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NOTE / NOTE Non-enzymatic degradation of chlorofluorocarbon 113 using cyanocobalamin under anaerobic conditions David M. Bagley, Ian G. Sutherland, and Brent E. Sleep
Abstract: Chlorofluorocarbon 113 (CFC113) was rapidly and completely degraded non-enzymatically in systems containing cyanocobalamin with titanium (III) citrate as the reductant. Hydrochlorofluorocarbon 123a (HCFC123a) accounted for up to 25% of the degraded CFC113. Chlorotrifluoroethene was also detected. Increasing concentrations of cyanocobalamin increased CFC113 removal rates asymptotically and also decreased the fraction of HCFC123a remaining. Key words: chlorofluorocarbon 113, cyanocobalamin, anaerobic degradation, non-enzymatic. Résumé : Le chlorofluorocarbone 113 (CFC113) a été rapidement et complètement digéré par action non enzymatique par des systèmes contenant de la cyanocobalamine et du citrate de titane (III) comme réducteur. L’hydrochlorofluorocarbone 123a (HCFC123a) représente jusqu’à 25 % du CFC113 digéré. Du trifluorochloréthylène a également été détecté. L’augmentation des concentrations en cyanocobalamine a augmenté les taux d’élimination du CFC113 d’une façon asymptotique et a également réduit la fraction résiduelle de HCFC123a. Mots clés : chlorofluorocarbone 113, cyanocobalamine, digestion anaérobie, non enzymatique. [Traduit par la Rédaction]
Introduction 1,1,2-Trichloro-1,2,2-trifluoroethane (CFC113) was widely used as a solvent for degreasing and dry cleaning, as a refrigerant, and for other typical CFC uses (CEPA 1997). Although usage has declined since production of CFC113 in the United States ceased in 1996, consumption of existing stocks continues (CEPA 1997). CFC113 has been detected in groundwaters at concentrations ranging from 6 µg/L to 2.7 mg/L (Lesage et al. 1990; Semprini et al. 1992), but is considered relatively non-toxic to humans and other organisms (WHO 1990). The State of California has set a public health goal for CFC113 in drinking water of 4 mg/L and a state maximum contaminant level (MCL) of 1.2 mg/L (CEPA 1997), but the US Environmental Protection Agency has not set a MCL for CFC113 in drinking water (USEPA 2001). CFC113 also inhibits the anaerReceived 3 July 2003. Revision accepted 30 March 2004. Published on the NRC Research Press Web site at http://jees.nrc.ca/ on 29 July 2004. D.M. Bagley,1 I.G. Sutherland, and B.E. Sleep. University of Toronto, Department of Civil Engineering, 35 St. George Street, Toronto, ON M5S 1A4, Canada. Written discussion of this note is welcomed and will be received by the Editor until 30 November 2004. 1
Corresponding author (e-mail:
[email protected]).
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obic degradation of chlorinated ethenes, which are common groundwater contaminants. Inhibition of tetrachloroethene and cis-1,2-dichloroethene degradation occurred in the presence of as little as 0.15 mg CFC113/L (Bagley et al. 2004). CFC113 has been detected at inhibitory levels at sites containing chlorinated ethenes (Lesage et al. 1990; Semprini et al. 1992). Therefore, removal of CFC113 is required at sites containing mixtures before chlorinated ethene removal via anaerobic biodegradation can commence. CFC113 degrades under anaerobic conditions, but only slowly (Bagley et al. 2004; Denovan and Strand 1992; Lesage et al. 1992). In anaerobic microcosms, CFC113 degraded to both 1,2-dichloro-1,2,2-trifluoroethane (HCFC123a) and chlorotrifluoroethene (CTFE), with HCFC123a degrading further to monochlorinated chlorofluorocarbons (Lesage et al. 1992). Slow biodegradation of CFC113 to HCFC123a and trace amounts of CTFE was also observed in cultures that had been enriched to degrade tetrachloroethene (Bagley et al. 2004). Corrinoids such as the nickel-containing coenzyme F430 , iron-containing hematin and the cobalt-containing vitamin B12 , have been well studied as degradation catalysts for chlorinated aliphatic compounds. Investigations of vitamin B12 reactivity were originally conducted in the late 1960s/early 1970s (Wood et al. 1968; Schrauzer et al. 1969; Hill et al. 1971). The anaerobic degradation of the chlorinated methanes carbon tetrachloride and chloroform by hematin (Klecka and Gonsior 1984), cobal-
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amins (Krone et al. 1989b), and coenzyme F430 (Krone et al. 1989a) led to expanded work on other compounds such as chlorinated ethenes and benzenes (Gantzer and Wackett 1991) and the chlorofluorinated methanes CFC11, CFC12, and CFC13 (Krone et al. 1991). CFC113 was observed to slowly degrade non-enzymatically in the presence of hematin (Lesage et al. 1992) but other corrinoids have not been tested. The objective of this work was to identify the potential of cyanocobalamin to facilitate the rapid degradation of CFC113 non-enzymatically.
Materials and methods Experiments were conducted in 122-mL vials containing 70 mL of 3.8 µM titanium (III) citrate solution (prepared with 3.6 mL of 15 % titanium (III) chloride (Fisher Scientific), 4.5 g NaHCO3 , and 1.6 g sodium citrate per liter) and capped with Teflon™ -lined rubber septa. The pH was adjusted to 7.5 with 1.0 N NaOH. The vials were prepared in a glove box with a 80% N2 /10% CO2 /10% H2 atmosphere and wrapped in foil to exclude light. CFC113 (Sigma Aldrich) was added from a saturated aqueous solution (approximate concentration of 170 mg/L) to provide from 1 to 5 µmol per bottle. Bottles were agitated after CFC113 addition for 1 h at 200 rpm on an orbital shaker at 22 ◦ C after which initial CFC113 measurements were conducted and cyanocobalamin (CnCbl) was added. The temperature remained controlled at 22 ◦ C throughout the experiments. Cyanocobalamin (99%, Sigma Aldrich) was added from stock solutions prepared in distilled, deionized water in appropriate volumes (6.7 to 1340 µL) to provide aqueous concentrations in the bottles from 10 to 2000 nM. CFC113 and HCFC123a (Caledon Laboratories) were measured via gas chromatography on a Hewlett Packard 5890 II gas chromatograph (GC) equipped with a flame ionization detector (FID) and a 30 m × 0.53 mm × 0.3 µm film Vocol column (Supelco). The GC was operated with 8 mL/min N2 flow rate and a temperature program of 35 ◦ C for 2 min followed by a 20 ◦ C/min ramp to 110 ◦ C, which was held for 2 min. Headspace samples (20 µL) were manually injected. The detection limits were 20 nMol per bottle for CFC113 and 30 nMol per bottle for HCFC123a. Mass spectrometry to identify CFC113 degradation products was conducted by the York-Durham Regional Environmental Laboratory (Pickering, Ontario). The dimensionless Henry’s law constants at 22 ◦ C (mol compound per liter in the gas per mol compound per liter in the liquid) for CFC113 and HCFC123a were 12.1 and 0.93, respectively, as determined using a method modified from Gossett (1987). These constants were used to determine total molar amounts of CFCs from headspace measurements, with the assumption that the gas and aqueous phases were in approximate equilibrium over the course of the experiments.
Results CFC113 was rapidly degraded non-enzymatically in the presence of cyanocobalamin with 16% of the degraded CFC113
J. Environ. Eng. Sci. Vol. 3, 2004 Fig. 1. Non-enzymatic removal of CFC113 in the presence of 10 nM cyanocobalamin. Average of triplicates shown. 5.0 4.0 3.0
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Fig. 2. Non-enzymatic removal of CFC113 with 10 nM cyanocobalamin added to one set of triplicate bottles after 17.5 h. Average of triplicates shown. 1.50 1.25 1.00 0.75 0.50 0.25 0.00 0.0
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recovered as HCFC123a (Fig. 1). In the absence of cyanocobalamin, little CFC113 was removed, while after cyanocobalamin was added, CFC113 removal occurred rapidly with 25% of the degraded CFC113 recovered as HCFC123a (Fig. 2). Cyanocobalamin apparently served to catalyze CFC113 removal as 10 nM cyanocobalamin (providing 0.7 nMol per bottle) facilitated the degradation of over 4.5 µmol of CFC113. Increasing the cyanocobalamin concentration increased the fraction of CFC113 degraded within 3.5 h from 63% with 10 nM cyanocobalamin to 100% with 2000 nM cyanocobalamin (Fig. 3a). The maximum (initial) CFC113 degradation rate increased asymptotically from 0.65 µmol h−1 bottle−1 when the cyanocobalamin concentration was 10 nM to 1.32 µmol h−1 bottle−1 when the cyanocobalamin concentration was 1000 nM and 1.36 µmol h−1 bottle−1 when the cyanocobalamin concentration was 2000 nM. HCFC123a production occurred concurrently with CFC113 degradation (Fig. 3b). At the lowest two cyanocobalamin concentrations, 19% of the degraded CFC113 was recovered as HCFC123a. At the highest two cyanocoba© 2004 NRC Canada
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297 Fig. 4. Unquantified products of non-enzymatic CFC113 degradation as a function of cyanocobalamin (CnCbl) concentration. (a) CTFE production (b) unidentified compound production.
CTFE (peak area units)
Fig. 3. CFC113 Removal and HCFC123a production as a function of cyanocobalamin (CnCbl) concentration. (a) CFC113 removal (b) HCFC123a production.
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lamin concentrations, the fraction of HCFC123a produced declined significantly (Fig. 3b). The initial rates of HCFC123a production were equal to those for the lower cyanocobalamin concentrations but after 1 h either the rate of HCFC123a production declined or the rate of HCFC123a degradation equaled the production rate (for 1000 nM cyanocobalamin) or exceeded the production rate (for 2000 nM cyanocobalamin). At least two additional compounds were produced during CFC113 degradation, producing responses at 0.98 and 1.06 min (Fig. 4). Gas chromatography – mass spectroscopy analysis indicated that CTFE was a degradation product and based on its production pattern, the peak at 1.06 min was presumed to be CTFE. Attempts to identify the compound(s) at 0.98 min were unsuccessful.
Discussion Cyanocobalamin used in this study produced more rapid and complete CFC113 degradation than did hematin in the study of Lesage et al. (1992). In the Lesage et al. study, aqueous CFC113 concentrations of approximately 8.5 µM degraded slowly, about 25% removal within 6 d. CFC113 was not completely removed in hematin systems, even after 90 d, although the more complex leachate systems that included biological ac-
Unidentified (peak area units)
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tivity achieved full CFC113 removal within about 30 d (Lesage et al. 1992). Another difference between the two studies was the reductant used. Lesage et al. (1992) used Na2 S and cysteine as the reductants, while this study used titanium (III) citrate under a 10% hydrogen atmosphere. Hematin was observed to be much less effective at catalyzing carbon tetrachloride degradation than cyanocobalamin, however, even when titanium (III) citrate was used as the reductant (Chiu and Reinhard 1995), and perhaps the same is true for CFC113. Cyanocobalamin derivatives with titanium (III) citrate as the reductant were shown to catalyze degradation of the chlorofluorinated methanes CFC11, CFC12, and CFC13 as well (Krone et al. 1991). The results shown in Fig. 3b indicate that HCFC123a is a product of CFC113 degradation in the presence of cyanocobalamin. These results also indicate that HCFC123a degradation is influenced by the ratio of CFC113 to cyanocobalamin. Accounting for partitioning between the liquid and the headspace, the actual initial aqueous CFC113 concentration for the bottles in Fig. 3a and 3b was approximately 4.3 µM (4300 nM). This provided a significant excess of CFC113 (initial CFC113 to cyanocobalamin molar ratios of 430 and 43) for the low© 2004 NRC Canada
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est two cyanocobalamin concentrations. The initial CFC113 to cyanocobalamin molar ratios for the highest cyanocobalamin concentrations, however, were only 4.3 and 2.15. As CFC113 degradation occurred, the aqueous CFC113 concentrations decreased below the cyanocobalamin concentrations at approximately 2.5 h in the bottle containing 1000 nM cyanocobalamin and within 1.3 h in the bottle containing 2000 nM cyanocobalamin. These times correspond well with the times at which net HCFC123a formation stopped in the bottles. One hypothesis that explains the HCFC123a results in Fig. 3b, then, is that CFC113 has a greater affinity for the cyanocobalamin site than HCFC123a, but as excess cyanocobalamin sites became available due to CFC113 removal, HCFC123a degradation could occur more rapidly. Suflita et al. (1983) observed that more highly chlorinated compounds would be preferentially biodegraded in mixtures of chlorinated compounds. It is not known what compounds were produced by HCFC123a degradation. The compound(s) eluted on the GC at 0.98 min could have been a degradation product of HCFC123a, perhaps 1-chloro-1,2,2-trifluoroethane (HCFC133) and (or) 1-chloro-1,1,2-trifluoroethane (HCFC133b). This compound(s) was present only in the bottles with the two highest cyanocobalamin concentrations, where HCFC123a degradation was observed. Both HCFC133 and HCFC133b were identified as degradation products from HCFC123a by Lesage et al. (1992). Becker and Freedman (1994) and Hashsham et al. (1995) showed that cyanocobalamin addition enhanced the anaerobic biodegradation of chloroform and carbon tetrachloride, respectively. More recently, Lesage et al. (2001) indicated that in situ addition of cyanocobalamin as vitamin B12 is practical as a method for accelerating remediation of chlorinated solvents. In subsurfaces contaminated with CFC113, supplemental cyanocobalamin may help facilitate CFC113 removal in addition to removal of other constituents. Another remediation approach would be to stimulate the growth of organisms that contain cobalamin such as methanol-enriched methanogenic cultures that contain a large number of cobalamincontaining methyltransferases (Zou et al. 2000). Alternatively, subsurfaces contaminated with CFC113 could be bioaugmented with cobalamin-rich anaerobic organisms. In sites where mixtures of contaminants must be removed, the spatially-segregated strategy proposed for removing carbon tetrachloride and chloroform in conjunction with tetrachloroethene (Bagley et al. 2000) may work. After producing a CFC113-free zone, the subsurface could then be appropriately enriched for the degradation of other constituents. The next steps are to confirm that cobalamin-rich cultures can indeed facilitate CFC113 removal and to identify the conditions that optimize such removal. Further work is also required to characterize the products of CFC113 degradation, and their rates of degradation. Some of these products, such as CTFE, may be more toxic than CFC113 (Lesage et al. 1992). Therefore, CFC113 remediation strategies will only be feasible if the CFC113 degradation products are either readily degradable or of low toxicity. The impact of other halogenated organic
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compounds on the degradation rates of CFC113 should also be investigated.
Acknowledgements This research was financially supported by the Centre for Research in Earth and Space Technology (CRESTech), an Ontario Centre of Excellence, the Canadian Chlorine Coordinating Committee (C4), and the Canadian Chemical Producers Association (CCPA).
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