Hydroxylated and Methoxylated Brominated Diphenyl Ethers in the

Department of Environmental Chemistry, Department of Botany, and Department of Applied Environmental Science (ITM), Stockholm University, SE-106 91 St...
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Environ. Sci. Technol. 2005, 39, 2990-2997

Hydroxylated and Methoxylated Brominated Diphenyl Ethers in the Red Algae Ceramium tenuicorne and Blue Mussels from the Baltic Sea A N N A M A L M V A¨ R N , * , † G O ¨ RAN MARSH,† LENA KAUTSKY,‡ MARIA ATHANASIADOU,† A° K E B E R G M A N , † A N D LILLEMOR ASPLUND§ Department of Environmental Chemistry, Department of Botany, and Department of Applied Environmental Science (ITM), Stockholm University, SE-106 91 Stockholm, Sweden

Methoxylated polybrominated diphenyl ethers (MeOPBDEs) and hydroxylated PBDEs (OH-PBDEs) have recently been identified in fish and wildlife from the Baltic Sea. Both OH-PBDEs and MeO-PBDEs are known natural products, while OH-PBDEs also may be metabolites of PBDEs. The aim of the present study was to determine if the red macroalga Ceramium tenuicorne could be a source for MeO- and OHPBDEs in the Baltic environment. Blue mussels (Mytilus edulis) from the same area were also investigated for their content of MeO- and OH-PBDEs. Seven OH-PBDEs and four MeO-PBDEs were present both in the red macroalga and the blue mussels. The mussels also contained a monochlorinated OH-tetraBDE. One of the compounds, 6-methoxy-2,2′,3,4,4′,5-hexabromodiphenyl ether, has never been reported to occur in the environment. The identification was based on comparison of relative retention times with reference standards, on two gas chromatographic columns of different polarities, together with comparisons of full-scan electron capture negative ionization (ECNI) and electron ionization (EI) mass spectra. It is shown that MeOPBDEs and OH-PBDEs are present in algae, but at this stage it could not be confirmed if the compounds are produced by the alga itself or by its associated microflora and/or microfauna.

first observed in seals and fish, have been identified as methoxylated polybrominated diphenyl ethers (MeO-PBDEs) (6-9). Also, several hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have been identified in blood plasma from Baltic salmon (7, 8). The origin of these OH- and MeOPBDEs is under discussion, and two major sources have been suggested. First, it is clear that PBDEs are metabolized to OH-PBDEs in rats, mice, and fish (10-14), but their potential for formation in invertebrates or in the abiotic environment is unknown. Second, a large number of brominated compounds have been found to be produced by nature (15-17). Both MeO- and OH-PBDEs have been found in, e.g., sponges collected in the marine environment from the Southern Hemisphere (18, 19). One MeO-PBDE (2-methoxy-2′,3,4′,5tetrabromodiphenyl ether) has also been identified in the green alga Cladophora fascicularis collected in Japan (20). Natural production of other types of brominated compounds such as brominated phenols, e.g., 2,3-dibromo-4,5-dihydroxybenzyl alcohol (lanosol) and polybrominated diphenyl methanes have previously been described in several red macroalga species collected along the Swedish West coast (21, 22). These brominated substances were suggested to be produced by enzymatic bromination in the red algae (23). The relatively high concentrations of MeO- and OH-PBDEs in Baltic salmon blood, together with their structures, indicate that there are additional sources besides PBDE metabolism of the OH-PBDEs and MeO-PBDEs detected in Baltic biota (7, 8). The aim of the present study was to investigate if Baltic filamentous macroalgae are a source of OH-PBDEs and MeOPBDEs in biota from the Baltic Sea and if these types of compounds may accumulate in blue mussels living intermingled with the algae.

Materials and Methods Chemicals. Dichloromethane (DCM; Riedel-de Haen, Seelze, Germany) and n-hexane (Fisher Scientific, Leicestershire, U.K.) were of pesticide grade. Methyl tert-butyl ether (MTBE) of HPLC grade was supplied by Rathburn Chemicals (Walkerburn Scotland, U.K.) and was glass distilled prior to use. Hydrochloric acid, sulfuric acid, phosphoric acid, ascorbic acid, potassium hydroxide, and sodium chloride were all of pro analysis quality and purchased from Merck (Darmstadt, Germany). Acetone of pesticide grade was purchased from Merck. Silica gel (0.063-0.2 mm) was obtained from Ma-

Introduction The Baltic Sea has been influenced by human activities during the past half-century, which has led to eutrophication (1, 2) as well as high levels of environmental pollutants, e.g., polychlorinated biphenyls (PCBs), 2,2-bis(4-chlorophenyl)1,1,1-trichloroethane (DDT), and related compounds (3, 4). More recently brominated flame retardants (BFRs), e.g., polybrominated diphenyl ethers (PBDEs), have been reported in all compartments, in the Baltic Sea (5). In addition to BFRs, several other brominated compounds are present in environmental samples from the Baltic Sea. Some of these, * Corresponding author phone: +46-8-163781; fax: +46-8-163979; e-mail: [email protected]. † Department of Environmental Chemistry. ‡ Department of Botany. § Department of Applied Environmental Science. 2990

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FIGURE 1. Scheme for the extraction and cleanup of the algae and mussels. 10.1021/es0482886 CCC: $30.25

 2005 American Chemical Society Published on Web 03/31/2005

FIGURE 2. GC-MS (ECNI) chromatograms of bromide ions (m/z 79, 81). The chromatograms to the left are the methylated phenolic fractions from red algae (a) and blue mussel (c). To the right the neutral fractions of the red algae (b) and blue mussels (d) are presented. Chromatograms e and f (salmon blood sample) have previously been published (8) and are only given herein as comparison. The peak marked UC is a OH-/MeO-monochlorotriBDE (8). The peak marked d3 in the chromatograms shows a mass spectrum of a brominated compound containing three bromine atoms with a molecular ion at m/z 418 earlier reported by Malmva1 rn et al. (30). cherey-Nagel (Du ¨ ren, Germany). Diazomethane was prepared in house from N-methyl-N-nitroso-p-toluenesulfonamide (Diazald) (24) obtained from Sigma-Aldrich (Steinheim, Germany). The 2,2′,3,4,4′,5′-hexabromodiphenyl ether (BDE138), used as retention time standard, and the methoxylated halogenated diphenyl ethers standards were synthesized as described elsewhere (8, 25, 26). Instruments. Gas chromatography-mass spectrometry (GC-MS) analyses were performed on a single-quadrupole, Finnigan MAT SSQ 710, coupled to a Varian 3400 gas chromatograph and a split/splitless injector operated in the splitless mode. Two fused-silica capillary columns were used for the MeO-PBDE analysis. A nonpolar column, CP-Sil 8CB

(30 m × 0.25 mm i.d. and 0.25 µm film thickness) from Chrompack (EA Middelburg, The Netherlands) was programmed as follows: 80 °C (2 min), 10 °C min-1 to 300 °C (10 min). The injector and transfer line temperatures were 260 and 270 °C, respectively. The second GC column was the polar SP-2331 (30 m × 0.25 mm i.d. and 0.2 µm film thickness) from SUPELCO (Bellefonte, USA). The column temperature program was as follows: 80 °C (1 min), 20 °C min-1 to 200 °C (1 min), 3 °C min-1 to 270 °C (12 min). Both the injector and transfer line temperatures were 260 °C. Helium was used as carrier gas. Two different ionization techniques were used: electron ionization (EI) and electron-capture negative ionization (ECNI). For ECNI, methane 5.0 (AGA, Stockholm) VOL. 39, NO. 9, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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(10 mL), and DCM (24 mL) was used as the mobile phase. The solvent volume of the extract was reduced to approximately 100 µL before being transferred to a new column. DCM was replaced with n-hexane, and the volumetric standard BDE-138 was added prior to GC-MS analysis. Procedure solvent blanks were processed in parallel to the samples to control for any systematic contamination. The samples were kept in the dark during the extraction and cleanup to minimize debromination. A second alga sample was extracted and cleaned up to avoid any potential oxidative formation of OH-PBDEs. Hence, also the partitioning with potassium hydroxide was omitted. The alga was extracted as described elsewhere (27) with an addition of ascorbic acid (700 mg) (29). The organic solvent volume of the extract was reduced and transferred to a test tube. The residue was treated with water (1 mL) and extracted with n-hexane/MTBE (9:1, 1 mL). The organic phase was combined with the first extract, and diazomethane (3.5 mL) was added. The analytes were cleaned as described above (Figure 1) prior to GC-MS (ECNI) analysis. Mussels. Mussel tissue (35 g fresh weight, without shells) was extracted and cleaned in a similar way as the alga (Figure 1). A larger volume of acetone (30 mL) was added to make sure that the proteins were denatured and to promote efficient extraction of the more polar lipids.

Results

FIGURE 3. Structures of the compounds identified in red algae, blue mussels, and salmon (8), marked with a, m, and s, respectively. The identification numbers of corresponding OH-/MeO-PBDEs are the same (Figure 2) since the OH-PBDEs are methylated prior to GC-MS analysis. was used as buffer gas. The electron energy was 70 eV, and the ion source temperature was 150 °C for both MS techniques. The spectra were scanned from 32 to 750 m/z in ECNI and from 50 to 750 m/z in EI. The chromatographic data were recorded by an ICIS data system (Finnigan MAT, USA). All glassware was heated in an oven (300 °C overnight) prior to use. A DISP 25 homogenizer (Labassco AB, Partille, Sweden) was used to homogenize the samples. Samples. The red macroalgae, Ceramium tenuicorne (Ku ¨ tz.) Waern, were collected at 3-4 m depth close to Asko¨ island (58°49-50′ N, 17°37-38′ E), which lies in the Southern Stockholm archipelago, in the northern Baltic Proper. The blue mussels (Mytilus edulis) were collected at the same site and depth as the algae. Both the mussels and algae were sampled at the end of June 2000 and were kept frozen at -20 °C until analysis. Extraction and Cleanup Procedure. Algae. The extraction of the algae (80 g fresh weight) was based on a method described by Jensen et al. (27) with the exception that diethyl ether was replaced with MTBE (Figure 1). After extraction and gravimetric determination of the extracted material, phenolic compounds were isolated from neutral compounds by partitioning with potassium hydroxide as described earlier (28). The fractions that contained phenol-type compounds were derivatized with diazomethane in diethyl ether (1 mL) (28). The cleanup procedure is illustrated in Figure 1. Two different columns were used; the first contained activated (300 °C overnight) silica gel impregnated with sulfuric acid (2:1 (w/w), 1 g), while the second was packed with activated silica gel (1 g). The columns were prewashed with n-hexane 2992

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Mass chromatograms (ECNI) of compounds isolated in the phenolic and neutral fractions of the red macroalga and the blue mussels are shown in Figure 2a-d. The structures of seven OH-PBDEs and four MeO-PBDEs were identified in the alga. In the blue mussels, seven OH-PBDEs, one OHchlorotetraBDE, and four MeO-PBDEs were present. The OHPBDEs, in the phenolic fraction of the algae, were as follows: 2′-OH-BDE-68 (peak 2, in Figure 2a), 6-OH-BDE-47 (3), 6-OHBDE-90 (8), 6-OH-BDE-99 (9), 2-OH-BDE-123 (10), 6-OHBDE-85 (11), and 6-OH-BDE-137 (12). The structures are shown in Figure 3. The same OH-PBDEs were also present in the phenolic fraction of the mussel sample together with 6′-Cl-2′-OH-BDE-68 (peak 6, Figure 2c). The corresponding MeO-PBDEs detected in the neutral fraction of both algae and mussels were as follows: 2′-MeO-BDE-68 (2), 6-MeOBDE-47 (3), 6-MeO-BDE-85 (11), and 6-MeO-BDE-137 (12) (Figure 2b,d). PBDE congeners were only present in trace concentrations in the neutral fraction of the alga and mussel samples, while MeO-PBDEs were abundant. The identification of the OH- and MeO-PBDEs in alga and mussel samples was made by comparison of relative retention times (RRTs), of sample peaks and 26 synthesized reference standards (8, 26) versus BDE-138 on two GC columns, a nonpolar (CP-Sil 8CB) and a polar (SP-2331) column. The RRT data of the MeO-PBDEs and the methylated OH-PBDEs analyzed are shown in Table 1. The RRTs were determined from full-scan ECNI chromatograms by recording the ions m/z 79 and 81. The difference in RRT between each reference standard and the corresponding MeO-PBDE in the samples was less than (0.001 on the nonpolar column (CP-Sil 8CB) and less than (0.003 on the polar column (SP-2331). The ECNI mass spectra for the OH- and MeO-PBDEs were compared between samples and the synthesized standards. All the identified OH- and MeO-PBDEs in the samples had matching ECNI spectra. In addition, two OH-chlorotetraBDEs of unknown structure (Figure 2) were present in the phenolic fraction of the mussel, and one was present in the alga sample. In the neutral fraction of the mussel sample one MeO-chlorotriBDE was also detected. The detection was done by interpretation of the MS fragmentation and the isotope pattern generated. The corresponding methoxylated chlorinated tri- or tetraBDE

TABLE 1. Relative Retention Times (RRTs) of the Methylated OH-PBDEs Detected in the Phenolic Fraction and the MeO-PBDEs Detected in the Neutral Fraction of the Red Alga and Mussel Extracts on a CP-SIL 8CB and a SP-2331 GC Column, Recording by ECNI m/z 79, 81a SP-2331b

CP-SIL 8CB phenolic fractions RRT

phenolic fractions RRT

MeO-PBDE

abbreviation

ID no.

algae

mussels

ID no.

algae

mussels

2′-methoxy-2,3′,4,5′-tetraBDE 6-methoxy-2,2′,4,4′-tetraBDE 6′-chloro-2′-methoxy-2,3′,4,5′-tetraBDE 6-methoxy-2,2′,3,4′,5-pentaBDE 6-methoxy-2,2′,4,4′,5-pentaBDE 2-methoxy-2′,3,4,4′,5-pentaBDE 6-methoxy-2,2′,3,4,4′-pentaBDE 2,2′,3,4,4′,5-hexaBDE 6-methoxy-2,2′,3,4,4′,5-hexaBDE

2′-MeO-BDE-68 6-MeO-BDE-47 6′-Cl-2′-MeO-BDE-68 6-MeO-BDE-90 6-MeO-BDE-99 2-MeO-BDE-123 6-MeO-BDE-85 BDE-138 6-MeO-BDE-137

2 3 6 8 9 10 11

0.824 0.836

0.824 0.836 0.870 0.900 0.903 0.935 0.947 1.000 1.044

2 6 3 8 9 10 11

0.587

0.587 0.658 0.669 0.724 0.731 0.836 0.946 1.000 *

MeO-PBDE 2′-methoxy-2,3′,4,5′-tetraBDE 6-methoxy-2,2′,4,4′-tetraBDE 6-methoxy-2,2′,3,4,4′-pentaBDE 2,2′,3,4,4′,5-hexaBDE 6-methoxy-2,2′,3,4,4′,5-hexaBDE a

abbreviation 2′-MeO-BDE-68 6-MeO-BDE-47 6-MeO-BDE-85 BDE-138 6-MeO-BDE-137

12

0.900 0.903 0.935 0.947 1.000 1.044

12

0.669 0.724 0.731 0.836 0.946 1.000 *

CP-SIL 8CB

SP-2331

neutral fractions RRT

neutral fractions RRT

ID no.

algae

mussels

ID no.

algae

mussels

2 3 11

0.824 0.836 0.947 1.000 1.044

0.824 0.836 0.947 1.000 1.044

2 3 11

0.587 0.669 0.946 1.000 *

0.587 0.669 0.946 1.000 *

12

12

b

The congeners are given in elution order on each column. The RRTs for the MeO-PBDEs marked with an asterisk (*) were not possible to calculate since they did not elute from the polar column. BDE-138 was used for calculation of the RRTs.

standards were not available to point out the identity of these compounds. EI and ECNI mass spectra of the methylated 6-OH-BDE85 and 6-OH-BDE-137 as detected in the red alga are shown, as an example of MeO-BDE fragmentation, in Figures 4a,b and 5a,b, respectively. The full-scan EI spectrum of the methylated 6-OH-BDE85 is dominated by the molecular ion [M]+ and the [M - Br2]+ fragment ion. In addition a [M BrCH3]+ fragment ion is present, characteristic of MeO-PBDEs substituted with a methoxy group in a position ortho to the diphenyl ether bond (6, 8, 31). This fragment ion originates from a brominated dibenzo-p-dioxin formed by the loss of a CH3Br fragment. The brominated dibenzo-p-dioxin structure undergoes further fragmentation and forms a [M - CH3Br - COBr]+ fragment ion, characteristic of the fragmentation of brominated dibenzo-p-dioxins (6). The bromine ions m/z 79 and 81 [Br]- and m/z 159, 161, and 163 [HBr2]- dominate the ECNI spectrum of the methylated 6-OH-BDE85 (Figure 4b), while no molecular ion can be observed. However some fragment ions above 163 could be seen such as [M - HBr]- mixed with [M - Br]-, [M - Br2]- mixed with [M-HBr2]-, [M - CH3Br1-3]-, and [M - CH3OBr]-. Due to cleavage of the diphenyl ether oxygen bond the [M - C6HBr3OCH3]-, [M - C6H3Br2CH3]-, and by H-transfer the [M + H - OC6H3Br2]- fragment ions were formed. The latter fragment ions, m/z: 249 and 342, indicate a substitution of two bromine atoms in one phenyl ring (m/z 249) and a methoxy group and three bromine atoms in the other phenyl ring (m/z 342) (8). The full-scan EI spectrum of the methylated 6-OH-BDE137 (Figure 5a) is characterized by its molecular ion (m/z 668) and a six bromine isotope pattern as well as by an intense ion at m/z 574 matching the fragment ion [M - BrCH3]+. In Figure 5b the corresponding ECNI spectrum is shown. The spectrum is dominated by the ions [Br]- and [HBr2]-. Other important ions are [M - HBr]- mixed with [M - Br]-, [M H2Br2]- mixed with [M - HBr2]-, [M - Br1-3CH3]-, and [M - Br2OCH3]-. Further fragment ions, representing the cleavage of the ether linkage, were also observed: (m/z 249) [M - C6Br4OCH3]- and (m/z 342) [M - Br + H - C6H3Br2CH3]and/or [M - Br + H2 - OC6H3Br2]-.

The OH-PBDE pattern resembles that of the OH-PBDEs in the phenolic fraction independent of ascorbic acid addition or not. It was therefore concluded that the OH-PBDEs are not artificially produced during extraction or cleanup of the alga samples.

Discussion This study shows that the red macroalga (Ceramium tenuicorne) is a possible source of OH-PBDEs and MeOPBDEs detected in Baltic fish, seal. and seabirds (6-9, 32, 33). It is evident that blue mussels living together with the red algae are taking up these compounds (Figure 2). Besides the red algae and blue mussels presented in this work, other macroalgae were also analyzed for OH- and MeO-PBDEs (unpublished). However, these species contained smaller amounts of OH- and MeO-PBDEs. It should be noted that clear seasonal variations in biologically active secondary metabolites have been reported for many macroalgal species (34). To the best of our knowledge, OH- and MeO-PBDEs have never been detected in any alga species from the Baltic Sea or any red alga elsewhere, even though red algae are known to produce a number of different types of monoaryland diarylbromophenols (15-17, 21, 22, 35). However, a MeOPBDE (2′-MeO-BDE-68) has been isolated from the green alga (Cladophora fascicularis) collected in Japan (20). One of the compounds, 6-MeO-BDE137 has never before been reported to occur in the environment. The other eleven OH- and MeO-PBDEs have been detected in environmental samples and/or isolated as natural products as specified in Table 2. The OH- and MeO-PBDE congeners (i.e., 6-OH-/MeOBDE-47, 6′-OH-/MeO-BDE-49, 2′-OH-/MeO-BDE-68, and 6-OH-/MeO-BDE-90) previously reported in Baltic fish, seal, and seabird species (6-9, 32, 33, 51) have both differences and similarities compared to those reported herein for alga and mussel samples. The identification of the OH- and MeOBDEs in the samples was based on comparison of mass spectra (EI and ECNI) and RRT of 26 synthesized reference standards (8, 26). Chromatograms of the phenolic and neutral fractions from Baltic salmon plasma previously investigated VOL. 39, NO. 9, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. EI (a) and ECNI (b) mass spectra of the methyl derivatives of 6-OH-BDE-85, originating from the phenolic fraction from the alga sample.

TABLE 2. Overview of Literature in Which Species OH-PBDEs and MeO-PBDEs, Identified in This Work, Previously Have Been Detected compound

species (ref)

marine spongea (19, 36-38); fish:b salmon (8), common carp, largemouth bass (14) 6-OH-BDE-47 marine spongea (36-39); tunicatesa (40, 41); fish:b salmon (7, 8), common carp, largemouth bass (14), human plasmab (Sweden) (42) 6′-Cl-2′-OH-BDE-68 fish:b salmon (8) 6-OH-BDE-90 fish:b salmon (8), largemouth bass (14) 6-OH-BDE-99 marine spongea (43); fish:b salmon (8), largemouth bass (14) 6-OH-BDE-85 marine spongea (36, 39, 44); fish:b largemouth bass (14) 2-OH-BDE-123 marine spongea (44); fish:b largemouth bass (14) 6-OH-BDE-137 marine spongea (19, 45) 2′-MeO-BDE-68 marine spongea (46, 47); green algaa (20), fish:b salmon (8) mammals:b whales, dolphins, dugong, seals (48, 49); human milkb (50) 6-MeO-BDE-47 marine spongea (18); fish:b salmon (7, 8), largemouth bass (14), pike (51); birds:b white-tailed sea eagle (32) 6-MeO-BDE-85 fish:b largemouth bass (14)

2′-OH-BDE-68

a Isolated, structurally identified by NMR, and confirmed as natural products. b Detected in environmental samples and identified by the comparison of authentic reference standards with GC-MS.

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(8) are shown in Figure 2e,f to visualize these similarities and differences. Starting with the neutral fraction, the following MeO-PBDEs were reported in Baltic salmon: 6-MeO-BDE47 (3), 6′-MeO-BDE-49 (1), 2′-MeO-BDE-68 (2), and 6-MeOBDE-90 (8) (8). Of these compounds, 6′-MeO-BDE-49 and 6-MeO-BDE-90 were not detected in the alga and mussel samples. However, 6-MeO-BDE-85 and 6-MeO-BDE-137 were found in the alga and mussel samples but not in the Baltic salmon sample (Figure 2e,f). In the phenolic fraction 6′-OH-BDE-49 (1) and 4′-OHBDE-49 (4) are present in the salmon but not in the alga and mussel samples (Figure 2). However, 4′-OH-BDE-49 present in the salmon plasma sample is believed to be a metabolite of BDE-47 (8). It is also notable that 6-OH-BDE-99, which is relatively abundant in the alga and the mussel samples, is present at a relatively low concentration in the salmon plasma sample (Figure 2). A comparison of monochlorinated OHand MeO-PBDEs between species is difficult to perform since several of these compounds are not yet structurally identified. It is rather obvious that the OH- and MeO-PBDE patterns are similar in the red algae and the blue mussels since they live together. The differences in the OH- and MeO-PBDE patterns when compared to the salmon (Figure 2) may be explained by their bioavailability, uptake, elimination, metabolism, and selective retention. However, a geographical variation may also have an influence since the salmon (7) have been caught far from where the red algae and mussels were collected. Even if the OH-PBDEs may be of anthropogenic origin, several arguments point in the direction of natural production

FIGURE 5. EI (a) and ECNI (b) mass spectra of methyl derivatives of 6-OH-BDE-137, originating from the phenolic fraction of the alga sample. being the most likely source for the major proportion of these compounds. To the best of our knowledge, none of the identified OH-/MeO-PBDEs are industrially produced. All the identified OH- and MeO-PBDEs have the hydroxy/ methoxy group in an ortho position to the diphenyl ether oxygen and a 2,4-dibromine substitution in the nonhydroxylated/nonmethoxylated phenyl ring, and it is less likely that only these congeners should be formed from industrial production or processing. On the other hand, this type of substitution is common for OH- and MeO-PBDEs that have been isolated and reported as natural products (see Table 2). Six of the seven OH-PBDEs (excluding the OH-PBDEs containing chlorine) in the alga and mussel samples have previously been reported as natural products. All reported natural MeO- and OH-PBDEs have the methoxy/hydroxy group in the position ortho to the diphenyl ether oxygen and have also been reported to be monosubstituted with one bromine in the 2-bromo or 4-bromo position in the nonmethoxylated/nonhydroxylated phenyl ring (19, 36, 52). Besides natural origin, biotransformation of PBDEs is a potential source of OH- and MeO-PBDEs. However, this work supports the natural origin of the identified OH- and MeOPBDEs rather than PBDE metabolism for two reasons. First, the OH- and MeO-PBDEs were present in much greater concentrations than the PBDEs in both the alga and mussel samples. No exact quantitative approach was applied, but still it was possible to estimate the concentrations of MeOPBDEs in the red algae to be in the lower nanogram per gram (wet weight) range, while the concentrations of OH-PBDEs were about 100 times higher. Second, as previously discussed

(8), several of the OH- and MeO-PBDEs lack relevant PBDE precursors, e.g., 2′-MeO-/OH-BDE-68 and 6-MeO-/OH-BDE90 as well as those that contain a chlorine atom (8). In the present study one additional type of structure was identified that lacks relevant PBDE precursors, i.e., 6-MeO-/OH-BDE137. It should however be remembered that certain OHPBDEs previously reported in fish most likely are metabolites of PBDEs (8, 14). Also, certain chemical structures may occur as both natural products and metabolites of PBDEs, i.e., 6-OH-BDE-47, which will make it difficult to determine the origin of certain OH-PBDEs. A possible approach to establish if the origin is antopgenic or natural could be to investigate the 14C content of the compounds. A high 14C content indicates a natural origin of the substances (53, 54). Several observations discussed above point toward a biogenic source of OH- and MeO-PBDEs in the Baltic Sea. However, it is not possible to tell if the OH- and MeO-PBDEs are produced by the red macroalga itself or its associated microfauna or microflora. It is also possible that OH- and MeO-PBDEs are produced by organisms not associated with the algae and that they are transported through the water to the algae. In the case of the natural OH-PBDEs isolated in marine sponge (Dysidea herbacea), it has been suggested that the producer is the symbiotic filamentous cyanobacterium (Oscillatoria spongeliae) (55). Further work is required to unambiguously determine the biogenic source(s) of OHPBDEs and MeO-PBDEs. For example, free living cyanobacteria need to be investigated to establish their potential contribution of these compounds to the Baltic Sea environment. VOL. 39, NO. 9, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Acknowledgments Ioannis Athanassiadis is gratefully acknowledged for the GCMS work, Michael McLachlan for improving the language, and Ulrika O ¨ rn for synthesis of the BDE-138 standard. The Swedish EPA, the Carl Tryggers Foundation, and the Swedish Research Council FORMAS financially supported the project.

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Received for review November 2, 2004. Revised manuscript received February 14, 2005. Accepted February 15, 2005. ES0482886

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