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Environmental pollutants and especially endocrine disrupting chemicals (EDCs) are implicated as one of the drivers of the amphibian declines. To advance the understanding of the risks of EDCs to amphibians, methods to determine endocrine-linked adverse effects are needed. The aims were to 1) develop a partial life-cycle assay with the model frog Xenopus tropicalis to determine endocrine perturbation and adverse developmental effects, and 2) determine effects of propiconazole in this assay. Propiconazole is a pesticide with multiple endocrine modes of action in vitro. Its potential endocrine activity and adverse effects in amphibians remain to be elucidated. Tadpoles were exposed to 0, 33 and 384 μg propiconazole/L during critical developmental windows until completed metamorphosis. At metamorphosis, a sub-sample of animals was analysed for endpoints for disruption of estrogen/androgen (sex ratio, brain aromatase activity) and thyroid pathways (time to metamorphosis). The remaining individuals were kept unexposed for 2 months post-metamorphosis to analyze effects on sexual development including gonadal and Müllerian duct maturity and gametogenesis. At metamorphosis, brain aromatase activity was significantly increased in the high-dose group compared to control. In both propiconazole groups, an increased proportion of individuals reached metamorphosis faster than the mean time for controls, suggesting a stimulatory effect on the thyroid system. At 2 months post-metamorphosis, testis size, sperm and Müllerian duct maturity were reduced in the low-dose males, and the liver somatic index in males was increased in both propiconazole groups, compared with controls. In conclusion, our results show that propiconazole exposure caused endocrine perturbations and subsequent hepatic and reproductive effects evident at puberty, indicating persistent disruption of metabolism and male reproductive function. Our findings advance the development of methodology to determine endocrine and adverse effects of EDCs. Moreover, they increase the understanding of endocrine perturbations and consequent risk of adverse effects of azoles in amphibians., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

There is an increasing concern regarding the risks associated with developmental exposure to endocrine disrupting chemicals and the consequences for reproductive capability. The present study aimed to refine the Xenopus (Silurana) tropicalis test system for developmental reproductive toxicity by characterising molecular and histological features of sexual development, and to explore effects of exposure to the progestagen levonorgestrel (LNG). Larvae were exposed to LNG (0, 3, 30, 300 ng/L) over the first three weeks of development, encompassing the beginning of gonadal differentiation. mRNA levels of amh (anti-Müllerian hormone), amhr2 (amh receptor 2), ipgr (intracellular progesterone receptor), mpgr beta (membrane progesterone receptor beta), and cyp19a1 (cytochrome p450 19a1) were quantified in larvae and juveniles (4 weeks post-metamorphosis). Relative cyp19a1 and amh expression was used as a molecular marker for phenotypic sex of larvae. Gonadal and Müllerian duct development were characterised histologically in juveniles. Compared to controls, LNG exposure increased the expression of amh and ipgr in male larvae. In juveniles, mpgr beta expression was increased in both sexes and amhr2 expression was decreased in males, implying persistent effects of developmental progestagen exposure on amh and pgr expression signalling. No effects of LNG on the gonadal or Müllerian duct development were found, implying that the exposure window was not critical with regard to these endpoints. In juveniles, folliculogenesis had initiated and the Müllerian ducts were larger in females than in males. This new knowledge on sexual development in X. tropicalis is useful in the development of early life-stage endpoints for developmental reproductive toxicity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Synthetic progesterone (progestins) and estrogens are widely used pharmaceuticals. Given that their simultaneous unintentional exposure occurs in wildlife and also in human infants, data on mixture effects of combined exposures to these hormones during development is needed. Using the Xenopus (Silurana) tropicalis test system we investigated mixture effects of levonorgestrel (LNG) and ethinylestradiol (EE2) on hormone sensitive endpoints. After larval exposure to LNG (0.1nM), or EE2 (0.1nM) singly, or in combination with LNG (0.01, 0.1, 1.0nM), the gonadal sex ratio was determined histologically and hepatic mRNA levels of genes encoding vitellogenin (vtg beta1) and the estrogen (esr1, esr2), progesterone (ipgr) and androgen (ar) receptors were quantified using quantitative PCR. All EE2-exposed groups showed female-biased sex ratios and increased vtg beta1 mRNA levels compared with the controls. Compared with the EE2-alone group (positive control) there were no significant alterations in vtg beta1 levels or in sex ratios in the co-exposure groups. Exposure to LNG-alone caused an increase in ar mRNA levels in females, but not in males, compared to the controls and the co-exposed groups, indicating that co-exposure to EE2 counteracted the LNG-induced ar levels. No treatment related impacts on the mRNA expression of esr1, esr2, and ipgr in female tadpoles were found, suggesting that these endpoints are insensitive to long-term exposure to estrogen or progestin. Due to the EE2-induced female-biased sex ratios, the mRNA expression data for the low number of males in the EE2-exposed groups were not statistically analyzed. In conclusion, our results suggest that induced vtg expression is a robust biomarker for estrogenic activity in exposure scenarios involving both estrogens and progestins. Developmental exposure to LNG caused an induction of hepatic ar mRNA expression that was antagonized by combined exposure to EE2 and LNG. To our knowledge this is the first study to report effects of combined exposures to EE2 and LNG during the period of sexual programming., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Most amphibians breed in water, including the terrestrial species, and may therefore be exposed to water-borne pharmaceuticals during critical phases of the reproductive cycle, i.e. sex differentiation and gamete maturation. The objectives of this paper were to (i) review available literature regarding adverse effects of hormonally active pharmaceuticals on amphibians, with special reference to environmentally relevant exposure levels and (ii) expand the knowledge on toxicity of progestagens in amphibians by determining effects of norethindrone (NET) and progesterone (P) exposure to 0, 1, 10 or 100 ng l(-1) (nominal) on oogenesis in the test species Xenopus tropicalis. Very little information was found on toxicity of environmentally relevant concentrations of pharmaceuticals on amphibians. Research has shown that environmental concentrations (1.8 ng l(-1)) of the pharmaceutical oestrogen ethinylestradiol (EE2) cause developmental reproductive toxicity involving impaired spermatogenesis in frogs. Recently, it was found that the progestagen levonorgestrel (LNG) inhibited oogenesis in frogs by interrupting the formation of vitellogenic oocytes at an environmentally relevant concentration (1.3 ng l(-1)). Results from the present study revealed that 1 ng NET l(-1) and 10 ng P l(-1) caused reduced proportions of vitellogenic oocytes and increased proportions of previtellogenic oocytes compared with the controls, thereby indicating inhibited vitellogenesis. Hence, the available literature shows that the oestrogen EE2 and the progestagens LNG, NET and P impair reproductive functions in amphibians at environmentally relevant exposure concentrations. The progestagens are of particular concern given their prevalence, the range of compounds and that several of them (LNG, NET and P) share the same target (oogenesis) at environmental exposure concentrations, indicating a risk for adverse effects on fertility in exposed wild amphibians.

Levonorgestrel is a synthetic progesterone commonly used in pharmaceuticals (e.g., in contraceptives). It is found in sewage treatment plant effluents at concentrations up to 30 ng/L and was recently shown to pose a threat to egg laying in fish. Information on the susceptibility of adult amphibians to progestin toxicity is lacking. The present study aimed to 1) characterize progestogenic effects on the full cycle of oogenesis (egg development) in frogs and 2) determine female amphibians' susceptibility to reproductive impacts from progestogenic compounds in the environment. Sexually mature female Xenopus tropicalis were exposed to levonorgestrel via the surrounding water for 7 days (0, 51, or 307 ng/L) or 28 days (0, 1.3, 18, 160, or 1240 ng/L). Their ovaries were analyzed histologically with respect to frequencies of immature (in early meiotic prophase I), previtellogenic, vitellogenic, mature, and atretic oocytes. The 28-day exposure caused reduced proportions of oocytes at immature, vitellogenic, and mature stages, and increased proportions of previtellogenic oocytes compared with the control. The lowest tested concentration, 1.3 ng/L, increased the proportions of previtellogenic oocytes and reduced the proportions of vitellogenic oocytes, indicating inhibited vitellogenesis. The present study shows that progestin concentrations found in the aquatic environment impaired oogenesis in adult frogs. Our results indicate that progestogenic effects on oocyte development include interrupted germ cell progression into meiosis and inhibited vitellogenesis. Considering the crucial role of oogenesis in female fertility, our results indicate that progestogenic pollutants may pose a threat to reproduction in wild amphibian populations.