Effects of endocrine disruptors in zebrafish (Danio rerio) as revealed with the fish sexual development test

Abstract

So-called “endocrine disrupting chemicals” (EDCs) are ubiquitous in the environment, especially in aquatic systems close to industrial and agricultural areas. Disturbances of the hormonal balance between estrogens and androgens are one central target of EDCs. Especially during sexual differentiation of organisms, the consequences of such a misbalance may easily lead to population-relevant effects. Thus research into EDCs has been massively intensified over the last decades. Since, at international levels, there is high interest to establish validated test systems for the evaluation of endocrine effects, the OECD has initiated a progran to develop test guidelines for the detection of EDCs. Within this scope, the present work forms part of a validation exercise for the so-called “fish sexual development test” (FSDT), an extension of the existing OECD test guideline 210, the fish early life-stage toxicity test as an OECD guideline. The main idea is to establish an in vivo test for EDCs, which is relatively straightforward, cheap and on the one hand more rapid than full life-cycle tests, but, on the other hand, more sensible than the currently discussed 21-day-screening test. In the present study, the two EDCs prochloraz and 4-tert-pentylphenol were selected as exemplary substances to be tested in the FSDT. Prochloraz is a fungicide that inhibits the enzyme aromatase and therefore the synthesis of steroids. 4-tert-pentylphenol belongs to the group of alkylphenols, which are known to interact with the estrogen receptor, and, therefore, are classified as pseudoestrogens. Zebrafish (Danio rerio) were exposed to these chemicals in flow-through systems for 60 days post-hatch, a period in which zebrafish finalize their sexual differentiation. Within this sensitive phase the sexual development can easily be disturbed by EDCs. After exposure, the zebrafish were measured and weighed, the gonads were examined by means of histological analysis, and vitellogenin concentrations were measured in heads and tails via ELISA. Both chemicals caused a shift in sex ratio (prochloraz: masculinization; 4-tert-pentylphenol: feminization), underdevelopment of the gonads, inhibition of growth and altered vitellogenin production. For the two substances examined, the sex ratio and the evaluation of maturity stages were both the most sensitive and the most relevant endpoints. Thus, even though reproductive and population-relevant effects could not be documented by the FSDTs, the test clearly had at least predictive power for what could have been seen in full life-cycle tests. Therefore, the present study corrobates the conclusion that the FSDT is an adequate test system for the evaluation of EDCs and that its further validation as an OECD test guideline is definitely required.