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From tht dtsk of WILLIAM RICHARD r >JO 4w e-P'V Z P&JLp* eJ-f'^-Xii u'-n u-*+4 dj.J*~jjj*****. . m-^cv-vi i 7^- Corz HONS 201600 PLfAft NANOLf PLtAM *OY HtTOHN X ron rood inpommayion *tTU*N M H COMMfMTi vou* *AQ TH9M M See attached comment V. R. Richard IN LOCAYION T3A AYf 3/24/75 HONS 201601 The l-intlm*' of Chlorinated Dihenzoftiruns in ajajiane.se I'olychlorinaled lii|dien vl Sample by John A. C. ItoACii and (imvin It. I'nm:HANTz (\hrnurnt 1`rrinutinyy Aufld if111/ /Irujr t lint nit ti r in i,,n 11 > tittn ftnit' l21IW4 _ Tbc presence of tetra- endpcntachlorlnared dlbcnznfnrtn* end hexa- end hrptachlorlnated naphthalene* In two samples of polychlorinated biphenyl mixtures (PCD) of European origin bos been reported by VOS *t al. (1970a). Their interest in 0 content analysts of tlie two rCS samples for impurities was stimulated by previous work in which toxic effects observed in chlclte were attributed to these two samples (VOS et si., 1970b), Ksnechlor 400 (KC-400), s commercial PCS preparation of Japanese origin, boa bean identified as the causative agent of Yusho, a human poisoning Incident resulting from the Ingestion of rice oil contaminated with KC-400 (KUKATSDNE at al., 1972). We have analyzed a sample of KC-400 made available to na from the Kancgafuchl Chemical Industrial Co.^, Ltd. through Dr. It. Dlumentlial of the Food and Drug Admin la tratlon to determine whether potentially hazardous chemicals, particularly chlorinated dlbenzofurana (Cl-DRF), were present as contaminants. It was the objective of this analysis to obtain, wliara possible, structural information oh these contaminants andean estimate of their con centration in the KC~400. Experimental A hexane solution of the KC-400 was gas chromatographed. using an electron capture detector, on a column of 60-100 mesh Chromosorb WIIP coated with 107. DC-200 and on a column of 15% QF-1 and 10% DC-200 each coated separately on 80-100 mesh Chrouosorb WHP and mlxod In a 1;1 weight ratio. Hi# KC-400 (chlorine content 467.) showed only minor differences in peak locations and relative peak heights from Aroclor 1248 (chlorine content 48%), a PCS of American manufacture. Therefore, Aroclor 1248 to which a'refarenca mixture of dl-, trl- and tetrochlorlnated dlbenzofurana (prepared by ehlorlnatlon of dlbenaofuran in tetrachloroathylene with iron powder as catalyst) had bean added, was used by Porter to davlae a procedure to separate Cl-DBF from PCS (PORTER, 1971b). This procedure, aa uaad in the preeent study. Is outlined In Figure 1. . ' ' . in RulffOn a{ Putlfnnnirn(ftl f4 j. V*|. IX, Nw. 1 ^ if74 by New twk Jw. HOMS 201602 . * ( t j t e P fi 1( Cl KC-400 in hexane solution FlortsU Column PCB in eluate A (discard) Cl-DBF in eluate B / Alumina Micro Column (POUTER, 1971a) PCB In eluate 1 (discard) Cl-DBF in aluata 2 Figure 1. Procedure for Separation of Chlorinated Dlbensofursns from Chlorinated Biphenyls in KC-600 The Florisll (8 g of 60-100 mesh PR grade) was activated by heating at 130*C ^ 12 hr and cooled to room temperature In a desiccator. Before the sample was added to the column, the Florlsll column was prevashed with about 25 ml hexane. The KC-600/ hexane solution (< 2 ml) was placed on the Florisll, which was contained In a 10 nan l.d, x 300 mm glass column fitted with a Teflon stopcock and medium porosity fritted disk. The column was eluted with two eluants in succession at the rate of about 5 >1/ min. Eluant A was 150 ml hexane. Eluant B was 100 ml 5% ethyl ether in hexane. Eluate B was concentrated to about 1 ml before the sample was placed on the alumina micro column. The micro coluan was a disposable plpet of roughly 5 on bora into which a small wad of glass wool had been Inserted to retain the aluminum oxide. Merck alumina, which had been heat-activated exactly like the Florisll, was poured into the column to a depth 0 2.5 cm. Sample or eluting solvent was than added to the column from a disposable plpet or a large glaaa syringe. Eluant 1 was 10 ml 11 methylene chloride In hexane; eluant 2 wee 6 ml 20% amthylene chloride In hexane. Additional PCB waa removed by con centrating eluate 2 to about 1 ml and subjecting It to a second 139 HONS 201603 t alumina micro column cleanup, (Imply repealing Lite tlumlna column procedure This procedure was flrat (ppUcd to duplicate 2 portions of KC-400. Electron capture gat ehtomatography of eluate 2 ahow.d a gas chromatographic peak In each (ample that had a relative retention time matching that of a tetrochlorodlbcnr.ofuran on both a 101 OV-101 and a 1:1 10* DC-200 + 15* QF-1 column under the following conditions: glass column, 6 ft. i l m id., column temperature 200*C, Injection temperature 223'C; carrier gas. . nitrogen at 120 nl/mln.; detector, trillion source (concentric type), operated In electron capture mode at 210*c, Detector voltage vai adjusted so that 1 ng heptachlor epoxide causes 1/2 full scale recorder deflection tt 1 x 10"9 AFS. the reference material for this retention time comparleon wee the tetrachlorodlbenzofuran component In the mixture of chlorin ated dlbenxofurans. To obtain sufficient material from the KC-400 for combined gas chromatogrsphy/mass spectrometry (CC/MS) analysis, the pro cedure wee scaled up to permit cleenup of 8 mg portions of KC-400. Cleaned up eluatas (aluate 2) ware than combined to give two composite samples, each equivalent to epproxlmately 100 mg KC-400. Theta two compoaltes were analysed by CC/MS (Flnnigan 1015 quadrupole mast spectrometer) and the resulting data were examined for evidence of the pretence of compounds that were not chloroblphenyle, A reagent blank of equivalent alee waa also analyzed by CC/MS. The reegent blank contained none of the compounds found In the samples. The qualitative result* of the KC-400 analyses are sunuarlztd In Table 1. The Identification of the compounds listed In Table 1 waa based on e comparison of the mass spectra of the sample compounds with the mass spectra of the dt-, trl-, and tetrachlorluated dlbeneofuran components In the reference mixture end on comperleon with meat spectral data on chlorinated dlbenxofurans In the published literature (VOS at el., 1970a; FIRESTONE at el., 1972; FLItMER at el., 1973), The amount of tetrechlorodlbenzofuren contained In the reference mixture wae approximated by compering lta sdcrocouiomatrlc CC response with the responae given by e known -quantity of e tetreehlorodlbensodloxln of known structure. The tetraehlorodlbenxodloxln wea considered an appropriate microcoulome.trle reference compound beceua# of lta atructural similarity to tetrachlorodlbenaofuran. The electron capture GC response* of the tetrachloro component In the synthetic mixture end In the KC-400 extracts ware than compared to quentltete the tetrachlorodlbenzo_ furen In the KC-400. The KC-400 wea estimated to contain - approximately 1 ppm tetrechlorodlbenzofuren. M0 t HOMS 201604 TABU 1 Compounds Present In Knncchlor 400 After Cleanups Hose (No, of Cl) end Fragmentation Dote Compound Present Molecular Weight of Compound 256 (3C1) ;H-2Cl trlchloroblphenyl 256 270(3C1);M-(CO+Cl) trlchlorodlbenzofuran 270 290(4C1);M-2C1 tetrachloroblphcnyl 298 (5C1) ;M-2C1 pentachloronaphtholene 304 (4C1);M- (CO+Cl), M-(CO+3Cl) tetrachlorodlbenzofuran 312(5C1);M-C1 unidentified; perhaps methylpentachloronaphthalene or pentachlorophenyl cyclopentadlene 290 298 304 312 338(4 or 5C1); M-(CO+3C1) Indlcatcd possible pantachlorodlbensofuren 338 ^Listed In order of elution from 32 0V-101, 6 ft. x 2 an l.d. glass column at 220*C, carrier flow 20 ml He/min.; the same MS evidence vaa found In both sample* examined for all entrlea but the lest (possible pentechlorodlbentofuren) for which e second determination was not attempted. Summary Chlorinated dlbensofurene, including 1 ppm of tatrechlorlnated dlbensofuren(s), end pentechloronephthelene have been identified by combined gea chromatography/mess spectrometry in a polychlori nated biphenyl of Jepaneso manufacture (KC-400). The question of vhathar chlorinated dlbeneofurans In KC-400 may hava contributed to tha reported Yuaho Inc Id ant la thua raised. ' Acknowledgement the authors vlah to thank Sandra Harder for aaalatanea In tftm preparation of tha samples for CC/MS analysis. The reference mixture of dl-, trl-, and tctrechlorlnaced dlbcnsofurans was kindly provided by Sr. Albert Foliland, Food and Drug Adminlatration. Refersncaa FIRESTONE, D., J. RESS, N. L. BROWN. R. F. BARROW, and J. N. DAMICO; J. Asa. Offtc. Anal. Cham. 5, 85 (1972). Ml HONS 201605 KURATSUHE, H.. T. YOSHIMURA. J. MATSUZAKA, and A. YAMACUCIU: Environ. Health Pa re pace, Iu No. 1, 119 (1972). PLIKMER, J. R. , J. M. RUTH, and E. A. WOOLSCN: J. Agr. Food Chen. 21, 90 (1973). PORTER, M. and J. A. BURJCE: J. Aaa. Of fie. Anal. Chen. 54. 1426 (1971a). " PORTER, M.: Prlvace Comunicatlon (Feb,, 1971b). VOS. J. C.. J. H. KOEMAN, H. L. VAN DER MAAS, M. C. TEN NOEVER DE BRAIN, and R. H. DE VOS: Fd. Cosmet. Toxicol. S, 625 (1970a). VOS, J. C. and J. H. KOQ4AN: Toxicol. Appl. Pharmacol- 17, 656 (1970b). ~ B42 MOMS 201606