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Analytical Chemistry Special Study 71-3 Job N o . 1348006 DETERMINATION OF POLYCHLORINATED BIPHENYL RESIDUES IN WHITE LEGHORN CHICKENS FROM A TOXICITY, REPRODUCTION AND RESIDUE STUDY WITH AROCLOR 1242, AROCLOR 1254, AND AROCLOR 1260 INTRODUCTION An integral part of the overall Aroclor Environmental Program in volved obtaining a more complete understanding of the environmental behavior and impact of our polychlorinated biphenyl (PCB) products. In order to accomplish this a number of laboratory feeding studies with beagle dogs, albino rats, and white leghorn chickens were initiated at Industrial Bio-Test Laboratories (IBT). This report covers the results of the analysis of biological sam ples from IBT's study IJ7300, "Toxicity,Reproduction, and Residue Study with Aroclor 1242, Aroclor 1254 and Aroclor 1260 in White Leghorn Chickens" and a supplementary IBT study #8746 with only Aroclor 1242. The specific objectives were to establish the relationships be-, tween tissue storage levels and exposure period, exposure level, degree of chlorination, and any observed toxic effects as well as determine the homolog distribution of the retainea PCb residues. Enough chickens were also included in these studies to determine the rate at which the PCB residues declined when the birds were put on PCB-free diets. SUMMARY PCB residues from the Aroclor products fed were observed at all exposure levels (1, 2, 4, 8, 10 and 100 ppm), at each sacrifice period (24 and 26 weeks), and in all of the samples (muscle, liver, fat, egg and chicks) analyzed. t The PCB residue levels found in the tissues decreased as the exposure level was reduced and as the chlorine content of the product fed decreased. Significant differences in the PCB residue levels were observed between males and females, and between the muscle, liver and fat tissues. Significantly lower PCB residue levels were observed . when the birds were put on a recovery diet that contained no t PCBs. \ The homolog distributions of all of the products fed were altered. The alterations were most pronounced with Aroclor 1242 and least with Aroclor 1254 and Aroclor 1260. -C0WFIPEWT7AL- AQrt 005729 Analytical Chemistry Special Study 71-3 Page 2 __________ DETAILS Analyses of the tissues were carried out according to Analytical Chemistry Method No. 71-35 with the exception that the chemical clean-up procedure was necessary only with the chick samples. Standard solutions of the material fed were used to prepare cali bration curves for the quantitation of the PCB residues. In the initial residue studies all of the samples were analyzed. In subsequent tissue residue studies, including these chicken studies, a statistical approach was employed to minimize the number of samples requiring analysis. In this manner, both the elapsed time and manpower requirements were significantly reduced with little real loss in either accuracy or precision. The ap proach involved subjecting the results from the residue analysis of a balanced subset of samples, along with the appropriate study variables (tissue type, exposure period, exposure level, sex, and Aroclor fed) and the most probable variable interactions to multiple regression statistics. In this way an interpolation equation was generated for predicting the PCB residues. The initial equation was then smoothed by deleting data more than two standard deviations from the predicted values and the variable interaction terms not significant at the 95% confidence level. The final equations were then used to calculate the PCB residue levels in the unanalyzed tissues. The validity of each predicting equation was established in the following manner. The respective residual standard deviations of each equation were compared to those obtained for the equations developed for the studies in which all of the tissues were analyzed. The 95% confidence limits calculated for these studies from the log of the residual standard deviations indicated that in all studies the actual values determined by analysis were within *0.5-2 tiroes the prediced values. The equations resulting from the statistical analysis used to predict the complete sets of PCB residue levels are shown in Tables III and VI. Figures I thru III are semi-logarithmic plots of PCB residue levels vs. time for the different exposure levels for each of the Aroclor products tested. The plots clearly illustrate a considerably slower rate- of recovery for the liver tissue. Ap parently the liver is the primary site for metabolism and/or collection point for elimination of the test material from the animal. Shown in Table VII are biological half lives for the Aroclor pro ducts tested in chicken tissues. Once again significant differences -COWFIPENTIAL- ADM 0 0 5 7 3 0 Analytical Chemistry Special Study 71-3 Page 3______ between tissues were observed. Also apparent are differences in the rate of recovery with the Aroclor product fed. Calculations based on average body weight, feed consumption and lipid content indicate that only 11% of the total Aroclor 1242 ingested was retained by the animal. The corresponding figures for Aroclor 1254 and Aroclor 1260 were 28 and 49% re spectively. Figure IV, which is a plot of PCB residue level vs. degree of chlorination, further illustrates the differences in storage level due to test material fed. It demonstrates that as the degree of chlorination decreases the PCB residue level decreases. Figure V is a semi-logarithmic plot of P.CB residue level vs. exposure level for each sex for the three Aroclor products fed. This figure illustrates the bias, which increases with degree of chlorination, toward higher PCB residue levels in the males. The higher PCB residue levels in males were also observed in the recovery sacrifice samples in the same ratios. Chromatograms I, II and III compare typical tissue extracts from the residue and final sacrifices with the material fed for Aroclor 1242, Aroclor 1254, and Aroclor 1260. The alterations were greatest with Aroclor 1242 and least with Aroclor 1260. It ic apparent uper. inspection of the chromatograms that the lower chlorinated isomers are metabolized and/or excreted quite rapidly. Further comparisons between the residue and final sacrifice extracts indicate that some of the higher chlorinated isomers also undergo metabolism and/or excretion but at much slower rates. Chromatogram IV is comparison of chicken tissue, egg, and chick extracts in the case where Aroclor 1242 was fed. Once again, it is apparent that the lower chlorinated homologs are readily metabolized and/or excreted. The gas chromatographic conditions employed do not completely resolve the Aroclor products into their component homologs. Nevertheless, the numbers above the chromatographic peaks in dicate the dominant homolog(s) in that peak. TOXICITY Some toxic effects were observed with the groups fed Aroclor 1242 at 10 ppm and 100 ppm, andvAroclor 1254 at 100 ppm. Consequently a supplementary study with'Aroclor 1242 at the 2, 4 and 8 ppm exposure levels was initiated. The toxic effects, primarily re lated to hatchability, are discussed in detail in the referenced Industrial Biotest Laboratories reports. -CONFIDENTIAL- r ADM 0 0 57 3 1 Analytical Chemistry Special Study 71-3 Page 4_________ REFERENCES Toxicity, Reproduction, and Residue Study with Aroclor 1242, Aroclor 1254 and Aroclor 1260 in White Leghorn Chickens," Industrial Bio-Test Laboratories Report (IBT #J7300). "Toxicity, Reproduction,and Residue Study with Aroclor 1242 in White Leghorn Chickens," Industrial Bio-Test Laboratories Report (IBT #8746). Residue Study - Aroclor and White Leghorn Chickens, Statistics Special Study 71-24. MONSANTO INDUSTRIAL CHEMICALS CO. Applied Sciences St. Louis, Missouri 3-73 W. M. Mees, E. S. Tucker, V7. J. Litschgi, J. Cowell ADM 0 0 57 32 TABLE I A ' ~JoX/crfy ftdpro'/uc.f/o/7 ////{'S/af&e. Sfttafy of'ffrac/op //?h/A/fd fe^Aor/? CA/cfons /Aj Cf/ofo/7 7fssue.s (k fd f f if e /jf f j0 js/jJ L___: tfcTtdut. \5ser/fice 3 0 / < ^ 7 /O0j*f*t r.r rp ? <? ? < ? ? fl/tick. - O.Oi 2 007 Cft*tk. Stcrtftce* < ? f? < ?f a.a s /s7 ht4 2*/ 3sJ wX m **k. .& |hr/; 3rS\//M W/f Jw/< /st 2J *r/c 3wn/|\wm'/t sst&/ Sacs'/fidz** /jy*f/ /OP/**** / 00y>j>w /OOytf*. *30 Dj %/ /%3c.c.av.*~ /%*'/&7 <? ? <? J TO ass a/ s /r*clct Ln /* !/:/ 3 n x/z ! / .V */.**i*-. 2 of / fa /23/ vT 38 of) on 0 .0 2 0-2 _L : a? 2 Vs 3 0.6 o.s 26 30 9 2*/ Y 8 OJ .Sr A* /V- 0.8 SS/ /s9 >.7 So 0 .0 ? 1 /oZ ____________ 1_____________ 2 o.s o.V 0.9 27 / 73 08 7S 36 /! /. o.z // 0.0& 0 .8 ovy 2 -Sit' /*:/ :/26o -------------- /? /S f ?* / 6 /3 o.oy 0 .2 //of / --*.7___\' - /3 8 /3 1 /o /:/. i ->* `|'VV 43 COC we'? % TABLE II 1S T V 7-300 TTx/dfy, Teprrx/vcSc* a/).777ss/7ae SfuTy oS/Yrvc/or- 3 b/i/Ye. epAt/r? (73/c.T.-. P C 3 /r? CA/c/i^S? 77**i/<CS ( ,7 , 7 iVs/aTT ii /cs/S*se.sS^cs'//'/C-C^ \30 jD'ySCA/cjLS^CS/fiC!".f4*z/?/. 2l//'*KjO;Sr.i/'r./-.*<AwX 2i+nA,/\ti\V33-/tf2 y5u/*A0tmVYY, fas/ \5r^s-/r.c. JfT-- /Oy/y>~! /Typ"/ /<7yy~' 3/*r~f /TOyyy*s /(OTA 7/J/nf /Oyp*/ /Sr, <? ? ? ? si? ft9 <?? 30 Djo /&.&>*/v /^r/r<y ? <3 ? 0 flT-c/c /3 70/ 9 32 A^c//-J-trtf' ' 52 jY 72 *' 6 70 3o-- 23 /i 32^2 57 30 22 /O'/3/0 Y r.fs 37 V72 33 S S 38336/2/,'^2f/c/twZ , s. 5/7Z'/o 2 /JsxJa,/ 16 Mi Y /2S*//Tr en IV r ... 70 7J//0 iV (0 s 6t 7 // 73 '3S3 C-G31 /V j/o .02r s 32S 7}/0// _/*.z' G 6> 6> /3 h: 3/' f2t,0a : / ` /es 36 rse?/3J7 / , .r 32 /*/& 33 7 US 73 // /* AJh 00573<t >\ 1> O.) O' Reprafucf/br? J/?7 TesiJfe. S fuJ y o f f/rc/or /s? h//?/7e yfor/? CAfefi $ 'O ^ vVt- Cfl4 V. 'Ci r<. *5 ` * ^s: '"N S' ^ v* 5 NVi Ce* *s V . ti 'S? I K \* JJ ** s' ' s t> I^ 0 ft V \c ^ <0 u ^x "N Eq CVl V? i ? * 5x 4* .! 4 i A vl <3 $ ADH 0 0 5 7 3 5 I T ^J 7300 * `v) VJ n .V 3 - Oi. $ \'*J` ' V X\ I * t 1 s. I* V, .Vv 'J *0 C\JN V oVs' '\. i (v IlL oN' *1i $1 NN A/H* U.* \ I XXJ ,b' >V i x ? * \) sM ^s sy <* TABLE IV I J. 1 t1 5 n \^V*Jjv' .~Ns ** \j ^i s ^N! *1 i V m 'V *< u\J K1 ffsi > .? *0 w V) $ s\?J Ot 'V > w *0 <5 is Oi. $1 s K$ Xi <VJ fV M Ck. ^x. VH. >> S; !\0 P Vs. Ji Ox "v. >1 b ,\XJ. *% x^ ** < 5 , \ i^ ^ <1 ADM 005 7 3o TABLE V I3r*87lt t Toxicity, /TiprotfifC7'/<?f7 jtjcT /Residue. S /'aty o fP roz/cr- m T/'A ife- ey/>orst C /r/cf'fn ppm P C 3 /r? C/ifcAe/.9 Z/sGc/es f?e3ii/t/e. 'SjCS'Sf/CCZ- s3oZhy 2/'/>*: s/fp^t r />*"' 2p*p* ///>'* 1 ? <f? si* <?i f **+**'*-, t-r s ? y 8 flvd* .'/ir /jf K* p `),r 30 / s /? I 73 3 S /3 /7 9 lU jf/-y v & kvX' iv-t hr UJ'/\s,A/v, Sr// *A# K 'i 1lv^' { *W' 1H'X: iv/r |i/4: *vX /v/W ~/>/>"* r / y 9"* ipy?s>/ fs CjLpy*"/ <$// - erf ? c??- cfJi /9 7/ i /t / '/ v jr.; X5* <? 25" & 2<? > o3 j i U05737 k I -L *VJ. \3 fe Vi V) 'X3 55 0 VJ N s Mc $4 0*. VX% s *0 Vis M *0 <Vi I ** 1 * 1 'to. ?b ' N \ * S i fcv \ 2i > A t * $* n X1 NN s 6 S8 it) QXs * S"ii X it) 4 1 1 >s I0 ~ <*) ? $1 $ s X X '5} k 63 I ? VJ w m $ SN >s \jss & .y S: Oi 'XS <*> > V$ <S h s i. * s * \a *5 4 3S 4 6 04. ' * VS. V Ok 4 1 'VJ. K " $ \> V* N3 '0J 4 cJ 04. "Vu n \> 5! fc Ok 'X , CV ND OJ r* V) ,T) \3 V i*i %4 04. *b x *> 58 NN "> | T? .N*X , X v ** x V x? j 1$ a ADH 005738 Material Aroclor 1242 TABLE VII BIOLOGICAL HALF-LIFE Tissue Biological Half-Life Muscle, Fat 27.6 days Liver 245.4 days Aroclor 1254 NN Muscle, Fat Liver 21.4 days 69.3 days Aroclor 1260 * *1 Muscle, Fat J.ivpr 19.3 days 51.0 days t@ AOW 0057^3  C , h ' f / ;/ J- 'i'i- :ii '$ r o /tc r T2j/ 2 ' j n / J ... : . ' . ' 4\ I : i t'O. C-/&/~ \/Z$2. ;. i______i . . <r~ 1T 4 ./Jti&g, . . '. -4+f - /r><^/ar /F-/2. F c / ?+6 AQM 005745 I %- <s% Chr~<p's/1>J*# .fsj; -LL /'T ^T +* ' Cf, /ro c /o r / '/2 1 / m 7 / w v / c y*T Si: *7? XT tcW* .v s k s t ^ r A ;/ 4 - v. ADM it