Environ. Sci, Technol. 1982. 16. 367-369
Engineering Research Center Report 76-1; US. Army Corps of Engineers: Belvoir, VA, 1976; p 50. Hanks, K. S. J. Ariz. Acad. Sei. 1976, 11, 3. Robbins, J. A.; Edgington, D. N.; Kemp, A. W. L. Radiological and Environmental Res. Div. Ann. Rep. ANL-76-88, Part 111;Argonne National Laboratory: Argonne, IL, 1976;
Literature Cited Ferrante, J. G; Dettman, E. H.; Parker, J. I. Report ANL/ES-85; Argonne National Laboratory: Argonne, IL, 1980; p 61. “Standard Methods for the Examination of Water and Waste Water”,14th ed.; APHA, AWWA, and WPCF New York, 1975. “Methodsfor Chemical Analysis of Water and Wastes”;U.S. Environmental Protection Agency: EPA-625-6-74-003a,
p 87.
Howard, D. L.; Frea, J. I.; Pfister, R. M. Proc. Conf. Great Lakes Res. 1971, 14, 236. Zapotosky, J. E.; White,W. S. Report ANL/ES-87;Argonne
1974; p 298.
Swinnerton, J. W.; Linnenbom, V. G.; Cheek, C. J. Anal. Chem. 1962,34,483. Swinnerton, J. W.; Linnenbom, V. G.; Cheek, C. J. Anal. Chem. 1962,34, 1509. Dettmann, E. H. Report ANL/ES-86; Argonne National Laboratory: Argonne, IL, 1980; p 21. Lawrence, M.; Schereer, E. Technical Report 520; Fisheries and Marine Service, Freshwater Institute: Winnipeg, Canada, 1974; p 47. Herbert, D. W. M.; Merkens, J. C. Int. J. Air Water Pollut. 1961, 5, 46. “Water Quality Criteria 1972”; NAS, NAE, USEPA Ecol. Res. Series,EPA-R3-73-033 Washington, D.C., 1973;p 594. Sherk, J. A.; O’Connor, J. M.; Neumann, D. A. Coastal
National Laboratory: Argonne, IL, 1980; p 150. Frea, J. I.; Ward, T. E.; Mallard, G. E. Report 485; Water Resources Center: Ohio State University, Columbus, OH, 1977; p 153. Teal, J. M. “Fate and Effect of Petroleum Hydrocarbons in Marine Ecosystems and Organisms”;NOAA, USEPA: New York, 1977; p 71.
Received for review July 13, 1981, Accepted February 25,1982. This study was financially supported by the US.A r m y Corps of Engineers and the U.S. Environmental Protection Agency. Work was performed under the auspices of the US.Department of Energy.
Biotransformation of PCB by Natural Assemblages of Freshwater Microorganisms Michael P. Shlarist and Gary S. Sayler” Department of Microbiology and Graduate Program in Ecology, University of Tennessee, Knoxville, Tennessee 379 16
Natural mixed-microbial populations in lake water were found capable of oxidizing 2-chlorobiphenyl but not 2,4‘-dichlorobiphenyl. No evidence was found to indicate that these populations were able to oxidize a PCB mixture 54% chlorine by weight. Oxidation of 2-chlorobiphenyl resulted in the accumulation of two biotransformation products, chlorobenzoic acid and chlorobenzoylformic acid (chlorophenylglyoxylic acid). The results indicate that biodegradation of PCB congeners may result in the accumulation of environmentally stable chlorinated biotransformation products in aquatic environments.
Introduction A number of reports have cited the ability of isolated heterotrophic bacteria to grow on and in some cases degrade polychlorinated biphenyls (PCB) (1-7).There are also reports that natural assemblages of microorganisms are capable of PCB biodegradation (8-12), but little information exists on the environmental biodegradative fate of PCB. Chlorinated biodegradation products have been suggested for trichlorobiphenyl in marine samples (8). A recent report has suggested that the terminal fate of p chlorobiphenyl in a marine environment is mineralization accompanied by accumulation of benzoic acid (13). The objective of this brief report is to demonstrate that an alternate fate for PCB exists in aquatic environments. Problems associated with assessing the biodegradative fate of PCB in the environment are numerous and relate to low solubility, volatilization, photodecomposition, and sorption/desorption to living organisms and particulate ‘Present address: Department of Biology, University of Massachusetts, Boston Harbor Campus. 0013-936X/82/0916-0367$01.25/0
matter. In addition, the position and degree of chlorination of the numerous congeners in commercial PCB mixtures, such as Aroclor 1254, modulates the biorecalcitrance of the PCB substrate. Consequently, gas-liquid chromatographic analysis of residual PCB following routine biodegradation assessment for removal of the substrate from terrestrial or aquatic samples is inexact and can only yield information pertaining to the potential interaction of the PCB with biotic components of a natural sample. As an example of this point, Table I represents a preliminary examination of PCB removal from aquatic samples exposed to PCB. These data are based on residual PCB quantitation using a previously described biodegradation assay, hexane extraction, and electron-capture GC analysis for PCB (14, 15). These results indicate a significant interaction of PCB with microbial populations present in a variety of temporally and spatially segregated samples from Center Hill Reservoir, T N (16). These data and the gas chromatographic profiles from which they are generated do little to assess the actual fate of PCB. In a further attempt to delineate the fate of PCB, the ability of microbial populations to mineralize PCB to COz was investigated. Some 10-mL Center Hill Reservoir samples were placed in 70-mL serum bottles to which 10 pL of repurified (Florisil column chromatography) [U14C]PCB (New England Nuclear, Boston, MA; 31.3 mCi/mmol, 54% chlorine by weight in mixture) in acetone was added. The reaction vessels were supplemented with 10 mg of prewetted powder XAD-4 resin (Supelco Inc., Bellefont,e,PA) to inhibit PCB volatilization. (Preliminary experiments indicated that XAD-4 resin had no effect on microbial growth and that naphthalene was mineralized in the presence of XAD-4 resin in the same experimental system.) l4COZliberated from the PCB substrate was collected in a center-well NaOH C02 absorber. The ex-
0 1982 American Chemical Society
Environ. Sci. Technol., Vol. 16,No. 6, 1982 367
Table I. Aroclor 1254 Biodegradation in Center Hill Reservoir Samples, 1977a % PCB degradation in samples taken site
Feb Mar A P ~ June Aug 1 N D ~ 62.6 c loc 53.5 c 7 70.3 c 14 ND 2 13.8 5 7 35.0 i 7 18.8 i 15 51 5 28 ND 3 ND 47.7 c 33 37.4 t 18 69.8 i 18 ND 4 42.4 c 21 6.8 c 10 11.4 c 11 66.7 c 30 ND . 10.8 c 33 41.5 c 39 53.8 c 6 ND 5 47.7 i 28 ND 81.2 c 1 6 71.2 i 20 70.4 c 36 ND 6 Values expressed as % PCB degradation relative to sterile controls, one month incubation at 25 "C. * 1 standard deviation calculated from triplicate replication,
Dec ND 43.6 c 9 ND 37.3 c 19 69.8 c 8 ND
Not determined.
Flgure 1. Mass spectrum of chlorinated metabolite tentatively identified as chlorobenzoylformic acid isolated from Center Hill Reservoir water amended with 2-chlorobiphenyl (peaks at m l e , 167, 150, and 149 represent a phthalate Contaminant).
perimental samples and autoclaved sterile control and acetone control samples were incubated for various time intervals up to 96 days at room temperature in the dark. Under the experimental conditions used, no 14C02was detected throughout the entire incubation period, indicating that for complex mixtures of PCB, mineralization was insignificant with respect to adsorption, volatilization, and cellular accumulation, which occur kinetically at a much faster rate (17). Based on the results of other studies (1-4,8,18-22), if metabolic transformation of PCB were occurring, various chlorinated acidic products should accumulate in samples exposed to PCB. For analytical purposes, high concentrations of two lesser chlorinated biphenyls were employed to determine if biodegradation was occurring with accumulation of refractory metabolites. Center Hill Reservoir samples were placed in 1000-mLcotton-stoppered Erlenmeyer flasks, each containing 50 mg of Celite, a diatomaceous earth. The flasks were dosed with a 10-mL acetone solution of 2-chlorobiphenyl or 2,4'-dichlorobiphenyl (Analabs Inc.), purified by Florisil column chromatography, at a final concentration of 40 mg L-l. Sterile control samples and experimental samples were incubated at 25 "C in the dark for 3 and 8 months, respectively, for the mono- and dichlorobiphenyl. Following incubation, the samples were adjusted to pH 1.5 with concentrated HC1 and twice extracted with pesticide grade ethyl acetate (Fisher Scientific Co.). The extracts were combined, reduced to a 10-mL volume by vacuum rotoevaporation, and further reduced to 1 mL by evaporation with N2 gas. Chemical standards and extracts were spotted on LQF Chromoflex, fluorescent thin-layer chromatography plates (Kontex, Vineland, NJ). Plates were developed in benzene/methanol/acetic acid (90:9:1 v/v) and viewed under ultraviolet light. Spots were removed and dissolved in ethyl acetate for analysis by a computerized direct-probe mass spectrometer (Hewlett-Packard 5980A; electron impact, 70 eV). Components of the 2,4'-dichlorophenyl extract migrated as a single yellow spot, R f 0.91, on TLC as compared to the 368
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--ae W V
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CHLOAOBENZOYL FORMIC ACID
139
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Flgure 2. Mass spectrum of authentic chlorobenzoylformic acid.
parent substrate, R f 0.99. This component was resolved as two peaks by mass spectrometry (MS); the first was identified as 2,4'-dichlorobiphenyl (M+, m l e 222) and the second was an unidentified peak containing no chlorine atoms (M', m l e 278)-this latter peak was tentatively identified as a phthalate based on a matching with the NIH library of reference mass spectra. It is most likely that the phthalate is a laboratory contaminant. The extracts of the 2-chlorobiphenyl samples were resolved as three components, Rf 0.98, 0.68, and 0.54. The first component (Rf 0.98) comigrated with the 2-chlorobiphenyl standard and was confirmed by the mass spectrum (M+, m / e 188). The second component (Rf 0.68) demonstrated molecular ions at m l e 139 and 111 with characteristic chlorine doublets and was tentative identified as chlorobenzoylformic acid from the mass fragmentation pattern (Figure 1). This identification was confirmed by mass spectra comparison with a benzoylformic acid standard (Aldrich Chemical Co.) and a chemically synthesized chlorobenzoylformic acid (23) (Figure 2) and by IR and NMR spectra. The third component (Rf 0.54) comigrated with chlorobenzoic acid on TLC. This identification was confirmed by MS (M+, m l e 156) and comparison to a chlorobenzoic acid standard (Analabs Inc.) (Figure 3). No attempts were made to determine the isomeric forms of either the chlorobenzoylformic acid or the chlorobenzoic acid. The results of the studies reported here indicate that chlorobenzoic acids can be expected to accumulate during
39
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8 Figure 3. Mass spectra of
o-chlorobenzoic acid standard and the metabolite isolated from Center Hill Reservoir water amended with 2-chkwobiphenyl.
the degradation of the lesser chlorinated biphenyls in the environment. These results validate the predictions from pure culture studies demonstrating chlorobenzoic acids as stable PCB biodegradation products. The isolation of chlorobenzoylformicacid (chlorophenylglyoxylicacid) as a biotransformationproduct of 2-chlorobiphenyl by natural aquatic microbial communities is a novel observation. Chlorobenzoylformic acid was postulated as a possible product resulting from the ortho fission of a chlorobiphenyl-2,3-diol (18). It may also hypothetically arise through a 3,4-meta fission of the chlorobiphenyl-2,3-diol, a mechanism proposed for the oxidation of biphenyl (21), and the oxidation of a chlorophenylpyruvic acid intermediate to the chlorobenzoylformicacid. However, these mechanisms have received little support in the literature, and the 1,2-meta fission of biphenyl-2,3-diol as proposed by Catelani et al. (22) has received general acceptance. Reports of the isolation of benzoylbutyric acid resulting from the oxidation of biphenyl (19) and a chlorobenzoylpropionic acid from the oxidation of chlorobiphenyl (20) as well as our results support the 1,Zmeta ring-fission mechanism with sequential oxidation of the ring-fission products to chlorobenzoic acids. These results support the hypothesis that aquatic ecosystems have detectable potential for PCB biodegradation. Since the Center Hill Reservoir environment appears to be free of PCB contamination, it is suggested that PCBcontaminated environments will demonstrate a greater potential for biodegradation as microbial populations respond to selective pressure. This biodegradation potential can result in the environmental accumulation of stable chlorinated degradation products.
Literature Cited (1) Ahmed, A.;Focht, D. D. Can. J. Microbiol. 1973,19,47-52. (2) Furukawa, K.; Matsumura, F. J. Agric. Food Chem. 1976, 24, 252-256. (3) Furukawa, K.; Matsumura, F.; Tonomura, K. Agric. Biol. Chem. 1978,42,545-548.
(4) Furukawa, K.; Tonomura, K.; Kamibayash, A. Appl. Environ. Microbiol. 1978,35,223-227. (5) Kaiser, K. L. E.; Wong, T. S. Bull. Environ. Contam. Toxicol. 1974,11, 291-296. (6) Lui, D. Water Res. 1980,14, 1467-1475. (7) Sayler, G. S.;Shon, M.; Colwell, R. R. Microb. Ecol. 1977, 3, 241-255. (8) Carey, A. E.; Harvey, G. R. Bull. Environ. Contam. Toxicol. 1978,20,527-534. (9) Clark, R. R.; Chian, E. S. K.; Griffin, R. A.Appl. Environ. Microbiol. 1979,37,680-685. (10) Kaneko, M.; Morimoto, K.; Nambu, S. Water Res. 1976, 10,157-163. (11) Tucker, E. S.;Saeger, V. W.; Hicks, 0. Bull. Enuiron. Contam. Toxicol. 1976,14,705-713. (12) Tulp, M. T. M.; Schmitz, R.; Hutziner, 0. Chemosphere 1978,7,103-108. (13) Reichardt, P. B.; Chadwick, B. L.; Cole, M. A.; Robertson, B. R.; Button, D. K. Enuiron. Sci. Tech. 1981,15,75-79. (14) Shiaris, M. P.;Sherrill, T. W.; Sayler, G. S. Appl. Environ. Microbiol. 1980,39,165-171. (15) Sherrill, T. W.; Sayler, G. S. Appl. Environ. Microbiol. 1980, 39, 172-178. (16) Sayler, G. S.;Lund, L. C.; Shiaris, M. P.; Sherrill, T. W.; Perkins, R. E. Appl. Environ. Microbiol. 1978,37,878-885. (17) Kalmaz, E.V.;KaJmaz, G. D. Ecol. Modell. 1979,6,223-251. (18) Ballschmiter, K.; Unglert, C.; Neu, H. N. Chemosphere 1977,7,51-56. (19) Ohmori, T.; Ikai, Y.; Minoda, Y.; Yamada, K. Agric. Biol. Chem. 1973,37,1599-1605. (20) Yagi, 0.; Sudo, R. J. Water. Pollut. Control Fed. 1980,54, 1035-1043. (21) Lunt, D.; Evans, W. C. Biochem. J. 1970,118,54-55. (22) Catelani, D.; Colombi, A.; Sorlini, C.; Trecanni, V. J.Biochem. 1973,134,1063-1066. (23) Sayler, G. S.;Reid, M. C.; Pagni, R. J.; Smith, R.; Rao, T. K.; Epler, J. L.; Morrison, W. D.; DuFrain, R. Arch. Environ. Contam. Toxicol. 1982,in press.
Received for review August 15,1980.Revised manuscript received May 21,1981. Accepted February 16,1982.This investigation was supported by NIEHS Grant ESO 1521-03. G.S.S. is supported by a NIEHS Research Career Development Award. Environ. Sci. Technol., Vol. 16, No. 6, 1982 389
