Synthetic Phenolic Antioxidants and Their Metabolites in Mollusks

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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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Environmental Science & Technology

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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

4 5

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

9

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

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

10

100085, China

11

2

12

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

20

Chinese Academy of Sciences

21

Beijing 100085, China

22

Tel./Fax: 86-10-6291 6113

23

E-mail: [email protected]

24 25

1

ACS Paragon Plus Environment


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Abstract

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

28

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

29

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

30

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

31

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

32

distribution and bioaccumulation of SPAs in mollusks were examined. The

33

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

34

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

35

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

41

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

48

and bioaccumulation potentials of SPAs and their metabolites in invertebrate species

49

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

55

commonly

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2-tertbutyl-4-methoxyphenol (BHA),

57

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

58

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

59

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

60

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

61

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

62

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-

63

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

64

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

65

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

70

(BHT-COOH)

71

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

74

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

75

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

through

oxidation

(BHT-OH),

of

the

p-methyl

3


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

group,

and

to


76

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

77

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

78

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

79

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

80

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

81

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

82

concentrations of SPAs and their metabolites in environmental matrices enhances the

83

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

85

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

86

in organisms are urgently needed.

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

88

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

89

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

92

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

93

monitoring of variation in environmental conditions.

94

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

95

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

96

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

97

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)

100

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

105

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

107

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

108

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

109

size of mollusks.

110

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

111

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

112

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

113

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

114

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

115

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

116

bioaccumulation potential of SPAs and their metabolites in invertebrate species

117

collected from a coastal marine ecosystem.

118 119

Materials and Methods

120

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

121

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

122

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

123

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

124

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

125

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



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126

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

127

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

128

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

129

was

130

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

131

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

132

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

133

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

134

solutions by appropriate dilution.

supplied

by

Fisher Chemical (Hampton,

NH,

USA).

HPLC

grade

135

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

136

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

137

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

138

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

139

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

140

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

141

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

143

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

144

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

145

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

146

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

147

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

148

(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

152

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

153

stored at -20 °C until analysis.

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

Scientific TRACE 1300 Series gas chromatograph coupled with a Thermo Scientific

172

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

173

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

174

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

175

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



176

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

177

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

178

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

179

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

180

shown in Figure S1.

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

202

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

203

concentrations: 25

204

Ln concentration = a + (b × TL)

(1)

205

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

206

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

207

TMF =

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

218

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

220

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

221

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

(2)

222

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

223

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

224

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

225

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



226

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

227

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

228

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

229

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

230

(MQL/2) for data analysis.

231

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

232

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

233

between target compound concentrations. The relationship between the nature

234

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

235

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

236

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

237

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

238 239

Results and Discussion

240

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

241

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

244

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

245

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,

248

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

249

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

250

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) >

311

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

312

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

313

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

314

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

315

BHT metabolites is needed.

316

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

317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

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



326

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

327

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

328

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

329

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

330

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

331

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

332

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

333

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

334

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

335

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

336

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

337

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

338

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

339

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

340

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

341

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

342

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

343

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

344

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

345

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

346

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

347

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

348

DG (2.09 %).

349

Principal component analysis (PCA) was performed to determine potential

350

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)

352

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

353

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

354

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

355

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

356

the abovementioned result of correlation analysis (Table S5).

357

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

358

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

359

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

360

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

361

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

362

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

363

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

364

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

365

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

366

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

367

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

368

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

369

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

370

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

371

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

372

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

373

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

374

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

375

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



376

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


Page 16 of 33

Page 17 of 33


400

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



425

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


Page 18 of 33

Page 19 of 33


450

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

Synthetic Phenolic Antioxidants and Their Metabolites in Mollusks

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