Stereoisomer-specific Trophodynamics of the Chiral Brominated

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Stereoisomer-specific Trophodynamics of the Chiral Brominated Flame Retardants HBCD and TBECH in a Marine Food Web, with Implications for Human Exposure Yuefei Ruan, Xiaohua Zhang, Jian-Wen Qiu, Kenneth M.Y. Leung, James C.W. Lam, and Paul K.S. Lam Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02206 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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Environmental Science & Technology

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Stereoisomer-specific Trophodynamics of the Chiral Brominated

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Flame Retardants HBCD and TBECH in a Marine Food Web, with

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Implications for Human Exposure

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Yuefei Ruan†, Xiaohua Zhang‡, Jian-Wen Qiu§, Kenneth M.Y. Leung , James

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C.W. Lam†,‡,*, Paul K.S. Lam†, ,*

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‖

⊥

†

State Key Laboratory in Marine Pollution (SKLMP), Research Centre for the Oceans

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and Human Health, Shenzhen Key Laboratory for the Sustainable Use of Marine

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Biodiversity, City University of Hong Kong, Hong Kong SAR, China

9

‡

Department of Science and Environmental Studies, The Education University of Hong of Kong, Hong Kong SAR, China

10 11

§

12

‖ The Swire Institute of Marine Science and School of Biological Sciences, The

Department of Biology, Hong Kong Baptist University, Hong Kong SAR, China

University of Hong Kong, Hong Kong SAR, China

13 14

⊥

Department of Chemistry, City University of Hong Kong, Hong Kong SAR, China

James C. W. Lam*

Paul K. S. Lam*

Department of Science and

State Key Laboratory in Marine

Environmental Studies, The Education

Pollution, Department of Chemistry,

University of Hong Kong, Hong Kong

City University of Hong Kong, Hong

SAR, China

Kong SAR, China

Tel: +852-2948-8537

Tel: +852-3442-6828

Fax: +852-2948-7676

Fax: +852-3442-0303

E-mail: [email protected]

E-mail: [email protected]

[email protected] 15

1

ACS Paragon Plus Environment


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ABSTRACT

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Stereoisomers of 1,2,5,6,9,10-hexabromocyclododecane (HBCD) and 1,2-dibromo-4-

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(1,2-dibromoethyl)-cyclohexane (TBECH) were determined in sediments and 30

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marine species in a marine food web to investigate their trophic transfer. Lipid content

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was found to affect the bioaccumulation of ΣHBCD and ΣTBECH in these species.

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Elevated biomagnification of each diastereomer from prey species to marine mammals

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was observed. For HBCD, biota samples showed a shift from γ- to α-HBCD when

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compared with sediments and technical mixtures; trophic magnification potential of

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(‒)-α- and (+)-α-HBCD were observed in the food web, with trophic magnification

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factors (TMFs) of 11.8 and 8.7, respectively. For TBECH, the relative abundance of γ-

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and δ-TBECH exhibited an increasing trend from abiotic matrices to biota samples;

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trophic magnification was observed for each diastereomer, with TMFs ranging from 1.9

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to 3.5; the enantioselective bioaccumulation of the first eluting enantiomer of δ-TBECH

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in organisms at higher TLs was consistently observed across samples. This is the first

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report on the trophic transfer of TBECH in the food web. The estimated daily intake of

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HBCD for Hong Kong residents was approximately 16-times higher than that for the

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general population in China, and the health risk to local children was high based on the

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relevant available reference dose.

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KEYWORDS

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HBCD; TBECH; Enantiomer; Food web; Trophic Transfer; Biomagnification

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INTRODUCTION

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Concerns over brominated flame retardants (BFRs) have been growing since the

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1990s, as many exhibit environmental persistence, bioaccumulation, and toxicity, in

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wildlife and humans.1,2 Numerous studies have reported that two kinds of extensively

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used

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hexabromocyclododecane (HBCD), are globally distributed in the environment and

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have been found in different environmental matrices, including biota, indicating the

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liver and thyroid hormone homeostasis as the main targets for toxicity.3–5 For most other

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BFRs, such as 1,2-dibromo-4-(1,2-dibromoethyl)-cyclohexane (TBECH), however,

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only limited data are available on their production and use, exposure status, and

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toxicological information.6–8 Considering that all formulations of commercial PBDE

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mixtures and HBCD technical products were recently included under the Stockholm

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Convention on Persistent Organic Pollutants (POPs) in Annex A for global elimination,

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research is shifting toward the use of the other BFRs as alternatives, as these alternatives

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have not been ascertained to be safer for environmental and public health.9,10

BFRs,

polybrominated

diphenyl

ethers

(PBDEs)

and

1,2,5,6,9,10-

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Several kinds of BFRs, including HBCD and TBECH, can exhibit optical activities

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as chiral compounds. For instance, HBCD theoretically includes 6 diastereomeric pairs

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of enantiomers and 4 meso forms, and commercial mixtures mainly consist of α-

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(~12%), β- (~9%), and γ-diastereomers (~79%);11–13 TBECH exhibits stereochemical

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diversity as 4 diastereomeric pairs of enantiomers, and the technical formulation

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contains equimolar amounts of α- and β-diastereomers.14 Many studies have revealed

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that HBCD exhibits high diastereo- and enantio-selectivity in organisms.3,15,16 A

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diastereomeric shift from γ- to α-HBCD was frequently observed in biota at higher

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trophic levels (TLs), whereas the preferential bioaccumulation of (−)-α-HBCD was

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revealed more often in certain animal species, independent of biological factors, such 3



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as body composition, age/sex variation, nutritive condition, and tissue distribution.15–17

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Although TBECH is increasingly detected in the environment and biota globally,

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TBECH concentrations are primarily quantified as the sum of all stereoisomers

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(ΣTBECH), due to difficulties in the chromatographic separation of every

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diastereomer.18–20 No study has investigated TBECH enantiomeric patterns in any

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environmental matrix, except our recent work on cetacean blubber.21 The trophic

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transfer of POPs and POP-like pollutants in food webs is a vital component for

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assessing their ecological and health risks. Biomagnification of HBCD has been

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observed through correlation between the TL and the concentration in organisms.22–30

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However, most studies do not fully consider the precise predator-prey feeding

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relationships, and even fewer focus on the trophodynamics of different HBCD

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enantiomers.31,32 Up to now, the biomagnification potential of TBECH is still

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uncharacterized.

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Our recent study reported on the occurrence of HBCD enantiomers and meso isomers

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as well as TBECH enantiomers in two resident species of marine mammals ― finless

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porpoises (Neophocaena phocaenoides) and Indo-Pacific humpback dolphins (Sousa

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chinensis) ― in the South China Sea.21 Elevated accumulation of these chiral BFRs

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with the preferential enrichment of specific stereoisomers was observed in these marine

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top predators. To build on this earlier work, the present study aims to (1) determine the

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occurrence and trophodynamics of specific HBCD and TBECH stereoisomers in the

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marine food web with the marine mammals as apex predators and (2) assess the food

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web magnification potentials of these stereoisomers and the potential role of diet on

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their levels in the marine mammals. Particular emphasis is placed on understanding the

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behavior and fate of chiral BFRs in marine ecosystems.

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MATERIALS AND METHODS 4


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Sample Collection. A total of 34 surface sediments were collected from Hong

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Kong waters in June, 2012, from sampling sites corresponding to the Environmental

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Protection Department’s regular sediment monitoring stations.33 Marine organisms,

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including 5 mollusk species, 6 crustacean species, and 19 fish species, were collected

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via bottom trawling in Hong Kong waters from July to November, 2012 (Table 1).

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These marine species, together with the two previously studied cetacean species from

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this region, constitute a marine food web pertaining to low-trophic-level marine lives

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to the marine top predators.34 The sampling area covered the western, southern, and

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eastern Hong Kong waters. Further details for the samples and sampling sites are

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presented in Table S1 and Figure S1 in the Supporting Information (SI).

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Sample Preparation and Instrumental Analysis. Details of the standards,

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reagents and solvents can be found in the SI. Sample extraction was accomplished using

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previously established methods with modest modifications, as described in the SI.

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HBCD stereoisomers were analyzed by liquid chromatography‒tandem mass

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spectrometry using an Eclipse Plus C18 column (4.6 mm i.d. × 100 mm, 3.5 μm)

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coupled to a NUCLEODEX β-PM column (4 mm i.d. × 200 mm, 5 μm) for separation,

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and TBECH enantiomers were determined using gas chromatography–mass

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spectrometry using a CHIRALDEX B-TA capillary column (30 m × 0.25 mm i.d., 0.12

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µm film thickness) for separation. More information on the instrumental analysis is

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provided in the SI.

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Quality Assurance and Quality Control (QA/QC). The limit of detection

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(LOD) was defined as an instrument signal-to-noise ratio (SNR) of ≥ 3:1. Detailed

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QA/QC measures for calibration curves, procedure blanks, and matrix recovery tests

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are given in the SI. The LODs of the samples ranged from 0.05 to 0.2 ng/g dry weight

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(dw) in sediments and 0.3 to 0.8 ng/g lipid weight (lw) in biota for HBCD stereoisomers, 5



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and from 0.1 to 0.5 ng/g dw in sediments and 0.8 to 1.8 ng/g lw in biota for TBECH

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stereoisomers. Matrix recoveries ranged from 83% to 110% for sediments and 72% to

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106% for bio-samples. Surrogate recoveries ranged from 78% to 95%, and the

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concentration data were corrected by the surrogates.

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Stable Isotope Analysis and TL Determination. Information on the stable

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isotope analysis is given in the SI. TLs of individual organisms were determined based

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on the assumption that zooplankton occupies TL 2.0 using the following equation:

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TL consumer = 2.0 + (δ15N consumer – δ15N primary consumer) / 3.8

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where δ15N primary consumer is the stable nitrogen isotope value of zooplankton (9.7) and

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3.8 is the isotopic trophic enrichment factor.35,36

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Biota–Sediment Accumulation Factor (BSAF), Bioaccumulation Factor

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(BMF), and Trophic Magnification Factor (TMF) Calculations. The BSAF

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calculation was based on the assumption that sediment is the dominant exposure

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pathway for benthic organisms using the following equation:

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BSAF = C biota (ng/g, lw) / C sediment [ng/g, total organic carbon (TOC)]

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where C biota is the average lipid-normalized concentration in benthos and C sediment is the

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TOC-normalized concentration in sediments.37 BMF was defined as the ratio of the

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average lipid-normalized concentration between predator and prey.37 TMF was used to

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describe the biomagnification, and the calculation was based on the correlations

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between average lipid-normalized concentrations and TLs using the following equation:

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ln C biota (ng/g, lw) = a + b × TL

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where a and b represent the constant and the slope of the linear regression, respectively.

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Slope b was used to calculate TMF using the equation TMF = e b.38

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Estimated Daily Intake (EDI) Assessment. The consumption rates (CRs) of

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marine crustaceans, mollusks, and fishes in the Hong Kong population were obtained 6


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from a dietary survey of the general population conducted in Hong Kong in 2005-2007

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and were 27.51 g wet weight (ww)/day, 23.17 g ww/day, and 65.98 g ww/day,

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respectively.39 The average body weight (BW) was 66.2 kg for adults and 21.1 kg for

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children in China.29 All the marine organisms investigated in this study are popular

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seafood items in Hong Kong. To evaluate human exposure to HBCD and TBECH via

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seafood consumption, the EDI for local residents was calculated using the following

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equation:

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EDI = CR × C biota (ng/g, ww) / BW

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Statistical Analysis. Statistical analysis was performed using Microsoft Excel

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2016 to generate descriptive statistics, and all other statistical procedures were

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conducted using IBM SPSS Statistics 20.0 and OriginLab OriginPro 2015. Different

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data treatments for values below the LOD, i.e., excluding values below the LOD, using

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1/2 the LOD instead, or setting those values to zero, was confirmed to have no effect

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on the conclusion and interpretation of the results. Thus, values below the LOD were

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replaced with 1/2 LOD, as was done in our previous studies.21,40 Enantiomer fractions

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(EFs) were used to describe enantiomeric patterns. Methods used for statistical analysis

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and detail information on the EF calculation are provided in the SI.

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RESULTS AND DISCUSSION

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Levels of ΣHBCD and ΣTBECH in Sediments and Biota. ΣHBCD and

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ΣTBECH were detected in most of the sediment samples. ΣHBCD was detected in all

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the sampled biota species, while ΣTBECH was detected in 26 of the 30 investigated

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species. These results indicate the widespread occurrence of HBCD and TBECH in the

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marine environment of Hong Kong. No significant spatial differences (p > 0.05) were

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found in the concentrations of ΣHBCD or ΣTBECH in biota species with similar TLs

7



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among different sampling areas (SI Table S1). Thus, concentrations from the same

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marine species were pooled for data analysis. ΣHBCD and ΣTBECH concentrations in

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sediments (dw) from different sampling areas and in each marine species (dw and lw)

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are summarized in Table 1 and illustrated in SI Figure S2.

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In Hong Kong waters, surface sediment concentrations were between < 0.1 (below

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the LOD) and 239 ng/g dw for ΣHBCD, and were between < 0.5 and 5.10 ng/g dw for

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ΣTBECH. ΣHBCD levels were approximately 1 order of magnitude higher than those

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of ΣTBECH. Considering the median concentrations, surface sediments from central

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waters accumulated more ΣHBCD and ΣTBECH than the other areas. The correlation

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between TOC content and sediment concentration was not significant for both ΣHBCD

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and ΣTBECH (p > 0.05), implying the minor role of TOC in shaping the sediment

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partitioning of HBCD and TBECH. These findings suggest a domination of local

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sources of HBCD and TBECH from human activities accounting for their occurrence

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in sediments from the study region. A strong positive correlation was found between

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the ΣHBCD and ΣTBECH concentrations in sediments (p < 0.0001), indicating their

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probably similar emission source and/or pathway. Following the method of Liu et al.

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(2014),41 for the Hong Kong’s 12 coastal council districts relevant to our sampling sites,

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a significant positive correlation was found in this study between the population density

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and the average BFR (ΣHBCD + ΣTBECH) concentration (p < 0.05). Since the 1980s,

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the dominant industry in Hong Kong has shifted from manufacturing to services.

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Therefore, BFRs in sediments from Hong Kong waters may have arisen from

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urbanization (e.g., using BFR-containing products). Overall, ΣHBCD concentrations in

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sediments from Hong Kong waters in this study were relatively high compared with a

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previous study on their occurrence in sediments from the Pearl River Delta (PRD)

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region collected in 2013, whereas ΣTBECH concentrations in our study were slightly 8


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lower compared with that study.42

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In biota, the lipid content varied largely between the sampled marine species, and the

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values ranged from 4.9% to 29%. The concentrations of ΣHBCD and ΣTBECH in these

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biota on dry weight basis were significantly positively correlated with the lipid contents

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(Pearson’s r = 0.695 and 0.498, respectively, p < 0.01) (SI Figure S3), indicating that

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lipid content plays a vital role in the bioaccumulation of HBCD and TBECH.

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Accordingly, the concentrations below are expressed in ng/g lw. No significant

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difference in ΣHBCD concentrations was found among fishes, crustaceans, and

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mollusks (p > 0.05), while ΣTBECH levels were significantly higher in mollusks than

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the other groups (p < 0.05). Tao et al. reported higher levels of ΣTBECH than ΣHBCD

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found in wild fish species including mackerel and tuna.10 In the present study, ΣHBCD

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concentrations in all three biota groups were all significantly higher than those of

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ΣTBECH (p < 0.05, SI Figure S2), in conformity with a previous finding of the lower

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bioaccumulation potential of TBECH than HBCD in zebrafish (Danio rerio) after

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dietary exposure.43 This may also reflect the higher production/use of HBCD than

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TBECH in the PRD in recent years.

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Data for HBCD levels in marine organisms have been documented for a number of

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areas and species. In marine fishes and invertebrates, the reported HBCD levels ranged

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from low ng/g lw up to 5,200 ng/g lw, with high levels typically found in recent years

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in Asian countries, especially China.3,29,30,44 The ΣHBCD concentrations in bio-samples

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in our study (3.01–93.2 ng/g lw) were approximately 1 order of magnitude higher than

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those in marine fish samples collected along the Chinese coastline in 2008 (0.57–10.1

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ng/g lw),45 but were 1–2 orders of magnitude lower than those reported in marine

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species collected from several estuaries in Bohai Sea, East China.29,30,46 Overall, the

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ΣHBCD levels in marine organisms in our study are among the moderate levels reported 9



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worldwide. Very limited information is available for TBECH in wild aquatic species.

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The ΣTBECH concentrations in bio-samples in our study (n.d.–13.9 ng/g lw) were

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slightly higher than those reported in Nordic mussels and fishes (up to 1.6 ng/g ww)

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and in Canadian Arctic beluga blubber (0.9–11.3 ng/g lw).47,48 The current results

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suggest that marine organisms in the marine environment of Hong Kong are susceptible

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to considerable TBECH exposure.

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BSAFs and BMFs in Marine Organisms. In this study, BSAFs were determined

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for the estimation of contaminant uptake by biota from sediment, and BMFs between

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prey species and marine mammals were calculated to further investigate the

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accumulation of contaminants from diet to top predators. The dataset of the marine

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mammals was published previously.21 The assessed BSAFs for crustaceans and

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mollusks are given in SI Table S2, while the identified prey list and calculated BMFs

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for the two cetacean species are given in SI Table S3.

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The BSAFs of ΣHBCD and ΣTBECH ranged from 0.02 to 0.28 and from 0.02 to

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0.08, respectively, i.e., none of these crustacean and mollusk species exhibited a BSAF

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greater than 1. The BSAF ranges of ΣHBCD for invertebrates in our study overlap with

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those reported in marine bivalves from South Korea (0.01–0.15),49 but are much lower

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than those determined from a laboratory study on bivalves assembled from brackish

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coastal bays (log BSAF > 1.25).50 This discrepancy is likely ascribed to the large

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variations between field and laboratory exposure. At the present time, there are no

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available BSAFs of TBECH for comparison.

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Considering the BSAFs of HBCD diastereomers (α, β, and γ), α-HBCD contributed

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the most to the bioaccumulation of ΣHBCD in each invertebrate species, whereas γ-

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HBCD always accounted for the least. This provides a pathway for α-HBCD to be the

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most readily transferred HBCD diastereomer in the marine food web as shown in this 10


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study. For TBECH, no significant differences in BSAFs were found among the four

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diastereomers. However, this does not necessarily indicate an identical bioaccumulation

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potential for different TBECH diastereomers in these organisms, as BSAF represents

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an integrated consequence of various processes, including bioavailability, uptake and

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depuration, as well as bioisomerization.15

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The feeding habits of finless porpoises (N. phocaenoides) and Indo-Pacific

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humpback dolphins (S. chinensis) in Hong Kong waters were identified in two previous

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studies via the examination of stomach contents collected from stranded cetaceans.51,52

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These marine mammals are the top predators in this investigated food web, and there is

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some dietary overlap between the two cetacean species, sharing some important prey

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items (e.g., anchovies and croakers). Notwithstanding, porpoises and dolphins have

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dissimilar prey preferences, which can be reflected in their distribution around Hong

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Kong: porpoises prefer cephalopods and usually appear in deeper and clearer saline

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waters (mainly in the southern and eastern waters of Hong Kong), whereas dolphins

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prefer demersal and shoaling fishes of the murky and brackish waters of estuaries, i.e.,

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mostly in northwestern waters of Hong Kong close to the Pearl River Estuary (PRE).53

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Among the collected marine species in our study, 11 pairs of predator-prey relationships

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were identified for each cetacean species (SI Table S3).

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For the predator-prey pairs of porpoises, the BMFs of ΣHBCD and ΣTBECH were

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all above 1 and ranged from 15.0 to 192 and from 2.23 to 19.8, respectively, suggesting

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the biomagnification of HBCD and TBECH from the studied prey to porpoises. Much

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higher BMFs were observed for the predator-prey pairs of dolphins (SI Table S3), with

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values ranging from 325 to 4,160 for ΣHBCD and from 9.80 to 111 for ΣTBECH. This

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signifies that dolphins have greater potential to accumulate HBCD and TBECH via

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food chains as compared to porpoises. For the 8 common preys between porpoises and 11



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dolphins, much higher BMFs were observed in ΣHBCD than in ΣTBECH. Therefore,

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similar to the BSAF results, ΣHBCD exhibited higher biomagnification potential than

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ΣTBECH in cetacean species. Future studies should examine the influence of other

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biological factors, such as age/sex variations and nutritive conditions.

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Considering BMFs for HBCD diastereomers (α, β, and γ), the BMFs of α-HBCD

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were the largest, in accordance with this diastereomer predominating cetacean species,

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as reported in our previous study.21 Although the BMFs of β-HBCD also suggest its

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high biomagnification potential, it was detected in marine mammals at relatively low

269

levels, possibly because this diastereomer is present in relatively small quantities in

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technical products and is susceptible to biotransformation.54,55 For TBECH, the BMFs

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of α- and β-TBECH were significantly lower than those of γ- and δ-TBECH (p < 0.01).

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The large BMFs of γ- and δ-TBECH are in agreement with the rapidly increasing

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proportions of these diastereomers in cetacean species, as described in our previous

274

study.21 In comparison, although the BMFs of α- and β-TBECH suggest their relatively

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“low” biomagnification potential, these diastereomers still prevailed in cetacean

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samples, partially because α- and β-TBECH are generally the exclusive components in

277

commercial mixtures.

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Trophic Transfer in the Marine Food Web. To assess the trophic transfer of

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HBCD and TBECH in the investigated food web, regression analysis was conducted

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between the lipid-normalized concentrations (ln-transformed) and the TLs, and TMF

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was derived from the slope of the regression line. Only species with detectable

282

contaminants were selected for this analysis. Thus, 32 species and 28 species (each

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including two cetacean species) were included in the TMF calculation for HBCD and

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TBECH, respectively. The results of the regression analysis are shown in Table 2. Note

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that TMF > 1 indicates that the contaminant is biomagnified. 12


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The increase in the ΣHBCD concentration with the TL was statistically significant,

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with a TMF of 7.9 (p < 0.0001). In addition, the concentrations of (±)-α-, (+)-α-, and

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(–)-α-HBCD were significantly positively correlated with the TL (p < 0.0001), with

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TMFs of 10.3, 8.7 and 11.8, respectively, indicating the strong preferential

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biomagnification of α-HBCD in this food web. There were no significant correlations

291

in the concentrations of (±)-β- and (±)-γ-HBCD with the TL. The trophodynamics of

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HBCD enantiomers in this food web are consistent with the observation in the eastern

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Canadian Arctic marine food web and in a freshwater food web from an electronic

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waste recycling site in South China.31,32 Compared with the reported TMFs of α-HBCD

295

(mostly 2~3) in other studies,23,25,26,28–32 our TMFs were approximately 4-fold higher

296

and comparable to the most biomagnified organic contaminants [e.g., 2,2',3,3',4,4'-

297

hexachlorobiphenyl (PCB-128) and oxychlordane, with reported average TMFs of 8.72

298

and 10.31, respectively].56 Our results indicate that α-HBCD can be substantially

299

magnified in the present food web and transferred to top predators. Although β- and γ-

300

HBCD were not trophic-magnified, the BMFs observed in this study indicate their

301

biomagnification potential. It is important to note that this is the first investigation on

302

the trophodynamics of HBCD in a subtropical marine food web.

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ΣTBECH was also found to be trophic-magnified in the investigated food web, with

304

a TMF of 2.2 (p < 0.05). Additionally, the concentrations of α-, β-, γ- and δ-TBECH

305

were significantly positively correlated with the TL (p < 0.05), with TMFs of 2.2, 1.9,

306

3.4 and 3.5, respectively, suggesting the possible preferential biomagnification of γ- and

307

δ-TBECH in this food web. However, the obtained TMF can be influenced by

308

interspecies ecological (e.g., variations in food intake) and organismal parameters such

309

as metabolism, reproductive status, migration, and age.57 This is the first study that

310

demonstrates the trophic transfer of TBECH in a food web. There are no available field 13



311

data on TMF of TBECH for further comparison. The observed TMF for ΣTBECH in

312

the present study was much lower than that of ΣHBCD, revealing the lower

313

bioaccumulation potential of TBECH in organisms at higher TL. However, the TMFs

314

of TBECH diastereomers were comparable to the abovementioned TMFs of α-HBCD

315

in other studies, which is likely due to the difference in food web configuration. It

316

should be noted that the range of TLs in our study (with apex predators included) was

317

broader than that in most of other studies, possibly resulting in a higher TMF obtained.

318

Reports on TMFs for non-PBDE and non-HBCD BFRs are scarce. Trophic

319

magnification was observed for hexabromobenzene (HBB) in a freshwater food web

320

from an electronic waste recycling site in South China, with a reported TMF of 1.76,

321

while trophic dilution (TMF < 1) through this food web was found for 1,2-bis(2,4,6-

322

tribromophenoxy)ethane (BTBPE) and pentabromotoluene (PBT).32,58 Trophic dilution

323

for BTBPE was also observed in the Lake Maggiore (Italy) food web.27 In the Lake

324

Winnipeg (Canada) food web, BTBPE concentrations were not significantly correlated

325

with the TL, while decabromodiphenyl ethane (DBDPE) was biomagnified with a

326

reported TMF of 8.6.23 The above findings indicate that several unregulated BFRs, such

327

as TBECH, HBB, and DBDPE, can have high trophic magnification potentials. The

328

magnification of TBECH diastereomers provides clear evidence of an expanding class

329

of bioaccumulative substances that urgently require assessment for their potential

330

ecological risks.

331

HBCD and TBECH Diastereomeric Profiles and Enantiomeric Patterns.

332

Figure 1 shows the diastereomeric compositions in the collected samples for all

333

examined HBCD and TBECH diastereomers. The samples were sorted into six groups:

334

technical products, sediments, mollusks, crustaceans, fishes, and cetaceans. For HBCD

335

[Figure 1 (A)], the γ-diastereomer was the predominant component in technical 14


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products, accounting for 85.3% of ΣHBCD. In sediments and crustaceans, γ-HBCD still

337

exhibited the principal abundance, but the contribution of α-HBCD in sediments

338

(19.2%) and crustaceans (32.8%) was much higher than that in technical products

339

(8.5%). In mollusks and fishes, a diastereomeric shift in the relative abundance from γ-

340

to α-HBCD was observed, where α- and γ-HBCD accounted for approximately 75%

341

and 20% of ΣHBCD, respectively. In cetaceans (apex predators), α-HBCD showed a

342

superlative preponderance (97.1%), while γ-HBCD existed only in a trace proportion

343

(1.1%), similar to the compositions of β-, δ-, and ε-HBCD. The stereoselective

344

biomagnification changed the diastereomeric compositions in these marine organisms.

345

A significant positive correlation was observed between the α-HBCD proportion and

346

the TL (Pearson’s r = 0.416, p < 0.05), while significant negative correlations were

347

found for the proportions of β- and γ-HBCD (Pearson’s r = –0.406 and –0.403,

348

respectively, p < 0.05). The δ-HBCD meso form has been found in samples of fish and

349

birds, previously reported by other studies.59–61 However, apart from our previous study

350

on marine mammals,21 to the best of our knowledge, this is the first report on the

351

presence of ε-HBCD in biota. Although δ- and ε-HBCD were widespread in our

352

collected samples, they existed at trace levels in general, and no significant correlations

353

were observed between the proportions of these two meso isomers and the TL (p > 0.05).

354

The percentage of α-HBCD to ΣHBCD increased with the TL in these marine organisms.

355

Among α-, β-, and γ-HBCD, the α-diastereomer had the highest uptake efficiency, the

356

lowest depuration rate, and the longest half-life, which could explain the prevalence of

357

α-HBCD in higher TL organisms.62–64 Other explanations may include the rapid

358

biotransformation of β- and γ- to α-HBCD and/or the preferential metabolism of β- and

359

γ- over α-HBCD.54,55,65

360

For TBECH [Figure 1 (B)], α- and β-TBECH were almost the exclusive components 15



361

in technical products, accounting for 57.3% and 42.5%, respectively, of ΣTBECH. In

362

sediments and marine organisms, except cetaceans, the composition of β-TBECH

363

remained almost unchanged (roughly 40~45%), while the proportion of α-TBECH

364

decreased by nearly 10%, and the contribution of γ- and δ-TBECH (approximately 5%

365

and 7%, respectively) was much higher than that in technical TBECH products (each

366

0.1%). In cetaceans, the composition of α-TBECH remained almost unaltered

367

compared with that in sediments and organisms at lower TLs, while a sharp decrease

368

by over 10% in the proportion of β-TBECH was observed, along with roughly doubled

369

proportions of γ- and δ-TBECH. These findings were at variance with those reported in

370

Canadian arctic beluga blubber,48 in herring gull egg pools,66 and in UK supermarket

371

fish,10 where the proportions of β-TBECH were higher than those of α-TBECH. The

372

changes observed in the diastereomeric compositions of TBECH in our marine

373

organisms might result from stereoselective biomagnification. A significant negative

374

correlation was found between the β-TBECH proportion and the TL (Pearson’s r = –

375

0.583, p < 0.01), while significant positive correlations were observed for γ- and δ-

376

TBECH (Pearson’s r = 0.571, p < 0.01 and Pearson’s r = 0.633, p < 0.001, respectively).

377

No significant correlation was found for α-TBECH (p > 0.05). Overall, the relative

378

abundance of γ- and δ-TBECH exhibited an increasing trend from technical products to

379

abiotic matrices and finally to biota samples, and the higher TMFs of γ- and δ-TBECH

380

may allow their facile transfer to top predators. A previous study suggested that β-

381

TBECH was metabolized much faster than technical TBECH mixtures by human liver

382

microsomes.67 This could partially explain the sharp decrease in the proportion of β-

383

TBECH rather than α-TBECH in the studied marine mammals.

384

A pair of enantiomers for a compound has identical physicochemical properties, and

385

the integrity of racemates is preserved (i.e., EF = 0.5) when the enantiomeric pair is 16


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subjected to achiral interactions, such as abiotic environmental processes.59 Deviation

387

from an EF of 0.5 indicates an enantiomeric enrichment from biologically-mediated

388

processes. SI Figure S4 shows the EFs in the collected samples, which were sorted into

389

the same six groups as those for the diastereomeric profiles.

390

Although the preferential enrichment of specific enantiomers of HBCD occurred in

391

some samples of technical products, sediments, mollusks, and crustaceans [SI Figure

392

S4 (A)], the average EFs for each HBCD diastereomer did not significantly deviate

393

from the EF ranges in standard solutions (p > 0.05) and were considered racemic

394

residues of HBCD enantiomers. Most of the samples of fishes and cetaceans had

395

significantly lower EFs than the range in standard solutions (p < 0.05), indicating that

396

the statistically selective enrichment of (–)-enantiomers relative to (+)-enantiomers

397

occurred for α-, β-, and γ-HBCD in these species.

398

A previous study suggested that cytochrome P450 enzymes (CYP450) played a

399

prominent role in the enrichment of (−)- over (+)-α-HBCD via in vitro metabolism

400

rather than stereoisomerization by human liver microsomes.65 If this also occurs in vivo

401

for other mammals and fishes which share certain similarities in xenobiotic-

402

metabolizing CYP450 with humans,68 the result could partially explain the preferential

403

bioaccumulation of (−)- over (+)-α-HBCD in the marine fishes and mammals examined.

404

The selective enrichment of (−)-α-HBCD could also arise from the higher

405

bioaccumulation potential of (−)- relative to (+)-α-HBCD, and the comparatively higher

406

TMF reported in this study further lends support to this idea. Additionally, a plot of the

407

EF of α-HBCD versus the TL (Figure 2) reveals a small but significant (p < 0.05)

408

decrease in the EF with the TL and suggests the enantioselective enrichment of (−)-α-

409

HBCD. Possible mechanisms for the preferential enrichment of (−)-β- and (−)-γ-HBCD

410

are still unknown, and more studies are needed. 17



411

Samples of technical products, sediments, mollusks, and crustaceans exhibited

412

racemic residues of TBECH enantiomers (p > 0.05) [SI Figure S4 (B)]. In most of the

413

fish samples, only the EFs of δ-TBECH were significantly different from (higher than)

414

the EF ranges in standard solutions (p < 0.05), indicating that the preferential

415

enrichment of the first eluting enantiomer of δ-TBECH (E1-δ-TBECH) relative to the

416

second eluting enantiomer (E2-δ-TBECH) occurred in our fish species. In cetaceans,

417

the EFs of α- and δ-TBECH were significantly higher than the EF ranges in standard

418

solutions, while the EFs of γ-TBECH were significantly lower (p < 0.05), indicating the

419

preferential enrichment of E1-α-TBECH, E2-γ-TBECH, and E1-δ-TBECH in these

420

species, whereas no significant deviations were observed for β-TBECH (p > 0.05). Two

421

trends consistent across samples are the enantioselective bioaccumulation of E1-δ-

422

TBECH in organisms at higher TLs and the virtually unchanged β-TBECH

423

enantiomeric composition. The enantioselectivity of TBECH could be potentially

424

attributed to the differences in uptake, metabolism, and excretion in biota, which

425

necessitates further investigation. For the first time, the enantioselectivity of TBECH

426

in a food web was reported.

427

Human Exposure to HBCD and TBECH Diastereomers via Seafood

428

Consumption. Dietary intake is one of the main routes for human exposure to

429

chemical pollutants, including several BFRs.4 In Hong Kong, seafood is one of the main

430

food sources for local residents. Accordingly, a preliminary estimation of the dietary

431

exposure of the Hong Kong population to HBCD and TBECH diastereomers through

432

seafood consumption was conducted based on the dataset of mollusks, crustaceans, and

433

fishes acquired from the present study. It should be noted that the EDI for children

434

might be overestimated because the consumption rate is the average for the local

435

population, and the local population may intake HBCD and TBECH through 18


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consumption of other foods or other pathways such as inhalation, dust ingestion, and

437

dermal absorption (occupational exposure is not expected to be significant in Hong

438

Kong).69

439

As shown in Table 3, the total EDIs of ΣHBCD via seafood consumption for children

440

and adults were 4.07 and 1.30 ng/kg/day, respectively. This estimated exposure for local

441

adults in our study was approximately 16-times higher than the value for the general

442

Chinese population via aquatic animal-origin food consumption (0.08 ng/kg/day),70 but

443

was slightly lower than that for the Scottish population via shellfish consumption (5.9–

444

7.9 ng/kg/day),71 while was comparable to that for the Japanese population via fish

445

consumption (1.3–3.7 ng/kg/day),72 and was much higher than that for the Dutch

446

population via seafood consumption (0.06–0.17 ng/kg/day).73

447

The human health risk from dietary exposure of HBCD can be assessed using the

448

hazard quotient (HQ) approach, which is derived by comparing the EDI with a

449

reference dose (RfD) (HQ = EDI/RfD).40 A HQ below 1 indicates little or no health

450

concern. Based on a no-observed-adverse-effect-level (NOAEL) of 450 mg/kg/day in

451

terms of increased liver weights and abnormal fatty accumulation in rats with an

452

uncertainty factor of 3000, the US National Research Council suggested an oral RfD

453

for ΣHBCD of 150,000 ng/kg/day.74 Following this method, a NOAEL of 35

454

μg/kg/week in terms of impaired lipid and glucose homeostasis in mice fed a high-fat

455

diet was used to derive the more sensitive RfD (1.67 ng/kg/day) used in this study.75

456

Consequently, the HQs for the local children and adults in this study were 2.44 and 0.78,

457

respectively, indicating the higher risk of ΣHBCD exposure to children through seafood

458

consumption. However, this preliminary assessment has certain limitations, and the

459

RfD could be refined when more toxicity data become available. The major

460

diastereomeric fraction of human intake from the present food web was α-HBCD 19



461

through the consumption of crustaceans and fishes, while mollusk consumption mainly

462

contributed to the intake of γ-HBCD. However, most toxicity data were established

463

from technical mixtures, which contain much more γ-HBCD, and very few studies have

464

reported the differentiation in toxicity between individual HBCD stereoisomers.16

465

Given that α-HBCD seemed to cause the least cytotoxicity in liver cells across

466

studies,76,77 the actual human health risk posed by ΣHBCD may be overestimated.

467

The total EDIs of ΣTBECH for children and adults via seafood consumption were

468

0.78 and 0.25 ng/kg/day, respectively, where the major diastereomeric fraction of

469

human intake was α- and β-TBECH (nearly 90%). Our results of α- and β-TBECH via

470

fish consumption for the Hong Kong residents (4.78 and 4.12 ng/day, respectively,

471

where BW was excluded for comparison) were higher than those reported for the

472

Swedish mothers (3.5 and 0.60 ng/day, respectively).78 Based on a NOAEL of 20.35

473

mg/kg/day in terms of mild renal lesions and inflammation in rats with an uncertainty

474

factor of 3,000,74,79 an oral RfD for ΣTBECH of 6,800 ng/kg/day was proposed in this

475

study. The calculated HQ of ΣTBECH was

Stereoisomer-specific Trophodynamics of the Chiral Brominated

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