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jr-v---* f~ U-gM' ^x.lZf^Sji'axnin`rl.,1- ZM. CREENHALCH U, ilitrockem. J., Jitw York, 1961, -all. London, 1963, ana, 196S, pp. 102, I Journal of ChromeUographv, 75 (1973) 3*9-236 0 Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands ' CHROM. 6367 ELIMINATION PATTERN OF AROCLOR 1254 COMPONENTS IN THE BOBWHITE , GEORGE E. BAGLEY* and EUGENE CROMARTIE . Bureau of Sport Fisheries and Wildlife, Patuxent Wildlife Research Center, Laurel, Md. 205/0 (V.S^i.) > (Received August 31st, 1972) ' 1 f SUMMARY .' ' The gas-liquid chromatographic profile for Aroclor 1254 did not maintain its integrity in bobwhite quail fed Aroclor 1254 for 14 days and clean food 14 or 42 days thereafter. Absorption of all components occurred at essentially the same rate, as F. shown by analysis of quail sacrificed within an hour after a single oral dose of Aroclor. ? Acetonitrile-hexane partitioning altered the peak pattern of the Aroclor 1254 r ? standard. 0 B Significant alteration of certain polychlorinated biphenyl isomers appeared to take place. Dechlorination was clearly apparent, and isomeric transformation is suggested. INTRODUCTION The piesence of polychlorinated biphenyls (PCBs) in the environment, their toxicity, and some of their physiological effects have been described for various species. The PCBs are industrial compounds marketed in the United States under the trade name Aroclor (Monsanto). Other registered names are Clophen and Phonochlor. They all contain a complex mixture of closely related chlorinated biphenyls varying in chlorine content. Aroclor 1254, which was used in this study, contains 54",, chlorine* as indicated bv the last two digits in its numerical designation. Some other Aroclors are designated 1232, 1242, 124S. 12<K), and 1262. Their uses are numerous and varied. There are 210 possible isomers and 102 most likely*. Bagi-kv ft a/.3 identified iS isomers in Aroclor 1254 by gas-liquid chromatography-mass spectrom etry (GLC-MS). Koeman 7 4/.* showed the presence of ri isomers in Arindor I2tx>. and Sissons and Wiilti4 have shown that <>o compounds are present in Aroclor 1254. Analysis of PCIVs is routinely accomplished by GLC with electron capture * I "resent address: Environinent.il Protection Agency. Pesticides Program, Washington, D.C. 20240. U.S.A. 1 DSW 032873 pi f 11 , 1 " . . - > f l,llt STLCOPCB4016835 120 C. E. BAGLEY, E. CROMAKTIE detection. Tlic commonly occurring chlorinated pesticides such as DDT and its metabolites are determined similarly, and as lx>th occur in fish and wildlife tissues, the PCBs must be separated in order to obtain reliable analytical data. Several methods have been used*-1. There has been less process in quantitation of PCBs and perhaps there are as many methods used as there arc analysts. The problems of quantitation arc related to the complexity of the closely related conqvmcnts of the Aroclors. In our laboratory, we use a semiquantitative thin-layer chromatographic procedure as reported by Mvlheux ct al.*. This method is applicable to all Aroclors in the 1200 series. Other investigators have used GLC peak heights of several peaks and even a single peak. Rote and Myrphy10 used a detector-response curve to determine the amount of each chlorinated component in the PCB standards. A review of various methods for quantitating PCB was presented by Peakau. and Lincer11. In analysis of bobwhite (Colinus Virginiamis) given a treated diet of Aroclor 1254, we observed that the ratios of the Aroclor peaks to each other differed from those in the Aroclor 1254 standard. Some peaks were relatively higher and others lower than in the standard. We set up the present study to observe elimination patterns of Aroclor 1254 components in relation to time. Changes in relative peak heights can greatly influence the value of a quantitative method, for if tiie integrity of the Aroclor GLC profile is not maintained, then only a method, which accounts for these changes, will give quantitatively dependable results. EXPERIMENTAL Diet treatment of bobwhite . Aroclor 1254 was dissolved in corn oil and then mixed with dry feed in a ratio of 2 parts of solution to qS parts of feed. The concentration of Aroclor 1254 in solution was such that, when mixed with feed, a diet containing 300 p.p.m. of Aroclor resulted. Early in May 1970, 30 bobwhite, i-ycar-old males, raised at the Patuxent Wildlife Research Center, were placed in 6 cages {5 birds per cage) and maintained on turkey starter mash for 1 week prior to start of treatment. Following 1 week of acclimation, 3 cages of birds were provided ad libitum access to the treated diet and 3 cages were provided the same commercial diet free of toxicant but containing 2 parts of com oil to qS parts of feed. On day 14 after start Of treatment 3 birds were sacrificed. The remaining birds were then placed on clean feed; 3 were sacrificed on day 28 and the remaining 9 on day 56. Control birds were sacrificed at the same rate and interval as treated birds. At the end of test, 3 birds from the control group were each given orally 1 capsule dose of Aroclor 1254 in corn oil equivalent to 500 mg/kg body weight and were sacrificed after 1 h. All birds, except those sacrificed, survived the test period. Preparation of sample The carcass (after removal of skin, wings, liver, feet, and gastrointestinal tract) was ground and mixed in a Hobart food cutter. A 30-g aliquot was mixed with odium sulfate and extracted 7 h with redistilled hexane in a Soxhlet apparatus. Aroclor I W'lmle live with sodiw panitionin of the unt analyzed k Apparatus An l column (9 Carrier gas flash hcatt ionization GLC-.1/5 < Tota group sacr volume of at approxi set at the injected w 109 mg of of the bird thereafter. To e 300 pg of ` on a Floris was made apparatus Aroclor 12 Relative pet Kela peak No. 5 highest pea tained wit hi each chromr as described resvlts a; Resu peaks lieigi acetonitrile Aroclor as; effect of no and detenu "w* ii" DSW 032874 STLCOPCB4016836 X. CHOMARTIE JDT and its jdlife tissues, oata. Several xps there are nn are related rr laboratory, -reported by juries. Other _ single peak. 3 amount of znnethods for 2t of Aroclor raftered from ~ and others rxion patterns z heights can rarity of the mts for these 1 | j j . , j ; ; ; > : ' j ; I 3d in a ratio _j in solution rior resulted. rit Patuxent r maintained rg 1 week of red diet and r. containing 3 birds were sacrificed on zit same rate : group were j 500 mg, kg rd, survived ' ; 1 1 l j j 1 j 1 I j astinal tract) mixed with .r. apparatus. AROCLOR 1254 COMPONENTS IN THE BOBWHITE 221 Whole livers, which weighed approx. 3-4 g, were ground in a Waring blender along with sodium sulfate and extracted. Extracts were cleaned up by acetonitrilc-licxane partitioning and eluted on a Florisil column as previously described11. A roo-g sample of the untreated feed was extracted and cleaned up by the same procedure and analyzed by GLC. ( Apparatus ' '' ' An LKB Model 9000 GLC-MS apparatus was used for analysis. The spiral glass column (9 ft. X 0.25 in.) was packed with 5% OV-17 on 60-S0 mesh Gas-Chrom Q. Carrier gas was helium, flowing at a rate of 40 ml/min. Operating temperatures were: flash heater, 230; GLC oven. 220; separator, 240; and ion source, 290s. The ionization potential was 70 eV, trap current 60 pA, and accelerating voltage 3.5 kV. GLC-MS analysis .. Total ion current (TIC) chromatograms were made for two birds from each group sacrificed, using a 30-g aliquot sample extract made to equal volume. The volume of sample injected was varied in order to maintain the height of peak No. 5 at approximately the same level in all chromatograms. This level was arbitrarily set at the level produced by 6 pg of Aroclor 1254 standard. The amounts of sample injected were as follows: 30 mg of the original sample of tire capsulc-dosed birds; 109 mg of the sample of birds fed treated diet for 14 days; and 90 mg of the samples of the birds fed treated diet for 14 days and untreated feed for either 14 or 42 days thereafter. To evaluate possible effects of the clean-up procedure on the Aroclor standard, 300 fig of technical Aroclor 1254 was partitioned by acetonitrile-hexane and eluted on a Florisil column in the identical procedure, used for the bird tissues. The eluate was made to appropriate volume and a portion was injected into the GLC-MS apparatus to produce a response on the TIC chromatogram approximating 6 ftg of Aroclor 1254 standard. . Relative peak heights ' Relative peak heights in TIC chromatograms were calculated by choosing peak No. 5 as reference and assigning it a value of 100. This was almost always the highest peak, and its relative depreciation appeared orderly. All peaks were main tained within the linear range of the recorder, and the height of the reference jx'nk in each chromatogram was kept similar by changing the amount of the sample injected, as described above. . RESULTS AXD DISCUSSION ' ' ". Results arc shown in Table I and in Figs. xa-f. Table 1 compares the relative peaks heights in the Aroclor 1254 standard, the Aroclor 1254 standard subjected to acetonitrile-hexane partitioning, and the quail carcass extracts. The change in the Aroclor as a result of partitioning is believed to t>c duo to the distribution coetlicient effect of acetonitrile- hexane. Stalling el ul.13 also observed this effect in their work and determined /> values for all |xvaks in Aroclor 1234. .' Tl V DSW 032875 STLCOPCB4016837 M2 C. E. BACLEY, E. CROMARTIE TADLE I RELATIVE PEAK HEIGHTS* IN TIC CHROMATOGRAMS Sample Peak So. Ia3 4s 67 a 9 to MX 12 0.49 0.5# 0.69 0.7S 0.S3 0.94 x.06 i.ij r.27 1,40 i.6r i.$r So. ofCl' 4 4 4.5 5 5 5 5 6 6 6 6 6 Aroclor4 1254 (S) Aroclor* 1234 (P) Extract C* Extract D* Extract Ek Extract F1 IOI 34 49 So YOO *3 39 68 73 30 jo 34 1*5 4# 57 9 IOO 32 44 71 *5 *7 31 31 US 48 93 15 33 15 96 45 IOO too 3* V49 0 . 75 31 7" 63 28 18 33 22 33 39 34 O *4 0 IOO 0 "133 *3 63 36 12 48 *5 O 26 0 zoo 0 34 O 77 66 14 86 Peak So. 5 -- 100%. * p,p'~tVE -- 1.0. . . So. of Cl in PCB. _ a S -- standard. *P standard partitioned bv acetonitrile-hexane and eluted on Florisil. 1 Carcass of quail administered 1 capsule of Aroclor 1254. 500 mg/kg body weight. For details, see text. * Carcass of quail sacrificed atday 14. For details, see text. A Carcass of quail sacrificed atday 28. For details, see text. * Carcass of quail sacrificed atday 36. For details, see text. Aroclo 2G Fig. 1. TIC chromatograms. GI.C conditions: q ft. * 25 in. column, 5% OV-i;; oven temperature, tio*, and helium How.rate. 40 inl.'min. (a) Aroclor 1254 standard; (b| Aroclor 1254. acvtonitrilehexanc partitioned, standard; (c| carcass extracts of quail administered a single capsule dose of Aroclor 1254 at 500 mg/kg body weight; (d| carcass extracts of quail sacrificed alter 14 days' diclarv dosage at 400 p.p.m. of Aroclor 1254: (e) carcass extracts ol quail sacrificed after 14 days' <1 ietarv dosage at joop.p.m. of Aroclur 1254 followed by 14 days of untreated food; (f) carcass extracts of quail sacrificed after 14 days' dietary dosage of 300 p.p.m. of Aroclor 1 234 followed by 42 days of untreated food. sf ^ 1 une soiwv r il||r*R^t DSW 032876 tt STLCOPCB4016838 tdi. --Vi . : ...tTp-^-`^| Miiairifi .: *r liMIM-- m a* #23 ! I ! I rrrt temperature. -4, acrlonitrilexapnule ilnw of . after 14 <l.iy*' ziil'imt after 14 crated food; (f) al Aroelor ujt l --~ (Continued <>#1 f*. DSW 032877 in'* ..... ....... m jaT STLCOPCB4016839 224 G. . BAGLEY, E. CR0MART1E Aroclor 12 1 Fig. l. (continued). Relative {x-ak heights anti chromatograms for the tissue extract of birds given Aroclor as a single capsule dose were similar to those of the Aroclor standard after partitioning (Table 1 and Figs, la and r). This suggests that all components of the Aroclor were readily absorbed. Krlative jn-ak heights in chromatograms for birds sacrificed 14 (day 'J<) and 42 (day 5O) days post-treatment (Figs, ic and f) show a methodical elimination of certain components and an increase in others by comparison with the chromatograms for the birds sacrificed after 14 days'dosage (Fig. id). After 42 days of untreated food, only four major components remained, peaks Nos. 5, 9, 10, and 12. Peaks 10 and 12 increased significantly. Peaks Ix-ymid 12 showed no significant change. j | ! ! ! j 1 1 | J 1 1 ----- -------- it Fig. t. (conti / The c certain con in tissue sai chlorinated Jsonicn:oti( lsoiiu reported. 1 place. Peak peak No. S. Quant identical an matugraph, decrease of plained sun Gm:' found that standard. "1 Peak cldorobiph* is observer treatment. After 42 >' Dechlorina eliminated TIC treatment Ireatmenl DSW 032878 STLCOPCB4016840 :Z, E. CROMARTIE AROCLOR 1254 COMPONENTS IN THE DOBWH1TE S 225 i ra -rt of birds given ~nr standard after mpononls of the -lgrntns lor birds _c ami f) show a others by corndosage (l ip. id), -ed, jvaks Nos. 5, zid 12 sliowed no Fig. 1. (continued). The chromatograms show that a dynamic system is at work, slowly removing certain components while increasing others. MS data confirmed all peaks obserred in tissue samples to be identical to the ones in the Aroclor 1254 standard. No foreign chlorinated isomers were detected. Isomerization of PCB Isomeric transformation of PCB in natural or biological systems has not been reported. The evidence presented here strongly indicates that isomerization is taking place. Peaks Nos. 10 and 12 containing 6 chlorines show a distinct increase while peak No. S, also containing 0 chlorines, shows a gradual decrease. Quantitation of individual peaks was not attempted; however, when an identical amount of extract for each sample group was injected into the gas chro matograph, the distinct pattern of disappearance of some peaks and increase or decrease of others was as apparent as in Table I. Thus the changes cannot be ex plained simply by faster elimination of certain components. Grant d al.u, in their study of the metabolism of Aroclor 1254 in male rats, found that the GLC-electron capture pattern of the residues was different from the standard. They concluded that all components were not metabolized at the same rate. Peak No. 3 in the Aroclor standard is composed of a tetrachloro- and j*entachlorobiphenyl. the latter in much greater proportion. A similar j>eak composition is observed for the cap>ule-dosed birds, and the birds sacrificed alter 14 days ol treatment. By 14 days post-treatment dechlorination apparently had occurred. After 42 da vs the |>eak had increased and was essentially the tetrachloropheuyl. Dechlorination of the ivntaehloro component is probable, for if the (vntachloro was eliminated, the jvak height should decrease significantly. TIC chromatograms of the liver samples from the birds sacrificed after 14 days' treatment were similar to the Aroclor 1254 partitioned standard. At 42 days |ttreatment the components of Aroclor 1254 were hardly in evidence; however, several DSW 032879 STLCOPCB4016841 226 C. E. BAGI.liY, E. CKOMAUTir. large peaks (out of tlic linear range) were observed. Two major |H'aks apjKared at relative retention times (Jij) of 0.44 ami o.JSa (with retention time of />,/>'-DI)lC equal to 1) and masked the Aroelor profile. MS analysis showed these to l>e butyl esters of short-chain fatty acids. It is susjx'eted, since the tcm|K'rature was 240, that these were thermal degradation products of highcr-moU-cular-weight li|H>idal compounds. These comjx'unds were observed only in the liver of treated birds, and might be related to the "fatty degeneration" observed by Vos and Kor.M.vN15 in the livers of PCB-dosed chickens. No significant peaks were observed in extracts of livers of control birds. Analysis of a 100-g sample of feed showed approx. 0.02 p.p.m. of DDT. None was detected in birds. . ACKNOWLEDGEMENTS We wish to thank Messrs. Robert Heath, James Spann, and Elwood Hill of this Center for providing the birds, mixing the feed, and offering general council in conducting the feeding study. REFERENCES 1 Technical Bulletin PL-J06, Monsanto Co.. St. Louis. Mo. 2 S. JENSEN, B. Nccci and G. Widmark, Institute of Analytical Chemistry, University of Stockholm, Sweden, unpublished notes sent to O.E.C.D. members, September, 190S. j G. E. Bagley. \V. L. Reicmel and E. Cromartie. J. Ass. Ojhc. Anal. Chon., 53 (1970) 231. 4 J. H. Koeman, M. C. ten Noever de Bravw and K. 11. DE Vos, Saline (Loudon). 221 (1909) 1126. 5 D. Sissons and D. Welti, J. Chroniatogr., 60 (1971) 15. 6 J. Armour and J, Burke, J. Ass. Ottic. Anal. Client.. 53 (>97) 761. 7 L. M. Reynolds, Bull. Environ. Contain. Toxicol.. 4 (1969) 128. 8 A. Y. Holden and K. Marsden. Xattire, 216 (1967) 1274. 9 B. M. Muliiern, E. Cromartie, W. L. Reiciiel and A. A. Beusle, J. Ass. Ojfic. Anal. Chnn.. 54 ('970 54S. 10 J. \V. Rote and P. G. Mvrpuv. Bull. Environ. Contain. Toxicol.. 6 (1970 377- 11 D. B. Peakall and J. L. Lincf.r. BtoUcimce, 20 (1970) 95S. 12 \V. L. Reichel, T. G. Lamont, E. Cromartie and L. N. Locke, Bull. Environ. Contain. Toxi col., 4 (1969) 24 13 D. L. Stalling, K. C. Tindle and J. L. Johnson, J. Ass. Oftc. Anal. Client., 35 (1972) 32. 14 D. L, Grant, \V. E. J. Phillips and D. C. Villeneuve. Bull. Environ. Contain. Toxicol., 6 (1971) 102. 13 J. G. Vos and J. H. Koeman. Toxicol. Afipl. Pharmacol., 17 (1970) . \ JvHruat t*f C Klscvjvr N ivttcihc l*i ciikom. (>319 l'KIClWRATl VIC 15 DRV-t'OLl'MN CM C. V. YISWAN ATII.W Jtiof/u ini.illv Pip.11 hurt A Iisfro'ian Siitionnl C1 (Received August :5th SUMMARY A simple ebre lipids has been des 1,2-diacyl-sn-glyccr1 (2-aminoethylpho>p Telrahymcna pyrifoi INTRODUCTION The concurrc AICP lipids' and g lipids of the proto?.! Kapovlas' and In (TLC), using solve minimal amounts < isolation of G-AICP 1 sn-glyccro-3-(2-amii glyccro-3-^2-aminoe el alisolated pure methanolvsis of a AND Horn" reporte their yields were \ In our Inborn described by |.\n fractionation of tie not only murli larf previously, but al diaryl G-AICP. Tl in detail. DSW 032880 STLCOPCB4016842