Environ. Sci. Technol. 1993, 27, 2207-2212
Metabolite Detection as Evidence for Naturally Occurring Aerobic PCB Biodegradation in Hudson River Sediments Wllllam P. Flanagan' and Ralph J. May General Electric Corporate Research and Development, P.O. Box 8, Schenectady, New York 12301-0008
Although aerobic microbial biodegradation of polychlorinated biphenyls (PCBs) has been widely demonstrated in the laboratory, there is little direct evidence that this process occurs naturally in the environment. A clear indicator of naturally occurring aerobic PCB biodegradation would be the presence of intermediate metabolites such as chlorobenzoic acids (CBAs) in contaminated sediments. CBAs have been detected in contaminated sediment cores, and their concentration profiles were correlated with PCB depth profiles. From the congener distribution pattern of the CBAs detected, it is extremely unlikely that these compounds were derived from eit,her the breakdown of chlorinated herbicides or the carboxylation of phenols. No CBAs were detected in sediment samples not contaminated with PCBs. In addition, other metabolites which are less prone to source ambiguity, and including 2,3-dihydro-2,3-dihydroxy-2'-chlorobiphenyl 2,3-dihydroxy-2'-chlorobiphenyl,have also been detected. To our knowledge, these findings represent the first detection of metabolites of aerobic PCB biodegradation in contaminated environmental samples.
Introduction Polychlorinated biphenyls (PCBs) are a family of compounds (congeners) consisting of a biphenyl nucleus carrying from 1 to 10 chlorine atoms. The widespread release of these materials into the envirqnment prior to a ban on their use in the 1970s is an issue of public and regulatory concern due to their relative persistence in the environment, their ability to bioaccumulate in fatty tissues, and the consequent potential health risks (1). Once widely thought to be recalcitrant, PCBs have since been shown to biodegrade via two distinct microbially mediated mechanisms: anaerobic reductive dechlorination, involving the removal of chlorine atoms from PCBs in the absence of oxygen (2,3);and aerobic biodegradation, involvingthe oxidative destruction of PCB molecules through a series of degradation intermediates (4, 5 ) . Evidence for ongoing anaerobic PCB dechlorination in aquatic sediments was obtained from the analysis of modified congener distribution patterns at contaminated sites (6-8). Subsequent investigations showed that similar selective shifts in PCB congener distributions could be reproduced in the laboratory and that these shifts were indeed microbially mediated (2, 3). Aerobic PCB biodegradation by a variety of naturally occurring bacteria has been extensively studied in the laboratory (4,5,9-11), but relatively little is known about this process in the environment. In one study, congener pattern modifications observed in surface sediment deposits were attributed to aerobic PCB biodegradation (81,although these results could alternativelybe explained by nonbiologicalprocesses such as selective dissolution or partitioning of congeners
* To whom correspondence should b e addressed. 0013-936X/93/0927-2207$04.00/0
0 1993 American Chemical Society
(12). Thus far, no conclusive evidence for aerobic PCB biodegradation in the environment has been reported. The enzymatic pathway for aerobic PCB biodegradation has been studied in a number of bacterial systems (5,1315). The predominant mechanism occurs via a 2,3dioxygenase attack resulting in the formation of a chlo(Figure 1).This rinated 2,3-dihydro-2,3-dihydroxybiphenyl product is further degraded to a chlorinated 2,3-dihydroxybiphenyl, followed by enzymatic cleavage of the hydroxylated ring to ultimately form the corresponding chlorobenzoicacid (CBA) and a five-carbon fragment. This paper describes the results of efforts to detect CBAs and other metabolites of aerobic PCB biodegradation in contaminated sediments collected from the upper Hudson River. The presence of such metabolites is a clear indication of naturally occurring aerobic PCB biodegradation (16) and has important implications regarding the fate and assessment of PCBs in the environment.
Experimental Section
Site Description and Sample Collection. PCBs were released into the upper Hudson River from capacitor manufacturing operations at Hudson Falls and Fort Edward, NY, between 1946 and 1977 (12). The originally deposited PCBs consisted primarily of Aroclor mixture 1242 (containing ca. 17% mono- and dichlorinated biphenyls) with a small amount of Aroclor 1254. The PCBs in this region have since undergone significant environmental dechlorination (6-8) such that, although overall sediment PCB levels have decreased with time (12),the PCB mixtures now contain a greater proportion of monoand dichlorinated biphenyls (ca. 62-73%). These environmentally altered PCB mixtures have been shown to undergo aerobic PCB biodegradation both in laboratory studies (10, 11) and in a recently completed large-scale field study of stimulated in situ aerobic PCB biodegradation (17). The Hudson River north of Hudson Falls, NY, contains essentially no PCBs. Four sediment cores were collected from a PCBcontaminated region spanning from 3.2 to 4.7 km downstream of Fort Edward (Figure 2). Sample coring was accomplished using a 2-in. (5.08-cm) diameter sediment corer (WildcoModel 2420-G55) equipped with plastic tube inserts. A separate insert was used for each sample core. Upon removal from the river, the sediment cores were immediately frozen on dry ice, sectioned at l-in. (2.54-cm) intervals, and stored at -20 "C prior to extraction and analysis for PCBs and CBAs. Cores HR1 and HR2 were collected on Sept 3,1991; core HR3 was collected on Jan 8, 1992; core HR4 was collected on Oct 30, 1991. Non-PCB-contaminated regions were also sampled. A series of 10sediment grab samples (ca. 0.4 L) was collected on Feb 20, 1992, from noncontaminated regions of the upper Hudson River spanning from ca. 15 km upstream of Fort Edward, NY, to 0.6 km upstream of Lake Luzerne (Figure 2). An 11th grab sample was collected from nearby Environ. Sci. Technol., Vol. 27, No. IO, 1993 2207
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Flgure 1. Biodegradation of chlorobiphenyls by the 2.3dioxygenase pathway. (A) Po~chlorinaledbiphenyl (PCB): (5)chlorinaled 2.3dihydro2.3dlhydrovbiphenyl; (CJchlorinated 2.3dinydrowbiphenyI, (DI chlorinated 2-hydroxy-6-oxo-6phenylhexa-2.4dienoic acld: (E)chlorinatedDenloic acid and a five-CarDon fragment. The chlorooenzo c acids are tunher oegraoed to caroon dioxide. water, chloride Ion. and biomass by a variety 01 microorganisms present in river sediments (22-24)
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Flgure 2. Sediment sample collection locations In the upper Hudson River. Closed circles represent seclioned sediment cores: open circles represent sediment grab samples (top 10-13 cm of sediment). Direction of river flow is from northwest to southeast. The region downstream of HudsonFalls contains PCBcontaminatedareas: upstream of Hudson Falls is generally not contaminated with PCBs.
Great Sacandaga Lake (not shown). In addition, a nonPCB-contaminated sectioned sediment core (HR5) was collected Jan 8,1992, ca. 19 km upstream of Fort Edward (Figure 2). Samples were transported on ice and stored a t -20 "C prior to extraction and analysis for PCBs and CBAs. PCB and Chlorobenzoic Acid Analyses. Two serial extractions were performed on each sediment sample. Thawed sediments (2-3 g of wet weight) were placed in glassscrew-capvials, along with sodium metasilicate added as a dispersant (1mL of a 3% solution; pH 12.2). The PCBswereextractedbyadding3.00iO.OImLofahexane/ acetone (9010) extraction solvent containing 0.505 p g l mL 4-fluorobiphenyl(4-FBP)as an internal standard. The vials were tightly capped with Teflon-lined lids, placed on a reciprocating shaker for 18 h, and centrifuged a t 700g for 20 min. The organic phase containing the PCBs was removed to a clean glass vial. The organicisediment interface was rinsed three times with 1-mL aliquots of anhydrous ethyl ether. The removed extracts were combined with the rinsates, gently concentrated to 3 mL under a nitrogen stream, and analyzed for PCBs via congener-specificgas chromatographyimassspectrometry (GCIMS). The method detection limit for PCB analysis was 1 pgig of sediment. The sediments were further treated to extract the CBAs. SampleswereacidifiedwithHCl (ImL;6N) andextracted 2208
Envlron. Scl. Technol., VoI. 27, No. 10. 1993
with 2.00 + 0.01 mL of anhydrous ethyl ether containing 0.644 pgimL 4-fluorobenzoic acid (4-FBA) as an internal standard. The vials were tightly capped with new Teflonlined lids, placed on a reciprocating shaker for 18 h, and centrifuged a t 700g for 20 min. The organic phase containing CBAs was removed and derivatized with pentafluorobenzyl bromide (Pierce) (18) prior to CBA analysis via congener-specific GCIMS. The nominal method detection limit for CBA analysis was 1 ngig of sediment. 3- and4-CBAwerenotresolvable bythismethod due to coelution, although 3-CBA should not be a significant PCB degradation product based on the PCB congener distributions in these sediments. Gas Chromatography/Mass Spectrometry. A Hewlett-Packard Model 5890 Series I1 gas chromatograph equipped with a 30 m X 0.25 mm (i.d.1 column of 0.25 mm DB-1 phase (J&W Scientific, Folsom, CA) and a 1-m uncoated fused-silica precolumn (0.53 mm i.d.; J&W Scientific) was connected to a Hewlett-Packard Model 5971A quadrupole mass selective detector operated a t an electron energy of 70 eV. A splitless injection was used, with a 1-min delay before purge. The injection volume was 1pL. Helium a t a linear gas velocity of 25 cm/s was the carrier gas. The injector and transfer line were maintained a t 270°C. The ion source pressure was maintained at 2.8 X IO" i 1.0 X 10" Torr. The GC temperature profiles were, for PCB analyses: 90 "C for 2 min, 20 ' C h i n to 120°C, 4°C/min to 270 "C, hold for 7 min; for CBAs and other PCB biodegradation metabolites: 90 "C for 2 min, 20 "Cimin to 150 "C, 6'Cimin to 270 'C, hold for 5 min. Selected ion monitoring was used to quantify each individual PCB and CBA congener. PCB quantification ions included m/z 188, 222, 256, 292, 326, 360, 396, 430, and 464, with a dwell time of 300 ms. A calibration standard consisting of Aroclor 124212541260 (702010) plus the internal standard 4-FBP was injected before each analysis set and once for every five injections. CBA quantification ions included m/z 139, 181, and 336 for monochlorinated CBAs, and m/z 173, 181, and 370 for dichlorinated CBAs, with a dwell time of 300 ms for each. A calibration standard consisting of 2-; 4-; 2,6-; 2,5-; 2,4-; 2,3-; 3,4-; and 3,5-CBA, along with the internal standard 4-FBA, was injected before each analysis set and once for every five injections (Figure 3). Clean glass beads (ca. 2 g) were routinely extracted as a CBA method blank using the same procedures and solvents as for the samples. Retention times and response factors for PCB and CBA analyses were updated hased on the calibration standard injections. The internal standards 4-FBP and 4-FBA were used to normalize against variations in sample handling
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during the PCB and CBA extraction procedures, respectively. Other PCB Biodegradation Metabolites. To facilitate a search for additional PCB biodegradation metabolites, a larger (ca. 4-L) sediment grab sample (top 10-13 cm) was collected on May 22, 1992, from a PCBcontaminated site ca. 3.2 km downstream of Fort Edward. The major portion of this sample was air-dried, sieved through no. 20 mesh (850 pm), and homogenizedovernight on a roller apparatus. The remaining sediment portion (ca. 0.4 L) was stored at -2OOC prior to extraction and analysis. A 10-g subsample of the dried homogenized sediment was combined with 10 mL of 1 N HC1 and 10 mL of anhydrous ethyl ether and placed on a reciprocating shaker overnight. Following centrifugation at 700g for 20 min, the ether extract was removed and the extraction process was repeated twice. The ether extracts were combined and gently evaporated to ca. 0.5 mL under a nitrogen stream. Separation and removal of nonpolar compounds was accomplished by adding the ether extract to a cyanopropylbonded silicasolid-phase extraction tube (LCCN; Supelco) that had been prewashed with 6 mL of hexane. Following application, the ether extract was solvent exchanged by gently evaporating to dryness under a nitrogen stream. The nonpolar organic fraction was eluted with 3 mL of hexane at a slow, dropwise rate and discarded. The polar organic fraction containing the PCB biodegradation metabolites was eluted dropwise with 6 mL of acetonitrile. The acetonitrile fraction was evaporated to ca. 2 mL, derivatized (60 OC;15 min) with 0.1 mL of bis(trimethylsily1)trifluoroacetamide(BSTFA; Supelco), and analyzed via GC/MS in scanning mode (splitless
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Figure 4. PCB and chlorobenzolc acid depth profiles In Hudson River sediment core HR 1. (A) Total polychlorinated biphenyls (solid bars); mono- and dichlorlnated biphenyls (open bars). (B) Monochlorlnated benzoic acids: 2-chlorobenzoic acid (solid bars)and 3-/4-chlorobenzoic acid (open bars). (C) Dlchlorinated benzoic acids: 2,5dichlorobenzoic acid (solid bars) and 2,4dichlorobenzoic acid (open bars). Data on all three plots represent the average of two analyses wlth ranges shown.
injection; mass range 65-420 amu; 1scan/s). This sediment sample was also analyzed for PCBs and CBAs as described above. Two portions of the dried homogeneous sediment grab sample were extracted and analyzed, with identical results. Similar extraction and analysis of the nondried sediment aliquot was also performed. The identification of PCB biodegradation metabolites was based on GC/MS comparisons with reference metabolites produced by Pseudomonas s p . LB400 strain FM905, a mutant PCB-degrading bacterium which acand 2,3cumulates the 2,3-dihydro-2,3-dihydroxybiphenyl dihydroxybiphenyl intermediates (13, 19). 2,3-Dihydro2,3-dihydroxy-2'-chlorobiphenyland 2,3-dihydroxy-2'chlorobiphenyl were produced using 2-chlorobiphenyl as substrate. Culture broth containing accumulated levels of these intermediates was acid-extracted into ether and derivatized with BSTFA as described above. Retention Environ. Sci. Technol., Vol. 27, No. I O , 1993 2200
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Flgure 5. 2,3-Dlhydro-2,3dihydroxy-2’-chlorobiphenyl in PCB-contaminated upper Hudson River sediment. During acid extraction, 2,3dlhydro2,3dlhydroxy-2‘-chlorobiphenyldehydratesto form 2-hydroxy-2‘-chloroblphenyl and 3-hydroxy-2’-chloroblphenyl. (A) Mass spectrum of trlmethylsllyl (TMS)-derlvatized 2-hydroxy-2‘-chlorobiphenyl reference metabolite produced by PCBdegrading mutant Pseudomonas sp. LB400 strain FM905 using 2-chiorobiphenyl as a substrate. GC retention time was 12.7 1 min. (B) Mass spectrum of TMSderivatized 2-hydroxy-2’-chloroblphenyl in river sediment extract. GC retention time was 12.73 min. (C) Mass spectrum of PMSderivatized 3-hydroxy-2‘-chlorobiphenyl reference metabolite produced by Pseudomonas sp. LB400 strain FM905 using 2-chlorobiphenyl as a substrate. GC retention time was 14.65 min. (D) Mass spectrum of TMSderivatlzed 3-hydroxy-2’-chloroblphenyl In river sedlment extract. GC retention time was 14.68 min. Note that mass spectra and GC retentlon times for both compounds in river sediment extracts closely match those of their respective reference metabolites. Characteristic ions are indicated with an asterlsk. The GC elution order of 2-hydroxy-2’-chlorobiphenyl and 3-hydroxy-2’-chloroblphenyl was determined by analogy wlth the elution order of 2-hydroxyblphenyl and 3-hydroxybiphenyl standards.
times and mass spectra for the trimethylsilyl (TMS) derivatives of these compounds were characterized via GC/ MS in scanning mode as described.
Results and Discussion Sensitive GC/MS analysis of PCB-contaminated sediment cores revealed systematic profiles of CBAs that generally correlated with PCB depth profiles, as shown for core HR1 in Figure 4. The CBA congeners observed were consistent with the expected PCB degradation products. The PCB mixtures in these sediments consisted of 60-70% mono- and dichlorobiphenyls. 2-; 2,2’-; 2,3’-; and 2,4’-chlorobiphenyl represented 55-60% of the total PCBs. All of these can be degraded to 2-CBA (20, 211, which accounts for the large proportion of this metabolite observed in these sediments. 4-CBA can be derived from the degradation of 4- and 2,4’-chlorobiphenyl. 2,4- and 2,5-CBA are expected degradation products of 2,2’,4-; 2,3‘,4-; and 2,4’,4-~hlorobiphenyland 2,2’,5-; 2,3’,5-; and 2,4’,5-~hlorobiphenyl,respectively. Additional cores (HR2, HR3, and HR4) exhibited monochlorinated CBAs (2- and 3-/4-CBA, ranging from 20 to 400 ng/g) which were similarly correlated with PCB depth profiles (data not shown). The extremely low CBA levels in these sediments (2-4 orders of magnitude lower than PCB concentrations) were consistent with laboratory studies indicating that CBAs 2210
Environ. SCl. Techno!., VOl. 27, No. 10, 1993
are readily biodegradable under aerobic conditions (2224) and would most likely not persist subsequent to formation. Aerobic CBA-degrading microorganisms isolated from upper Hudson River sediments showed good degradative competence in the laboratory against a range of CBAs, including 2-; 3-; 4-; 2,4-; 2,s-; 2,6-; and 3,5-CBA (25). We speculate that the presence of CBAs in aerobic surficial sediment layers (estimated top 2-5 cm) may indicate ongoing PCB biodegradation, whereas CBAs in underlying anaerobic sediments represent past aerobic PCB biodegradative activity. Additional microbial processes, including anaerobic dechlorination and mineralization of CBAs (26,27),may subsequently occur in the underlying anaerobic zones. The distribution and concentration of CBAs in these sediments may also be affected by mass transport considerations (i.e., dissolution into the water phase or redistribution within the sediment phase due to diffusion or natural disturbance) which were not characterized in this study. In addition to formation via PCB biodegradation, it is conceivable that CBAs could enter the environment via the breakdown of chlorinated herbicides (24) or alkylbenzenes (28), or by anaerobic carboxylationof chlorinated phenols (29). Several independent approaches were used to address this potentially confounding issue. To establish a control, 10 sediment grab samples and one sectioned sediment core (HR5) were collected from
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with reference metabolites produced by Pseudomonas sp. LB400 strain FM905 (13, 19). Positive identification of chlorobiphenyl metabolites in the sediment extracts was based on GC retention times and mass spectra which closely matched those of the known reference metabolites. Meaningful information contained in the mass spectra included characteristic molecular and fragment ions, relative abundance of ions, and chlorine isotope patterns. The presence of extraneous ions was due to the coelution of low-level compounds inherently present in complex environmental and microbiologicalsample extracts. The results shown were obtained via the analysis of a dried homogeneous sediment sample which contained PCBs (455 pg/g of sediment), 2-CBA (348 ng/g of sediment), and 3-1 4-CBA (47 ng/g of sediment). Positive detection was confirmed through the analysis of both a second dried homogeneous sediment aliquot and a frozen wet archived portion of the same sediment grab sample. Sediment samples collected from non-PCB-contaminated regions of the upper Hudson River exhibitedno evidence of either of these PCB biodegradation metabolites. The initial investigations reported here focused on the detection of 2-chloro metabolites due to the abundance of 2-; 2,2'-; 2,3'-; and 2,4'-chlorobiphenyl in these sediments. Future research activities will focus on the development of methods to detect a broader range of metabolite congeners and to quantify the sediment concentrations of these important metabolites.
Conclusions
non-PCB-contaminated regions of the upper Hudson River (Figure 2), along with one sediment grab sample collected from nearby Great SacandagaLake (notshown). NOCBAs were detected in any of the sediment samples that were not contaminated with PCBs (nominalCBA detection limit 1 ng/g of sediment). Aerobic biodegradation of complexPCB mixtures would likely result in the formation of a variety of corresponding CBA congeners. In contrast, CBA formation via herbicide or phenol transformations would likely produce single congeners. The detection of both mono- and dichlorinated CBAs in core HR1 (Figure 4B,C) further supports a connection with PCB biodegradation. In addition, the pattern of mono- and dichlorinated CBAs in this core match almost exactly the CBA distributions produced during the recent Hudson River aerobic PCB biodegradation field experiment (17, 25). It is unlikely that herbicide degradation or phenol carboxylationmechanisms would result in this particular arrangement of CBA congeners. Further evidence for aerobic PCB biodegradation in upper Hudson River sediments was obtained from the detection of intermediate PCB biodegradation metabolites which, unlike CBAs, retain the biphenyl ring and are therefore less prone to source ambiguity. Sediment analyses revealed qualitative evidence for the presence of (Figure both 2,3-dihydro-2,3-dihydroxy-2'-chlorobiphenyl 1B and Figure 5) and 2,3-dihydroxy-2'-chlorobiphenyl (Figure 1C and Figure 6), based on GC/MS comparisons
The detection of CBAs and other metabolites of aerobic PCB biodegradation in contaminated upper Hudson River sediments provides persuasive evidence that aerobic PCB biodegradation occurs naturally in the environment. This finding is consistent with previous laboratory studies indicating that aerobic PCB-degrading bacteria with broad congener specificities are widely distributed in contaminated soils and sediments (9). Further effort will be required to conclusively determine whether the PCB biodegradation metabolites observed in this study represem evidenceof ongoing aerobic biodegradative activity, remnants of past activity, or both. In addition, further studies are needed to elucidate the natural rate of this important biodegradative process. The interpretation of documented decreases in PCB concentrations in upper Hudson River fish, water, and sediment samples (12) requires consideration of all natural PCB loss mechanisms. This study suggests that aerobic PCB biodegradation, along with the complementary anaerobic reductive dechlorination previously reported (6-8), may be playing an important role in the removal of PCBs from the upper Hudson River. In addition, metabolite detection as described in this work may be generally useful as a tool for assessing the ultimate biodegradative fate of PCBs or other xenobiotic compounds in the environment. This approach, which relies on previous detailed laboratory studies of microbial metabolism, is capable of providing conclusivequalitative evidence for naturally occurring biodegradation (16).
Acknowledgments The authors wish to thank Frank J. Mondello for culturing the Pseudomonas s p . mutant strains; Angelo A. Bracco for assistance with sediment sample collection; and Environ. Scl. Technol., Vol. 27, No. IO, I993 2211
Daniel A. Abramowicz, Donna L. Bedard, Herman L. Finkbeiner, Mark R. Harkness, Terry K. Leib, and Frank J. Mondello for helpful discussions and comments on the manuscript. Literature Cited (1) Safe, S. In Halogenated Biphenyls, Terphenyls, Naptha-
lenes, Dibenzodioxins, and Related Products; Kimbrough, R., Ed.; ElsevierJNorth Holland Publishers: Amsterdam, 1980; pp 81-107. (2) Quensen, J. F., III; Tiedje, J. M.; Boyd, S. A. Science 1988, 242, 752-754. (3) Van Dort, H. M.; Bedard, D. L. Appl. Environ. Microbiol. 1991,57, 1576-1578. (4) Furukawa, K. In Biodegradation and Detoxification of
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1094-1102. (12) Interim Characterization and Evaluation: Hudson River
Received for review February 16, 1993. Revised manuscript received June 18, 1993. Accepted June 28, 1993.'
PCB Reassessment RI/FS, Phase I Report. EPA Work Assignment No. 013-2N84; Contract No. 68-S9-2001;Vol.
Abstract published in Advance ACSAbstracts,August 15,1993.
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