Short-term Reproduction Assay Research Articles

Is Seven Days Enough? Comparing A 7-Day Exposure to the Classical 21-Day OECD TG 229 Fish Short-Term Reproduction Assay in Fathead Minnow.

The OECD (Organisation for Economic Co-operation and Development) test guidelines (TG) 229-fish short-term reproduction assay (FSTRA) is one of the gold standard methods used to identify endocrine disrupting chemicals (EDCs). While informative, the FSTRA's 5-6week duration makes it difficult to use routinely. Prior studies have shown that EDCs' impact on fecundity, vitellogenin (VTG) and steroid levels can be detected after less than 1week of exposure suggesting the FSTRA could be shortened. This study compares both 7- and 21-day FSTRAs using fathead minnows (Pimephales promelas) for three known EDCs: 17α-ethinylestradiol (EE2; 40ng/L), 17β-trenbolone (TRB; 50µg/L), and propiconazole (PRP; 500µg/L). All three compounds led to arrested fertility after 24h of exposure, except for the 7-day EE2 treatment which still decreased reproduction. Moreover, independently of time of exposure, EE2 induced VTG production in males, and decreased estrogen levels in females and testosterone levels in males. In contrast, TRB-induced VTG production in males, while the levels were not different from controls in females even though testosterone levels increased, and masculinization was observed. Finally, PRP led to a decrease in VTG levels which was only significant during the 21-day exposure, and surprisingly, no effect on steroid levels were observed despite its known effects on steroidogenesis. For two of the three EDCs tested, both times of exposure led to similar outcomes supporting the shortening of the FSTRA to seven days. This proposed 7-day FSTRA could be used to screen EDCs in routine monitoring of environmental samples.

Application of a new approach method to assess the hazard of complex legacy contaminated groundwater mixtures on fathead minnows in outdoor mesocosms

Assessing the environmental risks of contaminated groundwater presents significant challenges due to its often-complex chemical composition and to dynamic processes affecting exposure of organisms in receiving surface waters. The objective of this study was to characterize the effects of groundwater collected from a legacy contaminated industrial site, in fish under environmentally relevant conditions. A 21-day fish short-term reproduction assay was conducted in outdoor wetland mesocosms by exposing adult fathead minnows (Pimephales promelas) to graded concentrations of groundwater (1 %, 3 %, and 6 %). Offspring were held in mesocosms up to four days post-hatch to apply a new approach method (NAM), the EcoToxChip™, to explore whether traditional apical endpoints could be predicted using an alternative mechanistic approach. None of the groundwater concentrations used in this study were lethal to fish. There was greater cumulative number of eggs produced at the highest concentration of exposure. However, no abnormal histological appearance was observed in the liver and gonads of fish and no significant effect was observed in the relative expression of genes, tubercle counts, and erythrocyte micronuclei counts compared to the negative control. Food availability in the mesocosms was also assessed and the abundance of zooplankton increased in all groundwater-treated mesocosms. Fathead minnow findings are in contrast to those obtained from previous controlled laboratory studies that revealed significant genotoxicity, hepatotoxicity, and reprotoxicity of the same mixtures. Several factors could explain these observations, including the aging of groundwater in mesocosms before fish addition resulting in photo- and biodegradation and binding to sediments of toxic components. Our static exposure scenario likely underestimated realistic exposure scenarios where groundwater inflow to surface water is generally semi-continuous. Nevertheless, focused transcriptome analysis using EcoToxChips also observed greater toxicity during previous laboratory tests compared to mesocosm scenarios, and thus, our results support the use of this NAM in the ecological risk assessment of contaminated groundwater.

Development of the integrated fish endocrine disruptor test-Part B: Implementation of thyroid-related endpoints.

Given the vital role of thyroid hormones (THs) in vertebrate development, it is essential to identify chemicals that interfere with the TH system. Whereas, among nonmammalian laboratory animals, fish are the most frequently utilized test species in endocrine disruptor research, for example, in guidelines for the detection of effects on the sex hormone system, there is no test guideline (TG) using fish as models for thyroid-related effects; rather, amphibians are used. Therefore, the objective of the present project was to integrate thyroid-related endpoints for fish into a test protocol combining OECD TGs 229 (Fish Short-Term Reproduction Assay) and 234 (Fish Sexual Development Test). The resulting integrated Fish Endocrine Disruption Test (iFEDT) was designed as a comprehensive approach to covering sexual differentiation, early development, and reproduction and to identifying disruption not only of the sexual and/or reproductive system but also the TH system. Two 85-day exposure tests were performed using different well-studied endocrine disruptors: 6-propyl-2-thiouracil (PTU) and 17α-ethinylestradiol (EE2). Whereas the companion Part A of this study presents the findings on effects by PTU and EE2 on endpoints established in existing TGs, the present Part B discusses effects on novel thyroid-related endpoints such as TH levels, thyroid follicle histopathology, and eye development. 6-Propyl-2-thiouracil induced a massive proliferation of thyroid follicles in any life stage, and histopathological changes in the eyes proved to be highly sensitive for TH system disruption especially in younger life stages. For measurement of THs, further methodological development is required. 17-α-Ethinylestradiol demonstrated not only the well-known disruption of the hypothalamic-pituitary-gonadal axis, but also induced effects on thyroid follicles in adult zebrafish (Danio rerio) exposed to higher EE2 concentrations, suggesting crosstalk between endocrine axes. The novel iFEDT has thus proven capable of simultaneously capturing endocrine disruption of both the steroid and thyroid endocrine systems. Integr Environ Assess Manag 2024;20:830-845. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Development of the integrated fish endocrine disruptor test (iFEDT)-Part A: Merging of existing fish test guidelines.

There has been increasing interest in endocrine-disrupting chemicals (EDCs) among scientists and public authorities over the last 30 years, notably because of their wide use and the increasing evidence of detrimental effects on humans and the environment. However, test systems for the detection of potential EDCs as well as testing strategies still require optimization. Thus, the aim of the present project was the development of an integrated test protocol that merges the existing OECD test guidelines (TGs) 229 (fish short-term reproduction assay) and 234 (fish sexual development test) and implements thyroid-related endpoints for fish. The integrated fish endocrine disruptor test (iFEDT) represents a comprehensive approach for fish testing, which covers reproduction, early development, and sexual differentiation, and will thus allow the identification of multiple endocrine-disruptive effects in fish. Using zebrafish (Danio rerio) as a model organism, two exposure tests were performed with well-studied EDCs: 6-propyl-2-thiouracil (PTU), an inhibitor of thyroid hormone synthesis, and 17α-ethinylestradiol (EE2), an estrogen receptor agonist. In part A of this article, the effects of PTU and EE2 on established endpoints of the two existing TGs are reported, whereas part B focuses on the novel thyroid-related endpoints. Results of part A document that, as expected, both PTU and EE2 had strong effects on various endocrine-related endpoints in zebrafish and their offspring. Merging of TGs 229 and 234 proved feasible, and all established biomarkers and endpoints were responsive as expected, including reproductive and morphometric changes (PTU and EE2), vitellogenin levels, sex ratio, gonad maturation, and histopathology (only for EE2) of different life stages. A validation of the iFEDT with other well-known EDCs will allow verification of the sensitivity and usability and confirm its capacity to improve the existing testing strategy for EDCs in fish. Integr Environ Assess Manag 2024;20:817-829. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Estetrol has a lower impact than 17α-ethinylestradiol on the reproductive capacity of zebrafish (Danio rerio)

Natural and synthetic oestrogens are commonly found in aquatic ecosystems. The synthetic oestrogen 17α-ethinylestradiol (EE2) is widely used in oral contraceptives and its ecotoxicological effects on aquatic organisms have been widely reported. The natural oestrogen estetrol (E4) was recently approved for use in a new combined oral contraceptive and, after therapeutic use, is likely to be found in the aquatic environment. However, its potential effects on non-target species such as fish is unknown. In order to characterize and compare the endocrine disruptive potential of E4 with EE2, zebrafish (Danio rerio) were exposed to E4 or EE2 in a fish short-term reproduction assay conducted according to OECD Test Guideline 229. Sexually mature male and female fish were exposed to a range of concentrations, including environmentally relevant concentrations of E4 and EE2, for 21 days. Endpoints included fecundity, fertilization success, gonad histopathology, head/tail vitellogenin concentrations, as well as transcriptional analysis of genes related to ovarian sex steroid hormones synthesis. Our data confirmed the strong impact of EE2 on several parameters including an inhibition of fecundity, an induction of vitellogenin both in male and female fish, an alteration of gonadal structures and the modulation of genes involved in sex steroid hormone synthesis in female fish. In contrast, only few significant effects were observed with E4 with no impact on fecundity. The results suggest that the natural oestrogen, E4, presents a more favorable environmental profile than EE2 and is less likely to affect fish reproductive capacity.

Ibuprofen: Fish Short-Term Reproduction Assay with Zebrafish (Danio rerio) Based on an Extended OECD 229 Protocol.

A study was conducted to understand the potential for ibuprofen to impact the hypothalamus-pituitary-gonadal endocrine axis resulting in disruption of fish reproduction. The Good Laboratory Practice study was conducted according to the Organisation for Economic Co-operation and Development 229 Protocol, Fish Short-Term Reproduction Assay, and extended an additional 4 d to evaluate hatching success in the F1 generation. Test organisms were exposed to nominal test concentrations of 0.5, 2.4, 11.5, 55.3, and 265.4 µg ibuprofen/L and a negative control (dilution water). To strengthen the statistical power of the study, twice the number of replicates were used in the negative control versus individual treatment levels. A 21-d pre-exposure to identify groups of actively spawning fish was immediately followed by a 36-d exposure. Results for apical endpoints of survival, growth, and reproduction (fecundity and fertility), as well as the biomarker vitellogenin in the F0 generation and time to hatch and hatching success in the F1 generation are presented. Based on mean measured exposure concentrations and effects on fecundity in the F0 generation and hatching success in the F1 generation, overall no-observed-effect concentration and lowest-observed-effect concentration for the present study were 55.2 and 265.9 µg ibuprofen/L, respectively. Results from the present study indicate a lack of endocrine-mediated reproductive effects in zebrafish at environmentally relevant concentrations of ibuprofen. Environ Toxicol Chem 2020;39:1534-1545. © 2020 SETAC.

Refinement of an OECD test guideline for evaluating the effects of endocrine disrupting chemicals on aromatase gene expression and reproduction using novel transgenic cyp19a1a-eGFP zebrafish

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 μg/L). In addition to “classical” endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 μg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 μg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.

Detection of biomarkers to differentiate endocrine disruption from hepatotoxicity in zebrafish (Danio rerio) using proteomics

Measurement of specific biomarkers identified by proteomics provides a potential alternative method for risk assessment, which is required to discriminate between hepatotoxicity and endocrine disruption. In this study, adult zebrafish (Danio rerio) were exposed to the hepatotoxic substance acetaminophen (APAP) for 21 days, in a fish short-term reproduction assay (FSTRA). The molecular changes induced by APAP exposure were studied in liver and gonads by applying a previously developed combined FSTRA and proteomics approach. We observed a significant decrease in egg numbers, an increase in plasma hyaluronic acid, and the presence of single cell necrosis in liver tissue. Furthermore, nine common biomarkers (atp5f1b, etfa, uqcrc2a, cahz, c3a.1, rab11ba, mettl7a, khdrbs1a and si:dkey-108k21.24) for assessing hepatotoxicity were detected in both male and female liver, indicating hepatic damage. In comparison with exposure to fadrozole, an endocrine disrupting chemical (EDC), three potential biomarkers for liver injury, i.e. cahz, c3a.1 and atp5f1b, were differentially expressed. The zebrafish proteome response to fadrozole exposure indicated a significant regulation in estrogen synthesis and perturbed binding of sperm to zona pellucida in the ovary. This study demonstrates that biomarkers identified and quantified by proteomics can serve as additional weight-of-evidence for the discrimination of hepatotoxicity and endocrine disruption, which is necessary for hazard identification in EU legislation and to decide upon the option for risk assessment.

Control performance of fish short term reproduction assays with fathead minnow (Pimephales promelas)

The fish short-term reproduction assay (FSTRA) is an in vivo screen to assess potential interactions with the fish endocrine system. After a 21-day exposure period vitellogenin (VTG) and secondary sexual characteristics are measured in males and females. Egg production and fertility are also monitored daily throughout the test. This paper presents data from 49 studies performed to satisfy test orders from the United States Environmental Protection Agency's Endocrine Disruptor Screening Program. Data Evaluation Records were used to collate the typical control variability and performance of test parameters in FSTRAs conducted in different laboratories with fathead minnow (Pimephales promelas). We also examine the statistical power of FSTRA endpoints and assess whether available historical control data (HCD) assist evidence-based interpretation of the endpoints. Statistically significant inter-laboratory differences were found for all endpoints except survival. HCD could therefore be usefully developed on a laboratory-by-laboratory basis to aid interpretation of new study data. Reliable HCD ranges could be developed for survival, body weight/length, gonadal somatic index, fertilisation success, and male tubercle score, and used in association with stated test acceptability criteria to interpret FSTRA data. In contrast, high intra- and inter-laboratory control variability for VTG and fecundity means that HCD for these endpoints are of limited use during study interpretation.

Evaluating the role of fish as surrogates for amphibians in pesticide ecological risk assessment

Ecological risk of chemicals to aquatic-phase amphibians has historically been evaluated by comparing estimated environmental concentrations in surface water to surrogate toxicity data from fish species. Despite their obvious similarities, there are biological disparities among fish and amphibians that could affect their exposure and response to chemicals. Given the alarming decline in amphibians, in which anthropogenic pollutants play at least some role, investigating the risk of chemicals to amphibians is becoming increasingly important. Here, we evaluate relative sensitivity of fish and larval aquatic-phase amphibians to 45 different pesticides using existing data from three standardized toxicity test designs: (1) amphibian metamorphosis assay (AMA) with the African clawed frog (Xenopus laevis); (2) fish short-term reproduction assay (FSTRA) with the fathead minnow (Pimephales promelas); (3) fish early life stage test (ELS) with fathead minnows or rainbow trout (Oncorhynchus mykiss). The advantage of this dataset over previous work is that the underlying studies are consistent in exposure method, study duration, test species, endpoints measured, and number of concentrations tested. We found very strong positive relationships between fish and frog lowest adverse effect concentrations (LOAEC) for survival [Spearman's rank correlation (rs) = 0.88], body weight (rs = 0.86), and length (rs = 0.89) with only one out of 45 chemicals (propiconazole) exhibiting 100-folder greater sensitivity in frogs relative to fish. While our results suggest comparable toxicity for pesticides between fish and aquatic-phase amphibians under these test conditions, further research with a greater diversity of amphibians and exposure scenarios will help determine the relevance of these results across species and life stages.

A combined FSTRA-shotgun proteomics approach to identify molecular changes in zebrafish upon chemical exposure

The fish short-term reproduction assay (FSTRA) is a common in vivo screening assay for assessing endocrine effects of chemicals on reproduction in fish. However, the current reliance on measures such as egg number, plasma vitellogenin concentration and morphological changes to determine endocrine effects can lead to false labelling of chemicals with non-endocrine modes- of-action. Here, we integrated quantitative liver and gonad shotgun proteomics into the FSTRA in order to investigate the causal link between an endocrine mode-of-action and adverse effects assigned to the endocrine axis. Therefore, we analyzed the molecular effects of fadrozole-induced aromatase inhibition in zebrafish (Danio rerio). We observed a concentration-dependent decrease in fecundity, a reduction in plasma vitellogenin concentrations and a mild oocyte atresia with oocyte membrane folding in females. Consistent with these apical measures, proteomics revealed a significant dysregulation of proteins involved in steroid hormone secretion and estrogen stimulus in the female liver. In the ovary, the deregulation of estrogen synthesis and binding of sperm to zona pellucida were among the most significantly perturbed pathways. A significant deregulation of proteins targeting the transcriptional activity of estrogen receptor (esr1) was observed in male liver and testis. Our results support that organ- and sex-specific quantitative proteomics represent a promising tool for identifying early gene expression changes preceding chemical-induced adverse outcomes. These data can help to establish consistency in chemical classification and labelling.

Reproductive effects on freshwater fish exposed to 17α-trenbolone and 17α-estradiol.

17α-Trenbolone and 17α-estradiol are principal metabolites in cattle excreta following the administration of Synovex® ONE, which contains trenbolone acetate and estradiol benzoate. As part of the environmental assessment of the use of Synovex® ONE, data were generated to characterize the effects of 17α-trenbolone and 17α-estradiol on the reproduction of freshwater fish. These substances are known endocrine disruptors, so the purpose of testing was not to clarify these properties but to identify concentrations representing population-relevant effects for use in risk characterization. The short-term reproduction assay was conducted with 17α-trenbolone using the fathead minnow (Pimephales promelas) and the medaka (Oryzias latipes) and with 17α-estradiol using the fathead minnow. Adverse effects on the population-relevant endpoints of survival and fecundity were used to establish the no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC) for each study. For 17α-trenbolone, adverse effects on fecundity of the fathead minnow occurred at 120 ng/L; this was the LOEC, and the NOEC was 35 ng/L. 17β-Trenbolone did not adversely affect survival and fecundity of medaka at the concentrations tested, resulting in a NOEC of 110 ng/L and a LOEC of >110 ng/L. 17α-Estradiol did not adversely impact survival and fecundity of the fathead minnow at the concentrations tested, resulting in a NOEC and LOEC of 250 ng/L and >250 ng/L, respectively. Environ Toxicol Chem 2017;36:636-644. © 2016 SETAC.

妊馬由来エクインエストロゲン類の汚染実態解明と生態影響評価

Limited data are currently available on the occurrences and toxicological implications of equine estrogens in aquatic ecosystems. In the present study, we determined concentrations of equine estrogens, including equilin, equilenin, and their 17α- and 17β-dihydro analogues, in the river water collected from Hokkaido, Japan. Among six equine estrogens measured, equilin (2.72±2.22ng/L) concentrations were the highest in the river sample, followed by 17α-dihydroequilin (2.17±2.20ng/L). This study was the first to detect the equine estrogens in the river water collected from Hokkaido, Japan. We further investigated transcriptional profiles of estrogen-responsive genes, such as vitellogenins (Vtg1 and Vtg2), choriogenins (ChgL and ChgH) and estrogen receptor subtypes (ERα, ERβ1, and ERβ2), in the liver of male medaka (Oryzias latipes) exposed to six equine estrogens (1—300ng/L) for 3 days. Our quantitative RT-PCR analyses revealed that the expression levels of hepatic Vtg, Chg and ERα genes in male medaka responded to various types and concentrations of equine estrogens. The estrogenic potencies of the chemicals were in the order of equilin>17β-estradiol>equilenin>17β-dihydroequilin>17β-dihydroequilenin>17α-dihydroequilin>17α-dihydroequilenin, suggesting the higher lowest-observed effective concentration (LOEC) of 17β-estradiol (LOEC : 30ng/L) than that of equilin (10ng/L). Furthermore, we investigated the reproductive and transgenerational effects of equilin in paired medaka exposed to 10, 100, and 1000ng/L for 21 days. The short-term reproduction assay demonstrated that equilin (100 and/or 1000ng/L) adversely affected the reproduction (fecundity and/or fertility) of adult medaka. In F1 generation fertilized eggs, the hatching of embryos in the 100 and 1000ng/L treatment groups also showed adverse effects, suggesting the endocrine-disrupting potential of equilin.

In response: challenges for statistical evaluation of ecotoxicological experiments--an industry perspective.

In response: challenges for statistical evaluation of ecotoxicological experiments--an industry perspective.

Application of endocrine disruptor screening program fish short-term reproduction assay: Reproduction and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus) exposed to Bermuda pond sediment.

A modified tier 1 Endocrine Disruptor Screening Program (EDSP) 21-d fish short-term reproduction assay (FSTRA) was used to evaluate the effects of sediment exposure from freshwater and brackish ponds in Bermuda on reproductive fecundity and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus). Reproductively active male and female fish were exposed to control sediment and sediment from 2 freshwater ponds (fathead minnow) and 2 marine ponds (killifish) contaminated with polyaromatic hydrocarbons and metals via flow-through exposure for 21 d. Reproductive fecundity was monitored daily. At termination, the status of the reproductive endocrine system was assessed by the gonadosomatic index, gonadal histology, plasma steroids (estrogen [E2], testosterone [T], and 11-ketotestosterone [11-KT]), steroidogenic enzymes (aromatase and combined 3β/17β -hydroxysteroid dehydrogenase [3β/17β-HSD]), and plasma vitellogenin (VTG). Decreased reproductive fecundity, lower male body weight, and altered endocrinological measures of reproductive status were observed in both species. Higher plasma T levels in female minnows and 11-KT levels in both male and female minnows and female killifish exposed to freshwater and brackish sediments, respectively. Decreased female E2 and VTG levels and gonadal cytochrome P19 (aromatase) activity were also found in sediment exposed females from both species. No effect on female 3β/17β-HSD activity was found in either species. The FSTRA provided a robust model capable of modification to evaluate reproductive effects of sediment exposure in fish.

A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals.

The fish short-term reproduction assay (FSTRA) is a key component of the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), which uses a weight-of-evidence analysis based on data from several assays to identify the potential for chemicals to act as agonists or antagonists of the estrogen or androgen receptors (ER and AR), or inhibitors of steroidogenic enzymes. The FSTRA considers a variety of mechanistic and apical responses in 21-d exposures with the fathead minnow (Pimephales promelas), including plasma steroid and vitellogenin (VTG; egg yolk protein) concentrations, secondary sex characteristics, gonad size and histopathology, and egg production. Although the FSTRA initially was described several years ago, recent data generation associated with implementation of the EDSP highlighted the need for more formal guidance regarding evaluation of information from the assay. The authors describe a framework for interpretation of FSTRA data relative to perturbation of endocrine pathways of concern to the EDSP. The framework considers end points individually and as suites of physiologically related responses relative to pathway identification. Sometimes changes in single end points can be highly diagnostic (e.g., induction of VTG in males by ER agonists, production of male secondary sex characteristics in females by AR agonists); in other instances, however, multiple, related end points are needed to reliably assess pathway perturbation (e.g., AR antagonism, steroid synthesis inhibition). In addition to describing an interpretive framework, the authors demonstrate its practical utility using publicly available FSTRA data for a wide range of known and hypothesized endocrine-disrupting chemicals. Environ Toxicol Chem 2014;33:2529-2540. Published 2014 Wiley Periodicals Inc., on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Challenges and Approaches to Conducting and Interpreting the Amphibian Metamorphosis Assay and the Fish Short‐Term Reproduction Assay

The amphibian metamorphosis assay (AMA) and the fish short-term reproduction assay (FSTRA) are screening assays designed to detect potential endocrine activity of a test substance. These assays are included in a battery of assays in Tier 1 of U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program. Based on our laboratory's experience with these two assays, we have noted several challenges in the conduct and interpretation of the AMA and FSTRA, including, but not limited to, diseased/parasitized test organisms, failure to meet some guideline performance criteria, and issues selecting and maintaining test concentrations. Various approaches are described for addressing the challenges associated with both the conduct and interpretation of these assays. Historical control data for both the AMA and FSTRA are presented to further understand background occurrences of histopathological phenomena and variability associated with the measured endpoints in these assays. In the historical control database for the AMA, wet weight on day 7 was the most variable endpoint (coefficient of variation = 26%), while developmental stage on day 21 was least variable (coefficient of variation = 0.47%). In the FSTRA, vitellogenin concentrations were the most variable endpoint (coefficient of variation = 47-84%), while fertility was the least variable endpoint (coefficient of variation = 1.5%) among historical controls.

Verification of responses of Japanese medaka (Oryzias latipes) to anti‐androgens, vinclozolin and flutamide, in short‐term assays

Various testing methods for the detection of the endocrine disruptive activities of chemicals have been developed in freshwater fish species. However, a few relatively easier specific methods for detecting anti-androgenic activities are available for fish. The aim of this study was to verify the papillary process in Japanese medaka (Oryzias latipes) as an indicator of the anti-androgenic activity of chemicals. Japanese medaka were exposed to two types of anti-androgenic compounds, vinclozolin and flutamide, using two short-term assays; one was conformed to the existing short-term reproduction assay using adult fish (adult test) and the other was a test based on the same methods but using juvenile fish at the beginning of exposure (juvenile test). Significant decreases in male papillary processes were observed in the juvenile test treated with the highest concentration of both antiandrogens (640 µg l(-1) vinclozolin and 1000 µg l(-1) flutamide); however, no significant effects were observed in the adult test. Consequently, our results indicate that papillary processes in Japanese medaka can be used as the end-point for screening the anti-androgenic activity of chemicals using juvenile fish for a specific period based on the existing short-term reproduction assay.

CROSS‐SPECIES CONSERVATION OF ENDOCRINE PATHWAYS: A CRITICAL ANALYSIS OF TIER 1 FISH AND RAT SCREENING ASSAYS WITH 12 MODEL CHEMICALS

Many structural and functional aspects of the vertebrate hypothalamic-pituitary-gonadal (HPG) axis are known to be highly conserved, but the significance of this from a toxicological perspective has received comparatively little attention. High-quality data generated through development and validation of Tier 1 tests for the U.S. Environmenal Protection Agency Endocrine Disruptor Screening Program (EDSP) offer a unique opportunity to compare responses of mammals versus fish to chemicals that may affect shared pathways within the HPG axis. The present study focuses on data generated with model chemicals that act (primarily) as estrogen receptor agonists (17α-ethynylestradiol, methoxychlor, bisphenol A), androgen receptor agonists (methyltestosterone, 17β-trenbolone), androgen receptor antagonists (flutamide, vincolozolin, p,p'-DDE), or inhibitors of different steroidogenic enzymes (ketoconazole, fadrozole, fenarimol, prochloraz). All 12 chemicals had been tested in the EDSP fish short-term (21 d) reproduction assay and in one or more of the four in vivo Tier 1 screens with rats (uterotrophic, Hershberger, male and female pubertal assays). There was a high concordance between the fish and rat assays with respect to identifying chemicals that impacted specific endocrine pathways of concern. Although most chemicals were detected as positive in both rat and fish assays, eliminating data from one class of vertebrate or the other would weaken the battery. For example, the effects of competitive inhibitors of steroid hormone synthesis were far more obvious in the fish assay, whereas the activity of androgen receptor antagonists was clearer in mammalian assays. The observations are significant both to the cross-species extrapolation of toxicity of HPG-active substances and the optimization of screening and testing frameworks for endocrine-disrupting chemicals.

An evaluation of 2,4-dichlorophenoxyacetic acid in the Amphibian Metamorphosis Assay and the Fish Short-Term Reproduction Assay

2,4-Dichlorophenoxyacetic acid (2,4-D) was evaluated in both the Amphibian Metamorphosis Assay (AMA) and the Fish Short Term Reproduction Assay (FSTRA). In the AMA, tadpoles were exposed to mean measured 2,4-D concentrations of 0 (water control), 0.273, 3.24, 38.0 and 113mg acid equivalents (ae)/L for either seven or 21 days. In the FSTRA, fathead minnows were exposed to mean measured 2,4-D concentrations of 0 (water control), 0.245, 3.14, 34.0, and 96.5mg ae/L for 21 days. The respective concentrations of 2,4-D were not overtly toxic to either Xenopus laevis tadpoles or fathead minnows (Pimephales promelas). In the AMA, there were no signs of either advanced or delayed development, asynchronous development, or significant histopathological effects of the thyroid gland among 2,4-D exposed tadpoles evaluated on either day seven or day 21 of the exposure. Therefore, following the AMA decision logic, 2,4-D is considered “likely thyroid inactive” in the AMA with a No Observable Effect Concentration (NOEC) of 113mg ae 2,4-D/L. In the FSTRA, there were no significant differences between control and 2,4-D exposed fish in regard to fertility, wet weight, length, gonado-somatic indices, tubercle scores, or blood plasma concentrations of vitellogenin. Furthermore, there were no treatment-related histopathologic changes in the testes or ovaries in any 2,4-D exposed group. The only significant effect was a decrease in fecundity among fish exposed to 96.5mg ae 2,4-D/L. The cause of the reduced fecundity at the highest concentration of 2,4-D tested in the assay was most likely due to a generalized stress response in the fish, and not due to a specific endocrine mode of action of 2,4-D. Based on fish reproduction, the NOEC in the FSTRA was 34.0mg ae 2,4-D/L.