysis, the sample appears to contain interfering substances which obscure the analyses for CDDs and CFDs, high performance liquid chromatograph (HPLC) cleanup of the extract is accomplished, prior to further GCMS analysis.
10. HPLC Cleanup Procedure7.
10.1 Place approximately 2 ml of hexane in a 50 ml flint glass sample bottle fitted with a Teflonlined cap.
10.2 At the appropriate retention time, position sample bottle to collect the required fraction.
10.3 Add 2 ml of 5% (w/v) sodium carbonate to the sample fraction collected and shake for one minute.
10.4 Quantitatively remove the hexane layer (top layer) and transfer to a microreaction vessel.
10.5 Concentrate the fraction to dryness and retain for further analysis.
11. GC/MS Analysis.
11.1 The following column conditions are recommended: Glass capillary column conditions: SP2250 coated on a 30 m long x 0.25 mm I.D. glass column (Supelco No. 23714, or equivalent) with helium carrier gas at 30 cm/sec linear velocity, run splitless. Column temperature is 210oC. Under these conditions the retention time for TCDDs is about 9.5 minutes. Calibrate the system daily with a minimum three injections of standard mixtures.
11.2 Calculate response factors for standards relative to 37ClTCDD/F (see Section 12).
11.3 Analyze samples with selected ion monitoring of at least two ions form Table 3. Proof of the presence of CDD or CDF exists if the following conditions are met:
11.3.1 The retention time of the peak in the sample must match that in the standard, within the performance specifications of the analytical system.
11.3.2 The ratio of ions must agree within 10% with that of the standard.
11.3.3 The retention time of the peak maximum for the ions of interest must exactly match that of the peak.
11.4 Quantitate the CDD and CDF peaks from the response relative to the 37CL-TCSS/F internal standards. Recovery of the internal standard should be greater than 50 percent.
7For cleanup see also method #8320 or #8330, SW846, Test Methods for Evaluating Solid Waste, Physical/Chemical Methods (1982).
11.5 If a response is obtained for the appropriate set of ions, but is outside the expected ratio, a coeluting impurity may be suspected. In this case, another set of ions characteristic of CDD/CDF molecules should be analyzed. For TCDD a good choice of ions is m/e 259. For TCDF a good choice of ions is m/e 241 and 243. These ions are useful in charterizing the molecular structure to TCDD or TCDF. For analysis of TCDD good analytical technique would require using all four ions, m/e 257, 320, 322, and 328, to verify detection and signal to noise ratio of 5 to 1. Suspected impurities such as DDE, DDD or PCB residues can be confirmed cy checking for their major fragments. These materials can be removed by the cleanup columns. Failure to meet criteria should be explained in the report, or the sample reanalyzed.
11.6 If broad background interference restricts the sensitivity of the GC/MS analysis, the analyst should employ cleanup procedures and reanalyze by GC/MS. See Section 10.0.
11.7 In those circumstances where these procedures do not yield a definitive conclusion, the use of high resolution mass spectrometry is suggested.
12.1 Determine the concentration of individual compounds according to the formula:
A x As
Concentration, ug/gm = _____________
G x Ais Rf
A=ug of internal standard added to the sample8.
G=gm of sample extracted.
As=area of characteristic ion of the compound being quantified.
Ais=area of characteristic ion of the internal standard.
8The proper amount of standard to be used is determined from the calibration curve (See Section 6.0).
9If standards for PCDDs/Fs and HxCDDs/Fs are not available, response factors for ions derived from these congeners are calculated relative to 37CLTCDD/F. The analyst may use response factors for 1,2,3,4 or 2,3,7,8TCDD, 1,2,3,4,7PeCDD, or 1,2,3,4,7,8HxCDDs/Fs, respectively. Implicit in this requirement is the assumption that the same response is obtained from PCDDs/Fs containing the same numbers of chlorine atoms.
Response factors are calculated using data obtained from the analysis of standards according to the formula:
As x Cis
RF = _____________
Ais x Cs
Cis=concentration of the internal standard.
Cs=concentration of the standard compound.
12.2 Report results in micrograms per gram without correction for recovery data. When duplicate and spiked samples are analyzed, all data obtained should be reported.
12.3 Accuracy and Precision. No data available at this time.
TABLE 1: GAS CHROMATOGRAPHY OF TCDD
Column (min.) (ug/kg)1
Glass capilary 9.5 0.003
1Detection limit for liquid samples is 0.003 ug/l. This is calculated from the minimum detectable GC response being equal to five times the GC background noise assuming a l ml effective final volume of the 1 liter sample extract, and a GC injection of 5 microliters. Detection levels apply to both electron capture and GC/MS detection. For further details see 44 FR 69526 (December 3, 1979).
TABLE 2: DFTPP KEY IONS AND ION ABUNDANCE CRITERIA1
Mass Ion abundance criteria
51 3060% of mass 198.
68 Less than 2% of mass 69.
70 Less than 2% of mass 69.
127 4060% of mass l98.
197 Less than 1% of mass 198.
198 Base peak, 100% relative abundance.
199 59% of mass 198.
275 1030% of mass 198.
365 Greater than 1% of mass 198.
441 Present but less than mass 443.
442 Greater than 40% of mass 198.
443 1723% of mass 442.
1J.W. Eichelberger, L.E. Harris, and W.L. Budde, 1975. Reference compound to calibrate ion abundance measurement in gas chromatographymass spectrometry. Analytical Chemistry 47:995.
TABLE 3: LIST OF ACCURATE MASSES MONITORED USING GC SELECTEDION MONITORING, LOW RESOLUTION, MASS SPECTROMETRY FOR SIMULTANEOUS DETERMINATION OF TETRA,PENTA, AND HEXACHLORINATED DIBENZOpDIOXINS AND DIBENZOFURANS
Class of chlorinated dibenzodioxin or dibenzofuran
Number of chlorine substituents (x)
Monitored m/z for dibenzodioxins C12H8x O2Clx
Monitored m/z for dibenzofurans C12H8x OCL2
Approximate theoretical ratio expected on basis of isotopic abundance
1Molecular ion peak.
2Cl4labelled standard peaks.
3Ions which can be monitored in TCDD analyses for confirmation purposes.
APPENDIX XI: PAINT FILTER TEST
"Test Methods for Evaluating Solid Waste, Physical/Chemical Methods," EPA Publication SW846 [Second Edition, 1982 as amended by Update I (April, 1984), and Update II (April, 1985)]. See Appendix III for instructions on how to obtain copies of this publication.
STATUTORY AUTHORITY: 38 M.R.S.A. §1301, et seq.
EFFECTIVE DATE: July 1, 1980
AMENDED: March 23, 1983
June 20, 1983
February 10, 1985
November 30, 1986
March 16, 1994
(ELECTRONIC CONVERSION): May 4, 1996
AMENDED: January 23, 2001
MINOR CORRECTIONS: March 5, 2001
AMENDED: November 3, 2002
July 20, 2004 - filing 2004-272
February 8, 2012 – filing 2012-12
March 11, 2015 – filing 2015-030
Chapter 850: Identification of Hazardous Wastes