Temporal Trends of Parabens and Their Metabolites in Mollusks from

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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

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Environmental Science & Technology

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Temporal Trends of Parabens and Their Metabolites in Mollusks from the

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

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Implications for Human Exposure

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Chunyang Liao1,2 and Kurunthachalam Kannan1,*

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

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

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

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

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

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*Corresponding author: K. Kannan

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Wadsworth Center

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Empire State Plaza, P.O. Box 509

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Albany, NY 12201-0509

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Tel: 1-518-474-0015

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Fax: 1-518-473-2895

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E-mail: [email protected]

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For submission to: ES&T

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ACS Paragon Plus Environment


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

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

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

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

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

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

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

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

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

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

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

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

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

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environments.

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

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INTRODUCTION

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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of parabens in marine organisms is lacking.

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

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

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

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

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

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

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collected from a coastal marine ecosystem.

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MATERIALS AND METHODS

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

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


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

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

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

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

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

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

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International, Dubuque, IA, USA).

C6-EtP,

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C6-PrP,

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C6-BuP, and

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C6-MeP,

C6-4-HB, were from Cambridge Isotope Laboratories

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

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

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

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

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

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

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

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including Neptunea cumingi, Ruditapes philippinarum, Venerupis variegate, Sinonovacula

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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into a HPLC-vial for analysis.

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Instrumental Analysis. Target compounds were analyzed using a Shimadzu Prominence

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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),

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

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

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

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

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

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

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

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

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

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Reported concentrations were corrected by the recoveries of internal standards;

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C6-MeP

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

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d4 for BzP, HepP-d4 for HepP, and

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

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

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

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

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

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

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

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to prevent carry-over of analytes between samples.

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

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

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

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

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

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

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

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

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

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analysis.

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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organochlorines, polybrominated diphenyl ethers, hexabromocyclododecanes, and chlorinated

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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of OH-MeP.

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Elevated concentrations of paraben metabolites have been reported to occur in

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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found among the sampling sites (p > 0.05).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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mollusks from the Bohai Sea in recent years.

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

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

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

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

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

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

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

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

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

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

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

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

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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


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Page 15 of 30


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



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


Page 16 of 30

Page 17 of 30


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



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


Page 18 of 30

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417

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418 419 420 421 422

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582 583

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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


Page 25 of 30

Range


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



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


Page 27 of 30


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


10


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


Page 28 of 30

Page 29 of 30


(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



640 641

TOC ART

642

643 644 645 646

30


Page 30 of 30

Temporal Trends of Parabens and Their Metabolites in Mollusks from

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