Environ. Sci. Technol. 1996, 30, 1115-1119
Bacterial Transformation of Pyrene in a Marine Environment XING-FANG LI, XIAO-CHUN LE,† CHRISTOPHER D. SIMPSON, WILLIAM R. CULLEN,* AND KENNETH J. REIMER Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
The bacterial transformation of pyrene in an estuary environment, Kitimat Arm, British Columbia, Canada, was studied. A metabolite was separated from marine sediment and pore water samples and was identified as cis-4,5-dihydroxy-4,5-dihydropyrene, based on a comparison of the metabolite with the authentic standard, by using both high-performance liquid chromatography and capillary gas chromatography with mass spectrometry detection. The presence of this key metabolite of dioxygenase-mediated transformation of pyrene, along with previous pyrene degradation studies using cultures isolated from the same sediment samples, suggests a possible in situ bacterial transformation of pyrene in the Kitimat Arm environment.
Introduction Hydrocarbon-degrading microorganisms are ubiquitous in most ecosystems (1, 2); however, it is often very difficult to prove that transformation, degradation, and mineralization actually occur in the environment because it is difficult to distinguish contribution from biotic and abiotic processes under uncontrolled conditions in the natural environment (3). In contrast, laboratory assays can provide definitive evidence for microbial degradation, and sterilized samples can be used to determine abiotic losses. Thus contributions from microbial degradation can be differentiated from abiotic loss from a mass balance study performed in a sealed vessel. Such studies have led to a better understanding of biodegradation of organic compounds (1, 4-13). Results obtained from laboratory studies have also been applied to in situ bioremediation of gasoline-contaminated aquifers and oil-spilled soils (1, 2, 14-20). In our previous reports (21, 22), we have shown that microorganisms enriched from seawater and sediment samples from Kitimat Arm, B.C, Canada, are capable of utilizing phenanthrene and pyrene. The microbial degradation of pyrene was further confirmed by the production of metabolites and 14CO2 from 14C-labeled pyrene (22). It * To whom correspondence should be addressed: Telephone: (604) 822-4435; Fax: (604) 822-2847. † Present address: Environmental Health Program, Department of Public Health Sciences, Faculty of Medicine, University of Alberta, Edmonton, AB, Canada T6G 2G3.
0013-936X/96/0930-1115$12.00/0
1996 American Chemical Society
now remains to be established whether or not if the microorganisms are able to carry out the transformation in the natural environment. It is very important to obtain this information in order to understand the fate of PAHs in a particular environment of study. Three approaches have been recommended to obtain evidence for in situ biodegradation (3, 23, 24). These include (i) quantitative determination of the pollutant of interest in samples collected at different times to show a decrease in its concentration over time; (ii) laboratory based microbial degradation studies under conditions that mimic the environment to show the potential of biodegradation in the field; and (iii) searching for a particular metabolite of biodegradation in samples collected from the field. It is very difficult to demonstrate microbial degradation in Kitimat Arm by monitoring the loss of PAHs in the samples, because PAHs are being continuously added to the environment from the nearby aluminum smelting plant. Without knowing the amount and nature of the PAH input, it is impossible to estimate any biotic loss of PAHs. Furthermore, weathering and other abiotic processes simultaneously occur and contribute to changes in the concentrations of PAHs in samples. For these reasons, two other approaches, laboratory microbial degradation and the determination of a target transformation metabolite in the sample, appear to be useful to evaluate the possibility of microbial transformation in the Kitimat Arm environment. cis-4,5-Dihydroxy-4,5-dihydropyrene is a well-established metabolite of pyrene from bacterial degradation (5, 25). We have obtained the same metabolite in the previous studies by using the cultures enriched from Kitimat Arm samples (21, 22). Further mineralization of the metabolite was observed only after pyrene was degraded to low level. Consequently, if the bacterial transformation that occurs in the field is similar to that in laboratory studies, cis-4,5dihydroxy-4,5-dihydropyrene is expected to be present in the samples from Kitimat Arm, where there is a continuous input of pyrene. For this reason, effort was made to detect cis-4,5-dihydroxy-4,5-dihydropyrene in samples from Kitimat Arm. The finding of this metabolite in the field along with previous laboratory culture studies provide an evidence for the presence of the in situ bacterial transformation of pyrene in Kitimat Arm.
Experimental Section Instrumentation. A high-performance liquid chromatography (HPLC) system, consisting of a Waters 600E multisolvent delivery unit, a system controller, and a U6K sample injector, equipped with a UV/visible spectrophotometer (Waters Lambda-Max Model 481), and a scanning fluorescence detector (Waters Model 470) were used for all HPLC analysis. Two reversed-phase analytical HPLC columns (Supelco LC-PAH and GL Scientific ODS-2, 250 mm × 4.6 mm i.d.) and a preparative HPLC column (Waters RCM, 100 mm × 25 mm i.d.) were used for separation. A GC/MS system (VG 7070E) and a GC/FID system (HP 5890) were used to analyze the acetylation products of the authentic standard, cis-4,5-dihydroxy-4,5-dihydropyrene, and the bacterial transformation metabolites of pyrene. A capillary column, DB-5 (30 m × 0.25 mm i.d., 0.25 µm) was
VOL. 30, NO. 4, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1115
FIGURE 1. Sampling sites in Kitimat Arm (53°50′ N, 128°50′ W), British Columbia, Canada.
used for the separation. The GC/FID system was also used for the determination of pyrene. Reagents. Authentic standard, cis-4,5-dihydroxy-4,5dihydropyrene, was generously provided by Dr. C. E. Cerniglia of National Center for Toxicological Research, Food and Drug Administration (25). HPLC-grade acetonitrile and methanol (Fisher Scientific) were used in all HPLC analyses. Analytical reagent-grade acetylation reagents (acetic anhydride and pyridine) were obtained from Aldrich. All other reagents used were of analytical reagent grade or better. Samples. The present study focused on the northern part of Kitimat Arm (British Columbia, Canada), where an aluminum smelter is located. The aluminum smelter is the major source of PAH input to this environment, because of pyrolysis of the pitch and tar in the smelting processes. All the samples used in this study were obtained from the locations shown in Figure 1. Surface sediments were collected from the top 5 cm by using the grab sampling method. Samples were immediately placed in sterile glass jars (500 mL) and stored at 4 °C before being processed for analysis. The sample jars were completely filled with sediment samples to reduce the amount of oxygen. The STN2, K5a, and KA7 samples were collected in May 1991. They were freeze-dried and stored at 4 °C before they were extracted for the determination of pyrene and cis-4,5dihydroxy-4,5-dihydropyrene. The six CD samples (CD-1 to CD-6) were obtained in November 1993. One set of the C-sed samples were collected in June 1992 and were directly inoculated into the mineral salt media to generate cultures that were used for the studies of phenanthrene and pyrene degradation (21, 22). One year later, another set of the C-sed samples were obtained from a similar location to investigate the presence of cis-4,5-dihydroxy-4,5-dihydropyrene. The wet sediment, without any pretreatment, was directly used for solvent extraction of the metabolite. These samples were analyzed within 3 days.
1116
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 4, 1996
The depth (in meter) of water above the sediment is 2 (STN2), 0.5 (CD1), 3 (CD2), 2 (CD3), 7 (CD4), 15 (CD5), 30 (CD6), 94 (K5a), and 119 (KA7), respectively. Sediment samples CD-1, STN2, and C-sed were dark and muddy and had a distinct sulfurous smell. All other sediment samples were brown and sandy. Determination of Pyrene. Concentration of pyrene in sediment samples was obtained by using a method for the determination of PAHs (26). Briefly, a 10-20-g sample of a freeze-dried sediment was Soxhlet extracted with 150 mL of HPLC-grade dichloromethane for 6 h. The extract was then condensed to almost dryness, adsorbed onto dry Na2SO4, and quantitatively transferred and washed onto a Florisil cartridge with two portions of 1 mL of hexane. The 1.2 cm diameter cartridge had been slurry packed with 3 g of Florisil (Fisher Scientific; 60-100 mesh, 2% deactivation with distilled water) in hexane and was used for cleanup purposes, to separate aromatic compounds from other components in the sample. The column was eluted with 15 mL of hexane followed by 25 mL of a 1:1 mixture of hexane/dichloromethane. The PAH-containing fraction was rotary evaporated to dryness and redisolved in 2 mL of toluene. A 2-µL aliquot of the toluene solution was injected onto a capillary column (PTE5, 1 m × 0.32 mm i.d., from Supelco) for GC/FID analysis. The GC oven temperature was programmed as follows: initial temperature of 100 °C for 4 min; 5 °C/min to 200 °C, held for 3 min; 8 °C/min to 250 °C, held for 5 min; and finally 12 °C/min to 300 °C, held for 20 min. The FID detector was maintained at 320 °C and was operated at an air flow of 300 mL/min and a hydrogen flow of 30 mL/min. Concentration of pyrene in several sediment samples is (in µg/g dried sediment) 40 (STN2), 34 (CD1), 18 (CD2), 0.6 (CD3), 1.2 (CD5), 3.0 (CD6), 3.4 (K5a), and 30 (KA7), respectively. Identification and Determination of cis-4,5-Dihydroxy4,5-dihydropyrene. Initial studies were carried out using a freeze-dried sediment sample, STN2. A portion of 50 g of this sediment was treated with 200 mL of acetonitrile with constant mechanic shaking for 8 h. The supernatant acetonitrile extract was decanted and filtered through a filter paper (Whatman 4) to remove the particles. The filtrate was concentrated to approximately 16 mL on a rotary evaporator at 35 °C and then further condensed to 5 mL under a gentle N2 stream. The extract was filtered through a 0.45-µm membrane filter, and an aliquot of 10 µL of filtrate was subjected to HPLC analysis. A reversed-phase C18 column (Supelco LC-PAH, 250 mm × 4.6 mm i.d.) was used for separation. A mixture of methanol and water (55:45) was used as the mobile phase, and the flow rate was 1.0 mL/min. A peak at the retention time of 10.9 min corresponding to cis-4,5-dihydroxy-4,5-dihydropyrene was observed. However, the extract also contained a great number of other components. In order to separate the peak at 10.9 min from other late-eluting components and to reduce the eluting time, the extract (1 mL) was first separated on a preparative column (Waters RCM, 100 mm × 25 mm i.d.); 55% methanol in water was used as the mobile phase at 13.8 mL/min. The fraction eluting between 3 and 14 min was collected, because standard cis-4,5-dihydroxy-4,5-dihydropyrene was found to elute in this range of retention time. The fractions collected from several sample injections were pooled together and concentrated to 0.5 mL. An aliquot (10 µL) of the concentrated solution was then injected onto the
analytical column (Supelco LC-PAH) and eluted by using methanol/H2O (55:45) at a flow rate of 1.0 mL/min. The component that eluted at 10.9 min, corresponding to cis4,5-dihydroxy-4,5-dihydropyrene, was collected, pooled, and concentrated to 0.2 mL for further characterization. When this purified fraction was analyzed on another analytical column (ODS-2, GL Scientific), by using the same mobile phase, a peak at 25.6 min was detected, which matched the authentic standard cis-4,5-dihydroxy-4,5dihydropyrene. This purified fraction (10.9 min) was also used to study the UV absorption and fluorescence properties of the metabolite by using HPLC/UV/fluorescence detection. A series of replicate HPLC/UV/fluorescence determinations were carried out. The detection wavelength was manually changed at 5-nm intervals after each determination. Chromatographic peak intensities of the metabolite at a series of wavelengths were obtained. By plotting peak intensity vs wavelength, a spectrum (e.g., absorption between 200 and 320 nm) of the metabolite was obtained. The fraction collected from the preparative column (Waters RCM) was acetylated with the treatment of acetic anhydride and pyridine (27). The procedures for the acetylation and determination of the products were the same as those previously used for the standard and the metabolite isolated from the cultures (22). Briefly, 0.3 mL of the metabolite-containing solution was evaporated to dryness, and the residue was treated with acetic anhydride (20 µL) and pyridine (10 µL). This mixture was placed in a warm water bath at 60 °C for 30 min. Then the reagents were evaporated to dryness under a gentle N2 stream. The residue was dissolved in 100 µL of toluene, of which 2 µL was analyzed by using GC/FID and GC/MS. The same treatment was also applied to the whole acetonitrile extract without preseparation. However, the expected product was not resolved from the other major components, and thus GC/MS did not provide useful mass spectra for individual compounds. Therefore, it is necessary to preseparate the metabolite on the preparative column from the other components in the acetonitrile extract in order to obtain complete separation and a good mass spectrum for the metabolite by using GC/MS. Other sediment samples (10 g) were extracted with acetonitrile (three times with 50 mL). These extracts were evaporated down to 1 mL and were analyzed by using HPLC to screen the presence of the metabolite and to estimate its concentration. The mobile phase for the initial 15 min was 55% methanol in water and then linearly ramped to 100% methanol within 20 min. The mobile phase remained at 100% methanol for a further 50 min before being changed to 55% methanol in water. The concentration of cis-4,5dihydroxy-4,5-dihydropyrene in each sample was estimated from the peak area measurement of the peak at a retention time of 10.9 min, calibrated against the standard cis-4,5dihydroxy-4,5-dihydropyrene. These procedures were also used to analyze the extracts of the pore water samples (1 L each) after they were extracted by using ethylacetate (approximately 200 mL) for three times. The extracts were concentrated (rotary evaporation) to 0.5 mL prior to HPLC analysis.
Results and Discussion Bacterial transformation of PAHs usually involves the use of dioxygenase enzymes (4, 5, 28): both atoms from the molecular oxygen are incorporated into the aromatic
FIGURE 2. HPLC with fluorescence detection for (a) sediment sample STN2; (b) cis-4,5-dihydroxy-4,5-dihydropyrene standard; and (c) 1:1 mixture of a and b. The peak due to cis-4,5-dihydroxy-4,5dihydropyrene is marked with an asterisk (*). HPLC column: Supelco LC-PAH, 250 mm × 4.6 mm i.d.; mobile phase: 55% methanol in water.
compound. This initial oxidation reaction results in the formation of dihydroxy-dihydro derivatives of PAHs with a cis-conformation (25, 28-32). Subsequent reactions lead to ring cleavage, the formation of small metabolites such as catechol, phthalic acid, salicylic acid, and protocatechuic acid (4, 28, 32, 33), and further mineralization (13, 28, 32). In a detailed study of the degradation of pyrene by Mycobacterium sp. PYR-1, Heitkamp et al. (25) isolated several metabolites from the culture, including cis-4,5dihydroxy-4,5-dihydropyrene. Cerniglia (5) further proposed two pathways for the bacterial degradation of pyrene, one of which involved the formation of cis-4,5-dihydroxy4,5-dihydropyrene. This initial oxidation product is believed (5, 25) to be a key metabolite from dioxygenasemediated bacterial transformation of pyrene. We also obtained this metabolite from the degradation of pyrene using cultures obtained from the Kitimat Arm environment (22). The metabolite was further mineralized to give CO2 and H2O. However, only after the concentration of pyrene became very low did the metabolite concentration gradually decrease to undetectable levels. Therefore, there is the possibility that cis-4,5-dihydroxy-4,5-dihydropyrene is present at a detectable level if there is a continuous supply of pyrene available for bacterial degradation. The concentration of cis-4,5-dihydroxy-4,5-dihydropyrene, therefore, was estimated in sediment samples obtained from Kitimat Arm. There is no known natural source of cis4,5-dihydroxy-4,5-dihydropyrene. Thus, the presence of this compound together with laboratory culture studies would be strong evidence for the bacterial transformation of pyrene in the environment. Figure 2a shows a chromatogram of an acetonitrile extract obtained from a sediment sample, STN2. A fluorescence detector was used for detection with excitation and emission wavelengths of 260 and 370 nm, respectively.
VOL. 30, NO. 4, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1117
A similar chromatogram was also obtained from this sample when UV detection at 254 nm was used. The small peak at retention time 10.9 min (Figure 2a) corresponds to that of the standard, cis-4,5-dihydroxy-4,5-dihydropyrene (Figure 2b). Analysis of a mixture of the sample and the standard, at a 1:1 ratio, shows a single peak eluted at 10.9 min (Figure 2c). From the peak height and peak area measurement, the recovery of the spiked standard is found to be approximately 95%. The chromatograms in Figure 2 demonstrate that the HPLC retention time of the compound of interest in the sample matches that of cis4,5-dihydroxy-4,5-dihydropyrene. The fraction of 10.9 min retention time was collected after eluting from both the preparative and analytical HPLC columns and was further characterized by using another reversed-phase HPLC column (ODS-2, from GL Scientific). This general purpose reversed-phase C18 column retains the compound stronger, giving a retention time of 25.6 min. Standard cis-4,5-dihydroxy-4,5-dihydropyrene also elutes at 25.6 min, in agreement with the result of Heitkamp et al. (25). A mixture of the sample and the standard (1:1 ratio) also elutes at the same retention time, suggesting that the compound in the sample matches the standard. Standard of trans-4,5-dihydroxy-4,5-dihydropyrene is not available to us. However, the cis and trans forms can be well resolved under the present HPLC conditions, as demonstrated by Heitkamp et al. (25). A direct UV/visible spectral scan of the sample did not provide useful information because of the insufficient sensitivity. In addition, the impurities in the fraction made it difficult or even impossible to obtain a UV/visible spectrum corresponding to the trace amount of the compound of interest. Therefore, a series of replicate HPLC/UV determinations of the same sample were carried out, only changing the detection wavelength after each determination. By plotting the peak intensity vs wavelength (200-320 nm at 5-nm intervals), a UV absorption spectrum was obtained. The UV spectra obtained from the sample and cis-4,5-dihydroxy-4,5-dihydropyrene have similar absorption maxim (220 and 260 nm) as those previously reported (25). The identical HPLC retention time and similar absorption spectra between the compound of interest and the standard cis-4,5-dihydroxy-4,5-dihydropyrene strongly suggest that this compound is cis-4,5dihydroxy-4,5-dihydropyrene, one of the common metabolites from the bacterial transformation of pyrene. Gas chromatography/mass spectrometry (GC/MS) was used to further confirm the identification. The fraction collected from the preparative column was acetylated (27). The chromatogram of the mixture of the acetylation products and the mass spectrum of the acetylated metabolite are shown in Figure 3. The chromatographic retention time (Figure 3a) and the mass spectrum (Figure 3b) of the compound are identical to those of the diacetate of the cis-4,5-dihydroxy-4,5-dihydropyrene authentic standard. A mass spectrum of the authentic standard can be found in ref 25. The mass spectral peak at m/z 320 is due to the diacetate of cis-4,5-dihydroxy-4,5-dihydropyrene (M•+). The major fragment ions are designated as follows: 260 (loss of CH3COOH from M•+), 218 (loss of CH2dCO from m/z 260), and 189 (loss of HCO• from m/z 218). These results strongly support the presence of cis-4,5-dihydroxy4,5-dihydropyrene in the sample. A number of sediment samples from various locations were analyzed by using HPLC/UV/fluorescence detection
1118
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 4, 1996
FIGURE 3. GC/MS analysis of the sediment sample STN2 after cleanup and acetylation treatments, showing the gas chromatogram (a) and mass spectrum (b) of the acetylation products. The peak marked with an asterisk (*) is identified as the diacetate of cis-4,5-dihydroxy4,5-dihydropyrene.
to screen the presence of this metabolite and to estimate its concentration. Sediment samples obtained from near the shore including STN2, C-sed, and CD1 were also found to give a chromatographic peak corresponding to cis-4,5dihydroxy-4,5-dihydropyrene. Based on the assumption that this peak is due exclusively to this compound, the concentration of cis-4,5-dihydroxy-4,5-dihydropyrene was estimated to be approximately 10-20 ng/g. The concentrations of pyrene in these samples were determined by using GC/FID and were found to be 34-40 µg/g. The CD3 and K5a sediment samples obtained further away from the shore in Kitimat Arm contain much lower concentrations of the metabolite (0.5-1 ng/g) and pyrene (0.6-3 µg/g). The metabolite was not detectable in sediment sample KA7. The detection limit for the determination is 0.5 ng/g for a 10-g sediment sample. No cis-4,5-dihydroxy-4,5-dihydropyrene was detected in seawater samples (below detection limit of 5 pg/mL). This is because pyrene tends to associate with sediment due to the hydrophobicity of pyrene, and there is not enough pyrene available in water for bacterial transformation to produce a detectable amount of the metabolite. A small amount of cis-4,5-dihydroxy-4,5-dihydropyrene (10-15 pg/ mL) was detected in the pore water of CD1 and CD3. This concentration is much lower than that in the sediment from the same site, probably because both pyrene and cis4,5-dihydroxy-4,5-dihydropyrene have low solubility in water and tend to associate with the sediment. We have previously shown that pyrene-utilizing cultures can be isolated from sediment samples that were obtained from Kitimat Arm. Samples from the same location were used in the present study. By using various analytical techniques, we have now identified cis-4,5-dihydroxy-4,5dihydropyrene, a key oxidation product from the bacterial transformation of pyrene, in these samples. These results suggest a possible in situ bacterial transformation of pyrene in the Kitimat Arm environment.
Acknowledgments This study was funded by the Natural Sciences and Engineering Research Council (NSERC) and the Department
of Fisheries and Oceans, Canada. The authors thank Mr. G. Hewitt for his technical support. They also thank Dr. C. E. Cerniglia of National Center for Toxicological Research, Food and Drug Administration for the cis-4,5-dihydroxy4,5-dihydropyrene standard. The NSERC of Canada and the Canada’s Killam Trust are thanked for a postgraduate fellowship (X.F.L.) and a Killam Predoctoral Fellowship (X.C.L.).
Literature Cited (1) Prince, R. C. Crit. Rev. Microbiol. 1993, 19, 217-242. (2) Pritchard, P. H.; Mueller, J. G.; Rogers, J. C.; Kremer, F. V.; Glaser, J. A. Biodegradation 1992, 3, 315-335. (3) Madsen, E. L. Environ. Sci. Technol. 1991, 25, 1663-1673. (4) Gibson, D. T.; Subramanican, V. In Microbial Degradation of Organic Compounds; Gibson, D. T., Ed.; Marcel Dekker: New York, 1984; pp 181-252. (5) Cerniglia, C. E. Biodegradation 1992, 3, 351-368. (6) Evans, W. C. Nature 1977, 270, 17-22. (7) Smith, M. R. Biodegradation 1990, 1, 191. (8) Foght, J. M.; Fedorak, P. M.; Westlake, D. W. S. Can. J. Microbiol. 1990, 36, 169. (9) Simkins, S.; Alexander, M. Appl. Environ. Microbiol. 1984, 47, 1299-1306. (10) Pritchard, P. H.; Bourquin, A. W. Adv. Microbiol. Ecol. 1984, 7, 133-215. (11) Alexander, M. Science 1981, 211, 132-138. (12) Herbes, S. E.; Schwall, L. R. Appl. Environ. Microbiol. 1978, 35, 306-316. (13) Bauer, J. E.; Capone, D. G. Appl. Environ. Microbiol. 1988, 54, 1649-1655. (14) Wilson, S. C.; Jones, K. C. Environ. Pollut. 1993, 81, 229-249. (15) Bragg, J. R.; Prince, R. C.; Harner, E. J.; Atlas, R. M. Nature 1994, 368, 413-418. (16) Pritchard, P. H.; Costa, C. F. Environ. Sci. Technol. 1991, 25, 372-379. (17) Bartha, R. Microbiol. Ecol. 1986, 12, 155-172.
(18) Rittmann, B. E.; Johnson, N. M. Water Sci. Technol. 1989, 21, 209-219. (19) Dibble, J.; Bartha, R. Soil Sci. 1979, 125, 56-60. (20) Bourquin, A. W. Hazard. Mater. Control 1989, 2, 16-59. (21) Cullen, W. R.; Li, X.-F.; Reimer, K. J. Sci. Total Environ. 1994, 156, 27-37. (22) Li, X.-F.; Cullen, W. R.; Reimer, K. J.; Le, X.-C. Sci. Total Environ. 1995, 177, 17-29. (23) National Research Council. In Situ Bioremediation: When Does It Work?; National Academy Press: Washington, DC, 1993. (24) MacDonald, J. A.; Rittmann, B. E. Environ. Sci. Technol. 1993, 27, 1974-1979. (25) Heitkamp, M. A.; Freeman, J. P.; Miller, D. W.; Cerniglia, C. E. Appl. Environ. Microbiol. 1988, 54, 2556-2565. (26) Simpson, C. D.; Cullen, W. R.; Quinlan, K. B.; Reimer, K. J. Chemosphere 1995, 31, 4143-4155. (27) Brooks, C. J. W.; Cole, W. J.; Borthwick, J. H.; Brown, G. M. J. Chromatogr. 1982, 239, 191-216. (28) Cerniglia, C. E.; Heitkamp, M. A. In Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment; Varanasi, U., Ed.; CRC Press: Boca Raton, FL, 1989; pp 41-68. (29) Catterall, F. A.; Murray, K.; Williams, P. A. Biochim. Biophys. Acta 1971, 237, 361-364. (30) Jeffery, A. M.; Yeh, H. J. C.; Jerina, D. M.; Patel, R. T.; Davey, J. F.; Gibson, D. T. Biochemistry 1975, 14, 575-584. (31) Ensley, B. D.; Gibson, D. T.; Laborde, A. L. J. Bacteriol. 1982, 149, 948-954. (32) Cerniglia, C. E. In Biochemical Toxicology; Hodgson, E., Bend, J. R., Philpot, R. M., Eds.; Elsevier/North-Holland: New York, 1981; p 321. (33) Evans, W. C.; Fernley, H. N.; Griffiths, E. Biochem. J. 1965, 95, 819-831.
Received for review May 3, 1995. Revised manuscript received October 23, 1995. Accepted November 21, 1995.X ES950321O X
Abstract published in Advance ACS Abstracts, February 1, 1996.
VOL. 30, NO. 4, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1119
