Environ. Sci. Technol. 2005, 39, 7784-7789
Tetra- and Tribromophenoxyanisoles in Marine Samples from Oceania JOACHIM MELCHER,† DANIEL OLBRICH,† GO ¨ RAN MARSH,‡ VLADIMIR NIKIFOROV,§ CAROLINE GAUS,¶ SIMON GAUL,† AND W A L T E R V E T T E R * ,† Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, D-70599 Stuttgart, Germany, Department of Environmental Chemistry, Stockholm University, Svante Arrhenius va¨g 12, SE-10691 Stockholm, Sweden, Department and Institute of Chemistry, St. Petersburg University, 198504, Universitetskii pr., 26, St. Petersburg, Russia, and National Research Centre for Environmental Toxicology, University of Queensland, 39 Kessels Road, Brisbane, Queensland 4108, Australia
Some methoxylated polybrominated diphenyl ethers (MeO-BDEs) are known halogenated natural products (HNPs) and are frequently detected in higher organisms of the marine environment. In this study we demonstrate that a prominent MeO-BDE, previously detected in marine mammals from Australia, is identical to 3,5-dibromo-2-(2′,4′dibromo)phenoxyanisole (BC-3, 6-MeO-BDE 47). Up to 1.9 mg/ kg of 6-MeO-BDE 47 was present in cetaceans from Australia, 0.2-0.3 mg/kg in two crocodile eggs from Australia, but concentrations of 1 or 2 orders of magnitude lower were found in shark liver oil from New Zealand and in marine mammals from Africa and the Antarctic. Concentrations of 6-MeO-BDE 47 in samples from Australia were in the same range as anthropogenic pollutants such as PCB 153 and p,p′DDE. Along with 6-MeO-BDE 47 and the known HNP 4,6dibromo-2-(2′,4′-dibromo)phenoxyanisole (BC-2, 2′-MeO-BDE 68), several tribromophenoxyanisoles (MeO-triBDE) were present in tissue of Australian cetaceans. To determine their structure, abiotic debromination experiments were performed using 6-MeO-BDE 47 and 2′-MeO-BDE 68 and superreduced dicyanocobalamine. These experiments resulted in formation of eight MeO-triBDEs, all of which were detected in the cetacean samples. Five of these eight MeO-triBDEs could be identified based on two standard compounds as well as gas chromatographic and mass spectrometric features. It was also shown that the first eluting isomer (compound 1), 6-MeO-BDE 17 (compound 2), and 2-MeO-BDE 39 (compound 5) were the most prominent MeO-triBDEs in the Australian cetacean samples. The concentrations of the MeO-triBDEs in two cetacean samples were 0.20 and 0.36 mg/kg, respectively. Although the reductive debromination with dicyanocobalamine resulted in a different congener pattern than was found in the marine mammals, it could not be excluded that the tribromo * Corresponding author phone: +49 711 459 4016; fax: +49 711 459 4377; e-mail
[email protected]. † University of Hohenheim. ‡ Stockholm University. Present address: Department of Environmental Chemistry, IIQAB, CSIC, Jordi Girona, 10-18, 08034 Barcelona, Spain. § St. Petersburg University. ¶ University of Queensland. 7784
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congeners of 6-MeO-BDE 47 and 2′-MeO-BDE 68 in the samples were metabolites of the latter.
Introduction A wide variety of halogenated natural products (HNPs) of marine origin are produced by diverse marine organisms (1-3), and HNPs are increasingly recognized as critical residues in foodstuff (e.g., fish) and environmental samples (e.g., marine mammals and birds). Some HNPs (heptachloro1′-methyl-1,2′-bipyrrole Q1 (4), the dibromotrichloro monoterpene MHC-1 (5), the tetrabromophenoxyanisole BC-2 or 2′-MeO-BDE 68 (6), and hexahalogenated 1,1′-dimethyl-2,2′bipyrroles (7)) have been detected in a range of fish and marine mammal samples at concentrations sometimes exceeding those of PCBs, DDT, and other anthropogenic pollutants (6, 7). Initially, it could not be determined whether the source of polybrominated phenoxyanisoles is of natural or anthropogenic origin, since they could arise from biotransformation of anthropogenic brominated diphenyl ethers (8, 9). However, the identification of sponges as a natural producer of these compounds in the habitat of the marine mammals with high polybrominated phenoxyanisole concentrations and low BDE concentrations provides evidence for a natural origin (6). This was recently confirmed by radioisotopic measurements of MeO-BDEs isolated from blubber of marine samples (10). Previous investigations indicated that cetaceans from Queensland, Australia contained particularly high concentrations of the HNPs (6, 11). Subsequently, Q1 was identified as heptachloro-1′-methyl-1,2′-bipyrrole, BC-2 as 4,6-dibromo-2-(2′,4′-dibromo)phenoxyanisole, BC-10 as 1,1′-dimethyl-3,3′,4,4′-tetrabromo-5,5′-dichloro-2,2′-bipyrrole, and BC-11 as 3,5-dibromo-2-(3′,5′-dibromo-2′-methoxy)phenoxyanisole (2′,6-diMeO-BDE 68) (4, 6, 12). BC-3 was tentatively identified as a tetrabromophenoxyanisole (11, 13). Natural products chemists have identified 3,5-dibromo2-(2′,4′-dibromo)phenoxyanisole (6-MeO-BDE 47) in sponges from the Indian Ocean (14). This compound was recently found to be abundant in samples from the Baltic Sea and the Pacific Ocean (15). This corresponds well to our detection of BC-3 in samples from Europe, Africa, and the Antarctic (11, 13). In this study we used an authentic synthesized standard for the verification of the structure of BC-3 and screened the samples for additional MeO-BDEs.
Materials and Methods Samples and Chemicals. Australian samples were obtained from Queensland, Australia. Commercial shark liver oil from New Zealand was sourced from Lovely Health (Auckland, New Zealand). Additional sample extracts were obtained and purified as described elsewhere (13). BC-2 (16), 3,5-dibromo2-(2′,4′-dibromo)phenoxyanisole (6-MeO-BDE 47) (17), 5-bromo-2-(2′,4′-dibromo)phenoxyanisole (6′-MeO-BDE 17) (17), and 3-bromo-2-(2′,4′-dibromo)phenoxyanisole (2′-MeO-BDE 28) (18) were weighed into calibrated flasks and stepwise diluted to concentrations ranging from 1 pg/µL to 10 ng/µL (calibration range). PCB 153 and p,p′-DDE were available as certified single standards (LGC Promochem, Wesel, Germany). The recovery standard perdeuterated R-HCH (RPDHCH) was synthesized as described elsewhere (19). Cyclohexane (purest; Merck, Germany), ethyl acetate (Acros, Germany), n-hexane (Unisolv; Merck, Germany), and isooctane (ECD tested for pesticide analysis; Acros, Germany) were used as solvents. Silica gel (60, purest, for column chromatography) was obtained from Merck, Germany. 10.1021/es051090g CCC: $30.25
2005 American Chemical Society Published on Web 09/07/2005
Sample Cleanup. Samples, except blubber and oils, were lyophilized prior to extraction, followed by accelerated solvent extraction (ASE, Dionex), gel permeation chromatography, and adsorption chromatography on modified silica. Details are reported elsewhere (20, 21). Abiotic Anaerobic Transformation. Anoxic debromination experiments were performed to determine the structure of MeO-triBDEs. The procedure was carried out using the conditions described by Ruppe et al. (22). All steps were executed under anoxic conditions using an anaerobic chamber. A volume of 0.5 mL of a 0.866 µM dicyanocobalamine solution diluted with 4.5 mL of a TRIS-HCl buffer (pH ) 7.5) was superreduced (Co(III) to Co(I)) by adding 100 µL of a buffered 0.19 M titanium(III) citrate solution. n-Hexane solutions of 5.4 mg of 6-MeO-BDE 47 or 5.8 mg of 2′-MeO-BDE 68 were added, and the vials were capped, shaken for a short time and stored under dark, anaerobic conditions at room temperature. After 4 days, the vials were opened and purged for ∼10 min with synthetic air (Linde, Germany) to stop the transformation process. Lipophilic transformation products were extracted twice with n-hexane, and the combined organic phase was dried over sodium sulfate. Once concentrated to 1 mL, it was analyzed by GC/MS. Gas Chromatography. GC/MS analyses were performed with a 3800 gas chromatograph connected to a 1200 triple quad mass spectrometer (Varian, Darmstadt, Germany). GC conditions were reported elsewhere (21). A solvent delay of 5 min was used to protect the filament. In the gas chromatography/electron capture negative ion (GC/ECNI-MS) mode, methane (purity 4.5) was used as reagent gas at a pressure of 8 Torr in the ion source. To overcome coelution of various analytes, the following temperature program was used for quantification: 70 °C (2 min), 30 °C/min to 210 °C (1 min), 2 °C/min to 240 °C (2.33 min), 10 °C/min to 300 °C (10 min). In the selected ion monitoring mode, m/z 79, 81, 159, and 161 were measured throughout the run. In the full scan mode, m/z from 70 to 550 were scanned twice a second. In the electron ionization mode (GC/EI-MS), a mass range from m/z 200 to 550 was chosen for the recording of full scan spectra. Additional GC/EI-MS analyses were also performed with a 3400/Saturn 4D iontrap GC/MS system (Varian, Darmstadt, Germany). The split/splitless injector was operated in splitless mode for 2 min and kept at 225 °C. The carrier gas (helium, purity 5.0) was used at a constant pressure of 22.5 psi. A chiral β-BSCD column (30 m, 0.25 mm i.d., 0.25 µm df; BGB Analytik, Adliswil, Switzerland) was installed in the GC oven. The GC oven temperature program was the following: 80 °C (1 min), increasing at 10 °C/min to 180 °C (2 min), and at 5 °C/min to 220 °C (39 min). The transfer line was heated to 230 °C. The manifold was set to 230 °C, and the multiplier voltage was set at 1800 V. At a filament emission current of 80 µA, the mass range was scanned from m/z 200 to 550 twice a second after a solvent delay of 5 min. A volume of 1 µL of sample extract was injected for each run. GC/ECD analyses were performed with Hewlett-Packard 5890 series II systems equipped with 7673A autosamplers, ECDs, and split-splitless injectors operated in the splitless modes recently described in detail (21). GC/ECD was used for quantification of PCBs and p,p′-DDE in the samples. Retention and MS Data of the Reference Standard 2′MeO-BDE 68 and BC-2 in Samples (First Mentioned in Parentheses) and the Reference Standard 6-MeO-BDE 47 and BC-3 in Samples (Second Mentioned in Parentheses). Retention times on CP-Sil 2 (46.0/46.8 min), CP-Sil 8 (39.5/ 40.2 min), CP-Sil 8, 10% C18 (49.2/50.9 min), CP-Sil 8, 20% C18 (56.7/59.0 min), β-BSCD (42.8/47.6 min), GC/EI-ITMS, M+, [M-CH3Br]+, [M-Br2]+ (100:57:48/100:20:17), GC/ECNIMS, M-, HBr2-, Br- (1:8:100/not detected:14:100).
FIGURE 1. GC/EI iontrap mass spectra of 6-MeO-BDE 47 (BC-3) obtained from (a) a reference standard and (b) an extract of the blubber of a bottlenose dolphin (T. truncatus) from Australia.
Results and Discussion Identification of BC-3 as 6-MeO-BDE 47. Both BC-3 in the samples and the authentic 6-MeO-BDE 47 standard had identical retention times on five capillary columns of different polarity and mass spectra (see Materials and Methods). In the GC/EI-MS mode, M+ (m/z 512), [M-Br-CH3]+, and [M-Br2]+ were found in the same ratio in both standard and samples (Figure 1). However, the relative abundances of these ions were different to those found for the isomer 2′-MeOBDE 68 (see Materials and Methods). The GC/ECNI-MS full scan spectrum of 6-MeO-BDE 47 showed fragment ions for [Br]-, [HBr2]-, and [M-HBr2]- in the known ratio for BC-3 (11). GC/ECNI-MS of 2′-MeO-BDE 68 showed a weak molecular ion, which is in contrast to BC-3, where none was detected. On the basis of these parameters, it is established that BC-3 has the structure 6-MeO-BDE 47 (Figure 1a). BC-3 or 6-MeO-BDE 47 was previously detected in environmental samples from Europe (9), Australia, Antarctica (11, 13), and Asia (15). Quantification of 6-MeO-BDE 47. Previous semiquantitative investigations based on the GC/ECD response of PCB 153 indicated high 6-MeO-BDE 47 concentrations in biota from Australia (11). The availability of an authentic standard allowed for the exact quantification of 6-MeO-BDE 47 in the marine samples. An equimolar mixture of 2′-MeO-BDE 68 and 6-MeO-BDE 47 standard resulted in identical GC/ECD, GC/EI-MS (full scan mode), and GC/ECNI-MS (full scan and SIM mode) responses of both analytes. In the GC/EIMS-SIM mode, m/z 516 is suggested for quantification and m/z 514 and/or m/z 518 for verification (Figure 1a). Due to the higher relevance of m/z 516 in the GC/EI-MS of 6-MeOBDE 47, its GC/EI-MS-SIM response was higher by a factor of ∼1.7 compared to that of 2′-MeO-BDE 68. We also determined a virtually identical ECD response of the two MeO-tetraBDEs and PCB 153 (∼0.95). Therefore, literature data based on the ECD response of PCB 153 (see above) could now be verified as quantitation values (Table 1). The highest concentrations of 6-MeO-BDE 47 (up to 1.9 mg/kg fat) were found in cetaceans from Australia (6, 11). This suggests that a considerable population of a natural producer of 6-MeO-BDE 47 must have been in contact with members of the food web of the analyzed dolphins (see discussion below). Indian Ocean sponges (Dysidea sp.) were previously described as a possible natural source of 6-MeOBDE 47 (14). However, 6-MeO-BDE 47 was not identified in the Australian sponges recently described as the producer of 2′-MeO-BDE 68 (BC-2) and 2′,6-diMeO-BDE 68 (BC-11) (6). In the samples of the Australian marine mammals analyzed, 6-MeO-BDE 47 was present at lower concentrations VOL. 39, NO. 20, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Concentrations [µg/kg Lipids] of 6-MeO-BDE 47, p,p′-DDE, PCB 153, and Ratios of 6-MeO-BDE 47 and 2′-MeO-BDE 68 in Marine Biota species
origin
n
6-MeO-BDE 47
common dolphin (D. delphis) bottlenose dolphin (T. truncatus) melonhead whale (P. electra) pygmy sperm whale (K. breviceps) humpback dolphin (S. chinensis) humpback dolphin (S. chinensis), brain crocodile eggs shark liver oil (commercial) mussel tissue pilot whale monk seal (M. monachus) weddell seal (L. weddelli) striped dolphin (S. coeruleoalba) striped dolphin liver (S. coeruleoalba) bottlenose dolphin (T. truncatus) minke whale (B. acutorostrata) Baird’s beaked whale (B. bairdii) seals
Australia Australia Australia Australia Australia Australia Australia New Zealand Mexico Faeroe Islands West Africa Antarctica Japan Japan Japan Japan Japan Baltic Sea
1 1 1 1 1 1 2 1 1 1 5 8 1 1 1 1 1 2
980 1910 790 540 980 46 200-240 4 -b -b e30c e3c 25d 250d 710d 32d 54d 95-160e
a
Averaged over all samples.
b
6-MeO-BDE 47/ 2′-MeO-BDE 68
Only qualitative determinations were carried out. c Data from (6).
0.22 0.17 0.65 0.23 0.54 0.44 ∼3.5a ∼2.3 ∼0.3 ∼12 ∼3.2a ∼4.3a 0.93d 0.86d 0.32d 0.50d 13.2d d
p,p′-DDE
PCB 153
590 1980 610 1120 960
310 1060 120 100 1530
260-1370 174 -b -b e130c
-b -b e160c 25 76 180 7.3 1.0
Data from (15). e Data from (8).
FIGURE 2. GC/ECNI-MS chromatograms (CP-Sil 8, m/z 79 and 81 extracted from the TIC) of sample extracts from (a) common dolphin (D. delphis) and (b) humpback dolphin (S. chinensis), and the MeO-triBDE formed by reductive debromination of (c) 2′-MeO-BDE 68 and (d) 6-MeO-BDE 47. Numbers were assigned with increasing retention time. compared to 2′-MeO-BDE 68 but at equal or higher concentrations compared to the major anthropogenic POPs in the samples, i.e., p,p′-DDE (with the exception of the pygmy sperm whale) and PCB 153 (with the exception of the IndoPacific humpback dolphin) (Table 1). Interestingly, brain of the Indo-Pacific humpback dolphin contained ∼5% of the concentration found in blubber (Table 1). The varying concentrations and ratios of 6-MeO-BDE 47 and 2′-MeOBDE 68 in individuals may be due to different bioavailability, uptake, elimination, metabolism, and selective retention of the HNPs but also may be owing to different ages, feeding behavior, and distribution of the investigated species; however, they might also indicate the presence of different natural producers found in different habitats that enter the food web of the cetaceans in a different way. Further support for the latter can be seen in the results obtained from crocodile eggs. The two investigated crocodile samples contained 20-40% of the concentrations of 6-MeOBDE 47 determined in the marine mammals, but 6-MeOBDE 47 was much more abundant than 2′-MeO-BDE 68 (Table 1). In shark liver oil samples from New Zealand, 6-MeO-BDE 47 accounted only for 40 BDE standards clarified that nonortho BDEs (BDE 35, BDE 37, BDE 77, and BDE 126) did not form the fragment ion at m/z 159 either. Thus, we conclude that formation of m/z 159 requires an o-Br substituent on one of the rings. On the basis of this evaluation, compound 5 must be 2-MeO-BDE 39. Figure 5 shows the proposed mechanism for the formation of m/z 159 which yields a neutral dibromomethoxydibenzofuran species. This ion (m/z 159) was recently structurally described by Landrum et al. (30). Note that the full scan GC/ECNI-MS spectra also showed the corresponding [M-159]- fragment ions at m/z 353 (MeOtetraBDEs) and m/z 275 (MeO-triBDEs), along with the GC/ ECNI-MS spectra of relevant BDE congeners. In the GC/ECNI-MS mode, a tetrabromo byproduct of the BC-2 synthesis was shown to form an abundant ion at m/z 264, which has been interpreted as a [C7H6Br2O]- or [dibromoanisole + H]- fragment ion (16). This fragment ion can be used to determine the distribution of the Br substituents on both phenyl moieties. On the present system, the [C7H6Br2O]- ion (m/z 264) was also found in the GC/ ECNI-MS of 2′-MeO-BDE 68 and 6-MeO-BDE 47 (previously not detected (16)), whereas 2′-MeO-BDE 28 showed the expected monobromo fragment ion [C7H7BrO]- at m/z 186. Accordingly, compound 7 (the tribromo byproduct of the synthesis of BC-2 (16)) must also carry one bromine on the anisole ring, since it forms no ion at m/z 264 (2 Br) but does form an ion at m/z 186 (1 Br). Therefore, compound 7 is either 2′-MeO-BDE 25 or 6′-MeO-BDE 25, but not 2-MeOBDE 34. 2-MeO-BDE 39 (compound 5) and 2-MeO-BDE 34 differ only in the position of the bromine on the left ring, as displayed in Figure 4. Since compound 7 is either 2′-MeOBDE 25 or 6′-MeO-BDE 25, 2-MeO-BDE 34 must elute before 2-MeO-BDE 39 (compound 5). Thus, a bromine substituent in the para position on the left ring caused a longer retention time compared to a substituent in the ortho position. This feature is also generally found for several pairs of BDE congeners (BDE 1 < BDE 3; BDE 8 < BDE 15; BDE 17 < BDE 28 (27)). Therefore, 6-MeO-BDE 17 is compound 2 and 6-MeO-BDE 28 is compound 6 (Figure 4). 7788
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The use of gas chromatographic and mass spectrometric data allowed the assignment of structures to five of the eight tribromo congeners produced by reductive debromination of 6-MeO-BDE 47 and 2-MeO-BDE 68 (Table 2). Parts a and b of Figure 3 illustrate that compound 1, 6-MeO-BDE 17 (compound 2), and 2-MeO-BDE 39 (compound 5) were the most abundant MeO-triBDEs in the Australian samples. It was also shown that 2-MeO-BDE 39 (compound 5) was more abundant than 6′-MeO-BDE 17 (compound 4) which coeluted on CP-Sil 8 (compare Figures 2a and 3a). This is in agreement with GC/MS data. Only 6′-MeO-BDE 17 (compound 4) and not 2-MeO-BDE 39 (compound 5) did form the [M-2Br]+ fragment ion, and this ion was not detected in the investigated samples at the corresponding retention time. Note also that the peak pattern in Figure 2, parts a and b, was different to that in Figure 3, parts a and b. This is due to the lower selectivity of the GC/ECNI-MS method for tribromo-MeOBDEs which can be seen from the fact that more peaks were detected in GC/ECNI-MS compared to the number in the GC/EI-MS chromatograms. Thus, the additional compounds are brominated but do not form the molecular ion of MeOtriBDEs. The different peak abundances in GC/ECNI-MS and GC/EI-MS also demonstrate that compound 6 was interfered with an unknown brominated compound in Figure 2a, which can more clearly be seen in Figure 2b. Therefore GC/EI-MS-SIM provides a higher selectivity for the quantification of MeO-triBDEs in environmental samples. However, only two reference standards were available, and since MeO-BDEs were found to have varying proportions of the molecular ion, GC/ECNI-MS was finally used in this study. In two samples, the concentrations of the MeO-triBDEs were approximately 0.20 and 0.36 mg/kg (Table 2). This represents 20-40% of the amount of 6-MeO-BDE 47 in the respective samples. Individual congeners ranged from 5.5 to 96 µg/kg (Table 2). Both the relative concentrations between the two samples studied, as well as the relative abundance of individual tribromo congeners, varied. For instance, all MeO-triBDEs except 6-MeO-BDE 17 were more abundant in the common dolphin. Environmental Relevance of the Findings. The high abundance of 2′-MeO-BDE 68 and 6-MeO-BDE 47 in Australia and the waters around Japan indicate production hot spots of these compounds in these locations. It is also worth mentioning that in Australia, 2′-MeO-BDE 68 and 6-MeOBDE 47 appear to be mostly related to sponges, whereas red algae were identified as a natural source in the Baltic Sea (24). Interestingly, these compounds have also been found in fish from freshwater lakes in Northern Europe (31). Our abiotic transformation experiments demonstrated that MeOBDEs may be metabolized as well. However, the reductive
debromination of 2′-MeO-BDE 68 and 6-MeO-BDE 47 led to different peak patterns compared to that found in the cetaceans. The most abundant debromination products of 2′-MeO-BDE 68 (compounds 3 and 7) and 6-MeO-BDE 47 (compound 4) played only a very minor role in the marine mammals, and the most prominent MeO-triBDEs in the cetaceans (see above) were only formed in traces by the corrinoids. Clearly, metabolism could have occurred in the food chain or in the mammals, hence similar or even identical peak patterns in mammals and our transformation experiments cannot be expected. It is noticeable that only two MeO-tetraBDEs but eight MeO-triBDEs, all structurally related to the MeO-tetraBDEs, were present in the dolphins. The lack of identification of several MeO-triBDEs by natural products chemists indicates that, at least to some degree, metabolism of 2′-MeO-BDE 68 and 6-MeO-BDE 47 has played an important role in the formation of MeO-triBDEs. Their formation, however, may not have occurred in the dolphins but could also have occurred during the transfer from the natural producer to the marine mammals via the food chain.
Acknowledgments We are grateful to Jochen F. Mu ¨ ller (National Research Centre for Environmental Toxicology, Cooper Plains, Australia) for his help in gaining access to the Australian samples. W.V. thanks the German Scientific Foundation (DFG) for financial support of this research.
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Received for review June 9, 2005. Revised manuscript received July 29, 2005. Accepted August 2, 2005. ES051090G
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