Gonadal intersex and high prevalences of the female phenotype have been observed in fish populations in urbanized areas. Environmental estrogens discharged in sewage treatment plant effluents may be responsible for feminization of fish but many compounds with the potential to induce these responses occur in effluents, including natural and synthetic estrogen hormones, degradation products of alkylphenol ethoxylate surfactants, and plasticizers. In this study, the estrogen hormones 17 alpha-ethinylestradiol, 17 beta-estradiol, estrone, and estriol induced intersex (i.e., testis-ova) and altered sex in Japanese medaka (Oryzias latipes) when these fish were exposed to nanogram per liter concentrations of test compounds from hatch to approximately 100 d after hatch. A mix of nonylphenol mono- and diethoxylate induced a weak response and a mix of nonylphenol mono- and diethoxycarboxylate did not give a response in this assay at microgram per liter concentrations, indicating that these degradation products of nonylphenol ethoxylates have little or no estrogenic activity in fish. Bisphenol A induced testis-ova in medaka exposed to a concentration of 10 micrograms/L, but diethylhexyl phthalate did not induce a response. Results with the medaka assay were consistent with estrogenic responses in the yeast estrogen screening assay. Analyses of monitoring data reported in the literature indicate that concentrations of estrogen hormones detected in the final effluents of sewage treatment plants are generally greater than the lowest-observed-effect levels for alterations to gonadal development in medaka.

Gonadal intersex and high prevalences of the female phenotype have been observed in fish populations in urbanized areas. Environmental estrogens discharged in sewage treatment plant effluents may be responsible for feminization of fish but many compounds with the potential to induce these responses occur in effluents, including natural and synthetic estrogen hormones, degradation products of alkylphenol ethoxylate surfactants, and plasticizers. In this study, the estrogen hormones 17 alpha-ethinylestradiol, 17 beta-estradiol, estrone, and estriol induced intersex (i.e., testis-ova) and altered sex in Japanese medaka (Oryzias latipes) when these fish were exposed to nanogram per liter concentrations of test compounds from hatch to approximately 100 d after hatch. A mix of nonylphenol mono- and diethoxylate induced a weak response and a mix of nonylphenol mono- and diethoxycarboxylate did not give a response in this assay at microgram per liter concentrations, indicating that these degradation products of nonylphenol ethoxylates have little or no estrogenic activity in fish. Bisphenol A induced testis-ova in medaka exposed to a concentration of 10 micrograms/L, but diethylhexyl phthalate did not induce a response. Results with the medaka assay were consistent with estrogenic responses in the yeast estrogen screening assay. Analyses of monitoring data reported in the literature indicate that concentrations of estrogen hormones detected in the final effluents of sewage treatment plants are generally greater than the lowest-observed-effect levels for alterations to gonadal development in medaka.

The estrogenic isoflavone compound genistein recently has been found in the effluents of sewage treatment plants and pulp mills, and the related compound equol has been detected in the runoff from agricultural fields treated with hog manure. Waterborne exposures of Japanese medaka (Oryzias latipes) to equol from soon after hatch to approximately 100 days posthatch induced gonadal intersex (i.e., testis-ova) in males at incidences of 10 and 87% in equol treatments of 0.4 and 0.8 µg/L, respectively. Exposure to the highest test concentration of genistein, 1,000 µg/L, also caused a low incidence (i.e., 12%) of gonadal intersex in male medaka. The ovaries of female medaka from both equol and genistein treatments showed delayed oocyte maturation, atretic oocytes, an enlarged ovarian lumen, proliferation of somatic stromal tissue, and primordial germ cells; responses were concentration dependent. Alterations to externally visible secondary sex characteristics occurred in medaka exposed to both equol and genistein. In treatments with 1,000 µg/L genistein, 72% of male medaka (as identified by the gonadal phenotype) showed feminized secondary sex characteristics. Gonadal intersex and alterations to secondary sex characteristics have been noted in several fish populations around the world. This laboratory study indicates that isoflavone compounds should be considered candidate estrogenic compounds that may be involved in the alteration of sexual development in feral fish populations.

The sheepshead minnow (Cyprinodon variegatus Lacépède), an estuarine fish species, was exposed to 17alpha-ethynylestradiol (EE2) at nominal test concentrations of 0.2, 2, 20, 200, 400, 800, 1,600, and 3,200 ng/L. Fish were exposed for up to 59 d, from subadult stages to sexual maturity, under flow-through conditions. The exposure period was followed by an evaluation of reproductive success and survival of progeny. The reproductive success of exposed sheepshead minnows, as determined from data on egg production from two subsequent spawning trials, was reduced in fish exposed to 200 ng/L EE2 and, in one spawning trial, in the 20-ng/L treatment. Hatching success was reduced in the progeny of fish exposed to 200 ng/L EE2, but survival was good among fry that successfully hatched. Histological examination indicated generalized edema, damage to gill epithelia, hepatic toxicity, fibrosis of the testis, and evidence of sex reversal, including testes-ova and spermatagonia-like cells in ovaries. The maximum acceptable toxicant concentration (MATC) for gonadal development in males was within the normal range of EE2 concentrations in sewage treatment plant effluents. The exposure regimen and choice of test organism, combined with histological examination, allowed independent evaluation of ecologically significant acute, reproductive and estrogenic endpoints. Estrogen receptor-mediated effects occurred at concentrations where reproductive effects were measurable under standard reproduction assays. The sheepshead minnow appears to be a sensitive in vivo model for partial life-cycle testing of compounds that have the potential to disrupt the endocrine system as well as reproduction in estuarine and coastal marine fish species.

Abstract-Various isomers and metabolites of DDT disrupt endocrine systems and gonadal development in fish and wildlife, and o,pЈ-DDT has been shown to be an relatively potent estrogen agonist. In this study, we exposed Japanese medaka (Oryzias latipes) to o,pЈ-DDT using two exposure protocols: direct exposure of early life stages to aqueous solutions from 1 to 100 d posthatch and exposure of female medaka to aqueous solutions, followed by mating with unexposed males to produce offspring that were exposed through mechanisms of maternal transfer. In treatments with direct aqueous exposures, an intersex condition of the gonad (testisova) was observed in male medaka exposed at early life stages to nominal o,pЈ-DDT concentrations of 50, 10, and 5 g/L, indicating that this estrogen agonist can alter gonadal development when exposure occurs continuously over the period of gonadal differentiation. Comparisons with previously published data on the induction of testis-ova by exposure to nonylphenol (NP) and octylphenol (OP) indicated that the relative potencies for induction of intersex in medaka are o,pЈ-DDT Ͼ NP ഠ OP, which is not consistent with the relative estrogenic potencies of OP Ͼ NP Ͼ o,pЈ-DDT observed in the yeast estrogen screening (YES) assay. In the maternal transfer protocol, no testis-ova were observed in the offspring, although there was some delay in time to hatch of the offspring. Medaka exposed by maternal transfer showed no other toxicological responses during early life stages, but when treated fish reached sexual maturity, the females showed more advanced development of oocytes. In addition, when medaka exposed by maternal transfer were subsequently exposed at 10 months of age to 17␤-estradiol (12 mg/L), there was a significantly greater induction of hepatic vitellogenin in DDT-exposed males in comparison to control males, indicating that exposure to estrogenic chemicals during early life stages may potentiate vitellogenin induction following exposure events later in the life of the fish.

Alkyl-phenanthrenes are a class of compounds present in crude oil and toxic to developing fish. Most research on alkyl-phenanthrenes has focused on retene (7-isopropyl-1-methyl-phenanthrene), but little is known about the chronic toxicity of related congeners to the early life stages of fish. This project is the first to describe the chronic toxicity of a series of alkyl-phenanthrenes to the embryos of Japanese medaka (Oryzias latipes) using the partition-controlled delivery (PCD) method of exposure and is the first to establish a relationship between toxicity of alkyl-phenanthrenes and log P. With PCD, test concentrations were maintained by equilibrium partitioning of test chemicals from polydimethylsiloxane (PDMS) films containing various concentrations of C1 to C4 phenanthrenes. Log film:solution partition constants (log K(fs)) and aqueous solubility limits were determined for each alkyl-phenanthrene. The prevalence of abnormalities in fish embryos increased in an exposure-dependent manner, with median effective concentration (EC50) values lower than experimental solubility limits of the compounds, and typical of environmental concentrations. Alkyl-phenanthrenes were more toxic to medaka embryos than unsubstituted phenanthrene, with effects resembling those of dioxin and indicating a specific receptor-based mechanism of toxicity. These results extend conclusions for the Exxon Valdez oil spill, suggest a specific mechanism of toxicity for alkyl-phenanthrenes, and provide a model for assessing the risks of mixture toxicity.

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