Environ. Sci. Technol. 1993,27,530-538
Reductive Dechlorination of Polychlorinated Biphenyls in Anaerobic Sediments Alfredo C. Aider,+-'Max M. Haggblom,*~§~'1 Stephanie R. Oppenheimer,tJ and L. Y. Young*rt*§sIl
Department of Microbiology and Institute of Environmental Medicine, New York University Medical Center, New York, New York 10016 To better understand the conditions which control PCB dechlorination in anaerobic sediments we examined the microbial activity in two freshwater sediments, Hudson River (NY) and Silver Lake (MA), and one estuarine sediment, New Bedford Harbor (MA). Two different reducing conditions were established, methanogenic and sulfidogenic; in addition, the effect of added organic substrates and the differences in the fate of preexisting and freshly added PCBs (100-400 ppm Aroclor 1242 or 1260) were examined. The most rapid dechlorination occurred in the methanogenic cultures established with Hudson River sediment to which Aroclor 1242 was added and supplemented with a fatty acid mixture. Meta and para chlorines were preferentially removed, resulting in accumulation of ortho-substituted mono-, di-, and trichlorobiphenyls. In cultures established with New Bedford Harbor sediment, dechlorination was slower and not as extensive, with di-, tri-, and tetrachlorobiphenyls accumulating. Dechlorination was observed with spiked Aroclor 1242 and with preexisting PCBs alone. In Silver Lake sediment cultures spiked with Aroclor 1260, dechlorination with accumulation of tri- and tetrachlorobiphenyls was observed. Dechlorination was slower than that for the Hudson River cultures. Both the Hudson River and Silver Lake cultures showed no additional dechlorination for the preexisting PCBs. The addition of fatty acids did not enhance activity in the New Bedford Harbor or Silver Lake cultures. No activities were detected under sulfate-reducing conditions with any of the sediments. Introduction Contamination of the environment by PCBs was first reported by Jensen in 1966 (I), and their presence and persistence has been a matter of concern ever since. Since 1929, -5.7 X lo8 kg of PCBs has been commercially produced, the majority in the United States (Monsanto tradename, Aroclor), and -2 X 10s kg, representing 35%) has been estimated to remain in mobile environmental reservoirs (2). The voluntary and legislative restrictions instituted since 1971 on the production, sale, and use of PCBs in closed systems and the banning of PCBs for future use has decreased the anthropogenic loading, which has resulted in lower levels of these compounds in sediments (3). Dispersion of these chemicals from source regions to global distribution occurs through atmospheric transport and subsequent deposition. The technical PCB products consisted of complex mixtures of various chlorinated homologues and isomers. These different congeners differ in their physical-chemical 'Department of Microbiology. t On leave from the Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), CH-8600 Dubendorf, Switzerland. Institute of Environmental Medicine. 11 Present address: Center for Agricultural Molecular Biology, Rutgers University, P.O. Box 231, Cook College, New Brunswick, NJ 08903.
Present address: Center of Marine Biotechnology, University of Maryland, Baltimore, MD 21202. 530
Envlron. Sci. Technol., Vol. 27, No. 3, 1993
and biological characteristics, leading to congener-specific toxicity ( 4 , 5 )and environmental fate. PCBs are characterized by their low solubility in water and low vapor pressures leading to high partition coefficients to abiotic and biotic particles. In aquatic systems PCBs can be removed through adsorption to or partitioning into abiotic settling particles and organisms with subsequent sedimentation. The sediments act as an important sink, but through resuspension and mixing by aquatic organisms, the surface sediment can also act as a source for these contaminants, increasing the residence time of PCBs in the ecosystem (6, 7). Because of their hydrophobicity, halogenated organic compounds such as PCBs tend to transfer to and accumulate at environmental interfaces and partition, for example, into anaerobic habitats where reductive dehalogenation, i.e., the reductive cleavage of the carbon-halogen bonds, may be an important environmental biotransformation process. In 1984, Brown et al. (8)reported an altered PCB congener distribution in anaerobic sediments from the upper Hudson River in New York State and from Silver Lake, an urban pond in Massachusetts. Their observations indicated a loss of meta and para chlorines and accumulation of ortho-substituted, lower chlorinated biphenyls (CBs), and they proposed this to be a biological process occurring in situ (8-10). Alternatively, it was suggested that a selective partitioning mechanism could result in the observed enrichment of mono- and dichlorobiphenyls (II), leading to some debate (12,13). Biologically mediated reductive dechlorination of PCBs was confirmed, however, in laboratory experiments by Quensen and co-workers (14,15). Environmental dechlorination of PCBs has subsequently been suggested to have occurred in situ in freshwater, estuarine, and marine sediments (16). Confirmation in other laboratory experiments of reductive dechlorination of PCBs continues to accumulate (17-20). To our knowledge, reductive dechlorination of PCBs has been observed only under methanogenic conditions. It has been suggested (21) that the dechlorination of chloroaromatic compounds observed under methanogenic conditions, but inhibited under sulfate-reducing conditions, may be due to sulfate competing more effectively than the chloroaromatics for the electrons. On the other hand, anaerobic dehalogenation has been shown to occur in the presence of sulfate (22-24) and degradation of chlorophenols can be coupled to sulfate reduction (24). Sulfate is usually the predominant electron acceptor for anaerobic metabolic processes in anaerobic marine Sediments or other sulfate-rich habitats (25). In marine environments a wide variety of halogenated aliphatic and aromatic compounds are produced biologically by marine organisms (22, 26). Therefore, anaerobic marine sediments may potentially allow for the selection and enrichment of anaerobic dehalogenating organisms. It has been reported for the anaerobic pure culture, Desulfomonile tiedjei, that dechlorination of 3-chlorobenzoate is an energy (ATP) generating mechanism (27, 28). Under conditions in the environment where electron acceptors are limiting, organisms with an ability to use PCBs in this capacity may be selected for or enriched (IO).
0013-936X/93/0927-0530$04.00/0
0 1993 Amerlcan Chemical Society
In order to better understand the conditions that control PCB dechlorination in anaerobic sediments, we examined the microbial activity in two freshwater sediments, Hudson River and Silver Lake, and one estuarine sediment, New Bedford Harbor. Two different reducing conditions were examined, methanogenic and sulfidogenic. In addition, the effect of added organic substrates and the differences in the fate of preexisting and freshly added PCBs were studied.
Experimental Section Sediments. In our study, three different sediments contaminated with PCB point discharges from industrial sources were used as inoculum: Hudson River, site H7, NY (HR); New Bedford Harbor, MA (NBH); and Silver Lake, MA (SL). Samples were collected from the top 20 cm of the sediment and kept in sealed glass jars until used. The HR sediment is a sandy silt with a moderate content of organic carbon historically contaminated with Aroclor 1242. Although it can vary, the average contaminant concentration of the samples is -50 pg/g (8-10). The particle size distribution showed that the coarsest sediment fraction (>53 pm) makes up to 89% of the sediment mass (M, Harkness, personal communication). The NBH sediment (Acushnet River estuary) is black, silty mud, rich in organic carbon and historically contaminated with a mixture of Aroclors 1242 and 1254 along with hydrocarbon oil. Contaminant concentrations, again, vary and have been reported to be 0.3-3700 pg/g (16,29,30). The samples we collected had approximately 400-500 pg/g. The salinity of the water in the estuary was 26%. New Bedford Harbor, Cr, 620 f 20; Pb, 550 f 30; Cd, 15 f 0.5; Zn, 1440 f 40. The SL sediment consists of a black, oily, methanogenic mud which was historically contaminated with what is believed to have been almost completely Aroclor 1260 (-300 pg/g) (9,IO). The sample of SL contained -200 pg/g PCBs. Metals. The analysis method was adapted from ref 31. Approximately 5 g of oven-dried (80 "C) sediment was mixed in an acid-washed 250-mL beaker with 30 mL of concentrated nitric acid (Fisher Scientific, trace metal grade). A glass watch glass was placed on the beaker, and the sample was refluxed for 24 h on a hot plate. After being refluxed, the samples were boiled down to - 5 mL and 1mL of 30% hydrogen peroxide was added to each sample in 0.2-mL increments. Each sample was then boiled down to near-dryness and resuspended in 10 mL of 2% nitric acid. After the samples were filtered using glass fiber filters, each sample was diluted to a final volume of 25 mL using 2% nitric acid. Metal concentrations in the acid extracts were measured by flame atomic absorption spectrophotometry using an IL atomic absorption Video 12 spectrophotometer equipped with deuterium background correction and analyzed for zinc (A = 213.9 nm), cadmium (A = 228.8 nm), and lead (A = 283.3 nm) using an air/acetylene flame. Chromium (A = 357.9 nm) was measured using a nitrous oxide/acetylene flame. Measured concentrations (in pg/g) in the sediments were as follows: Hudson River, Cr,460 f 90; Pb, 170 f 20; Cd, 8 f 1;Zn, 220 f 10. New Bedford Harbor, Cr, 620 f 20; Pb, 550 f 30; Cd, 15 f 0.5; Zn, 1440 f 40. Silver Lake, Cr, 940 f 30; Pb, 1680 f 290; Cd, 170 f 5; Zn, 5490 f 40. Reagents. All of the solvents used for PCB analysis were distilled in glass grade (Burdick & Jackson Lab. Inc.). Octachloronaphthalene,Aroclors 1242,1254,and 1260, and single chlorobiphenyls were purchased from Ultra Scientific (North Kingston, RI). Establishment of Cultures. Strict anaerobic techniques were followed through the study. A 35% (v/v)
-
sediment inoculum was added to a methanogenic (32)or sulfidogenic (24) mineral salts medium, supplemented with vitamins and trace elements. A volume of 50 or 100 mL was transferred to deoxygenated 65- or 160-mL flasks, respectively, with C02/N2(30/70) as the head space gas, and sealed with Teflon-coated butyl stoppers and aluminum crimp sealers. The rationale for using a vitaminsupplemented mineral salts medium was to provide optimal conditions for microbial activity. Methanogenic and sulfate-reducing conditions were selected by addition of an excess of carbonate or sulfate, respectively. Sterile controls were prepared in the same way and autoclaved twice. To the sediments of Hudson River and New Bedford Harbor was added 100 ppm Aroclor 1242 in 0.26 g/L acetone, and to Silver Lake sediments 400 ppm Aroclor 1260 in 1.06 g/L acetone. We also established background cultures in medium without spiked PCBs to study the fate of preexisting PCB contamination in the sediments. All cultures were carried out in duplicate with the exception of autoclaved controls and those inoculated with Silver Lake sediment because of the small amount of inoculum available. A fatty acid mixture consisting of acetate, propionate, butyrate, and hexanoic acid was added as a carbon source to one set of cultures, initially to 500 mg/L (6.44 mM) and then monthly to 250 mg/L (3.22 mM): acetate, 0.85 mM; propionate, 1.37 mM, butyrate, 0.57 mM; hexanoic acid, 0.43 mM. This fatty acid mixture was added to serve as a growth substrate for the anaerobic population as well as an electron donor for the reductive dechlorination of the PCBs. Acetate, propionate and butyrate are likely products from the fermentative decomposition of biomass in sediments and can be used by a wide spectrum of anaerobic bacteria as carbon sources and electron donors. Relatively low concentrations were chosen in order to minimize selection for fatty acid degraders and overgrowth of the cultures. All the cultures were incubated statically at 30 "C in the dark. Samples were taken periodically over 17 months; for each sample the bottle was shaken vigorously, the stopper removed, and 2 mL of sediment slurry removed with an inverted Pasteur pipet. The head space was flushed with N2/C02 and the culture bottle recapped. The samples were frozen until extraction and analysis. Gas production was measured with an air-tight syringe and the head space gas was monitored for methane using a gas partitioner (Model 1200; Fisher Scientific Co., Springfield, NJ) equipped with a thermal conductivity detector, as described elsewhere (24). Analytical Procedures. The sample vial was used as the extraction vessel. After the solids settled, the supernatant was removed and octachloronaphthalenewas added as an internal standard. Then the sediments were repeatedly extracted on a mechanical shaker: first with hexane/acetone (1/1)overnight and then with hexane for 4 h. The aqueous supernatant and all solvent phases were combined. Acetone was removed by reverse partition into water, the hexane extract concentrated to 1mL under a gentle stream of N2 or argon, and dried sodium sulfate added to immobilize the aqueous layer. Sample cleanup was performed in a micro Florisil (Sigma) column (-0.2 g in a disposable Pasteur pipet). PCBs were eluted from the Florisil with 6 mL of hexane. Elemental sulfur was removed by the addition of tetrabutylammonium sulfate and sodium sulfite (33),and samples were diluted if necessary. PCB analysis was performed on a HewlettrPackard (Model 5890 Series 11)gas chromatograph equipped with a DB-5 fused-silica column (J&W; 30 m X 0.32 mm i.d., Envlron. Sci. Technol., Vol. 27,
No. 3, 1993 531
Table I. Congener Assignment and Average Number of Chlorines for Each Chromatographic Peak
peak no.
IUPAC no.
congener name (CBs)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 3 4,lO 7, 9 6 8, 5 19 12, 13 18, 15 17 27,24 16, 32 34,54 29 26 25 28,31, (50) 33,21, 20, 53 22, 51 45 46 52, 73 49 47,48, 75 65, 62 35 44, (104) 37, 42,50 64, 71, 41, 73 96 40, (103, 57) 100, 67 58, 63 74,94 70, 76, (61) 66,95, 102 91,98, 55 56,60 92, 155 84,89 101, 90 99 150, 112, 119 83, 109 97, 86, (152) 87, 111, 115 85, (116)
2 4 2-2, 26 24,25 2-3 23, 2-4 26-2 34, 3-4 25-2, 4-4 24-2 26-3, 236 23-2, 26-4 35-2, 26-26 245 25-3 24-3 25-4, 24-4, (246-2) 34-2, 234, 23-3, 25-26 23-4, 24-26 236-2 23-26 25-25, 26-35 24-25 24-24, 245-2, 246-4 2356, 2346 34-3 23-25, (246-26) 34-4, 23-24, 236-3 236-4, 26-34, 234-2, 25-35 236-26 23-23, (246-25, 235-3) 246-24, 245-3 23-35, 235-4 245-4, 235-26 25-34, 345-2, (2345) 24-34, 236-25, 245-26 236-24, 246-23, 234-3 23-34, 234-4 235-25, 246-246 236-23, 234-26 245-25, 235-24 245-24 236-246, 2356-3, 246-34 235-23, 2346-3 245-23, 2345-2, (2356-26) 234-25, 235-35, 2346-4 234-24, (23456)
0. ortho:
m
av Cllbiphenylg 0 m+P 1 0
2 1 1 1
3 0 1
2 2 2 2.5 1 1 1 1 1.5 2 3 3 2 2 2 2 0 2 1.33 1.75 4 2 2 1 2 1 2.33 2.33 1 3 3 2 2 2.67 2 2 1.67 2
0 1 0 1 1 1 0 2
1.5 1 1 1 1
2 2 2 2
1.75 1.5 1 1 2 2
2 2 3 2 2.34 2.25 1
2 2.5 3 2.5 3 2.34 2.34 3 2.5 2 3 3 2.66 3 3 3.33 3
IUPAC no.
congener name (CBs)
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
136 77,110 82 151 135, 124, 144 107, 108, 147 149, 106,123 118 134, 143, 114 122, 131, 133 146, 161 132, 105, 153 141,179 130 137, 176 138, 163 158 129 178 175 187, 182 128 183 167 185 174,181 177 171, 156,202 173 200,204 172, 192, 197 180, 193 191 199 170, 190 198 201 196,203 189 195 208 207 194 205 206 209
236-236 34-34, 236-34 234-23 2356-25 235-236,345-25, 2346-25 235-34, 234-35, 2356-24 236-245, 2345-3, 345-24 245-34 2356-23, 2345-26, 2345-4 345-23,2346-23, 235-235 234-245, 2346-35 234-236, 234-34, 245-245 2345-25, 2356-236 234-235 2345-24, 2346-236 234-245, 2356-34 2346-34 2345-23 2356-235 2346-235 2356-245, 2345-246 234-234 2346-245 245-345 23456-25 2345-236, 23456-24 2356-234 2346-234, 2345-34,2356-2356 2345-23 2346-2356, 23456-246 2345-235, 23456-35, 2346-2346 2345-245, (2356-345) 2346-345 23456-236 2345-234, 23456-34 23456-235 2345-2356 2345-2346, 2356-2356 2345-345 23456-234 23456-2356 23456-2346 2345-2345 23456-345 23456-2345 23456-23456
81
82 83 84 85 86 87 88 89 90 91 92 93
4 1
2 3 2.33 1.67 1.67 1 2.33 2
2 2
3 2 3 2 2 2 3 3 3 2 3 1 3 3 3 2.67 2 4 2.67 2 2 4 2 3 3 3.5 1 3 4 4 2 2 3 4
2 3.5 3 3 3.34 3.66 3.66 4 3.34 3.67 4 3.67 3.5 4 3.5 4 4 4 4 4 4 4 4 5 4 4 4 4 4 4 4.66 5 5 4 5 5 5 4.5 6 5 5 5 6 6 6 6
+ D. meta + Dara.
film thickness 0.25 pm), a 63Nielectron capture detector, and a HP7673 automatic sampler. Helium was the carrier gas (head pressurep60 Wa; h e a r velocity, 0.26 m/s), with nitrogen as the make-up gas. The injector and detector temperatures were 270 and 340 "C, respectively. The column was held at 50 OC for 1min, ramped at 40 "C/min to 150 OC, 1.50 "C/min to 250 "C, and 10 OC/min to 280 "C,and finally held for 5 min. Quantitation. Compounds were identified by comparing the retention times with a known standard PCB mixture. Our quantitation is based on a PCB mixture of Aroclor 1242 (7 mg/L), Aroclor 1254 (3 mg/L), Aroclor 1260 (3 mg/L), 2-chlorobiphenyl (0.35 mg/L), and octachloronaphthalene (0.6 mg/L). Peak identities and quantities in the standard mixture were determined by Northeast Analytical, Inc. (Schenectady, NY) on a DB-1 column. Identification was also made by comparison with published chromatograms on a SE-54 column (34). Due to differences in the resolution of our DB-5 column and the DB-1 column with which the quantitation of the 532
peak no.
av Cl/biphenyl' o mCp
Environ. Sci. Technoi., Vol. 27, No. 3, 1993
standard was made, in some cases we had to combine peaks that were separated on the DB-1 column, while in other cases we obtained resolution of congeners not separated on DB-1. The congener assignments for all chromatographic peaks are given in Table I. Response factors for each peak were determined daily and used for quantitation and calibration with the standard mixture. The concentrations of PCBs recovered were calculated as mole percent based on the assumption that the biphenyl moiety remains intact and that complete removal of all the chlorines does not occur. This point was also assumed by other investigators in evaluating the reductive dechlorination of Aroclors (14,15,17). Since we observed no loss of total PCBs over the extended incubation period (17 months), this assumption is reasonable. For data calculations coeluting congeners and homologues were assumed to be present in equal proportions. The initial rates of dechlorination as nanomoles of chlorine released per milliliter of culture per week were calculated by linear regression analysis from the change
Table 11. PCB Homologue Distribution in Sediment Cultures from Hudson River, New Bedford Harbor, and Silver Lake" month
di
mono
tri
homologue, mol % fSD tetra penta
hexa
hepta
octa
Hudson River preexisting PCBs -FA +FA +Aroclor 1242 -FA
0.4 0.4 0.6 0.5
f 0.1 f 0.02 f 0.08 f 0.07
0.1 f 0 0.1 f 0.01 0.1 f 0.03 0.1 f 0.02
7.1 1.5 6.5 1.0
0.7 0.3 0.7 0.2
f 0.01
0.1 f 0.07 0.1 f 0.01 0.1 0.01 0.1 f 0
2.6 1.3 2.4 1.3
f 0.2
f 0.2
12.6 f 0.4 4.8 f 0.01 12.0 f 0.1 4.9 f 0.2
30.3 f 0.2 19.4 f 0.3 30.4 f 0.6 19.3 f 0.1
11.0 f 0.1 3.8 f 0.2 11.3 f 0.6 3.7 f 0.03
2.0 0.9 2.1 0.9
f 0.06 f 0.02 f 0.08
31.1 32.0 29.6 30.3
f 2.2 f 0.03 f 0.4 f 0.5
41.7 f 2.9 41.1 f 0.7 37.7 f 0.6 39.1 f 0.5
20.2 f 0.1 19.4 f 0.4 23.1 f 0.5 21.7 f 0.6
5.4 f 0.4 5.7 f 0.2 7.1 f 0.4 6.6 f 0.6
0
9.0 39.2 10.0 40.8
f 1.3
22.2 36.6 24.0 35.6
34.0 f 0.7 16.3 f 0.8 33.6 f 0.9 16.8 f 0.6
26.9 f 1.3 6.0 f 0.9 25.0 f 2.5 5.6 f 0.5
11 +FA
1.1f 0.2 1.3 f 0.04 1.8 f 0.1 1.6 f 0.2
0 11 0 11
0
11
f 0.8 f 1.9 f 1.3
f 1.1 f 1.4 f 2.2 f 0.1
f 0.5 f 0.4 f 0.6
f 0.1
f 0.08 f 0.02 f 0.02
*
New Bedford Harbor preexisting PCBs -FA +FA +Aroclor 1242 -FA +FA
f 0.07
15.7 23.4 16.6 22.9
f 0.8 f 0.4 f 0.3 f 0.5
37.9 47.5 38.3 48.3
1.2 f 0.02 1.7 f 0.2 0.9 f 0.3 1.2 f 0.1
17.0 25.8 16.9 26.2
f 0.1
38.1 f 0.3 48.2 f 0.4 37.8 f 0.5 48.5 f 0.1
0 11 0 11
0.7 1.0 0.9 0.8
0 11 0 11
f 0.04
f 0.04 f 0.04
f 0.3 f 0.5 f 0.2
f 0.1 f 0.4 f 0.1 f 0.5
29.9 21.5 29.5 21.5
f 0.01 f 0.01 f 0.02
f 0.06 f 0.05 f0
f 0.01
0.5 0.3 0.4 0.3
f 0.06
0.4 0.2 0.4 0.2
f 0.01 f 0.01 f0
f 0.03 f 0.01 f 0.01
f0
Silver Lake preexisting PCBs -FA +FA +Aroclor 1260 -FA +FA
0 11 0 11
14.0 12.8 13.0 11.5
24.0 22.9 23.8 19.5
28.5 28.6 27.8 28.6
13.0 14.2 13.9 15.5
8.8 9.1 9.7 10.1
6.9 7.3 7.0 8.5
3.9 4.2 4.1 5.1
0.9 0.9 0.9 1.2
0 11 0 11
9.0 6.8 5.1 4.3
15.5 12.3 9.3 8.0
18.6 25.0 11.4 23.5
10.3 24.8 7.3 30.3
12.8 8.8 15.9 8.7
16.4 8.6 23.5 9.0
13.6 9.9 21.5 11.5
3.0 3.0 5.1 3.9
+FA, with addition of fatty acids; -FA, without fatty acid addition.
in the average number of meta + para chlorines over time. The error for the mean dechlorination rate was calculated by error propagation of the standard deviation of the dechlorination rate of the individual cultures. The significance of the differences between the mean dechlorination rates of fatty acid-amended and unamended cultures was determined by the Student's t test assuming the population variances unknown and unequal. Nomenclature. The systematic numbering of congeners is used as a synonym for the corresponding PCB compound and follows the convention proposed by Ballschmitter and Zell(35) and adopted by IUPAC, Peak numbers and congener names are given in Table I.
Results and Discussion
Hudson River. The most rapid change occurred in the cultures established with Hudson River (HR) sediment under methanogenic conditions and supplemented with the fatty acid mixture. Within 1-2 months, dechlorination of the added Aroclor 1242 was observed and resulted in a decrease of tri-, tetra-, and pentachlorobiphenyls with a corresponding increase in mono- and dichlorobiphenyls. The sterile controls showed no change over the entire time period of the experiments. Figure 1illustrates the mole percent of PCBs represented by each chromatographic peak at the start of the incubation period (Figure 1A) and after 11months of incubation (Figure 1B). The changes are very readily seen after incubation with loss of the higher peak numbers, representing the higher chlorinated congeners, and significant increase in the lower chlorinated congeners. These changes can also be observed by taking
the difference in mole percentages between month 0 and month 11 for each of the congener peaks. As illustrated in Figure lC, peaks which decreased fall below the line designated 0, while those which increased fall above 0. Congeners which increased consist mainly of 2-CB (1, +24%), 2-2-CB/26-CB (4/10, +15%), 26-2-CB (19, +2.0%), and 4-CB (3, +7.0%), with a decrease of all other peaks, e.g., 25-2-CB/4-4-CB (18/15, -6.1%), 24-2-CB (17, -1.7%), 23-2-CB/26-4-CB (16/32, -2.6%), 25-4-CB/24-4CB (28/31, -8.3%), 34-2-CB/234-CB/23-3-CB/25-26-CB (33/21/20/53, -3.7%), 23-25-CB (44, -2.1%), 34-4-CB/ 23-24-CB/236-3-CB (37/42/59, -1.9%), 25-34-CB/345-2CB (70/76, -2.4 % ), 24-34-CB/ 236-25-CB/ 245-26-CB (66/95/102, -2.4%), and 23-34-CB/234-4-CB (56/60, -2.4%). The extent of PCB transformation in the HR sediments after ll-months incubation is summarized in Table 11. The PCB congeners are grouped into homologues based on the number of chlorine substituents per biphenyl molecule. They are summarized as the distribution of the homologues from the start of the incubation (month 0) and at month 11. For the HR sediment cultures with added Aroclor, a marked increase of the mono- and dichlorobiphenyls and a decrease of tri-, tetra-, and pentachlorobiphenyl were observed. The presence or absence of amended fatty acids did not affect the extent of degradation. No loss of the mono- or dichlorobiphenyls was observed in any of these cultures up to incubation periods of 17 months. In addition, as noted in Table 11,incubation with or without fatty acid amendment did not change the preexisting (in situ dechlorinated) PCBs in the HR sediments. Environ. Sci. Technot., Vol. 27, No. 3, 1993 533
A
35 30
E 2
however, show a relatively rapid rate of dechlorination of the meta and para positions. Approximately 65% of the meta + para chlorines were removed in 2 months, after which no additional dechlorination occurred (Figure 2A). The sediment cultures not supplemented with fatty acids displayed a substantially slower rate of dechlorination. As shown in Figure 2B, it took 11months for the same extent (-65%) of dechlorination to take place. For the fatty acid-amended cultures, the dechlorination rates were calculated for the initial 2 months, and for the unamended cultures, the rates were based on the initial 7 months (initial PCB concentration 990 nmol/mL). The fatty acid-amended cultures exhibited an initial dechlorination rate of 93 f 8 nmol mL-' week-l, while the dechlorination rate in the unamended cultures was 29 f 9 nmol mL-l week-l. Amendment with the fatty acid mixture, thus, significantly enhanced the dechlorination rate (a= 0.05, p = 0.01). We observed only limited amounts of methane in the cultures established for determining background activity (data not shown), which indicates that any background carbon in the Hudson River sediments was recalcitrant or unavailable. Fatty acid supplementation was observed to greatly stimulate methane production. The observation that addition of fatty acids enhanced the dechlorination of added Aroclor suggests that dechlorination in the HR may be limited by the availability of an electron donor and/or carbon source. In summary, the resulting congener pattern observed after incubation of the added Aroclor 1242 is similar to the preexisting PCB pattern found in the sediments (see Table 11). Thus, under these conditions the ortho-substituted mono-, di-, and trichlorobiphenyls remain biorefractory as previously shown (14,15). In addition, no further transformation of the preexisting PCBs was observed. We also noted that no transformation of PCBs was observed in cultures established under sulfate-reducing conditions, even after incubation periods of up to 17 months, suggesting that sulfate inhibited reductive dechlorination. New Bedford Harbor. Dechlorination of the PCBs under methanogenic conditions was also observed in sediment cultures from New Bedford Harbor (NBH). It took 2-4 months before any activity was seen, and unlike the HR sediment cultures, dechlorination of the preexisting PCBs occurred, as well as of the added Aroclor 1242. The sterile controls remained unchanged. These cultures also differed from the HR in the congener profile resulting from dechlorination and in the observation that the presence or absence of fatty acids yielded no difference in activity. Of particular interest is the observation that the preexisting PCBs in NBH can be dechlorinated, indicating that extensive biotransformation of the sediment PCBs has not taken place in situ. Results from NBH cultures with preexisting PCBs and with added Aroclor 1242 were
1
0
Month
20
10
0
20
40
30
60
50
90
80
70
Peak number
35
,
0
B
20
10
30
40
50
80
70
60
90
Peak number
c Difference
-10
4 18.15
0
10
30
20
40 50 60 Peak number
80
70
90
Flgure 1. Mole percent of PCBs In Hudson River sediment cultures with added Aroclor 1242 (100 ppm) and fatty acids as an auxiliary carbon source: (A) at month 0; (8)after 11 months of incubation; (C) difference between months 0 and 11.
The removal of chlorines at the ortho and meta + para positions, shown as the average number of chlorines per biphenyl residue in the HR cultures spiked with Aroclor 1242, is illustrated in Figure 2. There was no apparent ortho dechlorination in any of the sediment cultures, and the sterile controls remained unchanged (Figure 2C). Sediment cultures with the addition if fatty acid mixture, 1.6
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Flgure 2. Removal of chlorines from the ortho (0)and meta ipara (0)positions in Hudson River sediment cultures with Arocior 1242: (A) with fatty acids added as a carbon source: (B) without fatty acid addition: (C) sterile control.
534 Envlron. Sci. Technol., Vol. 27, No. 3, 1993
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Flgure 3. Qas chromatograms of preexlstlng PCBs after an Incubation time of 0 and 11 months in sediment cultures from New Bedford Harbor.
qualitatively and quantitatively very similar. Figure 3 shows the gas chromatograms of the preexisting PCBs at time 0 and 11 months from NBH. The change in the preexisting PCB congener distribution is illustrated in Figure 4. The mole percents of each chromatographic peak are shown at the initiation of the incubation, designated month 0 (Figure 4A) and after 11 months of incubation (Figure 4B). The differences, as shown by peaks which increased and which decreased from month 0 to 11, are illustrated in Figure 4C. In these cultures, we observed a decrease of tetra-, penta-, and hexachlorobiphenyls and an accumulation of di-, tri-, and tetrachlorobiphenyls, mainly 2-3-CB (6, +1.8%), 23-CB/2-4-CB (5/8, +1.7%), 25-2-CB/4-4-CB (18/ 15, +4.0%),236-CB/26-3-CB (24/27, +1.7%), 25-3-CB (26, +2.1%), 24-3-CB (25, +2.3%), and 24-4-CB/25-4-CB (28/31, +1.7%). A notable aspect was the accumulation of the tetrachlorobiphenyls 25-25-CB/ 26-35-CB (52/73, +1.6%) and 24-25-CB (49, +1.0%). A decrease of mainly 34-2-CB/234-CB/23-3-CB/25-26-CB (33/21/20/53, -1.3%), 23-25-CB (44, -1.9%), 34-4-CB/ 23-24-CB/236-3-CB (37/42/59, -1.2%), 25-34-CB/345-2CB (70/76, -2.1%), 24-34-CB/236-25-CB/245-26-CB (66/95/102, -1.7761, and 23-34-CB/234-4-CB (56/60, -1.1%) was observed. This activity can be assigned to a conversion of 34-chlorophenyl (CP) groups to 3-CPs, 23CPs to 2-CP, 245-CPs to 25-CPs, and 234-CPs to either 24-CPs or 23-CPs. The extent of dechlorination did not significantly change with incubation of up to 16 months (not shown). The PCB homologue distribution in the NBH sediment cultures at month 0 and at month 11 are summarized in Table 11. A similar homologue pattern and extent of dechlorination was observed for both the preexisting and for the added Aroclor in these cultures. Although the di- and
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Environ. Sci. Technol., Vol. 27, No. 3, 1993 535
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Flgure 5. Removal of chlorlnes from the ortho (0) and meta para (e)positions in New Bedford Harbor sediment cultures with preexisting PCBs: (A) with fatty acids added as a carbon source; (B) without fatty acid additlon; (C) sterile control with added Aroclor 1242.
the ortho position was detected, and sterile controls remained unchanged. Dechlorination of PCBs was not observed under sulfate-reducing conditions for any of the cultures with up to 17-months incubation. In contrast to the HR sediment, the NBH sediment is rich in degradable organic carbon since a high level of methane was noted in our background cultures to which no additions were made. Available organic carbon, therefore, did not appear to be limiting in this sediment and a source of electron donors for reductive dechlorination was present which precluded the need for the addition of fatty acids. Dechlorination of the preexisting PCBs in situ may not have progressed in this sediment since sulfate, present in marine environment, inhibited dechlorination activity. The overall rate and extent of PCB dechlorination in NBH cultures were less than in HR. The reasons for this are unclear at this time but may be due to (1) differences in the microbial populations, (2) inhibition by components in the sediment, e.g., high levels of oils (16) or heavy metals (see Experimental Section), and/or (3) bioavailability due to differences in the sediment matrix. Considerable differences in the rates and extent of dechlorination have been observed between sampling sites and within sediment cores in New Bedford Harbor (36). Silver Lake. Silver Lake (SL) sediment cultures were established with and without Aroclor 1260 and with and without amendments of fatty acids. Dechlorination was observed to be slower than that for the HR cultures. Similar to what was observed for the HR cultures, however, no dechlorination was noted for the preexisting PCBs in the sediment, Under methanogenic conditions, the cultures with added PCBs showed loss of tetra- through heptachlorobiphenyls with accumulation of some tri- and tetrachlorobiphenyls which were not present in the original spiked Aroclor 1260. Figure 6 illustrates the mole percent of each chromatographic peak in cultures to which Aroclor 1260 and fatty acids were added. Month 0 is shown in Figure 6A and month 11in Figure 6B, and the difference between the two time points in the congener profile as seen by increasing and decreasing peak is shown in Figure 6C. The changes show mainly a decrease of 2434-CB/236-25-CB/24526-CB (66/95/102, -2.4%), 245-25-CB/235-24-CB (101/90, -1.6%), 34-34CB/236-34-CB (77/110, -1.3%), 2356-25CB (151, -1.3 %), 235-236-CB/345-25-CB/2346-25-CB (135/ 124/144, -1.5%), 236-245-CB/2345-3-CB/345-24-CB (149/ 106/ 123, -3.8%), 234-236-CB/234-34-CB/245-245CB (132/105/153, -4.5%), 2345-25-CB/2356-236-CB (141/179, -1.6%), 234-245-CB/2356-34-CB (138/163, -4.4%), 2356-245-CB/236-246-CB (187/182, -1.2%), 2345-236-CB/23456-24-CB(174/181, -1.3%), 2345-245-CB (180, -2.7%), and 2345-234-CB/23456-34-CB (170/190, 536
Environ. Sci. Technoi., Voi. 27, No. 3, 1993
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Flgure 6. Mole percent of PCBs in Silver Lake sediment cultures with added Aroclor 1260 (400 ppm) and fatty acMs as an auxiliary carbon source: (A) at month 0; (8) after 11 months of incubation; (C) difference between months 0 and 11.
-1.4%) and an increase of mainly the following peaks: 23-2-CB/26-4-CB (16/32, +2.4%) 245-CB (29, +1.3%), 34-2-CB/234-CB/23-3-CB/25-26-CB (33/21/20/53, +4.0%), 23-4-CB/24-26-CB (22/51, +5.8%),24-25-CB (49, +3.9%), 2424CB/245-2-CB/246-4-CB (47/48/75, +12%), and 246-24-CB/245-3-CB (100/67, +3.0%). Note that
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Figure 7. Removal of chlorines from the ortho (0)and meta 4- para (0)positions In Sliver Lake sediment cultures with Aroclor 1200: (A) with fatty acids added as a carbon source: (B) sterile control.
nearly all enriched tetrachlorobiphenyls had a 24 substitution pattern, indicating a preference for a meta dechlorination. In contrast to the cultures of NBH sediment no enrichment of 25-25-CB/26-35-CB (52/73) was observed, but instead, an accumulation of 24-25-CB (491, 24-26-CB (51), and 24-24-CB (47) as coeluting peaks. Dechlorination continued up to month 17, at which time monitoring was stopped. The PCB homologue distribution for all the SL sediment cultures are summarized in Table I1 for month 0 and month 11. SL cultures with added PCBs show an increase of tri- and tetrachlorobiphenyls and a decrease of penta-, hexa-, and heptachlorobiphenyl congeners. No activity was noted for the preexisting PCBs, and no dechlorination of the mono- or dichlorobiphenyl congeners was observed. Figure 7 illustrates chlorine removal from the ortho and meta + para positions over time for SL cultures with Aroclor 1260 and amended with fatty acids. No change in the sterile controls occurred over 11 months of incubation. In the active cultures, however, by the end of month 11,nearly 30% of the meta + para chlorines was removed. The average number of meta para chlorines per biphenyl molecule decreased from about 2.9 to 2.1 over the incubation period. As in NBH sediment cultures, addition of an auxiliary carbon source had no effect on the dechlorination in SL sediment cultures. High background levels of methane were also noted in these cultures suggesting that available carbon is not limiting and additional carbon provided no benefit. Similar to the other two sediment sources, sulfate-reducing conditions yielded no activity.
+
Conclusions The numerous dechlorination patterns for PCBs observed in different sediments and sites (8-10,14-17) reflect
the complexities of the process. Yet to be understood are the effects and the importance of different biological factors and their interactions with physical-chemical factors at the different sites. It has been suggested that different microbial populations may be responsible for generating specific dechlorination patterns (15). Recent studies, indeed, support this view and show that pasteurized and ethanol-treated sediment select for organisms which mediate dechlorination at the meta position, suggesting that spore formers are important in this process (37). These treatments also eliminated the para-dechlorinating activity. Thus, position and congener specificity may be population specific and these populations can have different environmental needs. The sites examined in this study are different ecosystems, a river, an estuary, and a small lake. Each site also has different contaminant conditions, different Aroclors, metal concentrations, and cocontaminants such as oil and grease (16). Available organics also vary, and all these factors will affect the microbial population and activity. Physical-chemical factors are also numerous and varied at each of these sites. For example, adsorption of PCBs to sediment matrices may play an important role by decreasing bioavailability. Sorption processes have been shown to affect biodegradation of organic chemicals in aqueous systems (e.g., ref 38 and literature therein) as well as to serve as a mechanism for a volatile solute to be entrapped in soil micropores as a consequence of long exposure time and high initial concentrations (39). On the other hand, the presence of oils and grease may increase bioavailability to microbes by enhancing desorption during the shaking to disperse the added Aroclors when the cultures are prepared for sampling. The congener octanol/water partition coefficients (log Kow)of PCBs range from an average of 4.5 for the mono-CBs to 8.1 for the hepta-CBs (40), so that nearly all PCBs should be associated with the particulate phase. Thus, it would seem that differences of the matrix would play a less important role in affecting bioavailability of PCBs and the biotransformation kinetics could be the major rate-limiting step for determining the fate of different congeners in different sediments. Despite site and sediment differences, a dechlorinating microbial population is present in all three environments investigated. This is evidenced by the observation that dechlorination to some degree took place in the samples with the added Aroclors with all three sediments. In the Hudson River, the dechlorination pattern of the preexisting PCBs resembles that of added Aroclor 1242 in our samples after dechlorination had taken place. This supports the observation of others (14,15) and the reasoning that the preexisting PCBs in the Hudson River have already undergone extensive reductive dechlorination in situ (8-10). The preexisting PCBs in New Bedford Harbor did undergo dechlorination in the laboratory, suggesting that conditions in situ are adverse to the microbial process observed in the laboratory. It may be that a continuous input of sulfate with tidal flushing inhibits dechlorination. Morris et al. (41) recently reported that addition of sulfate to PCB-dechlorinating methanogenic consortia from HR inhibited dechlorinating activity. Our observations that no PCB dechlorination occurred with the sulfidogenic cultures established with HR, NBH, or SL sediments support this point. In addition, the total measured heavy metal concentration was more than 3 times higher in NBH (2625pg/g of sediment) than in HR (858 pg/g). The Silver Lake samples yielded no activity on the preexisting PCBs and indeed may be limited by abiotic factors such as heavy Envlron. Sci. Technol., Vol. 27, No. 3, 1993 537
metals (8280pg/g of sediment) and mineral oils. Reductive dechlorination is a slow process; added PCBs under laboratory conditions show half-lives of several weeks to months, while under field conditions, half-lives of months up to several years of the existing PCBs are estimated (7, 36). Our experiments were designed to provide optimal conditions and all necessary nutrients for microbial activity and thus may not reflect the activity in the environment. Since no ortho dechlorination was observed and virtually all congeners present in technical PCBs have at least one ortho chlorine, complete PCB dechlorination did not occur. The only anaerobic ortho dechlorination reported has been observed with the addition of the single congener 2,3,5,6-tetrachlorobiphenyl (19). Enhancement of these anaerobic microbial transformations will require further knowledge not only of the microbiology but also of the physical-chemical factors such as adsorption and desorption which can affect the microbiology. Acknowledgments
We thank Paul Capel (US. Geological Survey, St. Paul, MN) for his suggestions on the PCB analytics, Mark Harkness (General Electric Co., Schenectady, NY)for the measurements of the organic material in the New Bedford Harbor sediment, John F. Brown, Jr., (General Electric Co., Schenectady, NY) for helpful comments on the dechlorination patterns, Katherine Squibb (New York University Medical Center, Institute of Environmental Medicine, Tuxedo, NY) for the metal analysis, and Maire Heikkinen for help with statistics. Literature C i t e d Jensen, S. New. Sci. 1966, 32, 612. Eisenreich, S. J. In Sources and Fates of Aquatic Pollutants; Hites, R. A., Eisenreich, S. J., Eds.; Advances in Chemistry 216; American Chemical Society: Washington, DC, 1987; pp 394-469. Rapaport, R. A.; Eisenreich, S. J. Enuiron. Sci. Technol. 1988,22, 931-941. McKinney, J. D.; Chae, K.; McConnell, E. E.; Birnbaum, L. S. Enuiron. Health Perspect. 1985, 60, 57-68. McFarland, V. A,; Clarke, J. U. Enuiron. Health Perspect. 1989,81, 225-239. Larsson, P. Nature 1985,317, 347-349. Eisenreich, S. J.; Capel, P. D.; Robbins, J. A,; Bourbonniere, R. Enuiron. Sci. Technol. 1989, 23, 1116-1126. Brown, J. F., Jr.; Wagner, R. E.; Bedard, D. L.; Brennan, M. J.; Carnahan, J. C.; May, R. J. Northeast. Enuiron. Sci. 1984, 3, 167-179. Brown, J. F., Jr.; Bedard, D. L.; Brennan, M. J.; Carnahan, J. C.; Feng, H.; Wagner, R. E. Science 1987,236,709-712. Brown, J. F., Jr.; Wagner, R. E.; Feng, H.; Bedard, D. L.; Brennan, M. J.; Carnahan, J. C.; May, R. J. Enuiron. Toxicol. Chem. 1987,6, 579-593. Bush, B.; Shane, L. A.; Wahlen, M.; Brown, M. P. Chemosphere 1987, 16, 733-744. Brown. M. P.; Bush, B.; Rhee, G. Y.; Shane, L. Science 1988, 240, 1674-1675. Brown, J. F., Jr.; Wagner, R. E.; Bedard, D. L. Science 1988, 240, 1675-1676.
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Received for review June 29,1992. Revised manuscript received November 2,1992. Accepted November 3,1992. This work was supported in part by E A WAG (A.C.A.), General Electric Co., and the U.S. Environmental Protection Agency.
