3-(4-Methylbenzylidene) camphor induced reproduction toxicity and antiandrogenicity in Japanese medaka (Oryzias latipes)

Journal Pre-proof 3-(4-Methylbenzylidene) camphor induced reproduction toxicity and antiandrogenicity in Japanese medaka (Oryzias latipes) Mengmeng Liang, Saihong Yan, Rui Chen, Xiangsheng Hong, Jinmiao Zha PII:

S0045-6535(20)30417-3

DOI:

https://doi.org/10.1016/j.chemosphere.2020.126224

Reference:

CHEM 126224

To appear in:

ECSN

Received Date: 10 October 2019 Revised Date:

11 February 2020

Accepted Date: 13 February 2020

Please cite this article as: Liang, M., Yan, S., Chen, R., Hong, X., Zha, J., 3-(4-Methylbenzylidene) camphor induced reproduction toxicity and antiandrogenicity in Japanese medaka (Oryzias latipes), Chemosphere (2020), doi: https://doi.org/10.1016/j.chemosphere.2020.126224. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

CRediT author statement Mengmeng Liang: Investigation, Formal analysis, Writing-Original Draft Saihong Yan: Methodology, Formal analysis, Writing-Review & Editing, Funding acquisition Rui Chen: Investigation Xiangsheng Hong: Investigation Jinmiao Zha: Project Administration, Supervision, Conceptualization, Funding acquisition

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3-(4-Methylbenzylidene) camphor induced reproduction toxicity and

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antiandrogenicity in Japanese medaka (Oryzias latipes)

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Mengmeng Lianga,b,c, Saihong Yana,b,c, Rui Chena,b,c, Xiangsheng Honga,b,c, Jinmiao

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Zhaa,b*

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a

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

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b

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

Key Laboratory of Drinking Water Science and Technology, Research Center for

Beijing Key Laboratory of Industrial Wastewater Treatment and Reuse, Research

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100085, China

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c

University of Chinese Academy of Sciences, Beijing 100085, China

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* Corresponding author: Jinmiao Zha

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Address: Key Laboratory of Drinking Water Science and Technology, Research

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Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18

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Shuangqing Road, Haidian District, Beijing, 100085, China.

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Tel: +86-10-62849107

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Fax: +86-10-62849140

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Email: [email protected]; [email protected]

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1

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Abstract

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To assess the toxic effects of 3-(4-Methylbenzylidene) camphor (4-MBC) at

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environmentally relevant concentrations on the reproduction and development of

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Japanese medaka (Oryzias latipes), adult paired medaka (F0) were exposed to 5, 50,

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and 500 µg/L 4-MBC for 28 d in the current study. The fecundity and fertility were

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significantly decreased at 500 µg/L 4-MBC (p < 0.05). Histological observations

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showed that spermatogenesis in F0 males was significantly inhibited at 50 and 500

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µg/L 4-MBC, similar to the effects obtained with all treatments of plasma

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11-ketotestosterone (p < 0.05). Moreover, the plasma vitellogenin and estradiol levels

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in F0 females were significantly increased at 5 µg/L 4-MBC (p < 0.05). All the

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transcripts of hypothalamic-pituitary-gonadal (HPG) axis-related genes tested in the

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brains and gonads of males were significantly increased at all treatments, similar to

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the effects obtained for erα, erβ and vtg in the livers and in contrast to those found for

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arα in the livers (p < 0.05). Equal numbers of embryos were exposed to tap water and

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4-MBC solutions. Significantly increased times to hatching, decreased hatching rates

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and decreased body lengths at 14-day post-hatching (dph) were obtained at 500 µg/L

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4-MBC treatment (p < 0.05). The cumulative death rates at 14 dph were significantly

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increased with all the treatments (p < 0.05). Therefore, our results showed that

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long-term exposure to 50 and 500 µg/L 4-MBC causes reproductive and

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developmental toxicity and thus provide new insight into antiandrogenicity and the

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mechanism of 4-MBC in Japanese medaka.

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Keywords: UV filter, Development toxicity, Endocrine disruption, Histopathology, 2

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Transcripts

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3

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1. Introduction

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In recent years, ultraviolet (UV) filters have been increasingly used in cosmetics

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at concentrations up to 10% for skin protection (Wnuk et al., 2017). Some UV filters

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are also used as components of personal care products, such as creams, shampoos,

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lipsticks, soaps and perfumes, to confer stability and durability to the products

50

(Langford et al., 2015). Due to their increasing use, UV filters are increasingly

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entering aquatic environments either directly by being washed off from skin and

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clothing or indirectly via wastewater or swimming pool waters (Langford and Thomas,

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2008; Santos et al., 2012). Therefore, UV filters are frequently detected in

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wastewaters, lakes, rivers, and coastal areas, and the benzophenone-3 (BP3),

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3-(4-methylbenzylidene) camphor (4-MBC), and octocrylene (OC) concentrations in

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these habitats have reached several µg/L (Balmer et al., 2005; Langford and Thomas,

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2008). Although human studies have revealed that the acute and subchronic systemic

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toxicities of these compounds are rather low (Okereke et al., 1995), their effects on

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nontarget organisms have received considerable attention due to their relatively high

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concentrations and environmental stability in aquatic environments.

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4-MBC, which is an organic camphor derivative, has been widely used as a UV

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filter in sunscreens due to its highly effective absorption of UVB (Bachelot et al.,

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2012). The usual concentrations of 4-MBC in cosmetic products range from 0.5 to 4%

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(Orsi et al., 2006). The complete removal of 4-MBC through sewage treatment

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processes is difficult, and the removal efficiency is within a range of only 38 to 77%

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(Tsui et al., 2014a). Due to its high lipophilicity (log Kow = 4.95) and environmental 4

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stability, 4-MBC can bioaccumulate in fish at levels similar to those obtained for

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persistent

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dichlorodiphenyltrichloroethane (Daughton and Ternes, 1999; Gago-Ferrero et al.,

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2012). Thus, UV filters have been detected at concentrations higher than 2 ppm in

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lipid tissues of fish, and their bioaccumulation factors are greater than 5000 (21 µg/kg

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in the whole organism in response to a UV filter concentration of 0.004 µg/L in water)

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(Brausch et al., 2011). Additionally, 4-MBC has been found in the muscle tissues of

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brown trout (Salmo trutta fario) at concentrations up to 1800 ng/g of lipid weight

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(Buser et al., 2006). Moreover, 4-MBC has been frequently detected in effluents from

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wastewater treatment plants, and the maximum concentrations range from 207 to

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1851 ng/L in China (Ramos et al., 2016). In Switzerland, the concentrations of

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4-MBC reach 1.14 µg/L in surface waters in summer and 6.5 and 2.7 µg/L in influents

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and effluents, respectively (Balmer et al., 2005; Rodil et al., 2009).

organic

pollutants,

such

as

polychlorinated

biphenyls

and

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Previous studies have reported that 4-MBC exerts potential adverse effects on

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aquatic organisms; for example, 4-MBC affects neuronal, muscular (Li et al., 2016)

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and cardiac (Quintaneiro et al., 2019) development in zebrafish embryos and induces

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abnormal swimming behavior and AChE and LDH activities in S. senegalensis larvae

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(Araújo et al., 2018). Wang et al. (2016) indicated that 4-MBC exerts

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endocrine-disrupting effects and affects reproduction in vertebrates and invertebrates.

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4-MBC also induces oxidative stress and apoptosis in Tigriopus japonicus, resulting

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in developmental, reproductive and even lethal toxicity (Chen et al., 2018). Despite

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some information regarding its toxicity in vitro and in aquatic invertebrates, the 5

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developmental and reproductive effects of 4-MBC at environmentally relevant

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concentrations on aquatic vertebrates have not been sufficiently documented.

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Japanese medaka (Oryzias latipes) is small and easy to culture in the laboratory

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due to its short life cycle, ability to breed throughout the year, and high fertilization

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and hatching rates (Chiffre et al., 2014). In particular, their embryo development can

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be easily observed (Barjhoux et al., 2012), and the gender of adult medaka can be

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distinguished according to secondary sexual characteristics (Hirakawa et al., 2012;

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Papoulias et al., 2014). Therefore, previous studies have selected medaka as a model

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for the evaluation of endocrine-disrupting chemicals (Khalil et al., 2013; Lei et al.,

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2013).

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This study aimed to evaluate the toxic effects of 4-MBC at concentrations that

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include environmentally relevant concentration on the reproduction of Japanese

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medaka. As a result, several endpoints, including histological changes, plasma steroid

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hormone and vitellogenin levels, and transcripts of hypothalamic-pituitary-gonadal

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(HPG) axis-related genes, were evaluated. The study also aimed to investigate the

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underlying mechanism of 4-MBC in fish.

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2. Materials and methods

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2.1. Chemicals

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4-MBC (CAS No. 36861-47-9, purity > 99%) was purchased from Alfa Aesar

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China Chemical Co. (Shanghai, China). Anhydrous ethanol, chloroform and

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isopropanol were purchased from Sinopharm Chemical Reagent Co. (Shanghai, 6

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China). Bouin’s solution was obtained from Solarbio Life Science (Beijing, China).

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Hematoxylin and eosin were purchased from Beyotime (Shanghai, China), and

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TRNzol universal RNA reagent was procured from TIANGEN Biotech Co. (Beijing,

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China). The stock solutions were dissolved in anhydrous ethanol in brown bottles and

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stored in the dark at 4°C.

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2.2. Test fish

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The stock of Japanese medaka (d-rR) used in the present study originated from

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the Laboratory of Freshwater Fish at the Bioscience Center of Nagoya University,

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Japan, and has been utilized to assess the potential adverse effects of chemicals in our

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laboratory for more than 16 years. The fish were raised under a flow-through system

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and in dechlorinated tap water (pH 7.2-7.6; hardness 44.0-61.0 mg/L CaCO3) at 25 ±

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1°C with a 16-h photoperiod (Zha et al., 2006). The fish were raised with commercial

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food pellets (Trea, Germany) and newly hatched brine shrimp (Artemia nauplii).

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2.3. Subchronic exposure to 4-MBC

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After acclimation for 3 weeks under the same conditions as the maintenance

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conditions of the stock, 300 breeding pairs of adult medaka (aged approximately 3-4

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months) were selected for the 28-d exposure test according to their spawning quantity

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and egg viability. Pairs of fish were randomly divided into five different treatments

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(water control, solvent control (containing 0.01% ethanol), and 5, 50 and 500 µg/L

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4-MBC). Three replicates of each treatment were used in the experiment, and each

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replicate treatment consisted of 20 pairs of fish. Five pairs were randomly placed into

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five 1-L beakers for evaluating the fecundity and fertility and fifteen pairs were 7

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randomly placed in an 18-L glass aquarium for other endpoints. The fish were

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subjected to the same feeding regimen throughout the exposure period. Every day, the

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exposure solutions were renewed, and the residue was cleaned. After 28 d of exposure,

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15 pairs of adult fish in each aquarium were anesthetized with MS-222 buffer (Sigma,

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USA). Blood were drawn at first, and then the different tissues (gonad, liver, brain)

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from 30 fish were sampled on ice.

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The newly spawned eggs from the pairs of fish in the 1-L beakers belonging to

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each group were collected as soon as possible within 4 h after fertilization, and the

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egg number was recorded during the last week of exposure. The collected eggs were

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separated and transferred to H2O2 solutions (0.9%) for 10 min. The fertilized eggs

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during each treatment were selected under a dissecting microscope, and the number of

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fertilized eggs was recorded. Half of the collected fertilized eggs were then placed in

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dechlorinated tap water, and the other half were subjected to continuous exposure to

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the same concentrations of 4-MBC as their parents. The hatchability, time to hatching

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and gross abnormality rate were calculated. The types of gross abnormalities

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investigated in this study were described by Nimrod and Benson (1998). The hatched

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larvae were raised for 14 d to measure the whole body length. Because no significant

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differences were found between the solvent control and the water control in all the

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experiments, the results obtained for the exposure groups were compared with those

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found for the water control. All experimental procedures used in the current study

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were performed in accordance with terms detailed in the Guide for the Care and Use

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of Laboratory Animals. 8

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2.4. Chemical analysis

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The concentration of 4-MBC was measured by liquid chromatography-tandem

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mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) in the positive

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mode according to Rodil et al. (2008). The limit of detection (LOD) and limit of

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quantification (LOQ) were 2.7 ng/L and 9 ng/L, respectively. Water samples were

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collected before and after the exposure solutions were renewed. Three replicates were

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included in in the experiment. The measured 4-MBC concentrations were 4.21 (86%),

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42.5 (88%), and 424 (90%) µg/L, which are higher than 80% of the nominal

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concentrations, and the nominal concentrations are used throughout the text.

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2.5. Gonadosomatic and hepatosomatic indexes

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The gonadosomatic index (GSI) and hepatosomatic index (HSI) of 20 adult fish

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(female:male = 1:1) in each replicate (n=3) of each treatment were calculated as

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follows:

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GSI (%) = (gonad weight (g)/body weight (g))×100

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HSI (%) = (liver weight (g)/body weight (g))×100

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2.6. Histopathological analysis

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Gonads from 20 adult fish (female:male = 1:1) in each replicate (n=3) of each

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treatment were excised, fixed in Bouin’s solution for 48 h and then dehydrated in

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different concentrations of ethanol (70-100%) (Yan et al., 2018). After dehydration,

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the samples were embedded in paraffin and cut into 4-5 µm thick slices, and the slices

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were stained with hematoxylin and eosin (H&E). The results were observed with a

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BX53 optical microscope and analyzed using cellSens Standard imaging software 9

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(Olympus, Tokyo, Japan).

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2.7. Plasma VTG, E2, and 11-KT measurements

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Blood samples from 30 adult fish (female:male = 1:1) in each replicate (n=3) of

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each treatment were obtained in heparinized microcapillary tubes and immediately

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centrifuged at 3000×g and 4°C for 15 min to obtain the plasma (Chen et al., 2016).

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The plasma VTG, E2 and 11-KT levels were then immediately measured using

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enzyme-linked immunosorbent assay (ELISA) kits (VTG and E2: Cusabio, Wuhan,

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Hubei, China; 11-KT: Nanjing Jiancheng, Nanjing, Jiangsu, China) according to the

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manufacturer’s specifications. Three replicates of each treatment were included.

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2.8. Real-time PCR

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Samples of various tissues (brains, livers and gonads) from 10 adult fish (female:

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male = 1:1) in each replicate of each treatment were collected for the isolation of total

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RNA and the synthesis of cDNA according to Chen et al. (2016), and three replicate

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samples of each treatment were obtained. The purity of total RNA was determined by

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the OD260/OD280 ratio, which was measured using a MultiskanTM GO microplate

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spectrophotometer (Thermo Scientific, Waltham, MA, USA). Information for the

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forward and reverse primers used in the present study is listed in Table S1. Real-time

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PCR using a 20-µL reaction solution, which consisted of SYBR Green Master Mix

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(Vazyme Biotech, Nanjing, China), Rox Reference Dye 2, forward and reverse primer,

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was performed with a 7500 real-time PCR system (Applied Biosystems, CA, USA).

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The results were calculated using the delta-delta Ct method and were normalized to

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the rpl7 mRNA expression level (Zhu et al., 2013). 10

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2.9. Statistical analysis

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The results are presented as the means ± standard errors of the mean (S.E.M.s).

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The normality and homogeneity of variance of the data were checked using

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Kolmogorov-Smirnov and Levene’s tests. All statistical analyses were conducted by

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one-way analysis of variance (ANOVA) followed by Tukey’s HSD test and Duncan’s

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multiple comparisons test using SPSS 19.0 (SPSS, Chicago, IL, USA). P < 0.05 was

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regarded to indicate a significant difference. The figures were drawn using OriginPro

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8.0 (OriginLab, Northampton, MA, USA).

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3. Results

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3. 1. Reproductive and developmental effects

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No significant changes in the fecundity and fertility of adult medaka were

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observed with the 5 and 50 µg/L 4-MBC treatments (Fig. 1). However, the 500 µg/L

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4-MBC treatment significantly decreased the fecundity and fertility compared with

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the control treatment (p < 0.05, Fig. 1).

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As shown in Table 1, the hatchability and morphological abnormality rates were

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not significantly changed during exposure to dechlorinated tape water. However, after

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parental exposure to 50 µg/L and 500 µg/L 4-MBC, a significantly increased time to

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hatching and a significantly reduced body length (14 dph) were observed in

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dechlorinated tap water (p < 0.05). The cumulative death rates in dechlorinated tap

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water were significantly increased after parental exposure to 5, 50 and 500 µg/L

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4-MBC (p < 0.05). No significant changes in the morphological abnormality rates 11

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were observed during continuous exposure to the same concentration of 4-MBC as

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that used for the parental exposure (Table 1). The hatching rates and body length (14

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dph) were significantly decreased during continued exposure to 500 µg/L 4-MBC,

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which was contrary to the results obtained for time to hatching (p < 0.05). The

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cumulative death rates were significantly increased during continued exposure to all

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4-MBC concentrations (p < 0.05).

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3.2. Growth and development

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No significant changes in the body weight and body length of adult medaka

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were obtained with any of the treatments compared with the control groups (Table S2).

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However, the GSI of adult female medaka was increased significantly by the 5 and 50

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µg/L 4-MBC treatments (p < 0.05, Table S2), which was similar to the results

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obtained for the HSI of adult male medaka with the 500 µg/L 4-MBC treatment (p <

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0.05, Table S2).

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3. 3. Histopathology

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The ovaries are composed of primary oocytes (POs), previtellogenic oocytes

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(PVOs), vitellogenic oocytes (VOs), mature oocytes (MOs) and degenerating

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vitellogenic oocytes (DOs) (Fig. 2A-D). No marked changes in the percentages of

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oocytes at each stage were observed in the ovaries with any of the treatments (Fig.

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2E). However, a descending trend for PO and increasing trends for DO and MO were

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observed in the ovaries with all the treatments (Fig. 2E). The testes mainly consist of

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spermatogonia (SG), spermatocytes (SC), spermatids (ST) and spermatozoa (SZ) (Fig.

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2F-J). The percentage of SZ in the control testes was approximately 27% and was 12

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significantly reduced by treatment with 50 and 500 µg/L 4-MBC (p < 0.05, Fig. 2J).

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In addition, the percentage of ST was significantly decreased by 500 µg/L 4-MBC (p

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< 0.05, Fig. 2J). Exposure to 50 µg/L and 500 µg/L 4-MBC induced significant

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increases in the proportions of SC and SG, respectively, compared with those found in

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the control group (p < 0.05, Fig. 2J).

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3. 4. Plasma steroid hormone and vitellogenin levels

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The plasma VTG concentrations in the females were significantly increased by

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the 5 and 50 µg/L 4-MBC treatments (p < 0.05, Fig. 3A). In addition, the plasma E2

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concentration after treatment with 5 µg/L 4-MBC and the plasma 11-KT

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concentration after treatment with 500 µg/L 4-MBC were also significantly higher

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than those found in the control group (p < 0.05, Fig. 3C, E).

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In the males, no significant changes in the plasma VTG and E2 levels were

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obtained with any of the treatments compared with the control (Fig. 3B, D). However,

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significant decreases in the plasma 11-KT concentrations were observed after

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treatment with 50 and 500 µg/L 4-MBC (p < 0.05, Fig. 3F).

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3. 5. Transcripts of genes related to the HPG axis

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The levels of androgen receptor (arα), estrogen receptors (erα and erβ),

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cytochrome P450 aromatase 19b (cyp19b), follicle-stimulating hormone b (fshb), and

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luteinizing hormone b (lhb) in the brain were determined, and the results showed that

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the levels of all of these genes were significantly increased in female medaka after

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treatment with all 4-MBC concentrations (p < 0.05, Fig. 4 and Table S3). In addition,

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the levels of arα, erα and erβ in the males were significantly increased after exposure 13

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to all 4-MBC concentrations (p < 0.05, Fig. 4 and Table S3). Similar to the results

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obtained for lhb with the 50 and 500 µg/L 4-MBC treatments, marked increases in the

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levels of fshb and cyp19b were obtained in the males after treatment with 5 µg/L

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4-MBC (p < 0.05, Fig. 4 and Table S3). However, compared with the control group,

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no significant changes in the levels of cyp19b and fshb were obtained after the 50 and

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500 µg/L 4-MBC treatments or in the lhb level after the 500 µg/L 4-MBC treatment

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(Fig. 4 and Table S3).

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The levels of arα and steroidogenic acute regulator (star) in the livers of the

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males and females were significantly inhibited by the 500 µg/L 4-MBC treatment (p <

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0.05, Fig. 4 and Table S3). Moreover, the level of vtg was significantly increased in

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the females after the 5 and 50 µg/L 4-MBC treatments and in the males after all

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4-MBC treatments (p < 0.05, Fig. 4 and Table S3). Significant increases in the levels

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of erα and erβ were observed in the females after the 50 µg/L 4-MBC treatments and

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in the males after the 50 and 500 µg/L 4-MBC treatments (p < 0.05, Fig. 4 and Table

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S3).

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In the testes, the levels of erα, erβ, cytochrome P450 aromatase 17α (cyp17α),

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3b-hydroxysteroid dehydrogenase (hsd3b), star, follicle stimulating hormone receptor

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(fshr) and luteinizing hormone receptor (lhr) were significantly increased with all

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4-MBC treatments, which was similar to the results obtained for arα and vtg with the

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50 and 500 µg/L 4-MBC treatments (p < 0.05, Fig. 4 and Table S3). The levels of star

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and lhr in the ovaries were significantly upregulated with all 4-MBC treatments,

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which was contrary to the results obtained for vtg with all 4-MBC treatments (p < 14

287

0.05, Fig. 4 and Table S3). Significant upregulation of the levels of arα, erβ and

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cyp17α in the ovaries was obtained with the 500 µg/L 4-MBC treatment, which was

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contrary to those obtained for fshr with the 5 µg/L 4-MBC treatment (p < 0.05, Fig. 4

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and Table S3).

291 292

4. Discussion

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4-MBC, an organic UV filter, is considered an endocrine-disrupting chemical

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(Wang et al., 2016). Previous studies using in vitro assays have revealed that 4-MBC

295

has estrogenic activities (Schlumpf et al., 2001; Schlumpf et al., 2004a). However,

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Nashev et al. (2010) reported that 4-MBC has antiandrogen activities in HEK 293

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cells. Due to these contradictions, the adverse effects of 4-MBC at environmentally

298

relevant concentrations on Japanese medaka were determined. Our results

299

demonstrated that 4-MBC can cause antiandrogen activities and shows reproductive

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and developmental toxicity in Japanese medaka.

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4.1 Reproductive and developmental toxicity

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The cumulative number of eggs spawned (fecundity) is frequently used as a

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predictor of population effects (Overturf et al., 2015), and disturbances in sex steroid

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hormones and the mRNA levels of ER/AR can adversely affect reproduction in fish

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(Ji et al., 2013;Yan et al., 2018). The current study showed that the fecundity of paired

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medaka was significantly reduced by 500 µg/L 4-MBC treatment, and this effect

307

might be due to the increased 11-KT levels in females, which indicates the

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reproductive toxicity of 4-MBC on medaka. These findings are similar to the results 15

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obtained for medaka exposed to low levels of benzophenone-3 (less than 90 µg/L) for

310

28 d, which included decreased fish fecundity, effects on plasma sex steroid hormone

311

and VTG levels, and altered steroidogenic gene expression (Kim et al., 2014). Similar

312

effects were also observed in fathead minnows exposed to 74 and 285 µg/L

313

3-benzylidene camphor (3-BC) for 21 d (Kunz et al., 2006) and benzophenone-2

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(BP-2) at concentrations higher than 1.2 mg/L for 15 d (Weisbrod et al., 2007). As

315

Overturf et al. (2015) reported, some UV filters can impair fish reproduction. For

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example, BP-3 significantly reduces the number of eggs produced by each female

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Japanese medaka (Coronado et al., 2008; Kim et al., 2014), which is consistent with

318

the results obtained with 4-MBC in our study. In addition, 4-MBC exhibits

319

reproductive toxicity in Long-Evans rats by delaying male puberty and affecting the

320

reproductive organ weights of adult female and male offspring (Durrer et al., 2007;

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Schlumpf et al., 2001; Schlumpf et al., 2004b). In the current study, a significantly

322

delayed hatching time was obtained with the 50 µg/L and 500 µg/L treatments,

323

increased cumulative death rates were obtained with all 4-MBC treatments, and

324

decreased hatching rates of F1 embryos were obtained with the 500 µg/L 4-MBC

325

treatment (p < 0.05). Similarly, at concentrations equal to or higher than 5 mg/L,

326

4-MBC induced developmental delays and abnormal development in embryos by

327

affecting the heartbeat and delaying the hatching time (Torres et al., 2016). Moreover,

328

4-MBC also leads to a few developmental malformations in frog (Pelophylax perezi)

329

eggs (Martins et al., 2017). The body lengths of F1 larvae at 14 dph were also

330

significantly reduced (p < 0.05), which was similar to the results obtained for the 16

331

larval length of zebrafish embryos exposed to 4-MBC at a concentration higher than 2

332

µg/L (Torres et al., 2016). Our findings suggested that 4-MBC caused both

333

reproductive and developmental toxicity in medaka embryos and larvae.

334

4.2 Histopathological analysis

335

Histopathological observation of fish tissue has been used as an important

336

method for assessing the effects of environmental contaminants (Stentiford et al.,

337

2003). In the current study, the percentages of follicles at different stages in ovarian

338

tissues were not significantly changed by any of the 4-MBC concentrations, which is

339

consistent with the results obtained for the GSI of females and shows that 4-MBC

340

does not affect the gonad histopathology. However, a significant decrease in the

341

relative proportion of mature spermatozoa in the testes was obtained after treatment

342

with high levels of 4-MBC (50 and 500 µg/L), and no significant changes in the GSI

343

of adult male medaka were obtained; these findings were similar to the results

344

obtained for testicular tissues after treatment with 100 µg/L BP-3 (Christen et al.,

345

2011). The histological effects of 4-MBC indicated that 4-MBC inhibited testicular

346

development, which is in line with the results obtained for fish exposed to other UV

347

filters, namely, 3-BC (Kunz et al., 2006b) and BP-2 (Weisbrod et al., 2007). Therefore,

348

the suppression of testicular development by 4-MBC revealed that this chemical

349

might exert antiandrogenic and reproductive effects on Japanese medaka.

350

4.3 Plasma VTG, E2 and 11-KT levels

351

Sex steroid hormones play a very important role in the assessment of

352

reproductive effects in fish (Wang et al., 2013). VTG is produced in the liver after 17

353

stimulation with estrogen and is regarded as a biomarker for assessing the disrupting

354

effects of chemicals on the endocrine system (Miracle et al., 2006; Nilsen et al., 2004).

355

No significant changes in the levels of VTG and E2 in the males were observed,

356

which indicated that 4-MBC did not induce estrogen activity in males. VTG is

357

affected by ER signaling along the HPG axis and is related to the E2 concentration

358

(Yan et al., 2018), which is well explained by the consistent changes in the E2 and

359

VTG levels observed in male and female medaka in the present study. Similarly, no

360

significant induction of VTG was observed in juvenile fathead minnows after

361

exposure to low aqueous concentrations of 2-ethyl-hexyl-4-trimethoxycinnamate

362

(EHMC) (Kunz and Fent, 2006). In contrast, 4-MBC (0.039, 0.39 and 3.9 mM)

363

increased the plasma VTG levels in male medaka in a dose-dependent manner, which

364

might be due to the higher concentration and antiandrogen activity of 4-MBC (Inui et

365

al., 2003). Therefore, the current study revealed that 4-MBC at environmentally

366

relevant concentrations interfered with the generation of sex hormones and induced

367

antiandrogen activity in medaka .

368

4.4 Transcripts of HPG axis-related genes

369

As shown in Fig. 5, gonadotropins (FSH and LH) in the pituitary have major

370

impacts on the gonads in terms of steroidogenesis and gametogenesis by binding to

371

FSHR and LHR (Kwok et al., 2005; Yan et al., 2018). The transcript levels of nuclear

372

hormone receptors in fish can be affected by endogenous estrogens or androgen, as

373

reported by Park et al. (2014). In the current study, the levels of all tested genes, with

374

the exception of star and arα in the livers, were significantly upregulated in the males. 18

375

Similar results for the levels of vtg and erα were previously reported by Inui et al.

376

(2003) and Kunz et al. (2006a). In female fish, VTG is generally synthesized in the

377

liver under the control of estrogen (Girish et al., 2014).

378

estrogens/androgen can regulate the expression of nuclear hormone receptors in fish,

379

which induced the expressions of genes involved in HPG axis. In the presence of

380

external stimuli, the hypothalamus of fish produced GnRH that bound to GnRHR in

381

the pituitary to produce gonadotropins (FSH and LH), which acted on the gonads

382

(Liang et al., 2014). GnRHs are involved in regulating reproductive activity and can

383

be affected through negative feedback mechanisms of sex hormones in vertebrates.

384

Therefore, the contrasting effects of 4-MBC on plasma VTG with transcription of

385

VTG in females might be due to the negative feedback of ER/AR signaling or the

386

different fshr mRNA levels (Liang et al., 2014). Testosterone synthesis is related to

387

the expression of cyp17α, and this chemical can be converted into E2 by cyp19α

388

expression (Martinez-Arguelles et al., 2013), which explains the increased E2 and

389

11-KT levels observed in females in the present study. Although stimulated by FSH

390

and LH (Ji et al., 2013), 11-KT can also be indirectly affected by cyp17α via changes

391

in the E2 and T levels (Chen et al., 2016), which might lead to decreased 11-KT

392

expression in males with increased cyp17α levels. Moreover, the levels of arα were

393

significantly decreased in the livers of both male and female medaka following

394

exposure to 50 and 500 µg/L 4-MBC, consistent with the results obtained for 4-MBC

395

using yeast assays (Kunz and Fent, 2006). Sex steroid levels are associated with brain

396

aromatase activity, and estrogens can significantly change cyp19b expression (Diotel 19

Endogenous

397

et al., 2010), whose upregulation can contribute to regulation of the ER/AR signaling

398

pathway (Chen et al., 2016). Similarly, 4-MBC also reportedly has antiandrogenic

399

activity toward AR in the AR CALUX® cell line (Ma et al., 2003; Schreurs et al.,

400

2004) and in HEK 293 cells (Nashev et al., 2010). Therefore, our results demonstrated

401

that 4-MBC showed antiandrogenic activity and induced related changes in ER/AR

402

signaling along the HPG axis.

403 404 405

5. Conclusion The changes in the fecundity and fertility of Japanese medakas (Oryzias latipes)

406

exposed to different concentrations of 4-MBC observed in the current study suggest

407

that 500 µg/L 4-MBC exhibits reproductive toxicity in medaka. Moreover, the effects

408

on larval growth indicate that 4-MBC at all treatments exerts toxic effects on the

409

development of medaka. Furthermore, 4-MBC exerts antiandrogen effects on medaka

410

at 50 and 500 µg/L levels, as demonstrated by histopathology observation and

411

measurement of the plasma sex steroid hormone levels and transcripts of HPG

412

axis-related genes. Our results indicate that 4-MBC may pose an ecological risk to the

413

fish population in aquatic environments.

414

Acknowledgments

415

The authors are grateful to the National Natural Science Foundation of China

416

(21677165), the Major International Joint Research Project of the National Natural

417

Science Foundation of China (51420105012) and the China Postdoctoral Science

418

Foundation (2018M641496) for providing financial support. 20

419 420 421

Declaration of interest The authors declare no conflicts of interest.

422

21

423

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600

Gene Expression of Endocrine Biomarkers in Early Life Stages of Medaka (Oryzias latipes). 27

601

Environ. Toxicol. 30, 648-655.

602

28

603

Legend of Figures and Tables

604

Fig. 1. Fecundity and fertility of eggs from paired mature medaka in the last week of the 28-day

605

4-MBC exposure period. The values are shown as the means ± S.E.M.s (n=3). The asterisk symbol

606

(*) denotes a significant difference (p < 0.05) compared with the water control, as determined by

607

ANOVA.

608

Fig. 2. Light micrographs of gonad tissues from mature medaka in the 4-MBC exposure experiment

609

stained with hematoxylin and eosin. A-D show the results for ovarian tissues, and F-I show the results

610

for testicular tissues. Water control (A, F), 5 µg/L treatment (B, G), 50 µg/L treatment (C, H) and 500

611

µg/L treatment (D, I). (E) Percentages of the numbers of follicles at different stages in the water

612

control and exposure groups. (J) Percentages of the areas of sperm at different stages in the water

613

control and exposure groups. The data are shown as the means ± S.E.M.s (n = 3), and the asterisk

614

symbol (*) denotes a significant difference (p < 0.05) compared with the negative control, as

615

demonstrated by ANOVA. PO: primary oocyte; PVO: previtellogenic oocyte; VO: vitellogenic oocyte;

616

MO: mature oocyte; DO: degenerating vitellogenic oocyte; SG: spermatogonia; SC: spermatocyte; ST:

617

spermatid; SZ: spermatozoa.

618

Fig. 3. Plasma steroid levels of female (A, C and E) and male (B, D and F) medakas at 28 d. A and

619

B: plasma VTG concentration; C and D: plasma E2 concentration; E and F: plasma 11-KT

620

concentration. The data are expressed as the means ± S.E.M.s (n = 3). Significant differences (p <

621

0.05) between the control and experimental groups identified by ANOVA are denoted by different

622

letters.

623

Fig. 4. Heat maps of the expression of selected genes involved in the HPG axis of medaka in

624

response to exposure to different concentrations of 4-MBC. All the results were compared to those 29

625

found with the water control.

626

Fig. 5. Schematic summary of the transcriptional response involving the HPG axis in medaka to

627

4-MBC exposure. The directions of the changes in gene transcription in different tissues of

628

medaka treated with 4-MBC are shown in different colors. The genes included in the analysis are

629

related to selected endocrine pathways along the HPG axis. The red color indicates gene

630

upregulation, the blue color indicates gene downregulation, and the gray color indicates no

631

significant change in genes across the three concentrations.

632

Table 1 Hatchability, time to hatching, morphological abnormality rate and body length (14 dph)

633

of F1 embryos from F0 medaka in dechlorinated tap water and subjected to continuous exposure

634

to 4-MBC.

30

Table 1 Hatchabilities, time to hatching, morphological abnormality rates and body length (14 dph) of F1 embryos from F0 medaka in dechlorinated tap water and in continued exposure to 4-MBC. Concentration (µg/L) F0

Hatchability (%)

Time to hatching (days)

Morphological abnormality rates (%)

Accumulative death rates (%)

Body length (mm)

F1

Control

0

85.7 ± 6.6

13.6 ± 1.3

2.7 ± 1.9

13.2 ± 2.6

5.7 ± 0.5

5

0

88.4 ± 4.0

14.8 ± 1.2

1.1 ± 2.3

56.4 ± 7.6*

5.5 ± 0.5

50

0

78.0 ± 9.6

18.3 ± 1.5*

1.6 ± 3.1

57.5 ± 7.4*

4.4 ± 0.5*

500

0

81.3 ± 23.9

18.4 ± 1.1*

8.3 ± 16.7

61.3 ± 8.4*

4.6 ± 0.3*

Control

0

80.9 ± 11.1

15.8 ± 2.5

4.2 ± 1.2

14.0 ± 4.8

5.6 ± 0.6

5

5

84.3 ± 11.5

15.5 ± 1.8

9.4 ± 3.4

55.9 ± 6.8*

5.7 ± 0.4

50

50

76.7 ± 2.9

17.0 ± 1.8

4.9 ± 4.3

56.5 ± 5.8*

5.4 ± 0.8

500

500

43.2 ± 15.9*

22.5 ± 2.8*

0

65.8 ± 11.1*

4.6 ± 0.4*

Data expressed as mean ± S.E.M. of each treatment (n = 3). The asterisk (*) indicates statistically significant difference from the control (p < 0.05) by ANOVA analysis.

Highlights ● 4-MBC exhibited reproductive toxicity and antiandrogenicity in Japanese medaka. ● 4-MBC significantly decreased plasma 11-ketotestosterone levels in males. ● 4-MBC induced transcriptomic responses in HPG-axis of madaka. ● 4-MBC significantly inhibited spermatogenesis at 50 and 500 µg/L treatments.

Declaration of interests ■ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: