Document NQ2k3jXDjNkvOLROq1Nvw6nQ

EVIDENCE OF TETRACHLORODIBENZOFURAN (TCDF) IN AROCLOR 1254R AND THE URINE OF RATS FOLLOWING DIETARY EXPOSURE TO AROCLOR 1254R by August Curley* VIrlyn W. Burse2 Ralph W. Jennings3 Ellen C. Villanueva1* Renate D. Kimbrough5** Formerly: Environmental Protection Agency Chamblee Toxicology Laboratory 4770 Buford Highway Chamblee, Georgia 30341 Environmental Protection Agency, Research Triangle Park, N.C. 27711. 2U.S. Consumer Product Safety Commission, 1330 West Peachtree' Street, N.W., Atlanta, Georgia 30309, Environmental Protection Agency, Region IV, 1421 Peachtree Street, N.E., Atlanta, Georgia 30309. Vhe Coca-Cola Export Corporation, P.0, Drawer 1734, Atlanta, Georgia 30301. 5Center for Disease Control, Toxicology Branch, 1600 Clifton Road, N.E., Atlanta, Georgia 30333. * Portions of this paper were presented at the 163rd National ACS Meeting, Division of Pesticide Chemistry, April 10, 1972, Boston, Mass. ** Correspondence and reprint requests; Dr. R. D. Kimbrough. MOMS 212431 ABSTRACT Urine of rats following prolonged exposure to a polychlorinated biphenyl (Aroclor 125+**) and the Aroclor 1254** itself were analyzed for the presence of other chlorinated moieties by mass spectrometry. Ir. urine pentachloroblphenyl (M-l/e 324), the fragment resulting from loss of 70 mass units, and a molecular ion at M+/t* 304 with an isotopic cluster indicative of 4 Cl in addition to other PCD isomers belcw M+/e 230 were observed. One fraction (V) of the Aroclor 1254** florisil elution itself contained various PCD isomers and the molecular ion M+/e 304 with an isotopic cluster indicative o.f 4 Cl. This molecular ion M+/e 304 from urine as well as Aroclor 1254** compared favorably with the standard tetrachlorodibenzofuran and was also present in methylated urine of rats fed Aroclor 1254**. INTRODUCTION Recent reports and past findings indicate that impurities found in some technical products may result in disease following exposure. Chloracne, X-disease and the chick edema factor are classic examples of disease resulting from exposure to chlorinated compounds, contaminants in the same, or both*. Chlorinated dibenzodioxins (CDD) and chlorinated dibenzofurans (CDF) have been the most implicated as contaminants of polychlorinated biphenyls (PCB) VOS e al. accounted for differences in the toxicities of three commercially available PCB preparations, namely: Phenoclor DPG, French; Clophen A60, German; and Aroclor 1260**, U.S. by the presence of two polar compounds in the third fractions (25% Et^O in hexane) of the French and German products, namely tetra and pentachlorodibenzofurans. Chlorinated dibenzofurans, including the tetrachlorodibenzofuran as well as pentachloronaphthalene were also identified in a Japanese PCB (Kanechlor 400)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 Ute urine of rats following prolonged exposure to Aroclor 125UR. It also reports evidence in support of the presence of a similar compound in Aroclor 12S41* itself. HONS 212*32 EXPERIMENTAL In the first study sixLeen-hour urine camples were collected from eight male Sherman strain rats. Seven had been on a dietary level of 100 ppm Aroclor 1254K (between 13-5 mg/kg/day) for times varying from 4-58 days. One had been on a dietary level of 500 ppm Aroclor 1254K (about 25 mg/kg/day) for 252 days. These rats were started on the experimental dLet 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 nt and was extracted separately with hexane at a pH of about 7 and :luted 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 a second study, seven-day urine samples were collected from four female rats that had been fed Aroclor 12 54**, 100 pom (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 50 mis of extract was centrifuged and the supernatec combined. The extract was evaporated to IS mis and partitioned with acetonitrile as described by Mills . Prior to mass spectral analysis the sample was methylated using a procedure similar to that of Stanley6. Aroclor 1254, 1.7 grams, was dissolved in 300 mis of hexane. The chromatography column had an I.D. of approximately 34 mm and was filled with 180 grams of PR grade activated florisil. The column was pre-washed with hexane and the Aroclor standard wan added in 300 mis hexane. The fractions and volumes collected are listed in Table I. Analysis of each fraction using Coulson Conductometry revealed a general pattern Indicative of Aroclor 1254. TABLE I Elution of Aroclor 1254** from Florisil Elute Hexane 5% Et,0 in Hexane 25% Et20 in Hexane 50% Et20 in Hexane 50% EtjO in Hexane Vol. (mis) 1200 1200 200 mis each 500 mis 500 mis Fraction I II III, IV, V, VI, VII, VIII IX X RESULTS AND DISCUSSION The mass spectrum resulting from the analysis of authenic 2,3,7,8 tetrachlorodibenzofuran (TCDF) is shown in figure 1. The molecular ion M+/e 338 with a chlorine isotopic cluster indicative of 5 is the pcntachlorodibenzofuran (TCDF) obtained as an impurity during the synthesis of TCDF. The low intensity ion at H+/e 275 Is the fragment resulting from the loss of 63 mass units from PCDF^ The molecular AQNS 212433 r ion at M+/e 304, TCDF, contains the base peak. The fragmentography of TCDr is characterized by the loss of 63 mass units (C0C1) to yield the fragment at mVc 241 and subsequent loss of 70 mass units (2C1) to yield MVe 171. Doubly charged ions were observed at M+2/e 15? and M+2/e 120.5 with isotopic clusters synonomous with those of their singly charged counterparts at M*/e 304 and M+/e 241 respectively. The characteristic loss of C0C1 has been observed in the fragmentation of similarly structured compounds, namely the chlorinated dioxins and higher chlorinated dibenzofurans Curley et al,^. The urine spectrum from the first study (Fig 2A) shows the presence of pentachlorobiphenyl (M+/e 324) and the fragment resulting from the loss of 70 mass units to yield M+/e 254. Low intensity ions at M+/e 288 and M+/e 290 were observed. The molecular ion at M+/e 304 contains an isotopic cluster indicative of 4 Cl with one major fragment at M+/e 235 indicative of the loss of 2 Cl. Observation of the spectrum below MVe 230 revealed that it was attributed entirely to PCB's. Fraction V from the Aroclor 1254* florisil elution (Fig 2B) contains the hexachlorobiphenyl (M+/e 358), pentachlorobiphenyl (M+/e 324) in addition to tri and tetrachlorobiphenyl fragments (M+/e 28a and M+/e 253) resulting from the loss of 2 Cl from 358 and 324 respectively. Also present in the spectrum is the molecular ion M+/e 304 with an isotopic cluster indicative of 4 Cl. This ion compares favorably with TCDF. The low intensity of the ion at 304 precluded observance of any fragmentation. .Figure 2C shows the spectrum produced by urine that had been methylated. The spectrum indicates the presence of a monomethoxy derivative of pentachlorobiphenyl (M+/e 354) with major fragment ion* indicative ofs (1) loss of methyl and carbon monoxide (M+-43) to yield M+/e 311 and (2) subsequent loss of methyl and carbon monoxide and two chlorines (M+-113) to yield HVe 241. This spectrum also contains a molecular ion with an isotopic cluster indicative of four chlorines at M+/a 304. Hass spectral evidence does not establish unequivocally the presence of TCDF. However, it can be said that the molecular ion, 304, is not the result of fragmentation of a higher molecular weight component of the isomeric commercial Aroclor 1254* mixture. There is e noticeable difference in the relative intensities ot the molecular ions, H+/e 304, in the urine and standard Aroclor 1254*. The emount 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 was low necessitating a greatly amplified normalized spectrum. Hutzinger et_ l.have reported the presence of hydroxylated biphenyls in urine resulting from metabolism in addition to the presence of oxygen derivatives resulting from irradiation of Aroclor 1254* films; spectra in both instances show the presence of H+/e 306 with an isotopic pattern indicative of four chlorines. MONS 212434 This author and co-workers have observed a molecular ion matching the 106 species reported by Hutzinger, however its fragmentation has been characterized by the predominance of the fragment resulting from the loss of 70 mass units. The molecular ion M+/c 304 was also present in methylated urine obtain'd from 4 female rats that had consumed Aroclor 1254R for eight months (Fig ?C). This finding Indicates that a 304 tetrachloro > omponent is present in the urine and at a trace level in the analy ed commercial PCB preparation. A trace level of the 304 tetrachloro component was also present i^ the analyzed commercial PCB preparation. One would have to ccnsider the possibility of such a compound existing in the PCB preparation from a review of the purification process. This process consists of distillation of the crude material at reduced pressures (about 50 mm Hg) and elevated temperatures (150C-300C) in the presence of a few tenths of 1% of lime or sodium hydroxide, Papageorge10, Hubbard1*, and could lead to hydroxylation and the subsequent loss of HC1 could lead to a dibenzofuran derivative. legend Figure 1 Mass Spectrum resulting from the direct probe analysis, at 30 C and 70 eV, of authenicated 2,3,7,0 tetrachlorodibenzefuran; see Figure 2A for other conditions. Figure 2A Mass spectrum resulting from GC-MS analysis of 98 mis of rat urine. LKB 9000 GC-MS, mass marker *0.3 mass unit. GC column temperature, 209C; flash heater 235Ci glass coiled column, 6'xl/4" 1.5 OV-17/1.95 QF-1 on 60/80 mesh chromosorb "W" H.P., A.W. , DMCS; carrier gas (He) 30 psi and 45 cc/min.; separator, 320Cj source 290C; energy, 70 V; accelerating voltage, 3.5 KV; trap current, 60 uA; box current, 50 uA; leak current, 8 uA. Figure 2B Maas spectrum resulting from GC-MS analysis of Fraction V 25% Et-0/Hex of Aroclor 1254*. LKB 9000 GC-MS, other conditions see fig. 2A, Instrument calibrated with PFK. .. - Figure 2C Mass spectrum of 220 mis urine following methylation with diazomethane. Mass spectral conditions see fig. 2A, ACKNOWLEDGMENT The 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 the authenticated 2,3,7,Q totrachlorodibenzofuran. HONS 212435 u , REFERENCES KIMBROUGH, R.D. , Arch, Environ. Hlth. , 25, 125 (1972). VOS, J.G., KOF.MAM, J.H., VAN DER MAAS, H.L., TEN NOEVER DE BRAUW, M.C., and DE VOS, R. II., Fd. Cosmet. Toxicol., 025 (1970). ROACH, J.A.C. and POMERANTZ, I.H., Bull. Environ. Contain. Toxicol-, L2, 338 (1974). CURLEY, A., BURSE, V.W., GRIM, M.E., JENNINGS, R.W., and LINDER, R.E., Environmental Res., 4^ 981 (1971). MILLS, P.A,, J. Ass. Off. Agri. Chem., 42, 734 (1959), STANLEY, C.W., J. Agr. Food Chem., 14, 321 (1966). CURLEY, A., JENNINGS, R.W., BURSE, V.W,, VILLANUEVA, E.C., .Pesticide Chemistry, 4^ 71 (1974). HUTZINGER, 0., NASH, D.M., SAFE, S., DErREITAS, A.S.W., NORSTROM, R.J., WILDISH, D.J. , and ZITKO, V,, Science, 178, 312 (1972). HUTZINGER, 0., SAFE, S., and ZITKO, V., Environmental Hlth. Perspectives, No. 1, 15 (1972). Personal Communication. W. B, PAPAGEORGE, June 22, 1973. HUBBARD, H.L. , Encyclopedia of Chemical Technology, 5^ 289 (1965). / MOMS 212*36 FIGURE J 3 a> 360 HONS 212437 $ 1 I l 1 i I I i 1 [ W2 FIGURE 2A s lh "l-- --I------- 270 280 290 300 310 320 M+/e 324 100-1 FIGURE 2B 3 CM 0-J-- 230 T240 T* 270 280 356 HONS 212*3^ fc. Jf HONS 212**0 intensity %