Environ. Sci. Technol. 2009, 43, 2288–2294
Halogenated Bipyrroles and Methoxylated Tetrabromodiphenyl Ethers in Tiger Shark (Galeocerdo cuvier) from the Southern Coast of Japan K O I C H I H A R A G U C H I , * ,† YOHSUKE HISAMICHI,‡ YUICHI KOTAKI,§ YOSHIHISA KATO,| AND TETSUYA ENDO‡ Daiichi College of Pharmaceutical Sciences, Fukuoka 815-8511, Japan, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido 061-0293, Japan, School of Marine Biosciences, Kitasato University, Ofunato, Iwate 022-0101, Japan, and Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, 1314-1 Sanuki Kagawa 769-2193, Japan
Received October 24, 2008. Revised manuscript received January 26, 2009. Accepted February 04, 2009.
Naturally produced halogenated bipyrroles (HBPs) and methoxylated tetraBDEs (MeO-tetraBDEs) are lipophilic and persistent, and therefore may bioaccumulate with higher trophic levels. In this study, the livers of tiger shark (Galeocerdo cuvier) collected from the southern coast of Japan were investigated for size-related bioaccumulation of natural HBPs and MeO-tetraBDEs in comparison with anthropogenic PCBs and PBDEs. Heptachloro-1′-methyl-1,2′-bipyrrole (Cl7-MBP) and hexahalogenated 1,1′-dimethyl-2,2′-bipyrrole (Br4Cl2-DBP) were present at similar concentration ranges (4-4400 ng/g lipid) in the liver and increased with increasing body length. Two MeOtetraBDEs, 6-methoxy-2,2′,4,4′-tetrabromodiphenyl ether (6-MeOBDE47), and 2′-methoxy-2,3′,4,5′-tetrabromodiphenyl ether (2′MeO-BDE68) were present at 4- to 6-fold higher concentrations (88 and 58 ng/g lipid, respectively) than BDE-47. In mature tiger sharks, 2,2′-dimethoxy-3,3′,5,5′-tetrabromobiphenyl (2,2′diMeO-BB80) was present at a median concentration of 330 ng/g lipid. Concentrations of 6-MeO-BDE47 were positively correlated to body length (P < 0.01), but no such correlation was observed for 2′-MeO-BDE68 and 2,2′-diMeO-BB80. The concentration ratios (patterns) of PBDE-like natural products in tiger sharks were largely different from that found in other species, such as the bull shark (Carcharhinus leucas), the silvertip shark (Carcharhinus albimarginatus), and the sandbar shark (Carcharhinus plumbeus). The present study suggests that the concentrations of natural HBPs in the liver are size (age)dependent whereas MeO-tetraBDEs have species-specific biomagnification potentials.
Introduction In recent years, new bioaccumulative organohalogen compounds, proposed to be of natural origin, have been detected * Corresponding author phone: +81-92-541-0161; fax: +81-92553-5698; e-mail:
[email protected]. † Daiichi College of Pharmaceutical Sciences. ‡ Health Sciences University of Hokkaido. § Kitasato University. | Tokushima Bunri University. 2288
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in marine biota from different sites throughout the world. In some cases, the levels of natural brominated products are comparable to or higher than those of anthropogenic persistent organic pollutants (POPs). The major natural lipophilic components described to date in the Pacific are heptachlorinated methylbipyrrole (MBP) (1), hexahalogenated dimethylbipyrroles (DBPs) (2), methoxylated tetrabromodiphenyl ethers (MeO-tetraBDEs) (3), and dimethoxylated tetrabromobiphenyl (diMeO-tetraBB) (4). MBP has been found in the ppm range in marine mammals and in the ppb range in fish, seafood, and even in human milk (5). DBPs have also been found in the ppm range in marine mammals (6) and in the ppb range in fish composites in Canadian markets (7). On the other hand, MeO-tetraBDEs have been found in algae (8), sponges, fish (3), and marine mammals (9). Recently, diMeO-tetraBB has been detected in whale products (liver and blubber of dolphins) from Japan (4) and also in dolphins and dugongs from Australia (10). Their sources are thought to be substances produced by marine bacteria (11). Due to their similar lipophilicity to PCBs, these compounds likely biomagnify within higher trophic organisms, such as humans, via the food chain (12). Sharks are one of the top marine predators and are a long-lived species that can accumulate high levels of organohalogens via the food web (13). The contaminants pose significant health risks such as endocrine disruptions (14) and developmental neurotoxicity (15) to animals at higher trophic levels. Positive correlations between contaminant concentration and body size (age) have been studied (16, 17) due to the increasing body burden of contaminants in aging predators. Thus, a higher organohalogen concentration in animals may be a consequence of increased accumulation with age. However, only limited information is available regarding the size- and species-dependence of organohalogen concentrations in sharks. The aim of this project was to determine the contamination trends of naturally occurring POPs, as compared with anthropogenic PCBs and PBDEs in sharks from Japanese ecosystems. Here, we report the occurrence and levels of HBPs and MeO-BDEs, the relationship between body length and liver concentration in the tiger shark (Galeocerdo cuvier), and species-dependent concentration among four species of sharks collected from the southern coast of Japan.
Materials and Methods Sampling. Four species of sharks were culled off the coast of Ishigaki Island, Japan (Figure 1) in August 2007. A total of 52 liver specimens were collected from 42 tiger sharks (Galeocerdo cuvier), eight silvertip sharks (Carcharhinus albimarginatus), one bull shark (Carcharhinus leucas), and one sandbar shark (Carcharhinus plumbeus). All sharks were measured (total length), weighed, and sexed. No age estimates were available. In general, tiger sharks reach sexual maturity at a length of approximately 260 cm, and silvertip sharks range in length at maturity from 250 to 300 cm (18, 19). On this basis, 35 individual tiger sharks (17 male and 18 female sharks with a length range of 119-258 cm) analyzed in this study were estimated to be immature, whereas the remaining seven individuals (two male and five female sharks with a length range of 267-320 cm) were estimated to be mature. All eight silvertip sharks (two male and six female sharks with a length range of 97-129 cm) and the sandbar shark (a 129 cm-female) were considered to be immature. The bull shark (a 280 cm-female) was estimated to be mature. We selected the liver for matrix to analyze, since shark livers are 10.1021/es802999k CCC: $40.75
2009 American Chemical Society
Published on Web 03/04/2009
FIGURE 1. Sampling location of sharks, Ishigaki Island, Okinawa in the Pacific Ocean. lipid-rich and comprise about one-fourth of a shark’s weight. All samples were stored at -20 °C until analysis. Chemicals. One of the DBP congeners, 5,5′-dichloro-1,1′dimethyl-3,3′,4,4′-tetrabromo-2,2′-bipyrrole (Br4Cl2-DBP), was prepared according to the method of Gribble et al. (20). We synthesized 2,3,3′,4,4′,5,5′-heptachloro-1′-methyl-1,2′bipyrrole (Cl7-MBP) using the method of Wu et al. (21). Standards of the five MeO-BDE analogs, 2′-methoxy-2,3′,4,5′tetrabromdiphenyl ether (2′-MeO-BDE68), 6-methoxy2,2′,4,4′-tetrabromodipheyl ether (6-MeO-BDE47), 2′,6dimethoxy-2,3′,4,5′-tetrabromodiphenyl ether (2′,6-diMeOBDE68), 2,2′-dimethoxy-3,3′,5,5′-tetrabromobiphenyl (2,2′diMeO-BB80), and 4′-methoxy-2,3′,4,5′,6-pentabromodiphenyl ether (4′-MeO-BDE121) were kindly provided to us from Dr. G. Marsh (Stockholm University). Two internal standards, 2,2′,3,4,4′,5′-hexabromodiphenyl ether (BDE-138) and 4′MeO-BDE121, were used for the determination of PBDEs and natural organohalogens, respectively. Sample Clean-up. The procedure was performed according to a modification of our previous method (6). Accurately weighed samples (1-2 g) were cut into small species and mixed with a 10-fold volume of anhydrous sodium sulfate. The mixtures were wet-packed with dichloromethane (DCM)/n-hexane (1:1) into a glass column (2 cm, i.d.). The filtered extracts were then concentrated and the lipid contents were determined gravimetrically. A portion of each lipid sample (100 mg) was spiked with an internal standard solution of BDE138 and 4′-MeO-BDE121 (20 ng each per 100 mg lipid). The lipids were then removed by gel permeation chromatography (Bio-Beads S-X3; Bio-Rad Laboratories), with elution with DCM/n-hexane (1:1) for the organohalogen residues. The eluate containing target organohalogens was concentrated to dryness and purified over an activated silica gel S-1 column (1 g; Wako Pure Chemical Industries Ltd.), with elution with 12% DCM in n-hexane (15 mL). The eluate was reduced to 500 µL and subjected to gas chromatography/mass spectrometry (GC/MS). Identification and Quantification. Analyses of natural organohalogens were performed using a gas chromatograph (GC, Agilent 6890N) equipped with a mass-selective detector (5973i) in electron-ionization and selected ion monitoring mode (EI-SIM). The GC was equipped with an HP-5MS column (30 m × 0.25 mm, 0.25 µm film thickness, J&W Scientific Inc.), and all ions with m/z in the range of 50-650 were recorded in full scan EI mode to select the target ions.
Helium was used as a carrier gas at a constant flow rate of 1.0 mL/min. The injector and transferline temperatures were 250 and 280 °C, respectively. The GC oven program was as follows: after injection at 70 °C (1.5 min), the temperature was increased at a rate of 20 °C/min to 230 °C (2 min), then at a rate of 4 °C/min to 280 °C (20 min). The total run time was 35 min. In the SIM mode, the following channels (m/z values) were used. For the PCBs (14 congeners): m/z 292 for no. 52, m/z 324 for nos. 99, 101, 105, 118, m/z 362 for nos. 138, 146, 149, 153, m/z 394 for nos. 170, 180, 183, 187, and m/z 430 for no. 194. For the PBDEs: m/z 486 for nos. 47, 138 (IS), 153, 154, m/z 408 for nos. 28, 99, 100. For the halogenated bipyrroles: m/z 386 for Cl7-MBP and m/z 544 for Br4Cl2-DBP. For the MeO-tetraBDE analogs: m/z 516 for 2′-MeO-BDE68 and 6-MeO-BDE47, m/z 530 for 2,2′-diMeO-BB80, m/z 546 for 2′,6-diMeO-BDE68 and m/z 594 for 4′-MeO-BDE121 (IS). The concentration of total PCBs (∑PCB) was given from the sum of 14 congeners, whereas the concentration of total PBDEs (∑PBDE) was given from the sum of six congeners. Quality Control. The limit of quantification (LOQ) using EI-GC/MS was determined using a S/N of 10, and ranged from 1 to 50 pg on the column for all analytes. Levels of natural POPs in laboratory blanks were all below the method detection limits (MDL) (0.2- 0.8 ng/g for all analytes). Solvent blanks did not contain any of the analytes under investigation, indicating no carryover effect between GC/MS runs. Standard material, SRM 1588b cod liver oil (NIST), was used for quality confirmation of PCB and PBDE concentrations. Our data were consistent with the certified values (relative standard deviation 5-11%, n ) 5). The recovery of natural POPs and internal standards from blubber was assessed by spiking with approximately 10-200 ng of each isomer and carrying these spikes through the entire extraction method. Recoveries of all analytes ranged from 85 to 102%. Procedural blanks were analyzed simultaneously with every batch of 10 samples to check for interference or contamination from solvents and glassware. All reported concentrations were calculated by comparing their peak areas relative to the internal standard. Statistical Analysis. One-way analysis of variance (ANOVA) was used in this study. Relationships between concentration and body size were assessed by linear regression using SPSS ver. 14.0 (SPSS Inc.). The correlation between concentrations of natural POPs was verified by applying the Spearman correlation coefficient (r). To avoid sex-related bias, the Student’s t-test was applied between mature and immature tiger sharks after verifying that there was no significant difference in sex distribution between the two groups. The statistical significance was set at P < 0.01.
Results and Discussion Size-Dependent Bioaccumulation of PCBs and PBDEs. In the liver of tiger shark, the ΣPCB concentration ranged from 72 to 11 000 ng/g lipid (median ) 480 ng/g) (Table 1). The predominant congeners were CB-153, followed by CB-138, -180, and -118, which their combined sum constituting 58% of ΣPCB (14 congeners) (Supporting Information (SI) Table S1). The PCB levels in tiger sharks were within the same range as that found in blue shark (Prionace glauca) and kitefin shark (Dalatias licha) from the Mediterranean Sea (22), bonnethead shark (Sphyrna tiburo) from the U.S. east coast (23), and bamboo shark (Chiloscyllium plagiosum) from the southern waters of Hong kong, China (24). On the other hand, the ΣPBDE concentrations in tiger shark ranged from 4 to 650 ng/g lipid (median ) 26 ng/g) (Table 1). PBDE congener was dominated by BDE-47, accounting for 50% of ΣPBDE (six congeners). The residue pattern was in the following order: BDE-154 > BDE-100 > BDE-99 (SI Table S2). The present levels in tiger sharks were comparable to those in skipjack tuna (Katsuwonus pelamis, up to 53 ng/g lipid) (25), in fur seals (Callorhinus ursinus, 0.33-100 ng/g lipid) (26), and VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Body Size and Liver Concentrations (ng/g lipid) of Organohalogen Compounds in Tiger Shark (Galeocerdo cuvier), Silvertip Shark (Carcharhinus albimarginatus), Sandbar Shark (Carcharhinus plumbeus), and Bull Shark (Carcharhinus leucas) from Ishigaki Island, Japan tiger sharka immature (n ) 35) median
range
length (cm) weight (kg)
190 91
120-258 20-280
CB-153 ΣPCB (14 congeners)b BDE-47 ΣPBDE (6 congeners)c Halogenated Bipyrroles Cl7-MBP Br4Cl2-DBP Methoxylated TetraBDEs 2′-MeO-BDE68 6-MeO-BDE47 2′,6-diMeO-BDE68 2,2′-diMeO-BB80 ΣMeO-tetraBDEd
95 380 12 19
mature (n ) 7) median
range
total median
silvertip shark
sandbar shark
bull shark
immature (n ) 8)
immature (n ) 1)
mature (n ) 1)
median
range 129 15-40
129 35
280 270
17-870 72-2800 2-100 4-180
Body Size 267-320 190 114 310-600 100 25 Concentration (ng/g Lipid) 2000e 900-3200 120 72 6800e 3600-11000 480 280 100e 56-370 13 6.2 220e 110-650 26 12
37-150 120-520 4.3-14 8.1-30
130 280 11 17
11 000 35 000 440 850
56 73
15-780 4-740
740e 1600e
420-2300 980-4400
74 100
13 18
3.5-50 2.9-130
4.5 6.6
410 1300
87 44 2.6 25 160
16-830 12-220 0.7-46 10-2300 60-2700
130 130e 6.0 330 550
71-240 92-260 2.2-12 87-560 270-1000
88 58 3.3 30 200
120 25 5.6 35 190
53-260 18-60 0.9-21 20-60 100-330
24 20 3.0 16 63
600 230 3.8 230 1100
305 410
a Grouping of mature and immature sharks was conducted based on the samples below or above 260 cm in body length. ΣPCB is the sum of 14 congeners (nos. 52, 99, 101, 105, 118, 138, 146, 149, 153, 170, 180, 183, 187, 194). c ΣBDE is the sum of 6 congeners (BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154). d ΣMeO-tetraBDE is the sum of 2′-MeO-BDE68, 6-MeO-BDE47, 2′,6-diMeO-BDE68, and 2,2′-diMeO-BB80. e The difference of concentrations between immature and mature shark livers was significant at P < 0.01. b
TABLE 2. Correlation Coefficients (r) between Concentrations of Halogenated Contaminants and Body Size (Weight and Length) in Tiger Sharksa body length body weight body length CB-153 ∑PCB Cl7-MBP Br4Cl2-DBP BDE-47 ∑PBDE 2′-MeO-BDE68 6-MeO-BDE47 2′,6-MeO-BDE68
0.943*
CB-153
∑PCB
Cl7-MBP
0.797* 0.749*
0.801* 0.751* 0.999*
0.686* 0.585* 0.886* 0.886*
Br4Cl2DBP 0.734* 0.654* 0.896* 0.911* 0.826*
BDE-47
∑PBDE
2′-MeOBDE68
6-MeOBDE47
2′,6-diMeOBDE68
2,2′-diMeOBB80
0.752* 0.656* 0.922* 0.918* 0.749* 0.828*
0.738* 0.643* 0.928* 0.925* 0.964* 0.838* 0.995*
-0.028 -0.134 0.161 0.159 0.339 0.179 0.158 0.180
0.752* 0.403* 0.615* 0.613* 0.794* 0.577* 0.661* 0.653* 0.613*
-0.028 -0.111 0.153 0.152 0.228 0.153 0.084 0.111 0.856* 0.452*
0.084 -0.011 0.217 0.211 0.310 0.176 0.271 0.281 0.409* 0.447* 0.551*
a Values represent the correlation coefficient for the two variables. Asterisks indicate significant correlations (P < 0.01). ΣPCB is the sum of 14 congeners (nos. 52, 99, 101, 105, 118, 138, 146, 149, 153, 170, 180, 183, 187, 194). ΣBDE is the sum of 6 congeners (BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154). ΣMeO-tetraBDE is the sum of 2′-MeO-BDE68, 6-MeO-BDE47, 2′,6-diMeO-BDE68, and 2,2′-diMeO-BB80.
odontocetes (7.5-640 ng/g lipid) (4) from the Asia-Pacific waters, although the levels were lower than those found in bull sharks (C. leucas) (1630 ng/g lipid) from the Mediterranean Sea (13). In size-related bioaccumulation study, a thorough interpretation requires samples from several size-classes. The present data provides new knowledge on contaminants in shark species where data has been scarce. In general, the body length at birth ranges from 51-76 cm in tiger sharks. Males reach sexual maturity at 226-290 cm, while females become mature at 250-325 cm (19). On this basis, the PCB and PBDE concentrations between two size-classes (immature 260 cm) of tiger sharks were significantly different (P < 0.01). The ΣPCB and ΣPBDE concentrations were significantly correlated to body weight (r ) 0.80 and 0.74, P < 0.01) as well as to body length (r )0.75 and 0.64, P < 0.01), respectively (Table 2). No significant differences was observed between males and females (P > 0.72) in each class. In order to simplify the analysis of the size-dependent bioaccumulation, we examined the relation2290
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ships between body length and the concentrations of the most predominant congeners, CB-153 and BDE-47 (Figure 2). The relationship was significantly positive (P < 0.01), implying that the liver concentrations increase with age (growth). This finding is consistent with the prediction based on diet studies which showed a positive correlation between age, size and PCB concentrations in marine pelagic food web (17, 27). Thus, it could be expected that tiger sharks have size (age)-dependent biomagnifications of PCBs and PBDEs. Halogenated Natural Products in Tiger Sharks. To our knowledge, this is the first report to quantify halogenated natural products in shark species from the Pacific Ocean. The lipophilic fraction including PCBs and PBDEs was analyzed for a family of natural HBPs, which were dominated by Br4Cl2-DBP with a 1,1′-dimethyl-2,2′-bipyrrole skeleton, followed by Cl7-MBP with 1′-methyl-1,2′-bipyrrole skeleton. Br3Cl3- and Br3Cl2-DBPs were also detected as minor DBP homologues in several samples (SI Figure S1). For natural PBDE-like organohalogens, only two methoxylated tetraBDEs
FIGURE 2. Relationships between body length and CB-153 or BDE-47 concentrations in the liver of tiger shark (n ) 42).
FIGURE 3. Relationships between body length and Br4Cl2-DBP or Cl7-MBP concentrations in the liver of tiger shark (n ) 42).
(2′-MeO-BDE68 and 6-MeO-BDE47), one dimethoxylated tetraBDE (2′,6-diMeO-BDE68) and one dimethoxylated tetraBB (2,2′-diMeO-BB80) were detected in all shark livers investigated. The Cl7-MBP concentration ranged from 15 to 2300 ng/g lipid (median ) 74 ng/g) in the liver, which were in the same ranges as the levels of CB-153 (Table 1). The Cl7-MBP level was lower than that previously found in the blubber of bottlenose dolphins (690-14 000 ng/g lipid) from Australia (10), but was 2 orders of magnitude higher than that found in Antarctic fish liver (up to 5 ng/g lipid) (28). With regards to an effect of size upon concentration, the Cl7-MBP level was 5-fold higher in large-size (n ) 7) than in small-size (n ) 35) sharks, indicating that the concentration increases with growth (age). The concentrations found in tiger shark liver were positively correlated to body length, body weight and also concentrations of PCBs and PBDEs (P < 0.01, Table 2). This study therefore suggests that natural Cl7-MBP can biomagnify with higher trophic levels in this marine ecosystem, along with anthropogenic PCBs and PBDEs. Br4Cl2-DBP concentrations ranged from 4 to 4400 ng/g lipid (median ) 100 ng/g) in the liver, which were also at comparable levels to CB-153. Our recent surveys have shown that whale products (bottlenose dolphins) near this area have accumulated up to 40 µg/g lipid of Br4Cl2-DBP (6), whereas bluefin tuna (Thunnus thynnus) and groupers (Serranidae) have been shown to contain up to 180 ng/g lipid of Br4Cl2DBP (29). As compared to the previous data, the present results verified that the halogenated bipyrroles are widely distributed in this ecosystem and the levels in tiger sharks are moderate. For size-related bioaccumulation study, Br4Cl2DBP concentrations were positively correlated to body length, body weight and also PCB concentrations (P < 0.01, Table 2), and the findings were similar to the results for Cl7-MBP (Figure 3). However, the distribution of Br4Cl2-DBP may be limited to some areas of the Pacific. For example, Br4Cl2-DBP has been found in bottlenose dolphins (up to 4150 ng/g lipid) from Australia (12) as well as in California sea lions (44-660 ng/g wet) (30) along with comparable levels of CB-153, while it has been present at quite minor amounts (up to 14 ng/g lipid) relative to PCBs in beluga whales from Alaska (2). In the Asia-Pacific, Br4Cl2-DBP has been abundant in Baird’s beaked whales from the Pacific coast of Japan (6), but virtually not detected in the same species from the Sea of Japan as well as in fish from Micronesia (data not shown). These findings suggest that the release of Br4Cl2-DBP is regionally limited and source-specific.
Among the MeO-tetraBDE analogs, 2′-MeO-BDE68 ranged from 16 to 830 ng/g lipid (median ) 88 ng/g), whereas 6-MeOBDE47 ranged from 12 to 260 ng/g lipid (median ) 58 ng/g) (Table 1). Both concentrations were 4- to 6-fold higher than those of BDE-47. The present MeO-tetraBDE levels in tiger sharks appear to be higher when compared to the previous data from cod liver in the Arctic (up to 17 ng/g lipid) (31), from herring in the Baltic (up to 34 ng/g lipid) (32), from California sea lions (up to 12 ng/g wet; it was estimated to be 24 ng/g lipid) (30), from cetaceans in the Mediterranean Sea (up to 808 ng/g lipid) (33) and from whale products in Japanese market (up to 970 ng/g lipid) (4), but lower than data from cetaceans in Oceania (790-1900 ng/g lipid) (34). For the relationship between body size and organohalogen concentration (Figure 4), 6-MeO-BDE47 was significantly correlated to body length (r ) 0.40, P < 0.01), but no such correlation was observed for 2′-MeO-BDE68 (r ) 0.13, P ) 0.40) (Table 2). In addition, 6-MeO-BDE47 was significantly correlated to BDE-47 (r ) 0.66, P < 0.01), but no such correlation was observed for 2′-MeO-BDE68 (r ) 0.16, P ) 0.32) (Figure 5). The different distributions between 2′-MeOBDE68 and 6-MeO-BDE47 may be explained by their different dietary exposure, uptake efficiency, or metabolic process (e.g., biotransformation from the precursor) under field conditions. In addition, the positive correlation between liver concentrations of 6-MeO-BDE47 and BDE-47 may imply that both have similar biomagnification potentials. Although they both are thought to be derived from different source, it cannot be excluded that 6-MeO-BDE47 may be formed by the metabolism of BDE-47 to 6-OH-BDE47, which is subsequently followed by methylation in the environment, or that BDE-47 may be naturally produced. MeO-PBDEs have never been commercially produced or have been reported as byproduct in industrial processes (12). We are unaware of any data that MeO-tetraBDEs are the precursor of or the products from OH-tetraBDEs, as both OH- and MeO-tetraBDEs have been isolated in red algae, blue mussels and salmon blood from the Baltic Sea (3, 8). It has been suggested that there are numerous sources such as the sponge Dysidea herbacea from the Indian Ocean (35) and sponge Dysidea dendyi from the Pacific (36), green alga Cladophora fascicularis from the Pacific (37), and red alga Ceramium tenuicorne from the Baltic Sea (8). These observations and our results suggest that both 2′-MeO-BDE68 and 6-MeO-BDE47 originate from some natural sources which enters the food web via a different pathway from that of VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Relationships between body length and concentrations of three natural MeO-tetraBDE analogs in the liver of tiger shark. (a) 6-MeO-BDE47, (b) 2′-MeO-BDE68, and (c) 2,2′-diMeO-BB80. The subset graph in (c) represents the relationship in mature (267-320 cm) tiger shark. industrial PBDE products, and which biomagnify with different potentials. For the other MeO-BDE analogs, 2′,6-diMeO-BDE68 was found at 0.7-46 ng/g lipid in all samples investigated. The concentration was positively correlated to 2′-MeO-BDE68 (Table 2), suggesting that these are derived from the same biological source and may have similar biomagnification potentials. The precursor could be 2′,6-dihydroxy-BDE68 or 6-hydroxy-2′-methoxy-BDE68 present in the marine sponge Phyllospongia dendyi from the Palau Island, Micronesia, which contain few percent (mg/g wet) of dihydroxylated tetra-, penta-, and hexaBDEs and their methoxylated homologues (38). Another MeO-BDE analog, 2,2′-diMeO-BB80 showed a unique distribution. The liver concentration ranged from 10 2292
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FIGURE 5. Correlations between concentrations of BDE-47 and three MeO-tetraBDE analogs in the liver of tiger shark (n ) 42). x-axis: BDE-47 concentration (ng/g lipid), y-axis: concentrations (ng/g lipid) of (a) 6-MeO-BDE47, (b) 2′-MeO-BDE68, and (c) 2,2′-diMeO-BB80. to 2300 ng/g lipid (median ) 30 ng/g). The highest concentration (2300 ng/g lipid) was found in a 150 cm-length (immature) shark, and the second highest (1100 ng/g lipid) was also found in a 160 cm-length shark. The concentrations were not correlated to body length, although they tended to increase with size in the mature stage (Figure 4). For the relationship between concentrations of each compound, 2,2′diMeO-BB80 exhibited a weak correlation with 2′-MeOBDE68 (r ) 0.41, P < 0.01) and 6-MeO-BDE47 (r ) 0.45, P < 0.01) (Table 2). The deviations in concentrations within the immature stage suggest that the release of the compound is source-specific. The present concentrations appear to be among the highest in fish species from the Asia-Pacific region, because they are comparable with the levels found in whale products (12-800 ng/g lipid) (4). However, the levels were slightly lower than those found in dolphins from
dependent and species-dependent biomagnifications in sharks from the coastal ecosystem. The changes in the concentration ratios of these natural organohalogens may be explained by the hypothesis that the natural products produced in different habitats enter the food web in different ways, and the size-dependent diet-shift of sharks may influence their bioaccumulation in sharks. Although possible sources of HBPs are still unclear, MeO-tetraBDE analogs are expected to bioaccumulate in the shark liver via a specific dietary exposure to prey items derived from sponges or algae in the North Pacific ecosystem. Thus, these may lead to human exposure through seafood consumption. Continuous monitoring is required in order to gain a better understanding of the biomagnification processes.
Acknowledgments FIGURE 6. Concentrations (mean, ng/g lipid) of BDE-47 and three MeO-tetraBDE analogs in tiger shark (n ) 7, mature), tiger shark (n ) 35, immature), and silvertip shark (n ) 8, immature); Asterisks indicate significant differences of concentrations between immature and mature tiger shark and between tiger shark and silvertip shark (P < 0.01). Australia (250-4100 ng/g lipid) (12), demonstrating that the abundance of 2,2′-diMeO-BB80 varies by regions. It is unlikely that 2,2′-diMeO-BB80 would be a metabolite of BB-80, because BB-80 has never been detected in marine mammals. This product possibly bioaccumulates within the food chain after methylation of 2,2′-dihydroxy-BB80, which has been shown to occur in a marine bacterium Pseudoalteromonas (39). Species-Dependent Bioaccumulation. In order to investigate the species-dependent bioaccumulation potential of natural organohalogens, we compared the concentrations found in the liver of four species of tiger shark (G. cuvier), silvertip shark (C. albimarginatus), bull shark (C. leucas), and sandbar shark (C. plumbeus) found in the shallow waters of coastal areas (at depths to 90-130 m) close to shore. C. leucas commonly enters estuaries, or harbors and readily occurs in freshwaters, whereas G. cuvier generally prefers murky waters. They feed on sea turtles, rays, other sharks, bony fish, small sharks, dolphins, squid, various crustaceans, and carrion (19). C. albimarginatus and C. plumbeus reside in shallow waters, and move out to deeper water as they increase in size (19). They feed primarily on benthic, reef, and pelagic fishes. Most of organohalogens were abundant in G. cuvier than in C. albimarginatus. Although it is difficult to compare C. leucas (n ) 1) with G. cuvier, the levels of MeO-tetraBDEs were higher in C. leucas, but Br4Cl2-DBP and Cl7-MBP concentrations were relatively low in C. leucas (SI Table S3). This may be due to the low bioavailability or degradation of the bipyrroles in C. leucas. Otherwise, their concentrations may be influenced by factors such as habital use, diet or differences in metabolic capacity between C. leucas and G. cuvier. The species-dependent bioaccumulation was also observed for PBDE-like natural products (Figure 6). The mature G. cuvier exhibited the highest concentration of 2,2′-diMeOBB80 (median ) 330 ng/g lipid), whereas C. leucas accumulated the highest concentrations of 2′-MeO-BDE68 (600 ng/g lipid) and BDE-47 (440 ng/g lipid) (Table 1). While C. plumbeus exhibited similar concentrations of MeO-tetraBDEs and PBDEs, C. albimarginatus accumulated approximately 20-fold higher concentrations of 2′-MeO-BDE68 (median ) 120 ng/g lipid) than BDE-47 (Figure 6). These results suggest that the distribution of 2′-MeO-BDE68 and 2,2′-diMeO-BB80 is species-specific in the Asia-Pacific region. In conclusion, the present study has demonstrated that natural HBPs and MeO-tetraBDE analogs exhibit size-
This research was financed by Grants-in-Aid from the Japan Society for the Promotion of Science (B17404006, B19310042, B20404006). We are grateful to Dr. Go¨ran Marsh (Stockholm University) for kindly supplying the standard reference compounds.
Supporting Information Available Further details on the concentration of 14 PCB congeners (Table S1), 6 PBDE congeners (Table S2), and naturally occurring brominated compounds (Table S3) quantified for each of the 52 liver specimens, and GC/MS/SIM profiles of minor brominated compounds detected in a shark liver (Figure S1) are provided. This material is available free of charge via the Internet at http://pubs.acs.org.
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