Document GKXN9k8OQZn95xV9rvdpVEyOr

Ok'lEPGMEOTS und L. >) Cflrpp.nter for collecting Guy tor providing access to the we are grateful to L. F. Stickel aanuarript. FT.RENCES 'c. 55, 66 (1956). N, and D. M. SW1NEF0RD: J. Mamm. AMDNTs In manuscript. L S. LOCKE, A. A. BEL1SLE, T. E. M. MULHBRN, R. M. PROUTY, and D. l. J. (In pros). 1974). ' : . ) i * l i \ ! l i I i N Determination of Chlorinated Dibenzofuruns in Kanechlors and "Yusho Oil" by Jinya Naca>am\. Masanomi Kdratsi'ni: D*pr(mnc of Public Health Futulty of Mtulidriit Kyushu Onii vrsity fuknakn, Japan and Yoshjto Masuda Oniichi College of Pharmaceutical Sciences Fttkuakn, Japan Polychlorinated biphenyls (PCB) are widely distributed in the environment, especially in the tissues of fish, wild lives and man. They are also known as the causal agent of a mass food poisoning which occured among more than 1,000 persons in western Japan in I960. The disease induced by this poisoning is called "Yusho", namely oil disease, because the patients were proved to have consumed a commercial rice oil contaminated with a large amount of Kanechlor100 (KC-400), a brand of PCB of Japanese make (TSUKAMOTO et al., 1969). Feeding experiment using broilers demonstrated, however, that the rice oil taken by patients with Yusho were twice or more toxic than expected from the amount of KC-400 presented in the rice oil (IKEDA, 1972). This suggested that the "Yusho oil" must contain some more toxic substance or substances besides PCB. VOS et al. (1970), on the other hand, found by the chick embryo assay that toxicity of commercial PCB preparations is greatly affected by their contaminants, polychlorinated dibenzofurans (PCDF). Bauer et al. (1961) also demonstrated that structurally related polychlorinated dibenzodioxins (PCDD) were extremely toxic and acnegenic to man. Approximately 1 ppm of PCDF was estimated to contain in KC-400 (ROACH and POMERANTZ, 1974) . All these facts necessitated an analysis of Kanechlors and the rice oil taken by patients with Yusho for their possible content of PCDF and PCDD. EXPERIMENTAL In order to separate PCB, PCDF and PCDD from the "Yusho oil", 5 - 10 g of the oil was saponificated with ethanol containing an excess amount of sodium hydroxide for 1 hour at 80C and the n-hexane extract of the reaction mixture was chromatographed on a column of silica gel (Wakogel S-l, activated by heating at 130*C for 3 hours) with 100 ml of n-hexane. The eluate was concentrated to a small volume. 9 Bulletin of Environment*! Contamination A Toxicology, 'ol. I), No. I S (976 by Sprinfcr-Verlai New York Inc. MONS 087369 This concentrated eluate and samples of KC-300, 400, 500 and 600 (0.1 - 0.5 g) were chromatographed respectively, on a column of alumina (300 mesh, Wako Pure Chemical Ind. Ltd., 5 g) activated by heating at 150C for 12 hours, using as eluents, first 20 ml of n-hexane, then 120 ml of n-hexane containing 20 % carbon tetrachloride, 10 ml of n-hexane and finalLy 30 ml of n-hexane containing 20 % methylene chloride. The last eluate was evaporated to dryness, dissolved in n-hexane and subjected to gas chromatography with an electron capture detector (Beckman GC 72-5) and gas chromatography/mass spectrometry (JEOL D-100) for qualitative and quantitative determination of PCDF and PCDD. When the separation of PCDF and PCDD from PCB was not adequate, a similar chromatographic fractionation of the last eluate was repeated using a smaller amount of alumina (2 g) and a smaller volume of the same solvent systems, prior to the determina tion of PCDF and PCDD. Amount of individual di-, tri-, tetra-, penta-, hexa- and hepta-chlorodibenzo- furans was calculated from respective peak heights comparing with those of tetra-, penta- and hexa- chlorodibenzofurans which were synthesized by chlori nation of dibenzofuran. In this determination, the individual peaks were confirmed by GC-MS and their heights were assumed to have the same sensitivity. The total amount of PCDF was also determined by the perchlorination method. The PCDF from each sample were chlorinated to octachlorodibenzofuran (OCDF) with BMC reagents (HUTZINGER et al., 1972). The amount of OCDF was determined by ECD-GC comparing with standard OCDF (Analabs, Inc.) and converted to the amount of tetra-, hexa- or hepta-chlorodibenzofuran which was the main component of PCDF in the sample. Possible formation of PCDF from PCB during the saponification process was denied by comparing the determined amount of PCDF in KC-400 with that of PCDF in a sample of saponified vegetable oil containing KC-400. The recovery of PCDF in the whole analyti cal procedure was examined by analyzing 10 g of vegetable oil containing 1 pg of PCDF, finding more than 90 t. RESULTS AND DISCUSSION Presence of PCDF was demonstrated in all samples of Kanechlors and "Yusho oils" analyzed, as shown in Table 1 and Figure 1, but PCDD was not detected. As might be expected, Kanechlors with higher chlorine contents such as KC-500 and 600 contained more chlori nated dibenzofurans than those with lower chlorine contents. The concentration of total PCDF in KC-400 10 MQNS 087370 ,d samples of KC-300, 400# /ere chromatographed >f alumina (300 mesh, Wako ij) activated by heating at *3 eluents, first 20 ml of hexane containing 20 % of n-hexane and finally uj 20 1 methylene chloride, itocl to dryness, dissolved :o gas chromatography with n* (Beckman GC 72-5) and >ctrometry (JEOL D-100) for /i? determination of PCDF ition of PCDF and PCDD from nilar chromatographic *luate was repeated using a (2 g) and a smaller volume j, prior to the determinanount of individual di-, - and hepta-chlorodibenro!respective peak heights tra-, penta- and hexavere synthesised by chloriIn this determination, the Umed by GC-MS and their yn the same sensitivity, is also determined by the fhtt PCDF from each sample lorodibenzofuran (OCDF) with * il. 1972). The amount of ;GC comparing with standard Snvorted to the amount of drodibonzofuran which was in the sample, jPCDF from PCB during the {denied by comparing the in KC-400 with that of PCDF Vegetable oil containing PCDF in the whole analyti, by analysing 10 g of *>W of PCDF, finding more } DISCUSSION loironstroted in all samples \\3" analyzed, as shown in 'COD was not detected. As n* with higher chlorine l 000 contained more chloriio*q with lower chlorine ion of total PCDF in KC-400 Iv in r-i f"*- w> CN m mm in 0o *X*X aw OP s P VI mw uo P <H d xi oat o U sromoeue# < to u ,XTO oqsnjt,, il HOMS 037371 C a lc u la te d from peak h e ig h ts , b : C a lc u la te d by p e rc h lo rin a tio n m ethod. H\ t lu Jit., .L _ Ik II 200 230 300 % Figure X. GC-MS of Tetrachlorodibenzofuran A: Kanechlor-400, B: "Yusho oil", Ci Authentic specimen. watt estimated to be 18 ppm, the highest of the Kanechlors tested. GC peak patterns of PCDF present in three samples of the "Yusho oil" were very similar each other consisting of tri- to hexa-chlorodibenzofurans and their concentrations were 5 ppm. The PCB concentration in the "Yusho oil" analyzed by us was about 1,000 ppm. Therefore, PCDF concentration in the PCB in the oil was calculated to be 5,000 ppm. In other words, PCDF in the oil seemed to have been 250 times concentrated as compared with its concent ration in KC-400. 'VOS et al. (1970) showed in their chick embryo assays that PCDF was far more toxic than PCB. According to ARAKI's (1974) animal experiment, hepatic enzyme-inducing activity of PCDF was 170 times that of KC-400. In view of all these facts, the authors consider that the toxic role of this specific type of contaminants in the "Yusho oil" should not be dismissed In the causation of Yusho, even if their quantity was small in the "Yusho oil". 12 HONS 087372 9 300 f|trachlorodibenzofuran br-400, B: "Yusho oil", |c specimen. to, the highest of the Kanefcatterns of PCDF present in ,no oil" were very similar Jri- to hexa-chlorodibenzoations were 5 ppm. The PCB ho oil" analyzed by us was ore, PCDF concentration in Iculated to be 5,000 ppm. he oil seemed to have been compared with its concent** i al. (1970) showed in their PCDF was far more toxic than (1974) animal experiment, ctivity of PCDF was 170 times cof all these facts, the i toxic role of this specific he "Yusho oil" should not be n of Yusho, even if their "Yusho oil". REFERENCES 1. ARAKI, Y.: Fukuoka Acta Medica 6_5, 61 (1974). 2. BAUER, H., K.H. SCHULTZ, and U. SP1EGELBERK: Arch. Gewerbepathol. Gewerbehyg. ljJ, 538 (1961). 3. HUTZINGER, 0., S. SAFE, and V. ZITKO: Intern. J. Environ. Anal. Chem. 2, 95 (1972). 4. IKEDA, y.: J. Food Hyg. Soc. Japan 13, 359 (1972). 5. ROACII, J.A.G., and I.H. POMERANTZ j Bull. Environ. Contam. Toxicol. 12^ 330 (1974). 6. TSUKAMOTO, H. et al.: Fukuoka Acta Medica, 60, 496 (1969) . -- 7. VOS, J.G., J.H. KOEMAN, H.L. VAN DER MAAS, M.C. TEN NOEVER DE BRAUW, and R.H. DE VOS: Food Cosmet. Toxicol. 8, 625 (1970). 13 MGNS 087373