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Larval exposure to environmentally relevant concentrations of triclosan impairs metamorphosis and reproductive fitness in zebrafish
Reproductive Toxicology 87 (2019) 79–86
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Larval exposure to environmentally relevant concentrations of triclosan impairs metamorphosis and reproductive fitness in zebrafish Amanda Stenzel, Heidi Wirt, Alyssa Patten, Briannae Theodore, Tisha King-Heiden
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University of Wisconsin – La Crosse, Department of Biology and River Studies Center, 1725 State Street, La Crosse, WI, 54601, United States
ARTICLE INFO
ABSTRACT
Keywords: Triclosan Reproduction Thyroid hormone Metamorphosis Zebrafish
Developmental exposure to endocrine disruptors can cause organizational changes resulting in latent and transgenerational disease. We exposed zebrafish to environmentally relevant concentrations of triclosan during the critical period of metamorphosis and somatic sex differentiation to determine effects on metamorphosis and reproduction. We use biological and morphological biomarkers to predict potential modes of action. Larval exposure to environmentally relevant concentrations of triclosan was sufficient to cause adverse effects in adults and their offspring. TCS exposure delays metamorphosis and impairs fecundity and fertility. Offspring from TCSexposed fish show decreased survival and delayed maturation, but their reproductive capacity is not altered. Delays in metamorphosis in conjunction with morphological indicators suggest that toxicity may result from lowered thyroid hormones in parental fish. This work illustrates the importance of evaluating the latent effects of early exposure to environmental contaminants, and that further studies to evaluate the effects of triclosan on the thyroid axis are warranted.
1. Introduction There is increased public concern about the adverse effects of environmental contaminants that disrupt the endocrine system on the health and well-being of both humans and wildlife [1,2]. Since hormones control most physiological processes (e.g., growth, development, metamorphosis, and reproduction), exposure to endocrine disruptors can impact on the overall health of an organism. For example, chronic exposure to an estrogenic compound caused the feminization of males and ultimately a complete collapse of a population of fathead minnows [3]. Endocrine disruptors within personal care products enter the environment through sewage waste effluents [4]. As such, these compounds have become ubiquitous in the environment, particularly in aquatic systems, posing risk to wild fish populations [5,6]. Triclosan (TCS) is a broad-spectrum antibacterial and antifungal agent added to a variety of personal care products, which at low concentrations inhibits bacterial fatty acid biosynthesis [7]. It is one of the most commonly identified personal care products found in aquatic ecosystems, with surface concentrations ranging widely from 0.0014 to 86 μg/L [8–14]. Given its hydrophobicity, TCS tends to bioaccumulate in various aquatic species [15]. Its risk quotient (ratio of maximum environmental concentration to predicted no-effect concentrations) suggests that it poses a significant risk to aquatic organisms [8,16]
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which is supported by experimental work. Several studies demonstrate the potential risks from the developmental toxicity of TCS in fish [17–21]. Evidence that TCS may disrupt fatty acid biosynthesis in vertebrates is lacking, with most evidence suggesting that TCS acts as an endocrine disruptor [9,22,23]. Its mode of action has been difficult to ascertain as the specific hormone pathway impacted may be a reflection of dose [24]. In fish, TCS has been shown to disrupt reproductive hormones exerting weak androgenic effects in medaka [25], estrogenic effects in medaka and Western mosquitofish [26,27], and anti-androgenic effects in zebrafish [28]. TCS has also been shown to inhibit the thyroid axis by disrupting thyroid hormone biosynthesis [29–32]. Only one study, however; has evaluated the reproductive toxicity of TCS in fish. Ishibashi et al [26] showed that chronic (21 days) exposure of adult medaka to 20–200 μgTCS/L had no impact on fecundity, fertility, or health of offspring. While TCS may not cause activational effects in adults to impair reproduction, it is possible that exposure to TCS during critical stages of development could cause organizational changes resulting in reproductive impairment. The concept that alterations in developmental programing of structural and physiological relationships by environmental factors results in life-long changes in function and increased susceptibility to disease has resulted in a paradigm shift in how we evaluate the reproductive toxicity of endocrine disruptors [33]. TCS
Corresponding author. E-mail address: [email protected] (T. King-Heiden).
https://doi.org/10.1016/j.reprotox.2019.05.055 Received 10 August 2018; Received in revised form 1 May 2019; Accepted 9 May 2019 Available online 15 May 2019 0890-6238/ © 2019 Elsevier Inc. All rights reserved.
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has the potential to alter hormone pathways important in metamorphosis and reproduction following exposure to higher concentrations of TCS, yet the long-term effects of early exposure to environmentally relevant concentrations of TCS remain unresolved. Evaluating the potential consequences of exposure to endocrine disruptors during critical periods of development is nearly impossible to address in wild fish populations. Laboratory studies using small fish can provide the framework for predicting transgenerational risk to wild fish populations [34,35]. The zebrafish is the ideal species to screen for potential mechanisms of action using a variety of morphological and molecular biomarkers [36,37], are commonly used to evaluate the toxicity of endocrine disruptors [35,38,39], and the role of estrogens, androgens, and thyroid hormones in zebrafish development, metamorphosis, and reproduction are known [40–43]. Here we describe the effects of exposure to environmentally relevant concentrations of TCS during the critical period of metamorphosis and somatic sex differentiation in zebrafish. Our primary goals were to identify effects of early exposure to TCS on metamorphosis, somatic sex differentiation and reproduction of exposed fish and their offspring. We use a variety of biological and morphological indicators of reproductive and thyroid hormone disruption to predict the potential mode of action of TCS at sub-lethal concentrations.
metamorphosis (21–35 dpf). These concentrations of TCS were selected to be well-below the LC50 (˜400 μg TCS/L for embryo/larval zebrafish, [17,45]), and ˜340 μg/L for adult zebrafish [46],), and to correspond to concentrations found within surface waters [8,12]. Since endocrine disruptors are less likely to influence actions of sex-determining genes, but rather impact somatic sex differentiation resulting in shifts in sex ratios and impaired reproduction [43,47,48], the age at exposure was chosen to encompass the process of somatic sex differentiation [43] as well as metamorphosis in zebrafish [49]. 2.3. Impacts on survival, growth, maturation and reproductive fitness Three groups of fish (25 fish per group) were exposed to each concentration of TCS. Two replicate experiments were performed for a total n = 6 groups for each TCS concentration. Following exposure to TCS, fish were raised to adulthood in TCS-free water. Mortality was recorded daily for survival analysis. At 35 dpf, a subset was immobilized in 3% methylcellulose and photographed laterally using an Optronic MicroFire camera mounted on a Leica MZ16 stereomicroscope (8–10 fish per group depending upon survival; n = 49–55 per treatment). Images were used to determine the proportion of fish that completed larval development (% juveniles) as indicated by resorption of the lateral fin fold, pigmentation of stripes and tail, notochord flexion, and forking of the tail fin as illustrated in Fig. 1 [49,50]. Specifically, fish were classified as “larvae” if melanophores along ventral myotomes were present but dispersed, xanthophores were missing, and the caudal fin was rounded or slightly forked but lacked melanophore and xanthophore stripes. Fish were classified as “juvenile” if melanophores along ventral myotomes were organized into distinct stripes, xanthopohores were dispersed in the second dorsal and anal fin, and the caudal fin was distinctly forked with distinct melanophore and xanthophore stripes. At 3 months (following 55 days depuration), standard length was measured to assess growth and sex determined by external morphology. At this time, males and females were placed into separate tanks. Sex was confirmed via standard histological analysis post-spawning on a subset of fish (4 male and 4 female/group n = 48 except for the highest treatment group 2 male and 2 female/group n = 24). Fish were euthanized with 3-aminobenzoic acid ethyl ester (MS-222, Sigma, St Louis, MO). Head and tails were removed, and fish were fixed with 10% Zn formalin followed by decalcification with Cal-ExII. Tissues were dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin. Fish were sectioned at 5 μm and gonads were evaluated from 2 sections at 2 levels. At 6 months (following 145 days depuration), remaining fish from all treatment groups were spawned in groups (in a ratio of 1 female to 2 males) once weekly for a total of 4 spawns to assess reproductive capacity. Males and females were kept separate except for spawning, and there were 3-4 spawn groups per exposure group. Eggs were collected 3 h after simulated dawn, and assessed when eggs were approximately 4–6 hours old to determine fecundity (average number of eggs released/ female) and fertility (average proportion of fertilized eggs as evidenced of developmental progression past the 2-cell stage). Gamete quality was determined by assessing survival of offspring through 24 hpf. Offspring (F1) were pooled from the final spawn and a subset raised in TCS-free water as described above (n = 16/group for a total n = 96/ concentration) to determine transgenerational effects. Survival was monitored through completion larval development through adulthood (6 months). At 5 and 35 dpf, a subset of fish was photographed laterally as described above (3 from each replicate experiment; n = 18/concentration). Lateral images were used to assess growth (standard length at 5 and 35 dpf), to identify signs of abnormal development (5 dpf), and to determine whether fish had completed larval development (proportion of juveniles at 35 dpf as described above). At 6 months of age, F1 fish were spawned in groups as described above once weekly for a total of 4 spawns to assess fecundity and fertility as described above.
2. Materials and methods 2.1. Chemicals and test species Analytical grade triclosan (TCS, ≥ 97%; CAS 3380-34-5) was obtained from Sigma-Aldrich Co. (St. Louis, MO) and dissolved in acetone as a vehicle. High-performance liquid chromatography (HPLC) with ultraviolet detection (Agilent Technologies 1260 HPLC) was used to confirm the concentrations of 1000X stock solutions. Dosing solutions were made by diluting stock solutions in buffered zebrafish water (60 mg/L Instant Ocean, pH7) ensuring that acetone was < 0.01%. To determine whether 50% water changes were sufficient to maintain relatively constant concentrations of TCS, six containers containing dosing solutions were maintained under experimental conditions (temperature, light: dark cycle, and 50% daily renewal) without fish. One mL samples were collected prior to 50% renewal at 2, 11, and 15 days, and concentrations determined by HPLC-UV. Concentrations were approximately 0.48 ± 0.253, 5.4 ± 1.052, and 46.14 ± 5.689 μg TCS/L, indicating that 50% water changes were sufficient to maintain relatively constant exposure concentrations. Since we did not confirm actual exposure concentrations for each experiment, we refer to nominal concentrations throughout. The University of Wisconsin – La Crosse Animal Care and Use Committee approved all animal husbandry and experimental procedures. An established line of zebrafish (AB strain) were used for all experiments. Eggs were collected from mass spawns and fish raised according to Westerfield [44] at 25-26 °C in reconstituted deionized water (60 mg/L Instant Ocean, pH 6.8–7.2) and a 14:10 light-dark cycle. Dissolved oxygen was maintained at 5–8 mg/L and densities were maintained at 30–40 fish/L. During exposures, fish were raised in beakers of static water; dead fish were removed twice daily and 50% water changes were performed daily. Following exposure to TCS, fish were moved to recirculating systems maintained following the same water quality parameters described above. Throughout experiments, fish were fed twice daily with 48-hr hatched brine shrimp and flake food. 2.2. Overview of exposure An overview of the experimental design is presented in Fig. 1. Zebrafish were exposed for a total of 15 days to 0 (vehicle control), 0.4, 4, or 40 μg TCS/L via static waterborne exposure with 50% daily renewal during the period of somatic sex differentiation and 80
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Fig. 1. A summary of the experimental design. Timing for exposure to TCS and when endpoints of toxicity were assessed are indicated with respect to age (days post fertilization) and developmental milestones. Metamorphosis images modified from [49]).
Females exposed to 0.4 or 4 μg TCS/L released approximately 50% fewer eggs per spawn, while surviving fish exposed to 40 μg TCS/L released approximately 87% fewer eggs per spawn (Fig. 3A). Fertility was reduced in all fish exposed to TCS (Fig. 3B). The quality of offspring was also reduced following developmental exposure to TCS. Fewer fish survived through 24 hpf, and free-swimming larvae (5 dpf) from TCSexposed fish were slightly smaller (˜11% smaller than larvae from control fish), but did not show overt signs of impaired development. Survival through juvenile stages (35 dpf) was significantly reduced in all offspring from fish exposed to 0.4 to 4 μg/L TCS; no offspring from parents exposed to 40 μg/L survived through juvenile stages (Fig. 4A). The proportion of surviving fish that completed metamorphosis was reduced (Fig. 4B). All remaining offspring survived through adulthood. Reproductive capacity of F1 was highly variable, but not significantly different from control (Fig. 4 C, D).
2.4. Morphological indicators for estrogen disruption Sex ratios and gonad development of parental (exposed) fish were used as morphological indicators of disruption of estrogen signaling. Histological sections were of insufficient quality to establish a maturity index; however, impacts on gonad development (presence of mature ovary, mature testis, or presence of an ovo-testis) were determined. 2.5. Morphological indicators for thyroid hormone disruption Lateral images of exposed parental fish and their unexposed offspring taken at 35 dpf were used to the measure relative position of pectoral fins (snout to pectoral fin distance) and pelvic fins (pelvic to anal fin distance) as biomarkers for thyroid hormone disruption as described by Sharma et al., [51]. All distances are normalized to standard body length.
3.2. Morphological biomarkers for estrogen disruption
2.6. Data Analysis
Fish exposed to TCS do not show morphological signs of disruption in estrogen signaling. Sex ratios were altered in fish exposed to 40 μg/L TCS (Fig. 2D); however, given the high morality in this treatment group, these results could be biased. All ovaries and testes appeared healthy and mature, and no indications of an ovo-testis was observed.
Cumulative survival rates were determined using Kaplain-Meier survival analysis with a log-rank significance test. All other data were evaluated for homoscedasticity (Leven Median test) and for normality prior to one way analysis of variance (ANOVA) and Tukey’s post hoc test to determine treatment-related effects. The level of significance for all analyses was p < 0.05.
3.3. Morphological biomarkers for thyroid hormone disruption
3. Results
Fish exposed to 0.4 or 4 μg TCS/L during early development show morphological signs of reduced thyroid hormone concentrations. Relative snout to pectoral distances were significantly increased and relative pelvic to anal fin distance was reduced (Fig. 5 A, B). It is unclear whether impacts on metamorphosis result from reductions in thyroid hormones following exposure to 40 μg TCS/L since the snout to pectoral distance was increased but the pelvic to anal fin distance was not reduced. While fewer surviving offspring complete metamorphosis, morphological biomarkers of F1 fish do not indicate this is the result of thyroid hormone disruption (Fig. 5 C, D).
3.1. Exposure to TCS impacts growth, maturation, and reproductive fitness Fish exposed to 40 μg TCS/L during the period of metamorphosis and sex differentiation showed significant mortality (Fig. 2A), and surviving fish exposed to TCS were approximately 10% smaller compared with control fish at 35 dpf (Fig. 2B). Completion of metamorphosis was impaired in all fish exposed to TCS (Fig. 2C). All fish completed maturation by 3 months and were of similar size (19.2 ± 3, 18.5 ± 4, 18.7 ± 6, and 17.8 ± 9 mm for 0, 0.4, 4, or 40 μg TCS/L, respectively). Surviving fish exposed to 40 μg/L had shifts in sex ratio towards males (Fig. 2D). 81
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Fig. 2. Dose-related effects of TCS exposure on survival, metamorphosis, and sex maturation. Effects of exposure to TCS from 21 to 35 dpf (indicated by box) on survival (A), growth in juveniles (B), completion of metamorphosis (C) and sex ratios (D). In D, * indicates that significant mortality was seen in this treatment group. All data represent the mean ± SEM. An asterisk or letters denote significant differences (p < 0.05).
concentrations of TCS during critical periods of development reduces reproductive fitness, in addition to slowing metamorphosis. 4.1. Impacts on survival and latent toxicity While others report that embryonic exposure to TCS results in latent mortality [46,52], we saw significant mortality only in the highest treatment group during exposures with no additional mortality observed post-exposure. Using a study by [46] as reference, steady state in zebrafish larvae was likely reached within one week of exposure, and if a similar depuration rate occurred, residual TCS probably remained in zebrafish larvae exposed to ≥ 4 μg TCS/L through 55 days of depuration. The lack of latent mortality reported here may reflect differences in the age at exposure. Since mortality stabilized before the end of TCS exposure, is also possible that chronic exposure to TCS initiated the appropriate physiological responses that allowed fish to acclimate and initiate repair mechanisms as suggested by [53]. The rate of metamorphosis and overall growth was impaired slightly following exposure to ≥ 0.4 μg TCS/L, but all surviving fish eventually completed metamorphosis and maturation. At least in female zebrafish, the distance between pelvic and anal fins is associated with swimming speed and performance [54], suggesting that changes in the relative position of paired fins observed here could impact swimming performance and contribute to reduced growth rates following exposure to ≥ 0.4 μg TCS/L. This is supported by our other work that shows that embryonic exposure to the same concentrations of TCS impairs foraging efficiency of larvae [52], which could account for the initial decreased growth rate that is able to recover in a laboratory setting. Schnitzler et al [30] also report that sheepshead minnow exposed to TCS show a developmental delay in metamorphosis, and TCS has been shown to delay metamorphosis in frogs [55–57]. While seemingly innocuous, a delay in metamorphosis can have profound impacts on wild fish populations. Remaining in this energetically costly and vulnerable state for longer periods of time can impact larval growth and survival, maturation, the ability to adapt to new habitats [58–60], and can even reduce fecundity in adults [59]. It is unclear whether TCS is able to alter sex differentiation in fish. While there was a marginal bias towards males following exposure to
Fig. 3. Impacts of early exposure to TCS on reproduction. (A) Dose-related effects on fecundity. (B) Dose-related effects on fertility. All data represent the mean ± SEM. Letters denote significant differences (p < 0.05).
4. Discussion We lack even the basic information related to the consequences of early exposure to personal care products like TCS that are capable of disrupting the endocrine system. And while several lines of evidence indicate that TCS exposure has the potential to impair reproduction in fish, we are the first to show that exposure to environmentally relevant 82
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Fig. 4. Survival, metamorphosis, and reproductive success in offspring from parents exposed to TCS. Dose-related effects on survival (A), metamorphosis (B), fecundity (C) and fertility (D). All data represent the mean ± SEM. Letters denote significant differences (p < 0.05).
100 μg TCS/L, early exposure to TCS did not cause a significant shift sex ratios in medaka, [25]; however, these fish were exposed to TCS prior to the completion of sex differentiation. In this study, zebrafish larvae exposed to 0.4 or 4 μg/L TCS showed no skews in sex ratios or alterations in gonad development. Observed male-biased alterations in sex ratios following exposure to 40 μg/L are confounded by high mortality with gonad development of surviving fish appearing normal. Zebrafish
have polygenetic sex determination which is highly dependent upon a balance of sex hormones driving the formation of a testis from the ovary via apoptosis at 23–35 dpf [43,61,62]. The actions of endocrine disruptors are less likely to affect actions of the sex determining genes, but rather actions of somatic cells during the process of sex differentiation leading to shifts in sex ratios [43,63]. Exposure to estrogen after the first morphological signs of sex differentiation is initiated (21 dpf) has
Fig. 5. Morphological indicators of thyroid hormone disruption. Dose-related impacts on thyroid hormone-dependent distances (normalized to standard length of fish) in parental fish exposed to TCS (A, B) and their offspring (C, D). All data represent the mean ± SEM. Letters denote significant differences (p < 0.05). 83
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been shown to result in female-biased sex ratios [64]. As the sex differentiation process progresses, the likelihood that this process will be disrupted is reduced, but it is still possible to shift the sex of adult zebrafish [65,66]. It is possible that TCS could shift sex ratios in zebrafish if exposure to TCS had been initiated at earlier stages of the gonad differentiation process and extended through the completion of somatic differentiation. For example, Wnt signaling and recruitment of primordial germ cells initiate as early as 14 dpf, and if altered can cause a shift in sex ratios [61]. Further, alterations in sex ratios are not always permanent [66,67]. Since sex ratios in this study were not determined until after depuration, it is possible that impacts on sex differentiation were missed. A more careful evaluation of the potential for TCS to alter gonad differentiation in fish may be warranted given that TCS has been shown to alter this process in frogs [68].
medaka was reduced, but they did not assess their long-term survival. Offspring mortality would likely be greater in a natural setting, where fish must compete for resources and escape predation or disease. There is growing concern regarding the potential for transgenerational epigenetic inheritance of disease following exposure to endocrine disruptors during critical periods of development (see [79] for review). Several studies suggest that TCS is capable of altering methylation patterns [80,81] as well as alter microRNA expression [72], suggesting the potential for a transgenerational inheritance of disease following exposure to TCS. Our data support this hypothesis in that offspring from TCS-exposed parents show reduced survival and slowed metamorphosis; however, morphological indicators suggest this transgenerational effect does not seem to result from heritable alterations in the thyroid hormone axis. As with their parents, fish from TCS-exposed fish showed delays in maturation, and while fecundity and fertility were highly variable, they were not reduced compared with control. Perhaps life-long exposure to the parental fish could have resulted in impaired reproduction in offspring as has been seen in Xenopus [80]. Impacts on unexposed F2 fish in conjunction with molecular endpoints would be necessary to confirm a true transgenerational effect with respect to the effects on sublethal exposure to TCS on metamorphosis.
4.2. Impacts of TCS exposure on reproductive fitness This is the first report in fishes that TCS exposure impairs reproductive fitness. While impacts on fertility were subtle, in combination with reductions in egg production and reductions in offspring survival and growth, it is clear that early exposure to TCS reduces reproductive fitness in zebrafish. Gamete quality (measured here by survival through 24 hpf) is also impaired, which can have a profound effect on reproductive fitness [69]. Reproductive toxicity seen in this study does not appear to result from impaired oogenesis or spermatogenesis, which is supported by other work showing that adult fish exposed to TCS also do have altered gonad development [26,70]. Exposure to high concentrations of TCS has been shown to be hepatotoxic [71], potentially impair lipid metabolism in zebrafish embryos [23], and to alter vitellogenesis and the hypothalamus-pituitary-ovarian axis in females [72], which could account for our observed reductions in egg production. Impacts on fertility could also result from the potential estrogenic effects of TCS [26,27] resulting in, for example, reductions in sperm counts as found in mosquito fish [27]. However, based upon our morphological indicators (sex ratios, gonad development), it does not appear that TCS significantly disrupts estrogen signaling at these concentrations. We plan to initiate additional studies to confirm that TCS does not alter key genes regulated by estrogen at these concentrations in the near future. It is possible that TCS exposure is altering reproductive behaviors, accounting for our observed reductions in fecundity and fertility. Behavior can be altered by exposure to concentrations of contaminants much lower than are required for substantial morphological changes [73–75]. Even subtle changes in reproductive behaviors can influence population dynamics [6]. TCS has been shown to alter behaviors in larval fish [9,18], and adult male fathead minnows exposed to TCS showed reductions in nest defensive behaviors which are key behaviors in their reproductive strategies [70]. In zebrafish, behaviorally aggressive males have greater fertilization success [76–78], and dominance rank impacts the number of viable eggs that can be laid by females [78], leaving the potential for TCS to impair reproduction via alterations in aggressive behaviors. Further evaluation of gonad development, gamete quality, sperm production and motility, and reproductive behaviors are necessary if we are to understand which component of the reproductive system is most sensitive to early exposure to TCS.
5. Conclusions This is the first study to show that early exposure to environmentally relevant concentrations of TCS impairs metamorphosis and reproductive fitness in fish. While we were unable to establish the lowest observable effect concentration (LOEC) for all endpoints of reproductive toxicity or metamorphosis for TCS in zebrafish, our data suggests it is lower than 0.4 μg TCS/L. Using reported surface concentrations of TCS [8,9,82,83] and that our estimated NOEC is < 0.4 μg TCS/L, the risk quotient (RQ, ratio of maximum environmental concentration to predicted no-effect concentration) would suggest that TCS poses medium to high risk for reproductive failure in environments containing > 0.04 μg TCS/L and > 0.4 μg TCS/L, respectively, and that fish living in concentrations < 0.04 μg TCS/L have a low risk for reproductive failure [16]. The molecular mechanisms underlying TCS-induced toxicity in fish remains unclear, but our work supports the mounting evidence that TCS alters the thyroid hormone pathway. The Toxic Ratio (TR, quotient of the predicted LC50 via baseline toxicity QSAR and the experimental LC50) for TCS indicates that it would have a narcotic or uncoupling mode of action [84,85]. However, several known endocrine disruptors also have TRs within the range that would indicate baseline toxicity, suggesting that endocrine disruptors may act as baseline toxicants in acute toxicity tests [84], and TRs may not always indicate modes of action relevant to effects seen during chronic exposures through the juvenile stages [84–86]. TCS exposure leads to a reduction in circulating thyroid hormones by disrupting its biosynthesis [31,32], and may also disrupt the hypothalamus-pituitary-thyroid axis [29,30]. Thyroid hormones regulate the transition from larval to juvenile stages [87,88], and a functioning thyroid hormone axis is also essential for successful reproduction in zebrafish [89–91]. Disruption of thyroid hormone during critical stages of development could explain the delays in metamorphosis, lack of an observed shift in sex ratios at sublethal concentrations, and impaired reproductive capacity observed in this study. We continue to explore potential molecular indicators that TCS disrupts the thyroid hormone pathway in our on-going work.
4.3. Transgenerational effects This work suggests that early exposure to TCS can impair health of offspring even if they are not further exposed to the contaminant (beyond as gametes). It is also possible that offspring were exposed to TCS via maternal transfer. Even in a laboratory setting, survival of offspring from TCS-exposed fish was significantly reduced; no offspring from parents exposed to 40 μg/L survived through adulthood. Ishibashi et al [26] report that hatching success of offspring from TCS-exposed
Declaration of interests None Acknowledgements We thank Cole Fuchs for assistance with reproduction assays and Dr. 84
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Nadia Carmosini for measuring TCS in our stock and dosing solutions. This work was funded by UWL Faculty Research Grants, UWL Undergraduate Research grants and the UWL River Studies Center.
[29] [30]
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Larval exposure to environmentally relevant concentrations of triclosan impairs metamorphosis and reproductive fitness in zebrafish Amanda Stenzel, Heidi Wirt, Alyssa Patten, Briannae Theodore, Tisha King-Heiden
T
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University of Wisconsin – La Crosse, Department of Biology and River Studies Center, 1725 State Street, La Crosse, WI, 54601, United States
ARTICLE INFO
ABSTRACT
Keywords: Triclosan Reproduction Thyroid hormone Metamorphosis Zebrafish
Developmental exposure to endocrine disruptors can cause organizational changes resulting in latent and transgenerational disease. We exposed zebrafish to environmentally relevant concentrations of triclosan during the critical period of metamorphosis and somatic sex differentiation to determine effects on metamorphosis and reproduction. We use biological and morphological biomarkers to predict potential modes of action. Larval exposure to environmentally relevant concentrations of triclosan was sufficient to cause adverse effects in adults and their offspring. TCS exposure delays metamorphosis and impairs fecundity and fertility. Offspring from TCSexposed fish show decreased survival and delayed maturation, but their reproductive capacity is not altered. Delays in metamorphosis in conjunction with morphological indicators suggest that toxicity may result from lowered thyroid hormones in parental fish. This work illustrates the importance of evaluating the latent effects of early exposure to environmental contaminants, and that further studies to evaluate the effects of triclosan on the thyroid axis are warranted.
1. Introduction There is increased public concern about the adverse effects of environmental contaminants that disrupt the endocrine system on the health and well-being of both humans and wildlife [1,2]. Since hormones control most physiological processes (e.g., growth, development, metamorphosis, and reproduction), exposure to endocrine disruptors can impact on the overall health of an organism. For example, chronic exposure to an estrogenic compound caused the feminization of males and ultimately a complete collapse of a population of fathead minnows [3]. Endocrine disruptors within personal care products enter the environment through sewage waste effluents [4]. As such, these compounds have become ubiquitous in the environment, particularly in aquatic systems, posing risk to wild fish populations [5,6]. Triclosan (TCS) is a broad-spectrum antibacterial and antifungal agent added to a variety of personal care products, which at low concentrations inhibits bacterial fatty acid biosynthesis [7]. It is one of the most commonly identified personal care products found in aquatic ecosystems, with surface concentrations ranging widely from 0.0014 to 86 μg/L [8–14]. Given its hydrophobicity, TCS tends to bioaccumulate in various aquatic species [15]. Its risk quotient (ratio of maximum environmental concentration to predicted no-effect concentrations) suggests that it poses a significant risk to aquatic organisms [8,16]
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which is supported by experimental work. Several studies demonstrate the potential risks from the developmental toxicity of TCS in fish [17–21]. Evidence that TCS may disrupt fatty acid biosynthesis in vertebrates is lacking, with most evidence suggesting that TCS acts as an endocrine disruptor [9,22,23]. Its mode of action has been difficult to ascertain as the specific hormone pathway impacted may be a reflection of dose [24]. In fish, TCS has been shown to disrupt reproductive hormones exerting weak androgenic effects in medaka [25], estrogenic effects in medaka and Western mosquitofish [26,27], and anti-androgenic effects in zebrafish [28]. TCS has also been shown to inhibit the thyroid axis by disrupting thyroid hormone biosynthesis [29–32]. Only one study, however; has evaluated the reproductive toxicity of TCS in fish. Ishibashi et al [26] showed that chronic (21 days) exposure of adult medaka to 20–200 μgTCS/L had no impact on fecundity, fertility, or health of offspring. While TCS may not cause activational effects in adults to impair reproduction, it is possible that exposure to TCS during critical stages of development could cause organizational changes resulting in reproductive impairment. The concept that alterations in developmental programing of structural and physiological relationships by environmental factors results in life-long changes in function and increased susceptibility to disease has resulted in a paradigm shift in how we evaluate the reproductive toxicity of endocrine disruptors [33]. TCS
Corresponding author. E-mail address: [email protected] (T. King-Heiden).
https://doi.org/10.1016/j.reprotox.2019.05.055 Received 10 August 2018; Received in revised form 1 May 2019; Accepted 9 May 2019 Available online 15 May 2019 0890-6238/ © 2019 Elsevier Inc. All rights reserved.
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has the potential to alter hormone pathways important in metamorphosis and reproduction following exposure to higher concentrations of TCS, yet the long-term effects of early exposure to environmentally relevant concentrations of TCS remain unresolved. Evaluating the potential consequences of exposure to endocrine disruptors during critical periods of development is nearly impossible to address in wild fish populations. Laboratory studies using small fish can provide the framework for predicting transgenerational risk to wild fish populations [34,35]. The zebrafish is the ideal species to screen for potential mechanisms of action using a variety of morphological and molecular biomarkers [36,37], are commonly used to evaluate the toxicity of endocrine disruptors [35,38,39], and the role of estrogens, androgens, and thyroid hormones in zebrafish development, metamorphosis, and reproduction are known [40–43]. Here we describe the effects of exposure to environmentally relevant concentrations of TCS during the critical period of metamorphosis and somatic sex differentiation in zebrafish. Our primary goals were to identify effects of early exposure to TCS on metamorphosis, somatic sex differentiation and reproduction of exposed fish and their offspring. We use a variety of biological and morphological indicators of reproductive and thyroid hormone disruption to predict the potential mode of action of TCS at sub-lethal concentrations.
metamorphosis (21–35 dpf). These concentrations of TCS were selected to be well-below the LC50 (˜400 μg TCS/L for embryo/larval zebrafish, [17,45]), and ˜340 μg/L for adult zebrafish [46],), and to correspond to concentrations found within surface waters [8,12]. Since endocrine disruptors are less likely to influence actions of sex-determining genes, but rather impact somatic sex differentiation resulting in shifts in sex ratios and impaired reproduction [43,47,48], the age at exposure was chosen to encompass the process of somatic sex differentiation [43] as well as metamorphosis in zebrafish [49]. 2.3. Impacts on survival, growth, maturation and reproductive fitness Three groups of fish (25 fish per group) were exposed to each concentration of TCS. Two replicate experiments were performed for a total n = 6 groups for each TCS concentration. Following exposure to TCS, fish were raised to adulthood in TCS-free water. Mortality was recorded daily for survival analysis. At 35 dpf, a subset was immobilized in 3% methylcellulose and photographed laterally using an Optronic MicroFire camera mounted on a Leica MZ16 stereomicroscope (8–10 fish per group depending upon survival; n = 49–55 per treatment). Images were used to determine the proportion of fish that completed larval development (% juveniles) as indicated by resorption of the lateral fin fold, pigmentation of stripes and tail, notochord flexion, and forking of the tail fin as illustrated in Fig. 1 [49,50]. Specifically, fish were classified as “larvae” if melanophores along ventral myotomes were present but dispersed, xanthophores were missing, and the caudal fin was rounded or slightly forked but lacked melanophore and xanthophore stripes. Fish were classified as “juvenile” if melanophores along ventral myotomes were organized into distinct stripes, xanthopohores were dispersed in the second dorsal and anal fin, and the caudal fin was distinctly forked with distinct melanophore and xanthophore stripes. At 3 months (following 55 days depuration), standard length was measured to assess growth and sex determined by external morphology. At this time, males and females were placed into separate tanks. Sex was confirmed via standard histological analysis post-spawning on a subset of fish (4 male and 4 female/group n = 48 except for the highest treatment group 2 male and 2 female/group n = 24). Fish were euthanized with 3-aminobenzoic acid ethyl ester (MS-222, Sigma, St Louis, MO). Head and tails were removed, and fish were fixed with 10% Zn formalin followed by decalcification with Cal-ExII. Tissues were dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin. Fish were sectioned at 5 μm and gonads were evaluated from 2 sections at 2 levels. At 6 months (following 145 days depuration), remaining fish from all treatment groups were spawned in groups (in a ratio of 1 female to 2 males) once weekly for a total of 4 spawns to assess reproductive capacity. Males and females were kept separate except for spawning, and there were 3-4 spawn groups per exposure group. Eggs were collected 3 h after simulated dawn, and assessed when eggs were approximately 4–6 hours old to determine fecundity (average number of eggs released/ female) and fertility (average proportion of fertilized eggs as evidenced of developmental progression past the 2-cell stage). Gamete quality was determined by assessing survival of offspring through 24 hpf. Offspring (F1) were pooled from the final spawn and a subset raised in TCS-free water as described above (n = 16/group for a total n = 96/ concentration) to determine transgenerational effects. Survival was monitored through completion larval development through adulthood (6 months). At 5 and 35 dpf, a subset of fish was photographed laterally as described above (3 from each replicate experiment; n = 18/concentration). Lateral images were used to assess growth (standard length at 5 and 35 dpf), to identify signs of abnormal development (5 dpf), and to determine whether fish had completed larval development (proportion of juveniles at 35 dpf as described above). At 6 months of age, F1 fish were spawned in groups as described above once weekly for a total of 4 spawns to assess fecundity and fertility as described above.
2. Materials and methods 2.1. Chemicals and test species Analytical grade triclosan (TCS, ≥ 97%; CAS 3380-34-5) was obtained from Sigma-Aldrich Co. (St. Louis, MO) and dissolved in acetone as a vehicle. High-performance liquid chromatography (HPLC) with ultraviolet detection (Agilent Technologies 1260 HPLC) was used to confirm the concentrations of 1000X stock solutions. Dosing solutions were made by diluting stock solutions in buffered zebrafish water (60 mg/L Instant Ocean, pH7) ensuring that acetone was < 0.01%. To determine whether 50% water changes were sufficient to maintain relatively constant concentrations of TCS, six containers containing dosing solutions were maintained under experimental conditions (temperature, light: dark cycle, and 50% daily renewal) without fish. One mL samples were collected prior to 50% renewal at 2, 11, and 15 days, and concentrations determined by HPLC-UV. Concentrations were approximately 0.48 ± 0.253, 5.4 ± 1.052, and 46.14 ± 5.689 μg TCS/L, indicating that 50% water changes were sufficient to maintain relatively constant exposure concentrations. Since we did not confirm actual exposure concentrations for each experiment, we refer to nominal concentrations throughout. The University of Wisconsin – La Crosse Animal Care and Use Committee approved all animal husbandry and experimental procedures. An established line of zebrafish (AB strain) were used for all experiments. Eggs were collected from mass spawns and fish raised according to Westerfield [44] at 25-26 °C in reconstituted deionized water (60 mg/L Instant Ocean, pH 6.8–7.2) and a 14:10 light-dark cycle. Dissolved oxygen was maintained at 5–8 mg/L and densities were maintained at 30–40 fish/L. During exposures, fish were raised in beakers of static water; dead fish were removed twice daily and 50% water changes were performed daily. Following exposure to TCS, fish were moved to recirculating systems maintained following the same water quality parameters described above. Throughout experiments, fish were fed twice daily with 48-hr hatched brine shrimp and flake food. 2.2. Overview of exposure An overview of the experimental design is presented in Fig. 1. Zebrafish were exposed for a total of 15 days to 0 (vehicle control), 0.4, 4, or 40 μg TCS/L via static waterborne exposure with 50% daily renewal during the period of somatic sex differentiation and 80
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Fig. 1. A summary of the experimental design. Timing for exposure to TCS and when endpoints of toxicity were assessed are indicated with respect to age (days post fertilization) and developmental milestones. Metamorphosis images modified from [49]).
Females exposed to 0.4 or 4 μg TCS/L released approximately 50% fewer eggs per spawn, while surviving fish exposed to 40 μg TCS/L released approximately 87% fewer eggs per spawn (Fig. 3A). Fertility was reduced in all fish exposed to TCS (Fig. 3B). The quality of offspring was also reduced following developmental exposure to TCS. Fewer fish survived through 24 hpf, and free-swimming larvae (5 dpf) from TCSexposed fish were slightly smaller (˜11% smaller than larvae from control fish), but did not show overt signs of impaired development. Survival through juvenile stages (35 dpf) was significantly reduced in all offspring from fish exposed to 0.4 to 4 μg/L TCS; no offspring from parents exposed to 40 μg/L survived through juvenile stages (Fig. 4A). The proportion of surviving fish that completed metamorphosis was reduced (Fig. 4B). All remaining offspring survived through adulthood. Reproductive capacity of F1 was highly variable, but not significantly different from control (Fig. 4 C, D).
2.4. Morphological indicators for estrogen disruption Sex ratios and gonad development of parental (exposed) fish were used as morphological indicators of disruption of estrogen signaling. Histological sections were of insufficient quality to establish a maturity index; however, impacts on gonad development (presence of mature ovary, mature testis, or presence of an ovo-testis) were determined. 2.5. Morphological indicators for thyroid hormone disruption Lateral images of exposed parental fish and their unexposed offspring taken at 35 dpf were used to the measure relative position of pectoral fins (snout to pectoral fin distance) and pelvic fins (pelvic to anal fin distance) as biomarkers for thyroid hormone disruption as described by Sharma et al., [51]. All distances are normalized to standard body length.
3.2. Morphological biomarkers for estrogen disruption
2.6. Data Analysis
Fish exposed to TCS do not show morphological signs of disruption in estrogen signaling. Sex ratios were altered in fish exposed to 40 μg/L TCS (Fig. 2D); however, given the high morality in this treatment group, these results could be biased. All ovaries and testes appeared healthy and mature, and no indications of an ovo-testis was observed.
Cumulative survival rates were determined using Kaplain-Meier survival analysis with a log-rank significance test. All other data were evaluated for homoscedasticity (Leven Median test) and for normality prior to one way analysis of variance (ANOVA) and Tukey’s post hoc test to determine treatment-related effects. The level of significance for all analyses was p < 0.05.
3.3. Morphological biomarkers for thyroid hormone disruption
3. Results
Fish exposed to 0.4 or 4 μg TCS/L during early development show morphological signs of reduced thyroid hormone concentrations. Relative snout to pectoral distances were significantly increased and relative pelvic to anal fin distance was reduced (Fig. 5 A, B). It is unclear whether impacts on metamorphosis result from reductions in thyroid hormones following exposure to 40 μg TCS/L since the snout to pectoral distance was increased but the pelvic to anal fin distance was not reduced. While fewer surviving offspring complete metamorphosis, morphological biomarkers of F1 fish do not indicate this is the result of thyroid hormone disruption (Fig. 5 C, D).
3.1. Exposure to TCS impacts growth, maturation, and reproductive fitness Fish exposed to 40 μg TCS/L during the period of metamorphosis and sex differentiation showed significant mortality (Fig. 2A), and surviving fish exposed to TCS were approximately 10% smaller compared with control fish at 35 dpf (Fig. 2B). Completion of metamorphosis was impaired in all fish exposed to TCS (Fig. 2C). All fish completed maturation by 3 months and were of similar size (19.2 ± 3, 18.5 ± 4, 18.7 ± 6, and 17.8 ± 9 mm for 0, 0.4, 4, or 40 μg TCS/L, respectively). Surviving fish exposed to 40 μg/L had shifts in sex ratio towards males (Fig. 2D). 81
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Fig. 2. Dose-related effects of TCS exposure on survival, metamorphosis, and sex maturation. Effects of exposure to TCS from 21 to 35 dpf (indicated by box) on survival (A), growth in juveniles (B), completion of metamorphosis (C) and sex ratios (D). In D, * indicates that significant mortality was seen in this treatment group. All data represent the mean ± SEM. An asterisk or letters denote significant differences (p < 0.05).
concentrations of TCS during critical periods of development reduces reproductive fitness, in addition to slowing metamorphosis. 4.1. Impacts on survival and latent toxicity While others report that embryonic exposure to TCS results in latent mortality [46,52], we saw significant mortality only in the highest treatment group during exposures with no additional mortality observed post-exposure. Using a study by [46] as reference, steady state in zebrafish larvae was likely reached within one week of exposure, and if a similar depuration rate occurred, residual TCS probably remained in zebrafish larvae exposed to ≥ 4 μg TCS/L through 55 days of depuration. The lack of latent mortality reported here may reflect differences in the age at exposure. Since mortality stabilized before the end of TCS exposure, is also possible that chronic exposure to TCS initiated the appropriate physiological responses that allowed fish to acclimate and initiate repair mechanisms as suggested by [53]. The rate of metamorphosis and overall growth was impaired slightly following exposure to ≥ 0.4 μg TCS/L, but all surviving fish eventually completed metamorphosis and maturation. At least in female zebrafish, the distance between pelvic and anal fins is associated with swimming speed and performance [54], suggesting that changes in the relative position of paired fins observed here could impact swimming performance and contribute to reduced growth rates following exposure to ≥ 0.4 μg TCS/L. This is supported by our other work that shows that embryonic exposure to the same concentrations of TCS impairs foraging efficiency of larvae [52], which could account for the initial decreased growth rate that is able to recover in a laboratory setting. Schnitzler et al [30] also report that sheepshead minnow exposed to TCS show a developmental delay in metamorphosis, and TCS has been shown to delay metamorphosis in frogs [55–57]. While seemingly innocuous, a delay in metamorphosis can have profound impacts on wild fish populations. Remaining in this energetically costly and vulnerable state for longer periods of time can impact larval growth and survival, maturation, the ability to adapt to new habitats [58–60], and can even reduce fecundity in adults [59]. It is unclear whether TCS is able to alter sex differentiation in fish. While there was a marginal bias towards males following exposure to
Fig. 3. Impacts of early exposure to TCS on reproduction. (A) Dose-related effects on fecundity. (B) Dose-related effects on fertility. All data represent the mean ± SEM. Letters denote significant differences (p < 0.05).
4. Discussion We lack even the basic information related to the consequences of early exposure to personal care products like TCS that are capable of disrupting the endocrine system. And while several lines of evidence indicate that TCS exposure has the potential to impair reproduction in fish, we are the first to show that exposure to environmentally relevant 82
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Fig. 4. Survival, metamorphosis, and reproductive success in offspring from parents exposed to TCS. Dose-related effects on survival (A), metamorphosis (B), fecundity (C) and fertility (D). All data represent the mean ± SEM. Letters denote significant differences (p < 0.05).
100 μg TCS/L, early exposure to TCS did not cause a significant shift sex ratios in medaka, [25]; however, these fish were exposed to TCS prior to the completion of sex differentiation. In this study, zebrafish larvae exposed to 0.4 or 4 μg/L TCS showed no skews in sex ratios or alterations in gonad development. Observed male-biased alterations in sex ratios following exposure to 40 μg/L are confounded by high mortality with gonad development of surviving fish appearing normal. Zebrafish
have polygenetic sex determination which is highly dependent upon a balance of sex hormones driving the formation of a testis from the ovary via apoptosis at 23–35 dpf [43,61,62]. The actions of endocrine disruptors are less likely to affect actions of the sex determining genes, but rather actions of somatic cells during the process of sex differentiation leading to shifts in sex ratios [43,63]. Exposure to estrogen after the first morphological signs of sex differentiation is initiated (21 dpf) has
Fig. 5. Morphological indicators of thyroid hormone disruption. Dose-related impacts on thyroid hormone-dependent distances (normalized to standard length of fish) in parental fish exposed to TCS (A, B) and their offspring (C, D). All data represent the mean ± SEM. Letters denote significant differences (p < 0.05). 83
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been shown to result in female-biased sex ratios [64]. As the sex differentiation process progresses, the likelihood that this process will be disrupted is reduced, but it is still possible to shift the sex of adult zebrafish [65,66]. It is possible that TCS could shift sex ratios in zebrafish if exposure to TCS had been initiated at earlier stages of the gonad differentiation process and extended through the completion of somatic differentiation. For example, Wnt signaling and recruitment of primordial germ cells initiate as early as 14 dpf, and if altered can cause a shift in sex ratios [61]. Further, alterations in sex ratios are not always permanent [66,67]. Since sex ratios in this study were not determined until after depuration, it is possible that impacts on sex differentiation were missed. A more careful evaluation of the potential for TCS to alter gonad differentiation in fish may be warranted given that TCS has been shown to alter this process in frogs [68].
medaka was reduced, but they did not assess their long-term survival. Offspring mortality would likely be greater in a natural setting, where fish must compete for resources and escape predation or disease. There is growing concern regarding the potential for transgenerational epigenetic inheritance of disease following exposure to endocrine disruptors during critical periods of development (see [79] for review). Several studies suggest that TCS is capable of altering methylation patterns [80,81] as well as alter microRNA expression [72], suggesting the potential for a transgenerational inheritance of disease following exposure to TCS. Our data support this hypothesis in that offspring from TCS-exposed parents show reduced survival and slowed metamorphosis; however, morphological indicators suggest this transgenerational effect does not seem to result from heritable alterations in the thyroid hormone axis. As with their parents, fish from TCS-exposed fish showed delays in maturation, and while fecundity and fertility were highly variable, they were not reduced compared with control. Perhaps life-long exposure to the parental fish could have resulted in impaired reproduction in offspring as has been seen in Xenopus [80]. Impacts on unexposed F2 fish in conjunction with molecular endpoints would be necessary to confirm a true transgenerational effect with respect to the effects on sublethal exposure to TCS on metamorphosis.
4.2. Impacts of TCS exposure on reproductive fitness This is the first report in fishes that TCS exposure impairs reproductive fitness. While impacts on fertility were subtle, in combination with reductions in egg production and reductions in offspring survival and growth, it is clear that early exposure to TCS reduces reproductive fitness in zebrafish. Gamete quality (measured here by survival through 24 hpf) is also impaired, which can have a profound effect on reproductive fitness [69]. Reproductive toxicity seen in this study does not appear to result from impaired oogenesis or spermatogenesis, which is supported by other work showing that adult fish exposed to TCS also do have altered gonad development [26,70]. Exposure to high concentrations of TCS has been shown to be hepatotoxic [71], potentially impair lipid metabolism in zebrafish embryos [23], and to alter vitellogenesis and the hypothalamus-pituitary-ovarian axis in females [72], which could account for our observed reductions in egg production. Impacts on fertility could also result from the potential estrogenic effects of TCS [26,27] resulting in, for example, reductions in sperm counts as found in mosquito fish [27]. However, based upon our morphological indicators (sex ratios, gonad development), it does not appear that TCS significantly disrupts estrogen signaling at these concentrations. We plan to initiate additional studies to confirm that TCS does not alter key genes regulated by estrogen at these concentrations in the near future. It is possible that TCS exposure is altering reproductive behaviors, accounting for our observed reductions in fecundity and fertility. Behavior can be altered by exposure to concentrations of contaminants much lower than are required for substantial morphological changes [73–75]. Even subtle changes in reproductive behaviors can influence population dynamics [6]. TCS has been shown to alter behaviors in larval fish [9,18], and adult male fathead minnows exposed to TCS showed reductions in nest defensive behaviors which are key behaviors in their reproductive strategies [70]. In zebrafish, behaviorally aggressive males have greater fertilization success [76–78], and dominance rank impacts the number of viable eggs that can be laid by females [78], leaving the potential for TCS to impair reproduction via alterations in aggressive behaviors. Further evaluation of gonad development, gamete quality, sperm production and motility, and reproductive behaviors are necessary if we are to understand which component of the reproductive system is most sensitive to early exposure to TCS.
5. Conclusions This is the first study to show that early exposure to environmentally relevant concentrations of TCS impairs metamorphosis and reproductive fitness in fish. While we were unable to establish the lowest observable effect concentration (LOEC) for all endpoints of reproductive toxicity or metamorphosis for TCS in zebrafish, our data suggests it is lower than 0.4 μg TCS/L. Using reported surface concentrations of TCS [8,9,82,83] and that our estimated NOEC is < 0.4 μg TCS/L, the risk quotient (RQ, ratio of maximum environmental concentration to predicted no-effect concentration) would suggest that TCS poses medium to high risk for reproductive failure in environments containing > 0.04 μg TCS/L and > 0.4 μg TCS/L, respectively, and that fish living in concentrations < 0.04 μg TCS/L have a low risk for reproductive failure [16]. The molecular mechanisms underlying TCS-induced toxicity in fish remains unclear, but our work supports the mounting evidence that TCS alters the thyroid hormone pathway. The Toxic Ratio (TR, quotient of the predicted LC50 via baseline toxicity QSAR and the experimental LC50) for TCS indicates that it would have a narcotic or uncoupling mode of action [84,85]. However, several known endocrine disruptors also have TRs within the range that would indicate baseline toxicity, suggesting that endocrine disruptors may act as baseline toxicants in acute toxicity tests [84], and TRs may not always indicate modes of action relevant to effects seen during chronic exposures through the juvenile stages [84–86]. TCS exposure leads to a reduction in circulating thyroid hormones by disrupting its biosynthesis [31,32], and may also disrupt the hypothalamus-pituitary-thyroid axis [29,30]. Thyroid hormones regulate the transition from larval to juvenile stages [87,88], and a functioning thyroid hormone axis is also essential for successful reproduction in zebrafish [89–91]. Disruption of thyroid hormone during critical stages of development could explain the delays in metamorphosis, lack of an observed shift in sex ratios at sublethal concentrations, and impaired reproductive capacity observed in this study. We continue to explore potential molecular indicators that TCS disrupts the thyroid hormone pathway in our on-going work.
4.3. Transgenerational effects This work suggests that early exposure to TCS can impair health of offspring even if they are not further exposed to the contaminant (beyond as gametes). It is also possible that offspring were exposed to TCS via maternal transfer. Even in a laboratory setting, survival of offspring from TCS-exposed fish was significantly reduced; no offspring from parents exposed to 40 μg/L survived through adulthood. Ishibashi et al [26] report that hatching success of offspring from TCS-exposed
Declaration of interests None Acknowledgements We thank Cole Fuchs for assistance with reproduction assays and Dr. 84
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Nadia Carmosini for measuring TCS in our stock and dosing solutions. This work was funded by UWL Faculty Research Grants, UWL Undergraduate Research grants and the UWL River Studies Center.
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