Document 3N31VJZg5vgpwg1Nb2gZVL7pJ

% te * f# GENERAL ELECTRIC DIOXIN AND FURAN 6-3-93 PRODUCTION NPC # 783596-784088 GENP# 010785-011277 i CORPORATE ENVIRONMENTAL PROGRAMS GENERAL ELECTRIC COMPANY FAIRFIELD, CONNECTICUT 06431 July 22, 1985 Dr. Daniele DuPont Centre de Sante 1 REQ 1800 Montee Ste-Julie Yarennes, PQ JOL 2 P0 CANADA Dear Dr. DuPont: As we discussed by phone, I am sending you a copy of the program for the dioxin/furan meeting in September. It appears to me to be a very comprehensive program and should be useful for someone making decisions about such chemicals. Very truly yours, SBH :cas Enclosure S. B. Hamilton, ManagerEnvironmental Science & Technology 783596 GENP 010785 783597 GENP 010786 GENERALSfP ELECTRIC GENERAL ELECTR IC COMPANY fairficld. Connecticut oa*ai r S T E P H E N B. HAM ILTO N. JR . AMAII)IMVMaNNINWl SCIENCEAHOTECHNOLOGY August 1, 1985 Ms- Suzanne Rudzinski, Chief Chemical Regulation Branch U.S. Environmental Protection Agency Washington, DC 20460 Dear Ms. Rudzinski: Thank you for the opportunity to review the-package of documents relating to the control of halogenated dioxins and furans that may be produced inadvertently in the manufacture of chemicals- I will address the questions as you have listed them, below. 1- The rationale for developing the list of chemicals seems to be reasonable. The application of the rationale in the list of chemicals has led to some apparent inconsistencies, for example: tetrabromophthalic anhydride (632-79-1) is classified under 2a < (polybrominated phenols and derivatives) which does not seem appropriate. Tetrachlorophthalic anhydride (117-08-8) appears to be correctly classified as "3.11 Does the Group # defined in the document, "Development of List--.," correspond to the "Priority" classification in Table I? If so, the assignment of Priority number sometimes disagrees with the Group #. For example, there are no chemicals given Priority 2d, but 344-07-0, 392-56-3 (both assigned 2a) fit the description- Finally, unless data exists to the contrary, the inclusion of halogenated aliphatics and titanium dioxide in Group 3 seems inappropriate. On the other hand, mono-halogenated phenols should perhaps be elevated to the list for testing because of the possibility that they may be contaminated with higher halogenated phenols. 2- I.can think of no other chemicals that should be tested. 3. I do not see major problems per se in providing only a skeleton of an analytical framework and relying on industry to fill the gap. However, certain necessary guidance is absent from the skeleton. There is no direction as to whether the analytical scheme be devised to determine all PCDFs and PCDDS, only tetra through octa, or only 2,3,7,8- substituted congeners, which are considered to be the structures having the greatest biological activity. 783598 GENP 010787 Ms. Suzanne Rudzinski - 2- August 1, 1985 Is the analysis supposed to be quantitative for unique positional isomers, or only for total tetra, penta, etc., substituted dipxins and*furans? The more specific and the more comprehensive the analysis must be, the more development work, especially in terms of labeled and unlabeled standards, the more time-consuming and costly the analyses must be, and greater the probability that incompatible and unusable information will b"e generated. Re the development of analytical standards: This is a mammoth job since every individual structure quantitatively analyzed should have both a labeled and unlabeled standard for determination of response factors and recoveries. The introduction of the concern about mixed bromo and chi oro structures greatly enlarges the problem. I recommend that this latter-concern be dropped, except for the testing of mixed bromo and chi oro chemicals. I further recommend that EPA take the lead in synthesizing and making available the required standards. - Re the development of cleanup methods. Each chemical represents an individual case and will require development work. Most will probably be solvable - some may be quite recalcitrant and require special considerations. Re reliable data and comparable results. This answer depends to a large extent on the availability of analytical standards and on EPA's simplifying and prioritizing the analytical task. Such a prioritization scheme might be to require the quantitative analysis of the most probable furans and dioxins and a sample representative of the biologically active congeners, such as the 2,3,7,8tetra substituted dioxins and furans. This would greatly simplify the task and improve chances of getting reliable and comparable data. 4. The sensitivity of the mass spectrometer is limited to one picogram (1x10"'2 gram) for each individual structure to be analyzed. At one ppb (lxl0"9), the sample could be analyzed by injecting one mg (lxlO"3 ), the maximum sample size that can be injected into a GC, into a GC/MS system. At 0.1 ppb, the sample would have to be concentrated tenfold. This question boils down to one of cleanup. It should also be recognized that at the ppt level, background contamination could also be a factor. 5. There are probably 4 or 5 laboratories worldwide that could perform these tests on the simple cases now. The limiting factor is availability of standards. 6. The sampling and QA plans seem adequate. 783599 GENP 010788 Ms. Suzanne Rudzinski -3- August 1, 1985 7. General Electric has developed methodologies not referenced in the Draft Final Report dated June 3, 1985, by D. Steele and J. Stanley, the objective of our work was to develop quantitative analyses of PCB fluids for the most biologically active tetra, penta, and hexa chlorinated PCDF structures having the 2,3,7,8substitution backbone. The work involved the synthesis of all tetra through octa unlabeled PCDFs and C-14 labeled samples of those structures to be quantitatively analyzed. The analytical scheme involved GC-GC-MS instrumental analysis, in some cases preceded by cleanup procedures to extract the furans from the PCBs. This work is described in two papers enclosed for your interest (Target Compound Analysis by Two-Dimensional Gas Chromatography-Mass Spectrometry and isomer Specific Analysis of Selected Chlorodibenzofurans), both by W.V. Ligon and R.O. May. This analytical approach was also applied to 2,3,7,8-TCDD in hexachlorophene (7-30-4; 2,2`-Methylenebis (3,4,6-trichlorophenol), which is on your list, and provided a quick, quantitative result (see also Determination of Chlorodibenzofurans and Chlorodibenzodioxins using Two Dimensional Gas Chromatography-Mass Spectrometry, by the same authors, enclosed). 8. I have no additional information to provide regarding biological testing systems. I am familiar with the work of Gierthy, et al, at New York State and believe that this work shows real promise since it detects the strongest biological activity of these chemicals, i.e., keratinization. I believe that EPA should devote research funds to the area of biological testing since it could lead to more meaningful data about the toxic potency represented by the impurities in the tested chemical. I hope these comments will prove useful, and I stand ready to assist further if desired. Very truly yours, SBH:cas Enclosures S. B. Hamilton, ManagerEnvironmental Science & Technology 783600 GENP 010789 Journal o f Chromatography, 294 (1984) 87-98 Elsevier Science Publishers B.V.`, Amsterdam -- Printed n The Netherlands ii CHROM. 16,672 ISOMER SPECIFIC ANALYSIS OF SELECTED CHLORODIBENZOFURANS WOODFIN V. LIGON, Jr.* and RALPH J. MAY General Electric Company, Corporate Research and Development. Schenectady. N Y 12301 (U.S.A.) (First received December I3th, 1983: revised manuscript received February 16th, 1984) SUMMARY Two-dimensional gas chromatography has been used to provide full chro matographic resolution of three chlorodibenzofuran isomers. The materials studied were: 2,3,7,8-tetrachlorodibenzofuran, 2,3,4.7,8-pentachlorodibenzofuran and 1,2,3,7,8,9-hexachlorodibenzofuran. The materials were detected using medium reso lution mass spectrometry. INTRODUCTION Chlorination of dtbenzofuran can potentially yield a total of 135 unique prod ucts. These products vary both in degree of chlorination and in the positional sub stitution of chlorine atoms on the basic ring structure. Certain members of this group which have a 2,3,7,8 substitution pattern are known to have measurable physiological effects in a number of animal species. Other members of the group show little bio logical activity. For this reason, any attempted evaluation of the potential physio logical activity of a given chlorination mixture which is based solely on chemical analysis must utilize techniques which allow quantification of the active components. Analyses of these mixtures by gas chromatography-mass spectrometry (GCMS) is the method of choice because of the ability of this technique to deal with complex mixtures and small amounts. Such analyses have been reported by a number of workers1*. Such work has failed, however, to provide unambiguous chromato graphic resolution of the most interesting species.'Without effective separations, it is not possible to ensure that the observed signals accurately reflect the amounts present. If reliable quantification cannot be ensured correlations of analytical data with clin ical or epidemiological data are meaningless. In this paper, we report the chromatographic resolution of three important components of the set: 2,3,7,8-tetrachlorodibenzofuran, (2378-TCDF); 2,3,4,7,8-pentachlorodibenzofuran, (23478-PenCDF)'* and 1,2,3,7,8,9-hexachlorodibenzofuran, (123789-HexCDF). Two-dimensional GC methods were used to obtain the separa tions. Analysis was by mass spectrometry. 0021-9673/84/503.00 1984 Elsevier Science Publishers B.V. 783601 GENP 010790 Jautnai o f Chromatography. 294 ( 1984) 77-86 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands CHROM. 16.671 TARGET COMPOUND' ANALYSIS BY TWO-DIMENSIONAL GAS CHROMATOGRAPHY-MASS SPECTROMETRY WOODF1N V. LIGON. Jr.* and RALPH J. MAY General Electric Company. Corporate Research and Development. Schenectady. N Y 2201 ( U.S.A.) (First received December 13th, 1983; revised manuscript received February I6ih. 1984) SUMMARY The use of two-dimensional gas chromatography' for the analysis of specific target compounds in complex matrices in combination with mass spectrometry has been investigated. The combination of a high capacity, high polarity packed first gas chromatography column followed with a low capacity. low polarity, high resolution second column has been found useful. A component of interest is switched from the first column into a cold trap and then flash-evaporated into the second column. This combination allows part per billion analyses in complex mixtures such as soil extracts, crude oils, and biological extracts without any prior sample cleanup whatever. Two important advantages arise naturally therefore from this approach: a significant re duction in analysis time and a major improvement in the specificity of the analysis. The method offers a relatively inexpensive yet powerful alternative to mass spectrometry-mass spectrometry. INTRODUCTION In recent years considerable interest has been focused on the development of analytical methods for part per billion (109) and even part per trillion (1012) analyses of selected organic species. Quantification o f such materials in unusually complex matrices such as soil and biological extracts has been especially widely studied. The analyses which have been devised for 2.3,7.8-tetrachlorodibenzodibxin in environ mental samples, are representative of the current analytical approach to such prob lems'*-. Typically a method will involve an arduous multistep purification procedure, including, for example, liquid chromatography and gel permeation chromatography. These preliminary isolation steps are then usually followed by mass Spectrometry or gas chromatography-mass spectrometry (GC-MS1 for detection and quantification.* In the present paper, we show that two-dimensional GC can eliminate much and in many cases all requirements for sample cleanup even in very complex matrices. * Noie by the Editor. The authors insisted on calling this **two dimensional'' against the recom mendations or our reterees (see also l. M. Hals. J. Chrumatoyr.. 187 (1980) 466) and against the judgement of ihe Editor. 0021-9673/84/303.00 (fj 1984 Elsevier Science Publishers B.V. 783602 GENP 010791 Determination of Chlorodibenzofurans and Chlorodibenzodioxins Using TwoDimensional Gas Chromatography-Mass Spectrometry. Woodfin V. Ligon, Jr.* and Ralph J. May General Electric Company Corporate Research and Development Schenectady, New York, 12301 ABSTRACT Polychlorinated biphenyl fluids, flyash extract, and hexachlorophene have been successfully analyzed for 2,3,7,8-tetrachlorodibenzofuran and 2,3,7,8tetrachlorodibenzodioxin at part per billion levels by two dimensional gas chromatography-mass spectrometry without resort to preliminary isolation procedures. Detailed experimental procedures are provided. Systematic errors, fundamental limitations and interferences are documented. INTRODUCTION Chlorodibenzofurans and chlorodibenzodioxins are known to occur in the environment and in certain industrial materials at low levels. Techniques for the determination of these materials have generally involved multi-step isolation procedures followed by gas chromatography-mass spectrometry (GC-MS). The work of Harless et al is typical (1). A review of various methods has been published (2). We wish to report that for certain classes of sample, important congeners of each of these materials, can be determined at part per billion (ppb) levels by two-dimensional-GCMS (GC-GC-MS) without the use of 783603 GENP 010792 Citinophere, Vol. 15, Nos.9-12, pp 1265-1271, 1986 Printed In Great Britain 0045-6535/86 $3.00 + .00 .pergamon Journals Ltd. PYROLYSIS MCD COMBUSTION OF AROCLOR 1254 CONTAMI BATED 01 ELECTRIC FLUIOS SUbe.-t Addis Electric Power Research Institute DIOXIN 85-5th International Symposium on Chlorinated Dioxins and Related Compounds ABSTRACT Pyrolysis and combustion products were determined for several levels of PCB contamination in mineral oil and other dielectric fluids. Yields of PCOFs were roughly proportional to the quantity of PCB 1n the feed. BACKSIOMM) The electric utility Industry as a major purchaser of PC8 s In the past has been left with a legacy of PCB problems of two different dimensions. Although the reality of PCB as a prob lem Is still being debated and evaluated, the Industry 1s retrofllUng or replacing its PCB equipment at a steady pace. A short time ago. there were about 40,000 (_1J PCB transformers 1n utility hands with perhaps an equal or larger number owned by nonutllTty'entities. Several well publicized PCR fires U,_31 in the United States have accelerated the effort toward removal nf the askarel (generic term for PCB or PC8/tr1-/tetrachlorobenzene1 equipment. Two options are open for elimination of PCBs 1n transformers. The first is retroftiling and the second 1s replacement of the transformer. Each method has Its adherents and its detractors. RetrofllUng,. 1n the absence of further improvement of the technology, takes more than a year before a transformer can be reclassified as uncontaminated (1n the United States, below 50 ppm PCB). During most of this time, PCB concentration 1n the transformer fluid Is still above 500 ppm because even 1f one uses the best available technology, between t and 5s of the old liquid remains behind after draining a transformer as thoroughly as possible. It 1s estimated that this residual takes approximately 3 months to come to equilibrium with the replacement fluid. The dilution process must therefore be repeated several times before an appropriately low PCB level 1s reached. We understand that there is considerable,research presently underway to speed up the removal of this trapped material; however, the work has not yet come to fruition. \ Where physically possible, complete replacement of the transformer appears to be the easy way out. However, disposal of the transformer requires draining of the PCB followed by flushing with a solvent. The liquids must then be destroyed in a licensed Incinerator while the transformer carcass must go to a certified landfill. Certified landfills 1n the USA are rapidly disappearing and 1t 1s also considered possible that these may become the next generation of problem cleanup sites. A second area of concern 1s the roughly ?,000,000 (11 mineral oil transformers, which over the years have Inadvertently become contaminated wiTh PCBs at the 50 ppm level or higher. About lOt of these are contaminated above 500 ppm and must be treated as PCB transformers. This pair of problems has fostered a need to learn more about the pyrolysis and combustion of PCR, both 1n Its concentrated state, and at various levels of contamination 1n retrofill fluids, such as silicone and mineral oil. PCS 1s also of Interest as a contaminant 1n tetrachloroethylene, because it 1s a potential retrofill fluid, although 1t 1s more likely to be used as a replacement fluid. WORK P U N A project has been sponsored and partially funded by the Electric Power Research Institute (EPRI1 for the New York State Department of Health to study pyrolysis and combustion 1265 genpoiv m 783604 1266 products of PCBs, both ss a concentrated fluid and as a contaminant 1n mineral oil and aseveral replacement fluids. In-this project we define pyrolysis as the high-temperature decomposition of fluid 1n an oxygen-depleted atmosphere. Combustion 1s defined as ox1 datiqn In the presence of an open.Oame and an excess of air. The wort plan includes the investigation of Aroclor 1254, "neat," as well as at a 50, 500, and 5000 ppm Impurity level 1n mineral oil, silicone and tetrachloroethylene. Since a mixture of tr1- and tetrachlorobenzene Is frequently found as diluent In askarels, these two compounds, both separately and as a blend, also are being Investigated. EQUIPMENT 6 PROCESS Pyrolysis Pyrolysis trials as described by Eadon (4) were conducted using a simple thermostatically controlled apparatus, capable of accommodating a 6-cm diameter metal block within its 9-cm-long heated region. In order to minimize hazards and disposal problems, yet permit sufficient product formation to facilitate detection of PCDDs and PCDFs, pyrolyses were performed on 100 ul samples. In an attempt to differentiate this work from other earlier and ongoing Investigations, as well as to simulate more accurately certain catastrophic Incidents, pyrolyses were conducted at atmospheric pressure. The necessity of containing the starting materials and nongaseous pyrolysis products 1n an open system led to the use of an 8 mn ID x 0.5 m glass tube, sealed at one end, and mounted vertically. The material to be pyrolyzed was deposited at the sealed end, then inserted into a tight-fitting hole in the preheated metal block 1n the heating apparatus. Typically, the liquid refluxed up the inner walls of the tube; the length of the tube and Its comparatively large unheated volume kept the reflux level well below the open end in all experiments. To assure containment, the topmost 5 cm of the tube was chilled in dry 1ce and the end of the tube was connected to a charcoal trap. No visible material was trapped 1n the chilled region, and excellent mass balances were generally observed. Combust1on The design of the combustion apparatus was also constrained by the necessity of assuring that all -discharged gases pass through a trapping system capable of efficient removal of PCBs and any potentially toxic products. The combustion chamber consists of a 1 m quartz tube with a 2 2 - TM 10. One end of the chamber accommodates a modified blast burner and inlet tube through which the end of a 1/16" x 24" (1.6 x 635 mm) syringe needle 1s Introduced via airtight connections. The Inlet tube and needle are mounted to allow sample introduction into the flame of the blast burner. Sample addition Is accom plished using a 10-ml syringe mounted in a worm syringe drive. The combustion chamber 1s mounted In a furnace capable of main Figure 1: Pyrolysis Equipment taining temperatures of 100-1000C in three Independently controlled zones. The effluent of the combustion chamber is passed through a water filled Implnger, then through an XAD-2 packed adsorbent tube which in turn is attached through an orifice to a vacuum U n e . XAD-2 has been shown here and elsewhere to be an efficient trapping agent for polychlorinated dihenzofurans (PCDcs) and dihenzo-p-dioxins (PCDDs). 783605 \ *V.iV\ Collection <--------- Syringe needle 1267 Syringe needle and makeup gas -B-ur-n-e-r [MUM Figure 3: Feed Syringe ANALYSIS Because of the hazardous nature of the compounds to be prepared and to check out equipment expeditiously using rapid analytical techniques, the trials were approached stepwise. Initial runs 1n each case were made with unchlorinated biphenyl to determine the suitability of the.equipment and to find a first approximation of proper operating conditions. These runs were followed by trials using individual PCB congeners known to form specific PCDF mixtures. It was anticipated that the relatively simple products formed could be analyzed by capillary GC/EC or GC/FID after chromatographic cleanup. This technique was successful, except in the pyrolysis of contaminated mineral oil. The mineral oil Itself produced a complex mixture, and U was necessary, after appropriate up-front cleanup, to resort to the use of GC/MS from the start. Having thus optimized as far as possible operating conditions and analytical procedures, runs were finally made using Aroclor 1254 as the test fluid. 783606 GENP 010795 1268 Figure 4: Combustion Product Trap RESULTS Pyrolysis of Aroclor 1254 1n Mineral 011, Silicone, and CjCl^ Under the conditions chosen for this project, conversion of PCBs to PCOFs in the trial runs reached an approximate maximum around 550C. Most of the subsequent runs with Aroclor 1254 were then made at this temperature setting. Where other temperatures were used, they have been noted In the text or tables. All runs were of 15 minutes duration. To minimize'run-to-run experimental variations found 1n the preliminary work, and to produce larger samples for analysis, a series of six runs was made at each set of conditions. The six runs of each set were always made during a single day and combined randomly (prior to cleanup) Into two composites for analysis. Results are tabulated in Tables 1 and 2. Note I TABLE 1: POEF Foraed (r^Graa of B itture1 pyrolyied: Aroclor 1254 In Insulating Fluids Sanple Description 47582 42583 50854 50855 50856 50857 50058 50859 45298 44295 42571 100X Aroclor 1254 100X Aroclor 1254 5,000 ppa^ In alneral o11 5,000 ppa In aln ertl ol 5Q0 ppa 1n alneral o l 500 ppa In alneral ol 50 ppa 1n a ln e ra l'oil 50 ppa 1n alneral ol 5,000 ppa^i* In silicone 5,000 ppa^ in tetracA1oroetny1tne native al* In alneral ol Added Found 2378* TCDF 1,300 965 15.0 11.8 1.6 1.1 - 9.9 1.2 9.3 9.1 Total TCDF 8.200 5,500 45.0 .33.0 - 3.1 . 107.0 2.9 - 1237B2 Base Peak Total PeCDF PeCDF PeCQF - . 16,0 - - 10.000 17.0 62.0 165 17.2 55.0 171 1.8 6.7 17.2 1.9 s.a 18.2 0.4 1.1 . 3.1 0.9 1.7 3.4 4.5 . 70.0 4.8 - 28.0 8ase Peak HiCQF 8,000 5,100 87.0 60.0 6.3 6.8 l.S 1.3 2.2 5.5 Total mCQF 8,000 5.100 162 205 16.3 18.1 3.5 . 6.9 12.5 17:2 (23478) 17.5 * 32.4 (234678) 23.0 Total neCQF 60 - 24.0 14.0 < 10 2.2 - - G.15 0.9 QCOf , . <2 0.23 * _ - 56 (0C30) 47 fl) Cach nlnera 1 o l sap1e rtgrtsents a composite of trirte separate pyrolyses Mcn ere cornai neo prier ta analysis co Inlalit ruruto-run variation. Ali pyrolyses t t S50*C For 15 ninutes. (?) Includes n e lu te rs en 08-5 column. (3) fiWC. (4) Chlorinated Fluorenesf?) at li Mener levai. (5) 600*C. (fi) Minera) ol splked ttn PCDFs to test Hthod. 783607 GENP 010796 .* 'V + 1269 TAOl 2: r a r Famed (ng) per S ra of *Aroclor 1254 fyrolyied In fmuUtlng Fluid S*2 Description 42582 42583 50854 50855 50856 50857 50859 50859 45298 44295 IMS Aroclor 1254 10OX Aroclor 1254 5,000 ppml In Mineral oil 5,000 ppm tn Mineral oil 500 ppa In Mineral oil 500 poa 1n Mineral oil 50 ppM In Mineral oil 50 ppa In Mineral oil 5,000 ppm* in 1 1 1leone 5.0C0 ppm* In tttra c n loroeuiy 1en 2376* Total Tccr TCOF 1,300 965 3,000 2,360 3,200 2,200 1,960 240 1,200 5,500 9,000 6,600 6,200 21,400 580 12378* B ut Path PeCOF PeCOF 3,400 3,440 3,600 3,800 8,000 18.000 900 960 12,400 11,000 13,400 11,600 22,000 34,000 - Total PeCOF 16,000 10,000 33,000 34,200 34,400 36,400 62,000 68,000 14,000 5,600 lata Peek Total ncor HiCPF 8,000 5,100 17.400 12,000 12.600 13.600 30,000 26,000 440 1.100 8.000 5,100 32,400 41,000 - 33,600 36,200 70,000 1,380 2,500 Total HeCDF 60 4,aoo 2,800 4,400 30 ISO oeor 54 46 - (1) Each atneriI alt iimle represent! i esiooslte of three tepiriti pyrolyses. Then were coaetned prior to inalytis to Minimize run-to-run variation. All pyrolyses it 55Q*C for 15 minutes. (2) Includes coeluteri on OB-S coluai. (3) 650`C. (A) 600*C. that Tables 1 and 2 report the same data but expressed in different form. Table 1 shows ng PCDF formed per gram of total mixture pyrolyzed, while Table 2 shows ng PCD? per gram of Aroclor 1254. It may be seen that conversions of PCBs to PCDFs are substantially identical for all congeners and chlorination groups measured for 5000 and 500 ppm, and are less than an order of magnitude different for 1001 PC8 . Conversion appears to increase slightly at 50 ppm, but this may be the result of measuring error due to analytical difficulties at this level. Pyrolyses in silicone and tetrachloroethylene show conversion efficiencies qualitatively similar to mineral oil' for tetra- and penta-CDF, but are apparently dropping off for higher chlorination levels. Added work must be done to confirm these levels. Analysis of the pyrolysis products of PCBs 1n both silicone and tetrachloroethylene yielded qualitative indications of several products related to PCDF/PCDD. In silicone, compounds tentatively identified as chlori nated fluorenes were found in the HS scans. Also, 1n pyrolyzing 1?.3,4,5 pentachlorobiphenyl 1n silicone, a series of compounds CH. (which may be methylated chlorinated fluorene) was found. Chlorine atoms plus methyl-groups total four 1n each case (Figure 5). a TetracWoroethytena adduct ci P C S a Methylated - chlorinated Iluorene Pyrolysis of 2-chloroblphenyl in silicone has fluorene as a major product. This has been compared with an authentic standard. Other * compounds listed above as products 1n the silicone and discussed in the tetrachloroethylene work below will be compared with authentic standards before their presence is considered confirmed. Polychlorinated fluorene Polychlorinated btphenylene Figure 5: Compounds Related to PCDF/PCDD dNiTO 783608 o )---- o -<1 VO >*' `'T.v/ ;x T-.'.' v -1, 1270 In the combustion and pyrolysis of 1254 1n tetraehloroethylene, preliminary work shows that some different tetra- and penta-CDFs ire being formed, compared to "neat" 1254 or 1254 1n mineral oils. Chlorinated fluorene may also be forming. In addition, a compound that may be a reaction product of PCB and tetrachloroethylene 1s possible. Combustion of Aroclor 1254 In Mineral 011, Silicone and C?C14 Conversion for the Isomer groups studied (Table 3) was near optimum for a combustion wall temperature of 550*C and a 3-second residence time. With feed solutions of mineral oil containing Aroclor 1254 at 50, 500, and 5,000 ppm, tr1-, tetra-, and penta-CDF conversion in ng/g PCB fed fell within a narrow range for each of the Isomer groups. Under all conditions, hexa- and hepta-CDF formation was not detectable. Combustion trials with PC8s 1n silicone were not completed successfully. Large quant ities of S10- formed. The finely divided S10g tended to plug the equipment, particu larly the feed needle and the sample collection train. In C.CI^ solution, combustion yields were similar to those found with mineral oil for the -Cl3 Isomers. There were substantially greater yields for the more highly chlori nated isomers, reflecting chlorination of lower chlor inated COFs. Combustion 1n CjCl, under oxygen depletion provides a substantial yield of blphenylenes. TABU 3: Cortwstlon of Aroclor 1254 In ImuUtlng Fluids at SWC * 3 Seconds Solvent Mineral o u Mineral 011 Mineral 011 C2C1a C2C1| c2ci4 sn ic o ftti3) 1254 in sol. PCB's Coabusted destroyed vg/M) X 5000 82-88 500 87-92 SO 88-90 5000 68 500 92 50 72 , ... I PCOFt Fonryd*1* w Cl, CiT 0.72 0.58 0.17 .(2) .(2) 0.4 0.33 0.084 .(2) 0.3 0.3 0.056 _J2) .12) 0.44 t.so 1.35 0.33 o.so 0.24 0.7 0.66 0.23 0.70 0.44 1.32 1.0 0.38 0.16 __ (1) Sued on PCS 1n feed. (2) Non* detected. (3) Runt unsuccessful. Equipment plugged with SI0. OTHER WORK IN PROGRESS Pyrolysis and combustion of tr1- and tetrachlorobenzene are underway In the laboratory. These results will be of value In assessing the potential fire products from askarels where the PC8 Is diluted with these solvents. Preparation of certain synthetic chemicals such as substituted fluorenes is being undertaken. These will be used to confirm the structure of several of the unknowns that appeared during the GC/MS analysis. DISCUSSION Part of the original Impetus 1n this project was to determine whether formation of partial oxidation products of PCB was linear with Increasing dilution in a solvent. This question appears to be answered. Within the constraints of variations in analytical conditions and recoveries during cleanup and analyses of extremely small quantities of material, the linearity should be considered good; 1n most cases, there was no more than *a factor of 10 variation 1n a range of concentrations from 50 ppm to 100X of the askarel tested. Improvements 1n the pyrolysis and combustion schemes during the course of the project provided this degree of resolution. No doubt, this can be Improved upon with further sophistication 1n the work. The results of these laboratory trials are found to be significantly different from those of other workers In the field (-9.). This fact brings with It a word of caution 1n applying any of these results to real-world PCB fires except for use as guiding principles because each real PCB fire, under completely random conditions, 1s different from all others. Even GENP 010798 783609 1271 awithin given H r e , there will be an Infinite range of competing reactions. It can be assumed that only a small portion of the combustion process has optimum conditions, and that the combustion process varies with time, leading to the partial destruction of the various - acombustion by-products. Thus, all of the research being done must be considered as setting a boundary or worst-case condition that gives general direction to the Investigation of the real world. A number of new avenues for exploration have been seen here and 1n the work of others. Among the more significant ones are the finding of relatively large quantities of other chlorinated polycyclic aromatic compounds (PCAs). A second 1s the need for a more rapid amethod of analysis. A bioassay designated the Flat-Cell Assay (10), based on a change 1n 1n vitro growth and morphology of line of skin cells has been Tfiown to be very sensitive and relatively specific for the more toxic of the PCOF and PCDO compounds. The biological mechanism of this effect is considered to be related to the development of chloracne 1n humans after 2,3,7,8-TCDF exposure and 1s thus, a relevant end point. This method 1s already applicable to certain products of combustion. It 1s being modified to detect and assay these products In the presence of solvents such as mineral oil, which currently Interfere with the test. Calibration against a range of chlorinated PCAs would follow. ACKNOWLEDGMENTS The help of K, Aldous, R. Briggs, G. Eadon, 0. Hllker, K. Kidd, A. Narang, R. Narang, P. O'Keefe, and R. Smith are gratefully acknowledged. REFERENCES 1. RPC. Volume H I - Report of the Study of PCBs 1n Equipment Owned by the Electric Utility Industry^ Prepared for the Edison Electric institute, Washington, D.C., February 1982. 2. N. O. K1m and G. Eadon. The Binghamton State Office Building. Workshop Proceedings: PC8 By-product Formation, EPRI CS/EL-4104, July ,1986, p. 5-1. 3. R. L. Wade. Utilization of Quantitative Risk Assessment Techniques In the Development of Decontamination Standards (A Case tudy - San Francisco, California). Ibid, p. 5-16. 4. G. Eadon. Pyrolysis and Combustion of Mineral OilI, Tetrachloroethylene and Silicone 011 Mixtures Containing Aroclor 1254. Ibid., p. 3-24. ' 5. S. E. Swanson, M. D. Erickson, and L. Moody. Products of Thermal Degradation of Dielectric Fluids. Prepared for the U.S. Environmental Protection Agency, Washington, DC, Interim Report No. 2, May 1985. 6 . H. R. 8user, H-P. Bosshardt, and C. Rappe. Formation of Polychlorinated dbenzofurans (PCDFs) from the Pyrolysis of PCBs. Chemosphere A 11 l9>8, pp. 109-119. 7. H. R. Buser and C. Rappe. Formation of Polychlorinated Oibenzofurans (PCDFs) from the Pyrolysis of Individual Isomers. Chemosphere 8(3), 1&79, pp. 157-174. 8 . B. Dellinger, W. Rubey, D, L. Hall, and S. L. Mazer. Laborary Investigation of the High-Temperature Formation and Destruction of PCDFs. Workshop Proceedings: Pt9 By product Formation, e Pki Cs /EL-4104, July 1985, p. 3-17. 9. C. Rappe, S. Harklund. and L-0. Kjeller. Formation of PCDFs from PCBs. Ibid., p. 3-28. '-- ` 10. J. F. Glerthy and I). Crane. In Vitro Bioassy for D1ox1n-L1ke Activity Based on Alterations In Epithelial Cell Proliferation and Morphology. Fundamentals in Applied Toxicology, Vol. V, 1975, 1n press. GENP 783610 O o -J VO PYROLYSIS AND COMBUSTION OF CHLORORSANICS IN DIELECTRIC FLUIDS George Eadon, Ph.D. Wadsworth Center for Laboratories and Research New York State Department of Health Albany, New York Numerous laboratory simulations and several real world Incidents have demonstrated that, under certain conditions, heating of concentrated solutions of polychlori nated biphenyls (PCBs), or trl-* and tetrachlorobenzenes (TCBs) can produce signifi cant concentrations of polychlorinated dibenzodioxins ( P C D D s ) and/or polychlori nated dlbenzofurans (PCDFs). However, at the outset of this EPRI-supported pro ject, little was known about the thermal behavior of these compounds when diluted to the 5000-50 ppm concentration range with various retroflll fluids or askarel substitutes. This laboratory has selected two heating regimens for study with the goal of choosing conditions that were distinct from each other as well as those used by earlier and contemporary investigators and that might bear some similarity to potential real world events. At the outset, however, it was recognized that no feasible sequence of laboratory experiments could effectively model the range of possible real world incidents. One set of experiments ("Pyrolysis") involved heating 100 mg solutions of the mixture of interest in long vertically mounted pyrex tubes sealed at one end. The sealed end containing the sample was inserted for 15 minutes into a snugly fitting thermostatically controlled metal block, maintained at constant temperature in the range of 500C - 650C. Typically under these conditions, the solution refluxed vigorously about half-way up the tube; additional precautions were taken to assure that all materials were retained in the tube. The initial experimental design called for pyrolysis of neat Aroclor 1254 and 5000 ppm solutions of Aroclor 1254 in mineral oil, silicone oil, and tetrachloroethylene at a range of temperatures sufficient to establish the optimum for PCDF formation. Then* 5000, 500, and 50 ppm solutions of Aroclor 1254 in the three fluids were pyrolyzed at the optimal conditions. Similarly,'1,2,4-trichloroben zene, 1 ,2,3,4-tetrachlorobenzene and the 2:1 mixture were pyrolyzed neat and as 5000, 500, and 50 ppm solutions in the three dielectric fluids. The result of greatest practical Interest was the observation that the yield of PCDF, expressed as ug PCDF/g mixture pyrolyzed, decreased by a factor of 100 or more as PCB concen tration was decreased from 1,000,000 to 5000 ppm, and that yields decreased monotonlcally as PCB concentrations were lowered from 5000 to 50 ppm. Much larger decreases were observed as TCB concentrations were lowered to 5000 ppm; in fact, no PCDF or PCDD could be detected in pyrolysates of 5000, 500, or 50 ppm solutions. An alternative way to discuss the PCB and TCB results is in terms of PCDF or PCDD yield per gram of PCB or TCB pyrolyzed. This mode of interpretation, though of leas practical significance, can facilitate*understanding of the mechanisms of PCDF and PCDD formation. On this basis, PCDF yields are only moderately influenced by 783611 GENF 010800 dilution of Aroclor 1254 from 1,000,000 to-5000 ppm with mineral oil or silicone oil. The data contain equivocal evidence for Improved yields from 50 ppm Aroclor 1254 solutions in mineral oil as compared to 500 or 5000 ppm, and clearer evidence for this phenomena in the corresponding silicone oil solutions. This result implies the existence of a destructive pathway for PCB and/or PCDF that is higher than first order for PCB, Interestingly, no evidence for PCB conversion to PCDF was noted in the 5000 ppm TCE solutions; destruction of pre-existing PCDF in the PCB appeared predominant. The TCB data, expressed again as yield PCDF or PCDD per gram of TCB pyrolyzed, suggests that at 5000 ppm concentrations, the rate or yield limiting step must be kinetically blmolecular in TCB concentration. Combustion experiments were performed by motorized syringe-drive Introduction of solution into a natural gas fueled flame mounted in a one meter horizontal quartz tube Itself mounted in a thermostatically controlled oven. The entire gas flow was drawn through a series of traps arranged to ensure complete removal of organics. Some interesting observations Include the fact that combustion of TCE alone can form very small amounts (ng/ml) of dlbenzofurans. ( The pyrolysis of TCE Itself at 650 C also formed low amounts of FCDFs.) The solvent/chloroorganic combination that was most prone to PCDF formation during these combustion experiments was Aroclor 1254 in TCE. Aroclor 1254 in TCE, mineral oil, or silicone oil gave yields of PCDF (expressed as ug PCDF/g PCB) which varied only modestly, if at all, over the 5000-50 ppm concentration range. Yields from combustion of 5000 ppm solutions of TCBs In mineral oil, hexane, or silicone oil were markedly lower than obtained from the corresponding PCB solutions, again suggesting the rate or yield limiting step may be second order in TCB and thus sharply affected by dilution. In con trast, TCB In TCE forms considerable PCDF on combustion, probably resulting from reaction of one TCB derived molecule with multiple TCE-derived molecules. The observation that combustion of PCB or TCB in TCE forms higher yields of PCDFs than the corresponding combustion in mineral oil or silicone oil is in contrast to results obtained during pyrolysis. In those experiments, pyrolysis of Aroclor 1254 in mineral oil or silicone oil clearly gave higher yields than the corresponding reaction in TCE. This dichotomy illustrates what may be the most Important result of these studies. Qualitatively very different results can be obtained from a particular chloroorganlc/dlelectrlc combination depending on the experimental parameters and heating reglmem used. cTK Tqm 783612 O o ooo SCIENTIFIC AMERICAN C 783613 7fi0n r SCIENTIFIC Established 1845 l i m r u n i ^ T February 1986 Volume 254 Number 2 AMERICAN ________ Dioxin Concern that this material is harmful to health or the environment m ay be misplaced. Although i t is toxic to certain animals, evidence is lacking that i t has any serious long-term effect on human beings by Fred H. Tschirley Dioxin. The word evokes a variety 1 of reactions. Much of the pub lic worries that the compound will cause poisoning even at minimal exposure. Toxicologists, knowing the severe toxic effects of dioxin in experi mental animals but being uncertain about comparable serious effects on people, call for more research. Regula tors, who must make decisions based on this conflicting evidence, are left wondering what to do. As one who has spent many years studying the dioxin issue, I hope in this article to provide a useful p'TSpective for making judg ments about the potential hazard of the material. Dioxin is actually a name for a fami ly of chemical compounds. The name refers to their basic structure: two oxy gen atoms joining a pair of benzene rings. Substitution of chlorine atoms for hydrogen atoms on the rings pro duces a chlorinated dioxin, of which there are many. The chlorinated di oxin of interest here is 2^J,7,8-tetrachlorodibenzo-p-dioxin, usually ab breviated to TCDD. It is a by-product of the manufacture of trichlorophenol, which serves in the manufacture of two herbicides (the best-known being 2,4,5-trichlorophenol, or 2,4,5-T, one of the ingredients of Agent Orange) and the antibacterial agent hexachlorophene. TCDD is also produced in a variety of combustion processes. The legacy of Agent Orange, Tunes Beach, Seveso, several industrial acci dents and other instances of human exposure to significant amounts of TCDD is widespread concern about adverse health effects resulting from minimal exposure to the material. Vet none of the many studies directed at this question have -demonstrated that TCDD causes severe chronic human effects. Moreover, not one human death has been attributed to TCDD, even though exposure has been high in a number of cases. The issue points up the broader problem of the difficulty faced by regulators who must make judgments on the basis of incomplete scientific knowledge on the one hand and public fear on the other. TCDD was first recognized in 1957 as a contaminant of 2,4,5-T, when 31 workers involved in the manufacture of the herbicide in West Germany de veloped the dermatologic affliction now called chloracne. It is a skin erup tion resembling acne and takes its name from, the fact that it is caused by exposure to various chlorinated organ ic chemicals. General awareness that TCDD is a `potential hazard to health and the environment arose in 1970, when a House subcommittee held a hearing on "Effects of 2,4,5-T on Man and the Environment.'* The hearing dealt among other things with a study by the Bionetics Research Institute showing that 2,4,5-T caused birth defects in animals. Testimony suggested that the teratogenic component of 2,4,5-T may have been TCDD. In the sample of 2,4,5-T tested by Bionetics the di oxin occurred as a contaminant at an extremely high concentration: 27 8 parts per million. Since then there has been a steady accumulation of information about the sources of TCDD, its environ mental fate and its toxic effects. One well-defined source is the formation of TCDD during the manufacture of 2,4,5-T. The amount of TCDD formed increases as the temperature of the re action and the pH (degree of alkalini ty) increase. In 1977 investigators in the Neth erlands reported that polychlorinat ed dibenzo-p-dloxins (PCDD's) were present in the fly ash from a munici pal incinerator. Typically such an in cinerator burns, among other things, organic wastes containing chlorine. Similar reports soon came from Switz erland, Canada and Japan. It was be lieved the compounds resulted from the condensation of chlorophenols. Later quantitative data demonstrated from a wide variety of combustion sources the presence of TCDD's that could not be explained on the basis of preexisting polychlorinated phenols. These findings led R. R. Bumb of the Dow Chemical Company and 12 of his co-workers to put forward in 1980 the hypothesis that PCDD's can result from trace chemical reactions in fire. The hypothesis has been challenged because the reactions have not been defined. Nevertheless, PCDD's have now been found in the effluent and ash of so many combustion processes that there is no longer serious argument about their formation during combus tion, even though the precise nature of the process remains obscure. Moreover, TCDD has been specifi cally identified in soil and dust from numerous places, in soot from the chimneys of wood furnaces, as resi dues in river fishes (some from riv ers whose watersheds do not have in dustrial operations known to form 29 783614 fO R fiT fi JrKT7T r \ +,AOArn r k PLASTIC TARPAULINS cot much of the open arcs of the Dinmond Alkali Caw plant In Newark* N-J became the land may be contaminated by a chlorinated dloxlnt 2J*7*StetmcbIorodlbeaxoi>dloxta* usually abbreviated as TCDD. From 1943 through 1963 the company manufactured chemicals at the plant* Including the hcrhl* clde 2*4,5-T. TCDD Is a by-product of the manufacture of 2,4,5-T. Federal and state officials had the tarpaulins put down as a precautloa became TCDD is toxic, to a number of animals tested in expertinents. The material has not been shown to have severe cbvoaCc fleet* on human beings. It does produce some short-term efix-cts. 30 I TCDD), as residues in the eggs of her ring gulls and recently in adipose tissue from more than 100 people in Canada, the U.S. and Vietnam, It appears that TCDD is a ubiquitous chemical, par* ticularly in industrialized nations. One might then ask why it was not detected sooner. For one thing, no one looked for it seriously before the 1970's. At the time it would not haige been found anyway, except in unusual circumstances, because analytic chem ists were only able to detect concentra tions of a few parts per million. Since then the detecting equipment has im proved at least a millionfold, so that concentrations of a few parts per tril lion are now detected routinely. In ad dition dilution and the destruction of TCDD by light may reduce concentra tions to undetectable levels. As analyt ic technology improves, one can ex pect that TCDD will be found in many more sites than are currently known. In places where TCDD is protected from light it is an extremely per sistent material. In the early 1970's it was thought the half-life of TCDD (the time required for half of a given amount to be degraded) was about a year; later studies in the U.S.- suggest ed the half-life might be as long as three years. Recent 'reports from Italy raise the possibility that the half-life of TCDD in soil might be 10 years or even more. Although precise data are meager, CHEMICAL STRUCTURE of a dioxin and of TCDD is depicted. The dibenzo-p-dloxia nolecaie (d) consist! of two benzene rin^ joined by two oxygen atoms (light color). From one chlorine atom to eight atoms can substitute for hydrogen atoms (gray) attached to car bons (black) at tfaa numbered positions to form any of the 75 chlorinated dioxins. The chlo rinated dioxin known as TCDD (A) is shown with its four chlorine atoms (dark color). TCDD is known to be strongly held by most soils. The strength of the binding is inferred from the fact that known with the mold to speed up the degrada as high as the guinea pig's. Its intra concentrations of TCDD applied .to tion process. peritoneal level, 3,000 micrograms soil have remained near the surface. Sunlight degrades TCDD rapidly by per kilogram, was about 5,000 times as Even in the sandy soil examined in splitting off the chlorine atoms. The high as the oral dose for the guinea pig. Florida the concentration of TCDD reaction requires a hydrogen donor, Tin the upper 15 centimeters was as high which is usually available in water or as 1,500 nanograms per kilogram 10 in the wax on leaves. Experiments by he reasons for the extreme range of acute (short-term) toxicity may re to 12 years after application. Donald G. Crosby of the University of late to the relative speed of clearance In the most heavily contaminated California at Davis showed that 40 from the body. In the hamster half of a zones near Seveso TCDD has been percent of the TCDD layered on a dose is removed within 15 days, com found at a depth of 136 centimeters. glass plate remained after six hours in pared- with 30 days for the other spe Moreover, the concentrations well be sunlight; the amount was from 25 per cies tested. Even though the hamster is low the surface were slightly but sig cent to negligible when the material much less sensitive than the guinea pig, nificantly higher in 1977 than they was applied as drops oh leaves of the an intraperitoneal LDsq of only.3,000 were in 1976, soon after the accident rubber plant, but on a loam soil the micrograms per kilogram signifies an took place. The presence of soil fis figure was 85 percent extremely toxic material: the toxicity sures does not adequately explain this TCDD is a highly toxic chemical in is comparable to that of the insecti unexpected vertical distribution. experimental animals. The first ani cide parathion. The processes that degrade TCDD mal-toxicity test performed is usually Many essentially acute symptoms in soil are poorly known. Microorgan a determination of the LDbq: the dose have been observed in human beings. isms .do degrade the substance, but at that kills half of a test population. Be They include chloracne, digestive dis a low rate. S. D. Aust of Michigan tween 1973 and 1978 the LDm of orders, effects on some essential en cState University found a wood-decay TCDD was determined for eight spe zyme systems, aches and pains of mus .5-T. sexo- ing fungus (the white mold Phanerochaete chryosporium) that breaks down TCDD without observable mortality cies. The guinea pig was by far the most sensitive species tested: its LDsq for an oral dose was .6 microgram per cles and joints, effects on the nervous system and psychiatric effects. These symptoms have been transitory except t)*-* roak of the organism. The rate of degrada kilogram of body weight. The hamster for a few severe cases of chloracne. y feet. tion is low, but it is conceivable that was the least sensitive animal tested: its Additional tests have measured contaminated sites could be inoculated oral-dose LDsq was about 1,900 times the chronic, or long-term, effects of c > 31 c ( 783616 TCDD in rodents, rabbits and nonhu- man primates. Chloracne, the most I sensitive indicator of human exposure to TCDD, has also appeared in rab bits, nonhuman primates and hairless mice. The skin and body can become dry and scaly. A few species lose hair. Some nonhuman primates lose finger nails and toenails without apparent ev idence of pain. TCDD also causes reproductive ef fects in experimental animals. Cleft palate and abnormalities of the kidney were caused in the offspring of ex posed mice at dosage levels of from one nanogram to three nanograms per kilogram of body weight per day. Sim ilar doses in rats caused the death of fetuses. In monkeys a dose of 1.7 nano grams per kilogram per day for two years caused abortions in four out of seven pregnancies. TCDD is a proved carcinogen in rats and mice. Although the test results vary somewhat, there is fairly good agreement among tests by different in vestigators. The liver is the primary target in both rats and mice, although the brain, the respiratory system and the thyroid gland have also been in volved in a few studies. It is important to recognize that an oncogenic re sponse was reported only after the ani mal had ingested high doses of TCDD over a long period of time. Rats showed no oncogenic response at dos age levels of from one nanogram to 1.4 nanograxns per kilogram per day; mice showed none at dosage levels ranging from one nanogram to 30 nanograms. Moreover, a study by R. J. Kociba of Dow Chemical and his co-workers showed that rats tolerated a daily dose of one nanogram per kilogram per day for two years without showing toxico logic effects. y STEFS IN FORMATION of TCDD daring' the m u m h cta n of 2^4,5-trictdoraphcuol (TCF) i n show, (TCP it uMd to make tbo herbicides 2,4,5-T and Silvex and the antibac terial ageat hexachlorophcu*) la the proccm of making TCP a molecule of 1AA5-tetr> chlorobenzene h hydrolyzed (J) la the pretence of cawtic soda to form 2^5-trichloropheoata (2%Two molecnlca of tho phenate (J) combina to form TCDD (4). They loao atoms of aodlnaa (whit*) and chlorine. The higher the temperature and the alkalinity are, the great er la the amount of TCDD that la formed. When the mixture temperature b above ISO do gm a Ccbha, a hot-forming reaction occurs) it baa been the cause of Industrial accidenta. N evertheless, the findings from the tests on animals intensified con cern about the effects of TCDD on people. On several occasions people have been exposed to "high" levels of TCDD. In this context "high" is a rela tive term because with one exception-- a group of prisoners who volunteered for tests with TCDD--the amount of the material to which a person was ex posed is not accurately known. The criterion I employ here to distinguish high from low exposure is whether or not the exposure results in chloracne. The case of the prisoners is im portant because known amounts of TCDD were applied to their skin. In the first experiment 60 volunteers were treated with concentrations ranging from1200 to 3,000 nanograms (from three to 114 nanograms per kilogram for a 70-kilogram person), and the > 32 finon r a 783617 lonhu. riost Psure n rab- iV"'~ 1 4' t finger:ntev- ve efCleft .idney if - from is per Simth of lanor two ut of irais :sults good it in* nary 0ugh and 1 in - -tant reani- :d d Rats doa* > 1.4 ft[m'^-- 1 of cers ose day :co- the onon pie ; of la- ed of ;xhe ah or ie. n* of In re 18 m m ie dose was repeated two weeks later. The dosages chosen were those that had caused chloracne when they were SPECIES GUINEAPIG (MALE) ' ROUTE ORAL LDm (microgrsrr* p*f kilogram) .6 applied to the ears of rabbits. None of the volunteers developed chloracne, and no other symptoms were ob served. The second experiment in volved 10 volunteer prisoners who GUINEA PIG (FEMALE) RABBIT (MALE.FEMALE) RABBIT (MALEJ^EMALE) RABBIT (MALE.FEMALE) ORAL ORAL ERMAL INTRAPERITONEAL 2.1 11S 275 252-500 were treated with 107,000 nanograms MONKEY(FEMALE) ORAL <70 of TCDD per kilogram. Eight of them developed chloracne, but no other symptoms were noted. From these experiments one can conclude only that TCDD does cause RAT (MALE) RAT (FEMALE) MOUSE (MALE) MOUSE (MALE) ORAL ORAL ORAL INTRAPERITONEAL 22 45-500 <150 120 chloracne in humans when the dose is DOG (MALE) ORAL 30^300 sufficiently high but that people are less sensitive than rabbits. The tests did not identify a threshold for the development of chloracne in human beings--* piece of information that DOG (FEMALE) FROG HAMSTER (MALE,FEMALE) HAMSTER (MALE.FEMALE) ORAL ORAL ORAL INTRAPERITONEAL >100 1.000 1,157 3,000 would be of great value. II The number of people who have ACUTE TOXICITY of TCDD in experimental animals is ascertained on the basis of the been exposed to high levels of TCDD LDsoi the d o u (In micrognnts per kilogram of body weight) that kills half of a test group. cannot be determined accurately, but it must be in the thousands. Alistair Hay of the University of Leeds has es acne (184 cases, 164 of them children tute of Occupational Safety and Health timated that in the chemical industry under the age of' 15), headaches and reported that two of the seven people alone about 2,000 workers have had digestive upsets, but no long-term ef had in fact died of cancers other than high exposure. Low levels of exposure fects such as birth defects and chromo soft-tissue sarcoma. Moreover, the ex have undoubtedly been experienced somal damage have been identified. It posure of three others to TCDD could by people who handle the herbicides is too early to tell whether the inci not be documented. Such findings fall 2,4,5-T and Silvex, in which TCDD dence of cancer is abnormal. far short of being hard evidence for the was a contaminant; by,Vietnam veter An accident in a Monsanto plant in proposition that TCDD causes soft- ans exposed to Agent Orange (50 per Nitro, W.Va., in 1949 exposed more tissue sarcoma. cent of which was 2,4,5-T); by resi than 200 workers to TCDD. Of 122 Other studies also fail to support dents of Times Beach, Mo., where who developed chloracne, 121 were Hardell's hypothesis. In the state of waste oil.that contained TCDD was monitored for the next 30 years. The Washington no consistent pattern of spread on the ground in several places; total number of deaths in that group death due to soft-tissue sarcoma was I by chemical-industry workers making did not differ significantly from that found among occupations in which the products that include the material, expected in the population at large, workers would have been exposed to and by many thousands of people who and there were no excess deaths due TCDD, A study in Finland found no have eaten food (notably fish) contain to cancer or diseases of the circulato cases of the disease among 1,900 peo ing trace amounts of TCDD or have ry system. Similar findings have been ple who applied herbicides, nor was been exposed to fallout from combus made after other industrial accidents, their death rate from any natural cause tion processes that form TCDD. The except for two in which an excess of different from that of the total male total number of individuals with such deaths from cancer was found in small population in Finland. The U.S. Air low exposures probably runs well into groups of the people exposed. Force, in its Ranch Hand study of the millions. A particular type of cancer (soft-tis about 1,200 military personnel who sue sarcoma, a generic term for more sprayed Agent Orange in Vietnam, The possibility of chronic effects than 100 different types of rare cancer) found no cases of soft-tissue sarco from exposure to TCDD causes has become a focus of concern because ma. Finally, examinations by the Vet far greater public concern than that of of a survey of Swedish forestry work erans Administration of 85,000 self- acute effects. An aspect of this prob ers by Lennart Hardell of the Uni selected veterans showed fewer cases lem about which little is known is the versity of Umea. He concluded that of these cancers than the national av effect of protracted exposure to low their exposure to 2,4,5-T (and thus to erage would suggest. levels of the material, as might occur TCDD) had caused six times the nor Reproductive effects are also a sub in an occupational setting or from inci mal incidence of soft-tissue sarcoma. ject of concern because of the animal dental exposures to, for example, the This study led to an investigation findings. The most celebrated case al fallout of combustion effluents or to of chemical-plant workers in the U.S. leging such effects in humans is com fish that contain low levels of TCDD. who had been exposed to 2,4,5-T and monly known as the Alsea II study, A look at some of the major expo other chemicals. Seven apparent cases made by .the U.S. Environmental Pro sures to TCDD, approximately in or of soft-tissue sarcoma were discov tection Agency. The study reported a der of their severity, reveals few if any ered, raising the level of concern sub link between the spraying of 2,4,5-T unambiguous chronic effects. The in stantially. Subsequent events have em on foliage and spontaneous abortion dustrial accident at Seveso in 1976 ex phasized the difficulties in accurate di among pregnant women in Alsea, Ore. posed some 37,000 people of all ages agnosis of soft-tissue sarcoma and in This study has come in for much to considerable amounts of TCDD. accurate identification of exposed indi criticism; notably by an interdiscipli A relatively small number of them viduals. At a conference in 1983 Mari nary group at Oregon State Universi showed transient effects such as chlor lyn A. Fingerhut of the National Insti ty. The group concluded that an assc- 33 (JW V/ ,y I ciation between herbicide spraying and spontaneous abortion could not be shown from the data relied on by the agency. Other studies--in , Australia. Hungary, New Zealand and the U .S.failed toJ&nd a link between the use of 2,4,5-T and birth defects. Because of the extreme acute toxicity and the multiple chronic effects of TCDD in animals, regulatory agencies have had to consider what to do in or* der to protect people from exposure to the material. Such agencies must ex* trapolate animal data to human beings in all but a few instances, in spite of the fact that the validity of this type of extrapolation has not been ascer tained. Compounding the difficulty is the lack of a simple, accurate method for determining whether and at what level TCDD occurs in the tissues of exposed individuals. (The present test requires a surgical procedure to obtain samples of the liver and fat tissues where TCDD resides.) Without such information, a dose-response relation cannot be established. TCDD has been called the most tox ic synthetic chemical known to man. If its acute toxicity'' the guinea pig, and even the rat and the mouse, is the criterion, the statement is probably correct. If Its considerably lower toxic ity to the hamster is the criterion, how ever, the statement would surely not be true. Vet there is no need to quib ble: TCDD is unquestionably a chemi cal of supreme toxicity to experimen tal animals. Moreover, severe chronic effects from low dosages have also been demonstrated in experimental animals. Therefore the concern about its effects on human health and the en vironment is understandable. When toxic chemicals are at issue, a regulatory agency has few options be yond extrapolating animal data to hu DATE 1949 WORKERS EXPOSED 250 LOCATION OP ACCIDENT Monsanto plant in Nitro, W.Va. REMARKS 122 cases of efttoraene being studied; 32 deaths v. 46.4 expected; no excess deaths from malignant neoplasms or circulatory disease 1953 73 BASF plant in Lucfwigshafan 55 cases of chtaracne, 42 severs; 17 deaths v. 11 to 25 expected (four gastrointestinal cancers and twooat-con lung cancers); most common injuries were impaired senses and liver damage 1950 7 flhons-Poulenc 17 cases of chforacne, also elevated lipid plant in and cholesterol levels in the blood Granoblo 1963 106 1964 81 1965-69 78 I960 ? 1968 90 1978 156 NVPhilips plant in Amsterdam 44chtorecne cases (42 severs), of whom 21 also had internal damage or central-nervoussystem disturbances; eight deaths (six possible myocardial infarctions); some symptoms of fatigue DowChemical plant in Midland. Midi. 49 cases of chtorocrw; four v. 7.8 expected deaths; three cancer deaths v. 1.5 expect ed. one* soft-tissue sarcoma Continuing leaks in Spoiarie plant near Prague 78 casas of chioracne; five deaths; many of the 50 workers studied tor more than 10 years have hypertension, elevated Mood teveta of lipid and cholesterol, prediabetes; significant amounts of severe liver and newofogie damage Rhono-Pouianc plant tn Granoblo Coalite 4 Chemie* plant In Derbyshire ICMESA plant in Savoao, Italy 21 chioracne cases 79chioracne emsee; onedeath fromooronary ' thrombosis Workers are being studied along with exposed townspeople; more than 500 residents treated for presumed toxic symptoms;134 confirmed chioracne cases; overall mortality rate normal INDUSTRIAL ACCIDENTS h a n exposed more than 804 workers to ilg n iic u l amounts of TCDD. The accident at Sereso in 1976 also exposed some 37,000 residents of nearby commoaltfen The data are-hosed on a study made by tbe American Medical Association. mans. Vet health effects on humans are rarely proved in the case of environ mental chemicals to which the public is variably exposed at subacute levels that can only be estimated (and then only in the crudest approximation). A case in point is aflatoxin, the product of a mold that develops commonly in stored oilseed crops such as peanuts. In animal tests aflatoxin is one of the most potent carcinogens known, but it has not yet been proved to have this effect in human beings. Diversity in reaction to stimuli is a hallmark of biological organisms. Re actions to toxins are no exception to the general rule. People may be more, less or equally sensitive to a given tox in than an experimental animal is. Ex trapolation is neither art nor science; it is simply the most rational way to as sess a hazard in the absence of defin itive data. Hence regulatory actions continue to be based on the animal data even when the human data, al though they are not definitive, may be sufficiently compelling to allow a scientific judgment that the hazard to people has been overestimated. That appears to be the case with TCDD. Investigators are in general agreement that TCDD is less toxic to humans than it is to experimental ani mals, but the available information is not sufficiently compelling to stimu late a change in regulatory posture toward either more or less restriction of exposure to the material. 1 suspect that the direct evidence of TCDD's ef fects on humans will never be either more or less comp- ng than it now is. The public's perception of a toxin is an important determinant of the pos ture taken by a regulatory agency. The public has heard a great deal about both the acute and the chronic effects of TCDD on experimental animals but little about the substantial body of data showing that human beings are less sensitive. The initial reports of TCDD's acute toxicity, followed by reports of its carcogenicity and repro ductive effects, have instilled a public fear that probably cannot be dispelled even by adequate information about the countervailing experience with hu man beings. The regulatory agency is therefore left in the position of having to deal with not only the available evi dence but also the public's fear. The U.S. Environmental Protection Agency has responded to the pub lic's fear with a number of regulations intended to control the formation and release of TCDD and to limit individ ual exposure to it. Those regulations could be made stronger or weaker on the basis of new evidence. What the 34 3 1I i 1 I I -G ENP 010808 ins are iviron- 'pubtic levels agency has not done--and might be said to have a responsibility to do--is to try to dispel the public's fear on the basis of the evidence that exposure to low concentrations of TCDD in the en $180 million, and many people believe the settlement was an admission of guilt by the chemical companies that manufactured Agent Orange. Appar ently few people know of Federal matters before all the major studies now in progress have been completed. Additional expenditures of both time and money have been made by chemi cal companies, private organizations vironment appears not to have serious Judge Jack Br Weinstein's statement to and government agencies. The total chronic effects on human beings. the attorneys for the plaintiffs that "in outlay is a tremendous amount for an mly in anuts. of the but it e this The TCDD case is further exacer bated by its relation to the defoliation program in Vietnam, an unpopular program in an unpopular war. The many and diverse health effects al leged by Vietnam veterans to have no case have you shown causality for the health effects alleged." A troublesome mafter exemplified by the TCDD issue is the appropriate utilization of scientific resources. A. L. Young of the Office of Science and issue of questionable importance. Two years ago a conference on di oxin at Michigan State concluded that the TCDD case is relatively less im portant than a number of other issues and that the nation's limited scientific li is.a j been caused by exposure to Agent Or Technology Policy has calculated that resources should be devoted to the is s. Re ange have been widely publicized. The more than a billion dollars will have sues posing a greater threat. On the ion to j public is generally aware that the com- been spent by the Federal Government basis of the evidence turned up so far, more, \ plaints were settled out of court for for research and other dioxin-related the conclusion is still valid. a tox- s. Ex- nee; it to as- defin- itions aimai a, al- may ow a rd to with neral cic to I ani on is imu sture 6080T0<M3> idler w is; .in is pos- The bout re c t s nais 'Ody ings arts d by pro- blic lied lOUt huy 'ing evi- :ion ubons ind /idons on the DECOMPOSITION OF TCDD takes place when the ultraviolet reaction are shown. In soil, where sunlight cannot get at the is sonlight splits off the molecule's chlorine atoms. Six slept In the el, TCDD tends to be quite persistent, enduring as long as 10 783620 35 CHEMICAL MANUFACTURERS ASSOCIATION REceIVE M)G 2 B 198! S. E. HAMlUT August 26, 1986 TO: Dibenzofurans/Dibenzodioxins Program Panel FROM: C . Stack n. ..v-v'T'---- RE: Recent Filings and Documents For your files the following is enclosed: * Panel comments on the EPA Health Assessment Document on Dibenzofurans and cover letters to Drs. Mukerjee and Byrd; * Panel comments on EPA's Toxicity Equivalency Factors Scheme and cover letter to Dr. Yosi.; * The Panel's "Considerations for Assessing Potential Human Risk from Exposures to 2 , 3 , 7 , 8-Tetrachlorodibenzodioxin" sent .` to all of above; * The April 1986 draft of EPA's "interim Procedures for Estimating Risks Associated with Exposures to Mixtures of Chlorinated Dibenzo-p-dioxins and Dibenzofurans (CDDs and DCFs)" 783621 Formerly Manufacturing Chemists Association--Serving the Chemical Industry Since 1872. 2501 M S treet. NW W ashington. DC 20037 Telephone 202/887-1 100 Telex 89617 (CMA WSH) CHEMICAL MANUFACTURERS ASSOCIATION GERALDINE V. COX. Ph.O. Vice President Technical Director August 25, 1986 BY HAND Terry F. Yosie, Director Science Advisory Board, A-101-F Environmental Protection Agency 401 M Street, S.W. Washington, D.C. 20460 Dear Mr. Yosie: The Dibenzofurans and Dibenzodioxins Panel of theChemical Manufacturers Association (CMA) requests the opportunity to make a five-minute presentation at the Science Advisory Board's (SAB's) Dioxin Toxic Equivalency Methodology Subcommittee meeting on September 8-9, 1986. Dr. James Wilson will make the presentation on behalf of the Panel and will comment on EPA's "Interim Procedures for Estimating Risks Associated with Exposures to Mixtures of Chlorinated Dibenzo-p-Dioxins and Dibenzofurans (CDDs and CDF's), the subject of the SAB Subcommittee meeting. We are submitting with this letter a copy of the Panel's written comments on EPA's Toxic Equivalency Factor (TEF) Scheme for predicting risks from chlorinated dibenzo dioxins and dibenzofurans. Dr. Wilson's presentation will briefly summarize these comments. We are also enclosing with this letter (Attachment I) a copy of a working paper prepared by the CMA Panel entitled "Considerations for Assessing the Potential Human Risk from Exposures to 2,3,7,8-Tetrachlorodibenzodioxin." EPA's Toxic Equivalency Factor Scheme represents an effort by EPA to develop risk values for a number of chlorodibenzodioxins and chlorodibenzofurans in relationship to the toxicity of 2,3,7,8-TCDD. It thus is obviously important that EPA accu rately and reliably determine that underlying T C D D value. GENP 010811 783622 Formerly Manufacturing Chem ists A ssociation-- Serving the Chemical Industry Since 1872. - 2501 M Street, NW Washington, D C 20037 Telephone 202/887-1260 Telex 89817 (CMA Terry F . Yosie, Director August 25, 1,986 Page 2 The Panel's working paper discusses the variance between :PA's 19 85 risk assessment of TCDD and assessments of other international expert panels. It concludes that EPA over estimates the potency of TCDD by approximately three orders of magnitude. Because the underlying TCDD risk is an in tegral part of the TEF Scheme, we invite the Subcommittee's attention to this second document. We are sending copies of these comments directly to all members of the Toxic Equivalency Factor Methodology Subcommittee. If there are any questions, please call Dr. Carol Stack (887-1196) of my staff. Sincerely yours Enclosures cc w/ enes.: Geraldine V. Cox, Ph.D. Vice President Technical Director Members of the TEF Methodology Subcommittee 783623 GENP 010812 A ttach m en t I COMMENTS OF THE CHEMICAL MANUFACTURERS ASSOCIATION DIBENZOFURANS/DIBENZODIOXINS PANEL ON E P A 1S TOXICITY EQUIVALENCY FACTORS (TEF) SCHEME August 25, 1986 783624 COMMENTS OF"THE CHEMICAL MANUFACTURERS ASSOCIATION DIBENZOFURANS/DIBENZODIOXINS PANEL ON E P A 1S TOXICITY EQUIVALENCY FACTORS (TEF) SCHEME The Chemical Manufacturers Association (CMA) Dibenzofurans/Dibenzodioxins Panel urges the Scientific Advisory Board to recognize two features of the Toxicity Equivalency Factor (TEF) schemes used by EPA to estimate relative toxicity of different polychlorinated dibenzodioxins and dibenzofurans (PCDDs and PCDFs). First, none of them is truly valid, nor strongly supported by scientific evidence. The relative activity of different compounds in the dibenzodioxin and dibenzofuran series varies, from species to species and from biological endpoint to biological endpoint, in ways that are not predictable with our present state of knowledge. Second, the TEF system proposed by Barnes and Beilin, which EPA has used in several regulatory contexts, provides an approximation that can be useful for risk management purposes until more precise information can be developed. At the same time, several downward adjustments to the values attributed to certain congeners by the scheme are necessary to make it more consistent with the available data base. I. ALL TEF SCHEMES LACK SCIENTIFIC VALIDITY. No strong scientific support exists for a TEF scheme for chlorinated dibenzodioxins and dibenzofurans. We focus on the chlorodibenzofurans in this discussion, because they GENP 010814 783625 2 make up a substantial fraction of the FCDD/PCDF compounds present in hot exit gas streams. However, similar remarks also apply to the chlorodibenzodioxins. The compound 2,3,7,8-tetrachlorodibenzofuran (TCDF) is closely similar to 2,3,7,8-tetrachlorodibenzodioxiri (TCDD) in size and shape, and according tc the paradigm currently accepted by many scientists working in this field, should exhibit similar biological activity. This follows from the theory- of receptor-modulated toxicity, in which binding to a receptor protein is the first step in the process that eventually leads to the very unusual toxicities exhibited by these compounds. In his cell-culture systems, Dr. William Greenlee of the Chemical Industry Institute of Toxicology indeed found 2,3,7,8-TCDF exhibits the same potency as 2,3,7,8-TCDD. On the other hand, in several systems studied by Safe, et a l ,, TCDF exhibits only one-tenth to three-tenths the activity of TCDD. Birnbaum, et a l . , also found in the B6C3F1 mouse that 2,3,7,8-TCDF had one-thirtieth the activi ty of TCDD when the endpoint was hydronephrosis or cleft palate in the fetuses of treated dams. Birnbaum and her co-workers also observed that the relative activities of these two compounds in that mouse strain parallels the relative rates at which they are metabolized and cleared from the body, and that clearance activity was higher in pregnant mice. 783626 3 Accordingly, at a minimum, the relative activities of different compounds in the dibenzofurans series depend on two factors -- the intrinsic receptor-binding activity and the clearance rates in the organism involved. Other factors not yet identified may also contribute. Greenlee's cellculture system is capable of little or no metabolic activ ity, and expresses something nearer to the intrinsic activ ity than do most other systems yet studied. Conversely, the studies of Birnbaum and her colleagues at NIEHS show the maximal effects of clearance rate on activity. Safe and his co-workers, as well as many others, have made numerous attempts to derive a self-consistent, regular relationship between the activities of PCDDs and PCDFs so that reliable predictions could-be made. These efforts are doomed to failure. EPA should not be encouraged to pursue this line of inquiry. II. THE EPA TEF SCHEME NONETHELESS HAS REGULATORY RISK MANAGEMENT UTILITY AND THUS SHOULD BE AMENDED SLIGHTLY TO REFLECT AVAILABLE DATA MORE CLOSELY. CMA recognizes that from time to time circumstances will arise that require risk management decisions to be made on health risks posed by mixtures of PCDDs and PCDFs. Because good data will often not be available on a mixture close in composition to the one at issue, use of a TEF scheme can be justified. GENP 010816 783627 4 "CMA supports use of the Barne-s-Be 11 in TEF scheme for regulatory risk management purposes. Among the several schemes which have been proposed, this one best reflects the complexity of the toxicologic issues and makes use of more of the information available than other schemes. At the same time CMA urges the Science Advisory Board to recommend minor revisions to this EPA scheme so that it will better account for well-known toxicity differences in the tetrachlorodibenzodioxins and tetrachlorodibenzofurans. The present scheme assigns a single "toxicity equiva lent" value for all TCDDs other than the 2,3,7,8-isomer. Existing data indicate, however, that the non-2,3,7,8 tetrachlorodibenzodioxins are not equivalently potent. It is well known that certain non-2,3,7,8 -TCDD isomers, such as the 1,3,7,8-isomer, exhibit activity in several in vitro tests. (Few in vivo results have been reported). The value assigned by Barnes-Bellin for all other TCDDs may be appropriate for these compounds. However, it is also well known that other members of this series, such as 1,3,6 ,8 - T CDD, exhibit activities orders of magnitude lower than 2,3,7,8 -TCDD . In guinea pigs , for instance, the L D 5 0 for 2,3,7,8 -TCDD is 0.5 to 2.0 ug/kg, while the L D 5 0 for 1,3,6 ,8 -TCDD is >1.5 x 10 yg/kg, that is greater than 15 grams 1,3,6 ,8 -TCDD per kg body mass. Probably two-thirds of the 22 different TCDDs will be found, like 1,3,6 ,8 -TCDD, to be practically non-toxic. GENP 0 10817 783628 5 "If all of the nearly non-toxic TCDDs were rare and the more toxic isomers common, a scheme that treated all TCDDs equivalently would have little adverse consequence. However, the 1,3,6 ,8-/1,3,7,9-TCDD pair together predominate among tetra isomers in environmental sources of mixed PCDDs and PCDFs. At least these two TCDD isomers should be assigned a TEF value of zero. Similar considerations apply to the tetrachlorodibenzofurans. If these compounds are to be lumped in one category in a TEF scheme, the values assigned should reflect the fact that in most mixtures the non-toxic isomers make up the majority. Similarly, the data now available on pentachlorodibenzofurans should be reviewed to see if the BarnesBellin value assigned to this class reflects their activity. Finally, CMA urges the SAB to recognize that the composition of PCDDs/PCDFs on incinerator-derived particu lates to which humans are ultimately exposed differs marked ly from the compositions measured in hot gas streams. It is likely that the particulate composition is nearly enough constant that biological effects of this mixture could be measured directly. This would, of course, obviate the need for any indirect TEF estimate of toxicity. GENP 010818 783629 CHEMICAL MANUFACTURERS ASSOCIATION G E R A LD IN E V. COX. Ph.D. Vice President Technical Director August 22, 1986 BY HAND Dr. Daniel Byrd, III Science Advisory Board, A-101-F Environmental Protection Agency 499 South Capitol St., S.W. Washington, D.C. 20460 Dear Dr. Byrd: The Dibenzofurans and Dibenzodioxins Panel of the Chemical Manufacturers Association (CMA) requests the opportunity to make a ten to fifteen minute presentation at the September % , 1986, meeting of the Halogenated Organics Subcommittee of the Environmental Health Committee of the Science Advisory Board (SAB) to be held in Kansas City. The presentation will be made on behalf of the Panel by Dr. James Wilson. The Panel has submitted comments to Dr. Debdas Mukerjee of EPA's Office of Environmental Criteria and Assessment on the revised draft Health Assessment Document for Chlorinated Dibenzofurans, which will be the subject of the SAB meeting on September 2. A copy of those comments is attached to this letter. At the SAB meeting, Dr. Wilson intends to summarize briefly CMA's concerns about the draft Health Assessment Document. ENP 010819 783630 Formerly Manufacturing Chem ists Association-- Serving the Chemical Industry Since 1872. 2501 M Street, N W Washington, DC 20037 Telephone 202/887-1260 Telex 89617 (CMA W SH ) Dr. Daniel Byrd, H i August 22, 1986 Page 2 We are sending copies of the CMA Panel comments on the draft Health Assessment Document directly to members of the Halogenated Organics Subcommittee. If there are any questions, please call Dr. Carol Stack (887-1196) of my staff. Sincerely yours, Enclosure cc w/enc.: Vice President-Technical Director Dr. Debdas Mukerjee Members of the Halogenated Organics Subcommittee 783631 GENP 010820 za CHEMICAL MANUFACTURERS ASSOCIATION GERALDINE V. COX, Ph.O Vice President Technical Director August 22, 1986 VIA FEDERAL EXPRESS Dr. Debdas Mukerjee Project Officer for Polychlorinated Dibenzofurans Environmental Criteria and Assessment Office Environmental Protection Agency 26 W. St. Clair Street Cincinnati, Ohio 45268 Re: Health Assessment Document for Polychlorinated Dibenzofurans (51 Fed. Reg. 26052, July 18, 1986) Dear Dr. Mukerjee: The Chemical Manufacturers Association Dibenzofurans and Dibenzodioxins Panel (CMA) appreciates this opportunity to comment on the June 1986 External Review Draft of the "Health Assessment Document for Polychlorinated Dibenzo furans" prepared for the Office of Air Quality Planning Standards. C M A 1s comments focus upon the risk assessment in Chapter 8 of the document and the underlying health data upon which this risk assessment is based. We find that the risk assessment overestimates potential risks to chlorinated dibenzofurans by: (1) Misinterpreting the toxicology data on which it relies (the Murray three-generation 2,3,7,8-TCDD study) to conclude that 0 . 0 0 1 g/kg/day is a Lowest-Observed-Adverse-Effect Level (LOAEL), when in fact that dose was a No-Observed-Adverse Effect Level (NOAEL); and NP 010821 783632 Formerly Manufacturing Chem ists Association-- Serving the Chemical Industry Since 1872. 2501 M Street, NW Washington, DC 20037 Telephone 202/887-1260 Telex'89617 (CMA W SH) Dr. Debdas Mukerjee August 22, 1986 Page 2 (2) Underestimating exposures to chlorinated dibenzofurans of persons involved in the Yusho, Japan, incident, thus overestimating potency per unit exposure. In addition, we find that the document overstates by a factor of approximately 1 0 0 the likely dibenzofuran concentration in polychlorinated biphenyls found in the United States. Each of these points are detailed in our Comments (Attachment I). The draft document does not attempt to determine a unit potency value for potential carcinogenicity of any chlorinated dibenzofurans. CMA agrees development of any such value would be inappropriate given the absence of any*long-term studies indicating chlorinated dibenzofurans are carcinogenic. Nonetheless, we are aware that a number of scientists have proposed development of such potency values by analogy to the carcinogenic potency of 2,3,7,8 -TCDD. We invite your attention to this document. > We would appreciate your sending copies of our comments to members of the peer review panel that met in late May to discuss the chlorinated dibenzofurans criteria document. If there are any questions, please call Dr. Carol Stack of my staff (202/887-1196). Sincerely, Geraldine V. Cox, Ph.D. Vice President-Technical Director Enclosures - cc w/encs.: Members of the EPA Science Advisory Board Environmental Health Committee Halogenated Organics Subcommittee GENP 010822 783633 A ttach m en t I COMMENTS OF THE CHEMICAL MANUFACTURERS ASSOCIATION ON EPA'S HEALTH ASSESSMENT DOCUMENT fc*OR CHLORINATED DIBENZOFURANS August 22, 1986 GENP 010823 783634 COMMENTS O'F" THE CHEMICAL MANUFACTURERS A SSOCIA TION ON EPA'S HEALTH ASSESSMENT DOCUMENT FOR CHLORINATED DIBENZOFURANS Table of Contents Page INTRODUCTION .............................................. 1 I. THE DOSE OF 0.001 ug/kg/DAY IN THE MURRAY THREE-GENERATION TCDD REPRODUCTIVE STUDY WAS A NOAEL, RATHER THAN A LOAEL ............ 2 II. HUMAN DATA FROM THE YUSHO INCIDENT CONFIRM THAT THE REFERENCE DOSES FOR 2,3,4,7,8 -PeCDF AND 2,3,7,8-TCDF DERIVED FROM ANIMAL DATA ARE QUITE. PROTECTIVE 1.................. 5 A. Detailed Data Exist on Human Exposures to and Effects of PCDFs in the Yusho Incident ................... 6 B. EPA Misinterprets the Yusho Data ............................................ 8 C. Properly Interpreted, the Yusho Data Show an Acceptable Lifetime Average Daily Dose For Total PCDFs of 500 Picograms/Kilogram Day ............................................ . 11 III. THE CONCENTRATION OF CHLORINATED DIBENZOFURANS IN, AND POTENTIAL EXPOSURES TO, PCBS ARE OVERESTIMATED ... ;......... 17 REFERENCES ..................................... .......... 22 GEHP 0 1 0 8 2 4 783635 COMMENTS OF THE CHEMICAL MANUFACTURERS ASSOCIATION ON EP A 'S HEALTH ASSESSMENT DOCUMENT FOR CHLORINATED DIBEN2QFURANS INTRODUCTION EPA's Health Assessment Document for Chlorinated Diben zofurans (HAD) concludes with a risk assessment that derives Reference Doses (RfDs) for two chlorinated dibenzofurans -- 2 , 3 , 4 7 , 8 -PeCDF and 2,3,7,8 -TCDF. These RfDs are derived by analogy to results of a three-generation reproductive study, not of any chlorinated dibenzofuran, but rather of 2,3,7,8TCDD. EPA also compares the values determined from the TCDD data base to data from the Yusho incident in which humans ingested chlorinated dibenzofurans. The Agency further de termines some potential exposure routes, including exposures from polychlorinated biphenyls contaminated with chlorinated dibenzofurans. The CMA Dibenzofurans/Dibenzodioxins Panel finds flaws in each of these three aspects of the HAD. CMA considers the appropriateness of deriving any Ref erence Doses for chlorinated dibenzofurans from data on TCDD to be less than optimal. Great uncertainty exists in at tempting to estimate risks of one chemical based on data from another chemical. At the same time, we recognize that EPA has often in recent years used a Toxic Equivalency Fac tor (TEF) scheme in risk management decision-making for es timating the potency of dibenzofurans and dibenzodioxins GENP 010825 783636 2 other than 2,3,7,8-TCDD given the"*limi ted data on these oth er compounds. Employment of a TEF scheme i's appropriate for some reg ulatory purposes, but it always must be recognized that the use of such schemes is merely a surrogate and should not be misinterpreted as a conclusion that there is in fact evi dence that non-tested chemicals cause the predicted effects. Accordingly, we discuss in these comments the' manner in which- EPA employs the TCDD data to reach conclusions about chlorinated dibenzofuran potency.. As detailed below, we find the Agency has misinterpreted the TCDD data on which it relies and that the effect is to calculate an RfD that is at least ten times too low. We also comment on the Agency's employment of the human data from the Yusho incident. As our detailed discussion shows, the Agency's incorrect interpretation of these data leads to an overestimate of risk by more than 1 0 times and further demonstrates the likelihood the RfD derived from the TCDD animal data may be too low. Finally, we comment on the exaggerated estimate of chlorinated dibenzofuran exposures from potential exposures to polychlorinated biphenyls. The HAD both incorrectly states the contamination level found in U . S . commercial PCB products and inappropriately constructs exposure scenarios that fail to take into account the significant decline in potential PCB exposures that has occured over the past 15 783637 GENP 010826 3 years since manufacture was voluntarily curtailed and legis latively prohibited. I. THE DOSE OF 0.001 yg/kg/DAY IN THE MURRAY THREE-GENERATION TCDD REPRODUCTIVE STUDY WAS A NOAEL, RATHER THAN A LOAEL. Central to EPA's development of a Reference Dose (RfD) for chlorinated dibenzofurans is its declaration (at p. 8-4) that the three-generation Murray et al_. (1979) study found a LOAEL for 2,3,7,8-TCDD of 0.001 yg/kg/day. To the con trary, 0.001 yg/kg/day was a NOAEL in this study. The erroneous interpretation of the Murray TCDD study is based solely on statistical re-evaluation of the data by Nisbet and Paxton (1982) and is contrary to the interpreta tions of the study rendered by the original authors (Murray et a l . 1979), the FIFRA' Scientific Advisory Panel (1979), the Ontario Ministry of the Environment Expert Panel on Dioxins (1984) , and essentially all experts who have evaluated that study. The overwhelming scientific opinion is that the Murray study defined the dose level of 0.001 yg TCDD/kg/day to be a NO A E L . A NOAEL of 0.001 ug TCDD/kg/day as defined in the M ur ray multi-generation rat study also agrees with the NOAEL of 0.001 yg TCDD/kg/day established in the most definitive lifetime study, by Kociba et al. (1978), which subsequently has been used by most regulatory agencies in the derivation of long-term human exposure control limits for TCDD. As 783638 4 discussed in Attachment II to today's submission by CMA the same NOAEL has also been demonstrated in a variety of other toxicology studies of 2,3,7,8 -TCDD. (See Attachment II, Section II). Accordingly, if one uses a long-term or lifetime expo sure level of 0.001 ug TCDD/kg/day as a NOAEL, application of the 3-fola and 20-fold estimates by EPA of the lesser degrees of toxicity for 2,3,4,7,8 -PeCDF and 2,3,7,8 -TCDF, would lead to estimation of lifetime NOAELs for 2,3,4,7,8PeCDF and 2,3,7,8 -TCDF in the rat of 0.003 ug PeCDF/kg/day (3 times the TCDD value) and 0.02 ug TCDF/kg/day (20 times), respectively. The RfD values for human exposure to 2,3,4,7,8 -PeCDF and 2,3,7,8 -TCDF can thus be established by application of a composite uncertainty factor of 100 to the NOAEL projected for the rat species. This uncertainty factor of 100 is con sidered scientifically appropriate to represent a factor of 1 0 for extrapolation from animal to man and another factor of 1 0 to account for possible interhuman response variabili ty. Thus, the RfD value for 2,3,4,7,8 -PeCDF would be 0.03 nanograms/kg/day (30 picograms/kg/day). For 2,3,7,8 -TCDF, the RfD value would be 0.2 nanograms/kg/day (200 picograms/ kg/day) . The human LOAEL for 2,3,4,7,8 -PeCDF of 0.007 .ug/kg/day estimated on p. 8 - 6 of the HAD from Yusho data could also be used to derive short-term and long-term human exposure \ GENP 010828 783639 5 control 'limits. 1 / As the LOAEL* of 0.007 ug/kg/day was es timated for a 71-day period of exposure in humans, applica tion of an uncertainty factor of 1 0 will estimate a short-term (71 day) NOAEL RfD for 2,3,4,7,8 -PeCDF of 0.0007 ug/kg/day (or 700 pg/kg/day) for humans. To extrapo late from the 71-day short-term RfD to lifetime RfD, appli cation of an additional 1 0 times uncertainty factor would lead to a lifetime RfD of 70 pg/kg/ day for 2,3,4,7,8 -PeCDF. This calculated lifetime RfD value of 70 pg/kg/day derived from EPA's human exposure estimates for 2,3,4,7,8 -PeCDF is quite similar to the calculated RfD value of 30 pg/kg/day derived from the chronic lifetime animal toxicity data on 2.3.7.8 - TC D D , using the 3-fold TEF estimated by EPA for 2.3.4.7.8 - PeCDD. II. HUMAN DATA FROM THE YUSHO INCIDENT CONFIRM THAT THE REFERENCE DOSES FOR 2,3,4,7,8 -PeCDF AND 2,3,7,8TCDF DERIVED FROM ANIMAL DATA ARE QUITE PROTECTIVE.___________________ To attempt to confirm the accuracy of the Reference Dose developed from the 2,3,7,8 -TCDD three-generation study, the criteria document compares the animal-derived value to a determination of the LOAEL for 2,3,4,7,8 -PeCDF in the 1_/ We note in Section II of these comments, however, that this value is not appropriate reflection of the Yusho data of human effect levels. GENP 010829 783640 6 Western Japan poisoning incident'."" In so doing, however, EPA incorrectly assesses that incident. As detailed below, the human data suggest a lifetime RfD for total PCDFs in the Yusho episode of 500 picograms/kg/day, thus corroborating the appropriateness of RFD values of at least 30 and 200 pg/kg/day for 2,3,4,7,8 -PeCDF and 2,3,7,8 -TCDF, as derived from the animal data discussed above. A. Detailed Data Exist on Human Exposures to and Effects of PCDFs in the Yusho Incident. Persons in southern Japan ("Yusho" - 1968) and Taiwan ("Yu-Chen" - 1976) ingested cooking oil that had been become contaminated with heat-transfer fluids during processing. Originally thought to result from the chlorobiphenyls present in the oil, Yusho symptoms have now bean shown by Japanese investigators to be largely due to PCDFs present at ppm levels in the oil. (Masuda, et a l . , 1985; Chen et a l . , 1985; Miyata, e al_. , 1985; Kashimoto, et a_l. , 1985; Kunita, et a l . , 1985.) More than 1,000 patients with the Yusho syndrome had been identified in Japan by 1971; a larger number were af fected in Taiwan. People intoxicated in both incidents ex hibited a complex of symptoms very similar to what has been called "chloracne" when seen in industrial situations in the United States and Europe. (Kimbrough 1983; Kuratsune, et al., 1972.) G E N P 010830 783641 To estimate the amount ingested by persons with Yusho symptoms, Kuratsune, et a l . , surveyed 146 users of the con taminated oil, which was produced February 5, 1968, and was distributed in 16.5 kg cans. They estimated the level of consumption of contaminated oil from cooking and eating hab its. The population was divided into three dose groups: those who consumed: (1 ) less than 720 ml, (2) between 720 and 1,4 40 ml, and (3) more t.han 1,4 40 ml, of the contaminat ed oil. Ten of 80 in the low-dose group were without symp toms; all 6 6 in the mid- and high-dose groups exhibited ei ther "light" or i"severe" symptoms. Thirty-one of 80 in the low-dose group (38.8%), 31 of 45 in the mid-dose group (68.9%), and 18 of 21 in the high dose group (85.7%), exhib ited "severe" symptoms. From these data and knowledge of the concentration of PCDFs in the oil that was ingested, an approximate E D 5 0 value for grade III Yusho (clinical chloracne) can be estimated. 2 / The concentration of PCDFs in the contaminated oil has been studied by Buser, et al. , (1978), Miyata, et al. , (1985), Morita, et a l ., (1972). Each group used comparable 2/ By Kuratsune1s terminology, "light" symptoms correspond to clinical grades I and TI Yusho symptoms, while "severe" corresponds to clinical grades III and IV. Grade III corresponds approximately to chloracne as it would have been diagnosed by physicians seeing workers exposed to TCDD in 2 ,3,5-trichlorophenol production accidents. ENP 010831 783642 8 techniques -- medium-to-high resolution gas chromatography with mass spectrometric detection, and their results are comparable. All reported concentrations of total PCDFs in the range of 1.9 to 7.4 mg/kg oil. The high report of Miyata, t al_. , comes from a sample described as "5 February 1968 production"; this matches the date on which the oil consumed by patients surveyed by Kuratsune, et a l . , was re ported to have been produced and shipped. Samples described as having been produced on February 9 and 10 , 1968 , were estimated to contain 1.9 and 2.3 mg/kg PCDFs, respectively. The sample examined by Morita, e^ a l . , was described as hav ing been produced February 12, 1968. Buser, et a l . , gave no description of the origin of the sample they examined. Be cause of the coincidence of production dates, and the evi dent decline in PCDF concentration with time over the period from February 5 to February 9-12, 7.4 mg PCDF/kg rice oil is a reasonable estimate of the concentration of these agents in the oil ingested by the survey population. B . EPA Misinterprets the Yusho Data. As a means of checking the reliability of the P.FD de termined from animal data, EPA employs the Yusho data. In so doing, however, it fails to employ the relevant data cor rectly. EPA's calculation is based on the following factors: GENP 010832 783643 9 (1) minimum quantity to cause a grade III response of 1 2 1 ml; (2) a mean latency period of 71 days; (3) PCDF concentration in the oil of 3.85 ppm; and (4) a concentration of 2,3,4,7,8 -PeCDF of 8.4% of to tal PCDFs in the oil. Based on these factors, EPA concludes the LOAEL for 2,3,4,7, 8 -PeCDF was 0.007 ng/kg/day, thus leading it to conclude that the LOAEL it derived from the animal studies of 0.003 ug/kg/day is reasonable. E P A 's calculations contain several errors. First, no source is given for the value of 121 ml that is cited as "the minimim quantity ... that led to an ob served effect ... As detailed above, in the studies re ported by Kuratsune, et al_. (1972), only estimates of the amount of oil ingested were given, and the lowest range cit ed was "less than 720 ml." Over the period of ten or so days during which the contaminated rice-bran oil was pro duced, the concentration varied by at least a factor of three. Data from the Yusho reports show that oil processed February 5, 1968 had about 7.4 ppm of total PCDFs and that this had fallen to about 2 ppm by February 10. If it were not possible to relate the amount of oil ingested to the effects seen, use of the mean value would be acceptable. However, since good data are available on a population known to have ingested oil produced on a particular date, oil for GENP 010833 783644 which an analysis is available, the concentration given for this known oil should be used. Second, in using these data from the Yusho episode, the LOAEL value can be considered an approximation of the dose level that gave low incidence of the effects seen in studies of the population. Use of a single extreme value, as EPA has done, however, is improper. It incorrectly mixes risk management judgments into the risk assessment. When proper dose-response information can be extracted from the avail able data, as is the case here, it should be used to calcu late a population mean dose for the effect. Third, the work of Birnbaum (1985) and others has shown that the effects of compounds in the chlorinated dibenzofuran class are additive. In a series of experi ments, effects seen were directly proportional to the sum of the products of concentration of each compound times its relative activity in producing that effect in the animal being tested. Thus it is entirely incorrect to ignore the contributions to Yusho symptoms of compounds such as 1 ,2 ,3,7,8 -penta, 2 ,3,4,6 ,8 -penta, and 1 ,2 ,4,6 ,7,8 -hexa chlorodibenzofurans. Individually, their contributions may be less than that of the 2 ,3,4,7, 8 congener, but their total contributions may well exceed those of the latter. As no data are available to estimate reliably the relative activi ties of these compounds in whole animals, no attempt should GENP 010834 783645 11 be made to estimate the relative"' contribution of a single compound to the activity of this mixture. 3/ We discuss next appropriate use of the Yusho data to determine acceptable daily intakes for total chlorinated dibenzofurans involved in the Yusho episode. We encourage EPA to review this assessment closely. i C. Properly Interpreted, the Yusho Data Show an Acceptable Lifetime Average Daily Dose of 500 Picoqrams Total PCDFs/Kiloqram/Day. Combining the Yusho analytical, consumption and effects i data demonstrates the following Effective Doses (EDs): (1) From the low-dose group: 4/ E D 3 9 < (0.72 1) (0.91 kg oil/1) (7.4 mg PCDF/kg oil)/60 kg body weight = 81 ug/kg. I E D 3 g < 81 ug/kg. 3_/ In addition, it is known that the rate at which a com pound is metabolized and thus deactivated affects its activ ity in the whole animal. In a situation such as Yusho, where a large number of very similar compounds are present, the activity of individual compounds cannot be assessed be cause enzymes that would deactivate them if present alone may preferentially react with more reactive compounds also present. Because easily-metabolized compounds are present in the body along with the more toxic, activity of the less easily metabolized 2 ,3,4,7,8 -pentachlorodibenzofuran may be greater than it would appear to be when tested alone. 4/ We assume that the rice-bran oil of Yusho has the same specific gravity as cottonseed and corn oils (0.91 kg / 1 ) and that the mean body weight of the Yusho population studied by Kuratsune, et al., was 60 kg. GENP 010835 783646 12 (2) From the mid-dose group: 81 < E D 6 9 < {1.44 1) (0,91 kg/1) (7.4 mg/kg oil/60 kg) = 162 yg/kg. 81 ug/kg < E D 6 9 < 162 yg/kg (3) From the high-dose group: E D 8 g > 162 yg/kg. From analyses of these data by a normal probit method, an estimate of the E D 5 0 value for grade III Yusho can be made: 46 < E D S 0 < 105 ug/kg. 5/ It would be desirable to relate the activity of this mixture to that of some single standard, such as 2,3,7,8 -TCDD. However, that is problematic in this case, for three reasons. First, no isomer-specific quantitative analysis of Yusho oil has been published. The results of 3user, et: a l ., suggest that more than 30-tetra-, penta-, and hexachlorodibenzofurans were present in the oil. Second, the relative activities of these different congeners are unknown, even in in vitro systems. Finally, such a congener assessment would require de termining the contribution of chlorobiphenyls and -quaterphenyls to the activity since these were present in 5/ An E D qi estimate can also be made: 2.6 to 10 ug/kg. However, we have more confidence in the E D 5 0 value, as it is almost independent of the form of the dose-response curve assumed. GENP 010836 783647 13 the oil at more than one hundred 'times greater concentra tion, and Birnbaum, et al. (1985) , as previously noted, have shown that some compounds from these classes contribute to effects from PCDFs in an additive manner. It is reasonable to conclude that at least some of the PCDFs present in the Yusho oil would be more active than this aggregate dose-response estimate suggests, but it is unlikely that any would be more than ten times more so. Using the estimated low ED3 0 value from the above analysis (46 ug/kg) as a point of departure for standard-setting, we thus incorporate, as detailed below, an extra order of magnitude for this uncer tainty. Employing the E D 5 0 value of 46 yg/kg, short-term and lifetime health advisory levels can be 'developed from the Yusho data. Each is explained below. Single Dose Health Advisory (SDHA) The above estimates of Effective Doses in the Yusho incident were made from exposures that occurred over a peri od of about 50 days. This period approximates the estimated half-life for these compounds in the body (King, et al^. , 1983). Because of poorer absorption and more rapid excre tion at higher dose rates, a single dose of twice that accu mulated at Yusho would have approximately the same effect. Because human data are used as the basis here for de termining a single dose health advisory (SDHA) guideline, an GENP 010837 783648 14 uncertainty factor of 1,000 applied to the E D 5q would be a quite conservative approach. One factor of ten is applied because the base dose is not a NOAEL? a second factor of ten is applied to take into account conservatively the distribu tion of susceptibilities in the population; and a third fac tor of ten to allow for uncertainties in the activity of the various PCDFs present in the Yusho oil, as mentioned above. Thus: SDHA = 2 x E D 50/lOOO = (2) (46)'/1000 = 0.09 ug total PCDF/kg body weight _5 = 9 x 10 mg total PCDF/kg, or 90 ng , PvCDF total/kg Short-Term Health Advisory (STHA) For cumulative exposure over a 10 to 100 day period, one one-thousandth of the low E D 5 0 dose at Yusho can be con sidered to be protective of human health for determination of a short-term health advisory (STHA) guideline. Thus: STHA = E D 5 0 /IOOO = 0.046 ug total PCDF/kg body weight. _5 = 4.6 x 10 mg total PCDF/kg, or 46 ng total PCDF/kg Long-Term (Lifetime) Health Advisory (LADD) A lifetime health advisory or lifetime acceptable daily dose (LADD) should be estimated taking possible accumulation of chlorinated dibenzofurans into account. That is, the intake required to reach serum concentrations equal to those 783649 GENP 010838 15 implied by the STHA dose should be estimated. However, analysis of Yusho data shows that such an analysis would require more data on human pharmacodynamics than is now available. Absent these data, use of an additional conser vative uncertainty factor of 100 applied to the STHA is ap propriate. Here, that is appropriately represented by LADD = STHA/100. Thus: 6 / LADD = 0.046 ug total PCDF/kg body weight/100 _-7 = 4.6 x 10 mg total PCDF/kg/day, or 460 total PCDF pg/kg/day.*10 _6 / In 1973 FDA estimated the mean oil intake of all Yusho patients to be 15 grams/day for 50 days, or 750 grams. The mean PCDF concentration is not known, but we know it to be greater than about 2 ppm (the minimum concentration reported) . Thus the mean intake of PCDFs was > (2 x 10 6) (750 g) = >1.5 x 10~ 3 g = >1.5 mg. FDA assumes a mean body weight of 50 kg, and further assume that one-tenth the mean dose could be taken as a tolerable dose of PCBs. (This logic was used to derive a tolerance level for PCBs.) Following similar logic, we could define a tolerable dose for PCDFs to be >1.5 x 10~ 3 g/50/10 = 3 x 10 5 gm PCDF/kg = 3 ug/kg. FDA reasoned that a total accumulation of less than the dose so calculated would cause no effect. This quantity was divided by 675 days (22 months) to obtain an acceptable average daily intake (AADI). Applying a similar procedure here, one would obtain an LADD of 4 x 10 9 gm PCDF/kg/day = 4 ng/kg/day. Our lower value differs from FDA's in three respects: we started with a higher "effect" dose (46ug/kg vs. 30 ug/kg) and included a much more conservative uncertainty factor to arrive at a tolerable accumulated dose (0.046 ug/kg vs. 3 ug/kg), but allowed a shorter time to reach equilibrium body burden. On balance, we believe an LADD of 500 pg/kg/day to be quite protective of human health. G E N P 010839 783650 16 "'This LADD of 460 picogram's/kg/day derived from the Yusho data for total polychlorinated dibenzofurans is higher than the LADDs derived both by analogy to the TCDD animal data (30 and 200 picograms/kg/day for 2,3,4,7, 8 and 2,3,7,8 , respectively, see page 4 above) and by calculations based on what EPA says is the LOAEL in the Yusho incident (70 picograms/kg/day for 2,3,4,7,8 , see page 4 above). It thus supports the clear adequacy of 30 to 70 and 200 picograms/kg/day RFD values derived for 2,3,4,7,8 -PeCDF and 2,3,7,8-TCDF from animal data. III. THE CONCENTRATION OF CHLORINATED DIBENZOFURANS IN, AND POTENTIAL EXPOSURES TO, PCBS ARE OVERESTIMATED. Section 4 of the HAD also requires comment, especially in light-of the misuse of some of the exposure information set forth in other sections of the report, most notably Sec tion 6.3. The Section 4 discussion of chlorinated dibenzofuran (PCDF) contamination of polychlorinated biphenyls (PCBs) is not relevant to the situation in the United States. American PCB products have consistently been shown to contain extremely low levels of PCDFs, compared to normal foreign materials, let alone the thermally stressed materi als involved in the Yusho and Yu-Cheng episodes. Numerous exaggerations and misleading statements need to be eliminat ed from the H A D . 783651 17 'The errors on this issue begin in the HAD Chapter 4 discussion of potential exposures of PCDFs. On page 4-11/ the statement that less than ppt levels of PCDFs were de tected in Aroclor 1016 is misleading. The correct statement would be that no PCDFs were detected in Aroclor 1016, at a detection limit of X ppt. The next sentence is also errone ous, in that Bowes, et al. , did not have in 1975 the analyt ical capability to detect 2,3,7,8 -TCDF or 2,3,4,7,8 -PeCDF in PCB formulations. At that time, a's acknowledged on page 3-6, there were neither columns available to separate 2,3,7,8 -TCDF from coeluting isomers nor standards to deter mine the retention time of potential interferents. On page 4-12, the statement that the composition of Kanechlor 400 resembled that of, Aroclor 1248 is also errone ous and misleading, especially since the statement implies that the Aroclor product contained the same level of contam inants as "Yusho" fluid. In fact, the fluid responsible for the Yusho incident did not even resemble Kanechlor 400, hav ing been thermally stressed and exposed to oxygen, as later acknowledged expressly in the same paragraph. On page 4-12, the HAD attributes a 2,3,7,8 -TCDF concen tration of 1.25 ppm to a thermally stressed heat exchange fluid, but notes in the next sentence that at least one oth er isomer coeluted under the conditions used. Nonetheless, the 1.25 ppm value is used in Section 6.3 to calculate expo sure to PCDFs from any exposure to PCBs.- GENP 010841 783652 18 All of pages 4-13 is a compilation of "suspecteds", "probablies" and "may haves". This section of the HAD thus exaggerates the contribution of PCB exposure to potential effects of PCDFs. The calculations in Section 6.3 of the HAD, purporting to estimate maximum exposure levels associated with PCB ex posure, are not realistic estimations, even assuming the desirability of developing a worst-case scenario. First, the dietary intake value, stated to represent present intake is taken from an eight-year-old paper. The quoted paper relies on data obtained before the voluntary cessation of PCB manufacture in the United States, and thus bears little relation to current potential exposures. More recent EPA and FDA evaluations clearly reflect the fact that PCB levels in fish from the Great Lakes are decreasing. A more serious error, and substantial exaggeration, is the use of 2,3,7,8-TCDF concentration in PCBs of 1.25 ppm. This value is not representative of 2,3,7,8-TCDF concentra tions in American PCB products and in fact represents the reported concentration in a severely heat'-stressed and oxygen-exposed Japanese product, used under conditions known to maximize PCDF concentrations in PCBs. (See Rappe, C., et a l ., 1985). Nor is it correct to maintain that this material is "similar to Aroclor 1248". Japanese PCB products have consistently been shown to contain higher concentrations of PCDFs, even ignoring the GENP 010842 783653 19 issue'of heat stress. The same author who reported the val ue of 1.25 ppm in the heat-stressed Japanese product has also reported levels of PCDFs in truly representative Ameri can PCB products. (Rappe, C. e_t al. , 1985). The 2,3,7,8TCDF concentrations in Aroclor 1254 and Aroclor 1260 were reported as 19 and 13 ppb respectively. Although these data are presented in Table 4-8 of the HAD, they are later ig nored in the exposure assessments. Accordingly, the HAD exaggerates potential PCDF exposures due to PCBs by a factor of almost 100 by using data not representing PCBs that could be the source of exposure to the American public. Using v more realistic values for 2,3,7,8-TCDF in American PCB prod ucts reduces the value for stored 2,3,7,8-TCDF to about 0 . 0 0 2 ng/day, if one assumes no excretion, another exagger ated assumption. The same factor of 100 should be applied to all of the other calculated exposure levels in Section 6.3 of the HAD. The entire section thus needs to be rewritten to reflect representative exposures, even in developing worst case sce narios so popular in EPA risk assessments. The mention of exposure based on pentachlorophenol in gestion on page 6-19 is also misleading given the fact that the rest of the paragraph is a discussion of exposure to 2,3,7,8-TCDF resulting from exposure to PCBs. In the next paragraph, PCDD exposure is discussed, if only to conclude that "no firm conclusions could be reached." The rest of GENP 010843 783654 20 the paragraph is pure speculation--,- typified by the doublet "might possibly." The second paragraph on page 6-20 contains further ex amples of unwarranted speculation. Although generation of PCDFs as by-products of the incomplete combustion of PCBs in fire-related situations has been demonstrated in the labora tory and is substantiated by measured levels of PCDFs in building fires, the other exposure scenarios in the para graph, are not realistic and certainly do not support the statement that "exposures should be investigated." The use of PCBs in casting waxes, carbonless copy papers, and heat transfer systems was voluntarily curtailed by 1972 and was expressly banned under TSCA in 1979. Such exposure scenari os are thus very unlikely. 7/ In sum, a consistent pattern exists in Chapters 4 and 6 of the HAD of over-estimation of the contribution of PCDF contamination of PCBs to potential human exposures. A com plete rewrite of these sections of the report is necessary to reflect accurately both the actual concentrations of 2 / In addition, there is an apparent misunderstanding of the chemistry and physical properties of PCDFs. The HAD evidences apparent surprise at the fact the PCDFs were not extracted by water from latex nipples, "unlike pentachlorophenol." Pentachlorophenol is a polar material with a measurable solubility in water, while PCDFs are insoluble in water -- thus it should clearly not. be surprising that water is not a good extractant for PCDFs. GENP 010844 783655 21 PCDE^s in commercial U.S. PCBs and-- the limited current poten tial for human exposures to PCBs. Unless such changes are made in the HAD, it will inaccurately express unwarranted cause for concern about PCDF/PCB exposures. GENP 010845 783656 22 REFERENCES Birnbaum, L. S., et al. , "Toxic Interaction of Specific Polychlorinated Biphenyls and 2,3,7,8-Tetrachlorodibenzo-pdioxin Increased Incidence of Cleft Palate in Mice," 72 T o x . Appl. Pharm. 292 (1985). Buser, H. R. , et a l . , "Polychlorinated Dibenzofurans Found in Yusho Oil and in Used Japanese Polychlorinated Biphenyl," Chemosphere 439 (1978). Chen, P. H., et al. , "Polychlorinated Biphenyls, Dibenzofurans and QuaterphyenyIs in Toxic Rice-Bran Oil and in the Blood and Tissues of Patients with PCB Poisoning (Yu-Cheng) in Taiwan," 59 Env, Health Perspects., 59 (1985). FIFRA Scientific Advisory Panel, Review of Notices of Intent to Hold FIFRA Section 6(b)(2) Hearing on 2,4,5-T and Silvex (Sept. 27 , 1979) . Kashimoto, T., fet a_l., "PCBs, PCQs and PCDFs in Blood of Yusho and Yu-Cheng Patients," 59 Env. Health Perspects., 73 (1985) . Kimbrough, R. , in R. Tucker, A. L. Young, and A. P. Gray, eds., Human and Environmental Risks of Chlorinated Dioxins and Related Compounds (New York: Plenum Press, 1983) . King, F. G., et ad., "Physiological Model for the Pharmacokinetics of 2,3,7,8-Tetrachlorodibenzofuran in Sev eral Species," 67 Tox. Appl. Pharm. 390 (1983). Kociba, R. J. et a l . , "Results of a Two Year Chronic Toxici ty and Oncogenicity Study of 2,3,7,8-Tetachlorodibenzop-dioxin (TCDD) in Rats," 46 Toxicol. Apol. Pharmacol., 279 (1978) . Kunita, M., et a l . , "Biological Effect of PCBs, PCQs and PCDFs Present in the Oil Causing Yusho and Yu-Cheng," 59 Env. Health Perspects. 79 (1985) . Kuratsune, M. , et a_l. , "Epidemiological Study on Yusho, A Poisoning Caused By Ingestion of Rice Oil Contaminated With A Commercial Brand of Polychlorinated Biphenyls," 46 E n v . Health Perspects. 119 (1972). Masuda, Y., et al., "PCB and PCDF Congeners in the Blood and Tissues of Yusho and Yu-Cheng Patients," 59 Env. Health Perspects. 53 (1985) . GENP 010846 783657 23 Morita*, M. , et al. , "Detailed Examination of Polychlorinated Dibenzofurans in Polychlorinated Biphenyl Preparations and Kanemi Yusho Oil," 18 Bull. Environ. Contam-Tox 67 (1977). Murray, F. J., et a l ., "Three-generation Reproduction Study of Rats Given 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) In the Diet," 50 Toxicol. App. Pharmacol. 241 (1979), Nisbet, I.C.T. and M. B. Paxton, "Statistical Aspects of Three-generation Studies on the Reproductive Toxicity of TCDD and 2,4,5-T," 36 Ann. Stat. 290 (1982). Rappe, C., et a l . , Workshop Proceedings: PCB By-Product Formation, EPRI CS/EL-4104, July 1985. ENP 0W847 783655 A ttachm ent II CONSIDERATIONS FOR ASSESSING POTENTIAL HUMAN RISK FROM EXPOSURES TO 2,3,7,8 -TETRACHLQRODIBENZQDIOXIN A Working Paper of the Dibenzofurans/D.ibenzodioxins Panel of'the Chemical Manufacturers Association August 22, 1986 GENP 010848 783659 CONSIDERATIONS FOR ASSESSING POTENTIAL HUMAN RISK FROM EXPOSURES TO 2 ,3,7,8 -TETRACHLORODIBENZODIOXIN Table of Contents Page INTRODUCTION .............................................. I. EPIDEMIOLOGY STUDIES HAVE NOT DEMONSTRATED THAT TCDD CAUSES CANCER OR OTHER LIFJ-THREATENING DISEASES IN HUMANS ... ............................. II. LIFETIME STUDIES OF TCDD IN ANIMALS SHOW A CONSISTENT NO-OBSERVED-ADVERSE-EFFECT LEVEL OF AT LEAST 1 NANOGRAM/KILOGRAM/DAY ............... i A. Animal Toxicity Studies Have Adequately Defined the NoObserved-Adverse-Effect Levels for Non-Cancer Effects of TCDD ............... B. Lifetime Animal Studies of Chronic Toxicity/Carcinogenicity Have Established a No-ObservedAdverse-Effect Level for TCDD of at Least 1 Nanogram/Kilogram/Day .......... C. The Animal Bioassays and Other Studies on the Mechanism of Action of TCDD Show the Carcino genic Response Observed in Animals Is Reflective of a Promotion Rather than a Genotoxic Mechanism, Indicating Lifetime Human Exposure Control Limits Are Most Appropriately Derived on an "Uncertainty" or "Margin of Safety" Basis .............................. 1 4 6 7 9 11 REFERENCES ................................................ 18 GENP 010849 783660 CONSIDERATIONS FOR' ASSESSING POTENTIAL HUMAN RISK FROM EXPOSURES TO 2,3,7,B-TETRACHLORODIBENZODIOXIN A Working Paper of the Dibenzofurar.s/Dibenzodioxins Panel of the Chemical Manufacturers Association INTRODUCTION Numerous regulatory initiatives are being driven by the Environmental Protection Agency's (EPA's) assessment of the potential human risk posed by 2 ,3, 7,8 -tetrachlorodibenzodioxin (TCDD). Because EPA's Cancer Assessment Group has determined that a 1 in ,1 0 0 0 , 0 0 0 0 cancer risk exists for a Lifetime Average Daily Dose (LADD) as low as 6 femtograms ( 6 x 10 1 5 grams)/kilogram/day (EPA 1985), numerous regulatory initiatives have sought to control TCDD to extremely low levels. By contrast, many other nations have derived human ex posure control limits of 1 to 1 0 picograms ( 1 to 1 0 x 1 0 ~ 1 2 grams/kilogram/day). It is therefore important to recog nize, as detailed in this paper, the relevant scientific evidence that demonstrates the advisability of revising EPA's LADD to fall in line with the views of international experts. EPA has also employed a Toxicity Equivalency Factor (TEF) scheme to determine control limits for other chlori nated dibenzodioxins, as well as for chlorinated dibenzofurans. The Agency has further indicated the same GENP 010850 783661 2 sch'me may be applied to brominated dibenzodioxins and dibenzofurans. Because the TCDD toxicity determination is the base number from which these other control limits are derived, it is vitally important that the TCDD assessment be scientifically appropriate and technically supportable. The driving regulatory force of E P A 's extremely low recommended control limit for TCDD is symptomatic of a gen eral over-concern about potential human toxicity of chlori nated dibenzodioxins and dibenzofurans. The United States and Europe have expended huge resources in an effort to de termine if there has been any impact on human health of ex posures to such substances, but have not found any obvious concerns given current limited exposures. Scarce and valuable resources are being devoted to examine non-obvious and less major concerns with full knowledge that zero impact can never be scientifically proven. There will always be uncertainty in the numbers, and additional effort can only reduce the uncertainty by small increments -- never elimi nate it. At the same time, EPA is implementing ever more stringent rules and requirements for dibenzodioxins and dibenzofurans under FIFRA, TSCA, RCRA, and CERCLA. CMA is concerned that there is no plan for deciding when society has reached a point of diminishing return on the use of national resources investigating alleged human problems related to dibenzodioxin and dibenzofuran exposure. Before further substantial expenditures are mandated, EPA GENP 010851 783662 3 should reassess the health criteria upon which these regula tory initiatives are based. As one reviewer (Tschirley 1986) recently concluded: What the agency has not done -- and might be said to have a responsibility to do -- is to try to dispel the pub lic's fear on the basis of the evidence that exposure to low concentrations of TCDD in the environment appears not to have serious chronic effects on human beings. The importance of EPA's current determination of TCDD toxicity is illustrated by the fact that the control limits employed by EPA are about 1,000 times more restrictive than the levels determined by other international expert groups who have reviewed the same evidence. Specifically, its TCDD recommended human exposure control level is 160 to 1,600 times more restrictive than the levels recommended by var ious non-U.S. regulatory agencies and expert panels, includ ing a Swiss expert group (Schlatter, et a l . , 1985); a Cana dian expert panel (Ontario Criteria Document 1985) ; a West German expert group (Appel, et a l ., 1985); and a Dutch ex pert group (Van der Heijden, ej: al. , 1982). The EPA control limits are also about 10 times more restrictive than a U.S. Food and Drug Administration assessment of the same data (FDA 1983) . Accordingly, the CMA Panel on Dibenzodioxins and Dibenzofurans has prepared this Working Paper discussing the GENP 010852 783663 4 scientific evidence that has led-other expert groups to dis agree with EPA's assessment of T C D D . In this paper, we: (1) Note the extensive human data on exposures to TCDD and their results, which find some toxic effects (especially chloracne) in highly exposed manufacturing workers and in dustrial accident victims, but no consistent data indicating any carcinogenic or other life-threatening diseases in hu mans . _C2) Review the extensive animal data on TCDD with spe cial emphasis on the lifetime studies that evaluated chronic toxicity and carcinogenic potential. We also review the mechanistic and dose-response data, relied on heavily by other review panels, that indicate TCDD carcinogenicity is most appropriately considered a threshold-based phenomenon to which an "uncertainty" or "margin of safety" factor can be applied in deriving an acceptable human LADD. I. EPIDEMIOLOGY STUDIES HAVE NOT DEMONSTRATED THAT TCDD CAUSES CANCER OR OTHER LIFE-THREATENING DISEASES IN HUMANS. In the late 1970's concern that TCDD impurities in var ious pesticides caused cancer, especially soft tissue sarcomas, was raised by a series of Swedish epidemiology studies (Hardell 1981; Hardell, ^t al. , 1982; Hardell and Sandstrom 1979; Eriksson, et al., 1981; Hardell and Eriksson 1981; Axelson 1980). More recent data from studies G E N P 010853 783664 5 of similarly exposed pesticideapplicators in New Zealand (Smith, et a l . , 1983; Smith, t al. , 1984) and Vietnam (U.S. Air Force 1983 ; U.S. Air Force 1985) and forestry and agricultural workers in Sweden (Wiklund & Holm, 1986) have found no such association. In addition, critical scientific review of the original Swedish studies, as well as examination of results of stud ies of manufacturing workers exposed to TCDD in the U.S. (Cook, et aJL. , 1980; Ott, e_t al. , 1980; Zack and Suskind 1980; Zack and Gaffey 1983) and in Europe (Theiss, et a l ., 1982; Dallderup and Zellenrath 1983), have led most reviewers to conclude that there is no significant epidemiology evidence of an association between TCDD and human cancer or other life-threatening disease. The Expert Committee (including D r s . 0. Kutzinger, G. L. Plaa, S. Safe, E. Y. Spencer and B. Birmingham) that re ported to the Ontario Canada Ministry of Environment in its Scientific Criteria Document on PCDDs and PCDFs, for exam ple, concluded (at 1-13): Epidemiological studies to date do not present sufficient evidence to establish a causal relationship between exposure to PCDDs or PCDFs and a chronic human health effect relating to carcinogene sis, coronary disease or impairment of the immune system. The American Medical Association's Council on Scientif ic Affairs (1984) similarly concluded that although many GENP 010854 783665 - 6- adverse effects of dioxins had been found in 'exposed ani mals : Except for chloracne, -8. TCDD has not demonstrated comparable levels of bio logic activity in man; that is to say, no long-term effects on the cardio vascular and central nervous systems, the liver, the kidney, the thymus and immunologic defenses, and the reproduc tion function -- in the male, female or offspring -- has been demonstrated. It is true that even this extensive body of epidemiolo gic data cannot prove an absence of risk for low level TCDD exposures, but no such body of data ever will exist. On the other hand, these data provide assurance that significant adverse human effects are unlikely at low exposures. These data, moreover, caution strongly against exaggerated inter pretation of the laboratory animal evidence, as discussed next, to establish guidelines for human exposure limits. II. LIFETIME STUDIES OF TCDD IN ANIMALS SHOW A CONSISTENT NO-OBSERVEDADVERSE-EFFECT LEVEL OF AT LEAST 1 NANOGRAM/KILOGRAM/DAY. Given the absence of data indicating long-term debili tating effects of TCDD exposure in man, most expert review groups and governments have sought to establish human con trol limits based on the substantial animal toxicity data bas'e.* TCDD has been tested in a variety of toxicity tests using various animal species. GENP 0 1 0 8 5 5 783666 7 ''The animal studies considered most appropriate for de termining lifetime exposure control limits for man are the lifetime animal studies evaluating chronic toxicity and carcinogenic potential. These long-term animal studies con sistently have shown a no-observed-adverse-effect level (NOAEL) of at least 1 nanogran/kilogram/day. The preponder ance of data thus supports derivation of a lifetime human exposure control limit of approximately 1 0 picograms/kilogram/day based on the existence of'these NOAELs and appli cation of a 1 0 0 times "uncertainty" or "margin of safety" factor. A. Animal Toxicity Studies Have Adequately Defined the No-ObservedAdverse-Effect Levels for NonCancer Effects of T C D D . TCDD has been administered to a variety of species in standard toxicologic studies. Apart from the lifetime ani mal studies discussed next, the studies of reproductive and teratogenic effects are most significant to human health risk assessment. The Ontario Expert Committee summarized those studies (Ontario Criteria Document 1985 at 3-132): . Data from teratological (reproductive effects) studies in rats and mice indi cate that 2,3,7,8-Ti+CDD causes embryolethality and/or fetotoxicity. A 3-generation reproduction study of rats was associated with decreased fertility and neonatal survival in the first and second generations. GENP 010856 783667 8 ` NOELs for these teratogenic and repro ductive effects over the range 0 . 0 0 1 to 0.03 ug 2,3,7,8 -T^CDD/kg have been re ported . As the Ontario Report further notes, TCDD has been founa to affect the fetus only at doses equivalent to those which produce adult toxicity. An additional margin of safety should be inherent in any extrapolation from TCDD animal results given the data indicating the lesser sensitivity of humans. Reviews by both Neal (1983) and Tschirley (1986) found man to be less sensitive to acute TCDD effects than tested laboratory ani mals, including^rats, mice and guinea pigs. Data concerning actual human exposures to TCDD confirms this analysis. Perhaps the most valuable human data for evaluating teratogenic,- fetotoxic, and reproductive risk are those obtained in the Seveso region following the 1976 acci dent. Unlike other human exposures to TCDD, Seveso involved severe exposure to women of child-bearing age. The Seveso exposures caused no increase in spontaneous abortion, birth defects, or any abnormalities in the growth and development of children in utero at the time of the accident. (Marni, et a l ., 1982.*) In addition, the Kligman study which demon strated that humans voluntarily exposed to TCDD by dermal application suffered no adverse effects at doses well above those which produce chloracne in test rabbits, also suggests that people are less sensitive than test animals. GENP 010857 783668 9 "'Taking all of these consid'exations into account, the application of a 1 0 0 -fold "uncertainty" or "safety margin" for TCDD to the NOAEL defined in the animal studies is a conservative approach to establishment of lifetime human control limits. B. Lifetime Animal Studies of Chronic Toxicity/Carcinogenicity Have Established a No-ObservedAdverse-Effect Level for TCDD of at Least 1 Nanogram/Kilogram/Dav. Almost all reviews of the evidence of TCDD carcinogen icity in animals have focused on the Kociba, et^ aJ. (1978) i study in Sprague-Dawley rats. This study, in agreement with other bioassays conducted in other rat strains and in mice, established a lifetime NOAEL for chronic toxicity/ carcinogenic response of at least 1 nanogram/kilogram/day. The Kociba. study is considered the most pertinent animal study for deriving long-term human exposure control limits because it was conducted with state-of-the-art procedures to evaluate the dose-response pattern for both chronic toxicity and potential carcinogenicity. Detailed pathologic examination in the Kociba study of an extensive list of tissues from rats given the high dose level of 0 . 1 yg/kg/day revealed the presence of toxicologic lesions. The liver was the primary target organ. Only at this highest and overly-toxic dose level, which exceeded the maximal tolerated dose and shortened the*lifespan of female GENP 010858 783669 10 rats, was there a carcinogenic response following lifetime treatment. At the intermediate dose level of 0.01 ug/kg/day, there was no definite carcinogenic response, despite the fact that this dose was high enough to produce some toxicity. In the primary target organ, the liver, the maximal response was the formation of hyperplastic nodules originating from the liver cells. At the lowest dose level of 0.001 ug/kg/day, there was no carcinogenic response, and no other adverse effect was associated with this lifetime exposure to T C D D . NCI bioassay results in Osborne-Mendel rats are in agreement with the results observed in the Sprague-Dawley rats in the Kociba study. In the NCI study, TCDD induced liver neoplasms in female rats only at the high dose of 0,07 ug/kg/day. No carcinogenic effects were observed at a daily dose of 0.0014 ug/kg/day. TCDD has also been administered to mice in long-term studies. The report is ambiguous (Toth 1979), but even if a true carcinogenic response was produced at the middle dose level of 0.7 ug TCDD/kg/week, this dose is equivalent to the daily dose of 0 . 1 ug/kg/day demonstrated to be carcinogenic to rats in the Kociba study. The Toth study found no carcinogenic effects in mice at the lower dose of 0.03 ug/kg/day. Upon reviewing these lifetime studies, the Canadian experts concluded (at 1 -1 1 ): 783670 11 Those studies involving" oral administra tion (2,3,7,8 -T4 CDD is most toxic using this route of exposure) have reliable dose-response data and indicate clear NOELs (0.001 to 0.0014 ug/kg/day) for tumour incidence. In general, 2,3,7,8 -T^CDD administration was associated with relatively few histologically different tumour types. The onset of these tumours appeared late in the study and generally at similar time to onset of similar, but less fre quent, tumors in the control animals. Although 2,3,7,8 -TlCDD has been found to be carcinogenic in rats and mice, it is difficult at this stage to predict the ability of this compound to induce tumours in humans. In sum, long-term cancer studies of TCDD in several strains of both rats and mice have shown neither chronic toxicity nor carcinogenicity at lifetime doses of 1 nanogram/kilogram/day. C. The Animal Bioassays and Other Studies on the Mechanism of Action of TCDD Show That the Carcinogenic Response Observed in Animals Is Reflective of a Promotion Mechanism Rather than a Genotoxic Mechanism, Indicating Lifetime Human Exposure Control Limits Are Most Appropriately Derived on an "Uncertainty" or "Margin of Safety" Basis. Recent reviewers have deliberated on the proper inter pretation for human risk assessment of carcinogenic respons es from administration of TCDD to animals. Each of these assessments has focused on the absence of genotoxic poten tial and have identified an indirect promotion mechanism as the likely cause of the. tumors seen in laboratory animals. GENP 010860 783671 12 Accordingly, these reviewers have'all applied an "uncertain ty " or ''margin of safety" analysis to the NOAELs found in the definitive and sensitive long-term animal studies to determine human control limits. Many scientists, including Pereira (1985) of EPA, have recognized the need for differentiation between carcinogens that mechanistically interact directly with DNA (genotoxic) and those that do not (non-genotoxic) . Initiators are genotoxic agents that penetrate the cell nucleus and damage or alter the DNA genetic material of the cell. By contrast, promoters or modifying agents produce a carcinogenic response as an indirect effect of organ toxicity or by impair ing the body's defense mechanisms against carcinogenic ini tiators . Current theory of some scientists assumes that the genotoxic potential of initiators may indicate the lack of a threshold for carcinogenic effects. For promoters, on the other hand, it is generally agreed that a threshold for the carcinogenic response exists (Pereira 1985). This threshold would be indicated by the dose-response data defining the NOAEL for organ toxicity or impairment of bodily defense mechanisms. In the absence of detailed quantitative infor mation about the exact mechanism of action, most authorities thus recommend, in line with an increasing scientific con sensus, using a conservative "uncertainty" or "margin of safety" factor to protect human health. GENP 010861 783672 13 "These considerations apply* "to TCDD, as it is generally agreed it is not a cancer initiator. Short term assays in dicate TCDD is not genotoxic. Various mechanisms of TCDD carcinogenicity have been investigated, each of which points to the conclusion that TCDD is acting as a promoting or mod ifying agent, for example, through tissue damage found at the same doses at which a carcinogenic response is observed. TCDD does not covalently bind to DNA or RNA (Poland and Glover 1979), does not induce DNA repair synthesis (Althaus, et a l ., 1982), and is essentially devoid of any mutagenic activity (Wassom 1977; Kociba 1984). In an in vivo cytogenetics screening study conducted for FDA, Green and Moreland (1975) found no chromosomal aberrations in the bone marrow of male rats treated with TCDD. A subsequent study by Green, et al., (1977), limited by the fact that controls were not used, reported "only weakly positive" results, while an earlier study by Khera and Ruddick (1973) was nega tive for dominant lethal mutations in Wistar rats. In addition, Poland and Glover (1979) tested the DNA binding potential of TCDD in the liver of Sprague-Dawley rats and found that the maximum potential DNA binding by TCDD is 10,000 to 1,000,000 times less than that of most chemical carcinogens. They thus concluded, "it is unlikely that TCDD-induced oncogenesis is through a mechanism of covalent binding to DNA and somatic mutation." GENP 010862 783673 14 Taken together, these studies provide strong evidence that TCDD does not interact with or damage cellular genetic material to produce heritable genetic changes. TCDD is therefore unlikely to cause cancer via a genotoxic mecha nism. As the Ontario Expert Committee concluded (at 1-10): 2,3,7,8 -TuCDD does not form DNA adducts, i.e., it does not chemically bind to DNA. Therefore it is unlikely to cause mutations by chemically changing DNA directly. This compound has been extensively test ed in in vitro microbial mutagenicity tests, such as the Ames test, with main ly negative results. More limited in formation is available from in vitro and in vivo mammalian test systems for mutagenicity and clastogenicity. On the other hand, considerable evidence indicates that TCDD acts through one or more n'on-genetic mechanisms as a promoting or modifying agent. These mechanisms depend upon the occurrence of TCDD-induced toxicity or physiological alterations which, in turn, enhance or promote the growth of tumors. Dose levels below those needed to produce toxicity or physiological alterations are unlikely to increase tumor incidence. The lifetime study of TCDD rats by Kociba, et_ a l . (1978), for example, showed a carcinogenic response only at dose levels that induced toxicity. As detailed above, the lowest dose level (0.001 yg/kg/day) was a NOAEL for such toxicity, and no carcinogenic effects were observed. It is GENP 010863 783674 15 well-'known that recurrent tissue-injury and repair commonly leads to tumors in affected organs of laboratory animals. Thus, one very likely mechanism for TCDD production of tu mors is recurrent tissue injury and regenerative repair within the affected organ. The non-genetic mechanisms involved in the intermediate toxic or physiological effect occurred only above the 0 . 0 0 1 ug/kg/day NOAEL defined in the lifetime toxicity studies. Accordingly, with lifetime exposure* levels at or below 0.001 ug/kg/day, one would expect no intermediate toxicity or physiological alteration and therefore no tumor promotion. \ The subsequent study by Pitot, et al. (1980) , supports the non-genotoxic interpretation. Pitot tested "the hypo thesis that the liver cancer associated with chronic admin istration of TCDD might arise from the promoting activity of the compound, presumably stimulating cells already sponta neously initiated by dietary and other environmental car cinogens." The strain of "rats and the TCDD dosage regimen" used "were chosen to resemble closely the conditions" in the Kociba study. Dr. Pitot concluded: "TCDD is a potent pro moting agent for hepatocarcinogenesis." In addition to the promotion of liver tumors in rats reported by Pitot, another study by Poland, et al. (1982) reported TCDD to be a promoter of skin tumors in hairless mice after treatment with known cancer-initiating agents GENP 010864 783675 16 such as N-methyl-N-nitrosoguanidine (MNNG) or dimethylbenzonthrocene (DMBA) . Another more recent study by Aberr.ethy, et a l . (1984) reported TCDD to be a promoter of the trans formation of C 3 H/10T 1/2 cells that had been initiated with MNNG. Overall, these studies provide compelling evidence that TCDD acts as a cancer promoter in animal bioassavs. In sum, strong evidence exists that TCDD acts not by a genotoxic mechanism but rather by one or more non-genetic mechanisms that promote or enhance tumor growth. As the Canadian expert group concluded: Results of short term genotoxicity tests suggest that 2,3,7,8 - T 4 CDD is not genotoxic in a classical sense and does not act directly on the chromosomes or the DNA molecule itself. Consequently, 2 ,3,7,8 - T ^ D D appears to produce tumours in rodents by an indirect mechanism. The Ontario expert committee thus concluded that an acceptable LADD for TCDD could be. derived by applying an "uncertainty" or "margin of safety" factor of 1 0 0 to the NOAEL of 1 nanogram/kg/day. This LADD of 10 picograms/ kg/day is, as noted above, consistent with other Swiss, West German and Dutch expert conclusions. In sum, based on detailed review of the available data, international experts have determined the appropriate level of concern for lifetime exposure to TCDD is 1,000 times higher than EPA has determined. Given the many Agency ini tiatives being driven by this unduly high estimate of TCDD - G E K P 010865 783676 17 potency, it is imperative t h a t -EPA reassess its 1985 deter mination. GENP 010866 783677 REFERENCES Abernehy, et a l . , "The Effect of TCDD Upon Transformation, Initiation and Promotion of C3H/10T 1/2 Cells," Abstract, 15th Annual EMS Meeting (1984) . Althaus, F. R . , et al., "Chemical Quantification of Unsched uled DNA Synthesis in Cultured Hepatocytes as an Assay for the Rapid Screening of Potential Chemical Carcinogens," 42 Cancer Research 3010 (1982). AMA Council on Scientific Affairs, "Health Effects of "Agent Orange" and Polychlorinated Dioxin Contaminants: An Update," American Medical Association (1984). Appel; et al., 5th Intern. S-vthd. on Chlorinated Dioxins, (1985) . Axelson, 0., et a l . , "An Updated Epidemiologic Investiga tion of S w e d i s h (Railroad Workers," 6 Scandinavian J. Work Environmental Health 73-79 (1980). Cook, R. R . , et al., "Mortality Experience of Employees Exposed to 2,3,7,8 -Tetrachlorodibenzo-p-Dioxin (TCDD)," 22 J. Occup. Med. 530 (1980) .' Dalderup, L. M . , and D. Zellenrath, "Dioxin Exoosure: 20 Year Followup," 1 Lancet 1134 (1983). EPA, "Health Assessment Document for Polychlorinated Dibenzofurans," (June 1986). Eriksson, M . , et al. , "Soft Tissue Sarcomas and Exposure to Chemical Substances: A Case-Referent Study," 38 British J. Industr. Med. 27 (1981) . Green, S., et: a_l., "Cytogenetic Evaluation of Several Dioxins In The Rat," 33 Toxicol. Appl. Pharmacol. 161 (1975) . Green, S., et al_. , "Cytogenetic Effect of' 2,3,7,8 -Tetrachlorodibenzo-p-dioxin On Rat Bone Marrow Cells," 6 FDA By-Lines 292 (1977) . Hardell, L., "Relation of Soft Tissue Sarcoma, Malignant Lymphoma and Colon Cancer to Phenoxy Acids, Chlorophenols and Other Agents," 7 Scandinavian J. Work Environmental Health 119 (1981). GENP 010867 783678 19 Hardell, L., and M. Eriksson, "Soft Tissue Sarcomas, Phenoxy Herbicides, and Chlorinated Phenols," 2 Lancet 250 (1981). Hardell, L . , and A. Sandstrom, "Case Control Study: Soft Tissue Sarcomas and Exposure to Phenoxyacetic Acids and Chlorophenols," 39 British J. Cancer 711 (1979). Hardell, L., and M. Eriksson, "Soft Tissue Sarcomas, Phenoxy Herbicides, and Chlorinated Phenols," 2 Lancet 250 (1981). Hardell, L., et al., "Epidemiological Study of Nasal and Nasopharyngeal Cancer and Their Relation to Phenoxy Acid or ChloroDhenol Exposure," 3 British J. Industr. Med. 247 (1982) . Khera, K. S. and J. A. Ruddick, "Polychlorodibenzo-pdioxins: Perinatal Effects -and the Dominant Lethal Test in Wistar Rats," in Blair, E. H. (ed.) Chloroaioxins-Origins and Fate (1973). Kociba, R. J., et al., "Results of a Two Year Chronic Toxicity and Oncogenicity Study of 2,3,7,8 -Tetrachlorodibenzo-p-dioxin (TCDD) In Rats," 46 Toxicol. Appl. Pharmacol. 279 '(1978). Kociba, R . , "Evaluation of the Carcinogenic and Mutagenic Potential of 2,3,7,8-TCDD and Other Chlorinated Dioxins," at 73, jin Banbury Report 18: Biological Mechanisms of Dioxin Action, (1984). Marni, E., et a_l., "Birth Defects Register in Seveso: A TCDD-Polluted Area," at 174 in Plans for Clinical and Epidemiologic Follow-up After Area-Wide Chemical Contamin ation (Washington: National Academy Press, 1982). Neal, R., Interim Report of the Missouri Dioxin Task Force, 40-41 (1983). Ontario Ministry of the Environment, "Scientific Criteria Document for Standard Development," (1985). Ott, M. G . , et a l ., "A Mortality Analysis of Employees En gaged in the Manufacture of 2,4,5-trichlorophenoxyacetic Acid," 22 J. Occup. Med. 47 (1980). Pereira, M.A., "Mouse Liver Tumor Data: Assessment of Carcinogenic Activity," 1 Toxicol, and Indust. Health 311 (1985). GENP 010868 783679 20 Pitot, H. C., et a_l, , "Quantitative Evaluation of the Promotion by 2,3,7,8 -Tetrachlorodibenzo-p-dioxin of Heptacarcinogenesis From Diethylnitrosamine," 40 Cancer .Res. 3616 (1980). Poland, A. and E. Glover, "An Estimate of the Maximum In Vivo Covalent Binding of 2,3,7,8 -tetrachlorodibenzo--dioxin to Rat Liver Protein, Ribosomal RNA, and DNA," 39 Cancer Res, 3341 (1979) . Poland, A. et al_. , "Tumour Promotion By TCDD In Skin of HRS/J Hairless Mice," 300 Nature 271 (1982). Schlatter, C . and H . Poiger, "Assessment of Contamination with 2,3,7,8 -TCDD," Institute for Toxicology, University of Zurich (1985). Smith, A. H., et a_l. , "The New Zealand Soft Tissue Sarcoma Case-Control Study: Interview Findings Concerning Phenoxyacetic Acid Exposure," 12 Chemosphere 565 (1983). Smith, A. H. , _et a_l., "Soft Tissue Sarcoma and Exposure to Phenoxyherbicides and Chlorophenols in New Zealand," 73 J . Natl, Cancer Inst. 1111 (1984). "Statement by Sanford A. Miller Before the Congressional Subcommittee on Natural Resources, Agriculture Research and Environment," U.S. Food and Drug. Administration, 1983. Theiss, A. M . , et al . , "Mortality Study of Persons Exposed to Dioxin in a Trichlorophenol Process Accident That Occurred in the BASF AG on November 17, 1953," 3 Am. J. Ind. Med. 179 (1982) . Toth, K., et a l . , "Carcinogenicity Testing of.Herbicide 2,4,5-Trichlorophenoxyethanol Containing Dioxin and of Pure Dioxin in Swiss Mice," 278 Nature 548 (1979). Tschirley, F.H., "Dioxin," 254 Scientific American 29 (Feb. 1986). USAF School of Aerospace Medicine, "Project Ranch Hand II: An Epidemiologic Investigation of Health Effects of Air Force Personnel Following Exposure to Herbicides. Baseline Mortality Study Results" (Brooks Air Force Base, Texas: United States Air Force, June 30, 1983). USAF School of Aerospace Medicine, "Project Ranch Hand II: Mortality Update-- 1984" (Brooks Air Force Base, Texas: United States Air Force, 1985) . GENP 010869 783680 21 Van der Heijden, et a l . , "Evaluation of the Carcinogenicity and Mutagenicity of 2,3,7,8 -Tetrachlorodibenzo-p-dioxin (TCDD); Classification and No-Effect Level," Report DOC/LCM 300/292, Dutch State Institute of National Health (1982). Wassom, e_t al, , "Review of the Genetic Toxicology of Chlorinated Dibenzo-p-Dioxins," 47 Mutation Research 141 (1977/1978). Wiklund, K. and L. Holm, "Soft Tissue Sarcoma Risk in Swedish Agricultural and Forestry Workers," 76 JNCI 229 (1986) . Zack, J. A., and R. R. Suskind, "The Mortality Experience of Workers Exposed to Tetrachlorodibenzodioxin in a Trichlorophenol Process Accident," 22 J. Qccup. Med. 11 (1930) . Zack,'J. A., and W. R. Gaffey, "A Mortality Study of Workers Employed at the Monsanto Company Plant in Nitro, West Virginia," at 575 in Human and Environmental Risks of Chlo rinated Dioxins and Related Compounds, eds. Tucker, R. E., et a l . (New York: Plenum Press, 1983). GENP 010870 783681 INTERIM PROCEDURES; FOR ESTIMATING RISKS ASSOCIATED WITH EXPOSURES TO MIXTURES OF C H LORINATED D I B E N Z O - p-DIOXINS ANO OIBENZOF'JRANS (CDOs AND CDFs) Prepared for the Risk Assessment Forum U.S. Environmental Protection Agency Washington, DC April 1986 AUTHORS Judith S. Sellin, Ph.D. Office of Solid Waste and Emergency Response Donald G. Barnes, Ph.D. Office of Pesticides and Toxic Substances TECHNICAL PANEL Donald Barnes, Ph.D. (OPTS)* Steven Bayard, Ph.D. (ORD) Irwin Baumel, Ph.D. (OPTS) Judith Beilin, Ph.D. (OSWER) David Cleverly, M.S. (OAQPS) Frank Gostomski, Ph.D. (ODW/OWRS) Charalingayya Hiremath, Ph.D. (ORD) Paul Milvy, Ph.D. (OPPE) Abe Mittelman, M.S. (OSWER) Debdas Mukerjee, Ph.D. (ORD) Charles Nauman, Ph.D. (ORD) Hugh Spitzer, 8.S. (ORD)* Jerry Stara, Ph.D., D.V.M. (ORD) RISK ASSESSMENT FORUM STAFF Dorothy Patton, Ph. D., J.D., Executive Director (Acting) Alan Ehrlich, Ph. D., Executive Secretary ^Technical Panel Co-Chairman GENP 010871 783682 DRAFT-- DO NOT QUOTE OR CITE This is a draft document for review purposes only and does not constitute Agency policy. Mention of trade names or commercial produc does not constitute endorsement or recommendation for use. GENP 010872 11 783683 TABLE OF CONTENTS I. SUMMARY.........................................................-. 1 II. THE NEED FOR A PROCEDURE FOR ASSESSING THE RISK ASSOCIATED WITH EXPOSURE TO COMPLEXMIXTURES OF CDDs/CDFs. . . . 2 III. APPROACHES TO HAZARD ASSESSMENT FOR CDD/CDFMIXTURES ............ 4 A. The Ideal Approach -- Long-Term, Whole-Animal Toxicity Assay of Mixtures.............................................. 4 B. A Promising Approach -- Short-Term, Biological Assay of Mixtures ...................................................... 5 C. A Reductionist Approach -- Additivity.of Toxicity of Components .................................................... 5 D. An Interim Approach -- 2378-TCDD Toxicity Equivalence Factors (TEFs) ................................................ 5 IV. THE 2378-TCDD TOXICITY EQUIVALENCE FACTORS (TEFs) APPROACH TO ASSESSING THE TOXICITY OF COMPLEX MIXTURES OF CDDs/CDFs . . . 7 V. APPLICATIONS TO RISK ASSESSMENT.................................... 13 VI. COMPARISON OF TEF APPROACH WITH RESULTS OF BIOLOGICAL TESTING............................................................. 15 VII. RESEARCH N E E D S ........................... .. ................. . 17 REFERENCES............................................................... 19 T A B L E S ....................................................................23 APPENDIX A: NOMENCLATURE................................................ A-l APPENDIX B: COMPARISON OF DIFFERENT APPROACHES TO CALCULATING 2378-TCDD EQUIVALENTS ........................ B-l * G0 0\0*13 iii 783684 INTERIM PROCEDURES FOR ESTIMATING RISKS ASSOCIATED WITH EXPOSURES TO \JM I X T U R E S Of C H L O R I N A T E D D I B E N Z O - p - D I O X I N S * A N D - D I B E N Z O F U R A N S (CDOs and CDFs) I. SUMMARY The U.S. Environmental Protection Agency (EPA) is often confronted with the need to determine the risks associated with exposure to materials such as soot, incinerator fly ash, industrial wastes, and soils which contain complex mixtures of chlorinated dibenzo-p-dioxins (CDDs) and chlorinated dibenzofurans (CDFs). Recognizing the public and toxicol og-ical concern generated by these chemicals and the significant gaps in our ability to evaluate the human health potential of these compounds by existing procedures, the CDD/CQF Technical Panel of the m Risk Assessment Forumt(Forum) is reconnending an interim method to aid in the assessment of the human health risks posed by mixtures of CDDs/CDFs until data gaps are filled. The Technical Panel has reviewed' a spectrum of approaches for making such assessments, consistent with EPA1s Guidelines for the Health Risk Assessment of Chemical Mixtures and has concluded that a direct biological assessment of the toxicity of complex mixtures of CDDs/CDFs is preferred. A validated bioassay that can plausibly be applied to such mixtures is not now available, although promising research is in progress in the area. An alternative approach involves - explicit analysis and toxicological determination of each of the constituent CDD/CDF congeners. The data required for such an approach also need to be developed and are not likely to be generated soon. The Forum therefore concludes that, as an interim science policy measure, a reasonable estimate of the toxic 1/ See Appendix A for the nomenclature and conventions used in this paper. GENP 010874 l 783685 risks associated with a mixture of CDDs/CDFs can be made by taking into account the distribute on' of CDD/CDF congeners or homolugues and the likely relative toxicity of these compounds. This document describes the recommended interim procedure for generating the "2378-TCDO equivalence" of complex mixtures of CDDs/CDFs, based on congener- or homologue-specific data, and for using such information in assessing risk. (The recommendations are summarized in the rightmost column of Table 1.) The Forum acknowledges that this procedure is not based on a thoroughly established scientific foundation. Instead, the approach represents a consen sus recommendation for interim science policy, subject to change as additional data are available. The approach is judged to be applicable to mixtures of * CDDs/CDFs, but should not be construed as being applicable as well to mixtures of other chemicals. The basis of this approach, i.e., the assignment of toxicity equivalence factors (TEFs) is subject to revision as new scientific data become available in the future. Consequently, risk assessors' and risk managers are urged to use informed discretion, noting specific problems on a case-by-case basis, when deciding to which situations the procedure can be applied. The Forum urges the support of research to broaden the scientific basis for this approach and develop the more preferred approaches. II. THE NEED FOR A PROCEDURE FOR ASSESSING THE RISK ASSOCIATED WITH EXPOSURE TO COMPLEX MIXTURES OF CDDs/CDFs During the late 1970s, the Agency was faced with assessing the human health significance of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). 783686 2 GENP 010875 In preparation for the cancellation `hearings for the herbicides 2,4,5-trichlorophenoxyacet'ic_ acid_(2,4,5-T) and Silvex, 'the'Agency generated risk assessments for several toxic responses for 2,3,7,8-TCDO. The quantitative cancer risk assessment developed by the Carcinogen Assessment Group was later adapted for use in the Water Quality Criteria (WQC) Document for 2,3,7,8-TCDD (U.S. EPA, 1984a). In addition to carcinogenicity concerns, the WQC document contains an assessment of systemic toxicity based on reproductive effects resulting from exposure to 2,3,7,8-TCDD. Later, it became clear that exposure situations exist in the country which involve more than 2,3,7,8-TCDD alone. Data on emissions from combustion sources (e.g., hazardous waste and municipal waste incinerators) and contents of waste m from certain industrial production processes indicate that the majority of the 75 CDOs and 135 CDFs can be detected in the environment. In recent years, the reporting of at least homologue-specific data for the CDDs and CDFs has become commonplace, and the Agency has taken some steps to address the significance of these findings. For example, the current draft of the Health Assessment Document for Polychlorinated Dibenzo-p-Dioxins prepared for the Office of Air Quality, Planning, and Standards (U.S. EPA, 1984b) contains a quantitative risk assessment for a mixture of hexachlorodibenzo-p-dioxins (HxCDDs) based on carcinogenicity studies conducted by the National Cancer Institute. These concerns have also led to regulatory action; e.g., several industrial wastes containing tetra-, penta-, hexachlorodioxins, and -dibenzofurans were recently designated by the Agency as EPA hazardous wastes. Faced with increasing amounts of isomer- and homologue-specific data, and recognizing the significant potency and structure-activity relationships exhib- GENP 010876 3 783687 ited in in v u o and in vitro studies of CDDs and CDFs, the Technical Panel per ceives a need to address more generally the potential risks posed by the con geners other than 2,3,7,8-TCDD and the mixture of HxCDDs. 2j Detailed considera- 7 ation of the toxicity of the vast majority of the CDDs/CDFS is limited by the lack of a complete toxicological data base on most of the congeners. Further, it is unlikely that many long-term test results will be available soon. For example, research on 2,3,7,8-TCDD has been under way for more than two decades at an estimated cost of more than one' hundred million dollars. Although this chemical has been investigated to a much greater extent than any of the other CDDs/CDFs, unanswered questions remain. Therefore, the Forum believes that an interim science policy, position should be adopted for use in assessing risks associated with CDD/CDF mixtures, until more definitive scientific data are available. III. APPROACHES TO HAZARD ASSESSMENT FOR CDD/CDF MIXTURES A. The Ideal Approach -- Long-Term, Whole-Animal Toxicity Assay of Mixtures Under ideal conditions, an assessment of the toxicity of a mixture of chem icals is best accomplished by direct evaluation of its toxic effects, e.g., by determining the effects of chronic exposure in an experimental animal (U.S. EPA, 1985). Such an assessment is time-consuming and costly and would theoretically 2/ In the early 1980s, the Agency developed a method for an approximate assessment of the risks of the emission of CDDs/CDFs associated with the high-temperature incineration of PCBs and combustion of municipal waste (U.S. EPA,- 1981; U.S. EPA, 1982); see Table 1. The procedure presented in this document is a refinement of that approach. A comparison of a variety of methods Is included in Appendix B. GENP 010877 4 783688 have to be performed for each of the many-mixtures of environmental importance. Therefore, this idealized approach would cause unacceptable delays in addressing the potential health risks associated with exposures to CDD/CDF mixtures. B. A Promising Approach -- Short-Term, Biological Assay of Mixtures An alternative, and perhaps more achievable, approach to hazard assessment of a mixture is a short-term assay that indirectly provides a measure of the mixture's potential toxicity. In the case of mixtures containing CDDs and CDFs, short-term assays are under development that directly determine the 2.3.7.8- TCOD-like response which could be used as a measure of the toxicity of the mixture as a whole. Such assays, which take advantage of the similar toxic end points induced by CDDs and CDFs, and have been used to assess the potential health hazards of exposure to CDD/CDF-contaminated soot from PCB fires {Eadon, 1982; Gierthy and Crane, 1984; Gravitz et al., 1983), and for predicting the potential toxicity of incinerator fly ash (Rizzardini, et al., 1983; Sawyer, et al., 1983). The development of such "mixture assays" is progressing rapidly. While additional work is required to more fully validate the assay findings for specific toxic end points, especially chronic effects, and aspects of pharmacokinetics need to be considered, an increasing body of data demonstrate correlations with subchronic effects of CDDs/CDFs (Safe et al., 1985). The Forum, recognizing the importance of this approach in implementing its regulatory strategy for 2.3.7.8- TCDO-like chemicals, strongly encourages research in this area. C. A Reductionist Approach -- Additivity of Toxicity of Components In the absence of a fully developed "mixture assay," the components in a mixture of CDDs and CDFs could theoretically be identified and quantified GENP 010878 5 783689 by analytical chemists. Then the toxicity of the mixture could be estimated by adding the, toxicity contributed by each of its components. In the case of most environmental mixtures, however, this method would be of limited value since congener-specific analyses for the 75 CDOs and 135 CPFs potentially present in the mixture are seldom available. In addition, there is little information available on the toxic potency of most of these congeners. Therefore, this approach is not viable at this time; nor is it likely to be feasible in the near future. D. An Interim Approach -- 2378-TCDD Toxicity Equivalence Factors (TEFs) The Forum recommends a fourth alternative for estimating the risks associ ated with exposure to complex mixtures of CDDs/CDFs. In this approach, as in " approach C above, information is obtained on the concentrations of homologues and/or congeners present in the mixture. Then, using the available toxicolog ical data and reasoning on the basis of structure-activity relations, the sig nificance of the exposure to each of the components is estimated and expressed as an "equivalent amount of 2378-TCDD." Combining this information with hazard information on 2,3,7,8-TCDD, and assuming additivity of effects, the risks associated with the mixture of CDDs/CDFs can be estimated if exposure is known. Key to the approach are the 2378-TCDD Toxicity Equivalence Factors (TEFs) which are derived in Section IV. The general approach using TEFs as outlined here is not unique; several organizations have used similar approaches (see Table 1). At one extreme, all CDDs/CDFs could be assumed to be as toxic as 2,3,7,8TCDD (all TEFs =1 ). This position is not recommended since the limited long term data (2-year cancer bioassays) on 2,3,7,8-TCDD and a mixture of 2378-HxCCDs 6 783690 (and the.greater bedy of short-term data on many CDOs/CDFs) indicate that such an assumption" is overly conservative. At the other extreme one could totally ignore the presence of CDDs/CDFs other than those for which adequate long-term data are available (most TEFs = 0). This position is not recommended in light of the similar toxic properties of several of these compounds and the structureactivity relationship demonstrated for effects resulting from less than lifetime exposures. Instead, the Forum recommends that the TEF procedure presented below be adopted as a matter of science policy on an interim basis, subject to revision as new experimental data become available. Based on the available scientific information, the Forum believes that this approach represents an appropriate means of approximating the potential risk of exposure to mixtures of CDDs and CDFs for purposes of risk management. The approach will enable the Agency to deal with many, but not all, of its problems; e.g., assigning priority to Superfund sites, estimating the extent to which a hazardous waste site should be cleaned up, guiding decisions on which manufacturing wastes can be delisted as EPA hazardous wastes, and estimating risks associated with the emission of CDDs/CDFs from combustion sources. The remainder of this document discusses the TEF approach in greater detail, illustrates its use in risk assessment, and identifies additional research, the results of which would provide information for adjustments to this interim approach.IV. IV. THE 2378-TCDD TOXICITY EQUIVALENCE FACTORS (TEFs) APPROACH TO ASSESSING THE TOXICITY OF COMPLEX MIXTURES OF CDDs/CDFs 2,3,7,8-TCOD is one of 75 CDDs. Exceptionally low doses of this compound elicit a wide range of toxic responses in many animals., e.g., adverse reproduc- GENP 010880 7 783691 tive effects, thymic atrophy, and a "wasting syndrome" leading to death. The EPA's Carcinogen Assessment Group (CAG) has determined that there is sufficient evidence-to conclude that 2,3,7,8-TCDD and a mixture of two 2378-HxCDOs are probable human carcinogens. The CAG quantitative assesssment indicates that these chemicals are among the most potent animal carcinogens evaluated by the Agency to date. Limited data suggest that some of the other CDDs may have other toxic effects similar to those of 2,3,7,8-TCDD, again at very low doses. Moreover, these toxicity concerns are not restricted to CDDs. Limited experimental data, supplemented by structure/activity relationships in in vitro tests that are correlated with in vivo toxic effects of CDFs indicate that some of these compounds exhibit "2,3,7,8 -TCDD-like" toxicity (3andiera et al., 1984;Okey et al., 1984; Safe et al., 1985), The biochemical mechanisms leading to the toxic response resulting from exposure to CDDs and CDFs are not known in detail. However, experimental data have accumulated which suggest that an important role in the -development of systemic toxicity resulting from exposure to these chemicals is played by an intracellular protein, the Ah receptor, the putative product of a gene locus designated Ah, This receptor binds halogenated polycyclic aromatic molecules, including CDDs and CDFs. It has been postulated that the. Ah locus controls several pleiotropic responses: a limited, but widely expressed gene complex that includes the structural genes for AHH expression, and, in a few organs, such as skin and thymus, a second gene complex regulating cell proliferation and differentiation (Knutson and Poland, 1980; Neal, et al., 1982; Greenlee et al., 1985a). In several mouse strains, the expression of toxicity of 2,3,7,8-TCDOrelated compounds, including cleft palate formation, liver damage-, effects on GENP 010881 8 783692 body weight gain, thymic involution, and chioracnegenic response, has been cor related with their binding affinity for the Ah receptor, and with their ability to induce several enzyme systems, some of which have been linked to the expres sion of carcinogenicity (Poland and Knutson, 1982; Bandiera et al., 1934; Hadhukar et al., 1984; Poland et al., 1985; Safe et al., 1985; Vickers et al., 1985). Structure-activity studies also link the enhanced in vitro cell differen tiation caused by these compounds to the presence of the Ah receptor (Greenlee et al., 1985b). However, it has also been noted that the cytosolic receptor concentration alone may not be the sole determinant of the capacity for AHH induction (Neal, m 1985; Okey and Vella, ,1984). In interspecies comparisons there are poor correlations between the amount of cellular Ah receptor, its ability to bind 2,3,7,8-TCOD, and AHH induction (Denison and Wilkinson, 1985; Gasiewicz and Rucci., 1984; Neal, 1935); and in the mouse the development of TCDD-induced liver toxicity cannot be ascribed solely to the presence of the Ah receptor (Greig et al., 1984). A recent review concludes that although there are inconsistencies across species in the Ah receptor's being the sole mechanism of toxicity of CDDs and CDFs, the data suggest that the binding of these compounds to the receptor is in some way related to some of the biological effects they cause in experimental animals (Neal, 1985) Table 2 summarizes information on a variety of end points elicited by CDDs/ CDFs: acute toxicity, carcinogenicity, reproductive effects, receptor binding, enzyme induction, and in vitro cell transformation. For ease of comparison, the data are normalized to unity for 2,3,7,8-TCDD. For example, 2378-HxCDDs have about 5% the Ah receptor binding strength of 2,3,7,8-TCDD. Their repro GENP 010882 9 783693 ductive toxicity and carcinogenic potency are, respectively, about 1% and 4% that of 2,3,1,8-TCOD. Kociba and Cabey (1935) recently presented similar data. The structure/activity generalizations based on the data in Table 2 sup port the generalizations in the literature concerning the congeners that are most likely to be of toxic concern (Poland and Knutson, 1982;- Gasiewicz and Rucci, 1984; Bandiera et al., 1984). That is, congeners that are substituted in the lateral 2, 3, 7, and 8 positions are likely to exhibit toxic effects at lower doses than other congeners. This includes the 15 tetra-, penta-, hexaand heptachlorinated CDDs and CDFs listed in Table 3. 3/ 2/ The Technical Panel is aware that some investigators (e.g., Grant, 1977; 01ie et al, 1983; Commoner et al., 1984; and Ontario, 1982, 1934) have broadly defined congeners of concern to include those tri- to hepta- congeners which are substituted with at least three chlorines in the four lateral (2, 3, 7, and 8 ) positions. The toxicity data (Table 2) do not argue strongly for this extended range of concern. Further, the increased level of complexity invoked by including these additional congeners is to suggest a greater level of accuracy and resolu tion than the Technical Panel believes is presently warranted by the TEF approach. The Technical Panel is also aware that receptor binding data suggest a rela tively high potential toxicity for 1,2,4,6,7-PeCDF, Examination of stereochemical models show that the 4 and 6 positions of CDFs exhibit partial overlap with the lateral chlorine groups of 2,3,7,8-TCDD (Bandiera et al., 1984). However, this increased receptor binding activity is not reflected in an increased potency of 1,2,4,6 ,7-PeCDF as an enzyme inducer (cf. Table 2), an end point which has been shown to correlate with subchronic toxicity (Safe et al., 1985). Therefore, the Technical Panel is treating 1,2,4,6*,7-PeCDF as a "non-2378-congener" at this time; however, additional data could lead to a change in this position. 1,2,3,6 ,7- and 2,3,4,6 ,7-PeCDF are almost as potent as 2378-PeCDF in the induction of AHH activity in human lymphoblastoid cells in vitro (see Table 2). However, because this assay seems to yield relative potencies that do not agree with other short-term tests, and because dose-response'data are not available for this assay, these data are not included in the overall evaluation at the present time. 10 783694 The "2378-TCDO equivalence factors" (TEFs) listed in Tables 1 and 3 were assigned usirvg the-following criteria: 1. Information on carcinogenic potency based on long-term animal studies takes precedence. 2. Where carcinogenic activity has not been demonstrated, information on systemic effects is used in the following manner: a. Data on reproductive effects take priority because of the signif icance of the end point in humans and because the estimated human exposure levels potentially resulting in reproductive and carcin ogenic effects are very similar. b. Data on acute lethality is accorded very low priority since a cor relation between LD5QS chronic effects has not been established for these compounds. c. The data currently available on systemic effects (e.g., body or oraan weight losses, hepatotoxicity) are not sufficient for use in developing TEFs. Such data are used to provide qualitative support for these assignments. 3. In the interpretation of the data from in vitro test systems, more weight is placed on data from receptor binding interaction and oxida tive enzyme induction, because for these effects the correlations with in vivo systemic effects (irrmunotoxicity, body'weight loss) are strong est. The above criteria were applied as follows*. 1. Since the primary concern is with chronic effects, the relative carcinogenicity responses (Table 2) for 2,3,7,8-TCDD and the mixture of two 2378-HxCDDs 4/ were used to generate the TEF for 2378-PeCDO. The TEF for 2378-PeCDD (0.5) is the arithmetic mean of the carcin ogenic potency values for 2,3,7,8-TCDD (1) and 2378-HxCDDs (0.04). Data on receptor binding, enzyme induction, and cell keratinization generally support this value. 2. 2,3,7,8-TCDF is assigned a TEF of 0.1 primarily because it is 1 to 2 orders of magnitude (OMs) less potent than 2,3,7,8-TCDD in reproductive toxicity tests. Also, it is about one OM less potent than 2,3,7,8-TCDD in the in vitro tests. 3. The 2378-PeCDF congeners are assigned a TEF of 0.1 due to the responses seen in in vitro tests. Greater reliance was placed on the animal 4/ See Appendix A, item 6 , for explanation of notation. 11 GENP 010884 783695 enzyme indaction studies due to the more significant correlations observed between this end point and subchronic responses than have been observed with the receptor binding end point. The human cell data were accorded less weight because these experiments were conducted at only one exposure concentration. 4. Because in vitro data in general show HxCDFs to be about one tenth as potent as PeCDFs, their TEF is assigned a value of 0.01 (0.1/10). Further, the data generally suggest that CDFs are somewhat less toxic than the analogous CODs, Therefore, the TEF for 2378-HxCDFs should be less than that of the 2378-HxCDDs (0.04). 5. The 2378-HpCDDs and 2378-HpCDFs are assigned TEFs 3 OM less than that for 2,3,7,8-TCDD because the enzyme induction potencies of these congeners differ from that of 2,3,7,8-TCDD by about this factor. 6 . Based on the data in Table 2, the nonr2373-substituted isomers are 1 to 3 OMs less potent than the 2373-substituted isomers. Since these data are limited to in vitro systems, a factor of 0 .0 1 is ap plied to the non-2378-substituted, as compared to the 2378-substituted congeners. - With the exception of 2,3,7,8-TCDD, the 2378-HxCDOs, and 2378-TCDF, the TEFs are not based on the results of major animal (reproductive, carcinogenic) studies. Generally, TEFs are based on estimates of the relative toxicity in in vitro tests whose relationship to the chronic effects of concern is largely presumptive. However, as discussed above, studies on systemic effects continue to reinforce the view that the short-term assays provide important fundamental information on the toxicity of the CDDs/CDFs. In summary, the Forum concludes that there is a sufficiently plausible basis for the TEF approach of estimating risks associated with exposures for CDDs/CDF and recommends that the Agency adopt the approach, on an interim basis, as a matter of science policy. The TEFs should be revised as additional scientific information is developed. It should be noted that this general approach to estimating such CDD/CDF risks has been taken by other regulatory groups (see Table 1 and Appendix B). GENP 010885 12 783696 V. APPLICATIONS ID RISK ASSESSMENT In general, an assessment of the human health risk of a mixture of CDDs and CDFs, using the TEF approach, involves the following steps: 1. Analytical determination of the CDDs and CDFs in the sample, 2. Multiplication of congener concentrations in the sample by the TEFs in Table L to express the concentration in terms of 2378-TCDD equivalents. 3. Summation of the products in step 2 to obtain the total 2378-TCDD equivalents in the sample. 4. Determination of human exposure to the mixture in question, expressed * in terms of 2378-TCDD equivalents. I 5. Combination of exposure from step 4 with toxicity information on 2,3,7,8- TCDO (usually carcinogenicity and/or reproductive effects) to estimate risks associated with the mixture. In cases in which the concentrations of the 15 congeners of concern are known 2378-TCDD Equivalents = (TEF of each 2378-CDD/CDF congener x the concentration of the respective congener) + (TEF of each non-2378 CDD/CDF congener x the concentration of the respective congener) Samples of this calculation for several environmental mixtures are provided in Table 4. In cases where only the concentration of homologous groups is known, i.e., no isomer-specific data are available, different approaches are possible. For example, the assumption that the 2378-congeners of concern constitute all of the CDDs and CDFs present in the mixture is likely to provide an upper-bound, most conservative estimate of the toxicity. Alternatively, one could assume 13 - GEKP 010386 783697 that the-occurrence of each of the congeners, in the mixture has equal probabil ity (Olie et al., 1983; Commoner et al., 1984). For instance, 2,3,7,8-TCDO is one of 22 possible TCDDs and would constitute about 4% of a mixture of isomers occurring with equal probability. In other situations particular knowledge of chemical reaction parameters, process conditions, and results from related studies (e.g., congener distributions in emissions from combustion sources) might enable one to estimate the relative occurrence of 2378-congeners. How ever, one must be careful to explicitly explain and justify whatever assumptions are made. Table 5 illustrates the results obtained using different methods to estimate the proportion of 2378 to non-2378 isomers in the absence of analytical data for individual isomers. The calculated 2378-TCDD equivalents can then be used to assess the health risk of a mixture. As an explicit example, consider a municipal solid waste (MSW) combustor whose particulate emissions, the CDD/CDF mixture in question, are the same as the electrostatic precipitator (ESP) catch cited in columns 5 and 6 of Table 4. The sample is estimated to contain 32 ppb 2378-TCDD equivalents; i.e., 32 picograms of 2378-TCDD equivalents per milligram of mixture. Suppose that an exposure analysis indicates that a person living downwind from the incinerator receives an average daily dose of 1 ng of the mixture/kg body weight resulting from inhalation (i.e., without consideration of other possible routes of exposure). This exposure estimate is combined with the upper-bound carcinogenic potency of 2,3,7,8-TCDD (1.6 x 105 per mg/kg-day [U.5. EPA, 1984c]) to generate the upper 95% limit of the excess risk of develop ing cancer (from inhalation exposure alone) for a person living downwind from the facility emitting the mixture under consideration, assuming lifetime expo sure: GENP 010887 14 783698 Upper 95% limrt of excess cancer risk resulting from inhalation exposure = [potency] x [exposure] = [1.6 x IQS per mg 2 ,3,7,8-TCDD/kg-day] x [32 pg TCDD/mg mixture x 10~9 mg 2,3,7,8-TCDD/pg x 1 ng mixture/kg-day x 1 0 - 6 mg mixture/ng mixture] VI. COMPARISON OF TEF APPROACH WITH RESULTS OF BIOLOGICAL TESTING A limited number of in vivo and in vitro approaches have been employed in assessing the toxicity of complex mixtures of CODs and CDFs. While the results from these- attempts are not definitive, it is instructive to compare those results with the results from the TEF approach proposed here. Eadon et al. (1982) investigated the toxicity of CDD/CDF-contaminated soot associated with a fire involving PC3-containing electrical equipment. Using the results from acute in vivo toxicity (LD5 0 ) studies in which the soot was the test substance, the researchers determined that it had the acute toxic ity expected of material containing about 50 times the amount of 2,3,7,8-TCDD actually found by GC/MS analysis. Table 5 illustrates the results of employing the TEF approach through three different procedures, each of which depends upon the results of GC/MS analysis of the soot. In the first instance (A, in Table 5), the analytical data have been consolidated to totals within a homologous class. These con centrations are treated as if they consisted completely of 2378-members of the class and, therefore, are multiplied by the TEF appropriate for the 2378-mem bers of the class. The resulting estimate of 2378-TCDD equivalents by this procedure is about 80. 783699 15 In_procedure-S the assumption is made that the occurrence of each of the c o n f e r s in" a homologous class is equally probable; e.g., the concentration of 2,3,7,8-TCDD is 1/22 (about 5%) of the concentration of the total TCDDs. This approach leads to an estimate of the total 2373-TCDD equivalents of 8 . A rather unique data base exists in the case of the soot from this fire in that an extensive isaner-specific analysis of the sample is available (as cited in Des Rosiers, 1984). Therefore, the full array of TEFs from Table 1 (using the current EPA recommendations) can be applied. This procedure (C in Table 5) results in an estimate of roughly 50 for the total 2378-TCDD equivalents in the sample. As might be expected, the most conservative of these procedures, A, leads ,, to the highest estimate. Approach 3 (using theoretical probability of occur rence) leads to an estimate that is about 10-fold lower than the isomer-specific results C, reflecting the fact that the 2378-congeners are present in somewhat higher than "equal probability" proportions in this particular soot sample. Given the complexity of the analysis involved, the approximate nature of tne TEF method, and the vagaries of the assay, a major feature of note in Table 5 regarding the soot samples is that the results of procedures A, B, and C span a range of only one order of magnitude and bracket the bioassay estimate. In a separate study, Sawyer et al . (1983) published results of honologuespecific COD and CDF concentrations in fly ash from four municipal solid waste combustors which are amenable to treatment by the TEF methodology. In addition, extracts from the fly ash samples were analyzed by three bioassay techniques (AHH induction, EROD induction, and receptor binding). These data suggest that the TEF approach is likely to be a useful interim tool for the rough (order of magnitude) estimation of the toxicity of complex GENP 010889 16 783700 mixtures of CDDs _and CDFs. The availability of additional data comparing the results of an-alyti-eal and biological assays'will enable a conclusion regarding the preferred method of estimating TEFs (e.g., method A or B of Table 5). VII. RESEARCH NEEDS The Forum recommends that research be conducted that will enable the devel ment of assays to directly assess the toxicity of mixtures of CDDs and CDFs. In addition, research should be conducted in order to provide a firmer basis for, and to guide appropriate modification of the TEF approach. The following areas of research are appropriate for these purposes: 1- Validation and completion of the in vitro test data such as those listed in Table 2. 2. Investigation of the relationships between short-term in vivo and 1 n vitro tests and the toxic end points of concern; i.e., carcinogenicity, reproductive toxicity, immunotoxicity, and other significant human health effects resulting from CDD/CDF exposure. 3. Determination of the toxic potencies of metabolites of CDDs and CDFs in in vitro tests, relative to the potencies of the parent compounds. As pointed out by several reviewers, this would enable a refinement of the TEF approach. 4. Investigation of additional short-term assays which can test the mech anistic hypotheses underlying the TEF approach. 783701 17 GENP 010890 REFERENCES Bandiera, 5.; et al . (1933) Competitive binding of the cytosolic tetrachlorodibenzo-p-dioxin receptor. Biochem. Pharmacol. 32:3303-3813. Bandiera, S.; et al. (1984) Polychlorinated dibenzofurans (PCOFs): effects of structure on binding to the 2,3,7,8-TCDO cytosolic receptor protein, AHH induction and toxicity. Toxicology 32:131-144. Bradlaw, J.; et a l . (1979) Induction of enzyme activity in cell culture: a rapid screen for detection of planar polychlorinated organic compounds. J. Assoc. Off. Anal. Chem. 62:904-916. Bradlaw, J.; et al. (1930) Comparative induction of aryl hydrocarbon hydroxy lase -activity in vitro by analogues of dibenzo-p-dioxin. Cosmet. Toxicol. 13:627-635. Commoner, B .; et a l . (1984, Nay 1) Environmental and economic analysis of * alternative municipal solid waste disposal technologies. I. An assessment of the risks due to emissions of chiorinated'dioxins and dibenzofurans from proposed New York City incinerators. Cooper Engineers. (1984) Air emissions and performance testing of a dry scrubber (quench reactor) dry Venturi and fabric filter system operating on flue gas from combusion of municipal solid waste at Tsushima, Japan. Czuwa, J.M.; Hites, R. (1984) Environmental fate of combustion-generated poly chlorinated dioxin's and furans. Environ. Sci . Technol . 15:444-450. Dencker, L. ; et al . (1985) Fetal thymus organ culture as an in vitro model for the toxicity of 2,3,7,8-tetrachlorodibsnzo-p-dioxin and its congeners. Mol. Pharmacol. 27:133-140. Denison, M.S.; Wilkinson, C.F. (1985) Identification of the Ah receptor in selected mammalian species and induction of aryl hyrocarbon hydroxylase. Eur. J. Biochem. 14:429-435. Des Rosiers, P. (1934) PCBs, PCDFs, and PCDDs resulting from transformer/ capacitor fires: an overview. Proc. 1983 PC3 seminar. Research project 2028, Electric Power Research Institute. Palo Alto, California. Eadon, G . ; et al. (1982) Comparisons of chemical and biological data on soot samples from the Binghamton State Office Building. (Unpublished report) . Gasiewicz, T.A.; Rucci, G. (1984) Cytosolic receptor for tetrachlorodibenzop-dioxin. Evidence for a homologous nature among various mammalian species. Mol. Pharmacol. 26:90-98. GENP 010891 18 783702 Gierthy, J. F, ; C-p-ane, D. (1984) Reversible inhibition of in vitro epithe'ial cell' proliferation by 2,3,7,8-tetrachl-orodibenzo-p-dioxin. Toxicol. Appl . Pharmacol. 74":91-98. Gierthy, J.F.; Crane, D. (1935) In vitro bioassay for dioxin-like activity based on alterations in epithelial cell proliferation and morphology. Fundam. Appl. Toxicol. 5:754-759. Grant, D.L. (1977) Proc. 12th annual workshop on pesticide residues analysis. Winnipeg, Canada, p. 251. Grav'itz, N.; et al . (1933, Nov. 1) Interim guidelines for acceptable exposure levels in office settings contaminated with PCB and PCB combustion product Epidemiological Studies Section, California Department of Health Services. Greenlee, W. F.; et al. (1985a) Evidence for direct action of tetrachlorodibenzo-p-dioxin on thymus epithelium. Toxicol, Appl . Pharmacol. 79:112120. . Greenlee, W. F.; et al . (1935b) Toxicology of chlorinated aromatic hydrocar bons in animals and humans: in vitro approach to toxic mechanisms. Environ. Health Perspect. 60:69-76. t Greig, J.3.; et al. (1984) Incomplete correlation of 2,3,7,8-tetrachlorodi benzo-p-dioxin hepatotoxicity with Ah phenotype in mice. Toxicol. Appl. Pharmacol. 74: 17-25. Hassoun, E. et a l . (1934 ) Teratogenicity of 2,3,7,8-tetrachlorodibenzofuran in the mouse. J. Toxicol. Environ. Health 14:337-351. Knutson, J.; Poland, A. (1930) Keratinization of mouse teratoma cell line XB produced by 2 ,3,7,8-tetrachlorodibenzo-p-dioxin: an in vitro model of toxicity. Cell 22:27-36. Kociba, R.J.; Cabey, 0. (1985) Comparative toxicity and biologic activity of chlorinated dibenzo-p-dioxins and furans relative to 2,3,7,3-tetrachlorodibenzo-p-dioxin (TCDD). Chemosphere 14:649-660. Lamparski, L.L.; et al. (1984) Presence of chlorodibenzo-p-dioxins in a sealed 1933 sample of dried municipal sewage sludge. Chemosphere 13:361365. Madhukar, B.V.; et al . (1984) Effects of in vivo administered 2,3,7,8-tetrachlorodibenzo-p-dioxin on receptor binding of epidermal growth factor in the hepatic plasma membrane of rat, guinea pig, mouse, and hamster. Proc. Natl. Acad. Sci. USA 81:7407-7411. McKinney, J.; McConnell, E. (1982) Structural specificity and the dioxin receptor. Perg. Ser. Environ. Sci. 5:367-381. GENP 010892 19 783703 Moore, J.A. ; et aL, (1979) Comparative toxicity of three halogenated dibenzofurans in guinea pigs, mice, and rhes'LTS monkeys. Ann. N.Y. Acad. Sci. 320: 151-1637 Murray, F.J.; et al . (1979) Three-generation reproduction study of rats given 2,3,7,8-tetrachl orodibe.nzo-p-dioxin in the diet. Toxicol. Appl. Pharmacol. 50:241-252. Nagayama, J.; et al . (1985a) Inducing potency of aryl hydrocarbon hydroxylase in human lymphoblastoid cells and mice by polychlorinated dibenzofurans Environ. Health Perspect. 59:107-112. Nagayama, J.; et al. (1935b) Genetically mediated induction of aryl hydrocar bon hydroxylase activity in human lymphoplastoid cells by polychlorinated dibenzofuran isomers and 2,3,7,3-tetrachlorodibenzo-p-dioxin. Arch. Toxicol, 55:230-235. Neal, R,A..; et al . (1982 ) The toxicokinetics of 2,3,7,8-tetrachl orodibenzo-pdioxin in mammalian systems. Drug Metab. Rev. 13:355-385. Neal, R.A. (1935) Mechanisms of the biological effects of PCSs, polychlorina-* ted dibenzo-p-dioxins, and polychlorinated dibenzofurans in experimental animals. Environ', Health Perspect. 60:41-46. Okey, A.B.; Vella, L.M. (1984) Elevated binding of 2 ,3,7,3-tetrachl oro-d iba.nzo-p-dioxin and 3-methyl chol anthre.ne to the Ah receptor in hepatic cytosols from phenobarbital-treated rats and mice. Biochem. Pharmacol. 33:531-538. Okey, A.B.; et al . (1984) Ah receptor in primate liver: binding of 2,3,7,8tetrachlorodibenzo-p-dioxin and carcinogenic aromatic hydrocarbons. Can. J. Physiol. Pharmacol. 52:1292-1295. 01ie, K. ; et al . (1933) Formation and fate of PCDD and PCDF from combustion processes. Chemosphere 12:627-636. Ontario Government. (1982, Dec. 16) Chlorinated dioxins and chlorinated dibenzofurans. Ambient air guideline. Health Studies Service, Ministry of Labour. Ontario Government. (1984, Dec.) Scientific criteria document for standard development. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Ministry of the Environment. No. 4-84. December, Poland, A.; et al. (1979) Mechanism of action of dioxins. Ann. N.Y. Acad. Sci. 320:214-230. Poland, A.; Knutson, J. C.` (1982) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: an examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22:517-554. GENP 010893 20 783704 Poland, A.; et al . (1985) Studies on the mechanism of action of halogenated aromatic hydrocarbons. Clin. Physiol. Biochem. 3:147-155. Rappe, C. (1984) Analysis of polychlorinated dioxins and furans. Environ. Sci. Technol. 18:78A-90A. Rizzardini, M.; et a l . (1983) Toxicological evaluation of urban waste incinera tor emissions. Chemosphere 12:559-564. Safe, S.; et al. (1985) Polychlorinated dibenzofurans: quantitative structureactivity relationships. Chemosphere 14:675-684. Sawyer, T-; et al. (1983) Bioanalysis of polychlorinated dibenzofuran and dibenzo-p-dioxin mixtures in fly ash. Chemosphere 12:529-534. Schwetz, 8 .A.; et al . (1973) Toxicity of chlorinated dibenzo-p-dioxins. Environ. Health Perspect. 5:87-89. Swiss Government (Bundesamt fur Umvieltschutz, 3ern). (1932) Environmental pol lution due to dioxins and furans from chemical rubbish incineration plants. Schriftenreighe Umweltschutz. No. 5. Tong, H.Y.; et al. (1984) Identification of organic compounds obtained from incineration of municipal waste by HPLC and GC/MS. J. Chromatogr. 285: 423-441. U.S. Department of Health and Human Services. (1983, Apr. 29) Levels of con cern foi* hexa- (HCDD), hepta- (HpCDD) and octachlorodi6enzo-p-dioxins (OCDD) in chickens and eggs. Memorandum. U.S. Environmental Protection Agency. (1931, Nov. 19) Interim evaluation of health risks associated with emissions of tetrachlorinated dioxins from municipal waste resource recovery facilities. Office of the Administrator. U.S. Environmental Protection Agency. (1982) PC8 disposal by thermal destruc tion. National Technical Information Service, Springfield, VA. PB 82-241860. U.S. Environmental Protection Agency. (1984a, Feb.) Ambient water quality criteria for 2,3,7,8-tetrachlorodibenzo-p-dioxin. EPA-440/5-34-007. U.S. Environmental Protection Agency. (1984b,) Health assessment document for polychlorinated dibenzo-p-dioxins. EPA-60Q/8-84-014F. U.S. Environmental Protection Agency. (1984c, June) Assessment of emissions of specific compounds from a resource recovery municipal refuse incinerator. Office of Toxic Substances. EPA-560/5-84-002. U.S. Environmental Protection Agency. (1984d, Dec.) Thermal degradation products from dielectric fluids. EPA-560/5-84-009. 21 783705 U.S. Environmenca.L.Protection Agency. (1985, Jan. 9) Proposed guidelines for health risk assessment of chemical mixtures and request for comments: notice. " Federal Register 50:1170-1176. Vickers, A.E.M.; et a l . (1985) Mechanism of action of toxic halogenated aro matics. Environ. Health Perspect. 59:121-129. Weber, H.; et al. (1984) Teratogenicity of 2,3,7,8 -tetrachlorodibenzofuran (TCOF) in mice. Toxicol. Lett. 20:183-188. GENP 010895 22 783706 \ Basis/ canpound (Basis) TABLE 1. SOME APPROACHES TO ESTIMATING RELATIVE T0XIC1T1ES OF PCDDs AND PCDFs 1 Swiss3 Grantb 0Hec Commoner^ New York Statee Ontario^ F0A9 Enzyme ld50 Various - Various ef fects effects EPA , EPA1 current CAh 1981 recommend. iA Vari ous effects Mono thru di tri 2378-TCDD other TCDDs 2378-PeCDDs other PeCODs 2378-HxCDDs other HxCDDs 2378-HpCODs other HpCDDs OCDO 0 0 l 0.01 0.1 0.1 0.1 0.1 0.01 0.01 0 0 0 1 0 0.1 0 0.1 0 0.01 0 0 0 0 1 0 1 0 0.03 0 0 0 0 0 1 1 0.01 1 0.01 1 0.01 1 0.01 0 0 0 1 0 0 0 0.02 0.02 0.005 0.005 <0 .0 0 0 0 1 0 0 1 0 1 0 1 0 1 0 1 00 00 1 1. 1 0.01 0 0.5 f 0 0.005 0 0.04 0 0.0004 0 0.001 0 0.00001 00 Swiss Government, 1982. bfirant, 1977. c01ie et a l 1983. ^Commoner et al., 1984. eEadon et al., 1982. ^Ontario, 1982. 9U.S. DHHS, 1983. hfiravitz et al ., 1983. ilJ.S. EPA, 1981. (continued on the following page) k GENP 010896 GENP 010897 TABLE 1. (conti nuoti) Basi s/ compound (Basis) 2378-TCDFs other TCDFs 2378-PeCDFs other PeCDFs 2378-HxCOFs other HxCDFs 2378-HpCDFs other HpCDFs OCOF Swiss Grant*5 01 iec Commoner4* New York Statee - Ontario^ FDA9 Enzyme LD50 Various effects Various effects 0.1 0.1 0.33 0.02 0 0.1 0 0 0.0002 0 0.1 0.1 0.33 0.02 0 0.1 0 0 0.0002 0 0.1 0.1 0.01 0.02 0 0.1 0 0 0.0002 0 0.1 0.01 ' 0 0 00 0.02 0.0002 0 0 0 000 0 I EPA EPA1 current CAh 1981 recommend. Various effects 1 0 0.1 0 0 0.001 1 0 0.1 0 0 0.001 1 0 0.01 0 0 0.0001 1 0 0.001 0 0 0.00001 0 00 TAULE 2 . P O T E N C IE S OF D I O X I N S IIE L A T IV E TO 2 , 3 . 7 , 8 - T C D O Chemical Guinea pig ld50 Carcino genicity Oh producti ve/ teratogenic cf fects Receptor hindi ng CDDs: Mono thru trl 237H-TC0D TCDDs 2370-PeCDD PeCDDs 2378-HxCDOs HxCUDs 2378-HpCDflS HpCODs OCDD la < .Q01 .673 .002 .03a -- .0043 ,002a -- lb -- -- .04b -- _, -- -- a HcKtnney and HcConnel 1, 1902; Houre et al., 1979 b U.S. EPA. 1984a. c Rirray et al., 1979; Schwetz et al., 1973; Ueber et al.. 1904. d Randlera et a l 1963. e Knutson and Poland. I960. Ie .1 < .001k _ -- .Qlc -- .0 0 1 -.0 1e lfi <.01-.l6e Ie -- ,05e -- -<.ooomk --- f Dradlaw e t a l ., 1979. 9 Rradlaw e t a l . . I960. b\ llaruliera et a l . . hassnun et a l . . I9R4.. 1904. j Gler lhy and Crane, 1905 k Weber e t a l . , 1984. Animal cel Is Induction AMU EROO Human cel Is Cell keratin. l Flat (XII) cell assay Immune toxlci1 In. vitr. <.0 0 1 f ie < .0U1-.029 im .0 2 -.29 <.0019 .001-.19 <.oniy '.00?-.0049.f -- <.001f <.noif 19 *- -- -- --- .01e ' ^1 Ie <.noi-.oie .5'i -- .005e -- _ -- |J -- _ -- __ -- _ -- -- -- .00' ln .p -- __ -- -- _ -- -- I Poland et al., 1979. n Nagayama et al., 1985a,h. n Poland et a l 1 9 / i i . 0 Oenckcr et al., 1905. P Greenlee et a I., 1985b. (table continued on the fullowing page) 868010 3 CO 3 CD k t GENP 010899 Chemical Guinea pig *-D 5Q Carclnogenicity Kono thru trt 2378-TC0F TCDFs 23/8-PeCDF 12467-PeCDF PeCOFs 2378-HxCDFs HxCDFs 2378-HpCDFs HpCDFs -- .28;,5a -- .017* -- _ " -- -- --- -- -- -- Reprodnrtf ve/ teratogenic effects TABLE (c o n tin u e d ) Receptor binding Eniymc Induction Aim Animal cells Human cel Is EH00 Cell keratin. Flat i (XH) cel 1 aisay Immunotoxfclt in vTtro -- .3-.l3l.lt -- -- _ -- _,, -- .001-. 02*1 ,h c.OUld <.001*1 .3e;.24h;.4l .001-.0 Sd.e .0 1 -.4f.h.m <.0 0 ld;.04m .4*i ,4m .13*1;.7*;.fil <. 3*1;.4m .0*n .lbh .0 0 1 -.ld.e .0021 -- <.UUl-2ii,n,m .&ra .04-.&e.h .OUie.h .Ob-.2l.m .UUlm ;.0021 -- -c.OOlh .004 y c.OUlf -- -- .H .005*1 <.0 0 lh . 0011 .l-.fil .0061 -- .OUie .06e -- -- _ -- _ -- __ "~ -- . 1J -- -- __ -- __ 1 -- .10.IP -- -- -- ,, -- _ -- 26 ' 783710 T A B L E 3. C D D / C D F I S O M E R S OF M O S T T O X I C C O N C E R N S Isomer Dioxin TEFb Dibenzofuran Isomer TEF b 2,3,7,8-TCDD 1,2,3,7,8-PeCDD 1,2,3,-4,7,8-HxCDO 1,2,3,7,8 ,9-HxCDD 1,2,3,6,7,8-HxCDO 1,2,3,4,5,7,8-HpCDD 1 0.5 0.04 0.04 0.04 0.001 2,3,7,8-TCDF 0.1 1.2.3.7.8- PeCDF 2.3.4.7.8- PeCDF 0.1 0.1 1,2,3,4,7,8-HxCDF 1,2,3,7,8,9-HxCDF 1,2 ,3, 6 ,7 ,8 -HxCDF . 2,3,4,6,7,8-HxCDF 0.01 0.01 0.01 0.01 1.2.3.4.6.7.81.2.3.4.7.8.9- HpCDF Q.QQ1 HpCDF 0 .0 0 1 a In each homologous group, the relative toxicity factor for the isomers not listed above is 1 /1 0 0 of the value listed above. ^ TEF = Toxicity Equivalence Factor = relative toxicity assigned. 783777 27 GENP 010900 r) GENP 010901 TARLE 4. PCDDs/PCDFs IN SOME ENVIRONMENTAL SAMPLES Isomer TCDDs PeCDDs HxCDDs HpCDDS OCDD TCDFs PeCDFs HxCDFs HpCDFs OCDF Air parties. St. Louise con/F Tcon TEF cone. eqts. (ppb) 1 0.2 0.2 0.5 1 0.5 0.04 1 .2 0.048 0 .001 25 0.025 0 170 -- 0.1 -- -- 0.1 -- -- 0.01 -- -- 0.001 -- -- 0 -- -- MSW ESP dust0 con/F TCDO cone. eqts. (ppb) 55 10 5 160 6.4 120 0 .1 2 260 -- 40 4 80 8 280 2 . 8 160 0.16 40 - - Lake sediment0 COD/F TCDD cone. eqts. (PPb) 00 0 .1 0.05 0.34 0.014 0.5 0 .001 1.3 -0.13 0.013 0.14 0.014 0.38 0.004 1.13 0 .0 0 1 0.14 -- 1982 Mi lorganite^ COO/F cone. TCDD eqts. (ept ) 206 206 -- -- 2768 110.7 7600 7.6 60000 -- -- --- -- -- -- -- -- -- -- I MSW f]y ashf Ontario Oslo COO/F TCDO - co n / F TCDI cone. eqtq. .1 . cone. eqt' (ppt) (PPt ) 541 541 ND -- 467 234 11 5.! 591 24 51 2 434 C.43 119 0. 467 186 -- \ Total TCDD eqts. 0.08 32 0.10 324 799 7.: aU .S . EPA . 1984d, "Cooper Engineers, 1904 . cRappe, 1984. `M.amparski et al ., 1984. C?uwa and Hites, 1984. ^Tong et al., 1984. 90es Rosiers, 1984. (continued on Lh** following pagr T A B L E 4. ( c o n t i n u e d ) Thermal degradation prods, from dielectric fluidsa Run Run 8 -13-40 8-30-61 ASKL CDD/F TCDD CDD/F TCDD Isomer TEF cone. eqts. cone. eqts. ________ina)___ (ug) TCDDs 2378 other PeCDDs 2378 other HxCDDs 2378 . other HpCDOS 2378 other OCDD 1 0.01 0 0.5 0 0.002 0.04 0 0.0004 0.001 0 0.00001 00 00 00 00 0 330 0 37 0 0 0 0.33 0 Jap. MSWb Commercial CPs Soot from PCU fire9 -- i-- n------- Pt. A TEF Pt. B TEF 246TCPC PCPC CDD/F TCDD. cone. eqts. CDD/F TCDD cone. eqts. [1b/HMBTU(x10-6 )] CDD/F TCDD CDD/F TCDD cone. eqts. cone. eqts. (ppm) (ppm) CDD/F TCDD cone. eqts. (PPm) 0.1 0.1 0.07 0.035 0.04 0 .0 0 2 0.02 0.01 <0 .0 0 1 0 0.58 0.58 <0 .1 0.47 0.24 <0 . 1 0.36 0.014 <1 0.08 <0 .0 0 1 0.04 0 <1 <1 <0 .1 <0 .1 -- 2.5 0.1 175 0.18 500 0.6 0.6 Q.6 0.01 1 2.5 1.25 2.5 0 .0 1 l.l 0.04 3.6 0 30 40 2 (continued on the following page) 783713 GENP 010902 GENP 010903 TABLE 4. (continued) Thermal degradation prods, from dielectric fluids8 dap. MSWh Commercial CFs Sopt from PCI! fire(J Run Run 8-13-40 t-30-61 ASKL Ft. A TEF Ft. B TEF 246TCFC PCPC i 1---- CDD/F TCDO COO/F TCDO Isomer TEF cone . eqts. cone . eqts. ("9) (ug) COO/F TCDO CDO/F TCOO cone. eqts. cone. eqts. f1h/MMBTU(10 -6 )] CDO/F TCDO CDD/F TCDD cone. eqts. cone. eqts. (ppm) (ppm) CDD/F TCDD cone. eqts. (ppm) TCDFs u O 2378 0 .1 690 69 1400 140 other 0 .0 0 1 PeCDFs 1.31 0.131 ' 1.25 0.126 1.5 0.15 <0 .1 12 1.2 16 0 .0 1 2378 0 .1 43 4.3 6400 640 0.38 0.038 0.46 0.046 17.5 1.75 <0 .1 368 35.8 other 0 .001 312 0.3 HxCOFs 2378 0.0 1 7 0.07 910 9.1 0.06 0.006 0.06 0.006 36 3.6 <0.3 670 6.7 other 0.0001 295 0.03 HpCDFS 2378 0 .001 0 0 29 0.029 0.01 <.001 0 . 0 2 <.001 4.8 0.005 19 0.019 285 0.29 other 0 .00001 1/2 0 OCDF 0 00 3. 4 0 0.004 0 0.01 0 <1 -- 25 40 -- Total TCDD eqts 73 789 0.3 1.02 5. 5 0.3 46 0000 -"4 TAULE 5. USE OF THE TEF APPROACH Isomer PCB fire soota MSW fly ashb i ,,1__ Sample 1 Sample 2 CDD/F TCDD eqts. " CDD/F TCDD eqts. -------- T T 1------CDD/F TCDD eqts. cone. (PPfn) - cone. (PP) cone. (ppb) Propn. (ppm) (ppb) (ppb) TEF factor AC BC CC AC Be AC Be Total TCDDs 2378 TCDDs other TCDDs 1 1 0.01 1 0.05 0.95 1.2 1.2 1.2 u> Total PeCDDs 0.5 1 5.0 2378 PeCDDs 0.5 0.07 5.0 other PeCDDs 0.005 0.93 5.0 Total HxCDDs 2378 HxCDDs other HxCDDs 0.04 0.04 0.0004 1 0.3 0.7 4.7 4.7 4.7 Total HpCDDs 2378 HpCDDs other HpCDDs 0.001 0.001 0.00001 1 0.5 0.5 7 7 7 1.2 0.2 0.6 __d -- 2.5 0 . 2 1.3 ---- 0.2 0.1 -- ---- -- -- 85 85 85, 213 213 213 354 354 354 184 184 184 85 4.3 0.8 107 7.0 1.0 14.2 4.3 0.1 0.2 0.1 --" 2.7 2.7 2.7 6.6 6.6 6.6 11.6 11.6 11.6 5.7 5.7 5.7 2.7 0.1 -- 3.3 s: 0 . 2 0.5 0.1 -- _ -- a Des Rosiers, 1984, assuming only homologue-specific concentrations are known (for isomer-specific analyses; see Table IV). b Sawyer et al., 1983. c A = estimated assuming 2378-isomers constitute 100% of a homologous group. B = estimated assuming occurrence of all isomers in a homologous group is equally probable (thus using the --004 C-4O proportionality factor in column three). C = estimated by utilizing isoiner-specific analyses (see Table IV). d Values rounding off to less than 0 .1 are omitted. t cn (continued on the following page) .t GENF 010905 KU>l TABLE 5. (conti n u e d ) Isomer PCI1 fire soota j MSW fly ashb i Sample l <iample ^ CDD/F TCDD eqts . CCO/F TCDD eqts. CCD/F TCDD eqts. cone. (ppm) cone. (PPM cone. (PPb) Propn. (ppm) (ppb) (ppb) TEF factor Ac Bc Cc AC BC AC BC Total TCDFs 2378 TCDFs other TCDFs 0.1 0.1 0.001 1 0.03 0.97 Total PeCDFs 2378 PeCDFs other PeCDFs 0.1 0.1 0.001 1 0.07 0.93 Total HxCDFs 2378 HxCDFs other HxCDFs 0.01 0.01 0.0001 1 0.25 0.75 Total HpCDFs 2378 HpCDFs other HpCDFs 0.001 0.001 0.00001 1 0.50 0.50 Total TCDD equivalents (TEF): TEF estimate: * AHH bioassay: EROD bioassay: Receptor binding assay: Acute toxicity bioassay : 28 28 28 670 670 670 965 965 965 460 460 460 2.8 67 9.7 0.5 0.1 1.2 ---- 4.7 35.8 0 . 6 0.3 2.4 6.7 0.1 0 . 2 0.3 ---- 209 209 209 549 549 549 1082 1082 1082 499 499 499 20.9 0.6 0.2 54.9 3.8 0.5 10.8 2.7 0.1 0.5 0.2 -- 84 9 46 -- ---- -- ---- ---- -- 58 "-- 294 26 4 5 32 7.0 7.0 7.0 17.8 17.8 17.8 32.1 32.1 32.1 10.9 10.9 10.9 0.7 --- 1.8 0.1 -- 0.3 0.1 -- _ -- -- 91 -- -- -- -4-- 783716 (continued on the following page) Table 5. (continue d) --_ MSW fly ashb Sample 3 Sample 4 Isomer TCDD eqts. (ppb) Propn. CDD/F TEF factor cone. Ac Bc (ppb) CDD/F cone. (ppb) TCDD eqts. (ppb) Ac Bc Total TCDDs 2378 TCDDs other TCDDs 1 I 0.01 1 0.05 0.95 12.9 12.9 12.9 12.9 0.6 0,1 2.4 2.4 2.4 0 .1 2.4 -- Total PeCDDs 2373 PeCDDs other PeCDDs Total HxCDDs 2378 HxCDDs other HxCDDs 0.5 0.5 0.005 0.04 0.04 0.00d4 1 0.07 0.93 1 0.3 0.7 3 7.5 37.5 37.5 75 75 75 18.8 3 1.3 0.2 0.9 -- 7.9 4.0 7.9 0.3 7.9 -- 9.7 0.4 m 9.7 0 .1 9.7 -- Total HpCDDs 2378 HpCDDs other HpCDDs 0.001 0.001 0.00001 1 0.5 0.5 41.9 41.9 41.9 9.1 9.1 -- 9.1 -- Total TCDFs 2378 TCDFs other TCDFs 0.1 0.1 0.001 1 0.03 0.97 8.2 8.2 8.2 0.8 -- 4.4 0.4 4.4 -_ 4.4 -- Total PeCDFs 2378 PeCDFs other PeCDFs 0.1 0.1 0.001 1 0.07 0.93 19.8 19.8 19.8 2.0 0.1 -- 21.0 2.1 21.0 0.1 21.0 -- Total HxCDFs 2373 HxCDFs other HxCDFs 0.01 0.01 0.0001 1 0.25 0.75 38.7 38.7 38.7 0.4 0.1 -- 21.6 21.6 21.5 0.2 0.1 -- Total HpCDFs 2378 HpCDFs other HpCDFs 0.001 0.001 0.00001 1 0.50 0.50 20.6 20.6 20.6 16.6 -- 16.6 -- 16.6 --- Total TCDD equivalents (TEF): TEF estimate: AHH bioassay: EROD bioassay: Receptor binding assay Acute toxicity bioassay: 38 2 4-- 5 -- 65 -- 9 0.7 2 _-- 2 -_ 11 _ -- -- GENP 010906 783717 T A 3 L E 6. E X A M P L E OF A C A L C U L A T I O N OF T C D D E Q U I V A L E N T S W E R E ISOMER-SPECIFIC CONCENTRATIONS ARE A7AILA3LE Isomer 2,3,7f8-TCDO Other TCDDs 1,2,3,7,8-PeCDO 1,2,3,6 ,7,8-HxCDO 1,2,3,7,8,9-HxCDO 1,2,3,4, 7-,8-HxCDD Other HxCDDs 2,3,7,8-TCOF 1,2,3,7,8-PeCDF 2,3,4,7,8-PeCDF 1,2,3,6,7,8-HxCDF 1,2,3,7,8,9-HxCDF 1,2,3,4,7,8-HxCDF 2,3,4,6 ,7,8-HxCDF Other HxCDFs Concentration (ppb) 0.5 20 0.10 0.05 0.5 ' 0.1 50 10.5 0.1 0.01 0,01 0.01 0.01 0.01 1 Relative potency TCDD equivalents (ppb) 1 0.01 0.5 0.04 0.04 0.04 0.004 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.001 Total 0.5 0.2 0.05 0.002 0.02 0 .0 0 * 0.02 1.05 0.01 0.01 0.001 0.001 0.001 0.001 0.001 1.8 GETSfp 010907 34 783718 Homologus TCDOs PeCDDs HxCDDs TCDFs PeCDFs HxCDFs _ TA3LE 7. CALCULATION OF TCDD EQUIVALENTS WHERE ONLY HOMOLOGUE-SPEC IF IC ANALYSES ARE AVAILABLE Concentration Cppb) 0.5 0.5 13 100 150 \ 10 Relative potency 1 0.5 0.04 0.1 0.1 0.1 Total TCOD equivalents (PPb) 0.5 0.25 0.42 10.0 15.0 10.0 36.0 35 GENP 010908 783719 APPENDIX A NOMENCLATURE The following terminology and abbreviations are used in this document: 1. The term "congener" refers to any one particular member of the same chemical family; e.g., there are 75 congeners of chlorinated dibenzo-p-dioxins. 2 . The term "homologue" refers to a group of structurally related chem icals that have the same degree of chlorination. For example, there are eight homologues of CDDs, monochlorinated through octoch.l ori nated. 3. The term "isomer" refers to substances that belong to the same homol ogous class. For example, there are 22 isomers that constitute the homologues ,, of TCDDs. 4. A specific congener is denoted by unique chemical notation. For example, 2,4,8,9-tetrachlorodibenzofuran is referred to as 2,4,8,9-TCDF. 5. Notation for homologous classes is as follows: Dibenzo-p-dioxin D Dibenzofuran F No. of halooens Acronym Examole 1 through 8 M D Tr T Pe Hx Hp 0 CDDs and CDFs 2,4-DCDD 1,4,7,8-TCDD1 6. Dibenzo-p-dioxins and dibenzofurans that are chlorinated at the 2,3,7, and 8 positions are denoted as "2378" congeners, except when 2,3,7,8-TCDD uniquely referred to; e.g., 1,2,3,7,8-PeCDF and 2,3,4,7,8-PeCDF are both referred to as "2378-PeCDFs." GENP 010909 A-l 783720 APPENQI-X-3 COMPARISON OF DIFFERENT APPROACHES TO CALCULATING 2378-TCDD EQUIVALENTS Table III lists a number of different approaches to calculating 2378TCDD toxicity equivalents. Five of the approaches (those that deal with 4position 2378-substituted congeners, but not the 3-position substituted con geners) were applied to 1. The data in Table IV. 2. Some of the data included in Table I of the Report of the Citizens Advisory Committee on Resource Recovery in Brooklyn (March, 1985), produced by Ketcham and the Mt. Sinai School of Medicine. A summary comparison of the relative results is found in Table B-l, with the supporting tables attached: (Note that the units of mass emission are not the same for all of the facilities. Therefore, comparison of absolute numbers between facilities may be invalid.) These data indicate that, in general, the methods used by the Swiss government, New York State, and the U.S. EPA (the 1981 approach and the 1935 proposal) all generate results which are within an order of magnitude of each other. This suggests that, within the range considered, the results are not particularly sensitive functions of the TEFs selected. The procedure recommended by the state of California, however, gives results which are roughly an order of magnitude higher than those generated by the other approaches. In general, the greater the contribution from the TCDDs the greater the similarity in the results of the methods. This is due to the fact that all methods assign a TEF of 1 for 2,3,7,8-TCDD (and 1 to all TCDDs, when isomer-specific analyses are not available). Because higher chlorinated GENP 010910 B-l 783721 CDDs and CDFs- contribute significantly to the total, the disparity is greater between the CA results and those produced by the other methods, since CA assumes that all 2378-substitjted CDDs/CDFs are as potent as 2,3,7,8-TCDD, The other methods acknowledge, to one degree or another, the reduced toxicity in higher chlorinated species; c f . Table 2. GENP 010911 B-2 783722 TABLE B-l. RELATIVE 2378-TCDD EQUIVALENTS2 Source EPA `85 EPA '81 Swiss NY CA St. Louis ai r part. 1 PCB fire soot (isomer-speci fie) 1 MSW ESP dust I Lake sediment 1 Mi 1organite 1 Oslo MSW flyash 1 Ontario MSW flyash 1 Jap. plant A ,1 Jap. plant B 1 Albany 1 Wright-Pat. (best) 1 Wright-Pat. (worst) 1 0.3 0.03 0.2 -- 0 . 6 -- 0.8 0.3 0.6 0.3 0.2 0.4 1 2 40 4 3 .30 3 2 30 2 2 30 2 0.9 30 1 04* 20 1 23 1 27 0.8 2 3 0.4 2 5 2 3 20 2 2 20 Calculated using the Toxicity Equivalence Factors shown in Table 1. GENP 010912 B-3 783723 I60I( ia o TABLE B-2. CALCULATION OF 237U-TCDD T0XICI1T EQUIVALENTS FOR ST. LOUIS AIR PARTICULATES USING HOMOLOGUE-SPECIFIC DATA Compound CDD/F cone. (ppb) Mono to trl X 2378-TCDD TCODs 0.2 0 2378-PeCDD PeCDDs 1 0 2378-HxCDD HxCDDs 1.2 0 2378-HpCDD HpCDDs 25 0 OCDD 170 EPA 1985 TEFs TEs (ppb) EPA 1981 TEFs its (ppb) 0 00 0 1 0.01 0.2 1 01 0.2 0 0.5 0.005 0.5 0 00 0 0 0.04 0.0004 0.048 0 00 0 0 0.001 0 .0 0 0 0 1 0.025 0 00 0 0 0 00 0 Switzerland TEFs TEs . (ppb) New York TEFs TEs (ppb) 0 0 00 l 0.01 0.2 .0 1 0 0.2 0 0.1 0.1 1 1 0.1 0 00 0.1 0.12 0.03 0.036 0.1 0 00 0.01 0.01 0.25 0 *0 0 0 0 0 0 00 California , TEFs TES 1 (ppb) 00 ,V JI 1 0.2 00 11 00 1 1.2 00 l 25 00 00 Mono to tri X 0 2378-TCDF TCDFs NA 0 0 .1 0 .0 0 1 2378-PeCDF PeCDFs NA 0 0 .1 0 .0 0 1 2378-HxCDF HxCOFs NA 0 0.01 0 .0 0 0 1 2378-HpCDF HpCOFs NA 0 0 .0 0 1 0.00001 OCDF NA 0 Total 2378-TCDD equivalents 0 0 0 0 0 0 0 0 0 0 0.7 0 0 0 0 0 0 0 0 0 0 00 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 00 00 0.2 0 000 0 0.33 0 1 0 000 0 0.33 0 1 0 000 0 0.01 0 1 0 000 00 00 00 k 0.7 01 00 00 1.2 0 0 0 0 0 0 0 0 0 0 27.4 783724 Ta b l e b -3. c a l c u l a t i o n of 237o -t c d d t o x i c i t y e q u i v a l e n t s f o r pc b f i r e s o o t USING ISOMER-SPECIFIC DATA GENP 010914 Compound Mono to tri 2378-TCDD TCDDs 2378-PeCDD PeCODs 2378-HxCDO HxCDDs 2378-HpCDD HpCODs OCDD CDD/F cone. (PPTM) X 0.6 0.6 2.5 2.5 1.1 3.6 3 4 2 EPA 1985 TEFs TEs (ppm) EPA 1981 TEFs TEs (ppm) 0 00 0 1 0.01 0.6 0.006 1 1 0.6 0.6 0.5 0.005 1.25 0 0.0125 0 0 'o 0.04 0.0004 0.044 0 0.00144 0 0 0 0.001 0.00001 0.003 0 0.00004 0 0 0 0 00 0 Switzerland TEFs TEs (ppm) 00 1 0,01 0.6 0.006 0 .1 0.25 0 .1 0.25 0.1 0.11 0 .1 0.36 0.01 0.01 0.03 0.04 00 New York TEFs ~ TEs (ppm) 00 1 0.6 00 1 2.5 00 0.03 0 0.033 0 00 00 00 California TEFs TEs (ppm) 1 00 1' 0 0.6 0 1 2.5 00 1 1.1 00 13 00 01 0 Mono to tri X 0 2378-TCDF TCDFs 12 16 0.1 0.001 2378-PeCDF PeCDFs 358 312 0.1 0.001 2378-Hx COF HxCDFs 670 295 0.01 0.0001 2378-HpCOF HpCOFs 285 172 0.001 0.00001 OCDF 40 0 Total 2378-TCDD equivalents 00 1.2 0.016 0 0 35.8 0.312 0 0 6.7 0 0.0295 0 0.285 0 0.00172 0 00 46 00 0 0 0.1 0 0.1 1.2 1.6 0 0 .1 35.8 0 0 .1 31.2 0 0 .1 67 0 0 .1 29.5 0 0 .1 28.5 00 0 00 0 ft 1 .2 196 0 0.33 0 0.33 0 0.01 0 0 0 0 0 3.96 0 118.14 0 6.7 0 0 0 0 132 0 1 0 1 0 1 0 1 0 0 0 12 0 358 0 670 0 285 0 0 1332 O m *2 *TJ O H-1 O VO U\ wi cr> M 00 CO rCTo> , .-f TADLE B -4. CALCULATION OF 2378-TCDD TOXICITY EQUIVALENTS FOR MSW ESP DUST USING IIOMOLOGUE-SPECIFIC DATA AND 2378 TEFs V Compound CDD/F conc. _ (PPb ) Mono to tri X 2378-TCDD TCDOs 5 0 2378-PeCDD PeCDDs 10 0 2378-HxCDD HxCDDs 160 0 2378-HpCDD HpCDDs 120 0 CDD 260 EPA 1985 TEFs TE s (ppb) 00 1 0.01 5 0 0.5 0.005 5 0 0.04 0.0004 6.4 0 0.001 0.00001 0.12 0 00 EPA 1981 TEFs" Ts (PPb) Swi tzerland TEFs TE s (ppb) 0 00 0 1 51 5 1 0 . 0.01 0 0 0 0.1 l 0 0 0.1 0 0 0 0 .1 16 0 0 0.1 0 0 0 0.01 - 1.2 0 0 0.01 0 0 00 0 New York TFs TEs (ppb) 00 Cali fornia TEFs TEs (ppb) 1 00 1 5 1 -1 000 5 0 1 10 1 10 000 0 0.03 0 4.8 0 1 0 160 0 0 0 1 ' 120 000 0 0 00 0 Mono to tri X 0 2378-TCDF TCDFs 40 0 . 1 0 .001 2378-PeCDF 80 0 . 1 PeCDFs 0 0 .0 0 1 2378-HxCDF HxCDFs 280 0 0.01 0 .0 0 0 1 2378-HpCDF HpCDFs 160 0 0 .0 0 1 0.00001 OC DF 40 0 Total 2378-TCDD equivalents 0 4 0 8 0 2.8 0 0.16 0 0 31 0 0 0 0 0 0 0 0 0 (T 00 0 0 .1 0 0.1 0 0 .1 0 0 .1 0 0 .1 0 0 .1 0 0 .1 00 00 ft 5 0 4 0 8 0 28 0 16 0 ~0 79 0 0.33 0 0.33 0 0.01 0 0 0 0 0 13.2 0 26.4 0 2.8 0 0 0 0 62 0 1 0 l 0 1 0 1 0 0 0 40 0 80 0 280 0 160 0 0 855 _ TABLE B-5. CALCULATION OF tj?8-TCDD TOXICITY EQUIVALENTS FOR LAKE SEDIMENT USING HOMOLOGUE-SPECIFIC DATA ENP 010916 w -si 00 C-NOj ro Compound Mono to tri 2378-TCDD TCDDs 2378-PeCDD PeCDDs 2378-HxCDD HxCDDs 2378-HpCDD HpCDOs OCDD CDD/F cone. _(PPb) X 0 0 0.1 0 0.34 0 0.5 0 1.3 EPA 1985 TEFs TEs (ppb) EPA 1981 TFs TEs (ppb) 0 00 0 1 0.01 0 0 1 1 0 0 0.5 0.005 0.05 0 0 0 0 0 0.04 0.0004 0.0136 0 00 0 0 0.001 0.00001 0.0005 0 00 0 0 0 00 0 Switzerland TEFs TEs (ppb) 00 1 0.01 0 0 0.1 0.01 0.1 0 0 .1 0.034 0.1 0 0.01 0.01 0.005 0 00 New York TEFs TEs (ppb) 00 California JEFs TEs (PPb) i 00 1 0 1 l 0 0 0 00 1 0.1 1 0.1 0 0 00 0.03 0 0.0102 0 l 0 0.34 0 0 0 1 0.5 0 0 00 0 0 jo 0 Mono to tri X 0 2378-TCDF TCDFs 0.13 0 0.1 0.001 2378-PeCDF PeCDFs 0.14 0 0.1 0.001 2378-HxCDF HxCDFs 0.38 0 0.01 0.0001 2378-HpCDF HpCDFs 1.13 0 0.001 0.00001 OCDF 0.14 0 Total 2378-TCDD equivalents 00 0.013 0 0 0 0.014 0 0 0 0.0038 0 00 0.00113 0 00 00 0.1 00 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 n.i 00 00 0 0 0.013 0 0.014 0 0.038 0 0.113 0 0 1.2 0 0.33 0 0.33 0 0.01 0 0 0 0 00 0.0429 l 00 0.0462 1 00 0.0038 l 00 01 00 00 0.2 0 0.13 0 0.14 0 0.38 0 1.13 0 0 2.7 GENP 010917 TABLE B-6. CALCULATION OF ^78-TCDD TOXICITY EQUIVALENTS FOR MILORGANITE USING HOMOLOGUE-SPECIF1C DATA Compound CDD/F cone. (ppt) EPA 1985 TEFs TEs (ppt) Mono to tri X 0 0 2378-TCDD 206 1 TCDDs 0 0.01 206 0 2378-PeCDD PeCDDs 0 0.5 0 0.005 0 0 2378-HxCDD HxCDDs 2768 0 0.04 0.0004 110.72 0 2378-HpCDD HpCDDs 7600 0 .0 0 1 0 0.00001 7.6 0 OCDD 60000 0 0 EPA 1981 TETs TEs (PPt) 00 1 206 10 00 00 00 00 00 00 00 Switzerland TEFs TEs (PPt) 00 1 0.01 206 0 0.1 0 0.1 0 0 .1 276.8 0.1 0 0.01 O.OL 76 0 00 New York Cali forni a TEFi TEFs TEs (ppt) (ppt) i 000 0 1 206 ' -il 000 206 0 10 1 000 0 0 0.03 0 83.04 0 1 0 2768 0 0 0 1 7600 000 0 0 0 jo 0 Mono to tri X 0 00 00 2378-TCDF TCOFs NA 0 .1 0 0.001 00 00 0 0.1 0 0.1 2378-PeCDF NA 0 .1 00 PeCOFs 0 0.001 00 0 0.1 0 0.1 2378-HxCDF NA 0 .0 1 0 HxCDFs 0 0.0001 0 0 0 0 0.1 0 0.1 2378-UpCDF NA 0 .0 0 1 0 HpCDFs 0 0.00001 0 00 0 0.1 0u OCDF NA 0 00 Total 2378-TCDD equivalents 324 00 k 206 0 0 0 0 0 0 0 0 0 0 559 00 0.33 0 0 0 0.33 0 0 0 0.01 0 0 0 00 00 00 2B9 00 10 00 10 00 10 00 10 00 00 10600 GENP 010918 ` TABLE B-7. CALCULATION OF 2378-TCDD TOXICITY EQUIVALENTS FOR OLSO MSW FLY ASM USING HOMOLOGUE-SPECIFIC HATA ; ' Compound Mono to tri 237B-TCDD TCDDs 2378-PeCDD PeCDDs 2378-HxCDD HxCDDs 2378-HpCDD HpCODs OCDD CDD/F cone. (ppt) X NA 0 11 0 51 0 119 0 186 EPA 1985 TEFs Tts (ppt) EPA 1981 TFs T s (ppt) 0 00 0 1 0.01 01 01 0 0, 0.5 0.005 5.5 0 00 0 0 0.04 0.0004 2.04 0 0 0 0 0 0.001 0.00001 0.119 0 00 0 0 0 00 0 Switzerland TLFs " Tes (ppt) 00 1 0.01 0 0 0.1 1.1 0.1 0 0 .1 5.1 0.1 0 0.01 0.01 1.19 0 00 New York TEFs TE? , Cal 1forni a TEFs TEs (ppt) 1 (ppt) 0 0 0 1 o ' 'i 0 00 0 0 1 11 1 11 000 0 0.03 0 1.53 0 1 0 51 0 0 0 1 119 .0 0 0 0 0 0 !0 0 Mono to tri X 0 00 2378-TCDF NA 0 .1 00 TCDFs 0 0.001 0 0 2378-PeCDF NA 0 .1 00 PeCDFs 0 0.001 0 0 2378-HxCDF NA 0 .0 1 0 HxCDFs 0 0.0001 0 0 0 2378-llpCDF NA 0 .0 0 1 0 HpCDFs 0 0.00001 0 0 0 QCUF NA 0 00 Total 2378-TCDD equivalents 7.7 00 0 0.1 0 0.1 0 0.1 0. 0.1 0 0.1 0 0.1 0 0.1 00 00 0 0 0 00 0 0.33 0 1 0000 0 0.33 0 1 00 0 0 0 0.01 0 1 0000 0 00 1 0 000 000 0 7.4 12.5 0 0 0 0 0 0 0 0 0 0 181 783729 TAULE 8-8. CALCULATION .OF 237b .COD TOXICITY EQUIVALENTS FOR ONTARIO MSW FLY ASM USING HOMOLOGUE-SPECIF IC DATA VO -vl 03 00 oa Compound Mono to tri 2378-TCDD TCDDs 2378-PeCDD PeCDDs 2378-HxCDD HxCODs 2378-HpCDD HpCDDs OCDD CDD/F cone. (ppt) X 541 0 467 0 591 0 434 0 467 EPA 1985 TEFs TEs (ppt) EPA 1981 TEFi TEs (ppt) 0 00 0 1 0.01 541 0 1 1 541 0 0.5 0.005 233.5 0 0 0 0 0 0.04 0.0004 23.64 0 0 0 0 0 0.001 0.00001 0.434 0 00 0 0 0 00 0 Switzerland TEFs TEs _ (ppt) 00 1 0.01 541 0 0 .1 46.7 0.1 0 0 .1 59.1 0.1 0 0.01 0.01 4.34 0 00 New York TEFs Ts (ppt) 00 1 541 00 1 467 00 0.03 0 17.73 0 00 00 00 Cali fornia TEFs TEs (ppt) i 00 l.i 0 541 0 1 467 00 1 591 00 1 434 00 b0 Mono to tri X 0 2378-TCDF TCDFs NA 0 0.1 0.001 2378-PeCDF NA 0 .1 PeCDFs 0 0.001 2378-llxCDF NA 0 .0 1 HxCDFs 0 0.0001 2378-HpCDF NA 0 .0 0 1 HpCDFs 0 0.00001 OCDF HA Total 2378-TCDD equivalents 0 0 0 0 0 0 0 0 0 799 0 0 0 0 0 0 0 0 0 0 00 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 00 541 0 0 0 0 0 0 0 0 0 651 0 0.33 0 0.33 0 0.01 0 0 0 0 0 0 0 0 0 0 0 0 1026 0 1 0 1 0 1 0 1. 0 0 0 0 0 0 0 0 0 0 0 2023 TABLE B-9. CALCULATION OF 2378-TCui) TOXICITY EQUIVALENTS FOR HSW AT JAPANESE PLANT A USING IIOMOLOGUE-SPEC 1F 1C DATA GENP 010920 Compound Mono to tr1 2378-TCDD TCDDs 2378-PeCDD PeCDDs 2378-HxCDD HxCDDs 2378-HpCDD HpCDDs 0CL1D CDO/F EPA 1985 cone. 3 TEf T TEs& EPA 1981 TEf s TEsa X 0.1 0 0.07 0 0.04 0 0.02 0 0.01 0 1 0.01 0.5 0.005 0.04 ' 0.0004 0.001 0.00001 0 00 0.1 l 01 0.035 0 0 0 0.0016 0 00 0.00002 0 00 00 0 0.1 0 0 0 0 0 0 0 0 Switzerland TEFs TEsa 0 1 0.01 0.1 0.1 0.1 0.1 0.01 0.01 0 0 0.1 0 0.007 0 0.004 0 ' 0.0002 0 0 New York TEFs TEsa 00 Cal 1fornla TEFs TEsa i 00 1 0.1 i * - 0.1 0 0 00 1 0.07 l 0.07 0 r 00 0.03 0 0.0012 1 00 0.04 0 0 0 1 0.02 0 0 00 0 0 00 Mono to tri X 0 2378-TCDF TCDFs 1.31 0 0.1 0.001 2378-PeCDF PeCDFs '0.38 0 0.1 0.001 2378-HxCOF HxCDFs 0.06 0 0.01 0.0001 2378-HpCDF HpCDFs 0.01 0 0.001 0.00001 OCDF 0.004 0 Total 2378-TCDO equivalents 00 0.131 0 0 0 0.030 0 0 0 0.0006 0 00 0.00001 0 00 00 0.3 00 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 00 00 0.1 0 0.131 0 0.038 0 0.006 0 0.001 0 0 0.3* 0 0.33 0 0.33 0 0.01 0 0 0 0 00 0.4323 l 00 0.1254 1 00 0.0006 1 00 01 00 00 0.7 0 1.31 0 0.38 0 0.06 0 0.01 0 0 2.0 Units = lb/MM BTU(XIO-S) TABLE B-10. CALCULATION OF 2378-TCL ,'dXICITY EQUIVALENTS FOR MSW AT JAPANESE PLANT B USING IIOMOLOGUE -SPEC 1FIC DATA Compound *---i w IM-- Mono to tri 2378-TCDD TCDDs 2378-PeCDD PeCDDs 2378-HxCDD HxCDDs 2378-HpCDD HpCDDs OCDD CDD/F EPA 1985 conc.a TEFs TTs'a EPA 1981 TEFs TEsa X0 0.58 " 1 0 0.01 0.47 0 0.5 0.005 0.36 0 0.04 0.0004 0.08 0 0.001 o.oooot 0.04 0 00 0.58 0 1 1 0.235 0 0 0 0.0144 0 00 0.00008 0 00 00 0 0.58 0 0 0 0 0 0 0 0 Mono to tri X 0 2378-TCDF TCDFs 1.25 0 0.1 0.001 2378-PeCDF PeCDFs 0.46 0 0.1 0.001 2378-HxCDF HxCDFs 0.06 0 0.01 0.0001 2378-HpCDF HpCDFs 0.02 0 0.001 0.00001 OCDF 0.01 0 Total 2378-TCOD equivalents 00 0.125 0 0 0 0.046 0 0 0 0.0006 0 00 0.00002 0 00 00 1.0 0 0 0 0 0 0 0 0 0 0 0.6 Switzerland TEFs TEsa New York TEFs TEsa Californla TCF TEsa 0 1 0.01 0.1 0.1 0.1 0.1 0.01 0.01 0 0.58 0 0.047 0 0.036 0 .0.0008 0 0 1 0 1 0 0.03 0 0 0 i 00 0.58 0 l.i 0 0.47 0 1 0 0.0108 1 00 01 00 0 0.58 0 0.47 0 0.36 0 0.08 0 0 0 00 0 i 0 0 0 0 00 0.1 0.125 0.33 0.4125 l 1.25 0.1 0 0 0 00 0.1 0.046 0.33 0.1518 1 0.46 0.1 0 0 0 00 0.1 0.006 0.01 0.0006 1 0.06 0.1 0 0 0 00 0.1 0.002 0 0 1 0.02 0 0 0 0 00 0 0 0 0 00 b 0.8 1 .6 3.3 aLinills = lb/MM BTU(XiO-Sj 783732 GENP 010922 ,r TABLE B-ll. CALCULATION OF 2378-1CDD TOXICITY EQUIVALENTS FOR MSW AT ALBANY . USING IIOMOLOGUE-SPECIFIC DATA -w 783733 Compound Mono to tri 2378-TCDD TCDDs 2378-PeCDD PeCDDs 2378-HxCOO HxCODs 2378-HpCDD HpCDDs OCDD CDD/F cone. (ng/m3) EPA 1985 TEFi TEs (ng/m3) EPA 1981 TEFs TEs (ng/m3) X0 00 0 0.45 1 14 0 .0 1 0.45 0.14 1 1 0.45 14 97 0.5 48.5 0 0 0.005 0 0 0 0 53 0.04 2.12 0 0 0.0004 0 0 0 0 71 0 .0 0 1 0.071 0 0 0.00001 0 0 0 0 10 0 00 0 Switzerland TEFs Tits (ng/m3) 0 1 0.01 0. 0.45 0.14 0 . 1 9.7 0.1 0 0 .1 5.3 0.1 0 0.01 0.01 0.71 0 00 New York Tf-Fs TEs (ng/m3) 00 1 0.45 00 1 97 00 0.03 0 1.59 0 00 00 00 Cali fornia TEFs TEs (ng/m3) i 00 1 * -1 0.45 00 1 97 c0 1 53 00 1 71 00 0 \ 0. Mono to tri X 0 2378-TCDF TCDFs 2.1 33 0.1 0.001 2378-PeCDF 21 0 . 1 PeCDFs 0 0.001 2378-HxCDF HxCDFs 4 0.01 0 0.0001 2378-HpCDF HpCDFs 1 0.001 0 0.00001 OCDF 20 Total 2378- TCDO equivalents 0 0.21 0.033 2.1 0 0.04 0 0.001 0 0 54 0 0 0 0 0 0 0 0 0 0 00 0 0 0 00 0 0.1 0 0.1 0.21 3.3 0.33 0 0.693 0 1 0 2.1 0 0 0.1 0 0.1 2.1 0.33 6.93 1 21 0 0 0 00 0 0.1 0 0.1 0.4 0.01 0.04 1 0000 4 0 0 0.1 00 0.1 0 0 1 1 0 0 0 00 00 0 ft 14 22 00 107 00 250 $ ? 5 o \. 'o "O jO txs 1 Ms> i TABLE B-12. CALCULATION OF 237u .CuD TOXICITY EQUIVALENTS FOR WP AFB (BEST) USING UOMOLOGUE-SPECIFIC OATA Compound Mono to tri 2378-TCDD TCDDs 2378-PeCDD PeCODs 2378-llxCDD HxCOOs 2378-UpCDD HpCDDs ' OCDO CDD/F cone. (nq/m3 ) EPA 1985 TEFs TEs (nq/m3 ) EPA 1981 TEFs ' TEs (nq/m3 ) X0 00 0 0.4 1 0 0.01 0.4 1 01 0.4 0 0.4 0.5 0 0.005 0.2 0 00 0 0 I 0.04 0.04 0 0 0.0004 0 0 0 0 3 0.001 0.003 0 0 0.00001 0 0 0 0 30 00 0 Swi t2erland TEFs TEs *(nq/m3) 00 1 0.01 0.4 0 0 .1 0.04 0.1 0 0.1 0.1 0.1 0 0.01 0.01 . 0.03 0 00 New York TEFs TEs (nq/m3 ) 00 1 0.4 00 1 0.4 00 0.03 0 0.03 0 00 00 00 Cali fornia TEF s; TE s (nq/m3 ) i 00 1 .< 0 0.4 0 1 0.4 00 11 00 13 00 0; 0 Mono to tri X 0 00 2378-TCDF TCDFs 8 0.1 0 0.001 0.8 0 00 2378-PeCDF PeCDFs 3 0.1 0 0.001 0.3 0 00 00 03 2378-HxCDF HxCDFs 03 P* 2378-llpCDF HpCDFs 4 0.01 0 0.0001 0.04 0 0 0 9 0.001 0.009 0 0 0.00001 0 0 OCDF 20 00 Total 2378- TCDD equivalents 1.8 00 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 00 00 0.4 k G 0 0 0 00 0.8 0.33 2.64 1 0000 8 0 0.3 0.33 0.99 1 0000 3 0 0.4 0.01 0.04 1 0000 4 0 0 0 19 0 0 0 00 0 0 0 00 3.0 4.5 28.8 GENP 010924 TABLE B-13. CALCULATION OF 23. J,CDD TOXICITY EQUIVALENTS FOR WP AFB (WORST) USING HOMOLOGUE-SPECIFIC DATA ' ' ", i Compound CDD/F cone. (ng/m3) Mono to tri X EPA 1985 TEFs HIT 1[ng/m3 ) 00 EPA 1981 TEFs TEs (nq/m3) 00 Switzerland TEFs TEs (ng/m3) 00 New York TEFs-- TEs (ng/m3) 00 California TEFs TI? (ng/m3 ) 1 00 2378-TCDD TCDDs 41 0 0.01 41 01 41 4 1 4 4 i - 4 0 0.01 0 0 0 0 0 2378-PeCDD PeCDDs 3 0.5 0 0.005 1.5 0 00 0 0.1 0 0.1 0.3 1 3 I 0000 3 0 2378-HxCDD 6 0.04 0.24 0 HxCDDs 0 0.0004 0 0 bd *L--In 2378-HpCOD HpCDDs 32 0 0.001 0.00001 0.032 0 00 0 0.1 0 0.1 0.6 0.03 0.18 1 0000 0 0.01 0.32 0 0 1 0 0.01 0 0 0 0 6 0 32 0 OCDD ` 16 0 00 00 0 0 0 0: 0 Mono to tri X0 00 2378-TCDF TCDFs 31 0 . 1 0 0.001 3.1 0 00 2378-PeCDF 15 0 .1 1.5 0 PeCOFs 0 0.001 00 *V| 00 C-vOj OCOl 2378-HxCDF 23 0 .0 1 0.23 0 HxCDFs 0 0.0001 0 0 2378-HpCDF 93 0 .0 0 1 0.093 0 HpCDFs 0 0.00001 0 0 OCDF 80 00 Total 2378-TCDD equivalents 11.0 00 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 00 00 b 4 0 3.1 0 1.5 0 2.3 0 9.3 0 0 21.4 0 0.33 0 0.33 0 0.01 0 0 0 0 0 10.23 4.95 0 0.23 0 0 0 0 22.6 0 1 0 1 0 1 0 1 0 0 0 31 0 15 0 23 0 93 0 0 207 S T E P H E N B. HAM ILTON. J R . M M O - M V IO M t*IN T *L a e ilH C C A IO TCC M N Q IO O T GENERAL ELECTRIC GENERAI. ELECTRIC COMPANY FA IR FICL Q , C O N N E C T IC U T 0 A 3 I January 12, 1987 Dr. Ruth Arisman Rt. 1, Box 21 Burkville, AL 36725 Dear Ruth: Here are the two items on inhibition of dioxin toxicity by PCBs. I think they are pretty exciting. I am also enclosing an abstract of a paper given by Steve Sa f e j a s t October in Montreal. The bottom line conclusions on PCB health effects are very similar to those drawn by Seymour Friess back in 1982-83. Safe, as you may recall, has been one of EPA's main consultants on PCB toxicity, and what he says on the subject should be very influential. I will plan on taking you up on your offer to see the Burkville plant sometime when I'm traveling to the South. Very truly yours SBH:cas Enclosures 783736 GENP 010925 0971 SO CIETY OF TOXLCOLOGY ABSTRACT FORM FOR THE 1987 ANNUAL MEETING 783737 Any correspondence regtiding your abstract muti reference the abota number. Fonowfcrnutforheading. LeavedoublespacebetweenheadingMd abstract TextofabstractmutibeInglepaced.Therawill bechargeof$25loranytbtiraeithatmust beretypedLLeave no margins-- typemutt bewithin rectangle.Single space, elitetype, exceptfordouble spacebetweenbody of textand authors. Deadline for receiptIn Executive Secretary's office Is October 24,1986. Follow Instructions accompanying this form. Submit original and one copy only. Original mutt be on (his form. Proof carefully. - TERATOGENICITY OF 2,3,7,8-TETRACHLORODIBENZO- p-DIOXIN: ANTAGONISM BY AROCLOR 1254: J.M. Haake, F. Mayura, Phillips and S a f e , Departments of Veterinary Physiology and Pharmacology and Veterinary Public Health, Texas A&M University, College Station, TX. 1. Nam * and address of presenting author: Mamas Ms. J.M. Haake______________ . Organization: T e x a s ^ University________ Address: C H eSe of Veterinary Medicine The dose-response teratogenic effects of 2,3,7,8-tetrachlrodibenzo-p-dioxin (TCDD) were determined in the C57BL/6J mouse. Ad ministration of a single dose of 2,3,7,8-TCDD (20 ug/kg) in corn oil to the pregnant female on day 10 resulted in 70% cleft-palate and 85% hydronephrosis in the fetuses. In con trast, administration of the commercial poly chlorinated biphenyl, Aroclor 1254 (750 '/kg), to the pregnant mice did not result ^ny cleft palate or hydronephrosis in the .etuses. Cotreatment of the pregnant C57BL/6J mice with Aroclor 1254 (750 umol/kg) and 2,3,7,8-TCDD (20 ug/kg) resulted in only 3% fetal cleft palate and was significantly lower than the 70% fetal cleft palate ob served using 2,3,7,8-TCDD' alone. These re sults are comparable to other studies in our laboratory which demonstrate that Aroclor 1254 antagonizes receptor-mediated responses to 2,3*7,8-TCDD. ( S p o n s o r e d b y N.I.H. ES03554.) College Station Ct TX State 77843 Zip Code U.S.A. Country T, . , 4 0 9 Telephone I ______ 845-9181 2. Membership status:____ SO T member ' y Non-member MensAfatfrarfjn w l f t a f y SqTimnerM anfc^ror1 tpon j w J b ric w p h f. fltp M ortti, g* SOT m Dr. S. Safe Signature of SO T member sponsor Typed name of SO T member sponsor Undergraduate and graduate students and postdoctoral fellows are also requested to check one of the spaces below: v ____ Undergraduate -- Graduate ____ Postdoctoral 3. The Program Committee reserves the right to assign papers to eithera platform or postersession. Please indicate your preference below and whetheryou wish to withdraw your paper if your choice cannot be m et Platform ^ Poster_______ Either_______ W ithdraw _______ If choice cannot be met l. Select from the list below the session topic that best suits your paper. Specify your first and second choice or specify another possible session topic area under"other." specify only two choices. SESSION TOPICS _____ 1. General/Methods -------- 2. Aquatic/Environmental -------- 3. Molecular/Celluiar -- 4. Biotransformation/Disposition _ 5. Reactive intermediates :------- 6. Genotox/Mutagenesis --2_-- _ _ 7. Oncogenesis 8. Reproductrve/Teratology -------- 9. Cardiovascular/Renal . 10. Inhalation .1 1 . Hematologic . 12. DermaL/Ocular . 13. Hepatic/G! system Nervous system: . 14. Neurochemtstry . 15. Neuropathology I 16. Hlectrophysiology . 17. Behavior _____ 18. Immune system _____ 19. Endocrine system _____ 20. Metals ______ 21. Pesticides . 22. Solvents ' __I __ 23. Halogenated hydrocarbons _____ 24. Food/Drugs _____ 25. Communicating Concepts _____ 26. OTHER:_______________ (one topic only) ReturnthisformbyOctober24,1988,toeProgramCommittee,c/oExecutiveSecretary,SOT, 1133FifteenthStreet,N.W.,Suite20,WnWngton,D.C,20005(202/233-SS3S1. Su b m it ib ,t r a c t form end one ca o v O N LY . Do m ) M d a w u v o ttu r m im h im i. t i n . . . . . - ...h --m . j . .--------------- -- -- rrpNP 010926 P cB s may counteract dioxin, chemist ays ttm r /A iIf you lake a boHi In PCIIs, has concluded (h a t m any of vpii might notice aome effect. Maybe a little acne-type ik in ' JACK those relatively* harmless 'PCIIs actually can`work as a protec trouble which will go away. M ILLER tion, If not an outright antidote, . Then again, you may ace noth* log. Any exposure less extreme limn that prohahly won't do an y -1 Science against the bad chlorodloxlns. " And, yes, Ihls does work against 2378-TCpp, the worst of thing to you at all, eoy scientist^ expert In these things. * * * the bunch. I.aat week the Ontario envl-* There, now -- `do* we have. Vour altenlloii? It's taken years for the gener Four more of those 75 clilorodloxlns are somewhat toxic, and the oilier 65 seem to have no bad ro n in e n t ministry stag ed a forum In Metro on dioxins In theenvironment to a full house, and some of. what the crowd heard be dumped Into the-environ even a Utile harm. ment. . -Then he switched to the tub* Hut If there's an accidental Jecl ol chlorodloxlnst and drop; exposure, like that spill of PCf)s ped his bombshell. *" Horn a truck on the Trans-Cana , "In our studies of the effects ol da Highway In Ontario som e the few toxic forms of then lime ago,' ll'i nothing to worry chemicals, we felt there must hi about. some antl'dloxlns at work In tin People driving th e ir c a ra body. And we found that `then through (he stuff were not In were. any danger. The near-panic that "We have now developed s followed-that spill, which wound number of compounds that give up wjth a long stretch of pave this effect on laboratory lest anl al public to become convinced effect whatever on humans.' te em ed to surprise. m any of. ment* being dug up and carted mals. When administered alon* th a t polychlorinated biphenyls T h a t w o rst chlorodloxln of them.*'.' away, waa ridiculous and pure with the w ont of the chlorodlox are deadly poisons, 10 ll prob Ihem all (worst for humans, that ably will lake Just ns many more Is) c a r r ie s th e cumbersome Government advisor hysteria, Safe said. lns. they have cut the Incidents ' Several years ago. there were of harm by BOper cent." years to convince Hie public that chemical till a of. 237B-TCUO, Safe w as brought up from bad m a n poisonings, one-In . One of tliose magic antl-dloxin Ibis baa all been blind, uiinues- w hich la a h o rt *for* 2376- Texas to be keynote speaker. Ill Japan and another In Korea, of' compounds Is Aroclor 1251. Honing,* gross misinformation, le t r a c h lororilhenzo-para-d loxln credentials are good. H e1 t people who consumed rice oil What's Aroclor 1254? "It's a -these people say.* (Hie name alone la enough Id Uellevllle native who studied at Contaminated w ith chemical m i x t u r e of I'CHs. I t's well llut If that's what the Job calls scare you off). Queen's University and Him Oir- There were PCHs In the cheml known. It's been avallahle com lor, let's atari. None of lids Information; up -forrt on his wsydo becoming an cals, and Hie outcome was that mercially for yeart.'*3aie si Id. Oh, and while we're at It -- till now , Is new. It h as been advisor1on these bub|rcli fo r all PCIIs (there are several) were Any more bulletins? Well, II you'vn heard of dioxins?-.Or to generally missed by the media both the UiK pni) Canadian gov labelled deadly. may turn opt Hist you can also use the more common term,* and the public, but lota of ton ernments. In (act, Sale eald. some of the light dloxlnjwlth dioxin. Some ol "deadly dioxins?" tcliemlsla have known It all PCOa, he la id , a r e almost oilier components In the cheml- those 65 noh-toxlc chlorodloxlns * Six poisonous lung. ' . totally harmless to people. They cnls must have done the damage, - seem to offer Ihe same.sort of Uul here's something that Is may he damaging to aome plants because he has studied PCUs rolectlon from 2376-TCUD as GWell, thn proper term In this now. Heararch under chemist or oilier Ilia lorms In the food* exposure cases fo r years and. te PCIIs do. *. rase Is clilorlnnlml dlhxlns, al Stephen Safe at Texas A anil M chain,* and -they do not break found you practically have to Stay tuned to the good-newt though It's etpinlly correct.I? University, aqon to bp published, down readily, so they should not drown a human In the tuff to do channel. call ifiem clilornilloxlns. We make the distinction be-, danse Ihnre e re thousands of other dioxins that are not lm. vnlvetl w ith chlorine, w hich means they are not pnrl of Ihls family of chemicals llmt people w o rry so m uch ohoul, even though they can all properly, be THE TORONTO > F O R E called dioxins. Well, th e re n re 75 known ' 'chlorodloxlns. And yes, of those 10/ 27/86 75, six are considered harmful enough to people to be called polf ` sons, and one of those six Is rated as the third most polson- 783738 mir substance (it Hie world for (uurtaris (n farm of botulism 14 fhc Number f poison, but that's another sloryj. GEjs/j> 0/0927 '86 1^07 14:43 314-694-6398 MONSANTO STLOUIS 002 186IEEE MONTECH l Confrence Conference sur on Im SPC et PCSa end Iss fluides de replacement remplacement # 29 fluids September s2e9pt-emObcrteoib-erTe1r,1o9ct8o6bre 1986 HEALTH EFFECTS CF PCBs AND RELATED SUBSTANCES Stephen H. Safe, Kathy Farrell, Roy Bannister Michael Kelley, Stelvlo Handlers and Grit Mason Veterinary Ifcysiology and Pharmacology Texas A6M University, College Station, TX 778*13 Polychlorinated biphenyls (PCBs), dibenze-pdloxins (PCDDs) and dibenzofurans (PCDFs) are - halogenated arotnatlo chemicals which have been used as Industrial compounds (PCBs); or have been Identified as by-products In the production of organic compounds or their combustion (PCBs, PCDOs and PCDFs) (1,2). These chemicals are characterized by their chemical stability, resistance to chemical and biological breakdown and high lipophilieity. These chemical properties contribute to the adverse environmental effects of the class of compounds which have been widely identified in the global ecosystem and preferentially bioconcentrata in fish, wildlife and humans(3>. Since the commercial PCBs are complex mixtures of isomers and congeners, it is not suprising that the PCS composition of extracts from environmental samples and occupationally exposed humans Is also complex. Moreover It is also apparent that the relative composition of the individual PCS compounds which have been identified in various matrices are highly variable and do not resemble the distribution patterns observed fbr the " immereial PCS produots (U), It is apparent from /oral studies that the environmental persistence d bioaccumulation of individual PCBs are dependent on several factors Including their degree of chlorination, lipophillcity, volatility, and blodegradabllity. Hazard assessment of commercial and environmental PCS mixtures requires analytical data on the composition of these mixtures and .the toxicology of Individual PC3 isomers and congeners and their interactive effects. PCBs and Related Halogenated Aryl Hydrocarbons - Toxicology and Structure - Activity Relationships (SARs). " Toxloology Although the acute LDcq values fbr commercial FCB mixtures are relatively high, it is apparent from subacute and chronic exposures studies that much lower dose levels of some PCS mixtures elicit diverse biologic and toxic effects which include C2>! , 1. the development of skin lesions (which resemble human chioracne) in aeveral laboratory animal species md humois; 2. imnunotoxic effects; 3. a wasting syndrome; 4. reproductive and fetal toxicity; 5. carcinogenesis (Initiation and promotion) ' porphyria and hepatotoxlolty; 7* the Induction of diverse drug-metabolizing vzyoes including aryl hydrocarbon hydroxylase fH), a cytochrome P-^SO-dependcnt monooxygenaae zyme. It was also evident that the spectrum of toxic effects elicited by PCBs quantitatively were similar to thoee observed after treatment with several related classes of halogenated aryl hydrocarbons which include the dlbenzofurans (PCDFs)(2,55. Several studies with individual PCBs indicated that the ero 3 3 *4,4-tetri-, 3 , 3 4 , 4 ,5-penta, and b'^S'-hexacftlorcbiphenyl are minor to trace components of the commercial PCS formulations and are unlikely to be the sole contributors to the toxicity of these mixtures (2,4), Our research has focused on the identification of tho toxlo PCBs, the determination of their presence or* absence in commercial and environmental PCB mixtures and the use of this data to develop risk-assessment of PQs. PCBs - SARs The observed correlation between the toxioiEy end"the AHH induction potencies for the toxic halogenated sryl hydrocarbons suggests that the induction assay can be used as indicator of potential PCS toxicity. Our research initially focused on the aotivlby of PCBs as Inducers oif AHH in immature male Wlstar rats, and rat hepatoma H-4II E cells in culture. A comprehensive study of all 209 PCBs was not feasible. However, several reports indicated that the most potent AHH Inducers are substituted at both para and two or more aeta positions. Initial studies confirmed that only 4 of these compounds (3,3'r4,4*-tatra-, 3,4,4',5-tetra-, 3,3',4,4',5-penta- and 3*3\4,4*,5,S'-hexaehloroblphanyl) induced and its associated eytochrome P-450 isozyme and allotted the:toxlo effects assooieted with halogenated aryl hydrocarbons. In subsequent research we synthesized and reported the biologic and toxic effects of all the monoortho chloro-substituted analogs of the 4 highly toxic coplanar PCBs (See Figure 1). The results demonstrated that this group of 8 PCB congeners elicited a spectrum of biologic and toxlo effects comparable to these observed for the coplanar PCBs although it was apparent that the introduction of the ortho-chloro substituent substantially reduced the activity of these compounds (2,6). The monoortho oopimar PCBs have been identified in the oommeroial PCBs and environmental extracts and appear to be the major toxic components of these mixtures (4). 783739 aa MX U.<3 a A T V at ao tUM'i1 . /"'tT^X I1 A d A A j""v '86 10/07 14:44 314-694-68981 PCBs = Humin Toxicology Human populations have wen- exposed to PCBs and PBBa via three major pathways, I.e*, environmental, occupational, and --identaL The effects' of (PC8s and PBBs on humans ten determined by studying the occupationally .tdentally exposed individuals, since it has ... acknowledged that these two groups have been exposed to the highest levels of PCBs, The major sffects and symptoms observed during the Yusho poisoning In Japan and Taiwan include chloracno and levarai related dermal changes, ocular damage, Increased serum triglycerides, reproductive srobleas, alterations in steroid metabolism, increased serum (and adipose tissue) levels of PCBs, md immunologic effects (7). In contrast, only a Tew of these symptoms have been noted.in the xctpatlonally exposed workers. It is not olesr t*iy :his latter group does not present more* severe symptoms of FCB poisoning since the serum levels of markers exposed to PCBs are comparable and often iigh*r than the levels observed in victims of csldental poisonings. The toxic Yusho oil contained a mixture of lolychlorlnatad biphenyls (PCBs) which were lontaminated by other halogenated aromatics .ncludlng the highly toxic PCDFa. It has been `sported (7,8} that the PCBs and PCCFs which persist n the liver of victims still suffering from Yusho olsoning Include the following compounds; ^SM'.S-penta-, 2,2'A 4,5,5'-, 2,2',3*.M r,fi nd 2f3,3'A4',5-hexaf 2,2,3,4,4',5,S'- and 2',3,3'f4,4*,5-hapttaehlorabiphenyls and the ,3,7, 8-tetra-, 1,2,(1,7,2-, 1,2,3,7(8- and ,3,4,7,8-penta- and 1,2,3,4,7,S-hexaohlorodlbanzourins. All of these PCBs and PCDFs have been ynthenlied in my laboratory and reconstituted bo 'mate their composition in humot livo- (8). A Ison of the dose-response effects of the -- *r..jtituted PCB and PCDF mixtures In causing eight loss, thymic atrophy and the induction of ytochroae P-448-dependent monooxygenases Indicated nat the PCDF mixture was at least 700 times more ctive than the PCBs. Since the ratio of PCBs/PCEFs erststlng in Yusho patients1 blood and liver was sss than 600: 1 and 5*1 respectively, the results iggest that the PCCFs are the major etiologic agent i Yusho poisoning. Comparable studies on the ixicity of the PCB and PCEF frootlons isolated from tJaho oil confirm that the PCDFs were by far the ost toxic fraction present and must make the major sntrlbutlon to the toxicity of the contaminated Ice oil (9>* These results explain the toxic >sponse differences in the YUsho and occupationally (posed humans. YUshg oil contained relatively high Weis of toxic PCCFs whereas most commercial PCSa ^ntain lower levels of these toxic impurities. *sd on these results, risk assessment of wironmental PCS exposure should be extrapolated "cm epidemiologic evaluation of occupationally (posed Individuals* Since workers exposed to high >vis of PCBs exhibit relatively few adverse health 'foots this would suggest that the potential Averse human health affects resulting from ivirannental exposure to PCBs are very low. MONSANTO STLOUIS @003 REFERENCES Cl] 0. Hutzinger, S. Safe, and V. itko, The .Chemistry, of PCBs, Cleveland. Ohio. CRC .>:e;' m , ------ [23 S. Safe, "Polychlorinated biphenyls (PCBs) and polybrcmlnated biphenyls (PBBs); biochemistry, toxicology and mechanism of action," CSC Crit. Rev* Toxicol,* Vol. 13, PP*319-394, 193C C33 S. Safe, "Halogensted hydrocarbons and aryl hydrocarbons identified In human tissues,".). Toxicol* Environ. Chem. Rev*. Vol. 5. dd.1?3- U 5 , 1585.--------------------------- [41 3. Safe, L. Safe and H. Hull in:' "Polychlorinated biphenyls (PCBs) - congener- specific oialysls of a commercial mixture and s human silk extract" - J. Asrlo. Food Chem., Vbl. 33, pp.24-29, 1985. . C5] 3. Safe, "Comparative toxicology and mechanism of aotlon of polychlorinated dihenzo-p-dioxins and-dibenzofurans", Ann. Rev. Pharmacol* Toxicol.. Vol. 26, pp 37l-379""OT6 C63 5. Safe, 2. Bandlara, T. Sawyer, L. Robertson, A. Parkinson, P.E. Thames, D. Ryan, L.M. Reik. W. Levin, H. A. Denomme, and T. Fujita, "PCBs: Structure-Unction relationships and mechanism of action," Environ. Health Parspect., Vol* 60, PP.*7-50rT953 C7J H. (Curatsune and R.E. Shapiro (Editors), PCB Poisoning in Japan and Taiwan* Haw York,"ATan R. Uss, Inc., tef. [33 S. Sandleri, K. Farrell, G. Mason, K. Kelley, H. Romkas, R. Bannlstar, and 5. Safe, "Comparative toxioitiss of the polychlorinated - dibanmofuran (PCDF) and biphenyl (PCS) mixtures which persist in YUsho victims", Cheaoophera, Vol. 13; pp. 507-512, 1984. ----- ---- C93 H. Kunlta, T. Kashlmato, H. Mlyato, 3. FVikushima, 2. Horl and H* Ghana, "Causal agents of YUoho"* Amer. J. Ind, Med*. Yol. 5. 4558, 1 9 8 4 , --------- 783740 GENP 010929 21 G E N E R A L ^ ELECTRIC GENERAL ELECTRIC COMPANY F A IflF IC L D , C O N N E C T IC U T O C 4 3 I l<* S T E P H E N G. HAM ILTON. J R . . . o c a - chvimohhcht*^ aciCMcc *o T tcw oiooT November 24, 1986 12031 373 *3310 FEDERAL EXPRESS Dr. Stephen H. Safe, Professor of Toxicology Texas A&M University Veterinary Physiology and Pharmacology Highway 60 College Station, TX 77843-4466 Dear Dr. Safe: Thank you for the papers on PCBs as dioxin antagonists. I find them extremely interesting and would .like to see this work progress further to look at the carcinogenic response, and also look at the effectiveness of certain individual congeners. Regarding our meeting on December 1, I will be accompanied by Dr. Joseph Rodericks of Environ, who has personal knowledge of the PC8 regulatory process, and Dr. John F. Brown, Manager of GE's Health Research at our Corporate Research and Development Center. We will be arriving at the College Station Airport at 11:50 a.m. and departing at 4:45 p.m., same day. I would like to start the meeting by summarizing the reason's why we think that a new approach to quantitating PCBs in fish, i.e., quantitation of-specific . toxic congeners rather than total PCBs is more scientifically sound than FDA's current approach. I would then like to discuss with you the kind of toxicity testing that might make this a convincing argument to the Agency. I would also like to discuss toxicity tests that would demonstrate the differences between the Aroclors that were released to the environment, and those largely dechlorinated mixtures found in the sediments of certain rivers. For your interest, I am enclosing a copy of chromatograms of Aroclor 1242 and of PCBs found in Hudson River sediments. I trust we will also have time to discuss your dioxin "antagonist" work as well. We should have a very interesting and productive meeting. 783741 Sincerely yours, SBH-.cas Enclosure ce: Dr. J.F. Brown - GENP 010930 I D cJtt i0 r** M l lfx* /h /'/** * S^ a . Aroclor 1242 *---ft-AJl____ K. ._k u j j k - l LLokTXA-'AAA__J<-^A^(k_-- ft___ A^_ t jure 1. Capillary gas chromatograms of PCB specimens illustrating patterns ;t commonly seen in upper Hudson sediments: (a) Aroclor-1242 standard, :) "Pattern B," dechlorinated Aroclor 1242 from Core 18-6, 16-17" 783742 GENP 010931 Toxicology, 46 (1987) 29--42 Elsevier Scientific Publishers Ireland Ltd. AROCLOR 1254 AS A 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN ANTAGONIST: EFFECTS ON ENZYME INDUCTION AND IMMUNOTOXICITY R. BANNISTER, D. DAVIS, T. ZACHAREWSKI, I. TIZARD and S. SAFE Departments of Physiology and Pharmacology and Microbiology and Parasitology, College of Veterinary Medicine, Texas A A M University, College Station, T X 77843 (U.S.A.J (Received March 10th, 1987) (Accepted April 24th, 1987) An international journal concerned w ith the effects o f chem icals on living system s t(II i ELSEVIER SCIENTIFIC PUBLISHERS IRELAND LTD 783743 GENP 010932 : /v: j^ ntei^natjona^jouirial'c;oncemed with theTlfScts \ of chemicals on living systems A I Toxicology is a journal for the publication of original scientific papers an the biological effects arising from the administration of chemical compounds, principally to animals, tissues or cells, but also to man. Such compounds include industrial chemicals and residues, chemical contaminants, consumer products, drugs, metals, pesticides, food additives, cosmetics, and additives to animal feeding stuffs. Preference will be given to investigations dealing with the mechanisms of action of toxic agents. Papers describing molecular interactions with cailular and ganetic pro cesses will be welcomed. Quantitative toxicological studies will be published -that ara of relevance to risk assessment and regulatory management of exposure hazards and safety evaluation. This applies.particularly to carcinogenicity, mutagenicity, embryotoxicity and related areas, ds well as to alternatives to the use of animals in toxicological experimentation. Epidemiological studies bearing toxicological significance to man fall-within; the zcope of the journal. The Editors would also welcome the submission of concise end pertinent reviewsoncurrent issues in toxicology. * T, M A N A G IN G E D IT O R S : H.P. W IT S C H I, O ak Ridge# T N , U .S;A .; K.J. N E T T E R , Marburg, F.R .G . - . ' . E D IT O R IA L B O A R D ../r*^ V: F . Bergiund, Stockholm R . Henderson, Albuquerque. NM W .O. B e rn , Om sha, NE J.W . Bridgea, G uildford A .B . S u ckp itt. D avis, CA X A . Castro, Huenoe A ires J .B . Hook, Philadelphie. PAP J L de le Igiesie, Arm A rbo r, M l R ib o n * . Reeearcft.Trlangte P w te NC ` > R ; K a to , Tokyo'.- M.G . Cherian, O ntario , - V-'. PCehrar,Atm lnrT^ T-.T ^ - . N. Cham off, Hereereh T ria n tfe P a rk , N C : K .K o JIm e / T O k y o ^ :* * '.^ 'fif y . - . ] J.W . D aniel. U td e addow-- / ' ff.~ Lausaartifc:BrirresN/ G . D lrheim er, Stredsourg ,, D . Ecotoiehon. Montreal ' le w rC re M IIW O iW .K jV : .C XT^ to ow iG roton , C t* ' .. P-S. B ia s , Redbridge ~ EjC. Foulket, Gncinnetl, p H ,/ :'i Eowodre ( H scataw ey. S . G a rra ttlfii, M ilan ^ ihh ; f t ochhoi ai / . . . ~ P J . H kkinen, an d n n at^ O H ?; ^ S . Ham son. B irm in g h a m jtti-T^-W- - ; .P ,i3- ? -' -. A J S . M cLer_red1/.Lo..nd-,tXvr-^ .^ - -">. P. M ilas. Morgantown, WV G .J. M ulder, Lsidan R . Neal. Research Triangle P ark. NC F . Oaach, M ains W .E:Pariri> , Bedford Ki^ taM tr, D avis, C A ID , Schm ehl, Heidelberg V . fflttn o , Rom e L .L .S m ith , M acelesfiald F .M . S u llivan , London M : TValnen Moale n . Gshreston , T X ' H .T U chmen n D ip lew is. Parts ' ULUshtefco, BarHrr; - ; r.W k tirM * . M idland, M l. ` P ltm n sb d , Neuharberg A J J . W orden, Cambridge G . Zb ln den ,Schwarzen bach , O Elsevier Sciantific P u b lh b m lreten d L td . . ' V i;; / Copyright resented. N o-partofcdtle publication m ey b r reproduced; stored.in e re tria v d system , or tran tm in ad , in any form or by any Pmeans, electronic, m echanicaf, photocopying, recording. orotherw ire w ithout perm ission In w riting from the publisher, Elsevier S cie n tific Publishers Ireland Ltd . . ' , - . . . . . . : -i,*.-. 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Is granted b y Elsevier S cie n tific Publishers Ireland Ltd.- fo r libraries and o th e r u rert' registered w ith .'tffC fe p y rig h t Q aersnee. Center (C C C l'T ransactlonei 03 50 37Reporting Serelce, provided that the bare fee o f S J p e r co p y, p fta lJX p er pegs ts.peid directty-to C C C , Congrere St.^Selem , 01970 0300 483 87 03 01 00 'MA , - X/ /S ^ -. . U ** '* .'r..v.'-' `." - .o..., -J.-.: V ' * - ' ! s.--. . GENP 010933 783744 Tox Els AB IM R. I Def Vet (Rei (Ac. su: cyt last I anc fea teil pot 4-11 mai litt! duc mie jjint TCI ago Arc 2,3, of 1 cres Thi: Add Abbi rufic polyactiv tetra 0300 l Prit Toxicology, 46 (1987) 29--42 Elsevier Scientific Publishers Ireland Ltd. AROCLOR 1254 AS A 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN ANTAGONIST: EFFECTS ON ENZYME INDUCTION AND IMMUNOTOXICITY R. BANNISTER, D. DAVIS, T. ZACHAREWSKI, I. TIZARD and S. SAFE Departments of Physiology and Pharmacology and Microbiology and Parasitology, College o f Veterinary Medicine, Texas A & M University, College Station, T X 77843 (U.S.A.) (Received March 10th, 1987) (Accepted April 24th, 1987)*I SUMMARY 2,3,7,8-Tetrachlorodibenzop-dioxin (TCDD) and Aroclor 1254 induced the cytochrome P-450 dependent monooxygenases, aryl hydrocarbon hydroxy lase (AHH) and ethoxyresorufin 0*deethylase (EROD) in rat hepatoma H-4II E cells and C57BL/6J mice. It has been proposed that both Aroclor 1254 and 2,3,7,8-TCDD induce these enzymes via a common mechanism which features initial binding to the aryl hydrocarbon (Ah) cytosolic receptor pro tein. The major difference between these compounds was the relative potency (i.e. 2,3,7,8-TCDD > Aroclor 1254). Cotreatment of rat hepatoma H4-II E cells or C57BL/6J mice with a dose of 2,3,7,8-TCDD which submaximally induces AHH and EROD and a dose of Aroclor 1254 which exhibited little or no induction activity resulted in significant antagonism of the in duction effects of 2,3,7,8-TCDD. For example, cotreatment of C57BL/6J mice with 2,3,7,8-TCDD (15 nmol/kg) and Aroclor 1254 (25, 75 and 150 pmol/kg) resulted in up to 23% antagonism of AHH induction by 2,3,7,8TCDD. Moreover, cotreatment with a higher dose of the 2,3,7,8-TCDD agonist (30 or 50 nmol/kg) partially reversed some of the antagonism by Aroclor 1254. In vivo antagonism was observed only at Aroclor 1254/ 2,3,7,8-TCDD molar ratios of 1667:1, 5000:1 and 10 000:1. Administration of 2,3,7,8-TCDD (3.72 nmol/kg) to C57BL/6J mice resulted in a 76% de crease in the splenic plaque form in g cell response to sheep red blood cells. This T-cell mediated immunotoxic effect of 2,3,7,8-TCDD segregates with AAbdbdrreesvsiaatiollncso:rrAeshp, oanrdyel nhcyedrtooc: aSr.bSoanf;eA. HH, aryl hydrocarbon hydroxylase; EROD, ethoiyreso- rufin O-deethylase; HCBP, hexachlorobiphenyl; PCBa, polychlorinated biphenyls; PCDDs, polychlorinated dibenzo-p-dioxins; PCDFs, polychlorinated diberuofurans; SARs, structure- activity relationships; 2,3,7,8-TCDD, 2,3,7,8-tetrachlorodibenzop-dioxin; TCDF. 2,3,7,8tetrachlorodibenzofuran. 0300-483X/87/S03.50 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland 29 GENP 010934 783745 the Ah locus. In contrast, administration of 5, 15, 75 and 150 pmol/kg of Aroclor 1254 resulted in impairment of the immune response only at the highest dose level. However, cotreatment.of mice with 2,3,7,8-TCDD (3.72 nmol/kg) and Aroclor 1254 (5, 15 or 75 junol/kg) resulted in no significant decrease in the plaque forming cell response and complete protection from the immunotoxicity of 2,3,7,8-TCDD. Cotreatment of the mice with Aroclor 1254 (75 fimol/kg) and a higher dose of the 2,3,7,8-TCDD agonist resulted in partial reversal of the protective effects of Aroclor 1254, The in vitro and in vivo data suggest that within specific antagonist/agonist dose ratios, Aro clor 1254 can antagonize at least 2 Ah receptor-mediated effects of 2,3,7,8TCDD, namely AHH induction and immunotoxicity. K ey words: 2,3,7,8-TCDD; Aroclor 1254; Antagonism. INTRODUCTION 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aryl hydrocarbons elicit a number of common toxic and biologic responses including chloracne and other dermal lesions, porphyria, reproductive effects and teratogenicity, endocrine disorders, tissue-dependent hypoplastic and hyperplastic responses, immunotoxicity, a wasting syndrome and the induc tion of diverse enzymes including several cytochrome P-45O-dependent monooxygenases [1--4]. The overall mechanisms associated with the toxic effects elicited by 2,3,7,8-TCDD have not been delineated; however, it has been proposed that initial binding of the toxin (or related agonist) to a cyto solic (aryl hydrocarbon, Ah) receptor protein is a common obligatory step in initiating the pleiotropic responses noted above [1--7]. Although the Ah receptor protein has not been fully characterized or- pur ified, there is considerable evidence which supports a receptor-mediated mechanism of action for 2,3,7,8-TCDD and related compounds. F.or example, the Ah receptor exhibits saturable high affinity binding with [3H]2,3,7,8TCDD (K d = 0.7 nM) [5,7]; the competitive receptor binding affinities of polychlorinated dibenzo-p-dioxin (PCDD), biphenyl (PCB) and dibenzofuran (PCDF) isomers and congeners are structure-dependent and illustrate the highly stereoselective interaction between the receptor protein and different classes of chemical agonists [2,8--11]. The most avid ligands for the recep tor are approximate isostereomers of 2,3,7,8-TCDD and are substituted in their lateral positions. Genetic studies with responsive C57BL/6 (high recep tor levels) and non-responsive DBA72J (low receptor levels) inbred mice and their backcrosses demonstrate that tissue responsiveness to 2,3,7,8-TCDD is, in part, dependent on receptor levels. Moreover, mouse genetic inbreed ing studies have shown that teratogenicity, porphyria, body weight loss, immunotoxicity and the induction of cytochrome i^-450, segregate with the Ah locus [6,12--19]. Quantitative in vitro and in vivo structure-activity rela- 30 tionsl diver; obser hydro the ir loss a Pre stratf recep triphe S m il; 2,4,6,i recep but a: H-4-i: and e antag Previe (a6ls.6o xe ses in as a 2 6 mice MATEI Chemi 2,3, greate 1254 ' chasec Benzoi droxyl All ot [3H]2,2 1,6[3H. the pn In vivt Mai Labor; hardwf (Teklal h dark admini treatec 783746 tionships (SARs) have recently been reported for several structurally diverse PCDDs, PCDFs and PCBs [9--11,20], Comparable SARs were observed for cytosolic receptor binding,- -the in vitro induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD), the in vivo induction of these same monooxygenase enzymes, body weight loss and thymic atrophy (in the rat). Previous studies with agonists for the estrogen receptor have demon strated that some compounds which exhibit moderately high estrogen receptor binding affinities but low estrogenic activities (e.g. estriol and some triphenylethylene analogs) can antagonize the effects of estradiol [21,22]. Similarly, recent studies in our laboratory have shown that both 1,3,6,8- and 2,4,6,8-tetrachlorodibenzofuran (TCDF) exhibit relatively high cytosolic receptor binding affinities (1.25 X 10~7 M and 1.5 X 10"* M, respectively) but are only weakly active as inducers of AHH and EROD in rat hepatoma H-4-II E cells in culture [23]. Cotreatment of the cells with 2,3,7,8-TCDD and either of the 2 PCDF isomers demonstrated that both compounds antagonized the induction of the monooxygenases by 2,3,7,8-TCDD. Previous studies [8] have reported that the commercial PCB, Aroclor 1254 also exhibited moderate binding affinity to the cytosolic receptor protein (6.6 x 10"6 M) but was a weak agonist for most receptor-mediated respon ses in vivo and in vitro [24]. This paper reports the activity of Aroclor 1254 as a 2,3,7,8-TCDD antagonist in rat hepatoma H-4-II E cells and in C57BL/ 6 mice. i MATERIALS AND METHODS Chemicals 2,3,7,8-TCDD and ethoxyresorufin were synthesized in this laboratory to greater than 99% purity as determined by gas chromatography [10]; Aroclor 1254 was a gift from Dr. O. Hutzinger, NADPH and NADH were pur chased from the United States Biochemical Corporation (Cleveland, OH). Benzo[a]pyrene was purchased from Sigma Chemical Co.; DNA-grade hydroxylapatite was purchased from Bio-Rad Laboratories (Richmond, CA). All other chemicals were of the highest quality commercially available. [3H]2,3,7,8-TCDD was synthesized in the laboratory by the chlorination of l,6pH2]dibenzo-p-dioxin followed by chlorination and HPLC purification of the product (> 98% pure) [5], In vivo enzyme induction studies Male C57BL/6J, 3--4 weeks of age, were obtained from the Jackson Laboratories, Bar Harbor, ME. The mice were housed in plastic cages with hardwood bedding, allowed free access to Teklab Laboratory Rodent Chow, (Teklab, Madison, WI) and water and maintained on a diurnal 12-h light/12h dark cycle. Aroclor 1254 or 2,3,7,8-TCDD were dissolved in com oil and administered by intraperitoneal injection (10 ml/kg). The C57BL/6J mice treated with Aroclor 1254 and/or 2,3,7,8-TCDD were sacrificed after 72 h. 31* - GENP 010936 783747 Corn oil (10 ml/kg) served as a vehicle control for all studies. Livers were perfused via the hepatic portal vein with ice cold saline containing 0.1 mM -- EDTA, weighed and placed in beakers-containing homogenization buffer. Livers were washed and then homogenized using a Potter-Elvehjem homo* genizer in 4 volumes of 0.25 M sucrose with 0.1 mM EDTA. A microsomal pellet was prepared by centrifuging the homogenate at 10 000 g for 20 min and then centrifuging the resultant supernatant at 100 000 g for 1 h. The pellet was resuspended in homogenization buffer and stored at - 80C until used for the enzyme assays. Ethoxyresorufin o-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) were determined as previously described [25,26]. Protein concentrations were determined [27] using bovine serum albumin as a protein standard. All data are expressed as means S.D. Sig nificance was determined by a two-way ANOVA and Dunnet's t-test [28]. In vitro enzyme induction Rat hepatoma H-4-II E cells were grown in m inim um essential medium without ribo* or deoxyribonucleotides but with L-glutamine. The medium was supplemented with 10% fetal calf serum, 10% calf serum, gentamycin . sulfate (50 pg/ml) and Fungizone (2.5 pg/ml) and cultures were maintained in a humidified 5% C02 atmosphere at 37C. At confluency stock cultures were trypsinized and seeded in 25-cm2Coming culture flasks at a density of 0.6 X IQ6 cells/flask (Day 1). On Day 2, the spent medium was aspirated off and replaced with fresh medium containing the test compound in DMSO to a final DMSO concentration of 0.5%. Dose-response induction studies with at least 6 different concentrations of each inducer were carried out. On Day 3, the cultures reached confluency and were harvested. Cultures were washed 3 times with PBS (pH 7.4) and scraped off with a rubber policeman in Tris-sucrose (0.05--0.02 M) buffer (pH 8.0). The cell suspensions were centrifuged and resuspended in buffer. After protein determination [27], the cell suspensions were adjusted to a final protein concentration of 1 mg/ml and whole cell suspensions were assayed for enzyme activity. EROD and AHH activities were measured fluorimetrically [25,26] and the ECS0 values determined as previously described [9--11]. Receptor binding assays Hepatic cytosol ~3--5 mg/ml) was incubated with different concentrations of pH]2,3,7,8-TCDD and Aroclor 1254 (100, 500 and 667 nM) for 2 h at 20C. Each assay was performed in duplicate. A hydroxylapatite slurry (Bio Rad Lab; 3 parts gel in 5 parts phosphate or HEDG buffer) was freshly pre pared. The phosphate buffer was 50 mM Tris--HC1, 1 mM KH2P04. After incubation, 200 pi aliquot of the cytosol was transferred to a second tube containing -250 pi of the hydroxylapatite slurry. The tubes were incubated on ice for 30 min with shaking every 10 min. The mixture was then resus pended in 2 ml phosphate buffer and centrifuged at 800 g for 2 m in. The hydroxylapatite pellets were washed 3 additional times with phosphate buffer (2.0 ml buffer) and then resuspended in 1.0 ml absolute ethanol. The32 32 GENP 010937 783748 hydroxylapatite-ethanol solution was vortexed and transferred to scintilla tion vials and any remaining hydroxylapatite was washed into the vial with an .additional 1 ml of ethanol; 10 ml of scintillation cocktail was added and samples were counted for radioactivity. Saturation binding of [3H]2,3,7,8TCDD with C57BL/6J hepatic cytosolic receptor protein was determined in the presence of different concentrations of the competitive ligand, Aroclor 1254 and the results are summarized in Fig. 2. Plaque form ing cell (PFC) assay The method employed was the "Cunningham" variant of the Jerae PFC as say [29,30], C57BL/6J mice at 8 weeks were purchased from the Jackson Laboratories, Bar Harbor, ME. Upon arrival the animals were allowed to acclimate for 5--7 days before treatment. On day 1 the animals were in jected (i.p.) with the appropriate chemical as described for the enzyme induction studies (Aroclor 1254 and/or 2,3,7,8-TCDD) and sheep red blood ells (SRBC, 4 x 106 cells) were injected after 5 days. The animals were sac rificed by cervical dislocation 10 days after treatment with the chemicals and the spleens were removed and placed individually in 60-mm petri dishes containing 10 ml of cold Eagle's MEM media supplemented with Hank's salts (KC Biological). Each spleen was then teased through a 70-^m nylon screen (Spectrum Medical, Inc.) which was saturated with 2 ml of the Eagle's media. The screens were washed of remaining cells by the addition of 8 ml of media. The cell solution was then spun at 220 g for 10 min and the pellets resuspended in 2 ml of fresh media. Appropriate dilutions of the suspensions were then made with 0.1 ml of a dilution mixed with 0.2 ml of a 20% SRBC solution and 0.1 ml of a 1--5 guinea pig serum dilution. Through capillary action, microliter aliquots of the mixtures were placed in "Cunningham" slide chambers and incubated for 60 m in at 37C. Viable cell counts were determined by Eosin Y staining. RESULTS 2,3,7,8-TCDD evoked a dose-dependent induction of AHH and EROD in rat hepatoma H-4-II E cells at concentrations of 10-12--10-8 M. At higher dose levels (> 10~8 M), 2,3,7,8-TCDD was toxic to the cells and this was evi denced by a dose-dependent decrease in the AHH induction maximum with increasing concentrations of 2,3,7,8-TCDD (Fig. 1). The dose-dependent in crease and subsequent `decrease of AHH and EROD induction has previously been reported for other halogenated aromatics [24], Moreover, for some weak agonists such as Aroclor 1254, cellular toxicity is noted at con centrations which are insufficient to elicit m a x im u m induction of the cytochrome P-450-dependent monooxygenases (i.e. see Fig. 1). Cotreatment of the rat hepatoma cells with a non-toxic concentration of Aroclor 1254 33 GENP 010938 783749 120 'C Fig. 1. Dose response induction of AHH (pmol/mg protein/min) in rat hepatoma H-4-II E cells by Aroclor 1254 (O) and 2,3,7,8-TCDD {x ). Cellular toxicity by Aroclor 1254 and 2,3.7,8-TCDD was observed at concentrations > 10"M and 10"M, respectively. (1 x 10'7 M) and a concentration of 2,3,7,8-TCDD which elicited a submaximal AHH and EROD induction response (1 x 10'9 M) resulted in the an tagonism of the induction activity of 2,3,7,8-TCDD by Aroclor 1254 (Table I). Cotreatment of the cells with the same concentration of Aroclor 1254 (1 x 10~7 M) and a higher concentration of 2,3,7,8-TCDD (2.25 X 10'8M) par- TABLE i EFFECTS OF 2,3,7,8-TCDD, AROCLOR 1254 AND 2,3,7,8-TCDD PLUS AROCLOR 1254 (COTREATED) AS INDUCERS OF AHH AND EROD IN RAT HEPATOMA H-4-II E CELLS IN CULTURE Inducer Concen tration (M) ERODb AHHb Solvent (control) Aroclor 1254 2,3.7,8-TCDD 2,3.7,8-TCDD Aroclor 1254 plus 2,3.7,8-TCDD Aroclor 1254 plus 2,3,7,8-TCDD _ 1 X 10T 1 X 10" 2.25 X IO" 1 X 10-7 1 X 10- 1 x IO*7 2.25 X 10- 0 0 74.0 A 20.1 77.9 a 19.9 46.8 A 3.99* 65.6 A 2.21 0 0 155.9 14.6 179.0 A 19.7 105.7 11.7" 117.0 7.36 `Significantly lower IP < 0.01) than observed after treatment with 2,3,7,8-TCDD (1.0 x 10~* M) alone. `'pmol/mg protein/min. 34 c z i| cC Fig- pres the . in tl tivei abse of p coeff prep tiah sna pati Aro acts T clor mal* ERC 2,3/ thyr of A kg i GENP 010939 783750 Fig. 2. Double reciprocal plot analysis of the saturation binding of [3H]2,3,7,8-TCDD in the presence of different concentrations of Arodor 1254 (100, 500 and 667 nM). The y intercepts of the double-reciprocal plots of the saturation binding of [3H]2,3,7,8*TCDD with the Ah receptor in the presence of 100, 500 and 667 nM Arodor 1254 were 0.0091, 0.0088 and 0.0092, respec tively. The linear correlation coeffidents were 0.9925, 0.9984 and 0.9995, respectively. In the absence of Arodor 1254, the y intercept of the double-reciprocal plot of the saturation binding of [3H]2,3,7,8-TCDD with the Ah receptor was 0.0074 and the correlation linear correlation coefficient was 0.9899 (data not shown). The incubations were carried out using hepatic cytosol preparation (3--5 mg/ml) and a range of [3H]2,3,7,8-TCDD concentrations (from 0.8 to 11 nM). tiaUy overcame the antagonism by Aroclor 1254. Double reciprocal plot analysis of the saturation binding curves of [3H]2,3,7,8-TCDD and rat he patic cytosolic receptor in the presence of different concentrations of Arodor 1254 is summarized in Fig. 2 and demonstrates that Arodor 1254 acts as a competitive antagonist for the receptor-2,3,7,8-TCDD interaction. Table II summarizes the dose-response effects of 2,3,7,8-TCDD and Aro dor 1254 as inducers of hepatic microsomal AHH and EROD in immature male C57BL/6J mice. Maximal induction of AHH (4.06 nmol/mg/min) and EROD (4.86 nmol/mg/min) were observed at a dose level of 50 nmol/kg of 2,3,7,8-TCDD. No overt toxirity (e.g. body weight loss, hepatotoxidty or thymic atrophy) was observed at this dose level. In contrast, administration of Arodor 1254 to C57BL/6J mice at doses of 5, 25, 75, 150 and 400 mol/ kg significantly induced EROD only at the highest dose of Arodor 1254 35 GENP 010940 783751 TABLE li THE DOSE-RESPONSE EFFECTS OF 2,3,7,8-TCDD AND AROCLOR 1254 AS INDUCERS OF HEPATIC MICROSOMAL AHH AND EROD IN MALE C57BL/6J MICE (n = 5) Compound Com dii 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD Arodor 1254 Aroclor 1254 Arodor 1254 Arodor 1254 Arodor 1254 Dose (fimol/ kg) __ 0.001 0.003 0.010 0.015 0.03 0.05 5 25 75 150 400 ERODb 0.06 0.004 0.33 0.08` 1.39 0.54* 3.31 0.50` 3.96 0.16* 4.19 0.30* 4.86 0.61* 0.06 0.006 0.05 0.03 0.06 0.01 0.09 0.04 0.25 0.05 AHHb 0.21 0.08 0.64 x 0.16* 1.67 0.71* 3.36 0.07* 3.56 0.43* 3.67 0.30* 4.06 0.37" 0.18 0.06 0.41 0.06* 0.39 0.06* 0.40 0.08" 0.55 0.07* `Significantly different (P < 0.01) from the com oil controls, ^nmol/mg protein/min. TABLE III l COTREATMENT OF C57BL/6J MICE WITH AROCLOR 1254 AND 2,3,7,8-TCDD: EFFECTS ON AHH AND EROD INDUCTION (n = 5) Inducers Dose (pmol/kg) EROD* AHHC Arodor 1254 plus 2,3,7,8-TCDD Arodor 1254 plus 2,3,7,8-TCDD Arodor 1254 plus 2,3,7,8-TCDD Arodor 1254 plus 2,3,7,8-TCDD Arodor 1254 plus 2,3,7,8-TCDD Arodor 1254 plus 2,3,7,8-TCDD Arodor 1254 plus 2,3,7,8-TCDD '5 0.015 25 0.015 75 0.015 150 0.015 400 0.015 25 0.03 25 0.05 3.79 0.58 3.05 0.47" 3.09 0.34* 3.22 0.07b 3.96 0.45 4.19 0.39 4.93 0.25 3.34 0.42 2.84 * 0.33* 2.88 0.21* 3.09 0.27* 3.55 0.18 3.78 0.25 4.10 0.20 Significantly lower *tP < 0.01) b(P < 0.05) than induction values observed after treatment with 2,3,7,8-TCDD (15 nmol/kg) alone; the induction values for 2,3,7,8-TCDD [0.015, 0.03 and 0.5 jimol/kg) are shown in Table II. "nmol/mg protein/min. 36 GNP 010941 783752 and marginally increased AHH at doses above 25 unol/kg. Table III sum marizes the effects of coadministration of Aroclor 1254 (5, 25, 75, 150 or .400 fimol/kg) with a dose of 2,3,7,8-TCDD (15 nmol/kg) which elicited a submaximal AHH and EROD induction response. Cotreatment of 2,3,7,8TCDD with 25 or 75 pmol/kg of Aroclor significantly inhibited the induction of AHH and EROD by 2,3,7,8-TCDD. Coadministration of Aroclor 1254 (25 imoi/kg) with higher doses of 2,3,7,8-TCDD (30 and 50 nmol/kg) reversed the antagonistic effects of Aroclor 1254. Table IV summarizes the effects of 2,3,7,8-TCDD and Aroclor 1254 on the T-cell dependent plaque*forming cell (PFC) assay after exposure of TABLE IV EFFECTS OF 2,3,7,8-TCDD, AROCLOR 1254 AND 2,3,7,8-TCDD PLUS AROCLOR 1254 ON THE PLAQUE FORMING CELL ASSAY IN MICE PRETREATED WITH SHEEP RED BLOOD CELLS Treatment (dose) PFC/spleen x 10s PFC/10 viable spleen cells % Control (PFC/spleen x 10*) Com oil* 2,3,7,8-TCDD {3.72 nmol/kg)b t (11.2 nmol/kg) Aroclor 1254 (5 fimol/kg) (15 jtfnol/kg)b (75 pmol/kg) (150 pmal/kg) 1.61 0.17 0.38 * 0.03c 0.20 0.03c 1.57 0.11 1.60 0.87 1.57 0.23 1.20 0.22 562 76 156 40 62 * 12c 561 140 465 114 594 35 394 35 100 24 12 98 100 98 75 2,3,7,8-TCDD (3.72 nmol/kg) plus Aroclor 1254 (5 junol/kg) 2,3,7,8-TCDD (3.72 nmol/kg) plus Aroclor 1254 (15 nmol/kg)b 1.70 * 0.30 1.49 0.08 491 * 62 480 48 106 93 2,3,7,8-TCDD (3.72 nmol/kg) plus Aroclor 1254 (75 junol/kg) 1.66 0.28 558 52 103 2,3,7,8-TCDD (3.72 nmol/kg) plus Aroclor 1254 (150 ymol/kg) 0.92 0.08= 372 a 81c 57 2,3,7,8-TCDD (11.2 nmol/kg) plus Aroclor 1254 (75 anol/kg) 0.58 0.10 "11 animals. b8 animals; all other 4 animals/group. 'Significantly different IP < 0.01) than the com oil controls. 170 10c 36 37 GEN? 010942 783753 C57BL/6J mice to sheep red blood cells. Administration of 2,3,7,8-TCDD (3.72 and 11.2 nmol/kg) resulted in only 24 and 12% PFCs/108 spleen cells respectively, compared to the com oil-treated animals. In contrast, adminis tration of Aroclor 1254 at dose levels of-5, 15, and 75 pmoL/kg did not significantly impair the immune response although administration of a higher dose of Aroclor 1254 (150 pmol/kg) did evoke some decrease in the plaque fo rm in g cell response. Cotreatment of the mice with 2,3,7,8-TCDD (3.72 nmol/kg) and 5, 15, or 75 pmol/kg Aroclor 1254 resulted in significant protection of these animal! from the immunotoxic effects of 2,3,7,8-TCDD (Table IV). Significant antagonism of 2,3,7,8-TCDD was also observed in animals cotreated with the highest dose of Aroclor 1254 (150 unol/kg) which was also immunotoxic in this assay when administered alone. Coadministra tion of a higher dose of 2,3,7,8-TCDD (11.2 nmol/kg) with Arcolor 1254 (75 pmol/kg) reversed some of the antagonistic effects of the commercial PCB mixture. DISCUSSION This study describes the in vitro and in vivo interactive effects of 2,3,7,8TCDD and Aroclor 1254, 2 compounds which' individually elicit a number of common biologic and toxic effects in diverse animal species. For example, both of these halogenated aryl hydrocarbons induce the same monooxygen ase enzymes (e.g. AHH and EROD) and their associated cytochrome P-450 isozymes, cause body weight loss, hepatotoxicity and porphyria, thymic atrophy and imfnunotoxicity [1--3,241. In addition, 2,3,7,8-TCDD and Aro clor 1254 competitively bind to the Ah or 2,3,7,8-TCDD receptor protein [8]. These results support the proposed common mechanism of action for both compounds; however, it is apparent from this study and others that the major differences between Aroclor 1254 and 2,3,7,8-TCDD are their toxic and biologic potencies. The results summarized in Tables I and II clearly illustrate the quantitative differences between 2,3,7,8-TCDD and Aroclor 1254 as inducers of AHH and EROD. In rat hepatoma cells, the EC50 for 2,3,7,8-TCDD as an inducer of AHH and EROD was 9.78 x 10~11 and 1.90 x 10"10 M respectively whereas the EC50 for Aroclor 1254 as an inducer of these enzymes was > 10"4 M. Moreover in C57BL/6J mice, 2,3,7,8TCDD was > 50 000 times more potent than Aroclor 1254 as an inducer of hepatic microsomal AHH and EROD. 2,3,7,8-TCDD and Aroclor 1254 and several PCB congeners are immunotoxic and inhibit the plaque forming an tibody response in C57BL/6J mice challenged with sheep erythrocytes [13-- 17,311. The EDMfor inhibition of this response by Aroclor 1254 in C57BL/ 6N mice was between 250 and 500 mg/kg [31] whereas the EDWfor 2,3,7,8TCDD was <1.2 pg/kg [14]. Previous in vitro studies have reported that 1,3,6,8- and 2,4,6,8tetrachlorodibenzofuran antagonize the induction of AHH and EROD in rat hepatoma H-4-II E cells in culture [23]. Like Aroclor 1254, both of these iso mers exhibit moderate competitive receptor binding affinities and are weak 38 783754 inducers of AHH and EROD in the cell culture system. Table I summarizes the interactive effects of 2,3,7,8-TCDD and Aroclor 1254 in the rat hepatoma H-4-II E system and at Aroclor 1254/2,3,7,8-TCDD concentration ratios of 100:1 there was a significant inhibition of the induction activity of 2,3,7,8-TCDD by Aroclor 1254. Moreover at a higher dose of the agonist (2.25 x 10*4 M) some of the inhibitory effects of Aroclor 1254 were over come. A comparable interaction study was carried out in C57BL/6J mice. The mice were cotreated with a dose of 2,3,7,8-TCDD which elicited a submaximal induction response for hepatic microsomal AHH and EROD (15 nmol/kg) and one of several dose9 of Aroclor 1254. The results showed the Aroclor significantly reduced the induction of AHH and EROD by 2,3,7,8TCDD (15 nmol/kg) only at doses of 25, 75 and 150 pmol/kg. The role of Aroclor 1254 as a 2,3,7,8-TCDD antagonist was further supported by the effects of cotreating the anim als with the most effective "inhibitory" dose of Aroclor 1254 (25 pmol/kg) and higher doses of 2,3,7,8-TCDD (30 and 50 nmol/kg) (Table III). These results showed that at higher dose levels of the agonist the partial antagonist activity of Aroclor 1254 was decreased com plementing the results obtained in the in vitro studies. It was also apparent that Aroclor 1254 was a more effective partial, antagonist of the induction responses by 2,3,7,8-TCDD in cell culture. Significant antagonism of AHH was observed at Aroclor 1254/2,3,7,8-TCDD ratios of 100:1 in cell cultures whereas antagonism was only observed in the mice at ratios between 1667:1 and 10 000:1. Several reports have shown that 2,3,7,8-TCDD and related compounds are immunotoxic and impair many T-cell mediated functions [13--17,31-- 34], Moreover the T-cell dependent plaque forming cell response to sheep red blood cells is one of the most sensitive indicators of exposure to toxic halogenated aryl hydrocarbons and studies in genetically inbred mice have provided strong evidence that this response is Ah receptor-mediated. Table IV summarizes the immunosuppressive effects of 2,3,7,8-TCDD and Aroclor 1254 in C57BL/6J mice using the plaque forming cell assay response to sheep red blood cells. 2,3,7,8-TCDD was highly immunotoxic whereas doses of 5, 15 and 75 nmol/kg of Aroclor 1254 were inactive in this assay system. This latter result was not surprising since Aroclor 1254 was previously reported to be inactive at doses of < 125 mg/kg (382 pmol/kg) [31]. Cotreat ment of the mice with 2,3,7,8-TCDD (3.72 nmol/kg) and different doses of Aroclor 1254 (5, 15 or 75 pmol/kg) resulted in complete protection from the immunotoxic effects of 2,3,7,8-TCDD by Aroclor 1254 (5, 15 or 75 tmol/kg). Moreover, the antagonistic effects of Aroclor 1254 (75 ^mol/kg) can be par tially overcome by a higher dose of the 2,3,7,8-TCDD agonist (11.2 nmol/ kg). The interactive effects of 2,3,7,8-TCDD, a potent Ah receptor agonist, and Aroclor 1254 suggest that this commercial PCB mixture can antagonize at least 2 Ah receptor mediated responses, namely immunotoxicity and the induction of cytochrome Pj-450-dependent monooxygenases (AHH and EROD). These effects are dependent on the dose of both the antagonist and 39 GENP 010944 783755 agonist and their respective antagonist/agonist ratios. For antagonism of AHH and EROD induction by 2,3,7,8-TCDD in C57BL/6J mice, an Aroclor 1254/2,3,7,8-TCDD ratio of 1667:1 gave the maximum antagonism (23%) of th EROD induction response. In contrast, Aroclor 1254/2,3,7,8-TCDD ratios from 1340:1 to 20 160:1 resulted in complete protection from the immunotoxic effect of 2,3,7,8-TCDD (3.72 nmol/kg). The results demonstrate that the "window of antagonism" for Aroclor 1254 is clearly dependent not only on the absolute and relative doses of Aroclor 1254 and 2,3,7,8-TCDD but on the specific receptor-mediated response which is being measured. However, it was observed that for Aroclor 1254/2,3,7,8-TCDD ratios which result in antagonism by Aroclor 1254 of AHH and EROD induction (in vivo and in vitro) and immunotoxidty, an increased dose of 2,3,7,8-TCDD over came some of the partial antagonism by Aroclor 1254. These results, coupled with the double-reciprocal plot analysis of the in vitro binding data (Fig. 2) suggest that the antagonist activity of Aroclor 1254 is related to the competitive inhibition of 2,3,7,8-TCDD binding to the Ah receptor and is not due to cellular toxicity caused by Aroclor 1254. A previous study has reported that 2,3-dimethyI-5-i-butyl-l,3-benzodioxole (DBBD) inhibits EROD induction by 3-methylcholanthrene [35J. However, it is unlikely that the mechanism of antagonism for this com pound involves competitive inhibition of receptor binding since DBBD exhibits a low binding affinity for the Ah receptor [36], A more recent study suggested that the activity of DBBD may be associated with down regula tion of the Ah receptor [37]. A recent paper [38] reported that l-amino-3,7,8trichlorodibenzo-p-dioxin was also an effective antagonist for 2,3,7,8-TCDD induced myelotoxicity (in vivo) and enzyme induction (in vitro). They also proposed that this compound acts as a competitive antagonist and their results complement the data reported in this study which demonstrates that Aroclor 1254 antagonizes 2,3,7,8-TCDD-mediated enzyme induction and immunotoxicity in C57BL/6J mice. It is interesting to note that the relatively high ratios of Aroclor 1254/ 2,3,7,8-TCDD which result in antagonist activity are comparable to the ratios of PCBs/PCDFs or PCBs/PCDDs which have been identified in the analysis of human tissues and environmental samples [39,40], It is conceiv able that environmental or accidental exposure to these mixtures within certain exposure levels may result in some protection from the highly toxic PCDDs and PCDFs by PCBs and this possibility is currently being investi gated in our laboratory. ACKNOWLEDGEMENTS The financial assistance of the National Institutes of Health (ES-03843) and the Texas Agricultural Experiment Station is gratefully acknowledged. The experimental assistance of Ms. B. Keys and Mrs. S. Safe is also appreciated. 40 g e h p 01945 783756 R 1 2 3 4 5 6 7 8 9 i; 1 1: i: ll; u 17 18 19 REFERENCES 1 A. Poland and J.C. Knutson, 2,3.7,8-Tetrachlorodibenzop-diorin and related halogenated aromatic hydrocarbons: Examination of the of toxicity. Annu. Rev. Pharma col., 22 (1982) 517. 2 A. Poland, W.F. Greenlee and A.S. Kende, Studies on the mechanism of action of the chlorinated dibenzo-p-dioxins and related compounds, Ann. N.Y. Acad, So., 320 (1979) 214/ 3 S. Safe, Comparative toxicology and mechanism of action of polychlorinated dibenzo-p- dioxins and dibenzofurans. Annu. Rev. PhannacoL ToxicoL, 26 (1986) 371. 4 D.W. Nebert, H.J. Eisen, M. Negishi, M.A. Lang and L.M. Hjelmeland, Genetic mecha nisms controlling the induction of polysubstrate monooxygenase 1/M50) activities, Annu. Rev. PhannacoL ToxicoL, 21 (1981) 431. 5 A. Poland, E. Glover and A.S. 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Hodgson, 2,2-Dimethyl-5-t-butyI-l,3-benzodioxole: an unusual inducer of microsomal enzymes. Biochem. Pharmacol., 33 (1984) 3941. 36 J.C. Cook and E. Hodgson, The induction of cytochrome P-450 by isosafrole and related raethylenedioxyphenyl compounds. Chem.-Blol. Interact.. 54 (1985) 299. 37 J.C. Cook and E. Hodgson. Cytochrome P-450 induction by 3-methylcholamhrene and its antagonism by 2,2-dimethyl-5-r-l,3-benzodioxole. Biochem. Pharmacol., 35 (1986) 167. 38 M.I. Luster, L.I. Hong, R. Osborne, J.A. Blank, G. Clark, M.T. Silver. G.A. Boorman and W.F. Greenlee, 1-Amino-3,7,8-trichlorodibenzo-p-dioxin: a specific antagonist for TCDD-induced myelotoxicity. Biochem. Biophys, Res. Commun., 139 (1986) 747. 39 D.L. Stalling, R.J, Norsemen, L.M. Smith and M. Simon, Patterns of PCDD. PCDF and PCB contamination in Great Lakes fish and birds and their characterization by principal component analysis. Chemosphere,-14 (1985) 627, 40 J.J. Ryan, Variation of dioxins and furans in human tissue. Chemosphere, 15 (1986) 1585, 41 T. Sawyer and S. Safe, PCB isomers and congeners: induction of aryl hydrocarbon hydrox ylase and ethoxyresorufin O-deethylase enzyme activities in rat hepatoma cells. Toxicol. Lett.. 13 (1982) 87. 42 GENP 010947 783758 Manuaerip -- Author Editor, ind au Europe of Mart -- should -- must b< -- should -- should Review Ar -- Review authors r t E rL i k v v fc j r- LJ : ' *! K1 Animal Ex -- It is ex an ethic Address - Author and cor Summary The art Kay words A list c script. I ducing activity Figures - Line drt pro videi - Half-tor Colour to the a Tables -- should t References -- should t Referen referent -- In the n 1. A. G flor 2. A. Fr 3. T.A. Comr -- Female -- For jou Lancer. Msnuaertpt To avoid th 1 A ft pape 2 The be ct 3 The (i) tc () c 4 A cc TOX shou 5 Each This be at Advert bem Aevert ii MU US UVC: T.( 1ST Toxicology Letters, 38 (1987) 299-306 Elsevier 299 TXL 01844 AROCLOR 1254 AS AN ANTAGONIST OF THE TERATOGENICITY OF 2,3,7,8-TETRACHLORODIBENZO-jD-DIOXIN (Aroclor 1254; 2,3,7,8-TCDD; antagonism) J.M. HAAKE", S. SAFE', K. MAYURAb and T-D. PHILU PSb aDepartment o f Physiology and Pharmacology, bDepartment o f Public Health, College o f Veterinary Medicine, Texas A&M University, College Station, TX 77843 (U.S.A.) (Received 17 April 1987) (Revision received 30 May 1987) (Accepted 6 June 1987) SUMMARY Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 20^g/kg) to pregnant C57BL/6J mice (on day 10) resulted in 62% fetuses with cleft palate per litter without any observable maternal toxicity. In contrast, .Aroclor 1234 administered at a dose of 730 ptnol/kg was not teratogenic. Cotreatment of the pregnant mice with both Aroclor 1254 (244 mg/kg)and 2,3,7,8-TCDD (20/ig/kg) resulted in an 8.2% incidence of cleft palate per litter. In contrast, Aroclor 1254 did not afford any protection from the teratogenicity of dexamethasone in C57BL/6J mice. Previous studies have shown that Aroclor 1254 can act as a partial antagonist of the microsomal enzyme induction and immunotoxic effects of 2,3,7,8-TCDD in C57BL/6J mice and this paper demonstrates that the commercial polychlorinated biphenyl mixture also antagonizes 2,3,7,8-TCDD-mediated teratogenicity in this strain of mice. INTRODUCTION Polychlorinated biphenyls (PCBs) are widely used industrial compounds which have been identified in almost every component of the global ecosystem including fish, wildlife and human adipose tissue, blood and milk [1-4]. During the past few years, the more toxic haiogenated aryl hydrocarbons, namely the polychlorinated Address for correspondence: S. Safe, Department of Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, U.S.A. Abbreviations: Ah, aryl hydrocarbon; AHH, aryl hydrocarbon hydroxylase; PCBs. polychlorinated biphenyls; PCDDs, polychlorinated dibenzo-^dioxins; PCDFs, polychlorinated dibenzofurans; TCDD, tetrachlorodibenzo-p-dioxin. 0378-4274/87/S 03.50 1987 Elsevier Science Publishers B.V. (Biomedical Division) 783759 dibcn/.ofurans (PCDFs) ami dibcnzo-p-dioxins (PCDDs) have also been identified in ihc environm ent |5 -8 | and in human tissue including milk samples |9-I6J. Since the PCD Fs and PCD Ds arc industrial or com bustion byproducts 117-19], (he levels o f these com pounds arc usually > IO'-FoTtl lower in most environm ental samples. M oreover, high resolution analyses o f the human milk and adipose tissue have con firmed the presence o f 2,3,7,8-iclrachlorodibenzo-/>-dioxin ( I'CDD), the most toxic halogcnatcd aryl hydrocarbon and several other toxic 2,3,7,8-subsiitutcd PCDDs and PCDFs (9-lA|. The toxicity and mechanism of action orhalogcnaled aryl hydrocarbons including PCBs, PCDDs and PCDFs have been extensively investigated |20-26|. Laboratory anim al studies show that these com pounds elicit a num ber oT common toxic responses including: body weight loss, thymic atrophy and imm uuotoxicily, teratogenicity and reproductive problems, hepatotoxicity and porphyria, car cinogenicity, effects on the endocrine system and derm al toxicity. All o f these responses arc not evoked in any single animal species; however, body weight loss and thymic atrophy are the signs o f toxicity most frequently observed in laboratory animals. Most incidents involving environmental or accidental human exposure to the toxic PCD D s and PCD Fs arc associated with concom itant exposuie to higher levels o f PCBs and these arc generally rcflcclcd in hum an tissue levels in which the PC IJs/P C D D s, PCD Fs ratios arc >1000:1. For example in the Y ushoand Yu Cheng poisoning in Japan and Taiwan respectively, several thousand individuals were poisoned with rice oil contam inated with PCBs and PCDFs (27,28]. The ratios oT P C B s/P C D F s in the contam inated oil and the blood o f exposed individuals were generally in ihc order of 184-667:1 and 1500-1890:1 respectively |27|. Since the in teractive effects of concurrent exposure to halogcnatcd aryl hydrocarbons is not well understood, this study reports teratogenicity o f combined exposure to 2,3,7,8-T C D D and Aroclor 1254, a commercial PCB form ulation. MATERIALS AND METHODS Chemicals 2,3,7,8-TC D D was prepared in our laboratory and the purity was > 99*70 as determ ined by gas-liquid and high-pressure liquid chrom atographic analysis. Dexam ethasone was purchased from Sigma Chemical C o., St. Louis, MO. Aroclor 1254 was a generous gift o f Mr. B. Chittim , Wellington Environm ental Consultants, Guelph, O nt., Canada. !f A nim al treatment M ature male C57B L/6J mice and virgin female C57B L/6N mice (20-25 g) were purchased from H arlan Sprague-Daw ley, Indianapolis, IN and m aintained on food and water ad libitum at (Ire Texas A&M University L aboratory Animal Resources 783760 GENP 010949 ' 301 iim) Research facility. Al the end o f a 7-day acclimation period, females were mated with the males overnight. The following morning, females were checked for the presence of vaginal plugs. The presence of a vaginal plug was denoted as day 0 o f pregnancy. I:cmalcs with vaginal plugs were weighed and marked. Pregnancies were confirmed by measuring weight gain. The darns were treated by oral gavage with either coin oil (10 m l/kg), Aroclor 1254 (244 m g/kg), 2,3,7,8-TCD D (20 gg/kg), Aroclor 1254 (244 m g/kg) followed by 2,3,7,8-TCD D (20 jtg/kg), dexamthasone (90 m g/kg), or Aroclor 1254 (244 m g/kg) followed by dexamthasone (90 m g/kg). All these com pounds or mixtures were adm inistered in corn oil (10 m l/kg) by oral gavage. A roclor 1254 alone was adm inistered on day 9 o f pregnancy; corn oil and 2.3.7.8- TCOD were given on day 10 and dexamthasone on day 13. The animals receiving 2 chemicals were given A roclor 1254 on the day prior to either 2,3,7,8TCDD or dexam thasone, i.c. Aroclor 1254 on day 9 and 2,3,7,8-TCD D on day 10 or Aroclor 1254 on day 12 and dexam thasone on day 13 o f pregnancy. Controls were either untreated or treated with corn oil (10 m l/kg). On day 17, the females were killed with CO2 or by cervical dislocation. The weights of the dams and their respective liver weights were recorded. The uterine horns were exposed and the number o f resorptions, dead and live fetuses were counted. Live fetuses were remov ed from the uterus, weighed and examined for gross abnormalities. The fetuses were fixed in Uouin's solution for al least I week and examined for internal soft tissue anomalies as previously described [29,30). Statistical differences were determined using an analysis oT variance procedure (ANOVA). RMSUt.TS A N D DISCUSSION Table I summarizes the effects o f 2,3,7,8-TCD D , A roclor 1254, 2,3,7,8-TCDD plus Aroclor 1254, dexamthasone and dexamthasone plus Aroclor 1254 on C57IL/6.I females and their fetuses. No apparent signs o f maternal toxicity were observed in any o f the treatm ent groups. The percentages o f fetuses with hydrone phrosis per litter for the treatm ent groups were: untreated (1.3 4.3); corn oil (5.7 9.9), Aroclor 1254 (33.4 18.0); 2,3,7,8-TCD D (87.8 20.4); A roclor 1254 + 2.3.7.8- TCDD (96.0 M.0); dexamthasone (3.2 7.3); and A roclor 1254 + dexamethasone (3.7 5.5). The dose o i A roclor 1254 (224 m g/kg) caused con siderable hydronephrosis and, not surprisingly, did not antagonize the effects or 2.3.7.8- TCDD. H ydronephrosis is a more sensitive indicator o f exposure to toxic halogcnatcd aryl hydrocarbons [31,38,39J and at a dose level o f 244 m g/kg, Aroclor 1254 acts as an agonist for this response. At a dose of 20/xg/kg, 2,3,7,8-TCDD causcd 61.8% fetal cleft palate per litter whereas Aroclor 1254 (244 m g/kg) did not cause any cleft palate in Ilie litters. In contrast, hydronephrosis was observed in animals exposed to 2,3,7,8-TCDD or A roclor 1254 (data not shown). Colreatm ent o f the mice with a teratogenic dose o f 2,3,7,8-TCD D (20 ng/kg) plus Aroclor 1254 (244 m g/kg) resulted in only 8.2(7o fetal d e ft palate per litter which was significantly 3()2 'Significantly different from corn oil-treated group at P<0.05. " `Values with such signs are significantly different from each other at f, <0.05. n *" K> rj 41 it II n oo u. * V tx d so */S q d HM <N q oo d Vi oo II q d --i r&i | O"" Z, oo eO L r> 00 d =D. -5 * -- ,bm " * 41 41 41 -- N -* od IN N? O Nq d v It H q OO dd 12.30 2.23'3 7.78 i 9.3 vo d -- oo d " S-- ft 41 41 3 *- os 00 oo od d 00 o oo * CM n r- U; n q -g _ d d d E ^ 8 l* M<j 'S 41 41 41 cx 8 u q 00 r*l r i c VO vs d __ tu CU 00 E^ 5 -& 5? 00 q? m n +4 41 41 oo O ? vi r> n vi v> Tt q d vs 41 41 -- IN rj m oo Os i fi Os q d v> +1 M vs n -t d r* n os VI N> -- * d - 4 t 41 f* Vi Uq d VS Oo IN N dd r# d 41 41 41 OS o r** o o dd O d oo o f*# dd 41 ii ft > oo dd 'o<uao-o;J Z = o' Li. V) uaLUUl.. I --o - n " O c ^ ,,?H iBc O 3,, Mr-u*Qu7\_jMwo4<--,,VNMniv0 ^x-WEi. noQ* ri ~3| n OoQ -C l H 5 S0 5V jw_* --U55is--u J _TvU-,iT, *-T n*E OEb O " - fj ? 5 __ o^t> T~ E t 4 rJ DO ri < < Q < plus dxamthasone (90 mg/kg)__________ 9 0.64* r 0.21 tabi 783761 GENP 010950 ' 303 decreased from the teratogenic effects observed with 2,3,7,8-TCDD alone. These results contrast with previous studies on the interactive effects of individual PCBs and 2,3,7,8-TCD D as teratogens (31J. For example, the teratogenicity of 2.3.7.8- TCDD was unaffected by cotrcalmcnl with 2 ,2 ',4,4',5,5'-hcxachlorobiphenyl whereas 2 ,3 ,3 ',4 ,4 ',5-hexachlorobiphenyl potentiated the teratogenicity or 2.3.7.8- TCDD. Previous reports have shown that 2 ,3 ,3 ',4 ,4 ',5-hexachlorobiphenyl evokes several typical 2,3,7,8-TCDD-like responses such as the induction of cytochrome P-450c and related monooxygenases, body weight loss, thymic atrophy and imrnunotoxicity (32-36], Moreover, (his PCB congener binds with moderate af finity to the Ah receptor protein (37|. In contrast, 2,2',4,4',5,5'-hexachlorobiphcuyl docs not elicit Ah receptor-m ediated effects in laboratory animals (20,33). Subsequent studies on the interactive effects of 2,3,7,8-TCDD and 2,3 ,3 ',4 ,4 ',5 licxachlorobiphcnyl as teratogens suggest that the interactive effects are additive (38) and recent work shows that the interactive effects o f 2,3,7,8-TCDD and 2.3.7.8- ictrachlorodibcnzoruran (TCDF) as teratogens are also additive |39J. Although A roclor 1254 is a complex mixture o f PCB isomers and congeners, this mixture, like 2 ,3 ,3 ',4 ,4 ',5-hexachlorobiphenyl, also elicits a broad spectrum o f Ah receptor-mediated responses including immunotoxicity, thymic atrophy, body weight loss, porphyria and the induction of cytochrome P-450c and related monooxygenases and exhibits m oderate affinity Tor the Ah receptor protein (20,32-37]. In fact, both Aroclor 1254 and 2 ,3 ,3 ',4 ,4 ',5-hcxachlorobiphenyl arc weak Ah receptor agonists compared to 2,3,7,8-TCDD and their competitive binding af finities Tor the hepatic Ah receptor are com parable )37J. One m ajor difference be tween 2 ,3,3' ,4 ,4 ' ,5-hexachlorobiphenyl and A roclor 1254 is the relative potency of the former com pound. For example, the in vivo ED jo values for body weight loss, thymic atrophy and hepatic microsomal aryl hydrocarbon hydroxylase in the rat for 2 ,3 ,3 ',4 ,4 ',5'-hcxachlorobiphenyl arc 180, 180 and 7 pm ol/kg )36); the corre sponding values for Aroclor 1254 are at least 10 limes higher (40). Moreover, Aroclor 1254 exhibits a competitive Ah receptor binding affinity only 500 limes lower than 2,3,7,8-TCDD whereas the toxicity and enzyme induction activity o f the latter com pound is at least 10' times greater in the rat (41). Although a direct com parison of the teratogenic potency o f Aroclor 1254 and 2,3,7,8-TCD D has not been made, (he EDjo for teratogenicity (cleft palate) in this study was < 20 /ig/kg whereas the EDjo for Kancchlor 500 in the ddY strain of mice was >2000 ftm ol/kg [42]. We therefore propose (hat Aroclor 1254 may act as a partial 2,3,7,8-TCDD antagonist by competing for (he Ah receptor and thus lowering the cellular levels of the Ah receptor available for interaction with the potent agonist, 2,3,7,8-TCDD. Evidence which supports this hypothesis include the following: (a) colreatment of C57B L/6J mice with A roclor 1254 and 2,3,7,8-TCD D at A roclor 1254/2,3,7,8TCD D ratios >1000:1 showed that Aroclor 1254 can significantly antagonize the in duction oT 2 Ah receptor-m ediated responses, namely (he induction o f aryl hydrocarbon hydroxylase (AH H ) and the plaque-form ing cell response to sheep 31)4 erythrocytes 143]; (b) Aroclor 1254 also antagonizes ilic induction o AMU by 2,3,7.8-T C D D in rat hepatom a M-4-II E cells in culture J4.1I; (c) double-reciprocal plot analysis o f the Ah rcceptor-2,3,7,8-TCD D binding isotherms in the presence o f difrerent concentrations o f A roclor 1254 gave data which arc consistent with com petitive inhibition o f 2,3,7,8-TCD D -rcceptor binding by A roclor 1254 |4 3 |; (d) AroclOr 1254 does not act a s a non-specific teratogen antagonist since A roclor 1254 does not antagonize the teratogenicity of dexamethasone, a synthetic glucocorticoid horm one analog which acts through initial binding with the glucocorticoid receptor (see T able I). These results further extend the spectrum o f 2,3,7.8~TC'DD-mcdialcd responses which can be antagonized by A roclor 1254. M oreover since hum an environm ental exposure to the toxic PCDD and PC D F congeners occurs in concert with sim ultaneous exposure and bioaccum ulation o f PCIJs |l-4 ,9 -lfiJ , it is possible that the non-toxic environmental PCB levels may affo rd some measure o f protection from these toxins since the environmental ratios o f P O I/lo x ic PCDDs + PCDFs |7 -9 ,I2 -1 5 ) are comparable to ratios in which A roclor 1254 exhibits antagonistic ac tivity (this paper; (43J). Current studies in our laboratory arc focused on further delineating (he mechanism o f the interactive effects o f commercial PCBs and in dividual PCB congeners with the toxic 2,3,7,8-substilulcd PCDDs and PCDFs. ACK NOW LlijtXiUMENTS Financial assistance was provided by the N ational Institute o f Health (ES-03843), the Texas Agricultural Experiment Station and the Chester J. Reed Endowment. 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EN P 010952 783763 EPRI 'ectric Power esearch Institute Topics: PC 8 Chemical analysis Transformers Capacitors PCDF-PCDD Insulating oil EPRI EL/EA-5443 Volume 1 Project 2028-7 Final Report October 1987 Analysis of Polychlorinated Dibenzofurans and Polychlorinated Dibenzo-p-Dioxins in Transformers and Capacitors Volume 1: PCDF/PCDD at New York State Health Department Prepared by New York State Department of Health Albany, New York OE-tfP 010953 783764  Use of the 13C compounds allowed successful separation of the com ponents in several closely eluting pairs of physiologically active P C D F and less-active compounds. Analyses of samples from utility equipment suggested that the P C D F -P C D D present-did not result from high-load, high-temperature operation. Volumes 1 and 2 describe several analytic techniques, with the appen dix of Volume 2 including a report on the analysis by the University of Umea in Sweden, as well. Volume 3 summarizes all the laboratory techniques, presents comprehensive statistical analyses, and provides an executive summary. EPRI P E R S P E C T IV E This research is a landmark in P C D F -P C D D analysis. One important result is the synthesis of new P C D F s for use as spiking compounds in gas chromatography-mass spectrometry analysis. The ability to sepa rate many of the active P C D F compounds from more innocuous materi als is also of great value. The high correlation of results from several laboratories indicates that researchers can exercise great flexibility in selecting techniques and developing facilities. PROJECTS RP2028-7, RP2028-6, RP2028-10 E P R I Project Managers: Gil Addis; Jacques Guertin Electrical System s Division; Environment Division Contractors: New York State Department of Health; Battelie Columbus Laboratories; Research Triangle Institute For further information on E P R I research programs, call E P R I Technical Information Specialists (415) 855-2411. GENP 010954 783765 Analysis of Polychlorinated Dibenzofurans and Polychlorinated Dibenzo-p-Dioxins in Transformers and Capacitors Volume 1: PCDF/PCDD at New York State Health Department EL/EA-5443, Volume 1 Research Project 2028-7 Final Report, October 1987 Prepared by ' NEW YORK STATE D EPAR TM EN T O F HEALTH Wadsworth Center for Laboratories and Research Corning Tower The Governor Nelson A. Rockefeller Empire State Plaza Albany, New York 12201 Principal Investigators D. R. Hilker G. A. Eadon K. M. Aldous R. M. Smith R W. O'Keefe H. Valente S. Connor J. Jurusik Prepared for Electric Power Research Institute 3412 Hiliview Avenue Palo Alto, California 94304 EPRI Project Managers G. Addis Transmission Substations Program Electrical Systems Division J. Guertin Land and Water Quality Studies Program Environment Division GEKP 010955 783766 ORDERING INFORMATION Requests for copies of this report should be directed to Research Reports Center (RRC), Box 50490. Palo Alto. C A 94303, (415) 965-4081. There is no charge for reports requested by EPRl member utilities and affiliates. U.S. utility associations. U.S. government agencies (federal, state, and local), media, and foreign organizations with which EPRl has an information exchange agreement. On request, R R C will send a catalog of EPRl reports. Electric Power Research institute and EPRl are registered service marks of Electric Power Research Institute. Inc. Copyright S 1987 Electric Power Research Institute. In c All rights reserved. NOTICE This report was prepared by the organization(s) nam ed below as an account of work sponsored by the Electric Power Research Institute In c (EPRl). Neither EPRl, members of EPRl, the orgamzation(s) named below, nor any person acting on behalf of any of them: (a) makes any warranty, express or implied, with respect to the use of any information, apparatus, method, or process disclosed in this report or that such use may not mfnnge privately owned rights: or (b) assum es any liabilities with respect to the use of, or for dam ages resulting from the use of. any information, apparatus, method, or process disclosed in this report Prepared by New York State Department of Health Albany, New York 783767 GENP 010956 ABSTRACT Seven sam pLes o f in -s e r v ic e u t i l i t y f lu id s w ere a n a ly z e d f o r n in e te e n d if f e r e n t p o ly c h lo r in a t e d d ib e n z o fu ra n (P C D F) and p o ly c h lo r in a t e d d ib e n z o - -d io x in (PCD D ) c o m p o u n d s . A f t e r s p i k i n g w i t h f i v e d i f f e r e n t 13 C - l a b e l e d i n t e r n a l s t a n d a r d s , t h e sa m p le s w ere c h r o m a t o g r a p h ic a lly s im p lif ie d ( s e m i-a u t o m a t e d ). A n a ly s e s w ere co n d u cte d w ith h ig h r e s o lu t io n gas ch ro m a to g ra p h y-m a ss s p e c tro m e try (C C -M S) u s in g a 50-m D B -5 o r an O V -2 2 5 f u s e d - s i l i c a c a p il l a r y c o lu m n . G e n e ra te d c a l ib r a t io n p lo t s f o r e ig h t d if f e r e n t in d iv id u a l com pounds w ere lin e a r o v e r fo u r o rd e rs o f m a g n itu d e c o n c e n t r a t io n s . A n a ly s e s in t r i p l i c a t e p ro v id e d some in s ig h t on r e p r o d u c ib ilit y o f m ethod. I S p ik e d sam pLes w ere a n a ly z e d w ith an a v e ra g e e r r o r o f 2 8 .9 2 , in d ic a t in g ch a t fo r che cho sen a n a ly te s in c h is ty p e o f o il y m a t r ix , th e m ethod was q u it e re p r o d u c ib le and a c c u r a t e . The r e la t iv e s ta n d a rd d e v ia t io n was le s s ch an 30 2 f o r m ore th a n 9 22 o f th e a n a ly t e d e t e r m in a t io n s . In g e n e r a l, th e s tu d y show ed t h a t th e sa m p le s o f i n - s e r v i c e u t i l i t y f l u i d s h a v e c o n s id e r a b ly d if f e r e n t PCDF-PCDD p r o f i l e s . M axim um c o n c e n tra tio n s o f te tra c h lo ro d ib e n z o fu ra n (TC D F) and t e t r a c h lo r o d ib e n z o -g -d io x in (T C D D ) w e re 1 . 2 a n d 0 .0 0 1 m g /k g , r e s p e c t i v e l y . M axim um c o n c e n t r a t io n s o f o c t a c h lo r o d ib e n z o f u r a n (O CD F) and o c t a c h lo r o d ib e n z o - -d io x in (OCDD) w ere 40 and 0 .0 2 m g /k g , r e s p e c t iv e ly . And two f l u i d s c o n t a in e d b a r e ly d e t e c t a b le q u a n t it ie s o f a n y PCDD o r P CD F (m axim um c o n c e n t r a t io n : 0 .0 0 3 m g /k g ) . 783768 i i i GENP 010957 CONTENTS S e c tio n 1 IN TR O D U CTIO N 2 METHODS In -S e r v ic e F lu id S am p le s - PCDD and PCDF S ta n d a rd s Sam p le E x t r a c t io n and E x t r a c t P r e p a r a t io n H ig h R e s o lu t io n C a p illa r y G as C h ro m a to g ra p h y/L o w R e s o lu t io n M ass S p e c tro m e try (H R G C/LR M S) H ig h R e s o lt it io n G as C h ro m a to g ra p h y /H ig h R e s o lu t io n M ass M ass S p e c t ro m e t ry (H RGC/H RM S) 3 D ISC U SSIO N P r e lim in a r y B a s e lin e S tu d y D ata C a lib r a t io n and Q u a lit y A ssu ra n ce D ata B a s e lin e D ata In -S e r v ic e L iq u id s 4 REFERENCES APPEN D IX A IN -S E R V IC E L IQ U ID S BACKGROUND IN FO RM ATIO N A PPEN D IX B ANALYTE CA LIB RA TIO N CURVES A PPEN D IX C ANALYTE RESPONSE FACTOR CURVES APPEN D IX D TA B LES OF RESU LTS A PPEN D IX E BAR GRAPHS OF RESULTS APPEN D IX F T A B LE S OF R E C O V E R IE S , R E L A T IV E R E C O V E R IE S AND D ETECTIO N L IM IT S Page 1 -1 2 -1 2 -1 2 -1 2 -2 2 -3 2 -4 3 -1 3 -1 3 -2 3 -5 3 -7 4 -1 A -l B -l C -l D -l E -l F -l GENP 010958 V 783769 ILLUSTRATIONS F ig u re 2 - 1 T y p i c a l MSD Raw D a t a 3 - 1 E x a c t M ass C h ro m a to g ra m s S h o w in g th e R e t e n t io n T im e W indow f o r TCDD 3 -2 H ig h R e s o lu t io n M ass S c a n s & S u m m arie s 3 -3 In t e r fe r e n c e in th e D e te rm in a tio n of 23 78 -T C D D in S p ik e d A r o c lo r 12 6 0 3 -4 H ig h R e s o lu t io n M ass Chrom atogram o f S p ik e d A r o c lo r 12 6 0 3 - 5 H i g h R e s o l u t i o n M a s s D a t a f r o m t h e 2 , 3 , 7 , 8- T C D D G . C . P e a k f r o m t h e S p ik e d A r o c lo r Sam p le Page 2 -4 3 -3 3 -4 3 -10 3 -11 3 -12 GENP 010959 v ii 783770 TABLES T ab le 2 -1 H -P 5890 Gas Ch rom atograph Te m p e ratu re Program 2 -2 C a rlo -E rb a Gas Chrom atograph 4160 T e m p e ratu re Program Page 2 -3 2 -5 783771 GENP 010960 lx SUMMARY M ach c o n c e rn h a s been a tta c h e d to th e o c c u rre n c e o p o ly c h lo r in a t e d d ib e n z o fu ra n (PCD F) and p o ly c h lo r in a t e d d ib e n z o - -d io x in (PCDD) in th e e n v iro n m e n t. S e v e ra l e n v iro n m e n ta l c a ta s tro p h e s w ere p r e c ip it a t e d by th e id e n t if ic a t io n and q u a n t it a t io n o f t h e s e two c la s s e s o f c o m p o u n d s. 2 , 3 , 7 , 8- t e t r a c h l o r o d ib e n z o - - d io x in ( 2 , 3 , 7 , 8-T C D D ) , o ne o f 2 1 0 p o s s ib le com pounds w it h in t h e s e c l a s s e s , i s g e n e r a ll y re g a rd e d a s th e m ost t o x ic m an -m ade com p ound . M uch o f th e c o n t r o v e r s y g e n e ra t e d a t th e Lo ve C a n a l w as a r e s u l t o f f in d in g 2 , 3 , 7 , 8-T C D D . T h e F e d e r a l g o v e rn m e n t l i t e r a L l y p u rc h a s e d th e tow n o f T im e s B e a c h , M is s o u r i when 2 , 3 , 7 , 8-T C D D w as fo u n d w id e ly d is p e rs e d in L the s o il . A tra n s fo rm e r f ir e in th e S ta te O f f ic e B u ild in g in B in g h a m to n , New Y o r k s p r e a d h ig h c o n c e n t r a t io n s o f PCD F and PCDD t h r o u g h o u t th e b u ild in g ' s e ig h te e n s t o r ie s . C o n s e q u e n tly , the b u ild in g h as been c lo s e d s in c e F e b ru a ry 19 8 1 w it h o n g o in g c le a n u p c o s t s o f s e v e r a l m il lio n d o l la r s . Th e B in g h a m to n O f f ic e B u ild in g d is a s t e r h e lp e d to e m p h a siz e t h a t PCDF and PCDD c o n ta m in a tio n is a p o t e n t ia l u t i l i t y p ro b le m . T h is u n fo rtu n a te e v e n t and o c h e r r e s e a r c h r e v e a le d t h a t PCDF an d PCDD c a n be fo u n d in some t r a n s f o r m e r s a s a r e s u l t o f c o n ta m in a tio n , se v e re o p e ra tin g c o n d it io n s , o r f ir e . And h u n d red s o f th o u sa n d s o f A s k a re L and p o ly c h lo r in a t e d b ip h e n y l (P C B )-c o n ta m in a te d m in e ra L o i l tra n s fo rm e rs a re s t i l l in u se in th e U n ic e s S t a t e s . An o b v io u s a lt e r n a t iv e to r e p la c in g c u r r e n t ly o p e r a t in g e l e c t r i c a l e q u ip m e n t s u s p e c te d o f c o n t a m in a t io n i s to id e n t if y th e e q u ip m e n t w ith th e g r e a t e s t e n v ir o n m e n tal c o n c e n tra tio n s o f PCDF and PCDD. T h is w ill re q u ire an u n d e rs ta n d in g o f the f a c t o r s th a t c o n t r o l th e d e g re e o f c o n ta m in a tio n . I t w ill a ls o be n e c e s s a ry to h ave a c c u ra te m ethods o f m easurem ent. E s t a b lis h in g a c c u ra te m easurem ent m ethods fo r PCDF and PCDD in PCB m a t r ic e s is a g o a l o f t h is s t u d y . In a d d it io n , th e s tu d y m e a su re s c o m p o u n d -s p e c if ic c o n c e n t r a t io n s o f PCDF and PCDD in r e l a t i v e l y w e L lknow n in s u la t in g f l u id s fro m com m only u se d e l e c t r i c a l e q u ip m e n t. T h is d e te r m in e s th e m easurem ent p r e c is io n . A ls o , t h is w ork c o n s t it u t e s p a rt o f a la r g e r in t e r la b o ra t o ry stu d y th a t w ilL p ro v id e in s ig h t re g a rd in g th e a c c u ra c y and m easurem ent m ethods f o r a s s e s s in g PCDF an d PCDD c o n ta m in a tio n in u t i l i t y e q u ip m e n t. S - l GENP 010961 783772 S e v e n d if f e r e n t p ie c e s o f e l e c t r i c a l e q u ip m e n t ( i . e . , lo a d c e n t e r n e tw o rk t r a n s fo rm e rs, c a p a c ito r s , p r e c ip it a t o r s , d is t r ib u t io n tra n sfo rm e rs, and a rc fu rn a c e t r a n s f o r m e r s ) w ere a n a ly z e d f o r n in e t e e n PCDF and PCDD co m p o un d s. H is t o r ic a l in fo rm a t io n su ch as m a n u fa c tu re r, a g e , u s e , and m a in te n a n c e was c o lle c t e d on e a ch p ie c e o f e q u ip m e n t. P r io r to a n a ly s is , th e f lu id sam pLes w ere c h r o m a t o g r a p h ic a L ly s im p lif ie d , c o n c e n t r a t in g th e PCDF and PCDD f r a c t io n s w h ile e lim in a t in g in t e r f e r in g co m p o un d s. E a ch f l u id sam p le was p re p a re d in t r i p l i c a t e an d e a c h e x tr a c t was c h e m ic a lly a n a ly z e d by g as ch ro m ato g ra p h y-m ass s p e c tro m e try (G C -M S ). In a d d it io n , t h r e e common in s u la t in g f l u id s ( m in e r a l o i l , A r o c io r 1 0 1 6 , A r o c lo r 12 6 0 ) w ere s p ik e d w ith known q u a n t it ie s o f com pounds o f in t e r e s t . T h e n , th e s e w ere c o n c e n tra te d and s u b je c te d to a s im ila r " c le a n u p " p ro ce d u re as u sed fo r th e sa m p le s from th e in - s e r v ic e e q u ip m e n t. T h is la t t e r a n a ly t ic a l p ro c e d u re s e rv e d as q u a lit y c o n t r o l fo r th e a n a L y s is o f unknown sa m p le s. S p ik e d sa m p le s w ere a n a ly z e d w ith an a v e ra g e e r r o r o f 2 8 .9Z in d ic a t in g t h a t fo r th e ch o sen a n a ly te s in t h is ty p e o f m a t r ix , th e m ethod was q u it e re p ro d u c ib L e and a c c u r a t e . T h e r e l a t i v e s t a n d a r d d e v i a t i o n w a s l e s s t h a n 3 0 Z f o r m o re c h a n 92% o f th e a n a ly t e d e t e r m in a t io n s . In g e n e r a l, th e s tu d y show ed th a c th e sa m p le s o f i n s e r v ic e u t i l i t y f l u i d s h a v e c o n s id e r a b ly d if f e r e n t P C D F -P C D D p r o f i l e s . M axim um c o n c e n tra tio n s o f te tra c h lo ro d ib e n z o fu ra n (TC D F) and te t r a c h lo r o d ib e n z o -2-d io x in (T C D D ) w e re 1 . 2 a n d 0 .0 0 1 m g /k g , r e s p e c t i v e l y . M axim um c o n c e n t r a t io n s o f o c t a c h lo r o d ib e n z o f u r a n (O CD F) and o c t a c h lo r o d ib e n z o - -d io x in (OCDD) w ere 40 and 0 .0 2 m g /k g , r e s p e c t i v e l y . And two f l u i d s c o n t a in e d b a r e ly d e t e c t a b le q u a n t i t i e s o f a n y PCDD o r PCD F (m axim um c o n c e n t r a t io n : 0 .0 0 3 m g /k g ) . 783773 S -2 GENP 010962 Section 1 INTRODUCTION Considerable interest has been focused on the analysis of various environmental samples for the closely related families of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). This interest originated with the documented occurrence of the most toxic isomer, 2,3,7,8-tetrachloro-dibenzo-pdioxin (2,3,7,8-TCDD). This highly toxic species has been detected in Lake Ontario fish (1_), in human adipose tissue (2), and turtles (3) , landfills (4) and municipal incinerators (5_),(6_),C7_) and the combustion products of askarels and transformer fluids (8). Rappe has presented a comprehensive review of the occurrence of 2,3,7,8-TCDD and related isomers (9). Increased activity in this 1n 1*5 area stimulated the' availability of pure native (AXC) and labelled (A C) PCDD and PCDF compounds for use as analytical standards. This development coincided with investigations of improved compound separation techniques and was driven by the known fact that other PCDD and PCDF compounds have significant toxicity (10),(11),(12),(13)- PCDD and PCDF compounds have been detected in Utility Askarel fluids and combusted or pyrolyzed Utility Askarel fluids (3.)(9.),(8.). The occurrence of these contaminants in dielectric fluids raises several questions. Fundamental to this work is the question, "What are the predominant isomers and their concentrations in utility fluids in equipment now in use?". Once the specific compounds and their concentrations are known the relative health risk posed by continued use of these fluids can be assessed. To this end seven samples of dielectric fluids from in-service electrical equipment and three baseline samples were analyzed for the presence of 19 different compounds or groups of isomers. The samples were analyzed using five different C-labelled internal standards. In all, 13 different standards were synthesized and used for calibration and quantitation. Fourteen different individual PCDD and PCDF compounds were quantitated along with the total concentration of tetra- through hepta-CDF and tetra-CDD congener classes. The determination of each analyte was replicated three times for each sample in order to generate an average concentration, standard deviation of the concentration, and GENP 010963 i-i 783774 p e rce n t r e la t iv e sta n d a rd d e v ia tio n ( c o e f f ic ie n t of v a r ia t io n ) . T h re e o f the ten f lu id 's a m p le s w ere s p ik e d w ith known am ounts o f in d iv id u a l com pounds as a ch e c k on th e a c c u r a c y o f t h is m etho d. F u rth e rm o re , th e same te n f l u id sa m p le s w e re s u b je c te d to an in t e r la b o r a t o r y ro un d r o b in a n a ly s is . The r e s u lt s of th a t stu d y w i l l be p u b lis h e d e ls e w h e re . T h u s, b e s id e s p r o v id in g an i n i t i a l v ie w o f PCDD and PCDF compound c o n c e n t ra t io n s in in -s e r v ic e u t i l i t y f lu id s t h is w ork re p re s e n t s th e m ost r ig o r o u s stu d y o f PCDD and PCDF com pounds in an e n v iro n m e n ta l m a t r ix . 783775 1-2 GENP 010964 S e c c i n 2 METHODS IN -S E R V IC E FLU ID SAMPLES Sam p les o f in - s e r v ic e d ie l e c t r ic f lu id s w ere c o lle c t e d by v a r io u s u t i l i t i e s and s h ip p e d to th e B a t t e lle C o lum bus L a b o r a t o r ie s . BCL w as r e s p o n s ib le f o r a c c e s s io n in g th e f lu id s and d e v e lo p in g a c a ta lo g u e of b a ckg ro u n d in fo rm a tio n . The ty p e o f e q u ip m e n t fro m w h ic h e a ch in - s e r v ic e f l u id u se d in t h is s tu d y w as o b t a in e d is lis t e d b e lo w : 1 . 19 6 2 Load C e n te r N etw ork T ra n sfo rm e r 2 . Load C e n te r N etw ork T ra n sfo rm e r 3 . S u b s ta tio n D is t r ib u t io n T ra n sfo rm e r 4 . 1946 Load C e n te r N etw ork T ra n sfo rm e r 5 . P r e c ip it a t o r 6 . 1976 A rc F u rn a ce T ra n sfo rm e r 7 . C a p a c ito r A l i s t in g o f a cc u m u la te d b a ck g ro u n d in fo r m a t io n on each o f th e se p ie c e s o f e q u ip m e n t i s g iv e n in A p p e n d ix A . I n a d d it io n t h r e e t y p ic a l v i r g i n d ie l e c t r i c f lu id s w ere b lin d ly s p ik e d to t e s t th e m ethod a c c u r a c y . T h e se f lu id s w e re : 1 . M in e ra l o il 2 . A ro c lo r 10 16 3 . A r o c lo r 1260 PCDD AND PCDF STANDARDS PCD D a n d P C D F s t a n d a r d c o m p o u n d s i n c l u d i n g 13 C - l a b e l l e d s t a n d a r d c o m p o u n d s w e r e s y n t h e s iz e d by R a d ia n C o r p ., A u s t in . The s ta n d a rd s w ere d is s o lv e d in s o lv e n t a t th e v a r io u s c o n c e n tra tio n le v e ls and sh ip p e d in s e a le d g la s s v i a l s . Seven v ia l s w ere re c e iv e d each c o n t a in in g a l l o f th e f o llo w in g com pounds: 2 . 3 . 7 . 82 . 3 . 7 . 81 . 2 . 3 . 7 . 82 . 3 . 4 . 7 . 8- TCDD TCDF PnCDF PnCDF 2 , 3 , 7 , 8- T C D D - ^ C , 5 2 . 3 . 7 . 8- T C D F - 1 3 C 1 2 1 . 2 . 3 . 7 . 8 - P n C D F -13 C 12 2 -1 010965 783776 1 ,2 , 3 , 4, 7 ,8 -H x C D F 2 , 3 r 4 , 6 , 7 , 8-H x C D F 1 , 2 , 3 , 4 , 6 , 7 , 8-H p C D F 1 , 2 , 3 , 4 , 6 , 7 , 8, 9-O C D F 1 , 2 , 3 , 4 , 7 , 8- H x C D F - 1 3 C , 2 ' ** 1 , 2 , 3 , 4, 6 ,7 ,8 , 9 -O C D D -1J C 12 Each v ia l c o n ta in e d a l l o f th e n a t iv e com pounds a t one of th e fo llo w in g c o n c e n tra tio n le v e ls : 7 .5 0 0 n g /m l 2 .5 0 0 n g /m l 500 n g /m l 15 0 n g /m l 40 n g /m l 10 n g /m l 2 .5 n g /m l lo Each s ta n d a rd s o lu t io n c o n ta in e d a l l o f the A JC -co m p o un d s a t 500 n g /m l e a c h . SAM PLE E X T R A C T IO N AND E X T R A C T P R E P A R A T IO N S am p le s o f e l e c t r i c a l in s u la t in g f lu id s and m in e r a l o i l d is s o lv e d in h e x an e w ere r e c e iv e d fro m R a d ia n C o rp . in s e a le d v i a l s . Th e v o lu m e o f a p o r t io n o f e a ch v i a l was m easu red and e x tr a c t e d by a d d in g 25 m l o f a c e to n e and m ix in g t h o r o u g h ly . H e x a n e ( 2 5 m l) w as a d d e d and th e s a m p le w a s . s t ir r e d and e x t r a c t e d f o r a m in im u m o f 1 h . To rem ove PCBs and n o n p la n a r a ro m a tic s and is o la t e one f r a c t io n c o n t a in in g a l l t e t r a - to o c ta -C D F and -CD D -th e sam p le e x t r a c t was c h ro m a to g ra p h e d on a c id a lu m in a , P X -2 1 ca rb o n and n e u t r a l a lu m in a . A preprogram m ed H a m ilto n v a lv e c o n t r o l system d e sig n e d in our la b was used to r e p r o d u c ib ly s e le c t s o lv e n t s and c h ro m a to g ra p h ic co lu m n s (J J ). The r e c o v e r y o f a l l t e t r a - to o c ta -C D F s and -C D D s ( a s v e r i f ie d w it h a v a ila b le s t a n d a r d s ) fro m an in d iv id u a l c h ro m a t o g ra p h ic co lu m n is >95%. Th e sam p le e x t r a c t w as a p p lie d to an a c t iv a t e d a c id ic a lu m in a co lu m n , fo llo w e d by 30 m l o f 3% CH2C 1 2 in h e x a n e . The CD F/CD D f r a c t io n was e lu t e d w ith 70 m l o f 50% CH2C 1 2 in h e x an e o n to a c a rb o n co lu m n fo llo w e d by 50 m l o f 10 % b e n ze n e in h e x a n e . Th e CD F/CD D f r a c t io n w as e lu t e d in th e r e v e r s e d ir e c t io n w ith 30 m l o f 50% x y le n e in h e x an e o n to a n e u t r a l a lu m in a c o lu m n ( r e p la c e s x y le n e w ith a v o l a t il e s o lv e n t ) fo llo w e d by 30 m l o f 3% CH2C 1 2 in h e x a n e . Th e f i n a l p u r if ie d CD F/CD D f r a c t io n was e lu te d w ith 70 m l o f e a s ily v o la t iliz e d 50% CH2C 1 2 in hexane and c o n c e n tra te d by h e a t in g to 10 0 C and th e n vacuum e v a p o r a t in g to 4 -1 0 p i ( 8 ) . P r io r to GC/M S, e x t r a c t s w ere sto re d in the d a rk in s e a le d 15 0 p i b o r o s ilic a t e c a p illa r y tu b e s. GENP 010966 2-2 783777 H IG H R ESO LU TIO N C A P IL L A R Y GAS CHROMATOGRAPHY/LOW R ES O LU TIO N MASS SPECTRO M ETRY(H RGC/LRM S) A l l sam p le e x t r a c t s w ere a n a ly z e d w ith a H e w le t t -P a c k a r d 5970 M ass S e le c t iv e D e te c to r. A p o rtio n o f each e x t r a c t was in je c t e d in t o th e s p l it - s p l i t l e s s in je c t io n p o r t ( 2 5 0 C ) and e lu t e d th ro u g h a 50m (0.25m m i.d . ; 0 . 3 3 p m f il m t h ic k n e s s ) H e w le t t -P a c k a r d 5% p h e n y l m e th y l s ilic o n e fu s e d s i l i c a c a p il la r y c o lu m n . The fo llo w in g te m p e ratu re program was u se d : T a b le 2 -1 H -P 5890 GAS CHROMATOGRAPH TEM PERATURE PROGRAM Level 1 2 3 4 5 In it ia l Temp (C 0 19 0 In it ia l Tim e (m in ) 1.0 0 R ate (C /M in ) F in a l Temp (C ) F in a i Tim e (m in ) 5 .0 220 16 .0 0 5 .0 23 5 7 .0 0 5 .0 250 18 .0 0 5 .0 270 7 .00 5 .0 300 24 .0 0 To tal Tim e (m in ) 2 3 .0 0 3 3 .0 0 5 4 .0 0 6 5 .0 0 9 5 .0 0 The g a s ch ro m ato g rap h was in t e r f a c e d d ir e c t ly to th e s o u rc e o f the m ass s e le c t iv e d e te c to r and the t r a n s f e r lin e and io n so u rc e w ere h e ate d to 2 7 5 C and 15 0 C , r e s p e c t iv e ly . The m ass s e le c t iv e d e te c to r was o p e ra te d in th e s e le c t iv e io n m o n ito rin g (S IM ) m ode. The mlz v a lu e s c h a r a c t e r is t ic o f n a t iv e TCDD, TCDF, PnCDF, H xCD F, OCDF and OCDD and T C D D -13 C 1 2 , T C D F -13 C 1 2 , P n C D F -1 3 C 1 2 , H x C D F -l 3 C 12 and O CD D- 13 C ^2 w ere m o n ito re d a t v a r io u s tim e s d u r in g th e 95 m in u te ru n s* Th e tim e w indow s u se d f o r m o n it o r in g th e d if f e r e n t c o n g e n e r c la s s e s (TC D D , TC D F , e t c . ) w ere d e te r m in e d b y th e p r e v io u s i n j e c t i o n o f e it h e r w indo w s t a n d a r d s (T C D D ) o r a m ix t u r e o f co m b u sted P C B . Raw d a ta w ere c o lle c t e d in t o and p ro c e s s e d b y an H -P Q u ic k s ilv e r d a ta sy ste m . The d a ta syste m was used to g e n e ra te e le c t r o n ic c o p ie s o f r e p o r t s o f r e t e n t io n tim e s and a re a s a t e a ch m /z v a lu e m o n ito re d ( F ig u r e 2 - 1 ) . A d d it io n a l custom so ftw a re was used to id e n t if y G .C . p eaks and q u a n tit a te a n a ly te l e v e l s . T h is s o f t w a r e w as w r it t e n b y NYSDOH s t a f f ' ( H . V a le n t e ) and r u n s on a n IBM PC o r X T . The H -P re p o rts a re in p u t in A S C II and t r a n s f e r r e d by u s in g a GENP 010967 2-3 783778 Ret Tine 30.827 32. IS7 34.650 35.521 35.9!7 35.521 35.757 37.141 37.784 38.322 38.844 39.113 39.552 40.088 40.506 41.446 41.982 44.896 45.282 Mass 305.90 anu Area 107155 22962 7919 44918 8558 129702 80014 508644 1S l 286 96838 38905 38741 73974 2030 220688 83751 12165 3228 11243 F ig u r e 2 - 1 : T y p i c a l MSD Raw d a t a . T h e r e t e n t i o n t im e w in d o w f o r T C D F i s sh o w n . The Q u ic k s ilv e r d a ta syste m a u t o m a t ic a lly in t e g r a t e s th e g as c h ro m a to g ra p h ic p eaks and la b e ls e ach w ith i t s r e t e n t io n tim e . Th e l i s t o f r e t e n t io n tim e s f o r each m ass m o n ito re d is g e n e ra te d by a s e p a ra te p ro g ra m . GENP 010968 2 -4 783779 c u sto m iz e d H -P P a s c a l o p e ra tin g syste m (s e n d e r) and th e, p rog ram c a lle d K e rm it ( r e c e iv e r ) . H IG H R ESO LU TIO N GAS CHRO M ATO GRAFHY/HIGH R ESO LU TIO N MASS SPECTR O M ETR Y ( H R G C/H R M S) Some e x t r a c t s w ere a n a ly z e d w it h a K r a t o s M S -50 h ig h r e s o lu t io n G C /M S /D S . I n t h is syste m , p o rt io n s o f the e x t r a c t s w ere in je c t e d in to th e o n -c o lu m n in je c t io n p o rt o f a C a r l o - E r b a 4 16 0 h ig h r e s o l u t i o n g a s c h r o m a t o g r a p h an d e lu t e d t h r o u g h a 50m (0.20m m i.d .;0 .3 3 u m f il m t h ic k n e s s ) H e w le t t -P a c k a r d 5Z m e th y l p h e n y l s il ic o n e fu se d s i l i c a c a p il la r y co lu m n . The o ven was te m p e ra tu re program m ed as f o llo w s : T a b le 2 -2 CA R LO -ER B A GAS CHROMATOGRAPH 4 16 0 TEM PERATURE PROGRAM Level 1 2 3 In it ia l Temp (C ) 19 0 In it ia l Tim e (m in ) 1.0 R ate (C /M in ) 5 .0 5 .0 5 .0 F in a l Temp (c) 220 F in a l Tim e (m in ) 16 .0 0 235 7 .0 0 300 5 0 .0 0 To tal T im e (m in ) 23 .00 33 .0 0 86.00 The g as ch ro m a to g ra p h was in t e r f a c e d to th e M S-50 th ro u g h a d ir e c t l i c e h e a te d to 2 5 0 C . The io n so u rc e te m p e ra tu re was 2 5 0 C . The h ig h r e s o lu t io n m ass s p e c tro m e te r w a s .o p e ra t e d in e it h e r o f two SIM ty p e m o d e s. I n n o rm a l m u lt ip le p e a k m o n i t o r i n g m o d e (N M PM ), t h e m/ z v a l u e s o f a p a r t i c u l a r a n a l y t e a n d i t s c o rre s p o n d in g in t e r n a l s ta n d a rd w ere o b se rv e d a t 10 ,0 0 0 RP (10 X v a lle y ) (10 .) by in t e g r a t in g a l l th e s ig n a l d e t e c t e d w it h in 50 ppm o f t h e ir e x a c t m ass v a lu e s . I n h ig h r e s o l u t i o n m u l t i p l e p e a k m o n it o r in g m ode (HRMFM) t h e m a ss s p e c t r o m e t e r i s o p e ra te d a t th e same h ig h r e s o lv in g pow er b u t th e a n a ly t e and in t e r n a l s ta n d a rd e x act m asses are p r o f ile d . The d ata system a d ju s ts the a c c e le r a t in g and e le c t r ic s e c to r v o lt a g e s to fo c u s a m ass s l i g h t l y b e lo w th e m ass o f in t e r e s t . The co m p u te r th e n t r ig g e r s a 0 .3 s a n a lo g s c a n th ro u g h a 3 0 0 ppm s e c t io n o f th e m ass ra n g e w h ich in c lu d e s th e e x a c t m ass o f th e io n o f in t e r e s t ( 1 1 ) . In b o th io n m o n ito rin g m odes, the raw d a ta w ere a c q u ire d and p ro c e sse d by th e K ra to s D S -5 5 d a ta sy ste m . Q u a n t it a t io n was done m a n u a lly by c a lc u la t in g c o n c e n tra tio n s u s in g peak a re a s o b ta in e d from th e D S -5 5 o u tp u t. GENP 010969 2-5 783780 S e c tio n 3 D ISC U SSIO N PR ELIM IN A R Y B A S E LIN E STUDY DATA In an i n i t i a l p h ase of t h is p r o je c t , f iv e f lu id s w ere a n a ly z e d . T h e se f iv e f lu id s in c lu d e d th e A r o c lo r s 10 16 , 12 4 2 , 12 6 0 , t r i - and te tra c h lo ro b e n z e n e s , and m in e ra l o i l . Each of th e se f lu id s was s p ik e d w ith fo u r 13 C -la b e lle d in t e r n a l s ta n d a rd s , i . e . 2 , 3 , 7 , 8- T C D F - 1 3 C 1 2 , 1 , 2 , 3 , 7 , 8- P n C D F - 1 3 C 1 2 , 2 , 3 , 7 , 8- T C D D - 1 3 C 1 2 , 0 C D D -1 3 C 1 2 . N a t iv e 1 , 2 , 3 , 4 , 7 , 8-H x C D F a n d 1 , 2 , 3 , 4 , 6 , 7 , 8-H p C D F w e r e a l s o a d d e d s i n c e t h e R e la b e lle d v e r s io n o f th e s e com pounds w as n o t a v a ila b le a t th e tim e th e sa m p le s w ere p re p a re d . A d d it io n a lly , one o f th e f lu id s , aged m in e ra l o il , was s p ik e d w ith " b l in d " l e v e l s o f c e r t a i n n a t iv e PCD D a n d P C D F co m p o u n d s a n d a p p r o x im a t e ly 50 0 ppm each of A ro c lo r 126 0 and t r i - and te tra c h lo ro b e n z e n e and 100 ppb each of 1 , 2 , 3 , 4 , 7 , 9 - H x C D F , 2 , 3 , 4 , 7 , 8- P n C D F , 1 , 2 , 3 , 4 , 8- P n C D F a n d 2 , 3 , 4 , 8- T C D F . T h e r e s u l t s of th e se p r e lim in a r y a n a ly s e s a re g iv e n in A p p e n d ix D T a b le s D -l and D -2 . The le v e ls o f PCDD and PCDF com pounds added as a b lin d s p ik e to the aged m in e r a l o i l sam p le w ere q u a n t it a t e d w it h r e a s o n a b le a c c u r a c y . T h re e o f th e f o u r com pounds s p ik e d w ere th e same com pounds a s th e in t e r n a l s ta n d a rd s u se d f o r q u a n t it a t io n . 2 . 3 . 7 . 8- T C D D , 2 , 3 , 7 , 8- T C D F a n d 1 , 2 , 3 , 7 , 8- P n C D F w e r e q u a n t i t a t e d b y c o m p a r in g t h e a r e a s f o r t h e s e i s o m e r s w i t h 2 , 3 , 7 , 8- T C D D - R ^ 2 , 2 , 3 , 7 , 8- T C D F - R C | 2 , a n d 1 . 2 . 3 . 7 . 8- P n C D F - J C | 2 r e s p e c t i v e l y . T h e p e r c e n t a g e e r r o r i n t h e d e t e r m i n a t i o n o f th e le v e ls o f th e se n a t iv e is o m e rs w as 7 . 5 Z , 7QZ and 0Z r e s p e c t iv e ly . A lth o u g h t h e l e v e l o f 1 , 2 , 3 , 7 , 8-P n C D F i s r e p o r t e d a s 1 4 0 p p b , t h i s i n c l u d e s 1 0 0 p p b o f 1 . 2 . 3 . 4 . 8- P n C D F , w h i c h h a d b e e n a d d e d . T h e s e tw o i s o m e r s w e r e n o t s e p a r a t e d o n th e S P 23 3 0 fu s e d s i l i c a c a p il l a r y co lu m n w it h th e c o n d it io n s t h a t w e re u s e d . 2 . 3 . 4 . 8- TCD F w as added to th e m in e r a l o i l sam p le b u t w as n o t s p e c i f i c a l l y d e te rm in e d . I t was d e te c te d as p a rt o f th e t o t a l TCDF th a t was re p o rte d ( T o t a l TC D Fa 12 0 p p b ). Th us th e p e rce n ta g e e r r o r in th e d e te rm in a tio n o f T o t a l T C D F ( 2 , 3 , 7 , 8 - a n d 2 , 3 , 4 , 8- T C D F ) i s 2 1 Z . T h e d e t e r m i n a t i o n o f 1 , 2 , 3 , 4 , 7 , 8-H x C D F (a s T o t a l HxCDF) is g ro s s ly in e r r o r . The t o t a l HxCDF added was 220 ppb and 110 0 ppb w as d e t e c t e d . T h e H xCDF a d d e d b y t h e a d d it io n o f 500 ppm o f A r o c lo r 12 6 0 and th e c h lo ro b e n z e n e s is n e g lib le and does n o t a cc o u n t f o r t h is d is c r e p a n c y . The G E N F 010970 3-1 783781 e x act cau se o f t h is e r r o r is unknown but s im ila r e rro rs have been re co rd e d w he n e ve r a com pound is d e te rm in e d u s in g a h 'I n t e r n a l s ta n d a rd n o t o f th e same co n g e n e r c la s s . T o t a l PnCDF and t o t a l HpCDF w ere no t d e te rm in e d and OCDF was in a d v e r t e n t ly d e s tro y e d in th e c le a n -u p . The s p ik e d N u jo l w h ic h re p r e s e n t s a p u r if ie d a lip h a t ic h y d ro ca rb o n m a trix was p re p a re d in t h is la b o ra to ry and p ro v id e s an a d d it io n a l ch e ck on the a c c u ra c y o f th e d e te r m in a tio n s . A l l o f the compounds added to the aged m in e r a l o il w ere added to th e N u jo l. In term s o f a c c u ra c y the r e s u lt s w ere s im ila r . In a l l c a se s e x ce p t t o t a l HxCDF, the a b s o lu te v a lu e o f the r a t io o f the d if f e r e n c e betw een th e s p ik e d le v e l and th e m easu red le v e l was .9 0 o r le s s . As b e fo re t o t a l PnCDF and OCDF le v e ls w ere no t d e te rm in e d . The r e s u lt s q u o te d f o r th e A r o c lo r and c h lo ro b e n z e n e m ix tu re s a g re e w ith la t e r d e te rm in a tio n s of th e se m ix tu re s (se e s e c t io n on In -s e r v ic e liq u id s ) . In g e n e ra l th e d e t e c t io n lim it s f o r a l l th e s e r e s u lt s w ere <1 p p b . The d a ta in th e se p r e lim in a r y e x p e rim e n ts w ere a c q u ire d a t h ig h r e s o lu t io n u s in g t h e HRMPM m ode o f io n m o n i t o r i n g . T y p i c a l d a t a a r e sh o w n i n F i g u r e s 3 - 1 a n d 3 - 2 . The a re a o f th e m ass peak shown in F ig u r e 3 -2 is used to c a lc u la t e th e am ount of 2 , 3 , 7 , 8- T C D D . A l t h o u g h l e a s t s u s c e p t i b l e t o i n t e r f e r e n c e s , t h i s i n s t r u m e n t a l te c h n iq u e a d d re sse s th e la r g e s t amount o f d a ta and is le a s t a u to m a te d . C A L IB R A T IO N AND Q U A L IT Y ASSU RAN CE DATA A c a lib r a t io n c u rv e f o r e ach o f e ig h t o f th e a n a ly te s o f in t e r e s t was c o n s tru c te d fro m HRGC/LRM S d a t a . Th e c u r v e f o r 2 ,3 ,7 ,8 -T C D D i s show n in A p p e n d ix B F ig u r e 1 . The lo g o f th e r a t io o f th e a re a o f n a t iv e 2 ,3 ,7 ,8 -T C D D to ^ C - la b e lle d TCDD is p lo t t e d v e r s u s th e lo g o f the c o n c e n t r a t io n o f n a t iv e 2 ,3 ,7 ,8 -T C D D . S e v e n s o lu t io n s c o n t a in in g c o n c e n t r a t io n s o f n a t iv e 2 ,3 ,7 ,8 -T C D D o f 2 . 5 , 1 0 , AO, 15 0 , 500, 2500 and 7500 p g /p l and a co n sta n t ^ C -la b e lle d 2 ,3 ,7 ,8 -T C D D c o n c e n t r a t io n o f 500 p g /u l w ere u sed to c o n s tr u c t t h is c u r v e . The same c o n c e n tra tio n s o f th e o th e r a n a ly t e s and in t e r n a l s ta n d a rd s w ere u se d in o th e r c u rv e s (A p p e n d ix B F ig u r e s 2 - 8 ) . T h e se c u rv e s d e m o n stra te a lin e a r it y o f re s p o n s e o f a lm o st f o u r o rd e rs o f m a g n itu d e . The p ercen tag e f i t ("10 0 x ( c o r r e la t io n c o e ff ic ie n t ) ^ ) o f the le a s t sq u are s lin e in e a ch o f th e c u rv e s c o n s t r u c t e d w it h lo w r e s o lu t io n m ass s p e c t r o m e tr y d a t a i s : 783782 3-2 GENP 010971 2 . 3 . 7 . 8 - TCDD - 9 9 .7 3 9 8 ~2', 3 , 7 , 8- T C D F - 9 9 . 7 6 1 4 1 . 2 . 3 . 7 . 8- PnCDF - 9 9 .6 3 5 7 2 . 3 . 4 . 7 . 8 - PnCD F - 9 9 .0 4 8 3 1 . 2 . 3 . 4 . 7 . 8 - HxCDF - 9 9 .7 2 6 2 2 . 3 . 4 . 6 . 7 . 8- H xCD F - 9 9 .3 7 5 6 1 . 2 . 3 . 4 . 6 . 7 . 8 - HpCDF - 9 9 .5 8 4 8 OCDF - 9 9 .76 8 5 DS55 HI GH RESOLUTI ON HPH RUNNflHE 45344 DATE 2/ 6/85 TIHE 22i38 RETN TI HE e e i 3 e eiise 82i3e 3i3e 94130 1 1 Ifr------- -------- -------- 1---------------- -------- -- ---------- 1-------- -------- ------- -- --1 TIC 3 1 9 .8 9 6 4 i\ 190V.-31226768 HI NATIVE 2378-TCOQ A N2 .. 3^33.9335 ANATIVE 2378-TCDO H3 Cl3-LABELLED 2378-TCDO A.i i 1 i 1 " 1 -- n -- " i - -- 41 91 121 161 F ig u r e 3 - 1 : E x a c t M ass C h ro m a to g ra m s sh o w in g th e r e t e n t io n t im e w indow f o r TC D D . T h r e e 10 0 mmu. m a ss w in d o w s w e re m o n it o r e d o n e e a c h c e n t e r e d on m /z 3 2 1 .8 9 3 6 and m /z 3 19 .8 9 6 5 r e s p e c t iv e ly f o r n a t iv e TCDD and one c e n te re d on m /z 3 3 3 .9 3 3 8 f o r i J C n - U b e l l e d - 2 3 7 8- T C D D . GENP 010972 3-3 783783 I g s s t mich csouu'f iqm m m PCB* SUBNOTION FORT UMM0MC S3* |BTC */ S'S MSS 319.0046 us: mi- 13.30SCMCNS sSrei7aBneMnto8RMQRU0H0C0S 103.30-00108 0 1.4UU o r 7.2 TOT SASCU1MC SflM S3 ITCH 0RCH TtHC 22:30 J10.JV"Ji F ig u r e 3 - 2 : H ig h R e s o l u t io n Maas S c a n s & S u n n n a rie s. T h is f i g u r e shows th e sum of the m ass s c a n s w h ic h w ere ta k e n d u r in g th e .e lu t io n o f th e 23 78 -T C D D p eaks shown in F ig u re 3 - 1 . GENP 0 1 0 9 7 3 3-4 783784 Each _curve a ls o shows the 99% c o n fid e n c e in t e r v a l f o r the slo p e and in t e r c e p t of the le a s t sq u a re s r e g r e s s io n l i n e . T h e se c a l c u la t io n s w ere based on e q u a tio n s g iv e n in a r e c e n t ly p u b lis h e d book on s t a t i s t i c s fo r c h e m is t s ( 1 2 ). C a lib r a t io n c u rv e s d e riv e d from h ig h r e s o lu t io n m ass s p e c t ro m e tr ic data(HRGC/HRM S) f o r some o f th e a n a ly t e s a re drawn in A p p e n d ix B F ig u r e s 9 - 1 3 . Th e c u r v e s w ere c o n s t r u c t e d u s in g th e same s o lu t io n s as d e s c r ib e d a b o v e . T h e y a ls o d e m o n s tra te l in e a r it y o ver a p p ro x im a te ly fo u r o rd e rs of m agn itud e. The percentage f i t of the le a s t squares lin e in each of the curves co n stru cted w ith h ig h r e s o lu tio n mass sp ectro m etry d ata i s : 2 . 3 . 7 . 8 - TCDD - 9 9 .539 5 2 . 3 . 7 . 8 - TCDF - 99.4736 1 . 2 . 3 . 7 . 8 - PnCDF - 97.0347 1 . 2 . 3 . 4 . 7 . 8 - HxCDF - 99.949 1 . 2 . 3 . 4 . 6 . 7 . 8 - RpCDF - 9 9 .1 0 1 2 Response fa c t o rs were c a lc u la te d d a ily from d ata c o lle c t e d from the in je c t io n of a p o rtio n of one of the stan d ard s o lu t io n s . The re sp o n se f a c t o r s (RF) a re used in the f o llo w in g e q u a tio n to c a lc u la t e th e c o n c e n t r a t io n of a n a ly t e in the sam p le: CA " ( A N/ A I S ) ( R F ) ( C j S ) (3 -1) w here, CA * c o n c e n t r a t i o n o f a n a l y t e i n t h e s a m p le C^s 3 c o n c e n tra tio n of in t e r n a l sta n d a rd added to the sam ple b e fo re the c le a n -u p p ro ce d u re 3 a re a counts of the s ig n a l due to the a n a ly te A-is 3 A re a co un ts of th e s ig n a l due to th e . in t e r n a l sta n d a rd RF - Response f a c t o r - ? I S / S N SIS 3 A rea counts per u n it mass of the 3 Area counts per u n it mass of the in te rn a l standard n a t iv e compound Id e a lly , fo r g re a te st p r e c is io n , the resp o nse fa c t o rs should not v a ry from a n a ly s is to a n a ly s is . The d a y -to -d a y v a r ia t io n in response fa c to rC e x p re sse d as a re a r a t io ) can be d e m o n strate d by co m p arin g the d a i l y a re a r a t io s to the a re a r a t io s generated fo r the c a lib r a t io n c u rv e . In A ppendix C F ig u re 14 , the area r a t i o s f o r t h e 2 , 3 , 7 , 8- T C D D , i n t h e s t a n d a r d i n j e c t i o n a n a l y z e d o n e a c h d a y o f t h e p r o je c t a re p lo tte d on the c a lib r a t io n c u rv e . The a re a r a t io s fo r o th e r a n a ly te s a r e p l o t t e d i n F i g u r e s 1 5 t h r o u g h 2 1 A p p e n d i x C . F o r 2 , 3 , 7 , 8- T C D D o n l y o n e a r e a r a t i o f e l l o u t s id e th e 99% c o n f id e n c e i n t e r v a l . S in c e no c r i t e r i a had been 783785 s p e c i f i c a l l y e s t a b l i s h e d , t h i s a r e a r a t i o (and c o n s e q u e n tly , re s p o n se f a c t o r ) was a d ju s t e d b ased upon the a v e ra g e o f a re a r a t io s g e n e ra te d by in je c t io n s on the p re v io u s and fo llo w in g d ays. E rra n t r a t io s o b ta in e d fo r o th e r a n a ly te s were t r e a te d in t h is same f a s h io n . T h is was c o n s id e re d a c c e p t a b le b ecau se i t was observed th at the area r a t io s flu c t u a t e d o n ly r a r e ly and rand om ly. These la rg e a p p a re n tly random changes in the a re a r a t io s c o n s tit u t e d the g r e a t e s t f a c t o r r e s p o n s ib le fo r any la r g e r e l a t i v e s ta n d a rd d e v ia t io n s in the sam ple d e te r m in a t io n s . At the tim e of a n a ly s is no m echanism had been e s t a b lis h e d to e f f i c i e n t l y ch e ck th e d a i l y re sp o n se f a c t o r s of so many a n a ly t e s . In f u t u r e w ork, d a il y ch e cks o f the re sp o n se f a c t o r s c o u ld be made by in c o r p o r a t in g more c u r r e n t ly a v a ila b le autom ation in to the m ethodology. In stru m e n t m a n u fa ctu re rs are c o n t in u a l l y d e v e lo p in g s o ftw a re and one c o u ld e x p e c t th a t among th e d e v e lo p m e n ts w o u l d b e a n e f f i c i e n t a n d r e l i a b l e c h e c k o f t h e r e s p o n s e f a c t o r s . We c a n e n v i s i o n a r e l a t iv e ly easy method of c h e c k in g the resp o n se f a c t o r s a g a in s t the c a lib r a t io n cu rve u s in g the r e l a t i v e ly g e n e ra l and w id e ly a v a ila b le L o tu s 1 - 2 - 3 so ftw a re p a c k a g e . When m any r e s p o n s e f a c t o r s c a n be e a s i l y c h e c k e d , a c r i t e r i a f o r th e r e p r o d u c ib ilit y of re sp o n se f a c t o r s c o u ld be in c lu d e d as an a n a l y t ic a l s p e c i f i c a t i o n b e f o r e a n a l y s i s com m ences. L i k e l y c r i t e r i a c o u ld be t y p i c a l c o n fid e n c e in t e r v a ls such as the 90, 95 o r 99Z c o n fid e n c e in t e r v a l o r a p e rce n ta g e of the average response fa c to r c a lc u la te d from the responses generated d u rin g the c o n stru c tio n of the c a lib r a t io n curve. A n a l y s is of b la n k sam p le s i s a f i n a l q u a l i t y a s s u r a n c e p o in t r e q u ir in g some d is c u s s io n . A la b o r a t o r y b la n k was g e n e ra te d by c a r r y in g a sam ple of the s o lv e n ts used in the a n a ly s is thro ug h the c le a n -u p p ro c e d u re . T h is sam ple was s p ik e d w ith the a p p ro p ria te in t e r n a l stan d a rd s. The in t e rn a l stan d a rd s were d etected w ith re a s o n a b le r e c o v e r ie s . None o f the a n a ly t e s of in t e r e s t w ere d e te c te d . The th re e b a s e lin e f lu id s , w h ich c o n s tit u t e d th re e of the ten sam ples in the p r o je c t , were a n a ly z e d to q u a n t if y any of th e PCDD and PCDF a n a ly t e s w h ic h m ig h t be p re se n t b e fo re th e b lin d s p ik in g le v e l s w ere added. None of th e a n a ly t e s were d e te c te d in the m in e ra l o il o r A ro c lo r 10 16 . However, the re c o v e ry of in t e r n a l stan dards s p ik e d in t o th e s e sa m p le s w as lo w . Some o f t h e a n a ly t e s w ere d e te c t e d i n A r o c lo r 12 6 0 . The r e s u lt s of th ese a n a ly se s a re ta b u la te d in A p pend ix D T a b le s 3 , 4 and 5 and a re u se d in c o n ju n c t io n w it h the r e s u l t s o f the s p ik e d B a s e lin e f l u i d s to determ ine the a ccu ra cy of the a n a ly s is . BA SELIN E DATA The a c t u a l p r o je c t c o n s is te d of the a n a ly s is of ten sam ples o f v a r io u s u t i l i t y f l u i d s . Of th e s e te n s a m p le s , s i x w ere d i e l e c t r i c f l u i d s ta k e n fro m i n - s e r v i c e 3 -6 783786 e l e c t r ic a l equipm ent. Th ree of the sam p les-w ere m in e ra l o i l , A r o c lo r 1 D 1 6 , and A r o c lo r 1260 th a t were sp ik e d b e fo re a n a ly s is w ith both the in t e r n a l sta n d a rd s and b lin d le v e ls of v a r io u s n a t iv e PCDD-PCDF a n a ly t e s . The r e s u lt s of th ese a n a ly z e s are ta b u la te d in A ppendix D T a b le s 3 -5 . The average p ercen t e rro r is 2 8 .9 % . In two c a s e s , an a n a l y t e was m e a s u re d w h e re no ne h ad b een s p i k e d . I n t h e s p i k e d A r o c l o r 1 0 1 6 , 1 , 2 , 3 , 4 , 7 , 8- H x C D F w a s d e t e c t e d e v e n t h o u g h i t h a d n o t been s p ik e d . In sp ik e d A r o c lo r 126 0 , 1 , 2 , 3 , 7 , 8, 9-HxCDF was d e te cte d but not sp ik e d . I t is im p o ss ib le to d eterm ine w ith these r e s u lt s i f th e se compounds were p resen t in the u n sp ik e d f lu id s s in c e the d e te c tio n lim it in the u n sp iked f lu id s is g re a te r than the le v e l detected in the sp iked f lu id s . Th e V e r r o r f o r the d e t e r m in a t io n of OCDF in th e s p ik e d A r o c lo r 12 6 0 was n o t in c lu d e d in th e 2 8 .9 % a v e r a g e p e r c e n t e r r o r c a l c u l a t i o n . Th e s p ik e l e v e l was so low r e l a t i v e to the n a t iv e amount o f OCDF in A r o c lo r 12 6 0 t h a t the c o m p a riso n of the sp ik e d to m easured le v e l was not a re a s o n a b le in d ic a t io n of a c c u r a c y . The % e r r o r f ig u r e was e lim in a t e d from the a v e rag e based upon the s i m i l a r i t y in m agnitude of the sp ik e d le v e l and the sta n d a rd d e v ia t io n fo r the t r i p l i c a t e d e t e r m in a t io n (com pare the OCDF v a lu e s in T a b le D - 1 5 ) . The average % e rro r in each m a trix is lis t e d at the bottom of T a b le s D -l, D -2 and D - 3 . The % e r r o r in the OCDF d e t e r m in a t io n in A r o c lo r 12 6 0 ( T a b le D -3 ) was not in c lu d e d in the lo c a l average as e x p la in e d above. The la rg e average % e rro r in the case of A r o c lo r 126 0 i s due to the la r g e e r r o r in the d e te r m in a tio n of 2 , 3 , 4 , 6 , 7 , 8- H x C D F a n d 1 , 2 , 3 , 4 , 6 , 7 , 8- H p C D F . T h i s l a r g e e r r o r c o u l d b e a c c o u n t e d f o r by a low r e c o v e r y o f th e se compounds in th e b la n k A r o c lo r 12 6 0 . R e c o v e ry , as m easured in t h is re p o rt , r e f e r s to the re c o v e ry of the in t e r n a l sta n d a rd used to q u a n tita te a p a r t ic u la r a n a ly te and not to the re co v e ry of the a c tu a l a n a ly te . F o r t h e s e two c o m p o u n d s t h e i n t e r n a l s t a n d a r d w a s 1 , 2 , 3 , 4 , 7 , 8- H x C D F . T h i s i s o m e r may be d if f e r e n t enough th a t s l i g h t d if f e r e n c e s in th e p e rfo rm a n c e of the chro m ato g rap h ic system w i l l cause la rg e d if f e r e n c e s in re c o v e ry . T h is phenomenon may a cco u n t f o r many of the la r g e r e l a t i v e s ta n d a rd d e v ia t io n s o b se rv e d in t h is study. IN -S E R V IC E LIQUIDS The re s u lts of the t r ip lic a t e a n a ly s is of each of the seven in -s e r v ic e d ie le c t r ic f l u i d s a r e t a b u la t e d i n A p p e n d ix D, T a b l e s D-6 to D - 1 5 . T h e s e r e s u l t s a re 783787 g r a p h i c a l l y d is p la y e d in A p p e n d ix E, F ig u r e s 1 th ro u g h 1 0 . I n e a ch t a b le the r e s u lt fo r each r e p lic a t e fo r each a n a ly te is lis t e d alo ng w ith the average of the th ree r e p lic a t e s , the stan d ard d e v ia tio n and the percent r e la t iv e stan d ard d e v ia t io n ( c o e f f ic ie n t of v a r ia t io n ) . A n a ly te s th at were not d e te cte d a re in d ic a te d by the d e te c tio n lim it e n clo se d in p a re n th e se s. In a l l ten sam ples, the c o n c e n t r a t i o n s r a n g e f r o m 0 . 1 5 p p b o f 2 , 3 , 7 , 8- T C D F i n t h e S u b s t a t i o n D i s t r i b u t i o n T r a n s f o r m e r (A p p e n d ix D, T a b l e D - l l ) to 3 9 ,6 0 0 ppb o f OCDF i n th e Lo a d C e n t e r Netw ork T ra n s fo rm e r (A p p e n d ix D, T a b le D -6 ) . F o r a l l th e a n a ly t e s in a l l the sam ples, o n ly 15 averag e v a lu e s (o ut of 18 0 ) had p e rce n t r e la t iv e stan d a rd d e v ia t io n s o ve r 3 0 2 . None o f th e se a n a ly s e s in v o lv e d the m ore t o x ic compounds n a m e l y 2 , 3 , 7 , 8 - T C D D o r 2 , 3 , 7 , 8- T C D F . T h e l a r g e s t r e l a t i v e s t a n d a r d d e v i a t i o n s (RSD) o ccu rre d in the a n a ly s is f o r T o t a l TCDF o r an iso m er in one of the h ig h e r c h lo r i n a t e d c o n g e n e r c l a s s e s . Some had p e r c e n t r e l a t i v e s t a n d a r d d e v ia t io n s as low as 1 % . S ix o f the a n a ly z e s w it h g r e a t e r th a n 30% RSD w ere d e t e r m in a t io n s o f t o t a l co n g e n e r c o n c e n t r a t io n s , two w ere d e t e r m in a t io n s of c l o s e l y e l u t in g PnCDF is o m e r s and s e v e n w ere d e t e r m i n a t io n s q u a n t i t a t e d a t < 10 p p b . The d e te c t io n lim it s and r e c o v e r ie s are ta b u la te d in A p p end ix F T a b le 1 th ro ug h 1 0 . T a b l e s 1 1 t h r o u g h 2 0 l i s t % r e c o v e r y v e r s u s 2 , 3 , 7 , 8- T C D D - ^ C ^ 2 * T b e d e t e c t i o n l i m i t s w e re c a l c u l a t e d b a s e d u p o n a b a c k g r o u n d r e s p o n s e o f 1000 c o u n t s in the d ilu t e d sam ple e x t r a c t . Thus the fo llo w in g e q u a tio n was u se d : D e te ctio n L im it (1000/A ^g) (RF) (10 0 ) (3 -2) w here, A jg - area co unts due to the in t e r n a l stan d ard RF s iS^ N * re sp o n se f a c t o r S ig - are a co unts per u n it mass of in t e r n a l stan dard * a re a co u n ts per u n it mass of n a t iv e compound A b ackg ro u n d a re a co u n t o f 1000 was ch o se n b e ca u se t h is s iz e peak was t y p ic a l f o r G .C . peaks w hich are ju s t d is t in g u is h a b le from background by the d e te c to r and com puter com bined. S m a lle r n o is e peaks a re d is t in g u is h a b le in the d e te c to r o utp u t but th ese are not g e n e r a lly in t e g ra t e d by the com puter and it s in t e g r a t io n a lg o rith m . Furtherm ore 1000 counts is a c o n se rv a tiv e g e n e ra l e stim a te of the area n o is e o bserved ju s t p r io r to the e lu t io n o f the in t e r n a l sta n d a rd s based upon the 2 .5 pg/m l stan dard s o lu tio n used fo r the c a lib r a t io n c u rv e s . G E N P 010977 3-8 783788 Some a p p a r e n t d i s c r e p a n c i e s may a r i s e w hen c o m p a rin g t h e s e d e t e c t i o n l i m i t v a l u e s w i t h t h e r e s u l t s ( a n d d e t e c t i o n l i m i t s ) l i s t e d i n A p p e n d ix D T a b l e s D-6 t h r o u g h D - 1 5 . H o w e v e r, t h e b a s i s f o r an y p a r t i c u l a r c a l c u l a t i o n i n t h e s e two g r o u p s o f t a b le s may be d if f e r e n t . The d e t e c t io n l i m i t c a l c u la t io n s in A p p e n d ix F T a b le s 1 t h r o u g h 10 a r e b a s e d on th e p r e v io u s e q u a t io n f o r d e t e c t i o n l i m i t s w h ic h i n c l u d e s the a re a co unt o b se rv e d f o r each in t e r n a l s ta n d a rd in a d ilu t e d e x t r a c t . Most of th e sa m p le e x t r a c t s w e re d i l u t e d b e c a u s e m ost c o n t a in e d one o r two a n a l y t e s w h ic h w ere h ig h ly c o n c e n tra te d r e l a t i v e to th e o th e r a n a ly t e s . Many o f the v a lu e s lis t e d in A ppendix D T a b le s A thro ugh 13 were g e n e ra te d from d ata c o lle c t e d on the co n cen trated e x tra c ts befo re they were d ilu t e d . In one sam ple, sp ik e d A r o c lo r 12 6 0 , the r e p lic a t e a n a ly s is of 2378-TCD D g en erated the fo llo w in g v a lu e s : 6 1 .7 , 6 5 .5 and 3 2 .0 ppb. A p o s s ib le reason fo r the d i s c r e p a n c y b etw een t h e f i r s t two d e t e r m i n a t io n s and th e t h i r d i s an i n t e r f e r e n c e i n t h e 2 , 3 , 7 , 8- T C D D - ^ C j^2 i n t e r n a l s t a n d a r d p e a k e l u t i n g a t 4 2 . 1 9 4 m i n u t e s ( s e e f ig u r e 3 - 3 ) . To c h e c k f o r th e o c c u rre n c e o f an in t e r f r a n t and g e n e ra te th e most a c c u ra te q u a n tit a tio n , the t h ir d e x tr a c t was re a n a ly z e d by h ig h r e s o lu t io n G .C ./h ig h r e s o lu t io n M .S.C see F ig u re 3 -4 ) . d u r i n g t h e e l u t i o n o f t h e 2 , 3 , 7 , 8- T C D D - 1A3 The mass p r o f ile of the scans a cq u ire d in t e r n a l s ta n d a rd shows the p re se n ce . of an in t e r f r a n t (se e F ig u r e 3 - 5 ) . The s m a ll peak in the p r o f il e was used w ith a t y p ic a l re sp o n se f a c t o r to q u a n t it a t e 2 ,3 ,7 ,8 -T C D D a t a le v e l o f 106 ppb. GENP 010978 3-5 783789 Ion 322 . 0 0 amu. i 2 00 SPIKED 1000 AROCHLOR BQQ 1 2 0 0 2379-TCQO * EGG 40&1 2GG1 Q J< 36 38 4G 4 2 4-4 46 Ti me fir,in. ' 30" INTERNAL STANDARD 2000 INTERFERENCE C 1000- 0-* 36 _ _ /V_ _ J <i 36 40 42 Time f min. 1 - J __ f 44 46 F ig u re 3 -3 . In t e r fe r e n c e in the d e te rm in a tio n of 2378-TCD D in sp ik e d A r o c lo r 126 0 . The in t e r n a l sta n d a rd G .C . peak appears to have a c o e lu t in g in t e r fe r e n c e w h ich w ould cau se the d e te r m in a tio n of 2 , 3 , 7 , 8-TCDD to be coo low . GENP 010979 3-10 783790 00:30 R E T E N T I O N T I M E ( m i n u t e s = s e c o n d s ) 03:30 06:30 09:30 12:30 Fig u re 3 -4 . H ig h r e s o lu t io n Mass Chrom atogram of s p ik e d A r o c lo r 12 6 0 . N o tic e the same p a t t e r n o f G .C . p e a k s as in th e low r e s o l u t io n d a t a . T h is a llo w s , in a sense, the a n a ly s is of is o b a r ic components of the in t e r n a l stan d a rd G .C . peak in the low r e s o lu t io n m ass d a ta . ENP 010980 3 -11 783791 MASS 3 2 1 . 8 9 3 5 SWEEP 3000 (PPM) SCANTIME 0 .3 (SECS) SCANS 1 1 6 -1 2 3 100% IN TEN SITY 91565 MASS 3 3 3 .9 3 3 5 SWEEP 300 (PPM) SCANTIME 0 .3 (SECS) SCANS 1 1 6 -1 2 3 100% IN TEN SITY 28 18 79 F ig u r e 3 - 5 . H ig h r e s o l u t i o n m ass d a ta fr;om th e 2 ,3 , 7 ,8 - T C D D G .C . p e a k fro m the s p ik e d A r o c lo r sa m p le . The mass scan of the in t e r n a l sta n d a rd io n shows a c o e lu t in g in t e r f e r e n c e r e s p o n s ib le f o r th e low d e t e r m in a t io n o f 2 ,3 ,7 ,8 -T C D D . GENP 010981 783792 Section 4 REFERENCES G ^ 0' 09 783793 1. P . W . O ' K e e f e ; C . M e y e r ; D . R . H i l k e r ; K . A l d o u s ; B . J e l u s - T y r o r ; K . D i l l o n ; "R. D o n n e l l y ; E . H o r n a n d R . S l o a n . ' " " A n a l y s i s o f 2 , 3 , 7 , 8 - T e t r a c h l o r o d i b e n z o -p -d io x in in G reat Lakes F is h ." Chemosphere 1 2 , 19 8 3 , p. 325 2 . P.W . A lb ro and et a l . "M ethods f o r the Q u a n t it a t iv e D e te rm in a tio n of M u lt ip le , S p e c if ic P o ly c h lo rin a te d D ib e n z o -p -d io x in and D ib e n zo fu ra n Isom ers in Human A d ip o s e T is s u e i n th e P a r t s - p e r - T r i l l i o n R a n g e ." A n a l y t i c a l C h e m is try . November 19 85, p. 2 7 1 7 . 3 . J . J . R yan; B. P . -Y . Lau ; J .A . H a rd y; W .B. S to n e ; P . O 'K e e fe and J . F . G ie r t h y . " 2 , 3 , 7 , 8 -T e t ra c h lo r o d ib e n z o -p -d io x in and R e la te d D io x in s and Fu rans in Snapping T u r t le ( C h e ly d ra S e r p e n t in a ) T is s u e s from the Upper S t . Law rence R iv e r." Chem osphere. V o l. 15 1986, p. 537. 4. R.M . S m ith ; P.W . O 'K e e fe ; K.M . A ld o u s ; D .R . H ilk e r and J . E . O 'B rie n . " 2 , 3 , 7 , 8 -T e t r a c h lo r o d ib e n z o -p -d io x in in Sedim ent Sam ples from Love C a n a l Storm Sew ers and C r e e k s ." E n v ir o n . S c i, T e c h . . V o l. 17 19 8 3, p, 6. 5. K . O lie ; P .L . Verm eulen and 0 . H u t z in g e r . " C h lo r o d ib e n z o -p -d io x in s and C h lo r o d ib e n z o f u r a n s A re T r a c e Com ponents o f F l y Ash and F l u e Gas o f Some M u n ic ip a l In c in e ra t o rs in the N e th e rla n d s." Chem osphere. V o l. 6 19 7 7, p. 455 . 6 . H .R . B u se r; H -P . B o ssh a rd t and C. Rappe. "F o rm a tio n of P o ly c h lo r in a t e d D ib e n zo fu ra n s XPCDFs) from the P y r o ly s is of P C B s." Chem osphere. V o l. 7 19 78, p. 109. 7 . H .R . B u se r; H -P . B o ssh ard t; C . Rappe and R. L in d a h l. " Id e n t if ic a t io n of P o ly c h lo r in a t e d D ib e n z o fu ra n Iso m e rs in F ly Ash and PCB P y r o ly s e s ." Chem osphere. V o l. 7 19 78, p. 4 19 . 8. R.M . S m ith ; P.W . O 'K e e fe ; D .R . H il k e r ; B. J e lu s -T y r o r and K.M . A ld o u s . " A n a ly s is fo r 2 , 3 , 7 , 8 -T e tra c h lo ro d ib e n z o fu ra n and 2 , 3 , 7 , 8 -T e t r a c h lo r o d i b e n z o -p -d io x in in a Soot Sam ple from a T ran sfo rm e r E x p lo s io n in Bingham ton, New Y o r k . " C h e m o s p h e re . V o l . 1 1 1 9 8 2 , p . 7 1 5 . 9. C . Rappe. " A n a ly s is o f P o ly c h lo r in a t e d D io x in s and F u r a n s ." Ch em o sph ere. V o l. 18 No. 3 1984, p. 78A. 10 . A. P o la n d ; E. G lo v e r and A .S . Kende. " S t e r e o s p e c if ic , H ig h A f f i n i t y B in d in g of 2 , 3 , 7 , 8 -T e tra c h lo ro d ib e n z o -p -d io x in by H e p a tic C y to s o l." J . B io l. Chem .. August 25, 1976, p. 4936. 1 1 . E . E . M c C o n n e ll; J . A . M o ore; J . K . H a s e ra n and M.W. H a r r i s . " T o x ic o l o g i c a l Assessm ent of H e xa ch lo ro b ip h e n yl Isom ers and 2 , 3 , 7 , 8 -T e tra c h lo ro d ib e n z o fu ran in C h ic k s ." T o x ic o l. A p p l. P h a rm a co l.. A p r il 19 76, p. 65. 1 2 . J . A . M oore; E . E . M c C o n n e ll; D.W . D a lg a r d and M.W. H a r r i s . " C o m p a r a tiv e T o x ic it y o f T h re e H alo gen ated D ib e n z o fu ra n s in G u in e a P ig s , M ice and Rhesus M onkeys." Ann. N .Y . A cad. S c i . . V o l. 320 19 79 , p. 1 5 1 . 1 3 . J . E . H u ff; J .A . Moore; R . S a r a c c i and L . T o m a tis. "Long-Term H azards of P o ly c h lo r in a t e d D ib e n z o d io x in s and P o ly c h lo r in a t e d D ib e n z o fu ra n s ." E n v ir o n . H e a lth P e rs p e c t. . November 1980, p. 2 2 1 . GENP 010983 4-2 783794 14 . S t a t e -o f -c h e -A r t R eview ; PCDDs and-PCD Fs In U t i l i t y F l u i d . P a lo A lt o , C a l i f . : E le c t r ic Power R esearch In s t it u t e , November 19 8 3. C S -330 8 -F R . 1 5 . P .W . O 'K e e f e ; R .M . S m it h ; D . R . H i l k e r ; K'.M. A ld o u s a n d W. G i l d a y . "A Sem iautom ated C lean u p Method fo r P o ly c h lo r in a t e d D ib e n z o -p -d io x in s and P o ly c h lo rin a te d D ib en zo furan s in E n viro n m e n ta l Sam p les." In C h lo rin a te d D io x in s and D ib e n zo fu ra n s in the T o t a l Environm ent I I , L . K e it h , C.Rappe and G. Choudhary, E d s .; 1985, pp. 1 1 1 -1 2 4 . 16 . J .R . Beynon. Mass Sp ectro m etry and I t s A p p lic a tio n to O rg a n ic C h e m is try . Am sterdam : E ls e v ie r P u b lis h in g C o ., 1960, pp. 5 1 -5 4 . 1 7 . D .R . H ilk e r ; K.M . A ld o u s ; R.M . S m ith ; P.W . O 'K e e fe ; J . F . G ie r t h y ; J . J u r u s lk S.W . H ib b in s ; D. S p in k and R . J . P a r i l l o . " D e te c tio n o f S u lf u r A n alo g of 2 ,3 ,7 ,8 -T C D D in the En viro n m en t." Chem osphere, V o l. 14 19 8 5, p. 12 7 5 . 1 8 . R . C a u l c u t t and R . B o d d y . S t a t i s t i c s f o r A n a l y t i c a l C h e m i s t s . New Y o r k : Chapman and H a l l , 19 8 3 , p. 54. 783795 4 -3 GENP 010984 Appendix A IN -S E R V IC E L IQ U ID S BACKGROUND INFORMATION Type of equipm ent: Load C e n te r Netw ork T ra n sfo rm e r BCL L a b o ra to ry Code: IS L -0 2 -A V o lta g e R a tin g : 7 5 0 KVA 12 KV to 4 8 0 /27 7 V L iq u id Volum e: 2650 lb s . Y ear: 1962 P C B / A s k a r e l T y p e : A r o c l o r 1 2 6 0 29Z - T 3 C B 29 Z - T 4 C B 2X PCB L e v e ls ( f o r O i l F i l l e d ) : A d d it io n a l Equipm ent I d e n t if ic a t io n : 6 5 C max. r i s e Len g th o f A c t u a l S e r v ic e : 20 y r s . M ajor M a in te n an ce : Recorded F a ilu r e in S e rv ic e : Was F l u i d C h a n g e d a t F a i l u r e : Any Record of Tem perature E x c u rs io n s : Are M aintenance R ecord s A v a ila b le : Any O ther P e rtin e n t In fo rm a tio n : Typ e o f equipm ent : Load C e n te r N etw ork T ra n s fo rm e r BCL La b o ra to ry Code: IS L -0 3 -A V o lta g e R a t in g : 500 KVA 13 KV to 213 V L iq u id Volum e: ? Y e ar: 1953 P C B /A s k a r e l T yp e : A r o c lo r 126 0 70Z - T3CB 29X - T4CB IZ PCB L e v e ls ( f o r O i l F i l l e d ) : A d d it io n a l Equipm ent I d e n t if ic a t io n ; Length of A ctu a l S e rv ic e : 31 y r s . M ajo r M ain ten an ce: Recorded F a ilu r e in S e rv ic e : Was F l u i d Changed a t F a i l u r e : A -l. ?a3?96 Any Record or Tem perature E x cu rsio n s: A re M aintenance R ecords A v a ila b le : Any Other P e rtin e n t In fo rm a tio n : Type of equip m ent: S u b s ta tio n D is t r ib u t io n T ra n sfo rm e r BCL L a b o ra to ry Code: IS L -0 4 -0 V o ltag e R a tin g : L iq u id Volum e: 200 g a l. Y e a r: P C B /A skarel Type: PCB L e v e l s ( f o r O i l F i l l e d ) : 1 0 0 ppm A d d it io n a l Equipm ent X d e n t if ic a t io n : 65 C max. r is e L e n g th o f A c t u a l S e r v ic e : 20 y r s . M ajor M a in te n an ce : Recorded F a ilu r e in S e r v ic e : M ajo r a r c in g and in - s e r v ic e f a ilu r e Was F l u i d Changed a t F a i l u r e : Any Record of Tem perature E x c u rsio n s: A re M aintenance R ecords A v a ila b le : Any Other P e rtin e n t In fo rm a tio n : Type of equip m ent: Load C e n te r Netw ork T ra n sfo rm e r BCL L a b o ra to ry Code: IS L -0 8 -A V o lta g e R a t in g : 500 KVA 12 KVA to 213 V L iq u id Volum e: 3 17 5 lb s . Y e a r: 1946 P C B /A sk a re l T yp e : A r o c lo r 1260 about 67% - T3CB 30% - T4CB 2% PCB L e v e ls ( f o r O il F i l l e d ) : A d d it io n a l Equ ipm ent I d e n t i f i c a t i o n : 55 C m ax. r i s e Length o f A c tu a l S e rv ic e : 28 y r s . M ajo r M a in te n a n ce : None Noted Recorded F a ilu r e in S e rv ic e : Was F l u i d Changed a t F a i l u r e : Any Record of Tem perature E x c u rsio n s: A re M aintenance R ecords A v a ila b le : Any Other P e rtin e n t In fo rm a tio n : Carbon p resen t throughout the A skarel Type o f equip m ent: P r e c ip it a t o r BCL L a b o ra to ry Code: IS L -1 7 -A 733797 o E T i P 010986 A -2 V o lta g e R a tin g : 480 to 5 3,5 0 0 V L iq u id Volum e: 143 g a l. Y e a r: PCB /A skarel Type: PCB L e v e ls ( f o r O il F i l l e d ) : A d d it io n a l Equipm ent I d e n t i f ic a t i o n : S e r i a l No. TH 5 8 7 5 , 50 KVA Length of A c tu a l S e r v ic e : 1947 - 19 74 (27 y r a .) M ajor M a in te n an ce : Recorded F a ilu r e in S e rv ic e : Any Record of Tem perature E x cu rsio n s: Are M aintenance R ecords A v a ila b le : Any O ther P e rtin e n t In fo rm a tio n : F u ll load r is e of 4 5 C . Type of equip m ent: A rc Fu rn ace T ra n sfo rm e r BCL L a b o ra to ry Code: IS L -2 3 -0 V o ltag e R a tin g : 12470/280 L iq u id Volum e: 4500 Y e a r: 1976 P C B /A skare l Type: M in eral O il PCB L e v e l s ( f o r O i l F i l l e d ) : 1 5 0 ppm A d d it io n a l Equipm ent I d e n t if ic a t io n : 750 0 /8 750 KVA, 4 .7 % im p ., N o -lo a d Se co n d ary tap ch a n g e r, FOA, 6 5 C r i s e in s u la t io n . Length of A ctu a l S e rv ic e : 8 y r s . M ajo r M a in te n an ce : Top co ve r of tra n s fo rm e r and tap chang er removed each y e a r f o r in t e r n a l v is u a l in s p e c tio n and check of c o n n e c tio n s. Recorded F a ilu r e in S e r v ic e : None Was F l u i d Changed a t F a i l u r e : Any Record of Tem perature E x c u rs io n s : None o ccu rre d A re M ain ten ance R ecord s A v a ila b le : Yes Any O ther P e rtin e n t In fo rm a tio n : O il ch e m ica l te s t and gas chrom atography te s ts run y e a rly . Type of equip m ent: C a p a c ito r BCL L a b o ra to ry Code: I S L - 2 7 - ? V o lta g e R a t in g : 7200 KVAR 100 L iq u id Volum e: 4" x 1 3 .5 " x 22" Y e a r: P CB /A skarel Type: PCB L e v e ls ( f o r O il F i l l e d ) : 783798 A d d it io n a l Equipm ent I d e n t if ic a t io n : Length of A ctu a l S e rv ic e : ? M ajo r M a in te n an ce : Recorded F a ilu r e in S e rv ic e : Was F l u i d Changed a t F a i l u r e : Any Record of Tem perature E x cu rsio n s: A re M aintenance R ecords A v a ila b le : Any O ther P e rtin e n t In fo rm a tio n : 783799 GENP 010988 A-4 Appendix B ANALYTE CALIBRATION CURVES F ig u re s B -l thro ugh B -8 . C a lib r a t io n cu rve s co n stru c te d w ith d ata o b ta in e d from the low r e s o lu t io n m ass s e le c t iv e d e t e c t o r . A lo n g w it h th e le a s t sq u a re s l i n e the 99% c o n fid e n c e i n t e r v a l i s a ls o p lo t t e d . F ig u re s B-9 through B -1 3 . C a lib r a t io n cu rves c o n stru c te d w ith data o b ta in e d from the h igh r e s o lu tio n mass sp ectro m eter. 783800 B -l FIGURE B - 1:EPRI BASELINE PROJECT 2 3 7 8 -T C D D C ALIBRATIO N C U R V E ttuIo O tri % T) o t-- * O vo vo STA N D A R D S O L 'N S LOG(CONC in pqa/ul or ppb) ------- LST S Q S c 9 9 % CON O O FIGURE B -- 2: EPRI BASELINE PROJECT s 2 3 7 Q -T C D F CALIBRATIO N C U R V E % hd o o MD MD 0-v0! CO 0o0 ro L0 G (C 0 N C in pg9/ul or ppb) STANDARD SO L'N S ------- LST S O S & 9 9 % CON FIGURE B-3: EPRI BASELINE PROJECT 1 2 3 7 8 -- P n C D F CALIBRATIO N CURVE. CId O )-- k O L O C ( C O N C in pgs/ul or ppb) vo S T A N D A R D S O L 'N S ------- LST S Q S & 9 9 % CON K> O M FIGURE B-- 4: EPRI BASELINE PROJECT o i-- i O vo 'sO OJ 2 3 4 7 8 - P n C D F CALIBRATIO N C U R V E 'UtItl -COI CO oo o STANDARD SO L'N S L0G (C 0N C in pga/ul or ppb) ------- LST S Q S 8c 9 9 % CON FIGURE B - 5 : EPRI BASELINE PROJECT 1 2 3 4 7 8 -- H xC D F C ALIBRATIO N C U R V E OOId' O M 3 O 1-- I O vo oGoO 0 01 STANDARD SO L'N S L0 G (C 0 N C in pga/ul or ppb) ------- LST SQ S 8c 9 9 % CON FIGURE B --6: EPRI BASELINE PROJECT 2 3 4 6 7 8 -- H x C D F CALIBRATIO N C U R V E O t-- k O VO VD U\ DIJ --4 000000 OCT) STANDARD SO L'N S L 0 G ( C 0 N C in p g s / u l) --------- L S T S Q S 8c 9 9 % C O N FIGURE B -7 : EPRI BASELINE PROJECT 1 2 3 4 6 7 6 -- H p C D F C ALIBRATIO N C U R V E w1 00 O (-- * O LOG(CONC in pga/ut or ppb) STANDARD SO L'N S ------- LST S Q S Sc 9 9 % CON Os FIGRE B -- S: EPRI BASELINE PROJECT STANDARD SO L'N S i Figure B-3: CALIBRATION CURVE FOR 2,3,7,8-TCHD CD A M T C0N C .)RATIO CoDo o CD GENP 010998 GENP 010999 Figure -10; CRLIERRTION CURVE FOR 2,3,7,8-TCDF P U T .CONC .)RPTIO Figure E-- 11: COL IBROT ION CURVE FOR l 8-PnCDF o M I-- CC Cii ui i_ i LL </> U Ckl S 00 PMT. ( CONC . ) Rfl T10 GENP 011000 GENP 011001 F i q u r e E-ls COLIERRTI ON CURVE FOR 1 , 2 , 3 , 4 , 7 , 8 - Hx CDF Least squares curve fit o I-- a: u w l_l CL tA Ld C 3 . CO CD ro AMT . (CONC . ) RATIO CT .iiL- I1_ 1 O I l ^ U I C L> X ^ a GENP 011002 eisesz RMT. C 0 H C . ) RATIO A p pend ix C ANALYTE RESPONSE FACTOR CURVES F ig u re s C - l through C -8 . Area r a t io s from d a ily response fa c t o r d e te rm in a tio n s p lo tte d onto the c a lib r a t io n cu rv e s. G EN P0i 1003 783814 FIGURE C -- 1:EPRI BASELINE PROJECT 2 3 7 8 - T C D D DAILY R E S P . FA C T O R S nI L O G ( C O N C in pgs/ul) O X DAILY R ESP. FACTORS LST SQ S & 9 9 % CON O 4^ oe X DAILY RESP. FACTORS LOG(CONC in p q s /u l or ppb) LST SQS & 99% CON FIGURE C --3;EPRI BASELINE PROJECT 1 2 3 7 8 -- PnCDF DAILY RES. FACTORS 0-1e* O W 2 l-Tj O O O LST SQS 8c 99% CON LOG(CONC in p g s /u l or ppb) V X DAILY RES. FACTOR a\ FIGURE C --4: EPRI BASELINE PROJECT 0U11 + DAILY RESP. FACTORS LOG(CONC in p g s /u l or ppb) LST SQS Sc 99% CON FIGURE C -5 : ERRI BASELINE PROJECT 12 3 4 7 8 -- HxCDF DAILY R E SPO N SE FACTORS O01' O M o oo LOG(CONC in pg9/ul or ppb) oo D + DAILY RESP. FACTORS ------- LST SQ S & 9 9 % CON O w o S CvIlJ FIGURE C - 6 : EPRI BASELINE PROJECT 2 3 4 6 7 8 -- HxCDF DAILY R E SP O N SE FACTORS -J C0D0 00 ro o + DAILY RESP. FACTORS L0G(CNC in p g a /u l or ppb) LST SOS & 99% CON FIGURE C -- 7: EPRI BASELINE PROJECT 1234678 --HpCDF DAILY RESP. FACTORS C1i OD O U O >-- * LOG(CONC in p g a /u l or ppb) O X DAILY RESP. FACTORS LST SQS & 99% CON 1 O FIGURE C --8: EPRI BASELINE PROJECT tri o O cc O < LJ Cl < O -N] 0C0O 00 Nro) X DAILY RFSP. FACTORS LOG(CONC in p g a /u l or ppb) LST SQS Sc 99% CON A ppendix D TA B LES OF R ESULTS TABLE D-L BASELIN E SAMPLES P R E L IM IN A R Y R E S U L T S OF S P E C I F I C COMPOUND A N A L Y S IS SAMPLES CONC. 23 7 8-TCDD ppb ( D .L .) RET. TIM E (m in .) RATIO U) CONC. 237 8-TCDF ppb ( D . L .) RET, TIM E (m in .) CONC. 12 3 7 8-PCDF ppb ( D . L .) RET. TIM E (m in .) A ro clo r 1016 E -729 -0 9 -0 1 -- 0 .1 4 ( 0 .12 ) 22.34 0 .4 0 (0 .0 8 ) 23.41 A ro clo r 1016 E729 -0 9 -0 1 0 .5 1(0 .19 ) 17.63 77 0 .3 4 (0 .0 8 ) 2 2 .4 8 0 .3 2 (0 .2 4 ) 23 .4 8 ND1 ND1 A ro clo r 1242 0 .4 5 (0 .0 3 ) E729 -10 -0 1 106 1 5 0 ( 1 .6 ) 22.34 1000(25) 6 2 (1.4)2 2 3 .0 3 A ro c lo r 1260 E72 9 -11-0 7 .3(0 .72) 17 .35 79 19 0 (1.7 ) 2 2 .15 220(5) 23 .20 T ri/T e tra C l Benz E72 9 -12 -0 1 0 .4 6 (0 .13) 17 .2 9 44 0 .4 3 (0 .0 5 ) 2 2 .2 7 0 .6 3 ( 0 .0 9 ) 23 .2 4 Aged M in e ra l O il E 729 -0 8 -0 1 4 3(0 .0 1) 17.5 5 80 3 4 ( 0 . 2 7 ) 22.4 4 140* 2 3 .5 2 B lin d Sp ike L e v e ls 40 20 40 B lank 0 .9 3(0 .0 6 ) 17 .5 8 83 0 . 1 4 ( 0 . 0 5 ) 2 2 . 5 3 N .D .C 0 .4 1) 23.55 N u jo l Sp ike 10 (0 .0 8 ) 17.5 8 91 1 2 ( 0 . 5 8 ) 22.4 5 24 (0 .3 4) 2 3 .4 8 *Sum o f 1 , 2 , 3 , 7 , 8 -PnCD F and 1 , 2 , 3 ,4 , 8-PnCD F 1 . ) ND * N o t D e t e c t e d i n a s e c o n d a n a l y s i s 2 . ) The r e s u lt s o f an a lt e r n a t iv e in t e r p r e t a t io n o f the h ig h r e s o lu t io n mass d a ta . GENP 011012 D-L 783823 TABLE D-2 E P R I BA SELIN E STUDY PRELIM INARY TOTAL CONGENER GROUP RESULTS SAMPLE TOTAL TCDD PPb (D .L .) A ro clo r 1016 E729 -0 9 -0 1 N .D .CQ .6) A ro clo r 1242 E 729 -10 -0 1 . N .D .C 0 .2) A ro c lo r 1260 E72 9 -11-0 N .D .(1.0 ) Chlo ro benzenes E 729 -12-0 1 N .D .(0 .2) Aged M in e ra l O il E729 -0 8 -0 1 42 B lin d S p ike Le v els 40 T o ta l-S p ik e d 40 B lank N .D .(0 .4 ) N u jo l 11 (10 )* TOTAL TCDF ppb (D .L .) 0 .4 2 1900 940 3 .0 95 20 120 0 .75 22 (10 )* TOTAL PENTA CDF Ppb (D .L .) 7.3 584 932 12 .5 -- 40 240 -- -- TOTAL HEXA CDF ppb (D .L .) 3 50 140 5500 1000 1100 120 220 28 420 (10 )* TOTAL HEPTA ppb (D .L .) 92 130 2970 91 -- 0 0 -- 19 (10 )* OCDF ppb (D .L .) N .D .(12 ) 23 00 NA NA NA 0 160 NA NA * S p ike L e v e l NA - N ot A v a i l a b l e 783824 D-2 GENP 011013 TABLE D-3 E P R I B A S E L IN E P R O JE C T : A N A L Y S IS OF AGED M IN ERA L O IL TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD BLANK AGED MINERAL O IL ( in ppb) (0 .6 1) (1.0 3) (10 2.3 ) (16 4 ) (58 .3) (0 .9 3) (58 .3) S P IK E D MIN O IL ( in ppb) 178 282 400 356 3 .3 7 18 .8 (2 .1) S P IK E D LEVEL (in, ppb) XERROR 200 300 47 5 400 NS 25 NS 11.0 6 .0 15 .8 11.0 NA 2 4 .8 NA 2 ,3 , 7, 8-TCDD (0 .9 3) 18 .8 25 24 .8 2 , 3 , 7 , 8-TCDF (0 .6 1) 178 200 1 1 . 0 2 ,3 , 4, 8 -TCD F (0 .6 1) 9 1.3 100 8.7 1 , 2 , 3 , 7 , 8-PnCDF (1.0 3) 13 5 150 10 .0 1 , 2 , 3 , 4, 8 -P n C D F (1.0 3) (0 .0 6) . NS NA 2 ,3 ,4 , 7, 8-PnCDF (1.0 3) 14 1 150 6 .0 1 , 2 ,3 ,4 , 7, 8-HxCDF (10 2 ) 227 250 9 .2 1 , 2 , 3 , 4 , 7, 9-HxCDF (10 2 ) (0 .22) NS NA 1 , 2 , 3 , 7 , 8, 9-H xCD F (10 2 ) (0 .22) NS NA 1 , 2 , 3 , 6 , 7 , S-HxCDF (10 2 ) 4 1.5 50 1 7 .0 2 , 3 , 4 , 6 , 7 , 8-HatCDF (12 0 ) 160 17 5 8.6 1 , 2 , 3 , 4, 6, 7, 8-HpCDF (16 4 ) 354 400 1 1 . 5 AVE. Z ERR 12 .5 1 . ) Numbers in p a r e n t h e s e s in d i c a t e d e t e c t io n l i m i t s i n ppb 2 . ) Average d e te c tio n lim it of three d e te rm in a tio n s 3 . ) NS n o t s p i k e d ; NA n o t a p p l i c a b l e 4 . ) 2 , 3 , 7, 8 -T C D F v a lu e i s th e sum o f 2 , 3 , 7 , 8 - and 2 , 3 , 4 , 8 -T C D F c o n ce n tra tio n s. GENP 011014 D-3 783825 TABLE D-4 EPRI BASELINE PROJECT: ANALYSIS OF AROCLOR 1016 TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD BL AROCLOR S P IK E D 1016 AROCL 10 16 ( in ppb) ( in ppb) SPIKED LEVEL ( in ppb) (0 .4 1)1 (0 .50 ) (2 2 .2 ) (36) (7.9 ) (0 .58 ) (16 ) 63 312 212 627 120 48 .2 (2 .1)2 53 316 185 526 13 2 53 NS3 % ERROR -18 .9 1.3 -14 .6 -19 .2 9 .1 9 .1 NA3 2 , 3 , 7 , 8-TCDD (0 .58 ) 2 ,3 ,7 , 8-TCD F (0 .4 1) 2 ,3 ,4 , 8-TCD F (0 .4 1) 1, 2 ,3 , 7 , 8-PnCDF (0 .50 ) 1 , 2 , 3 , 4, 8-PnCDF (0 .50 ) 2 ,3 ,4 , 7, 8-PnCDF (0 .52) 1 , 2 , 3 , 4, 7 ,8 -H x C D F (22) 1 , 2 , 3 , 4 , 7, 9-HxCDF (22) 1 , 2 , 3 , 7 , 8 , 9 - H x C D F - ( 2 2 ) 1 ,2 ,3 ,6 , 7,8-H xCD F (22) 2 ,3 ,4 , 6, 7,8-H xCD F (26) 1 , 2 , 3 , 4 ,6 ,7 , 8-KpCDF (36) 48 .2 53 62.3 53 (0 .7 1)2 NS 159 158 (0 .3 8 )2 NS 148 158 3.9 8 NS (0 .26 )2 NS (0 .26 )2 NS 5 6 .5 53 151 132 625 526 AVE % ERR 9 .1 -17 .5 NA -0 .6 NA 6 .3 NA NA NA -6 .6 -14 .4 -18 .8 11 1 . ) Numbers in p a re n th e s e s in d ic a t e d e t e c t io n l i m i t s 2 . ) Average d e te c tio n lim it of th re e d e te rm in a tio n s 3 . ) NS n o t s p i k e d ; NA n o t a p p l i c a b l e 783826 E H P 0 l 1015 TABLE D-5 E P R I B A S E L IN E PR O JEC T SAM PLE: A N A L Y S IS OF AROCLOR 12 6 0 TOTAL TOTAL TOTAL TOTAL OCDF TOTAL OCDD TCDF PnCDF HxCDF HpCDF TCDD SPIKED AROCL 126 0 ( in ppb) 1613 13 20 2473 2390 5037 64 (4 .7 8 )1 ' 2 SPIKED BLANK ( in ppb) 1093 412 1253 732 403 64 NA-1 S P IK E D LEVEL ( in ppb) 1137 222 834 111 222 NSJ % ERROR 3 `? NA^ NAJ NA A - 8 1 .54 4 2 .3 NAJ 2 , 3 , 7 , 8-TCDD 2 , 3 , 7 , 8-TCD F 2 , 3 , 4 , 8-TCDF 1 , 2 , 3 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 8-PnCDF 2 , 3 , 4 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 7 , 9-HxCDF 1 , 2 , 3 , 7 , 8 , 9-HxCDF 1 , 2 , 3 , 6 , 7 , 8-HxCDF 2 , 3 , 4 , 6 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 64 1210 436 205 140 294 (0 .5 )1 ' 2 38 336 297 845 64 1 1 1 1033 1027 NA 1 9 4 127 111 6 1 NS 119 111 647 500 NA NS 3 8 NS 336 27 8 260 56 358 111 AVE X ERR 4 2 .3 -0 .6 NA -14 .4 NA -7 .2 -29 ,4 NA NA -20 .9 -3 6 4 .3 7 -2 2 2 .54 75 1 . ) Numbers in p a re n th e se s in d ic a t e d e te c t io n lim it s 2 . ) Average d e te c tio n lim it of th re e d e te rm in a tio n s 3 . ) NS - n o t s p ik e d ; NA n o t a p p l i c a b l e 4 . ) These v a lu e s are d isc u sse d in the te x t GENP 011016 D-5 783827 TABLE D-6 EPRI BASELINE PROJECT SAMPLE 1953 LOAD CENTER NETWORK TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 232 1930 6 840 10 10 0 39000 (1.3 2 ) 11.8 10 DET. 2 121 1690 6570 8610 37700 (6 2.2) 9 10 .4 DET. 3 337 1760 6520 8880 42100 (2.76 ) 14 .3 17 AVE. 230 17 93 6643 9196 39600 12 SD 10 8 .1 12 3 ,4 17 2 .1 7 93.9 2234 3 CV 47 6 .9 2.6 8 .5 5 .6 25 2 , 3 , 7 , 8-TCDD (1.3 2 ) 2 , 3 , 7 , 8-TCD F 4 8 .9 2 , 3 , 4 , 8-TCDF 16 .5 1 , 2 , 3 , 7 , 8-PnCDF 19 .2 1 , 2 , 3 , 4 , 8-PnCDF 919 2 , 3 , 4 , 7 , 8-PnCDF 101 1 , 2 , 3 , 4 , 7 , 8-HxCDF 4790 1 , 2 , 3 , 4 , 7 , 9-HxCDF ( 2 .3 4 ) 1 , 2 , 3 , 7 , 3 , 9-HxCDF ( 2 .3 4 ) 1 , 2 , 3 , 6 , 7 , 8-HxCDF 360 2 , 3 , 4 , 6 , 7 , 8-HxCDF 5 1.2 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 3890 (6 2.2) 4 2 .9 29 .4 705 107 4610 (4 .14 ) (4 .14 ) 367 4 2.4 3280 1 . ) Average of s ix (6) re s u lts (2.76 ) 67.7 5 3 .17 15 .6 772 105 46 20 (5) (5) 362 40.6 3990 2 1.4 798 .7 10 4 .3 4673 363 4 4 .7 3720 12 .9 4 24.3 7 .16 10 9.5 3 .0 6 10 1.2 33 13 .7 2.9 2 .2 3 .6 1 5.6 7 3 84.3 1 12 .7 10 .3 783828 GENP011017 D-6 TABLE D-7 EPRI BASELINE PROJECT SAMPLE: LOAD CENTER NETWORK TRANSFORMER TOTAL TOTAL TOTAL TOTAL OCDF TOTAL OC DD TCDF PnCDF HxCD HpCDF TCDD DET. 1 228 430 983 2400 6 800 C O .3 5 ) (27.4 ) DET. 2 18 2 303 897 1940 8000 (0 .4 3) (26 .6 ) DET. 3 518 435 93 8 1850 7010 (7 .1) (2 7 .1) AVE. 30 9 .3 3 8 9 .3 9 3 9 .3 2063 7 270 SD 18 2.2 74 .8 43 295 6 4 0 .9 CV 5 8 .9 19 .2 4.6 14 .3 8.8 2 , 3 , 7 , 8-TCDD 2 , 3 , 7 , 8-TCDF 2 , 3 , 4 , 8-TCD F 1 , 2 , 3 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 8-PnCDF 2 , 3 , 4 , 7 , 8-PnCDF 2 , 3 , 4 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 7 , 9-HxCDF 1 , 2 , 3 , 7 , 8 , 9-HxCDF 1 , 2 , 3 , 6 , 7 , 8-HxCDF 2 , 3 , 4 , 6 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF (0.0 5) 6 8 .6 29 .4 8 8 .8 6 0 .6 619 1.5 3 7 .9 3 3 .9 43 15 .2 662 (0 .12) 6 1.1 6 .95 3 0 .7 3 8 .1 3 9 .6 596 1.5 4 17 .7 2 2 .2 4 4 .6 9 .1 545 (2) 9 0 .9 2 8 .9 7 2 .3 59 .4 610 1.8 3 6 .1 3 1.5 4 2 .3 12 .3 631 7 3 . 5 1 5 . 5 21.1 29 .7 6 6 .4 5 3.2 6 0 8 .3 1.6 29 .9 0 .9 3 2 5 .9 11.8 11.6 0 .16 7. 3 .13 39 22.2 1.9 10 25 4 3 .3 12 .2 6 12.7 1.18 3 .0 5 6 0 .6 2 .7 25 GENP 011018 D-7 783829 TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD TABLE D-8 EPRI BASELINE PROJECT SAMPLE: CAPACITOR DET. 1 686 368 3 9 .7 8 .3 13 .4 CO .16) (1.1) DET. 2 700 351 5 1.9 12 .5 15 .3 C O .3 9 ) C O .3 8 ) DET. 3 942 346 42 9 .51 16.6 CO.83) (0 .29 ) AVE. 776 355 4 4 .5 10 .1 15 .1 SD 144 11.5 6.48 2 .1 6 1.6 0 CV 15 3 .3 15 21 11 2 .3 .7 .8- TCDD 2 .3 .7 .8- TCDF 2 . 3 . 4 . 8 -TCDF 1 . 2 . 3 . 7 . 8 - PnCDF 1 , 2 , 3 , 4 , 8-PnCDF 2 , 3 , 4 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 7 , 9-HxCDF 1 , 2 , 3 , 7 , 8 , 9-HxCDF 1 , 2 , 3 , 6 , 7 , 8-HxCDF 2 , 3 , 4 , 6 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF CO .16 ) 197 8 .3 11.5 6 1.3 16 .5 0 .3 0 .19 3.4 1.7 3 .4 C O .3 9 ) 226 4 4 .1 9.6 7 .7 6 1.5 2 1 .5 0 .3 6 0 .3 6 4 .3 6 2 ' 4.8 CO. 83) 273 8 .3 7 8 .7 7 1.1 15 .8 0 .38 0 .24 3.9 4 1.7 5 3.38 232 38.40 8 .7 5 7 9 .3 6 4 .6 3 13.5 3 0 .3 5 0 .2 6 3 3 .9 1.8 17 3 .8 6 0 .7 3 1.9 7 5.6 0 4 .5 5 0 .0 4 0 .0 9 0 .48 0 .16 0.8 1 7 8 .4 21 8 .7 34 12 33 12 8 .9 21 GENP 011019 D-8 783830 TABLE D-9 EPRI BASELINE PROJECT S A M P L E : PRECIPITATOR TRANSFORMER TOTAL TOTAL TOTAL TOTAL OCDF TOTAL OCDD TCDF PnCDF HxCDF HpCDF TCDD ' DET. 1 1500 10500 10500 2000 190 (0 .0 5) 1.0 4 DET. 2 953 10200 10900 2020 187 0.42 0.98 D ET.3 1100 10800 10700 2100 181 0 .9 8 1.0 1 AVE. 118 4 10500 10700 2040 186 SD 28 3.1 300 200 5 2 .9 4.6 1.0 1 0 .0 3 CV 2 3 .9 2.9 1.9 2.6 2 .5 3 2 , 3 , 7 , 8-TCDD 2 ,3 ,7 , 8-TCD F 2 ,3 ,4 , 8-TCDF 1 , 2 , 3 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 8-FnCD F 2 ,3 ,4 , 7, 8-PnCDF 1 , 2 , 3 , 4 , 7 , 8-HxCDF 1 ,2 ,3 ,4 , 7,9-H xCD F 1 , 2 , 3 , 7 , 8, 9-H xCD F 1 , 2 , 3 , 6 , 7 , 8-HxCDF 2 , 3 , 4 , 6 , 7, 8-HxCDF 1 ,2 ,3 ,4 , 6,7,8 -H p C D F CO.05) 480 799 307 1930 43 90 22 .5 162 1030 366 690 CO.02) 349 101 796 256 1920 4410 21.6 177 1030 404 678 CO.09) 3 95 818 317 1970 4360 22 175 1000 398 700 408 66.5 804.3 293 .3 1940 4387 22 171.3 1020 389.3 6 89.3 11.9 32.7 26.5 25.2 0.45 8.2 17.3 20.4 11 16.3 1.5 11.2 1.4 0.6 2.1 4.8 1.7 5.2 1.6 GENP 011020 D-9 783831 TABLE D-10 EPRI BASELINE PROJECT SAMPLE: 1976 ARC FURNACE TRANSFORMER TOTAL TOTAL TOTAL TOTAL OCDF TOTAL OCDD TCDF PnCDF HxCDF HpCDF T CDD DET. 1 0 .5 3 1.7 2 1.7 8 0 .5 1 0 .4 6 (0.0 9) 3.0 2 DET. 2 0 .38 1.9 4 1.14 0 .23 0 .3 (0 .0 8 ) 3 .3 8 D ET.3 0 .31 2 .1 4 2 .4 3 0 .19 0 .24 (20) 3 .8 AVE. 0 .41 1.9 3 1.7 8 0 .31 0 .3 3 3 .4 SD 0 .11 0 .21 0 .64 0 ,17 0 .11 0 .3 9 CV 28 11 36 56 34 11 2 , 3 , 7 , 8-TCDD 2 , 3 , 7 , 8-TCDF 2 , 3 , 4 , 8-TCDF 1 , 2 , 3 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 8-PnCDF 2 , 3 , 4 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 7 , 9-HxCDF 1 , 2 , 3 , 7 , 8 , 9-HxCDF 1 , 2 , 3 , 6 , 7 , 8-HxCDF 2 , 3 , 4 , 6 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF (0.0 9) 0 .2 0 .21 (0 .16 ) 0 .4 5 1.0 8 (0 .2) 0 .22 0 .25 (0 .21) (0 .28 ) (0 .0 8 ) 0 .24 (0 .54 ) 0 .2 6 (0 .13) 0 .51 0 .54 (0 .0 6 ) 0 .11 0 .13 (0 .14 ) (0 .0 8 ) (20) 0 .2 3 0 .223 0 .2 3 (0.0 8) 0 .4 1 1.13 (0 .0 3) 0 .28 0 .27 (0 .0 7) (0.0 9) 0 .2 3 0.46 0 .9 17 0.20 3 0 .217 0 .0 2 0 .0 3 0.0 5 0 .33 0 .0 9 0.0 8 9 .3 11 11 36 42 35 783832 GENP 011021 D-10 TABLE D - l l EPRI BASELINE PROJECT SAMPLE: SUBSTATION DISTRIBUTION TRANSFORMER TOTAL TOTAL TOTAL TOTAL OCDF TOTAL OCDD TCDF PnCDF HxCDF' HpCDF TCDD DET. I 0 .15 (0 .34 ) 0 .5 6 (0.65) (0.6 3) (0 .6 1) 20 .8 DET. 2 0 .18 (0 .6 ) 0 .52 (0 .23) (2.4 ) 0 .19 16 .8 DET. 3 0 .2 3 (0 .53) 0 .71 (0 .21) (3.8 ) 0 .1 17 .1 AVE. SD 0 .19 0.0 4 0.6 0 .1 18 .2 2 .2 3 CV 22 16 .7 12 2 , 3 , 7 , 8-TCDD 2 , 3 , 7 , 8-TCDF 2 ,3 ,4 , 8-TCDF 1 , 2 , 3 , 7 , 8 - PnCDF 1 , 2 , 3 , 4 , 8-PnCDF 2 , 3 , 4 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 7 , 9-HxCDF 1 , 2 , 3 , 7 , 8 , 9-HxCDF 1 , 2 , 3 , 6 , 7 , 8-HxCDF 2 , 3 , 4 , 6 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF (0.6) 0 .15 (0 .34 ) (0 .34 ) (0 .34 ) 0 .5 6 (0 .21) (0 .21) (0 .21) (0 .32) '0 .6 5 (0 .12) 0 .18 (0.0 9) (0.0 9) 0 .0 9 0 .5 2 (0 .19 ) (0 .19 ) (0 .19 ) (0 .2) (0 .23) (0.0 9) 0 .13 (1.5 ) (0 .0 7) (0 .0 7) 0 .11 0 .5 3 (0 .16 ) (0 .16 ) (0 .16 ) (0 .18 ) (0 .21) 0 .15 0 .54 0.0 25 0 .0 2 16 3 .7 GENP 011022 D-ll 783833 TABLE D-12 EPRI BASELINE PROJECT SAMPLE: 1962 LOAD CENTER NETWORK TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. I 553 1170 1760 1780 3960 (1.16) 18.7 DET. 2 552 1130 17 80 1580 4410 (0.82) 24.2 DET. 3 512 1100 1820 1610 4730 (0.71) 28.4 AVE. 53 9 1133 1790 16 57 4366 SD 23.4 35.1 30.5 107.8 3 87.7 23.8 4.9 CV 4.3 3.1 1.7 6.5 8.9 20.6 2.3.7.8- TCDD 2.3.7.8- TCDF 2,3,4, 8-TCDF 1.2.3.7.8- PnCDF L,2,3,4, 8-PtiCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 1.2.3.4.7.9- HxCDF l,2,3,7,8, 9-HxCDF 1 . 2 . 3 . 6 . 7 . 8 - HxCDF 2.3.4.6.7.8- HxCDF 1.2.3.4.6.7.8- (1.16) 195 59.4 230 170 1000 (0.89) (0.89) 64.9 70 HpCDF815 (0.82) 184 42.4 60.8 216 151 1030 (0.86) (0.86) 65.2 58 761 (0.71) 182 187 61.4 206 173 1110 (0.59) (0.59) 61.7 56 784 60.5 217 164.6 1047 63.9 61 787 7 1 12 11.9 56.9 1.9 7.6 27 3.7 1.7 5.5 7.2 5.4 3 12 3.4 783834 D-12 O B ii? 1 ' 1 TABLE D-13 EPRI BASELINE PROJECT SAMPLE: SPIKED AGED MINERAL OIL TOTAL TCDF TOTAL PnCDF TOTAL HxCDF ' TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 145 251 3 96 354 3.49 17 C5.09) DET. 2 195 312 455 341 3.55 21.3 (0.49) DET. 3 194 283 350 372 3.06 18.2 (0.67) AVE. 178 282 400 356 3 .37 18.8 SD 28.6 30.5 52.6 15.6 0.27 2.219 cv 16.1 10.8 13.2 4.4 7.9 12 2,3,7,8-TCDD 2,3,7,8-TCDF 2,3,4, 8-TCDF 1,2,3,7,8-PnCDF 1,2,3,4,8-PnCDF 2,3,4,7, 8-PnCDF 1,2,3,4,7,8-HxCDF 1,2,3,4,7,9-HxCDF 1,2,3,7,8,9-HxCDF 1,2,3,6,7,8-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF 17 146 121 (0.04) 127 224 (0.53) (0.53) 42.1 150 351 21.3 195 91.3 153 (0.07) 158 259 (0.07) (0.07) 47.4 161 341 18.2 192 18.8 2.219 12 177.7 27.5 15.5 132 (0.07) . 138 199 (0.05) (0.05) 35.1 170 369 135 141 227 41.5 160 353.7 16.3 15.7 30.1 6.2 10 14.2 12.1 11.1 13.3 14.9 6.3 4 GrENP 011024 D-13 783835 TABLE D-14 EPR I BA SELIN E PROJECT SAMPLE: SP IK ED AROCLOR 10 16 TOTAL TOTAL TOTAL TOTAL OCDF TOTAL OCDD TCDF PnCDF HxCDF HpCDF TCDD ' DET. 1 6 0 .8 276 201 57 8 133 3 9 .6 (1.4 4 ) DET. 2 6 2.7 336 209 712 107 50 .4 (0 .4 1) DET. 3 65.5 3 24 225 591 121 5 4 .5 (4 .52) AVE. 63 312 212 6 27 120 .3 48 .2 SD 2 .4 3 1.7 12 .2 7 3 .9 13 7 .7 CV 3 .8 10 .1 5 .8 11.8 10 .8 15 .9 2 , 3 , 7 , 8-TCDD 2 , 3 , 7 , 8-TCDF 2 ,3 ,4 , 8-TCDF 1 , 2 , 3 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 8-PnCDF 2 , 3 , 4 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 7 , 9-HxCDF 1 , 2 , 3 , 7 , 8 ,9-H xCD F 1 , 2 , 3 , 6 , 7 , 8-HxCDF 2 , 3 , 4 , 6 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 3 9 .6 6 0 .8 13 8 (0 .30 ) 122 2 .5 6 (0 .21) (0 .21) 5 8 .5 140 575 50 .4 60.5 (0 .72) 171 (0 .0 9 ) 159 3 .8 (0 .0 5) (0 .0 5) 5 4 .6 150 709 5 4 .5 65.5 4 8 .2 6 2.3 16 8 (0 .75 ) 163 5 .6 (0 .52) (0 .52) 5 6 .5 162 592 159 148 3.9 8 6 .5 15 0 .7 625 7.7 15 .9 2 .8 4.5 18 .2 11.4 2 2 .6 1.5 2 15 .3 38 2 3.5 11 7.3 73 11 GETSTP 011025 D-14 783836 m TABLE D - l 5 EPRI BASELINE PROJECT SAMPLE: SPIKED AROCLOR 1260 TOTAL TCDF TOTAL PnCDF TOTAL H xCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 1630 1330 2490 2470 4850 65.1 (3.73) DET. 2 1620 1290 23 80 2300 5090 65.5 (0.72) DET. 3 1590 13 40 2550 2400 5170 106 (9.89) AVE. 1613 1320 2473 23 90 5037 79.9 SD 20.82 26.46 86.2 85.4 166.5 23 CV 1.3 2 3.5 3.6 3.3 30 2 , 3 , 7 , 8-TCDD 2 , 3 , 7 , 8-TCDF 2 , 3 , 4 , 8-TCD F 1 , 2 , 3 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 8-PnCDF 2 , 3 , 4 , 7 , 8-PnCDF 1 , 2 , 3 , 4 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 7 , 9-HxCDF 1 , 2 , 3 , 7 , 8 , 9-HxCDF 1 , 2 , 3 , 6 , 7 , 8-HxCDF 2 , 3 , 4 , 6 , 7 , 8-HxCDF 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 6 5.1 1210 207 139 304 1228 (0 .4 1) 3 7 .8 339 313 865 6 5.5 1220 436 203 139 288 1209 (0 .0 9 ) 3 5 .8 321 286 805 10 6 1 1200 7 9 .9 1210 23 30 10 0 .8 3 206 14 2 291 1284 (0.99 ) 3 9 .9 347 291 865 205 140 29 4.3 1240 2 .0 8 1.7 3 8 .5 3 8 .9 9 3 7 .8 336 297 845 2 .0 5 13 .3 2 14 .3 6 3 4 .6 4 1 1.2 2 .9 3 .1 5 .5 4 4 .9 4 .1 1 . ) A n alyzed by high re s o lu tio n m .s. GENP 011026 D-15 783837 A ppendix E BAR GRAPHS OF R ESU LTS F ig u re s E -l through E -10 . B ar p lo t s of the r e s u lt s . The numbers lis t e d alo ng the a n a ly te a x is in each of these fig u r e s re p re se n t the fo llo w in g isom ers or groups of iso m e rs: 1 . T o ta l TCDF 2. T o ta l PnCDF 3 . T o t a l HxCDF 4. T o t a l HpCDF 5. OCDF 6. T o t a l TCDD 7 . OCDD 8. 2 , 3 , 7 , 8-TCDD 9. 2 , 3 , 7 , 8-TCDF 10 . 2 , 3 , 4 , 8-TCDF 1 1 . 1 , 2 , 3 , 7 , 8 - PnCDF 1 2 . 1 , 2 , 3 , 4 , 8-PnCDF 1 3 . 2 , 3 , 4 , 7 , 8-PnCDF 14 . 1 , 2 , 3 , 4 , 7 , 8-HxCDF 1 5 . 1 , 2 , 3 , 4 , 7 , 9-HxCDF 16 . 1 , 2 , 3 , 7 , 8 , 9-HxCDF 1 7 . 1 , 2 , 3 , 6 , 7 , 8-HxCDF 18 . 2 , 3 , 4 , 6 , 7 , 8-HxCDF 19 . 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF In the F ig u r e s a b ra c k e t is used to in d ic a t e th a t most of the in d iv id u a l isom er d e te rm in a tio n s were m u lt ip lie d by a f a c t o r of 10 b e fo re they were p lo t t e d . Th u s, f o r exam ple the c o n c e n t ra t io n of 1 2 3 4 7 8-HxCDF in the f i r s t f ig u r e is 4 .7 x 10J ppb. G B H P I1 0 2 7 E -l 783838 FIG E -- 1 ; LOAD CENTER NETW. TRANSFORM ER AVERAGE ANALYTE CONC.S PII 4M O w o \7~y\ AVERAGE CONC. O to oo [ V X ] STANDARD DEV. f FIG E-- 2: LOAD CNTR NETWORK TRANSFORM ER GENP 011029 _ [ / /\ AVERAGE CONG. ANALYTE STANDARD DEV. 4 FIG E--3: CAPACITOR AVERAGE ANALYTE CONC.S GENP 011030 1 7 7 1 AVERAGE CONC. ANALYTE ____ j \ \ J STANDARD DEV. FIG E - 4 : PRECIPITATOR TRANSFO RM ER ro _[771 AVERAGE CONC. ANALYTE [771 STANDARD DEV. -HpCDF F FIG E - 5 : ARC FURNACE TRANSFORMER AVERAGE ANALYTE CONC.S 783843 GENP 011032 _X7~7\ AVERAGE GONG. ANALYTE STANDARD DEV. FIG E--6: SUBSTATION DISTRIBUTION TRANSF AVERAGE ANALYTE CONC.S GENP 011033 ___ 1 7 7 1 AVERAGE CONG. ANALYTE (7 7 3 STANDARD DEV. FIG E -- 7: LOAD CNTR NETWORK TRANSFO RM ER AVERAGE ANALYTE CONC.S GENP 011034 _ 1 7 7 1 AVERAGE CONG. ANALYTE | \ \ J STANDARD DEV. FIGURE E -- 8: SP IK ED MINERAL OIL AVERAGE ANALYTE AND SPIKE CONC.S . GENP 011035 i t --yi l/ V j AVERAGE CONC. ,, ANALYTE fX X l SPIKED CONC. Y_7__7_7_X STAND. DEV. FIGURE E -- 9: SP IK ED A R 0 C L 0 R 1 0 1 6 AVERAGE ANALYTE AND SPIKE CONC.S o-"oJ 0C0O -p*. -vj GENP 011036 FIGURE E -- 10: SP IK E D AROCLOR 1260 AVERAG E ANALYTE A N D S P IK E CONC.S PII s CO OD Co _ / \ AVERAGE CONC. _____ ANALYTE | \ \ | SP IK E D CONC. HpCDF F ______ X ///A STAN D . DEV. A ppendix F TABLES OF RECO VERIES, R E L A T IV E R ECO VERIES AND D ETECTIO N L IM IT S GENP 01W38 783849 TABLE 1 RECOVERIES AND DETECTION LIMITS EPRI BASELINE PROJECT SAMPLE: 1953 LOAD CENTER NETWORK TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 %REC DET LIM . (.in p p b ) 43 2 . 1 47 2 . 1 55 2 . 1 55 2 .9 24 10 .2 43 3 . 0 24 20.3 DET. 2 %REC DET LIM . ( in ppb) 16 4 .3 19 4.0 24 3 .7 24 3 .7 8 23.5 16 6 .3 8 46.9 DET. 3 %REC DET LIM . ( inl p p b ) 3 15 .1 10 5 .2 14 4 .5 14 6 .1 7 19 .0 1 4 7.4 7 3 8 .1 2 ,3 ,7,8 -TC D D 43 2,3 ,7 ,8 -T C D F 43 2 ,3 ,4 , 8-TCD F NA 1 , 2 , 3 , 7 , 8-PnCDF 47 1 , 2 , 3 , 4 , 8-PnCDF 47 2 , 3 , 4 , 7 , 8-PnCDF 47 1 , 2 , 3 , 4 , 7 , 8-HxCDF 55 1 , 2 , 3 , 4 , 7 , 9-HxCDF 55 1,2 ,3 ,7 ,8 ,9 -H x C D F 55 1 , 2 , 3 , 6 , 7 , 8-HxCDF 55 2 , 3 , 4 , 6 , 7 , 8-HxCDF 55 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 55 1 . ) NA * n o t a p p l ic a b le 3 .0 2 .1 NA 2 .1 2 .1 2 .2 2 .1 2 .1 2 .1 2 .1 2 .5 2 .9 16 6 .3 16 4 .3 16 0 .1 19 4.0 19 4.0 19 4 .2 24 3 .7 24 3 .7 24 3 .7 24 3 .7 24 4 .4 24 5 .1 1 47 . 4 3 15 .1 NA NA 10 5 .2 10 5 .2 10 5 .5 14 4.5 14 4 .5 14 4 .5 14 4.5 14 5.3 14 6 .1 783850 GENP 011039 TABLE 2 RECOVERIES AND DETECTION LIMITS EPRI BASELINE PROJECT SAMPLE: 1946 LOAD CENTER NETWORK TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 ZREC DET LIM . ( in ppb) 29 1 . 2 28 1 . 5 31 1.5 31 2.0 22 5.6 35 1.6 22 10 .9 DET, 2 %REC DET LIM . ( in ppb ) 18 2 .1 _ 17 2 .5 18 2 .7 18 3 .5 7 17.9 18 3 .2 7 34.6 DET. 3 %REC DET LIM . (in ppb) 10 2 .9 27 1 . 3 30 1.3 30 1 .7 18 5.9 4 19 .0 18 1 1 . 4 2 , 3 , 7 , 8-TCDD 35 2 , 3 , 7 , 8-TCD F 29 2 , 3 , 4 , 8-TCD F NA 1 , 2 , 3 , 7 , 8-PnCDF 28 1 , 2 , 3 , 4 , 8-PnCDF 28 2 , 3 , 4 , 7 , 8-PnCDF 28 1 , 2 , 3 , 4 , 7 , 8-HxCDF 3 1 1 , 2 , 3 , 4 , 7 , 9-HxCDF 3 1 1 , 2 , 3 , 7 , 8 , 9-HxCDF 3 1 1 , 2 , 3 , 6 , 7 , 8-HxCDF 3 1 2 , 3 , 4 , 6 , 7 , 8-HxCDF 3 1 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 3 1 1.) NA not applicable 1.6 1.2 NA 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.7 2.0 18 3 .2 18 2 .1 18 0 .2 17 2 .5 17 2 .5 17 2 .5 18 2 .7 18 2 .7 18 2 .7 18 2 .7 18 3 .0 18 3 .5 4 19 .0 10 2 .9 NA NA 27 1 .3 27 1 .3 27 1 .3 30 1.3 30 1 .3 30 1 .3 30 1.3 30 1 .5 30 1 .7 GENP 011040 783851 TABLE 3 R E C O V E R IE S AND D E T E C T IO N L I M IT S EPR I BASELINE PROJECT SAMPLE: C A P A C IT O R TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 %REC DET LIM . ( in ppb) 35 0 .1 34 0 .2 38 0 .2 38 0 .2 35 V 61 0 .7 0 .1 35 1 .4 DET. 2 %REC DET LIM . ( in ppb) 17 0 .2 16 0.2 19 0 .2 19 0 .2 23 0 .3 18 0 .2 23 0 .6 DET. 3 %REC DET LIM . ( in ppb) 2 1.3 55 0 .1 63 0 . 1 63 0 . 1 59 0 .1 5 0.9 59 0 .3 2 , 3 , 7 , 8-TCDD 61 2 , 3 , 7 , 8-TCD F 35 2 , 3 , 4 , 8-TCD F NA 1 , 2 , 3 , 7 , 8-PnCDF 34 1 ,-2 ,3 ,4 , 8-PnCDF 34 2 , 3 , 4 , 7 , 8-PnCDF 34 1 , 2 , 3 , 4 , 7 , 8-HxCDF 38 1 , 2 , 3 , 4 , 7 , 9-HxCDF 38 1 , 2 , 3 , 7 , 8 , 9-HxCDF 1 , 2 , 3 , 6 , 7 , S-HxCDF 38 38 2 , 3 , 4 , 6 , 7 , 8-HxCDF .38 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 38 1 . ) NA * n o t a p p l i c a b l e 0 .1 0 .1 NA 0 .2 0 .2 0 .2 0 .2 0 .2 0 .2 0 .2 0 .2 0 .2 18 0 .2 17 0 .2 17 0.3 16 0 .2 16 0 .2 16 0 .2 19 0 .2 19 0 .2 19 0 .2 19 0 .2 19 0 .2 19 >0.2 F, -4k 5 0.9 2 .1.3 NA NA 55 0 .1 55 0 .1 55 0 .1 63 0 . 1 63 0 . 1 63 0 . 1 63 0 . 1 63 0 . 1 63 .0 .1 783852 GENP 011041 TABLE 4 RECOVERIES AND DETECTION LIMITS EPRI BASELINE PROJECT SAMPLE: PRECIPITATOR TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD ' OCDD DET. 1 ZREC DET LIM . ( in ppb) DET. 2 ZREC DET LIM . ( in ppb) 16 4 .2 . 24 1.5 17 4.3 28 1 . 5 19 4 .4 29 1 .6 19 5 .7 29 2 .1 14 15.0 17 7 .1 16 6 .5 26 2 . 3 14 30.6 17 14 .5 DET. 3 %REC DET LIM . ( in ppb) 21 2.2 32 1.6 34 1.7 34 2.3 24 6 .2 56 3 . 3 24 12 .7 2 , 3 , 7 , 8-TCDD 16 2 , 3 , 7 , 8-TCD F 16 2 , 3 , 4 , 8-TCD F NA 1 , 2 , 3 , 7 , 8-PnCDF 17 1 , 2 , 3 , 4 , 8-PnCDF 17 2 , 3 , 4 , 7 , 8-PnCDF 17 1 , 2 , 3 , 4 , 7 , 8-HxCDF 19 1 , 2 , 3 , 4 , 7 , 9-HxCDF 19 1 , 2 , 3 , 7 , 8 , 9-HxCDF 19 1 , 2 , 3 , 6 , 7 , 8-HxCDF 19 2 , 3 , 4 , 6 , 7 , 8-HxCDF 19 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 19 1 . ) NA n o t a p p l i c a b l e 6 .5 4 .2 NA 4 .3 4 .3 4 .3 4 .4 4 .4 4 .4 4 .4 4 .7 5 .7 26 2 .3 24 1 .5 24 0 .2 28 1 . 5 28 1 . 5 28 1 . 5 29 1.6 29 1.6 29 1.6 29 1.6 29 1 .7 29 2 . 1 56 3 .3 21 2 .2 NA NA 32 1.6 32 1.6 32 1.6 34 1.7 34 1.7 34 1.7 34 1.7 34 1.9 34 2.3 GENP 011042 F-5 783853 TABLE 5 RECOVERIES AND DETECTION LIMITS EPRI BASELINE PROJECT SAMPLE: 1976 ARC FURNACE TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 %REC DET LIM . ( in ppb) 67 0 .1 81 0 .1 82 0 .1 82 0 .2 63 0 .5 74 0 .1 63 1 .0 DET. 2 %REC DET LIM . ( in ppb) 37 0 .1 51 0 .1 51 0.0 51 0 .1 24 0 .4 45 0 .1 24 0 .8 DET. 3 %REC DET LIM . ( in ppb) 78 0.05 128 0.03 123 0 .0 4 123 0 .0 5 119 0 .11 0 .4 28 .12 119 0 .19 2 , 3 , 7 , 8-TCDD 74 2 , 3 , 7 , 8-TCD F 67 2 , 3 , 4 , S-TCD F NA 1 , 2 , 3 , 7 , 8-PnCDF 81 1 , 2 , 3 , 4 , 8-PnCDF 81 2 , 3 , 4 , 7 , 8-PnCDF 81 l i 2 , 3 , 4 , 7 , 8-HxCDF 82 1 , 2 , 3 , 4 , 7 , 9-HxCDF 82 1 , 2 , 3 , 7 , 8 , 9-HxCDF 82 1 , 2 , 3 , 6 , 7 , 8-HxCDF 82 2 , 3 , 4 , 6 , 7 , 8-HxCDF 82 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 82 1 . ) NA - n o t a p p l ic a b l e 0 .1 0 .1 NA 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .2 45 0 , 1 37 0 .1 37 0 .7 51 0 .1 51 0 .1 51 0 .1 51 0.0 51 0.0 51 0.0 51 0.0 51 0 .1 51 0 .1 0 .4 78 NA 128 128 128 123 123 123 123 123 123 28 .12 0 .0 5 NA 0 .0 3 0 .0 3 0 .0 4 0 .0 4 0 .0 4 0 .0 4 0 .0 4 0.0 4 0 .0 5 783854 F -6 GENP 011043 TABLE 6 RECOVERIES AND DETECTION LIMITS EFRI BASELINE PROJECT SAMPLE: SUBSTATION DISTRIBUTION TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD ` OCDD DET. 1 ZREC DET LIM . (in ppb) 36 0 .2 48 0 .2 60 0 . 1 60 0 .2 66 0 . 1 38 0.3 66 1 . 0 DET . 2 Z R E C 1DET L I M . (in ppb) 69 0 .0 4 80 0 . 0 4 36 0 .0 9 36 0 .1 2 4 2.14 80 0 . 0 5 4 2 1.6 1 DET .3 ZREC DET LIM . (in ppb) 76 0 .0 4 66 0 .0 5 33 0 .1 1 33 0 .1 3 7 1.33 73 0 .0 7 7 2.54 2 , 3 , 7 , 8-TCDD 38 2 , 3 , 7 , 8-TCD F 36 2 , 3 , 4 , 8-TCD F NA 1 , 2 , 3 , 7 , 8-PnCDF 48 1 , 2 , 3 , 4 , 8-PnCDF 48 2 , 3 , 4 , 7 , 8-PnCDF 48 1 , 2 , 3 , 4 , 7 , 8-HxCDF 60 1 , 2 , 3 , 4 , 7 , 9-H xCD F 60 1 , 2 , 3 , 7 , 8 , 9-HxCDF 60 1 , 2 , 3 , 6 , 7 , 8-H xCD F 60 2 , 3 , 4 , 6 , 7 , 8-H xCDF 60 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 60 1 . ) NA * n o t A p p l i c a b l e 0 .3 0 .2 NA 0 .2 0 .2 0 .2 0 .1 0 .1 0 .1 0 .1 0 .2 0 .2 80 0 . 0 5 69 0 .0 4 69 0 .0 4 80 0 . 0 4 80 0 . 0 4 80 0 . 0 4 36 0 .0 9 36 0 .0 9 36 0 .0 9 36 0 .0 9 36 0 .1 0 36 0 .1 2 73 0 .0 7 76 0 .0 4 NA NA 66- 0 . 0 5 66 0 .0 5 66 0 .0 5 33 0 .1 1 33 0 .1 1 33 0 .1 1 33 0 .1 1 33 0 .1 1 33 0 .13 GEKP 011044 783855 TABLE 7 RECOVERIES AND DETECTION LIMITS EPRI BASELINE PROJECT SAMPLE: 1962 LOAD CENTER NETWORK TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 ZREC DET L1M . ( in ppb) DET. 2 ZREC DET LIM . (in ppb) 42 2.0 45 1 . 4 51 2.0 . 47 1.6 51 2.3 40 2 . 2 51 3 .3 40 3 . 1 30 8.6 29 26 .7 46 2 . 8 50 2 .0 30 19 .4 29 1 2 4 .7 DET. 3 ZREC DET LIM . ( in ppb) 55 1 . 2 62 1.3 60 1 . 5 60 2 . 2 26 7 .7 59 1 . 7 26 1 7 . 3 2 , 3 , 7,8-TCD D 46 2 , 3 , 7 , 8-TCD F 42 2 , 3 , 4 , 8-TCD F NA 1 , 2 , 3 , 7 , 8-PnCDF 51 1 , 2 , 3 , 4 , 8-PnCDF 51 2 , 3 , 4 , 7 , 8-PnCDF 51 1 , 2 , 3 , 4 , 7 , 8-HxCDF 51 1 , 2 , 3 , 4 , 7 , 9-HxCDF 51 1 , 2 , 3 , 7 , 8 , 9-HxCDF 51 1 , 2 , 3 , 6 , 7 , 8-HxCDF 51 2 , 3 , 4 , 6 , 7 , 8-HxCDF 51 1 , 2 , 3 , 4 , 6 , 7 , 8 -HpCDF 51 1.) NA not applicable 2 .8 2.0 NA 2.0 2.0 1.8 2.3 2.3 2.3 2.3 2.6 3 .3 50 45 45 47 47 ' 47 40 40 40 40 40 40 2.0 1.4 0 .2 1.6 1.6 1.5 2 .2 2 .2 2 .2 2 .2 2 .5 3 .1 f-8 59 55 NA 62 62 62 60 60 60 60 60 60 1.7 1.2 NA 1.3 1.3 1.1 1.5 1.5 1.5 1.5 1.7 2 .2 783856 0U045 TABLE 8 RECOVERIES AND DETECTION LIMITS EFRI BASELINE PROJECT SAMPLE: SPIKED AGED MINERAL OIL TOTAL TCDP TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 ZREC DET LIM . ( in ppb) 50 0 .5 55 0.6 55 0 .7 55 1 . 1 49 3 .6 54 0.8 49 6 .3 DET. 2 ZREC DET LIM . ( in ppb) 45 2 .7 44 3 .2 43 3 . 7 43 4 . 8 33 1 3 .1 47 4 .0 33 12 3 .6 DET. 3 ZREC DET LIM . ( in ppb) 39 0 .1 43 0 . 1 40 0 . 1 40 0 . 1 27 0 .5 40 0 . 1 27 0 .9 2 , 3 , 7 ,8-TCD D 54 2 , 3 , 7 , 8-TCD F 50 2 , 3 , 4 , 8-TCD F NA 1,2 ,3 ,7 ,8 -P n C D F 55 1,2 ,3 ,4 ,8 -P n C D F 55 2,3,4 ,7,8 -P n C D F 55 1 , 2 , 3 , 4 , 7 , 8-HxCDF 55 1 . 2 , 3 , 4 , 7 , 9-HxCDF 55 1 , 2 , 3 , 7 , 8 , 9-HxCDF 55 1 , 2 , 3 , 6 , 7 , 8-HxCDF 55 2 , 3 , 4 , 6 , 7 , 8-HxCDF 55 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 55 1 . ) NA - n o t a p p lic a b le 0 .8 0 .5 NA 0.6 0 .6 0 .6 0 .7 0 .7 0 .7 0 .7 0 .8 1.1 47 4 .0 45 2 .7 45 0 .2 44 3 .2 44 3 .2 44 3 .2 43 3 . 7 43 3 . 7 43 3 . 7 43 3 . 7 43 4 . 1 43 4 .8 40 0 . 1 39 0 .1 NA NA 43 0 . 1 43 0 . 1 43 0 . 1 40 0 . 1 40 0 . 1 40 0 . 1 40 0 . 1 40 0 . 1 40 0 . 1 GETJPO11046 783857 TABLE 9 RECOVERIES AND DETECTION LIMITS EFRI BASELINE PROJECT SAMPLE: SPIKED AROCLOR 1016 TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 ZREC DET LIM . ( in ppb) 52 0.3 68 0 .2 77 0 .2 77 0 .4 103 0 .5 57 0 .4 103 1 .4 DET. 2 ZREC DET LIM . ( in ppb) 32 2.2 37 2.0 ` 36 2 .4 36 4 .0 37 7 .0 34 3 .1 37 10 0 .7 DET. 3 ZREC DET LIM . ( in ppb) 30 0 .9 44 0 .8 42 1 .0 42 1 .4 40 2 . 2 42 1 . 1 40 5 .0 2 , 3 , 7 , 8-TCDD 57 2 ,3 ,7,8 -T C D F 52 2,3,4 ,8 -T C D F NA 1 , 2 , 3 , 7 , 8-PnCDF 68 1 , 2 , 3 , 4 , 8-PnCDF 68 2 , 3 , 4 , 7 , 8-PnCDF 68 1 , 2 , 3 , 4 , 7 , 8-HxCDF 77 1 , 2 , 3 , 4 , 7 , 9-HxCDF 77 1 , 2 , 3 , 7 , 8 , 9-HxCDF 77 1,2 ,3 ,6 ,7 ,8 -H x C D F 77 2,3 ,4 ,6 ,7,8 -H x C D F 77 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 77 1 . ) NA * n o t a p p l i c a b l e 0 .4 0 .3 NA 0 .2 0 .2 0 .2 0 .2 0 .2 0 .2 0 .2 0 .3 0 .4 34 3 .1 32 2.2 32 2.3 37 2.0 37 2.0 37 2 .1 36 2 .4 36 2 .4 36 2 .4 36 2 .4 36 3 .2 36 4 .0 42 1 .1 30 0 .9 NA NA 44 0 .8 44 0 .8 44 0.7 42 1 .0 42 1.0 42 1.0 42 1.0 42 1 .1 42 1 .4 783858 G ENP 011047 TABLE 10 RECOVERIES AND DETECTION LIMITS EPRI BASELINE PROJECT SAMPLE: SPIKED AROCLOR 1260 TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD ' OCDD DET. 1 XREC DET LIM . ( in ppb) 52 0.5 58 0 .6 64 0.6 64 0.8 57 1 . 8 16 1 0.3 57 3 . 1 DET. 2 ZREC DET LIM . ( in ppb) 75 2 .4 80 2 . 7 .91 2.8 91 3 .8 72 9.4 112 2.5 72 74.6 DET. 3 ZREC DET LIM . ( in ppb) 63 4 .6 60 6 .0 61 6 .8 61 9 .4 45 2 4 .8 98 4 . 8 45 4 2 .3 2,3 ,7,8 -T C D D 16 1 0.3 1 1 2 2 .5 2 , 3 , 7 , 8-TCD F 52 0 .5 75 2 .4 2 , 3 , 4 , 8-TCD F NA NA 7 5 1 . 0 1 , 2 , 3 , 7 , 8-PnCDF 58 0 .6 80 2 . 7 1 , 2 , 3 , 4 , 8-PnCDF 58 0.6 80 2 . 7 2,3 ,4 ,7,8 -P n C D F 58 0 .6 80 2 . 7 1 , 2 , 3 , 4 , 7 , 8-HxCDF 64 0.6 91 2 .8 1,2 ,3 ,4 ,7 ,9 -H x C D F 64 0.6 91 2,8 1 , 2 , 3 , 7 , 8 , 9-HxCDF 64 . 0.6 91 2 .8 1 , 2 , 3 , 6 , 7 , 8-HxCDF 64 0 .6 91 2.8 2 , 3 , 4 , 6 , 7 , 8-HxCDF 64 0 .6 91 2 .9 1 , 2 , 3 , 4 , 6 , 7 , 8-HpCDF 64 0 .8 91 3 .8 1 . ) NA n o t a p p l i c a b l e 98 4 . 8 63 4 .6 NA NA 60 6 .0 60 6 .0 60 5 .8 61 6 .8 61 6 .8 61 6.8 61 6 .8 61 7 .1 61 9 .4 GENP 011048 783859 TABLE 11 RECOVERIES RELATIVE TO 2378-TCDD-13C12 EPRI BASELINE PROJECT SAMPLE: 1953 LOAD CENTER NETWORK TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 ZREC ZREC vs 13C-TCDD 43 100 47 109 55 128 55 128 24 56 43 100 24 56 DET . 2 ZREC ZREC vs 13C--TCDD 16 100 19 119 24' 150 24 150 8 50 16 100 8 50 DET . 3 ZREC ZREC vs 13C -TCDD 3 300 10 1000 14 1400 14 1400 7 700 1 100 7 700 2,3,7,8 -T C D D 43 2 , 3 , 7 , 8-TCD F 43 2,3,4 ,8 -T C D F NA 1 , 2 , 3 , 7,8-PnC D F 47 1 , 2 , 3 ,4,8 -P nC D F 47 2 , 3 , 4 , 7,8-PnC D F 47 1 , 2 , 3 , 4 , 7 , 8-HxCDF 55 1 , 2 , 3 , 4 , 7 , 9-HxCDF 55 1 , 2 , 3 , 7 , 8 , 9-HxCDF 55 1 , 2 , 3 , 6 , 7 , 8-HxCDF 55 2 , 3 , 4 , 6 , 7 , 8-HxCDF 55 1 , 2 , 3 , 4 , 6 , 7 , 8 -HpCDF 55 1 . ) NA n o t a p p lic a b le 100 100 NA 109 109 109 128 128 128 128 128 128 16 100 16 100 16 100 19 119 19 119 19 119 24 150 24 150 24 150 24 150 24 150 24 150 F-12 1 100 3 300 NA NA 10 1000 10 1000 10 1000 14 1400 14 1400 14 1400 14 1400 14 1400 14 1400 783860 GENP011049 TABLE 12 RECOVERIES RELATIVE TO 2378-TCDD-13C12 EPRI BASELINE PROJECT SAMPLE: 1946 LOAD CENTER NETWORK TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD l OCDD DET. 1 ZREC ZREC vs 13C -TCDD 29 83 28 80 31 89 31 89 22 63 35 100 22 63 DET. 2 ZREC ZREC vs 13C -TCDD 18 100 17 94 18 100 18 100 7 39 18 100 7 39 DET. 3 ZREC ZREC vs 13 C-TCDD 10 1000 27 2700 30 3000 30 3000 18 1800 1 100 18 1800 2 3,7,8-TCDD 35 2,3,7,8-TCDF 29 2,3,4,8-TCDF NA 12,3,7,8-PnCDF 28 1,2,3,4,8-PnCDF 28 2,3,4,7,8-PnCDF 28 1,2,3,4,7,8-HxCDF 31 1,2,3,4,7,9-HxCDF 31 1,2,3,7,8,9-HxCDF 31 1,2,3,6,7,8-HxCDF 31 2,3,4,6,7,8-HxCDF 31 1,2,3,4,6,7,8-HpCDF 31 1 . ) NA n o t a p p l ic a b l e 100 83 NA 80 80 80 89 89 89 89 89 89 18 100 18 100 18 100 17 94 17 94 17 94 18 100 18 100 18 100 18 100 18 100 18 100 1 100 10 1000 NA NA 27 2700 27 2700 27 2700 30 3000 30 3000 30 ' 3000 30 3000 30 3000 30 3000 a B N p o ,, OSg F -13 783861 TABLE 13 RECOVERIES R ELA TIVE TO 2 3 7 8 -T C D D -13 C 12 EPRI BASELINE PROJECT SAMPLE: C A P A C IT O R TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 ZREC ZREC v s 13C -TCDD 35 57 3 4 56 38 62 38 62 35 57 61 100 35 57 DET. 2 ZREC ZREC vs 13C -TCDD 17 94 1 6 89 19 " 106 19 106 23 1 2 8 1 8 100 23 1 2 8 DET. 3 ZREC ZREC v s 13C -TCDD 2 40 55 110 0 63 12 6 0 63 1260 59 1 1 80 5 100 59 118 0 2,3,7,8-TCDD 61 2,3,7,8-TCDF 35 2,3,4, 8-TCDF NA 1,2,3,7, 8-PnCDF 34 1,2,3,4, 8-PnCDF 34 2,3,4,7, 8-PnCDF 34 1,2,3,4,7, 8-HxCDF 38 1,2,3,4,7,9-HxCDF 38 1,2,3,7,8, 9-HxCDF 38 1,2,3,6,7,8-HxCDF 38 2,3,4,6,7,8-HxCDF 38 1,2,3,4,6,7,8-HpCDF 38 1 . ) HA not a p p lic a b le 100 57 NA 56 56 56 62 62 62 62 62 62 18 100 17 94 17 94 16 89 16 89 16 89 19 106 19 106 19 106 19 106 19 106 19 106 F -u 5 100 2 40 NA NA 55 1100 55 1100 55 1100 63 1260 63 1260 63 1260 63 1260 63 1260 63 1260 783862 G E N P o u 051 TABLE "l 4 RECOVERIES RELATIVE TO 237 8-TCDD-13C12 EPRI BASELINE PROJECT SAMPLE: PRECIPITATOR TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 %REC %REC vs 13C-TCDD 16 100 17 106 19 119 19 119 14 88 16 100 14 88 DET. 2 %REC %REC vs 13C-TCDD 24 92 28 108 29 112 29 112 17 65 26 100 17 65 DET. 3 %REC %REC vs 13C-TCDD 21 38 32 57 34 61 34 61 24 43 56 100 24 4 2,3,7,8-TCDD 16 2,3,7,8-TCDF 16 2,3,4, 8-TCDF NA 1,2.3,7,8-PnCDF 17 1,2,3,4, 8-PnCDF 17 2,3,4,7, 8-PnCDF 17 1,2,3,4,7,8-HxCDF 19 1,2,3,4,7,9-HxCDF 19 1,2,3,7, 8,9-HxCDF 19 1,2,3,6,7,8-HxCDF 19 2,3,4,6,7,8-HxCDF 19 1,2,3,4,6,7, 8-HpCDF 19 1 . ) NA not a p p lic a b le * 100 100 NA 106 106 106 119 119 119 119 119 119 26 100 56 100 24 92 21 38 24 92 NA NA 28 108 32 57 28 108 32 57 28 108 . 32 57 29 112 34 61 29 112 34 61 29 112 34 61 29 112 34 61 29 112 34 61 29 112 34 61 GENP 011052 p ,, 783863 TABLE 15 RECOVERIES RELATIVE TO 237 8-TCDD-13C12 EPRI BASELINE PROJECT SAMPLE: 1976 ARC FURNACE TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 %REC 2REC vs 13C-TCDD 67 91 81 109 82 111 82 111 63 85 74 100 63 85 DET. 2 %REC %REC vs 13C-TCDD 37 82 51 113 51 113 51 113 24 53 45 100 24 53 DET. 3 %REC %REC vs 13C-TCDD 78 19500 128 32000 123 307 50 123 30750 119 297 50 0.4 100 119 297 50 2,3,7,8-TCDD 74 2,3,7,8-TCDF 67 2,3,4, 8-TCDF NA 1,2,3,7, 8-PtiCDF 81 1,2,3,4, 8-PnCDF 81 2,3,4,7,8-PnCDF 81 1,2,3,4,7, 8-HxCDF 82 1,2,3,4,7,9-HxCDF 82 1,2,3,7, 8,9-HxCDF 82 1,2,3,6,7,8-HxCDF 82 2,3,4,6,7,8-HxCDF 82 1,2,3,4,6,7,8-HpCDF 82 1 . ) NA * no t a p p lic a b le 100 45 100 0.4 100 91 37 82 78 19500 NA 37 82 NA NA 109 51 113 128 32000 109 51 113 128 32000 109 51 113 128 32000 111 51 113 123 30750 111 51 ' 113 123 307 50 111 51 113 123 307 50 111 51 113 123 307 50 111 51 113 123 30750 111 51 113 123 30750 F'16 . 783864 GENP 011053 TABLE 16 RECOVERIES RELATIVE TO 2 3 7 8-TCDD-13C12 EPRI BASELINE PROJECT SAMPLE: SUBSTATION DISTRIBUTION TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 %REC 2REC vs 13C-TCDD 36 95 48 126 60 158 60 158 66 174 38 100 66 174 DET. 2 2REC ZREC vs 13C-TCDD 69 86 80 100 36 45 36 45 45 80 100 45 DET. 3 %REC %REC vs 13C-TCDD 76 104 66 90 33 45 33 45 7 10 73 100 7 10 2,3,7, 8-TCDD 38 2,3,7,8-TCDF 36 2,3,4, 8-TCDF NA 1,2,3,7,8-PnCDF 48 1,2,3,4, 8-PnCDF 48 2,3,4,7,8-PnCDF 48 1,2,3,4,7, 8-HxCDF 60 1,2,3,4,7,9-HxCDF 60 1,2,3,7, 8, 9-HxCDF 60 1,2,3,6,7, 8-HxCDF 60 2,3,4,6,7,8-HxCDF 60 1,2,3,4,6,7,8-HpCDF 60 1.) NA * not applicable 100 95 NA 126 126 126 158 158 158 158 158 158 80 100 69 86 69 86 80 100 80 100 80 100 36 45 36 45 36 45 36 45 36 45 36 45 73 100 76 104 NA NA 66 90 66 90 66 90 33 45 33 45 33 45 33 45 33 45 33 45 E N p 0 ,, 054 783865 TABLE 17 RECOVERIES RELATIVE TO 237 8-TCDD-13C12 EPRI BASELINE PROJECT SAMPLE: 1962 LOAD CENTER NETWORK TRANSFORMER TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 %REC IREC vs 13C-TCDD 42 91 51 111 51 111 51 111 30 65 46 100 30 65 DET. 2 %REC %REC vs 13C-TCDD 45 90 47 94 40 80 40 80 29 59 50 100 29 59 DET. 3 %REC %REC vs 13C-TCDD 55 93 62 105 60 102 60 102 26 44 59 100 26 44 2,3,7,8-TCDD 46 2,3,7,8-TCDF 42 2,3,4, 8-TCDF NA 1,2,3,7,8-PnCDF 51 1,2,3,4, 8-PnCDF 51 2,3,4,7,8-PnCDF 51 1,2,3,4,7, 8-HxCDF 51 1,2,3,4,7,9-HxCDF 51 1,2,3,7, 8,9-HxCDF 51 1,2,3,6,7, 8-HxCDF 51 2,3,4,6,7, 8-HxCDF 51 1,2,3,4,6,7, 8-HpCDF 51 1.) NA - not applicable 100 91 NA 111 111 111 111 111 111 111 111 111 50 100 45 90 45 90 47 94 47 94 47 94 40 80 40 80 40 80 40 80 40 80 40 80 59 100 55 93 NA NA 62 105 62 105 62 105 60 102 60 102 60 102 60 102 60 102 60 102 753866 GENP 011055 TABLE 18 RECOVERIES RELATIVE TO 237 8-TCDD-13C12 EPRI BASELINE PROJECT SAMPLE: SPIKED AGED MINERAL OIL TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 %REC %REC vs 13C-TCDD DET. 2 %REC %REC vs 13C-TCDD DET. 3 %REC %REC vs 13C-TCDD 50 93 55 102 55 102 55 102 49 91 54 100 49 91 45 96 44 94 43 91 43 91 33 70 47 100 33 70 39 98 43 108 40 100 40 100 27 68 40 100 27 68 2,3,7,8-TCDD 54 2,3,7,8-TCDF 50 2,3,4, 8-TCDF NA 1,2,3,7,8-PnCDF 55 1,2,3,4,8-PnCDF 2,3,4,7, 8-PnCDF 55 55 1,2,3,4,7, 8-HxCDF 55 1,2,3,4,7,9-HxCDF 55 1,2,3,7, 8, 9-HxCDF 1,2,3,6,7,8-HxCDF 55 55 2,3,4,6,7,8-HxCDF 55 1,2,3,4,6,7, 8-HpCDF 55 1 . ) NA not a p p lic a b le 100 93 NA 102 102 102 102 102 102 102 102 102 47 100 45 96 45 96 44 94 44 94 44 94 43 91 43 91 43 91 43 91 43 91 43 91 40 100 39 98 NA NA 43 108 43 108 43 108 40 100 40 100 40 100 40 100 40 100 40 100 GENP011056 F-19 783867 TABLE 19 RECOVERIES RELATIVE TO 237 8-TCDD-13C12 EPR1 BASELINE PROJECT SAMPLE: SPIKED AROCLOR 1016 TOTAL TCDF TOTAL PnCDF TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 ZREC ZREC vs 13C-TCDD 52 91 68 119 77 13 5 77 135 103 181 57 100 103 181 DET. 2 2REC %REC vs 13C-TCDD 32 94 37 109 36 106 36 106 37 109 34 100 37 109 DET. 3 %REC %REC vs 13C-TCDD 30 71 44 105 42 100 42 100 40 95 42 100 40 95 2,3,7,8-TCDD 57 2,3,7.8-TCDF 52 2,3,4, 8-TCDF NA 1,2,3,7, 8-PnCDF 68 1,2,3,4,8-PnCDF 68 2,3,4,7,8-PnCDF 68 1,2,3,4,7, 8-HxCDF 77 1,2,3,4<7,9-HxCDF 77 1,2,3,*7, 8,9-HxCDF 77 1,2,3,6,7.8-HxCDF 77 2,3,4,6,7,8-HxCDF 77 1,2,3,4,6,7, 8-HpCDF 77 1.) NA not applicable 100 91 NA 119 119 119 135 135 135 135 135 135 34 100 32 94 32 94 37 109 37 109 37 109 36 106 36 106 36 106 36 106 36 106 36 106 F-20 ' 42 .100 30 71 NA NA 44 105 44 105 44 105 42 100 42 100 42 100 42 100 42 100 42 100 783868 GENP 011057 TABLE'"20 RECOVERIES RELATIVE TO 237 8-TCDD-13C12 EPRI BASELINE PROJECT SAMPLE: SPIKED AROCLOR 1260 TOTAL TCDF TOTAL PnCDF .TOTAL HxCDF TOTAL HpCDF OCDF TOTAL TCDD OCDD DET. 1 m e ZREC vs 13C-TCDD 52 32 58 36 64 40 64 40 57 35 161 100 57 35 DET. 2 ZREC ZREC vs 13C-TCDD 75 67 80 71 91 81 91 81 72 64 112 100 72 64 DET. 3 ZREC ZREC vs 13C-TCDD 63 64 60 61 61 62 61 62 45 46 98 100 45 46 2,3,7,8-TCDD 161 100 112 100 2,3,7, 8-TCDF 52 32 75 67 2,3,4, 8-TCDF NA NA 75 67 1,2,3,7,8-PnCDF 58 36 80 71 1,2,3,4, 8-PnCDF 58 36 80 71 2,3,4,7,8-PnCDF 58 36 80 71 1,2,3,4,7, 8-HxCDF 64 40 91 81 1,2,3,4,7,9-HxCDF 64 40 91 81 1,2,3,7, 8,9-HxCDF 64 40 91 81 1,2,3,6,7,8-HxCDF 64 40 91 81 2,3,4,6,7,8-HxCDF 64 40 91 81 1,2,3,4,6,7, 8-HpCDF 64 40 91 81 1.) NA * not applicable 98 100 63 64 NA NA 60 61 60 61 60 61 61 62 61 62 61 62 61 62 61 62 61 62 G E N p 011058 F-21 783869 rhtf REPORT SUMMARY SUBJECTS Hazardous and toxic waste management ITransmission substation design and operation / Distribution substations TOPICS PCB Chemical analysis Transformers Capacitors PCDF-PCDD Insulating oil AUDIENCE Environmental managers IDistribution engineers Analysis of Polychlorinated Dibenzofurans and Polychlorinated Dibenzo-p-Dioxins in Transformers and Capacitors Volum es 1-3 PCDF and PCDD-- by-products of PCB pyrolysis and combus tion-- apparently cause many of the health effects originally attributed to PCB. In parallel and round-robin studies, indepen dent laboratories found that normal equipment operation and arc ing failure cause little if any oxidation and do not significantly modify PCB fluids. BACKGROUND Partial oxidation of askarels-- mixtures of polychlorinated biphenyls (PCB) and tri- and tetrachlorobenzenes-- produces polychlorinated dibenzofurans (PCDF) and polychlorinated dibenzo-p-dioxins (PCDD). However, analytic techniques for separating the active components in these mixtures had not matured prior to this study. Information had also been insufficient on the quantity and types of P C D F and P C D D in as-manufactured PCB and on the formation of these by-products during normal operation or arcing failure in utility equipment. Because of these uncertainties, utilities and regulators have assumed the worst-case PCDF-PCDD formation when there is a PC Brelated accident, thereby overstating the potential toxicity. EPRI report ' EUEA-4858 describes portions of the research performed in these projects. OBJECTIVE To improve techniques for measuring PCDF/PCDO in the presence of PCB. APPROACH Five laboratories (including two whose studies are discussed in report EUEA-4858) performed round-robin gas chromatography-mass spectrome try analyses of PCDF-PCDD samples using specially prepared carbon-T3 (13C) PC DF spiking compounds. After the first set of analyses, each labora tory modified its techniques and analyzed a larger group of samples from utility equipment. Another organization statistically analyzed all the labora tory results. RESULTS These three volumes report the second group of PCDF-PCDD analyses. Results from all participating laboratories correlated remarkably well. Seme of the results follow. ERRI EUEA-54439 Vaia. 1-3 783870  Use of the 13C compounds allowed successful separation of the com ponents in several closely eluting pairs of physiologically active PCDF and less-active compounds. Analyses of samples from utility equipment suggested that the PCDF-PCDD present-did-not result from high-load, high-temperature operation. Volumes 1 and 2 describe several analytic techniques, with the appen dix of Volume 2 including a report on the Analysis by the University of Umea in Sweden, as well. Volume 3 summarizes all the laboratory techniques, presents comprehensive statistical analyses, and provides an executive summary. EPRI PERSPECTIVE This research is a landmark in PC DF-PCDD analysis. One important result is the synthesis of new PCDFs for use as spiking compounds in gas chromatography-mass spectrometry analysis. The ability to sepa rate many of the active P C D F compounds from more innocuous materi als is also of great value. The high correlation of results from several laboratories indicates that researchers can exercise great flexibility in ' selecting techniques and developing facilities. PROJECTS RP2028-7, RP2028-6, RP2028-10 EPRI Project Managers: Gil Addis; Jacques Guertin Electrical Systems Division; Environment Division Contractors: New York State Department of Health; Battelle Columbus Laboratories; Research Triangle Institute For further information on EPRI research programs, call EPRI Technical information Specialists (415) 855-2411. 783871 GENP 011060 Analysis of Polychlorinated Dibenzofurans and Polychlorinated Dibenzo-p-Dioxins in Transformers and Capacitors Volume 2: Formation of PCDF and PCDD in Askarel and Contaminated Mineral Oil Equipment EL/EA-5443, Volume 2 Research Project 2028-6 Final Report, March 1988 Prepared by BATTELLE MEMORIAL INSTITUTE Battelle Columbus Division. 505 King Avenue Columbus, Ohio 43201*2693 Principal Investigators W, M. Cooke F. L. DeRoos With contributions from U M E UNIVERSITY Department of Organic Chemistry Ume, Sweden S-901 87 Principal Investigator C. Rappe Prepared for Electric Power Research Institute 3412 Hillview Avenue Paio Alto, California 94304 EPRI Project Managers G. Addis Transmission Substations Program Electrical Systems Division J. Guertin Land and Water Quality Studies Program Environment Division 783872 ORDERING INFORMATION Requests for copies of this report should be directed to Research Reports Center (RRC), Box 50490, Palo Alto, CA 94303, (415) 965-4081. There is no charge for reports requested by EPRI member utilities and affiliates, U.S. utility associations, U.S. government agencies (federal, state, and local), media, and foreign organizations with which EPRI has an information exchange agreement. On request. RRC will send a catalog of EPRI reports. E le an c Power Research Institute and E P R I are registered service m arks of Electnc Power Research Institute, In c Copyngnt 1988 Electnc Power Research institute In c All rights reserved. NOTICE This report w as prepared by 8A TT ELLE as an account of work sponsored by the Electric Power Research Institute. In c (EPRI), Neither EPR I. m em bers ot EPR I. BATTELLE. nor any person acting on benalf ot any ol them: (a) m akes any warranty, express or implied, with respect to the u se of any information, apparatus, method, or process disclosed in this report or rn a such use m ay not infringe pnvaiefy owned rights: or (b) assum es any liabilities with respect to the use of, or for dam ages resulting from the use of. any information, apparatus, method, or process disclosed in this report Furtherm ore if any item (for exam ple a model, prototype or test-piece) is delivered pursuant to this Agreem ent IT S H A L L C A R R Y N O W AR R AN T Y O R G U A R A N T E E W H A TSO EVER IN C LU D IN G W A R R A N T IES O F F IT N E S S FO R P U R P O SE O R O F M ER C H A N T A B ILIT Y .' 783873 ABSTRACT A study was conducted by five independent laboratories to evaluate methods for the measurement of trace levels of polychlorinated dibenzofurans (PCOFs) and polychlorinated dibenzo-p-dioxins (PCDDs) in utility dielectric liquids. Each laboratory evaluated a different analytical method. The method evaluated by Battelle involved spiking with labelled internal standards, analyte enrichment by column chromatography, and analysis by high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). Baseline analyses of five dielectric fluids were conducted by each laboratory as an initial test of their methods. The variability of results between the five methods was within expectations, and the five methods were deemed suitable for round robin evaluation. In the round robin evaluation, the five methods were used to analyze 10 liquid samples, including seven utility dielectric fluids. The liquid samples were spiked with native PCDD/PCDF to estimate accuracy. The Battelle results were generally within 70 percent of the spike value except for HxCDF which exhibited a strong positive bias. This result was considered an outlier. The Battelle results were consistently lower than the average results obtained for the five laboratories. Overall, the methods proved to be both rugged and reliable when used with labelled quantification and recovery standards. The in-service liquids used in this study were found to have relatively low levels of PCDDs and trace amounts of PCDF. Liquids from transformers with more than 25 years of service had higher PCDF levels than liquids from newer transformers. Liquids from utility appliances exposed to thermal and electrical stresses did not have elevated PCQD or PCDF levels. PCDF levels increased as the Aroclor number (and relative amount of chlorination) increased. GENP 011063 i11 783874 ACKNOWLEDGMENTS The authors would like to thank the EPRI technical management team led by Dr. Gilbert Addis. Dr. Addis and hts colleagues, Dr. Jacques Guertin, and Dr. Ralph Komai, guided this program and provided encouragement to all the participants. Dr. Thomas Rouse (General Electric Company) is acknowledged for his contributions concerning utility use of dielectric liquids and development of the engineering stress test protocols. The authors are grateful for the technical assistance provided by Battel le scientists who developed the methods, and performed the analyses reported in this document. Sample preparation was coordinated by Joe Hatchel, data computation was conducted by Susan Watson, and mass spectral analyses were performed by Dan Aichele, Charles Sueper, and Michael Zimmerman. We would also like to thank our colleagues who contributed the parallel analyses to this interlaboratory collaborative study. They are Dr. Sydney Gordon (Illinois Institute of Technology Research Institute), Drs. George Eadon and David Hilker (New York State Department of Health), Ms. Maureen Kilpatrick (Radian Corporation), Dr. Edo Pellizzari (Research Triangle Institute), and Dr. Christoffer Rappe (University of Umea). GENP 011064 v 783875 CONTENTS Section 1 INTRODUCTION 2 EXPERIMENTAL ACQUISITION OF IN-SERVICE SAMPLES ANALYTICAL TECHNIQUES SAMPLE PREPARATION AND SPIKING ANALYTE ENRICHMENT ANALYSIS QUALITY ASSURANCE RECOVERY OF INTERNAL STANDARDS QUANTIFICATION CALIBRATION CURVE DETECTION LIM IT S BASELINE ANALYSES IN-SERVICE ANALYSES 3 BASELINE ANALYSES BACKGROUND RESEARCH STANDARDS USED FOR SPIKING RESULTS Chemical A nalyses Baseline Recovery Data Method R e lia b ilit y 4 SUPPLEMENTAL ANALYSES CROSS CHECK ANALYSES ANALYSES FROM THE UNIVERSITY OF UMEA 5 ROUND ROBIN ANALYSES OF IN -SERV ICELIQUIDS Accuracy Estimate Comparison with P a ra lle lAnalyses 6 DISCUSSION GEKP011065 vl 1 783876 Pagi 1-1 2-1 2-1 2-2 2-2 2-4 2-5 2-5 2-5 2-6 2-7 2-8 2-8 2-8 3-1 3-1 3-2 3-4 3-4 3-4 3-10 4-1 4-1 4-1 5-1 5-1 5-13 6-1 Section METHODS IN-SERVICE LIQUID ANALYSES SUMMARY APPENOIX A APPENDIX B APPENDIX C IN-SERVICE LIQUID QUESTIONNAIRE ANALYTICAL CURVE DATA FOR THE CROSS CHECK ANALYSES LETTER REPORT FROM UMEA Pace 6-1 6-2 6-3 A-l B-l C-l 783877 v i l i GENP 011066 ILLUSTRATIONS Figure 4-1 C a lib ra tio n curve fo r 2,3,7,8-te trach lorod ibe n zofu ran over the range 40 pg to 2.5 ng GENP 011067 1x 783878 TABLES Table 2.1 EQUIPMENT RECOMMENDED FOR SAMPLING FOR PCOF AND PCDD ANALYSES 2.2 SURVEY OF LIQUID SAMPLES AVAILABLE FOR THE IN-SERVICE TRANSFORMER LIQUIDS STUDY 3.1 BASELINE TEST LIQUIDS 3.2 CONCENTRATION OF PCOF/PCDD STANDARDS SPIKED INTO TRANSFORMER LIQUIDS 3.3 BATTELLE FURAN AND DIOXIN DATA FOR THE BASELINE ANALYSES STUDY 3.4 IIT R I FURAN AND DIOXIN DATA FOR THE BASELINE ANALYSES STUDY 3.5 NYSDOH FURANAND DIOXIN DATA FOR THE BASELINE ANALYSES STUDY 3.6 RADIAN FURANAND DIOXIN DATA FOR THE BASELINE ANALYSES STUDY 3.7 A8SOLUTE RECOVERIES OF LABELLED STANDARDS IN THE BASELINE STUDY 3.8 FIVE LABORATORY RESULTS OF ANALYSIS OF AGED MINERAL O IL 5.1 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE IS L -0 2 -A 5.2 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE IS L -0 3 -A 5.3 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE IS L -0 4 -0 5.4 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE IS L -0 8 -A 5.5 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE IS L -1 7 -A 5.6 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE IS L -2 3 -0 5.7 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE IS L -2 7 5.8 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE EZ29-53-01 5.9 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE E729-53-02 5.10 CHLORINATED FURAN AND DIOXIN RESULTS - SAMPLE EZ29-53-03 5.11 ACCURACY DETERMINATION USING SPIKED 8ASELINE LIQ U ID ANALYSES 5.12 MEAN FURAN RESULTS FOR SEVEN UTILITY DIELECTRIC FLUIDS Page 2-2 2-3 3-1 3-3 3-5 3-6 3-7 3-8 3-9 3-11 5-2 5-3 5-4 5-5 5-6 5-7 5-8 5-9 5-10 5-11 5-12 5-14 783879 xl GENP 0U068 SUMMARY SYNOPSIS This report describes a study to develop reliable methods for the chemical analysis of polychlorinated dibenzo-p-dioxins (PCOD) and polychlorinated dibenzofurans (PCOF) in utility dielectric liquids. Prior to this study a great deal of data was available on levels of PCDO and PCDF in utility fluids; but since the methods used to obtain this data had never been rigorously challenged in a statistically valid study, the reliability of the existing data hases was suspect. Current analytical methods are affected by high levels of polychlorinated biphenyl (PCB). Since PC8 is an interfrent in most methods proposed for the analysis of PCDD/PCDF, and many existing utility test appliances contain PCB as the major dielectric fluid or as a contaminant of other fluids (due to residual PCB), investigation of reliable analytical methods was warranted. Five independent laboratories participated in this program. Each laboratory employed a different analytical procedure to measure trace levels of PCDF and PCDD in dielectric liquids. The method used by Battel!e involved spiking with labelled internal standards, analyte enrichment by multiple column chromatography, and analysis by high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). BASELINE ANALYSES The first phase of this study, baseline analyses, involved comparing methods for analyzing difficult matrices; e.q.. Aroclor. Baseline analyses of five dielectric fluids were conducted by each laboratory as an initial test of their methods. The variability of results among the five methods was within expected levels. The five analytical procedures proved to be rugged and reliable when used with isotopically labelled, quantification and recovery standards. Use of labelled standards is especially important in the Askarel and mineral oil matrices since the high organic burden found in these materials requires secondary partitioning to remove interfering PCB and background hydrocarbons. GENP011069 S-l 783880 I The relative average deviation is the absolute difference between individual results and the mean (or true value), divided by the number of determinations. For samples such as Aroclor 1242, Aroclor 1016, and mineral oil, the average deviation for baseline samples was often less than 50 percent. This result was found in individual laboratory replicate analyses and in grouped data from different laboratories. All five methods were deemed suitable for use in the second phase of the program. ROUND ROBIN STATISTICAL ANALYSIS The second phase of the study involved a more severe test, a round robin statistical evaluation that employed field samples collected from in-service utility applications. In the round robin evaluation, the five independent laboratories used different methods to analyze 10 liquid samples, including seven utility dielectric fluids. The in-service dielectric liquid samples were collected from four Askarel-filled network transformers, one arc furnace transformer, one electrostatic precipitator (ESP) transformer, and one capacitor. The Battel!e results were generally within 70 percent of the spike value except for hexachlorodibenzofurans, which exhibited a strong positive bias. This result was considered an outlier. The Battelle results were consistently lower than the average of results obtained for data from all five laboratories. All samples of the in-service liquids tested had relatively low amounts of PCDD. This result supports the hypothesis that dioxins are not a major constituent of Askarel and PCB-containing fluids. Except for the mineral oils, trace amounts of polychlorinated dibenzofurans were found in all of the Aroclor samples tested. The mineral oil samples had extremely low amounts, if any, of PCOF. Even the inservice liquid from Transformer 4, a network distribution transformer that was involved in a high-energy excursion, had minimal amounts of PCDD and PCDF. Transformer 4 had visible carbon, and major signs of scorching on the case and in the area of the electrodes. The Askarel transformers with more than 25 years of service tended to have higher levels of PCDF than the appliances put into service for a shorter time. It is possible that manufacturing controls improved as the production of Askarel matured in the 1960s such that Askarel was produced with lower levels of PCDF in the asdelivered fluids. g ENP011070 S-2 Two utility appliances were selected by the^EPRI Technical Advisory Committee for special attention: an Askarel-filled ESP transformer and an oil-filled arc furnace transformer. These two samples were the "worst case" for testing service stress on the dielectric fluids. The arc furnace had essentially no PCDF or PCDD, and the PCDF levels in the ESP transformer appeared to be dictated by the age of the unit (27 years of service) and the fluid composition (Aroclor 1260). This extended period of stress did not appear to elevate the PCDF levels above typical Aroclor 1260 levels. One capacitor was tested in the in-service liquid screening program. It was found to have relatively low levels of PCOF compared to the other Askarel-filled units. CONCLUSIONS The in-service liquids used in this study were found to have minimal levels of PCDD and only trace amounts of PCDF. Liquidsfrom transformers with more than 25 years of service had higher PCDF levels than liquids from newer transformers. Liquids from utility appliances exposed to thermal and electrical stresses did not show elevated PCDD or PCOF levels, indicating that high-energy events (electrical and thermal excursions) do not appear to generate PCDD or PCDF in utility electrical appliances that employ PC8 . PCOF levels increased as the Aroclor number (and relative amount of chlorination) increased. This study was successful indeveloping a set of methodologies that reliably determine trace levels of PCDF and PCDDin a very difficult matrix -- chlorinated organic oil. The development of commercially available reference standards, and the statistical validation of reference methods that can be used to analyze utility fluids was a major contribution .to the quality and defensibility of data. The method verification program showed that a range of instrumentation can be successfully used to measure trace levels of PCDF and PCDD in utility fluids. In general, the methods employing low-resolution mass spectrometry (LRMS) require additional column chromatography to isolate PCDF/PCDD-enriched fractions for final HRGC/LRMS analysis. The precision of blind, spiked samples was superior to that expected by experienced analysts who had previously performed these analyses. The availability of labelled quantification standards helped improve method performance and lowered data scatter. GENP 011071 S-3 783882 Section 1 INTRODUCTION The Electric Power Research Institute initiated this program to investigate the chemistry and analysis of polychlorinated dibenzofurans (PCDF) and polychlorinated dibenzo-p-dioxins (PCDD) as they relate to the utility use of Askarel, a dielectric liquid which contains polychlorinated biphenyl (PCS), frequently diluted with tri/tetra chlorinated benzene. Askarel is sometimes contaminated with traces of PCDF/PCDD. The program had three goals; (I) to develop reliable, well characterized analytical methods to measure PCDF/PCDD, (2) to measure PCDF/PCDD levels in typical dielectric liquids used in utility operations, and (3) to investigate possible PCDF/PCDD production in unusual transformer events such as high energy arcing. This report describes analytical methodology and results of chemical analyses conducted by Battel!e Columbus Division in support of EPRI Research Project 20286 , "Formation of PCDD and PCDF in Askarel and Contaminated Mineral Oil Equipment". Chemical laboratories of four additional organizations conducted parallel measurements during this program: the Illinois Institute of Technology Research' Institute, the New York State Department of Health, Radian Corporation, and the University of UMEA (Sweden). The data presented in this report were replicated by these four laboratories and separate reports are available from each of the study participants. Combined data from the replicate analyses conducted by the five collaborating laboratories (including Battelle), are presented in a summary report prepared by the Research Triangle Institute. The summary report examines pooled data and gives a rigorous statistical analysis, including variability and reliability of analytical results reported by laboratories routinely conducting PCDF/PCDD analyses in Askarel, mineral oil, and related sample matrices. Reliable analytical methods are especially difficult to develop for Askarel since PCB constitutes the major interference in most PCDF/PCDD analytical methods. In Askarel, the normal PCB concentration is 60-70 percent by weight. Thus, Askarel presents perhaps the most difficult matrix for these analyses. Parts-per-billion (ppb) levels of PCDF, and even lower concentrations of PCDD must be measured. When this program was initiated in early 1984, the accuracy of PCDF analyses was hampered by a lack of accurate quantification standards. A major product of this 1-1 GENP 011072 study was' the synthesis of a series of ^C-labelled PCDF standards that allowed use of sensitive isotope dilution techniques to measure PCDF. Quantification and recovery measurements based on labelled standards permit the chemist to measure accurate mass spectral response ratios for naturally occurring PCDF (and PCDD) compared to the response factors of 1abel1ed standards spi ked into the sample matrix. The method development phase involved testing methods proposed by the five participating laboratories; Battelle, the Illinois Institute of Technology, Research Institute (IITRI), the New York State Department of Health (NYS), Radian Corporation, and the University of Umea. These five methods were challenged by analyzing PCDF/PCDD in baseline liquids, a series of five typical sample matrices: Aroclor 1260, Aroclor 1242, Aroclor 1016, chlorobenzene, and mineral oil containing approximately 50 ppm of Aroclor 1260. The baseline Askarel liquids contained native PCDF/PCDD, and the mineral oil was spiked with labelled PCDF. The presence of PCDF/PCDD in typical utility liquids was investigated by measuring characteristic compounds and congener groups in samples provided by EPRI member utilities. A report was generated by the General Electric Company which outlined the main types and distribution of PCB containing liquids'(1). A repository was established at Battelle where samples of transformer oils provided by several EPRI members were stored. A questionnaire was developed, and a data base was accumulated on the repository samples. Seven of these liquids were selected by the EPRI Technical Advisory Committee. The seven samples, along with three control liquids, were analyzed by the participating laboratories. The seven "in-service" liquids selected for analysis represented either high energy operation, or utility applications that typify PCB usage. The data collected in the in-service liquid study were analyzed by chemists and statisticians at the Research Triangle Institute (RT1)., A separate report will be generated by RTI that summarizes the results of all five laboratories. In summary, the independent methods worked well, indicating that good results can be expected when appropriate quality control procedures are applied. Arcing experiments were conducted by General Electric. Transformer test units were fabricated out of the normal materials of transformer construction. These test units were exposed to high electric energy such as might be expected in catastrophic transformer failure. In the engineering tests, final PCOF/PCOD levels were either the same or lowered from initial levels, indicating that short term GENP011073 1-2 783884 electrical discharges are not a primary-- source of production for PCOF/PCDD in transformer liquids. 783885 1-3 GENP 011074 Section 2 EXPERIMENTAL ACQUISITION OF IN-SERVICE SAMPLES The selection of in-service dielectric fluids for this study was coordinated by the EPRI Technical Advisory Committee with the assistance of Dr. Thomas Rouse of the General Electric Company. It was important to select a series of in-service liquids that represented a range of utility applications. In the early planning for this program, Or. Rouse prepared a criteria document that discussed the distribution of Askarel among different types of electrical devices, the relative chemical stresses expected from these devices in normal usage, the dispersion of Askarels, heating processes in transformers, and situations that represent the worst case for Askarel usage. He estimated that the United States has 140,000 Askarel-filled transformers in service, 30 million oil-filled transformers, and 510 million power capacitors. The oil-filled transformers constitute a large number; however, only about 50,000 are considered PCB-containing (>500 ppm (w/w)), and another approximately 1 million are PCB-contaminated (50-500 ppm). Since PCDF and PCDD are minor constituents of PCB, PCB-containing and PCB-contaminated transformers are expected to contain very low concentrations of PCDF/PCDD (perhaps not measurable). Dr. Rouse published an EPRI report (1) in which he recommended establishing a repository of 30-40 in-service samples that would be reviewed by the technical advisory group to select a final set of In-service liquids for exhaustive testing by the methods developed in this program. The samples recommended by Or. Rouse for inclusion in the final test group are given in Table 2-1. In order to acquire suitable samples for this study, several participating utility representatives were asked to survey their records on available samples, searching for dielectric materials that met the criteria established by Dr. Rouse. To prescreen the available samples, a questionnaire was developed and a data base of questionnaire information maintained. A copy of the questionnaire is given in Appendix A. 783886 2-1 TABLE 2..L EQUIPMENT RECOMMENDED FOR SAMPLING FOR PCOF ANO PCDO ANALYSES ASKAREL-FILLED Load Center/Network Transformers Rectifier Transformers Precipitator Transformers Capacitors 8-10 1-2 2 4 OIL-FILLED WITH PCB Load Center/Network Transformers 4 After gathering completed questionnaires on a large number of candidate fluids, a repository was established, that contained the samples listed in Table 2.1. As shown in Table 2.2, suitable dielectric liquids were collected from each of the categories of interest. A final selection was made using information in the repository data base, and the*materials listed in the last column of Table 2.2 were selected for inclusion in the in-service testing program. ANALYTICAL TECHNIQUES The following method description gives the procedures that were used by Battel!e to analyze all Askarel and mineral oil samples in this program. The samples preparation procedures, the spiking protocols, the instrumental conditions, and the equations used to calculate final results are given here. The oil samples (five "baseline1' samples) were spiked with labelled internal standards prior to distribution to the participating laboratories. The sample preparation and spiking procedures for the baseline liquids are given in a separate report in this series prepared by Radian Corporation (Austin, TX). SAMPLE PREPARATION AND SPIKING The seven in-service liquids were diluted to a concentration of approximately 2 pg/mL with hexane prior to spiking with internal standards. Aliquots of each liquid were weighed by difference into 100 mL volumetric flasks which were then filled to the mark with DIstilled-in-Glass hexane. The flasks were placed on a 783887 2-2 GENP 0U076 O o *^4 IV) i 00 CCOO CO CO orSURVEY LIQ U ID SAMPLES Utility ___________Appliance___________ CAPACITORS Capacitor 1 Capacitor 2 Capacitor 3 TRANSFORMERS Arc Furnace 1 Arc Furnace 2 CIRCUIT BREAKER Circuit Breaker LOAD CENTER NETUORK TRANSFORMERS Network Load Center 1 Network Load Center 2 Network Load Center 3 Network Load Center 4 Network Load Center S Network Load Center 6 Network Load Center 7 Network Load Center 8 Network Load Center 9 Network Load Center 10 Network Load Center 11 Network Load Center 12 Network Load Center 13 Network Load Center 14 Network Load Center IS Network Load Center 16 Network Load Center 17 Network Load Center 18 Network Load Center 19 Network Load Center 20 PRECIPITATORS Precipitator 1 Precipitator 2 Precipitator 3 Service Life (Years 1 1 7 ? 8 8 30(Est.) 30(Est.) 21 28 20 19 20 19 31 30(Est.) 30(Cst.) 2Q(Est.) l5(Est.) 10(Est.) 20(Est.) 20(Est.) 20(Est.) 10(Est.) 20(Est.) 20(Est.) 20(Est.) 27 TAnif 2.2 A U A IlA Rl C ron Tile 1N -SCRVIC TRAKSrORHEft IIJ IIIO S STUDV Rat inq Dielectrtc PCfi (ppm) Voluiae (CaKJ Field Code Laboratory Code 7200 KVAR 7200 KVAR ? ?7 Aroclor 1242 --- ? ISL-28 7 1SI-27 41159-11-13 7 1SL-01-A -- 12470/280 KVA Mineral O H 12470/280 KVA Hiera! Oil 161 ISO 4.500 4,500 ISL-24-0 ISL-23-0 4115911-15 34.5 KV Mineral 011 403 360 ISL-29-0 450 KVA Mineral 011 248 7 ISL-OS-O 1500 KVA Aroclor 1242 -- 500 1SL-06-A -- 1000 KVA Inerteen -- 3780 (Ib) 15L-07-A -- 500 KVA Aroclor 1260 -- 3175 (Ib) 1SL-08-A 41159-11-12 750 KVA * Aroclor 1260 - - 2650 (Ib) ISL-02-A 41159-11-17 500 KVA Aroclor 1260 - - 7 1SL-09-A - - 750 KVA Aroclor 1260 -- 240 1SL-10-A -- 500 KVA Arador 1260 3200 (Ib) I5L-U-A -- 500 KVA Aroclor 1260 ? ISL-03-A 41159-11-11 243 KVA Pyranol 195 ISL-12-A -- 559 KVA- Pyranol -- 370 ISL-13-A -- 313 KVA ? -- 195 ISL-14 -- 339 KVA Pyranol -- 360 ISL-15-A -- 126 KVA Pyranol -- 490 ISL-I6-A -- 11 KVA Inerteen -- 235 ISL-18-A 11 KVA Chlorestol -- 272 1SL-19-A -- 11.5 KVA Inerteen -- 282 ISL-2Q-A -- 11.5 KVA Inerteen 184 ISL-21-A -- 11.5 KVA Chlorexdol 277 ISL-22-A -- ? Mineral Oil 100 200 1SL-04-0 41159-11-16 440/53.5 KVA 440/53.5 KVA 440/53.5 KVA 7 ? 7 94 1SL-26 -- 94 1SL-25 143 ISL-17-A 41159-11-14 mechanical shaker and mixed overnight. After approximately 12 hours of mixing, the flasks were removed from the shaker and spiked with the five isotopically labelled internal standards. The internal standards were: 2 ,3 ,7 ,8 -TCDD-13Ci2 2 ,3,7,8TCDF-13C12, l,2,3,7,8-Penta-C0F-13C 12, l,2,3,4,7,8-Nexa-CDF-13C12, and Octa-CDD13Ci 2 . ANALYTE ENRICHMENT One-milliliter aliquots (containing *250 mg of the original fluid) of each sample were transferred to separatory funnels and diluted with 49 mL of hexane. The hexane solutions were washed with 10-ml portions of concentrated sulfuric acid until the washes were only slightly colored. A maximum of five acid washes were used. Following the acid washes, each oil was washed with 20 ml of distilled water to remove any residual acid. The hexane solutions were transferred to glass collection tubes and concentrated to approximately 2 mL using gentle streams of nitrogen gas. The hexane solutions w'ere transferred to multilayered silica gel columns containing activated silica gel, 44 percent concentrated sulfuric acid on silica gel, and 33 percent 1M sodium hydroxide on silica gel. The purpose of these columns was to remove acidic and basic compounds from the solutions as well as materials that were easily oxidized. The silica gel support provided a large surface area for contact with the sample solutions thus improving the cleanup efficiency. The PCDD/PCDF isomers were eluted from the columns using 70 mL of hexane and the entire eluents, including the original sample volume, were collected. The el uates from the multi1ayered silica gel columns were concentrated to approximately 1 mL using gentle streams of nitrogen and transferred to the top of columns containing 5 g of activated basic alumina. These columns were eluted using hexane, hexane/methylene chloride (97:3, v/v) and hexane/methylene chloride (1:1, v/v) as elution solvents. The 1:1 hexane/methylene chloride eluates were collected, solvent exchanged into hexane, and transferred to the top of alumina columns containing approximately 2 g of activated basic alumina. These columns were eluted with hexane, hexane/methylene chloride (97:3, v/v), and hexane/ methylene chloride (1:1, v/v). The 1:1 hexane/methylene chloride eluates were collected, concentrated to near dryness with nitrogen gas, and spiked with 10 ng of l,2,3,4-TC00-13Ci2- These solutions were stored at 0C until they were analyzed. Several, of the sample solutions were still too contaminated with interferences to be analyzed. These solutions ' (41159-11-12,13,14,17 and 729-53-02,03) were i 2-4 GENP 011078 783889 chromatographed through florisil columns to provide additional analyte enrichment. Each of the florisil columns was prepared using 5 g of activated florisil (60-100 mesh) which was slurry packed with hexane. The sample solutions were diluted with approximately 5 mL of hexane and transferred to the florisil columns. The columns were eluted with hexane, hexane/ethyl ether (94:6, v/v), and hexane/methylene chloride (25:75, v/v). The 25:75 hexane/methylene chloride fraction was collected and concentrated to approximately 20 pL. These solutions were stored at 0C until they were analyzed. ANALYSIS The sample solutions were analyzed and quantified for PCDD/PCDF using combined capillary column gas chromatography/high resolution mass spectrometry (HRGC/HRMS). The HRGC/HRMS consisted of a Carlo Erba Model 4160 Gas Chromatograph interfaced directly into the ion source of a VG Model 7070 high resolution mass spectrometer. The chromatographic column was a 60 M OB-5 fused silica column using helium carrier gas at a flow velocity of 30 cm/sec. The mass spectrometer was operated in the electron impact (El) ionization mode at a mass resolution of 9000-12000 (M/ M, 10 percent valley definition). All HRGC/HRMS data were acquired by multiple-iondetection (MID) using a VG Model 2035 Data System. QUALITY ASSURANCE The operation of the HRGC/HRMS was evaluated each day by analyzing standard mixtures of selected PCDD/PCDF compounds. These were used to define the chromatographic elution windows, the compound resolution of the chromatographic column, and to verify the quantitative accuracy of the analyses. The mass focus accuracy of the MID unit was evaluated at least every four hours by observing selected ion masses from perfluorokerosene (PFK) which was introduced continually into the ion source as a reference compound. Mass focus stability was assured by using a reference PFK "lock mass'1 to correct for any mass focus drift. Native spike and laboratory method blank samples were also processed during the extraction and cleanup of the samples. The native spike samples were used to evaluate the accuracy of the quantification while the laboratory method blank sample was used to demonstrate freedom from laboratory contamination. 783890 RECOVERY OF INTERNAL STANDARDS The recoveries of the internal standards were calculated by comparison to an external standard, l,2,3,4-TCD0-^Ci2 which was added following the column cleanup steps. Relative response factors were determined from triplicate analyses of a GENP 011079 2;5 standard mixture containing the labelled internal standards and the 1,2,3,4-TCDD^ 2 * The equation used to calculate the recoveries was: Recovery (%) Ais x Ors x 100 Ars x Qis x Rf Where: Ais - Sum of integrated areas for internal standard; Qrs - Quantity of recovery standard in ng; Qis - Quantity of internal standard in ng; Ars - Sum of integrated areas for recovery standard; and Rf - Response factor. QUANTIFICATION The PCDO/PCOF isomers and congener class concentrations were determined by comparing the sum of the two ion masses monitored for each class to the sum of the two ion masses monitored for the corresponding internal standard. It was assumed that the response factors for each of the individual isomers in a class were the same as the response factors calculated for the specific isomers present in the standard solutions. The response factors were calculated from six replicate analyses of standard solutions at four concentrations. The sum of the two most intense ion masses in the molecular ion region of eachisomer were used to generate the response factors.. The formula used for quantifying the PCDD/PCDF isomers was: Quantity/Saraple (ng/g) - A i s V l ^ x W Where: Quantity - Total quantity in ng of target isomer or congener class Ac - Sum of integrated areas for the target isomer or congener class Qis - Quantity of internal standard in ng Ais - Total integrated areas for the internal standard Rf " Response factor. 783891 Each pair of resolved peaks in the selected-ion-current chromatograms was evaluated manually to determine if it met the criteria for a PCDD or PCDF isomer. By examining each pair of peaks separately, quantitative accuracy was improved over what 1s obtained when all of the peaks in a selected chromatographic window are averaged. When averaged data are used, it Is possible for pairs of peaks with high and low chlorine isotope ratios to produce averaged data that meet the chlorine isotope ratio criterion. For example, two pairs of peaks having chlorine isotope ratios of 0.56 and 0.96, both outside of the acceptable range, would have an 2-6 GENP 011080 average, ratio of 0.76. By checking the isotope ratio of these peaks separately, they would be eliminated from the class total. The criteria that were used to identify PCDO and PCDF isomers were: 1. Simultaneous responses at both ion masses. 2. Chlorine isotope ratio within +/-!$% of the theoretical value. 3. Chromatographic retention times within windows determined from analyses of standard mixtures. 4. Signal-to-noise ratio equal to or greater than 2.5 to 1.0. The individual isomers for which an isotopically labelled analog was available included the additional criterion that they eluted within 2 seconds of the internal standard. The limit of detection (LOD) was calculated for samples in which isomers of a particular chlorine congener class were not detected. The formula used for calculating the LOO was: L00/g (ng) He x Qis x 2.5 His x Rf x W Where: LOD = Single isomer limits of detection for a congener class in ng; He = Height of congener class isomer; Qis - Quantity of internal standard in ng; His * Peak height of internal standard; Rf * Response factor; and W - Weight in grams of sample. CALIBRATION CURVE Calibration curves were generated for each of the native/isotopically labelled isomer pairs. The calibration standards were analyzed at least five times at each of four concentration levels between 0.04 and 2.5 parts-per-million (ppm). The response factors were calculated by dividing the sum of the areas for the two most abundant ion masses in the molecular ion cluster of each native PCDD/PCDF isomer by the sum of the areas for the corresponding ion masses from the isotopically labelled standard. The isomer pairs for which, response factors were calculated included: GENP 011081 2-7 783892 2.3.7.8- TCDD vs 2,3,7,8-TCDD-J3Ci2 --2,3,7,8 -TCOF ys 2,3,7,8 -TCDF-13Ci2 -- OCOO vs 0CD0-}3Ci2 OCDF vs OCOO-13Ci2 1.2.3.7.8 - Penta-tDF vs 1,2,3,7,8 -Penta-CDF-33Ci2 2.3.4.7.8- Penta-COF vs l,2,3,7,8-Penta-C0F-13Ci2 1.2.3.4.7.8- Haxa-CDF vs 1,2,3,4,7,8-Hexa-CDF-J3Ci2 2.3.4.6.7.8- Hexa-COF vs l,2,3,4,7.8-Hexa-COF-13C12 1.2.3.4.6.7.8- Hepta-CDF vs 0CD0-13Ci2 It was assumed that each of the isomers of a particular chlorine congener class had the same response factor. DETECTION LIMITS Detection limits were calculated for samples that did not contain PCDD/PCDF isomers in a particular class. These detection limits were calculated using the quantification equation with the addition of a factor of 2.5 to account for the signal-to-noise criterion. The noise level was measured as the average of the peak to peak noise that occurred in the baseline of the particular MID channel. If at all possible, the noise was measured at the retention time of the isotopically labelled internal standard. The height of the internal standard peak was measured to the average of the noise that occurred on the peak top. The reported limits of detection are for single PCDD/PCDF isomers and have been corrected for recovery losses during sample workup. BASELINE ANALYSES The three baseline samples, Aroclor 1260, Aroclor 1254, and Aroclor 1016, were analyzed using the methodology described above. The Aroclor 1016 presented the greatest analytical problem due to the relatively high amount of the sample that passed through the column cleanup steps. Even after the four column cleanup steps, a noticeable PCB background was evident 1n the single ion current chromatograms. IN-SERVICE ANALYSES The seven in-service samples were also analyzed using the methodology described above. 783893 GENP 011082 2-8 Section 3 BASELINE ANALYSES This section describes the baseline analyses performed by all five laboratories participating in this study. The baseline analyses were designed as a rigorous blind analysis of authentic Aroclor and mineral oil samples. If the proposed analytical methods performed well in the baseline tests, the additional steps of method validation* and screening of in-service utility fluids could be performed with confidence in the reported results. Consequently, the baseline analyses were critical to the success of this program. BACKGROUND RESEARCH Before the baseline analyses could be performed, five test liquids were obtained by Radian Corporation and a combination of native and labelled (13C) PCDF/PCDD were spiked into the test liquids to ensure a rugged test of the proposed analytical techniques. The test liquids selected for the baseline study are listed in Table 3,1. Dielectric Fluid Aroclor 1016 Aroclor 1242 Aroclor 1260 Chlorobenzenes Aged Mineral Oil TABLE 3.1 BASELINE TEST LIQUIDS Narrow boiling point fraction of Aroclor 1242. 42% Cl (w/w) 60% Cl (w/w) -Synthetic Mixture 1.2.4- Trichlorobenzene (51.2% (w/w)) 1,2,3-Trichlorobenzene (13.4% (w/w)) 1,2,4,5-Tetrachlorobenzene ( 2.6% (w/w)) 1.2.3.4- Tetrachlorobenzene (28.4% (w/w)) Pentachlorobenzene ( 4.4% (w/w)) -Spiked Aroclor 1260 (500 ppm (w/w)) Tri/Tetrachlorobenzene Mix Listed Above (500 ppm (w/w)) g ENP01W83 3-1 7 8 3 8 9 4 RESULTS Chemical Analyses The results of chemical analyses performed by Battelle on the baseline samples are given in Table 3.3. All results are given as parts-per-billion by weight in the original oil (ng/g). These data are interesting because they show the expected trends in the Aroclor PCDF/PC0O concentrations. PCDD are not found at the detection limit of the high resolution mass spectrometer used for this analysis, approximately 10 ppb (w/w) in the mineral oil samples. Trace levels of PCDF are observed with the concen trations increasing as the level of chlorination increases in the supporting Aroclor. The PCDF levels increase as the Aroclor number (and relative amount of chlorination- increase), Aroclor 1260 >Aroclor 1242 >Aroclor 1016. Even though Aroclor 1016 is similar in composition to Aroclor 1242, the wider distribution of PCB congener groups in Aroclor 1242 would produce higher quantities of the high chlorination number PCB, 6-10 chlorine atoms per molecule, which are believed to be the precursors of PCDF when HC1 elimination is the route for PCDF formation. An increase in PC0F concentrations is shown in Table 3.3 as the total high chlorine PCB concentration increases. Total PCDF levels increase from 280 ppb for Aroclor 1016, to 1,710 ppb for Aroclor 1242, to 10,4 ppm for Aroclor 1260. Data for three of the participating laboratories are given in Tables 3.4 through 3.6. These data show the same trends observed in the Battelle data. The measured levels of spiked PCDF/PCDF were also similar among the reporting laboratories. 8aseline Recovery Data The recovery of labelled standards is used to determine how much of the native PCDF/PCOO present 1n the sample 1s lost during the sample preparation steps of candidate methods. Since the internal quantification standard corrects for method losses, the reported value should be accurate within the measured precision of the method; however, excessive loss of analytes during sample preparation will have a profound affect on both method sensitivity, and precision. Recoveries for six labelled PC0F/PC0D standards spiked into the baseline samples are given in Table 3.7. These data are interesting because they show the outliers that can affect Individual data points. 783895 3-4 TABLE 3.3 BATTELLE FURAN ANO DIOXIN DATA FOR THE BASELINE ANALYSES STUDY (ppb (w/w)) 011085 Comoound Aroclor Aroclor Aroclor Tri and Tetra 1016 1242 1260 Chlorobenzene Mineral Oil Run No. 1 Mineral Oil Run No. 2 Polychlorinated Dibenzofurans Total TCDF Total PnCDF Total HxCDF Total HpCDF OCDF Total PCDF 5 190 85 NR* 280 990 400 84 184 52 1710 640 2100 3100 2100 2500 10440 0 2 82 100 NR 184 119 115 270 228 278 246 128 113 152 157 947 859 Polychlorinated Dibenzodioxins U cin Total TCDD OCOD 0 00 NR NR NR 0 NR SDecific Molecules and Unresolved Molecular Clusters 32 34 NR NR 2,3,7,8-TCDD 2,3l7,8-/2,3,4,8-TCDF 1,2,3,7,8-/1.2,3,4,8 -PnCDF 2,3,4,7,8-PnCDF 1,2,3,4,7,8-HxDF I,23,7,a,9-ltxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF 0 00 0 84 150 0 54 480 0 00 190 84 1700 NR NR NR NR NR NR 85 147 680 0 0 0 0 82 NR NR 100 'vj CD * NR=Not Reported. 03 32 34 119 115 170 143 98 95 246 262 NR NR NR NR 113 120 TABLE 3.4 IITRI EURAM AND 010XIN DATA FOR THE BASELINE ANALYSES STUDY (ppb (w/w)) ComDound Total TCDF Total PnCDF Total HxCDF Total HpCOF OCDF Total TCDD CDD 2,3,7,8-TCDD 2,3,7,8-/2,3,4,8-TCDF 1,2,3,7,8-/l2,3,4,8-PnCDF 2,3,4,7,8-PnCDF 1,2,3,4,7,8-HxCDF 1,2,3,7,8,9-HxCDF 2,3,4,6,7,8 -HxCDF 1,2,3,4,6,7,8-HpCDF Aroclor 1016 30 42 92 81 20 0 0 0 8 11 0 76 0 0 58 Aroclor 1242 866 353 105 109 127 0 0 0 227 ' 42 51 63 0 0 77 Aroclor 1260 612 1037 1433 1985 4154 0 1 0 193 231 88 601 0 0 684 Tri and Tetra Chlorobenzene 0 0 103 98 0 0 1 0 0 0 0 103 0 0 98 Mineral O H Aaed 70 165 207 40 393 42 0 42 70 112 52 206 0 0 68 GENP 011086 -owo4oo CD -J 8ono a n a o TABLE 3.5 NYS00H FURAN AMO DIOXIN DATA FOR TUE BASELINE ANALYSES STUDY (ppb (w/w)) Comoound Total TCDF Total PnCDF Total HxCDF Total HpCDF OCDF Total TCDD OCDD 2,3,7,8-TCDD 2,3,7,8/2,3,4,8-TCDF 1 ,2 ,3 ,7 , 8 - / l,2 , 3 ,4 8-PnCDF 2 , 3 , 4 , 7 , 8 - PnCDF 1,2,3,4,7,8-HxCDF 1,2,3,7,8,9-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF Aroclor 1016 1 7 350 92 0 0 0 0 0 0 0 0 0 0 0 Aroclor 1242 1900 584 140 130 2300 0 0 0 150 1000 0 0 0 0 0 Aroclor 1260 940 '9 9 6 5500 2430 NA 0 0 7 190 220 0 0 0 0 0 Tri and Tetra Chlorobenzene 3 13 1000 91 0 0 0 0 0 1 0 0 0 0 0 Hineral Oil Acted 95 0 1100 0 0 42 0 43 34 140 0 0 0 0 0 oo 0CO0 C0O0 TABLE 3.6 RADIAM FURAN AND DIOXIN DATA FOR THE BASEUNE ANALYSES STUDY (ppb (w/wj) Comoound Total TCDF Total PnCDF Total HxCDF Total HpCDF OCDF Total TCDD OCDD 2,3,7,8-TCDD 2,3,7,S-/2,3,4,B-TCDF 1,2,3,7,8-/l2,3,4,8-PnCDF 2,3,4,7,8-PnCOF 1,2,3,4,7,8-HxCDF 1,2,3,7,8,9-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF Aroclor 1016 0 0 97 80 0 0 0 0 0 0 0 97 0 0 80 Aroclor 1242 0 0 60 46 24 0 0 0 0 0 0 60 0 0 46 Aroclor 1260 0 477 872 807 1786 0 0 0 0 477 0 872 0 0 807 Tri and Tetra Chlorobenzene 0 0 161 170 0 0 386 0 0 0 0 161 0 0 170 Mineral Oil Aqed 125 226 178 36 126 0 0 0 125 159 66 177 0 0 36 GENF o11089 2.3.7.8- TCDF-I3C|2 1.2.3.7.8- PCDF-12C12 2.3.7.8- TCOD-i3C)2 OCOO-13Ci2 I,2,3,4,7t8-HxCOF-13C]2 ],2,3,4,6,7,8-HpCDF *Outliers C3O CooO TABLE 3.7 ABSOLUTE RECOVERIES OF LABELLED STANDARDS IN THE BASELINE STUDY (UNIVERSITY OF UMEA) Spike Level jppbl Aroclor Aroclor Aroclor Chloro- Mineral 1016 1242 1260 benzenes Oil 100 93 95 95 97 116 100 111 134 140 97 114 100 67 86 86 101 118 100 102 89 157 86 121 100 83 90 317 n o NA 100 57 45 1224* 68 NA One effect is evident in these data, the recoveries from chlorobenzene and mineral oil appea'r to be superior to recoveries fronrArodors. The Aroclors contain the major interferents in the analysis of PCOF and PCDD. It appears that there may be matrix dependent difficulties in the analytical preparation of complex samples. Method Reliability The baseline data are grouped to show the relative performance of the four laboratories reported here. A compilation of data for aged mineral oil, baseline sample results is given in Table 3.8. Method reliability is compared by computing the Student's-t variability for four independent determinations around the mean value, the known spiked amount of standard PCOF/PCDD. In the four determinations shown the Student's-t variability of the mean varies from 20 to 167 percent. This is the degree of expected scatter in a group of four PCDD/PCDF determinations* conducted by independent laboratories using the Student'st distribution to approximate variance at the 95 percent confidence level. 783901 GENP 011090 GENP 011091 TABLE 3.8- FOUR-LABORATORY RESULTS OF ANALYSIS OF AGED MINERAL OIL (ng/g (w/w)) Compound Total TCDF Total PnCDF Total HxCDF Total HpCDF OCDF Total TCDD 2,3,7,8-TCDD 2,3,7,8/2,3,4,8-TCDF 1,2,3,7,8-/1,2,3,4,8 -PnCDF 2,3,4,7,8-PnCDF UI) 1,2,3,4,7,8-llxCDF 1,2,3,4,6,7,8-HpCOF Battelle 115 228 246 113 157 34 34 115 143 95 262 120 1TTRI 70 165 207 40 393 42 42 70 112 52 206 68 NYSDOti 95 0 1100 0 0 42 43 34 140 0 0 0 - Radian 125 226 178 36 126 0 0 125 159 66 177 36 Grouo Mean 101 155 433 47 169 30 30 86 139 53 161 56 Native Spike Level Confidence Interval I95%1 200 109% 160 167% 100 20% 100 41% ; 0'-0J CO CoMO Section 4 SUPPLEMENTAL ANALYSES CROSS CHECK ANALYSES To validate the analytical curve for each method proposed by the participating laboratories, a series of calibration runs were performed to establish the variability, linear dynamic range, and the limit of detection for each instrument. The calibration range is different from the method range. The validation of the calibration curve range only deals with standard solutions eluted through a tuned gas chromatograph or mass spectrometer (GC/MS) system. The quantities used to develop the calibration curve were 40 pg, 150 pg, 500 pg, and 2.5 ng. Each concentration level was analyzed with five replicates. The analytes used for the calibration curve validation were 2,3,7,8-TCDD, OCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PCDF, 2,3,4,7,8-PCDF, 1,2,3,4,7,8-HxCDF, 2,3,4,6,7,8-HxCDF, and 1,2,3,4,5,7,8-HpCOF. An illustration of the type of calibration plot generated in this study is given in Figure 1, which shows the standard curve for 2,3,7,8-TCDF. The complete set of calibration curve plots, along with statistical parameters describing calibration precision are given in Appendix B. The standard deviation and the relative standard deviation at each point show the variability of response which increases at higher concentrations. The calibration curves were linear over the range of concentrations measured. ANALYSES FROM THE UNIVERSITY OF UMEA Under a subcontract to Battelle, the University of Umea participated in the baseline and round robin analyses. A letter report of the data from Umea is included as Appendix C. 783903 4-1 GENP 011092 G E N P '011093 -nJ 0GJ0 Figure 4-1. Calibration curve for 2,3,7,8-tetrachlorodibenzofuran over the range 40 pg to 2.5 ng COO Section 5 ROUND ROBIN ANALYSES OF IN-SERVICE LIQUIDS The main thrust of this study was to evaluate a series of congeners and individual molecular species in typical in-service utility transformer and capacitor liquids. Ten (10) liquid samples, including the seven utility dielectric fluids, were analyzed for PCDO/PCDF using the methods developed in this program. All five participating laboratories contributed independent data sets, which will be statistically analyzed by an independent contractor, the Research Triangle Institute. The results of the Battelle analyses are given in Table 5.1 to 5.10. Accuracy Estimate The in-service analyses were designed with a quality control check on accuracy of reported PCDD/PCOF 'concentrations. The "Baseline" liquids included with actual utility operating fluids were spiked with known amounts of several PCDD/PCDF standards. These known spike concentrations allowed an estimate of accuracy using the mean of three determinations performed by each participating laboratory compared with the "true" or spiked concentration. The "Baseline" analyses revealed that the aged mineral oil sample and the Aroclor 1016 had minimal concentrations of the PCDD/PCDF standards present as contaminants. Thus, native PCDD/PCDF were suitable spike materials. The Battelle results for in-service liquids are presented in Table 5.11 for the mineral oil and Aroclor 1016 samples. Table 5.11 gives the spike levels and compares the percent difference in spiked levels with measured levels of PCDD/PCDF. The Battelle results are generally within +70 percent of the spike value except for one notable exception. HxCDF exhibited a strong positive bias. Most of the elevated HxCDF levels were 2,3,4,6,7,8-HxCDF. The HxCDF measured levels were 438 percent higher than the HxCDF spike levels 1n mineral oil, and 1325 percent higher in Aroclor 1016, even after subtraction of the native HxCDF found in the earlier baseline investigations. These HxCDF data are deemed to be outliers. 783905 5-1 GENP 011094 TABLE 5.1 CHLORINATED FURAN AND DIOXIN RESULTS (ppb (w/w), ng/g) SAMPLE ISL-02-A Congener Determination Pet 1 Pet 2 Pet 3 Mean Relative Standard Standard Deviation Deviation PCDF Congeners Total TCDF Total PnCDF Total HxCOF Total HnCDF OCDF 360 69 8,100 1,600 4,300 230 72 19,000 1,400 4,800 290 71 20,000 2,000 5,100 293 71 15,700 1,667 4,733 53 1 5,389 249 330 18% 2% 34% 15% 7% Total PCDF Furan Comoounds 14,429 25,502 27.,461 22,464 5,738 26% 2,3,7,8-/2,3,4,8-TCDF 2 ,3,4,7,8-PnCDF 1,2,3,7,8-/1,2,3,4,8-PnCDF 1,2,3,4,7,8-/1,2,3,4,7,9-H*CDF 1,2,3,6,7,8 -HxCDF 2,3,4,6,7,8-HxCDF 1 2,3,4,6,7,8-H0CDF 1,2,3,4,7,8,9-HpCDF 190 300 69 2,700 760 1,100 180 280 Dioxin Congeners 140 260 72 6,400 2,300 3,400 230 250 180 290 71 6,400 2,700 3,800 320 320 170 283 71 5,167 1,920 2,767 243 283. 22 17 1 1,744 836 1,190 58 29 13% 6% 2% 34% 44% 43% 24% 10% Total TCDD OCDD PCDD Comoounds NA NA NA NA NA NA 18 18 10 15 4 25% 2,3,7,8-TCDD NA NA NA NA NA NA OU095 783906 TABLE 5.2 CHLORINATED FURAN AND DIOXIN RESULTS (ppb (w/w), ng/g) SAMPLE ISL-03-A Congener Determination Pet I Pet 2 Pet 3 Mean Relative Standard Standard Deviation Deviation PCOF Conaeners Total TCDF Total PnCDF Total HXCDF Total HpCDF OCOF Total PCDF Furan Compounds 170 2,300 52,000 3,800 22,000 110 2,100 11,000 13,000 28,000 40 720 9,700 11,000 22,000 170 1,707 24,233 9,267 24,000 80,270 54,210 43,460 59,313 53 702 19,641 3,951 2,828 15,455 50% 41% 81% 43% 12% 26% 2 ,37 ,8-/2,3 4 ,8-TCDF 31 2 ,3 ,4,7,8-PnCDF 130 l,2,3,7,8-/l,2,3,4,8-PnCDF 35 12,3,4,7 ,8-/1,2,3 ,4,7 ,9-HxCDF 29,000 1,2,3,6,7,8-HxCDF ' 6,000 2,3,4,6 ,7,8-HxCDF 1,800 1,2 ,3,4,6,7,8-H0CDF 1,100 1,2,3,4,7,8 ,9-HpCDF 1,200 Dioxin Conqeners 30 130 49 6,100 1,100 240 4,500 2,600 40 110 26 5,100 1,200 210 3,100 2,600 34 123 37 13,400 2,767 750 2,900 2,133 4 9 9 11,038 2,287 743 1,395 660 13% 8% 26% 82% 83% 99% 48% 31% Total TCDD OCDD PCDD Compounds NA NA NA NA NA NA IS 12 NA 10 . 7 75% 2,3,7,8-TCDO NA NA NA NA NA NA 783907 gEWP 011096 TABLE 5.3 CHLORINATED FURAN AND DIOXIN'RESULTS (ppb (w/w), ng/g) SAMPLE ISL-04-0 Conoener Determination Det 1 Det 2 Det 3 PCDF Congeners Total TCDF Total PnCDF Total HXCDF Total HdcDF OCDF NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Total PCDF NA NA NA Furan Cmpounds 2 ,3 ,7,8-/2,3 ,4 ,8-TCDF NA NA NA 2,3,4,7,8-PnCDF NA NA NA 1,2,3,7,8-/! ,2 ,3,4 ,8-PnCDF NA NA NA 1,2 ,3,4,7,8-/1,2 ,3 ,4,7,9-HxCDF NA NA NA 1,2,3,6 ,7 ,8-HxCDF NA NA NA 2,3,4,6,7,8-HxCDF NA NA NA 1,2,3,4,6 ,7,8-HaCDF NA NA NA 1,2,3,4,7,8,9-HpCDF NA NA NA Dioxin Congeners Total TCDD OCDD PCDD Compounds NA NA NA NA 13 11 2,3,7,8 -TCDD NA NA NA Mean NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 8 NA Relative Standard Standard Deviation Deviation NA NASS NA NASS NA NAS NA NAS NA NASS NA NASS NA NASS NA NASS NA NASS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS 6 71S NA NAS GENP011097 783908 TABLE 5.4 CHLORINATED FURAN AND DIOX'lN RESULTS (ppb (w/w), ng/g) SAMPLE ISL-Q8-A Determination Congener__________ Pet 1 Pet 2 P e t 5 PCDF Congeners Total TCDF Total PnCDF Total HXCDF Total HqCDF OCDF Total PCDF Furan "Compounds 62 79 1,500 1,500 4,000 7,141 64 230 1,300 2,100 4,800 8,494 99 310 990 2,700 4,900 8,999 2 ,3,7,8-/2 ,3,4,8-TCDF 20 20 30 2,3,4,7,8-PnCDF 39 41 37 1,2,3,7,8-/1,2,3,4,8-PnCDF 22 25 34 1,2,3,4,7,8-/1,2,3 ,4,7,9-H*CDF 880 740 640 1,2,3,6,7 ,8-HxCDF 54 50 36 2,3,4,6,7,8-HxC0F 47 25 43 1,2,3,4,6,7,8-H0CDF 350 530 750 1,2,3,4,7,8 ,9-HpCDF 340 450 580 Dioxin Congeners Total TCDD OCDD PCDD Compounds NA NA NA NA NA NA 2 ,3,7,3-TCDO NA NA 'NA Mean Relative Standard Standard Deviation Deviation 75 206 1,263 2,100 4,567 8,211 17 96 210 490 403 784 235 465 175 235 92 105 23 5 39 2 27 5 753 98 47 8 38 10 543 164 457 98 205 45 195 135 175 255 305 215 NA NA NA5 NA NA NA5 NA NA NAS 783909 g E N P 011098 TABLE 5.5 CHLORINATED FURAN AND DIOXIN RESULTS (ppb (w/w), ng/g) SAMPLE ISL-17-A Conaener Determination Det 1 Det 2 Det 3 Mean Relative Standard Standard Deviation Deviation PCDF Congeners Total TCDF Total PnCDF Total HXCDF Total HqCDF OCDF Total PCDF Furan Compounds 970 11,000 14,000 3,100 150 580 6,700 36,000 2,800 170 610 6,500 16,000 4,200 220 720 8,067 22,000 3,367 180 29,220 46,250 27,530 34,333 177 2,076 9,933 602 29 8,455 252 26* 45* 182 16* 25* 2,3 ,7,8-/2,3 ,4,8-TCDF 2 ,3,4,7,8-PnCDF l,2,3 ,7,8-/1,2 ,3,4,8-PnCDF 1,2,3,4,7,8-/1,2,3,4,7 ',9-H*CDF 1,2 ,3,6 ,7 ,8-HxCDF 2,3,4,6 ,7,8-HxCDF 1 ,2,3,4,6,7,8 -H0CDF 1,2,3,4,7,8 ,9-HpCDF 400 2,300 870 4,700 1,800 620 920 740 Dioxin Congeners 380 2,400 900 7,200 3,200 6,800 560 860 350 1,800 780 4,100 2,300 930 1,300 990 377 2,167 850 5,333 2,433 2,783 927 863 21 262 51 1,342 579 2,832 302 102 5* 12* 6* 25* 24* 102* 33* 12* Total TCDD OCDD PCDD Comoounds NA NA NA NA NA 18 NA NA NA NA* 8 141* 2.3,7,8-TCDD NA NA NA NA NA NA* GENPOJI099 5 -6 7 8 3 9 10 TABLE 5.6 CHLORINATED FURAN AND DIOXTN-RESULTS (ppb (w/w), ng/g) SAMPLE ISL-23-0 Conqener Determination Det 1 Det 2 Det 3 PCDF Conqeners Total TCDF Total PnCDF Total HXCDF Total HnCDF OCDF NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Total PCDF NA NA NA Furan -Compounds 2 ,3,7 ,8-/2,3,4 ,8-TCDF NA NA NA 2 ,3 ,4,7,8-PnCDF NA NA NA l,213 t7 I8-/l,2,3I4 t8-PnCDF NA NA NA 1,2 ,3,4,7,8-/1,2 ,3 ,4 ,7 ,9-HxCDF NA NA NA 1,2,3,6,7,8-HxCDF NA NA NA 2,,3,4,6,7,8 -HxCDF NA NA NA 1,2 ,3,4,6,7 ,8-HdCDF NA NA NA 1,2,3,4,7,8 ,9-HpCDF NA NA NA Dioxin Conqeners Total TCDD OCDD NA NA NA 12 NA NA PCDD Compounds 2,3,7,8-TCOD NA NA NA Mean NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 4 NA Relative Standard Standard Deviation Deviation NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS NA NAS 6 141S NA NAS 783911 5-7 GENP 011100 TABLE 5.7 CHLORINATED FURAN AND DIOXIN RESULTS (ppb (w/w), ng/g) SAMPLE ISL-27 Conqener Determination Det 1 Det 2 Det 3 PCDF Conaeners Total TCDF Total PnCOF Total HXCDF Total HnCDF OCDF K 580 570 350 250 270 220 140 130 160 26 23 6 46 44 10 Total PCDF 1,042 1,042 746 Furan Compounds 2,3,7,8-/2,3,4,8-TCDF 300 280 270 2 *3 ,4,7 ,8-PnCDF 71 74 53 i,2,3,7,8-/i,2,3,4,a-pncoF 580 570 350 1,2 ,3,4,7,8-/1,2 ,3 ,4,7 ,'9-HxCDF 37 16 31 1,2,3,6,7,8-HxCDF 2,3,4,6 ,7,8-HxCDF 15 15 27 25 17 39 1,2,3,4,6,7,8-HdCDF 792 1,2,3,4,7 ,8,9-HpCDF 551 Dioxin Conqeners Total TCDD OCDD NA NA NA NA NA NA PCDD Compounds 2,3,7,3-TCDO NA NA NA Mean Relative Standard Standard Deviation Deviati 500 106 21% 247 21 . 8% 143 21 9% 20 10 33 17 51% 50% 943 140 ' 15% 283 12 66 9 500 106 28 9 19 6 27 9 63 4' 2 4% 14% 21% 32% 30% 34% 50% 45% NA NA NA NA NA% NAX NA NA NAX g e n p 011101 783912 TABLE 5.8 CHLORINATED FURAN AND DIOXIN RESULTS (ppb (w/w), ng/g) SAMPLE EZ29-53-Q1 (Spiked Mineral Oil) Conoener Determination Det 1 Det 2 Det 3 Mean Relative Standard Standard Deviation Deviati PCDF Congeners Total TCDF Total PnCDF Total HXCDF Total HnCDF OCDF 220 160 310 550 680 6,600 530 33 NA NA 190 190 260 373 390 2,557 220 261 31 24 127 2,862 205 1 13% 34% 112% 79% 141% Total PCDF Furan* Compounds 1.740 7.343 1,063 3,382 2,815 83% 2,3,7,8-/2,3,4,8-TCDF 2 *3,4,7,8-PnCDF 1,2,3,7,8-/1,2 ,3,4,8-PnCDF 1.2,3,4 ,7 ,8-/1,2 3;4,7 t9-HxCDF 1(2 ,3,6 ,7,8-HxCDF 2,3,4,6,7,8-HxCDF l,2,3,4,6,7,8-HoC0F 1,2,3,4,7,8,9-HpCDF 220 160 170 370 140 170 310 550 140 120 200 5,900 530 33 NA NA 190 190 120 220 140 150 260 373 76 112 140 2,080 220 261 NA NA 24 108 14 127 27 2,701 205 NA 13% 49% 9% 34% 24% 130% 79% NA% Dioxin Congeners Total TCDD OCDD PCDD Compounds 19 19 23 20 NA NA 1 NA 2 9% NA 141% 2.3,7,8-TCDD 19 19 23 20 2 9% 783913 GEKP 011102 TABLE 5.9 CHLORINATED FURAN AND DIOXIN-RESULTS (ppb (w/w), ng/g) SAMPLE E729-53-02 (Spiked Aroclor 1016) Determination Congener__________ Pet 1 Pet 2 Pet 3 PCDF Congeners Total TCDF Total PnC0F Total HXCDF Total HqCDF OCOF 57 600 5,400 65 110 67 500 2,300 150 120 66 460 780 340 120 Total PCDF 6,232 3,137 1,766 Furan Comoounds 2 ,3,7 ,8-/2 .3,4 ,8-TCDF 57 2 ,3,4 ,7,8-PnCDF 410 l,2,3,7,8-/l,2,3,4,8-PnCDF 180 1,2 ,3 ,4,7,8-/1,2,3,4,7,9-HxCDF NA l,2,3,6,7t8-HxCDF 100 2 ,3 ,4,6 ,7,8 -HxCDF 5,200 1,2,3,4,6,7,8-HpCDF 65 1,2,3,4,7,8,9-HpCDF NA 67 310 180 NA 84 2,100 150 NA 66 260 190 NA 81 680 180 NA Dioxin Congeners Total TCDD OCDD 46 46 44 NA NA NA PCDD Comoounds 2 ,3,7,8-TCDD 46 46 45 Mean Relative Standard Standard Deviation Deviation 63 520 2,827 185 117 3,712 4 59 1,923 115 5 1,868 7% 112 682 622 4 502 63 327 183 NA 88 2,660 132 NA 4 62 5 NA 8 1,887 49 NA 72 192 32 NA2 92 772 372 NA2 45 1 22 NA NA NA2 46 NA 12 GENP 011103 5-10 - 783914 TABLE 5.10 CHLORINATED FURAN AND DIOXIN "RESULTS (ppb (w/w), ng/g) SAMPLE E229-53-03 (Spiked Aroclor 1260) Congener Determination Det 1 Det 2 Oet 3 Mean Relative Standard Standard Deviation Deviation PCDF Congeners Total TCDF Total PnCDF Total HXCDF Total HnCDF OCDF 1,400 1,100 20,000 1,700 5,000 1,700 1,300 4,700 3,000 4,900 970 1,200 11,000 660 2,600 1,357 1,200 11,900 1,787 4,167 300 82 6,279 957 1,109 222 72 532 542 272 Total PCDF 29,200 15,600 16,430 20,410 6,225 30% Furan Compounds 2 ,3,7,8-/2 ,3,4,8-TCDF 1,200 2 ,3 ,4,7 ,8-PnCDF 590 1,2 ,3,7 ,8-/1,2 ,3,4,8-PnCDF 1,400 1,2,3,4,7,8-/1,2,3,4,7,9-H*CDF 1,900 1,2,3,6 ,7,8-HxCDF 1,200 2 ,3,4,6 ,7 ,8-HxCDF 10,000 1,2,3,4,6,7,8 -HnCDF 55 1,2,3,4,7,8 ,9-HpCDF 300 1,300 410 1,170 1,800 93 770 850 360 880 800 970 1,600 780 6,500 49 180 1,127 627 1,357 1,767 691 5,757 318 280 179 194 300 125 456 3,805 376 75 162 312 222 72 662 662 1182 272 Dioxin Congeners Total TCDD OCDD PCDO Compounds n o 100 80 97 NA 12 NA 4 12 132 6 1412 2 ,3,7 ,8-TCDD n o 100 80 97 12 132 ' 783915 5-11 GENP011104 TABLE 5.11 ACCURACY DETERMINATION USING SPIKED BASELINE LIQUID ANALYSES GENP 011105 s.! 2 Total TCDF Total PnCDF Total HxCDF Total IlpCDF OCDF Total PCDF Total TCDD OCOD Mineral Oil E729-53-01 Spike Amount Percent(1) Level Found Di fference 200 190 300 373 475 2557 .400 261 01 -5 24 438 -35 1375 3382 146 25 2 00 -92 Aroclor 106 E729-53-02 Spike Level Amount Found Percent Di fference 53 63 316 520 185 2827 526 185 132 - 117 19 65 1428 1212 3712 206 53 45 00 -15 Corrected(Z) Aroclor 1016 F729-53-02 63 515 2637 100 117 3432 45 0 2.3,7.8 -TCDD 2 ,3,7.8 -/2 .3,4,8 -TCDF 25 2 200 190 -92 -5 53 46 53 63 -13 19 46 63 l,2.3,7.e-/1.2.3,4.8-PnCDF 150 150 0 158 183 16 183 2,3,4,7,8 -PnCOF 150 220 47 158 327 107 327 1 2.3,4.7,8 -/ 1.2,3,4.7,9-HxCOF 250 373 49 00 -- -190 1.2.3.6,7,8-HxCDF 50 0 -100 53 aa 66 88 2,3,4,6,7,8-HxCDF 175 2080 1089 132 2660 1915 2660 1,2,3,4,6 ,7,8 'ttpCDF 400 261 -35 526 132 -75 132 1.2,3.4.7.8 ,9-IlpCDF -- 0 -- -- 0 -- -85 (1) Percent Difference 3 100 (Measured Value - True Spike)/True Spike (2) Corrected by subtracting the Aroclor 1016 "Baseline- PCDD/PCDF concentrations from the measured values. Corrected Percent Di fference 19 63 1325 -81 -11 183 -15 -13 19 16 107 -- 66 1915 -75 -- 783916 ILitfi r - j11g1 Analyses i reSu1ts are compared with the results reported by the other four *"* Rttfi* narticipating in this program in Table 5.12. The data presented in i=QiratofiesforPdIthe other laboratories are taken from a draft statistical report (2) *,s ta ^ Ed0 pellizari (Research Triangle Institute, Research Triangle Park, ^ Sei1 meeting of the five participating laboratories held in Washington, O.C. i.C) n November 19, 1986. -e Sattelle results averaged over three replicate d t a&le 5.12 where the average reported values by the f erminatlons are Pointed In inducting parallel analyses are also given. co,,aboratin9 laboratories he two sets of data compare favorably; except for hexachlorinated furans, the i t t e l l e results are consistently lower overall than the data contributed by other o r a t o r i e s . The general trends in the data such as very low values for PCDD/PCDF n m in e r a l oil samples was apparent in all the reported data. The 30-year-old etworfc transformer containing Askarel (Sample Number 3 in Table 5.12) had the ighest reported PCDF level of all the in-service samples tested, approximately 32 pm (w/w) using the average of all reported determinations. 5-13 783917 O B N ? 01im 1A lii f 5 , 12 HFAN FURAN RFSUl IS I0R St VIN U T U M Y OlilfCIRlC n u i n s ( i 0 l U0<lM 3Q Network Transformer No. 1 Network Transformer No. 2 Network Transformer No. 3 Network Trans former No. 4 Arc furnace Jrans former Prec ipilator Transformer Capacitor Sample Identifier Fluid Years Service Field Code laboratory Code Askarel 20 1SL'02-A 17 Askarel 2B 1St OG A 12 Askarel 30 ISl 03 A 11 Mineral Oil UNK ISl -04 0 16 Mineral Oil UNK ISl 23 0 15 Askarel 27 1SL-17-A 14 Askarel UNK ISL-27 13 Dattclle furan Results Total 1COF lota) PnCDF Total HxCDF Total HpCDF OCDf Ui) Total PCDF 293 71 15700 249 330 16643 17 96 1263 490 403 2269 53 702 24233 3951 2828 31767 0 0 0 0 0 0 0 177 0 2076 0 22000 0 602 0 29 106 21 143 10 17 0 24084 297 Combined Furan Results ill Total ICDr Total PnCDF Total HxCDF Total HpCDF OCDF 415 991 4886 1277 3346 128 279 1012 1822 6244 158 1672 10824 65B7 21830 1 45 80 15 14 1 782 586 4 7746 221 2 12931 67 8 1767 12 18 188 21 Total PCDF 10914 9486 41071 154 34 23413 906 Percent Difference in furan Conaener Results (21 52% -76% -23% (3) t3) 6% -67% 0003 (1) Combined Mean = Average reported values from the five participating laboratories. CO (2) Percent Difference - (Combined Mean - BaLlelle Value)/Combined Hejo X 100% 00 (3) Zero value division not possible. Section 6 DISCUSSION This study produced a great amount of insight into the variability of analytical methods applied to trace analysis in a complex dielectric fluid matrix. PCB constitute the principal class of interferents for modern mass spectrometric methods when used to analyze polychlorinated aromatic species such PCDF and PCDO. Several conclusions can be drawn concerning performance of the methodology and the accurate screening of in-service dielectric fluids. METHODS' The analytical procedures proved to be both rugged and reliable when used with labelled quantification and recovery standards. The quantification standards are used to calculate errors from extraction and sorbent partitioning losses. This is especially important in the Askarel and mineral oil matrix since the high organic burden found with this matrix requires secondary partitioning to remove interfering PCB and the background hydrocarbons from mineral oil. Random outliers were observed in this study. One example was the elevated levels of hexachlorodibenzofuran found in the in-service analyses. This kind of acute error can arise from a mistake in the gravimetric preparation and dilution of the labelled recovery standard. Another possible cause for this random error is selective partitioning losses during sample clean up. Observing this type of spurious result indicates that the methods should be used with enough quality control checks to "flag" this type of error during the analysis. Using a second labeled internal standard to quantify the mass recovery of the quantification standards would identify this problem if it is related to quantification standard losses.. The observed data variability in this program was smaller than the participants had anticipated before the program began. In this very difficult matrix, a chlorinated oil, most analysts would predict a variability of one to two orders of magnitude (IOx to lOOx scatter around the true value). In most cases, the relative standard devia-tions were found to be less than 100 percent. The relative average deviation is the absolute difference between' individual results and the mean (or true value), divided by the number of determinations. For samples such as the Aroclor 1242, 6-1 783919 Aroclor 1016, and mineral oil, the average deviation was most often less than 50 percent.--This result was seen in individual-laboratory replicate analyses and in grouped data from different laboratories. IN-SERVICE LIQUID ANALYSES The in-service liquids tested in this program were found to have relatively low amounts of PCDD in all samples tested. This result supports the hypothesis held by many chemists who have investigated Askarel and PCB-containing fluids that dioxins are not a major constituent of these mixtures. Except for the mineral oils, polychlorinated dibenzofurans, however, were found in trace amounts in all of the samples tested. The mineral oil samples were found to have extremely low amounts of PCDD/PCDF if any were found at all. Even the in-service liquid from Transformer 4, a network distribution transformer that was involved in a high energy excursion, was found`to have minimal amounts of PCDD and PCDF. Transformer 4 had visible carbon, and major signs of scorching on the case and the area of the electrodes. The Askarel transformer? with more than 25 years of service tended to have higher levels of PCDF than the appliances put into service for a shorter time. It is possible that manufacturing controls improved as the production of Askarel matured in the 1960s such that Askarel was produced with lower levels of PCDF in the asdelivered fluids. It would be interesting to obtain replacement Askarel with a known lot number which matched a sample with extended service life. Analyzing samples of an unused Askarel and comparing the PCDF levels in a sample subjected to heavy load and environmental stress for an extended period would challenge the theory that utility operation contributed to elevated PCDF levels in Askarel fluids used in transformer and capacitor applications. Two utility appliances were selected by the EPRI Technical Advisory Committee for special attention. An Askarel-filled electrostatic precipitator (ESP) transformer, and an oil-filled arc furnace transformer. Both of these applications involve extended thermal and electrical excursions during daily operation. These two samples were the "worst case" for testing service stress on the dielectric fluids. The arc furnace had essentially no PCDF or PCDD, and the PCOF levels in the ESP transformer appeared to be dictated by the age of the unit (27 years of service) and the fluid composition (Aroclor 1260). This extended period of stress did not appear to elevate the PCDF levels above typical Aroclor 1260 contamination levels. One capacitor was tested in the in-service liquid screening program. It was found to have relatively low levels of PCDF compared to the other Askarel-filled units. 6-2 ^83920 The observed distribution of PCDF seemed to follow a trend of higher concentrations of PCDF.arising with the highest degree of,,chiorination Aroclor. The Aroclor 1260 with 60 percent chlorine by weight tended to have higher levels of PCDF in every comparable sample than Aroclor 1242, or Aroclor 1016, both of which have approximately 42 percent chlorine in the base Aroclor. A second effect was observed in these data, the relative concentrations of PCDF congeners in any sample tends to favor the analogs with higher chlorination as the chlorination number of the underlying Aroclor increases. For instance the proportion of hexa, hepta, and octachlorodibenzofurans are higher in Aroclor 1260 than Aroclor 1242. If PCDF are formed by direct internal condensation with the addition of oxygen, HC1 elimination can occur. This reaction would preferentially produce higher levels of chlorination in the product PCDF when high degrees of chlorination occur in the reactant PCB. SUMMARY This study was successful in developing a set of methodologies that reliably determine trace levels of PCDF and PCDD in a very difficult matrix -- chlorinated organic oil. The development of commercially available reference standards and the statistical validation of reference methods that can be used to analyze utility fluid was a major contribution to the quality and defensibility of data in this field. The method verification program showed that a range of instrumentation can be successfully used to measure trace levels of PCDF and PCDD in utility fluids. In general, the methods employing low resolution mass spectrometry require additional column chromatography to isolate PCDF/PCDD-enriched fraction for final gas chromatography. The precision of blind spiked samples was superior to the variance expected by experienced analysts who had previously performed this analysis. The availability of labelled quantification standards helped improve method performance and lower data scatter. Although the in-service liquids tested in this program were a very limited set, there were interesting findings about the type and distribution of polychlorinated dioxin and furan species in the systems tested. Dioxin levels were very low, or not measurable in the liquids analyzed. The mineral-oil-filled equipment had levels that were found only at the quantification limits of the methods employed. Aroclor 1260 appears to have higher levels of PCDF with enrichment of the higher chlorine-number analogs, when compared to Aroclor 1242, 1016, or Aroclors with fewer chlorine atoms per molecule than 1260. This data base can not support the 6-3 GENP011110 783921 premise that the age of Askarel transformers tends to favor higher PCDF levels in older transformers since only four units were-investigated. GENP 011111 6-4 783922 Appendix A IN-SERVICE LIQUID QUESTIONNAIRE 783923 GENP 011112 Respondant: GENERAL INFORMATION QUESTIONNAIRE EPRI IN SERVICE LIQUIDS STUOY (RP 2028) Samples provided Askarel Load Center/Network Transformers Rectifier Transformer Precipitator Transformer Capacitor Oil Filled Load Center/Network Transformer Number Shipped GENP 011113 A -1 783924 DATA SHEET FOR EACH SAMPLE EPRI IN SERVICE LIQUIDS STUOY fRP 2Q28) General Information Voltage Rating: Liquid Volume: Manufacturer and Year: PCB/Askarel Manufacturer: (if available) PCB/Askarel Type: PCB Levels: (for oil filled, if known) Additional Equipment Identification: (name plate data) Equipment Maintenance History Length of Actual Service: Major Maintenance: ____ Recorded Failure in Service: 783925 GENP 011114 A-2 DATA SHEET FOR EACH SAMPLE Was Fluid Changed at Failure: Any Record of Temperature Excursions: Are Maintenance Records Available: Any Other Pertinent Information About This Unit or the Sample Liquid: GEtvp 0 l A-3 783926 Appendix B ANALYTICAL CURVE DATA FOR THE CROSS CHECK ANALYSES 783927 GENP 011116 COCEHTOUICH EPI. COMPOSITE STO CJflvE. . . 7 .a.Ttr*COF -VALUES 34 9999 1*9 9990 499 9990 3499 9900 T-VAtUCS 0. 07* 0. 32** 1. 147* 9. 8409 0 0802 0. 324* 1. 1*09 9. 4**0 X-VALUE Y-VACUC STO OEV 0 07** 0 334 1. 1*07 9. 7829 X9C1 0 0910 0 3430 1 1278 9*90 AEI. E. 0 08*8 0 3**1 1 133* 4 4339 A. ft. C. OATA WITH ftESPECT TO TV* V-VALUE* 40. OOOl 190. OOOO 900. 0000 2900. OOOO 0. 0039 0. 33** 1. 1431 9. 4247 0. 0099 0. 0043 0. 0117 0. *019 *. * 2. 70 1. 01 11. 08 0. 008* 0. 014* 0. 0189 0 4910 DATA WITH RESPECT TO Tt A 1 0 M M 1 0 N LINK 40. 0001 190. OOOO 900. OOOO 2900. OOOO 0. 0833 0.33** 1. 1431 9. 247 0. 0271 0. 0139 0. 099 0 *01* 32. 3* 3. 49 3. 4* 11. 08 0.0427 0. 0212 0. 0714 0. 4911 A slope op 0. 0031* orvca AN INTCftCCPT OP 0. 020*8 A SLOPE OP 42. 02300 0IVE8 AN INTCFCCCT OP 4 998*9 THE OATA HA8 A COftftCLATtON COEFFICIENT OP 0 44443 0 0090 0. 008 0. 010* 0. 9449 0. 0249 0. 0122 0 0412 0. 9449 GENP 01 u 17 B-l 783928 EPai. COMPOSITE STO CURVE. 1.2.J,J .3-PtnllCQF 624.39 1249.99 CtHXHTRATKM 1974.99 2499.99 i aa aa s iB1 COMPOSITE STO CURVE, 1,2,3, ;(a-P<nciCOF X-VALUES r-v4*_uE9 40 0000 130.0000 4 4 4 (fl 344 4400 0 0484 0 3347 I 3183 A. 3080 0 1031 0 3333 1 2171 A. 3310 X-VALU* y-w al uc STO DEV 0 0461 0. 3343 1 3301 3. 9300 4301 0 0434 0 3931 1 1449 9 3030 0. 0934 0 3603 1 1469 9. 3400 4E9. E. ft ft. E. i : MMiMMiVUl DATA WITH RESPECT TQ TH* y -VALUSS 40. 0000 130. 0000 900. 0000 3900. 0000 0. 0003 0. 3441 1. 1033 9. 8333 0. 0344 0. 0119 0 034 0 41*9 'DATA WITH RESPECT TO THE REGRESSION LINE 44 14 3. 33 3. 39 7 19 0. 0434 0. 01E3 0. 043 0. 499 40. 0000 190. 0000 900. 0000 3900. 0000 0 0409 0 3441 1 1833 9 1333 0 0419 0 0133 0 0444 0 4149 91 91 3. 94 3. 74 7 19 A SLOPE OF 0.00333 OtVES AN INTERCEPT OP -0. 00197 A SLOFf OF 434 03700 OIVES AN INTEftCECT OF 0 67917 THE DATA HAI A COFftELATTON COEFFICIENT OP 0. 4444* 0. 0494 0 0144 0. 0709 0. 49* 0. 0337 0. 004 0. 0334 0 3944 0 0393 0 0109 0 0380 0. 3944 783929 E N P o u , I8 EPOI. COMPOSITE STD CUPVE, 2.3.4,7 .S-PenuCOF ,: : mpos;te STD CURVE. 2.3,4, 7 ,3-?entjC3F i - vauS Y-vALues 0. 0 0 00 (90. 0000 4* * * * * o a *** **oo 0. 0 7 03 0. 3 * * 1. 3397 7 4339 0. 0 7 4 3 0 4409 1. 4380 7 4430 S-VACUS Y-VACUC 9T0 n v 0. 07 14 0. 4 4 7 3 l. 4*33 7. 0 * * 0 RIDI 0 08*3 0 3384 1 1333 9 1*30 411. K. 0. 08 10 0. 3 3 * 4 1. 1 1 (4 9. 3 * * 9 A4 Data with ( p ie r to rxe y- vacu*i 40. 0000 190. 0000 900. OOOO 3900. 0000 0 0773 0. 4009 1. 3403 *. 3040 0 0080 0 09*4 0 3019 1 13** DATA WITH MC8AKCT TO TMC nceaessiON u k 10. 33 14 0* 19. 03 17 3* 40. 0000 190. 0000 900. OOOO 3900. OOOO 0 0773 0 *009 1 3403 *. 9040 0. 03** 0 09** 0 3090 1 13** 47 *3 14. 13 19 3* 17 3* a sloak oa o. ooaM a i ve a am imtcmccat oa o ooao* * SLOAC OF 34 30*00 OtvCS am (NTEACCCT 0A >2 0*303 TH DATA MA A CQRNCUATION CQCAAICISNT OA 0 ****9 0. 0113 0. 07*7 0. 3890 l. 9*74 0. 0931 a osai 0 3*00 1. 9*79 0. 0099 0. 0383 0. 1370 0. 7*78 0. 0391 0 0389 0 13*4 0. 7*7* GENP 011119 B-3 783930 EPRt. COMPOSITE STO CURVE. 1.2.3.4.7 .8-lfeuCOF' 9.98 624.99 1249.99 CONCENTRATION 1974.99 2499.99 CPH. COMPOSI" S " CURVE. 1.2.3.4..a-*t**CDF X-VACUCS Y-VACUE3 40. 0000 190. 0000 447 4440 3444 4400 0 0703 0 3137 1 178A 9 339 0 0777 0. 3101 l 0739 9. 77*3 X-VACUI Y-VACUt STO 0EV 0 070 0 3073 1 3033 9 *700 ftSDS 0 014* 0 iva i 0977 * *0*0 - Rea. e. 0 0733 0 3707 1. 0371 4 3130 ARC. SATA MtTM IttWCCr TO T X Y - V A C U 40 oooo 190. OOOO 900. OOOO 3900. OOOO 0. 07*4 0. 3439 1 1149 9 *973 0. 00A 9 0 0447 0 0A74 0 97*1 0. 37 13 04 6 09 1. 19 0. 0103 0. 0707 0 10* 1. 9790 o a t a w i t h nespeer t o t k a c c a c s s i o n c i n c 40. OOOO 190 OOOO 900. OOOO 3900. OOOO 0 07*4 0 3439 1 1149 9 4073 0 0144- 0 0499 0 0600 97*1 39 44 13 30 6. 17 li. 19 0. 0314 0. 0730 a. toe* 1. 9790 a scope o p 0.00317 GIVES AH INTEPCCPT op 0 009*7 A SCOPI UF *99 9**00 Stvs a h INTE9CECT OP -3 99*9 THC DATA HA A COPPSLATICN .c o e f f i c i e n t OP 0 47470 0. 009 0 0403 0 0*04 0 3774 0 0174 0 0410 0 0630 0 8974 783937 GEJS*P O l i 1 2 0 EPRI. COMPOSITE STO CURVE. Z.J.I../.a-HeuCOF 624.39 1249.39 COCEHTMTICM 1874.39 2499.93 EPRI. .CMPGS'." STD CURVE. 2.1.4 .6.7 ,a-***C3F 1 v a l u e s Y-VALUEf 40. 0000 130. 0000 *44 4440 2444. 4400 0. 094 0. 2S3* l. 03E4 9. 4239 0. 0*37 0, 2740 1 08*3 9. 9*73 VALUE V-V4LUS STO DEV 0. 0973 0. 2294 0. 4733 9. 3113 USDS 0. 0710 0. 3733 1. 0334 4 3340 1(9. (. 0. 0713 0. 249* 1. 0949 7 3400 * *. e. DATA U1TH RESPECT TO THE V-VALUES 40. 0000 190. 0000 900. OOOO 2900. 0000 0. 0*47 0. 304* 1. 0393 3. 3437 0. 00*3 Q 0403 0. 0414 t. 0942 4 4S 13. 14 4 03 14 4* DATA MXTH RESPECT TO T X REGRESSION L I X 40. OOOO 190. OOOO 900. OOOO 2900. OOOO 0. 0*47 0. 304* 1. 0393 3. 9437 0 02*0 0. 0424 0. 0*24 1.0942 3. 29 14 0* 6. 09 14 4* A SLOPE OF 0. 0 0 M 3 0IVE9 AN INTERCEPT OP >4. 04** A SLOPE OP 443. *3000 QtVCS AN INTERSECT O P 30, 74330 t h e d a t a m s A CORRELATION COEFPICIENT o p 0. 44443 0. 0042 0. 03*4 0. 0343 1. 9403 0 0330 0. 0*0* 0. 0194 1. 3404 0. 0032 0. 0323 0. 0334 0. 9749 0. 0200 0 0347 0. 0909 0 9749 ENP 011121 8-5 783932 ERRI, COMPOSITE STO CURVE. 1 .2 .J .4 .6 .'.3 -H D tJ C D F AS AE S oAri erri. *.ow>os: te STO CURVE. 1.2.3.4.6.T.8-''oeiC3F X-VAUUES T-VAUUE3 *0. 0000 130. OOOO 34*4440 4440 t*oo 0 3*73 1 3*7* 3. 3070 33. 3370 0.3037 1. 1393 4 3144 33. 3704 0. 3409 1 413* 3. 4303 3*. 01*9 1 -VACUO Y-VACUC STO OCV RSDS 0 330* 0 8433 4 0030 33. 0300 1 . C. 0. 30*9 1. 1*00 3. 7310 31. 7000 9, 4. DATA UfTM H K SP fC T TO TM> Y-VALUCS 40. 0000 130. 0000 900.0000 3900. 0000 0. 33t4 l. 1437 3. 4*07 31. 4093 0. 03*0 0. 191* 0. 9*79 3. 7*43 13. 4* 19. 31 31. 44 17 04 DATA W1TM A E S W T TO THC RS04CSS10M L1NC 40. OOOO 130. OOOO 300. OOOO 3300. OOOO 0. 3914 l. 1437 3.4*37 31.4099 0.1399 0 30*1 0 8419 3. 7449 93. 90 17 37 33. *0 17. 04- a score or o oom 3 e t v n am in t e r c e t t or -o a sis** suore or 113.30*00 o iv cs am i n t c t c s c t or 33.0*010 TMO DATA MAO A C0A9CCAT1CN COETTICieNT QT 0 44499 0. 0337 0. 3971 1.3*04 3. 4300 0. 3143 0. 3339 1. 04* 3. 4309 0. 0074 0. 0431 0.14*9 0. 0*7* 0. 0314 0. 0479 0. 30** 0. 9*77 783933 8-6 GENP 011122 ERRI. C O S 'OSI TE STO CURVE, 2,3.7 .S-TstriCOO coMXKrmncN EPRt, COMPOSITE STO CURVE. 2,3.7.fl-TttrtCOO X-VALUI9 Y-VACUXS 40 0000 190.OOOO T T atto 2444 TTOO o ooa 0. 2480 o. T7oa 4 8330 0. 0403 Oi 2833 0 T934 4 0339 X-VALX V-VALUC STO OCV 0 041T 0. 2703 0 .Al 17 4 7199 49 0 0749 0 2798 0 0476 4 4340 RE I. C. 0 0677 0 2817 0 4212 4. 1340 4. R. data m it m n c s p ic t -re t x y- v a c u w 40. 0000 190. 0000 900. 0000 2900. 0000 0. 0*43 0. 2740 0. 4282 4. 4440 0. 00*4 0. 0041 0. 0271 . 0. 40*4 ATA uitm r c s m c c t TS TX R I M K 9 I Z 0 M LINK 17 2. 14 2. 41 12. 20 a.0104 0. 0044 0. 043* 0. 4942 40. 0000 190. OOOO 900. OOOO 2900.oooo 0. 0443 0. 2740 0.4212 . a *a o 0.0233 0. 0121 0o:.04404437 39. 10 4 42 9. 30 12. 20 0. 0341 0. 0202 0. 0774 0. 4942 > su** op a. ooiAA o iv u am [n t c r c c p t o p -0. 03401 A SLOPI OP 900. 33400 SCUCI AN tMTCMCCCT V 17 09430 T X DATA HAI A COPRO-ATION CBIPPICIENT OP 0 TATA3 0. 0047 0. 0042 0. 0282 a. 4144 0.0234 0.0130 0.0444 0.4190 GEN P 01U 23 B-7 783934 EPSti corposiTE STO cunc< ocoo a.ea 624.99 1249.99 1874.99 2499.99 COCDnKiTIW . CF*I. COlOSITE s t o cu*ve. ocoo l-VALUCO 0.0000 190.0000 4 t * t t o 3474. 4700 r-vALues 0. 0 7 7 0 * 0. 93*4 a. 2410 la. 3**9 0. 1027 0. 9040 a. 0311 la. 9443 I-VALU* r-VM.UK STO DCV o. i o s a 0. 9411 a. i*39 la. m *9 *901 0. 1933 0 4443 a. 4300 4 *000 Ats. C. 0. 1919 0. 9901 a. 1320 4 1020 * * e. OATA W1TM ftS IF K C T I O T M V -V M .U K 1 40. 0000 190. QQOO 900. 0000 2900 OOOO 0. 1347 0. 9944 2. 1209 10. *141 0. 03*0 0. 0713 0. 33*3 1. M S * 31. 4 13. 09 10. 00 17. 30 0. 0433 0. 1137 0. 3*33 3. 4090 DATA MITH N n P t e r TO THt *10*1901 ON LINK 40. 0000 190. 0000 900. 0000 2900. 0000 0. 1347 0. 9944 3. 1309 10. *141 0. 03*4 0. 0774 0. 2243 1 8004 31 9* 13. 4* 10. 0 17. 30 0. 0*33 0. 133* 0. 3*33 3. 4090 A SLOOK QP 0. 00437* O IV K S A M IN T C T C tP T o r -O 07799 A SLOFK QF 227. 47100 OIVKS AN IHT EO C KCT 0F 17 *43*0 T > * DATA MAS a c o r r c l a t i o n c o k f f i c i k n t o r o. 44447 0. 0134 0. 0337 0. 1097 0. 0707 a. 0183 0 0397 0. 1097 0. 0707 GENP Oli 124 B- 8 783935 Appendix C LETTER REPORT FROM UMEA GENP 011125 783936 Umei tlniversitet 901 87 CJmea 86-06-23 Analys results EPRl-pro jekt 2028 Formation of PCDD and PCDF in askarel and contaminated mineral oil equipment. Contents. Normalized raw counts for regression lines. Levels in baseline liqids Levels in inservice liqids: TCDF/TCDDs PnCDF/PnCDDs and HxCDF/HxCDDs HpCDF/HpCDDs and OCDF/OCDDs GC/MS Conditions. Clean-up By Lars-Owe Kjeller UmeA Universitet 901 87 ume4 Sweden. 783937 C-l GENP 011126 LARS-OWE KJELLER Une Universitet 901 87 UMEA Sweden Ume 85-06-18 EPRI-prodjekt 2028 Calibration curve, raw counts normalized for ^C-internalstandard. Concentration injected p g 2.3.7.8-TCDF 2.3.7.8--TCDD 1.2.3.7.8-PnCDF 2.3.4.7.8-PnCDF 1.2.3.4.7.8-HxCDF % 2.3.4.6.7.8-HxCDF 1.2.3.4.6.7.8-HpCDF OCDF 10 9.5 3.4 6.0 4.9 3.9 1.7 1.4 WO 10 12.4 7.9 9.5 8.5 7.8 4.6 3.9 ND 10 10.8 7.0 8.1 7.5 7.3 4.5 3.8 ND Concentration injected pg 2.3.7.8-TCDF 2.3.7.8-TCDD 1.2.3.7.8-PnCDP 2.3.4.7.8-PnCDF 1.2.3.4.7.8-HxCDF 2.3.4.6.7.8-HxCDF 1.2.3.4.6.7.8-HpCDF OCDF 40 38.5 18.0 24.8 17.8 19.1 8.8 5.8 0.9 40 42.3 27.6 35.0 29.0 27.3 18.2 13.0 2.0 C-2 40 42.6 27.8 37.8 29.0 27.3 18.2 13.0 1.5 783938 G B N P 011127 Concentration injected 2.3.7.8- TCDF 2.3.7.8- TCDD 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 2.3.4.6.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF OCDF Concentration injected 2.3.7.8- TCDF 2.3.7.8- TCDD 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 2.3.4.6.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF OCDF 150 160.9 100.1 133.4 100.7 105.3 51.7 39.3 5.6 150 155.1 94.2 124.5 98.2 82.2 43.1 33.7 2.1 150 160.1 105.7 137.9 118.1 103.3 68.9 53.4 6.7 500 545.3 370.9 470.0 403.5 339.8 214.3 182.4 23.5 500 570.0 371.0 471.0 401.0 338.0 203.0 177.0 17.0 500 550.9 359.9 467.7 402.1 341.9 216.2 181.9 17.4 Concentration injected 2.3.7.8- TCDP 2.3.7.8- TCDD 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 2.3.4.6.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF OCDF 2500 2791.7 1925.0 2412.8 2012.5 1780.1 1118.9 973.4 166.4 250Q 2769.4 1806.1 2384.7 1979.0 1760.3 1145.8 889.8. 125.3 C-3 2500 2767.9 1924.3 2413.1 2061.7 1793.3 1100.5 952.1 169.1 783939 GENP 011128 Concentration injected pg 2.3.7.8-TCDF 2.3.7.8-TCDD 1.2.3.7.8-PnCDF 2.3.4.7.8-PnCDP 1.2.3.4.7.8-HzCDF 2.3.4.6.7.8-HxCDP 1.2.3.4.6.7.8-HpCDF OCDP 7500 __ 7500 8731.9 8593.7 5966.9 5480.0 7401.8 7264.2 6219.0 5897.8 5565.6 5502.2 3532.6 3577.6 3067.2 2790.8 513.6 419.3 7500 9037.3 5735.6 6989.2 5980.2 5494.7 3638.1 2768.4 433.1 - Characterization for the regression line y = a*x + b y = amont of Dioxin/furans. x = normalized counts. i Residual a b standard deviation. 2.3.7.8-TCDP 0.853 29.89 135.7 2.3.7.8-TCDD 1.306 18.28 117.5 1.2.3.7.8-PnCDP 1.037 9..24 77.5 2.3.4.7.8-PnCDP 1.240 6.76 76.1 1.2.3.4.7.8-HxCDF 1.357 24.50 38.0 2.3.4.6.7.8-HxCDF 0.487 -21.26 34.1 1.2.3.4.6.7.8-HpCDF 2.595 31.83 159.6 OCDP1 0.061 -4.44 22.6 1 10 pg cone, not included. y- 783940 g e n p 011129 783941 01 in a >-d o LARS-OWE KJELLER UmeA universitet 902 05 UMEA Sweden UmeA 06-06-17 ERR 1-prodject 2020 Levels of Jioiina and dihensofurans in inservice liquid isaZai.1 2 Alt or. 41159-11- TCDP 1160 1379/1370 1147 1466 1247/1367 1346 1346/1248 1246/1237/1266/1476/1369 1234/2349 1236/1467/2466/1236 1349 1278 1267/1279 1469 1249 2368 2467 1239/2347 1269 2376 2348 2346 2367 3467 1289 TOT TCDP BBC l3C-2378-TCDP 11 <1.1 5.4 4.0 5.4 8.6 2.3 05 14 5.4 2.3 <1.1 9.9 4.0 <1.1 <1.1 6.6 4.5 9.0 <1.1 16 20 <1.1 24 <1 .1 <1.1 240 45 12 13 14 15 16 17 0.46 2.0 1.4 1.9 2.1 2.2 11 6.7 1.4 4.2 <0.14 6.4 2.5 <0.14 <0.14 4.6 0.81 3.9 <0.14 14 7.0 <0.14 9.5 <0.14 <0.14 86 50ft 11 5.0 1 10 lilt Int 2.6 1 30 95 32 9.1 <0.03 29 7.6 <0.03 <0.03 Int 37 140 <0.03 a.e 66 <0.03 8.B <0.03 <0.03 740 47 1.2 6.9 6.1 23 27 4.8 170 61 9.7 15 <0.32 110 16 <0.32 <0.32 41 31 68 <0/32 170 120 <0.32 77 <0.32 <0.32 960 54 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 <0.04 NO 65 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 ND 67 <0.34 <0.34 3.0 3.0 14 5.4 75 31 5.4 8.4 <0.34 56 13 <0.34 <0.34 16 2.3 13 <0.34 12Q 40 <0.34 64 <0.34 <0.34 470 81 TCDD BBC 13C-2378-TCOO No TCDD isomers dcLect e d . Detection limit 0.2 ng/g 41 46 49 50 56 44 26 Att nr. 41159-11- PuCPP 11468 12468 21479 11478 11479/12168 12478 12479/13467 12467 23469/12347 11469 12348/12370 12146 12179 12167 12469/21489 11489 12169 21468 12349 12489 23470 12389 23467 T O T FnCfiP n i BBC l3C-1237B-PnCDP O) PnCDD 783942 G E N P O0 1i1l 1 3 1 UiCDF 123468 124678/134679 114678 124679 121479/123470 123670 124689 123467 123679 123469/123689 123709 121489 214670 TOT HxCDP BBC 33C-123470-H*CDP HsCDO 11 12 13 14 IS 16 17 <1.1 27 <1 .1 <1.1 21 100 <1.1 76 160 <1 .1 1100 36 <1.1 200 <1.1 <1.1 <1.1 <1.1 <1.1 21 110 <1.1 7.0 2100 56%. 2.6 6.6 1.9 <0.17 24 57 3.1 11 14 <0.17 100 4.3 4.3 22 <0.17 <0.17 <0.17 1.9 <0.17 4.1 37 <0.17 6.5 310 47% 2.0 7.0 3. <0.04 <0.04 lnt 2.0 16 15 <0.04 13 .1.6 0,98 29 <0.04 <0.04 <0.04 3.5 <0.04 21 40 1.5 6.0 160 33 200 65* <0.35 490 2600 81 360 340 <0.35 1000 <0.35 1 10 1200 49 49 <0.35 66 <0.35 1100. 1900 ai 200 10000 52% 57% <0.06 <0.05 <0.05 <0.05 <0.05 <0.05 <0.<iS <0.05 <0.05 <0.05 <0.0$ <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 NO 73% <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 ND 63% <0.36 30 <0.36 <0.36 54 260 16 58 60 <0.36 320 6.0 18 130 <0.36 <0.36 2.0 10 <0.16 4.0 660 <0.36 50 1600 86% No FnCDD isomers detected. Detection limit 0.2 ng/g 410 490 800 <1.7 5100 530 310 1100 30 190 100 330 120 9700 49% 23 43 86 1l 570 47 93 70 10 18 17 34 18 1000 50% 0.35 0.86 4.8 0.35 14 2.3 2.5 1.6 1.6 <0.05 0.19 1.5 0.75 31 100 380 1500 160 4200 1000 3100 460 110 670 230 1400 470 12000 51% 56% <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.06 <0.10 <0.10 <0.10 ND 78% <0.07 <0.07 <0.07 <0.07 <0.07 <0.07 <0.07 <0.07 <0.07 <0.07 <0.12 <0.12 <0.12 ND 110 72 490 15 1300 81 68 230 15 52 36 49 73 2600. 69% 95% No llxCUD isomers detected. Detection limit 0.2 ng/g Atfc n r. 41159-11HpCDP3 1234679 1234679 1234669 1234789 TOT BpCDF BpCDO OCDF 12346799 OCDQ 12346789 REC l3C-OCDD 11 12 13 14 15 16 17 150 1000 16 40 150 1000 60 460 400 2500 1.7 <0.07 1.2 0.27 3.2 880 170 610 470 2100 <0.06 <0.06 <0.06 <0.06 NO <0.07 <0.07 <0.07 <0.07 NO 22 1.8 15 3.3 43 No HpCDD Isomers detected. Detection limit 0.5 ng/g 20Q004 10000 5.4 100 <3.3 <3.8 5400 <10 7.7 <0.73 <4.7 <1.2 8.5 <5.2 NO 4SI 411 691 431 411 1001 ^ All quanti.cations are made with the ^ C - c o m p u n d s in RADIAN calibration standards set. 3 C o r e c t i o n for recovery is made. 3 Corection for recovery made with ^ C - 1 2 3 4 7 8 - H x C D F spike. 4 The values ar not corected for recovery. 783943 C-7 GENP 011132 High Resolution Gas Chromatography and Mass Spectrometry Conditions Column SUPELCO SP-2330, Fused Silica Length 60 m Diameter 0.25 mm Film Thickness 0.20 urn GC-Conditions HP 5890 with Helium as carrier gas Column pressure 40 psi Temperatur program: 10QC for 2 min splittless, to 180C with 20C/min, to 250C with 3C/min, hold at 250C for 30 min. Injections volym 1-3 ul MS-Conditions Instrument: VG 12-250 Low Resolution Quadrupole Electrone Energi 37 eV Source Potential 9V Repeller Voltage 9V Amplifier Gain Low sens SIR Multiplier Voltage 2000 V Tuning Calibrant PFK Source Temp. 200 C Transfer Line Temp 250 C 783944 C-8 GENP 011133 Clean-up technique for the determination of PCDP in askarel.and miniral oil 783945 GENP 011134 C-9 LA R S-O W E K J E L L E R -Umea Universitet 901 87 UMEA Sweden rJmea 86-06-18 EPRI-prodject 2028 Levels of dioxins and dibensofurans in baseline liquids Att nr. E729-53- 2.3.7. 8-TCDF 2,3,4,8-TCDF Tot. TCDF's REC 13C-2378-TCDP 2.3.7,8-TCDD Tot. TCDD's REC 13C-2378rTCDD 1.2.3.4.8- / 1.2.3.7.8- PnCDP 2.34.7.8-PnCDP Tot. PnCDF's REC 13C-12378-PnCDP 1.2.3.7.8-PnCDD Tot. PnCDD's 1.2.3.4.7.9-/ 1.2.3.4.7.8--HxCDF 1.2.3.6.7.8-HxCDF 1.2.3.7.8.9-HxCDP 2.3.4.6.7.8-HxCDF Tot. HxCDP1s REC 13C-123478-HxCDF 1.2.3.4.7.8-HxCDD 1.2.3.6.7.8-HxCDD 1.2.3.7.8.9-HxCDD Tot. HxCDD's l r2,3,46i7,8-HpCDP Tot. HpCDF13 Tot. HpCOO'3 OCDP OCDD REC ^^C-OCDD 01 89 100 190 60% 14 14. 67% 160 160 320 59% <1.5 ND 290 31 <2.1 170 490 59% <2.7 <2.7 <2.7 ND 450 450 ND ND2 ND2 ND2 C-10 02 03 62 <0.8 62 60% 38 38 58% 1300 ND1 2900 67% 92 92 62% 190 180 370 63% <1.3 ND 400 370 1800 58% <1.6 ND <1.1 40 <1.1 110 150 66% <2.2 <2.2 <2.2 ND 630 630 ND 56 <10 . 76% 1400 370 <1.8 350 2300 69% <2.4 <2.4 <2.4 ND 1000 2300 ND 3800 <8.1 110% GENP 011135 783946 The values are given in ng/g ND = Hot. Detected. ND^ - Not detected due to interferens from the big 2,3,7,8-TCDF peak. 7 ND - Not detected , no singnal for 13C-0CDDn. GENP 011136 c-n 783947 0026'49AX/88/030229*09M2.00/0 Ccpynitit i by Th Aw k u Soem y (at Phvm K ekoe tn d ExpatioMnul Thtnputiea Ail m ftu o f raprodunton m u y fana rwd. m o l l o ; Lak yHAMiACOLOQT, 34;22-237 Kinetic and Equilibrium Studies of Ah Receptor-Ligand Binding: Use of [125l]2-lodo-7,8-dibromodibenzo-p-dioxin C H R IST O P H E R A. BRA0F1ELD, A N D R EW S. KEN DE, and ALAN PO LAN D McArdt* Laboratory for Cancar Research, University of W isconsin, Madison, W isconsin 53706 (C.A.8., A.P.) and Department of Chemistry, University of Rochester, Rochester New York 14627 (A.S.K.) Received Oecember 14,1987; Accepted Apri 12,1988 SUMMARY In this report, we have used the radioligand p asl]2-iodo-7,8dibromo-dibenzo-p-cJioxin to describe the kinetics of ligand bind ing to the Ah receptor prepared from C578L/6J m ouse liver. The higher specific activity of this radioligand (2176 Ci/mmol), com pared with the usual tritiated ligand [1.6-3H]2,3,7,8-tetracfitorodibenzo-p-dioxin (58 Ci/mmol) permitted the study of ligandreceptor interactions at much lower com ponent concentrations. For this radioiodinated ligand, Scatchard analysis of saturation binding curves, determined at six different protein concentra tions, indicated that the apparent equilibrium dissociation con stant, K 0, w as directly related to the dilution of the receptor preparation; for example, at 1160 pq of proteifl/ml, K 0 * 1 . 6 x 10 "' m ; at 36 **g of protein/ml, K 0 * 1.2 x 10~`' m . Extrapolation of this function to infinite receptor dilution yielded K 0 =* 6 x lQ -,a w. The addition of 70 *iQ/ml of bovine serum albumin to a receptor preparation of 30 ug of protein/ml produced a 10-fold decrease in the slope of the Scatchard plot (i.e., 10-fold increase in the apparent K0). Conversely, enrichment of the receptor by high performance liquid chrom atography led to an increased slope and thus decreased estimate of K0. The association rate constant (*,), calculated from the integrated second-order rate equation, w as 2.8 x 10' m - ' h r"' and, from the initial velocity equation, had a value of 5.25 x 10' m - ' hr- '. The dissociation rate constant w as biphasic, consisting of a predominant fast com ponent with a rate constant of 0.36 hr-1 (*_,) and a slower com ponent with a rate constant of 4.2-9.4 x 1Q-3 hr- ' (k .2). Higher protein concentrations produced a decrease in estimates of k, but not k - t or k . 2. The K 0 determined from the ratio of the kinetic rate constant, *_>/#, * 6.9 x I0 " ,a m , is in excellent agreement with that derived from the results of equilibrium binding experiments extrapolated to infinite dilution, K0 = 6 x 10"'a m . The decrease in K 0t observed in equilibrium binding studies upon dilution of the receptor preparation, is best ex plained by a more accurate classification of `free' radioligand at lower protein concentrations. Finally, ligand binding to the A h receptor is best described by a tw o-step process, the formation of an initial complex, characterized by rapid ligand dissociation, which undergoes transformation to a second distinct complex displaying a much slow er ligand dissociation rate. 783948 Planar halogenated aromatic hydrocarbons elicit a variety of biological effects, including induction of cytochrome P-450 isozymes, epidermal hyperplasia and metaplasia, thymic invo lution, taratogenesis, tumor promotion, and lethality (1). These diverse biological responses are believed to arise from the altered gene expression that ia consequent to the high affinity, stereospecific binding of planar halogenated aromatic hydro carbons to a soluble protein known as the Ah receptor (1, 2). Like the steroid hormone receptors, the Ah receptor is rou tinely found in the high speed supernatant fraction ("cytosol") This papar is dedieuad to Or. ElUabtth Millar. This work was supported in part by th* National Instituts of Enviranmantal Hcilth Scianca Grant ES-01S84, National Cancar Institut* Cora Grant-07179, and National Cancar Institut# Postdoctoral Trainine Grant T32-CA09020. of tissue homogenates. After ligand binding and a poorly de fined "transformation"1step, the ligand-receptor complex dis plays an increased affinity for polyanions, such as DNA (S-G), and functions as a transcriptional activator (7, 3). The A h receptor also bears many physicochemical similarities to steroid hormone receptors, including a high molecular weight "aggre gate" form in low salt buffers (Af, 2.S-2.8 X 103) that dissociates to a low molecular weight "monomeric subunit" form in high salt buffers (Af, approximately l x 10s) (9). For the Ah receptor present In the hepatic cytosol of the C57BL/6 mouse and the Sprague-Dawley rat estimates of sedimentation coefficients, 1Racaptor transformaban is d(insd in this report u an alteration in th phyaiochamicml nature of th* lifand-racaptor compita, which multa in an incrtaaad affinity for DNA or othtr polyanionic matrice*. ABBREVIATIONS: TCC, 2.3.7,8-tetrBCri*orudlbenzo-^H3ioxin; MOPS, 3-(N-<norprioino)propenesutionic acid; K0, equilibrium dissociation constant; (L|, concentration of unbound ligand; [A], concentration of untigandod receptor, [(A), concentration of receptor-ligand complex; [LflL. reesptaligand complexat equkbrium; [LR'], concentration of activated RL complex; (Ljr. total raotoigafid concentration; [R]r, total receptor concsntxoon: 8mmi. receptor concentration per ml; second-order rate constant of association; Jr.,, fast component dissociation rate constant; k .2, slew component dissociation rata constant; HPLC, high performance liquid chromatography; TEA, trietftyiamine; r*, half-life of dissociation; ASPfT. <to55% ammonium sulfate precipitate; BCA, pidncftoninic add; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide,________ (TF.NP 011137 230 Bradffeidefs/. Stokes radii, molecular weights, and frictional and axial ratios a vortex mixer (30 sec). The organic phase was aspirated into a syringe (9) are all within-the ranges described for steroid hormone receptors (e.g., for the low salt aggregate form of steroid hor mone receptors the ranges reported are: sedimentation coeffi cient, 8-10 S; Stokes radius, 7-10 nm; molecular weight, 2-3,5 x 10*; frictional ratio, 1.45-2; and axial ratio 8-20; for a complete listing see Ref. L0). .(gas syringe, pressure-locked plunger tip, P010032; Pierce Chemical Co., Rockford, IL) transferred to a 1-ml sealed conical vial (Reacti-vtal 13221. Pierce Chemical Co.) containing 10 mg of magnesium sulfate, 75 m! of dichloromethane, and a microstirring bar, and the mixture was stirred for 45 min to dry the organic phase. [ll'I]2-iodo-7,3-dibromodibenzo-p-dioxin was formed from [(11I]2- amino-3-iodo-7,8,dibromodibenzo-p-dioxin by generation of the aryl- We recently reported the synthesis and use of an `" [-labeled diazonium salt, which produces the aryl radical with subsequent ab azidodibenzo-p-dioxin as a photoaffinity ligand for the Ah straction of hydrogen from tetrahydrofuran to form the deaminated receptor (11). The use of `'"'I-labeled dioxin congeners is ap product (13), This was accomplished as follows. The dried dichloro pealing because of their higher specific activity and counting efficiency compared with tritiated ligands. We now report the synthesis of a reversible radioligand, (i:*Il2-iodo-7,8-dibrotnodibenzo-p-dioxin. and its use to characterize the ligand binding kinetics of the murine hepatic Ah receptor. methane solution was aspirated and transferred to a L-ml conical vial containing 250 m1of anhydrous tetrahydrofuran and 67 Mmol of n-butyl nitrite. The reaction mix was then heated at 55* with stirring. After 15 min, the vial was opened and the solvent evaporated, with heating, under a stream of nitrogen gas, venting the vial into a funnel (2 cm diameter) attached to two serially connected charcoal filters in line Materials and Methods with negative airflow. This system trapped moat of the volatile radioi odine in the first charcoal filter and minimized release into the labo R e a ge n ts ratory hood ventilation system. The reaction mix solute was dissolved in 50 m1of methanol/water (92:8) and the '''[-labeled product purified TCDD was a gift of Dow Chemical Co. (Midland, MI); 2,3,7,8- by HPLC as detailed below. tetrachlorodibenzoruran was a gift from Dr. David Firestone (Food and Drug Administration, Washington. D.C.). Activated charcoal, grade PX-21. was a gift from Amoco Research Corp- (Chicago, ID. BactoGelatin was from Difco Laboratories (Detroit, MI). Magnesium sulfate was from Fisher Scientific Co. (Fair Lawn, NJ). Ammonium sulfate (ultra-pure) was from Schwart/Mann '(Cambridge, MA). Methanol (HPLC grade) was from Burdick and Jackson Laboratories, Inc. (Muskegan, MI). Glycerol was from J. T. Baker (Philliptburg, NJ). EDTA was from EM Scientific (Cherry Hill, NJ). Carrier-free Na1" ! (NE2033L) was from New England Nuclear (North Billerica, MA). BSA, dithiothreitol, d-mercaptethanol, sodium azide, chloramine T, MOPS (free acid and sodium salt), TEA, and TEA hydrochloride were pur chased from Sigma Chemical Co. (St. Louis, MO). Dichloromethane (reagent grade), tetrahydrofuran (anhydrous, 99.9% pure), n-butyl ni trite (97% purel. p-dioxane (anhydrous 99+% pure), and dimethyl sulfoxide (anhydrous, 99% pure, stored under N? gas) were purchased from Aldrich Chemical Co. (Milwaukee, WI). BCA Protein Assay Reagent was purchased from Pierce Chemical Co. (Rockford, IL). A n alysia and Purification of the R ad ioligan d Unlabeled 2-iodo-7,8-dibromodibenzo-p-dioxin, synthesized as above on a milligram scale, was used as an analytical standard. The unlabeled compound was characterized by 1) low resolution mass spectrometry: m/e (more than 40% of the base peak) 467.9 (100% M*), 469.9 (46%), 466 (50%), 71 (55%), and 43.1 (60%); and 2) reverse phase HPLC; using a C-1S column, 4.6 x 250 mm, 5 Mm particle (Altex Scientific Inc., Berkeley, CA), and an isocratic solvent system (methanol/water, 92:8) with a flow rate at l ml/min. The compound was detected by its UV absorbance at 254 nm and eluted with a retention time of 24 min. The radiolabeled reaction mix was subjected to HPLC separation as above. Approximately 60% of the radioactivity injected eluted with a UV absorption peak that had a retention time identical to that of the uniabeled compound (i.e., 24 min), in this methanol/water HPLC system, the potentially significant reaction product contaminants, 2amino-7,8-dibromodibenzo-p-dioxin, 2-iodo-3-ammo-7,8-dibromodibenzo-p-dioxin, and 2,3-dibromodibenzo-p-dioxin. elute with retention B u ffe r* times of 6.3,11.6, and 13.0 min. respectively, Reinjection of the 24-mir. MN represents the stock buffer which contains 25- nM MOPS and 0.02%sodium azide, pH 7.5 (at 4*). MEN is the stock buffer plus I mM EDTA. MdENG and MDENG represent MEN with the addition of 10% <w /vt glycerol, plus the addition of either 10 mM d-mercaptoethanol or 1 mM dithiothreicoL eluate (from above) also cochromatographs with authentic 2-iodo-7.8dibromodibenzo-p-dioxin using an 9:1 acetonitrile/water isocratic sol vent system (retention time, 22 min; 98% radiochemical purity). The overall yield of radioligand, judged by incorporation of 13iI into tht purified product, ranges from 40 to 50%. The specific activity v&s. assumed to be equivalent to that of carrier-free NamI, (2176 Ci/mmoL Radlosynthasia of [T" q2lodo-7,g-dlbreroodibanao-p-dtagin The synthesis was performed in a room dedicated to radiosynthesis, equipped with a high flow chemical hood, with the use of sealed vials and airtight syringes to minimize escape of radioiodine. The entire procedure was monitored with a y-radiation detector. 4,831 dpm/fmal). The radioligand was stored at 6 x 10*dpm/ml in pdioxant in a capped test tube protected from light. For use in binding experiments, an aliquot of the stock solution was dried under a stream of nitrogen gas and redissolved in DMSO at the appropriate concentra tion. Synthesis of (11JI]2-amino-3-iodo-7,8-<ilbroaio<iibenxo-pdioxin (Fig. 1). To 5 mCi of carrier-free Na,:aI (s350 mCi/ml in 6 Recaptor Preparation m M NaOH, 15 mI of water) in a septum-sealed conical vial was added Cytosol. C57BL/6J mice were purchased from The Jackson Labo 2.5 nmol of 2*amino-7.8-dibromodibenzo-p*dioxin (11) in 25 pi of ratory (Bar Harbor, ME) and bred in our laboratory. Adult male end methanol. 1.13 Mmol of sulfuric acid in 10 #1 of methanol/water (9:1), female mice were killed by cervical dislocation and their liven wera and 25 nmol of chloramine T in 5 of methanol (12). The iodination removed, rinsed with ice-cold KC1 (150 mM), homogenized in 9 volumes reaction was complete in 30 min and terminated by the addition of 500 of M0ENG buffer, and centrifuged at 10,000 x g for 20 min at 4*. The Mgof sodium metabisulfite and 5 pmol of sodium hydroxide in 35 pi of posunitochondrial supernatant was carefully removed to avoidcontim- water. The reaction product, (l,t!I]2-anuno-3-iodo-73-dibromodibenzo- ination by the surface lipid layer and subjected to eentrifiigarion ex :-dioxin, was extracted with 250 m1of dichloromethane with the use of 105,000 x g. The supernatant (approximately 10 mg of protein/mi) was 783949 f, o f o NM, <ti I. H. Chioriifim* T NM, Cuulnimn & , TMf 1* Mr. li 0 0 Pig, 1. Radkjsynttwsia of ['Ml]2-kxk>7,8^tvornodibenzo-p-dioxri, THF, tetrahydrofuran. T7~MT> A 1 1 1 n o Ah Receptor Kinetic* 231 separated from surface lipida and tha microsomal pellet and stored at Softw are -90' for uso. in.binding experiments or until processed further. Weighted nonlinear curve-fitting estimates of binding parameters ASPPT. The frozen cytosolic fraction was thawed and placed in an were determined as described (15, 16) using a commercially available ice bath with slow stirring. A saturated solution of ammonium sulfate software package for the IBM-PC (Kinetic. EBDA, Ligand, Lowry; in MEN buffer was added to the cytosolic preparation, to a final Elsevier Biosoft, Cambridge, England). concentration of 40%, over a period of 30 min and stirred for an additional 30 min. The solution was centrifuged at 10,000 x g for 20 Results min. and the supernatant fraction was removed, slowly brought to an ammonium sulfate concentration of 55%. stirred for an additional 30 min. and centrifuged at 10,000 x g, and the supernatant was discarded. The ASPPT was resuspended in 55% ammonium sulfate in MflEN, and aitquau equivalent to 0.5 g wet weight of liver (15 mg of protein) were placed in 12 x 75 mm borosilicate cubes (VWR Scientific. San Francisco, CA) and spun at 5000 x g. The supernatant was removed by careful aapiration, and the pellets were stored in stoppered tubes at -80* until use. HPLC-anion exchange-enriched receptor preparation. HPLC-anion exchange chromatography was performed on a Mono-Q HR 5/5 column (5 x 50 mm; Pharmacia, Uppsala. Sweden). All column parts, external tubing, injection loop, and buffers were placed on ice during the entire run. The HPLC instrumentation was identical to chat outlined in the purification of the radioligand (above). The sample lASPPT fraction, 200-900 *igof protein) was dissolved in 1-2 ml of 20 mMTEA buffer pH 7.5 (4') and injected into a 2-ml sample loop. The elution buffers used were the following; buffer A, 20 m M TEA. pH 7.5; and buffer B, 20 m M TEA, pH 7.5, plus 500 m M NaCI. The linear gradient program ran from 100:0 (A:B) to 40:60 over 8 min. then to 10:90 over 10 min, followed by isocratic elution (10:90) for 5 min. The 16-IS-min fractions were collected and pooled for uso in binding studies. These fractions appear at the beginning of a large UV-absorbing peak and thus some variation in purification was observed between runs. Protein concentrations'were determined by the method of War burg and Christian (14) or by the use of BCA Protein Assay using BSA as standard. Equilibrium binding analysis. Equilibrium saturation binding of [li']2-iodo-7,8-dibiomodibenzo-p-dioxin to the he patic Ah receptor of C57BL/6J mice, determined at three different protein concentrations, is shown in Fig. 2. Analysis of saturation binding curves by the method of Scatchard ( 17) is presented in Figure 3 and Table 1. With progressive 2-fold dilutions of the receptor preparation, the estimate of maximal binding sites per ml, Bmm, (x axis intercept), shows approxi mately 2-fold decreases, i.e., mg of protein remains con stant (Table 1; Fig. 3). Unexpectedly, the slopes of the Scat chard plots (-1 /Ko) increased from 2.6- to 1.2-fold with each 2-fold dilution (i.e., the estimate of Ko decreased from L.6 x 10~l to 1.2 x 10"" M, as the protein concentration decreased from 1160 to 36 Mg/ml). Hill coefficients were 1.0 at all receptor dilutions, indicating that site-site cooperatively did not contribute to the increase in the slope of Scatchard plota observed with dilution tlS). Scatchard and Hill analyses of saturation binding data were reproducible and of good linear fit, with coefficients of variation below 30% and with correlation coefficients of at least 0.SS (Table 1). Regression analysis of the relationship between the procein concentration of the ASPPT and the estimate of K0 as deter mined by Scatchard analysis suggested a simple linear function (Fig. 4). Extrapolation of the least squares fit of the line to the y intercept (infinite dilution of protein) yielded an estimate of JCo of 6 3 X 1(T,J M. Binding Experim ents Binding experimenta were carried out at 4*. The frozen ASPPT pellets or cytosolic fractions were dissolved and diluted in ice-cold MDENG buffer to the appropriate protein concentration. The radioli gand and the competing unlabeled ligand (2,3,7.S-tatnchlorodibenzofuran) were dissolved in DMSO and added to tha buffer/receptor solution so that the final concentration of solvent was 6 m! of DMSO per mi of buffer. Binding reactions were performed in 1.0-ml volumes (12 x 75 mm borosilicate test tubes), or in 50-mi volume (Ehrlanmeyer flasks), from which 1.0-tnl sample were transferred to the smaller tube. The binding reaction was terminated by the addition of a 0.5-mi volume of an ice-cold charcoal/gelatin suspension (3%/0.3% in MN buffer). The suspension was then stirred vigorously on a vortex mixer (2 sec), followed by incubation for 10 min at 4*. The charcoal/gelatin waa then sedimented at 2000 x g for 10 mis at 4*. A 1.0-ml aliquot of the supernatant fraction of each tube waa transferred to 12 x 75 mm polypropylene tubes (for higher counting efficiency, 75%), and tha bound radioligand waa quantified in a MINAX1 Saries-5000 7 -counter (United Technologiee/Packard Instrument Co., Downcri Grove, ID. Sample counting times were adjusted to achieve a counting error of 3%. Total radioligand, (L]t, was defined as tha concentration of radioli gand in solution after the 16-hr incubation. Total radioligand bound was defined as the radioligand in solution after charcoal adsorption. Two experiments were conducted to describe more clearly the relationship between protein concentration and the in crease in apparent binding affinity that waa observed with dilution. First, when the Ah receptor concentration mg of protein) in the ASPPT fraction waa enriched 5- to 9-fold by HPLC-anion exchange chromatography, the estimated Kr, was approximately 4-fold lower than the K0 determined on the corresponding ASPPT fraction (Table 1; compare 20 mg/ml wet weight values). Second, saturation binding was described on an ASPPT preparation (30 ug/mi) in the absence or presence of 70 Mg/ml of a highly purified protein, BSA (Fig. 5), Scatchard analysis of the binding data described a 10-fold increase in the apparent Ko when BSA waa present. Despite the large effect of BSA on the apparent K0, the estimate of flTM, was only slightly increased in the presence of the additional protein, approxi mately 1.3-fold. It should be noted that this ASPPT preparation was from a different batch than that used to generate the data in Table 1 and Figs. 2, 3, and 4. This preparation yielded a slightly greater receptor concentration per milligram of protein. Binding kinetics. For a simple bimolecular reaction gov erned by the law of mass action, the K0 of ligand binding is equal to the rate constant of ligand-receptor dissociation di vided by the second-order rate constant of ligand-receptor association: Nonspecific binding waa defined as the amount of radioligand bound R + RL (l) in tha presence of a 200-fold molar excess of 2J,73-tttrachlorodiben- zofuran. Specific binding was defined as tha difference between total radioligand bound and radioligand nonapecifically bound. Unbound ("free") radioligand was defined as the difference between total radi oligand and total bound radioligand. and K..o - --A-i (2) GRNP 0111-^0 232 SradfltM ef at. M ug/m i l4 S u g /m l SSO us/m i 783951 111u o Rg. Z Equilibrium binding of [,33l]2-iodo-718-dibromodibnzo-o-j>oxm. The receptor preparation (ASPPT. 36 (A), 145 (B). or 580 (C> ag of protan/ mi] was incubated at 4a for 16 hr. with concentrations of radioligand ranging from 2.5 to 250 fmoi/mt. Total, specifically bound, and nonspecificaily bound radioligand were determined as outlined in Matenais and Methods. Each point represents the average of duplicate determinations. TABLE 1 Summary of saturation binding experiments performed on the Ah receptor at various protein concentrations Tha saturation binding isotherm s to r me radioingand and Aft receptor m neoattc cytosol, the A S P P T fraction of cytosol, and the H PLC-enncned preparation of ine A S P P T fraction were determ ned over a range of protem concentrations. The binding param eters K 0, and B m m were calculated by Scatenarci analysis of binding data as desentnd in the legend to Fig. 2 or by H * analysis of binding data as descnbed (18). Values represent the mean the standard deviation, except m me ca se of a single data point or duplicata data pomta, for w hich onty the mean is given. Correianan coeffloents were at least 0.98 for both Scatenarci and Miti plots. Tha standard deviation of the Hti coefficient did not exceed 0.02. EquvMrtw tt wexyn Protan X 0 8 Hicoettaam m gfnt Mfl/fflf fm e ifm q a tfm tm Rg. 3. Scatchard analysis of equilibrium binding data generated at vanous dilutions of receptor. Equilibrium saturation binding curves of ('"t]2-iodo-7,8-dibroniodibenzO"P-dioxin were determined at vanous di lutions of the ASPRT fraction (as descnbed in Fig. 2). The binding parameters K0 (-1 /stops of the line) and 3mm (receptor concentration/ ml; the x intercept) were calculated from a weighted least squares fit of a plot of specifically bound raciotigand/free radioligand versus spsaficatfy bound radioligand (16, 17). When more than two plots were generated at a given protem concentration, the plot that yielded tha median K0 value was chosen for presentation. Eq. 2 provides an estimate of the K0, independent of that calculated from equilibrium binding studies. Association ra te constant. The rata constant of associa tion was calculated from the time course of ligand-receptor ASPPT 1.25 36 12 2.5 72 14 4 5 145 1 9 *3 10 290 4 9 * 9 20 560 92 40 1160 160 HPLC-Enricfted 6.7 6 11 14 9 12 20 25 22 30 46 22 Cytosol 0.62 42 7 1.25 65 13 2.5 170 24 141 114 21 110* 14 121 * 7 121 131 780 611 684 1100 95 113 80 1.0 1.00 0.97 0.98 0.98 0.98 1.00 0.99 0.98 0.98 1.00 1.00 0.99 1 5 4 3 1 2 1 1 1 1 1 2 1 binding, via the integrated second-order rate equation, as de scribed by Weiland and Molinoff (19): ."Lf[LRl, [fL[LMlrIL-R[IL,R-)[[LLRRJ1MRlrl]J equilibrium, (f2],, and ligand-receptor complex, [Z], at var ious time intervals, t. The advantage of this method is that no constraints are placed upon the concentrations of ligand ol- receptor used in the binding studies. By this method, dilution ` f e - H (3> of the protein concentration by 18-fold resulted in a 37-fold increase in the calculated rate constant of association (/tt = 7.6 Substitution of lnV for the left side of the equation and re 3 Tl arrangement yields the equation of a straight line, with slope InY7time. Thus, can be calculated from the relationship: jP' * slope [LRl [L]AR)t (4) [LR]* x 10* to 3.0 x 10to M"` hr"'; Fig. 8, A and B, and Table 2). In contrast, at a fixed protein concentration a 13-fold change in total radioligand concentration did not altar the association rate constant (La., kx 3.0 and 2.6 x 10l m~1 hr"'; Fig. 6, E and C, and Table 2). The association rate constant was also estimated from theinitial binding velocities (u0) under conditions in which [L]r This method allows determination of from estimates of the and (R]r > [LR], so the free radioligand and free receptor concentrations of total ligand, [L\r, total receptor, (R]r (ob concentrations are approximated by [L\r and [R]r > re sp e c tained from Scatchard analysis), ligand-receptor complex at tively. 783952 Ah Receptor Kinetic 233 O to - ,O Fig. 5. The effect of BSA on me slope of me Scatchard plot. Equilibrium saturation binding curves were generated, as described m Fig. 3, for an ASPPT sample of 30 ^g of protem/mi w rth and without the addition of 70 Mg/m< BSA. Calculated binding parameters: plus BSA, K0 - 8.3 x 10'" m, Bmm 9.2 fmoi/mi: without BSA, Kq - 7.3 x i0 *'a m, a * . 7.4 fmol/m where: fraction equal to protein concentrations of 1160 and 72 and radioligand concentrations of 5.1 x 10~M m and 4.1 x 10` ,J u (250,000 and 20.000 dpm/rrt). Each point represents the average of two detet- mirtations. The data are presented as a plot of bound radioligand as a function of time(A1. S i, and Cl), and as a plot of In/ (left side of Eq. 3) as s function of tone (A2, B2, and C2). The rate constant of association was determined from the slope of In? versus time plots as described in Eq. 4 and is inset (units of u " hr*'). rearranging gives: Vo m X| [R ]rL r] (5) *. * v 0/[ R ] r { L lr (6) Plotting initial binding velocity as a function of receptor or radioligand concentration, with the other variable fixed, allows the determination of k\. A plot of initial binding velocity versus receptor concentra tion yielded a hyperbolic plot (Fig. 7A). The correlation coef ficient of the plot (for a linear fit) using ail data points pre sented is 0.94. By elimination of the values obtained above a receptor concentration of 1 x 10*" M and inclusion of the theoretical y intercept of (0,0),--a correlation coefficient of 0.99 is obtained and an association rate constant of 5.5 x 10` M '1 h r '1is calculated (i.e., calculation of A, from the asymptote of the hyperbola). The initial binding velocity as a function of radioligand concentration, at a fixed receptor concentration of 6.8 x 10"u M, is shown in Fig. 7B. This plot yielded a good linear fit and an estimate of the second order rate constant of association equal to 5 x 10l M~khr"1. Dissociation ra te constants. As shown in Figs. 8 and 9, the dissociation of the ligand-receptor complex does not follow a simple first-order process. The dissociation profile is inde pendent of the receptor concentration or the type of prepara tion. The data is best fit by a two-component exponential curve (y *Ae*-1.1+ Be*-*).3The initial rapidly dissociating component comprises approximately 75% of the total complex and has a 1Our multa indieata that a atiatically battif fit wu obiamec by using & biasponantial modal ovar a monoaxponantal ona tp < 0.001). We used pam a! F t i aa deacribad by Munin and Rodbaid (19). No Smhar improvmenc was. obtainad whan attampta wata mada co fit cha data to a tntxponantit mcccl ip < 0.98), altboueb tbia and biabar ordar axponantial modal* cannot mkd cuc by tba data pmantad. 234 SradftoM * *t. TABLE2 binding has appeared. The kinetic and equilibrium studies in Summary of kinetie data Rat* constants wr* calculated u descneed m tna fast and expressed as me man and. wnara posswe. tna standard error. The aquttortum dissooatxsn con* stint. x,,. was detemwwd tiy i) Scatenard anirysts of tna saturation omamg isatnamt at infinite citutton (Rg. 3) or 2) as a ratio of tna dissociation rata constant. (0.36 nr") and tna association rata constant from atner tna integrated rata equation or tna initial binding velocity metnod. The dissociation rata constants presented asove nave not seen corrected tor receptor-figand complex destruction rates, tfius they are tne sum at dissociation and destruction rates (sea Results). this report challenge the following two assumptions implicit in past binding studies: 1) that both specifically bound radioligand and free radioligand concentrations can be determined accu rately at high protein concentrations and 2) that ligand-A/i receptor binding can be described by a simple reversible bimo- lecular model (Eq. 1). The most common method of analyzing equilibrium satura L, flr ftj tion binding data is the linear transformation of Scatchard K m" nr" (17): Association rate constant Integrated 2nO-order 4.1 X I0 ',a 8.2 x 10*'* 2.8x10' 5.1 x 1Q-" 8.2 x 10-'3 3.0 x 10' [LR\ [RW - [LR] [.L1 * K0 5.1 x 10'" 1.5x10*' 7.0x10 Initial velocity method or Ligand Receptor 2.1 x 10"" Varied 5.5 x 10' Varied 6.8X10-'* 5.0x10' PifCstt ft.} Pifcent [Boundl [Free] - AO [Bound] + A q (8 ) nr" nr" x I0J Dissociation rate constant ASPPT 0.37 0.02 78 4 4.3 0.4 22 1 Cytosol 0.36 0.05 77 7 9.4 0.8 23 2 Warmed cytosol 0.36 0.04 72 5 7.2 0.5 28 1 K0 A plot of the ratio of specifically bound ligand/free ligand as a function of specifically bound permits one to estimate K D and total binding sites, []r>Analysis of equilibrium binding data by the Scatchard equation requires that a number of criteria be met aa follows: 1) the concentration of specifically bound and free ligand are measured accurately at equilibrium; 2) both Equilibrium dissociation constant Scatchard (infinite dilution) ' 6.0 3 x IQ-'* ligand and receptor are homogenous species; 3) binding obeys the law of mass action as described in Eq. 1 and 2; and 4) no Kinetic (initial velocity) 6.9 x 1 0"3 cooperativity exists. Kinetic (integrated 2nd order) 13.0 x 10-* C riterion 1: M easurem ent of free and specifically bound ligand ia accurate. The accuracy of Ka and iissociation rate of 0.36 h r '1 (t., = 1.9 hr). The second slower values generated from Eq. 3 is dependent upon the accuracy of .ssociating component (25%) has a rate constant of 4.3 to 9.4 the determination of bound and free. Estimates of bound radi x 10"' hr"1U-, = 74-161 hr) (Table 2). The rate constant for oligand are fairly straightforward and can be supported by "degradation"* of the receptor-ligand complex at 4* is - 2 X physicochemical characterization of the ligand-receptor com 10"' hr"1, monoexponential for over 120 hr. and not signifi* plex (e.g,, density gradient centrifugation, size exclusion chro cantly affected by protein concentration. Thus, the second matography, anion exchange chromatography, etc.). Con component of "dissociation" is a composite, representing both versely, the concentration of free radioligand ia most often ligand-receptor dissociation and degradation. The rate constant determined indirectly as the difference between total radioli for degradation of the unoccupied receptor ia --4 x 10"3 h r '1 gand in solution and total bound radioligand. In Ah receptor- and is monoexponential for over 100 hr. ligand binding studies, free is defined operationally as the As shown in Table 2, Ko values calculated ae the ratio of amount of radioactivity that can be adsorbed to charcoal (20), kinetic constants, k^,/ki (where &_i is the dissociation rate that can be removed from hydroxyapatite by extensive deter constant of the major rapid dissociating pool and k\ is estimated gent washing (21), or that does not sediment in a sucrose from the integrated second-order equation dr initial velocity gradient (22). By these methods, radioligand, bound to low method), are 6.9 x 10'13 and 13.0 x 10"13 M, which agree very affinity nonspecific binding sites, that dissociates during char well with the Kn determined by equilibrium saturation data coal adsorption, hydroxyapatite washing, or long-term centrif extrapolated to infinite dilution, 6.0 X l(T,a m . ugation will bo misclassified as free radioligand. This systematic D iscu ssio n misclassification will decrease the ratio of bound/free ligand and hence decrease the slope of Che Scatchard plot (i.e., - 1 / Due to its high receptor affinity, high biological potency, commercial availability, and environmental importance, TCDD has become the most studied Ah receptor ligand. Despite exten sive characterization of the Ah receptor using (3H1TCDD or other ligands, no detailed kinetic analysis of ligand-receptor K o ), although it may not significantly compromise estimates of Bmaa (18, 23, 24). One would expect the misclassification of nonspecifically bound radioligand aa free radioligand to be greatest, and thus the overestimation of Ko to be greatest, at higher protein concentrations. We believe this is the most plausible explanation of the data presented in Figs. 3-5 and 'Th* degradation rata of the ligand-receptor complex was determined by incubation of tha ASPPT fraction 172, 14S, and 290 Mg of procain/taU with radioligand with or without 200-fold excaas 2J,?,S-tetrachioredibenxofunn at 4* ^ 16 hr tax described in tha legend to Pig. 6), Specific binding was determined iquota removed at tima inttrvala from IS to 130 hr. Tha degradation rata of - jnocr- pied receptor waa m imated by sampling a receptor preparation stored at 4' itttr vsrioua urns intervals. The receptor sample waa incubatad with radioligand with or without 'i00-fotd excess 2A7,B*tatrachla(odibenxofuran at 4* for 16 hr. and specifically bound radioligand waa determined. The degradation rates were estimated from the plots of the log of specific binding versus tima. Table 1. Using [3H1TCDD, Fane! and Safe (25) have noted a rela tionship between protein concentration and K0 estimates gen erated from Scatchard analysis of saturation binding ckus_ Charactarization of Ah receptor ligand binding with a radioli gand of modest specific activity, such as fHITCDD, necessi tates the use of protein concentrations greater than 1 mg/nd (20-22, 25). Thus, Ko values generated previously for (3H? 783953 GENP 01114?. Ah Receptor Kinetic 235 -otCUJonD1 Fig. 7. Initial binding velocity of (,JV]2-+odo-7,8-dibromodibefizo-p-dioxin to the hepatic Ah receptor estimate of the association rate constant. The ASPPT fraction in MDENG buffer was incubated with radioligand in 50-ml Ehhenmeyer flasks. At the indicated times intervals. 1.0-mi aliquots were transferred to txjrosilicate tubes containing 1000-foW molar excess of 2.3.7.8-tetrachlorodibenzofuran (600-fold for nonspecific binding) to prevent further binding of the radioligand and total, specifically, and nonspecifically bound radioligand was determined. Receptor concentrations were determined by duplicate Scatchard analyses. A, Initial binding velocity as a function of Atreceptor concentration. Receptor concentrations of 1.7 x 10"'2m (), 3.4 x 10"'* m (), 6.1 x I0~,a m (O), 8.5 x i0 -,a m (0 ). 1.7 x 10"" m (). and 2.4 x 10*" m () were incubated witn a fixed radidigsnd concentration, 2.7 x 10'11 m . The initial binding velocity was determined for each receptor concentration from the slope of the plot of specifically bound radioligand as a function of time (inset). The initial binding velocities were then plotted as a function of receptor concentration. The rare constant of association was calculated from the slope of the linear portion of the line(asymptote of the hyperbola) divided by the ligand concentration (see Materials and Methods). B, Initial binding velocity as a function of radioligand concentration. A fixed concentration of receptor. 6.8 x TO"'* m. was incubated witn radioligand concentrations of 5.6 x 10"'* m (O), 1.1 x 10"" m (p), 2.0 x 10"" m (), and 3.1 x 10~" m (.). The initial binding velocity was calculated from the slope of specifically bound radioligand as a function of time (inset). The initial velocity was plotted as a function of radioligand concentration. The rata constant of association was calculated from the slope of this line divided by the receptor concentration. TCDD are probably overestimates. Using a competitive binding assay with [l*'I]2-iodo-7,8*dibromodibenzo-p-dioxin as the ra dioligand and low protein concentration, we estimate the Ko for TCDD to be approximately 4 x 10-Ia M.4 Misclassification of nonspecifically bound radioligand as free radioligand has been described in a number of other receptor systems that bind hydrophobic ligands: estrogen (23), proges terone (24), and thyroxine (26). In these systems, misclassification errors have been minimized by the use of methods that better estimate free and bound radioligand, e.g., equilibrium dialysis (23, 24) or centrifugal ultrafiltration-dialysis (27). Be cause our radioligand adsorbs strongly to dialysis tubing and filter membranes,* we provided support for radioligand misclassification by the following: 1) demonstrating that addition of a purified protein (BSA) decreases, whereas receptor enrich ment increases, the slope of the Scatchard plot (Fig. 5 and Table 1), and 2) eliminating the possibility that the rate con stants of association or diaaociation were altered by dilution. We observed that, like K0t the apparent association rata constant also varies as a function of the concentration of receptor, or more likely, protein (Figs. 6 and 7A). Association rate constant estimates are also dependent upon accurate de termination of free radioligand. From the initial velocity rata equation (Eq. 6), it follow that for a given ligand concentration a plot of Voas a function of receptor concentration will yield a linear plot, with a slope equal to k,[L]r . The hyperbolic shape *C. A. Bnditeld ad A. Poland, manuscript in preparation. *C. A. Bradfiaid. and A. Poland, unpublished obaarvanona. of the curve generated (Fig. 6A) indicates that kilLlr is decreas ing as the concentration of the receptor preparation increases and supports the idea that free ia being overestimated at higher receptor concentrations. An alternative explanation is that kK is actually increasing with dilution of the receptor. Because k{ is a function of diffusion rate and the energy of activation for the binding step (28), an increase in ki with dilution implies that a decrease in the energy of activation for ligand binding is also occurring with dilution. A decrease in the energy of acti vation for the forward reaction should also result in a decrease in the energy of activation for the dissociation reaction and lead to a increasing k-i with dilution. Because is not affected by protein concentration (Fig. 7 and discussion below), the hyperbolic nature of the curve depicted in Fig. 6A is best explained by an overestimation of free radioligand at the higher protein concentrations. Criterion 2: Both ligand and receptor are homogenous binding species. The time course of ligand-receptor dissocia tion is biphasic, suggesting two Ligand-receptor species that have dissociation rate constants differing by a factor of at least 60-fold (Figs. 8 and 9 and Table 2). The dissociation of pH] TCDD from the rat Ah receptor has previously been described as "irreversible" (25). Our preliminary comparisons between the dissociation kinetics of tbs Ah receptor-radioligand com plex in the Harlan-Sprague Dewtey rat end C57BL/6J mouss indicate significant difference.5 C riterion 3: .Binding obey the law of mass, action a? described in Eq. 1. The linear functions generated from plo:s of initial binding velocity ( uQ) versus cither the ligand c.- / n - n x m r\* 236 BradftoMta*. 783955 receptor concentrations (Fig. 7) indicate that the binding re action is first order with respect to both radioligand.and recep t o r and thus second order overall (formation of the ligand receptor complex) (28). This lends support to the assumption that one molecule of ligand binds to one molecule of receptor. Biphosic ligand-receptor dissociation is not in agreement with the equilibrium binding scheme presented in Eq. 1. Bi- phasic ligand-receptor dissociation curves have been observed for ligands bound to the estrogen (29, 30), progesterone (31), glucocorticoid (32), and androgen (33) receptors. For these steroid hormone receptors, the slow dissociating species (be lieved to represent a "transformed" receptor species) has an increased affinity for polyanionic matrices such as DNA (33-- 35) and an altered molecular mass as compared with the fast dissociating species (36, 37). In these systems, in which the ratio of the fast to slow component can be altered by increasing the ionic strength, heating, etc., we did not observe a significant HOURS change in the ratio of the fast and slow components by heating Fig. S. Dissociation of tfta (1J5!]2-kx3o-7,8-dit>rornodibenzo-p-dioxin Ah a 30* for 30 min, dilution, or ammonium sulfate precipitation. receptor complex at 4: the effect of protein concentration, fladlotigand- Ligand binding to the Ah receptor appears to conform to the receptor complexes.were preformed by incubation of radioligand and receptor for 16 hr at 4 in 50-ml Ehrienmeyer flasks. Fractional receptor model proposed for the steroid hormone receptor (32) in which: occupancy of radioligand was between IS and 18% for all protein concentrations. This was achieved by the following protein (receptor) and radioligand concentrations: 1160 pg/ml (150 p m ) and 62 pM; 290 nQjntt (35 pM) and IS pM; and 72 ig/ml (8.2 pM) and 4 pM: After equilibrium binding had been obtained, a 1000-fold excess of 2.3.7,8- L + R L R & LR' L + R' *-i *-ii *-i and thus at equilibrium: (9) tetrachtorodibenzofuran was added to prevent rebinding of radioligand (time zero). Bound radioligand was determined, at the indicated times, on 1,Q*ml aliquots. Nonspecific binding was determined by the addition [L] [R] [L ] + ( 'I ( 10) of a 200*fotd excess of 2,3.7.8-tetrachJorodibenzofuran during the prebinding stage, followed by the addition of an 600-fold excess at time zero. All values were determined in duplicate. Results are presented as specifically bound radioligand/radioligand specifically bound at time zero, C riterion 4: No site-site cooperatively exists. Hill coef ficients were 1.0 at all dilutions; there is no indication of cooperativity in this system. Hill coefficients of approximately plotted as a function of time. 1.0 have previously been reported for a number of mammalian species (25, 38). In this report, analysis of the Ah receptor-ligand binding, facilitated by a new high specific activity radioligand has re vealed important findings. 1) Estimates of the apparent K0t as determined by Scatchard analysis, are a function of protein, concentration and attributable to a systematic overestimaticr. of free ligand. The true K0 (from equilibrium saturation at infinite dilution and ratio of kinetic constants k - J k x) is ap proximately 100 times lower than previous estimates, 2) The Ah receptor ligand complex can exist as two distinct species, which have different dissociation rates, and thus ligand binding does not conform to a simple reversible mass action model, as has been assumed previously. FTg. 9. D issociation s of (ia^-iod o>7.0-dtbrom odlb en zp-diexin from different receptor preparations at 4#. D issociation rates w era determ ined a s described in fig . 6. Cyroeof (85 Mg of prctaai/m l) and A S P P T (72 g/ rm) were preineubated wrtn 10 p m radioligand for 16 hr at 4* before dissociation rate determ inations. Heared cytosoi w s s preincubated for 18 hr at 4 * a s above, follow ed by a 30-rmn incubation at 30*. then recooled to 4* (30 min) before d isso o a tio n rate determina tion. Kinetic rate con stan ts w ere calculated by the nonlinear curve fitting technique, constrained to a biexponential model, as described (16). Reference L. Poland, A., and J. C. Knutson. 2,3,7,8-Ttrsehlorodib#nzo-p-dioxin and re laud haloganatad aromatic hydrocarbons: lamination of the mechanism of toxicity. Annu. Rmv. Pharmacol Toxicol. 22:517-554 (1952). 2. Whitlock, J, P. Tha peculation of cytochrome P-450 gene xpreuion. Annu. Acu. Pharmacol ToxtcoL 28:333-369 (1986). 3. Greenlea, W. F-. and A. Poland. Nuclear uptake of (he 2^,7,3-utrachlorodibanxn-p-dioxin in C57BL/& and DBA/2J mice. J. Biol Cham. 254:98149821 (1979). 4. Okey. A. B., G. P. Bondy, M. E. Mason, G. F. KahL H. J. Eiaan. T. M. Guanthar, and D. W. Nebeit. Regulatory gene product of the Ah locu: charsetafixation of tha cytosolic inducer-receptor complex and evidence fot iu nuclear translocation. J. Biol Cham 254:11638-11648 (1979). 5. Hannah. R. R., J. Lund, L Poedinger. M. Giilner, end J.-A. Characttruatioa of tha DNA-binding properties of tha receptor for 2,3,7,8tetraehlorodibenco.p-dioiin. Ear. J, Biochtm. 158:237-242 (19&8). 6. Gaeiewicx, T. A,, and P. A. Bauman. Heterogeneity of tha rat Ah receptor and evidence for transformation in uuro and in moo. J. Biol Chem. 2&&21162120(1987). 7. Jones, P. B. C., L. K. Bunin, J. M. Fixher, and J. P. Whitlock, Control of gene xpreuion by 2,3.7,S-utrachlorodibenxo-p-dunin: multiple dioxm-re-' rVPKTP 0 1 1 1 4 4 Ah Rcptor Kinetics 2 3 7 sponsive dormiri 5'-wand of Uva cytochrome Pt*460 gene. J. BioL Chtm 291:6647-6650 (1966). 3. Giguare, V., S. M. Hcllenbarg, M. G. Roean/eld od R. M. Evasa. Functional domains of the human glucocorticoid receptor. Cell 40:646-652 (1986). ____ 9. Oanison, M. S,, L. M. Valla, and A, B. Okay. Structure and function of tha Ah receptor for 2.3.7,6*tauichlorodibefuo<p-diosin.J. BioL Cham. 261:39373993 <1986), 10. Vedackia, W. V. Steroid honnone receptor itructura in normal and naoplaatic cell, in Hormonally Rnporuutt Tumor* (V. P. Hollander, ed). Academic Presa. New York, 4-35 (19851. 11. Poland. A., . Giovar, F. H. Ebitlno. and A. S. Kanda. Phocoaffimty labeling of the AH receptor. J. BioL Chtm. 261:6352-6365 (19861. 12. Danny, J. B.. and G. Blobal. '" [-Labeled croaalinking reagent chat it hydro* phytic, photoactivatable, and claavable through an axo linkage. Pnx. Hatl Acad. ScL USA 81:5286-5290 (1984). 13. Cadogan. J. I. G., and 0. A. Molina. A aimple and convenient deamination of aromatic amines. J. Chtm. Soc. Perhint Trans 1541-542 (1973). 14. Warburg. 0. and. W. Chriatian. laoliarung und kriatalliiation dea garungtftrmenu anolaaa. Biochem Z. 310:384-421 (1942). 15. Munson. P. J- and D- RodbsnL Ligand: a versatile computerized approach for characterization of ligand-binding systems. AnaL BitxHtm 107:220-239 (1980). 16. McPherson. G. A. Analysis of radioligand binding experiments: a collection of computer programs for tha IBM PC. J. PharmacoL Methods 14:213-228 1985). 17. Scstchard G. Tha attractions of proteins for small molecule and iona. Ann. .V. Y Acad. Set. 51:660-672 (1949). IS. Bennet, J. P.. and H. 1. Y&mamura. Neurotranamittar. honnone, or drag receptor binding methods, in Hcurotranjmuttr Receptor Binding !H. I. Yamamura. S. J. nne, and M. J. Kuhar, eds). Raven Press. New York. 61-90 11983) . 19. Weiland, G. A., and P. B. Molinoff. Quantitative analysis of drag-receptor inunctions. I. Determination of kinetic and equilibrium properties. Life ScL 29:313-330(1981). 20. Poland. A., and E. Glover. 2.3.7.8-Tetrmchlorodibenzo-p-dioiin: segregation of toxicity with tha Ah locus. MoL Pharmacol 17:88-94 (1979). 21. Gaaiswicz, T. A., and R- A. Neal. The examination and quantitation of tissue cytosolic receptors for 2.3.7,S-tetrachlorodibenzo-p-dioxm using hydroxyl- apatite. A/tui. Biochem 124:1-11.(1982). 22. Manchester, D. K.. S. K. Gordon. S. L. Galea, E. A. Roberta, and A. B. Okay. Ah receptor in human placenta: stabilization by molybdate and characteriza tion of binding of 2,3,7>S-tetchlorodiben20*p-dioxin,3-mthylcholenthrene, and benzolajpyrene. Cancer Re*. 47:4861-4868(19871. 23. Sitteh, P. K. Receptor binding studies. Scitnct (Wash. D. C.J 223:191-193 11984) , 24. Ssiduddin. S.. and H-. P. Zaasenhaua. in vitro and in vivo enhancement of progesterone binding to the uterine progesterone receptor by cortisol. Biochemistry 10:2829-2834(1977). 23. Farrel, K.. and S. Safa. Absence of positive cooperativity in the binding of 2.3.7,3-tetrflchlorodibanxo-p-dioxin to iu cytosolic receptor protein. Biochem. J. 244:339-548 (1987). 26. Seelig, S-. H. L Schwartz, and J. H. Oppanheimar. Limiutions in the conventional analysts of the interaction of triiodothyronine with solubilized nuclear receptor sites. J. BioL Chem 230:2154-2161 (1981). 27. Hammond. G. 1--J- A. Niaktr, L. A. Jones, and P. K. Siiteri. Estimation of the percentage of free steroid in undiluted serum by centrifuge! ultrafiltration dialyaia. J. BioL Chtm 296:5023-5026 (1980). 28. Meitea. L An introduction to chemical kinetics, in An fntroduenon to Chemical Equilibrium and Kineticj. Pergamon Press, New York. 33-76 (ISSli. 29. dBeeasts-iBteeslproemcemptee,uMre.,uJte. rFinriee.sE, aunr.dJT. .BEiordchoes.mIn1te7r:a4c2t3io-4n3s2sn(1tr9e70T1o. escradtol et 30. Best-Belpomme. MTMJ. Mester, H. Weintreub, and E.-E. Bauliau. Oestrogen receptors in chick oviduct: characterization and subcallular fractionation. Eur. J. Biochem 57:337-347 (1975). 31. Wolfaon, A.. J. Master. Y. Chang-Ran. and E.-E. Baulieu. Non-activated form of the progesterone receptor from chick oviduct: characterization. Btochem Biophyt. Ret. Common. 96:1577-1384 (1980). 32. Pratt, W. B., J. L. Kaina, and D. V. Pratt. Tha kinetics of glucocorticoid binding to tha soluble specific binding protein of mouse fibroblasts. J. Biot. Chem 250:4384-4591(1975). 33. de Boer, W,, M. Lindh, J. Bolt, A. Brinkmann. and E. Mulder. Characteriza tion of the calf uterine androgen receptor and iu activation to the deoxyri bonucleic acid-binding state. Endocrinology 118:851-861 119861. 34. McBlain, W. A.. D. 0. Toft, and G. Shyamata. Transformation of mammary cytoplasmic glucocorticoid receptor under cell-free conditions. BiochemistTM 20:6790-6798(1981). 35. Yang, C. R., J. Mester, A. Wolfson. J. M. Renoir, and E.-E. Baulieu. Activation of the chick oviduct progesterone receptor by hepann in the presence or absence of hormone. Btochem. J. 208:399-406 (1982). 36. Weichman B. M.. and A. C. Notides. Estradiol-binding kinetics of the activated and nonactivated estrogen receptor. J. BioL Chtm. 252:8856-6862 (1977). 37. Muller, R. EL, D. M. Beebe, E. Bercel. A. M. Traish. and H. H. Wotiz. Estnoi and estradiol interaction* with the estrogen receptor in two and in vitro. -J. Steroid Biochem 20:1039-1046 11984). 33. Gaaiewicx. T. A., and G. Rucei. Cytosolic receptor for 2.3.7,8-cetrachlorodibanzo-p-dioxin: evidence for a homologous nature along mammalian species. MoL PharmacoL 20:90*98 (1984). Send p rin t roquaata to: Alan Poland, McArdie Laboratory for Cancer Re search, University of Wisconsin, Madison, Wl 53706. GENP 011145 783956 ERRI Electric Power Qoarch Institute Topics: PCB Chemical analysis ` Transformers Capacitors PCOF-PCDD Insulating oil EPRI EUEA-5443 Volume 3 Project 2028-10 Final Report June 1988 A nalysis of Polychlorinated Dibenzofurans and Polychlorinated Dibenzo-p-Dioxins in Transformers and Capacitors Volume 3: Sum m ary and Statistical A nalysis of the Round-Robin Test Prepared by Research `Mangle Institute Research "Mangle Park, North Carolina GENP 011146 783957 REPORT SUMMARY SUBJECTS Hazardous and toxic waste management /Transmission substation design and operation / Distribution substations TOPICS PCS Chemical analysis ' Transformers Capacitors PCDF-PCDD insulating oil AUDIENCE Environmental managers / Distribution engineers Analysis of Polychlorinated Dibenzofurans and Polychlorinated Dibenzo-p-Dioxins in Itansfformers and Capacitors Volum es 1-3 PCDF and PCDD-- by-products of PCB pyrolysis and combus tion-- apparently cause many of the health effects originally attributed to PCB. In parallel and round-robin studies, indepen dent laboratories found that normal equipment operation and arc ing failure cause little if any oxidation and do not significantly modify PCB fluids. BACKGROUND Partial oxidation of askarels-- mixtures of polychlorinated biphenyls (PCB) and tri- and tetrachlorobenzenes-- produces polychlorinated dibenzofurans (PCDF) and polychlorinated dibenzo-p-dioxins (PCDD). However, analytic techniques for separating the active components in these mixtures had not matured prior to this study. Information had also been insufficient on the quantity and types of PCDF and PCDD in as-manufactured PCB and on the formation of these by-products during normal operation or arcing failure in utility equipment. Because of these uncertainties, utilities and regulators have assumed the worst-case PCDF-PCDD formation when there is a FC-Brelated accident, thereby overstating the potential toxicity. EPRI report EUEAr4858 describes portions of the research performed in these projects. OBJECTIVE To improve techniques for measuring PCDF/PCDD in the presence of PCB. APPROACH Five laboratories (including two whose studies are discussed in report EUEAr485fl) performed round-robin gas chromatography-mass spectrome try analyses of PCDF-PCDD samples using specially prepared carbon-13 (13C) PCDF spiking compounds. After the first set of analyses, each labora tory modified its techniques and analyzed a larger group of samples from utility equipment. Another organization statistically analyzed all the labora tory results. RESULTS These three volumes report the second group of PCDF-PCOD analyses. Results from all participating laboratories correlated remarkably well. Some of the results follow. EPRI EUEA.5443 Vais. 1-3 GENP 0 11147 783958 Use of the 13C compounds allowed successful separation of the com ponents in several closely eluting pairs of physiologically active PCDF and less-active compounds. Analyses of samples from utility equipment suggested that the PCDF-PCDD present did not result from high-load, high-temperature operation. Volumes 1 and 2 describe several analytic techniques, with the appen dix of Volume 2 including a report on the analysis by the University of Umea in Sweden, as well. Volume 3 summarizes all the laboratory techniques, presents comprehensive statistical analyses, and provides an executive summary. EPRI PERSPECTIVE This research is a landmark in PCDF-PCDD analysis. One important result is the synthesis of new PCDFs for use as spiking compounds in gas chromatography-mass spectrometry analysis. The ability to sepa rate many of the active PCDF compounds from more innocuous materi als is also of great value. The high correlation of results from several laboratories indicates that researchers can exercise great flexibility in selecting techniques and developing facilities. In addition, new work has started (EPRI project RP2028-21) in which the best features of each of the five analytic methods will be assembled to provide a single recommended method. PROJECTS ' RP2028-6, RP2028-7, RP2028-10 EPRI Project Managers: Gil Addis; Jacques Guertin Electrical Systems Division; Environment Division Contractors: New York State Department of Health; Battelle Columbus Laboratories; Research Triangle Institute For further information on EPRI research programs, call EPRI Technical Information Specialists (415) 55-2411. 783959 GElSiP 011U8 Analysis of Polychlorinated Dibenzofurans and Polychlorinated Dibenzo-p: Dioxins in Transformers and Capacitors Volume 3: Summary and Statistical Analysis of the Round-Robin Test EUEA-5443, Volume 3 Research Project 2028-10 Final Report, June 1988 Prepared by RESEARCH TRIANGLE INSTITUTE Analytical and Chemical Sciences Post Office Box 12194 Research Triangle Park, North Carolina 27709 Principal Investigators E. D. Pellizzari R. Perritt C. A. Clayton I D. Hartwell Prepared for Electric Power Research Institute 3412 Hillview Avenue Palo Alto, California 94304 EPRI Project Managers G. Addis Transmission Substations Program Electrical Systems Division J. Guertin Land and Water Quality Studies Program Environment Division 783960 ORDERING INFORMATION Requests for copies of this report should be directed to Research Reports Center (RRC), Box 50490, Palo Alta CA 94303. (415) 965-4081. There is no charge for reports requested by EPRI member utilities and affiliates, U.S. utility associations. US. government agencies (federal, states and local), media, and foreign organizations with which EPRI has an information exchange agreement On request, RRC will send a catalog of EPRI reports. Electric Power Research Institute and E P R I are registered service m arks of Electric Power Research Institute Inc. Copyright 1988 Electnc Power Research institute. In c All rights reserved. NOTICE This report w as prepared by trie organizations) nam ed below as an account ol work sponsored by the Electnc Power Research Institua In c (EPHI). Neither EPRI. m em bers ol EPRI. the organization nam ed below, nor any person acting on behalf of any of them: (a) m akes any warranty, express or implied, with respect to the use ol any information, apparatus, method, or process disclosed in this report or that such use m ay not infringe privately owned rights: or (b) assum es any liabilities with respect to the use of. or for dam ages resulting from the use of. any information, apparatus, method, or process d isd ose d in this report Prepared by Research Triangle Institute Research Triangle Park. North Carolina 783961 GENP 011150 ABSTRACT An Interlaboratory study Involving five Independent laboratories was conducted to quantitatively assess accuracy and precision of polychlorinated d1benzo--d1ox1n (PCOD) and polychlorinated dlbenzofuran (PCDF) congener analysis in three spiked and seven in-service utility fluids. Data was obtained using capillary gas chromatography/mass spectrometry for 16 different PCDD and PCDF compounds. Five different ^-labeled Internal standards were added to the samples and the samples were purified prior to analysis. Each laboratory's generated calibrations for the individual compounds were linear for concentrations over four orders of magnitude. Analyses were performed in triplicate on an extract to provide insight to instrumental reproducibility. The median and range of percent accuracy for four of the five laboratories was 102 and 56-149, respectively, across all chemicals measured 1n the two spiked samples. The fifth laboratory had a median and range of 107 and 8-3939, respectively. The median and range of percent reproducibility (coefficients of variation) for three of the four laboratories was 8.2 and 0-46, respectively, across all 10` synthetic and in-service samples and all compounds measured. The fourth laboratory had a median and range of 30 and 2-159, respectively. The fifth laboratory did not report replicate analyses on each extract and thus reproducibility for instrumental analysis could not be calculated. The largest variability (accuracy and precision) was attributed to the different types of samples Q>90%) in three of the four laboratories while the fourth had as much as 63.1% variability from the sample type analyzed and as much as 36.9% due to Instrumental measurement error. At the approximate 95% prediction Intervals, the analysis of these 10 samples by any one of the five laboratories would be expected to produce variability in the results for each PCDD or PCDF congener measured ranging from a factor of two to 300. Of the five participating laboratories, the analytical method employed by Lab-D gave for the most part the best accuracy and precision while Lab-C was the poorest. GENP 011151 in 783962 ACKNOWLEDGMENTS The authors extend their sincere appreciation to the EPRI technical management team led by Dr. G. Addis and his colleagues, Dr. J. Guertln, and Dr. R. Komal who provided adroit guidance to this program and encouragement to all the participants. Dr. T. Rouse (General Electric Company) 1s thanked for h1s contributions concerning utility use of dielectric liquids. We would like to also thank our research comrades who contributed the analytical data for this Interlaboratory collaborative study. They are Dr. S. Gordon (Illinois Institute of Technology Research Institute), Drs. G. Eadon and Dr. Hllker (New York State Department of Health), Dr. M. Kilpatrick (Radian Corp.), Dr. M. Cooke (Battelle Columbus D1v.), and Dr. C. Rappe (U. of Umea). GENP 011152 V 783963 CONTENTS Section 1 INTRODUCTION 2 CHEMICAL METHODOLOGY Selection of Samples from Electric Equipment for PCDF and PCDD Analysis Acquisition of In-Service Samples Synthesis of Standards Preparation of PCDD and PCDF Standards Sample Extraction and Extract Preparations Capillary Gas Chromatography/Mass Spectrometry Analysis Quality Control 3 RESULTS AND DISCUSSION OF STATISTICAL ANALYSIS Evaluation of Calibration Model Results of Round Robin 4 REFERENCES . APPENDIX A EVALUATION OF CALIBRATION REGRESSION USED BY U. UMEA APPENDIX B DATA USED IN COMPARISONS BETWEEN LABS APPENDIX C PROJECT* SUMMARY Page 1-1 2-1 ' 2-1 2-1 2-1 2-10 2-11 2-16 2-38 3-1 3-1 3-5 4-1 A-l B-l C-l GENP 011153 VI i 783964 Figure 1 2 3 4 5 6 7 8 9 10 11 12 13 ILLUSTRATIONS Overview of Analytical Protocol Sample Clean-up Procedure: Round Robin Analysis Summary of Major Elements of IITRI's GC/MS Analysis Protocol Selected Ion Current Profile for Mass 322 From GC/MS Analysis of Performance Check Solution Containing TCODs as Noted in The Plot, Using a 60 m SP-2330 Fused Silica Capillary Column Selected Ion Current Profile for Mass 304 From GC/MS Analysis of Performance Check Solution Containing TCDFs as Noted 1n The Plot, Using a 60 m SP-2330 Fused Silica Capillary Column TCDD Test Mixture Chromatogram on OB-5 TCDF TestMixture Chromatogram on DB-5 PnCDF Test Mixture Chromatogram on DB-5 HxCDF Test Mixture Chromatogram on OB-5 TCDD Test Mixture Chromatogram on SP-2331 TCDF Test Mixture Chromatogram on SP-2331 PnCDF Test Mixture Chromatogram on SP-2331 HxCDF Test Mixture Chromatogram on SP-2331 Page 2-12 2-14 2-19 2-20 2-21 2-25 2-26 2-27 2-28 2-30 2-31 2-32 2-33 0 lU S4 783965 TABLES Standard Solutions Baseline Fluid Samples - GC/MS Crosscheck Baseline Fluid Samples - Round Robin Analysis Analytical Sequence 1n GC/MS Determination of PCDFs and_PCDDs HRGC-MS Analytical Conditions Conversion Factors Calibration Curves: LAB-A Calibration Curves: LAB-B Calibration Curves: LAB-0 Calibration Curves: LAB-E Coefficients of Variation by Compound, Lab, and Sample Minimum, Median, and Maximum CVs By Lab and Sample Over All Compounds Percent Accuracy for Spiked Samples By Compound, Sample, and Lab Minimum, Median, and Maximum Percent Found (Accuracy) For All PCDF and PCDD Congeners 1n Spiked Samples 1 and 2 Variance Components By Compound And Lab Variance Components By Compound For Lab-0 Standard Deviations, Their Ranks, and Bartletts Test By Compound and Sample Levene's Test of Differences Among Labs By Compound Over Samples Approximate 95 Percent Prediction Intervals By Compound And Sample Page 2-3 2-7 2-8 2-22 2-29 3-2 3-2 3-3 3-3 3-4 3-6 3-10 3-11 3-15 3-16 3-21 3-22 3-26 3-27 GENP 011155 xi 783966 Table B-r Data Used in Comparisons Between-Labs i Analytical Procedures Employed by Round Robin Labs 2 Samples Analyzed 1n Round Robin Study 3 Example of Measurements Performed for D1ox1ns/Furans By A Lab on One Sample (Lab-B, Sample 14) - PPB 4 Percent Accuracy for Spiked Samples for 2,3,7,8-TCDD By Sample and Laboratory 5 Percent Accuracy for Spiked Samples for 2,3,4,6,7,8-HXCOF By Sample and Laboratory 6 Median and Range of Percent Accuracy Across All Compounds For Spiked Samples 7 ' Coefficients of Variation for 1,2,3,7,8 + 1,2,3,4,8-PNCDF By Compound and Laboratory 8 Coefficients of Variation for 1,2,3,4,6,7,8-HPCDF By Compound and Laboratory 9 Median and Range of Coefficients of Variation Across All Compounds By Laboratory and Sample 10 Variance Components for 2,3,7,8-TCDD Across All Samples (1,2,3,11,12,13,14,17) 11 Variance Components for 2,3,4,6 ,7,8-HXCDF Across All Samples (1,2,3,11,12,13,14,17) 12 Variance Components By Compound For Lab-0 13 Levene's Test of Differences Among Labs By Compound Across Samples 14 Approximate 95 Percent Prediction Intervals (PPB) Page B-2 C-3 C-5 C-5 C-7 C-7 C- 8 C- 8 C-10 C-10 C-ll C-ll C-12 C-14 C-15 783967 GENP 011156 SUMMARY EPR1 has sponsored a four-year research program to evaluate the analysis of selected Individual compounds of polychlorinated d1benzo-p-d1ox1ns (PCDDs) and polychlorinated dlbenzofurans (PCDFs) 1n dielectric fluids. These are Impurities often associated with askarel mixtures of polychlorinated biphenyls (PCBs) and trl/tetrachlorobenzene (TCB). They have been known to form either 1n the PCB manufacturing process or as a result of excessive heat (fires). This program included the synthesis of new natural (^C) and Isotopically labeled (*3C) PCDD and PCDF analytical standards. Standards and spiked test samples were prepared by one of. the laboratories and distributed to the four other participating laboratories as part of a round robin study. The samples were then analyzed as unknowns. The impetus for the studies has been the wide range of toxicities (4-5 orders of magnitude) seen for the Individual compounds in animal tests and the difficulty of quantitatively analyzing for some of these chemically similar compounds of dissimilar toxicity, often in the presence of a great excess of PCBs. PCBs themselves are a group of compounds with chemical behavior relatively similar to PCDF/PCDD during sample cleanup stages. It should be remembered that the major object of the study was to evaluate and upgrade methods of analysis; analysis of field samples was an important secondary output. The study involving five participating laboratories, was conducted in two phases. Phase I was designed to located glaring discrepancies In the techniques, and Phase II to statistically Improve results of the different laboratories on a series of unknown samples. Phase I involved the analysis of PCDFs and PCDDs 1n five spiked basic samples: Aroclor-1016, Aroclor-1242, Aroclor-1260, a tri- and tetrachlorobenzene mixture, and aged mineral oil; and four In-service dielectric fluids. Each participating laboratory used Its best available 1n-house extraction and analysis techniques (Gas Chromatography/Mass Spectrometry, GC/MS). Since validated analytical methods did not exist before this program was initiated, this phase of the program was necessary to assess any deficiencies and optimize the analytical methodology. GENP 011157 S-l 783968 The*isotopically labeled standards were needed to calibrate the efficiency of cleanup and to determine the sensitivity (response) of the MS for the various , Individual compounds of PCDF and PCDD being analyzed. After evaluating the results of Phase I and Implementing several Improvements to the methodology, Phase II was Initiated. Phase II was designed to provide quantitative data on PCDF and PCDD compounds In selected In-service dielectric fluids and to make a statistical Interlaboratory comparison of these data. The specific alms were 1) to determine the accuracy of analysis by each participating laboratory for a selected 11st of PCDFs and PCDDs 1n spiked samples, 2) to determine the precision of analysis by each laboratory for PCDFs and PCDDs In spiked and In-service utility samples, 3) to perform an interlaboratory comparison and thus a comparison of methods employed for accuracy and precision, and finally 4) to measure the levels of PCDFs and PCDDs in a .few selected in-service utility samples.- This study suggests that there 1s no strongly preferred analytical procedure (among those using a combination of extractlon/chemical cleanup, gas chromatography, and mass spectrometry) for measuring compound specific concentrations of PCDF and PCDD. For spiked samples, originally free of PCDF and PCDD, five different methods (corresponding to five different participating laboratories) yielded concentrations accurate to within +203S (except one laboratory measured 2,3,4,6,7,8-hexachlorinated dibenzofuran [2,3,4,6 ,7,8-Hg-CDF] with an order of magnitude error). Since standards for all 150 PCDF and PCDD compounds were not available, it was not possible to know the accuracy of measurement on the unknown field samples. Only the precision of measurement could be ascertained. Considerable variation in measurement precision was observed between laboratory procedures. For example, measurement of very low concentrations (1 to 10 ng/g range) showed an order of magnitude variation, whereas measurement In higher concentration range (100 to 10,000 ng/g) were considerably more precise. Again, a specific HgCDF measurement was an exception, having as much as four orders of magnitude variation. For total individual congener classes, concentration measurements had only one order of magnitude variation. What range of concentrations were found 1n the field samples? The maximum concentration for 2,3,7,8-Tetra-COD was 32 ng/g and the maximum concentration of 2,3,7,8-tetra-CDF + 2,3,4,8-tetra-CDF was 459 ng/g. The highest concentration found was for one sample of the much less critical octachlorlnated dibenzofuran at 65,000 ng/g. These concentrations are reported with a statistical 95 confidence level. 783969 S-2 GENP01J158 SECTION 1 INTRODUCTION Because of the concerns about the health effects of polychlorinated biphenyls (PCBs) their production was banned and regulation Instituted 1n 1976 by the Toxic Substance Control Act (TSCA), PL 94-469. Certain totally enclosed electrical uses of PCBs are allowed for the remainder of the useful life of existing electrical equipment. Many of these adverse health effects appear to be attributable to trace contaminants such as polychlorinated dibenzofurans (PCDF) and polychlorinated dibenzo-jD-dioxins (PCDD) in the PCBs. Th Electric Power Research Institute (EPRI) inititated a program to investigate the chemistry of PCDF and PCDD as they may relate to use of Askarel, a dielectric liquid, which contains PCBs and potential trace contamination of PCDF/PCDD. This prqgram had three primary objectives: (1) to develop and optimize analytical methods and quantitatively characterize these methods for measuring PCDF/PCDD in dielectric fluids, (2) to apply these methods to measurement of PCDF/PCDD levels in typical dielectric fluids used in utility operations, and (3) to investigate possible PCDF/PCDD formation in simulated transformer events. As part of the objective to evaluate methodology, the program included the synthesis of native and isotopically labeled PCDD and PCDF analytical standards (1) to allow for analysis of selected individual congeners of PCDDS and PCDFs. Analytical standards and spiked matrix samples were prepared by a contract laboratory and then distributed to four other participating laboratories as part of a round robin study. These samples were analyzed blind by five independent contract laboratories - Illinois Institute of Technology Research Institute (IITRI), New York State Department of Health (NYSDOH), Radian Corp., Baitelie, Columbus Division, and U. Umea. The method evaluation study was conducted in two phases. Phase I involved the analysis of PCDF's and PCDD's in five spiked baseline samples; for example, Aroclor-1016, Aroclor-1242, Aroclor-1260, tr1- and tetrachlorobenzene mixture, aged mineral oil, and four in-service dielectric fluids (1-4). Each participating laboratory used their best available in-house extraction and analysis techniques (1-4). Since validated methods for PCDD and PCDF analysis did not exist before this program was Initiated, this phase was necessary to assess any deficiencies and to optimize the analytical methodology. After Phase I results were evaluated and 1-1 783970 several Improvements were Implemented, then Phase II was undertaken. This report covers the effort under Phase II. Phase II was designed to yield quantitative data on PCDD and PCDF congeners in selected In-service dielectric fluids and to make a statistical Interlaboratory comparison on these data. The specific alms were (1) to determine the accuracy of analysis by each participating laboratory for a selected 11st of PCDO's and PCDF's 1n spiked samples, (2) to determine the precision of analysis by each laboratory for PCDF's and PCDD's 1n spiked and in-service utility samples, (3) to perform an Interlaboratory comparison of methods for accuracy and precision, and finally (4) to measure the levels of PCDF's and PCDD's 1n a few selected In-service utility samples. Although this report primarily addresses the statistical analysis of PCDD and PCDF congener data from the five laboratories, a section on chemical methodology is included to allow the reader to examine the similarities and differences in the analytical procedures employed by each laboratory. These procedures are also described in more detail In the reports from the five individual laboratories (14). 783971 1-2 GENP 011160 SECTION 2 CHEMICAL METHODOLOGY SELECTION OF SAMPLES FROM ELECTRICAL EQUIPMENT FOR PCDF AND PCDD ANALYSIS The rationale for the selection of transformers and capacitors to be sampled for PCDF and PCDD analysis was previously developed and reported elsewhere (5). Since the analysis 1s time consuming and difficult, the Intention was to choose a small number of samples, which would yield the greatest amount of information about the large population of equipment in use. Also, the samples were chosen to provide a potentially wide spectrum of challenge to the analytical methodology which was being -statistically evaluated in the Round Robin Study. ACQUISITION OF IN-SERVICE SAMPLES The acquisition of in-service samples was performed by one of the participating laboratories and the> procedures employed have been reported (4). The samples selected for use 1n the Round Robin Study are shown in Table 2. Two baseline samples were spiked with a selected number of congeners at known concentrations into mineral oil and Aroclor 1016, respectively. A third sample, Aroclor 1260, was spiked at known levels with these same congeners but it also contained endogenous PCBF's and PCDD's. Samples 11-17 (Table 2) were in-service utility samples that were selected to represent the various types of utility uses (5). In some cases the dielectric was Aroclor 1260, or mineral oil containing PCBs. Dielectric fluids from transformers, capacitors, or precipitators were selected for this Round Robin Study. Some of the devices had been In use for many years (Table 2). SYNTHESIS OF STANDARDS Because 13c Isotope 1s present 1n a natural abundance of 1.1%, synthetically enriched compounds can be prepared for use as analytical Internal standards. Isotoplcally enriched compounds can be clearly distinguished from native substances by mass spectrometry. For this reason, Radian Corporationprepared 13c-labeled an^ unlabeled (native) PCDD and PCDF congeners as analytical'standards for use by all laboratories participating 1n the Round Robin Study (1). Subsequently, analytical standard solutions were also prepared of these authentic PCDD and PCDF congeners and distributed by Radian Corp. GENP 011161 2-1 783972 Standard Materials Radian synthesized the following materials (1): 1.2.3.7.8- Pentachlorodlbenzofuran (PnCDF) 1.2.3.4.7.8- Hexachlorodlbenzofuran (HxCDF) 1.2.3.4.6.7.8- Heptachlorodlbenzofuran (HpCDF) 2 .3 .7 .8 - Tetrachlorod1benzofuran-l3Ci2 (TCDF-13C) 1.2.3.7 .8- Pentachlorod1benzofuran-l3Ci2 (PnCDF-i3C) 1.2.3.4.7.8- Hexachlorodlbenzofuran-l3Ci2 {HxC0 F-l3C) 'Several congeners 1n Radian's PCDF/PCDD Standards Inventory were also donated to the program. These congeners were: 2.3.7 .8- Tetrachlorod1benzo-2 -d1ox1n 2.3.7.8- Tetrachlorodi benzo--di ox1n-13Ci2 2.3.7.8- Tetrachlorodlbenzofuran Octachl orodl benzo-j)-dioxin-13Ci2 2.3.4.8- Tetrach1orodi benzofuran 2.3.4.7.8- Pentachlorodibenzofuran 1.2.3.6 .7.8- Hexach1orodi benzofuran 2.3.4.6 .7.8 - Hexachlorodi benzofuran Octachlorodlbenzofuran A mixture of congeners was obtained from Professor Christoffer Rappe of the University of Umea, Sweden. This semi-quantitative mixture, the "toxic cocktail1', contained the following Isomers: l,3,4-Tr1chlorod1benzofuran 2.3.4.8 - Tetrachlorodlbenzofuran 2 .3.4.7.8- Pentachlorodi benzofuran 1.2.3.4.8- Pentachlorodibenzofuran 1,2,3,4,7,9-Hexachlorodibenzofuran 1.2.3.4.6 .7.8 - Heptach1orodi benzofuran Octachlorodibenzofuran All the compounds prepared or obtained by Radian were made up as stock spiking solutions at 50 ^g/mL or 100 /tg/mL. These solutions were further diluted to produce standard solutions for distribution to the participating analytical laboratories. Table 15 lists the standards provided by Radian. Baseline Liquids To determine the accuracy and precision of PCDO/PCDF measurements, a selection of baseline liquids were re-analyzed 1n the Round Robin Analysis portion of the program (1). The Baseline liquids (Table 16) were dissolved 1n hexane at levels of 0.18 - 0.20 grams/mL and spiked with measured levels of ^3C-labeled congeners. In this instance, however, a selection of native congeners were spiked "blind" at levels known only to the person performing the spiking. Additionally, a set of samples were prepared with no native congener spikes 1n order to determine background PCDD/PCDF levels. Table 17 describes these samples and reveals the spiking levels. The seven "In-Service" Liquids were spiked with l3C-labeled 783973 2-2 GENP 011162 TABLE 15. STANDARD SOLUTIONS3 I.D. No. E729-14-02 E729-06-04 E729-15-02 E729-16-02 E729-05-04 E729-06-02 E729-05-01 E729-05-02 E729-05-03 E729-06-01 E729-07-02 E729-55-01 Description 2,3,7,8-Tetrachlorodlbenzofuran (TCDF)-13C 2,3,7,8-Tetrachlorodlbenzo-p-d1ox1n (TCD0)-13C 1,2,3,7,8-Pentachlorodlbenzofuran (PnCDF)-13C Octachlorodlbenzorp-d1ox1n (OCDD)-13C 2,3,7,8 -Tetrachlorodlbenzofuran 2,3,7,8-Tetrachlorodi benzo-p-d1ox1n 1,2,3,7,8 -Pentachlorodlbenzofuran 1,2,3,4,7,8-Hexach1orodlbenzofuran (HxCDF) 1,2,3,4,6,7,8-Heptachlorodibenzofuran (HpCDF) Radian PCDF/TCDD Mixture 2,3,7,8-TCDF 2,3,7,8-TCDD 1,2,3,7,8-PnCDF l(2,3,4,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF Professor Rappe's "Toxic Cocktail" 1,2,4-TrCDF 2,3,4,8-TCDF 2,3,4,7,8-PnCDF 1-,2,3,4,8-PnCDF 1,2,3,4,7,9-HxCDF 1,2,3,4,6,7,8-HpCDF OCDF Calibration Mixture I Native Congeners 2,3,7,8-TCDD 2,3,7,8-TCDF 1,2,3,7,8-PnCDF 2,3,4,7,8-PnCDF 1,2,3,4,7,8-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF OCDF Labeled Congeners 2,3,7,S-TCDD-l3Ci2 2,3,7,8-TCDF-13c 12 Concentration 100 ng/mL 100 ng/mL 100 ng/mL 40 ng/mL 100 ng/mL 100 ng/mL 100 ng/mL 100 ng/mL 100 ng/mL 100 ng/mL 100 ng/mL 100 ng/mL 100 -ng/mL 100 ng/mL "116 ng/mL "100 ng/mL "100 ng/mL "100 ng/mL "100 ng/mL -100 ng/mL "160 ng/mL 7.5 /ig/mL 7.5 /ig/mL 7.5 /ig/mL 7.5 /ig/mL 7.5 /ig/mL 7.5 /ig/mL 7.5 /ig/mL 7.5 /ig/mL 500 ng/mL 500 ng/mL GENP 011163 2-3 783974 I.D. No. E729-55-03 E729-55-04 TABLE 15 (cont'd.) Description 1.2 .3 .7 .8- PnCDF-13Ci2 1.2.3.4.7.8- HxCDF-l3Ci2 0CDD-13Ci2 Calibration Mixture II Native Congeners 2.3.7.8- TCDO 2.3.7.8- TCDF 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 2.3.4.6.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF OCDF 'Labeled Congeners 2,3,7r8-TCDD-13c12 2,3f7,8-TCDF-13c12 1.2.3.7.8- PnCDF-13c12 1.2.3.4.7.8- HxCDF-13c 12 0CDD-13c 12 Calibration Mixture III Native Congeners 2.3.7.8- TCDD 2.3.7.8- TCDF 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 2.3.4.6.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF OCDF Labeled Congeners 2.3.7.8- TCD0-13c12 2.3.7.8- TCDF-13Ci2 1.2.3.7.8- PnCDF-13c12 1.2.3.4.7.8- HxCDF-13c12 0C0D-13c 12 Concentration 500 ng/mL 500 ng/mL 200 ng/mL 2.5 /ig/mL 2.5 /ig/mL 2.5 /ig/mL 2.5 /ig/mL 2.5 /ig/mL 2.5 /ig/mL 2.5 /ig/mL 2.5 /ig/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 200 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 200 ng/mL 2-4 783975 GENP01164 TABLE 15 (cont'd.) I.D. No. E729-55-05 E729-55-06 E729-56-01 Description Calibration Mixture IV Native Congeners 2,3,7,8-TCOD 2,3,7,8-TCDF 1,2,3,7,8-PnCOF 2,3,4,7,8-PnCDF 1,2,3,4,7,8-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF OCDF Labeled Congeners 2I3,7,8-TCDD-13Ci2 2,3,7,8-TCDF-l3Ci2 l,'2,3,7,8-PnCDF-l3C12 l,2,3,4,7,8-HxCDF-l3C12 0CDD-13C12 Calibration Mixture V Native Congeners 2,3,7,8 -TCDD 2,3,7,8-TCDF 1,2,3,7,8-PnCDF 2,3,4,7,8-PnCDF 1,2,3,4,7,8 -HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6 ,7,8-HpCDF OCDF Labeled Congeners 2,3.,7,8-TCDD-13c 12 2,3,7,8-TCDF-13c 12 l^.SJ.S-PnCDF-^Cxa l ^ . S . ^ . S - H x C D F - l 3^ 0CDD-13C!2 Calibration Mixture VI Native Congeners 2,3,7,8 -TCDD 2,3,7,8-TCDF * GENP 011165 2-5 783976 Concentration 150 ng/mL 150 ng/mL 150 ng/mL 150 ng/mL 150 ng/mL 150 ng/mL 150 ng/mL 150 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 200 ng/mL 40 ng/mL 40 ng/mL 40 ng/mL 40 ng/mL 40 ng/mL 40 ng/mL 40 ng/mL 40 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 200 ng/mL 10 ng/mL 10 ng/mL 1.0. No. E729-56-02 TABLE 15 (cant'd.) Description 1,2,3,7,8-PnCDF 2,3,4,7,8-PnCDF 1,2,3,4,7,8-HxCOF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8 -HpCDF OCDF Labeled Congeners 2,3,7,8-TCDD-13c12 2,3,7(8-TCDF-13C12 l,2,3,7,8-PnCDF-13c12 l,2 ,3 ,4 ,7 ,8-HxCDF-l3Ci2 0CDD-13Ci2 Calibration Mixture VII 'Native Congeners 2,3,7,8 -TCDD 2,3,7,8-TCDF 1,2,3,7,8-PnCDF 2,3,4,7,8-PnCDF 1,2,3,4,7,8-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6 ,7,8-HpCDF OCDF Labeled Congeners 2,3,7,8-TCDD-13c 12 2,3,7,8-TCDF-13c 12 l,2,3l7,8-PnCDF-l3Ci2 l,2,3,4,7,8-HxCDF-13Ci2 0CDD-13Cj2 aSee Ref. 1. for details. 2-6 Concentration 10 ng/mL 10 ng/mL 10 ng/mL 10 ng/mL 10 ng/mL 10 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 200 ng/mL 2.5 ng/mL 2.5 ng/mL 2.5 ng/mL 2.5 ng/mL 2.5 ng/mL 2.5 ng/mL 2.5 ng/mL 2.5 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 500 ng/mL 200 ng/mL 783977 GENP 011166 TABLE 16. BASELINE FLUID SAMPLES - GC/MS CROSSCHECK4 ID No. E729-09-01 E729-10-0 E729-11-01 E729-12-01 E729-08-01 E729-08-01 Description Spike Spiking Level Aroclor 1016 2.3.7.8- TCDF-13C 1.2.3.7.8 - PnCDF-13C 2.3.7.8- TC0D-13C o c d o -13c 1.2.3.4.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF 100 ng/g 100 ng/g 100 ng/g 40 ng/g 100 ng/g 100 ng/g Aroclor 1242 2.3.7.8- TC0F-l3C 1.2.3.7.8- PnCDF-l3C 2.3.7.8- TCDD-13C o c d d -13c 1.2.3.4.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF 100 ng/g 100 ng/g 100 ng/g 40 ng/g 100 ng/g 100 ng/g Aroclor 1260 2.3.7.8 - TCDF-13C 1.2.3.7.8- PnCDF-l3C 2.3.7.8- TCDD-l3C o c d d -13c 1.2.3.4.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF 100 ng/g 100 ng/g 100 ng/g 40 ng/g 100 ng/g 100 ng/g Tri-Tetrachlorobenzenes 2.3.7.8- TCDF-l3C 1.2.3.7.8- PnCDF-13C 2.3.7.8- TCDD-l3C 0CDD-13C 1.2.3.4.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF 100 ng/g 100 ng/g 100 ng/g 40 ng/g 100 ng/g 100 ng/g Aged Mineral 011 2.3.7.8- TCDF-13C 1.2.3.7.8- PnCDF-l3C 2.3.7.8- TCDD-13C o c d d -13c 2.3.7.8- TCDD 2.3.7.8- TCDF 1.2.3.7.8- PnCDF 1.2.3.4.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF 100 ng/g 100 ng/g 100 ng/g 40 ng/g 40 ng/g 20 ng/g 40 ng/g 120 ng/g 0 ng/g Professor Rappe''s MTox1c Cocktail" Aged Mineral Oil OCDF 1,2,3,4,7,9-HxCDF 2.3.4.7.8- PnCDF 1.2.3.4.8- PnCDF 2.3.4.8- TCDF 1,3,4-TrCDF Aroclor 1260 -*160 ng/g -100 ng/g -100 ng/g -100 ng/g -100 ng/g -116 ng/g 510 fig/q Tr1-Tetrachlorobenzenes 530 /tg/g aSee Ref. 1 for details. g e n p o u 167 2-7 783978 TABLE 17. BASELINE FLUID SAMPLES - ROUND ROBIN ANALYSIS3 ID No. E729-53-01 E729-53-02 E729-53-03 Description Mineral Oil Aroclor 1016 Aroclor 1260 Spike Aroclor 1260 Tr1/Tetrachlorobenzenes 2.3.7.8- TCDD-l3C 2.3.7.8- TCDF-l3C 1.2.3.7.8- PnCDF-^C 1.2.3.4.7.8- HxCDF-13C 0CD0-13C 2.3.7.8- TCDD 2 .3.7.8- TCDF 2.3.4.8- TCDF 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 1.2.3.6.7.8- HxCDF 2.3.4.6.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF OCDF 2,3,7,8-TCDD-}3C 2.3.7.8- TC0F-l3C ,, 1.2.3.7.8- PnCDF-^3C 1.2.3.4.7.8- HxC0F-13C 0CDD-13C 2.3.7.8- TCDD 2.3.7.8- TCDF 2.3.4.8- TCDF 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 1.2.3.6.7.8- HxCDF 2.3.4.6.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF OCDF 2,3,7,8-TCDD-l3C 2.3.7.8- TCDF-l3C 1.2.3.7.8- PnCDF-*3C 1.2.3.4.7.8- HxCDF-l3C 0CDD-13C 2.3.7.8- TCDD 2.3.7.8- TCDF 2.3.4.8- TCDF 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 1.2.3.6.7.8- HxCDF 2.3.4.6.7.8- HxCDF 1.2.3.4.6.7.8- HpCDF OCDF Aroclor 1260 Tr1/Tetrachlorobenzenes Spiking Level 640 /ig/g 1250 ^g/g 100 ng/g 100 ng/g 100 ng/g 100 ng/g 40 ng/g 25 ng/g 100 ng/g 100 ng/g 150 ng/g 150 ng/g 250 ng/g 50 ng/g 175 ng/g 400 ng/g 0 ng/g 100 ng/g 100 ng/g 100 ng/g 100 ng/g 40 ng/g 53 ng/g 53 ng/g 0 ng/g 158 ng/g 158 ng/g 0 ng/g 53 ng/g 132 ng/g 526 ng/g 132 ng/g 100 ng/g 100 ng/g 100 ng/g 100 ng/g 40 ng/g 111 ng/g 833 ng/g 194 ng/g 111 ng/g 111 ng/g 500 ng/g 278 ng/g 56 ng/g 111 ng/g 222 ng/g 680 /jg/g 1320 ^g/g 783979 GENP 011168 TABLE 17 (cont'd.) ID No. E729-54-02 E729-54-03 E729-54-04 Description Mineral 011 Aroclor 1260 Aroclor 1016 ... Spike 2.3.7.8- 1000-130 2.3.7.8- TC0F-13c 1.2.3.7.8- PnCDF-13c 1.2.3.4.7.8-Hx CDF-13c 0C0D-13C 2.3.7.8- TCDD-^c 2,3,7f8-TCDF-13C 1.2.3.7.8-PnCDF-l3C l^.S/./.S-HxCOF-^C 0CDD-13C 2.3.7.8 - TCDD-^3C 2.317.8- TCDF-13C 1.2.3.7.8- PnCDF-l3C 1,2,3.4-,7,8-Hx CDF-13C 0C0D-13C aSee Ref. 1 for details. Spiking Level 100 ng/g 100 ng/g 100 ng/g 100 ng/g 40 ng/g 100 ng/g 100 ng/g 100 ng/g 100 ng/g 40 ng/g 100 ng/g 100 ng/g 100 ng/g 100 ng/g 40 ng/g GENP 011169 2-9 783980 congeners at Battelle Memorial Institute, Columbus, Ohio, using stock solutions provided by Radian Corporation (4). PREPARATION OF PCDD AND PCDF STANDARDS Calibration Standards) PCDD and PCOF standard compounds Including l3C-labelled standard compounds, dissolved 1n solvent, were received at each laboratory sealed In glass vials. The seven vials each contained all of the following compounds: 2.3.7.8- TCDD 2.3.7.8- TCDF 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 2.3.4.6 .7.8- HxCDF 1,2,3,4,6 ,7,8 .9-0CDF 2.3.7.8 - TCDD-" C12 2.3.7.8- TCDF-l3Ci2 1.2.3.7.8- PnCDF-^3Ci2 1.2.3.4.7.8- Hx CDF-13C i2 lr2 ,3 ,4 ;6 ,7 ra,9-0CDD-I3Ci2 Each vial contained all of the native compounds at one of the following concentration levels: 7.500 ng/mL 2.500 ng/mL 500 ng/mL 150 ng/mL 40 ng/mL 10 ng/mL 2.5 ng/mL Each standard solution contained all of the *3C compounds at 500 ng/mL each. Baseline Samples Three baseline samples (Aroclor 1016, Aroclor 1260, and aged mineral oil) were prepared at Radian Corporation as solutions in n-hexane at a concentration of about 0.2 g/mL. Each sample was spiked with 100 ng/g of each of the five 13C-compounds listed above. Each sample was also spiked with "blind" amounts of the following unlabeled compounds: 2.3.7.8- TCDD 2.3.7.8- TCDF 2.3.4.8- TCDF 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.3.4.7.8- HxCDF 1.2.3.6.7.8- HxCDF 2.3.4.6.7.8- HxCOF 1.2.3.4.6.7.8- HpCDF 0CDF In addition, the mineral oil sample was spiked with 625 iq/q Aroclor 1260 and 1250 jiqlq tr1-/tetrachlorobenzenes. 783981 2-10 G E N p 01117Q In-Service Samples Seven In-service samples were fluids-consisting of five askarels and two mineral oils, all of which were prepared at a concentration of approximately 200 mg/mL 1n n-hexane. Each sample was spiked at Battel!e with the five Isotoplcallylabeled Internal standards at concentrations of 100 ng per gram of oil. SAMPLE EXTRACTION AND EXTRACT PREPARATIONS For this Round Robin Study each participating laboratory employed their best available 1n-house extraction and analysis techniques which are shown 1n Table 1. Because the methods varied between the laboratories, a parameter being statistically tested 1n the Round Robin Study, a detailed description of each is presented here. Procedures Employed by NYSDQH f3) The volume of a portion of samples of electric insulating fluids and mineral oil was measured and extracted by adding 25 ml of acetone and mixing thoroughly. Hexane (25 mL) was added and the sample was stirred and extracted for a minimum of 1 h. To remove PCBst and nonplanar aromatics and to Isolate one fraction containing all tetra- to OCDFs and OCDDs the sample extract was chromatographed on acid alumina, PX-21 carbon and neutral alumina. A preprogrammed Hamilton valve control system designed at NYS00H was used to reproduclbly select solvents and chromato graphic columns (3). The recovery of all tetra- to OCDFs and OCDDs (as verified with available standards) from an Individual chromatographic column was >95%. The sample extract was applied to an activated acidic alumina column, followed by 30 mL of 3% CH2CI2 1n hexane. The CDF/CDD fraction was eluted with 70 mL of 50% CH2CI2 In hexane onto a carbon column followed by 50 mL of 10% benzene 1n hexane.The CDF/CDD fraction was eluted in the reverse direction with 30 mL of 50% xylene In hexane onto a neutral alumina column (replaces xylene with a volatile solvent) followed by 30 mL of 3% CH2CI2 1n hexane. The final purified CDF/CDD fraction was eluted with 70 mL of easily volatilized 50% CH2CI2 1n hexane and concentrated with first boiling water and then vacuum to 4-10 pi (3). Extracts were stored in the dark for GC/MS in sealed 150 pi borosilicate capillary tubes. Procedures Employed by IITRI (2) The extraction and cleanup procedure which was employed was based on the general method described by Albro and coworkers (2) for the fractionation and class determination of complex mixtures of chlorinated aromatic compounds. An overview 1s shown In Figure 1. Briefly, the procedure consisted of the following major steps. GENP 011171 2-11 783982 Figure 1. Overview of Analytical Protocol (Ref. 2) 7 8 3983 2-12 GENP 011172 1. To the sample (ca. 0.1 g), spiked with a mixture of 13C-labe1ed PCDF. and/or PCDO Internal standards, about-30 ng of 2,3,7-tri-CDD was added to serve as a ''carrier" compound. 2. The sample was chromatographed on a gel permeation column (20 g Sephadex LH-20), eluting with 50% methylene chlor1de-1n-methanol and collecting the 60-150 mL fraction, to separate halogenated aromatic compounds from any alphatlcs present. 3. About 10 il of propylene glycol were added as a "keeper" compound, to prevent sample loss during roto-evaporat1on. 4. After roto-evaporat1on, the halogenated aromatic fraction was chromatographed on an A-540 basic alumina column (20 grams topped with a layer of anhydrous sodium sulfate), eluting with 180 mL of 2% methylene chloride-in-hexane, to elute the PCB fraction. 5. Congeners eluted with 20% methylene chlor1de-in-hexane (200-220 mL), to recover PCDFs, PCDDs, polychlorinated quadriphenyls, and less chlorinated PCBs. 6 . About 10 /*L of propylene glycol were added, then the fraction was concentrated to near dryness and loaded onto a 10 g EM-1078 acidic alumina column, eluting with 80 mL of 1% methylene chlor1de-1n-hexane, to purify the PCDF/PCDD fraction. The eluate was discarded. 7. The purified PCDF/PC0D fraction was eluted with 20% methylene chloride-in hexane (120 mL). 8 . The extract was concentrated to 25 pi immediately before analysis using toluene. 9. When necessary, the extract was purified further by loading it in hexane onto a small (1 cm) column of Carbopack C mixed with Cellte 545, eluting with 2 mL hexane, 1 mL of 50% methylene chlor1de-1n-cyclohexane and 1 mL of methylene chlor1de:methanolbenzene (75:20:5). The column flow was reversed and 8 mL of toluene were collected. Procedures Employed by Radian (1) The extraction technique used by Radian for the Round Robin analysis phase of this study was a modified Smith-Stall1ngs/EPA Region VII clean-up (1). Figure 2 describes the clean-up procedure used for these dielectric samples. An aliquot of the sample (approximately 0.25 g) was transferred to a column consisting of 2.5 cm sodium sulfate (on bottom), 5 g silica gel type 60, (70-230 mesh), 6 g basic silica gel (35% potassium hydroxide), and 10 g acid silica gel (40% sulfuric add). This column was eluted with 170 mL 1:1 methylene chlor1de:cyclohexane directly onto a 5.0 cm column of 1-9 /*m carbon (PX-2) on glass fibers. The silica gel was discarded and the carbon column washed with 25 mL 1:1 G E N p 011173 2-13 783984 Part 1 Modified Smith-Stallings Clean-Up 150 m L 1:1 Methylene chloride: cyclohexane Discard silica gel 25 m L 1:1 Methylene chloride: cyclohexane 50 m L 75:20:5 Methylene chloride: methanol: benzene Invert carbon column 50 m L Toluene (collect) Ir--Io Acid silica gel 10g Basic silica gel 6g Silica gel 5g Sodium sulfate V ^ " f0<N3O Carbon/glass libers "Ni CD CO CO 00 07 T Figure 2. Sample Clean-up Procedure Part 2 "Region 7" Clean-Up 0 A cid silica gel 5g Silica gel 1g Acid alumina 69 Solvent exchange the toluene to hexane Transfer hexane extract to silica gel colum n 60 m L hexane Discard silica gel 20 m L hexane 20 m L 20% methylene chloride: hexane (collect) Concentrate Round Robin Analysis (Ref. 1) methylene chlor1de:cyc1ohexane and 50 mL 75:20:5 methylene chloride:methanol: benzene. The eluted solvents were discarded. The PCDDs and PCDFs were eluted from the carbon column with 50 mL toluene. The toluene was evaporated to dryness on a rotary evaporator. The dried extract was redissolved In hexane and transferred to a column of 5 g ac1d-1mpregnated silica gel and 1 g silica gel (lower layer). This column was eluted with 60 mL hexane directly onto a 6 g a d d alumina (AG 4, 100-200 mesh) column. The silica gel was discarded and the a d d alumina was washed with another 20 mL hexane. The eluted hexane was discarded. The PCDDs and PCOFs were eluted off the alumina with 20 mL 20% methylene chlorlde/hexane. The volumeof this eluate was reduced to 1-2 mL with a stream of nitrogen. The extract was transferred to a 1 mL conical vial and taken to dryness. Each sample was processed 1n triplicate (this was the only lab which performed sample processing in replicate). Procedures Employed by Battel!e (4) One milliliter aliquots (containing 250 mg of the original fluid) of each sample was transferred to separatory funnels and diluted with 49 mL of hexane. The hexane solutions wer^e washed with 10 mL portions of concentrated sulfuric acid until the washes were only slightly colored. A maximum of five acid washes were used. Following the acid washes, each oil was washed with 20 mL of distilled water >to remove any residual acid. The hexane solutions were transferred to glass collection tubes and concentrated to approximately 2 mL using gentle streams of nitrogen gas. The hexane solutions were transferred to multilayered silica gel columns containing activated silica gel, 44 percent concentrated sulfuric acid on silica gel, and 33 percent 1M sodium hydroxide on silica gel. The PCDO/PCDF isomers were eluted from the columns using 70 mL of hexane and the entire eluates, Including the original sample volume, were collected. The eluates from the multilayered silica gel columns were concentrated to approximately 1 mL using gentle streams of nitrogen and transferred to the top of columns containing 5 g of activated basic alumina. These columns were eluted using hexane, hexane/methylene chloride (97:3, v/v) and hexane/methylene chloride (1:1, v/v) as elution solvents. The 1:1 hexane/methylene chloride eluates were collected, solvent exchanged Into hexane, and transferred to the top of alumina columnns containing approximately 2 g of activated basic alumina. These columns were eluted with hexane, hexane/methylene chloride (97:3, v/v), and hexane/methylene chloride (1:1, v/v). The 1:1 hexane/methylene chloride eluates were collected, concentrated to near dryness with nitrogen gas, and spiked with 10 ng of 1,2,3,4-TC0D-I3ci2. These solutions were stored at 0*C until they were analyzed. GENP 011175 2-15 783986 For samples still too contaminated with Interferences to be analyzed, they (41159-11-12,13,14,17 and E729-53-02,03) were chromatographed through florlsil columns to provide additional analyte enrichment. Each of the florlsil columns was prepared using 5 g of activated florlsil (60-100 mesh) which was slurry packed with hexane. The sample solutions were diluted with approximately 5 mL of hexane and transferred to the florlsil columns. The columns were eluted with hexane, hexane/ethyl ether (94:6, v/v), and hexane/methylene chloride (25:75, v/v). The 25:75 hexane/methylene chloride fraction was collected and concentrated to approximately 20 pi, These solutions were stored at 0*C until they were analyzed. CAPILLARY GAS CHROHATOGRAPHY/MASS SPECTROMETRY ANALYSIS Procedures Used by NYSDOH (3) Low Resolution Mass Spectrometry-- All sample extracts were analyzed with a Hewlett-Packard 5970 Mass Selective Detector. A portion of each extract was Injected into the split-splitless injection port (250*C) and eluted through a 50 m (.25 mm i.d.; .33 pm film thickness) Hewlett-'Packard 5% phenyl methyl silicone fused silica capillary column. The following temperature program was used: H-P 5890 Temperature Program Level 1 2 3 4 5 Initial Temp. 190 Initial Time 1.00 Rate (*C/min) 5.0 5.0 5.0 5.0 5.0 Final Temp. 220 235 250 270 300 Final Time 16.00 7.00 18.00 7.00 24.00 Total Time 23.00 33.00 54.00 65.00 95.00 783987 The gas chromatograph is interfaced directly to the source of the mass selective detector and the transfer line and 1on source are heated to 275*C and 150*C respectively. The mass selective detector was operated 1n the selective ion monitoring (SIM) mode. The m/z values characteristic of native TCDD, TCDF, PnCDF, HxCDF, 0C0F, and 0CDD and TCDD-l3Ci2 . TCDF-l3Ci2 , PnCDF-13C12, HxCDF-l3C12 and 0CDD*3Ci2 were monitored at various times during the ninety-five (95) minute runs. The time window used for monitoring the different congener classes (TCDO, TCDF, etc.) were determined by the previous Injection of either window standards (TCDD) or a mixture of combusted PCS. Raw data was collected Into and processed by an H-P Quicksilver data system. The data system was used to generate electronic copies of E N P o n 1 75 reports of retention times and areas at each m/z value monitored. Additional custom'software was used to Identify GC peaks- and quantitate analyte levels. This software was written by NYSDOH staff and processed on an IBM PC or XT. The H-P reports were input in ASCII and transferred by using a customized H-P Pascal operating system (sender) and the program called Kermit (receiver). Each sample extract was instrumentally analyzed 1n triplicate. High Resolution Mass Spectrometry-- Some extracts were analyzed with a Kratos MS-50 High Resolution GC/MS/DS. In this system portions of the extracts were Injected Into the on-column injection port of a Carlo-Erba 4160 high resolution gas chromatograph and eluted through a 50 m (.20 mm l.d.; .33 /*m film thickness) Hewlett-Packard 5% methyl phenol silicone fused capillary column. The oven was temperature programmed as follows: Level 1 2 3 Initial Temp. 190#C H-P 5890 Temperature Program Initial Time 1.0 Rate (*C/min) 5.0 5.0 5.0 Final Temp. 220 235 300 Fi nal Time 16.00 7.00 50.00 Total Time 23.00 33.00 86.00 The gas chromatograph 1s Interfaced to the MS-50 through a direct line heated to 250*C. The ion source temperature was 250*C. The high resolution mass spectrometry was operated in either of two SIM type modes. In normal multiple peak monitoring peak (NMPM) the m/x values of a particular analyte and its corresponding internal standard were observed at 10,000 RP (10% valley) by Integrating all the signal detected within 50 ppm of their exact mass values. In high resolution multiple peak monitoring mode (HRMPM) the mass spectrometry is operated at the same high resolving power but the analyte and Internal standard exact masses are profiled. The data system adjusts the accelerating and electric sector voltages to focus a mass slightly below the mass of interest. The computer then triggers a 0.3 sec analog scan through a 300 ppm section of the mass range which includes the exact mass of the 1on of interest (3). In both ion monitoring modes the raw data was acquired Into and processed by the Kratos DS-55 data system. Quantitation was done manually by calculating concentrations using peak areas obtained from the DS55 output. E N P 0 1 JJ77 2-17 783988 Procedures Used by IITRI (2) Samples and standards were analyzed"using combined capillary column gas chromatography/!ow-resolut1on mass spectrometry (HRGC/LRMS or GC/MS). Figure 3 summarizes the major elements of their analysis protocol. The gas chromatograph, a Varlan 3700, was equipped with a 60 m x 0.25 mm l.d. fused-s1Hca SP-2330 (Supelco) column. Samples were injected 1n the splltless mode, with the column programmed limit for 60 minutes. The SP-2330 column had been shown to separate the 2,3,7,3-TCDD from all other tetra-1somers, and to partially separate the 2,3,7,8-TCDF from the 2,3,4,8-TCDF (2). However, 1t was unable to resolve 1,2,3,7,8-PnCDF from 1,2,3,4,8-PnCDF and 1,2,3,4,7,8-HxCDF from 1,2,3,4,7,9-HxCDF. These Isomers can be separated to some extent on less polar columns, such as OV-17 or DB-5 (2). An example of the chromatographic resolution obtained using the SP-2330 column 1s shown in Figure 4 for 2,3,7,8-TCDD and several closely-eluting tetradioxin isomers. The plot clearly shows that this column was isomer-specific for 2,3,7,8TCDD. Figure 5 shows the separation obtained between the 2,3,7,8- and 2,3,4,8-TCDF isomers. Here the resolution was not as good as 1n the previous case, but is nonetheless sufficient to permit quantification of the two Isomers 1n most cases. All data were acquired by software-controlled multiple 1on detection using two ion masses from the molecular ion cluster for each of the PCDF and PCDD levels of chlorination. Two Ions were also monitored for each internal standard which permitted the calculation and comparison of the isotope ratios with their theoretical values to verify their identity. Several additional ion masses were monitored to indicate possible interferences from chlorinated diphenyl ethers (2). The presence of these compounds could give rise to fragment ions with the same masses as those monitored for the PCFs. Therefore, the absence of a co-response for the diphenyl ether Ion mass when a PCDF compound shows a positive response was taken to Indicate that the signal for the PCDF was "real'*. A listing of the masses that were monitored, along with the theoretical isotope ratios, 1s given 1n Table 18. In order to obtain maximum sensitivity and selectivity during an experiment, the run was divided into 4 adjoining time windows. Each window contained up to 16 1on masses (for specific native and labeled PCDFs and PCDDs, and chlorinated diphenyl ethers), and the sample dwell times and Interchannel delay times were chosen to give the most sensitive and rapid cycle time. Since the GC column used in this work did not yield complete separation of each chlorination level of PCDFs/PCDOs from the other groups 1n a given time windows, some of the Ion masses were Included 1n more than one window to ensure that all Isomers from a particular congener class were monitored. 2-18 G E N P O in 78 783989 GC/MS ANALYSIS (IITRI) HRGC/LRHS - UNIT MASS RESOLUTION <-1,000 - SP-2330, OV-17 COLUMNS SELECTED ION MONITORING - TWO ION MASSES MONITORED PER PCDF/PCDD CONGENER CLASS IDENTIFICATION CRITERIA - COINCIDENT GC RETENTION TIMES - CORRECT INTENSITY RATIOS FOR MONITORED IONS QUANTIFICATION - DIRECT COMPARISON WITH ADDED INTERNAL STANDARDS - MS RESPONSE FACTORS FROM STANDARDS Figure 3. Summary of Major Elements of IITRI's GC/MS Analysis Protocol (Ref. 2) 2-19 783990 ssx> ANALYSIS NAME'. D R 0 0 C 0 0 3 > 0 3 3 X )P E M 0 0 0 2 .M IS j 1 U 0 3 . 0 WINDOW 1 T IT L E ! TCOO P E MIX # 2 l l U L S P L IT L E S S I N J ;S P 2 3 3 W # 1 9 4 2 > J 1 . 8KU OPERATOR T C 0 0 . I W ; 7 , SCAM SPEED 6 0 0 . 0 MSEC SPC 5 SAMPLE ID DATE 5 - N 0 U - 8 4 1 0 '2 5 > 1 9 COMMISSION C 0 8 7 S 6 GENP 011180 Figure 4. Selected ion current profile for mass 322 from GC/MS analysis of performance check solution containing TCDDs as noted in the plot, using a 60 m SP-2330 fused silica capillary column (Ref. 2). I-6 6 E8 Z ANALYSIS NAME' D R 0 0 < C 0 0 3 .0 3 3 3 C T M 0 1 0 0 1 .M IS il ` U 0 3 . 0 WINDOW* 1 T IT L E 1 CHROM TEST MIX H 1 -2 0 0 P G 'U L )1 U L S L I S P 2 3 3 0 < 4 1 2 2 7 8 > > 8 3 - 2 3 0 .1 5 0 * 4 1 OPERATOR' C Y T M 'W l* 3 2 0 0 .W 2 -3 1 6 7 .W 3 < 5 7 3 0 ,W 4 -3 7 3 0 SPC' 3 O SAMPLE 1 0 ' DATE' 2 - J A N - 8 6 0 8 ' 3 4 14 5 COMMISSION M A S SM E N O PR IPE .M W >2 Co ^-4 rrioo 783992 Figure 5. Selected ion current profile for mass 304 from GC/MS analysis of performance check solution containing TCDFs as noted on the plot, using a 60 m SP-2330 fused silica capillary column (Ref. 2 ). TABLE 18. ANALYTICAL SEQUENCE IN GC/HS DETERMINATION OF PC D Fs AND PCODs3 Temp Cycle Time Start Stop GC Column Prog Rate Time Window (min) (min) Temp (C) (C/min) (ms) 1 14:00 26:00 85 15 3200 197 2 26:00 35:30 250 3167 195 3 35:30 56:00 250 -- 3750 372 4 56:00 66:00 250 -- 3750 534 Illl Ions Monitoredc Compounds (m/z) Monitoredb Mass 1 Mass 2 Isotope Ratio (Mass 1/Mass 2) TCOF 13C-TC0F TCDO 13C-TC00 PnCDF l3C-PnC0F PnCDO HxDPE3 HpOPE* 303.9 315.9 319.9 331.9 337.9 349.9 355.9 373.8 407.8 305.9 317.9 321.9 333.9 339.9 351.9 357.9 -- -- 0.77 0.77 0.77 0.77 0.61 0.61 1.54 -- -- PnCOF PnCDFb PnCOD PnC00b HxCOF 13C-HxC0F HxCDFb 13C-HxC0F HxCDFb HxCOO HxCDDb HpDPE3 00PEa 337.9 274.9 355.9 290.9 373.8 385.9 310.9 389.8 310.9 389.9 326.9 407.8 443.8 339.9 -- 357.9 -- 375.8 387.9 -- 391.8 -- 391.8 -- -- -- 0.61 1.54 1.23 1.23 -- 1.23 1.23 -- -- HxCOF HxCOO HpCOF HpCOD 00PEa NOPE3 373.8 389.9 407.8 423.8 443.8 477-7 375.8 391.8 409.8 425.8 -- -- 1.23 1.23 1.03 1.03 __ -- OCOF 0C00 13C-0CDD DOPE3 441.7 457.7 469.8 511.7 443.7 459.7 471 .8 ---- 0.88 0.88 0.88 -- ' aSee Ref. 2. bHxPE, HpDPE, OOPE. NOPE. DOPE designate hexa-, hepta-, octa-, nona-. and decaeh loresiphenyl ethers' resp. cMass eonitored to identify fragment ion (M-C0C1). 783993 2-22 GENP 011182 The major criteria which were used for confirming the presence of specific PCDFs/PCDDs In the samples analyzed were -the^foil owing: (1) correct retention times for each PCDF/PCOD of Interest relative to the appropriate 1sotop1callylabeled Internal standard(s), (2) Intensity ratio for (M)+/(M+2)+ within 10X of theoretically-expected ratio (Table 18), and (3) signal-to-no1se response of each PCDF/PCOD of Interest greater than 2.5:1 for both 1on masses monitored. Each sample extract was Instrumentally analyzed In triplicate. Quant1f1cat1on"Idea1lv. each of the 210 separate Isomers of PCDF and PCDD should be quantified using the Instrument response of the corresponding labeled Internal standard as a reference. Since only a limited number of these standards were available 1n practice, the approach generally followed was to use an appropriate set of internal standards which Included representative Isomers from each chlorinated class of PCDFs and PCDDs, and further assumed that the data obtained for these was representative of all Tsomers 1n each group. Relative response factors were determined from the analysis of standard solutions containing the Internal standrds and isomers representative of each chlorination class. Response factors at IITRI were calculated from the following \ equation: where Ax - A-|s = Qis = Qx 3 = AxQls/A1sQx sum of the Integrated Ion abundances of the masses for the unlabeled compound sum of the Integrated Ion abundances of the masses for the appropriate labeled compound amount of the appropriate labeled compound (ng) amount of the labeled compound (ng) Quantification of PCDFs and PCDDs 1n the sample extract was achieved by calculating the ratios of the mass spectral responses obtained for the ions characteristic of the labeled PCDFs/PCDDs to those of the appropriate internal standards, corrected for differences 1n response factor. The equation used for quantification was: where W = Rp a Concentration (ng/.g) - (Ax x Q1s)/A1s x w x RF weight of sample (g) response factor Because the labeled Internal standard was added prior to sample extraction and analysis, and the Internal standard was quantified at the same time as the native components, any losses of PCDD/PCDF Incurred during the analysis were accounted for by the above approach. Detection limits, 1.e. the minimum detectable concentrations required to produce a signal 2.5 times the average background signal, were calculated for each GENP 011183 2-23 783994 PCDF/PCDD congener class. For each class, the average width of the baseline noise- band- -for the native compounds and the peakJielght for a known concentration of the associated 13c-labeled analog were measured manually. Measurements of the noise band were made 1n a region-of the selected 1on current profile that was free of Interferences and as close as practicable In the plot to the peak for the corres ponding 13c-iabeled compound. The detection limit DL was calculated using the relationship: DL - (2.5) (Ax x Qis)/Als x RF x W) where Ax * height of the noise band of the selected mass for the unlabeled compound Ais 3 height of the peak corresponding to the labeled compound of the same congener class. Procedures Used by Radian (1) GC/MS analysis of the sample extracts was performed on a Hewlett-Packard 5985/87 in the selected 1on monitoring mode for quantitation and identification of PCDD/PCDFs. Each sample was extracted in triplicate and each extract was instrumental!y analyzed in triplicate yielding nine determinations on each sample. The first series of extracts were run on a Hewlett-Packard 50 m, Ultra-2 column (a methylphenyl sill cone coating equivalent to DB-5). On the Ultra-2 column, an initial temperature of 80*C was used. The second and third determinations were performed using a J&W 60 m, 0.32 mm I.O., 1 pm film, DB-5 column. The GC/MS conditions are shown on Table 19. Dried sample extracts were dissolved in 50 pL of hexane and a 1.0 /tL volume was injected onto a cool SGE on-column Injector. Each of the three extracts was analyzed a fourth time using a Supelco SP-2331, 60 m, 0.32 mm I.D., 0.2 pm film capillary column to separate 2,3,7,8-TCDF and 2,3,4,8-TCOF. For this separation, samples were evaporated to dryness and then reconstituted with 40 /tL of nonane. A two-stage temperature program of 170*-200* at 20Vm1n followed by 200*-275* at 4*/m1n was used. Chromatographic Test Mixture Part of this project Involved separating the more toxic PCD0/PCDF Isomers from coeluters. The chromatographic test mixture prepared at Battel!e Memorial Institute contained the following analytes at 200 ng/L each: 1.3.6.8- TCDD 1.2.8.9-TCDD 1,2,3,4-TCDD 2.3.7.8- TCDD 1.3.7.8- TCDD 3.4.6.7- TCDF 2.3.4.8- TCDF 1.2.3.8- TCDF 2.3.7.8- TCDF 1.2.3.4.8- PnCDF 1.2.4.6.8- PnCDF 2.3.4.6.7- PnCDF 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1.2.4.6.7.8- HxCDF 2.3.4.6.7.8-HxCDF 1.2.3.4.7.8- HxCDF 1.2.3.6.7.8- HxCDF 783995 This test mixture was run dally to evaluate the chromatography. Chromatograms of all the analytes 1n the test mixture are shown on Figures 6 through 9 for DB-5 and 2-24 GEfl? OUI4 rIo rtno -4 0GO0 Figure 6. TCDD Test Mixture Chromatogram on DB-5 (Ref. 1) CO CO CD 783997 rcino a CO On Figure-7. TCDF Test Mixture Chromatogram on DB-5 (Ref. 1) rrIoo Figure 8. PnCDF Test Mixture Chromatogram on DB-5 (Ref. 1) 783998 GENP 011188 Figure 9. HxCDF Test Mixture Chromatogram on DB-5 (Ref. 1) TABLE 19. HRGC-MS ANALYTICAL CONDITIONS3 i. i Hass Spectrometer: Data System: Source Temperature: Internal Source Pressure: Mode: Electron Energy: Electron Multiplier Voltage: Resolution: Interface Temperature: San Mode: Cycle Time: Column: Carrier Gas: Carrier Linear Velocity: Injector: Interface to MS: GC Program: Injection Volume: Tailing Factor: Hewlett-Packard 5985 with 5987 Upgrade HP1000 RTE/6 250*C 3 x 10"5 Torr Electron Impact 70 eV 2650-2800 volts Unit 275*C Selected Ion Monitoring 0.5 sec 60 meters x 0.32 mm I.D., J&W D8-5 fused silica capillary column, 1.0 /*m film Hydrogen at 5 ps1 35 cm/sec Cool 0n-column (65*C) SGE Source Coupling at 275*C Initial hold of 0.5 min at 65*C then 65*-200* at 30*/min, then 200*-310* at 4*/min 1 fiL 0 .7-2.0 aRef. 1 . Figures 10 through 13 for SP-2331. One analyte, 1,2,4,6,8-PnCDF, could not be found 1n the chromatographic test mixture. On DB-5, 2,3,7,8-TCDF and 2,3,4,8-TCDF were not resolved, but on SP-2331 this pair could be separated with a 65% valley. On SP-2331, 1,2,3,4,8-PnCDF and 1,2,3,7,8-PnCDF could not be resolved, but on DB-5 this pair could be separated with a 55% valley. Actual samples were more difficult to resolve than the chromatographic test mixture. The analytes 1,2,3,4,8-PnCDF and 1,2,3,7,8-PnCDF could not be resolved In many cases, particularly when the 1,2,3,4,8-PnCDF concentration was much higher than the 1,2,3,7,8-PnCDF concentration. In one sample (41159-11-14), the GENP 011189 2-29 784000 \ / roOi\ji 00 oo o6x n o a * > iso o o 14000 13000 12000 IIOOO IODOO oooo eooo 7000 oaoo 9000' 4000 3000J 2000 lOOO o' II.o j,),i,*-Tcno I TCOD 1,2,1,4-TCOD F fio o L t Joo { foo L* !. fI reo i : 50 t f"40 I ! 1*30 S u p c lc n S P - 2 3 3 1 , 60 m, (1 .3 ? mm 111, 1 |im f i l m . It.imjit to 2110*, ilie n 2 0 0 * - 2 / 5 * H 4 */ m ln . 20 L t ho ' r ^ i ' r r i T f T T i n t i - j t -j - i r 1 i- 12. O 13: O 14. 0 r l * | I - t r j - n : 'r | ` n l | i n i | r i -^ ` O 13. a is. a |7. o ib .o Figure 10. TCDD Test Mixture Chromatogram on SP-2331 (Ref. 1) GENP 011191 ruio> "si 00 Figure 11. TCDF Test Mixture Chromatogram on SP-2331 (Ref. 1) orOo GENP 011192 Figure 12. PnCDF Test Mixture Chromatogram on SP-2331 (Ref. 1) <\ GENP 011193 ruCiOo "si CD Figure 13. HxCDF Test Mixture Chromatogram on SP-2331 (Ref. 1) OO fc. 2,3,7,8-TCDF and 2,3,4,8-TCDF pair could not be resolved accurately due to a coeluting Interference. Several of the'Tn-servIce liquid extracts were reported by Radian to be very complex and required the removal of 12 to 18 inches of the front of the column after every injection to give adequate resolution for subsequent samples. Column degradation for DB-5 and SP-2331 could be observed from the daily test mixture chromatograms over the course of this work. Calibration Four C W / C13 area ratio calibration curves were generated during this study, the first on Ultra-2, the second and third on OB-5 and the fourth on SP-2331. Concentrations of 40, 150, 500, 2500, and 7500 ng/mL were calibrated for the first three calibration curves with each calibration point being the average of three determinations. The fourth calibration curve on SP-2331 also was determined in triplicate for concentrations of 40, 150, 500, and 2500 ng/mL. Calibration curves were*generated and statistical analysis performed for the following analyte/C*3 internal standard sets: 2.3.7.8- TCDD 2.3.7.8- TCDF 2,3,7,8-TCDD-l3C 2,3,7,8-TCDF-l3C 1.2.3.7.8- PnCDF 2.3.4.7.8- PnCDF 1,2,3,7,8-PnCDF-l3C 1.2.3.4.7.8- HxCDF 2.3.4.6.7.8-HxCDF 1.2.3.4.6.7.8- HpCDF l^^^^.S-HxCDF-^C 0CDF 0CD0-13C Identification of PCDD/PCDFs Identification of native PCDFs and PCDDs was performed by measuring chlorine isotope ratios and monitoring the presence of the fragment Ions from loss of C0C1. For PCDFs, two chlorine Isotope peaks were monitored 1n addition to the quantitating 1on. Only the most abundant chlorine Isotope peak was monitored for the first series of extracts. The acceptance criteria for PCDD/PCDF positive Identification required agreement better than +10% between the theoretical and experimentally determined chlorine Isotope ratios. The presence of either a second chlorine Isotope peak or a H-C0C1 peak also was necessary for positive identification. In the few cases where there was substantial Interference in the 1on profile of the largest chlorine isotope peak, the second largest chlorine Isotope peak was used for evaluation of the Isotope ratio acceptance criteria. In a few cases, a +15% chlorine isotope ratio was accepted. If a peak was not within +10% of the criteria on one determination but was within +10% on previous determinations, the determination was accepted. GENP 011194 784005 Quantitation Quantitation was performed by Isotope dllutlon using the ^-labeled Internal standards added before extraction. Concentrations were determined from C*2/cl3 ratios of sample analyte to Internal standard and a linear fit to the five-point calibration curves. A separate calibration curve was generated for 2,3,4,7,8PnCDF. All other PnCDFs were quantitated from the 1,2,3,7,8-PnCOF calibration curve. A separate calibration curve was produced for 2,3,4,6,7,8-HxCDF. All other HxCDFs and HpCDFs were quantitated from the 1,2,3,4,7,8-HxCDF calibration curve. OCDD and OCDF were quantitated from the 0CDD-13C calibration curve. Congener class total concentrations were calculated by summing concentrations of each peak which passed the Identification criteria for the congener class. Surrogate recoveries were determined using the mean 500 ng/mL response factor from the calibration checks made at the start and end of the analyses. One sample (41159-11-13), had a severe Interference which coeluted with the TCDF-13c peaks. TCDFs 1n this sample were quantitated verus the area of the PnCDF13c quantitating 1on with a normalization factor of 1.1 (the average ratio of TCDF13C to PnCDF-l3C response). \ Detection Limits Instrumental detection limits were defined as the analyte concentration which gave a 3:1 signal-to-no1se ratio. Method detection limits for all analytes were approximately 3 ng/g except 0C0F and OCDD which were 10 ng/g due to peak broadening at the later retention times. The lowest point on the calibration curves, 40 ng/mL, corresponds to a sample concentration of 8 ng/g. Procedures Used by Battel!e (4) Analysis-- The sample'extracts were Instrumentally analyzed in triplicate and quantified for PCDD/PCOF using combined capillary column gas chromatography/h1gh resolution mass spectrometry (HRGC/HRMS). The HRGC/HRMS consisted of a Carlo Erba Model 4160 Gas Chromatograph Interfaced directly into the 1on source of a VG Model 7070 high resolution mass spectrometer. The chromatographic column was a 60 M DB-5 fused silica column using helium carrier gas at a flow velocity of 30 cm/sec. The mass spectrometer was operated in the electron Impact (El) Ionization mode at a mass resolution of 9000-12000 (M/AM, 10 percent valley definition). All HRGC/HRMS data were acquired by mult1ple-1on-detect1on (MID) using a VG Model 2035 Data System. Recovery of Internal Standards-- The recoveries of the Internal standards were calculated by comparison to an external standard, l,2,3l4-TCDD-^3c^2 which was added following the column cleanup steps. Relative response factors were determined from triplicate analyses of a GENP 011195 2-35 784006 standard mixture containing the labelled Internal standards and the 1,2,3,4-TCDD13Ci2- The equation used to calculate the.recoveries was: A1s x Qrs x 100 Recovery (*) - ^ x Ql> Where: A^s * Sum of integrated areas for internal standard; Qrs Quantity of recovery standard 1n ng; Qjs Quantity of Internal standard 1n ng; Ars * Sum of Integrated areas for recovery standard; and Rf =* Response factor. Quantification The PCDD/PCDF Isomers and congener class concentrations were determined by comparing the sum of the two 1on masses monitored for each class to the sum of the two 1on masses monitored for the corresponding Internal standard. It was assumed that the response factors for each of the individual isomers in a class were the same as the response factors calculated for the specific isomers present in the standard solutions. The response factors were calculated from six replicate analyses of standard solutions at four concentrations. The sum of the two most intense 1on masses In the molecular 1on region of each Isomer were used to generate the response factors. The formula used for quantifying the PCDD/PCDF Isomers was: Ac x Q1s .Quantlty/Sample (ng/g) =' . x Rf x w Where: Quantity * Total quantity 1n ng of target Isomer or congener class Ac Sum of Integrated areas for the target Isomer or congener class Qjs Quantity of Internal standard 1n ng A-jg Total Integrated areas for the Internal standard Rf * Response factor. Each pair of resolved peaks 1n the selected-1on-current chromatograms was evaluated manually to determine 1f It met the criteria for a PCDD or PCDF Isomer. By examining each pair of peaks separately, quantitative accuracy was Improved over what 1s obtained when all of the peaks 1n a selected chromatographic window are averaged. When averaged data are used, it 1s possible for pairs of peaks with high and low chlorine Isotope ratios to produce averaged data that meet the chlorine Isotope ratio criterion. For example, two pairs of peaks having chlorine isotope ratios of 0.56 and 0.96, both outside of the acceptable range, would have an average ratio of 0.76. By checking the isotope ratio of these peaks separately, they would be eliminated from the class total. QENP 011196 784007 The criterion that were used to Identify PCDO and PCDF Isomers were: 1. Simultaneous responses at both ton-masses. 2. Chlorine Isotope ratio within +/-15X of the theoretical value. 3. Chromatographic retention times within windows determined from analyses of standard mixtures. 4. Signal-to-no1se ratio equal to or greater than 2.5 to 1.0. The Individual Isomers for which a 1sotop1cal1y labelled analog was available Included the additional criterion that they eluted within 2 seconds of the Internal standard. The limit of detection (LOD) was calculated for samples 1n which Isomers of a particular chlorine congener class were not detected. The formula used for calculating the LOD was: LOD/g (ng) = Hc x H^s Q1s x 2'5 x Rf x W Where: LOD = Single isomer limits of detection for a congener class 1n ng; Hc - Height of congener class Isomer; Qis * Quantity of internal standard 1n ng; His 3 Pea^ height of Internal standard; Rf 3 Response factor; and W = Weight 1n grams of sample. Calibration Curve Calibration curves were generated for each of the nat1ve/1sotop1cally labelled isomer pairs. The calibration standards were analyzed at least five times at each of four concentration levels between 0.04 and 2.5 parts-per-m1ll1on (ppm). The repsonse factors were calculated by dividing the sum of the areas for the two most abundant ton masses 1n the molecular Ion cluster of each native PCDD/PCDF Isomer by the sum of the areas for the corresponding ton masses from the Isotoplcally labelled standard. The Isomer pairs for which response factors were calculated Included: 2.3.7.8- TCDD vs 2,3,7,S-TCDD-13C12 2.3.7.8- TCDF vs 2,3,7,8-TCDF-l3Ci2 OCDD vs 0CDD-13c 12 OCDF vs 0CD0-13Ci2 1.2.3.7.8- Penta-CDF vs l,2,3,7,8-Penta-CDF-l3C12 2.3.4.7.8- Penta-CDF vs 1,2,3,7(8-Penta-CDF-13Ci2 1.2.3.4.7.8- Hexa-CDF vs l,2,3,4,7,8-Hexa-CDF-13Ci2 2.3.4.6.7.8- Hexa-CDF vs l,2,3,4,7,8-Hexa-CDF-13Ci2 1.2.3.4.6.7.8- Hepta-CDF vs 0CDD-13C12 GENP 011197 2-37 784008 It was assumed that each of the Isomers of a particular chlorine congener class had the-same response factor. Detection Limits Detection limits were calculated for samples that did not contain PCDD/PCDF Isomers 1n a particular class. These detection limits were calculated using the quantification equation with the addition of a factor of 2.5 to account for the signal-to-no1se criterion. The noise level was measured as the average of the peak-to-peak noise that occurred 1n the baseline of the particular MID channel. If at all possible, the noise was measured at the retention time of the Isotopically labelled internal standard. The height of the internal standard peak was measured to the average of the noise that occurred on the peak top. The reported limits of detection are for single PCDD/PCDF Isomers and have been corrected for recovery losses during sample workup. Baseline Analyses-- The three baseline samples, Aroclor 1260, Aroclor 1254, and Aroclor 1016 were analyzed using the methodology described above. The Aroclor 1016 presented the greatest analytical problem due to the relatively high amount of the sample that passed through the column cleanup steps. Even after the four column cleanup steps, a noticeable polychlorinated biphenyl (PCB) background was evident in the single ion current chromatograms. In-Service Analyses-- The seven In-service samples were also analyzed using the methodology described above. QUALITY CONTROL Each participating laboratory followed their own extensive quality control program which has been previously reported (1-4). GENP 011198 2-38 784009 SECTION 3 RESULTS ANO DISCUSSION OF STATISTICAL ANALYSIS EVALUATION OF CALIBRATION MODEL The calibration data from four of the five participating laboratories In the Round Robin Study was evaluated 1n terms of the regressions employed, 1.e. their appropriateness and linearity over the range of Interest for PCDD and PCDF congener analysis (data from Lab C was not available for regression analysis). For each of the labs, the calibration model 1s: L0G10 (y) = a + b L0G10 (x) Where x => concentration ^"response ratio for LAB-A I peak area ratio for LAB-B y 3 \ area ratios for LAB-D ^normalized counts for LAB-E Exponentiating, the model becomes y a 10a . xb Since b is approximately equal to one for each lab and compound, the model can be simplified to y = CX where C = 10a Tables 20 to 24- give the estimates of C for each lab and compound. Where ca x The Intercepts 1n Tables 21 through 24, except for 2,3,7,8-TCDF (Table 24) were all significantly different from zero at the 0.01 level. Thus, 1f laboratories employed regression models using a zero Intercept, considerable error 1n the calibration would result. For example, the regression model used by Lab-E was Inappropriate (See Appendix A). Discussions with the Lab-E Project Director at this laboratory Indicated that the Isotopic response ratios of 13C/12C-PCDF and PCDD congeners were used for quantification, thus, the results reported did not employ the reported regressions. All other laboratories used regression models 1n which the regression was not forced through the zero Intercept. GENP 011199 3-i 784010 -- 2,3,7,8-TCDD 2,3f7,8-TC0F 1,2,3,7,8-PNCDF 2,3,4,7,8-PNCOF 1,2,3,4,7,8-HXCDF 2,3,4,6,7,8-HXCOF 1,2,3,4,6,7,8-HPCDF OCDF TABLE 20. CONVERSION FACTORS LAB-A --- LAB-B LAB-D 0.002227 0.001916 0.001661 0.001713 0.001947 0.002217 0.002677 0.0009404 0.003117 0.003795 0.003915 0.003916 0.003657 0.006736 0.004121 0.000611 0.001994 0.002293 0.002513 0.002601 0.002211 0.002275 0.001679 0.001395 LAB-E 0.6901 1.0950 0.8904 0.7414 0.6701 0.4082 0.3281 0.0459 X -< Il II TABLE 21. CALIBRATION CURVES: LAB-A , ,Average Response Factor 500 x Concentration>; LOGio (Concentration) 2,3,7,8-TCDD 2,3,7,8-TCOF 1,2,3,7,8-PNCDF 2,3,4,7,8-PNCDF 1,2,3,4,7,8-HXCDF 2,3,4,6,7,8-HXCDF 1,2,3,4,6,7,8-HPCDF OCDF NUMBER OF OBSERVATIONS 6 6 6 6 6 6 6 6 INTERCEPT -2.704** -2.731** -2.819** -2.702** -2.769** -2.632** -2.600** -2.906** **S1gn1f1cantly different from zero at .01 level. SLOPE 1 .0 2 0 * * 1.005** 1.016** 0.973** 1.023** 0.991** 1.011** 0.949** R2 0.9994 0.9997 0.9999 0.9996 0.9999 0.9998 0.9995 0.9980 GENP 011200 784011 TABLE 22. CALIBRATION CURVES: LAB-B Y * LOGiq (Averac*e Response'Factor x Concentration^ X = LOGio (Concentration) NUMBER OF OBSERVATIONS INTERCEPT 2,3,7,8-TCDD 25 -2.475** 2,3, 7,8-TCDF 25 -2.432** 1,2,3,7,8-PNCDF 25 -2.418** 2,3,4,7,8-PNCDF 25 -2.414** 1,2,3,4,7,8-HXCDF 25 -2.468** 2,3,4,6,7,8-HXCDF 25 -2.184** 1,2,3,4,6,7,8-HPCDF 25 -2.410** OCDF 25 -3.470** * Significantly different from zero at .05 level. ** Significantly different from zero at .01 level. SLOPE 0.985** 1.005** 1.005** 1.003** 1.014** 1.006** 1.011** 1.091** TABLE 23. CALIBRATION CURVES: LAB-0 Y LQGiq (Average Area Ratio) X = LOGiq (Concentration) NUMBER OF OBSERVATIONS INTERCEPT 2,3,7,8-TCDD 2,3,7,8-TCDF 1,2,3,7,8-PNCDF 2,3,4,7,8-PNCDF 1,2,3,4,7,8-HXCDF 2,3,4,6,7,8-HXCDF 1,2,3,4,6,7,8-HPCDF OCDF 4 4 4 4 4 4 4 3 -2.691** -2.609** -2.600** -2.636** -2.634** -2.617** -2.778** -2.762** * Significantly different from zero at .05 level. ** Significantly different from zero at .01 level. SLOPE 0.996** 0.988** 1.000** 1.020** 0.991** 0.989** 1.001** 0.966** R2 0.9991 0.9997 0.9994 0.9996 0.9996 0.9991 0.9994 0.9918 R2 0.9999 0.9999 0.9999* 0.9995 0.9999* 0.9999* 0.9999* 0.9999* GENP 011201 3 -3 7 8 4 0 12 TABLE 24. CALIBRATION CURVES: LAB-E Y LOGjo (Ravi "Counts) X LOGio (Concentration) 2,3,7,8-TCDD 2,3,7,8-TCDF 1,2,3,7,8-PNCDF 2,3,4,7,8-PNCDF 1,2,3,4,7,8-HXCDF 2,3,4,6,7,8-HXCDF 1,2,3,4-,6,7,8-HPCDF OCDF NUMBER OF OBSERVATIONS 18 18 18 18 18 18 18 18 INTERCEPT -0.286** 0.004 -0.144** -0.230** -0.259** -0.547** --0.668** -1.762** SLOPE 1.047** 1.014** 1.037** 1.038** 1.032** 1.058** 1.067** 1.142 * Significantly different from zero at .05 level. ** Significantly different from zero at .01 level. R2 0.9945 0.9994 0.9977 0.9960 0.9958 0.9893 0.9875 0.9779 GENP011202 3-4 784013 The slopes were very nearly unity and the of the regression Indicated excellent linearity over the three orders of magnitude concentration range'used for calibrating the Instrumentation.(Tables 21-24). RESULTS OF ROUND ROBIN Statistical analysis of the PCDD and PCDF concentration data first required the establishment of a standard reporting format for all of the laboratories. To satisfy this requirement, the following data conversions were performed: 1) For LAB-C 1,2,3,6,7,8-HXCDF was excluded because 1t was not reported separately but 1n combination with 1,2,3,4,7,8-HXCDF. 2) If measured amounts were not reported, then detection limits were substituted, If available. 3) For those labs which separately reported 2,3,7,8- and 2,3,4,8- TCDF, or 1,2,3,7,8- and 1,2,3,4,8-PCDF, or 1,2,3,4,7,8- and 1,2,3,4,7,9-HXCDF, the reported concentrations of the pairs, were added together (in order to conform with the reporting format of the other laboratories). Table B-l in Appendix B gives a listing of the data. Measured concentrations (ng/g) for each of the individual instrumental determinations of a single extract are shown, along with the means and standard deviations of the determinations, by laboratory, sample, and compound. Since LA8-E reported only one instrumental determination of a single extract for each sample and compound, only that measured concentration Is given. LAB-0 reported results for three extracts for each sample with three instrumental determinations for each extract. These extract results are listed separately. LAB-A reported six determinations for OCDO in sample 11 and 1,2,3,7,8,9-HXCDF in sample 12. Large ranges in concentrations are evident -- for example, by comparing the means among compounds within a sample 1 for LAB-A 1,2,3,4,6,7,8-HPCOF has an average concentration of 354 while OCDD has an average concentration of 2.08. Also, the standard deviations (precision) show great variation in magnitude. Because of this variation 1n standard deviations, equal variances could not be assumed. Since the standard deviations tended to Increase as the concentration levels Increased, basic data transformations of taking logarithms and square roots were tried. These, unfortunately, did not correct the problem. This limited the types of statistical tests that could be performed since many depend on the equality of variances. The coefficients of variations (CVs) which represents precision of the analytical methods for the labs, excluding LAB-E, are given 1n Table 25. Only those concentrations reported as measured amounts rather than detection limits were Included 1n the calculations. There appears to be little relation between the measured concentration and the CV, 1.e., the CV does not tend to Increase or decrease as the measured concentration Increases. These CVs are summarized 1n GENP011203 3-5 784014 TABLE 25. COEFFICIENTS OF VARIATION BY COMPOUND, LABf AND SAMPLE Sample LAB-A LAB-B COMPOUND = TOTAL TCDF 1 16.1 9.4 2 3.8 0.9 3 1.3 3.1 11 47.0 8.6 12 58.9 4.9 13 18.5 12.1 14 23.9 30.0 15 27.6 a 16 21.7 17 4.3 22.3 COMPOUND = TOTAL PNCDF 1` 2 3 11 12 13 14 15 16 17 10.8 10.2 2.0 6.9 19.2 3.2 2.9 10.9 8.8* 3.1 3.1 0.7 5.8 9.7 4.7 10.8 23.7 61.6 22.5 COMPOUND = TOTAL HXCDF 1 13.1 2 5.8 3 3.5 11 2.6 12 4.6 13 14.6 14 1.9 15 36.2 16 A6.8 17 1.7 1.0 2.3 2.1 14.1 3.7 27.5 4.0 54.0 14.0 COMPOUND = TOTAL HPCDF 1 4.4 10.9 2 11.8 20.4 3 3.6 16.8 11 8.6 19.2 12 14.3 20.2 13 21.4 39.4 14 2.6 14.1 15 56.2 62.9* 16 i 58.9 17 6.5 22.6 LAB-C LAB-D-1 15.8 8.? 27.0 61. 27.7 26. 30.1 22.2 5.6 2.4 6.6 13.7 6.8 11.3 10.5 11.9 41.5 13.9 8.3 50.4 56.9 10.2 31.5 10.8 7.6 0.8 11.8 15.4 13.6 17.5 28.5 0 12.1 137.1 83.3 64.6 99.3 20.3 10.7 55.3 42.0 1.3 1.7 10.9 11.9 8.5 22.5 12.4 44.1 47.0 9.2 96.2 76.1 65.6 52.2 28.6 62.4 21.9 0 18.3 9.7 3.2 2.7 18.3 13.9 o 19.4 38.7 70.1 15.1 LAB-0-2 6.5 4.2 4.4 12.0 3.1 11.5 3.6 9.4 3.6 2.7 10.6 2.0 2.6 6.5 3.1 11.1 1.0 0.9 30.3 1.8 5.7 18.4 6.2 5.6 0 1.3 5.1 4.3 5.6 0.8 5.3 3.5 18.2 12.8 0 5.4 LAB-D-3 1.9 1.0 1.3 6.2 18.3 12.6 2.1 0.6 20.9 1.1 5.0 0.2 2.3 8.0 1.2 8.8 1.7 4.8 1.9 1.8 5.1 26.9 1.0 24.1 0.3 2.4 6.3 7.1 5.0 4.2 , 1.9 10.0 0 1.2 784015 GENP 011204 _, Sample LA8-A LAB-B TABLE 25 (cont'd.) LAB-C --- LAB-D-1 COMPOUND = TOTAL OCDF 1 7.9 i 2 10.8 45.8 3 3.3 30.6 32.6 11 5.7 23.8 14.4 12 8.8 12.7 10.8 13 10.7 60.7 14 2.5 40.3 20.0 15 34.1 16 17 8.9 8.8 8.5 26.5* 1.6 2.9 13.6 12.2 39.0 4.5 4.8 47.8 12.3 COMPOUND = TOTAL TCDD 1 11.8 2 16.0 3 3.3 14 56.6* 16 43.9* COMPOUND = OCDD 17.0 2.2 9.2 2.5 15.8 2.3 3.0 1.7 11 12 13 25.1** 19.0 14.5 18.1 6.9 91.m7 m 14 3.0 46.6 m 15 11.5 24.4 16 12.2 9.9 11.8 17 20.5 28.5 30.1 COMPOUND = 2,3,7,8-TCDD 1 11.8 17.0 11.4 2 16.0 2.2 2.5 3 3.3 9.2 15.8 2.3 3.0 1.7 COMPOUND = 2,3,7,8 + 2, 3,4,8-TCDF 1 15.5 9.4 15.8 2 4.5 0.9 8.7 3 0.8 1.0 19.5 11 24.3 45.7 16.4 12 21.1 4.3 24.7 13 16.5 4.4 15.6 14 16.3 9.1 6.7 15 9.3 t 16 16.4 17 3.7 22.0 15.6 5.6 2.4 1.1 9.6 11.5 9.9 9.7 14.5 LAB-D-2 4.1 6.0 8.5 2.4 4.4 11.8 2.6 27.4 3.6 4.0 2.3 5.3 . 8.3 4.0 2.3 5.3 6.5 4.2 3.8 0.0 5.4 5.6 2.1 6.4 LAB-D-3 5.5 5.4 5.0 4.1 4.2 16.8 4.7 2.1 4.3 3.1 1.6 4.3 3.1 1.6 1.9 1.0 2.2 2.9 15.9 2.2 1.2 1.1 GENP 011205 3-7 784016 TABLE 25 (cont'd.) Sample LAB-A LAB-B LAB-C ~ COMPOUND = 1,2,3,7,8 + 1,2,3,4, 8-PNCDF 1 12.0 2.1 11.5 2 11.5 1.8 3.1 3 0.9 4.1 13.6 11 12.9 6.3 31.6 12 26.1 4.8 23.1 13 8.4 7.1 12.8 14 3.8 4.6 7.3 15 11.7 16 61.1 17 4.0 14.9 2.2 COMPOUND = 2,3,4,7.a- PNCDF 1. 2 3 11 12 13 14 15 16 17 11.1 15.3 2.9 2.9 22.2 8.7 1.4 11.0 14.1* 7.2 , 4.2 1.8 4.4 5.8 10.1 6.3 5.8 60.1 13.7 60.1 23.4 37.8 9.4 5.1 17.2 14.8 7.3 COMPOUND = 1,2,3,4,7, 8 + 1,2,3, 4,7,9-HXCDF 1 2 13.2 40.2 0.6 34.6 * 36.7 m 3 3.2 3.5 8.6 11 2.1 13.9 100.9 12 1.9 6.6 16.0 13 17.0 23.1 38.6 14 0.6 1.2 30.8 15 35.8 16 5.8 59.2 17 5.4 14.1 43.0 COMPOUND = 1,2,3,7,8, 9-HXCDF 3 5.4 80.9 11 82.3 12 27.1** 20.3 13 33.2 36.5 14 4.8 15.7 88.5 15 42.4 16 61.9 17 * 53 3 LAB-D-1 1.4 1.3 4.0 10.4 16.6 6.3 15.6 11.5 13.8 0.4 0.7 32.4 22.0 10.9 20.1 22.0 1.3 1.9 8.0 10.1 7.5 9.9 57.3 38.6 10.3 4.7 11.0 6.9 14.0 4.8 LAB-D-2 3.0 2.1 6.8 2.1 4.5 7.5 17.3 ( 18.4 2.1 3.3 5.2 3.7 3.2 1.8 1.3 4.9 2.4 2.0 1.7 0.6 2.3 16.3 4.3 0.8 5.1 4.4 5.4 24.1 26.9 . * 2.3 LAB-D-3 0.7 1.0 3.7 2.2 2.9 0.0 5.7 4.5 2.1 1.4 2.8 2.7 5.3 2.5 1,9 4.9 3.7 3.0 1.7 4.7 8.4 3.4 27.2 19.2 0.9 1.5 2.5 1.5 15.7* 1.3 6.6 G E N F 0 1 1206 3-8 784017 TABLE 25 (cont'd.) Sample LAB-A LAB-B COMPOUND = 1,2,3,6,7 8-HXCOF 1 14.9 0.0 2 3.4 2.5 3 4.0 3.2 11 1.0 19.6 12 2.7 5.3 13 12.3 27.2 14 1.7 2.6 15 34.9 16 m 64.9 17 3.0 2.0 COMPOUND = 2,3,4,5,7 8-HXCDF 1 6.2 1.5 2 7.3 4.2 3 4.8 4.7 11 12.7 13.4 12 25.0 19.5 13 8.8 31.5 14 5.2 13.7 16 . 51.3 17 12.3 19.2 COMPOUND = 1,2,3,4,6 7,8-HPCDF 1 4.0 10.9 2 11.7 20.4 3 4.1 12.1 11 10.3 17.8 12 9.9 18.7 13 21.1 31.6 14 1.6 11.5 15 60.6* 16 53.4 17 3.4 23.9 LAB-C 159.1 86.9 80.9 121.3 30.6 41.2 125.1 m 52.7 96.2 76.1 144.9 58.9 36.9 60.9 39.9 29.2 LAB-0-1 4.1 5.3 5.2 7.0 8.1 7.6 7.9 2.2 0.4 1.2 15.3 21.m7 11.2 5.8 9.7 3.2 1.4 17.6 14.9 20.4 21.7 57.4 14.9 LAB-0-2 5.9 2.7 9.3 20.8 7.9 10.2 14.7 22.5 1.6 0.4 11.4 4.0 3.2 10.2 26.2 1.6 5.1 4.3 5.4 0.7 5.5 7.5 17.6 6.7 ft 4.8 LAB-O-3 8.1 7.6 2.0 1.3 27.6 16.0 8.7 1.3 3.7 2.1 0.5 5.4 23.6 2.0 5.5 2.4 6.3 5.7 4.0 4.9 1.2 10.3 1.6 * Based on two rather than three determinations. ** Based on six rather than three determinations. 3 No measurable value reported, 1.e., not detected, therefore no CV was calculated. GB^P 0U207 3-9 784018 1 Table 26 showing the minimum, median, and maximum CVs over all compounds.by laboratory and sample. Overall, Lab-C shows the largest median and maximum values for CVs, but there 1s also variation among the other laboratories.*27 TABLE 26. MINIMUM, MEDIAN, AND MAXIMUM CVs BY LAB AND SAMPLE OVER ALL COMPOUNDS MINIMUM LAB-A LAB-B LAB-C LAB-0-1 LAB-D-2 LAB-D-3 MEDIAN LAB-A LAB-B LAB-C LAB-D-1 LAB-D-2 LAB-D-3 MAXIMUM LAB-A LAB-B LAB-C LAB-D-1 LAB-D-2 LAB-D-3 12 4.01 0.00 11.36 1.25 0.98 0.71 3.45 0.67 2.55 0.40 0.41 0.96 11.78 4.25 41.53 4.85 ' 4.00 2.40 11.15 2.25 13.87 2.44 2.70 3.08 16.06 16.99 159.06 26.52 6.53 20.86 40.29 45.81 86.90 5.30 4.29 7.64 Sample 3 11 12 13 0.83 1.02 8.33 0.74 2.04 1.31 0.99 5.83 9.36 6.97 0.00 0,17 1.90 3.71 5.13 6.84 0.58 1.55 3.25 4.41 10.20 6.25 1.75 0.00 3.26 4.56 29.82 2.68 5.39 2.21 8.63 14.10 58.92 13.60 2,08 2.53 19.21 6.62 23.94 12.22 4.54 4.92 15.54 17.63 31.23 11.08 7.53 8.24 5.42 30.59 144.88 11.82 30.30 7.11 46.96 45.73 121.26 32.44 20.80 6.21 58.89 20.24 56.85 22.05 7.86 27.56 33.18 39.41 62.39 39.04 24.12 26.91 14 17 0.57 1.24 6.68 4.48 1.28 0.98 1.71 1.96 2.16 4.84 0.83 0.34 2.86 12.62 30.49 12.39 14.65 1.93 4.34 19.22 22.18 11.94 4.90 2.07 56.57 46.63 125.10 28.47 26.89 16.84 20.47 28.49 53.35 22.04 22.51 8,84 The spiked versus measured amounts 1n samples 1 and 2 are compared 1n Tables 27 and 28. The percent accuracy by compound, lab, and sample for the arithmetic mean, the geometric mean, the minimum, and the maximum are given 1n Table 27. The percent accuracy was determined by calculating the summary statistic, dividing by the spiked amount, then multiplying by 100. For example, % accuracy mean * ffiea-- x 100 spike With the exception of Total PNCDF; Total HXCDF; 2,3,4,7,8-PNCDF; 2,3,4,6,7,8-HXCDF, and 1,2,3,4,6,7,8-HPCDF for LAB-C, these numbers generally fall 1n the 80 to 120 range. To summarize the percent found, Table 28 gives the minimum, median, and maximum percent found by laboratory. Percent found expresses each determination as a percentage of the spiked amount similar to the formula above. LAB-C had by far 3-10 GEKP 011208 784019 GETSP 011209 Ar IthM tlc Hear COKPOUW - TOTAL ICOF Lab-A lab-6 Lab-C Lab-0 Ext 1 Lab-D Ext 2 Lab-0 Ext 1 Lab-E 19.0 It.5 95.0 it.a 92.1 92.7 95.0 CHPOUW - TOTAL PHCOF Lab-A Lab-6 Lab-C Lab-0 Ext 1 Lab-D Ext 2 Lab-0 Ext 3 Lab-E 94.0 99.1 124.4 96.1 102.3 ti.a IOfi.7 COMPOUM) - TOTAL HXCDF Lab-A Lab-B Lab-C Lab-0 Ext 1 Lab-0 Ext 2 Lab-0 Ext 1 Lab-E 04.3 9t.2 53B.2 102.3 TOI .2 99.a 103.2 COKPOUNO - TOTAL HPCDF Lab-A Lab-6 Lab-C Lab-0 Ext | Lab-0 Ext 2 Lab-0 Ext 3 Lab-E 90.9 91.6 65.3 102.9 100.4 93.0 112.5 COMPOUM) - OCDF Lab-A Lab-B Lab-C Lab-0 Ext 1 Lab-0 Ext 2 Lab-0 Ext 3 Lab-E _M -- -- -- -- -- -- TABLE 21. PERCENT ACCURACY FOR SPIKED SAMPLES BY COMPOUND. SAMPLE, AND LAB Supla 1 CaoMtr le Mean Hintaun HaxlxuB Ar Ithnetle Mean Saxpla 2 Geoaetric Mean Hlnlxua 0.2 71.1 94.2 aa.i 92.5 92.1 95.0 93.6 99.7 n a .o 96.6 102.3 16.4 106.7 72.5 71.5 00.0 4.0 la o 91.0 95.0 13.7 97 7 16.7 00.3 90.3 66.7 106.7 97.5 07.0 110.0 94.0 99.5 94 5 95.0 104.0 103.3 103.3 101 0 105.7 99.3 106.7 110.9 115 7 119 5 110 2 112.6 113 0 - 117.0 90.7 96.6 164.6 95.1 98.2 96.2 117.1 IIB.S 115.7 119.2 110.2 112.5 113.6 IIT.O 9B.4 9B.6 163.5 95.1 98.2 96.2 117.1 114.7 115.1 107.5 US. 1 101.5 113.2 III.0 7.3 90.1 145.1 94.3 96.5 94.9 117.1 3 .t 91.2 253.7 102.3 101.2 99.1 103.2 73.7 97.3 02.1 101.5 100.4 97.9 103.2 95.0 99.2 I3B9.5 103.5 (02.3 101.1 103.2 114.4 122.2 1527.9 102.9 105.9 105.2 81.1 114.3 122.1 1152.3 102.9 105.9 105.1 61.1 100.6 120.0 421.6 101.6 104.9 99.5 01.1 0.9 91.2 39.2 102. f 100.3 93.1 112.5 _ --- -- -- -- -- 05.3 00.S 0.3 97.0 102.0 91.5 112.5 __ -- --- -- -- ~ 93.0 100.0 132.5 114.5 113.0 96.0 112.5 __ --- -- --- -- 119.2 100.5 35.2 104.4 114.1 99.9 119.0 91.2 102.5 96.0 92 9 97.0 42.4 M e,7 99.0 20.3 104.3 114.1 99.7 119.0 90.0 94.7 -- 96.0 92.9 96.9 42.4 100.9 76.a 12.4 102.5 109.7 94.3 19.a 0 i.i 53.3 -- 94.7 09.4 93.2 42.4 Haxiaua 123.6 117.0 126.4 120. 117.0 115.1 117.0 106.3 99.4 109.9 95.9 101.3 96.1 117.1 121.6 125.4 2910.9 104.9 106.5 100.6 01.1 135.4 112.7 64.t 100.2 119.4 106.7 119.1 loo.a 149.2 -- 97.7 97.0 101.0 42.4 GENP 011210 Sxxple 1 A riihactic CeoMtrfc Ltb Mean HaAn Minimi COWOUND - TOTAL TCDO Lab-A lab-8 LAb-C Lab-O Ext I Lab-0 Ext 2 Lab-0 Ext 3 Lab-E 75.3 62.7 10.4 98.7 100.0 92.0 56.0 75.0 1.9 10.4 98.6 99.9 91.9 56.0 68.0 68.0 10.4 96.0 96.0 9E.0 56.0 COKPOUHO OCDO Lab-A LAb-B LAb-C lAb-0 Ext 1 Lab-0 Ext 2 Lxb-0 Ext 3 Lxb-E -- -- -- -- -- -- COMPOUND - 2.3.7.8-TCD0 -- -- -- -- -- -- -- -- -- -- -- -- LAb-A iib-e LAb-C Lab-0 Ext 1 Lab-0 Ext 2 Lab-0 Ext 3 Lab-E 75.3 82.7 81.3 98.1 100.0 92.0 56.0 75.0 119 81.0 98.6 99.9 91.9 56.0 68.0 68.0 76.0 96.0 96.0 88.0 56.0 COMPOUND 2.3.7.8 2.3,,4,8-TCDF Lab-A Lab-6 Lab-C Lab-0 Ext 1 Lab-0 Ext 2 lab-0 Ext 3 Lab-E 88.8 78.5 95.0 88.8 92.7 92.1 94.5 II 78.3 94.2 88.7 92.5 92.1 94.5 73.0 73.5 80.0 84.0 8.0 91.0 94.5 COMPOUND - 1.2.3.7.8 * 1,,2.3,4.8-PKCOF lab-A Lab-B Lab-C Lab-0 Ext 1 Lab-0 Ext 2 Lab-0 Ext 3 Lab-E 90.3 101.6 100.0 91.6 91.6 93.3 106.7 9.8 101.5 99.6 97.5 91.6 93.3 106.7 80.7 100.0 91.3 96.0 09.3 92.7 106.7 Table 27. ( c a n t'd .) Ma x i m i A rith iet(c Mean Salile 2 Geoietric Mean Minima 85.2 96.0 10.4 100.0 104.0 96 a 56.0 90.9 101.3 85.5 95.6 95.6 91.7 71.7 90.1 101.2 85.5 95.6 95.6 91.7 71.7 74.7 100.0 83.0 92.5 94.3 90.6 71.7 -- -- -- -- -- -- 85.2 96.0 92.0 100.0 104.0 96.0 56.0 97.5 87.0 110.0 94.0 99.5 94.5 94.5 102.0 104.0 113.3 98.7 94.7 94.0 106.7 -- -- -- -- -- -- 90.9 101.3 65.5 95.6 95.6 93.7 71.1 117.5 115.7 119.5 118.2 112.6 111. 8 MS.5 100.9 104.0 116.0 99.4 97.0 98.5 130.1 -- -- -- -- -- 90.1 101.2 65.5 95.6 95.6 93.7 71.7 117.4 115.7 119.2 118.2 112.5 113.0 118.5 100.4 104.0 116.0 99.4 97.0 90.5 120.3 -- -- -- -- -- -- 74.7 100.0 13.0 92.5 94.3 90.6 71.7 114.2 115.1 107.5 115.1 107.5 113.2 118.5 87.5 101.9 113.9 98.1 95.6 97.5 120.3 Maxima 102.1 103.8 86.8 98.1 98.1 96.2 71.7 -- -- -- -- -- -- 102.8 103.8 6.8 98.1 98.1 96.2 71.1 123.6 117.0 126.4 120.8 117.0 115.1 118.5 108.3 105.1 120.3 100.6 99.4 99.4 120.3 TABLE 27 ( c o o t'll.) Saapla 1 Saapla 2 TlzltO<lM3D ArittiMttc C tow tric Lab Hean Haan Hlntsua Haxlaua Arithmetic Hean Geometric Hean Hinlnum Haxiaua COHPOUW - 2 .2 .4 .T.I-PNCOF Lab-A Lab-0 Lab-C Lab-0 Ext 1 Lab-0 Ext 2 Lab-0 Ext 2 Lab-E 24.0 96.0 146.7 96.0 ioa.7 101.4 106.7 93.6 91.9 130.9 95.4 109.7 104.4 t06.7 4.7 94.7 eo.o 90.7 107.3 102.0 106.7 105.3 102.7 246.7 104.0 111.3 106.0 106.7 93.7 93.0 206. S 90.9 99.4 91-9 113.9 92.9 93.0 203.1 90.9 99.3 93.9 113.9 77.2 91.1 164.6 90.5 91.5 92.4 113.9 103.2 94.3 259.5 91.1 103.2 94.9 113.9 COHPOUW - 1 .2 .2 .4 .7 .1 * 1.2.2.4.7.9-KXCDF Lab-A Lab-fl Lab-C Lab-0 Ext 1 Lab-0 Ext 2 L*b-0 Ext 2 Lxb-E 91.0 95.9 104.0 106.4 103.1 104.4 116.0 90.5 95.9 99.4 106.4 103.0 104.4 116.0 79.6 95.2 60.0 104.9 100.4 100.0 116.0 103.6 96.4 128.0 107.2 105.2 107.2 116.0 ---- ---- ---- ---- ---- ---- -- "" -- -- -- -- -- -- -- -- -- -- -- COHPOUW I.2.I.7.6.1-KXCDF Lab-A Lab-0 Lxb-C Lab-0 Ext I Lab-0 Ext 2 Lxb-0 Ext 2 Lxb-E _ -- -- -- -- -- _ -- -- -- -- -- -- _ -- -- -- -- -- -- _ -- -- -- -- -- -- _-- ---- ---- ---- ---- ---- "" -- -- -- -- -- -- -- -- -- -- -- -- COHPOUW - 1.2.3.6.7,6-HXCOF Lxb-A Lab-A Lab-0 Ext 1 Lab-0 Ext 2 Lab-0 Ext 2 Lab-E 3.1 124.0 102.7 97.2 6.0 62.0 2.4 124.0 102.6 97.2 15.9 62.0 70.2 124:0 96.0 94.0 2.0 62.0 94.1 124.0 106.0 104.0 94.0 62.0 106.7 117.6 106.6 106.9 113.2 75.5 106.6 . 117.6 106.7 106.9 113.0 75.5 103.0 115.1 103.0 103.1 103.1 75.5 110.4 120. 115.1 109.4 120.6 75.5 COMPOUND - 2.3.4.6.7.6-HXC0F Lab-A 91.6 91.5 5.7 97.1 114 1 113.9 106.1 122.7 Lab-6 91.5 94.5 93.1 96.0 123.0 122.9 116.9 121.6 Lab-C 1106.6 313.5 10.0 3371.4 2015.2 1478.0 515.2 3939.4 -"I 0Jb0. Lab-0 Ext 1 Lab-D Ext 2 Lab-0 Ext 3 98.1 101.3 90.7 96.1 101.3 98.7 96.6 99.4 97.1 100.6 102.3 99.4 100.5 105.6 102.0 100.5 105.6 102.0 100.0 105.3 97.7 100.6 106.1 104.5 Lab-E 97.1 97.1 97.1 97.1 63.3 63.3 13.3 63.3 Saapla 1 A rfthaattc Gaoaatrlc Lab Haan Haan Hinlaua COMPOUND - 1. 2.3.4.t.7.a-HPCOF Lab-A Lab- Lab-C Lab-0 Ext 1 Lab-0 Ext 2 lab-0 Ext 3 Lab-E II. 4 1. 65.3 102.1 lO t.4 93.1 112.S a t.4 11.2 39.2 102.1 101.3 93. 112.5 as.3 10.5 a .i 9T.0 102. a 91.5 112.5 *Baln* H a lt of d tlK llo n . TABLE 27 (confd.) Haxtaua A rfrhM lic Haan Saapla 2 Geoaetric Haan Hlnlaua 92.3 100.0 132.5 114.5 113.8 9S.0 112.5 118.9 100.5 35.2 104.4 114.1 99.9 119.0 110.4 99.0 20.3 104.3 114.1 99.7 119.8 109.3 76.a 12.4 102.5 109.7 94.3 119. Haxlaua 134. 112.7 64. C 101.2 119.4 106.7 111. OIJ GENP 011212 TABLE 28. MINIMUM, MEDIAN, ANO MAXIMUM PERCENT FOUND (ACCURACY) FOR ALL PCDF AND PCDD CONGENERS IN SPIKED SAMPLES 1 AND 2 LAB LA8-A LAB-B LAB-C LAB-0 LAB-D LAB-D LAB-E Minimum 68.0 68.0 8.2 80.7 88.0 66.7 56.0 Sample 1 Median 87.8 96.0 94.2 98.7 100.8 94.5 103.2 Maximum 105.3 124.0 3371.4 114.5 113.8 107.2 116.0 Minimum 74.7 55.3 12.4 90.5 89.4 90.6 42.4 Sample 2 Median 106.8 112.0 120.3 100.8 104.9 98.7 113.9 Maximum 135.4 149.2 3939.4 120.8 119.4 120.8 120.2 the largest ranges. Among the other laboratories, LAB-B had the highest maximum values, while LAB-E had the lowest minimums. To provide a better comparison of the analytical method performance among the laboratories, the following steps were taken: 1) Samples 15 and 16 were dropped from further analysis because few compounds were found above the detection limit. 2) To eliminate the missing data for LABS-B and C caused by nonreporting of detection limits, the following values, based upon detection limits reported by LAB-D, were imputed: Compound______ Limit of Detection (nq/q) Total TCDD 3 0CDD 10 2,3,7,8-TCDD 3 2,3,4,7,8-PCDF 3 Analyses of variance were then run on the altered data to determine the sources of variation. Table 29 gives the total variance by compounds for each laboratory and then breaks 1t down Into sample (different types analyzed) and error (instrumental measurement) variances. Since LAB-E reported only one observation per compound and sample, its variance could not be broken down, but the total variance can be compared with the other labs. Generally, the TCDF, PNCDF, and OCDO compounds showed smaller variances, while OCDF showed the largest variances. There was some variability among laboratories 1n amount of total variance with LAB-E having a tendency to be higher, but no clear cut patterns emerge. Lab-C had the largest measurement error for almost every congener. The greatest percent of variation, however, was due to sample differences as reflected In comparing percent sample variance with percent measurement error variance.- OU^ 3-15 784024 TOTAL TCDF LAB-A LAB-B LAB-C LAB-D Ext.-l LAB-D Ext.-2 LAB-D Ext.-3 LAB-E TOTAL PNCDF LAB-A LAB-B LAB-C LAB-D Ext.-l LAB-D Ext.-2 LAB-D Ext.-3 LABrE TOTAL HXCOF LAB-A LAB-B LAB-C LAB-D Ext.-l LAB-D Ext.-2 LAB-D Ext.-3 LAB-E TOTAL HPCDF LAB-A LAB-B LAB-C LAB-D Ext.-l LAB-D Ext.-2 LAB-D Ext.-3 LAB-E OCDF LAB-A LAB-B LAB-C LAB-D Ext.-l LAB-D Ext.-2 LAB-D Ext.-3 LAB-E TABLE 29. VARIANCE COMPONENTS BY COMPOUND AND LAB Variance Components Measurement Total Sample Error As Percent of Total Measurement Sample Error 1.23 1.24 1.33 1.52 1.31 1.37 1.66 1.14 1.22 1.22 1.51 1.30 1.36 _a 0.09 0.02 0.12 0.01 0.01 0.01 - 93.0 98.3 91.1 99.4 99.5 99.5 - 7.0 1.7 8.9 0.6 0.5 0.5 - 1.55 1.37 1.57 1.71 1.55 1.60 1.90 1.54 1.361.42 1.68 1.55 1.59 - 0.01 0.02 0.15 0.02 0.00 0.01 - 99.4 98.9 90.3 98.6: 99.7 99.5 - 0.6 1.1 9.7 1.4 0.3 0.5 - 3.34 2.96 3.50 4.42 3.18 3.38 4.20 3.33 2.94 2.86 4.41 3.16 3.36 - 0.01 0.01 0.65 0.01 0.02 0.01 - 99.8 99.5 81.6 .99.7 99.4 99.7 - 0.2 0.5 18.4 0.3 0.6 0.3 - 4.20 4.19 0.01 99.7 0.3 3.19 3.15 0.05 98.5 1.5 4.65 4.06 0.59 87.3 12.7 6.06 6.04 0.02 99.7 0.3 3.76 3.75 0.01 99.8 0.2 5.55 5.49 0.06 98.9 1.1 5.35 - -- 10.94 6.50 6.46 8.52 6.41 8.19 9.86 10.93 6.43 6.30 8.48 6.40 8.19 - 0.01 0.07 0.16 0.04 0.00 0.00 - 99.9 98.9 97.5 99.5 99.9 99.9 0.1 1.1 2.5 0.5 0.1 0.1 784025 GETSn? 011214 TABLE 29 (cont'd.) TOTAL TC0D LAB-A LAB-B LAB-C LAB-0 Ext.-l LAB-D Ext.-2 LAB-D Ext.-3 LAB-E OCDD LAB-A LAB-B LAB-C . LAB-0 Ext.-l LAB-0 Ext.-2 LAB-D Ext.-3 LAB-E 2,3,7,8-TCDD LAB-A LAB-B LAB-C LAB-D Ext.-l LAB-D Ext.-2 LAB-D Ext,-3 LAB-E 2,3,7,8 + 2,3,4,8-TDCF LAB-A LAB-B LAB-C LAB-0 Ext.-l LAB-D Ext.-2 LAB-D Ext.-3 LAB-E 1,2,3.7.8 + 1,2.3.4,8-PNCDF La b -a * LAB-B LAB-C LAB-D Ext.-l LAB-D Ext.-2 LAB-D Ext.-3 LAB-E VarlanceTComponents Measurement Total Sample Error 5.61 2.22 2.24 2.25 2.30 2.19 7.52 4.34 2.22 2.24 2.25 2.30 2.19 - 1.27 0.00 0.00 0.00 0.00 0.00 - 2.73 0.24 0.59 _ _ _ 0.84 2.15 0.15 0.53 _ _ - 0.58 0.09 0.06 - - - 8.05 5.46 2.12 2.25 2.30 2.19 7.52 6.87 5.45 2.12 2.25 2.30 2.19 - 1.18 0.00 0.00 0.00 0.00 0.00 - 1.13. 1.32 1.70 1.57 1.43 1.43 1.65 i.ii 1.29 1.67 1.56 1.42 1.43 - 0.02 0.03 0.03 0.01 0.00 0.00 - 1.70 1.54 2.10 1.94 1.92 1.65 2.02 1.69 1.54 2.08 1.93 1.91 1.65 - 0.02 0.00 0.03 0.01 0.01 0.00 - As Percent of Total Measurement Sample Error 77.4 99.8 99.8 99.9+ 99.9+ 99.9+ - 22.6 0.2 0.2 0.0 0.0 0.0 - 78.7 63.1 89.2 21.3 36.9 10.8 - 85.4 99.9 99.8 99.9+ 99.9+ 99.9+ - 98.0 97.6 98.4 99.5 99.8 99.8 - 99.0 99.7 98.8 99.5 99.5 99.9 - 14.6 0.1 0.2 0.0 0.0 0.0 2.0 2.4 1.6 0.5 0.2 0.2 1.0 0.3 1.2 0.5 0.5 0.1 GENP 011215 784026 2,3,4.7.8-PNCDF LAB-A LAB-B LAB-C LAB-0 Ext.-l LAB-0 Ext.-2 LAB-0 Ext.-3 LAB-E 1,2,3,4,7.a + 1,2,3.4,7,9-HXCDF LAB-A LAB-B * LAB-C LAB-0 Ext.-l LAB-0 Ext.-2 LAB-0 Ext.-3 LAB-E 1,2,3,7,8,9-HXCDF LAB-A LAB-B LAB-C LAB-0 Ext.-l LAB-0 Ext.-2 LAB-0 Ext.-3 LAB-E 1,2,3,6.7.8-HXCDF LAB-A LAB-B LAB-C LAB-0 Ext.-l LAB-0 Ext.-2 LAB-0 Ext.-3 LAB-E 2,3,4.6.7.8-HXCDF LAB- LAB-B LAB-C LAB-0 Ext.-l LAB-0 Ext.-2 LAB-0 Ext.-3 LAB-E TABLE 29 (cont'd.) Variance Components Measurement Total Sample Error As Percent of Total Measurement Sample Error 1.27 1.20 1.65 1.30 1.32 1.29 1.76 1.26 1.20 1.58 1.26 1.32 1.29 - 0.01 0.01 0.08 0.04 0.00 0.00 - 98.9 99.5 95.4 97.0 99.9 99.9 - 1.1 0.5 4.6 3.0 0.1 0.1 - 6.47 7.25 4.48 5.93 5.69 5.78 8.71 6.44 7.22 4.24 5.93 5.69 5.78 - 7.86 3.50 4.68 4.88 4.46 4.86 6.06 7.48 3.50 4.04 4.86 4.44 4.85 - 2.99 2.48 - 3.01 2.32 2.85 3.79 2.99 2.46 - 3.00 2.30 2.83 - 3.27 2.62 4.53 3.11 1.77 1.85 4.27 3.25 2.59 3.18 3.10 1.76 1.84 - 0.03 0.03 0.24 0.01 0.00 0.00 - 0.38 0.00 0.65 0.02 0.02 0.01 - 0.01 0.01 0.00 0.02 0.02 - 0.01 0.03 1.38 0.01 0.01 0.01 - 99.6 99.6 94.6 99.9 99.9 99.9+ - 95.2 99.9 86.2 99.7 99.6 99.8 - 99.8 99.4 _ 99.9 99.2 99.3 - 99.5 99.0 69.6 99.6 99.2 99.5 - 0.4 0.4 5.4 0.1 0.1 0.0 - 4.8 0.1 13.8 0.3 0.4 0.2 - 0.2 0.6 0.1 0.8 0.7 - 0.5 1.0 30.4 0.4 0.8 0.5 - 3-la " 784027 GENP 011216 1,2,3,4,6,7,8-HPCDF LAB-A LAB-B LAB-C LAB-0 Ext.-l LAB-D Ext.-2 LAB-D Ext.-3 LAB-E aBelow limit of detection. TABLE 29 (cont'd.) Variance Components Measurement Total Sample Error As Percent of Total Measurement Sample Error 4.05 3.01 3.97 4.43 3.32 4.00 5.28 4.04 2.97 3.10 4.41 3.32 3.94 0.01 0.04 0.87* 0.02 0.01 0.06 99.8 98.7 78.1 99.6 99.8 98.5 ' 0.2 1.3 21.9 0.4 0.2 1.5 GENP 011217 3-19 784028 For LAB-0, the variance component for repetitive extraction of the same sample could be included because data for three extracts was reported (see Table 30). Here as before, at least 95X of the variance was 1n the samples with extract and Instrumental measurement error contributing little. To gain further understanding of the differences 1n variance, Table 31 gives the standard deviations (the square root of the variances) by compound, sample, and laboratory (recalling that each lab used different analytical procedures as described 1n Section 2.0). The laboratories were then ranked by compound and sample according to the size of the standard deviation, 1.e. for TCDF 1n Sample 1F LAB-0-3 had the smallest standard deviation while LAB-C had the largest. Generally, LAB-C had larger standard deviations (and therefore poorer accuracy and precision from their analytical method). While the other laboratories, excluding LA8-C, show no large differences, LAB-D tended to be lower (and therefore the best accuracy and precision from their analytical- method). Also shown are the results of Bartlett's tests for homogeneity of variance among laboratories, by compound and sample (6). Here again, there 1s variability 1n the results. For some of the compound-sample combinations, the labs were significantly different at the .01 level while for other combinations the labs were not significantly different at the .05 level. Table 32 gives the results of Levene's test for homogeneity of variance among laboratories (over samples), by compound (6). For every compound, the laboratories were significantly different from one another at the .05 level, and for all but one compound (1,2,3,6,7,8-HXCDF), they were significantly different from one another at the .01 level. Table 33 provides approximate 95-percent prediction Intervals of levels reported from future analyses of the samples. The seven laboratories (LAB-D Included three times) were treated as a random sample from a large population of laboratories, and a variance component, denoted as VL, of the log concentrations due to laboratories (and therefore analytical method used) was thus estimated. A measurement error variance component, VM, was also estimated. This component 1s the wlthln-extract variance of the log concentrations. Data from the LAB-E were not used In determining this component of variance, since there were no replicate observations. The two components were used to calculate the prediction intervals via the following formula: where exp {x * 2V), x * mean of the log concentrations (across laboratories and extracts), and V - square root of (VI + VM). 784029 3-20 TABLE 30. VARIANCE COMPONENTS BY COMPOUND FOR LAB-D Compounds Variance Components Total Sample Extract Error As Percent of Total Sample Extract Error Total TCDF 1.40 Total PNCDF 1.62 Total HXCDF 3.66 Total HPCDF 5.13 OCDF 7.73 Total TCDD 2.25 OCDD 0.00 2,3,7,8-TCDD 2.25 2,3,7 r8 + 1.48 2,3,4,8-TCDF 1,2,3,7,8 + 1.84 1,2,3,4,8-PNCDF 2,3,4,7,8-PNCDF 1.31 1,2,3,4,7,8 + 5.80 1,2,3,4,7,9-HXCDF 1,2,3,7,8,9-HXCDF 4.73 1,2,3,6,7,8-HXCDF 2.73 2,3,4,6,7,8-HXCDF 2.25 1,2,3,4,6,7,8-HPCDF 3.92 1.38 1.61 3.59 5.00 7.41 2.25 0.00 2.25 1.47 1.82 1.30 5.80 4.71 2.69 2.16 3.87 0.01 0.00 0.05 0.09 0.30 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.01 0.02 0.08 0.02 0.01 0.01 0.01 0.03 0.02 0.00 0.00 0.00 0.00 98.9 99.2 98.3 97.6 95.9 99.9+ 99.9+ 99.9+ 99.5 0.01 99.2 0.01 0.00 98.9 99.9 0.01 0.01 0.01 0.03 99.5 98.6 96.1 98.7 0.6 0.0 1.3 1.8 3.9 0.0 0.0 0.0 0.2 0.4 0.0 0.0 0.2 0.8 3.4 0.5 0.5 0.8 0.4 0.6 0.2 0.0 0.0 0.0 0,3 0.4 1.1 0.1 0.3 0.5 0.5 0.7 H 2l9 3-21 784030 TULE 31. STANDARD DEVIATIONS. THEIR RANKS. ANO 8ARTLETTS TEST BY COMPOUND AND SAMPLE Standard Oavlationi --- S avi LAS-A LAfl-S LA6-C LA8-0-1 ' LAB-0-2 LAB-O-3 COMPOUND - TOTAL TCDF 1 2S.5B 14.80 30.00 10.02 12.10 3.51 2 2.36 0.58 5.51 1.53 2.52 0.56 3 20.82 49.32 366.92 96.15 65.51 20.55 11 106.01 8.33 65.06 19.67 20.31 10.44 12 162.17 6.35 20.61 4.51 3.06 19.60 13 143.93 60.92 130.00 97.12 92.67 109.61 14 283.06 216.61 217.03 92.48 30.75 16.45 17 23.39 78.17 65.06 59.67 39.55 3.21 COMPOUND TOTAL PNCOF 1 30.51 9.29 155.03 21.94 11.14 54.93 2 31.75 2.08 72.11 2.52 8.39 3.48 3 26.46 66.37 100.00 166.96 134.52 67.88 11 123.42 166.86 860.31 312.66 41.07 3.52 12 74.81 15.26 177.30 42.45 7.09 7.23 13 11.53 27.10 25.17 46.48 20.64 26.51 14 300.00 1722.88 2542.31 2686.65 275.13 113.15 17 35.12 218.48 64.29 161.04 131.23 109.55 COMPOUND - TOTAL HXCOF l " 52.63 4 .SI 3504.6 2 12.22 5.29 2354.6 3 96.22 48.17 7669.6 n 172.14 913.30 24055.4 12 43.02 36.37 257.0 13 6.43 17.35 15.3 14 200.00 413.63 12165.5 17 30.55 283.28 6600.8 COMPOUNO - TOTAL HPCOF 6.08 3.21 271.01 828.67 85.04 3.61 1217.54 206.65 4.73 1.73 622.22 126.26 49.90 9.45 590.93 30.29 7.94 9.29 42.15 131.05 44.66 11.93 96.11 8.06 1 15.57 2 73.90 3 85.44 11 793.87 12 295.01 13 2.16 14 52.92 17 107.66 COMPOUNO - OCOF 40.00 107.96 175.84 1592.80 345.56 8.14 290.13 422.94 251.02 140.60 1172.40 4838.73 600.00 12.40 737.11 305.51 40.13 17.32 64.84 1855.90 332.58 0.00 387.56 322.10 22.03 25,77 147.14 92.60 125.01 0.58 370.24 139.41 9.02 32.96 148.19 548.43 95.04 4.04 40.78 28.04 1 0.27 0.00 4.56 0.56 0.00 2 3 13.01 166.53 62.00 829.67 1357.89 2.08 114.04 5.03 221.20 7.00 228.29 11 2260.53 9644.25 3464. |0 3667.22 879.17 1420.07 12 640.96 1049.04 493.29 617.62 127.60 232.46 13 14 1.61 0.00 4.56 173.66 20.23 36.06 6.51 M S 1.15 8.62 22.74 36.67 17 387.66 546.51 404.15 596.67 14.29 89.69 87 LA-A 5 4 fftt* 2 6 ft 6 6 ft 6 ft 2 ffttft 4 5 1 ft* 3 ft* 5 ftft 1 3 1 ftft 5 ft 5 ftfftt 3 3 2 2 ft 2 ft 3 ft 2 ftft 4 ft 2 ft 3 3 3 ft 2 2 ffttft 3 4 ft 2 3 ftft 4 4 ft 1 3 LAB'S 4 1 3 1 3 1 4 6 1 1 2 4 3 5 4 6 I 3 2 5 4 6 3 5 4 5 5 4 5 5 3 6 1.5 5.0 5.0 6.0 6.0 1.0 6.0 5.0 Paniti LAS-C LAB-0-1 62 63 65 53 52 53 53 54 63 62 46 65 64 36 56 25 63 6: 64 64 65 5l 65 64 6S 61 61 65 64 61 65 45 5 I 61 45 35 65 42 46 LAS-0-2 3 5 4 4 1 2 2 3 2 4 5 2 1 2 2 4 2 1 5 1 4 3 4 2 3 2 3 I 2 2 4 3 4.0 2.0 3.0 1.0 1.0 2.5 3.0 1.0 LAS-0-3 1.0 2 1 2 4 4 1 1 5 3 3 1 2 4 1 3 4 4 1 2 3 4 1 1 1 3 4 2 1 4 1 1 1.5 3.0 4.0 2.0 2.0 2.5 5.0 2.0 011220 3-22 784031 table 3: t c w d . ) Standard Oaviatlons Sangla LAS-A LAB-B LAfi-C LAB-0-1 LAS-O-2 COMPOUND TOTAL TCOO 1 2.22 3.51 0.58 1.00 2 7.70 1.13 1.15 1.53 1.15 3 2.09 9.02 15.28 1.73 5.51 11 34.97 0.00 0.00 0.00 0.00 12 3.87 0.00 0.00 0.00 0.00 13 0.34 0.00 0.00 0.00 0.00 14 0.47 0.00 0.00 0.00 0.00 17 0.24 0.00 0.00 0.00 0.00 COMPOUND OC00 1 2.61 0.00 00 2 2.14 0.00 00 3 4.G8 0.00 00 11 3.03 2.52 4.16 0 0 12 0.40 7.64 0.00 0 0 13 0.44 0.58 0.00 0 0 M 0.03 4.04 4.62 0 0 T 4.86 5.51 4.52 0 0 : o m p o u n o 2.3.7. 8-TCDQ 1 2.22 3.51 2.31 0.58 1.00 2 *7.70 1.13 1.15 1.53 M S 3 2 09 9.02 15.28 1.73 5.51 11 34 74 0.00 0.00 0.00 o.oa 12 1 11 0.00 0.00 0.00 0.00 13 0.34 0.00 0-00 0.00 0.00 U 0 04 0.00 0.00 0.00 0.00 17 0.24 0.00 0.00 0.00 0.00 COMPOUNO 2.3,7. 8 2.3.4.8-TCbf 1 27.47 14.80 30.00 10.02 12.10 2 2.80 0.58 5.51 1.53 2.52 3 10.00 11.59 219.39 12.22 42.03 11 12.94 16.46 5.51 3.46 0.00 12 15.50 1.73 5.77 3.06 1.73 13' 38.35 4.58 41.63 25.01 12.06 14 66.46 25.11 25.17 32.15 7.09 17 7.00 28.73 26.46 27.51 1L02 COMPOUNO - 1.2.3. 7.8 I ,2 .3 .4 . 8-PNCOP 1 16.27 3.21 17.32 2.08 4. 16 2 18.29 2.89 5.77 2.00 3.21 3 3.06 13.58 32.15 12.74 20.23 11 105.71 58.50 11.59 104.41 21.13 12 25.10 5.51 6.24 19.86 4.58 13 1.51 1.53 1.12 1.00 1.15 14 42.12 49.15 62.45 221.59 210.79 17 11.04 42.23 L.53 37.40 49.00 LAS-O-3 1.00 1.53 1.53 0.00 0.00 0.00 0.00 0.00 a 0 0 0 0 0 a 0 1.00 1.53 1.53 0.00 0.00 0.00 0.00 a.ao 3.51 0.58 25.42 1.15 5.51 5.77 4.00 2.00 1.00 1.53 12.17 22.81 3.00 0.00 56.04 14.47 BT LAB-A LAS-6 45 *63 35 63 63 63 63 63 5 5 5 5 * 5 5 4 5 2.5 2.5 2.5 4.0 6.0 6.0 5.0 6.0 46 63 35 63 63 63 63 63 5 5 * 1 5 6. 5 6 2 4.0 1.0 2.0 6.0 1.5 1.0 3.0 6.0 ** 5 t* 6 1 *6 *6 5 1 2 3 3 4 4 3 6 2 5 Ranks LAfl-C LAB-0-1 LA-0-2 1 1.5 5 6.0 2 3.0 3 3.0 3 3.0 3 3.0 3 3.0 3 2.5 1.5 4.0 3.0 3.0 3.0 3.0 3.0 0 2.5 2.5 2.5 6.0 2.0 2.5 2.5 2.5 2.5 6.0 2.0 4.0 2.0 2.5 2.5 2.5 2.0 2.5 2.5 2.0 2.0 5.0 I 1.5 5 6.0 2 3.0 3 3.0 3 3.0 3 3.0 3 3.0 3 2.5 L5 40 3.0 3.0 3.0 3.0 3.0 62 63 63 43 53 64 45 45 3.0 4.0 5.0 1.0 1.5 3.0 2.0 3.0 62 52 63 15 45 32 46 14 4 4 5 2 2 4 2 6 LAB-O- 2.5 4.0 1.0 3.0 3.0 3.0 3.0 3.0 2.5 2.5 2.5 2.0 2.5 2.5 2.0 2.0 2.5 40 10 3.0 3.0 3.0 3.0 3.0 1 2 4 2 4 2 I 1 1 1 2 3 1 1 3 3 GENP 011221 3-23 784032 TABLE 31 (cont'd.) Standard Deviations _ __ Sapl UB-A UB-fl LAB-C LAB-0-I LA8-D-2 LAB-0-3 COMPOUND - 2.3.4.7,8-PNCOF ' 1 15.72 6.24 132.29 19.92 3.46 3.21 2 22.61 2.65 76.36 0.58 5.20 2.01 3 8.90 11.59 237.14 2.01 14.01 6.08 11 3.06 6.51 11.55 34.82 4.00 3.06 12 11.79 4.62 2.00 7.94 1.15 2.06 13 S.60 4.00 11.31 6.51 1.00 1.53 14 26.46 96.00 321.46 312.67 21.13 33.05 IT 11.93 22.61 20.62 44.66 9-64 9.29 COMPOUNO 1.2.3 ,4.7.6 1.2.3.4.7,9-WtCOF 1 30.12 1.53 95.4 3.46 6. II 9.64 2 1.71 0.51 0.00 0.00 0.00 3 39.45 33.72 152.6 22.74 23.86 35.23 11 99.90 517.84 13519.3 394.71 80.89 83.93 12 11.59 35.80 120.5 67.98 3.06 25.87 13 3.12 4.93 10.1 t .53 0.58 2.08 14 25.02 46.74 1644.2 419.97 660.20 148.33 IT 56.70 253.24 2294.2 145.23 11.53 13.32 COMPOUND 1.2.3 .7 .8 .9-MXCQF 1* 0.27 0.00 0.00 0.00 2 0.24 0.00 0.00 0.00 3 2.05 558.64 1.53 1.73 0.58 11 1.36 0 647.66 28.62 11.66 6.61 12 6.11 0 9.45 2.52 2.08 0.58 13 0.09 0 6.93 1.15 1.53 1.00 U 8.14 31 3665.66 173.14 289.63 15.72 17 0.16 0 1024.30 2.89 1.53 4.51 COHPOUNQ 1 .2 .3 .G.7 .8-HXC0F 1 6.17 0.00 2 1.96 1.53 3 13.32 12.06 11 3.61 100.72 12 1.18 4.36 13 0.46 2.08 14 17.32 29.69 17 1.94 2.31 2.08 3.06 15.72 22.28 3.00 0.00 63.66 4.51 2.89 1.53 27.30 64.49 3.51 0.58 110.39 18.61 3.46 4.58 6.00 4.04 8.08 0.58 110.53 6.03 COMPOUKO 2 .3 .4 ,6 .7 .B-HXCOF I 10.02 2.52 3308.35 3.79 2.89 2.31 2 11.02 6361 2311.45 0.58 0.58 4.93 3 14.36 14.29 4659.68 3.79 39.58 6.43 11 5.67 10.56 909.45 30.05 8.01 1.00 12 3.05 3.06 11.72 5.00 1.15 2.08 13 0.16 1.19 11.14 0.00 1.15 2.52 14 20.43 62.55 3461.99 105.72 227.28 19.73 17 7.27 13.45 1457.17 6.03 1.73 8.00 87 LAB-A US-6 ** 4 ** 5 3 # -- 1 6 4 ft* 2 3 3 3 4 4 4 3 4 5 ** 5 5 5 ** 3 *2 4 ft 1 t* 3 1 4 3 5 4 5 2 5 4 4 ft 4 ft* 2 ft* 5 2 ft* 1 * 2 1 1 1 3 1 5 3 3 fftt* 1 1 2 ft 1 1 1 2 2 5 4 5 2 2 ft 5 5 ft 4 ft 2 3 2 ft* 2 ft* 4 2 4 3 4 4 4 3 5 Ranks LAB-C LAB-0-1 LAB-O-2 65 61 61 56 25 65 65 46 2 4 5 3 1 1 1 2 62 2 61 64 65 62 64 64 3 2 2 1 1 1 5 1 2 2 52 65 64 64 64 64 2 2 3 4 3 5 5 3 23 41 45 34 23 13 34 35 64 62 61 65 65 61 64 63 3 1 5 3 1 3 1 UB-0-3 1 2 2 2 3 2 3 l 4 2 4 2 3 3 3 2 2 2 1 3 2 3 2 5 4 5 I 2 5 i i i 1 3 2 1 2 5 1 2 GENP011222 3-24 784033 TABLE 31 (e o n t'd .) Standard Deviations . __ Saagle LAS-A LAB-6 LAS-C LAS-D-1 LAB-O-2 LAB-0-3 COHPOUNO 1.2.3.4 6 ,7 ,8-HPC0F 1 14.19 40.06 251.02 40.13 22.03 9.02 2 72.95 107.96 140.60 17.32 25.77 32.98 3 34.64 61.56 460.74 10.69 42,67 42.03 11 314.32 698.55 1708.80 672.92 29.02 166.79 12 60.62 111.97 200.33 101.36 37.00 31.97 13 o.ai 3.06 3.53 0.00 0.58 4.04 U 11.02 96.70 370.05 133.06 119.93 8.72 17 27.10 250.47 70.95 153.61 59.37 17.93 BT LAB-A LAB-9 ft** 2 ft* 4 ft 2 ft* 3 3 3 fftt* 2 2 4 5 5 5 5 4 6 6 Ranks LAB-C LAB-0-1 65 61 61 64 64 51 45 45 LAB-0-2 3 2 4 1 2 2 6 3 LAS-0-3 1 3 3 2 1 6 1 1 * B a r tle tt's te s t shows sig n ific a n t difference a t the .05 level. * B a r tle tt's te s t shows sig n ific a n t difference a t the .01 level. i GENP 011223 3-25 784034 TABLE 32. LEVENE'S TEST OF DIFFERENCES AMONG LABS BY COMPOUND-OVER SAMPLES Compound F-Value Degrees of Freedom Total TCDF Total PNCDF Total HXCDF Total HPCOF OCDF Total TCDD OCDD 2,3,7,8-TCDD 2,3,7,8 and 2,3,4,8-TCDF 1,2,3,7,8 and 1,2,3,4,8-PNCDF 2,3,4,7,8-PNCDF 1,2,3,4,7,8 and 1,2,3,4,7,9-HXCDF 1,2,3,7,8,9-HXCDF * 1,2,3,6,7,8-HXCDF 2,3,4,6,7,8-HXCDF 1,2,3,4,6,7,8-HPCDF 8.89** 11.15** 23.12** 17.39** 6.97** 17.82** 12.81** 14.96** . 6.60** 4.39** 9.76** 17.80** 16.10** 2.96* 39.93** 22.80** 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 * Significantly different at the .05 level. ** Significantly different at the .01 level. GENP 011224 3-26 784035 TABLE 33. APPROXIMATE 95 PERCENT PREDICITON INTERVALS BY COMPOUND..AND SAMPLE3 Compound Total TCDF Total PNCDF Total HXCOF Total HPCDF OCDF ' Total TCDD OCDD 2,3,7,8-TCDD 2,3,7,8 + 1,2,3,4,8-PNCDF 1,2,3,7,8 + 1,2,3,4,8-PNCDF 2,3,4,7,8-PNCDF 1,2,3,4,7,8 + 1,2,3,4,7,9-HXCDF 1,2,3,7,8,9-HXCDF 1,2,3,6,7,8-HXCDF 2,3,4,6,7,8-HXCDF 1,2,3,4,6,7,8-HPCDF 12 144 57 222 67 206 220 431 517 155 40 1862 2118 103 137 1105 1532 2 67 89 208 6 40 60 60 11 59 40 15 40 30 60 144 57 221 67 123 138 168 193 99 90 248 321 182 1 347 15 <1 34 31 72 40 1041 <1 25 45 72 24 1912 103 137 1104 1530 Sam ple 3 11 12 1046 57 37 2321 362 321 1004 1682 1125 3079 143 590 798 3041 10654 21535 726 1320 819 1581 4341 41116 1467 3090 2280 9641 3822 6436 64743 10453 63 <1 <1 132 21 14 275 32 19 30 63 <1 <1 132 25 20 977 24 15 1355 61 81 226 41 425 7913 27 264 164 84 618 145 28 59 825 2138 1838 12579 447 809 21 965 8869 4 167 254 218 412 603 20 97 55 3975 26 879 9 68 96 580 3258 16405 418 976 13 389 1247 179 442 11 198 2 52 7 75 <1 15 <1 70 <1 13 90 459 8 30 46 80 13 36 <1 61 2 10 1 51 1 17 14 532 1389 '5846 13417 5897 21657 1391 3486 96 457 <1 20 1 43 <1 32 251 459 742 1577 1324 2389 3144 5860 64 7800 561 1385 196 2882 482 1130 17 249 729 562 1999 610 13977 242 11063 442 24465 <1 10 5 32 <1 11 119 241 ;74 7|46 106 403 486 5023 <1 6136 43 134 10 2237 79 5652 aValue$ are In ppb. 01U25 GB'H? 3-27 784036 With about 95 percent confidence of being correct (1.e. 1 our of 20 measurements having a chance of being outside of this range), each of the reported Intervals is expected to cover a future laboratory's analytical results for the given compound and sample. This assumes that the concentrations are log normally distributed. Note that the data are not adequate for assessing this assumption; also the data for generating these Intervals are extremely limited and can be sensitive to spurious observations. Many of .the Intervals are so wide that they are likely to be of little use. If the analytical method used by Lab-C was omitted, these Intervals would improve, but a quantitalve estimate of this improvement would be difficult to determine since the amount of data for statistical analysis would be Insufficient. 3-28 784037 SECTION 4 REFERENCES 1. A. W. Nichols, M. P. Kilpatrick, E. D. Hardin and D. A. Hayes. Polychlorinated Dlbenzofurans (PCDF) and Polychlorinated D1benzo-g-D1ox1ns (PCDD) 1n Utility Transformers and Capacitors. Vol. 3, Palo Alto, CA: Electric Power Research Institute, February, 1987. EPRI EL/EA-4858,. 2. S. M. Gordon and M. Hiller. Polychlorinated Dlbenzofurans (PCDF) and Polychlorinated D1benzo-jj-D1ox1ns (PCDD) 1n Utility Transformers and Capacitors. Vol. 2. Palo Alto, CA: Electric Power Research Institute, December, 1986. EPRI EL/EA-4858. 3. D. R. Hllker, G. A. Eadon, K. H. Aldous, R. M. Smith, P. W. O'Keefe, H. Valente, S. Conner, and J. Jur1s1k. Analysis of Polychlorinated Dibenzofurans and Polychlorinated D1benzo-g-D1ox1ns 1n Transformers and Capacitors. Vol. 1. Palo Alto, CA: Electric Power Research Institute, October, 1987. EPRI EL/EA5443. 4. W. H. Cooke and F. L. DeRoos. Formation of PCDD and PCDF 1n Askarel and Contaminated Mineral 011 Equipment. Palo Alto, CA: Electric Power Research Institute, July, 1987. 5. T. 0. Rouse. Polychlorinated Dlbenzofurans (PCDF) and Polychlorinated Dibenzojj-D1ox1ns (PCDD) 1n Utility Transformers and Capacitors. Vol. 1. Palo Alto, CA: Electric Power Research Institute, October, 1986. EPRI EL/EA-4858. 6. G. W. Snedecor and W. G. Cochran. Statistical Methods. The Iowa State University Press, 1980, p, 252-254. E N P 011227 4-1 784038 APPENDIX A EVALUATION OF CALIBRATION REGRESSION USED BY U. UMEA GENP 011228 784039 RESEARC H TRIANGLE INSTITUTE Center 1or Medical. Environmental and Energy Statistics S e p ie rtx r 2, 1906 KE2CRMOM TO: Edo fe llix z a x l FROI: Andy Clayton SUaJECT: Problems with Calibration of Dioxin/Turan Data RETD&a: Analys results ETU-pradjekt 2028 (6/23/86) TVo problems with the calibration procedure used by the Urea Universitet laboratory are noted. These are the following: 1. The regression oi x on y is performed, where x is the (injected) amount of dioxin/furan, and y is the (observed) normalized count. Although there has been iruch controversy over the past 20-25 years concerning whether i t is more appropriate to regress x or. y or y on x, I believe that most statisticia n s a^ e e that regressing y on x is the preferred approach. 2. Ordinary least squares (CCS)' is used to f i t the calibration curve (assmed to be a straight lin e). This' is a roch more serious problem. OLS is appropriate when the magnitude of the measurwnent errors is approximately the same across the entire range of concentrations of in terest. This is clearly not the situation here. As an example, consider the data for 2376-Tar. When x-7500, the y values range from 6593.7 to 9037.3, a difference of 443.6. Or. the other hand, when X"10, the range of observed counts is only 2.9. Clearly, the measurement error increases with increasing concentration, so that CLS is inappropriate. The severity of the problem is demonstrated by the estimates obtained for 2376-TOT. Using the values of a and b from the referenced document, i t is seen th at a normalized count of 10 units will yield a predicted concentration of (.853)(10) 29.B9 36.42 pg. examination of the calibration data indicates that the true concentration should be roughly 10 pg. For lever concentrations (say, less than 200 pg), CLS has thus produced a curve that (in this case) results in a significant overestimation of the true concentration. It is precisely in th is range where the transformer data occur. One solution to the above problems is to perform the calibration ourselves and to apply the new calibration to normalized counts obtained iron the reported concentrations and the reported curve parameter estimates. Sere imprecision will occur due to the fact that the concentrations are reported to only gw significant d ig its. Alternatively, we can request the rav (normalized count) data, perform, an appropriate calibration, and re-estimate the concentrations for the transformer. FteslOtlce Box 12194 Resaarch Triangle Park.North Carolina 27709 Telephone 919541-622? GENP 011229 A-2 784040 APPENDIX B DATA USED IN COMPARISONS BETWEEN LABS GENP 011230 784041 Saapl 1 Lab KY5D0K Z NVSDQH KYSDOH TABLE B-l DATA USED IN COMPARISONS BETWEEN UBS Coapaund - --- TOTAL TCDr TOTAL PNCOP TOTAL HXCOr TOTAL h p c d f OCOF TOTAL t c o o ocoo Z.3.T.-TCOO 3,3,7,* * 2.3,4,8-TCDF .z.3 .t ,a < ],3,3,.a-Piicor z.a.a..s-pttcop 1.3.3.4>?.* 1,3.3.4.7,0-MCOf 1.2.3.7.8.9-HXCOF 1.2,3.6,7,8-KXCOF Z ,3,4.B .7.t-KXCOF 1.Z.3.4.S.T.B-HPC0F TOTAL TCDF TOTAL PNCOF TOTAL HXCOF TOTAL HPCDF OCOF TOTAL TCOO OCDD 2.3.7.B-TCOD 2.3.7,8 - 2.3.4,8-TCOf 1.Z.3.T.B * 1,2,3,4.8-PNCDP 2.3.4,7,8-PNCDF 1.2.3.4.7.B 1,2,3,4,7,B-HXCDF 1.2,3.7,8.9-HXCOF 1.2.3,6,7,8-HXCDF Z,3.4.8,7,8-HXCOF 1,2.3,4.8.7,8-HPCDF TOTAL TCOF TOTAL PNCOF TOTAL HXCOF TOTAL HPCDF OCOF TOTAL TCOO OCDD 2.3.7.a-TCTD Z.3.7.B 2,3,4,8-TCOF 1.2.3.7,8 1,2.3.4,8-PNCDF Z.3.4.7.B-PNCDF 1.2.3.4.7.8 * 1,Z ,3,4.7.9-HXCOF 1,2.3.7.8.9-HXCDP 1.2.3.8.7,8-HXCDF 2.3.4,8.7,8-HXCDF 1,2.3.4,8,7.a-HPCOP Dat. 1 143.00 331.00 398,00 334.00 3.49 17.00 3. IT. W 148.00 121.00 127.00 333.00 0.33 42.10 130.00 331.00 80.00 278.00 301.00 378.00 133.00 39.80 1.44 39.60 60.80 138.00 122.00 3.77 0.21 38.30 140.00 373.00 1630.00 1330.00 3490.00 2470.00 4830.00 83.10 3.73 63.10 1310.00 348.00 304.00 1228.00 37.80 339.00 313.00 803.00 Del. 2 193.00 312.00 433.00 341.00 3.33 21.30 0.49 21.30 193.00 133.00 131.00 239.00 0.07 47.40 161.00 341.00 82.70 338.00 209.00 712.00 107.00 30.40 0.41 30.40 80.30 171.00 139.00 3.83 0.03 34.80 130.00 709.00 1820.00 1290.00 3380.00 2300.00 3090.00 83.30 0.72 83.30 1220.00 342.00 288.00 1209.00 33.80 321.00 288.00 803.00 Del. 3 194.00 263.00 330.00 372.00 3.06 18.20 0.87 11.20 192.00 132.00 139.00 199.00 0.03 33.10 170.00 389.00 83.30 324.00 223.00 391.00 121.00 34.30 4.32 34.30 83.30 189.00 163.00 6.12 0.32 38.50 162.00 392.00 1390.00 1340.00 2330.00 2400.00 5170.00 81.70 9.89 81.70 1200.00 380.00 291.00 1283.00 39.90 347.00 291.OO 883.00 Mian 178.00 282.00 400.00 358.00 3.37 18.80 2.08 18.60 178.00 133.00 141.00 228.00 0.22 41.50 180.00 334.00 63.00 312.00 212.00 827.00 120.00 48.20 2.12 48.20 62.30 139.00 148.00 4.24 0.26 36.50 131.00 825 00 1613.00 1320.00 2473.00 2390.00 3037.00 64.10 4 78 64.10 1210.00 343.00 294.00 1241.00 37 80 336.00 297.00 843.00 Std. Dv. 28.80 30.50 32.60 13.80 0.27 2.22 2.81 2.22 27.30 18.30 13.70 30.10 0.27 6.17 10.00 14.20 2.38 31.70 12.20 73.90 13.00 7 70 2. 14 7 70 2.8Q 18.30 22.60 1.71 0.24 1.93 11.00 73.00 20.80 26.30 86.20 83.40 167.00 2.09 4.68 2.09 10 00 3.06 8.30 39 40 2.03 13 30 14 40 34.60 g u n 784042 -Saapla Lab 11 KYSDOH 12 KYSDOH 13 NYSDOH TABU B-l (cant'd.) Coapound TOTAL TCOF TOTAL PNCOF TOTAL HXCDF TOTAL HPCOP OCOF TOTAL TCOO OCOO 3,3,7,1-TCOO 3.3.7. 2,3,4.-TCOF 1.2.3.7. 1,2,3.4.FNCOF 3.3.4.7.-FNCOF 1,2.3.4,7. 1.2.3,4.7.S-taC0F 1.3.3.T.B.B-HXCOF 1.2.3.8.7,8-HXCDF 3.3,4.6.T ,1-HXCDF 1.2.3.4.8.7.8-HPCDF TOTAL TCOF TOTAL PNCOF TOTAL HXCOF TOTAL HPCDF OCOF TOTAL TCOO OCDD 2.3.7.S-TCOO 2,3.7.B 2,3.4,8-TCDF 1.2.3.7.8 1.2,3,4.S-PNCDP 2.3.4,7.8-PNCOF 1.2.3.4.7.B * 1.2.3.4.7,9-HXCDF 1.2.3.7,8.9-KXCOP 1.2.3,8,7.B-HXCOF 2.3.4.8,7.S-HXCOP 1,2,3,4.8.7.8-HPCDF TOTAL TCOF TOTAL PNCOF TOTAL WtCDF TOTAL HPCDF OCOF TOTAL TCDD ano 2.3.7.B-TC00 2.3.7,B - 2,3.4.8-TCOF 1.2.3.7.8 1.2,3,4,B-PNCOF 2.3.4.7.B-PNCOF 1.2,3,4.7,B 1,2.3.4.7,9-HXCOF 1.2,3 .7 .8.B-HXCDF . 1.2.3.8,7,B-HXCOF 2.3.4.8,7,B-HXCDF 1,2,3,4.8,7.8-HPCDF Dat. 1 232.00 1030.00 8840.00 10100.00 30000.00 1.32 11. 0 10.00 1.32 4.00 3.00 101.00 47*2.00 2.34 380.00 51.20 3100.00 221.00 430.00 083.00 2400.00 8800.00 0.38 27.40 0.05 88.60 116.00 80.60 621.00 37.90 33.90 43.00 15.20 682.00 886.00 388.00 30.70 8.30 13.40 0.18 ' 1.10 0.18 107.00 19.80 61.30 16.60 0.19 3.40 1.70 3.40 Dat. 2 121.00 1800.00 8570.00 6610.00 37700.00 82.80 0.00 10.40 62.20 42.00 734.00 107.00 4614.00 4.14 367.00 42.40 3280.00 182.00 303.00 807.00 1040.00 8000.00 0.43 28.80 0.12 81.10 88.80 30.60 508.00 17.70 22.20 44.80 9.10 545.00 700.00 351.00 51.00 12.50 15.30 0.30 .0.38 0.30 226.00 17.30 61.50 21.00 0.36 4.38 2.00 4.80 Dat. 3 337.00 1760.00 8520.00 8880.00 42100.00 2.76 14.30 17.00 2.78 67.70 78.00 103.00 4623.00 5.00 382.00 40.80 3900.00 511.00 435.00 038.00 1850.00 7010.00 7.10 27.10 2.00 00.90 101.00 30.40 812.00 36.10 31.50 42.30 12.30 631.00 042.00 348.00 42.00 0.51 a.eo 0.83 0.29 0.83 273.00 17.10 71.10 18.20 0.24 3.94 1.75 3.38 Mean 230.00 1703.00 6643.00 0197.00 30800.00 22.20 12.10 22.10 53.20 20.00 104.00 4877.00 3.3 383.00 44.70 3720.00 300.00 380.00 030.00 2083.00 7270.00 2,83 27.00 0.72 73.50 98.10 53.20 810.00 29.90 43.30 12.20 813.00 776.00 335.00 44.50 10.10 15. 10 0.46 0.59 0.46 232.00 18.10 64.60 IS.30 0.28 3.90 1.82 3.88 Std. Dav. 108.00 123.00 172.00 794.00 2281.00 35.00 3.03 34.70 12.00 108.00 3.06 00. so 1.30 3.61 5.67 384.00 182.00 74.80 43.00 295.00 641.00 3.87 0.40 1.11 15.50 25.10 11.60 11 .80 8.11 1.16 3.05 60.80 144.00 11.50 6.48 2.18 1.81 0.34 0.44 0.34 38.40 1.51 5. SO 3.12 0.09 0.46 0.16 0.81 GENP 011232 B-3 784043 Supla Lab II KYSDQM 13 HYSDOH IB NY5DOH TABIE B-l (cont'd.) Coapound TOTAL TCOF . TOTAL FNCOF TOTAL HXCDF TOTAL HPCOF OCDF TOTAL TCOO OCOO 2.3.7,1-70 2,3.7, 2.3,4.1-TCOF 1 .2.3.7.a * i.2,3.4.a-m csr a .3 .i.7 .a-w co p 1,2.3.4,7.a l,2.3.4,7.f-KXCOF l,a.3.T.a.9-KXC0F 1.2.3.6,7,8-KXCOF 2 , 3 , 4 . 0 . 7 , 6 -HXCDF 1.2.3.4,0.7.a-HPCOP TOTAL TCDF TOTAL PHCDF TOTAL HXCDF TOTAL HPCOF OCDF TOTAL TODO OCOO 2 , 3 , 7 ,8-TCDQ 2 .3 ,7 .S 2 .3 .4 ,S-TCOF 1.2.3,7.a 1.2,3,4,1-PHCDP 2.3.4. T.a-PfICBF I . 2 .3.4.7.a 1.2,3,4,7,9-KXCOP 1.2.3.7.8.9-HXCDF 1.2.3.6.7.S-HXCDF 2 ,3 .4 ,6 ,7 .B-HXCDF 1 , 2 , 3 , 4 , 6 , 7 .S-HPCOF TOTAL tcof TOTAL PNCOF TOTAL HXCDF TOTAL hfcof OCDF TOTAL TODO OCOO 2 . 3 .7 .B-TCDO 2.3.7.a 2 ,3 ,4 ,9-TCDF 1.2.3.7.B 1.2,3,4,8-PNCOP 2.3.4.7,1-PMCDF 1.2.3.4.7.6 1.2,3.4,7,9-KXCOP 1 .2 ,3 ,7 ,0 ,9 - KXCOF 1 .2 .3 .0 ,7 ,6-HXCDP 2 .3 .4 .0 .7 ,6-HXCDP 1 . 2 ,3 ,4 ,6 ,7 ,6 -HPCOF Dt. 1 1300.00 10300.00 10900.00 2000.00 ISO.00 0.03 1.04 0.03 410.00 1104.00 1930.00 4113.00 102.00 1030.00 306.00 090.00 0.33 1.72 1.76 0.31 0.46 0.09 3.02 0.09 0.20 0.37 0.45 1.26 0.22 0.23 0.21 0.28 0.13 0.34 0.36 0.63 0.63 0.61 20.30 0.60 0.13 0.66 0.34 0,77 0.21 0.21 0.32 0.63 IXt. 2 933,00 10200.00 10900.00 2020.00 117.00 0.42 0. 9a 0.02 349.00 1032.00 1920.00 4432.00 177.00 1030.00 404.00 378.00 0.38 1.B4 1.14 0.23 0.30 o.oa 3.36 0.08 0.24 Q.39 0.31 0.80 0.11 0.13 0.14 0.08 0.18 0.60 0.32 0.23 2.40 0.19 18. ao 0.12 0.18 0.18 0.09 0.71 0.19 0.19 0.20 0.23 Dat. 3 1100.00 10600.00 10700.00 2100.00 MI .00 0.96 1.01 0.09 395.00 1133.00 1970.00 4382.00 173.00 1000.00 398.00 700.00 0.31 2.14 2.43 0.19 0.24 20.00 3.80 20.00 0.23 0.31 0.41 1.16 0.26 0.27 0.07 0.09 0.23 0.33 0.71 0.21 3.80 0.10 17.10 0.09 0.13 0.14 0.11 0.69 0.16 0.16 o.ia 0.21 Main 1184.00 10300.00 10700.00 2040.00 166.00 0.40 1.01 0.03 408.00 1098.00 1940.00 4409.00 171.00 1020.00 389.00 689,00 0.41 1.93 1.76 0.31 0.33 6 72 3.40 6.72 0.22 0.38 0.46 1.01 0.20 0.22 0.14 0 13 0.19 0.49 0.60 0.36 2.28 0.30 16.20 0.27 0.13 0.33 0.16 0.72 0.19 0.19 0.23 0.36 Std. Dev. 283.00 300.00 200.00 32.90 4.38 0.47 0.03 0.04 68.30 42.10 26.30 23.00 6.14 17.30 20.40 11.00 0.11 0.21 0.63 0. 17 0.1) 11.30 0.39 11.30 0.02 0.04 0.05 0.36 0.09 0 06 0.07 0,11 0.04 0.13 0.10 0.25 1.39 0.27 2.23 0.29 0 03 0.30 0.14 0.04 0 03 0.03 o.oa 0.23 784044 B-4 GENP 011233 -Sup] Lab Compound . Dt. 1 Det. 3 Det. 3 Main Std. Dev. o^*o*oo oooro *occ >oo* aoifo 'o*'o *cenv* )t -'o* nnocooor> **pdtD O n r) ti o c **f*m ov am* 0 o o o H O "omo * P CM 0 non n O IMO on-* QN Q9 A O O 1O v) o n o a ^ o a co Oa3 O 00 91 o NO 000000000000*0000 o o o o o d i o o o o t > ( B r )0 ^ t** o i MoOe <oto-*d0rO- o * H r*O *t***^o^^c*o * v i 0 t>iiN Fe|t*nn* r - r t P) t P) " - ee N So o o o o c t a r t Q O Q to O O o o o o r * o o O o O O O ve f - f d O O Q O O t* O O_ O_ f t *e C_ H --0 ,( IoO h O * O 0 h" --0 --0 "0 f d Oo *t* Oo J *d * 0 f-0t-M0rife(tr-0*v o o o o o o o o o o o rrtO O O O t'N t'P JO O tf n n mM OoO --oo Oh 0 fe 0 gU S*SX *5 -S S 8 -8 *Ss K > Q it * a. a i % h0h *h<j <H*j<H<J M*Jp uh4) aa ht.9.h.f..ir..r.t.t.*.f.i..fn..i..t.n.f..t.n.f..*?..r.o.t. E P hO h o N ^ * h -d | J * OO V fj*^ n u* io fl N k h (b g -* -q a o *rth.^L >U (jX - O X X K I n --u X X X O X I I * hkk a N O , 0 0 h 0e 0 Q Q Q O 4J ............................................... a a o a UGU o h Q h 0 h h ou U U U 3B K I L U I ........................................U Z ^ILX X I- 0 M e * h O h k S X r4k k k V -a o o "i o -3 a r (L C A D u i-- M - * h (fi o J J J J . J M..*.r.).*.f.).(..t n..* .n rb t^ Q ^ a n n n n rtc trtn N P P b O U p U .......................... e . e h h h h O h O < tN fd N fe* d * N - *hJ- h4hJh h h o n n i t f l w N N n rl <0 i ci t O-----O-hH Oh Oh OU HO OO f t <' t *** * t .....f...d....-....*....H......*....d.... Svt i P LO ^oTorfo h- LO oaI GENP 011234 784046 co i o\ Q nj o LP ** ................................. a n -o<a4x0xxQ5itft tXC>i oa *dSnttJ ** -uM* OO x n n n ^ iM * SO DO XX X i U i * -I S s HHHH X*o XX Ka): nH nona oda* ........................................... n o n o o o | i l PNMMUNUUt ) HO*4HH .u.....*.....u.....u......u.....*....i....J.....-....t....-....a.D >r* > r> r >p l...>..a...i....^...*. i4>4fitO D -j g a o a *4 ...................... n i a 9 *o m o P XI IXIX ft OX ft*-U o xa nXnx nXn a4i *- Xaa a - Oa XX X M. u i *n4 Xh Xm3 Xs2 S nono oa o X U *H *n * sa m M n h M m U 9 ^ OHMHH no do d N b I M M M U M U U O H O <-| H H H ..................................................... o > U tfU *U *M P 99 0^**1499 H ......................* in 9- 4- 494<Of t H ..............................i n ?439S950*93r4rM Hi j a * o O O xnnnM M 9 Xo o d - *- XXXM u 9 rXinn SH O OO X X -(I -* 4n * o IX 3 ?3 Ss " o ^ i* * M O ^ M^ o oo o o o o oo o o o M01 (fi lai ft* MMU N -I MO 9 M0 O M^ ^ M9 9 ft* O 9 I 9 O -I 10 -4 I* ft40*9^ H U9 *9ftO< 9O OOOOO ooooo UUU 9*1 f<t9^f9t* Oo Q ooo 9!?9MU fNtO 4O9 *^lO9 9U9m9 0V OOQOQ9000000 oooooooooooo u 9 -4 9 U9 0MN 9 me 9 O9 M M U9 * 0 O O 9 MU 0 MU M O MN A 0O 0O 0O 0O 9O 0O 0O 0O '' N0 9 nob M 1M9M0 *ft>M*^- oo oooo ooooo A m OCJm 4O O O N 9 0 19 0 9 oO oO oO oO Oo 090 9 99 SOO oo O ft*4P V MN M m 9 M9 4 O 9 O O O9 O oooooo * 0 tt * a 00*94 ooooo ooooo ooo ooo N9* UNMMNp(UM4 94 9 49 009 OP9 N*M0O0P9 99 N 0-4 0 O tt1* o ft*f1t* Oo o4 oO oO oO oU 4U0M A Oo AA NN MM 9 4 M9 0 ooooo ooooo * 4 M 00 * m 0 M9 9 O 4 O OO O9 u oooooo 404 oooou ooooo UU O <9 ooo ooo 9 N M 4 a u M9 0 900 it* * a * 9 4 0 4 N 4 CDH0 oooI 4__0___0 0 0 0 oooooooooooo 90 * 0 * 0M o4 OOoO)U O9 N9 M0 40 O0 0* *O *0 0 90 999 9 OOO A0 0 9 O 009m * 9 ft^ A *0040 OO O O U ooooo 0 mh m *N 9 U** ooo 4 9 0 AOOOOOM0 U o o o o j o *j o o o o o Std. Dev. Maan Dal. 3 Oat. 2 Dat. 1 ' Caapound Lab Saapia S o p le Lb 13 1ITR 14 IITRI is u n ti TABLE B-l tcont'd.) Caspaund TOTAL 7CDT TOTAL PNCDF TOTAL HXCDP TOTAL KPCOF OCOF TOTAL TCOD OCDO 2.3.7,1-TCOD 2.3.T. 2,3,4,1-TCOF 1.2.3,7.0 1.2.3.4.0-PHCDF 2 ,3 .4 ,7,1-PNCOP 1.2.3.4.7.1 1 .2 ,3 ,4 ,7.9-HXCDF 1 ,2 ,3 ,7 ,0 ,9 -HXCDP 1.2.3..7.B-HXCDF 2 ,3 ,4 .6 ,7 .0-HXCOF 1 ,2 ,3 ,4 ,9 .7 .0-HPCDF TOTAL TCflP TOTAL PHCOF TOTAL HXCDP TOTAL HPCDF OCOF TOTAL TCOD OCDO 2.3.7.S-TC0D 2.3.7.9 * 2.3.4,8-TCDP 1.2.3.7.6 1.2,3.4.S-PNCOF 2,3.4.7.8-PNCDP 1.2.3.4.7.8 1.2.3,4.7.9-HXCDP 1.2,3,7,0,9-HXCDP 1.2.3.8.7.8-HXCDP 2.3.4.8,7.8-HXCDP 1,2,3.4.8.7.8-HPCDF > TOTAL TCOF TOTAL PNCDF TOTAL HXCDP TOTAL HPCDF OCOF TOTAL TCOO OCDD 2.3.7.8-TCDD 2.3.7.8 2.3.4.8-TCOF 1.2.3,7.0 1,2.3,4, 8-PNCDP 2,3.4.T.9-PNCDP 1.2.3.4.7.8 * 1.2,3.4.7.9-HXCDF 1.2.3.7.8,9-KXCDF 1.2.3,8.7.8-HXCDF 2.3.4.0. 7.B-HXCDP 1.2.3.4.0. 7.8-HPCDF Dat, 1 872.00 279.00 34.00 17.00 42.00 9.00 0.80 103.00 23.00 83.00 19.00 8.00 3.00 9.00 970.00 9238.00 10598.00 1723.00 293.00 8.00 0.30 297.00 1115.00 1772.00 3602.00 174.00 1193.00 387.00 728.00 Det. 2 473.00 233.00 32.00 13.00 42.00 8.00 0.30 99.00 22.00 39.00 18.00 7.00 3.00 7.00 817.00 8389.00 9918.00 2196.00 82S.00 13.00 0.50 248.00 1039.00 1304.00 3711.00 187.00 1133.00 472.00 903.00 Det. 3 401.00 222.00 03.00 30.00 42.00 8.00 0.30 108.00 20.00 67.00 27.00 10.00 3.00 13.00 378.00 8002.00 10680.00 2284.00 374.00 3.00 0.30 8.00 1023.00 1728.00 3773.00 233.00 1137.00 309.00 884.00 Keen 302.00 250.00 83.00 20.70 42.00 8.33 0.30 104.00 21.70 83.00 21.30 7.87 3.87 9.67 721.00 7276.00 10394.00 2038.00 431.00 8.67 0.50 276.00 1039.00 1894.00 3783.00 198 OO 1162.00 436.00 839.00 Std. Dev. 80.90 27,10 17.30 8.14 0.00 0.38 0.00 4.38 1.33 4.00 4.93 2.08 1.13 3.06 217.00 1723.00 414.00 290.00 174.00 4.04 0.00 23.10 49.20 98.00 46 70 31 00 29 70 62.60 96 70 13.00 31.00 13.00 I. 00 3.00 1 0.30 1.00 18.00 3.00 31.00 10.00 1.00 3.00 0.30 7.00 1.00 31.00 8.00 1.00 3.00 0.30 1.00 16.00 9.00 31.00 10.30 1.00 3.00 0.30 7.00 1 00 3.66 0.00 2.32 0 00 0.00 0.00 0 00 3.00 2.00 0.50 2.30 2.29 784047 B-7 n-'RATP m 10^9 s 3m 3 i(rT TABIE B-l ( c o n t'd . ) ........ a>**>*> >.M.....U....M......M.....U.....M.....U.....U....QoHnD qH Ho Ho Hq u ( j u u *i ^ r r*r*r*r* m Nh h ^n m n m O ^O HdHH ..Mu.........*..U.........u..M........u...M.......(....j..M.....*..*.U..u.-.^..-.U4..>OrUi> oD rH-n**1 O>o rda>*Hrq*Hra>*Hr* r.N...U..r.M....M.r...M.r...4."..J..rM....?U**Ufo 8O> a? O8aH Ma 3MaH r* r r r r xffntlnOMoDt MI *nO H^ S..-xDg0*...i....xOSn^..>^i.....x.x.v..ntO....u..*.^..on......nox.n...^.ml-t>il-* * oh a *U 1 H r-i* T 1i x^x*n4 * ............ i od an oo nd*a md.a.....^......^...o..*...^.....aAimti nh i i i z *a x x x n --9 I X MM C3- - t o d - - n ^ imo m u 9 -o4 oa xT >X*7a} Hn on oa tnioi .*..............*.....^... a i -......i..1.o...4...o......1.o...........X`.g. * M dnO aI xX Xx xS nD -< u x no nan rMi - ^ i >u 4Q 4 ^ 4 U- *9 H0I> nh ti o xv xx xt oh nn n d o*4d"*o^ ^ 3 * 3* a 4 CID ?8 383 oo U *4 uO OO OO OO u M M M^ A N M OOOO OO O O O OOOOO ui li M U Mli OOOO OOOO OOOO <-* D *c -I M 9*0 OOO OOO M li MU M M MtJI M Oo NON 000090 OOOOOO A MN M O 10 U U * A m M|) ( | u mm m ooooo OOOOO U *-* U M* M - W UN<ltlti^H(IU 9 ooppopoooo OOOOOOOOOO 90 (i Mm (0 -I * tt OOOO OOOO 910 oo UO PO OO U U MM M -4 X N OOOOO OOOOO OOOOO a 9 099 OOOO OOOO OOOO Nm M -4 M O ** 9 OOO OOO hh *Nm (j 410 * li H A U N OU 000090 OOOOOO nm mo U * <4 O * 0)00-4* OOOOO OOOOO N h mw U h M ti 01 (RHMlRtt |0l RpU H U 4 OOOOOOOOOO OOOOOOOOOO OOOO OOOO N 4 * OO OO OO M M H u M * >0 l i 0 0 0 0 9 0*4 O O O O O*1 OOOOOOO MUWH U V 10 OOOO OOOO OOOO H gM 09 OOO OOO I0 H M W ( i U (0 h tt 4 a O U 000090 OOOOOO tH M A *J f i O U ^ M -| Mo ci O O OOOOO OOOOO H Il m * A* N 94000**909901 OOOOOOOOOO OOOOOOOOOO ^ U li O m *4 w tl 999 OOOO fi UO Aa Ho 00 OO li U H H 01fi U (0 N 0 0 0 0 0 0 14 O Oo O L-4 0000090 li NUI UH 0 9-1 H -4 (J O OOOO OOOO M H IH 4 0 OOO OOO 9 --4 OHNH fi O c (i 10900 *-- y <4 N -4 y O 01 U MO 0 MN Mu O 000099 OOOOOO OOOOO OOOOO H yM N Ny V A M y a 0 ti 4 O M 0 H O U l y i i *4 0 -4 o 0 -9009-*O Q 9 OOOOOOOOOO 0-100 OOOO ezixo a is ^o i yu 90 MA OO OO m 0 UMy l* N 4 D li AOUOU OOOO* * n yy m 9099 **990 OOOO OOOO u 9 0910 099 ooH h U N A fi 999009 O N 4 OU OOOO O Hi fi fi AU h 4 49900 0 O OO M OOOOO h my 0 *9 * OUUCMAOOIi * UIRUH40OU OOO 4 O O o O O y wm _ AH Oh OOo O OOOO Supla Lab 3 BATTELLE 3 BATTELLE 11 BATTELLE TABU B-l (eont'd.) Coapound TOTAL TCDF TOTAL FNCOF TOTAL HXCOP TOTAL HPCDF OCDP TOTAL TCOn OCDO 3.3.7.6-TCDD 3.3.7. 3,3.4.l-TCOF 1.3.3.7.1 1.2.3,6,l-PKCDF 2,3.4.T.I-PKCDF 1,3.3.4.7.0 * 1.2.3.4.7,9-HXCOF 1.3,3,7.,9-HXCOF 3,3.4,0,7,1-HXCOF 1.2.3.4.8.7.8-HPCDF TOTAL TCOF TOTAL PNCDP TOTAL HXCOF TOTAL HPCDF OCOF TOTAL TCDD OCCD 2.3.7.B-TCO Z.3.7.B * 2.3,4.8-TCOF 1.2,3,7.8 1,2.3,4,8-PHCOF 3.3,4,7.B-PKCF 1.3.3.4.7.8 * 1.2,3,4,7,9-KXCOF 1,2.3.7,8,9-HXCDF 2.3.4.8.7,8-HXCOF 1,2,3.4.6.7,8-HPCDF TOTAL TCOF TOTAL PNCOP TOTAL HXCOF TOTAL HPCDF OCOF TOTAL TCDD OCDO 2 ,3.7.8-TCOD 3.3.7,8 3.3.4.8-TCOF 1.2.3.7.8 > 1.2.3.4.B-PNCOF 2.3.4,7.8-PNCOF 1.2.3.4.7.8 1,2,3,4,7,9-KXCDF 1.2.3.7.8,9-HXCDF 2.3.4.8.7.8-HXCOF 1,2,3,4.8.7.8-HPCOF Hat. 1 37.00 300.00 3400.00 38.00 f 48.00 48.00 37.00 180.00 410.00 3300.00 63.00 1400.00 1100.00 20000.00 1700.00 3000.00 110.00 110.00 1200.00 260.00 390.00 1900.00 1200.00 10000.00 33.00 170.00 2300.00 32000.00 3800.00 22000.00 18.00 31.00 33.00 130.00 29000.00 2000.00 1800.00 1100.00 Dat. 2 67.00 300.00 2300.00 130.00 48.00 48.00 87.00 180.00 310.00 3100.00 130.00 1700.00 1300.00 4700.00 3000.00 4900.00 100.00 100.00 1300.00 200.00 410.00 1800,00 93.00 770,00 830.00 110.00 2100.00 11000.00 13000.00 28000.00 12.00 30.00 49.00 130.00 8100.00 430.00 240.00 4300.00 Dat. 3 86.00 460.00 780.00 340.00 44.00 44.00 88.00 190.00 280.00 680.00 340.00 970.00 1200.00 11000.00 680.00 2800.00 80.00 80.00 880.00 230.00 880.00 1800.00 780.00 8300.00 49.00 40.00 720.00 9700.00 11000.00 22000.00 0.00 40.00 26.00 110.00 3100.00 860.00 210.00 3100.00 Kaan 83.30 320.00 2637.00 183.00 43.30 43.30 63.30 183.00 327.00 2880.00 183.00 1337.00 1200.00 11900.00 1787.00 4167.00 98.70 96.70 1127.00 237.00 627.00 1767.00 691.00 3737.00 318.00 107.00 1707,00 24233.00 9267,00 24000.00 10.00 33.70 38.70 123.00 13400.00 1030.00 730.00 2900.00 Std. Dav. 3.31 72.10 2333.00 141.00 1.13 1.13 3.31 5.77 78.40 2311.00 141.00 387.00 100.00 7890.00 1172.00 1338.00 13.30 13.30 219.00 32.10 237.00 133.00 539.00 4660.00 461.00 63.10 860.00 24053.00 4839.00 3464.00 9.17 3.31 11.60 11.30 13519.00 848.00 909.00 1709.00 784049 B-9 FLD 004645 I Lab BATTELLE SATTELLE BATTELLE TABLE B-l (eont'd.) Coapound ..... TOTAL TCDF TOTAL PNCDF TOTAL HXCDF TOTAL HPCDF OCDF TOTAL TCOO OCDD 2,3.7.B-TCOO 2.3.7.B * 2,3.4,6-TCDF 1.2.3.7,B 1,2,3,4.S-PHCOF 2.3.4,7.B-PNCDF 1.2.3.4.7.8 * 1,2.3.4,T,9-KXCOF 1.2.3,1,8,9-HXCDF 2.3,4,8,7,8-HXCOF 1,2.3,4.8,7.B-HPCOF TOTAL TCDF TOTAL PNCDF TOTAL HXCDF TOTAL HPCDF OCDE TOTAL TCDD OCDO 2,3.7,S-TCDD 2.3.7.8 2.3.4.8-TCDF 1.2.3.7.S 1,2,3.4,8-PNCDP 2.3.4,7,8-PNCDF 1.2.3.4.7.8 * 1,2,3,4,7,9-HXCDF 1.2.3.7.B.9-HXCDF 2,3.4. 6.7,8-HXCDF 1.2,3,4.8,7.8*HPCDF TOTAL TCDF TOTAL PNCDF TOTAL HXCDF TOTAL HPCDF OCDF TOTAL TCDD OCDD 2.3,7.S-TCDD 2.3,7.8 2.3.4.8-TCDF I.2.3.7.B * 1.2.3,4.S-PNCDF 2.3.4.7.8-PNCDF 1.2.3.4.7.B 1.2.3.4.7.9-HXCDF 1,2.3.7,8.9-KXCDP 2.3.4.6,7,S-HXCDF 1.2.3,4,6,7,8-HPCDF Dat. 1 82.00 78.00 1300.00 1900.00 4000.00 Dat. 2 64.00 230.00 1300.00 2100.00 4800.00 Dat. 3 98.00 310.00 990.00 2700.00 4800.00 Xaan 73.00 206.00 1263.00 2100.00 4387.00 Std. Dav.. 20.80 117.00 237.00 800.00 493.00 20.00 22.00 38.00 880.00 34.00 47.00 330.00 sao. 00 230.00 140.00 28.00 48.00 20.00 23.00 41.00 740.00 30.00 23.00 330.00 370.00 270.00 130.00 28.00 44.00 30.00 34.00 37,00 640.00 38.00 43.00 730.00 330.00 220.00 180.00 3.60 10.00 23.30 27.00 39.00 733.00 46.70 38.30 943.00 300.00 247.00 143.00. 19.90 33.30 3.77 8.24 2.00 121.00 9.45 11.70 200.00 130.00 25.20 15.30 12.40 20.20 300.00 10.00 , 71.00 37.00 13.00 23.00 7.10 280.00 8.30 74.00 18.00 13.00 17.00 8.30 970.00 11000.00 14000.00 3100.00 130.00 380.00 8700.00 36000.00 2800.00 170.00 18.00 400.00 870.00 2300.00 4700.00 1700.00 620,00 920.00 380.00 900.00 2400.00 . 7200.00 8800.00 6800.00 960.00 220.00 7.90 53.00 31.00 27.00 39.00 1.60 610.00 6300.00 16000.00 4200.00 220.00 330.00 780.00 1800.00 4100.00 2600.00 930.00 1300.00 267.00 8.73 66^00 26.00 19.00 27.00 5.80 720.00 8067.00 22000.00 3367.00 180.00 18.00 377.00 890.00 2167.00 9333.00 4367.00 2783 00 927.00 41.60 1.12 11.40 10.80 6.93 11.10 3.33 217.00 2342.00 12166.00 737.00 36.10 25.20 62.40 321 00 1644.00 3866.00 3482.00 370.00 784050 .. GENP 011239 B - 10 Std. Dev. Mean Det. 3 o o o o ri n o o o Onmo <QO Oo oo Om n n (V IO ^ n OM ci o^M nht rO* o oa oo oo oo oO to QO OO OO OO OO OcrtiMfh-n roo* r0*> fncs' iwn roh Oc* oci nnn o apii rhc-odn^i o oo o o o oo o o ooooo ai r* Oo oO o o Nm o *4 ^r* oCi oOOoho ooonci OOOO O aft o) oQ oO oO n oo oo Do oo oo Oo Oo oV wt*(oOoo oo oo o OOoo oa ooooo oooooo o oh oO Oo Oo ri Det. 2 Det. 1 Coapound auH3mC^l* m rt taf b aUft. - * a a &. fQt. PO I I n*-ua *xK Kaci OOf* Xta I I * Og h Ic * h-o* P ^ r- V c < < < <m *<j o * - * - * * elEi am ta ta ta ta a q p o Uu Q u K X ta H ta X X on fi h* n, * -o a N ta rt U U n' re<ua XX XK a- z *i Q N ta ft O ou i *- * a h * e h oc u i ............................... H aoeh f* C j J J J j r -.r..-..n....^...n...n......r..>.... eie es sa .,*u t a h H a h O n P i f i n N N n n h ta X X n n h ta V ** Ntari o U Ua n ri u * XX XX o8H u oaVinf*t*>f"'zc"t-ai r^-."co-*oo- <_3 fa<ip| a >.' .h.f.l..f.i.f.i . ?-H aoO HfoaOcoCil.nM.^^n.Wr.rii.cei.ec.Ni Lab Saapje in o tcfTo- O ci ca .i 10.00 17.00 177.00 1*8.00 f*ovoooo<iai ONDO n n h od N N (? T4 *4 O -4*4r*OOOCTOtm* ronoO BonoA O A O h U) ^ NNi Hi nna OOOOOOOOOOOOOOOO t-oooot-or*ro NHOffhOQONh r>n n h -t lf> oo oo oo oo oo o oo oo oo oo oo oo ro- oo oo o ^ ffl o h Nrt OOtl b h r>n i* nnD Nd^ dannn f ot <H NMh,>m4wipn*) 1i1 o oo oo oo oo oo oo oo o ooooooo oo oooao oooooooooooooooo oooooooooooooooo piNion^h-OMnHKvnaffri rt p i iv n n ooo o o nn*om otnoh*f)aa o on ^o tei ^ n h r * >4 n ici ali oa oo oooo n Vi ^ mopfifi nfntapn*iaafMoi OpoipQiOr*f*pli fn.fM(.Dv. DI-,O_inVO, t^.f*V. AO_ oo oo oo oo Oo Oo OO 11 <vi n a * o a m pi pi o o o o o ip* r*o >*a^ai nfi Of> 0aaNpin oo oo oo oo oo oo oo oo oo oo oo oOoOoooooo na ioOOafoifiOOOpOi oO*p-oi(Oi fP)i PiMNi (VO p* Pi <- ^) N*pfpi i-fi Dinpro'i* * r- 8 .. 3 s <; * E* r-' . au aa X Pu eH* - fi i. mi n- f i a ^ h uQ *n\f*8 8 Sx Oor*-i - i 8oK, a aSI cI SIc -cr- u h * a f* o;r- t* o O h ^ M 3 a h *fi a fi n ii a fi JB 6 8 ^*, rr* ^! r " p ^, h P o Wpi NHpi i p| *i u*a*te* ote, OU Bu Qu Qa 0h3aKKX*X *Ph Qtacfii-<Nn-b**XoOa.,pN-iQaXXaUl.aaUxXcibaUXMoI.U*&hl* , UH00<10-ha.....V^......o....M....r......-..O....r......c......a.c......tOe,SCgh ua 1 j j g j 4 h i * n * n f ) o f f ) j 4 hph4 Hhp4hh4phhDO4o Hhuu. OQ<offalluPN*.l.-..f..i.*l..f.P...l...t..-....i...i..v...P....i.....iv.....p....i.....Vi.....f.....f..l....i.r..N.*.............0hh4 04hh ` h8 ^*- fT te c*f.<n*v t en- apai uoauouu xaau. n** *oa xx xx xx d oara* Xcl0 a bi l *h au ............................ aUh h0 * 0 h0.i0.*.ha..h0.h.>.0.0.e J...h.h..o.v.n.n..r t.v.n. h0hOa..f..nl..N..n.-..i.n.N...f.p.i.i..p...i.P*i fol p*ni 16.00 TOTAL TCOf XADIAN EXT1 C \l ioo ^r oo n. GENP 011241 RADIA EJCTl TOTAL TCDP 147.00 102.00 123.00 144.00 19.70 OOOOOOOOOOOOOO 0000000*0400(100 (i o*o1h0 O o o n *o*(o1 Oo rNi NOf()ib n oto oc o 0 OOOODO C OY OMO O O Kltnooooooootaoioooo * N*nOr (t1iH O o o f i o i h DN u pi m ^o n 4 OOOOOOOOOOOOOOOO n0u30i000000p)1^000 tO- o* n o o o o o mf^* u ) - * 4 0 o o OOOOOOOOOOOOOOO ooooooooooooooo h o Oo a OObbbOOOb rOt bO * *< O r* n o o N O N o n o M o ri o 1 *.040-*0Cft 4 rt OOOOOOOOOOOOOOOO oooooooobooor*ooo bnONOOOP0O0N0bHN 0 M o 0i a non * o N ^ n 0b o n n OOOOOOOOOOOOf - OOO oooor-ooooot-nvooo n0OnnoON0O)Onr)O<1 0 o n * > * * * > r* nn oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo ObOno<iM0aNbc4O b b o a h no00*ooo 0 0 0 c- i 0 n n rt o r* 1 c M < * o o0 o0 o0 0o 0o 0a 0o 0o 0o 0o 0o 0o 0o 0o 0o 0o i0 iei bA Oof<*9 no n 4 b b f t i A i a o i *0 n r* 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0 o0 n*n on n o n h ^ b0 o^d in h n i i n n c* S oO oO Oo oQ oO O0 O0 O0 0O 0O 0O 0O 0O 0O baano4^D0Cn^ao h oo 0 4 f ) 0 r t 4 t < n 4 nb n n 0 *4 n n n r ri n b ><f0* 0 O0 O0 O0 O0 0O O0 O0 0O 0O 0O 0O 0O 0O 0O 0O 0O 000ObnonoNio*oan o ^ 0 o r* i n * n N * non b b- hn b oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo 0O0nbnonb4onnnn b^ * b ^ r* ci n So o o o o q o o o o o o q o OOOOO O O O O OO O OO n rt a n o r t 0 b * c * b r t O n M wm n r t t i b b n o n (i rt ri b o rt 0 ri 1 n N N b *4 A * 0 oooooooooooooooo oooooooooooooooo c* f * r ^0 Q n o n 0 r4 ai~*br-r) bn0 4 b - N O nnnnrin n n 0 rt -*0 b (*0 0o 0o 0o 0o 0o 0o oo OO ooo ooo o o o o o o o o o o N a o n n n o n b 0 e m n n iin n n < 1 * -1 1* h n or* n U. _ gSS3 8 O Q Xp H8 " 0T h *oI nrift.rtik6 8b k& XOu O *~eKftS sI sI *i Obi S, A 0 b ti. SU ............................f t p a o h I0 0 b 0 b b 0 g li a.u uaa b b V b O H D. X H riftfthU --- Gp ua uo sa ' X(1 r t ( J N I ft B O 0 0 b ft0 0 b 0 b b 0 bb j u b b f* n n *rt i S8 * "I " *!" *! b O b O f i Ci r t f t r t PAMNr t << b. <j j J J J b b n * n n n 0 n sb H HOb bpHOOUbh--Oa n n ri NNrt n n Mn n N^rt^N* n * f81t.- a, 0 b *Cl ftW 0fntl Qf t inr-tMokUKofUt<MaI* KuI ft k ft aOnn* rt (Xt>Lft0xi xl l0%*bo ft,aoQ a o .............. guu^j g H * V0 b 0 b b O b ft X X H 0 0 b b b O 0 * 0 0<JJ j J< * J a b..b....0.......(...i..n...n....*...n... ^ S f P ^ 5 M - ^h H h H Q H a n 0 r i n ( * r i r i 0 n O1CT0) ff I CO G E N P 011242 3t4. Mil *4 *i 000000^)0000 O O O o o< o oo oo **oIDod t*l oo 9 O,, w* f l Hn*O lhI'). M ri o i OCflOl-rtOrt HHnno w * 0 o 4 o4 V n n n e ^ n o r t n r i o o n r t r t H Main 0*1. 3 0t. 2 0*e. I H 0 r t hO n o o r) nn Q* dp r* *o h r tQ O f <"* fit)H--oP 0 r O t CM* M OOOOOOQQQPPPQO ~oooooooooooo 9 n n ri oooooooo oooooaoo' r n o M *f)o o rto n N n o rt ooooo V O OO o n o n c 0 i choct n< fiondo OOOMOMDI * NVON 0d*4^0OO Oi0O n t*0o n op* 1 On nOmP mOmOmOn Dc i O O Oo Oo Oo Oo Oo Oo Qo Q OoOoOoOoOo ft* o a a HIS nm ft ft f t ft f t f t U f-C8f t*^SSu5uAi ^ ESSSo auO re- &Ei o...o.I...I...Io...*.^.. h t ftfthQr-ho pf tupuaua Qu N * f.t.f.t.ft..O.f.t.h H t ......^......r.-...i.'*o Ka n n1hft_nn!c-cnf*ot M*n -i.f t. g<'>j3Sj<^3j&fatj&3 au<j- <t-l-nnN^n-NNrl<10< ft. 8 ouhftpOai- fIt * ft ft* frt o- M a, &ft uO * - a O o a. ft ft ft ft P Q pU uK uK u& H ft S ft - - O i xKxKxKq I O- E I I * QM4 f t 0 Of t Ch O (J . -* * PP hI * ft..f.t...r.>..f.t..H...h....f.t h ftf t hr- t - 0Of t 5Jo oJ< g Jo3 au, 3*48...h.r..)..hn...r.n.<..r...1..No..r..o.4*..Nn..f...t..f.o.*. -H H E UXJ E 3< i i 2O ** tf> ft> Compound Su p] * Lib GENP 011243 ion VW;U0cLHHo aii -0J0 oaaii .......................................n o n d o o o M fO N M U M U U B ^D H H iiH .....................................................O > *9 > > W> u^U u^-t-i r* r r p r ............................. i o^i -4c*-4Ao* h ...........................i n Hn *Xp sx 1a9t Hn b n n no o 000*9 - Ill - C3 9 X X X O *- U x*I 9nX0n0nX0*X** ^Ou- f*-t o - in u 9 9 n u n *9 *U* -* *on4 * * - - - . . . o o nOoH oHdH Q^ .^...U....m....M....m.......i..u...u.. u....D. H DH >HH> >H U*vuu^u*4H r* p r p f 1 aoiM^^-lOlV ....................................... i -o- h- h* (bh1o g h n H 0 V ( Onu * MD -&i SXl iXXtXX a0ne~. w-- o xob0n0noM-* D M* - r t 9 Tl T] W 0* uo - 9 U ** HO H n o 0 X XT) H t S Q nono 000*9 *9*9*9 8^3|3gg jj MUMMMUMUU 0 H 0 ^ H H H ...........................................a UUUU*U>4H r* *aa-4*4<49CI B '-I l i 9 9 ........................... ri -M gM9I xMtl tlxM 0l j | 0nXI-*-M* Oo xnnnM ^M * 7 0 0 0* * ri 7 *9 *9 M U o od^ S 3 ! H 0 in o i o u "9 U**> *0* H0n *9 3 Ts *3'* m *-*- ** MU M B I k> M * O Oj oO a oMu ^U^HM* OBbU sss:o o o o o oo o oIB*OO*U- OU P U B ^ h O m O N I oooooooo Oo oo oO oO oO oO oI o oOooOoDoOoOoDoO HK U M ft) M * ** 0 MU ** - 40 *U0* 0U0m B M ^ U H U O U O U ft> O M B o ooO ^ ^- n BMg MC U tfOt I U BU *t lNU^t lOl *D* O( l Ol M> NO( I9 * 0 Uh1l o o O O OO O O O O O O O O O O oooooooooo MM NM ,----- , ^ ( j M M 0t*o*O<ftOUA H A f t U O H O H O U O U O O O H H *- M a - g U A ' (IfUt(JCIUOi(DftON*OlftUl>O)OBIOOMI UDI WM*- M m m m U B O B OO ^ OOOOOODUOOOb OOOOOOOOOOOOOOOO M mHOHI a MMu a OM U cMm A Ng fnil aM bO**OOOiUft)MO^(ftUlMag OB tt t iggOOOO OM MM g gI ^OOUMUOOB UH tfOMl MNM0fi)UftMM 0<*0MftHN o a> B O M A M M O O O ( f t O l M # * ObBD^Baoooonou o o H -* B U M N* Jat O * M 0 **HgH hO O O O fo4MH o ~b Ou oO oUu^ OnOoO- O4 *O( O oOoOoOoOoOoOo o TABLE B-l (cont'd.) RADIAN EXT2 TOTAL TCDP 1407.00 1S3S.00 1474.00 1473.00 S3.SO oo oo oo oo ^1 o0 *^ 1o0oNoooQnh ro) of )oh >0)M0l1p4hN N N W r0* o (onoo) oOoooooooonto>imo tnfoi &o iMr ts^i oo P it '(n in Q )O o O '-'T ^ n N o h OOOOOOOOOOOODQO ooooooooooooooo 0)(DO*4OO*O0r)Oi --*f* Mrt h pNa N N 4 -----------r- n o O O r> O t- N P) OI -H N a r m ooooooooo o ooo ooooooooo o ooo o f4 *>9 i Mn *O<^ n ^ r FttAo MN oND n Mnr P)) A0 0 `0 rt n Kf) 0 n o n d * 4 v o n 0 fof*tir-MV?ronr- OOilOO-oO0Ot-0fl000 o * N o M *o n tt *4 ci n i " ooooooo r9- 99 m9 0o' a oa ooo OOOOQOPOQOQOOOO oooooo ooooooooo 0 HPhO v4O 0 O i n n i-N 0 n o h *4 h h n h w n * oooooooo h N e rt t .. i ? * o0. oI t r 0. * a Otr)0*( 4'080u0Btu - - p MMM I ^ r) *4 u X X z a 0o O_ Op n * iS . *.d...i.D......t..'. Ji g H 0 0 Q h ^ 0 h 00^^0hC0* J40 ...h.M..i .t .n .n.n.^..n Ha h g n n n r ) N n ( n c eg ^ HO NN* 404p440r f auH oy- ^ n 0* Qi--(0M--* O0, --C*-auUK*ua0X 0 uaK oO xa. 000 Qa Nn- -4 0UX 0AI 0XI 0IH- 0a p a o u < ........................... QU u U a H 0 0 H 0 H O EKQ. U I *-'** H 0 X H 0 H h h 0 0 V H^J f4J*<H^ <UH0J<H Qo Hn..n.h..w..o..n...p...ni...o..N..n...n.t..rn..i. E HhHOHOrPi Ft ( Fi r t p- ni ' 4p o p o o u ..................................................... QU Otf--(O-*a0N-- no 04aKU0aKU0aXU XX X uX0<0 0 0 0H 88 tn nn ^p 3 t h H?j gu. jh< Qa ho. h n *nn*r i N n rt n N ee ho-QNN**N^Fi-rH co ot o \P ff "sf- <N *-- 4 o - i m TABLE B-l (cant'd.) oonQ toaooom ocnttm A h <0 H O o O ' i H O l T l O n H i n M o O ti O rt O O O C p< ** v 4 0 M O H O ooooooooaooooooa hO O O hO O O O O ^ O O aO O n0 r(f-1 o4 O r N irt o N n 0 0 0 h *4 C4 n <m k0) O n Q hP4 r n < N N0 *4 oov^ noooooovoooo 0 0 < n 0 v > 0 0 0 0 0 0 tf) 0 0 0 0 0 0 0 *^ 0 0 0 0 0 0 0 0 0 0 ^ oooooooooooont> ovo o o ^ o o a o a n a h tio n o *a Oh N io o o o o in v ir n * v - i h n w d ^ <n *-44 c- en r* oooooooooooooaoo ooooooooaooooooo c a ) n N o o o o o * r 5 n * o o oO 1 Ma -( "* * n r t* r * io r t fl O N-4 ipo o o^4 t ' O oooooooooooooooo o o o t^ h o o o o o o n o oa o floo^ h nonfinrirtofirtfl o --* ^ 80 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ooooooooooooaoo n o n m * * h4 rn o o n ^ n o4^v t D n d i i n H4 O on M oooooooooooooooo oooooooooooooooo q ft a o n D 0N O 0 *4 h vfirt<B*r-a* oooooooooooooooo OOOOOOOOOOOOOOOO r t n*4o n t ' m w no n n o n -4n n n o o <h. oqq o o o q po o o o o q o o OOOOOOOOOOOOOOOO 1* 0 0 0 0 N * r* n 0 0*4 0 0*000 rtrtion 0 00 0n n ' oooooooopoaooooo OOOOOOOOOOOOOOOO VAN *M r t F 4 O 0 n n o o t* o 0 0 a rt Fi o*4r t 0 t*r<t` 0 * f f nrtnri*^ ort n <f-i 0p* * f t t - a r > oooooooooooooooo oooooooooooooooo n n H n 0a n n o r<4> n n n * r-4i n i i > -* oopopooopopooopo oooooooooooooooo v0 0f b0 4n 4M ^ n o i 0i f *O4 0h n o o N<* h r-n OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO 0M 0o oo 00v *c4 vh n >4 o*4n o 0h 0^ r f- tf tv *a0 o t -o 0i n n t f ( 0 9 t> OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO n n Po4 * Q0 n a4 n n n n n<P4n n f i **4 k8O2DSO& H CL X X 3 e i 8E1 *0: SVa rnV,*-k.-nik8 ak h8k8UI n-8*2i a- e 111- a r, 5L m at m t~ a f- ^ I- i- C H l-h|>ioV jo M - n o n f i i i |g| h a n n n n M n r i o N P i j n H n --n -- ki kt k . a a p quuu 8E5S 1 * R*- *T:a a O OtHJ o On*-* * --l ipi n* - uX * n*-U0KhxIiUxKtIokUIKxah,X*ou^E0 u h t 0 B...^..........r...*....h.....0... ftft.a f0t aft a uu u p P* n ft. 0- O* fi*aft fat fpt, Q O_ 0**-f pyxt - iUKk1OKx1U1MxX*I pp f i l f .t.0...0..0..0...|.*. h 88 i ^ r-' V XKft p ................ H ft X X p 0 0 t > f * * h 0 0 * j M .4ijj MHHP H J Qn nhr*hf'on *r..dn.on.tn.n*.nr.rt.* Hj< JH< J0h P? fJPt b4*Ja...nh...0.h..N.O...i..ti..r...t.f.n.t...Nn....0*...N... H p E P o h 8 N W 4 4 * 4 N H 8 8 8 8 8 8 8 4' rf - M- - - - - 5r t tWK h X U) 5 3 2< i i O IO^fr) 0N-0 <0 \r<N o i co TABLE a-1 C c o n t 'd . ) GENP011247 oooor-ooooooooooo oooor-ooooooooooo oooor-ooooooooooo oooooooooo^onono eonor)oooootfiointfi* ft n ^out vo ono- o o n O^t c^n H H-*i i o^H o<io loonooDoooOrftfovno n ^ H t f t o ^ o ^ D ^ n ^ o n r * oO oO oO oO oO Oo Oo oO Oo Oo oQ Oo Oo oO Oo oO n o n o o<4 n nn o n o c o o n n oo oo oo oo oo oo oo oo oo oo oo oo on oh oo oo FC^*r^t**tnnt t<O*>cOoDn-o o r^*>tr*M0t -n9u)>n--9t *^c4i t0^o^oMrt OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO #nMnh^^hnor toNnHor oi nn<oMnhnIt^UNo n ^n fr-t ^)f oooooooooooooooo OOOOOOOOOOOOOOOO n n h n r* n ri < i o n f o n n n n dN oooooooooooooooo oooooooooooooooo A ^o o io n D U ttc^i'-h in o n Ft h N 4 D O A 4 4 h 4 V p n to n F* M * fi n n r) -- >i n +* oooooooooooooooo oooooooooooooooo NOt-ononwN {iM n<^e Ot*hrtM fiO( 4b h nvn fi n n n fi n SOOOOOOOQOOOOOOO OOOOOOOOOOOOOOO d ti d d n r) r t d d n o o o n r ) r* oooooooooooooooo OOOOOOOOOOOOOOOO nc^UMonan h a n M i o "* o o - r) ^H oN cNn fi n n --* ^n oooooooooooooooo oooooooooooooooo mNO^ONOdann^^# r- md n ^n < oooooooooooooooo oooooooooooooooo r) d n n dn n *H- d t i i m e d n n r t oooooooooooooooo oooooooooooooooo MM H n ^ n n n nnn - ^ oooooooooooooooo OOOOOOOOOOOOOOOO o o i o o o o n o o o n r ^- oh o m * r> ^ p n m mo *. 5 B*- n (1 -1 I3t M,-a5ua 8_ Iir* 4 Xa*a at oI o1 t* 688 * o m*- 0 ) c*h * r* Illgggg<HJQa hnj dtj- 1 dd 0 d n d n d d n d o-n * N*oEWkat n - 8 s-fin. - ^g g U _ pt n. ^Do UpX UamUa au n 4&i .a.a.r* H (O h t* I0 0 0 h h h 0 0 * ...................- * j JjiJ J i * h d n ( in r i hHH EPS HO Ua h0 OU d n d d d d o i d N oo * ^ !- y n o**Nnk H c-( piJ\aUhnOia,X8 - p * KX < hlk. Pi n U * XXX a - X li* f4&0V0h hiOao ao i * * * g u u ^ g h e a h r* h X X h 00hr-h00 J4 <J4 4*j <j h. X*1a h..h....n.......n....r..t..d.......n... hH hHO HQ UdtiPtciddnfi hSPoPS' - liHrl-cin 00 i G GENF 011248 CD I-- CD -*l oo o4^ Ul CD MN NM U MbU U * u b) (J ^ P -4'-4 r* h r r r *4 4 -4i 9 Oft * <4 -4 a <b i tmi Xi o i X o *D 4 M nZ *--u 3 o at n Sn xo m SO D O n *)N u -I H isl US 4.N..b...N...^...N...U.>..M...U....U> UIn Ho On HdHd" Hq M*d y*uu u *u -4 -i .Ol.4..-4.t.t..I fit V * Hr> ) a O io 9 D t P> o - I > Z D zzae i MXX na * - w * xti na on on -^ H M- ** oa ^ ^ *M - p y** *B O*f4t 5 3S T M SS " 8 rfrrrfrf.-R 38333 M U M b W P M U U D4Df*lR U V'u (I u * U *%|1| f **f P *a*9ij4l ^4*-I 4<4i!iO 8*4 S n ftX jCt) o o p ^ y a THBS8r? iS B A r-V * uo ^ n *p PP PR N*>lOP M oooooooooooooooo 4 UN Mp u M p p u p M p g v*i:* UM 0ti OOOOPOOQOOOQOOOQ oooooooooooooooo >ft u4 (Pp mO OttOAO4 OP MO OUIPM9U0 O0 Oo- oO- oO- Qo Oo Oo Oo Oo Qo Oo oO oO oO oQ oO P M VO4M OMo oU aN opmMD* m *P (pi pO aO 4 u d u n u m * o O u q u m <i u o o oo o ' o o o o - M g Mf) MM 4a oMoP b i PH Op pi uMM 0 M 0Oo 0^ 4MMy 4(D pwpapNMMUoypa^Mbi OOO P oooo p mmm pm mp b u n r nm p* rMou a c w u o p a o - o m u a * o Q d p O O p Q O O OQOOOO ooooooooooooooo MUg Ow(t>Qgu*wNlBa4pMMgpopu Om V HBN) p A. _U o- o- o- o- D U_R P B I 0 h m _ O * ^ P * S M -OOOOOOOOOOOOOOOOOOOOOOOOOOO OO OO P R ^ U B t Pt p^ AM *p m i 9a P pm tt P SS o S S S o 3 o o o o oo oo oo m4 PwOuL>At0oM0BMm MPo UP U M U M P 0 (i M M OM 9A CO -I U h) M O tO A A M ^lU ttP O P tfP O U P p u o oo o yp b io o p p o oooooooooooooooo - i M * p y b ) | t j M o o oMo Pa M(J u o" OOOP0OAMOPOOQOP OO Pm o AOPOQOOOOm M A A O P A M P m O m C D A M ^ O O ftO B M O N fcR O O i O M p OUOBOOOOCJ OUOOOOO O O P ) M OM O m <4i) 0 O ttu o o oO U 9 O O O U ACPUBOOUJOOOO0I A 0.S8 SO.30 S1.00 SO.00 00.00 TOTAL TCDT RADIAN EXT3 TABLE B-l (cont'd.) o n oh ao oo oo oa oo ^n a O O O0 ^i oi * tQoOo Oo rh' oOino Ao oOo4)i o o I f) M I A O N f* O l f l r i V r t O O O l A O H N H O N Y OOOOOOOOOOOOOOON o o r- o t* h .O...h....h.... ^ _ -+ oooooaoo t-onorirtr*o nortrtnNrt o o rt n O* O* "O i Oo Oo OO Oh Oi 'O Or rt )Ohrf ti U) (4 G Q i 0 n hh h _ .. h OQ v h. M_ O__M, . veonn^moO - flO* W (7 O l r < C I) ( *4 <0 O i t l> o o o o o Oo o o B " W s *> oo oo oo oo oo oo o o O o o o O a O o O o O o O o O O *o oOM nth*rBO) Bon n ortn n 4 __O_ D O_ - r>Of-N*-tii^ o g oo ao o o- r- n fl Br) o n n o b n Ok r* n n i "* h N 0 N h9 nnn ooo n 9 n OrtD hntmftDtfoiOrftff0B)Nri-oO " o4' oa <H - Bo ne oo 4 b n o n nn ro)nhnOnhn nM BO rt n G Nh rt B B nm OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO Qa KOB4f ) ((4o nrto nhH NQ aIOrIVf Hrt O t* r> 4 oooooooooooooop 9 000000000000000 iriiOah ^OnaA ornt i ABNl Nr tinnBNfcJ Bn oh hO Or t dr t r t nBhnnvah 8 . 5 8 p: 8Ph*.iB fr Ik m4i fnd bN^hSiSk 3b yxa i S ni -8E <.g g 5 6 S 8 S h * B B *bW B EES III O4 1. J M...*...f....4...B...n...f....4..B... HOuQoh au n...n....N.....n....B......t..i...n.n n 8 *fa. I ifa. I* fa. U f a fa. f a apa UUU a o O r i * a fl a fl fl r> u .................................. ||- i a a r . a i > i . i o hSX h D o t-r-^t'o o * j P J J J i-hniPinntrt hM -H oi-Q nnnnN N K nN p o O p U O U ..................................................... P P h h O H G N N H N * 4 r 4 r4 N *4 far fa. fa. fa o p o ouuu U X X Su a p P JJJJ J Mh h H h oooquo H H hH O h 8 Xa |o fa. I 8 *- T H *4 (1 O B B* *o a o SCifafa. fa. O 4Bfa, rt^uuB g n4*- rt- pO(XL fl arK iKsfllMfl I i* S B BMA I* ^ B I *..*> flflhhihflfly hhn^nnnfrt O oCO h0-0 a <N oOJ I CO S u p le . U b 15 RADIAN EXT3 1S RADIAN tXT3 17 RADIAN EXT3 TABLE B -l (c o n t 'd . ) Coepaund TOTAL TCDF TOTAL PNCOF TOTAL HXCDF TOTAL KPCOF OCDF TOTAL TCDD OCOD 3,3.7,i-TCDD 3.3.7,fl * 2.3.4.0-TCDF 1,3,3,7.1 * 1,3.3,4,0-PKCDF 2.3.4.7,8-PRCDF 1,3,3.4.7,0 1.2.3,4.7,9-HXCDF 1,2,3.7,B ,9-HXCDF 1,2.3,0,7,0-HXCOr 2,3,4,0,7,8-HXCDP 1,2,3,4,0,7,-HPCDF TOTAL TCDF TOTAL PNCDF TOTAL KXCOF TOTAL HPCOF OCDF TOTAL TCDD OCDD 2,3.7,8-TCDD 2,3,7.a * 2.3,4,8-TCDP 1,2,3,7,8 1.2,3,4,0-PNCOF 2,3,4. 7.0-PNCDF 1,2.3.4.7,S * I,2,3,4,7,9-HXCDF 1.2.3,7.8,9-HXCDF 1.2.3.6.7,8-HXCDF 2,3,4,0,7,B-HXCDF 1,2,3,4,0,7,8-HPCDF TOTAL TCDF TOTAL PNCDF TOTAL HXCDF TOTAL HPCDF OCDP TOTAL TCDD OCDO 2,3.7,0-TCDO 2.3.7,8 * 2.3,4,8-TCDF 1.3.3.7.8 * 1,2,3,4,8-PNCDF 2.3.4,T.S-PNCDF 1.2.3.4.7.8 1,3,3,4,7,9-HXCDF 1.2,3.7.8,9-HXCDF 1,2.3.0,7,8-HXCDF 2,3,4,0,7,8-HXCDP 1.2,3,4,0,7,O-HPCDF Det. 1 *3.00 3.00 28.00 43.00 03.00 3.00 10.00 3.00 3.00 3.00 3.00 31.00 3.00 3.00 3.00 23.00 3.00 3.00 8.00 3.00 10.00 3.00 10.00 3.00 3.00 3.00 3.00 11.00 3.00 3.00 3.00 3.00 813.00 1263.00 2300.00 2413.00 4412.00 3.00 10.00 3.00 182.00 315.00 187.00 1408.00 64.00 70.00 100.00 1173.00 Det. 2 3.00 3.00 20.00 51.00 100.00 3.00 10.00 3.00 3.00 3.00 3.00 19.00 3.00 3.00 3.00 24.00 7.00 3.00 3.00 3.00 10.00 3.00 10.00 3.00 7,00 3.00 3.00 8.00 3.00 3.00 3.00 3.00 512.00 1119.00 2374.00 2382.00 4354.00 3.00 10.00 3.00 184.00 313.00 182.00 1432.00 06.00 75.00 103.00 1143.00 Det. 3 3.00 3.00 11.00 35.00 100.00 3.00 10.00 3.00 3.00 3.00 3.00 21.00 3.00 3.00 3.00 27.00 3.00 3.00 3.00 3.00 10.00 3.00 10.00 3.00 3.00 3.00 3.00 8.00 3.00 3.00 3.00 3.00 518.00 1334.00 2380.00 2383.00 4236.00 3.00 10.00 3.00 186.00 339.00 200.00 1424.00 73.00 63.00 113.00 1141.00 Mean 3.00 3.00 22.00 80.30 97.30 3.00 10.00 3.00 3.00 3.00 3.00 23.70 3.00 3.00 3.00 24.30 4.33 3.00 4,07 3.00 10.00 3.00 10.00 3.00 4.33 3.00 3.00 9.00 3.00 3.00 3.00 3.00 514.00 1239.00 2365.00 2380.00 4334.00 3.00 10.00 3.00 184.00 322.00 190.00 1421.00 a. 30 09.30 109.00 1152.00 3 M . Dev. 0.00 0.00 5.29 5.03 4.62 0.00 0.00 0.00 0.00 0.00 0.00 6.43 0.00 0.00 0.00 2.52 2.31 0.00 2.69 0.00 0.00 0.00 0.00 0.00 2.31 0.00 0.00 1.73 0.00 0 00 0.00 0.00 3.21 110.00 a. oa 2B.00 89.70 0.00 0.00 0.00 2.00 14.50 9.29 13.30 4.51 6.03 0.00 17.90 \ GENP 011250 784061 APPENDIX C PROJECT SUMMARY GENP 011251 784062 PROJECT SUMMARY INTRODUCTION --- During the past four years Electric Power Research Institute (EPRI) has sponsored a research program to evaluate methodology for the analysis of selected Individual congeners of polychlorinated dlbenzodloxlns (PCDDs) and polychlorinated dlbenzofurans (PCDFs) In dielectric fluids. This program Included the synthesis of native and 1sotop1cally labeled PCDD and PCDF analytical standards (1). Analytical standards and spiked matrix samples were prepared by a contract laboratory and then distributed to four other participating laboratories as part of a round robin study. These samples were analyzed blind by five Independent contract laboratories. The study was conducted 1n two phases. Phase I involved the analysis of PCDF's and PCDD's in five spiked baseline samples; for example, Aroclor-1016, Aroclor-1242, Aroclor-1260, tr1- and tetrachlorobenzene mixture, aged mineral oil, and four in-service dielectric fluids (1-4). Since validated analytical methods did not exist before this program was initiated, this phase of the program was necessary to assess any deficiencies and optimize the analytical methodology. Each participating laboratory used their best available in-house extraction and analysis techniques (1-4). After evaluating these results and implementing several improvements to the methodology, then Phase II was initiated. Phase II was designed to provide quantitative data on PCDF and PCDD congeners 1n selected in-service dielectric fluids and to make a statistical interlaboratory comparison of the analytical methods. The specific aims were (1) to determine the accuracy of analysis by each participating laboratory for a selected list of PCDF's and PCDD's 1n spiked samples, (2) to determine the precision of analysis by each laboratory for PCDF's and PCDD's 1n spiked and In-service utility samples, (3) to perform an Interlaboratory comparison and thus a comparison of methods employed for accuracy and precision, and finally (4) to measure the levels of PCDF's and PCDD's 1n a few selected In-service utility samples. METHODS Five Independent laboratories participated 1n the Interlaboratory study. Each laboratory was provided with aliquots of the three baseline and seven In-service utility samples (4). Also, native and isotoplcally labeled PCDD and PCDF analytical standards were prepared by one laboratory (4) and distributed to each participating laboratory. Subsequently, each laboratory employed their best GENP 011252 C-2 784063 available In-house extraction and analysis techniques (Table 1). Each laboratory added.jfJve carbon-13 labeled PCDD or PCDF congeners as internal standards to the sample. Some laboratories employed a 11qu1d-11quid extraction step, either hexane/acetone or hexane/sulfurlc ac1d/water, whereas others applied aliquots of the sample directly to a chromatographic column. All laboratories performed a purification step using open column chromatography (Table 1). For example, Lab-A used acid alumina, PX-21 carbon and neutral alumina chromatography, respectively, for purification; Lab-8 used LH20, basic alumina and addle alumina; and Lab-C used a combination of sulfuric acid s111ca/sl11ca sodium hydroxide followed by basic alumina and florlsll. Lab-D used a sandwich of acid silica, basic silica and silica followed by PX-21 carbon, a d d s1l1ca/s1l1ca and then addle alumina; Lab-E used sulfuric a d d Impregnated silica, PX-21 carbon and florlsll. Various solvent evaporation techniques were also used. Specifically, Lab-A used heat and vacuum^Lab-B used rotary evaporation, while Labs-C and -D used nitrogen blowdown. During extract analysis, Lab-A used both high and low resolution MS, In the electron impact mode. Lab-B> D and E used only low resolution mass spectrometry while Lab-C used exclusively high resolution mass spectrometry. All laboratories employed high resolution gas chromatography (HRGC), although different stationary phases and GC operating parameters were used. Calibration curves were generated over the linear response range of each gas chromatograph/mass spectrometry system at five concentrations. All labs used the same initial stock calibration solution prepared by one laboratory containing eight native and five isotopically labeled PCDD's and PCDF's (1). The samples analyzed in the round robin study are shown In Table 2. Two samples were spiked with a selected number of congeners at known concentrations Into mineral oil and Aroclor 1016, respectively. A third sample, Aroclor 1260, was spiked at known levels with these same congeners but It also contained endogenous PCDF's and PCDD's. Samples 11-17 (Table 2) were In-service utility samples that were selected to represent the various types of utility uses (5). In some cases the dielectric was Arochlor 1260, or mineral oil containing PCBs. Dielectric fluids from transformers, capacitors, or precipitators were selected. Some samples represented devices that had been In use for many years (Table 2). A large database was produced 1n this study. An example of the measurements that were performed for dioxins and furans by Lab-B on sample No. 14 1s shown 1n Table 3. All of the five participating laboratories reported either measurable values or limits of detection for each of these compounds 1n each of the ten samples. With the exception of one laboratory, each laboratory performed triplicate Instrumental analysis on a single extract from each sample. The mean and standard deviation were calculated (Table 3). Lab-E reported only one C-3 784064 G E N P 011253 TABLE 1. ANALYTICAL PROCEDURES EMPLOYED BY ROUND ROBIN LABS Step Lab-A 1 . Add standards 2. Extraction 3. Chromatography + Hex/acetone Acid alumina PX-21 carbon Neutral alumina 4. Solvent evaporation 5. Analysis ^ and vacuum HR6C/LRHS (El) HRGC/HRMS (El) Lab-B Lab-C + LH-20 Basic alumina Addle alumina Roto-evaporation + Hex/H2S04; H20 S1l1ca/H2S04silica/ NaOHsilica Basic alumina (2Xs) * Florisil N2 blow-down HRGC/LRMS (El) HRGC/HRMS (El) Step 1 . Add standards 2. Extraction 3. Chromatography 4. Solvent evaporation 5. Analysis Lab-0 + - A d d sH1ca/bas1c si11ca/s111ca PX-21 carbon Acid s1Hca/s1l1ca A d d alumina N2 blow-down HRGC/LRMS (El) Lab-E + - H2S04-silica PX-21 carbon Florisil HRGC/LRMS (El) GENP 011254 C-4 784065 Sample No. 1 2 3 11 12 13 14 15 16 17 TABLE 2. SAMPLES ANALYZED IN ROUND ROBIN STUDY Dielectric Spiked mineral oil Spiked Aroclor 1016 Spiked Aroclor 1260 Aroclor 1260 Aroclor 1260 ? 7 Mineral oil (150 ppm PCB) Mineral oil (100 ppm PCB) Aroclor 1260 Utility Appliance - Transformer (LCN)a Transformer (LCN) Capacitor Precipitator Transformer (Arc furnace) Transformer (LCN) Transformer (LCN) Service Life (yr) - - - 31 28 ? 27 8 7 20 aLCN = load circuit, network. TABLE 3. EXAMPLE OF MEASUREMENTS PERFORMED FOR OIOXINS/FURANS BY A LAB ON ONE SAMPLE (LAB B. SAMPLE 14)- PPB Compound 2.3.7.8-TCDD Total TCQO OCOD 2.3.7.B 2.3.4.8-TCOF Total TCOF 1.2.3.7.8 + 1.2.3.4.8-PNCOF 2.3.4,7,8-PNCOF' Total PNCOF 1.2.3.4.7.8 1.2,3.4,7.9-HXCDF 1.2.3.7.8.9-HXCOF 1.2.3.6.7.8-HXCDF 2,3.4.6.7.8-HXCOF Total HXCDF 1.2,3.4.6.7.8-HPCOF Total HPCOF OCOF 1 (0.5) (0.5) 8 297 970 1115 1171 9236 3802 174 1195 387 10.598 728 1725 293 Measurement 23 (0.5) (0.5) (0.5) (0.5) 13 5 248 282 617 576 1039 1023 1584 1726 6589 6002 3711 3775 187 233 1155 1137 472 509 9918 10.666 905 884 2196 2254 626 374 Mean (0.5) (0.5) 8.7 276 721 1059 1694 7276 3763 198 . 1162 456 10.394 839 2058 431 S.O. (0.0) (0.0) 4.0 25 217 492 96 1723 47 31 30 63 414.0 97 290 174 *() * Limit of detection. 784066 C-5 GE N P 011255 measurement on each sample extract. Lab-0, on the otherhand, extracted each sample .three times and then performed three. Instrumental determinations on each of the extracts, thus producing nine measurements per sample. RESULTS AND DISCUSSION Accuracy of Measurement The accuracy of measurement for specific selected congeners, for each labora tory and by sample matrix was examined. To gain insight Into measurement accuracy, samples were spiked with known amounts of each congener and the percent accuracy of the mean of triplicate measurements was calculated and expressed as the mean divided by the spiked amount times one.hundred. Table 4 shows an example of these data for just one chemical, 2,3,7,8-TCDD. These statistical data were obtained for each compound measured. The percent accuracy for spiked samples for 2,3,7,8-TCDD by sample and laboratory is shown. Lab-D performed triplicate extract1on/pur1f1cation on each of the samples which also provided an estimate of precision for the entire analytical procedure. The mean value of the accuracy for 2,3,7,8-TCDD 1s given In Table 4 and the range for all detennlnations 1s shown 1n parenthesis. For these two samples the percent accuracy 1s 1n general very good. Lab-E only performed a single determination and experienced a lower percent accuracy than the other four laboratories. Accuracy of analysis for 2,3,4,6,7,8-hexachlorodlbenzofuran congener is shown 1n Table 5. The percent accuracy of measurement for this PCDF, by laboratory and sample is given 1n Table 5. The accuracy of determination was quite good for four of the five laboratories. Lab-C reported more variable recoveries with a range of 80-3371 1n sample No. 1. Data such as these were obtained for each congener to determine the percent accuracy 1n two samples. With the exception noted above, acceptable accuracies were obtained. A third sample also was spiked at known concentrations with these congeners, but there were endogenous levels present 1n the Aroclor 1260. Thus, the percent accuracy could not be calculated. The median and range of percent accuracy across all the chemicals that were spiked Into the two samples 1s sutranarlzed 1n Table 6. As Indicated, the accuracy Is reasonable 1n most cases with a few exceptions where the range was quite large. The determination of the hexa CDF congeners by Lab-C yielded this ' large overall variability. Precision of Measurement Similarly to accuracy, the precision of measurement by congener, laboratory and sample matrix was also Investigated. The coefficients of variation for the determination of 1,2,3,7,8- plus 1,2,3,4,8-pentachlorodibenzofuran (not chromatographlcally resolved) by compound and laboratory are given 1n Table 7. In C-6 OENP 011256 784067 Laboratory A B C D-l D-2 D-3 E TABLE 4. PERCENT ACCURACY FOR SPIKED SAMPLES FOR 2,3,7,8-TCDD BY SAMPLE AND LABORATORY Sample 1 75 (68-85) 83 (68-96) 81 (76-92) 99 (96-100) 100 (96-104) 92 (88-96) 56a Sample 2 91 (75-103) 101 (100-104) 85 (83-87) u 96 (92-98) 96 (94-98) 94 (91-96) 72a aS1ngle determination. Laboratory A B C D-l D-2 D-3 E TABLE 5. PERCENT ACCURACY FOR SPIKED SAMPLES FOR 2,3,4,6,7,8-HXCDF BY SAMPLE AND LABORATORY Sample 1 92 (86-97) 94 (93-96) 1189 (80-3371) 98 (97-101) 101 (99-102) 99 (97-99) 97a Sample 2 114 (106-123) 123 (119-129) 2015 (515-3939) 100 (100-101) 106 (105-106) 102 (98-104) ' 83 aS1ngle determination. 784068 c-7 GENP 011257 Laboratory A B C 0-1 D-2 D-3 E TABLE 6, MEDIAN AND RANGE OF PERCENT ACCURACY ACROSS ALL COMPOUNDS FOR SPIKED SAMPLES Sample 1 Sample 2 88 (68--105) 96 (68-124) 94 (8-3371) 99 (81-114) 101 (88-114) 94 (67-107) 103 (56-116) 107 (75-135) 112 (55-149) 120 (12-3939) 101 (90-121) 105 (89-119) 99 (91-121) 114 (42-120) Sample 1 2 3 11 12 13 14 15 16 17 TABLE 7. COEFFICIENTS OF VARIATION FOR 1,2,3,7,8 + 1.2,3,,4,8-PNCDF BY COMPOUND AND LABORATORY Laboratory No. A B C D-l D-2 12.0 11.5 0.9 12.9 26.1 8.4 3.8 11.7 4.0 2.1 1.8 4.1 6.3 4.8 7.1 4.6 61.1 14.9 11.5 3.1 13.6 31-6 23.1 12.8- 7.3 2.2 1.4 1.3 4.0 10.4 16.6 6.3 15.6 11.5 3.0 2.1 6.8 2.1 4.5 7.5 17.3 18.4 0-3 0.7 1.0 3.7 2.2 2.9 0.0 5.7 4.5 ge^ 0112^ C-8 784069 general across all the samples, the coefficients of variation (1.e., precision) were qtHte low, Lab-0 performed triplicate extractions on each'baseline and utility sample and then performed triplicate instrumental determinations on each extract. Good precision for both extraction and measurement were observed. Another example 1s shown 1n Table 8 for 1,2,3,4,6,7,8-heptachlorodlbenzofuran by compound and laboratory. For this particular congener, a greater variability was observed amongst Labs-B and C. The median and range of coefficients of variation across alj_ compounds by laboratory and samples are given 1n Table 9. A small range in the coefficients of variation was observed except for Lab-C which had relatively poor precision. Components Contributing to Variability A more detailed statistical analysis was performed to determine the contribu tion of Instrumental measurement, method extractlon/purlficatlon, and sample type to the-total variability of a reported value for a PCDD or PCDF congener. The variance components for a set of measurements made on 2,3,7,8-TCOD across all except two samples, samples 15 and 16 (most data were non-measurable values), are listed 1n Table 10. The total variance and the percentage of the total variance apportioned to either the sample or the Instrumental measurement step are shown. The greatest variance 1s attributed to the sample type as compared to the Instru mental measurement step. Lab-A, as compared to the other laboratories, exhibited a higher percentage of variability for the measurement step (14.6%). The remain ing three laboratories exhibited the major variability (99+%) from the sample type. Table 11 gives the variance components for 2,3,4,5,6,7,8-hexachlorodibenzofuran across all sample types analyzed. Again, the total variance and the percent of the total variance for each of the laboratories are given for the sample and the measurement step. Except for Lab-C, the predominant variance Is associated with the sample type. For Lab-C, 30% of the variability 1n these results was associated with the Instrumental measurement step. The variance components by congener for Lab-D are depicted 1n Table 12. Lab-0 performed triplicate extraction on the utility sample and triplicate Instrumental determinations on each sample extract. Thus, It was possible to apportion the variability due to sample type, extraction, and measurement. The largest contri bution to variation 1n the reported values was associated with the types of sample or sample matrix analyzed. Variability resulting from the extraction steps was smaller while an insignificant variation was attributed to the Instrumental deter mination step. The measured values between each lab were tested to determine whether they were significantly different from one to another. The Levene's test of GENP011259 784070 Sample 1 2 3 11 12 13 14 15 , 16 17 TABLE 8. COEFFICIENTS OF VARIATION FOR 1,2,3,4,6,7,8-HPCOF* BY COMPOUNDAND LABORATORY Laboratory No. A B C D-l D-2 4.0 11.7 4.1 10.3 9.9 21.1 1.6 3.4 10.9 20.4 12.1 17.8 18.7 31.6 11.5 60.6 53.4 23.9 96.2 76.1 144.9 58.9 36.9 60.9 39.9 29.2 9.7 3.2 1.4 17.6 14.9 20.4 21.7 57.4 14.9 5.1 4.3 5.4 0.7 5.5 7.5 17.6 6.7 4.8 0-3 2.4 6.3 5.7 4.0 4.9 1.2 10.3 1.6 TABLE 9. MEDIAN AW) RANGE OF COEFFICIENTS OF VARIATION ACROSS AU. COMPOUNDS BY LABORATORY ANO SAMPLE Sard It Loartory 1 23 )1 12 13 14 17 A i l . a (4-16) 11.2 (3-40) 3.3 (1-5) 8.6 (1-47) 19.2 (2-59) 15.5 (3-33) 2.9 (1-57) 4.3 (2-201 S A.2 (0-17) 2.2 4.6 (1-30) 14.1 16-46) 6.6 (4-20) 17.6 (4-39) 12.6 (1-47) 19.2 (2-28) C 41.5 (11-159) 13.9 (2-87) 29.8 (8-145) 48.9 (9-121) 23.9 (5-57) 31.2 (10-62) 30.5 (7-125) 22.2 (2-531 0-1 4.8 (1-27) 2.4 (O-S) 2.7 (1-12) 13.6 (7-32) 12.2 (7-22) 11.1 (6-39) 12.4 (4-29) 11.9 (5-22) D-2 4.0 (1-7) 2.7 (0.4) 5.4 (2-30) 2.1 (0-21) 4.5 (1-8) 7.5 (2-24) 14.6 (1-271 4.9 (1-23) 0-3 2.4 (1-211 3.1 (1-8) 2.2 (1-7) 2.5 (0-6) 4.9 (2-28) 8.2 (0-27) 1.9 (1-171 2.1 (0.9) ?8 4 0 7 i C-10 G B i* o I1 2 w TABLE 12. VA RIA N C E COMPONENTS BY COMPOUND FOR L A B -0 Compound Total 2.3,7,S-TCOO 2.25 Total TCOD 2.25 OCOO 0.00 2.3.7.8 + 2.3.4.6-TCDF 1.48 Total TCOF 1.40 1.2.3.7.8 + 1.2.3.4.8-FNCOF 1.84 2,3,4.7.8-PNCOF 1.31 Total PNCDF 1.62 1.2.3.4.7.8 + 1.2,3,4.7,9-HXCQF 5.80 1.2.3.7.8,9-HXCOF 4.73 1.2.3,6,7,8-HXCOF 2.25 2.3.4.6.7.8-HXCOF 2.25 Total HXCDF 3.66 1.2,3.4.6,7,8-HPCDF 3.92 Total HPCOF OCDF 5.13 7.73 Variane Components Sanpl Extraction 2.25 0.00 2.25 0.00 0.00 0.00 1.47 0.00 1.38 0.01 1.82 0.01 1.30 0.00 1.61 0.00 5.80 0.00 4.71 0.01 2.16 0.08 2.16 0.08 3.59 0.05 3.87 0.02 5.00 0.09 7.41 0.30 Mas. 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.03 0.03 0.03 Sanpl 99.9* 99.9+ 99.9 99.5 98.9 99.2 98.9 99.2 99.9 99.5 96.1 96.1 98.3 98.7 97.6 95.9 * of Total Extraction 0.0 0.0 0.0 0.2 0.6 0.4 0.0 0.0 0.0 0.2 3.4 3.4 1.3 0.5 1.8 3.9 Meas. 0.0 0.0 0.0 0.3 0.5 0.4 1.1 0.8 0.1 0.3 0.5 0.5 0.4 0.7 0.6 0.2 784072 GENP 011261 C-12 Laboratory A B C 0-1 D-2 D-3 E. TABLE 10. VARIANCE COMPONENTS FOR 2,3,7,8-TCDD ACROSS ALL SAMPLES (1,2,3,11,12,13,14,17) Variance Components Total Sample Measurement 8.05 6.87 1.18 5.46 5.45 0.00 2.12 2.12 0.00 2.25 2.25 0.00 2.30 2.30 0.00 2.19 ' 2.19 0.00 7.52 - -- % of Total Sample Measurement 85.4 14.6 99.9 0.1 99.8 0.2 99.9+ 0.0 99.9+ 0.0 99.9+ 0.0 i Laboratory A B C D-l 0-2 D-3 E TABLE 11. VARIANCE COMPONENTS FOR 2,3,4,6,7,8- HXCDF ACROSS ALL SAMPLES (1,2,3,11,12,13,14,17) Variance Components Total Sample Measurement 3.27 3.25 * 0.01 2.62 2.59 0.03 4.53 3.15 1.38 3.11 3.10 0.01 1.77 1.76 0.01 1.85 1.84 0.01 4.27 - - % of Total Sample Measurement 99.5 0.5 99.0 1.0 69.6 30.4 99.6 0.4 , 99.2 0.8 99.5 0.5 | - 784073 C-ll GBNP 01262 differences amongst the laboratories by compound across the samples was applied to the data (6). For available data, the F value for these measurements and the degrees of freedom were calculated and are shown 1n Table 13. In every case, the values reported by each of the laboratories were significantly different from one to another at the 95% confidence level for each of the congeners except 991.2.3.6.7.8- hexachlorodlbenzofuran, which was significantly different at the % confidence level. This test indicates that each laboratory reported statistically different quantitative values for each of these congeners 1n these samples. Prediction of Variability 1n Future Measurements In practical terms, what would be the predicted range of values if the analyses were repeated by a laboratory using one of the methods previously employed. Shown in Table 14 are the approximate 95% prediction Intervals for the laboratories if they were to repeat the analysis for each of these chemicals in the same utility samples. In other words, if-one of the five laboratories were to repeat the analysis for 2,3,7,8-TCDD 1n sample No. 1, the reported value would fall within a range of 15-30 ppb. If 20 determinations were made, one value would be outside of this range. For 2,3,7,8-TCDD a factor of two variation would be anticipated. In sonfe cases, the variability for other congeners may be as much as 20-fold or more. This variability does not appear to correlate to the congener concentrations actually present in the samples. For example, even though, in samples 11-17 the amount of 2,3,7,8-TCDD 1s quite low and the variability may be as much as a factor of 2O730 as indicated here, the reported values for 1.2.3.7.8.9- hexachlorodibenzofuran could vary over the range of 1-8869. In sam ples 11-17, a large variability In levels for the Individual isomers are predicted as well as for the total, for example, the total hexachlorodlbenzofuran. 784074 C-13 GENP 011263 TABLE 13. LEVENE'S TEST OF DIFFERENCES AMONG LABS BY COMPOUND ACROSS SAMPLES Compound 2.3.7.8Total TCDD TCOD OCDD 2.3.7.8 + 2,3,4,8-TCDF Total TCDF 1.2.3.7.8 + 1,2,3,4,8-PNCDF 2.3.4.7.8- PNCDF Total' PNCDF 1.2.3.4.7.8 + 1,2,3,4,7,9-HXCDF 1,2,3,7,8,9-HXCDF 1.2,3,6,78-HXCDF 2 3,4,6,7,8-HXCDF Total HXCDF 1.2.3.4.6.7.8- HPCDF Total HPCDF OCDF F-Value 14.96s 17.82s 12.81s 6.60s 8.89s 4.39' 9.76s 11.15s 17.80 ir k 16. 10*' 2.96* 36.93 k i t 23.12s 22.80s 17.39s1 6.97* Degrees of Freedom GENP 01J264 C-14 784075 TABLE 14. APPROXIMATE 95 PERCENT PREDICTION INTERVALS (PPB) Compound 1 2.3.7,8-TCOD OCOD 1530 159 2.3.7.0 2.3,4,0-TCDF 144221 1.2.3.7.8 + 1.2.3,4.0-PNCDF 123168 2.3.4.7.8-PNCOF 99.248 1.2.3.4.7.8 1,2,3.4.7.9-HXCOF 182347 1.2.3.7.8.9-HXCDF 134 1.2.3 .6.7-HXCDF 3172 2.3.4.6.7.8-HXCDF 401041 1.2,3.4,6,7.8-HPCOF ' 1031104 OCDF 289 Total TCDF 144222 Total PNCDF 206431 Total HXCOF 1551862 Total HPCDF 1031105 Total TCDD 660 2' 3 40- 6360 132 1- 240 32 57- 97767 1355 138193 226425 90- 164.321 618 1- 82515 1838 1- 225 965 45- 25472 412 241912 553975 1371530 963.258 67- 2280208 6436 57- 1046 67 2321 220517 10041682 402118 79810,654 1371532 8194341 40- 6360 132 11 12 I- 125 20 7- 519 30 24- 1561 81 417913 27264 84- 28145 59 213812.579 447809 18869 4167 218603 2097 26- 9879 68 580-16.405 964164.743 418976 382210.453 57- 37362 321 11253079 304121.535 143590 7261320 1581- 1467- ' 41.116 3090 1- 121 14 13 113 170 90459 830 4680 1336 161 2010 151 117 775 3891247 179442 11198 252 115 14 * 17 1- 132 11 1- 543 32 251459 119241 7421577 74746 - 13242389 106403 31445860 4865023 647800 16136 5611385 43131 1962882 102237 4821130 795652 96- 442457 24.465 532 1389 249 729 584613.471 5621.999 589721.657 61013.977 13913483 24211.063 1- 120 10 784076 C-15 O B N t> 0 l I2 6 s Chemosphere, Vol.l7,No.4, pp 627-631, 1988 Printed in Great Britain cc- t:r j f -rm 0045-6535/88 93.00 + .00 Pergamon Press pic \j' * woURCES OF DIOXINS IN THE ENVIRONMENT: A STUDY OF FOODS AND PCDFs IN ANCIENT, FROZEN ESKIMO TISSUE Arnold Schecter^*, Albert Deicing N.C.A. Weerasinghe2 , Saleh Arghestanl2 and Michael L. Gross2* ^Department of Preventive Medicine, Clinical Campus at Binghamton, State University of New York Health Science Center/Syracuse, 88 Aldrich Ave. Binghamton, New York 13903 2*Midwest Center for Mass Spectrometry, Dept, of Chemistry, University of Nebraska, Lincoln, NE 68588. ^Department of Anthropology, University Center at Binghamton, State University of New York, Binghamton, New York 13901 ABSTRACT According to the trace chemistries of fire theory, small amounts of polychlorodibenzodioxins (PCDDs) , including the most toxic 2,3,7,8-tetrachlorodibenzodioxin, and .polychlorodibenzofurans (RCDFs) are generated from combustion of natural products. The implication is that PCDDs and PCDFs have existed in nature prior to the large scale manufacture of chemicals that has occurred during the past few decades. However, on the basis of Hites'* et al. studies of lake ,sediment, it has been concluded that nearly all PCDDs and PCDFs are either synthetic in origin, or arise from the incomplete combustion of synthetic chemicals. In the present work, few if any PCDDs and PCDFs were found in human tissue samples and other samples that have been preserved' for greater than 400 years. The samples were from a Barrow, Alaska, Inupiat Eskimo household that was destroyed by an ice overflow, trapping two women. The analytical chemistry evidence is consistent with the Czuczwa and Hites" conclusion that the majority of PCDD/Fs in the environment today are of anthropogenic origin. INTRODUCTION Polychlorodibenzodioxins (PCDDs) and polychlorodibenzofurans are highly toxic chemicals which are persistent in the environment. Relatively recently, it was proposed that the PCDDs and PCDFs including the highly toxic 2,3,7,8-TCDD are produced in small or trace amounts by combustion of natural. 627 . GENP 011266 0028-M3.X,M/IMOM2-QTJM.OO/0 Copynjtnt c by Tha Am*nc*a Socmy far PhArraacotocr ta d E r p m w fiu l Thafapautui All n * h u o r r t p r e d u n i o n i n a n y fa r m w t v a d VOlCCt/lAX PHAAM4C0L0CV. 3 4 :8 8 3 -8 8 8 A Competitive Binding A ssa y for 2,3,7,8-Tetrachlorodibenzo-pDioxin and Related Ligands of the Ah Receptor C H R IST O P H E R A. BR A O FIELO and ALAN PO LA N D McArdie Laboratory for Cancer Research, University at Wisconsin, Madison, Wisconsin 53706 Received May 16. 1988; Accepted August 4,1988 SU M M ARY A sensitive competitive binding a ssa y for the detection of 2,3.7,8tetrachlorodibenzo-p-dioxin (T C D D ) and other ligands of the Ah receptor w as developed using a stable preparation of the Ah 'receptor, the 4 0 -5 5 % ammonium sulfate fraction of liver cytosol from C57BL/6J-m ice. and the radioligand [133l]2-iodo-7.8-dibromodibenzo-p-dioxin (specific radioactivity, 2176 Ci/mmol. and binding affinity, K 0 = 6.5 pM), Conditions are described which maximize assay precision and sensitivity, while minimizing sam ple counting time, ensuring ligand solubility, and permitting at tainment of binding equilibrium fgr com peting ligands. A ssa y conditions were developed to allow calculation of the binding affinity for com peting ligands and to ensure that an unknown competitor could be quantified in terms of `T C D D binding equiv alents.* Standard assa y conditions consisted of incubation of 6 pM radioligand and 18-20 pM Ah receptor with 5-1000 pM TCDD, in a 1-mi volume, for 16 hr at 4. Statistical analysis of the standard curve of bound radioligand versus the log of the con centration of com peting T C D D indicated the minimal detectable concentration of T C D D to be 10 pM (3.2 pg in a 1-ml assay a < 0.01). The simplicity, sensitivity, and reproducibility of this com petitive binding assa y should prove useful a s a screen to detect planar haiogenated aromatic hydrocarbons and other ligands ov the A h receptor. The availability of this '" Wabeled dioxin con gener also permitted the characterization of Ah receptor-ligand binding over a range of ligand and receptor concentrations not possible with currently available 3H-tigands. 2.3,7,8-TetrachIorodibenzo-P'dioxin is one of the most potent small molecule toxins known (1-3) and serves as the prototype for a large number of planar haiogenated aromatic hydrocar bons which elicit their biological effects by a common mecha nism (e.g., certain isomers of haiogenated dibenzo-p-dioxins, dibenzofurans, azo(xy benzenes, and biphenyls). The risk pre sented by the widespread dispersion of these compounds into the environment is a function of 1) their toxic potency, 2) their resistance to chemical and biological degradation (4), and 3) their lipophilicity and hence potential for accumulation in the food chain (5). Analysis of trace concentrations of these compounds in en vironmental and biological samples has been made possible through recent advances in chromatographic and mass spectral technologies. Current methodologies involve varying degrees of sample preparation, separation of isomers by liquid or gas chromatography, and identification and quantification of con- Thu work was supported in part by the National Institute of Environmental Health Sciences Grant ES-01884. National Cancer Institute Core Grent*07lT5, and National Cancer Institute Postdoctoral Training Grant T32-CA09020. genera by mass spectrometry. Extremely low concentrations of TCDD in environmental samples, picogiam per gram levels (i.e., parts per. trillion), are now routinely quantified using this technology (6). Despite the sensitivity of mass spectrometerbaaed methods, their use is limited by coat and availability of instrumentation. Bioasaays have played a prominent early role in the. identi fication and monitoring of chlorinated dibenzo-p-dioxins: e.g., the formation of pericardial edema in the newborn chick (7), and the production of chloracne in the rabbit pinna (8). Mere recently, cell culture bioassays (e.g., the induction of aryl hy drocarbon hydroxylase activity in rat hepatoma cells (8), and keratinization in XB/3T3 cell cultures (10)] have achieved remarkable sensitivity, 10 pg of TCDD, but have not gained widespread use. Radioimmunoassays have been developed to detect TCDD, 2,3,7,8-tetTachlorodibenzofuian, and chlorinated biphenyls with detection limits approaching 25 pg (11-13). The limited use of these radioimmunoassays may be attributable to 1) the need to characterize each antisera for its reactivity Coward a large number of isomers and cross-reacting com- ABBREVIATIONS: TCDD. 2.3,7.8-tetrachiorodibenzo-p-dioxin; MO PS. 3-(N-morpnoiino)propanesuttorrie add; EDTA, {ettiyienedinitrttohtgtrzaceac add); K0, equilibrium dissociation constant; L, ligand (labeled or unisteied}; /?, receptor RL. receptor-ratfioagand complex; R L ', transformed recapt*-radiofigand complex; [L]r. total radtodgand concentration; [R]r, total receptor concentration; 8m*.. concentration of receptor determined by Sczxch&c analysis; B, bound radioligand at equilibrium: S 0. receptor-bound radioligand in me presence of no competing ligand; S ,, receptor-bound rccic*;gsnc m the presence of competing ligand-, C. competing ligand; [C V concentration of `free* competing ligand at equilibrium; ft,,, dissociation rate constant; NSB, nonspecific binding (radioligand binding in me presence of a 200-1000-fold excess of 2.3,7,8-tetracniorodibenzofuran): EC, EC, EC, competing ligand concentration which produces 20%, 50%. or 80% reduction in specific binding, respectively; f*, half-life of dissocanon. 682 GENP 011267 784078 784079 Binding Aaaay for TCOD 68- pounds, 2) difficulties in the solubility of radioligand (or anti detail (18), and 2) the receptor concentration in this preparation i gen), and 3) the limited availability of the radioligands. TCDD~and related halogenated aromatic hydrocarbons'are all approximate isostereomers and produce a characteristic slightly enriched compared to cytosol, the lipid contamination is re duced, and binding characteristics are unchanged for over 1 year whe: stored at -80*. pattern of morphological and biochemical changes. The biolog ical effects elicited by these compounds appear to result from their stereospecific binding to a soluble protein, the A h recep tor, and the resultant gene expression which is initiated by this ligand-receptor complex (14-17). In support of this model: for chlorinated dibenzo-p-dioxins, the apparent binding affinities (K ,, ) of congeners for the A h receptor correspond to their rank- Mathematical and Statistical Theory Used to Optimize Aaaay SaniM vity E quilibrium b lad in g . The binding of a radioligand. L. to it receptor, R, law of mass under action, conditions where the eoqfueilqiubirliiubmriudmis,soccainatbioendceosncrsitbaendt. bKy 0t.ht-: equal to receptor the [,] *ratciooncoefntthraeticoonnosftitfureeentrsa:di(oAli]ga*ndc; oanncden[RtrLat]io-n cf fra, concen ordered potencies to produce biological responses (e.g., induc tration of radioligand-receptor complex. Classically, this interaction i* tion of monooxygenase activity, lethality, chloracne, and tumor ` represented as: promotion). Secondly, in mice there is a genetic polymorphism in the A h locus that determines the receptor. Strains which R fls L U) express a high or low affinity receptor are more or less sensitive to the effects of agonists, respectively, and a variety of toxic Ko \R L\ 12 1 responses (e.g., thymic involution, porphyria, epidermal hyper keratosis, and teratogenicity) have been shown to segregate with the A h allele which determines the high affinity receptor (for a review-see Ref. 16). Since these halogenated aromatic hydrocarbons produce their biological effects by virtue of their binding to the A h receptor, a competitive binding assay of sample and radiola beled ligand for A h receptor occupancy would appear to offer a direct and simple assay system. As an analytical method, a competitive radioligand-A/i receptor binding assay has a num We have recently examined the binding of the Ah receptor from C57BL/6J mouse liver and (l=1I]2-iodo-7,8-dibroraodibenzo-p*dioxin (18) and found it to be best characterized by the following model: R + L RL R L' s= R' + L i3) where R L ` is a a much slower ldigisatnindcdt ifsosromciaotfiothneralicgeacnhda-nrecdeopetsoRr Lco. mplex, which has Under the binding conditions employed lAh receptor from C57EL/ 6J mice, complex, iRncLu',biastiloenss at th 4*. an for 25% 16 of hr), the the total transformed receptor-ligand bound ligand; and equations ber of advantages: 1) theoretical foundation based on the law 1 and 2 provide a rough approximation of equilibrium binding.1 of mass action, 2) elimination of the biological variability inherent in the use of whole animals or cells, and 3) rapid screening not requiring expensive instrumentation. The sensitivity, and hence utility, of A h receptor-based com petitive binding assays has been limited to date by the low specific activity of available radioligands (e.g., (:lH]TCDD, 58 Ci/mmol). We have previously reported the synthesis and binding kinetics of a new radioligand of the A h receptor, [ri'Il Optimization of a com petitive radioligand bound to receptor, [AL] bi or nBd,inatg assay. a fixed The quantity concentration of of radioligand and receptor, is progressively decreased by the addition of increasing concentrations of unlabeled competitor. C, which competes for receptor occupation. The concentration of radioligand bound, as a function of unlabeled ligand added, describes a competitive binding curve and can be derived from the following relationship: 2-iodo-7,8-dibromodibenzo-p-dioxin (18). This radioligand pos sesses a high affinity for the A h receptor [Ko m 6.5 pM) and a high specific activity (2176 Ci/mmol). We now report the use of this radioligand in the development of a highly sensitive competitive binding assay for Uganda of the A h receptor. where ( ] r " concentration of total ligand and (A]r - concentr^ion of total receptor. A major goal in developing a competitive binding assay ia to optimize the precision adjusting the and sensitivity concentrations of of the assay. radioligand T[hLi)st is accomplished by and recepto:- [ |T. Rational Ben o n and Yalow (20) and Ekina and co-workers (19) independently developed theoretical soluciona to calculate optimum reagent concen Our approach to the development of this competitive binding assay trations. Although these solutions differ in their definitions of sensitiv consisted of: 1) synthesis of s radioligand of high specific activity and ity and considerations of error, they predict similar optimal conditions. phirgehp arreacteiopntoor fatfhfieniAtyh; 2) characterization of receptor 3) use of com a stable petitive and reproducible binding theory to approximate optimal conditions; and 4) refinement of the conditions Beraon and Yalow define conditions which yield maximal sensitivity to ( those which ]r) 0.33 to give an 0.5, [A]r ini0ti.a5lKroat.iaonodf[bLo)ur nthdattoaptoptraolarcahdeisolziegraon.dEk(irn./s by experiment. Radioligand and co-workers 0.5; [A)r 5 counting error, 1do.e2rf5winKheiocohpatnaimdppa[rloc]aorcnhdi[tAi2o.)2nr5s*aKs0o.t5,howKshoeewnanhcdicohn[Lsyi]idreel=dr*in0g, only with (spTechieficseancstiitvivitiytyoof fthaecoramdpioeltiigtiavnedbxindIKinog]iartss(a1y9)i.sTphruops,ourtsieonoaflftiaosIt]h2ei-o7d0o%-T,.SK-doibmrom1.o7diKbeonzfoo-rp-TdCioDxiDn)(2s1h7o6ulCdi/minmcroeal,secoausnstaiyngseefnfsicitiievnictyy approximately 6-fold compared to use of [1,6-JH]TCDD (58 Ci/mmol, counting efficiency -40% ). increasing experimental error. These derivations assume simple mass action ing to and the Aeqhuirleicberiputmor conditions which only approximate ligand bind (i.e., equations 1, 2, and 4). Additionally, they are dapendant upon experimental aa well as counting errors. Therefore, we used these theoretical values to determine initial ranges of receptor and radioligand concentrations and further optimized assay sandtivi^y empirically. Receptor Preparation The 40-55% ammonium sulfate precipitate fraction of hepatic cy tosol from C578L/6J mice was chosen because 1) we have recently characterized the kinetics of radioligand binding to this preparation in 'This aiaumpticn u necessary since the optimization theory esad w-ss derived from Eqa. 1, 2. and 4. The aatumption u supported by the h c i chi; optimal sensitivity * u achieved in accordance with (ha theoretically optimal reacent concentration ran*. Isaa "Results" and "Discussion"). GENP 011?6R 8 8 4 Bredflaid and Poland Quantification of competing Uganda with differing receptor Affinity. A host af-halagenated aromatic hydrocarbon isomers are ligands for the Ah receptor, but differ widely in their binding affinities. We foresee this competitive binding assay as being useful in the quantification of the sum of these compounds present in environmental eotr abLio(lo1g9)icaalnsdamRpoldebsa, ridn terms of **TCDD and Lewald (21) binding equivalents." Ekins have examined the complex relationship between the relative binding potency of coolimgapnedt,itoLr,(wC,itwh iathffianniteyqKuitl,i)b. riWumhednistshoeciaaftfiionnitycoonfsttahnet aKnc. uniabeled and radi radioligand la equal to or greater than that of the competing ligand (and the ratio of bboinudnidngrapdoitoelnicgyanids /nfreeaerlyraldiinoelaigr a(ni.de.,isECle*s*s*/tEhCan*u,0*.5)K, vth/Ke 0e)~stSiminactee of we have chosen a radioligand with a receptor affinity approximately one- half that of TCDD, it is very likely the estimate of binding equivalency of unknown ligands will always be nearly linear. We have chosen to optimize the assay sensitivity for detection of TCDD; one may enhance the sensitivity of detecting a known ligand of lower affinity by adjusting the concentration of receptor (/?]r and radioligand []r (19). T im e for equilibrium . To accurately estimate the binding equiv alency of a competing unlabeled ligand, it is important that binding equilibrium be achieved for both the uniabeled compound and the radioligand. The time required to achieve equilibrium binding is a function of the dissociation rate constant of the slower dissociating ligand. Since the radioligand has a very high affinity for the Ah receptor, comparable to or greater than that of any known competing ligand, we assumed that 5 times the half-life of its dissociation [ft. 1.9 hr (18)] or 3.5/-i is a good approximation of equilibrium conditions for all potential ligands (22). C alculation of dissociation co n stan ts. The equilibrium dissocia tion constant of a competing ligand (Ac) is moat commonly calculated >y the Cheng-Prusoff equation (23), which is applicable to situations .sere These conditions are not met in competitive binding assays which have 0.5). We estimated been the Ko,ptoimf cizoemdpfeotrinsgenlisgitainvditsy Ii.e.. from B0/[L ]r " 0.3EC* values by che equations of Linden 124) which place no constraints on the fraction of radioligand or competitor bound. First the concentration of ``free" competing ligand is calculated; Ko K0 + {L\t + [ S W then [Cr] is substituted into the following equation to determine K& Materials and Methods R eagents. (l*Tl2-Iodo-7t9-dibromodibenxo-p-dioxm (217601/ mmol) was synthesized and purified u described previously (18). TCDD was a generous gift of Dow Chemical Co. (Midland. MI), 2,3,7,8Tetrachlorodibenzofuran and 2.3-dichlorodibanzo-p-dioiin were a gift from Drs. David Firestone and Albert Pohland (Food and Drug Admin istration, Washington, DC). [UC]TCDD (specific activity 114 mCl/ mmol), and 2,3-dibromodibenzo-p-dioxin were synthesized as described (25,26). 3,4,3' ,4' -Tetrachlorobiphenylether waa a gift from Dr. Andrew ` inda (University of Rochester). Estradiol was purchased from Cal- ham (San Diego, CA). Pregenolone-16a-carbonitrile waa purchased .. .m Upjohn Diagnostics (Kalamazoo, MI). Sodium phtnobarbital waa purchased from Merck Chemical Division (Rahway, NJ). Active char coal, grade PX-21, was a gift from Amoco Research Carp. (Chicago, ID . Bacto-Gelatin was purchased from Difco Laboratories (Detroit, MI). Glycerol was purchased from J. T, Baker (Phillipsburg, NJ). EDTA was purchased from EM Scientific (Cherry Hill. NJ). Testos terone, cortisol, Na-L-thyroxins, dithiothreitol. d-mercaptoethanol. so dium azide, and MOPS (free acid and sodium salt) were purchased from Sigma Chemical Co. (St. Louis, MO). p-Dioxane (anhydrous 99+% pure) and dimethyl sulfoxide (anhydrous, 99% pure, stored under N- gas) were purchased from Aldrich Chemical Co. (Milwaukee. WI). B uffer*. MN represents the stock buffer which contains 25 mM MOPS and 0.02% sodium azide, pH 7.5 (at 4'). MEN is the stock buffer plus 1 mM EDTA. MdENG and MDENG represent MEN with the addition of 10% (w/v) glycerol, plus the addition of either 10 mM d-mercaptoethanol or 1 mMdithiothreitol (respectively). R eceptor p re p a ra tio n . The 40-55% ammonium sulfate precipitate of C57BL/6J mouse hepatic cytosol was prepared as described previ ously (IS). S ta n d a rd aaoay protocol. To a series of 12- x 75-mm borosilicate assay cubes was added 5 jd of dimethyl sulfoxide containing TCDD standards (0, 5, 10, 20, 40, 60, 100, 200, 500, and 1000 fmol) or unknowns. The frozen 40-55% ammonium sulfate precipitate 115 mg of to p a rotein/tube) was concentration of 1d5is0snoglvoefdpirnotiecien-c/molld MDENG buffer (equivalent to an and diluted Ah receptor concentration of 18-20 fmol/ml). The radioligand in dimethyl sulfoxide was added to the solution of receptor to a concentration of 8.0 fmol/ml (-40.000 dpm/ml. added as a l jil stock solution/ml of buffer) before dispensing 1 ml of the solution to each assay tube (without vortexing). The tubes were then incubated at 4* for 16 hr. The assay was terminated by the addition of a 0.5 ml of suspension of charcoal/gelatin (3%/0.3%. w/v) in MN buffer with vigorous stirring on a vortex mixer for 3 sec. followed fuged a t 2b0y0i0ncxubgaftoiorn10fomr 1in0 min a at 4*. t 4*C. The tubes were A 1-ml aliquot of the then centri supernatant fraction of each tube was transferred to 12- x 75-mm polypropylene tubes (VWR Scientific, San Francisco, CA), and the bound radioligand was quantified in a MINAXI Series-5000 gamma scintillation counter (United,Technologies/Packord Instrument Co., Downers Grove. IL). Sample counting times were adjusted to obtain a counting error of 3% (range of standard wQe.rle-2a.0namlyizne,dcionunqtuinadgruepffliicciaetnec. yToofta7l5r%ad).ioEliagcahnds.am[ Lp\lre, and was determined by the transfer of 0.67 ml of assay solution directly to the polypropylene tubes for counting (no charcoal/gelatin is added to these tubes). D ata an aly sis. The y-scintillation counter was connected by a serial communication port to an IBM-XT personal computer equipped with software for analysis of radioimmunoassay data and quality con trol (SECURIA PLUS, Packard Instrument Co., Downers Grove, IL). Total radioligand. [L]r, waa defined as the concentration of radioli gand in solution after the 16*hr incubation. (This value is generally 10% lower than the concentration of radioligand originally added to the tubes, i.sM8 pM.) NSB was defined as the amount of radioligand bound in the presence of 8 nM 2,3,7,3-tetrachJorodibenzofuran (1000- fold molar excess). The total radioligand bound at each concentration of competing ligand 5-1000 fmol of TCDD/ml) minus the NSB was defined os the specific binding; Bo " specific binding in absence of competing ligand and S , specific binding at 'i ' concentration of competing ligand. To generate a standard curve, the response parameter, BJBn, i.e., the ratio of specific binding at each concentration of TCDD relative to the specific binding in the absence of competing ligand, was plotted verso* the log of the concentration of TCDD. For data analysis we chose to fit these results, via an four-parameter logistic modsl iterative curve-fitting described by DeLeon teetcahLniq<u2e7,).tioTthhee concentration of ligand is unkown samples is quantified os "equiva lent#" of TCDD (in picamolar concentration or ferncomoles per milli liter), by interpolation of the fractional bound response metameter on s , - NSB 'syfl.- 1 - ((C1/C,,)J NSB where 5 is slope of log logit plot. 011269 784080 5thsetasntadnadradrddecvuiravtieo.nOs uftrloiemrathweermeedaenfi(nned* as 4 values varying at each point). more than Radioligand' The variation~5etween assays was monitored by following a variety ' of quality control parameters for the standard curve, which included: l) fl... (specifically bound ligand in the absence of competing ligand: 2) B../Lr. (specifically bound ligand as a fraction of total radioligand per milliliter); .1) NSB: 4) estimates of EG.-, EC*, and EC (competing ligand concentration which produces a 20%. 50% and 80% reduction in specific binding, respectively;), 5) the calculated slope of the four- parameter logistic model ii.e,, slope of the logit-log plot), and 6) a control standard yielding a TCDD concentration of 45 pM. 2.4 pM naTniha a aa Binding Aaaay for TCDD . 685 7.7 pM Results A representative saturation binding isotherm of [`*'I]2-iodo7,8-dibromodibenzo-p-dioxin binding to the Ah receptor in the 40-55% ammonium sulfate precipitate fraction of liver cytosol is shown in Fig. 1A. The data in Fig. IA were transformed by the method of Scatchard (28) to generate the plot described in Fig. IB. From an average of three experiments the concentra tion of binding sites, = 120 7 fmol/mg of protein, the apparent Kn = 16 3 pM, and the correlation coefficient =* 0.99 0.00. The Hill coefficient of these plots is 0.97 0.02 with a correlation coefficient = 0.99 0.00 (29). As outlined under "Rationale," the approximate conditions for optimal precision and sensitivity of a competitive binding assay are; L) R,/[LJr 0.33-0.5; 2),[R]r 1.25 K{) and [L\r 2.25 K. In Fig. 2, are displayed a series of competitive binding curves generated at three radioligand concentrations ([ ]r =* 2.4, 7.7, and 23 pM) and three receptor concentration ([2]r 7, 28. and 112 pM), with TCDD as the competing unlabeled ligand. The initial B,,/[L]T ratios (in the absence of unlabeled ligand) varied from 0.2 to 0,7. For a given radioligand concen tration, decreasing the concentration of the receptor decreased the initial R./[,]r and increased the initial slope of the com petitive binding assay. As a test of the relative assay sensitivity obtained at differing R g. 2. Effect of radioligand and receptor concentration on binding curves generated with TCDD as competing ligand. Assays were incubated at 4 for a period of 16 hr (see text). All data points are the result af quadruplicate determinations. Ordinate: total radioligand bound divided by total radioligand in solution. Abscissa: concentration of unlabeled competitor (TCDO, picomolar concentration). Receptor concentration: 112 pM (); 28 pM (O); and 7 pM (). Left, total radioligand concentration, [L]r. is 2.4 pM (11,600 dpm/mi); center, total radioligand concentration is 7.7 pM (37,200 dpm/mi); right,-total radioligand concentration is 23 pm (11T,000 dpm/mi). TABLE 1 Statistical power at the competitive binding assay Statistical power (see footnote 3) of various assay conditions to detect tne decrease in radioligand bound in tne presence of 5 pm TCDO. as compared to radioligand bound in me absence of TCDO (i.e.. Bn). Ekms at a/. (19) have snown mat tne sensitivity ol a competitive tending assay is equivalent to tne error mdo divided by tne initial slope of me assay curve, i bound/.! competing ligand added. The statistical power (1 - d) was calculated using the Student's f value wim a s 0.0i (30. 31). Haacigand Concanm oon Slittaci Power fiaceoior Concentration 7pu 2aOU 112DM pu 2.4 0.99 0.10 0.00 7.7 0.95 0.30 0.00 23 0.60 0.10 0.10 Binding Isotherm Scatchard Analysis Total Radioligand (fm al/m l ) Total Sound (fm al/ml) Fig. 1. A. Equilibrium binding of [<MH24odo-7.8-dibnofnodibenzfrp-dlown to me 40-55% ammonium sulfate preaprtat* fraction of hepatic cytosol. The receptor preparation (145 *g of protein/ml) waa incubated at 4* for -16 hr. with increasing concentrations of ratfoMgand (3-70 pM). Nonspacrtlc binding was estimated in the presence of a 200-fold excesa of 2.3,7.9-tetrachtorodibenzofuran. Each pomt represants me average of two determinations. 0. Scatcnard analysis of equiiibhurn binding data from a . The binding parametere were caicuiatad by a linear least squares estimate of specifically Bound/free radioligand versus specifically bound radioligand. The K0 and 8mm results (inset) are derived from the experi ment shown, me mean and standard deviations from three experiments are K0 - 10 3 p* ano 8mm * 18 X 3 fmoi/mi (120 17 fmol/mg of protein). reagent concentrations, we compared the statistical power1of these assay conditions to detect a 5 pM concentration of TCDD. As shown in Table 1, the power of the assay increased with a reduction of receptor concentration, at a receptor concentration of 7 pM, the power, increased as radioligand concentration decreased. TCDD and related halogenated aromatic hydrocarbon have very limited solubilities in aqueous solution. We examined the concentration of radioligand that remained in solution, after a 16-hr incubation at 4at aa a function of the concentration of the receptor preparation used. Aa seen in Fig. 3, only at protein concentrations of 150 jsg/ml ([&lr * 18 pM) or greater, did at least 95% of the added radioligand remain in solution. While maxim! sensitivity is achieved at receptor concentrations of 7 pM, we chose to use the higher concentration (18 pM, 150 ug/ ml) to increase the solubility of competing ligands. Based on the observations presented above, the standard conditions for the competitive binding assays were chosen to be: radioligand concentration, []r, of 7.2-7.7 pM 0*4 x io* dpm/mi added initially), a receptor concentration of 18-20 phi. 1The slatinici) power of an assay (30) is the probability that ths deon.-^ in bound radioligand, due to the presence at 5 pH TCDO. will be detected .hen compared to fi* (Student's t value, a a 0.01), Thua, aaaay conditions which yield the greatest statistical power are the conditions which yield the greats ssey sensitivity. Statistical power is equal to l --d (where d is the probability of making a type (I statistica] error) (311. GENP 011270 7R4081 686 Bradfleid and Poland COMPETITIVE BINDING CURVE Fig. 3. Effect of protein concentration on radioligand solubility. The radioligand (8.3 pm) was incubated at 4 with varying concentrations of the receptor preparation (0. S, 10. 19. 38, 75. 150. 300, 600, and 1200 ug of protein/ml. After incubation for 16 hr. total radioligand in solution, [L]t, was determined as described in the text. Each point represents the average of two determinations. (approximately 150 jig of protein/ml), and an incubation time of 16 hr at 4*. A typical standard curve for the competitive binding of TCDD determined under these conditions is shown in Fig. 4. The initial total bound ligand is 17,176, NSB is 1996 dpm, initial specifically bound ligand. Bo, Is 15,130 dpm and B../[L\t =*0.40. The concentration of TCDD which produces a reduction in specifically bound radioligand equal to one-half the initial value (ECW) is 41. 3 pM (Table 2). Under the standard assay conditions the minimal detectable concentra tion of TCDD which reproducibly produced a statistically sig nificant reduction in bound radioligand is 10 pM (10 fmol/ml 3.2 pg/ml, a s 0.05). Thus, to ensure ligand solubility with an increase in [Brl, we have reduced sensitivity about 2-fold. By reducing the assay volume to 0.25 ml, one can decrease the minimal detectable concentration to 0.8 pg of TCDD (data not shown). The advantage of the 1.0-ml incubation volume it greater bound radioactivity, and thus reduced counting time per sample to achieve the same precision. We have previously found that [l:ul]2-iod0-73*dibromodtbenzo-p-dioxin has a biphasic dissociation from the Ah recep tor, with the major component (75%) having a dissociation rat constant, =0.36/hr at 4' (7% - 1.9 h r '1) (18). Thus, binding equilibrium for competing ligands with dissociation rata con stants equal to or greater than that of the radioligand should be achieved by 5 x or 10 hr (22) (sea Rationale). Competing ligands with slower dissociation rates than the radioligand [presumably TCDD, since its K0 ia approximately one-half that of the radioligand (Table 3)] may require more than 10 hr to reach binding equilibrium. To confirm that binding equilibrium was obtained for TCDD by 16 hr, we examined the competitive binding curves at 4* as a function of Incubation time (Fig. 5). The initial bound radioligand B0 and slope of the competitive binding curved reached a maximum by 16 hr, remained virtually unchanged between 16 and 46 hr, and then progressively de clined. The decrease in Boat longer incubation times is probably attributable to degradation of receptor with time. The accuracy of the assay is dependent on tbe standard, i.e., the accuracy in weighing and dilution of small amounts of TCDD. We checked this by determining the competitive bind- TCDO (pM) Fig. 4. Competitive binding assay. Standard curve with TCDD as com peting ligand. Assay was run, as described in text, with increasing concentrations of TCOD, a total radioligand concentration of 7.2 pk; (added 8.2 pM),and a receptor concentration of 18 pm.Incubation was for 18 hr at 4. Ordinate: 8,18a, specifically bound radioligand in the presence of a given concentration of TCDO divided by specificity bound radtotigand in me absence of TCDD. Abscissa: concentration of TCDD (picomoiar concentration). The first data point is the interpolated concen tration of So, as calculated by the four parameter logistic model (see text). For this standard curve; EC - 13 pM, EC * 40 pw, EC - 138 pw, So divided by total radioligand - 0.36. NSB divided by total radioli gand - 0.06. 784082 TABLE2 Quality control parameters of tha competitive binding essay standard curva The standard curve for TCDO competition with radictajand for the Ah receptor was detarmned aa described w ttr `Mitenaie and Metnods.' Quality control parame ters were compered from assays usng freshly prepared radioligand (n - 9). and using radioagand after 75% radiodecay (120 days, n - 1). Values are means = standard deviation. Al parameters are desorbed in the text. NO. not oeteoninec. PWTMT *Ntw*Radagmt NoRadoony flaoctgctS after75% Raastecay SoJLr 0.38 0.06 NSB/Lr 0.06 0.01 , ECn (pw) 14 3 EC (pw) 41 3 EC (PM) 140 20 Logit slope 1.3 0 .1 45 pM TCOD (control pool) 42 6 40pm f'crrcoo * 40 2 0.34 OiOS 14 45 160 ' 1.2 . 48 NO ` G R N P011271 784083 c 0 Tj O <1 Binding Assay (or TCOO 6B7 TABLE3 Relative binding potencies for Ifgends of ttia Ah receptor Tna competitive boding assay was performed using oonratfloiaoeied congeners, under standard conditions as described under 'Matenatt and Methods.' The [fl|r was 20 d m . and |L]r - 7-7 6 pw. The true K0 ol me radiofigand was assumed to be 6 5 pu (18). Tne EC is me total ligand concentration wfiicfl produced a 80% reduction in me specific binding of the radioligand; the relative binding patency is g r ]an,-u. m/g r -K>-rrHi- The *c lor each comoound was detenmmod by the method ot unden (Eqs. 8 and S. flef. 24) and me relative K<* Kc compamor/Xe TCOO. CofflMtitar sc* flWM EC* Rem* 2.3.7,3-Tetraciiiorodibenzo-p-dioxin 2-iodo-7 .a-Dibromodibenzo-p-dioxin 2.3.7,8-Tetracbiorodibenzofuran 2.3-Dibromodibenzo-p-dioxin 2.3-DicMioroOibenzo-p-Oioxm fit 41 58 85 580 6400 1 1.4 2.1 14 160 fi* 6.8 11 18 140 1600 1 1.7 2.6 20 230 benzo-p-dioxin in the radioligand stock solution. The K t,of the 2,3-dibromodibenzo-p-dioxin was estimated fay competitive binding to be approximately 140 pM (see below). We tested whether radioligand decay products might compete for receptor binding and alter the competitive binding curve. Assays using the stock solution of radioligand 120 days after its synthesis (75% of '*T radiodecay), showed no significant deviation in the quality control parameters compared co freshly prepared radi oligand (Table 2). Thus, purification of the radioligand by high performance liquid chromatography does not appear nec essary for at least 120 days after synthesis. Using the standard assay conditions outlined above, compet itive binding curves were generated for a variety of halogenateddioxin congeners (examples are given in Fig. 6). The relative binding affinities of these congeners, as indicated by estimates of ECui and Kn (Table 3), are in agreement with relative binding affinities, and biological potencies which have been published previously (Le., TCDD > TCDBF > 2,3-dichlorodibenzo-pdioxin > 3,4,3\4'-tetrachlorobiphenylether (10,25)]. Addition ally, testosterone, phenobarbital. thyroxine, estradiol, cortisol, and pregnenalone-16a-carbonitrile at levels as high as 0.3 uM (i.e., 10Atimes the concentration of radioligand) did not com pete with the radioligand for Ah receptor occupancy (data not shown). Discussion In this report, we describe a sensitive competitive binding assay for the detection and characterization of ligands of the Ah receptor. Since the sensitivity of such an assay is a function of the binding affinity and specific activity of the radioligand, considerable attention has been devoted to the selection, label- Ftg. 5. Effect of incubation time on the competition binding curve. The assay was run as descnbed in the text, with increasing concentrations of TCOO. a total radioligand concentration of 7.5 pm and a receptor concentration of 18 pm. The competition binding curves were analyzed after varying incubation times (key is inset) at 4*. Ordinate: radkatigand ing, and binding kinetics of this compound. [li'I]2-iodo*7,Sdibromodibenzo-p-dioxin (18). The higher specific acitivity of this radioligand (2176 Ci/ mmol) and the greater counting efficiency of 7-emission ovei- specifically bound in disintegrations per miiiititer. Abscissa: concentration /3-emission increases the ratio of counts per minute per femto- of uniabeied TCOO (ptcomoiar concentration). Each value is the result ot mole of radioligand about 2 orders of magnitude as compared tnpiieate determinations. to pH]TCDD (SBCi/mmoi). T h e1"I*labeled ligand offers other minor advantages: 1) it has high radiochemical purity resulting from the easy separation of the iodinated product from the ~ `:oir=: starting material, reaction intermediates, and radiodecay prod ucts, 2) specific activity is determined as that of the Na[liT] used for synthesis, and 3) after 75% radiodecay, a stock solution - of the `"'I-ligand produces the same competitive binding curve, - am : indicating that radiodecay products do not significantly affect receptor-ligand binding (Table 2). In a previous report (18), we described the effects of protein concentration on the quantification of "free" radioligand and Concentration of Competitor (M l on the calculations of K0 and [R]r - We suggested that at protein concentrations above 70 pg/ml, "nonspecifically bound" radi Fig. 8. Competition by various compounds for mo specific binding ot i'" l]2-kxio-7,8-dibrornodibenzo-p-djQxjn to tho Ah receptor. The assay conditions are identical to thosa described in Fig. 5. All values are the result of quadruplicate determinations. Compounds were dissolved in dimethyl sulfoxide and added to the incubation in 5 total volume. oligand is misclassified as "free" radioligand, leading to signif icant overestimation of Ko by Scatchard analysis. By minimaing misclasaification errors we calculated the Ko of [li'I]2-iodo7,S-dibromodibenzo-p-dioxin to be approximately 6.5 pw. Folthe competitive binding assay, a concentration of 150 ig/rd ing of [UC]TCDD. Three independent analyses of a (HC]TCDD was employed to ensure ligand solubility. At this protein con standard, quantified by ^scintillation spectrometry to a con centration Scatchard analysis of the saturation binding isoth centration of 40 pM. gave a value of 40 2 pM from the erm yielded an apparent K0 of 16 pM (Fig. 1), suggesting that standard curve (Table 2). some misclasaification of nonspecifically bound radioligand as li'I decays to tellurium with a half-life of 60 days. Radiodecay free radioligand is occurring. The effect of this misclassification of the iodinsted ligand may produce unlabeled 2,3-dibromodi- ~appears to be minimal, as the calculation of Kc for 2-iodo-7,- 680 BndttaM and Poland dibromodiben2o-p-dioxin (i.e., 11 pM) 9 only 1.7-fold greater than the previously determined estimate of K0 for the `' "'I analogue (i.e., 6.5 pM). This 1.7-fold overeetimation of Kc suggests that the true Kc for TCDD ia approximately 4 pM, 100-fold lower than previous estimates for this compound (25). It has been previously proposed (32) and demonstrated (33) that a competitive binding assay, using the Ah receptor and radioligand, could be used to screen for the presence of com* peting ligands present in the environment. The extraordinary sensitivity of the present assay, attributable to the high specific activity of the radioligand, revised estimate of receptor affinity, and optimization of assay sensitivity, make it feasible to screen environmental samples for TCDD and related halogenated aromatic hydrocarbons: e.g., chlorinated dibenzo-p-dioxin, -dibenzofuran, and -biphenyl isomers. Environmental samples may also contain polycyclic aromatic hydrocarbons, many of which are also ligands of the Ah receptor. These compounds can be eliminated from analysis by the appropriate sample cleanup. The method of sample cleanup depends on the sample matrix (e.g,, biological tissue, soil, water, etc.) and the presence of interfering substances. The competitive binding assay does not provide Chemical identification of competing ligands, but an estimate of their concentrations as "TCDD-binding equiv alents" (see Rationale and Table 3). Samples judged to have sufficient concentrations of TCDD-binding equivalents, could be subjected to mass spectrometry for chemical identification. Finally, one of the most exciting uses of this competitive binding assay, is to screen biological tissue extracts for the postulated endogenous ligand of the A h receptor. References 1. Poland. A., and A. Kendo. 2,3,7,8-Tetrachlorodibcnro-p-dioxin: environmen tal contaminant and molecular probe, fed. Proc. 33:2404-2411 (1976). 2. Schweiz. B. A,. J. M. Norris. G. L Sparechu. V. K. Rowe, p. J, Gehring. J. L. Emerson, and C. G. Gerbig. Toxicology of chlorinated dibenzo-p-dioiins. Environ. Health Perspect. 5:87-99 119731. I Kuciba. R. I.. 0. G. Keyes, J. C. Beyer. R. M. Carreon, C. E. Wade, D. A. Dittenber. R. P. Kalnms. L. E. Frauson. C. N. Park. S. D. Barnard. R. A. Hummel, and C. G. Humision. Results of s two-year chronic toxicity and oncogenicity study of 2.3,7.S-tatrachlQrodibenz0-p-dioxM in rata. Toxicol. AppL PharmacoL 43:279-303 (1978). 4. Kearney. P. C., E. A. Woolaon, A. R. laanaae. andC. S. Hailing. Tetraehlo* rodibenzodioxin in tha environment: source, fate, and decontamination. Environ. Health Penpect. 3:273-277 11973). 5. Bickel. M. H,, and S. Muehfbach. Pharmacokinetic* and ecodisposition of polyhalogenatad hydrocarbons: aspects and concepts. Drug Metah. Rtv. 11:149-19011980). 6. Smith. L M.. D. L. Stalling, and J. L Johnson. Determination of part-partrillion levels of polychlorinated dibenzofuran* and dioxins in tnvironmental samples. AnaL Chem 35:1830-1842(1984). 7. Higginbotham. G. R.. A. Huang, D. Firestone. J. Verm, J. Reas, and A. D. Campbell. Chemical and toxicological evaluations of isolated and synthetic chloro derivatives of dibenzo-p-dioxin. Nature (Land.) 220:702-703 (1968). 8. .Jones, E. L,, and H. Krixek. A technic for testing acnegtnic potency in rabbits, applied to the potent acnegsn, 2.3,7.3-ietrachiorodibenxo-p-dioun. J Invest. Dermatol 30:311-317 (1962). 9- Bradlaw, .J. A., and J. L Cssuriine. Induction of enzyme activity in cell culture: a rapid screen for detection of planar polychlorinated organic.compounds. J. Assoc. Offie. Anal Cham. 82:904-916, (1979). 10. Knutson. J. C.. and A. Poland. Karacinizstion of mouse teratoma call line XB produced by 2.3.7,8-tetrachtorodjbenzo-p-diozin: an in vitro model of toxicity. Cell 22:27-36 (1980). 11. Albro, P. W,, M. I. Luster. K. Chae, S. K. Chaudhary. G. Clark, L. D. Lawson. J. T. Corbett, and J. D. McKinney. A radioimmunoassay for chlonnatec dibenxo-p-dioxins. Toxicol AppL PharmacoL 50:137-146 11979). 12. Luster. M. L. P- W. Albro. K. Chae. L. D. Lawson. J. T. Corbett, and J, D McKinney. Radioimmunoassay for quantitation of 2.3.7.8-teirachlordibenzofuran. AnaL Chem. 52:1497-1300 (1980). 13. Luster. M. I., P. W. Albro; G. Clark. K. Chae, S. K. Chaudhary, L. D. Lawson. J. T. Corbett, and J. D- McKinney. Production and characterization at ' antisera specific for chlorinated biphenyl species: initiation of & radio immunoassay for aroclors. Toxicol AppL PharmacoL 30:147-155 (1979). 14. Goldstein. J. A. Structure-activity relationships for the biochemical effects and the relationships to toxicity, in Halogenated Biphenyls, Terphsn\ls Naphthalenes, Dibemodioxmt and Related Products iR. Kimbrough, ed.l Elsevier/North-Hollsnd Biomedical Press. Amsterdam 11980). 15. Poland, A., W. F. Greenlee, and A. 5. Kende. Studies on the mechanism oi action of the chlorinated dibento-p-dioxins and related compounds. Ann. N't Acad. Sci. 320:214-230 11979). 16. Poland. A., and J. C. Knutson. 2.3.7.8-Tetrachlorodibenzo-p-dioxin and re lated halogenated aromatic hydrocarbons: examination of the mechanism ot toxicity. Anno. Rev. PharmacoL 22:317-554 (1982). 17. Jones, P. B. C.. L K. Curtin, D. R. Gaieaxzi. and J. P. Whitlock. Control of cytochroma Pt-450 gene expression: Analysis of a dioxin-responsive enhancer system. Proc. NatL Acad. Set. USA 83:2802-2806 11986). 18. Bradfield. C. A.. A. S. Kendt, and A. Poland. Kinetic and equilibrium studies of Ah receptor-ligand binding: use of {TMl]2-iodo-7,8*dibromodibenzo-pdioxin. Mol PharmacoL 34:229-237 (1988). 19. Ekina, R. P.. G. B. Newman, and J. L. H. O'Riordan. Theoretical aspects of 'saturation' and radioimmunoassay, in Radioisotopes in Medicine: [n Vitro Studies |R. L Goswitz and B. E. P. Murphy, eds.). U.S. Atomic Energy Cammisiion, Oak Ridge, TN (1968). 20. Berson. S. A., and R. S. Yalow. Quantitative aspects of the reaction between insulin and insulin binding antibody. J. Clin, Invest. 38:1996-2016 119591. 21. Rodbard. D., and J. E. Lewald. Computer analysis of radioligand assay and radioimmunoassay data. Acta EndocnnoL 64(suppl. U7):79-103 (1970. 22. Motulsky, H. J.. and L. C. Mahan, The kinetics of competitive radioligand binding predicted by the law of m m action. .Wof. Pharmacol. 25:1-9 <1964). 23. Cheng, Y--C., and W, H. Prusoff. Relationship between the inhibition con stant (Ki) and the concentration of inhibitor which causes 30 percent inhi bition <IM) of an enzymatic reaction. Biochem. PharmacoL 22: 3099-3106 (1973). 24. Lindan, J. Calculating the dissociation constant of an unlabeled compound from the concentration required to displace radiolabel binding bv oO1^. J. Cyclic. NucL Res. 8:163-172 (1982). 23. Poland. A., E. Glover, and A. S. Kendt. Stereoapecific, high affinity binding of 2,3,7,3-tetrachlordibenzo-p-dioxin by hepatic cytosol. J BioL Chem. 231:4936-4946(1976). 26. Kende, A. S.. J. J. Wade, D. Ridge, and A. Poland. Synthesis and Fourier transform carbon-13 nuclear magnetic resonance spectroscopy of new toxic polyhalodibenzo-p-dioxina. J. Org. Chem. 39:931-937 (1974). 27. DeLean, A., P. J. Munson, and D. Rodbard. Simultaneous analysis of families of sigmoidal curves; application to bioassay, radioligand assay, and physio logical dosa-reaponae curves. Am. J, PhyswL 233:E97-E102 11978). 28. Seatchard, G, The attractions of proteins for small molecules and ions. Ann. NY Acad. ScL 51:660-671, (1949). 29. Bannet, J. P.. and H. I. Yamamura. Neurotransmitter, hormone, or drug recaptor binding methods, in Neurotnnamitter Receptor Binding IYama mura, H. I., Enna. S. J. and Kuhar. M. J., eds.). Raven Press, New York 11985). 30. Owen, D. B. The power of Student's t-teat. Am. Statist. Assoc. J. 60:320-333 (1965). 31. Box. G, E. P., W. G. Hunter, end J. S. Hunter. Statistics for Experimenters. John Wiley it Sane, Inc.. New York (1978). 32. Poland, A., end E. Glover. 2,3,7,8-Tetnchlorodibento-pani-dioxin and en zyme induction, in Chlorinated Phenoxy Acids and Their Dioxins tC. Ramel. <kL). Ecologieai Bulletins. Swedish Natural Science Research Council, Berlinga. Lund, Sweden (1978). 33. Toftgard. R-, G. Lofroth. J. Carlatedt-Duke, R. Kurl, and J.-A. Gustafsson. Compounds in urban air compete with 2,3.7.8-tatrachlorodibenzo-p-dioxin for binding to tha receptor protein. Chem-BioL Interact. 46:335-346 (1983). Send reprint requests to: Dr. Alan Poland, McAidle Laboratory for Cancer Research. University of Wisconsin. Madison, W! 53706. GENP 011273 784084 ASKAREL:' REQUIEM FCR AS CID RELIABLE David C. iords,. Weyerhaeuser Cs. Tacana, Washington Abstraer For several decades, askarel containing poly chlorinated biphenyls (PCB's) was .considered an excellent Insulating flu id . In the span of a few short years, i t has cone under attack as a pernicious and ubiquitous environmental contaminant. Regulations concerning th is m aterial are evolving ra p id ly , and a to ta l ban is u ltim ately expected. S p ill prevention, control and disposal are required to preclude high economic loss as well as adverse p u b licity . A lter nates to the use of askarel include new flu id s th at oust also be reviewed fo r environmental a c c e p ta b ility . A. Traces of two very sim ilar chemicals (see Figure 3), polychlorinated dlbeiizofuran and polychlorinated dibenzodioxln, have been found in the tis su e s of animals which have absorbed PCB's. The m ateria ls may be present in the o rig in al PCB or may be metabolized from ?C3 by the animal. These compounds are known to be highly toxic. 5. ?C3's are ubiquitous and are apparently mobile ir. the atmosphere. They have even been found in snow a t Che A ntarctic. Introduction U ntil ra th e r re cen tly , our society used many chemicals with seemingly few environmental problems. In a short span of time, several factors contributed to a su b stan tial change in the environment. A rapidly expanding population, enjoying Che f r u its of the most advanced technology and g reatest affluence in the h isto ry of mankind, created a demand for more goods, often basedton a host of new chemicals and m aterials. Consequently, genuine and serious environmental problems were becoming common. In the l a t t e r paye of the 1960's, there was a ev mood of so cial awareness and activism . Con currently, the rapid advances in technology also included sig n ific a n t improvements in the means of detecting and measuring eoneaoleancs at remarkably l o w level 5 . The stage was sec for the sudden change in the sta tu s of a sk a re l, an old re lia b le . PC3's - The Problem 6. They are being found a t sig n ific a n t le v e ls in human tissu es and 1q m other's milk. About can years ago, PCB's were f i r s t is o la te d and Identified as a possible contaminant c o n trib u tin g to problems in animals. At that time, th is chemical was in wide use. About h alf was u tiliz e d as the p r i mary constituent of askarel in e le c tric a l equipment (askarel also contains trichlorobenzene as a so lv en t). The remainder of the PCB was used in p la s tic s , p a in ts , carbonless paper, and many other products and processes. Since few problees had ever been noted with the m aterial, any scrap or waste was in d isc rim i nately sewered or placed in dumps, as most chemicals had been handled for quite some time. One of the ch a ra c te ristic s chat made PC3's or askarel a ttra c tiv e was i t s s ta b ility ; but th is v ery permanency also meant th at the m aterial' remained v irtu ally intact for decades. It is generally conceded today that th is residual of PCB's w ill be present as a contaminant far decades, regardless of complete elim ination from the market place. Polychlorinated biphenyls are produced by su b stitu tin g one to ten chlorine acorns for hydrogen atoms on the biphenyl s tru c tu re shown in Figure 1. There are 209 possible combinations or isomers; for example, there are twenty-four d iffe re n t possible arrangements for the molecule containing three hlorine atoms. One of these trlchlorobiphenyls is illu s tr a te d by Figure 2. "ie manufacturing process re s u lts in a loosely .-or.rrtiled mixture of various amounts of manv of *na lenca the c h s ra c ts rls tlc s of a t a r c l c i i i r batch is 'h e sum of the several individual rnmuenancs. some general conclusions about these .'.oeponccn nre: 1. They eause abnorm alities in c e lls , organs and glands i'f aar.v animals. This occurs a t ra th e r low lev d s .' the chemical. 2. The macerlal is c u ite stable and barslv re a c ts, leqrade*. ir Jecompose* under normal circumstances; It is quite insoluble In water. It is heavier than water and is ingested by irrcm-feeamg animals. Animal tend to bioacturu le te the cruin.ica1, i . e . . It la absorbed read ily end excreted alowLy, with a re su ltan t high level becoming stored .n tissu es. I n i t i a l Concrol E fforts PCB's because of th e ir excellent s t a b i l it y and thermal properties, have been used as a h e a t-tra n s fe r flu id in several industries. Is Japan during 196S, a food product was contaminated with PCB's from a heac-exehange operation and more chan 12C0 people were a f f litte d , some rath er severely, with what was called '"Tusho" disease. This outbreak, plus ocher incidents, and a rapidly expanding research e f f o r t Identifying PCB'e as a potential detrim ent, Led the sola U.5. producer (Monsanto In d u strial Chemicals Comaany) to voluntarily r e s tr ic t sales to d o se d e le c tric a l systems. This step removed about half the PCB's from the maTkec, and began the elim ination of the mast common uses whereby PCB's were being introduced co the environment, such as through carbonless o ffle a papers. Of course, outdated capacitors and the lik e continued co be thrown on the Junk p ile . I n 'la te 1975, the D.5. Environmental P ro tectio n Agency sponsored a PCS conference wtilch was been technical and p o litic a l. The technical program wax enlightening; there was l i t t l e doubt that hard s c ie n tific evidence identifying C3's as a major conearn was developing quickly. The p o litic a l aspect of the conference came from two sources. F ir s t, the GENP 011274 784085 36 1 LAS *77 A N N U A L public secto r ws allowed to sake presentations, the m ajority of vhleh predictably were emotional and often based an misinformaeion. . The second p o litic a l aspect arose as representatives iron various govern mental agencies - FDA, In te rio r, NIOSH. Com erct, e tc . - made statem ents to explain past indifference and to scourge the plaguo of the 7 0 's, PCS's. There was l i t t l e doubt th a t the bureaucracy f e lt safe in taking a stand, and th at regulation or r e s tr ic tio n was imminent. There is strong public support for control since the mass of the populace is. technically naive and thoroughly frightened by the constant d is closure th at everything from lic o ric e to maraehino ch erries say be mutagenic, terato g en ic, or carcino genic. In th is context, and knowing th at there were soae su b s titu te m aterials forthcoming, the equipment manufacturers began phasing out of askarel and into new flu id s . N aturally, the flu id supplier (Monsanto) recognized chat the market was disappearing and made the decision to* term inate a l l production of ehe ehemlcal la te r th is year. Thus today, th ere are three questions to be'answered: 1. What should be expected concerning the ask a relf ille d equipment now in service? % 2. Exactly what Is available in the future? I 3. What assurance is there th a t ehe course taken new Is c o rre c t: can th is happen again with a substitute sacerial? Anticipated Pressure I Ir these times ofOSHA, IAS, FDA, I?A, Consumer Protection Agencies ana the lik e , i t Is apparent th at users of equipment containing askarel can expect the following:j 1. S pecific re g u la tio n s w ill be published for askarel handling, p ro tectio n , d isp o sal, e tc . For example, sne s ta te is ;In the process of promulgating a reg u latio n chae Includes mandatory labeling of equipment containing more chan two pounds of PCS. 2. Users w ill ba asked to Inventory askarel on the plant premises and to |in d ic a te the lo catio n of equipment in reference to waterways. i 3. Monitoring of fluents w ill be required; th is is already being specified in 7TPDES permits in soae locales. | 4. P rotective measures, such as dikes, w ill be made SanaatoC7. 3. Ic 'u y be required to include askarel in the s p ill clan* which most plants already have. *. Any accidental rip lll w ill have to be rep o rted , even i f i t was apparently completely controlled on sne p lan t s i t e . I t the s p ill should Involve more chan a c e rtain q u an tity , a regulatory agency may v is it the alee. 7. I f a s p ill should' escape to a waterway, recovery of the lo s t askarel w ill be required. There have been same, spedC ie (end very texpensive) Instances where chia has been don In the past. 9-; There w ill be a ban on new in sta lla tio n s of > a sk a re l-fille d equipment, which w ill be rather | meaningless since Industry is already voluntarily abandoning the market. Sue there w ill ultim ately also ba a ban forbidding the continued use of e x istin g equipment. This coo may prove to be meaningless [ because the unavailability of askarel for topping out or re tr o f illln g transformers may re su lt in early retirem ent of most a sk a re l-fille d equipment. There are su b stitu tes available for topping out, but there is a lim it to their use. Loss Prevention and Control j U ntil quite recen tly , askarel s p ills were v ir tu a lly Ignored. As atten tio n focused on the problem, many incidents came to lig h t. S p ills or losses are known to have occurred under these circumstances: 1. As a consequence of moving or handling. 2. Overloaded or fau lty capacitor. 3. Overloaded and overheated transformer fluid through the re lie f valve. which lj o s e s I Changing out carbonized askarel from a shorted transform er. 5. Leaks from valves on the transformer easing. ' 1 ^ fs 6. Loss when sampled for d ielec tric or m oisture, te s t. 7. Improper disposal of sample. | 3. Misuse - mistaken for another chemical. This has occurred when waste askarel was placed in a used drum without relab elin g . | 9. Damage to drums or equipment by other equipment such as f o r k lif ts . 10. Freak incidents, such as an ic ic le piercing a transform er coaling fin , or a capacitor skewered by the arrow of a fru strated bow hunter. Two actions should be taken Immediately to pre clude such occurrences. A rather detailed Inventory should be taken of every askarel transformer and| capacitor aa shown In Figure 4. While most p la n ts have some form of inventory for e le c tric a l purposes, they do not include information which w ill allow jah evaluation of the environmental risk . I t is suggested th at o il transformers be Included in the la v a to r y since even these present potential environmental j c o s ts . Inactive units or stored flu id s should also be noted The second most immediate action la to inform or educate the plant s t a f f . There are cwo o b jectives (1) to prevent or control s p ills , and (2) to ensure that personnel are not confused or frightened by inadequate knowledge of a potential problem. I t is suggested that an appropriate label - larg e and bright - be placed on askarel equipment. I t I is also necessary to ensure chat contract personnel are informed to look for these labels, and i t may b[ advisable to specify in writing the re sp o n sib ility for s p i l ls caused by a contractor. | j 784086 GENP 011275 ii After teaking an inventory of askarel equipment, waters, the boctea is dredged. The material f i r s t decisions muse be made whether to invest in pre*section or sp ill control for a particular ices. This n a tte r is a rb itra ry , but i t Is suggested that the icons be rated by v u ln e ra b ility to dasage, quantity of a sk a re l. and proximity to a waterway. Thus the larg er transform er located by a road with an adjacent running ditch going toward the riv er passes through a se ttlin g chamber, then through a sand f i l t e r , and fin a lly through an activated carbon column. All of the wee solids require disposal. \ Obviously, every effo rt should be made to avoid a s p ill of th is nature; the cost is horrendous.' The fin al b i l l for cleaning ehe s p ill of 265 gallons Inca the Duwaaish Waterway was 5500,000l w arrants more consideration than the sm aller unit insid e a building away from any t r a f f i c . Proper disposal of vasca askarel, scrap equip ment, or solids wastes Is rather well defined. i T h e'p o ten tial for a co stly s p ill ean be reduced by preventive measures. For example, place fences or poses around exposed equipment; a lig h t roof say be added to prevent dasage from ice or dropped to o ls. Relocation of the equipment Is an a lte rn a te . A plug should be threaded into the drain valve of a trans former. F in a lly , many s p ills could be prevented by operating equipment a t the design racing instead of 1102 plus. Askarel say be transported as a liq u id or gelled ; with molecular sieves. About twenty f a c ilitie s j ara available for burning or la n d f ill. Burning requires quite high temperatures to ensure complete destruction, and the exhaust gases must be scrubbed with caustic co remove the hydrochloric acid that! Is emitted. -Landfill can only be in certain approved s ite s . Transportation of FCB's co a disposal sice requires special preparation and handling. In one incident Involving a s p ill from a 1500 kVA t r a n s - ! In ev itab ly , equipment w ill f a l l or freak ac ci dents w ill occur; several steps may be taken to con ta in sp ille d a sk a re l. Dikes can be in sta lle d around equipment preferably with a capacity s lig h tly greater former, transportation and incineracin cost $2,200. In another incident involving the rupture of a pole- mounted capacitor, transportation and burial cost $150. than the la rg e st volume of askarel in one u n it. Xf the lo catio n is outdoors In a wee e lim a tt, a device Environmental A cceptability of Alternase Compounds known as an imbiber valve, whieh contains molecular I siev es th at pass water but reeain PC3's, can be in Several alte rn a te flu id s are now available in s ta lle d when the dike is constructed. Fasele shields new transformers and capacitors. can be placed around pole-mounted capacitors to r e s tr ic t a s p ill to a lesser area. Silicone-based fluids are offered for liquid- j fille d transformers. All evidence thus far indicates, A ll drain holes In v au lts should be plugged, Char the m aterial is quite innocuous. Ic should be ( and ramped s i l l s placed a t doors. All jo in ts should be sealed with a silic o n e putty, arid concrete block w alls should be sealed with silic o n e paint or an noted rhac unlike askarel, the silico n e fluid has a specific gravity le ss than one, and any s p ill would have to be created as an ordinary o il s p i l l. j equivalent coaclng. Should askarel be s p ille d , ehe precise amount of cleanup or reclamacin Is a rb itra ry - chare are no standards y et. Ac the time of the s p i l l in the Obtaining detailed environmental information from the vendors on th a ir new capacitor flu id s has been j d i f f ic u lt, and much of i t is not comparable. For j example, one vendor w ill report on the toxic effec ts Duwaaish Waterway (S ea ttle) in 1971, the E?A term i nated the operation when the PC3 content was reduced to 77 p a rts per t r i l l i o n ; th is should in d icate th at a thorough co llectio n la expected. on a certain fish species on the b asis of a 16 day t e s t , while another uses a 96 hour te s t . All the new m aterials o ffer d e fin ite Improvements over PCB'i however. On the basis of a ll the daca-coxiclty, blodegradabilicy, so lu b ility , e tc . - whieh th is C ollection of v is ib le or sesnding askarel can be w riter has been able to obtain, "the three available in itia te d with an absorbent compound. Rags with so l flu id s should be environmentally ranked: vent such ss trlehlorobenzene should bs used to wipe down equipment, poles, e tc . Soil should be removed Host acceptable - Isopropyl biphenyl to a depth exceeding the obvious le v e l to which the flu id soaked, and ra d ia lly beyond ehe l a s t v is ib le signs of f lu id . Boards o r ehe Ilk a should be re Second - butylsted chlorodiphenyl oxide placed. All solid wastes - rags, gloves, so il, shovels, e tc . ss well ss liquids should bs see aelde Third - di - 2 - ethylhexylphthalate for proper disposal. (a phchalate ester) Should a s p i l l e a te r ah In tern al sewer, the water flow should be terminated and askarel colleeeed from the low spots. If the sever discharges into a sewage p la n t, most of the PCS th a t is carried In the water w ill attach to suspended matter in the treatment p lan t and s e t t l e . Of course, these se ttle d m aterials usually end up In s la n d f ill eventually, but at le a s t most of the PCS apparently remains Immobilised. The worst s itu a tio n , of course, is If a s p ill taeers a waterway in ta c t. Koone has an answer for Che s itu a tio n where Che contaminant enters e fa st scream and disperses widely. For lakes or ra th e r s e ttle One suggested a lte rn a tiv e to the replacement of askarel transform ers has been co r e t r s f i l l with the silicone-based flu id s. This procedure Involves draining out a l l passible askarel, followed by flush ing with triehlorobcnzena. A ce rtain amount of askarel w ill be retained In pockets, insu latio n , e tc .; the residual PCS content Is estimated co be 1-3X, and w ill be very dependent upon the amount of eare taken and number of- flushings performed. I t should be noted that a ll flushing fluids w ill require disposal by methods approved for PCS'a. y* a.S C d x t,*; ^ 7'? 784087 0^ ^ .*-/,7 * -V 38 3 1 r f* i In th is age o t environmental awareness and social ac tiv ism , many chemical substances w ill ba s c ru ti nized in te n tly . Regardless of i ts re la tiv e impact, PCS Is currancly being cited as the leading hazardous ch caical which varrar.es co n tro l. Industry faces a new round of re g u latio n s, rep o rts, and coses. As w ith any such issu e, the coat ean be base controlled by gathering inform ation, making uncsoeional judg ments, and planning to take the appropriate action before the citatio n s are issued. \J \ JM FQW9 i * QtOft^fi MeltCJit S p e c ific a lly , these steps are recotraended: 1. Define the problem in the plane. Locate each item and assass the p ro b ab ility of dacage or io ss. Estim ate the ccst of equipment protection and s p ill control. 2. Cat inform ation from the vendors on th e ir new equipment, including very specific data to back any d a le s of environmental properties. figft 2 figuft 2 - l - 2 '. r **ncMcrD'0'itnyl 3. .Ear transform ers, determine the efficacy of r e tr o f i l li n g and the probable a v a ila b ility of capping flu id . I4 4. Proceed with protection, te tro fillin g , or replacement on a scheduled b asis. In oany instances, the choice w ill not be obvious, and I t is suggested th a t i t is b e tte r to e rr on the side of greater environmental protection. 3. In some instances where only a few pieces of equipment ara Involved, consider that to ta l elim i nation of aakarel may cost less chan the hidden costs of continually reporting, inspecting, etc. $^ Noona ean be completely sure that the steps taken w ill preclude a co stly s p i l l of ehls hazardous chemi c a l. Should one occur, however, the plane that has evinced awareness and e ffo rt w ill fare b e tte r with th e regulatory agencies and the public than a plane which ignored the problem of PCB's. \A, Tttracfthn Oibmnfvrai ftg t 2 - PotnM PCB Mrtaokt? Trn(*i*ri T** 1 U x m Las i tut u o ' t. nrt I iMlll 3 4 J W M M JW* Sfr. VMt tiM et c*M cr WA 1300 ISO M . 1000 Iatl*tlSl nie oi. IM II M in t i l l *1 Nttnilil m,h. M i U lug.,. aaphalc irt* 2 il<Hi r.tdiii Mt' i u r w *f fnvtraewaneal Steal!ivlc? M I i m . 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