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Temporal changes and stereoisomeric compositions of 1,2,5,6,9,10hexabromocyclododecane (HBCD) and 1,2-dibromo-4-(1,2-dibromoethyl) cyclohexane (TBECH) in marine mammals from the South China Sea Yuefei Ruan, James C.W. Lam, Xiaohua Zhang, and Paul K.S. Lam Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05387 • Publication Date (Web): 04 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 2018

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Environmental Science & Technology

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Temporal changes and stereoisomeric compositions of

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

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1,2-dibromo-4-(1,2-dibromoethyl) cyclohexane (TBECH)

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in marine mammals from the South China Sea

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Yuefei Ruan†, James C.W. Lam*,†,‡, Xiaohua Zhang‡, Paul K.S. Lam*,†,§

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†

(HBCD)

and

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

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

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

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‡

Hong of Kong, Hong Kong SAR, China

10 11 12

Department of Science and Environmental Studies, The Education University of

§

Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China

James C. W. Lam*

Paul K. S. Lam*

Department of Science and

State Key Laboratory in Marine

Environmental Studies, The Education

Pollution, City University of Hong

University of Hong Kong, Hong Kong

Kong, Tat Chee Avenue, Kowloon,

SAR, China

Hong 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]

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1

ACS Paragon Plus Environment


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ABSTRACT

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Stereoisomeric compositions of 1,2,5,6,9,10-hexabromocyclododecane (HBCD) and

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1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) were investigated in the

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blubber of two species of marine mammals, finless porpoises (Neophocaena

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phocaenoides) and Indo-Pacific humpback dolphins (Sousa chinensis), from the South

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China Sea between 2005 and 2015. The concentrations of ΣHBCD in samples of

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porpoise (n = 59) and dolphin (n = 32) ranged from 97.2 to 6,260 ng/g lipid weight

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(lw) and from 447 to 45,800 ng/g lw, respectively, while those of ΣTBECH were both

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roughly two orders of magnitude lower. A significant increasing trend of ΣHBCD was

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found in dolphin blubber over the past decade. The diastereomeric profiles exhibited

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an absolute predominance of α-HBCD (mostly > 90%), while the proportions of four

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TBECH diastereomers in the samples appeared similar. A preferential enrichment of

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the (−)-enantiomers of α-, β-, and γ-HBCD was found in most blubber samples.

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Interestingly, the body lengths of porpoises showed a significant negative correlation

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with the enantiomer fractions of α-HBCD. Significant racemic deviations were also

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observed for α-, γ-, and δ-TBECH enantiomeric pairs. This is the first report of the

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presence of TBECH enantiomers in the environment. The estimated hazard quotient

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indicates that there is a potential risk to dolphins due to HBCD exposure.

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INTRODUCTION

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Approximately one third of organic flame retardants on the market are categorized as

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brominated flame retardants (BFRs).1 One of the most extensively used BFRs in

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consumer products — polybrominated diphenyl ethers (PBDEs) — have been

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identified as global pollutants, and technical mixtures containing these compounds

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have been restricted worldwide.2,3 Thus, there has been a shift to the use of

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alternatives. Two types of alternatives, namely, 1,2,5,6,9,10-hexabromocyclododecane

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(HBCD) and 1,2-dibromo-4-(1,2-dibromoethyl) cyclohexane (TBECH), have been

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regarded as possible replacements for PBDEs.4 Both of these compounds have similar

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applications in expanded and extruded polystyrene.5–7 Nevertheless, HBCD has been

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ubiquitously found in the environment, with very high concentrations (up to ppm

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levels) frequently observed in marine mammals and seabirds.8,9 Since 2013, HBCD

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has been listed under the Stockholm Convention on Persistent Organic Pollutants

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(POPs) under Annex A for global elimination;10 however, its use is still permitted, and

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it has a global annual production of more than 30,000 metric tons.11 TBECH has been

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produced since 1970s, but information on its production is very limited and was,

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formerly reported between 4.5 and 226 metric tons/year in the USA.12,13 Frequently

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detected in environmental matrices, TBECH has been an emerging concern due to its

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toxicity as the first potent environmental androgen receptor (AR) agonist, which was

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found to be as potent as dihydrotestosterone in human AR activation.14 As indicated

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by studies on indoor dust from European regions, TBECH could be a possible HBCD

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replacement in the investigated area.15–17

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These two BFRs can exhibit optical activities as chiral compounds. HBCD can

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generate up to 6 pairs of enantiomers and 4 meso-forms, while TBECH can yield up

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to 4 pairs of enantiomers.18–20 Technical products of HBCD (tHBCD) usually consist 3



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of three diastereomeric pairs of enantiomers, α- (~12%), β- (~9%), and γ- (~79%)

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racemates, with trace amounts of others such as δ- and ε-meso-isomers (~0.5%).21–24

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Only one manufacturer (Albemarle Corporation. Charlotte, NC, USA) reported the

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commercial formulation of TBECH (tTBECH), and it contains equimolar amounts of

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α- and β-racemates with trace amounts of γ- and δ-diastereomers generated in a

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high-temperature operation.20 With respect to the environmental relevance of their

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stereoisomerism, a shift toward enrichment of α- over β- and γ-HBCD along trophic

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levels has consistently occurred in food webs.25 Few studies have confirmed the

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presence of HBCD meso-isomers in the environment.26 The prevalence of the

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preferential bioaccumulation of (−)-α-HBCD can be observed more often in certain

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animal species, but this seems to be independent of other biological factors such as

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body composition, age/sex variation, nutritive condition, and tissue distribution.8,25,27

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There is still very limited knowledge of the fate of these chiral contaminants in the

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environment, particularly TBECH, which has been primarily reported as the sum of

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all stereoisomers (ΣTBECH).28,29 Information about the environmental occurrence of

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TBECH enantiomers has not yet been reported.

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Although stereoisomers can exhibit different environmental fates and toxicities, the

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stereoisomerism of environmental contaminants has not yet received sufficient

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attention.30 The understanding of the toxicities of individual HBCD or TBECH

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stereoisomers is far from adequate. In general, in vitro studies have found that all

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(+)-HBCD appeared more toxic than their corresponding (−) forms, while γ- and

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δ-TBECH were more potent in AR agonism than α- and β-TBECH.14,31,32 In vivo

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studies on juvenile rainbow trout (Oncorhynchus mykiss) and adult zebrafish (Danio

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rerio) have evidenced a stronger potency of γ-HBCD,33,34 while in fertilized eggs of D.

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rerio, a more pronounced embryo toxicity of γ/δ-TBECH than α/β-TBECH was

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observed.35

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Marine mammals, particularly those from coastal regions, have been suggested to

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serve as important sentinel species for tracking spatio-temporal dynamics of

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environmental contamination.36 Several BFRs have been linked with detrimental

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health effects in marine mammals, such as the decrease in offspring survivorship.37

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According to our previous studies, two resident marine mammal species in the South

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China Sea, finless porpoises (Neophocaena phocaenoides) and Indo-Pacific

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humpback dolphins (Sousa chinensis), were susceptible to exposure to high levels of

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PBDEs and other BFRs.38 However, the stereoisomerism of these chemicals has not

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been considered, especially regarding their environmental fate and potential risks to

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these cetaceans.

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To better understand the current status of the exposure and the risks posed by the

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chiral BFRs, the present study aimed to (1) identify and quantify HBCD and TBECH

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stereoisomers using novel methods for the investigation of the possible temporal

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trends (2005‒2015) and physiology-related differences in the accumulation of these

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contaminants in marine mammals from the South China Sea and (2) study the changes

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in the stereoisomeric compositions of the chiral BFRs in marine mammals and

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evaluate their potential risks to the cetaceans. To our knowledge, this is the first study

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that demonstrates the environmental occurrence of TBECH as well as HBCD

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meso-isomers in marine mammals in the Asian region, as well as the first report on the

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identification of ε-HBCD in organisms and the detection of TBECH enantiomers in

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the environment.

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MATERIALS AND METHODS

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Materials and Sample Collection. The standards used in this study were

5



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purchased from Wellington Laboratories (Guelph, Ontario, Canada). The details of

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standards, solvents, and reagents are described in the Supporting Information (SI).

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The blubber samples of finless porpoises (n = 59) and Indo-Pacific humpback

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dolphins (n = 32) were obtained from stranded cetaceans collected by the Agriculture,

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Fisheries and Conservation Department in Hong Kong, China, between 2005 and

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2015 (SI Table S1). Once in the laboratory, the samples were freeze-dried and stored

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at −50 ºC until further processing.

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Extraction and Cleanup. Analyses of blubber samples were accomplished by

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using previously established methods with modifications.38 Briefly, ~1 g of blubber

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sample was ground with anhydrous sodium sulfate and spiked with surrogates of 10

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ng each, and then the sample was extracted via pressurized fluid extraction. Each

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extract was purified for lipid removal by gel permeation chromatography and then

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further purified by elution through anhydrous sodium sulfate, activated aluminum

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oxide, and activated silica gel. The final reduced extract was spiked with internal

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standards of 10 ng each before it was concentrated to dryness under a gentle nitrogen

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stream and, finally, reconstituted to 100 µL in methanol or n-hexane.

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Instrumental Analysis. Determination of HBCD stereoisomers was performed

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using liquid chromatography‒tandem mass spectrometry (LC–MS/MS) consisting of

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an Agilent (Palo Alto, CA, USA) 1290 Infinity LC system coupled to an AB SCIEX

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(Woodlands, Singapore) QTRAP®3200 system with an electrospray ionization (ESI)

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interface in negative multiple reaction monitoring (MRM) mode. An Agilent

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(Lawrence, Kansas, USA) ZORBAX Eclipse Plus C18 column (4.6 mm i.d. × 100

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mm, 3.5 µm) coupled to a Macherey-Nagel (Düren, Germany) NUCLEODEX β-PM

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column (4 mm i.d. × 200 mm, 5 µm) was applied for chromatographic separation.

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More details about LC–MS/MS determination can be found in the SI. 6


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Determination of TBECH enantiomers was performed using gas chromatography–

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mass spectrometry (GC–MS) with an Agilent (Shanghai, China) 7890A GC system

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coupled to an Agilent (Santa Clara, CA, USA) 5975C inert XL MSD with a triple axis

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detector in negative chemical ionization mode. A Supelco (Bellefonte, PA, USA)

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CHIRALDEX B-TA capillary column (30 m × 0.25 µm i.d., 0.12 µm film thickness)

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was used for chromatographic separation. The GC oven program is listed in SI Table

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S2. More details about GC–MS determination can be found in the SI.

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Quality Assurance and Quality Control (QA/QC). The QA/QC was

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composed of procedure blanks and reference samples per batch of 10 samples. A

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9-point standard calibration curve, with concentrations ranging from 5 to 2,000 ng/mL,

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was used for the quantification of each stereoisomeric analyte. The regression

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coefficients (r) were ≥ 0.995 for all calibration curves. Concentrations of analytes

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were expressed as ng/g lipid weight (lw). Recoveries of spiked standards in the

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reference samples, including eel muscles and corn oil, can be found in the SI.

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Recoveries of spiked surrogates in the cetacean blubber were all in the range of 71%

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to 97%. Recoveries of each native standard and surrogate are listed in SI Table S3.

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The limit of detection (LOD) was defined as an instrument signal-to-noise ratio (SNR)

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of ≥ 3:1. The method LODs of each HBCD and TBECH stereoisomer are listed in SI

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Table S4.

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Statistical Analysis. Statistical analysis was performed using Microsoft Excel

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2016 to generate descriptive statistics, with all other statistical procedures conducted

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using IBM SPSS Statistics 20.0 and OriginLab OriginPro 2015. For the calculation of

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geometric mean and arithmetic mean, it was confirmed that different data treatments,

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i.e. excluding values below the LOD, using ½ the LOD instead, or setting those

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values to zero, had almost no effect on the conclusion and interpretation of the results. 7



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Thus, values below the LOD were replaced with ½ LOD, in the same way as our

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previous studies.38,39 Methods used for statistical hypothesis and correlation analysis

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are described in the SI. The detection frequencies of HBCD and TBECH

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stereoisomers in the samples are listed in SI Table S5. Enantiomer fractions (EFs)

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were used to describe enantiomeric patterns,40 and the details of EF calculation can be

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found in the SI.

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RESULTS AND DISCUSSION

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Levels of ΣHBCD and ΣTBECH in the Cetacean Blubber. ΣHBCD was

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found in all blubber samples, whereas the detection frequencies of ΣTBECH (65.9%)

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were much lower than those of ΣHBCD (SI Table S5). The concentrations of ΣHBCD

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in the samples of porpoise and dolphin ranged from 97.2 to 6,260 ng/g lipid weight

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(lw) and from 447 to 45,800 ng/g lw, respectively, while those of ΣTBECH ranged

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from below the limit of detection (LOD) (< 0.8) to 125 ng/g lw and from < 0.8 to 362

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ng/g lw, respectively (Table 1). Concentrations of both ΣHBCD and ΣTBECH in

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porpoises were much lower than those in dolphins (p < 0.001), probably partially due

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to the difference in the spatial distributions between these two species — dolphins

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exploit prey species common in the estuary, while porpoises rely more on pelagic

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habitats for food.41,42 As indicated by our previous studies, compared to the habitat of

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porpoises, the habitat of dolphins is closer to the coastal regions of the Pearl River

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Estuary, where the aquatic environment is more contaminated by different brominated

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flame retardants.38,39 However, species-specific differences in contaminant uptake

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from diets, accumulation, and metabolism should not be neglected and these deserve

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for further investigation. Concentrations of ΣTBECH were roughly two orders of

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magnitude lower than those of ΣHBCD in both porpoises and dolphins, in accordance

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with previous findings of a lower bioaccumulation potential of TBECH than HBCD in

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zebrafish (Danio rerio) after dietary exposure.43 This also suggests the much higher

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production/use of tHBCD than tTBECH in the Pearl River Delta (PRD). Positive

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correlations between the levels of ΣHBCD and ΣTBECH were observed in the two

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species of marine mammals (Pearson’s r = 0.500, p < 0.0001 and Pearson’s r = 0.680,

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p < 0.0001 for porpoises and dolphins, respectively), indicating that there may have

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been a similar production/use pattern between tHBCD and tTBECH, a potential

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HBCD replacement, in the PRD over the past decade. Notably, though HBCD has

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been listed under the Stockholm Convention on Persistent Organic Pollutants (POPs)

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for global elimination since 2013, the control on the production and use of HBCD in

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China has not come into force until December 26, 2016 (SI Figure S3). In addition,

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TBECH can be used as a substitute for HBCD, the regional market demand for

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TBECH has kept increasing. Thus, it is not surprising that positive correlations

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between the levels of ΣHBCD and ΣTBECH were observed.

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The sex, life history and biological factors of marine mammals have been reported

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to affect the accumulation pattern of some legacy organochlorines and PBDEs, where

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the contaminant burden in adult males and juveniles has been generally higher than

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that in adult females

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placental transfer and/or lactation and metabolism capacity with age and/or dilution

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with growth. In the present study, the effects of the marine mammal’s sex and age on

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contaminant levels were also examined (Table 1). The levels of both ΣHBCD and

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ΣTBECH in porpoises varied greatly with developmental stages. Significantly higher

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levels of ΣHBCD were observed in adult males and adult females than in juveniles (p

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< 0.01), and significantly higher levels of ΣTBECH were observed in adult males than

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in adult females and juveniles (p < 0.05). Compared to age classes, sexual differences

44–47

. The main reasons for this finding are suggested to be

9



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appear to have little influence on ΣHBCD accumulation in porpoises. Compared with

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adult females, adult males had higher mean concentrations, but this difference was not

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significant (p > 0.05). The accumulation pattern in porpoises indicates a combined

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consequence of maternal offloading and increased bioaccumulation capacity with age.

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In dolphins, however, no significant difference in the levels of either ΣHBCD or

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ΣTBECH was found among juveniles, adult males, and adult females (p > 0.05), and

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similar finding of POPs was also reported in cetaceans from Korean waters.47 The

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reason for this finding is not entirely clear, but the finding suggests that HBCD loss

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through maternal transfer may be lower in dolphins. However, the compensation via

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continuous bioaccumulation long after reproduction for the loss of HBCD through

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maternal transfer should not be neglected.

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Additionally, the current status of HBCD and TBECH was examined on a global

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scale. The highest ΣHBCD concentrations detected in marine cetaceans collected

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from different regions are summarized in SI Table S6. The highest concentration in

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marine cetaceans reported to date was observed in blubber samples of dolphin

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investigated by the present study, suggesting a heavier use of HBCD and a greater

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contamination burden in the PRD than in other regions. Likewise, because TBECH is

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a novel BFR and due to its limited records in environmental matrices, the biological

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tissues of concern were extended to those of any types of organisms ever reported (SI

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Table S7), and the highest ΣTBECH concentration observed to date was also found in

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dolphin blubber from the PRD. However, the environmental fate of TBECH is still

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poorly understood and further study is needed.

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Temporal Trends of ΣHBCD and ΣTBECH in Porpoises and Dolphins.

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Time-series analysis was conducted on the present data set of blubber samples, and

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the concentrations were used to perform a log-linear regression. To attenuate the 10


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differences between the sexes caused by maternal transfer and possible age-related

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dissimilarities, adult males with body lengths greater than 120 and 200 cm for

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porpoises and dolphins, respectively, were selected for this temporal-trend analysis

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(SI Figure S1). Individuals with body lengths smaller than 120 and 200 cm were

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categorized as juveniles for porpoises and dolphins, respectively.38,39 Consequently,

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the dataset of adult males shows a clear upward trend between 2005 and 2015.

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Specifically, the concentrations of ΣHBCD (p < 0.05) and ΣTBECH (p < 0.01) were

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both found to exhibit a significant increasing trend in dolphin blubber. Although the

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size of the dataset was limited, the log-linear regression slope of ΣTBECH in adult

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male dolphins (Pearson’s r = 0.745) was higher than that of ΣHBCD (Pearson’s r =

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0.816), suggesting a possible rapid growth in tTBECH consumption in the PRD over

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the past decade.

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The ΣHBCD concentrations are not statistically different between the juveniles and

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adult dolphins and between males and females; thus, pooling of more individual

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samples is possible. The complete dataset (SI Figure S2) shows that the

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concentrations of ΣHBCD exhibited a significant increasing trend in dolphin blubber

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between 2005 and 2015 (Pearson’s r = 0.567, p < 0.001), with an estimated annual

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rate of 8%; an increasing trend in ΣHBCD levels was observed in porpoise blubber,

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but this trend was not significant (p > 0.05). In comparison, the concentrations of

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ΣTBECH appeared to remain steady in the blubber of both porpoise and dolphin

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between 2005 and 2015, which possibly reflected a relatively low exposure of these

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animals to TBECH within this period. However, the levels were higher in recent years

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(as indicated by the log-linear regression slope), which may suggest the increasing

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usage of tTBECH.

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Based on one of our previous studies on PBDEs and several PBDE alternatives (but 11



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excluding HBCD and TBECH) in blubber samples of the same mammal species

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collected between 2003 and 2012, we found a similar contamination level between

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PBDEs and ΣHBCD (within the same order of magnitude) over the past decades.38

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Note that the large-scale production and uses of tHBCD in China began around 2000

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and proliferated after 2005.11 However, HBCD has been banned for production, use,

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import and export in China since December 26, 2016 with a five-year exemption for

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the use in EPS/XPS foam in building insulation; after that period, its use is planned to

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cease by 2021. Between 2005 and 2015, the production of tHBCD in China over time

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increased dramatically (SI Figure S3). It is interesting to observe that the production

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of tHBCD in China had an increasing trend that was similar to the ΣHBCD

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concentrations found in dolphin blubber. In this case, dolphins may act as a potent

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biological indicator to reflect the regional contamination status and trends.

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Diastereomer Distribution Patterns in the Cetacean Blubber. The

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stereoisomerism of HBCD has recently gained much interest. Because TBECH has

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been indicated as a potential replacement for HBCD in expanded polystyrene for

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building insulation, it is possible that this contaminant will be of global concern in the

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future.4,16 TBECH also contains stereoisomers. However, knowledge on the

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stereoisomerism of TBECH in the environment is still very limited. In this study, α-,

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β-, γ-, δ-, and ε-HBCD, as well as α-, β-, γ-, and δ-TBECH, were considered for

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diastereomeric identification.

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The α-diastereomer of HBCD was detected in all blubber samples, accounting for

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95.3% and 98.2% (mean values) of ΣHBCD in porpoises and dolphins, respectively

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(Figure 1A). The results shown in SI Figure S4 further demonstrated that α-HBCD

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dominated the HBCD profiles in marine mammals among the studied physiological

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traits. For β-, γ-, δ-, and ε-HBCD, in general, their detection frequencies in porpoise 12


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blubber were lower than those in dolphin blubber (SI Table S5), while the

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ε-meso-isomer, on average, possessed the least proportions in both marine mammal

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species.

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In porpoise blubber, significantly higher proportions of δ-HBCD were found in

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adult males than in adult females (p < 0.01), but when juveniles and adults were

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pooled, the HBCD diastereomeric compositions did not show any sexual differences

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(p > 0.05) (SI Figure S4A); in comparison, in terms of developmental stage, the

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proportions of α-HBCD in juveniles were significantly lower than those in adults (p
0.05). However, the ratios of α-HBCD to

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γ-HBCD were much higher in dolphins than in porpoises (p < 0.01), suggesting a

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possibly higher bioaccumulation potential of α-HBCD in dolphin blubber.

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For TBECH diastereomeric compositions, α-TBECH had the highest mean values,

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accounting for 35.3% and 35.8% of ΣTBECH in porpoises and dolphins, respectively;

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δ-TBECH exhibited the lowest mean values, accounting for 15.5% and 17.3% of

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ΣTBECH in porpoises and dolphins, respectively (Figure 1B). TBECH diastereomeric

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compositions in blubber showed certain species differences between porpoises and

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dolphins. The proportions of β-TBECH in porpoise blubber were significantly higher

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than those in dolphin blubber (p < 0.05). Although the difference was not significant,

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γ-TBECH in porpoises had a lower mean value than in dolphins (p > 0.05). Hence, a

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higher potential for the preferential bioaccumulation of γ-TBECH and/or the

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biotransformation from β-TBECH may have occurred in dolphins compared to 13



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porpoises. For both marine mammal species, their sex and life history did not appear

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to affect the compositions of TBECH diastereomers (p > 0.05).

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Diastereomer Proportions of HBCD and TBECH across Different

309

Studies. In the present study, the diastereomeric compositions of a specific tHBCD

310

were ascertained in a product of technical mixtures purchased from Toronto Research

311

Chemicals (Toronto, Ontario, Canada), and the mixtures were composed of 8.47%,

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5.69%, 85.3%, 0.28%, and 0.22% of α-, β-, γ-, δ-, and ε-HBCD, respectively, similar

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to the HBCD commercial/technical formulations previously reported. Dozens of

314

studies published elsewhere have demonstrated that in abiotic matrices, γ-HBCD was

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the prevailing diastereomer, which was called a technical mixture-like diastereomeric

316

pattern, whereas in biotic matrices, especially for organisms at higher trophic levels

317

such as the apex predators, α-HBCD usually had an absolute predominance, or was

318

even the exclusive diastereomer detected. Studies indicated that this pattern possibly

319

resulted from diastereoselective uptake and/or metabolism.8,25 However, there are

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very few records on δ- and ε-meso isomers in environmental matrices, which is

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probably not attributed to their absence in the environment, but rather a result of the

322

very limited number of studies addressing this issue.8 In our study, the diastereomeric

323

patterns of α-, β-, and γ-HBCD in blubber of porpoise and dolphin coordinated well

324

with the diastereomer-specific enrichment pattern referred to above, and, δ- and

325

ε-HBCD found in our samples may reflect a combined consequence of

326

bioaccumulation and biotransformation (including bioisomerization).

327

For TBECH, specifically, a product of technical mixtures was also purchased from

328

Toronto Research Chemicals for diastereomeric identification. The proportion of

329

α-TBECH was ~15% higher than that of β-TBECH, and trace amounts of γ- and

330

δ-TBECH were detected with the resultant proportions of 0.13% and 0.12%, 14


Page 14 of 37

Page 15 of 37


331

respectively. This finding is different from the diastereomeric profiles of tTBECH

332

reported in other studies, with equimolar amounts of α- and β-racemates consistently

333

quoted from Arsenault et al. (2008).20 To date, complete TBECH diastereomeric

334

profiles in the environment have not been reported. Interestingly, in this study,

335

compared with the diastereomeric composition in TBECH technical mixtures, a

336

preferential enrichment of γ- and δ-TBECH, rather than α- and β-TBECH, could be

337

clearly observed in the blubber of the two marine mammal species. To our knowledge,

338

this was the first study to report a diastereoselective bioaccumulation of TBECH

339

stereoisomers in the environment.

340

Enantiomer Fractions (EFs) of HBCD in the Cetacean Blubber. The

341

elution order of (+)- and (−)-enantiomers of α-, β-, and γ-HBCD has been determined

342

in previous studies using the same NUCLEODEX chiral column as that used in the

343

present study.48,49 TBECH also contains enantiomeric pairs, and the enantioselective

344

degradation of α-TBECH has been examined as an indication of microbial processing

345

by Wong et al. (2012), who reported a shift in degradation preference from the second

346

elution enantiomer (on the BGB-176MS column) to its antipode.50 However, the

347

chromatographic elution order of any TBECH enantiomeric pair as (+)- and

348

(−)-enantiomers has not been confirmed. Here, as previously mentioned, the

349

concentration ratio of the first-eluting enantiomer (E1) on the CHIRALDEX B-TA

350

column and the total concentration of both enantiomers (E1 + E2) was used for

351

calculation. The resulting EFs of each pair of enantiomers in the blubber of porpoise

352

and dolphin are shown in Figure 2.

353

All blubber samples showed that the EFs of α-HBCD enantiomers negatively

354

deviated from the racemic standards with statistical significance (p < 0.01) (Figure

355

2A), with averages of 0.371 ± 0.044 and 0.387 ± 0.034 in porpoise blubber and 15



356

dolphin blubber, respectively, which evidenced a strongly preferential enrichment of

357

(−)-α-HBCD. The mean EFs of β- and γ-HBCD were also far below 0.5, with

358

averages of 0.418 ± 0.137 and 0.419 ± 0.063, respectively in porpoise blubber, and

359

0.357 ± 0.136 and 0.282 ± 0.107, respectively in dolphin blubber (p < 0.05),

360

suggesting a predominance of all (−)-HBCD enantiomers (in terms of α-, β-, and

361

γ-HBCD) in these marine mammals.

362

Physiological traits, including development stages and sexual differences, affected

363

the enantioselective pattern of α-HBCD and γ-HBCD in blubber (SI Figure S5). For

364

α-HBCD, a similar pattern of enantiomer-specific accumulation was observed within

365

the same categories of physiological traits in both marine mammal species.

366

Significant differences in the EFs of α-HBCD were found between adult males and

367

adult females and between juvenile males and adult males (p < 0.05), where the

368

lowest values of the EFs of α-HBCD occurred in adult males. For γ-HBCD, another

369

pair of enantiomers, the lowest mean EFs occurred in the dolphin blubber of juvenile

370

males. In accordance with the extremely high levels of α-HBCD found in blubber

371

(1,420 ± 1,320 ng/g lw and 7,580 ± 9,340 ng/g lw for porpoises and dolphins,

372

respectively), a possible explanation for the specific pattern observed above is the

373

maternal transfer of (−)-α-HBCD from mature females to their calves via gestation

374

and lactation, resulting in higher amounts of (−)-α-HBCD in adult males. The

375

stereoisomer-specific maternal transfer of persistent hydrophobic contaminants has

376

also been reported by several studies.51,52

377

Correlations between Enantiomer Fractions (EFs) of HBCD and Body

378

Lengths of Cetaceans. Studies have reported that a decreasing EF trend with

379

increasing age and/or animal body length was found for α-HBCD in the blubber of

380

Atlantic white-sided dolphins (Lagenorhynchus acutus) from the eastern coast of the 16


Page 16 of 37

Page 17 of 37


381

United States,51 for α-HCH in the blubber of ringed seals (Phoca hispida) from the

382

Northwest Polynya,53 and for PCB-95 in the blubber of bowhead whales (Balaena

383

mysticetus) from the northern coast of Alaska.54 For both porpoises and dolphins,

384

there was no difference in the EFs of α-HBCD among juvenile males, juvenile

385

females and adult females (p > 0.05); therefore, pooled samples of males and females

386

are possible for the assessment of the potential interaction between enantiomeric

387

distribution and body length as a surrogate for growth (SI Figure S6A). As a result,

388

the body length of porpoises (p < 0.0001), rather than that of dolphins (p > 0.05), was

389

significantly inversely correlated (Pearson’s r = ‒0.575) with the EFs of α-HBCD in

390

blubber. In other words, a steady excess of (‒)-α-HBCD in blubber was found as the

391

porpoise body length increased, suggesting that enantiomer-specific accumulation of

392

this enantiomer in porpoises gradually occurs over time.

393

Several studies have evidenced the dietary changes during early life stages of

394

certain marine mammals and the subsequent changes in the accumulation patterns of

395

persistent hydrophobic contaminants.45,55–57 With respect to the porpoises’ sex, the

396

correlation between the EFs of α-HBCD in blubber and body length was further

397

examined by using first-order linear regression, and interestingly, the EFs of α-HBCD

398

were not correlated with the body length of female porpoises (p > 0.05) but were

399

highly correlated with that of male porpoises [Pearson’s r = ‒0.706, R2 (linear) =

400

0.485, p < 0.0001]. This finding suggests that the EFs of α-HBCD may remain

401

constant in these female marine mammals during their life spans, which is probably a

402

cumulative consequence of a gradual dietary change from maternal dependency

403

before weaning to independent foraging for small prey in juveniles as they grow. In

404

the present study, because no correlation was found between the concentrations of

405

α-HBCD and animal body lengths in either porpoises or dolphins (p > 0.05), the

17



406

stereoselective metabolism of α-HBCD was not likely to account for the significant

407

decline in EFs as the porpoise body length increased, which further supports the

408

above-mentioned implications of the maternal transfer of (−)-α-HBCD from mature

409

females, possibly accompanied by the gradual change in diet from milk to prey in

410

these juvenile marine mammals.

411

For γ-HBCD, considering that for both porpoises and dolphins, there was no

412

difference in the EFs of γ-HBCD among juvenile males, juvenile females and adult

413

females (p > 0.05), pooled samples of males and females are also possible for

414

evaluating the potential interaction between enantiomeric distribution and body length

415

(SI Figure S6B). In contrast to the decreasing trend of the EFs of α-HBCD with

416

increasing body length, the body length of dolphins (p < 0.05), rather than that of

417

porpoises (p > 0.05), was significantly positively correlated (Pearson’s r = ‒0.575)

418

with the EFs of γ-HBCD in blubber. Thus, a continuous preferential elimination of

419

(‒)-γ-HBCD in blubber was found as the dolphin body length increased, implying that

420

enantiomer-specific depuration of this enantiomer by dolphins occurs over time. With

421

respect to the sex of the dolphin, the correlation between the EFs of γ-HBCD in

422

blubber and the body length was further examined by using first-order linear

423

regression, and identical to the case of α-HBCD in porpoises, the EFs of γ-HBCD

424

were not correlated with the body length of female dolphins (p > 0.05), but were

425

highly correlated with that of male dolphins [Pearson’s r = 0.798, R2 (linear) = 0.604,

426

p < 0.01]. This result indicates that the EFs of γ-HBCD may remain constant in

427

female dolphins during their life spans. Sex-specific enantioselective accumulation

428

and/or depuration of either γ-HBCD enantiomer in dolphins may be a possible

429

explanation for this phenomenon.

430

Enantiomer Fractions (EFs) of TBECH in the Cetacean Blubber. For 18


Page 18 of 37

Page 19 of 37


431

TBECH enantiomers, in the present study, almost all blubber samples showed that the

432

EFs of α-TBECH positively deviated from the racemic standards with statistical

433

significance (p < 0.05), with averages of 0.587 ± 0.073 and 0.569 ± 0.053 in porpoises

434

and dolphins, respectively (Figure 2B). This finding indicates that a preferential

435

accumulation of E1-α-TBECH occurred in the blubber of these marine mammals.

436

Interestingly, for dolphins, although no difference was found in the EFs of α-TBECH

437

among juveniles, adult males and adult females (p > 0.05), the EFs of α-TBECH in

438

adult males (indicated by the mean and median values) appeared to have the greatest

439

deviation from the racemic standards (SI Figure S7). Therefore, similar to the case of

440

α-HBCD, the maternal transfer of E2-α-TBECH from mature females to their

441

offspring was likely to occur in dolphins.

442

In comparison, no enantioselective accumulation of β-TBECH was observed in

443

either porpoises or dolphins (p < 0.05). Note that EFs could be calculated only for

444

samples with the concentration of a specific TBECH diastereoisomer above the

445

method LOD. In this study, the detection frequencies of the γ- and δ-enantiomer pairs

446

in dolphins were both too low (~30-50%) to compare their EFs among different

447

physiological traits with sufficient precision. However, a preferential enrichment of

448

the E2-γ-enantiomer and the E1-δ-enantiomer seemed to be consistent for both

449

porpoises and dolphins (Figure 2B). Additionally, in porpoises, significantly higher

450

proportions of E1-δ-TBECH were observed in females than in males (p < 0.05). To

451

date, the toxicological significance of the enantioselective bioaccumulation of

452

TBECH stereoisomers is unknown, which warrants urgent investigation.

453

Preliminary Evaluation of the Hazards of Chiral BFRs to Marine

454

Mammals. In this screening assessment, hazard quotients (HQs) were determined

455

based on best-case and worst-case scenarios (HQbcs and HQwcs, respectively) to 19



456

provide a preliminary quantitative hazard evaluation of ΣHBCD and ΣTBECH to

457

porpoises and dolphins. HQs were calculated by dividing the measured environmental

458

concentration (MEC) [ng/g wet weight (ww)] by the lowest levels of estimated “body

459

burden” at which critical harmful effects were observed in mammalian toxicological

460

studies (hereafter referred to as tentative critical concentration, TCC).

461

The no-observed-adverse-effect levels (NOAELs) are available for several

462

mammals (rodents) with the investigated contaminants.58,59 In the absence of

463

dolphin/porpoise-speciﬁc toxicity data, by applying a body weight scaling factor

464

following the method of Lam & Lam (2015),60 these NOAEL values were converted

465

into TCC, which were used in the present risk assessment. In addition, an uncertainty

466

factor of 10 was applied to the calculation to account for the cross-species

467

extrapolation involved in the assessment process, and the whole-body concentration

468

was estimated to be 1/5 the blubber concentration.60–62 Because the extrapolation

469

between species and tissue types involves many uncertainties, the results of this

470

assessment will require further refinement when more data becomes available. The

471

corresponding HQs of ΣHBCD and ΣTBECH for porpoises and dolphins in this study

472

are summarized in SI Table S8. It is important to note that the hazard of HBCD to

473

finless porpoises and TBECH to aquatic organisms was evaluated for the first time.

474

The results of the assessment for ΣHBCD revealed that the HQbcs values for the

475

marine mammals were below unity, while the HQwcs values for both porpoises and

476

dolphins exceeded unity, suggesting that the whole-body concentrations of ΣHBCD

477

pose potential risks to these marine mammals. The HQs of one dolphin individual

478

exceeded 1, suggesting a moderate hazard to 3.13% of the investigated population of

479

dolphins from HBCD exposure, while not any porpoise individual had a HQ value

480

greater than 1, indicating that porpoises are much less threatened by exposure to 20


Page 20 of 37

Page 21 of 37


481

HBCD. Nearly 14% and 47% of the investigated populations of porpoises and

482

dolphins, respectively, were at risk (or had a low to moderate hazard) due to HBCD

483

exposure. HBCD may have induced potential detrimental effects from mild impaired

484

oligodendroglial development at the adult stage by long-term maternal exposure in

485

porpoises and dolphins in this studied region. In contrast, none of the HQs of

486

ΣTBECH detected exceeded a value of 0.01, thus posing no hazard (in terms of mild

487

renal lesions and inflammation) to porpoises and dolphins. Nonetheless, a study on

488

TBECH should be ongoing, as this chemical is a possible replacement for HBCD.

489

HBCD stereoisomers have been demonstrated to exhibit different biological

490

activities, thus resulting in different toxic effects on organisms. The stereoisomers

491

may interact additively, synergistically or antagonistically, even within the same

492

toxicity endpoint. Accordingly, the TCCs of tHBCD used in the preliminary hazard

493

evaluation may overestimate or underestimate the actual risks posed by ΣHBCD (a

494

mixture of varying proportions of HBCD stereoisomers) for porpoises and dolphins.

495

Different cytotoxicities for the three pairs of HBCD enantiomers were demonstrated

496

on human HepG2 cells (SI Table S9),32 and a set of stereoisomer-based threshold

497

values were derived based on the difference in toxic potency among the enantiomers

498

and were used for comparison with the MECmax values of individual HBCD

499

enantiomers in blubber.

500

Among the MECmax values of six HBCD enantiomers in porpoise blubber (SI Table

501

S10), only the MECmax value of (+)-α-HBCD (1,030 ng/g ww) exceeded the

502

significant toxic level of the identical enantiomer in terms of the increase of LDH

503

leakage. Similar to the result of porpoises, only the MECmax value of (+)-α-HBCD in

504

dolphin blubber (3,330 ng/g ww) exceeded the significant toxic level. Nevertheless,

505

the MECmax value of (−)-α-HBCD in dolphin blubber (4,750 ng/g ww) was very close 21



506

to the significant level of the identical enantiomer. As suggested by Zhang et al.

507

(2008), the γ-diastereomer was likely to exert the most significant toxicological

508

effects.32 Based on the above findings, the actual risks posed by ΣHBCD may be

509

overestimated, as indicated by using the HQ approach in the present study.

510

Likewise, the threshold values of the cytotoxicity for TBECH diastereomers to

511

human HepG2 cells (SI Table S11) could be used for comparison with the MECmax

512

values of individual TBECH diastereomers in blubber. The potency of TBECH

513

diastereomers in the activation of the human androgen receptor was indicated by

514

Khalaf et al. (2009) to follow the order of γ-, δ-, α-, and β-TBECH.14 Among the

515

MECmax values of four TBECH diastereomers in cetaceans (SI Table S10), the

516

MECmax value of γ-TBECH in porpoise blubber exceeded the EC50 value of 50:50

517

γ/δ-TBECH, while the MECmax values of γ- and δ-TBECH in dolphin blubber were

518

much higher than the EC50 values of 50:50 γ/δ-TBECH and 25:75 γ/δ-TBECH,

519

respectively. Given that γ-TBECH appears to be the most potent human androgen

520

receptor activator among the four TBECH diastereomers, its EC50 value should be

521

lower than that of 50:50 γ/δ-TBECH. For this reason, porpoises and dolphins may

522

suffer a certain hazard level in terms of androgen agonism from γ-TBECH exposure,

523

and this risk posed by exposure to ΣTBECH may increase for the marine mammals in

524

the studied region.

525

According to the above results, the influence of stereoisomerism on the

526

environmental fate and effects of chiral chemicals can introduce substantial

527

uncertainty into a risk assessment of their potential hazards. Further investigations of

528

the exposure of these marine mammals to chiral contaminants could focus on whether

529

a specific enantioselective trend matches that of their primary prey.

22


Page 22 of 37

Page 23 of 37


530

ASSOCIATED CONTENT

531

Supporting Information. Additional information on materials and methods.

532

Tables containing biological information for individual porpoises and dolphins,

533

recoveries of each native standard and surrogate, method LODs and detection

534

frequencies of each stereoisomer, concentrations of ΣHBCD and ΣTBECH in

535

biological tissues reported, and toxicological effects caused by HBCD and TBECH

536

stereoisomers. Figures showing data of the annual production of tHBCD in China,

537

temporal distributions in adult males, compositions of HBCD diastereomers in

538

porpoise blubber, and EFs of TBECH categorized by different physiological traits.

539

ACKNOWLEDGEMENTS

540

This project was supported by the General Research Fund (CityU 11100614 and

541

11338216) and Early Career Scheme (EdUHK 28300317) from the Hong Kong

542

Research Grants Council. The authors thank the Agriculture, Fisheries, and

543

Conservation Department, Hong Kong SAR Government and Ocean Park

544

Conservation Foundation, Hong Kong (OPCFHK) for their assistance with blubber

545

collection.

546

23



547

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786

Table 1. The concentrations (displayed with three significant digits) of ΣHBCD and ΣTBECH in the blubber of porpoises and dolphins

787

categorized by these marine mammals’ age and sex [shown as mass fractions (ng/g lipid weight)]. The structural formula of each analyte is

788

shown in the first column.

Analyte

ΣHBCD

Species (n)

Porpoise (59)

Dolphin (32)

ΣTBECH

Porpoise (59)

Dolphin (32)

Age Class

Minimum

Median

Maximum

Mean ± SD

Adult male

394

1,780

6,260

2,010 ± 1,460

Adult female

97.2

1,150

5,110

1,670 ± 1,590

Juvenile

129

509

1,720

657 ± 426

Adult male

447

2,650

12,600

4,590 ± 4,050

Adult female

997

5,410

45,800

11,500 ± 12,500

Juvenile

1,760

3,290

26,900

5,910 ± 6,640

Adult male

< 0.8

45.4

125

43.6 ± 31.5

Adult female

< 0.8

11.2

93.5

21.4 ± 28.7

Juvenile

< 0.8

19.5

60.2

21.2 ± 22.5

Adult male

< 0.8

33.5

193

56.2 ± 58.2

Adult female

8.08

101

341

110 ± 92.0

Juvenile

< 0.8

16.7

362

81.7 ± 110

789 34


Page 35 of 37


790 791

Figure 1. Stereoisomeric compositions of (A) the five HBCD diastereomers, (B) the

792

four TBECH diastereomers in the blubber of porpoises and dolphins (“*”: p < 0.05;

793

“**”: p < 0.01; “***”: p < 0.001).

794

35



795 796

Figure 2. EFs of (A) the three pairs of HBCD enantiomers, (B) the four pairs of

797

TBECH enantiomers in the blubber of porpoises and dolphins, respectively (“*”: p

Temporal Changes and Stereoisomeric ... - ACS Publications

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