Effects of 17α-ethinylestradiol in a fathead minnow (Pimephales promelas) gonadal recrudescence assay
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).
References (67)
- et al.
Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in The Netherlands
Sci. Total Environ.
(1999) - et al.
In vitro estrogenicity of the catechol metabolites of selected polychlorinated biphenyls
Toxicol. Appl. Pharmacol.
(1999) - et al.
Feminisation of young males of the common carp, Cyprinus carpio, exposed to 4-tert-pentylphenol during sexual differentiation
Aquat. Toxicol.
(1998) - et al.
Toxicity of selected priority pollutants to various aquatic organisms
Ecotoxicol. Environ. Saf.
(1983) - et al.
Measurement of vitellogenin-mRNA in primary cultures of rainbow trout hepatocytes in a non-radioactive dot blot/RNase protection assay
Sci. Total Environ.
(1999) - et al.
Effects of 17α-ethinylestradiol on the expression of three estrogen-responsive genes and cellular ultrastructure of liver and testes in male zebrafish
Aquat. Toxicol.
(2003) - et al.
Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas)
Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol.
(2001) - et al.
Gamete viability as an indicator of reproductive endocrine disruption in fish
Sci. Total Environ.
(1999) - et al.
Development of a sensitive E-screen assay for quantitative analysis of estrogenic activity in municipal sewage plant effluents
Sci. Total Environ.
(1999) - et al.
Input/output of estrogenic active compounds in a major municipal sewage plant in Germany
Chemosphere
(2000)
Effects of the mammalian antiandrogen vinclozolin on development and reproduction of the fathead minnow (Pimephales promelas)
Aquat. Toxicol.
Effects of waterborne exposure of 17β-estradiol on secondary sex characteristics and gonads of fathead minnows (Pimephales promelas)
Aquat. Toxicol.
Effects of waterborne exposure to 4-nonylphenol and nonylphenol ethoxylate on secondary sex characteristics and gonads of fathead minnows (Pimephales promelas)
Environ. Res.
A QSAR study of the toxicity of amines to the fathead minnow
Sci. Total Environ.
Adverse reproductive effects in male fathead minnows (Pimephales promelas) exposed to environmentally relevant concentrations of the natural oestrogens, oestradiol and oestrone
Aquat. Toxicol.
A proposed multigeneration protocol for Japanese medaka (Oryzias latipes) to evaluate effects of endocrine disruptors
Sci. Total Environ.
17α-ethinylestradiol affects reproduction, sexual differentiation and aromatase gene expression of the medaka (Oryzias latipes)
Aquat. Toxicol.
A low viscosity embedding medium for electron microscopy
J. Ultrastruct. Res.
Feminized responses in fish to environmental estrogens
Toxicol. Lett.
Behaviour and occurrence of estrogens in municipal sewage treatment plants—I. Investigations in Germany, Canada and Brazil
Sci. Total Environ.
Overview of a workshop on screening methods for detecting potential (anti-)estrogenic/androgenic chemicals in wildlife
Environ. Tox. Chem.
Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas)
Environ. Tox. Chem.
A yeast estrogen screen for examining the relative exposure of cells to natural and xenoestrogens
Environ. Health Perspect.
PCBs as environmental estrogensturtle sex determination as a biomarker of environmental contamination
Environ. Health Perspect.
Developmental effects of endocrine disrupting chemicals in wildlife and humans
Environ. Health Perspect.
Identification of estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening
Environ. Sci. Technol.
DDT-induced feminization of gull embryos
Science
Factors affecting the development of testis-ova in medaka, Oryzias latipes, exposed to octylphenol
Environ. Tox. Chem.
Induction of testis-ova in Japanese medaka (Oryzias latipes) exposed to p-nonylphenol
Environ. Tox. Chem.
Serum vitellogenin levels and reproductive impairment of male Japanese medaka (Oryzias latipes) exposed to 4-tert-octylphenol
Environ. Health Perspect.
Organisation versus activationthe role of endocrine-disrupting contaminations (EDCs) during embryonic development in wildlife
Environ. Health Perspect.
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