Heiko L. Schoenfuss
Dalma Martinovic
Peter W. Sorensen

This study assessed the effects of exposure to low
disruption in fishes (Purdom et al., 1994; Harries et al., levels of estradiol (E2), as they have been found in 1997; Routledge et al., 1998). Numerous studies have some treated sewage effluents, on nest holding ability assessed the effects of E2 and associated xenoestrogens and sperm quality, two measures likely related to on wild fish (Folmar et al., 1996; Jobling et al., 1998; reproductive success in fathead minnows (Pimephales Lee et al., 2000; Folmar et al., 2001), fish that were promelas), a species with paternal nest care. Male caged (Purdom et al., 1994; Harries et al., 1996; fathead minnows were exposed for 21 days to either 50 Harries et al., 1997; Routledge et al., 1998; Rodgers- ng/L (E2) in 25 µl/L ethanol, an E2 concentration Gray et al., 2001), and on fish in the laboratory found in the effluent of some British and Canadian (Kramer et al., 1998; Panter et al., 1998; Miles- sewage treatment plants (STP), or to a 25 µl/L ethanol Richardson et al., 1999; Korte et al., 2000; Bjerselius et control (aqueous flow-through exposure). After al., 2001). All of these studies found induction of the exposure, groups of six males from one treatment were yolk protein vitellogenin (VTG) in exposed male fish, placed into an observation aquarium with females and as well as other effects such as gonadal abnormalities a limited number of spawning sites and allowed to (Gimeno et al., 1998; Jobling et al., 1998; Miles- spawn for four days. Plasma vitellogenin (VTG) Richardson et al., 1999; Rodgers-Gray et al., 2001), concentrations, the ability to acquire and defend a nest changes in behavior (Bayley et al., 1999; Bjerselius et site, and sperm quality were compared between E2 al., 2001), and sperm production (Gimeno et al., 1998; exposed and control males. E2 exposure resulted in a Bjerselius et al., 2001). However, little is known about significant increase in VTG concentrations in E2 the reproductive effects of exposure to E2 at these low exposed males, but did not affect nest holding ability or concentrations. In a previous study (Schoenfuss et al., sperm quality. These preliminary results indicate little in press), we found that 50 ng/L E2 exposure resulted effect of 21-day exposure to levels of E2 that match the in minor reductions in spawning behavior and sperm highest reported E2 loads in STP effluent, and parallel production in male goldfish (Carassius auratus), which our findings from a previous study using goldfish. have no parental care. Based on these results, we However, caution is warranted in extrapolating these designed a new experiment to determine whether results to populations of wild fish, as other factors such exposure to similar E2 concentrations would be more as the overall estrogenic potency of effluent, the detrimental to fish with greater paternal involvement in duration and timing of exposure to estrogenic reproduction. E2 exposure concentrations were chosen compounds, and the competition between fish for to match the highest E2 concentrations reported in spawning opportunities need to be estimated. sewage effluent in Great Britain (Desbrow et al., 1998; Rodgers-Gray et al., 2000) and Canada (Ternes et al., INTRODUCTION
1999). In this study we exposed male fathead minnows (Pimephales promelas), a species with paternal nest The steroid 17ß-estradiol (E2) is an important hormone care, to low levels of E2 and assessed the effects of in endocrine pathways of fishes (Arcand-Hoy & exposure on the fishes ability to hold nest sites and to Benson 1998) and occurs frequently in effluent discharge of municipal sewage treatment plants (STP) (Desbrow et al., 1998; Ternes et al., 1999; Rodgers- The fathead minnow is widely used in toxicological Gray et al., 2000) where it can cause endocrine studies (Ankley et al., 1998) and reproduces in the laboratory. Fathead minnows breed year round with females and allowed to spawn. Male behavior was the male guarding a cavernous nest site until eggs observed, after which plasma VTG concentrations, and hatch. The ability to defend a nest is determined by the individual’s social status in which dominant males, identifiable by their tubercles, dorsal pad, and bright banding pattern, establish successful breeding territories (Cole & Smith, 1987). Smith (1974) For each trial, 16 spermiating fathead minnows were suggested that these morphological characteristics are netted from a stock aquarium and distributed evenly associated with elevated androgen levels. The male among four 20 L aquaria. Aquaria were aerated, defends the nest site continuously until hatching as shielded on three sides, and fish were maintained eggs are quickly eaten by other fathead minnows in the otherwise as in the stock aquaria. Males were held for absence of the nest-guarding male (Sargent, 1989). 21 days in either (i) 50 ng/L E2 dissolved in 25 µl/L Estrogen exposure could increase circulating E2 levels ethanol, or in (ii) a 25 µl/L ethanol control. in male fish and suppress androgen levels, by altering Experiments with guppies (Poecilia reticulata) and neuroendocrine feedback loops (Trudeau et al., 1993; goldfish (Bayley et al., 1999; Schoenfuss et al., in Jobling et al., 1996; Ankley et al., 1998). press) found no effects of ethanol exposure on spawning behavior and sperm production. Both We expect nest holding ability to be a sensitive treatments were administered via continuous flow- indicator of reproductive success as the opportunity to through delivery (280 ml/min). A peristaltic pump spawn is conditional upon possession of a nest site continuously supplied two aquaria with an E2 stock (Unger, 1983; Huntingford & Torricelli, 1993). We solution, while the other two aquaria received ethanol also expect sperm production to serve as a sensitive at concentrations similar to the one used to prepare the and relevant measure of the effects of estrogen E2 stock solution. E2 was extracted weekly from E2 exposure as sperm production is important in externally exposure aquaria and bi-weekly from the control fertilizing fish (Petersen & Warner, 1998). A reduction aquaria. Water samples were concentrated using C18 in sperm quantity (which normally increases in SepPaks (Waters Corporation, MA), and than association with interactions with ovulating females measured with an E2 ELISA test kit (Cayman (Stacey & Sorensen, 1986; Dulka et al., 1987; Chemicals, MI). Aquarium concentrations of E2 were DeFraipont & Sorensen, 1993)) or quality can lead to approximately 50 percent of the 50 ng/L nominal reduced fertilization rates (Nakatsuru & Kramer, 1982; concentration at 24 ± 3.6 ng/L E2 (mean ± standard Zheng et al., 1997; Suquet et al., 1998; Cosson et al., error). E2 concentrations in the ethanol control aquaria 2000). By exposing fathead minnows to E2 we addressed two questions. (1) Is sperm quality in fathead minnows compromised by E2 exposure? (2) Are E2 exposed male fathead minnows able to After 21 days, groups of six male fish from the same treatment were moved from the exposure aquaria into 160 L observation aquaria (day one). Each observation MATERIALS & METHODS
aquarium contained six mature female fathead minnows and three nest sites (3” PVC pipe sections) to ensure that only nest sites are limiting reproductive success. In preliminary studies performed with Fish for this study were bred and reared at the unexposed males in a similar setting, males routinely University of Minnesota. Fish were held in 28°C water occupied all nest sites within 24 hours. Males were (flow-through, 300 ml/min) from an in-house well, at a marked using a combination of the same three latex constant photoperiod (16 h:8 h light:dark, 0800 lights colors that were injected subcutaneously. A pre - on), and were fed frozen brine shrimp (San Francisco experiment found no effects of latex injection on the Bay Company, CA) twice daily ad libitum. reproductive ability of male fathead minnows. Male fathead minnows were observed twice each morning (days two through five) for five-minute An experiment was conducted to assess the effects of intervals. During the observation period the nest E2 exposure on nest holding ability and sperm quality holding male in each of the three nest cavities was in male fathead minnows. In four trials, males were identified. Each nest defended by a male fathead exposed for 21 days to either E2 or an ethanol control. minnow was scored as a “nest holdin g event” for the After exposure, groups of males were placed with treatment. Data from all four replicates were pooled resulting in a total of 48 “nest holding events” per a Fisher’s Exact test. All calculations were performed aquarium and treatment (three nests x four observation with the Prism 2.0 statistical package for the Macintosh On the fifth day, fish were anesthetized with 0.1% phenoxyethanol (Sigma, St. Louis, MO), placed upside-down into a grooved sponge, and then milt Plasma VTG concentrations in E2 exposed male (sperm and seminal fluid) was drawn into a capillary fathead minnows were significantly higher than in tube by applying light abdominal pressure following control males (p<0.01, Mann-Whitney U test; Table 1). established protocols (Stacey & Sorensen, 1986). Care Despite VTG induction in exposed males, sperm was taken not to contaminate the sample with feces or quality did not differ between E2 exposed and control urine. For storage, 1 µl of milt was transferred into males and the MPrV was virtually identical (p>0.05, 100 µL of isoosmotic sperm extender following Mann Whitney U test, Table 1). The ability to hold published protocols (Chao et al., 1987). For the sperm nest sites also did not differ between E2 exposed and analysis, 10 µl of sperm extender solution was control fish. The number of nest holding events was suspended in 100 µl distilled water (1:2000 final similar for E2 exposed and control male fathead dilution) and vortexed. The drop in osmotic pressure minnows (p>0.05, Fisher’s Exact test; Table 1). E2 activated the sperm sample (Billard et al., 1995), which exposed male fathead minnows were holding nest sites was then analyzed using a Hamilton-Thorn mo tility during all 48 observations, while control males held analyzer (Danvers, MA). Mean Progressive Velocity nests during 44 out of 48 observations. As males were (MPrV), a measure of sperm velocity along its path color-coded it was also possible to identify changes in was used to characterize sperm quality (Kime et al., the identity of the nest holder from day to day. 1996). Each sperm sample was analyzed three times However, very few incidents in which the nest holding and values were averaged to determine the MPrV for male changed from one day to the next were observed each fish. MPrV was calculated only for those fish DISCUSSION
Our preliminary results indicate that three-week After completion of sperm analysis, blood was exposure to 50 ng/L E2, a concentration exceeding E2 collected from the caudal vasculature, stored on ice, loads in the effluent of at least two of the few STP and centrifuged at 10,000 rpm for one minute. Plasma effluents that have been analyzed for E2 loads, was transferred into aprotinine coated microcentrifuge consistently induced VTG production in mature male tubes and stored at –20°C for later analysis. Plasma fathead minnows. The reproductive consequences VTG concentrations were determined using a fathead associated with this physiological effect appear minor minnow-specific ELISA following published protocols under the experimental conditions presented in this (Parks et al., 1999). For the statistical analysis VTG study. These results match our earlier findings in concentrations below detection limit were reported as goldfish, for which ten week exposure to E2 at similar concentrations results in VTG induction but had relatively minor effects on spawning behavior and sperm production (Schoenfuss et al., in press). Ours is the first study to evaluate the effects of aqueous Sperm quality and plasma vitellogenin concentrations exposure to E2 at concentrations that simulate E2 loads were analyzed using a nonparametric Mann-Whitney U in some STP effluents on nest holding ability and test as some of the data were not normally distributed. The proportional nest holding data were analyzed using Plasma vitellogenin (VTG) concentrations, sperm quality, and nest holding ability in male fathead minnows exposed to E2 or a control. The E2 exposure protocol used in this study is notable (2001) saw an increase in sperm numbers in guppies because the absolute exposure concentration in this exposed to 30 ng/L E2. In this experiment we were study (24 ng/L) is below the highest E2 loads reported unable to measure sperm quantity as the small size of for STP effluent. Although the E2 concentration in this the fathead minnow renders it impossible to strip study was higher than effluent E2 concentration consistently all milt while avoiding contamination by reported for U.S. STP effluents, some studies have reported E2 concentrations in STP effluent as high as 88 ng/L in Great Britain (Desbrow et al., 1998, Although we did not find any impairment of nest Rodgers-Gray et al., 2000) and 64 ng/L in Canada holding ability and sperm quality in male fathead (Ternes et al., 1999). Thus, our exposure protocol minnows exposed to E2, we remain cautious about represents a high, but still realistic exposure scenario. extrapolating these laboratory results to populations of Furthermore, E2 is but one of many estrogenic wild fish. Indeed, ongoing studies in our laboratory compounds that have been found in STP effluent, and suggest that intra -species interactions might be an the overall estrogenic potency of the effluent could be important component in evaluating the effects of E2 much greater than simulated in this experiment. exposure. Furthermore, STP effluent consists of complex mixtures of chemicals, many of which have The plasma VTG concentrations found in E2 exposed been shown to be estrogenic to fish and their combined male fathead minnows in this study match those action may exceed our exposure concentrations. Our reported by Parks et al., (1999), in fathead minnows understanding of the effects of STP effluent on fish exposed to E2 via injection. Despite VTG induction, populations is still rudimentary and further studies will the fish appeared healthy and active and in each be necessary to determine the effects of estrogen and replicate spawning behavior could be observed within xenoestrogen exposure on all life stages of fish. 24 h in both treatments. Nest holding ability of E2 Answering these questions will allow us to determine exposed fathead minnows was also not affected by E2 the effects of effluent exposure on wild fish exposure, with E2 exposed males holding all nest sites in all replicates of the experiment. The ability to defend a nest site is crucial for the reproductive success of male fathead minnows as eggs in any unguarded ACKNOWLEDGEMENTS
nest are quickly eaten by other fathead minnows (Sargent, 1989). Our results are similar to those The authors acknowledge funding from the National reported in two studies on guppies that did not find Sea Grant College Program. We thank Gary Ankley reproductive impairment at 30 ng/L E2 exposure (Toft and the staff at the Duluth EPA laboratory for help & Baatrup, 2001), and saw no behavioral impairment with setting up a fathead minnow breeding colony and at 100 ng/L E2 exposure (Bayley et al., 1999). In the VTG assay. We are grateful to Mats Troedsson contrast, a previous study conducted in our laboratory (School of Veterinary Medicine, University of (Schoenfuss et al. in press) found a significant decline Minnesota) for making the computer assisted motility in reproductive behavior in male goldfish exposed to analyzer available to us, and Kate Loseth for providing 50 ng/L E2 for ten weeks. Bjerselius et al., (2001), also found behavioral impairment at E2 exposure concentrations exceeding 1 µg/L E2, but that E2 exposure level is far above any recorded for STP Heiko L. Schoenfuss, Dalma Martinovic, and Peter
MPrV, a measure of sperm quality did not vary W. Sorensen, Department of Fisheries, Wildlife, and
between E2 exposed and control fish. Sperm quality Conservation Biology, 200 Hodson Hall, 1980 Folwell has been suggested previously (Kime et al., 1996; Avenue, University of Minnesota, St. Paul, MN 55108 Kime 1998; Kime & Nash, 1999) as a tool to analyze the effects of EDCs on the reproductive fitness of fish. However, despite the strong physiological response (VTG induction) in male fish exposed to E2, sperm REFERENCES
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