’ the Dioxin-like Potency of HO- and MeO- Analogues of PBDEs
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Article pubs.acs.org/est Dioxin-like Potency of HO- and MeO- Analogues of PBDEs’ the Potential Risk through Consumption of Fish from Eastern China Guanyong Su,† Jie Xia,† Hongling Liu,† Michael H. W. Lam,§ Hongxia Yu,*,† John P. Giesy,†,‡,§ and Xiaowei Zhang*,† † State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, China State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, China § Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada ‡ S Supporting Information * ABSTRACT: Polybrominated diphenyl ethers (PBDEs) and their analogues, such as hydroxylated PBDE (HO-PBDEs) and methoxylated PBDE (MeO-PBDEs) are of interest due to their wide distribution, bioaccumulation and potential toxicity to humans and wildlife. While information on the toxicity/biological potencies of PBDEs was available, information on analogues of PBDEs was limited. Dioxin-like toxicity of 34 PBDEs analogues was evaluated by use of the H4IIE-luc, rat hepatoma transactivation bioassay in 384well plate format at concentrations ranging from 0 to 10 000 ng/mL. Among the 34 target analogues of PBDEs studied here, 19 activated the aryl hydrocarbon receptor (AhR) and induced significant dioxinlike responses in H4IIE-luc cells. Efficacies of the analogues of PBDEs ranged from 5.0% to 101.8% of the maximum response caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD-max) and their respective 2,3,7,8-TCDD potency factors (RePH4IIE‑luc) ranged from 7.35 × 10−12 to 4.00 × 10−4, some of which were equal to or more potent than some mono-ortho-substituted PCBs (TEF-WHO = 3 × 10−5). HO-PBDEs exhibited greater dioxin-like activity than did the corresponding MeO-PBDEs. Analogues of PBDEs were detected mostly in marine organisms. Of these 11 detected analogues of PBDEs, 6 were found to have measurable dioxin-like potency. Though some analogues of PBDEs exhibited significant dioxin-like potency as measured by responses of the H4IIE-luc transactivation assay, concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (PBDEs analoguesTEQH4IIE‑luc), calculated as the sum of the product of concentrations of individual PBDE and their RePH4IIE‑luc, were less than the tolerance limit proposed by European Union and the oral reference dose (RfD) derived by U.S. Environmental Protection Agency, respectively. (Hazard Quotients (HQ) < 0.005) Additional investigations should be conducted to evaluate the toxic potencies of these chemicals, especially for 2′-MeO-BDE-28, 4-HO-BDE-90, 6-HO-BDE-47, and 6-MeO-BDE-47, which had been detected in other environmental media, including human blood. 1. INTRODUCTION Previous studies have shown that PBDEs and their analogues can interact with some endocrine nuclear receptors such as estrogen receptors (ER), androgen receptor (AR) and thyroid hormone receptor (ThR). Furthermore, HO-PBDEs were more potent than their postulated precursor PBDEs and corresponding MeO-PBDEs.7−11 Because of its structural similarity to other polyhalogenated aromatic hydrocarbons such as polychorinated biphenyls (PCBs), PBDEs have been suggested to be potential agonists of the Aryl hydrocarbon receptor (AhR). To test this hypothesis, several in vivo or in vitro experiments have been conducted, and a weak response of Due to their performance and cost-effectiveness, polybrominated diphenyl ethers (PBDEs) have been used for many years as flame retardants in various commercial products, such as furniture, textiles, plastics, paints, and electronic appliances.1,2 Due to their persistence and potential to bioaccumulate,3 PBDEs have been detected in various environmental matrixes and concentrations have been increasing continuously.4 Hydroxylated polybrominated diphenyl ethers (HO-PBDEs) and methoxylated polybrominated diphenyl ethers (MeOPBDEs) have been observed in tissues of wildlife and humans and have been suggested to be biotransformation products of PBDEs,.5,6 This is especially true for 6-HO-BDE-47, 5-HOBDE-47, and 5′-HO-BDE-99. Concerns have been raised about the potential toxicity of these PBDEs analogues and their modes of molecular toxicity. © 2012 American Chemical Society Received: Revised: Accepted: Published: 10781 June 12, 2012 August 20, 2012 September 6, 2012 September 6, 2012 dx.doi.org/10.1021/es302317y | Environ. Sci. Technol. 2012, 46, 10781−10788 Environmental Science & Technology Article Figure 1. Structures of 34 PBDEs analogs. (19 HO-PBDEs are marked with a red frame, and 15 MeO-PBDEs are marked with a dark-blue frame.). Here we report the first evidence that analogues of PBDEs have measurable potency as AhR-agonists and might elicit dioxin-like toxicity. Concentrations of PBDE and their analogues were determined in freshwater and marine fishes from East China. Finally, the potential risk of these analogues of PBDEs through dioxin-like mechanism was assessed. AhR has been observed.12,13 However, the presence of brominated furans, which were impurities in PBDEs was the likely reason for these apparent potencies.14,15 PBDEs did not activate the AhR, but AhR-mediated effects of tetrachlorodibenzo-p-dioxin (TCDD) could be reduced during coexposure to PBDEs and TCDD. This chemical activity effect is likely due to the fact that PBDEs can interact with the AhR but not bind with sufficient avidity to produce AhR-mediated signaling. However, an investigation of the potency of the HO- and MeOanalogues of PBDEs had not been conducted. These two classes of analogues of PBDEs have been detected in various environments media, including human blood.5,6,16,17 MeO-PBDEs have been known to be produced naturally by marine organisms.18,19 There are contradictory reports on sources of HO-PBDEs analogues. Both in vitro and in vivo exposures have shown that HO-PBDEs might be formed due to biotransformation of various PBDEs.20 However, recent research has demonstrated that HO-PBDEs, especially 6-HOBDE-47, can also be generated from naturally occurring MeOPBDEs.21−23 Specifically, demethylation of 6-MeO-BDE-47 was the primary transformation pathway that resulted in formation of 6-HO-BDE-47 in the small fish, Japanese medaka, while the previously hypothesized formation of HO-PBDEs from synthetic BDE-47 did not occur.21 2. MATERIALS AND METHODS 2.1. Chemicals. PBDEs (BDE-17, -28, -71, -47, -66, -100, -99, -85, -154, -153, -138, -183, -190), C13-BDE-139, C13-2-HOBDE-99 and 13C-PCB-178 used for quantification were purchased from Cambridge Isotope Laboratories (Andover, MA). Analogues of PBDEs, including 19 HO-PBDEs and 15 MeO-PBDEs (Figure 1), were synthesized in the Department of Biology and Chemistry of City University of Hong Kong following the previously published methods.24 Purities of the synthesized compounds were determined to be higher than 98%. The results of proton NMR and electrospray LC-MS/MS of the intermediates and end products, the synthesis procedure was confirmed to not generate brominated dioxin and/or furans.8 2.2. H4IIE-luc Cell Culture and Bioassay. For the first time a high throughput, 384-well plate method was used to determine the relative potencies of various PBDE and their analogues. Rat hepatoma cells that had been stably transfected 10782 dx.doi.org/10.1021/es302317y | Environ. Sci. Technol. 2012, 46, 10781−10788 Environmental Science & Technology Article Figure 2. Dioxin-like effects of 34 PBDEs analogs in the AhR transactivation assay using stable H4IIE-luc reporter cells. Cells were treated with 0.0024−10 mg/L of 34 target PBDEs analogs. Values represent mean ± SD of three independent experiments and are presented as the percentage of the response, compared with 100% activity defined as the maximum activity achieved with TCDD. 2.4. Chemical Analysis. Details of the instrumental analyses, including “Identification and Quantification of PBDEs and their analogues”, “Instrument Conditions”, “Quality Assurance/Quality Control”, and “TEQBIO testing for each biological sample” are provided in the SI. 2.5. Data Analysis. Relative potency factors (RePs), expressed as g TCDD/g chemical, were calculated for each analogue of PBDEs as the quotient of the 20% effect concentration (EC20) for TCDD divided by the EC20 of individual PBDE and analogues of PBDEs.29 TCCD equivalents (TEQ) for each sample were calculated as the sum of the product of concentrations of individual analogues of PBDEs by their respective RePs as follows: with an AhR-responsive luciferase reporter gene construct (H4IIE-luc) was used to study AhR activity of PBDEs analogues.25,26 Potencies of individual analogues of PBDEs were determined by use of previously published methods.27,28 Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium at 37 °C with 5% CO2 and 99% humidity. On the first day, 79 μL of cell solution at a concentration of 7.5 × 104 cells/mL was added to each well of a 384-well plates. To avoid cross-contamination, each chemical treatment was bordered by one blank column. A volume of 79 μL of medium was also added into each well of blank columns. On the second day, cells were dosed with serial dilution of chemicals stock solutions (2 × 106 ng/mL) with dimethylmethane (DMSO) as solvent. Stock solutions were diluted with cell culture medium by 20-fold, and then 0.8 μL of the diluted solution was added into each well of 384-well plate to make to a final dose at 0.5% v/v. Three replicates were conducted per treatment, including TCDD standards. Each control and each standard concentration were averaged for all plates within a given experiment. For chemicals and TCDD standards, 7 (0−10 000 ng/mL) and 10 (0−1.61 ng/mL) concentrations were used, respectively. On the fifth day, cells were lysed and luciferase activity mediated by AhR receptor was assayed by use of a commercial kit (Promega Corporation, Madison, WI) in a microplate reader (BioTek Instruments Inc., Winooski, VT). 2.3. Sampling. Six fishes (the sharpbelly (Hemicculter leuciclus), the yellow catfish (Pelteobagrus f ulvidraco), the crucian carp (Carassius auratus), the bigmouth grenadier anchovy (Coilia macrognathos bleeker), the oriental sheatfish (Silurus spp), and the common carp (Cyprinus carpio)) were collected from the lower Yangtze River. Five marine fishes (the razor clam (Sinonovacula constrzcta), the spotted sicklefish (Drepane punctata), the elongate ilisha (Ilisha elongate), the bigeyed flathead (Suggrundus meerdervoortii), the small yellow croaker (Pseudosciaena polyactis)) were collected from Yellow Sea. All samples were transported to the lab on ice and were maintained intact at −20 °C until dissection for subsequent identification and quantification of PBDEs and their analogues. Details of the samples are given in Supporting Information (SI) (Table S1). i=1 TEQ = ∑ concentrationi × RePi n Following EPA superfund guidance terminology, hazard quotients (HQ) were calculated as the ratio of the exposure estimate to effects concentration considered to represent a “safe” environmental concentration or dose. For quantification, statistical analyses were performed by use of SPSS 13.0 for Windows (SPSS Inc., Chicago, IL). Spearman rank correlation was used to examine the strength of associations between parameters (including mass and length of individual fish, the concentrations of individual target compounds). Mann− Whitney U nonparametric tests were used to compare the difference between/among groups. Concentrations of analytes in fishes are presented as the mean and range. Figures were generated with ChemBioDraw Ultra 11.0 (Figure 1), Microsoft Office Excel 2007 (Figure 2 and SI Figure S3), OriginPro 8 (Figure 2) or with R software (version 2.14.1) (SI Figures S1 and S2). The R code for these analyses is available upon request. 3. RESULTS 3.1. Method Robustness. A 384-well plate format for the H4IIE-luc assay was used here for the first time, and the robustness of this modified method was evaluated. Exposure of H4IIE-luc cells to AhR agonists results in induction of luciferase activity that is a function of duration of exposure, dose, and 10783 dx.doi.org/10.1021/es302317y | Environ. Sci. Technol. 2012, 46, 10781−10788 10784 0.11 0.06 0.07 0.10 0.00 0.04 ND 0.15 ± 0.02 0.02 ± 0.01 1.99 ± 1.08 1.88 × 10−4 2.42 ND 1.18 ± 0.29 0.16 ± 0.00 12.66 ± 3.42 9.67 × 10−5 4.20 ND NA 1.18 ND ND 2.14 ± 0.21 2.41 × 10−5 3.54 ND ND ND ND 6.26 ± 1.91 ND ND ND ND ND 0.15 ± 0.00 ND ± 0.04 0.16 ± 0.03 2.33 × 10−5 1.65 ND NA 2.79 ND ND ND 0.04 ± 0.00 ND ND ND ND ND ND ND ND 0.93 ± 0.33 ND 1.81 ± 0.51 17.67 ± 3.25 ND 0.57 ± 0.06 0.17 ± 0.02 0.71 ± 0.54 20.35 ± 19.28 48.78 ± 8.79 17.25 ± 1.94 ND ND Hemicculter leuciclus ND 0.09 ± 0.04 ND 0.14 ± 0.01 ND ND ND ND ND 0.03 0.01 0.00 0.00 0.00 ± 0.08 ± 0.03 ± 0.06 ± ± ± ± ± 8.21 ± 4.01 ND ND 0.09 0.39 0.02 0.04 0.06 ND 0.86 0.23 0.14 ND 2.15 Pseudosciaena polyactis ND ND 1.49 ± 1.19 2.28 ± 0.13 ± 0.01 ± 0.16 ± 0.23 ± 0.10 11.09 ± 0.73 0.06 0.06 0.13 0.54 ± 0.01 20.88 ± 0.56 ± ± ± ± ± 0.20 ND ND ND 1.44 ND ND ND ND 6.56 8.52 6.72 ND 0.16 Suggrundus meerdervoortii ND ND ND ND 0.93 ND ND ND 0.16 0.74 0.43 4.82 ND ND Ilisha elongata ND ND ± 0.83 ± 0.74 ± 0.54 ± 2.17 ± ± ± ± ± ± ND 0.12 0.06 0.18 0.27 0.01 0.15 ND ND 3.94 3.59 6.35 ND 2.16 33.42 ± 8.14 ± 0.01 ± 0.04 ± 0.55 ± 0.90 Drepane punctata 0.18 ± 0.02 3.81 ND ND 6.08 ND ND 2.10 ND ND ND ND ND 0.85 Sinonovacula constrzcta ND NA 6.57 ND ND ND ND ND ND ND ND ND 3.58 ± 2.24 ND 0.11 ± 0.00 4.63 ± 1.02 0.05 ± 0.02 ND ND ND 12.03 ± 1.85 21.14 ± 2.10 8.71 ± 1.32 ND ND Pelteobagrus f ulvidraco ND NA 4.68 ND ND ND ND ND ND ND ND ND ND ND 0.14 ± 0.02 1.12 ± 0.05 ND ND ND ND 3.99 ± 1.03 6.42 ± 1.54 39.31 ± 6.03 36.51 ± 2.14 ND Carassius auratus ± ± ± ± ± 0.37 0.03 0.17 0.00 0.01 ± 0.04 ± 0.02 ND 1.68 × 10−5 2.86 ND ND ND ND ND ND ND ND 6.99 ± 1.04 ND ND 0.15 0.16 ND ND ND ND 0.90 0.34 0.25 0.03 0.11 Coilia macrognathos bleeker Yangtze River samples ND NA 6.60 ND ND ND ND ND ND ND ND ND ND ND 0.25 ± 0.11 5.04 ± 1.31 ND ND ND ND 38.92 ± 3.09 60.00 ± 4.34 32.04 ± 1.87 ND ND Silurus spp ND NA 0.86 ND ND ND ND ND ND ND ND ND ND ND 1.56 7.84 ND ND ND ND 7.22 nd 5.86 ND ND ± 1.52 ± 1.43 ± 0.03 ± 0.12 Cyprinus carpio a “ND” means not detected, and “NA” means not achieved. bAll concentrations had been represented with mean and standard error. (ng/g lip). cThe unit of TEQ was “pg/g wet weight”; TEQ CHEM (PBDEs analoguesTEQH4IIE‑luc) was calculated as the sum of the product of concentrations of individual analogues of PBDEs by their respective RePs; TEQBIO (raw extractTEQH4IIE‑luc) represent the TCDD equivalents of raw extract of biological samples as measured by the H4IIE-luc cells. BDE-17 BDE-28 BDE-71 BDE-47 BDE-66 BDE-100 BDE-99 BDE-85 BDE-154 BDE-153 BDE-183 BDE-190 2′-MeO-BDE68 6-MeO-BDE47 6-MeO-BDE90 3-MeO-BDE100 2-MeO-BDE123 6′-MeO-BDE17 4-MeO-BDE90 2′-MeO-BDE28 6-HO-BDE-47 2′-HO-BDE68 4-HO-BDE-90 TEQCHEM TEQBIO chemicals Yellow Sea samples Table 1. Concentrations of PBDEs and HO- and MeO-Analogues of PBDEs in Fishes from the Yangtze River and Marine Organisms from the Yellow Sea, China Environmental Science & Technology Article dx.doi.org/10.1021/es302317y | Environ. Sci. Technol. 2012, 46, 10781−10788 Environmental Science & Technology Article BDE-90), were detected. (SI Figure S3 and Table 1) Concentration of ∑PBDEs in marine fish (1.8−2.3 × 101 ng/g lipid with mean of 1.2 ng/g lipid) was 56.7 fold lower than those in freshwater fish. These results suggest that fishes in the Yangtze River were more affected by synthetic chemicals than those from the marine environment. Unlike freshwater organisms, analogues of PBDEs were detected in each of the marine fishes, especially 2′-MeO-BDE-68, 6-MeO-BDE-47, and 4-HO-BDE-90, which were detected in 80% of samples. 3.4. TEQCHEM and TEQBIO in Samples. In order to assess the potential for adverse effects of PBDEs and their analogues on humans and wildlife in the future, PBDEs analoguesTEQH4IIE (TEQCHEM) for each organism were calculated as the sum of the product of concentrations of individual analogues of PBDEs by their respective RePs, which ranged from NA (NA means not achieved) to 1.88 × 10−4 pg.g−1 wet weight (Table 1). Among these testing organisms, the analoguesTEQH4IIE in spotted sickle fish was highest. Raw extractTEQH4IIE (TEQBIO) were tested to be from 0.86 to 6.60 pg pg.g−1 wet weight (Table 1). The ratio of TEQCHEM and TEQBIO (TEQCHEM/ TEQBIO) were calculated to be from NA to 7.77 × 10−5. strength of binding of ligands to the AhR (Figure 2). Each point of the curve represents the mean of three replicates and its standard error, and also represents the ratio of mean luciferase response relative to the maximum response to 2,3,7,8TCDD (TCDDmax). The mean EC50 for luciferase induction by 2,3,7,8-TCDD was 5.4 ± 0.7 pg/mL (16.9 ± 2.2 pM). When the maximal response induced by chemicals exceeded the standard deviation (expressed as % TCDDmax) of the mean DMSO blank response (0% TCDDmax) by at least 3-fold, the chemical was deemed to have significant AhR-mediated potency. 3.2. Relative Potencies of Analogues of PBDEs Relative to 2,3,7,8-TCDD. Among the HO-PBDEs tested, 68% (13 of 19 HO-PBDEs) exhibited significant AhR-mediated potency relative to 2,3,7,8-TCDD in H4IIE-luc cells. The OHBDE's that exhibited significant potency included 6′-Cl-2′-HOBDE-7, 2′-HO-BDE-28, 2′-HO-BDE-68, 6-HO-BDE-47, 5-Cl6-HO-BDE-47, 6-HO-BDE-85, 6-HO-BDE-90, 2-HO-BDE123, 4-HO-BDE-90, 6-HO-BDE-137, 3-HO-BDE-100, 2′-HOBDE-66, and 2′-HO-BDE-25. (Figure 2) At the maximal tested concentration of 10 000 ng/mL, 6′-Cl-2′-HO-BDE-7, 2′-HOBDE-28, 6-HO-BDE-47, and 6-HO-BDE-85 caused significant cytotoxicity to H4IIE-luc cells, and the TCCD-max for these chemicals was 2500 ng/mL. (SI Table S3) Similarly, 6 of the 15 MeO-PBDEs that were tested exhibited significant AhRmediated potency. These MeO-PBDEs included 2′-MeOBDE-28, 6-MeO-BDE-47, 5-Cl-6-MeO-BDE-47, 6-MeO-BDE85, 2-MeO-BDE-123, and 6-MeO-BDE-137, which accounted for 40% of the tested MeO-PBDEs. (Figure 2) Dose−response curves of four PBDEs analogues that exceeded 50% TCDDmax, including 6-HO-BDE-47, 5-Cl-6-HO-BDE-47, 6-HOBDE-137, and 5-Cl-6-MeO-BDE-47, were also fitted, which indicated that these chemicals exhibited significant, concentration-dependent, AhR-mediated potency as determined in H4IIE-luc cells (SI Figure S1). 3.3. Concentrations of PBDEs and their Analogues. 3.3.1. Freshwater Fishes. Concentrations of 13 PBDEs and 34 PBDEs analogues were quantified in six fishes from the Yangtze River, China. Eleven PBDEs (BDE-17, BDE-71, BDE-47, BDE66, BDE-100, BDE-99, BDE-85, BDE-154, BDE-153, BDE-183, and BDE-190), and two analogues of PBDEs (2′-MeO-BDE-68 and 6-MeO-BDE-47), were detected. (SI Figure S3 and Table 1) The analogues of PBDEs, 2′-MeO-BDE-68, and 6-MeOBDE-47, were detected only in bigmouth grenadier anchovy, which is a migratory fishes that resides in the Yangtze River estuary and migrate back to the Yangtze River to spawn. Concentrations of ∑PBDEs ranged from 1.8 to 1.4 × 102 ng/g lipid with mean and median values of 6.8 × 101 ng/g lipid and 6.9 × 101 ng/g lipid, respectively. Concentrations of four PBDEs congers, including BDE-47, BDE-154, BDE-153, and BDE-183, were detected most frequently and exhibited the greatest concentrations and percentages of the four individual congeners that contributed to total PBDE (∑PBDEs) were calculated: BDE-47: 9.0%; BDE-154: 20.5%; BDE-153: 34.0%; BDE-183: 25.7%. 3.3.2. Marine Fishes. Concentrations of 13 PBDEs and 34 PBDEs analogues were quantified in five fishes from the Yellow Sea, China. Eleven PBDEs (BDE-17, BDE-28, BDE-71, BDE47, BDE-66, BDE-100, BDE-99, BDE-85, BDE-154, BDE-153, and BDE-183), 8 MeO-PBDEs (2′-MeO-BDE-28, 2′-MeOBDE-68, 6-MeO-BDE-47, 6-MeO-BDE-90, 3-MeO-BDE-100, 2-MeO-BDE-123, 6′-MeO-BDE-17, and 4-MeO-BDE-90) and 3 HO-PBDEs (6-HO-BDE-47, 2′-HO-BDE-68, and 4-HO- 4. DISCUSSION Slight alterations in structures of chemicals can alter the potency to bind to biomolecules. Based on 6 homologous pairs of HO- and MeO-substitued BDE, including 2′-HO-BDE-28 and 2′-MeO-BDE-28, 6-HO-BDE-47, and 6-MeO-BDE-47, 5Cl-6-HO-BDE-47 and 5-Cl-6-MeO-BDE-47, 6-HO-BDE-85 and 6-MeO-BDE-85, 2-HO-BDE-123 and 2-MeO-BDE-123, 6-HO-BDE-137 and 6-MeO-BDE-137, the maximum response relative to TCDDmax caused by HO-PBDEs was greater than that caused by MeO-PBDEs, which indicated that HO-PBDEs exhibited greater potencies to induce AhR activity than did MeO-PBDEs. The maximum potency of four chemicals, including 6-HO-BDE-47, 5-Cl-6-HO-BDE-47, 6-HO-BDE137, and 5-Cl-6-MeO-BDE-47, exceeded 50% of TCDDmax, even though the respective analogous PBDEs did not result in significant activation of the AhR-mediated responses. (SI Figure S1) These results are consistent with the observation that addition of a MeO- or HO group can result in greater potency as AhR agonists.30 This conclusion is supported by comparing relative potencies of BDE-47, 6-MeO-BDE-47 and 6-HO-BDE47.31 ReP values were calculated for each of the HO- and MeOsubstituted BDE relative to 2,3,7,8-TCDD (SI Table S3). ReP-H4IIE of dioxin-like analogues of PBDEs ranged from 7.35 × 10−12 to 4.00 × 10−4. ReP-H4IIE for 6-MeO-BDE-85, 6′-Cl-2′HO-BDE-7, 5-Cl-6-MeO-BDE-47, 6-HO-BDE-47, 6-HO-BDE137, 6-HO-BDE-85, and 5-Cl-6-HO-BDE-47 ranged from 2.56 × 10−5 to 4.00 × 10−4, which were equal to or greater than 2,3,7,8-TCDD Equivalents suggested by the World Health Organization (TEFWHO) reported for mono-ortho-substituted PCBs, which were assigned a value of 3 × 10−5. Of the substituted analogues studied here, 5-Cl-6-HO-BDE-47 exhibited the greatest relative potency, which was almost equal to that for OCDD and OCDF. Concentrations of analogues of PBDEs, regardless of whether they are natural products or come from synthetic BDE, are greater in marine organisms.18,22 For this reason, marine organisms might pose greater risks to humans via the diet than would freshwater organisms. Among those 11 detected analogues of PBDEs, 6 exhibited AhR-mediated potency. These included: 2′-HO-BDE-68, 2-MeO-BDE-123, 10785 dx.doi.org/10.1021/es302317y | Environ. Sci. Technol. 2012, 46, 10781−10788 Environmental Science & Technology Article exhibit significant concentration-dependent AhR-mediated potency. Thirty eight OH-PBDE and 25 MeO-PBDEs have been detected in the environment or tissues of humans (SI Figure S2 and Table S4). Of the 63 analogues of PBDE, 15 exhibited measurable dioxin-like potency. Four analogues, including 2′MeO-BDE-28, 4-HO-BDE-90, 6-HO-BDE-47, and 6-MeOBDE-47, have been detected in various environmental samples, human samples and this study, and exhibit significant AhR agonist potency as measured in H4IIE-luc cells. This is especially true for 6-HO-BDE-47 and 6-MeO-BDE-47, which have been shown to be of natural origin in marine organisms and have been quantified in various environment media. Since analogues of PBDEs exhibited dioxin-like potency and concentrations in the environment that are sufficient to cause adverse effects, these chemicals should be considered when assessing the total potency of mixtures in the environment. 2′-MeO-BDE-28, 4-HO-BDE-90, 6-HO-BDE-47, and 6-MeOBDE-47. Among those analogues of PBDEs that were detected in fishes, 2′-MeO-BDE-68, 6-MeO-BDE-47, 2′-MeO-BDE-28, 6-HO-BDE-47, and 2′-HO-BDE-68 had been previously identified and confirmed to be natural products of either marine sponges or their associated filamentous cyanobacteria, red or green algae.23 6-MeO-BDE-90 and 6′-MeO-BDE-17 have been observed in marine wildlife, but have not been classified as natural products. 4-HO-BDE-90 has been detected in blood serum of humans.6 For these four substituted BDE that have been observed to occur in algae or sponges, 2′-HOBDE-28 and 6-HO-BDE-85 have been determined to be of natural origin.23 Analogues of PBDEs might be concentrated in the marine environment by fishes. These results were consistent with previously published results.19 Concentrations of ∑PBDEs in organisms studied here, were generally less than those in biota from other locations around the world, but equal to those reported by Gao et al (mean: 44.04 ng/g lipid).32 Analogues of PBDEs were identified to be naturally occurring AhR ligands. Generally, AhR ligands were classified into two categories: synthetic and naturally occurring. PCBs PCDD/Fs and PAHs had been known to be synthetic, dioxin-like compounds. However, recent work has focused on naturally occurring compounds with the hope of identifying endogenous ligands. After exposure to PBDEs analogues, AhR of H4IIE cells was activated, which indicated that PBDEs analogues should also be listed as naturally occurring dioxin-like compounds. Most importantly, PBDEs analogues, especially MeO-PBDEs, can be accumulated by organisms because of their large solubility in lipids,23 unlike the other naturally occurring dioxin-like compounds, derivatives of tryptophan33 or tetrapyrroles.34 Based on the calculated PBDEs analoguesTEQH4IIE, risks posed by marine organisms were greater than freshwater fishes. Taking into account risk related to consumption of fishes, the European Union (EU) had proposed tolerance limits of 8 pg TEQWHO g−1 wet weight for fish and fishery products,35 which is relatively greater than that in the marine fishes studied here. Assuming that a 60 kg adult would eat 1 kg sea fish, the total daily dietary intake (TDI) from PBDEs analogues was estimated to be approximately 3.13 × 10−3 pg PBDEs analoguesTEQH4IIE‑luc /kg bw-day), which is also less than the oral reference dose (RfD) of 7 × 10−1 pg/kg-day for TCDD derived by U.S. Environmental Protection Agency.36 This RfD was based on the results of two epidemiologic studies: sperm concentration and motility in men, and thyroid stimulating hormone levels in newborn infants. The HQ from PBDEs analogues in marine fishes was calculated to be 0.005. Though concentrations of PBDEs analoguesTEQH4IIE in individual fishes were less than the reported tolerance limits, humans could be exposed to some analogues of PBDEs that are of natural origin via seafood and thus should be further evaluated in vivo for their toxicity. Raw extract TEQH4IIE (TEQBIO) for each biological sample was determined to be from 0.86 to 6.60 pg pg.g−1 wet weight, which was lower than samples from other locations37,38 and less than the tolerance limit of 8 pg TEQ g−1 wet weight for dioxin and dioxin-like compounds in fish proposed by the European Union. The ratio of TEQCHEM and TEQBIO (TEQCHEM/ TEQBIO) were calculated to be from NA to 7.77 × 10−5. These results suggested that the contribution of PBDEs analoguesTEQH4IIE‑luc to the total TEQ in fishes was very little, no more than 0.1%, though some OH- and MeO- analogues do 5. PERSPECTIVE For the first time, it has been reported that naturally occurring analogues of PBDEs could exhibit measurable AhR-mediated potency. While the ReP values of the OH- and MeO-analogues of PBDE are in general less than those of PCDD/DF and PCBs, they can contribute significant proportions of the total concentration of AhR-mediated potency in marine organisms. In China, wild fish are considered beneficial to human health and marine algae and plants are thought to be nutritionally rich, and thus relatively large quantities are consumed by people. It is still too early to reject this “ancient wisdom”, however, additional work should be conducted to assess the balance between the toxicity and benefit of these compounds naturally occurred in dietary source in East China. ■ ASSOCIATED CONTENT S Supporting Information * Supporting Information includes additional information as noted in the text including Tables S1−S4 and Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author *Phone: 86-25-89680623; fax: 86-25-83707304; e-mail: yuhx@ nju.edu.cn (H. Y.); howard50003250@yahoo.com (X. Z.). Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 20737001 and 20977047) and National Science and Technology Major Project (No. 2008ZX08526-003). The research was also supported by a grant from National Natural Science Foundation of China (No.21007025) and from Major State Basic Research Development Program (No. 2008CB418102). G.S. was supported the Shanghai Tongji Gao Tingyao Environmental Science & Technology Development Foundation (STGEF). J.P.G. was supported by the program of 2012 “High Level Foreign Experts” (#GDW20123200120) funded by the State Administration of Foreign Experts Affairs, the P.R. China. J.P.G. was also supported by the Canada Research Chair program, an at large Chair Professorship at the Department of Biology and 10786 dx.doi.org/10.1021/es302317y | Environ. Sci. 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Polychlorinated dibenzo-p-dioxins, dibenzofurans, biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents in fishes from Saginaw Bay, Michigan. Environ. Toxicol. Chem. 1997, 16 (4), 713−724. (38) So, M. K.; Zhang, X.; Giesy, J. P.; Fung, C. N.; Fong, H. W.; Zheng, J.; Kramer, M. J.; Yoo, H.; Lam, P. K. Organochlorines and dioxin-like compounds in green-lipped mussels Perna viridis from Hong Kong mariculture zones. Mar. Pollut. Bull. 2005, 51 (8−12), 677−87. 10788 dx.doi.org/10.1021/es302317y | Environ. Sci. Technol. 2012, 46, 10781−10788 Dioxin-like Potency of OH- and MeO- Analogues of PBDEs’ the Potential Risk through Consumption of Fish from Eastern China Guanyong Su1; Jie Xia1; Hongling Liu1; Michael H. W. Lam2;Hongxia Yu1*; John P. Giesy1,2,3; Xiaowei Zhang1* 1 State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, China 2 Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada 3 State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, China Authors for correspondence: School of the Environment Nanjing University Nanjing, 210089, China Tel: 86-25-83593649 Fax: 86-25-83707304 E-mail: yuhx@nju.edu.cn (Hongxia Yu) howard50003250@yahoo.com (Xiaowei Zhang) 1 Chemical Analysis Procedures 1.1 Identification and Quantification of PBDEs and their analogues Concentrations of individual polybrominated diphenyl ethers (PBDE), and hydroxylated brominated diphenyl ethers (OH-BDE) were determined by application of an adaptation of the methods1. After measuring the length and weight of individual fish, the edible fillet was removed, lyophilized and homogenized. Approximately 5.0 g of dry sample, to which surrogate standard - 13C-BDE-139 and C13-2-HO-BDE-99 was added, was extracted by accelerated solvent extraction (ASE, Dionex ASE-350, Sunnyvale, CA, USA). Extraction was conducted with n-hexane / dichloromethane (DCM) (1:1) as the first extraction solvent at a temperature of 100 ℃ and pressure of 1500 psi, and then the samples were extracted with n-hexane/methyl tert-butyl ether (MTBE) as the second extraction solvent at a temperature of 60 ℃ and pressure of 1500 psi. Two cycles were performed for each solvent and duration of each cycle was 10 min. The extract was concentrated by rotary evaporation to 10 mL, and 2 ml of extract was taken out for gravimetrically lipid content determination. An aliquant of 4 mL of 0.5 M potassium hydroxide (KOH) in 50% ethanol was added to the concentrated extract. Phenolic compounds were separated from the neutrals into an aqueous layer of KOH. The aqueous phase was extracted with 8mL of n-hexane three times (neutral fraction), followed by acidification with 1.5 mL of 2 M hydrochloric acid. Then phenolic compounds were extracted three times with n-hexane/MTBE (9:1; v/v). For neutral chemicals, the extract was concentrated to near dryness and dissolved in 10 ml of dichloromethane and hexane (V:V=1:1) and acidified with 10 ml of H2SO4 to remove the fat. PBDEs and MeO-PBDEs were back extracted with a total of 30 mL dichloromethane and hexane (V:V=1:1) in 3 separate 10 mL extractions. The organic solvent containing PBDEs and MeO-PBDEs was concentrated and passed through a silica gel column for further clean up. The silica gel column was packed with glass-wool, activated silica gel (0.25 g), 44% (w/w) acid silica gel (1.0 g), silica gel (0.25 g), and anhydrous sodium sulfate (0.30 g) from bottom to top in a disposable Pasteur pipette 2. The fraction containing PBDEs and MeO-PBDEs was eluted with 15 mL hexane followed by 15 mL n-hexane/dichloromethane(1:1). The elution was concentrated by rotary evaporation and further concentrated to near dryness under a gentle nitrogen flow. Then, 9.6 ng of 13 C-PCB-178 was added as the internal injection standard and made up to 100 µL with hexane prior to GC/MS analysis. For the extract containing the phenolic compounds, the extract was concentrated to near dryness by rotary evaporation and transfer into a 15 ml blown glass vials with 3 ml of n-hexane. The organic solvent containing HO-PBDEs were dried under a gentle nitrogen flow. And then the derivatization process was conducted according to previously published methods 1. The aqueous solution was extracted with 6 mL of n-hexane three times, and the extracts were subjected to the silica gel chromatography as described above. The column was eluted with 30 mL n-hexane/DCM (1:1), and the elution was concentrated by rotary evaporation and further concentrated to near dryness under a gentle nitrogen flow. Then, 9.6 ng of 13C-PCB-178 was added as the internal injection standard and made up to 100 µL with hexane prior to identification and quantification by use of GC/MS. 1.2 Instrument Conditions Concentrations of 13 PBDEs and 34 PBDEs analogs were determinzed by use of a Thermo Scientific TSQ Quantum GC (USA), coupled with an Agilent DB-XLB column (15 m × 0.25 mm × 0.25 µm, USA). The mass spectrometer detector was operated in electron impact ionization (EI) mode. analyzed in selected reaction mode (SRM) mode. were processed by SRM modes. Samples and standards were Quantification and qualification The precursor ion and product ions selected in SRM mode for each chemical were based on the mass spectrum of the standard solution. Detailed information about precursor ion, product ions, ions ratio and collision energy are given in Supporting Information (Table S2). 1.3 Quality Assurance/Quality Control QA/QC was conducted by performing laboratory blanks, GC/MS detection limit (based on 3S/N) and standard spiked recoveries. Concentrations of target analytes in laboratory blanks were less than 5% of the sample minimum concentration, which demonstrated that samples were free from contamination. The limit of detection (LOD) was defined as the concentration that would result in a signal-to-noise ratio of 3. LOD based on 2.0 g of dry sample and instrument sensitivity, varied from congener to congener, from 30.3 to 123.4 pg/g dry wt. Concentrations less than the LOD were assumed to be not detected in calculating summary statistics. For samples where concentrations of a congener were less than the LOQ, they were reported as not detected. Before sample analysis, matrix spike (n=4) for each target compound had been evaluated. And recoveries ranged from 74.3 to 125.2% for PBDEs and their analogs, respectively. 13 C-labeled BDE-139 and 13 To ensure accuracyof analytical procedures, C-labeled 2-HO-BDE-99 was used as the internal standard for neutral (PBDEs and MeO-PBDEs) and phenolic compounds (HO-PBDEs), respectively. Recoveries of the 13C-labeled BDE-139 internal standard were between 85.1 and 111.2%. 2 TEQ BIO testing for each biological sample The procedures of biological samples for H4IIE-luc testing were similar to those described by with some modifications3. Approximately 10.0 g of dry sample was extracted by accelerated solvent extraction (ASE, Dionex ASE-350, Sunnyvale, CA, USA). Extraction was conducted with dichloromethane (DCM) 4 solvent at a temperature of 100 ℃ and pressure of 1500 psi. performed for each sample and duration of each cycle was 10 min. as the extraction Two cycles were The extract was then concentrated to approximately 5 ml using a rotary evaporator under reduced pressure. To avoid the fat’s toxicity to cells, the 5 ml extract was acidified with 5 mL concentrated H2SO4 to remove the fat5. And the target compounds were back extracted with a total of 30 mL dichloromethane in 3 separate 10 mL extractions. Finally, the extract were collected and concentrated to 150 µL for AhR activity testing. Cell culture and bioassay had been described in section “2.2 H4IIE-luc Cell Culture and Bioassay” of the manuscript. Supporting Table 1 Samples Information Samples n Location Time Mass (g) Length (cm) Sinonovaculaconstrzcta 17 Yellow Sea 2011.02.21 8.3-12.1 5.5-7.5 Drepanepunctata 2 Yellow Sea 2011.02.21 200/189.55 22/20.5 Acanthogobius hasta 14 Yellow Sea 2011.02.21 8.1-15.2 6.5-10.9 Suggrundusmeerdervoortii 1 Yellow Sea 2011.02.21 537.65 43.5 Pseudosciaenapolyactis 2 Yellow Sea 2011.02.21 288.83/295.58 24/24.5 HemicculterLeuciclus 16 Yangtze River 2011.06.16 13.6-43.2 10.0-14.0 Pelteobagrusfulvidraco 32 Yangtze River 2011.06.16 11.7-32.6 10.0-14.0 Carassiusauratus 10 Yangtze River 2011.06.16 39.9-81.5 10.0-14.0 CoiliamacrognathosBleeker 9 Yangtze River 2011.06.16 30.0-65.8 20.0-26.0 Silurusspp 2 Yangtze River 2011.06.16 671.3/554.3 44/41 Cyprinuscarpio 3 Yangtze River 2011.06.16 885.3/426.1/420.7 33/26/26.5 Supporting Table 2 Ion pairs, abundance ratio and collision energy of selected reaction mode. Ion pairs for Quantification and Qualification Chemicals Collision Energy (eV) Parent Ion Product Ion Abundance Ratio BDE-17 245.88 245.88, 138.85 100/30 20 BDE-28 245.88 245.88, 138.86 100/12 20 BDE-71 325.66 216.79, 218.94 92/100 30 BDE-47 325.66 216.79, 218.95 61/100 30 BDE-66 325.66 216.79, 218.96 100/93 30 BDE-100 405.63 296.60, 405.63 100/44 30 BDE-99 405.63 296.60, 405.64 100/40 30 BDE-85 405.63 296.60, 405.65 100/28 30 BDE-154 483.64 483.64, 402.57 42/100 30 BDE-153 483.64 483.64, 402.58 18/100 30 BDE-138 483.64 483.64, 402.59 5/100 30 BDE-183 563.73 563.73, 485.15 5/100 30 BDE-190 563.73 563.73, 485.15 5/100 30 2’-HO-BDE-7 263.85 155.48, 127.37 70/100 15 3’-HO-BDE-7 401.70 198.25, 183.19 100/10 15 6’-Cl-2’-HO-BDE-7 297.96 126.14, 189.17 100/20 30 6’-HO-BDE-17 341.71 126.30, 235.49 100/4 30 5-Cl-6-HO-BDE-47 455.70 456.51, 347.31, 349.44 100/30/40 20 4-HO-BDE-90 578.56 578.93, 443.86, 390.96 100/40/26 15 2’-HO-BDE-66 419.77 420.25, 313.24 40/100 20 2’-HO-BDE-25 341.88 126.33, 235.49 100/4 30 2’-HO-BDE-28 341.90 233.39, 235.29, 342.52 38/100/1 20 2’-HO-BDE-68 419.75 313.33, 311.33 100/50 20 6-HO-BDE-47 419.76 313.41, 420.45 20/100 25 4’-HO-BDE-49 500.64 365.82, 364.24 100/4 25 6’-Cl-2’-HO-BDE-68 455.71 347.22, 456.14 52/100 20 6-HO-BDE-90 499.64 392.58, 390.99 100/54 30 6-HO-BDE-85 499.60 390.99, 340.09 100/40 25 6-HO-BDE-137 513.52 297.88, 470.69 2/100 25 2’-MeO-BDE-28 435.57 342.12, 340.12 100/44 25 2’-MeO-BDE-68 515.45 422.14, 420.06 40/100 30 6-MeO-BDE-47 515.47 422.14, 420.06 10/100 30 4’-MeO-BDE-49 515.47 356.17, 516.26, 501.12 100/4/12 15 6’-Cl-2’-MeO-BDE-68 549.43 456.17, 454.13, 434.33 55/100/1 25 6-MeO-BDE-90 435.57 420.89, 392.91, 339.95 44/100/22 25 6-MeO-BDE-85 593.38 499.68, 433.95 100/4 25 6-MeO-BDE-137 673.31 579.69, 577.59, 513.83 20/10/100 25 6’-MeO-BDE-17 435.56 341.95, 339.94 50/100 25 5-MeO-BDE-47 515.42 356.12, 516.25 100/2 15 5-Cl-6-MeO-BDE-47 549.40 455.92, 453.88, 390.00 100/80/80 20 3-MeO-BDE-100 433.56 418.92, 390.97 100/20 20 4-MeO-BDE-90 593.35 578.78, 433.84 100/48 15 2-MeO-BDE-123 593.35 499.84, 497.81 100/62 30 13 C -BDE-139 495.49 335.78, 415.01 100/30 30 C13-2-HO-BDE-99 511.57 351.82, 402.02, 403.91 60/80/100 30 C13-PCB-178 405.62 370.73, 335.86 80/100 20 Supporting Table 3 Responses caused by OH- and MeO-PBDE in the H4IIE-luc assay, relative to the maximum response to 2,3,7,8-TCDD (TCDD-max) and their respective 2,3,7,8-TCDD equivalency factors (RePH4IIE-luc). Chemicals TCDD DMSO Control 6'-Cl-2'-HO-BDE-7 2'-HO-BDE-28 2'-HO-BDE-68 6-HO-BDE-47 5-Cl-6-HO-BDE-47 6-HO-BDE-85 6-HO-BDE-90 2-HO-BDE-123 4-HO-BDE-90 6-HO-BDE-137 3-HO-BDE-100 2'-HO-BDE-66 2'-HO-BDE-25 2'-MeO-BDE-28 6-MeO-BDE-47 5-Cl-6-MeO-BDE-47 6-MeO-BDE-85 2-MeO-BDE-123 6-MeO-BDE-137 Test Concentrations TCDD-max RePH4IIE-luc (ng/ml) 0 2500 2500 10000 2500 10000 2500 10000 10000 10000 10000 10000 10000 10000 10000 10000 10000 10000 10000 10000 100.00% 0% 13.20% 12.70% 5.00% 52.70% 101.80% 42.20% 6.80% 31.30% 16.40% 56.20% 18.10% 35.20% 9.80% 25.70% 14.50% 59.40% 37.10% 9.60% 28.00% 5.40×10-05 1.30×10-06 1.27×10-10 7.63×10-05 4.00×10-04 2.20×10-04 7.35×10-12 3.32×10-06 7.23×10-07 1.91×10-04 8.96×10-07 3.92×10-06 1.99×10-07 2.18×10-06 1.71×10-07 6.48×10-05 2.56×10-05 2.23×10-08 2.68×10-06 Supporting Table 4 PBDEs analogs detected in other publications. Number Samples Tissue Chemicals Reference 1 Human Serum 6 2 Human Breast Milk 3 Human Blood 4´-HO-BDE-17, 6-HO-BDE-47, 3-HO-BDE-47, 4´-HO-BDE-49, 4-HO-BDE-42 4-HO-BDE-90 2’-MeO-BDE-28, 4’-MeO-BDE-17, 2’-MeO-BDE-75, 6-MeO-BDE-47, 2’-MeO-BDE-74, 6’-MeO-BDE-66, 4’-HO-BDE-17, 2’-HO-BDE-75, 6-HO-BDE-47, 2’-HO-BDE-74, 6’-HO-BDE-66 4-HO-BDE-42, 3-HO-BDE-47, 5-HO-BDE-47, 6-HO-BDE-47, 4’-HO-BDE-49, 5’-HO-BDE-99, 6’-HO-BDE-99 4 Human Serum 9 5 Human 4’-HO-BDE17, 5-HO-BDE47, 6-HO-BDE47, 4’-HO-BDE49 6-HO-BDE-47 5-Cl-6-HO-BDE-47, 5-Cl-6-MeO-BDE-47, 6’-HO-BDE-49, 6’-MeO-BDE-49, 4’-HO-BDE-49, 3’-Cl-6’-HO-BDE-49, 6’-Cl-2’-HO-BDE-68, 6’-Cl-2’-MeO-BDE-68, 6-MeO-BDE-90, 6-HO-BDE-47, 2’-MeO-BDE-68, 6-MeO-BDE-47, 2’-HO-BDE-68, 6-HO-BDE-99, 2’-HO-BDE-68, 6-HO-BDE-47, 3-HO-BDE-47, 5-HO-BDE-47, 4’-HO-BDE-49, 4-HO-BDE-42, 6-HO-BDE-90, 6-HO-BDE-99, 6-HO-BDE-85, 2-HO-BDE-123 2’-MeO-BDE-28, 4-MeO-BDE-42, 6-MeO-BDE-47, 3-MeO-BDE-47, 4’-MeO-BDE-49, 6-MeO-BDE-90, 6-MeO-BDE-99, 4-HO-BDE-42, 6-HO-BDE-47, 3-HO-BDE-47, 4’-HO-BDE-49, 6’-HO-BDE49, 2’-HO-BDE-68 6’-HO-BDE-49, 6-HO-BDE-47, 4’-HO-BDE-49 6′-HO-BDE-49, 2′-HO-BDE-68, 2′-HO-BDE-75, 6-HO-BDE-90, 6-MeO-BDE-17, 2′-MeO-BDE-28, 4-MeO-BDE-42, 5-MeO-BDE-47, 6-MeO-BDE-47, 6′-MeO-BDE-49, 6′-MeO-BDE-66, 2′-MeO-BDE-68, 6-MeO-BDE-90, 6-MeO-BDE-99 7 8 10 11 6 Salmo Blood 7 Fish Plasma 8 Glaucous Gulls and Polar Bears Plasma 9 Bald Eaglet Plasma 10 Beluga whales Blood, Milk and Blubber 11 Harbour seals and harbour porpoises Serum 2’-MeO-BDE-68, 6-MeO-BDE-47 12 Bird Serum 3-HO-BDE-47, 2'-HO-BDE-68, 4'-HO-BDE-17, 6-HO-BDE-47, 4'-HO-BDE-49, 3-MeO-BDE-47, 6-MeO-BDE-47 Blood 6-HO-BDE-47, 6-MeO-BDE-47 18 Plasma 3’-HO-PBDE-7, 6’-HO-PBDE-17, 2’-HO-PBDE-28, 4’-HO-PBDE-17, 3’-HO-PBDE-28, 6’-HO-PBDE-49, 19 13 14 Japanese amberjack and scalloped hammerhead shark bottlenose dolphins 12 13 14 15 16 17 2’-HO-PBDE-68, 6-HO-PBDE-47, 3-HO-PBDE-47, 5-HO-PBDE-47, 4’-HO-PBDE-49, 4-HO-PBDE-42, 6-HO-PBDE-90, 6-HO-PBDE-99, 4-HO-PBDE-90, 2-HO-PBDE-123, 6-HO-PBDE-85, 6-HO-PBDE-137 15 1. ringed seals Liver and Plasma 16 Water 17 Sediment 18 Red Alga and Cyanobacteria 19 Blood of Japanese Terrestrial Mammals 20 Human Blood 21 Marine Sponges and Fish Samples 2′-HO-BDE-68, 6-HO-BDE-47, 3-HO-BDE-47, 6-HO-BDE-90, 4′-HO-BDE-49 6′-HO-BDE-49, 2′-HO-BDE-68, 6-HO-BDE-47, 3-HO-BDE-47, 5-HO-BDE-47, 4′-HO-BDE-49, 4-HO-BDE-42, 6-HO-BDE-90, 6-HO-BDE-99, 4-HO-BDE-90, 2-HO-BDE-123, 6-HO-BDE-85, 6-HO-BDE-137 6-HO-BDE-47, 2-HO-BDE-68, 5-HO-BDE-47, 4-HO-BDE-49, 3-HO-BDE-47 2’-HO-BDE-68, 6-HO-BDE-47, 6-HO-BDE-90, 6-HO-BDE-99, 2-HO-BDE-123, 6-HO-BDE-85, 6-HO-BDE-137, 2’-MeO-BDE-68, 6-MeO-BDE-47, 6-MeO-BDE-85, 6-MeO-BDE-137 2'-HO-BDE-28, 2'-HO-BDE-68, 6-HO-BDE-47, 5-HO-BDE-47, 4-HO-BDE-49, 3-HO-BDE-154 3-OH-BDE-100, 3'-OH-BDE-100, 3-OH-BDE-99, 4'-OH-BDE-101, 3-OH-BDE-154, 3'-OH-BDE-154, 3-OH-BDE-153, 4-OH-BDE-187, 4'-OH-BDE-17, 4-OH-BDE-42, 6-OHBDE-47, 3-OH-BDE-47, 4'-OH-BDE-49, 4-OH-BDE-90 2'-MeO-BDE68, 6-MeO-BDE47, 2,2'-diMeO-BB80, 2',6-diMeO-BDE68, 2'-OH-BDE68, 6-OH-BDE47, 2,2'-diOH-BB80, 2',6-diOH-BDE68 20 21 22 23 24 25 26 Wan, Y.; Wiseman, S.; Chang, H.; Zhang, X. 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