Document EqJyJYrOXxaZ6GodojQQByLKR

r - I?* JAN 5 m 77' iSiftr-oc* , TR 77-522 HM'O'lM- iw-5UTIWS NfcT,D' , u torn f.wn-*h#41or ; rCl. *c nuMlslUMl " a"* i0Tm ncl ta so.'.' c p " , ,, 0i th* aulhor* *ilhoui th Prn`,*sl0B fit** -- mtcn jEsrefiJs f DECOMPOSITION OF CHLORINATED AROMATICSJ MICROBIOLOGICAL DECOMPOSITION OF POT.Ynn/tRTNATED BIPHENYLS fPCBl; rcul III. CHLORINATED BENZOIC ACIDS AS PCB METABOLITES J> [Abbau von chlorierten Aromatan: Mikrobiologischer Abbau der polychlorierten Biphenyle (PCB). III. Chlorierte BenzoesHuren ala Metabolite der PCB] by K.jJJallBchmiter, Ch. /Unglert and H.J.^Neu Department of Analytical Chemistry, University of Ulm Published inYchemogphere, M.J, pp. 51-56, 1977 \ L----- stL *--------- J The decomposition [1] of polychlorinated biphenyls (1)* by monoxy genases progresses across the intermediate stage of arenoxides (2](2) by spontaneous isomerization to phenylphenols [3] (.3), or by enzymatic hydration across phenylcyclohexadlene-transdiols (4) to o-dihydroxy-biphenyls (7), whose ring openings lead one to expect a complex system of chlorophenylcarbonic acids (8) [4,5,6](Fig, 1). By a renewed reaction, the phenyl phenols can also lead to dihydroxy-blpbenyls [3]. *[Underlined numbers in parentheses refer to numbered groupings in Fig. 1, Numbers in brackets Indicate references. -- Translator] Translated for NERC-Library, EPA, from the original German, by -E0 KANNER ASSOCIATES, P.0. Box 5187, Redwood City, California 94063, (415) 365-3046, August 1977. DSW 031575 i i. $ STLCOPCB4015537 t Alternatively, If a reaction with dloxygenasea to 1,2-dioxetanes (5) follows as the primary Intermediate step In PCB decomposition, then phenylhexadiene-cls-dlols (6) are to be expected (Fig. 1) [7]. These aromatize easily to o-dihydroxy-biphenyls (7), with chlorophenyl-carbonic acids () again to be expected as the result. If one applies the general scheme suggested by Dagley et al. [4] for microbial decomposition of aromatics, as a working hypothesis for poly chlorinated biphenyls, then in addition to chlorobenzolc acids (9) and chlorophenylaeetlc acids (10), chloroacetophenones (11) should develop as phenyl-replacing metabolites. Furthermore, ehlorophenyl-chloroplcollulc acids (12) and chlorolutldlnlc acids (13) are to be expected as artifacts [5], depending on the presence of ammonium ions in the nutrient solution after meta-spllttlng QSW 031576 i i OOH of the o-dihydroxyphenyle (phenyl pyrocatechlns) and pyrocatechlna by a non-enzymatic closing of Che rings of the nuconic acid semialdehydes. The aromatic carbonic acids will be structurally determined by: 1) the type of o-dihydroxy-biphenyls formed, and 2) the type ot ring opening of the o-dihydroxy-biphenyls. The basic equivalence of the 2,3-position in biphenyl with the 5,6-position is eliminated by axially asymmetric chlorine substitution and then leads to different resulting products, depending on the formation of the o-dihydroxy-biphenyl. The same applies for the formation of 3,4-biphenylols, which are to be distinguished from the 4,5-biphenylols. Various ring openings are possible for a given dihydroxy-biphenyl. The possibility of an ortho-splitting of the pyrocatechlnes is Increased by two different kinds of meta-splitting during decomposition of chlorin ated o-dihydroxy-biphenyls. For the case of 2,3,3',4,5-pentachlorobiphenyl, assuming the formation of 2*,3*-dihydroxy-biphenyl, the result of ortho splitting and of the two types of meta-spllttlng is presented here (Fl~ 2, following page). Without taking into account the Intermediary stages (e.g. isomerisation. lactone formation), tha accumulation products to be discussed as metabolites 1 t:. Instance are tetrachloride derivatives of benzole acid, of phenylscetlc acid, of phenyl-pyroracaalc acid and of 2-phenylmalonic acid. 3 DSW 031577 STLCOPCB4015539 a ci *0-0 C, | a Cl' OH OH Cl Cl a^^-CHj-COOH a H COOH X Cl COOH CICHj-COOH Fig. 2. Carbonic adds as possible metabolites of 2,3,3',4,5-pentachlorobiphenyl after formation of 2,3,A,5,5'-pentachloro-2',3'-dihydroxy-biphenyl. Key: a. X-splittlng b. Decomposition together vith chloromalelc acid and chloroacetlc acid. Materials and Apparatus The PCB isomers (Analabs, KEN Chemicals, Drelelchenhaln: Bayer Co., Leverkusen), the chlorobentolc acids and chlorocinnamic acids (Fluke, Heu-Ulm and Aldrich Europe, Dttsceldorf), as veil ae the solvents, for reaction (Merck, Darmstadt), vers used as obtained. To concentrate the solutions, superpurlfled nitrogen was used. The gas-chromatographic experiments were performed with a Carlo Erba Model 2300 gas chromatograph, vith macrosplitter, Ni-63 electron capture detector and temperatu-e DSw 031578 A STLCOPCB4015540 1) Fig, 3. Methylesters of monochloro- and dichlorobenzoic acids (90C, Carbovax 20 M, 50 m, glass) control. The class capillaries (SB 30, 27 m, l.d. 0.25 mm, TZ^ - 42; Carbovax 20 M. 50 m, l.d. 0.20 on, TZ* ^ wer cured according to modified data from the literature. Electronic reten tion time determination and surface evaluation was performed vlth a \ Spectra Physics Autolab System 1 integrator. l A Carlo Erba Model 2300 gas chromatograph vlth splitter connected I to a magnetic field mass spectrometer, vacuum generators, mlcromaas 15 F (pump delivery 1300 Lfsec.) vlth a UV recorder was available as a ga* chromatograph-mass spectrometer combination vlth a glass capillary column I * [Expansion unlcnovn -- Translator,] 5 DSW 031579 STLCOPCB4015541 Table 1. Retention indices of methyl esters of chlorobenzoic acids, referred to trichloroacetic acid esters of the n-alcohols (Cn > 5); R1 of trichloroa:etic acid-n-hexylester ^ 1400. rtff. J SK 30 (130C> 2 cMorb*Aio*lur^ <lt J-Ch lor banco* slur a1*" (2) 4-Chlorbanzoeituraa ^ (1) 2,4-Dlchlorbanaoaalura b 2 v$-DlchXorbn*otiAur* 3,6-DlchlorbanzoaaSurS^ (6) (7) <B) 3 f4^DichlorbcncoiIur* (5) j,5-Dichlorb*nto*iurt y (4) 12*9,5 1384,4 1280,3 1424,3 1436,6 1394,4 1445,G 1411,7 2,),4,S-Tatrachlorbanzoaz<ura ____________ ------------------------------------ 1652,1* crarbowax 2oh(90C) 1495,3 1430,3 1416,3 1600,0 1616,3 1634,3 1577,7 1500,0 -- Stationary liquid OV 101, Temperature 100*C Key: a. Chlorobenzoic acid b. Dichlorobenzoic acid c. Tetrachlorobenzoic acid and a direct connection over a Pt-Ir capillary. The El* source was run with 25 V. The chlorobenzoic acids were identified gas chromatographically, with glass capillary columns and an EC detector, as their methyl esters (Fig. 3 and Table I), by relating their retention indices to authentic substances and by mass fragmentography with the eld of the gee chromatograph * mass spectrometer combination. Reference substances for the calculation of retention indices with the use of the EC detector were the trichloroacetic acid esters of normal * IExpansion unknown -- Translator.) OSW 031580 6 ) 1 STLCOPCB4015542 C alcohol (n > 6), which were synthesized according to standard by II " csterizatlon with trichloroacetyl chloride. These homologs can be used for retention Index determination with either a flame Ionization detector or an electron capture detector. For the trichloroacetic acld-n-hexyl c"tcr one gets, with reference to the n-alkanes, a retention index value of 1345.6 at 120C on SE 30; and a value of 1494.6 at 90#C on Carbovax 20 M. To simplify evaluation, a retention Index of 1400 was assigned to trichloroacetic acid-hexyl ester. Culture Conditions A mixed culture was used as microorganisms, obtained by water extrac tion of a garden mold via an intermediate enrichment by growth on benzene as a C-source, with the use of a standard phosphate salt solution (pH 6.t0) with additional trace elements. This mixed culture contained mainly gram negative bacilli with polar cilia, gram-positive cocci and paramecla. Decomposition experiments with PCB isomers were performed with benzene as an additional C-source. The benzene could enter the culture medium by diffusion from a paraffin bottom stratum [8](2 g benzene/20 g paraffin hlstoplast, melting point 56*, Serve Heidelberg, per 2 t culture medium), On the lower half of the wall of a 2-liter steep-aided bottle, 1-5 ag PCB Isomers were applied as a film from 20 ml acetone. After only 4 days, the culture medium was found to be saturated with the PCB Isomers. The cultures were Incubated for 2, 4 and 6 weeks at 28* - 32*C. After suspen sion of the culture on aromatic acids as described, 200 ml samples were processed [9]. DSW 031581 7 STLCOPCB4015543 Result The PCB isomers listed in Table 2 were examined for their decompo- eablllty to chlorobenzoic acid. The chlorobenzoic acids obtained aa metabolites are Hated. TABLE II. PCB Isomers 2-Chlor-blphanyl* 4-Chlor-blphanyl* 2,2 1-DlchXor-blphanyl ^ 2,4 -Dletalor-bXphanyl ** 2.2 * , S-Trlchlor-blphaayl^ J,4,4-Trlchlor-blph*nyl ^ 2,4,4 '-Trlchlor-blphanyl ft 2,3.3 *,4,5-PantachIor-blphaayl*' 2,3,4,41.S-PantachXoE-blphanyl^ Clophan X 30 (liytt) Benzoic acids as PCB metabolites ... 4-Chlorbancoaakura * -- P 2 *5-0 IchlorbtnioMiurf; 4-Chlorliinto*luri J#4*0iobiorbu* rKOBlUr 4*ChUrb*AXoB4ut8 2 *4-Dlchlorbo- totiAur* 2.3.4.5-Tatr*chiorbanoa*lura^' 2.3.4.5-Tatrachlorbanaoaatara? . tt 4-Chlorbansoaatura, 2,4-Diehlorbai.toaalura Keyi a. Chlorobiphenyl b. Dichlorobiphenyl c. Trichlorobiphenyl d. Pentachlorobiphenyl e. Chlorobenzoic acid f. Dichlorobenzoic acid g. Tetrachlorobenzoic acid During decomposition of 2f4,4'-trichlorobiphenyl, 4-chloroben*oic acid and 2,4-dichlorobenzoic acid were concentrated in the ratio of 5:2. The results confirm the findings of other authors on the decomposi tion of 4-chlorobiphenyl to 4-chlorobenzoic acid [10,11]. The concentra tion of chlorobenzoic acid during decomposition of PCB isomers agrees with the finding that 4-chloro-, 2,4-dichloro-, 3,4-dichloro-, 2,5-diehloro-, and 2,3,4,5-tetrachlorobenzoic acids are not noticeably decomposed by ground bacteria in the course of 60 days [12]. ,, , .... . 8 l DSW 031582 STLCOPCB4015544 For 2-chlorobenroic acid, with ground bacteria, both a comparatively fast metabolism [12] anJ complete stability [13] have been observed. Our ovm experiments on the microbial transformation of 2-chloro- and 4-chloroben,,oic acids, with quantitative determination of the Isomeric benzole acids by means of gas chromatography, yielded almost the same decomposition rate for both aclde. Likewise, decomposition experiments with 2,4-dichloro- and 2,5-dichlorocinnamic acid, under the conditions of the PCB experiments, yielded an accumulation of 2,4-dichloro- and 2,5- dichlorobenzoic acid, respectively, which confirms the results of the decomposition of 2,2',5-trichloro- and 2,4,4'-trichloroblphenyls, respectively. Clophen A 30R (Bayer) (42Z Cl) Is 80Z composed of PCB Isomers with / chlorine in 2-, 4-, 2,2'-, 2,4'-, 2,2',3-, 2,2',5-. 2,3,4*-, 2,4,4'-, 2,4',5-, 2,2',3,5'-, 2,2',4,5'- and 2,2',5,5'- position. According to the above findings, 2-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-- and 2,5-dlchlorobenzoic acid should thus preferably develop. But In the decomposition of Clophen A 30^ (Bayer), only 4--chloro-- and 2,4--dlchloro-- benzoic acid have been detected In the culture medium to dnte. The absence of both 2-chlorobenzoic acid and 2,5-dichlorobenzoic acid is conspicuous. In the capillary chromatogram from acid fraction [9], however, there are further acid methyl esters with higher boiling points, and their Identification le In progress. Since the slow microbial breakdown of polychlorinated biphenyls (PCB) Is demonstrably followed by an even slower breakdown of certain chlorobenzoic acids, one may easily conclude that as a result of the well-known DSW 031583 STLCOPCB4015545 global presence of PCB, chlorinated benzoic acids are likewise present globally as environmental chemicals. Acknowledgments Our thanks to the Bunde3miniaterium fUr Forschung und Technologie iCerman Federal Ministry of Research and Technology] for the financing of this study in the context of the coordinated research program on "Polychlorinated Biphenyls in the Environment." The University of Ulm supported the experiments with a special grant from their research support fund. Our thanks to Bayer AG for supplying PCB isomers. Bibliography 1. SundstrBm, G., 0.' Hutzinger and S. Safe, Chemosphere 5. 267 (1976). 2. Jerina, D.M., J.W. Daley, Science 185, 573 (1974). 3. Safe, S., 0 Hutzinger, D. Jones, J. Agr. Food Chem. 23. 851 (1975). 4. Dagley, S., P.J. Chapman, D.T. Gibson, J.M. Wood, Nature 202, 775 (1964). 5. Gibson, D.T., Science 161, 1093 (1968). 6. Cain, R.B., R.F. Bilton, J.A. Darrah, Biochem. J. 108. 797 (1968). 7. Gibson, D.T., B. Gschwendt, W.K. Yeh, V.M. Kobal, Biochemistry 12. 1520 (1973). B, Gibson, D.T., G.E. Cardlni, F.C. Maseles, R.E. Kallio, Biochem. 9. 1631 (1970). 9. Ballschmiter, K., U. Niederschulte, H. Thama, H.J. Neu, Chemosphere 5, 367 (1976). 10. Ahmed, M., D.D, Focht, Can, J. Microbiol. 19, 47 (1973). 11. Ohniori, T., T. Ikal.Y. Mlnoda, K. Yamada, Agr. Biol, Chem. 37. 1599 (1973). 10 DSW 031584 \ !> STLCOPCB4015546 12. McRal* I.C., M. Alexander, Foqd..Che^ 13> 72 (1965). 13. KnacVraua, H.J., Chera. Zeltunft 99, 213 (1975). Jf. ~ir~ OSW 031585 11 STLCOPCB4015547