tetrabromobisphenol a (tbbpa) and hexabromocyclododecanes (hbcds) in …

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Tetrabromobisphenol A (TBBPA) andhexabromocyclododecanes (HBCDs) in tissues of humans,

dolphins, and sharks from the United States

Boris Johnson-Restrepo a,1, Douglas H. Adams b, Kurunthachalam Kannan a,*

a Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health,

State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USAb Cape Canaveral Scientific, Inc., 220 Surf Road, Melbourne Beach, FL 32951, USA

Received 15 May 2007; received in revised form 24 September 2007; accepted 2 October 2007Available online 26 November 2007

Abstract

Concentrations of tetrabromobisphenol A (TBBPA) and a-, b-, and c-isomers of hexabromocyclododecanes (HBCDs) were deter-mined by liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS) in human adipose tissue obtained in New York City,and in three marine top-level predators – bottlenose dolphin, bull shark, and Atlantic sharpnose shark – collected from coastal watersof Florida, USA. The overall mean concentrations (mean ± SD) of TBBPA and HBCDs were 0.048 ± 0.102 and 0.333 ± 0.571 ng/g lipidwt in human adipose tissue samples, 1.2 ± 3 and 7.38 ± 18 ng/g lipid wt in bottlenose dolphin blubber, 9.5 ± 12 and 77.7 ± 128 ng/g lipidwt in bull shark muscle, and 0.872 ± 0.5 and 54.5 ± 88 ng/g lipid wt in Atlantic sharpnose shark muscle. Overall mean concentrations ofHBCDs were 5–10-fold higher than mean TBBPA concentrations, in all of the samples analyzed. The highest concentrations of TBBPAand HBCDs were detected in the bull shark muscle at concentrations of 35.6 and 413 ng/g, lipid wt, respectively. Concentrations ofTBBPA and HBCDs, after log-transformation, were significantly correlated with each other in human adipose tissue and bottlenose dol-phin blubber, but not in bull shark muscle samples. In the human adipose tissue samples, the concentrations of HBCDs were 3–4 ordersof magnitude lower than the concentrations of polybrominated diphenyl ethers (PBDEs) previously reported for the same set of tissuesamples. Concentrations of HBCDs in human samples from the United States were 1–5-fold lower than the concentrations reported fromseveral European countries. HBCD concentrations in bottlenose dolphins from the United States were 1–2 orders of magnitude lowerthan the concentrations reported for other cetacean species from Europe. The present report is the first to determine levels of TBBPA andHBCDs in humans, bottlenose dolphins, and sharks from the United States.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Brominated flame retardants; PBDE; TBBPA; HBCD; Dolphin

1. Introduction

Brominated flame retardants (BFRs) are incorporatedas additives or by chemical reactions with polymers, forthe purpose of reducing the flammability of the final man-ufactured product. Polymers that contain BFRs, including

polyurethane foams, high-impact polystyrene, acrilonitri-le–butadiene–styrene, and polyvinyl chloride, are exten-sively used in products such as building materials,electronic goods, carpets, upholstery textile, and car panels.The most widely used BFRs (Fig. 1; BSEF, 2005) in theUSA and Canada are polybrominated diphenyl ethers(PBDEs), followed by tetrabromobisphenol A (TBBPA),and then by hexabromocyclododecanes (HBCDs). Themarket demand for BFRs in the USA and Canada was esti-mated to be 54000 metric tons per year (BSEF, 2005).PBDEs and HBCD occur at measurable levels in humanand wildlife tissues due to their persistent and lipophilic

0045-6535/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.chemosphere.2007.10.002

* Corresponding author. Tel.: +518 474 0015; fax: +518 473 2895.E-mail address: [email protected] (K. Kannan).

1 Present address: Environmental and Computational Chemistry Group,Department of Chemistry, University of Cartagena, Cartagena,Colombia.

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Available online at www.sciencedirect.com

Chemosphere 70 (2008) 1935–1944


properties (Loganathan et al., 1995; de Wit, 2002; Hites,2004; Hoh and Hites, 2005; Law et al., 2005; Covaciet al., 2006).

There are eight HBCD diastereoisomers found in thetechnical HBCD mixture, comprising three pairs ofracemic enantiomers of (±) a-(11.8%), b-(5.8%), andc-(81.6%) isomers and two meso forms of d-(0.5%) ande-(0.3%) isomers (Heeb et al., 2005). When the HBCD tech-nical mixture is heated at temperatures of between 160 and200 �C, a-HBCD prevails due to thermal stereoisomeriza-tion (Peled et al., 1995). Liquid chromatography–tandemmass spectrometry (LC-MS/MS) is routinely used in theanalysis of HBCD stereoisomers (Budakowski and Tomy,2003). The occurrence of HBCDs has been reported inenvironmental and biological samples collected from sev-eral European countries (Remberger et al., 2004; Zegerset al., 2005; Covaci et al., 2006; Law et al., 2006). However,prior to this study, no information on the occurrence ofHBCDs and TBBPA in humans, bottlenose dolphins,and sharks collected from the United States was available.In this study, we report the concentrations of HBCDs andTBBPA in human adipose tissue obtained in New YorkCity, and in three top-level marine predators, namely, bullshark, Atlantic sharpnose shark, and bottlenose dolphin,all collected from Florida coastal waters between 1991and 2004. Concentrations of HBCDs and TBBPA inhumans, dolphins, and sharks were compared with PBDEconcentrations reported earlier for the same set of samples(Johnson-Restrepo et al., 2005a, 2005b). Relationshipsamong TBBPA, HBCDs, and PBDE concentrations inthe human and marine predator samples were investigated.Also, the influence of biological/demographic factors (age,gender, and ethnicity of donor) on the concentrations of

TBBPA and HBCDs in the human adipose tissues wasexamined.

2. Materials and methods

2.1. Sampling

Human adipose tissue samples (n = 20) were obtained inNew York City during 2003–2004, from patients whounderwent liposuction surgery. The samples were devoidof personal identifiers; the only demographic informationavailable to this study was age, gender, ethnicity, occupa-tion, and date of collection. Samples were stored in pre-cleaned glass bottles at �20 �C until analysis. InstitutionalReview Board (IRB) approval for the analysis of humanadipose samples was obtained from the New York StateDepartment of Health. Further details on the human sam-ples have been provided previously (Johnson-Restrepoet al., 2005a).

Blubber from bottlenose dolphin (Tursiops truncatus;n = 15) and muscle tissues from bull shark (Carcharhinus

leucas; n = 13) and Atlantic sharpnose shark (Rhizoprion-

odon terraenovae; n = 3) were analyzed in this study. Bot-tlenose dolphins were collected from the coastal watersof Florida during 1991–2004 by Hubbs-Sea WorldResearch Institute, and the Southeastern Marine Mam-mal Stranding Network. Details regarding gender, length,and weight of the dolphins and sharks analyzed in thisstudy have been reported in a previous publication(Johnson-Restrepo et al., 2005b). Shark samples werecollected from estuarine waters of the Indian RiverLagoon and adjacent offshore coastal waters of Floridain 2004.

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(-) -HBCD(1R,2R,5S,6R,9R,10S)

(-) -HBCD(1R,2R,5S,6R,9S,10R)

(-) -HBCD(1S,2S,5S,6R,9R,10S)

TBBPA

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Fig. 1. Chemical structures of a-, b-, and c-HBCD isomers and TBBPA.

1936 B. Johnson-Restrepo et al. / Chemosphere 70 (2008) 1935–1944


2.2. Chemicals

TBBPA (99% purity) was obtained from TCI America(Portland, OR, USA). Stock solutions (50 lg/ml) of a-,b-, and c-hexabromocyclododecane (HBCD) isomers werepurchased from Wellington Laboratories (Guelph, ON,Canada). All solvents and reagents used in the extractionand clean-up procedures were of HPLC grade. Ammoniumacetate and anhydrous sodium sulfate were obtained fromSigma–Aldrich (Milwaukee, WI, USA).

2.3. Chemical analysis

The method used for the analysis of TBBPA and HBCDisomers was similar to that reported earlier (Morris et al.,2006; Law et al., 2006), with some modifications. Briefly,20 g of muscle tissue or 3 g of blubber/adipose tissue wereground with anhydrous sodium sulfate and extracted withdichloromethane and hexane (3:1; 400 ml) for 16 h in aSoxhlet apparatus. The extract was concentrated to11 ml, and a portion of the extract (1 ml) was used forthe measurement of lipid content by gravimetry. An ali-quot of the remaining extract (4 ml) was purified by pas-sage through a gel permeation chromatography (GPC)column (380 mm · 22 mm i.d.) packed with Bio-beads S-X3 (Bio-Rad Laboratories, Hercules, CA, USA). A mix-ture of 50% hexane in dichloromethane was used as themobile phase at a flow rate of 5 ml/min. The first 100 mlof the eluate were discarded, and the following 150 ml frac-tion, which contained TBBPA and the HBCD isomers, wascollected. The extract was treated with concentrated sulfu-ric acid (5 ml), and then rinsed with purified water (5 ml).The extract was filtered through a nylon syringe filter(Cameo 30N, 0.22 lm, 30 mm; Osmonics, Minnetonka,MN, USA). The solvent was evaporated to dryness undera gentle stream of nitrogen, and then reconstituted with100 ll of methanol.

2.4. Identification and quantification

Extracts were injected into an Agilent 1100 Series highperformance liquid chromatograph (HPLC; Agilent Tech-nologies, Palo Alto, CA, USA) coupled to a API 2000 elec-trospray triple quadrupole mass spectrometer (ESI-MS/MS; Applied Biosystems, Concord, ON, Canada) for thedetermination of TBBPA and HBCD isomers. Analyteswere separated by passage of the extracts (10 ll) through aC18 column (100 · 2.1 mm, 5 lm particle size; ThermoHypersil, Keystone Scientific, Bellefonte, PA, USA). Themobile phase was 10 mM ammonium acetate/methanolstarting at 60% methanol, at a flow rate of 250 ll/min. Thegradient was increased to 100% methanol in 4 min, held for5 min, reversed back to 60% methanol in 11 min, and thenheld for 5 min. The ESI-MS/MS was operated in an electro-spray negative ion mode; the optimized MS/MS parametersfor TBBPA and HBCD isomers were as follows: nitrogencurtain gas, 10 psi; ion spray voltage,�4500 V; declustering

potential, �60 V; temperature, 300 �C; dwell time, 300 ms;collision energy, �100 eV for TBBPA and �50 eV forHBCD isomers. Target compounds were determined bymultiple reaction monitoring (MRM). The ions wereselected at the ([M�H]�) transition of m/z 640.6! 79/640.6! 81 for a-, b-, and c-HBCD isomers, and m/z540.9! 79/540.9! 81 for TBBPA. HBCDs denotes thesum of a-, b-, and c-hexabromocyclododecane isomers.

2.5. Quality assurance/quality control

Procedural blanks were analyzed with every set of 10samples to check for interferences and contamination.The reported concentrations were corrected for blank val-ues. Procedural blanks for human samples contained onaverage, 0.130, 0.198, and 0.497 pg for a-, b -, and c-HBCD isomers, respectively, and 0.018 pg for TBBPA.Method quantitation limits (MQLs) were calculated as10-times the standard deviation of blank measurements.MQLs were 1.3, 2.8, 3.6, and 0.33 pg/g, wet wt, for a-HBCD, b-HBCD, c-HBCD, and TBBPA, respectively.The analytical procedure was validated by spiking knownconcentrations of target compounds (at three different lev-els bracketing the concentrations found in biological sam-ples) into sodium sulfate and passing the standardthrough the entire procedure to calculate the recoveries.Recoveries of target compounds through the analyticalprocedure were (mean ± SD; n = 6) 93.2 ± 6.3% forTBBPA, 87.1 ± 11% for a-HBCD, 96.6 ± 4.2% for b-HBCD, and 90.7 ± 8.5% for c-HBCD. Labeled standardsof HBCD isomers or TBBPA, were not available for useas international standards, at the time of sample analysis(Tomy et al., 2005; Dodder et al., 2006; Morris et al.,2006). To examine the effect of ionization suppression orenhancement by sample matrix, extracts from dolphin,shark, and human tissues, prior to instrumental analysiswere split into two halves. One half of the extract wasinjected to measure concentrations in the samples. Theother half was spiked with 1 ng each of TBBPA, a-HBCD,b-HBCD, and c-HBCD and injected into the LC-MS/MS.Response of the target compounds spiked into the sampleswas compared against the external calibration standardsprepared in methanol, to test for matrix effects. Recoveriesof target compounds are shown in Table 1. Concentrationsof TBBPA and HBCD isomers were calculated by compar-ison of peak areas for samples with the peak areas in a six-point external calibration curve. Mean of blank peak areawas subtracted from the sample peak area for the determi-nation of concentrations. Reported concentrations werenot corrected for the recoveries.

2.6. Statistical analysis

Data are presented as mean ± standard deviation, on alipid weight basis. Compounds with reported concentrationsbelow MQLs were assigned zero for the calculation of meanand median. For the human samples, the non-parametric

B. Johnson-Restrepo et al. / Chemosphere 70 (2008) 1935–1944 1937


Wilcoxon–Mann–Whitney rank sum test was used to com-pare median concentrations of TBBPA and HBCD by gen-der or ethnicity. Linear regression was performed betweenTBBPA and HBCD concentrations, as well as betweenTBBPA/HBCD concentrations and the age of humandonors. For all comparisons, a value of P < 0.05 was consid-ered significant. Pearson’s or Spearman’s correlation coeffi-cients were computed for determining associations amongvariables; 95% confidence intervals (CIs) were estimatedbased on bias-corrected accelerated (BCA) percentile boot-strap method, which provides more accurate results whenhandling skewed data (Efron and Tibshirani, 1993; Hender-son, 2005). To assess whether the correlation coefficientswere significantly different from zero or not, all 95% CIswere calculated at 2000 resamplings. The estimated correla-tion coefficient was considered significant, when the 95% CIdoes not overlap with a value of zero. The Kolmogorov–Smirnov goodness-of-fit test was used to examine the distri-bution of concentrations of TBBPA and HBCDs. Skeweddata were log-transformed for further analysis. Before thedata were log-transformed, values below the respectiveMQLs were each replaced with a value equal to ½ of the cor-responding MQL value. A value was considered as an out-lier if it falls greater than 1.5 times the distance from theupper or lower boundaries of the interquartile range. Thestatistical software package R (release 2.4.1) was used forall statistical calculations (R Development Core Team,Vienna, Austria, 2006).

3. Results and discussion

3.1. Human adipose tissue

TBBPA was above the limit of quantitation of 0.0033 ng/g lipid wt in 69% of the human adipose tissue samples ana-lyzed. TBBPA concentrations in human tissue ranged from<0.0033 to 0.464 (mean: 0.0479 ± 0.102) ng/g lipid wt (Table2). Sum of a-, b-, and c-HBCD isomers were found in 85% ofthe human tissue samples at concentrations ranging from<0.0026 to 2.41 (mean: 0.333 ± 0.571) ng/g lipid wt (Table2). The overall mean concentration of HBCDs in adipose tis-sue was an order of magnitude greater than the mean concen-tration of TBBPA. The mean concentrations of TBBPA andHBCDs in the adipose tissue samples were 3–4 orders ofmagnitude lower than the mean concentration previouslyreported for PBDEs in the same set of samples (Johnson-

Restrepo et al., 2005a). Even though the usage of TBBPAin the United States is over 6 times higher than that ofHBCDs, (BSEF, 2005), the finding of higher HBCD concen-trations than TBBPA concentrations can be explained by thedifferences in the usage and bioaccumulation potential of thetwo BFRs. HBCDs are additive BFRs, and TBBPA is a reac-tive BFR. TBBPA is bound chemically to the polymer struc-ture; thus, the leaching/release of TBBPA into theenvironment is limited (de Wit, 2002). In addition, TBBPAis rapidly metabolized by the mammalian liver and elimi-nated into bile (Schauer et al., 2006). The finding of concen-trations of PBDEs higher than HBCD concentrations inthese human samples can be explained by the large commer-cial production and usage of PBDE as an additive BFR in theUSA during the past three decades. The production ofPBDEs was 2–12-fold greater than that of TBBPA andHBCDs in 2001 (BSEF, 2005).

Very few studies have reported the occurrence ofTBBPA and HBCDs in human samples. To our knowl-edge, this is the first report of concentrations of TBBPAand HBCDs in human tissues from the USA. The meanconcentration of HBCDs in adipose tissue samples fromNew York City was 1–5-fold lower than the concentrationsreported for human tissue samples from several Europeancountries. For instance, the mean concentration of HBCDsin breast milk collected in 2004 from Swedish mothers was0.39 ng/g, lipid wt (Fangstrom et al., 2006); this concentra-tion is similar to that in the adipose tissues in our study.Another breast milk study of Swedish mothers in 2003showed 3-fold higher mean concentration (Lopez et al.,2004) than the concentration found in our study. Concen-trations of HBCDs in serum from Dutch mothers collectedfrom 2001 to 2002 ranged from <0.160 to 7 ng/g, lipid wt,with a mean of 1.1 (Weiss et al., 2004); the mean concentra-tion was 3 times greater than the mean concentration inour adipose tissue samples. HBCD concentrations havealso been reported in breast milk and plasma samples fromMexican mothers in 2003 (Lopez et al., 2004), at concentra-tions ranging from 0.7 to 5.4 ng/g, lipid wt, with mean con-centrations of 1.2 and 2.1 ng/g, lipid wt, in breast milk andplasma, respectively. These mean concentrations were 4–6times higher than the mean concentration found in ourhuman adipose tissue samples. The lower usage of HBCDsin consumer and industrial products in the USA than inEurope is reflected in the geographical variation in HBCDconcentrations in human tissue samples.

Table 1Recoveries (%), standard error (SE), and relative standard deviation (RSD%) of TBBPA, a-HBCD, b-HBCD, and c-HBCD spiked into human,bottlenose dolphin, and shark tissues and passed through the procedure

Tissues n TBBPA a-HBCD b-HBCD c-HBCD

Mean SE RSD (%) Mean SE RSD (%) Mean SE RSD (%) Mean SE RSD (%)

Human 5 65.5 2.2 7.6 96.4 5.6 37 85.7 2.8 7.3 89.3 4.2 11Bottlenose dolphin 5 86.4 8.7 22 75.7 4.8 6 70.8 4.0 13 86.8 5 13Bull shark 5 62.8 7.5 27 87.2 7.5 19 85.9 8.0 21 65.7 7.1 24Atlantic sharpnose shark 3 65.5 4.9 8.6 96.5 5.6 13 85.7 6.2 7.3 70.2 4.5 11



There was no significant correlation between TBBPA orHBCD concentrations and the age of donors in our study(Spearman’s rank correlation, 95% CI). A lack of age-depen-dent increase in concentrations has previously been reportedfor PBDEs, and for synthetic musk fragrances, in this set oftissue samples (Johnson-Restrepo et al., 2005a; Kannanet al., 2005). The lack of correlation between TBBPA orHBCD concentrations and the age is indicative of metabo-lism and excretion of these contaminants. No significant dif-ference was found in the concentrations of TBBPA andHBCDs, between male and female donors (TBBPA,P = 0.15; HBCDs, P = 0.37). The concentrations were alsonot statistically different when concentrations of TBBPAor HBCDs were compared between Caucasians and Hispan-ics (Fig. 2; TBBPA, P = 0.3; HBCDs, P = 0.59). The highestconcentration of TBBPA, 0.464 ng/g, lipid wt, was found ina 30-year old Hispanic female teacher, while the highestconcentration of HBCDs, 2.41 ng/g, lipid wt, was found ina 29-year old Caucasian female aesthetic worker. The lackof significant gender- or ethnicity-related differences in theconcentrations suggests that human exposure to TBBPAand HBCDs is derived from general sources, such as indoorair and/or diet (Remberger et al., 2004). It should also benoted that the number of samples analyzed is small, and fur-ther studies are thus needed to examine the sources and dis-tribution of TBBPA and HBCDs.

Concentrations of TBBPA and HBCDs in human adi-pose tissues are log-normally distributed (Fig. 2; Kolmo-gorov–Smirnov test, P > 0.05). Pearson’s correlationcoefficient for log-transformed concentrations of TBBPAand HBCDs showed a positive and statistically significantcorrelation between the two BFRs in human tissues(R = 0.48; P < 0.05). Nevertheless, neither TBBPA concen-trations nor HBCD concentrations were significantly corre-

lated with PBDE concentrations. Although the sources ofexposure to TBBPA and HBCDs in humans are not welldocumented, dietary intake is thought to be an importantpathway. Our analysis of styroform plates and containersshowed the presence of TBBPA and HBCDs. The lack ofcorrelation between the concentrations of PBDEs andTBBPA/HBCDs indicates differences in pharmacokineticsof the three BFRs in the human body.

Profiles of a-, b-, and c-HBCD isomers in adipose tissuesamples from New York City are shown in Fig. 3. Studieshave shown that the isomer pattern of HBCDs can varydepending on the animal species (Gerecke et al., 2003).Some reports have suggested that b- and c-HBCD stereo-isomers can be more extensively metabolized in organismsthan can a-HBCD isomer (Zegers et al., 2005). Thermalisomerization can also result in the enrichment of a-HBCDduring the polymer extrusion process, which involves hightemperatures of 200–226 �C (Larsen and Ecker, 1988). In apooled serum sample from an elderly population in Swe-den, the most abundant stereoisomer was a-HBCD (Weisset al., 2006). c-HBCD was the dominant isomer present inour adipose tissue samples, accounting for 83% of the totalHBCD concentrations, followed by a-HBCD (17%)(Fig. 3). Thomsen et al. (2007) found high concentrationsof c-HBCD than the other HBCD stereoisomers in serumof workers from an industrial polystyrene plant in Scandi-navia. b-HBCD stereoisomer was not detected in any of thehuman adipose tissue samples analyzed in this study. How-ever, 35% of the human tissue samples contained a-HBCDconcentrations higher than the concentrations of c-HBCD(Fig. 4), suggesting that some individuals are exposed toelevated concentrations of a-HBCD. Further studies areneeded to elucidate the relationship between HBCD iso-mers found in humans and those found in source materials.

Table 2Concentrations of TBBPA, HBCDs, and PBDEsa (ng/g, lipid wt) in human adipose tissues from New York City, USA

TBBPA (ng/g, lipid wt) RHBCD (ng/g, lipid wt) RPBDEs (ng/g, lipid wt)a

N Mean ± SD Median(range)

%Positive

Mean ± SD Median(range)

%Positive

N Mean ± SD Median(range)

%Positive

Overall 20 0.0479 ± 0.102 0.0152(<0.0033–0.464)

69 0.333 ± 0.571 0.111(<0.0026–2.41)

85 52 399 ± 1410 77.3(17–9630)

100

Gender

Female 16 0.049 ± 0.115 0.0087(<0.0033–0.465)

75 0.316 ± 0.608 0.111(<0.0026–2.41)

81 40 253 ± 639 85.7(20–4060)

100

Male 4 0.0434 ± 0.027 0.0449(0.0089–0.0749)

100 0.400 ± 0.458 0.25(0.0651–1.04)

100 12 885 ± 2640 68.7(17–9630)

100

Ethnicity

Caucasian 10 0.0189 ± 0.0247 0.0066(<0.0033–0.068)

56 0.491 ± 0.768 0.118(<0.0026–2.41)

90 35 518 ± 1700 80(26–9630)

100

Hispanic 9 0.0739 ± 0.149 0.0235(<0.0033–0.465)

89 0.182 ± 0.217 0.120(<0.0026–0.627)

78 7 236 ± 322 53(20-961)

100

AfricanAmerican

1 0.1040 0.1040 100 0.101 0.101 100 9 100 ± 75 73(17–296)

100

Fat (%) 67.2 ± 9.3 (52.2–88.6).Donor age (year) 32.5 ± 8.8 (21–51).

a Johnson-Restrepo et al., 2005a; sum of 11 PBDE congeners (28, 30, 47, 85, 99, 100, 153, 154, and three unidenfied congeners).



3.2. Shark and dolphin tissues

Concentrations of TBBPA, and a-, b-, and c-HBCD inmuscle tissues from two shark species and bubbler frombottlenose dolphins collected from the coastal waters ofFlorida are reported in Table 3. Muscle tissues from bullshark contained the highest concentrations of TBBPA(35.6 ng/g, lipid wt) and HBCDs (413 ng/g, lipid wt).TBBPA was found in all the samples analyzed, at concen-trations ranging from 0.056 to 8.48 (mean ± SD;1.2 ± 3 ng/g, lipid wt) in bottlenose dolphin, from 0.035to 35.6 (mean ± SD: 9.5 ± 12) ng/g, lipid wt in bull shark,and from 0.495 to 1.43 (mean ± SD: 0.872 ± 0.5) ng/g,lipid wt, in Atlantic sharpnose shark. HBCD isomers were

also detected in all samples, at concentrations ranging from0.46 and 72.6 (mean ± SD: 7.38 ± 18) ng/g, lipid wt in bot-tlenose dolphin, from 9.15 to 413 (mean ± SD: 77.7 ± 128)

0.0 0.2 0.4 0.0 1.0 2.0

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Fig. 2. (a) Frequency distribution of TBBPA and HBCD concentrations in human adipose tissue samples from New York City, USA. (b) Box-and-whisker plot of TBBPA and HBCD concentrations stratified by ethnicity in human adipose tissue samples from New York City, USA.

α-HBCD γ-HBCD

Composition (%)0 20 40 60 80 100

Bottlenose dolphin

Atlantic sharpnose shark

Bull shark

Human

β-HBCD

Fig. 3. a-, b-, and c-HBCD stereoisomer composition (%) in humanadipose tissue, bottlenose dolphin blubber, and bull shark and Atlanticsharpnose shark muscle from the USA.

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αβ

γ

Fig. 4. LC-MS/MS chromatograms of a-, b-, and c-HBCD stereoisomers(a) a standard containing concentrations of stereoisomers (b) humansample from New York City, showing higher proportion of a-HBCD (c)another human sample from New York City, showing higher proportionof c-HBCD.



ng/g, lipid wt in bull shark, and from 1.83 to 156(mean ± SD: 54.5 ± 88) ng/g, lipid wt, in Atlantic sharp-nose shark (Table 3). HBCD concentrations in musclefrom the two shark species were an order of magnitudegreater than the concentrations found in blubber from bot-tlenose dolphin. PBDE concentrations previously deter-mined in the marine predator samples were 1–2 orders ofmagnitude greater than the concentrations of TBBPAand HBCDs analyzed in this study (Johnson-Restrepoet al., 2005b). This pattern is similar to that found forhumans, and can be explained by high usage of PBDEsin the United States for over 30 years.

HBCD concentrations in the liver from white-sided dol-phin collected from Massachusetts in 1993–2000 rangedfrom 2.92 to 68.3 ng/g, with a median of 18.1 ng/g, lipidwt (Peck et al., 2005); this median is an order of magnitudehigher than the concentrations found in the blubber frombottlenose dolphin in our study. The mean concentrationof HBCD in bottlenose dolphin analyzed in this studywas 1–2 orders of magnitude lower than the concentrationsreported for cetacean species collected in European andAsian coastal waters (Kajiwara et al., 2006; Zegers et al.,2005). Mean concentrations of HBCDs in common dol-phins collected in 2001–2002 from coastal waters of Spain,France, and Ireland were 2 orders of magnitude higher thanthe mean concentration in our bottlenose dolphins (Zegerset al., 2005). Concentrations of HBCDs in finless porpoisescaught in 2000–2001 from the Chinese coast ranged from 21to 55 ng/g lipid wt, with a mean concentration of 35 ng/glipid wt (Kajiwara et al., 2006); this mean concentrationwas an order of magnitude greater than the mean concen-tration found in our bottlenose dolphins. Concentrationsof HBCDs in harbor porpoises collected from coastalwaters of the UK were as great as 21 lg/g, lipid wt; the lat-ter is the highest concentration reported for HBCDs to date(Law et al., 2006). HBCD concentrations were also reportedin skipjack tuna from several off-shore waters of the Asia–Pacific region at concentrations ranging from <0.100 to45 ng/g, lipid wt (Ueno et al., 2006). HBCD concentrationsin muscle tissues from skipjack tuna were in a range similarto the concentrations found in blubber from bottlenose dol-phin, but lower than the concentrations found in muscle tis-sue from bull shark and Atlantic sharpnose shark.

Very few studies have reported the occurrence ofTBBPA in wildlife. The range of TBBPA concentrationsfound in marine predators from Florida was similar tothe range reported for harbor porpoises from the UK(<5–35 ng/g, lipid wt; Law et al., 2006).

We performed a pairwise correlation among TBBPA,HBCD, and PBDE (Johnson-Restrepo et al., 2005b) con-centrations in bottlenose dolphin and bull shark, afterlog-transformation of concentrations. The results sug-gested positive and statistically significant correlationsamong the concentrations of three BFRs in bottlenose dol-phins (Fig. 5). However, pairwise relationships among thethree BFRs in bull shark showed only weak correlationsand were not statistically significant (Fig. 5).T

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Among the three isomers of HBCDs in marine preda-tors, a- and b-HBCD isomers were found in all samplesanalyzed, while c-HBCD was detected in 87% of the sam-ples. a-HBCD was the most abundant isomer in bottlenosedolphin, followed by c-HBCD and b-HBCD, accountingfor 48%, 37%, and 15%, respectively, of the total HBCDconcentrations (Fig. 3). Nevertheless, c-HBCD was themost abundant isomer in the two shark species, followedby a-HBCD and b-HBCD, accounting for 76%, 17% and7%, respectively, of the total HBCD concentrations. Thedifferences in the profiles of HBCD isomers between sharksand dolphin may suggest differences in metabolism, sourcesof exposure and tissue-specific distribution (i.e., blubberversus muscle).

Recent studies have recognized that there are large dif-ferences in the isomer profile of HBCDs, between envi-ronmental samples and technical mixtures (Covaci et al.,2006). The isomer pattern in environmental samplesshowed that a-HBCD is the predominant stereoisomer,although it only accounts for �12% in the technical mix-ture (Heeb et al., 2005). Profiles of HBCD stereoisomerscan vary depending on the species, type of tissues ana-lyzed, and sampling locations (Law et al., 2003; Tomyet al., 2004; Janak et al., 2005; Ueno et al., 2006). Skip-jack tuna collected in the Pacific ocean contained a-HBCD as the most abundant isomer, but the compositionof a- and c- isomers varied depending on the samplinglocation (Ueno et al., 2006). Harbor porpoise collectedin the UK contained a-HBCD at 38% of the total HBCDconcentrations (Law et al., 2003) while later studies foundthat this isomer at 99% of the total HBCD concentrations

(Law et al., 2006). a-HBCD was the predominant isomerin marine fish collected from the Western Scheldt estuaryin Belgium (Janak et al., 2005). a-HBCD was the predom-inant isomer in lake trout from Lake Ontario (82% of thetotal HBCDs) (Tomy et al., 2004). Profiles of HBCD ster-eoisomers in the environment can be related to biologicaland physicochemical properties, including species-depen-dent metabolic isomerization, biodegradation, and sourcesof exposure.

In summary, this is the first report of TBBPA andHBCD concentrations in human, bottlenose dolphin,and shark tissues from the United States. Concentrationsof HBCDs were greater than those of TBBPA in all of thesamples analyzed. Concentrations of HBCDs in dolphinsand sharks were on the order of ng/g, lipid wt, whilethe concentrations in human adipose tissue samples wereon the order of pg/g, lipid wt. Concentrations of HBCDsand TBBPA were significantly correlated in human andbottlenose dolphin tissue samples. Although the concen-trations of PBDEs in human samples from the UnitedStates were 10–100 times higher than the concentrationsreported for European countries, HBCD concentrationsin human samples from the United States were 1–5-foldlower than the concentrations reported for Europeancountries. Similarly, HBCD concentrations in dolphinsand sharks from the United States were lower than theconcentrations reported in other marine species fromEuropean countries. The profile of HBCD isomers inhumans and marine predators varied considerably, sug-gesting species-dependent metabolism and exposuresources.

0

6

6 6

2

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y = 0.25x + 4.9 R = 0.14; P = 0.64 2

4

6

8

10

0

Log HBCDs (ng/g, lipid wt)

Bull shark

y = 0.71x + 0.73R = 0.80; P < 0.01

y = -0.19x + 1.6R = -0.33; P = 0.28

-4

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0

2

4

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Bottlenose dolphin

Bull shark

0

y = 0.56x + 5.9R = 0.65; P< 0.01

y = -0.40x + 5.5 R = -0.39; P = 0.19

0

2

4

6

8

10

-2 -1 20

Bull shark

Bottlenose dolphin Bottlenose dolphin

3

2

1

Log TBBPA (ng/g, lipid wt)

Log

PB

DE

s (n

g/g,

lipi

d w

t)

Log TBBPA (ng/g, lipid wt)

1

Log

HB

CD

s(ng

/g, l

ipid

wt)

Fig. 5. Pairwise relationships among TBBPA, HBCD, and PBDE concentrations in bottlenose dolphin blubber and bull shark muscle tissues from Floridacoastal waters, USA. PBDE concentrations were taken from the reference Johnson-Restrepo et al., 2005b.



Acknowledgements

We thank Cape Canaveral Scientific, Inc. for their assis-tance in collecting shark samples. Thanks to G. Burgess, T.Curtis, and D. McGowan for additional bull shark sam-ples. We also thank M. Stolen and W. Durden of HSWRIand the Southeastern Marine Mammal Stranding Networkfor bottlenose dolphin samples from the IRL collected un-der Letters of Authorization from the National MarineFisheries Service.

References

Bromine Science and Environmental Forum (BSEF). Major BrominatedFlame Retardants Volume Estimates. Available at: <http://www.bsef.com> (accessed 04.04.2005).

Budakowski, W., Tomy, G., 2003. Congener-specific analysis of hexa-bromocyclododecane by high-performance liquid chromatography/electrospray tandem mass spectrometry. Rapid Commun. MassSpectrom. 17, 1399–1404.

Covaci, A., Gerecke, A.C., Law, R.J., Voorspoels, S., Kohler, M., Heeb,N.V., Leslie, H., Allchin, C.R., deBoer, J., 2006. Hexabromocyclo-dodecanes (HBCDs) in the environment and humans: a review.Environ. Sci. Technol. 40, 3679–3688.

Dodder, N.G., Peck, A.M., Kucklick, J.R., Sander, L.C., 2006. Analysisof hexabromocyclododecane diastereomers and enantiomers by liquidchromatography/tandem mass spectrometry: chromatographic selec-tivity and ionization matrix effects. J. Chromatogr. A 1135, 36–42.

de Wit, C.A., 2002. An overview of brominated flame retardants in theenvironment. Chemosphere 46, 583–624.

Efron, B., Tibshirani, R.J., 1993. Better bootstrap confident intervals(chapter 14). An Introduction to the Bootstrap. Chapman & Hall, NewYork, pp. 178–201.

Fangstrom, B., Athanassiadis, I., Strid, A., Odsjo, T., Guvenius, D.,Noren, K., Bergman, A., 2006. Temporal trends of PBDEs and HBDDin milk from Stockholm mothers, 1980–2004. Organohalogen Compd.68, 774–777.

Gerecke, A.C., Kohler, M., Zenegg, M., Schmid, P., Heeb, N.V., 2003.Determination of alpha hexabromocyclododecane (HBCD) isomer inSwiss fish at levels comparable to polybrominated diphenyl ether(PBDEs). Organohalogen Compd. 61, 155–158.

Heeb, N.V., Schweizer, W.B., Kohler, M., Gerecke, A.C., 2005. Structureelucidation of hexabromocyclododecanes-a class of compounds with acomplex stereochemistry. Chemosphere 61, 65–73.

Henderson, A.R., 2005. The bootstrap: a technique for data-drivenstatistics. Using computer-intensive analyses to explore experimentaldata. Clin. Chim. Acta 359, 1–26.

Hites, R.A., 2004. Polybrominated diphenyl ethers in the environment andin people: a meta-analysis of concentrations. Environ. Sci. Technol. 38,945–956.

Hoh, E., Hites, R.A., 2005. Brominated flame retardants in theatmosphere of the East-Central United States. Environ. Sci. Technol.39, 7794–7802.

Janak, K., Covaci, A., Voorspoels, S., Becher, G., 2005. Hexabromocy-clododecane in marine species from species from the Western ScheldtEstuary: diastereomer- and enantiomer-specific accumulation. Envi-ron. Sci. Technol. 39, 1987–1994.

Johnson-Restrepo, B., Kannan, K., Rapaport, D.P., Rodan, B.D., 2005a.Polybrominated diphenyl ethers and polychlorinated biphenyls inhuman adipose tissue from New York. Environ. Sci. Technol. 39,5177–5182.

Johnson-Restrepo, B., Kannan, K., Addink, R., Adams, D.H., 2005b.Polybrominated diphenyl ethers and polychlorinated biphenyls in amarine foodweb of coastal Florida. Environ. Sci. Technol. 39, 8243–8250.

Kajiwara, N., Isobe, T., Ramu, K., Tanabe, S., 2006. Temporal trendstudies on hexabromocyclododecane (HBCD) in marine mammalsfrom Asia–Pacific. Organohalogen Compd. 68, 515–518.

Kannan, K., Reiner, J.L., Yun, S.H., Perrotta, E.E., Tao, L., Johnson-Restrepo, B., Rodan, B.D., 2005. Polycyclic musk compounds inhigher trophic level aquatic organisms and humans from the UnitedStates. Chemosphere 61, 693–700.

Larsen, E.R., Ecker, E.L., 1988. Thermal stability of fire retardants: III,decomposition of pentabromochlorocyclohexane and hexabromocy-clododecane under Processing Conditions. J. Fire Sci. 4, 139–159.

Law, R.J., Allchin, C.R., Morris, S., Jepson, P.D., 2003. Persistentorganohalogen compounds in marine mammals stranded or bycaughtin the UK. Organohalogen Compd. 62, 224–227.

Law, R.J., Kohler, M., Heeb, N.V., Gerecke, A.C., Schmid, P.,Voorspoels, S., Covaci, A., Becher, G., Janak, K., Thomsen, C.,2005. Hexabromocyclododecane challenges scientists and regulators.Environ. Sci. Technol. 39, 281A–287A.

Law, R.J., Bersuder, P., Allchin, C.R., Barry, J., 2006. Levels of the flameretardants hexabromocyclododecane and tetrabromobisphenol A inthe blubber of harbor porpoises (Phocoena phocoena) stranded orbycaught in the UK, with evidence for an increase in HBCDconcentrations in recent years. Environ. Sci. Technol. 40, 2177–2183.

Loganathan, B.G., Kannan, K., Watanabe, I., Kawano, M., Irvine, K.,Kumar, S., Sikka, H.C., 1995. Isomer-specific determination and toxicevaluation of polychlorinated biphenyls (PCBs), polychlorinated/brominated dibenzo- p-dioxins (PCDDs/PBDDs), dibenzofurans(PCDFs/PBDFs), polybrominated biphenyl ethers (PBBEs) andextractable organic halogen (EOX) in carp from the Buffalo River,New York. Environ. Sci. Technol. 29, 1832–1838.

Lopez, D., Athanasiadou, M., Athanassiadis, I., 2004. A preliminarystudy on PBDEs and HBCD in blood and milk from Mexican women.The third international workshop on brominated flame retardants.Toronto, Canada, pp. 482–487.

Morris, S., Bersuder, P., Allchin, C.R., Zegers, B., Boon, J.P., Leonards,P.E.G., de Boer, J., 2006. Determination of the brominated flameretardant, hexabromocyclodocane, in sediments and biota by liquidchromatography–electrospray ionisation mass spectrometry. TrendsAnal. Chem. 25, 343–349.

Peck, A.M., Tuerk, K.J., Kucklick, J.R., 2005. Hexabromocyclododecanediastereoisomers and enantiomers in white-sided dolphin blubber andliver tissues. Organohalogen Compd. 67, 1259–1262.

Peled, M., Scharia, R., Sondack, D., 1995. Thermal rearrangement ofhexabromocyclododecane (HBCD). Advances in OrganobromineChemistry II. Elsevier Publishers, New York, pp. 92–99.

Schauer, U.M., Volkel, W., Dekant, W., 2006. Toxicokinetics of tetrab-romobisphenol A in humans and rats after oral administration.Toxicol. Sci. 91, 49–58.

R Development Core Team, 2006. R: A language and environment forstatistical computing. R Foundation for Statistical Computing,Vienna, Austria. ISBN 3-900051-07-0, URL <http://www.R-project.org>.

Remberger, M., Sternbeck, J., Palm, A., Kaj, L., Stromberg, K.,Brorstrom-Lunden, E., 2004. The environmental occurrence ofhexabromocyclododecane in Sweden. Chemosphere 54, 9–21.

Tomy, G.T., Budakowski, W., Halldorson, T., Whittle, D.M., Keir, M.J.,Marvin, C., MacInnis, G., Alaee, M., 2004. Biomagnification of alpha-and gamma-hexabromocyclododecane isomers in a Lake Ontario foodweb. Environ. Sci. Technol. 38, 2298–2303.

Tomy, G.T., Halldorson, T., Danell, R., Law, K., Arsenault, G., Alaee,M., MacInnis, G., Marvin, C.H., 2005. Refinements to the diastereo-isomer-specific method for the analysis of hexabromocyclododecane.Rapid Commun. Mass Spectrom. 19, 2819–2826.

Thomsen, C., Molander, P., Daae, H.L., Janak, K., Froshaug, M., Liane,V.H., Thorud, S., Becher, G., Dybing, E., 2007. Occupationalexposure to hexabromocyclododecane at an industrial plant. Environ.Sci. Technol. 41, 5210–5216.

Ueno, D., Alaee, M., Marvin, C., Muir, D.C., Macinnis, G., Reiner, E.,Crozier, P., Furdui, V.I., Subramanian, A., Fillmann, G., Lam, P.K.,



Zheng, Muchtar, M., Razak, H., Prudente, M., Chung, K.H., Tanabe,S., 2005. Distribution and transportability of hexabromocyclodode-cane (HBCD) in the Asia–Pacific region using skipjack tuna as abioindicator. Environ. Pollut. 144, 238–247.

Weiss, J., Meijer, L., Sauer, P., Linderholm, L., Bergman, A., Athanas-siadis, I., 2004. PBDE and HBCD levels in blood from Dutch mothersand infants – analysis of a Dutch Groningen infant cohort. Organo-halogen Compd. 66, 2677–2682.

Weiss, J., Wallin, E., Axmon, A., Jonsson, B.A.G., Akesson, H., Janak,K., Hagmar, L., Bergman, A., 2006. Hydroxy-PCBs, PBDEs, and

HBCDs in serum from an elderly population of Swedish fishermen’swives and associations with bone density. Environ. Sci. Technol. 40(20), 6282–6289.

Zegers, B.N., Mets, A., vanBommel, R., Minkenberg, C., Hamers, T.,Kamstra, J.H., Pierce, G.J., Boon, J.P., 2005. Levels of hexabromo-cyclododecane in harbor porpoises and common dolphins fromWestern European Seas, with evidence for stereoisomer-specificbiotransformation by cytochrome P450. Environ. Sci.Technol. 39,2095–2100.


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