Document MJ69ZYDooVqykgo1BdM6EjX0V

ji, bL CRBENHALCH 1ms. Microthem. J., jiw York, 1961, .dl, Loudon, 196a. SOB. 1968, pp. IQJ, j I Journal of Chromatography, 75 < 1073) i>0->i6 q Elaevier Scicntihc publishing Company, Amsterdam--Printed in The Netherlands CHROM. 6367 ELIMINATION PATTERN OF AROCLOR 1254 COMPONENTS IN THE BOBWHITE . GEORGE E. BAGLEY* *sr> EUGENE CRO.MARTIE , Bureau of Sport Fisheries and Wildlife, Patuxent Wildlife Research Center, Laurel. Md. 108to (U.S.A.) ' (Received August 31st, 1972) SUMMARY The gas-liquid chromatographic profile for Aroclor 1254 did not maintain its integrity in bobwhitc 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 shown by analysis of quail sacrificed within an hour after a single oral dose of Aroclor. j! Acctonitrile-hexanc partitioning altered the peak pattern of the Aroclor 1254 standard. . .. i> P Significant alteration of certain polychlorinated biphenyl isomers appeared to take place. Dechlorination was clcnrlv apparent, and isomeric transformation is suggested. INTRODUCTION The piesencc of polychlorinated biphenyls (PCBs) in the environment, their toxicity, and some of their physiological effects have been described for various species. The PCBs arc industrial compounds marketed in the United States under the trade name ArucJni (Monsanto). Other registered names are Cloplieu and I'henochlor. They all contain a complex mixture of closely related chlorinated biphenyls var\ mg in chlorine content. Aroclor 1254, which was used in this study, contains 54'`,, chlorine1 as indicated by the last two digits in its numerical designation. Some other Aiodors are designated 1232, 124:, 124S. i2(o, and ub2. Their uses are numerous and varied. There are 210 possible isomers and 102 mo>t likely2 H\r.t.i*v d ill1 identified 18 isomers in Aroclor 1234 by gas-liquid rlir<miatography-mav? spectrom etry (GLC-MS). Koojan 17 4//.* showed the presence of u isomers in Atoelor uoo, and Sissons and \Yi:ltj5 have show 11 that () compounds are present in Aroclor 125 j. Analysis of 1'OJs is routinely accomplished by GLC with electron capture * I*rtknt aildres* Enviromrn-nr.il I'mtcctinn Agency. IVshcnk* Program. WajUunylon. 1>.C. 20140. l .S.A. HONS Oti23i,6 220 C. E. BACLEY, E. CKOMART1E detection. Hie commonly occurring chlorinated jx'sticides sucli as DDT and its metabolites are determined similarly, and as bo;-:Yoccur in fish and wildlife tissues, the PCDs must lx* separated in order to obtsiv.- reliable analytical data. Several methods have been used*-*. There has been less progress in quantitation of TCBs 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 components of the Aroclors. In our laboratory, we use a semiquantitative thin-layer chromatographic procedure as reported by MlLHERN ct at.*. This method is applicable to all Aroclors in the izoo series. Other investigators have ued GLC peak heights of several peaks and even a single peak. Rote and Mnti'HY10 used a detector-response curve to determine the amount of each chlorinated component in lire PCB standard.-A review of various methods for quantitating PCB was presented by Peakall t .. .Lincer11. In analysis of bobwhite (Colinus Virginia**., -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 1554 standard. Some peaks were relatively higher and others lower than in the standard. We set up the present rludv 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 the integrity of the Aroclor GLC profile is not maintained, then only a method, which accounts for these changes, will give quantitatively dependable results. EXPERIMENTAL b Diet treatment of bobichite . Aroclor 1254 was dissolved in corn oil and then mixed with dry feed in a ratio of 2 parts of solution to 9S parts of feed. The concentration of Aroclor 1254 in solution was sucli that, when mixed with feed, a diet containing 300 p.p.m. of Aroclor resulted. Early in May 1970, 30 bobwhite, i-vcar-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 ol` 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 dirt free of toxicant but containing 2 parts of com oil to 9S parts of feed. On day X4.-fUter start Of treatment 3 birds were sacrificed. The remaining birds were then placed on clean feed; 3 were sacrificed on day 2S and the remaining 9 on day 56. Control hods were sacrificed at the same rate and interval as treated birds. At the end of tcsi>j birds from the control group were each given orally 1 capsule dose of Aroclor 125:,So corn oil equivalent to 500 mg/kg body weight and were sacrificed after 1 h. All bi;d,-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-R aliquot was mixed with sodium sulfate and extracted 7 li with redistilled hexane in a Soxhlcl apparatus. Aroci.or ! Whole live with sndiuj partitiooin of the unt analyzed b A pparntus An L column (g : Carrier gas flash heate ionization | CLC-MS a Total group sacn volume of at approxii set at the 1 injected we 109 mg of t of the bird* thereafter. To c^ 300 ftg of t on a Florist was made apparatus t Aroclor 125 Relative pea Rclat peak No. 5 highest pea tanird with, each ebrom; as dot ribcd Kl sl l.TS AM KcmiIi peaks heigh acetonitrileAroclor as a effect of are' and dt In mi: MQNS 082317 . CROMARTIE DDT and its idlife tissues, uta. Several m there are :n are related 7 laboratory, -reported by aeries. Other .single peak. amount of -jncthods for rt of Aroclor affered from r and others yon patterns : heights can .ZTity of the: at* for these sd in a ratio j in solution :ior resulted. ;:e Patuxent . maintained :g x week of ted diet and : containing j birds wen* sacrificed on .ie same rate : group weu* -> 500 mg, kg td. survived stinnltract) mixed with . apparatus. AHOCLOR 1254 COMPONENTS IN THE BOBWHITE til 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 acetonitrile-hexane partitioning and eluted on a Horisil column as previously described1*. 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 (y 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 mJ/min. Operating temperatures were: flash heater, 230; GLC oven, 220'; separator, 240; and ion source, 290'. The ionization potential was 70 eV, trap current 60 /<A, 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 arbitrarilv set at the level produced by 6 of Aroclor 1254 standard. The amounts of sample injected were as follows: 30 mg of the original sample of the capsule-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 leed for either 14 or 42 days thereafter. To evaluate possible effects of the cleun-up procedure on the Aroclor standard, 300 /jg of technical Aroclor 1254 was partitioned by acetonitrile-hexane and eluted on a Horisil column in the identical procedure, used for the bird tissues. The eluate was made to appropriate volume and a portion was injected into the C.I.C-MS apparatus to produce a response on the TIC chromatogram approximating b /<g 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 orderlv. All peaks were main tained within the linear range of the recorder, and the height of the reference |x-nk in each chromatogram was kept similar by changing the amount of the sample injected, as descriU'd above. RESULTS AND IHSCTSSION .. - Results me shown in Table I and in Figs. la-f. Table l compares the relative )icnks heights in the Aroclor 1254 standard, the Aroclor 1254 standard Mihjected to acetonitrile-hexane partitioning, and the quail carcass ex traits. The change in the Aroclor ns a result of partitioning is believed to he due to the di>trihution coefficient effect of acetonitrile hexane. StaU.isc. %t ul.n al>o observed this died in their wmk and determined /> values for all peaks in Aroclor 1254. MUNS 082318 222 C. E. BAGIEV, E. CR0MAIIT1E TADLE1 RELATIVE PEAK HEIGHTS* IN TIC CHROMATOGRAMS Simple Peak No. t$3 4J 67 8 9 to it 17 *b o-49 0.58 0.69 0.78 0.83 094 x.06 t.rj r.37 r.40 e.6r i.8t No. of CP 4 4 4.5 5 3 3 3 6 6 6 6 6 Aroclor4 1254 (S) Aroclor* 1254 (P) Extract C1 Extract D Extract E" Extract F* 101 34 49 80 100 *3 39 68 73 30 30 34 4 57 98 too 32 44 7 65 27 3 3 Ji8 a 3 96 too P 49 75 70 28 33 33 93 S *5 43 too <0 39 5 63 28 21 39 54 0 *4 0 too 33 *3 63 3< 12 48 5 0 2b 0 100 T 34 0 77 66 `4 86 Peak No. 5 * ioo%. * />,p'-DDF. - i.o. .. * No. of Cl in PCB. . S m standard. Pm standard partitioned by acetonitrile-hexane and eluted on Florisil. Carcass of quad administeredI capsule of Aroclor 1234. 500 mg,'kg body weight. For details, sec teat. Carcass of quail sacrificed at day 14. F01 details, sec text. * Carcass of quail sacrificed at day rS, For details, see text. 1 Carcass of quail sacrificed at day 56. For details, sec text. AliOCUH 26 FJg. 1. TIC chromatograms. C.LC conditions: o ft. v 25 in column, s% OV-i 7 ; o\ en temperature, IJJO*. and helium llow-ratc, 40 inPnun. (a) Aroclor i-*M standard; (b| Arnclor 1 .-34. aectnmtrilehoxanv partitioned, standard; (>-} carcass i-Mraets of 11u.itl administered a simile capsule ilusc of Aroclor t.*54 at 300 mg kg body weight; (ill carcass extracts of quad saenticed alter 14 days' dietarv dosage at too p.p.m. of Arm lor 1254; le) carcass extracts ol quad sacrilued after 14 days' dietary do-.me at 300jvp.n1. of Aroclor 1/54 followed liv 14 davs ol untreated loud, (f) carcass exir.irts of quail saimiied alter 14 day.s' dietary dosage of 300 p.p.m. of Armlur 1234 followed by 44 days of untreated food. I ~rrr*r' MOWS 084319 ts I m lrtn|H'Tturr, _l. a.'itonitnli'- T|IMlU' do'll1 of tior 14 ih\V Jilicnl nflrr 14 .i-ali'd (f) -<f Anxlor U34 MUMS 082320 324 C. E. BAGIEV. E. CROMABTIE Arocloa 135 Relative peak heights anil chromatograms for the tissue extract of birds given Aroclor as a single capsule dose were similar to those of (lie .\roclor standard after partitioning (Table 1 and Figs, in ami c). This suggests that all components of the Aroclor were icadily absorbed. Relative peak heights in chromatograms for birds sacrificed 14 (day ami 42 (day $(>) days post-treatment (Figs. ie and f) show a methodical elimination of certain components and an increase in others by com parison with the chromatograms for tlie hiids sacritued alter 14 day-.' dosage (Wg. id). After 42 davs of untit ated loud, only four major component remained, peaks Nos. 5, 9, 10, and 12. Teaks 10 and 12 increased significantly. Peaks beyond j2 showed no significant change. -- The ch certain cotnj in tissue sin chlorinated : IsomcriziiiioI some' reported. T). place. Peaks peak No. $, Quant identical an inatogiaph, decrease of plained simj Gran found that l stand,ird l! Peak c/dombipho is oliNitsed tU'.llllH'Ilt After 42 ilDeehliuiiial elimin.iled, TIC c treatment t treatment t HQNS 08,2321 t\ *. CR0MART1E AROCLOn I254 COMrONHNTS IN THE DODWRITE 22$ r "1 of biuU given t stnmlaid after moments of tin* grants for birds . v and f) show a nthets In- coin* diwage (l'ig. id), .d, peak* Nos. 5. girt li showed no Fif. .. (eonlinned). The chromatograms show that a dynamic system is at work, slow ly removing certain components while increasing others. MS data confirmed all peaks observed in tissue samples to lx.* identical to the ones in the Arodor 1254 standard. No foreign chlorinated isomer? were detected. Isomerization 0/ PC 11 Isomeric transformation of I'CB 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 0 chlorines show a distinct increase while peak No. S, also containing 6 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 peak? and increase nr decrease of others "as ns apparent as in Table I. Tims the changes cannot lx? ex plained simply bv faster elimination of certain components. Grant ,7 ol.il, in their study of the metabolism of Arodor 1254 in male rats, found that the Gl.C-elet trim capture pattern of the residues was diiieieni from the Standard. They concluded that all components were not metabolized at the same rate Peak No, 3 in the Aroclor standard is compos'd of a totraeldoro- and pontachlorobiphcnvl, the latter in much greater prupotiimi. A similar peak i itinpo-iiion is observed for the capsule-dosed bird', ami the birds sacrificed alter 14 days of treatment. Hv 14 days post-treatment dechlorination apparently ii.ul occurred. After 42 dav> the jvak had increased and wa cs.emiallv the tetraeliloropheiiv 1. Per blot mat in u of the ]>entael dot o 1 omponent is pv< >bal>le, for it the pent.u liloro was eliminated, the |vak height should decrease significantly. TIC chromatograms of the Iher sample' `m the birds sacrificed after 14 da\s' treatment were similar to the Amelor 1234 partitioned staml.ud. At 42 days po-ttreatnunt the components of Ar*e|or 2234 were lianllv in evidence ; however, several MONS 082322 226 C. E. BAREEV, E. CBOMAKTlE large peaks (out of the linear range) were olcrved. Two major jvaks apjK'ared at relative retention limes (Itj) of 0.44 ami o.S' (with retention time of /*./*' DDU equal to I) ami marked the Aroelor profile. MS analysis showed the-e to In* Imtyl etterb of short-chain fatty acids. It > -usjv.v <!, since the temperature teas '4O0, that these were thermal degradation produets of higher-moUeular-weiglit hpoidnl compounds. These coiu|>oumls were observed only in the liver of treated bittls, and might lu* related to tin* "fatty degeneration" observed by Vos and Koi'.man14 in the livers of PCB-dosed chickens. No significant ]x-aks were observed in extracts of livers of control birds. Analysis of a ioo-g sample of feed showed approx. 0.02 p.p.m. of DDT. None was detected in birds. ACKNOWLEDGEMENTS We wish to thank Messrs. Rodert Heath, James Spann, and Eiwooo Hill of this Center for providing the birds, mixing the feed, and offering general council in conducting the feeding study. REFERENCES I Technical bulletin PL-3o6, Monsanto Co.. St. Louis. Mo. a S. Junsrn, 11. Nvcci ash Cl. WinMAHK. liiHtilutc of Analytical Chemistry, University of Stockholm. Sweden, unpublished notes sent to U.E.C.D. mvmbvis, September, 1968. 3 G, E. liAGl.f'V, U-. L. Kcichrl am/ F.. (. rou.artm:. J. -its. Ottit. Aturl. Chrut., 53 (1 g;o) 231. 4 J. H. Koeman, M. C. tex Noever or. Hk.trw and K, H. DE Vos, .Vo/mr (London). 221 1126. 3 1). Sissons and D. Welti. J. Chf'iinrtorr.. 00 (1971) 15. 6 ). AftMOVR AND J. llVRKL, J. A st. (>'/u. A lltli. Chi lli.. .S3 (970) 7*>*< 7 L. M. Kevnolds. Hull. Environ. Contain. 7\/.vt<'/., 4 (1909) 128. k A. V. ]Iolui:n anu 1v. .Marspen. Sotnic, 216 (191,7) 1274. 9 11. M. Mcliierx. E. Ckomartie, \V. L. Keiciiel and A. A. IIelisle, J. Ass. Offu. .Inn/. Chew.. 54 197) 54*10 J. \V. Roil anu F. G. Mirnniv. Uutl. Environ. Contain. Tokico!.. 6 (1971) 377. 11 D. H. Feak.ll and ]. L. Linckk. HmScicntr. 20 (1970) 958. 13 \V. L. Kliciill, T. G. Lamost. E. Ckomartie anu L. N. Locke, Hull. Environ. Contain Ton1- >/., 4 (1909) 24. 13 I). L. Stalling. K. C. Tindle and J L. Johnson, J. Ats. Opt. Anal. Chew., 53 (1972) V14 1>. L. Grant, W. t. J. Phillis** am* 1). C Villenecve, Hull. Environ. Contain. Toxicol.. 6 (1971) >02. 13 j. G. Vos and J. H. Koeman. Toxicol. Af>pt. Pharmacol., 17 (1970) O56. Journal >>( ( In <' ICIlH-VIVt N It UtllK 1*1 OIKOM. bjlf) PKKIUKATIVK 15 PRY-t OLVMN Cll C. V. VtSW \N \TIIAN /tinr/ii > r>\ Ihf'nhitfH Atitlia'ian A oiiouiii I'tt (Received Aiijut j$th. SUMMARY A simple chro lipids has been ilex i,2-Hiaryl-s>i-glycrTf (2*aminneth\ lpho?pI Tdrahvniciiii fyn/or IXTHOIM-CTluN The concurret AEP lipid- and pi lipids of the proto*.* KAi'Ot LAS* and Tim (TLC), u*mg xilvcis minimal am-amts n isolation of (i-.\KI> i>i-glvcero.-').(2-amii glvceio-3-i.' aminnr ct a/.1 iM*latcd punmethnnoK-i> <>f a t ANU Mom* reporter their \ icM m1ie t lu nnr l.ilxiiat described In J u P fractionation of the not only much largpreviou-lv, but nl* diart 1 f.-AnV lit. in detail. HONS QZZ323