Environ. Sci. Technol. 1994, 28, 830-635
Microbial Reductive Dechlorination of Trichlorobiphenyls in Anaerobic Sediment Slurries William A. Williams'
Environmental Laboratory, General Electric Corporate Research and Development, P.O. Box 8, Schenectady, New York 12301
* Phone: (518) 387-7835; fax: (518) 387-7611; e-mail address:
[email protected].
The studies of Aroclor dechlorination by microorganisms in sediment slurries have clearly established that PCB dechlorination in the environment can be reproduced in the laboratory (6-11). However, most of these studies have not provided information about the sequence of chlorine removal or how the position of chlorines on the phenyl rings influences the dechlorination sequence. One difficulty has been the complexity of identifying the dechlorination products of each congener in a sediment sample containing an Aroclor, since many of the products are also substrates. Examination of individual congener dechlorination could be useful in characterizing PCBdechlorinating microorganisms. There are examples of specific dehalogenation sequences of other halogenated aromatic compounds that have implied distinct microbial populations. Boyd and Shelton (14)observed at least two distinct chlorophenol-degrading populations in sludges acclimated to either 2- or 3-chlorophenol. Fathepure and co-workers (15) reported that hexachlorobenzene was dechlorinated to tri- and dichlorobenzenes in anaerobic sewage sludge via two routes distinguished by the sequential removal of chlorines from the aromatic ring. They speculated that these separate, sequential transformations could be the result of two distinct microbial populations or a product distribution based on the chemical reactivity of the chlorines on the ring (15). Two papers have described the fate of single PCB congeners added to sediment slurries and have furthered the understanding of PCB reductive dechlorination, since the congeners and their dechlorination products were identified (9,13).Abramowicz et al. (9)identified a specific sequential dechlorination pathway for 2,3,3',4,4'-pentachlorobiphenyl(2,3,4-3,4-CB) in anaerobic sediment slurries. Van Dort and Bedard (13) identified two dechlorination pathways (a meta and a sequential ortho and meta chlorine removal) by the acclimation times and the sequences of chlorine removal from added 2,3,5,6-CBand its dechlorination products. In the present study, microbial dechlorination of six trichlorobiphenyls in PCBcontaminated sediment slurries is characterized by the acclimation times and the dechlorination sequences. The six trichlorobiphenyls selected represent all possible configurations for three chlorines on one phenyl ring. From their studies of environmental PCB dechlorination, Brown and co-workers ( 4 )noted that the removal of chlorines on one ring is influenced not only by the chlorination pattern on the target ring but also by the number and position of chlorines on the second ring. Aroclors 1254 and 1260 consist mainly of penta-, hexa-, and heptachlorobiphenyls that have one or two trichlorophenyl rings. Laboratory studies of Aroclor dechlorination using microorganisms from Hudson River, Silver Lake, or Wood Pond sediment showed selective losses of meta, para, or both meta and para chlorines from some but not all congeners (6-11,16). In the current study, I selected PCB congeners with all the chlorines on a single ring to assess how the chlorine configuration on the target ring influences microbial
630 Environ. Sci. Technol., Vol. 28, No. 4, 1994
0013-936X/94/0928-0630$04.50/0
To understand how the number and arrangement of chlorines on a phenyl ring affect polychlorinated biphenyl (PCB) dechlorination, anaerobic microbial reductive dechlorination of six trichlorobiphenyls is characterized by the acclimation time before dechlorination and the sequence of chlorine removal. Each of the six trichlorobiphenyls with all chlorines on one phenyl ring was incubated at 22-25 "C in slurries of PCB-contaminated sediment from the Hudson River (NY), Silver Lake (MA), and Woods Pond (MA). In each slurry, the chlorine between the other two chlorines on the phenyl ring was usually removed from the trichlorobiphenyl first. Approximately the same acclimation time (3 weeks) and dechlorination sequences were observed in every slurry for trichlorobiphenyls with adjacent meta and para chlorines. All meta and para but no ortho chlorines were removed in Hudson River sediment slurries. Generally only one meta or para chlorine was removed in Silver Lake and Woods Pond sediment slurries, leaving a terminal, dichlorobiphenyl dechlorination product. However, orthodechlorination of 2,4,6-trichlorobiphenyl occurred in the Woods Pond and Silver Lake samples. The results indicate that the sediments share some microbial PCB dechlorination characteristics, but the differences probably indicate several, distinct, microbial populations. Introduction Polychlorinated biphenyls (PCBs) are stable, waterinsoluble, industrial chemicals that were banned from production in the United States in 1977. They consist of a biphenyl nucleus carrying 1-10 chlorine atoms and are separately known as congeners. PCBs were produced and sold as congener mixtures given the trade name Aroclors. Some of these mixtures were released into the environment, and because of their potential health effects, remediation of the contaminated sites is an important issue. Microbial biodegradation of PCBs is a potential means of remediating a contaminated site without substantial changes to the environment. Aerobic microbial biodegradation of PCBs is well established (for a review, see ref 1). Brown and co-workers (2-5) proposed that the changes in congener distribution of PCB-contaminated aquatic sediments resulted from reductive dechlorination of PCBs mediated by microorganisms. In addition, they proposed that the distinctive patterns of PCB transformation seen in the sediments resulted from several different microbial dechlorination systems that are distinguished by congener selectivity. Dechlorination of Aroclors (6-11) and of single PCB congeners (9, 12, 13) in sediment slurries has also been demonstrated in laboratory experiments. All these reports suggest that there are different PCB-dechlorinating microorganisms with characteristic specificities for PCB dechlorination.
0 1994 Amerlcan Chemical Society
dechlorination, while eliminating influences by chlorines on the other ring. The similarities and differences in trichlorobiphenyl dechlorination are characterized among slurries of PCBcontaminated sediment from the ,upper Hudson River, Silver Lake, and Woods Pond. The sediment from the upper Hudson River near Fort Edward, NY, is composed of sand, silt, a small amount of brown humic matter, and extensively dechlorinated Aroclor 1242 (meta and para chlorines were removed, leaving predominately orthosubstituted mono- and dichlorobiphenyls; refs 2, 4, and 5). Silver Lake in Pittsfield, MA, though separate, is connected by a spillway to the Housatonic River that flows through western Massachusetts and western Connecticut. The Silver Lake sediment is a black organic silt that has high concentrations of PCBs (originally a mixture of Aroclor 1254 and 1260) and hydrocarbon oil (2, 4, 7). Extensive environmental dechlorination of the PCBs from the ortho, meta, and para positions has occurred in this sediment (2, 4). Neither the sediment nor the PCBs in Silver Lake wash into the Housatonic River. Woods Pond is an impoundment on the Housatonic River located downstream 10.5 mi (ca. 16.9 mi) of Silver Lake in Lenox, MA. The sediment is a mixture of black humic matter, sand, and silt contaminated with a hydrocarbon oil and Aroclor 1260 (13). There is a slight amount of Aroclor 1260 dechlorination with the loss of meta and para chlorines (13,16).This investigation reveals that the three sediments share some PCB reductive dechlorination characteristics but differ considerably in others. Most likely these differences represent several distinct microbial populations.
Materials And Methods Sediment Collection Sites. PCB-contaminated sediments were collected from the Hudson River downstream of Fort Edward, NY, at river mile 193.5 (the H7 site in ref 5), from the Housatonic River at Woods Pond (Lenox, MA), and from Silver Lake (Pittsfield, MA). The sedimenta were collected in screw-top, glass vessels sealed with aluminum foil-lined lids or sealed stainless steel cans, topped with water from the collection site, and stored at 4 "C. The PCB concentrations were approximately 50, 100, and 4000 ppm in the Hudson River, Woods Pond, and Silver Lake sediment samples, respectively. Sediment Slurry Preparation. Slurries were prepared and sampled within a flexible, controlled-environment chamber (CoyLaboratory Products, Ann Arbor, MI) containing an oxygen-free atmosphere (197 % Nz, 1 3 % Hz). Slurries (300-500 mL) of wet sediment and revised anaerobic mineral medium (RAMM, ref 17) reduced with 0.1 % L-cysteine hydrochloride (Sigma Chemical Co., St. Louis, MO) were prepared; the ratio of wet sediment to RAMM was 2:3 (vo1:vol). While the slurries were stirred magneticallyto suspend the sediment, 30-mLaliquots were transferred to 50-mL serum vials (Wheaton, Millville,NJ). A trichlorobiphenyl stock solution (70 mM in acetone) was added to each serum vial; the final concentrations of the congener and acetone were 350 pM and 0.5%, respectively. After PCB addition, each vial was vortexed for 2 min. Duplicate 1-mL samples were taken immediately after vortexing as time zero points and were stored at -20 "C until analysis. All vials were crimp-sealed with Teflon-lined, butyl rubber septa (Wheaton, Millville, NJ) and removed from the chamber. A group of four vials was
Table 1. Observed Acclimation Times before Dechlorination of Trichlorobiphenyls in Three PCB-Contaminated Sediment Slurries origin of sediment in slurry trichlorobiphenyl
Hudson River
Woods Pond
Silver Lake
2,3,4-CB 3,4,5-CB 2,4,5-CB 2,3,6-CB 2,3,5-CB 2,4,6-CB
21 days 18 days 21 days 21 days 18 days 21-28 daysa
21 days 21 days 21 days 35 days 12 weeks 24 weeks
22 days 22 days 22 days 56 days 14 weeks >52 weeksb
Dechlorination was observed in one sample at 21 days and in the duplicate sample at 28 days. *No dechlorination by 1 year of incubation, but one of the duplicate samples showed dechlorination when sampled at 143 weeks of incubation.
prepared for each combination of added congener and sediment; two vials of each group were immediately autoclaved for 3 h. The autoclaved vials represented the control samples for each group. All vials were incubated stationary a t room temperature (22-25 "C) in the dark. PCB congeners (>99% purity) and Aroclor 1242 (neat) were purchased from Accustandard, Inc. (New Haven, CT). Sampling and PCB Analysis. The vials were sampled as sediment groups in the anaerobic chamber every 7-9 days for the first 8 weeks and then at selected time points. Each vial was vortexed for 30 s, immediately uncapped, and a 1-mL sample was removed and placed in an 8-mL screw-top, glass vial using a Gilson pipetman with a cutoff, 1-mL, plastic tip. The sample vials were sealed with Teflon-lined, foam-backed, screw caps. The PCBs were extracted from each slurry sample by shaking at room temperature for 114 h with 5 mL of diethyl ether and -0.5 mL of mercury. The mercury was added to precipitate the elemental sulfur in the sediment sample. The extracts were analyzed by capillary gas chromatography (GC) on a fused silica, capillary column (30 m by 0.25 mm id.) coated with a 0.25-pm bonded liquid phase of DB-1 (polydimethylsiloxane, J&W Scientific, Folsom, CA) with an electron capture detector (ECD) at 300 "C as previously described ( 4 ) . Selected PCBs (the trichlorobiphenyls described in Table 1; the dichlorobiphenyls 2,3-CB, 2,4CB, 2,5-CB, and 2,6-CB; and the monochlorobiphenyl 2-CB)were identified either by chromatographing the pure congeners (the trichlorobiphenyls) to determine the retention times m, for the other congeners, by comparison with a 10ppm Aroclor 1242standard for which the relative retention times had been published (4, 5, 18). The congeners 2,4-CB and 2,5-CB are not resolved using the DB-1 capillary column. Therefore, some samples were analyzed by GC/ECD using a fused silica, capillary column (50 m X 0.25 mm id.) coated with a 0.2-pm film of C-87 (Chrompack, Inc., Raritan, NJ). Splitless injections of 1 pL were chromatographed using an isothermal gradient at 210 "C and a helium gas velocity of 25 cm/s at a back pressure of 18 psi. The dechlorination sequence of an added trichlorobiphenyl was determined by the appearance of the dichlorobiphenyl and monochlorobiphenyl dechlorination products in conjunction with the disappearance of the trichlorobiphenyl. For this information, quantitation was not necessary since predominantly one sequence of chlorine removal was observed for each trichlorobiphenyl in each sediment slurry. Therefore, for analysis of each trichloEnviron. Sci. Technol., Voi. 28,
No. 4, 1994 631
robiphenyl, except 3,4,5-CB in Hudson River sediment slurries, the GC/ECD peak areas of the trichlorobiphenyl and each dechlorination product were compared on a percentage basis. The percentage of a particular congener was assigned by dividing its peak area by the sum of the peak areas for the trichlorobiphenyl and the dechlorination products. Since the ECD response factor for a monochlorobiphenyl is considerably less (530-fold) than a trichlorobiphenyl, the percentage analysis provided a low estimate of the true dechlorination extent. Nonetheless, the dechlorination sequence for each trichlorobiphenyl was readily determined. It was assumed that the disappearance of each congener was due to reductive dechlorination only. Biphenyl was not detectable under the GC conditions using an ECD and, hence, could not be included in the peak area percentage. In Hudson River sediment slurries, 3,4,5-CB and its dechlorination products, including biphenyl, were measured by gas chromatography/mass spectrometry (GUMS) using a Hewlett Packard 5890/ 5971A GC/MS with a DB-1 coated, fused silica, capillary column using the chromatography conditions previously described ( 4 ) . Since the concentrations of 3,4,5-CB and its dechlorination products were significantly higher than the background PCBs in the slurries, only the total ion chromatograph of each sample was analyzed. The mass selective detector was operated in the scan mode. A linear three point calibration curve (0.1-10.0 pM range) for 3,4,5CB, 3,5-CB, 3,4-CB, 3-CB, 4-CB, and biphenyl was developed using the pure congeners and biphenyl. The curve was used to determine the relative molar distribution of the congeners and biphenyl in each sample. Results Every sediment slurry that was not autoclaved had a significant amount of gas bubbles (presumed to be methane) by 2-3 weeks, regardless of when dechlorination was observed. Many samples were frothing and had an audible head pressure when opened. In separate but similar slurry experiments, significant amounts of methane were detected and measured by gas chromatography of the headspace gas in the vials (data not shown). Gas bubbles appeared in the sediment slurries within 5 days after each sampling; this occurred for 4-8 weeks. Acclimation Times before Dechlorination of Trichlorobiphenyls. In their review of microbial reductive dehalogenation, Mohn and Tiedje (19) describe an acclimation time in an undefined culture as the initial period of an incubation during which reductive dehalogenation is not detectable. In the current study, the time when the first dechlorination product was detected in a sediment slurry was recorded as the acclimation time (Table 1).In the Hudson River sediment slurries, the acclimation times ranged from 18 to 28 days for all the trichlorobiphenyls. Once dechlorination occurred in any sediment slurry, 175% of the trichlorobiphenyl was dechlorinated within three sampling periods (21 days). The acclimation times of the trichlorobiphenyl dechlorination products 2,4-CB or 2,5-CB ranged from 7 to 21 days before 2-CB first appeared; the acclimation times varied between duplicate samples. In Woods Pond and Silver Lake sediment slurries there were significant differences among the acclimation times of the added trichlorobiphenyls (Table 1). The congeners 632 Environ. Sci. Technol., Vol. 28, No. 4, 1994
Table 2. Reductive Dechlorination of Trichlorobiphenyls in Three PCB-Contaminated Sediment Slurries
trichlorobiphenyl 2,3,4-CB2,3,5-CB2,3,6-CB 2,4,5-CB2,4,6-CB 3,4,5-CB
--
Hudson River sediment slurries first second third product product' product' 2,4-CB2,5-CB 2,6-CB 2,5-CB2,6-CB 3,5-CB
2-CB 2-CB 2-CB
- 3-CB
Silver Lake and Woods Pond sediment slurries first second product product'
2,4-CB 2,5-CB 2,6-CB 2,5-CB 2,4-CBbiphenyl 3,5-CB
4-CB
' A blank indicates that further dechlorination of the di- or monochlorobiphenyl product was not observed.
2,3,4-CB, 3,4,5-CB, and 2,4,5-CB had the shortest acclimation time (21 days). The acclimation times were longer for 2,3,6-CB, 2,3,5-CB, and 2,4,6-CB, which are listed in order of shortest to longest acclimation time. No dechlorination of 2,4,6-CBwas observed in Silver Lake sediment slurries by 12 months of incubation, but >55 % of the 2,4,6CB was dechlorinated to 2,4-CB and 4-CB in one of the duplicate samples when they were checked again at 30 months. In most of the autoclaved sediment slurries, dechlorination of the trichlorobiphenyl did not occur. However, dechlorination of 2,4,5-CBwas observed in the autoclaved Woods Pond sediment slurries by 10-12 weeks of incubation. Both the duplicate samples had a significant amount of gas bubbles (like the samples that were not autoclaved) concomitant with dechlorination. Dechlorination Sequences of Trichlorobiphenyls. Dechlorination of every trichlorobiphenyl occurred in the Hudson River sediment slurries (Table 2). In each sample, all meta and para chlorines were removed from the added trichlorobiphenyl. No ortho-dechlorination was observed. There was a preferential sequence of chlorine removal from each trichlorobiphenyl with two or more meta and para chlorines; in these samples, 1 9 5% of one dichlorobiphenyl was produced relative to the other. From the congeners 2,3,4-CB and 3,4,5-CB, both of which have three adjacent chlorines on one phenyl ring, the chlorine in the center position was removed first. From 2,4,5-CB, the para chlorine was removed first and from 2,3,5-CB, the meta chlorine adjacent to the ortho chlorine was removed first. Since 3,4,5-CBdoes not contain ortho chlorines, all three chlorines were removed in the Hudson River sediment slurries resulting in the accumulation of biphenyl (Figure 1). A dechlorination from trichlorobiphenyl to biphenyl occurred in the sequence 3,4,5-CB to 3,5-CB to 3-CB to biphenyl. A small amount (5%) of the 3,4,5-CB was dechlorinated in the sequence 3,4,5-CBto 3,4-CBto 4-CB to biphenyl. In Silver Lake and Woods Pond sediment slurries, for each trichlorobiphenyl except 2,4,6-CB, the first chlorine was removed from the same position as in Hudson River sediment slurries (Table 2). However, in contrast to the Hudson River sediment slurries, only one chlorine, meta or para, was removed (i.e., the dichlorobiphenyl was a terminal product). Ortho-dechlorination of 2,4,6-CBwas observed in both Woods Pond and Silver Lake sediment slurries, but in the Silver Lake sediment slurries this dechlorination occurred some time between 12 and 30 months in only one of the duplicate samples. Both ortho
100
-7
- / \\ /A \
E 80
/pp/ -3 BIPHENYL -C 35-CB
a,
3
-rn
40
+345-CB
d 20 0 0
10
20
30 40 Incubation Time (Days)
50
60
Figure 1. Stepwise dechlorinatlon of 3,4,5-trichiorobiphenyi (3,4,5CB) in PCB-contaminated Hudson River sediment slurries. The data were obtained by GC/MS as described under Materials and Methods. Each datum point is an average of the duplicate samples;the numeric difference between any duplicate samples was 5 4 % . The congener 3,4-CB was detected at 5 1% in each sample and was not included in the graph.
chlorines were removed from 2,4,6-CB producing 4-CB; there was no para-dechlorination. In both the unheated and the autoclaved Woods Pond sediment slurries, the para chlorine was removed from 2,4,5-CB.
Discussion Characterization of Microbial Trichlorobiphenyl Dechlorination Activities. Microbial dechlorination of each trichlorobiphenyl is characterized by the acclimation time and the isomeric arrangement of the chlorine atoms (Tables 1and 2). For congeners containing adjacent meta and para chlorines (2,3,4-CB,3,4,5-CB, and 2,4,5-CB),the acclimation times were roughly equivalent in all the sediment slurries. Furthermore, the position, meta or para, of the first chlorine removed also was the same for these congenersin all the sediment slurries. It is unclear whether this correlation is due to biological parameters (i.e., similar enzymes)or chemical parameters (i.e., dechlorination order is dictated by molecular structure). It is difficult to compare the dechlorination of some of the congeners among the different sediment slurries because there is no good reference (e.g., an organism count). The only common feature among the sediment slurries was the volumetric ratio of wet sediment to RAMM. There were several differences in the characteristics of the trichlorobiphenyl dechlorination in the sediment slurries. One difference was that the microbial population in the PCB-contaminated Hudson River sediment mediated removal of all meta and para chlorines (e.g., dechlorination of 3,4,5-CB to biphenyl), whereas the microorganisms in the more highly chlorinated PCB-contaminated Silver Lake and Woods Pond sediments reduced all the trichlorobiphenyls, except 2,4,6-CB, to only dichlorobiphenyls (Table 2). Another difference was that the acclimation times of congeners that do not contain adjacent meta and para chlorines (2,3,6-CB, 2,3,5-CB, and 2,4,6-CB) were longer in Silver Lake and Woods Pond sediment slurries relative to Hudson River sediment slurries (Table 1).The acclimation times of these congeners were also significantly different within each sediment group (e.g., acclimation times of 8,14, and >52 weeks for 2,3,6-CB, 2,3,5-CB, and 2,4,6-CB, respectively, in Silver Lake sediment slurries).
The dechlorination of 2,4,6-CB in Woods Pond and Silver Lake sediment slurries is unique when contrasted with its dechlorination in Hudson River sediment slurries and the dechlorination of the other trichlorobiphenyls in slurries of the same sediments (Tables 1 and 2). Both ortho chlorines were removed from 2,4,6-CBin the Woods Pond and Silver Lake sediment slurries contrasted with only para-dechlorination in the Hudson River sediment slurries. The ortho-dechlorination of 2,4,6-CB occurred after significantly longer acclimation periods than the dechlorination of the other trichlorobiphenyls. Orthodechlorination of PCBs in Silver Lake sediment ( 4 ) and of 2,3,5,6-CB in Woods Pond sediment slurries (13) has been documented. In both cases, the ortho-dechlorination was not exclusive. Moreover, only one of the ortho chlorines was removed from 2,3,5,6-CB, and the extent of ortho-dechlorination in Silver Lake sediment was not determined. In the present study, the microorganisms in the Silver Lake and Woods Pond sediments did not remove the lone para chlorine from 2,4,6-CB, yet removal of lone para chlorines from 2,4,6-CB and 2,4,6-trichlorophenylsubstituted congeners of Aroclors by microorganisms in these sediments was described elsewhere (16). It is not known how the PCB concentration or other contaminants in the sediments influence the acclimation time or the sequence of a trichlorobiphenyl dechlorination. Quensen et al. (20) have demonstrated that oil addition to sediment slurries, similar to the oil contamination in Silver Lake sediment, will increase the acclimation time before PCB dechlorination. Although, the similar acclimation times and dechlorination sequences of 2,3,4-CB, 2,4,5-CB, and 3,4,5-CB in all three sediment slurries (Tables 1 and 2) suggest that inhibition does not play a major role in trichlorobiphenyl dechlorination. Hence, the long acclimation periods for 2,3,5-CBand 2,4,6-CBin Woods Pond and Silver Lake sediment slurries seem more likely due to undefined microbial ecological factors (i.e., population dynamics, carbon and energy sources, etc.). Complete dechlorination of 3,4,5-CB to biphenyl was observed in Hudson River sediment slurries (Figure 1). This establishes that complete dehalogenation of PCBs to biphenyl is possible, at least by microorganisms from Hudson River sediment. Therefore, potentially toxic congeners in Aroclors, such as those that do not contain ortho chlorines (e.g., 3,4-4-CB,3,4-3-CB, and 3,4-3,4-CB), could be completely dechlorinated by microorganisms in Hudson River sediment. Brown and co-workers (3, 4 ) noted that these congeners were environmentally dechlorinated in PCB-contaminated aquatic sediments. Dechlorination of 2,4,5-CBwas observed in autoclaved Woods Pond sediment slurries after a 10-12 week acclimation period (3-4 times longer than the acclimation period in the unheated samples). It is possible that autoclaving did not kill all the microorganisms capable of dechlorinating PCBs or that some of them are resistant to or activated by autoclaving (120 "C for 3 h). It is also possible that dechlorination in the autoclaved samples was due to microbial contamination from the unheated samples within that group of sediment slurries. The dechlorination of 2,4,5-CB in the autoclaved and the unheated samples might represent the same microbial population, since only the para chlorine was removed in each sample. Trichlorobiphenyl Reductive Dechlorination Reactivity Comparisons. In their study of environmental Environ. Scl. Technol., Vol. 28, No. 4, 1994 633
~
~~~
~~-
Table 3. Chlorine Atom Reactivity Sequence for Anaerobic Dechlorination of PCBs by Microorganisms in Hudson River, Silver Lake, and Woods Pond Sediments
order"
position of chlorine on phenyl ring
refsb
doubly-flanked meta chlorine (2,3,4-to 2,4-; 2,3,4,6-to 2,4,6-) A, B, Br, Qb, W doubly-flanked para chlorine (3,4,5- to 3,5-; 2,3,4,5- to 2,3,5-) B, Br, Qa, Qb, W singly-flanked para chlorine (3,4- to 3-; 2,4,5- to 2,5-) A, B, Br, &a, Qb, W singly-flanked meta chlorine (2,3,5- to 2,5-; 2,4,5- to 2,4-) B, Br, V, Qb, W unflanked meta or para on di- or tri-substituted ringC(2,5- to 2-; 2,4-, to 2-; 2,4,6- to 2,6-) A, B, Br, &a, Qb, W isolated meta or para chlorinec (3- or 4- to biphenyl) A, Br, W singly-flanked ortho chlorined (2,3,5,6- to 2,3,5-) V unflanked ortho chlorine on di- or tri-substituted ringd (2,4,6-to 2,4- to 4-) W isolated ortho chlorine (2- to biphenyl) not observed a An approximate ranking by both acclimation times observed in laboratory experiments and dechlorination patterns observed in laboratory and environmental samples. b References: A, Abramowicz et al. (9);B, Bedard et al. (16); Br, Brown et al. (3-5); Qa, Quensen et al. (7);Qb, Quensen et al. (6); V, Van Dort and Bedard (13);W, Williams (this study). Upper Hudson River sediment only. d Silver Lake and Woods Pond sediment only.
PCB dechlorination in sediments from Silver Lake and the Hudson River, Brown and co-workers (4) noted that "the patterns of congener reactivity fell into two broad categories: o,m,p-dechlorinations found in Silver Lake sediment, which remove chlorines from ortho, meta, and para positions, with congener reactivities primarily determined by reduction potential; and m,p-dechlorinations found in Hudson River sediment, which remove chlorines from meta and para positions only, with relative reactivities determined mainly by molecular shape". The trichlorobiphenyl dechlorination data tend to support the conclusions of Brown et al. (4),with one exception (discussed below). In Hudson River sediment slurries, trichlorobiphenyl dechlorination was influenced by the spatial configuration of the chlorines on the target ring. For example, complete meta- and para-dechlorination occurred including dechlorination of 3-CB to biphenyl, but orthodechlorination of 2,6-CB or 2-CBwas not observed despite their reduction potentials that are relatively less negative than that of 3-CB (4, 21). In most cases, microbial dechlorination of a trichlorobiphenyl in a Silver Lake or a Woods Pond sediment slurry was simply correlated to the trichlorobiphenyl chemical reactivity (4,21),since a dichlorobiphenyl was the terminal dechlorination product. This suggeststhat PCB congeners with reduction potentials below a specific point are not dechlorinated in these sediments. An exception to this hypothesis is the ortho-dechlorination of 2,4,6-CB. Orthodechlorination of 2,4,6-CB and 2,4-CB occurred, but dechlorination of 2,3-CB,2,5-CB,and 3,5-CB did not, even though these congeners have reduction potentials that are relatively less negative than those of 2,4,6-CB or 2,4-CB (4,21). This result suggests that microorganisms in Silver Lake and Woods Pond sediment have reductive dechlorinating activities that are not completely influenced by the reduction potential of a PCB congener but also by its molecular shape. A general chlorine atom reactivity sequence for microbial reductive dechlorination of PCBs in the three sediments is proposed in Table 3 from the data of this study and of other investigators (3-7, 9, 13, 16). The sequence of chlorine removal from a PCB is associated with the isomeric arrangement of the chlorines on the phenyl rings such that a chlorine (meta or para) flanked by two other chlorines is preferentially removed first (e.g., 2,3,4-CBwas dechlorinated to 2,4-CB, 3,4,5-CB was dechlorinated to 3,5-CB; Table 2). A similar observation was made by Fathepure and co-workers (15) from their studies of hexachlorobenzene reductive dechlorination in anaerobic 634
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sewage sludge. They noted that a chlorine would be removed if it was between two other chlorines until the molecule had no adjacent chlorines (i.e., 1,3,5-trichlorobenzene was a terminal dechlorination product). As shown in Table 3, a chlorine with one or two flanking chlorines on a phenyl ring of a PCB is more reactive to dechlorination than an unflanked or isolated chlorine. However, note that the order of reactivity displayed in Table 3 is a general ranking by both acclimation times and dechlorination patterns. The reactivity of a chlorine for dechlorination in any of the sediments depends on both the chemical and microbiological conditions. Conclusions. Two conclusions are drawn from the dechlorination data summarized in Table 3. Meta and para chlorinesof a PCB are roughly equivalent in reactivity, but ortho chlorines are significantly less reactive than meta or para chlorines. The reactivity of a meta or para chlorine of a PCB is dependent upon the number and position of the chlorines on the same phenyl ring, such that a doublyflanked chlorine is more reactive than a singly-flanked chlorine, which is more reactive than a unflanked chlorine, which is more reactive than a lone chlorine (a monochlorophenyl ring). This suggests that reduction potential and molecular shape both contribute to the order and extent of a PCB reductive dechlorination by microorganisms in the sediment slurries. Further research on how the position and extent of chlorination of a PCB affect microbial reductive dechlorination is necessary. Future dechlorination studies could focus on single congenerswith chlorines on both phenyl rings such as tetrachlorobiphenyls containing a trichlorophenyl and a monochlorophenyl ring (e.g., 2,3,4-2-CB, 2,3,5-2-CB, 2,3,6-2-CB, etc.); these experiments would also address how chlorination on one phenyl ring affects dechlorination on the other phenyl ring. Acknowledgments
I thank Ralph J. May for his expert GC/MS analysis. I also thank Donna L. Bedard and John F. Brown, Jr., for helpful discussions and comments on this manuscript. Literature Cited (1) Abramowicz, D. A. In Critical Reviews in Biotechnology; Steward, G. G., Russell, I., Eds.; CRC Press, Inc.: Boca Raton, FL, 1990; Vol. 10, pp 241-251. (2) Brown, J. F., Jr.; Bedard, D. L.; Brennan, M. J.; Carnahan, J. C.; Feng, H.; Wagner, R. E. Science 1987,236,709-712. (3) Brown, J. F., Jr.; Wagner, R. E. Enuiron. Toxicol. Chem. 1990,9,1215-1233.
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Abstract published in Advance ACSAbstracts, February 1,1994.
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