Environ. Sci. Technol. 1993, 27, 714-719
Long-Term Study of the Anaerobic Dechlorination of Aroclor 1254 with and without Biphenyl Enrichment G-Yull Rhee,’tt*$ Roger C. Sokol,t** Brian Bush,+*$and Charlotte M. Bethoney?
Wadsworth Center for Laboratories and Research, and School of Public Health, State University of New York at Albany, New York State Department of Health, P.O. Box 509, Albany, New York 12201-0509 The reductive dechlorination of Aroclor 1254 by microorganisms eluted from Hudson River sediments was investigated. The rate, extent, and pathway of dechlorination were compared in biphenyl-enriched and nonenriched sediments over a 24-month period. The greatest dechlorination activity took place within the first 5 months of incubation, where -30% of the total chlorines was removed in both treatments. Dechlorination activity slowed substantially so that by the end of 24 months only an additional 8 and 2 % of the total chlorines were removed in the nonenriched and biphenyl-enriched sediments, respectively. Aroclor 1254 was dechlorinated primarily with the loss of chlorines meta to the biphenyl link. It ranged from 64 to 66% in the biphenyl-enriched and nonenriched sediments, respectively. The more extensive overall dechlorination in the nonenriched sediments was characterized by the loss of 40% of the para chlorines compared with only a 12 % loss in the biphenyl-enriched sediments. However,dechlorination was congener-specific as evidenced by the recalcitrance of two peaks 2,4,2’,4’- + 2,4,5,2’-chlorobiphenyl (CB) and 2,4,2’,5’-CB. The difference in paradechlorination was also in fact due primarily to the inability to dechlorinate 2,4,4’-CB with biphenyl enrichment. These results suggest that environmental factors may alter the congener specificityof dechlorination. In both treatments, several parent congeners persisted at low levels after 24 months, suggesting the existence of a threshold concentration below which no dechlorination took place.
Introduction Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants. Improper disposal practices resulted in the discharge of large quantities of PCBs into soils, river and lake sediments, and landfills. Concerns over their toxicity and bioaccumulation potential have emphasized the need to remediate these contaminated sites. Laboratory studies have demonstrated that PCBs can be reductively dechlorinated by sediment microorganisms in the anaerobic environment (1-7). This biotransformation process converts highly chlorinated PCB congeners into congeners containing fewer chlorines. Dechlorination proceeds primarily through the selective removal of meta and para chlorines although ortho dechlorination has recently been reported (8). However, recent work suggests that the chlorine removed is determined by the pattern of chlorine substitution on the particular phenyl ring rather than the substitution position with respect to the biphenyl link per se (4, 7). Reductive dechlorination is an important step toward the bioremediation of PCBs since it reduces the chlorine + Wadsworth Center for Laboratories and Research. 4
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content of PCB mixtures thereby reducing their bioaccumulation potential and, in some respects, toxicity (9). However, very little is known about the microorganisms or the metabolic pathway(s) involved in the reductive dehalogenation of PCBs. A more complete knowledge of biotransformation pathways is essential to better understand the fate of these compounds in the environment. Dechlorination of PCBs in anaerobic environments is generally considered to be slow, with major change occurring over a period of months or years. Although there is ample evidence to show that PCBs can be reductively dechlorinated, few studies have conducted long-term incubation experiments. Except for the study of Rhee et al. (6),where some samples were incubated for as long as 12 months, the majority of the studies incubated samples for much shorter durations (1-3). Because of the environmental significance of anaerobic dehalogenation, it is important to try and enhance dechlorination rates. Biphenyl, the structural analog of PCBs, seemed to enhance disappearance of PCBs in Hudson River sediments under anaerobic conditions (IO). Similarly, the biodegradation of PCBs by aerobic bacteria was also enhanced with biphenyl enrichment (11). The purpose of the following study was to compare the longterm rate and pathway of dechlorination of Aroclor 1254 by Hudson River sediment organisms with and without biphenyl enrichment.
Materials and Methods PCB-free, air-dried, sieved sediments from Owasco Lake, NY, were spiked with Aroclor 1254 in hexane to yield a total PCB concentration of 400 wg/g on a sediment dryweight basis. I4C-Labeled Aroclor 1254 (Amersham, St. Louis, MO) was added as a tracer. After the hexane was evaporated, the PCB-spiked sediments were made into slurries by adding biologically reduced synthetic minimal medium (12)in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) with an NdCOdHz atmosphere (85:5:10). The minimal medium contained the redox indicator resazurin at a final concentration of 0.0001% . To ensure the homogeneous distribution of PCBs, the sediment slurry was stirred overnight with a magnetic stirrer. Batch incubations were prepared by dispensing 20 mL of the sediment slurry to 50-mL serum vials (Wheaton Scientific, Millville, NJ) and sealingthe vials witha Teflonlined rubber septum and aluminum crimp seal in the anaerobic chamber. The vials were autoclaved, and cystine sulfide (0.025% final concentration) was added to further reduce the sediments. Three days after the addition of the cystine sulfide, all vials, except the controls, were inoculated with a 0.5-mL aliquot of the supernatant of a slurry prepared from Hudson River sediments collected at Fort Miller, NY. Treatments consisted of sediments under nitrogen (nonenriched) and sediments plus biphenyl under nitrogen (biphenyl-enriched). Biphenyl (1000 pg/ 0013-936X/93/0927-0714$04.00/0
0 1993 American Chemical Society
g) was added to the sediments at the time of PCB contamination. Vials requiring an N2 atmosphere were flushed with filter-sterilized N2 gas. All vials were incubated at room temperature (23 “C) without shaking since previous work showed no difference with and without shaking (unpublished results). Each treatment, including the control, was set up in triplicate for each sampling period. Vials were removed for sampling following 5 months of incubation and approximately every 3 months thereafter for 24 months. Incubation was terminated by placing the vials in a cold room (4 “C) until their extraction. I4C radioactivity was counted in a liquid scintillation counter (LKB Rackbeta). The measurement of methane in the headspace was determined with a gas chromatograph equipped with a thermal conductivity detector and Porapak Q column. The sediments were extracted and analyzed as described previously (4). Data analysis was conducted on 39 major congener-containing chromatographic peaks.
Results Dechlorination of Aroclor 1254 by Hudson River microorganisms was clearly evident after 5 months of incubation, in both biphenyl-enriched and nonenriched sediments. As dechlorination proceeded, the concentration of the higher chlorinated congeners decreased, with a concomitant appearance of the lower chlorinated congeners (Figure 1). Despite the dramatic shift in congener composition, no significant decrease in the total molar concentration of chlorobiphenyls (CBs) occurred in the experimental treatments over the 24-month incubation period. 14C counts showed that all the radioactivity was in the hexane fraction, suggesting that all the transformation products were hydrophobic. Dechlorination of Aroclor 1254in both nonenriched and biphenyl-enriched sediments was accompanied by methane production. Control sediments showed no loss of PCBs or change in the congener pattern. Both the rate and extent of dechlorination of Aroclor 1254 varied between the biphenyl-enriched and nonenriched sediments. The nonenriched sediments dechlorinated faster and more extensively than the biphenylenriched sediments (Figure 2). The greatest dechlorination activity took place by the first sampling at 5 months, were -30% of the total chlorines was removed in both treatments. Dechlorination activity slowed substantially so that by the end of 24 months only an additional 8 and 2% of the total chlorines were removed in nonenriched and biphenyl-enriched sediments, respectively. The time course of dechlorination over 24 months could be described by first-order kinetics, yielding rate constants of -0.016 and -0.011 Cllmonth (P< 0.05) for the nonenriched and biphenyl-enriched treatments, respectively. Analysis of the PCB homolog distribution over time showed that the extent and pattern of dechlorination was different in the two treatments (Figure 3). After 5 months of incubation, both treatments showed a similar dramatic decrease in hexa- and pentachlorinated homologs. However, there was a pronounced difference between the two treatments in the accumulation of the tri- and dichlorinated homologs. The clearest difference was observed in the accumulation of dichlorinated homologs; in the nonenriched sediments there was a steady increase in the dichlorinated homologs, while in the biphenyl-enriched
sediments relatively little accumulated after the initial 5-month sampling period. The final extent of dechlorination in the nonenriched sediments was greater than in the biphenyl-enriched sediments, as illustrated by the higher final levels of mono- and dichlorinated biphenyls. A detailed examination of the chromatograms showed that, after 5 months of incubation, 11 dechlorination products were found to accumulate in the nonenriched sediments while only 9 were found in the biphenyl-enriched sediments (Figure 1). Although the amounts of the congeners were different, all nine dechlorination products of the biphenyl-enriched sediments were found as products in the nonenriched sediments. Dechlorination proceeded further in the nonenriched sediments, accumulating a greater proportion of lower molecular weight congeners compared with the biphenyl-enriched sediments. By the end of 24 months, the nonenriched sediments had accumulated four new dechlorination products (2,3,6-+ 2,6,2’CB, 2,5,2’-CB, 2,2’- + 2,6-CB, 2-CB) while the biphenylenriched sediments had accumulated only one new product (2,3,6- 2,6,3’-CB; Figure 1). In addition to the smaller number of dechlorination products, the biphenyl-enriched treatment accumulated a much greater proportion of parasubstituted congeners, in particular 2,5,4’- + 2,4,4’-CB (Figure 1). Aroclor 1254 was dechlorinated primarily with the loss of meta chlorines in both treatments (Figure 4). By the end of 24 months, 64 and 66 % of the chlorines in the meta position had been removed in the nonenriched and biphenyl-enriched sediments, respectively. The more extensive overall dechlorination in the nonenriched sediments (38 vs 32%) was therefore due to the greater removal of para-substituted chlorines. In the nonenriched sediments, 40% of the para chlorines was removed while in the biphenyl-enriched sediments only 1 2 % was removed after 24 months of incubation. Little or no dechlorination occurred from the ortho positions in either treatment. The large difference in para-dechlorinating capability between the two treatments was primarily due to the inability to further dechlorinate the product 2,4,4’-CB in biphenyl-enriched sediments (2,4,4’-CB was separated from 2,5,4’-CB with an Apiezon L column). In the biphenyl-enriched sediments, 2,4,4’-CB accumulated to a much greater extent than in nonenriched sediments and did not undergo further dechlorination (Figure 5B). The absence of accumulation of its potential products indicated that this might be an end product. By contrast, in nonenriched sediments 2,4,4’-CB, once produced, underwent further dechlorination to produce 2,4’-CB through the removal of a para chlorine (Figure 5D). These data suggest that biphenyl enrichment altered the congener specificity of dechlorination. The inability to further dechlorinate 2,4,4’-CB was contrasted by 12 % overall para dechlorination in this treatment. These results indicate that dechlorination is specific to the chlorination pattern of the congeners. Interestingly, 2,5,4’CB, once produced, did not undergo further dechlorination in either treatment (Figure 5C). When the dechlorination pathway was traced back to account for the production of 2,4,4‘- and 2,5,4’-CBs, two potential parent congeners were identified (2,4,3’,4’- and 2,5,3’,4’-CBs) on the basis of their C1 substitution pattern and concentration in the original Aroclor mixture. In addition, the dechlorination sequence of Aroclor 1242also pointed to these congeners as the parent compounds (4). An examination of dechlorination products indicated that
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A
Figure 1. Mole perwnt 01 PCB congeners of Aroclw 1254 in (A) control and in nonenriched and biphenyknriched sediments following (8)5 and (C) 24 months of incubation wim sediment microorganisms from the Hudson River.
2,5,3’-CB, 2,4,3’-CB, 2,3’,4’-CB, and their subsequent dechlorination products 2,4’-CB and 2,3’-CB could be potential products of these parent congeners in the nonenriched sediments. These five products have a common characteristic in that they are largely potential products of para dechlorination of the two parent congeners. In contrast to the nonenriched sediments, only three potential transformation products (2,3’,4’-CB,2,4,3’-CB, 2,4’-CB) were identified in the biphenyl-enriched sediments (Figure 1B). Furthermore, the concentrations of these congenerswas insufficient to account for the observed 710 Envimn. Scl. Temnal.. Vol. 27. NO. 4, 1993
reduction of parent congeners, 2,4,3’,4’-CB and 2,5,3’,4’CB. Therefore, the most probable transformation route was through meta dechlorination at the 3’ position, resulting in a large accumulation of 2,5,4’-CB and 2,4,4‘CB in the biphenyl-enriched sediments. Thesedataagree withthe observationon 2,4,4’-CB thatbiphenylenrichment inhibits para dechlorination by Hudson River microorganisms. In both the nonenriched and biphenyl-enriched sediments, two chromatographic peaks containing tetrachlorinated dechlorination produds (2,4,2’,4’- 2,4,5,2’-CB, 2,4,2’,5’-CB), once produced, did not undergo further
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However, several of these congeners persisted at low levels even after 24 months of incubation. After an initial decrease during the first 5 months of incubation, the concentrations of 2,3,4,2’,4‘,5’- 2,3,4,6,3’,4’-CB, 2,4,5,2’,5‘CB, and 2,4,3’,4’-CB remained constant throughout the remaining 24-month incubation period (Figure 7). These results suggest that these residual concentrations might represent threshold levels, below which dechlorination could no longer take place. The residual concentration of each congener was similar, regardless of treatment.
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Figure 3. Change in homolog dlstributlon In nonenriched (V)and biphenyl-enriched (0)sediments over a 24-month Incubation period. Error bars are the standard devlatlon of triplicate samples; where not shown, the error bars are smaller than the symbols.
dechlorination, suggesting that these were terminal products (Figure 6). Although 2,4,2’,4’-CB seemed to decrease after 20 months of incubation, no significant difference in its concentration was found across the 24-month incubation period (ANOVA p > 0.05). This congener represented 21 and 24% (on a molar basis) of all of the PCBs recovered after 24 months of incubation in the nonenriched and biphenyl-enriched sediments, respectively. Similarly, once formed, the concentration of 2,4,2’,5’-CB remained constant across the 24-month incubation period. The almost complete loss of the parent hexa- and pentachlorinated congeners after the first 5 months of incubation discounts the possibility of a dynamic equilibrium between their production and further transformation. As dechlorination proceeded, the levels of most of the highly chlorinated congeners decreased below detection.
Discussion Sediment microorganisms from the Hudson River were able to reductively dechlorinate Aroclor 1254to a mixture composed largely of tetra-, tri-, and dichlorinated congeners. In contrast to earlier observations on the in-place PCBs from Hudson River sediments ( I I ) , biphenyl enrichment did not decrease the concentration of Aroclor 1254. However, biphenyl enrichment did decrease both the rate and extent of dechlorination. The effect of longterm incubation on the population dynamics or viability of sediment microorganisms in a closed system is unclear. However, dechlorination activity was still evident at 24 months in the present study. The closed nature of the experimental system may also not be much different from deep undisturbed anaerobic layers in sediments. Environ. Sci. Tschnoi., Vol. 27, No. 4, 1883
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Figure 5. Accumulation of dechlorination products 2,4,4'-CB, 2,5,4'CB, and 2,4'-CBin nonenriched (V)and biphenyl-enriched(0)sediment.s. Coeluting congeners (A) 2,5,4'-CB and 2,4,4'-CB were quantitatively separated (B C) on an Apiezon column. Additionally, analysis on this column revealed that 2,3-CB, which coelutes with 2,4'-CB (D) on a DB-5 column, was not present.
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In these experiments, the dechlorination of Aroclor 1254 by Hudson River microorganismsproduced a pattern quite different from that reported by Quensen et al. (3). After 24 months of incubation in this study the major dechlorination peaks were 2,4'- + 2,3-CB, 2,5,4'- + 2,4,4'-CB, and 2,4,2',4'- + 2,4,5,2'-CB, whereas in the Quensen et al. ( 3 ) study, after 4 months of incubation they were 2-CB and 2,2'- + 2,6-CB. However, the experimental protocols of 718
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the two studies were quite different. Although both studies used inocula from the Hudson River, the sediments were collected from different locations, Fort Miller vs Hudson Falls, NY. Probably the most significant difference is the way PCBs and microbial inocula were introduced. In the present study, hexane-solubilized PCBs were spiked to dry PCB-free sediments. After the hexane was evaporated, the sediments were made into slurries by adding reduced minimal medium. After heat sterilization, a microbial inoculum was added. By contrast, Quensen et al. (3)first added microbial inoculum into PCB-free sediment slurries. When the slurries were biologically reduced, PCBs were then introduced using acetone as a carrier solvent. Although the acetone concentration was very low, PCB availability between the two studies may have been different. The availability of PCBs may have a direct effect on dechlorination rates (13). The two studies also differ in the gas atmospheres and media compositionsused. Whatever the reason, dechlorination appears to be highly sensitive to the sediment environments although it is unclear from the present study whether the physical and chemical characteristics, associated microbial populations, or both are responsible for the differences. The dechlorination pattern obtained with Hudson River inoculum in these experiments is similar to environmental dechlorination pattern H described for sediments from the Acushnet Estuary (14). The PCBs deposited into the
estuary originally consisted of Aroclors 1242 and 1254. Although we show a much more extensive dechlorination in our experiments, both patterns have many of the same di- and trichlorinated congeners accumulating, such as 2,4- 2,3’-CB, 2,5,2’-CB, 2,4,2’-CB, 2,6,4’- + 2,3,2’-CB, and 2,4,4’-CB. It is also interesting to note that the difference in 2,4,4’-CB accumulation between biphenylenriched and nonenriched treatments is similar to the difference in 2,4,4’-CB accumulation between sediment patterns H and H’ (peak 24 in ref 14). The addition of biphenyl inhibited further dechlorination of 2,4,4’-CB, thus reducing overall para dechlorination. It is possible that the availability of this additional substrate could have altered the composition of the dechlorinating population, thereby changing the dechlorination specificity. Recently, Ye et al. (15) were able to obtain enrichment cultures with different preferential dechlorination after pasteurizing or treating the Hudson River inocula with ethanol. The effects of biphenyl on the rate, extent, and pathway of Aroclor 1254dechlorination also suggested the variable nature of dechlorination as affected by sediment conditions. It was most interesting to note that 2,4,4‘-CBmight be a terminal product under biphenyl enrichment whereas under nonenriched conditions it was further dechlorinated to di- and probably to monochlorinated congeners. However, 2,5,4’-CB appeared to be a final product under both conditions in the present experiments. The accumulation of this congener under nonenriched conditions is also quite interesting, since the para chlorine was removed from 2,4,4/-CB and the meta chlorine was more readily dechlorinated in general than the para chlorine under the same condition. These results reconfirm that dechlorination is determined by chlorination pattern of C1 rather than the position of C1 per se (4, 7). The recalcitrance of the tetrachlorinated congener peaks (2,4,2’,4/-+ 2,4,5,2’-CB,2,4,2’,5’-CB) may also be explained by the substitution-pattern-specific nature of dechlorination. Both of these congeners were also found to accumulate following the dechlorination of Aroclor 1254 in the study by Quensen et al. (3). In addition, these same congeners were also found to be major components in the sediments from the Acushnet Estuary ( 1 4 ) . The results of the present experiment provide additional support to show that dechlorination is congener-specific. The further transformation of dechlorination products appeared to be more difficult as reflected by decreasing dechlorination rates with time. These findings suggest that it is hazardous at best to extrapolate results from one congener to another. Furthermore, when the sensitivity of dechlorination to sediment conditions is considered, in situ dechlorination in contaminated sediments can be understood only through direct investigation of in-place congeners in their own sediments. Constant residual concentrations of certain congeners for nearly 20 months after their initial decrease strongly suggest that these levels may be lower thresholds, below which no further dechlorination can take place. A constant residual concentration was also found in a study of the single congener 2,4,5-CB using different approaches (7). These levels may be specific to congeners and more importantly may vary with sediment compositions. The threshold concentration may be a potential limit to biotransformation.
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Conclusions Microorganisms eluted from Hudson River sediments anaerobically removed -38% of the C1 from Aroclor 1254 during 24 months. With biphenyl enrichments, they removed only 32 % and dechlorination products were also qualitatively different. Biphenyl seemed to inhibit para dechlorination, but it was primarily due to the accumulation of 2,4,4’-CB without further transformation. Dechlorination was congener-specific or specific to the C1 substitution pattern rather than to the C1 substitution position. This specificity explains the accumulation of certain dechlorination products such as 2,4,2‘,4‘- 2,4,5,2’CB and 2,4,2’,5’-CB. Even after 24 months of incubation, several parent congeners persisted at constant low concentrations, suggesting the existence of a threshold concentration below which no dechlorination may take place. The existence of a threshold indicates that, even with a successfulbioremediation of PCBs by dechlorination, lowlevel contamination of some highly chlorinated congeners may still persist.
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Acknowledgments This study was supported by the Hudson River Foundation (Grants 00988A024and 02186B0151,the New York Center for Hazardous Waste Management (Grant 150W009),and US.EPA (GrantR816903010). We thank Alan DeNucci, Rebecca Slezak, and Bryan Michon for their excellent technical support.
Literature Cited Nies, L.; Vogel, T. M. Appl. Environ. Microbiol. 1990,56, 2612-2617. Quensen, J. F., 111; Tiedje, J. M.; Boyd, S. A. Science 1988, 242. 752-754. Quensen,J. F., 111;Boyd, S. A.; Tiedje, J. M. Appl. Environ. Microbiol. 1990, 56, 2360-2369. Rhee, G.-Y.; Bush, B.; Bethoney, C . M.; DeNucci, A,; Oh, H.-M.; Sokol, R. C . Environ. Toxicol. Chem., in press. Rhee, G.-Y.; Bush, B.; Bethoney, C. M.; DeNucci, A.; Oh, H.-M.; Sokol, R. C . Environ. Toxicol. Chem., in press. Rhee,G.-Y.;Bush,B.;Bethoney,C.M.;Sokol,R.C. Environ. Toxicol. Chem., in press. Rhee,G.-Y.;Sokol,R.C.;Bethoney,C. M.;Bush,B.Enuiron. Sci. Technol., in press. Van Dort, H. M.; Bedard, D. L. Appl. Environ. Microbiol. 1991,57, 1576-1578. Smith, L. M.;Schwartz,T. R.; Feltz, K. Chemosphere 1990, 21,1063-1085. Rhee, G.-Y.;Bush, B.; Brown, M. P.; Kane, M.; Shane, L. Water Res. 1989,23, 957-964. Brunner, W.; Fain, H.; Sutherland, H.; Focht, D. D. J. Emiron. Qual. 1985, 14, 324-328. Balch, W. E.; Fox, G. E.; Magrum, L. J.; Woese, C . R.; Wolf, R. S. Microb. Rev. 1979, 43, 260-296. Mohn, W. W.; Tiedje, J. M. Microbiol. Rev. 1992,56,482507. Brown, J. F., Jr.; Wagner, R. E. Environ. Toxicol. Chem. 1990,9, 1215-1233. Ye, D.; Quensen, J. F., 111.; Tiedje, J. M.; Boyd, S . A. Appl. Environ. Microbiol. 1992, 58, 1110-1114.
Received for review August 6, 1992. Revised manuscript received December 10, 1992. Accepted December 14, 1992 .
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