Comparison of ethinylestradiol and nonylphenol effects on reproduction of Chinese rare minnows (Gobiocypris rarus)

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Abstract

Breeding pairs of rare minnows were exposed to 5, 10, 20 μg/L 4-nonylphenol (NP) or 4 ng/L 17α-ethinylestradiol (EE2) for 21 days. Among reproductive parameters investigated, fecundity, fertility, and laying interval were significantly affected by 4 ng/L EE2, but were not affected at any exposure level of NP. These effects coincided with male feminization, ova-testis, increased plasma VTG in both males and females as well as the accumulation of eosinophilic material in kidneys and livers. Histopathologic evaluation revealed severe kidney lesions in both EE2 and NP-exposed fish. The bioassay succeeded in demonstrating that EE2 was several orders of magnitude more potent than the industrial chemical NP. The rare minnow was more sensitive to EE2 than medaka, but comparable to zebrafish in terms of sensitivity. The results of the present investigation indicated that a short-term reproductive bioassay is feasible using the Chinese rare minnow as the test species.

Introduction

Adverse effects of xenobiotic chemicals capable of disrupting the endocrine systems of wildlife species are of increasing concern (Nichols et al., 1999; Solé et al., 2003; Liney et al., 2005). The capacity to mimic natural steroids is found in diverse chemical groups including organochlorine pesticides, polychlorinated biphenyls, dioxins, alkylphenols, phthalates (Jobling et al., 1996; Groonen et al., 1999) as well as natural and synthetic steroids (Ternes et al., 1999; Liney et al., 2005).

Prominent among the alkylphenols, 4-nonylphenol (NP) enters the aquatic environment as a degradation product of nonylphenol ethoxylates (NPEO) used as nonionic surfactants in a variety of industrial and agricultural processes and as cleaning agents (Tolls et al., 1994). Field surveys have revealed that nonylphenols are distributed at low μg/L levels in the aquatic environment (Tsuda et al., 2001). More specifically, the US Geological Survey reported that NP was one of the most frequently detected compounds with a median concentration of 0.8 μg/L (Kolpin et al., 2002). Nonylphenols have been found to range from <0.2 to 12 μg/L in polluted British river waters (Blackburn and Waldock, 1995) and <0.3 to 45 μg/L in the Swiss river waters (Ahel et al., 1994). Several studies have demonstrated that NP affects teleost reproduction. Noteably, NP induces the female-specific, egg-yolk precursor vitellogenin (VTG) in males (Jobling et al., 1996; Ishibashi et al., 2006), causes testis-ova (Gray and Metcalfe, 1997), and decreases fecundity and fertility (Ishibashi et al., 2006).

17α-ethinylestradiol (EE2), a synthetic estrogen, has an extensive distribution in the aquatic environment. Median concentrations of EE2 ranged from 1 to 17 ng/L in sewage treatment effluents and ranged from below the detection limit to 15 ng/L in surface waters (Ternes et al., 1999; Komori et al., 2004; Liney et al., 2005). EE2 has been shown to affect elements of the steroid hormone signaling pathway in vitro (Thorpe et al., 2003; Van den Belt et al., 2004) and disrupt endocrine systems in vivo (Van den Belt et al., 2002; Nash et al., 2004; Palace et al., 2006). Among EE2 effects in fish are VTG induction in males (Seki et al., 2002), the reduction of yolk-filled oocytes in females (Van den Belt et al., 2001), and organ toxicity (Hahlbeck et al., 2004) in addition to decrease fecundity and fertility (Nash et al., 2004).

The present investigation was designed to compare the reproductive effects of two representative endocrine-disrupting chemicals (EDCs), NP and EE2, at environmentally relevant exposure concentrations. The study also attempts to investigate possible mechanistic links to organ toxicity at low exposures through histopathologic evaluation. A third objective is to examine the suitability of a short-term reproductive bioassay using the Chinese rare minnow in the context of low-level exposure to endocrine modulators.

Short-term reproduction bioassays have been developed for several species including fathead minnow (Pimephales promelas) and medaka (Oryzias latipes) (Harries et al., 2000; Ankley et al., 2001; Seki et al., 2002) and are considered pertinent to the evaluation of effects of EDCs. The Chinese rare minnow belongs to the family Cyprinidae and its natural distribution is limited to the upstream waters of the Yangtze River, Sichuan Province, China. Rare minnow are small (30–80 mm in total length) and easy to culture in the laboratory. They have a relatively short life cycle, spawning hundreds of eggs with high fertilization and hatching rates. Males and females are easily distinguished in adults according to secondary sexual characteristics (Wang, 1992). Rare minnows have been used previously for aquatic toxicity testing (Zhou et al., 1995) and our previous work has reported short-term toxic effects, such as histological alterations in rare minnow exposed to EE2 and NP (Zha et al., 2007).

Section snippets

Chemicals

Reagent 17α-ethinylestradiol (EE2) (98% purity) was obtained from Sigma (St. Louis, MO, USA). Technical 4-nonylphenol (NP) (Sigma-Aldrich, Deisenhofen, Germany) consisted of 98% NP isomers (90% 4-NP, 10% 2-NP) and 2% dinonylphenol. To prepare stock solutions, EE2 and NP were diluted in HPLC-grade acetone and then mixed with distilled-deionized water. Final exposure concentrations were achieved by means of a dilution apparatus constructed of polytetrafluorethylene (PFTE) and isoversinic tubes

Mortality, growth, and somatic indices

During the experiment period, no adult fish died and growth in the toxicant-exposed groups was not different to that of controls (data not shown). Adult GSIs were significantly different between controls and males exposed to 10 μg/L NP (Fig. 1). Significantly increased HSI in females occurred only at 4 ng/L EE2 (Fig. 1). The RSI in males was increased significantly at all exposures and in females was increased in the 10 and 20 μg/L NP and EE2 groups (Fig. 1).

Plasma vitellogenin (VTG) concentrations

After 21 days of exposure to NP or EE2,

Discussion

The 21 days low-level exposure of breeding rare minnows succeeded in demonstrating adverse effects of two representative EDCs. Large differences in toxic potency and qualitative differences in response to the EDCs were revealed. Not only standard reproductive parameters (e.g., fecundity, fertility, embryo development, survival, and growth of larvae), but also other types of toxic endpoints (e.g., target organ effects, VTG biomarker) were evaluated. It clarifies that short-term assay presented

Conclusion

The results of the present investigation indicate that a short-term reproductive bioassay is possible using the Chinese rare minnow as the test species. The bioassay succeeded in demonstrating that the synthetic estrogen EE2 was several orders of magnitude more potent than the industrial chemical NP. Among reproductive endpoints, fecundity, fertility, and the interval between spawnings were significantly affected by 4 ng/L EE2. These effects coincided with male feminization, ova-testis,

Acknowledgments

We gratefully acknowledge the support of National Basic Research Program of China (2007CB407304) and the National Natural Science Foundation of China (20677075, 50538090).

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    Any studies in this paper involving rare minnow (Gobiocypris rarus) were conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare.

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