Environ. Sci. Technol. 1994, 28, 2054-2064
Reductive Dechlorination of Polychlorinated Biphenyls in St. Lawrence River Sediments and Variations in Dechlorination Characteristics Roger C. Sokol, 0-Seob Kwon,? Charlotte M. Bethoney, and G-Yull Rhee'
Wadsworth Center for Laboratories and Research, New York State Department of Health and School of Public Health, State University of New York at Albany, Albany, New York 12201-0509 Sediment cores taken near three industrial plants on the St. Lawrence River (Reynolds Aluminum, General Motors, ALCOA) showed evidence of in situ dechlorination at all sites except at Reynolds 001. The extent of dechlorination varied widely from site to site, ranging from 2 to 45%, based on the average number of Cl's per biphenyl. The absence of dechlorination at Reynolds 001 was not due to the lack of competent microorganisms but appeared to be associated with a high level of cocontaminants. There was no correlation between sediment PCB concentrations and the extent of dechlorination. Even though the three sites were primarily contaminated by the same Aroclor, laboratory dechlorination assays with single congeners and Aroclor 1248 revealed significant differences in dechlorination characteristics, suggesting wide difference in dechlorinating populations among the three sites. The differences in dechlorination pattern between native sediments and laboratory sediments suggested the involvement of sediment characteristics in the selection of dechlorinating populations.
Introduction The St. Lawrence River along the northeast border of New York State has been contaminated by polychlorinated biphenyl (PCB) pollution from at least three suspected industrial sources. A major contributor has been the General Motors Central Foundry (GM),which used a PCBbased hydraulic fluid in their die-casting machines from 1959to 1974. The wastewater treatment system generated PCB-contaminated sludge, which was disposed of at several sites near the plant. Significant off-site migration from these areas has contaminated the St. Lawrence River and its tributaries. As a result of this PCB contamination, the GM site was placed on the National Priority List for Superfund Sites in 1983. Two other major industrial sources of PCBs into the U.S. side of the St. Lawrence River are Reynolds Metal Co. (Reynolds) and the Aluminum Corporation of America (ALCOA), located 1and 3 mi west of the GM site, respectively. The sources are believed to be leakage of PCB-containing hydraulic fluids at ALCOA, and heat-transfer fluids at Reynolds. Once thought to be recalcitrant, many halogenated hydrocarbons including PCBs are known to be reductively dehalogenated in anaerobic environments (see citations in refs 1 and 2). Evidence for PCB dechlorination has been found in many environments including the Hudson River (2-5), New Bedford Harbor (6, 7), Silver Lake (8), Sheboygan River (9),and Waukegan Harbor (10). In most cases, m- and p-chlorines were preferentially removed, although ortho dechlorination has been reported ( 6 1 1 ) . There is evidence to suggest that different PCBcontaminated sites contain microbial populations with + Department of Environmental Science, Inje University, Kyongnam, 621-7419, Korea. 2054
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different dechlorinating competence; under identical incubation conditions, microorganisms from the St. Lawrence River produced only 2,5-chlorobiphenyl (2,5CBP) from 2,4,5-CBP whereas Hudson River microorganisms yielded predominantly 2,4-CBP and a small proportion of 2,5-CBP (12). Recently Ye et al. (13)were able to separate two physiologically distinct groups of dechlorinating microorganisms on the basis of their sensitivity to heat and ethanol treatments. Dechlorination products were also found to vary with the supply of Hz (12, 14) or pyruvate (15),which also suggested the activity of different dechlorinating populations. The purpose of the present study was to assess the occurrence of in situ PCB dechlorination and to determine the dechlorination competence of sediment microorganisms from three sites in the St. Lawrence River. The occurrence of in situ dechlorination was evaluated by congener-specific analysis of the PCBs present in the sediments. The dechlorination competence of sediment microorganisms from the three sites was evaluated with laboratory studies using PCB-free sediments spiked with 2,3,4-CBP, 2,4,5-CBP, or Aroclor 1248, the primary PCB mixture contaminating the study area. The sediments were also analyzed for the presence of other cocontaminants that might influence the dechlorination process.
Materials and Methods Sample Collection. Sediment core samples were collected from the St. LawrenceRiver with a4.8-cm (inside diameter) plexiglass sampling tube. Cores were collected in June and October 1992 adjacent to active or former discharge points at three industrial sites (Figure 1; sampling site numbers are State Pollutant Discharge Elimination Sites designated by the New York State Department of EnvironmentalConservation). Cores were returned to the laboratory and stored at 4 OC until time of extraction and analysis. A sediment sample was removed from a silty horizon approximately 10 cm below the surface of each core. PCB Extraction. PCBs were extracted from sediments with acetone and hexane under ultrasonication as described previously (2). Elemental sulfur was removed by treating the extracts with tetrabutylammoniumhydrogen sulfate and sodium sulfite (16). Sample cleanup was performed in a chromatographic column containing 10 g of 4% deactivated Florisil (Sigma, 60-100 mesh) topped with 1 g of anhydrous sodium sulfate. The column was eluted with 65 mL of hexane, and the first 50 mL was collected. When necessary, the eluent was concentrated in a Kuderna-Danish condensing apparatus. Metals and PAH Analysis. The concentration of metals in the sediments was measured using the inductively coupled plasma (ICP) emission spectroscopy technique following nitric/hydrochloric acid digestion (17). Polycyclic aromatic hydrocarbon (PAH) were isolated from 0013-938X/94/0928-2054$04.50/0
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sediments by Soxhlet extraction with toluene and chromatographically separated from other organic chemicals using Whatman LPS-1 (13-24 pm) silica gel by eluting with 20 % methylene chloride/hexane. PAH concentrations were determined by capillary gas chromatography/ low-resolution mass spectrometry using deuterated PAHs as internal standards. Laboratory Dechlorination Studies. All experimental procedures were performed with strict anaerobic techniques. PCB-free, air-dried, sieved sediments from Owasco Lake, NY, were spiked with either Aroclor 1248, 2,3,4-CBP,or 2,4,5-CBP(AccuStandard,New Haven, CT) in hexane to yield a total PCB concentration of 300 pg/g on a sediment dry-weight basis. After the hexane was evaporated, the PCB-spiked sediments were made into slurries by adding biologically reduced synthetic minimal medium (18), containing the redox indicator resazurin (0.0001% 1, in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) with an Nz/COz/Hz atmosphere (85:5:10). To ensure a homogeneous distribution of PCBs, the sediment slurries were stirred overnight in the anaerobic chamber 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 the slurry was further reduced by adding sodium sulfide (0.025%). The vials were then sealed with a Teflon-lined rubber septum and aluminum crimp caps in the anaerobic chamber. They were autoclaved and, except for the controls, inoculated with the supernatant of sediment slurries (0.5 mL). Sediments collected from three different industrial discharge points in the St. Lawrence River, Reynolds 001, ALCOA 002, and General Motors (GM 001) (see Figure 1 and above), were used as inoculum. Inocula were prepared by mixing samples of each sediment with reduced minimal medium. The sediment slurry was mixed and allowed to settle for approximately 15 min. A 0.5-mL aliquot of the super-
natant was used to inoculate the vials. All experimental and control vials were set up in duplicate and incubated statically at room temperature. Duplicate experimental and control vials were removed from incubation periodically over 24 weeks. At each sampling, the entire contents of each vial was extracted as described above and previously (2). Gas Chromatographic Analysis. PCB analysis of extracts from both the sediment samples and the laboratory dechlorination studies was performed by use of one of two gas chromatographs (Hewlett-Packard 5890A or 5890II), each equipped with a 63Ni electron-capture detector (ECD), a H P 7673 autosampler, and a HP 3396 integrator. Aroclor 1248 samples were analyzed on a 25 m X 0.2 mm Hewlett-Packard Ultra I1 fused silica capillary column (0.11-pm film thickness) while a 30 m X 0.25 mm Apiezon-L column (Restek, Bellefonte, PA; 0.2-pm film thickness) was used to analyze 2,3,4-CBP and 2,4,5-CBP samples. GC conditions were as follows. Helium was used as the carrier gas and argon/methane (955%) as the makeup gas. The injector and detector temperatures were 250 and 300 "C, respectively. The oven program for the HP Ultra I1 column had an initial temperature of 100 "C, which was increased at a rate of 10 OC/min to 160 "C and then increased at a rate of 3 "C/min up to 250 OC, with maintenance of that temperature for 20 min. The oven program for the Apiezon-L column consisted of an initial temperature of 90 "C increased at a rate of 10 OC/min to 150 OC and increased at a rate of 3 "C/min up to 220 "C, which was maintained for 25 min. PCBs were quantitated on the H P Ultra I1 column with a calibration standard containing equal amounts of Aroclors 1221,1016,1254, and 1260 (0.2 pg/mL of each in hexane). Peaks were identified and calibrated according to response factors published by Schulz et al. (19) and Mullin et al. (20). The Apiezon column was calibrated with a mixture of weighed single-congener standards Environ. Sci. Technol., Vol. 28,
No. 12, 1994 2055
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PCB CONCENTRATION (ppm) Flgure 2. Total Cl’s per biphenyl vs total PCB concentration in each core sampled. Dashed line represents the average number of Cl’s in an Aroclor 1248 standard. A and V indlcate Reynoldscores with some Aroclor 1260. (The average number of CI per biphenyl in Aroclor 1260 is 6.1.)
(AccuStandard, New Haven, CT) containing all possible dechlorination product permutations. Following an initial calibration, the integrator was recalibrated every sixth sample with the appropriate calibration mixture. The data from the ECDs were collected and processed with a ChromPerfect chromatography data system (Justice Innovations, Palo Alto, CA) on a microcomputer. Dechlorination patterns were evaluated by calculating the mole percentage of each identified PCB containing peak and by calculating the average number of m-, p-, and total chlorines per biphenyl. In all these calculations, coeluting congeners were assumed to be present in equal proportions.
Results PCB Dechlorination in St. Lawrence River Sediments. The analysis of the sediment cores showed that total PCB concentrations varied considerably between the sampling sites, ranging from 2 to 7956 ppm (sediment dryweight basis) in the 30 cores collected (Figure 2). In general, cores collected adjacent to the GM 001 discharge had the highest concentrations and ranged from 51 to 7,956 ppm. Congener analysis revealed that the sediments were primarily contaminated with Aroclor 1248,coinciding with the records of PCB purchases by the three industrial plants. (In some cores, however, the congener pattern of Aroclor 1260 was also detected; see below.) Substantial changes in the relative distribution of PCB congeners with respect to Aroclor 1248 were found in most sediment samples. These changes are readily seen in the GM O O i samples, where there are significant losses of the higher chlorinated congeners and a significant increase in the lower chlorinated ones (Figure 3). Most significant decreases were found in the parent congeners 3,4,3’,4’- + 2,3,6-, 2,4,5,2’,4‘-, 2,5,3’,4‘-, 2,4,5,4’-, 2,3,2‘,3‘-, 2,3,6,4‘-, 2,3,6,3’- + 2,3,2’,4’+ 3,4,4’-,and 2,3,2’,5’-CBP, with an accumulationof largely 2058
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+
2-, 2,2’- + 2,6-, 2,3’-, 2,4’- 2,3-, 2,4,2’- 4,4‘-, and 2,5,4‘+ 2,4,4’-CBP. A similar pattern of dechlorination was also observed at all other sites except Reynolds 001. At Reynolds001, the congener pattern was little different from that of Aroclor 1248 (Figure 4). In addition, several cores from this site showed an abundance of higher chlorinated congeners not present in Aroclor 1248. This congener profile closely matched that of an Aroclor 1260 standard, suggesting that Aroclor 1260 might have also been released at some time (Figure 4). This Aroclor 1260 pattern also showed little alteration. The extent of dechlorination, expressed as the average number of Cl’s per biphenyl, varied widely among sites and among cores from within a site. Dechlorination resulted in the removal of between 2 and 45% of the total chlorines originally present in Aroclor 1248 (Figure 2). No correlation was found between sediment PCB concentration and the extent of chlorine removal (Figure 2). In situ dechlorination at most sites resulted in the removal of m- and p-chlorines. A plot of the average number of m- vs p-chlorines from each sediment core revealed that at most sites dechlorination occurred preferentially at the meta position relative to the para (Figure 5). However, there was no evidence of ortho dechlorination at any of the sites. To determine whether the lack of in situ dechlorination at the Reynolds 001 site was due to the absence of competent microorganisms, we carried out a dechlorination assay, which we have successfully used to determine dechlorination activities in earlier studies of Hudson River sediments (2, 12). The assays were performed by transferring microorganisms eluted from Reynolds 001 sediments into vials containing sediment spiked with 2,3,4CBP. After 6 weeks of incubation, 2,3-CBP and 2,4-CBP along with a small amount of 2-CBP were identified (Figure 6). After 12 weeks of incubation, the 2-CBP peak further increased. Autoclaved controls showed no change throughout the experiment. These data indicate that the absence of dechlorination at the Reynolds 001 site was not due to the lack of competent microorganisms. Similar dechlorination assays also showed the presence of active organisms at all the other sites examined (see below). To determine whether the absence of dechlorination at the Reynolds 001 was related in part to the presence of cocontaminants, the concentrations of metals and PAHs were determined in sediments from each samplinglocation. The Reynolds 001 site had the highest overall concentration of metals in comparison to the other locations; in particular, this site had very high concentrations of AI (93 000 pg/g) and Zn (637 pg/g) (Table 1). These levels are significantly higher than at any other site. Therefore, they could potentially be responsible for inhibiting dechlorination activities. The concentration of Cu was also high (161 pg/g) but not much different from the GM 002 site (154pg/g), which showed extensive transformation. PAH concentrations were also significantly higher at the Reynolds 001 compared with the GM 001 (Table 2) and other sites. The concentrations at other sites were similar to that at the GM site. Laboratory Evaluation of Dechlorination Characteristics. To determine the competence of dechlorinating microorganisms from the various sites, laboratory dechlorination experiments were conducted using sediment eluates as inocula in vials containing clean sediment spiked with either 2,3,4-CBP or 2,4,5-CBP. These assays
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CONGENERS Flgure 3. Mole percent of PCB congeners In a Aroclor 1248 standard, sediments from the General Motors 001 site, and the ALCOA 002 site.
demonstrated that all sites harbored dechlorinating microorganisms, but with significant differences in their dechlorination competence. Sediment microorganisms from the GM and ALCOA sites dechlorinated 2,3,4-CBP to 2,4-CBP. No production of 2,3- or 2-CBP was evident even after 8 months of incubation. By contrast, those from the Reynolds site produced 2,3- and 2,4-CBP with subsequent production of 2-CBP. Similarly, there was also a difference in the dechlorination of 2,4,5,-CBP; organisms from the GM site dechlorinated 2,4,5-CBP to 2,4-, 2,5-, and 2-CBP whereas those from the Reynolds site produced only 2,5-CBP (Figure 7). However, none of the sediment organisms were capable of ortho dechlorination. To further elucidate these site-specific dechlorination characteristics, the dechlorination of Aroclor 1248 was investigated. Both the rate and extent of Aroclor dechlorination were greatest with inoculum from the Reynolds site. The time course of dechlorination, expressed in terms of the average number of Cl’s per biphenyl, showed that 10% of the total chlorines were removed after 6 weeks and 29% after 24 weeks with organisms from the Reynolds sediments (Figure 8). Organisms from GM and ALCOA sediments were slower, removing only 7 and 3% after 6
weeks and 20 and 18% after 24 weeks, respectively (Figure 8). Dechlorination of Aroclor 1248 by sediment microorganisms from all three sites occurred through the removal of m- and p-Cl’s. No ortho dechlorination was observed with any of the inocula as was the case with the single congeners. Organisms from Reynolds sediments reduced the average number of rn-Cl’s by 59% (from 1.28 to 0.53) and that of p-Cl’s by 32 % (from 1.05 to 0.71) on an average between 18 and 24 weeks. A similar pattern of chlorine removal was seen with microorganisms from ALCOA sediments, where m- and p-Cl’s were reduced by 35 (from 1.28 to 0.83) and 20% (1.05 to 0.841, respectively, during the same period. Thus the organisms from these two sites showed greater removal of m-Cl’s. By contrast, microorganisms from the GM site exhibited a preference for para; the average number of m-Cl’s only decreased by 23 % (1.28 to 0.99) compared with p-Cl’s, which decreased by 37% (1.05 to 0.66). The differences in dechlorination competence can be seen clearly in a plot of the average number of m-Cl’s per biphenyl vs p-Cl’s over the time course of incubation (Figure 9). The plot illustrates the dechlorination process of each inoculum, beginning at the upper right and Environ. Scl. Technol., Vol. 28, No. 12, 1994 2057
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CONGENERS Figure 4. Mole percent of PCB congeners in a Arocior 1248 standard, Reynolds 001 sediments, Reynolds 002/003 sedlments, Aroclor 1260, and a second core from Reynolds 001.
progressing toward the lower left. The dotted line represents a proportional removal of m - andp-Cl’s. It is 2068
Environ. Sci. Technol., Vol. 28, No. 12, 1994
clear from the figure that sediment organisms from both the Reynolds and ALCOA sites preferentially removed
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chlorines from the meta position. By contrast, dechlorination by the GM inocula occurred preferentially at the para position. A part of these observed differences could have been contributed by our assumption that coeluting congeners are present in equal proportion. However, a detailed examination of individual congeners showed that microorganisms from the GM site primarily reduced the parent congeners 2,4,5,4’-, 2,5,3’,4‘-, and 2,4,3’,4’-CBP, yielding congeners rich in m-Cl’s such as 2,3’-, 2,5,3’-, and 2,4,3’-CBP, and that parent congeners such as 2,3,2’,5’and 2,5,2’,5’-CBP,showed little change (Figure 10). This pattern of dechlorination was reflected as the preferential removal from the para position. On the other hand, the predominant dechlorination product by organisms from the ALCOA site was 2,5,4’- + 2,4,4’-CBP,which is rich inp-Cl’s (Figure 11). Additional analysis of this peak (using the Apiezon-Lcolumn) showed that each of these coeluting congeners was present in almost equal proportion. The organisms also dechlorinated 2,3,2’,5’-CBP, which GM organisms were unable to dechlorinate. Another difference was an accumulation of 2,4,3’-, 2,5,3‘-, 2,4’- 2,3-, and 2,2’- + 2,6-CBP. Sediment microorganisms from the Reynolds site showed the most extensive pattern change. In addition to the parent congener 2,3,2’,5’-CBP, they dechlorinated 2,5,2‘,5‘CBP, which organisms from both ALCOA and GM sites were unable to transform. In contrast to microorganisms from GM and ALCOA sediments, 2,5,2’-CBP did not accumulate but was further dechlorinated. The congener profile also showed a high accumulation of the low molecular weight congeners such as 2-, 2,2’- + 2,6-, 2,4 2,5-, 2,3‘-, 2,4‘- + 2,3,2,6,2‘-,4,4‘- 2,4,2’-, 2,3,2‘- + 2,6,4‘-, and 2,5,4’- + 2,4,4’-CBP (Figure 12). These results indicate that the appearance of meta or para dechlorination was due to the inability to dechlorinate specific congeners and/ or the accumulation of products which are rich in either p- or rn-Cl’s.
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Flgure 6. Gas chromatograms (Apiezon-L column) from the 2,3,4CBP dechlorination assay using microorganisms eluted from Reynolds 001 sediment: (Top panel) Autoclaved control showing 2,3,4-CBP. (Center panel) Sample after 6 weeks of incubation showing parent congener (2,3,4-CBP) and three products: 2,4-CBP, 2,3-CBP, and 2-CBP. (Bottom panel) Sample after 12 weeks of incubatlon showlng stronger peak for 2-CBP.
Discussion Little evidence of in situ dechlorination was found at the Reynolds 001 site. Interestingly, however, the microorganisms from this same site exhibited the most rapid and extensive dechlorination in the laboratory dechlorination studies. These results indicate that the absence of dechlorination at the Reynolds 001 site was not due to the lack of competent microorganisms. I n situ dechlorination may have been limited by the high levels of metals and/or PAHs found at this site. The sediments at the Reynolds 001site also had a “tarlike”consistency, probably resulting from the release of coal tar pitch, used in the production of carbon electrodes, from the plant. This “tar” (of which PAHs are a component) may sequester the PCBs, limiting their bioavailability to the dechlorinating organisms. I t has been suggested that dehalogenation of brominated biphenyls was inhibited by cocontaminants such as oil and grease, which limited their bioavailability (21).These cocontaminants were obviously not lethal to competent sediment microorganisms at their ambient levels, since assays demonstrated active dechlorination. Thus, their effects were limited only to the inhibition of in situ dechlorination in native sediments. Environ. Sci. Technol., Vol. 28, No. 12, 1994
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Table 1. Concentrations of Selected Metals in the Sediments Surveyed
location Reynolds 004 Reynolds 002i003 Reynolds 001 GM 001 GM 002 GM Cove ALCOA 002 Grasse River
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Cd
Cr
18300 6760 93000 4800 8400 12400 8640 7500
