Contaminant-induced feminization and ... - Semantic Scholar

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Environmental Research 100 (2006) 3–17 www.elsevier.com/locate/envres

Contaminant-induced feminization and demasculinization of nonmammalian vertebrate males in aquatic environments Matthew R. Milnes, Dieldrich S. Bermudez, Teresa A. Bryan, Thea M. Edwards, Mark P. Gunderson, Iskande L.V. Larkin, Brandon C. Moore, Louis J. Guillette Jr. Department of Zoology, 223 Bartram Hall, P.O. Box 118525, University of Florida, Gainesville, FL 32611, USA Received 4 November 2004; received in revised form 2 April 2005; accepted 8 April 2005 Available online 23 May 2005

Abstract Many chemicals introduced into the environment by humans adversely affect embryonic development and the functioning of the male reproductive system. It has been hypothesized that these developmental alterations are due to the endocrine-disruptive effects of various environmental contaminants. The endocrine system exhibits an organizational effect on the developing embryo. Thus, a disruption of the normal hormonal signals can permanently modify the organization and future function of the male reproductive system. A wide range of studies examining wildlife either in laboratories or in natural settings have documented alterations in the development of males. These studies have begun to provide the causal relationships between embryonic contaminant exposure and reproductive abnormalities that have been lacking in pure field studies of wild populations. An understanding of the developmental consequences of endocrine disruption in wildlife can lead to new indicators of exposure and a better understanding of the most sensitive life stages as well as the consequences of exposure during these periods. r 2005 Elsevier Inc. All rights reserved. Keywords: Endocrine-disrupting contaminants; Feminization; Development; Reproduction; Endocrinology; Sexual dimorphism; Steroids; Fertility

1. Introduction Many chemicals introduced into the environment by humans adversely affect embryonic development and the functioning of the vertebrate reproductive system. It has been hypothesized that ubiquitous environmental contaminants can induce developmental alterations through disruption of the endocrine system. The endocrine system exhibits an organizational effect on the developing embryo, altering gene expression and dosing. Thus, a disruption of the normal hormonal signals can permanently modify the organization and future functioning of the reproductive system. The development of the male reproductive systems is a common target of endocrine-disrupting contaminants (EDCs) (see Fig. 1). Studies in wild, free-ranging male mammals, reptiles, amphibians, and fish have documenCorresponding author. Fax: +1 352 392 3704.

E-mail address: [email protected]fl.edu (L.J. Guillette Jr.). 0013-9351/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2005.04.002

ted depressed plasma androgen profiles, altered spermatogenesis, altered penis/gonopodium development, and altered male behavior. Laboratory-based exposure studies have provided powerful documentation that similar alterations can be induced in developing male embryos exposed to ecologically relevant concentrations of various estrogenic and antiandrogenic EDCs. Further, although the mechanism of sex determination can vary among species, endocrine control of the testis and the role of androgens in male secondary sex development and functioning is highly conserved among vertebrates, indicating that wildlife are effective and important sentinels of human public health.

2. Reproductive tract—form and function The effects of EDCs on testicular and reproductive tract development and function have been investigated

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M.R. Milnes et al. / Environmental Research 100 (2006) 3–17

Fig. 1. Endocrine-disrupting contaminants can alter the biology of males at many levels of the endocrine system, besides the interaction with cellular-level receptors. Alterations of neuroendocrine function (e.g., gonadotropin-releasing hormone (GnRH) or gonadotropins (Gn)), serum-binding protein concentrations (SBP), and modification of liver biotransformation of hormones would alter the endocrine biology of males, leading to feminization or demasculinization.

in many fish species, but rarely in amphibians or reptiles. Most studies have focused on the effects of estrogens such as estradiol-17b (E2) or ethynylestradiol on sexual development. There are several studies on the effects of individual estrogenic and antiandrogenic contaminants, but noticeably few that look at the effects of chemical mixtures on the male reproductive tract. Environmental contaminants are known to affect sexual differentiation of the gonad, timing of sexual maturation, gonadosomatic index (GSI), and reproductive tract and testis morphology. Here, we present examples of each of these in fish and then present examples from the amphibian and reptilian literature. 2.1. Primary sex determination When sex ratios are skewed in fish populations exposed to environmental estrogens or antiandrogens, the ratio is typically biased in favor of females. Sex ratios can be affected in the developing embryos of exposed parents or in juvenile fish exposed during the period of sex determination and differentiation. Exposure to E2 during sexual differentiation increased the frequency of ovarian differentiation in Cyclopterus lumpus (Martinrobichaud et al., 1994), Odontesthes bonariensis (Strussmann et al., 1996), and Silurus asotus (Kim et al., 2001). Similarly, populations of Japanese medaka (Oryzias latipes) exposed during development to

the estrogenic contaminants octylphenol (Knorr and Braunbeck, 2002) and bisphenol A (Yokota et al., 2000) exhibited female-biased sex ratios. More females than expected were also observed in juvenile guppies (Poecilia reticulata) exposed during development to the antiandrogens vinclozolin and p; p0 -DDE (Bayley et al., 2002). Heavy metals such as mercury have also been shown to cause feminization of the gonads in fish (Matta et al., 2001). Intersex is a condition that results from disrupted gonadal differentiation and typically describes the simultaneous presence of ovarian and testicular tissue in the same gonad. In feminized males, oocytes may be scattered throughout the testis or occur within distinct regions of ovarian tissue that are well delineated from testicular tissue (Nolan et al., 2001). Transgenic zebrafish assays in which transactivation of the estrogen receptor was linked to expression of the luciferase reporter gene have shown that estrogen receptor activity is very high during sexual development and could be one reason that the period of gonadal differentiation may be especially susceptible to disruption by estrogenic compounds (Legler et al., 2000). Experimental research has shown that estrogens administered during the period of gonadal differentiation can induce intersex in a variety of fish species such as Japanese medaka (Knorr and Braunbeck, 2002; Metcalfe et al., 2001), carp (Cyprinus carpio) (Gimeno et al., 1998a, b), and sheepshead minnow (Cyprinodon variegatus) (Zillioux et al., 2001). Several xenoestrogens have also been shown to induce development of intersex gonads. Examples include 4tert-pentylphenol in carp (Gimeno et al., 1998a, b) and medaka (Gronen et al., 1999), and bisphenol A in medaka (Yokota et al., 2000). Examples of wild fish populations exhibiting intersex gonads include flounder taken from Tokyo Bay (Hashimoto et al., 2000); rainbow trout (Tyler and Routledge, 1998) and gudgeon (Gobio gobio) (van Aerle et al., 2001) collected from rivers in the UK polluted with estrogenic sewage treatment effluent; wild barbel from polluted portions of the Po River in Italy (Vigano et al., 2001), and lake whitefish (Coregonus clupeaformis) collected from the St. Lawrence River in Quebec, Canada (Mikaelian et al., 2002). The lake whitefish had elevated tissue levels of polychlorinated biphenyls (PCBs), chlorobenzenes, pesticides, and trace metals. Similar effects were observed in shovelnose sturgeon from the Mississippi River below Saint Louis, where fish tissues and roe were found to be contaminated with chlordane, PCBs, and p; p0 -DDE (Harshbarger et al., 2000). 2.2. Sexual maturation Whether exposure to EDCs causes gross gonadal changes such as intersex or more subtle changes such as

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delayed maturation depends on the exact timing of exposure. For example, sea bass (Dicentrarchus labrax) exposed to estrogens after sex determination exhibited delayed sexual maturation characterized by a dosedependent reduction of the surface of the testicular lobules in males (Blazquez et al., 1998). Alterations in the timing of sexual maturation can significantly affect reproductive fitness, especially if maturation is delayed. EDCs have been shown to both delay and speed up the timing of sexual maturation, although delays are more often reported. For example, delayed sexual maturation has been observed in perch exposed to bleached kraft mill effluent in the Baltic Sea (Sandstrom, 1994), maturing trout exposed to estrogenic alkyl phenolic chemicals (Sumpter, 1995), juvenile guppies experimentally exposed during development to the antiandrogens vinclozolin and p; p0 -DDE (Bayley et al., 2002), and wild roach collected from UK rivers contaminated with large volumes of treated sewage effluent (Jobling et al., 2002a). Alternatively, Colombo and Grandi (1995) reported that European eels underwent early maturation after exposure to 17-b-methyltestosterone. This precocious puberty was characterized by early differentiation of Leydig and Sertoli cells and early formation of the spermatic duct. 2.3. Gonadosomatic index Typically, the GSI is reduced after exposure to estrogenic or antiandrogenic contaminants. Reduction in GSI may be due to inhibition of testicular development at puberty, suppression of normal seasonal growth of the testes, or atrophy, although authors do not always note the distinction among these and may use the terms interchangeably. Suppression of testicular development has been shown in fathead minnows collected from a lake that was experimentally treated with ethynylestradiol (Palace et al., 2002), adult fathead minnows exposed to bisphenol A (Sohoni et al., 2001), and sea bass exposed to estrogens during the period of gonadal differentiation (Blazquez et al., 1998). In addition, low GSI has been induced experimentally in adult male guppies (Poecilia reticulata) exposed to E2 or the xenoestrogen 4-tertoctylphenol (Toft and Baatrup, 2001), and the antiandrogens vinclozolin or p; p0 -DDE (Baatrup and Junge, 2001a). Reductions in GSI have also been observed in wild perch exposed to bleach kraft mill effluent in the Baltic Sea (Sandstrom, 1994), and wild roach and caged and wild rainbow trout living in English rivers contaminated with treated sewage effluent containing estrogens (Jobling et al., 2002a; Tyler and Routledge, 1998). 2.4. Morphology A wide range of histological alterations have been noted in the gonads of fish exposed to EDCs. For

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example, mature fathead minnows exposed to environmentally relevant doses of E2 or 4-nonylphenol (a weak estrogen agonist) exhibited testicular lesions that included loss of germinal cells, presence of necrotic aggregates of germ cells at various stages of spermatogenesis, presence of germ cell syncytia, and proliferation and hypertrophy of the sertoli cells to the extent that some seminiferous tubules were partly or totally occluded (Miles-Richardson et al., 1999a, b). Electron microscopy of the seminiferous tubules and Sertoli cells revealed phagocytic cells in the lumina of the seminiferous tubules, as well as enlarged, distended Sertoli cells containing phagolysosomes. These phagocytic elements contained the remnants of spermatids, spermatozoa, lipids, and cellular debris. In addition, the cytoplasm of some sertoli cells was distended with myelin figures and necrotic spermatozoa. The authors noted that not all lesions were permanent once fish were removed from estrogenic exposure. In another example, flounder captured from a river that receives a high volume of estrogenic treated sewage effluent exhibited testes with poor lobular structure or rounded/truncated testicular lobes (vs. normal lobes, which are elongated), thickening of the connective tissue surrounding the testis, discoloration of the gonads, vacuolation of the lobule wall, presence of amorphous eosinophilic precipitates, remains of degenerated spermatozoa in the lobular lumen, and fibrosis that resulted in an absence or reduction of germ cells (Lye et al., 1997, 1998). Testicular fibrosis has also been observed in sheepshead minnow (Cyprinodon variegatus) exposed to 17-b-ethynylestradiol during sexual maturation (Zillioux et al., 2001), and male fathead minnows captured from a lake experimentally treated with ethynylestradiol (Palace et al., 2002). Fibrosis, vacuolation, atrophy of the germinal epithelium, and reduced seminiferous lobule diameter were also observed in mature male common carp (Cyprinus carpio) exposed to the xenoestrogen 4tert-pentylphenol or E2 during spermatogenesis (Gimeno et al., 1998b). Hypertrophy of Sertoli cells has been reported in male platyfish (Xiphophorus maculatus) treated with nonylphenol and E2 (Kinnberg et al., 2000), and hypertrophied compact connective tissue was observed in European eel stimulated with 17-b-methyltestosterone (Colombo and Grandi, 1995). Toft and Baatrup (2001) noted that Sertoli cells were cuboidal in control fish, but squamous and containing numerous phagocytized spermatozoa in guppies (Poecilia reticulata) exposed to 4-tert-octylphenol and E2. There was an increase in apoptotic/necrotic cells in the testicular interstitial tissues and seminiferous tubules of swordtails (Xiphophorus helleri) treated with nonylphenol, bisphenol A, and their mixture (Kwak et al., 2001). Finally, summer flounder (Paralichthys dentatus) exposed to E2 accumulated excessive hyalin material (part of which was vitellogenin) in the testes, an outcome that was

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associated with the presence of dead germ cells and proliferation of spermatogonia (Folmar et al., 2001). A variety of reproductive duct malformations have been described for intersex roach. Most intersex roach have a female-like reproductive duct (based on the presence of an ovarian cavity) as well as zero, one, or two sperm ducts within a single gonad (Jobling et al., 2002b; Nolan et al., 2001). According to Nolan et al. (2001), the ovarian cavity is distinguished by its characteristic ciliated epithelial cell lining. However, in intersex fish, this lining sometimes appears on both dorsal and ventral edges of the ovarian cavity, whereas in normal females the lining occurs only on the ventral edge. Sperm duct(s) may be blind-ended (terminating before the opening of the genital pore), blocked, or reduced, or they may form part of the ovarian cavity wall (Jobling et al., 2002b; Nolan et al., 2001). Femalelike reproductive duct and ovarian cavity formation were induced experimentally in juvenile male roach treated with graded concentrations of sewage effluent, known to contain E2, estrone, and the alkyl phenolic chemicals—4-octylphenol, 4-nonylphenol, and nonylphenol mono- and di-ethoxylates (Rodgers-Gray et al., 2001). Similarly, when juvenile male carp were exposed to 4-tert-pentylphenol or E2 during sexual differentiation, oviduct development in place of the vas deferens was observed, in addition to the occurrence of intersex (Gimeno et al., 1998a). Finally, when Toft and Baatrup (2001) observed an increase in the number of ejaculated sperm cells collected from guppies (Poecilia reticulata) exposed to 4-tert-octylphenol and E2, they hypothesized that the unexpected increase could be due to duct blockage and sperm entrapment. Sperm were only released under the pressure of palpation during stripping. Compared to fish, the effects of EDCs on amphibian or reptile development are not as well understood. However, the available studies indicate that amphibians and reptiles are subject to similar disruptive effects of environmental contaminants. For example, skewed sex ratios, in favor of the female phenotype, have been observed in African clawed frogs (Xenopus laevis) exposed during metamorphosis to environmentally relevant doses of ammonium perchlorate (Goleman et al., 2002), E2, 4-nonylphenol, bisphenol A, butylhydroxyanisol, and octylphenol (Kloas et al., 1999). In addition, hermaphroditism occurred in Xenopus exposed during metamorphosis to environmentally relevant doses (0.1–200 ppb) of the herbicide atrazine (Hayes et al., 2002). Partial or complete ovarian development was also induced in a dose-dependent manner in male tadpoles of Rana rugosa exposed during sex differentiation to dibutyl phthalate or E2 (Ohtani et al., 2000). Many species of turtles and all crocodilians experience environmental sex determination where the incubation temperature is the primary sex-determining factor (Lang

and Andrews, 1994). Current research suggests that chemically induced sex-reversal is mediated through interaction with steroid receptors or alterations in steroidogenic enzymes. Natural and synthetic estrogens have been shown to induce ovarian development at male-producing temperatures (sex-reversal) in the redeared slider (Trachemys scripta) (Bergeron et al., 1999; Sheehan et al., 1999), the American alligator (Alligator mississippiensis) (Crain et al., 1997; Lance and Bogart, 1994), and the sea turtle (Lepidochelys olivacea) (Merchant-Larios et al., 1997). However, Merchant-Larios et al. (1997) noted that E2-induced ovaries were smaller than temperature-induced ovaries. Incomplete sex-reversal indicated by formation of ovotestes at maleproducing temperatures was observed in European pond turtles after exposure to tamoxifen or a combination of tamoxifen and E2 (Dorizzi & Mignot, 1991). Xenobiotics shown to induce sex-reversal include trans-Nonachlor, cis-Nonachlor, Aroclor 1242, p; p0 -DDE, and chlordane in the red-eared slider (Willingham and Crews, 1999), and trans-Nonachlor, TCDD, and several metabolites of DDT in alligators (Guillette and Crain, 2000). In addition to alterations in sex determination, environmental contaminants are capable of disrupting hormone-dependent development of sexually dimorphic traits and reproductive structures in reptiles. For instance, reduction in the sexually dimorphic ratio of precloacal length to posterior lobe of the plastron (PPR) was reported in several PCB-contaminated populations of common snapping turtles (Chelydra serpentina) (de Solla et al., 1998). This reduction in PPR was significant enough to cause researchers to incorrectly identify males as females based on secondary sex characteristics. Abnormal testis morphology has been observed in alligators collected from Lake Apopka, a central Florida lake contaminated with several known EDCs (Guillette et al., 1994). Symptoms included poorly organized seminiferous tubules, many of which were lined with a cuboidal epithelium or contained cells with bar-shaped nuclei. Neither of these characters was present in the testes of alligators from a reference site. Few studies have examined the endocrine control of copulatory organ development and growth in nonmammalian species. What is known is that, as in mammals, the development and growth of the phallus of reptiles and the gonopodium of fish is androgen-dependent (e.g., for reptiles see Raynaud and Pieau, 1985; fish, see van Tienhoven, 1983). Thus, if these species were exposed to EDCs with antiandrogenic activity, the androgendependent phallus would be developmentally altered and feminized. This could occur not only through the actions of environmental antiandrogens, but also through the actions of estrogens as well. Male alligators living in contaminated lakes exhibit reduced plasma testosterone concentrations as well as


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dose-dependent fashion (Doyle and Lim, 2002). We have recently reported that male mosquitofish from Lake Apopka, a lake in which alligators display reduced plasma testosterone concentrations and phallus size, also exhibit reduced gonopodial length and altered tissue concentrations of testosterone (Toft et al., 2003). Altered phallic development is an important marker of endocrine disruption in male vertebrates.

3. Gamete production and fertility

Fig. 2. Mean (7SE) penis size in normalized populations of subadult alligators from seven lakes in central Florida. Those animals from contaminated lakes (e.g., Apopka, Okeechobee) exhibited reduced size compared to those from reference lakes (e.g., Monroe, Orange, Woodruff). Adapted from Guillette et al. (1999).

reduced phallus size (Guillette et al., 1996, 1999; Pickford et al., 2000) (Fig. 2). Further, unlike male alligators from reference lakes, juvenile males from contaminated lakes, such as Lake Apopka, exhibit no relationship between plasma testosterone concentrations and phallus size (Guillette et al., 1999). Turtles from contaminated sites in the Great Lakes region of the US exhibited a reduction in the ratio of precloacal length to the posterior lobe of the plastron (PPR), a sexually dimorphic character in snapping turtles (de Solla et al., 1998). The PPR measurement is indicative of phallus size in male turtles and these data suggest a reduction. The modification in PPRs of males from contaminated sites resulted in a large degree of overlap with PPRs of females in contrast to the strong sex dimorphism seen in this measurement in animals from a reference site (de Solla et al., 1998). Phallus development and growth is androgen-dependent in turtles as reported for other reptilian males (see Pickford et al., 2000). Our observations are consistent with those reported for mammals exposed to antiandrogenic contaminants (Gray et al., 2001). In addition to crocodilians and turtles, mosquitofish (Gambusia sp.) from contaminated sites have also undergone alterations in secondary sex characteristics. The androgen-dependent anal fin of males is referred to as the gonopodium and serves as the intromittent organ for internal fertilization. It was observed that male mosquitofish inhabiting sewage-contaminated waters in Australia had significantly smaller gonopodial length than males from nonexposed reference populations (Batty and Lim, 1999). This phenomenon was not observed in a population exposed to sewage in a small US stream; these fish also displayed no signs of estrogen exposure such as elevated plasma vitellogenin concentrations (Angus et al., 2002). Exposure to estrogenic substances can reduce gonopodial development in a

Direct measures of male fertility include the assessment of spermatogenesis, sperm delivery, and fertilization. These are complex processes that involve several steps and depend on proper reproductive anatomy and hormonal balance. Therefore, a given observation, such as low spermatocrit, may be due to a number of causes, which, in turn, may be difficult to elucidate. However, measures of male fertility are important because they provide tangible evidence of an endpoint with direct implications on health and sustainability of wild populations. 3.1. Spermatogenesis Disruption of spermatogenesis has been described in several ways, including changes in proportions of sex cell types in the testis, reduction in ejaculated sperm cell number, arrest of spermatogenesis, reduction in number of primordial germ cells, dead germ cells, proliferation of spermatogonia, progressive disappearance of spermatozoa and spermatogenic cysts, delayed spermatogenesis, reduced sperm motility, and absence of running milt. General decreases in spermatogenesis and ejaculated sperm counts have been observed in several species, including goldfish treated with E2 (Schoenfuss et al., 2002), adult zebrafish after 24 days of laboratory exposure to 17-b-ethynylestradiol (Van den Belt et al., 2002), adult Japanese medaka exposed to 4-tert-octylphenol (Gronen et al., 1999), swordtails (Xiphophorus helleri) exposed to nonylphenol (Kwak et al., 2001), and adult and sexually developing juvenile guppies exposed to vinclozolin and p; p0 -DDE (Baatrup and Junge, 2001a; Bayley et al., 2002). Van den Belt et al. (2002) noted that spermatogenesis recovered when fish were removed to clean water. Reduced spermatogenesis was also noted in English flounder captured from waterways contaminated with treated sewage effluent (Lye et al., 1998). Lye et al. (1997) reported an absence of running milt in these flounders, although they noted that milt could be forced out by stripping. A similar absence of milt, despite presence of sperm in the testes, has been observed in wild roach captured from English rivers receiving large volumes of treated sewage effluent (Jobling et al.,

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2002b). This effect may be due to physiological dysfunction or to physical occlusion of the spermatic ducts (see section on duct development above). In addition to the lack of running milt, the roach also exhibited delayed or inhibited spermatogenesis and reduced sperm motility (percentage of motile sperm and curvilinear velocity), sperm density, and milt volume (Jobling et al., 2002a, b). In male mosquitofish, Toft et al. (2003) reported that fish collected from Lake Apopka, FL (USA) had fewer sperm cells per milligram testis when compared to fish from less contaminated lakes nearby. In adult fathead minnows exposed to bisphenol A, Sohoni et al. (2001) observed changes in the proportions of sex cell types in the testis, suggesting an inhibition of spermatogenesis. Similarly, male carp (Cyprinus carpio) exposed as adults and maturing juveniles to 4-tertpentylphenol or E2 showed a reduction in the number of primordial germ cells per gonadal section and inhibited spermatogenesis (Gimeno et al., 1998a, b). In the adults, these effects co-occurred with the progressive disappearance of spermatozoa and spermatogenic cysts and a higher incidence of histopathological alterations with time. Similar effects were noted in platyfish (Xiphophorus maculates) exposed to nonylphenol or E2 (Kinnberg et al., 2000). Treated platyfish testes showed a decrease in the number of spermatogenic cysts associated with Sertoli cell hypertrophy. In addition, platyfish normally package their sperm in spermatozeugmata, but treated fish exhibited free spermatozoa in their efferent ducts. Moreover, summer flounder (Paralichthys dentatus) exposed to E2 accumulated excessive hyalin material in their testes (Folmar et al., 2001). Based on immunoreactivity assays, part of this material was identified as vitellogenin. The proteinaceous accumulation disrupted spermatogenesis and was associated with reduced testicular mass/atrophy, dead germ cells, and proliferation of spermatogonia. 3.2. Fertility Successful production of viable offspring is possibly the most important endpoint in endocrine disruption studies. Nonetheless, it is rarely measured. The few studies that are available suggest that exposed fish produce fewer embryos. Endpoints in offspring production tests include the number of eggs fertilized and/or hatched or offspring born to viviparous species. For example, a dose-dependent decrease in fertilization rate was observed when male Japanese medaka exposed during development to octylphenol and E2 were mated with control females (Knorr and Braunbeck, 2002). A similar decrease in fertilization rate as well as an increase in the number of abnormally developing embryos was noted in Japanese medaka exposed as adults to 4-tertoctylphenol (Gronen et al., 1999). Likewise, wild

intersex roach collected from waterways contaminated with treated sewage effluent exhibited a reduced ability to fertilize eggs to produce viable offspring (Jobling et al., 2002b). In that case, both parents were exposed. In a study of adult male guppies (Poecilia reticulata) exposed to 4-tert-octylphenol and E2, treated males produced fewer offspring than untreated fish, based on number of offspring fathered (Toft and Baatrup, 2001). In a rare study of transgenerational effects, Matta et al. (2001) exposed adult Fundulus heteroclitus to methylmercury and the PCB mixture Aroclor 1268. Offspring of the mercury-exposed parents had reduced fertilization success. Offspring of parents exposed to low concentrations of Aroclor also had reduced fertilization success compared to controls, but higher fertilization success at moderate Aroclor concentrations. This last example illustrates the common observation that biological effects can vary significantly with dose, but the effects are not always linearly dose-dependent. Low and intermediate concentrations of EDCs can be more harmful than high concentrations, possibly due to receptor down-regulation at high doses.

4. Endocrinology Mechanistically, EDCs have been shown to affect an organism through receptor-mediated actions, altered hormone synthesis or degradation, and binding to plasma proteins (Guillette and Crain, 2000; NRC, 1999). Many studies of EDCs have focused on the estrogenicity of pesticides such as o,p0 -DDT (Fry and Toone, 1981; vom Saal et al., 1995; Vonier et al., 1996) and sewage effluent (Harries et al., 1997; Purdon et al., 1994). Other studies indicate that environmental contaminants such as p; p0 -DDE (metabolite of DDT) (Kelce et al., 1995) and the fungicide vinclozolin (Gray et al., 1994) are capable of antiandrogenic actions. Furthermore, the exact mechanism(s) through which each of these chemicals works remains unclear, but it appears that circulating steroid concentrations, steroidogenesis, and hepatic steroid metabolism are susceptible to chemically induced alterations and these effects are species- and dose-dependent. Because the endocrine system regulates so many physiological processes, any perturbations in this system can have profound impacts on reproductive and/or developmental processes. 4.1. Plasma steroids Alterations in circulating steroid concentrations have been documented in numerous aquatic vertebrates exposed to EDCs. Depressed plasma androgens and/or elevated E2 are often characteristic of males exposed to antiandrogenic or estrogenic xenobiotics. Summer flounder (Paralichthys dentatus) experimentally exposed


to estradiol exhibited reduced plasma testosterone (T) and elevated plasma E2 (Mills et al., 2001). Similarly, flounder treated with o,p0 -DDT and octylphenol showed a reduction in plasma T concentrations. The octylphenol-treated flounders also had an initial increase in plasma E2 concentration (Mills et al., 2001). In white sturgeon (Acipenser transmontanus) from the Columbia River, plasma T and 11-ketotestosterone concentrations were negatively correlated with p; p0 DDE levels found in the liver (Foster et al., 2001). Male largemouth bass (Micropterus salmonoides) living downstream from a coal-fired electric plant and a chemical manufacturing plant in the Escambia River had lower plasma T concentrations than fish from a nearby reference site (Orlando et al., 1999). Likewise, lake whitefish exposed to bleached kraft mill effluent and white sucker exposed to pulp mill effluent have depressed circulating T concentrations (Munkittrick et al., 1992, 1994). Whole body testosterone concentrations were lower in male mosquitofish from Lake Apopka compared to Lake Orange (a reference site) during the month of January, when T and E2 concentrations normally peak (Toft et al., 2003). Similar alterations in circulating steroid concentrations have been reported in aquatic vertebrates other than fish. When exposed to 25 ppb atrazine, the African clawed frog exhibited a 10-fold decrease in testosterone level (Hayes et al., 2002). Several studies that compared steroid concentrations in American alligators from multiple Florida lakes have shown that plasma testosterone is depressed in male alligators from lakes contaminated with organochlorine pesticides and organic nutrients (Crain et al., 1998; Guillette et al., 1994, 1999). In addition, male alligators from highly contaminated Lake Apopka have exhibited elevated plasma E2 when compared to reference site alligators (Milnes et al., 2002). An extensive literature review reveals there has been little research documenting nonsteroidal hormones that exhibit sexually dimorphic patterns that are affected by EDCs. However, exposure to hormonally active compounds or the resulting changes in plasma steroid concentrations can affect circulating concentrations of other biomolecules. A well-documented example is the presence of the yolk protein precursor, vitellogenin (Vtg), in males, which is normally only present in females. In a study by Palmer and Palmer (1995) production of vitellogenin was induced experimentally following injection of several xenobiotic estrogens in the red-eared slider turtle and African clawed frog. The induction of Vtg has been shown in several fish including medaka (Cheek et al., 2001) and summer flounder (Mills et al., 2001) exposed to o,p0 -DDT and rainbow trout exposed to nonylphenol and PCBs (Flourit et al., 1995). Mosconi et al. (2002) reported the presence of Vtg in several species of frogs following

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exposure to 4-nonylphenol. The metabolic cost associated with production of this large protein and its potential impacts on reproduction in males have yet to be investigated in depth. 4.2. Steroidogenesis The numerous cases documenting alterations in circulating steroid concentrations has led researchers to examine steroidogenesis in contaminant-exposed vertebrates. Much of the work has focused on steroidogenic enzymes that function downstream from the conversion of cholesterol to pregnenolone such as 3bHSD and various cytochrome P-450s. These studies generally rely upon whole tissue or steroidogenic tissue homogenates incubated in culture medium alone or supplemented with a steroid substrate such as progesterone or androstenedione. Following incubation, steroids in the steroidogenic pathway are measured to determine relative enzyme activity. The steroidogenic pathway appears to be affected by numerous pharmaceutical, agricultural, and industrial contaminants. Exposure to various hormonally active compounds can result in decreased androgen production (demasculinization) or increased estrogen production (feminization) in males. For example, exposure to the phytoestrogen genistein resulted in decreased testicular testosterone (T) production in medaka with a comparable reduction in plasma T concentration (Zhang et al., 2002). Similarly, goldfish exposed to bleached sulfite mill effluent showed a reduction in testicular T and 11-ketotestosterone synthesis when compared to control males (Parrot et al., 1999). In male alligators obtained from eggs collected from Lake Apopka, FL, Guillette et al. (1995b) found increased testicular E2 production compared to males from a reference population. One mechanism for disrupting the steroidogenic pathway is to alter activity or expression of specific enzymes. The enzyme responsible for the conversion of androgens to estrogens, aromatase (CYP 19 or P450arom), is usually expressed in a sexually dimorphic manner in various tissues including the gonads, liver, and brain. The herbicide atrazine was shown to increase gonadal aromatase activity in male alligators to levels similar to those on control females following embryonic exposure (Crain et al., 1997). In the frog, Xenopus laevis, exposure to atrazine during larval development resulted in hermaphroditic males with demasculinized larynges (Hayes et al., 2002). It was hypothesized that atrazine induced aromatase activity, as evidenced by the decrease in plasma testosterone. In addition to direct suppression or induction of steroidogenic enzymes, exposure to estrogenic compounds could interfere with normal feedback mechanisms on steroidogenic enzymes. In male medaka, brain aromatase activity increased in a

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dose-dependent manner following E2 exposure (Melo and Ramsdell, 2001). Although not well studied in nonmammalian systems, environmental contaminants have been shown to alter transcription and translation of the steroidogenic acute regulatory (StAR) protein. The StAR protein is responsible for mediating the transfer of cholesterol from the outer to the inner mitochondria membrane. This must occur to facilitate the first step of steroidogenesis, the conversion of cholesterol to pregnenolone (Stocco and Clark, 1996). Post-transcriptional disruption of StAR protein expression was observed following treatment of MA-10 (mouse Leydig tumor) cells with the antifungal drugs econazole and miconazole (Walsh et al., 2000a). Alternatively, the insecticides, Lindane (organochlorine) and Dimethoate (organophosphate) inhibit steroidogenesis by disrupting StAR protein transcription (Walsh and Stocco, 2000; Walsh et al., 2000b). Future research should focus on the effects of environmental contaminants on StAR protein expression in nonmammalian, in vivo systems. 4.3. Hormone biotransformation Sexually dimorphic patterns of steroid and drug biotransformation are documented in fish, amphibians, reptiles, birds, and mammals (Gunderson et al., 2001; Gustafsson and Ingelman-Sundberg, 1974; Gustafsson et al., 1983; Khan et al., 1998; Legraverend et al., 1992; Pampori and Shapiro, 1993; Parks and LeBlanc, 1998; Saito et al., 1997; Snowberger et al., 1991). In rodents, these patterns are regulated by the persistence (females) or prolonged suppression (males) of growth hormone in the trough period after a growth hormone surge (Legraverend et al., 1992). These patterns are imprinted during development by sex steroids. Certain enzymes are indicative of masculinized or feminized patterns of steroid or drug biotransformation though differences exist within and among species (Gustafsson and Ingelman-Sundberg, 1974; Gustafsson et al., 1983; Niwa et al., 1995). In mice, the STAT5a and STAT5b genes have been shown to play a role in the sexually dimorphic growth hormone release patterns and sex specific cytochrome P-450 activities observed (Park et al., 1999). The liver plays a key role in steroid, drug, and xenobiotic biotransformation in all vertebrates. Fish, amphibians, reptiles, birds, and mammals all possess cytochrome P-450 enzymes that are involved in the biotransformation of xenobiotics, drugs, and hormones (Ertl and Winston, 1998; Gustafsson et al., 1983; Pampori and Shapiro, 1993; Snowberger and Stegeman, 1987; Stegeman et al., 1997). Phenobarbitol (PB), 3methylcolanthrine, and b-naphtholflavone are inducers of cytochrome P-450 activity in mammals and are often used to distinguish between CYP 1 and CYP 2 families of enzymes. These inducers have been demonstrated to

induce cytochrome P-450 activity in fish, amphibians, and reptiles, though the families of enzymes induced and patterns of induction often differ from those observed in mammalian models (Ertl and Winston, 1998; Khan et al., 1998; Novi et al., 1998). To our knowledge, the regulation of sexually dimorphic patterns of steroid, drug, and xenobiotic biotransformation reported in fish, reptiles, and birds has not been investigated, though sex steroids are suspected as the inducers of the reported dimorphism (Gunderson et al., 2001; Pampori and Shapiro, 1993; Sanderson et al., 1997; Snowberger et al., 1991). Hepatic biotransformation of steroids could serve as a useful endpoint to examine the impacts of hormonally active xenobiotics on aquatic wildlife populations. Hepatic biotransformation enzymes tend to have broad substrate specificity. There are several theories as to why these enzymes evolved as they did, but the underlying theme is that broad substrate specificity increases an animal’s ability to respond to a diverse range of chemicals in the environment. These enzymes not only biotransform a diverse range of environmental compounds but also inactivate endogenous substrates such as androgenic and estrogenic steroids. Given that hepatic biotransformation of steroids is regulated by sex steroids in mammals and imprinted during development, this system is potentially susceptible to perturbations by hormonally active xenobiotics. These compounds could act organizationally by altering the normal imprinting processes during critical points in development that would then be manifested in juvenile and adult enzyme activities (masculinized or feminized patterns) (Guillette et al., 1995a). These compounds could also have a transitory effect by inducing enzymes that not only biotransform the xenobiotics but also play a role in the inactivation of endogenous substrates such as steroids (Guillette and Gunderson, 2001; Gunderson et al., 2001). A combination of these mechanisms of action could also take place. The functional result of these alternations would lead to alterations in the circulating concentrations and/or clearance of endogenous substrates, such as testosterone, estradiol, or thyroxine, and thus perturb the normal signaling mechanisms that coordinate physiological processes for the organism. Numerous examples in the literature experimentally demonstrate modulation of enzymes, many involved in the biotransformation of sex steroids and other hormones, by exogenously injected hormones or xenobiotics. In rats, DDT has been shown to induce the malespecific isozyme CYP 3A2 in females, marginally effect the male-specific isozyme CYP 2C11 in males, and induce PROD and BROD (Sierra-Santoyo et al., 2000). Endosulfan and the fungicide ketokonazole have both been shown to modulate hepatic enzyme activity in mice. Endosulfan exposure led to increased clearance of


androgenic metabolites with no significant decrease in serum testosterone concentrations (Wilson and LeBlanc, 1998). In contrast, ketokonazole induced significant decreases in serum testosterone, testosterone hydroxylase activity, and gonadal testosterone synthesis. The changes in serum testosterone in this case could be explained by altered gonadal synthesis and not the differential inhibition of hepatic hydroxylase enzymes (Wilson and LeBlanc, 2000). Atrazine, an herbicide commonly used on monocot crops because of its ability to inhibit photosynthesis in broad-leafed plants, has been shown to decrease CYP 2C11 (associated with testosterone 2a-hydroxylase activity), increase MROD, EROD, and PROD, and have no effect on testosterone 6-b-hydroxylase (associated with CYP 3A) or testosterone 7-b-hydroxylase in rats (Hanioka et al., 1998). Estradiol has been demonstrated to depress EROD, testosterone 6-b-hydroxylase, and estradiol 2-hydroxylase in winter flounder (Snowberger et al., 1991). These experiments are intriguing in light of the fact that field studies sampling alligators and fish from a contaminated site in Florida exhibit alteration in hepatic biotransformation enzymes and altered steroid concentrations. Female brown bullheads collected from Lake Apopka, contaminated with organochlorine compounds, exhibit lower glutathione-s-transferase expression and activity than females from a reference lake, Lake Woodruff. On the other hand, females from Lake Apopka in turn exhibit elevated androgens when compared to Lake Woodruff females (Gallagher et al.,

Fig. 3. Total testosterone hydroxylase and oxidoreductase activity measured in juvenile animals collected from Lake Woodruff (reference), Lake Apopka (contaminated), and Lake Okeechobee (moderately contaminated). *Denotes difference between males and females within that site. Adapted from Gunderson et al. (2001).

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2001). Juvenile alligators from Lake Woodruff exhibit a sexually dimorphic pattern of testosterone hydroxylase and oxido-reducatase activity (Fig. 3). This pattern is not observed in Lake Apopka alligators or alligators collected from two sites on Lake Okeechobee and one site in the northern Everglades (Gunderson et al., 2001). Previous studies have reported altered testosterone concentrations in alligators collected from Lake Apopka and Lake Okeechobee when compared to Lake Woodruff animals (Guillette et al., 1999). The plasma testosterone and estradiol concentrations measured in animals from the three sites in south Florida (Lake Okeechobee and the northern Everglades) did not correlate with hepatic testosterone biotransformation enzyme activity or urine metabolites for atrazine (Gunderson et al., unpublished data).

5. Neuroendocrinology The neuroendocrine system transforms external and internal environmental signals along with internal endogenous endocrine feedback into multiple endocrine signals that coordinate gametogenesis, reproductive cycles, reproductive behavior, fertilization, and parental behaviors. Therefore, altered steroid biosynthesis pathways could disrupt the hypothalamic–pituitary complex via feedback mechanisms and/or direct agonist/antagonist actions resulting in alterations in gonad morphology and reproductive ability (Jalabert et al., 2000). In vertebrate males, gonadotropin-releasing hormone (GnRH) secreted from neurons in the preoptic-anterior hypothalamus (POA) and ventral telencephalon is the principal regulator of pituitary release of gonadotropins. This nucleus interfaces the hypothalamus and limbic system and is the primary control of male typical behaviors (Norris, 1997). In many species, the size of the POA is sexually dimorphic, usually larger in males, and dimorphic between intraspecific males displaying alternative sexual phenotypes (Crews, 2003). In a series of experiments, Khan and Thomas treated male Atlantic croaker (Micropogonias undulatus) with the PCB Aroclor 1254 through diet for 30 days during gonadal recrudescence (Fig. 4). This exposure was found to decrease serotonin (5-HT) and dopamine concentrations and increase related neurotransmitter metabolites in both the POA and medial and posterior hypothalamus (Khan and Thomas, 1997). Furthermore, exposure caused decreases in GnRH content in the POA and pituitary GnRH receptors and pituitary gonadotropic response to a GnRH analogue. Aroclor 1254 was shown to impair hypothalamic 5-HT metabolism through inhibition of hypothalamic tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, thereby disrupting pituitary gonadotropin secretion via the 5-HT-GnRH pathway, leading to

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in brain aromatase activity during the temperature sensitive period of sex determination. Additionally, this exposure increased the ratio of female hatchlings at the male-producing temperature and treated male hatchlings exhibited significantly lower testosterone levels than controls in response to FSH administration. These results suggest that alterations in sex steroid hormone levels observed in animals exposed to contaminants might result from alterations of neuroendocrine axis control of gonadal sex steroid hormone production.

6. Behavior Fig. 4. Effects of Aroclor 1254 exposure on hypothalamic–pituitary–gonadal axis of male Atlantic croaker (Micropogonias undulatus). Adapted from Khan and Thomas (1997, 2000, 2001).

impairment of gonadal growth (Khan and Thomas, 2000, 2001). The possibility that these results were a consequence of a treatment-induced decline in GnRH release was explored. Experiments using p-chlorophenylalanine, an irreversible TPH inhibitor that mimicked the effects of PCB on the GnRH system, followed by administration of exogenous 5-hydroxytryptophan restored 5-HT to control levels and was shown to prevent the deleterious neuroendocrine effects associated with the PCB. In addition, GnRH implants prevented the PCB-induced decline in GnRH receptors and restored the LH response to a GnRH analog (Khan and Thomas, 2001). Therefore, it was concluded that TPH is one of the targets of PCB neurotoxicity and a decrease in 5-HT availability results in disruption of the stimulatory 5HT/GnRH pathway controlling LH secretion. LH secretion is typically higher in sexually mature croaker compared to early recrudescing fish. Following Aroclor 1254 exposure during testicular maturation, Khan et al. (2001) reported GnRH content of the POA, pituitary GnRH receptor density, and LH secretion similar to those of recrudescing male croaker. They hypothesized that PCB exposure disrupted maturation of the GnRH-LH system during gonadal recrudescence through GnRH neurons and/or indirectly by altering neurotransmitters that modulate GnRH function, such as 5-HT. In turtles, Willingham and Crews (2000) showed that low dosages of hormonally active chemicals could have neurological effects during development. The sex of redeared slider turtles is temperature-dependent and embryos incubated at female-producing temperatures have higher levels of aromatase activity in the brain than those incubated at male temperatures. However, embryos incubated at male-producing temperatures treated in ovo with Aroclor 1242 showed a significant increase

The mechanisms of hormonal control over certain behaviors related to reproduction and/or survival have not been completely elucidated, but sex steroids have clearly been shown to impact reproductive behavior. For example, castrated male three-spined stickleback fish treated with methyltestosterone resumed nestbuilding, courtship, and parental care behaviors that were otherwise drastically reduced or eliminated after castration (Hoar, 1962a, b; Smith and Hoar, 1967; Wai and Hoar, 1963). By similarly treating female guppies with dietary methyltestosterone, the entire male courtship repertoire can be induced in females (Landsman et al., 1987). The ability of males to properly display and communicate to the opposite sex is critical for breeding success in numerous species. Display rates and intensity have been correlated with higher fecundity, such as sperm numbers in male guppies (Matthews et al., 1997). Hormones are correlated with the mating displays of guppies and the secretion of pheromones in goldfish, but various EDCs can alter the performance of these reproductive behaviors. Vinclozolin, flutamide, and p; p0 -DDE act as antiandrogens that bind competitively to the androgen receptor, preventing the transcription of androgen-dependent genes. In adult male guppies exposed to all three of these chemicals, the number of sigmoid displays toward females were significantly reduced, although a negative or neutral dose–response was indicated (Baatrup and Junge, 2001b). Thus, lower levels of vinclozolin (1 mg/mg vs. 10 mg/mg), DDE (0.1 mg/mg vs. 1 mg/mg), and flutamide (1 mg/mg vs. 10 mg/mg) resulted in more pronounced inhibition of reproductive displays than the higher doses. Baatrup and Junge (2001b) suggest that this may be caused by tissue-dependent differences in AR affinities for antiandrogens, or lipophilic characteristics of the chemicals causing them to rapidly impact high-lipid-content tissues, such as the brain. Juvenile males exposed to sublethal doses of these chemicals result in delayed maturation, reduction in body size at maturation, and both vinclozolin and the highest doses of DDE caused significant reductions in the final body size of male guppies (Bayley et al., 2002). The duration and number


of male displays were reduced in males exposed to vinclozolin and the high-dose flutamide treatments. All behaviors measured in male guppies were recovered after several weeks in uncontaminated tanks. The male’s display color area was not impacted, but all three chemicals reduced the orange color intensity (Bayley et al., 2002). Coloration in male guppies has been identified as an androgen-dependent characteristic (Pandey, 1969). 17-b-Estradiol and the xenoestrogen 4-tert-octylphenol (OP) act to feminize male guppies by decreasing the rate and intensity of sexual display toward females (Bayley et al., 1999). These chemicals similarly impacted male coloration by decreasing the orange area and intensity. Guppies removed from OP exposure to clean water for 3 months had a return of color intensity, but not color area. In male Japanese medaka fish (Oryzias latipes), 6 months of sublethal OP exposure from day one post-hatch resulted in decreased approaches, circles, and successful copulations with females (Gray et al., 1999). Adult male Japanese medaka that received E2 (3 or 30 mg/g BW) daily for 2 weeks in their diet showed a striking reduction in reproductive behavior, with some behaviors (dancing, floating, crossing, and mating) eliminated completely (Oshima et al., 2003). Approaches and copulatory attempts made toward females were also reduced in a dose-dependent manner (20, 100, and 500 ng/L) by Gambusia holbrooki juvenile males exposed to E2 during sexual maturation (Doyle and Lim, 2002). Other androgen-dependent aspects of reproductive behavior, such as those related to paternal care and nest-building (Guderly, 1994), can be impacted by exposures to estrogenic compounds (Arcand-Hoy and Benson, 1998; Wibe et al., 2002). For example, threespine sticklebacks injected with E2 (2.0 mg/g) exhibited delayed nest-building behavior and reduced paternal care, such as fanning, gluing, and guarding of the nest (Wibe et al., 2002). A hormone–pheromone system has been described in goldfish (Carassius auratus), involving 17a,20b-P and prostaglandin F2a and its metabolites. These hormones functions as critical cues for synchronizing spawning (Bjerselius et al., 1995; Dulka et al., 1987; Liley, 1982; Partridge et al., 1976; Sorensen et al., 1988; Stacey, 1987). Exposures of male goldfish to 24–28 days of E2 resulted in deceased following, pushing, and courting behaviors (Bjerselius et al., 2001). Schoenfus’s et al. (2002) similarly found a clear reduction of male spawning behavior in goldfish exposed to E2 for 10 weeks, but only small, nonsignificant decreases in spawning behavior with estrogenic sewage treatment plant effluent.

7. Conclusions Contaminants can alter the development of male characters such as steroidogenesis, spermatogenesis,

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hormone biotransformation, and neuroendocrine functioning. Although a database exists for fishes for some aspects of the work described here, it is largely the result of uncoordinated studies using varying biomarkers and exposure scenarios. For aquatic amphibians and reptiles, the database is very poor and largely represents work done on a few populations. Even given these limitations, it is clear that contaminants from various sources, including industrial pollutants, agricultural pesticides, and sewage effluents, impact males from a diversity of species in all aquatic systems, including rivers, streams, and lakes and near marine ecosystems. Future studies must focus on (1) the development of coordinated studies that can provide cross species comparisons in similar ecosystems and exposure scenarios, (2) the development of tools, whether molecular, physiological, or behavioral that will allow more extensive testing and analysis of response, and (3) the development of key aquatic ecosystems study sites that would be identified for long-term study of species with known exposures (e.g., the Laurentian Great Lakes, central Florida lakes, and the rivers of Great Britain and Japan), and differing ecosystem compositions (fresh vs. marine; temperate vs. tropical). These studies would begin to provide a picture of sensitive endpoints as well as providing insight into the consequences of disruption of male activity at the population and community levels.

Acknowledgments Work from our lab was supported by grants from the US Environmental Protection Agency to LJG (CR821437 and R824760-01-0). All works conducted at the University of Florida involving experimental animals and contributing to this review was performed under the guidelines specified by the Institutional Animal Care and Use Committee. References Angus, R.A., Weaver, S.A., Grizzle, J.M., Watson, R.D., 2002. Reproductive characteristics of male mosquitofish (Gambusia affinis) inhabiting a small southeastern US river receiving treated domestic sewage effluent. Environ. Toxicol. Chem. 21, 1404–1409. Arcand-Hoy, L.D., Benson, W., 1998. Fish reproduction: an ecologically relevant indicator of endocrine disruption. Environ. Toxicol. Chem. 17, 49–57. Baatrup, E., Junge, M., 2001a. Antiandrogenic pesticides disrupt sexual characteristics in the adult male guppy (Poecilia reticulata). Environ. Health Perspect. 109, 1063–1070. Baatrup, E., Junge, M., 2001b. Antiandrogenic pesticides disrupt sexual characteristics in the adult male guppy (Poecilia reticulata). Environ. Health Perspect. 109, 1063–1070. Batty, J., Lim, R.P., 1999. Morphological and reproductive characteristics of male mosquitofish (Gambusia affinis holbrooki) inhabiting sewage-contaminated waters of New South Wales, Australia. Arch. Environ. Contam. Toxicol. 36, 301–307.

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