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V4ji? n / 'i*; v ENVIRONMENTAL HEALTH PERSPECTIVES ...y *, ...Experimental Issue nalApril 1972 ``f ;.; '%-L-^Zz > * - ,, HONS 208101 department of health education and welfare public health service national institutes of health RETHEBDA MARViANO 20014 I am pleased to publish in this format the excellent papers presented at the December 1971 Con ference on PCB's sponsored by the National Institute of Environmental Health Scium-rR at the tjumt Roost Conference Center in Roufemont, North Carolina I have long been impressed by the need for a journal which would provide --a vehicle for the rapid publication of research findings on environmental factors which might have broad impact on human health, --a forum for exploration in context and perspective of basic research on environmental constituents, --a focus for those aspects of environmental research which have implications for human health; and --a medium for publication and exchange of adequately documented negative findings from research into these topics. It is my hope that Environmental Health Perspectives will fulfill these goals This is the first of four issues to be designated "experimental " If these are successful, we plan to publish regularly We hope that you will assist us, especially with these first four issues, to refine and improve our efforts so that the journal will become a respected vehicle for communication We invite your comments, suggestions, and contributions Subsequent issues will contain papers on a diversity of topics prepared specifically for publication, u well as contributions to s single topic conference like those of this issue In addition, we hope that our readers will make use of Environmental Health Perspective ss a forum for expression of relevant opinions in order that all of us working in thiB field may maintain a current view of findings and practices of importance to environmental health. Future issues will devote space to your letters on pertinent topics, to reports of research and to commentaries which help to improve our perspectives on the en vironment and its effects on human health. Dr. Lee and Dr. Falk, along with our distinguished Board of Editors, welcome your comments and contributions. I am confident that with their guidance and your advice and interest, Environmental Hmmlth Perspective* will be able to take b permanent place among the journals of major importance David P. Rail, M D , Ph D Director, National Institute of Environmental Health Sciences MOHS 208102 BOARD OF EDITORS "Douglas H K. Lee, M.D , Ph D "Hans L. Falk, Ph.D. Lawrence Fithbein, Ph D James R Fouts, Ph D. David W Gaylor, Ph D. Dll- Robert Coyer, Fh.D mil John A Moore, D.V.M, *C<y*dttor* STAFF ich j Robert M Namovicz, Managing Editor Judith EdmondB, Ralph Hester, Elizabeth James, ital Earl Linthicum, Lucille Reaves inn om l tn land ion, that ssut `Jce^ H'llt (*11- and iftlf nci` DHEW Publication No (NIH) 72-218 ENVIRONMENTAL HEALTH PERSPECTIVES a publication of the National Institute of Environmental Ileal ihKrienoce, National JnaUtutee of Health, Department of Health, Education and Welfare. Opinion* expreeeed herein, however, are the opinion! exclusively of their author* end an not neceaanty endorsed nor ehtred by any of the above afendea. ENVIRONMENTAL HEALTH PERSPECTIVES Eiperntuti! turn Rsmfcv m, April, 1171 Address Communications to, Manasm* Editor, ENVIRONMENTAL HEALTH PERSPECTIVES National Institute of Environmental Health Seienoee Box 12233 Research Triangle Park, North Carolina, 27700 Reprint* of Artirlee Should be Requested from the Principle Author I'nlea*Stated Otherwise HONS 208103 i AROCLOR u s registered trademark of the Monsanto Company. KANECHLOR m a trademark of the Kanegafuchi Chemical Induitry CLOPHEN u trademark of Bayer A6 of Germany PHENOCLOR ia a trademark nf Prodelec of France They are referred to in their several formulations throughout the papers in this publication many times without further acknowledgments. No endorse ment is intended by this statement SUBSCRIPTION INFORMATION The first four issues of EndreRimataf Health Perspectives are available in single copies at no charge Tboee receiving copies by mail are included on a mailing list which will be used for distribution of these four experi mental issues. Information about opportunities for future subscript ions will be supplied when svsiisble 11 HONS 208104 CONTENTS Paqt Editorial. Perspective on PCBs ..... J. W. Cook. Some Chemical Aspects of Polychlorinated Biphenyls (PCBs) 0. Hutzinger, S. Safe and V. Zitko. Photochemical Degradation of Chloro-biphenyls (PCBs) . .... 1. C. T. Nisbet and A. F. Sarofim. Rates and Routes of Transport of PCBs in the Environment R. W. Rlsebrough and B. de Lappe. Accumulation of Polychlorinated Biphenyls in Eco systems ... . V. Zitko, O. Hutzinger and P. M. K. Choi. Contamination of the Bay of Fundy--Gulf of Maine area with Polychlorinated Biphenyls, Polychlorinated Terphenyls, Chlorinated Di- lienxodioxins, and Dibenzofurans . G. D. Veith. Recent Fluctuations of Chlorobiphenyls (PCBs) in the Green Bay, Wisconsin, Region . G. F. Fries. Polychlorinated Biphenyl Residues in Milk of Environmentally and Experimentally Contaminated Cows , . M. Kuratsune and Y. Masuda. Polychlorinated Biphenyls in Non-carbon Copy Papier P. E. Trout. PCB and the Paper Industry--A Progress Report F. Berglund. Levels of Polychlorinated Biphenyls in Foods in Sweden . II. A. Price and R. L. Welch. Occurrence of Polychlorinated Biphenyls in Humans A. R. Yob*. Levels of Polychlurinated Biphenyls in Adipose Tissue of the General Population of the Nation . . D. I. Hammer, J, F. Finklea, L. E. Prieater, J. E. Keil, S. H. Sandtfer and K. Bridbord. Polychlorinated Biphenyls Residues in the Plasma and Hair of Refuse Workers A. C. Kolbye. Food Exposures to Polychlorinated Biphenyls.. R. B. Dahlgren, R. L. Linder and C- W'. Carlaon. Polychlorinated Biphenyls: Their Effects on Penned Pheasants .. ... D. B. Peakall, J. L. Linear and S. E. Bloom. Embryonic Mortality and Chromosomal Alter ations Caused by Aroclor 1254 m Ring Doves. , . .. J. G. Vos. Toxicology of PCBs for Mammals and for Birds ... M. Kuratsune, T. Yoahimura, J. Matsuzaka and A. Yamaguchi. Epidemiologic study on Yusho, a Poisoning Caused by Ingestion of Rice Oil Contaminated with a Commercial Brand of Polychlorinated Biphenyls ... M. Kuratsune. An Abstract of Results of Laboratory Examinations of Patients with Yusho and of Animal Experiments .. .... D. II. Norbaek and J. R. Allen. Chlorinated Aromatic Hydrocarbon Induced Modifications of the Hepatic Endoplasmic Reticulum: Concentric Membrane Arrays , J. Biiman, H. C. Cecil and S. J. Harris. Biological Effects of Polychlorinated Biphenyls in Rats and Quail . .. E. P. Lichtenstein. PCBs and Interactions with Insecticides . R. lfooplngarner, A. Samuel and 1). Krause. Polychlorinated Biphenyl Interactions with Tissue Culture Cells . . .. .. 1). Stalling and F. L. Mayer. Toxicities of PCBs to Fish and Environmental Residues L. K. Cutkomp, II. H. Yap, D. Deaaiah and R. B. Koch. The Sensitivity of Fish ATPases to Polychlorinated Biphenyls . .. i 3 15 21 39 47 51 55 61 63 67 73 79 83 85 89 103 105 119 129 137 145 151 155 159 165 ill ~ HONS 208105 -tn . - W. B. Kin ter, L. S. Merkens, R. H. Janicki and A. M. Guarino. Studies on the Mechanism of Toxicity of DDT and Polychlorinated Biphenyls: Disruption of Osmoregulation m Manne Fish J.E. Kell, C. D. Graber, L. E. Pries ter and S. H. Sandifer. Polychlorinated Biphenyls (Aroclor 1242). Effects of Uptake on E colt Growth. . H. P. Tomberga. The PCB Situation in Germany . N. IVelaon. Comments on Research Needs 175 179 181 Perspective on \ The discovery at polychlorinated biphenyls (PCBs) in ftsh front, the Baltie Sea attracted, widespread attention among scientists. Prior to '' \ \ \ that eyent In. 1966 the many formulations of \ \ PCBs--tsome \210 isohiers are theoretically \A glance at the table of contents for the first possible;--were (thought Vo be UBed largely in laahe of Environ mental fteallh. Perspective* controlled or closed systems. That they were gives, the reader some idea abodt the quantity of present ip hah, and were later discovered In birds, research being done to specify the extent of the added to ithe concern since the inevitable la^t link PCBs story Furthermore, while the spectrum of m the food chain was man h\msclf. \ work presented is comprehensive, it is neither Concerns were lleiglitcned Oyen further by tlie all-inclusive nor definitive. Rather, our hope is knowledge that one of the primary characteristics that this volume will contribute a new perspective winch made PCBs to useful during the put 40 to the study of PCBs and will help as well to years was ^heir extreme stability!under a variety engender a renewed vigilance about other "inert" of circumstances. Furthermore, t|>eir structural chemicals which are so pervasive a part of the resemblance to the persistent drgano-clilorine \environment of man. pesticides raised other questions about whether ' The reports which .follow were originally all of our `worries oUer these pesticides were prepared for a Conference on PCBs, sponsored by accurate or whether, ae some suspected, PCBs NIEHS at the request of dn Interdepartmental were the real, cause of that concern \ Task Force on the subject, Tt Conference was Alert epidemiologists and other scientists in held at the Quail Roost Conference Center in Japan confirmed our expectations of the worst in Rougeihont, North Carolina, on December 20-21, 1969 with their startling discovery and description 1971. The papers presented at that meeting of Yuslio, a hupnan disease paused by consumption appear here substantially as they werfrpresented of large quantities of PCB-contam mated rite oil Your comments on them arc indeed welooine April 1972 HONS- 208107 1 Environmental Hwailh Pmpvxiim Some Chemical Aspects of Polychlorinated Biphenyls (PCBs) by J. William Cook* In the past few years considerable scientific information has appeared on the chemistry and biological effects of the PCBs and related ma lerials The amount of chemical investigation, and especially the biological effects investigation that can be done on these products, is almost limitless Even though the PCBs show a great deal of inertness, a property very useful in their industrial applications, they certainly do change in most instances before they are inadvertently consumed by man. It is important that we learn more about the nature of these products and the changes they undergo. 1 believe that the choice of scientific investigation that will provide effective answers is going to be made by close cooperation between the scientists in the field of chemistry and those in the various biological disciplines. I hope that conferences like this one will help give us guidance as to the most fruitful avenues to follow in re search and wilt assist in the interpretation of the data obtained. It is simpler to acquire data than to interpret them. No attempt has been made to make or present a complete survey of literature on chemistry of PCBs. Instead, I have chosen to discuss in a general way some facets of the chemistry which 1 think are interesting and relevant to the subject of this conference. For the most part I have drawn upon illustrations from our FDA labora tories because of the easily available material. There are a number of statements in the liter ature and the public press which liken PCBs to * Director, Division of Chemical Technology, U 3. Food and Drug Administration, BF-420, 200 C Street, S W , Waahington, D C 20204 DDTs If thla were the case, we would need to do little research in chemistry of PCBs, because the chemistry of the DDTs and related compounds is well known. Even so, all the biological effects of DDTs are by no means known or fully under stood even though there has been much work during the past 25 years In contrast to the three well known isomers of DDT--that is, the p,p', o,p', and o.o'-dichlorocompounds, the term polychlorobiphenyls or PCBs encompasses a very complex heterogeneous series of chemicals. When manufactured in a certain way, they form technical products which have enjoyed tremendous industrial use over the past 40 years because of the unique chemical and physical properties of the complex mixtures The exact chemical composition of these mix tures has not been determined. There probably was no incentive to determine the real nature of ail the components as far as the industrial value of the products was concerned, but now that it has been found that there are untoward biological effects from these products and that their residues are widely distributed, there is need for more knowledge than is now available There are also statements in the literature which in effect assert that PCB analytical methods and limits for residues should apply to specific chemicals within the series rather than to PCB undefined. Stated differently, if the toxicity of the PCBe is mainly due to certain compounds in the mixtures, then we could be justified in developing methodology to separate, quantitate, and evaluate these toxic compounds We will need to know more about the chemistry and biological effects before we can be so specific. April 1972 HONS 208109 3 Until we hive developed date which, on the one band, show that essentially all components have about the same biological effects or, on the other hand, show that some are greatly different from others, we have no real scientific basis on which to make a choice Of oourse there is evidence that some industrial PCB products contain polychlorodibenzofurana We need to know how and where these form Do they form somehow in the environment, and if so, do they survive through the food chain to man? If so, are there great differences in biological effects among the large number of possible polychlorofurans as is the case among the polychlorodionsT If these compounds are present m foods, we certainly should try to identify them soon, but if they are like the dioxins, they will be difficult to measure in food. PCBs are manufactured in France, Germany, Great Britain, Italy, Japan, Russia and U S.A. Assuming that the starting raw product is only biphenyl, and assuming that no reaction takes place except chlorination of biphenyl during manu facture, 210 different chemicals are theoretically possible from the 10 positions where chlorine can add on as shown m Fig 1. 0 1 2 Chlorine* . oa I ring 4 5 Chlorines on A ring 0 I____ l____ 2____4____ L 13 6 6)1 6 IS IB 9 3 21 36 IS 6 21 IB 6 63 t Ftounz 1 Structural formula for biphanvl and theoretically poamble Kiel for chlorination. It is not certain how many of these do appear in significant amounts in the technical products or in food. It is possible that a few molecules of each is present in each of the technical products. It is important to point out that, in the in dustrial products, the PCBs are associated with chloroterpbenyls. There art three possible terphenyla (the o, m, and p) as starting materials in contrast to one biphenyl os a starting material, and also there art 14 possible points instead of 10 at which each of the 3 isomers may be chlori nated. Therefore, these compounds could re>m!t in a considerably more complex mixture than the PCBs. The chloroterpbenyls can be associated with the PCBs in the ecosystem, and they are not readily determined by the usual methods of anal ysis for PCBs and the pesticides Later some ga-, chromatograms will be shown that illustrate some of the relationahipa The industrially important products are formed by chlorination of biphenyl to a given chlorine content. Products which contain 21, 42, 48, 54 and 60% chlorine hy weight appear to Is' the most important ones The end point of the desired average chlorine content is found by making ;<cific gravity or softening point measurement.'! on samples withdrawn during chlorination The crude products so formed arc generally purified to re move color and traces of hydrogen chloride and ferric chloride, largely by distillation Mixing of distillate Is also used to obtain uniform material of the desired composition (I) Various factors during the manufacturing proc ess can affect the proportion of the various isomers and the proportion of compounds of higher or lower chlorine content than the average (1) Therefore products manufactured by different processes could yield products which give some what different amounts of various components If some components have different biological effects than others, then different samples would produce unequal effects. Figure 2 shows gas chromatograms of two PCBs; A is Aroclor 1221, and B is 1232. These and the following gu chromatograms (Figs 3-8) were made by the conditions used in FDA labora tories The patterns generally look like those we see from other laboratories, so the products seem to be relatively uniform, except for Aroclor 1232 This appears not to have been a fully consistent product in the past Figure 3 shows more in the senes A is 1212, and B is 1248. 4 Environmental Health Perspectives HONS 208110 the m<ed with bio tcrlatenals Daterial, it-ciwJ of c chlond result than the socmU'd ' are not of analonir go* tic aomc ! fonni'd chlorine , 48, TM > be the : desired ;ing t|ricnts mi he crude (1 to re ride and imng of material ng proc1 isomers igher or c (I) different re somenents If il effects produce of two 2 These igs 3-8) 1 leboraIhoso we cts seem lor 1232 insistent is 1242, pectives )t> III --,--%-- I I I I I I I'T' > to Fiovu 3 Gm chromatograms of PCfit A ii 1242 and B is 1248. TIME (MINUTES) y >oee 2 Gm chromatograms of PCBs. A ti Arcelor 1221 Bio 1232. Figure 4 shows three more in the series; A is 12M, B is 1260, and C is 1262. Note that there is an overlap of peaks. This does not prove that they always represent the same compounds, but m ail probability the major part of the peaks is the same. Those which appear in the lower series probably would have been chlorinated to higher chlorine content if the product had stayed in the reaction chamber longer. Generally, as the peaks drop off on the left of the chromatograms, the ones on the right ineresse, or new ones appear. In alt probability there are a few molecules of every possible structure present in each product Only the predominant ones are in high enough concentrations to give the detector responses. Also note that the time the PCBb are in the gas Fiaune 4 Gm chromatograms of PCBs A is 1234. B ie 1200 and C is 1202 April 1972 _ MONS 206111 S chromatograph under these conditions is between 0 and 50 minutes. Figure 5 shows & mixed biphenylterphenyl product (Aroclor 4465), note that some come out early, and some come out late Some of the early H1 " ones look like 1260 and 1262 in Fig 4 Figure 6 shows a polychloroterphenyI-(Aroclor 5442) Note that there are peaks which come out as early as portions of Arodor 1242 and 1248. The proportion of the early eluters is relatively low; however, if accumulation through the food chain occurred, they may appear as PCBs Figure 7 shows a comparison between the gas chromatograms of Kanechlor 400 from Japan and Aroclor 1248 from USA This is presented to show that the two products look remarkably alike, even though one was made across the sea The one from Japan may be manufactured by the same processes as the U.S. one. A FlOUU 7 Comparison of gu chromatogram* of Kanechlor 400 (Japan) and Aroclor 124H (USA) o 9 10 IS 20 23 30 39 40 *9 90 39 40 49 TO 7 9 40 49 TlWC(MINUTES) Fiovne 5 Gm chromatogram of a mixed btphenyl-lerphenyl product (Arockti 4465) 9 Tiwimwffui Fiovita 6 Gm chromatogram of a polyehloroterpbenyl (Aroclor 5442) Ftaoac 6 Companion of gas chromatograms of Aroclor 1254, Chlophen A-60 (Germany) and Aroclor 1200 Figure 8 shows a comparison between the re sponse of Aroclor 1260 and Clophen A-fiO from Germany (2). 6 Environmental Health Perspectives MOHS 208112 All the gas chromatograms shown above were produced utilising an electron capture detector. This is an extremely good detector in many re spects, however, care must be used in attempting to interpret data from it. First, it is not specific for any given type of compounds It is generally sensitive to halogenated compounds, but many nonhalogenated ones also respond Equally im portant, the electron capture response of halo genated compounds is not always in relation to the amount of chlorine. For instance, the response to carbon tetrachloride is 2,000 times greater than to ethylene dichlonde--that is four chlorines in one case, and two in the other. This is an extreme example, but it points out the need for caution in interpreting response from unknown peaks. That is, a peak may or may not represent a halogenated compound, and it may represent a large or small amount To be certain that any peak is a chlori nated compound, confirmatory tests are required Using electron capture, amounts can only be esti mated by comparison to the response of a known amount of the exact same compound. The parameters of operating an electron capture detector also influence the responses. For instance, the voltage applied to the detector affects the relative responses between peaks. Table 1 shows the relative responses of some individual tetrachlorobiphenyls to an electron capture detector (3). Table 1, Retention time and electron-capture detector reeponee of cblorobfpbenyle Compound Relative retention tune (p,p'-DDE-1,00) Relative reeponee per ng;fcstandard deviation (p,p'-DDE-l 0(1) 2>2*,4, cblorobtplMnyl 3,3',4,4'.Wti. chiorobipbanyl 2,2',6,6'-tetrachlorobipbenyl 3,3',5, S'-letrachlorobipbenyl 2,3,4,5-tetrachlorobi phenyl 2,3,5, (Metrechlorobiphenyl 2,3,4,5.0- pentachlorobipheny! 0 St 0 90 0.33 0 70 0 09 0 51 l 00 0 206 0 003 0 7700 027 0 0403 0 0016 0 6250.045 0 7150 010 0 505 0 010 1 300 017 Fiooax 0 A comparison of response of 1254 utilising different detectors The upper curve it electron capture, lower curve is electrolytic conductivity detector Figure 9 shows the relative response from 1254 utilising the same gas chromatographic conditions but different detectors, the upper curve is electron Table 2. Physical properties of ehlorobiphenyls Compound Melting point, C Boiling poiot, C (mm Hg) *2-chtoro3-chloro- 4-chkwo2,2'-diehlofo3,3'-dichlorD*4,4'-dichloro3,5-dichloro2,5-dchloro3,4-dichloro2,3-dichtorp- 2,4'-dichtoro3,4,3', 4'-tetrachloro3,4,2\5'-tetrachloro2,6,2', O'-telnchloro*2,5,3', 6'-tetrachioro2,4,2', 4'-telrachloro2,5,2', 5'-tetrecUoro2,4,5,3',4'-pente- ohloro2t3t4,5^2',4't5 - heptachloro- 34 267-268, 154(12) 89 284-285 76 125(14) 61 29 322-324, 320-326 148 315-319 36 166(10) 171(15), 182(30) 49 185-200(15) 172 (30) 44 172 230(50) 103 198 162 83 65 179 195-220(10) 240-280(20) Present rn commercial nurture* Reference (except )--Hubbard April 1972 HONS 208113 7 capture, and the lower is the electrolytic con ductivity detector. The latter is more nearly re sponsive to the amount of chlorine; therefore, it should more nearly represent the relative amount of the various components. Use of a microcoulometrio detector may also provide a better quantitative estimate. Coulometry should yield results which would permit an exact measure of the chlorine. However, the cur rently available microcoulometric equipment does not give strictly quantitative response. Both microcoulometry and electrolytic conductivity are more specific for halogen-containing compounds than the electron capture detector, and accord ingly they can be used as a means to be mom certain that the products detected contain chlorine Not enough is known about the chemistry of the individual components and their relation to the whole industrial product Some of the individual compounds are shown in Tabic 2 (1) It can be seen that the physical properties vary quite widely Note the melting points of all the compounds listed; they are solids at room temperature. Yet when the commercial produets are manufactured by chlorination of biphenyl, the mixtures formed vary from liquid to soft, sticky noncrystalhne products with a chlorine content of 60%, low-melting resinous Table 3. The retention Indloe^, chlerine numbers and structures of the major PCB constituents In Aroclor 1X54. <4) Peak No. 22 23 24 20 32 33 36 30 41 42 43 44 45 48 30 52 55 56 SB JU, 1004 2010 2022 2069 2119 2136 2150 2175 2191 2203 2207 2228 2238 Chlorine No * NMR determined 4 2,5-2' ,5' 4 4 2,3-2', 5' (5) S 2,5-2',3',6' 5 2,3-2',.T, 6' 4 2,5-3',4' 5 5 2,5-2',4',5' (6) 5 2,4-2', 4`, 5' 5 2,3-2',4',5' 5 2,5-2',3',4' 5 3.4-2',3',6' -- 2264 2299 2321 2356 2356 2390 ' 6 6 5 5 6 6 (7) 2,3,6-2',4',5' 2.3.4-2', 3', 6' 3,4-2', 4', 3` 3,4-2', 3', 4' 2.4.5-2', 4', 5' 2,3,4-2', 4', 5' Alternative predictions Structure R 1" 2-2', 3', 5' 2-2',4', 5' 2,4-2',5' 3-2',4'. 6' 2,4-2',4' 2,6-2',3',6' 2,6-2\3',5' 1996 2012 2010 2021 2027 2017 2092 2,5-2', 3', 5' 2,4-2', 3', 5' 3,5-2',4',6' 2,6-2',3',4',6' 2,3,6-2',3`,6' 2,3-2',3',5' 2159 2175 2175 (2175) 2172 2189 2,4-2',3',4' 2,3-2', 3', 4' 2,6-3'.4',5` 3,4-3',5' 2,3,5-2',4',6' 2.4-2', 3', 4',' 2226 2240 2240 2238 2240 (226.1) 2,5- 2', 3', 4', 5' 3,5-2',3',4',6' 2,;i-2,3',4',5' 2,4 ,6-3',4' ,5' 2,3,5-2'. 3', S' ,0' (236(1) (2357) (32*111 21188 (23)10) * Cl No between bracket#, Chlorine number of minor constituent not associated with accurate It T k R I. between brackets Predicted R.I using Table VIII of Ref 4a 8 Environmental Health Perspectives HONS 206114 e more ilonne itry of lion to shown hysical nelting solids nercial Jon of liquid with & Minous In r* M 12 10 II 27 17 B2 ae 76 76 76) 72 SB 40 40 3K *0 i) SO) 57) tan KM on) active# products containing between 00 and 05% chlorine, and partly crystalline or crystalline products of much higher melting points containing more than 05% chlonne Thus, the commercially-made mix tures have at least some physical properties that are quite different from any of the individual constituents This is probably one of the very important factors in the industrial utility of the products The same phenomenon probably has a bearing on the survival of many of the compounds through a host of possible conditions through the ecosystems. So it is remarkable that the end products in food, which we will see later, look as much like the original industrial product as they do. A number of workers have identified the pre dominant number of chlorine atoms in the major components of some of the peaks. Others have made significant approaches to determine or pre dict the placement of the chlorines on the biphenyl rings (4), (5), (6). Sissons and Welti (4) used a SCOT column and a flame iomaation detector to obtain the graph shown in Fig. 10 from 1254. Note that there are 69 identifiable peaks. They did further purifi cation, and then by utilising MS, NMR, and factors such as retention time, they identified the major peaks of 1254 as shown in Table 3. Note that the retention times do not necessarily relate to number of chlorines. These researchers also made use of retention times and other infor mation to predict the composition of a large number of the other peaks. To attempt to be certain of the structure of the minor peaks would be tremendously complicated. It takes a significant amount of a chemical to make NMR analysis. It would be of great value to have available simple biological testa that would give a clue as to which peaks, major or minor, would be of enough sig nificance to warrant full-scale chemical- and bio logical-effect investigations. If it were found that all peaks contributed essentially equally to the biological effects (and there are certainly many biological effects), then there would be no need to perform a great amount of chemistry. On the other hand, if we determine that they vary in biological effects as widely as their propensity to capture electrons, then we would have to devise means to and measure these individual chemicals. It is known that PCBs can be dechiorinated and oxidised by photochemical effects (7) 1 look forward to the discussions by Dr Hutzinger on this subject I want only to point out that these reactions may complicate the process of evaluation of both chemistry and toxicology If the dechlori nation is simply a reversal of chlorination, so that the higher chlorinated products revert to exactly the same compound in the lower chlorinated products, then the problem is not more compli cated. If new and different chemicals form, then the problem is greater. If there is significant photo-oxidation through the food chain, then we have the potential for chioropbenols and even the potential for the for mation of polychiorodibensofurans as furthrr chemicals to evaluate. I hope that we hear dis cussion on these chemical problems during this conference. Man has been exposed to residues of PCBs in his food in many forms; that is, from the original industrial product introduced accidentally into his food to cases where these compounds have been subjected to almost all conditions of food chain reactions, photochemical reactions, etc Some of these conditions are: A. PCB directly into food in rice oil (Japan) B. PCB in paperboard transfers to food C. PCB into animal food we eat animal tissue D PCB into environment into fish we eat fish E. PCB into environment into fish fish into animal feed we eat animal tissue The first example is that of PCB accidentally put into rice oil. The product consumed by man may have all the ingredients formed during the manufacturing process of the PCBs, plus any reaction that may be induced by food processing. In the second example the food will have only those components which migrate from the pack aging material to the food, either through the vapor phase or by contact, or both. If any polar compounds are present, they may not migrate so April 1972 HONS 208115 9 Ftooiti 10 Aroclor 1254 solution chromatographed on in Apieion L SCOT column at 20.V emplnying h flame ionisation detector (4) readily, If the packaging material contains fat or wax, some of the higher chlorine compounds may not transfer as readily as the lower ones. However, there is the mutual effect of all on each, discussed earlier, which makes it difficult to predict. Some controlled experiments should be done. In the third situation the whole industrial product contaminates the animal feed, and we eat the animal tissue. The animal appears to modify the product, because the gas chromatograms from tissue extracts do not look exactly like the in dustrial product. In the fourth example the product or products have gotten into the environment, then into fish or other marine products, and we eat the fish or marine product. Here, there could be an accumu lation of a number of different Aroclora or similar products, each of which has been subjected to vanous biochemical reactions through the food chain and to vanous other effects BUch as photo chemical reactions When we eat the fish, we eat the PCBs or metabolites, the end products of whatever reactions may have taken place. And in the last example as in the previous one, the fish is fed to a domestic food animal--we cut the animal tissue The residues that we find in food products unv look a good deal like the original industrial prod ucts or like a mixture of those products Figure 11 illustrates some food products, turkey and chicken tissues and cows' milk, which have been found to contain PCBs. Figure 12 shows that the turkey sample had a product which looked most nearly like 1260 How ever, it can be seen that the proportion of peaks is different, and also a couple of peaks are missing Figure 13 shows that the chicken sample looked like 1248; yet there arc some quite important differences. Figure 14 shows some gas chromatograms from 1260 and the residues in muscle tissue from swine fed the 1260. Note that there are differences, they arc not as great as in the case of the turkey tissue Figure IS gives the results from a sample of coho salmon from Lake Michigan A is before separation of pesticides from PCB, B is PCB after 10 Environmental Health Perspectives MONS 208116 :0U8 one, --we eat ictu may ial prod- V turkey ich have tic had a 30 Howof peaks missing le looked nportant ma from en swine they y tissue, unple of 8 liefore ~B after wettvea HliiauelalilkkailEKItllBC Fima* 11 Gag chromatogram* of PCB* from some food product*. Fiona* 13. A companion of gas chromatogram* from chicken and Arodor 1248 and 1260. Fiounc 12 A comparison of ga* chromatogram* of PCB from turkey and Arodor 1248 and 1200 Fionas 14. A comparison of gaa chromatogram* from Arodor 1200 and residue* in muide tmue from swine fed Arodor 1200 April 1972 HONS 208117 11 Fiotms IS. Compansun of gat chromatogram or Vrorlnr 1254 residue in rat blood following feeding of Am, W |2vi Piovu IS. Gh chromatograms of simple from Minna from Lake Michigan A is before separation of pealicidea from PCB, B is PCB after separation and C is DDT pesticides. separation, and C shows the DDT pesticides present. The FCBs here look most like 1254. Many of the samples of wildlife reported by most workers yield chromatograms which look like 1254. Figure 16 shows the residue in rat blood follow ing the feeding of 1254. The peak, which dis appeared, is a pentachloro compound. The ma terial in the rat blood looks more like 1254 than does the material from coho salmon. Veith (8) made a study of the PCBs in the Milwaukee River and out into Lake Michigan. He found that the lower chlorine content products disappeared more rapidly than the higher chlori nated ones. It may be that the residues in food products, which come from a long food chain, do not originate entirely from the industrial product which is similar to the residue but may be the result of a concentration of the higher chlori nated units originally in the lower chlorine content products, the lower chlorinated units of which have largely disappeared. So even though these industrial products are generally stable and persistent, they are altered by microorganisms, in animal systems, by light, and probably in plant systems. Some units dis appear and some concentrate We need to develop some short term in vitro and in vivo bioaNsay that can be used to give clues for more thorough and detailed examination. The chemist has tools and techniques to obtain almost any degree of sophistication required by the biologist. REFERENCES 1. Hubbard, H L 1064 Encyclopedia of Chemn ,il Technology, lutencience Publishers, John Wilcv md Sons, Inc., New York 2 Zitko, V and Choi, P M K 1971 Fisheries Revircli Board of Canada, Tech Rpt No 272 3. Zitko, V , Hutunger, O and Safe, S 19*1 Ketmi m<ii times and eiectron-caplure detector response* of umnindividua! chiorobiphenyls. Bull Environ Coiilniinir Toxicol 6' 160 4a Simons, D and Welti, D 1971 Structural idem,In i tion of polychlorinated biphenyls in commrnnl mixtures by gas liquid chromatograph* mu I, rr magnetic resonance, and mass gpeetromelr* .1 Chromatogr SO 15 b Jensen, 8 1970 PCB Conference, Seplemlier J'l, I'1711 Wsnner-Gren Center. Stockholm, 11 12 Environmental Health Perspective* HOIKS 208118 S Wrljl), R-G and McCall, A C 1971 Paper presented at 11,2nd American Chemical Society, Washington, D C , N-pumber 13, 1071 C Tan A C and Voa, H. H de 1971 Characterisation of four major components in a technical polychlorinated In [i hen vl mixture Environ Sci Tech 5 1216 7 Safe. S and Hutlinger. O 1971. Polychlorinated hiphenyl* photolysis of 2,4,6,2\4`,8'-heiiachlorobi. phenyl. Nature 232. 641 8 Veith, G D 1970. Environmental Chemistry of the Chlorob'.phenylg in the Milwaukee River Doctoral Thetis, University of Wisconsin i-H I - 1J t I i of Arortor wlor 12'*4 product y be the r chloricontent sf which lucts are e altered by light, nuts disi develop bioaaaay thorough tuts tools legrcc of Chrmirii] IViley am) I Kceeurrh Rrlrnf inn n of aoroc Contumm idenlihriiommrrciul , ii'iclcnr netrv J 211, 107(1 pectives April 1972 13 MONS 20*119 EnvirvtmMiJ Haith PtrtptOiva Photochemical Degradation of Chlorobiphenyls (PCBs)* by 0, Hutzinger and S. Safet and V. Zitko** Polychlorinated biphenyls (PCBs) are stable towards oxidation, hydrolysis and other chemical reactions which conceivably occur in the environ ment Although there is little direct evidence ai nibble, it is generally assumed that PCBs, particularly those with a high chlonne content, arc nNo quite resistant to metabolic change (her the past years photochemical degradation ''bun ii to be a possible major route of en vironmental breakdown for a number of pesticides (1-4) Until relatively recently {.)), however, sim ple cliloroaromatic compounds have not received much attention. The limited interest of photochenusts in compounds of this type can perhaps be explained by the commonly held view that there is generally little cleavage of the C-Cl bond in cliloroaromatic compounds (C) We have previously shown (7) that 2,2',4,4', 6,G'-hexachlorobiphenyl, when irradiated at 3100 A in hexane, pbotolyies rather readily to give products which are formed by loss of chlorine, rearrangement and condensation. We now wish to prrsent a first report of our studies od the photochemical stability of a number of chlorobiphcnyl isomers in hexane as well as on the breakdown of chlorobiphenyls under natural and simulated "natural conditions", with particular emphasis on polar products Considerable experience in laboratory irradi ations of pesticides has accumulated (1, 3, 3) and an elaborate "weathering chamber", which also allows controlled irradiation, has been described laflutHl u NllCC No 12W9 t Atlantic Kegfonft! Lulwratorv, National Research I nunc it of Canaria, Halifax, Nova Scotia * Environment Canada, Fisheries Services, St Andrtvt^, Nrw Unirinwu k (9). "Natural conditions", however, are difficult to simulate for water-insoluble compounds such as the PCBs. Even in sunlight, irradiation experi ments in solution are artificial since "unnatural" organic solvents have to be U9cd Irradiations of chlorobiphenyls in thin films, m the gas phase (both in the presence of water or water vapor) or in aqueous suspension are likely to be closer to the natural environmental conditions to which PCBs are actually exposed Eqtipment art Methrts Gas chromatographic (glc) separations were car ried out with a Hewlett Packard model 5750 chromatograph using a flame ionization detector and an 8 ft X Yk in stainless steel column con taining 3% SE-30 on acid washed Chromosorb W (60/80 mesh) Temperature was isothermal or programmed as indicated. Quantitative data were obtained with a Packard A79G1 gas chromato graph equipped with a 6 ft X Yt m 4% SE-30 column and an electron capture detector using the conditions previously described (10) Combined glc mass spectral data were record rd with a Hewlett Packard model 5750 instrument, using a 6 ft X ^ in glass column, coupled to a DuPont/CEC 21-491 mass spectrometer via a single stage jet separator A DuPont/CEC 21-110B mass spectrometer equipped with a direct introduction-constant tem perature probe (11) was used for obtaining spectra of individual samples The infrared (ir) spectra (thin films) were re corded on a Perkin Elmer 237 spectruphotumrtcr Thin-layer chromatography (tic) was carried out on precoated Merck silica thin layer plates (F-254) and plates (100 X 20 cm) eoated with April 1972 MONS 208120 15 Merck silica gel (F-254, 0 9 mm thickness) Sol vents used were: hexane and (A) 1,1,1-tnchloroethane-hexane (5:1), (B) hexane-acetone (3:1), (C) benaene-ethyl acetate (12:1), (D) bensenechloroform (2:3) The chlorobiphenyls were syn thesised as described before (12,13). Aroclor 1254 was a gift of Monsanto Co. Sources of ultraviolet light for the irradiation experiments were as follows: (i) Sun: direct sunlight at Halifax, N S during the penod July to September 1971. (li) Photochemical reactor: Bayonet (the Southern N.E. Ultraviolet Co.) using 16 R.P.R. 3100 lamps (3100 A) The temperature was maintained at 14 2 (iii) Blackligbt: four General Electric F40BL fluorescent lamps mounted with reflec tive backing ca. 15 cm above the sam ples. The temperature at this distance wss 30-33 (iv) Sunlamp: General Electric 275 W "Sun lamp", one per diah at ca. 25 cm dis tance (temp. 35-40*). For irradiation of chlorobiphenylwater vapor, four lamps per 500 ml quarts tube were mounted horitontally at ca. 10 cm. distance. Expefimints uri Rawto Stabilities of ChlorobipbenyU at 3100 A In Hexane Solution Time study of 2,3" ,5 tS'-tstrachlorobiphmnyl decomposition--2,2' ,5,5'-Tetrachkrobiphenyl (50 mg) in hexane (300 ml) wss irradiated in the Rayonet reactor with Nt bubbling through the solution 50 ml Aliquots were removed from the reaction vemel at 2, 4.6, 17, 4] and 89 h. Good quantitative data could not be obtained for the 89 h sample, probably because much of the material had polymerised. Chromatograms ob served after injecting equal amounts of these ali quots and using a flame ionisation (F.I.) detector are shown in Fig. 1. Percentages of starting ma terial remaining after different irradiation times are also presented in Fig. 1. These data were ob tained by measuring the area under the peak with retention time identical to the starting material Frotme 1 Irradiation of 2,2',5,5'-tetreehion>biphenvt fnr different Uma chromatogram* end percent ttartmg mitcn&i remaining. utiiig tm electron capture (EC) detector Mass spectral and glc data for the 41 h sample arc given in Fig. 2. Comparative stability of chlorobiphenyl isomers--A 0.1% solution (3 ml) of the chloro biphenyl hexane was flushed for 3 mm with nitrogen, stoppered and irradiated in the Rayone( reactor for 24 h Tbe volume of the solution was removed from the quarts reaction cel! and, after rinsing with hexane and evaporation of some of the solvent, adjusted to 3 ml Equal amounts of the irradiated solution and the original solution Fiotnii 2 Chromatogram and rntas ipertral data for the 41 h aample Oven temperature 150* teothermal, at 3d min beginning of programming (4*/mm). A number of further peaka were obeerred after 40 min 16 Environmental Health Perspectives SONS 206121 t too 98 90 6? 5 i 3* HlTiHJH*m phenvl for t starting [>r. Maas nple are iphinyi t chloroiin with Rayonet tion was nd, after some of ounts of solution \ I Table 1. Rabtl'e Photochemical Labili lie* of wmi CUorobfphenyl Isomer* Compound Starting material remaining after 24 h at 3100 A rn Hexane Tet rue htombipbeoyls 3, 1 J.ft.fl'2 2` r',S's.j'.M'.s.s'- Hcs.irhlorobiphenyl 2.2*,3,3',4,4't 6,5'- OctnHilorobiphenyl % 32 2U 33 3e <i were injected into the gas chromatograph, and the areas under the peak corresponding to the starting material compared (Table 1). No immedi ate' correlation can be seen between stenc hind rance of o-chlorine substitution or uv spectra (14) and photochemical lability, although chlorobiphenyls with high chlorine content seem to de grade more rapidly. Stability oj Aroclor 1254--Aroclor 1254 (20 mg) in hexane (3 ml) was irradiated for 4 h at 3100 A in the presence of air Examination of the irradiated mixture and standard Aroclor 1254 by glc shows gross differences with tendencies toward shorter retention times (Fig 3) Irradiations in Sunlight; Under Simulated "Natural" Conditions and in Solvent Mixtures Containing Water Aroclor 12541(Aqueous dioxans suspension at pH 9--A suspension of Aroclor 1254 (2 g) in a mixture of dioxane (150 ml) and water (350 ml) containing sodium bicarbonate (0.5 g) was irradi ated in the Rayonet reactor for 24 h with air bubbling through the mixture After this penod another portion of sodium bicarbonate (0 5 g) was added, and the irradiation continued further for 24 h The mixture was worked up as shown in Scheme 1. Preliminary mass spectral data for the crude nonpolar fractions one, two and three indi cated the presence of only chlorobiphenyls since no ions containing oxygen corresponding to hydroxychlorobiphenyls or chlorodibenzofurans were detected. The mass spectrum of the crude CHLOROBI PHENYL EXPOSED TO UV, EXTRACTED WITH ETtCR AT fH2, PREPARATIVE TIC (ZX HEXANE) A /' tr h ila for ihn trial, at 36 number ol ' f TIME (mm} Fioimr 3 Chromatogram of Aroclor 1254 before end after irradiation Glc conditions of (10) pec live* ApriJ 1972 RF 0,7-0.9 "HYDROXY" FRACTION RF 0,0-0.1 "CARBOXY" FRACTION Schixe 1 Beparation-scheme for irradiated chlorobiphenyls HONS 208122 17 0L',,yv> *H.0 Frauitx 4 Partial mm spectrum (70 eV. probe temper* attire 35*) of "hydroxy" fraction from Arocior 1354 irradiation expenmen t I The chlorine itotope pattern for the CJ, end Clr compound! are distorted due to imp unltee or fragment ion) "hydroxy" fraction, part of which is shown in Fig 4, indicated the presence of compounds formed by the addition of water to chlorobiphenyls. Hydroxy and carbonyl stretching fre quencies in the infrared spectrum of the crude "carboxy" fraction (Fig. 5) are indicative of the type of compounds present in this mixture. Arocior 1254 //; Irradiation at thin film-- Arocior 1254 (0 5 g) was dissolved in benzene (3 mi), and this solution spread over a circular pyrex dish (diameter: 35 cm). The bensene was allowed to evaporate slowly in a way to ensure formation of a uniform Aroclor 1254 film Water (3 ml) was added, and the dish was covered with plastic film Water was frequently replaced during the Blacklight one-week irradiation The irradi ated product was fractionated according to Scheme 1 Preliminary mass spectra of the crude nonpolar fractions 1 and 2 (in this experiment no material FlopXX 8 Partial mam spectra (70 eV, probe temperature 35*) at "hydroxy" fraction from Aroclor 1254 irradiation experiment fl (top)) end atanderd Arocior 1254 (bottom looa <340 not ahown) corresponding to 3 was detected) showed only the presence of chlorobtphenyls A mass spectrum of the crude "hydroxy" fraction (Fig. 6) showed ions which can be most easily explained as re sulting from hydroxychlorobiphenyls The ir spec trum of the crude "carboxy" fraction was almost identical to that from experiment I (Fig 5) Irradiation* of 2,2',5 ,S'~tetrachlorobiphenyl--Some information on the types of prod ucts formed from 2,2' ,5,5'-tetrachlorobiphcnyl on irradiation under a variety of conditions is given in Table 2. Samples were fractionated as Fjouhe 5 Partial infrared spectrum (thin film) of `'carboxy" fraction from Arocior 1254 irradiation ex penmen 11 Fiotra* 7 Chromatogram for 2.2\5,5'-letraehlorobiphenyl irradiation expenment II Oven tempefaiun' programmed from 150* at 10*/imn until 310 A number at further peak* were observed after 20 nun 18 Environmental Health Perspectives HONS 208123 ft AftOCLO* 'EM -r" MO Mnperfttura irradiation 4 (bottom. t only the >ctruni of ) showed ed u ro te ir specss almost (.5). .htorobit of prodjbiphenyl ditions ib mated as mn trochtorobtempermturc A number pectlvM Table 2. Photochemical Decomposition of I,S',5,5'-Tetrachlorobiphenyl. Experiment number Irradiation conditions uv source Tim* Types of products formed* h "Nonpolar" product* "Polar" products Retention lime* "Hydroxy-" "Csrboxy-" Short tong ` i Aqueous suspension sun (+ emulsifier} 2 mo 2 Thin film 3 Thin film sun sub lamp 2 mo 24 h 4 Thin film block light 1 wk S Dioxan-2N NsOH solution reactor 16 h 0 Aqueous dtoxsne solution + FeClt reactor 16 h 7 Vapor (refluxing with water) sun lamp 16 h - (+) (+) (+> -- (+) + + + + + + + (+) (+>" (+) (+) + + (+} + +4 + + + + + + - preoent, (+) - present in email quantities, - - not detected For further description of products, etc we text r Glc retention time shorter or longer than starting material (2,2', 5,5'-tetrochlorobiphenvi), 4 See Fig 7 described in Scheme 1 The nonpolar fraction was analyzed by glc (e.g- Fig. 7). "Carboxy'-type compounds were indicated in all experiments by the chromatographic properties of a portion of the reaction products which were similar to the corresponding fractions from the Aroclor 1254 irradiation experiments I and II (i e , R/<0 1 in solvents A-C; high R/ in aqueous methanol mixtures) The presence of "hydroxy''-type compounds was indicated by: (i) chromatographic comparison of the ap propriate fractions with authentic hy droxy- and dihydroxybipbenyls in solvent systems A, (Rf 0.3-0.8); B, (R, 0 3-0 7) and C, (R/ 0.4-0.8), and (ii) by reacting the "polar" fractions with dansyl chloride (1-dimetbylaminonaphthalene-5-sulfonyl chloride) and compar ing the chromatographic properties (sol vent D; Rf ca. 0,75 for monohydroxy and ca. 0-4 for dihydroxy compounds) and in situ fluorescence excitation (350 nm) and emission (525 nm) wavelength with those of dansyl derivatives of au thentic hydroxy and dihydroxybiphenyls (see reference 15 for the general method) Disussitn The photolysis of chlorobiphenyls reveals a number of degrodative reactions which occur on irradiation in sunlight and a number of laboratory conditions In hexane, the most prominent reaction was the progressive reductive dechlorination of these compounds as well as formation of polymeric materials, Irradiation of Aroclor 1254 in hydroxylic solvents at pH 9 yields compounds corresponding to the addition of water to the FOB molecules and a more polar carboxylic acid fraction The same sample irradiated os a thin film does not yield the water-addition products, but mass spec trometry reveals the presence of new hydroxylated species. This experiment also gives a more polar "carboxylic" fraction Data for a number of differ ent irradiations of 212',5,5'-tetraehlorobiphenyl indicate that dechlorination, formation of poly mers, and carboxylic products as well as hydroxyiation, does occur. April 1972 MONS 208124 19 The present photolytic work thus reveals de composition of PCBs by both oxidative and re ductive pathways. REFERENCES 1 Crosby, D 0 and Li M V 1969 In P C Kearney and L> D Kaufman, (eda ) Degradation of Herbicidra, Dekker, New York, 321 2 Croaby, D (J 1970 In Peatieadea in the Soil, Sym posium at Michigan Slate Umveriity, Eaat Lanaing, Kfl 3 Plimmer J R 1070 The photochemistry of halogenated herbicide* Residue Review* 33 47 4 Roaen, J. D 1971 In S D Fauat and J V Hunter, (eda } Organic Compounds in Aquatic Environment*, Dekker, New York, 425 5 Croabv, D. 0 and Hamadmad, N 1971 The photomluction of pentachlorobenaene* J Agric Food Chem 19 1171 0 Pmhey, J. T and RigHv, R D G 1969, Photo reduction of chloro- and bromo-aromatic compound* Tetrahedron Letter* No 16 1267 7 Hefe, S and Hutamger, O 1971 Polychlorinated biphenyla photolyaia of 2,4,6,2',4',6-hexachIorobiphenyl Nature 232 641 8 Croahy, D G 1969 Experimental apprnachei to peaticide photodecompoaition Residue Reviews 25 I 9 Tbomaa, R 0 and Fiahbein, L 1966 Design and utility of an environmental weathering device Chem Anal 55 90 10 Zilku. V , Hutamger. O and Safe, 9 1971 Retention Limec and electron-capture detector response* of aomr individual cJilurubiplwnyla Bull Environ Contamm Toxicol 6- 160 11 Jamiaaoti, W D and Maaon, F G 1970 Vtide-range controHed-temperative direct-introduction prole for maaa spectrometer ample*. Rev 8ci lustrum 41 778 12 Huuinger. O., Safe. S and Zitko, V 1971 Polvchlonnated biphenyl*, ayntheai* of nome individual chloroblphenyi* Bui! Environ Contamm Toxicol n 209 13 Safe, S and Hutringer, 0 (In preea) The mass spectra of polychlorinated biphenyl*. J Chem Soc (C) 14. Saeki. 8 et al 1971 The isolation and structure elucidation of the main component* of Kannechlor 400 (Chlorinated Biphenyls) Fukuoka-fgaku-Zaastn 62 20 15. Frei, R W and Lawrence, J F 1971 Fluongenic labelling of carbamate with danayl chloride I Study of reaction condition* J Chromatogr. 61 174 20 Environmental Health Perspectives HONS 208125 ippmachM u> 2ri | D*n nd tk* Chm 71 Raientioc onw of mn* n Conumm 1 W ide-range >n prof* fur Inztruro 11 1 Pol ychl'in '*duJ chlornwol 6 2011 mut spertm (C). nd itructure Juiechlor 400 u-Zuahi 02 Fluonnetnr kle I atuilv 174 pec live* nwnniu( Health Ptrtpaenvn Rates and Routes of Transport of PCBs in the Environment by tan C. T. Nisbet* and Add F. Sarofimf Pith chlorinated biphenyls (PCBs) are one memIx r of a class of chlorinated organic compounds which give nse to concern, because of their wide (ii'.|K'r-iil and persistence in the environment and icmtcnry to accumulate in food chains, with poskihlc adverse effects on animals at the top of the food webs, including man In the past, attention has lua n concentrated on chlorinated hydrocarbon IteMioidcB, such as DDT, dieldnn, heptachlor and HCt! (hexachlorocyclohexanc) More recently al ien! mu has been focused on PCBs and on chlori nated dibensodiouns Likely candidates for future alteniiun arc hcxachlorobenzene, chlonnated dibrnrofurans, and chlorinated phenols. In the past, such compounds have generally been studied indi vidually As the number of compounds giving rw io concern increases, there is an urgent need So cvinblish uniform monitoring schemes and gem ndizrd models describing environmental irnii'iort and bioaccumulation which will be ap plicable to alt compounds with these properties Models such as the global monitoring scheme outlined by the SC HI* study (1), and the global truif-port model outliniil recently by Woodwell el sil (2), arc needed in tinier to help identify sources of environmental contamination, to es tablish acceptable levels of discharge, and to estimate the effectiveness of different control strategics. This paper summarises the fragmentary knowl edge available about production, uses, ami losses of PCBs, and attempts to define the major routes of s ransport anil reservoirs of PCBs in the environ- * Itcsenrrli Dnpartment, MiuNnchuflctlH AilUuImn Snticlv, Lincoln, M*a*chusetti 01773 1 DepHrOnent of Chemical Enainrenn*, Mimaachusetts limtituu of Technology, Cunbndge, Mssuchuseiu 02136 ment It discusses only production and uses within North America, and distribution of PCB residues m North America and adjacent seas (the eastern half of the North Pacific Ocean and the western half of the North Atlantic) It is expected that the picture will be generally similar for other areas where PCBs have been used extensively (i e , industrialized regions, primarily m the North Temperate Zone), with minor differences depend ing on local patterns of use and disposal However, references to studies made in Europe and Japan are made in this paper where they help to fill gaps in the North American picture Although we attempt to cover ail the important environmental aspects of the use and dist nbution of PCBs, much of the discussion in this paper is extremely speculative One of its main purposes is to point out the most important gaps in knowl edge which must be filled before firm estimates can be made of present and future levels in the environment Fratatian and lisa in Nartfe Amrica The sole manufacturer of PCBs in North America is the Monsanto Company Commercial mixtures are sold under the trade name Arnclor and are distinguished by numbers, in which the first two digits 12 specify polychlorinated bi phenyls and the last tno digits the approximate percentage of chlorine in the mixture <i) In November, 1971, Monsanto released figures for U.S. domestic sales of Aroeiorn during the period 1963-70, summarized here in Figs 1 and 2, \ihere they are divided according to category of use and grade of Aroclor. Prior to Monsanto's voluntary reduction of sales in September, 1970, approximately 60% of HONS 208126 Fhjdsl 1 U.H ilomrsiK tain of PCBa by category 1254 and above, comprised approximately 3<>tT of sales pnor to 1971 Their share of the reduced sales in 1971 is expected to fall to 22% Aroclor 1242 is being replaced with Aroclor 1016 baung a similar composition but with isomers containing 5 or more chlonne atoms removed The breakdown by grade of the current uses of PCB is electrical capacitors, mainly Aroclor 1016 with limited usage of Aroclor 1221 and Aroclor 1254; electrical transformers, Aroclors 1242, 1254 and 1260; vacuum pumps, Aroclor 1248 and 12.14, and gas transmission turbines, Aroclor 1221 and 1242, The breakdown by PCB grade of former uses include Aroclors 1232, 1242, [248, 1254, and 1260 for hydraulic fluids, Aroclor 1242 for heat transfer systems, Aroclors 1248, 1254, 1260, 1262, and 1268 for plasticisers in synthetic resms, Aroclors 1221, 1232, 1242, 1248 and 1254 for ad hesives, Aroctors 1221, 1232, 1242, 1248, 1254 and 1268 for plasticizers m rubbers; Aroclors 1242, 1254 and 1268 for wax extenders; Aroclore 1231 and 1260 for dedusting agents; Aroclor 1254 for pesticide extenders, inks, lubricants, and cutting oils; and Aroclor 1242 for carbonless reproducing paper. sales were for closed-system electrical and heat transfer uses, 25% for plasticiser applications, 10% for hydraulic fluids and lubricants, and less than 5% for miscellaneous applications such as surface coatings, adhesives, printing inks, and pesticide extenders Exports by Monsanto have averaged 13% of domestic sale* over the period 1963 to 1970. Imports are thought to be small, comprised primarily of ptasticsiers in resinB and adhesives, transformer oils and capacitor fluid in electrical equipment. Exports to Canada should be of the order of 7% of IT S sales and are included in this modet of North America. Monsanto has restricted sales of Aroclors based on consideration of either the possibility of con tamination of food products or its inability to control or monitor possible losses into the environ ment The fraction of sales for use in confined systems, primarily electrical applications, will in crease to approximately 90 percent m 1971 and to 100% in 1972 The higher chlorinated Aroclors, Fiovxe 2 U.S domestic sales by PCB grmle 22 Environmental Health Perspectives MGNS 208127 sly 30% of ie reduced %> Aroclor H6 having containing mt uses of odor 1016 id Aroclor 1242, 1254 and 1254, 1221 and of former 1254, and 2 for heat 260, 1202, jc reams; 54 for ad1254 and lore 1242, dore 1254 1254 for id cutting producing 1221 tnule peclives Data on production outside the U S are not available. It has been estimated (4) that the Japanese production iB 26 million pounds per year, 40-50% for capacitors, 15% for transformer oil, 10-15% for heat transfer fluid, 5% for plasti cisers, 15% for carbonless duplicating paper, and 5--10% for export. ttontts iRto tin tnrironmtnt As in the case of most industrial chemicals, loss figures for PCBs are practically non-existent Pos sible routes into the environment include- (1) leaks from sealed transformers and heat exchangers; (2) leakB of PCB-contaming fluids from hydraulic systems which are only partially sealed; (3) spills and losses in the manufacturing either of PCBs or PCB-containing fluids, (4) vaporization or leaching from PCB-containing formulations; (5) disposal of waste PCBs or PCB-containing fluids. Examples of losses by each of these proposed routes include leaks from faulty heat exchangers in the contaminated Japanese rice oil (5) and contaminated chicken feed {Holly Farms) inci dents; leakage of hydraulic fluid from an air com pressor in Escambia Bay, Florida (6); the indirect evidence of possible losses during manufacture provided by the high level of PCBs in catfish m waters near Anniston, one of the two sites at winch PCBs are manufactured in the US (7); the leaching of PCBs from ribs by sileage (7) and the use of waste electrical insulator containing PCBs as solvent in herbicide treatment, of power rights-of-way near Martinsburgh, West Virginia (7). Other unreported disposal of scrap un doubtedly occurs into sowers (8) Statistical informntion on such losses is not available but could lx- generated by an accounting of PCB inventories ami disposal by major users. To meet the problem of scrap disposal, Monsanto has set up a disposal system with a capacity of 10 million pounds per year for their customers. Within a year of an nouncement of the service, 500,000 pounds of waste PCBs had accumulated at the disposal rite, where it was held in storage, pending the com|>le1 ion of an incinerator (9) Another waste disposal problem is that of PCBconlainmg products including the PCB-impregnuted paper in capacitors, caulking compounds, and carbonless dupbeating paper. It has been postulated that the PCBs may be vaporized during incineration. Based on studies into the incineration of PCBs and pesticide residues, mu nicipal incinerators meeting design guidelines of a residence time of 2 seconds at 2000F are expected not to be a significant source of PCBs (9, 10) Poorly operated commercial and municipal in cinerators, small domestic and apartment inciner ators, and open-burning dum|w may, however, be major sources of emission, but data are not presently available Another problem is that of leaching from dumps Again, data are scarce, but analysis (11) of stagnant water close to a sanitary landfill indicated levels below the detection limit of 4 pob. Quantitativi Estimates of Rates of loss into tho Eavironiitont It is unlikely that reliable quantitative estimate* of past losses of PCBs into the environment will ever become available However, m order to assess the biological significance of the PCBs now in the environment, it is necessary to make some nu merical estimates, however rough The following calculations are intended to provide order-ofmagmtude estimates of rates of loss. Figure 3 shows qualitatively some of the many possible routes of loss into the environment Estimates of the rates of loss and disposal of Aroclors in 1970 will be based on rough guesses of the useful service life m different applications Transformers are fairly permanent, installations and it is therefore estimated that only 10% of sales of transformer fluid is to replace oil that was scrapped and that the remaining 90% is for new units. The useful life of capacitors, especially those used in small items such as fluorescent, light ballasts, is expected to be under a decade, and it is therefore estimated that the rate at which the capacitors are discarded, primarily into landfill dumps, is equal to half the rate of production (Figure 1 indicates a doubling period for sales of about 10 years) A similar figure is assumed for the scrapping of heat exchangers, but it is ex pected that the replacement of fluid decomposed under extreme thermal conditions would have led, in the past, to additional direct losses, pninunly into sewers It is estimated that the rate of vapori zation of plasticizer amounts to 10 to 20% of April 1972 HONS 208128 23 Aovlg into the o>r ra> !K(1 Or lefetit,?! *Tf vhviroAmont Kiocu 3. Possible route* of PCBt into the environment sales (12). Since many plastic objects have rela tively short useful lives the rate of disposal of plasticisers into dumps is assumed to equal the residual 80 to 90% of sales. Hydraulic fluids and lubricants arc rarely re-used, and it is therefore assumed that a major fraction of these fluids used for this purpose, together with those going into the miscellaneous applications, were scrapped at rates approximately equal to those of correspond ing sales On the basis of these gross estimates, it appears that only about 20% of the 1970 sales in North America, some 7X 10* tons, represented a net in crease in the amount of PCBs in service, in transformers, heat exchangers and capacitors The remainder is assumed to have been discharged into the environment, l-2x 103 tons by evapo ration of plasticisers, 4-5X103 tons by leaks and disposal of hydraulic fluid and lubricant plus small amounts by disposal of heat transfer and trans- Environmental Health Perspective* HONS 208129 Title 1. Gross Estimates of Rate* of Input and Accumulation of PCBa la North Amorioa in 19T0. Cnlejcory of input Rates PCB Grade (tone/year) \aportrn1ion of pUaticuera Viporimtion during open tiuminx Leak* unit disposal of indu^rml fluids Dnimsed 1>V incineration and open liuminj Dupo.ul in dumps and limlfiJIft Accumulation m service 1-2 X 10* Mawlv 1348 to 1200 4X10* Mainly 1242 4-5X10* 1242 to 1200 3X10* Main v 1242 1 8X10' 1242-1200 7X10* 1242-1254 Reservoir Suit (I'vctuding dumps) Ocean* (adjacent to North Amottcn) Fr<"li wHler {diwrived or m xiMpcintion) Fmili water sediment Btolu Accumulation (torn) PCEs 1 5X10* 1 5X10* 10* 2X10* <10* 2 DDT 3X10* 10* --10* 7 <10* former oils, and 22X10* tons by disposal in in cinerators, dumps and sanitary landfills. Of the laliei, wc estimate (13) that 10 to 20% (3X10* ton-) were destroyed by burning, and 2% (4X10' ton*-) were vaporized, mainly by open burning of mu' scrap, auto components, and material in dumps. butu inti thi EnvkcsRtflt The total rate of loss of PCBs is thus estimated to have been of the order of 1,5 to 2 X10* tons/year trio the atmosphere, 4 to 5X10* tons/year into fresh and coastal waters, and 1.8X104 tons/year into dumps and landfills The input into sods via the use of Aroclors as pesticide extenders is be lieved to have been small, less than 10 tons/year, on the basts of reports of purchases for this pur pose The unauthorised and unrecorded use of wrap PCBh oh pesticide extenders is difficult to estimate but ha* probably been small. Direct discharge into the oceans, eg by dumping of hydraulic fluids and lubricants from ships, has probably been of relatively small magnitude, but may have been locally significant (49) Most of the PCBs discharged into the atmos phere will have been Aroclor 1248 to 1260 vapor ized from plastic resins, augmented primarily by 1242 vaporized from burning dumps The dis charge into waters will be heavily weighted by the Aroclors used as hydraulic fluids and lubri cants and iB therefore likely to have included a mixture of Aroclors 1242 to 1280. The residual in dumps will have a large fraction of the Aroclor 1242 production. Hitts if Traaspwt within the Enviremmit The modes of transport of the PCBs within the environment are complex, Vaporized PCBs will be partially adsorbed on particulates, transported with the prevailing winds, and deposited on land or water by particle sedimentation or ram-out PCBs introduced into water streams may be ad sorbed by the waterborne particulates or the benthos; the adsorbed PCBs will diffuse into the bottom sediment, rediasoive in the water stream or be entrained with sediment eroded from the bottom surface. The problem is further complicated by the assimilation, transport and degradation of PCBs by the biota. A major fraction of the PCBs dis carded in dumps iB encapsulated in sealed con tainers or plastic resins The rate of loss from these will therefore be low until the confining material is degraded and the PCBs released The PCBs will then slowly diffuse through the sur rounding soil. In principle, the rates of transport may be calculated from knowledge of the physico chemical properties of the PCBs and the pertinent data on atmospheric conditions, particulate trans port, hydraulic dispersion, bottom sediment trans port, and biological degradation rates. At present, the data are too incomplete and the interactions between the different elements in the environment too complex to attempt a formulation of but the crudest of transport models. The similarity be tween the properties of the PORs and DDT, summarized m Table 2, permits rough estimation of some of the otherwise unknown routes and rates of transport of PCBs from the corresponding information on DDT. In Fig 4, a generalized model for the distribution and transport of the PCBs is outlined, without any indication of the transport down spatial gradients within the dtfTer- April 1972 HONS 206130 25 Table 2 Comparative Pbyaica) Propertiea of PCBs and DDT (Kef. 9. 23, 94, 95) Molecular weight 1242 Range* 154-358 Average 2(52 % Chlorine Solubility* m HiO (nt 20'C ppb) Vapor pressure* at 38C (mm, tig) Vapor pressure at 20*C (mm Hg) 42 200 HP* lCr*' 1248 222-358 288 48 100 Aroclor 1254 290-392 324 54 50 3 7X10-* 0X10* 3XI0"" 3 6x 10"** 1260 324-400 370 60 25 Est 2X10-' DDT 352 152 07 2 X l<)-" 1 ,5X10-* * Bawd on cnnatil uents prevent in amount* of 1 percent or more t The Inch vnlues of the solubilities and vapor pressures of the PCB* are a consequence of the heaw * righting given the lower chlorinated species. * Extrapolated eot compartments of the environment An at tempt is made in the following sections to estimate fluxes between aud within each compartment Air Transport The estimated emission of 1500 to 2500 tons a year of PCBs, mainly by vaporization and open va*rtw M mcrfwrpttOn Ficu'ri, 4 Environmental transport model burning, is expected to be concentrated in urban areas By analogy to DDT (17, 18} it i*. expected that most of the airborne PCBs will be adsorbed on particles Since roughly three quaiU'p. of total suspended particulates (TSP) in urban nreas are of non-agncultural origin (19}, and the omission of TSP within cities is of the order of 2XlOf tons per year (`20), this gives rise to a rough estimate of PCB level on urban particulates of 50 to 80 ppm. The half-life of particulates in the air will de pend greatly on the site of particles to winch the PCBs are attached and the extent of atmospheric precipitation. (19) It is estimated that most of the vaporised PCBs will be deposited within 2 or 3 days, mostly onto the land mass and cna-sial waters surrounding urban areas The -mall amounts of PCB attached to fine particulate-- will reside in the atmosphere for extended periods and be transported to remote areas Based on the above assumptions, the rate of terrestrial net input of PCBs by serial fallout in North America will be a little less than the rule of vaporisation, about 1000 to 2000 tons/ycar The vapor pressure of Aroclor 1254, the dominanl air borne contaminant, is close to that of DDC (2lj, and it is estimated, by analogy to DDC (17) that the likely half-life of PCBs in soil is of the unit t of five years. The PCBs removed from the vn! 26 Environmental Health Perspectives HONS 208131 DDT 352 352 60 07 -- xir ixio-' hung given I in urban i expected adsorbed rs of total areas are s emission of 2X10' > a rough cuiates of ir will dcwhich the mospheric lost of the mi 2 or it id coastal 'lie small ulutes will mods and te rslc of fallout in the rate of 'year The jinan l iur3DC (21), l (17) that ' the order n tile soil IMCtitH will become adsorbed to particulates, mostly of relatively short range, and be redeposited and vaporised one or more times until they reach the coW By analogy to DDE (1, 2), the redistri bution of PCBs by this mechanism is expected to hnv o resulted m the redepostuon into the ocean of one quarter of the PCBs that originally fall out in the terrestrial environment The deposition p*tIrrn will be influenced by the sue distribution of purl teles to which the PCBs arc attached, with a major fraction ending in coastal waters and a small amount being transported into remote regions It is expected that a negligible fraction of the PCBs reaching the oceans will be revaponxed, thnr concentration will be too low VI ater Transport On account of the low water solubility and high specific gravity of PCBs, it is expected that jTKi-1 of the PCBs discharged into the environ ment will be resting as sludges or adsorbed in the sediment at the bottom of rivers or lakes near their jxunt of discharge, and that transport m stn uns will be primarily by means of waterborne particles Evidence for this postulate is indirect, roii-i'ting mainly of the strong adsorption of P( Hs by surfaces (22), the observation of PCB concentrations as high as five times the solubility htnii (fi), and the evidence that chlorinated hydro carbon pesticides are removed from contaminated lakes by adsorption in sediments and codepoBition villi algal bloom (24). The transport of PCBs within rivers is as iWnlied previously, by solution and readsorption in the sediment and by sediment transport The duU on partition coefficient between sediments ami water and on difTusivity in bottom sediment arc not available so that the transport modets (2Ti) developed for rivers cannot yet be applied The only measurements reported on PCBs m ground sediment are for Escambia Bay (G, 26) The PCB concentrat'on m the sediment 10 miles downstream from a discont nued source of PCB loss v ss found to remain constant over a year suggc-ting that the PCBs are strongly adsorbed to sediment Pile amount of PCBs transported to the ocean in `-elution or suspension in river water is esti mated as 200 tons/year (49), a small amount compared to the estimated input into rivers Irri gation waters amount to about of the total river flow and are expected to carry a corre spondingly tower amount, about 13 tons year, of PCBs to the terrestrial environment Dredging and Dumping Dredging of inland rivers and harbors may lead to significant transfer of PCBs from con taminated sediments, especially when the dredge spoils are dumped at sea. According to the Council on Environmental Quality (27), some 13 X 10* tons of polluted dredged spoils are dumped at sea annually off the United States, 90% off the Gulf and Atlantic coasts A nigh figure for PCB levels in polluted sediments is 30 ppm, the highest level recorded in the Escambia Bay incident away from the immediate vicinity of the discharge (26) If all the polluted dredge spoils were contaminated to this level, the total rate of transfer of PCBs to the oceans would be around 400 tons 'year, mostly into the Atlantic Ocean and Gulf of Mexico Based on the general level of contamination a more reasonable estimate of rate of transfer would be only 20 tons/ycar. Biota Flux The total quantity of PCBs stored in migratory animals is very small (see below), and their move ments cannot account for a significant fraction of environmental transfer of PCBs The movements with the greatest local significance are probably the migrations of seabirds from the northern to the southern hemisphere, these might account for the movement of about a ton/year (28) Human fishing is estimated to remove only 1-2 ton-year from the eastern North Pacific, the western North Atlantic and the Great Lakes (30) Separation and Transformation of PCB isomers in the Environment There are three processes hy which the compo sitions of PCB mixtures, may change after release into the environment Fractionation In general, the water solubilities and vapor pressures of PCB isomers decrease with increasing April 1972 NONS 208132 27 Table 3. Percent Low in Area of Seven Chroma togram Peak* of Aroclor 1JS4 After Steam Heating for 15 and M Minutes. [From Ref. 23[ Peak 25 Minutes 60 Minutes 1 66 2 41 3 22 4 40 5 14 fi n 70 63 74 73 54 51 15 33 chlorine content, although the decreases are not uniform (Table 2) (31) Hence the processes of evaporation, co-distillation and dissolution in water are expected to fractionate mixtures of PCBs, the lower isomers being much more mobile Tlus has been confirmed for oo-distillation by the results of Freed (21, 23) (Table 3), which show that, after 60-min Bteam heating, the ratio of the first to the sixth of the seven major chromato graphic fractions of Aroclor 1254 was reduced by a factor of 5 Thus environmental transport mechanisms involving these processes are ex pected to reduce the proportions of the lower isomers in environmental samples near to the point of release, and to augment them in samples from remoter areas Photolysis and Chemical Decomposition Photoiytic decomposition of certain PCB isomers has been reported by Hisebrough et al (32, 33), and Safe and Hutstnger (34 , 35). The results of the latter suggest that in natural sunlight some higher isomers are more eaady broken down than the lower isomers (35). Hence photolysis is expected to reduce the proportions of at least some of the higher isomers. Photoiytic dechlori nation is also expected to give rise to lower isomers, including some which may not be present in commercial mixtures (35). Because of their high stability in industrial use, we assume that other forms of chemical (nonbiological) decomposition of PCBs are very slow in the environment. Metabolism and Excretion Published studies of the dynamics of PCBs in rats and birds (36-39) indicate that the higher isomers are generally taken up and or retained more efficiently In each case the proportions 0[ pentachloro- and lower isomers found in the ani mals' tissues were generally lower than those m the Aroclor mixture to which they had lieen exposed, but there appears to have I Hen little differentiation of the higher isomers except for one hexachloro-txnmer (36-38) It is nt clear m each case whether the deficiencies of the lower isomers resulted from metabolism oi differential excretion, but it is likely that both occurred Because of their higher solubility in water {see above), the lower isomers would be expei ted to lie excreted more easily than the higher isomer* In one study on rats, loss of PCBs from i hr both was marked!} retarded by adminixtiatinii nf car bon tetrachloride (.36), suggesting mtc t ft-rcnci with metabolism in the liver In anothei xtmly with rats, hexachbro-ixomen were found in dis proportionate amounts in the urine (37). nig. gesting that the tetra- and pentachloiu-i-ouiei' were metabolised and the hc'tachloro-i.xomri-* ex creted in part, while the hcptachloro-isomeix wen differentially retained Two studies of met.il>oio-ni of PCBs m fish (40, 41) gave incon^i-ti (it iexult* but showed no marked tendency foi the lughei isomers to accumulate differentially | j Evidence from Environmental Samples Y'eith (42) has found that the proportiuu of higher isomere increases downstream in soun \\ i*. consin rivers and that certain isomers character istic of lower Aroclors disapjiear Since the lower isomers arc unlikely to be differentially returned in sediments (see above), this suggests that ilicy are decomposed rapidly in the river environment Evidence of biodegradation of pesticides under anaerobic conditions (43) suggests Ihe possibility that the degradation of the missing lower ixometx may occur primarily by microbial metabolism in the bottom sediment We recommend that highm priority be given to this aspect of dceompoxttmi! of PCBs A number of writers have reported that P( M samples extracted from animals generally mat elt Aroclors 1254 or 1260. but that the lowest !*oinciare sometimes relatively deficient or missing peciaily in animals high in food chains (38, Ml Since substantial quantities of Aroclors 1242 and 28 Environmental Health Perspectives HONS 206133 Tablr 4. Relative Pub Height* in Cas-LIquld Chromatogram* of Extract* from Sewage Slud|. IFrotn Ref. 451 > rrtcnl.mi umr 0 fi'l o si 1 00 1 -'1 1 i :i 2 (JO 2 :t4 2 S3 .7 33 3 70 Sludge A 38 03 61 104 100 92 32 19 21 10 5 Aroclor 1254 30 72 59 no 100 92 26 12 10 8 4 Sludge B 39 24 17 51 100 66 70 53 84 29 25 Aroclor 1260 3 14 4 30 100 69 70 55 87 30 23 1248 must have been released into the environ ment (see above), these observations suggest that a large proportion of the lower isomers (those with 4 or fewer chlorine atoms) is missing from the animal samples. However, few critical data have been published. In one set of data from tewage sludge (45) (Table 4), none of the lower isomers is clearly deficient In published chromato grams for birds (38, 46, 47) (Table 5), only the lowest isomers (tetrachlorobiphenyls) and one higher isomer were markedly reduced in com parison to Aroclor 1254 In human adipose tissue (48), however, pentachloro- and hexachloroisoinere were reduced or missing TLe higher isomer reduced in the bird samples (38, 46) is probably that reported as deficient in samples from the Gulf of California and Quebec and found to be degraded by ultraviolet light (32, 33). With this one exception, we conclude tentatively that higher isomers (pentachlorobi phenyls and higher) are not significantly differenti ated as they pass through food chains up to fish and birds. Mammals appear to be able to excrete and/or metabolise penta- and hexachloro-isomera. The lack of the lower isomers in samples from relatively contaminated areas could be accounted for either by metabolism or by their greater mo bility. However, Risebrough and Berger (33) found that lower isomers were relatively deficient also in samples from fish in a remote lake in northern Canada This suggests that differential metabolism is the primary mechanism in the environmental differentiation of isomers, outweighing the effects of differential photolysis and differential mobility, which would be expected to have reduced the proportion of higher isomers in remote samples Conclusions and Comments on Separation of PCB Isomers in the Environment We conclude that most PCB ieomers with four or fewer chlorine atoms have been degraded in the environment, possibly by microbial action De composition of penta- and hexachloro-isomers ap pears to occur in birds and mammals, but this will have affected only a negligible fraction of the PCBs in the environment Accordingly, we assume (3) that some 75% of the Aroclor 1242 released into the environment, 60% of Aroclor 1248, 20% of Aroclor 1254 and 5% of Aroclor 1260, have disappeared. The above discussion has been confined to a general comparison of lower and higher isomers, because of the scarcity of data on relative pro portions of individual isomers in environmental samples. In fact, the physical and chemical prop erties and biodegradabihty of PCB isomers depend on the positions of substitution as well as the total chlorine content, More precise data on the Table 5. Relative Peak Areaa (Peak 11w144} for Chromatogram of Extract of Eagle Carcaae and Aroclor 1254. (From Ref. 47] Paak No Relative retention nme* carcass Aroclor 1254 1 0 39 2 0 49 3 0 63 4 0 58 5 0 70 6 0 79 7 0.83 8 0 97 9 1 04 10 1.23 11 1 48 12 1 72 13 2 01 14 2 32 IS 2 86 16 3 38 75 34 8 55 0 15 7 58 6 28 0 111 0 68 7 91 6 92 5 100 0 62 2 28 7 18 4 32 6 15 4 68 100 0 42 3 21 0 191 5 32 2 127 5 66 5 105 2 139 5 100 0 75 5 23 6 42 6 38 8 22 2 p,p'-DDE-1.00 April 1972 MOMS 206134 29 exact quantities of individual isomers in environ mental samples would permit more precise con clusions about the sources and routes of contami nation. We urge that numerical characterisation of individual peaks should be published whenever possible Cumulative Input lute the EavireMMt From an extrapolation of the sales curve m Fig. 1 to sero production m 1030, it is estimated that the cumulative sales in North America over the period 1930-1970 were 5 X10* tons Assuming that the proportions of sales for different usee were simi lar throughout the period, the cumulative looses may be estimated as about 3 X10' tons into the air, 0X10* tons into fresh and coastal waters, and 3X10* tons into dumps and landfills. Using the results of the premous section, it may be estimated that roughly one-third of the PCBs released into the air and one-half of those released into water have now been degraded. It is difficult to estimate the extent of degradation in dumps, because some of the POBr there may still be in sealed containers, but we assume in any case that leaching from dumps into fresh waters has so far been negligible Of the PCBs released into the air, we have assumed that most would have been adsorbed Table 6. Range of concentration* of PCB* in --n|L from the lrieb Sea and Firth of Clyde, October 19W and *ub*equently (Ref. SS|, All Spu* are in ppm except tboee for aeaaater. Sample nze pen IDDT> 8etvatcr Zooplankton MusmIi Norway lobster Herring Whiting' muscle liver Cod muscle liver Other fish Rasorbili livers Guillemot livers birds found dead birds shot 53 7 r 2011 33 154 18 15 5 ft S3 7 < 10 ppt < 1 -3 pnt 0 01-0 03 0 01 0 05-0 0 01 0 <11-0 1 n oj-2 u 0 01-0 1 om io 0 01-0 4 0 01 1 O-T 0 1 0-2 0 0 J-l 8 4 5-50 0 4-0 52 2 J-l! 0 t-1 0 n tw-i o 2 -44 5 -13 49 2 -R80 5 0-2 1 7-26 (1 1-0 8 Theee figures, u given in the original onirre. include dieldnn as well as DDT and ita metabolite*. I ml dieidrm compnaed only o( the total in the caae of the gmllennrti onto particulates soon after volatilization and would therefore have fallen out relatively close to the source By analogy with DDT residues, PCBs in soil would be expected to have a half-life of the order of 5 years (2, 17) Again u<-ing the analogy with DDT and its metabolites, we may estimate that roughly one-quarter (1) of the total (i.e., 5X10* tons) will have been transferred into the sea, mainly into the Atlantic Ocean, and the remainder (1.5X10* tons) distributed over ter restrial North America, with a negligible fraction having fallen into fresh waters. Other inputs to the sea arc difficult to estimate but have probably been of the order of 10* tons (49) The remainder (2X10* tons) is assumed to have accumulated in lakes and rivers. FcamtE 5. Ocean sub-model PCB Livils and x DDT/PCB Ratios in tha Environmoat In this section we compare the above estimates of cumulative input into the environment with measurements of PCB levels m physical and bio logical samples The purposes of this comparison are: (I) to use the observed distribution of PCB* m the environment to check the consistency of our 30 Environmental Health Perspectives HONS 208135 K** (o un,r|n ly*. Scotland ll. AH SguN, feawalcr IDDl. o <11 (i <ji U OMI 1 0 m-i o (i i 1 0-2 <1 0 4-0 ',2 2 J-1J 0 CM-1 0 5 -]J 1 7-20 0 Ml M purr?, incluilf but difldrin be (Ollllonint.i kation and lively close Jr rcBidut s, f a half-Ufc using the la, we may If the total [erred into and the over terc fraction !inputs to t probably Hmaindcr pulated in estimates lent with and bionpanson PCBs in y of our Ifrrritm ,wl <,,ti|lrmr>l (CHS llffn While tndr,1 Eagle lirrtvl tnuacle limit! W'' Mean Baltic Sea Range N IDDT/PCB Mean Stockholm Archipelago Range N IDDT/PCB 43 68 30 250 (1 9-86) (0 5-23) (16-44) (140-360) (40) (18) (3) (9) 14 25 34 23 52 51 30 0400 <3 4-7 0) (3 3-8 5) (16-56) -- (15) (4) (3) (!) 14000 910 540 (8400-17000) (49(M500) (250-800) <4> (3) (5) 0G 15 57 15 !8 21 19 models .if environmental transport, (2) to define ilie tt pi's and locations of input to the environ ment Hindi arc responsible for the most senous contamination of the biota. I pinko and Biological Magnification of PCBs in Food Chains A In i ro fraction of the reported measurements of PC 1! concentrations in environmental samples have In'on from animats, especially fish and birds jugli hi marine food chains. Figure 5 outlines sclieiiL.itically a model for the transport of PCBs within a simple marine ecosystem Laboratory experiments (6, 41, 55) indicate that aquatic in vertebrates and fish can accumulate PCBs to level-' between 3X10* and 7X10* times higher limn those m the ambient water. It is not yet clear whether concentrations of PCBs are con sist mil ly increased as they pass up food chains from invertebrates to fish and from fish to fish (">7) However, PCB levels are clearly magnified ml In n food chains involving birds and mammals bv a factor on the order of 10 to 100 at each step (32,44, 56, 57, .58). Hence, in the long food chains cliainctcristic of marine systems (59), the lex'els ir 1 lie tup predators may be 107 times higher than those in the ambient water (Tables 6 and 7) In extreme rases, such as the Btarved guillemots in Table (>, or the eagle fat in Table 7, the concen tration factors may be as high as 10* or 10* HetiiUbe of the extreme variability observed both within find between samples, it is difficult to use measured levels in animals as a direct measure of levels in the environment However, it is possible to compare levels in the same species or in eco logically equivalent species to define spatial gra dients in contamination (29, 44) ZDDT/PCB Ratios Another approach (32,56) utilises the similarity in properties between the persistent PCBs and the persistent metabohtes of DDT, primarily DDE. (In the remainder of this section, DDT and its metabolites DDE and DDD are grouped to gether as ZDDT) Experiments suggest that PCBs and ZDDT are accumulated by aquatic animals to an extremely similar degree from ambient water (41), and measurements in the same areas indicate that the ratios ZDDT/PCB are very similar in animals from different levels in the food chain (Tables 6 and 7) (32,56, 75). Hence the ratio ZDDT/PCB in environmental samples, even in animals at the top of food chains, can be used to infer the ratio in the substrate, at least to order or magnitude (32, 56). Then the extensive infor mation about the environmental distr button and transport of ZDDT (2, 17) can be used to make inferences about the distribution and transport of PCBs. Air The only numerical measurements available of PCB levels in air are some estimates of levels in total suspended particulates (TSP) in four U S cities between 1968 and 1970 (E P A , unpub lished data). The mean level of PCBs was about 50 ppm, with levels in one city substantially April 1972 HONS 208136 31 higher. This is at least consistent with our earlier estimate based on rates of transport in the en vironment. Risebrough et al (60) were unable to detect PCBs in a sample of marine air off the California coast This suggests that long-range (inter-continentai) transport of PCBs in air may be negligible. tamination, meet of the human intake of PCB* appears to be \n fish (68), whereas 2DDT is much more widely distributed in the diet (72) If the fish and shellfish portions of the diet arc excluded the SDDT/PCB ratio would probably S>e greater than 10 Similar data have Ijeen reported from Sweden, where the SDDT/PCB ratio in total diet samples and m human milk is about 7 (73) Land We have estimated the cumulative net input into terrestrial North America as of the order of 1.5X 10* tons For comparison, the total quantity of IHDT remaining in terrestrial North America is of the order of 3X10* tons, as estimated by two methods: total application minus estimated total losses to the sea (1); mean levels of 2-3 ppm in a wide variety of treated soils, and leaser amounts in untreated soils (2, 17, 61) Hence the continental mean SDDT/PCB ratio should be of the order of 20 However, PCBs are expected to be distributed primarily around urban areas and downwind from them, whereas ZDDT is still concentrated in the agricultural and forested areas where it was applied (61). A nation-wide monitoring scheme using starlings (Slurnus rulpam) and woodcocks (Philoheia minor) is in progress Early results show mean PCB levels in pooled samples of fat from woodcock wings from eastern states in the range 4-7 ppm (62). SDDT levels from these pools are not yet available, but fat from woodcocks in Canada con tains 6-130 ppm, depending on the local history of application (29). Woodcocks feed on earth worms and should be a good indicator for soil concentrations. Other data from terrestrial birds may be biased towards low SDDT/PCB ratios, because the birds were obtained in industrialised regions. The median ZDDT/PCB ratio in a small sample of terrestrial birds from California was 4 (32) Ter restrial birds of prey m five European countries have very high levels of PCBs, with SDDT/PCB ratios in the range 0.3-6 (63-67) In human food, market basket surveys in the United States suggest a SDDT/PCB ratio of about 4 (68). Ratios in human milk (69, 70) and human blood plasma (7l) are in the same range. However, except for sporadic instances of con Fresh Waters Measurements of PCB levels m water are avail able only for relatively polluted river, and bays and are summarized in note (50) Table 8 summa rises PCB levels and SDDT/PCB ratios in fresh water fish and birds PCB levels are both abso lutely and relatively high in industrial rivers and in the Great Lakes; within the Great Lakes there is a gradient in SDDT/PCB ratios from west to east. Levels of PCBs are lower in the three samples from lakes in less industrialized areas listed at the bottom of Table 8. These data are consistent with our conclusion that roost PCB* in fresh water systems result from local discharge* by in dustries, but that there is also widespread aerial fallout of small quantities Based on our assumptions of mean concentra tions in fresh waters (50), we estimate that the water of the Great Lakes contains a quantity of PCBs of the order of 100 tons, the total quantity Table t. PCB Level* and XDDT/PCB ratio* in North American Freeh Water Vertebrate*. Area Mean PCB level in fish (ppm) IDDT/PGB ratio in fish and/or (wUeatmg birds Itefer- U.S industrialized men Lake Ontario L*k Michigan Lake Huron (Oeorfian Bay) Lake Superior Onts.no Lake Nipigon USA Prune Lake* N Quebec Lake Minto 1-213 19 2U -- -- 01 <0 1 01 0 005-0 35 0 08-0 2 0 4-1 55 74 5.3 0 8 7") 2 4 7r> 1-4 7") 2 7U i )3 32 ~FnvimnnientaJ Health Perapectives HONS Z0S137 of PCD* T is much '2) If the excluded, be greater rted from total diet 73). are availand bays 6 8ummam freshoth abso lvers and decs there west to e samples d at the sonsistent in fresh W by md aerial sneentrathat. the antity of quaotity tn North Refer ence u other lakes would be negligible The quantity med in river water would be only of the order of 3 tone It is difficult to estimate the total quantity in the biota, but since the Great Lakes' fisheries probably remove less than a ton of PCBs per year (30), the total quantity in fish is probably only of the order of 10 tons These quantities are negligible in comparison to the estimated cumulaUve input of 2X10* tons after allowing for degradation Hence we conclude that almost all the PCBs that have been released into fresh waters are adsorbed onto bottom sediments There are no data to indicate the rate at which they are being released into water and into the biota or being biodegraded It is expected that PCB levels in ground water will be negligible, since any PCBs in water per colating through the soil should be adsorbed onto anil particles (77). The Sea According to our estimates, the total input of PCBs into the seas around North America has been of the order of 1.5 X104 tons, mostly into the Atlantic Ocean, in part by localised discharge and in part by aerial fallout. For comparison, the total input of 2DDT into the oceans haa been estimated as of the order of 5X10* tons (1), of which a disproportionate quantity, probably more Table 9. PCB Level* and IDDT/PCB ratio* in Marine Vertebrate*. Ares Mean PCB XDDT/PCB level in fish ratio in fish, Refer(ppm) fab-eating bird* ence and/or mammals Long Island Hound B*y of Fuady Atlantis Ocean 1.2 0S 01 0 08-0 18 04 0 2-0 5 78 29, 79, 80 57 Puget Sound San Francisco Bay Californian Coast Gulf of California Gulf of Putami 0 16 0 l-l 2 0 02-1 -- -- 1 1 56 1-3 32,56 5 32, 56 10 32 1-2 32 Pacific Ocean (Galapagos k Hawaii) 0 03 >10 56 than 10* tons, would have fallen into the North Atlantic Ocean, because of the heavy use of DDT in North America (17) Thus, on these argu ments, thB total quantity of 2DDT m the oceans would exceed that of PCBs by several times, especially in the eastern North Pacific However, 2DDT has been introduced over large areas (1,2), so that it should be more uniformly distributed than the PCBs, the discharges of which are localised near industrialised coasts. Available measurements of PCB levels and 2DDT/PCB ratios, summarised in Table 9, are generally consistent with these predictions, al though the pattern is less clear than that in Table 8, in part because of the wide variability between species (cf Tables 8 and 7). The highest PCB levels and the lowest 2DDT/PCB ratios have generally been in areas close to industrial ac tivity At least in the Pacific, PCB levels decrease and 2DDT/PCB ratios increase with increasing distance offshore Data on marine birds summa rised by Keith and Gruchy (29) show that these offshore gradients exist m both the Atlantic and Pacific, and show further that PCB leveiB m the Atlantic seabirds are 5-10 times higher than those in the same or ecologically equivalent species in the Pacific. The correlation of high PCB levels with industrial activity has been demonstrated most clearly in Europe, where it has been observed along the coasts of Sweden (85, 81), the Nether lands (38) and Great Britain (82). The only major discrepancy from this general picture is the observation (57) of extremely low 2DDT/PCB ratios (of the order of 0.01) in zoo plankton over a wide area m the North Atlantic Ocean. 2DDT/PCB ratios in fish in the same area were of the order of 0.2 to 0.5 It is almost inconceivable that aenal fallout of PCBs could have exceeded that of 2DDT by such large factore over such a large area, hence to explain the dis crepancy, it is necessary to assume either selective removal of 2DDT or a local source of PCBb A likely local source is dumping or leakage from ships. PCBs introduced into the ocean in this way would frequently be mixed with oil, would tend to mix with surface slicks (83), and hence would be peculiarly subject to uptake by plankton Further study is necessary to confirm this sug gested explanation or otherwise explain the anom alous low 2DDT/PCB ratio in the Atlantic Ocean April 1972 HONS 20B138 33 Assuming the standing crop of fish in the eastern North Pacific to be of the order of 10* tons, and that in the western North Atlantic to be of the order of 5XlOr tons (derived from 59), we may estimate the corresponding loads of PCBs to be of the order of 10 and 15 tons respectively As suming the standing crop of plankton to be five times larger than that of the fish (1, 2), the corre sponding load of PCBs might be of the order of 100 tons In the case of ZDDT, it has been esti mated that the total load in manne plants may be several times larger than that m manne ani mals (2); this may be true for PCBs also, but we know of no measurements to support it In any case it seems unlikely that more than a few X10* tons of PCBs are stored in the manne biota of the area considered, a very small fraction of the total quantity introduced This fraction may be significantly greater than the corresponding frac tion for IDDT (1), because of the extraordinarily high levels of PCBs in the zooplankton. We know of no measurements of PCB levels in sea water However, as in the case of fresh water, it seems likely that most of the PCBs would be attached to sediments or to floating particles Observations on sediments (6, 26) are in accord with this. As for ZDDT, the deep oceans represent an ultimate sink for PCB residues There is no infor mation from which to estimate the rate of transfer of either to the deep oceans. SuffiMryanlCtKluteas According to the estimates made in this paper, the PCBs released into the North American en vironment in the past are now concentrated in three major compartments in the environment: (a) buried in landfill dumps (roughly 3X10* tons, without allowing for degradation); (b) attached to sediments m rivers and the Great Lakes (roughly 2X10* tons); (c) attached to sediments on the continental ahelf (roughly 10* tons). A further substantial quantity (of the order of 2 X10* tons) has been widely distributed over the land and sea by aerial fallout and by disposal from ships. Atl the numerical estimates are expected to be valid to order of magnitude only Transfer of PCBs within the environment is expected to take place by the following main routes: (a) volatilization, aerial transport on par ticulates, and fallout, (b) leaching from dumps, (c) sediment transport in rivers and in the hn!bw sea; (d) sedimentation in the ocean Uptake and transport by the biota is probably & quantitatively unimportant route of transfer of PCBs hut is of major biological significance Virtually no evi dence is available on the rate of transport lw any of these routes, but it is expected that all are very slow In particular, PCBs in sedum ni> in rivers and lakes are likely to move downstream and augment those in the shallow sea for a long penod into the future As a result of Monsanto's restrictions on distri bution, several inputs into the environment are likely to have been sharply reduced As existing products containing PCBs are scrapped, the re maining inputs into the North American environ ment are expected to decline gradually over t penod of the order of ten years As in the case of DDT and its metabolites, the total quantities of PCBs accumulated by the biota are an extremely small fraction (less than one percent) of those in the environment This raises the possibility that the dissemination of relatively small fractions of the PCB production into sensi tive areas may be primarily responsible for locally high levels of contamination of the biota Evaluation of the long-term effects of the accumu lation of PCBs and of the change in use patterns and production will require the development of environmental transport models more sophisti cated than those currently in use, together with the requisite data. The following aspects of environmental trans port of PCBs should have high priority for futurr research. 1. Properties and Rates Data are needed on the solubilities and vapor pressures of indi vidual isomers; on the partition coeffi cients between water and sediments, water and fats, on the rate of photochemical decomposition and aerobic and anaerobic biodegradation, diffusion coefficients in sediment 2 Routes The magnitude of leaching from dumps and aenal transport of PCBs must be established by direct measurements of concentrations m ground waters, airborne 34 Environmental Health Perspective* HONS 20B139 Ion par I dumps, I shallow fikc and Itativrly ut IB of [no evif by any ail arc hcnlo in stream ' a long diatnbent arc I existing the re~ pnviron- over a Dtlves particulates, and rainfall Data are needed on the magnitude of industrial losses in the past. 3 Transport Models Data are needed on the transport of PCBs in contaminated sedi ments in nvers and shallow seas, including the dynamics of uptake from shallow sedi ment and louses to the ocean floor 4 Lewis. Additional data, including quanti tative evaluation of the fractionation of isomers, are needed for fresh waters, sods, terrestrial plants and animals 5 Bioaccumulalion model Inasmuch as the l>enod since the first commercial appli cation of PCBs is less than the lifetime of a number of species, including man, and the use and discharge pattern has been variable, there is a need to project the long-range impact of the PCBs on the different elements of the ecosystem. l'lmphaBis should be on coordinated approaches to sampling and modelling rather than a statistical accumulation of data. REFERENCES 1 SCF.P (Study of Critical Environmental Problems). 1970 Man's Impact on the Global Environment MIT Press Cambridge. Maaa 2 Wood well, G M . Craig, P P and Johnson, H A. 1971 DDT in the biosphere where does it go' Science 174 1101 3 Detailed analyses of the commercial Aroclon have re cently been given by Stalling. D L,, andJ N Huckms, 1971, Journal of the Association of Official Analytical Chemists, 54 801-807, and by Webb, R G., and A. C McCall, 1971 Identities of Polychlorinated Biphenyl (PCB) Isomers in Aroclon Paper presented at 162nd National Meeting of the American Chemical Society, Washington, DC., September 13. The approximate compactions of four of the major Amciors aa reported by R. Webb (PCB Newsletter, 28 July 1971) are for 1243; 3% mono-, 13% di-, 28% tn-r 30% te*r-, 22% penta-, and 4% hexachlorobiphenyls, for 1248, 2',, u 18% tn-r 40%, tetri-, 36% penta-, and 4%, hexschlorobiphenvls, for 1254, 11% telra-, 49% penta-, 34% hexa-, 0%, heptachlorobiphenyls, and for 1260, 12% pent*-, 38%. hexa-, 41% septa-, 8r, octa and 1% nonachlorobiphenyls A slightly different composition for Aroclor 1242 has been given recently by Monsanto (W B l'apagcorge, personal communication, 1972) 1% mono-, 16% di-, 49% tn-, 25% tetra-, 9% penta-, and 1 % hcxa-clilorobipheiiyl 4 Isono, -N 1970 Jishu Kora 1(1) GO, 1(4) 58 (in Japanese) 5. Kuratsune, M et al 1971 Yusho, a poisoning caused by rice Oil contaminated with chiorobiphenvls Un published report. Department of Public Health, Kyushu University (See p J19, this issue ) 6 Duke, T W , Lowe, J I , and Wilson, A J )97(1 A polychlorinated luphenv] (Aroclor 1254) in the muer sediment, and biota of Eacambia Bey, Florida Bull Environ Contamm Toxicol, 5 171 Tlie reference ascribes the loss to leaks from heat exchanger* but information obtained subsequent to public., aon indi cates thst the losses were from sn air compressor 7. F D.A 1970. Supplement l Stalus report on the chemistry and toxicology of |>olrch]orinsted hiphcntls (PCB) or Aroclon Decemher 1970 Unpublished report. 8. Schmidt, T T., Risebrough. R W snd Cress. F 1971 Input of polychlorinated biphenyls into Cali fornia coastal waters from urban sewage outfalls Bull Environ Contamm Toxicol 6 235 9Papageorge, W B and Wheeler, E P, 1971 (Mon santo). Personal communication, 8 February 10 Peterson, A 1970 PCB--Possibilities of destruction. Pp, 82-87 in PCB Conference, Wenner-tiren Center, Sept 29, 1970. Stockholm National Swedish Environ ment Protection Board 11 Ltdgett, R. A and Vodden, H A 1970 PCB--the environmental problem Pp 88-06 in PCB Conference. Wenner-Gren Center, Sept 29, 1970 Stockholm National Swedish Environment Protection Board 12. The lose of Aroclon from selected plasticized resins maintained at 87'C for 24 hours over activated char coal is reported in Ref 84 For five Aroclor plasticizers m polyvinyl chloride the fraction of the plasticizer lost was 19% for Aroclor 1248, 14% for Aroclor 1254, snd 11% for Aroclor 1260, for three Aroclor plasticizer* in polyvinyl acetste emulsion adhesives the percentage losses were 4 8% for Aroclor 1221, 4 3*0;, for Aroclor 1232, snd 4 0% for Aroclor 1242 These 24-hour tests st elevated temperatures are equivalent to operation at ambient temperature over a one-year period for an Aroclor 1242 to a two-year period fur an Aroclor 1254. if it u aseumed that the loss rate is proportional to vapor pressure These results provide the basis fnr estimated loss of 10 to 20 percent of the plssticner over the useful lifetime of a plastic resin It in of course recognized thst the fraction of plasticizer vaporized will vary greatly between objects depending on ahiiiic, size, snd ease of diffusion of the plasticizer to the surface 13 Approximately 190X10* tons of municipal waste and an equivalent amount of industrial solid waste are dis posed of annually in the U S A month' m dumps Some 4 7 X VO* tons of industrial solid waste are dumped at sea (27) Of the remainder 18x10* inns are burned in municipal incinerators. 14X10* tons tn commercial incinerators, and 14x10* tens m open burning dumps (14) Emission of PCBs from these sources may be gauged indirectly from measurements of the ennsaion of comlmstiblc matter and pohnuclcnr aromatics From measurements reported by A D April 1972 HONS 208140 35 Little (15) end Hem end Engdahl (16) it is estimated that 6 25 percent of solid waste is emitted by muni cipal incinerators and up to 20 percent bv open burn ing If it is assumed that the fraction of FCBs in the organic mutter evolved from these sources is equal to the fraction of PCBs in the solid waste, the rate uf emission of PCBs will lie of the order of two tons per year from municipal incinerators ami 200 Vons/year from open burning dumps Commercial incinerator* and burning of automobile component (each contribute a total quantity of polynuclear aromatic compounds (s rough gauge of incomplete burning) similar to that emitted by open burning (14) The estimate of 200 lons/year of PCBs from the burning of commercial waste and aulo components is baaed on the relative magnitude of combustible pollutants from these dif ferent aources, weighted by the eatimate of the frac tion of total PCB production disposed of in open dumps. These estimates are highly speculative but are considered sufficient to discount the burning of wastes sa the major aource of PCB emissions 14 Hangebrsuck, R P , Lahndan, D J von, and Meeker, J E 1967 Sources of Polynuclear Hydrocarbons in the Atmosphere, U S Dept of HEW, P H S Publ No 999-AP-33 15 Arthur D Little, Inc 1970 Systems Study of Air Pollution from Municipal Incineration, Report to Division of Process Control Engineering, N A P.C A HEW, under Contract CPA-22-69-23 16 Hem. G M and Engdahl. R. B 1959 A Study of Effluents from Domestic Gas-Fired Incinerators, American Gas Association. 17 Edwards, C A 1971 Persistent Pesticides in the Environment Chemical Rubber Co , Cleveland, Ohio ift Stanley, C W et al, 1971 Measurement of the at mospheric levels of pesticides. Environ Sci and Technol 5. 430 tit Air Quality Criteria for Particulate Matter 1969 U S. Public Health Publication AP-4B 20 Council on Environmental Quality. 1970. Environ mental Quality Fust Annual Report of C E.Q. Washington, D C. 21 Freed, V. H. 1971. Personal communication 22. Evidence for the turfact adsorption of PCBs is pro vided by an experiment m which one gram of woodbum was added to 100 ml of a aolution saturated with Aroclor 1254. The concentration of the PCBa in the eupematanl liquid was found to be below the detection limit (23). Additional evidence is provided (iy the expenmenis reported by J. Eichelberger (PCB Newsletter, July 1971) He observed that when 10 ag of an Aroclor, ranging from 1232 to 1268, is added u> one liter of river water approximately 00 percent is ad sorbed on the walls of the container or the silt in the river water 23 Freed, V H 1971 Preliminary Notes on the Study of Physical-Chemical Properties of Polychlorinated Biphenvl Aroclor 1254 as Related to its Environmental Behavior, Personal communication October 7, 1971 24 Veith, G D and Lee G F 1971 Wafer rhemistn of toxaphene--Rule of lake sediments Environ So Technol 5' 230 25 Shih, C and Glovna, E F I960 Influence nf Sed, ments on Transport of Solute" Joum il of thr Hi draulic Division Proc Amer Soe Civil Lug 95 | ;4r 26. Nimmo. D R et al 1971 Polvrhlonniiieil Lipheml absorbed from sediments bv Fiddler Crnlis and Pint Shrimp Nature 231 50. 27 Council on Environmental Quality 1070 Own Dumping A National Policy Washingtun D r 28 For example, Wilson's Petrels (Octa?oie\ ocett,nnn\ collected m the western North Atlantic neraged ,i; ppm of PCBs (29) 10* birds each weighing 40 g and contaminated to this average level would csrrv 0 If tons of PCBs 29. Keith, J A and Gruchy, I, M 1971 Residue levrO nf chemical pollutants in North American wildlife Cn Wildl Serv Pesticide See MS Report 22 30. The marine fisheries remove some 10' tons of fish per year, at mean PCB levels assumed to be o( the order nl 0.2 ppm The Greet Lakes fisheries remove some K>` tons of fish per year, at mean PCB levels assumed to lie of the order of 5 ppm (Statistical Alistrnct of the United States, Tables 8 and 9) Only part nf the catch of marine fish is landed in North America The largest component of imports of fish into North America a fiah meal from Peru, where PCB levels are probahly relatively low (Ruebrough, this symposium 39) 31 Evidence of fractionation of isomers on solution is pro vided by V H Freed (Some Physical Chemical Properties of the Polychlorinated Biphenyls-- Aroclor 1254, unpublished manuscript, 1971) The chromstogram peaks of the lower isomers m the extract of the water soluble fraction were higher by factors up to ten than the corresponding peaks in the standard Mtsolute solubility of individual isomers is reported by Hoover. T B 1971 Water solubility of PCB isomer" PCB Newsletter, third issue (28 July 1971) 32. Ruebrough, R. W. et al. 1968 Polychlorinated bi phenyls in the global ecosystem Nature 220 1098 33 Ruebrough, R W and Berger, D D 1971 Eudence for aerial fallout of polychlorinated biphenvU (PCBI in the eastern Canadian arctic Can Wildl Serv Pesticide ser MS. Report 23. 34 Safe, S and Hutimger, O 1971 Polychlorinated bi phenyls photolysis o( 2,4,6,2',4',9'-hexnch)ornl>iphenvl Nature 232 641 35 Hutsmger, 0 and Safe, S 1972 Photochemical be havior of chtorobiphenyla (PCB) This symposium 15 36 Grant. D L., Phillips, W E J and Villeneuve D C 1971 Metabolum of a polychlorinated biphsnvl (Aroclor 1254) mixture in the rat Bull Enuron Contamin and Toxicol 6 102 37 Curley, A et al 1971 Polychlorinated biphenil" distribution and storage in body fluids and tissues of Sherman rats Unpublubed MS Environment il Protection Agenrv *6 Environmental Health Perspectives HONS 208141 mutrv nf Iron a., of SediI the Hv ICS 1347 I hipbeni I "<1 Pink Ocean |C eeamnn) raged 3S fO R end m-v 0 14 level, of ife Can | fish per order nf one jo* limed to t of the catch i large** Iienca i* robabii ) i ia proIhemiral [Amclnr gomato. of the 1 to ten llieolute p cover, PCB ted In- m ndenc* (PCB) Serv led bilorobi- :al beoeiulti DC bhenvl pviron Linvls lues of Rental live 3K Koeman, J H , ten Noever de Brnuw, M C. and Vo*, K H de I960 Chlorinated biphenvl, in fish, inueeel, and bird* from the River Rhine and the Nrtherlanda coaatal area Nature 221 1126 3li Heath, R. Q et al, 1070 Effect* of polychlorinated Inplienvla on bird* Paj>er read at XVI Congreasus International!, Omithoiogirue, Den Haag, September 1070 40 Mailing, D, L 1971 Preliminary report in PCB New* letter, No 3, 28 Julv 1071 41 Hon son, D J et al 1971 Chronic toxicit), uptake, and retention of Aroclor 1254 in two eetuanne hahee Hull Environ Cnntamin Toxicol 6 113 42 Vnth, 0 D 1972 Chiorobiphenyis (PCB*) m Wieconein natural water, Thia avmpn*inm, 61 43 a C.ueni), W D and Beard. W E 1067 Anaerobic Biodegradation nf DDT to ODD in Soil, Science 166 1110 h MarRae, 1 C , Raghu, K Rnd Caatro, T F 1967 Pematencc and biodegradation of four common lanmert of bentene hexachlomle m aubmerged aoiLs J Agric FoudChem 15 911 c Raghu, K and MaeRae, 1 C 1006 Biodegradation of the gamma isomer of bentcnc hexachloridc in aubmerged aoila Science 154 203 44 Jenaen, S ct at I960 DDT and PCB in marine ani mals from Swedish water* Nature 224 247 45 Holden, A V 1970 Source of polychlorinated bi phenyl contamination m the marine environment, Nature 22K 1220 41' Holme*, D C , Simmon*, J H and Tatton, J 0'G PHu Chlorinated livdrocarlKMis in British wildlife V a* ore 2US 227 47 Hnglcv, G E, Reichel, W. L and Cromartie, E. 1970 Idcntihi ntion of polychlorinated biphenyls in two bald eagle* l>y combined gas bipod chromatog raphy-man* Hpeetmmctry J Assoc 0(1 Anal. Chem, 5;i 251 4s Iliros, F J , Walker, A C and Medliery, A 1970. I'nlvchlnnnated biphenyl, in human adipose tissue Hull Environ. Contamm Toxicol 5 317 49 The total input of PCB, into the oceans from North American rivers is estimated at of the order of 2X10* ion*/year, by assuming that one-tenth of the total runoff from the continent (3X101* blert/year) is in highly polluted rivers with a mean level of 500 ppt, (pari* per trillion] one-lhird m alightly polluted nvers with a mean level of 60 ppt, and the remainder at much lover level* (SO) Sediment transport to the ocean i> unlikely to have amounted lo a* much as 10 perccni of the annual input to fresh waters, (i e , no more than 2X10* tona/year after allowing for degradation) Sew age outfall* may arcount for a few x 10* tons/year, but this estimate depend, on a single measurement of a high r.mrenirauoii in Loa Angeles (B1 which may not have been representative Some 4M10 ton, year of sewage "bulge, itre dumped off the Atlantic and Gulf const* of the II b (27) if till* contained the levels til PCB* (0 2-14 ppm) found in British sewage sludge (45), thm would amount lo no more than 50 tona of PCBs Dumping of polluted dredge apoils w,, estimated in the text to contribute no more than 20 tona/year Accidental leak, from coastal mduatriex might be of the order of lb1 tons/year, baaed on measurements in sediments following the Escambia Bav incident (6, 26) Deliberate dumping at sea i estimated to account for 2-3 percent of industrial solid wastes in the '' S \ (13) if it accounts for the same fraction of industrial disposal of scrap PCBs, thi* would amount to leaa than 100 tons/year, after allowing for degradation Losses from ships may amount to a fswxio* tnns/year. baaed on the likely contribution (5-10'.J>) of shipping to total uses c.' hydraulic and lubricating fluids Losses from aircraft are assumed to be small Moat of these figures art probably somewhat high, so we estimate the total input to the oceans from these sources ss lea* than 10* tons/year Only some 10-20 percent of this total is likely to have entered the Pacific Ocean 50 Only scattered data on concentrations of PCBs in water have been published Water entering a Swedish water works contained 0 5 ppt (parts per trillion) (86) Industrial nvers in Maine, Massachusetts, Wisconsin, and Japan contained levels ranging from 0 2 to 2 8 ppb (42, 51-53). Measured levels in Green Bay and Milwaukee Harbor, Wisconam, were in the range 20-70 ppt (42, 52) Other estimates can be derived indirectly from I DDT/PCB ratios in aquatic fauna (see text) In Lake Michigan, where DDT levels are of the order of 1-3 ppt (64), SDDT/PCB ratio* in fish are of the order of 0 4-1 (55) In San Francisco Bay, the IDDT/ PCB ratio in the biota n 1-2 ( 32, 56), and the level of I DDT m the San Joaquin River, the main input to the bay, ia about 100 ppt (32) For the rough calculations of storage and outflow made in this paper, we use these data as a basis to adopt 0.5 ppt as a plausible back ground level for unpolluted fresh waters, 5 ppt for the Great Lakes, 50 ppt for slightly polluted nvers and polluted bays, and 500 ppt for highly polluted nvers 51 Ikilainen, A 1971 PCB--Pest or Pesticide7 Paper read at All New England Conference on Pesticide Monitonng and Control, 13-14 December 1971 New England Regional Commission 52. Veilh, G D and Lee, G F 1970 A review of chlori nated biphenyl contamination in natural waters Water Res 4 265 53 Fupwars, K 1971 Research reports on environmental contamination bv polychlorinated biphenyl m our laboratory. Unpublished report, Public Health Inst of Kyoto Citv 54 Reinert, R E 1970 Pesticide concentrations in Great Lakes fish Pestic Monit J 3 233 55. Stalling, D and Mayer, F L 1972 Toxicibe* of PCBs to fiah and environmental residues in fish. This symposium, 159 56 Riaebroiigh, R. W I960 Chlorinated hydrocarbon* in the global ecosystem. In G G Berg A M W Miller (ed* ) Chemical Fallout, Charles C Thomas, Spring field, Illinois, 5 57 G Harvey (personal communication, December 1971) has found mean levels of the order of 200 ppb in April 1972 HONS 208142 37 sooplankton over large tree* of the North Atlantic Ocean Levels in fish caught in the same srea were consistently somewhat lower, of the order of 100 ppb However, plankton-eating birds of the western North Atlantic had much higher levels, J --35 ppm (29), 58 Holdgate, M W (ed ) 1970 The seabird wreck of 1909 in the lnsh Sea Unpublished report (with suppl). Natural Environment Research Council London 59 Rvther, J H 1969 Photosynthesis and fish production in the sci Science 106 72 60 Risebroufh, R W et al 1968 Pesticides transat lantic movements in the Northeast Trades Scisnce 195 1233 61. Stevens. L 3 , Collier, C. W and Woodham, D W 1970 Monitoring pesticides in soils from areas of rsgufsr, limited, and no peeticide use. Peatic Momt. J 4- 345. 62 Stickel, L P 1071 Personal communication 63 Koeman, J H , Vink, J A J. and Goetj, J J M de. 1969 Causes of mortality in birda of prey and owls in the Netherlands in the winter of 1968-1969. Ardea 57-67 64 Preatt, I., Jefferies, D. J and Moore, N W 1970 Polychlorinated biphenyls in wild birds in Britain and their avian toxicity Environ Pollut, 1 3 65 Johnels, A G. 1970. PCB--Occurrence in Swedish wildlife Pp 29-42 m PCB Conference, Wenner-Gren Center, September 29, 1970 National Swedish En vironment Protection Board. Stockholm 66 Helmincn, M 1970 PCB--experiences in Finland. Pp 26-28 in PCB Conference, Wenner-Gren Center, September 29 1970 National Swedish Environment Protection Board, Stockholm 67 Karlog, 0, Kranl, I and Dalgaard-Mikkalsen, S. 1971 Residues of polychlorinated biphenvls (PCB) and organochlonn* insecticides m tiv*r tuwue from terrestrial Danish predatory birds Acta Vet Scand 12 310 68 Kolhve, A C 1972, Food exposures to polychlorinated biphenvls This symposium. 85 69 Kwebrough, K and Brodine, V 1969 More tetters in the wind Environment 12 10 70. Secretary's Commission 1969. Report of the Secre tary's Commission on pesticides sod their relationship to environmental health, Washington, D C U.S Dept of Health, Education A Welfare. 71 Creason, J P et si 1971 An in-hnnse technics! report on polychlorinsted biphenyl residues in human plumt Unpublished report, Bureau of Air Prvjlulion Scitnoi 72 Comeliussen, P E 1970 Pesticide residues in toul diet samples (V) Peetic Momt J 4 80 73 WeatoO, G , Norin, K and Andersson M !0;o Lcveli of organochtorine pesticides and pnl\chlorinated hi phenyls in margarine, vegetable oils and some foods of animat origin on the Swedish mBrkei m 1 i*o~-1Be, Vlr Foda, 22 10 74 Gilbertson, M and Revnolda L M 'In pnn; Hexachlorobensene (HCB) in the egg' nf Common Terns in Hamilton Harbor, Ontario Bull Environ Contimin. Toxicol 75. Postupilaky, S 1971. Toxic rhemicals and declining Bald Eagiea and Cormorants in Ontario Canadian Wildlife Service Pesticide Section MS Report 20 76 Anderson, D W et al 1969 Significance of chlori nated hydrocarbon residues to breeding pelicans and cormorants Can Field-Naturalists 83 80 77 Edwards, W M and Harrold, L L 1970 Agricul tural Pollution of Water Bodies. Ohio J Sci 70 50 78 Hays, H and Riaebrough. R W 1972 Pollutant concentrations in abnormal young terns from Long Island Sound Auk 89 (In press) 79 Zitko, V , Hutiinfer. 0 and Choi, P M K 1972 Contamination of the Bay of Fund)--Gulf of Maine area with polychlorinated biphenyls, polychlorinsted terphenyls, chlorinated dibenxodioxins, and dibenxnfurans 1 his symposium, 47. 80. Gaaktn, D E, Holdnnet. M and Frank It 1071 Orgsnochlortoe pesticide residues in Harbor Purjioiiiee from the Bay of Fundy region Nature 233 400 81 Westbt), G and Nortn, K 1970. Levels of urgani'chlonne pesticides and polvchlonnated biphrmh in fish caught in Swedish water areas or kept fur sale in Sweden, 1967-1970 Vir Foda, 22 93 82 Bailey, S. 1971 Report of third meeting of f K PCB Discussion Group PCB Newsletter, No, 3, Juh 1971 83 Seba, D B. and Corcoran. E F 1069 Surface 'licks as concentrators of pesticides in the marine ennron, ment Peatic. Momt J 3 190 84. Monsanto 1968 Aroclor Plasticisers Technical Bulletin 0/PL-300 85 Beilsteins' Handbuch fur Orgamschen Chemie 1004 5 606, Vierte A ullage 38 Environmental Health Perspectives HONS 208143 Enrironmmuai Hiaith Pmp^ai, Accumulation of Polychlorinated Biphenyls in Ecosystems by Robert W. Risebrough and Brock de Lappe* Chlonnaied biphenyl compounds have become ubiquitous components of the global environment They were first detected in species of Swedish wildlife (1, 2, 3) and were subsequently found in seals from the North Atlantic (4), in a bird of prrv in Great Britain (5), in fish and birds from the Netherlands (6), and in both terrestrial and marine species of wildlife in western North Amer ica (7) In the Arctic they arc present in the polar brars {Thalarctos martfimua) of Hudson Bay (8) and in fresh-water fish of tundra lakes that receive no pollutants save those that are present in the fallout from the atmosphere (9). In the Southern Hemisphere, they were found in all bird species analyzed that inhabit waters north of the Ant arctic Convergence but could not be detected in a number of samples of wildlife resident in Ant arctica south of the Convergence (10) In this paper Borne of the available residue data are summarized within the framework of a prelimi nary formulation of a mass balance equation for the global distribution of polychlorinated bi phenyls Commercial manufacture of PCBs was begun m the United States in 1929 (11), and environ mental input may be assumed to have begun at that time. Current levels of PCBs in major sinks, such as the oceans, are evidently a function of many parameters, including the proportion of the total amount of PCBs produced over the past forty years that has so far entered the environ ment, pathways of dissemination, rates of degra dation to other compounds, partition coefficients between water and lipid fractions of organisms * Ik*Jega Manut L*l*>r*LJ>rv, University of California, O Bo* 247, Bodega Bay, California 94923 and the rates of permanent deposition in the sedi ments The determination of accumulation rates in major sinks and the prediction of levels to be expected in the future, given estimates of global production and of environmental input, constitute a challenging problem in environmental science A mass balance equation that would predict future levels of PCBs in the sea might profitably be modeled on the approach recently used by Weiss et al. (12) in considering the amount of mercury introduced to the sea as a result of the activities of man Like PCBs, mercury in its various chemical forms is not essential to orga nisms but may be accumulated by them Both groups of compounds move through the environ ment by water and air transport and are mobilized from pools that exist in a variety of industrial products, m addition to the pool of mercury in the earth's crust. The transfer of mercury from the continents to the oceans in river runoff was esti mated to be at most 2.8 X 10* grams per year, considerably less than the estimated rate of ex change between the continents and the atmos phere, between 2 5X10" and 1 5X10" grams per year The tatter figures were obtained from meas urements of mercury in rainwater of the 1930's and in uncontaminated glacial ice The amount of mercury produced by man per year is currently on the order of 10M grams, of which one third was estimated to enter the atmosphere Other sources of atmospheric mercury include the burning of fossil fuels, cement production and mining oper ations, but the integrated input resulting from man's activities in industry and agriculture was less than that released into the atmosphere by natural processes The doubling of the mercury content in dated layers of snow from a Greenland April 1972 HONS 08144 39  . m w liiiffaii< rfrtfiTtr 'iifflBUBI ice sheet over the pwt severs] decades was at tributed to an increased disturbance of sedimen tary rocks as a result of human disturbance (12) Such an approach yielded the prediction that the current mercury content of marine organisms is at most no more than double that present in pre-technological times (12) The only synthetic pollutant for which a com parable mass balance approach has been formu lated is the insecticide DDT, together with its derivatives (13) Over the past fifteen years about fix 10w grams per year of DDT have been manu factured in the United States, of which a consider able fraction is exported The total global pro duction per year was estimated to be in the order of 10" grams (13), of which all may he assumed to be released into the environment Analyses of DDT concentrations in river waters indicated that only a small fraction of the DDT applied to the land would reach the sea by river transport. The few data available for DDT concentrations in precipitation indicated, however, that as much as one quarter of the global production and use could be transported to the sea through the atmosphere (13,14) Yet the total amount of DDT compounds estimated to be in pelagic marine organisms was only 10* grains Evidently ad ditional measurements of DDT in marine precipi tation and in the water and organisms of the mixed layer are required before the mass balance approach can be used to predict future levels of DDT compounds in the oceans. The ubiquity of the PCB compounds and their relatively high concentrations in marine organisms of certain arcus require that an approach now be made toward the formulation of a mass balance equation in the global environment, as a necessary step in the implementation of a program that would prevent the buildup of unacceptable levels. At the request of a number of environmental scientists concerned about the possibility that PCB levels would continue to increase in the sea, the Monsanto Company has recently released the total production figures for their Aroclor PCB products from 1960 through 1970, with estimated figures for production in 1971, Total U-S pro duction of the combined total of all PCB products rose steadily between 1960 and 1970, from ap proximately 2X101* grains in I960 to approxi mately 4 X10" grams in 1970. Estimated pro duction figures for 1071, however, show a shaqi drop from those of 1970 and are equivalent to 1960 levels Approximately half of this amount u UBed in "closed system'' applications as electneai insulating liquids The domestic sales for all other uses, including heat transfer systems, hydraulna and lubricants, plasticisers, etc , rose from ap. proximately 5X10* grams in i960 to about 1 5X10" grams in 1970, but estimated sales m 1971 were far below the 1960 figures A consider able fraction of PCBs used in the "open system1 applications can be expected eventually to enter the environment. U.S. export sales showed ap proximately no change between 1960 and 1965 and were in the order of 2X10* grams per year but have since risen to about 5X10* grams (15) Of the other industrialized countries that produce chlorinated biphenyl compounds, notably France, Germany, Italy, Japan, and the Soviet Union, production figures are apparently readily available only from Japan where PCBs are manufactured by two companies, the Kanegafuchi Chemical Industry and the Mitsubishi-Monsanto Chemical Company, with combined production figures on the order of 1010 grams per year, somewhat less than the estimated 1971 total U S. production of 2 0X10" grams In 1970, 60% of the PCBs pro duced in Japan were used for insulating Quids of capacitors and transformers, 15-20% for the sol vent in carbonless reproducing paper, 10% for heat-transfer media, and 5-10% as plasticizer* and for export (16) The figures made public by Monsanto were for total PCB production only. The chlorine compo sition of the PCBs detected in marine svstemB may vary widely between that in waste water outfalls and in the tissues of fish-eating birds (17, IS). It is therefore essential to have pro duction figures for each class of PCBs in order to understand PCB movements into and through marine ecosystems. These have been presented m the previous paper. Combined U.S and Japanese figures for 1970 were, therefore, on the order of 5X10" gram-, A reasonable estimate for global production figures, including the PCBs manufactured in the Soviet Union and Western Europe, is therefore on the order of 10" grams per year, equivalent to those of DDT Few data are yet available that permit calcu- 40 Environmentai Health Perspectives HONS 208145 Itzers P anon* of the Amounts of PCBs transferred to the Holden (19) found that about one ton of pCBs per year was entering the estuary of the CUdr a* a component of crude sewage sludge from the Glasgow district Approximately a ton of PCBs was also entering the estuary of the Thtnie1- ns a component of sewage sludge from London Comparable amounts of PCBs were found to be entering the Pacific Ocean from each of nrvcrul waste water outfalls m California (17) Thr evidence for atmospheric dissemination of PCBs derives from their virtually universal distri bution in the global environment, including resi dent Arctic species (9) and from the detection of PCB peaks in all samples of rain water obtained over a year st seven widely distributed sites in Great Britain (14) These PCBs were not quanti tated Permanent snow fields provide a historical rrcord of PCB deposition with time The possi bility therefore exists for measuring the total amount of PCBs transferred from the atmosphere to >mow fields receiving fallout from the major wind systems Measurements of PCB concentrations in the mixed layer of the ocean or in deeper waters have not vet. been made Concentrations in collections of zooplankton from the Atlantic, however, are surprisingly high. Samples from eleven stations mi r the Continental Shelf and Slope areas east of tin northeastern United States contained a median ronrentration of 57 ppm PCBs, with a range from 2 to 250 ppm, in the extractable lipids, defined as ail materials extracted in a Soxhlet apparatus with 2:1 hexane:acetone. Median dry and wet weight concentrations were on the order of 2 and 0.2 ppm respectively Comparable values were obtained `n samples from two stations near Bermuda and from two stations in the South Atlantic in the wind path from the industrialised areas of Brasil. Concentrations of PCBs in two samples from the eastern South Atlantic were lower by an order of magnitude (20), Because of wide differences in the total mass of sooplankton per unit volume of water, the values obtained in analysing soo plankton samples are an imperfect indication of the local burden of PCBs Moreover, partition coefficients between PCB concentrations in sea water and in zooplankton might be expected to duixtnd on the nature of the lipids and other non polar substances in which PCBs accumulate Quantitation procedures for PCBs have not yet been standardised In many waste water and biological samples, however, the profile of PCB peaks closely matches one of the commercial preparations, and accurate quantitation can then be made using the height of any one of the promi nent peaks The Atlantic zooplankton and fish samples from Long Island Sound (21) had a PCB composition that closely matched that of Aroclor 1254. Other environmental samples, particularly birds (10,18), frequently have a PCB composition that is intermediate between 54 and 60 per cent chlorine. An arbitrary decision must therefore be made which may affect the values obtained by a factor as much as 2 or more. Most of the PCB values reported in papers from our laboratory have been obtained by comparing the total area of all PCB peaks emerging after p ,p'-DDE to the total areas of the same peaks in chromatograms of the Aroclor 1254 standard These values may be somewhat too high Samples may contain a lower proportion of the PCB compounds with fewer chlorine atoms than does the 1254 standard Also, the biphenyl molecules with more chlonne atoms have a greater ability to capture electrons and give, in general, proportionately greater responses in tbe electron capture detector per molecule of biphenyl. Thus, two eggs of the Common Tern, Sterna furundo, from the colony on Great Guli Island, Long Island Sound, were quantitated for PCBs using two different methods The areas of PCB peaks emerging after DDE were first com pared with the 1254 standard and concentrations of 308 and 214 ppm in the egg lipids were calcu lated, respectively When areas were compared with the total areas of the same peaks in chro matograms of the Aroclor 1260 standard, the PCB values obtained were lower by a factor of 2 and were, respectively, 145 and 98 ppm. The column used m this instance was 5% QF-1 on Chromosorb W, 80-100 mesh, acid washed, HMCS treated Under the conditions employed, the inte grated areas of all peaks of chromatograms of equal amounts of Aroclor 1242, 1254, and 1260, respectively, increased in the ratio 1.00:1 68:2 46 (22). The different quantitation procedures used by various laboratories may be expected to yield, therefore, somewhat different estimates of PCB concentrations. It is reasonable to assume, how- April 1972 HONS 209146 41 Table 1. PCB In Marine Fish and in Fifth from an Arctic Tundra Labe* Locality, reference Species ppm, ppm, fresh extracted weight lipid or fat Tokyo Bay Sea ban Laieclabrax japontca 1 734 120 Nova Scotia Atlantic salmon Banks (23) Salma solar Atlantic hemng dupes Aorenguj Atlantic mackerel Scomber tcombru* Cod Gadus morhua 0 45 0 320 54 0 35 0 02 -- -- -- Swedish West Cod Cosat (2) Godus morhua Fish oil 0 02 -- 73 07 Bailie S#a (2) Atlantic hemnf Clupca hareityus Cod Cadia merhua Atlantic salmon Salmo solar Fish oil 0 27 0 03 0 30 -- 68 11 29 35 North Atlantic (24) Flying Fifth 0 001- Cypseluru* cxttfiens 0 007 0 405 Dolphin Coryphaena Atppurui Shark (liver) Cardtarhtmts lotiQtmamix Meftopelagie Fifth ( hauiiodut da not 800-900 meters 0 01 03 0 010 06 10 06 1 573 Long Island Atlantic round hemng Sound (21) Elntmcu* leret Atlantic atlverode Mwmd\a menui\o Atlantic mackerel Scomber scombrus 12 3.2 12 30 52 79 California Northern anchovy Ouulal Engruuhi matdaz Waters (25) Jack mackerel 7'rnehurui lyttiTnrtncuj Bluefin tuna 7'Aunnu# thyw\va i0 0 02 0 04 Table I--Continued Locality, rsferenoc Species ppm, ppm, fresh ejlrtrt*d * eight lipid or fit Peruvian Coastal Sardina. peruana Sardtuops sagax Waters (26) Caballa Scomber japon icu Bomto Sorda tarda Tiburdn uul Lake Mmto Ungava, Quebec (9) Pnonace ylauca Lake trout ta/refiNua nQmayeush n in 0 0.' 1) 01 a os 0 0400 0G 2 (1 01 11 4 1 112 0 (-)-Concentrations in parts per million ever, in comparing these results, that differences of an order of magnitude would yield useful infor mation on geographical distribution In Table 1 are summarized some of the reported PCB residue data in fish from representative areas of the oeeans and, for companion, PCB values obtained m lake trout, Salvehm<* namaycutk, from a remote Arctic lake Among these, highest concentrations were in the fish from Tokyo Bay and from Ix>ng Island Sound (Table 1) These bodies of water can be expected to receive significant amounts of PCBs from urban 'cwage outfalls A comparison of the residues in three species, the Atlantic salmon, Sabiio salar. the Atlantic hemng, CIvpea karengus, and cod, (iadut nwrhita, from the Nova Scotia banks and from Swedish waters shows that both areas have com parable levels of PCB contamination That PCBi have penetrated below the mixed surface lover of the North Atlantic is shown by their prc'-cncr in fish characteristic of deeper waters obtained at depths between 800 and 000 meters PC13- vine also detected in the fish from the coastal waters of Peru. The levels in lake trout from Lake Mnito in the Arctic region of Quebec were higher tlmn expected Lakes in this area arc shallow with rocky bottoms, almost no vegetation, and wry low amounts of organic matter It is interesting that levels in the Atlantic hemng, a fattv fi-.li, from both the Nova Scotia banks and the Bultu 42 Environmental Health Perspectives HONS 208147 Ppm, irtrartcd lipid or fat 00 Tablt 2. PCB raolduee in petrels and ehearwaten. Mean arithmetic concentration* in part* per million. Spw'^i rtftttnce Petri 1(27) OctQUiHirftma. homoehroa beach's Pi ird (28) OtmninlK'nta leuchorhoa Hlurk Petrel (T) Loomcfo'tUi mtianuj Lewi Pcirei (7) Htiocupictta microtoffui Fuimnr (7) /"iJmarux fflactolu Pmk-ftHi(w) Shearwater (7) Puijffipi/i cTtniapu* Soo(\ Mjerirwutfr (7) Pufitinx gnxeui Slender-lulled Shearwater Pvflttut* /PfM*tro6iru (7) Ribon1*- Petrel (10) Awhile* ocean icua (limit IVtrel <IO> SfarrttHcdeM pifauteux Snow lWrrl (10) VtiftKiromn titreri Breeding area Non-breeding area Tueue (N) California coastal California coastal Whole body (9) waters waters E (G) Dt}i California, Eastern Pacific Pacific Coast Clulf nf Cntffnmia -- E (D Whole body (8) (Iwlf of California -- Whole body (3) Alaska Eaetera Pacific Whole body (3) Chile New Zealand Western Mexico, Whole body (1) northern Pacific Northern Pacific Whole body (2) Australia Northern Pacific Whole body (1) Hallett Station, Antarctica Palmer Station, Antarctica Palmer Station, Antarctica Hallett Station, Antarctica Auotralian aeaa Whole body (10) North Atlantic Whole body (9) Southern ocean* ei(3) Antarctic ice-pack Whole body (10) ppm. fresh weight 21 37 10 04 23 04 11 21 21 33 02 0 08 ppm, liquid bpwJ 200 (120-320) 240 (130-370) 3AO -- -- -- -- -- -- 11 0 190 35 03 trere mnt.Klora.bly higher, by an order of magni tude, than levels in the cod on a fresh weight lifiM-', nil hough the cod occupies a liigher potation in tin- food web On a fat haaia, however, residues in both species in the Baltic were comparable. These data suggest that amounts and kinds of lipids may affect the retention of PCBa, modifying the trophic accumulation predicted by the classical food chain concentration theory. Consistent with this hypothesis are the higher PCB residues meas ured m extractable lipids of the North Atlantic plankton than in the lipids of fish from the same area. Tabic 2 includes a representative selection of PCB residues so far measured in species of petrels and shearwaters. These birds are strictly oceanic, rarely approaching land except to breed on islands or undisturbed coastal areas They do not dive for their food as do the fish-eating species but rat her feed upon plankton and other organic ma il'rial obtained at the surface of the sea Within a given marine ecosystem, their burdens of both PCB and DDT residues may be higher than those in fish-eating species such as the Brown Pelican, Pelccaniui occidenialis, or the Osprey, Pnndion haliaetu* (7) Many specieB range widely, breeding in one hemisphere and spending the remainder of the year in the other As expected, lowest levels were found in the Snow Petrel, Pagodroma mvea, a species resident in the Antarctic pack-ice region High residues were found in tissues of the WdBon's Petrel, Oceamtei oceaniciw, from the population that breeds on the Antarctic Peninsula and mi grates to the North Atlantic during the southern winter months. Comparable levels were found in the Ashy Petrel, Oceanodroma konwhroa, a species that remains largely or entirely within the Cali fornia coastal waters. High PCB concentrations were also found in an egg of a Leach's Petrel, Oceanodroma Unehorhoa, obtained on the lslas Coronados, Baja California, Mexico, m the vicinity of San Diego, California (Table 2) Table 3 summarises the PCB data obtained from analysis of eggs of the Brown Pelican over a April 1972 HONS 206148 43 r " ------- - Table 3. PCB teiiduei In egg* of the Brown Pelican PeleeaniM occtdenislis, from North and South America. Mean arithmetic concentration! in parti per million of the egg lipid (301. Breeding area Sample ppm PCB, sire lipid Peru Pftnamti Venezuela Jamaica Flond. 4 colonies Gulf of California San Benito* Western Baja California Sen Martin. Western Baja California Los Coronados Anacapa Island, California 5 14 04 45 4 19 ft7 71 44 10 39 6 72 2ft 266 85 210 substantial portion of its range in both North and South America This species feeds only upon marine fish obtained in coastal waters As expected, levels in the South American populations are sub stantially lower than those in North America. Concentrations in Florida tend to be about three times lower than those in California This species docs not occur along the coast of the heavily industrialized areas of the northeast United States, but m these areas the Osprey, another fish-eating species, may contain PCB residues five to ten times higher than those in the Florida populations of Brown Pelicans (29). The PCB or associated compounds are a possible cause of embryonic mortality observed in the Osprey populations of these areas (29) The major difficulty in the design of a moni toring program to determine whether PCB resi dues are increasing or declining in a given sink is the lack of a statistical design that would es tablish how many individual specimens should be analyzed in order to determine the parameters of the pollutant distribution at a desired confidence level. Nor hss it been established which species of organisms are most suitable for such monitoring programs. The distribution of residues of chlori nated hydrocarbon insecticides and their deriva tives within a population is frequently nonGaussian, although transformation of residue concentrations to their logarithms yields a distri bution that is sufficiently Gaussian for the use of established statistical methods (.11, 12) Confi. dence intervals, therefore, can be established for such parameters as the means of logarithms of residue concentrations The confidence interval however, cannot be transformed back tn the onginal distribution There apparentK me no satis factory statistical methods that would determine the Confidence intervals about the arithmetic or geometric mean of measured concentrations (32) Moreover, one hundred or more analyses of indi vidual specimens might be necessary to determine the precision of the logarithm of the geometrical mean within a range equivalent to 10% of the value of the geometric mean (31, 32) The two major research problems that are emerging from the occurrence of PCBs in the environment appear to be first, the determination of the accumulation rates in sinks, so that the concentrations found there might be related to input levels, and second, the determination of the effects of PCBs or their derivatives upon organisms and ecosystems at each contamination lei el It might be hoped that s fraction of the considerable sums of money that would be spent at both national and international levels to ''monitor" the environment will be diverted to programs designed to answer the significant questions rather than to assemble tables of random numbers, for which there is no pressing need AcLnowledgmen ts Research was supported by Grant NPF-GX28743 from the International Decade of Ocean Exploration Program, National Science Foun dation. REFERENCES 1 Jensen, S 1966 Report of a new chemical hszrd .New Scientist 32 612 2 Jenien, S et *1 1969 DDT and PCB m marine ninmals from Swedish wnters Nature 221 217 3 Widmark, O 1967 Possible interference S<\ ililurinated biphenyls J Assoc Off Anal Chem .'81 KHiH 4 Holden. A V and Marsden K 1967 Orgmrot hl'-rme pesticides in seals and porpoises Nature-'ll: 1271 5 Holme*, D C , Simmons, J H nnd Tnttnn J Ot, 1967 Chlorinated hydrocarbon* m Hriti-.ii wildlife Nature 216 227 6 Koeman, J H , Ten Ncever tie Hrauw M ( rtnl Vo*. R H tie I960 Chlorinated biphem!- t-i `1-6 mussels and birds from the River Rhine in I 11.,Netherlands coastui area Nature 221 1 _M 44 Environmental Health Perspective* HONS 2081*9  Rwlimneh, R W et ai 1068, Tolythlonnatod bi' nhrnib in the global ecosystem Nature 22t) 1098 ,, cantilmn Wildlife Service, Ottawa 1971 Personal cnmmiimnition from C Jonkel d Ril.r..ith R W and Berner, D D 1971 Evidence for sen il fallout of polychlorinated hiphenvb (PCB ) in ihrnoterH Canadian Arctic Manuscript Report* No U, Ponticnle Section, Canadian Wildlife Service Oil an u 10 Rinehrough, R W and Carmignam. U M (Inpreaai Chloriinted hvdrocnrimna in Antarctic birda ProeeedMt* nl 'he Colloquium Conservation in Antarctica 11 Hnlihanl, H L 1964 Chlorinated inphenvl and related ramiKiinids In R, , Kirk and D F Otherm fed* 1 Eiinclnpedia of Chemical Technology 5 289 Second rev cd 12 \\hh.\ H V , Koidc. M and Goldberg, E D 1971 Mrrrurv in a Greenland ice aheet evidence of recent input l>t man Science 17+ 692 13 Nuti'iiia! Academy of Science 1971 Chlorinated hy drncnrliona ni the marine environment Waahington, Dt 14 lurrunt, K R and Talton, J O'G 1968 Organo- clilonnc peaticidea m rainwater in the British Isle* Nut are 219 725 l.i Mnnwnto Induatrml Chemical* Companv 1971 St lamia, Miwouri, Public Relation* Department, New* Ri'lnuc, November TO 10 loam, N. Department nf Biology, Tokyo Metro politan University, Tokyo (Personal communicnton). 17 s limuit, T,, Riaelirouffh, R W and Gross, F 1971. Input of polychlorinated biphenyl* into California mutt at water* from urban sewage outfall* Bull himmn Contnmm Toxicol 6 235 18 Hmclirough. R. W, 1971 Determination uf pulvi hUirinatcd biphenyls tn environmental samples Inttniationul Svmpo*mm on Identification and Measure ment uf Environmental Pollutants Ottawa Mi Hidden, A V 1970 Source of polychlorinated bi phenyl contamination in the marine environment Nature 228 1220 20. Risebrough, R. W et nl PolychtnnnaLed biphenyls in marine xooplankton (In preparation) 21 Hays, H and Risebrough, R W (In press) Pollutant concentration* in abnormal young terns from Long Island Sound. Auk 22 Risebrough, R W and Lnppe, B de Unpublished observations 23 Zltko, V 1971- Polychlorinated biphenyls end orgunnchlonne pesticides in some freshwater end marine fishs* Bull Environ. Contemn. Toxicol 6' 464 24 Harvey, G. R. 1971. Progress Report--August 10, NSF-IDOF, Grant GX-2B334. Vaughan T Bowen, Principal Investigator, Woods Hols Oceanographic Institution. Woods Hole, Massachusetts 25 Risebrough, R. W. 1969 Chlorinated hydrocarbons in marine ecosystems In M W Miller and G G Berg (eds ) Chemical Fallout. Charles C Thom**, Spring field. Illinois 5 26 Risebrough, R. W et ti Chlorinated hydrocarbons in ths Peruvian coastal ecosystem (In preparation). 27 Coulter, M C. and Risebrough, R W. Shell thinning in eggs of the Ashy Petrel (Oaanodromit AomocAroa) from the Farallon Islands (In preparation) 28 Risebrough, R W 1971 Artifacts of man chlorinated hydrocarbons. In D. W Wood (ed ) Man's Impinge ment on the Ocean* John Wiley and Sons, New York 250 29 Spilxer, P and Risebrough, R W Persistent pollut ants and reproductive failure* of Ospreys (Fonriion haluxetut) (In preparation). 30 Risebrough, R. W et al Oceanic pollution reproduc tive failures of Brown Pelicans (Pefscsnu* occufentaiw) (In preparation). 31 Holden, A V 1970 International cooperative study of organochlorine pesticide residue in terrestrial and aquatic wildlife, 1967/1968 Pestic Monit. J 4 117 32 Risebrough, R, W. et al. (In press) DDT residues in Pacific marine fish Pestic Momt J April 1972 HONS 208150 45 Environmental Health Pmpacttvn Contamination of the Bay of Fundy--Gulf of Maine area with Polychlorinated Biphenyls, Polychlorinated Terphenyls, Chlorinated Dibenzodioxins, and Dibenzofurans by V. Zitko*, 0. Hutzingert, and P. M. K. Choi** Tlir procedure used to determine polychiorins(cd biphenyls (PCBs), polychlorinated terphenyls (PCI's), chlorinated dibenzodioxins, and chlori nated dibenzofurans is outlined in Table 1. It is based on the cleanup-chromatography of Holden and Marsdcn (1), modified as described (2). Most PCBs arc eluted in fraction I. Fraction II contains some PCBs, p ,p'-DDE, and PCTs, whereas heptaclilor exposide, dieldnn, endrin, p,p'-DDD, and P ,]>'-DDT are eluted in fraction III Fractions I and 11 arc further chromatographed on alumina, essentially according to Porter and Burke (3), to determine chlorinated dibenzodioxins and dibenzo furans. PCTs, hexe- and octachlorodibenzodioxm, and oct achlorodibenzofuran were quantified by gas chromatography on 3% OV-21Q on Chromosorb WAW 60/80 in a 6 ft X 4 mm column at 200C. A 4% SE-30 column was used to quantify PCBs, chlorinated hydrocarbon pesticides, 2,3,7,8-tetraihlorodibensodioxin, di-, tri*, and tetrachlorodibcnzofuran Recovery of PCBs and chlorinated hydrocarbon * Environment Canada. Fiahenea Research Board lbulogicd Station, St. Andrew New Brunswick t Atlantic Regional Laboratory, National Research Council of Canada, Halifax, Nova Scotia ** McMaater University, Hamilton, Ontario, Canada pesticides m this procedure was generally better than 95% (2). Recovery of PCTs was 90% Re sults of the analyses of commercial fishmeal, spiked with chlorinated dibenzodioxins and dibenzofurans arc presented in Table 2 PCTs were found only in eggs and fatty tissues of hemng gulls in levels of 0 1 and 1 4 /tg.'g wet weight, expressed as Aroclor 5460, respectively This indicates that PCTs are not as widely spread in the environment as PCBs Only a limited number of samples were analyzed for chlorinated dibenzodioxins and dibenzofurans The samples included muscle and liver of white shark (Care/iarodon carchanas), eggs of doublecrested cormorants (Phaiacrocorax auntm), and herring gulls (Larus argenialus), commercial her ring oil, and groundfish herring fishmeal No residues of chlorinated dibenzodioxins and di benzofurans were found. Detection limits of the individual compounds are estimated in Table 3 The absence of chlorinated dibenzodioxins and dibenzofurans from aquatic animals is encourag ing. However, these compounds are extremely toxic, and a more detailed investigation is neces sary The analytical technique should be further improved to achieve lower detection limits The levels of PCBs and chlorinated hydro carbon pesticides found in fishes, double-crested April 1972 MQNS 206151 47 Table 1. Procedure for dclemliiini PCBs, PCTi, chlorinated dlbenzodloxln*. end chlorinated dibeninfuraaa. Chromatography nn alumina (800*C 4 h, 5% H,0> Chromatography on Sthcar* (130*C overnight, 3% H|0 ; Fraction I I (20 ml 2% CH.Cl, in hexane) Fraction I (10 ml hexane) Fraction I II (10 ml 20% CH.C1, in hexane) Chromatography on alumina (130*C overnight) Fraction II I (20 ml 2% CH.C1, in hexane) Fraelinn II (20 ml hexane) Fraction II II (10 ml 20% CHiOt in hexane) Fraction III (10 ml 10% ether in hexane) PCB, C,C1, HCDD faster isomer TCDD, OCDD OCDF P,P -DDE HCDD slower isomer TCDD, CDF TC DD - 2,3,7, B-tetrachlorodibeniodioxui HCDD, OCDD- hexa-, octachlorodibenaodioxin, CDF-mixture of di-, tri-, and tetrachlorodibeniofuran OCDF - octachloradibetuof uran. cormorants, and herring gulls from the Bay of Fundy-Gulf of Maine area are summarised in Table 4. The concentration of PCBs is generally higher than that reported for the same or similar species from other areas, such ss the Baltic Sea (4), California coastal watere (5), Britain (6), and Western Canada (7) It is interesting to note that the levels of PCBs found in fishes from the Bay of Fundy-Gulf of Table 1 Analysis of spiked iahmeel asmplee, jig/g wet weight. TCDD* HCDD* OCDD* Sample Added Found Added Found Added Found 1 1 18 0 90 1.08 1 IS 1 30 1 29 2 0 1W 0 04 1 08 1 04 0 OS 0 57 3 0 29 0.33 0 20 0 31 0 32 0 SO CDF* OCDF Added Found Added Found 4 1 21 1 41 0 0 80 0 62 (1 0 30 0 28 0 91 O 45 0 23 0 90 0 38 0 14 Maine area are about one order of magnitude higher than those in fishes from the Not a Scotia banks (2) Determination of PCB residues in Atlantic salmon (iSaimo solar) also indicates that elevated levels of PCBs are associated with coastal areas. Thus muscle of salmon caught off Greenland contains 0.20 og/g of PCBs, whereas in specimens landed in Canada PCB levels are from 0 45 to 0.62 ftg/g on wet weight basis In all samples the peak pattern of PCB residues resembles quite closely that of Aroclor 1254. and the results are expressed as Aroclor 1254 However, fish from lower and intermediate levels of the food web contain relatively lower amounts of hexachiorobiphenyls (4th, 5th, and 6th major Aroclor 1254 peak) and higher amounts of tetra- and Tabla 3. Detection limit* of chtorineted dlbencodioun* end dibeuxofuran* Compound Detection limit, ug/g vet weight TCDD* CDF* HCDD* OCDD* OCDF* 0 04 0 02 0 02 0 01 0 01 See Table 1 * See Table 1 48 Environmental Health Perspectives HONS 208152 Ibenaofur omer, D.OCDF mer, stive Yffclc 4. PCB and chkrlnpltj hydrocarbon pcpthUn in aquatic animals from the Bay of Fundy-Gulf of Maine area. Bpeeiea Tissue PCBs #g/g wet weight (as Aroclor 1254) p.p-DDE Other Herrin* r/upm harengiu Msrkerrl Scomber wornbn* Plaier Ilippofiotunda plalnmntUt White link* Urophynt Inau Ocean perch cSehaatae mannas Cod frnrfuj mochuo So raven Hemtlnpleru* amnafttu Bluffin Luni Thunniu fAfrmw Herring oil Ftshmpdl DiHiMr~crested cormorant I'b.alacrocerax aun/wi whole fish mooele muscle muode muscle muscle muscle viacera muscle liver muscle liver muscle eggs muscle liver Subcut. fat Abdoiu fat eggs muscle liver Subcut fat 0 34 0 35 0 38 0 44 0 32 0 55 0 21 0 73 0 07 1 07 0 77 218 1 54 3 55 0 54 43 5* 17 2` 3 38 2 13 38 52 12 6* 5 54* 5 06 6 50 75 0 06 0 07 0 01 0 03 0 <v; 0 04 0 06 0 30 -- 0 48 335 0 15 2 27 0 19 29 4* 8 6* 8 40 4 16 164 162 5 67* 2 83* 2 07 2 08 26 DDT 0 24 DDT 63 DDD 43 DDT 0 37 ppntachJorobipbenyfa {let and 2nd major Aroclor 1254 peak) than high trophic level white and silky shark (Carcharodm carchariat and CareAar/wnus Jalctformu, respectively) and aquatic birds. At lantic salmon parr fod Aroclor 1264-oontaminated diet in the laboratory also contain relatively higher amounts of tetra- and pentaehlorobipbenyls. This may indicats that fish more efficiently absorb the lower chlorinated biphenyls. On the other hand, these compounds may be metabolised and ex creted, so that a preferential accumulation of higher chlorinated biphenyls occurs in the food chains. The fractions of bexachiorobiphenylB at different trophic levels are summarised in Table 5. The toxicological significance of environmental levels of PCBs and their rate of accumulation in the environment are not known. In spite of this, PCBs and other industrial halogenated hydro carbons should be considered potentially harmful. Table 5. Fraction of bexacblorebipbenyls at different trophic level*. Sample Aroclor 1254 Fish, wild Atlantic salmon parr on Aroclor 1254contaminated diet Silky shark White shark Eggs of double-crested cormorants and herring gulli Fraction of hexachlorobiphenyla in terms of Aroclor 1254 peak heights, 4+5+6 l + 2+4 + 5 + f> 0 56 0 38 0 41 0 74 0 71 0 72 Numbers refer to the major Aroclor 1254 peaks April 1972 MONS 208153 49 and their release into the environment should be limited as much as possible A cknowledg men is Wc thank our colleagues at the Biological Sta tion, St Andrews, New Brunswick, Canada, for samples of fish and Dr D Firestone, Food and Drug Adrmnistiation, Washington, D C , U S A., for samples of chlorinated dibeniodioxins and dibenaofurons. We thank Mrs Madelyn M. Irwin for typing the manuscript REFERENCES 1 Hidden, A V and Maraden, K 1 SOS) Single-sti** clean-',ip nf animal tissue extracts for org&nochlonne residue anatvsis J Chromatogr 44 481 2 Zitko, V 1971 Polychlorinated luphemls and n^.-- chlorine pesticides in some (reshmuer and nann, fishes Bull Environ Contamm Toxicol & a64 3 Porter, M, L and Burke, J A 197) Separatum three rhlorodihenso *-dioxins from wmie poUrlilon rated btphtnyi* by chromatoeraphv on an alumina* oxide column J Assoc Off Ansi Chem 74 1426 4 Jensen. S et a) 196S DDT and PCB m marine animals from Swedish waters Nature 224 247 5 Kisebrough, R 1969 Chlorinated h v drocsrlswii ,* marine ecosystems In M W Mitler ,mil G G Bet* (eds ) Chemical Fallout Charles C Thomas hpnn* field, Illinois 6. Piaett, I , Jeffnsa. D J and Moore N U HIM P0|vchlorinated biphenvli rn wild birds in Brilum and tbeu avian toxicity Environ Pollut 1 3 7 Reynolds, L M. 1971 Pesticide residue anshsm m the presence of polychlorobiphenyla (PCB) Iteaidue Renews 34 27 50 Environment*] Health Perspective* MONS 208154 |<j Qttln. taarjijM irttion nf olyrhluri aluminum |42fl h mirrmU ftn rn If! Bor* 1 Rprin*. jo Pul V fid llifir I in llir idm- Enrinnmmul HtailM Pmpteltva Recent Fluctuations of Chlorobiphenyls (PCBs) in the Green Bay, Wisconsin, Region' by Gilman 0. Veith, Ph.0.f introduction During the past six years, Wisconsin and Michi gan have maintained a major stocking program for trout and salmon in Lake Michigan m an effort to re-establish a sport fisheries, and to coni rol nuisance fish, such as the alewife in 1969, Veil It and Lee (l) found these large predators contained chlorobiphenyls (PCBs) at concentra tion- ranging from 10 to 25 (ig/gm (fresh weight-- as Arocior 1254) Furthermore, the coho salmon eggs contained 12 to 17 ug/gm PCBs (fresh weight) which, based on the results of Johansson, et it! (2) suggest that the PCBs may contnhute to t lie poor reproductive success of the stocked fish in Lake Michigan. The seizure of fish from Lake Michigan, because of the presence of chlonnak'd pesticides and PCBs at concentrations greater than that regarded as safe for human consumption, has demonstrated that the continu ous input of trace quantities of these chemicals muv jeopardize major food resources for man, if proper management of hazardous chemicals is not practiced. There are numerous examples of the aqueous transport of PCBs originating from industrial and sanitary wastes (3, 4). Veith and Lee (5) reported the presence of PCBa in sanitary wastes of all * ContnbutKM of the Water Chemistry Program, Delurtment of Civil and Environmental Engineering, Ur.iwrwiv of Wisconsin, Madteon, $3700 Thu work was luyrixjried under the University of Wisconsin Sea Grant I'nigrum, the Department of Civil and Environmental kiiicmeenng, and the Engineering Experiment Station t Aaistsnt Professor of Civil and Environmental En~ piircring, University of Wisconsin, Madison, Wisconsin 53700 cities and villages studied in the Milwaukee River (Wisconsin) watershed Dube (6) has found similar results in a study of PCBs in municipal wastes from southeastern Wisconsin. All of these data were obtained pnor to or shortly after the volun tary partial ban on PCB sales by Monsanto Com pany This paper presents the preliminary data from studies of the levels and sources of PCBs in nvera discharging into Lake Michigan--Green Bay in northeastern Wisconsin Green Bay (Figure 1) is an 120-mile long elon gated bay in the northwest comer of Lake Michi gan The major rivers tributary to lower Green Bay are the Fox (mean discharge of 11X10* m9 day-1), which enters at the southwest end of the Bay, and the Oconto (2X10* m* day"'), Peshtigo (2X10* m* day-1) and the Menominee (9 X10* m9 day-1), all of which enter along the northern shore of the Bay (7). The Fox and Menominee Rivers receive the wastes from the cities of Green Bay and Marinette (Wisconsin)--Menominee (Michi gan), reepectively. The Oconto River receives the wastes from Oconto Falls and Oconto, and wastes from the city of Peshtigo are discharged into the Peshtigo River. The Big Suamioo and Penaaukee Rivers primarily drain agricultural and forested land and do riot receive any major municipal wastes above the respective sampling sites Field Sampling The procedures used m this study have been discussed in detail elsewhere (5). The collection of water was conducted with all-glass systems to April 1972 HONS 20S155 51 Fkh.'ki I LorMion oi Green Bay, Lake Michigan and major river* tributary to the Bay preclude possible interaction of the sample with rubber, plastic, or polyvinyl chloride surfaces. Samples were collected from the river by sub mersing s weighted, glass carboy <20 1) at a depth of 0 2 m in the center of the main channel Pre servatives were not added, as samples were cooled and extracted withiD 24 hours of collection Extraction Water samples (20)) were batch-extracted twice with hexane id 21 separatory funnels. The hexane portions were combined, dried with anhydrous NatSO, and concentrated to 15 ml for cleanup m a gentle air stream Liquid Chromatography The cleanup of extracts for gas chromatographic analysis was conducted with liquid chromatog raphy on Florisil, as described by Reynolds (8) and Hughes et si (9) The media (Kensington Scientific) was extracted in a Soxhlet extractor for 24 hours with an ascotrope of hexane and acetone (1:59) to remove organic impurities. The azeotrope was evaporated from the Florisil at 105C, and the Florisil was heated to 650eC for 2 5 hours for activation. The Flo nail column for preliminary cleanup was prepared by vibrating 19 g of Flonsil ltAo a 25 mm o d. glam column, which was fitted with a glass fnt and Teflon stopcock The column * topped with 10 g anhydrous sodium sulfate to prevent deactivation of the Flonsil by traces Q{ water in the extract. The extracts were placed 0D the column and eluted with hexane (200 ml) to recover the PCBs, if present With some samples, it was necessary to reehromatograph the hexane eluate from the 25 am o.d. Flonsil on a smaller diameter Florisil oohinm (9 mm i d ) to isolate the PCBs from organic interference* If necessary, the PCBs were separated from the DDT group of pesticides through chromatography on silicic acid (10, 11) Instrumentation The analyses of water extracts were conducted on an Aerograph 1745-20 gas chromatograph equipped with concentric tube electron capture detectors ('H, 250 raC) and a 50:50 effluent splitter for simultaneous analysis with electron capture and Same ionisation detectors. Analytical GLC columns consisted of 20 m X 18mm glss.* coils which were packed with either OV-101 (3 percent), OV-lOl/XE-flO (3:3 percent), or OV-101/QF-1 (3:4.5 percent), coated onto Gas Chrom Q (720/140mesh) The carrier gas (purified Nj) was maintained at 21 ml min'1; and the in jector, column, and detector temperature's were 250C, 180C, and 220C, respectively Analysis The commercially prepared PCBs m the USA (Monsanto Company, St Louis, Mo) exict as seven complex mixtures under the name "Aroclor", which range in chlorine content from 21 to 62 percent. When the PCB mixtures are chromato graphed with GLC, the mixtures of isomers p re duce both resolved and superimposed peaks and are somewhat charactensed by the GLC finger print presented as relative peak heights and re tention times. Because of the complexity of the mixtures, determinations have been quantitative) \ defined by comparing the area of a sample chro matogram to the area of a known quantity of the commercial mixture which most closely resemble that of the sample. 52 Environmental Health Perspectives MOHS 206126 ' cleanup >risil into tied with lumn wf^j uilfate to traces of placed on 0 ml) to sample, e hexane 1 smaller o isolate ercessary, T group ID BlIlCIC aductcd tograph capture effluent lectron alytical 31 glass 101 (3 t), or jo Gas lunfieri the in- ires Table 1. Variation* of Cblorobtphenyl* in Rtxcra in Ibe Green Bay Study Area. River 12/29/70 Concentration (ef/1 a* Aroclor 1254)'1 5/21/71 7/20/71 8/0/71 Peshligd River, Peshtigo Onn'o River, Oconto Peiunnkee River, Pen**nine Bik Stiamir-O. SuUiuco Fox River, Green Bay 0 31 0 45(3) <0 01 <0 01 0 I8b (3) 0 38 0 16 <0 01 <0 01 0 20 <0 01 <0 01 <0 01(1) <0 01(1) 0 10 <0 01 <0 01(3) <o oi:d <0 01(1) 0 15 Mean at two analyse* unless noted otherwise k fo\ River samples analysed as Aroclor 1248. Late eluting components present near municipal waste treatment outfall Explicit chemical confirmations for the presence uf PCBs in each sample were not possible in this study PCBs with similar retention volumes and relative peak heigbte were confirmed in the fish from the Milwaukee River using i.r and mass spectrometry (5). The analyses do not preclude the possible presence of other chemicals which may have similar chemical properties, such as the chloronaphthalenes. The recoveries of PCBs from water samples which were "spiked'' with Aroclor 1254 and 1260 were approximately 80 to 85 percent. Although tlif relative error at 1 gg/1 was about 8 percent, the relative error in replicate analyses of nvpr wa(fn, at the 0.1 pg/1 level may increase to 15 to 20 percent, Riififts aid OisnttsiM The results of the monitoring of five river* in the Green Bay area for PCBa during 1971 are presented in Table 1. The concentration of PCBs in tlie Pcnaaukee and Big Suamioo Rivers re mained below the determinate limit of 0.01 Mg/1 throughout the period of the study. The PCBs in the Pcshtigo River below the city of Peshtigo wen- approximately 0.3 to 0.4 pg/1 (as Aroclor 1254) until May 21, 1971, but decreased to below the determinable limit by July 20, 1971. PCBs m the Oconto River below the city of Oconto were present at a concentration of 0.45 >ig/l (as Aroclor 1254) on December 29,1970, but decreased during the spring of 1971 and were also below the de terminable limit on the July 20 sampling date In cunt last, no trends in the concentratioiib of PCBs tbe Fox River were observed, and the levels were found to be above 0 1 mg/1 (as Aroclor 1248) throughout the study It should be noted that the chromatograms of the PCB mixtures in the Oconto and Peshtigo Rivers were most similar to that of Arocloi 1254, whereas the mixtures in the Fox River were predominately Aroclor 1248, with minor components similar to those in Aroclor 1254 sometimes apparent The initial purpose in monitoring the tributaries of Green Bay was to relate the composition and concentration of PCBs in the major tributaries to those in the water from the Bay and to examine possible changes in the composition of PCBs in tbe sediments The data obtained f rom the initial monitoring, which shows that the PCB concen trations in two of the major tributaries decreased sharply to below the determinable limit in 1971, suggests that the self-imposed restrictions on the sale of PCBs by Monsanto Company in 1970 and 1971 may be having an immediate effect in some river systems. Continued monitoring of the Fox River will be conducted to determine if the levels of PCBs ansing from this highly industrial area will also diminish in the near future Although the analyses of water from Green Bay indicated the concentrations of PCBs varied from 0 07 ug/1 (as Aroclor 1248) at Long Tail Point near the city of Green Bay to 0 04 g/1 approxi mately 35 miles northeast of the city near Sturgeon Bay, the variations introduced hy windmixing and the inability to measure small concentration differences below the 0 05 Mg/I level may make it impossible to determine if the decline in PCBs m tributaries will be reflected in the water from the open Bay in the near future Consequently, April 1972 MQNS 208157 53 the sampling program has been expanded to in clude fish and other organisms of the Bay which contain itgfgm quantities of the PCBe REFERENCES ! Veith C D and Lee, G I" 197] Chlorobiphenyts (PCBs) in fish from Lake Michigan Proceedings of the 14th Great Lake* Research Conference. Toronto (In press) 2 Johansson, N , Jenaen, S and Olmon, M, 1970 PCS Conference, Swedish Salmon Institute, Stockholm, September 29, P- 59 3 Veith, C, D 1970 Ph D thesis (Water Chemistry) University of Wisconsin. Madison, 180 4 Duke. T W, Low*, } I end Wilson, A, i 1970 A FCB (Arodor 12M) in the water, sediment, and biota in Escambia Bay, Florida, Bull. Environ. Contamin Toxicol 5 171 5 Veith, G D and I^e, G. F 1971 ChlorobipKemU (PCBs) in the Milwaukee River Water Reseat 5. U07 6 Dube, D, 1971 M 3 thesis (Water Chemistry) Uaiversity of Wisconsin, Madison p 44 7 Ahmabrak, W F and Rsgotikie R 4 1170 Miitng Processes m Green Bay Proc 13th Great Lakpi ftM Coni 930 8. Reynolds, L M. 1989 PCBs and ihcir interference with pesticide residue analysis Bull Em iron Contamin Toxicol. 4` 128 9 Hughes, R A , Vsith, G D and bee. G i 1970 Gas chromatographic analysis of tors phene m nstural water*. 6sh and lake sediments Water Rc 4 547 10. Amour, J A and Burke, J A 19*0 Methods oi separating PCBs from DDT and its analogs J Assoc Off Anal Chem 53 761 11 Remert, R E 1970 Fishery Biologist. Great Lakes Fisheries Laboratory, Ann Arbor, Michigan (Unpublished) 54 Environmental Health Perspectives HONS 208158 Environmental Health Pertfirmart Polychlorinated Biphenyl Residues in Milk of Environmentally and Experimentally Contaminated Cows byG. F. Fries* Polychlorinated biphenyl (PCB)f residues have i*n found in the milk of cows. In some instances ;iic residue levels exceeded the FDA guideline ii 1 ppm in milk (equivalent to 5.0 ppm in milk, in), and the milk was removed from the market. The major source of PCB residues m milk is trorlor 1254 that has been used in coatings for concrete silos. Aroclor 1254 is unaltered in silos, tnd most of the contamination is adjacent to the mils (1,2) Wc have observed a number of farms with rCB-l rented silos and have fed Aroclor 1254 to cows under controlled conditions This paper nimmnnzes our major findings. Mature and Occvrrinci of ftosMuis The PCB recovered from silage is identical to standard Aroclor 1254 (1). However, many of the components with short gas chromatographic re lent mn times do not occur in milk residues (Fig 1). The comjionents with short retention times are apparently less chlorinated than those with long retention times. Our observations in cows are similar to observations in other species (3). A complication in analyzing environmental sam ples is a possibility of interference by DDT and some of its related compounds. We dehydro- ' I'mted Ststos Department of Agriculture, Animal Science Hcacarcli Diviaion, AUS, Belleville, Maryland 207(1.1 t In this paper the term PCB it interchangeable with ArerJor 12.14 or, in the rase uf milk and body fat, reaidues dmred from Aroclor 1214 chlorinated the samples to remove possible inter ferences of DDD and DDT DDE did not interfere, because those components of Arocloi 1254 with the same retention time as DDE did not occur in significant amounts in milk (Fig. 1). We obtained milk tank samples from six farms with PCB-cont&mlnated silos. The PCB levels in milk fat are summarized in Table l In all cases PCB concentration in milk fat exceeded the FDA guideline, at least part of the time, when silage was fed, What the silage was not fed, levels of PCB in milk fat were always lower than the guidelines. The farms represent a variety of feeding and management conditions The results suggest that any dairyman with a contaminated silo will prob ably exceed the guideline at least some of the time during the year. While a few higher levels have been reported for herds removed from the market, most were within the range of our obser vations. The number of forma with PCB-con taminated silos is not known However, PCB contamination of milk la probably more prevalent than the regulatory record would auggest. Comparison of ME aMi PCB We have observed a herd that was simultane ously contaminated with high levels of DDE and Aroclor 1254 from a silo. By a fortuitous set of circumstances, the cons had been removed from the sources of both contaminations at about the same time. A detailed description of this work has been prepared (4). April 1972 MONS 208X59 55 1 Table I. Arempe rataa of decline of DDE and PCI concentration In milk fat of wn la 'nou -`nr, eflartitioo.' Group Cowv DDE PCD Mkd )*ct*tion Early lactation Late lactation All (No) ie 9 31 (dsy-*) 0 0145 0 0107 0 0147 0 0134 (day-1) 0 0147 0 OIQU 0 0140 0 0134 * The value* an for tbe eneetaat. k, in the linear lom 0f the firet-order aquation UC>lnC,-kt where C the Bilk fat concentration, C, i* tbe initial cooceiu.ro.lion, ud t M tune in day*. Titled by iaaataquarea flom I Gw ckronutopuni of Amclor 1254 ituulud and residuea in milk. The lit X ^ in. o.d column vpeeked with 10% DCt-200on GaackroovQ A05% argon-5% methane mixture used w earner [H and the column temperature wea 200*C. mately four moothe. After three weeks the decline in concentration m linear, when plotted on semilog graph paper, suggesting first order kinetic*. First-order rate constants were calculated for both DDE and FCBs for the individual cows. A summary is presented in Table 2. The overall average of the rate constants for PCB was identi cal to that for DDE. The average DDE rate constant was similar to the rate that we have previously reported (5). There wse a tendency for cows in early lactation to have lower rate constants than the other groups of cows. The average concentration of PCBs in milk fat was 19.3 pg/g when the silage feeding stopped. This concentration declined to 10.3 pg/g within three weeks We continued to sample the indi vidual cows at three-week intervals for spproxi- IIS a UUI t un Tsble 1 PCB nmcMtratiooa in oUk fat from firm* with contaminated silo** Farm 11/10 12/7 1/21 3/24 5/n ()*/*> 1 9 6 S.S 6 7 NS. 7 0 3 7.6 fi 7 e 7 o e 3 B 3 N.8.* 1 6 7 4 S 0 t.l 4 NB. t 0 1 4 1 i ft 0 i s t 7 6 t 10 3 10 s e N.S 1 0 e 2 5 2 t 9 Silage waa not being fed wbeo italicized aamptea were obtained, i Not Sampled ss(MrJt Fiocne 2. Relalionabip of tbe ratae of decline of DDE and PCB concentration in milk fat of individual cow Mid, early, and late are etagea of lactation (4) 56 Environmental Health Perspectives MOHS 208160 Tablr 3. Cffeat of phenobarbltal and phcnobarbital with aril voted carbon on the concentration of DDE and PCB in milk fai.* Treatment Cows (No ) Concentration ------------------------- Initial Final (rtS/g) Final Initial SD DDE Cont nil Ptieno- barbital Pheno- hnrlutaHCerbon 5 10 0 n e 5 2 0 45 0 05 0 5 4 3 0 45 0 15 8 0 3 7 0 46 0 20 PCB Con tm! Fheno- 5 10 3 7 4 0 38 0 08 barinUl Pkeno- 10 17 7 7 0 0 40 0 11 barlntal+ Oarunn 0 22 3 8 5 0.37 0 14 * Observation |>enod wa* 6 weeita. PhenobarbitaJ was led at A grama per riav for the first three week* and ac tivated carbon at one kilogram per day for the 6 week* Fiounc 4 Concentration of PCB in milk fat after feeding Aroelor 1254 stopped Each point is an avenge of nine cowa Relationship of the rate constants for ODE and PCBs within individual cows is presented in Fig. 2. The correlation (r * 0.82) between the rate constants of the two compounds was sig nificant (P<0,01), The usual linear regression was calculated, and the intercept did not differ sig nificantly from sero (6). Therefore, the regression in Fig. 2 was recalculated, forcing the intercept through sero- The regression coefficient, 0.974, was essentially unity. In some cases there wss considerable variation between the rate constants for DDE and for Table 4. Concentration of PCB in milk fat and body fat of oowm fed 300 mg/day PCB far M days. Time Milk fat Body fat Fioi nv 3 Concentration of PCB in milk fat of cowa fed 2(i(i mg/day A roclor 1254 for 60 dayt Each point u an average of nine cowa (daye) 30 60 00 120 (/*) 45 6*8 4* 66 7*8 4 24 0*3 0 19 4*2 4 Standard deviation 21 9*8 5 64 0*12 2 40 3*6 4 28 8 *6 2 i/i April 1972 HONS 2OB161 57 Table 5. Normalised conci-mritlon of chlorinated hydrocarbon compound* in milk fat and bodv row* after continuous feeding 1 ^l j Compound Reference Observations (No) Milk fnt 20 days (ng/g> Milk ful 60 rix'v <**/'*' RvhIi 'ini lift itai i ikift g \roelur 125-4 DDE Dieiclrin DDD p.p'-DDT'' o,p,-DDT'' (5) <7, 8) (7, 8) (5) (5) (7, 8) (9) (9) 9 0 20 0 33 3 0 21 O 20 12 0 27 -- 12 0 19 -- 3 0 003 0 077 3 0 036 0 12 0 030 -- 4 0 106 -- 4 0 023 -- 0 20 (1 2.1 _ -- 11 1141, tl IUI -- - * Value* nr* normaluod to an intake of 1 ntg/d&y * Value* are sum of DDD and DDT PCBs within a given cow The rate constants for the two compounds within a given cow were tested statistically (6), but the differences were not significant The usefulness of phenobarbital alone, or in combination with activated carbon, m acceler ating the reduction in milk concentration of DDE and PCBs was tested using three groups of cows. Neither phenobarbital nor phenobarbital in combi nation with activated carbon had an effect on the relative reduction in concentration of either com pound (Table 3) We have previously reported a small effect of phenobarbital on DDE, but we did not consider it of practical significance (?) The absence of an effect here is consistent with that interpretation. The failure of activated car bon, even in combination with phenobarbital, to affect the reduction of DDE and PCB concen trations is consistent with our previous interpre tation of laboratory experiments (S). Aroclw 1254 ftHtai Study We have carried out a controlled feeding study uaing nine Holstein cows The cows were fed 200 mg per day Aroclor 1254 for 60 days Milk samples were obtained periodically during the 00-day feeding period and the subsequent 60-day period Body fat samples were obtained by biopsy at 30-day intervals The cows were selected to have a range in both the stage of lactation and level of milk production However, preliminary examination of the results indicates that there wax little difference in the residue levels due to these factors Therefore, only average data for all cows are presented The average level of PCBs in the milk fut during the feeding period is presented in Fig 3 The standard deviation of any gnen point did not exceed 10% of the mean The corn entradon of PCBs increased rapidly and approached a "steady state'' at about 40 to 60 days expected from our field studies, the shape of the <ur\e is similar to that for other chlorinated hydiutarhon pesticides, particularly DDE {.">) The decline in PCB concentration in the milk for the 60 days after feeding stopped is presented in Fig. 4 This curve is quite typical of pro\ sous findings with DDE and was resolved into a twocomponent first-order system (5) The equation for the curve normalised to an initial concen tration of 1.0 ag/g is C0.52r'+0 48e'fl <**1 Where C is concentration, e is the base of the natural logarithms, and t is days The constants are similar to our piex ious o!ner vations for DDE (o) While the rate fnt the second component in tins study was less ihm the values found in the field, it was within the range of values that one might expect The level of PCB m body fat at larious times is presented in Table 4 Foi reference parpo-emilk fat levels at these times are also preseiifeil 58 Environmental Health Perspective* HONS ZOBi.62 While the PCBs were being fed, the level in milk fat was higher then that m body fat. When feeding stopped, milk fat levels dropped below and appear to reflect body fat levels. We have conducted a number of studies with chlorinated hydrocarbon pesticides under con ditions similar to those of this PCBs study A summary of levels in milk fat at 20 and 60 days and body fat at 60 dayB with normalised intakes is presented in Table 5. The levels of PCBs are similar to levels of DDE and dteldrln in corre sponding samples In contrast DDD, p,p'-DDT, and o,p'-DDT are transferred to the body fat and milk fat at much lower rates than DDE and PCBs ConclHtfvns Farms having silos that have been treated with PCB-contaimng paints will frequently have resi dues of PCBs in milk which exceed the FDA guidelines. The behavior of this residue in the cows is similar to DDE and other chlorinated hydrocarbon pesticides resistant to metabolic degradation The only practical countermeasures appear to be to decontaminate the silos sufficiently to remove most of the PCBs or to discontinue the use of these silos completely. Limited field experi ence and the similarity of PCBs to DDE suggest that there are no practical procedures to minimize the transfer of PCBs from the diet to the milk or to speed up the elimination of PCBs from cows REFERENCES 1 Fries, G F 1972 Degradation of chlorinated hjrtrocarbons under anaerobic conditions Ad wince* |N Chemistry Senes (In press) 2 Skreatny, R F , Ilemken R \V and Domugh H \Y 1971 Silo sealants ss a source of polychlorobipheni i (PCB) contamination of animal feed Bull Environ Contamm Toxicol 6' 400 3 Peakall, D. B and Lincer, J L 1970 Polvchlorinated biphenyls--another long-life widespread chemical in the environment BioSeience 20 958 4 Fries, G F, Marrow, G S and Gordon. C H 1972 Similarity of a polychlorinated lilphenv! (Aroclor 12.11) and DDE in rate of elimination from cows Bull En viron Contamm Toxicol (In press) 5 Fries, G. F., Marrow, G 3 and Gordon, C H 1969 Comparative excretion and retention of DDT snslngs by dairy cows J Dairy Sci 52 1800 6 Snedecor. G W and Cochran, W G 1967 Rut mitral Methods. (6th ed ) Iowa State Cmv Press, Ames 7 Fries, G F et al 1971 Effects of microsomal enrimc, inducing drugs on DDT and Dieldrm elimination from cows J Dairy Sci. 54 364 6 Fries, G F et al 1970 Effect of activated carbon on elimination of organochlonne pesticides from rats and cows, J. Dairy Sci 53 1632 9, Fnes, G F, Marrow, G S and Gordon, C H 1972 Excretion of o,p'-DDT in milk of cows J Dain Sci 54 1870 April 1972 HONS 208163 59 Environmental llwoixh PrrifNOim Polychlorinated Biphenyls in Non-carbon Copy Paper by Masanori Kuratsune and Yoshito Masuda* In nn effort to clarify the distribution of poly chlorinated biphenyls (PCBs) in our environment, it nn* suggested by Dr T Hiraya nia, National Canon Center, [Japan] that PCBs or their re lated compounds might be used for production of noii-<"irbon copy paper Three of 4 Japanese brand1' of the paper on the market were, there fore, i ollectcd and examined for PCBs The paper ronsi'ts of three different sheets, namely upper, middle, and lower ones These sheets of paper were separately extracted with acetone in a Soxhlet apparatus for 3 days The extracts were examined by gas chromatography with an elec tron capture detector, using 3 different liquid phases, namely r>% SE-30, 5% QF-1, and '3.5% DECS coated on Chromosorb W AW DMCS. The gas chromatographic patterns observed were all identical with that of Kanechlor-300 (a com mercial brand of PCBs) containing 43% of chlo rine (Fig 1) The infrared absorption spectra and mos-s-spectra of the extracts also demonstrated the presence of Kanechlor-300. The approximate concentration of PCBo was 2-6% in the upper and middle sheets and 002% in the lower sheets as shown in Tabic 1. To estimate how much PCBs stick to fingers by handling the copy papers, a set of 96 precut sheets of the same sise paper, consisting of 32 sheets of each of the upper, middle, and lower onus, was counted by turning over all the sheets one by one by fingers After counting, the fingers u ere washed with n-hexane once and the resulting * Department of PuMic Health. Faculty of Medicine, Kuislm I'mvprmty and Department of Analytical CheraiHtrt Danclii College of Pharmaceutical Sciences, Fukuoka, Japan n-hexane washings wpre examined for PCBs The amount of PCBs in the washings, calculated as Kanechlor-300, ranged from 11.40 to 52.68 pg in 5 subjects (Table 2). It was proved thus that PCBs in the paper easily stick to fingers when handled It was also demonstrated that only one third of the PCBs stuck to fingers can be removed by ordinary hand washing with soap and water (Table 2) In addition to the above domestic brands of [Japanese] paper, a few other samples of non carbon copy paper were analysed for PCBs One of them was a brand made by one of the leading paper makers m Britain and was obtained as packed m its original container in May, 1971 Analysis showed that both upper and middle Tsbla 1, Amounts of PCB in carbonless copying paper. Brand Kind erf paper Weight of paper <g/l0(> cm*) Amount of acetone extract* <">/*) Amount of PCB found* <mg/g) Upper 0 47 A Middle 0 50 Lower 0 51 Upper 0 48 B Middle 0 55 Lower 0 51 Upper 0 40 C Middle 0 53 Lower 0 50 117 0 125 8 23 4 40 3 37 8 70 8 59 6 61 0 28 2 04 7 63 8 0 24 30 7 26 0 0 28 31 6 22 2 0 20 Weight of extract per weight of paper * Weight of PCB calculated as Kanechlor-300 per weight of paper April 1972 HDNS 208164 61 sheets of this make contained PCBs The second sample! which was of American make and ob tained as packed m its original container in June, 1971, also contained a considerable amount of PCBs Another sample of paper, which was sent Table 3. Amounts of PCB found from fingers gin t counting % sheets of paper. ' Subject Amount of I'd)* W*j|ied mil-, n-hexsne is*/ W iNiinJ %iih n-lioxanc aflpr h*mj Wiiatimg *Hh t 1 2 3 4 s Avenge Standard <lw*bmn 39 7fi 13 flO 11 40 52 GA 44 30 22 10 oo if* in 10 47 t 50 4* r> 25 41 20 tin lilt Calculated ae Kanechlor-HXl Figure 1 CMehromsUigraias of kMMehloi-900 (upper) hiuI tin acetone extract of rapjnni paper (lower) Column Claee catunut (3mX2m) containing S'o SE-30 on cHreeeaeorh W AW DMCS. Column temperature- 1*C to us for examination by a company m Thailand, however, did not contain PCBs We Merc told that it is also of American make. A sample of non-carbon copy paper which was used by the U.S Printing Office in 1907 was obtained from the American Consulate in Fukuoka and analysed for PCBs. It also contained a large amount of PCBs. Needless to say, the free use of PCBs for this kind of very popular copy paper necessarily lead* to the uncontrollably heavy, direct exposure of the general population to it In view of its chemical and pathological characteristics, we consider that the use of PCBs for copy paper should be discon tinued in any country m order to prevent another occurrence of "Yusho" The Japanese makers of the paper are said to have acted quickly along this line in April, 1971 To our regret, however, there have been no control measures set up by the government against the use of 1`CBh and a considerable amount of the PCB-polluted papers are still widely used in Japan 62 Environmental Health Perspectives HOMS 2061*5 by i " cr Wo lad line pap pre: dne T sear nati mak teria hesr lam! used conti in tr quid. as le* carb* Pt of th' syste Th assoc soure hoaru liber-* adopt of cat the P Ttu in i>> inter*' Dm turn "i April Environmental Health PmpeOtt PCB and the Paper Industry-- A Progress Report by Paul E. Trout* The paper industry's experiences with PCBs Here chronologically reviewed at a recent "PCB Workshop" sponsored by the Grocery Manu facturers of America, Inc (1). This history goes back somewhat over a yeai, at which time two paperboard manufacturers were apprised of the presence of PCBs in folding cartons for packaging dried fruit These two manufacturers immediately began searching for the source of the PCB contaminahon, screening all converting materials used in making cartons from the paperboard. These ma terials, including printing inks, varnishes and ad hesives, proved not to be the source of the con tamination. Examination of the fibrous materials used in the paperboard showed the source of PCB contamination to be some of the paperstocks used in manufacturing the paperboard. Certain high quality white and colored paperstock grades, such m ledger papers, were found to contain carbonless csrbon papers which were the source of the PCBs PCBs at that time were used as one component of the dye carrier in the carbonless carbon paper system The American Paper Institute, the industry association, alerted the paper industry to this source of PCB contamination. Combination paper board manufacturers, those who use recycled fibers in paperboard manufacture, immediately adopted operating procedures to eliminate the use of carbonless carbon papers and thus to minimise 1 he PCBs in the paperboard. This action unquestionably was essential Yet, in one respect, it does run counter to the national interest and the goals of the solid waste program * Director, Environmental Control, Container CorporaIwm nf Amcncii, OhLcs, Penn&Ylvitnia 10456 of the Environmental Protection Agency. The combination paperboard industry now uses 70% of all paper that is recycled in the United States. Additionally, the paper industry is being implored by government agencies to recycle more wastepaper in order to help combat our mounting solid waste problem Continuing research by the industry also showed that virgin fiber grades of paper and paperboard sometimes contained measurable amounts of PCBs, even though no carbonless carbon papers were used in their manufacture Since PCBs have been established to be widespread m both our air and water environment (2, 3, 4,5), the presence of PCBs in virgin fiber products is felt to be a reflection of these background environmental levels. The Monsanto Company, sole domestic manu facturer of PCBs, withdrew PCBs from sale for other than closed system applications as of Sep tember 1, 1970. The manufacturers of carbonless carbon papers discontinued the use of PCBs in their product on June 1, 1971. These actions, together with the paper industry's elimination of carbonless carbon papers from paperboards used for food packaging, have resulted in marked re ductions m the levels of PCBs in combination paperboard. They are also expected to show' de creasing environmental or background PCB levels in the future. Under the sponsorship of The American Paper Institute (A.P.I), research is currently in progress at Haileton Laboratories, Inc to study the mecha nism by which PCBs migrate from paperboard to various food products and, also, to survey the levels of PCBs in United States pulps, papers and paperboards The former study is only well begun so I am April 1972 HONS 208T66 63 Piuduct Product weight or Carton product ratio Catculfctrd ppm PCB in looti product for 13', transfer Ifnm canon# containing 5 J>ptn 10 ppm Fioure 1 Cumulative-distribution o( PCB in 676 awn. plea of paper and paperboard unable to present any meaningful data on mi gration to you at this time. The survey study is progressing smoothly, however, with approxi mately (V76 samples having been analyzed to date The results of these analyses are shown as a cumulative-distribution in Fig 1, They have not yet been classified according to paper or paper board type, so represent a broad spectrum of virgin and reclaimed paper and paperboard ma terials. Th cumulative distribution shows 94% of ail the samples to have PCD contents less than 10 ppm; 86% less than 5 ppm; and 46% less than 0.5 ppm (not detectable). A second survey study ib being sponsored by the Boxboard Research and Development Associ- Fiouitt 2 Effect of fumjh control on PCB content of combination paperboard, Rice Uncooked cereal Candy gum drop* Spaghetti Ranine 42 28 14 n a 6 48 16 6 IS 1S 0 048 0 045 0 053 0 07 0 096 0 08 0 056 0 06 0 136 0 052 0 13 0 08 0 07 0 09 0 16 0H 0 17 0 11 0 16 0 22 U 32 0 13 0 09 0 10 0 22 0 ifll 0 22 0 26 0 18 U 20 0 4', 0 17 Q 44 ation at The Institute of Paper Chemistry This study is concerned only with recycled fiber paper boards. Preliminary data from this study indicate a mean PCB value of 3.6 ppm and an average PCB value erf 6.4 ppm That the prompt action of the paper industry has indeed been effective in reducing PCB levels in food packaging materials can be Been from Fig. 2. Figure 2 depicts the change in PCB content with time for several grades of recycled fiber paperboard, where sufficient data were available to permit reconstruction erf such a history Similar histories could be developed for other food-packag ing grades. TTub however would not add materially to our understanding of the problem more than is already seen from Fig, 2 Although we do not yet have the results of the A.P.I. migration study, preliminary research by one A.P I. member indicates that PCB migration from paperboard to high fat-content products does not exceed 33% of the total PCB m the paper board, even m the absence of any protective barrier between the paperboard and the product Based on this 33% migration factor and PCB levels of 5 to 10 ppm as being common in paper and paperboard, we can estimate expected levels of PCBs in products packaged directly therein Environmental Health Perspectives HONS 20814? avirwiniiit/ Hfaith Pmptatvtt Levels of Polychlorinated Biphenyls in Foods in Sweden by Fredrik Berglund, M. D,* PCBs have been used in industry in Sweden 6incc 1929, initially only in electric transformers and capacitors, later also in paints, lacquers etc The importation of PCBs to Sweden in 1970 was close to 501) tons Approximately half of this amount, was exported to other countries in electric, capacitors The use of PCBs in dyes was 65 tons in 1968 but. was voluntarily diminished by in dustry to 35 tons m 1970 The Swedish Riksdag (parliament) has enacted the 1971 PCB Act, snthorning the government to prescribe addi tions regarding importation, production, offering for sale and handling of PCBs, but detailed regu lations have unfortunately not yet been published. Tin* high degree of persistence and lipid solu bility of the PCBs have led to their accumulation m some foods of animal origin In Sweden they now necur in the natural environment roughly in the same amounts as the chlorinated pesticides (1) Tlic special hydrographical conditions of the Baltic fa\ or the accumulation of PCBs in certain species and areas. The water exchange with the North Sen is very limited because of the thresholds (8 m and IS m) and the narrowness of the Danish Sounds. The wide archipelagos at some places also make possible in some localities high concen trations of pollution, e.g., outside of Stockholm Table 1 briefly summarise* some of the earlier data, illustrating these points. The difference in levels of total DDT equivalents and PCBs be tween the Swedish west coast and the Baltic is more than 5-foid in Pish oil and almost 10-fold m * K'rofesror and Iread, Food Research Department Xutiormi huotl Arlmimatmtion, 104 0] Stockholm 60, SatHltn, as of January i<>72k former head. Department of I'ikkI Hygiene, same address herring The levels in fat are relatively high in all species shown The role of the food chain is shown by the high levels in seal and by the ex tremely high levels in the white-tailed eagles from the Stockholm archipelago Their muscle fat con tained about 2.5% of total DDT metabolites and 1 4% of PCBs, The Stockholm archipelago is evi dently heavily contaminated fcf lielou) The very high levels of DDT and of PCBs in muscle fat compared with the levels in eggs are most probably due to sampling from unrelated individuals The muscle samples were from sick birds, whereas the eggs derived from apparently healthy birds There are only few data from seal The levels of 2DDT and of PCBs per kg fal show fair agree ment between seal pup and seal milk {from the stomach of one of the pups) The samples were analyzed by gas chromatoggraphy and electron capture detection, combined with treatments with sulphuric acid and potassium hydroxide, The principle PCB components were found to have 7-14 chlorine atoms, with an in crease of those with higher chlorine content as the PCBs passed along the food chain Analyses of PCBs were later taken up by Westoo d: Nordn (2) After extraction and routine clean-up, the extracts were separated by thin-layer chromatography into two groups, one of which contained PCBs and p,p'-DDE, o,p'-DDT, o,pDDE The PCBs were determined by gas chroma tography of this group, before and after oxidation of the organochlonno pesticides Some of the PCBs were analyzed by gas chromatography without decomposition of the organochlorme com pounds Recoveries averaged 96-101% for PCBs added to purified fish extracts before application to thm-iaycr plate (2). April 1972 HONS 2OB169 67 Table 1. Avene* level* of organocblorme compound* in H>m( Svediifa marine orgn>*m* I^im Modified (mu (1), Specie* Locality Fat content SDDT n>i/kg fat mg/kg PCB mg/kg f.if tl Herring Fish oil White tailed eagle IBUIKie K Seal Seal pup Seal milk W coast BalUc (IS) W coeel Baltic Stockholm <41 Stockholm (5} Gull of Bothnia (2) Gulf of Finland (2) Gulf ol Finland {1 > * 4% 100% 100% 1.5% 5 6% 54% 60% 31% ie 17 2 le 26 000 1 000 120 42 36 68 330 63 25 11 0 7'. if \ 7 i 14 000 MO 1J 0 "i 4 - >* 190 i9 11 Table 1 Lctdi of DOT* DDE. DDD and PCB* In some fiab in Sweden 1967-1969. Modified from (21 Fish (n) Range DDT DDE DDD PCB mg/kg n n n n <4% fat freshwater <4% fat saltwater >4% fat saltwater (87} 0-0 1 SO 84 87 Hr 0 1-1 0 1 3 i (65} 0-0.1 57 59 65 0* 0 1-1.0 3 8 ? (34) 0-0 1 9 14 23 !3 0 1-1 0 24 20 li !9 1 0-3 0 i 1 Table 3- Level* of organochlorine compounds In Sab In Sweden 1967-1970. Modified from (31. Species Locality (No of samples) Fat content 2DDT mg/kg PCHi mg'kg Herring, Clupse Anrsngu# Eel. AnfutUa anfudla Cod, G'adw morrAw Cod liver Salmon, iSoimo taler pike, Stax tvctux Baltic (72) S Baltic, deep-froxan (18) Baltic (43) W. coeaMll) Baltic (30) Baltic (7) W coast (3) W coast(5) Lake* (5) Lake* (37) 3-20% 3-20%) 25% <2% 40-70% 10-15% 10-15% <2% 1 I7 06! 1 40 070 0 50 038 0 26 05b 0 05 (0-0 2) 10-22 2-5 0 06 t 04- 07) 2 63 72 0 00 10-0 3) 0 49* Olil 0 27* 11)7 0 28* uiu 0 43* )J4 0,02 (0-0 l> 2-5 2-4 Trace 0 58* 177 <0 1 (0-0 61 68 Environmental Health Perspective* HONS 208170 TUr 4, Levels of DDT. DDE end PCD* in some foods in Sweden 1967-1969. Modified from 44). Food fn) Range DDT DDE PCB* mg/kg n n n Margarine imi 0-0 l 115 115 115 Vegetable oils (197) o-o i 196 197 167 O !-! 0 I Beef (34) 0-0 1 34 34 34 Lstnli fat (10) 0-0 1 10 10 10 Pork (107) 0-0 1 101 104 105 0 1-1 0 6 3 1 Chicken (93) 0-0 I 93 93 93 Hen (50) 0-0 t 60 49 50 0 1-0 3 I Egg white* (28) 0-0 1 28 28 28 Egg yolk* (1855 0-0 1 178 176 183 0 1-1 o 8 e 2 1 0-6 0 1 1 * Six in web simple The level* of JDDT and of PCBs depended on the fat content of the fish and on geographical location. Regular salmon, Salmo solar, with 10-15% fat, had an average DDT level of 0.08 mg/kg on the west coast and 2 6 mg/kg in lakes, and of PCBs only trace# on the west coast hut 0.6 mg/kg in the lakes. Herring and eel, both with fairly high fat contents, had high levels. The highest PCB levels in Hah were found in 2 small samples of herring from the Stockholm archipelago, 2.8 and 2.5 mg/kg, and in 4 yellow eels close to Gothen burg, 1.5 mg/kg. Five chars, Saimo talvfltntu, in Lake Vkttern had an average fat content of 7% and PCB level of 1.3 mg/kg (not included in Table 3). Cod and pike, both with lees than 2% fat, usually had very low DDT and PCB levels Cod liver, on the other hand, with 40-70% fat, had high levels of DDT and PCBs. Because of its high level* of DDT, cod liver from the Baltic waa declared unlit for human consumption by the Swedish health authorities this year (1071) Some of the geographical variation of PCB and DDT levels m herring is shown in Fig, 1. PCB levels are highest outside of Stockholm, whereas DDT is higher off Gotland (16) and Blekinge (18-20). Other PCB-contanunated fish mentioned above were the eels caught off Gothenburg '34 and the chars in Lake Vattem (37) In other foods the levels of DDT, DDE and PCBs were much lower than in fiah (4), as shown in Table 4 With few exceptions, ail the samples of the foods listed contained less than 0 I mg kg of each compound. Egg yolk showed some high levels, but PCB levels in whole eggs would still not be expected to exceed 0 1 mg/kg Table 5 shows the results of analyses on con a milk, butter, and human milk Analyses on cow's milk showed DDT less than 01, DDE less than 0.3 and PCBs less than 0 5 mg/kg on a fat basis Analyses on a much huger number of samples of butter revealed levels of DDT below 0 13, of DDE below 0.1 and of PCB below 0.1 mg, kg, also on a fat basis The difference between cow's milk and butter in terms of DDE and PCBs is probably only a matter of sensitivity of the method--there is no reason to suspect losses of DDE and PCBs in the churning process In the analysis of human milk there was no need to look for "human butter", since the levels of DDT, DDE and PCBs were much higher than m cow's milk. The DDT levete in whole milk show fair agreement with those in the U S (5) and England (6). The PCB levels average 05 mg/kg fat or 16 ng/kg whole milk. This average would probably only represent the Stockholm area, since all the samples originated from a "human milk donor center" m Stockholm Recently Weatoo et al (7) also discovered PCBs m some samples of cereal products The origin was found to be the packaging materia! On thin-layer chromatography and on gas chroma tography, the PCBs behaved as PCBs of type Clophen A 60. In several samples of one kind of imported children's food, the PCB levels exceeded 1 mg/kg In one sample of this product, which had been stored at room temperature for two years, the level of PCB had increased to 11 mg/kg. The sale of this children's food m Sweden was stopped in July, 1971. The main intake of PCBs via the food in Sweden thus occurs by eating fish The average consumption of fish in Sweden 1961-1969 was 17 kg/person/year--and of crustaceans and mol luscs, about 1 kg/year In terms of fish flesh Westm (8) calculated an intake for 1965 of 35 g per person and day. Of this, about 2 g represent 70 Environmental Health Perspectives HONS 20817Z Tublt 5. Lintk of DDT, DDE and Pr.Bi in butter, row'* IT!ilk end human milk in Sweden IM7-19M. Calculated from (4). Eomi(n) Cm'i milk (11) Butipr (154) Human milk (22) Fat ~3% >81% 3 25 0 11% DDT <0 1 mg/kg fat <n 13 mg/kg fat 1 24 0 07 mg/kg fat 403 0 M/kg milk DDE <0 3 mg/kg fat <0 1 mg/kg fat 2 08 0 13 mg/kg fat 07e o /kg milk DDT+DDE 3 32 0 19 mg/k* fat 1068 8 sg/kg milk PCBs <0 5 mg/kg fat <0 1 mg/kg fat n S00 039 mg/kg fat lfl2 5 */kg milk freshwater fish, 1 g caviar, and 1 g shellfish and molluscs together The average intake of PCBs therefore probably does not exceed 1 fig per day but may well exceed 10 ug/day in people eating herring from the Baltic 3 times a week. A suckling baby ingests about 0 15 litre of mother's milk per kg body weight daily in the 2nd and 3rd months of life, with an average PCB level of 15 ug/kg milk. This corresponds to 2.4 Hg/kg body weight per day. Thus we have a situ ation similar to that with DDT with the suckling baby getting the highest exposure Also, it is known from Japan that PCBs pass the placental barrier (9). Conclusions. Proper food processing should not produce PCB levels higher than 0.1 mg/kg food, except in fish with more than 2% fat in edible portions, Human milk has low levels of PCBs, but due to the high intake of milk, the suckling baby w ill experience the heaviest exposure. To avoid further food contamination in the future, the use of PCBs should only be allowed in dosed systems and under most careful super vision. Less dangerous substitutes should be looked for in industry. REFERENCES l. Jensen, S. et tl 1909 DDT and PCB in marine anun&h from Swedish waters Nature 224 247 2 Westhfl, G. and Nortn. K 1970 Determination of organochlonne pesticides and polychlorinated biphenyls in animal foods. Acta Chem Scand 24 1639 3 Weetofl, G and Norfn, K 1970 Levels of orgennchlonne pesticides and polychlorinated biphen\ls in fish caught in Swedish water areas or kept for sale in Sweden VlrFoda22 93 4. WestGo. G.. Norfn, K. and Anderason, M 1970 Levels of organochlonne pesticidee and polychlorinated bi phenyls in margarine, vegetable oils, and some food products of animal origin on the Swedish market in 1907-1909 VlrF6da 22' 9 5. Qutnby, G. E,. Armstrong, J F and Durham. W F 1905 DDT in human milk Nature 207 726 0 Egan. H. et al. 1905 Organochlonne pesticidee residues in human fat and human milk Br Med J 2.06 7 WestOO, G . Norfn, K and Anderason, M 1971 Levels of organochlonne pesticides and polychlorinated bi phenyls in some cereal products. Vlr FGda 23 341 8. Weatm, S. I. 1909. Reviewed in F Berglund et al 1971. Methyl mercury in fiah. A toxicologic-epidemio. logic evaluation of naks. Report from an expert group Nord. Hyg. Tidakr., Suppl 4 252 9 Tsukamoto, H et at. 1909. The chemical studies on detection of toxic compounds in the nee bran oils used by the patient* of Yuaho Fukuoka Acta Med 60 497 April 1972 HONS 208173 71 Ejtmronmtniai Htallh Ptripmmt Occurrence of Polychlorinated Biphenyls in Humans* by Harold A. Price and Robert L. Wetchf Jo 1067 the Human Monitoring Survey (1) rat established by the Pesticides Program of the US Department of Health, Education and Welfare (now, Division of Pesticide Community Studies, E.P A.) to determine on a national scale the levels and trends of certain more commonlyused pesticide chemicals in the general population. Initially, this program was limited to the iden tification and measurement of chlorinated hydro carbon residues in human adipose tissue and blood serum. The Michigan Department of Public Health has been one of several state health laboratories participating in the analysis of sam ples, collected by die State Services Branch of the Divibion of Pesticide Community Studies, from hospitals located in approximately 30 states. For the purpose of obtaining poolable data, the state laboratories use methodologies recommended by the I'cmne Primate Laboratory, E.P.A. (Florida), which is responsible for methodology develop ment, the operation of the quality control pro gram, and serves as a repository of reference standards Since 1906 our laboratory has been conducting routine analysis of human adipose samples for organochlorine pesticide residues by gaa chro matography, utilising electron capture detection. In many instances, we observed numerous un * The raveatigatiofM reported herein were supported through a contract with the Division of Peetictdee Com munity Studies, Office of Pesticide*, Environmental Pro tection Agency, Chambtee, Georgia. The viewa expreaaed herein are thoee of the inveaUgatoia and do not neoesaarily reflect the official viewpoint of the Enviromental Protec tion Agency. t Michigan Department of Public Health Peelipdea Laneing, Michigan 48914. identified peaks recurring in a repeated pattern on chromatograms, some eluting much later than p,p'DDT Our attention became focused on polychlorinated biphenyls (PCBs), after the Perrine Primate Laboratory alerted laboratories involved with the human monitoring program and community studies on pesticides of the possible interference of pesticide residue analysis by these compounds. This was prompted by early reports of research workers in Europe and the United Statee finding PCS residues in animal and environmental media (2, 3, 4). Early confirmation of some of these compounds had been obtained with a combined gaB chromatograph-mass spec trometer from extracts of fish, sea birds, conifer needles, and some samples of human depot fat (,5) In March, 1969, one of Michigan's state labora tories found 19 ppm DDT m canned coho salmon during routine residue analysis. Our residue analysis on a similar sample seemed to corroborate this finding. However, considerable interference by "artifact'' peaks was evident on the chro matogram, causing difficulty in both qualitative and quantitative analysis (2, 6) of pesticide residues To a lesser extent, similar peaks had been observed with some human adipose samples These unknown peaks were ascertained later as polychlorinated biphenyls when compared with several Monsanto Aroclors by electron capture gas chromatography and imcrocoulometry Prior to receipt of Monsanto Aroclors, we found a readily available source of a PCB indicator, e g., residue analysis report forms One of us had known by previous experience that carbonless copy paper has on one side a coating of nucrocapsules containing an Aroclor solution of a colorless dye (16). An extract of this paper pro April 1972 HONS 208174 73 duced a chromatogram which twitched a con siderable portion of the interference observed with residues in coho salmon and human adipose tissue. During the year following the coho, salmon incident, our laboratory found substantial levels of polychlorinated biphenyl* in several samples of human adipose tissue collected through the Human Monitoring Survey program Three of these examples are given here to illustrate our findings MatiriilSHri EtyfipiMit Materials and glassware used for extraction of samples and preparation cleanup of extracts are described fully elsewhere (7, 8). A Micro-Tek Model 220 gas chromatograph (Tracor, Inc ) was used. It was equipped with two parallel-plate, tritium-source electron capture detectors, Vycor glass inserts installed at inlet ports to accommodate off-column injection, and U-shaped Pyrex glass columns (6 ft. X H id. o.d,). Column packings (9) supplied by the Perrine Primate Laboratory consist of 1.5%OV-17/ 1.95%QF-1 (Column 1) and 4%SE-30/6%QF-l (Column 2) on 80/100 mesh Chromosorb W HP. Operating temperature* of columns, detectors, and inlet were 200C, 210*C, and 225*C, respec tively. Carrier gas was nitrogen at flow rate* of 65 ml/min through column 1 and 80 ml/min through column 2 Reference standards of 1200 senes Aroclora and organochlorm* pesticides were obtained from the Pesticide* Repository, Perrine Primate labora tory, E.P.A , Perrine, Florida. Aroclora, products of Monsanto Company, are designated by num bers, such as 1260 which represent* * mixture of chlorinated biphenyls having an average chlorine conteot of 60%. For thin-layer chromatography, glass plates (Analtech, Inc.) precoated with aluminum oxide G, 250 microns thick, impregnated with 3-3% silver nitrate were used. MttNri The analytical scheme used for the preparation of these samples was the modified Mills procedure (8) One to three grains of tissue were extracted with hexane, partitioned with acetonitrile, and subjected to fractionation and cleanup by FlonaJ column chromatography All of the PCB com pounds were eluted m the first fraction (6% ether in petroleum ether), and this volume of eluate (200 ml) was reduced in a Kudmia-Daiuah concentrator to 10 ml. A 5-l aliquot nas injected into the gas chromatograph The extracts ..ert. diluted sb required to obtain peaks within the linear range of the detector For certain selected samples, the polychlo rinated biphenyls were separated from the organochlorine pesticide residues by application of the method of Armour and Burke (10) The 6% Floriail eluate was chromatographed on the silicic acid-Cehte column with 250 ml of petro leum ether, which eluted the PCBs The organochlorine pesticides were recovered next after elution with 200 ml of acetonitrile-hexanemethylene chloride (1:19:80) Rtsvlts Mrf Ditcmion In Fig. 1, electron capture chromatograms of human adipose tissue extract M69.305 (8% Florisil eluate) and a standard solution of Aroelor 1260 are presented to illustrate that some of the "artifact" peaks in this tissue sample match those in the PCB standard. On the 4%SE-30 6%QF-1 Fiona* 1 GLC-EC chromatogram of Arcelor 1260 ' I 5 ng) and human adipose sample M61I.105 (JLC condi tions' 6'Xlf" i% SB-30/6% QF-1 column, oven (eni|i 2O0`C, and nitrogen do* 75 mt/mm 74 Environ mental Health Perspectives HONS 208175 s 10 robiphenyls to decachlorobiphenyl were confirmed by combined gas chromatography-mass spec trometry (11). Fig. 2 shows that tissue extract M69308 con tains some components which are present in Aroclor 1254. Approximately 20 ppm PCBs as Aroclor 1254 mss determined by gas chroma tography, Biros (11) confirmed the presence of pentachlorobiphenyls and hexachlorobiphenyls in addition to p.p'DDE. The largest unlabeled peak following Peak 5 in sample M69308 ) p,p'DDE. The level of p ,p'DDT in this sample was too low for confirmation by mass spectrometry, and its peak is masked by Peak 14 of Aroclor 1254 on the SE-30/QF-1 column used in this illustration In the spring of 1970, we found approximately 100 ppm PCBs in a sample of subcutaneous adipose tissue (M70227) which was obtained from a hospital in southeastern Michigan The subject was a white male, age 77, whose demise followed a pulmonary thrombo embolism. of An electron capture chromatogram of the 6% Floriail column eluate of this sample is illustrated in Fig. 3 and is compared with a chromatogram of Aroclor 1260. This extract contuns late eluting Fioum 2. GLC-EC chromatogram of Aroclor 1254 (3 i isomers that are also components of Aroclor ng) end human adipose sample M60308. Peek 1' It 2,2' 5,ft'-tetrerhkirobiphenyl, tod petit 3 it 2,2',4.5,6'-pentachlorobiphenyl GLC conditions 6'xX'' 4% SE-30/8% QF-1 column, oren tamp 200*C, tad nitrogen flow 73 ml/rou. column, p.p'DDT it completely masked by Peak 8 of Aroclor 1260; however, no interference it encountered with the quantitation of p,p'DDE, On the basis of a major peak following P.p'DDT, the level of PCfie in this sample was estimated to be seven parts per million as Aroclor 1200 For estimating PCB levels by gas chro matography, we uae Peak 12, preferably cm 1 .5%OV-17/1.95%QF-l column, as a reference in Aroclor 1260 which has the following retention lime relative to aldrin (Rt - 1.00): Column 0V-17/QF-1 SE-30/QI'-l Aroclor Peak p ,p'DDE 6.4 4.S3 2.23 1.82 p,p'DDT 4.18 3.12 In this sample, PCBs ranging from hexachio- Fiocub 3. GLC-EC chromatogram of Aroclor 1260 (2 l ng) and hu^an adiooee sample M70227 (30 5 Mg, A% Flonsil fraction) GLC conditions 6' x W`% SE-30/6% QF-1 column, oven teinp. 200*C, and nitrogen flow 80 ml/min. April 1972 HONS 208176 75 I l kOK i * < 4 5 If I* Ficon* 4. GLC-EC chnjio*U)icriiii ihowm* separation of PCB (A) and ortwnorhkonne pesticides (B) by method of Armour and Burke GLC conditions same ft* Fig. 3. Weight of sample injected 30 5 Mg. 1268, which makes matching to any particular Aroclor difficult. Fig. 4 shows the separation of PCBs from organochlonne pesticides by the method of Armour and Burke (10), although some of the p, p'DDE eluted in Fraction A in this ewe. On the basis of a chromatogram of the second eluate from the silicic acid-Celite column resulting from a subsequent effort, residues of 0.37 ppm p,p'DDT and 0.81 ppm p,p'DDE were found in the fat of this sample. Ueiag the combined gas chromatograph-mass spectrometer, Biros (12) confumed the presence of p,p'DDE m M70227 as well as ten polychlorinated biphenyls ranging, from hexachloro- to nonachloro-compounds. These compounds are found m Aroclors 1260, 1202, and 1268. Further examination of other tissues of the same autopsy case revealed a good correlation of PCB levels when compared on the basis of extractable lipids as demonstrated in Table 1 These data are good estimates calculated from the height of a peak corresponding to Peak 12 of Aroclor 1260 on the OV-17/QF-1 column. PCB peaks in M70227 appear to be a complex mixture of several Aroclors not usually observed in human adipose tissue Efforts to determine the source of PCBs m sample M70227 have not been successful Indica tions are that this individual was exposed lo PCBs during his lifetime As PCB1. enter a biological system, the less chlorinated isomers nneither metabolised or excreted, which changes the composition of original Aroclors, so that chro matograms of these mixtures from biological samples do not match exactly those of lefercnce standards This has also been obsmed with PCBs in M70227. In fact, more than 4,000 human adipose samples examined in our laborator\ ha\e produced chromatograms that do not exactly match those of standard Aroclor solutions We have found the distribution of PCBs in samples, such as M70227, to be uniform throughout the sample as received, indicating that these samples have not been contaminated by PCBs during handling As for all samples, M70227 was collected from an autopsy examination through the co operation of pathologists, according to directions specified by the State Services Branch of the Division of Pesticide Community Studies, E P \ As many investigators of PCBs htu e found, one of the principal sources of polychlorinated biphenyls in the biological system is the food chain (5, 13, 14). Coho salmon, for example, had been a source of PCBs in food until it exceeded the action level of 3 ppm. Another possible avenue of human exposure to polychlorinated biphenyls may be indicated by their presence m some house du^t (vacuum sweepings). Our examination of house dust from residences of occupationally-exposed population of southwestern Michigan revealed that several samples contained up to 180 ppm PCBs, mainly Aroclor 1234 Table X. Level* of PCB** in Various Tissue* Related to Sample M70227' Tissue S Adipotc* S Adipose 9 Adipose P Adipose Liver Kidney rA Lipid ext'd PCU level ppm Wet weight lltAl* f* at lnw? 41 1 63 6 32 S 44 1 0 OS 0 06 7", IDO 50 50 r 0 7'. 18il LiO lit 125 76 --Environmental Health Perspective* Table 2. Distribution 9f PCD Level* in Adipose Tiaaue of i he Genera] Population--Analyst* by Gas Chromatography laTVpl, ppm Samples Percent of total Nsgnh'C Trarc t( < 1 0 l o-2 n >2 il 4 20 109 55 6 72 36 7 11 5 6 A* mentioned previously, levels of poly chlorinated biphenyls in human tissue extracts illustrated in Figs. 1, 2, and 3 were determined by electron capture gas chromatography using a major peak after p,p'DDT (Peak 12) as a refer ence peak for Aroclor 1260. Since April, 1971, our laboratory has run semiquantitative analysis of polychlorinated biphenyls in samples of human adipose tissue by both electron capture gas chromatography and thin-layer chromatography. The latter based on total chlorine is an adaptation of a procedure reported by Mulhem et al., (7, 15). After gas chromatographic analysis is completed, the remaining extract is evaporated, and pesticide residues are dehydrochlorineted with ethanolic potassium hydroxide and oxidiied with chromic oxide. The concentrated extract of PCBs remain ing is spotted on a thin-layer plate, and the developed spots after exposure to ultraviolet light are compared with several spots of standard Aroclor 1260 at different concentrations. The limit of detection is 250 ng of Aroclor 1260. Results in Tables 2 and 3 indicate that both methods provide similar information, but thin-layer chroma tography is preferred. On the basis of samples received and analysed by our laboratory, these data show the distribution of PCB levels in adipose tissue of the general population. Tsble 3. Distribution of PCB Levels io Adipose Tissue of the General Population--Analysts by Thin-Layer Chroma tofrephy Level, ppm Negative 1 race to < 1 0 1 0-2 0 >2 0 Samples Percent of total 20 10 3 87 44 g 70 36 1 17 8 8 Summary According to samples of human adipose tissue received by our laboratory, 41-45% of the general population have levels of 1.0 ppm or more (wet weight) polychlorinated biphenyls with isomers, from Aroclors 1254, 1260, 1262, and 1268 The presence of PCBs ranging from peutachlorobiphenyls to decachlorobiphenyl has been con firmed in selected samples, M69305, M6930S, and M70227, by the use of combined gas chroma tography-mass spectrometry. fckflOW!ri|MM*tS We thank Dr Anne R, Yobs of the Division of Pesticide Community Studies, Environmental Protection Agency, Chamblee, Georgia, for pro viding the human adipose tissue samples described in this study, and Dr. Henry F. Enos for con sultation and advice. We thank also Dr Ronald G. Webb erf the Southeast Water Laboratory, Athens, Georgia, for providing samples of a pure tetrachlorobiphenyl and a pentachlorobiphenyl REFERENCES 1. Yobs, A R 1967. Pesticides in people Pestic Momt J 1. 6 2. Rmbrough, R. W. et al 1968. Polychlonnsted bi phenyls m the global ecosystem. Nature 220 109B 3 Holmes, D. C , Simmons, 1 H. and Tatton, J O G 1967 Chlorinated hydrocarbons in British wildlife Nature 216: 227. 4 Koeman, J H., ten Noever DeBrauw, M C and Voe, R- H de I960, Chlorinated biphenyls in fish, mussels, and bird* from the River Rhine and the Netherlands coastal area Nature 221. 1120 S. Widmsrk, G 1967. Poeeible interference by chlorinated biphenyls J Assoc Off Anal Chem 50 1069 6 Reynolds. L. M 1969 Polychlorobiphenyls (PCBs) and their interference with pestiridp residue analysis Bull. Environ Contain Toxicol 4 128. 7. Pemne Pnmate Laboratories 1971 Analysis of Pesti cide Residues in Human and Environmental Samples Manual of Analytical Methods, Environmental Pro tection Agency, Pemne, Florida 8 Mills. P A..Onley,J. H and Gaither, R A a 1963, Rapid method for chlorinated pesticide resi dues in non-fatty foods J. Assoc Off Anal Chem 46 186 b 1970 Methods whirh detect pesticide residues in human and environmental media Pesticide Anal Manual, Food and Drug Admin , Washington, D C., Ill Sec 211 April 1972 HONS 208178 77 Levels of Polychlorinated Biphenyls in Adipose Tissue of the General Population of the Nation by Anne R. Yobs, M.D.* Polychlorinated biphenyls (PCBs) have been reported in many different parts of the environ ment in this country (1-3). The purpose of this report is to present preliminary results from a monitoring program in which these materials are routinely sought and quantitated. The Human Monitoring Survey, established in 19G7 by the Pesticides Program, DHEW (now Division of Pesticide Community Studies, EPA), determines, on a continuing basis, the exposure to pesticides experienced by the general population by measuring levels of pesticide residues present in tissues or excreted m urine. Initially, tech nological limitations and resource restrictions limited the Survey to the identification and measurement of those chlorinated hydrocarbon residues which are stored in measurable amounts in mammalian adipose tissue, or which can be measured in blood, these residues being a reflection of past exposure. The initial program plan baa been and continues to be reviewed frequently with reference to technological and/or research developments to identify other groups of pesticides and other materials of interest, which can be incorporated with slight modification of the existing program. Samples are collected through the direct co operation of pathologists, who are in hospital or private practice or who are in public service as city or county medical examiners. Samples are Chief. State Services Drench. Division ot Pesticide Community Studies, Office of Pesticides Programs, CP. Environments] Protection Agency, 4770 Buford Highway, Chnmlilco. Ca. 30341 collected from both sexes in all age and racial groups. Adipose samples are collected from tissues removed for therapeutic surgery or from post mortem examinations, the latter including people who have died accidentally (trauma) or during relatively brief hospitalisation. No samples are accepted from institutions for long-term care of the chronically ill patient All samples are analysed by laboratories established under contract with State Health Departments or at community study projects m Michigan, Florida and Colorado. These labora tories use analytical methodologies specified by the program on the advice of technical consultants and are required to maintain acceptable levels of performance, as demonstrated in both inter- and intra-laboratory quality control programs. Tech nical consultation for analytical aspects of the Survey is provided by the Chemistry Branch, Perrins Primate Laboratory, EPA, under the direction of Henry F. Enos, Ph D., Chief. In I960, with the increased interest in poly chlorinated biphenyls, it was recognised that PCBs probably occur in several segments of the environment and that their presence would distort analytical values for certain pesticides, especially elected DDT residues, The monitoring labora tories were therefore asked to report all samples suspected of containing PCBs by gas-liquid chromatography. A reporting system was de veloped based on rough quantitation, as follows negative, present in usual amounts, present m greater or lesser amounts than usual Samples collected in late 1968 and 1969 showed that PCBs were indeed present m a significant portion April 1972 HONS 20ISO 79 of the general population of the country over a wide geographic distribution Positive samples were reported from 14 States, including Michigan, New York, Minnesota, California, Massachusetts, Georgia, Kentucky, Illinois, North Carolina, South Dakota, Ohio, Louisiana, Delaware and Arkansas. To insure that proper identification was being made, two samples with higher levels were confirmed by combined gas liquid chroma tography-mass spectroscopy (4) These two cases are discussed in another paper in this symposium (5) Based on these preliminary findings and certain aa yet unpublished information about the effect of PCBs on the accuracy of measurement of residues of DDT (Enos, H F, personal com munication), high priority was given by the Perrine Laboratory to the development and evaluation of an analytical method which would separate PCBs from pesticides permitting accurate quantitation of pesticides and confirmation of PCB levels. The resulting methodology is af urther modification of the Mille-Olney-G&ither procedure in which adipose tissue is subjected to extraction by petroleum ether, acetonitrile partitioning, and Flonsil cleanup A portion of the resulting 6% ethyl ether/petroleum ether eluate, in oonoentrato form, is treated with KOH to effectuate de hydrochlorination of DDT and DDD to their olefins, thus eliminating the problem of separating these pesticides from the PCBs. Oxidative treat ment is then applied to convert any interfering DDE to p, p'-dichlorobensophenone which has an Rf value different from the PCBs. The PCBs are then determined by thin layer chromatography (6) . On April 15, 1971, all monitoring laboratories in the Human Monitoring Survey adopted this TU 1. laeUcac* ef pelychlorinated biphenyls in adipate af Usa yenaral population of the nation. Reported Analysed No. % No. % Total Negative Trice- <. 1 0 ppm 1-2 ppm >2 ppm 637 100 0 314 40 3 125 19 6 165 25 0 33 5 2 688 100 0 236 34 2 220 33 3 188 27 3 36 5 2 Table 2. Descriptive statistics of samples anal vied Negative and trace< I 0 ppm No `r. 7'otal number Sex Male Female Race White Nonwhite Samples from Therapeutic surgery Postmortem Diagnostic group Neoplasms 140-209 Circulatory system 390-450 Gsitromleetmal system 530-565 Liver A gall bladder 570-575 Pancreas 577 t?nn*ry system 580-505 Congenital Anomalies 740-750 Accident* A violence numbers All other 430 221 218 381 58 05 344 77 121 41 *1 0 11 5 16 145 -- 430 too 0 50 3 40 7 86 8 13 2 21 6 78 4 17 5 27 6 10 0 48 00 25 11 36 33 0 -- 90 0 > ( 0 ppm \rt 108 inn n i >7 M 1 71 15 `1 151 "G n 47 23 7 2>t 14 h ISO 85 4 111 10 7 til 30 8 10 8 t n :t o > 1 0 7 ,1 5 i 05 7 35 50 2U 8 -- ---- 108 w y methodology for the analysis of all adipose samples with the lowest limit of sensitivity being 1.0 ppm. Aliquots of all samples in which meas urable amounts of PCBs are found are saved for future confirmation by mass spectroscopy. limits and Discission Analytical results for 637 samples have been reported and are summarised in Table 1 (.11 additional samples have been analysed but hat c not as yet been reported). Of the 037 samples reported, 198 (31.1%) contained measurable amounts of PCBs and 125 (19 6%) contained trace amounts, with the balance being negative These samples were collected from 40 pathologists in 38 cities distributed over 18 States and the District of Columbia. The States are Arkansas, Illinois, Louisiana, Oklahoma, South Dakota, Environmental Health Perspective* HONS 208181 Tennessee, Oregon, Pennsylvania, Florida, Kansas. Maine, Georgia, North Carolina, Califomis, Michigan, New York, Ohio and Kentucky Positive samples came from every hospital, city nd state sampled It, therefore, appears that ihwe mnirnals are widely found in the population Details of distribution by sex and race, collection procedure, and diagnostic groupe are summarised in Table 2 The difference between figures for the positive and negative groups does not appear to be significant, although no statistical evaluation fats been attempted because of the preliminary nature of this report. Since a statistical design has been established and is being followed for sample collection for the Human Monitoring Survey, in due time valid statistical analysis will be possible snd mil then be performed. Summit Polychlorinated biphenyls have been found in measurable amounts in 31 1% of 637 samples of human adipose tissue collected from the general population as a part of the Human Monitoring Survey Sample collection involved 18 States and the District of Columbia Positive samples were obtained from every State sampled REFERENCES 1 Gustation, C G 1970 PCBs--prevalent end persistent Environ. Sci Technol 4.814 2 Risebrough, R, N etal 1968. Polychlorinated biphenyls m the global ecosvstem Nature 220 1008 3 Duke, T W, Lowe. J I and Wilwm, A J 1970 A polychlorinated biphenyl (Aroetor 1254) in the water, sediment, and biota ut Escambia Bay. Florida Bull Environ. Contamin Toxicol. 5 171. 4 Btroe. F J 1970 Polychlorinated biphenyl* in human adtpoae tissue, Bull Environ. Contamin Toxicol 1 317 5 Price, H A Occurrence of polychlorinated biphenyls m humans Env Hllh Perep This issue 73 6 Thompson, J F (ed.) 1971. Analysis of pesticide residues in human sod environmental samples for use by Community Studies Projects and Pesticide Monitor ing Laboratories. Pesticides Program Section 0 Perrme Pnmate Research Laboratories. Environmental Pro tection Agency, Femne, FI* April 1972 MONS_ 206182 81 Environmental Health Pertpectovn Polychlorinated Biphenyl Residues in the Plasma and Hair of Refuse Workers by D. I. Hammer*, J. F. Finklea, L. E. Priester, J. E. Keil, S. H. Sandifer, K. Bridbord Refuse workers may be exposed to polychlo rinated biphenyl (PCB) residues from the burning of automobiles, tires, transformers and wire insulation. Paired plasma and scalp hair specimens were obtained from 37 refuse burners and 54 controls (lumberyard and waterworks workers), PCU residues were dehydrochlonnated and assayed by gas chromatography with a Niu electron capture detector Rough PCB quantita tion w os achieved by integrating the area under a fnc peak "fingerprint" associated with the PCB fractions, Arochlor 1254 and 1260 Unwashed hair specimens were all negative for PCBe. Detectable ` Division ol Health Eftact* Research, EPA National Envir lies Ctr . Research Triangle Park, N C. 27709, plasma PCB levels were found in 81% (32/37) of the refuse burners but only m 11% (6/54) of the controls. Median PCB Levels for those with detectable amounts were similar m the t o groups, refuse workers--2.6 ppli, controls--3 7 ppb Maximum plasma PCB levels were 14.1 ppb in the refuse workers and 20 2 in the controls. The higher frequency of measurable plasma PCB levels in refuse workers may reflect their increased PCB exposure from incinerated ma terials. Similar median plasma values for both groupe reaffirm the role of blood as a transport rather than a storage tissue. As expected, scalp hair is of no utility in estimating PCB body burdens. April 1972 MONS 209183 83 Envuvnmmutl Ilniih P<fipfUne Food Exposures to Polychlorinated Biphenyls by Albert C. Kolbye, Jr., M.D.* Ttic PCn family of industrial chemicals has been used in a number of useful and beneficial s)'h for more than 40 years. PCBs have been used in capacitors, as transformer fluids, as heat transfer agents, in plasticizers and adhesiveE, and in sealants and printing inks. Needless to say, they have no place in the nation's food supply The rant if ir community has been aware of the potential hazard of the PCBs since at least 1966 Reports in the foreign literature at that time indicated the detection of this chemical in fish and birds However, the status of the analytical detection method fur organochlorine pesticide residues in reference to PCBs was not perfected until early 1969, after it was discussed in FDA's pesticide analytical workshop and the former Division of Pesticides improved the analytical method, so that it was satisfactory for routine analysis. We know that as a toxic substance PCBs are a potential but not immediate health hazard and that their environmental background level is not high We do not know how long-term exposure to PCBs might afTect human health, and we cannot ycl explain the inconsistent presence of the chemical in certain areas of the environment. We ha\e been faced recently with PCB adulteration of food from accidental sources and from un saleable environmental sources. The exact routes of contamination are subject to speculation 3ume theories point to the industrial wastes from plants using PCB, leaching of PCB from plastic objects in waste disposal (burning at waste incineration plants or run-off from landfills into streams), and Deputy Director, Bureau of Food*, U S Food and Drug Admimutretioli, ttuhmgton, D C 20204 accidental use of PCB as solvents for which they are not intended (vehicles for spray preparations etc) Some of FDA's earliest findings of PCBs m foods occurred during the fall 1969 when PCBs were encountered m coho salmon and milk. In August, 1969, Baltimore District found an adulterant identified as PCBs in 5 of 12 milk samples from West Virginia. During a six-month followup investigation, it was learned that PCBs were commonly used as a heat transfer agent in electrical capacitors in the area from which the milk samples were collected Further investigation revealed that the electric company had allowed a right-of-way sprayer to utilize a transformer oil base for defoliant spraying Vegetation collected from the power line nght of way was tested and found to be positive for PCBs It was eventually concluded that the apparent misuse of tlie trans former fluid containing PCBe for defoliant spray ing operations adjacent to dairy pasturage was responsible for the milk contamination. Ae a result, in November, 1970, the FDA undertook a specific survey of the incidence of the PCBs in the milk supply. The Monsanto Company informed FDA on July 16, 1971, that large amounts of fish meal may have been contaminated with PCBs (Aroclor 1242) during pasteurization of fish meal at the East Coast Terminal, Wilmington, North Carolina. PCBs were used as the heat exchange fluid The U. S. Department of Agriculture informed FDA on July 19, 1971, that Holly Farms, a large North Carolina poultry producer, had discovered the presence of PCBs in its poultry, resulting from an investigation to determine the cause of reduced hatch&bility in eggs. The firm's investigation April 1972 HONS 208164 as implicated the fish meal ingredient aa the causa tive agent. The FDA tested the processed fish meal and confirmed that fish meal on hand was con taminated with PCBs. Continuing investigation indicated the leak began on April 30 and continued through July 10 Approximately 10.000 tons of fish meal were shipped during that period. The contamination was caused by a leak in the heat exchange equipment, which used PCBs in the heat exchange fluid Individual hah meal samples examined contained from 14 to 30 ppm PCBs, Fish meal, the direct subject of this contamina tion incident, is a minor component of poultry and fish feed Investigations conducted by FDA and US DA focused attention on the contaminated meal as a possible source of PCB residues in poultry, eggs and commercially raised catfish The Food and Drug Administration visited primary and subconsignecs and initialed followup sampling of AbIi feeds, commercial fish, and eggs, when the contaminated fish meal was implicated USDA was informed when investigation indicated eggs were being distributed to egg breakers The firm recalled outstanding stocks of fish meal on July 21 and approximately 1,500 tons of fish meal were returned to the plant. Sampling of poultry for PCB residues was instituted promptly by USDA who advised that no lots of broiler poultry were found exceeding the 5 ppm interim guideline for PCB residues Follow-up sampling on eggs was initiated on August 3, 1071 As of September 29, ]g7| samples of eggs were analysed in the f(J|]ou '* with 71 containing residues in excess of n ^ (range 0.6-4 2). PPTM We seised five shipments of fish feeds m thi States of Louisiana, Georgia, and Mississippi contained from 0 0 to 4 ppm PCBs p>vt,, samples of catfish, which had Iwcn fed u,e f0ntaminatcd feed, contained PUB re-nduci wnhm the 5 ppm guideline, ranging from 0 3 3 0 ppm In addition, we seised a shipment of tlm ron. laminated fish meal from East Coast Terminal that had not been recalled and contained in exreo of 350 ppm PCBs Since November, 1969, the FDA has analvwd all raw agricultural commodities sampled under tbe pesticide surveillance program for PCQs u weft as pesticide residues Over 17,000 sample* have been collected and analysed In the past is months, 684 samples of fish, cheese, milk, shell eggs and hsh by-products, out of 3,505 samples of these commodities, hsve been found positive for the PCBs. PCBs were encountered most frequently in fish m 363 of the 670 samples ranging from trace levels to 35 29 ppm. PCBs were detected in fresh water fish including catfish, chubs, and smelt, and also salt water species including porgies, sen front, bluefish, bonita and sardines PCBs have onlv been detectable in shellfish at trace levels The results of the pesticide program for PCBs for that 18-month period are as follows Fob Cheese Milk Shell eggs Fish by-product PCB in selected food commodities--7/1/76 to 4/30/71. Number of samples examined Number of samples positive Percent positive Low PCB levels (plim) high 670 1344 041 550 -- 363 91 69 161 13 54 T 6T 7T 29 T --T 35 29 !o 27 $ 3 74 50 A\cfnR& 1 H7 4- 2 :t <) r 1 17 Total (excluding fish by-product) -- 3505 -- 684 -- 19 ____ 1U Aiiove result* reflect both surveillance and compliance* samples Average residue levels do not include values indicated ns "true* " T indicates "trace" which in norma) arnili v* imuld lie less Until nppnnsimalclv l) 1 ppm hah, 1-1 3 ppm milk or cheese fat, 0 2 ppm eggs * Compliance samples reflect 1 ollowup of violative samples bancs biased towards lugh levels 86 Environmental Health Perspectives MOMS 208185 The FDA total diet studies for 60 market biakete representing a total of 720 composite ample* during FY 70 and FY 71 show 22 com posite samples to contain PCB residues ranging from a trace to 0.36 ppm These positive findings nere found in the meat, fish, poultry, dairy, and the grain and cereal composites. The 0.36 ppm PCB value reported in the total diet study was found to be caused by migration of PCBs from the grayboard container and dividers to packaged bredded wheat Subsequent information gathered on the oc currence seemed to point to the use of reclaimed, or recycled paper in the manufacture of the card board packaging material used for these food packages. Consequently FDA initiated on September 8, 1971, a Survey of PCBs in Food and Food Packaging Material. This survey was designed to cover selected food categories nationwide to determine the extent of food contamination by PCB-containing cardboard packaging materials. As of November 12, 1971, we had complete information from 12 of the 17 districts showing results on a total of 584 samples. The packaging material analysed in this survey included the following types of components: oml nc Figuri 1 Survey--PCB in Food and Food Packaging Material Cumulative Analytical Findings--Sept. 8Nov 5, 1971 Type* of component* comprising packaging malarial analysed during survey Inner wrapper* Waxed paper Cellophane Foil Paper Piastre Dividers Paper eupa (as used for cookie* and candy) Piastre trays Paper trays Outer Wrappers Foil Piastre Cellophane Paper Other Cellophane Fiber eartona windows Plastic bags Paper envelopes Metal can ends Plaatic can ends Plastic tear strip* Tea bags Fionas 2 Survey--PCB in Food and Food Packaging Material Cumulative Analytical Findings--Sept 8Nov. 5, 1971. awina Some of the finding* from this survey as received os of that date are shown in the following Figs. 1. 2, 3, 4, 5, G and 7 Fiocac 3 Survey--PCB in Food and Fond Packaging Materia! Cumulative Analytical Findings--Sept 8Nov 5, 1071 April 1972 HONS 208186 ~ 87 Flams 4 Survey--PCB in Food ind Food Ptckifing Metenai Cumulative Analytical Findings--Sept. 8Nov 5, 1971. JBU --> Fionas & Surrey--PCB in Food end Food Pecksging Material Cumulative Analytical Finding*--Sept. 8Nov S, 1971 Fioriti 6 Survey--PCB in Food end Food Packaging Mtlend Cumulative Analytical Finding*--Sept. 8Nov. 6, 1971, Flooitc 7 Survey--PCB m Food and Food Packtgint Material Cumulative Analytical Findings--Sept 8Nov i, 1971. It is significant (1) that no PCBs have thus far been reported in fresh fruits and vegetables, (2) that PCBs are found in fish quite frequently, (3) that in addition to poultry, PCBe are also found in milk and eggs, and (4) the PCBs have now been found in various food commodities apparently as the result of migration from their packaging components. At this point the signifi cance of these findings has not been determined. With the conclusion of the PCBs in Food and Food Packaging Material Survey, FDA has embarked on a program to determine the level of PCBs in animal feeds. Following the PCBs in feed program, FDA will further evaluate PCBs in milk by collecting milk for .both bottling and manufacturing use on a nationwide basis Specific attention will be then given to PCBs in shell eggs. FDA's current working levels for PCB residue findings are based cm specific occurrences of "accidental" contaminations. They are 5 ppm in the edible portion for fid); 5 ppm on a separate fat basis for poultry; 0.2 ppm in whole milk (5 ppm in the fat). Action hsa been taken against animal feeds and whole eggs associated with the Cast Coast Terminal incident at the 0.S ppm level These levels should not be construed as guidelines permitting consumption of foods containing these amounts of PCBs. If the FDA finds that the frequency of PCB contamination in the nation's food supply increases, the present working guide lines for PCB residues will be substantially lowered. <8 Environmental Health Perspectives MONS 208187 Environmental Health Pertpoatm Polychlorinated Biphenyls: Their Effects on Penned Pheasants* ty Robert B. Dahlgren and Raymond L. Linderf and C. W. Carlson^ Mnfcctton Polychlorinated biphenyls (PCBs) have been uwful industrial products since the beginning of ihrir commercial production in 1929. The first irfiort of PCBs occurrence in wildlife was made Irom the work of Sbren Jensen, a Swedish Chemist, m the New Scientist in 1960 (1) Since then PCBs have been reported to be widespread in the world's ecosystem, building up in food chains as his been reported for organochlorme insecticides. The chemical structure of PCBs and their action on organism*, although leas toxic, are similar to that of DDT. The contamination of human milk, poultry feed, and all classes of wild vertebrates, hit crested a concern for the need to determine possible harmful effects of PCBs. Funds tor the study were eupplied by the Bureau of Sport Fiahenee and Wildlife through the South Dakota Cooperative Wildlife Heaearcb Unit, supported jointly by the South Dakota Department of Game, Fiah, and Parka, the Bureau of Sport Fiahenee anil Wildlife, the South Dakota State University; and the Wildlife Management 1intitule Tiasue analytea were made by the Denver Wildlife Research Center and by Dr. Yvonne A Qnechua, Experiment Station Biochemistry Department. Dr Robert J Bury, Veterinary Science Department, conducted nectniwtea and histopathologic studies. Dr. W. L- Tucker. Ei|irnment Station Statistician, gave statistical advice Wild pheasants wen eoDeeted by Fred E. Hartman and John J Knt, Psnwylvama Game Commission; and Rnlvrt D Feldt, Indiana Department of Natural Re source*. This work is frum a doctoral dissertation st South Dakota State University by the senior author. t Amistant Leader and Leader, respectively, South Dakota Cooperative Wildlife Research Unit, South Dakota State University, Brookings, 8 D ATOM } Professor of Animal Science, and Leader, Poultry Re search and Extension. South Dakota State University, Brookings, S.D. S700S Because of the economic importance of the pheasant (P/uutanus colchxcut), it was chosen as the experimental animal using PCBs in this study The objectives were (1) to determine the patterns of absorption, storage, and excretion for PCBs, and (2) the effects of PCBs on reproduction, behavior, and survival. Since many published reports indicated that the chromatograms of PCBs in wildlife most nearly resemble those of Aroclor 1254, this was the PCB product chosen for study. The use of brand names in this paper does not indicate an endorsement of any product. Mstirists art Mittwds Aroclor 1254 (supplied by the Monsanto Co ) was mixed (w/w) in a 1:9 dilution with com oil and weighed into No, OO sue gelatin capsules to within 2.5 percent of the required weight Capsules were administered into the esophagus using a glass tube. Capsules containing only com oil were administered to control birds. Analyses for residues were made by Dr Yvonne A. Greicbus, Experiment Station Biochemistry Department, South Dakota State University, and the Bureau of Sport Fisheries and Wildlife laboratory at the Denver Wildlife Research Center Analytical procedures used at the South Dakota State University laboratory are described by Dahlgren et a!. (2) and procedures of the Denver Laboratory by Dahlgren et al. (3) Hens for breeding experiments were purchased in the winters of 1970 and 1971 (30 and 34 respec tively) from the South Dakota Pheasant Co of Canton, and cocks were raised from birds pre viously obtained from the Canton source All birds used for breeding were about 1 year of age. April 1972 HONS 208188 89 In late January they were placed under 16 hours daily of artificial light to stimulate breeding Both sexes were fed a pheasant breeder ration (Zip Feed Mills, Sioux Falls) One tabiespoonful of oyster shells was added to the food cup of each hen weekly Cocks were kept in breeder cages (30X36X18 inches high), and hens were kept m individual cages (12X18X12 inches high) de signed to facilitate handling and identification of eggs- Temperatures were kept at near 65 F in colder months. Eggs were collected daily, in dividually numbered, set weekly for 15 weeks in a forced-draft incubator, and hatched in pedigree cages In 1970, cocks were first dosed with 35 mg PCBe on February 13, and weekly thereafter for a total of 17 times Hens were first dosed 1 week later, February 20. Breeding was commenced February 16 Eggs were gathered for the first weekly egg group from March 7-13 and gathered last 15 weeks later, June 13-19 In 1971, the same sequence was followed, except that the first dosing began February 19 for cocks and February 25 for hens, and breeding began March 1 Eggs were gathered for 15 weekly egg groups beginning March 6 and ending June 18. Within 36 hours after hatching, chicks were taken from the incubator, wmgbanded, and one wing was removed to the alula. They were then placed on a visual cliff (4) and given 5 minutes to jump to cither the visually deep or shallow side. Chicks from this study and those hatched from a concurrent dieldrjn study were placed together in brooders and fed a turkey pre-starter diet (Zip Feed Mills) until they were 6 weeks of age. At 6 weeks, they were placed outside in 16X16 foot pens and fed a pheasant grower ration (Zip Feed Mills). Several times in summer and fall, clucks were caught by hand by the same person, and wingband numbers were recorded The number of birds caught in each category in the first and last half of aK birds caught was compared with an expected 50:50 distribution using chi-square analysis Each pen was a closed unit, and birds of several groups caught predominantly in the first half caused birds of other categories to be caught in the last half. Weights to the nearest gram were taken of all adult breeders weekly Chicks were weighed to the nearest gram at 6 weeks of age To study effects of PCBs in combination um, dieidrm, cocks and hens from 0 to 9 months oitl were stratified by sex and weight mid randomise) to five groups of 22 birds each They were capsules containing either (I) 4 mg technics) grade dieldnn, (2) 8 mg dieWtrsn, { r '>0 mg !'( tj, (4) 100 mg PCBs, or (">; 50 mg PCBs and 4 mr dieldnn together Ten doses were given one capsule each 3 3 days for 5 necks Three and one-half days after the last capsule was gum, all surviving birds were sacrificed The Denver Wildlife Research Center anaiv as) for residues of PCBs in pooled luers from three road-killed pheasants collected near Washington Pennsylvania; six road-kiJled pheasants eollrrird southeast of Lancaster, Pennsylvania six plicaants shot east of Garv, Indiana, six phcasunisshot in Benton County, Indiana and six phe.xsanis shot near Brookings, South Dakota All of dvr above collections were made m the spring of 1971 fUsnItt and Discussion Effects on Reproduction The averages of eggs laid per hen per da\ among hen treatment groups were significantly different in 1970 (5). With the use of an orthogonal test the rate of egg production among hens gnen U j mg PCBs was not found to be lower (P>005) than the rate of the control group, but the rate of the 50-mg group was lower (P<0 01) than the rates of the other groups. In 1971, the same pattern was evident (Table 1), there were signifi cant differences (P<0.05) among hen groups The production of hens given 12 5 mg a ns not lower (P>0.05) than that of controls, while production of 50-mg hens was lower (P<001i Egg production was lower in groups where cocks were given PCBs, but this is not to be expected, and we cannot explain it Differences among groups in egg fertihtv in 1970 were significant (P<0.05), however, variation m fertility did not follow a pattern related to I'C'H dosage, and, thus, the results mav not haw ,tnv biological meaning (5) In 1971, there were no significant differences in fertility among groups (Table 1) ` Hatchabihty in both 1970 (5) and 1971 v.is highest in control groups and lowest m gtoup'. m 90 Environmental Health Perspective* HONS 208189 -fcv - T*bte 1. Reproductive atalialice from control pheasant* and pheasant* given PCBs 1970-71 Trcntment group* No eggs per hen per day No eggs set in incubator Fertile egge No Percent Fertile egge hatched No Percent Fertile pjot pipped but not hitched No Percent iirn D-db 0-12 5 0-50 25-0 25-12 5 25-50 IM 0-0 0-12 5 0-50 25-11 25-12 5 25-50 0 621 0 450 0 292 0 335 0 302 0 108 0 337 0 430 0 328 0 405 0 385 0 228 277 101 36 220 128 58 130 50 37 168 91 64 170 71 40 08 63 64 168 82 49 199 128 04 148 96 65 233 170 73 288 153 53 95 42 44 74 73 87 68 32 04 73 80 50 70 41 05 65 70 64 50 51 53 no 68 109 71 10 24 06 29 23 8 16 9 10 18 25 10 16 67 20 16 44 85 15 10 5 12 Ivnrh treatment group had five hens except that the 25-12 5 mg group had nine hens in 1971 ' The first number is the weekly PCB level in m* given to cocks, the second, that for hens tthith hens had received 50 mg PCBs, In 1970, lhc.sc differences tested by chi-square were not significant (P>0.05), while in 1971 hatchability wits reduced (P<001) among hens given PCBs. Significant, differences (PcO.fKi) were found in the number of eggs that were pipped but not hub bed for hen groups in both 1970 arid 1971 Apparently, the administration of PCBs to the hen adversely affected the viability of the embryo *< bill clung McLaughlin ct al. (6) injected both 10 and 2.1 mg of Aroclor 1242 into chicken eggs and found only 0-5 percent hatchability with growth retardation, edema, and beak deformities in embryos lCggRhcll thickness was measured only in 1970, and no significant differences (P>0.0o) in eggshell tlmktsess were found using analysis of variance (.-pi Eggshells from hatched eggs (without mem branes) laid by control hens averaged 0.200 02 mm standard deviation, and those from all hen gitrnps receiving PCBs averaged O.23Q02 mm Eggshells from unhatchcd eggs averaged 0.3J0 02 mm for control hens, and 0 31 0 02 mm for hens receiving PCBs. Dahlgren and Linder (7) found the eggshell thickness of pheasants to be unaffected by weekly administration of capsules containing C nig dieldnn Heath et al (8) fed 25 ppm of Aroclor 1254 to mallards (Anas platyrhyncitos) through two breed ing seasons They also fed bobwhite quail (Cohnus mrgtntanua) 50 ppm l'CBs and at a joint level of 25 ppm PCBs and 15 ppm DDE for one reproduc tive season They found no effects on egg produc tion, cracked eggs, eggshell thickness, embrvonation, embryos alive at 3 weeks, normalitv of hatchlings, and normal hatchlings alive at L4 d&vs. Scott et al. (9), who fed 0, 0 5, 1 0, 10 0, and 20.0 ppm Aroclor 1248 for 8 weeks to chickens in full egg production, found no reduction in egg production on the lowest levels of PCBs after 8 weeks, however, they noted a 10 percent reduction was associated with 10 ppm, and a 13 percent reduction was associated with 20 ppm. They reported that 10 ppm Aroclor 121S reduced hatchability of chicken eggs by 8 percent after 4 weeks and 44 pei rent after 8 weeks The "20 ppm level nearly eliminated all hatchability. Most embryos died at 21 davs of development, tnanv after pipping They also found that eggshell strength was not affected when as much as 20 ppm was fed Effects on Behaviur Seventy-cine 11-week old hens given one 210-mg Table 2. Behavior on the visual cliff of chick* hatcheP from control pheasant* and pheasant* liven PCB'e 1970-71, Treatment group No that jumped to visually deep Bid 1070 1071 No that bO visually shallow idt 1970 1971 No not jumping within 5 minutes 1970 1971 0-00-12 5 0-5O 25-0 25-12 5 25-50 29 37 37 5 Hi 5 If) 40 35 43 42 34 fl 30 33 67 22 78 5e 87 85 59 15 17 77 61 The first number is the weekly PCB level m mg given to cock*, the second, that for hens capsule in the evening of the first day of testing appeared weak at the end of the following day After receiving the second capsule, they sat with feathers fluffed, and some tremormg was noticed. During the 24 hours before death, they often showed tremors, particularly when disturbed. Shortly preceding death, birds were comatose and died without tremors. Eleven 11-week old hens given 20 mg daily were hypcrexcitable after 4 days of PCB treatment, after 10 days they sat with feathers fluffed. They exhibited weakness with occasional tremors about 30 days after dosage began Eleven hens 11 weeks old given 10 or 20 mg daily appeared to have fewer body feathers after 30 days than controls, although they did not appear to peck one another more often. Flick et al. (10) reported feather loss in chicks of the domestic chicken given PCBs, yUual Cliff Behavior--In 1070, when chicks were placed on a visual cliff for up to 5 minutes, significant differences (P<0 01) among group* were found in their behavior using chi-square analysis (5); offspring of hens given SO mg jumped to the visually-deep side of the cliff more often than offspring in other groups In 1971, no signifi cant differences in behavior between groups were noted (Table 2), When data from both years were combined, no significant differences (/>>0.05) were found Baxter et al. (11) reported that pheasant chick behavior in a visual-cliff test was apparently affected by dieldrin given their parents Response to Rand Catching--Results of catching young pheasants by hand were snalvied by comparing the number of birds, from each treatment group caught in the first half and ian half of all birds caught to an expected number equaling 50 percent of the birds m that group for example, 16 out of 20 young in the rategorv where both parents had received PCBs were caught m the first half of all birds caught on July 22, 1970 (Table 3), The 16 caught in the first half and 4 caught in the second half were compared by chi-square analysis to an expected 10 (half the total category of 20), resulting in a highly signifi cant difference (/><0.01), Since each pen mss i closed unit and the PCB-tre&tment birds were penned with other young from a concurrent dieldrin study, it must be remembered that birds of several groups caught predominantly in the first half would cause birds of other groups to be caught in the last half. It is important to compare not only how the PCB birds were caught in comparisoa to an expected distribution, but also how categories compared with one anothet The com parisons are shown in Table 3 as ratios These indicated that the ability of penned pheasants to avoid hand capture was significantly less in 1976 offspring where both parents had been guen PCBs, This is identical to the findings of Dahlgren et a] (12) for offspring of parents administered dieldrin. However, in 1071, the responses of oil spring of treatment groups were similar to controls. Data for 1970 and 1971 were combined and * contingency table chi-square technique was used to determine the effect of PCB treatment on the response to hand catching between groups The response was similar whether PCBs were given to either the cock or hen but was significantly different (P<0 05) when both parents received PCBs from that where only one parent received PCBs During catching, pheasants were herded around and around the pen, and often the catcher mbs able to capture those birds which ran back directly toward the pursuer. However, there was no attempt made to determine or to quantify the elements of behavior that would explain why offspring of birds treated with PCBs were easier to catch. 92 Environmental Health Perspectives HONS 208191 `jVTt,- ; :tiva l,Mr 3, Effects of fCJlm on band capture of penned pheasants. 1970-71. (Number* repreaent bird* caught in |kf (nl, j,,if af all bird* caught) number* in parentheses repreaent one-half the number of that category id tbe pen- Chi-square era* uaed to compare number* actually caught with half of the number* in each category.) !Jrtcn of i npturr No of hutches caught No of penft Both Parents receiving PCB Hens only Cocks only Neither u>:n Julv 22 Julv 2*1 Aug M Aug 11 Sri>l I Or! 15 Nov 2! Dcr 5 IS 14 i9(to;`* 1-9 15 19(11 5)'* 1-10 18 11(12 5) 1-12 20 21(15 5)* 1-14 22 25(18)* 1-15 23 20(18) i-15 13 14(11 5) 1-15 13 11(11) All InTO catches combined Ratio, lW 137(106)** 1 6 11*71 Juft -'1 Sq>! 1-3 Nep< 21-23 0r( 7-11 Ort t`1-22 2*5 1-7 17 27(19 5)* 1-15 28 75(70 5) 1-15 28 71(70 5) 1-15 28 33(37) 1-15 28 34(35 5) 1-15 28 30(30 5) 8(8 5) 7 00) 12(12 5) 17(14) 19(15 5) 14(16) 14(22 5) 14(11 5) 105(100 5) 1 91 23(24,5) *1(37) 38(36) 33(35 5) 23(33 5)* 29(32 5) 7(10 5) 0(12) 12(13.5) 18(17 5) 22(20) 25(17 5)* 8(10 5) 12(10 5) 113(112) 1 98 25(21 5) 30(36 5) 37(36) 27(36) 35(34 5) 23(28 5) 11*(10 5) 8(13 )* 8(13)* 19(18) 1409 5) 9(14) 12(11 5) 6(10)** 85(110)** 2 59 12(14 5) 21 (22) 24(21 5) 19(20 5) 17(21) 19(18) All IfiTI catches combined Kafni, 1971 270(204 5) 1 96 187(199) 2 13 187(192) 2 05 112(117 5) 2 10 All 1970-71 catches Ratio, 1070-71 407(370 S)** 1 82 202(299 5) 2 05 300(304) 2 03 197(227 5)** 2 31 Total* of row number* may exceed one-half total birds because odd numbers of birds in pens were rounded higher ' ibnia in pen/bird* caught in fuel half *(P<0 05) ' (P<0 01). Table 4, Weight* and survival for tbe lift 6 week* of offspring from control pheasant* and pheasant* given PCBs, 1970-71. Treatment group Average weight at 6 week* (g) 1970 1971 No. of chick* to brooder 1970 1971 No. of chicks alive after 6 weeks 1970 1971 Percent survival 1970 1971 Bo(H years )MJ* 0-12 5 0-50 25-0 25-12 5 25-50 396 429 378 435 303 425 389 428 403 458 344 373 73 65 84 64 29 50 72 115 90 100 40 10 49 50 49 45 3 37 50 91 30 72 96 67 77 72 58 70 *4 10 74 51 69 79 75 60 66 64 22 60 30 * 71ic first number is tbe weekly PCBlevel in mg given to cocks, the second, that for hens April 1972 HON3 ZU3192 95  -L..v ------------ ------ .---------------- .-- ------ - -`-v. Kuaiaiiiifiini run, 71 Group No of voun* tlive m fill* Percent survival from 6 weeks of age to fall*1 Percent jut\j Wil h&trlttnft to ftlip 1070 1971 11)70 1971 Both Year? 1970 B^(h 1*171 'i ean fM)4 0-12 5 0-50 25-0 25-12 5 25-50 20 35 23 36 0 20 21 57 18 5 44 41 72 57 47 SO 63 0 78 72 42 63 55 60 S3 75 44 67 53 27 ',5 41 27 Ml 40 0 JR 37 29 50 42 36 54 4k 10 40 1ft * December 5, 1970, November 2-5, 1971 Using the number of chicks ihve at ter 6 weeks, Table 4, 1 Using the number o( chicks to brooder, Tsble 4 * The first number is the weekly PCB level m mg given to cocks, the second, that for hens Effects on Survival and Weights of Offspring Survival--Chick survival was determined for treatment groups in 1970 (5) and 1971 during the first 6 weeks of age while chicks were kept in brooders (Table 4). The survival o chicks was not related to whether cock parents received PCBs, but chi-square analysis showed that significantly more (P<0.01) deaths occurred among chicks hatched from hens receiving SO mg PCBs in 1970. These differences were not evident in 1971 data. When data from both years were combined, there was a significant difference (P<0.01) between offspring where hens received 0 and 12 5 mg PCBs and between offspring where hens received 12.5 mg and 50 mg PCBs (P<0.01) Survival of young pheasants in outdoor pens was measured in December, 1970, and November, 1971 (Table 5). Survival of offspring from 6 weeks of age to the fall in both years appeared to be unaffected by level of treatment in either year. When overall survival from hatching to the fall in both years was considered, the overall survival of offspring from hens given 50 mg was significantly less (P<0 05) than that of the other groups. The lower rates of survival lor the 50-mg groups were due to the effect on early survival, not that from mortality of birds older than 6 weeks of age. No meaningful departures from the expected 50:50 sex ratios in the treatment groups were deter mined (Table 6), Apparently PCBs did not affect the survival of one sex more than another Wright--Weights of chicks from liens on 50 mg PCBs weekly were loner (P<0 01) ate weeks of age (5) than those of other groups in 1070 (Table 4) In 1971, weights of offspring of 50-mg hens were lower (P<0 01) than that of lontroh. while weights of offspring of 12,5-nig hem were higher (P<0,0]), When data from both years were combined, there was no relationship betnecn weight and treatment level Although we did not adequately determine that Table 6. Number* of cock* end hrns elite in November that were offspring of rontrol pheasant! and pheasants given PCB'e, 1970-71. (Uhi-|uer* was used to compare numbers of rerb sea il,>e ,lh an espected 50: SO distribution. 1 Group 1970 November 21 Cocks Hens 1971 INnvemher 2-5 Cocks Hens 1070-7! Combined Cocks Hem 0-00-12 5 0-50 25-0 25-12 5 25-50 All PCB group* 11 11 14 11 01 5 16* 13 6 11 33 35 t" 17 15 20 13 17 33 22 30 20* 31 100 80 28 2* 20 31 >! 18 18 t.H 1') 4 -i 122 115 * The first numlier ]R the Weekly Jcvet m me given to cocks, the second, that for hens *{P<0 051 **{P<0 0t) >f rcD- 94 Environmental Health Perspectives HON5 208193 PCB via the egg depressed the weight of offspring, McLaughlin et si. (6) mentioned growth retarda tion of embryos as an effect of PCBs injected into the yolk sac of chicken eggs. We fed no pheasant flocks PCBs, but Flick et ai (10) found that l-dsy-old chicks fed Aroclor 1242 at 200 and 400 ppm had depressed growth by the second week of feeding and that the growth depression was related to the level fed. Vos and Koeman (13) fed PCBs to 1-day-old cockerels and found bodyneight depression from 400 ppm Aroclor 1260 Platonow and Funnel) (14) found that 1-day-old cockerels fed 250 ppm Aroclor 1254 had depressed body weights between the sixth and ninth week of iheir feeding trial; this depression was associated uith reduced feed consumption. Rehfeld (15) also found depressed weight gains in 1-day-old chicks fed sublethal levels (10-50 ppm) of Aroclor 1254; chicks fed 30 and 50 ppm for 2.5 weeks and then fed a dean diet recovered from the growth depres sion, while chicks fed 40 and 50 ppm for 5 weeks nd then fed a clean diet for 8 weeks did not show a recovery from the growth depreesion Body weight* and Mortality In Birds GWen PCBs Adult hens in both the 1970 and 1971 breeding experiments were unaffected m body weight by administration of as much as 50 mg PCBs in single capsules weekly. It was characteristic, howc\ or, of birds that died on PCB treatment to slop eating and die within several days. Hens 11-weeks old given a 210-mg capsule of PCBs in the evening ate very little the following da\ and appeared weak by the end of that day (3) After receiving the second capsule 24 hours sub sequent to the first, birds sat with feathers duffed. sonic tremonng was noticed, and no food was consumed. Birds of both sexes 6-9 months of age gi\m 100 mg or 50 mg PCBs every 3.5 days for 5 weeks continued to eat, as did 11-week-old hens rcjx-atedly given either 10 or 20 mg daily Seott e< ul (9) found no effect on feed consumption when laying ehickons were fed up to 20 ppm Arodor 1248, and no mortality that could be attributed to treatment l'restt et al (16) found no effect on weight of Bengalese finches (LoneAurn sliinta) when they were fed up to 400 ppm Aroclor 1254 Tor 56 days Birds 11-weeks old given daily capsules con taining 210 mg PCBs died within 1 3 and 5 9 days, the 16 designated to be analysed upon death lived longer than the other birds in the experiment, from 2.2-S.9 days, averaging 3 81 0 dav stand ard deviation (3) They loet from 15-37 percent of their initial weight before death All control birds lived. Correlations of the initial weight, days to death, and percentage weight loss were ob tained for 53 birds in this experiment. Initial weight was correlated with days to death (r--0.699, P<0.01); heaviest birds lived longest The birds which were heaviest initially lost the greatest percentage of their weight before death {r=0,589, P<0,01). Birds that lived the longest lost the greatest percentage of their weight hefore death (r--0.744, P<0.01) Time of death of the II birds that were not given PCBs, but were starved, ranged from 2 3 days to 8.6 days, aver aging 3.91.8 days. They loet from 27-51 percent of their weight by the time of death (3) Of the birds given 50 or 100 mg every 3.5 days for 5 weeks, 4 of 22 died in the 50-mg group and 7 of 22 died in the 100-mg group. In addition, two birds in the 100-mg group were so weak they were near death at the conclusion of the experiment. Mortality of birds given 10 mg began 30.6 days after the first capsule was given, the ninth bird died after 179.3 days. The other seven birds died between 50.3 and 00.8 days after initial treatment. The tenth bird of this group, still alive after 8 months of treatment, was sacrificed (3) In tlie 20-mg group, the first bird died 39.6 days and the last bird 54 1 days after capsules were firet given. The average number of days to death was 46 15 3 days (3). Mortality was light in breeding experiments in 1970 and 1971 In 1970, only 2 hens died from among the 30 hens and 10 cocks under study. both hens had received 50 mg PCBs weekly (5). In 1971, among 34 hens and 14 cocks in the study, three hens died, two that had received 12 5 mg and one that had received 50 mg weekly. Tucker and Crabtree (17) reported that 2000 mg/kg of either Aroclor 1242, 1254, 1260, or 1268 given to mallards caused no mortality or symp toms Prestt et al (16) estimated that 254 mg/kg/day given to Bengalese finrhes would give 50 percent mortality at 56 days. Heath et a) (3) reported that Aroclor 1254 liad an LCW of 1090 April 1972 HONS 20M.94 95 ppm when fed for 5 days an pert of the diet to pheasants Vos and Koeman (13) reported that, of 20 chicks fed 400 ppm for 60 days, only three died. In the present study, 210 mg PCBs daily were given to 11-week-old hens. By the time the average bird died, it had received 840 mg PCBs, the first to die received 420 mg, and the last to die, 1260 mg. These same figures for 11-week-old hens on 20 mg daily were 940 mg, 800-1100 mg, and for birds given 10 mg daily (one was sacrificed after surviving 8 months) were >830 mg, 310-2410+ mg Thus, for a group of pheasants given PCBs at 10 mg or more daily, the most susceptible in dividuals would probably die with 300-400 mg Aroclor 1254, the average bird would die with a cumulative dose totaling from 800 to 950 mg; and the least vulnerable would die after receiving 1200-2410+ mg PCBs Absorption, Storage, and Excretion Dahlgren et al. (2) reported that retention of PCBs in the bodies of four hens, each given single 50-mg capsules and sacrificed 28 days later, averaged 40.5 rag in each hen. Levels of PCBs were highest m al) tissues at 12 hours after capsule administration and declined most rapidly in liver at 24 hours. Throughout their experiment, levels were highest in liver, followed by brain and muscle. Brain tissue contained more PCBs than muscle per unit weight, but, havmg a higher lipid percentage than muscle tissue, brain tissue had a smaller concentration of PCBs per unit of lipid. Analysis of whole bodies of four hens that received 12.5 mg PCBs weekly for 17 weeks re vealed that they retained from 37 to 56 percent of the administered dose, while hens on the 50-mg level retained 60 to 82 percent of the administered dose The hens on the higher dose averaged about six times as much PCBs in their bodies as the hens on the lower dose, although they had been given only four times as much PCBs (2). A total of 48.7 mg of PCBs in the whole body plus that excreted in eggs and feces over 26 days was accounted for after administration of a 50-mg capsule (2). The PCB-com oil mixture was readily absorbed in the gut of the pheasant, calculations showed that a maximum amount excreted unsbsorbed. was 6 percent of the 50 mg given, and as much as 98 percent may have been absorbed. Five of the hens that had received 12 5 Bi weekly for 16 weeks in the 1971 breeding expery. ment were sacrificed 1 week following the ministration of the last capsule in the series Tfe ppm PCBs in the bodies of these five hens wen 17,6, 18.6, 24.4, 28 3, and 30 5, averaging 23 S ppm Three months later, three other liens of the same group, which had been kept raged, sacrificed The analyses of their bodies shoved 8 9, 12.0, and 20.0 ppm PCBs, averaging 13 6 ppm PCBs After 0 months on a clean diet, three other birds siroihu'Iy treated had whole-body values of 18.3,19.5 and 25.0, averaging 20 9 ppm The apparent rise in PCB concentration at 6 months may be due to sample variation and, or the physiological state of the hens sampled It ts obvious that the rate of excretion of PCBs relatively slow These birds were analysed st the Denver Wildlife Research Center, Scott et al. (9) found that chicken bens given up to 20 ppm Aroclor 1248 in the diet for 8 weeks lost less than 50 percent of the stored PCBs after 4 weeks on either a standard diet or low-energy diet He also found that a low-energy diet followed by s high-energy diet did not affect reduction of PCBs over tune. Prestt et al. (16) fed 1500 mg Aroclor over 56 days to Bengalese finches and were able to recover only 9 percent of the amount fed, when they analysed one bird from the experiment Excretion Via the Feces--Excretion of PCBs in feoes was relatively low as reported by Dahlgren et al. (2) An average of 4.0 mg per single 50 mg dose was excreted in the feces of four hens over 28 days, the feces from these birds were analysed as a pool Two other hens excreted 2.2 and 2 9 mg per 50-mg dose over a 28-day period, the feces from these two hens were analysed separately PCBs in the pooled feces of the four hens were at a peak during the first week and declined to relatively lower levels thereafter. They also found that excretion in the feces was highest during the first 24 hours in the two other hens given single 50-mg capsules, and less than 1 mg PCBs appeared in the feoes the first day. Variability in excretion between these two hens was low An average of 2.6 mg per hen was passed in the feces of the two hens by the end of `28 days Excretion Via the Egg--In four hens given 50-mg capsules when egg laying was declining, levels of PCBs in the eggs were lowest in the first % Environmental Health Perspectives HONS 206iSS 2.5 mg expcri. he ad * The * were S 23 S of the were honed t 13 C three ' ppm ppm at G id/or It w Be ii t the io up dost er 4 dm! by h CBs dor e to hen :b Ten mg ver ed t*'g ces I)1 at to ud he tie td in of fO ;n trek, highest during the second week, and dedined thereafter The average excretion per hen via the egg was calculated to be 4.2 mg PCBs (2) Excretion of PCBs via the egg could be higher than excretion through the feces when hens are tn full egg production. A single egg laid between 1 ind 2 weeks after a hen was administered a single capsule containing 50 mg PCBs was shown to contain 1 5 mg of PCBs This egg was one of two laid that week by one hen Heath ct al (8) reported that two mallard eggs from tlie second reproductive season, when birds had been on 25 ppm Aroclor 1254, had 56 ppm and 33 ppm PCBs, wet weight. Peakall (18) reported that ring dove (Streptopelta nutria) eggs taken from birds given 10 ppm Aroclor 1254 m the diet averaged 4.81 108 ppm standard error The actual amounts in mg of PCBs in ring dove (18), mallard (8), and pheasant eggs in the present study would be far below the 10 mg injected by McLaughlin et al. (6) into chicken eggs The 10 mg resulted in poor hatcliability, edema, and beak deformation. Scott et al (9) found that PCBs deposited in chicken eggs were less than 0.5 ppm after 8 weeks with 0.5 and 1.0 ppm Aroclor 1248 in the diet. They found Levels of over 3 ppm after 8 weeks with 10 ppm in the diet and levels of about 6-7 ppm in eggs of hens on 20 ppm. These values are much lower than those found in the present study from 3-4 weeks after administration of a single capsule of 50 mg Aroclor 1254 Their results, showing a drastic reduction in hatchability associated with 7 ppm or less residue m eggs, are not comparable to our findings with pheasants. Differences with the experimental animal or the Aroclor product used may have resulted in the gross differences in findings between our studies. Heath et al. (8) reported a nearly four-fold difference between bobwhite and Japanese quail in the LCw, thus species differences may be important. Hcsidues in eggs of wild birds have been deter mined by several authors from diverse collection points. Anderson et al. (19) found PCBs in all egg pools of cormorants {Phaiacrocorax aurUus) and pelicans (Pclecanus erylkrorbynchos); cormorants were shown to have an estimated 8 ppm in eggs and pelicans 06 ppm. Jensen et al. (20) reported 48 ppm in the egg of a heron (Ardeo cinerea) collected in Sweden, and 8-21 ppm from 9 guillemot (Una aalge) eggs from the Baltic Sea Risebrough et al. (21) reported that an egg of a peregrine falcon (Falco pereyriims) contained 10 ppm, 8 black petrel (Loomelama melanta) eggs had an average of 1 ppm; 5 eggs of a bam owl (Ti/io alba) had < 1 ppm; and a golden eagle egg (Aquila chrysaeiot) had <1 ppm. Dustman et al (22) reported median measurements of egg resi dues of bald eagles to be 1.65 ppm for Alaska and 9 7 for those from all other states. They further reported median egg levels of 15.9 ppm for the osprey IPandton haliaekun) of Connecticut and 2 5 ppm for those in Maryland; 5-6 ppm in brown pelican (P occidental**) eggs from different areas, and 5 ppm in eggs of royal terns (Thalatseut maximus). Prestt et al- (16) found residues ranging from 6-80 ppm in 363 eggs from 28 species of both land and water birds collected in Britain. Most of these had less than 10 ppm, but those which had higher levels included a single egg of the great crested grebe (Podteep* erwtata*), 40 ppm; one egg among 101 of the heron that had 80 ppm, while the arithmetic mean was 5 ppm; one egg of a moorhen (Gallinula Mrropus) with 15 ppm, among 13 samples that averaged 2.4 ppm, and a single egg of a great skua (Slerocoranut skua) that had 25 ppm. Particularly in view of findings that about 5 ppm was associated with nearly complete negation of hatchability in the chicken (9), the above findings in some wild birds are alarming. Apparently, though, as earlier pointed out, there must be considerable differences among species in (1) the rate at which they deposit PCBs in the egg, and (2) what a particular ppm range may mean in associated deleterious effects. Residue Level* in Tissues We analyzed tissues from seven different groups of birds: (1) 16 birds given 210 mg PCBs daily that were designated for analysis upon death, (2) five additional birds on 210 mg daily that died, (3) nine birds that were killed at intervals for matching with the 16 designated to die, (4) pooled samples of birds killed 12 hours and 24 hours after receiving a single capsule containing 210 mg PCBs, (5) pooled samples of four birds dying and from four birds surviving capsules con- taimng 50 and 100 mg PCBs, (0) a pooled sample of four birds dying on 20 mg and 10 mg PCBs, and (7) a pooled sample erf two control birds (3) Birds that died from daily doses of 210 mg PCBs had brain levels that ranged from 320 to 770 ppm wet weight and averaged 520 ppm110 ppm standard deviation Liver residup levels were much more variable than brain levels. They ranged from 390 to 9,300 ppm and averaged 2,50Q2,000 ppm wet weight Muscle residues were also relatively more variable than brain residues, as they ranged from 51 to 290 ppm, and averaged 14G53 ppm. Tissue levels from the birds killed for matching overlapped with ranges in the birds that died on 210 mg, but less so for brain than for liver or muscle ranges. Brain residue levels of nine birds sacrificed for matching ranged from 280 to 500 ppm, and averaged 37065 ppm, livers ranged from 1,000 to 5,000 ppm, and averaged 1,9001,300 ppm, and muBcle ranged from 58 to 110 ppm, and averaged 8317 ppm. Brain showed less variability than other tissues. Further, bnun was the only tissue independent of other parameters (initial weight, percent weight loss, days to death, and lipid content in brain, liver, and muscle) when testing correlations of all possible parameters using birds that died from capsules containing 210 mg PCBs This lack of correlation with other parameters indicated the usefulness of the brain in assessing toxic levels of PCBs. The relationship of the liver with other parameters such as original bird weight and days to death tended to negate its usefulness. Further, the interrelationships of liver parameters with those of muscle, even though it is not known how many of these relationships were meaningful, tended to negate the usefulness of muscle as an indicator tissue. Brain levels of PCBs were generally higher in birds that died than in birds that were sacrificed at the amae time for matching of brain residue levels, although there was some overlap of the two grou ps. A brain residue level of 400 ppm separated the bulk of the birds (86 percent) that died from most (67 percent) of those that were sacrificed There appeared to be a relationship between days to death and brain residue level, however, this was not significant (P>0 05) A pooled sample of four birds that died on treatment with 100 mg PCBs given each 3.5 days had 320 ppm m the bt,n 1 tissue, while a pooled sample of four sacrificed ! birds in this group had 59 ppm. A pooled sample of three birds that died on treatment with 50 m* PCBs given every 3 5 days had 3.50 ppm in the brain, while a pooled Bam pie of four sacrificed from this group had 34 ppm Pooled brain tissues from four birds that died on daily doses of 10 and ! 20 mg had 360 and 380 ppm PCBs, respectnch no data were available for survivors of the* groups. These latter data indicated that 300 ppm might be better than 40D ppm as a separation point that would indicate death from PCBs and that, when smaller amounts of PCBs nere received by the birds over a period of time, the spread m brain levels between birds that died and survnrd might be greater Ratios of residue levels using wet-weight ppm values among brain, liver, and muscle in dead and sacrificed birds overlapped considerably, particu larly in brain:liver and brain:muscle ratios However, hver:muscle ratios appeared to be smaller in birds dying on 210-mg doses. If one establishes 19 as a liver:muscle ratio, 3 of 9 birds sacrificed on the 210-mg doses had smaller ratios and only 4 of 18 birds dying had higher ratios However, it is necessary to gather more data to determine if this ratio is useful in diagnosing the cause of death as PCB toxicosis. Vos and Koeman (13) found that, when 1-davold cockerels were fed 400 ppm Phenochlor DP G. Clophen A60, and Aroclor 1260 in the diet for 60 days, the chicks had brain residue levels ranging from 70 to 700 ppm among II birds that died on treatment and 40 ppm for one chick which sur vived. Liver levels in their experiment were more variable than brain levels and ranged from 120 to 2,900 ppm among 28 birds that died and from 210 to 340 ppm among four survivors Seven of their 28 birds that died had liver residues of less than 250 ppm, while four of 11 birds had less than 300 ppm. It appears from their data that liver might be as useful as brain for diagnosing cause of death Their liver: brain ratios were similar to those of the present study, but, since they had only one sur vivor from which brain and liver were analysed, overlap could not be evaluated Rehfeld (lot reported that 30-50 ppm Aroclor 1254 given to 1-day-old cockerels resulted in liver residues of 300-500 ppm Prestt et al (16) reported a range nl in w i- r r\" ii. Ill' te rn H in li. n m r ir I. 1> 1 f l 0 t 1 1 ( 1 1 c M Environmental Health Perspective* HONS 208197 ^ 3^34 ppm in livers of survivors and 70-697 pheasants given 20 mg daily and in the 10-mg -,pm in luer* of Bengalese finches dying during birds except for one that survived 8 months and iitstment with Aroclor 1254, and that liver level was sacrificed (3) Splenic atrophy was char tis correlated with the amount received acterized by almost complete absence of lymphatic /'C001) Prcstl et al (16) stated that the liver:brain nodules and an increase in the relative abundance of red pulp. In one of the birds given 20 mg, loei rrsuiups should be compared and that brain of necrosis were found in lymphatic nodules ppm liver ppm X 100/1 was three times higher in Prestt et al. (16), using the Bengalese finch, Bengalese finches that died during their experi reported that kidneys were larger in birds that ment than jn birds killed at the end. Data in the died from PCBs than in controls. Flick et al present study showed a complete overlap m brain (10), using chickens, mentioned both enlarged ppm/livcr ppm X 100/1, birds that died during adrenals and kidneys from PCB treatment In testing had a range in ratios of 5 4 to 112 8, while our study, kidneys were larger in pheasants given matching birds sacrificed had 10 0 to 37 0 10 or 20 mg daily, but neither kidney nor adrenal Relatively little sampling of brain tissue for enlargement was visually detected during ne PCB residues has been done in wild birds Dust cropsy MeCune et al (23), using Aroclor 1242 man et a), (22) reported that a sick bald eagle had with chickens, mentioned both enlarged livers and 230 ppm PCBs in its brain, and that PCBs may kidneys in birds given PCBs. Platonow and have contributed to its death. Jensen et al (20) Funnell (14) and Rehfeld (15), using 1-day-old reported that three white-tailed eagles (Haluuetus chicks, reported enlarged livers with dietary olbictlla) had a bruin residue range of 26-70 ppm intake of Aroclor 1254 Grant et al (24) reported PCBs, averaging 47 ppm; muscle residues ranged enlarged livers and decreased spleen size over a from 150-240 ppm, averaging 160 ppm. Rise- period of time in the rat Flick el al. (10) and Vos brough et al (21) found 0 04, 1 5, 21, and 34 6 and Koeman (13) reported small spleens in their ppm PCB in the brains of four peregrine falcons. studies. Although hydropencardjum was found in Prtsfl ct al. (16) reported liver residues ranging varying degrees by many of the authors cited, it from 0 to around 900 ppm from a wide variety of was found only rarely in the present study Vos British birds; arithmetic averages ranged from and Koeman (13) found that Phenodor DP 6 and 0 5 ppm in the buzzard (Buito buieo) to 98 ppm in Clophen AGO caused much more liver necrosis the heron. The tissue levels reported above were and hydropericardium than Aroclor 1260, this below those at which mortality occurred in the was probably due to contaminants (25) present study, except that the mUBcle level for the w lute-tailed eagle was higher than those in PCBs in Combination with Dieldrin this study associated with death in the pheasant Mortality among pheasants of both sexes 6-9 from PCBs. However, these levels from wild months old given dieldrin, PCBs, or a combination birds do constitute substantial percentages of the of the two, varied with the level of chemical brain and liver levels in the present study where administered (Table 7) Among the 22 pheasants death occurred from PCBs. on each level, 3 died with 4 mg dieldrin per capsule, eir and 6 died with 8 mg dieldrin. The Bame propor an Histopathologic Effects tions held true for PCBs, since 4 died with 50 mg 00 PCBs per capsule, and 9 died with 100 mg PCBs ;ht Dohlgren et al. (3) found that PCBs decreased When 50 mg PCBs and 4 mg dieldrin were :h weights of heart and spleen at all treatment levels administered together, a total of 9 birds died. ho (P<0.01). PCB treatment increased weights of None of 11 control birds died during this period ir- kidney and liver in birds given 10- and 20-mg of time. These data suggest that effects of PCBs d, doses (P<0 01), but no effect was seen in the and dieldnn together are additive, not synergistic 5) 210-mg group. Starved birds had smaller hearts Heath et al (8) found that the joint toxicity of to (P<0 0.r>) and livers (P<0.01) than controls Aroclor 1254 and DDE given to Japanese quail of Splenic atrophy, as described by Flick et al. was additive, and found no evidence of synergism I* (10) and Vos and Koeman (13) was found in all in their joint effect April 1972 HONS 208198 99 Table 7. Mortality occurring among SI pheatanu of both Mm that were fr-9 months of age when PCBe nil dhlilrin were administered separately and in combination. No mortality ociurred among 11 control birds. Group treatment No of death* m week 1 234 5 Total death* 50 mg PCD* 100 mg PCBs 4 mg Dieldnn 1 00 1 2 0 3 0 4 2* 3 00 00 8 mg Dieldnn 300 1 2 50 m* PCBs and 4 mg Dieldnn 1 2204 4 ft 3 e There two birds were near death at the end of the experiment Residue* in Wild Bird# A pooled sample of six pheasant livers taken from wild South Dakota pheasants had <0-1 ppm PCBs A pooled sample of three livers collected near Washington, Pennsylvania, had <0 1 ppm PCBs, and a pooled sample of six livers collected southeast of Iancaster, Pennsylvania, had 2 0 ppm PCBs. A pooled sample of sis livers collected east erf Gary, Indiana, had 0 5 ppm PCBs, and a pooled sample of six livers taken from pheasants in Benton County, Indiana, had 1.5 ppm PCBs. These relatively low levels in livers of wild pheasants taken both from industrial areas and rural areas indicate these pheasants were probably exposed to small amounts of PCBs. Preett et al. (.16) found that residue levels m birds in Britain were related to their food habits. They found the most PCBs in livers of fresh-water fish-eating birds (up to about 900 ppm); bird-feeding raptors had up to 70 ppm; birds which eat mammals had up to 50 ppm, birds with a mixed diet of mammals, birds, and carrion had up to 15 ppm; and those which eat insects had 0-1 ppm. Swwmtt ttd Caicliutau PCBs given to laying pheasant hens adversely affected egg production, hatchability, and viability of the embryo about the time of hatching but did not affect fertility or eggshell thickness. The chief effects on reproduction occurred through the PCBs taken in by the hen, not by the cock. A subtle effect on behavior was indicated ^ studies on the visual cliff and of the abibn d offspring to avoid hand capture These behviWli effect* have been reported for birds with org*^ chlorine insecticides and could be deleterious to , wild species, in that instinctive patterns nerea.nr, for survival are involved Behavioral differences may be affected through PCB administration to the cock as well as through the hen, as shown b; the study of hand catching, this implies that the effect is, though unexplained as to mechanism more than through the physical presence of the PCBs in the egg-yolk lipids The presence of PCBs in egg lipids is probably responsible for the observed effect of increased mortality m young during the first 6 weeks of life, and in a possible depression of weight at 6 weeks of age Only early survival erf chicks was affected, as no effect wu noted in survival from 6 weeks of age through the fall Sex ratios of young pheasants sun, iving to the fall showed an expected 50;50 distribution, thus PCBe did not affect the survival of one sex more than another. Large doses of PCBs, 210 mg daily, effected a loss of appetite, but lesser doses tested did not affect feed consumption until just prior to death, The death of birds given PCBs was not attri butable to the starvation that occurred in the few days prior to death, because birds dying from PCBs did not loee aa much weight as birds which were not given PCBs but were starved Further starved birds did not show the histopathologic effects observed in PCB-treated birds such u degeneration of liver cord cells and depletion of lymphatic nodules in the spleen. PCBs were (1) rapidly turd readily absorbed into the pheasant's body, (2) stored in the lipid fraction, and (3) excreted slowly m feces and eggs. Excretion via the egg may be an important means of ridding the body of PCBs for the hen, but the PCBs in the egg yolk lipid may be dangerous for the offspring because of altered behavior and lowered survival both erf the embryo and hatched young. Brain tissue may be s valuable indicator of PCB toxicosis, level of 300-400 ppm. or more were associated with death due to PCBs Marked splenic atrophy was the most consistent char acteristic noted among several organ parameters checked in birds that died from PCBs Enlarged 190 Environmental Health Perspective* HONS 20*199 bdofyt and livers were also useful characters in ^tempting to diagnose PCB toxicosis. PCB and dieldrin were not, when combined, nsepstic m their joint toxicity to pheasants. liven from wild pheasants collected in Penn^Ivama, Indiana, and South Dakota, did not sowd 2 ppm PCBs, indicating relatively low- contamination. REFERENCES , jeuen.S I960 Report of a new chemical haxard. New Scientist J2 612 1 Dahlgren, R B , Greichus, Y. A and Linder, R L. 1071 Wioragc sad excretion of polychlorinated bi phenyls in the pheasant. J Wildlife Manan 35 823 1 Dahigrrn, R B et al 1972 Residue levels and hislopathology in pheasants given polychlorinated bipbrnrlr J Wildlife Manag 36 (In press) l Tallanro, R B and Farrell. W M 1964 Studies of visual ilrpth perception an effect of early experience on chicks on a visual cliff. J. Comp. Physiol. Psychol 57 94 5 Dahigrrn, K B. and Linder, R. L 1971 Effects of pollrhlnrinaled biphenyls on pheaaant reproduction, hehavmr, and survival J. Wildlife Manag 35' 315. 4 McLaughlin. J. et al 1963. The injection of chemicals into the yolk sac fertile eggs prior to incubation aa a loxicitv teat. Toxicol Appl Pharmacol & 760 ? Dahlgren, R B and Linder, R. L. 1070. Eggshell thickness in pheasants given dieldrin J. Wildlife Manag 34 220 l Hastli R G et al 1970 Effecta of polychlorinated biphenyls on birds Proceedings XV International OtnitlHiIngieal Congress, The Hague--1970 t Scott, M. et si. 1971 Results of experiments on the effects of PCBs on laying ban performance. Proceed ings 1971 Cornell Nutrition Conference for Feed Manufacturers, Cornell University, Ithaca, N Y. 56. 10 Flick. D F, O'Dell, R. G. and Childs, V. A 1965. Studies of the chick edema diesass. 3. Similarity of symptoms produced by feeding chlorinated biphenyl. Poult Sc. 44 1460 11 Baxter. W. L, Linder, R. L. and Dahlgren, R B. 1969 Dtekirin effecta tn two gensrations of panned hen pheasants J. Wildlife Manag. 33: 96. 12 Dahlgren. R B , Linder. R L and Ortman, K K 1970 Dieldnn effects on susceptibility of penned pheaaants to hand capture J Wildlife Manag 34 957 13. Vos, J G and Koeman, J H 1970 Comparative toxicologic study with polychlorinated biphenvls m chickens with special reference to porphyria, edema formation, liver necrosis, and tissue residues Toxicol Appl Pharmacol 17 656 14 Platonow, N S and Fimnell. H S 1971 Anti-androgenio-hke effect of polychlorinated biphenyls in cockerels. Vet, Ree 88 109 15 Rehfeld, B M 1971 The effect of snalathion, poly chlorinated biphenyls, and iron on growing chicks Dissertation Abstracts International 1971 31- 73W7-B (Abstr) 16 Preatt, I . Jeffries, D J and Moore, N W 1970 Polychlorinated biphenyls m wild birds in Britain and Lheir avian toxicity, Environ Potlut t 3 17 Tucker, R K . and Crabtree, D G. 1970 Handbook uf Toxicity of Pesticides to Wildlife U S. Department of Intenor, Bureau of Sport Fisheries and Wildlife, Resources Publication No 84. 18. Peakall, D, B. 1971 Effect of polychlorinated biphenyla (PCBs) on the eggshells of ring doves Bull Environ Contamin. Toxicol 61 100. 19. Anderson, D. et al 1969. Significance of chlorinated hydrocarbon residues to breeding pelicans and cor morants. Can Field-Naturalist 83' 91 20. Jensen. S. 1969 DDT and PCB in marine animals from Swedish waters Nature 224 247 21. Rnebrough, R. W. et al. 1968 Polychlorinated bi phenyls in the global ecosystem. Nature 220 1098 22. Dustman, E H. et al. 1071. The occurrence and sig nificance of polychlorinated biphenyls in the environ ment Trans North American Wildlife and Natural Rasourees Conf 36 118 23. McCune, E L , Savage, J. E. and O'Dell, B L 1662 Hydropenoardium and ascites in chicks fed a chlori nated hydrocarbon Poult Sci. 41 295 24. Grant, D L,, Phillips, W E J and Villeoeuve, D C 1971. Metabolism of polychlorinated biphenvl (Arc elor 1254) mixture in the ret Bull Environ Contamin Toxicol. 6: 102. 25. Voe, J. G. et al. 1970. Identification and toxicological evaluation of chlorinated dibentofuran and chlorinated naphthalene in two commercial polychlorinated bi phenyls FoodCoamet Toxicol. 8'625 April 1972 MONS 208200 101 Environmental Health Penpoctieet Embryonic Mortality and Chromosomal Alterations Caused by Aroclor 1254 in Ring Doves by David B. Peakail, Ph.D., Jeffery L Lincer, M.S.* and Stephen . Bloom, Ph.D.f The breeding success of Ring Doves {Streploprha nsona) fed Aroclor 1254 at 10 ppm was determined over two generations. Six pairs of birds were fed PCBs for three months, then allowed to incubate their clutch of two eggs Eggs ere then collected again for six months, at which time the birds were allowed to incubate snot her clutch Results are given in Table 1 The So* -hatching success of the second generation mis due to heavy embryonic mortality. The age of emhryomc death varied considerably but was mainly in the range of 3-8 days. This finding is at variance with that of Scott et at. (1), who found a significant decrease in hatchability of chicken eggs in the first generation with 10 ppm Aroclor 1248 for eight weeks and almost complete failure at both four and eight weeks at 20 ppm Aroclor 1248. In this case mortality occurred immediately before hatching. Eggshell thinning was not observed in either the first or second generation (2, 3). This is in agreement with the findings of Dahlgren and Linder (4) for the pheasant (Phaitamu coichiau) and Heath et aL (5) for the Mallard (Ana* plalyrhyndwt). Cytogenetic studies were performed on 24 dove embryos at 3-6 days of incubation. Six embryos were from dove pairs not fed PCBs (control), 17 embryos were from PCB-fed (10 ppm in diet) psirs and one embryo was irradiated with 155 ` Section of Ecology and Systematica, Cornell University t Department of Poultry Science, Cornell University, Tthere, New York 14850 X-rays (positive control). Relative frequencies of chromosome aberrations (aneuploidy, polyploidy, breakage, rearrangements) were recorded for the largest 8 chromosome pairs occurring in metaphase cells of allantoic sac and limb bud origin An average of 365 metaphases were examined per embryo. In control and PCB-treated groups primarily chromatid, some lsochromatid and 1 rearrangement was observed Mean aberration rates were as follows: control *0 8% (range = 0-2 0%); PCB-treated -1.8% (range -0-9 4%), positive control--18 0% The chromosome re arrangement occurred in a PCB-treated embryo 13 of 17 PCB embryos had aberration rates exceeding the mean control rate, and 4 PCB embryos exceeded the highest control rate with frequencies of 2 4%, 2.6%, 3.1% and 9 4% These TsUs 1- Breeding success of Ring Doves fed 14 ppm PCBs ever two generations. No of pun No of No of No of eggs eggs young laid batched fledged Pre-experiment let generation PCBs 2nd generation PCBs 2nd generation Control 6 0 6 0 24 22 22 24 24 24 20* 4 2** 24 22 22 * One pair failed to produce any eggs during the period of the experiment ** Fledging looses consisted of one young found outside the nest at one day of age and one deformed birrl killed at the age of three weeks April 1972 MOMS 20820t 103 Pre-etna* (n -- 4} Post-stress ("5) Muscle 8 12 3 (4 1-9 9) J72 9*38 3 (120 (1-227 0) Brain S 5*1 6 (4 8-8 0) 293 0*27 6 (254 0-340 2) Liver 15 3*12 6 (3 6-27 5) 1118*267 (937 3-1688 0) Fst 730 1 *211 r. <481 2-1069 41 ** Figure* are means, standard deviatioae, and range *** No fat present remits are indicative of a possible clastogenic (chromosome breaking) action of PCBs in dove embryos. Further studies are warranted to define more precisely the conditions under which PCBe might be mutagenic, clastogenic and possibly teratogenic to avian and mammalian embryos Residue analysis on eggs laid at various times from the start of the experiment showed that the levels increased for three months when an approxi mately steady level of 50 ppm (dry weight) was reached (6). The second generation eggs had similar levels; thus increased embryonic mortality was not caused by increased levels of PCBe. Tbe most striking finding of the seabird "wreck'' in the Irish Sea in 1069 was the elevated concen tration of PCBs in the liver (7). In birds shot as controls the concentration was only 0 4 ppm (wet weight), whereas birds found dead had 40 ppm m the liver In an attempt to evaluate these findings, we subjected to stress five first generation birds after they had been exposed to Aroclor 1254 in their diet for six months. The stress was applied by reducing the food intake severely so that the birds lost approximately 10% body weight per week. Control birds were also stressed in a similar manner No significant difference was found in either the time to death nor in the weight loss to death between controls and experimental groups. The organ levels of the first generation birds sacrificed at tbe end of the breeding experi ment can be compared to organ levels after starva tion (Table 2). A massive increase of levels as a result of mobilisation of fat is clearly seen in all tissues studied The fact that liver levels ranged from 937-1688 ppm at the time of den(h strong!;, suggests that the 40 ppm recorded in the hvers ol seabirds in the Irish Sea was not responsible for the death of the seabirds Increased occurrence of abnormal j oiing in two species of tern has been reported front Long Island (8). In most cases, the levels of PCBs found in the muscle tissue of these abnormal birds were higher than those found in adult Rmg Doves during our experiments. It possible that the chromosomal aberrations noted in our study could be the cause of these abormaUties REFERENCES 1 Scott, M L ft tl 1971 Result* of experiments on the effects of PCDs on laying hen performance Proe 1971 Cornell Nutrition Conf . November 56 2 Peakall, D B 1971 Effect of polychlorinated lupheml* (PCBs) on the eggshells cf Ring Doves Bull Environ Contsmm Toxicol. 6 100 3 Peakall, D B Unpublished data 4. Dahlgren, R B and Linder R L 1671 Effects of polychlorinated biphenyls on pheasant reproduction behavior, and survival J Wildlife Manag 35 315 5 Heath, R. 0. et al (In press) Effects of polychlormsted biphenyls on birds. XV Cong, of lot OrmOiol Den Haag. September 1970 6. Lmrer, J L and Peaks!]. O B Unpublished dnta 7 Holdgate, M W (ed ) 1970 The seabird wreck nl 1969 in the Irish Sea (Unpublished report with supplements) Natural Environmental Research Council, Great Britain 8 Hays, H and Riaebrough, R W 1972 Pollutum concentrations m abnormal young terns, from Long Island Sound Auk 89 19 104 Environmental Health Perspective* t HONS 208202 i rongly renj of >le for n (wo [atami in thr lighi-r g OUr nmal cause W ttlr . 1#71 wnyl* iron ill of :Uon. mini Den 19flil mt) Ire* l lint Long I i Environmental Health Pertpaetivn Toxicology of PCBs for Mammals and for Birds byJ.G.Vos* Introtaction Ip the early days of its use, little work was done on the toxicology of the PCBs, and this was only m relation to the risks of occupational exposure As will be shown many more studies were made is soon as it appeared that the extremely stable PCB'e became a threat to the environment and its wildlife, and accidents occurred of acute poisoning in man and animals. These studies have been made with material with different contents of chlorine, from different manufacture, and--as we can now say in retrospect--with different and unknown contents of toxic im purities. For this reason the toxicological infor mation of PCB's is difficult to summarize, but since the character of some important impurities has recently been elucidated, it is well to discuss these first in order to be able to consider their contribution to the overall toxicity of the dif ferent preparations studied These studies wen started because of the analogy between the effects of the PCBs (1-6) and some toxic effects associated with toxic factors in crude chlorophenols and in "toxic fat". Effects of the latter am liver damage (7), chloracne (7,8) sad edema formation (0). toxicity of three commercial PCB samples (con taining 60% chlorine on the average). Phenoclor DP6 (sample I), Clophen A60 (sample II) and Aroclor 1260 (sample III) These three mixtures showed a marked resemblance in their gas chroma tograms and mass spectra (10) In this compara tive feeding test m une-day-old chicks (11), it was found that 100% mortality, subcutaneous and abdominal edema, and centrolobular liver necrosis occured in only two groups (fed the samples I and II). Hydropericardium (Fig. 1), a common effect of these two mixtures, was only occa sionally seen in the chicks fed the sample III The Chimlcil. TnlMlMfcil art PitWtficN IdtRtlflcatiM Mi fvakatteR of Toxic Inpiritiot io Technical PCB PropwitiHS The first indication of the presence of toxic impurities was obtained in a comparison of the * Institute of Veterinary Pathology and Inititute of Veterinary Pharmacology and Toxicology, State Uni versity of Utrecht, The Netherlands Fionas 1. Hydropericardium m a chick fed 400 ppm of aomple 1. April 1972 HONS 208203 105 Table 1. Mortality, and Pathologic Observations of Chicka Fad 400 ppm PCB for to Day*. PCB sample I II 111 Control Number of Number of birds with edetnt. Kuintjer of N deaths bird* ith Hydro* Abdominal Subcutaneous liver neerosu pentfirdivstn 24 24 IS ft 5 0 22 22 20 9 7 9 20 3 3 0 0 0 20 0 0 0 0 0 mortality in this group was only 15% (Table 1) The excretion of coproporphyrin and protopor phyrin in the feces was increased Examination of tissues under Wood's light showed the presence of red fluorescence indicating porphyrins in the liver and other tissues of especially the birds that died. This hepatic porphyria was found in all three experimental groups. In the subsequent study (12) by means of column and gas-chromatography the presence of relatively more polar compounds was demon strated in the 25% diethyletter fraction of samples I and II. In a chick embryo assay (Table 2), the difference in toxicity between the three samples was confirmed; the high toxicity of the 25% diethylether fraction of sample II is demon strated by the similarity between the mortality levels in the group injected with 3 5 sample II/ egg and the group injected with the 25% diethyletheT fraction from 3.5 mg sample 11/egg. Mass spectiometric analysis revealed that Table I. Chick-Embryo Assay of Throe PCB samples and tbs tf% Diethylether Fraction from Sample II PCB sample Dose (**/) Number af eggs treated Percentage hatch of fertile eggs I 11 III Friction from taJBpto 11 Ethanol control Untreated control 35 3.5 35 35 36 a 35 0 035 0 -- 15 20 15 15 15 15 15 20 20 0 5 80 0 7 92 100 94 90 identical chlorinated compounds were present in the 25% diethylether fraction from samples 1 and II but not in that from sample III They included compounds with mass number 304 and 338. The proposed identity of these compounds, tetrachlorodibensofuran and pcntachlorodibeniofuran, is indicated by the following chemi cal-analytical, pathological, and toxicological data From exact mass measuremen s it was found that the formulae of these peaks were CijH<0"C1i and CttHtC^CU The formulae of the fragment ions 24] and 275 were CnH(wClj and CnHi*Cl, From this data it can be concluded that the parent ion has a preferential fragmentation for the loss of a single CICO unit Mass spectra were compared with the spectrum of a chick edema factor 1,2,3,7,8,9-hexachlorodibenxo-p-dioxm The mass spectrum of this compound has a similar fragmentation pattern, i.c. the loss of two suc cessive mass units of 63 suggests a loss of two CICO units. With microcoiilometnc analysis a certain maximum level could be indicated. This maximum level was found to be five ppm of the compound with mass number 338 in sample 11 and 20 ppm in sample I. Polychlorinated dibenzofurans are strong hepatotoxic and acnegenic compounds Tn- and tetrochlorodibenzofurans in a single oral dose of 0 5 ] 0 mg/kg caused severe and often lethal liver necrosis in rabbits (7,13), and application to the ear resulted in chloracne. Tetrachlorodibcnzodioxin was about 10 times more toxic (7) In jection of the 25% diethylether fraction obtained from 35 mg sample II into the aircell resulted in 100% mortality (Table 2) It can be calculated that the maximum dose/egg is 0 2 ag pentachlorodibenzofuian (taking five ppm as the ma\i- 106 Environmental Health Perspectives HONS 208204 mum value). This confirms the order of toxicity toxicity of 2,4,5,2',4',5'-hexachlorobiphenyl found by Higginbotham et &1 (9) in the case of From the increased fecal excretion of copropor chloro-dibenxodioxms Tetra and pentachlori- phyrin in both experimental groups (Table 3), it dibcnzofurans were considered responsible for is very likely that PCBs themselves are responsible the higher toxicity of samples 1 and II for the porphyrogenic action of crude prepara Confirmation is also obtained from the sub tions. From the presence of slight skin lesions sequent comparative toxicity study in rabbits induced by 2,4,5,2' ,4',5-hexachlorobiphenyl (14) Again samples I and II were more toxic; the when compared with the Aroclor sample, it can liver and skin lesions were more severe Por be concluded that the major acnegenic action of phyria, especially of the liver, was present in all crude mixtures comes from a possible contamina three groups (Fig 2) A remarkable finding was tion with chlorinated dibensofurans It can also the intense red fluorescence of small foci inside be concluded that PCBs, themselves have a it hepatic cells They probably represent nuclei. This slight acnegemc action. Liver damage was es I was confirmed in an additional cell culture experi sentially the same after treatment with both y ment with the Aroclor sample and with 2,4,5,- 2,4,5,2',4',5-hexachlorobiphenyl and the d 2',4',5'-hexachlorobiphenyl (This experiment was Aroclor mixture The conclusion that the liver carried out by my colleague Dr J. O Wit of the injury, caused by crude preparations, is pre Biochemical Section). dominantly due to the contaminants, is based Application of the 25% dicthylether fraction on the differences in liver toxicity between the J on the akin of rabbits resulted also in differences three PCB preparations (11,14) in toxicity (Fig. 3). So the presence of the hep&to- The probable contribution of polychlorinated i toxic and acnegemc polychlorinated dibenzofurans dibenzofuran (PCF) and pure polychlorinated as impurities in samples 1 and II was found to biphenyl (PCBs) in the toxicity of crude prepara t be established. tions is summarized in Table 4 A proper evalua In another experiment (15), the toxicity of the tion of toxicity data and residue data can be Aroclor (60% Cl) sample was compared with the hindered by the possibility that PCB samples may I ' - Fiecac 2, Fluorescence of porphyrins under ultraviolet light in livers from rabbits treated 'with 60% chlorinated PCB s, 1, Aroclor, 2, Clophen, 3, Pheaoclor, and 4, Control liver (14). April 1972 HONS 208205 107 Fiouits 3. Response of lh made of lh rabbit's tr titer topical application of the 23% diethylsther fruitions from tech nical PCB's Hoemotoxylin and eoaui. X90. (a) Skin of control animal treated with ethanol Note the hair follicle at 1, eeabaceous gland tissue at 2, and cartilage at 3. (b). Ear skin of the animal treated with the fraction from sample III Some hyperplem* and hyperkeratosis of the follicular epithelium can be seen <e) Ear skin of the rabbit tree fed with the fraction from sample II Considerable hyperplasia and hyperkeratosis of the follicular epithelium (d) Ear skin of the rabbit treated with the fraction from sample I Part of a section that shows the most severe lesion The gravity of the response was in general the same as seen in (c) Note the cystic dilated hair follicle with prominent hyperplama and hyperkeratosis of ths follicular and epidermal epithelium (14) 108 Environment*! Health Perspectives HONS 208206 differ in on important respect: the presence of loxic impurities The possibility of distinguishing between the effects of PCBs and their impurities can be further improved by using pure isomers with known positions of the chlonne atoms. Mortality, Uvar Elficts, Eferni Formation and Othor Edicts These data, as presented by different authors, are summarised in Tables 5, 6, and 7. As can be seen in Table 5, the acute and subacute toxicity data of PCB's are poor The established values are high. Semichronic oral toxicity studies are ummanxed in Table 6 Dermal and inhalation studies are given in Table 7 TabteS. Coproporpbyrln Content! (f/| Dry Weight) of Feces of Rabbits Treated with FOB for 4 weefca, and of Centroli.* 2,4, ii 12', 4', S'-He*iwhtorobipheflyi Aroclor Control (60% Cl) 45 0 e2 20 8 36 0 Mean 27 0* 24 1 50 26 4 16 0 1ft 8` 45 37 36 35 38 * Figures are the contents of feces, collected from the cecum of the individual animate. 1 Significantly different from controls, P <0 025 It is very probable that the results of these Btudiea may have been influenced by the presenoo of polychlorodibenzofurans or other toxic im purities. For example, in the study of Rehfeld et al. (23) general edema was already found in chicks fed 30 ppm Aroclor 48% Cl, while Kohanawa and co-workers (22) noted edema formation at the 100 ppm level of another 48% chlorinated mixture. Mortality in the latter study was also lower (Table 6). Liver Effect* The most important liver effects, summarised in Tables 5, 6, and 7, are weight increase, fatty degeneration, hyalin degeneration and necrosis Increased liver weights, as noted in several studies, Table 4. Probable Contribution of Polychlorinated Dlbensofuran (PCF) and Pure Polychlorinated Biphenyl (PCS) in the Toxicity ef Crude PC8 Mlrture*. Chtor* tone Edema Liver Hepatic forma. damage porphyria tion Polychlorinated Dibeaiofuran Polychlorinated Biphenyl ++ ++ ++ ++ ++ Table 5. Acute and Subacute Oral Toxicity StudJea ef PCB Preparation!. Preparation Animal Treatment Mortality Liver effect! Reference! Unknown Aroclor 54% Cl Mouee Rat Aroclor 42, 54, 60, and 68% Cl 42% Cl Mallard Rat 42% Cl Guinea pig 65% Cl Rat nngle doee of LD60 approx 3000 mg/kg ingle done of 0% Increase of weight end 500 mg/kg lipid; potentiation of CCU toxicity single doee of 0% 2000 mg/kg 20 daily doee* of 0% in 3 Hyalin bodiee in liver ceUa 138 mg monthi 2 doees of 69 mg 100% between Fatty metamorphoau, 1 week apart 11 end 29 central atrophy days 6 daily doeee of 70% in 14 Iocreaee of weight; cell 300 mg day! veiling- hyalin granulea (16) (17) (18) (10) (10) (20) April 1972 MOWS ^208207 109 Table 4. Semtchronlc Oral Toxicity Studies of PCB Preparation!. Prep*ration Animal Tresunent Mortality Liver effect* Other t Refer emr-s 65%, Cl Rat 48% ci 48% Cl Cynoraolgus monkey Squirrel monkey 48% Cl Mouse Aroclor Chicken 42% C! Amlor Chicken 42% Ct 48' {, Cl Chicken Aroclor Chicken 48% Cl Aroclor Chicken 34% a Aroclor Bengalese S4% Cl finch Phcondor J&PUI696 ao% ci quail Doses of 50 mt every atcunri day From 641 mg uj 40 days to 348 mg in 236 dan From 320 mt m 40 day* to 67 mg in 48 dsyt 80% in 5 weeks not given not given Duly dove* of 0 001 ml for 13 to 26 wsakt 0% 100, 200. 400, and and 1000 ppm in diet for A week* 0, 0, 50, 90, end 00% respectively 200 and 400 ppm m diet for 3 weeks 0 and 12% respectively l, 5, 10, 25, 50, 100. 300, 800, 1200, 2400, and 4800 ppm in diet for 20 days 10, 20, 30, 50, 100, cpnd 150 ppm tn diet for 4 5-5 week* 0% from 1 to 100 ppm; 100% from the 100 ppm level 3Alter weeks: 0, 0, 30,90 snd 20%; at the end 0, 0, 80, 80, and 80% respectively 250 and 150 ppm in diet for 8 to 13 weeks Estimated doee rate at 50 days of 254 mg/kg/ day 2000 ppm ut diet 250 ppm 100% hetween 3 and 10 weeks; 500 ppm some mortality at tbs end 50% 100% between 6 and 55 days 3J% weight increase, cell swelling, hyalin globules Enlargement, SER proliferation Main ciiifte of <JeMh pneumonia w Enlargement, SER diarrhea Main cause of death proliferation pneumonia or SER proliferation diarrhea, palpe bral edema in Enlargement, SER proliferation HER reduction, 1 animal Skin loss of hair, erosion and ulceration after myelin figures; increase of micro 3 months bodies, lysosomee and lipid Enlargement; damage at the Edema formation from 200 ppm; at higher levels high levels internal haemorrhage and tubular dilatation in kidneys Pronounced edema tt 400 ppm, en- larged kidneys, small spleen, defeathenng and dermatitis Edema formation 100 ppm level Enlargement General edema and depression of the secondary sexual characteristics from the 30 ppm level 500 ppm at end comb weights 20(old and testes weights 2-fold lower than controls Hydropericardium in some birds Hydropericardium (21: (211 (5) (5) (22) (23) (24) (25) (10) 110 Environments! Health Pertpeclivn HONS 208206 Table 7. Dermal Toiiclty and Inhalation Studies of PCB Preparation*. Prrpsrit ion Animal Treatment Mortality Liver effect* Skin effects Refer ence* o'"; ci Guinea pig 42(7 Cl tUbbit Aroclor Rabbit \rorior Rat w;0 Cl 11 daily akin application* of .74 5 mg 100% between 11 and 21 daye Sit in application at alternate day*, total doae from 946 to 1980 mg Dally akin appli cation* of 0 3, 0 6, and 0.9 g 100% between 17 and 98 daye High doee died be fore liver Decreel* developed Inhalation of 0 57 mg/cubic meter for 16 hour* for 37 to 134 daye 0% Fat, central atro phy, perinuclear basophilic granu lation, focal necrosis in a few animals Fatty degeneration; central atrophy Moderate doses' mottled liver, aubacute yellow atrophy, fatty degeneration, and marked necrosis Pale and yellow, cell swelling, hyalin degenera tion; potentiation of CCL and CiHiOH toxicity Occasional thicken ing of the epidermis Thinning of pnckle cell layer and thickening of outer eonufied layers Reddening, forma tion uf small papules and bisters, finally deequamatir.n of external epidermal layers (19) (10) (26) (20) arc well explained by the proliferation of smooth surfaced membranes of the endoplasmic reticu lum (SER) as was found by Nishizumi (21) in mice and monkeys and by Norback and Allen (27) m rats The latter workers found a prolifera tion of the SER in rats fed PCB for 1 to 5 weeks. Concomitant with the structural changes, the activities of measured drug metabolising enzymes (nitroreductase and aromatic hydroxylase) were increased The induced level of drug metabolising activity persisted ns the proliferation of the SER decreased and concentric arrays pervaded the cytoplasmic reticulum (27). These concentric membrane arrays, probably representing the hyalin bodies described by Bennet et al (20) and Miller (10), could have an enzymatic func tion similar to that associated with the SER (27) Similar formations, the so-called myelin fig ures, were demonstrated m mouse liver by electron microscopy; in monkey liver they were not found (21). In both mouse and monkey liver a proliferation of the SER was found. In our comparative donnal toxicity study (15) in rabbits with 2,4,5,2',4',5'-hexachlorobiphenyl and Arodor (60% Cl), the light microscopic findings included necrosis, hydropic degeneration (Figs. 4 and 5) as well as a peripheral and perinuclear shift of cell organelles (Fig. 5) and focal cyto plasmic hyalinUation. In electron microscopy, the shift was found to be due to a proliferation of the SER resulting in a displacement of rough surfaced membranes (RER) and mitochondria. The focal cytoplasmic hyalin degeneration, often seen in hydropic cells, was recognized as tightly packed tubules of proliferated SER (Fig 6) This very probably represents hypertrophic, hypoactive SER, Subletbal effects caused by induction of hepatic enzymes have been noted by several authors Increased steroid metabolism in pigeon liver homogenates has been demonstrated by Riaebrough et al. (28) Liucer and Peakalt (29) confirmed the effect of PCB on the hormone metabolism in birds at very low dose levels They fed kestrels for 5 months with Arodor 54% and 62% Cl at levels of 0.5 and 5.0 ppm. The higher April 1972 HONS 204209 111 Fiona . Liver damsge in killed rsbbit treated with 120 mg Aroclor (00% Cl), 5 times per week, for 28 devNote the oeotrolobular necroai* (1) and the hydropic celle (2) at the margin of the necrotic and vital tissue Haemoionyiin ami mu. X160. Fionas 6(a) Liver cells of a control rabbit Clear areas may represent negative images of glycogen (arrow) Tolmdme blue, X049 (b) Liver cella of an Aroclor treated killed rabbit Note the hydropic cells (1) and the perinuclear ant! peripheral displacement of cell organelles with sometimes hyalin foci (2) inside hepatic cells Toluidine blue y <no 112 Environmental Health Perspectives HONS 208210 Fwrwt A H\<iropic liver cells from the tune animal u seen in Figure 5b showing s luge number of vacuole* (V) end t penvKruottr localization of mitochondria (arrow), Note the strong proliferation of the SER, consisting of tightly packed tubules Ursnyl sestet* sod lend citrate. X5600. dote being roughly equivalent to 2 mg/kg PCBs lo each kestrel. A dose dependent in vitro break ' down of estradiol to a more polar metabolite occurred in the hvera from kestrels fed either Aroctor 1251 or Aroclor 1262. No such conversion 1 took place in the livers of the control birds The ' increase in hepatic enryme activity correlated with an increase in cytoplasmic RNA, os was measured cytophotometrically. A shortened sleep ing time after treatment with hexobarbitol. and 1 enhanced in vitro rates of aniline hydroxylotion and p-nilroaniBolc demethylations were demon strated by Street and coworkers (30). These f authors also found on increase of these effects j with increasing chlonne content of the different 4 PCB preparations (Aroclor 21 to 68% Cl). Using enzyme induction os parameter, no effect levels of some PCB preparations were established in the rabbit, rat, and Japanese quail. Oral administration of Aroclor 21% CL and 54% Cl (1.0 and 10 mg/kg) for 28 days to pregnant rabbits resulted m liver enlargement and in creased activities of the drug metabolising en zymes aniline hydroxylase and aminopynne n-demethylase at the 10 mg/kg level of the 54% chlorinated Aroclor. The no-effect level for enzyme induction in the pregnant rabbit appeared to lie between 1.0 and 10 mg/kg in the case of Aroclor 54% Ct, and higher than 10 mg/kg for Aroclor 21% Cl (31). Another parameter for enzyme induction was used by Komatsu and Tanaka (32) They found that the hexobarbitol induced sleeping times in rats were reduced by pretreatment with PCBs April 1972 MONS 208211 113 r J The minimal effective dose was 5 mg/kg for 3 days with Kanechlor 400 (48% Cl) and 2 mg/kg can be responsible for reduced serum albumm levels for 3 days with the higher chlorinated Kanechlor 500. The porphyrogenic action of PCBs was As mentioned in Table 4, the edema formation is probably due to the presence of polychlorodi- further evaluated in a study with Japanese quail (33). The results (Table 8) indicate that the bensofurans. In our study the edema formation by the 00% chlorinated Aroclor sample was a. hepatic porphyria is closely associated with an minimal at the 400 ppm level. As can be seen increase of mitochondrial ALA synthase activity. in Table 6, chick edema-like lesions were noted A significantly increased activity of this enjyme at low feeding levels and were caused by lower was already noted after administration of daily chlorinated Aroclora. Therefore the presence of doses of 1 mg/kg Aroclor 60% Cl for 1 week. toxic impurities in these Aroclor samples has to Mean PCB content of the liver at that dose was be considered 1.41 ppm A less sensitive parameter is tissue fluorescence due to excess quantities of porphyrins Liver fluorescence was only seen at the 100 mg/kg Otlser Effect* level. It develops, probably, only in animals An interaction of PCBs with duck hepatitis showing clinical symptoms, such as loss of weight. virus was found by Friend and Trainer (35) A similar finding was done in the prior experi Ten-day-old ducklings were fed a 54% chlorinated ment with chickens (11). Aroclor mixture at levels of 25, 50 and 100 ppm. The birds suffered no apparent clinical intoxica Edema Formation tions. Five days later they were challenged with duck hepatitis virus, and they suffered signifi The most striking finding in birds is the ac cantly higher mortality than birds which were cumulation of fluid. The pathogenesis of the not exposed to PCBs. edema formation is discussed by Flick and co Effects of PCBs on the lymphoid system were workers (6) The primary site of the edema caus noted in some studies. Feeding of PCBs to chick ing factor could be the heart by increasing the ens resulted in Bmall spleens (6,11) Lymphopenia, permeability of the vascular bed, leading to atrophy of the cortex of the thymus, and a re cardiac congestion. Pulmonary edema could be duction in the number of germinal centers in the result of the cardiac congestion. The pul spleen and lymph nodes was found in rabbits monary edema might be followed by a flow of fluid into abdominal and subcutaneous air sacs. (14). Therefore, an immunosuppressive action could be present. In an experiment with guinea l Decreased serum protein values (34) could also pigs, this was established (36) Feeding of 10 contribute to the edema formation. Liver damage ppm Aroclor 60% Cl, for 8 weeks resulted in a Table I. Formation of l-Amlnoevtiltalc Acid by Uer Mitochondria, Lirer Residue*, and Tlwuc Fluwwcenw In Female Japanese Quail Orally Dated with PCB for Seven Day*. Aroclor <60% Cl) (mg/kg body weight) ALA formed (ra* mole* ALA/g hver/hr) PCB content liver (ppm) - Tueue fluorescence incidence Macroscopic Microscopic (liver) 0 01 1 10 100 6 46*1 64 8 76 4 06 10 60*1 21' 17 3*6.4* 119 * Mean valuec*SD, S bird* per group * Significantly different from control*, PS00). * Pooled sample* 0 15' 0 45*0 27 l 41*0 67 27 0*9 4 478 *2M 0/5 0/5 0/5 0/6 3/5 0/5 0/5 0/5 0/5 2/5 114 Environmental Health Perspective* MONS 208212 i Fiquhc 7 Representative treu of tetanus toxoid stimulated popliteal lymph nodes of guinea pigs (a) Large number of antibody forming relie in a control animal (b) Reduced number of antibody forming cells m an animal fed 10 ppm Aroclor (60% Cl) for 8 weeks Direct fluorescent antibody technique Cryostat sections x 375. (36) iits ***. decreased number of antibody-forming cells in the popliteal lymph node, after stimulation of the humoral lymphoid system with tetanus toxoid (Fig 7). This suppression may explain the higher sensitivity of PCB-fed ducklings for duck hepatitis virus (35) In a comparative toxicity study in guinea pigs, an indication for an effect of PCB (Clophen and Aroclor 60% Cl) on the cell-mediated immunity was obtained. Feeding of these mixtures at 50 ppm levels for 6 weeks resulted in a decreased number of circulating lymphocytes (unpublished data). An estrogenic activity of PCBs (Aroclor 21-48% Cl) was demonstrated by Bitman and Cecil (37). The estrogenic activity was evaluated using the lS-hr glycogen response of the immature rat uterus after a single subcutaneous injection. The minimum effective dose was 8 mg. The higher chlorinated PCB mixtures were inactive at die 8 mg level In the above mentioned subacute feeding study of 60% chlorinated mixtures in guinea pigs, we found significantly increased uterus weights in the PCB treated animals. Both increased steroid metabolism, as mentioned by Behfeld and coworkers (23), and the estrogenic activity could be responsible for the depression of secondary sexual characteristics (decreased development of comb and wattles) noted in cockerels (24). Administration of AtocIot (54% Cl) at levels of 12 5, 25, and 50 mg/kg body weight during the first 28 days of gestation had embryotoxic effects in the rabbit (31) Edema and beak deformities in chicken embryos have been deacribed after yolk-sac injection of 10 and 25 mg 42% chlori nated Aroclor, resulting in respectively 95 and 100% embryonic mortality (38). An effect of PCB on the nervous system was noted by Ogawa (39). Oral administration of PCB (0.3-Q.5 ml/kg/duy) to rats for 14 or 21 days, resulted in marked or moderately impaired motor function, decreased motor conduction velocity and loss of large nerve fibres. He con cluded that PCB caused neuropathy in rats. Csnduslm Because of the possible presence of poly chlorinated dibensofurans (PCF) or other toxic impurities in crude PCB preparations, it is difficult to interpret many of the toxicitv studies Pure samples are required for comparatne in vestigations Also the fate of the toxic impurUw, in the environment has to be determined As presented here, PCBs have several subleihal effects, such as microsomal enryme induction, porphyrogenic action, estrogenic acimty, and immunosuppression Since porphyria <wems to be an effect of PCBs themselves and not from PCF, the induction of ALA synthase could be used as criterion m the approximation of a no effect level (at least for the 00% chlorine type of PCBs) The no-effect level could he about 0 1 mg/kg (mean PCB content of the liver m Japa nese quail about 0.2 ppm) This is in the same order of magnitude as found m the other studies Additional research needed to determine fully the significance of these sublethal effects. More over, chronic and reproduction studies are neces sary. The present results also make clear that manufacture of commercial PCB mixtures that are free from impurities is urgently requested Acknowledgmen t The author gratefully acknowledges the helpful suggestions and critical reading of Prof H van Genderen, Head of the Institute of Veterinary Pharmacology and Toxicology Many thanks are also due to colleagues of the working party of the Institute of Veterinary Pathology and the Institute of Veterinary Pharmacology and Toxi cology: Dr. J. H. Koeman, Mr H L van der Maas, and Dr. J. G. Wit The author also thanks the students who studied for their degree in biological toxicology and Mr. M C ten Noever de Breuw, Mr. R. H. de Vos, and Dr. R J C Kleipool of the Central Institute for Food and Nutrition Research, T. N. O., Zeist. REFERENCES 1 Jones, J. W and A1Jen, H S 1936 An acneform dermatergoais Arch Derm Syphil 33 1022 2. Schwarts. L 1636. Dermatitis from synthetic mint and waxes Amer. J. Public Health 26 566 3 Meigs, J K , Album, J. J and Kartm, B L 1954 Chloracne from an unusual exposure to Aroclor. J Amer Med. Asaoc 154 1417 4 Puectnelli, V. 1954. Dell'acne clones Med Lavoro 45 131 5. McCone, E L. Savage. J E and O'Dell, B L 1962 Hydropericardium and ascites in chicks fed a chlori nated hydrocarbon. Poultry Science 41 295 116 Environmental Health Perspective* HONS 200214 i t t n,fk D F , O'Dell, R. G. end Child., V A IBM Mtidir* nf the chick edema disease 3 Similarity of pmptiini- produced by feeding chlorinated biphenyl Poult No 44 14C0 : Hiurr II Srhuli, K. H and Spiegelberg, U- 1961 neruflulic Vergiflungen bei der Heretellung von (lilorjihrnol Vrrbindungen Arch. Gewerbepathol (,ccri*4i\g IS 538 t ttchrlnihm, P 1959 Uber Gefahren beim Umgang mit Chloricrtcn Phennlen Dtach GeaundheiUw 14 014 0 Hwil'olham, G R et al 1066. Chemical and toxico- Initirnl evaluations of leolaled and synthetic chloro- dcnv ut ivee of dibenso-p-dioxm Nature 220 702 10 hoemiiii, J H , Ten Noever de Brauw, M C and Vo, H H de I960 Chlorinated biphenyls m fish, mussels, Find birds from the River Rhine and the Velherhinds coaetal area Nature 221 1126 It Vim J G and Koeman, J, H 1070. Comparative toxicologic etudy with polychlorinated biphenyl in chickens with special reference to porphyria, edema (nrmuiion, liver necroeie, and tissue residues i'oxicol. Api'l Pharmacol 17 656. 12 Vm J G et nl 1070 Identification and toxicological evaluation of chlorinated dibensofuran and chlonnntnf naphthalene in two commercial polychlonnated Uphrnvla Food Coemet Toxicol 8 625 13 Hofmann. H. T 1957 Neuere Erfahrungen mit hoch- Imiirhen CMorkolilenwasaeratolfen. Naunyn- N'lnmedebergs Arch Exp Pathol Pharmakol 232 228 14 \n< J G and Deems, R. B 1071. Dermal toxicity studies of technical polychlonnated biphenyls and frictinns thereof in rabbits. Toxicol. Appl Pharmacol. l`l 617 15 Vi is, J. (I , and Nolenboom-Him, E. Comparative toxicity studies of 2,4,5,2`l4',5'-hexachlorobiphenyt and a polychlorinated biphenyl mixture in r&bbita (Submitted for publication). 16 Tiinaka, K et al I960. Experimental subacute poison* ing liv chlorobiphenyls, particularly the influence on the serum lipida in rata Fukuolta-Ifaku-Zaasbi 60 544. 17, Grant, D. L , Phillips, W, E. J and ViHemeuve, D. C. 1971. Melalxolism of a polychlonnated biphenyl (Aroclor 1254) mixture in the rat. Bull Environ Gontamin Toxicol. 6. 102. IK Tucker, R. K. and Crabtree, D G. 1970. Handbook of Toxicity of Pesticides to Wildlife. U.S Dept, of In terior, Bureau of Sport Fisheries and Wildlife Re sources Pub. No. 84. p. 19 ID Miller. J W. 1944. Pathologic changes in animals ex posed to s commercial chlorinated diphenyl Public Health Rpts. 59. 1085 20 Bennett. G A , Dnnker, C K and Warren, M F 1938 Morphological changes in the livers of rsts re sulting from exposure to certain chlorinated hydro- carlvin* J Induet Hrg. Toxicol 20 97 21 Nishmimi, M 1970 Light and electron microscope study of chlomhiphenyl poisoning. In mouse and monkey liver Arch. Environ Health 21 620. 22 Kohanswn, M et ai 1069. Poteoiung due to an oily by-product of rice-bran similar to chick edema disease II Tetrmchlorodiphenyl ss toxic substance Nat Inst Anim Health Quart 0 220 23 Rehfeld, B M , Bradley, R L and Sunde, M L 1971. Toxicity studies on polychlonnated biphenyls in the chick I. Toxicity and symptoms Poul Sei 50 1090 24 Platonow, N S. and Funnell, H S 1971 AnU androgemc-bke effect of polvchlormated biphenyls in cock erels Vet, Ree 88 109 25. Preset. I , Jefferies, V J. and Moore, N. W 1970 Polychlonnated biphenyls in wild birds in Bntam and their svicji toxicity Environ Pollut 1 3. 26. Wedel, H von, Holla, W A and Denton. J 1943 Observations on the toxic effects resulting from expos ure to chlonnated naphthalene end chlorinated phenvls with suggestions for prevention Rubber Age 5J 410 27 Norback, D. H and Allen. J R 1970. Emvmatic and morphubgic alterations of hepatic endoplasmic reticulum induced hv a chlorinated aromatic hydro carbon Fed Froc 29 816 2& Risebrough, R W et al 1968 Polychlonnated bi phenyls in the global ecosyatem Nature 220 1098. 29. Linear, J. L and Peakall, D B 1970 Metaliolic effects of polychlonnated biphenyls in American kestrel Nature 228 783 30 Street, J C et al 1969 Comparative affects nf poivchlonnated biphenyls and organochlonnc pesticides in induction of hepatic microsomal ensyme Presented at ACS meeting September 8-12, New York 31. Villeneuve, D C et al 1971 Effects of PCB adminis tration on microeoma! ensyme activity in pregnant rabbits Bull. Environ Contamin Toxicol 6 120. 32 Komatsu. F. and Tanaka, K 1971 Shortening of hoxoberbilal sleeping time and change of serum tri glyceride level in chlorobiphenyls-intoxicated rats Fukuoks-lgaku-Zssshi, 62 35 33. Vos, J. G et al. 1971. Polychlorinated biphenyls as inducers of hepatic porphyria in Japanese quail, with special reference to I aminolevulinic acid synthetase activity, fluorescence, and residues in the liver Toxicol Appl Pharmacol 20 232 34. Flick, D. F. and O'Dell, R G 1968 Studies of the chick edema disease 6 Preventive treatment with oral diuretics, Poult So 47 821 35 Friend, M , and Trainer, D O 1970 Polychlonnated biphenyl interaction with duck hepatitis virus Sci ence 170. 1314 36 Voe, J G and Roij, Th de Immunosuppreanve activity of a polychlorinated biphenyl preparation on the humoral immune response in guinea pigs Toxicol Appl Pharmacol (In press) 37 Hitman, J and Cecil, H. C 1970 Estrogenic activitv of DDT analogs and polychlorinated biphenylB J Agnr Food Chem 18 1108 38. McLaughlin, J et al 1963 The injection of chemicals into the yolk sac of fertile eggs prior to incubation as a toxicity test Toxicol Appl Pharmacol 5 760 39 Ogawa, M 1971 Electrophvsioiogical and histological studies of experimental chlorobiphenyl poisoning Fukunkg.lgaku-Zasahi 62 74 April 1972 M0NS 208215 117 Environmental Health PmptaiM Epidemiologic Study on Yusho, a Poisoning Caused by Ingestion of Rice Oil Contaminated with a Commercial Brand of Polychlorinated Biphenyls* by Masanori Kuratsune, Takesumi Yoshimura, Junichi Matsuzaka, andAtsukoYamaguchi In October, 1968, an epidemic of a peculiar skin disease similar to chloracnc was reported in Fukuoka-Ken (Fukuoka prefecture), Japan. The epidemic waa later proved to have spread not only over Fukuoka-Ken but also over 20 other prefectures in the western part of Japan (Fig 1) it produced 1,057 patients according to the latest tabulation (August, 1971) by the Ministry of Welfare. Soon after the epidemic was announced, a study group was organised by the staff of the faculties of medicine, pharmaceutical sciences, agriculture, and engineering of the Kyushu University and by the staff of the local health departments, to clarify the cause of the epidemic and to effectuate its control (1). We participated in the study group and conducted, as members of its epidemiologic study subgroup, an extensive epidemiologic investigation following the basic methodology of epidemiology (2) Fortunately, the cause of the epidemic was soon demonstrated to be the ingestion of a brand of rice oil contami nated with a commercial brand of polychlori nated biphenyls, and the disease was called "Yusho", namely oil disease. Although our observations and experiences are confined to the Yusho patients seen in FukuoluKcn, we believe that reporting of them will help many people in the world who are deeply con cerned about the chronic deleterious effects cf PCBs Tile outline of our findings will, therefore, be presented, referring in addition to some other important observations reported by the clinical and chemical study subgroups of the Yusho study group and to those collected from other sources. Clinical Symptoms The most common initial symptoms experi- * Prepared bv Department of Public Health, Faculty of Medicine, Kyiuliu University, Fukuoka, Japan Table 1. Initial symptoms Goto el at Initial symptoms Patient* No Kwelhng of upper eyelids, increased rye dwclmrtp; Acne-form eruption, follicular arcentuibtion Edematous swelling ol limbs Langmshmeni Dislurliances in digestive canal Numbness and other neurological signs Pigmentation of skin Total 52 38 3 45 33 l 9 88 4 29 4 29 e 68 13 9 8 I3C. 100 0 enced by 136 patients with Yusho were increased eye discharge and swelling of upper eyelids (38.3%), followed by acne-form eruption and follicular accentuation (33 1%), and pigmenta tion of the skin (9 6%) (3) (Table 1) With others which appeared later, the subjective symptoms of Yusho as stated by 189 patients diagnosed up to October 31, 1968, are summarised in Table 2. Dark brown pigmentation of nails and skin, follicular accentuation, acne-form eruption, in creased eye discharge, increased sweating at palms, and feeling of weakness were the most notable symptoms (2) Descriptive Epidemiologic Studies First, 325 patients seen in Fukuoka-Ken from October, 1968, to January, 1969, were analysed in order to know their distribution characteristics. One of the most important characteristics readily noted was a distinct familiar aggregation. The 325 patients belonged to 112 families. As shown m Fig. 2 and Table 3, 99 percent of these patients were affected during 1968, while the remaining stated that they became ill in December, 1967. Fifty-five percent of the patients were concen trated in the 3 months from June to August, and ao significant difference was noted between sexes in monthly distribution (Table 3) For geographi cal distribution of the patients, crude incidence rates of Yusho were calculated for 3 large cities and for the jurisdictional areas of tbe 22 local health departments of Fukuoka-Ken. Excepting 10 health departments where no cases of Yusho were reported, the rates varied considerably from 1.0 to 58.9 per 100,000 Examination of uch geographical distribution failed to indicate am common socioeconomic or environmental factor which might be associated with the disease The 325 patients consisted of 158 males and lf>7 females, indicating that both sexes were equalK affected More than 90 percent of them wtrr younger than 50 years (Tabic 4) Age- and *ev specific incidence rates again indicated no sig. nificant sex difference but lower n.ks for both males and females in the ago group over 60 years (Table 5) Analytical Epidemiologic Studies When our study started, a commercial brand of rice oil produced by K company (abbreviated as K rice oil) m Kitakyushu-Shi (Kitakyushu city), Fukuoka-Ken, had vaguely been suspected as a possible cause of the disease, because moat patients with Yusho seemed to have used it Table 2. Percent distribution of symptom* of Yusho reported by 119 patients examined before October 11, 1968. Symptoms Male* Fctiule* (N-8'l) (N -100) Dark brown pigmentation of nails Distinctive hair follicles Increased sweating at palms Acnelike akin eruptions Red plaques on limbs Itching Pigmentation of akin Swelling of limbs Stiffened soles in feet and palms of hands Pigmented mucous membrane Increased rye discharge Hyperemia of conjunctiva Traneient visual disturbance Jaundice Swelling of upper eyelids Feeling of weakness Numbness in limbs Fever Ht&ftng difficulties Spiuun of limbti Headache Vomiting Diarrhea 83 1 64 0 SO 6 87 8 20 2 42 7 75 3 20 2 24 7 56 2 88 8 70 8 56 2 Uz 71 9 58 4 32 6 16 9 18 0 79 30 3 23 6 19 1 75 0 58 0 55 0 8! 0 16 0 52 0 72 0 41 0 29 0 47 (I 8.1 0 71 0 53 0 11 U 74 0 52 0 JO 0 19 6 10 11 80 39 0 28 0 17 0 120 Environmental Health Perspectives MOMS 20821? y from f such te nv fact or o The (J 167 qu||v were d M'\0 Sir` both 1 years brand mtcd yushu wctcd most sd it Ituiho artl. taler 100) i0 I0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ,0 io .0 0 0 0 l*re 1*1 1969 Fioru 2 Dwtnbution of patient* by aex and month when aymptuma appeared. Therefore, a thorough investigation was under taken to determine whether the patients had actually taken such special brand of oil before being affected. This was achieved by examining lot numbers appearing on the remaining con tainers of the oil used by some of the patients, hipping records of K company, and purchase and sale records at wholesale oil dealers' offices and at retail stores It was soon disclosed that all the patients had used K rice oil, either canned (16.5 kg) or bottled (1 65 kg) Furthermore, an astonishing fact became evident. No matter where they lived, 166 of 170 patients who used only the canned K nee oil had used a very specific oil, produced or shipped by the company on February 5 and 6, 1968 (Table 6). For those who used bottled K nee oil, date of production or shipment could not be confirmed, because they had no old bottles at home. Examination of all available records concerning shipping, sale, and purchase at K company, wholesale dealers and retail stores, however, clearly indicated a pos sibility that 143 of 155 patients who used bottled K rice oil only had used a very specific nee oil produced or shipped from February 5 to 15, 1968. This was because Buch oil had been shipped to and had reached the retail stores from which they had purchased their oil (Table 6). Thus, nearly all of the patients had had a very peculiar common experience of use or possible ure of a rice oil produced or shipped by one company in a specific period of time An attack rate as high as 63.9 percent was noted for those who con sumed the specific canned oil. In an additional Burvey, we examined whether those who regularly used K rice oil, but did not use the specific K rice oil produced or shipped Table t. Distribution of patient* with yuabo dlafnoaed by January SO, 1969, by *ei and mouth when symptom* appeared. Month Male* Number Percent Female* Number Percent Total Number Percent Before 1969 1968 January February March Apnl May June July AutUKt September October November December 2 13 0 00 4 25 8 51 15 9 5 18 11 4 23 14 8 27 17 1 34 21 5 17 10 8 8 51 1 06 1 06 2 12 0 00 6 36 12 7 2 10 6 0 15 9 0 33 19 8 32 19 2 30 18 0 12 7 2 14 8 4 1 06 0 00 4 l2 0 00 10 3 1 20 6 2 25 7 7 33 10 2 56 17 2 59 18 1 64 19 7 29 8 9 22 6 8 2 06 1 03 Total 158 100 0 167 100 0 325 100 0 April 1972 MONS 208218 121 Thl* 4, DialrUnitlon of 325 pattern* with yutho dlagnaaed by January JO. 10W. b> h> .ad t|, A*e croup (years) t>-9 10-19 20-29 30-39 40-49 60-59 60-69 70-79 Total Males Number Percent 37 23 4 38 24 1 28 17 7 30 19 0 11 7 0 9 57 4 25 1 05 158 100 0 Females Number Percent 27 16 2 28 16 8 36 21 6 39 23.4 23 13 S 11 6 6 3 I8 0 00 167 100 0 Toul Number Purest 64 19 7 66 20 3 64 19 7 69 21 I 34 10 S 20 6 2 7 22 l 03 325 100 0 is the penod in question, were free of the disease or not. A group of 113 persons of 29 households living in an apartment house had purchased canned K rice oil as a unit from one dealer and divided it among themselves fairly regularly from December, 1907, to September, 1968, except for the penod from January to April, 1968, when no bulk purchases were made. Their disease experience in 1968 was carefully explored through investigation of their medical records at hospitals and doctors' offices. No case of Yusho was found among them All these results suggested that Yusho was caused by use of the K rice oil that was produced or shipped from K company on February 5 and 6, 1968, or soon thereafter Nevertheless, some other factors or agents might have been the primary or secondary cause of the epidemic. Therefore two case-control studies were done. In one, the per- sonal backgrounds of the patient* and matched controls were compared In the other, the use tA oil and fata in the households of the patients and the controls were compared. 121 patients and their 121 healthy controls, (53 males and 68 females) matched by age, \, and place of residence to each of the patients, were selected and asked 60 questions conccrmnjt their occupations, medical background, general health status, habits, customs, diet, pets, and other characteristics of their lues A* ghovtn in Table 7, only one of the 60 personal factors examined, namely habit of "eating fried foh or tempura nearly every day", was significant!) more commonly seen among the patients than among the controls In the latter case-control study, 69 households with Yusho patients were matched 1>\ place of residence with 207 control households without Table S. Ape-apecigc Incidence rates for Yuabo, by Kt of patient*. Incidence rate* per 100,000 Age group (yean) --------------------------------------------------- Malts Female* 0-9 10-19 20-29 30-39 40-49 50-59 60-09 70 and over Toul 11 4 89 91 06 54 54 35 17 83 86 33 10 3 11 9 03 72 24 00 81 Table 6, Rice oil used by Yu*bo patient*, No patient* SpcetficftUotus of oil lifted Type Date of proportion or shipment 166 (51 1) Canned 4 (1 2) Canned Februarj 5 and 6, 1968 Unknown 143 (44 0) Bottled 12 (3 7) Bottled Unknown, but uae of oil shipped from February S t o 15 1998 is very probable, Unknown 325 (100 0) 122 Environmental Health Perspectives HONS 208219 fable 7, Reaulta of um nwitrol eludy. Items investigated Vnrtlf I" I"1' tlVrgii m iivpirm tjlrrgu I" nOwr drugs }huo(t nl home l.Lmg li'itli everyday (him* pet- a* home Iiung in Iimiw smaller thnn 60 m' liner 'linn' Ceeee O' /I Controls (b 50 00 T5 84 7 73 0 18 3* 66 9 75 42 66 85 5 70 6 36 5* 66 1 llnmlling agricultural ehrtnicale Taking rod liver oil luting viumin pills Tiding ether reetorative drugs Wntrr supply available at home 25 10 8 23 2 fl l 81 3 60 83 18 3 75 74 7 Dining nut orcaaumallv Dining llm name meals with families hid grwii vegetables dailv Drink milk nearly everyday Take Imllcr nearly everyday Esi rggi nearly everyday Eal fried foods or tempura nenrli everyday Eli foods prepared with oil nesrli everyday Eil lisle nearly everyday Talr muvonneiee nearly everyday Eil instant "rnhmen" or chineee noodle nearly everyday 28 1 88 8 63 1 49 0 22 4 64 7 22 4* 21 6 21 6 10 6 10 8 30 6 89 6 38 9 39 0 24 9 59 8 11 6* 29 1 29 1 10 8 10 0 [KUOS such patients A distinct difference was noted between the two groups only for regular use of ricr-bran oil, namely 96 percent of the patients' households affirmed it while only 31 percent of the control households did (Table 8). Thus, the case-control studies clearly indicated that none of the factors tested except use of K rice oil could account for the disease. Dose-Response Relationship To prove a causal relationship, a definite doaeretponse relationship is needed A rough estimate of the quantity of the specific K rice oil consumed Id each patient and his family members was made disregarding their age, sex, amount of fuud intake, find possible loss of oil during and after cooking (4) Eighty of the 146 users of the specific canned K rice oil in question were believed to have con sumed, individually, less than 720 ml For these 80 light users, the attack rate of Yusho was 88 percent, while for those who were estimated to have used more than 720 ml, it was 100 percent (Table 9) It was also demonstrated that the proportion of severe cases of Yusho clearly in creased with the amount of oil consumed While the clinical severity of the disease was not found to differ significantly between the sexes, it did differ considerably according to age, as shown in Table 10 and Fig 3. The proportion of severe cases among those aged 13 to 29 was significantly larger than that of other age-classes Therefore, each of the three groups of users, with different levels of oil intake, was standardized for age using the age composition of the whole 146 users as standard The figures, however, hardly changed from those shown in Table 9, Thus, a clear doseresponse relationship could be demonstrated, even though the estimates of the dose w**re inac curate (4), Toxic Agent In view of all these epidemiologic observations, we concluded that the K rice oil of specific pro duction or shipments had caused Yusho, Now, why was the oil toxic? The chemical study sub group (chief- Prof H Tsukamoto, Faculty of Pharmaceutical Sciences, Kyushu University) demonstrated that the canned K rice oil produced or shipped on February 5, 1968, and used by some of the patients contained about 2,000 to Tabte 8. Results of caie-control study on oils uied> Fat or oil Cases No of % households Controls No of f* ,4 households Butter Margarine Sesame oil Rape>*eed oil Rice-bran oil Lard Othu oils 35 50 7 44 ft3 R 21 30 5 ID 14 5 66 95 7 12 17 4 13 18 8 105 50 7 127 61 4 85 41 1 77 37 2 64 30 9 3K 18 4 117 56 5 Caws 69 patient households Controls 207 non-patient households April 1972 HONS 208220 123 Table 9. filiation between tbe amount of tbc K rice oil uaed by patients and their clinical aeventv Amount of oil Non-affected ------------------------------------ No. % Light cases ---- --------------------------- No. % Revere caeca ------------------------------------ No % Tout ------------------- __ Wo % Leas than 720 mi 720-1,440 ml More thus 1,440 ml 10 0 0 02 0) (0 0) (0 0) 39 (49 0! 14 (31 0) 3 04 0) 31 13P 0) 31 (09 0) 18 (06 0) 80 ooo m 4.1 ono hi 21 OOO Oi 3,000 ppm of Kanechlor 400, (Kanegafuchi Chemical Industrial Co Ltd) a brand of poly chlorinated biphenyls (chlorine content. 48%) (5) Id this discovery, Prof. K In&g&mi of the De partment of Food Technology, Faculty of Agri culture, Kyushu University, and his associates made meet admirable contributions as members of the chemical study subgroup. The study subgroup also demonstrated that the oil was not contaminated with toxic agents such as Cu, Ni, Zn, Co, As, Hg and Pentachlorophenol. Further more, it found that most of the components of Kanechlor 400, particularly those corresponding to the peaks of higher retention times in the gas chromatograms, were retained in the sebum, subcutaneous fat, mesenterium, mesenteriolum, extraperitoneal adipose tissue, appendix vermiformis, heart, sternal marrow, small intestine, trachea, and other organs of patients, and can be transported into the fetus through the pla centa (5,6,7). To examine whether only the K rice oil produced or shipped in the period in question was contaminated with Kanechlor, the chemical study subgroup analyzed 109 ssmpfet of the bottled rice oil which had been shipped between October, 1967, and October, 1968 Gas chromatographic analysis revealed that a significant contamination of the bottled oil ar limited only to those produced or shipjied between February 7 to 10, 1968 (5) No analysis could he made of the bottled K nee oil produced or shipped on February 5 and 6 because of the lack of sample* Similarly, 479 random samples of bottled K nee oil were analysed for chlorine content by the X-ray fluorescence method with a count mcler Again, only the samples of February 7 to 10 contained a large amount of chlorine (maximum 462 ppm). None of the oils shipped m other months were contaminated with more than t trace amount of chlonne Thus, the results of chemical studies completely coincided with those achieved by epidemiologic approaches By the way, the Kanechlor had been used at the K Company in the equipment for heating the proc essed oil at a reduced pressure in order to remove the odorous matters of tiie rice ml (tig 4) It is Table 10. Clinical eevertty by age, Amount of 1 uaad (ml) Age Number of patient* by clinical grade ------------------------------------------------------------------------------------------------------------------------------ Non-affected Light* Severe* Total <720 720-1440 0-12 J3-29 30- Total 0-12 13-29 30- Total 3 (16 7) 2 (S 3) 5 (13 1) 10 (12 5) 0 (0) 0 (0) 0 (0) O (0) 13 (72 2) 7 (29 2} 19 (50 0) 39 (48 8) 5 ( 50 0) 1 (6 7) 8 (40 0) 14 (31 2) 2 (11 1) (5 (62 5) 14 (36 9) 31 (38 7) 5 (50 0) 14 (93 3) 12 (90 01 31 (98 8} is i wo oi 24 OOO 0] 38 1100 0) 80 (100 0' 10 OOO 01 15 OOO Oi 20 OOO O' 45 OOO rn * "Light" correspond* to grade* [ and U while "severe'1 to grades III and IV ueed by Goto el a), 124 Environmental Health Perspectives HONS 208221 9J.3 a,. - 770 720-1. MO m. Finriu 3 Percentage of severe ca*e of Yuaho by age believed that it must have leaked from the heatup pipe and contaminated the oil, because small openings were discovered in the old pipe. Amount of Kaneehlor 400 Ingested by Patients One hundred forty-six patients who were proved to have used the contaminated canned K nrr oil of February 5 and 6 by our investiga tion were used for the estimation of the amount of Kaneehlor 400 ingested As stated, the approxi mate amount of the oil consumed could be calcu lated for each of the patients. It was on average about 800 ml. Since the concentration of Kaneehlor 400 in the oil was 2,000 to 3,000 ppm, the Average amount of Kaneehlor 400 ingested by a patient was estimated to be about 2 g (4). Similarly, the minimum dose of Ksnechlor 400 consumed by s patient was estimated to be about 0 5g Babies Bom to Patients and Non-affeeted Wives of Patients Thirteen women consisting of 11 with Yusbo and 2 unaffected wives of patients were shown to huve delivered 10 live born and two stillborn babies from February 15 to December 31, 1908 (8,10,11). As shown in Table 11, nine of them had unusually grayish, dark-brusTi stained slun and similar pigmentation of the gingiva and nail* wan noted in five of them. Increased eye discharge wns also notable in most of them Histological examination of a stillborn fetus showed a marked hyperkeratosis and atrophy of epidermis and cystic dilatation of hair follicle, especially at the bead. A marked increase of melanin pigments in the basal cells of epidermis was also noted (9) Since no such symptoms at birth or stillbirth has been experienced in Japan dunng the penod in which their incidence among the patients was extremely high, and also since none of the mothers bad had any unusual experiences, for instance, m use of drugs or in health status, these unusual phenomena could be considered to have been caused by ingestion of the contaminated oil However, a clear dose-response relationship be tween the oil consumption and auch phenomena could not be shown because of the limited number of caws. The amount of the contaminated oil consumed during pregnancy ranged from 0 3 to Rice bran Extracted with s-icwc Defatted bra* Cwce oil After ismjval of solvent, treated with sccum tetawcsphate AW CENTRIFUGED _____ L GuT Oil Treated wim IMH N*0H LAVER (tUTRALIZED WITH "Dark oil* Oil I Wed with water iATCI FILTERED Color rewved wit* SUITE PORCELAIN CLAY Deodorized by seating at over 200 C at T TO A * Ita Final products Fionas 4 Flow sheet of nee oil production. April 1972 HONS 208222 125 Table 11. Btbln born tn patient* end unaffected w|* of patient* and tbelr uac of K rice ail Amount of oil Pregnancy 4other Age need period No. during when oil pregnancy ueed Grad*o f clinical aevanty of mother Delivery (L) Rex Small of for- Stained Stained Stained baby data mkia gmgiva oail Increased eye Neonatal discharge jaundice 1 28 0 3 3rd tnmeater 3 Normal M No r -- - 4- 2 24 7 ? 3 Normal F No 3 22 1 4 Later half Normal Forcep M No of 2nd + 7 -- 4- + 4- + + tnmeater * 27 0 7 2nd 1 Normal M Ye* 4- 4- tnmeater 5 2 0 7 lit 3 Normal F No + 4- 4- 4+ ? 4 tnmeater 6 20 1.4 Later half of 2nd 3 Noma! M No + 4- 4- + tnmeater 7 30 1 1 Whole 1 Normal M Yea 4- -- 8 23 7 Whole 3 Normal M 30 + -- -- 9 33 T 7 7 Caesarean M Yea 4- 4- 4- aection 444- 7 4 ? 10 Z 0 3 Early half Normal Normal F No 7 4 of 2nd tnmeater 11 2Q 7 7 2 Normal M No + 4 + 13 33 2 6 Early half 4 Stillbirth 7 7 + 7 7 4- 4 of 2nd 13 25 7 tnmeater 7 3 Stillbirth F Ye* 4- 7 7 Table IS. of patient* with Yuabo.* Grade of clinical eventjr* Improved CluuoeJ condition# Station*) Worsened 0 1 2 3 4 Total l# m v 30 (81 7) 23 (82 3) 12 (82 2) 0 (0) 81 (80 0) 0 (25.0) 15 (27 6) 10 (30 4) 10 (43 5) 10 (100 0) 58 (3ft 5) 2 (8 3) 12 (20 7) 5 (11 4) 1 (4 3) 0 (0) 20 (12 0) * Calculated from the figure* reported by Toahitam tad Kitamur*. O' Pbyncal complaints without ehin-leaion*- 1' Pigmentation of the akin and the mucous membrane. 2 Comedo formation 3 Aoneforre eruption*. 4 Extensive diatnbution of acnefonn eruption* Total 24 (100 0> W (100 0) 44 (100 01 23 (100 0) 10 (100 0) 159 (100 0) 126 Environmental Health Perapectivea HONS 206223 r, htrri (Table 11) (8) Twelve of 13 fetuaes smaller than the national standards, and 4 tlmm were small-for-dates babies (8,10) As Krt1" older, the stam in their skin gradually jjeil il 1) N'o evidence has so far been obtained mh regard to any physical and mental retarda,no of thr babies, but wc suggest that a parji-ulnrlv careful and prolonged follow-up observaiion should Ire maintained for their future (8). l,roih of tffected Children To examine whether Yuaho disturbs children's frontli, the affected school children, 23 boys and IP girls, were compared in 1907, 1968, and 1909 with their 719 healthy classmates matched by The gains of the affected boys in both height ami weight decreased significantly after the poisoning, while the affected girls showed no definite change in this respect (12} Current State of Patients More than 3 years have passed since the cpitlemn was reported. It is a heart-breaking fact dial many patients with Yusho are still tortured lw the Mckncss because no curative treatments hue bren discovered yet In summer of 1970, a muss clinical examination of the Yuslio patients was carried out in Fukuoka-Ken, and the clinical slate of the patients examined were compared mth their previous findings observed in summer of IfKitl. As shown in Table 12, about a half of the 159 patients, for whom the comparison was feasible, were Bhown to have clinically improved signs, while the remaining half showed no such faior.-ilile signs, and more than 10 percent of the patients were even worsening (13), It should be noted, furthermore, that even many of those who appeared to be clinically improving had several serious complaints, such as persistent headache, gcnci id fatigue and feekng of weakness, numbness in limbs, weight loss, and others (14), All these fact's dearly indicate that recovery from Yusho is extremely difficult. To cure them as quickly as possible and to prevent another epidemic of Yusho, all possible world-wide cooperative efforts are highly desired, From the middle of February to the end of March, 1968, an epizootic of a strange disease closely resembling chick edema disease occurred involving more than two million chickens in the western part of Japan (15,18) More than 400,000 chickens were reported to have died The disease was characterized by such clinical signs as labored breathing, droopiness, ruffled feathers, high mortality and decreased egg production (15,18). Autopsy revealed a marked subcu taneous edema, hydropericardium, ascites, and pulmonary edema, muscular ecchymoais in the thorax or inside of the thigh, and yellowish mottled appearance of the liver (15,17,18) An epizootiological investigation demonstrated that the disease was caused by feeding chickens with specific lots of commercial brands of formula chicken feeds manufactured by two companies In view of the fact that the K nee oil used by Yusho patients had been contaminated with Kanechlor 400, the toxic feeds were aualyzed for chlorinated hydrocarbons and proved to contain Kanechlor 400 (16,19,20) The experimental reproduction of the disease by administering this chemical mixture was successful (16,20,21). The reason the chicken feeds were contaminated with Kanechlor 400 could be reasonably understood by the following facts, both of the chicken feed manufacturers hsd been using "Dark oil" pro duced by the K company (Fig. 3) as an ingredient of their formula feeds, and they had actually used "Dark oil" purchased from the company from February 6 to 27, 1968. The remaining sample of "Dark oil," used for production of the toxic formula feeds, was also shown to contain about 1,300 ppm of Kanechlor 400 (16,20). Thus, it became clear that both the epidemic of Yusho and the epizootic of the disease among chickens had been very closely associated, being caused by intake of the oils contaminated with Kanechlor 400 It is very unfortunate, however, that the epidemic of Yusho could not have been pre vented even though a large scale epizootic of such an unusual chicken disease had preceded the incidence of Yusho by more than 6 months REFERENCES 1 Katcuki, 6 1969 Foreword, Reports of the Study Group for "Yusho'' (Chlorobiphenvla Poisoning), Fukuoka Acts Med 60 403 2 Kurmtsune, M et at 1969 An epidemiologic studv on "Yusho" or chlorobiphenyls poisoning Ibid. 90 513 3. Goto, M luid Higuchl, K 19GB The symptomatology April 1972 MONS 208224 127 of Yusho (chlorabiphenyls poisoning) in dermatology, Fukuoka Acta Med 00 409 4 Yoahimura, T 1971 Epidemiological analysis of ''Yuaho'' patients with special reference to sex, age, clinical grades and oil consumption Ibid 62 104 5 Tsukamoto, H ct a! 1969 The chemical studies un detection of toxic compounds in the rice bran oils used by the patients of Yusho, 1 bid 60 496 6. Kojima, T 1971 Chlorabiphenyls in the sputa and tissues Ibid 62 26 7 Kijuchi, M. et al 1971. Two autopsy cases of chronic rhlorobiphsnyIs poisoning Ibid 62 89 8 Yamsguchi, A , Yoahimura, T and Kuratsune, M 1971 A survey on pregnant women having consumed nee oil contaminated with chlorabiphenyls and tbsir babies. Ibid 62 ] 17 9 Kikuchi, M et al 1969 An autopsy case of stillborn of chlorobiphenyli poisoning Ibid 60 489 10. Tiki, I , Hisansgi, S and Amsgase, Y 1969 Report on Yuaho (chlorobiphenyli poisoning) pregnant women and their fetuses. Ibid 60 471 11 Kutiatsu, I et al 1971 A chlorabiphenyls induoed fetopathy. Ibid 62' 139. 12. Yoahimura, T 1971 A case control study on growth of school children with Yusho Ibid 62' 109 13 Tbshitam, S and Kitamura, K 1971 Clinical observa tion of Yusho (chlorabiphenyls poisoning), especially further study of Its dermstologicnl findings l|Ml| (p 132 ' 14. Okumura. M , Hirayama, C and ! smn M ]a:t Study of Yuaho (ehlorobiphentls pinioning i m etunril examination Ibid 62 123 15 Kohanawa, M et al 1969 PoiMimng .luc p, an ,,,j, by-product of nce-bran similar to i lm k edema diwuv I Occurrence and toxicit; teat \,,il |n.| Health Quart. 9:213 16 Kohanawa, M et ai I960 Poisoning doc u, an ,,i by-product of nce-bran similar to t-lnrl, Hi-ma ihwm II Tetrachlorobiphenv! as toxic aid isism r :i,,,; i, jjn 17. Shpya, S. etsl 1969 Psthologicul r-tnngr'.. nf pommint m chickens due to dark-oil, n mh In priori of rr bran. Ibid 9 229 18. Koga, K et al 1970 Studies on llie time prtnnplr m a certain by-product of rire oil rendering I I'cumlm on the chick Japan J Zooteehmrnl , n -j-jij 19 Koga, K et al 1970 Studies on thr tour prmeiplr in a certain by-product of rtrr ml rendering IJ Amu, of a toxic subatance Ibid 41 4.<o 20 Koga, K et fcl 1971 Sturts thp tiuir prmriplr rn i. eertim by-product of rep nil rendering I1J rnstrit* of Chlonnftted biph*ny) nn ehick< ititri wmnnviriAnl revulte of I--III Ibid 42 ]G 21. Goto, K , Stknjtuchi, K and Ogawn K l Hvdro- pencanlauro uny of nee oil caused 'i nsho nnH of Kuechior 400 in chickens Ibid <10 Vi 1 128 Environmental Health Perspectives HONS 208225 m]\ !tln< nm* I 'MJ *h*< Iline Dr< f IN Ml s f in Vf*\ * in ir\ Bf'lf k mi Kill f I i An Abstract of Results of Laboratory Examinations of Patients with Yusho and of Animal Experiments by Masanori Kuratsune* An extensive clinical study of Yusho and many snimitl experiments were carried out by the Yusho study group in Kyushu University in order to Department of Public Health, Faculty of Medicine. Kyushu University, Fukuoka, Japan clarify the mechanism erf toxic actions of Kanechlor Most of the findings have been pub lished in Japanese with brief English abstracts I hope this abstract will help in the understanding of the important observations obtained by these studies. Tabic 1, Laboratory findings of patlenta with Yusho. Item fitol (1) 1 Red blood ceils 2 Leucocytes 3. Blood platelets 4. Coagulation time S Prothrombin time fl Blood sedimentation rate 7 Hemoglobin Blood (!1) serum liptda 1 Tojl lipid 2 Tnglyoendec Laboratory findings Decreased in moat seven cases. Increased m moat severe cases. Normal Normal Normal Normal except for a few cases Decreased in moat severe cases Increased 728.0*199 8 (mg %) 9 eases of grade IV* Increased 180.8*88 2 (mg %) 9 eases of grade IV* 89-817 (mg %) 24 cases More than 300 (mg %) 9 out of 24 cases 189*94 (mg %) 32 adult patients Fifteen percent of the patients showed unusually high levels (more than 300 sag %) of triglycerides Reference 3 1 3 3 3 3 1, 3 3 1, 3, 4, 8 3 4 4 8 20 f 0-9 10-19 20-29 30-39 40-49 80-39 60- Total No. CUM 82 89 68 88 88 28 18 396 TG>300 No case 18 17 8 10 6 2 2 80 % 34 6 19 1 74 11 8 10 3 72 12 5 15 1 April 1972 HONS 208226 129 Item 3. Cholesterol 4 Phospholipids 6 Serum lipoprotein 4 Fatty acid eoroposi tion of serum lipids Table 1--Continued. Laboratory findings Referetifi Normal 181 43 (mg 27 adult patients Normal 8 1 1 8 (mg %) 27 adult patients fl/n-Jiproprotem ratio increased Woman with Yusbo, aged 24' 2.0 Woman with Yusbo, aged 00 4 4 Man with Yusho, aged 35. 3 5 Analysis of cholesterol eaten, triglycendee, free fatty acids, and phospho lipids showed no abnormal components, sleept that increase of oleic acid and decrease of palmitic acid were seen in eerum-free fatty acids. Let els of glucose and free fatty acuta m blood were determined 30, 60, 90, 120, 150, and 180 minute* after oral administration of 50 g glucose Two types of variation of levels were noted Type A Blood glucoee increased and later decreased Fatty acids hardly changed Type B Blood glucose increased and did not return to normal even after 180 minutes after glucose administration. Fatty acids decreased markedly after administration Palmitic acid decreased by glucoee administration more markedly in patients than in normal persona PH LA values (FFA aEq/mm/ml) 10 minutes after heparin injection in 10 normal women aged 18 to 20 in years were 0 201 0 058 Four of 5 female patients showed PHLA 10 minute values lower than 0 143, namely the mean--1 S D value for control, while 2 of 8 male patients had simitar low values Five patients with Yuaho were examined. Mean value was 41 1% for patients which was significantly lower than control (p-0 02) 18 8 8 Table 1 --Continued* Item Laboratory findings Reference 2 Cl 2 Ke 4 Cu 5 Zn 4 20 5 (mEq/1) 17 Yusho children Normal Tended to decrease 66 0 300 (jig %) 0 cases of grade IV* Decreased 9034 (*g %) SO adult patients 8320 (ag %) 17 Yusho children Increased in esvere cases 183 961 0 (u* %) 9 cases of grade IV* Norm*! iW (l ) truymtt ] Alknline phosphatase, serum 2 Lactate dehydrogennec (LDH), serum 3, Tranaaminsaes GOT GPT Increased slightly in severe cases 10.3 3.5 (King-Armstrong unite) 65 adult patients 17 44 9 (King-Armetrong units) 11 Yusho children 14 9 9 26 (King-Armstrong units) 9 cases of grade IV* Normal in most cases 278 65 (Karmen units) 24 adult patients 28424 (Karmen units) 5 Yusho children Normal 24 19 (Karmen units) 70 adult patients 3623 (Kerman units) 12 Yusho children 23.0 7 7 9 cases of grade IV* 28 010 2 cases of grade IV* 24.78 8 cases of grade III 20.0 7 3 cases of grade 11 Normal in moat cases 22.611 9 7 cases of grade IV* 2316 (Karmen units) 48 adult patients 2111 (Karroin units) 10 Yusho children Mean 30.1 cases of grade IV* Mean 24.1 cases of grade III Mean 16.0 cases of grade II Blond (17) otheri 1. BronuuVilein (BSP) tent 2 leterue index 3 l"1 resin sponge uptake (Triosorb teat) Delayed in several adult cases 6.9S.19 (%) 8 cases of grade IV* Norms] 3 3 0.66 8 cases of grade IV* Normal Cnne ] Protein 2 Glucose 3 Urobilinogen 4 Vnnary neutral 17-ketoeteroids Negative Negative Negative in most cases Elevation of ratio of etiocholanolone/androsterons was noted, indicating adrenocortical hyperfunction but urinary excretion of total 17-ketosteroidi was in normal range. Etiochol /androst 2 N ormal women aged 28 0.0, 10 2 Normal men aged 20 and 28 0 4, 0.8 2 Yusho women aged 35' 1 7, 2.0 2 Yusho men sged 37 and 39 0.9, 2 6 3. 8 3 8 3 3 3 1,3 8 8 H j 3, 8 8 8 3, 8 8 8 3 1 1 1 3, 8 3 8 8 1 1 1 3 3 3 3 3 3 8 8 April 1972 MOWS 20*228 131 To Ip I 17-kftoKteroids 23 adult Yuoho women 5 7*1 8 (mg/U*y) 13 adult ^ usho men 7 3 2 8 (mg/day) Urinary total I7.kelo*terids {17-K.S) anil |7-hvdrox) corticosteroid (17-OHCS) were determined in SO male and 45 female patients Posit i\r correlation waa found between 17-KS and 17-OHCtf. 42',i of patients exceeded normal range in both steroids m males and females Analysis of major components of urinary 17-KS waa made in 0 male (I!) to 57 yearn in age) and 7 female (10 to 51 year* in age) patients For androsterone etiocholanolone, and dchydmepiandroatemnr, female patient* seemed to be lower than control, while male patients seemed to be higher Normal (mg/davl Patient* (mg/dnv) M Pregnanediol Androsterone Etiocholanolone Dehydroeplandrosterone 0 70 0 17 2 95*1 02 1 83 0 55 1 55*0 75 0 84*0 46 3 28 1 19 2 04 0 84 1 96*! 18 Total 6 41 8 7 9 3 2 Determined by Ztmioermtnti reacttun 10 0*2 3 11 6*3 2 F Pregnanediol Androaterone EUochoUnolone Deh yd roepiandrosterone 0 95 0 55 2 02 0 97 1 35 0 48 1 010 40 1 34*0 91 1 32 *0 60 1 03 *0 40 0 93 *0 4 8 Total 4 4*1 7 3 8*1 0 Determined by Zunmermann reaction 6 1*2 4 7 21 9 C Ammo acid* Fatty and analysis of aens and okmfat I. Change like malarial ootlected from acne eruptions and fat in the sweat of patients Two patients were examined, A remarkable increase in excretion waa noted for taurine (5,641; 8,103 nM/day), tf-aminoiaobutyric acid (247, 1.042 pM/day), glycine (1,865,2,997 pM/day), and hiatidme (743, 1,170 iiM/day). Increase waa also noted for threonine, serine and Tyrosine mynatic palmitic paimitoleic stearic oleic linoleic linolemc cheeae-ltke % 27 21 5 10 4 16 3 30 4 16 3 04 Fat in sweat Face % 11 7 41 2 23 5 11 8 11 8 Body r*0 91 41 0 22 7 91 18 1 8 132 Environmental Health Perspectives HONS 208229 Item ^ n*li" nrne and nun't naho acne Table I--Continued. Laboratory findings Yuaho acne contained more stearic and oleic acid* than did non-Yusho acne Linoleic acid was present only in the former The former contained 10 times more cholesterol than the latter myrirtic palmitic pfcJmitolejc stearic oleic linoleic Cheese like materials from ordinary acnes of many persons % from acnes of members of a Yuaho family 12 % Cc' 11 6 41.2 17 0 13 7 16 5 DOM 30 22 1 10 7 16 6 31 S 16 1 83 23 4 61 31 2 23 .5 75 Reference 8 2 Skin Stvrtilogteal finding* Otorfwioiaryn^ologuxil h'<d\ngn (Vtfliir Iifns Reduction of rough endoplasmic reticulum and hypertrophy of smooth endoplaamic reticulum Mitochondria ahowed morphological hetero geneity M any filamentous inclusions were present in the matrix Giant mitochondria were frequently encountered Twenty-four biopsy specimens from 18 patients were examined. Marked hyperkeratosis and cystic dilation of hair follicles, marked increase of melanine in basal cells of epidermis. Slowing of sensory nerve conduction velocities of radial and sural nerves was noted in 9 of 23 patients examined. Motor nerve conduction velocities of ulnar and tibia! nerves were shown to be unaffected except for 2 patients in whom they decreased. No audiometnc abnormality and no disturbed vestibular function Hypersecretion of meibomian gland; abnormal pigmentation of conjunctiva, no particular lesion in intraocular tissues. Increase of melanine granules was noted electronmicroecopicelly in cytoplasm of epithelial cells, especially in basal cell* of conjunctiva. Innumerable electron dense particlei. 300 A400 m diameter, were in |jte cytoplasm of basal cells * Grade IV means clinically most severe cases 5, 23 9 b 7 Table 1, Findings of animal experiments Eipenmen tal Results Reference 1 2 ml of olive oil containing 1% Kanechlor 400 orally given to rabbits daily for 10 <iavi 2 Oral administration of Kanechlor 400 to female mice. rvnomolgns monkevs. and Increase of serum triglyceride* and slight increase of total cholesterol Enlargement of liver; alight fatty metamorphosis of liver. swelling of sebaceous gland Increase of smooth endoplasmic reticulum and decrease of rough endoplasmic reticulum m 4 (Uiawa et ai ) 10 (Niahisumi et al ) April 1972 HONS 208230 133 Table 4--"Continued, v Experimental Result* Reference squirrel-monkeys (or a month liver or longer 3 Oral administration of 0 l g of Loss of body weight, hepatomegaly, and marked increase m ij Kaneehlor per K( per day to serum lipid components were noted Serum triglyceride# (1 unaka et a] ) rata (or 4 week*. reached a level aa high as 10 times the control level Serum cholinesterase activity was not affected. 4 Oral administration of the nee Hyperkeratosis, cystic dilatation of hair follicle and awsai- (2 oil uaed by a Yuaho patient to gland, marked hepatomegaly, fatty degeneration of liver (langmoi et al hairless mice 5 Distribution and elimination of component* of Kaneehlor 400 orally administered in a Each component of Kaneehlor 400 was equally absorbed from gastro-intestinal tract Concentration of Kaneehlor 400 in km one day after administration waa twice that in liver and 13 {Ycshimura et ij ) single doae of 2 0 mg/body to kidney Kaneehlor 400 was retained in skin more than in female mice other tissues. Tetraehlorobiphecyl* were almoet completely eliminated from tissues in three to four weeks, but penta- and hexa-cblcrobiphenyla were retained 9 to 10 weeks after administration e Distribution and excretion of Radioactivity in akin, adipose tissue, liver, adrenal gjand and 14 'H-Kaneehlor orally adminis gastrointestinal tract was higher than in plasma 3 days after (Yoshimura et *1 ; tered m a single doae of 500 administration Radioactivity was noted in some tiaeuss after sc or 25 mg/bodv to Wutar 8 weeks Urinary excretion of Kaneehlor waa limited, while Ktng male rata weighing about 150 g. 70% of the doee given was in feces A small portion of radio activity excreted during the first day waa in the phenolic fraction More phenolic metabolites and less unchanged com ponents were excreted in the feces on the second and third day r Effect of oral administration Kaneehlor 400, 500, 600 Hepatomegaly Microsomal protein 15 of Kaneehlor 400, 500, 600 per gram of liver increased in proportion to chlorine content (Fujitn cl #1 j and their components, 0 2 ml of Kaneehlor Amount of cytochrome P-450 aod specific ac of a solution of 6.8 mg/0 2 tivities of O-dealkylaae (p-nitrapbenetol), N-detnethylaae ml/100 g of body weight, (p-ehloro-N-QKthylandine), and aniline hydroxylase, in daily lor 3 days, on liver mi creased, particularly with Kaneehlor 500. crotomes of male rata. 4,4'-diehlorobiphenyl Slight increases seen. 3,4,2*,t'-tetracblorobiphenyll' Increases noted, but leas than 3,4,3', V-tetrachlorubiphenyl/ with Kaneehlor 400 3,4,3',4'-tetrachlon>biphenyl was s more potent inducer than the other# 2,4,5,3',4'-pentach!orobiphenyl' Most marked increases were noted in cyt P450 and specific activities of 3 enzymes. 3,t,6,3',4',6'-hexachk)robiphenyl, The increase was larger then that induced with the tetra chlorobiphenyls but leas than that with the pentachlorobiphenyl Pbenobarbital Resembling Kaneehlor 400 in enzyme induction capability The above mtcroaomal entyme activities and the microsomal components started to increase 12 hours after administration of 3,4,3',4'-t*Cr*ehiobipheny], reaching the maximum after about 1 week. Thereafter, activity levels tended to decrease but dad not return to normal even after 6 weeks Mirrosomss, particularly their membranes, contained the largeet amount of H*-Kaneehlor-400 administered orally, as compared with mitochondria or lysosomes. 8 Oral administration of Kane- Hexobarbjtal sleeping time waa markedly shortened by preehlor 400 or 500 to female rats treatment with Kaneehlor 400 or 500 in rat* Minimum effec- (Komatsu et c! ) iu Environmental Health Perspectives HONS 208231 Table --Continued. Experimental Remit* Reference (Minter King), about 100g lit weight, m order to see efTecta of lhe administration on htlobarbiiol sleeping ftme 9 Ora! administration of daily doer of 0 5, 2 5, 5, 90 nag of Kanechlor to rata for 13 to 21 days live dose was daily 5 mg and 2 mg/Kg for 3 days for Kane chlor 400 end 500, respectively When 40 mg/Kg of Kanechlor 400 or 500 was given daily for 3 days, the effect lasted for 3 weeks by the former and for 0 weeks by the latter Ethionme inhibited the shortening of hexobarbital sleeping time by Kanechlor Anabolic steroids, chloroquine, glutathione, <r-mercaptopropionylglyrtne, essential phosphollptde, diphenyl, and mila dies me did not modify the sleeping time reduction by Kanechlor Growth was inhibited by the treatments. No weight increase at daily dose of 5 mg, and a weight decrease at 50 mg. Plasma triglycerides did not increase at daily dose up to 5 mg, and slightly exceeded the control at dose of 90 mg, while plasma cholesterol and phospholipid increased definitely by increas ing amount of Kanechlor. 17 {Nagai et al ) Daily doae Kanechlor (mg) Tnglvcende M% Cholesterol mg % Phospholipid mg % Control 05 25 60 50 0 100+20 98+19 89+21 89+18 130+37 83+12 118+20 102+11 125+28 178+18 4 70.8 5 8+1.7 7 2+1 2 7 1+1 3 13 4+2 2 10 Onil administration of Frac tion A and B of Kanechlor 400 to rata, each daily dose 1 mg on every fourth day for 40 dnvs, 10 mg total Kr A Boiling point 123-- !24*C/0.01 mmllg, 3.12 rhSonne atom* per biphenyl average. Fr B Boiling point 140142'C/OOt mmHg, 3.S4 rhSonne atoms per biphenyl average 11 L'put in Ivtr Ural administration of daily dose of 8 ml of olive oil con taining 1% Kanechlor for 3 and 11 day* to 2 female rabbita The administrations hardly affected concentration of triglyc eride, cholesterol, phospholipid in liver except for daily ad ministration of 50 mg Kanechlor, which caused slight increase of triglyceride and cholesterol, They slightly increased tnfSvcendc in akin but hardly affected cholesterol and phos pholipid. A definite reduction of triglyceride in perirenal adipose tissue was noted. Animals grew in weight with Fr A but lost weight with Fr. B, which caused marked atrophy of abdominal adipose tuarnc Lipid content of adipose tueue around kidney decreased when Fr. B waa given but did not when Fr A given Plasma tri glyceride decreased with Fr. B but did not with Fr A. 17 (Nagiu et al ) Animal Control Fr. A Fr B Triglyceride sM % 13018 128 10 832t Cholesterol mg % 00 5 00+7 57+5 Phospholipid mg % 6 7+0 4 5 1+04 5 2+0 3 Total lipid and glycendee of liver increased, but concentration of serum glycerides was normal after edmmistration for 3 consecutive days, but increased abnormally after tdmmulration for 11 consecutive days. 18 (Ito et al.) Lipid Control (n - 1) Poisoned Poisoned for 3 days for 11 days April 1972 M0NS 208232 135 Glycerides Cholesterol CGP Lecithin Lytotocithin Sphingomyelin ECP SCP PI Glycoltpid Others Serum glyceridei (mg %) 7 1% 63 22 5 \ 72 49 is e 90 68 52 15 9 J 52 24 3% 61 30 0 39 6 65 25 3% 10 8 97 76 48 14 4 45 51 67 11 I 1,890 12. Oral administration of daijy doae 0.5 or 0 3 rag/Kg of Kanechlor 400 each to 40 adult Wistar rata. Marked or moderately impaired motor function, decreased motor conduction velocity, early stage of segmental demyetination and low of large nerve fibers were noted 2t REFERENCES 1. Goto, M and Higuchi, K I960 The symptomatology of Yuaho (ehlorobtphenyb poiaoning) in dermatology, Fukuoka Acta Med S0` 409 2 Ikui, H , Sugi, K and Uga, 9 1960 Orular aigne of chronic chtorobiphenyla poiaoning. Ibid 60 432 3 Okumura, M and Kntauki. 3 i 960 Clinical observa tion on Yuaho (chlorobipbenyla poiaoning) Ibid 60 440 4. Usawa, H. et aL 1969 Hyperglycendemia reaulting from intake of nee oil contaminated with chlorinated biphenyl*. Ibtd 60 449. 5. Hirayama, C , Inaa. T. and Yamamoto, T 1969 Fine etructurai changea of the liver in a patient with chlorobiphenyl* intoxication. Ibid SO: 455 A Kurorwa, Y.( Mum, Y. and Santa, T 1909. Neuro logical and nerve conduction velocity atudie* on 23 patients with chlorobipbenyU poiaoning Ibid 60 4A2. 7. Monmitau, T, et el. 1969 Otnrhmolaryngologioal findings of Yu*ho (ehlorobiphenyl* poisoning) Ibid 60 464. 8. Nagai, J. et al. 1969. Cluucochemical investigation of eUorobiphenyla poisoning--especially on the serum lipid analysis of the patient*. Ibid SO' 475 9. Kikuchi, M and Haahimoto, M. I960 Hiatopathological studies of skin lesions of patients with chlorobiphenyls poisoning, Ibid 60. 484 10 Niahisumi, M , Kohchi, S. and Kuratsune, M I960 An experimental study of "Yusho" or chlorobiphenyls poisoning (preliminary report) Ibid 60- 539 11 Tanaka, K et al I960 Experimental subacute poison ing of chlorobiphenyls, particularly the influence on the serum lipids in rats. Ibid 60 544 12 Inagami. K et al 1969 Experimental xtudt of hairier mice following administration of nee ml used hi a ''Yuaho" patient HhH 410 * 548 13 Yoahimura. H and Oshima. M 1971 `bullies on the tisane distribution and elimination of went I compo nents of KC-400 (chlorobipbenyla) in mice Hod 62 r> 14 Yoahimura H et al 1971 Stud me on l lie Insue dis tribution and the urinary and fecal excretion n/ 'H-Kanechlor (chlorobiphenjli) in rats Ibid tif 12 16, Fujita. S et al 1971 Effect of biphenyl chlorides on rat liver nucroeomes Ibid 62 30 16. Tanaka, K 1971 Shortening of hexobarbita! sleeping time and change of serum triglyceride level in rhlorobiphenyls-intoxieated rats. I bid 62 35 17 Nagai, J. et al 1971 The influence of chlomlophenyls (Kanechlor) administration on the orgsn lr)uds in rati Ibid 62 . 42. 18 Ito, Y, Usawa, H and Notomi, A 1971 Lipid com position of the rabbit liver poisoned *ith rhlorobiphenyl* Ibid 62 48 15 Nagai, J et al 1971, Colorimetric and gnxrhrcmatngraphic determination! of urinary 17.keii>temids Ibid 62- 51 20 Utawa, H et al 197! Clinical and ex|ierimental atudia on the hyperglycendemia induced bv will m gestion of chlorinated biphenyl*. Ibid 62 66 21 Ogswa, M. 1971 Eleetrophysioiogtcsl tnd histological studies of experimental chlorobiphenyls poisoning Ibid 62 74 22 Y'amamoto, T , Hirayama. C and Irisa, T 1971 Some observations on the fine structure of the mitochondria in hepatic cells from a patient with chlurobiphtnvls intoxication Ibid 62 86 136 Environmental Health Perspectives HONS 208233 Entimuntntal HwmUh Ptriptaian Chlorinated Aromatic Hydrocarbon Induced Modifications of the Hepatic Endoplasmic Reticulum: Concentric Membrane Arrays* by D. H. Norback and J. R. Allenf A number of commercial compounds having chlorinated aromatic hydrocarbon structures are used extensively for medical{3), induetnal(4) and agricultural (5) purposes. Quantities of certain parent chlorinated aromatic hydrocarbons and metabolites have been repeatedly detected as residues in the food supply and tissues of birds, fish.und mammals(6-9) including man{10) Chlor inated diphenyl-p-dioxins have been identified as contaminants of many commercial products, such as < crtain edible fata, herbicides and disinfectants. It is suggested that the compound results from the conjugation erf commercial chlorinated phenols(ll). Polvchlonnated polyphenyls are among the most abundant chlorinated hydrocarbon global pollu tants Their contamination erf the environment is a result of the widespread commercial use erf these compounds for their properties of insulation, adhesiveness, thermoplasticity, chemical inertness, and insolubility. Following this investigation of sequential bio chemical and ultrastructural alterations within the liver produced by three chlorinated aromatic hydrocarbons -- chlorinated diphenyl-p-dioxin, polychlorinated biphenyls (PCBs), and a highly chlorinated triphenyl (PCT)--certain similarities of biochemical and ultrastructural effects of the compounds were observed The livers of rats fed the chlorinated aromatic hydrocarbons consis tently contained numerous multi-layered concen tric membrane arrays, proliferated smooth * Portions of this psper were previously reported (1,2). I Department of Pathology, University of Wisoonsin Medical School, Madison, Wisconsin 59700 endoplasmic reticulum (ER), and altered micro somal enayme activity. Materials ind Methods Separate groups of male Sprague-Dawley rats initially weighing 100 grams woe fed diets containing the following chlorinated aromatic hydrocarbons: 1% PCTs (Aroclor 5460), 0 02% PCBe (Aroclor 1254), and 0.002% chlorinated diphenyl-p-dioxin. respectively. At regular inter vals the rats were deprived of food for 24 hours and sacrificed by exsangumation The livers were weighed and portions were removed for electron microscopy. This tissue was immediately placed in veronal acetate buffered osmium tetroxide and cut into small cubes. Following one and one half hours of fixation, the tissues were dehydrated in a graded series of ethanol, cleared in propylene oxide and embedded in an Epon-Araldite mixture Sections were cut at a thickness of 0.5-0.8 microns for tight microscopic evalustion, and thinner sections at a thickness of approximately 60 millimicrons were cut and stained with uranyl acetate for electron microscopic examination The remaining portions of the livers were perfused through the portal vein with isotonic KC1, weighed, and homogenised at0 with two volumes of the salt solution. Microsomes were isolated from the poet mitochondrial supernatant obtained by centrifugation at 9,000 X G for 20 minutes, washed by homogenisation with KC1 and re centrifuged, resuspended in KC1 equal to three volumes of the origins! liver material, and immediately assayed for the activities of aromatic April 1972 HONS 208234 137 hydroxylase(12), nitroreductase(l2), N-demethylaae(12), nitrophenyl acetate hydrolysis (esterase) (13), and glucose-6-phosphatae(14), RNA(lo), phospholipid(16), and cholesterol(16) concentra tions 'Mere determined from samples thathad been frozen at -- 80 Protein determination^.") on alkaline-waahed(lg), microsomal material pro vided est.imation of membrane protein in the microsomal suspensions, which is subsequently referred to as microsomal protein Results The rats from the dioxin and PCT groups readily ate the experimental diets and attained at least 80% of the weight of the controls at three weeks. Hypertrophy of the liver, varying from a moderate enlargement, following chlorinated diphenyl-p-dioxin ingestion, to an increase in the relative liver weight of 8 grams per hundred grams body weight, following PCB and PCT ingestion, was observed. Neurological side effects, including tremors, convulsions, or sedation, Mere not observed The rats from the dioxin and PCT group remained on the diet for four weeks without apparent extra-hepatic effects other than the small decrease in weight gamed The rets from the PCB group did not gain weight and were sacrificed at 8 days The ultrastructural alterations within the hepatocytes of rats from the three c'.permien'.a! groups were similar and will be considered collectively The changes were primarily limited to the endoplasmic reticulum and consistently affected all experimental animals The hepatocytes contained numerous multi-layered, concentric membrane arrays and proliferated smooth ER Preceding the formation of the concentric arrays, the parallel cistemae of the rough ER, representa tive of the control hepatocytes (Fig I) were reorganized into sinuous patterns thut encom passed lipid droplets, mitochondria and other organelles (Fig 2) Annular profiles consisting of paired membranes with ribosomes attached to the Fiooite I Elements of tbe rough endoplumie reticulum are characteristically arranged in parallel lamellae in hepstoo tes of rata fad a control diet ( X 6,630) 1 Environmental Health Perspective* HONS 208235 cr Fim'Mt 2 Fnllnwing the mention of ft chlorinated aromatic hydrocarbon, the parallel membrane* are rcorpsanjftpd into of sinuous cisternae that encompass lipid droplets, mitochondria, and other organelles following ingestion of chlorinated te diphenyl-p-dioxin, X 15,500) si central surface of the inner membrane and the were also evident following light microscopic peripheral surface of the outer membrane were also evaluation of the toluidine blue-stained sections prevalent (Figo 3 & 4), Shortly after the develop Within three weeks, each experimental group ment of the annular formations, concentric annuli developed multi-layered membrane arrays com composed of van&ble numbers of degranulated posed of several units of concentric annuli (Fig 6). paired membranes were observed (Fig. 5). These Lamellar profiles of paired membranes resembling structures composed of multi-layers of membranes flattened cisternae partially circumscribed the I u L` Floras 3 Membranes of the endoplasmic reticulum arc also represented aa annular profiles in bepatocytee of the expenmeau.1 cal (following ingestion of cltlorinAtod diphenyl-p-dioxin, X 24,300) April 1972 HONS 208236 139 activities of glucoae-d-phosphatace and hydroxylase were reduced. The ciuymatjc (.hang* following PCB ingestion were parallel to thone of the PCT group. Disafuiw Ingestion of the chlorinated aromatic h\<W carbons by rats induces liver hyportropln vnth proliferation of the ER and formal ion of large concentric membrane arrays within the cytoplasm of the hepatic cells. These changes measured after 21 days PCT ingestion result in increased hepatic function. An increase in total acm dy of cacli entyme investigated is evident when the neari) three-fold liver hypertrophy and more than doubted quantity of microsomal protein per gram liver is considered. Associated with the enrymstic alterations are an increased ratio of phospholipid to protein and a decreased ratio of cholesterol to protein Increased ensyme activity and prolif erated smooth ER are commonly produced by a Floras 4. The concentric annuli with attached nbceome* are apparently derived from the rough eadoplaanuc reticulum (following ingestion of chlorinated dipbenylp-dioxm; X 35,600) annular units. The outer membrane pairs of the lamellae were continuous with elements of the smooth ER and rough ER. Riboeomes were occasionally attached to the surfaces of the outer membrane pairs of the multi-layered arrays. The concentric membrane arrays increased m sise and complexity in direct relationship to the time the rats remained on the experimental diets. Associated with the modified membrane struc tures were alterations m entyms activity and biochemical composition of the microsomal fraction of these cells {Table 1). Following the ingestion of PCTb for 21 days, there was an increase in microsomal protein per gram of liver. Composi tional change* of the membrane material were indicated by the reduction in cholesterol to protein ratio and slight increase in phospholipid to protein ratio The increase m esterase activity was parallel to that of protein, whereas, increases in Ndemethylation and mtroreduction were greater than the increase in membrane material Specific Florae 5 Larger concentric membrane arrays con'(si "t numerous dieltnct rings of paired membranes mgealjoa of chlorinated diphenyl-p-dtoviri, x ,1a inn 140 Environmental Health Perspectives HONS 20S23? fiounz B Following ingestion of polychlorinated tnpheny! for three weeks extensive eojicentne membrane imvs develop within the hepitlocytw The membrsne arrays encompsss lipid droplets, mitochondria, and other cellular organelles <X 3,700) j: wide variety of agents including phenobarbital, of paired membranes. The concentric annuli are F however, the phosphohpid/protem are unaltered similar to the compositional units of the multi i and cholesterol/protein increased (18). Thus the layered membrane arrays This similarity suggests l~ membranes low in cholesterol and high m phos- that the reorganised cisteraae of the rough ER are pholipid composition, which are characteristic of incorporated into the arrays of the concentric 5 chlorinated trip henyl induced microsomei, likely paired membranes Continuity of these arrays represent the concentric membrane arrays. with the rough and smooth ER further support a Structures similar to concentric membrane postulate of a rough ER derivation i arrays have been observed following the adminis- Enzymatic and compositional changes of the ' tr&tion of numerous other compounds including microsomes suggest the modification of the ER v phenobsrbital (16), thiohydantoin(20), tnpara- may be correlated to several probable functions 11 nol(21), aflatoxin(22), ethionine(23), carbon tetra- Consistent with the increased proportion of chlonde(24), Rnd ammo aso dyes(2o). An phospholipids within the membrane structure is evaluation of the sequential changes that occurred the proposal that the proliferated membranes following consumption of the chlorinated aromatic provide a site for storage, for isolation, or for hydrocarbons suggests that arrays of concentric contact with membrane enzymes of the hydro paired membranes originate from cisternal ele phobic chlorinated aromatic hydrocarbon The ments of the ER Modification of the rough ER increased proportion of phospholipid enhances the included reorganization of parallel cisteraae into suitability of the membrane to sequester the sinuous cistemae and concentric annular profiles foreign material The proximity of the concentric April 1972 HONS 200230 141 Table 1. Hepatic alteration* in rat* fi chlorinated trtphcoyl foe 21 day*. Cont Exp Liver weight (g/100 g body weight) 3 00* 0 28 Microsomal protein 20 1**0 5 (mg/g liver) KNA 180* 33 (eg)* Photphollpid 455* 24 (eg)* CbDlestetol 44 0*8 9 (mg)* GIucoee-6-photphataac 3 98* 0 51 (nmol P04/16 rain',* Eatereae 9 753 0 (pmol a-nitrophenol/mm ) Aromatic hydroxylaie (m>unol f aoiino- 30 2*8 2 phenol/30 min,)* N-demethylaee 103'19 (mpmol formaldehyde/ 30 mm )* Nitroreductase 32 6*6 8 (rnmnol p amino- bento*te/hr ) 8 63 0 79 54 54 1 63 74 3 50830 17 9l 5 1 080 08 10 31-21 6 06 0 29 1867 45 4 6 2 * Difference between mean and following mean it sig nificant (p < 001) * Difference between mean and following mean it sig nificant tp< 01) * per mg protein. membrane arrays to lipid droplets, also a probable location of the lipid soluble compound, provides accessibility of the lipid component of the membrane to the compound. Hydroxylation activity present within the microtomes converts a lipid soluble hydrophobic compound into a form which has the potential to bind with activated glucuromde (uridine diphoephste glucuronic acid) to form a glucoeiduroaate, The resulting water soluble conjugate could then be excreted through the biliary or urinary system, Although the specific activity of this entyme is reduced, the overall activity within the liver is substantially increased over the control value. Cholesterol within the membrane structure has been related to the rigidity of the membrane by its ability to condense the unsaturated fatty acids of the phoepholipidB(26). Lack of cholesterol suggests a less stable structure or, alternatively, regions available for insertion of other compounds, such as the chlorinated aromatic compounds among the fatty acids and the phospholipids Thu* the eniymatic and compositional alterations of the newly formed membranes suggest a membrsne suited for localising the chlorinated aromatir hydrocarbon for possible sequestration of meta bolites for excretion (Supported by NIH Grant ES00472) RSFERENCES 1. Norback, D. H and alien, J R KH3i> Morphogenmn of toxic faMaductd concentric membrane arrive in tit bepatocytee Lab Invest 30 338 2. Nnrback, D, H. and Allen, J R (In press) Chlon. noted triphenyl-indticed extension* of the hcpaiie endoplasmic reticulum Proc. Soc, Exp Biol *od Med 3 Chlortetracvcltne ChJorpromaame Chloropropamide 4 Aroclor Product*, Moneanto Co , St Louu, Mneoun 5 DDT (), 1, l-Tnchlnfo-2,2-bi*;* ehlorophenvl) etta- *ne 2, A, p(2, A-Dichlorophenoxy) acetic acid Chlorbenaide. 8 Woodwell, G. M , Wurster, C F and lamcson, P A 1007 DDT residue* m an Ea*t Coast estuin a caw of biological concentration of a persistent insecticide Science 136- S21 7. Rwebrough, R W it il 1968 Polychlorinated bi phenyls in the global ecosystem. Nature 220 1W8 8. Holmee, D C, Simmon*, J H and Tatton, J O'G 1967. Chlorinated hydrocarbons m British wildlife Nature 2|6 227 9. Holden, A V and Metsden, K 1967 Organochlorio* pesticide* in eeala and porpoteee. Nature 216 1274 10 Biros, F J , Walker, A C and Medberr. 4 1970 Polychlorinated biphenyl* m human adipoee tiaeue Bull Environ Cantnmin. Toxicol. 5. 317 11 Pltmmer, J R and Klingebie), V I 197] Riboflavin photosenaitised oxidation of 2,4-dichforophrnol n eeesment ol possible chlorinated dioxin formation Science 174 407 12. Entyme incubation condition* and product reactions according to Gram, T E,, Roger* L A ami Fouls J K. 1067. J Pharmacol. Exp Ther 155 470 name 3 nM p-nitrobenioic acid, 100 oM aniline and 0 m,\l aminopyruic 13 3 5 mM a-nitrophenyl acetate incubated at p!! 6 5 in citrate buffered solution, change in ahaorbnnci at 420 m* measured 14 40 mM gluiuee-d-phoephalaae incubated at pH fl 5 in citrate buffered aolution, inorganic photphaic mm*, ured according to Bagmaki, ft S , Foa, P and v5h k ti 1967 Clin Chem 13 326 15 Munro, H N and Fleck, A 1966 In D Click <ed) Method* of Biochemical Analysts, Interscienrc Publiahers, New Y'ork, 14 113 16 Lipid extracted according to Polrh, J Lee* M nod Sloane Stanley, G H 1957 J, Biolog Chem 22i, 497. 142 EnvironmentaI Health Perspective* HONS 208239 the the the int IlK tu rn on lt|r 0ti iri (h id A i*r * t; v Digmlion of organic material and determination of inorganic phosphate according to Bagtnaki, E $ et at, Ref 14, Cholesterol aepamted according to Shin, Y S i mdlice, J C 1062, Clin Chem 6 508; and quantified according to Click D , Fell, B F and Sjolin, K 1064 Ann! Chem ,16 1110 17 Determined bv method o( lown, O H. et al 1B56 J Biiilog. Chem 103 265 with bovine aerum albumin u alandard Ik Glaumann, H nnd Dallner, F 1068 Lipid competition and turnover of rough and am noth microaomal memhranea in rat liver J Lipid Rea 0 720 19 Burger, P. C and Herdann, T B 1066, Pbenobarhitalmiluced fine atructural changes in rat liver, Amer. J, Path 48 703 30 Herdnrn, P B and Kaltmliarh, J P 1065 Electron mirroacope studies on enxtme activitv nnd the isola tion of thinhvrfftnt/nn-inHnced myelin figures in rat liver J Cell Biol 25 485 r k 21 Hruban, Z , Swift, H and S]sacra, A. 1065 Effect of Triparanol and Diethanolamine on the fine structure of hepntocytcs nnd pancreatic acinar cclle, Lab Invest 14 1652 22 Svobodn, D , Grady, H J and Higgwaon, J 1066 Aflatoxm U in rat and monkey liver Amer J Path 40 1023 23 Steiner, J W , Miyai, K and Phillips, M J 1964 Electron microscopy of membrane particle nrriiig in liver cells of ethionine-in toxicated rata Amer J Pathol 44 169 24 Stenger, R J 1006 Concentric lamellar formations m hepatic parenchymal cells of carbon tetrachloridetreated rats J Ultraatr Res 14 240 25 Smuckler, E A and Arcasoy, M 1060 In G W Richter and M A Epstein (eds) International Review of Experimental Pathology. Academic Preen, New York, 7- 305 26 Deenen, L L M van 1066 Phospholipids and bio membrane Progress Chem Fata. Lipids 8' 1 U e in i i i* * ( \ n [} ki 5i iK h \ ii k i April 1972 HONS 208240 143 ^Hfiroftwintgl Hpmlth Piripcrtturt Biological Effects of Polychlorinated Biphenyls in Rats and Quail by Joel Bitman, Helene C. Cecil and Susan J. Harris* Polychlorinated biphenyl* (PCBa) have been administered to rats and to quail to discover the nature of the biological effects which these environmental pollutants produce in mammals and birds. Biological effects evaluated were1 (1) estrogenic activity of the PCBa in rats, (2) alterations of pentobarbital metabolism induced by the PCBs as judged by sleeping time of Japanese quail, (3) alterations of liver vitamin A, liver lipids snd liver weight induced by Aroclor 1242 in rats and quail, and (4) egg production of Japanese quail Ertricink Activity Estrogenic activity was assessed by determining the stimulation of the uterine glycogen response of the immature rat uterus 18 hours after administra tion of the test compounds) The potency of active compounds is reported in terms of the minima', subcutaneous dose which will increase glycogen to a level significantly different from control. In a senes of 11 polychlorinated biphenyls snd terphenyls, the compounds containing up to 48% chlorine were estrogenically active (Table 1). A polychlorinated terphenyl containing 42% chlorine was found to be 8 times mere active. This is a low level at estrogenic activity and probably arise* from hydroxyl*ted metabolites during in vivo metabolism. * United States Department of Agriculture, ARS, Belleville, Maryland 20705 Efficts ob Pontobarbital Motaboliui h Japanis* Quail The polychlorinated biphenyls have been demonstrated to stimulate liver ensyme systems which metabolise barbiturates in rats(2) We utilized a sleeping-time test with pentobarbital in quail as a means of determinmg effects of poly chlorinated biphenyls and terphenyls on liver microsomal ensyme activity The liver is the major site for the metabolism of foreign chemicals, and u standard dose of pentobarbital will produce a standard sleeping time. Stimulation of the liver enzymes which metabolize the barbiturate will be reflected in a shorter sleeping time, and conversely, any inhibition of the liver enzymes will be reflected in a longer duration of pentobarbital hypnosis (i) The PCBs and PCTs were tested for their acute or short-term effects (48 hours) and for longer-term effects at 3 and 7 days At appropriate times after administration of the PCBs or PCTs, sodium pentobarbital waa injected intramuscularly at a dosage rate of 50 mg/Kg body weight. Birds were considered asleep as long as they could not right themaelves when placed on their backs A single dose of Aroclor 1268 or Aroclor 5460 administered orally at a dosage level of 100 mg/ Kg to mature male or female Japanese quail initially increased sleeping times (Fig. 1) At 18 hours, however, sleeping time* were less than untreated control values in the mate quail, and by 48 hours, the PCBs and PCTs had stimulated the liver enzymes which metabolize pentobarbital, so that sleeping times were only about 50% of control sedation times In the female quail, there was little effect of the single oral dose after 5 hours In both male and female quail, Aroclor 1268 appeared April 1972 HONS 208241 145 Table 1. Eetrogenic Activity of Polychlorinated Biphenyl (PCB) and Terphenyl (PCT) Compound*. PENIOSARS1IM SlEEPlNG Tit in Qu*u X'nne Minimum effective dose mg PCB Aroclor 1221 21% Chlorine PCB Aroclor J2J2 32% Cl PCB Aroclor 1242 42% Cl PCB Aroclor 124a 48% Cl PCB Aroclor 1254 f>4% Cl PCB Aroclor 1200 80% Cl PCB Aroclor 1282 82% Cl PCB Aroclor 1288 08% Cl PCB Aroclor 4485 80% PCB, 4P% poivrhiormateil tnphenvi 1 PCT), 65% Cl PCT Arorior 6442 42% Cl PCT AtocIot S40O 80', Cl 8 8 8 8 Inactive Tnictive Inactive Inactive Inactive 1 Inactive to exert greater effects than Aroclor 5460 The diphasic pattern elicited suggests that the PCBa and I'CTs first inhibited the eniymea which melnbohxe pentobarbital, but then, at later times, stimulate the induction of greater ensymic activity. Continuous feeding for 3 and 7 days at levels of 100, 300, and 02.3 ppm ad libitum stimulated the metabolism of the standard dose of pentobarbital HOURS POST INJ HOURS POST <NJ Fionas 1 Sleeping time* of quail fed l'CR 12W or n fi 5400 Each point rrpresrnl* n\ iiuml (Fig. 2) Sleeping times were about 30-70r~( of control levels, thus indicating great ci metabolism of the barbiturate Higher dose levels everted greater effects and. again. Aroclor libs appeared to be more stimulatory than Arocloi ,3-tOf) Sleeping times were not decreased to as gieat an e\lem m the female quail as in the male The sleeping time pattern elicited by PCBs and PCTs is in marked contrast to that "Inch the organochtonne pesticide analogs of DDT produre in quail(3), DDT and its analogs produce an EFFECT OF AROCLORS ON STEEPING TIME IN QUAIl PC5 1266 100 90 80 70 60 50 40 MALE PCT 5460 female Flaunt 2 Sleeping time* of quail fed PCB 1208 or PCB 5400. Each point represent* six quail 14b Environmental Health Perspective* MONS 208242 f Table ! Liver Change* in Male and Female Rat* after Feeding Ad Libitum for Two Month* Diet* Containing 100 ppm Aroelor 1142. t ______________ _________________ rfj Treatment Liver wt Liver lipid Vitamin A n I8E %SE iif/f fiver mg/liver 1 Control Arodor 1242 Conin'! J Aroelor 1242 p<001 50 p < 0 00J I pcoooi. Female rale 10 6 4 2 3 I5 04 479 23 4 54 29 10 7 4 3* 3 46 08b 244 15* 2 26 11* Male ret* 10 12 2 4 9 14 it 12 564 20 6 82 22 s 13 0.S 7 12 18* 270I5* 3 46 21' `CM initial inhibition of pentobarbital metabolism, and possibility that PCBs would produce simitar effects sleeping times are longer during tbe first week of was investigated in rats by feeding Aroelor 1242 of feeding in males and during the first 3 weeks of for 2 months at a dietary level of 100 ppm sni feeding in female quail. (Table 2). In both male and female rats there was oil ivI Effects on liver Vitamin A in Rati ml Quail an increase in liver weight and liver lipids There was a very striking decrease of about 50% in the hi: liver concentration and content of vitamin A in Repeated exposure of rats to DDT results in Aroelor 1242, fed to male Japanese quail for 2 degenerative changes in liver tissue, an increase in months at a dietary level of 100 ppm, produced id liver sise and an increase in liver lipids(4), and a similar effects, an increase in liver weight and ie decrease in liver storage of vitamin A (5). The 'c n J55 6 or LIVE* VJT * CONC IN QUA* FEMALES LAVING TOTAL LIVE* V1T A IN OLMIL FEMALES LAVING 3,0 6 z 75 w&at 50 25 0 DDT PCI 13*3 DDT PCI J343 DDT PCB 1343 Fiocre 3 Liver vitamin A concentration) of quml fed 100 ppm DDT or PCB 1242 for two monthi Groups contained 14 to 20 quail 1143 1343 13*3 Floe a* 4. Total liver vitamin A of quail fed 100 ppm DDT or PCB 1242 for two month*. Group* contained 14 to 20 quad. April 1972 MOMS 208^.43 147 iff-*** EGG PRODUCTION LOW CALCIUM DAYS FiquRe 5. Egg production of Japanese quail fed PCB 1242 A verier production for three day period! in plotted for group* of 1ft quail lipids, and a large decrease in liver vitamin A (Fig 3 and 4) Female quail that were laying did not show consistent changes During the ovulatory cycle, large amounts of vitamin A stores are mobilised to provide the vitamin A for the yolk of the eggs Because of these large, almoet daily FUU CALCIUM DAYS Fionas 7 Egg production of Japanese quail fed PCB 1254 on a low calcium diet Average production (or throe day period* n plotted for group* of 16 quail DAY 5 Fionas 6. Egg production of Japan*** quail fed PCB 1254 on full calcium diet Average production for three day period* n plotted for groups of 16 quail. changes, variability in liver vitamin A levels is great. In order to determine whether PCBs exert similar effects in female quail, a group of young females were kept in the dark, from the time they matured (39 days of age), and fed Aroclor 1242 for 2 months. Under these conditions, egg-laying is inhibited, and the cyclic mobilisation of vitamin A for deposition in the egg yolk does not occur In the birds kept in the dark, Aroclor 1242 caused about a 50% reduction in liver vitamin A (Figs 3 and 4) Effects ii E(t Pretectal The effects of Aroclor 1242 and Aroclor 1254 on egg production was studied in Japanese quail Aroclor 1242 was incorporated into an adequate calcium laying mash at a level of 100 ppm for 00 days. Egg production (Fig, 5) was similar for controls and Aroclor 1242-fed birds during the first 40 days (>90%) Average egg production in the Aroclor 1242-fed quail declined to about 71^ during the next 3 weeks. The number of mem branous and broken eggs in the Aroclor 1242 group was over 2 times that of the control quail Environmental Health Perspective* HONS 206244 Aroclor 1254 was fed at a level of 100 ppm in materials containing compounds which differ so both a full calcium (2.55%) and low calcium greatly in chlorine content and m chemical and (0 (ifl%) diet Egg production was not suppressed physical properties More extensive research on by the PCBs in either case (Figs 6 and 7), individual PCB compounds must be undertaken to i although there was a negative effect of the low assess fully the biological effects of polychlorinated calcium diet. There were no differences in the biphenyls on mammals and birds numbers of broken or membranous eggs attribut able to Aroclor 1254 REFERENCES A depression in egg production of White Leg 1 Bltmnn, J and Cecil, H C 1070 Estrogenic sctivitv of horn hens fed Aroclor 1248 was recently reported by Scott et al.(6). Heath et al.(7), however, found little effect of Aroclor 1254 on eggshell thickness or reproduction in mallards or bobwhite quail. An DDT analogs and polychlorinated biphenyls J Agrrc Food Chem, 18 1108 2 Street. J C et el 1969 158th Fall Meeting American Chemical Society, New York City Abntrset Sept 7-12, 1909 effect upon egg production and reproduction of Aroclor 1242 and Aroclor 1254 has been previously reported in White Leghorn chickens(8). Recently, Prakall{9) found no effects upon eggshell weight in Ring doves fed 100 ppm Aroclor 1254 Thus, there 3. Bitman, J etil 1971. Comparison of DDT effect on pentobarbital metabolism in rats and quail J Agnc Food Chem 19 333 4 Laug, E P et al 1950 Liver cell alterations and DDT storage in the fat of the rat induced bv dietary level cf 1 to 50 ppm DDT J Pharmacol Exp Ther 08 268 is a notable lack of consistency in the published 5 Phillips, W E J 1903 DDT and the metabolism of reports of the effects of PCBs cm egg production and reproduction in birds Vitamin A and Carotene in the rat Can J Biochcm Physiol 41 1793 6 Scott, M, L. et si 1971 Proc, 1971 Cornell Nutrition CaocIvsiM Conf Feed Manufacturers, Buffalo, New Fork, Novem ber 2-4, 1971 7 Heath, R G et al 1970 Pror XV Tnt Omithol [!H A study was made of the effect of PCBs on Cong , The Hague Aug 30-Sept 5, 1970 E J Brill estrogenic activity, liver barbiturate metabolism, Leiden In cr vitamin A and egg production using a limited 8 Industrial BIO-TEST Laboratories, Inc Rpt No J7300, June 4, 1970 ]h number of PCB materials. It appears that the 9. Peak all, D B 1971 Effect of polvchlorinaled hiphenvls *rt biological effects of specific PCB compounds are (PCBs) on the eggshells of King Doves Bull Environ tig different, as might be expected with this series of Cont&min. Toxicol 6 100 Cy or is A 'le ut h in :i ,0 0 >r ie n P Envuwimafttai //m^A Pifipninwi PCBs and Interactions with Insecticides by E. Paul Lichtenstein* The effect* of polychlorinated biphenyl plasti cizers, like those of many other environmental chemicals ("synthetic chemicals to which man is exposed by either voluntary or involuntary means''), on biological systems are not too well understood. When research by our group at the University of Wisconsin indicated in 1969 (1) that several of the Aroclor compounds increased the toxicity of DDT and dieldrin to insects, their potential biological interaction with environ mental chemicals was indicated, Since then further studies were conducted in our laboratory to investigate the effects and interactions of environmental chemicals on human cells in tissue culture by determining the toxicity of some of thceo chemicals on HeLa cells and skin fibro blasts (2) Aroclor 1254 (a polychlorinated bi phenyl plasticiser), carbary 1, parathion, DDT, several insecticide metabolites, aspirin and caf feine were used to determine their effects on cell growth and synthesis oi proteins and nucleic acids. The dosage causing a 50% inhibition in culture growth was determined, and the interac tion or combined effects between these chemicals relative to their effects on protein and nucleic acid synthesis was studied. Both cell types re sponded nearly equally to the presence of an individual chemical. Aspirin and caffeine were 8 to 20 times less toxic than the other chemicals studied, while Arcolor 1254 was as toxic as DDT (Fig. 1). Parathion was more toxic than its me tabolite paraoxon. Para-nitrophenol was as toxic as parathion. 1-Naphthol was less toxic than its parent compound carbaryl. The toxicitiea of DDT and DDE were dependent upon ceil type. Little significant interaction on nucleic acid or protein synthesis was seal (2). * Department of Entnmologv, University of Wisconsin, Madison, Wisconsin 53706. Utilizing insects as the test objects, the bio logical interaction between plasticizers and some insecticides was also investigated (1) Because of the structural similarity to DDT-related com pounds, 11 plasticizers were tested for their toxicological interaction with dieldrin and DDT Many of these polychlorinated biphenyl com pounds were toxic to Drotophda mdanoqatter M eigen and house flies, Aftuea domtihca L, but to a lesser extent than dieldrin or DDT. Their toxicity increased with a decrease in their chlorine content Dieldrin, DDT, or any one of the less chlorinated and more volatile plasticizers (Aroclor 1221, 1232, 1242, 1248, 1254) were toxic to both test insects (Table 1), While these polychlori nated biphenyls contain increasing amounts of chlonne, their toxicity towards the insects de creased Dieldrin was the most toxic of the com- Fiocnx 1 Dosage-response curves obtained after 48-hour exposure of human celi cultures to various concentrs* lions of p,p'-DDT, Aroclor 1254 (PCB). aspirin and caffeine (Ref 2) Mule' ial None' Dieldrin p,pr-DDT Aroclor 1221 Aroclor 1232 Aroclor 1242 Aroclor 1248 Aroclor 1254 Afoclor ) 260 Table 1. Effect of plaaticlcere or i oaacticidn on iaaert ntoitajitiea. D meianogaster Applied* CT 'V mortality <*> 24 hr 48 hr Applied' (**/* fly) 01 30 200 800 200 800 200 800 200 800 2000 2000 00 31 84 24 58 00 57 02 00 35 64 04 40 73 00 15 45 00 00 ln 25 0 500 1000 500 1G0U 500 1000 500 1000 1000 1000 Onfii'a i 2t W y 43 |M 3n 17 Vi 13 13 m n * Applied in 8 ml hexane to *-oi flue jari. D, mlanogtuter exposed to dry residue. k Applied topically to houae fliee in 2 ulilsr of acetone. Weight of I fly 20 mg. Control* were treated with solvent* only pounds, as measured by the percent mortality of the insects within a 24- or 48-hr exposure period. Approximately 25 and 30 times more DDT than dieldrin, and 8000 and 1000 times more plasti cisers than dieldrin had to be used to obtain similar mortalities of D. meianogaster or of house flies, respectively, within a given time of expoeure to those chemicals. The more highly chlorinated and less volatile plasticisers were nontoxic even when used at dosages of 2000 pg with D. melanogaster and 20 pg/Ry (1000 pg/g fly) with house flies. Table 2 summarises the results obtained after the insects had been exposed to sublethal dosages (200 sg for D. meianogaster, 10 pg/house fly) of either one of the plasticisers (Aroclor 1221, 1232, 1242, 1218 or 1254) and in combination with dieldrin or DDT. Though no appreciable insect mortalities were observed with plasticisers alone, they significantly increased the toxic effects of both dieldrin and DDT This effect was most noticeable with DDT on house flies, and differ ences observed were highly significant. After a 24-hr exposure period, 38% of the house flies had died after only DDT had been applied However, all the insects were killed when DDT was applied in combination with any one of these plasticisers The polychlorinated biphenyls also increased the toxic effect of DDT with D mdanogaprr, but to a leaser extent. The most striking effect was observed with Aroclor 1221 Aroclor 1280, 1262, 1268, 1265, 5442 and 5460 applied with dieldrin or DDT to glass surfaces to which D melanogasUr were exposed reduced the toxicity of the insecticide, resulting in mortalities of 30% to none within 48 hr (1). In particular, though, a substantial increase in the toxicity of organophosp horns msectieida to houseflies was achieved with polychlorinated biphenyl plasticisers (3). Aroclor 1248--although nontoxic by itself to house flies at dosages of 10 jig/fly--significantly increased the toxicitv of the oxygen analogs of five organophosphorus insecti cides after simultaneous topical application to houseflies. Both p,p'-DDT and p,p'-DDE, applied at sub-Jethai dosages, also increased the mortality of houseflies due to paraoxon from 2 to 60 and 73 per cent, respectively The polychlorinated bi phenyl plasticiser, applied simultaneously with parathion, however, decreased the toxicity of parathion to houseflies but increased mortalities when applied to flies one-half hour after the appli cation of the insecticide The plasticiser apparently inhibited the conversion of parathion to its toxic 152 Environmenial Health Perspectives HONS 2082A7 Ttbk I. Combined lfect* or plasticisers end inariileidks on tneecli. m-irtulitie* of insects after exposure to r inM'i lictde Dicldnn ji.p'-DDT 1) metanogatier after 48 hr* Vdrt (i <1 08 6 87 4* 59 *r, 035` (i 813' 84 6' 'tj 5*2 779 .1' If 72 4 83 6* 77 3* ,:a (1 103* 473- House flies' after 24 hr V'Or (1 275 38 8 i^l 4 'J LJ 4 II 49*3' 27 5 47 5* IOC* 100* 98 34 0 5811* 10C< : 11 24 0 10C* > ficlnviQiutir' were exposed in 4-o* glass jars to the In riser of the chemicals plasticisers, 200*g, i M*, DU I', 3Lt,i* htwwv tan * about plasticisers contained 250 *g of " fl)| ffffll " liiffrrrnrf.'. <>)>tained between treatment! with the -- |utr onli niul those with insecticide plus plasticiser i- aniticaiii at tin c 1%, d "0. l't, or e"5% level ' la-Rurnlh wen applied topically Hi 2 jiliter of acetone nrflir* plimbnser* 500 jig/g flj ; dleldrin, 0.75 *g/g DDT, 15 ft e fly Control only acetone was applied 1 <!!! of 1 8\ - 2l m*. ititatton product, paraoxon, and the breakdown ,unioxou into nontoxic compounds. This was uv indicated l>> the recovery of larger amounts of "C-paraoxon and smaller amounts of water soluble 'Mil-products from flies treated simul taneously with Aroclor 1248 plus paraoxon, in companion to tlioee which had been treated with ,4C-paraoxon only Utilising the 10,000XG supernatant from housefly homogenates, the addition of Aroclor 1248 with '*C-paraoxon in vitro also resulted--after 2 hours incubation at 30*C--in higher recoveries of "C-paraoxon and in a reduction of the appearance of water soluble HC-producte. Identical results were obtained with subcelluiar fractions (10,000XG superna tant) from flies that had been treated with Aro clor 1248 eighteen hours before homogenate preparation. No difference was observed in the degradation of paraoxon or the appearance of water soluble products with Aroclor applied to the living flies or to the 10,000xG supernatant of homogenates from untreated flies (3). Since plasticisers are in the environment, their potential effects on biological systems, especially in combination with other synthetic chemicals, should not be disregarded. REFERENCES 1 Lichtenstein, E. F et si. 1009. Biological interaction between plasticisers and insecticides J Econ. Entomol 82. 761. 2 Litterst, C L. and Lichtenstein. E. P 1971 Effects and interactions of environmental chemicals on human cells in tissue culture Arch Environ Health 22 454 3 Fuhremann, T W and Lichtenstein, E. P. 1972 In crease in the toxicity of orgsnophoephorua insecticide* to houseflies due to polychlorinated biphenyl plasti cisers. Toioeol Appl Pharmacol (In press). in 1971 HONS 20824S 153 Environmental Hnith Polychlorinated Biphenyl Interactions with Tissue Culture Cellst 5f Roger Hoopingarner, Albert Samuel, and David Krause* The ncccssfiil use of tissue culture cells in , firal work is now almost GO years old. How the applicability or the "fitness" of these to demonstrate biological principals is still s*rd It is well known that many of the cell - War little resemblance to the parent tissue urftiin cells Yet isolated functional cells of 5.l\-propiii<nied clonai strains, that can per il specialited, organ-specific functions for Biped periods, have been produced in nearly ilcvs numbers (1). The loss of specific func- has been ascribed to (i) selective overgrowth connect he tissue cells, (n) a phenotypic mpr m the cultured cells, or (ni) inadequate harmful environmental conditions (2) In W to cirrumvcnt these problems, primary lines are used and tested before the line hi<t>ts' to culture conditions through aneuiih or other means Tiieuse of tissue culture cells for the examinan of environmental chemicals has been rela'wh recent (3) The effects measured have - ii) inhibitory dose, (ii) toxic dose, (iii) '""io\iv effects, and (iv) chromoaome aberra'ii' Samuel (4) has used both established and ,-.-nin cell hues also to study the metabolism i certain pesticides. Lrtterst and Lichtenstein uvinp Hel.ii cells and a cell line of non-malig^.ii origin found that the interaction of DDT v I l`( H> [ tioelor 1234) that had been fmind m * i' "us hIiwiu They concluded that the various ` t c.'iii rilmi i,,r, from the Michigan Agricultural Expenf' * '1 ilmti ( trnclc :iKJ2) J'his research wax iiipfiorted i 'o lo fiiml' proMiled hv the Food and Drug Admini- 1 Ui..[, lull W imrtcr eontrnrt FDA 71-2SS lx 'irinu of, [ l.HiHtniilogY MichignnSuitr Fnneraitv, * Mulligan 48S2i interactions of chemicals with the cell lines had no correlation to intact animals The use of human primary cell lines was en hanced greatly with the discovery of the mitotic stimulus of the mucoprotcin phytohaemaggiutinm (.3). The leucocyte culture techniques developed by Moorehead et al (G) proved to be most suit able for m vitro cel! studies of human cells and chromosomes The method also allows an in vivo check for chromosome effects (7) and, as such, may offer a comparison of in vivo with in vitro effects on essentially the same cell. In the studies to be reported here, w-e used Chinese hamster cells (quasi-diploid epithelial cells, CH-461) and primary human lymphocyte cells. The Chinese hamster cell line has a genera tion time of approximately 24 hours. They were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum. The lymphocyte cultures were obtained from blood of laboratory volunteers and were grown m chromosome medium 1A from Grand Island Biological Company. The cell cycle time varies with individual donors but is approximately 24 hours after a "lag" period of from 24 to 48 hours prior to activation. The PCBs were obtained from the Monsanto Company and were used without alteration The gas chromatography of the PCBs was ac complished using a 50 foot XO 02 inch SCOT column of Apienxon L at 200C Detection was hy hydrogen flame ionisation detector The growth suppression of Chinese hamster cells with PCBs and DDT are shown in I'ig 1 These data are comparable to those found by Litterst and Lichtenstein (3) though they did not directly study an lDt0 using Iwth chemicals ytl WT2 155 (0 lO HOURS so Fior*t 1, Growth of Chinow Hun*t*r otllft with PCB and DDT simultaneously. The toxic doae of the chemical waa several times greater than is usually found biologically. It ia of some interest to note the disintegration of ceils in the period from 24 to 30 hours, when the cells were being streseed from culture condi tions. This same phenomenon can be Been from the differences between 24 and 30 hours in Table 1. Hus Table gives the relative growth informa tion for the different PCBs. As you can see, there waa a steady drop in oell numbers as one de creases the percentage of chlorine. The Aroclor 1016 is s distillation product of 1254, which essen tially excludes the biphenyls of five chlonnee Tahto 1. Population* of Chlneae Hamster Cell* Cultured with SO ppm of Various PCB'* PCB 24 bn* 30 hn Non* 1280 1254 1248 1221 1018 355 0 275 8 23 5 S !88 1 188 4 4I 1 364.4 7! 7 S1 7 61 4 11 3 4 10 4 * Average of ten replicates Count of oelb/unit ocular grid area. 156 Fionas 3 Gas Chroma topersm of \rockir 1232 U M # * *| ti Ficuss 4 Gas Chromatogram nf Aroclor 1242 44 >H a* W Flocal 5 Gas Chromatogram of \rodor 1254 Environmental Health Perspectives HONS 208250 Table 2. Human Lymphocytes Treated with 100 ppm Arodpr 1254.* Pmod of PCB Mitotic Treatment Index Satellite Aaeociation Chro matid Gap Chro matid Breaks Tut 1 0-24 bre, .032 9 5 1 44-52 hre, 009 15 3 1 riomt 6 Gm Chromatogram of Aroclor 1010. 40-52 hra 017 3 4 0 Acetone check 022 e 3 0 Control cf above The product is still a biphenyl, but 012 4 5 1 t U't it is derived differently, the prefix is changed, Tut t 11 -vn 12 to 10 Baaed on the percent chlorine, 0-24 hr*. 017 10 1 0 l-wtor 1016 falls out of the pattern of toxicity. 44-62 hrs 007 4 4 0 ,, uni look at the gas chromatographic patterns, 49-52 hr*. 012 4 2 1 * toxicity is somewhat of an anomaly. For Acetone check 009 4 3 0 'ample, the Figures 2 through 6 are the chroma* Control 008 8 2 1 trims for the various PCBs using the support* tied open tubular, or S.C.O.T., columns, * 50 cells analyssd/tt/lreatinent Thr pattern of 1016 was very much like that end for 1232 or 1242 yet 1016 was several fold we toxic to the tissue culture ceils. It would ippmr that there may be certain components at ire in higher proportion in the 1016 product, uid these components (or component) are causing iir increase in toxicity. We have fractionated *et of the chromatographic peaks into their -Hmponent parts and are currently assaying them > determine the relative toxicity of each. The human lymphocyte culture* are of course > primary cell line, and in fact we examine the 'rile cylologtcally at the first m vitro division. The celts are shown diagramatically in Fig 7 Within the body, the- lymphocyte is in a diffferentiated state Go. These cells are then acti vated or transformed into a division cycle with phytohemagglutinin. Sasaki and Norman (8) have shown that the small lymphocyte finishes its transformation in 24 to 48 hours after stimu lation and begins a penod of cell proliferation with a generation time of 22 hours, Gi, 5, Gi, and mitosis are 6, 11, 3, and 2 hours, respectively We treated these cells with Aroclor 1254 at 100 ppm for the first 24 hours and again during the last eight (S plus Gi), and the last three hours (mitosis). During the first 24 hours, the cells would be in some stage between Go and Gi. The cells were then examined cytologically for chromo some aberrations for the various stage treatments The results are shown m Table 2 PCBs seem to have no effect on chromosomes under these conditions. The results of this study would indicate that established tissue culture lines are more sensitive than primary cultures to at least one PCB mix ture. The various PCB mixtures are generally more toxic as one decreases the percentage of chlorine. The distillation product 1016, however, was more toxic to these cells than was indicated ficrim 7 Diagram of Cultured human lymphocytes at firet in vitro division from the percent chlorine content or from its gas chromatographic pattern At 100 ppm of April W2 * HONS 208251 157 1254 to human lymphocyte cultures, there was no apparent effect to the chromosome integrity as measured by cytologtcal evidence Tissue culture systems may not always be indicative of whole body conditions. However, these systems do offer some uniform conditions for many definitive studies of environmental chemicals With substantial data for comparative tests they may be good indicator systems. The human lymphocytes system also offers the com parative cytological test for both m vivo or in vitro exposure. REFERENCES 1 Yasumura, Y,, Taahjian, A H and Sato, C H. 1966 Establishment of lour functional, clonal strains of animal cell* in culture Science 154 1186 2 Eagle, H 1905 Metabolic controls m cultured mam malian cell* Science 148 142 3s Gubliks, J 1965 Res|miie uf cell cultures tu insecti cides 11 Chronic toxicity and induced resistance Prw, So<- Exp Biol Med 120 108 b Gablika, J 1965 III Mli-red riiweptil.ilm i. virus and diphtheria toxin l'rm |i, vi- 120 172 c Gabliks, J and Friedman I, l'*"i j .. cultures to maeftifulcv | Anile tnsinu i.. i cells Proc Soc Ex|i ]lu<| Mnl 1211 lui d Litterst, C L and I,u iilcnlrm V I' l11" I t* and interaction* uf cm iron mental chemirnl. mh n > celt* in tissue culture treh Enurnn if,ilil, .*_ ( , e Hoopingaroer, R and Mourner A \t I'tTn L.n,].t. cyte chromosome annhi r>f pe'lirnle m>~ i dividual* VII fnt Cong Plum Pruteriiun l\r,. 4 Samuel, A, 1971 Effect of malathion Dt) tt'l i}| w md mimintliui tnwc culture PhD Thr-i^ State University 5. Nowell, P C i960 Phytohaemnggiutinin on miut of mitosis in cultures uf normal human leunv,,r. Cancer Res 20 462 6. Mootehead, P S eta! 1960 Chrotnovimr j>rrj>inii,.<. of leucocytes cultured from human peripheral t>|...| Exp Cell Res 20 613 7 Krause, D, H 1970 The rvlolngiral efieri- of p-iin ir on human chromosomes Hu Theus Ms hem -im University. 8 Saaakl, M S. and Norman A l%ti Proliferate* -l human lymphocytes m culture Nuturc 210 .Hi. 158 Environmental Health Per.pecti'e* MOHS 206252 Toxicities of PCBs to Fish and Environmental Residues by David L. Stalling and Foster Lee Mayer, Jr, Polychlorinated biphenyle (PCBs) have been and in fish and wildlife from many parts of hr orld at levels which may adversely affect rustic organisms and interfere with pesticide --iduc analyses (l, 2, 3, 4) The environmental rurrcncc, uses, and present toxicological aspects < PCBs were recently reviewed by Peakall md Imccr (.')), Gustaffson (8), and Rlsebrough Ti PCBs have gas chromatographic characm-iirs similar to many organochlorme insect i- Jensen (8) first identified unknown gas hromatographic peaks as PCBs in extracts of pike and an eagle which were analysed for organo'hlonnc insecticides. The Monsanto Company is the sole manufidurer of PCBs in the United States (6) and markets eight formulations of chlorinated bi phenyls under the trademarks Aroclor 1221, 1i1-\ 1242. 1248, 1254, 1260, 1262, and 1268. The latter two digits designate the percent chlorine cf each formulation. Aroclor 1248 and iJM arc the materials produced in greatest quantities and are used as a dielectric fluid in capacitors and in closed-system heat exchangers '.>) Aroclor 1242 is used as a hydraulic fluid, and Aroclor 1260, as a plasticiser. Chlorinated lerphenyh. are marketed under the trademark Wlor 5442 and 5460. A mixture of bi- and 'ctlihcnvis is designated Aroclor 4465, The 'Innwnlo Company has restricted the sale of I'CBj, for uses in which disposal of the end produris cannot be controlled, such as the use of I'tlh us plasticisers (0) "ChrmiM and Fishery Biologist, respectively. FiohI'vticJr IWurth Laboratory, Bureau of Sport Fisheries 'i i\ ildiifi Columbia, Missouri 85201 Cfeanlcai Compasitiaa and Analysis PCBs occur m the aquatic environment as mixtures of chlorinated biphenyl isomers (10, 11). The biphenyl structure may be substituted with one to ten chlonne atoms, and over 200 com pounds are possible (fi) The isomer composition and chromatographic characteristics of each formulation have been described by Stalling and Huckins (12) and Bagley (13). The PCBs can cause serious interference in gas chromatographic (GC) determination of chlorinated insecticides (4). Analysis of PCBs is best accomplished by GC after separation of PCBs and pesticides during sample cleanup using silicic acid column cleanup (14) No standardised GLC method has been proposed for the analysis of mixtures of PCB formulations in environmental samples. The solubility of these formulations in water have not been precisely determined but is reported in the range of 100-1000 i*g/l (9) Considerable research remains to be done cm the structure of the isomers of each technical materia! and how environmental residues of various Aroclor* are to be reported. Envirinniitat PCB RnMvii Analyses of 40 fish (cross-check sample) from the 1970 National Pesticide Monitoring Program were made using sample preparation techniques which separated PCBs from pesticides and other industrial residues (Table 1). These analyses employed GC and gas chromatography-maas spectrometry (GC-MS) for confirmation. The samples were prepared using gel permeation chromatography (23), and PCBs were separated from the pesticides with silicic acid (14) Only April 1972 WONS 208253 159 Table 1. Composition of PCB residue* In (elected flab (ample* from the 1970 National Peeilcidt n--,. Monitoring Program. Iw River Location Species PCB residue aa Arocior 1i, pc (Hg/g whole |..j, 1232 1248 12.-.4 ,200 Ohio Ohio Ohio Ohio Yotoo Hudson Allegheny Delaware Cape Faar Lake Ontario Misaiaaippi Merrtmoc Cincinnati, 0 Cincinnati, 0 Marietta, O Marietta, O Redwood, Mias PoughVwpwrp. N Y Natrona, Pa. Camden, N J Elisabeth Town, N C Port Ontario, N Y Memphis, Tenn Lowell, Maas Carp White crappie Channel catfiah Channel catfish SmaUmouth buffalo Goldfish Walleye White perch Gitsard shad White perch Drum 10.2 15.9 38 15 6 72 8.6 -- -- 18 9 12 9 11 2 13 8 75 17 23 52 ___ 173 52 80 -- -- -- 75 42 27 It 12 6 14 32 25 68 20 4 r> 45 G1 60 .1 ft 4 ft 4A 4 f 30 11 12 34 32 JJ | fl j`4 |U l 1th one of the forty samples contained less than 1 ng/g PCB residue. The composition of PCB residues in a lake trout and coho salmon were examined by GC-MS and found to be a mixture of Arocior 1248 and 1254 (Figure 1), and residues of PCBs were in excess of the "DDT" residue levels (Table 2). Widespread pollution of the major waterways has occurred, and appreciable PCB residues exist in fish, In view of the PCB bioconcentration in fish, PCB levels as high as 5 pg/1 may exist in the waters of the Hudson River. These levels are certain to have adverse effects on aquatic organisms Tixlcity m4 BiOKcufflKlition Fish Chronic and acute toxicity studies on aqmlir organisms are quite limited Due to the lou solubility of PCBs, tests to obtain 9G-hr L('M values do not adequately reflect their toxtnuc' to fish (Table 3), For Arocior 1221-1268, 90-lir LCm values ranged from 1,170 to 50,000 ag 1 for cutthroat trout (15). The acute oral toxicity of Arodors 1242, 1248, 1254, and 1260 u greater than 1500 mg/kg in rainbow trout Intermittent-flow bioassays of Arocior I24J, 1248, and 1254 to bluegills resulted in 15-d*) Table 2. Pesticide end PCB Keeiduce In a Coho Salmon and a Lake Trout from Lake Michigan. Residue* to ng/g whole fieh Dieldnn p.p' isomers DDE DDD DDT o,p` women --------------------------DDE DDT Total PCB (Arocior)' 'DDT" ------------------------------------ - 1248 125* Tola! Lake trout1 Coho salmon1 Old 0 10 13 0 43 18 08 5.2 23 OB 18 22 7 12 0 16 0 28 n 04 06 83 67 67 13 1 1 Calculated from ratios of characteristic 1248 and 1254 peaks 1 Lake trout (33 L)--taken 10-14-6B. 990 mm, 3590 gm. immature M Taken in 2 5 fathome in Little Bay de Nw Michigan 1 Coho salmon (7Ce)--taken 9-3-69. 975 mm, 5718 gm F. Token in 17-19 fathoms4 miles NW of Manistee, Michigan 160 Enrtr*----*! Health Perspectives MONS 208254 I,( l r> tii 'it u a t jlm vsluea of 54, 76 and 204 Mg/1, respectively 'iblc 4). Exposure* to channel catfish gave -dy LCo values of 107, 127, and 741 Mg/1 for , *me Aroclore. All LCso values decreased jiificwitiy with longer exposures. The 20-day Xu values for Arodors 1248, 1254, and 1260 we 10, 155, and 245 Mg/1 for bluegitls and 300, od 296 Mg/1 for Aroclore 1254 and 1260 with -jnnel catfish at 20C. The toxicity of Aroclor tS to bluegills and channel catfish increased io-foid at 27C. The toxicity of the PCBs to c u mvereely related to percent chlorine (higher wlor number). la chronic tests to determine the effects of tmclor 1242 and 1254 on fathead minnow reprorjctioo, all bsh exposed to greater than 8.3 Mg/1 Table 3. Acute loxiclty- of several Aroclore to selected fiahee. Aroclor Species Temperature 96-hr LCh (C) <M/n 1221 1232 1242 1248 1254 1250 1262 1268 1248 1254 1248 1254 Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Channel catfish Channel catfish Bluegills Bluegilt* 8 9* 8B 86 89 8B 8B 89 89 18 3` 18 3 18 3* 18 3 1,170 2,500 5,430 5,750 42,500 60,900 50,000 60,000 8,000 12,000 278 2,740 1 Static bioaisay. * Aikalmity, 159 ppm, pH 7 6 Alkalinity, 35 ppm; pH 7 1 *wru 1 Composition of PCB reakiues in fish Arrows and tnanalM designate the most characteristic peaks of the Amdor Alt numbered peaks in ehromatocruna of *<e eitrerU are PCBe These numbers designate the sunmrr of chlorine atoms in the compounds as deunained l,y mass spectrometry Temperature proptm rite 4"C/min, He 9 psig, SO'X0 020" id SE8COT column, PE-2708 GC-MS of each Aroclor died. Reproduction occurred at and below 8.3 Mg/1 Aroclor 1242 and at and below 2.8 Mg/1 Aroclor 1254 (17). Accumulation of Aroclor 1248 and 1254 by bluegiils chronically exposed to 2-10 Mg/1 was from 26,300 to 71,400 times the exposure levels for both PCBs. Concentration factors were not strongly dependent upon water expoeure levels, but a direct correlation erf water concentrations and whole-body residues may exist. No major modification of the PCB isomer ratios was ob served in the tissue residues, and no new com ponents were identified (18). Coho salmon fed Aroclor 1254 for 240 days at concentrations of 14.5 to 14,500 Mg/kg body weight per day (dietary concentrations of 0.4 to 680 Mg/g) accumulated whole-body residues which were 0.9 to 0 5 times the exposure levels (300 mg/kg highest residue value). Growth rates were not affected; however, all fish exposed to the highest treatment died after 240 days' ex posure, and thyroid activity was stimulated m all but the lowest treated group (19) Direct water exposures appear to represent a greater haaard to fish than dietary exposures, although both contribute to tissue residues How ever, in the environment the kinetics of residue uptake from dietary sources could be more im portant, since PCBs have a high affinity for **H 1972 MONS 208255 161 Table 4. Intermittent-flow bioauayt of Aroclor against three 6ehe* ' ' ' "------- Aroclor Specie* 5 days 1254 1200 DDT 1242 1248 1254 1260 1242 1248 1254 1260 1248* 1248* Rainbow trout Bluegilla Channel catfish niuegills Channel catfish 156 -- 2 26 154 307 -- -- -- -- -- -- 137 -- 1 Temperature, 20C; alkalinity, 260, pH, 7 4. * Temperature, 27*C 10 days 8 240 0 87 72 160 443 -- 174 225 -- -- 76 94 15 days _ 94 0 26 54 76 204 -- 107 127 741 -- -- 57 20 Hn\ % _ 21 -- -- 10 135 245 -- -- 300 296 -- -- 25 dti\< __ _ _ __ 54 212 _ _ 113 , 166 _ -- Ml .1 r.i _ _137 - sediment* and PCBs can readily enter food chains (20, 21) LC# value of 3 Mg/1 compared to a 5-dnv !,(, ,, of 1 Mg/1 for DDT (22) ' Aquatic Invertebrates Daphnia magna. Aroclor 1248 is the most toxic of the Aroclor senes to Daphnux, and pre liminary studies indicate that levels above 5 Mg/I are not safe for reproduction (17). After 48 hours' exposure to 300 Mg/1, Daphma concen trated Aroclor 1254 by 48,000 times (22). Gammanu pteudolimnaeut; The level id Aroclor 1248 that did not affect reproduction was com parable to that for daphmds, ca 5 Mg/1 (17). The 96-hr LCU values for Aroclor 1248 and 1254 were 52 Mg/1 and 2,400 Mg/1. respectively (Table 5). Scud were more sensitive to Aroclor 1242 (96-hr LCm -- 10 Mg/1)- After exposing another species of scud (Gammanu fatcutiua) to 1 6 Mg/1 Aroclor 1254 for 14 days, the PCBs were con centrated 27,000 times the exposure level. No further increase in PCB residue resulted after an additional 21 days of exposure (22). Orconecles non Crayfish were more susceptible to Aroclor 1242 (7-day LC0"30 Mg/1) than they were to Aroclor 1254 (7 day LCu-80 Mg/1) PCB residues in crayfish did not reach equilibnum after a 28-day exposure to Aroclor 1254, and the uptake was linear during this period (22) Palaemonete* kadtakensis Glass shrimp were very sensitive to Aroclor 1254, having a 7-day Marine Organisms Immature pink shrimp were extremely aensitnr to Aroclor 1254 exposures Fiftj-one percent of the shrimp died within 15 davs when continu ously exposed to 0 94 pg; 1 Aroclor 1234 (23) Mortalities in two estuarine fishes {Lagodmi rhomboide* and Lnoslomu* rcuU/turus) expose! for 14 to 45 dayB to Aroclor 1254 were obsened at 5 Mg/1- Fish mortalities were not observed it 1 Mg/1 during the same exposure time (24) PCI) concentration factors in these fish were similar to uptake by freshwater fish (1-5X10* times the exposure levels). Correlation of Reproduction and PCB Residues In preliminary investigations, Jensen et >1 (25) reported a possible relationship between PCB residues in salmon eggs and egg mortal it' in Sweden On a fat basis, PCB residues m egg' ranged from 7 7 to 34 Mg g and mortal it i ranged from 16 to 100 percent A regression analysis of their data resulted in a coefficient of correlation of 0 85, and the correlation was significant at I* .001. 162 Environmental Health Perspective* HONS 206256 i Tabic 5. Aroclor and DDT Imlrllji tn lnerttbritet> Compound Organism1 Binassay type* Exposure (da vs) LC (<4C/l) \fnflor I2-r)4 Uwlor 1251 DDT imrlor 1242 Imflw 1242 ^rnfioT 124# liwlor 1254 DDT DDT trader 1254 DDT DDT Iradtir 1242 Iradtir 1254 Imrior 1242 4mdor 1254 DDT I Crayfish Crayfish Crayfish Scud Scud Scud Scud Scud Scud Class shrimp Class shrimp Class shrimp Dragonfly Dragonfly Damsel fly Damselfly Damselfly nUitic atic continuous-flow static continuous-flow continuous-flow static static static continuous-flow continuous-flow static continuous-flow static static continuous-flow continueus-flow static 7 30 t 100 7 SO 4 100 4 10 10 5 0 4 52 4 2,400 4 32 5 0 fl 7 30 5 10 ,) 1 3 7 800 7 j,ooo 4 400 4 200 4 541 1 Cm lisli (OromeelM nan), Scud (Gammaru* Jatciatiu), Claw shrimp (Palaemonete* kadvikattix), Dragonfly (Afa- trtmia tjr), Damselfly (Itchrtura rtriicahi) Temiternturt, 15 0*C, alkalinity, 35 ppm, pH, 7 1 III Summary determine more definitely the exposure levels which do not adversely effect aquatic organisms I'l The omirronce of high levels of PCB residues The problem of PCB residues in industrained I id aquatic organisms (fish and invertebrates) as areas can be greatly improved by dose monitor complex mixtures of Aroclor formulations has ing of effluents and by identification of those H'l been determined by GC analysis and confirmed PCB uses which contribute to effluent contamina r>i b> GC-MS Suitable methods for sample cleanup tion. Elimination of widespread PCB residues in s' include gel permeation and silicic acid column fish at concentrations of 1-20 pg/g will require it chromatography. Agreement on methods for greater efforts, such as the control of PCBs from (<> reporting PCB residues is lacking. sewage treatment plants and other as yet un f Tlie highest concentrations of PCB residues in identified sources of PCBs into the aquatic freshwater fish occur in rivers which are asso environment. ciated with industrialised areas, i.e Hudson II Kiver (ca 200 pg/g whole body residue) and the Related Materials Ohio and Allegheny Rivers (ca 100 jig/g). Residue let els associated with these rivers approach Chlorinated napthalenes are recommended for it! PCB residues which were associated with fish uses Bimilar to those of the PCBs (27) These 11 mortalities in chronic continuous-flow exposures materials are recovered by the same analytical 11 1jOO-GOO ug >'g) procedures as the PCBs (28) and may be recog e- Concentration of PCBs by fish is greater than nised by their gas chromatographic elution pat W.OOd times the exposure levels. Similar concen terns if PCBs are not present The materials are *-i tration factors have been observed for inverte designated Haluwax 1000, 1031, 1099, 1013, n brate), Adverse effects on reproduction of aquatic 1014, and 1051 and are listed in an order of in I' organisms may occur at PCB concentrations of creasing chlorine content (27). Aquatic toxicity J ag 1 or leas l'urther studies are required to information is not presently available, nor has r* April 1972 MONS 208257 163 i the persistence and fate of theee materials been examined in the aquatic environment REFERENCES 1 Jensen, S et al 1969 DDT and PCB in marine animals from Swedish wsteri Nature 224 247 2 Holme*, D C., Simmon*. J H and Tatton, J O'G 1967 Chlorinated hydrocarbon* in Bntiah wildlife Nature 216 227 J Koeraan, J H , Ten Noarer de Brauw. M C. and Vos, R H de 1969 Chlorinated biphenyls in fish, mussels, and birds from the River Rhine and the Netherlands coaatal area Nature 221 1126 4 Risebrougb, R. W. et al 1968 Polychlorinated bi phenyls in the global ecosystem Nature 220 1098 5 Peakall, D B and Lmcer J L 1970 Polychlorinated bipbenyle another long-life widespread chemical in the environment BioSctence 20 958 6 Guatafaon, C G 1970 PCBs--prevalent and per sistent intensified research is needed to minimise their dangers Environ Sci Tech 4 814 7 Risebrough, R, 1970 More letters in the world. Environ 12 16. H Jensen, S 1966 Report of a new chemical haxard New Scientist 32 612 9 Papageorge, W B 1970. PCBs in the Environment, present in part at a meeting sponsored by the National Water Quality Laboratory, FWQA, Duluth, Minne sota, March 17, 1970 (Monsanto Company) IQ Stalling, D L and Johnson, J. L. 1971 Unpubliahcd data from laboratory cross-check analyses from the 1070 National Pesticide Monitoring Program, FishPesticide Research Laboratory 11 Stalling, D L. (In press) Analysis of Organoehlonna Residues in Fish--Current Research at the FiahPesticide Research Laboratory, Conference on Pesti cide Chemistry, February, 1971, Tel Aviv, Israel. 12- Stalling, D. L and Huckma, J. N. 1971. CC-MS characterisation of PCBs (Aroclora) and "Cl labeling of Aroclor 1248 and 1254. J Aaeoc Off Anal, Chem 54 801. 13 Bagley, O E., Reicbel, W L. and Cromartie. E. 1970 Identification of polychlorinated biphenyls in two bald eagle* by ootnbined gaa-ltquid chromatography mass spectrometry J Assoc Off Anal. Chem 53'251 14. Armour, J and Burke, J 1970. Method for separating polvchlorinated biphemls from DDT J Assoc Off Anal Chem 5.1 7b| 15 Swedberg, D 1070 S|vmnl rrimri n |U Bureau of Sport Fisheries sml Wildlife pnere, Sport Fishery Resesrrh Uexmrce pn|, | s Print Off (In press) ' 16 Mayer, F L ibid 17 Nebeker, A. 1971 Unpublished dais Nats.nnl tt ,i,, Quality Laboratory. EPv Dulmh Mwnr*.,i, 18 Stalling, D L. and Hurkms, J \ I npuUidird ,i,,. Fish- Pesticide Research Ijiioniion Missouri 19 Mehrie, P M amt (.mm 14 h I npiihlmhnl :i,,, Fiah-Pesttcide Resennh l.al>orntnr\ Miseoun 20. Duke, T W, Lowe, J I and W ilwm j no, Vertebrate insecticide resistance the in nim endnn effect on succinic dehydrogenase srliviti on endnn resistant and susceptible mosquitofish Bull Lminm Contamin. Toxicol 5 171 21 Nltnmo, D R. et al 1971 Polvchlonnnted Uipheml absorbed from sediments by fiddler crslw anil pink shrimp Nature 231 50 22 Sanders, H O 1970 Special Report on PCB* In Progress in Sport Fishery Research Bureau o( .'.port Fisheries and Wildlife, Resource Publication l 'Hd'ii (In preas) 23. Stalling, D L. Tmdle, R C and Johnson J L tin pens) Pesticide and PCB cleanup by gei permeation chromatography J Assoc Off An.d Chem 24. Hansen, D. J etal 1971 Chronic toxicity, uptake and retention of Aroclor 1254 in two eatsurine fiahes Bull Environ. Contamin Toxicol <> ill 25. Duke. T. W 1971 Unpublished data Gulf Bieeir Laboratory, Environmental Protection Agencv Liuif Breeie, Florida 26. Jensen, S , Johansson, N. and Olsson M 1970 PCB- Indicationa of Effects on Salmon PCB Conference Stockholm, September 20, 1970 .Swedish Salmon Research Inatitute-Report LF1 MEDD 7/1970 27 Halowax Chlorinated Naphthalenes, Technical Bui. letin. Koppers Co, Inc, Tsr Products Division Pittsburgh, Pennsylvania 28 Armour, J A and Burke, J A 197! Behavior of chlorinated naphthalenes in anah-ticu! method* for organoehlorme pesticides and polychlorinated In phenyls J Assoc. Off. Anal Chem 54 175 164 Environmental Health Perspective* MONS 208258 iril It. II > III i. .1 Ul'l nil rtt nit It ii *i ml It 11/ Int In rs The Sensitivity of Fish ATPases to Polychlorinated Biphenyls* by L. K. Cutkomp, H. H. Yap, D. Desaiah, and R, B. Kocht In spile of widespread concern about the pos able hazards of polychlorinated biphenyls (PCBs), little is known about the action of these materials Vtc have therefore examined the m vitro (1,2) ind m vivo (3) effects of PCBs on the adenosine triphosphatase (ATPase) enzyme system. We hivr found some effects which are similar, yet distinctive, to chemically related compounds of die DDT type. A background of earlier research tuLh DDT and closely related chemicals has shown the greatest inhibition on Mg,+ATPase and less on Na+-K+ATPase (4-7). Subsequent mesrcli showed that mitochondrial Mg*1*ATPase of fish and insects was most sensitive to DDT (8) The results from testing several PCBs on the ATPase enzyme system are presented in this manuscript Materials and Mittnds Tissues from bluegil! fish, Lepomtt tnachrocAtnw, were dissected, homogenised, and frac tionsted by centrifuging at 13,000 Xg for 20 min utes, and the sediments were resuspended tn 0.32 M sucrose, 1 mM EDTA, and 10 mM imidaxole. The tract ion obtained (B) contained mitochon drial and nerve ending particles. Each prepara tion nos appropriately diluted and the samples were quirk frosen in liquid nitrogen and stored t -20C, until the ATPase assay ATPase * I'lrimFcd by the Department of Entomology. Fwhanea Md University of Minnesota, St Paul, Minne sota jTilOl A review anil condenaaUon of publications (1), tZ). anil {!!) 1 flta!T Si lentist, Honeywell Corporate Reaearch Center, Hopkins Minnesota 55343 activity was determined using the enzymatic or continuous procedure in which catalysts pyruvate kinase and lactic dehydrogenase were present, and NADH (reduced nicotinamide adenine di nucleotide phosphate) is oxidised to NAD (nico tinamide adenine dmucleottde phosphate) for spectrophotometric determination at 340 nm The procedure is detailed m Pullman et si (9), and Frits and Hamrick (10), and in Yap and Cutkomp (11) Mg,+ATPase activity wsa meas ured when one mM ouabain was m the reaction mixture. Ouabain is a cardiac glycoside which specifically inhibits the Na*-K+ATPase Mg,+ATPsse was further separated into oligomycmsensitive (mitochondrial) and oligomycin- in sensi tive portions by adding 0.03 pg oligomycin (oligomycin A 15%, B 85%) per ml reaction mixture Oligomycin insensitive Mg,+ATPase is generally more prominent in microsomca (endo plasmic reticulum and plasma membrane) and is present to a small extent in mitochondria. Four PCB preparations (Aroclor 1221, Aroclor 1242, Aroclor 1254 and Aroclor 1268) and a polychlorinated terphenyl, Aroclor 5460 were investigated for effects on the ATPase systems Aroclors 1221, 1242 and 1254 were dissolved in ethanol, and Aroclors 1268 and 5460, in acetone, and added to the reaction mixture by slowly releasing the solution from a Hamilton microsynnge under the liquid surface of the vortex of a rapidly-stirred reaction mixture This pre vented sedimentation and promoted the forma tion of colloidal mixtures Reaction temperature was 37C, except for muscle homogenates, where the temperature was maintained at 27C The amount of solvent added (never exceeding 5 pi) with the PCBs had no effect on the ATPase April 1972 HONS 208259 165 t (ml FlQUftx ] Aroelor 1242 inhibition (Probit Units) of oligomycin--mnenaitive Mg** ATP*a*c from fish homogenates ATPsse Activity of uni rested in nmoles P< mg'1 protein hr-1 [or brain 14 00 03, kulney IB 1 0 02, liver 10 40 S, muscle 46 29,6, Unbroken lines computed, dotted line connects actual points activities. Four tissues of fish (brain, muscle, liver and kidney) were selected as enzyme sources Risufts Mtf Dlttutsiin Those PCBs tested in vitro were generally most effective as inhibitors of oligomycm-inseriaiUvc Mg'+ATPaoe, particularly from muscle homogenates Aroclors 1242 and 1254, which Fiouag 3 Aroelor 1221 inhibit ion (Probil Umh) of olign mycin--insensitive Mg'* ATPsse ftom fish homognmf, ATPsse activity of untreated in nmoles P, mg-1 imnrm hr"1 for bram 14 20 4; kidnei IT JJ o, lurr 10 7 0 07, muscle 63 7 5 5 are in the intermediate range of chlorination were more effective than 1221 and 1208 Com pared at 50% inhibition, the lalues uere 0 6 ppm for Aroelor 1242 (Fig 1), 1 2 ppm for \roclor 1254 (Fig. 2) and 2,1 ppm for 4roelor 1221 (Fig 3), the smallest value indicating the most effec tive. Mg'+ATPase from mitochondria (oligomycin-sensitive) was not affected as much bi the PCBs as by DDT-type compounds The h, for Aroelor 1242 was 2 0 ppm on mitochondrial Mg,+ATPase from muscle, 4.0 ppm from kidnei (Table 1 and ref. 2) and 3.5 ppm from bram Table 1. Percent Inhibition of mitochondrial Mg1' ATPaae activity by AROCLOR 1242 in Bah kidncv and muscle homogenates. Concentration (ppm) Per cent inhibition* Kidney Muscle Fiooa* 2 Aroelor 1254 inhibition (Probit Units) of ollgomycin--insensitive Mi" ATPsse from fish homogenates. ATPsm activity of untreated in nmoles Pi mg-' protein hr-1 for brain 14 20 4, kidney 17 33 0, liver 10 0 0 05, muscle 63 7 5,5 05 10 20 40 60 16 0 + 13 7 + 10 4 17 4 62 0 66 2 66 0 + 26 5 25 0 67 6 69 4 77 9 76 6 Untreated 5p ActSE 2t 2 2 5 56 1 1 *(-)-) Values represent the per cent rnrimr actu slion 166 Environmental Health Perspectives HONS 206260 T6f 2 Fifty percent iuMbitloi refute (in ppm) of lITuet determined from homogenates of blue gill U brain. Summarised from references 2 end 6, (Lower value* denote (Teeter effectiveness.) Al't'sscs /n iitro testa DDT Aroclor 1242 Totsl M*'4 Vliiochortdnal \(j OtipmycuInsecsiUve Mg*4 s*-K* 15 0 05 >7 6 (30%) >16 0 (30%) 83 3 5-5 6 (ms* 64Co) to 8 18 5 Fioom 5. Aroclor 1254 inhibition (Probit Units) o( mito chondrial Mg'* ATPaae from fish homogenates ATPaae activity of untreated m nmoles P( mg-1 protein hr"1 for brain 13 50.35; kidney 28 #0 85, liver 39,01 25 Fiousr 4 Arciclor 1242 inhibition (Probit Units) of mito chondrial Mr14 ATPssr from fish homogenates. ATPaae sHintv of tintrested m snwics P, mg"1 protein br"1 for brsm 13 2l 1, liver 29 1*0 004. Unbroken lines com puted, du tied line connects actual points. Table 3. Percent Inhibition of ATPsees in bomogrnaie* of mitochondria] fraction of kidney ef 4-month Aroclor 1242 treeted fst-Uead minnow ATPsees Mg*4 ATPaae Mitochondria] Ohgotnycin- Na4-K4 insensitive ATTsee 0 9 56 23 21 2 8 75 21 36 8 3 45 35 18 (Fig. 3). The It* for DDT was 0 5 ppm in brain (Table 2) In contrast to the effective inhibitors, two of the poorest inhibitors, Aroclor 1221 and Aroclor 1268 (see ref 2), showed some stimulation of Mg,+ATPase. Na+-K+ATPase from fish brain homogenates was inhibited by Aroclor 1242, but the concen tration required was about 5X that for mito chondrial Mg*fATPaae. Aroclor 1242 is compared with DDT in Table 2, showing this PCB to be a better inhibitor of Na+-K4ATPase, but poorer on mitochondrial Mg,+ATPase. The in vivo studies were conducted with fat head minnows, Pvwphales pramelaa, which were chronically exposed to Aroclor 1242 and 1254 for four months following hatching Comparisons of ATPaae activity showed considerable inhibi tion and some stimulation over a concentration range in which fish were exposed to 0 31, 0 03, 2.8 and 8,3 ppb. Aroclor 1242 compared with Aroclor 1254 showed the greatest inhibition or Mg,+ATPases, as it had m in vitro studies The inhibition in the chronic in vivo study was great est on mitochondrial Mg,+ATPaae from kidney (Table 3). Inhibition of mitochondrial Mg*+ATPase was also greatest in brain and liver homogenates Comparisons with muscle homog enates were not feasible in the small, chronically exposed minnows. The result contrasted with the inhibitory effect on oligomycm-msensitive April 1972 MOMS 208261 167 Mg*+ATPaae which occurred m vitro A maxi mum inhibition by Aroclor 1242 in treated fish (in vivo) was 75% on mitochondrial Mg,+ATPase and 35% on Na+-K+ATPase In contrast, Aroclor 1254 did not inhibit mitochondrial Mg,+ATPase in kidney but gave 30% Na+-K*ATPaae inhibi tion in the group of fish treated with 8.3 ppb (3). Results from brain tissues showed more consistent inhibitory effects than did other tissues Overall, PCB-treated fish showed more varia tion of enzymic effects than in vitro determiner tions Thus, although the precision of the results was greater in vitro than in vivo, both approaches seem desirable for assessing the mode of action In both cases the greatest effects were cm Mg*+ATPaae, with lesser effects on Na+-K+ATPase Of the Aroclors studied, those in the intermediate range of chlorination appeared to have the greatr est inhibitory effect on the ATPase system. Aroclor 1242 was a particularly good inhibitor. Ctncluslws The ATPase enzyme system in fish is adversely affected by all PCBs studied Mgt+ATPase is inhibited to the greatest extent m muscle, but effects m kidney, brain, and liver also occurred Fifty per cent inhibition occurred with less than 1 ppm up to a few ppm. The greatest effects, in vitro, were on ohgomycin-insensitive Mg,+ATPase, but on ohgomycin-sensitive (mito chondrial) Mg,+ATPase in vivo (chronicallyexposed fish). The most effective PCB studied, Aroclor 1242, required a somewhat higher con centration than DDT in comparative in vitro studies. AckaowMKiMRts Research support by grant of FWPCA, Environmental Protection Agency, 16030 ELZ Chronically exposed fish wrrr obtained fmm experiments from the National Water Qunlin Lab., Environmental Prutrctum Agenct, IJuluili Minn, through the court est of Dr Ainu \<.j,, r Monsanto Company kmclh Mtpplird o,, \r[,,j,,r preparations REFERENCES 1 Yap, H H et al 1971 Senmtivuy fi-d, \T1'hm n. polychlorinated biphenyl* Nature 241 <>1 2 Dsaaiah, D et al. 1972 Inhibition of ntigiiimnti sensitive ami insensitive mngnr*utm .iilcnoeinr ir, phoaphstsae activity in hsh (Pulvrtdnrmsied Hi phenyls) Biochem Pharmacol 21 (In press! 3 Koch, R B 1972 Polychlorinated Inpheny I* elm nl long-term exposure on ATPasr nrtivity m h-t Ptmephoiet pmmtlat Bull Enuron Cnntamin Tim col 7 (In press) 4 Koch, R B et al !9ll9 Chlorinated tivilrnrartaH, insecticides, inhibition nf rnlilnt hriun ATPase a< tivilies J Neurochem Hi 2b<> 5 Koch, R B,, Cutkomp, L K nntl Do, F M liHei Chlorinated hydroearlam insertimle inhibition of cockroach and honey hee ATPaee* Life ro-, mil, tmo 6 Koch, R B 1969/1970 Inhibition of nnimnl Iivki1 ATPaae activities by chlorinated bydnunrlinii pe*n cidea Chem-Biolog Interactions 1 199 7 Cutkomp, L K et a) 1971 ATPase activity m fish tissue homogenates and inhibitory effects of HI) I and related compounds Chem-Biolog Internet ions .s tn 8. Cutkomp, L K el al 1971 Inhibition of uligunyr,,,. sensitive (Mitochondrial) Mg1* ATPnse l,v DDT and selected analogs in hah and insect tissues Life do KHIl) 1201 9 Pullman, M E et nl I960 Partial resolution nf the enzymes catalyzing oxidative phosphorylation I Purification and properties of soluble dimtrophenolstimulated adenosine triphosphatase J liming Chem 235 3322 10 Fritz, P J and Hamrick. M F. IStfio Enzvmsluanalyais of adenosine tnpho*phntne Entvmnlogu 30 67 11 Y'ap, H H and Cutkomp, L K 1`ITH Artivitv sod rhythm of ATFasrs in larvae of the minuiuito, 4fn aspppft L Life Sci 9(11) 141D 168 Environmental Health Perspectives HONS 206262 Studies on the Mechanism of Toxicity of DDT and Polychlorinated Biphenyls (PCBs): Disruption of Osmoregulation in Marine Fish ih by W. B. Kinter,* L. S. Merkens,t R. H. Janicki.f and A. M. GuarinoJ lt> In 1970 studies were initiated at the Mount Materials and Metlieds ti Dcacri Island Biological Laboratory to explore morhumbmis of toxicity underlying the high Eels, Angudla rosirala, and kilhfish, Funduius wnsiln lty of bony fish, teleoeta, to organochlonne helerochlus, were captured in estuaries along the pollutants The starting point was several reports Maine coast They were adapted to and main that DDT [1,1, l-t,richloro-2,2-bia (p-chloro- tained in sea water for about three weeks before .1 K'l pben\t) ethane] inhibited Na,K-ATPaae (Na+, use Non-radioactive puriss grade p,p'-DDT was K4-iii in ated adenosine triphosphatase), as well as procured from Aldrich Chemical Co, Milwaukee, (I the knowledge that this eniyme appears to play a Wis 14C-DDT was obtained from Amersham/ rent nil role in osmoregulation by marine teleosts. Searle, Arlington Heights, 111 and had a specific In thr face of a desiccative environment, these fish activity of 24 mCi/mM. ll>l maintain body fluid hypotonicity (Fig. 1) by Aroclor 1221 was donated by Monsanto Co , |i drinking sea water, absorbing water coupled with St. Louis, Mo. Plasma milliosmolarity was deter 4.1 salts across the intestinal epithelium, and eventu mined by a standard freeling point depression al! \ Mrrcting the NaCl acroes the gill epithelium technique, while Na+ and K+ were analysed by n inlc ret inning the free water. The primary driving means of a flame photometer. Levels of UC-DDT I mcclmnism in both intestine and gill is the Na were determined as described previously (4). The -I pump with which Na, K-ATPase appears to be fractional "Na space of isolated gills was measured Df intimately involved For fuller discussion, see (2) according to the procedure of Kamiya (5) where and (:t), eel gills arc incubated for 1 hr at 15" in oxygenated ir After demonstrating that DDT did, in fact, sea water. Control gills were incubated with the inhibit Xu, K-ATPase activity in homogenates of solvent 0.5% N,N-dimethylformamide (DMF), w| intestinal mucosa and gill filaments from several and experimental gills were exposed to 50 ppm V. marine teleosts (2), attention was focused on the DDT in 0 5% DMF. Gill homogenates were nes \\ liter-adapted eel (Anguilla rotiraia), in which assayed for Na,K-ATPase activity as described osmoregulation has been well investigated. This eel enn also adapt to fresh water and can he used previously (2) Acute toxicity and osmoregulatory studies were conducted in kiltifuh using a simple for future DDT studies. static system For each experiment ten sea water-adapted fish (5 males and 5 females, each Ntulip* conducted t Mt, Deeert Island Biological weighing about 5 g) were placed in an aluminum Ijil-nrnirv, Salisbury Cove, Maine 04672. 1 v^ 1 Upstate Medical Center, Syracuse, New York I32t!1 ! 1 u w hom reprint requests should be sent Dr A. M CuHnuu, Bid* 10 Rm 6N !19, National Cancer Institute, brlUndu, Md 30014 or enameled metal container holding 2 liters of sea water and maintained at 14-16 throughout the exposure time In control containers 2 ml of ethanol were added to the sea water, in expenmental containers Aroclor 1221 (mixture of PCBs April 1972 HOWS 206263 169 d SALT WATER TELEOST Hypo-osmoltc to Medium (Angullia roitratat ' T`"* Flaama Gill Gut mucosa Brain Dili level- pt,ni 1- 2*1 0 22 2 2 2 li 8*0 8 11 2*1 1) * MeanjfcSE for 6 animals exposed lo 1 nnm of "C DDT for 6 hr Figure 1 Water and unit tronaport in * *all water teleoat Typically the mdliosmois/L ere 1,000 for sea water end 360 for fish body fluid* Adapted from (1) with 21% chlorine content) or p,p'-DDT was first dissolved in the ethanol These solutions formed a cloudy suspension when added to the sea water, and in addition some separation of the Aroclor was noted, making the actual concentrations unknown (Aroclor 1248 was not tested due to much greater separation). Blood was drawn by cardiac puncture with lightly hep&rinised glass capillary tubes Whole blood from 3 to 5 fish was pooled and centrifuged. The 0.08-0.1 ml of serum was diluted, and the oamolarity and concentration of Na and K ions were measured as for the eel. Table 3. The effect* of DDT on sodium space and Na.K-ATPaae in gills from aee water adapted eels (Anguilla rostrum 1.' Treatment Nn spacef \ I r.l'-f RCtl\ l\ J Control DDT, 50 ppm 0 |12o 010 0 162*0 0105 o jro ii; o 2,fn m( *MeanSE for 15 gills in each grrum for \n sjmir determination and 5 duplicate assays (icr group for tTl'mrr assays t Fractional Na apace after incubation nt l(or I )ir expressed as *N* gill filament to media ratio t amolei Pi/mg protein X hr | Significantly different (I'COOl) from nintrob Rmlts Ability to osmoregulate was followed in indi vidual eels (about 100 g) placed in IS* sea water (2 liters) containing 0-1% ethanol and 1 ppm DDT, a level which is fatal by 10 hr. In Table 1 it is seen that, at 0 hr, plasma milliosmolarity of Tmhla 1, Effect of p,p'-DDT on plunii oamolarity. odium and potaaaium in tea water adapted eel* (AngutUm rot Irala l.m Treatment Oamolarity Na* K* (moamola /L) (mEq/L) (mEq/L) Control p,p'-DDT 358:1:10 462 . 10| 183 2 2 40 3 192 .It 5 40 It * MeitniRE for 5-0 animals exposed to I ppm of DDT for 0 hr 1 Significantly different (P<001) from controls Table 4. Summary of DDT effect* on osmoregulation In aee water eel*. Study Result-: Gut Gill In pno at 6 hr 7 ppm DDT (1 ppm in Plasma osmoianty water)* In intro (50 ppm HtO absorption in media) from sacs de- creased 47T t Homogenate Na.K-ATPasc (50 ppm) inhibited W,t 22 jipm DDT increased 20', Na content of isolated gill- increMed -t-Vi Na.K-ATI'iisc inhibited f * DDT solubilised with ethanol (0 m ea nter; or N, N-dimethylfonnamide (fli'V in media anil 5', m homogenate) 1 Calculated from ref 3 170 Environmental Health Perspective* MONS 206264 Table 5. Acute loMlcity of ArtxJor 1221 and DDT in kiUlftab. Compound Initial level* 111 Kfl ff HVCI Tola) number pf fah Day 1 % Dead (cumulative) Day 2 Day 3 Dav 4 Cunlrnl (ethanol) 10 0 0 0 0 Amdor 1221 7 5 ppm 10 0 000 25 ppm 10 50 80 80 80 75 ppm 50 88 06 98 100 DDT IH 0 025 ppm 0 075 ppm 0 25 ppm 10 10 10 0000 40 50 50 50 60 80 00 00 0 75 ppm 10 100 100 100 100 * See description of alette system in text. mil trU DDT-treated fish had increased about 30% com* Considered all together (Tables 1,2, and 3 and pared to ethanol controls. Furthermore, in this summarized in Table 4), our eel data provide table one can see that, while much of this increase circumstantial evidence that disruption of osmo a as due to plasma Na, plasma K also doubled in regulation is a primary toxic effect of DDT, t e., these animals. In additional experiments with 1 the DDT levels in gut and gill of fatally-dosed fish ppm "C-DDT, the fish had taken up about half were high enough to have affected osmoregulatory the radioactivity by 6 hr. The levels of radio* Na transport under in vitro conditions. Both water activity for some tissues are indicated in Table 2 absorption from gut sacs and Na content of v and ranged from 7-22 ppm. Using isolated eel gills isolated gills are directly dependent on Na pump the fractional "Na space was found to be increaaed activity and can be altered similarly by obtaining significantly (Table 3) by 50 ppm DDT. Data in a highly specific Na,K-ATPase inhibitor More hr Table 3 also shows that Na,K-ATPase activity over, from the published (3) dose-response curve was significantly inhibited (38%) by 50 ppm for gut homogenate, 7 ppm DDT in mucosal DDT tissue should still inhibit Na,K-ATPase activity Table 6. Effecta of Aroclor 1211 and DDT on aerum oamolarity* Na and K in klUiflsh. ,n Compound and mosmois/liter initial level Na* mEq/liter K* mEq/liter Untreated 9'Hqvt tzpomrt Control Aroelgr 1321 7$ ppm DDT 0 25 ppm ll Hour ezpoouFf Control Amrior 1221 25 ppm 75 ppm Du r 0 (175 ppm 352 2 (18) 3663 (6)* 3M7 (9)t 380 9 (8)t 361 7 (6) 374 7 (6) 40710 (4)t 3633 (7) 173 2 (16) 171 2 (6) 173 3 (9) 178 4 (9) 1762 (6) 179*4 (6) 189 5 (4)t 180*5 (5) * Significantly different (P<0 05) from untreated controls, t Significantly different (P<0 05) from ethanol-treated controls. 3 60 2 (16) 4 30 2 (5)* 4 40 3 (9) 4 40 3 (9) 5 30 5 () 4 5*0 3 (6) 5 00 4 (4) 4 10 4 (9) April 1972 HONS 208265 171 by 45%, provided, of course, that tissue content on s wet weight basis is equivalent to homogenate content based on added DDT. The data in this same curve also indicate that Mg-ATPase is less sensitive to DDT than Na,K-ATPase. Certainly, until more information is available, the present data would appear to provide a partial explanation as to why a whole body burden of less than 10 ppm DDT is often lethal to hah It is also recognised that DDT may exert a primary toxic effect on the central nervous system; by 6 hr our fatally-dosed eels were hyperactive and easily stimulated to convulsions by touch. Such symptoms, however, could be secondary to alterations to osmolanty of plasma and cerebrospinal fluid. Last summer, because of the chemical similarity between the insecticide DDT and the industrial PCBs (polychlorinated biphenyls) pollutants in the environment, acute toxicity and osmoregula tory effects were compared using killihsh. The toxicity data, Table 5, show that, with respect to the acute lethal level in sea water, the Aroclor 1221 was less toxic than DDT by a factor of about 100. There was a "dose-response" in terms of the theoretical concentration in sea water and num ber of deaths for both Aroclor 1221 and DDT, but the actual dose which the fish received is unknown since the tissue levels were not measured. The osmolanty and Na concentration of the serum (Table 6) consistently increased (toward sea water values) in fish exposed to lethal levels of either Aroclor 1221 or DDT. The Aroclor and DDT levels selected for these serum-composition studies were those that were potentially lethal to most of the fish, but low enough to have killed only some at the time (6 or 24 hr) samples were taken from those still surviving. With the higher levels tested, most of the increases m serum osmolanty and Na were statistically significant when com pared to ethanol controls. Also, serum osmolanty and K were elevated in all ethanol-treated rontrols (P < 0.05, except for 34-hr osmolanty), this elevation may have been due to the ethanol or to confinement of the fish in the experimental contamers. Additional data (Table 7) show that in the extreme coses, when blood was sampled before the fieh died but after they had completely lost their righting ability and remained sideways, the serum osmolarity and TaWe 7. Ounolarlty, Na and K in individual pooled ample* of killifiah Krum * moiimoi* Compound Exposure sod level br liter Na* KmEq litrr Arodnr 1221 75 pptD 23-32 DDT 1 pptn P-10 SB0 428 430 440 208 210 207 210 81 8i 88 * Each sample represents eerum from ulroui j fish electrolyte concentrations were all increas'd well above normal. It should be noted that the osmolarity changes (Table* 6 and 7) cannot always be accounted for by changes in measured electrolyte*. DisciuiM Overall, these results suggest that lethal levels of Aroclor 1221 and DDT both decrease the ability of killifiah to osmoregulate to about the same degree. Also killifiah and eels seem about equally sensitive to DDT. Thus, inhibition of Xa(KATPaae by appropriate level* of Aroclor liyi was anticipated. Using a homogenate of King O'Norway (HemUnpierut americanus) intestinal mucosa 250 ppm of Aroclor 1221 inhibited the Na,K-ATPase by 51% and 22% in two experi ments. Moreover, recently-published work by Ysp et al. (6) showed that several of the Aroclor mixtures inhibited the eniyme from fish brain and kidney. Concerning the molecular basis for Na,KATPaee inhibition by DDT and PCBs, one may speculate that these lipophilic compounds interact with the phospholipid-activating component of this lipoprotein enzyme (7, S). It is well known that many lipophilic compounds, e g., cyclo hexanone, inhibit the eniyme and evidence of proton interaction between DDT and a phospho lipid, lecithin, recently ha* been reported (91 Moreover, other organochlorine insecticides, in cluding endrin, also inhibit Na,K-ATPase from fish brain (10), and etidnn has been observed to disrupt osmoregulation in both a marine and a fresh water teleoat (H, 12) Thus, it seems reasonable to predict that many organochlorine 172 Environmental Health Perspective* HONS 208266 pollutants will be found to disrupt osmoregulation in fish. REFERENCES 1 Prosser, C L and Brown, F A Jr 1961 Comparative AmTnel Phyriology (2nd ed ), Philadelphia, W B Saunders 2 Jnmcki, R H and Kinler, W B 1971 DDT inhibits Na`, K+, Mg'*-ATPase in the intestinal mueoeae and T-ilanf marine telenets Nature 233 14S 3 .lanicki, R. H and Klnter, W B. 1971 DDT Dis rupted oimoresulatcry events in the intestine of the eel Anguilla fnilrala adapted to sea water Science 173 1146 4 Guarmo, A M . Janicki, R. H. and Kmter, W B 1972 Distribution and metabolism of "C-DDT in the cel (Anguilla mtrata) after six hours of uptake from ambient water. Bull Mt. Desert Island Biol Lab 13' (In press) A Kamiva M 1967 Changes in ion and water transport in isolated (tills of rultured eel in course of salt adapta tion Annot Zool Japan 40 123 (i Yap, H , Desaiah, D and Cutkomp. C 1971 Sensi tivity of fish ATPeaes to polychlorinated biphenyls. Nature 233. 61 7 Tanaka, R , Sakamoto, T and Sakamoto, Y. 1971 Mechanism of lipid activation of Na, K, Mf-activeled adenosine triphosphatase and K, Mg-ectivated phos phatase of bovine cerebral cortex J Membrane. Btol 4 42 8. Wilson, W E,, Clements, S R. and Latuner, A 1971 Phosphatidyl serine protection of beam membrane! (Ns* and K*) ATPase from inhibition by fatty acids and chlorinated hydrocarbon pestsudce. Fed Proc 30- 673, 8 Tinsley, I., Haque, R and Schmadding, D. 1071 Binding cf DDT to lecithin. Scieoee 174 146 10 Cutkomp, L. et al 1971. ATPam activity m fish tissue homogenstes and inhibitory effects of DDT and related compounds. Chem -Biol Interacttone 3: 439 11. Eialer, R and Edmunds, P 1966 Effects of endnn on blood and tissue chemistry of a marine Bah. Trane Amer Fish Soe 95 153 12. Grant, B F and Mehrle. P M 1970 Chronic endnn poisoning in goldfish, CaroMius auratur. J. F*h Res Bd. Canada 27 2225 April 1972 HONS 208267 173 Environmental Health Ptrtpeaivta Polychlorinated Biphenyls (Aroclor 1242): Effects of Uptake on co/i Growth by Julian E. Keil, M.S.*, Charles D. Graber, Ph.D.f, Lamar E. Priester, Ph.D.i, Samuel H. Sandifer, M.D.* Introduction Polychlorinated biphenyls (PCBs), similar to DDT in structure and physiological effects (7), are of concern to the scientific community because of their ubiquity (8), persistence (11) and possible toxicity The alleged lack of biodegradabiiity of thibenvironmental pollutant has caused alarm and curtailment of this useful chemical. Work as early as 1963 by Cope (3) indicated that DDT, another chlorinated hydrocarbon, might be metabolised by Bcveral species of bacteria, Subsequently, other workers (2, 4, 9, 10) reported degradation of this insecticide by bacteria, fungi, and marine diatoms. Unpublished work performed in our laboratory (5) also indicated possible metabolism of certain PCB component* by Cyhndroiheca chwtmum, With the thesis established that DDT was indeed bio degradable and with evidence that PCBs may be biologically converted, the authors postulated that human flora may play an important part in the metabolism of DDT, PCBs, and other ecological contaminants. Experiment* were performed to study the effect* of PCBs m vitro on a facultative organism, E coh, common to human intestinal flora. This bacterium was also selected because it is the prime indicator of fecal contamination. Mitirlilt wrtlktJ*#* The study organism, Etchertchta colt, was* I Preventive Medicine Section, Medical University of tSnulli Carolina, 80 Barre Street, Charleston, S C 29401 f Microbiology Department, Medical University of S C I Environmental Health Laboratories. Stale Board of Health, Columbia, S C selected, because it is a common autochthonous, human intestinal aerobe, easily cultured, and is an accepted standard indicator of fecal contamination Initial work screened a wide range (0-1000 ppm) of PCB concentrations, against E. colt by use of impregnated paper filter discs similar to those used to test antibiotic sensitivity Impregnated discs with PCB concentrations of 0, 001, 01, 1,10,100, and 1000 ppm were placed on tripticase soy broth (TSB) agar plates which had been inoculated with E. colt. Experimentation indicated that 6 mm diameter discs cut from number 2 Whatman filter paper would absorb 02 ml of the acetone solvent The discs were dried prior to use. For the first experimental seneB (I), 20 one-liter Erlenmeyer flasks, each with 500 ml TSB, were inoculated with E, colt. Four flasks served as controls, 4 for each of 2 levels of PCB treatments, and 4 as acetone controls since acetone was the suspending vehicle for the toxicants. Additionally, 4 flasks were used as uridine controls. One flask, con taming only media, was used as a growth check, Each flask was vigorously agitated every 2 hours during the first and last S hours of culture time. At the conclusion of a 24-hour incubation period, flask contents were centrifuged, the result ing pellet lyophihzed, weighed and assayed for PCBs, nucleic acids and tntiated uridine ('Hu) uptake. A second experiment (Scries II), identical in treatments except for u ridme, was conducted to confirm stimulation patterns observed in Senes I. Serie* II measured lyophilized harvest weights only. A third trial (Series III), identical in design to April 1972 HONS 208268 175 l Fiouu 1. Sensitivity screening of E colt on T5B to 0, 001, 01, 1, 1, 10, 100, and 1000 ppm of A. DDT, B, PCB Senes I, assayed tntiated undine uptake after 5 hours sad 8 hours incubation time to determine if assimulatioo rates were affected by PCBs during the logarithmic growth phase Aroclor 1242 (PCB--42% chlorine) was used as the source of active ingredients in these experiments Samples of bacteria were extracted with nano grade hexane and a 5 *1 aliquot was injected into the gas chromatograph for analysis. Two-column systems were used a 6 foot X Vi inch O.D. glass column packed with 3% SE-30 on 60-80 ABS chromosorb W and 2% QF-1 on 60-80 Table 1. Lyaphlllaad Weight* ud Toxicant Uptake by Ecchafdcbda eotl Culture* Expoaed to PCB (BaetwiaJ S*ri* I). Treatswt PCB I */ul PCB 01*/ml Control Acetone control Undine control LSDa> Mean1 harveet Mean1 residue weight* (rogm) Ih/e) 86 0 eo 4 44 5 27 2 41 6 13 6 13 6 44 0 12 0 10 8 1 Mean of four replicate*. 1 L6D -- Leal significant at B5% probability level calculated from one way analysis of variance ABS chromosorb W. The operating parameters were: (1) inlet temperature 23fiC (2) column temperature 200C isothermal, (3) detector tem perature 240C, (4) flow-rate 70 cc per mm X, Qualification was accomplished by measurement of the absolute and relative retention times of 5 major peaks of the PCBs Quantitation was accomplished by determination of the area under the curve of the five major peaks Utilizing tins technique, recoveries of 92 4% of the PCBs were obtained, R.NA and DNA were measured using a modifi cation of Agranoff's procedure (1) Radio assays utilized the Nuclear-Cliicago Mark I Liquid Scintillation Spectrometer Rtuilts and Discussion As may be seen from Fig 1, concentrations up to 1000 ppm of PCBs impregnated into filter paper discs failed to inhibit E. coli growth on TSB agar As shown in Table 1 (Senes I), the addition of 01 and 1 jig/ml PCBs to TSB markedly stimu lated E col: growth above control levels This is consistent with other microorganism work (4, 6) showing that DDT and PCBs are concentrated up to 1000 times by marine diatoms. The data also suggest that the concentration factor mav be inversely related to dosage 176 Environmental Health Perspectives HONS 208269 Table 2 presents data from a duplicate experi ment (Senes II), wherein only harvest weights were studied. This second senes confirmed apparent stimulatory effects of all levels of PCBs. There were no differences between treatments in DN A and RNA leve Is af ter 24 hours' incubation The significance of this is not clear, since it had been anticipated that increased RNA levels would accompany growth increases However, it is thought that differences may have occurred earlier than the 24-hour time. Data showing undine uptake by PCB-treated E coli at several phases of growth are presented in Table 3. No significant differences in uptake between treatments and controls were evident except at 5 hours incubation time, when there was a significant diminution of *Hu uptake in PCBdosed bacteria. While not consistent with increased culture yields, the lack of change in uptake between chemically treated E. coli and controls does coincide with chemical assays indicating no increased levels of nucleic acids. Results from these experiments indicate that PCBs stimulate in vitro growth of E. colt. If this phenomena occurs in vivo further concern is justified since any environmental contaminant which affects indicator organisms may result in a distorted portrayal of the actual situation. AekBowttdftmmts This research was supported by the Community Pesticide Study of the Environmental Protection Agency--Contract #PH21-2017. Appreciation is also expressed for technical Tihlt 3, Lyophtllsed Weights of EtchoHchla coli Cultures Exposed to PCB (Bacterial Series II). Treatment Mean1 harvest weights (mgm) PCB 1 ag/ml PCB 01 ag/ml Control Acetone control LSDu1 186 4 163 .2 160 8 133 6 23 6 ' Mean of four replicates 1 LSDh - Least significant difference st 06% probability level calculated from one way analysis of variance Tahle 3. Tritiated Uridine Uptake by PCB Treated Escherichia coli Cultures During Exponent]*! and Decline Growth Phases. PCB 1 sg/nil PCB 01 ag/ml Undine control LSD,,< Counts per minute' 5 Houn 8 Hours 24 Hours 4342 4756 8020 2800 2250 2367 2675 979 2142 2462 2JSfl fW 1 Mean of four replicates 'Least significant difference at 95% probability level erlculated from one way analysis of variance facilities afforded by Training Grant No AM05078-NIAMD and NIH General Research Support Grant 5420 Monsanto Company kindly supplied the PCB used m this work REFERENCES 1 Agranoff, B W and Santen, R, J 196.J Studies on the estimation of deox > ribonucleic acid and ribonucleic acid in the rat brain. Biochem Biophvs 72 251 2 Chacko, C I., Lockwood. J L and Zabik, M 196ti Chlorinated hydrocarbon pesticides: degradation In microbes. Science 154 893 3 Cope, O B and Sanders H O 1963 Microorganism and Hydrocarbons U S D I Fish and Wildlife Circ Pesticides--Wildlife Studies 167 27 4. Keil, J. E, and Pnester, L E 1969 DDT uptake and metabolism by a marine diatom. Bull Environ Contamin Toxicol, 3 169 6 Kcil, J E. and Pnester, L E 1070 Unpublished work Medicai University of S C 6. Keil, J E, Pnester, L E and Sandifer, S H 1971 Polychlorinated biphenyl (Aroclor 1242) effects of uptake on growth, nucleic acids, and chlorophyll of a marine diatom. Bull Environ Contamin Toxicol 6 156 7 Lichtenstein, E P et al 1969 Biological interaction between plasticizers and insecticides J Eicon Entorool 62' 761 8 Risebrough. R W el si 1968 Polychlorinated bi phenyls in the global ecosystem Nature 220 1098 9 Slenevsen, J H V 1965 DDT metabolism in re sistant and susceptible stable-flies and in bacteria Nature 207 860 10 Wedemeyer, G. 1966 Dechlorination of DDT by aerobscler aerogenea Science 152 647 11 Wurster, C F and Wingate, D B. 1968 DDT resi dues and declining reproduction m the Bermuda petrel Science 159 979 April 1972 MONS 2D8270 177 d Eru'irvniruruaJ Health Perspective* The PCB Situation in Germany by H. P. Tombergs* A PCB problem has been known to exist in Germany since 1960. At the Swedish-German Symposium at Baden-Baden, Dr. Jensen, who first published about this problem in 1066, made a report entitled "PCB--the DDT Problem of Industrial Countries.'1 In September, 1970, there was a PCB conference in Stockholm One of the reports given there mentioned that a fatal case of biphenyl intoxica tion had occurred m Finland. The person was a 31-year-old man who had worked iu a plant which used biphenyl (not chlorinated) for impregnation. The exposure level was about 100 mg m'--10 times the MAC (maximum allowed concentration). He had been working in this industry for 11 years and suddenly developed liver insufficiency--i.e. hepatic necrosis, atrophy, and cirrhosis. General atrophy of the brain cortex was also found. Medical examinations of the other 120 persons working in this plant revealed three additional cases with liver damage (biopsy) and two with abnormal EEGs and impaired peripheral nerve function. In July, 1970, Acker (University of Milneter) was able to find PCBs in human milk at & level of 0.103 ppm, which means 3.5 ppm in the milk fat In human fat tissue he found an average of 5.7 ppm. A recent German investigation on rats (Benthe, University of Hamburg) reports the absorption and distribution of PCBs after one inhalation exposure to Pydroul A 200, a mixture of low chlorinated biphenyls Absorption as well as distribution were studied by measurements of PCB concentration in different organs. By this route very high absorption could be shown Depending on duration of PCB exposure, a rapid increase in West German Ministry of Youth, Family, and Health PCB concentration in the liver was observed After 15 minutes the concentration was more than 50 percent of the maximum concentration attained after two hours (70 ng/g tissue) Concentration in fat after 30 minutes of exposure was 14 Mg g tissue (about 27% of the liver concentration', whereas in brain tissue, only 9 Mg/g tissue (17% of the liver concentration) was found at that time. Depending on the amount of time which passed after the end of aerosol exposure, a rapid decrease of PCBs in the liver during 24 hours was measured and was accompanied by an increase in brain and fat. In the course of two days brain and liver concentrations fell to a minimum value, whereas in fat, a maximum value was attained (260 jig/g tissue) which remained at a constant level in the subsequent penod. There was no toxic fatty degeneration of liver under these experimental conditions. Seven weeks after exposure to low chlorinated PCBs (Tetraehlor), the compounds were found in the fat of rats. After three weeks, 70 percent of the maximum value was still detectable m the fat, and it should be noted that, in a stress situation, PCBs move from fat tissue into the liver. In addition to the inhalation route, studies were made of other methods of exposure The results follow: After mtrapentoneal injection of PCBs, a stimulation of the microsomal enzyme systems which are responsible for the metabolism of drugs was found by measuring the O-demethylation, and the increased enzyme activity persisted for a long time. Even after four weeks, the values were still twice the value of the control animals. Because the microsomal enzymes also metabolize adrenal hor mones, an effect on reproductive and adrenal hormones can be expected After long exposure (3 inhalations for 3 hours), April 1972 MONS 208271 179 a toxic effect on liver function was found trans aminase activity (SGOT, SGPT) had increased on an average by 50 percent Parallel to this increase of transaminase activity, the liver fat increased from a normal 3 9% to 8 7% We consider this a toxic effect After a subclimcal dose of tetrachlonnated hydrocarbon (a dose which does not cause a measurable amount of liver damage) mas given, the application of PCBs caused a high degree of liver toxicity, that means that, in the case of a subclimcal liver damage, PCB absorption mui be dangerous by causing liver dystrophy All these results will be published in detail in i Uc next issue of the Archuiet of Toxicology ISO Environmental Health Perspectives HONS 208272 Environmental Health Ptnpectim Comments on Research Needs by Norton Nelson* I will not attempt to summarise all of the excellent work presented at this meeting--such a summarisation would presume expertise m all of the varied fields which have been represented here, an expertise I do not possess. I believe it will be more useful for me to attempt a very personal assessment of those gaps which I see as now confronting us, in an attempt to identify those that are significant and merit attack. In short, "Where should we go from here?" First, a general statement. I was charged with a similar task last August at a conference convened by FDA on work done on PCBs within the federal government. The extent of additional work that has surfaced as the result of the present conference is remarkable. Clearly, many universities and governmental agencies are extremely interested in the PCBs and have been working intensely in the field in the lsst few years I will follow the order of presentation in the present conference roughly, but will assume the liberty of regrouping where this seems desirable Aitlysis Analytical technics have improved immensely in the last three to four yean since the disturbing peaks resembling DDT metabolities were identi fied on chromatograph traces. It now appears that analytical procedures of considerable specificity are now available and quite widely used. The confusions and uncertainty of several years ago appear to have been largely mastered. The procedures are in Borne degrees overly tedious and have senouB defects in respect to quantitation, especially because of the disparate response of some of the detector systems used in the gas Director, Institute of Environments! Medicine, New York University Medical Center, 550 First Avenue, New York, New York 10010. chromatographic analysis. The latter has made quantitation of mixed isomers difficult and some what uncertain. Clearly then, there is a need for improved quantitation in respect to mixed isomers and equally room for improved efficiency of the analytical technics There is a very substantial gap in reliable analytical technics for impurities such as the chlorinated dibenzofurans It appears fairly defi nite that such impurities are present in some preparations and may indeed significantly account for some of the toxicity. Accordingly, better means for identification and measurement of these im purities is required. Because they occur only in the part per million range in the PCBs themselves, which in turn occur in the biosphere in the part per million range, extreme and perhaps unattain able sensitivity would be required for their identifi cation in environmental samples It may, however, be very important to make the attempt. A closer and more attainable goal would be the develop ment of adequate technics for detection and meas urement of such impurities m the original PCBs themselves. In this connection, it may be desirable to look for biological assays as at least an intenm approach to this problem. Dr. Vos, in his excellent presenta tion, has presented work which suggests possi bilities in the use of btoassays in the assessment of impurities in PCBs This appears to be a very promising approach and should be pursued. It is proposed below that selected purified isomers of the PCBs and their impurities be made available for toxicological studies These would also be very useful m the further improvement of analytical technics Emiranmnntal Transport, Distribution and Attiratino Dr Sarofim and Dr Nisbet, in their paper, and April 1972 HONS 208273 1S1 Dr Risebrough, in his, have undertaken overall assessments of environmental sources, patterns of distribution and alteration of the PCBs. It has already been clear from earlier surveys reinforced by studies presented at this conference, that the PCBs arc very widely distributed and are almost ubiquitous in their presence at many levels in the biosphere It appears, however, that their distribu tion may be less uniformly global and more representative of local sources than DDT and its analogues The approaches used in the two papers noted represent a very good beginning and serve to define, albeit perhaps only in an initial way, some of the major features in the overall PCB budget Equally important, they serve to define gaps in our knowledge which may play decisive roles in developing a sound analysis of environ mental distribution patterns I note a series of items emerging as urgently needing clarification. 1 Further refinement of sources, including a Patterns of marketing and use Im provement here will require unproved access to such information from pro ducers and distributors b. Dumping practices, c Stability of semisequestered sources, e g , capacitors in dumps. 2 Better quantitation of discharge amounts by route into water and air 3. Appropriate analysis to determine the relative role of vapor versus particle borne PCB in aerial transport. 4 Rain out data in selected regions to assess the atmosphere as a transport route, by season and by region. Allied to tliis is the need for appropriate selective analyses of dated snow deposits for the develop ment of time trends. 5 Clarification of the importance of loca lised as opposed to generalized sources of PCB. As only one example of the many dilemmas, the Sarofim-Nisbet scheme re quires mid-ocean dumping to explain some high PCB values far at sea. Are such sources important'* 6. Clarification of the role of particle bound as contrasted to dissolved PCB in water transport 7 Development of a more efficient tnoni- toring strategy as a means of optimizing analysis and the development of global distribution patterns of PCB's In approaching the global problem, one must not overlook the importance of more localized systems of contamination Again a beginning towards this was illustrated in the paper of Dr Veith Studies of well defined localities can pro vide not only knowledge relevant to the specific units examined, but may permit extrapolation to other similar regions as components in the broader global scheme. These presentations made very clear the serious gaps arising from our ignorance of environmental patterns of alteration of the PCB's and their contaminants. This was brought out in the e\oellent report of Dr Hutzinger on photolysis of the PCB's Work such as this needs to be ex tended, and, at an appropriate point in their development, brought into more realistic rela tionship to the form in which the PCB's arc found in the atmospheric and aquatic environ ments, e.g., photolysis of PCB's os \ apor, on particulates, and so forth It is also very clear that attempts to deal with the transport and distribution problem are frustrated by la< k of understanding of biological alteration of PCB's, that is, the metabolism of the PCB's by biological systems at all levels Occarrtnet The insidious way in which PCB's have found their way into unexpected sources of exposure was illustrated by Dr. Kuratsune's report on carbonless copying paper. Although the state ment has been made that substitutes for PCB's in carbonless copying paper have now been found, the completeness of this substitution will require monitoring. It may also be desirable to check body burdens of those routinely using forms with carbonless copying papers Similarly, the reports of Dr Trout and Dr Kolbye, on the presence of PCB in papers used in food packaging, further illustrate the need for vigilance for detecting and preventing the escape of these materials into unanticipated places Although it appears that most of the major sources of PCB contamination in paper and card board have been identified and intercepted, this 182 Environmental Health Perspectives HONS 20827% optimum needs to be tempered by continuing scrutiny of food packaging material. The reports of Dr. Kolbye and Dr. Berglund, on the presence of PCB's m foods, suggest con siderable similarity in the general patterns of food contamination in Sweden and the US. In each instance, fish, particularly fresh water fish, appear to be the largest dietary source Milk has been found heavily contaminated on occasion. Hopefully, this is no more than episodic, re sulting chiefly from the contamination of silage from PCB's in silo lining paints as described by Dr. Pries. This mode of contamination clearly needs careful scrutiny and correction. Control will be simpler with major milk producers than with the smaller units, and particular attention should be given to the latter In a related matter, the possibility that human milk may be a sig nificant source of exposure of nursing infants needs further examination The data from FDA is perhaps heavily biased by including a large number of samples secured as part of compliance studies. A more orderly picture of PCB distribution in food will require a more systematic sampling pattern and this should be undertaken Similarly, food surveil lance is now hampered by inadequate technics for detecting the presence of possible impurities in the PCB's. As noted above, resolution of this issue will require either chemical analytic technics many orders of magnitude more sensitive than are now available or bio-assays as suggested above. Animal Ttxlcatoor The Biotest studies are now complete and were well summarised by Dr. Keplinger. A Bomewhat parallel study at Chamblee is still incom plete (personal communication. Dr. Renate Kimbrough), Examination of the complete data from the Biotest sad the Chamblee work will be required before a full assessment of these toxicity studies can be made. Several issues present them selves The extreme sensitivity of the mink, both in lethality (1), and in possible reproductive injury (2), raises an alert which will require resolution before we can take too much comfort from results showing lower toxicity in other mammals Re grettably, this conference did not include an up dated report on studies of mink. Although the Biotest report indicates no evidence for PCB carcinogenicity,one (possibly two cases) of bladder cancer may have been identified in the Chamblee study (Personal communication, Dr Renate Kim brough). These findings will require that we await the completion of the Chamblee study and examine the Biotest reports in more detail before we can further clarify the question of malignancy Reproduction seems to be one of the most vulnerable functions in PCB poisoning This is true in mammals as well as m other life forms, including both fish and birds. Again, the report on embryotoxicity in the rabbit (3) (not discussed here) suggests that this iBsue be further examined in mammals. It would also be desirable to relate reproductive effects to PCB burdens in wild populations. It is a remarkable circumstance that despite the interest in the problem, the ktnetics, distri bution, metabolism and excretion of PCB in animals are only very scantily understood. There is some data on half time of body burdens in the fat m the cow (Fries) and in the rat (4) but the information is still quite inadequate To jump ahead, it is unfortunate that such data did not emerge from study of the Yusho victims. These basic questions of metabolism, excretion and distribution need resolution and appropriate research should be put underway immediately The range of sensitivity to PCB is enormous, thus invertebrates show effects at levels of a few ppb (5). At the other extremr, E Coh appear to thrive at thousands of ppm (Keil). Fish, birds and mammals fall between these extremes, with fish and birds appearing to be more sensitive than mammals. Again, reproduction appears to be the most vulnerable funrtion In respect to birds, and m contrast to DDT, it appears that hatchability may be more important than egg shell thinning. This area of comparative toxicity from species to species is so large and uncharted that generali ties seem out of place. It would thus seem de sirable for those involved in the study of inter dependence within the biosphere to plan carefully the necessary studies to clarify these issues of comparative metabolic patterns by species \s suggested above, such studies may be of great consequence for determining overall patterns of transport and the ultimate fate of these materials in the environment April 1972 MONS 20*275 183 A problem that persists in confusing our under standing of the toxicity of the PCB'a is the un resolved question of contaminants and the dif ferent isomeric constituents of the various technical PCB's. This has led me to the strong conviction that it will be very desirable to aelect a modest numlier of representative isomers which should be synthesised and highly purified, and then carefully examined toxicologically. The selected iBomers should be chosen on the basis of the best judgment available as being likely to typify not only '`average" biological properties but also those corresponding to limiting or boundary biological actions in a qualitative as well as quantitative sense. Synthetic capabilities, we are told, are quite adequate to meet this challenge. Included in this should be well defined examples of the chkinnated dibenaofurans. Such an approach should permit a more rational plan ning of future toxicological work. These pure com pounds will also be indispensable in the further development of selected bio-assays. MtchmisiR >f Actta Voa has described five distinctive patterns of pathologic outcome, acnegenic, liver damage, porphyria, edema and immunosuppression which he believes are diffenngly contributed to by different chemical components in the crude PCB preparations. As just noted, the availability of purified trace contaminants and isomers would permit an extension and refinement of this im portant approach to an understanding of patho genesis A number of studies have thrown light on some intracellular actions; however, it can hardly be said, at this stage, that we have good suggestions as to the major underlying mechanisms of toxicity. Rather than to hastily examine the effect of PCB's on a myriad of in vitro test systems, it would seem wiser that these studies be carefully planned as aids to understanding the observed course of PCB poisoning in the mtset animal. Humu BedyBartai The reports from Dr, Yobs and Dr Price and the comments from Dr Hammer all point to the ubiquity of the presence of PCB's in humans in this country. PCB's occur ui the fat of all segments of the population and some gross features of population distribution have been identified This work needs to be extended In doing so, par ticular attention should be given to 1) more representative population sampling, and 2) stan dardisation on the basis of lipid content of the tissue. This latter appears to be especially im portant in relationship to plasma concentra tion. The lack of understanding of the kinetics of disappearance in humans hampers the interpre tation of some of this data Studies on primates could help clarify this issue YhsIh Dr. Kuratsune has presented us with a re markable example of the quick r&llyiug of medical and scientific talents to clarify the unfortunate accident leading to human PCB poisoning. The thorough and thoughtful studies of the staff of Kyushu University have provided excellent data on clinical effects and dose response in the Yusho episode It is rare that such accidents have been so systematically studied. This data will neces sarily play & major role in the assessment of the human hazard from the PCB's We in this country should be deeply grateful for their skill and thoroughness in studying this episode It would be extremely desirable to carry out de tailed systematic, long term, follow-up studies, perhaps every five years throughout the life of the exposed individuals. We hope that their desire to make such follow-up studies for the assessment of possible chronic effects will be fulfilled This country should be prepared to lend whatever collaboration it can in this very urgent matter fimral CimMints Extensive deposits of PCB's now exist through out the biosphere Even without further dis charge, these deposits will remam for some years, It will be very urgent for us to develop information on distribution and degradation in order to reach some predictions as to the probable persistence of these deposits The manufacturers of PCB in this country are now well alert to the problem and have announced a senes of steps intended to restrict uses to those which are con trollable. The success in achieving these reduc tions in usage will need monitoring In addition, 184 Environmental Health Perspectives MONS 208276 however, the socalled "controlled'' patterns of use, such aa large scale transformers and capaci tors, will in turn require monitoring against accidental discharge and leakage Substitution in the less controllable uses is apparently underway It will be very important in carrying out such substitution, not to replace a known hazard with an untried, unknown, pos sibly greater hazard. Accordingly, considerable care in effecting substitution will be needed. The approach used m the above discussions of concentrating on gape--things not done as op posed to those which have been done--brings the risk of implying that our ignorance is greater than it actually is. The many detailed and solid papers presented at this conference and as sembled here should serve as an adequate counter balance against such misunderstanding. Never theless, our ignorance is still too large. I have thus tried to identify some places where, I believe, we need additional understanding. Finally, I apolo gize for the necessity forced upon me by the shortness of time, of omitting from these remarks discussion of a number of important contribu tions at this conference. REFERENCES 1 Ringer. R , Johnson J end Hoopingamer, R 1971 Interagency Meeting on PCBe Dept Health. Educ A Welfare, Washington. DC . August 5. 1971 2 Aulench, R. J et al. 1971 Effecta on feeding coho salmon and other Great Lakes fish on mink reproduc tion Can. J Zool 49* 011. 3. Villeneuve. D. C st al. 1971 Effsote of PCB adminis tration on microsomal snsyme activity in pregnant rabbits Bull Environ. Contamin. Toxicol. 0. 190. 4. Grant, D L., Phillips. W E J and Vitlensuvs. D. C. 1971 Metaboham of a polychlorinated biphenyl {Arc elor 1204) mixture in the ret Bull Eoviroo. Cootamin Toxicol S' 102. 5. Duke, T. W , Lowe. J. ) end Wilson. A J. 1970 A polychlorinated biphenyl (Aroclor) 1254) tn the water, sediment, and biota of Escambia Bay, Florida. Bull. Environ. Contamin Toxicol. 5 171. 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