Synthesis and Characterization of 2, 3-Dibromopropyl-2, 4, 6

Jan 30, 2007 - The unknown compound UBC-1 previously described as the major organobromine contamination in the blubber extract of a hooded seal ...
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Environ. Sci. Technol. 2007, 41, 1590-1595

Synthesis and Characterization of 2,3-Dibromopropyl-2,4,6-tribromophenyl Ether (DPTE) and Structurally Related Compounds Evidenced in Seal Blubber and Brain ROLAND VON DER RECKE AND WALTER VETTER* Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, D-70599 Stuttgart, Germany

The unknown compound UBC-1 previously described as the major organobromine contamination in the blubber extract of a hooded seal (Cystophora cristata) from the Barents Sea was identified as 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE). DPTE, which is the main component of the brominated flame retardant (BFR) Bromkal 73-5 PE, was synthesized by electrophilic addition of bromine to allyl2,4,6-tribromophenyl ether (ATE). The chirality of DPTE was proven by gas chromatographic enantioseparation of the synthesized racemate. On the basis of GC/ECNI-MS ion chromatograms (m/z 79 and 81), DPTE was the dominating organobromine compound in blubber and brain samples of hooded seals and harp seals (Phoca groenlandica) from the Barents and Greenland Seas. The concentrations of DPTE in blubber and brain were up to 470 and 340 µg/kg wet weight. Next to DPTE, the natural dibromo-trichloromonoterpene (MHC-1), the anthropogenic BDE 47 and BDE 99, as well as ATE, 3,5-dibromo-2-(2′,4′-dibromo)phenoxyanisole (6-MeO-BDE 47), 2-bromoallyl-2,4,6tribromophenyl ether (BATE), and 4,6-dibromo-2-(2′,4′dibromo)-phenoxyanisole (2′-MeO-BDE 68) were present with decreasing relevance. BATE, which was detected for the first time in environmental samples, was synthesized from DPTE by E2 elimination. In brain samples of the harp seals, DPTE, ATE, and BATE were the most abundant organobromine compounds, whereas polybrominated diphenyl ethers (PBDEs) and MHC-1 were virtually absent. This indicated that DPTE, ATE, and BATE were able to penetrate the blood-brain barrier. The general co-occurrence of ATE and BATE in samples contaminated with DPTE support the hypothesis that these compounds are biotransformation products of DPTE. Anaerobic transformation studies of DPTE with super-reduced corrinoids resulted in the formation of ATE. Furthermore, 2,4,6-tribromophenol (TBP) and two other unknown minor transformation products were detected.

Introduction Many brominated compounds have been or are still used in fire prevention since the 1970s (1, 2). The persistence along with toxic effects reported for some brominated flame retardants (BFRs) has initiated intense research programs * Corresponding author phone: +49 711 459 24016; fax: +49 711 459 24377; e-mail: [email protected]. 1590

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on the distribution and fate of these compounds in the environment (3, 4). Important representatives of BFRs of environmental concern are polybrominated biphenyls (PBBs) (1), polybrominated diphenyl ethers (PBDEs) (1-3), hexabromocyclododecanes (HBCDs) (5), and tetrabromobisphenol-A (TBBPA) (4). A method frequently used for the quantification of BFRs is GC in combination with electron capture negative ion mass spectrometry (GC/ECNI-MS) in the selected ion monitoring (SIM) mode. Since brominated compounds usually form intense bromide ion isotopes (m/z 79 and 81), ECNI-MSSIM allows for the detection of all GC-accessible organobromine compounds in a sample (6, 7). However, not all peaks detected that way in environmental and food samples could be traced back to BFRs. In fact, some previously unknown peaks have been identified as halogenated natural products (HNPs). For instance, marine birds from Canada showed high concentrations of hexahalogenated 1,1′-dimethyl-2,2′-bipyrroles (HDBPs) (8), dolphins accumulated high concentrations of polybrominated phenoxyanisoles (911), hydroxylated and methoxylated polybrominated diphenyl ethers were found in salmon plasma from the Baltic Sea and whales (12, 13), and the naturally produced dibromotrichloro-monoterpene MHC-1 was detected in marine samples from all over the world (14). In addition, contamination with a hitherto unknown pentabrominated compound labeled UBC-1 was reported in the blubber extract of a hooded seal (Cystophora cristata) from the Barents Sea (7). On the basis of the GC/ECNI-MSSIM response of m/z 79, UBC-1 was the most abundant peak in this sample. Further abundant organobromines were the anthropogenic PBDEs and the HNP MHC-1 (7). At this point, it remained unclear if UBC-1 was an anthropogenic BFR or a marine HNP. GC/MS data of UBC-1 were similar to those of 2,3dibromopropyl-2,4,6-tribromophenyl ether (DPTE) (7, 15, 16). DPTE is the main constituent of the BFR Bromkal 73-5 PE (Chemische Fabrik Kalk), which was used in extrusion material for polypropylene (PP) and as an additive to acrylonitrile-butadiene-styrene copolymers (17-20). The aim of this work was to clarify the identity of UBC-1 and to investigate some aspects of its environmental properties (e.g., the reactivity against the super-reduced corrinoids).

Materials and Methods Chemicals and Standards. Allyl-2,4,6-tribromophenyl ether (ATE), 98% purity, was from Merck (Darmstadt). 4,6Dibromo-2-(2′,4′-dibromo)-phenoxyanisole (2′-MeO-BDE 68, commercially available as BC-2 from LGC Promochem) was synthesized in our research group (21). Octachloronaphthalene (Dr. Ehrenstorfer, Augsburg, Germany) was used as an internal standard for GC/MS analysis. Vitamin B12 (cyanocobalamin, CCA) was obtained from Sigma, and titanium(III) citrate was prepared from titanium(III) chloride (Acros) according to Ruppe et al. (22). Sodium thiosulfate, bromine, and potassium hydroxide were from Fluka. Isooctane (Suprasolv for gas chromatography) and n-hexane (organic trace analysis) were from Merck. Ligroin, methanol (both technical grade, glass-column distilled), trichloromethane, and acetonitrile (both p.a., Fisher Scientific) were also used as solvents. Nuclear Magnetic Resonance Spectroscopy (NMR). NMR analyses were performed with a Varian Inova 300 MHz spectrometer equipped with a 3 mm i.d. PFG probe. The 1H and 13C chemical shifts were referenced to the signal at δH/C 7.27/77.0 ppm of CDCl3 (CHCl3 impurities) relative to TMS. 10.1021/es062383s CCC: $37.00

 2007 American Chemical Society Published on Web 01/30/2007

FIGURE 1. Electrophilic addition of bromine to allyl-2,4,6-tribromophenyl ether (ATE) and formation of two enantiomers of 2,3-dibromopropyl2,4,6-tribromophenyl ether (DPTE) (left reaction) and E2 elimination of the primary bromine of DPTE and formation of 2-bromoallyl-2,4,6tribromophenyl ether (BATE) (right reaction). Synthesis of 2,3-Dibromopropyl-2,4,6-tribromophenyl Ether (DPTE). Allyl-2,4,6-tribromophenyl ether (20.0 g, 53.9 mmol) was dissolved in 70 mL of CHCl3 and cooled in an ice bath. A total of 3.0 mL (58.6 mmol) of bromine in 6.0 mL of CHCl3 was added dropwise within 30 min while the temperature was held at 0-5 °C. The mixture was brought to ambient temperature and stirred for another 30 min. Excessive bromine in the reaction mixture was removed by addition of an aqueous solution of sodium thiosulfate inducing the formation of insoluble NaBr. The organic phase was separated and desiccated (Na2SO4), and the solvent was removed in a rotary evaporator. The resulting bright yellow oil was recrystallized from ligroin (40-60 °C) at -18 °C. We obtained 21.9 g (41.2 mmol) of colorless needles that correspond with 76% yield. Both the 1H NMR spectrum (300 MHz) in CDCl3 (3.954.56 ppm, m, for five aliphatic hydrogens, and 7.70 ppm, s, for two aromatic hydrogens) and the 13C NMR spectrum (75 MHz) in CDCl3 (33.4-73.4 ppm for three aliphatic carbons and 118-152 ppm for six aromatic carbons) of the recrystallized product were in accordance with literature data (19). The synthesized product had a melting point of 41-42 °C, which closely matched the value of 42.5-43.5 °C reported by Raiford and Birosel (23). Stock solutions of DPTE were of >98% purity according to GC/EI-MS full scan measurements. Synthesis of 2-Bromoallyl-2,4,6-tribromophenyl Ether (BATE). DPTE (308.5 mg, 0.581 mmol) was dissolved in 20 mL of acetonitrile, and 25 mL of an aqueous solution of potassium hydroxide (5.32 mol/L) was added. The mixture was heated under reflux for 2 h, and then the mixture was brought to ambient temperature. The product was extracted with 40 mL of n-hexane in three portions. The organic phase was separated and desiccated (Na2SO4), and the solvent was removed in a rotary evaporator. The resulting colorless solid was recrystallized from methanol. A total of 118.7 mg (0.264 mmol) of colorless needles was obtained, which corresponds with 45% yield. Both the 1H NMR spectrum (300 MHz) in CDCl3 (4.62 ppm, s, for aliphatic CH2, 5.80 ppm, s (trans-allyl-CH2), 6.27 ppm, s (cis-allyl-CH2), 7.70 ppm, s, for two aromatic hydrogens) and the 13C NMR spectrum (75 MHz) in CDCl3 (75.8, 119, and 126 ppm for three aliphatic carbons and 118, 119, 135, and 152 ppm for six aromatic carbons) of the recrystallized product were in good agreement with data calculated using ACD/HNMR Predictor Version 2.51 (Advanced Chemistry Development Inc., http://www. acdlabs.com). Stock solutions of BATE were virtually free of impurities (>97% as determined with GC/EI-MS). Samples and Sample Cleanup. Analyses were performed with available sample extracts of blubber and brain of hooded seals (C. cristata) and harp seals (Phoca groenlandica) from the Barents Sea as well as harp seals from the Greenland Sea (24, 25). Information on age and gender was not available. Sample cleanup was as previously described. In brief, 2-10 g of blubber or brain of harp seals was heated with perchloric acid/acetic acid (1:1, v/v) for 4 h at 75 °C. Lipophilic compounds were extracted with 60 mL of n-hexane and treated with 10 mL of H2SO4, which was renewed daily during

the 5 days. Following that, a final purification step on 4 g of deactivated silica (30% water w/w, elution with 50 mL of n-hexane) was carried out (24). Hooded seal samples (blubber or lyophilized brain) were purified using microwave assisted extraction with n-hexane using weflon disks as microwave adsorbent. The extract was purified by adsorption chromatography on silica as mentioned previously (26). Octachloronaphthalene was added to the sample extracts prior to GC/MS measurements. Anaerobic Transformation Study with Corrinoids. Abiotic transformation of 10 µg (0.018 µmol) of DPTE with corrinoids was performed according to Gaul et al. (27). A total of 19.44 µmol of titanium(III) citrate was used to transfer 0.401 µmol of vitamin B12 (CCA) into super-reduced vitamin B12 (CCAs) (22, 27). 2′-MeO-BDE 68 was used as a recovery standard. Spiked controls without the strong reducing agent titanium(III) citrate showed no transformation of DPTE. All experiments were performed in duplicate in good correlation, and in the case of 24 h incubation, only a single measurement was available. As an addition, the internal standard octachloronaphthalene was used for GC/MS analysis. DPTE, ATE, and BATE were quantified using standard solutions, and concentrations of other organobromines were estimated by using the response factor of ATE. Known molecular masses (DPTE, ATE, and TBP) or assumed molecular weights for two unknown transformation products (labeled A and B) were used for mass balances. Gas Chromatography in Combination with Mass Spectrometry (GC/MS). All GC/MS analyses were performed with a CP-3800/1200 system (Varian). Helium 5.0 (Sauerstoffwerke Friedrichshafen) was used as carrier gas at a constant flow of 1 mL/min. GC conditions (DB5-like Factor Four VF 5-MS column, 30 m × 0.25 mm i.d. × 0.25 µm film thickness, Varian) were reported elsewhere except for a final isothermal phase of 4 min instead of 24 min (28). In the electron capture negative ion (ECNI) mode, methane 4.5 (Air Liquide) was used as a reagent gas at ∼8.5 Torr. The electron energy was set at 70 eV, and the ion source temperature was set at 150 °C. A scan time of 0.5 s/cycle was used, and the SIM peak width was 0.5 u. For GC/ECNI-MS analysis in full scan mode (m/z 70-550), the detector voltage was set at 1200 V. In the electron ionization (EI) mode, the electron energy was set at 70 eV, and the ion source temperature was set at 200 °C. In the EI-full scan mode (m/z 50-550), the detector voltage was set at 1200 V. Enantiomer Separation of DPTE by Gas Chromatography in Combination with Electron Capture Detection (ECD). A 5890A GC/ECD (Hewlett-Packard) was used in combination with a 7673A autosampler. Helium 5.0 was applied as the carrier gas at a constant column head pressure of 20 psi. The injector (splitless mode) and the ECD temperatures were set at 250 and 300 °C, respectively. Enantioseparation was obtained on a 25 m × 0.25 mm i.d. capillary column coated with 0.25 µm permethyl-β-cyclodextrin covalently bonded to dimethyl polysiloxane (β-PMCD, Chirasil-Dex). The GC oven temperature program for DPTE started at a temperature of 80 °C (hold time 1 min), which then was raised at 10 °C/min to 150 °C (hold time 192 min) VOL. 41, NO. 5, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. GC/EI (left panels) and GC/ECNI (right panels) full scan mass spectra of (a and b) allyl-2,4,6-tribromophenyl ether (ATE), (c and d) 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE), (e and f) 2-bromoallyl-2,4,6-tribromophenyl ether (BATE), as well as the transformation products by anaerobic treatment of DPTE (g and h) A and (I and j) B. and finally at 20 °C/min to 200 °C (hold time 7.5 min); the total run time was 210 min. With this oven program, DPTE eluted in the isothermal phase at 150 °C.

Results and Discussion Structure Confirmation of Synthesized DPTE. DPTE was not commercially available, and thus, it was synthesized by electrophilic addition of bromine to allyl-2,4,6-tribromophenyl ether (ATE) (Figure 1). ATE has been used as BFR (e.g., Bromkal 64-3 AE, Chemische Fabrik,Great Lakes PHE-65). The GC/EI-MS spectrum of ATE was characterized by the molecular ion (m/z 368), [M - Br]+ (m/z 289) as well as [M - 2Br]+ (m/z 210). Elimination of the allyl group led to [C6H2OBr3]+ (m/z 327) and [C6H3OBr3]+ (m/z 328) fragment ions. Additional elimination of CO resulted in the formation of five-membered ring compounds ([C5H2Br3]+ (m/z 299), [C5H2Br]+ (m/z 141), and [C5H2]+ (m/z 61); Figure 2a). The GC/ ECNI-MS spectrum of ATE was exclusively composed of [Br](m/z 79), [M - Br]- (m/z 289), and [M - Br + H]- (m/z 290) fragment ions (Figure 2b). ATE eluted with a similar retention time as lindane from the used Factor Four VF 5-MS column (Figure 3a). The addition of Br2 to ATE led to DPTE, which had a much higher retention time than the starting material (Figure 3b). The GC/EI-MS spectrum of DPTE (Figure 2c) showed only traces of the molecular ion (m/z 526), as well as [M Br]+ (m/z 447), [M - 2Br]+ (m/z 368), [M - Br - 2HBr]+ (m/z 287), and [M - 3Br]+ (m/z 289) fragment ions. The base peak [C6H3OBr3]+ (m/z 328) fragment ion was most likely formed 1592

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FIGURE 3. GC/ECNI-MS full scan chromatogram (m/z 79 and 81) of (a) allyl-2,4,6-tribromophenyl ether (ATE, 98%), (b) the synthesized and recrystallized product 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE), (c) blubber extract from a harp seal (P. groenlandica), (d) brain extract from a harp seal, and (e) the synthesized and recrystallized product 2-bromoallyl-2,4,6-tribromophenyl ether (BATE). Relative retention times based on DPTE (1.00): BDE 28 (0.87), BDE 47 (1.07), BATE (0.69), BDE 15 (0.66), ATE (0.45), and lindane (0.45). by elimination of the dibromopropyl group (m/z 199) and rearrangement of the γ-hydrogen from the molecular ion. This fragment ion represents TBP (Figure 2c). Similar to ATE,

FIGURE 4. GC/ECD chromatograms of the enantioseparation of 2,3dibromopropyl-2,4,6-tribromophenyl ether (DPTE) (10 ng/µL) on a 25 m β-PMCD column using (a) an isothermal elution temperature of 150 °C, (b) a temperature program ramp of 10 °C/min, and (c) a temperature program raise at 1 °C/min.

TABLE 1. Concentrationsa of ATE, BATE, and DPTE in Blubber and Brain of Harp Seals (P. groenlandica) and Ratios of ATE/DPTE and BATE/DPTE ATE BATE DPTE ATE/DPTE BATE/DPTE a

blubber (n ) 4)

brain (n ) 4)

5.4 - 9.1 4.9 - 6.5 322 - 470 0.018 ( 0.002 0.015 ( 0.004

3.1 - 10 1.9 - 8.0 130 - 340 0.030 ( 0.008 0.019 ( 0.01

µg/kg wet weight.

elimination of COH resulted in [C5H2Br3]+ (m/z 299), [C5H2Br]+ (m/z 141), and [C5H2]+ (m/z 61). A further characteristic fragment ion was [C3H4Br]+ (m/z 119). The GC/ECNI-MS spectrum of DPTE was dominated by [Br]- (m/z 79) and [Br2]- (m/z 158) fragment ions. At higher mass, there were [M - Br]- (m/z 447), [M - 2Br]- (m/z 368), [M - 3Br]- (m/z 289), [M - 3Br + H]- (m/z 290), [C6H2OBr3]- (m/z 327), [C6H3OBr2]- (m/z 328), and [C6H3OBr2]- (m/z 249) fragment ions detectable (Figure 2d). Further structural proof was obtained by the enantioseparation of DPTE. Electrophilic addition of bromine to nonchiral ATE leads to an asymmetric carbon on DPTE (Figure 1). According to the reaction mechanism, DPTE was formed as the racemate. Enantioselective GC was used for the separation of the DPTE enantiomers (Figure 4). Both retention time and GC/EI and GC/ECNI-MS spectra of DPTE were identical with those reported for UBC-1 (7). Spiking of DPTE into seal blubber extracts confirmed its identity with UBC-1. In addition to DPTE, MHC-1 (7), BDE 47, BDE 99, ATE, 3,5-dibromo-2-(2′,4′-dibromo)-phenoxyanisole (6-MeO-BDE 47), BATE, and 2′-MeO-BDE 68 were detected with decreasing abundance (Figure 3c). The dominance of DPTE over PBDEs was surprising since the latter substance class is usually the major contaminant arising from BFRs in adipose tissue of environmental samples (4). Surprisingly, DPTE was scarcely mentioned in reviews on BFRs (18, 20, 29). Only limited information is available about the amount and period of Bromkal 73-5 PE production. Furthermore, no manufacturer other than Chemische Fabrik Kalk has reported producing DPTE. The patent on DPTE as BFR from 1974 indicated that the production may have started in the mid-1970s (17). A GC/ECNI-MS full scan chromatogram (m/z 79 and 81) of seal brain extract was also dominated by DPTE (Figure 3d). However, MHC-1 and PBDEs were virtually absent, whereas ATE and BATE caused the second and third most abundant peaks. This indicated that DPTE, ATE, and BATE are able to penetrate the blood-brain barrier to a much higher degree than MHC-1 and PBDEs.

FIGURE 5. Time profile of the abiotic transformation of 2,3dibromopropyl-2,4,6-tribromophenyl ether (DPTE) to its metabolites allyl-2,4,6-tribromophenyl ether (ATE), 2,4,6-tribromophenol (TBP), as well as the unknown compounds A and B. Abundances on a molar basis of ATE and DPTE are based on standard solutions, and the transformation products TBP, A, and B were quantified by usage of the ATE response factor. Relative retention times based on DPTE (1.00): A (0.33), TBP (0.36), B (0.41), and ATE (0.45). Structure Determination of 2-Bromoallyl-2,4,6-tribromophenyl Ether (BATE). Because of the fact that ATE and BATE were abundant in all samples positively tested on DPTE and even enriched in brain, it was presumable that BATE was structurally related to ATE and DPTE. Furthermore, the occurrence of the [Br2]- fragment ion (m/z 158) and a retention time on a nonpolar DB5-like column between the tribrominated ATE and the pentabrominated DPTE (Figure 3e) indicated a tetrabrominated compound with the backbone of either ATE or DPTE, including one aliphatic carbon substituted with bromine. Since tetrabrominated analogues of ATE and DPTE were not commercially available, we attempted a synthesis of BATE. Electrophilic and radical addition of HBr using ATE led to different products, which did not match BATE (data not shown). However, treatment of DPTE with KOH finally led to BATE (Figures 1 and 3e). Both retention time and GC/ECNI-MS spectrum and spiking experiments confirmed the identity of BATE in the seal samples. The GC/EI-MS spectrum of BATE exhibited the molecular ion (m/z 446), [M - Br]+ (m/z 367), [M - 2Br]+ (m/z 288), [M - Br - HBr]+ (m/z 287), and [M - 3Br]+ (m/z 209). Additionally, [C6H3OBr3]+ (m/z 328), [C6H2OBr3]+ (m/z 327), [C5H2Br3]+ (m/z 299), [C5H2Br]+ (m/z 141), and [C5H2]+ (m/z 61) fragment ions similar to ATE were detectable, and [C3H4Br]+ (m/z 119) was formed equally to DPTE (Figure 2e). The GC/ECNI-MS spectrum of BATE was mainly composed of [Br]- (m/z 79) and [Br2]- (m/z 158) fragment ions. [M Br + H]- (m/z 368), [M - 2HBr]- (m/z 286), [M - Br - HBr](m/z 287), [M - 2Br]- (m/z 288), and [C6H2OBr3]- (m/z 327) fragment ions were detected with low abundance (Figure 2f). BATE has not been used as a BFR but was obtained by the thermolysis of DPTE (30). Note that BATE has been determined for the first time in environmental samples. Concentrations of DPTE, ATE, and BATE in Environmental Samples. The concentrations of DPTE in blubber and brain were up to 470 and 340 µg/kg wet weight, respectively (Table 1). In both tissues, ATE and BATE were more than 1 order of magnitude less abundant than DPTE, but the ratios of both ATE and BATE to DPTE were virtually constant within each sample matrix (Table 1). Unfortunately, we could not identify data of BFRs in seal brain in the literature. PCBs concentrations in brain of harbor seals (Phoca vitulina) account for 1-5% of the concentration in blubber (31). In brain samples of harp seals from the Greenland Sea, enrichment of DPTE was about 5-fold higher as compared to PCBs and PBDEs (data not shown). Additionally, other pairs of brain and blubber samples of harp and hooded seals VOL. 41, NO. 5, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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from the Barents Sea showed an enrichment of DPTE in the brain that was up to 30-fold higher as compared to PCBs and PBDEs. Despite their high abundance in our samples, data on DPTE and ATE are scarcely described in the literature. Oehme et al. detected an interference component of lindane in moss samples on a DB5-like GC column, which was tentatively labeled as unknown BFR (15). Both GC/ECNI-MS data and retention time clarified that this unknown BFR was ATE. However, DPTE was not studied by Oehme et al. (15). In the ECD chromatogram of the PCB fraction isolated from snoek fillet (Thyrsites atun) caught in the South Atlantic, DPTE caused the highest peak but was not quantified (32). Concentrations of DPTE in the North Pacific were from 0.3 to 5.6 µg/kg lipid weight (33). In sewer slime from German urban residential zones, amounts of ATE and DPTE were up to 50 and 1940 µg/kg dry weight, respectively (16). In these samples, DPTE was 1 order of magnitude more abundant than PBDEs. Despite these few data, the occurrence in samples from two oceans as well as in urban wastewater effluents documented the environmental relevance of DPTE. It might be speculated that DPTE and its metabolites ATE and BATE were frequently overseen when GC/EI-MS in the SIM mode was used or misinterpreted (e.g., as an unknown PBDE congener) when GC/ECNI-MS in the SIM mode (m/z 79 and 81) was applied. For instance, BATE eluted only slightly after BDE 15 using the GC conditions in this study. Anaerobic Transformation Study with Corrinoids. After 1 h incubation with CCAs, DPTE was almost completely transformed (Figure 5). The main product ATE amounted for 68% of the initial pool of DPTE. Together with two additional minor transformation products (i.e., the unknown compound labeled A and TBP), 85% of the initial pool could be detected on a molar basis. GC/EI-MS of compound A showed the assumed molecular ion (m/z 290), as well as [M - CH3]+ (m/z 275), [M - Br - CH3]+ (m/z 196), and [M 2Br]+ (m/z 132) fragment ions (Figure 2g). In GC/ECNI-MS, the assumed molecular ion (m/z 290) was the base peak, but [Br]- (m/z 79) was also detectable (Figure 2h). The isotope pattern of m/z 290 indicated that there are two bromine substituents on A and that the molecular formula was C9H8Br2O. After 24 h of incubation, the intermediate products ATE and A were again almost completely transformed, but no additional brominated compound except the unknown compound B was detected, Although the TBP concentration more than doubled, only 26% of the initial pool was detected on a molar basis. GC/EI- and GC/ECNI-MS spectra of the transformation product B were similar with A due to the same molecular ion (m/z 290) and identical fragment ions, which differed only by different relative abundances (Figure 2i,j). Furthermore, B eluted significantly later from the column than A (Figure 5). After 72 h of anaerobic incubation, all intermediate products were below the level determined after 24 h (Figure 5). Only 4.6% of the initial pool was detected on a molar basis. No further transformation product could be observed in the GC/ECNI-MS full scan chromatograms (m/z 79 and 81). Neither BATE nor any other tetrabromo compound was observable as an intermediate throughout the incubation phase. Thus, successive Br-H exchange as found for PBDEs (27) was not the dominating initial step in the anaerobic transformation of DPTE by super-reduced corrinoids. For DPTE, elimination of Br2 from the side chain giving the formation of ATE was the principal initial anaerobic transformation pathway. Metabolism, Fate, and Perspectives on DPTE. Our analyses indicate that ATE is the major metabolite of DPTE both in mammals and under anaerobic conditions using super-reduced corrinoids. ATE itself was also used as flame 1594

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retardant, which represents an additional source. However, the general co-occurrence of ATE in samples contaminated with DPTE and the relatively constant ratios of ATE/DPTE and BATE/DPTE support the hypothesis that residues of ATE found in the seals mainly originate from the transformation of DPTE. On the other hand, exclusive detection of BATE in the seal samples became possible by the usage of GC/ECNIMS, which was ∼100-fold more sensitive as GC/EI-MS. The chirality of DPTEsin contrast to ATE and BATEs offers opportunities for detailed studies on its enantioselective fate, a topic that is currently the focus of diverse scientific activities (34, 35). The methods for both enantioseparation as well as the sensitive and selective determination of DPTE described in this paper will allow for more detailed studies on the occurrence and fate of DPTE in the environment.

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Received for review October 5, 2006. Revised manuscript received December 20, 2006. Accepted December 20, 2006. ES062383S

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