Anaerobic Degradation of Decabromodiphenyl Ether - Environmental

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Environ. Sci. Technol. 2005, 39, 1078-1083

Anaerobic Degradation of Decabromodiphenyl Ether A N D R E A S C . G E R E C K E , * ,† PAUL C. HARTMANN,‡ NORBERT V. HEEB,† HANS-PETER E. KOHLER,‡ WALTER GIGER,‡ PETER SCHMID,† MARKUS ZENNEGG,† AND MARTIN KOHLER† Laboratory of Organic Chemistry, Swiss Federal Institute for Materials Science and Technology (Empa), U ¨ berlandstrasse 129, 8600 Du ¨ bendorf, Switzerland, and Swiss Federal Institute for Environmental Science and Technology (EAWAG), U ¨ berlandstrasse 133, 8600 Du ¨ bendorf, Switzerland

The environmental safety of decabromodiphenyl ether (BDE-209), a widely used flame retardant, has been the topic of controversial discussions during the past several years. Degradation of BDE-209 into lower brominated diphenyl ether congeners, exhibiting a higher bioaccumulation potential, has been a critical issue. Here, we report on the degradation of BDE-209 and the formation of octa- and nonabromodiphenyl ether congeners under anaerobic conditions. Sewage sludge collected from a mesophilic digester was used as the inoculum and incubated up to 238 days with and without a set of five primers. Following Soxhlet extraction and a liquid chromatography cleanup procedure, parent compounds and debromination products were analyzed by GC/HRMS. In experiments with primers, concentrations of BDE-209 decreased by 30% within 238 days. This corresponds to a pseudo-first-order degradation rate constant of 1 × 10-3 d-1. Without primers, the degradation rate constant was 50% lower. Formation of two nonabromodiphenyl ether and six octabromodiphenyl ether congeners proved that BDE-209 underwent reductive debromination in these experiments. Debromination occurred at the para and the meta positions, whereas debromination at the ortho position was not statistically significant. All three nonabromodiphenyl ether congeners (BDE-206, BDE207, and BDE-208) were found to undergo reductive debromination as well. No significant change of the BDE209 concentration and no formation of lower brominated congeners was observed in sterile control experiments. To our knowledge, this is the first report demonstrating microbially mediated reductive debromination of BDE-209 under anaerobic conditions.

Introduction Decabromodiphenyl ether (BDE-209) is widely used as a flame retardant in products such as electronic equipment and upholstered furniture. Its estimated annual global market demand was 56 100 t in 2001 (1). The environmental safety of BDE-209 (e.g., bioavailability, persistence, toxicity) is a * Corresponding author phone: +41 (1) 823 4953; fax: +41 (1) 823 4041; e-mail: [email protected]. † Empa. ‡ EAWAG. 1078

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subject of ongoing discussions. For example, it has been suggested that BDE-209 is highly persistent in sediments (1) and that formation of more toxic transformation products can be neglected in these systems. However, a European Union risk assessment (2) on BDE-209 concluded recently that there is a need for further information on the degradation of BDE-209 into more toxic and bioaccumulative compounds (e.g., reductive debromination to lower brominated congeners). We addressed this information gap by studying the fate of BDE-209 under anaerobic conditions in digested sewage sludge. The worldwide occurrence of tri- to decabromodiphenyl ethers has been reviewed recently (3). Tri- to heptabromodiphenyl ether congeners that are contained in technical PentaBDE and OctaBDE mixtures have been frequently found in biota, whereas BDE-209, being the dominating congener of technical DecaBDE (97-99%), has rarely been reported. The highest concentrations of BDE-209 in biological tissue samples were found in peregrine falcon eggs (up to 430 ng/g of lipid (4)). Concentrations in human blood samples from Mexico and from occupationally exposed Swedish workers were between 5 and 15 ng/g of lipid (5) and between 1 and 300 ng/g of lipid (6), respectively. However, BDE-209 has been frequently detected at concentrations exceeding 1000 ng/g dry weight (dw) in sediments (7, 8) and sewage sludge (9, 10). In Switzerland, average concentrations of BDE-209 in digested sewage sludge increased by a factor of 6 from 180 ng/g dw in 1993 to 1200 ng/g dw in 2002 (median concentration, n ) 8, same treatment plants in both sampling campaigns) (10). These data indicate that reservoirs of BDE209 are building up in the abiotic environment, whereas bioaccumulation of BDE-209 itself appears to be low. In fact, limited uptake, rapid elimination, and fast transformation of BDE-209 were shown in animal studies with rats, rainbow trout, and carp (11-13). Irreversible neurological effects were evoked in mice after a single oral dose at postgestational day 3 (but not after administration at day 10) (14). As the same effects were observed after administration of lower brominated diphenyl ether congeners at day 10 (15), it appears that BDE-209 needs to be metabolized to evoke these neurological effects. From a chemical point of view, BDE-209 is a labile molecule. BDE-209 reacts readily with nucleophiles (16), it is reduced by hydride reagents such as sodium borohydride (17), and it is rapidly photolyzed under UV-B and UV-C irradiation (18). On the other hand, BDE-209 is very hydrophobic; its estimated log Kow is on the order of 10. Free concentrations of BDE-209 in aqueous solutions are therefore very low and could be a rate-limiting factor for biologically mediated transformation processes. Within the past two decades, reductive dehalogenation has been described for a variety of halogenated compounds in anaerobic systems (19, 20). In such reactions, halogenated compounds serve as electron acceptors in respiratory or cometabolic processes. For example, polychlorinated and polybrominated biphenyls (PCBs and PBBs) were reduced in anaerobic enrichment cultures (21, 22). Bedard et al. were able to show that addition of certain chemically related compounds, called primers, may enhance the degradation of the target compound (23). For example, addition of 2,6dibromobiphenyl increased the degradation of PCBs in sediment (24). Only a few studies looked at polybrominated diphenyl ethers. Rayne et al. reported complete debromination of 4,4′-dibromodiphenyl ether (BDE-15) under anaerobic condition in a fixed film plug flow bioreactor (25). However, Schaefer and Flaggs (2, 26) did not find a statistically 10.1021/es048634j CCC: $30.25

 2005 American Chemical Society Published on Web 01/13/2005

significant change of concentrations for BDE-209 or 2,2′,4,4′tetrabromodiphenyl ether (BDE-47) within 32 weeks in an anaerobic sediment. To our knowledge, degradation of BDE209 has not yet been demonstrated in any anaerobic system. In this paper, we describe the transformation of BDE-209 into nona- and octabromodiphenyl ethers within a period of 238 days in experiments with sewage sludge as the inoculum. Heat-sterilized samples served as a negative control, whereas R-hexachlorocyclohexane (R-HCH) was added to the experiments as a positive degradation control. To stimulate the degradation, primers were added to all but three experiments. Two analogous experiments were carried out with two nonabrominated diphenyl ether congeners to elucidate the fate of these two potential BDE-209 degradation products.

Experimental Section Chemicals. Chemicals were obtained from the following sources: technical DecaBDE (98%) and tetrabromobisphenol A (97%) from Aldrich (Buchs, Switzerland), R-HCH (98%) from Dr. Ehrenstorfer GmbH (Augsburg, Germany), 4-bromobenzoic acid and hexabromocyclododecane (95%) from Fluka (Buchs, Switzerland), 2,6-dibromobiphenyl from AccuStandard (New Haven, CT), and decabromobiphenyl from Elf Atochem (Paris, France). Standards were purchased from Cambridge Isotope Laboratories (Andover, MA), [13C6]-γHCH, [13C12]BDE-126, [13C12]BDE-183, and [13C12]BDE-209 (numbers according to the IUPAC convention for PCBs), from Wellington Laboratories (Guelph, Canada), BDE-196, BDE197, BDE-206, and BDE-207, and from AccuStandard, BDE198, BDE-203, BDE-204, and BDE-205. Incubation. Glass serum bottles (100 mL), filled with a 1 cm layer of glass beads, were spiked with stock solutions of technical DecaBDE (10.0 nmol per sample). As a positive degradation control, R-HCH (10.5 nmol) was added. In standard experiments, five compounds were added as primers (4-bromobenzoic acid, 2,6-dibromobiphenyl, tetrabromobisphenol A, hexabromocyclododecane, and decabromobiphenyl, 9-11 nmol each). In two experiments, BDE-207 or BDE-206 was added instead of technical DecaBDE. Solvents were evaporated overnight. Starch (20 mg) and yeast (50 mg) were added immediately before the bottle was filled with 20 mL of freshly collected digested sewage sludge (sewage treatment plant in Du ¨ bendorf, Switzerland, serving 45000 people; mesophilic stabilizer operated at 37 °C, June 3, 2003, pH 7.6, 3% (w/w) dry weight, containing 58 nmol L-1 BDE-209). The bottles were tightly capped and incubated at 37 ( 1 °C for up to 238 days in the dark. Control experiments with heat-sterilized sludge (autoclaved at 120 °C for 60 min, twice 24 h apart) were performed simultaneously. After predetermined time periods, the bottles were opened and internal standards were added ([13C6]-γ-HCH, [13C12]BDE-183, and [13C12]BDE-209). Thereafter, the degradation was stopped by addition of 0.25 mL of concentrated sulfuric acid, and the sample was frozen until analysis. Analysis. The samples were thawed and transferred into Soxhlet thimbles. After air-drying, extraction was performed with a 1:1 mixture of hexane and acetone for 6 h in a Soxhlet apparatus. Sulfur was removed by gel permeation chromatography, and the extracts were split into two equal portions. Half was further purified on a silica gel column, and the other was set aside. GC analysis was carried out with a DB-1 equivalent stationary phase (PS347.5, 10 m × 0.28 mm, film 0.1 µm). Hydrogen at an estimated flow rate of 4 mL min-1 served as the mobile phase. The GC oven temperature program was as follows: isothermal at 110 °C for 1 min, 12 °C/min to 320 °C, and held at 320 °C for 5 min. The GCto-MS transfer line was held at 330 °C. Identification of octabromodiphenyl ethers (OctaBDEs) was performed on a DB-5 equivalent stationary phase (PS086, 20 m × 0.28 mm,

TABLE 1. Analytical Quality Assurance Parameters for Deca-, Nona-, and Octabromodiphenyl Ether Congeners limit of quantification (nmol)a oncolumn BDE-209 BDE-208 BDE-207 BDE-206 BDE-205e ΣOctaBDEs

46 × 10-6 naf 48 × 10-6 24 × 10-6 34 × 10-6 na

imprecision, CV (%)

per instrumental sampleb imprecisionc 0.023 na 0.024 0.012 0.017 na

6.9 6.8 3.1 6.2 6.9 8.3

imprecision of replicate samplesd 9 (at 10 nmol) ncg 40 (at 0.050 nmol) 15 (at 0.18 nmol) nc nc

a Based on S/N ) 10 in the chromatogram of a standard sample (500 fmol on-column per compound). b Sample size: 20 mL of sludge for incubation experiments; 5 g dry weight for the sample from the inlet and outlet of the anaerobic digester. c Based on five consecutive injections of the same sample (standard incubation, day 238). d Imprecision of replicate samples is estimated from the set of sterile control samples (n ) 6). e Data for BDE-205, an octabromodiphenyl ether, are shown as an example for an individual octabromodiphenyl ether congener. f na ) no standard available. g nc ) not calculated because some or all samples were below the limit of quantification.

film 0.15 µm). Hydrogen at an estimated flow rate of 2.3 mL min-1 was used as the mobile phase. The GC oven temperature program was as follows: isothermal at 62 °C for 1 min, 20 °C/min to 280 °C, 10 °C/min to 300 °C, 2 °C/min to 330 °C, and held at 330 °C for 10 min. The GC-to-MS transfer line was held at 350 °C. Chiral analysis of R-HCH was performed on a column coated with 33% 2,6-di-O-methyl-3-O-pentylβ-cyclodextrin (27) in OV-1701-OH (20 m × 0.25 mm, film 0.15 µm). Hydrogen at a constant pressure of 60 kPa was used as the mobile phase. The GC oven temperature program was as follows: isothermal at 110 °C for 1 min, 10 °C/min to 150 °C, held at 150 °C for 12 min, and 15 °C/min to 220 °C. In all GC/MS analyses, samples were injected cold oncolumn (1 µL out of a 250 µL final volume). Positive ion EI-MS signals were acquired on a MAT 95 high-resolution mass spectrometer (Thermo Finnigan MAT) in single-ion monitoring mode at an ionization energy of 60 eV and a mass resolution of 8000. For hepta-, nona-, and decabromodiphenyl ethers, the two most intense ions of the [M 2Br]+ cluster were monitored. OctaBDEs were monitored on the two most intense ions of the molecular ion cluster. Response factors for BDE-207 and BDE-206 versus [13C12]BDE-209 were determined and used for quantification. BDE208 was quantified with the mean of the response factors of BDE-207 and BDE-206. The mean response factor of BDE198, BDE-203, BDE-204, and BDE-205 versus [13C12]BDE-183 was used to quantify the sum of the (partially coeluting) octabromodiphenyl ethers. QA/QC. Limits of quantification were on the order of 30 × 10-6 nmol per on-column injection in GC and 0.02 nmol per sample (see Table 1). Only a few determinations of ΣOctaBDEs (summation of peak areas) and BDE-208 were below the limit of quantification (S/N ) 10), but these measurements were still above the limit of detection (S/N ) 3). Coefficients of variation (CV) were around 7% for multiple injection of the same sample. Sterile control samples exhibited a very low CV for BDE-209 and BDE-206 (Table 1) and demonstrated the high reproducibility of the incubation and analytical procedures. During incubation and analysis, exposure to light was kept as low as possible. Windows and fume hoods were covered with a UV filter foil (UV filter no. 226, Lee Filters, Burbank, CA). Incubation and sterile control experiments were processed at the same time under identical conditions. VOL. 39, NO. 4, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Amount of deca-, nona-, and octabromodiphenyl ether congeners (nmol per sample) versus time in the sterile control and incubation samples (with and without primers). P values represent significance of the linear regression of the amount versus time. Considering the low sample number, we did not calculate P values for experiments without primers.

Results and Discussion Microbial Activity. Gas production indicating methanogenic conditions was detected in all sample bottles. No gas formation was observed in sterile control samples. The apparent pseudo-first-order degradation rate constant for (()-R-HCH was 0.40 d-1 and compared well to the degradation rate constant observed by Buser et al. (28) of 0.47 d-1 for (+)-R-HCH and 0.17 d-1 for (-)-R-HCH in a similar experimental system (except incubation at 25 °C instead of 37 °C). The enantiomeric fraction (see ref 29 for the definition) changed from 0.52 at the beginning, to 0.47 after 20 h, and 0.40 at day 5. This change indicated biological involvement in the degradation of R-HCH. Thus, an active microbial community, involved in the degradation of our positive degradation control R-HCH, was present in the experiments. A slow disappearance of (()-R-HCH was also observed in sterile control samples (apparent pseudo-first-order degradation rate constant of 4.4 × 10-3 d-1) which was almost a factor of 100 slower than in incubation experiments and also approximately a factor of 10 slower than disappearance of (+)- and (-)-R-HCH in sterile control experiments reported by Buser et al. (28). Disappearance of BDE-209. The amount of BDE-209 decreased by 30% from 11.2 nmol (10 nmol of spiked + 1.2 nmol of BDE-209 residues contained in 20 mL of sludge) to 7.9 nmol within 238 days in the anaerobic experiments with primers (Figure 1b). The observed disappearance was significant at the 95% level (p ) 0.037 for linear regression of the amount versus time) and corresponded to a pseudofirst-order rate constant of 1 × 10-3 d-1, being more than 2 orders of magnitude lower than the apparent pseudo-firstorder degradation rate constant for (()-R-HCH. In the sterile control experiments, no significant change in the amount of BDE-209 occurred over time (p ) 0.41, Figure 1a). 1080

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Presence and Formation of Lower Brominated Congeners. The technical DecaBDE used in this work contained low concentrations of the three nonabromodiphenyl ethers (about 0.04% BDE-208 on a molar basis, 0.4% BDE-207, and 2% BDE-206, Figure 2a). Comparable ΣNonaBDE concentrations in technical DecaBDE were reported in the literature (2). No octabromodiphenyl ethers were detected in the used technical product (i.e., they were below the limit of detection of 0.005 nmol per sample). Hence, the sum of the 12 possible octabromodiphenyl ethers in the sample of the used technical mixture (10 nmol of BDE-209) was lower than 0.060 nmol (12 × 0.005 nmol) and therefore below 6000 ppm. For comparison, concentrations of ΣOctaBDEs in technical DecaBDE have been reported to be around 400 ppm (2). Two nonabromodiphenyl ether congeners (BDE-208 and BDE-207) as well as a number of octabromodiphenyl ether congeners were formed in the incubation experiments (Figure 1e,h,n). P values representing the significance of a linear regression of the amount (nmol per sample) versus time were p ) 2 × 10-4, 6 × 10-4, and 5 × 10-6 for BDE-208, BDE-207, and ΣOctaBDEs, respectively. The amount of BDE-208, formed by para debromination of BDE-209, increased more than 10 times (Figure 1) from an amount below the limit of quantification to 0.15 nmol. The amount of BDE-207 in the sample, resulting from meta debromination, rose from 0.024 to 0.16 nmol, and ΣOctaBDE increased from below the limit of quantification to 0.21 nmol. In total, at least 0.5 nmol of transformation products was formed within 238 days, and consequently at least 5% of the BDE-209 initially present (11.2 nmol) was reduced to lower brominated congeners. These results are reflected in Figure 2, showing the chromatograms of (i) the three nonabromodiphenyl ethers BDE208, BDE-207, and BDE-206 of incubation samples after 7, 63, and 238 days, respectively, (ii) the sterile control, and (iii) the technical DecaBDE product. Signals for BDE-208 and

FIGURE 2. GC/MS chromatograms of nonabromodiphenyl ethers (at m/z 719.42 and 721.42) contained in technical DecaBDE (a), formed in incubation samples (b-d), and present in the sterile control (e). The chromatograms illustrate the formation of BDE-208 and BDE207 over time under anaerobic conditions. BDE-207 do clearly increase relative to the signal of BDE-206 with increasing incubation time. Whereas the formation of BDE-208 and BDE-207 was very evident, formation of BDE-206 could not be shown conclusively. BDE-206 did not significantly increase (95% level) during the experiment, because either the formation rate for BDE-206 was too low or formation and further degradation resulted in a steady state. As we know that BDE-206 is

debrominated to octabromodiphenyl ethers (see below), the second hypothesis cannot be ruled out. However, for PCBs microbially mediated dehalogenation at the ortho position has also been less frequently observed than a reduction of the meta and para positions (22, 30). The mass balance between loss of BDE-209 and formation of products was not closed at 238 days. Whereas 3 nmol of BDE-209 disappeared, only a total of 0.5 nmol of transformation products was identified so far. There was no evidence for the formation of other PBDE congeners, such as heptabromodiphenyl ethers in the chromatograms. Possible explanations for this discrepancy are (i) formation of unidentified transformation products, (ii) formation of bound BDE-209 residues (nonextractable residues), and (iii) imprecision of the analytical procedure (see Table 1). In the sterile control experiments, neither nonabromodiphenyl ethers nor octabromodiphenyl ethers were formed (Figure 1d,g,j,m). Influence of Primers. A set of five primers (4-bromobenzoic acid, 2,6-dibromobiphenyl, tetrabromobisphenol A, hexabromocyclododecane, decabromobiphenyl) were added to the standard experiments to possibly increase the degradation rate for BDE-209. In three additional experiments, no primers were added, yet formation of lower brominated congeners was observed in these samples as well (Figure 1f,i,o). The observed rate of formation in these experiments was approximately half that in the standard experiments. Thus, as for the case of PCBs in sediment from Woods Pond (Lenox, MA) (24), degradation of BDE-209 was accelerated by the addition of primers, but was also taking place without them. Degradation of BDE-207 and BDE-206. Two experiments were carried out spiking digested sewage sludge with either BDE-207 or BDE-206, the two commercially available nonabromodiphenyl ethers, instead of technical DecaBDE (Figure 3). In both cases, degradation was indicated by the formation of OctaBDEs. No degradation rate constants were deduced, because the number of experiments was too small (i.e., one for BDE-207 and one for BDE-206). Interestingly, different octabromodiphenyl ether patterns were formed upon incubation of technical DecaBDE, BDE206, and BDE-207, indicated by six, three, and four peaks in the respective chromatograms (Figure 3b-d). As only 6 out

FIGURE 3. GC/MS chromatograms of octabromodiphenyl ethers (at m/z 799.35 and 801.35) of a mixture of six available octabromodiphenyl ether standard solutions (a) and of octabromodiphenyl ethers formed during incubation of technical DecaBDE (b), BDE-206 (c), and BDE-207 (d). Unequivocal assignment of chromatographic signals to individual congeners is not possible at this point, since coelution may occur and since only 6 out of 12 octabromodiphenyl ether congeners are currently available as reference materials. VOL. 39, NO. 4, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Concentrations of BDE-208 and BDE-207 Relative to the Concentration of BDE-206 in Sewage Sludge at Different Stages of a Mesophilic Digester (WWTP, Du1 bendorf, Switzerland) and during the Incubation Experiments nonabromodiphenyl ether relative concn (-) BDE-208

BDE-207

Full-Scale Reactor (Grab Samples) inlet 0.087 0.17 reactor 0.17 0.27 outlet (28 day residence time) 0.29 0.33 day 0 day 21 day 238

Incubation Experiment 0.02 0.19 0.56

0.21 0.27 0.64

BDE-206 1 1 1 1 1 1

of the 12 possible octabromodiphenyl ethers were available as analytical standards (Figure 3a), a complete identification of the formed octabromodiphenyl ethers was not possible. However, it is clear that debromination took place at various positions without any pronounced preference. Further, the signal at retention time tR ) 18.25 min, which only appears in the experiments with technical DecaBDE and not in the experiments with BDE-206 or BDE-207, corresponds to an unknown octabromo congener, which must result from debromination of BDE-208. Therefore, the nonabromo congener BDE-208 undergoes further degradation as well. On the basis of the early elution, which is characteristic for congeners without bromine substituents at the para position (31), we speculate that this signal corresponds to BDE-202 (2,2′,3,3′,5,5′,6,6′-octabromodiphenyl ether), the only octabromodiphenyl ether congener without bromine substituents at both para positions. Degradation in an Anaerobic Digester. A preliminary study was performed to investigate whether degradation of BDE-209 occurs also in full-scale anaerobic digesters. To this end, grab samples were taken at the inlet and the outlet of the anaerobic digester at STP Du ¨ bendorf, from which the inoculum for this study was taken. In accordance with the results of the incubation experiments, the concentrations of BDE-208 and BDE-207 increased relative to the concentration of BDE-206 between the inlet and outlet of the full-scale anaerobic digester (see Table 2). These results suggest that transformation of BDE-209 into BDE-208 and BDE-207 also took place in the full-scale reactor. However, given a residence time of 28 days in the reactor, one set of grab samples does not provide unequivocal evidence. Environmental Relevance. Under anaerobic conditions, BDE-209 underwent degradation to lower brominated diphenyl ethers. The observed debromination of BDE-209 at the para position led to lower brominated congeners, being different from lower brominated congeners contained in technical Penta- and OctaBDE formulations. In these products, all major congeners reported are brominated at the para and the para′ positions (an exception is BDE-17). Future studies with samples from anaerobic environments should look carefully to (early eluting) non-para-brominated diphenyl ether congeners. As degradation rates are expected to vary greatly between individual anaerobic systems and prevailing redox conditions (sludge, sediment, soil) (32), a valid rate constant for other systems cannot be deduced. However, our results provide clear evidence that BDE-209 is degradable under anaerobic conditions and that compounds with a higher bioconcentration potential are formed. This is an important finding, because the inventory of BDE-209 in anaerobic compartments (soil, sediment) is anticipated to increase over the 1082

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next few decades, given the continuous use and emission of BDE-209.

Acknowledgments Financial support for this study was provided by the Swiss National Science Foundation (National Research Programme NRP50 “Endocrine DisruptorssRelevance to Humans, Animals and Ecosystems”, Projects FLARE 4050-066536 and PHEBRO 4050-066566).

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Received for review September 2, 2004. Revised manuscript received November 10, 2004. Accepted November 15, 2004. ES048634J

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