Letter Cite This: Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/journal/estlcu
Adverse Effects of Triclosan and Binary Mixtures with 17β-Estradiol on Testicular Development and Reproduction in Japanese Medaka (Oryzias latipes) at Environmentally Relevant Concentrations Chen Wang, Yu Li, Guomao Zheng, Shiyi Zhang, Yi Wan, and Jianying Hu* MOE Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China S Supporting Information *
ABSTRACT: Considering triclosan (TCS) is ubiquitous in surface water and wild fish at relatively high concentrations, its adverse effects on gonadal development and reproduction were evaluated. After exposure for 100 days after hatching, the lowest observable effective concentration (LOEC) of TCS to significantly induce gonadal intersex in male Japanese medaka (Oryzias latipes) was 117.9 ng/L. Courtship frequency and hatching rates in male medaka were significantly inhibited, and the LOECs of TCS to impact courtship frequency and hatching rates were 117.9 and 17.2 ng/L, respectively. Male medaka were also exposed to binary mixtures of 2.2 ng/L 17β-estradiol (βE2) with 2.3 ng/L TCS and 2.4 ng/L βE2 with 117.9 ng/L TCS, and a more severe intersex induction and depressed mating behavior compared to those seen after exposure to only βE2 or TCS were observed. The adverse effects of TCS and binary mixtures with βE2 on testicular development and reproduction in fish at environmentally relevant concentrations are demonstrated here for the first time.
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INTRODUCTION As a broad-spectrum antibacterial, triclosan (TCS) is commonly used in disinfectants, soap, shampoo, toothpaste, mouthwash, fabrics for sport clothing, and plastic additives.1 TCS is found in 75% of antibacterial soaps at a content of 0.1− 0.3%.2,3 Given the high production volumes of personal care products containing TCS, significant amounts have entered the aquatic environment. TCS is one of the seven most frequently detected compounds in the aquatic environment across 30 states in the United States,4,5 and its concentrations in surface waters have reached 2.3 μg/L in the United States4 and 0.656 μg/L in China.6 High levels of TCS have also been found in the bile of wild fish (0.24−4.4 mg/kg of fresh weight) located downstream of wastewater treatment plants,7 in the bile of wild male bream (14−80 μg/L) from the North Sea Canal and the Dutch Dommel River,8 and in the plasma of fish (750 to >10000 pg/L of wet weight) from the Detroit River in the United States.9 TCS has been reported as an anti-androgen in vitro, with a potency 4.80-fold higher than that of flutamide, a model androgen receptor (AR) antagonist.10 As anti-androgens can prevent endogenous androgens from mediating their biological effects, potential adverse effects, such as testicular malformation and abnormal reproduction, can be expected to be exemplified in male fish following exposure to anti-androgens such as flutamide, fenitrothion, and 1,1-dichloro-2,2-bis(pchlorophenyl)ethylene (p,p′-DDE).11−13 While high levels of TCS have been quantified in U.S. rivers where a high incidence © XXXX American Chemical Society
of intersex (abnormal presence of ovarian cells in the testes of the same individual, i.e., testicular oocytes) was exhibited,14 the effects of TCS on testicular development and reproduction in male fish were poorly understood. In addition, wild fish are facing an increasing risk of exposure to mixtures of chemicals, such as estrogens and anti-androgens, which can induce gonadal intersex and cause reproductive abnormalities by different modes of action.13,15 Thus, it would be interesting to know whether TCS and binary mixtures of TCS with estrogens at environmentally relevant concentrations elicit any adverse effects on those reproductive end points. In the study presented here, the adverse effects of TCS on testicular development and reproduction were investigated in male Japanese medaka (Oryzias latipes). Exposure to binary mixtures of TCS and 17β-estradiol (βE2) at environmentally relevant concentrations was also conducted to better understand their combined effects. Our results demonstrated for first time the adverse effects of TCS and binary mixtures of TCS with βE2 on testicular development and reproduction in male medaka at environmentally relevant concentrations. Received: Revised: Accepted: Published: A
January 2, 2018 February 4, 2018 February 7, 2018 February 7, 2018 DOI: 10.1021/acs.estlett.8b00003 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
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Environmental Science & Technology Letters
Figure 1. Male medaka testes showing different intersex severity (A−F) and incidence of intersex (G) in male medaka after exposure to TCS and binary mixtures with βE2. An asterisk indicates p < 0.05. GFP indicates the occurrence of primary oocytes (PO) in the testis.
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MATERIALS AND METHODS Chemicals and Fish Maintenance. All details are provided in the Supporting Information. Experimental Design. We used the pMOSP1-EGFP transgenic Japanese medaka strain, developed to specifically indicate intersex by fluorescence measurement.15 On the first day after hatching (1 dph), medaka larvae were collected and exposed to waterborne TCS at nominal concentrations of 5, 50, and 500 ng/L. To investigate the binary effects with βE2, medaka larvae (1 dph) were also exposed to environmentally relevant concentrations of waterborne βE2 alone (4 ng/L) and of βE2 mixtures (5 ng/L TCS with 4 ng/L βE2 and 500 ng/L TCS with 4 ng/L βE2), together with a vehicle control [0.0005% dimethyl sulfoxide (DMSO)]. Concentrations in test containers were prepared by adding 100 μL volumes of stock solutions prepared in DMSO to 20 L of water, while 100 μL of DMSO alone was added in the vehicle control. All treatments consisted of two replicates. Fifty medaka larvae in each replicate were exposed in 5 L glass tanks filled with 4 L of exposure water for the first month, and then 20−25 male adults in each replicate were separated from the females by sex phenotype (shape of the anal and dorsal fin) as described previously16 and transferred to 12 L glass tanks filled with 10 L of exposure water to accommodate growth to 100 dph (Figure S2). The temperature was controlled at 25 ± 1 °C, and exposure water was renewed every 24 h using a flow-through exposure system. After exposure for 100 days (100 dph), nine males from each replicate (n = 18 for each treatment) were randomly selected to mate with one control female each (18 pairs; n = 18) in clean water for reproductive success tests, with six of the pairs recorded to assess the courtship and mating behaviors of male medaka. The remaining fish were anesthetized with tricaine methanesulfonate (MS-222) and then dissected. Intersex occurrence and severity index were determined by green florescence in the testes examined by fluorescence stereoscopic microscopy (Leica M165 FC) (Figure S1) as described previously.15 Evaluation of Reproductive Success and Behavior. From each treatment, 18 males were randomly selected and mated to one control female each in clean water. Among the 18 pairs from each treatment, six were recorded automatically by image analysis software (Video track, View Point Inc.) to evaluate reproductive behavior. Mating was defined as a spawning act, in which the male initiated contact with the female to oviposit by tail flipping.17 The number of matings was used to evaluate reproductive behavior. Courtship was defined
as a pursuing act, in which the male moved close to and circled the female,18 and the frequency of courtship ( f) was estimated by courtship number per minute, as described by the following equation: f=
c t
where c is the number of courtships within the period from interaction between a male and female to spawning and t is the time (minutes) from interaction between a male and a female to spawning. The eggs were collected for three consecutive days, and spawning (number of eggs per female per day), fertilization rate, and hatching rate were used as parameters for evaluating reproductive success. Fertilization rates were calculated as the number of fertilized eggs relative to that of spawned, and hatching rates were calculated as hatched fries relative to fertilized eggs. Determination of Plasma Sex Hormone Levels. Plasma samples were collected from three male fish of each replicate of exposure (n = 6 per treatment) using cardiac puncture, and the βE2, testosterone (T), and 11-ketotestosterone (11-KT) levels were determined by ultraperformance liquid chromatography and tandem mass spectrometry (UPLC−MS/MS), as described in our previous paper.19 Gene Expression Analysis. After exposure for 100 days, three liver samples from male fish from each replicate (n = 6 for each treatment) were isolated and immediately stored in RNAlater at −20 °C for subsequent RNA analysis. RNA extraction was performed with TRIzol reagent (Invitrogen) following the manufacturer’s guidelines. First-strand cDNA synthesis was performed using a MMLV kit (Promega), and relative quantification via real-time polymerase chain reaction (PCR) was performed using SYBR Green Fluorescence (Toyobo Life Science) according to the manufacturer’s guidelines. All primer sequences are listed in Table S1. Details are provided in the Supporting Information. Chemical Analysis. Solid-phase extraction (SPE) combined with UPLC−ESI-MS/MS were used to determine actual concentrations of TCS and βE2 in water for each exposure group every 10 days. The recoveries in the investigated samples were 87.2 ± 4.4% for TCS, 90.8 ± 8.8% for [13C]TCS, 85.8 ± 5.6% for βE2, and 84.8 ± 5.8% for βE2-d3. The limits of quantification at a signal-to-noise ratio of 10 were 0.06 ng/L for TCS and 0.2 ng/L for βE2. Details of the chemical analyses are provided in the Supporting Information. B
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Figure 2. Reproductive behavior of male medaka exposed to TCS and/or βE2. (A) Control female fish successfully spawned when mating with males in the control group. (B) Control female fish failed to spawn when mating with males in the exposure group. (C) Frequency of courtship. (D) Number of matings. Each group contains six replicates (n = 6). An asterisk indicates p < 0.05. “Spawning without eggs” means that the pair of fish had spawning behavior but no eggs were produced (Videos 1 and 2).
Considering that TCS coexists with βE2 in the aqueous environment and their anti-androgenic and estrogenic properties, medaka fish were exposed to binary mixtures of TCS and βE2 at environmental concentrations to assess their potential combined effects. In the 2.2 ng/L βE2/2.3 ng/L TCS coexposure group, gonadal intersex was significantly induced with an incidence of 11.1% (5 of 45; p = 0.048) with a severity of level 2 (Figure 1D,G). Considering that no significant alterations in intersex incidences were observed following exposure to only 1.7 ng/L βE2 (2.2%, 1 of 46, level 1) and only 2.1 ng/L TCS (0%, 0 of 42, level 0) (Figure 1G), these results suggested a potential synergistic effect between TCS and βE2. In the 2.4 ng/L βE2/121.5 ng/L TCS co-exposure group, the intersex incidence (13%, 6 of 42) was similar to that (13%, 6 of 42) induced in the corresponding exposure group with only 117.9 ng/L TCS (Figure 1G); however, an intersex (i.e., level 3) more serious than single-chemical exposure was observed (Figure 1E). Sex Hormone Levels and Hepatic VTG-1 Gene Expression. Sex steroid hormones play an important role in the testicular development of teleost fish. The alteration of sex hormone balance, such as excess βE2 or less 11-KT, can affect testicular development of male medaka.13,19 Thus, plasma βE2, 11-KT, and T concentrations were determined to investigate whether TCS at environmentally relevant concentrations could disrupt sex hormone balance. As no significant alterations in plasma βE2, 11-KT, and T concentrations were observed in the male fish exposed to TCS and/or βE2 (Figure S3A), gonadal intersex in male fish exposed to TCS and/or βE2 should be not caused by effects on concentrations of βE2, 11-KT, or T. A
Data Analysis and Statistics. Significant differences between the control and exposure groups in sex hormone levels, transcript levels, reproductive success, and reproductive behavior were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple test (95% confidence interval). LOEC is the lowest test concentration with a statistically significant effect. The differences in the incidence of gonadal intersex between the control and exposure groups were tested by a χ2 test. Differences at the p < 0.05 level of were considered statistically significant.
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RESULTS AND DISCUSSION Gonadal Intersex in Male Medaka. During the 100 day exposure, the measured concentrations of TCS and βE2 are environmentally relevant (Table S2). After exposure from hatching to 100 dph, TCS induced minimal to mild intersex (levels 1 and 2) at the tested exposure concentrations (Figure 1A−C,G, Table S3), and a significant incidence (13.0%) was observed in the 117.9 ng/L TCS exposure group (6 of 46; p = 0.028) compared with the control (Figure 1C). These results are similar to those observed in Japanese medaka chronically exposed to anti-androgenic p,p′-DDE, where the intersex severity indexes were also levels 1 and 2.13 Considering the relatively high concentration and ubiquitous occurrence in the field (a median concentration of 140 ng/L in 139 American streams across 30 states4 and a median concentration of 238 ng/L in the Zhujiang River tributary, China6), TCS could possibly induce intersex in wild fish, which will be worth investigating in the future. C
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behavior between conspecifics.17,27 As pheromonal signals from females, βE2 might attract males to chase females, which could weaken the inhibition of TCS on courtship behavior in this study. Previous research has indicated, for example, that 0.2−2 ng/L EE2 can induce male medaka to exhibit more courtship behavior toward female medaka.23 For mating behavior, all unexposed (control) males successfully enticed the female to spawn after one mating episode, whereas some pairs of fish in the exposure groups showed spawning behavior without eggs being produced (Figure 2D, Table S4). The mean frequency of mating to successful female spawning for exposed males was 1.2 ± 0.4 in the exposure groups with only TCS, which was not statistically significant compared with the control (1.0 ± 0.0) (Figure 2D). However, the mean frequency significantly increased to 1.8 ± 0.8 in the 2.4 ng/L βE2/121.5 ng/L TCS co-exposure group (Figure 2D), which was significantly higher than that in the control (1.0 ± 0.0) (p = 0.025) (Figure 2D, Table S4). Many studies have documented that estrogens could feminize the male fish and damage the reproductive capability of males,13,19,20 which might explain why βE2 strengthened the inhibition by anti-androgens, TCS, of mating behavior here. Technically, the male fish with less courtship desire and spawning capability would lose opportunities to find an exactly matching female. Under the experimental conditions, the exposed males had abundant opportunities to initiate contact with the control female, and therefore, the impact on reproductive success could be limited. In the field, however, wild adult male fish have relatively fewer chances to make contact with females at the appropriate time for reproduction, which might pose a relatively high risk of reproductive loss in the wild. Overall, this study demonstrated that TCS at environmentally relevant concentrations can cause intersex and depress the mating behavior of male medaka fish; the toxicity of TCS was enhanced by adding with βE2 at a concentration that could not induce adverse effects on intersex and reproductive behavior. Because the experiments started at the larval stage, not at the embryo stage, the potential adverse effects on sexual development may be stronger than that observed in this study. Anti-androgenic activity and estrogens (estrone and 17βestradiol) have been co-associated with intersex in a male roach (Rutilus rutilus) living in U.K. rivers, and a reduction in courtship has been observed in three-spined stickleback exposed to sewage effluents; however, no causative agents were identified in those studies.28,29 The results obtained in this work will be helpful for the causal analysis of intersex incidence and depressed reproductive behavior in wild fish, and understanding the ecological impact of endocrine-disrupting chemicals. Our work demonstrates the previously underappreciated impact of TCS, highlighting the need for better management and control of the application and discharge of TCS to protect the aquatic ecosystem.
similar phenomenon was also observed in our previous paper in which medaka were exposed to βE2 at measured concentrations of 1.6, 3.2, 6.4, 11.6, and 22.9 ng/L, and significantly induced plasma βE2 was observed at ≥6.4 ng/L. Vitellogenin (VTG) is a lipoglycoprotein precursor of egg yolk produced in female fish, and its production in male fish has been used as a physiological response to estrogenic activity.20 TCS can induce VTG in male medaka at a high concentration (20 μg/L), which has been attributed to the weak ER binding activity of its metabolites,21 as TCS elicited no ability in activating medaka fish ERα based on an in vitro cell-based ERαmediated bioassay.22 To explore whether the estrogenic mode of action contributes to the incidence of intersex induced by TCS at environmentally relevant concentrations, VTG-1 gene expression was assessed in the liver of male medaka. Compared with that of the control, no significant alterations in the expressions of the VTG-1 gene were observed in any of the exposure groups with only TCS (Figure S3B), suggesting that TCS did not elicit estrogenic activity at concentrations used in this study. In the binary mixtures, significant VTG-1 expression was observed only in the exposure group with only 1.7 ng/L βE2 along with 2.2 ng/L βE2/2.3 ng/L TCS and 2.4 ng/L βE2/121.5 ng/L TCS co-exposure groups, but without significance among those exposure groups (Figure S3B). These results indicated that TCS at the tested concentrations did not show an estrogenic mode of action, and the antiandrogenic activity of TCS via inhibition of AR and the estrogenic activity of βE2 via activating ER in male medaka could be involved in induction of intersex. Reproductive Inhibition. Intersex has been used as an important indicator for reproductive inhibition.13,18 Of reproductive success, hatching rates were significantly inhibited in the exposure groups with only 17.2 or 117.9 ng/L TCS and in the 2.4 ng/L βE2/121.5 ng/L TCS binary exposure group (Figure S4C). Considering reproductive behaviors are related to reproductive success, especially in ecological systems,23,24 courtship and mating behavioral parameters were further assessed. After exposure for 100 days, no significant variation in courtship or mating behavior was observed in the exposure group with only 2.1 ng/L βE2 (Figure 2C,D), which has also been reported in male medaka exposed to 0.2−10 ng/L 17αethinylestradiol (EE2).23 For courtship, no significant variation was found in the exposure groups with only 2.1 or 17.2 ng/L TCS, whereas the frequency of courtship (0.23 ± 0.13) in the exposure group with only 117.9 ng/L TCS was significantly lower than that of the control [0.84 ± 0.52; p = 0.032 (Figure 2C)]. Courtship is an important reproductive strategy in male fish, and the reduced courtship in male fish might lead to a failure in synchronization of mating behavior.25 For the initiation of courtship behavior of male medaka, endogenous androgens from males and pheromones from females were two “switches”. Androgens are major hormones that control male reproductive behaviors, such as courtship activity,26 and therefore, such inhibited courtship behaviors can be explained by the anti-androgenic activity of TCS via blocking interaction between the androgen and AR. It is worth noting that there was no significant variation in courtship frequency in the 2.4 ng/L βE2/121.5 ng/L TCS co-exposure group, which is different from the significant decrease found in the corresponding exposure groups with only 117.9 ng/L TCS (Figure 2C). It has been reported that fish use reproductive hormones such as βE2, 17,20β-dihydroxy-4-pregnen-3-one, androstenedione, and prostaglandin F2α as exogenous signals to synchronize reproductive
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.estlett.8b00003. Chemicals and reagents, fish maintenance, detailed methods for observation of intersex in male medaka, detailed methods for determination of actual chemical concentrations in exposure tanks, detailed methods for D
DOI: 10.1021/acs.estlett.8b00003 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
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gene expression analysis, primer sequences of genes used in real-time PCR, reproductive behavior of exposed male fish mated with the control female, intersex testis of transgenic medaka and light micrograph of the same testis by paraffin section with H&E staining, fish exposure design, plasma hormone levels and relative gene expression of VTG-1, and reproductive success, and images from videos of spawning with eggs and spawning without eggs (PDF) Video of spawning with eggs (WMV) Video of spawning without eggs (WMV)
AUTHOR INFORMATION
Corresponding Author
*College of Urban and Environmental Sciences, Peking University, Beijing 100871, China. Telephone and fax: 86-1062765520. E-mail:
[email protected]. ORCID
Yi Wan: 0000-0003-1312-6254 Jianying Hu: 0000-0003-1965-7494 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (41330637) and the International S&T Cooperation Program of China (2016YFE0117800).
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REFERENCES
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