Environ. Sci. Technol. 1984, 18, 608-610
Biochemical and Photochemical Processes in the Degradation of Chlorinated Biphenyls Robert M. Baxter” and Dale A. Sutherland
Environmental Contaminants Division, National Water Research Institute, Burlington, Ontario, Canada L7R 4A6 Resting suspensions of a pseudomonad isolated from activated sludge and grown with biphenyl as the sole carbon and energy source converted 2,4’-dichlorobiphenyl to a variety of products, including two monochlorobenzoic acids, two monohydroxydichlorobiphenylsprobably produced by dehydration of the corresponding dihydrodiols, and a yellow hydroxyoxo(chloropheny1)chlorohexadienoic acid. Irradiation of the metabolite mixture led to the disappearance of the yellow compound and appearance of two monochloroacetophenones. The same or very similar compounds, and analogous more highly chlorinated compounds, were produced from Aroclor 1242, a commercial polychlorinated biphenyl mixture. It is suggested that successive biochemical and photochemical processes may contribute to the degradation of chlorinated biphenyls in the environment. Introduction Mixtures of chlorinated biphenyls, known as polychlorinated biphenyls (PCBs), were used for many years as dielectric fluids, solvents, and plasticizers and for a variety of other purposes until the extreme resistance of some components of these mixtures to environmental degradation was recognized (1,2). Although the use of these substances has now been greatly curtailed in most parts of the world, considerable quantities still persist in the environment. There is also evidence that certain chlorinated biphenyls can be produced in nature by the bacterial metabolism of other contaminants (3). Not all chlorinated biphenyls are completely recalcitrant to environmental degradation. Some may be susceptible to photochemical reactions, particularly dechlorination (4). Biochemical degradation can also occur. Studies with mixed populations (5, 16, 28) and with pure cultures (17-27, 29, 30) have shown that some of the less highly chlorinated compounds can be degraded to a greater or lesser extent by a variety of microorganisms. Combined photochemical and biochemical degradation has also been reported (16); it was suggested that photochemical dechlorination may occur, yielding products more readily attacked by microorganisms than were the original chlorinated biphenyls. In procaryotic organisms the principal metabolic pathway appears to involve the addition of molecular oxygen to one ring to form a dihydrodiol, oxidation of this to a diphenol, 1,Qmeta oxidation of the diphenol with ring cleavage to form a chlorinated 2-hydroxy-6-oxo-6phenylhexa-2,4-dienoic acid, and degradation of the aliphatic part of this, probably in several steps, to yield a chlorobenzoic acid (17-23,25,26,29,30). With most pure microbial cultures capable of attacking chlorinated biphenyls this accumulates in the medium. Organisms capable of oxidizing chlorobenzoic acids exist, however, so suitable mixed cultures are capable of completely degrading certain chlorinated biphenyls to carbon dioxide, water, and chloride ion (16, 28). With certain chlorinated biphenyls and certain organisms the reaction sequence may be partially or completely blocked at some point before chlorobenzoic acid, allowing intermediates to accumulate. If it is the chlorohydroxy608
Environ. Sci. Technol., Vol. 18, No. 8, 1984
oxophenylhexadienoic acid that accumulates, this is immediately apparent because these compounds are bright yellow, with absorption maxima at about 400 nm (17-22). This suggested that combined biochemical and photochemical degradation might occur, biochemical reactions converting the substrates to products susceptible to further degradation by photochemical processes. Experimental evidence in support of this hypothesis is presented here. Materials and Methods Reagents. 2,4’-Dichlorobiphenyl was obtained from Ultra Scientific, Hope, RI. A gas chromatograph trace provided by the supplier showed only one component. Aroclor 1242 was obtained from Monsanto. Other reagents were obtained from general laboratory suppliers. Organism. This was a species of Pseudomonas (strain 7509) originally isolated from activated sludge (24). It was grown on the mineral medium of Furukawa & Matsumura (19) with solid biphenyl as the sole carbon source. The organisms were usually grown for 36-48 h with shaking, the cultures were filtered through a coarse sintered glass filter to remove biphenyl, and the cells were harvested by centrifugation and resuspended in potassium phosphate buffer (0.05 M, pH 7.4), to a concentration of about 2 mg dry weight/mL. Experimental Procedures. In a typical experiment, 0.1-1 mg of chlorinated biphenyl, dissolved in acetone, was added to 50 mL of bacterial suspension, and the mixture was shaken, usually overnight. It was then centrifuged, and all or part of the supernatant was acidified to pH 1-2 with hydrochloric acid and extracted with three 10-mL portions of ethyl acetate. The ethyl acetate solution was then dried over anhydrous sodium sulfate and concentrated, and the solutes were methylated with diazomethane. To study the photochemical transformation of the metabolites, a portion of the supernatant was placed in a 10or 25-mL cuvette and irradiated with light from a 200-W mercury-xenon lamp. The light was filtered through Pyrex glass to remove short wavelength ultraviolet and provide a spectral distribution similar to that of sunlight at the surface of the earth. The cuvette was mounted in a water-cooled holder, and the solution was stirred by a magnetic stirrer mounted vertically against the end of the cuvette opposite to the lamp. The light intensity at the face of the cuvette was about 250 mW cm-2. When the solution was virtually colorless (usually 1-3 h), it was acidified and treated as described above. A few experiments were carried out with a commercial PCB, Aroclor 1242 (manufactured by the Monsanto Co., St. Louis). A total of 0.1 mL of the mixture was added to 50 mL of bacterial suspension, and the same procedure was followed. The extracts were analyzed on a Tracor gas chromatograph using an OV-101 column. To attempt to identify the components, the samples were then transferred to toluene or methylene chloride and analyzed on a CarloErba gas chromatograph/Riber-Nermag R10-10 mass spectrometer using a 25-m OV-1 or DB-1701 capillary column.
0013-936X/84/09 18-0608$01.50/0
0 1984 American Chemical Society
t inn -- . ,
C-OCHq
I
139
Gz
$
[email protected];31, ,il , 13gll,i,
$6>
Ill
w
50
154
KZ
0 50
Figure 1. Mass spectrum of methylated derivatives of components extracted from acldlfied mixture of bacterlal metabolkes of 2,4'-DCB.
100
150
200
Flgure 3. Mass spectrum of product found after irradiation of mixed metabolites of 2,4'-DCB.
i 1
I
CI
?
l
o
0
1
'
I'S'
0
m
CI
Q c-0 I
CH3
0
250
300
Flgure 2. Mass spectrum of methylated derivative of component extracted from acidified mixture of bacterlal metabolites of 2,4'-DCB.
Results Metabolites. The largest number of experiments was carried out with 2,4'-dichlorobiphenyl, which was found to give a good yield of the yellow meta oxidation product. Among the metabolites two compounds yielding virtually identical mass spectra showing an apparent molecular ion at m l e 170 (Figure 1) were consistently found. This spectrum corresponds closely to that of a monochlorobenzoic acid methyl ester (23). The presence of these compounds indicates that the transformation of the meta oxidation product is only partially blocked in this organism. The occurrence of two such compounds indicates that the organism can attack either the 2-substituted or the 4-substitued ring. Another compound consistently found had the spectrum shown in Figure 2. This corresponds closely to that reported for methylated 2-hydroxy-6-oxo-6-(chlorophenyl)monochlorohexa-2,4-dienoicacid (23). An apparent molecular ion at m l e 314 can be observed, and the peaks at m l e 111, 139, 175,212, 240, and 255 are characteristic of this type of compound. Two compounds having apparent molecular ions of m l e 252 were detected. The more abundant of these had a base peak corresponding to the molecular ion and a large peak at m l e 209 (M+ - 43) and is probably a 3-methoxydichlorobiphenyl; the other had a base peak at m l e = 202 (M+ - 50) and is probably a 2-methoxydichlorobiphenyl. The 3-methoxy compounds characteristically fragment by loss of COCH, (M = 43) whereas the 2-methoxy compounds lose CH3C1 (M = 50) (32). With Aroclor 1242, which contains about half trichlorobiphenyls with smaller amounts of the dichloro and tetrachloro compounds (5), we again found two products with spectra like that in Figure 1. In addition, we found two compounds with similar spectra but with all the peaks shifted upward by 34 mass units, corresponding to dichlorobenzoic acid methyl esters. We also found two compounds with spectra similar to that in Figure 2 (although the molecular ion could not be seen) and two with
m
CI
m
Figure 4. Principal steps in the biochemical and photochemical degradation of 2,4'-DCB. (B) Biochemlcai; (S) spontaneous: (P) photochemical. The occurrence of I1 and 111 is inferred.
corresponding spectra shifted upward by 34 mass units, but without the expected molecular ion at m l e 348. Photodegradation Products. When a colored solution of metabolites was irradiated until the color had largely or totally disappeared, the compound of m f e 314 (Figure 2) derived from 2,4'-dichlorobiphenyl, and the corresponding products from Aroclor 1242, could no longer be detected. This was to be expected if it is assumed that these compounds are methylated derivatives of chlorinated 2-hydroxy-6-oxo-6-(chlorophenyl)hexa-2,4-dienoic acids, the substances responsible for the color. At the same time there appeared in the photolysis products of the 2,4'-dichlorobiphenyl metabolites two new compounds having mass spectra of the type shown in Figure 3. This resembles the spectrum of chlorobenzoic acid methyl ester, except that the apparent molecular ion occurs at m l e 154 instead of 170. This corresponds to the spectrum of a monochloroacetophenone (32), and we believe these two compounds to be 2- and 4-chloroacetophenone. Two compounds with the same spectrum were also produced from the metabolites of Aroclor 1242 as well as one compound with a similar spectrum shifted upward by 34 mass units, corresponding to a dichloroacetophenone.
Discussion Our organism appears to transform chlorinated biphenyls by the same metabolic pathway as those previously described (17-27,29,30) so the main steps in the combined biochemical and photochemical degradation of 2,4'-dichlorobiphenyl can be summarized as in Figure 4. 2,4'Dichlorobiphenyl (I) is converted to a dihydrodiol(I1) and then to a diphenol (111),which in turn is converted to a hydroxyoxo(chloropheny1)chlorohexadienoic acid (IV). Biochemically, this is then degraded to chlorobenzoic acid (V). I t is likely that monohydroxychlorobiphenyls (VI) are derived from I1 when the solution is acidified (21). Environ. Sci. Technoi., Vol. 18, No. 8, 1984 609
Photochemically, IV undergoes degradation of the side chain to produce a chlorinated acetophenone (VII). Photochemical reactions of ketones involving degradation of an aliphatic chain are well-known to occur in the gas phase and involve an intramolecular hydrogen abstraction referred to as a type I1 mechanism (33). It seems possible that some similar mechanism may be involved here. At least some of’the more highly chlorinated components of Aroclor 1242 appear to undergo analogous series of reactions. There seems little doubt that both biochemical and photochemical processes contribute to the degradation of chlorinated biphenyls in the environment. It also seems likely that the combined photochemical-biochemical process described by Kong and Sayler (16) may also play a part. We believe it is possible that the biochemicalphotochemical sequence of reactions described in this paper may also contribute. The reactions proceed at a significant rate at ambient temperature in aqueous solution, and the intensity and spectral distribution of the light required for the photochemical step are comparable to those of sunlight at the surface of the earth.
Acknowledgments We are greatly indebted to Dickson Liu for providing a culture of Pseudomonas 7509, to Michael E. Comba for analyzing our samples in the GC/MS and for advice on the interpretation of the spectra, and to John H. Carey for assistance and advice in photochemical matters. Registry No. 2,4’-DCB, 34883-43-7; Aroclor 1242,53469-21-9.
Literature Cited (1) Jensen, S. Ambio 1972, 1, 123-131. (2) Ballschmiter, K.; Zell, M.; Neu, H. 3. Chemosphere 1978, 7, 173-176. (3) Corke, C. T.; Bunce, N. J.; Beaumont, A.-L,; Merrick, R. L. J . Agric. Food Chem. 1979,27,644-646. (4) Bunce, N. J.; Kumar, Y.; Brownlee, B. G. Chemosphere 1978, 7, 155-164. (5) Tucker, E. S.; Saeger, V. W.; Hicks, 0. Bull. Environ. Contam. Toxicol. 1975, 14, 705-713. (6) Baxter, R. A.; Gilbert, P. E.; Lidgett, R. A.; Mainprize, J. H.; Vodder, H. A. Sci. Total Environ. 1975, 4, 53-61. (7) Sayler, G. S.; Shon, M.; Colwell, R. R. Microb. Ecol. 1977, 3, 241-245. (8) Ballschmiter, K.; Unglert, Ch.; Neu, H. J. Chemosphere 1977, 6 , 51-56. (9) Carey, A. E.; Harvey, G. R. Bull. Enuiron. Contam. Toxicol. 1978, 30, 527-534.
610
Environ. Sci. Technol., Vol. 18, No. 8, 1984
(10) Tulp, M. Th. M.; Schmitz, R.; Hutzingsr, 0. Chemosphere 1978, 7, 103- 108. (11) Sayler, G. S.;Thomas, R.; Colwell, H.R. Estuarine Coastal Mar. Sei. 1978, 6, 553-567. (12) Clark, R. R.; Chian, E. S. K.; Griffin, R. A. Appl. Enuzron. Microbiol. 1979, 37, 680-685. (13) Liu, D. Bull. Environ. Contam. Toxicol. 1981,27,695-703. (14) Reichardt, P. B.; Chadwick, B. L.; Cole, M. A.; Robertson, B. R.; Button, D. K. Environ. Sci. Technol. 1981,15,75-79. (15) Shiaris, M. P.; Sayler, G. S. Enuiron. Sci. Technol. 1982, 16, 367-369. (16) Kong, H.-L.; Sayler, G. S. Appl. Environ. Microbiol. 1983, 46. 666-672. (17) Ahned, M.iFocht, D.D. Can. J. Microbiol. 1973,19,47-52. (18) Ahmed, M.; Focht, D. D. Bull. Environ. Contam. Toxicol. 1973, 10, 70-72. (19) Furukawa, K.; Matsumura, F. J . Agric. Food Chem. 1976, 24, 251-256. (20) Furukawa, K.; Tonomura, K.; Kamibayashi, A. Appl. Enw o n . Microbiol. 1978, 35, 223-227. (21) Furukawa, K.; Tonomura, K.; Kamibayashi, A. Agric. Biol. Chem. 1979, 43, 1577-1583. (22) Furukawa, K.; Tomizuka, N.; Kamibayashi, A. Appl. Environ. Microbiol. 1979, 38, 301-310. (23) Yagi, 0.;Sudo, R. J . Water Pollut. Control Fed. 1980,52, 1035-1043. (24) Liu, D. Water Res. 1980, 14, 1467-1475. (25) Sylvestre, M. E a u Que. 1980, 13, 204-207. (26) Sylvestre, M. Eau Que. 1982, 15, 394-397. (27) Liu, D. Bull. Environ. Contarn. Toxicol. 1982,29,200-207. (28) Furukawa, K.; Chakrabarty, A. M. Appl. Environ. Microbiol. 1982, 44, 619-626. (29) Sylvestre, M.; Mass&,R.; Messier, F.; Fauteux, J.; Bisaillon, J.-G.; Beaudet, R. Appl. Environ. Microbiol. 1982, 44, 871-877. (30) Furukawa, K. In “Biodegradation and Detoxification of Environmental Pollutants”; Chakrabarty, A. M., Ed.; CRC Press Inc.: Boca Raton, FL, 1982; pp 33-57. (31) Jansson, B.; Sundstrbm, G. Biomed. Mass Spectrom. 1974, 1, 386-392. (32) Stenhagen, E.; Abrahamsson, S.; McLafferty, F. W. “Registry of Mass Spectral Data”; Wiley: New York, 1974; Vel-I, i p 1-826, (33) Turro, N. J. “Modern Molecular Photochemistry”; The Benjamin/Cummings Publishing Co.: Menlo Park, CA, 1978; pp 1-628.
Received for review July 25, 1983. Revised manuscript received February 1, 1984. Accepted February 1.3, 1984. Some of this material was presented at the Thirty-third Annual Meeting of the Canadian Society of Microbiologists, Winnipeg, Manitoba, Canada, J u n e 19-23,1983.
