1 15 2 Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 034 Folder The folder containing the original item. 0690 Series The series number of the original item. Series III Subseries I Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Gross, Michael L. Jackson O. Lay, Jr. Philip A Lyon Dixie Lippstreu Nancy Kangas Robert L. Harless Scott E. Taylor Aubry E. Dupuy, Jr. Source A related resource from which the described resource is derived Environmental Research Date A point or period of time associated with an event in the lifecycle of the resource 1984 Title A name given to the resource 2,3,7,8-Tetrachlorodobenzo-p-dioxin Levels in Adipose Tissue of Vietnam Veterans Subject The topic of the resource VA Dioxin Fat Assay Study ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/d545c9c9d00a290332e61879c72f6d4a.pdf 6c5c1d89ed5b7a0421ac73c9c035d398 PDF Text Text Item ID Number °1488 Author Gross, Michael L. Corporate Author Report/Article TltlB Typescript: Analysis for 2,3,7,8-Tetrachlorodibenzo-pdioxin in Adipose Tissue from Vietnam Veterans Journal/Book Title Year oooo Month/Day Color n Number of Images 15 DBSCrlptOn Notes Draft version. See item 690 for published article. Tuesday, May 15, 2001 Page 1488 of 1514 �DO. A FT A n a l s i s for 2, 3,7, in Adipose Tissue from Vietnam Veterans, by Michael L.Gross, Jackson O. Lay, Jr., Philip A. Lyon, Dixie Lippstreu and Nancy Kangas Department of Chemistry University of Nebraska Lincoln, NE 68588 Robert L. Harless and Scott E. Taylor U.S. Environmental Protection Agency Health Effects Research Laboratory, ETD, ACS, MD-69 Research Triangle P a r k , NC 27711 Aubry E. Dupuy, Jr. U.S. Environmental Protection Agency OPTS/OTS, Exposure Evaluation Division Field Studies Branch Toxicant Analysis Center Bay St. Louis, MS 39529 �— 2 -* ABSTRACT Tetrachlorodibenzo~£-dioxin ranging from (TCDD) has been detected at levels 20-173 parts-per-trillion in adipose tissue from three Vietnam veterans who were "heavily exposed" to Herbicide Orange. The detection corresponded to a single isomer, having the characteristics of 2,3,7,8-TCDD, and was validated in inter laboratory studies for two of the three tissue samples. Tissue samples from other Vietnam veterans and from controls also contained 2, 3, 7, 8-TCDD at levels below 20 parts-per-trillion. These findings are in accord with uptake and long term storage of 2,3,7,8-TCDD by veterans who experienced a heavy exposure to Herbicide Orange. Furthermore, they may signal accumulation of 2, 3, 7, 8-TCDD in some members of the U.S. population. Aerial defoliation in Vietnam carried out by the United States military from 1962-1970 made use of Herbicide Orange/ a 1:1 mixture of the herbicides contaminant, 2r4,5-T and 2,4-D. The 2,4,5~T 2, 3,1, 8-tetrachlorodibenzo-p_-dioxin contained (2, 3, 7, 8-TCDD) a at levels sufficient to produce a mean concentration of about 2 partsper-million in the defoliant. The ,.2f 3, 7, 8-TCDD, in addition to being extremely toxic, is known to be carcinogenic and teratogenic in animal 2 tests. Vietnam veterans now claim adverse chronic and delayed health effects related to their exposure to Herbicide Orange. These claims and recent publicity regarding the m i l i t a r y defoliation program have increased public awareness of the potential health e f f e c t s of exposure to 2,3,7,8-TCDD, especially among Vietnam veterans. �- 3 - . We report here the results of a preliminary study designed to determine the range of TCDD levels that might exist in the tissue of veterans. Specifically, we wished to determine if 2,3,1, 8-TCDD could be detected at levels above 1-5 parts-per-trillion. was chosen as the preferred sampling medium Adipose tissue because of its good accessibility (compared to other tissue) and because 2,3,7,8-TCDD is O known to accumulate in this tissue in other species. ' wished to examine whether any correlation exists A Secondly, we between exposure (assigned by the Veteran's Administration ) and observed levels. existence of a correlation could signal the need for The additional monitoring studies. In order to have confidence in the results generated from test samples, it is necessary to demonstrate 'analyses of TCUD at low ppt levels. laboratories involved in this the capability to conduct This has been demonstrated in the analytical work by means of blind validation studies in which coded spiked samples of beef fat and human milk were analyzed. ' Additional validation of the methodology used in this study for low levels of TCDD has been demonstrated at the 1100 part-per-trillion range using human and beef adipose tissue (see Table 1) . The Veterans" Administration supplied thirty coded samples of adipose tissue which had been surgically removed from the abdominal area of "exposed" veterans and control subjects who had not served in Vietnam. Additional tissue was taken from three U.S. Air Force officers with several years experience in TCDD research. Particular care was exercised, even in tissue removal, to avoid any potential contamination. The samples were placed in rigorously cleaned glass �- 4 - vessels, f r o z e n , shipped in dry ice, and stored in a f r o z e n state (20 C) . No disinfectants containing hexachlorophene or other trichlorophenol-based materials were used. Approximately 5 to 10 grams of tissue were used, when available, for each analysis. standard (either 37 A known amount, generally the adipose tissue. hydroxide followed hexane extract 13 Cl-2, 3,1, 8-TCDD or by extraction was involved the use of resolution employed gel, washed for internal C-2, 3, 7, 8-TCDD) was added to with hexane to with remove TCDD. concentrated The final sulfuric The acid, stage of clean-up three short-column liquid chromatography steps alumina, mass of The sample was digested in alcoholic potassium neutralized, d r i e d , and concentrated. (silica 2 ng, and Florisil). spectrometry quantitation (as of Q Gas described 2,3,7,8-TCDD chromatography/high previously 8 ' 10) was and coeluting isomers. Signal profiles were obtained at a mass resolution of 10,000 for m/z 321.8936, the most abundant molecular ion for TCDD, and for the internal standard mass by signal averaging for approximately 100 sec. commencing with the appearance of the co-eluting internal standard 2, 3, 7, 8-TCDD. / Extracts which contained materials giving signals greater than 2 . 5 times noise at the exact mass of TCDD (i.e. 321.8936 ± 0.0020) over the integration period discussed above were reanalyzed. second analysis, signal profiles of For the m/z 321.8936 and m/z 319.8965 were monitored over the elution period of 2,3,7,8-TCDD (determined by injection of standard solutions). if A positive detection was reported signals were observed above the detection limit ( 2 . 5 times noise) and if their intensity ratio was 1.0:0.78 + 0 . 1 0 , which is consistent �- 5 - with the presence of meeting all four chlorine atoms in the molecule. Samples criteria except the correct isotope intensity ratio have been considered to contain "not detectable" levels of TCDD. For these samples, we judged that the presence of TCDD is not disproved by the observation of an incorrect isotope ratio at these low concentrations; r a t h e r , the presence of TCDD is not confirmed. For the quantitation Nebraska, of all stage considered the Two of "heavily highest of analyses, samples were Midwest Center results) . contain first the for exposed" conducted at Mass three Spectrometry samples to both extraction the (see and U n i v e r s i t y of Table 2 for tissue from veterans Herbicide Orange 12 were found to levels of TCDD of detected in this study. The recovery (20%) of internal standard for the tissue sample of the t h i r d "subject was un-acceptably low. values, We consider the levels to be minimum and the higher level, in cases of replicate analysis, is judged to be more accurate. This is because the internal standard 37 signal for Cl-2,3,7,8-TCDD at m/z 327.8849 is susceptible to an 35 37 interference from m/z 327.8775 (C-, 2 H 5 Cl, C12) , a molecular ion of a polychlorinated biphenyl, which could enhance the intensity of the standard and lead to the calculation of too low a value for the TCDD using the internal standard method of quantitation. Three of five samples of tissue from veterans having "light exposure" were found to contain TCDD, as were tissue from two of the three Air Force officers who have done extensive research environmental and biological samples containing 2,3,7, 8-TCDD. with Seven of thirteen tissue samples from other Vietnam veterans were found to contain 2,3,7,8-TCDD or coeluting isomers at levels between 3 and 13 �- 6- parts-per-trill ion. Samples taken from veterans who had no service in Vietnam (controls) also showed low levels of TCDD (four of ten were judged to be "positive" at levels between 4 and 8 parts-per-trillion). In view of the limited isomer specificity 14 of the methodology used at the University of Nebraska (UN-L) certainty by interlaboratory validation these low levels, a subset of and/or reanalyzed at other the and the need to increase of reported detections tissue samples was reextracted laboratories (see interlaboratory validation was done in two ways. the first stage of analyses at at UN-L were Table 3) . The First, extracts from reanalyzed at the Environmental Protection Agency laboratory at Research Triangle Park (RTP) using capillary column gas chromatography/high resolution mass spectrometry (reported as UN-L/RTP in Table 3) . the tissues stored in the University of Nebraska transmitted to the EPA Toxicant Analysis Center and clean-up. The extracts were then Triangle Park laboratory for analysis 3) . Second, portions of laboratory were (TAG) for extraction forwarded to the Research (reported as TAC/RTP in Table All samples were coded at the University of Nebraska and their identities were not known to the workers at TAG or RTP. The tissues were extracted at TAG using methods similar to those employed at the University of Nebraska. the RTP laboratory made use of The quantitation conducted at capillary column GC and peak-top monitoring (rather than peak profiles) at a mass resolution of 600010,000, a method more specific for TCDD isomers than that employed at the University of Nebraska. All detections reported in Table 3 were for a material having an identical GC retention time as 2,3,7,8-TCDD �_ 7 — and no other isomers were detected at levels greater than 10% of the level corresponding to 2, 3,1, 8-TCDD. Some small degree of uncertainty remains that the material detected is the 2,3,7,8-TCDD isomer because standards for all 22 TCDD isomers were not available at the RTF laboratory. Based confirmed on the that interlaboratory two of three validation samples from studies,it veterans is now designated as having "heavy exposure" were found to contain measurable amounts of 2,3,7,8-TCDD at levels higher than any other participant's tissue in this study. is Adipose tissue from the third "heavily exposed" veteran a "possible positive"; i.e./ the positive detection made by RTF scientists was not validated by the group at Nebraska, presumably because of the problem with recovery. Both the TJSAF officers'" adipose tissues, which were examined in the interlaboratory validation, definitely contained 2,3,7,8-TCDD, but at lower levels than the tissue of "heavily exposed" veterans. Definite detections of 2,3,7,8-TCDD were made in the analyses of adipose tissue from other exposed veterans and veterans who never served in Vietnam (controls). The detection of 46 ppt for VA-21 is considered an aberrantly high value based on a comparison with four other analyses reported in Table 3. Ignoring this value, the highest level detected in these two groups (20 ppt) coincides with the 1owest detection made for the "heavily exposed" veterans. may signal base-line accumulation of TCDD in These observations some members of the general U.S. population. There is good agreement of the results obtained in both mass spectrometry laboratories particularly in terms of the assignments o£ �- 8 - whether the sample contains TCDD. Differences in concentrations were found for repeated analysis of samples containing the higher levels of TCDD. These d i f f e r e n c e s appear to be systematic; concentrations determined explained above. difficulty of by the Nebraska for example, the workers may be low as This lack of agreement is expected considering the performing concentrations. extractions and Accordingly, we do not analyses consider at these low the differences obtained for a given sample to be significant. Because TCDD could be detected in some control samples, a simple statistical analysis was used to test the significance of the 4 results. A contingency table for the hypothesis of independence was used' to test the supposition that "concentration levels of TCDD in adipose tissue of test Herbicide Orange". were used except individuals is unrelated to exposure to The exposure classifications as previously listed the Air Force scientists were excluded. concentrations of TCDD were classified in four categories: The (1) "less than four parts-per-trillion or not detected", (2) "4 to 10 parts-pertrillion", (3) "11 to 20 parts-per-trillion", and (4) "greater than 20 parts-per~trill ion". statistical The concentrations test were averages of. all detected" having detection limits used in the in both We did not use reports of greater positive detection made in separate analyses The "not detected" TCDD determinations made laboratories with the following exceptions. "not of than or equal to (see VA 8, 9, 19, 34) . assignments of samples having low recoveries of internal standard (VA 18 and 19, Table 2) were not used. Finally, determinations which appeared to be positive but yielded incorrect isotope ratios (see Table 2) were considered as "not detected" except �_ 9 — when another analysis showed that the third concentration "heavily classification exposed" veteran"*s positive" (VA 19) and tissue the of the other two "heavily considerably higher in all which fall was chosen to The encompass the which was considered "possible control concentration of TCDD (VA 2 0 ) . sample contained TCDD. sample having the highest The levels of 2,3,7,8-TCDD in tissues exposed" veterans were found to be analyses, and they were the only results in the f o u r t h category. The chi-square test showed that the hypothesis of independence was false at the 95% confidence level, indicating a correlation between degree of exposure to Herbicide Orange and tissue levels of 2,3,7,8-TCDD. In conclusion, Administration a<3 levels of Vietnam veterans "heavily exposed" 2,3,7,8-TCDD in designated by the Veteran's to Herbicide Orange carry low adipose interlaboratory analytical studies. tissue,as validated in The 2,3,7,8-TCDD levels found in these veterans are higher than for other exposed veterans or for the controls. The observations that the TCDD observed is a single isomer, and likely the 2,3,7,8-isomer found in 2,4,5-T, and that the levels in the "heavily exposed" group are higher than those observed in any of the other study cases are in accord with an interpretation that the exposure occurred in Vietnam. We emphasize that the confidence ascribed to this conclusion can'be increased (or decreased) by conducting a more thorough investigation which would include a larger Orange. sample of Vietnam veterans "heavily exposed" to Herbicide �- 10 REFERENCES i tion of Orange Herbicide by Incineration, Final^ ^ ^ Environmental Statement, 36 (Department of the Air Force, November 1974) . 2. For a review, see M.P. Esposito, T.O. Tiernan, F. E. Dryden, Dioxins (Research Reporting Series EPA-600/2-80-197, Industrial Environmental Research Laboratory O f f i c e of Research and Development, United States EPA, Cincinnati, Ohio, November 1980). 3. T.A. Gasiewicz, R.A. Nealy, 4. G. Reggiani/ The Estimation of the TCDD Toxic Potential ir± the Light of the Seveso Accident. Presented at the 20th Congress of the European Society of Toxicology, West Berlin (1978) . 5. L. E. Lee, P. Donahue, H. B. Greenlee, C. R. Mota, W. Schumer , P. A. Thomas, J r . , Science, submitted with this manuscript. 6. R. L. Harless, E.O. Oswald, M. K. Wilkinson, A. E. Dupuy, D.D. McDaniel and Han Tai , Anal. Chem. , 52_, 1239 (1980) . 7. R. G. Heath, Int erl a bo r at or y Method Validation Study for Dioxin, An Interim Report , (United states EPA, Human Effects Monitoring Branch, O f f i c e of Toxic Substances, Washington, DC, 1979) . 8. M. L. Gross, Tung Sun, P. A. Lyon, S. F. Wojinski, D. R. Bilker, A. E. Dupuy, R. G. Heath, Anal . Chem . , in press. 9. R. W. Baughman, M. Meselson, 5_, 27 (1973) . Fed. Proc. 37, 501 (1978) . Envi ronmental Jr., Health Perspectives^ 10. B. J. Kimble, M. L. Gross, Science, 207, 59 (1980) . 11. A number of method blanks (14) were extracted and analyzed with actual tissue samples so that one blank for approximately every three samples (including repeats) was examined. No TCDD was detected in the blanks at at an^average detection limit of 4 and a range of 1 to 11 parts-per-trillion. 12. The classification as to degree of exposure was made by the U . S . Veterans Administration (see reference 5) . 13. The packed column used in these studies has been evaluated by Dow Chemical Company scientists in terms of TCDD isomer specificity (see reference 15). In our hands 2, 3, 7, 8-TCDD, for example, could be separated from 1,3, 6, 8-TCDD with base line resolution. 14. Another interlaboratory study, TAC/RTP, was conducted prior to the two reported in table 3. Analysis of method blanks showed that the glassware in the TAG laboratory was contaminated w i t h 2 , 3 , 7 , 8 TCDD and other isomers at the time of the study. None of these results are reported h e r e . All glassware was replaced for the �- 11 later studies, which yielded the results given in table 3. Actual adipose samples VA-9 and 10 were presumably contaminated d u r i n g the time of the f i r s t extraction at TAG as they were found to contain 160 and 230 ppt respectively in the second TAC/RTP validation run (coded TCDD TAC/RTP ) . These results are also not reported in Table 3 because they are clearly not consistent w i t h results of other analyses of these samples. Samples VA-26 and VA31 were not present in the TAG laboratory at the time of contamination. They and additional portions of tissue samples for the t h i r d validation run (Coded TAC/RTP ) were transmitted to TAG after the laboratory contamination had been removed. 15. T. L. Nestrick, R. S. Lamparski, R. Stehl, Anal. Chem. (1979) . 51, 2273 16. E. W. Minium, Statistical Reasoning in Psychology and Education, (John Wiley and Sons, New Y o r k , 1970), p. 438. We thank Professor Y. L. Tong of the University of Nebraska-Lincoln for helpful discussion of the statistical methods. 17. We thank Dr. James Norman and Dr. Lawrence Hobson for helpful discussions. This work was supported by the National Science Foundation Regional Instrumentation Facility Program (grant number CHE7 8-185 72) , by the Veterans Administration, and by the U . S . Environmental Protection Agency. The contents of this paper, do not necessarily reflect the views or policies of any of these agencies. - �Table 1. Analysis of Control Samples for TCDD. Nature of Sample Human Human Human Human Fat Fat Fat Fat Sample Wt. (g) Spike (ng) Concentration1 Added (ppt) 14.80 14.80 14.70 15.55 2.00 2.05 2.00 2.00 0 6 16 38 9.91 10.44 1.00 1.00 2 96 Concentration Found (ppt) nd 9 20 41 3 Beef Fat Beef Fat 1. ppt = parts-per-trillion 2. recovery of internal standard 3. average of two analyses 4. average of three analyses 1 + .1 75 + 5 * Detection Limit 3 3 4 4 0.5 3 Percent Recovery 40 40 45 40 120 60 �Table 2. Results of Stage 1 Analysis of TCDD in Human Adipose Tissue1 VA- Code Number Concentration (PPt)2 Percent Recovery Ratio3 4 9 3 10 6 65 100+ 20 90 45 .85 .75 5 2 4 5 6 3 50 80 50 95 40 100 3 3 3 2 3 3 4 3 4 4 3 6 5 65 50 40 55 60 65 60 80 45 45 100+ 100 60 95 .90 .90 4 2 3 4 4 3 2 3 4 4 7 65 60 75 30 50 35 100 55 50 60 100 1.02 .92 .84 Detection Limit (ppt) "Heavily Exposed Veterans" 10 10 19 26 26 23 35 2 ND 99 63 "Lightly Exposed Veterans" 1 ND 13 ND 15 7 28 7 28 8 34 5 .77 .78 .85 "Possibly Exposed Veterans" 6 8 ' 9 11 12 14 16 24 24 25 25 27 29 30 5 51 ND 3 9 4 ND 5 5 12 10 ND 13 ND .77 .88 .74 .71 .78 .88 "Controls" 5 7 17 18 20 21 23 23 31 32 33 4 3 4,3" ND 5 6 8 6 7 4 14 .86 1.07 .78 .98 .74 .94 �Table 2 continued. VA Code Number Concentration (ppt)2 Percent Recovery Ratio3 50 85 50 Detection Limit (ppt) .77 .94 .76 "USAF Scientists" 2 3 4 5 4 6 2 1 2 1. Sample sizes ranged from 2.2 - 11. 6g for each extraction. Internal standard amounts used varied from 2.0 - 2.6ng/extraction. 2. ppt = parts-per-trillion; ND = not detected. 3. Ratio of intensities of m/z 320 and m/z 322. 4. Duplicate analysis of same extract. Acceptable values are 0.78 0.10. �Table 3. Results of Interlaboratory Validation Studies. VA Code UN-L/UN-L' UNL/RTP 36 UN-L/UN-L* TAC/RTP' 173 h "Heavily Exposed Veterans" VA-26 VA-10 VA-19 63,99 23,35 ND(3) e 86 20 ND(29) USAF Researchers VA-3 VA-2 4g 3" 10 5 .... .... 24 Other Vietnam Veterans VA-13 VA-8 VA-9 VA-15 VA-34 ND(2) 5 ND(3) 7 5 ND(0.2) 3 3 .. _ . ND(7) 5 h ____ 5 3 20 8 12 ND(3) -.«.__ .. .. . . . . ND(7) 18 ND(5) f .. .. Control s VA-17 VA-18 VA-21 VA-31 VA-20 4,3 ND(4)f 69 ND(4) 5 .... • 14 .... 46 9 19 20 Notes: (a) Extracted at UN-L/Analyzed at UN-L (from Table 2). The values given in parentheses are the detection limits. (b) Portion of the Extract from UN-L/Analyzed at RTP (c) Extracted at TAC/Analyzed at RTP (d) Another portion of tissue shipped from UN-L, extracted at TAC/Analyzed at RTP (e) Extracted at UN-L/ Analyzed at UN-L. Results obtained with knowledge of the code. (f) Poor recovery of internal standard (<40%). (g) Isotope ratio for m/z 320 and m/z 322 not correct, (h) See footnote 14 regarding these samples. � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 056 Folder The folder containing the original item. 1488 Series The series number of the original item. Series III Subseries II Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Gross, Michael L. Jackson O. Lay, Jr. Philip A. Lyon Dixie Lippstreu Nancy Kangas Robert L. Harless Scott E. Taylor Aubry E. Dupuy, Jr. Title A name given to the resource Typescript: Analysis for 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Adipose Tissue from Vietnam Veterans Subject The topic of the resource VA adipose tissue testing ao_seriesIII