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Invariant chlorine isotopic signatures during microbial PCB reductive dechlorination
Environmental Pollution 128 (2004) 445–448 www.elsevier.com/locate/envpol
Short communication
Invariant chlorine isotopic signatures during microbial PCB reductive dechlorination Nicholas J. Drenzeka,*, Timothy I. Eglintona, Carl O. Wirsenb, Neil C. Sturchioc, Linnea J. Heratyc, Kevin R. Sowersd, Qingzhong Wue, Harold D. Maye, Christopher M. Reddya a
Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA b Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA c Department of Earth and Environmental Sciences, University of Illinois, Chicago, IL 60607, USA d Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA e Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29412, USA Received 4 March 2003; accepted 12 September 2003
‘‘Capsule’’: No chlorine isotopic fractionation was observed for the microbial reductive dechlorination of 2,3,4,5-tetrachlorobiphenyl. Abstract In order to develop more robust insight into the natural attenuation of polychlorinated biphenyls (PCBs), the chlorine isotopic composition of residual 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB) was monitored as it underwent microbial reductive dechlorination to 2,3,5-trichlorobiphenyl (2,3,5-CB) in laboratory cultures. Reverse-phase high performance liquid chromatography (HPLC) was employed to isolate the former compound from the experimental matrix for d37Cl measurement. No detectable isotopic fractionation was observed over the 90 day incubation with sterile control, standard, and inoculated samples all exhibiting d37Cl values with a range of 0.5%. These results show that this type of biological activity can be discriminated from other transformations by the absence of a measurable isotope effect during microbial reductive dechlorination. The utility of HPLC isolation for compoundspecific d37Cl analyses of environmentally relevant species is also demonstrated. # 2003 Elsevier Ltd. All rights reserved. Keywords: Polychlorinated biphenyls; Chlorine isotopes; Reductive dechlorination; Organic contaminants; Bioremediation
1. Introduction The natural attenuation of polychlorinated biphenyls (PCBs) is increasingly becoming a subject of intense interest as a possible in situ remediation mechanism. In the United States, these compounds were manufactured as mixtures (called Aroclors) of 209 distinct congeners for use in industrial applications throughout a majority of the twentieth century (Erickson, 1997). Their contemporaneous and subsequent release into the environment is of substantial concern to native ecosystems as well as proximal human inhabitants. A suite of evidence already exists for the activity of aerobic and anaerobic * Corresponding author. Tel.: +1-508-289-3741; fax: +1-508-4572164. E-mail address: [email protected] (N.J. Drenzek). 0269-7491/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2003.09.006
bioattenuation by indigenous bacterial populations in natural settings (Bedard and May, 1996; Brown et al., 1984, 1987; Lake et al., 1992; reviewed by Wiegel and Wu, 2000). In anaerobic environments, PCBs have been shown to undergo microbial reductive dechlorination, wherein a chlorine is removed from one of the biphenyl rings and substituted with hydrogen (Abramowicz, 1990; Quensen et al., 1990; Wu et al., 2000). This process can repeat several times, resulting in extensively dechlorinated products that may then be susceptible to wholesale ring cleavage when exposed to oxic conditions during diffusive flux or bioturbation in sedimentary systems (Abramowicz, 1990; Master et al., 2002). Yet these reactions are difficult to resolve from other chemically discriminating processes such as abiotic degradation, evaporation, and sorption. Isotopic characterization of these and other reactions, however,
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has been demonstrated to be a promising technique in identifying, resolving, and quantifying these processes in contaminated systems (Drenzek et al., 2001; Jendrzejewski et al., 2001; Kelley et al., 1997; Numata et al., 2002; Sherwood Lollar et al., 2001). Chlorine isotopic analysis, conventionally reported in the delta notation, d37Cl [referenced here to standard mean ocean chloride (SMOC), with a 37Cl/35Cl value of 0.318988 (Rosenbaum et al., 2000)], has been successfully employed to assign synthetic origin and assess environmental fate of more volatile organic contaminants (VOCs) (Beneteau et al., 1999; Heraty et al., 1999; Huang et al., 1999; Jendrzejewski et al., 2001; Poulson and Drever, 1999) in addition to an array of other compounds (Drenzek et al., 2002; Lewandowicz et al., 2001; Reddy et al., 2002a,b). Thus, in order to more robustly characterize the existence and extent of the reductive dechlorination of PCBs in a particular system, we investigated the chlorine isotopic composition during the selective dechlorination of 2,3,4,5-tetrachlorobiphenyl (2,3,4,5CB) to 2,3,5-trichlorobiphenyl (2,3,5-CB). This particular substrate was chosen because tetra-, penta-, and hexa-chlorobiphenyl congeners constitute a significant fraction of the mass in most Aroclors and are relatively susceptible to in situ reductive dechlorination, and because 2,3,4,5-CB can be dechlorinated by a bacterial co-culture in defined medium without sediment (Wiegel and Wu, 2000; Wu et al., 2000).
2. Materials and methods A detailed procedure can be found elsewhere (Drenzek et al., 2001; Wu et al., 2002). Briefly, anaerobic culture tubes containing 15 ml of a pre-sterilized, sedimentfree, aqueous media (E-Cl) were inoculated with a mixed culture containing the highly enriched PCB dechlorinating bacterium DF-1, specific for removing doubly flanked chlorines under anoxic conditions. Sterile control tubes received no cell inoculum. Fifteen microliters of a 67 mg/ml solution of 2,3,4,5-CB (AccuStandard lot no. 981007LB-AC) in acetone was also introduced to the cultures, which were subsequently sealed under 4:1 N2:CO2 and incubated at 30 C in the dark. Two or three cultures were harvested at different periods over the course of 3 months and extracted with methyl-tert-butyl ether (MTBE). Reaction progress was monitored using a gas chromatograph equipped with a flame ionization detector (GC–FID). Selected extracts were then rotary evaporated to 100 ml, with the remaining MTBE allowed to passively evaporate under ambient conditions. The sample was immediately re-dissolved in 100 ml of acetone and the tube was rinsed once with an additional 100 ml of acetone. Both washes were pooled in 1.5-ml autoanalysis vials. Residual 2,3,4,5-CB was quantitatively
isolated on an Agilent 1100 Series high pressure liquid chromatograph (HPLC) equipped with a photodiode array detector operating under reverse-phase conditions. A Chrompack Chromsep Omnipher 5 C18 column (2504.6 mm), thermostated at 25 C, with an isocratic mobile phase of 90:10 acetonitrile:water achieved optimum peak resolution at a flow rate of 1.0 ml/min and a 100 ml injection volume. Separate reservoirs of isopropyl alcohol were injected after every sample injection in order to rinse the injector needle and flush the column, thereby eliminating memory effects. Continuous UV/vis spectra were acquired between wavelengths of 190 and 400 nm. Several compounds were detected, including the PCB congeners of interest and the culture vitamins. The HPLC flow downstream of the detector was diverted from the waste reservoir to 15-ml glass vials during the 2,3,4,5-CB elution interval and subsequently backextracted into pentane (2500 ml) by sonication in the presence of 5 ml ultra-pure, deionized water. Small (15 ml) aliquots were removed to confirm purity by GC– FID. A standard solution of 2,3,4,5-CB was prepared independently and measured for d37Cl in order to assess possible isotopic fractionation associated with the isolation procedure. The remaining material was transferred to pre-combusted 12 mm (I.D.)22 cm (length) Pyrex tubes, generally yielding 2 to 6 mmol of chlorine. The pentane was removed under a stream of nitrogen with 1–2 g of pre-combusted copper oxide subsequently added to each tube. The d37Cl analyses were then performed according to Holt et al. (1997), with a procedural error of 0.15%.
3. Results and discussion Approximately 90% conversion of 2,3,4,5-CB to 2,3,5-CB occurred over the 90-day incubation period, with the chlorine isotopic composition of residual 2,3,4,5-CB remaining relatively constant at 0.55 0.27% (n=9) over this interval (Fig. 1). These results are analogous to an absence of a carbon isotope effect for the same reaction reported previously (Drenzek et al., 2001), and are partly a function of mass spectrometric signal dilution by the three non-participating chlorines (i.e. those at the ortho- and meta-positions). 2,3,4,5-CB was the only compound isotopically monitored during this process as the only other species involved in the isotope dynamics was the evolved Cl , which especially in estuarine settings would prove impossible to independently discriminate from the much larger standing inventory of unaffiliated chloride. Additionally, there is no substantial difference in d37Cl values of the 2,3,4,5-CB standard ( 0.39%) and control samples ( 0.36 and 0.90%) (Fig. 1), indicating that minimal chlorine isotopic fractionation occurred during the quantitative isolation procedure. This
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4. Conclusions Bacterially mediated reductive dechlorination of 2,3,4,5-CB to 2,3,5-CB does not exhibit measurable chlorine isotopic fractionation, allowing for the potential to differentiate physiochemical processes from this important biological counterpart in contaminated settings. Moreover, HPLC has been established as an effective tool for quantitatively isolating single PCB congeners for compound-specific chlorine isotopic analysis from more complex matrices. Further investigation into the bulk and compound-specific isotopic characteristics in both unaltered and environmentally transformed Aroclor mixtures should help to more fully elucidate the ultimate fate of these potent compounds.
Fig. 1. Chlorine isotopic composition of microbial culture and sterile control 2,3,4,5-CB samples as a function of remaining substrate. Controls correspond to the time points at which 0 and 80% of the 2,3,4,5-CB in the cultures had been dechlorinated. The horizontal line denotes the d37Cl value of the 2,3,4,5-CB standard (not isolated via prep-HPLC). All d37Cl values represent the mean of four individual mass spectrometric measurements on each sample (instrumental error bars are smaller than their respective symbols).
demonstrates the effectiveness of reverse-phase HPLC as a preparative tool for isolating hydrocarbons destined for compound-specific chlorine isotopic measurements from experimental matrices, analogous to results reported for the preparation of various samples for carbon and nitrogen isotopic analyses (Bidigare et al., 1991; Caimi and Brenna, 1997; Kenig et al., 2000; Sachs and Repeta, 2000). If the maintenance of isotopic composition is symptomatic of biological reductive dechlorination reactions operating on other PCB congeners, then the depletion in bulk d37Cl compositions of altered Aroclors extracted from highly contaminated sediments (Reddy et al., 2000) suggests the activity of different environmental processes. This notion is further supported by invariant d37Cl values for a suite of individual congeners measured within Aroclors themselves (Xu et al., unpublished data), as this precludes the possibility that bulk depletions in environmental samples are simply a result of modified internal isotopic mass balance. In other words, the bulk d37Cl composition should be insensitive to the preferential loss, for example, of the less chlorinated (more soluble) congeners before sedimentary deposition. Rather, other factors that might produce an isotope effect such as sequential phase partitioning or chemical destruction are implicated. A means is thus provided to discriminate between alteration profiles produced from abiotic weathering (depleted bulk d37Cl) and reductive dechlorination (unaffected bulk d37Cl).
Acknowledgements N.J.D. acknowledges current support from the Stanley W. Watson Foundation and the National Science Foundation’s Research Experience for Undergraduates program (REU) while a Student Fellow at WHOI. This work was also funded by WHOI EPA Grant (R82816101) to C.M.R., the US Office of Naval Research, Harbor Processes Program, U.S. Department of Defense (N00014-99-1-0078 to H.D.M. and N00014-991-0101 to K.R.S.), and a Rinehart Coastal Research Center grant to C.M.R. This is WHOI contribution no. 11041.
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stable carbon isotopic fractionation during the reductive dechlorination of polychlorinated biphenyls. Environmental Science and Technology 35 (16), 3310–3313. Drenzek, N.J., Tarr, C.H., Eglinton, T.I., Heraty, L.J., Sturchio, N.C., Shiner, V.J., Reddy, C.M., 2002. Stable chlorine and carbon isotopic compositions of selected semi-volatile organochlorine compounds. Organic Geochemistry 33 (4), 437–444. Erickson, M.D., 1997. Analytical Chemistry of PCBs, second ed. Lewis Publishers, Boca Raton, FL. Heraty, L.J., Fuller, M.E., Huang, L., Abrajano Jr., T., Sturchio, N.C., 1999. Isotopic fractionation of carbon and chlorine by microbial degradation of dichloromethane. Organic Geochemistry 30 (8), 793–799. Holt, B.D., Sturchio, N.C., Abrajano, T.A., Heraty, L.J., 1997. Conversion of chlorinated volatile organic compounds to carbon dioxide and methyl chloride for isotopic analysis of carbon and chlorine. Analytical Chemistry 69 (14), 2727–2733. Huang, L., Sturchio, N.C., Abrajano Jr., T., Heraty, L.J., Holt, B.D., 1999. Carbon and chlorine isotope fractionation of chlorinated aliphatic hydrocarbons by evaporation. Organic Geochemistry 30 (8), 777–785. Jendrzejewski, N., Eggenkamp, H.G.M., Coleman, M.L., 2001. Characterization of chlorinated hydrocarbons from chlorine and carbon isotopic compositions: scope of application to environmental problems. Applied Geochemistry 16 (9–10), 1021–1031. Kelley, C.A., Hammer, B.T., Coffin, R.B., 1997. Concentrations and stable isotope values of BTEX in gasoline-contaminated groundwater. Environmental Science and Technology 31 (9), 2469–2472. Kenig, F., Popp, B.N., Summons, R.E., 2000. Preparative HPLC with ultrastable-Y zeolite for compound-specific carbon isotopic analyses. Organic Geochemistry 31 (11), 1087–1094. Lake, J.L., Pruell, R.J., Osterman, F.A., 1992. An examination of dechlorination processes and pathways in New Bedford Harbor sediments. Marine Environmental Research 33 (1), 31–47. Lewandowicz, A., Rudzin˜ski, J., Tronstad, L., Widersten, M., Ryberg, P., Matsson, O., Paneth, P., 2001. Chlorine kinetic isotope effects on the haloalkane dehalogenase reaction. Journal of the American Chemical Society 123 (19), 4550–4555. Master, E.R., Lai, V.W.-M., Kuipers, B., Cullen, W.R., Mohn, W.W., 2002. Sequential anaerobic-aerobic treatment of soil contaminated with weathered Aroclor 1260. Environmental Science and Technology 36 (1), 100–103. Numata, M., Nakamura, N., Koshikawa, H., Terashima, Y., 2002. Chlorine isotope fractionation during reductive dechlorination of
chlorinated ethenes by anaerobic bacteria. Environmental Science and Technology 36 (20), 4389–4394. Poulson, S.R., Drever, J.I., 1999. Stable isotope (C, Cl, and H) fractionation during vaporization of trichloroethylene. Environmental Science and Technology 33 (20), 3689–3694. Quensen III, J.F., Boyd, S.A., Tiedje, J.M., 1990. Dechlorination of four commercial polychlorinated biphenyl mixtures (Aroclors) by anaerobic microorganisms from sediments. Applied and Environmental Microbiology 56 (8), 2360–2369. Reddy, C.M., Heraty, L.J., Holt, B.D., Sturchio, N.C., Eglinton, T.I., Drenzek, N.J., Xu, L., Lake, J.L., Maruya, K.A., 2000. Stable chlorine isotopic compositions of Aroclors and Aroclor-contaminated sediments. Environmental Science and Technology 34 (13), 2866–2870. Reddy, C.M., Drenzek, N.J., Eglinton, T.I., Heraty, L.J., Sturchio, N.C., Shiner, V.J., 2002a. Stable chlorine intramolecular kinetic isotope effects from the abiotic dehydrochlorination of DDT. Environmental Science and Pollution Research 9 (3), 183–186. Reddy, C.M., Xu, L., Drenzek, N.J., Sturchio, N.C., Heraty, L.J., Kimblin, C., Butler, A., 2002b. A chlorine isotope effect for enzymecatalyzed chlorination. Journal of the American Chemical Society 124 (49), 14526–14527. Rosenbaum, J.M., Cliff, R.A., Coleman, M.L., 2000. Chlorine stable isotopes: a comparison of dual inlet and thermal ionization mass spectrometric measurements. Analytical Chemistry 72 (10), 2261– 2264. Sachs, J.P., Repeta, D.J., 2000. The purification of chlorins from marine particles and sediments for nitrogen and carbon isotopic analysis. Organic Geochemistry 31 (4), 317–329. Sherwood Lollar, B., Slater, G.F., Sleep, B., Witt, M., Klecka, G.M., Harkness, M., Spivack, J., 2001. Stable carbon isotope evidence for intrinsic bioremediation of tetrachloroethene and trichloroethene at Area 6, Dover Air Force Base. Environmental Science and Technology 35 (2), 261–269. Wiegel, J., Wu, Q., 2000. Microbial reductive dehalogenation of polychlorinated biphenyls. FEMS Microbiology Ecology 32 (1), 1–15. Wu, Q., Sowers, K.R., May, H.D., 2000. Establishment of a polychlorinated biphenyl-dechlorinating microbial consortium, specific for doubly flanked chlorines, in a defined, sediment-free medium. Applied and Environmental Microbiology 66 (1), 49–53. Wu, Q., Watts, J.E.M., Sowers, K.R., May, H.D., 2002. Identification of a bacterium that specifically catalyzes the reductive dechlorination of polychlorinated biphenyls with doubly flanked chlorines. Applied and Environmental Microbiology 68 (2), 807–812.
Short communication
Invariant chlorine isotopic signatures during microbial PCB reductive dechlorination Nicholas J. Drenzeka,*, Timothy I. Eglintona, Carl O. Wirsenb, Neil C. Sturchioc, Linnea J. Heratyc, Kevin R. Sowersd, Qingzhong Wue, Harold D. Maye, Christopher M. Reddya a
Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA b Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA c Department of Earth and Environmental Sciences, University of Illinois, Chicago, IL 60607, USA d Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA e Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29412, USA Received 4 March 2003; accepted 12 September 2003
‘‘Capsule’’: No chlorine isotopic fractionation was observed for the microbial reductive dechlorination of 2,3,4,5-tetrachlorobiphenyl. Abstract In order to develop more robust insight into the natural attenuation of polychlorinated biphenyls (PCBs), the chlorine isotopic composition of residual 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB) was monitored as it underwent microbial reductive dechlorination to 2,3,5-trichlorobiphenyl (2,3,5-CB) in laboratory cultures. Reverse-phase high performance liquid chromatography (HPLC) was employed to isolate the former compound from the experimental matrix for d37Cl measurement. No detectable isotopic fractionation was observed over the 90 day incubation with sterile control, standard, and inoculated samples all exhibiting d37Cl values with a range of 0.5%. These results show that this type of biological activity can be discriminated from other transformations by the absence of a measurable isotope effect during microbial reductive dechlorination. The utility of HPLC isolation for compoundspecific d37Cl analyses of environmentally relevant species is also demonstrated. # 2003 Elsevier Ltd. All rights reserved. Keywords: Polychlorinated biphenyls; Chlorine isotopes; Reductive dechlorination; Organic contaminants; Bioremediation
1. Introduction The natural attenuation of polychlorinated biphenyls (PCBs) is increasingly becoming a subject of intense interest as a possible in situ remediation mechanism. In the United States, these compounds were manufactured as mixtures (called Aroclors) of 209 distinct congeners for use in industrial applications throughout a majority of the twentieth century (Erickson, 1997). Their contemporaneous and subsequent release into the environment is of substantial concern to native ecosystems as well as proximal human inhabitants. A suite of evidence already exists for the activity of aerobic and anaerobic * Corresponding author. Tel.: +1-508-289-3741; fax: +1-508-4572164. E-mail address: [email protected] (N.J. Drenzek). 0269-7491/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2003.09.006
bioattenuation by indigenous bacterial populations in natural settings (Bedard and May, 1996; Brown et al., 1984, 1987; Lake et al., 1992; reviewed by Wiegel and Wu, 2000). In anaerobic environments, PCBs have been shown to undergo microbial reductive dechlorination, wherein a chlorine is removed from one of the biphenyl rings and substituted with hydrogen (Abramowicz, 1990; Quensen et al., 1990; Wu et al., 2000). This process can repeat several times, resulting in extensively dechlorinated products that may then be susceptible to wholesale ring cleavage when exposed to oxic conditions during diffusive flux or bioturbation in sedimentary systems (Abramowicz, 1990; Master et al., 2002). Yet these reactions are difficult to resolve from other chemically discriminating processes such as abiotic degradation, evaporation, and sorption. Isotopic characterization of these and other reactions, however,
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has been demonstrated to be a promising technique in identifying, resolving, and quantifying these processes in contaminated systems (Drenzek et al., 2001; Jendrzejewski et al., 2001; Kelley et al., 1997; Numata et al., 2002; Sherwood Lollar et al., 2001). Chlorine isotopic analysis, conventionally reported in the delta notation, d37Cl [referenced here to standard mean ocean chloride (SMOC), with a 37Cl/35Cl value of 0.318988 (Rosenbaum et al., 2000)], has been successfully employed to assign synthetic origin and assess environmental fate of more volatile organic contaminants (VOCs) (Beneteau et al., 1999; Heraty et al., 1999; Huang et al., 1999; Jendrzejewski et al., 2001; Poulson and Drever, 1999) in addition to an array of other compounds (Drenzek et al., 2002; Lewandowicz et al., 2001; Reddy et al., 2002a,b). Thus, in order to more robustly characterize the existence and extent of the reductive dechlorination of PCBs in a particular system, we investigated the chlorine isotopic composition during the selective dechlorination of 2,3,4,5-tetrachlorobiphenyl (2,3,4,5CB) to 2,3,5-trichlorobiphenyl (2,3,5-CB). This particular substrate was chosen because tetra-, penta-, and hexa-chlorobiphenyl congeners constitute a significant fraction of the mass in most Aroclors and are relatively susceptible to in situ reductive dechlorination, and because 2,3,4,5-CB can be dechlorinated by a bacterial co-culture in defined medium without sediment (Wiegel and Wu, 2000; Wu et al., 2000).
2. Materials and methods A detailed procedure can be found elsewhere (Drenzek et al., 2001; Wu et al., 2002). Briefly, anaerobic culture tubes containing 15 ml of a pre-sterilized, sedimentfree, aqueous media (E-Cl) were inoculated with a mixed culture containing the highly enriched PCB dechlorinating bacterium DF-1, specific for removing doubly flanked chlorines under anoxic conditions. Sterile control tubes received no cell inoculum. Fifteen microliters of a 67 mg/ml solution of 2,3,4,5-CB (AccuStandard lot no. 981007LB-AC) in acetone was also introduced to the cultures, which were subsequently sealed under 4:1 N2:CO2 and incubated at 30 C in the dark. Two or three cultures were harvested at different periods over the course of 3 months and extracted with methyl-tert-butyl ether (MTBE). Reaction progress was monitored using a gas chromatograph equipped with a flame ionization detector (GC–FID). Selected extracts were then rotary evaporated to 100 ml, with the remaining MTBE allowed to passively evaporate under ambient conditions. The sample was immediately re-dissolved in 100 ml of acetone and the tube was rinsed once with an additional 100 ml of acetone. Both washes were pooled in 1.5-ml autoanalysis vials. Residual 2,3,4,5-CB was quantitatively
isolated on an Agilent 1100 Series high pressure liquid chromatograph (HPLC) equipped with a photodiode array detector operating under reverse-phase conditions. A Chrompack Chromsep Omnipher 5 C18 column (2504.6 mm), thermostated at 25 C, with an isocratic mobile phase of 90:10 acetonitrile:water achieved optimum peak resolution at a flow rate of 1.0 ml/min and a 100 ml injection volume. Separate reservoirs of isopropyl alcohol were injected after every sample injection in order to rinse the injector needle and flush the column, thereby eliminating memory effects. Continuous UV/vis spectra were acquired between wavelengths of 190 and 400 nm. Several compounds were detected, including the PCB congeners of interest and the culture vitamins. The HPLC flow downstream of the detector was diverted from the waste reservoir to 15-ml glass vials during the 2,3,4,5-CB elution interval and subsequently backextracted into pentane (2500 ml) by sonication in the presence of 5 ml ultra-pure, deionized water. Small (15 ml) aliquots were removed to confirm purity by GC– FID. A standard solution of 2,3,4,5-CB was prepared independently and measured for d37Cl in order to assess possible isotopic fractionation associated with the isolation procedure. The remaining material was transferred to pre-combusted 12 mm (I.D.)22 cm (length) Pyrex tubes, generally yielding 2 to 6 mmol of chlorine. The pentane was removed under a stream of nitrogen with 1–2 g of pre-combusted copper oxide subsequently added to each tube. The d37Cl analyses were then performed according to Holt et al. (1997), with a procedural error of 0.15%.
3. Results and discussion Approximately 90% conversion of 2,3,4,5-CB to 2,3,5-CB occurred over the 90-day incubation period, with the chlorine isotopic composition of residual 2,3,4,5-CB remaining relatively constant at 0.55 0.27% (n=9) over this interval (Fig. 1). These results are analogous to an absence of a carbon isotope effect for the same reaction reported previously (Drenzek et al., 2001), and are partly a function of mass spectrometric signal dilution by the three non-participating chlorines (i.e. those at the ortho- and meta-positions). 2,3,4,5-CB was the only compound isotopically monitored during this process as the only other species involved in the isotope dynamics was the evolved Cl , which especially in estuarine settings would prove impossible to independently discriminate from the much larger standing inventory of unaffiliated chloride. Additionally, there is no substantial difference in d37Cl values of the 2,3,4,5-CB standard ( 0.39%) and control samples ( 0.36 and 0.90%) (Fig. 1), indicating that minimal chlorine isotopic fractionation occurred during the quantitative isolation procedure. This
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4. Conclusions Bacterially mediated reductive dechlorination of 2,3,4,5-CB to 2,3,5-CB does not exhibit measurable chlorine isotopic fractionation, allowing for the potential to differentiate physiochemical processes from this important biological counterpart in contaminated settings. Moreover, HPLC has been established as an effective tool for quantitatively isolating single PCB congeners for compound-specific chlorine isotopic analysis from more complex matrices. Further investigation into the bulk and compound-specific isotopic characteristics in both unaltered and environmentally transformed Aroclor mixtures should help to more fully elucidate the ultimate fate of these potent compounds.
Fig. 1. Chlorine isotopic composition of microbial culture and sterile control 2,3,4,5-CB samples as a function of remaining substrate. Controls correspond to the time points at which 0 and 80% of the 2,3,4,5-CB in the cultures had been dechlorinated. The horizontal line denotes the d37Cl value of the 2,3,4,5-CB standard (not isolated via prep-HPLC). All d37Cl values represent the mean of four individual mass spectrometric measurements on each sample (instrumental error bars are smaller than their respective symbols).
demonstrates the effectiveness of reverse-phase HPLC as a preparative tool for isolating hydrocarbons destined for compound-specific chlorine isotopic measurements from experimental matrices, analogous to results reported for the preparation of various samples for carbon and nitrogen isotopic analyses (Bidigare et al., 1991; Caimi and Brenna, 1997; Kenig et al., 2000; Sachs and Repeta, 2000). If the maintenance of isotopic composition is symptomatic of biological reductive dechlorination reactions operating on other PCB congeners, then the depletion in bulk d37Cl compositions of altered Aroclors extracted from highly contaminated sediments (Reddy et al., 2000) suggests the activity of different environmental processes. This notion is further supported by invariant d37Cl values for a suite of individual congeners measured within Aroclors themselves (Xu et al., unpublished data), as this precludes the possibility that bulk depletions in environmental samples are simply a result of modified internal isotopic mass balance. In other words, the bulk d37Cl composition should be insensitive to the preferential loss, for example, of the less chlorinated (more soluble) congeners before sedimentary deposition. Rather, other factors that might produce an isotope effect such as sequential phase partitioning or chemical destruction are implicated. A means is thus provided to discriminate between alteration profiles produced from abiotic weathering (depleted bulk d37Cl) and reductive dechlorination (unaffected bulk d37Cl).
Acknowledgements N.J.D. acknowledges current support from the Stanley W. Watson Foundation and the National Science Foundation’s Research Experience for Undergraduates program (REU) while a Student Fellow at WHOI. This work was also funded by WHOI EPA Grant (R82816101) to C.M.R., the US Office of Naval Research, Harbor Processes Program, U.S. Department of Defense (N00014-99-1-0078 to H.D.M. and N00014-991-0101 to K.R.S.), and a Rinehart Coastal Research Center grant to C.M.R. This is WHOI contribution no. 11041.
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