Gonadal Development in Smallmouth Bass (Micropterus Dolomieu) Reared in the Absence and Presence of 17-α-Ethinylestradiol.

Testicular oocytes in wild adult bass (Micropterus spp.) are considered a potential indication of exposure to estrogenic compounds in municipal, agricultural, or industrial wastewater. However, our ability to interpret links between testicular oocyte occurrence in wild fish species and environmental pollutants is limited by our understanding of normal and abnormal gonadal development. We previously reported low-to-moderate testicular oocyte prevalence (7%-38%) among adult male bass collected from Minnesota waters with no known sources of estrogenic compounds. In the present study, two experiments were conducted in which smallmouth bass (Micropterus dolomieu) fry were exposed to control water or 17-α-ethinylestradiol (EE2) during gonadal differentiation, then reared in clean water for an additional period. Histological samples were evaluated at several time points during the exposure and grow-out periods, and the sequence and timing of gonadal development in the presence of estrogen were compared with that of control fish. Testicular oocytes were not observed in any control or EE2-exposed fish. Among groups exposed to 1.2 or 5.1 ng/L EE2 in Experiment 1 or 3.0 ng/L EE2 in Experiment 2, ovaries were observed in 100% of fish up to 90 days after exposure ceased, and approximately half of those ovaries had abnormal characteristics, suggesting that they likely developed in sex-reversed males. Groups exposed to 0.1, 0.4, or 1.0 ng/L in Experiment 2 developed histologically normal ovaries and testes in proportions not significantly different from 1:1. These findings suggest that, while presumably able to cause sex reversal, juvenile exposure to EE2 may not be a unique cause of testicular oocytes in wild bass, although the long-term outcomes of exposure are unknown. Environ Toxicol Chem 2022;41:1416-1428. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Testicular oocytes in smallmouth bass in northeastern Minnesota in relation to varying levels of human activity.

Testicular oocytes (TOs) have been found in black bass (Micropterus spp.) from many locations in North America. The presence of TOs is often assumed to imply exposure to estrogenic endocrine disrupting compounds (EDCs); however, a definitive causal relationship has yet to be established, and TO prevalence is not consistently low in fish from areas lacking evident EDC sources. This might indicate any of a number of situations: 1) unknown or unidentified EDCs or EDC sources, 2) induction of TOs by other stressors, or 3) testicular oocytes occurring spontaneously during normal development. In the present study, we analyzed TO occurrence in smallmouth bass (Micropterus dolomieu) from 8 populations in northeastern Minnesota watersheds with differing degrees of human development and, hence, presumed likelihood of exposure to anthropogenic chemicals. Three watersheds were categorized as moderately developed, based on the presence of municipal wastewater discharges and higher human population density (4-81 per km2 ), and 5 watersheds were minimally developed, with very low human population density (0-1 per km2 ) and minimal built environment. Testicular tissues from mature fish were evaluated using a semiquantitative method that estimated TO density, normalized by cross-sectional area. Testicular oocyte prevalence and density among populations from moderately developed watersheds was higher than in populations from minimally developed watersheds. However, TO prevalence was unexpectedly high and variable (7-43%) in some populations from minimally developed watersheds, and only weak evidence was found for a relationship between TO density and watershed development, suggesting alternative or more complex explanations for TO presence in smallmouth bass from this region. Environ Toxicol Chem 2017;36:3424-3435. © 2017 SETAC.

Development of the Larval Amphibian Growth and Development Assay: Effects of benzophenone-2 exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of Environment to support risk assessment. The assay is employed as a higher tiered approach to evaluate effects of chronic chemical exposure throughout multiple life stages in a model amphibian species, Xenopus laevis. To evaluate the utility of the initial LAGDA design, the assay was performed using a mixed mode of action endocrine disrupting chemical, benzophenone-2 (BP-2). X. laevis embryos were exposed in flow-through conditions to 0, 1.5, 3.0 or 6.0 mg l-1 BP-2 until 2 months post-metamorphosis. Overt toxicity was evident throughout the exposure period in the 6.0 mg l-1 treatment due to elevated mortality rates and observed liver and kidney pathologies. Concentration-dependent increases in severity of thyroid follicular cell hypertrophy and hyperplasia occurred in larval tadpoles indicating BP-2-induced impacts on the thyroid axis. Additionally, gonads were impacted in all treatments with some genetic males showing both testis and ovary tissues (1.5 mg l-1 ) and 100% of the genetic males in the 3.0 and 6.0 mg l-1 treatments experiencing complete male-to-female sex reversal. Concentration-dependent vitellogenin induction occurred in both genders with associated accumulations of protein in the livers, kidneys and gonads, which was likely vitellogenin and other estrogen-responsive yolk proteins. This is the first study that demonstrates the endocrine effects of this mixed mode of action chemical in an amphibian species and demonstrates the utility of the LAGDA design for supporting chemical risk assessment. Copyright © 2016 John Wiley & Sons, Ltd.

Development of the Larval Amphibian Growth and Development Assay: effects of chronic 4-tert-octylphenol or 17ß-trenbolone exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid-mediated amphibian metamorphosis and reproductive development. During the validation phase, two well-characterized endocrine-disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4-tert-octylphenol (tOP) and xenoandrogen 17ß-trenbolone (TB). Xenopus laevis embryos were exposed, in flow-through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l-1 ) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l-1 ) until 8 weeks post-metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment-related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l-1 ) during metamorphosis. Müllerian duct development was affected after exposure to both chemicals; tOP exposure caused dose-dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Müllerian duct regression in males and complete regression in >50% of the females in the 100 ng l-1 treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine-active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow.

In the present study, a hypothesized adverse outcome pathway linking inhibition of thyroid peroxidase (TPO) activity to impaired swim bladder inflation was investigated in two experiments in which fathead minnows (Pimephales promelas) were exposed to 2-mercaptobenzothiazole (MBT). Continuous exposure to 1mg MBT/L for up to 22 days had no effect on inflation of the posterior chamber of the swim bladder, which typically inflates around 6 days post fertilization (dpf), a period during which maternally-derived thyroid hormone is presumed to be present. In contrast, inflation of the anterior swim bladder, which occurs around 14dpf, was impacted. Specifically, at 14dpf, approximately 50% of fish exposed to 1mg MBT/L did not have an inflated anterior swim bladder. In fish exposed to MBT through 21 or 22dpf, the anterior swim bladder was able to inflate, but the ratio of the anterior/posterior chamber length was significantly reduced compared to controls. Both abundance of thyroid peroxidase mRNA and thyroid follicle histology suggest that fathead minnows mounted a compensatory response to the presumed inhibition of TPO activity by MBT. Time-course characterization showed that fish exposed to MBT for at least 4 days prior to normal anterior swim bladder inflation had significant reductions in anterior swim bladder size, relative to the posterior chamber, compared to controls. These results, along with similar results observed in zebrafish (see part II, this issue) are consistent with the hypothesis that thyroid hormone signaling plays a significant role in mediating anterior swim bladder inflation and development in cyprinids, and that role can be disrupted by exposure to thyroid hormone synthesis inhibitors. Nonetheless, possible thyroid-independent actions of MBT on anterior swim bladder inflation cannot be ruled out based on the present results. Overall, although anterior swim bladder inflation has not been directly linked to survival as posterior swim bladder inflation has, potential links to adverse ecological outcomes are plausible given involvement of the anterior chamber in sound production and detection.

Cross-species sensitivity to a novel androgen receptor agonist of potential environmental concern, spironolactone.

Spironolactone is a pharmaceutical that in humans is used to treat conditions like hirsutism, various dermatologic afflictions, and female-pattern hair loss through antagonism of the androgen receptor. Although not routinely monitored in the environment, spironolactone has been detected downstream of a pharmaceutical manufacturer, indicating a potential for exposure of aquatic species. Furthermore, spironolactone has been reported to cause masculinization of female western mosquitofish, a response indicative of androgen receptor activation. Predictive methods to identify homologous proteins to the human and western mosquitofish androgen receptor suggest that vertebrates would be more susceptible to adverse effects mediated by chemicals like spironolactone that target the androgen receptor compared with invertebrate species that lack a relevant homolog. In addition, an adverse outcome pathway previously developed for activation of the androgen receptor suggests that androgen mimics can lead to reproductive toxicity in fish. To assess this, 21-d reproduction studies were conducted with 2 fish species, fathead minnow and Japanese medaka, and the invertebrate Daphnia magna. Spironolactone significantly reduced the fecundity of medaka and fathead minnows at 50 µg/L, whereas daphnia reproduction was not affected by concentrations as large as 500 µg/L. Phenotypic masculinization of females of both fish species was observed at 5 µg/L as evidenced by formation of tubercles in fathead minnows and papillary processes in Japanese medaka. Effects in fish occurred at concentrations below those reported in the environment. These results demonstrate how a priori knowledge of an adverse outcome pathway and the conservation of a key molecular target across vertebrates can be utilized to identify potential chemicals of concern in terms of monitoring and highlight potentially sensitive species and endpoints for testing.

Propiconazole inhibits steroidogenesis and reproduction in the fathead minnow (Pimephales promelas).

Conazoles are designed to inhibit cytochrome P450 (CYP) 14α-demethylase, an enzyme key to fungal cell wall formation. In vertebrates, conazoles may inhibit other CYPs, potentially disrupting processes like sex steroid synthesis. Propiconazole is a current-use pesticide that is among the first chemicals being tested in the U.S. Environmental Protection Agency endocrine disruptor screening program. Fathead minnows (Pimephales promelas) were exposed to 0, 5, 50, 500, or 1000 µg propiconazole/l in a 21-day study that evaluated apical reproductive endpoints (fecundity, fertility, hatch); measures of endocrine function and steroid synthesis, such as cholesterol, vitellogenin (VTG), and sex steroid (testosterone [T], 17ß-estradiol [E2]) concentrations in the plasma; and changes in gonadal expression of steroidogenic genes. Plasma E2 and VTG concentrations in females were reduced by exposure to propiconazole, and egg production was decreased in the 500 and 1000 µg/l treatment groups. These in vivo effects coincided with inhibition of E2 synthesis by ovary explants exposed to propiconazole in vitro. We also observed a compensatory response in females exposed to propiconazole, manifested as increased gonad weight and upregulation of genes coding for key steriodogenic proteins, including CYP19 (aromatase), CYP17 (hydroxylase/lyase), CYP11A (cholesterol side-chain-cleavage), and steroidogenic acute regulatory protein. Other than an increase in relative testis weight, effects on endocrine function in males were less pronounced than in females. This study provides important data relative to the potential endocrine activity of propiconazole in fish and, more generally, to the further delineation of pathways for the reproductive effects of steroid synthesis inhibitors in fish.

Effects of a glucocorticoid receptor agonist, dexamethasone, on fathead minnow reproduction, growth, and development.

Synthetic glucocorticoids are pharmaceutical compounds prescribed in human and veterinary medicine as anti-inflammatory agents and have the potential to contaminate natural watersheds via inputs from wastewater treatment facilities and confined animal-feeding operations. Despite this, few studies have examined the effects of this class of chemicals on aquatic vertebrates. To generate data to assess potential risk to the aquatic environment, we used fathead minnow 21-d reproduction and 29-d embryo-larvae assays to determine reproductive toxicity and early-life-stage effects of dexamethasone. Exposure to 500 µg dexamethasone/L in the 21-d test caused reductions in fathead minnow fecundity and female plasma estradiol concentrations and increased the occurrence of abnormally hatched fry. Female fish exposed to 500 µg dexamethasone/L also displayed a significant increase in plasma vitellogenin protein levels, possibly because of decreased spawning. A decrease in vitellogenin messenger ribonucleic acid (mRNA) expression in liver tissue from females exposed to the high dexamethasone concentration lends support to this hypothesis. Histological results indicate that a 29-d embryo-larval exposure to 500 µg dexamethasone/L caused a significant increase in deformed gill opercula. Fry exposed to 500 µg dexamethasone/L for 29 d also exhibited a significant reduction in weight and length compared with control fry. Taken together, these results indicate that nonlethal concentrations of a model glucocorticoid receptor agonist can impair fish reproduction, growth, and development.

Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment.

Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations. Here we discuss a framework designed for this purpose, the adverse outcome pathway (AOP). An AOP is a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment. The practical utility of AOPs for ecological risk assessment of chemicals is illustrated using five case examples. The examples demonstrate how the AOP concept can focus toxicity testing in terms of species and endpoint selection, enhance across-chemical extrapolation, and support prediction of mixture effects. The examples also show how AOPs facilitate use of molecular or biochemical endpoints (sometimes referred to as biomarkers) for forecasting chemical impacts on individuals and populations. In the concluding sections of the paper, we discuss how AOPs can help to guide research that supports chemical risk assessments and advocate for the incorporation of this approach into a broader systems biology framework.

Medaka (Oryzias latipes) for use in evaluating developmental effects of endocrine active chemicals with special reference to gonadal intersex (testis-ova).

Japanese medaka (Oryzias latipes) has been widely used for the evaluation of the toxicity of endocrine active chemicals (EACs) and other chemicals as well as for monitoring the adverse effects of effluent discharges in relation to sexual development and function. It is useful for these evaluations for many reasons including the following: 1) it has a short life cycle facilitating studies extending over long phases of development and over multigenerations, 2) it is easy to rear, 3) male and female phenotypes can easily be distinguished on the basis of secondary sex characteristics, and 4) a genetic marker (DMY) is available for identifying the true genotypic sex. Several biomarkers have been found to be useful for identifying the effects of exposure to estrogenic and androgenic chemicals in medaka and they include increased levels of hepatic vitellogenin (VTG) and testis-ova induction in males for exposure to estrogenic chemicals, and decreased levels of hepatic VTG in females and an altered morphology of dorsal and anal fins and formation of papillae for androgenic chemicals. In this paper, we present a critical analysis of the use of medaka as a test species for studies of endocrine disruption and report on the use of sex-related genetic markers and alterations in gonadal development, including the induction of testis-ova formation, for assessing the disruptive effects of EACs. In this paper, we focus on some of the more recent studies and findings.

Ketoconazole in the fathead minnow (Pimephales promelas): reproductive toxicity and biological compensation.

Ketoconazole (KTC) is a model pharmaceutical representing imidazole and triazole pesticides, which inhibit fungal growth through blocking a cytochrome P450 (CYP)-mediated step in ergosterol biosynthesis. Several of these fungicides have been shown to be reversible inhibitors of CYPs in vertebrates (primarily mammals), including CYP isoforms involved in the pathway that converts cholesterol to active sex steroids. In these studies, we assessed the effects of KTC on aspects of steroidogenesis and reproductive function in the fathead minnow (Pimephales promelas). Exposure of spawning adults to the fungicide for 21 d significantly decreased egg production at a water concentration as low as 25 microg/L. Despite evidence of reduced ex vivo testosterone production by gonads from KTC-exposed fathead minnows, circulating plasma concentrations of sex steroids (testosterone, 17beta-estradiol) were not affected. Exposure to KTC caused an increase in the gonadosomatic index in both sexes and, in males, the fungicide caused a marked proliferation of interstitial (Leydig) cells. In addition, mRNA transcripts for two key steroidogenic enzymes, cytochrome P450 side-chain cleavage (CYP11A) and cytochrome P450 c17alpha hydroxylase/17,20 lyase (CYP17), were elevated by exposure to KTC. Both the changes in transcript levels and proliferation of gonad tissue represent potential adaptive or compensatory responses to impaired steroidogenic capacity. Overall our data indicate that, although KTC does adversely affect steroidogenesis and reproduction in the fathead minnow, the fish can compensate to some degree to mitigate effects of the fungicide. This has important implications for the interpretation of data from tests with endocrine-active chemicals.

Strain difference in sensitivity to 3,4-dichloroaniline and insect growth regulator, fenoxycarb, in Daphnia magna.

Acute and reproductive toxicity tests were conducted on seven strains of Daphnia magna from six laboratories in five countries. 3,4-Dichloroaniline (DCA) and fenoxycarb were used as test chemicals. Acute toxicity tests revealed that estimated EC(50) (50% effective concentration) values for DCA varied by a factor of 2.1 among strains (310-640 microg/L), whereas the EC(50) values for fenoxycarb varied by a factor of 4 (210-860 microg/L). EC(50) values for reproductive toxicity tests with DCA ranged from 5.9 to 38 microg/L among strains. Fenoxycarb exposure induced the production of male neonates in all the strains used in the present study. Estimated EC(50) values for the induction of male offspring were highly variable among strains: sensitivity to fenoxycarb differed by a factor of approximately 23 overall (0.45-10 microg/L). The present pre-validation tests suggest that induction of male sex in neonates by a juvenile hormone analog is universal among genetically different strains. Decreased total numbers of neonates at increased concentrations of fenoxycarb as well as other juvenoids may, however, obscure the incidence of male neonates production in the 21-day reproduction tests due to the low statistical power.

Diagnostic criteria for proliferative thyroid lesions in bony fishes.

Thyroid proliferative lesions are rather common in bony fishes but disagreement exists in the fish pathology community concerning diagnostic criteria for hyperplastic versus neoplastic lesions. To simplify the diagnosis of proliferative thyroid lesions and to reduce confusion regarding lesion interpretation, we propose specific criteria for distinguishing hyperplastic from neoplastic lesions. Development of these criteria was based on the examination of a large series of proliferative lesions from Japanese medaka (Oryzias latipes), lesions from other small fish species, and a reexamination of the 97 cases of proliferative thyroid lesions from bony fishes deposited in the Registry of Tumors in Lower Animals. Specific diagnostic criteria are provided for all lesion categories including follicular cell hyperplasia (simple, nodular, or ectopic), adenoma (papillary or solid), and carcinoma (well- or poorly differentiated). These criteria should assist fish pathologists in describing and categorizing naturally occurring proliferative lesions from wild fishes, lesions that develop in laboratory fishes due to suboptimal culture practices or water quality, those in fishes used in toxicological assays, and captive aquarium fishes.

Small fish models for identifying and assessing the effects of endocrine-disrupting chemicals.

Endocrine-disrupting chemicals (EDCs), particularly those that affect the hypothalamic-pituitary-gonadal (HPG) axis of vertebrates, have become a focus of regulatory screening and testing throughout the world. Small fish species, principally the fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio), are used as model organisms for several of these testing programs. Fish are appropriate models for testing EDCs, not only from the perspective of existing ecological impacts, but also in terms of species extrapolation. Specifically, there is a significant degree of conservation of basic aspects of the HPG axis across vertebrates, which provides a technically robust basis for using results from fish tests to predict likely modes/mechanisms of action of potential EDCs in other vertebrates. Different experimental designs/endpoints for partial- and full-life cycle tests with fish that enable a consideration of a broad range of EDCs are described. Examples of results with specific chemicals in tests with the fathead minnow, medaka, and zebrafish are presented and discussed in terms of sensitivity and specificity for different classes of EDCs.

Use of multi-photon laser-scanning microscopy to describe the distribution of xenobiotic chemicals in fish early life stages.

To better understand the mechanisms by which persistent bioaccumulative toxicants (PBTs) produce toxicity during fish early life stages (ELS), dose-response relationships need to be understood in relation to the dynamic distribution of chemicals in sensitive tissues. In this study, a multi-photon laser scanning microscope (MPLSM) was used to determine the multi-photon excitation spectra of several polyaromatic hydrocarbons (PAHs) and to describe chemical distribution among tissues during fish ELS. The multi-photon excitation spectra revealed intense fluorescent signal from the model fluorophore, pentamethyl-difluoro-boro-indacene (BODIPY), less signal from benzo[a]pyrene and fluoranthene, and no detectable signal from pyrene. The imaging method was tested by exposing newly fertilized medaka (Oryzias latipes) eggs to BODIPY or fluoranthene for 6 h, followed by transfer to clean media. Embryos and larvae were then imaged through 5 days post-hatch. The two test chemicals partitioned similarly throughout development and differences in fluorescence intensity among tissues were evident to a depth of several hundred microns. Initially, the most intense signal was observed in the oil droplet within the yolk, while a moderate signal was seen in the portion of the yolk containing the yolk-platelets. As embryonic development progressed, the liver biliary system, gall bladder, and intestinal tract accumulated strong fluorescent signal. After hatch, once the gastrointestinal tract was completely developed, most of the fluorescent signal was cleared. The MPLSM is a useful tool to describe the tissue distribution of fluorescent PBTs during fish ELS.