Document qazJRg2DnnOz0YjXnJ00LOK5j

Food Exposures to Polychlorinated Biphenyls by Albert C. Kolbye, Jr., M.D. The FCB family of industrial chemicals has been used in a number of useful and beneficial way# for nioic than 10 years. PCBs have been used in capacitors, as transfoimer fluids, as heat transfer agenls, in plasticizers and adhesives, and in sealants and pi inting inks. Needless to say, they have no place in the nation's food supply. The sncDtific community has been aware of the potential ha mid of the PCIls since at least 1900. Deports in the foreign literature at that time indicated the detection of tills chemical in fish and birds. However, the status of the analytical detection method for organochlonne pesticide residues in reference to PCBs was not perfected until early 10G9, after it was discussed in lrD Vs pesticide analytical workshop and the former Division of Pesticides improved the analytics! method, so that ft was satisfactory for routine annlysis. AVc know lhat 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 affect human health, and we cannot .vrt explain the inconsistent presence of Ihe chemical in certain areas of the environment We have been faced recently with PCB adulteration of food fiom accidental sources and from untrarrablc cm ii onmcntal sources. The exact routes of contamination are subject to speculation. Some theories point to the industrial wastes from plants using PCB, loathing of PCB from plastic objects ui wablc disposal (burning at waste incineration plants or run-off from landfills into streams), and* *31c)m1> Dmclor, D.irr.u of Foods, US Foud and Uruf tjiimmtrohon, \\ n-hmalon, 1> C JOWH accidental use of PCB as solvents for which they arc not intended (vehicles for spiay preparations etc) Some of PDA's earliest findings of PCBs in foods occurred during the fall 1909 uiicn PCBs were encountered in roho salmon ami milk In August, 1969, Baltimore District found an adulterant identified aa PCBs in 5 of 12 milk samples from West Virginia During a art-month followup investigation, it was learned lhat 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 right of way was tested and found to be positive for PCBs. It was eventually concluded that the apparent misuse of the trans former fluid containing PCBs for defoliant spray ing operations adjacent to dairy pasturage was responsible for the milk contamination. As a result, m 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 llolly Farms, a large North Carolina poultry producer, bad discovered the pre-enco of PCBs in its poultry, resulting from an investigation to determine the cause of rfdiieed hatehabihty in eggs, The film's imesligatjon April 1972 85 HONS 08 L 389 `*'4** i UtlW >.l^.*J*T.,U'.. . jf miplirnled the fish meal ingredient as the causa tive agent The l'DA tested the processed fish meal ami confiimed that fish meal tin hand was con(niniunled with PCBs Continuing invesligahon indicated the leak he gnu on Apul 30 mid continued through July 1G Approximately 16,000 tons of fish meal vicie 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 fish men! samples examined contained from 14 to 30 ppm PCBs. Fish ineal, the direct subject of this contamina tion incident, is a minor component of poultry and fish feed Investigations conducted by FDA and USDA focused attention on the contaminated meal as a possible sou ice of PCB residues in poultry, eggs and commercially raised catfish. The Food nnd Drug Administration visited primary and eubconsignees and initiated followup sampling of fish 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 1,300 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 3 ppm interim guideline for PCB residues Follow-up sampling on eggs waa initiated on AuguM 3, 19*1 Vs of September JO, ;<)T| jji .samples of ogg-> were anwlv zrd m llie follow up, with 71 containing re-i<Uies> in cvcm of 0 j ppm (rnngc 0 0 4') We seized Inc shipment? of fish frciJs in llir Stales of Loum'ina, Geo'da. and .\Ii--i-.i[.pi Hu contained fiom 0 G to 4 7 ppm PCIfc JJcvcn samples of catfish, which had liccn frd tin enntaminated feed, cun lamed PCB residue'. willnn the 3 ppm guideline, ranging fiom 0 3-3 0 ppm In addition, ne seized a shipment of the iontnminated fish meal from East Coast 'lerrmnal that had not been recalled and contained in cxcc". of 350 ppm PCBs Since November, 1969, the FDA has analyzed all ran agricultural commodities sampled under the pesticide surveillance progiam foi PCBs as well as pesticide residues Cher 17,000 samples have been collected and analysed In the past IS months, 6S4 samples of fish, cheese, milk, shell eggs and fish by-products, out of 3,503 samples of these commodities, have been found positive for the PCBs FCBs were encountered most frequently in fish in 303 of the 070 samples ranging from trace levels to 33.29 ppm PCBs were detected in fresh water fish ineluding catfish, chubs, and smelt, and also salt water species including porgies, sea trout, bluefish, bonita and sardines PCBs have only been detectable in shellfish at trace levels 1 tic results of the pesticide program for PCBs for that lS-month period are as follows: PCB in aeleeted food eommodltim--7/1/70 to 9/30/71, Number of URipiw summed Number of empire positive Percent positive PCB levels Low (ppm) high Average Piah Cheese Milk Shell eggs Fwh by-product 670 1344 941 550 -- 303 1 SO Ml 13 HT T TT 2# T --T 31 39 10 17 8 3 74 50 1 67 75 2 27 55 1 17 Tots) (excluding fifth by-product) 3505 -- 084 -- 19 -- 1 14 Above results reflect both survtiilsncs and comphaaes* samples. Avenge residue levels dn not include values indicated at "trace" T indicates "trice", "Inch in normal analyses would tic Ins Uwn approximately O 1 ppm flsh, 1-1 5ppm milk orcheesefet; 02ppm eggs. * Compliance sa mples reflect follow up of violative samples hence biased towards high levels 86 Environmental Ilealllv Perspectives & ' `''Si *0NS G8I39Q Tlio FDA total diet studies for GO market baskete representing a totiil of 720 composite som[lies during FY 70 and FY 71 show 22 com posite samples to contain i'Cfi residues ranging fiam s trace to 0 3G ppin These poi(n c findings cic found m the meat, fish, poultry, daily, and the gram and coca I composites The 0 3G ppm PCB value reported in the total diet study it as found to bo caused by nugiation of PCUs from die gruyboard container and dividers to packaged shredded 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 m Food and Food Packaging Material Tins aurvey was designed to cover selected food categories nationwide to detsrniino the extent of foud 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 284 samples. The packaging material analysed in this survey included the following types of components: mj? maou* nc Tltienr I ?imei-rCH m Foot! and looil Pnrkifiiiig Material Cnmuiainu tnalyrusl Finding..-- ,Scpi 8Nov, 1H71, (Mimas nr Type* of components cotnprialng pnli|la| material uuiyacd during auney Inner wrp|iors Waxed piper Cellophane Foil Paper Plastic Divider* Piper tnpa (aa used for cookies and randy) Plastic trays Paper Irayt Outer Wrappers Ko.t riastle Cellophane Paper Other Cellophane Fiber cartons a inflow Pint ic bigs Paper envelope* Mclal ran ends Darin. i.m end* PI nfic le.ir slrip* Icabagi Klevaa t Survey--PCIl in Food and Food Packaging Material Cumulative Analytical Findings--Sept 8Jfov. 4, 1971. enaaauaim Pome of the findings from tins sur\ cy ai received ii' nf dial date are diowu in the follow mg Figs. I, 2, .1, 4, 5, G nml 7 Flo HIT. 3 Pur\cj~PCU in Food mid loot! Packaging Material Cumulative Vnabtu.nl 1 Hidings--?cpt 8 Nov 5. 1971 tpiil 1972 87 HONS 081391 Wfllttl MR anqft U fror KjQ L-J * (MOM* < WW.V Fjgc* 4 Sun rv--PCB in Food and Food Picksting Mstensl Cumulative Analytical Findingl--Sept 8Nov. 5,1071 umiowu. mtuM Figure 5 Survey--PCB In Food and Food Packaging Material Cumulative Analytical binding*--Sept. 8Nov. 5, 1071. net} ok. I*. Flccjil 6 survey --rCB irv Food and Food Packaging Material Cumulative Analytical Finding} Sept lNov 6,1971. Fic.ohe 7 Survey--PCB in Food and Food P*rL*(nng Material Cumulative Aoalytieal Finding*--Sept 8Nov. 5, 1971 It is significant (1) that no PCBs have thus far been repotted in fresh fruits and vegetables, (2) that PCBs are found in fish quite frequently, (3) that in addition to poultry, PCBs 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 front their packaging components. At this point the signifi cance of thei>e findings has not been determined With the conclusion of the PCBs in Food sad Food Packaging Material Survey, FDA has embarked on a program to determine the level of PCBs in animal feeds Following the I'CDs in feed program, TD.\ ttiU further evaluate PCBs m 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 on apecifis occurrence* of "accidental" contaminations They are 5 ppm in the ediblo portion for fish; i ppm on s separste fat basis for poultry; 0.2 ppm in whole milk (5 ppm in the fit). Action has been taken against animat feeds and whole eggs associated with the East Coast Terminal incident at the 0 5 ppm level These levels should not be construed ss guidelines permitting consumption of foods containing thc->e amounts of PCBs. 1/ 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 low ered. 88 Environmental Health Perspective} A MOMS 08,1392 Environmental Health Persftectiu*i Polychlorinated Biphenyls: Their Effects on Penned Pheasants* by Robert B. Dahlgren and Raymond L. Linderf and C. W. Carlson}: Introduction Polychlorinated biphenyls (PCBs) have been useful industrial products since the beginning of tlieir commercinl production in 1929 The first rrport of PCHs occurrence m wildlife was nude from the work of SSren Jensen, s Swedish Cliemist, in the Ntvo SaenM in 1906 (1). Since then l'CBs have been reported to be widespread in the world a ecosystem, building up in food chains as has been reported for organochlonno insecticides. The chemical structure of PCBs and their action on organisms, although less toxic, are similar to tlint of DDT The contamination of human mil);, poultry feed, and all classes of wild vertebrates, '<v crested a concern for the need to determine 'ossible harmful effects of PCBa. * Fund* for the ituty wer* supplied by the Bureau of 'iiti Kirhrnes and tVUdlifs through the South DskoU 1 ooperame tt ikDife Research Unit, supported jointly by Muth Dakota Department of Came, Fish, ind Parka, 'Itr Bureau of Sport Fisheries sod Wildlife; the South Iaioiji stale t immity; and the Wildlife Management .intitule Tmm i>1j*s *re made by the Denver " i*dlrf Reseateh Center and by Dr. Yvonne A Cneehus, l rpenmeni Station Bwheaualf) Department Dr Robert ' Hor.v Vetennery Seieneo Deportmenl, condurted etowies and hntopelhelecie itudies. Dr W L Tueter, l-'i*rmen( Station Slstolieisn, gave ilstuliesl odriec "iH iibe.issnta wire rofleelrd by Fred E. Hartman and 1 " i Kne PvnnvIvans Game Commission, and I'M 13 leldl, Indian* Department of Natural He. nv 1 bi work i fruin a dueiorvl ih'sertuliim i South 1 -tm l ii.verstv l,v the rrnmr stulior, 1 'i.ii bejdi r and Leader rew etilvelv, S.,uth "11 Cv^'vranr \Vilritalc ReHaith Vun, South 11 "M net nuertilv ilnotin*. S p ,7016 l r..:.. i of \iuntjl Nirnie, ,-uul LejJir, I'oultrv Re ' >.it Lvtiri.mil South D.itot.i Si ile L imrr.ilv >1 ' D iTOOC Because of the economic importance of the pheasant (Phasianus colchicui), it was chosen as the experimental animal using PCBa in this study The objective* were 0) to determine the patterns of absorption, storage, and excretion for PCBs, and (2) the effects of PCBx on reproduction, behavior, and survival Since many published reports indicated that the chromatograms of PCBs in wildlife most nearly resemble those of Aroclor 1234, this was the PCB produet chosen for study The use of brand names in this paper does not indicate an endorsement of any product. Mate rlils and Methods Aroclor 1234 (supplied by the Monsanto Co ) was mixed (w/w) in a 1:9 dilution with corn oil and weighed into No. 00 sue gelatin capsules to within 2 3 percent of the required "right Capsules were administered into the eaophigus using a glass tube Capsules containing only corn oil were sdministered to conttol bird* Analyses for residues w ere made by Dr Yvonne A Greiehus, Experiment Station Biochemistry Department, South Dakota State Unix ersnty, and lhe Bureau of Sport Fisheries and Wildlife laboratory at the Denver Wildlife Research Center Analytical procedures used at the South Dakota Stale University laboratory arc deseiibevl by Dahlgren ct al (2) and procedures of the Drnver Laboratory by Dnhlgrcii el al (3) Hens for breeding o'penmeiils weio pmrhivod in the mniers of 10"0 and 197 i <!0 and 34 icsfjr'eiivcty) from the South Dukor.i Pheasant Co of Canton, and cocks were raided from birds pre viously obtained from tlic Canton tourcc Ml birds used foi bleeding were about l year of age Wml 1972 89 HONS 061393 "'*'> 'Sm-inm- .1 tr>t n H--yp*>* <-y'ni~i-W'Vrjhiiitna n'twy ta*aiW<n*m,i.... n _. ln late January they wore pin red under 10 hours daily of artilinal light to stimulate breeding. Goth sexes were fed a pheasant breeder ration (Zip l-'ecd Mills, Sioux Tails), One tablespoonful of ojsler shells uas added to the food cup of each hen Meekly. Cocks were kept 1:1 btecdci cages (30X3UX1S inches high), and hens wcie kepi in individual cages (12X16X12 inches high) de signed to facilitate handling and identification of eggs. Temperatures ere kept at near Go F in colder months. Egga "ere 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 25 mg PCBs 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 30 hours after hatching, chicks were taken from the incubator, wmgbanded, and one wiog was removed to the alula. They were then placed on a visual cliff (4) and given 5 minutes to jump to either the visually deep or shallow aide. Chicks from this study and those hatched from a concurrent dieldrin study were placed together in brooders and fed a turkey pre-starter diet (Zip Feed Mills) until they were 0 weeks of age At 6 weeks, they were placed outside in 16X10 foot pens and fed a pheasant grower ration (Zip Feed Mills). Several times in summer and fall, chirks were caught by hand by the same person, and w ingbaod numbers were recorded. The number of birds caught in eadi category in the first and lost half of all 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 tlie nearest grain at 0 weeks of age To study effects of TCIls in combination mth dieldrin, eotks and hens from G to 9 months old were stiatificd In sex and weight and randonnrcd tn five groups of -U birds carh 1 licv were git cn capsules containing cithci (I) 4 mg tcdmic.il glade dictUrin, (2) 5 mg dicMiin, (3) 00 mg I'CHs, (4) 100 mg PCBs, or (0) 50 mg l'CUs and 4 mg dieldrin together. Ten doses wcic given, one capsule each 3 5 ditjs for 5 weeks Tluce and one-half davs after the last capsule was given, alt suiviving birds were sacrificed The Denver Wildlife Research Center anal wed for residues of PCBs in pooled livers from three road-kilted pheasants collected near Washington, Pennsylvania, six rood-kilted pheasants collected southeast of Lancaster, Pennsylvania, siv pheas ants shot cast of Gary, Indiana; six pheasants shot in Benton County, Indiana, and six pheasants shot near Brookings, South Dakota All of the above collections w ere made in the spi ing of 1971 Results and Discussion Effect* on Reproduction The a\ ernges of eggs laid per hen per day among hen treatment groups were significantly different in 1970 (5). With the use of an orthogonal test, the rate of egg production among hens given 12 5 mg PCBs was not found to be lower ((*>0 05) than the rate of the control group, but the rate of the 50-mg group was lower (PC0.01) than the rates of the other groups In 1071, 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 was not lower (P>0,05) than that of controls, while production of 50-mg hens was lower (PcOOl) Egg production was loner in groups where cocks were given PCBs, but this is not to be expected, and w* cannot explain it Differences among groups in egg fert ihty in 1970 were significant (P<003); however, venation in fertility did not follow a pattern related to i'CU dosage, and, thus, the results may not have anv biological meaning (5). In 1971, there were no significant differences in fertility among groups (Table 1), Hatclubility in both 1970 (5) and 1971 "as highest in control gioups and lowest in group* m 90 Environmental Health Perspective* 7llc I Rrprn<lH-`i'e from ronirol |>l>fl*unl and j>ltraanii gnen PCI)* 1970-71. TirWineftl group* hrij per dry in irrsbnnr Fcr<iJe rcgi \o Percent Ferufc ecu hatched No Pereenl Fertile re** | hul not h*ft lied \n Percent jn;y p-0` 0-12 5 0-50 2cr9 2o-!2 5 2o-50 l7l 0-0 0-12 5 a-JO 2.V0 2>J2 J 2.--SO 0 021 0 436 0 202 0 33J 0 3G2 0 198 0 337 0 430 0 328 0 405 0 380 0 228 277 101 38 220 128 53 136 50 37 IG8 flt 54 179 71 40 98 03 04 188 82 49 199 128 04 148 90 05 233 170 73 283 1 S3 53 9S 42 44 74 73 87 68 32 04 73 80 50 70 41 05 65 79 64 50 51 53 116 68 109 71 10 24 fl 0 29 23 8 16 9 10 18 23 10 10 07 20 19 44 85 13 10 3 12 Hid* IrcilnicntRroupliiw^ fhehn, evctptthM the 25-12 5 mg group hndnmt hen* in 197 \ * 7 be first number u the neckly PClHe\l m mg given tococii, the second, tht farhons *hirh lien* had received 50 mg PCBa. In 1670, ',!-f differences tested by chi-square were not 'ipufiranl (P>Q 05), while in 1971 hatchability *#f reduced (7><001) among Hen* given PCBa. Mpnficant differences (P<0 04) were found in the number of eggs that were pipped but not hatched for hen groups in both 1970 and 1971. tpiwrcntly, the administration of PCBa to the urn adversely affected the viability of the embryo at hatching. McLaughlin ct a) (8) injected both 10 and 23 mg of Aroclor 1242 into chicken eggs wid found only 0-5 percent hatchability with run ill retardation, edema, and beak deformities ui embryos. Eggshell thickness was measured only in 1970, and no significant differences (P> 0 05) in eggshell ilurkuess were found using analysis of variance Lcgslicil* from hatclied eggs (without memh'anes) laid by control hens averagsd 0.2<j0.02 mm standard deviation, and those from ail hen Puups reeeiv ing PCBs averaged 0.23 0 02 mm. LfRlietls from uuhatched egga averaged 0 3_'i0 02 mm for control liens, and 0.31 002 .un for iwna receiving PCBa. Dahlgren and Linder 11 found the eggshell thickness of pheasants lo be naficcicd by weekly administration of capsules winwiimg <5 mg dieldrin. Heath ct al. (8) fed 2.5 ppm of Aroclor 1254 to mallarda (Ano* plahjihonchos) through two breed ing seasons They also fed bobwhite quail (Cohnut virfiiuonia) 30 ppm PCBs and at a joint level of 25 ppm PCBs and 13 ppm DDE for one reproduc tive season. They found no effects on egg produc tion, cracked eggs, eggshell thickness, embryomtlon, embryos alive at 3 weeks, normality of hatchlings, and normal hatchlings sin e at 14 days 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 weeka; however, they noted a 10 percent reduction was associated with 10 ppm, and a 13 percent reduction waa associated with 20 ppm They reported that 10 ppm Aroclor 1248 reduced hatchability of chicken eggs by S percent after 4 weeka and 44 percent after 8 weeks. The 20 ppm level nearly eliminated all hatchability Most embryos died at 21 days of development; many after pipping They also found that eggshell strength was not affected when as much as 20 ppm was fed. Effects on Behavior Seventy-one U-wcck old hens given one 210-mg M>nl1972 91 v I I i I f I I TiiMeZ Behinroroii the'i.iiul cliff of ducks ImlcheP front control pliea.nntg anti pliciwiits gnrn I'CfVi 1970-71. '] mliuftil No 1 lit lumped to twnll\ 4*rt mt/o So that lumped (0 Mwtl'lll) wlnllnvs inlo Xo n t jumping itimi j minute 4 1070 ten 197 0 1971 1970 197] 0-0CM2 5 0-50 25-0 2.V12 5 zi-ao 29 \7 37 5 ID 5 16 40 35 43 .14 6 30 33 67 22 78 59 87 s5 58 15 17 77 61 The first number is the wetUy PCB level in mg given to cocks; the second, that for hens capsule in the evening of the first day of testing appeared v. ck at the end u( the following day. After receiving tlte second capsule, they sat with feathers fluffed, and some tremonng was noticed. JJurmg the 24 hours before death, they often showed tremors, particularly when disturbed. Shortly preceding death, birds were comatose and died without tremors. Eleven 11-weck old hens given 20 mg daily were hypercxcitablc after 4 days of PCD treatment, after 10 days they eat 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 ct al (10) reported feather loss in chicks of the domestic chicken given PCBs. Kmtut< CUff Behavior--In 1970, when chicks were placed on a visual cliff for up to 5 minutes, significant differences (PC0.01) among groups were found in their behavior using chi-square analysis (3); offspring of hens given 50 mg j umpe d to tlie visually-dcep side of the cliff more often than offspring in other groups. In 1071, no sigmficaut differences in behavior between groups were noted (Table 2). When data from both years were combined, no significant differences (P>0.05) wer* found. Baxter et al. (11) reported that pheasant chick behavior in a visual-cliff test was apparently affected by dieidrin given their parents. Response lo Hand fitlclung--It'vnu M catching } oung pheasants b> ham! wore jm,\ 7fd by coni pining the number of birds from c.uh ticatmrnt group caught in tlie first half and l.nt half of all birds caught to an rvpcctccl mmiinv equaling 50 percent of the birds in that group 1 or evil 111 pits. 1G out of 20 j oung in the cotegori whore both parents hud received i'CHs were caught 111 the first half ot fill birds caught on July 22, 10"0 (Table 3) The 10 caught in the first half and 4 caught 111 the second half were compared by chi-square analysis to an e spected 10 (half the total category of 20), resulting m a highly signifi cant difference (P<0 0i) Since each pen wns a closed unit and the PCB-treatment buds were penned with other young fiom a conctment dieidrin study, it must be remembered that birds of several groups caught predominantly m 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 PCD birds were emight in com parison to an expected distribution, but also how categories compared with one another 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 m 1970 offspring where both parents had been given PCBs. This is identical to the findings of Djhlgrcn ct al. (12) for offspring of parents administered dieldrui. However, in 1971, the responses of off spring of treatment groups were similar to controls. Data for 1970 and 1971 were combined and a contingency table chi-square technique was used to determine the effect of PCB treatment 011 tlie response to hand catching between groups The responbO was simitar whether PCBs were given to either the cock or hen but was significantly different (P<005) when both parents received PCBs from that where only one parent recen ed PCBs. During catching, pheasants w ere herded around and around the pen, and often the catcher was able to capture those birds w hich ran back directly toward the pursuer. However, there was no attempt made to determine or to quantify (be elements of behavior that would eapliun why offspring of birds treated with PCBs were easier to catch 92 Environmental Health Perspectives HONS 081396 r-i-/*. m* r-V* * ike Oril keif of all Wrfi caught i number* in pinnllmee represent one-half ilia numkcr of that category In tke pen. Ctii-squar* nam vtf to compare number# actually caught with half of the number# In each category.) D<i of capture No cl hatches caught No of pen* Both Parents receiving PCB Hens only Cock* only Neither 1970 July 22 July 29 Aug 13 Aug 19 Sept 1 OcL IS Nor 21 Dec S 1-8 14 18[10)* 1-9 15 19{ll 4)** 1-10 18 11(12 5) 1-12 20 21(15.3)* 1-14 22 2508)* 1-15 23 <) 1-15 13 14(11 5) 1-13 13 11(11) 8(8 5) 700) 12(12 3) 17(14) 19(13 5) 14(18) 14(12 5) 1401 5) 7(10 5) 902) 1203 5) 18(17 3) 23(20) 23(17 5)* 800 5) 12(10,5) 11*(10 3) 8(13 5)** 803)* 19(18) 14(19.5) 904) 12(11 5) 8(10)** All 1970 e# tehee combined IST(lOe)** Ratio, 1970* 1 55 1971 July 21 1-7 17 27(19 5)* Pept. 1-3 t-16 28 75(70 5) Sept 21-28 MS 28 71(70,5) Oct 7-11 1-1* 28 33(37) Ort 19-22 1-16 28 34(35 3) Nor, 2-3 1-15 28 30(30 3) 105(100 6) 1 91 23(24 5) 41(37) 38(38) 33(35.3) 33(33 5)* 29(32 5) 113012) 1 98 25(21 5) 39(38 8) 37(38) 27 <W> 35(34 5) 23(28 5) 85010)** 2 39 12(14 5) 21(22) 24(21 8) 19(20 5) 17(21) 1(18) All 1971 eetcbea eombaed Ratio, 1971 270(284.5) 1 98 187(199) 2 13 187092) 2.05 112(117 5) 2 10 All 1971-71 Celebes Ratio. 1970-71 407(370 3)** 1.83 202(298 5) 2 OS 300(304) 2 03 197(237 5)** 2 31 `ToteIt of row number* may exceed one-balf total birds beeaut* odd number# of birdt in pen* acre rounded higher. Bird* in pen/bird* caught 10 first half. *{P<0 OS). *(P<0 01), TaUa 4. Welghu and wrdtal for tha brat t weeks af offspring from control pheasants and pheasant* given PCBa, mO-71. Treatment group 0-0# 0-12 5 0-50 25-0 25-12 5 25-50 at 8 week# (|) 1970 1971 898 429 378 435 803 425 389 428 403 458 344 373 No. of chielce to brooder 1970 1971 73 85 84 84 29 50 72 115 50 109 40 10 Mo of ehwkt alive after 8 *eeha 1970 1971 49 50 49 45 3 37 50 91 30 72 98 Peneut sumivsl Bath 1970 1971 yetre 87 77 72 58 70 84 10 74 51 89 79 73 60 M 54 22 50 30 The first number it the weekly PCB lev cl in tggiven to cock#, tlw aecond, that lor hen# April 1972 93 WgggH JW /'t?' ^ HONS 08139? mtr Tl>le 5. Survival 10 III* (all of offvpi-irg of control phtt*nls end phrai.nl 1 gnrn Pf hi, 2970-71. Croup No of younf live m fftH* Percent eurviyal from 6 uceks of ig* to fall* Percent, suf' al h t'clting to fn|J' 1070 1971 Both 2070 197] Yeire Both 1970 1971 ^ nfa IMX 0-12 5 0-50 35-0 25-12.5 25-50 20 36 23 36 0 29 21 57 18 69 44 41 72 57 47 60 6** 0 78 72 42 63 65 60 42 75 44 67 53 27 A3 41 27 5& 40 0 53 37 29 50 42 36 54 43 to <0 1& * Decembers, 1870; November 1-5,1871 v Uing the number of etucka alive after 6 week*. Table 4 * Hung the number of chicks to brooder, Table 4 * The Orel number in the weekly PCB level in mg given to cocki; the aecond, thet for hena Effect* on Survival and Weight* of Offspring Survival--Chick survival was determined for treatment group* in 1970 (5) and 1971 during the first 0 weeks of age while chick* were kept in brooder* (Table 4). The survival of chirk* was not related to whether cock parents received PCB*, but chi-square analysis showed that significantly more (PCO.OI) death* 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 nas s significant difference (PcO.Ol) between offspring where hen* received 0 and 12.5 mg PCB* and between offspring where hens received 12.5 mg and SO mg PCBs (PCO.Ol). Survival of young pheasants in outdoor pans was measured in December, 1970, and November, 1971 (Table 5). Survival of offspring from 6 weeks of age to the fall in both yean appeared to be unaffected by level of treatment in either year. When overall survival from hatching to the fall in both yean was considered, the overall survival of offspring from hen* given SO mg was significantly less (7* <0.05) than that of the other groups. The lower rates of survival for 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. IPsighe--Weights of chicks irom hens on 50 mg PCBs weekly were loner (P <0 01) at 6 weeks of age (5) them those of other groups in 1970 (Table 4). In 1971, weights of offspring of 50-mg hens were lower (7,<0.01) than that of controls, while weights of offspring of 12.5-mg hens nere higher (P<0.01). Wien date from both years Here combined, there was no relationship between weight and treatment lei el, Although we did not adequately determine that Table 4. Number* of twill and ben* tllve In NovtmUr thfft wr# offiprinf of control phtmnti m>4 pbmanti fifen PCB*i* 1970-71. |Chi*iquar was Uifd to coniftf* nunibtn of rath ei aln* mill n expected SOiSOdJltribulioDv) Oroup 1970 KoTCrobof 21 Cocks Hen* 1971 November 2-3 Cocks Hens 1970-71 Combined Cocks- linn 0-0* 0-12 4 0-30 35-0 35-12 5 25-50 AH PCB froup* 11 n M 11 O1 5 to- 13 6 11 33 35 17 17 IS 20 13 17 S3 22 3 20* 3i 100 80 26 :i 20 31 13 IS 38 36 4D 26- 42 122 11-9 TU lint iwimbtr i* the 'wkh In el m mg if PCD, given lo torts; the second, that (or hem (P<0 05). **(P<0 01). 94 Environmental Health rtrspeclim HONS Off1390 PCBa vi the egg depressed the weight of offspring. McLaughlin et al (6) mentioned growth retarda tion of embryos as an effect of PCBs injected into the yolk s&c of chicken eggs. We fed no pheasant clucks PCBs, but Flick et tl. (10) found that 1-dny-old chicks fed Arocior 1242 st 200 and 400 ppm had depressed growth by the second week of feeding and that tlie growth depression was related to Die level fed. Vos and Koemsn (13) fed PCBs to 1-day-old cockerels and found bodyweight depression from 400 ppm Arocior 1260. Plalonow and Funneil (14) found that 1-day-old cockerels fed 250 ppm Arocior 1254 had depressed body w eights between the sixth and ninth w **k of their feeding trial; this depression was associated with reduced feed consumption, Relifeld (15) also found depressed weight gains in 1-day-old chicks fed sublcthal levels (10-50 ppm) of Arocior 1234; chicks fed 30 and 50 ppm for 2 5 weeks and then fed a clean diet recovered from the growth depres sion, while chicks fed 40 and 50 ppm for 5 weeks and then fed a clean diet for 8 weeks did not show a recovery from the growth depression. Body weights and Mortality in Birds Given PCBa Adult hens in both the 1970 and 1971 breeding experiments were unaffected in body weight by administration of aa much as 50 mg PCBs in single capsules weekly. It was characteristic, however, of birds that died oo PCB treatment to atop 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 day 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 fluffed; some immoring was noticed; and no food was consumed. Birds of both aexea 6-9 months of age given 100 mg or 50 mg PCBa every 3.5 days for 5 weeks continued to eat, as did 11-week-oM hens repeatedly given cither 10 or 20 mg dally, Scott ct a). (9) found no effect on feed consumption when laying chickens were fed up to 20 ppm Arocior 1248, and no mortality that could be attributed to treatment. Prestt et a). (16) found no effect on weight of BengaWve finches (LencAure alt tala) when they were fed up to 400 ppm Arocior 1254 for 60 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 2-5 9 days, averaging 3 81 0 day stand, ard deviation (3). They lost from 15-37 percent of their initial weight before death All control birds lived. Correlations of the initial w-eight, days to death, and percentage weight loss were ob tained for 53 birds in this experiment. Initial weight was correlated with daya to death (r-0 899, 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, PC0.01). Birds thst lived the longest lost the greatest percentage of their weight before death (r-0.744, P<0 01). Time of death of the 11 birda that were not given PCBa, but were starved, ranged from 2.3 days to 8.6 days, averaging3 91.8 days. They lost from 27-51 percent of 1 heir weight by the time of death (3). Of the birda 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 dkd 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 day*. The other seven birds died between 50.3 and 60.6 days after initial treatment. The tenth bird of this group, still alive after 8 months of treatment, was sacrificed (3). In the 20-rag group, the first bird died 39.6 days end the last bird 54.1 days after capsules were first given. The average number of days to death was 46.1 5.3 days (3). Mortality was light In breeding experiments in 1970 and 1971. In 1970, only 2 hens died from among tire 30 hens and 10 cocks under study; both hens had received 50 mg PCBa 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 Arocior 1242, 1234,1200, or 1268 given to mallards caused no mortality or symp toms. Prestt et at. (16) estimated that 254 mgAg/day given to Bengalese finches would give 50 percent mortality at 56 days. Heath et al. (8) reported that Arocior 1254 had an LC of 1090 April 1972 1 HONS 081399 Olrni, MW ppm when fed for 5 days as part of the diet to pheasants. Vos and Koeman (13) reported that, of 20 chicks fed 400 ppm for CO days, only three died. In the present study, 210 mg PCBs daily were given to 11-eek-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 birda 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 1234; 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 Dablgrcn et at. (2) reported that retention of PCBs in the bodies of four hena, each given angle 50-mg capsules and sacrificed 28 days later, averaged 40.5 mg in each hen. Levels of PCBs were highest in all tissues at 13 hours after capsule administration and declined most rapidly in liver at 24 hours. Throughout their experiment, levels were higheet in liver, followed by brain and muscle. Brain tissue contained more PCBs than muscle per unit weight, but, having a higher lipid percentage than muscle tissue, brain tissue bad 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 doae, while hena on the 50-mg level retained 60 to 82 percent of the administered dose. The hens on the higher doae averaged about six times as much PCBs in their bodiea as the hena on the lower doae, 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 28 days was accounted for after administration of a 50-mg capsule (2). The PCB-com oil mixture was readily absorbed in tlie gut of the pheaennt; calculations showed that a maximum amount excreted un absorbed was 6 percent of the 50 mg given, and as much as 9S percent may have been absorbed. Five of the hens that had received 12 3 mg weekly for 1G weeks in the 1971 breeding expenment were sacrificed 1 week following the ad ministration of the Inst capsule In the series The ppm PCBs in the bodies of these five hens u ere 17 6, 18 6, 24 4, 28 3, and 30 5, averaging 23 S ppm. Three months later, three other hens of the same group, which had been kept caged, were sacrificed. The analyses of their bodies showed 8.9, 12.0, and 20.0 ppm PCBs, averaging J3 6 ppm PCBs. After 6 months on a clean diet, three other birds similarly treated had whole-body ppm values of 18.3,19 5 and 25 0, averaging 20 9 ppm The apparent rise in PCB concentration at G months may be due to sample variation and/or the physiological state of the hens sampled It is obvious that the rate of excretion of PCBs is relatively slow. These birds were analysed at the Denver Wildlife Research Center. Scott et at. (9) found that chicken hens given up to 20 ppm Aroclor 1248 in the diet for 8 weeks lost lest 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 a high-energy diet did not affect reduction of PCBs over time. Prestt et al. (16) fed 1500 mg Aroclor over 56 days to Bengalese finches and ivere 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 fecee was relstively low as reported by Dahtgren 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 analyzed as s pool. Two other hens excreted 2 2 and 2 9 mg per 50-mg dose over a 2R-day period, the feces from these two hens were analyzed separately. PCBs in tiie pooled feces of the four hens were st a peak during the first week and declined to relatively lower levels thereafter. They also found that excretion in the feoea was highest during the first 24 hours in the two other hens given srngie 50-mg capsules, and less than 1 mg PCB* appeared in the feces the first day. Variability in excretion between these two hena was low An average of 2.6 mg per hen was passed in the fetes of the tv o bens by the end of 28 days Excretion Fia th Egg--In four hens given 50-mg capsules when egg laying was declining, levels of PCBs in the eggs were lowest in the first 96 Environmental Health Perspective* 0B1*0 kqhs highest during the rccond week, and de emed thereafter. The average excretion per hen six the egg was calculated to he 4,2 mg PCBs (2). Excretion of PCBs via tlie egg could be higher than excretion through the feces when hens are in full egg production. A single egg laid between 1 and 1 necks 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 ]id that week by one hen Heath et a], (8) reported that two mallard eggs from (he second reproductive season, when birds had bren on 25 ppm Aroctor 1254, had 56 ppm and 33 ppm PCBs, wet weight. Peskall (IS) reported that ring dove (5/repfopefio maria) eggs taken from birds given 10 ppm Aroctor 1254 in the diet averaged 4 81 108 ppm standard error The actual (mounts in mg of PCBs in ring do\c (IS), mallard (8), and pheasant eggs in the present study would be far below the 10 mg injected by McLaughlin et aj. (0) into chicken eggs. The 10 mg resulted in poor hetchability, edema, and beak deformation. Scott et ai. (9) found that PCBa deposited in thicken eggs were less than 0.5 ppm after 8 weeke with 05 and 1.0 ppm Arodor 1248 in the diet. They Tound levels of over 3 ppm after 8 weeka with 10 ppm in the diet and levels of about 6-7 ppm in eggs of hens on 20 ppm. Them value* ate much loser than those found in the present study from 3-4 weeks after administration of a single capsule of 50 mg Arodor 3254. Their results, showing a drastic reduction in hatdwbility associated with 7 ppm or lest residue in 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 bobwhitc and Japanese quad in the LCw; thus species differences may be important. Residue* 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 (PKalaerocarvx etiritus) and pelicans [PeUcanus trythrorhyncfios); cormorants vtre shown U> have an estimated 8 ppm in eggs and pelican* 0.6 ppm. Jensen et el. (20) reported 48 ppm in tlio egg of a heron (Arden ciuerea) collected in Sweden, and 8-21 ppm from 9 guillemot (Uria aalge) eggs from the Baltic Sea Risebrough ct at. (2l) reported that an egg of a peregrine falcon (Falta perepmw) contained 10 ppm; 8 black petrel (Loomelama melarna) eggs had an average of 1 ppm, 5 eggs of a bam 0"l (Tylo alba) had <1 ppm; and a golden eagle egg (Atfutla. cArysaelon) had <1 ppm Dustman et a! (22) reported median measurements of egg resi dues of bald eagles to be 1.65 ppm for Alaska and 97 for those from all other states They further reported median egg levels of 15 9 ppm for the osprey (Pandinn helvuUa) of Connecticut and 2 5 ppm for those in Maryland; 5-6 ppm in brown pelican (P. occtdtnialu) eggs from different sreas; and 5 ppm in eggs of royal terns (TAaforseu* maiimut). Prestt et al (16) found residues ranging from 0-80 ppm in 363 eggs from 28 species of both land and water birds collected in Britain. Moat of these bad less than 10 ppm, but those which had higher levels included a single egg of the great crested grebe (Paiieeps ersstatus), 40 ppm, one egg among 101 of the heron that had 80 ppm, while the arithmetic mean wsa 5 ppm, one egg of e moorhen (Gallinula chloroput) with 15 ppm, among 33 samples that averaged 2.4 ppm; and a single egg of a great skua (Sferocorari'ur tkua) that had 25 ppm. Particularly in view of findings that about 5 ppm was associated with needy complete negation of hetchability in the chicken (9), the above findings in some wild birds are alarming Apparently, though, es 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 Levels in Tissues We analysed tissue* from seven different groups of birds: (1) 16 birds given 210 mg PCBs dally that were designated for analysis upon death, (2) five additions! birds on 210 mg daily that died, (3) nine birds that were killed at intervals for matching with the 36 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- taming 50 and 100 mg PCBs, (6) a pooled sample of four birds dying on IN mg and 10 mg BCBs, and [7) a pooled sample of two control birds (3) Birds that dted from daily doses of 210 mg PCBs bad brain levels Dint mngcd from 320 to 770 ppin not weight nnd averaged 520 ppm*1.10 ppn\ standard deviation Lner residue levels were much moic vnnablc than biam levels They ranged from 390 to 9,300 ppm and averaged 2,300 *2,000 ppm wet weight Muscle residues were also relatively more variable than brain residues, as they ranged from 51 to 290 ppm, and averaged 140*53 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 370* 06 ppm; livers ranged from 1,000 to 5,000 ppm, and averaged 1,900*1,300 ppm; and muscle ranged from 58 to 110 ppm, and averaged 83*17 ppm Brain showed less variability than other tissues. Further, brain was the only tissue independent of other patameters (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 daya to death tended to negate its usefulness. Further, the interrelationships of liver parameter* with those uf muscle, even though it is not known how many of these -relationships were meaningful, tended to negate the uselutness of muscle as an indicator tissue. Brain levels of PCBs were generally higher in birds that died than m bird* that were sacrificed at the sans* time for matching of brain residue lev els, although there was some overlap of the two group*. 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 jnd lirain residue level, however, this was not significant (/'> 0 05), A pooled sample of four birds that died on treatment with 100 mg PCBs given each 3 5 days had 320 ppm in the I,run tissue, while a pooled sample of four s.irrifwr'l birds in tins gioup had 59 ppm A pooled satupli of three buds that died on treatment with 50 me PCBs given every 3 5 days lind 350 ppm in Hie bturn, while a pooled sample of (our saenfued from this group had 34 ppm Pooled bram Us-mei from four birds that died on daily doses of !0 nnd 20 mg had 300 and 3S0 ppm PCBs, respectively. no data were available for survivors of these groups Those latter data indicated that 300 ppm might be bettor than 403 ppm ss a separation point that would indicate death from PCBs and that, when smaller amounts of PCBs were rcccn ed by the bird* over a period of tune, the spread in brain levels between birds that died and sun n ed 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 brainrlner and bram muscle ratios However, liver: muscle ratios appeared to be smaller in birds dying on 210-mg doses, ff one establishes 19 aa 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 ratio.* However, it ia necessary to gather more dntn to determine if this ratio is useful in diagnosing the cause of death as FCB toxicosis. Vos and Koeman (13) found that, when 1-daiold cockerels were fed 400 ppm Phenochlor OP 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 11 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 130 to 2,900 ppm among 28 birds that died and from 210 to 340 ppm among four survivors Seven of Ihcir 23 birds that died had liver residues of less than 250 ppm, white four of 11 birds hod 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 An.xly red. overlap could not be evaluated Rehfrld (lit reported that 30-50 ppm Aroclor 1254 given to 1-day-old cockerels resulted in liver residues of 300-500 ppm. l'restt ct el. (16) reported a rtmge 98 Environmental Health Perspectives * -f - je- MONS 081402 of 3-034 ppm in liven of eurvivore and 70-897 ppm in liver* of BcDgoJcv; finches dying during treatment ith Aroclor 1234, *nd that liver level was correlated with the amount received (P<0 01). Presit et *1 (18) stated that tiro liver: brain residues should be compared and that brain ppni/livcr ppm X 100/1 was three times higher in Bengalese finches that died during their experi ment than in birds killed at the end. Data in the present study showed a complete overlap in brain ppm/liver ppm X 100/1 i birds that died during lest ing bad * range in ratio* of 5 4 to 1128, while matching bird* sacrificed had 10.0 to 37 0. Relatively little sampling of brain tissue for rCB residues ha* been done in wild birds Duet- man et al. (22) reported that a sick bald eagle had 230 ppm FCBs in its brain, and that PCBa may h*\ contributed to its death. Jensen et al, (20) reported that three white-tailed eagles (Haliaulut n/tncUfa) had a brain residue range of 29-70 ppm PCBa, averaging 47 ppm; muscle residues ranged from 150-240 ppm, averaging 190 ppm. Risebrougli et al. (21) found 0.04, 1.5, 21, and 34.9 ppm PCB in the brains of four peregrine falcons. Preslt t al. (10) reported liver residues ranging from 0 to around 600 ppm from a wide variety of British bird*; arithmetic average* ranged from 0 5 ppm in the burned (Btdeo buteo) to 98 ppm in flic heron The tissue level* reported above were below those at which mortality occurred in the present study, except that the muscle level for the white-tailed eagle w*a higher than thoee in this study associated with death In the pheasant from PCBa. However, there level* from wild birds do constitute substantia! percentages of the brain and liver levels in the present study where death occurred from PCB*. Histopathologic Effect* - A . Dahlgrcn et al. (3) found that PCBa decreased "rights of heart and spleen at all treatment level* U'<001). PCB treatment increased weights of kidney end liver in birds given 10- and 20-mg doses (PcO.Ol), but no effect was seen in the 't0-mg group. Starved bird* had smaller hearts (fJ<00$) and livers (P<0 01) then control*. Splenic atrophy, a* described by Flick et al. OO) end Vos mid Koemsn (13) wtu found in all pheasants given 20 mg daily and in the 10-mg birds except for one that survived 8 months and was sacrificed (3) Splenic atrophy was characterired by al most complete absence of lymphatic nodules and an increase in the relative abundance of red pulp: In one of the birds given 20 mg, loci of necrosis were found in lymphatic nodule* Prestt et al. (10), using the Bengalese finch, reported that kidneys were larger in birds that died from PCBa than in controls. Flick et al (10), using chickens, mentioned both enlarged adrenals and kidneys from PCB treatment. In our study, kidneys were larger in pheasants given 10 or 20 mg daily, but neither kidney nor adrenal enlargement was visually detected during ne cropsy McCune et al. (23), uaing Aroclor 1242 with chickens, mentioned both enlarged livers and kidneys in birds given PCBa Platooow and Funnell (14) and Behfeld (15), using 1-day-otd chicks, reported enlarged liver* with dietary intake of Aroclor 1254, Grant et *1. (24) reported enlarged liver* and decreased spleen se over a period of time in the rat. Flick et al. (10) and Vos and Koeman (13) reported amall apleena in their studies. Although hydropericardium eas found in varying degrees by many of the authors cited, it was found only rarely in the preaant study. Vos and Koeman (13) (bond that PbanopkeiP 0 and Clophen A00 caused much more five* necrosis and hydropericardium than Aroclor 1200; this was probacy due to contaminants (25). . PCB* In Combination with DisUria. . Mortality among pheasant*-of both sues 6-9 month*aid given dieklrin, PCB#;ora con&iustion of the tiro,' varied with the jevel at chtmical administered (Tah& TK Among the 22j*6easanLs on eaeh!evfc3 diadwith 4Hgddrinprcapsule, and 9 (had wTthf'mgifnUrin. The **nw( propor tion* held true lot PCBe, mice*died with 50 mg PCBa per capsule, amt 9 died with-HI 0% PCBs. When 50 mg PCB* arid 4 miwfiekfcin were administered tegether, total of 9 bird* died None of 11 coatto^^da died during diflt period of time. These data suggest that effecti'Cf PCBs and dieldrin together are additive, not synergistic Heath et al (8) found that the joint tovicity of Aroclor 1234 mid DDE given to Japanese quail was additive, and found no evidence of synergism in their joint effect. VU 1972 99 MGNS 081403 1 Tbl ?. occurring among 22 pheasants of A subtle effect on behavior ss indicated by both, Hit* th*t v-ett ^-4 month* of age when PCD* tn4 (lithlrin were ndniiniitifrd upirittlj1 n4 m combination* No mortality occurred among 11 studies on the visual cliff and of the ability of offspring to aioid hand capture These behavioral control birds. effects have been reported for birds with oegano- chlonne insecticides and could be deleterious to a Group treatment No of deaths in neck Total i 2 3 4 5 death* wild species, in that instinctive patterns necestsry for euiviva) are involved. Behavioral differences may be affected thiuugh PCB administration to SOmgFCBs l0 100 mg PCDa 03 4 mg Dwldnti 3 0 6 trf Pieldrw 3 0 GO rof PCBe and 4 oig Dwtirinn 1 3 0 12 4 0 4 2-9 00 0 0 12 3 5 20 4 0 the cock as well as through the hen, *a shown by 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 "Thu* two bird* wen near death at the and of the experiment observed effect of increased mortality in young during the first 8 weeks of life, and in a possible depression of weight at 0 weeks of age Only early Residues in 'WUd Bird* A pooled (*mple of six peasant livers taken from mid South Dakota pheasants had <0.1 ppm TCBa A pooled sample of three livers collected near Washington, Pennsylvania, had < 0.1 ppm PCBa, and a pooled sample of six livers collected southeast of Lancaster, Pennsylvania, had 2.0 ppm PCBs. A pooled sample of six livers collected east of Gary, Indiana, had 0 5 ppm PCBa, and a pooled sample of six livers taken from pheasants in Benton County, Indiana, had 1.5 ppm PCBs. These relatively low levels m liven of wild pheasants taken both from industrial areas and rural areas indicate these pheasants were probably exposed to small amounts of PCBs. Prestt et si (16) found that residue levels in birds in Britain were related to their food habits. They found the most PCBa in liver* of fresh-water fish-eating bird* (up to about 900 ppm); bird-feeding raptors had up to 70 ppm; birds which eat mammals had up to r>0 ppmjbirda with a mixed diet of mammal*, birds, and carrion had up to IS ppm; and those which eat insects had 0-1 ppm. survival of ehicks was affected, as no effect was noted in survival from 0 weeks of age through the fall. Sex ratio* of young pheasants surviving to the fall showed an expected 50:50 distribution, thus PCBs did not affect the survival of one sex more than another. Large doeea of PCBa, 210 mg daily, effected a loss of appetite, but leaser 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 PCBa did not lone as much weight as birdi which were not given PCBa but were starved Further, starred birds did not show the histopathologic effects observed in PCB-treated birds such as degeneration of liver cord cells and depletion of lymphatic nodules in tbe spleen PCBs were (1) rapidly and readily absorbed into the pheasant's body, (2) stored in the lipid fraction, and (3) excreted slowly in feces and eggs Excretion via the egg may be an important means of ridding the body of PCBa for the hen, but the PCBs in the egg yolk lipid may be dangerous for the offspring because of altered behavior and SunnuyariCOKtalNt lowered survival both of the embryo and hatched young. PCBa given to laying pheasant hens adversely affected egg production, hatchabllily, 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. Brain tissue may be a valuable indicator of PCB toxicosis; level* of 300-400 ppm or more were associated with death due to PCBa. Marked splenic atrophy was the moat conaiatent charocteriatic noted among several organ parameters checked in birds that died from PCBa Enlarged 100 Environmental Health Perspectives 1 kidneys and liver* were also useful character* in tempting to diagnose PCB toAicoeie. PCBs and dicldrin were not, when combined, rvnergirtic in their joint tOMcity to pheasant* Livers from wild pheasants collected in Penn* rvhania, Indiana, and South Dakota, did not exoeod 2 ppm PCB*, indicating relatively Jow)f\el contamination. REFERENCES I Jrnwfi, S 1900 Report of a new chemical hasaid New Scifnli*t 32. 012. 7 Dahlgrcn, R B , Greichus, Y. A, and Linder, R, L. 1072 Btnrago end excrttlon of polychlorinated bi> plienvl* in the pheasant J. Wildlife Mariag 33* 823 i Dalilgrvn, II B rt al. 1972 Residue levels end histo- pathology in phenainta given polychlorinated hi- l-lienyls J. Wildlife Minag 30 (In preaa) 4 TallarKO, R B. and Farrell. \V. M 1004. Studies of vwuil depth perception an effect of early experience on ducts on a visual cliff. J, Comp. Fbyrsiot. Psychol. 47 M. 4 Dalilgren, R. B. and Linder, R. L 1971 Effects of polvchlorinated biphenyl* on phnaeent reproduction, behavior, and aurvival J Wildlife Manag 34: 314. 6 McLaughlin, J. ct al. 2903 The Injection of chemicals into the yolk me of fertile eggs prior to incubation at a toueily teat. Toxieol Appl Pharmacol. 4: 700. 7 Dthlgren, R. B. and Ltadbr, R L 1970. Eggshell thick net* in phaeaanta given dieldrin J. Wildlife Manag 34:220. 5- Healli. R. C. et al. 1970 Effect* of polychlorinated biphenylt on bird*. Proceeding* XV International Ornithological Concrwa. The Hague--1970 9 Scott, M. et nl. 1971. Result* of experiment* an the effects of PCBs on laying hen perfommneo. Proceed ings 1971 Cornell Nutrition Conference for Fsad- Menufictann, Cornell Unlvereity, Ithaca, N Y. SO. Id Flick. D P, O'Dell, R. G. and Chad*, V. A. 1901? Studies of the chick admit dianaaa 2. Similarity of sjmptoms produced bf feeding ehlociulnd biphenyl Poult Sci. *4. 1480. -' " U Better. W L., Linder. R. L. rntf-Duhlgren. It 19G9 Dieldrin effect* in tern generation* of penned hen pheasants. J. Wildlife Manag. 38t4Mt- _ 12 Dahlgren, R B. Linder, R L and Ortmtn. K K. 1970 DieWrm effect* un aunrrptibilily of penned pheasants to hand capture J Wildlife Mini* 31 0*7 13 Vos. J G and Koeman, J H 1970 Comparative toxicologic study with polychlorinated hi phenyls ln chicken* with apccial reference to porphyria, edema formation, liver necronia, and tissue residue* Toxicol Appl Pharmacol 17 640 14 Platonow, N 6 and Funnell. H 8 1971 Anti andro genic-ilk* effect of polychlorinated biphenyls in cockerels Vet R*e 88 109. 14 Hehfeld, B M 1971. The effect of malslhion. palychlorinated biphenyls, and iron on growing chicks Dissertation Abstracts International 1971 31' 7397-B (Abate ), 16 JTeatt, I, Jeffries, D J. and Moore, N W 1970 Polychlorinated biphenyls m wild buds In Britain and their avian tmucity. Environ PoQut 1,3 17. Tucker. R. X, and Crabtree, D G 197a Handbook of Toxicity of Pesticides to Wildlife U S Department of Interior, Bureau of Sport Fisheries and Wildlife, Resource* Publication No. 84. 18. Fcakall, D B. 1971, Effect of polychlorinated bi phenyl* (PCBs) on the egg*belli of nng doves. Bull. Environ. Contains) Toxicol 8:100. 19. Andereon, D. et tl. 1909, Sign(Scant* of chlorinated hydrocarbon residues to breeding peliceo* and cor morants, Can. Field'Naturalist 83' 91. 2a Jensen, S. 1969 DDT and PCB ui marine animals from Swedish water*. Nature 3?i|9*7. 21. Riaebnwgh, H W. et *L IBM- Polychlorinated bjphanylo in She globaleeoayvUsn. Nature 220 1098. 22. Dustman, Kff. et if. 1071. Urn occurrence and ajg. niffeano* of psMHorinatad biphenylt in the environ ment. Trans, moth American Wildlife and Natural ftwounti Cm/< 36: U|* 22. MaCnne. E. U. Bang*, i. B. god O'Dell, B. L 1962. HydMporioardium and anritoa m stdeka fed a cbltindOnlbydrueatbaa. Poult. ML 41:29ft. 24. Wmt, ft!, HOmi W. X. J. and VSeneuva, D. C. * - 1S71. Metabolisct N pafothJoiinalad biphenyl (Aro- ckir 1204) mixture in tborai. fttlWylnn. Conlamin ^ijggi g: ipj, M?Vom*. O. at at Hf!i.1d*otifi<i*UM> sad lOKieologieal misluatioaonflflorihaled dlbansofuranmxi chlorinated naphthalene fee tww commercial polychlorinated bi' phenyls. Food Cconet. TenmoL-SiMft r J 4 1972 B!!1LIULI0| 101 MOWS 081405 Enrirmrimlal Iftotlh /Vipxduea Embryonic Mortality and Chromosomal Alterations Caused by Aroclor 1254 in Ring Doves by David B. Peakall, Ph.D., Jeffery L. Lincer, M.S. and Stephen E. Bloom, Ph.D.f The breeding success of Ring Doves (Slrtptopcha ruoria) fed Aroclor 1254 St 10 ppm was determined over two generations. Six pair* of birds were fed PCBs for three months, then allowed to incubate their clutch of two egg*. Eggs were then collected sgain for six months, at which time the birds were allowed to ineubete another clutch. Penult* are given in Table 1. The low-hatching niceess of the second generation was due to heavy embryonic mortality. The age of embryonic death varied considerably but was mainly in the range of 3-8 days. This finding is at variance with that of Scott et al. (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 week* at 20 ppm Aroclor 1248 In this case mortality occurred immediately before hatching. Eggshell thinning waa not observed In either the first or second generation (2, 3). This is in agreement with the findings of Dahlgren ted Linder (4) for the pheasant (P/ioetarttu ecUMieut) sod Heath et al. (5) for the Mallard (Ana* platyrhynzhot). Cytogenet ic studies were performed on 24 dove embryos at 3-0 day* of incubation- Six embryos "ere from dove pairs not fsd PCBa (control), 17 embryos were from PCB-fed (10 ppm in diet) pairs and one embryo was irradiated with 155 * Section of Ecology uid Systematica Cornell University t l>perlnient of Poultry Science, Cornell University, Itbscs, Kc* 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 metaphsse cells of allantoic sac and limb bud origin. Ah average of 365 metaphases were examined per embryo. In control and PCB-treated groups primarily chromatid, tome isochromatid and 1 rearrangement was observed. Mean aberration rates were as follow*: control -0 8% (range -- 0-2.0%); PCB-treated -1.8% (range -0-9 4%); positive control--180%. The chromosome re arrangement occurred in s 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 Table 1. Breeding aucoeea of Ring Doves fed 14 ppm PCBe ever two genetetions. Nad No of No, of No'of pen eta egga young laid hatched fledged Pre-axperiment lit RmcntaoB PCBi 2nd generation PCBa 2nd generstnn Control ft ft ft ft 34 32 33 . 34 24 34 20* 4 2** 34 22 33 ' One pur failed to produce any eggs during the period of the experiment. a* Fledging losses consisted of one young found oulaide the nett at one day of age and one deformed bird killed at the age of three eeks, April 1972 103 MGNS 08 Muscle Brain Lrcer lav Pre-alffsf <o 4) Port atreaa (n5) S J2 3 (4 1-9 9) 172 938 3 (120 0-227 0) 5 JSI e (4 8-8 0) 293 0*27 i (234 0-310 2) 13 3 12 0 '4 R --27 4) lnS257 'D!7 3-1SSS 0) 770 1 *31 1 0 (1SI 2 I0M *i Figures are means, standard deviation!, rod range No fat present result* are indicative of a passible clastogenic (chromosome breaking) action of PCBs in dove embryos. Further studies are warranted to define more precisely the conditions under which TCBs might be mutagenic, clastogenic and possibly teratogenic to avian and mammalian embryos. Residue analysis on eggs laid at various times from the atari of the experiment showed that the levels increased for three months when an approxi mately steady level of 50 ppm (dry weight) was reached (0). The second generation eggs had similar levels; thus increased embryonic mortality was not caused by increased levels of PCBs. The most striking finding of the seabird "wreck" in the Irish Sea in 1960 was the elevated concen tration of PCBs La the liver (7). In birds shot as controls the concentration was only 0 4 ppm (wet weight), whereas birds found dead had 40 ppm in the liver. In an attempt to evaluate these findings, we subjected to stress five first generation birds after they had been exposed to Arodor 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 strewed 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 group*. The organ levels of the first generation birds sacrificed at the end of the breeding experi ment cen be compared to organ levels after starva tion (Table 2). A massive increase of levels as a result of mobilisation of fat it dearly seen in all tissues studied. The fact that liver level* ranged from B37-16S8 ppm at the time of death strongly suggests that the 40 ppm recorded in the livers of seabirds in the Irish Sea was not responsible for the death of the seabirds. Increased occurrence of abnormal joung in two species of tern has been reported from 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 Ring Dot es during our experiments It is possible that the chromosomal aberrations noted Ln our study could be the cause of these abormalities. REFERENCES 1. genu. M. L et si 1971. Results of experiments on the effects of PCBs an laying hen performance. Inc 1971 Cornell Nutrition Conf, November SO 2. FeakalJ, D B 1971. Effect of pofj chlorinated biphenyls (FCBa) on Vht eggshells ct Ring Doves Du]] Environ Cotvtaaun. Toxicol 4:100 3. Peekaff, D B. Unpubllahed data. 4. Dahfgiea. R B and Linder, R 1, 1971 Effects of put}chlorinated btpbenyls on pheasant reproduction, behmor, aod nrmxtl. J Wildlife Msnag 34 314 4. Heath, R. <3. et el (In press) Effects of polj chlorinated biphenyls on birds, XV Cong of lnt Dmithol Den Haag September 1970. 0 Luicef. J L and Psakail. D. B. Unpublished data 7. Holdgate. M tV. (evl) 1970 The seabird tvrreti, of 1009 in the Irish Sea [Unpublished report mth supplements) Natural Environmental Research Council, Great Bntain. B. Have, H and Rbet)rough. R. IV 1972 Pollutant conttntrataona in abnormal joung terns, from Long Inland Sound ,4ulc 60: 19. 104 Environmental Bnltb Perspectives Eimrofurenfal HreitH Pmptaittt Toxicology of PCBs for Mammals and for Birds byJ.G. Vat* Introduction In the early days of its use, little work was done on the toxicology of the PCBs, and this was only in relation to the risks of occupational exposure As will be shown many more studies were made as soon as it appeared that the extremely stable PCB's became a threat to the environment and its wildlife, and accidents occurred of scute poisoning is man and animals These studies have been made with material with different contents of ehloriae, from different manufacture, and--as we can now say is retrospect--with different and unknown contents of toxie im purities. For this reason the toxicological infor mation of PCB's is difficult to summarise, but since the chanrV-r of some important impurities has recently bet u 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 were started because of the analogy between the effects of the PCBa (1-4) and some toxic effects associated with toxic factor* in crude chloropbenob and in "toxic fat". Effects of the latter are fiver damage (7), cbloracne (7,8) and edema formation (8). toxicity of three commercial PCB samples (contuning 60% chlorine on the average). Phenoclor DPO (sample I), Clophen A60 (sample ]I) and Arodor 1260 (sample HI). These three mixtures showed a marked resemblance in their gas chroma tograms and mass spectra (IQ). Is this compara tive feeding test in one-day-old chicks (11), it was found that 100% mortality, subcutaneous and abdominal edema, and centrolobular liver necrosis oocurcd in only two groups (fed the samples I and II). Hydropericardium (Fig. 1), a common effect of these two mixtures, wss only occa sionally seen in the chicks fed the sample 111. The ChemlexI, Toxfeologlwrl, ind Pathological Identification and Evaluation of Toxic Inipnrftlii b Technical PCB Preparations The first indication of the presence of toxic impurities was obtained in a comparison of the * Institute of Veterinary Pathology sad Institute at YeUnntry nurmscology and Toxicology, SUU Umvcnity of Utrecht, The Netherlands. April 1972 105 . *+ ^ iffx-rtfi+nvr^zcv} Table 1. Mortal! lr. and Pathologic Observation* of Chick* Fed <00 ppm PCf< for 00 Dy* pen sample Number of Number of birds wi(h tdtma *\ timber a( N birds wnh Hydra* Abdominal Subcutaneous Uver necToua pericardium I II HI Control 24 24 18 s 5 9 22 22 20 9 7 0 20 3 3 0 0 0 20 0 0 0 0 0 mortality in this group was only 15% (Table I). The excretion of coproporphyria 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 wae found in all three experimental groupe. In the subsequent study (12) by means of column and gas-chromatography the presence of relatively more polar compounds was demon strated in the 28% diethylether fraction of samples 1 and 11. In a chick embryo assay (Table 2), the difference ia toxicity between the three Btmples was confirmed; the high toxicity of the 25% diethylether fraction of sample 11 ia demon strated by the similarity between the mortality levels in the group injected with 3.5 sample 11/ egg and the group injected with the 25% diethylether fraction from 3.5 mg sample 11/egg. Mass epectrometnc analysis revealed that Tabic t Chick-Embryo Assay of Throe PCB aamplas and Iha SS% Dlcthylathvr Traction from Somplo 11 PCB sample Do** Number (rnc/mt) of egg* iroatad Percentage hatch of lerttlo eggs 1 II III Fraction fram sampls II Ethanol control Untreated control 35 3.9 3S 38 3.8 0.38 0.035 0 -- 15 20 15 15 15 15 15 20 20 0 5 80 0 7 92 too 94 90 identical chlorinated compounds were present in the 25% diethylether fraction from samples I and II but not in that from sample III They included compounds with mass number 304 ami 338. The proposed identity of these compounds, tetrachlorodibenrofuran and pcntnchlorodibensofuran, is indicated by the following chemi cal-analytical, pathological, and toxicological data. From exact mass meaauremen's it was found that the formulae of these peaks were CuH<OuCl, aod CnHiOuClt. The formulae of the fragment ions 241 and 275 were CoH^Oi and CuHi"CU. From this dats it can be concluded that the parent ion has a preferential fragmentation for the losa of a single C1CO unit. Mass spectra were compared with the spectrum of a chick edema factor 1,2,3,7,8,9-hexachlorodibenro-p-dto'cn The mass spectrum of this compound has a similar fragmentation pattern, i e the loss of two suc cessive mass units of 63 suggests a loss of two QCO units. With microeoulometric analysis a certain maximum level could be indicated Tins maximum level was found to be fivo ppm of the compound with mass number 338 in sample II and 20 ppm in sample 1. Polychlorinated dibemofurans are strong hepatotoxic and acnegenie compounds. Tri- and tetrachlorodibensofurans in a single oral dose of 0.5 -- 1.0 mg/Vg caused severe and often lethal liver necrosis in rabbits (7,13), and application to the ear resulted in ehloracne. Tetrachlorodibeiuodioxin was sbout 10 times more toxic (7). In jection of the 25% diethylether fraction obtained from 35 mg sample II into the aircell resutted in 100% mortality (Table 2). It can be calculated that the maximum dose/egg is 0 2 ag penischlorodibemo/urnn (taking fivo ppm as the mnu- 106 Environmental Health Perspectives mum value) This confirms the order of toxicity found by Higginbotham cl al. (B) in the case of toxicity of 2,4,5,2',A'l5'-hexachlorobiphcnyl From the increased fecal excretion of copropor- chloro-dibcneodioxios. Tetra and pcntachlori- phyrtn m both experimental groups (Table 3), it dibcnwfurans were considered responsible for the luglicr tonicity of samples I and 11. Confirmation is also obtained from the sub sequent comparative toxicity study in rabbits (14) Again samples I and I! were more toxic; the is very likety that PCBs ihemselvcs are responsible for the porphyrogenic action of crude prepara tions. From the presence of slight skin lesions induced by 2,4,5,2',4',5'-hexaehIorobiphenyl when compared with the Aroelor sample, it can liver and skin lesions were more set ere. Por be concluded that the major acncgenic action of phyria, especially of the liver, was present in all crude mixtures comes from a possible contamina three groupa (Pig. 2). A remarkable finding was tion with chlorinated dibentofurans. It can also the intense red fluorescence of small foci inside be concluded that PCBs themselves have a hepatic cclla. They probably represent nuclei. This slight acnegenic action Liver damage was es was confirmed in an additional cell culture experi sentially the same after treatment with both ment with the Aroelor sample and with 2,4,5,- 2,4,5,2',4',5'-hexachlorobiphenyl and the 2',4',5'-l)exadilorobiphenyl (This experiment waa Aroelor mixture. The conclusion that the liver carried out by my colleague Dr. J. G. Wit of the injury, caused by crude preparations, ia pre Biochemical Section). dominantly due to the contaminants, ia based Application of the 25% diethylether fraction on the differences in liver toxicity between the on the skin of rabbits resulted also in differences three PCB preparation* (11,14), . in (oxicity (Fig. 3). So the presence of the hepato- The probable contribution of polychlorinated toxic and acnegcnic polychlorinated dibentofurans dibensofuran (PCF) and pure polychlorinated as impurities in samples I and II waa found to biphenyl (PCBa) in the toxicity of crude prepara be established. tions is summarised in Table 4. A proper evalua In another experiment (15), the toxicity of the tion of toxicity data and reaidue data can be * Aroelor (60% Cl) sample waa compared with the hindered by the possibility that PCB samples may s*a-3$% "- iuk-.-z x- p^Yt ,,r I 1 Floras 3. Rwpmm of tin Inlida of tli* rabbit's etc sftar lopictl apptkstion of the 25% dwlhykwr fraction* from techmeal PCBY HaemoloaySn and min. X60 (a) Skin of control animal treated witli ethanol Note the hair follicle at I, aeabeceoua gland tissue at 2, and cartilage at 3 (b) Far akin of the animal treated with the friction from sample III. Some hyperplasia and hyperkeratosis of Ihe follicular epelhehom can he seen (c). Kir hm of the rabbit treated with the fraction from sample II Considerable hyperple.ua and hyperkeratosis of the Mhnitor epithelium (d) Her akin of the rabbit treated with the fraction tram sample I part of a aectnn that how* the moil severe lesion The gravity of the response wu in general the same as seen In (e). Kota the cystic dilated hair follicle vnlh prominent hyperplasia and hyperkeratosis of the follicular and epidermal epithelium (14). 108 Environmental Health Perspectives HONS 081411 differ in in important respect, the presence of toxic impurities. The possibility of distinguishing between the effects of PCBs and their unpurities enn be further improved by using pore isomers nilh known positions of the chlorine atoms. Mortality, Uvtr Effects, Edema Formation and Other Effects These data, M presented by different authors, arc summarised in Tables 5, 6, and 7, As can be seen in Table 5, the acute and subacute toxicity data of PCB'a are poor. The established value* are high. Semichronic oral toxicity studies are summarised in Table 6 Dermal and inhalation studies arc given in Tabic 7. Table t Coproporphyrin Contents (g/| Dr> Wright) of Fccea of Rabbi la Treated with PCB for 4 weeks, and of Control*.* 2.4 , 2', 4', 6'vHcxchlorobiphcn)I Aroclor Control <60% a> 45 O 63 20 6 39 6 Mean 37.9* 24.1 3.0 29.4 16 0 18.6* 48 37 36 3.3 38 * Figures era the contents of feces, collected from the cecum of the individual cnuntla. * Significantly different (ran controls, P 0.025. It is very probable that the results of these studies may have been influenced by the presence of poiyehlorodibcMofurans or other toxic im purities For example, in the study of Ihtbfeld et ai. (23) general edema was already found in chicks fed 30 ppm Aroclor 48% Cl, while Kohauawa and co-workers (22) noted edema formation at the 100 ppm level of another 48% chlorinated mixture. Mortality in the latter study was also tower (Table 6). Liver Effects The most important liver effects, summarised in Tables S, 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 Dtbrnaofuren (PCF) and Pure Polychlorinated Biphenyl (PCB) la the Tonicity of Crude PCB Mixtures. Dior* Edema Liver Hep*tie BCA* forma 4mih porphjm tion Polychlorinated Dibencofursa Polychlorinated Btphrnyi + + ++ ++ ++ ++ Table S. Acute end Subacute Orel Toxicity Studies ef PCB Preparation*. Preparilioo Animal Treatment Mortrlily Litct efccta RoTstoosm Unknown Aroclor 54% Cl Monas Rat Aroclor 43, 54, SO, and Mallard 63% a 43% a Hat 42% a Quinta pig 65% a Rat -smglo doss of IM0 approx. 2000 mg/kgt sroglo doco of 0% Incream of wsight and 500 mg/kg lipid; potentiation ef OCh toxicity ingle doso of 0% 2000 mg/kg 90 diSy doer* of 0% in 3 Hytli* bodiw n liver edit 136 mg month* 2 doecs of 69 mg 1 *ek apart 100% between Fatty nwUtnorpboeir, 11 and 29 central atrophy days 6 daily door* of 70% in 14 IaenuM of weight; call 300 mg dsys swelling hyabs granules (16) (17) (W> (19) (19) (20) Table 6. Bernlehronic Ora] Toticlty Alodial of PCB Preparation!. l'rfp.v rMtoo Animal Treatment Mortality Liver iffecu Other effects Refrr- 65% Cl Ant 48% Cl 48% Cl Cynomolfua monkey Squirrel mookey 48% Cl hlouao Arodor Chirkaa 42% Cl Arodor Chicken 42% Cl 48% Cl Chicken Arodor Chicken 48% Cl Doan of tO m| every aecond day From 641 mg id 40 dayi lo 348 mg in 238 day* From 320 tog in 46 dayi to 67 mg in 48 daya 00% in 0 ecki not given not given Daily doaw of 0X01 ml for 13 to 20 waeka 0% 100, 200. 400, and and 1000 ppm hi diet for 4 waaki O, 0, 50, BO, and 20% retpeetivdy 200 and 400 ppm in diet for 8 weak* Oind 12% napactnoiy 1, 5, Ilk 25, 50, 0% from 1 to 100 W0,300,800. ppm; 100% from 1201k 2400, ud the 100 ppm Icrel 4800 ppm a diet for 20 day* 10,20,30, 80,100, After 3 weeki: 0, Ik ltd ItOppm m 30,30 and 20%; diet lor 4.8-8 *ttb#ead0.fk week* 80, 00, and 80% napaethraly 33"To weight mcrea**t cel] welling; hyalin globules fnlerpmcnt; SER Mam cause of rie'ith proliferation pneumonia or diarrhea Enlargement; 8R Mam cause of death proliferation pneumonia or 9ER proliferation diarrhea, palpe bral edema in 1 animal Enlargement; BR Skirt low of hair, proliferation erosion ind RES reduction; utaerattun after myelin figure*; 3 month* increaee of auero- bodree, lyeoaome* and lipid Enlargement; Edam* formation damage at the from 200 ppm, at higher tevde high level* internal haemorrhage and tubular dilatation In kidneys Pronounced edema at iOOppn; an* Urged kidney*, melt spleen, defeathtnog and dermatitis Edema formation 100 ppm level Enlargement General edema and depreotion of the Meoodary texual characteristic* from the 30 ppm (2C) (31) (21) (21) W () (22) () Amtor Chicken %a And** % a finch Pbenoclor JipineM 00% Cl quad 280 tod ISO ppm is diet for 6 to 12 week* EaUmaladdoa* rata at 80 daya of 284 0|/k(/ day 2000 ppm in diet 280 ppm 100% be tween 3 and 10 week*; 300 ppm aom* mortality at lb* and to% 100% between 8 and 65 day* 800 ppm itcadcomb eights 20 fold end teste* atights 2-told lower than controls Hydropericardium to some birds (21) (2*> Hydropencardiutn (10) no Environmental Health Perspectives HQNS 081413 Table 7. Dtrntk] Tviitli/ inJ Inhalation StudiM of f*CD rreparationa. pttparel nn Animal Treatment Mortality Liver e/Teete bkm cffccla Refer ences IlCiCl Guinea PC %CI Rabbit Arodor Rabbit Aroftor Ret 05% a 11 doily akin ppticat ion* of 3* 5 coi 100% betn cm 11 and 31 day a Skin application *1 alternate dayi, total dot* from 94C to ISM mg IHiIy akin appli cation! of 0 3, 0 6, and 0 B g 100% betnaan 17 and 58 daya High dote died be fore liver neeroan developed Inhalation of 0 57 ug/cubie meter for IS hour* lor 37 to 134 daya 0% Fat, central atroph>, pcnnuelenr balophilie granu lation, focal necroaia In a faw ammala Fatty degeneration; central atrophy Moderate doaei mottled liver, lubecute j ellow atrophy, fatty degeneration, and marked necroria Pale and yellow, cell metling; hyalin degenera tion; potentuPoo of CCU nod C,H,OH tonicity thickfning of ihe p*derrai Thirtmnf of prickle teH Uyt tad tlncktmag o( outer comifird Jiyen Reddening, fonnnnon of imell pipub end blnlen; Anally desqutmeiicn of tvtarna) tpidarntai U)ert ire well explained by the proliferation of smooth surfaced membranes of tlie endoplasmic reticu lum (SER) as tu found by Ntehisumi (21] in mice and monkeys sod by Norback and Allen (27) In rats Tbc latter workers found a prolifera tion of the BEIl in rats fed PCS for 1 to 5 weeks. Concomitant with the structural changes, the activities of measured drug metabolising enxytnea (nitroreductase and aromatic hydroxylase) erne 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 yslm bodies described by Bonnet et aL (20) and Miller (12), could have an entymatie func tion similar to that associated with tbs SER (27). Sunilar formations, the so^alled myelin fig ures, were demonstrated In 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 dermal toxicity study (15) in rabbits with 2,4,3,2* ,4' ,5-hexachlorobiphenyl and Arcelor (60% Cl], the light microscopic findings included necrosis, hydropic degeneration (Figs. 4 and 5) aa well as a peripheral and perinudear shift of cell organelles (Fig. 5) and focal cyto plasmic hyiliniiation. In electron microscopy, the shift was found to be due to a proliferation of the SER resulting in a displacement of rough surfaced membranes (HER) and mitochondria The focal cytoplasmic hyalin degeneration, often seen in hydropie cells, was recognised aa tightly packed tubules of proliferated SER (Fig. 6). This very probably represents hypertrophic, hypoactive SER. _ SublethaJ effects caused by induction of hepatic enzyme* have been noted by several authors. Increased steroid metabolism in pigeon liver homogenates has been demonstrated by Rieebrough et el. (28). Lincer and Peakall (39) confirmed the effect of FCB 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 6.0 ppm. The higher Furaw 4. Liver damage In lulled rabbit treated with 130 me Arocior (#0% Cl), S timn per week, tor 28 day*. Not* the canWolobolar necroae (1) and the hydropic cells (3) at th* martin of the oecrotie and vital tissue. Haemotosylio and Mia. XI60. Fnttnis 8(a). Liver tells ol control rabbit. Clear area* may represent negative images of glycogen (arrow) Tohmhne blue. XD40 <b). Uvet tell* ol an Jtroetor treaiod hilled rabbit. Note the hydropic cells (1) and the pcrmuclm and penpherai displacement of cell organelles with sometime* hyalin loci (3) inside hepatic ceils. Tohiidioa blue XQ4Q 113 Environmental Health Peiepwllm HONS 081415 * Figcm ft Hydropic liver eellt from the nine inimtl u teen in Figure Sb (bowing ( Urge number of vacuole* (V) end prrivtcuolnr loc*lin.lton of raiVoebondni i*no> Sol* the strong p*cSiferVion of the 8ER, conneUng of tightly perked lubulet, Uranyl eeetele end lead ciU*U. X&SOO. do being roughly equivalent to 2 mg/kg PCBs (o each kestrel. A dose dependent in vitro break* down of estradiol to a more polar metabolite occurred in the liven from kestrels fed cither Arodor 1254 or Arodor 1252. No such conversion look place in the liven of the control birds. The increase in hepatic enzyme activity correlated milt an increase in cytoplasmic RNA, as wag measured cytopholomctrically. A ahortened sleep* mg time after treatment with hexobarbital, and enhanced in vitro rales of aniline hydroxylstion and p-nitroanisdtc demethyl&tiona were demon* si rated by Street and coworkers (30). These authors also found an increase of these effects ith increasing, chlorine content of the different l'CB preparations (Arodor 21 to G8% Cl). Using enyrrve induction u parameter, no effect levels of some PCB preparations were established in the rabbit, rat, and Japanese quod. Oral administration of Arodor 21% Cl and 54% Cl (1 0 and 10 mg/kg) for 28 days to pregnant rabbits resulted in liver enlargement and in creased activities of the drug metabolising en zyme* aniline hydroxylase and aminopyrine n-demethylaae at the 10 mg/kg level of the 54% chlorinated Arodor. The no-effeot level for eniyme induction in the pregnant rabbit appeared to lie between t.O and 10 mg/kg in the case of Arodor 54% Cl, and higher than 10 mg/kg for Arodor 21% Cl (31). Another parameter for enzyme induction was used by Komatsu and Tanaka (32). They found that the hexobarbital induced sleeping times in rats were reduced by pretreatment with PCBs. April 1972 as The minimal effective dose was 5 mg/ks for 3 days with Kanechlor 400 (48% Cl) and 2 mg/kg for 3 days with the higher chlorinated Kancchlor 5(X) The porphyrogome action of PCBs was further evaluated in a study with Japanese quail (33) The results (Table 8) indicate that the hepatic poiphyna is closely associated with an increase of imtochondnol ALA synthase activity A significantly increased activity of this cnsyme was already noted after administration of daily doses of 1 mg/kg Aroclor 60% Cl for 1 week. Mean PC1J content of the liver at that dose was 1.41 ppm. A less sensitive parameter Is tissue fluorescence due to excess quantities of porphyrias. Liver fluorescence was only seen at the 100 mg/kg level It develops, probably, only in animals showing clinical symptoms, such as loss of weight. A similar finding was done in the prior experi ment with chickens (11). Edema Formation The most striking finding in birds is the ac cumulation of fluid The pathogenesis of the edema formation Is discussed by Flick and co workers (0). The primary site of the edema caus ing factor could be the heart by increasing the permeability of the vascular bed, leading to cardiac congestion. Pulmonary edema could be the result of the cardiac congestion. The pul monary edema might be followed by a flow of fluid into abdominal and subcutaneous air sacs Decreased scrum proton values (34) could also contribute to the edema formation Laver damage can be responsible for reduced serum albumin levels. As mentioned in Table 4, the edema formation is probably due to the presence of polychlor-odi beiuofurans In our study the edema formation by the 60% chlorinated Aroclor sample was minimal at the 400 ppm level As can be seen m Table 0, chick edema-like lesions were noted at low feeding levels and were caused by lower chlorinated Aroclors. Therefore the presence of toxic impurities in these Aroclor samples hav to be considered Other Effect* An interaction of PCBs with duck hepatitis virus was found by Friend and Trainer (35) Ten-day-old ducklings were fed a 54% chlorinated Aroclor mixture at levels of 25, 50 and 100 ppm The birds suffered no apparent cluneal intoxica tions. Five days later they were challenged u rth duck hepatitis virus, and they suffered signifi cantly higher mortality than birds which were not exposed to PCBs. Effects of PCBs on the lymphoid system were noted in some studies. Feeding of PCBs to chick ens resulted in small spleens (6,11). Lymphopenia, atrophy of the cortex of the thymus, and a re duction in the number of germinal centers in spleen and lymph nodes was found in rabbits (14). Therefore, an immunosuppressive action could be present. In an experiment with guinea pigs, this was established (36). Feeding of 10 ppm Aroclor 60% Cl, for 8 weeks resulted in a Table 8. Formation of l-Aminocvulinlc Acid by Liver Mitochondria, Liver Residues, and Tissue Fluorescence In'Fcmala Japanese Quail Orally Dosed with PCB far Sevan Days. Aroclor (0% a) ALA formed (m* rooks Cmg/Vg body weight) ALA/g liver/hr) PCB coolant Ihw CPP) Tissue fluorescence incidence Macroscopic Microscopic (livtr) 0 4.1 i 10 100 4.44*1.64 g 76*4.00 10 40*1.21* 17 4*6 4 114 9* Mean value**SD, 5 bud* per group. I Sifniflcantly different from control*, P 0 01. * Pooled simples 0 If 0 45*0 37 1 41*0 47 27.0*4.4 474*244 0/4 0/5 0/5 0/5 3/4 0/5 0/5 0/6 0/5 2/5 114 Environmental Health Perspectives ,r. . . - ,. HONS 081417 Fici at 7 Hrpresent mvivc areas of tstanus toroid stimulated popliteal lymph nodes of guinea pigs () Large number of enlilmdy forming cells in a control animal (b). Reduced number of antibody forming cells in an animal fed 10 ppm Aroclor (00% Cl) for 8 wka Dircct fluureseent antibody technique Cryostat lections X 375 (30). April 1972 115 XONS 081418 ------------ ,-^a, - =* c-acSKgg^^ deereared number of antibody-forming cells in difficult to interpret many of the toxicity stucii? the popliteal lymph node, alter stimulation of Pure samples are required for comparable u the humoral lymphoid system m tth tetanus toxoid vestigatione Also the fate of the toxic impuntii (Fig. 7) This suppression may explain the in the environment has to be determined A higher sensitivity of PCB-fed ducklings (or duck presented here, PCBs have several sublet hi, hepatitis virus (35) In a comparative toxicity effects, such is microsomal enzyme induction study in guinea pigs, an indication for an effect porphyrogeme action, estrogenic activity, am of I'CB (Clophen and Aroclor 60% Cl) on the immunosuppression Since porphj n* seems !> cell-mediated immunity was obtained. Feeding be an effeet of PCBs themselves and not fro.i of these mixtures at 50 ppm levels for 6 weeks PCF, the induction of ALA synthase could bi resulted in a decreased number of circulating used as criterion in the approximation of s no lymphocytes (unpublished data). effect level (at least for the 60% chlorine type An estrogenic activity of PCBs (Aroclor 21-48% of PCBs). The no-effect level could be about 0 1 Cl) was demonstrated by Bitmen and Cecil (37), mg/kg (mean PCB content of the liver in Japa The estrogenic activity was evaluated using the nese quail about 0 2 ppm). This is in the same 18-hr glycogen response of the immature rat order of magnitude as found in the other studies uterus after a tingle subcutaneous injection. The Additional research is needed to determine fully rafoiraum effective dose was 8 mg. The higher the significance of these eublethal effects More chlorinated PCB mixtures were inactive at the over, chronic and reproduction studies are neces 8 mg level. In the above mentioned subacute sary. The present results also make deer that feeding study of 60% chlorinated mixtures in manufacture of commercial PCB mixtures that guinea pigs, we found significantly increased are free from impurities is urgently requested. uterus weights in the PCB treated animals. Both increased steroid metabolism, at mentioned by Acknowledgment Rehfetd and eoworkert (23), and the estrogenic The author gratefully acknowledges the helpful activity could be reaponaible for the depression suggestions and critical reading of Prof. H van of secondary sexual characteristics (decreased Genderen, Head of the Institute of Veterinary development of comb and wattles) noted in Pharmacology and Toxicology. Many thanks cockerels (24), are also due to colleagues of the working party Administration of Aroclor (54% Cl) at levels of the Institute of Veterinary Pathology and the of 12.5,25, and 50 mg/kg body freight during the Institute of Veterinary Pharmacology and Toxi first 28 days of gestation had embryotoxic effects cology-, Dr. J. H. Koemsn, Mr. H. L. van der in the rabbit (31). Edema and beak deformities Mass, and Dr. J. Q. Wit. The author also thanks in chicken embryos have been described after the students who studied for their degree in yolk-aae injection of 10 and 25 mg 42% chlori biological toxicology and Mr. M. C. ten Noever nated Aroclor, resulting in respectively 05 and de Brmuw, Mr. R. H. de Vos, and Dr. Hl.C 100% embryonic mortality (38). An effect of PCB on the nervous system was KIripool of the Central Institute for Food end Nutrition Research, T. N, O., Zeiat. noted by Ogawa (30). Oral adminiatration of PCB (O.3-0.5 ml/kg/day) to rat* for 14 or 21 hefexences days, rerulted in marked or moderately impaired motor function, decreased motor conduction velocity end lore of large nerve fibres. He eon- 1, Awv J- W and Aldan, B, K. IUt An u!e' dsnaalargosis Arch, Derm. Byphil 33 1013. 2. Schwarts. L. 103d. Dermatitis tram synthetic retnu sad wans. Arocr. J, Public Health 24. MS rinded that PCB cauesd neuropathy in rata. >. Meigs, ]. X, Alboa, J. J. sad Ktrtia, B L IMI Cbloreew from aa unusual exposure to Aroclor J ! CawlsslM Amat. Med. Aaane. 154.1417. 4, Puecioetli, V, 1054. Dell'ieno denes Mad Lavoio Because of the possible presence of poly chlorinated dibemofurass (PCF) or other toxic impurities in crude PCB preparations, it is 45:1*1. 6 McCone, L , Savage. J E. tod OTMl, B L l2 Hydropericardiuras and utiln ui chicks ted a chlori nated hydrocarbon. Poultry 8eieoee 41 205. 116 Environmental Health Perspective* MOWS 081419 t fink, D F, O'Dell. R G ud Child*, V A 19M. Siudw* ol tha chick edema dinette 3 Similarity of ivraplosm produced by feeding chlorinated biphenyl PouJLSci.U 1M0 7 Bluer, H, Sehulx, K H. ind Spiegelberg, t 1061. Benifticbe Vergiftungen bei der Heratellung von Chlorphar.ol-Verbindungea Arch Generbepathol Gewttbehyg IS 533 c Behrbchm, P 1956. Vber Ccfehrcn betm Urngang nut Chloftrf.cn Plienolen Dtach. Geeundheitsn 14 914 Q Higginbotham, G R cl el 1069 Chemical and toxico logical evaluations of twilled and tynlhetic chlorodeni ative* of dibeno-p-dioxin Nature 320' 101 10 Kocmin, J H, Tea Noeier da Breuw, M. 0 tod 1 o4, R. H. de 1149 Chlorinated biphenyls in hah, annuli, eod birdi from the River Rhine and the .VctberlafideeoeeUl ire*. Nature 321 1124. ]l \t*t, J G tod Kooman, J. H 1970 Comparative totieologic itudy with pohchlonaeted biphenvl in chicken* <ritk epeeutl reference to porphyria, edema formation; liver necrotic, and tiasue reiiduei Toxicol tppl Pharmacol 11 4S6. 1 os. J. G et al 1910 Identification and toxicological eitluation of chlorinated dihenaofurin end cblonnated naphthalene to ten commercial polychlorinated biphcny la Food Coamet. Toticol B 425 ,3 Hofmann, H. T 1947. Neuere Erfahrungen mil hoch- tntiaclwn Chlorkohlenwaieerstoffen Neunvn-cbraiedeberge Arch. Exp. Pathol Pharmakoi 132. 72S. 14 to>. J G. and Beeme, R. 8 1971. Dermal tonicity nudwa of techmeat polychlorinated biphenyls and fraetioo* thereof m rabbit*. Toxicol. Appi Pharmacol, in 617 13 \ei. J G, and Notenboom-Ram, E, Coraparativa toxicity atudiee of 2,4,3,2*, 4', d'.htxwhlorobipbtoyl and a polychlorinated biphenyl mature in rabbit*. (Subimiwd for publication]. Tinaka, X. at al. 1969. Experimental eubacute poiaonixg by chloeobipbeoyla, particularly the mSueaics oo the Mnta lipida in rata, FTikuoka-Igaku-ZasahL 60: M4 I* Grant, D. L, Phillips, W. E. J, tod Villeneuvt, D. C 1911. Metabolism of a polychlorinated biphenyl (Aroclor 1244) mixture U1 the rat. Bull. Environ. Coaumm Toxicol. 5: KB. .v Tucker, R, K, aod Crabtree, D G. 1910. Handbook of Toxicity of Poebeidee to Wildlife, U 8. Dept of fn* tenor. Bureau of Sport Plaberfee and Wiidlifo Rooureaa Pub, No. St. p. 19. l* Miller, J, TV 1944. Pathologic changa* in aniaala ea. paced to roismareutf ehlorfanted diphaoyt Pubhe Health Rpta. 49: 1064. Bennett, 0. A., Drink*. C. K. and Varna, M. P. 1934. Moephoiofieai abaages in the bvan of into re writing frmm oapeoure to certain ehtnnnated hydrw_ earboaa. J. Indust. Hyg, Toneol. 20- 97. : \iskieumf, M. 1910. Ugkt end electron ratomeeope tudy f eklorobiphenyl poisoning. In mouse end Arch Environ. Health 21 920. * Kohanawa, M et al 1969 Poisoning din to an oily b> product cf nce-oran sumilxr 'o chick edema disease I! Tetrichlorodiphenyl aa toxic 'u.atance Nat Inal Alum Health Quart P 220 23 RchfcW, B M . Bradley, R L and Sunde M L 1911 Tovcity itudiae on pclyrhlorinated biphenyla m the chick I Toxicity and a: mptom* Poul Sci 50 1090 21 Platonov, N S and Funnel! H S Hit Anti-androgenie like effect of polychlorinated biphenyls in cock erel* Vet Ree SS 109 25, Preeat f , Jefferiei, D 1 end Moore, .V tV 1070 Polychlorinated biphenyls in nild birds in Britain and their avian toxicity Environ Pollnt l 3 26 IVedel, H von. Holla, W A and Denton, J 1943 Observations on lha toxic effects resulting from expoeure lo chlorinated naphthalene mdrhlnnnetid phenyls with euggettiona lor prevention Rubber Age 53 419 27 Norback, D H and Mien, J El 1970 Eniyihetie and morpholigic slteraltont of hepatic endoplasmic reticulum induced by a chlorinated aromatic hydro, carbon Fed Pros 29 SIS, 28, Rircbrough, R W et el 19G8 Polvehlormeted biphemla in the global eeoty stem Nature 220 1098 29 Linear, J L and Peakall D ft 1970 Metelralic effects of polychlorinated biphenyls in American kestrel Nature 228 7S3. 30. Street. J C et al 1969 Comparative effects of poly, ehloiinited biphenyl* end organocMonne pesticides in induction of hepatic raicrooomal eniyme. Presented at 4CS meet ing September 8-12 New York31. Yilleneuve, D C et ai 1971 Effects of PCS adminis tration on rmcroeomsl tnayme activity in pregnant rabbits Bull. Environ. Corns min Toxicol 6 120 32 Komatsu. F and Tanaka, K 1971 Shortening of hexobarbitel sleeping time and change of serum tn(lyccnda levs! in chlotnbiphenyla-inloxicated rU Fukuoka-lgaku-ZaMhi, 62 34. 33 Voa, J. G. at al. 197) Polychlorinated biphenyls as inducers of hspiim porphyria in Japanaeo quail, with special reference to l-emmolevuhmc acid synthetase ertivity, Suoreecene*. and reetduea in tha hvar Toaieol App) Pharmacol. 20:232. 34. Flick, D. F. and O'GeU. ft. G 1968 Studies of tha chick edema disease 6. Preventive treatment with oral diuretics. Poult. Be. 47' 821 33. Friend. M,, and Trainer, O. O 1970. Polychlorinated biphenyl: mUnation wiOl duck hepatitis virus Bo* cnee 1T0: (314. 36. Voa, }. G and Rail. Th. da Jmmunoaiippn--ve aetinty of a polychkinMiad biphenyl preparation on the humoral iMraitn reaposse in guinea piga Toxicol Appl. Pbamacol. fin preae). 37. Bitman, J end Cadi. H C 1970 Eatiuganie activity of DDT analoge and polychlorinated biphenyls J. \gnc Food Cham. IS: 1106. 38. McLaughlin, J. at al, 1963 Tha infection of chomieala into tha yolk sac of fartila eggs prior to incubation aa a toxicity teat, Toxicol. Appl Pharmacol 5 760 39. Ogaera, M. 1971 Eleetrophyiiologicel and hiatoiogieal studies of experimental chlerobiphonyl poisoning Fukuokg.lgiku-Zaaahi 62: 74 *pm i97j U7 HONS 081420 Envirenmttual Htahh Ptnptetim 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, and Atsuko YamaguchJ Id October, 1968, an epidemic of a peculiar rkw disease similar to chloracnc was reported in Fukuoka-Ken (Fukuoka prefecture), Japan. The epidemic was later proved to have spread not only over Fukuoka-JCen but also over 20 other prefectures in the western part of Japan (Fig. 1). It produced 1,067 patients according to the latest tabulation (August, 1071) by the Ministry of Welfare. Soon after the epidemic was announced, a study group waa organised by the staff of the faculties of medicine, pharmaceutical sciences, agriculture, and engineering of the Kyushu University and by the ataff of the local health departments, to clarify the cause of the epidemic tad to effectuate it# control (1). We participated u the study group and conducted, as members of U epidemiologic study subgroup, an extensive epidemiologic investigation following the baric 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 ) usbo", namely oil disease. Although our observations end experiences are foufiued to tlie Yusho patients seen in Fukuokahen, we believe that reporting of Stem will help many people in the world who are deeply con- cerncd about the chronic deleterious effects cf PCBs. The 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 expert- l'n.`1>rni by Drpurlmenl of Public Health, Faculty of t-Jreuw hjuahu Vnivimty, Fukuoka, Japan Fiavaa l Number of paticnti arth Yuho by prefecture 'l*rii 1972 119 jiiMvTPiUJWti-lh.. HONS 081*41 ITfctde 1. Inkilil }mplomi Goto el el. Initial i>rri|a(onn Patienti No Swelling of upper eyelids, increased eye discharge Acne-tare tfupturn, fotlieuUr ncftnluelion Edematous tfveUinc of Umb* Lnpgiiwhmenl Disturbances in digestive canal Numlniwa sod other nouroiogrcil tgoa PiffraeMation of Lm Total 52 38 3 45 33 I 0 ac 4 20 4 20 0 86 13 0 6 too too O etioed by 338 patients *Uh Yusho were mereued 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 169 patients diagnosed up to October 31, 1963, are summarised in Table 3. Dark brown pigmentation of nails and skin, follicular accentuation, acne-form eruption, in creased eye discharge. Increased sweating st palms, and feeling of weakness were the moat notable symptom* (2). Descriptive Epidemiologic Studies First, 325 patients seen in Fukuoka-Ken from October, 1068, to January, J969, were analysed in order to know their distribution characteristics. On* of the moat important characteristics readily noted was a distinct familiar aggregation. The 325 patients belonged to 112 families. As shown in Fig 2 and Table 3,99 percent of these patients were affected during 1908, while the remaining stated that they became iti In December, 1967. Fifty-five percent of the patients were concen trated In the 3 months from June to August, and no 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 the 22 local health departments of Fukuaka-Ken. Excepting 10 health departments where no cases of Yusho were reported, the rates varied considerably from 1.0 to 58 9 pet 100,000 Kxaniination of such geographical distribution failed to indicate any common socioeconomic or environmental factor which might be associated with tire disease The 325 patients consisted of 158 malts and 1G7 females, indicating that both sexes were equally affected More than 90 percent of them were younger than 50 years (Table 4) Age- and sexspecific incidence rates again indicated no sig nificant sex difference but lower risks for both males and females in the age group over 60 years (Table 5) Analytical Epidemiologic Studies When our atudy started, a commercial brand of rice oil produced by K company (abbreviated as K rice oil) in Kitnkyvshu-Shi (Kitakyushu city), Fukuoka-Ken, had vaguely been suspected as a possible cause of the disease, because most patients with Yusho seemed to hsve used it Tabta !. Pinwl dUlribucton of ijmplomi of Yuihw reported by llffslttoli esnmined before October 31, IMS. Bydiploma Males Females (N-S9) (N-10Q) Dark brown pigroentatnn of naila Distinctive hair foflrclaa laereaaad sweating at palms Acnelika stem eruption! Red plaques on limb* Itching Pigmentation of slue Swelling at limbs Stiffened aolea in foot and palms of handa Fignwotsd mucoua mambrana Increased aya discharge Hyperemia of conjunctiva Traotuftt visual diaturbniiea Jtuitae* Swelling of upper eyelids Feeling of neatness Xumbnaa* to limbs Fever Hairing difficulties Spasm of limba Htadacba Vmartin* Dunha* 83 l 84 0 50 6 67 8 20 I 42 7 75 3 20 2 24 7 M2 8S 8 70 6 56 2 It 2 71 9 58 4 32 8 I# 18 0 79 JO 3 23 6 10 1 76 0 56 a SS 0 82 0 16 0 52 0 72 0 41.0 29 0 47 0 S3 0 71 0 55 0 1! 0 74 0 52 0 30 0 19 Q 10 0 80 30 0 28 0 17 0 120 Environmental Health Perspective* HONS Q814di ficrur z. Distribution oI petienta by w rod month .hen symptom* appeared Therefore, thorough mvettigation woo undcrtiWeti to detcna'm# whether the patients had actually taken uch special breed of oil before being affected. This wee achieved by examining lot number* appearing on the remaining con tainer* of the oil Deed by *ome of the patients, dupping record* of K company, and purchase and tale record* at wholesale oil detiers' office* and (t retail stores. It was soon disclosed that all the patients had used If nee oil, other canned (15 5 kg) or bottled (1 85 kg) Furthermore, an astonishing fact became evident No matter where they lived, 166 of 170 patients who uaed only the canned If rice oil had used a very specific oil. produced or shipped by the company on February 5 and 6, 1988 (Table 6) For those who used bottled If rice 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, rate, and purchase at K company, wholesale dealer* and retiul aborts, however, clearly indicated a pos sibility that 143 of 155 patients who used bottled K rice oil only bad used a very specific rice oil produced or shipped from February 5 to 15, 1968. This was because such 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 bad bad a very peculiar common experience of use or possible use 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 survey, we examined whether those who regularly used X rice oil, but did not use the specific K rice oil produced or shipped Table }. Distribution of pal lent* with yuaho diagnosed by January *0, I960, by *ex sad month when symptom* appeared. Month Male* Number Percent Female* Number Percent Total Number Parana Before IMS IMS January February Uuil April Miy JuAt July Auto* September October November December Tot it 2 1* 0 06 4 2t i 31 15 e.s IS 11.4 23 M a 27 17.1 34 21. 17 to a a 9.1 i o.e i o.e 155 100.0 2 1.7 0 00 a 3 12 7 1 to #0 18 9 0 33 t 3 12 10 2 10 11.0 12 7 l 14 8.4 t 0 0 00 ier too 0 4 1-2 0 00 to 3 I 20 5 2 25 7.7 13 10 3 M 17.2 30 IS 1 94 10.7 29 5 9 22 9.5 2 oe I 0$ 323 100 0 April 1972 121 e XONS 081*423 Table Dlilrlliullon of 323 piiianta with yuaho diagnosed by January 20, I960, by ee> and age group Age group (years) 0-9 10-10 20-29 30-39 40-49 80-59 00-49 70-79 Total Mule* Number Percent 37 23 4 38 24 1 28 17 T 30 IB 0 11 7 0 9 57 4 1s 1 03 158 100 0 Females Number Percent 27 1ft 2 28 1 3 3ft 21 8 39 23 i 23 13 8 11 ft A 3 18 0 00 187 100 0 Total Number Percent 64 19 7 66 2U 3 . 64 19 7 89 21 1 34 10 5 20 6 2 7 22 1 03 328 100 0 in the period in question, were free of the disease or not. A group of 113 persons of 29 households living m hi apartment house had purchased canned K rice oil as a unit from one dealer and divided it among themselves fairly regularly from December, 1067, to September, 1968, except for the period from January to April, 1068, 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 n-as produced or shipped from K company on February 5 and 6, 10C8, 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- sons) backgrounds of the patients and matthe controls were compared. In the other, the use < oil and fats in the households of the patients an the controls were compared. 121 patients and their 121 heslthy control: (53 males and 68 females) matched by age, at' and place of residence to each of the patient: were selected and asked 60 questions conremin their occupations, medical background, genera health status, habits, customs, diet, pets, tm other characteristics of their lives. As shown it Table 7, only one of the 60 personal factor examined, namely habit of "eating fried food or tempura nearly every day", was significant)more commonly seen among the patients thai among the control*. In the latter case-control study, 69 household with Yusho patients were matched by place o residence with 207 control households withou Table S- Afa-tpaci&e Incldorco rata* far Yuoho. by a of patient*. Incident* rate* per 100,000 Age group (years) ------------------------------------------ Male* Female* O-B 10-19 20-29 30-39 40-49 80-39 60-09 70 and over Tola) 11 4 8.9 9.1 o.e 84 i4 3.5 1.7 83 8,6 66 10 3 11 9 93 72 2.4 00 81 Table 6. gke oil used by Yusho patient*. No, patient* Specifications of oil used Type Dale of production or fthipmtn 166 (51 1) Conned Febnitiry & *nd ft. 4 (1 2) Canned UoLnown 143 (44 0) Bottled 13 (3 7) Bottled Unknown, but us* of oil shipped from February 5 to 15 lOoS is very probable. Unknown 32o (100 0) 122 Environmental Health Perapecii'f* HONS 081A2A T*l>ie 7. Rfinlta of cisc-control 1 tern* mvr-vlrj-tted C*ea COAtroU 0 AHcrgic-tn flub Allergic vo npmn Allergic (o other drugs l-irmng bath fnpihtiwv At liuitio Tnking linth everyday H'fvjnf |>Mi at bom# I.iv>ng m hou amnllcr Ib.m 00 m* door ipncQ so 00 73 7 73 0 IB 3* GO 9 75 42 60 83 6 70 36 V 60 1 1imid1 mil agricultural chemicals Taking cod liver od Taking vjMmin jiHIh 'Inking oiIict restorative drugs Water tuppty available at home 25 10 23 2 91 81 1 66 83 18 3 75 74 7 Dining out occasionally Liming the same meals w ith families bat green vegetables daily Drink mill* nearly everyday Tike butter nearly everyday bat eggs nearly everyday Eat fned foods or tempura nesrly everyday Ul foods prepared with oil nearly everyday Cat rail nearly everyday Tk# mayonnane nearly everyday Lat instant "rallmea" or duiieso noodle nearly everyday 2$ 1 88 8 63 1 49 0 22 4 M7 22 4* 21 6 21 6 10 8 10 8 30 8 89 6 38 9 39 0 24 9 59 8 u 29.1 29 1 10 8 10 0 p<0 0$ melt patients, A distinct difference tree noted l<ctneen lira two groups only for regular use of nre-bran oil, namely 98 percent of the patients' households affirmed it while only 31 percent of lie control households did (Table 8), Thus, tits mw-control studies clearly indicated that none of >l>c factors tested except use of K rice oil could recount for the disease. l>o<e-Respona Relationship To prove a causal relationship, a definite doserelationship is needed A rough estimate 1 ` 'Ik quantity of the specific K rice oil consumed - cscli patient and his family members ass made ''garding Iheirage, sex, amount of food intake, 'I possible loss of oil during and after cooking ' Liplity of the U6 users of the specific canned I( rice oil in question "ere believed to have con sumed, individually, less than 7JU ml For these RO light users, the attack rate of Yuslio wss R8 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 dui differ considerably according to age, as shown in Tabic 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 standardised for age using the age composition of the a hole 14R users as standard The figures, however, hardly changed from those shown m Table 9. Thus, a dear doseresponse relationship could be demonstrated, even though the estimates of the dose were inac curate (4) Toxic Agent In view of all these epidemiologic observations, we concluded Lhat 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.1 Prof. H. Tsukamoto, Faculty of Pharmaceutical Sciences, Kyushu University) demonstrated that the canned K rice od produced or shipped on February 5, 1988, and used by gome of the patients contained about 2,000 to Table S. Results of caae-control study on oila usod. ral or ou No. of % household* No of % households Butter Margarine Sename oil Rjipe-eeed oil Rice-bran oil Lard Other oils 33 30 7 44 63 8 21 30 5 10 14 5 08 OS 7 12 17 4 13 to a 103 SO 7 127 61 4 83 41 i 77 57 1 64 30 9 78 18 4 117 50 3 Caws 69 patient households Coni roll 207 non-patient households M'ril 1972 123 M0N5 Table *> Kelntlon bei*ren th amount of the K rice mi nsr<i by patient) nnd their rlmirai Amount of ol S'nii iffpeted No % Lifihtrisp* No % Soscrc t. No % l<Mu Nj % litas limn 720 ml 720-M10 tnl More them 1,110 ml 10 0 0 (13 0) (1) 0) ',0 0) 39 (40 0) 14 MI 0) J (11 0) .11 (JO 0) Jl i'G'J 0) id (SO 01 so f\ i} (l<\(} flj 21 ri 3,000 ppm of kanrehlor 400, (Kuncgafuchi Chenucnl Industrial Co Ltd ) a brand of poly chlorinated biphenyls (chlorine content. 4S%] (5) In this discovery, Prof K Inagami of the De partment of Food Technology, Faculty of Aguculturc, Kyushu University, and his associates made most admirable contributions os members of the chemical study subgroup The study subgroup also demonstrated that the nil was not contaminated 'nth toxic agents such as Cu, N'i, Zn, Co, As, Ilg and Penlachlorophenol Further more, it found that most of the components of Kanechlor 400, particularly those corresponding to the peaks of higher retention times in the gaa chromatograms, were retained in the sebum, ubcuUaeou* fat, mesentcrium, mesenteriolum, exteaperitooeal adipose tissue, appendix ver miformis, heart, sternal marrow, small intestine, trachea, and other organs of patients, and can be transported into the fetus through the pla centa (5,0,7). To examine whether only the K ricn oil produced or shipped in the period in question was contaminated with Kanechlor, the chemical study subgroup analyzed 100 rumpleof the bottled rice oil which had been shipped between October, 1067, and October, 196? Gns chromatographic analysis revealed that a significant contamination of (he hot tied oil wulimited only to those produced or shipped between February 7 to 10, 1968 (5) No an.aly sis could bmade of the bottled K rice oil produced or shipped on February 5 and 6 because of llic lack of sampleSimilarly, 479 random samples of bottled K no oil were analysed for chlorine content by the X-ray fluorescence method with a count meter Again, only the samples of lobrunry 7 lo in contained n large amount of chlorine (maximum 462 ppm). None of the oils shipped in other months were contaminated with more than a trace amount of chlorine Thus, the results of chemical studies completely coincided with tho-e achieved by epidemiologic approaches 13y tho 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 mutters of the rice oil (Fig 4) It )-> Table 10. Clinical verify by age. Amount of oil used (ml) Non-affected Number of patients by climes! grade tight* Severe* <790 720-1440 0-12 13-20 30Total 0-12 13-29 30- lolftl 3 (16 7) 3 (8 3) S (13 1) 10 (12 5) 0 (0) 0 (0) 0 (0) 0 (0) 13 (72 2) 7 (20.2) 10 (30 0) 30 (48 8) 5 (30 0) 1 (6 7) 8 (40 0) 14 (31 2) 2 (ll 1) 13 (02 3) 14 (30 0) 31 (38 7) 5 (50 0) 14 (93 3) 12 (SO 0) 31 (08 S) "Light'' rurre-pomls U* grade* I and II u tide "wvere'* to grades Hi and TV used l.y Goto el oi Total 18 (100 0) 24 (100 0) 38 (100 0) SO (100 0) 10 (100 01 13 (ICO 0) 20 (100 0) 43 (100 n) 124 Environmental Health Perspectives HONS 081426 Fiaf 3 PimoU** of *vtre s of Y usbo by **. believed that it roust have leaked from the heating pipe and contaminated the oil, because small opening! were discovered in the old pipe. Amount of Kaneehlor 400 Ingeoted by Pal tecta One hundred forty-six patients who were proied to have used the contaminated canned X rice oil of February 5 and S by our investiga tion were used for the estimation of the amount of Ksnechlor 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 Ksnechlor 400 io the oil was 3,000 to 3,000 ppm, iht avenge amount of Kaneehlor 400 ingested hv a patient was estimated to be about 3 g (4). Similarly, the minimum dose of Kanscblor 400 consumed by a patient was estimated to be about 05g babies Bom to Patients sod Non-alTeeled ^ fees of Patients ' Thirteen women consisting of 11 with Yusho and 2 unaffected wire* of patients were shown to hiNe delivered 10 live born and two stillborn habies from February 16 to December 31, 1968 (S, 10,11) As shown in Table 11, nine of them bad unusually grayish, dark-brown ataiasd skin and feftnlar pigmentation of tbe gingiva and nails as noted in five of them. Increased eye discharge "a* also riotsbio in most of them. Histological r\amiiiation of a stillborn fetus allowed a marked hyperkeratosis and atrophy of epidermis and cystic dilatation oi 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 st birth or stillbirth baa been experienced in Japan during tbe period in which their incidence among the patients was extremely high, and also since none of the mothers bad had any unusual experiences, for instance, In 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 such phenomena could not be shown because of the limited number of cases. The amount of the contaminated oil consumed during pregnancy ranged from 0 3 to Xicr saw EximcTCD Mim rr+ewe* Defatted swa After new*, or solvent. TREATED H|TK XXIUT r*TeWSFHMt. MD CSdOIFUGED MB LATER fcunuuBB w *t>nl 1973 Table lli Bible* bam la pktianu and unaffected wivea of patient* ami their uae of K rice oil. Amount Of Oil Pregnancy Grade of Mother Age used period clinical Delivery No during alien oil aeverdy pregnancy uaed of mother <L> Sea Smallof for- 6t*ined Stained Stained baby date lkm gingiva nail Increased eye Neonatal discharge jaundice 28 0 3 3rd 2 Normal M No 7 trimester + 3 24 T 7 3 Normal 7 No + _e + + 3 22 1 4 Later half Normal Force? M No T _-- + ++ of 2nd truaeater 4 27 0 7 2nd 1 Normal M Yet + + - tilmeattr + 7 t 2ft 0 7 lit 3 Normal F No + + + trimeatar ft 20 1.4 Later half ft Normal M No + -- + of 2nd + + + + tnmaatar 7 30 1 1 Whole s 33 ft 33 7 Whola 77 l Normal M Yea + -- ft Normal M 30 + s~ -- 7 Cieaaresn M Yea + + 4* eeetioa 10 3 0.3 tally hall Normal Normal T No 7 --- + + + -- 7 + 7 + of 2nd trimeatar ii 2ft 7 7 2 Normal M No + + + 12 33 2 ft KaHybetf 4 Stillbirth 7 7 + r r of 2nd + + trimeatar 13 25 7 7 3 Stillbirth F Yea + T T Table 12. ProgBoaU of potlente with Ytatbn.* Grad* of clinictl Improved Clinical condition* BUlieuI Womened 0 1 2 * 4 Total It (ftft 7) 30 (31.7) 23 (52 3) 12 (53.3) 0 (0) 1 (30 ft) ft (25 0) 1ft (27.8) It (3ft 4) 10 (43 3) 10 (100 0) M (3A ft) 2 (8.3) 12 (20.7) S <H 4) 1 (4.3) 0 (0) 20 (12.ft) * Calculated from the figurea reported by Toahitam and Kitamura. **0. Physical complaint* aritboul altio-leeiona. 1 Pigmentation of the aliia mod the mueoua membrane 2' Curoedo formation. 3; Ac inform eruption*. A Ealanaive distribution of acncfonn eruption*. Total 24 (100 0) 63 (100 0) 44 (lOO 0) 23 (100 0) 10 (100 0) 150 (100 0) 126 Envirotunertta] Health Perapcctlveo HOM5 0814*8 ,r, liters (Table 11) (8) Twelve of n fetuses 'fft smaller than the national M.imlnrtii, and 1 them were smnll-for-dalcs liable'. (8,10) \i ipv grew older, tlie vt,-im m their k\ gradnallv ,.-lrd (II) No evident"* lias so far been obtained regard to any physical oml mcuLHl retard,i(0n of the babies, but we, suggest that a pur'iilarK careful and prolonged follow-up observnl(1l, should be maintained for their future (ft) ('.rout h of A (Tec tod Children To examine whether Yusho disturbs duldrciTs c-owtli, tlie affected school children, 23 boys and ;d prls, were compared in 1967, 1968, and 1909 uiili tlicir 719 healthy classmates matched by u-\ The gains of the affected boys in both height > ni weight decreased significantly after the oifoiung, while the affected girls showed no ii'iiute change m this respect (12) < urren t State of Patients More than 3 years havo passed sinto the rinlemic was reported It is a heart-breaking fact ;hat many patients with Yusho are still tortured iv tlie sickness bccnusc no curative treatments hue been discovered yet. In summer of 1970, & mass clinical examination or the YuBho patients um earned out in Fukuoka-Ken, and the clinical -late of the patients examined were compared uith their previous findings observed in summer , 1969. As shown in Table 12, about a half of the 1 >9 patients, for whom the comparison was feasible, were shown to have clinically improved igns, while the remaining half showed no such mvorabks signs, and more than 10 percent of the patients were even worsening (13). It should be noted, furthermore, that even many of those who spiwsred to be clinically improving had several wnous complaints, such as persistent headache, pvueral fatigue and feeling of weakness, numbness ui limbs, weight loss, and others (14). All these mas clearly indicate that recovery from Yusho is rvtftmely difficult. To cure them as quickly as lossible and to prevent another epidemic of )vistio, ail possible world-wide cooperative efforts aw highly desired. 1 rom the middle of February to the end of ''larch, 1968, an epizootic of a strange disease iluselv resembling chick edema discsto occurred involving more than two million thickens in the western pint of Japan (15,18) More ill,in 400,000 chickens nric icportcd to have riicrl 'i he liwasc vva- charjticnci-tl by winW (linrinl signs ,iv labored bic,lilting, droopmc", rutiled feathers, high uioit.ility and detrensed egg production fl'i,IS) Mitopsv icvcalci! :i marked Mibrutancous edema, bydropoucnrdmni, asides, ami pulmonary edema, ninstular crihvmoMS in the thorax or inside of the thigh, and yellowish mottled spiiearar.ee of the iivcr ()>,17,1S) An epizooliological investigation ilrmonstrntcd 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 lice oil used by Yusho patients had been contaminated with Kanechkir 400, the toxic feeds were analyzed for chlorinated hydrocarbons and proved to contain Kancchlor 400 (16,19,20) The experimental reproduction of the disease by administering this chemical mixture was successful (16,20,21), The reason tlie chicken feeds were contaminated with Kancchlor 400 could be reasonably understood by the following facts-, both of the chicken feed manufacturers had been usiug "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 0 to 27, 1903 The remaining sample of "Dark oil," used for production of the toxic formula feeds, was also shown to contain about 1,300 ppm of Kaoechlor 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 Kmechlor 400, It is very unfortunate, however, that tlie epidemic ol Yusho could not have been pre vented even though a large scale epizootic of such an unusual chicken disease had preceded tlie incidence of Yusho by more than 6 months REFERENCES 1 KaUmki, S 1909 Forescrd, Reports of the Study Group for "Yusho" (Chlorubiplienyle Poisoning), Kukuolu Vet* Med 60: 403. 2 Kuralsune. M et J 1909 An epidemiologic study on "Yusho" or ehlorobiphcnvls poisoning Ibid 00 513 3 Goto. 11 end Higuchi, K IW Tlie symptomatology April 1972 127 SI MONS 081429 rrtffi -i*f.v* &* ^.v--*, 1 Kr*^ vaMwws--iyxtyv ,.'.^iM*hltbMB.lilris..sy.t.,*..,.------ --i|ll, ^ v ^ of Yuaho (chlorobiphen)'ls poisoning) in dermatology. Fukuoka Act* Med CO 4QQ 4 Yoihimuni, T 1071 Epidemiological analysis of "Yuiho'1 patient* with epecial reference to aex, age, clinical grades and oil consumption Ibid 62 104 t Tiukamelo, H et *1, 1069 The chemical studies on detection of toxic com pounds in the nee bran oil* used by the patients of Yueho, Ibid 60" 406 6, Ko|im, T |07l Chlorobiphanyla id the apula and tissue* Ibid 62. 26 7. Kijuchl, M. et el 1971. Two autopsy UM of chronic chlorobiphenylapoiaoiung lb*d #2 80 $ Yeraaguchi, A, Yoahimura, T. and Kuratsuna, M 1971. A survey on prepant women having coneumed rice oil contarainatad with chlorotnpbenyl* and their bebiee. Ibid62' 117. 9 Kikueh i, M. et al. 1969 An aulopty c*M of stillbom of chlorobiphenyl* poisoning Ibid 60 489. 10 Taki, I., Himnagai S. and AmigaM, Y. 1969 Report on Yueho (chlorobiphenyl# poisoning) pregnant women and their feline*. Ibid 60` 471 11. Kunatau, I. at aL 1971. A chlorobiphenyl* induced fetopathy. Ibid 62.139 12. Yoahimura, T. 1971. A cae* control etudy on growth of ichool children with Yuaho. ibid 62.109. 18. Toahitani, 6. and Kitamura, K 1971. Cllnicnl obaareotioa of Yuaho (chlorobiphanyla pouooing), especially further etudy of it* dermatological findings Ibid 0'1 132. 14 Okumura, M, Hirayimvi, C and I'saun. M 1971 Study of Yusho (clilorotuphenjls poisoning) m clinical examination Hud G2 123 15 tCohanawa, M at *1 1069 Puisomng due to an oilv by-product ol rlre brun similar to chicIs edema disease I Occurrence and toxicity test Mail Inst Amm Health Quart 9 213 16 Kohanawa, M. et *1 1909 Poisoning due to an oil by-product of rieo-bran similar to chick edema durase II Tatrachlorobiphenyl aa toxic auhatance Ibul 9 2'.>0 17 Shoya, S etal 1969 Pathological changes of poisoning in chickens due to dark-oil, an oily by-product of fire bran. Ibid 9- 229 18. Koga, K et at 1970. Studies on tha tone principle in a certain by-product of rice oil rendering I Tone.lien on the chick Japan. 1 Zootech meal Set 41 336 19. Koga. K alal 1970 Studie* on th* toaic principle in a certain by-product of rice oil rendering II A survey of * toaso substance Ihid 41 439 20. Koga, K. at al. 1971. Studies on the toxie principle in a certain by-product of rice oil rendering HI Toaicuy of chlorinated biphenyl on chick* and summarised results of I--III. Ibid 42 16 21. Goto, K., Bakaguchi, K. and Ogewa, K 1069 Hydro, paneanttum assay of nai ml which caused Yusho and of KaoedUor 400 in chickens. Ibid 60. 533 i 128 Environmental Health Perapcciiree HONS 081430 <rAj-' rtaiiriM faynuMhif rNtiihSMadfcihriMfltomai Environmaniei Htalih PtnptHim 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 animal experiments were earned out by the Yusho study group in Kyushu University in order to * Department of Public Health, Faculty of Medicine. Kyirebu University, Fukuoka, Japan. clarify the mechanism of toxic actions of Kanediior, 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. Tab!* 1. Laboratory finding* ot pattenti with Vubo. Item W(/) 1. Red blood cells 2. Leueocytm 3 Blood platelets 4 Coagulation tiano S, Prothrombin tuna . Blood sadunentaliou rate 7. Hemoglobin Blood (//) (tram lipids 1. Total lipid 2. Tnglyaeridts Laboratory Bodinga Decreased la moat lever* eases. Increased in most sever* ciaea. Normal Manual Normal Normal except for a fair eaaea ImtwmI 725.0*109.3 (mg %) 9 ease* of gnda IV* Incmasad 180.0*88 3 (mg %)9 eaaea of gnda IV* SIM!17 (mg %) 24 eaaea More than NO (sag %) 9 out of 34 eases 19*04 (mg %) 33 adult patients Fifteen percent of Um palitnti ahovred unusually high levels (more than 300 mg %) of thglyouidea * No. TO? 800 % tun No. case 0-9 10-19 30-29 30-39 40H9 30-39 00- S3 89 U 03 38 28 10 IS 34 t 17 19.1 5 7.4 10 11 S 0 10 3 3 73 3 12.3 Total 390 60 13.1 Referenda M 30 April 1972 129 HONS 0814*31 r TaUa 1---Conlioued. Item Laboratory findings Refrrsn 3 Cholesterol Norms] 161 43 (mg %) 27 edult pstisoU 4 Phospholipid# Normal 8 1 1 B (mf %) 27 adult patient# 6 Serum lipoprotein 0/*->lipr9prot*fl ratio increased. TVomsn wifh Yutbo, jfed 24. 241 TVoraan with Yuiho* sfed 80* 4 4 Min with Yuiho, tied 31 3-1 0 Fatty acid csmpooi- Anelylu of eholeeteroi eetere, triflycendee, free fatty ectdi, end photyho- ton of serum lipids Uptda showed no abnormal eompooooto, except that ineraaaa of oltic ae*d and dacraaaa of palnutie acid waraaaan in aatumdTee fatty acids. 7. Effect of (lutono Lei el* of (lueoee end free fatty aeide in blood were determined 30, 60, 60, ndmioalrotion on 120, 160, end ISO mmutae liter on) admbuetrntioa of 40 f (lueoee. wrius free titty iCidj Two typee of venation of levela vara notad. Type K: Blood (lueoee incralaad and liter deoreeled. Fatty end* hardly changed Type B: Blood (lueoee mcreaaed ind did not return to consul even after ISO minutaa after (lueoee admiautiatioB. Fatty icide decreneed markedly after admmutmtioa, Filnutie acid deereeled by (lueoee ednaintetretioa more markedly in patience then la normal peeeooai 3, Po#i<kc|irii) plfttcot PKLA value* (FFA Eq/mia/mi) 10 ns'nutv* after heparin injection m 10 lipofiroletn Up*M normal women aged 18 to 30 in years war* 0201 0.068 four of 5 octmty (PHLA) femil* patient* ebowed FHLA 10 miaut* viluee lower then 0 143, namely the mean--18.D. value for teuUel, while 2 of 8 male patient! had einular loer value!. B. Lecithin-eholeeUrol Five patient! with Yuiho were examined. Mean value waa 417% for icyHraiufenae (LCA) patwnta which ru ligniftcafltly lower than control fp-O.OO) Bleed (///) protein end efkm 1. ToUl oenun protein Normal in moat caaee 6 870,73 f! %) caeea ef (rede IV* 7.30.6 (l %) IS adult petnata 7.30.6 (| %) 2 Yuaho tbildtea 2. Serum tlbnata Normal 66 <5.47 (,,) > cnee of (nule IV* 66.4 4J (%> 33 adult patient! - 68.l6.8(%)8 Ymho ehildres 2. Serum (lobulin Incneie in evtiobulin in trvtr* eerne. No eigeifieaat thaete IB other fraction!. erfhhuKn 12.7 3.74 (%) > eaaee of (rede IV* 10.32 1 (%) 33 adult patient* 13 1 3.0 (%) S Yuihn children laereeea In i-flot>oliJi 6l.i (%) S3 adult palmate 6.010 (%) S Yuaho children 4. Non-protein nitrogen Normal OeeBpovuuU BM (IV) dettnlfm end nuMir 1 Na, K, C* Normal Ne and Ca ware m aormal rente but K M ehghtly deemaed. 4 0*0.4 {mPm/ll 53 adult patient* 1,8 8 8 4 8 20 20 3 8 8 3 S 8 3 3 B 8 & i & ISO Environmental Health Perspective* HOMS 08143^ Table 1--Continued. Item Laboratory findings Reference 2 Ci I Ft 4 Cu t. Zn 4 2*05(iuEq/1) 17 Yuaho children Normal Tended to decreese W 0 30 9 (,1 %) case* of grade IV* Decreeeed #0 34 (i* %) SO adult patients 63*20 (sg %) 17 Yuaho children Increased in severe rases I S3 9*61 0 (ng %) 9 cases of frade IV* Normal Stood ( V) enrymea 1 Alkaline phosphatase, scrum 2 Lactate deliydrogonote (LDH), aerum 3. Trencsinmasas GOT OPT Increased slightly in aevera eaaee. 10 3*3.5 (King-Armstrong units) 65 adult patients 17.t4.9 (King-Annetrong units) 11 Yuaho children 14 9*9.28 (King-Armstrong units) 9 eaaee of grade IV' Normal in moat cases. 278*05 (Karmen unite) 24 adult patients 284 *24 (Karmen units) 5 Yuaho children Normal 24*19 (Karmen units) 70 adult patients 38*23 (Kerman unite) 12 Yuehn children 230*7.7 9 cam of grade IV* 28.0*10.2 eaaee of grade IV* 24.7*8.8 eaaaa of grade III 200 *7J eaaaa of grade 11 Normal in moat eases 22.6*11,9 7 eaaaa ef grade IV* 23* 16 (Kerman units) 48 adult patients 21*11 (Kerman units) 10 Yuaho children Mean 30.1 eaaaa of grade IV* Mean 24.1 eaaea of grade 111 Mean 16.0 eaaaa of grade II Bleed (VI) otArre 1. Bromeutfskcia (BSP) teat 2 Icterus index 2 I"' resin sponge uptake (Trioaorh teat) Delayed in several adult eaaea. 3 9*5.19 (%) 8 eaaea of grade IV* Normal 3.3*0.88 eaaaa of grade IV* Normal Unni 1 Protein 2. Clucoeo 3 Urobilinogen 4 Urinarj neutral 17-keloalcroida Nagsthru Negative Negative in moat cases Elevation of ratio of etioehelanolona/aodrmterona was noted, indicating adrenocortical hyperfunction but urinary excretion of total 17-ketoateroidi u in normal rings. Etiochol./androst. 2 Normal somen aged 28- 0 6, 10 2 Normal men aged 20 and 28 0 4, 0 8 2 Yuaho women aged 35` 1 7, 2 0 2 Yuaho men aged 37 and 39 0 9, 2 6 3.8 3 8 3 3 3 1. 3,8 8 8 3 3,8 8 8 3, 8 8 8 3 1 3,8 3 3 8 1 1 1 3 I 3 3 8 3 8 1 V April 1972 131 HONS 001433 Table l~Continued. I*m Laboratory findings Hcfc.rer.ee Told 17-kctosteroid* 23 adult Yusho oomen 3 7* 1 S (mg/diy) 13 adult Yuaho men 7 3 2 8 (mg/day) Urinary total 17-hetosteroids (17-KS) and 17-hydroxycoriicosteraid (17-OHCS) nere determined in 30 male and 45 female patients Positive correlation was found between 17-KS and 17-OHCS 429c of patients exceeded normal range in both steroids in mo lea and tamales Analysia of major eomponanta of urinary 17-XS was mad* m 0 male (18 to 57 >sirs in age) and 7 female (16 to $1 yean in age) patients For eni'-nsterone, etmeholanolone, end dehydroepiaadroeterone, female pananta seemed to be lower than control, while main patient# oeetned to be higher Sex 17-Ka Normal (mg/day) Patients (mg/day) g iq M Pregnanedwl Androsterona Etioeholanolons behydroepundioatemne 0 70*0 17 2 95*1 02 1 83 0 55 1 55*0 75 0 84*0 46 3 38*1 18 2 64*0 54 1 86*1.18 Total DtUrmmMl by ZimfntrmtQn retcinn F Pregnanediot Androsterona Ftiocholanolone Dehydraepiasdmatarooa 0 41 8 10 6*2 3 0 830 55 2 02 0 97 1 35*0 48 1.01*0 40 7.9*3 2 II 6*3 2 1 34*0 91 1 32*0 60 1.03 *0 40 0 83 * 0 48 Total 4 4*1.7 3 8*1 0 DeUrcninad by Zimmcrrnum rMclion 8.1*9 4 7.3*1 9 S. Amino aeidi Two petlnnta irere examined. A remarkable inertaae us excretion not ooted for taurine (5.M1; 8,103 pM/day), g-aaunonobutyric acid (247; 1,042 M/day), glycine (1.803; 2,907 M/day), and histidine (743, 1,170 eM/day) tncxeaaa was also noted lor threonine, aertne, and Tyrosine, 8 FqUy octf axafyau tf OCXS ond ikinfti Cbeeaa-lika matonal collected from tea* eruptions and fat a the ewoat of patienta _________________________________ _____________________________________________ cheese-like material % - Fat in sweat Face % Body % 1 mynatic palaitM pataitolsie flt**n olew Imoltio linoltaia 2.7 21 5 10.4 10 3 30 4 18 3 04 11 7 41 2 23 5 11.8 11 8 91 41.0 22 7 81 18 1 132 Environmental Health Perspectives Tabl* 1--Continued. Item Lahorsloiy findings Reference 2 Yusho acne end on-Y lithe sens Yueho ecne coni allied more ilunc end olrie ecide then did non-Yusho acne. Lmoieic acid VII prevent only m the farmer The former conuined JO timer more cholesterol then the letter. Cheese like mslernli from ordinary acnea of many persons % from senes of meml-iera of e Yusho ftroily I2 %% myriatie paUmlie pnlnutofoc iteanc okie linoleie 11 fl 41 2 17 0 13 7 te s nona 30 23 1 10 7 i e 31 S 14 1 83 33 4 61 31 2 23 S 7J fliojilf I Liver 2. 6k la Kcunloeual finding* Otorhmote rynpaiogioal finding* Ocular ngnt Reduction Of rough endoplasmic reticulum and hypertrophy of smooth endopleemie reticulum. Mitochondria showed morphological haterogeneity. Many filamentous inclusions nere present in the matrix Giant mitochondria were frequently encountered. TWentyfour bsopay apecimena from IS patient! nere exunified Marked hyperktreloeie and eyttie dilation of hair follicles; marked increase of melasiine in basal celti of epidennia. BTowmf of sensory nerve conduction velocitiea of radial and aural nerves was noted in V of 23 patients examined. Motor nerve conduction velocities of ulnar and tibial nerves were abown to be unaffected except for 2 patients in whom they decreased. No audiometric abnormality and no disturbed vestibular function. Hypersecretion of meibomian gland; abnormal pigmentation of conjunctiva; no particular lesion in intraocular tissues. Increase of melanino granules waa noted eicctrononcroceopieaUy in cytoplasm of epithelial cells, especially In basal ceSt of conjunctiva. Innumerable electron dense particles, 300 400 A m dismeter, wan in the cytoplasm of basal telle. * Grade IV meant clinically most revert ce 5. 22 Experimental Takli 1, Pledle|i uf uloul experiments Results Reference 2 ml of olive oil centsininf 1 % Kanechlor 400 orally given to rabbits daily fee 10 days Oral administration of Kanechlor 400 to female mice, eyncmolgua monkeys, and Increase of serum tnglycendes and alight mcreaaa 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 In (Usawa et al.) 10 (Nuhitumiet al) April 1972 133 M0NS 081.435 Experimental Table 2--Conllnued Results Referent* equirref-monkeye for a month or longer 3 Or*) administration of 0 1 f of Kinnchlor per Kg per day to rets for 4 week! 4 Oral administration of the nee oil used by a Yuebo patient to hairless Dice. 6. Distribution and elimination of component* el Kanechlor 400 orally administered in a ringla dote of 2 0 mg/body to female mice. 6. Distribution and emotion of *H-Ksuechtor orally adminic lered m a single dote of 300 pc ot 25 ang/body to Witter Kmc mala rata * debut about ISO g. 7. Effect of oral administration or Kanechlor 400, SOD, COO and their components, 0.2 ml of a solution of 0.1 mg/03 sil/100 c of body weight, daily for 3 daya, on liver nucroeomee of male rata. bver. Loss of body weight, hepatomegaly, and marked increaae in aernm lipid eeeipontntc aara anted Serum wigl>rvmJei reached a level aa high is 10 times the control level Serum cholmesterese activity wee not effected Hyperkeratosis, cyitic dilatation of hair follicle and sweatgland, marked hepatomegaly, fatty degeneration of liver U (Tanaka et al ) 12 (tnagami el al ) Each component of Ktnechlor 400 was equally abaorbad from 13 gaatro-uileatinal tract. Concentration of Kanechlor 400 in O oshitnura eta] ) akm one day after adminiatratioa *aa twice that id livar and kidngy Kanechlor 400 wae retained in skin more than in othti tissues Telrwchlorobiphenyle ware ilmoat completely eliminated from tissues u three to four aeeke, hut paste- end hexa-chterohipbeoyla were retimed 9 to 10 weeks after administration. Redtoaetivity in ikin, adlpoae titans, liver, adrenal (land and 14 gaitromtaatioal tract waa higher (ban In pfaama 3 daye after (Yoshimurs et tl.) adisinietrction Radioactivity iraa noted in aomt tissues after g eeelce Urinary aacration of Kanechlor wea limited, while 70% of the doaa given waa m faces. A amall portion of radio- etivity escretad during the Cnt day waa in the phanolle fraction. More phenolic mctabolilec and leaa unchanged com ponents acre excreted in the fecce on the second and third day. Kanechlor 400, 300,000: Hepatomegaly Miereeomal protein 13 par gram of livar Increased in proportion to eMoriaa content (Fujila et al) of Kanechlor. Amount of cytochrome P-450 and pacific ac* tmtrea of O-dealkyiaM (p-aitraphenetel), N-demethytue (p-ebloro-N-methylandiiie}, and aniline hydro*} leee, in creased. partieuinrly with Kaeachlor 300. 4,4'-diehlorobiphteyl. Slight incraaeaa eaen 3,4,2',4'-ttruchlorobipbeii}i\: Increaaca noted, but leaa than 3,4,3'.4'-tefraehlorobiphanyl/ with Kanechlor 400. 3,4,3',4'-tetraehlorobiplienyl wee a more potent inducer than the otbett 2,4,6,5',4'-pent*efa)crobiphsny); Mast marked increaaca were noted in cyt. P450 and epaeftr aetivrtiae of 3 eniyme*. 3.4,3.3',4',3'4ie*achlotobipbyl: Hi* increase * as larger than that Induced with the tells ehlorobiphanyls but lace thin that with the pentachlorohiphonyl. Phenobarbetal: Reeembliog Kantehlor 400 in eniyme induction capability. The abova micmaoaaal enayme activities and the tmcroaomul eomponanta etarted to increaaa 12 hours after admuuatntion of 3,4,3',4'-tetraehlobipbenyf, teaching the maximum after about 1 weak. TherMfter, activity levels tended to decrease but did not return to normal even after 0 weeks. Microtomes, particularly Uieir membranes, contained fha largest amount nt HMCaaachlor-COO administered orally, at compared with matoeboudrin or tysowmae 3. Oral administration of Kano- Heaobarbitai sleeping tuna waa markedly shortened by pre- 10 chlor 400 or 600 tc female rata treatment with Kanechlor 400 or 500 in refs. Minimum eflec- (Komatsu et si 1 m Environmental Health Perspectives HONS 081^36 Table 3--Continued. Experimental Results Reference (W'utar King), about 100 f in right. m order <o m effacU of the administration on AcretarWof steeping lime V. Oral administration of daily doit of 0 4, 2 S, 5, 50 mg of Kanechlor to rata for 13 to 21 daya live doae net daily 4 mg and 2 mg /Kg for 3 daya for Kane- chlor 400 and 400. respectively When 40 mg/Kg of Kanerhlor tOO or $00 wae given deity for 3 deya, the effect lasted for 3 vetks by the former and for t weeka by the latter. Ethioninc inhibited the ahortemng of hexobarbital sleeping time by Keaechlor. Aneboltc eceroide, ehloroquine, glutathione, a-nwreaptopro- pmnylglyrine, eeaential phoepbolipidi, diphenyl, and aulfs- diaaiae did not modify the deeping time reduction by Kenechlor. Growth was inhibited by the treatmenta No weight inereaae at 17 daily dose of 4 mg, and a weight decreaaa at SO mg Plaerne (Nagel at al) tilth, ctridea did not inereaae at daily doae up to 4 mg, and alightly exceeded the control at doae of 40 mg, while plasma cbolnterol and phospholipid inereaaed definitely by inereaa- ing amount of Kenechlor. Daily dose Kenechlor (rag) Triglyceride pM% Choleaterol rag % Phospholipid rag % Control 0.4 34 S0 60.0 109*28 98+1# 89+21 69+13 130+37 83*13 118+28 102+11 1284-38 178+18 4.7*0 8 8 8+1.7 7 3+1.3 7 14-1 3 13 4+3 3 10. Oral adminialration of Frac tion A and B of Kanerhlor tOO to rata, each daily dose 1 n>( on ci. ary fourth day for 40 daya, 10 mg total. Fr A- Boiling point 123124'C/OOl mmllj, 3.12 ehlorina atoma per biphenyl average. FV. B Boiling point 140142*0/0 01 mmllg, 3.34 chlorine atoma per bipbanyl average. 11. Lipid in fur. Oral adminiatratkm cf daily dorc of 8 ml of olive od eea> taming 1 % Kenechlor for 1 and 11 daya to 2 female i*l)biu. The edminiitraliooe hardly affected concentration cf triglyc eride, eholealerol, phoepholipid in liver except for daily ad- mmutration of $0 mg Kaneeblor, which caused alight mcreess of triglyceride and choleaterol. They alightly meresaed tri glyceride in tkm but hardly affected choleaterol and phoe- phoiiptd. A definite reduction of triglyMndc in peiinoai adipose tissue was noted. Antmala grew In weight with Fr. A but loat weight with Fr B, 17 which caused marked atrophy of abdominal adipoae basue. (Nagni at ai) Lipid content of adipose tieeue around kidney decreased when Fr. B waa gives but did not when Fr. A given. Fleams to- glyctnde decreased with ft. B but did net with Fr, A, Animal Contml Fr. A Pr.B Triglyceride sM% 130*18 128*10 83*21 Cholesterol mg % 86*$ 88*7 $7*5 Pboapholipid rag % $.7*0.4 $.1*0.4 $.3*0 3 Toiftl lipid ind iljrwridN of ]iw inetMied. but eonctnintieii of atrum glyetndet was normal after administration for 3 consecutive daya, but increased abnormally after adminidra- tiou tor 11 eonaceutive daya 18 (Ito at ai.) Lipid Control (n-l) Poisoned Poisoned for 3 daya for 11 daya April 1972 135 M0NS 081A3 7 Experimental TiU* 1--Continued. Results Total lipid To/dry st 17 (n-l) 29 (o-l) 30 Glycerides Cholesterol CGP Lecithin LysolecithiD Sphmiomyeiin EGr SGF PI Glycohpid Other* 7 iCi 63 23 5 74 29 10 9 90 68 02 10 0 24 3% 61 30 0 39 6 25 3% 10 8 97 7t 48 14 4 45 51 67 11.1 Serum glycerides (g%) 02 45 1,690 12, Oral ndminiatretion of duly dote 0.6 or 01 mg/Kg of Knnochlor 400 encb to 40 edutt WiaUr rate. Marked or moderately impaired motor function, decraaad motor conduction Telocity, terly itage of tegmuntel deroyehnetion end lorn of large nerrt libera were noted, Reference 21 REFERENCES 13. Inagaai, K et el 1949. Eapenmantal itudy of hairleai 1. Onto, M. end Hifuthi, K. 1900. The aymplomatolofy of Yuabo (chlorobiphenyle poiaomng) in dermatology, mice following administration of rice oil used hy a "Yusho'' patient. IbidflO 048. * Fukuoka Acta Med 40' 409, 13. Ycehimura. H end Othima, M 1071 Studies <n the 2. Ikui, H,, 8u|i, K end Uga. 8. 1909 Ocular aigna of lieeue diMnbution end elimination of eevcrel compo chronic chlorobipheoyta powonmg. Ibid 40:433 nent! of KC-400 (chlorobiphenyle) in mice Ibid 43 0 5. Okumun, M end Xetruki, 8. 1949 Clinical obeerva- t4 Yoihimura, H. at al 1971 Studiea on tbe tmi dia- tion on Yutbo (chlorobiphenyle pouonrai), Ibid 40; 440 tnbution and tha urinary and fecal excretion ol *H-Knaechior (chlorobiphenyle) in ratr. Ibid 42 12 4. Uaawa, H, et el. 1949. Hyperjlyceridemia reeuitiag 15. Fujita, 8. et al. 1971. Effect of biphenyl chlondea on from intake of nee oil contaminated with chlorinated rat liver nucroaooea. Ibid92 30. biphenyl*. Ibid 40.449. 14. Tanaka, K 1971. Shorlenine ol hcxobarbitol iWpmg 0. Uireyama, C. Inea. T. and Yamamoto. T. 1909. Fine lime and ehnnge of aeruo tn|lycende level in chlcro- etructunl chan|ca of the liver in a patient with cbloro- taphenyb-intoueated rata Ibid 03:30. bipbenyl* intoucatioa Ibid 40: 404 17. Nagai, J etal 1971. The mflueocc of chlorobiphenyle 0. Kuroiwa, Y,, Muni, Y, and Santa, T. 1949, Neuro- (Kanochlor) idnuiuftration on the organ lipids in rata Ingieel and nerra conduction vsloeity etudiea on 33 Ibid 62:42. patient* with chiorobrphanyla poiaomng, Ibid 40- 443. 14 Ito, Y-, Uaawa, H. and Notnmi, A, 1971. Lpid com- T. Monuutm, T. et al. 1909. Olorhinblaryngologual peaition of the rabbit liver potioned with ehlorobi- Ondiogt of Yueho (eblorobipboByl* poieorusg). Ibid 90: phenyl*. Ibid 63: 44 444. 19 Nigu, J. et al. 1971 Colorimetric and gaachrensato- 6. Nagai, J, at al 1949. Omicocbemical inveatigaUoo of (rapluc datarmmationa of urinary 17-kctojteTOidj chloiobipbanyia poraoanag--cepecially on tha Mnun Ibid 62. 51. lipid analyse of tha petaeota. Ibid 40' 470. 9. Kiktech), M. and Haahimoto, M. 1949. Hietapetho- 20. Uiawa, R. et al. 1971 Clinical and experimental etudree on the hyperglyeendemi* induced by oral in- logieal etudm of akin Mona of patiaata with cUoro- gertton of chlorinated biphenyla. Ibid 63:86. VhkHTb aniannina Ibid 40. 444. 31. Ogawa, M. 1971 Eleetrophyaioiogical and hntotogical 10. Niakiaumi, M , Kohehi, & and Knnteuso, M. 1949. atudica of experimental cblorobiphenyla poisoning Aa caperimental itudy of "Yuaho" or eblorobiphanyla Ibid 62:74. pobonmg (preliminary report). Ibid 40.039 32. Yamamoto, T,, Hirayaaa, C andlriu, T 1971 Some 11. Tanaka, K. et it 1949. Experimental aubacut* poaontag of thlorobiphenyls, particularly tbe influence on observation* on the One itructura of the mitochondria in hepatic cello from a patient with chloiobiphenyti tbo eerum lipide in tala Ibid 80. M4 intoxication. Ibid 03. 85. 136 Environmental Health Perspective* *:- <", x4*w *i v.!-- *** .iatde,'ia'u>^Ar/J . * -w .. * tmici- HONS 081438 . -X , . IV 'V a. Enwfwuiwnia/ H*akh Pnpxnm Chlorinated Aromatic Hydrocarbon Induced Modifications of the Hepatic Endoplasmic Reticulum: Concentric Membrane Arrays* by 0. H. Norback and J. R. Allenf A number of commercial compound* bavin ( chlorinated aromatic hydrocarbon structures arc used extensively for medlcal<3), industrial^) and agricultural (5) purposes. Quantities of certain parent chlorinated aromatic hydrocarbon* and metabolites have been repeatedly detected a* residues in the food supply and tissues of birds, fish, and mammal(6-9) including man(lO). Chlor inated diphenyl-p-dioxini have been identified as contaminant* of many commercial products, such as certain edible fata, herbicides and disinfectants. Hi* *uggesled that the compound results from the conjugation of commercial chlorinated phenolsfll). Polychlorinated polypheny]* are among the most abundant chlorinated hydrocarbon global pollu tant* Their contamination of the environment it a result of the' widespread commercial use of these compounds for their properties of insulation, adhesiveness, thermoplasticity, chemical inertness, and insolubility. Following this investigation of sequential bio chemical and ultrsstnictural alterations within the liver produced by three chlorinated aromatic hydrocarbons -- chlorinated diphenyUp-dioxin, polychlorinated biphenyls (PCBs), and a highly chlorinated triphenyl (PCT)--certain similarities of biochemical and uHrastructursl effects of the compound* were observed, The livers of rats fed the chlorinated aromatic hydrocarbona eonsi*tently contained numerous multi-layered concen tric membrane arrays, proliferated smooth * Portions of this paper were previously reported (1. 2). 1 Department of Patlwloiy, Uoiveraity of Wiaconnn Medici) School, Madison, Wisconsin 637QC, endoplasmic reticulum (ER), and altered micro somal coxy me activity. Mitirials md Methods Separate group* of male Sprague-Dawley rats initially weighing 100 grama were fed diets containing the following chlorinated aromatic hydrocarbons. 1% TCT* (Aroclor 5460), 0 02% FCBs (Aroclor 1254), and 0 002% chlorinated diphenyl-p-dioxin, respectively. At regular inter val* the rata were deprived of food for 24 hour* and sacrificed by exaanguination. The livers were weighed and portions were removed for electron microscopy. This tissue was immediately placed in veronal acetate buffered osmium tetraxide and cut into small cubes. Following one and ons half hours of fixation, the tissues were dehydrated in a graded aeries of ethanol, cleared in propylene oxide and embedded in an Epoo-Araldite mixture. Sections were cut at a thickness of 0.5-0.E microns for light microscopic evaluation, and thinner sections at a thickness of approximately GO millimicrons were cut and stained with uranyl acetate for electron microscopic examination. The remaining porticos of the livers were perfused through the portal vein with isotonic ItCl, weighed, and homogenised at 0* with two volumes of the salt solution. Microeomes 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 original liver material, snd immediately assayed for the activities of aromatic April 1972 a liydraxylaae(12), nitroreductase(12), X-demethvlute(l2), nitrophenyl acetate hydrolysis (esterase) (13), and ghicose-C-phosphatase(l4). ItNA(lj), phospholipid(10), and cholesterol (10) concentra tions were determined from samples tliat.had been frozen at --80". Protein determinnUon(17) on slkaline-i\ashed(18), microsomal material pro vided estimation of membrane protein in the microsomal suspensions, which is subsequently referred to as microsomal protein Results The rata from the dioxin and PCT groups readily ate the experimental diets and attained at least 80% of the w eight 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 w eight of 8 grama per hundred grams body weight, following PCB and PCT ingestion, was observed. Neurological aide effects, including tremors, convulsions, or sednhon, were not observed The rats from the dioxin and PCI group remained on the diet for four weeks without apparent extra-hepatic effects other than the small decrease m weight gamed The rats fiom the PCB group did not gain weight and Here sacrificed at 3 days. The ultrastructural alterations within the hepatoeytes of rats from the three experimental groups were similar and will be considered collectively The changes were primarily [united to the endoplasmic reticulum and consistently affected all experimental animals The hepatoeytes 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 hepatoeytes (Fig 1) were reorganized into ainuous patterns that encom passed lipid droplets, mitochondria and other organelles (Fig. 2). Annular profiles consisting of paired membranes with ribosomes attached to the Fracas 1. Elements of the rou*h endoplasmic reticulum era cbarecterisltcaUy *rtn*ed ptraUel Umetlee in hepitocytM of lets fed a control dial (X 8,630) 138 ! .M-wJlidUibv'. '/tii - *y Environmental Health Perspectives HONS 081't'iO i iftiifc r'n a^niiii activities of gIucoie-6-plio^phatsse end aronint hydroxylase line reduced The enzymatic chant; following PCU ingestion were parallel (o those the PCT group Discission Ingestion of the chlorinated aromatic liytir. carbons by rats induces liver hypertrophy uu proliferation of the ER and formation of lari concentric membrane arrays within the cytoplm of the hepatic ceils. These changes measured aft21 days PCT ingestion result in increased hepat function An increase in total activity of cac enxyme investigated is evident when the near! three-fold liver hypertrophy and more tha doubled quantity of microeomal protein per grai liver is considered. Associated with the enzymati alterations are an increased ratio of phospholipi to protein and a decreased ratio of cholesteioi t protein. Increased ensyme activity and proiil rated smooth Eft are commonly produced by . Fiooiu 4. The concentric uuiuli with attached ribooome* re apparently derived from the rough endoptaumo reticulum (following ingatno of chlorinated diphenyip-dio>uii X3S.M0) annular unit* The outer membrane pain of the lamellae were continuous with elements of the month ER and rough ER. Ribosomes were occasionally attached to the surfaces of the outer membrane pain of the multi-layered arrays- The concentric membrane arrays increasad in else and complexity in direct, relationship to the time the rate remained on the experimental diete. Associated with the modified membrane struc tures were alterations in easyme setivity and biochemical composition of the microsomal fraction of these cells (Table 1). Following the ingestion of PCTs for 21 days, them was an increase in microsomal protein per gram of liver. Composi tional changes of the membrane material were indicated by the reduction in cholesterol to protein ratio and elight increase in phospholipid to protein ratio- The increase in esterase activity was parallel to tint of protein, whereas, increases in Ndemethylation and mtroreduction were greater than the increase w membrane material Specific Fioeat 5 larger concentric membrane artj con*i '>< numerous rheiipei Tmgl of paired membranee (following mgeetion of chlorinated diphenjl-p-diown; xlo.JtM,1 140 Environmental Health Pcnpcctim HONS __________ MMk Hfc Flaunt 0. Folia*ing insertion al polychlorinated tnphenyl for tbn* weeks enlensire concentric cnensbrson trays develop ithm the bepateeytas. Tbe tnembftoe srrnys mooapua lipid droplets, aitochondno, and other oellulu orjinellrt (X 3,700). wide variety of agent* including phenobarbetal; however, the phospholipid/protein are unaltered and cholesterol/protein uicreased(18). Thus the membrane* low in cholesterol and high in phos pholipid composition, which are characteristic of chlorinated triphenyl induced microaomes, likely represent the concentric membrane array*. Structure* similar to concentric membrane array* have been observed following the adminis tration of numerou* other compound* including phenobarbiUl(19), thiohyd*ntoin(20), triparenol(21), tfialoxin(22), ethionifle(23), carbon tetrachlondc(2t), and amino aso dyea(25). An evaluation of the sequential chant** that occurred following consumption <4 the chlorinated aromatic hydiocsibona suggests that arrays of concentric paired membranes originate from cisternal demtnls of the ER.. Modification of the rough ER included reorganisation of parallel eistemsc into sinuous ostemae and concentric annular profiles of paired membranes. The concentric annuli are similar to tbe compositional units of the multi layered membrane arrays This similarity suggest* that the reorganised cistern** of the rough ER are incorporated into tbe arrays of the concentric paired membranes. Continuity of them arrays with tbe rough and smooth ER further support a postulate of a rough ER derivation. Ensymatie and compositional changes of the microtome* suggest tbe modification of the ER may be correlated to several probable functions. Consistent with tbe increased proportion of phospholipid* within the membrane structure is the proposal that the proliferated membranes provide a site for storage, for isolation, or for contact with membrtne ensymes of the hydro phobic chlorinated aromatic hydrocarbon. The increased proportion of phospholipid enhances the suitability of the membrane to sequester the foreign material. The proximity of the concentric April 1972 141 MONS 061443 'vri-'M.V'- * Table I> Htptllt iliinllMi In ral fed chkmiuttd trlphenyl {or 11 days. Cont Ell)) cr weigh* (S/VUO g body weight) Mtcroom%l prolem 3.00**0 28 20 1**0 3 (mg/g liver) RNA 130* 33 (jO1 . Phospholipid 45V 24 (H? Gholwltrol (mgp Cluco**-6-phoephtese 44 0**8 9 3 08**0 it (nmol P04/15 mu)1 Esteraae 0 78*8 0 (paid p-oHrophtnol/mii),)* Arottatw hydroxyl*** 30 2*fl 2 < Bismol ***iinp* pheool/30 mt& )* N-dsmslhylase J03* 10 (mpmol formaldehyde/ 30 min.)' Nitroraductaea 32 64*8.8 (Op<do1 r'iaiD- bcovMte/lve)* 8 63 * 0 70 54 8*4 1 63 7*4 3 808 *30 IT 0*1.8 1 08 0 OS 10 3*1 31 8 06*0 29 168*7 48 4*6.3 Difference between omm and following own ia *<<ntftcani (p < 001). a Difference between mean and following man ia sig nificant (p< 01). per mg protein. membrane arrays to lipid droplets, also a probable location o( ths lipid soluble compound, provides accessibility of the lipid component of the membrane to the compound. Hydroxylation activity present within the nucroeomee converts a lipid soluble hydrophobic compound into a form which has the potential to bind with activated glucuronide (uridine diphosphate glucuronic acid) to form glucoeiduronete. The resulting water soluble conjugate could then be excreted through the biliary or urinary qretem. Although the specific activity of this eniyme is reduced, the overall activity within the liver i* substantially increased over the control value. Cholesterol within the membrane structure has been related to the rigidity of the membrane by ita ability to condense the unsaturated fatty acids of the phospholipid*(2C). Lock of cholesterol suggests a less stable structure or, alternatively, regions available for insertion of other compounds, such as the chlorinated aromatic compounds among : fatty acids and the phospholipids ThuJ t eniymatic and compositional alterations al t newly formed membranes suggest a membra suited for localising the chlorinated aroma', hydrocarbon for possible sequestration of met bolites for excretion (Supported by NIH Grant ES00472) REFERENCES 1. Norbeek, D. H and Allen, J K !069 Morpholine of toxic fat-induced concentric membrane unyi in r hepatocyte*. Lab Invest 20. 338 2. Norback, D H and Allen, J K (In press) Chlo, seled Iriphenyl-mdueed extensioni of the hipit endoplasmic reticulum. Pro* Soc Exp Bail aod Ms 3. Chloitettatyrehna ChtoqHonuioa Chtoroprepanuda 4. Aroclor Products, Mousanto Co, St Louie, Miueu, 8, DDT (1,1, l-Trichloro-2,l-b,s>-cbloropheoy|) etl ana. 2,4, o(2,i-Djchlocophenoxy) eettie ac Chlorbooaide. 8. Woodwall. G. M , wureter, C F- and Xeaaceon, P / 1067 DDT rroidoee in an East Coast estuary a tai of biological concentration of n persistent inaecttetd* Science ISO. 821. 7, Rnebrough. A W. et el 1068. Polyehlcrinsled b phenyl* in the global ecosystem Nature 220 1098 8. Holmes, D. C,, Simmons, J. H end Tntton, J 0'C 1087. Chlorinated hydrocarbon* m British eildltft Nature 216 227. 0 Holden, A. V. and Maixdett, K. 1067 Organochlonn pesticides in seals and porpoises Nature 218 1274 lft Biros, F. t, Walker, A C. and Medbtrry, A 1970 Polychlorinated biphenyls m human adipoM Uuue Bull Environ. Contain in Toxicol 6 317 J1 Pbmmer, J R, and Klmgebiel, V. I 1071. Biboftatu photaeeneitised oxidation of 2,4-dichIorophenol as aeeatneat of possible chlorinated dawn formation Science 174:407 12. Ensymt incubation conditions end product reaction, according to Cram, T E, Rosen, L. A end Fouls 1. R. 1087. J Pharmacol Exp Ther 188 470 usmy t mM a-n,'rmboojoie acid, 101) mM aniline and 6 mM amioopyrina. 13 3 $ mM p-oitrophcnyl acetate incubated at pH 8 5 in citrata buffered solution, change in absorbancy at 420 Ap measured 14. 40 mM glucose 8-phoephateee incubated at pH 6 3 in citrate buffered eolutloo, inorganic phosphate mj>mod according to Bagtneki, E S, Foa, P and Zak, B 1067. Clm Chcm 13 326. 18 Munro, H N and Fleck, A 1068 In. D Click (ed 1 Methods of Biaehcnucal Analysis, Inlrrtcianct Putr lichen, New York. It UJ 18. Lipid extracted according to Polcb. 3, Lees. M and Sloane Stanley, C H. 1057. J Bnlog Chem 226 401. 142 Environmental Health Perspectives MOMS G81AAA Diff!11 ion of organic luatrrial and determination of inorganic |>hoephiU according to Baginski, C 8, el el, Ref 14, Cholrelrrol tcpantnl according to 8hm, Y, 8. indLee, J. C 1942 Clin Owm 8'598, and quantified according toGlick. D , Fell, B F nnd 8]olin, K 1904 Anal Chtm 38' 1119 Determined by method of lwry, O i! d at. 1950 J Bioiog Chem 193 205 with bovine aerum albumin aa standard. Glaumann, H and Dallncr, F 19G8 Lipid cornpoaition and turnover of rough and amooth microeomel membraaaa in rat liver. J Lipid Rea. 9: 720 Burger, P. C, and [lerdaon, P. B 1940. Phenoharbitalinducrd fine atauetural changer in rat liver. Amtr. J Path 48- 793 50 Perdeon, P. B. and Xaltenliarh, J P 1DG3 Electron rnirroecope tludice on enavme activity end the uolation o( lliiohvdanloin-uidueed myelin figuree in rat liver. J Cell Bi'ol 25 485. 21. Hruban, Z, Swift, H and STeaere, A 1905 Effect of Triparmol end Diethanolamine on the fine etructure of hapetocytea and pancreatic acinar ceile. Lab. Inveat 14- 1052. 22 Svoboda, D, Grmlj, H i and Higgineon, J 1904 Affatoxm B in rat and monkey liver. Amer J, Path 49- 102J. 23 Sterner, J. W,, Miyai, K end Philhpe, M J. 1964 Electron microacopy of membrane-particle arrayt in liver Celia of etbionme-intoxicated rate. Amer J Pathol. 44: 149, 24 Stenger, R J 1944 Concentric lamellar formation! in hepatic parenchymal celti of carbon tetrachloridetreated rate J. Uitraalr Rea, 14 240. 25 Bmuckler, E A and Aroaaoy, M. 1949 In 0 W. Richter and M A Epstein (ads) Inlcmalionel Review of Eapertmental Pathology, Academic Press, New York, 7 305. 20. Deenen, L L. M van. I04M5. Phoapholtpida and bionrrfmbrane. Progress Chem Feta. Lipids 8 1. 9 * April 1972 Emdronmmtel HmUh Pmptaii Biological Effects of Polychlorinated Biphenyls in Rats and Quail by Joel Bitman, Helene C. Cecil and Susan J. Harris* Polychlorinated biphenyl* (PCB) have been administered to rata and to quail to discover the nature of the biological effect* which these environmental pollutant* produce in mammal* and birds Biological effects evaluated were; (1) estrogenic activity of the PCBe in rats, (2) alteration* of pentobarbital metabolism induced by the PCBa as judged by deeping time of Japanese quail, (3) alterations of liver vitamin A, liver lipids and liver weight induced by Aroclor 1242 in rat* and quail, and (4) egg production of Japanese quail. EitrspHlc Activity Estrogenic activity was assessed by determining the stimulation of the uterine glycogen response of the immature rat uterus 18 bows after administra tion of the teat eompound(l). lbs potency of active compounds is reported in terms of the minimal subcutaneous dose which will increase glycogen to a level significantly different from control. In a series of 11 polychlorinated biphenyls and terphenyla, the compounds containing up to 48% chlorine were eetrogenicelly active (Table 1). A polychlorinated terphenyl 42% ehlorine was found to be 8 times mom active. This is a low level of estngsaie activity and probably arises from hydrooyiated metabolite* during is vivo metabolism. * United State* Depanzneat at Agriculture, ARS, Beiteviile, Maryland 2070}. Effects on Pentobarbital Metabolism In Japutesa Quail The polychlorinated biphenyls have bmn demonstrated to stimulate liver enzyme system* which metabolise barbiturates in rata(2). We utilised a deeping-time test with pentobarbital in quail as a mean* of determining effects of poly chlorinated biphenyls and terphenyla on liver microsomal enzyme activity. The liver ia the major site for the metabolism of foreign chemicals, end a standard dose of pentobarbital wilt produce a etandard 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 enzyme* will be reflected in a longer duration of pentobarbital hypno*n(3). The PCBa end PCTi were tested for their acute or short-term effects (48 houn) and for longer-term effects at 3 and 7 days. At eppropriate times after administration of the PCBe or PCTs, sodium pentobarbital was injected intramuscularly at a dosage rate of 50 mg/Kg body weight. Bird* were considered asleep as long as they could not right themselves when placed on their backs A single dose of Arcelor 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, deeping time* were less than untreated control values in the male quail, and by 48 hours, the PCBa 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 tingle oral dote after 5 houn. In both male and female quail, Aroclor 1268 appeared April 1972 145 HONS a Tnlil* 1. Ealrogcnlc Artibty of Polychlorinated Biphenyl (I'CU 1 and 1 erphenyl (PCT) Comi>oiind. PCNTOSAUUAl SlttPlNG riMI IN Ouau Name Minimum <ffee(ng doat rci PCI! Aroclor 1221 21% Chlonna PCD Aroclor 1232 32% Cl PCB Aroclor 1242 42% Cl PCB Aroclor 1218 48% Cl PCB Aroclor 1234 S4% Cl PCB Aroclor 1260 60% Cl rCB Aroclor 1202 62% Cl PCB Aroclor 1268 68% Cl PCB Aroclor 4465 60% PCB, 40% polychlorinated triphenyl (PCT), 65% Cl PCI' Aroclor 5442 42% Cl PCT Aroclor 5460 60% C) 8 8 8 s Jnaetive Inactive Inactive Inactive Inactive 1 Inactive to exert greater effect* than Arcelor 5460 The diphasic pattern elicited suggest* that the FOB* ami I'CTe first inhibited the ensymea which metabolite pentobarbital, but then, at later times, etinnilatc the induction of greater enzymic activity, Contmuoue feeding for 3 and 7 days at level* of 100, 300, and 635 ppm ad libitum (timulated the metabolism of the standard doae of pentobarbital Fiona* 1. Bleeping timet of quail led PCB 1263 or PCD 5400. Kith point rxpreMftti u quail (Fig. 2). Sleeping times were about 50-70% of control levels, thus indicating greater metaholiim f the barbiturate. Higher dose levels everted greater effect* and, again, Aroclor 1263 appeared to be more stimulatory than Aroclor 5400 Sleeping time* were not decreased to as gi eat an extent m the female quad as in the male. The sleeping time pattern elicited by FCBs and PCTa is in marked contrast to that nhich the organoehlonne pesticide analogs of DDT produce in quail(3) DDT and its analogs produce sn EfffCf Of AR0008S ON SLEEPING riMf IN qvail PCS 1769 MALE fer 4440 female Fiona 2 Sleeping tltnea of quail fed PCB IMS wPCB Each point represent! ax quail 145 I Environmental Health Penpetlim TiWr 2. U'r Clin*M in M*[e tnd Female R*u f(er Feeding Ad Libitum for Tuo Months Dicta Containing >00 ppm Argdor 1242. Treatment Cm'ml Arorlor 12*2 ConlroJ Aroclor 1M2 Doer wt. Liver lipid Vilamio A a ISE 7cSE */* liver mg/liver Frmale rats 1010 0 4* 2 1 15 i 04 47823 4 54 22 7 4* 3- 3 4S 08k 244 15- 2 25 tl* Male rata s10 12 3-Jg 4 S 14 13 504 i.20 0 82 22 13 0.8 7 12 18' 270 15' 3 40 21' p<0 01. P<oaa. p<0 001. innal inhibition of pentobarbital metabolism, and seeping times are longer during the first week of feeding in males snd during the first 3 weeks of feeding in female quail. Effects on Liver Vitamin A iff Rats and Quali Repeated exposure of rats to DDT results in degeneratit e changes in liver tissue, an increase in liter use and an increase in liver lipids(4), and a decrease in liver storage of vitamin A (5). The possibility that PCBs w ould produce similar effects was investigated in rata by feeding Aroclor 1242 for 2 months at a dietary level of 100 ppm (Table 2). In both male and female rats there was an increase in liver weight and liver lipids. There w as a very striking decrease of about 50% in the liver concentration and content of vitamin A. Aroclor 1242, fed to male Japanese quail for 2 months at a dietary level of 100 ppm, produced similar effects, an increase in liver .weight and uva vn a conc in qua* 150 125 1 o IQO V 8 2 75 25 --l--------- "* 1 cor act nor rc 3 IMS 1 oor ki FierriL 3 Liver vitamin A Mhrtntrvtioni cf quail fed 00 ppoi DDT or PCS 1242 for two monthi Group* coulmoed 14 to 20 queil. 0 odi eci >342 ooi res nii cor rc 1242 Fionei S ToUlliverviUmmAof quail fed 100 ppm DDT OT PCS 1242 for two month*. Group* conUmed 14 to 20 quad. April 1972 147 MQNS 081448 (GC PSOOUCUON LOW CALCIUM DAVS Fiaosr G Eg* production o( Jupunts* quiul led PCB 1242 Average production lor three day periods is plotted for group* of 1C quint lipids, end a large decrease m 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, almost daily FULL CAICIUM DAYS Fiouac 7 Egg production of Japanese qn.iil fed PC [ 1154 on a low calcium diet. Average production lor thre, day pertodt is plotted for groups of 15 quail changes, variability in liver vitamin A levels u great. In order to determine whether PCBa 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 Ute cyclic mobilisation of vitnrmn A for depoeitkin in the egg yolk does not occu r In the birds kept in the dark, Aroclor 1242 caused about a 50% reduction in liver vitamin A (Figs 3 and 4) DAYS Flcuas B Egg production of Japanese quail fed PCB 1254 on a full cilcium diet Average production for three day periods la plotted for groupe 01 IB quail. ERretsonEff Produetlsii The eflecte of Aroclor 1242 and Aroclor 12.14 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 L>0 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 gioup was over 2 times that of the control quail 148 Environmental Health Perspective* HONS 081449 An.lor II'A wm fed at n level of 100 ppm in both full calcium (2,V>%) and low calcium (0 007,,) did Eng production was not suppressed by the PCH* in either ease (Kigs C and 7), aitlioupli Lhcrc 'ias a negative effect of tlic low isle min diet There were no differences in the i unibriK of biokcn or membranous eggs atlnbuti Die to Aroclor 1264 A depression hi egg production of While Leg,ioni liens fed Aroelor 1248 was recently reported >y Sr oil ct 1 (C) Heath el *1.(7), however, found little effect of Arocloi 1264 on eggshell thickness or '/'production in mallard* or bobwlpte quail An effect upon egg production and reproduction of Aroelor 1242 and Arotior 1264 has been previously repolcd in White Leghorn cluckeus(8) Recently, !'r*knll{9) found no effect*upon eggshell weight m King dove fed 100 ppm Aroclor 1254. Thu*, there is * notable lack, of consistency in Die published reporU or the effect* of PCBs on egg production and reproduction in birds. Conclusion A study was made of the effect of PCD* on esl rngemc activity, Ijver barbiturate metabolism, liver vitamin A and egg production using a limited number of PCH materials. It appear* that the biological effects of sjiecific PCB compounds are different, as might be expected with this sene* of materials containing compound1! tbicli differ so greatly in chlorine content and in clicniMMl and physical piopcrtics More czlcnsr. q research on individual PCB compounds must be undci taken to assess fully the hiologic.il effect!- of polychlorinated biphenyls on mammals and birds nEFEKIPNCFS 1 Hitman. J and Cecil. II C 1970 F'trogenir admit of DDT aiMlngs and polychlorinated Inphcnib J A,nc toad Chem 18 1)06 2 Slrecl, J C etui 1000 ]58lh Full Medina anw*ftr#n Chemical Society, New \ork Ot' Abstract 7-12, 1900 3 25)111)1111, J cl nl 1971 Comparison of DDT effect on pentobarbital roeUbohsm in ruts and quad J Agne Food Chon 19 333 4 Lauk, E P el si 1930 Liver tell slterahona and DD1 storage in the fat of the rat induced by dicterv level cT 1 lo 30 ppm DDT J Phermscot Eip Ther 8 ?GS 5 Phillip*, W IS J J9G3 DDT and the mcialjolism of YjUimn 8 and Carotene in the rat Can J Biochcm Physiol 411 1793 5 Scott, M L el al 1971 Proc 1971 Cornell Nutrition Conf Feed Manufacturers, Buffalo, New >ork, Novem ber 2-4, 1971 7 Heath, R G. et al 1970 Proc XV lnt OrnitbnJ Cong., The Hogue Aug 30-So|>t 5, 1970 E J Brill, Leiden 9 Industrial BIO-TEST Laboratories, Inc Rf* No J7300, June 4, 1970 9, PeakalL D B 1971 Effect of polychlorinated bt|>henv!a (PCBe) on the qtgsliellft of King Doves Dull Environ ConUimn. Toxicol. 6* 100 April 1972 HONS 081A50 Enuironffifniaj /fwitli Periptctitrt PCBs and Interactions with Insecticides by E. Paul Lichtenstein* The effects of polychlorinated biphenyl plsatl- Utilizing insects as the test objects, the bio citers, like those of many other environmental logical interaction between plasticisers and some chemicals ("synthetic chemicals to which man insecticides was also investigated (1) Because of is exposed by either voluntary or involuntary the structural similarity to DDT-related com means'1), on biological systems are not too well pounds, 11 plasticisers were tested for their understood. When research by our group at the toxicological interaction with dieldrin and DDT University of Wisconsin indicated in 1909 (1) Many of these polychlorinated biphenyl com that several of the Aroclor compounds increased pounds were toxic to Drosophila melanagasftr the toxicity of DDT and dieldrin to insecta, their Meigen and house flies, ifvsca domcsiica L., but potential biological interaction with environ to a leaser extent than dieldrin or DDT. Their mental chemicala was indicated. Since then toxicity increased with a decrease in their chlorine further studies were conducted in our laboratory content Dieldrin, DDT, or any one of the less to investigate the effects and interaction* of chlorinated and more volatile plasticisers (Aroclor environmental chemicals on human cells in tissue 1221, 1232, 1242, 124S, 1254) were toxic to both 0 culture by determining the toxicity of some of test insects (Table 1). While these polychlori these chemicals on HeL* cells and ekin fibro nated biphenyls contain increasing amounts of blasts (2). Aroclor 1254 (a polychlorinated bi- chlorine, their toxicity towards the insecta de plienyl plasticiser), carbaryl, parathion, DDT, creased. Dieldrin was the most toxic of the com- several insecticide metabolites, aspirin snd caf feine mere used to determine their effects on cell growth and synthesis oi proteins and nucleic rids. The doeage causing a 50% inhibition in culture growth was determined, and the inters* non or combined effects between these chemicals relative to their effects on protein and nucleic and synthesis was studied. Both cell types re sponded nearly equally to the presence of an individual chemical. Aspirin and caffeine were 8 lo 30 times less toxic than the other chemicals ftudied, while Arcolor 1254 was as toxic u DDT (fig l), Parathion was more toxic than its me tabolite paraoxon. Prs-nitrophenol was as toxic xs parathion. 1-Xaphthol was less toxic than it* parent compound carbaryl. The toxierties of DDT and DDE were dependent upon cell type, bulc significant interaction on nucleic acid or protein synthesis was seen (2). ` Department of Enlomolo**, Umveriiiy of Wbronain, 'l*diwn. \\ isconMQ 4370fl. Ficoxe 1 Dotage-response eurvea obtained after 45-hour espoture of human tell eultum to vtnoue concenuationa of p, p'-DDT, Arcelor 1244 (PCB), aspirin and ca/feinc (Kef 2). l I t 14 4 H 1 Table lv hITect of pUaticirers or Imtcticidci on insoot mortalitim. Mtitcnni 1) melanogBSier Applied* % mortality teg) 24 hr 48 hr Jloute Applied* (of/* By) cr mort tin-/ 24 hr None* Dieidrm p.p'-DDT Aroclor 1221 Aroclor 1232 Aroclor 1242 Arovkrr 1243 Aroclor 1254 A rockir 1280 0l 30 200 800 200 SOO 200 SOO 200 SOO 2000 2000 00 3! 84 24 58 n0 57 92 00 33 54 04 40 73 00 15 45 0 ft 00 10 25 0 500 1000 500 1000 $00 1000 600 1000 1000 1000 0 73 57 IT 43 If) 30 17 20 13 13 10 0 * Applied in 8 ml hexane to 4<o* flew jen P mdanogatut exposed to dry residue. * Applied topically to house Alee ui 2 *Utt? of acetone Weight of 1 fly**2D mg. * Control* were treated with solvent* only pounds, u measured by the percent mortality of the insects within s 24- or 48-hr exposure period. Approximately 25 and 30 times more DDT than diridnn, and 8000 and 1000 times more plasti cizers than dieldnn had to be used to obtain similar mortalities of D. melanofatUr or of house flies, respectively, within a given lime of exposure to those chemicals. The more highly chlorinated end less volatile plasticisers u-ere nontoxic even when used st dosages of 2000 ag with D. metanogasltr and 20 iig/fly (1000 jig/g fly) "nth house flies Table 2 summarises the results obtained after the inserts had been exposed to sublethal dosages (200 jig for D. mthmogatUr, 10 jig/house fly) of eithor one of the plasticisers (Aroclor 1221, 1232, 1242, 1248 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 dieidrm and DDT. This effect ivae most noticeable with DDT on house flies, and differ ences observed were highly significant. After a 24-hr expomue period, 38% of the house flies hod died after only DDT had been applied However, all the insects were killed when DDT ivos applied in combination u ith any one of these plasticizers The polychlorinated biphenyls also increased the toxic effect of DDT oath D mefanojosier, but to a looser extent. The most striking effect was observed with Aroclor 1221 Arocior 1260, 1262, 1268, 1265, 5442 and 5460 applied with dieldrin or DDT to gloss surfaces to which D mcianogasicr were exposed reduced the toxicity of the insecticide, resulting in mortalities of 30% to none within 48 hr (l). In particular, though, a substantial increase in the toxicity of organophospborus insecticides to houseflies was achieved with polychlorinated biphenyl plasticisers (3). Aroclor 1248--although nontoxic by itself to house flies at dosages of 10 iig/fly--significantly increased the toxicity of the oxygen analogs of five organophoaphorus insecti cides after simultaneous topics! application to houseflies Both p,p'-DDT and p,p'-DPE, applied at aub-ltbnl dosages, also increased the mortality of houseflies due to psrooxon from 2 to 60 and 73 per cent, respectively. The polychlorinated bi phenyl plasticiser, applied simultaneously with parathion, however, decreased the toxicity of parwthion to houseflies but increased mortal,lies when applied to flies one-half hour after the appli cation of the insecticide. The plasticizer apparently inhibited the conversion of parathion to its urnc 152 Environmental Health Perspectives HONS OS 14fid Tsbtc 2. Combin'd of plaatlcszere and trurctlUtJri on Insect*. % mortalitiea of insect! alter exposure to ArocJor No insecticide Ditldrt/i p,p'-DDT Softc' ii 1232 1212 1248 1234 .Van* 1221 lUi |24? 1248 1234 0 0 0 6*2 0 0 0 4 4 0 8 0 D mtlanogo*!# i/ur 45 hr* Qfl 6 59*6 B7*4* *3*5* 8J *3" 84*6" 77 9 83*6" 72*4 77*8" 10*3* 47* House lies' after 24 hr 27*6 38*8 43* 10C* 27*8 100* 47*8e *8*3* 88*11* 24*8 toe* too* 0 Wanefo-ifer were cxpoaad m 4-oa ftaaa ian to the dn durface dcpoait of the chemicals. iilssttciien, 20Qec; dieldna, 0 I Dti DDT. 8 0 at * fiminy jtn without plasticisert eentaioed 250 ji| of rcm oil each. Differences obtained batmen tnaloaeati irilt the UNtOode only and thoae with insecticide phis plasticiser art npiificaal at Ota e I %, d 0 1%, or *-- 8% level. ' Chemicals wera applied topically In 3 sitter of acetone t houseflies plasticisers, 600 atft fly: diddrin, 0.75 r*/c ft', DDT. 18 ag/c fly Control. only acetone wu applied Uuthlof 1 fly-20 mf. activation product, paraoxon, and the breakdown of paraoxon into nontoxic compounds. This was alto indicated by the recovery of larger amounts of "C-paraoxon and smaller amounts of water soluble ,*C-prodixrt from dies treated simul taneously with Aroclor 1283 plus paraoxon, in comparison to those which had been treated with "C-paraoxoa only Utiliting the 10.000XG supernatant from housefly homogenates, the addition of Aroclor 1248 with "C-peraoxon id vitro also resulted--after 2 hours incubation at 30*0--in higher recoveries of "C-paraoxon and in a reduction of the appearance of water soluble uC-producta Identical results were obtained with subcellulu fractions (10,000XG superna tant) from flies that bad been treated with Aro clor 1248 eighteen hours before homogenate preparation. No difference was observed in the degradation of paraoxon or tbs appearance of water soluble products with Aroclor applied to the living flies or to the 10,000 XG supernatant of homogenates from untreated flies (3). Since plasticisers ate in the environment, their potential effects on biological systems, especially in combination with other synthetic chemicals, should not be disregarded. REFERENCES 1. Uehteo*tn, E. P. at ai. IMP. Boloficei nrtersetwe between plastieisus and insecticides 1. Earn. Entomol 82:781. 3. Litterst, C. L. ad leehtcnstetn, S. P. 1871 Effects and interactions of eovtroemental cbeoieala on human cella m tiwua culture. Arch. Environ. Hsalth 22 184. 3, Fuhremans, T. W. and Lichtenstein, S. P. 1872 Increeee in tha toxicity of wginophetphorut insecticides to hotieefliee duo to polychlorinated biphenyl plasti cises*. Toxwol. Appl. PtsriMeoi. (In presa). t|>rij 1972 1S3 HONS Environmental Htahf, i'vnptciu+i Polychlorinated Biphenyl Interactions with Tissue Culture Cellst by Roger Hoopingarner, Albert Samuel, and David Krause* The successful use of tissue culture cells in biological work is now almost 00 years old. Howc\cr, Hie applicability or the "fitness" of these cells to demonstrate biological principals is still debated It is hell known that many of the cell lines bear little resemblance to the parent tissue or organ cells. Yet isolated functional cells of serially-propagated clonal strains, that can per forin specialised, organ-specific functions lor prolonged periods, have been produced in nearly limitless numbers (1). The loss of specific func tion hss been ascribed to (i) selective overgrowth by connective tissue cells, (ii) a phenotypic change in Lhe cultured cells, or fill) inadequate or harmful environmental conditions (2). In order to circumvent these problems, primary cell lines are used and tested before the line "adapts" to culture conditions through aneuploidy or other means. The use of tissue culture cells for the examina tion of environmental chemical* has been rela tively recent (3). The effects measured have been (i) inhibitory dose, (ii) toxic dose, (iii) cytotoxic effects, and (iv) chromosome aberra tions. Samuel (4) has used both established and primary cell lines also to study the metabolism of certain pesticides. Litterst and Lichtenstein `3} using HeLa cells and a cell line of non-maligiiant origin found that the interaction of DDT and PCBs (Aroclor 1234) that had been found in flics tits absent. They concluded that tbs various t A contribution from tbs Michigan Agricultural Eiperir"m! Siation (Attlcl* 3832). ThumeareH a mipportcd ll,rt b} fund, promled by the Food and Drug Admior- r.11011 Dl)i;tv under contract FDA 71.284. ` Deportment of Cntomology, Michigan StateVan oraity, b-il taming, Michigan 4SS23. interactions of chemicals with the celt lines had no correlation to intact animals. The use of human prin -ry cell lines was en hanced greatly with the d <-eovcry of the mitotic stimulus of the mucoprotcin phytohsemagglutimn (5). The leucocyte culture techniques developed by Moorehead et si. (6) proved to be most suit able for in vitro cell studies of human ceils 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 affects on essentially the tame cell. In the studies to he reported here, we 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 groom in Eagle's minimum essential medium supplemented with 10% fetal calf serum. The lymphocyte cultures ivere obtained from blood of laboratory volunteers and were grown in 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 X0.02 inch S C.O.T column of Apieoton L at 200*C. Detection was by hydrogen dame ionisation detector. The growth suppression of Chinese hamater ceils with PCBs and DDT are shown in Fig 1 These data are comparable to those found by Litterst and Lichtenstein (3) though they did not directly study an ID using both chemicals April 1972 1S5 \ * i I tr "Tv ' i IIIMIIES Fionas 2. Gaa Chromatogram ef Arodor 1221 Fiotnu 1. Growth of Chlneea Hamster eeOi with FCB ud DDT. simultaneously. The toxic does of the chemical was several timet greater than it usually found biologically. It is of some interest to note the disintegration of cells in the period from 34 to 30 hours, when the cells were being stressed from culture condi tions. This same phenomenon ean be seen from the differences between 24 and 30 hours in Table 1, This Table gives the relative growth informa tion for the different PCBs. As you can sea, there was a steady drop in cell numbers as one de creases the percentage of chlorine. The Arodor 1010 is e distillation product of 1254, which essen tially excludes the biphenyls of five chlorines Table 1. Pepulatleaa of Chinee* Hubiut Cells Cultured with U ppm ef Ycrtoue PCB'e FCB None 1260 1254 1948 1321 1016 34 bn* Bill 27 9.6 23 5 8 ISAS 1 lfl 4 4*1.1 SO bis 36*4.4 71.7 1.7 3*1 4 11*8.4 1*0 4 * Avanga ef ten repheatea. Count ef eeUs/uaU end area. 155 "m waarit h " d ` 1 * Flows 3. Caa Chromatotram of Arcelor 1232 S1W>|I Flows 4. Caa Chromatogram of Aroelor 1113, Flows 9 Gaa Chromatogram of Aiodcr 1134 Environmental Health Perspectives HONS 081.555 fc ,, ^ --------- -<** "irtfrfaB,"r Table I. Human Lymphovy lev Treated *ilb 100 ppm Aroclpr IZM.' Fanod of FOB Mitotic Treatment Index Satellite Associ ation Chro matid Gap* Chro me tid Brinks IBVt f Fietnic 0 Gm Chromatogram of Aroctar 101ft md above The produet is still a biphenyl, but since it ie derived differently, the prefix is changed, from 12 to 10. Baaed on the percent chlorine, Arodor 1016 falls out of the pattern of toxicity. If i ou look at the gM chromatographic patterns, One toxicity is eomeivhat of an anomaly. For csample, the Figures 2 through 6 are the chroma tograms for the various PCB* using the supportrotted open tubular, or B.C.O.T., columns The pattern of 1016 was very much like that found for 1232 or 1242 yet 1016 was several fold more toxic to the tissue culture cells It would PI>citr that there may be certain components shut arc in higher proportion in the 1010 product, and these components (or component) are causing the increase in toxicity. We have fractionated most of the chromatographic peaki into their romponent ports and are currently assaying them to determine the relative toxicity of each. The human lymphocyte cultures are of course a primary cell line, and in fact we examine the evils cylologically at the first in vitro division. 1i r 7, Diagram of Cultured human lymphocyte* at first in vitro division. Tat 1 0-24 bn 032 a 6 1 44-12 hn 009 14 3 1 40-52 h:l. 017 3 4 0 Acetone check 022 t 3 0 Control 012 4 5 l Tati 0-24 hrt .017 10 1 0 44-42 hr*. 007 4 4 0 4B-42 hrt. 012 4 2 1 Acetone check 009 4 3 0 Control 008 8 2 1 50 cells analyz'd/test/ueatmerit The cells are shown diagramatically in Fig 7. Within the body, the lymphocyte is in a dtffferentiated state G*. These cells are then acti vated or transformed into a division cycle with phytohemsgglutinin. Sasaki and Norman (8) have shown thst the small lymphocyte finishes its transformation in 24 to 48 hours after stimu lation and begins a period of cell proliferation with a generation time of 22 hours; Gi, S, Gr, and mitosis are 6, 11,3, and 2 hours, respectively. We treated these cells with Aroclor 1254 at 100 ppm for the first 24 boure and again during the last eight (S plus G>), and the last three hours (mitosis). During the first 24 hours, the cells would be in some stage between G and Gi The cells were then examined cytdogically for chromo some aberrations for the various stage treatments The results are shown in Table 2. PCBs seem to have no effect on chromosomes under these oondltloca. 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 es one decreases the percentage of chlorine. The distillation product 1016, however, was more toxic to these cells than was indicated from the percent chlorine content cr from its gas chromatographic patten* At 100 ppm of tpril 1972 1S7 HONS 081456 ----- --fiim 1 1254 to human lymphocyte cuIIiiict, thcie was no nppHicnt effect to the duornosome integrity ns mensiucil by cytologicnl evidence Tissue cultiuc systems may not always be indicative of whole body conditions Houevei, these systems do odor some untfoicn conditions foi many definitive studies of emit ortmcnlal chciiticols With substantial data foi comparative tents they niay be good indicator systems The human lymphocytes system also offers the compjuuiivc eytological test for both in vivo 01 in vitro exposure REFERENCES 1 Vosumura, Y, Til'll ]Min A II and Sato, G H I9G6 Establishment o( four functional, rlonal strains of unmet cells rn culture Science 154 1186. 2 Eagle, H 1966. Metaholic controls in cultured rnnmmslinn cells Science MS Z42 3n Gabiiha. J 1965 Response of cell cultures to insecti cides. It. Chronic toxicity snd induced resistance Prae. Soe Exp Biot Med. 120.>68 I. f.shlit- J I'll,-. Ill tliad Lr | i j i tini>-\wl <liiflilhrriu I'lviri Pi.k* v* j X(r |\i ' , l-l I7J < <s.5ihV't J uni innimiri la 1`lfj'j Kf|irn f\ ililturc* to uiM<hri<le I Vuilc <om< iu * . '. ir, , Cll- Piu< Xio latj* Mji.J MtJ I Jfl IM d l.atrri,C L unit 1 i'hCrnsIctn 1 V 1*171 f Ti" and inlerjrtinns or cUs ircmmeM l t il- wi Vji i t-t cells in tissue culture \rrh Lriuruh FJeii*!. 4>l t llotpngijrr>er. U md Mourner, \ W lOTn L irjiu >- eyte chromosome ntvdjsi*! o( prsltrale ct|uwu m rtmcliinls \ 11 (hi Con* PI mt Pruteifmn l'ir 4 Samuel, 4 lt>7l Effect of nmhtlnon o yrVttd and mantmrduia u-wue cultures IT\ t> Mr.n^in State University 5 Nowell, P Cc lOGO PhjtohnenviKgluUnm jm i. ,t t|.ir of mitosis in cultures of normal human leu Cancer Res ?0 4G2 8 Moorehesd, P S et aI 1960 Chromosome prrpiraitons of leucocytes cultured (com human jienphernl liJut'd Exp Cell Res 20 6J3 7. Krause, D If 1970 1 heeytolofcic.nl effects of nfstirnlr-, on human chromosome* M S Tries*, Michigut Si it? University 8 Sasaki. M S end Norman, A i960 Proliferation of humtn lymphocytes tn culture Nature 210 Xr2 15ft Environmental Health Perspei lives HONS 061437 Environment*! Hmith fVi^frU Toxicities of PCBs to Fish and Environmental Residues by David L. Stalling and Foster Lee Mayer, Jr.* Polychlorinated biphenyls (PCBs) have been found in fish and wildlife from many parte of the world at levels which may adversely affect aquatic organisms and interfere with pesticide residue analyses (1, 2, 3, 4). The environmental occurrence, uses, and present toxicological aspects of PCBs were recently reviewed by Peakatt and Lincer (5), Gustaffson (0). and Risebrough (7). PCBs havo gas chromatographic charac teristics similar to many orgsnochlorine insecti cides. Jensen (S) first identified unknown gae chromatographic peaks as PCBs in extracts of pike and an eagle which were analysed for organoclilorine insecticides. The Monsanto Company is the sole manu facturer of PCBs In the United States (6) and markets eight formulations of chlorinated bi phenyls under the trademarks Aroclor 1221, 1232, 1242, 1248, 1234, 1260, 1202, and 1268. The latter two digits designate the percent chlorine of each formulation. Aroclor 1248 and 1254 ere the materials produced in greatest quantities and are used as a dielectric fluid in capacitors and in closed-system heat exchangers (9) Aroclor 1242 is used as a hydraulic fluid, and Aroclor 1260, as a plasticiser. Chlorinated lerphenyls are marketed under the trademark Aroclor 5442 and 5460. A mixture of hi- and icrphcnyla is designated Aroclor 4405, The Monsanto Company has restricted the sale of PCBs (or uses in which disposal of the end pro ducts cannot be controlled, such u the use of PCBs $ plasticizers (6). * Chemist and Fishery Blolofis), respectively, Fish* 1>'| ickIc Heacarrh laboratory, Bureau of Sport Fiahtnea "id W ildlrfe, Columbia. Missouri 85201. Chemical Composition and Analysis PCBs occur in the aquatic environment as mixtures of chlorinated biphenyl isomers (10, II). The biphenyl structure may he substituted with one to ten chlorine atoms, and over 200 com pound* are possible (6). The isomer composition snd chromatographic characteristics of each formulation have been described by Bulling and Huckins (12) and BagLey (13) The PCBt can cause serious interference us 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 lor the analysis of mixtures of FCB formulations in environmental camples. The solubility of these formulations in water have not been precisely determined but is reported in the range of 100-1000 pg/1 (9). Considerable research remains to be done on ths structure of the ieomera of each technical material and how environmental residues of various Aroclora are to be reported. byinnmnUi PCE Residm Analyse* of 40 fish (cross-check sample) from the 1970 National Pesticide Monitoring Program were made using sample preparation techniques which separated PCBs from pesticides snd other industrial residues (Table 1). These analyses employed GC and gas chromatography-mass spectrometry (GC-MS) for confirmation. The samples were prepared using gel permeation chromatography (23), and PCBs were separated from the pesticides with silicic acid (It) Only tpril 1972 159 i t I 1 HONS 081^58 Table ! Companion of PCB realduea In iclectcd fih eatnpkf from tbo 1970 Notional Peiiicide fteatdue Monitoring Program. Kivor Location Species PCB residue ai Aroclor type (g/g whole U/dv) 1232 1248 1251 1260 total Ohio Ohio Ohio Ohio Yasoo Hudson Alleghany Delaware Capa Fwf Lake Oolsno MkMmippi Merrunse Cincinnati, 0 Cincinnati, O Marietta, O. Marietta, O Redwood, Mu*. Poughkeepsie, N,Y. Natrona, P Camden, N J libetb Town, N CPort Ontario. N,Y. Memphis. Teoo Lowell, M**t Carp White crappie Channel catfiah Channel catfish Smallmouth buffalo Goldfish Walleye White perch Giaaard abad Whit* perch Drum 10 2 15 38 15 B 72 8B -- -- 18 9 13 9 11 2 13 8 75 17 23 53 -- 173 53 80 -- -- -- 76 42 27 11 12 6 14 32 25 68 26 46 45 61 80 50 49 46 -- -- 4S 39 11 12 34 32 131 60 77 33 73 213 35 19 23 19 19 98 one of tho forty temples contained less then I pg/g PCB reeidue. The composition of PCB residue* in a lake trout and coho salmon were examined by GC-MS and found to be a mixture of Aroclor 1348 and 1354 (Figure 1), and residues of PCBe were in excess of the "DDT'' reeidue level* (Table 2). Widespread pollution of the major waterway* hoe occurred, end appreciable PCB residues exist in fish. In view of the PCB bioconcentration in fish, PCB levels as high a* 5 pg/1 may exist in the waters of the Hudson River. These levels are certain to have adverse effect* on aquatic organisms Toifcitjr and BtoaccuRulatlon Fish Chronic and acute toxicity studies on aquatic organism* are quite limited. Due to the low solubility of PCB*, testa to obtain 06-hr LC,. values do not adequately reAect their toxicities to fish (Table 3). For Aroclor 1321-1268, 96-hr LCi* value* ranged from 1,170 to 50,000 pg/l for cutthroat trout (15). The acute oral toxicity of Aiodors 1242, 1248, 1254, and 1360 us* greater than 1500 mg/kg in rainbow trout. Intermittent-flow bioassaya of Aroclor 1212. 1248, and 1254 to bluegills resulted in 15-day TubIt S. rmlclde and PCB Rttldutt In a Cobe Salmon and a Lake Trout from Lake Michigan. Residue* in *c/g whole fish DiaUrin p,p` woman DDE DDD DDT o,p' aomtn Total PCB (Aroclor)1 ------------------------------ DDT'---------------------------------- ------ DDE DDT 1248 1354 Total Laka trout* Coho salmon* 0.10 0.10 13.0 4.8 1.8 08 53 3.8 0- 1.8 0.4 0-5 23.7 83 13 0 67 lB 0 67 28 0 13 4 * Calculated from ratio* of characteristic 124$ and 1254 petit*. Lake trout (33 L)--taken 10-14-69. 690 mm, 3590 gm, immature M Taksn In 3 6 fathoms in Little Bijr d Noe. M icbigso. 1 Coho salmon (7Ca)--Ukan 9-3-69. 675 mm, 5718 gta F Tskaa in 17-19 fathom* 4 mils* NW of Manistee, Michigan 160 Environmental Health Perspective* , Ci, \lucs of 54, 76 and 204 pg/1, respectively 4)- Exposures to channel catfish gave l.'-diy LCm 'clues ol 107, 127, and 741 pg/1 for ,h( tame Arodors. All LC values decreased r.pmficandy with longer exposures The 20-day LC. i slues for Aroclora 124S, 1254, and 1260 pert 10, 135, and 245 sg/1 for bluegills and 300, xrid 1NG pg/l for Aroclora 1254 and 1260 with riianud catfish at 20*C. The toxicity of Arodor i_'4$ to bluegills and channel catfish increaaed i co-fold at 27*C The toxicity of the PCBs to Sth is inversely related to percent chlorine (higher treelor number). Id chronic testa to determine the effects of tree lor 1242 and 1254 on fathead minnow repro duction, all fish exposed to greater than 83 pg/l Table 3. Acute loikity' of sexrsl Aroclors to (elected Geliei, Aroclor Species Tempers ture 90-hr LCm (C) (xS/1) 1221 1232 1242 1248 1284 1260 1202 1268 1248 1254 1248 1234 Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Cutthroat trout Channel catfilth Channel catfish Blufgilli Bluegiilt 8 9* 89 89 89 89 89 89 89 18 3` 18 3 18 3* 18 3 1.170 2.500 5.430 5.750 42,500 60,900 50,000 50.000 6,000 12,000 278 2,740 1 Stitie bnaauy 1 Alkeliaity, 159 ppm; pH 7 8 1 Alkaiioity, 35 ppm; pH 7 1 IiccrxI Cotnpoii]gQofPCBrwduMin6ah-Atwwiand tronglei designate the moat chanetarietie paak t of the Arodor. All minilierod peaks la chtomatograms of foh extracts are PCBs. Them numbers design*te the number of chlorine atoms la tha compounds as da. (eraiu.ed by mass spectrometry. Temperature pro- *TM r.te, 4`C/mtn. Hs 8 pst|. W'XO.MO" JOSCOToolumn, FE-270SGC-M& of each Aroclor died. Reproduction occurred at and below 8.3 pg/l Aroclor 1242 and at and below 2.8 peg/t Aroclor 1254 (17). Accumulation of Aroclor 1248 and 1234 by bluegiUs chronically exposed to 2-10 pg/l was from 26,300 to 71,400 times the exposure levels for both PCBs Concentration factors were not strongly dependent upon water exposure levels, but a direct correlation of water concentrations end whole-body residues may exist. No major modification id the PCB isomer ratios was ob served in the tissue residues, and no new com ponents were identified (18). Coho salmon fed Aroclor 1234 for 240 day* at concentrations of 14 5 to 14,500 pg/kg body weight per day (dietary concentration of 0.4 to 580 rt/g) accumulated whole-body residues which wen 00 to 05 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 days1 ex posure, and thyroid activity was stimulated in all but the lowest treated group (19) Direct water exposures appear to represent a greater hesard 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 i i 1> 1 1 T*M< 4 tnifnmtlnl'Oi`V or Aro*lnr Agtlnil ihrte flaiiei ' A rot* tor 4^;>eeies 6 dtt\b 12W iroo miT 1242 1248 1254 1200 1242 1248 1254 1200 1248* 1248* R/unbow trout IMutgitla Cli a nnei catfish BluegjJJi Channel catfish 350 -- 2 26 i.r< 307 -- -- -- -- -- -- 137 -- * Teropralur. 20'C. aiiuliaiiy, 260, pH. 7 4 1 Ttmpwuture, 27*C fO rtftj i 8 240 0 67 72 tao 443 -- 174 225 -- -- 76 94 LCm Ur/1) f6 diys _ M 0 2G 54 70 204 -- 107 127 741 -- 57 20 da>a _ 21 -- -- 10 135 345 -- -- 300 296 -- -- 33 day* _ -- -- -- -- 54 212 -- -- 113 166 -- -- 30 dim _ _ -- -- -- -- IM -- -- -- 137 -- -- sediment* and PCDs can readily enter food chains (20, 21). LCia value of 3 yg/1 compared to a 5-day LCi, of 1 pg/1 for DDT (22) Aqunlie Invertebrates DapAma majna Aroclor 1248 is the most toxic of the Aroclor senes to Dophnta, and pre liminary studies indicate that levels above 5 Mg/1 arc not safe for reproduction (17). After 48 hour*1 exposure to 300 pg/1, Dnphnta concen trated Aroclor Plot by 48,000 times (22). Oammorvs pstvrlolmnaeus; Hie level of Aroclor 1248 that did not affect reproduction was com parable to that for daphnids, ca 5 wg/1 (17), The 9G-hr I.Ctt values for Atocior 1248 and 1254 were 52^ig/l and 2,400pg/1, respectively (Table 5). Send were more sensitive to ArocJot.1242 (00-lir 1/Cw-10 itg/1). After exposing another sfiecics of scud (Gaminarus /otcutfus) to 1.9 pg/1 Aroclor 1254 for 14 days, the 1'CBs were con centrated 27,000 tunes the exposure level. No further increase in l'CB residue resulted after an additional 21 days of exposure (22) Orconttltt mi$ Crayfish were more susceptible to Aroclor 1242 (7-day LCm-30 *jtg/l) than they were to Aroclor 1254 (7 day LCu80 yg/1). PCI) residues in crayfish did not reach equilibrium after a 2fUfay exposure to Aroclor 1254, and the uptake was linear during this period (22) Palaeninrchi kothakentn. Class shrimp were very sensitive to Aroclor 1254, having a 7-day Marine Organism* Immature pink ehnmp were extremely sensitive to Aroclor 1254 exposure* Fifty-one percent oi the ehnmp died within 15 days when continu ously exposed to 0.94 pg/1 Aroclor 1254 (25) Mortalities in two estuarine fishes (Lagodon rhombotdes and Lctosiomns zanthunu) exposed for 14 to 45 days to Arodor 1234 were obeen e<j at 5 (ig/1. Fish mortalities were not observed at 1 yg/1 during the same exposute time (24) PCD concentration factors in these fish were similar to uptake by freshwater fish (1-5X10* times the exposure levels) Correlation of Reproduction and PCI) Residues In preliminary investigations, Jensen et al (25) reported a possible relationship between PCI) lesidues m salmon eggs and egg mortality in Sweden. On a fat basts, PCI) residues m eggs ranged from 7 7 to 34 ng/g and mortality ranged from 16 to 100 percent. A regression analviis of their data resulted m a coefficient of corn-la lion of 0.S5, and the correlation was significant at l1 001. 162 Environmental Health Perspectives MCJVS 081*62 TuJ>]p 5 Arorlor And 1)D1 tunirrl) io in>prlp!>ra)c* t> -- A 1 1 Compound Or(H nism* BlO|t.S8> t \ l*CJ l'\l>osure C(li\ s) 1 1242 A *s^!nf 1 m f>4 S'tKlnr |2ol 'l'l Vrvl.T 1212 Irxlvr 1-'I2 mwW 124^ A F*clor 12.^4 PUT Pill AncTof 1251 nr i'in I'sclof 1242 Anvlhr l?34 1-cti.r 12-12 oiflfif 3254 ,'i>r 0\fWl Cm) fish Cf jj fish Crnyfoh Semi Scud Scud Scud $rd Sr ud CillM AtlDYnp ihnmp Glass shrimp Dragonfly Drasonfly Datnsclfly IJimscIflv Dimaelfly MMlC Malic conlinuou5-l1ow Bl^VlC conlinnouvflou continuous flo* BtfttlC static klahC conlirmouS'flon rpntinuoua-flon stfttrc con(MKus*flon static BlA(l contmuoui-flo* continuous* flow static 7 to 7 100 7 so 4 100 4 10 10 5 n 4 52 4 2,400 4 32 5 00 7 30 5 10 5 13 7 aoo 7 1,000 4 400 i 200 4 50 ' Ctav fi*h (Oreovrcfei iwu), Scud ((faytruanu /mcmOm), G1a ihfimp {Pahemvnctes kodurktuni*), Dragonflj (Kfo* 14 rp ), (/*eAr teKu-alw) 1 TVnipcrfltdrc, 15 0`C; aikilinrty, 35 ppio; pll, 7 3. ^ummAry The occurrence of High levels of PCD residues aquatic organisms (fish and invertebrates) as <simji|p'{ mixtures of Aroclor formulations has 'vn determined by GC analysis and confirmed GO-MS Suitable methods for sample cleanup iriudc gel permeation and silicic acid column r iromalogriphy. Agreement on methods for rv;x>n mg PCS residues is lacking. The highest concentrations.of PCB residues in itahwatei fish occur in rivers which are asso rted with industrialised areas, i e. Hudson liner (ca 200 jig/g vihole body residue) and the t'aio and Allegheny Rivera (ca 100 ag/g). Residue *" i*l? associated with these rivers approach i'Clt residues hieh were associated with fish iiiwtahtie* in chrome continuous-flow exposures ng/g). Concentration of PCBa by fish is greater than '"(*10 times the exposure lotels. Similar conccn "siion factors have been observed for inteiUi Adverse effects on reproduction of aquatic Tlsui-ms |i>o) OCCUI at PCB COllCOlltrations of 's I or 1cm. l'uithcr studies aic required to determine more definitely the exposure levels which do not adversely effect aquatic organisms The problem of PCB residues in mdustralizcd areas can be greatly improved hy close monitor ing of effluents and by identification of (hose PCB uses which contribute to effluent contamina tion Elimination of widespread PCB residues in fish at concentrations of 1-20 pg/g mil require greater efforts, such as the control of RGBs from sen age treatment plants and other as yet un identified sources of PCBs into the aquatic environment. Belated Materials Chlorinated napthalenes aic recommended for uses similar to those of the PCBs (27), These materials are recovered by the same aiiahliCftl procedures as the PCBs (2S) and may i>c recog nized by then gas chronmtogiaphir elution pat terns if PCBs arc not present- The materials air designated Halouax 1000, 1031, 1099, 1013, 101*, and 10A1 and arr listed in an older of incieasmg diloiinv content (27) Aquiilit (o\iu1y information is not piesently ft' jilable, nor has Vi! 1972 163 HONS 061.462 tho persistence and fiite of these materials been examined in the aquatic environment ltlFEHE!SC'E5 t Jcii'rn. S et id 1069 DDT nnd PCI) rn marine amriiAh frtfrn Swednh ntani Nature 224 247 2 Holmes, D C , Sunmum, J H amt 'Initon, J (VQ 1907. Chlorinated hydrocarbon* in British wildlife Nntnro 210 227 3 Koetnan, J fl , Ten Soever de Bran*, M, C and Vos, K 1) do HKj'i Chic rrnated biphenjls in fish, mussels, and birds fiom the ttiver Rhine and the Netherlands consul area Nature 221 1126 4 Kimbrough, R VV et al 19C8 Polychlorinated bi phenyls in the global ccosyitem Nature 220 1098 6 Peak a II, D 1) and Lmcer, J L 1970 Polychlorinated biphenyls another long-life widespread chemical in the environment LhaSaence 20 958 6 Qustafsqn, C O 1970. PCbs--prevalent end per sistent intensified research is needed to minimise their dangers Environ Sci Tech. 4 814 7 Rrsetarough. K 1970 More letters m the world Environ 12 16 8 Jensen, S. I960 Report of a new chemical hasard. New ScionLutt 32 612 0 Pniiageorga, W. B. 1070, PC Be in the Environment, preterit in pint at a meeting sponsored by the National Water Quality Laboratory, FWQA, Duluth, Minne sota, March 17, 1970 (Monsanto Company). 10 Stalling, D L. and Johnson, J. L 1971 Unpublished data from laboratory eroea-eheek analytes from the 1070 National Pesticide Monitoring Program, FhPcntK'ide Research Laboratory, 11 Stalling, D L (In pme), Analysis of Organoehlorioe Rnstdnea in Fish--Current Research at the Fhl'entiade Hosearch laboratory, Conference on Pesti cide Chemistry, February, 1971, Tel Aviv, Israel 12, Stalling, D L, nnd Huckins, J N. 1971 GC-MS character!Ml ion rf PCBi (Aroelore) and <*Ct labeling of Aroclor 12IS and 1254 J Assoc Off Anal. Chem 64 SOI. 13 Itajcloy, G K, Rachel, W L. and Cromartie, E. 1979, Identification of polychlorinated biphenyl* in two bakl eagles by combined fia-liquld chromatography mate t|>eciromttry J Awoe. Off Anal Chem 83. 251. 14. Armour, J end Burke, J 1970. Metliod for separating pofjrhlonmtctl taphernls from DDT m Us ambit* J Assoc Off An.il Chem 5t 7fil 13 Swedlirrg, D H70 bpcenf rrp<>rt nti P(,|U fn IhtTcnu of bport hishenue nnd Wrhllife, Program in Sport >nhery Research, Resource Tub. L' S Covt Print Off {In pres*) 16 Meyer, F L ibid 17 Nebeker, A 1971 Unpublished Onta NalmniiMt ater Quality LabarnUir}, EPA, Pultiih, Mimi^ola 18 Stalling, D L nnd Hnclona, J N' Unpublished (l,(n Fish-Pwiicwle Kf>virrh Ijilroratory, Cohnil>i Missouri 19 Mchrle. P M and Grant, H UnpnbiKhnl tbu Fab-Pcelicide Research laboratory, Columbia, Missouri 20 Duke, T W. Lowe, J I and Wdaon, A J 1970 Vertebrate insecticide resistance the in vitro eudnn effect on succinic dehydrogenase activity on endrm* resistant snd susceptible moaqnrtofish Bull Environ Contomin. Toxicol 5 171 21, Nimmo, D, R* et at 1971 Polychlorinated biphenvl absorbed from sedimenU by fiddler crabs and pink shrimp Nature 231 50. 22 Sanders, H. O 1970 Special Report on PCBs In Progress in Sport Fishery Research, Bureau of Sport Fisheries and Wildlife, Resource Publication, USGPO (Id press). 23 Stslhng, D L , Tindle, R. C and Johnwii, J L (In press). Pesticide and PCB eleanup by gel permeation chromatography J Assoc Off Anal Chem 24. Hansen, D J. etal. 1971 Chrome toxicity, uptake, amt retention of Aroclor 1234 in two estaunne fishes Dull Environ Contamm Toxicol 6 113. 25. Duke, T W, 1971. Unpublished data Gulf Breeze Laboratory, F4ivif0ttnratal Protection Agency, Gulf Breeze, Florida 20 Jensen, 3., Johansson, N and Olsson, M 1970 PC3ladications of Effects on Snlmon PCB Conference. Stockholm, September 29, 1070 Swedish Salmon Research Institute-Report LF1 ME>D 7/1970 27 Melons* Chlorinated Naphthalenes, Technical Bul letin, Kop|ierv Co, Inc, Tar Product* Division. Pittsburgh, Peniieylvania 26. Armour. J. A. and Burke, J A 197) behavior of chlorinated naphthalenes in analytical methods for organoehtonne pest miles and polychlorinated bi phenyls J. Aaaoc Off. Anal Chem 54 175. 164 Environmental Health Perspective# MCNS 081463 Environmt/Uei JftoJlh Fr*,ip+lCSlt<fi The Sensitivity of Fish ATPases to Polychlorinated Biphenyls* by L. K. Cutkomp, H. H. Yap, D. Desaiah, and R, B. Kochf In spile ol widespread concern about the pos . nlc hazards of polychlorinated biphenyls (PCBs), ;;1p if knotwi about tlicaction of these materials. Uc ]in\c therefore examined the m vitro (I, 2) fid in ' ivo (3) cITeeta ol PCBs on the adenosine i|)lios|ilmtac (ATPase) enzyme system. We iji\c found some effects tvhich are similar, yet e, to chemically related compounds of [he DDT type. A background of earlier research mill DDT and closely related chemicals has Kioaii the greatest inhibition on .\fg,+ATPase and less on Na+-K+ATPaso (4-7) Subsequent iwcsrch showed that mitochondrial Mg*vATPase of fish and insects was most sensitive to DDT x) The results from testing several PCBs on die ATPase ensyme system are presented m this manuscript. Matemis ind Method* Tissues from bluegil! fish, Lepomia mochtocAinu, were dissected, homogenised, and fracuoealed by centrifuging at i 3,000 Xg for 20 min ute, and the sediments were resuspended in 0.32 M sucrose, 1 mM EDTA, ud 10 mM imidazole. Die fraction obtained (B) contained mitochon drial and nerve ending particles. Each preparaHon uaa appropriately diluted and the samples "rc quick frosea in Liquid, nitrogen and stored M - 20*C. until the ATPisa assay ATPase l'r,)irrd l,y the Department of Entomology, F,henei viii Wildlife, University of MiniinoU, 6t. Paul, MimieH-la 55101 a review and condensation of poMirsUonv (1). '-).iid(3). t Slaff Rrienti-t, 11 nrf-yv ell Corporate He-earrli Center. hopUm, MmiiCHiU 553-I3. activity was determined using the enzymatic or continuous procedure in which catalysts pyruvate kinase and lactic dehydrogenase itcio presrnt, and KADII (reduced nicotinamide adenine dinuclcotide phosphate) is oxidized to XAD (nico tinamide adonine dmuclcotide phosphate) for spedrophotometric determination at 340 nm The procedure is detailed in Pullman ct al (9), and Frits and Hamrick (10), and in Yap and Cutkomp (II). Mg,+ATPase activity was meas ured when one mM ouabain waa in the reaction mixture Ouabain is a cardiac glycoside which specifically inhibits the Na+-I\+ATPasc Mgt+ATPase was further separated into ohgomyemsensitive (mitochondrial) and oligomycm-insensitive portions by adding 003 rig otigomycro (oiigomyein A 15%, B 85%) per ml reaction mixture. Oiigomyein insensitive Mg,+ATPae is generally more prominent in mierosomes (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 terphenyi, Aroclor 5460 were investigated for effects on the ATPase systems Arodora 1221, 1242 and 1254 wore dissolved m ethanol, and Arodors 1268 and 5460, in acetone, and added to the reaction mixture by slowly releasing the solution from a Hamilton micro syringe 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, Head ion temperature was 37"C, except for muscle homogenates, where the temperature was maintained at 27C The amount of solvent added (never exceeding 5 d) with the PCBs had no effect on tho ATPase April 1972 165 HONS 081464 Fkuihe l Arodor 1242 inhibition (Probit Units) of ollgo* tnyon--insensitive Mg1* ATPue from fkah bomogeoetei, ATPtse aclWiiy of untreated jo ^moles Pi mg'** protsm hr"1 for brsin 14 6*0 03; kidney 151 0 02; liver 104406; muscle 4G 29 6 Unbroken lines computed; dotted line connects ectusl points. activities. Four tissues of fish (brain, muscle, liver end kidney) were selected u eniyme sources. Results and Dlsessit Those PCBe tested in vitro were generally most effective as inhibitors of oligomycin-insensitive Mgs+ATPa*e, particularly from muscle homogenates. Arodors 1242 and 1254, which Floras S. Arodor 1221 inhibition (Probtt Units) ol oligom> tin insensitive M|l+ AXP.se from fiih honottton ATPm. activity of untreated In nmoles P, m|-1 protein hr< for brain 14 2*0 4, kidney 17.3*30, liver 107* 0 07; muscle *3,7JJ are in the intermediate range of chlorination, were more effective than 1221 and 1268 Com pared at 50% inhibition, the values ttere 06 ppm for Arodor 1242 (Fig. 1), 1.2 ppm for Arodor 1254 (Fig. 2) and 2 3 ppm for Arodor 1221 (Fig. 3), the smallest value indicating the most effec tive. Mg,+ATPaae from mitochondria (obgomycin-aensitive) was not affected as much by the PCBs as bjr DDT-type compounds The 1,, for Arodor 1242 was 2.6 ppm on mitochondrial Mg**ATPsse from muscle, 4 0 ppm from kidney (Table 1 and ref. 2) and 3.5 ppm from brain Table 1. Fernet inhibition of mitochondrial Mg*' Alpaca activity by AROCLOR 1242 in fish kidnsy and eauaela homogenates. Concentration (ppm) * Pit cent mbibitioo* JCidosy Muscle Floras 2. Arodor 1234 inhibition (Probit Units) of olifomytlft--insensitive Ml1'* ATPiseftom fish homogenates. ATI's*. activity of unlrtated in .molts Hi mi'1 proton hi-1 for brsin 14.30 4; kidney 17 34:3.0; liver 10.0* 0 03, muxk 63 7 *31. 0.3 1.0 20 40 ao 16.0 + 13 7 + 10 4 17 4 2 0 M2 06 0 +26 5 25 9 67 S 69 4 77 9 76 6 Uattested 6p. Act*8.E 2) 2 *2.5 56 *1 1 * {+ ) Values represent the per cent inijme scliv.tiun 166 Environmental Health Perspective* Table 2. Fifty percent inhibition valued (in ppm) of ATPases determined from homogenates of blue gill fih brain* Summarized from references 2 and 8. (Lower values denote greater effectiveness.) ATPases In, vitro tests DDT Aroclor 1242 *T WTOCMHMHI. MS ret nit Total Mb*'*' Mitochondrial MgJ+ Olivomycin- Insensitive Mg,+ Xa+-K+ 15.0 0.5 >7,5 (30%) >15.0 (30%) 8.3 3.5-5.6 (max. 64%) 10.8 18.5 COHCCMTt-TIVN (rr> I rth UWMUftlL Mt* ITP* Kl 111! ' COHCIHTIATIOM (PtM) Figcbe 4. Arcelor 1242 inhibition (Probit Units) of mito chondrial Mg,+ ATPase from fish homogenates. ATPase activity of untreated in mmoles Pj mg-1 protein hr"1 for brain 13.21.1; liver 29.ldr0.004. Unbroken lines com puted; dotted line connects actual points. Table 3. Percent inhibition of ATfases in homogenates of mitochondrial fraction of kidney of 4-month Aroclor 1242 treated fat-head minnows ATPases ppb of 1242 Mg,+ ATPase Mitochondrial Oligomycininsensitive Na+-K+ ATPase 0.9 56 23 21 2.8 75 21 36 S.3 45 35 IS Figure 5. Aroclor 1254 inhibition (Probit Units) of mito chondrial Mg,+ ATPase from fish homogenates. ATPase activity of untreated in mmoles Ps mg"1 protein hr"1 for brain 13.50.35; kidney 28.60.85; liver 39.01.25. (Fig. 3). The IB0 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 Mg2+ATPase. Aroclor 1242 is compared with DDT in Table 2, showing this PCB to be a better inhibitor of Na+-K+ATPase, but poorer on mitochondrial Mgs+ATPase. The in vivo studies were conducted with fat head minnows, Pimepkales promelas, which were chronically exposed to Aroclor 1242 and 1254 for four months following hatching. Comparisons of ATPase activity showed considerable inhibi tion and some stimulation over a concentration range in which fish were exposed to 0.31, 0.93, 2.8 and 8.3 ppb. Aroclor 1242 compared with Aroclor 1254 showed the greatest inhibition of Mg2+ATPases, as it had in in vitro studies. The inhibition in the chronic in vivo study was great est on mitochondrial Mgs+ATPase 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 oligomycin-insensitive April 1972 167 MONS 081466 ... ...............:-. - | ' , Mg'^ATPase which occurred m vitro. A maxi Chronical!} exposed fish ere obtained Iron mum inhibition by Aroclor 1242 in tieated fish experiments irom the Kationnl Witter Qualm (in vivo) ivos 75% on mitochondrial Mg'*ATPase Lnb , linvironmcnlol Protection Agenc), Duluth and 35% on NaMCATPftse In contrast, Aroclor Mmu through I lie courtesy of Dr Alan NMxhir 1254 did not inhibit mitochondrial Mg,+ATPase Monsanto Company kindly .supplied tlic Arodur in kidney but gave 30% NaM^ATPnse inhibi preparations tion in the group of fish treated '\ ith 8 3 ppb (3) Result* from brain tissues showed more consistent inhibitory effect* than did other KEFERf/NCES tissues. Overall, PCB-treated fish showed more varia 1 Y*[>, H H et al 1071 Senailivily of fish ATI'asesio pot}chlorinated hiphenvl* Mature 733 SI tion of enzymic effects than in vitro determina 2 Desuiah, D at ] 1072 Inhibition of oligomycin- tions. Thus, although the precision of the results ms* greater in vitro than in vivo, both approaches seem desirable for assessing the mode of action. In both cases the greatest effects were on Mg5+- ensilivt and insensitive magnesium edecovnn tr, phosphalaaa activity in fish (Polychlorinated Hi phenyls) Sioehem Pharmacol 2] (In press) 2. Koch, R B. 1972. Polychlorinated biphenyta rHwi of long-term tapoaure on ATPase activity in fish ATPase, with lesser effects on Na+-K+ATPase. PunapAoles premetas Bull Environ Con la mm Ton- Of the Aroclors studied, those in the intermediate range of chlorination appeared to have the great est inhibitory effect on the ATPase system. Aroclor 1242 was a particularly good inhibitor. eel 7- (In press), 4. Koch, R. B at al. IMS. Chlorinated hydrocarbon inaectlcidea: inhibition of rabbit bnin ATFiae actintiaa. J, Neurochem 10. 209 0. Koch, R. B., Cutkomp, L. K and Do, F M 1000 Chlorinated hydrocarbon insecticide inhibition rtf Ciacluslmt The ATPase ensyme system in fish is adversely affected by all PCBs studied. Mg'+ATPaae is cockroach and honey baa ATPaaaa Lite Sc i 8(11) 199 0. Koch, X. B. 1909/1970, Inhibition of animal tissue ATPaaa aelivitiee by chlorinated hydrocarbon peaucidaa. Chen-BMof Interactions 1- 199 inhibited to tbe greatest extent in muscle, but effects in kidney, brain, and liver also occurred 7. Cutkomp, L. K. et al. 1971. ATPaaa actinty in Salt tiaauc homogenates and Inhibitory effect* of DDT and Fifty per cent inhibition occurred with less than related compounde Chem-B+olog Interactions 3 439 8. Cutkomp. L. K. at al. 1971 Inhibition of oiigomyem- I ppm up to a few ppm. The greatest effects, in tensitive (Mitochondrial) Mg'* ATPase by DDT asd vitro, were on oligomycin-insensitive Mg*+- selected enalngi in Hah and inaect tissues Life Set ATPase, but on oligomycin-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. Ackncfftodgiratutt Research support by grant of FWPCA, 10(11) 1291. 9Pullman, M, E, at al I960. Partial resolution of the eniymea catalysing oxidativa phosphorylation 1 Purification and properties of soluble dimtropbenelilimulated adenosine triphosphatase. J Biolog Cliem 210' 3322 10. Frits, P. J. and Hamrick. M E 1969 Enzymatic analysis of adenosine triphosphatase Ensymolcgia 80:67. 11. Yap, H. H. and Cutkomp. L K 1970 Activity anil rhythm of ATFaaea in larvae of the mosquito, Af Environmental Protection Agency, 10030 ELZ nsfypfi L. Life Be! 9(11) 1419 168 Environment*! Health Penpeclur* .Ent'tftfftftifnfa! HW(/i /Vipfetiivu Studies on the Mechanism of Toxicity of DDT and Polychlorinated Biphenyls (PCBs): Disruption of Osmoregulation in Marine Fish by W. B. Kinter,* l, S. Merkens.t R. H. Janicki.t and A. M. Guarino$ In 1070 studies were initiated at the Mount Desert Island Biological Laboratory to explore mechanisms of toxicity underlying the high sensitivity of bony fish, teleoat*, to organochlonne pollutant!. The starling point was several reports that DDT |1 ,l,l-lrichloro-2,2-bia (p-chlorophenyl) ethane) inhibited Na,K-ATPaee (IsV, K*-aclivated adenosine triphosphatase), as well as the knowledge that this ensymo appears to play a central role in osmoregulation by marine teleosls. In the face of a desiccative environment, these Hah maintain body fluid hypotonicity (Fig. 1) by drinking sea water, absorbing water coupled with salts across the intestinal epithelium, and eventu ally secreting the NaCt across the gill epithelium a bile retaining the free water. The primary driving mechanism in both intestine and gi0 is the Na pump with which Na,K-ATPaee appears to be intimately involved. For fuller discussion, see (2) and (3). After demonstrating that DDT did, in fact, inhibit Na,K<ATPaae activity in homogenates of intestinal mucosa and gill filaments from several oisnnc teleoata (2), attention was focused on tbft sea a ater-adapted eel (A nguiiio rostrate), in which osmoregulation has been well investigated. This eel can also adapt to fresh water and ean be used for future DDT studies. Studw* conducted at Ml. Sunt Island Biological Laboratory, Salisbury Cove, Mains 04672. t SONY Upstate Medical CmUr, Syrmces, New York 11210. ! To *liom reprint rtqnoU should be sent; Dr. A. M. Gusmw, Bldg, 10, Rm. ONUS, National Cancer Institute, Htlhtadt, Md. 20014. Materiiis md Methods Bela, Anquxtta rostrate, and kilURsh, Fundvlut heterodttta, were captured in estuaries along the Maine coast. They were adapted to and main tained in aea water for about three weeks before use. Non-radioactive punas grade p,p'-DDT was procured from Aldrich Chemical Co., Milwaukee, Wis. ,4C-DDT was obtained from Amersham/ Searle, Arlington Heights, 111. and had a specific activity of 24 mCi/mM. Aroclor 1221 was donated by Monsanto Co., St. Louis, Mo. Plasma roillioemolarity was deter mined by a standard (reeling point depression technique, while Na* and K* were analysed by means of s flame photometer. Levels of "C-DDT were determined u described previously (4). The fractional "Na space of isolated gills was measured according to the procedure of Kamiya (5) where eel gills are incubated for 1 hr at 15* in oxygenated ea water Control gills were incubated with the solvent 0.5% N, N-dimethyIformamide (DMF), and experimental gills were exposed to SO ppm DDT in 0.5% DMF. Gill homogenates were assayed for Na,K-ATPaae activity as described previously (2). Acute toxicity and osmoregulatory studies were conducted in killifiah using a simple static system. For each experiment ten sea water-adapted flab (5 males and 5 females, each weighing about 5 g) were placed in an aluminum or enameled metal container holding 2 liters of sea water and maintained at 14-16* throughout the exposure time In control container* 2 ml of ethanol were added to tho aea water; in experi mental containers Aroclor 1221 (mixture of PCBs SALT WATER TfLfOST Hypo-osmotic lo Medium Table 2. "C-DDT leiels In st* water adapted eel, (MngulUa rotiro(s) .* Ti?*ue " "---- ------ --------------- ----DDT In els, ppm P2ama Cill Out mucota Brain 17 3*3 O 22 3*2 2 8 S*0 8 11 3*1 0 Mean3E for 9 snirash exposed lo 1 ppm of'`C-DDT for 8 hr. risen 1 Water and ash. transport in ft till water teleoet Tj picatly (he miUtonaoh/L ate 1,000 (of fee water end 330 for Rah body fluid* Adopted from (1). with 21% chlorine content) or p,p'*DDT wsi Ant dissolved in the ethanol. These solutions formed * 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 heparinised glass capillary tubes. Whole blood from 3 to 5 Ash was pooled and centrifuged. The 0,05-0.1 ml of serum was diluted, and the oamolarity and concentration of Na and K ions were measured as for the eel. Table B. Tht effect, of OUT on sodium space and Na.K-ATPaaa in gills from ate water adapted cell Idnfullla raerrsta),* Treatment Control DOT, 00 ppm Na tpacsf *- - ATFmo sctivit) f 3kv 0 112*0 010 0 37*0 07 0 102*0 0101 0 23*0 031 * Maa*8E tor IS gill* in each (roup lor Na specs determistUon end 5 duplicate asasysper a roup for ATPau assays. 1 Fractional Na apaee afiar incubation at 15* for 1 hr expraasad is "Na |iU filament to media ratio (emote* Pi/rnf protein X hr i Signiflrantly different (p <0 01) from controls Resells Ability to osmoregulate was followed in indi vidual eels (about 100 g) placed in 15* sea water (2 liters) containing 0.1% ethanol and 1 ppm DDT, a level which is fatal by 10 hr. In Table 1 it ia seen that, at 8 hr, plasma nuUiosmolarity of Table I. tffwl of p,p'-DDT on plasms osmolality, odium and potassium to ns water adapted eels {AnguUU rostra(el.* TmtaMot Otmolanty Ns* K+ (mo*rnolg./X) (mEq/i.) (mEg/L) Control p.p'-DDT U8*l0 t2mi 143*3 3 4*0.3 133 ASt 5 4*0 It Man*SE far 3-0 siumsts exposed to 1 ppm of DDT for S hr. f Significantly different (P<001) from control*. Table S. Summary of DDT effects on osmoregulation In sea water cels. Study Results Cut Ciill In tin at 0 hr 7 ppm DDT (1 ppm in Plasma molarity water)* In ntratSOppm H,0 absorptioO in media) from sacs ds- craasod 47%| Romossasts Na.X-ATPaaa (80 ppm) Inhibited 43%t 33 ppm DDT mcressad 39% Na content of isolated (ills increased 45% Na, K-ATPase inhibited 38% * DDT solubilised with ethanol (0 1% in sea water) or NiN-dunethylfunnamide (0.5% m media and t% <ft homogenatr) | Calculated from ref 3. 170 Environmental Health Perspective* HONS 08144*9 TiW 5. Acuta tonicity of Arotlot Ull and DDT in hitliSab. Compound ltuUal level* in c* water Total number Control (ethanol) Aroclor 1221 DDT 7 $ pprn 25 ppm 7$ ppm 0 025 pptt 0 075 ppm 0 25 ppm 0 75 ppm * Sac deecnplion ol static system in text. 10 to 10 JO 10 10 10 10 Day 1 0 0 50 88 0 40 80 100 7c Deid (euinulttive) Dsv 2 Dty 3 00 00 80 80 ee 98 00 50 50 80 90 100 100 Day 4 Q 0 80 100 0 50 90 100 DDT.treeled fish had increased about 30% com pared to ethanol control* Furthermore, in thii table one can ec that, while much oF thia increase ass due to plasma Na, plasma K alto doubled in these animals. In additional experiments with 1 ppm llC-DDT, the fish had taken up about half the radioactivity by 0 hr. The level* of radio activity for some tissues are indicated in Table 2 and ranged from 7-22 ppm. Using isolated eel gilla the fractional "Na apace was found to be increased significantly (Table 3) by 50 ppm DDT. Data in Table 3 also abowa that Na.K-ATPaae activity was significantly inhibited (38%) by 50 ppm DDT. Considered all together (Tables 1, 2, and 3 and summarized in Table 4), our eel data provide circumstantial evidence that disruption of osmo regulation is a primary toxio effect of DDT, t.s, the DDT levels in gut and gill of fatally-dosed fish were high enough to have affected osmoregulatory Ns transport under in vitro conditions. Both water absorption from gut tact and Na content of isolated gilla are directly dependent on Ns pump activity and can be altered similarly by obtaining a highly specific Na,K-ATPase inhibitor. More over, from the published (3) doee-response curve for gut homogenate, 7 ppm DDT in mucosal tissue should still inhibit X*,K-ATPase activity Tabls 4. Effect* of Aroclor 1121 and DDT on sarum eamolarity, N't and K In lillllfiab. Compound and initial level meemoie/litar Na* mEq/Uter K* mEq/liter Untreated S-litvr Kpotm Control Arodor 1221 7$ pptn DDT 0 25 ppm H-llevr exposare Control Aroclor 1221 25 pptn 75 pptn DDT 0 075 ppm 353 2 (18) 888 3 <*) 898 *7 (#>t 389*9 (8)t 381*7 () 374 7 (8) 40710 (4)4 388 3 (7) 1733 (18) 171 2 (8) 173 3 (9) 1784 (9) 178 2 <> 179*4 (6) 139*3 (4>f 180*5 (5) * Significantly different {P<0.0&) from untreated controls, t S<nificanlly different (P <0.03) from ethanol-lrealed controls. 3 8*0 2 (14) 4 3*0.2 (51* 4.4*0 3 {9} 4 4*0.3 (9) 5 3*0.5 (61* 4 5*0 3 (8) 5 0*0 4 (4) 4 1*0 4 (0) April 1972 171 HONS 081470 by 45%, provided, of course, that tissue con tent on a wet weight basis is equivalent to homogenate content baaed on added DDT. The data in this same curve also indicate that Mg-ATPose 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 aa to why a whole body burden of less than 10 ppra DDT is often lethal to fish It 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 oamolarity of plasma and cerebrospinal fluid. Lost summer, hecause 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 killifish, The toxicity data, Table 5, show that, with respect to ths acute lethal level in sea water, the Aroclor 1221 was less toxic than DDT by a factor of about 100 There was a `'doae-responae" In terms of the theoretical concentration in sea water and num ber of deaths for both Aroclor 1221 and DDT, but the actual doee which the fish received la unknown since the tissue levels were not measured. The oamolarity and Na concentration of the scrum (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 (0 or 24 hr) samples were taken from those still surviving. With the higher level* tested, meet of the increases in serum osmolarity and Na were statistically significant when com pared to ethanol controls. Also, serum oamolarity and K were elevated in all ethanol-treated controls (P < 0.05, except for 24-hr osmolarity); this elevation may have been due to the ethanol or to confinement of tbe fish in the experimental containers. Additional data (Table 7) show that in the extreme cases, when blood was sampled before the fish died but after they had completely lost their righting ability and remained sideways, the serum osmolarity and Table 7. Oamolarity, Na sod K in Individual pool'd aampleaof billiSah Hram.` rnosmoU Compound Exposure end level hr litr Ka* mEq/hUr K* mkqAiter Aroclor 1221 75 ppm 23-32 DDT 1 ppm -io 550 428 430 440 208 215 210 81 T5 83 8 * Each sample lepreseeta terum from about 3 fjh electrolyte concentrations were all increased well above normal. It should be noted that the oamolarity changes (Tables 6 and 7) cannot always be accounted for by changes in measured electrolytes. Discussin Overall, these results suggest that lethal levels of Aroclor 1221 and DDT both decrease the ability of killifish to oamoregulate to about the same degree. Also killifish and eels seem about equally sensitive to DDT. Thus, Inhibition of Ka,KATPase by appropriate levels o! Aroclor 1221 was anticipated. Using a homogenate of King O'Norway (Hemilripterut amerieanut) 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 Yap et *L (0) showed that several of the Aroclor mixtures inhibited the enzyme from fish brain and kidney. Concerning the molecular basis for Na,KATPase inhibition by DDT end PCSs, one may speculate that these lipophilic compounds interact with the phospholipid-activating component of this lipoprotein ensyme (7, 8). It is well known that many lipophilic compounds, e g, cyclo hexanone, inhibit the ensyme and evidence of proton interaction between DDT and a phospho lipid, lecithin, recently has been reported (9) Moreover, other organochlorine insecticides, in cluding endrin, also inhibit Na,K-ATPase from fish brain (10), and endnn has been observed to disrupt osmoregulation in both a marine and a fresh water teleost (11, 12). Thus, it seems reasonable to predict thet many organochlorine 172 Environmental Health Perspective* HONS 081471 frl' ----------........... ^ pollutant* ill be found to disrupt oamoregulalion in fi*h. REFERENCES 1 Prosser, C L ind Bfon, F A Jr J9QI Cooijiaralive Amnosl Physiology (2nd ed,)t Philsdclplus, W B. Sounder* J Janicti, R H and Kwler, W, B 1971 DDT inhibits \*4, K*, MgH'ATl'ua in t're intnalmtl nuconr ud (ills of marine teleosta Allure 233.143. 3 Jsoichi, K H end Kinter, W B 1971. DDT: Dis rupted osmoreguletery events in the intestine of (he eel An#villa rattrala. adapted to an nur. Sciatica 173 1149 4 Guinno, A. M., Janifki, R. H. and Kinter, W B. 1972 Distribution and metabolism of l<C-DDT in the tel {Anfuitlo rcitrafa) after nx houri o( uptake from ambient eater. Bull. Mt. Daeert Island Biol. Lab. 13: (In press). i Ksmiya, M. 1997 Changes in ion end water transport in itoleted (ills of cultured eel In course of salt adapta tion AnnoL Zool Japan. 40-123. C I ip, H , Deaaiah, D. and CuUomp, C. 1971. Benai- tivity of fi*h ATPaaea to polychlorinated biphenyls Nature 233 91 7 Taciki, R, Stliimoto, T and Sakamoto, Y 1971 Mechanism of lipid icttvalion of Na,K,ltfg-aeliviled adtnoeine triphosphatase end K. Mg activated plioaphatate of bovine cerebral eorter J Membrane. Biol 4 42 8. WiJron, VV E , Clemenli, S R and Latimer, A 1971. Phosphatidyl eenne protection of brain membrmnal (Ns* and K4) ATPate from inhibition by fatty acids and chlorinated hydrocarbon pesticides Fed Pipe 30:973. V Tinsley, I, Haquo, R. and Sclimeddiol, D 1971, Binding cf DDT to lecithin Science 174 143 10. Cutkomp, L. et si 197] ATPssa activity in ihh tissue bomogenates and inhibitory effects of DDT and related compounds Chem -Biol Interaction* 3 439 11 Eider, R and Edmunds, P 1999 Effects of endrtn on blood and tissue chemistry of a marine Rah Traot Amer Fish Roc 93: 153 12 Grant, B. F and Mehrle. P M 197(1 Chronic eodrin poisoning in goldfish, Caroinue auralus J. Fish Rea. Bd, Canada 27 2223 nmrnfflnfi Htaith PrrapctU4j Polychlorinated Biphenyls (Aroclor 1242): Effects of Uptake on E co/i Growth by Julian E. Kei!, M.S.*, Charles D. Graber, Ph.D.f, Lamar E. Priester, Ph.D4> Samuel H. Sandifer, M.D.* Introduction l'oh chlorinated biphenyls (PCBs), similar to PDT in structure and physiological effecU (7), are i't concern to the scientific community because of ilieir ubiquity (8), persistence (11) and possible n.vinly. The alleged lack of biodcgradability of this environmental pollutant hsa caused alarm and curtailment of this useful chemical. Work as early 190.1 by Cope (3) indicated that DDT, another rMorinated hydrocarbon, might be metabolised li\ ittcral species of bacteria. Subsequently, other uorkcre (3, 4, 9,10) reported degradation of this :uscc1ieidc by bacteria, fungi, and marine diatoms. I'npublislied work performed in our laboratory (5) indicated possible metabolism of certain PCB i omponents by CylvidrothtM dotlerium. With the ilicis established that DDT wan indeed bio`irentdablc and with evidence that PCBs may be l!llloRtcslly converted, the authors postulated that human flora may play an important part in the .iclabohsm of DDT, PCBs, and other ecological lomsminsnts. Experiments were performed to study the effects : I'CUs in vitro on a facultative organism, <fi, common to human intestinal flora. This bacterium was also selected because it ie the prime indicator of feeal contamination. Materials imt Mitbofe 1 lie study organism, Rtchtriehia calx, was * 1'iMrntne Mvdinns Reeijon, Mrdicsl University ol 'it. (.'mhnn, SU Borre Street, ClisrleMtm. S C. 24<0l. ''licnilniiliuiv l)f,iartnKnl. Medical University of S C. . l.i\iroiiii,er,t ,| Health Laboratories, .Stole Board of ' i Till Cu)onibi.t. S C selected, because it is a common autochthonous, human intestinal aerohe, 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 co/i 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 tnpucase soy broth (TSB) agar plates which had been inoculated with E. coft. Experimentation indicated that 9 mm diameter discs cut from number 2 Whatman filter paper would absorb .02 ml of the acetone sol rent The discs were dried prior to use. For the first experimental scries (I), 20 one-liter Erlenmeyer flasks, each with 500 ml. TSB, were inoculated with E. coft. 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, containing only media, was used as a growth check. Each flask was vigorously agitated every 2 hour* during the first and last 8 hours of culture time. At the conclusion of a 24-hour incubation period, flask contents were centrifuged, the result ing pellet lyophiliied, weighed and assayed fur PCBs, nucleic acids and tntiated undine ('Hu) uptake. A second experiment (Series II), identical in treatments except for undine, was conducted to coufinn stimulation patterns observed in Scries I. Series II measured lyophihsed harvest weights only. A third trial (Series III), identical in design to tpril 1972 175 MQNS OB147.3 Flower I. Sensitivity screening o( . tali on TSB to 0, 001, 01, 1, 1, 10, 100, Mid 1000 ppm of A, DDT, B, 1'C'U Scric* I, assayed tritiated uridine uptake after 5 hours and S hours incubation time to determine if assimulation rates were affected by PCBs during the logarithmic growth phase. Aroclor 1242 (]*CB--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 id aliquot was injected into tho gas chromatograph for analysis Two-column systems were used: a 0 foot X ]4 inch O.D. glass column packed with 3% SB-30 on GO SO ADS chromosorb W and 2% QF-1 on 00-80 table 1. Lyopbilisml Wrights and Toalnnt Uptake by JEseherfWife cell Culture* Exposed to PCU f Jleetertnl Series !) Treatment Mean1 harvest Mean' residue weights (rojro) 0*/l> FCB ] tig/nil I'Cll 01 m/iiiI Control Acetone control Undine control LSD*' *6 0 90 4 44 5 27 2 M0 U6 13 6 44 0 12 0 10 s > Mcitn of four refill nlcs * atptnifieanl nl 03% probability level enluilntcd from >e way mmIjms of variance ABS chromosorb W. The operating parameiers were. (1) inlet temperature 235'C, (2) column temperature 200*C isothermal, (3) detector tem perature 24Q*C; (4) flow-rate 70 re per/nun X, Qualification was accomplished by measurement of the absolute and relative retention tunes of 5 major peaks of the PCBs Quantitation accomplished by determination of the area under tlie curve of the five major peaks. Utilizing this technique, recoveries of 92.4% of the PCBs were obtained. llNA and DNA were measured using modifi cation of Agranoff's procedure (1) Radio utilised the Nucleor-Chicago Mark 1 Liquid Scintillation Spectrometer Results anil Discussion As may be seen from Fig 1, concentrations up to 1000 ppm of PCBs impregnated into filter paper discs failed to inhibit E. colt growth on TSB agar As shown in Table 1 (Series I), the addition of .01 and .1 itg/ml PCBs to TSB markedly cumu lated E eoh growth above control levels 1 ins is consistent with other microorganism work (4 0) showing that DOT and PCBs arc concentrated up to 1000 times by marine diatoms The data aUo suggest that the concentration factor nun he inversely related to dosage 176 NONS 081474 Environmental lleallh Perspective' Title 2 presents daU from a duplicate expon Tsble J, Tritisled Lrldint I'ptoke by PCB Treated ent (Senes II), "herein only harvest weight** ie studied. Thte second series confirmed EscAerie/ifo coil Culture* During Eiponenti.) and Decline Growth fbiH* apparent stimulatory effects of all levels of PCBs, There were no differences between treatments uj DNA snd RNA levels after 24 hours' incubation. The significance of thie is not clear, since it hsd Treatment Cbunti per minute1 5 Hours 8 Hours 24 Hcura teen anticipated that increased RNA levels would snvmpsny growth increase* However, it is thought that differenoea may have occurred earlier than the 24-hour tune. Data showing uridine uptake by PCB-treated PCB 1 |/ml PCB 01 ng/ini Undine oontrol LSD.1 4343 4756 5020 3800 2050 2367 2073 979 2192 2402 2389 859 E.toli at several phasea of growth are preeented in Table 3. No significant differences in uptake letreen treatments and controls were evident except at 5 hours incubation tune, when there was 1 Moan of four replicate * Least Mfnifesnt difference st 98% probability level calculated from one way analysis of variance a significant diminution of *Htr uptake in PCBdtwj bacteria. Whilo not consistent with increased culture yields, the lack of change in uptake tetween chemically treated E. toit and controls ones coincide with chemical assays indicating no facilities afforded by Training Orant No. AM03078-NIAMD and NIH General Research Support Grant 5420. Monsanto Company kindly supplied the PCS used in this work. increased levels of nucleic acids. Results from these experiments indicate that REFERENCES l'CBs stimulate in vitro growth of E. toii. If thia 1. Agrsnoff, B W and Santan, R. J 1M3 Studies on the phenomena occur* in vivo further concern is ssUBSlwn of deoxyribonucleic acid and nbonuelme i unified since any environmental contaminant add in tha rat brain Bnebtn Biophy* 73:351. which affect* indicator organism may result in a 2. Ctiseho, C. I., Lockwood, }. L- snd Zabik, M 1990. * diriorted portrayal of the actual situation. Chiunrated hydrocarbon pcalicidaa: degradation by microboo. Setence 154: S9). itkawle<iinant This research was supported by the Community IVatiride Study of the Environmental Protection Agency--Contract /PH21-2017. Appreciation is also expressed for technical laWf 3, Lyophltlud Weights of Jbahortofcle wB Cultures Exposed to PCB (Bacterial Swiss II). Treatment Mean1 2ha3rv4e*s*t w7e*ig9h1ts0 11 (mg> 1 k/bI ^ .01 ag/ml tlrut *wut oonttoi -'ll.' 1W.4 151.S ieo.i ise.e s.s Wren of four replicate LSD.- Llut sigiufaant differene* st *6%prebsbility r"tl "fwlatcd from oas way analysis of vsrfonca 3. Ceps, 0. B. snd Sanders, H. O. 19*3. Microorganism snd Hydrocarbons U.8.D I. Fish and Wildlife Circ. Paatiddee--Wildlife Studies 107:37. 4. Kil, i. S. and Pnestsr, L. E. IMS. DDT uptake snd metabolism by a marina diatom. Bull. Environ Contamin. Toxicol. 3:19S. 3. Jtal, J. E. snd Tncstar, L. E, 1970. Unpublished work. Medical University of 8. C. *. XeO, i, E., Prissier, L. E and Sandiler, 8. H 197] Polychlorinated biphenyl (Aroclor 1343)- effort* of uptake on growth, nucleic acids, and chlorophyll of s msrins diatom. Bull Environ. Canlatnu. Toxicol. 8: 15*. 7. liebtenetam, E 7 ltd. 19*9. Biological interaction between pleatirhera snd insecticide*. i. Eton. EntomoL 83: 7*1. 1 Risabrtugb, R. W. si si 1988. PolycMorineted bi phenyls in tbs global ecosystem. Nature 330: 1OT*. 9. Btenevasn, i. H. V. 1965 DDT metabolism In re alatent sod aoaeeptibte SaUsSn snd ui bacteria. Nature 307:880, 10. Wadmnayer, O. 1988. Dechlorination of DDT by aerobecter aerogenea. Scienea 153. 847. 11. Wuratar, C. 7. and Wingate, D B 1988 DDT reel, dues and declining reproduction in the Bermuda patrai Seines 159: 979. 4pri) lS7g 177 Enwirvnmmtml Health Pertpmxivn The PCB Situation in Germany byH.P. Tombergs* A PCB problem has been known to exist in Germany since 1000. At the 8wedisli-Gennan Symposium at Baden-Baden, Dr. Jensen, who first published about this problem in 1600, made a report entitled "PCB--the DDT Problem of Industrial Countries." In September, 1970, there vfa PCB conference in Stockholm. One of the reports ghen there mentioned that a fatal case of biplienyl intoxica tion bed occurred in Finland. The person was a 31-year-old man who had worked in a plant which used biphenyl (not chlorinated) for impregnation. The exposure level wss 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 strophy 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 EEGa and impaired peripheral nerve function. In July, 1070, Acker (University of Mfinster) was able to find PCBs in human milk at a level of 0.103 ppm, which means 3.5 ppm in the milk fat. In human fat tissue be found an average of 5 7 ppm. A recent German investigation on rate (Benthe, University of Hamburg) report* the ebaorption and distribution of PCBi after one inhalation exposure to Pydroul A 200, a mixture of low chlorinated biphenyls. Absorption aa well as distribution were studied by measurements of PCB concentration in different organa. By this routs 'try high absorption could be shown. Depending on duration of PCB exposure, a rapid increase in * Wert German M inintry of Youth. Family, and Health PCB concentration in the liver was observed After 13 minutes the concentration was more than 50 percent of tho maximum concentration attained after two hours (70 *g/g tissue) Concentration in fat after 30 minutes of exposure was 14 *g/g tissue (about 27% of the liver concentration!, whereas in brain tissue, only 9 >ig/g tiswe (17% of the liver concentration) was found at that time. Depending on the amount of time which passed after the end of aeroeol exposure, a rapid decrease of PCBs in the liver during 24 hours was measured and was accompanied by an incream in brain and fat. In the couiee of two days brain and liver concentrations fell to a minimum value, whereas in fat, a maximum value was attained (260 pg/g (issue) which remained at a constant level in the subsequent period. There wss no toxio fatty degeneration of liver under these experimental conditions. Seven weeks after exposure to low chlorinated PCBs (Tetrachlor), the compounds were found in the fat of rats. After three weeks, 70 percent of the maximum value was stBI detectable in the fat, and it should be noted that, in a stress situation, PCBs move from fat tiiaua into the liver. In addition to the inhalation route, studies wen made of other methods of exposure. The results * follow; After intraperitoneal injection of PCBs, a stimulilion of the microsomal ensyme systems which are responsible for the metabolism of drugs wss found by measuring the O-demethylation, and the increased ensyme activity persisted for a long time. Even after four weeks, the values were still twice the value of the control animals. Because the microsomal ensymes 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 179 MGAS 081476 a toxic effect on liver function was found: trans aminase activity (SCOT, SGPT) had increased on an average by 50 percent. Parallel to tins increase of tiansaminaae activity, the liver fat increased from a normal 3.9% to 3 7%. We consider this a toxic effect. After a aubclinical dose of tctrachlorinated hydrocarbon (a dose which doea not cause a measurable amount of liver damage) was given, the application of PCBs caused a high degree of liver toxicity; that means that, in the case of a aubclinical liver damage, PCB absorption may be dangerous by causing liver dystrophy All these results will be published in detail in the next issue of the A rchn-tx of Toxicology 1M Environmental Health Perspectives MONS 081477 Environmental Health Perspectives 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 in all of the varied He Ida which have been represented here, o 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 ratfronting us, in an attempt to identify those that are significant and merit attack. In ahort, "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 PCBe within the federal government. The extent of additional work that has surfaced u 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 last few years. I will follow the order oi presentation in the present conference roughly, but will sssumo the liberty of regrouping where this seems desirable. Analysis Analytical technics have improved immensely in the lsat three to four yean since the disturbing pesks resembling DDT metabolities sere identifled on chromatograph traces. It now appears that analytical procedures of considerable specificity arc now available and quite widely used. The confusions and uncertainty of several yean ago appear to have been largely mastered. The procedures an in some degrees overly tedious and hate serious defects in respect to quantitation, especially because of the disparate response of some of the detector systems used in the gee ' * Director, liMitute of Eavwsmnsotsl Medicine, New Vork t'oivcruty Medical Center, MO First Avenue, Naw Voik. Me* York 10019, chromatographic analysis. The latter has made quantitation of mixed iso mere 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. Thera ia a very substantial gap in reliable analytical technics for impurities such as the chlorinated dibensofurans. It appears fairly defi nite that such impurities sre 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 ia required. Been use 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 in the original PCBs themselves. In this connection, it may be desirable to look for biological assays as at least an interim approach to this problem. Dr. Vos, in hia excellent presenta tion, hu presented work which suggests possi bilities in the use of bioessay* in the assessment of impurities in PCBs, This appears to be a very promising approach sod 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 in the further improvement of analytical technics. Enrironntontal Transport, Distribution and Alteration Dr. SaroAm and Dr, Nisbet, in their paper, snd April 1972 l)r Itisohrough, in Vis have timln taken o'cmil nN^'vtiiK'ni' of c*u\ iioiuncntil bunic1-, pullem-. of distiiliuluiii nuct nilt`iution ol tti<> I'Otl-. II Im* illicit!} Ix-on tJi-nr iiotu cailici siirvi'v;, romfoi ted by suit I if' jii c^ulcd a I this toiifrirmo, that Hie I'CHsmr ici i widely dctulniUd and mo almost ubiquitous m their picsCine til Mom loveU in the liiuxpliorc It nppenis, houoier, that their rlistuhulion .... y lx" It's.' imifonnly global and moic ir|npsctil.itis u of local sources than DDT and its ftmil ortics The n]iprom.'lics used m (lie two papers noted irprcwul u vei> good beginning .md sene to define, (illicit perhaps only m an mitial way, some of tltc nmjor features in the overall l1CIS budget Equally important, tlicy sen c to define Rftjw in our knowledge -which may play decisive rules in developing a sound analysis of cnviionmental distribution patterns 1 note a senes of items emerging us urgently needing dnufication 1 Further refinement of sources, including. a. Patterns of marketing and use Im provement here will require improved access to such information fiont pro ducers and distributors. b. Dumping practices, c. Stability of scmiscqucstered sources, eg., capacitors in duni|M 2 Better quantitation of discharge amounts by route into water and air. 3 Appiopnalc analysis to determine the relative role of vapor versus particle borne PCB in aerial transport. 4 Bain out data in selected regions to assess tlic at mospiicrc as a transport route, by season and by region. Allied to this is the need for appropriate selective analyses of dated snow deposits for the develop ment of time trends. 0. Clarification of the importance of loca lised as opposed to generalised sources of PCB. As only ana example of the many dilemmas, the Sarofun-Xisbet scheme re quires mid-ocean dumping to explain some high l'CB values far at sea. Are such sources important? (1 Clarification of (lie role of particle bound as coutiidled to dissolu.nl PCB in water transport 7, Development of a mote efficient moni- taring strategy > n means of optimimi nnnhsis and tlic development of globe disliihvition jirvltcina of PCU's In nppio.u hmjc (he global pmblem, one mu, not oicilook tlic linpoitame of moie Soc.iIijdi systems of contamination Again a bcgniiimi tow aids this was illustrated in the paper of Dr Ycith Studios of well dchned localities can pro vide not only knowledge relevant to the spixjfi' units examined, but may permit extrapolation ti other similar regions as comixnients m the hrourle global scheme. These piesentations mode very* clear the seriou gaps ansing from our ignorance of environments patterns of alteration of the PCU's and then contaminants. This was brought out in the c\ cellcnt rejiort of Dr. HuUmger on photoly sis o the PCU's. Work such as this needs to be cs tended, and, at on appropriate point in then development, brought into more realistic rein tionslnp to the form in which the PCB's aic found in the atmospheric and aquatic environ ments, e g, photolysis of PCU's as vapor, nr particulates, and so foith It is also very cleat that attempts to deal with the transport and distribution problem are frustrated by lack oi understanding of biological alteration of PCB's, that is, the metabolism of the PCB's by biological systems at all levels. Occflfrencs 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 PCll's in carbonless copying paper have non been found, the completeness of this substitution will require monitoring. It may also be desirable to check body burden* of those routinely using forms with carbonless copying papers. Similarly, the reports of Dr. Trout and Di 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 tliat most of the major sources of I'CB contamination in paper and card board have been identified and intercepted, this 182 finviroiidienLai limit th Perspectives HONS 081479 optimism needs to be tempered by continuing scrutiny of food packaging material. The reports of Dr. Kolbye and Dr. Berglund, on the presence of PCB'a in food*, suggest con siderable similarity in the general patterns of food contsmination in Sweden end the U.S. In eech instance, fish, particularly fresh water fish, ppesr to be tiie largest dietary source. Milk has been found heavily contaminated on occasion. Hopefully, this ia no more than episodic, re sulting chiefly from the contamination of silage from PCB's in silo lining paints as described by Dr. Fries. This mode of contamination dearly needs csreful scrutiny end 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 it perhaps heavily biased by including a large number of samples secured as pert of compliance studies. A more orderly picture of PCB distribution in food will require s mom 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'a As noted sbove, resolution of this issue will require either chemical ennlytie technics many orders of magnitude more sensitive than are now available or bio-assays as suggested above. Antal Tufeoiitf The Biotest studies an now complete and wen well summarised by Dr. Kcplinpr. A somewhat parallel study at Cbamblee ia (tin incom plete (personal communication, Dr. Senate Kimbrough). Examination of the complete date from the Bioteet and the Cbarablea work will be required before a full assessment of these toxicity studies can b* mode. Several issue* present them selves. The extreme sensitivity of the mink, both in lethality (1), nndinpossible reproductive injury (2), raises an alert which will require resolution before we can taka too much comfort from results s)towing lower toxicity in other tramtask. Re grettably, this conference did not include en up dated report on studies of mink. Although the Bioteet report indicates no evidence for PCB carcinogenicity, one (possibly two cases) of bladder cancer may have been identified in the Cbamblee study (Personal communication, Dr. Renate Kim brough). These findings will require that we await the completion of the Cbamblee 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 ia true in mammal* as well as in other life forma, including both fish and birds. Again, the report on embryotoxicity in the rabbit (3) (not discussed here) suggests that this issue be further examined in mammals. It would also bo desirable to relate reproductive effects to PCB burdens in wild populations. It is a remarkable circumstance that despite the interest in the problem, the kinetics, distri bution, metabolism and excretion of PCB m animals are only very scantily understood. There is some data on half time of body burdens in the fat in 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 question* 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 (A). At the other extreme, E. Cdi 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 moat vulnerable function. In respect to birds, and in contrast to DDT, it appear* that hatchabQity may be more important than egg shell thinning Thia area of comparative toxicity from species to species is so large and uncharted that generali ties seem out of {dace. 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. As suggested sbove, such studies may be of great consequence for determining overall pettema of transport and the ultimate fate of these materials m the environment. April 1972 183 HO NS *' --th. 1 A problem that persist* in confuting our under funding of the toxicity of the FCB's it 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 select a modest number of representative isomers which should be synthesised sad highly purified, and then carefully examined toxicologtcally. The selected isomers should be chosen on the basis of the beet 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 ere told, are quite adequate to meet this challenge. Included in this should be well defined examples of the chlorinated dibenxofuraas. Such an approach should permit a more rational plan ning of future toxicological work. These pure com pounds will also be indispeoseble in the further development of selected bio-sorey. MethMlSMaftetha Vos baa described five distinctive patterns of pathologic outcome, aeaegenic, liver damage, porphyria, edema and immunoeuppreaaion which he believes are diffaringly contributed to by different chemical components in the crude PCS preparations. As just noted, toe 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 aid, at thin stage, that we have good suggestion* as to the major underlying meehanmns of tosadty. Rather than to hastily examine the effect of PCB's on a myriad of in vitro test systems, it would seem wiser that there studies be carefully planned as aide to uuJerstaiwIlng the observed course of PCB poiioataf In the intact snimaL Hum Bidjr Banka The reports from Dr. Yobs and Dr. Price and the comments from Dr. Hammer all point to the ubiquity of the presenee of PCB'a in humane in thin country. PCB's occur in the fated all regmenta of toe population and some gross features of population distribution have been identified Tins 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 plasms concentra tion. The lack of- understanding of the kinetics of disappearance in humans hempen the interpre tation of some of this data. Studies on primstes could help clarify this issue. Yasha Dr. Kuratsuae has presented us with a re markable example of the quick rallying of medical and ecientifie 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 Yusbo episode. It is rare that such accidents have been so systematically studied. This data will neces sarily {day a major rote in the assessment of the human haaard 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 systemstic, long term, follow-up studies, perhaps every fire yearn throughout the lifs of the exposed individuals. We hope that their desire to make such follow-up studies for the assessment of possible ehronie effects will be fulfilled. This country should be prepared to lend whatever collaboration it cam in this very urgent matter. fiMNnlCMIlMtS Extensive deposit* of PCB's now exist through out the biosphere. Even without further dis charge, there deposits wiE rerosin for some yean. 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 there deposits The manufacturers of PCB in this country are now well alert to the problem and have announced a series 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, 1U Environmental Health Perspective* HCWS 081481 owmr, the M-etlkd ''controlled" patterns of $t, such u Urge ccale tranifonnen and capncirjn, will in turn require monitoring against cckfental discharge tnd leakage. Substitution in th 1ms controllable uses U pparently underway. It will be very important i carrying out such substitutioo, not to replace known hasard with an untried, unknown, posibly greater haxard. Accordingly, coniiderabl* are in effecting aubstitutioo will be needed. The approach used in the above diacuaaiona of oncentratiug on gape--thing! not done as optoGcd to those which have been done--brings ha risk of implying that our ignorance la greater han it actually is. The many detailed and solid upera presented at this conference and aaeroblcd here should serve as an adequate oounteritlance spiral such misunderstanding. Neverheleas, our ignorance U still too large. I have thua ried to identify some places where, I believe, we <eed additional understanding. Finally, I apolo gise 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. snd Hoopmgnmer. R. 1071. Interagency Meeting on PCBa. Dept Health, Educ. t Welfare, Washington. D.C, August 5. IB71. 2. Aulerieh, R. J. et sL 1S71 Effects on feeding coho salmon and ether Orest lakes Hah on mink reproduc tion Can. J Zool 49-til. 3. ViltaMuvo, D. C. et al. 1971. Efforts of PC8 adminis tration on microsomal Smyrna activity in pregnant rabbits. Bull, Environ. Cootarnis, Toxicol- S: ISO. 4 Grant, D. L, Phillips, W. S. J. and ViUtasuve. D. CL 1971. Metabolism of a polychlorinated biphenyl (Arorlor 1354) mntuis in tha rat. BulL Eaviroo Contanun. ToskoL 8:103. 5. Duke. T. W., Lows. J. I. and Wilson, A. J. 1970. A pelycblerioated biphenyl (Aroclor) 1354) in tbs wafer, ediment, and biota of Eaeaiubla Bay, Florida. Bull. Environ. Cootamia. Toxicol. J: 171. i IF t t ( < it if.i, ftovMMMtfifT MiNfim e^neti JW I