An Environmental Estrogen Alters Reproductive ... - ACS Publications

This disruption in the reproductive hierarchy in group spawning fish, if it occurs in the wild, has potentially major implications for population gene...
0 downloads 0 Views 203KB Size
Download PDF
Environ. Sci. Technol. 2008, 42, 5020–5025

An Environmental Estrogen Alters Reproductive Hierarchies, Disrupting Sexual Selection in Group-Spawning Fish T O B I A S S . C O E , * ,† PATRICK B. HAMILTON,† DAVID HODGSON,‡ GREGORY C. PAULL,† JAMIE R. STEVENS,§ KATIE SUMNER,§ AND CHARLES R. TYLER† Ecotoxicology and Aquatic Biology Research Group, University of Exeter, Exeter, U.K., Centre for Ecology and Conservation, University of Exeter Cornwall Campus, Penryn, U.K., and Molecular Ecology and Evolution Group, University of Exeter, Exeter, U.K.

Received January 29, 2008. Revised manuscript received April 08, 2008. Accepted April 11, 2008.

There is global concern regarding the potential impacts of endocrine-disrupting chemicals (EDCs) on the health of wildlife and humans. Exposure to some estrogens, at concentrations found in the environment, impairs reproductive function and behavior. However, nearly all work on endocrine disruption has investigated the effects of exposure on individuals and there is an urgent need to understand impacts on populations. Many fish have mating systems with complex social structures and it is not known whether EDCs will exaggerate or buffer the reproductive skews caused by the dominance hierarchies that normally occur for these species. This study investigated the impact of exposure to the pharmaceutical estrogen ethinylestradiol (EE2) on reproductive hierarchies and sexual selectioningroup-spawningfish.Breedingzebrafishwereexposed to environmentally relevant concentrations of EE2, and effects were determined on reproductive output, plasma androgen concentrations (in males), and reproductive success through microsatellite analyses of the offspring. Reproductive hierarchies in breeding colonies of zebrafish were disrupted by exposure to EE2 at a concentration that did not affect the number of eggs produced. The effect was a reduction in the skew in male paternity and increased skew in female maternity. This disruption in the reproductive hierarchy in group spawning fish, if it occurs in the wild, has potentially major implications for population genetic diversity. Reproductive success in male zebrafish was associated with elevated plasma concentrations of the male sex hormone 11-ketotestosterone and this hormone was suppressed in EE2-exposed males.

Introduction There is concern regarding the release of man-made and naturally occurring estrogenic compounds into the environ* Corresponding author e-mail: [email protected]; tel: +44 (0)1392 264674; fax: +44 (0)1392 263700. † Ecotoxicology and Aquatic Biology Research Group, University of Exeter. ‡ Centre for Ecology and Conservation, University of Exeter Cornwall Campus. § Molecular Ecology and Evolution Group, University of Exeter. 5020

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 13, 2008

ment, and substantial evidence that these compounds are altering physiological function in wildlife (reviewed in refs 1 and 2). These estrogenic chemicals include natural steroid estrogens, pharmaceutical estrogens used in contraceptive and menopause treatments (e.g., ethinylestradiol (EE2) and equine estrogens), and a wide range of industrial chemicals, including plasticizers (e.g., phthalates), resins (e.g., bisphenols), and various pesticides and herbicides (3, 4). Disruptions in reproduction induced by exposure to some of these estrogenic chemicals include alterations in sexual differentiation, induction of intersex (5, 6), and a reduced reproductive output (7, 8). In mammals, exposure to some of these chemicals can induce trans generational effects through epigenetic mechanisms, via alteration of DNA methylation (9). In mammals exposure to estrogens has been shown to induce alterations in neurological development (10) and behavior (11, 12). In fish, exposure to the estrogenic alkylphenolic chemical octylphenol at concentrations between 25 and 50 µg/L has been shown to suppress courtship behavior and reduce reproductive success (13). Exposure to 17β-estradiol at concentrations between 1 and 10 µg/L has similarly been shown to suppress sexual activity and reproductive behavior in goldfish, Carassius auratus (14), and in mosquitofish, Gambusia holbrooki, at concentrations >20 ng/L (15). Most studies to date on fish (and on other animals) have focused on the effects of endocrine-disrupting chemicals (EDCs) on individuals, and to induce adverse reproductive effects, concentrations employed have generally exceeded those normally found in the ambient environment. However, for some potent environmental estrogens, such as EE2, longterm (multigenerational) exposure to environmentally relevant concentrations has been shown to cause reproductive failure (16–18). In an attempt to predict population-level effects of various EDCs in fish, modeling approaches have been adopted. Grist et al. (19) used a matrix population model (20, 21) and data from a long-term exposure of fathead minnow, Pimephales promelas, to EE2 (22) to predict a zero population growth rate for exposure to between 1.0 and 4.0 ng EE2/L. Similar modeling approaches applied to fish have predicted population decreases for exposures to nonylphenol at exposures between 17 and 27.5 µg/L (23) and to the synthetic androgen 17β-trenbolone at concentrations >0.027 µg/L (24). Despite these predicted population-level impacts of EDCs in fish, no study has addressed the effects of how EDC exposure might affect interactions between individuals in groups of fish and how this impacts breeding (16). Disruption of interactions between individuals, particularly during reproduction, could have significant population-level consequences through effects on parentage and sexual selection. In this study we investigated whether exposure to EE2, including at environmentally relevant concentrations, had an impact on the reproductive success and breeding hierarchy in colonies of zebrafish. Zebrafish were chosen for this study because of their well-characterized experimental conveniences, and because they have a group-spawning reproductive strategy, which is the most common breeding system in fish. There is also an extensive suite of DNA microsatellite markers available for parentage studies, and a wealth of data exists on the effects of EDCs in this model species (25). Furthermore, a basic understanding of the territorial and competitive breeding behavior of zebrafish, and how this affects their reproductive success, have been recently developed (26–28). EE2 was chosen as the test chemical as it is 10.1021/es800277q CCC: $40.75

 2008 American Chemical Society

Published on Web 05/22/2008

particularly potent as an environmental estrogen, inducing feminizing effects in fish at concentrations as low as 0.1-10 ng/L, via the water (16, 29, 30), including in zebrafish (17, 31–33). It is also believed to play a key role in the feminization of wild fish living in U.K. rivers (34, 35).

Materials and Methods Test Fish. Adult zebrafish were bred in the laboratory at the University of Exeter from an existing stock of wild Indian Karyotype (WIK) fish originally obtained from the Max Planck Institute, Tu ¨ bingen, Germany. Fish were maintained according to Santos et al. (36). Chemicals and Chemical Dosing. EE2 with a purity of 98% was obtained from Sigma, UK. A stock solution of 1 mg EE2/mL ethanol (100%) was diluted into 2.5 L dosing bottles at a concentration of 1 µg/L (for experimental exposure at 10 ng/L) and 0.2 µg/L (for experimental exposure at 2 ng/L). Tap water was filtered by reverse osmosis (RO; Osmonics E625 with cellulose membranes; GE Water and Process Technologies, Trevose, PA) and was then reconstituted with Analar grade mineral salts to concur with U.S. Environmental Protection Agency (EPA) guidelines on standardized synthetic freshwater. Conductivity was 304 ( 7 µS cm-1 and pH was 7.0 ( 0.05. Clean water was supplied to each tank at a rate of 2 L/hr, with flow rates checked daily. EE2 was delivered independently from the dosing bottles to the chemical and control tanks at a rate of 20 mL/hr. Nominal EE2 dosing concentrations were 2 and 10 ng/L. The final ethanol concentration was 1 µL/L. Experimental Procedure. Sixty-four adult zebrafish were sexed, according to Paull et al. (28), and randomly assigned to sixteen 15 L tanks (2 males and 2 females in each tank) and acclimatized for five days. This group size is found naturally in wild zebrafish (37). For the next eight days reproductive output (and parentage, see below) were determined for all 16 tanks, and this provided a reference on reproductive output and parentage in the individual colonies for the subsequent analysis of chemical treatment effects. Fish were then exposed for 17 days, with four replicate tanks for each of the exposure treatments (solvent control, 2 ng EE2/L and 10 ng EE2/L) and for the dilution water controls, and reproductive output was determined for all tanks and parentage assessed for the solvent controls and 10 ng EE2/L treatment tanks. Eggs were collected daily, 2-3 h after the artificial dawn, using an egg collection system attached externally to the lower section of the tanks that avoided disturbance of fish in the tanks, as described in Paull et al. (28), washed, and counted, and unfertilized/fungally infected eggs were discarded. Fertilized eggs were incubated in plastic containers for a minimum of 6 h to increase DNA quantity for microsatellite analysis and then fixed and stored in 100% ethanol for subsequent parentage analysis. On day 25, all fish were killed by a lethal dose of benzocaine and destruction of the brain, according to UK Home Office Animal Licence guidelines. The wet weight and fork length of each fish was recorded and a fin-clip was taken and stored in 100% ethanol for subsequent parentage analysis. Blood was collected from each fish using heparinized capillary tubes, centrifuged to separate the plasma from the blood cells, and then the plasma was stored at -80 °C. Water Chemistry. Water samples were collected from each tank on days 2, 10, 16, and 24 and stabilized with 5% methanol and 1% acetic acid. Samples were then extracted using SepPack C18 cartridges (Waters Corporation, USA). EE2 concentrations of the extracted samples were measured by gas chromatography-mass spectrometry by the Environment Agency’s National Laboratory Service, Nottingham (38). The detection limit was 0.1 ng EE2/L.

11-Ketotestosterone Quantification. 11-Ketotestosterone (11-KT) is one of the major androgens in male fish and is associated with dominant behavior (39). 11-KT was quantified from 1 µL of blood plasma using radioimmunoassay (RIA), according to the method described in Scott et al. (40). Parentage Analyses. 96 randomly selected, fertilized eggs from each solvent control replicate tank and each 10 ng/L EE2 replicate tank were sampled for parentage analysis over the whole study period and in direct proportion to the number laid on each day. Following a power analysis of parentage in the solvent control and 10 ng/L tanks to determine the minimum number required to significantly assign parentage, 16 randomly selected, fertilized eggs from the pre-exposure period were additionally sampled from all of the water control and 2 ng/L EE2 tanks for parentage analysis. The number sampled on each day was again in direct proportion to the total number of eggs spawned on that day. In total 896 embryos were genotyped. All adult fish were genotyped from fin clips collected at the termination of the experiment. DNA was extracted from the parental fins and from embryos using ammonium acetate precipitation (41). To develop a suitable suite of microsatellites for the study, we first screened 20 loci for use and from this, five, Z249, Z6104, Z9230, Z20450 (www.zfin.org), and Ztri1 (F: 5′-AACTCAAACAAACAGAGCTG-3′, R: 5′-ATAACACTTCCAGTTGACTG-3′ designed for this study) were chosen to assign parentage, as their PCR products could be pooled for simultaneous sequencer analysis. Full details of the PCR protocols used can be found in the online Supporting Information. Parentage was assigned with the program Probmax, version 1.3 (42). 94.2% of all the embryos tested could be assigned to a single parental pair or single male/female. Embryos unable to be assigned to a single parental pair were not included in the analysis. Data Analysis. Effects of exposure to EE2 on egg number were determined by comparing daily egg production for the pre- and during exposure periods, and differential egg count (number of eggs spawned after exposure minus number of eggs spawned before exposure). Once parentage had been assigned for all embryos, within each tank the number and proportion of eggs parented by each fish was calculated. This was done for all tanks (with the exception of one of the four 2 ng EE2/L replicate tanks, where microsatellite resolution was insufficient to assign complete parentage) for the first 8 days (pre-exposure) and for all solvent control and 10 ng/L exposed tanks over the whole 25 days of the experiment (i.e., including the EE2 exposure period). Analyses of parentage investigated how proportional success prior to exposure (used as a covariate) and treatment affected proportional success during exposure. All statistical analyses were conducted as generalized linear models with model simplification, using the software R 2.5.1. Standard model checks were used to verify normality and homogeneity of standardized residuals (43). Egg numbers were analyzed using a Gaussian error structure. Paternity and maternity analyses used binomial errors with the number of offspring as the denominator, and were corrected for overdispersion where necessary. Tests and error structures used are given with the corresponding results. Interactions between, and main effects of, explanatory variables were removed from generalized linear models (GLMs) sequentially using the criterion of nonsignificance with R ) 0.05. Where given, values are expressed as the mean ( one standard error.

Results Exposure Concentrations of Estrogens. Measured mean concentrations of EE2 were close to nominal at 2.38 ( 0.18 ng/L for the 2 ng/L exposure and 10.6 ( 0.73 ng/L for the 10 ng/L exposure. EE2 was undetectable (less than 0.1 ng EE2/L) in the water and solvent control tanks. VOL. 42, NO. 13, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5021

FIGURE 1. Total reproductive output of zebrafish breeding groups during exposure to EE2. Impacts of EE2 on Egg Output. The mean numbers of eggs produced per-female per-day pre-exposure in each treatment were as follows: control, 40.3 ( 13.6; solvent control, 51.0 ( 9.90; 2 ng/L EE2, 49.5 ( 16.6; 10 ng/L EE2, 41.2 ( 7.25. The mean numbers of eggs produced per-female per-day during the exposure period in each treatment were: control, 40.9 ( 9.69; solvent control, 57.0 ( 9.26; 2 ng EE2/L, 56.7 ( 12.0; 10 ng EE2/L, 43.4 ( 9.17. There was substantial inter- and intratank variability in egg output. Exposure to EE2 had no significant effect on the viable egg output in the breeding colonies of zebrafish. A linear model showed that across all tanks, there was a significant relationship between the number of eggs spawned pre-exposure and the number spawned during exposure (F ) 17.887, df ) 1,14, p < 0.001). Colonies of zebrafish producing higher numbers of eggs during the pre-exposure period similarly did so during the exposure period. However this relationship did not differ significantly between the different treatments (F ) 0.307, df ) 3,11, p ) 0.820), showing that exposure to EE2 had no effect on egg output (see Figure 1). There was no interaction between the number of eggs laid pre-exposure and the treatment (F ) 0.757, df ) 3,8, p ) 0.549). There did, however, appear to be a decline in egg production in the 10 ng EE2/L treatment group during the last 3-4 days of the exposure. Impacts of EE2 on Parentage (Reproductive Success). The effects of EE2 on reproductive success were assessed by comparing the reproductive success of individuals pre- and postexposure. For the pre-exposure period, across all tanks, the average proportion of eggs sired by the more successful male was 0.67 ( 0.04 and by the more successful female was 0.74 ( 0.05. Exposure to 10 ng/L EE2 had a significant impact on the male reproductive success in the breeding groups of zebrafish when compared to that of the solvent controls. A generalized linear model, using binomial errors, showed a significant effect of both the paternity prior to exposure (χ2 ) 6.961, df ) 1, p ) 0.008) and the treatment (χ2 ) 4.618, df ) 1, p ) 0.032) on the paternity during exposure. There was no interaction between the treatment and paternity prior to exposure (χ2 ) 0.706, df ) 1, p ) 0.401). This result shows that the reproductive success of the more successful males was significantly lower in the exposed males following exposure than in the control males, for any given level of pre-exposure parentage success (see Figure 2a). The proportion of offspring sired by the more successful male was on average 0.15 lower in fish exposed to 10 ng EE2/L compared with the controls. 5022

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 13, 2008

FIGURE 2. (a) Proportion of eggs sired by reproductively more successful male zebrafish before and during exposure to either a solvent control (open circles) or 10 ng EE2/L (closed circles). (b) Proportion of eggs sired by reproductively more successful female zebrafish before and during exposure to either a solvent control (open circles) or 10 ng EE2/L (closed circles). The numbers in brackets correspond to the number of embryos genotyped before and during exposure, respectively. In both cases, the lines given are the fitted success curves from the statistical models. The solid line corresponds to the control fish and the dashed line corresponds to the exposed fish. For females, individuals that were reproductively more successful in the breeding groups prior to the exposure were also more successful during the exposure period for both the solvent controls and 10ng/L EE2. However, there was a significant interaction between treatment and maternity prior to exposure, as determined by a generalized linear model using binomial errors (χ2 ) 14.269, df ) 1, p < 0.001). Thus, exposure to EE2 changed the proportional maternity between the pre-exposure and exposure periods, reinforcing the female success hierarchy relative to the solvent controls, as shown by the crossing of the fitted reproductive success curves in Figure 2b. 11-Ketotestosterone and Male Reproductive Success. Plasma 11-KT concentrations in males were 14.4 ( 2.33 ng/ mL in the dilution water controls and 18.8 ( 4.08 ng/mL in the solvent controls. Across the control and solvent control

FIGURE 3. Box-plot of plasma 11-KT concentrations in male zebrafish after exposure to 2 ng/L and 10 ng/L EE2 compared to the dilution water and solvent controls. The box is the median value, plus or minus a quartile of the y-value range. Whiskers are maximum and minimum y values. Open circles are significant outliers within the data. males, 11-KT concentrations were almost 2-fold higher in those males siring the greater proportion of offspring compared with those in subordinate males. Mean 11-KT concentrations were 24.0 ( 4.49 ng/mL in the reproductively more successful males and 12.7 ( 1.10 ng/mL in subordinate males. Plasma 11-KT concentrations in males exposed to 10 ng/L EE2 (Figure 3) were below the detection limit in 6 out of the 7 males tested, but were not significantly lower than the controls in fish exposed to 2 ng EE2/L.

Discussion Effect of EE2 Exposure on Egg Output. The measured concentrations of EE2 in the tank water established that the fish were exposed appropriately to the test EDC. Daily egg output in the controls and pre-exposure colonies was similar to those reported previously for this strain of zebrafish in similar breeding setups (36, 44) and the lack of any effect of EE2 on egg production in adult zebrafish is consistent with that found previously for an exposure to 5 ng EE2/L for a similar exposure period of 15 days (16). In contrast, a recent study by Santos et al. (45) showed 5 ng EE2/L resulted in significantly fewer viable eggs in breeding colonies of zebrafish. In that study, however, the exposure period was longer (21 days) and the reduction occurred during the last 4-5 days of the exposure. We similarly saw a trend of a decreased egg production in the last 3-4 days in the 10 ng EE2/L exposure and extending the time of this treatment would likely have resulted in a significant reduction in egg production. In another study that exposed zebrafish to 10 ng EE2/L for more extensive periods there was both a significant reduction in egg output and in the proportion of viable eggs (31). Effect of EE2 on Reproductive Success. For both the solvent control and 10 ng EE2/L exposed fish, reproductive success during the pre-exposure period was positively correlated with success during the exposure period, thus males that were more successful during the pre-exposure period were also more successful during the exposure period. Exposure to 10 ng EE2/L (a concentration found in some of the more polluted effluents (46)), however, had a significant effect on the reproductive success of the males that were “dominant” (in terms of their reproductive success) in breeding groups during the pre-exposure period, with a

suppression in their proportional parentage, relative to the control. This suppression in the males’ reproductive success therefore allowed the reproductively less successful male to sire a proportionally higher percentage of the offspring compared with the pre-exposure period, relative to the unexposed controls. For both the solvent control and 10 ng EE2/L exposed fish, female reproductive success during the pre-exposure period was positively correlated with success during exposure. Exposure to 10 ng EE2/L appeared to strengthen the female reproductive hierarchy, relative to the solvent control exposed fish. The mechanism for this strengthening of the female hierarchy remains unclear, but EE2 may directly influence female attractiveness, reproductive quality, or the outcome of competitive interactions between females. Alternatively, female reproductive success may be reinforced indirectly via the effects of EE2 on the male hierarchy: we speculate that dominant females may be the primary target of mating effort by previously reproductively subordinate males. Experiments are required to determine the male- and femalecomponents of these breeding dominance mechanisms. These findings potentially have highly significant implications as they suggest that exposure during the reproductive process to EDCs, such as EE2, can disrupt the process of sexual selection and this may have impacts on group and population biology. The suppression of reproductive success in previously more successful males may also act to increase and maintain genetic diversity in group spawning fish, by reducing the normal skew in reproductive success between competing male fish. Conversely, skews in female reproductive success may be reinforced by exposure to endocrine disruptors. This could lead to divergent, sex-specific changes in reproductive skew and fitness. As wild fish may be exposed for long periods of time, including life-long exposure in some instances, these effects are likely to be even more pronounced in populations of wild fish, particularly when considering the findings of this study were from an exposure of a relatively short duration. The change in proportional reproductive success in this study implies that EE2 had a greater negative impact on the more successful male fish compared to the subordinate fish. The differences in the degree of effect of the EE2 treatment may have resulted from changes in their behavior and/or differential impacts on their physiology. Previous studies have shown that exposure of fish to EDCs such as EE2 can alter reproductive behaviors. For example, exposure of the threespined stickleback (Gasterosteus aculeatus) to EE2 (albeit at relatively high concentrations of 15 ng EE2/L) resulted in a decrease in male-male aggressive behavior (47) and transient decrease in nesting behavior (48). However, these studies investigated changes in behavior only for males exposed individually, and did not consider the subsequent interactive effects of these exposures between individuals, as would likely occur in the environment; nor did they consider effects on reproductive success. Male behavior was observed during the spawning period, immediately after dawn throughout our study (on days 4, 5, 8, 10, 12, 14, and 25) to identify the behaviorally dominant males for all tanks. Dominant males were characterized by their activities in defending spawning territory, aggressive pursuits of the otherssubordinatesmale, and their external appearance (27, 49). Throughout the duration of the experiment, across all tanks, the behaviorally dominant male at the start of the experiment remained behaviorally dominant until the end of the study. We did not investigate subtle features of male-male, or male-female behavior interactions in this study but, for the 10 ng EE2/L exposure, the behaviorally dominant male became less aggressive with time of exposure (reduced number of chases of other male, fewer sparring between rival males). VOL. 42, NO. 13, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5023

These changes in male aggressiveness and reproductive success in behaviorally dominant males exposed to 10 ng EE2/L were associated with significant suppression in plasma 11-KT concentrations. 11-KT has been previously shown to be an important hormone in the determination of dominance in male fish (39, 50) and a similar result is also shown here for zebrafish; plasma concentration of 11-KT in reproductively more successful males was almost 2-fold higher than that in the reproductively subordinate males in controls. The dramatic suppression in plasma 11-KT on exposure to EE2 further highlights the high sensitivity of endocrine systems to exogenous estrogen and the vulnerability of evolutionary processes that are driven by hormones, such as sexual behavior and selection, to chemical exposures.

Acknowledgments We thank Jan Shears, Tessa Scown, Anke Lange, Amy Filby, and Alexander Scott for their assistance with the practical work and data collection. Funded by the UK Environment Agency, Department of the Environment, Food and Rural Affairs, European Social Fund and University of Exeter to C.R.T. and D.H. The authors declare they have no competing financial interests.

Supporting Information Available Experimental design graphic and full PCR protocols used. This information is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Tyler, C. R.; Jobling, S.; Sumpter, J. P. Endocrine disruption in wildlife: a critical review of the evidence. Crit. Rev. Toxicol. 1998, 28 (4), 319–361. (2) Vos, J. G.; Dybing, E.; Greim, H. A.; Ladefoged, O.; Lambre, C.; Tarazona, J. V.; Brandt, I.; Vethaak, A. D. Health Effects of Endocrine-Disrupting Chemicals on Wildlife, with Special Reference to the European Situation. Crit. Rev. Toxicol. 2000, 30 (1), 71–133. (3) Hayes, T. B.; Collins, A.; Lee, M.; Mendoza, M.; Noriega, N.; Stuart, A. A.; Vonk, A. Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. PNAS 2002, 99 (8), 5476–5480. (4) Oehlmann, J.; Schulte-Oehlmann, U.; Bachmann, J.; Oetken, M.; Lutz, I.; Kloas, W.; Ternes, T. A. Bisphenol A induces superfeminization in the ramshorn snail Marisa cornuarietis (Gastropoda: Prosobranchia) at environmentally relevant concentrations. Environ. Health Perspect. 2006, 114, 127–133. (5) Hirai, N.; Nanba, A.; Koshio, M.; Kondo, T.; Morita, M.; Tatarazako, N. Feminization of Japanese medaka (Oryzias latipes) exposed to 17 beta-estradiol: Effect of exposure period on spawning performance in sex-transformed females. Aquat. Toxicol. 2006, 79 (3), 288–295. (6) Pettersson, I.; Arukwe, A.; Lundstedt-Enkel, K.; Mortensen, A. S.; Berg, C. Persistent sex-reversal and oviducal agenesis in adult Xenopus (Silurana) tropicalis frogs following larval exposure to the environmental pollutant ethynylestradiol. Aquat. Toxicol. 2006, 79 (4), 356–365. (7) Robinson, C. D.; Brown, E.; Craft, J. A.; Davies, I. M.; Megginson, C.; Miller, C.; Moffat, C. F. Bioindicators and reproductive effects of prolonged 17β-oestradiol exposure in a marine fish, the sand goby (Pomatoschistus minutus). Aquat. Toxicol. 2007, 81, 397– 408. (8) Scholz, S.; Gutzeit, H. O. 17-alpha-ethinylestradiol affects reproduction, sexual differentiation and aromatase gene expression of the medaka (Oryzias latipes). Aquat. Toxicol. 2000, 50 (4), 363–373. (9) Anway, M. D.; Cupp, A. S.; Uzumcu, M.; Skinner, M. K. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 2005, 308 (5727), 1466–1469. (10) Corrieri, L.; Della Seta, D.; Canoine, V.; Fusani, L. Developmental exposure to xenoestrogen enhances spatial learning in male rats. Horm. Behav. 2007, 51 (5), 620–625. (11) Laessig, S. A.; Auger, A. P.; McCarthy, M. M.; Silbergeld, E. K. Effects of prenatal chlordecone on sexually differentiated behavior in adult rats. Neurotoxicol. Teratol. 2007, 29 (2), 255– 263. 5024

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 13, 2008

(12) Della Seta, D.; Minder, I.; Belloni, V.; Aloisi, A. M.; Dessi-Fulgheri, F.; Farabollini, F. Pubertal exposure to estrogenic chemicals affects behavior in juvenile and adult male rats. Horm. Behav. 2006, 50 (2), 301–307. (13) Gray, M. A.; Teather, K. L.; Metcalfe, C. D. Reproductive success and behavior of Japanese medaka (Oryzias latipes) exposed to 4-tert-octylphenol. Environ. Toxicol. Chem. 1999, 18 (11), 2587– 2594. (14) Bjerselius, R.; Lundstedt-Enkel, K.; Olsen, H.; Mayer, I.; Dimberg, K. Male goldfish reproductive behaviour and physiology are severely affected by exogenous exposure to 17[beta]-estradiol. Aquat. Toxicol. 2001, 53 (2), 139. (15) Doyle, C. J.; Lim, R. P. Sexual behavior and impregnation success of adult male mosquitofish following exposure to 17 betaestradiol. Ecotoxicol. Environ. Safe. 2005, 61 (3), 392–397. (16) Nash, J. P.; Kime, D. E.; Van der Ven, L. T. M.; Wester, P. W.; Brion, F.; Maack, G.; Stahlschmidt-Allner, P.; Tyler, C. R. Longterm exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish. Environ. Health Perspect. 2004, 112 (17), 1725–1733. (17) Fenske, M.; Maack, G.; Schafers, C.; Segner, H. An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish Danio rerio. Environ. Toxicol. Chem. 2005, 24 (5), 1088–1098. (18) Kidd, K. A.; Blanchfield, P. J.; Mills, K. H.; Palace, V. P.; Evans, R. E.; Lazorchak, J. M.; Flick, R. W. Collapse of a fish population after exposure to a synthetic estrogen. PNAS 2007, 104 (21), 8897–8901. (19) Grist, E. P. M.; Wells, N. C.; Whitehouse, P.; Brighty, G.; Crane, M. Estimating the effects of 17 alpha-ethinylestradiol on populations of the fathead minnow Pimephales promelas: Are conventional toxicological endpoints adequate? Environ. Sci. Technol. 2003, 37 (8), 1609–1616. (20) Caswell, H. Matrix Population Models, 2nd ed.; Sinauer Associates, 2001. (21) Hodgson, D. J.; Townley, S. Linking management changes to population dynamic responses: the transfer function of a projection matrix perturbation. J. Appl. Ecol. 2004, 41 (6), 1155– 1161. (22) Lange, R.; Hutchinson, T. H.; Croudace, C. P.; Siegmund, F. Effects of the synthetic estrogen 17 alpha-ethinylestradiol on the life-cycle of the fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 2001, 20 (6), 1216–1227. (23) Meng, Y. B.; Lin, B. L.; Tominaga, M.; Nakanishi, J. Simulation of the population-level effects of 4-nonylphenol on wild Japanese medaka (Oryzias latipes). Ecol. Model. 2006, 197 (3-4), 350– 360. (24) Miller, D. H.; Ankley, G. T. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17 beta-trenbolone as a case study. Ecotoxicol. Environ. Safe. 2004, 59 (1), 1–9. (25) Ankley, G. T.; Johnson, R. D. Small fish models for identifying and assessing the effects of endocrine-disrupting chemicals. Ilar J. 2004, 45 (4), 469–483. (26) Pyron, M. Female preferences and male-male interactions in zebrafish (Danio rerio). Can. J. Zool. 2003, 81 (1), 122–125. (27) Spence, R.; Smith, C. Mating preference of female zebrafish, Danio rerio, in relation to male dominance. Behav. Ecol. 2006, 17 (5), 779–783. (28) Paull, G. C.; Van Look, K. J. W.; Santos, E. M.; Filby, A. L.; Gray, D. M.; Nash, J. P.; Tyler, C. R. Variability in measures of reproductive success in laboratory-kept colonies of zebrafish and implications for studies addressing population-level effects of environmental chemicals. Aquat. Toxicol. 2008, In Press, Corrected Proof. (29) Larsson, D. G. J.; Adolfsson-Erici, M.; Parkkonen, J.; Pettersson, M.; Berg, A. H.; Olsson, P. E.; Forlin, L. Ethinyloestradiol - an undesired fish contraceptive. Aquat. Toxicol. 1999, 45 (2-3), 91–97. (30) van Aerle, R.; Pounds, N.; Hutchinson, T. H.; Maddix, S.; Tyler, C. R. Window of sensitivity for the estrogenic effects of ethinylestradiol in early life-stages of fathead minnow Pimephales promelas. Ecotoxicology 2002, 11 (6), 423–434. (31) Hill, R. L.; Janz, D. M. Developmental estrogenic exposure in zebrafish (Danio rerio): I. Effects on sex ratio and breeding success. Aquat. Toxicol. 2003, 63 (4), 417–429. (32) Orn, S.; Holbech, H.; Madsen, T. H.; Norrgren, L.; Petersen, G. I. Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone. Aquat. Toxicol. 2003, 65 (4), 397–411. (33) Van den Belt, K.; Verheyen, R.; Witters, H. Effects of 17 alphaethynylestradiol in a partial life-cycle test with zebrafish (Danio

(34)

(35)

(36)

(37)

(38)

(39) (40)

(41)

rerio): effects on growth, gonads and female reproductive success. Sci. Total Environ. 2003, 309 (1-3), 127–137. Katsu, Y.; Lange, A.; Urushitani, H.; Ichikawa, R.; Paull, G. C.; Cahill, L. L.; Jobling, S.; Tyler, C. R.; Iguchi, T. Functional associations between two estrogen receptors, environmental estrogens, and sexual disruption in the Roach (Rutilus rutilus). Environ. Sci. Technol. 2007, 41 (9), 3368–3374. Jobling, S.; Williams, R.; Johnson, A.; Taylor, A.; Gross-Sorokin, M.; Nolan, M.; Tyler, C. R.; Van Aerle, R.; Santos, E.; Brighty, G. Predicted exposures to steroid estrogens in U.K. rivers correlate with widespread sexual disruption in wild fish populations. Environ. Health Perspect. 2006, 114 (S1), 32–39. Santos, E. M.; Workman, V. L.; Paull, G. C.; Filby, A. L.; Van Look, K. J. W.; Kille, P.; Tyler, C. R. Molecular basis of sex and reproductive status in breeding zebrafish. Physiol. Genomics 2007, 30 (2), 111–122. Pritchard, V. L.; Lawrence, J.; Butlin, R. K.; Krause, J. Shoal choice in zebrafish, Danio rerio: the influence of shoal size and activity. Anim. Behav. 2001, 62, 1085–1088. Kelly, C. Analysis of steroids in environmental water samples using solid-phase extraction and ion-trap gas chromatographymass spectrometry and gas chromatography-tandem mass spectrometry. J. Chromatogr. A 2000, 872, 309–314. Borg, B.; Mayer, I. Androgens and behaviour in the three-spined stickleback. Behaviour 1995, 132, 1025–1035. Scott, A. P.; Mackenzie, D. N.; Stacey, N. E. Endocrine changes during natural spawning of the white sucker Catostomus commersoni. II. Steroid hormones. Gen. Comp. Endocr. 1984, 56, 349–359. Bruford, M. W.; Hanotte, O.; Brookfield, J. F. Y.; Burke, T. Multilocus and single-locus DNA fingerprinting. In Molecular Genetic Analysis of Populations: A practical approach, 2nd ed.; Hoelzel, A. R., Ed.; IRL Press: Oxford, 1998.

(42) Danzmann, R. G. PROBMAX: A computer program for assigning unknown parentage in pedigree analysis from known genotypic pools of parents and progeny. J. Hered. 1997, 88 (4), 333–333. (43) Crawley, M. J. Statistics - An introduction using R; John Wiley and Sons, Ltd: New York, 2007. (44) Schafers, C.; Teigeler, M.; Wenzel, A.; Maack, G.; Fenske, M.; Segner, H. Concentration- and Time-dependent Effects of the Synthetic Estrogen, 17R-ethinylestradiol, on Reproductive Capabilities of the Zebrafish, Danio rerio. J. Toxicol. Environ. Health A 2007, 70, 768–779. (45) Santos, E. M.; Paull, G. C.; Van Look, K. J. W.; Workman, V. L.; Holt, W. V.; van Aerle, R.; Kille, P.; Tyler, C. R. Gonadal transcriptome responses and physiological consequences of exposure to oestrogen in breeding zebrafish (Danio rerio). Aquat. Toxicol. 2007, 83 (2), 134. (46) Ternes, T. A.; Stumpf, M.; Mueller, J.; Haberer, K.; Wilken, R. D.; Servos, M. Behavior and occurrence of estrogens in municipal sewage treatment plants - I. Investigations in Germany, Canada and Brazil (vol 225, pg 81, 1999). Sci. Total Environ. 1999, 228 (1), 87–87. (47) Bell, A. M. Effects of an endocrine disruptor on courtship and aggressive behaviour of male three-spined stickleback, Gasterosteus aculeatus. Anim. Behav. 2001, 62, 775–780. (48) Brian, J. V.; Augley, J. J.; Braithwaite, V. A. Endocrine disrupting effects on the nesting behaviour of male three-spined stickleback Gasterosteus aculeatus L. J. Fish Biol. 2006, 68 (6), 1883–1890. (49) Spence, R.; Jordan, W. C.; Smith, C. Genetic analysis of male reproductive success in relation to density in the zebrafish, Danio rerio. Front. Zoo. 2006, 3, 5. (50) Cardwell, J. R.; Sorensen, P. W.; VanDerKraak, G. J.; Liley, N. R. Effect of dominance status on sex hormone levels in laboratory and wild-spawning male trout. Gen. Comp. Endocr. 1996, 101 (3), 333–341.

ES800277Q

VOL. 42, NO. 13, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5025