Temporal Trends of Parabens and Their ... - ACS Publications

Plaza, P.O. Box 509, Albany, New York 12201-0509, United States. 9 ... Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China...
0 downloads 0 Views 562KB Size
Subscriber access provided by READING UNIV

Characterization of Natural and Affected Environments

Temporal Trends of Parabens and Their Metabolites in Mollusks from the Chinese Bohai Sea during 2006-2015: SpeciesSpecific Accumulation and Implications for Human Exposure Chunyang Liao, and Kurunthachalam Kannan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02750 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Environmental Science & Technology

1

Temporal Trends of Parabens and Their Metabolites in Mollusks from the

2

Chinese Bohai Sea during 2006-2015: Species-Specific Accumulation and

3

Implications for Human Exposure

4

Chunyang Liao1,2 and Kurunthachalam Kannan1,*

5 6 7

1

8

Health Sciences, School of Public Health, State University of New York at Albany, Empire State

9

Plaza, P.O. Box 509, Albany, New York 12201-0509, United States.

Wadsworth Center, New York State Department of Health, and Department of Environmental

10

2

11

Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for

12 13 14 15

*Corresponding author: K. Kannan

16

Wadsworth Center

17

Empire State Plaza, P.O. Box 509

18

Albany, NY 12201-0509

19

Tel: 1-518-474-0015

20

Fax: 1-518-473-2895

21

E-mail: [email protected]

22 23

For submission to: ES&T

1

ACS Paragon Plus Environment

Environmental Science & Technology

24

ABSTRACT: Parabens are used as preservatives in many consumer products and human

25

exposure to these chemicals has been a public concern. In this study, mollusks (n = 186),

26

collected from the Chinese Bohai Sea during 2006-2015, were analyzed for six parabens and

27

their five metabolites. The total concentration of parabens was in the range of 2.66-299 ng/g dw

28

(geometric mean: 24.1). Methyl paraben and 4-hydroxybenzoic acid were the predominant

29

parent and metabolic parabens, respectively found in mollusks. Mollusk species, Mactra

30

veneriformis, Mytilus edulis, and Cyclina sinensis contained elevated concentrations of both

31

parent and metabolic parabens. A gradual increase in paraben concentrations was found in

32

mollusks collected between 2006 and 2012. Principal component analysis suggested the

33

existence of a common source for these chemicals in mollusks. Consumption of mollusks can

34

contribute to human exposures and we estimated daily intakes of parabens through the

35

consumption of mollusks. This is the first study to report temporal trends and accumulation of

36

parabens and their metabolites in a variety of invertebrate species from coastal marine

37

environments.

38

Key words: Parabens; Benzoic acid; Mollusks; Accumulation; Temporal trend; Human exposure

39 40

INTRODUCTION

41

The Chinese Bohai Sea, located in northeastern China, is a semi-enclosed inland sea

42

surrounded by the Liaodong Peninsula, North China Plain and Shandong Peninsula, and

43

encompasses several large cities near its shoreline. Approximately 18% of the Chinese

44

population inhabits along its shoreline, which is highly industrialized and contributes 28% of the

45

total gross domestic product (GDP) of China (1). The rapid socioeconomic growth in the Bohai

46

Rim has raised serious concerns with regard to the deterioration of the environment in this

47

region. Environmental issues of this coastal marine region have been widely studied (1-9). 2

ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

Environmental Science & Technology

48

Mollusks are invertebrate benthic organisms, with a wide geographical distribution both in

49

marine and freshwater environments. Because of their sedentary habit and low metabolic enzyme

50

activities, mollusks can bioaccumulate diverse contaminants (10,11). Furthermore, mollusks

51

have good tolerance to a wide range of contaminants, often coexist in polluted water and

52

sediment, and are also easily accessible. Due to these characteristics, mollusks have been used

53

for biomonitoring of a variety of contaminants in the Chinese Bohai Sea, including legacy

54

chemicals such as heavy metals, organochlorine pesticides, polychlorinated biphenyls and

55

dioxins (2-5), as well as emerging chemicals such as perfluorinated compounds, chlorinated

56

paraffins, and novel brominated flame retardants (1,6-9).

57

Parabens (alkyl esters of p-hydroxybenzoic acid) are a group of a homologous series of

58

chemicals widely used as antimicrobial agents for the preservation of cosmetics,

59

pharmaceuticals, and food products (12-14). Among paraben analogues, methyl (MeP), ethyl

60

(EtP), propyl (PrP), butyl (BuP), benzyl (BzP), and heptyl parabens (HepP) are commonly used.

61

In practice, MeP and PrP are often used in combination due to their synergistic antimicrobial

62

action (15). Parabens have been used as preservatives in over 22,000 cosmetic products at

63

concentrations of up to 0.4% (by weight) for a single compound or up to 0.8% for a mixture of

64

parabens (15). The use of parabens in pharmaceuticals varies from product to product, but is

65

generally below 1% (by weight) (16). Parabens have been used in food products for several

66

decades at concentrations of up to 0.1% (by weight), and several parabens, including MeP, PrP

67

and BuP, are directly added to food products as flavoring agents and adjuvants (17,18).

68

Studies have shown that 4-hydroxybenzoic acid (4-HB) and 3,4-dihydroxybenzoic acid

69

(3,4-DHB) are the common metabolites of several parabens in animals and humans (19-21).

70

Other metabolites of parabens include methyl protocatechuate (OH-MeP), ethyl protocatechuate

3

ACS Paragon Plus Environment

Environmental Science & Technology

71

(OH-EtP), and benzoic acid (BA) (22-27). Public concerns over the safety of parabens and their

72

metabolites have increased due to their estrogenic properties, as has been shown in both in vitro

73

and in vivo studies (14,15-17,28).

74

Occurrence of parabens and their metabolites in environmental matrixes, including house

75

dust, wastewater, sediment, and sewage sludge, is known (12,13,29,30). Parabens have been

76

reported to occur in foodstuffs and personal care products from China and the United States at

77

concentrations on the order of a few nanograms per gram and a few milligrams per gram of the

78

product, respectively (31-34). These chemicals have also been found in various human samples,

79

including urine, serum, placenta, breast tumor tissue and adipose tissue (35-38). Few studies

80

exist on the occurrence of parabens and their metabolites in wildlife such as marine mammals,

81

fish, birds, and invertebrates (18,23,24,26,39-41). Although these studies provide insight into the

82

bioaccumulation potential of parabens in marine ecosystems, information on the temporal trends

83

of parabens in marine organisms is lacking.

84

In this study, we collected mollusks from coastal areas along the Chinese Bohai Sea during

85

2006-2015 to investigate temporal trends of parabens and their metabolites. The bioaccumulation

86

potential of these substances was compared among 16 species of mollusk species analyzed.

87

Mollusks are consumed widely in coastal cities in China. Therefore, human dietary intake of

88

parabens through the consumption of mollusks was calculated. To our knowledge, this is the first

89

report on the occurrence and temporal patterns of parabens in a variety of invertebrate species

90

collected from a coastal marine ecosystem.

91 92

MATERIALS AND METHODS

93

Standards and Reagents. Six paraben analogues, MeP, EtP, PrP, BuP, BzP, and HepP as

94

well as their five metabolites, 4-HB, 3,4-DHB, OH-MeP, OH-EtP, and BA, were analyzed in this 4

ACS Paragon Plus Environment

Page 4 of 30

Page 5 of 30

Environmental Science & Technology

95

study. MeP, EtP, PrP, BuP, BzP, HepP and 4-HB were purchased from AccuStandard, Inc (New

96

Haven, CT, USA), and 3,4-DHB, OH-MeP, OH-EtP, BA, and formic acid (98.2%) were from

97

Sigma-Aldrich (St. Louis, MO, USA). Isotope labeled internal standards, including

98

13

99

(Andover, MA, USA); BzP-d4 and HepP-d4 were from CDN Isotopes (Pointe-Claire, Quebec,

100

Canada). HPLC grade of methanol and ethyl acetate were from Mallinckrodt Baker (Phillipsburg,

101

NJ, USA). Ultrapure water was prepared with the Milli-Q Ultrapure System (Barnstead

102

International, Dubuque, IA, USA).

C6-EtP,

13

C6-PrP,

13

C6-BuP, and

13

13

C6-MeP,

C6-4-HB, were from Cambridge Isotope Laboratories

103

Sample Collection. A total of 186 composite mollusk samples were collected from five

104

coastal cities, namely Tianjin, Shouguang, Penglai, Yantai, and Weihai, located along the Bohai

105

Sea each year (except for 2008) from 2006 to 2015 (Figure S1, Supporting Information). The

106

mollusk species analyzed were Neverita didyma (Nev), Rapana venosa (Rap), Mya arenaria

107

(Mya), Cyclina sinensis (Cyc), Chlamys farreri (Chl), Scapharca subcrenata (Sca), Meretrix

108

meretrix (Mer), Mytilus edulis (Myt), Crassostrea talienwhanensis (Ost), Amusium (Amu), and

109

Mactra veneriformis (Mac). Several other mollusk species were also sampled and analyzed,

110

including Neptunea cumingi, Ruditapes philippinarum, Venerupis variegate, Sinonovacula

111

constricta, and Moerella iridescens, but were not included in extensive data analyses due to

112

small sample sizes. Each species was identified by following the catalog of marine mollusks (42);

113

further details of mollusks are shown in Table S1 and Table S2 (Supporting Information).

114

Detailed procedure of sample collection and preparation has been described elsewhere (1-9).

115

In brief, after sampling, mollusks were depurated in filtered water for over 12 h and transported

116

to the laboratory on ice. After cleaning by tap water and then by ultrapure water, soft tissue was

117

collected by excision with a stainless steel scalpel. Each composite mollusk sample was obtained

5

ACS Paragon Plus Environment

Environmental Science & Technology

118

by homogenization of approximately 500-1500 g of wet soft tissue (consisting of 3-30

119

individuals). Approximately 558-5580 individuals were collected to prepare the 186 composite

120

mollusk samples. The samples were freeze-dried, homogenized, sieved and stored at -20 °C.

121

Sample Extraction. Mollusk samples were extracted by following the methods described

122

elsewhere, with some modifications (23,24,26,27). Briefly, ~0.5 g dry powdered mollusk sample

123

was weighed and placed in a 15-mL polypropylene conical tube (PP tube). A mixture of labeled

124

internal standards, 13C6-MeP, 13C6-EtP, 13C6-PrP, 13C6-BuP, BzP-d4, HepP-d4, and 13C6-4-HB (50

125

ng each), was spiked and equilibrated for 30 min at ambient temperature. The spiked sample was

126

extracted with 7 mL methanol by shaking in a mechanical shaker for 60 min and centrifuged at

127

4500×g for 5 min (Eppendorf Centrifuge 5804, Hamburg, Germany). The supernatant was

128

transferred into another PP tube. Extraction was repeated with 7 mL ethyl acetate and the

129

supernatant was combined. After freezing the extracts at -20°C overnight, they were immediately

130

centrifuged at 4500×g for 5 min, and the supernatant was collected, evaporated to approximately

131

1 mL under a gentle stream of nitrogen, and reconstituted to 10 mL with 0.2% formic acid in

132

water. The extract was loaded onto an Oasis MCX cartridge (150 mg/6 mL; Waters, Milford,

133

MA, USA) preconditioned with 6 mL methanol and 6 mL 0.2% formic acid in water. The

134

cartridge was rinsed with 12 mL of 20% methanol in water and 6 mL of water, and dried under

135

vacuum for 5 min. The analytes were eluted with 3 mL methanol and 3 mL ethyl acetate. The

136

eluate was concentrated to 1 mL under nitrogen, centrifuged at 4500×g for 5 min, and transferred

137

into a HPLC-vial for analysis.

138

Instrumental Analysis. Target compounds were analyzed using a Shimadzu Prominence

139

series LC-20AD system (Shimadzu USA, Canby, OR, USA) coupled with an Applied

140

Biosystems API 3200 electrospray triple-quadrupole mass spectrometer (ESI-MS/MS; Applied

6

ACS Paragon Plus Environment

Page 6 of 30

Page 7 of 30

Environmental Science & Technology

141

Biosystems, Foster City, CA, USA), operated under the negative ionization multiple reaction

142

monitoring (MRM) mode. The chromatographic separation of target compounds was achieved

143

by a Zorbax SB-Aq column (2.1×150 mm, 3.5 µm; Agilent Technologies Inc., Santa Clara, CA,

144

USA) serially connected to a Javelin guard column (Betasil C18, 2.1×20 mm, 5 µm; Thermo

145

Electron Corporation, Waltham, MA, USA). A 10 µL aliquot of the sample extract was injected

146

onto the analytical column. The mobile phase consisted of methanol (A) and 0.1% formic acid

147

in water (B) at a flow rate of 300 µL/min. The gradient elution program of the mobile phase is

148

shown in Table S3. The cone voltage, collision energy, and capillary voltage for the MS/MS

149

system were set at -30 V, -28 eV, and -4500 V, respectively; desolvation temperature was set at

150

450°C. Nitrogen was used as both curtain (flow rate: 20 psi) and collision gas (2 psi). The

151

compound-specific MRM transitions of target chemicals are presented in Table S4.

152

Quality Assurance and Quality Control (QA/QC). Because parabens are present in many

153

personal care products, the analyst refrained from using skin lotions and other products during

154

analysis. With each analytical batch of 60 samples, several procedural blanks (n = 3), spiked

155

blanks (n = 3), and spiked matrixes (n = 4) were included to evaluate the background

156

contamination and matrix effects arising from sample preparation and instrumental analyses.

157

Trace levels of MeP (0.313 ng/g), EtP (0.033 ng/g), 4-HB (4.88 ng/g), OH-MeP (0.182 ng/g),

158

OH-EtP (0.092 ng/g), and BA (43.5 ng/g) were found in procedural blanks. The concentrations

159

of target analytes detected in the procedural blanks were subtracted from sample values.

160

Absolute recoveries of target analytes (500 ng for 4-HB, 3,4-DHB and BA, and 50 ng for other

161

analytes) spiked into blanks and sample matrixes were in the range of 43.7% (3,4-DHB)-119%

162

(OH-EtP) and 62.5% (4-HB)-137% (OH-EtP), respectively. Nevertheless, the isotope dilution

163

method of quantification accounted for the low recoveries found for certain target analytes.

7

ACS Paragon Plus Environment

Environmental Science & Technology

Page 8 of 30

164

Recoveries of internal standards spiked into all samples ranged from 70.6 % (HepP-d4) to 87.1%

165

(13C6-EtP) (Table S5). Several samples (n = 11) were randomly selected for analysis in duplicate,

166

and the concentrations measured in duplicate samples were within ± 20% of the mean.

167

Reported concentrations were corrected by the recoveries of internal standards;

13

C6-MeP

168

for MeP and OH-MeP, 13C6-EtP for EtP and OH-EtP, 13C6-PrP for PrP, 13C6-BuP for BuP, BzP-

169

d4 for BzP, HepP-d4 for HepP, and

170

point calibration curve at concentrations ranging from 0.1 to 200 ng/mL (1000 ng/mL for 4-HB,

171

3,4-DHB, and BA) was used for the quantification of target analytes, and the regression

172

coefficient (r) of the calibration curve was >0.99. The limits of quantification (LOQs) were 2.0

173

ng/g for 4-HB, 3,4-DHB and BA and 0.2 ng/g for other target analytes, which were calculated

174

from the concentration of the lowest acceptable calibration standard and a nominal sample

175

weight of 0.5 g (Table S4). With every 20 samples, a midpoint calibration standard was injected

176

as a check for instrumental drift. A pure solvent (methanol) was injected after every 10 samples

177

to prevent carry-over of analytes between samples.

13

C6-4-HB for 4-HB, 3,4-DHB and BA (Table S5). An 11-

178

Data Analysis. All data are presented on a dry weight basis unless stated otherwise. Values

179

below the LOQ were assigned a value equal to the LOQ divided by the square root of 2

180

(LOQ/√2) for statistical analysis. Statistical analyses were performed with Origin (Version 8.0)

181

and SPSS (Version 18.0). A one-sample Kolmogorov-Smirnov test, used for determination of

182

normality of the data, showed that the data did not follow a normal distribution. Geometric mean

183

and median values were used to describe the results. Mann-Whitney U test, a non-parametric test,

184

was used for the comparison of concentrations between groups. Correlations among the natural

185

logarithmic concentrations of target analytes in mollusks were assessed by linear regression

186

analysis.

8

ACS Paragon Plus Environment

Page 9 of 30

Environmental Science & Technology

187 188

RESULTS AND DISCUSSION

189

Parabens in Mollusks. MeP was found in all mollusk samples (detection frequency [df]:

190

100%; n = 186) at concentrations ranging from 1.38 to 298 ng/g, dry wt, with a geometric mean

191

(GM) concentration of 19.1 ng/g (Table 1). The highest concentration of MeP (298 ng/g) was

192

found in a Rap species collected near Yantai city in 2012. Notable concentrations of MeP were

193

also found in Cyc (217 ng/g; [location/year] Penglai, 2015), Nev (109 ng/g; Weihai, 2012), Myt

194

(103 ng/g; Penglai, 2013), and Mer (102 ng/g; Shouguang, 2013). EtP was also frequently

195

(84.9%) detected in mollusk samples at a concentration range of below the LOQ (nd) to 18.2

196

ng/g, and the GM and median concentrations were 1.20 and 1.29 ng/g, respectively (for the

197

entire sample set). The GM and 95th percentile concentrations of PrP (df = 34.4%) were 0.289

198

and 34.4 ng/g, respectively. A positive correlation was found between the natural logarithmic

199

(ln) concentrations of MeP (ln[MeP]) and PrP (ln[PrP]) (p < 0.01; Figure 1a). This pattern is

200

consistent with what have been reported in our earlier studies, which showed a significant

201

positive correlation between MeP and PrP concentrations in foodstuff, indoor dust, sediment,

202

sewage sludge, and human urine (22,30-32,43,44). The correlation between MeP and PrP

203

concentrations suggests that these two parabens are often used in combination to improve the

204

antimicrobial activity in various products (15). The concentrations of PrP were higher than those

205

of EtP in some mollusks, whereas EtP concentrations were higher in other cases, a pattern

206

similar to that reported for marine animals and birds previously (23,24,26). Besides MeP and PrP,

207

EtP is also used in consumer products. BuP, BzP, and HepP were rarely detected in mollusks

208

( 0.05), which is similar to those reported earlier for

229

organochlorines, polybrominated diphenyl ethers, hexabromocyclododecanes, and chlorinated

230

paraffins (4,7,8). The five cities are located in the southern Bohai Sea and most of these cities are

231

highly industrialized and urbanized, particularly Tianjin (1). The measured concentrations of

232

parabens in mollusks from the Bohai Sea (ΣPBs mean: 32.9-43.8, ΣPBs range: 2.66-299 ng/g dw;

10

ACS Paragon Plus Environment

Page 11 of 30

Environmental Science & Technology

233

Figure S2a) were approximately 1-2 orders of magnitude higher than those found in mollusks

234

from the northern coast of Spain (nd-7.83 ng/g dw; ref. 41), and in fish from several rivers in

235

Spain (nd-92.1 ng/g dw; ref. 40). However, ΣPB concentrations measured in mollusks in our

236

study were lower than those found in liver, kidney, blubber and brain of marine mammals from

237

the United States coastal waters (where concentration of MeP was up to 865 ng/g wet wt in the

238

liver of bottlenose dolphin; ref. 23). This is probably attributable to the higher trophic level of

239

marine mammals (4,7,8, 26).

240

Paraben Metabolites in Mollusks. Among paraben metabolites analyzed, 4-HB and BA

241

were the two predominant compounds found in all mollusks (100%). The respective GM, median,

242

and range of concentrations for the entire sample set were 7540, 7180, and 978-161000 ng/g, dry

243

wt, for 4-HB, and 2840, 2730, and 344-49700 ng/g for BA (Table 1). The highest 4-HB

244

concentration (161000 ng/g) was found in a Myt species collected near the city of Penglai in

245

2013. It is interesting that this Myt sample also contained the highest BA concentration (49700

246

ng/g). 3,4-DHB was also frequently (91.9%) found in mollusk samples at GM, median, and

247

range of concentrations of 38.5, 47.8, and nd-4960 ng/g, respectively, which were 2 orders of

248

magnitude lower than those found for 4-HB and BA (p < 0.01). The detection frequency of OH-

249

MeP was high (71.5%), but its concentration was 3-4 orders of magnitude lower than those of 4-

250

HB and BA (GM: 0.553 vs 7540 and 2840 ng/g, respectively). OH-EtP was rarely found in

251

mollusk samples, at a GM concentration of 0.291 ng/g, comparable to or slightly lower than that

252

of OH-MeP.

253

Elevated concentrations of paraben metabolites have been reported to occur in

254

environmental matrixes, human samples, and pet food (22-27, 38). 4-HB, 3,4-DHB and BA

255

originate from multiple sources including biological transformation of parabens, whereas OH-

11

ACS Paragon Plus Environment

Environmental Science & Technology

256

MeP and OH-EtP are specific metabolites of MeP and EtP, respectively. 4-HB has been reported

257

to occur naturally in certain plants, including seagrasses (45). In our study, a significant positive

258

correlation was found between MeP and OH-MeP concentrations (p < 0.01; Figure 1c),

259

suggesting the existence of a common source for these chemicals (23,24,26). In other words, this

260

indicates that OH-MeP is a specific metabolite of MeP in mollusks. MeP and 4-HB

261

concentrations showed a positive correlation (p < 0.01, Figure 1b), which suggests a common

262

source for MeP and 4-HB and/or biotransformation/interconversion of MeP and 4-HB by

263

methylating and demethylating microorganisms in the aquatic environment (46). Nevertheless,

264

the concentrations of 4-HB (GM: 7540 ng/g) were significantly higher than those of the six

265

parent parabens determined (GM range: 0.144-19.1 ng/g; Table 1), suggesting the existence of

266

additional sources of 4-HB (23). There were no other significant correlations observed between

267

other parabens and the metabolites analyzed, which is consistent with the findings in our recent

268

study on trophic magnification of parabens and their metabolites in a marine food chain (26).

269

Positive correlations were also found among 4-HB, 3,4-DHB, and BA concentrations (Figures

270

1e-1g), suggesting similar sources of exposure for these compounds.

271

A similar accumulation pattern of ΣMBs (sum of five metabolites) was found among

272

various species of mollusks (Figure 2b). The highest ΣMB concentrations were found in Myt

273

(mean: 42700, median: 16900 ng/g, dry wt), followed by Mac (27100, 21500 ng/g), Cyc (25200,

274

17200 ng/g), and Mya (20000, 15300 ng/g). The ΣMB concentrations in Myt were 4-6 times

275

higher than those in Nev (mean: 7820, median: 7400 ng/g), Sca (9240, 8370 ng/g), and Rap

276

(10900, 8390 ng/g) (p < 0.01). The spatial distribution of paraben metabolites in mollusks was

277

also investigated. The mean ΣMB concentrations ranged from 13100 (Weihai) to 19600 ng/g

278

(Penglai), which were 3 orders of magnitude higher than the mean ΣPB concentrations (range:

12

ACS Paragon Plus Environment

Page 12 of 30

Page 13 of 30

Environmental Science & Technology

279

32.9-43.8 ng/g for the entire sample set; Figure S2b). No significant difference in ΣMBs was

280

found among the sampling sites (p > 0.05).

281

Temporal Trends. The mollusk samples were collected along the Chinese Bohai Sea each

282

year (except for 2008) from 2006 to 2015 (Figure S1). A gradual increase in MeP concentrations

283

was found in mollusk samples collected between 2006 and 2012 and, thereafter, the

284

concentrations remained stable or slightly decreased, except for 2014 (Table 1). The GM MeP

285

concentrations in mollusks collected between 2012 and 2015 were in the range of 31.3-45.0 ng/g,

286

which were 2-3 times higher than those found for 2006 (GM: 14.6 ng/g) (p < 0.05). A similar

287

temporal trend was observed for total paraben concentrations in mollusks. Because of their

288

broad-spectrum antimicrobial activities, parabens are added as ingredients in cosmetics,

289

pharmaceuticals, and foodstuffs (12-14). Rastogi et al. (47) reported that 77% of rinse-off and

290

99% of leave-on cosmetics contained parabens as preservatives. Based on the US FDA database,

291

the Cosmetic Ingredient Review Expert Panel estimated that the number of parabens used in

292

cosmetics in 2006 was 1.7 times higher than that it was in 1981 (15). It has been reported that

293

most paraben-containing products are produced in China or India (13). With the increase in the

294

usage of paraben-containing products, the discharge of parabens into the environment is

295

inevitably increasing. This may contribute to the gradual increase in MeP concentrations in

296

mollusks from the Bohai Sea in recent years.

297

The distribution of individual parabens (expressed as percentage of the total) was calculated

298

for mollusks collected every year. No obvious difference in the distribution profile of parabens

299

was observed from 2006 to 2015. MeP, EtP and PrP were the three dominant compounds,

300

accounting for 81.4% ± 17.1% (mean ± SD), 10.6% ± 13.4%, and 3.34% ± 6.28%,

301

respectively, of the total paraben concentrations (for the entire sample set; Figure S3a). This

13

ACS Paragon Plus Environment

Environmental Science & Technology

302

composition pattern of parabens in mollusks is in agreement with that reported for foodstuffs,

303

human urine, blood, and breast milk (31,32,35-37,48).

304

There were no obvious temporal trends for paraben metabolites found in mollusk samples

305

collected during 2006 to 2015. The highest concentrations of 4-HB were found in mollusks

306

collected in 2013 (GM: 18600, median: 18800 ng/g), followed by those collected in 2010 (12800,

307

12700 ng/g), 2007 (12400, 8090 ng/g), and 2006 (9410, 7130 ng/g) (Table 1). For BA, mollusks

308

collected in 2007 contained relatively higher concentrations (GM: 5400, median: 3810 ng/g) than

309

those collected in 2010 (5070, 4310 ng/g). The temporal variation in the total concentrations of

310

total paraben metabolites (ΣMBs) was similar to that found for BA. 4-HB and BA collectively

311

accounted for over 99% of the total paraben metabolite concentrations during 2006 - 2015

312

(Figure S3b).

313

Principal component analysis (PCA) was performed on the concentrations to identify

314

potential patterns for source apportionment of parabens in mollusks (Figure S4a). The first two

315

PCs collectively accounted for 100% (PC1 and PC2 accounting for approximately 95% and 5%,

316

respectively) of the total variance in concentrations of parabens and their metabolites. Almost all

317

mollusk species clustered in the form of an arc, suggesting the existence of a common source of

318

exposure to these chemicals. The coastal region of the Bohai Sea is highly industrialized and

319

urbanized (1). Several rivers flow into the Bohai Sea, carrying huge amounts of industrial and

320

domestic wastewater. Parabens present in mollusks are likely from the discharge of wastewater

321

(25). The PCA was also performed for the concentrations of parent and metabolic parabens in

322

mollusks (Figure S4b) and for various years (Figure S4c). The first two PCs were extracted,

323

which collectively explained almost 100% the total variance in the original data and all mollusks,

324

either from different locations or years, distributed on the PC plot in the form of an arc. These

14

ACS Paragon Plus Environment

Page 14 of 30

Page 15 of 30

Environmental Science & Technology

325

results further suggest that there exists a similar source of exposure of all species of mollusks and

326

for all sampling years.

327

Dietary Exposure Estimation. Based on the concentrations measured and the daily

328

ingestion rate of mollusks in China, we calculated the estimated dietary intake (EDI; ng/kg

329

bw/day) of parabens and their metabolites, as shown in eq 1:

330

EDI =

஼×஽஼ ஻ௐ

(1)

331

where C is the concentration of parabens and their metabolites in mollusks (ng/g), DC is the

332

daily consumption rate of mollusks in China (g/day wet wt), and BW is the body weight (kg). For

333

C, the GM and 95th percentile concentrations were used for average and high exposure

334

scenarios, respectively. For DC, the values were adopted from a nationwide survey in China (49).

335

For EDI calculation, we stratified the population into three age groups: toddlers (2-5 years),

336

children & teenagers (6-17 years), and adults (≥18 years); the corresponding BW for males and

337

females of different age groups was obtained from an earlier study (50). Infants (0-1 year) were

338

excluded from the EDI calculation, since this age group was assumed not to consume mollusks.

339

Detailed parameters used in the EDI calculation are presented in Table S6. It should be noted that

340

the concentrations of parabens and their metabolites in mollusks were converted from a dry

341

weight to a wet weight basis for EDI calculation, as the DC data were presented on a wet weight

342

basis (49). Based on the moisture content of mollusks reported in several previous studies (51-

343

53), we used a value of 80% moisture content for the conversion of concentrations from dry

344

weight to wet weight basis.

345

The daily intakes of parabens and their metabolites for the general population in China

346

through consumption of mollusks are summarized in Table 2. As there was no significant gender

347

difference in EDI values, we primarily describe the mean values calculated for males and

15

ACS Paragon Plus Environment

Environmental Science & Technology

348

females combined. The GM and 95th percentile EDIs of total parabens for toddlers were 3.87

349

and 18.3 ng/kg bw/day, respectively, which were higher than those estimated for children and

350

teenagers (3.24 and 15.3 ng/kg bw/day) and adults (2.48 and 11.7 ng/kg bw/day). Among

351

paraben analogues, MeP was the major contributor to ΣPB EDIs, and the daily intakes of MeP

352

(GM) were approximately 16 times higher those estimated for EtP, and 2 orders of magnitude

353

higher than those estimated for PrP, BuP, BzP and HepP for all age groups. An acceptable daily

354

intake (ADI) for the sum of three parabens (MeP, EtP and PrP) at 10 mg/kg bw/day was

355

recommended by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1974

356

(54). The GM EDIs of parabens were 7 orders of magnitude lower than the ADI for all age

357

groups. The daily intake of parabens from the consumption of mollusks in adult females

358

(EDI=2.42 ng/kg bw/day; Table 2) was much lower than that attributed to the use of cosmetics in

359

Chinese female adults (341000 ng/kg bw/day) (34).

360

The highest GM and 95th percentile EDIs of total paraben metabolites were found for

361

toddlers (GM EDI: 1740, 95th percentile EDI: 7770 ng/kg bw/day), followed by children &

362

teenagers (1460 and 6520 ng/kg bw/day) and adults (1110 and 4980 ng/kg bw/day) (Table 2).

363

Among paraben metabolites, 4-HB was the most predominant contributor to the ΣMB EDIs. The

364

daily intake of 4-HB (GM doses: 775-1210 ng/kg bw/day) was approximately 2.7-fold higher

365

than those of BA (292-456 ng/kg bw/day). An ADI value of 5 mg/kg bw/day was proposed for

366

BA by the WHO in 1997 (55). The GM and 95th percentile EDIs of BA calculated in our study

367

were 3-4 orders of magnitude lower than the ADI value. Considering the fact that mollusks are

368

only a part of the total diet, exposure of BA from the consumption of mollusks cannot be ignored.

369

Other source of BA exposure can also augment current EDI values. To date, there are no ADI

370

values proposed for 4-HB, 3,4-DHB, OH-MeP, and OH-EtP. However, as the EDI value of 4-HB

16

ACS Paragon Plus Environment

Page 16 of 30

Page 17 of 30

Environmental Science & Technology

371

was approximately 2.7-fold higher than that of BA, exposure to 4-HB through the consumption

372

of mollusks is a concern.

373

It should be noted that there exist several uncertainties in our exposure assessment. Owing

374

to the lack of species-specific daily consumption rates for mollusks, we used an average daily

375

seafood consumption rate for EDI calculations, which may over- or underestimate the actual

376

exposures. The concentrations of parabens and their metabolites widely varied even within a

377

single species of mollusk. For instance, 4-HB concentrations in Myt ranged from 7620 to 161000

378

(Figure 2b). The large variation in the concentrations of parabens and their metabolites in

379

mollusks adds uncertainty in our intake estimates.

380

In summary, widespread occurrence and high concentrations of parabens and their

381

metabolites were found in marine mollusks collected from the Chinese Bohai Sea during 2006-

382

2015. MeP and 4-HB were the predominant compounds found among parent and metabolic

383

parabens, respectively. MeP concentrations ranged from 1.38 to 298 ng/g dw, which were 2-3

384

orders of magnitude lower than those found for 4-HB (978-161000 ng/g dw). MeP and 4-HB

385

concentrations had a significant positive correlation, suggesting the existence of a common

386

source of exposure to these chemicals in mollusks. Among mollusk species, Mactra veneriformis

387

(Mac), Mytilus edulis (Myt), and Cyclina sinensis (Cyc) exhibited high bioaccumulation of both

388

parent and metabolic parabens. Paraben concentrations in mollusks increased during 2006-2012,

389

while no obvious temporal trend was found for paraben metabolites. Elevated concentrations of

390

4-HB in mollusks is a cause for concern in terms of human exposure. To our knowledge, this is

391

the first study to describe the occurrence and temporal trends of parent and metabolic parabens in

392

invertebrate species collected from coastal marine environments.

393

17

ACS Paragon Plus Environment

Environmental Science & Technology

394

SUPPORTING INFORMATION

395

Additional details as described in the text (6 tables and 4 figures). Denomination of the selected

396

mollusks (Table S1). Sample information of mollusks collected from several coastal cities along

397

the Bohai Sea, China during 2006-2015 (Table S2). HPLC gradient parameters optimized for

398

analysis of target compounds (Table S3). Molecular formula and MRM transitions monitored for

399

target compounds in mollusks (Table S4). Blank spike and matrix spike recoveries for target

400

compounds (Table S5). Per capita daily consumption rate of mollusks used for estimation of

401

human exposure to parabens and their metabolites (Table S6). Map showing the study area and

402

sampling sites (Figure S1). Spatial distributions of parabens and their metabolites in mollusks

403

collected from coastal sites along the Chinese Bohai Sea (Figure S2). Composition profiles of

404

parabens and their metabolites in mollusks collected from Chinese Bohai Sea during 2006-2015

405

(Figure S3).

406

collected from the Chinese Bohai Sea during 2006-2015 (Figure S4). The Supporting

407

Information is available free of charge on the ACS Publications website at DOI:

408

10.1021/acs.est.xxx.

Principal component analysis of parabens and their metabolites in mollusks

409 410

ACKNOWLEDGMENTS

411

All samples were analyzed at Wadsworth Center. This work was partly supported by the

412

National Natural Science Foundation of China (21522706 and 21677167) and the Thousand

413

Young Talents Program of China. We thank Prof. Guibin Jiang (Research Center for Eco-

414

Environmental Sciences) for sample collection. We also thank Dr. Jingchuan Xue (Wadsworth

415

Center) for help with sample analysis.

416

18

ACS Paragon Plus Environment

Page 18 of 30

Page 19 of 30

Environmental Science & Technology

417

REFERENCES

418 419 420 421 422

1.

Qu, G.; Liu, A.; Wang, T.; Zhang, C.; Fu, J.; Yu, M.; Sun, J.; Zhu, N.; Li, Z.; Wei, G.; Du, Y.; Shi, J.; Liu, S.; Jiang G. Identification of tetrabromobisphenol A allyl ether and tetrabromobisphenol A 2,3-dibromopropyl ether in the ambient environment near a manufacturing site and in mollusks at a coastal region. Environ. Sci. Technol. 2013, 47 (9), 4760-4767.

423 424

2.

Yang, R. Q.; Yao, Z. W.; Jiang, G. B.; Zhou, Q. F.; Liu, J. Y. HCH and DDT residues in mollusks from Chinese Bohai coastal sites. Mar. Pollut. Bull. 2004, 48, 795-805.

425 426 427

3.

Wang, Y.; Liang, L.; Shi, J.; Jiang, G. Study on the contamination of heavy metals and their correlations in mollusks collected from coastal sites along the Chinese Bohai Sea. Environ. Int. 2005, 31 (8), 1103-1113.

428 429 430 431

4.

Wang, Y.; Wang, T.; Li, A.; Fu, J.; Wang, P.; Zhang, Q.; Jiang, G. Selection of bioindicators of polybrominated diphenyl ethers, polychlorinated biphenyls, and organochlorine pesticides in mollusks in the Chinese Bohai Sea. Environ. Sci. Technol. 2008, 42 (19), 7159-7165.

432 433 434

5.

Zhao, X.; Zheng, M.; Liang, L.; Zhang, Q.; Jiang, G. Assessment of PCBs and PCDD/Fs along the Chinese Bohai Sea coastline using mollusks as bioindicators. Arch. Environ. Contam. Toxicol. 2005, 49, 178-185.

435 436 437

6.

Pan, Y. Y.; Shi, Y. L.; Wang, Y. W.; Cai, Y. Q.; Jiang, G. B. Investigation of perfluorinated compounds (PFCs) in mollusks from coastal waters in the Bohai Sea of China. J. Environ. Monit. 2010, 12, 508-513.

438 439 440

7.

Yuan, B.; Wang, T.; Zhu, N.; Zhang, K.; Zeng, L.; Fu, J.; Wang, Y.; Jiang, G. Short chain chlorinated paraffins in mollusks from coastal waters in the Chinese Bohai Sea. Environ. Sci. Technol. 2012, 46 (12), 6489-6496.

441 442 443 444

8.

Zhu, N.; Li, A.; Wang, T.; Wang, P.; Qu, G.; Ruan, T.; Fu, J.; Yuan, B.; Zeng, L.; Wang, Y.; Jiang, G. Tris(2,3-dibromopropyl) isocyanurate, hexabromocyclododecanes, and polybrominated diphenyl ethers in mollusks from Chinese Bohai Sea. Environ. Sci. Technol. 2012, 46 (13), 7174-7181.

445 446 447 448

9.

Liu, Y.; Ruan, T.; Lin, Y.; Liu, A.; Yu, M.; Liu, R.; Meng, M.; Wang, Y.; Liu, J.; Jiang G. Chlorinated Polyfluoroalkyl Ether Sulfonic Acids in Marine Organisms from Bohai Sea, China: Occurrence, Temporal Variations, and Trophic Transfer Behavior. Environ. Sci. Technol. 2017, 51 (8), 4407-4414.

449 450

10. Tanabe, S.; Subramanian, A. Bioindicators of POPs: Monitoring in Developing Countries. Kyoto University Press: Kyoto, Japan, 2006.

451 452

11. Isobe, T.; Takada, H.; Kanai, M.; Tsutsumi, S.; Isobe, K. O.; Boonyatumanond, R.; Zakaria, M. P. Distribution of polycyclic aromatic hydrocarbons (PAHs) and phenolic endocrine

19

ACS Paragon Plus Environment

Environmental Science & Technology

453 454

disrupting chemicals in south and southeast Asian mussels. Environ. Monitor. Assess. 2007, 135, 423-440.

455 456

12. Błędzka, D.; Gromadzińska, J.; Wąsowicz, W. Parabens. From environmental studies to human health. Environ. Int. 2014, 67, 27-42.

457 458

13. Haman, C.; Dauchy, X.; Rosin, C.; Munoz, J. F. Occurrence, fate and behavior of parabens in aquatic environments: a review. Water Res. 2015, 68, 1-11.

459

14. Darbre, P. D. Endocrine Disruptors and Obesity. Curr. Obes. Rep. 2017, 6 (1), 18-27.

460 461

15. Andersen, F. A. Final amended report on the safety assessment of methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, isobutylparaben, and

462 463 464 465 466

benzylparaben as used in cosmetic products. Int. J. Toxicol. 2008, 27, 1-82. 16. Soni, M. G.; Carabin, I. G.; Buradock, G. A. Safety assessment of esters of phydroxybenzoic acid (parabens). Food Chem. Toxicol. 2005, 43, 985-1015. 17. Elder, R. L. Final report on the safety assessment of methylparaben, ethylparaben, propylparaben and butylparaben. J. Am. Coll. Toxicol. 1984, 3, 147-209.

467 468 469 470 471

18. Ramaswamy, B. R.; Kim, J. W.; Isobe, T.; Chang, K. H.; Amano, A.; Miller, T. W.; Siringan, F. P.; Tanabe S. Determination of preservative and antimicrobial compounds in fish from Manila Bay, Philippines using ultra high performance liquid chromatography tandem mass spectrometry, and assessment of human dietary exposure. J. Hazard Mater. 2011, 192 (3), 1739-1745.

472 473

19. Ste-Marie, L.; Vachon, L.; Bemeur, C.; Lambert, J.; Montgomery, J. Local striatal infusion of MPP+ does not result in increased hydroxylation after systemic administration of 4-

474 475 476 477

hydroxybenzoate. Free Radical Biol. Med. 1999, 27, 997-1007. 20. Liu, M.; Liu, S.; Peterson, S. L.; Miyake, M.; Liu, K. J. On the application of 4hydroxybenzoic acid as a trapping agent to study hydroxyl radical generation during cerebral ischemia and reperfusion. Mol. Cell. Biochem. 2002, 234-235, 379-385.

478 479 480 481

21. Jewell, C.; Prusakiewicz, J. J.; Ackermann, C.; Payne, N. A.; Fate, G.; Voorman, R.; Williams, F. M. Hydrolysis of a series of parabens by skin microsomes and cytosol from human and minipigs and in whole skin in short-term culture. Toxicol. Appl. Pharmacol. 2007, 225 (2), 221-228.

482 483

22. Wang, L.; Kannan, K. Alkyl protocatechuates as novel urinary biomarkers of exposure to phydroxybenzoic acid esters (parabens). Environ. Int. 2013, 59, 27-32.

20

ACS Paragon Plus Environment

Page 20 of 30

Page 21 of 30

Environmental Science & Technology

484 485 486

23. Xue, J.; Sasaki, N.; Elangovan, M.; Diamond, G.; Kannan, K. Elevated Accumulation of Parabens and their Metabolites in Marine Mammals from the United States Coastal Waters. Environ. Sci. Technol. 2015, 49 (20), 12071-12079.

487 488

24. Xue, J.; Kannan, K. Accumulation profiles of parabens and their metabolites in fish, black bear, and birds, including bald eagles and albatrosses. Environ. Int. 2016, 94, 546-553.

489 490 491

25. Wang, W.; Kannan, K. Fate of Parabens and Their Metabolites in Two Wastewater Treatment Plants in New York State, United States. Environ. Sci. Technol. 2016, 50 (3), 1174-1181.

492 493 494

26. Xue, X.; Xue, J.; Liu, W.; Adams, D. H.; Kannan, K. Trophic Magnification of Parabens and Their Metabolites in a Subtropical Marine Food Web. Environ. Sci. Technol. 2017, 51 (2), 780-789.

495 496 497

27. Karthikraj, R.; Borkar, S.; Lee, S.; Kannan, K. Parabens and Their Metabolites in Pet Food and Urine from New York State, United States. Environ. Sci. Technol. 2018, 52 (6), 37273737.

498 499

28. Boberg, J.; Taxvig, C.; Christiansen, S.; Hass, U. Possible endocrine disrupting effects of parabens and their metabolites. Reprod. Toxicol. 2010, 30 (2), 301-312.

500 501

29. Eriksson, E.; Andersen, H. R.; Ledin, A. Substance flow analysis of parabens in Denmark complemented with a survey of presence and frequency in various commodities. J. Hazard

502

Mater. 2008, 156 (1-3), 240-259.

503 504 505

30. Liao, C.; Lee, S.; Moon, H.; Yamashita, N.; Kannan, K. Parabens in sediment and sewage sludge from the United States, Japan, and Korea: Spatial distribution and temporal trends. Environ. Sci. Technol. 2013, 47, 10895-10902.

506 507

31. Liao, C.; Liu, F.; Kannan, K. Occurrence of and Dietary Exposure to Parabens in Foodstuffs from the United States. Environ. Sci. Technol. 2013, 47, 3918-3925.

508

32. Liao, C.; Chen, L.; Kannan, K. Occurrence of parabens in foodstuffs from China and its

509

implications for human dietary exposure. Environ. Int. 2013, 57-58, 68-74.

510 511 512

33. Guo, Y.; Kannan, K. A survey of phthalates and parabens in personal care products from the United States and its implications for human exposure. Environ. Sci. Technol. 2013, 47 (24), 14442-14449.

513 514 515

34. Guo, Y.; Wang, L.; Kannan, K. Phthalates and parabens in personal care products from China: concentrations and human exposure. Arch. Environ. Contam. Toxicol. 2014, 66 (1), 113-119.

21

ACS Paragon Plus Environment

Environmental Science & Technology

516

35. Calafat, A. M.; Ye, X.; Wong, L. Y.; Bishop, A. M.; Needham, L. L. Urinary concentrations

517

of four parabens in the U.S. population: NHANES 2005−2006. Environ. Health Perspect.

518

2010, 118 (5), 679-685.

519 520 521

36. Frederiksen, H.; Jorgensen, N.; Andersson, A. M. Parabens in urine, serum and seminal plasma from healthy Danish men determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). J. Exposure Sci. Environ. Epidemiol. 2011, 21 (3), 262-271.

522 523 524 525

37. Schlumpf, M.; Kypke, K.; Wittassek, M.; Angerer, J.; Mascher, H.; Mascher, D.; Vökt, C.; Birchler, M.; Lichtensteiger, W. Exposure patterns of UV filters, fragrances, parabens, phthalates, organochlorine pesticides, PBDEs, and PCBs in human milk: correlation of UV filters with use of cosmetics. Chemosphere 2010, 81 (10), 1171-1183.

526 527

38. Wang, L.; Asimakopoulos, A. G.; Kannan, K. Accumulation of 19 environmental phenolic and xenobiotic heterocyclic aromatic compounds in human adipose tissue. Environ. Int.

528

2015, 78, 45-50.

529 530 531

39. Renz, L.; Volz, C.; Michanowicz, D.; Ferrar, K.; Christian, C.; Lenzner, D.; El-Hefnawy, T. A study of parabens and bisphenol A in surface water and fish brain tissue from the Greater Pittsburgh Area. Ecotoxicology 2013, 22 (4), 632-641.

532 533 534 535

40. Jakimska, A.; Huerta, B.; Bargańska, Ż.; Kot-Wasik, A.; Rodríguez-Mozaz, S.; Barceló, D. Development of a liquid chromatography-tandem mass spectrometry procedure for determination of endocrine disrupting compounds in fish from Mediterranean rivers. J. Chromatogr. A 2013, 1306, 44-58.

536 537 538

41. Villaverde-de-Sáa, E.; Rodil, R.; Quintana, J. B.; Cela, R. Matrix solid-phase dispersion combined to liquid chromatography-tandem mass spectrometry for the determination of paraben preservatives in mollusks. J. Chromatogr. A 2016, 1459, 57-66.

539 540

42. Qi, Z. Y.; Ma, X. T.; Wang, Z. R.; Lin, G. Y.; Xu, F. X.; Dong, Z. Z.; Li, F. L.; Lv, R. H. Mollusca of Huanghai and Bohai Sea. Agriculture Publishing House, Beijing, China, 1989.

541 542 543 544

43. Wang, L.; Liao, C.; Liu, F.; Wu, Q.; Guo, Y.; Moon, H. B.; Nakata, H.; Kannan, K. Occurrence and human exposure of phydroxybenzoic acid esters (parabens), bisphenol A diglycidyl ether (BADGE), and their hydrolysis products in indoor dust from the United States and three east Asian countries. Environ. Sci. Technol. 2012, 46 (21), 11584-11593.

545 546 547

44. Wang L, Wu Y, Zhang W, Kannan K. Characteristic profiles of urinary p-hydroxybenzoic acid and its esters (parabens) in children and adults from the United States and China. Environ. Sci. Technol. 2013, 47 (4), 2069-2076.

548

45. Olga Zapata, C. M. Phenolic acids in seagrasses. Aquat. Bot. 1979, 7, 307-317.

22

ACS Paragon Plus Environment

Page 22 of 30

Page 23 of 30

549 550 551

Environmental Science & Technology

46. Peng, X.; Adachi, K.; Chen, C.; Kasai, H.; Kanoh, K.; Shizuri, Y.; Misawa, N. Discovery of a marine bacterium producing 4-hydroxybenzoate and its alkyl esters, parabens. Appl. Environ. Microbiol. 2006, 72 (8), 5556-5561.

552 553 554

47. Rastogi, S. C.; Schouten, A.; de Kruijf, N.; Weijland, J. W. Contents of methyl-, ethyl-, propyl-, butyl- and benzylparaben in cosmetic products. Contact Dermatitis 1995, 32, 2830.

555 556 557 558

48. Sandanger, T. M.; Huber, S.; Moe, M. K.; Braathen, T.; Leknes, H.; Lund, E. Plasma concentrations of parabens in postmenopausal women and self-reported use of personal care products: The NOWAC postgenome study. J. Exposure Sci. Environ. Epidemiol. 2011, 21 (6), 595-600.

559 560

49. Zhai, F. Y. A prospective study on dietary pattern and nutrition transition in china. Science Press: Beijing, China, 2008.

561 562 563

50. Yang, X.; Li, Y.; Ma, G.; Hu, X.; Wang, J.; Cui, C.; Wang, Z.; Yu, W.; Yang, Z.; Zhai, F. Study on weight and height of the Chinese people and the differences between 1992 and 2002. Chin. J. Epidemiol. 2005, 26, 489-493 (in Chinese).

564 565 566

51. O’Connor, T. P. Concentrations of organic contaminants in mollusks and sediments at NOAA National Status and Trend sites in the coastal and estuarine United States. Environ. Health Perspect. 1991, 90, 69-73.

567 568 569

52. Bayen, S.; Gong, Y.; Chin, H. S.; Lee, H. K.; Leong, Y. E.; Obbard, J. P. Androgenic and estrogenic response of green mussel extracts from Singapore's coastal environment using a human cell-based bioassay. Environ. Health Perspect. 2004, 112 (15), 1467-1471.

570 571 572 573

53. Villaverde-de-Sáa, E.; Valls-Cantenys, C.; Quintana, J. B.; Rodil, R.; Cela, R. Matrix solidphase dispersion combined with gas chromatography-mass spectrometry for the determination of fifteen halogenated flame retardants in mollusks. J. Chromatogr. A 2013, 1300, 85-94.

574 575 576 577 578

54. JECFA (the Joint FAO/WHO Expert Committee on Food Additives). Toxicological Evaluation of Certain Food Additives with a Review of General Principles and of Specifications; 17th Report of the Joint FAO/WHO Expert Committee on Food Additives. World Health Organization Technical Report Series no. 539; FAO and WHO: Switzerland, 1974.

579 580 581

55. World Health Organization. Evaluation of certain food additives: forty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series No. 868. Geneva: WHO; 1997.

582 583

23

ACS Paragon Plus Environment

Environmental Science & Technology

Page 24 of 30

584

Table 1. Concentrations (ng/g dw) of Parabens and Their Metabolites in Mollusks Collected from the Chinese Bohai Sea during

585

2006-2015. MeP

EtP

PrP

BuP

BzP

HepP

GM

14.6

0.576

0.886

0.647

0.141

0.141

Median

12.2

0.472

1.24

0.141

0.141

P95

54.1

3.25

2.55

5.82

DF (%)

100

88.2

82.4

Range

3.83-56.1

nd-3.29

nd-4.03

ΣPBs

4-HB

3,4-DHB

OH-MeP

OH-EtP

BA

ΣMBs

19.3

9410

57.1

0.796

0.225

4160

14000

0.141

18.0

7130

54.7

0.755

0.141

3460

10700

0.141

0.141

59.7

55000

180

1.30

0.844

16200

70200

47.1

0

0

100

100

100

100

35.3

100

100

nd-7.04

nd

nd

4.81-60.6

3020-73100

18.7-331

0.489-1.81

nd-1.79

1310-18600

4370-89000

12400

58.1

0.717

0.141

5400

18400

2006 (n=17)

2007 (n=10) GM

16.9

0.570

0.801

0.237

0.141

0.158

21.3

Median

15.7

0.593

1.08

0.141

0.141

0.141

17.6

8090

47.1

0.635

0.141

3810

13700

P95

93.4

1.54

6.79

1.90

0.141

0.294

96.4

58100

242

1.71

0.141

14700

72400

DF (%)

100

90.0

70.0

20.0

0

10

100

100

100

100

0

100

100

Range

3.29-95.9

nd-1.63

nd-8.53

nd-2.11

nd

nd-0.420

6.62-96.7

4270-71000

25.8-243

0.282-1.80

nd

2410-17100

6700-82900

GM

22.6

1.03

0.645

0.204

0.141

0.141

26.8

8540

48.5

0.848

0.196

3970

12900

Median

23.4

0.864

0.948

0.141

0.141

0.141

27.2

8320

50.5

0.879

0.141

3400

12600

P95

99.4

2.36

1.98

5.17

0.141

0.141

103

18100

127

2.29

1.50

8670

27000

DF (%)

100

100

70.0

10.0

0

0

100

100

100

100

23.3

100

100

Range

4.84-147

0.367-6.21

nd-3.43

nd-13.9

nd

nd

6.76-150

3330-34100

11.7-242

0.246-2.61

nd-2.04

1480-16600

4840-41700

12800

89.1

0.661

0.256

5070

18300

2009 (n=30)

2010 (n=13) GM

17.3

1.88

0.181

0.256

0.141

0.141

21.9

Median

14.6

2.60

0.141

0.141

0.141

0.141

17.1

12700

87.1

0.673

0.141

4310

17700

P95

51.7

3.52

0.693

6.61

0.141

0.141

57.2

27100

413

0.999

1.53

22100

49600

DF (%)

100

92.3

15.4

15.4

0

0

100

100

100

100

38.5

100

100

Range

6.32-54.5

nd-3.59

nd-0.804

nd-7.60

nd

nd

10.2-63.5

3810-28500

11.2-703

0.462-1.05

nd-2.48

1650-22500

5480-51100

GM

11.4

4.34

0.433

0.176

0.141

0.141

18.8

7160

50.9

0.441

1.10

3210

10700

Median

8.68

4.38

0.546

0.141

0.141

0.141

17.0

6980

43.3

0.390

1.21

2790

9990

P95

94.5

13.3

1.33

0.141

0.141

0.141

103

13100

127

2.71

3.17

12600

28400

DF (%)

100

100

72.7

4.55

0

0

100

100

100

77.3

95.5

100

100

2011 (n=22)

24

ACS Paragon Plus Environment

Page 25 of 30

Range

Environmental Science & Technology

2.82-109

0.728-13.9

nd-2.02

nd-16.6

nd

nd

5.82-113

3450-21100

20.8-132

nd-3.67

nd-6.37

1260-19400

4750-32600

2012 (n=26) GM

45.0

0.629

0.141

0.150

0.141

0.141

46.9

5100

11.1

1.68

0.175

1730

7030

Median

39.8

0.824

0.141

0.141

0.141

0.141

41.6

4660

17.4

1.57

0.141

1460

6690

P95

161

2.24

0.141

0.141

0.141

0.141

164

10200

55.9

3.86

0.466

5820

13500

DF (%)

100

73.1

0

3.85

0

0

100

100

76.9

100

15.4

100

100

Range

12.8-298

nd-6.00

nd

nd-0.636

nd

nd

14.0-299

2610-17500

nd-107

0.471-4.59

nd-3.28

748-6940

3360-21100

2013 (n=12) GM

42.5

0.770

0.141

0.166

0.141

0.141

45.0

18600

114

1.07

0.488

8780

29400

Median

39.9

0.930

0.141

0.141

0.141

0.141

41.7

18800

201

1.19

0.141

7200

26200

P95

102

10.1

0.141

0.501

0.141

0.141

111

101000

2522

5.50

6.87

38900

132000

DF (%)

100

66.7

0

8.33

0

0

100

100

83.3

91.7

41.7

100

100

Range

14.4-103

nd-17.3

nd

nd-0.940

nd

nd

17.4-121

2910-161000

nd-4960

nd-8.17

nd-8.64

3350-49700

6290-215000

2014 (n=35) GM

8.25

0.989

0.182

0.141

0.141

0.152

11.6

6180

70.7

0.231

0.361

1550

8010

Median

8.31

0.937

0.141

0.141

0.141

0.141

10.2

6360

82.4

0.141

0.141

1480

7800

P95

29.9

11.7

0.707

0.141

0.141

0.141

50.4

12000

173

4.38

3.06

4840

18400

DF (%)

100

68.6

8.57

0

0

2.86

100

100

97.1

17.1

37.1

100

100

Range

1.38-71.7

nd-16.0

nd-34.9

nd

nd

nd-1.97

2.66-74.3

1160-17500

nd-257

nd-5.42

nd-3.66

366-25500

1520-38300

2015 (n=21) GM

31.3

2.99

0.158

0.141

0.175

0.141

37.6

4250

6.79

0.166

0.231

1560

6120

Median

29.6

3.07

0.141

0.141

0.141

0.141

33.4

4000

9.59

0.141

0.141

1140

6250

P95

151

16.2

0.141

0.141

1.17

0.141

166

17800

40.7

0.411

1.77

29900

47600

DF (%)

100

90.5

4.76

0

14.3

0

100

100

71.4

14.3

23.8

100

100

Range

4.97-217

nd-18.2

nd-1.36

nd

nd-1.38

nd

5.67-222

978-21200

nd-46.4

nd-0.832

nd-1.89

344-39100

1560-60400

All (n=186)

586

GM

19.1

1.20

0.289

0.193

0.145

0.144

24.1

7540

38.5

0.553

0.291

2840

10800

Median

18.3

1.29

0.141

0.141

0.141

0.141

23.6

7180

47.8

0.641

0.141

2730

10200

P95

109

10.7

1.83

3.65

0.141

0.141

114

27900

241

3.05

2.90

17000

48400

DF (%)

100

84.9

34.4

9.68

1.61

1.08

100

100

91.9

71.5

35.5

100

100

Range

1.38-298

nd-18.2

nd-34.9

nd-16.6

nd-1.38

nd-1.97

2.66-299

978-161000

nd-4960

nd-8.17

nd-8.64

344-49700

1520-215000

GM = geometric mean; P95 = 95th percentile; DF (%) = detection frequency (%).

25

ACS Paragon Plus Environment

Environmental Science & Technology

Page 26 of 30

587

Table 2. Estimated Daily Intakes (EDI, ng/kg bw/day) of Parabens and Their Metabolites,

588

via Ingestion of Mollusks, for Various Age Groups in China. MeP

EtP

PrP

BuP

BzP

HepP ΣPBs

4-HB

3,4-DHB OH-MeP OH-EtP

BA

ΣMBs

Geometric Mean Toddlers (2-5 years) Male 2.88 0.181 0.044 0.029 Female 3.24 0.204 0.049 0.033 Mean 3.06 0.193 0.046 0.031 Children & Teenagers (6-17 years) Male 2.61 0.165 0.040 0.026 Female 2.52 0.159 0.038 0.026 Mean 2.57 0.162 0.039 0.026 Adults (≥18 years) Male 2.01 0.126 0.030 0.020 Female 1.91 0.120 0.029 0.019 Mean 1.96 0.123 0.030 0.020

0.022 0.025 0.023

0.022 0.025 0.023

3.64 4.09 3.87

1140 1280 1210

5.83 6.55 6.19

0.084 0.094 0.089

0.044 0.049 0.047

430 483 456

1640 1840 1740

0.020 0.019 0.020

0.020 0.019 0.019

3.30 3.18 3.24

1030 997 1020

5.29 5.10 5.19

0.076 0.073 0.075

0.040 0.038 0.039

390 376 383

1480 1430 1460

0.015 0.015 0.015

0.015 0.014 0.015

2.53 2.42 2.48

793 757 775

4.06 3.87 3.96

0.058 0.056 0.057

0.031 0.029 0.030

299 285 292

1140 1090 1110

95th Percentile Toddlers (2-5 years) Male 16.5 1.62 0.277 0.552 Female 18.5 1.83 0.312 0.620 Mean 17.5 1.72 0.295 0.586 Children & Teenagers (6-17 years) Male 15.0 1.47 0.252 0.501 Female 14.4 1.42 0.243 0.483 Mean 14.7 1.45 0.247 0.492 Adults (≥18 years) Male 11.5 1.13 0.193 0.384 Female 10.9 1.08 0.184 0.366 Mean 11.2 1.10 0.189 0.375

0.021 0.024 0.023

0.021 0.024 0.023

17.2 19.3 18.3

4220 4740 4480

36.5 41.0 38.7

0.462 0.519 0.490

0.438 0.492 0.465

2570 2880 2730

7320 8230 7770

0.019 0.019 0.019

0.019 0.019 0.019

15.6 15.0 15.3

3830 3690 3760

33.1 31.9 32.5

0.419 0.404 0.411

0.398 0.383 0.390

2330 2240 2290

6640 6400 6520

0.015 0.014 0.015

0.015 0.014 0.015

12.0 11.4 11.7

2940 2800 2870

25.4 24.2 24.8

0.321 0.307 0.314

0.305 0.291 0.298

1790 1700 1740

5100 4860 4980

589 590 591

26

ACS Paragon Plus Environment

Page 27 of 30

Environmental Science & Technology

595 596 597

599

0 (c)

2

610 611 612

6 4 2 (e)

7

8

9 10 11 ln[4-HB]

616 617 618

ln[BA]

615

11 10 9 8 7 6 5

1

12

5

6

(d)

11 10 9 8 7 6 5

1

2 3 4 ln[MeP]

5

6

r=0.78 p=0

(f)

6

11 10 r=0.36 p=8.4E-7 9 8 7 6 5 0 2 4 6 ln[3,4-DHB]

7

8

9 10 11 12 ln[4-HB]

(g)

8

619

27

2 3 4 ln[MeP]

r=0.44 p=2.6E-10

0

r=0.58 p=1.1E-16

0

(b)

6

ln[BA]

ln[3,4-DHB]

8

613 614

5

10

606

609

3 4 ln[MeP]

r=0.35 p=6.2E-7

0

-1 1

12 11 10 9 8 7 6

5

1

605

608

4

-2

604

607

2 3 ln[MeP]

p=1.4E-8

ln[OH-MeP]

603

1

2 r=0.46

600

602

(a)

0

598

601

r=0.33 p=0.0047

ln[BA]

594

ln[PrP]

593

4 3 2 1 0 -1 -2

ln[4-HB]

592

ACS Paragon Plus Environment

10

Environmental Science & Technology

620

Figure 1. Correlations between natural logarithmic (ln) concentrations of parabens and their

621

metabolites in mollusks from Bohai Sea, China. (a) ln[MeP] vs ln[PrP]; (b) ln[MeP] vs ln[4-HB];

622

(c) ln[MeP] vs ln[OH-MeP]; (d) ln[MeP] vs ln[BA]; (e) ln[4-HB] vs ln[3,4-DHB]; (f) ln[4-HB]

623

vs ln[BA]; and (g) ln[3,4-DHB] vs ln[BA]. Note: only those samples with measurable levels of

624

target chemicals are presented.

625 626

28

ACS Paragon Plus Environment

Page 28 of 30

Page 29 of 30

Environmental Science & Technology

(a)

Concentration (ng/g)

300

200

100

0

Nev Rap Mya Cyc Chl Sca Mer Myt Ost Amu Mac

627

Mollusk species

628 220000

(b)

Concentration (ng/g)

200000

60000

40000

20000

0 Nev Rap Mya Cyc Chl Sca Mer Myt Ost Amu Mac

629

Mollusk species

630 631

Figure 2. Species-specific accumulation of parabens (a) and their metabolites (b) in mollusks

632

collected from Bohai Sea, China. The box plot shows fifth (lower whisker), 25th (bottom edge of

633

the box), 75th (top edge of the box), and 95th (upper whisker) percentiles of concentrations. The

634

lower and upper stars represent 1st and 99th percentiles of concentrations, respectively. The

635

arithmetic mean and median concentrations are given as the open square and the line within the

636

box, respectively. The dots are outliers. The sample numbers of mollusk species were 18, 41, 6,

637

11, 22, 21, 10, 12, 12, 19, and 5 for Nev, Rap, Mya, Cyc, Chl, Sca, Mer, Myt, Ost, Amu, and

638

Mac, respectively.

639 29

ACS Paragon Plus Environment

Environmental Science & Technology

640 641

TOC ART

642

643 644 645 646

30

ACS Paragon Plus Environment

Page 30 of 30