Abiotic Transformation of Toxaphene by Superreduced Vitamin B12

Environ. Sci. Technol. 2004, 38, 3063-3067

Abiotic Transformation of Toxaphene by Superreduced Vitamin B12 and Dicyanocobinamide S T E F F E N R U P P E , † A N K E N E U M A N N , ‡,§ GABRIELE DIEKERT,‡ AND W A L T E R V E T T E R * ,†,| Department of Food Chemistry, Friedrich-Schiller-University of Jena, Dornburger Strasse 25, and Department of Applied and Ecological Microbiology, Friedrich-Schiller-University of Jena, Philosophenweg 12, D-07743 Jena, Germany, Department of Technical Biology, University of Karlsruhe (TH), Engler Bunte Ring 1, D-76131 Karlsruhe, Germany, and Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, D-70599 Stuttgart, Germany

Toxaphene is a complex organochlorine pesticide mixture, residues of which are widespread in the environment. Previous studies with the isolated bacterium Sulfurospirillum (formerly Dehalospirillum) multivorans resulted in an effective anaerobic biotransformation of toxaphene. Since the bacterium contains a corrinoid derivative in the active center of the tetrachloroethene dehalogenase, we attempted to use superreduced corrinoids for abiotic transformation of toxaphene. The two corrinoids studied were dicyanocobinamide and cyanocobalamin (vitamin B12). Superreduced dicyanocobinamide mediated a rapid transformation of toxaphene. More than 90% of the initial pool was transformed within 6 h. The transformation was nonselective, and even the most persistent metabolite in environmental samples, the so-called dead-end metabolite 2-exo,3-endo,6-exo,8,9,10-hexachlorobornane (B6-923 or Hx-Sed) was transformed within hours. Superreduced cyanocobalamin was also able to transform toxaphene albeit at significantly lower velocity. The lack of transformation products detectable in gas chromatograms of hexanesextracted fractions of the assays suggests rapid, sequential dehalogenation and/or destruction of the C10-hydrocarbon backbone of the compounds of technical toxaphene.

Introduction The chloropesticide toxaphene (CAS Registry No. 8001-352) is a highly complex mixture obtained during the exhaustive chlorination of bicyclic monoterpenes. Several hundred compounds are found in the technical products which have been used worldwide since 1945 on a 1 million ton scale (1, 2). Despite ongoing efforts in the production of analytical standards (3, 4), still only ∼10% of the constituents of the compounds of technical toxaphene (CTTs) are structurally known. Technical products were marketed under trademarks * Corresponding author phone: +49 711 459 4016; fax: +49 711 459 4377; e-mail: [email protected]. † Department of Food Chemistry, Friedrich-Schiller-University of Jena. ‡ Department of Applied and Ecological Microbiology, FriedrichSchiller-University of Jena. § University of Karlsruhe (TH). | University of Hohenheim. 10.1021/es034994f CCC: $27.50 Published on Web 04/27/2004

 2004 American Chemical Society

FIGURE 1. Structure of 2-exo,3-endo,6-exo,8,9,10-hexachlorobornane (B6-923). such as Toxaphene (U.S.), Melipax (former GDR), Strobane (U.S.), and Polychlorocamphene (former USSR). Due to its heavy use, primarily in the 1960s and 1970s, several environmental compartments have been contaminated with toxaphene. For instance, concentrations in polluted areas could exceed 1 mg/kg sediment (dry weight) (5). Due to its persistence, toxicity, and tendency to bioaccumulate in higher organisms, toxaphene has been classified as a persistent organohalogen pollutant (POP), and it belongs to the “dirty dozen” (6). Although contamination from new applications is unlikely to occur, the high concentrations found in the environment along with the slow transformation rates are still of environmental relevance. For this reason, efforts have been undertaken to understand the fate of toxaphene (7-9). Such mechanistic studies are an appropriate approach for the development of strategies that allow for remediation of contaminated sites. The transformation of toxaphene in sediment, sewage sludge, and soil mainly occurs under anaerobic conditions. In these anoxic media, hexa- and heptachlorobornanes were the major CTT residues, and the principal recalcitrant compounds were identified as 2-exo,3-endo,6-exo,8,9,10-hexachlorobornane (B6-923; Figure 1) and 2-endo,3-exo,5-endo,6-exo,8,9,10-heptachlorobornane (B7-1001) (10, 11). These two CTTs may account for 80% of the CTT residues in contaminated sediments and soils. Recently, it was demonstrated that the isolated dehalorespiring bacterium Sulfurospirillum (formerly Dehalospirillum) multivorans was effective in the biotransformation of toxaphene; furthermore, the transformation occurred in a manner similar to that in anaerobic sediment and soil samples (9, 12). It is widely known that cofactors of bacteria contain metals such as iron and cobalt in the reduced form. This knowledge has been used for performing abiotic degradation experiments. Saleh and Casida transformed individual CTTs with reduced hematin systems (FeII/FeIII redox system) (13, 14). Maruya et al. used zerovalent iron for toxaphene degradation (15). The reductive dehalogenase of S. multivorans contains a corrinoid cofactor with cobalt (active form CoI) in the central position (16, 17). Minor transformation of CTTs with heatinactivated controls of S. multivorans suggested that abiotic processes accompanied the microbial biotransformation, particularly during the initial incubation period (12). Corrinoids were suspected to be responsible for this effect (12). The most well-known corrinoid derivative is vitamin B12 (Figure 2). In this study the abiotic transformation of toxaphene was examined using suitable corrinoids in the superreduced state (i.e., those having the central atom Co in the oxidation state +1). Superreduced (CoI) vitamin B12 (CCAs) has previously been used for the degradation of PCBs, hexachlorobenzenes, lindane, chloroform, and chloroethanes and -ethenes (18-23). Reductive dechlorination and reducVOL. 38, NO. 11, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Structure of cyanocobalamin (CCA, vitamin B12). DMB ) 5,6-dimethylbenzimidazole. tive elimination were found to be the major routes of transformation (19, 21). We also examined abiotic transformations of toxaphene using superreduced dicyanocobinamide (DCCs).

Material and Methods Chemicals. Melipax was found originally packed in a garden shed in Jena, Germany. The composition was similar to that of Toxaphene. Hereafter, toxaphene will be used as a synomym for Melipax, Toxaphene, and other trademarks. B6-923 was isolated using HPLC from a solution of the technical toxaphene (Melipax) (12). An authentic reference standard of B6-923 (commercially distributed by Dr. Ehrenstorfer, Augsburg, Germany) was obtained from H. Parlar (Technical University Munich, Germany). Other CTT standards were from Dr. Ehrenstorfer or LGC Promochem (Wesel, Germany). The internal standard perdeuterated R-HCH (RPDHCH) was synthesized according to ref 24. Cyanocobalamin (vitamin B12) and dicyanocobinamide (see the next section) were obtained from Sigma (Deisenhofen, Germany). All other chemicals used were of the highest available purity and were purchased from Aldrich, Fluka, and Merck (Darmstadt, Germany). IUPAC Nomenclature of Corrinoids and Abbreviations. Corrinoids are derivatives of the 15-membered ring system corrin (C19H22N4) with cobalt as the central atom. Corrin carries four reduced pyrrole units joined into a ring skeleton identical to porphyrin except one methylene group is missing between rings A and D (Figure 2). The derivative bearing a propionic acid ring on each ring and acetic acid on each ring except ring C, as well as several methyl groups as shown in Figure 2, is named cobinic acid, and its heptaamide is named cobinamide. The cobinamide derivative with two cyano axial substituents (R- and β-positions) of cobalt is CoR,Coβ-dicyanocobinamide (C50H72CoN13O8). Cobamides with 5,6-dimethylbenzimidazole (DMB) as the aglycon attached to the propionic amide on ring D by a glycosyl link from its N-1 to the C-1 of the ribose and additionally linked by a bond between the N-3 and CoR are named cobalamin (Figure 2). CoR-[R-(5,6-dimethylbenzimidazolyl)]-Coβ-cyanocobamide (C63H88CoN14O14P), also known as vitamin B12, is termed cyanocobalamin. The oxidation 3064

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state of cobalt is usually labeled as follows: vitamin B12, cyanocob(III)alamin; reduced vitamin B12r, cob(II)alamin; superreduced vitamin B12,s, cob(I)alamin. In this presentation, the abbreviation CCA was used for cyanocobalamin and DCC for dicyanocobinamide (C50H72CoN13O8). The oxidation state is indicated by a subscript: no subscript means CoIII, subscript “r” refers to reduced (CoII), and subscript “s” refers to superreduced (CoI). Preparation of Titanium(III) Citrate. Titanium(III) citrate (E°′[Ti(IV)/Ti(III)] ) -480 mV) was used as the reducing agent of the corrinoids. Solutions of titanium(III) citrate were produced in the anaerobic chamber. A 10 mL 0.4 M sodium citrate (pH 7.0) sample was added to 2 mL of 30% (w/v) titanium(III) chloride in 2 N HCl. By stepwise addition of 2 M sodium carbonate solution the prepared titanium(III) citrate solution was adjusted to pH 8.0 and filled up to 20 mL with distilled water (25). Preparation of Superreduced Cyanocobalamin (Vitamin B12, CCA) and Dicyanocobinamide (DCC). The cofactor concentrations were determined by UV/vis spectroscopy at 361 nm for CCA and 368 nm for DCC. Superreduced corrinoids (0.1, 0.5, or 5 µM in the case of DCC; 1 µM or 1 mM in the case of CCA) were prepared with titanium(III) citrate in 10 mL vials; the end point of the reduction was detectable by the color change of the solution (26). The vials were kept in a glovebox under strict anoxic conditions. Incubation Preparation and Sampling. In an anaerobic glovebox, 5 mL of superreduced DCC or CCA was transferred into 10 mL vials, and 55-220 µg (0.135-0.54 µM) of toxaphene (in 5 µL of n-hexane) or 18 ng (0.052 nM) of B6-923 (1 µL of n-hexane) was added. After incubation times of 2 h, 6 h, and 1, 2, 3, 6, and 7 days, the vials were opened, the internal standard R-PDHCH was added, and the entire sample was immediately transferred into a 100 mL conical flask and extracted with 2 × 10 mL of n-hexane (ultrasonic bath, 5 min). The combined n-hexane extracts were filtered through Na2SO4, and the volume was adjusted to 2 mL (technical toxaphene) or 1 mL (B6-923). A 1 µL sample of each extract was analyzed by GC/ECD and GC/ECNI-MS (27). Gas Chromatography/Electron Capture Detection (GC/ ECD). Analyses were performed with a dual GC/ECD system previously described in detail (9). A CP-Sil 2 and a CP-Sil 8/20% C18 were installed in the GC oven. Parameters discussed in this presentation were based on the CP-Sil 8/20% C18 column. Determination of toxaphene by ECD was carried out as described elsewhere (9). Quality Assurance/Quality Control (QA/QC). Duplicates were analyzed for each sample extracted at each period. The variations ranged from 0 to 25%. Sample blanks and spiked controls without titanium(III) citrate were carried out for every series and over the same time range as the experiments. Loss of CTTs in these controls was 95%. Further extraction with n-hexane did not yield any CTTs. Incubation of toxaphene and corrinoids without titanium(III) citrate as well as toxaphene and titanium(III) citrate without corrinoids resulted in recovery rates of ∼100% for toxaphene.

Results Experiments with Superreduced Dicyanocobalamin (DCCs). Incubations of toxaphene with 5 µM DCCs were accompanied with a very effective transformation of toxaphene (Figure 3). Within 6 h, more than 90% of toxaphene was transformed (Figure 3b). Quantitative recovery of the internal standard R-PDHCH proved that no toxaphene was lost during the sample preparation. It was also verified that incubations with nonreduced corrinoids (no titanium(III) citrate controls) or

FIGURE 3. GC/ECD chromatograms of (a) a toxaphene standard and (b) toxaphene after a 6 h treatment with DCCs. Equal amounts of the internal standard r-PDHCH (see markings in both chromatograms) illustrates the decrease in toxaphene abundance after 6 h.

FIGURE 4. Transformation profile of toxaphene with superreduced dicyanocobalamin (DCCs): (a) relative amount (percent of the initial ECD area) of toxaphene detected dependent on the reaction time and (b) distribution during different time intervals. the reducing agent alone (no corrinoid controls) did not transform toxaphene (see the QA/QS section). Thus, the transformation of toxaphene observed in our experiment was due to the reaction of CTTs with the superreduced corrinoid. Interruption of the incubation at different times only resulted in slightly different transformation rates (Figure 4a). Moreover, the range obtained from some duplicate samples at a certain time was similar to the variations over the entire experimental phase (Figure 4a, days 1 and 3). This indicates that the reaction proceeded to completion within the first few hours (Figure 4). For a closer inspection, areas of 5 min intervals in ECD chromatograms were separately integrated and compared (Figure 4b). The dominating hepta-, octa-, and nonachlorobornanes of toxaphene eluted between 35 and 50 min, while lower chlorinated homologues are found at shorter retention times (Figure 4b). For instance, the first heptachlorobornane (B7-1000) and octachlorobornane (B8-1413) in toxaphene eluted at 33.5 and 40.8 min, respectively. Lower chlorinated bornanes are more stable under anaerobic conditions, and anaerobic microbial transformations are usually accompanied with a shift of the peak pattern toward shorter retention times (5, 8, 9). To our surprise, such preferential enrichment of more persistent CTTs was not observed in the transformation of toxaphene with superreduced DCCs (Figure 4b). GC/ECNI-MS measurements did not identify additional penta- or hexachloro-CTTs. For further confirmation, the reaction of DCCs with neat B6-923 was

studied using the same experimental conditions. B6-923 (also known as Hx-Sed (11)) was found to be the most recalcitrant metabolite of toxaphene in anaerobic sediment, soil, and sewage sludge (7, 10, 11). Recent experiments with the dehalorespiring bacterium S. multivorans demonstrated that anoxic degradation of B6-923 was ∼50-500-fold slower than that of the mesostable (for instance B9-1679) and unstable CTTs (for instance B7-515 and B8-806) (9, 12). Incubation with 0.1 µM DCCs (i.e., only 1/50 of the DCCs concentration used above in the case of toxaphene) resulted in ∼90% transformation of B6-923 within 2 h. In agreement with data depicted from Figures 3 and 4, the velocity of the transformation of B6-923 was comparable with that of transformation of toxaphene. Consequently, all CTTs are likely to be transformed the same way. The degree of toxaphene transformation was dependent on the concentration of the corrinoid. Use of a 10-fold lower concentration of DCCs (0.5 µM instead of 5 µM) resulted in a slower transformation rate, particularly in the initial phase. However, after 7 days toxaphene was also transformed for the most part using the lower concentration of DCCs (Figure 5a). A similar dependence was also found for titanium(III) citrate (data not shown). The standard method (5 µM DCCs and 100 µL titanium(III) citrate solution) was used to study the transformation with three concentrations of toxaphene (55-220 µg or 0.1350.54 µM, corresponding to 11-44 mg/L). A near complete transformation of toxaphene at all three concentrations was observed, even though the percentage of transformation VOL. 38, NO. 11, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Parameters influencing the transformation of toxaphene with superreduced dicyanocobinamide (DCCs): (a) transformation of 55 µg (0.135 µM) of toxaphene with different concentrations of DCCs (0.5 and 5 µM); (b) remaining amounts (and percent of transformation) of CTTs after transformation of 55 µg (0.135 µM), 111 µg (0.27 µM), and 222 µg (0.54 µM) of toxaphene with 5 µM DCCs after 7 days.

FIGURE 6. Influence of the concentration of superreduced cyanocobalamin (CCAs; 1 mM, 1 µM) over a time range of 23 days. Asterisk: after 8 days, different, no control without titanium(III) citrate. achieved was slightly lower for the highest concentration (transformation of ∼90% instead of >97% in the lower concentrated toxaphene solutions) (Figure 5b). Experiments with Superreduced Cyanocobalamin (CCAs). Superreduced vitamin B12 (CCAs) was also able to transform toxaphene (Figure 6). However, the processing was significantly slower than with DCCs. More than 100-fold of the concentration of CCAs and an incubation time of 8 days were required to reproduce the efficiency of toxaphene transformation by DCCs after 6 h. At lower concentrations of superreduced vitamin B12 (CCAs), ∼30% of the initial pool of toxaphene was left (Figure 6). Figure 6 (right bars) illustrates that titanium(III) citrate alone did not degrade CTTs. Although the transformation with CCAs was slower than with DCCs, no enrichment of individual CTTs was observed throughout the experiment (data not shown).

Discussion The method used in this work was effective to achieve more than 90% transformation of toxaphene within a short time. To our knowledge, transformation of toxaphene with DCCs is the fastest nonselective process reported for toxaphene to date. A similar degradation rate was only observed for certain less stable CTTs when exposed to UV light (28). Woods et al. studied the transformation of PCBs by CCAs (superreduced vitamin B12) (19). In agreement with our results obtained with toxaphene, no accumulation of individual congeners was found during PCB transformation. However, a stepwise reductive dechlorination of 2,3,4,5,6-pentachlorobiphenyl to di-, tri-, and tetrachlorobiphenyls occurred (19). A stepwise transformation by reductive dechlorination, eliminination, and/or dehydrochlorination was also observed in studies with other halogenated compounds (21, 29, 30). In our experiments with toxaphene, the residue pattern remained unchanged so that stepwise transformation (leading to a decrease of late-eluting octa- and nonachloro-CTTs 3066

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in favor of early-eluting hexa- and heptachloro-CTTs) was not observed in the case of toxaphene. Marks et al. reported very fast dehalogenation of aaaeee-1,2,3,4,5,6-hexachlorocyclohexane (lindane) using DCC (22). This indicates that reduced corrinoids nonspecifically dehalogenate other organochlorine compounds as well. The lack of enrichment of earlier eluting CTTs in the gas chromatograms indicates a partial transformation or even mineralization of the toxaphene hydrocarbon backbones (chlorinated bornanes, camphenes, bornenes, and other monoterpenes). Unfortunately, we were not able to elucidate any products of this process. Note that the use of chloride salts during the formation of superreduced corrinoids prevented the determination of chloride that may be formed in this process. Moreover, the PCB transformation was significantly slower than the toxaphene transformation (80% transformation within 10 days vs >90% within 6 h with DCCs and ∼90% within 8 days with CCAs under comparable conditions) (19). DCCs proves to be a more potent toxaphene transformer than CCAs, and this may also be valid for PCBs since different processing rates are known for different corrinoids (31). The biotransformation rate of toxaphene with S. multivorans was of the same order of magnitude as the abiotic transformation with CCAs but less than that for DCCs (9). However, bacterial suspensions contain significantly lower amounts of corrinoids (low nanomolar range) than were used in this study. These results emphasize the particular suitability of S. multivorans for transformation of toxaphene (9, 12). Interestingly, this bacterium does not contain the typical corrinoid CCA in the reductive tetrachloroethene dehalogenase but norpseudovitamin B12 (31). Unfortunately, sufficient amounts of this corrinoid of the dehalogenase of S. multivorans were not available for further studies. However, abiotic dehalogenation of trichloroacetate showed that the reactivity of the norpseudovitamin B12 is far higher than the reactivity of DCCs or CCAs (31). Reactions with isolated corrinoids require strict anaerobic conditions and the addition of reducing agents such as titanium(III) citrate (32). This limits the application of the method to environments contamined with toxaphene and other organohalogens. However, on-site applications may be possible for remediation of selected highly contaminated sites. For instance, Mowder et al. demonstrated in-field in situ reductive, vitamin B12-catalyzed dechlorination (33). The method may also be used for supporting the bacterial transformation of toxaphene as was found for other chlorinated compounds (20). This is plausible in light of the fact that corrinoids are the catalytic center of the responsible dehalogenase (e.g., in the case of the reductive transformation of tetrachloroethene by S. multivorans (21, 34)). However, such considerations require further studies in the transformation of toxaphene, to elucidate the structures of the

degradation products formed by the treatment with superreduced corrinoids. Assuming that both corrinoids transform in the same way, the more reactive DCCs may be more suitable for effective transformation while the less reactive CCAs may be used to identify transformation intermediates in the vast degradation of toxaphene.

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Received for review September 10, 2003. Revised manuscript received March 15, 2004. Accepted March 17, 2004. ES034994F

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