Document babQ55YwxRwZxkJL5add9gBv3

Piqm (A# d**k ot W. B. PAPACEORCE M. Dleterlch R. E. Keller J, p. Mleure R. H. Munch W. R, Richard D. Wood 9/24/75 Note: Curley's paper was given at ACS meeting In 1972. Scott Tucker discussed with Curley doubts we had regarding true Identification. Bob Keller: Do we still have these doubts7 MOHS 20229? E. P. V/liwIw Evidence of Tcfraclilorocfihcnxofuran (TCDF) in Aroclor 1254", and ihe Urine of Kats Following Diclary Exposure (o Aroclor 1254"* by Aucust Curley'. VifiLTN W. Rukse', Ralph W. Jehnimcs', F.llzx C. Villanueva*, nd ItCMATC D- Kiaiukoucii'** Environmental 1`rotrrtinn Agency CAamblre Toriruloty !.t]boratury 4770 Ilufard Ihtthnor CMombltt, C JUJ4I INTRODUCTION Reesnt reports end pest findings Indicate thet impurities found in some technical products may result in disease following exposure. Chloracne, X-dismas* end the chick edema factor arc classic examples of disease resulting from exposure to chlorinated compounds, contaminants in the seme, or both1. Chlorinated dibenzodloxins (ODD) and chlorinated dibenzofurans (CDF") have been the most Implicated as contaminants Of polychlorinated biphenyls (PCB), VOS t al. accounted for differences in the toxiclrles of three commercially available PCB preparations, namely: Phenoclor DPG, French; Clophen ABO, German; and Aroclor 1260*, U.S. by the presence of two polar compounds in the third fractions (25% Et.Q in hexana) of the French and German products, namely tetra and pentaehlorodihenzofurans. Chlorinated dibenzofurans, including the tetrachlorodibenzofuran as well ss pentachloronaphthalene were also identified in a Japanese PCB (Ksnechlor too)3* Neither was found in the U. S. product, Aroclor 1260*. This paper reports the details of the mass spectral findings of a tetrachloro compound with a molecular weight of 304 in the urine of rats following prolonged exposure to Aroclor 1254*. It also reports evidence in support of the presence of a similar compc :nd in Aroclor 1254* itself. ^Environmental Protection Agency, Research Triangle Park, N.C. 27711. 2U,S. Consumer Product Safety Comnislion, 1330 West Peachtree Street, N.V., Atlanta, Georgia 30309- ^Environmental Protection Agency, Kagion XV, 1421 Peachtree Street, S.E-, Atlanta, Georgia 30309. *The Coca-Cola Export Corporation, P.0, Drawer 1734, Atlanta, Gaorgia 30301. 3Cntw for Disease Control, Toxicology Branch, 1600 Clifton Road, I.E., Atlanta, Georgia 30333. Portions of this psper ware presented et the 163rd Mstionsl ACS Keeting, Division of Pesticide Chemietry, April 10, 1972, Boston, Hass. *e Correspondence and reprint requests; Or. R, D- Kimbrough. 153 ViL 14, Nft.it im fry Vmrtt* TIIh, MOHS 202298 EXPERIMENTAL In the first study sixteen-hour urine samples were collected fmm eight male Sherman strain rats. Seven had been on a dietary level of 109 PPm Aroclor 1254** (between 13-5 g/kg/day) for tines varying from 4-58 days. One had been on a dietary level of 500 ppm Aroclor 1254** (about 25 mg/kg/day) for 252 days. These rats were started on the experimental diet when they were about 45 days old. The total Aroclor consumption of all rats over the entire period of exposure up to the time the urine was collected was 3,0 grams. Urine averaged 11 mis per rat and was extracted separately with hexane at a pH of about 7 and eluted from micro silica gel columns with a 1:1 mixture of benzene'.hexane according to the method of Curley et_ al_.**. Samples were combined prior to mass spectral analysis. In e second study, seven-day urine samples were collected from four female rats that had been fed Aroclor 1254**, 100 ppm (7.5 mg/kg/ day) for eight months. The pooled urine sample, 440 mis - pH 6.6, was extracted six times with 50 mis of diethyl ether-hexane (3:1). Each SO mis of extract was centrifuged and the supemates combined. The extract was evaporated to 15 mis and partitioned with acetonitrile as described by Mills5. Prior to mass spectral analysis the sample was methylated using s procedure similar to that of Stanley6. Aroclor 1254**, 1.7 grams, was dissolved in 300 mle of hexane. The chromatography column had an I.D. of approximately 34 mm arid was filled with ISO grams of PR grade activated florisil. The column was pre-washed with hexene end the Aroclor standard was added in 300 mis hexane. The fractions and volumes collected ere listed in Table I. Analysis of each fraction using Coulccn Conductometry revealed a general patters indicative of Aroclor 12 54**. TABLE I Elution of Aroclor 1254** from Florisil Elute ` Hexane 5% Et,0 in Hexane 25% EtjO In Hexane 30% EtjO in Hexane $0% EtjO In Hexane Vol. (mis) 1200 1200 200 mis each 500 mis 500 mis Fraction 1 II III, IV, IX X RESULTS AMD DISCUSS ION The mass spectrum resulting from the analysis of euthenic 2,3,7,9 tetreehlorodibenzofuran (TC0F) is shown in figure 1, The molecular iom H*/e 338 with a chlorine isotopic cluster indicative of 5 is the peotachlorodibensofuran (PCDF) obtained as an impurity during the synthesis of TCDF. The low intensity Ion at H /a 275 is the fragment resulting from the loss of 63 mass units from PCDF. The molecular 134 MONS 202299 ion at M+/e 304, TCDr, contains the base peak. The fragmentograpby of TCDr is characterized by the loss of 63 mass units fCOCl) to yield the fragment at Ve 241 and subsequent loss of 70 mass units (;c 1) to yield MVe 171. Doubly charged ions were observed at M+J/e i52 and H+2/e 120.5 with isotcpic clusters synonomous with those of their singly charged counterparts at MVe 304 and M*/t 241 respectively. The characteristic loss of C0C1 has been observed in the fragmentation of similarly structured compounds, namely the chlorinated dioxins snd higher chlorinated dibenzofurans Curley et al.7. The urine spectrum from the first study (Tig 2A) shows the presence of pentachlorobiphenyi (M+/e 324) and the fragment resulting from the loss of 70 mass units to yield Ve 254. Low intensity ions at MVe 288 and M+/e 290 were observed. The moleculer ion at MVe 304 contains an isotopic cluster indicative of 4 cl with one major fragment at H*/* 235 indicative of the loss of 2 Cl. Observation of the spectrum below M+/e 230 revealed thet it was attributed entirely to PCB's. Fraction V from the Aroclor 1254* fiorisil elution (Fig 2B) contains the hexechlorobiphenyl (MVe 358), pentachlorobiphenyi (D'Ve 324) in addition to tri and tetrachlorob I phenyl fragments (MVe 288 snd Nve 253) resulting from the loss of 2 Cl from 358 and 324 respectively. Also present in the spectrum is ths molecular ion MVe 304 with an isotopic cluster indicative of 4 Cl. This Ion compares favorably with TCDF. The low intensity of the ion et 304. precluded observance of any fragmentation. Figure 2C shows the spectrum produced by urine that had been ethylated. The spectrum indicates the presence of a monomathoxy derivative of pentachlorobiphenyi (K*/e 354) with major fragment lens indicative of: (1) loss of methyl end carbon monoxide (HV43) to yield M*/e 311 end (2) subsequent lose of methyl end carbon monoxide and two chlorines (If** 113 > to yield M+/e 241. This cpcctrum also contains a molecular ion with an isotopic cluster indicative of four chlorines at M*/e 304, Mass spectral evidence does not establish unequivocally the preeence of TCDF. However, it can be said that the molecular ion, 304, is not the result of fragmentation of a higher molecular weight eoag>onent of the isomeric commercial Aroclor 12S4R mixture. There ia a noticeable difference in the relative intensities of the molecular ions, H*Ve 304, in the urine and standard Aroclor 1254H. The amount of Aroclor represented in Fraction V is 1.7 grams, while the urine represents 3.0 grams consumed over an extended period. The urine sample enrichment in the analyzer tube waa low necessitating greatly amplified normalized spectrum. Hutsinger et el.*, hve reported the presence of hydroxylated biphenyls in urine resulting from metabolism in eddition to the presence of oxygen derivatives resulting from irradiation of Aroclor 1258* films', spectra in both instances show ths presence of MVe 306 with an isotopic pattsm indicative of feur chlorines. This author and co-workers have obaerved a molecular Ion matching the 306 species reported by Hutlinger, however its fragmentation haa been characterised by the predominance of the 1 MOWS 202300 Figure 1 Maas Spectrum resulting from the direct probe analysis, at 30C and 70 eV, of authenlcated 2,3,7,8 tetrachlorodibenzofuran; see Figure 2A for other conditions. MOWS 202301 Figure 2A fc Hass spectrum resulting from GC-MS analysis of S3 mis of rat urine. LKB 9000 GC-MS, mass marker *0.0 mass unit. GC column temperature, 209C; flash heater 235*C', glass coiled column, S'xlA" l.S 0V-17/1.95 QF-1 on 60/80 isesl. chromosorb "V" H.P. , A.W., DMCS; carrier gs# (He) 30 psl and 4S cc/min.; separator, 320*C; source 290*C; energy, 70 eV; accelerating voltage, 3.5 KV; trap currant, 80 uA; boa current, 50 uA; leak current, 8 uA. mauttm * |* * Figure 28 1 X 8 L 'i1 _ _ it.. fl.. . m tm m ni n m m tit tiig Ud 1*011 wie Hess epeetrum resulting fro* GC-MS analysis of Fraction v 2St Et-0/Hex of Aroelor 12SC** UCB 9000 GC-HS, other conditions sea fig. 2A. Instrument calibrated with me; 1S7 HONS 202302 fragment resulting from the loss of 70 mass units. The molecular ion M+/e 304 vas also present In methylated urine obtained from 4 female rats that had consumed Aroclor 1254^ for eight months (Fig 2C). This finding indicates that a 304 tetrachloro component is present in the urine and at a trace level in the analyzed commercial PCB preparation- A trace level of the 304 tetrachloro component was also present in the analyzed commercial PCB preparation. One would have to consider tha possibility of such a compound existing in the PCS preparation from a review of the purification process. This process consists of distillation of the crude material at reduced pressures (ebout SO am Hg) and elevated temperature* (150*C-300*C) in the presence of e few tenths of 1\ of. lime or sodium hydroxide, Papageorgei0, Hubbard**, and could lead to hydroxylation and the subsequent loss of HC1 could lead to a dibenzofuran derivative. ACKNOWLEDGMENT Tha authors are indeed grateful to Dr's. A. Poland, J.J. Wade and A.S. Kende, School of Medicine and Department of Chemistry, University of Rochester, Rochester, New York for a gift of tha. authenticated 2,3,7,8 tetrachlorodibenxofuran. REFERENCES KIMBROUGH, R.D. , Arch. Environ, tilth., 2S, 12S (1972). VOS, J.G., KOEKAN, J.H. , VAN DER MAAS, H.L., TEN NOEVER DE BRAUW, M.C., and DE VOS, ft. H., Fd. Cosnet. Toxicol., 625 (1970). . ROACH, J-A-G. and POKERANTZ, I.H., Bull. Environ. Contanin. Toxicol., 12, 33 (1974). CURLEY, A., BURSE, V.V., GRIN, M.E., JENNINGS, R.V., and LINDER, R.. Environmental Res., 4, 481 (1971). MILLS. FA., J. Ass. Off. Agri- Chaa., 42, 734 (19S9). STANLEY, C.V., J. Agr. rood Chaa., 14, 321 (19*6). CURLEY, A., JOININGS, R.V., BURSE, V.V. , VILLANUEVA, E.C., Pesticide Chemistry, 4_, 7i (1974). BUrZINSEX, 0., NASH, D.M., SATE, S., DEFREITAS, A.S.W., MORSTROH, R.J., WILDISH, D.J., and ZITXO, V., Science, 178, 312 (1972). HUTZIWGER, 0.. SATE, S., and ZITXO, V., Environmental Hlth. Perspectives, No. 1, IS (1972). Personal Communication. W. B, PAPAGE0RGE, June 22, 1973. HUBBARD, 8.L., Encyclopedia of Chemical Technology, 5, 289 (1965). 13* HOMS 202303