Dioxin-like Potency of HO- and MeO- Analogues of PBDEs’ thePotential Risk through Consumption of Fish from Eastern ChinaGuanyong 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 CheeAvenue, 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 theiranalogues, such as hydroxylated PBDE (HO-PBDEs) andmethoxylated PBDE (MeO-PBDEs) are of interest due to theirwide distribution, bioaccumulation and potential toxicity to humansand wildlife. While information on the toxicity/biological potenciesof PBDEs was available, information on analogues of PBDEs waslimited. Dioxin-like toxicity of 34 PBDEs analogues was evaluated byuse of the H4IIE-luc, rat hepatoma transactivation bioassay in 384-well plate format at concentrations ranging from 0 to 10 000 ng/mL.Among the 34 target analogues of PBDEs studied here, 19 activatedthe aryl hydrocarbon receptor (AhR) and induced significant dioxin-like responses in H4IIE-luc cells. Efficacies of the analogues ofPBDEs ranged from 5.0% to 101.8% of the maximum responsecaused 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 ofPBDEs were detected mostly in marine organisms. Of these 11 detected analogues of PBDEs, 6 were found to have measurabledioxin-like potency. Though some analogues of PBDEs exhibited significant dioxin-like potency as measured by responses of theH4IIE-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. EnvironmentalProtection Agency, respectively. (Hazard Quotients (HQ) < 0.005) Additional investigations should be conducted to evaluatethe 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

Due to their performance and cost-effectiveness, polybromi-nated diphenyl ethers (PBDEs) have been used for many yearsas flame retardants in various commercial products, such asfurniture, textiles, plastics, paints, and electronic appliances.1,2

Due to their persistence and potential to bioaccumulate,3

PBDEs have been detected in various environmental matrixesand concentrations have been increasing continuously.4

Hydroxylated polybrominated diphenyl ethers (HO-PBDEs)and methoxylated polybrominated diphenyl ethers (MeO-PBDEs) have been observed in tissues of wildlife and humansand have been suggested to be biotransformation products ofPBDEs,.5,6 This is especially true for 6-HO-BDE-47, 5-HO-BDE-47, and 5′-HO-BDE-99. Concerns have been raised aboutthe potential toxicity of these PBDEs analogues and theirmodes of molecular toxicity.

Previous studies have shown that PBDEs and their analoguescan interact with some endocrine nuclear receptors such asestrogen receptors (ER), androgen receptor (AR) and thyroidhormone receptor (ThR). Furthermore, HO-PBDEs weremore potent than their postulated precursor PBDEs andcorresponding MeO-PBDEs.7−11 Because of its structuralsimilarity to other polyhalogenated aromatic hydrocarbonssuch as polychorinated biphenyls (PCBs), PBDEs have beensuggested to be potential agonists of the Aryl hydrocarbonreceptor (AhR). To test this hypothesis, several in vivo or invitro experiments have been conducted, and a weak response of

Received: June 12, 2012Revised: August 20, 2012Accepted: September 6, 2012Published: September 6, 2012

Article

pubs.acs.org/est

© 2012 American Chemical Society 10781 dx.doi.org/10.1021/es302317y | Environ. Sci. Technol. 2012, 46, 10781−10788

AhR has been observed.12,13 However, the presence ofbrominated furans, which were impurities in PBDEs was thelikely reason for these apparent potencies.14,15 PBDEs did notactivate the AhR, but AhR-mediated effects of tetrachlorodi-benzo-p-dioxin (TCDD) could be reduced during coexposureto PBDEs and TCDD. This chemical activity effect is likely dueto the fact that PBDEs can interact with the AhR but not bindwith sufficient avidity to produce AhR-mediated signaling.However, an investigation of the potency of the HO- and MeO-analogues 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 bymarine organisms.18,19 There are contradictory reports onsources of HO-PBDEs analogues. Both in vitro and in vivoexposures have shown that HO-PBDEs might be formed due tobiotransformation of various PBDEs.20 However, recentresearch has demonstrated that HO-PBDEs, especially 6-HO-BDE-47, can also be generated from naturally occurring MeO-PBDEs.21−23 Specifically, demethylation of 6-MeO-BDE-47 wasthe primary transformation pathway that resulted in formationof 6-HO-BDE-47 in the small fish, Japanese medaka, while thepreviously hypothesized formation of HO-PBDEs fromsynthetic BDE-47 did not occur.21

Here we report the first evidence that analogues of PBDEshave measurable potency as AhR-agonists and might elicitdioxin-like toxicity. Concentrations of PBDE and theiranalogues were determined in freshwater and marine fishesfrom East China. Finally, the potential risk of these analogues ofPBDEs through dioxin-like mechanism was assessed.

2. MATERIALS AND METHODS2.1. Chemicals. PBDEs (BDE-17, -28, -71, -47, -66, -100,

-99, -85, -154, -153, -138, -183, -190), C13-BDE-139, C13-2-HO-BDE-99 and 13C-PCB-178 used for quantification werepurchased from Cambridge Isotope Laboratories (Andover,MA). Analogues of PBDEs, including 19 HO-PBDEs and 15MeO-PBDEs (Figure 1), were synthesized in the Departmentof Biology and Chemistry of City University of Hong Kongfollowing the previously published methods.24 Purities of thesynthesized compounds were determined to be higher than98%. The results of proton NMR and electrospray LC-MS/MSof the intermediates and end products, the synthesis procedurewas confirmed to not generate brominated dioxin and/orfurans.8

2.2. H4IIE-luc Cell Culture and Bioassay. For the firsttime a high throughput, 384-well plate method was used todetermine the relative potencies of various PBDE and theiranalogues. Rat hepatoma cells that had been stably transfected

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.).

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with an AhR-responsive luciferase reporter gene construct(H4IIE-luc) was used to study AhR activity of PBDEsanalogues.25,26 Potencies of individual analogues of PBDEswere 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. Toavoid cross-contamination, each chemical treatment wasbordered by one blank column. A volume of 79 μL of mediumwas also added into each well of blank columns. On the secondday, cells were dosed with serial dilution of chemicals stocksolutions (2 × 106 ng/mL) with dimethylmethane (DMSO) assolvent. Stock solutions were diluted with cell culture mediumby 20-fold, and then 0.8 μL of the diluted solution was addedinto each well of 384-well plate to make to a final dose at 0.5%v/v. Three replicates were conducted per treatment, includingTCDD standards. Each control and each standard concen-tration were averaged for all plates within a given experiment.For chemicals and TCDD standards, 7 (0−10 000 ng/mL) and10 (0−1.61 ng/mL) concentrations were used, respectively. Onthe fifth day, cells were lysed and luciferase activity mediated byAhR receptor was assayed by use of a commercial kit (PromegaCorporation, Madison, WI) in a microplate reader (BioTekInstruments Inc., Winooski, VT).2.3. Sampling. Six fishes (the sharpbelly (Hemicculter

leuciclus), the yellow catfish (Pelteobagrus fulvidraco), thecrucian carp (Carassius auratus), the bigmouth grenadieranchovy (Coilia macrognathos bleeker), the oriental sheatfish(Silurus spp), and the common carp (Cyprinus carpio)) werecollected from the lower Yangtze River. Five marine fishes (therazor clam (Sinonovacula constrzcta), the spotted sicklefish(Drepane punctata), the elongate ilisha (Ilisha elongate), the big-eyed flathead (Suggrundus meerdervoortii), the small yellowcroaker (Pseudosciaena polyactis)) were collected from YellowSea. All samples were transported to the lab on ice and weremaintained intact at −20 °C until dissection for subsequentidentification and quantification of PBDEs and their analogues.Details of the samples are given in Supporting Information (SI)(Table S1).

2.4. Chemical Analysis. Details of the instrumentalanalyses, including “Identification and Quantification ofPBDEs and their analogues”, “Instrument Conditions”, “QualityAssurance/Quality Control”, and “TEQBIO testing for eachbiological sample” are provided in the SI.

2.5. Data Analysis. Relative potency factors (RePs),expressed as g TCDD/g chemical, were calculated for eachanalogue of PBDEs as the quotient of the 20% effectconcentration (EC20) for TCDD divided by the EC20 ofindividual PBDE and analogues of PBDEs.29 TCCD equiv-alents (TEQ) for each sample were calculated as the sum of theproduct of concentrations of individual analogues of PBDEs bytheir respective RePs as follows:

∑= ×=

TEQ concentration RePn

i

i i

1

Following EPA superfund guidance terminology, hazardquotients (HQ) were calculated as the ratio of the exposureestimate to effects concentration considered to represent a“safe” environmental concentration or dose. For quantification,statistical analyses were performed by use of SPSS 13.0 forWindows (SPSS Inc., Chicago, IL). Spearman rank correlationwas used to examine the strength of associations betweenparameters (including mass and length of individual fish, theconcentrations of individual target compounds). Mann−Whitney U nonparametric tests were used to compare thedifference between/among groups. Concentrations of analytesin fishes are presented as the mean and range. Figures weregenerated with ChemBioDraw Ultra 11.0 (Figure 1), MicrosoftOffice Excel 2007 (Figure 2 and SI Figure S3), OriginPro 8(Figure 2) or with R software (version 2.14.1) (SI Figures S1and S2). The R code for these analyses is available uponrequest.

3. RESULTS3.1. Method Robustness. A 384-well plate format for the

H4IIE-luc assay was used here for the first time, and therobustness of this modified method was evaluated. Exposure ofH4IIE-luc cells to AhR agonists results in induction of luciferaseactivity that is a function of duration of exposure, dose, and

Figure 2. Dioxin-like effects of 34 PBDEs analogs in the AhR transactivation assay using stable H4IIE-luc reporter cells. Cells were treated with0.0024−10 mg/L of 34 target PBDEs analogs. Values represent mean ± SD of three independent experiments and are presented as the percentage ofthe response, compared with 100% activity defined as the maximum activity achieved with TCDD.

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Table

1.Con

centration

sof

PBDEsandHO-andMeO

-Analogues

ofPBDEsin

Fishes

from

theYangtze

River

andMarineOrganismsfrom

theYellowSea,China

YellowSeasamples

Yangtze

River

samples

chem

icals

Sinonovacula

constrzcta

Drepane

punctata

Ilishaelongata

Suggrundus

meerdervoortii

Pseudosciaena

polyactis

Hem

icculter

leuciclus

Pelteobagrus

fulvidraco

Carassius

auratus

Coilia

macrognathos

bleeker

Silurusspp

Cyprin

uscarpio

BDE-17

3.81

±0.90

0.12

±0.11

ND

ND

ND

0.93

±0.33

3.58

±2.24

ND

ND

ND

ND

BDE-28

ND

0.06

±0.06

ND

ND

ND

ND

ND

ND

ND

ND

ND

BDE-71

ND

0.18

±0.07

ND

ND

0.09

±0.03

1.81

±0.51

0.11

±0.00

0.14

±0.02

0.15

±0.04

0.25

±0.11

1.56

±0.03

BDE-47

6.08

±0.55

0.27

±0.10

0.93

±0.20

1.44

±0.01

0.39

±0.01

17.67±

3.25

4.63

±1.02

1.12

±0.05

0.16

±0.02

5.04

±1.31

7.84

±0.12

BDE-66

ND

0.01

±0.00

ND

ND

0.02

±0.00

ND

0.05

±0.02

ND

ND

ND

ND

BDE-100

ND

0.15

±0.04

ND

ND

0.04

±0.00

0.57

±0.06

ND

ND

ND

ND

ND

BDE-99

2.10

±0.04

ND

ND

ND

0.06

±0.00

0.17

±0.02

ND

ND

ND

ND

ND

BDE-85

ND

ND

0.16

±0.06

ND

ND

0.71

±0.54

ND

ND

ND

ND

ND

BDE-154

ND

3.94

±0.74

0.74

±0.06

6.56

±0.16

0.86

±0.08

20.35±

19.28

12.03±

1.85

3.99

±1.03

0.90

±0.37

38.92±

3.09

7.22

±1.43

BDE-153

ND

3.59

±0.54

0.43

±0.13

8.52

±0.23

0.23

±0.03

48.78±

8.79

21.14±

2.10

6.42

±1.54

0.34

±0.03

60.00±

4.34

ndBDE-183

ND

6.35

±2.17

4.82

±0.54

6.72

±0.10

0.14

±0.06

17.25±

1.94

8.71

±1.32

39.31±

6.03

0.25

±0.17

32.04±

1.87

5.86

±1.52

BDE-190

ND

ND

ND

ND

ND

ND

ND

36.51±

2.14

0.03

±0.00

ND

ND

2′-M

eO-BDE-

680.85

±0.01

2.16

±0.83

ND

0.16

±0.01

2.15

±0.04

ND

ND

ND

0.11

±0.01

ND

ND

6-MeO

-BDE-

470.18

±0.02

33.42±

8.14

ND

20.88±

0.56

8.21

±4.01

ND

ND

ND

6.99

±1.04

ND

ND

6-MeO

-BDE-

90ND

ND

11.09±

0.73

2.28

±0.13

ND

ND

ND

ND

ND

ND

ND

3-MeO

-BDE-

100

ND

ND

1.49

±1.19

ND

ND

ND

ND

ND

ND

ND

ND

2-MeO

-BDE-

123

ND

ND

ND

0.14

±0.01

ND

ND

ND

ND

ND

ND

ND

6′-M

eO-BDE-

17ND

0.15

±0.00

ND

ND

0.09

±0.04

ND

ND

ND

ND

ND

ND

4-MeO

-BDE-

90ND

ND

6.26

±1.91

ND

ND

ND

ND

ND

ND

ND

ND

2′-M

eO-BDE-

28ND

ND

ND

ND

0.04

±0.00

ND

ND

ND

ND

ND

ND

6-HO-BDE-47

1.18

±0.29

0.15

±0.02

ND

ND

ND

ND

ND

ND

ND

ND

ND

2′-H

O-BDE-

680.16

±0.00

0.02

±0.01

ND

ND

ND

ND

ND

ND

ND

ND

ND

4-HO-BDE-90

12.66±

3.42

1.99

±1.08

ND

2.14

±0.21

0.16

±0.03

ND

ND

ND

ND

ND

ND

TEQ

CHEM

9.67

×10

−5

1.88

×10

−4

NA

2.41

×10

−5

2.33

×10

−5

NA

NA

NA

1.68

×10

−5

NA

NA

TEQ

BIO

4.20

2.42

1.18

3.54

1.65

2.79

6.57

4.68

2.86

6.60

0.86

a“N

D”means

notdetected,and

“NA”means

notachieved.bAllconcentrations

hadbeen

representedwith

meanandstandard

error.(ng/glip).c The

unitof

TEQ

was

“pg/gwet

weight”;TEQ

CHEM

(PBDEs

analogues TEQ

H4IIE‑luc)was

calculated

asthesum

oftheproductof

concentrations

ofindividualanaloguesof

PBDEs

bytheirrespectiveRePs;TEQ

BIO

(raw

extract TEQ

H4IIE‑luc)representtheTCDD

equivalentsof

rawextractof

biologicalsamples

asmeasuredby

theH4IIE-luccells.

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strength of binding of ligands to the AhR (Figure 2). Eachpoint of the curve represents the mean of three replicates andits standard error, and also represents the ratio of meanluciferase response relative to the maximum response to 2,3,7,8-TCDD (TCDDmax). The mean EC50 for luciferase inductionby 2,3,7,8-TCDD was 5.4 ± 0.7 pg/mL (16.9 ± 2.2 pM). Whenthe maximal response induced by chemicals exceeded thestandard deviation (expressed as % TCDDmax) of the meanDMSO blank response (0% TCDDmax) by at least 3-fold, thechemical was deemed to have significant AhR-mediatedpotency.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-mediatedpotency relative to 2,3,7,8-TCDD in H4IIE-luc cells. The OH-BDE's that exhibited significant potency included 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, and 2′-HO-BDE-25. (Figure 2) At the maximal testedconcentration of 10 000 ng/mL, 6′-Cl-2′-HO-BDE-7, 2′-HO-BDE-28, 6-HO-BDE-47, and 6-HO-BDE-85 caused significantcytotoxicity to H4IIE-luc cells, and the TCCD-max for thesechemicals was 2500 ng/mL. (SI Table S3) Similarly, 6 of the 15MeO-PBDEs that were tested exhibited significant AhR-mediated potency. These MeO-PBDEs included 2′-MeO-BDE-28, 6-MeO-BDE-47, 5-Cl-6-MeO-BDE-47, 6-MeO-BDE-85, 2-MeO-BDE-123, and 6-MeO-BDE-137, which accountedfor 40% of the tested MeO-PBDEs. (Figure 2) Dose−responsecurves of four PBDEs analogues that exceeded 50% TCDD-max, including 6-HO-BDE-47, 5-Cl-6-HO-BDE-47, 6-HO-BDE-137, and 5-Cl-6-MeO-BDE-47, were also fitted, whichindicated that these chemicals exhibited significant, concen-tration-dependent, AhR-mediated potency as determined inH4IIE-luc cells (SI Figure S1).3.3. Concentrations of PBDEs and their Analogues.

3.3.1. Freshwater Fishes. Concentrations of 13 PBDEs and 34PBDEs analogues were quantified in six fishes from the YangtzeRiver, China. Eleven PBDEs (BDE-17, BDE-71, BDE-47, BDE-66, BDE-100, BDE-99, BDE-85, BDE-154, BDE-153, BDE-183,and BDE-190), and two analogues of PBDEs (2′-MeO-BDE-68and 6-MeO-BDE-47), were detected. (SI Figure S3 and Table1) The analogues of PBDEs, 2′-MeO-BDE-68, and 6-MeO-BDE-47, were detected only in bigmouth grenadier anchovy,which is a migratory fishes that resides in the Yangtze Riverestuary and migrate back to the Yangtze River to spawn.Concentrations of ∑PBDEs ranged from 1.8 to 1.4 × 102 ng/glipid with mean and median values of 6.8 × 101 ng/g lipid and6.9 × 101 ng/g lipid, respectively. Concentrations of fourPBDEs congers, including BDE-47, BDE-154, BDE-153, andBDE-183, were detected most frequently and exhibited thegreatest concentrations and percentages of the four individualcongeners that contributed to total PBDE (∑PBDEs) werecalculated: 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 YellowSea, China. Eleven PBDEs (BDE-17, BDE-28, BDE-71, BDE-47, BDE-66, BDE-100, BDE-99, BDE-85, BDE-154, BDE-153,and BDE-183), 8 MeO-PBDEs (2′-MeO-BDE-28, 2′-MeO-BDE-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) and3 HO-PBDEs (6-HO-BDE-47, 2′-HO-BDE-68, and 4-HO-

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 thanthose in freshwater fish. These results suggest that fishes in theYangtze River were more affected by synthetic chemicals thanthose from the marine environment. Unlike freshwaterorganisms, analogues of PBDEs were detected in each of themarine fishes, especially 2′-MeO-BDE-68, 6-MeO-BDE-47, and4-HO-BDE-90, which were detected in 80% of samples.

3.4. TEQCHEM and TEQBIO in Samples. In order to assessthe potential for adverse effects of PBDEs and their analogueson humans and wildlife in the future, PBDEs analoguesTEQH4IIE(TEQCHEM) for each organism were calculated as the sum ofthe product of concentrations of individual analogues of PBDEsby their respective RePs, which ranged from NA (NA meansnot achieved) to 1.88 × 10−4 pg.g−1 wet weight (Table 1).Among these testing organisms, the analoguesTEQH4IIE in spottedsickle fish was highest. Raw extractTEQH4IIE (TEQBIO) were testedto be from 0.86 to 6.60 pg pg.g−1 wet weight (Table 1). Theratio of TEQCHEM and TEQBIO (TEQCHEM/ TEQBIO) werecalculated to be from NA to 7.77 × 10−5.

4. DISCUSSIONSlight alterations in structures of chemicals can alter thepotency to bind to biomolecules. Based on 6 homologous pairsof HO- and MeO-substitued BDE, including 2′-HO-BDE-28and 2′-MeO-BDE-28, 6-HO-BDE-47, and 6-MeO-BDE-47, 5-Cl-6-HO-BDE-47 and 5-Cl-6-MeO-BDE-47, 6-HO-BDE-85and 6-MeO-BDE-85, 2-HO-BDE-123 and 2-MeO-BDE-123,6-HO-BDE-137 and 6-MeO-BDE-137, the maximum responserelative to TCDDmax caused by HO-PBDEs was greater thanthat caused by MeO-PBDEs, which indicated that HO-PBDEsexhibited greater potencies to induce AhR activity than didMeO-PBDEs. The maximum potency of four chemicals,including 6-HO-BDE-47, 5-Cl-6-HO-BDE-47, 6-HO-BDE-137, and 5-Cl-6-MeO-BDE-47, exceeded 50% of TCDDmax,even though the respective analogous PBDEs did not result insignificant activation of the AhR-mediated responses. (SI FigureS1) These results are consistent with the observation thataddition of a MeO- or HO group can result in greater potencyas AhR agonists.30 This conclusion is supported by comparingrelative potencies of BDE-47, 6-MeO-BDE-47 and 6-HO-BDE-47.31

ReP values were calculated for each of the HO- and MeO-substituted 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-BDE-137, 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 than2,3,7,8-TCDD Equivalents suggested by the World HealthOrganization (TEFWHO) reported for mono-ortho-substitutedPCBs, which were assigned a value of 3 × 10−5. Of thesubstituted analogues studied here, 5-Cl-6-HO-BDE-47 ex-hibited the greatest relative potency, which was almost equal tothat for OCDD and OCDF.Concentrations of analogues of PBDEs, regardless of

whether they are natural products or come from syntheticBDE, are greater in marine organisms.18,22 For this reason,marine organisms might pose greater risks to humans via thediet than would freshwater organisms. Among those 11detected analogues of PBDEs, 6 exhibited AhR-mediatedpotency. These included: 2′-HO-BDE-68, 2-MeO-BDE-123,

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2′-MeO-BDE-28, 4-HO-BDE-90, 6-HO-BDE-47, and 6-MeO-BDE-47. Among those analogues of PBDEs that were detectedin fishes, 2′-MeO-BDE-68, 6-MeO-BDE-47, 2′-MeO-BDE-28,6-HO-BDE-47, and 2′-HO-BDE-68 had been previouslyidentified and confirmed to be natural products of eithermarine sponges or their associated filamentous cyanobacteria,red or green algae.23 6-MeO-BDE-90 and 6′-MeO-BDE-17have been observed in marine wildlife, but have not beenclassified as natural products. 4-HO-BDE-90 has been detectedin blood serum of humans.6 For these four substituted BDEthat have been observed to occur in algae or sponges, 2′-HO-BDE-28 and 6-HO-BDE-85 have been determined to be ofnatural origin.23 Analogues of PBDEs might be concentrated inthe marine environment by fishes. These results wereconsistent with previously published results.19 Concentrationsof ∑PBDEs in organisms studied here, were generally less thanthose in biota from other locations around the world, but equalto those reported by Gao et al (mean: 44.04 ng/g lipid).32

Analogues of PBDEs were identified to be naturally occurringAhR ligands. Generally, AhR ligands were classified into twocategories: synthetic and naturally occurring. PCBs PCDD/Fsand PAHs had been known to be synthetic, dioxin-likecompounds. However, recent work has focused on naturallyoccurring compounds with the hope of identifying endogenousligands. After exposure to PBDEs analogues, AhR of H4IIEcells was activated, which indicated that PBDEs analoguesshould also be listed as naturally occurring dioxin-likecompounds. Most importantly, PBDEs analogues, especiallyMeO-PBDEs, can be accumulated by organisms because oftheir large solubility in lipids,23 unlike the other naturallyoccurring dioxin-like compounds, derivatives of tryptophan33 ortetrapyrroles.34

Based on the calculated PBDEs analoguesTEQH4IIE, risks posed bymarine organisms were greater than freshwater fishes. Takinginto account risk related to consumption of fishes, theEuropean Union (EU) had proposed tolerance limits of 8 pgTEQWHO g−1 wet weight for fish and fishery products,35 whichis relatively greater than that in the marine fishes studied here.Assuming that a 60 kg adult would eat 1 kg sea fish, the totaldaily dietary intake (TDI) from PBDEs analogues wasestimated to be approximately 3.13 × 10−3 pg PBDEs analogues-

TEQH4IIE‑luc /kg bw-day), which is also less than the oralreference dose (RfD) of 7 × 10−1 pg/kg-day for TCDD derivedby U.S. Environmental Protection Agency.36 This RfD wasbased on the results of two epidemiologic studies: spermconcentration and motility in men, and thyroid stimulatinghormone levels in newborn infants. The HQ from PBDEsanalogues in marine fishes was calculated to be 0.005. Thoughconcentrations of PBDEs analoguesTEQH4IIE in individual fisheswere less than the reported tolerance limits, humans could beexposed to some analogues of PBDEs that are of natural originvia seafood and thus should be further evaluated in vivo fortheir toxicity.

Raw extractTEQH4IIE (TEQBIO) for each biological sample wasdetermined to be from 0.86 to 6.60 pg pg.g−1 wet weight, whichwas lower than samples from other locations37,38 and less thanthe tolerance limit of 8 pg TEQ g−1 wet weight for dioxin anddioxin-like compounds in fish proposed by the EuropeanUnion. The ratio of TEQCHEM and TEQBIO (TEQCHEM/TEQBIO) were calculated to be from NA to 7.77 × 10−5. Theseresults suggested that the contribution of PBDEs analogues-

TEQH4IIE‑luc to the total TEQ in fishes was very little, nomore than 0.1%, though some OH- and MeO- analogues do

exhibit significant concentration-dependent AhR-mediatedpotency.Thirty eight OH-PBDE and 25 MeO-PBDEs have been

detected in the environment or tissues of humans (SI Figure S2and Table S4). Of the 63 analogues of PBDE, 15 exhibitedmeasurable dioxin-like potency. Four analogues, including 2′-MeO-BDE-28, 4-HO-BDE-90, 6-HO-BDE-47, and 6-MeO-BDE-47, have been detected in various environmental samples,human samples and this study, and exhibit significant AhRagonist potency as measured in H4IIE-luc cells. This isespecially true for 6-HO-BDE-47 and 6-MeO-BDE-47, whichhave been shown to be of natural origin in marine organismsand have been quantified in various environment media. Sinceanalogues of PBDEs exhibited dioxin-like potency andconcentrations in the environment that are sufficient to causeadverse effects, these chemicals should be considered whenassessing the total potency of mixtures in the environment.

5. PERSPECTIVEFor the first time, it has been reported that naturally occurringanalogues of PBDEs could exhibit measurable AhR-mediatedpotency. While the ReP values of the OH- and MeO-analoguesof PBDE are in general less than those of PCDD/DF andPCBs, they can contribute significant proportions of the totalconcentration of AhR-mediated potency in marine organisms.In China, wild fish are considered beneficial to human healthand marine algae and plants are thought to be nutritionally rich,and thus relatively large quantities are consumed by people. It isstill too early to reject this “ancient wisdom”, however,additional work should be conducted to assess the balancebetween the toxicity and benefit of these compounds naturallyoccurred in dietary source in East China.

■ ASSOCIATED CONTENT*S Supporting InformationSupporting Information includes additional information asnoted in the text including Tables S1−S4 and Figures S1−S3.This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: 86-25-89680623; fax: 86-25-83707304; e-mail: yuhx@nju.edu.cn (H. Y.); howard50003250@yahoo.com (X. Z.).NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the National Natural ScienceFoundation of China (Nos. 20737001 and 20977047) andNational Science and Technology Major Project (No.2008ZX08526-003). The research was also supported by agrant from National Natural Science Foundation of China(No.21007025) and from Major State Basic Research Develop-ment Program (No. 2008CB418102). G.S. was supported theShanghai Tongji Gao Tingyao Environmental Science &Technology Development Foundation (STGEF). J.P.G. wassupported by the program of 2012 “High Level ForeignExperts” (#GDW20123200120) funded by the State Admin-istration of Foreign Experts Affairs, the P.R. China. J.P.G. wasalso supported by the Canada Research Chair program, an atlarge Chair Professorship at the Department of Biology and

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Chemistry and State Key Laboratory in Marine Pollution, CityUniversity of Hong Kong, The Einstein Professor Program ofthe Chinese Academy of Sciences.

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Dioxin-like Potency of OH- and MeO- Analogues of PBDEs’ the

Potential Risk through Consumption of Fish from Eastern China

Guanyong Su1; Jie Xia

1; Hongling Liu

1; Michael H. W. Lam

2;Hongxia Yu

1*; John P.

Giesy1,2,3

; Xiaowei Zhang1*

1State Key Laboratory of Pollution Control and Resource Reuse & School of the

Environment, Nanjing University, Nanjing, China

2Department of Biomedical Veterinary Sciences and Toxicology Centre, University of

Saskatchewan, Saskatoon, SK S7N 5B3, Canada

3State 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 - 13

C-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 13

C-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. Samples and standards were

analyzed in selected reaction mode (SRM) mode. Quantification and qualification

were processed by SRM modes. 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. To ensure accuracyof analytical procedures,

13C-labeled BDE-139 and

13C-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 13

C-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 as the extraction

solvent at a temperature of 100 ℃ and pressure of 1500 psi. Two cycles were

performed for each sample and duration of each cycle was 10 min. 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.

Chemicals Ion pairs for Quantification and Qualification

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

C13-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 Test Concentrations

(ng/ml) TCDD-max RePH4IIE-luc

TCDD

100.00%

DMSO Control 0 0%

6'-Cl-2'-HO-BDE-7 2500 13.20% 5.40×10-05

2'-HO-BDE-28 2500 12.70% 1.30×10-06

2'-HO-BDE-68 10000 5.00% 1.27×10-10

6-HO-BDE-47 2500 52.70% 7.63×10-05

5-Cl-6-HO-BDE-47 10000 101.80% 4.00×10-04

6-HO-BDE-85 2500 42.20% 2.20×10-04

6-HO-BDE-90 10000 6.80% 7.35×10-12

2-HO-BDE-123 10000 31.30% 3.32×10-06

4-HO-BDE-90 10000 16.40% 7.23×10-07

6-HO-BDE-137 10000 56.20% 1.91×10-04

3-HO-BDE-100 10000 18.10% 8.96×10-07

2'-HO-BDE-66 10000 35.20% 3.92×10-06

2'-HO-BDE-25 10000 9.80% 1.99×10-07

2'-MeO-BDE-28 10000 25.70% 2.18×10-06

6-MeO-BDE-47 10000 14.50% 1.71×10-07

5-Cl-6-MeO-BDE-47 10000 59.40% 6.48×10-05

6-MeO-BDE-85 10000 37.10% 2.56×10-05

2-MeO-BDE-123 10000 9.60% 2.23×10-08

6-MeO-BDE-137 10000 28.00% 2.68×10-06

Supporting Table 4 PBDEs analogs detected in other publications.

Number Samples Tissue Chemicals Reference

1 Human Serum 4´-HO-BDE-17, 6-HO-BDE-47, 3-HO-BDE-47, 4´-HO-BDE-49, 4-HO-BDE-42 4-HO-BDE-90 6

2 Human Breast Milk 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

7

3 Human Blood

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

8

4 Human Serum 4’-HO-BDE17, 5-HO-BDE47, 6-HO-BDE47, 4’-HO-BDE49 9

5 Human 6-HO-BDE-47

10

6 Salmo Blood

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,

11

7 Fish Plasma 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

12

8 Glaucous Gulls and

Polar Bears Plasma

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

13

9 Bald Eaglet Plasma 6’-HO-BDE-49, 6-HO-BDE-47, 4’-HO-BDE-49 14

10 Beluga whales Blood, Milk

and Blubber

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

15

11 Harbour seals and

harbour porpoises Serum 2’-MeO-BDE-68, 6-MeO-BDE-47

16

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

17

13

Japanese amberjack and

scalloped hammerhead

shark

Blood 6-HO-BDE-47, 6-MeO-BDE-47 18

14 bottlenose dolphins 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

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 ringed seals Liver and

Plasma 2′-HO-BDE-68, 6-HO-BDE-47, 3-HO-BDE-47, 6-HO-BDE-90, 4′-HO-BDE-49

20

16 Water

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

21

17 Sediment 6-HO-BDE-47, 2-HO-BDE-68, 5-HO-BDE-47, 4-HO-BDE-49, 3-HO-BDE-47 22

18 Red Alga and Cyanobacteria 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

23

19 Blood of Japanese Terrestrial Mammals 2'-HO-BDE-28, 2'-HO-BDE-68, 6-HO-BDE-47, 5-HO-BDE-47, 4-HO-BDE-49, 3-HO-BDE-154 24

20 Human Blood

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

25

21 Marine Sponges and Fish Samples 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

26

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Supporting Figure 1 Responses and a fitted curve for TCDD and 4 analogues of PBDEs that resulted in luciferase expression that exceeded 50 % of

TCDD-max. Individual values and mean are plotted along with the fitted curve.

Supporting Figure 2 Number of PBDEs analogues detected in our study (marked with red “This Study”), with a dioxin-like activity (marked with

green “Dioxin-like Activity”), detected in the previous publications (marked with blue “Environment Samples”), and detected in human tissues

(marked with pink “Human Tissues”).

Supporting Figure 3 Profiles of concentrations of PBDEs (A) and HO- and MeO-analogues of PBDEs (B) in fishes from the Yangtze River and marine

organisms from the Yellow Sea, china.