Effects of 17α-ethinylestradiol in a fathead minnow (Pimephales promelas) gonadal recrudescence assay

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Abstract

To contribute to the development and evaluation of a practical and cost-effective in vivo testing system for endocrine disruption (specifically environmental estrogens), the effects of 17α-ethinylestradiol (EE2) were assessed in a gonadal recrudescence assay with the fathead minnow (Pimephales promelas). Mature male and female fathead minnows were kept first at 15°C on a 8 h light/16 h dark regime and then transferred to 25°C and a 16 h light/8 h dark regime to induce gonadal recrudescence. They were then exposed to various nominal concentrations of the synthetic estrogen EE2 (0, 0.1, 1, 3, 10, 100 ng/L). After 3 weeks of chemical exposure, effects on plasma vitellogenin (VTG), secondary sex characteristics, gonad growth (gonadosomatic index; GSI), and condition factor were assessed. Additionally, the effects on liver and gonad tissue morphology were investigated by means of light (LM) and electron microscopy (EM). Reproductive output (egg production) and fertilization rate were measured during a subsequent 3-week period in breeding adults maintained in clean water. Exposure to EE2 resulted in a significant decrease in GSI, condition factor, and number of batches of eggs and their fertilization rate at EE2 exposure concentrations between 10 and 100 ng/L. A reduction in the extent of parenchymatic areas in ovaries and ultrastructural changes in the livers of females could be detected at EE2 concentrations ⩾3 ng/L. The lowest observed effective concentration (LOEC) of EE2 for plasma VTG induction in both sexes and for ultrastructural changes in the testes and livers was 1 ng/L. A significant increase in the mean number of eggs spawned per pair occurred at EE2 exposure doses of 0.1 and 1 ng/L. However, at higher EE2 concentrations, a dose-dependent decrease in the mean number of eggs per pair was apparent. Therefore, the LOEC for a biological effect of EE2 in the fathead minnow using the selected endpoints in the recrudescence assay was 1 ng/L for biomarkers such as plasma VTG and number of tubercles, and 0.1 ng/L for an increased number of eggs spawned per pair.

Introduction

Many synthetic chemicals in the environment may act as endocrine disruptors (Bergeron et al., 1994; Colborn et al., 1993; Fry and Toone, 1981; Gimeno et al., 1998; Guillette et al., 1995; Routledge et al., 1998; Sumpter, 1995). In vitro bioassays have been developed to detect endocrine activity of single compounds and chemical mixtures found in environmental samples such as wastewater effluents and surface waters (Ankley et al., 1998; Arnold et al., 1996; Desbrow et al., 1998; Garner et al., 1999; Harries et al., 1997; Islinger et al., 1999; Milligan et al., 1998; Pawlowski et al., 2000; Soto et al., 1995). In vitro assays, however, do not necessarily consider aspects such as cellular differentiation, regeneration, biotransformation, and bioaccumulation. The use of in vivo systems is therefore indispensable to assess the true environmental risk of potential endocrine disruptors. Several full and partial life-cycle tests have been developed using zebrafish (Danio rerio), fathead minnow (Pimephales promelas), and medaka (Oryzias latipes), but most of these tests are both time-intensive and costly (Bulder et al., 2000; Gronen et al., 1999; Papoulias et al., 1999; Patyna et al., 1999; Scholz and Gutzeit, 2000). To minimize time and budget requirements, a fish gonadal recrudescence assay with the fathead minnow (P. promelas) has been recommended by the Endocrine Disrupter Screening and Testing Advisory Committee as a tier 1 screening test for endocrine disrupting chemicals (EDSTAC Final Report, 1998). In this assay, adult fish previously kept under winter conditions are transferred to summer conditions and simultaneously exposed to endocrine-disrupting chemicals to study the influence of (xeno) estrogens on gonadal recrudescence.

The fathead minnow is a fish specie widely used in toxicological research, and many data are available on its culture and reproductive behavior (Gunatilleka and Poole, 1999; Holcombe et al., 1983; Kovacs et al., 1995; Manner and Dewese, 1974; McMillan and Smith, 1974; Newsome et al., 1991). Its small size, rapid progression to maturity, and sexual dimorphism make the fathead minnow suitable and easy to handle for laboratory research including reproduction tests. In contrast to tropical fish such as zebrafish and Japanese medaka, the fathead minnow originates from temperate climates with definite seasonal changes in gonadal status, as is typical for many indigenous European fish species. This difference makes the fathead minnow a more appropriate species for use in risk assessment of the potential impacts of endocrine-disrupting chemicals on European and North American fish populations (Nicols et al., 1999).

Under natural conditions, the cold period during winter induces gonadal dormancy in the fathead minnow. In the recrudescence assay, this dormancy state is induced by keeping the fish under winter conditions for at least 4 weeks before the start of the test. The test design thus requires stock populations at the winter stage (for a maximum of ∼6 months). However, the experimental induction of dormancy before the actual test makes the test fairly independent of the natural breeding cycle and season (Miles-Richardson et al., 1999b) and also minimizes the seasonal variation of the gonadal status between individuals in a population (Jensen et al., 2001; Miles-Richardson et al., 1999b), thus facilitating standardization in the test fish. Despite this advantage, this test design has not been considered in recent studies with this fish species (Ankley et al., 2001; Knutson et al., 1997; Makynen et al., 2000; Miles-Richardson et al., 1999a; Panter et al (1998), Panter et al (2000), Panter et al (2002); Sohoni et al., 2001).

In this study, exposure to the potent estrogen 17α-ethinylestradiol (EE2) was used to evaluate the ability and sensitivity of the gonadal recrudescence assay to detect estrogenic effects with the fathead minnow. The endpoints measured included gonadal growth (gonadosomatic index, GSI=gonad wt/somatic wt), secondary sex characteristics, vitellogenin (VTG) induction, histological and ultrastructural changes of liver and gonads, and reproductive success (egg production and fertilization rate) subsequent to chemical exposure. The experimental protocol was designed to include EE2 exposure concentrations that are present in the aquatic environment: EE2 has been detected in sewage effluents from a few nanograms per liter up to 62 ng/L in Europe (Belfroid et al., 1999; Desbrow et al., 1998; Kuch and Ballschmiter, 2000; Ternes et al., 1999), up to 42 ng/L in North America (Ternes et al., 1999), and in surface waters at concentrations up to 5 ng/L (Huang and Sedlak, 2001; Stumpf et al., 1996). Several full life-cycle experiments with small fish species such as fathead minnow, zebrafish, and Japanese medaka have shown that, even at very low concentrations, EE2 has endocrine-disrupting effects such as induction of VTG, ovo-testes, and phenotypic sex reversal in males, and the appearance of atretic follicles and reduced egg production in females, as well as decrease in the fertilization rate (Kime and Nash, 1999; Länge et al., 2001; Papoulias et al., 1999; Schäfers et al., 2000; Scholz and Gutzeit, 2000)—all of which make it an ideal chemical to evaluate the recrudescence assay for its potential to detect estrogenic effects. A central aim of this study was to compare the data from the fathead minnow recrudescence test with those from other short- and long-term experiments that have exposed small fish to EE2.

Section snippets

Chemicals

17α-Ethinylestradiol (EE2, ⩾98% purity) and dimethyl sulfoxide (DMSO) were purchased from Sigma (Deisenhofen, Germany).

Fish

Breeding stocks of adult fathead minnows were provided by Reinhard Länge (Schering AG, Berlin, Germany). Mature male and female fathead minnows between 6 and 11 months of age were held under winter conditions (15±1°C water temperature and 8 h light/16 h dark regime) for at least 30 days before the start of the test.

Test conditions

The dilution water was a mixture of tap and demineralized water.

Chemical analysis

No EE2 could be detected in the controls (water, solvent). Measured EE2 concentrations varied between 20% and 32%, with values of 0.7 and 0.8 ng/L (nominal concentration: 1 ng/L) and 8.1 and 7.8 ng/L (nominal concentration: 10 ng/L) for male and female aquaria, respectively.

Condition factor and gonad growth

In the controls, no significant differences were observed in condition factors of females after 3 weeks’ exposure to EE2 concentrations of 0.1, 1, 3, and 10 ng/L (Fig. 1). At an EE2 concentration of 100 ng/L, however, there was a

Discussion

The development of sensitive and cost-effective in vivo screening and testing systems in fish for chemicals that mimic endogenous hormones, both natural and synthetic, is indispensable for risk assessment. With this in mind, the gonadal recrudescence assay proved to be highly sensitive to detect estrogenic effects of the potent synthetic estrogen EE2. With the exception of the optional measurement of plasma titers of sex steroids, all recommended endpoints mentioned in the EDSTAC proposal (

Conclusions

With respect to biomarkers of exposure such as plasma VTG and the number of tubercles on the heads of male fathead minnows, as well as more population-relevant endpoints such as egg and sperm production, the recrudescence assay with the fathead minnow seems to be suitable to detect changes that are due to estrogenic or estrogen-like stimuli even at environmentally relevant concentrations of EE2. The recrudescence assay with the fathead minnow should be evaluated in more detail with respect to

Acknowledgements

The authors are particularly grateful to Reinhard Länge and Melanie Zerulla (Schering AG, Berlin, Germany) for providing adult fathead minnows. This study was supported by the Federal Environmental Protection Agency of Germany under contract no. 29765001/01 and a personal grant from the Deutsche Bundesstiftung Umwelt (Osnabrück, Germany).

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