In response to the initial Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. This research highlighted key limitations associated with relying on tail resorption as a measure of anti/thyroid activity. The most critical limitation being that tail tissues of tadpoles at metamorphic climax are insensitive to perturbation by thyroid axis agonists/antagonists. To improve upon the initial proposal, we have conducted experiments comparing the sensitivity of pre-metamorphic (stage 51) and pro-metamorphic (stage 54) larvae to the model thyroid axis disruptors methimazole (control, 6.25, 12.5, 25, 50, 100 mg/l), 6-propylthiouracil (PTU) (control, 1.25, 2.5, 5, 10, and 20 mg/l), and thyroxine (T4) (0.25, 0.5, 1, 2, 4 microg/l). Exposures were conducted using two different experimental designs. For experimental design 1, tadpoles were exposed to methimazole or PTU starting at either NF stage 51 or NF 54 for 14 days. For experimental design 2, tadpoles were exposed to PTU or T4 starting at NF stage 51 or NF 54 for 14 and 21 days, respectively. Methimazole and PTU, which are thyroid hormone synthesis inhibitors, both caused a concentration dependent delay in larval development. As determined from this endpoint, there were only minor differences in sensitivity observed among the two stages examined. Further, both compounds caused concentration dependent changes in thyroid gland morphology. These changes were characterized as reduced colloid, glandular hypertrophy, and cellular hyperplasia and hypertrophy. Treatment failed to negatively affect growth, even in tadpoles that experienced significant metamorphic inhibition. T4 treatment resulted in a concentration dependent increase in developmental rate, as would be expected. Similar to studies with methimazole, there were no differences in sensitivity among the two developmental stages examined. These results indicate that tadpoles in the early stages of metamorphosis are sensitive to thyroid axis disruption and that development of a short-term, diagnostic amphibian-based thyroid screening assay shows considerable promise.

The perchlorate anion inhibits thyroid hormone (TH) synthesis via inhibition of the sodium-iodide symporter. It is, therefore, a good model chemical to aid in the development of a bioassay to screen chemicals for affects on thyroid function. Xenopus laevis larvae were exposed to sodium perchlorate during metamorphosis, a period of TH-dependent development, in two experiments. In the first experiment, stage 51 and 54 larvae were exposed for 14 d to 16, 63, 250, 1,000, and 4,000 microg perchlorate/ L. In the second experiment, stage 51 larvae were exposed throughout metamorphosis to 8, 16, 32, 63, and 125 microg perchlorate/L. Metamorphic development and thyroid histology were the primary endpoints examined. Metamorphosis was retarded significantly in the first study at concentrations of 250 microg/L and higher, but histological effects were observed at 16 microg/L. In the second study, metamorphosis was delayed by 125 microg/L and thyroid size was increased significantly at 63 microg/L. These studies demonstrate that inhibition of metamorphosis readily can be detected using an abbreviated protocol. However, thyroid gland effects occur at concentrations below those required to elicit developmental delay, demonstrating the sensitivity of this endpoint and suggesting that thyroidal compensation is sufficient to promote normal development until perchlorate reaches critical concentrations.

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.

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In response to various legislative mandates, the US Environmental Protection Agency (USEPA) formed its Endocrine Disruptor Screening Program (EDSP), which in turn, formed the basis of a tiered testing strategy to determine the potential of pesticides, commercial chemicals, and environmental contaminants to disrupt the endocrine system. The first tier of tests is intended to detect the potential for endocrine disruption mediated through estrogen, androgen, or thyroid pathways, whereas the second tier is intended to further characterize the effects on these pathways and to establish a dose-response relationship for adverse effects. One of these tier 2 tests, the Medaka Extended One Generation Reproduction Test (MEOGRT), was developed by the USEPA for the EDSP and, in collaboration with the Japanese Ministry of the Environment, for the Guidelines for the Testing of Chemicals of the Organisation for Economic Co-operation and Development (OECD). The MEOGRT protocol was iteratively modified based on knowledge gained after the successful completion of 9 tests with variations in test protocols. The present study describes both the final MEOGRT protocol that has been published by the USEPA and the OECD, and the iterations that provided valuable insights into nuances of the protocol. The various tests include exposure to 17β-estradiol, 4-t-octylphenol, o,p'- dichlorodiphenyltrichloroethane, 4-chloro-3-methylphenol, tamoxifen, 17β-trenbolone, vinclozolin, and prochloraz. Environ Toxicol Chem 2017;36:3387-3403. Published 2017 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.

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.

The medaka extended one-generation test (MEOGRT) was developed as a multigenerational toxicity test for chemicals, particularly endocrine-disrupting chemicals. Briefly, 3 generations of Japanese medaka (Oryzias latipes) are exposed to a chemical over a 20-wk period: 3 wk in the parental generation (F0), 15 wk in the first generation (F1), and 2 wk in the second generation (F2). The present study reports the first MEOGRT results concerning branched isomer mixtures of 4-nonylphenol (NP). Adult F0 medaka exposed to NP at 5 actual concentrations (1.27, 2.95, 9.81, 27.8, 89.4 µg/L) were unaffected in terms of reproduction, although vitellogenin in the male liver was increased dose-dependently at concentration of 2.95 µg/L and higher. In F1, in contrast, total egg (fecundity), fertile egg, and fertility decreased as NP increased; lowest-observed-effect concentrations (LOECs) for total egg, fertile egg, and fertility were 1.27, 1.27, 27.8 µg/L, respectively. In F1, but not in F0, secondary sex characteristics (i.e., anal fin papillae in males) were suppressed at 27.8 µg/L NP. Vitellogenin induction in adult male fish was slightly weaker in F1 than it was in F0, however. Gonadal sex abnormality and sex reversal occurred at 27.8 and 89.4 µg/L NP in F1 subadults. At 89.4 µg/L NP, all genotypic F1 males in breeding pairs had female phenotype, and some even demonstrated spawning. Concentrations of NP lower than 89.4 µg/L did not affect F2 survival or hatching. The highest detected NP level in environmental freshwater in Japan was approximately a half of the LOEC (1.27 µg/L for F1 fecundity); in other countries, however, environmental concentrations above the LOEC are reported, suggesting that NP may be affecting fish populations. Environ Toxicol Chem 2017;36:3254-3266. © 2017 SETAC.

Small aquarium fishes are increasingly used as animal models, and one of these, Japanese Medaka (Oryzias latipes), is frequently utilized for toxicity testing. While these vertebrates have many similarities with their terrestrial counterparts, there are differences that must be considered if these organisms are to be used to their highest potential. Testing commonly may employ either the developing embryo or adults; both are easy to use and to work with. We present here three main protocols to illustrate the utility and breadth of toxicity testing possible using medaka fish. The first protocol assesses neurotoxicity in developing embryos. The second protocol describes the sexual genotyping of medaka to evaluate toxicant effects on sexual phenotype after treatment with endocrine disrupting chemicals. The third protocol assesses hepatotoxicity in adult fish after treatment with a model hepatotoxicant. The methods run the gamut from immunohistology through PCR to basic histological techniques.