Tissue Concentrations ofPolybrominated Compounds inChinese Sturgeon (Acipensersinensis): Origin, HepaticSequestration, and MaternalTransferK U N Z H A N G , † Y I W A N , ‡

J O H N P . G I E S Y , ‡ , § , |

M I C H A E L H . W . L A M , | S T E V E W I S E M A N , ‡

P A U L D . J O N E S , ‡ A N D J I A N Y I N G H U * , †

Laboratory for Earth Surface Processes, College of Urban andEnvironmental Sciences, Peking University, Beijing 100871, China,Department of Veterinary Biomedical Sciences and ToxicologyCentre, University of Saskatchewan, Saskatoon,Saskatchewan, S7J 5B3, Canada, Zoology Department, Centerfor Integrative Toxicology, Michigan State University, EastLansing, Michigan 48824, and Department of Biology andChemistry, and State Key Laboratory in Marine Pollution, CityUniversity of Hong Kong, Kowloon, Hong Kong SAR, China

Received January 31, 2010. Revised manuscript receivedJune 3, 2010. Accepted June 29, 2010.

Information on concentrations of polybrominated compoundsin various tissues of wild fish is limited. Concentrations ofpolybrominated diphenyl ethers (PBDEs), methoxylated PBDEs(MeO-PBDEs), and hydroxylated PBDEs (OH-PBDEs) weremeasured in 12 organs and eggs of 17 female Chinese sturgeon(Acipenser sinensis). The highest concentrations of PBDEs(42.8 ( 39.4 ng/g ww), and MeO-PBDEs (135 ( 63.6 pg/g ww)occurred in adipose followed by liver (PBDEs: 25.0 ( 27.0ng/g ww, MeO-PBDEs: 32.3 ( 29.1 pg/g ww) and eggs (PBDEs:21.2 ( 19.4 ng/g ww, MeO-PBDEs: 120 ( 119 pg/g ww), andthe highest concentration of OH-PBDEs was observed in liver(185 ( 174 pg/g ww) and eggs (178 ( 294 pg/g ww). Thelack of in vitro transformation of 6-MeO-BDE47 or BDE47 bymicrosomes prepared from Chinese sturgeon liver suggests thatmost 6-OH-BDE47 was directly accumulated as a naturalproduct. Lipid-normalization revealed preferential accumulationof PBDEs in liver, and ratios of concentrations between eggsand liver were 0.10 ( 0.11 to 0.22 ( 0.26, which was lower thanthat for MeO-PBDEs (6-MeO-BDE47: 0.57 ( 0.60, 2′-MeO-BDE68: 0.65(0.85) and 6-OH-BDE47 (0.59(0.51). Concentrationsof PBDEs were negatively correlated with age, but nosignificant relationships between concentrations of OH-PBDEsor MeO-PBDEs and age were observed.

IntroductionPolybrominated diphenyl ethers (PBDEs) are a class of flameretardants used in a wide range of products such as textiles,

construction materials, and electronic equipment (1). En-vironmental occurrences (2–4), fates (5), and toxicities ofPBDEs (6–9) have been reported. Methoxylated polybromi-nated diphenyl ethers (MeO-PBDEs) and hydroxylated po-lybrominated diphenyl ethers (OH-PBDEs) are also toxic (10).The presence of OH-PBDEs in wildlife and humans is ofconcern due to their higher toxic potencies relative to PBDEsand MeO-PBDEs (11). Some OH-PBDEs bind to humantransthyretin with affinities 3-fold higher than the naturalligand thyroxine (T4) (12). Some OH-PBDEs such as 4′-OH-BDE17 can displace 17-� estradiol (E2) from the estrogenreceptor (ER)-R with a relative estrogenic potency ap-proximately 30% higher than that of E2 (13).

OH-PBDEs have been reported to originate from bothanthropogenic and natural sources. They have been reportedto be transformed from PBDEs by female Sprague-Dawleyrats (14) or from MeO-PBDEs by rainbow trout, chicken, andrat microsomes (15). OH-PBDEs and MeO-PBDEs have alsobeen reported to be synthesized by marine organisms suchas algae and sponges or their associated microorganisms(16, 17). There are reports that OH-PBDE can be formed invitro and in vivo from PBDE, but the literature is ambivalentwith some authors reporting transformation while othersdid not observe transformation. The proportions transformedare small even when exposures were large (5, 15, 18, 19).Purity of the exposure mixtures is always an issue in thesesorts of studies and the reported formation of OH-BDEs andMeO-BDE from PBDE may be an artifact. When the precursormaterials were confirmed to not contain OH-BDE or MeO-BDE no transformation was observed (15, 18). OH-PBDEsand MeO-PBDEs have been reported in many aquaticorganisms including marine and freshwater fish, birds, andhigher trophic level organisms such as marine mammalsincluding beluga whales, ringed seals, and polar bears (20–22).However, information on concentrations of OH-PBDEs andMeO-PBDEs is mainly restricted to blood, adipose, and liverof wildlife. There have been few studies of the toxicokineticsof OH-PBDEs, MeO-PBDEs, and PBDEs (23, 24), which couldhelp clarify the tissue concentration, maternal transfer, andage-related accumulation of these polybrominated com-pounds in organisms and help identify possible target organsto guide monitoring and studies of toxicity.

For oviparous organisms, such as some fish, transfer ofhydrophobic chemicals from females to eggs along with yolkproteins is a pathway of exposure of eggs (25). Organismsusually exhibit higher sensitivity to pollutant exposure duringearly life stage than adult life stages (26, 27), and variouseffects of polybrominated compounds on embryos have beenreported. Exposure of zebrafish embryos to BDE47 resultedin development of morphological, cardiac, and neural deficits(7). Bromkal 70-DE, a commercial mixture containingpredominantly BDE47, 99, and 100, delayed and alteredactivity level, fright response, predation rates, and learningability in subsequent life stages (8). Exposure of zebrafishembryos to 6-OH-BDE47 caused a wide range of develop-mental defects, including pericardial edema, yolk sac de-formations, reduced pigmentation, and lowered heart rateas well as delayed development, with an EC50 value of 25 nM(28).

The Chinese sturgeon (Acipenser sinensis) is a predatoryfish that can live for 40 years or longer and weigh as muchas 500 kg (29). Previous studies have shown that Chinesesturgeon can be exposed to pollutants and accumulateespecially high concentrations into their eggs (27, 30). In thisstudy, concentrations of 9 OH-PBDEs, 12 MeO-PBDEs, and11 PBDEs were measured in thirteen organs, including 8

* Corresponding author tel and fax: 86-10-62765520; e-mail:hujy@urban.pku.edu.cn.

† Peking University.‡ University of Saskatchewan.§ Michigan State University.| City University of Hong Kong.

Environ. Sci. Technol. 2010, 44, 5781–5786

10.1021/es100348g 2010 American Chemical Society VOL. 44, NO. 15, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 5781

Published on Web 07/07/2010

liver, 8 muscle, 6 heart, 7 gonad, 5 stomach, 7 intestine, 5adipose, 6 gill, 2 pancreas, 1 kidney, 1 gallbladder, 1 spleen,and 15 egg samples from 17 Chinese sturgeon. Tissue-dependent accumulations of the three classes of compoundswere examined and hepatic sequestration and maternaltransfer rates were calculated. Finally, age-related ac-cumulation was compared among PBDEs, OH-PBDEs, andMeO-PBDEs.

Materials and MethodsSample Collection. Because the Chinese sturgeon has beenlisted as a grade I protected animal in China since the 1980s,the capture of Chinese sturgeon was authorized strictly forscientific purposes only. Artificial propagation has beenimplemented to save this endangered species. This programcaptures adult Chinese sturgeon from the Yangtze River.Chinese sturgeon migrate from the East Sea and spendapproximately one year in the river before spawning. Afterpropagation, sturgeon were released back into the YangtzeRiver, but some did not survive. Eggs were collected beforepropagation and the other organs and tissues came from the17 sturgeon that died during artificial propagation in theperiod between 2003 and 2006. To avoid contamination ofstomach and intestine with gut contents, only the inner layerof the stomach and intestine were collected. This was possiblebecause of their large body size and because the musclelayer of the stomach and intestine are very thick. Aftercollection, tissues were frozen immediately at -20 °C untilanalysis. The ages of fish were determined by counting growthlayers in the cleithrum, as described previously (29). Thetotal number of sturgeon samples was 72, and the detailedinformation about the samples is given in SupportingInformation Table S1.

In Vitro Microsomal Incubations. Microsomes wereisolated from cultured two-year-old Chinese sturgeon, ac-cording to the method improved by Benedict et al. (31) andincluded dithiothreitol (DTT) in the homogenization, washand resuspension buffers to preserve catalytic activity ofreductases and deiodinases. Ethoxyresorunfin O-deethylase(EROD) activity and protein content of microsomes weredetermined simultaneously by use of a fluorescence kit(Genmed Scientific Inc., USA). The final reaction volumewas 100 µL and contained either 50 µL of the microsomalpreparation and 3 µL of exposure chemicals. Individualcongeners (BDE47, BDE99, BDE154, BDE183, and 6-MeO-BDE47) were used, and the concentration in the incubationmixture was 150 ng/mL. The protein concentration in thereaction vial was 5.2 mg/mL and the CYP1A1-catalyzed ERODactivity was 3.5 pmol/mg/min. Reactions were performed at37 °C for 20 h with constant agitation. Incubations withoutchemicals and without microsomes were used as negativecontrols to assess background contaminants and the pos-sibility of nonenzyme-mediated changes in chemical struc-ture. After the incubation, the samples were extractedimmediately for chemical analysis.

Quantification of Target Analytes and TransformationProducts in Tissues and Microsomes. The method used toquantify PBDEs, OH-PBDEs, and MeO-PBDEs has beendescribed previously (15) and details specific to this studyare given in Supporting Information.

Quality Assurance and Quality Control (QA/QC). Detailsof the QA/QC of the method have been described previously(15). Concentrations of all congeners were quantified by theinternal standard isotope-dilution method with mean relativeresponse factors determined from standard calibration runs.PBDEs and MeO-PBDEs were quantified in sample extractsrelative to 13C-PBDEs, and OH-PBDEs were quantified relativeto 6′-OH-BDE17. Recoveries of 13C-PBDEs and 6′-OH-BDE17were 74.9 ( 38.8% to 130 ( 60.0% and 98.5 ( 33.7%,respectively. All equipment rinses were carried out with

acetone and hexane to avoid sample contamination. Alaboratory blank was incorporated in the analytical proce-dures for every batch of 12 samples. The method detectionlimits (MDLs) were set to be the mean of the concentrationplus three times the standard deviation in the blank samplesin which BDE28, BDE47, BDE85, BDE154, BDE153, andBDE138 were detected. The MDLs for the other compounds,which were not detected in blank samples, were set to theinstrumental minimum detectable amounts. Based on anaverage sample with a wet mass of 10 g, the detection limitswere 0.4 pg/g ww for MeO-PBDEs; 2.0 pg/g ww for 2′-OH-6′-Cl-BDE7; 4.0 pg/g ww for 3-OH-BDE47, 5-OH-BDE47,2′OH-BDE68, 6-OH-BDE47, 4′-OH-BDE49, 2′-OH-6′-Cl-BDE68, 6-OH-BDE90, and 2-OH-BDE123; 1.6 pg/g ww forBDE28; 26.8 pg/g ww BDE47; 48.8 pg/g ww for BDE85; 22.4pg/g ww for BDE154; 3.2 pg/g ww for BDE153; 11.2 pg/g wwfor BDE138; and 0.4 pg/g ww for BDE66, BDE77, BDE100,BDE99, and BDE183. Purity of stock chemicals was checkedby measuring, and concentrations of potential transformationproducts in stocks were determined by quantification ofconcentrated standards.

Statistical Analysis. In this study, for those results lowerthan the MDL, half of the MDL was assigned to avoid missingvalues in statistical analyses. The average liver/adipose andegg/liver ratios calculated on a lipid weight basis werecalculated for individual PBDE and MeO-PBDE congenerand the egg/liver ratios base on wet weight was calculatedfor the 6-OH-BDE47 in Chinese sturgeon. All data analysessuch as linear regression were performed with SPSS 15.0.Statistical significance was defined as p < 0.05.

Results and DiscussionConcentrations among Tissues. Higher concentrations ofΣPBDEs were observed in adipose (42.8 ( 44.0 ng/g ww),liver (25.0 ( 27.0 ng/g ww), and eggs (21.2 ( 19.4 ng/g ww)(Figure 1). Detailed information on concentrations of PBDEs,OH-PBDEs, and MeO-PBDEs in tissues of 17 Chinese sturgeonare shown in Supporting Information Tables S2 and S3.Concentrations of ΣPBDEs in eggs of Chinese sturgeon (21.3( 19.4 ng/g ww) were higher than those of sturgeons fromAzerbaijan (Acipenser Huso huso), Bulgaria (Acipenser guelden-staedti), and Russia (Acipenser stellatus) (0.010-0.27 ng/gww) (32). Concentrations of PBDEs in skipjack tuna from theEast China Sea were relative high compared to other areasof the world (33). This is consistent with the concentrations

FIGURE 1. Concentrations of ΣOH-PBDEs, ΣMeO-PBDEs, andΣPBDEs in tissues of Chinese sturgeon. Due to the limitednumber of samples, kidney, spleen, and gallbladder are notpresented. The horizontal line represents the medianconcentration. The 25th and 75th percentiles define the boxesand the whiskers represent the 10th and 90th percentiles. Ofthe five stomach samples, concentrations of PBDEs in onesample exceeded three times the standard deviation so thatdata point was not included in calculating the median.

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of PBDEs observed in the Chinese sturgeon which is ananadromous fish that spends its first 14 years of life in theEast China Sea (29).

The highest concentrations of MeO-PBDEs were foundin adipose (135 ( 63.6 pg/g ww), eggs (120 ( 119 pg/g ww),and liver (32.3 ( 29.1 pg/g ww). Concentrations of 6-MeO-BDE47 in liver of Chinese sturgeon were lower than those inbeluga whales from Eastern Hudson Bay and the HudsonStrait (34) and in tuna (Katsuwonus pelamis) from the NorthPacific Ocean (15).

Both PBDEs and MeO-PBDEs were preferentially ac-cumulated in organs with higher lipid contents such asadipose (65.9 ( 13.8%), egg (35.1 ( 8.5%), and liver (12.7 (6.6%). When concentrations of PBDEs and MeO-PBDEs wereexpressed on a lipid weight basis, differences in concentra-tions among tissues were less (Supporting Information FigureS1). But, concentrations of PBDEs were still higher in liver(157( 114 ng/g lw) than in other tissues, which suggests thatthe lipid of tissues is not the sole factor influencing the tissueconcentrations of PBDEs in Chinese sturgeon.

The highest concentrations of OH-PBDEs occurred in liver(185 ( 174 pg/g ww) and eggs (178 ( 294 pg/g ww).Concentrations of 6-OH-BDE47, a major congener of OH-PBDEs, in liver (166 ( 182 pg/g ww) of Chinese sturgeonwere higher than those (17.8-44.0 pg/g ww) in liver of tuna(Katsuwonus pelamis) collected from the North Pacific Ocean(15). The tendency of OH-PBDEs to associate with liver maybe due to their biotransformation formation in this tissueand/or competitive binding to the major thyroid hormonetransport protein (TTR) (12), which is mainly produced andexpressed in liver (35). Among target tissues, gonad and heartcould be classified as richly perfused tissues, and adiposeand muscle as poorly perfused tissues (36). Concentrationsof OH-PBDEs in two richly perfused tissues (gonad: 42.8 (39.4 pg/g ww, heart: 46.1 ( 26.5 pg/g ww) were higher thanthose in poorly perfused tissues (adipose: 35.0 ( 40.1 pg/gww, muscle: 11.1( 14.3 pg/g ww). This observation indicatesthat blood flow could be an important factor to influence thetissue concentrations of OH-PBDEs. OH-PBDEs have beenshown to bind competitively to TTR leading to their ac-cumulation in blood (12, 37).

Relatively high concentrations of PBDEs (28.5 ng/g ww,n ) 1) and OH-PBDEs (138 pg/g ww) were observed in gallbladder. This indicates possible preferential disposition ofPBDEs and OH-PBDEs in bile, which could facilitate excretionof these compounds from Chinese sturgeon.

Patterns of Relative Concentrations. All of identifiabletri- to hepta-BDE congeners (BDE28, 47, 66, 77, 99, 100, 85,153, 154, and 183) were detected, and the patterns of relativeconcentrations were similar among tissues except for stom-ach (Figure 2). BDE47 (52.5 ( 13.8%) was the predominantcongener, followed by BDE154 (16.7 ( 8.4%), BDE100 (12.2(2.8%), BDE28 (6.1(2.1%), BDE153 (4.1(2.3%), and BDE99(2.4 ( 2.2%). In the stomach, the profile of PBDEs wasdominated by BDE183, accounting for 27.7 ( 27.5%, whichis largely different from those in the extra-hepatic tissues.While such a different pattern observed in stomach could beattributed to the molecule size of BDE183, which makes itdifficult to pass through the stomach wall or intestines intothe bloodstream and further to other tissues (38), an exposureexperiment with Juvenile lake trout (Salvelinus namaycush)showed that the assimilation efficiency of BDE183 (22.8%)was similar to those of BDE153 (33.3%), BDE 154 (47.0%),BDE47(21.8%), BDE99(31.3%), and BDE100 (41.9%) (39).Thus, the most likely explanation for the concentration ofBDE 183 observed in other organs would be biotransfor-mation. To better understand the potential biotransformationof BDE183 in Chinese sturgeon, in vitro studies of BDE183,BDE154, and BDE99 were conducted in microsomal fractionsof Chinese sturgeon liver (Supporting Information Table S4).

After a 20-h exposure, 94 ( 1% of BDE183 and 78 ( 1% ofBDE99 were biotransformed while concentrations of BDE154were comparable to the starting concentrations indicatingthat BDE183 is readily metabolized in Chinese sturgeon. Asignificant amount of BDE154 was formed by conversion ofBDE183 (73 ( 1.2%), while BDE47 was formed from BDE99(52 ( 2%). This result demonstrated that part of the BDE99and BDE183 were transformed to unquantified products orbecoming unextractable. This result is similar to thoseobserved when common carp (Cyprinus carpio) were exposedto BDE99 and BDE183 (40). Thus, it is likely that biotrans-formation was responsible for the relatively large BDE183/154 and BDE99/47 ratios in stomach and intestine relativeto other tissues (Supporting Information and Figure 2).Furthermore, application of these two ratios can be used toestimate the extent of biotransformation in other tissues.

Of the 11 MeO-PBDEs congeners monitored, 4-MeO-BDE17, 2′-MeO-BDE68, 6-MeO-BDE47, 5-MeO-BDE47, and5′-MeO-BDE100 were detected in eggs and liver, but theirconcentrations were lower than the detection limit instomach, pancreas, and spleen. 6-MeO-BDE47 was thepredominant congener, followed by 2′-MeO-BDE68 (Sup-porting Information Table S3). A similar pattern of relativeconcentrations was observed in organisms from the CanadianArctic marine food web and pike (Esox lucius) from theSwedish coast (34, 41). Generally, MeO-PBDEs are detectedin marine organisms at concentrations sometimes higherthan those of PBDEs, and concentrations of OH-PBDEs arelower than those of MeO-PBDEs (42–44). But in the tissuesamples of Chinese sturgeons, the concentration of MeO-PBDEs was 100-1000 times lower than those of PBDEs andcomparable to those of OH-PBDEs. A recent study foundthat concentrations (31 ng/g lw) of MeO-PBDEs in theanadromous anchovy (Coilia nasus) from the Yangtze Riverestuary were higher than those (9.1 and 14 ng/g lw) fartherupstream in the Yangtze River near the city of Nanjing (45).The Chinese sturgeon is an anadromous fish in the YangtzeRiver Basin. Chinese sturgeon were collected from theirspawning habitat in the middle-stem of the Yangtze River.The fact that Chinese sturgeon may have been in the riverfor as much as a year may have resulted in the lowerconcentrations of OH-PBDE and MeO-PBDEs.

Of the 9 OH-PBDEs monitored, 2′-OH-BDE68, 6-OH-BDE47, 5-OH-BDE47, and 4-OH-BDE49 were detected (Sup-porting Information Table S3). 6-OH-BDE47 was the pre-dominant compound in all tissues. This result is similar tothe pattern observed in blood plasma of bottlenose dolphinfrom Indian River Lagoon, Florida and fish collected fromthe Detroit River and Hudson Bay region of northeastern

FIGURE 2. Relative contribution (%) of seven major PBDEcongeners (% congener contribution on a mass per mass basis)in tissues of Chinese sturgeon.

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Canada (21, 34, 43). Relatively high 6-OH-BDE47 concentra-tions and comparable 2′-OH-BDE68 concentrations weredetected in eggs. 5-OH-BDE47 and 4-OH-BDE49 with higherfrequencies of detection were observed in liver (SupportingInformation Table S3). It was reported that OH-PBDEs witha hydroxyl moiety at the meta or para position have beenhypothesized to be biotransformation products of PBDEsbased on the results of in vivo exposures while OH-PBDEsof natural origin all have a hydroxyl group at the ortho position(14, 18). Since the hydroxyl groups in 5-OH-BDE47 and 4-OH-BDE49 are at the meta and para positions, respectively, theylikely would originate from biotransformation of PBDEs inliver rather than from accumulation of natural products (15).2′-OH-BDE68 and 6-OH-BDE47 which have a hydroxyl groupat the ortho position would be derived from natural products.There was a significant correlation between concentrationsof 6-OH-BDE47 and 6-MeO-BDE47 (R2 ) 0.534, p ) 0.003)(Supporting Information Figure S3) in the eggs of Chinesesturgeon. Such correlation was also observed in albatross(Thalassarche chlororhynchos, Phoebetria palpebrata, Thalas-sarche chrysostoma, Thalassarche cauta, and Thalassarchemelanophrys), and polar bear (Ursus maritimus) (15). Thisresult suggested that 6-OH-BDE47 would be from metabolicdemethoxylation of MeO-PBDEs as demonstrated by a recentstudy based on in vitro metabolism using rainbow trout(Oncorhynchus mykiss), chicken (Gallus gallus), and rat(Rattus norvegicus) microsomes (15). On the other hand,6-OH-BDE47 has also been observed in rats exposed to BDE47as metabolite (18). To further understand the origin of 6-OH-BDE47 in Chinese sturgeon, in vitro metabolism of 6-MeO-BDE47 and various PBDE individual congeners by microso-mal fractions of Chinese sturgeon was conducted (Table S4in Supporting Information). Concentrations of 6-OH-BDE47were lower than the MDL when dosing with PBDEs and6-MeO-BDE47 at concentration of 150 ng/mL. Consideringthat 6-OH-BDE47 and 6-MeO-BDE47 have been reported inthe marine environment (from formation in algae/sponges)and Chinese sturgeons spend most of their life in the sea, alarge proportion of 6-OH-BDE47 in Chinese sturgeons would

result from direct bioaccumulation of OH-PBDEs fromnatural sources.

Liver/Adipose Ratios. Ratios of lipid-normalized con-centrations in liver and adipose (liver/adipose) have beenused as in some physiologically based pharmacokinetic (PB-PK) models (46). The liver/adipose ratios based on lipidnormalized concentrations were 1.1 ( 1.0 for MeO-PBDEsand 2.6 ( 1.7 for PBDEs, indicating preferential depositionof PBDEs in liver. This result is similar to the hepaticsequestration of PCDD/Fs that has been observed in birds(47). Hepatic sequestration of PCDD/Fs is due to binding toproteins such as cytochrome P450 (CYP) enzymes (48, 49)via the aromatic hydrocarbon receptor (AhR) mediatedpathway. A statistically significant correlation (SupportingInformation Figure S4) was also observed between liver/adipose ratios of individual PBDEs and their reported AhRbinding affinities (r 2 ) 0.934, p < 0.001), suggesting that AhRmight be involved in the hepatic sequestrations. However,PBDEs are relatively weak AhR agonists relative to PCDD/Fsor nonortho, 2,3,7,8-substituted, PCB (50, 51), the actualmechanism(s) responsible for hepatic sequestrations ofPBDEs should be further studied.

Maternal Transfer of PBDEs to Eggs. Hydrophobicchemicals can be transferred from the female to eggs alongwith yolk proteins formed in the liver of the female parent(25). Ratios of lipid-normalized concentrations of PBDEs andMeO-PBDEs egg to those in liver (E/L) were used to assessmaternal transfer in Chinese sturgeon. Since lipid is not amajor factor affecting concentrations of OH-PBDEs indifferent tissues, wet weight concentrations were used tocalculate egg to liver ratios. The E/L ratios of PBDEs rangedfrom 0.10 ( 0.11 for BDE153 to 0.22 ( 0.26 for BDE28. Ratiosof 6-OH-BDE47 (0.59 ( 0.51), 6-MeO-BDE47 (0.57 ( 0.60),and 2′-MeO-BDE68 (0.65 ( 0.85) were higher. Previousinvestigations of maternal transfer of organochlorines inChinese sturgeon (30) provide an opportunity to comparethe E/L ratios of the polybrominated compounds with otherchemicals. The E/L ratios of PBDEs were low than those ofp,p-DDD (0.27( 0.15), p,p-DDE (0.30( 0.19), and HCB (0.98

FIGURE 3. Correlations among concentrations (pg/g ww) of PBDEs, OH-PBDEs, and MeO-PBDEs in eggs as a function of age.

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( 0.46), but those of 6-OH-BDE47, 6-MeO-BDE47, and 2′-MeO-BDE68 were higher than the E/L ratios of DDTs. It wasfound that the more PBDEs were hepatic sequestrated in theliver, the lower the ratio of E/L, indicating that hepaticsequestration of PBDEs would affect their maternal transferin Chinese sturgeon.

Maternal transfer can influence accumulation of pollut-ants in females, and generally higher transfer ratios will resultin lower accumulation by females than males (52). Whilethere is no correlation between age and concentrations ofOH-PBDEs or MeO-PBDEs, the concentration of ΣPBDEssignificantly decreased with age (Figure 3a). As shown inFigure 3b, c, and d the lipid normalized concentrations ofsix major PBDEs congeners (BDE28, BDE47, BDE99, BDE100,BDE153, and BDE154) also show a negative trend with ageof female Chinese sturgeon (p < 0.05 except for BDE99 andBDE154, Supporting Information Table S5). This result isdifferent from those for DDE and HCB which were positivelycorrelated with age of Chinese sturgeon (30). A similarnegative correlation between concentrations of PBDEs withage was observed in tissues of both male and female Harbourseals (Phocoena phocoena) (53). Since maternal transfer ratios(E/L) of HCB and DDE were higher than those of PBDEs,maternal transfer would not be the primary factor resultingin accumulation of PBDEs in Chinese sturgeon. The highermetabolism of PBDEs such as BDE99 and 183 is a more likelyfactor. In addition, rapid excretion could be another potentialfactor since higher concentrations were observed in gallbladder as discussed above.

AcknowledgmentsFinancial support for this study was obtained from theNational Basic Research Program of China (2007CB407304)and the National Natural Science Foundation of China(40632009). We thank Qiwei Wei, Yangtze River FisheriesResearch Institute, Chinese Academy of Fisheries Science,Ministry of Agriculture of China, for providing samples ofeggs and tissues from Chinese sturgeon. Portions of thisresearch were supported by a Discovery Grant from theNational Science and Engineering Research Council ofCanada (Project 326415-07) and a grant from WesternEconomic Diversification Canada (Projects 6578 and 6807).J.P.G. was supported by the Canada Research Chair programand an at large Chair Professorship at the Department ofBiology and Chemistry and State Key Laboratory in MarinePollution, City University of Hong Kong.

Supporting Information AvailableText, figures, and tables addressing (1) chemicals andstandards used in the analysis; (2) extraction and cleanup oftissues; (3) instrumental conditions; (4) details of Chinesesturgeon samples; (5) mean concentrations and ranges ofOH-PBDEs, MeO-PBDEs, and PBDEs in tissues of Chinesesturgeon; (6) percentages of brominated compounds relativeto the dosing concentration after metabolism with Chinesesturgeon microsomes exposed to PBDEs and 6-MeO-BDE47;(7) association of concentrations of PBDE congeners withage of Chinese sturgeon; (8) levels of MeO-PBDEs and ΣPBDEsin various tissues in Chinese sturgeon after lipid normaliza-tion; (9) ratios of concentrations of BDE99 to BDE47 (BDE99/47) and BDE183 to BDE154 (BDE183/154) in Chinese sturgeontissues; (10) relationships between the concentrations of6-OH-BDE47 and the concentration of 6-MeO-BDE47; (11)ratios of lipid-normalized ratio of concentration in liver tothat in adipose of Chinese sturgeon plotted versus Ah receptorbinding affinities. This material is available free of charge viathe Internet at http://pubs.acs.org.

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ES100348G

5786 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 15, 2010

SUPPORTING INFORMATION 1

For: 2

Tissue concentrations of Polybrominated compounds in Chinese Sturgeon (Acipenser 3

sinensis): Origin, Hepatic Sequestration, and Maternal Transfer 4

Kun Zhang1, Yi Wan

2, John P. Giesy

2,3,4, Michael H. W. Lam

4, Steve Wiseman

2, 5

Paul D. Jones2, and Jianying Hu

1* 6

1Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, 7

Peking University, Beijing 100871, China 8 2Department of Veterinary Biomedical Sciences and Toxicology Centre, University of 9

Saskatchewan, Saskatoon, Saskatchewan, S7J 5B3, Canada 10 3Zoology Department, Center for Integrative Toxicology, Michigan State University, East 11

Lansing, MI 48824, USA 12 4Department of Biology and Chemistry, and State Key Laboratory in Marine Pollution, City 13

University of Hong Kong, Kowloon, Hong Kong SAR, China 14

15

16

Tables 5 17

Figures 4 18

Words 819 19

20

This supporting information provides detailed descriptions of chemicals and standards used in 21

the analysis, extraction and cleanup of tissue samples and instrument condition. Figures, and 22

tables addressing: details of Chinese sturgeon samples (Table S1); Mean concentrations and 23

ranges of OH-PBDEs and MeO-PBDEs in tissues of Chinese sturgeon (Table S2); Mean 24

concentrations and ranges of PBDEs in tissues of Chinese sturgeon (Table S3); Percentages of 25

brominated compounds relative to the dosing concentration after metabolism with Chinese 26

sturgeon microsomes exposed to PBDEs and 6-MeO-BDE47 (Table S4); Association of 27

concentrations of PBDE congeners with age of Chinese sturgeon (Table S5); Tissue 28

distribution of MeO-PBDEs and ∑PBDEs in Chinese sturgeon after lipid normalization 29

(Figure S1); Ratios of concentrations of BDE99 to BDE47 (BDE99/47) and BDE183 to 30

BDE154 (BDE183/154) in Chinese sturgeon tissues (Figure S2); Relationships between the 31

concentrations of 6-OH-BDE47 and the concentration of 6-MeO-BDE47 (Figure S3); Lipid 32

normalized liver to adipose concentration ratios in Chinese sturgeon plotted versus Ah 33

receptor binding affinities (Figure S4). 34

35

Chemicals and Standards. Eleven PBDEs (BDE28, BDE47, BDE66, BDE77, BDE100, 36

BDE99, BDE85, BDE154, BDE153, BDE138 and BDE183), twelve MeO-PBDEs 37

(6-MeO-BDE17, 4-MeO-BDE17, 2’-MeO-BDE68, 6-MeO-BDE47, 5-MeO-BDE47, 38

4’-MeO-BDE49, 5’-MeO-BDE100, 4’-MeO-BDE103, 4’-MeO-BDE99, 4’-MeO-BDE101, 39

6-MeO-BDE90, and 6-MeO-BDE85), and nine OH-PBDEs (2’-OH-6’-Cl-BDE7, 40

6-OH-BDE47, 3-OH-BDE47, 5-OH-BDE47, 2’-OH-BDE68, 4’-OH-BDE49, 41

2’-OH-6’-Cl-BDE68, 6-OH-BDE90 and 2-OH-BDE123) were selected as target compounds. 42

PBDEs, 13

C-PBDEs, and eight MeO-PBDEs standards were obtained from Wellington 43

Laboratories Inc. (Guelph, Ontario, Canada). 3-OH-BDE47, 5-OH-BDE47 and 44

2’-OH-BDE68 were obtained from AccuStandard (New Haven, Connecticut, USA). 45

6-MeO-BDE17, 4-MeO-BDE17, 6-MeO-BDE90, 6-MeO-BDE85, 2’-OH-6’-Cl-BDE7, 46

6-OH-BDE47, 4’-OH-BDE49, 2’-OH-6’-Cl-BDE68, 6-OH-BDE90 and 2-OH-BDE123 were 47

synthesized in the Department of Biology and Chemistry, City University of Hong Kong, and 48

purities of all metabolites were >98%. Dichloromethane (DCM), n-hexane, methyl 49

tert-butyl ether (MTBE), acetonitrile and methanol were pesticide residue grade obtained 50

from OmniSolv (EM Science, Lawrence, KS, USA). Sodium sulfate, silica gel (60-100 51

mesh size), aluminum oxide (neutral, 150 mesh size), pyridine (anhydrous, 99.8%), methyl 52

chloroformate (MCF), potassium hydroxide (KOH) and hydrochloric acid (HCl) were 53

purchased from Sigma-Aldrich (St. Louis, MO, USA). For biochemical analyses, the 54

fluorescence kit was obtained from (Genmed Scientific Inc, USA), sodium phosphate dibasic 55

(Na2HPO4), sodium phosphate monobasic (NaH2PO4) and potassium phosphate monobasic 56

(KH2PO4), resorufin, ethylenediaminetetraacetic acid (EDTA), and dithiothreitol (DTT) were 57

obtained from Sigma-Aldrich (St. Louis, MO, USA). All other biochemical reagents, 58

including NADPH, were obtained from Sigma-Aldrich and were reagent grade or better 59

unless stated otherwise. 60

Extraction and Cleanup of Tissue Samples Details of our methods for identifying and 61

quantifying PBDE, OH-PBDEs and MeO-PBDEs have been described previously (26). 62

Tissues were freeze-dried, then approximately 1-3 g dry weight (dw) subsamples were spiked 63

with a mixture of 13

C-labeled PBDE (13

C-BDE28, 13

C-BDE47, 13

C-BDE99, 13

C-BDE100, 64

13C-BDE153,

13C-BDE154 and

13C-BDE183) and 6-OH-BDE17 surrogates, and extracted by 65

accelerated solvent extraction (Dionex ASE-200, Sunnyvale, CA). The extraction employed 66

two 10 min cycles, the first cycle was performed with n-hexane/dichloromethane (DCM) (1:1) 67

at 100 °C and 1500 psi, followed by a second cycle with n-hexane/methyl tert-butyl ether 68

(MTBE) (1:1) at of 60°C and pressure of 1000 psi. The two extraction fractions were 69

combined and rotary evaporated to near dryness. The extract was then transferred to 15 ml 70

glass tubes by 8 mL hexane, and 4 mL 0.5 M KOH in 50% ethanol was added. The aqueous 71

layer (KOH) was extracted with 8 mL of n-hexane three times (neutral fraction). After 72

extraction, 1.5 mL of 2 M HCl was added to 15 ml tubes and the phenolic compounds were 73

extracted with n-hexane/MTBE (9:1; v/v) three times (phenolic fraction). 74

The neutral fraction was concentrated to approximately 2 mL and loaded onto a column 75

of 1 g Na2SO4 and 8 g acidified silica (48% H2SO4) and eluted with 15 mL of n-hexane and 76

10 mL of DCM. The eluate was further purified on a neutral alumina column (4 g of sodium 77

sulfate, 4 g of neutral alumina, 4 g of sodium sulfate). The first fraction eluted from the 78

alumina column with 20 mL of hexane was discarded. The second fraction, which contained 79

PBDEs and MeO-PBDEs, was obtained by elution with 25 mL of 60% DCM in n-hexane. 80

The eluate was evaporated to dryness under a gentle stream of nitrogen, then 30 µl nonane 81

and 10 µl internal standards (13

C-BDE138) were added for analysis of PBDEs and 82

MeO-PBDEs. 83

After dried by a gentle stream of nitrogen, the phenolic fraction was re-dissolved in 480 84

µL of derivatization solvent (acetonitrile/methanol/water/pyridine (5:2:2:1; v/v/v/v)) and then 85

40 µL of methyl chloroformate (MCF) was added. The reaction mixture was shaken on a 86

vortex at room temperature for 1 h and then diluted with 1.2 mL of pure water. The aqueous 87

solution was extracted three times with 6 mL volumes of n-hexane. Extracts were 88

concentrated and subjected to acidified silica gel chromatography as described above, eluted 89

with 30 mL of n-hexane and 30 mL of DCM. The eluate was concentrated to 40 µL for 90

OH-PBDE analysis. 91

Instrumental Conditions. Identification and quantification of PBDEs congeners were 92

performed using a Hewlett-Packard 5890 series II high-resolution gas chromatograph 93

interfaced to a Micromass® Autospec® high-resolution mass spectrometer (HRGC-HRMS) 94

(Micromass®, Beverly, MD). Chromatographic separation was achieved on a DB-5MS 95

capillary column (30 m length, 0.25 mm ID, 0.1 µm film thickness, Agilent, Carlsbad, CA). 96

A splitless injector was used and the injector was held at 285°C. The interface temperature 97

was 320°C, and ion temperature was 285°C. The carrier gas was helium. The electron 98

ionization energy was 37 eV and the ion current was 750 µA. Data acquisition was 99

conducted in selected ion monitoring mode. For PBDEs, the temperature program was from 100

110°C (10min) to 250°C at the rate of 25°C/min, then increased to 260°C at the rate of 101

1.5°C/min, and then to 323°C (15 min) at a rate of 25°C/min. For OH-PBDEs, the 102

temperature program was from 150°C (2min) to 320°C (2min) at the rate of 10°C/min. For 103

MeO-PBDEs, the temperature program was from 150°C (2 min) to 245°C (2 min) at 2°C/min, 104

and then increased to 320°C (2 min) at 30°C/min. 105

SUPPORTING INFORMATION TABLE S1. Details of Chinese Sturgeon Samples. 106

Sample

Code

date of

collection

age

(year)

wt

(kg)

body

length (cm) Tissue collected

a

A0403 2005 24 260 280 E

A0406 2004 18 174 245 E, L, M, H, Go, St, P

A0408 2004 22 230 258 E

A0410 2004 17 140 246 E, L, M, H, Go, St, I, A, Gb

A0412 2004 24 230 287 E, L, M, H, Go, St, I, Gi, P

A0414 2004 25 263 285 E, L, M, I, A, Gi

A0438 2006 26 334 290 E

A0439 2006 21 223 262 E, L, M, H, Go, St, I, A, Gi, S

A0440 2006 18 176 250 E

A0441 2006 25 240 300 E

A0444 2005 23 224 270 E

A0445 2005 18 187 237 L, M, H, Go, I, Gi

A0447 2005 19 192 247 E, L, M, H, Go, I, A, Gi

A0449 2005 22 252 275 E

A0452 2005 23 207 282 E

A0500 2005 22 227 261 E

A0466 2003 24 254 285 L, M, Go, St, I, A, Gi, K

a) E: egg; L: liver; M: muscle; H: heart; Go: gonad; St: stomach; I: intestine; A: adipose; Gi: 107

gill; K: kidney; Gb: gallbladder; P: pancreas; S: spleen; 108

SUPPORTING INFORMATION TABLE S2. Mean Concentrations and Ranges of OH-PBDEs and MeO-PBDEs (pg/g ww) in Tissues of 109

Chinese Sturgeon. 110

Eggs Liver Gonad Adipose Heart muscle Intestine Stomach Gill Pancreas Gall bladder Spleen Kidney Chemicals

n=15 n=8 n=7 n=5 n=6 n=8 n=7 n=5 n=6 n=2 n=1 n=1 n=1

lipid content (%) 35 13 3.5 66 3.8 1.9 2.6 1.2 2.2 6.8 23 3.3 32

(24-50) (7.1-29) (1.2-6.3) (46-89) (2.3-5.9) (0.5-3.6) (1.0-5.4) (0.8-1.7) (1.4-3.4) (5.1-8.6)

6-OH-BDE47 180 170 35 17 38 5.2 15 7.7 8.5 7.1 58 64 19

(17-1200) (35-580) (18-54) (N.D.-38) (9.3-81) (N.D.-18) (N.D.-41) (N.D.-20) (N.D.-26) (5.4-8.8)

2-OH-BDE68 N.D. 2.9 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

(N.D.-7.7)

5-OH-BDE47 3.2 16 N.D. 14 N.D. N.D. N.D. N.D. N.D. N.D. 22 N.D. N.D.

(N.D.-17) (N.D.-39) (N.D.-62)

4-OH-BDE49 8.8 15 4.2 N.D. 4.4 N.D. N.D. N.D. N.D. N.D. 56 N.D. N.D.

(N.D.-75) (N.D.-59) (N.D.-17) (N.D.-16)

4-MeO-BDE17 0.6 1.2 0.3 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

(N.D.-3.2) (N.D.-4.1) (N.D.-0.7)

2'-MeO-BDE68 14 4.5 0.8 16 0.6 0.4 0.4 N.D. N.D. N.D. N.D. N.D. 1.9

(1.5-45) (N.D.-12) (N.D.-2.4) (7.1-29) (N.D.-2.4) (N.D.-0.9) (N.D.-1.4)

6-MeO-BDE47 110 25 6.3 120 3.3 1.3 1.5 N.D. 2.2 N.D. 12 N.D. 12

(15-370) (N.D.-69) (1.3-24) (36-210) (N.D.-9.5) (N.D.-3.7) (N.D.-6.9) (N.D.-6.7)

5-MeO-BDE47 0.5 0.7 N.D. 1.1 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

(N.D.-4.9) (N.D.-2.0) (N.D.-4.8)

5'-MeO-BDE100 1.2 1.4 N.D. 1.8 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

(N.D.-3.6) (N.D.-3.1) (N.D.-4.8)

aND, not detected. 111

112

113

114

SUPPORTING INFORMATION TABLE S3. Mean Concentrations and Ranges of PBDEs (ng/g ww) in tissues of Chinese Sturgeon. 115

116

Eggs Liver Gonad Adipose Heart Muscle Intestine Stomach Gill Pancreas Gall bladder Spleen Kidney Chemicals

n=15 n=8 n=7 n=5 n=6 n=8 n=7 n=5 n=6 n=2 n=1 n=1 n=1

BDE28 1.6 1.2 0.2 2.4 0.1 0.05 0.03 0.01 0.05 0.04 1.2 0.02 0.1

(0.2-4.0) (0.1-3.2) (0.05-0.5) (0.8-4.8) (0.05-0.3) (0.02-0.1) (0.01-0.1) (N.D.-0.01) (0.01-0.1) (0.03-0.1)

BDE47 14 15 1.7 29 1.7 0.5 0.3 0.1 0.4 0.5 20 0.2 0.7

(1.6-48) (0.3-49) (0.5-4.2) (4.1-85) (0.5-4.1) (0.2-1.3) (0.03-0.8) (N.D.-0.1) (0.04-0.8) (0.4-0.7)

BDE66 0.3 0.2 0.04 0.4 0.03 0.01 0.01 0.001 0.01 0.01 0.1 0.01 0.03

(0.04-1.0) (0.01-0.8) (0.0-0.1) (0.1-0.9) (0.01-0.1) (N.D.-0.03) (N.D.-0.02) (N.D.-0.004) (N.D.-0.03) (N.D.-0.02)

BDE77 0.005 0.01 0.002 0.009 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 0.002

(N.D.-0.1) (N.D.-0.03) (N.D.-0.005) (N.D.-0.03)

BDE100 2.5 3.8 0.3 5.7 0.4 0.1 0.1 0.02 0.1 0.1 4.4 0.1 0.2

(0.3-10) (0.1-13) (0.1-0.8) (1.0-16) (0.1-0.8) (0.1-0.3) (0.01-0.2) (N.D.-0.03) (0.01-0.2) (0.1-0.2)

BDE99 0.3 0.2 0.03 0.5 0.03 0.03 0.02 0.02 0.02 0.02 0.2 0.02 0.02

(0.02-0.7) (0.03-0.7) (0.01-0.1) (0.2-1.0) (0.02-0.1) (0.01-0.1) (0.01-0.04) (N.D.-0.04) (0.01-0.03) (0.01-0.02)

BDE85 0.2 N.D. N.D. N.D. N.D. 0.05 N.D. 0.1 N.D. N.D. N.D. N.D. N.D.

(N.D.-0.7) (N.D.-0.2) (N.D.-0.2)

BDE154 2.2 3.6 0.4 3.5 0.4 0.1 0.1 0.1 0.1 0.2 1.7 0.04 0.2

(0.3-5.1) (0.1-11) (0.1-1.0) (1.7-5.6) (0.1-0.8) (0.1-0.2) (0.02-0.2) (0.02-0.2) (0.02-0.3) (0.1-0.4)

BDE153 0.5 1.0 0.1 1.1 0.1 0.04 0.03 0.1 0.03 0.1 1.0 0.02 0.04

(0.1-1.3) (0.02-2.6) (0.03-0.2) (0.2-2.4) (0.04-0.2) (0.01-0.1) (N.D.-0.1) (N.D.-0.1) (N.D.-0.1) (0.03-0.1)

BDE183 N.D. 0.03 N.D. N.D. N.D. 0.01 0.05 1.3 0.01 0.04 N.D. N.D. N.D.

(N.D.-0.2) (N.D.-0.1) (N.D.-0.2) (N.D.-6.1) (N.D.-0.02) (N.D.-0.1)

aND, not detected. 117

SUPPORTING INFORMATION TABLE S4. Percentages of brominated compounds 118

relative to the dosing concentration after metabolism with Chinese sturgeon microsomes 119

exposed to PBDEs and 6-MeO-BDE47 (%) 120

Exposed group (chemicals)

BDE47a BDE99

a BDE154

a BDE183

a 6- MeO-BDE47

a

6-OH-BDE47 N.D.b N.D. N.D. N.D. N.D.

6-MeO-BDE47 N.D. N.D. N.D. N.D. 106 ± 7

5-MeO-BDE47 N.D. N.D. N.D. N.D. N.D.

4′-MeO-BDE49 N.D. N.D. N.D. N.D. N.D.

5′-MeO-BDE100 N.D. N.D. N.D. N.D. N.D.

4′-MeO-BDE103 N.D. N.D. N.D. N.D. N.D.

4′-MeO-BDE99 N.D. N.D. N.D. N.D. N.D.

4′-MeO-BDE101 N.D. N.D. N.D. N.D. N.D.

BDE28 N.D. N.D. N.D. N.D. N.D.

BDE47 99 ± 5 52 ± 2 N.D. N.D. N.D.

BDE66 N.D. N.D. N.D. N.D. N.D.

BDE100 N.D. N.D. N.D. N.D. N.D.

BDE119 N.D. N.D. N.D. N.D. N.D.

BDE99 N.D. 28 ± 1 N.D. N.D. N.D.

BDE85 N.D. N.D. N.D. N.D. N.D.

BDE154 N.D. N.D. 109 ± 6 73 ± 5 N.D.

BDE153 N.D. N.D. N.D. N.D. N.D.

BDE183 N.D. N.D. N.D. 6 ± 1 N.D. a Data represent mean±standard deviation of technical triplicates. N.D.: concentrations less 121

than the detection limit; dosing concentration was 150 ng/mL for BDE47, BDE99, BDE154, 122

BDE183 and 6-MeO-BDE47 as described in materials and methods..123

SUPPORTING INFORMATION TABLE S5. Association of concentrations of PBDE 124

Congeners (pg/g ww) with age of Chinese sturgeon: Ln [PBDE concentration] = a× age+ b 125

(R2, p value). 126

a b R2 p

BDE28 -0.115 9.49 0.51 0.03

BDE47 -0.163 12.7 0.66 0.01

BDE100 -0.161 10.9 0.69 0.01

BDE99 -0.175 8.86 0.45 0.05

BDE154 -0.098 9.51 0.37 0.08

BDE153 -0.164 9.10 0.56 0.02

∑PBDEs -0.158 13.0 0.65 0.01

127

Con

cen

trat

ion

s of

MeO

-PB

DE

s (p

g/g

lw

)

Co

ncentra

tion

s of P

BD

Es (n

g/g

lw)

GillStomach Intestine Heart

Gonad Muscle

AdiposeLiver Egg

900

600

300

0

MeO-PBDE

300

200

100

0

PBDE

N=15 N=8 N=5 N=8 N=7 N=6 N=7 N=4 N=6

Richly perfused organs Poorly perfused organs 128

SUPPORTING INFORMATION FIGURE S1. Levels of MeO-PBDEs and ∑PBDEs in 129

various tissues in Chinese sturgeon after lipid normalization. The horizontal line represents 130

the median concentration. The 25th

and 75th

centiles define the boxes and the whiskers 131

represent the 10th

and 90th

centiles.132

133 SUPPORTING INFORMATION FIGURE S2. Ratios of concentrations of BDE99 to 134

BDE47 (BDE99/47) and BDE183 to BDE154 (BDE183/154) in Chinese sturgeon tissues. 135

136

R

atio

s

Gil

l

Sto

mac

h

Inte

stin

e

Mu

scle

H

eart

Go

nad

Liv

er

Eg

g

6

4

2

0

BDE183/154

BDE99/47

Ad

ipo

se

137

138 SUPPORTING INFORMATION FIGURE S3. Relationships between the concentrations of 139

6-OH-BDE47 and the concentration of 6-MeO-BDE47 (R2=0.534, p=0.003) and BDE47 140

(R2=0.25, p=0.069). 141

1

10

100

1000

10000

100000

1 10 100 1,000 10,000

Log(Conc.) of 6-OH-BDE47 (pg/g ww)

Lo

g(C

on

c.)

(pg

/g w

w)

6-MeO-BDE47

BDE47

SUPPORTING INFORMATION FIGURE S4. Lipid normalized liver to adipose 142

concentration ratios in Chinese sturgeon plotted versus Ah receptor binding affinities. 143

2

3

4

5

0 10 20 30 40 50AhR EC50 (μM)

Liv

er/a

dip

ose

rat

io

2

3

4

5

0 10 20 30 40 50AhR EC50 (μM)

Liv

er/a

dip

ose

rat

io