Life-cycle exposure of fathead minnows (Pimephales promelas) to an ethinylestradiol concentration below 1 ng/L reduces egg fertilization success and demasculinizes males.

Forty-eight hours after fertilization, fathead minnow (Pimephales promelas) eggs were exposed to the synthetic estrogen 17alpha-ethinylestradiol (EE2) at nominal concentrations of 0.32 and 0.96 ng/L and measured concentrations of 3.5, 9.6, and 23 ng/L. The fish were observed through the larval, juvenile, and adult stages. Growth, secondary sex characteristics, the liver somatic index, the gonadosomatic index, and fecundity were examined after several lengths of exposure. No significant changes were seen in fry or juvenile growth from 8 to 30 days posthatch (dph). An increase in the ovipositor index (a female secondary sex characteristic) was the most sensitive early response at 60 dph and was seen in fish exposed to EE2 concentrations > or = 3.5 ng/L. Continuation of the EE2 exposure until 150 dph, through maturation and reproduction, allowed measurement of two sensitive end points: decreased egg fertilization and sex ratio (skewed toward females), both of which were significantly affected at the lowest EE2 concentration tested, 0.32 ng/L. The next most sensitive end point was demasculinization (decreased male secondary sex characteristic index) of males exposed to an EE2 concentration of 0.96 ng/L. The effects of low concentrations of EE2 (0.32 and 0.96 ng/L) were manifested in male fish (decreased male sex characteristics and reduced egg fertilization success), whereas female fish showed no changes in the gonadosomatic index. Exposure to higher EE2 concentrations negatively affected females, as shown by a reduced gonadosomatic index at 150 dph in fish exposed to > or =3.5 ng/L EE2. Although there were some end points that showed changes at 60 dph, the reproductive end points and external sex characteristics measured in mature fish at 150 dph were more sensitive, with response thresholds of EE2 ranging from 0.32 to 0.96 ng/L. The concentrations of EE2 that negatively affected fathead minnows were similar to or lower than those detected in many municipal wastewater effluents. In conclusion, life-cycle exposure of fathead minnows proved to be a very sensitive bioassay, and responses were seen at concentrations of less than 1 ng/L, which are environmentally relevant concentrations of EE2.

Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent.

It is now well established that there is a diverse array of chemicals discharged into the environment that can mimic or antagonise the action of hormones. These endocrine-disrupting chemicals (EDCs) can thus interact with physiological systems and cause alterations in development, growth and reproduction in wildlife that are exposed to them. As yet, however, there is little information on the relative sensitivities of different wildlife groups to these chemicals and/or mixtures of them (e.g. estrogenic effluents) and hence, there are fundamental shortfalls in our knowledge of the ecological importance of endocrine disruption in wildlife. In this study, the effects of exposure to individual estrogenic chemicals (17alpha-ethinylestradiol; EE2, bisphenol-A, and 4-tert octylphenol) and a mixture containing these chemicals (treated sewage effluent) on embryo production in the prosobranch mollusc, Potamopyrgus antipodarum, were studied and compared with the effects of EE2 and the same estrogenic effluent on vitellogenin induction and/or egg production in various species of freshwater fish (fathead minnow; Pimaphales promelas, rainbow trout (Oncorhynchus mykiss); Cyprinus carpio, carp; Cyprinus carpio). The lab-based studies demonstrated that all of the tested chemicals (known to be estrogenic and to cause reproductive effects in fish) also affected embryo production in P. antipodarum. Furthermore, exposure to EE2 induced similar reproductive responses in the snails as in the fathead minnow (Pimephales promelas), stimulating egg/embryo production at low doses (up to 1 ng/l in the minnow and 25 ng/l in the snail) and causing inhibitory effects at higher doses. A similar pattern of embryo production occurred in P. antipodarum when it was exposed to a graded concentration of treated sewage effluent containing mixtures of estrogenic EDCs and hence, the total number of new embryos produced by the snails increased steadily over the 9 weeks exposure period in treated snails. Plasma vitellogenin concentrations in two species of male fish (the rainbow trout and the carp) also increased over the same time period. These data indicate that both the nature of the response and the relative sensitivities to environmental estrogens in P. antipodarum and three different fish species fish are comparable. P. antipodarum is thus, potentially a sensitive test organism for assessing estrogenicity of chemicals with a relevance to their activity in vertebrates.

Short-term exposure to 17 alpha-ethynylestradiol decreases the fertility of sexually maturing male rainbow trout (Oncorhynchus mykiss).

The synthetic estrogen 17 alpha-ethynylestradiol (EE2) is a commonly used oral contraceptive that has been increasingly detected in sewage effluents. This study determined whether EE2 exposure adversely affected reproduction in sexually maturing male rainbow trout (Oncorhynchus mykiss). We exposed male trout to graded water concentrations of EE2 (10, 100, and 1,000 ng/ L) for 62 d leading up to the time of spawning. Semen and blood plasma samples were removed from each fish. Semen was used to fertilize groups of eggs from one nonexposed female. As a measure of fertility, eggs were incubated for 28 d after fertilization to determine the proportion that attained the eyed stage of embryonic development. Additional endpoints also measured included sperm motility, spermatocrit, gonadosomatic and hepatosomatic indices, testis histology, and circulating plasma levels of the sex steroids 17 alpha, 20 beta-dihydroxyprogesterone (17,20-DHP) and 11-ketotestosterone (11-KT). Exposure to 1,000 ng/L of EE2 caused complete mortality of the treatment group by day 57. Exposure to lower EE2 water concentrations (10 and 100 ng/L) caused an increase in sperm density, while a significant reduction in testis mass was observed only in the 100-ng/L exposure group. Most significantly, semen harvested from fish exposed to 10 and 100 ng/L EE2 caused an approximately 50% reduction in the number of eggs attaining the eyed stage of embryonic development. Plasma levels of 17,20-DHP in exposed fish were roughly twice the level of the controls, while levels of 11-KT were significantly reduced in fish exposed to 100 ng/L EE2. These results suggest that sexually maturing male rainbow trout are susceptible to detrimental reproductive effects of short-term exposures to environmentally relevant levels of EE2.