Synthetic Phenolic Antioxidants and Their Metabolites in Mollusks

Aug 8, 2018 - Synthetic phenolic antioxidants (SPAs) are a group of chemicals widely used in ... to 501000 ng/g (geometric mean: 3450 ng/g), accountin...
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Ecotoxicology and Human Environmental Health

Synthetic Phenolic Antioxidants and Their Metabolites in Mollusks from the Chinese Bohai Sea: Occurrence, Temporal Trend, and Human Exposure Xiaoyun Wang, Xingwang Hou, Yu Hu, Qunfang Zhou, Chunyang Liao, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03322 • Publication Date (Web): 08 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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Synthetic Phenolic Antioxidants and Their Metabolites in

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Mollusks from the Chinese Bohai Sea: Occurrence,

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Temporal Trend, and Human Exposure

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Xiaoyun Wang1,2, Xingwang Hou1,2, Yu Hu1,2, Qunfang Zhou1,2,

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Chunyang Liao1,2,*, and Guibin Jiang1,2

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1

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Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

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100085, China

11

2

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Beijing 100049, China

College of Resources and Environment, University of Chinese Academy of Sciences,

13 14 15 16 17 18

*Corresponding author: Dr. Chunyang Liao

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Research Center for Eco-Environmental Sciences

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Chinese Academy of Sciences

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Beijing 100085, China

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Tel./Fax: 86-10-6291 6113

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E-mail: [email protected]

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Abstract

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Synthetic phenolic antioxidants (SPAs) are a group of chemicals widely used in

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various daily necessities and industrial supplies. Little is known about the occurrence

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and bioaccumulation potential of SPAs in marine biota. In this study, five commonly

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used SPAs and their four metabolites were detected in mollusk samples (n = 274)

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collected from the Chinese Bohai Sea during 2006-2016 and the spatiotemporal

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distribution and bioaccumulation of SPAs in mollusks were examined. The

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concentrations of 2,6-di-tert-butyl-4-hydroxytoluene (BHT) ranged from 383 to

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501000 ng/g (geometric mean: 3450 ng/g), accounting for 79.4% of the total

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concentrations of SPAs and their metabolites (∑9SPAs). The mollusk species, Rapana

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venosa (Rap), contained higher levels of BHT than other species, suggesting that Rap

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could be used as a potential bioindicator for monitoring of the BHT pollution in the

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investigated region. The ∑9SPA concentrations in mollusks gradually increased with

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years and a significant positive correlation (r=0.900, p < 0.05) was found between

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∑9SPAs concentration and trophic level (TL) of the mollusks. The trophic

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magnification factor (TMF) value of ∑9SPAs was calculated as 16.1, suggesting a

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high biomagnification potential of SPAs in mollusks in the Chinese Bohai Sea. The

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estimated daily intake (EDI) of ∑9SPAs through dietary ingestion of mollusks was up

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to 602 and 789 ng/kg bw/day for adults and children and teenagers, respectively. The

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principal component analysis (PCA) result suggests that there exists a common source

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for three gallates (OG, DG and PG), and BHT metabolites in mollusks were mainly

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derived from degradation of BHT. This is the first study to report the occurrence of

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and bioaccumulation potentials of SPAs and their metabolites in invertebrate species

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from coastal marine environments.

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Introduction

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Synthetic phenolic antioxidants (SPAs) are a group of chemicals that can

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promote capture and neutralization of free radicals to mitigate potential damages. The

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commonly

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2-tertbutyl-4-methoxyphenol (BHA),

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gallates (DG).1,2 Due to their chemical stability, low cost and flexibility, SPAs are

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gradually used to replace natural antioxidants in more and more consumer products

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such as foodstuffs, cosmetics and plastics.3,4 The Organization for Economic

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Co-operation and Development (OECD) reports that BHT, the most frequently used

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SPA, is added in various kinds of consumer products, including food, medicine,

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cosmetics, rubber, plastics, fodder and printing ink, at concentrations up to 0.5%.2

used

SPAs

include

2,6-di-tert-butyl-4-hydroxytoluene

(BHT),

propyl (PG)-, octyl- (OG), and dodecyl-

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The toxicities of SPAs have been illustrated in a few studies. It showed that

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continuous feeding of 0.5 or 0.05% BHT enhanced the development of spontaneously

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occurring liver tumors in C3H mice.5 Both in vitro and in vivo exposure of BHA can

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disturb steroid hormone secretion in zebrafish gonad and cause endocrine disrupting

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effects, genotoxicity, and reproductive toxicitity.6-10 BHT can be metabolized to

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2,6-di-tert-butyl-4-(hydroxymethyl)phenol

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hydroxybenzaldehyde (BHT-CHO) and 3,5-di-tert-butyl-4-hydroxybenzoic acid

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(BHT-COOH)

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2,6-di-tert-butyl-1,4-benzoquinone (BHT-Q) through oxidation of the tert-butyl

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groups.11 Besides, BHT could be degraded to BHT-OH and BHT-CHO through

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photochemical reaction.12 It was reported that BHT metabolites may cause DNA

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damage directly or through H2O2 generation.13 Therefore, more and more attention

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has been paid on the toxicities of SPAs and their metabolites.

through

oxidation

(BHT-OH),

of

the

p-methyl

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3,5-di-tert-butyl-4-

group,

and

to

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SPAs and their metabolites have been reported to occur in various environmental

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matrices, including dust14-16, river water17,18, air19, sewage sludge20 and sediment16.

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SPAs and their metabolites were found in house dust from several countries at

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concentrations up to 121000 ng/g.14 Several SPAs and their metabolites were detected

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in sewage sludge samples from 33 cities in China at concentrations ranging from 183

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ng/g to 41000 ng/g, with an average value of 4960 ng/g.20 The evidence of high

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concentrations of SPAs and their metabolites in environmental matrices enhances the

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controversies on the application of these compounds. However, few data exist on the

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SPAs and their metabolites in organisms as well as their bioaccumulation potential in

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food chain. Therefore, more surveys on the occurrence of SPAs and their metabolites

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in organisms are urgently needed.

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The Chinese Bohai Sea is the sole inland sea of China with three sides bordered

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by the mainland. The coastal line of the Bohai Sea has a length of nearly 3800 km and

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a surface area of 77 × 109 m2. It receives a large amount of fresh water from over 40

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rivers, including Liaohe, Luanhe, Yellow and Haihe rivers. Although the ecosystem of

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the Bohai Sea is stable, it needs a long period of time to repair the marine

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environment once contaminated. Therefore, the Bohai Sea is a good place for

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monitoring of variation in environmental conditions.

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Mollusks have been widely used as monitoring organisms for a long time,

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because of their diversities, widespread distribution and high abundance in aquatic

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ecosystem, and convenience for collection. Besides, they are tolerant to many

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pollutants.21 Thus, mollusks can be used to examine the bioaccumulation potential of

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contaminants in the aquatic environment. In our previous studies, mollusks from the

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Bohai Sea have been used as bioindicators for persistent organic pollutants (POPs)

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and heavy metals.22,23 For examples, Mya arenaria (Mya), Mactra veneriformis (Mac), 4

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and Crassostrea talienwhanensis (Oyster, Ost) could be used as sentinels to reveal the

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short chain chlorinated paraffins (SCCPs) contamination, while Rapana venosa (Rap)

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could be applied as a potential bioindicator for mercury pollution in the coastal

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regions of China. Bioaccumulation of pollutants in mollusk is affected by different

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factors, such as bioaccumulation time, individual volume, etc. Mollusks with larger

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size usually have longer time to accumulate pollutants, whereas the growth dilution

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effect may also play an important role in the bioaccumulation of pollutants.24 It is of

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significance to examine the association of bioaccumulation potential of SPAs with the

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size of mollusks.

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In this study, several SPAs and their metabolites were determined in mollusk

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samples collected from the Chinese Bohai Sea during 2006-2016. The aim is to

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investigate the occurrence, bioaccumulation potential, and temporal trends of SPAs

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and their metabolites. On the basis of the measured concentrations, we estimated

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human dietary intake of SPAs and their metabolites through the consumption of

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mollusks. To our knowledge, this is the first time to determine the occurrence and

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bioaccumulation potential of SPAs and their metabolites in invertebrate species

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collected from a coastal marine ecosystem.

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Materials and Methods

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Chemicals and Reagents. The chemical names, CAS registry numbers, Log Kow

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values14 and molecular structures of the target analytes are shown in Figure 1. BHT,

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BHT-Q, BHT-OH, BHT-COOH, BHA and DG were obtained from Sigma-Aldrich (St.

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Louis, MO, USA). BHT-CHO, PG and OG were purchased from TCI America

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(Portland, OR, USA). Isotope-labeled internal standards 2,6-di(tert-butyl-d9)-4-methyl

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(phenol-3,5, O-d3) (BHT-d21), n-propyl 4-hydroxybenzoate-2,3,5,6-d4 (PrP-d4) and 5

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methyl 4-hydroxybenzoate-2,3,5,6-d4 (MeP-d4) were obtained from CDN Isotopes

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(Quebec, Canada). Milli-Q water (18.2 MΩ cm-1) was prepared by an ultrapure water

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system (Barnstead International; Dubuque, IA, USA). HPLC grade methanol (MeOH)

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was

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dichloromethane (DCM) and hexane (Hex) were supplied by J.T. Baker (Phillipsburg,

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NJ, USA). Oasis HLB cartridge (60 mg/3 mL) was purchased from Waters (Milford,

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MA, USA). Stock standard solutions (1000 µg/mL) were individually prepared in

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methanol, and the working solutions were weekly prepared from the stock standard

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solutions by appropriate dilution.

supplied

by

Fisher Chemical (Hampton,

NH,

USA).

HPLC

grade

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Sample collection. A total of 274 composite mollusk samples were collected

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from nine coastal cities along the Chinese Bohai Sea, including Dalian (DL), Yingkou

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(YK), Huludao (HLD), Beidaihe (BDH), Tianjin (TJ), Shouguang (SG), Penglai (PL),

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Yantai (YT), and Weihai (WH) in July and August each year (except for 2008 and

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2013) during 2006-2016 (Figure 2; Table S1, Supporting Information). Among the

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six mollusk species collected, two mollusk species were gastropods, including

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Neverita didyma (Nev) and Rapana venosa (Rap). The other four species were

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bivalves, including Amusium (Amu), Scapharca subcrenata (Sca), Mytilus edulis

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(Blue mussel, Myt), and Meretix meretrix (Mer). Their denominations are presented in

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Table S2. After sampling, mollusks were depurated in filtered seawater for 24 h

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before transported to the laboratory on ice. The soft tissues of mollusks were excised

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by stainless steel scalpel blades, and then thoroughly rinsed with Milli-Q water to

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remove extraneous impurities. Approximately 500-1500 g of wet soft tissue

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(consisting of 3-30 individuals) was homogenized to make one composite mollusk

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sample so that approximately 822-8220 individuals were collected to prepare the 274

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composite mollusk samples. All samples were freeze-dried, homogenized, and sieved 6

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through a sieve (80 meshes per inch). The processed samples were preserved in clean

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aluminum foil that was put in zip bags to avoid cross contamination and loss, and

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stored at -20 °C until analysis.

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Sample Preparation. Approximately 0.1 g of the sample (spiked with 50 ng of

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BHT-d21, MeP-d4, and PrP-d4) was weighted and put into a 15 mL glass tube. Then the

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sample was extracted with 5 mL DCM/Hex (3:1, v/v) by ultrasonication for 20 min at

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53 MHz and shaking for 20 min at 300 rpm. The mixture was centrifuged at 3300 rpm

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at 4 °C for 10 min (Eppendorf Centrifuge 5804, Hamburg, Germany). The supernatant

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was transferred into another glass tube. The extraction procedure was repeated three

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times. After extraction, the combined extract was evaporated and solvent-exchanged

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to 0.5 mL MeOH, and 0.5 mL water was added. Then the extract was filtrated through

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a 0.22 µm PTFE filter membrane (Waters, MA, USA) to remove lipid. After that, 9.5

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mL water was added into the sample. HLB SPE cartridge was preconditioned by 7 mL

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MeOH and 7 mL water. The sample was loaded onto the HLB cartridge, and then

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washed by 10 mL 5% MeOH/water solution. After drying for ∼30 min under vacuum,

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the HLB cartridge was eluted by 10 mL MeOH. The elute was collected and equally

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separated to two parts: one half was concentrated to 0.5 mL under a gentle stream of

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nitrogen for UPLC-MS/MS analysis and the other half was concentrated and solvent

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exchanged to 0.5 mL Hex for GC-MS/MS analysis.

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Instrumental Analysis. The measurement of BHT was performed on a Thermo

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Scientific TRACE 1300 Series gas chromatograph coupled with a Thermo Scientific

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TSQ 8000 Evo triple quadrupole mass spectrometer (GC-MS/MS; Thermo Fisher

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Scientific, San Jose, CA, USA). A DB-5 capillary column (30 m length × 0.25 mm i.d.

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× 0.25 µm film thickness) was used for separation. The initial oven temperature was

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set at 50 °C and held for 3 min, and increased at 20 °C/min to 240 °C and held for 5 7

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min. The ion source and transfer line temperatures were 250 °C and 280 °C,

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respectively. Quantitative determination by GC-MS/MS (electron ionization, EI) was

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performed in the selective reaction monitor (SRM) mode (Table S3). The total ion

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chromatogram (TIC) and extract ion chromatograms (EICs) of BHT and BHT-d21 are

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shown in Figure S1.

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An Exion LC AD ultra-high performance liquid chromatograph interfaced with a

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triple quadrupole 5500 mass spectrometer (UPLC-MS/MS; AB Sciex, MA, USA) was

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used for the determination of the SPAs and metabolites other than BHT. A symmetry

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shield CSHTM Phenyl-Hexyl UPLC analytical column (100 × 2.1 mm, 1.7 µm; Waters)

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connected with a BEH C18 guard column (5 × 2.1 mm, 1.7 µm; Waters) was used for

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chromatographic separation. Column temperature was set at 40 °C. 0.1% acetic

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acid/ultrapure water (A) and MeOH (B) were used as mobile phase. The gradient was

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initiated at a composition of 75:25 (A:B, v/v) and held for 0.5 min. Then the

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composition B was increased to 60% in 2 min and held for 2 min. After that, the

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composition B was increased to 100% in 1.5 min and held for 2.9 min. After

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immediately returning to the initial composition of 75:25 (A:B, v/v), the column was

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allowed to re-equilibrate for 2 min. The total running time for one sample analysis

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was 11 min. Flow rate of the mobile phase was set at 0.3 mL/min. Ion spray voltage

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was kept at -4.5 kV, and ion source temperature was set at 500 °C. Nitrogen was used

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as both curtain and collision gas. The MS/MS was operated in negative electrospray

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ionization (ESI) with the multiple reaction monitoring (MRM) mode. The MRM

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transitions of target chemicals are presented in Table S3, and the TIC and EICs of

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MeP-d4, PrP-d4 and the target compounds (except for BHT) are shown in Figure S2.

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Trophic Magnification Factor (TMF). TMF has been often used to show

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biomagnification potentials of organic contaminants in aquatic environment. In our 8

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study, the TMF value of SPAs in mollusks was calculated to evaluate the

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biomagnification of such chemicals, based on the relationship between TLs and SPAs

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concentrations: 25

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Ln concentration = a + (b × TL)

(1)

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where a is the y-intercept (constant). By using the equation (2) as shown following,

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TMF for SPAs was determined from the slope b of equation (1):

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TMF =  

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Quality Assurance and Quality Control (QA/QC). A procedural blank, two

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duplicate samples and two pre-extraction matrix spike samples were analyzed for

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every 20 samples. The standard calibration curve was evaluated by injecting 12

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concentration levels (0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 500 ng/mL) for all

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target compounds except BHT. The calibration curve standard of BHT was prepared

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at concentrations from 1 to 1000 ng/mL due to its relatively high concentrations in

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samples. Recoveries of SPAs in spiked mollusk matrices ranged from 61% for BHA to

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92% for BHT (Table S3). The variation in concentrations of randomly selected

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duplicate samples (n = 12) were all less than 20% of the mean. Quantification of SPAs

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was performed by an isotope-dilution method based on the response of BHT-d21 (for

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BHT), MeP-d4 (for BHT-Q, BHA, BHT-OH, BHT-CHO, and BHT-COOH), and

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PrP-d4 (for OG, PG and DG). For every 10 samples, a midpoint calibration standard

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was injected as a check for instrumental drift in sensitivity, and a pure solvent (MeOH

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or Hex) was injected to prevent carry-over of target analytes from sample to sample.

(2)

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Statistical Analysis. Method quantification limits (MQLs) were defined as the

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analyte concentrations corresponding to signal-to-noise ratio (S/N) of 10 in mollusk

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matrices by injecting 10 spiked samples. MQLs values were 0.4 ng/g for OG, 0.7 ng/g

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for BHT-CHO, 0.8 ng/g for PG, 0.9 ng/g for DG, 1.1 ng/g for BHA, 1.2 ng/g for 9

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BHT-Q and BHT-OH, 1.4 ng/g for BHT, and 1.9 ng/g for BHT-COOH, respectively

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(Table S4). N.D. (not detected) was defined as S/N < 3 and N.Q. (not quantified) was

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defined as S/N > 3 but < 10. In calculation of the detection frequency, samples with

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N.Q. levels were included. Both N.Q. and N.D. were replaced by half of the MQL

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(MQL/2) for data analysis.

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Origin Pro 2017 and SPSS Statistics 20 were used for statistical analyses.

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Spearman’s correlation coefficient (r2) was used for determination for correlations

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between target compound concentrations. The relationship between the nature

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logarithm concentrations and tropic levels (TLs) was evaluated using linear regression

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models. Mann-Whitney U test, a nonparametric test, was used for the comparison of

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concentrations between groups. The statistical significance level was set at p < 0.05.

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All data are presented on a dry weight basis unless mentioned otherwise.

238 239

Results and Discussion

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SPAs (BHT, BHA, OG, PG, and DG). BHT was detectable in all samples at

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concentrations ranging from 383 to 501000 ng/g (geometric mean (GM): 3450,

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median: 3100 ng/g), which accounted for the largest proportion (98.0 %) of the total

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concentrations of five SPAs (BHT, BHA, OG, PG, and DG; ∑5SPAs) (Table 1). BHT

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is the most commonly used SPA that is used in plastics, cosmetics and other materials

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to protect the products from oxidation.2 A few studies showed that BHT was the

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predominant compound among SPAs and their metabolites analyzed in different

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environmental matrices, such as sewage sludge samples (range: 51.7-30300 ng/g,

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detection frequency (df): 100%) and indoor dust samples (< 1.2-118000 ng/g,

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99.5%).14,20 BHT was detected in adipose tissue samples at concentrations of 230 ±

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150 ng/g in 11 residents of the UK and of 1300 ± 820 ng/g in 12 residents of the USA 10

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in 1970.26 BHA was found in 76.9% of mollusk samples in a concentration range of
Rap (906 ng/g) > Sca (642 ng/g) >

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Myt (637 ng/g) > Nev (451 ng/g), suggesting species-specific bioaccumulation

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capacity for BHT metabolites (Figure S3). It was reported that persistence of BHT

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metabolites was longer than or equal to that of BHT.20 Considering the widespread

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occurrence, long persistence and environmental health risks, more information on the

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BHT metabolites is needed.

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The temporal variations of ∑MTs in mollusks are shown in Figure 3. The

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highest GM concentration of ∑MTs (4810 ng/g) was found in mollusks collected in

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2011, followed by those found in 2010 (3580 ng/g) and 2006 (1690 ng/g). The GM

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∑MT concentrations in mollusks collected during 2014-2016 were 1-2 orders of

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magnitude lower than those found for 2006-2012. A generally increasing trend in the

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concentrations of BHT-OH, BHT-CHO and BHT-COOH in mollusks was observed,

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which is consistent with that found for BHT. The concentrations of BHT-Q in

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mollusks collected during 2014-2016 suffered a sudden decrease, probably due to the

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different environmental behavior of BHT-Q as compared to other BHT metabolites.

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BHT-Q is degradation product of BHT through oxidation of the tert-butyl groups, 13

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which is different with other degradation products of BHT (BHT-OH, BHT-CHO and

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BHT-COOH) through oxidation of the p-methyl group.11,12

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The total concentrations of five SPAs and their four metabolites (∑9SPAs).

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The ∑9SPA concentrations of mollusk samples, collected from nine coastal sites, were

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in the range of 833-505000 ng/g. The highest ∑9SPAs concentration of 505000 ng/g

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was found in a Rap sample collected from Penglai in 2016. In Huludao, the collected

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mollusk samples contained the highest ∑9SPAs at concentrations ranging from 2390

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to 55700 ng/g, with a GM value of 8090 ng/g, probably due to the continual pouring

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of wastewater into the Bohai Sea without disposal.24 The mollusk samples from

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Tianjin contained the lowest ∑9SPAs (range: 857-18200, GM: 4460 ng/g). As shown

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in Figure 2, the concentrations of ∑9SPAs in different species varied from sites. There

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were no obvious geographic differences found for ∑9SPAs, which is consistent with

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what reported in a previous study that no obvious difference in the SPAs

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concentrations in municipal sewage sludge samples collected from 33 cities in China

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was found.20 As shown in Figure 4a, the contribution of individual compounds to

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∑9SPAs varied with sampling locations. Among five SPAs and their four metabolites,

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BHT contributed to 79.4% of the total concentrations (∑9SPAs), followed by BHT-Q

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(17.8%) and DG (1.05 %). Other target compounds were minor and generally

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contributed to less than 1% of the ∑9SPAs. The contribution of nine target compounds

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to ∑9SPAs varied among mollusk species (Figure 4b). BHT was the predominant

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compound, accounting for from 51.5% of ∑9SPAs in Mer to 85.6% of ∑9SPAs in Rap

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(mean: 70.1 %); which was followed, in decreasing order, by BHT-Q (25.4 %) and

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DG (2.09 %).

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Principal component analysis (PCA) was performed to determine potential

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sources of SPAs and their metabolites in mollusks collected from the Chinese Bohai 14

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Sea during 2006-2016. As shown in Figure S4, the three gallates (OG, DG and PG)

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are clustered in the first quadrant, suggesting the existence of a common source for

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these chemicals. BHT and its metabolites (except for BHT-Q) are congregated in the

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fourth quadrant of scatter plot, which suggests that the concentrations of BHT

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metabolites in mollusks were mainly derived for BHT itself. This is consistent with

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the abovementioned result of correlation analysis (Table S5).

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Biomagnification of SPAs in Mollusks. According to the different sizes, Rap

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was divided into L (large) and S (small) classes. The GM concentrations of ∑9SPAs

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decreased with the size of Rap collected from the Bohai Sea (L vs S Raps: 7170 vs

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7430 ng/g), although the difference was not significantly different (p > 0.05; Table

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S7). The concentrations of all target compounds (except for BHA) decreased with the

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size of Rap. The growth dilution effect may play an important role in the

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bioaccumulation of SPAs in mollusks. The reduced trend of mercury with size was

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also found for the Rap samples collected from the same region.24 There needs more

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data to evaluate the concentration variation with the shells’ size of mollusks.

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The similar sample set has been analyzed in our previous studies to evaluate the

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spatial and temporal patterns of some legacy and emerging contaminants in the

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coastal water of the Chinese Bohai Sea, such as SCCPs22, mercury23,24, and

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polybrominated biphenyl ethers (PBDEs)30. In those studies, the trophic levels (TLs)

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of mollusks have been used to examine the relevance of accumulation of

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contaminants with the TLs of mollusk species, where TLs were calculated by their

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nitrogen isotope ratios (δ15N).22,31,32 We adopted the TLs of mollusk species and

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evaluate the biomagnification potential of SPAs and their metabolites in mollusks in

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the present study (Table S2). Significant linear correlation (r=0.900, p < 0.05) was

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found between the ∑9SPAs concentrations and TLs, suggesting the biomagnification 15

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potential of ∑9SPAs in mollusks. Significant positive correlations between the

377

concentrations of BHT and BHT-CHO with TLs (r=1.00 and 0.866, p < 0.05) were

378

also found (Table S5), which is similar to the patterns of mercury contents in

379

mollusks with the TLs of mollusk species.23

380

The TMF value of ∑9SPAs was assessed by linear regression analysis between

381

TLs of mollusks and the ∑9SPA concentrations measured (Figure 5). The high TMF

382

value of ∑9SPAs (16.1) further suggests the high biomagnification potential of SPAs

383

in the marine food chains of the Chinese Bohai Sea.

384

Human Exposure to SPAs via Ingestion of Mollusks. Seafood, including

385

mollusks, is daily consumed by the Chinese residents, especially those inhabiting in

386

coastal area. Whereas, little is known about human exposure to SPAs through

387

consumption of mollusks. Two age groups of population, viz., children & teenagers

388

(6-17 years) and adults (≥ 18 years), were taken account in the exposure assessment

389

in this study. The daily consumption rates of mollusks were assumed as 26.0 and 24.0

390

g/day for males and females of children & teenagers, and 33.0 and 27.5 g/day for

391

males and females of adults in China, respectively.33 The average body weights were

392

adopted from a previous study, which are 37.9 and 36.3 kg for males and females of

393

children & teenagers, and 62.7 and 54.8 kg for males and females of adults,

394

respectively.34 The concentrations of SPAs in mollusk samples need to be converted

395

from a dry weight to a wet weight basis for the calculation of daily intake, as the daily

396

consumption rates of mollusks were presented on a basis of wet weight.33 The

397

moisture content of mollusks of 80% was used for concentration conversion in

398

reference to earlier studies.35-37 We assumed that SPAs through ingestion of mollusks

399

were completely absorbed in the body. On the basis of the daily consumption rates 16

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and the concentrations measured, we estimated the daily intake (EDI, ng/kg bw/day)

401

of SPAs via consumption of mollusks, as shown in equation (3):

402

EDI =

××(%)

(3)

!"

403

where C is the GM concentration of SPAs in mollusks (ng/g dry wt); DC is the daily

404

consumption rate of mollusks (g/day); and BW is the body weight (kg).

405

The EDIs of SPAs and their metabolites for the general population in China from

406

mollusks are summarized in Table 2. Due to the elevated concentrations, BHT had the

407

highest EDIs, ranging from 347 to 474 ng/kg bw/day for children & teenagers and

408

adults, which were 3 orders of magnitude higher than those of BHA (range:

409

0.128-0.174 ng/kg bw/day). The EDIs of three gallates, DG (2.93-4.00 ng/kg bw/day),

410

OG (0.535-0.731 ng/kg bw/day) and PG (0.635-0.868 ng/kg bw/day), were

411

comparable. Of the four BHT metabolites, BHT-Q was predominant contributor to the

412

EDIs of BHT metabolites and the EDIs of BHT-Q (range: 23.7-32.4 ng/kg bw/day)

413

were 1-2 orders of magnitude higher than those of BHT-CHO (1.41-1.93 ng/kg

414

bw/day), BHT-COOH (1.72-2.35 ng/kg bw/day) and BHT-OH (0.450-0.615 ng/kg

415

bw/day). The EDIs values of ∑9SPAs from mollusks (602 ng/kg bw/day on average

416

for adult males and females) were higher than those of other contaminants, e.g.,

417

tetrabromobisphenol A (1.3 ng/kg bw/day for adults) and hexabromocyclododecane

418

(0.49 ng/kg bw/day for adults).39 Therefore, it is urgent to investigate the fate and

419

toxicity of SPAs to humans. The EDI values of ∑9SPAs for males (616 and 803 ng/kg

420

bw/day for adults and children & teenagers, respectively) were slightly higher than

421

those for females (588 and 774 ng/kg bw/day). For all males and females, the EDI

422

values for children & teenagers were much higher than those for adults (Table 2).

423

In summary, high concentrations and frequent detection of SPAs and their

424

metabolites were found in mollusks collected from the Chinese Bohai Sea during 17

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2006-2016. BHT was the predominant SPA analogue in mollusks and the

426

concentrations ranged from 383 to 501000 ng/g (GM:3450 ng/g). A gradual increase

427

in the total concentrations of SPAs and their metabolites (except for BHT-Q) in

428

mollusks with years was found. Among six mollusk species investigated, Rapana

429

venosa (Rap) could be regarded as the potential bioindicator of BHT due to high

430

bioaccumulation potential. The TMF value of ∑9SPAs (16.1) was considerably higher

431

than 1, suggesting biomagnification of SPAs in the marine food chains. The daily

432

intakes of ∑9SPAs through consumption of mollusks were 789 and 602 ng/kg bw/day

433

for children & teenagers and adults in China, respectively. The widespread occurrence

434

and bioaccumulation potential of SPAs warrant further studies on the toxicity of SPAs

435

and their metabolites.

436 437

Supporting Information

438

Number and sizes of the collected mollusk species. Denomination and trophic levels

439

of the selected mollusk species. Optimized MRM parameters. The method

440

quantification limits and recoveries from matrix spikes. Correlation analysis of target

441

compounds and trophic levels in mollusks. Concentrations of SPAs and their

442

metabolites in mollusks from several locations along the Chinese Bohai Sea. The GM

443

concentrations of target compounds with S and L size of Rap. The total ion

444

chromatogram (TIC) and extract ion chromatograms (EICs) of BHT and BHT-d21

445

from GC-MS/MS analysis. The TIC and EICs of MeP-d4, PrP-d4, BHA, BHT-Q,

446

BHT-OH, BHT-CHO, BHT-COOH, OG, DG and PG from UPLC-MS/MS analysis.

447

The total concentrations of five SPAs (∑5SPAs) and four BHT metabolites (∑MTs) in

448

different mollusk species from the Chinese Bohai Sea. Principal component analysis

449

of the nine target compounds in mollusks. The Supporting Information is available 18

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free of charge on the ACS Publications website at DOI: 10.1021/acs.est.xxx.

451 452

Acknowledgments

453

This work was jointly supported by the National Natural Science Foundation of China

454

(21522706 and 21621064), the Major International (Regional) Joint Project

455

(21461142001), and the Thousand Young Talents Program of China.

456 457

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Table 1. Concentrations of SPAs and their metabolites in mollusks (ng/g dry weight) collected from the Chinese Bohai sea BHT 2006 (n=26) GM 2340 median 2230 df c (%) 100 range 1050-5110 2007 (n=35) GM 2220 median 2260 df (%) 100 range 502-7630 2009 (n=25) GM 3040 median 2890 df (%) 100 range 1210-11300 2010 (n=29) GM 4450 median 4650 df (%) 100 range 383-51400 2011 (n=32) GM 2480 median 2130 df (%) 100

∑5SPAsa

∑MTsb

BHA

BHT-CHO

BHT-COOH BHT-OH

BHT-Q

DG

OG

PG

1.30