Document 1gZkXaXbbM4GXKzop129E6Bad

Tissue Residues from Subacute Oral Feeding of Polychlorinated Biphenyl Dielectric Fluids by Robert G. Kaley, Orville Hicks, William M. Mees, E. Scott Tucker, James P. Mieure, Frederick.R. Johannsen, and George J. Levinskas Monsanto Company 800 /V. Lindbergh Boulevard St. Louis, Mo. 63166 Since the identification of polychlorinated bi phenyl (PCB) residues as environmental contaminants, numerous studies have investigated the uptake, distri bution, and depuration of PCBs by various organisms (HUTZINGER et al. 1974). It has been established that the lower chlorinated PCBs are readily biodegraded and that resistance to biodegradation increases with in creasing degree of chlorination {BAXTER et al. 1975, METCALF et al. 1974, TUCKER et al. 1975). Monsanto Company, a producer of PCB fluids, marketed as Aroclor* products, has restricted sales to use as dielectric fluids in sealed systems such as capacitors and trans formers. Recently a new product, Aroclor 1016, has been introduced. Aroclor 1016 contains only about one tenth the level of the more resistant penta- and hexachlorobiphenyls as the functionally similar product Aroclor 1242. The purpose of the present study was to assess the tissue residue accumulation and depuration of Aroclor 1242 and Aroclor 1016 during a 30-day subacute feeding to albino rats. Monochlorobiphenyl (MCBP) and a 30%chlorinated biphenyl, MCS 1043, were included in this feeding study. During the course of this study, other workers (BURSE et al. 1974) reported that Aroclor 1242 accumu lated to a lesser extent than Aroclor 1016 in rat tis sues. This result was somewhat unexpected since, as mentioned above, the only difference in the products is the level of the more resistant, more highly chlorina ted biphenyl homologs, as shown in Table I. It is these more highly chlorinated species which have been consistently reported as environmental residues in wildlife (POLYCHLORINATED BIPHENYLS AND THE ENVIRONMENT 1972) . Registered Trademark of Monsanto Company 699 Bulletin of Environmental Contamination A Toxicology. Vol. |,V No. 6 1976 hy Springer-Verlag New York Inc. DSW 033786 TABLE I TYPICAL % COMPOSITION OF POLYCHLORINATED BIPHENYL PRODUCTS HOMOLOG MCBP Cl/BIPHENYL 0 ND 1 100 2 ND 3 ND 4 ND 5 ND 6 ND 7 ND 8 ND PERCENT (W/W) BY GC/MS BY HOMOLOG RESPONSE MCS AROCLOR AROCLOR 1043 1016 1242 0.1 <0.1 <0.1 22 1 1 72 20 16 6 57 49 ND 21 25 ND 1 8 ND <0.1 1 ND ND <0.1 ND ND ND USING AREA CORRECTION FACTORS ND = NONE DETECTED, <0.01% Experimental Eight batches of feed were prepared by mixing the four products at two levels each into Ralston Purina rat chow. The treated chow was fed ad. libitum to adult male Sprague-Dawley albino rats for an exposure period of 30 days. At predetermined intervals during the ex posure period (days 2, 5, 10, 20, and 30), five rats from each set were sacrificed. Fat, liver, and muscle were excised for analysis and composited by tissue for each group. Following the 30-day feeding period, all remaining rats were placed on the basal laboratory diet. At days 32, 35, 45, 75, and 105 of the study (days 2, 5, 15, 45, and 75 of recovery), tissues were sampled in a manner identical to that used during exposure. Samples were composited and quick-frozen in glass containers with aluminum foil-lined caps to minimize risk of con tamination. A control composite of tissues from rats fed only the basal laboratory diet was collected at each sampling date to determine possible interferences in the analyses of tissues from the exposed animals. During the period that rats were fed PCB-containing rat chow, their food intake and body weight gains were sim ilar to those of rats fed the basal laboratory diet alone. Feeding, sacrifice, and tissue excision were car ried out at Younger Laboratories, Inc., St. Louis, Mo. The samples were delivered to our laboratories for sam ple clean-up and analysis. The PCB residues were isolated from the tissues by solvent extraction. A weighed amount (approximately 10 g) of each composited tissue was placed in an Erlenmeyer flask and homogenized three times with 25 ml of 700 DSW 033787 pesticide grade hexanes and anhydrous sodium sulfate using a Polytron ultrasonic homogenizer (Brinkman In struments, Westbury, N.Y.). The combined supernatants and washings were filtered through anhydrous sodium sulfate and diluted to 100 ml with pesticide grade hexanes. A 5 ml aliquot of the hexanes solution was pipetted onto an alumina column (TUCKER et al. 1975) and the PCBs were eluted with 125 ml of pesticide grade hexanes. The column eluate was collected in a Kuderna-Danish evapora tive concentrator, a 3-ball Snyder condenser was attached, and the solution was concentrated for electron capture gas chromatographic analysis. The gas chromatographic conditions are listed in Table II. TABLE II Instrument: Column: Temperatures: Injection Port: Detector: Column: Carrier Gas: Purge Gas: Pulse Interval: Hewlett-Packard 5753A Research Gas Chromatograph (Ni-63 Pulsed Electron Capture Detector) 2 m X 1/4" glass column packed with 4% XE-60 on 80/100 mesh Chromosorb W-HP 230 C 300 C 140 C Isothermal for MCBP and MCS 1043 170 C Isothermal for Aroclor 1016 and Aroclor 1242 Helium, 60 ml/min 10% Methane in Argon, 120 ml/min 50 microsec A second 5 ml aliquot of the extract solution was pipetted into a tared 50 ml beaker. After evaporation of the solvent under a stream of nitrogen, the beaker and residue were reweighed to obtain the lipid weight of the aliquot. All residue levels are reported as parts per million (ppm) on a lipid weight basis. Calibration curves for each product were prepared by plotting detector response (total peak area) versus nanograms of material injected. Residue levels were determined by summation of the total area of the PCB peaks in the sample and use of the appropriate calibra tion curve. The calculations were done as follows: Residue (ppm) = ^ (Vj)(W) Where N = amount of PCB from calibration curve (ng) 701 DSW 033788 STLCOPCB4017750 Vf = volume of final concentrate (ml) Vj = volume injected (ul) W = lipid weight of original sample (g). Results and Discussion Plots of the data for the PCB adipose tissue resi dues for the chlorinated biphenyl products are shown in Figure 1. The values plotted represent the levels of each product found in the adipose tissue, calculated on a lipid weight basis. Ill MOMOCHlOROBIPHfUVI Ihl MCS IM) Id AROCIOR I2 Idl AR0C10R 1016 Figure 1: Adipose Tissue Residue Level Versus Time Plots for Products Investigated 702 DSW 033789 STLCOPCB4017751 The test error associated with the data points of Figure 1 was evaluated using the logarithms of the data points for which replicate determinations were performed. The 95% confidence limits for the determination of Aroclor 1016 and Aroclor 1242 were calculated to be 79% to 126% of the observed value of a data point. In the case of MCBP and MCS 1043 the test error limits were calcu lated to be 63% to 160% of the observed value. MCBP was fed at levels of 25 and 100 ppm. Figure la shows that MCBP reached a relatively stable level of about 3 ppm for the low dietary level and 9 ppm for the high dietary level immediately after feeding began. These levels were maintained throughout the 30-day ex posure period. Upon removal from the test diets, the tissue residue levels dropped to such low levels that no MCBP was detected in fat extracts five days after cessation of exposure. MCBP thus showed no tendency to accumulate or be retained in fat. When fed to rats at the same levels as MCBP, the behavior of MCS 1043 was similar to that of MCBP. It reached a plateau in fat immediately upon initiation of feeding and maintained that level throughout the ex posure period. This behavior is shown in Figure lb. Upon cessation of exposure the level of MCS 1043 in fat rapidly dropped to such an extent that it was not de tected after 15 days of the recovery period. Figure lc is the plots of the fat residue levels for the 5 and 25 ppm feeding levels for Aroclor 1242. The curves indicate that Aroclor 1242 accumulated in the fat and continued to build up throughout the expo sure period. The shape of the build-up curve suggests, however, that a plateau level was being approached. Upon removal of the test diet, the Aroclor 1242 level in the tissues declined rapidly. However, at day 75 of the recovery period, detectable levels of Aroclor 1242 remained in the tissues. The residues in the tissues sampled after the exposure period consisted of the more highly chlorinated homologs, as discussed below. The plots of the fat residue levels for Aroclor 1016 are shown in Figure Id. Aroclor 1016 accumulated more slowly than Aroclor 1242 during exposure, and de clined rapidly during recovery. Low levels of Aroclor 1016 remained in the tissues through day 75 of the re covery period. Figure 2 emphasizes the differences in response to the feeding of the products at the same dietary level. The difference in the behavior of MCS 1043 is obvious, since it did not accumulate in fat and rapidly declined 703 0$w 033790 STLCOPCB4017752 to below the detection limit when the animals were re turned to the basal laboratory diet. Figure 2: Adipose Tissue Residue Level Versus Time Plots for Aroclor 1242, Aroclor 1016 and MCS 1043 at 25 ppm Feeding Level As shown in Figure 2, the residue levels of Aroclor 1016 were lower than those of Aroclor 1242. A test was made to see if this difference was statistically signi ficant based on the estimated precision of the determi nations, The 95% confidence limits for Aroclor 1242 and Aroclor 1016 (79-126%) were superimposed on theoretical accumulation and recovery plots (based on multiple re gression analysis of the actual data) for the two pro ducts. These plots are shown in Figures 3a and 3b. The difference in residue levels for the two products is statistically significant at the 95% confidence level where the superimposed error limits on the regression curves do not overlap. As can be seen from the graphs, at day 17 of the study the difference between the resi due levels of Aroclor 1016 and Aroclor 1242 became sig nificant and remained so throughout the remainder of the exposure period and the entire recovery period. Thus, this study shows a significant difference in the tissue residue levels of Aroclor 1016 and Aroclor 1242. 704 DSW 033791 These findings differ from those reported by Burse et al. (1974). The feed levels in our study, 5 and 25 ppm, were chosen to be high enough to permit precise and ac curate determination of residue levels but low enough to have minimal effect on the health of the rats. Burse et al. did report some morphological effect in the livers of the rats at the 100 ppm exposure level used in their study. Figure 3: Regression Curves for Accumulation and Recov ery with Superimposed Error Limits for Aroclor 1242 and Aroclor 1016 Figures 4a and 4b are chromatograms of the extracts of the feed and fat tissue (30-day exposure samples) for Aroclor 1242 and Aroclor 1016, respectively. The chro matograms for Aroclor 1242 show that most of the early peaks seen in the extract of the feed have disappeared or decreased in relative size in the fat extracts, indi cating that the corresponding components were not re tained in the tissue. These peaks correspond to PCB homolo^s with a low degree of chlorination, i.e., diand trichlorobiphenyls. Some peaks corresponding to more highly chlorinated homologs increased in relative size, indicating a greater tendency for these homologs to accumulate. However, it should be noted that since relative electron capture detector response increases with increasing chlorine substitution on the biphenyl molecule, a peak corresponding to a tetra-homolog, for example, results from a much smaller absolute amount of PCB than a peak of equal area corresponding to a di homolog . In the chromatograms for the Aroclor 1016 extracts shown in Figure 4b, there is exact correspondence of 705 OSW 033792 peaks in the early portions of the feed and fat chroma tograms with peaks in the Aroclor 1242 chromatograms. The chromatograms differ markedly, however, in the re gion where the more highly chlorinated homologs elute. Examination of the 30-day exposure chromatogram for Aroclor 1016 shows that there was no accumulation of more highly chlorinated homologs analogous to that noted for Aroclor 1242. 30-0AY USSUt EXTRACT J RntNIION TIMC Figure 4: Chromatograms of Feed and Tissue Extracts of Aroclor 1242 and Aroclor 1016 Since no persistent residues of MCBP or MCS 1043 were detected in fat during this study, no chromato grams for these products are shown. Liver and muscle tissues from the 30-day high feed ing level of each material were also analyzed. The data for these analyses are shown in Table III. Summary The results of this study can be summarized as fol lows : (1) Monochlorobipheny1 and the 30%-chlorinated biphenyl MCS 1043 did not accumulate in the lipid reservoir of the rats when fed at 25 ppm and 100 ppm in the diet. 706 DSW 033 793 TABLE III 30-DAY EXPOSURE LIVER, MUSCLE, AND FAT RESIDUE LEVELS (LIPID WEIGHT BASIS) Product MCBP MCS 1043 Aroclor 1242 Aroclor 1016 Dietary Level (PPM) 100 100 25 25 Muscle (ppm) 7.4 8.4 42.0 31.3 Liver 9. l 15.9 60.4 46.3 Fat (ppm) 6.4 11.5 37.3 22.7 This result indicates that mono-, di-, and trichlorobiphenyls are readily metabolized and/or excreted under the conditions of this study. (2) Although a fraction of the ingested Aroclor 1242 and Aroclor 1016 was stored in the rats' lipid reser voir, most of this residue was depleted after the rats had been on the basal laboratory diet for several weeks. (3) Residues of Aroclor 1016 accumulated more slowly and to a significantly lesser extent than those of Aro clor 1242. During the recovery period these PCB resi dues decreased to lower values for Aroclor 1016. This result indicates that a product containing reduced amounts of the more highly chlorinated PCBs should have ' improved environmental compatibility. Acknowledgment The authors wish to thank J. D. Hinchen for the statis tical treatment of the data. References BAXTER, R. A., et al., Sci. Total Environ., A, 53 (1975). BURSE, V. W. , et al. , Arch. Environ. Health, 2_9, 301 (1974) . HUTZINGER, O., SAFE, S., and ZITKO, V., The Chemistry of PCBs, Cleveland, CRC Press, 1974. METCALF, R. L., et al.. Arch. Environ. Contain. Toxicol., 3, 151 (1975). POLYCHLORINATED BIPHENYLS AND THE ENVIRONMENT, National Technical Information Service, U. S. Department of Commerce, Springfield, Virginia 22151, (1972). COM-72-10419. TUCKER, E. S., et al.. Bull. Environ. Contam. Toxicol., Accepted for Publication, (1975). 707 DSW 033794