Exposure of Juvenile Roach (Rutilus rutilus) to Treated Sewage

Exposure of Juvenile Roach (Rutilus rutilus) to Treated Sewage Effluent Induces Dose-Dependent and Persistent Disruption in Gonadal Duct Development...
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Environ. Sci. Technol. 2001, 35, 462-470

Exposure of Juvenile Roach (Rutilus rutilus) to Treated Sewage Effluent Induces Dose-Dependent and Persistent Disruption in Gonadal Duct Development T R E V O R P . R O D G E R S - G R A Y , * ,†,‡ SUSAN JOBLING,† CAROLE KELLY,§ STEVEN MORRIS,§ GEOFF BRIGHTY,| MICHAEL J. WALDOCK,§ JOHN P. SUMPTER,† AND CHARLES R. TYLER‡ The Fish Physiology Research Group, Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH, U.K., CEFAS Fisheries Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex, CM0 8HA, U.K., National Centre for Ecotoxicology and Hazardous Substances, U.K. Environment Agency, Wallingford, U.K., and Environmental and Molecular Fish Biology Research Group, The Hatherly Laboratories, School of Biological Sciences, Exeter University, Exeter, EX4 4PS

Wild roach (Rutilus rutilus) have been found with intersex gonads in rivers throughout the United Kingdom. The incidence of intersexuality is strongly correlated with discharges of estrogenic treated sewage effluent into those rivers, and this has led to the hypothesis that estrogenic chemicals in effluents are feminizing wild male fish. In this study, early-life stage roach (50 days post hatch, dph) were exposed for 150 days to a graded concentration (0%, 12.5%, 25%, 50%, and 100%) of treated sewage (primarily domestic) effluent to examine the effects of exposure on sexual differentiation and development. Measurement of steroid estrogens and alkylphenolic chemicals in the effluent and a resulting dose-dependent induction of vitellogenin (VTG; a female-specific, estrogen-dependent plasma protein) confirmed that the fish had been exposed and responded to “estrogen” in the effluent. Exposure to treated sewage effluent induced feminization of the reproductive ducts in “male” roach in a dose-dependent manner (in fullstrength effluent, 100% of the fish had feminized ducts), indicating that the disruption of the gonad ducts seen in wild roach is the result of exposure to treated sewage effluents during early-life stages. There were no effects of treated sewage effluent exposure on germ cell development; therefore, no oocytes occurred in the testes of the feminized male roach. Subsequent, depuration of the effluent exposed fish in “clean” water for 150 days resulted in a reduction in plasma VTG but no alteration of the feminized ducts, indicating that the effect of the treated sewage effluent on reproductive duct development was permanent. The causality of oocytes in the testes of wild male roach therefore remains to be elucidated.

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Introduction It is well-established that chemicals discharged into the environment have the ability to modulate or disrupt the normal physiological function of exposed organisms. More recently, these chemicals (which can be either natural or synthetic) have been shown to interact with the endocrine system by binding to hormone receptors, altering hormone metabolism, and/or hormone transport. This type of environmental pollution has been termed endocrine disruption and has been hypothesized, controversially, to be responsible for the worldwide occurrence of reproductive and developmental abnormalities seen in some wildlife populations (reviewed in ref 1). In the past few years, there have been a number of studies documenting the widespread occurrence of sexual disruption in freshwater and marine fish species (2-6). An extensive study of wild roach (Rutilus rutilus), a cyprinid fish, found “males” with intersex gonads in rivers throughout the United Kingdom (3). These fish had gonads that contained both male and female germ cells at various stages of gametogenesis, “female-like” reproductive ducts (ovarian cavities), and elevated concentrations of plasma vitellogenin (VTG, a female-specific protein; 7). There was a strong correlation between the incidence of intersex roach and the proximity of these fish to estrogenic treated sewage effluent discharges (3). Together these findings suggest that the intersex condition has resulted from the feminization of males, but they do not provide direct evidence that this is the case. The phenotypic sex of gonochoristic fish species, such as the roach, develops after sexual differentiation of the gonads, which occurs during early life (8). Sexual differentiation usually starts shortly after hatch and can occur over a period of a few weeks to many months (depending on the species and the growth rate). It is a two-stage process, involving gonadogenesis (the development of the somatic tissues and duct of the gonad) and gametogenesis (the differentiation of the germ cells into male or female sex cells). Gonadogenesis usually precedes gametogenesis, although there can be much overlap between these processes (9). The genetic basis of sex differentiation in the roach is not known, although the sex ratio has been found to be approximately 50:50 in laboratoryreared populations (our own unpublished data). Likewise, the precise timing of sexual differentiation in the roach is unknown. However in a closely related species, the common carp (Cyprinus carpio), sexual differentiation has been reported to occur between 50 and 120 days post hatch (dph) (10). Estrogens play central roles in both gonadogenesis and gametogenesis in female fish (8), and this accounts for why the sexual phenotype of many fish species can be reversed by application of high doses of sex steroids (including estrogens) during the period of sexual differentiation (11). On the basis of this knowledge, it has been hypothesized that intersex fish found in U.K. rivers are feminized males that result from exposure to estrogenic treated sewage effluents during the period of sexual differentiation, i.e., during this window of heightened sensitivity of the gonad to sex steroids. Major estrogenic components that have been identified in treated sewage effluents include the natural estrogens (17β* Corresponding author telephone: +44 (0) 1895 274000, ext. 2106; fax: +44 (0) 1895 274348; e-mail: [email protected]. † Brunel University. ‡ Exeter University. § CEFAS Fisheries Laboratory. | U.K. Environment Agency. 10.1021/es001225c CCC: $20.00

 2001 American Chemical Society Published on Web 12/30/2000

FIGURE 1. Flow diagram of the experimental design. A pilot study was conducted to assess the chronology of sexual differentiation in the roach. Roach were sampled at 50 days post hatch (dph) for biochemical and histopathological analyses prior to exposure to treated sewage effluent (sampling point 1). Juvenile roach were reared in various concentrations of treated sewage effluent or river water or tap water, from 50 to 150 dph, and sampled for biochemical and histopathological analyses at 100 and 150 dph (samplings 2 and 3). Subsequently, fish reared in the full-strength effluent and in tap water were kept for a further 50 days and sampled for biochemical and histopathological analyses at 200 dph (sampling 4). Finally, fish kept in the full-strength effluent were depurated in tap water for 150 days, and tap water fish were reared in parallel for a further 150 days, until 350 dph, and both groups were sampled for biochemical and histopathological analyses (sampling 5). Details of the sampling protocols are in the text. estradiol and estrone), the synthetic estrogen 17R-ethynylestradiol (12), and estrogen-mimicking alkylphenolic chemicals (13). The concentrations of the natural estrogens alone in U.K. treated sewage effluents are sufficient to induce the observed effects on VTG synthesis in roach (14). In some rivers, alkylphenolic chemicals are also present at concentrations sufficient to induce VTG synthesis as well as to cause suppression of testicular growth (15). In laboratory studies, intersex or sex-reversed fish have been produced by exposure of eggs or larvae to environmental estrogens but only at concentrations considerably higher than those found in the ambient environment (16, 17). To date, no studies have demonstrated directly that treated sewage effluent affects sex differentiation. This study set out to establish the effects of exposure to treated sewage effluent discharged into U.K. rivers on sexual differentiation and development in juvenile roach.

Materials and Methods Establishing the Period of Cellular Gonadal Sexual Differentiation in the Roach. A pilot study was conducted to establish the timing of sexual differentiation (i.e., the development of the reproductive ducts and sex cells) in roach reared in clean water. Juvenile roach were reared in dechlorinated tap water and sampled for gonadal histopathology (as described below) at 50, 100, 150, and 200 dph. Experimental Design. On the basis of the pilot study, experiments were set up to investigate the effects of treated sewage effluent on sexual differentiation (see Figure 1): 50 dph roach were sampled before exposure to treated sewage effluent to assess their gonadal status (sampling point 1). They were then exposed from 50 to 150 dph to examine the effects of a graded concentration series of treated sewage effluent on development of the reproductive duct (100 dph, sampling point 2; 150 dph, sampling point 3). Fish exposed

to full-strength treated sewage effluent were exposed for a further 50 days (200 dph, sampling point 4) to investigate effects on germ cell differentiation. At 200 dph, fish exposed to the full-strength treated sewage effluent were depurated for 150 days to examine effects on the gonadal and vitellogenic responses induced by exposure to treated sewage effluent (350 dph, sampling point 5). Treated Sewage Effluent. The treated sewage effluent was derived from Chelmsford Sewage Treatment Works (STW), Chelmsford, Essex, U.K. This treated sewage effluent was chosen because it has been well-characterized with respect to its induction of estrogenic responses in fish (20, 26) and its estrogen steroid and nonylphenolic content (20). Chelmsford STWs receives influent with a population equivalent of 138 000, and the STW has both activated sludge and biological filter secondary treatments. The influent load is primarily domestic, although industrial inputs contribute 14% of the load. Effects of Exposure to a Graded Concentration of Treated Sewage Effluent on Sexual Differentiation (Exposure from 50 to 150 dph). Five 1-m3 tanks were supplied with one of a series of treated sewage effluent dilutions produced by mixing different percentages of effluent with river water. Nominal concentrations of treated sewage effluent were 100%, 50%, 25%, 12.5%, and 0% (river water only). The flow rate through each of the tanks totaled 10 L/min. An additional tank was supplied with dechlorinated tap water to act as an absolute control as the river water control might have contained treated sewage effluent from upstream sources. At the beginning of the experiment, each tank contained 1000 juvenile roach that were 50 days old (50 dph). All fish were fed commercial trout pellets (fine and medium grade, Calverton Fish Farm, U.K.) six times daily throughout the trial by automatic feeders (5% body weight/day). The tanks were aerated to ensure sufficient oxygen supply to support VOL. 35, NO. 3, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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the biomass of fish, and the temperature was monitored daily in all tanks. The roach were exposed to the graded concentration of treated sewage effluent for 100 days (from 50 to 150 dph). Sixty roach were sampled from each treatment at day zero (pretreatment) and after 50 and 100 days exposure. The lengths (mm) of all sampled fish were measured to determine growth rate. Thirty roach from each sampling were washed, weighed, and homogenized as described in ref 18, and the whole-body concentrations of vitellogenin were measured in individual fish. The remainder of the homogenates were used to analyze whole-body concentrations of 17β-estradiol. Because of the limited volume of the remaining homogenates and the low concentrations of 17β-estradiol in juvenile roach, it was not possible to measure concentrations in individual fish (homogenates). Concentrations of 17βestradiol therefore were measured in pooled homogenates from 5 fish to produce six pooled samples per treatment. The remaining 30 fish for each sampling were fixed for gonadal histopathology. Effects of Exposure to Full-Strength Treated Sewage Effluent on Sexual Differentiation (Exposure from 50 to 200 dph). Fish held in the full-strength (100%) effluent and the tap water control for 100 days were kept for a further 50 days (a total exposure of 150 days). At 200 dph, 60 roach were sampled from each treatment and analyzed for somatic growth, VTG induction, and gonadal histopathology as described above. Effects of Depuration in Clean Water for 150 Days on the Gonadal and Vitellogenic Responses Induced by Exposure to Treated Sewage Effluent. Fish held in the fullstrength treated sewage effluent for 150 days were transferred to clean water for a further 150 days (from 200 to 350 dph). After the depuration period, 50 fish were sampled from the depuration group, and 50 fish were sampled from the tap water controls. Growth (length) and gonadal histopathology were examined in all these roach, and the weight, gonadweight (mg), and whole-body concentration of VTG were measured in 20 fish from each treatment. Biological Sampling. At all sampling points, fish were sacrificed with a lethal dose of anaesthetic (2-phenoxyethanol; 1:1000). Whole fish were fixed for 24 h in Bouins and then stored in 70% industrial methylated spirits prior to histological processing. Fish sampled for VTG and 17β-estradiol analyses were placed in cryovials and frozen immediately in dry ice prior to transport and storage at -20 °C. Growth. Condition factor (K; a measure of the body form) was calculated for individual fish by the expression: K ) weight (g) × 100/(length cm)3. Gonadal growth (if applicable) was assessed using the gonadosomatic index (GSI), calculated for individual fish using the formula: gonad weight/(total body weight - gonad weight) × 100. Measurement of Vitellogenin. Homogenization and subsequent quantification of whole-body vitellogenin (using a carp VTG ELISA) was carried out as described in ref 18. Briefly, fish were homogenized in phosphate-buffered saline 0.05% Tween 20 and 1% bovine serum albumin, pH 7.4 (1 mL of buffer/1 g of fish), and after centrifugation at 9000g, the supernatant was removed and frozen at -20 °C. Measurement of 17β-Estradiol. Whole-body 17β-estradiol concentrations were measured by competitive radioimmunoassay (19) of homogenate extracts. The assay detection limit was 67 pg of 17β-estradiol/mL of homogenate. Briefly, supernatants from homogenized fish were diluted 1:5 with ethyl acetate (for 17β-estradiol extraction). They were then vortexed and centrifuged for 2 min at 2500g, and the ethyl acetate was transferred to clean tubes. The ethyl acetate was then removed by evaporation, and the 17β-estradiol was redissolved in 100 µL of steroid assay buffer (1 L of 0.05 M phosphate-buffered saline, 1 g of gelatin (type A: from porcine skin; Sigma), and 1 g of sodium azide). 464

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Gonadal Histopathology. Mid-body transverse sections were taken from whole, fixed roach by cutting off both the head and the tail either side of the dorsal fin. Blocks (3-5 mm) of the mid-body sections were then processed and embedded in paraffin wax. The samples were sectioned at 5 ( 1 µm, mounted, stained with Harris’ hematoxylin and eosin, and examined by light microscopy. The gonads from roach aged 350 dph (after depuration) were removed and examined macroscopically to determine sex and fixed in Bouins for 6 h prior to transfer into 70% industrial methylated spirits. The fixed gonads were subsequently dehydrated and three 3-5-mm transverse sections were cut, one each from the anterior, mid, and posterior portions of the gonad. The resulting six portions of gonad (three from each gonad) were embedded in paraffin wax, sectioned, and stained as described above. Gonadal sections from phenotypic males were examined for the presence of developing eggs (oocytes) in the testis and female-like reproductive ducts (ovarian cavities) (3). Phenotypic female gonads were assessed for any developmental abnormalities. Measurement of Natural and Synthetic Estrogens and Alkylphenolic Chemicals in the Full-Strength Treated Sewage Effluent. Coincident with the sampling of the fish, the full-strength treated sewage effluent was analyzed for the presence of natural and synthetic estrogens and alkylphenolic chemicals. Samples (5 L) of full-strength treated sewage effluent were collected to coincide with the times for biological sampling. The treated sewage effluent was analyzed for the presence of the natural steroids 17β-estradiol and estrone, the synthetic estrogen 17R-ethynylestradiol, and the alkylphenolic chemicals 4-octylphenol, 4-nonylphenol, and the nonylphenol mono- and diethoxylates. The methodologies used to measure these chemicals are described in refs 20 and 21. Briefly, the estrogenic chemicals were immobilized on a C18 silica-bonded solid-phase extraction column, eluted, and analyzed by GC/MS. Statistical Analyses. All analyses were carried out using SigmaStat V 2.0 (Jandel Scientific). Statistical significance was accepted at p ) 0.05, for all comparisons. Data sets found to lack variance homogeneity or not to have a normal distribution were either log-transformed prior to parametric analysis, or analyzed using nonparametric tests. Betweentreatment comparisons were carried out for all measured parameters, either by a one-way ANOVA (parametric) or Kruskal-Wallis test (nonparametric, for data that could not be normalized). Subsequent multiple comparison tests (where appropriate) were carried out using the StudentNewman-Keuls post-hoc test or using Dunn’s method (for parametric or nonparametric data, respectively). Possible relationships between VTG titer, treated sewage effluent concentration, and longevity of exposure were investigated using a multilinear regression analysis. The presence or absence of histopathological parameters (e.g., the presence of an ovarian cavity) was compared between treatments by χ2 analysis.

Results Establishing the Period of Gonadal Cellular Sexual Differentiation in the Roach. Histological analysis demonstrated that roach gonads were undifferentiated at 50 dph and that gonadogenesis (development of the reproductive ducts) occurred predominately between 50 and 150 dph. Female germ cell differentiation (the appearance of primary oocytes) took place between 100 and 200 dph, while male germ cell differentiation (the appearance of spermatogonia) was not apparent in the majority of males even at 200 dph. Concentrations of Natural and Synthetic Estrogens and Alkylphenolic Chemicals in the Full-Strength Treated Sewage Effluent. The results of the chemical analyses are summarized in Table 1. The natural estrogens, 17β-estradiol

TABLE 1. Measured Concentrations of Steroidal and Alkylphenolic (ng/L) Estrogens in Full-Strength (100%) Treated Sewage Effluent time (days exposure)

estrone

estradiol

ethynylestradiol

octylphenol

nonylphenol

nonylphenol mono + diethoxylates

0 50 100 150 mean ( sem

32 27 56 34 37 ( 6.5

4.0 8.8 4.4 6.3 5.9 ( 1.1

0.05). Whole-Body Vitellogenin Concentrations. After depuration in tap water for 150 days, whole-body VTG concentrations in roach previously exposed to full-strength effluent remained elevated as compared to controls (p < 0.001). The VTG concentration in the 100% effluent treated group was however significantly lower than it was at 200 dph (prior to depuration; p < 0.001; Figure 5). VTG concentrations in female and male roach from the control treatment were 310 ( 240 and 80 (

10 ng/mL, respectively. In fish previously exposed to fullstrength effluent, VTG concentrations were 1830 ( 520 and 1890 ( 540 ng/mL in feminized males (intersex) and females, respectively. Gonadosomatic Index (GSI). There were no differences in the GSI in either male or female roach previously exposed to treated sewage effluent as compared to control fish (p > 0.05). Gonadal Histopathology. Male fish had not completed sexual differentiation by the end of the effluent exposure period (200 dph); hence, the interpretation of the depuration study is complicated. At the end of the experiment (350 dph), after 150 days depuration, all roach from both the controls and the 100% effluent exposed fish had completed sexual differentiation such that their phenotypic sex could be ascribed. In the controls, phenotypic male and female gonads appeared histologically normal. Gonads in control male fish possessed a single sperm duct (vas deferens) with one point of attachment to the mesentery and spermatogonia A (and in some individuals, spermatogonia B germ cells) in typical cysts surrounded by somatic cells (presumptive sertoli and Leydig cells; Figure 6A). Gonads in control female fish possessed an ovarian cavity with two points of attachment to the mesentery, and primary oocytes (at the perinucleolar stage) and, occasionally, oogonia were observed in the interstices between the primary oocytes (Figure 6B). At 350 dph, despite 150 days depuration, all fish that had been exposed to full-strength treated sewage effluent (to 200 dph) and thus possessed female-like reproductive ducts retained these ducts regardless of germ cell sex (Figure 7; p < 0.001). The phenotypic males had a female-like ovarian cavity in an otherwise “normal” looking testis (containing spermatogonia and somatic cells as described for control fish; Figure 6C,D). In the controls, 70% of the population were normal males, but there were no normal males in the fish exposed to 100% effluent (all fish were either female or intersex). Females previously exposed to treated sewage effluent were indistinguishable from control females (Figure 6E). The percentage of phenotypic females was not significantly different between treatments (in the controls, 30% of individuals were females; while in the effluent exposed roach, 38% were females; p > 0.05; Figure 7).

Discussion Sexual differentiation and development in the roach appears to be a two-stage process common to many fish species where gonadogenesis precedes gametogenesis. In control roach, the formation of the female reproductive duct (ovarian cavity) took place between 50 and 150 dph, female germ cell differentiation (oogenesis) took place between 100 and 200 dph, and discernible (at the level of the light microscope) male germ cell differentiation (spermatogenesis) took place between 200 and 350 dph. These experiments clearly demonstrated that exposure of juvenile roach to treated sewage effluent from 50 to 200 dph resulted in the feminization of their reproductive ducts, while not obviously affecting development of the germ cells. The appearance of female-like reproductive ducts was shown to be dose-related, increasing with increased concentration of treated sewage effluent. After 100 days exposure (150 dph), the threshold concentration for inducing an effect on duct development was 23.6% effluent. Depuration of the roach previously exposed to full-strength treated sewage effluent in clean water for 150 days did not alter the incidence of female-like reproductive ducts, suggesting that the effect on the ducts was permanent. Female-like reproductive ducts have been reported in wild roach and gudgeon in U.K. rivers, and the incidence of these female-like reproductive ducts was strongly correlated with exposure to sewage effluent discharges (3, 4). The results of VOL. 35, NO. 3, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Gonadal histopathology of 350-day-old roach after exposure to 100% effluent followed by depuration: (A) control male, (B) control female, (C and D) feminized male, (E) effluent-exposed female. M, mesentery; T, testis; SD, sperm duct; S, spermatogonia; OV, ovary; OC, ovarian cavity; PO, primary oocyte; O, oogonia. Arrows mark point(s) of attachment of the gonad to the mesentery [100×]. previous data where it was established that exposure of maturing, adult roach to the same effluent did not induce the feminization of the reproductive ducts, even after 4 months continual exposure (20).

FIGURE 7. Percentage of female-like reproductive ducts and female germ cells in control roach and in roach previously exposed to 100% effluent, following depuration (350 dph). Asterisks refer to a comparison within a treatment: ***, p < 0.001. our study strongly suggest that female-like reproductive ducts found in wild intersex fish result from exposure of early-life stage roach (approximately between 50 and 100 dph) to treated sewage effluent. This hypothesis is supported by 468

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It is important to point out that the functional significance of the female-like reproductive ducts in feminized males is not known. The ovarian cavity in female fish has a multiplicity of functions. It has phagocytotic activity and contains many ciliated cells that direct the eggs along the oviduct toward the genital pore; microvilli; and a secretory epithelium function to maintain the ionic gradient of the ovarian fluid and the secretion of pheromones (22). In contrast, in males, the sperm duct stores spermatozoa (prior to release; 23) and contains steroidogenic cells that line the epithelium of the sperm duct (24). Exposure to treated sewage effluent also resulted in an acceleration of sexual differentiation. In fish exposed to fullstrength (100%) treated sewage effluent, formation of femalelike reproductive ducts occurred between 50 and 100 dph, while in controls, it occurred between 50 and 150 dph. Similarly, female germ cell differentiation appeared to be accelerated in fish maintained in the full-strength treated

sewage effluent for 100 days (assessed at 150 dph). After 150 days depuration (350 dph), however, there were no differences in the proportions of fish with female germ cells between the control and effluent exposed fish, indicating that germ cell development was precocious in the effluent exposed fish. The explanation for the accelerated rate of sexual differentiation observed in the effluent exposed roach is not known. The precocious development may have been due to the estrogens present in the effluent and/or the greater nutritional content of the effluent; these fish were also larger than the controls at 100 dph but not after 150 or 200 dph (100 or 150 days exposure). It has been reported previously that exposure to steroidal estrogens (e.g., 17R-ethynylestradiol) can affect the rate of sex differentiation in fish (25). As expected, VTG production was induced in the juvenile roach exposed to the treated sewage effluent, demonstrating that the effluent was estrogenic. After 50 days exposure (100 dph), the induction of VTG was dose-dependent, increasing with increasing concentration of treated sewage effluent, a finding in accordance with previous studies on this effluent (20, 26). After a further 50 days exposure (150 dph) however, the induction of VTG no longer appeared to be dose-related. Indeed, while concentrations of VTG in the 100% treated sewage effluent exposure group increased, whole-body concentrations of VTG in the fish exposed to 47.4, 23.6, and 12.0% effluent remained unchanged. Moreover, at day 100 of the study, concentrations of VTG in the fish exposed to river water alone were significantly elevated as compared to the concentrations of VTG at 50 days exposure. These data suggest that low concentrations of estrogens were present in the river water, which after a prolonged exposure (100 days) were sufficient to cause a significant induction of VTG. Previous studies demonstrated that the river water was not estrogenic to caged, adult rainbow trout (as determined by VTG induction, following a 3-week exposure; 26) or to adult roach (exposed for up to 4 months; 20). The reason for this difference is not known, but for the previous exposure studies the estrogenicity of the river water may have differed and/or juvenile fish (as used in the present study) may be more sensitive to estrogens as compared with adult fish. After 100 days exposure, concentrations of the female sex steroid 17β-estradiol (E2) were elevated in the fish exposed to 100, 47.4, and 23.6% effluent relative to the controls. It is possible that the fish in these treatments were sexually precocious (due to the accelerated sexual development described earlier); hence, the females within these groups may have started producing endogenous E2. It is also possible that exogenous estrogens present in the effluent entered and concentrated in the fish (natural and synthetic estrogens have been shown to bioconcentrate in fish, see ref 27). The precise chemical or chemicals responsible for the female-like reproductive duct formation are unknown. Female-like reproductive ducts have been induced in common carp exposed (during sexual differentiation) in the laboratory to estrogens, including both E2 and tert-pentylphenol (16), albeit at concentrations far higher than that found in the environment. Concentrations of DDT measured in bird eggs, from some heavily polluted areas, have been shown to be sufficient to cause development of a femalespecific oviduct in males exposed to DDT in the laboratory (28). Given the concentrations of estrogens in the effluent and the known effects of estrogens on reproductive duct development, it seems likely that an estrogenic chemical or chemical cocktail caused the induction of the female-like reproductive ducts in genotypically male fish. No gonads of fish exposed to treated sewage effluent contained both male and female germ cells; therefore, we have yet to determine when and how affects on germ cell differentiation are effected in wild intersex fish. It is possible that the effluent was not sufficiently potent or appropriate

(in chemical composition) to induce oocyte development within testes. It is also possible that exposure of the roach was not conducted sufficiently early during development to induce these effects [although the roach were sexually undifferentiated (histologically) when the exposure began, the genetic programs that determine sexual differentiation may operate earlier in development]. A further possibility is that the exposure may not have been sufficiently long to effect the development of the germ cells. In conclusion, exposure of juvenile roach to treated sewage effluent resulted in dose-dependent and persistent feminization of the reproductive ducts in male fish. It is thus likely that wild male roach with female-like reproductive ducts are the result of exposure to treated sewage effluent during earlylife. No effects were seen on germ cell differentiation. Therefore, the cause of oocytes in the testes of wild roach remains to be elucidated.

Acknowledgments T.P.R.-G. was funded by the U.K. Environment Agency, and S.J. was supported by a Natural Environment Research Council Grant to C.R.T. and J.P.S. We would like to thank other members of the Fish Physiology Research Group at Brunel University for their help during field sampling and Alan Henshaw of the U.K. Environment Agency, Calverton, U.K., for his help and advice in maintaining the fish. We gratefully acknowledge the use of the effluent exposure facilities provided by Essex and Suffolk Water and the technical assistance of Kathryn Walls.

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Received for review April 28, 2000. Revised manuscript received October 24, 2000. Accepted October 25, 2000. ES001225C