THE SHEEPSHEAD MINNOW AS AN IN VIVO MODEL FOR ENDOCRINE DISRUPTION IN MARINE TELEOSTS: A PARTIAL LIFE-CYCLE TEST WITH 17 -ETHYNYLESTRADIOL

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1 Environmental Toxicology and Chemistry, Vol. 2, No. 9, pp , SETAC Printed in the USA /1 $9.. THE SHEEPSHEAD MINNOW AS AN IN VIVO MODEL FOR EOCRINE DISRUPTION IN MARINE TELEOSTS: A PARTIAL LIFE-CYCLE TEST WITH 17 -ETHYNYLESTRADIOL EDWARD J. ZILLIOUX,* ISABEL C. JOHNSON, YIANNIS KIPARISSIS, CHRIS D. METCALFE, JEFF V. WHEAT, SCOTT G. WARD, and HUI LIU Florida Power and Light, 7 Universe Boulevard, Juno Beach, Florida 338, USA Golder Associates, 621 N.W. 23rd Street, Suite 5, Gainesville, Florida 32653, USA Environmental and Resource Studies Program, Trent University, Peterborough, Ontario K9J 7B8, Canada Toxikon Environmental Sciences, 16 Coastal Way, Jupiter, Florida, 3377, USA (Received 21 June 2; Accepted 17 January 21) AbstractThe sheepshead minnow (Cyprinodon variegatus Lacépède), an estuarine fish species, was exposed to 17 -ethynylestradiol (EE2) at nominal test concentrations of.2, 2, 2, 2,, 8, 1,6, and 3,2 ng/l. Fish were exposed for up to 59 d, from subadult stages to sexual maturity, under flow-through conditions. The exposure period was followed by an evaluation of reproductive success and survival of progeny. The reproductive success of exposed sheepshead minnows, as determined from data on egg production from two subsequent spawning trials, was reduced in fish exposed to 2 ng/l EE2 and, in one spawning trial, in the 2-ng/L treatment. Hatching success was reduced in the progeny of fish exposed to 2 ng/l EE2, but survival was good among fry that successfully hatched. Histological examination indicated generalized edema, damage to gill epithelia, hepatic toxicity, fibrosis of the testis, and evidence of sex reversal, including testes ova and spermatagonia-like cells in ovaries. The maximum acceptable toxicant concentration (MATC) for gonadal development in males was within the normal range of EE2 concentrations in sewage treatment plant effluents. The exposure regimen and choice of test organism, combined with histological examination, allowed independent evaluation of ecologically significant acute, reproductive and estrogenic endpoints. Estrogen receptor mediated effects occurred at concentrations where reproductive effects were measurable under standard reproduction assays. The sheepshead minnow appears to be a sensitive in vivo model for partial life-cycle testing of compounds that have the potential to disrupt the endocrine system as well as reproduction in estuarine and coastal marine fish species. KeywordsEndocrine disruption Estrogen Ethynylestradiol Reproductive effects Sheepshead minnow INTRODUCTION Concern is growing that many chemicals may disrupt the endocrine system through estrogenic effects at environmental concentrations much lower than those causing acute or chronic toxicity. Thus, a need exists for in vivo test methods based on definitive indicators for estrogenic effects that are distinct from typical endpoints used to measure toxic responses. Further, it is desirable that these tests show a direct cause-and-effect relationship for endpoints of ecological concern such as reproductive success. Endocrine disruptor compounds (EDCs) are substances thought to mimic, alter, or inhibit the action of endogenous hormones. It has been assumed that hormonal responses to xenobiotics, such as alterations to levels of sexrelated steroids or induction of vitellogenin synthesis in males, also cause effects of ecological concern. However, little direct evidence exists to establish these links [1]. Several classes of compounds have been shown to modulate endocrine responses, alter sexual development, or reduce reproductive success in fish, including alkylphenols [2], organochlorine pesticides [3,], endogenous estrogens [5,6], and phytosterols [6]. However, many xenobiotic substances have not been tested in in vivo assays to determine whether they are potential EDCs. Effects have been observed in fish populations that have been attributed to exposure to endocrine modulating substances. These effects include synthesis of vi- * To whom correspondence may be addressed (ed zillioux@fpl.com). tellogenin and reduced serum testosterone in male carp (Cyprinus carpio) captured near sewage treatment plants [7], a high prevalence of intersex gonads in roach (Rutilus rutilus) collected near sewage treatment plants [8], vitellogenin induction and intersex development in flounder (Platichthys flesus) captured in contaminated estuaries in the United Kingdom [9], altered serum steroid levels and delayed gonadal maturation in lake whitefish (Coregonus clupeaformis) near pulp mills [1], and masculinization of mosquitofish (Gambusia affinis) in streams downstream of pulp mills [11,12]. The U.S. Environmental Protection Agency s Endocrine Disrupter Screening and Testing Advisory Committee (ED- STAC) has recommended a suite of in vitro and in vivo screening assays to evaluate large numbers of chemicals for endocrine-disrupting activity. Two general requirements necessary to establish an EDC screening test acceptable for regulatory use are validation and standardization. An EDC is important to human or environmental health only to the extent that it may cause harm to normal functioning of organisms or populations. To achieve validation, therefore, an EDC indicator (e.g., MCF-7 breast cell proliferation, sex steroid or vitellogenin induction, or change in gonadal size) used in a screening test must be shown to be relevant to an endpoint of direct human or ecological concern. Standardization generally requires interlaboratory comparisons to demonstrate that the candidate test is reproducible between laboratories. At this time, no EDC screening test has met these requirements. Short- and long-term in vivo assays with primarily freshwater fish species 1968

2 Estuarine fish model for assessing estrogenic effects Environ. Toxicol. Chem. 2, have been recommended by the EDSTAC and proposed by the U.S. Environmental Protection Agency [13], but to date no in vivo screening methods have been identified and validated that are relevant to exposure of estuarine or marine fish to EDCs. Since many potential EDCs are likely to be released directly into estuarine or marine ecosystems (including pesticides and chemicals in oil dispersants, marine antifouling paints, and drilling muds), a need exists to develop in vivo screening tests for EDCs using estuarine or coastal marine teleost species. The goal of this work was to develop an assay for EDCs applicable to estuarine environments that would combine markers of hormonal effects with whole organism measurable effects of ecological significance. The study was designed to evaluate a partial life-cycle assay for EDCs with the sheepshead minnow (Cyprinodon variegatus Lacépède), an estuarine fish species, by exposing sexually immature fish to several aqueous concentrations of the synthetic estrogen 17 -ethynylestradiol (EE2). Our research design set forth the following three objectives: determine effects of an estrogenic chemical (17 -ethynylestradiol) on survival and reproduction of the sheepshead minnow under flow-through conditions during a partial life-cycle exposure; estimate the no-observed-effect concentration (NOEC), the lowest-observed-effect concentration (LOEC), and the maximum acceptable toxicant concentration (MATC) of EE2 for the sheepshead minnow based on biological effects endpoints such as survival, reproductive success, and histopathological effects; and develop an estuarine model for assessing threshold estrogen-mediated effects. Selection criteria for the test species included ease of culture, abundance in estuaries, a large toxicological database, and moderate sensitivity to toxicants so that acutely toxic effects generally would not be exhibited near toxicant concentrations at which thresholds for reproductive or estrogenic effects occur. The sheepshead minnow fits these criteria and also has been shown to be useful in developing assays for the induction of sex steroids [1]. In order to expand an existing biological assay (sheepshead minnow early life-cycle assessment), it was critical to first evaluate whether the proposed species was sensitive to and produced measurable effects from EDC exposures. The test chemical, EE2, was selected because it is a potent estrogen; it has been used previously as a positive control for aqueous exposures of fish [5,15]; it has been detected in samples of sewage and surface water in Europe, Canada, Brazil, and the United Kingdom [16 18]; and it has been implicated in the endocrine responses noted in fish downstream of discharges from sewage treatment plants (STPs) [8]. Vitellogenesis has been shown to be induced by EE2 in roach, carp, and male and female rainbow trout [18 2] when administered at very low concentrations in the test water supply. For commercial purposes, EE2 is produced as the principal active ingredient in the common oral contraceptive pill; it has been reported to occur in sewage treatment plant effluents up to 7 ng/l [16] and in water bodies receiving domestic effluents up to approximately 7 ng/l [18]. METHODS Fish Sheepshead minnows (Cyprinodon variegatus Lacépède) in the subadult stage, as indicated by lack of secondary sexual characteristics and confirmed through histological examination of the gonad, were obtained from AquaTox (Hot Springs, AR, USA) and were acclimated to standard conditions (16:8-h light: dark photoperiod, 2 25 salinity, and C temperature) for 1 d prior to test initiation. Fish were fed a commercial flake food (Zeigler Brothers, Gardners, PA, USA) throughout the acclimation period. The general health of the animals appeared good, and no evidence of fungal infection was observed during this period. Dosing The test substance, EE2, was obtained from Sigma Chemical Company (St. Louis, MO, USA [lot 5H716]). Nominal concentrations of EE2 were.2, 2., 2, 2,, 8, 1,6, and 3,2 ng/l, in addition to controls and solvent (triethylene glycol) controls. This exposure series was selected based on an earlier range-finding experiment that identified acutely toxic concentrations. Tests with the three lowest concentrations were conducted in a continuous flow-through exposure system in which test solutions were injected directly into test chambers. Tests at higher concentrations were conducted under flowthrough conditions using an intermittent flow-proportional vacuum siphon diluter system based on the design of Mount and Brungs [21]. Both exposure systems were constructed of glass, silicone adhesive, and silicone tubing and were calibrated to provide approximately nine water turnovers per day. Both systems were operated with EE2 for a period of at least 2 h prior to the introduction of fish in an attempt to saturate all adsorptive surfaces and minimize the in-test loss of test substance. The dilution water was natural saltwater pumped from a shallow well and treated by filtration with activated carbon, adjusted to a salinity of approximately 2 with carbon-filtered freshwater, and aerated vigorously prior to use. The dilution water was filtered through a 2- m filter prior to delivery into the diluter system. A primary stock solution of EE2 (27.7 mg/l, nominal), dissolved in triethylene glycol solvent, was prepared and delivered to the diluter for proportional delivery of treatments. The solvent control treatment delivered the same volume but contained only the solvent. The test substance stock was injected into the chemical mixing chambers of the dilution system at a rate of 3 l at each diluter cycle. Dilution water was added proportionally to deliver the appropriate concentrations to the test chambers in volumes of approximately 1, ml for each cycle of the diluter. In the case of the three lowest concentrations, stock solutions of EE2 dissolved in the solvent were prepared at appropriate concentrations (2.63,.263, and.263 mg/l, respectively) and were injected by a syringe pump using 6-ml plastic syringes at a rate of.78 l/min into filtered dilution water that was continuously flowing at a rate of 1 ml/min. Solvent concentrations were the same ( 7.8 l/l) in the solvent control and all test substance treatments. Stock solutions of EE2 were prepared weekly. The tests were conducted using duplicate test chambers consisting of 2-L glass tanks equipped with overflow tubes to maintain test chamber volumes of approximately 15 L of gently aerated test water. The test chambers were randomly positioned in water baths for control of temperature, which was maintained at 25.6 to 3.2 C throughout the F phase of the exposure. The photoperiod was maintained at 16 h light: 8 h dark with fluorescent lamps delivering a light intensity range of approximately 3 to 55 lux.

3 197 Environ. Toxicol. Chem. 2, 21 E.J. Zillioux et al. Fig. 1. Schematic of sheepshead minnow partial life cycle protocol showing exposure period and test duration and toxicological and histological endpoints. Sacrifices for histological analyses occurred at exposure day 17 for 1,6- and 3,2-ng/L treatments, day 2 for 8-ng/L treatment, day 3 for -ng/l treatment, and days 3, 57, and 73 for controls, solvent controls, and.2-, 2.-, 2-, and 2-ng/ L treatments. Spawning groups were set out at days 3 and 59. Experimental Fish were exposed for up to 59 d through subadult stages to sexual maturity at several concentrations of the test compound under flow-through conditions, followed by evaluation of standard reproductive success endpoints in addition to histological endpoints for toxicopathic and estrogen-mediated developmental effects. The experiments were conducted in two phases: the F phase, which included exposure of subadults and reproductive trials, and the F 1 phase, which included evaluation of fry hatched during the experiments. Figure 1 presents schematically the two phases, including exposure period and test duration, toxicological and histological endpoints, and event days, including initiation of spawning groups and sampling for histological analyses. Fish were fed daily. The diet consisted of commercial flake food during the initial acclimation. Following the acclimation period, the diet was changed from commercial flake food to frozen brine shrimp, which contained a higher lipid content. This dietary change increased the reproductive potential of the fish as they approached sexual maturity. Mortalities were monitored and dead fish removed daily. The F phase was initiated with continuous exposures of sheepshead minnows from the subadult stage of development through sexual maturity. Subadult minnows were selected at random from acclimation tanks and placed in duplicate test chambers (n 2 per chamber). A subsample of fish from the acclimation tanks were sacrificed at test initiation and examined histologically to confirm sexual immaturity. Any fish in the highest exposure groups (1,6 and 3,2 ng/l) remaining at day 17 were sacrificed for histological analysis since major toxic effects were observed at these concentrations. Treatments of minnows at all other exposure concentrations continued undisturbed and were monitored for survival and development of secondary sexual characteristics. A portion of sexually mature fish, recognizable by development of secondary sexual characteristics, were removed at day 3 of exposure from the exposure chambers and placed in spawning groups for the first reproductive trial. Fish not selected for the first reproductive trial continued in exposure chambers to day 59; at that time, the second reproductive trial was initiated. A subsample of exposed fish were sacrificed and fixed for histological analysis just prior to initiation of each reproductive trial. Any remaining fish were placed in dilution water alone until termination of the experiment on day 73, at which point all remaining fish were sacrificed for histological analysis. Length and weight measurements also were taken of fish sacrificed at days 3, 57, and 73. Reproductive trials consisted of one to two spawning groups for each treatment; each spawning group included two males and five females. Two reproductive trials, each lasting 1 d, were conducted during this study. The first reproductive trial included two replicate spawning groups per treatment; the second reproductive trial consisted of one spawning group per treatment. Spawning groups were placed in spawning chambers consisting of 1-L polyethylene pails with their bottoms removed and 7-mm plastic mesh attached to the bottom with silicone sealant. Each pail was placed inside another pail with 355- m Nitex mesh (Sefar, Depew, NY, USA) on the bottom. The spawning chambers were suspended in tanks and supplied with clean dilution water in intermittent cycles from a diluter system. These spawning chambers allowed the fertilized eggs to drop through the 7-mm mesh for easy retrieval from the second pail with the smaller mesh. Adult minnows were fed frozen adult brine shrimp three times daily throughout the reproductive trials. On a daily basis, the spawning chambers were removed and examined for eggs that filtered through the 7-mm mesh into the second pail. The number of eggs was recorded, and any abnormalities were noted. At the end of each 1-d reproductive trial, the adult fish were sacrificed, length and weight measurements were recorded, and the fish were fixed for histological evaluation. Reproductive success was evaluated according to the number of eggs produced per female reproductive day, determined by dividing the total number of eggs produced during each reproductive trial by the number of female reproductive days (i.e., 5 1 7). Egg hatchability and fry survival were monitored during the F 1 phase of the study. Newly spawned fertilized eggs were collected from each spawning group and placed in incubation chambers suspended in tanks supplied with dilution water through a diluter system as described for the reproductive trials. An attempt was made to collect a minimum of 5 eggs from each spawning group, but this was not possible in all treatments. Hatching success was monitored for 8 d, after which unhatched eggs were considered nonviable and discarded. Hatched eggs were kept in the same dilution water system and monitored for 7 additional days to determine fry survival. Newly hatched fry were fed two to three times daily with live, fatty acid supplemented brine shrimp nauplii obtained from Aquarium Products (Glen Burnie, MD, USA). Chemical monitoring Water temperature, salinity, ph, and dissolved oxygen were monitored throughout the study. Temperature was monitored hourly in the dilution water control with a thermister and data logger and continuously in the water bath with a minimum/ maximum thermometer. Salinity was measured daily in the dilution water control with an Aquafauna (Hawthorne, CA, USA) salinity refractometer. Dissolved oxygen (YSI Model 58 dissolved oxygen meter) and ph (Fisher Accumet 12 ph

4 Estuarine fish model for assessing estrogenic effects Environ. Toxicol. Chem. 2, meter, Fisher, Pittsburgh, PA, USA) were measured weekly in all treatments. Water samples were collected for EE2 analysis during the F phase from the control, solvent control, and all treatments at 2 ng/l and higher, beginning on the day before test initiation and continuing on a weekly basis until termination of exposures. Each 1,-ml control or treatment water sample was a composite of 5-ml samples collected from each of two replicates. Water samples were extracted with solid-phase extraction. Prior to sample extraction, C 18 solid-phase extraction cartridges (J.T. Baker BAKERBO, Hayward, CA, USA) were rinsed with two 3-ml volumes of high-pressure liquid chromatography (HPLC)-grade methanol followed by two 3-ml rinses with HPLC-grade deionized water using a vacuum manifold. The 1-L samples were then applied to the cartridges. The cartridges were not allowed to go to dryness during any of the previous stages. After the sample volume passed through each cartridge, coextractives were eluted with a solution of 2:8 HPLC-grade acetonitrile (ACN):H 2 O. Cartridges were then dried under vacuum for 1 min. The EE2 was eluted with a volume of 1 ml of methanol from the prepared cartridges into graduated vials. Following extraction, samples were concentrated under nitrogen to bring samples within the calibration range before analysis by HPLC. Because of the low EE2 concentrations in the.2-, 2.-, and 2-ng/L treatments, only the stock solutions were analyzed to confirm the respective dosing concentrations. Prior to HPLC analysis, the stock solutions were diluted or concentrated to bring the final injected sample into the calibration range for EE2. The HPLC analysis used the following chromatographic conditions: Zorbax RX-C 18 (5- m particle size); mm HPLC column; Shimadzu SPD-1AV ultraviolet/visible light detector (Shimadzu, Kyoto, Japan) operated at a wavelength of 28 nm; Shimadzu LC-6 HPLC pump (calibrated daily with external standard); HP 3396 series II integrator; Waters 71B HPLC autosampler (operated at an injection volume of 5. l); mobile-phase 35:65 ACN:H 2 O (flow rate 1.5 ml/min); standard calibration range 25 1,5 g/l; and a run time of 1 min. The HPLC-grade methanol and water were obtained from J.T. Baker Chemical (Phillipsburg, NJ, USA). The HPLC-grade ACN was obtained from J.T. Baker and Fisher Scientific (Pittsburgh, PA, USA). Histological evaluation Fish sampled prior to testing and on exposure days 17, 2, 3, 57, and 73 of the F phase were sacrificed by an overdose of MS-222 anaesthetic. Physical condition of all fish was noted. Fish sacrificed on days 3, 57, and 73 were measured for length and weight. The tails of the fish were removed with a scalpel, and each fish was placed in a tissue capsule and fixed in Calex decalcifying fixative (Fisher Scientific, Toronto, ON, Canada) for 8 h before washing in tap water and transferring to 7% ethanol. Prior to embedding, fish were removed from ethanol, and the posterior part of the fish was removed to the anal papilla with a scalpel. Fish were blocked in paraffin, sectioned longitudinally with a microtome (7- m sections), and stained with hematoxylin and eosin using standard procedures. Typically, four to eight sections were prepared per fish to ensure that a range of tissues, primarily the gonad, liver, kidney, and gill, could be evaluated during histological examination. In evaluating the condition of the ovaries of sheepshead minnows, the stages of oogenesis were classified as previtellogenic (early or late), vitellogenic (early or late), and postvitellogenic phases, according to a classification scheme developed for Japanese medaka (Oryzias latipes) by Iwamatsu et al. [22]. Classification criteria in this scheme include the size, appearance, and staining characteristics of the oocyte as well as the appearance of the granulosa cells in the surrounding ovarian follicle. Atresia of oocytes was recognized by the separation of the egg nucleus from the ooplasm and also by separation of the ooplasm from the oocyte membrane. In the testes, the stages of spermatogenesis were classified according to the presence of undifferentiated spermatogonia, spermatocytes, spermatids, and mature spermatozoa. General appearance and other morphological observations were recorded for all tissue sections evaluated. Data analysis Percentile data were analyzed using Fisher s Exact Test. Continuous data were analyzed using parametric analysis of variance of untransformed data, followed by Dunnett s multiple comparison test. Maximum acceptable toxicant concentrations for EE2 were calculated for toxicological endpoints as the geometric mean of the LOEC and the NOEC. F phase RESULTS Survival and growth. Survival observed in controls throughout the 59-d exposure period was 95%, while survival in the solvent controls was 97.5%. Measured concentrations of EE2 in the exposure chambers for nominal treatments of 1,6 and 3,2 ng/l varied between 91 and 16% of nominal. Results from these highest concentrations are of interest in documenting the acutely toxic effect at these dose levels; however, poor survival precluded obtaining data on reproductive effects at these concentrations. At day 17, when all fish at these concentrations were sacrificed for histological examination, survival was only 35% in the 1,6-ng/L treatment and 27.5% in the 3,2-ng/L treatment. Measured concentrations at nominal treatments of 2,, and 8 ng/l of EE2 varied between 3 and 1% of nominal, with the concentrations in the 2-ng/L treatment being consistently lower than expected (Table 1). The exposure concentrations for this toxicological study were calculated as average measured concentrations. For the controls and solvent controls, EE2 concentrations were below detection limits in all samples. In the three lowest treatments,.2-, 2.-, and 2-ng/L nominal concentrations of EE2, the stock solutions that were injected into test chambers were quite consistent and varied between 73 and 18% of the nominal values. Exposure concentrations in these test chambers could not be directly quantified by HPLC analysis at such low EE2 concentrations. Over the exposure period, temperatures in the test chambers were maintained between 25.6 and 28.7 C, and measured salinities were between 2 and 21, except for one day when salinity rose to 28 because of an equipment malfunction. Dissolved oxygen concentrations in test chambers were between.7 and 6.7 mg/l; ph varied between 7.8 and 8.. Data on the total cumulative survival of fish through the exposure period indicated that survival was severely reduced in the treatments at nominal concentrations of and 8 ng/l (5 and 3% survival, respectively, at day 2), leaving

5 1972 Environ. Toxicol. Chem. 2, 21 E.J. Zillioux et al. Table 1. Concentrations of EE2 measured in samples from the exposure chambers for the control, solvent control, and EE2 nominal exposure treatments of 2,, and 8 ng/l and in samples from stock solutions for EE2 nominal exposure treatments of.2, 2., and 2 ng/l (values shown for.2-, 2.-, and 2-ng/L treatments are the calculated final exposure concentrations). Samples were collected for analysis at approximately weekly intervals, beginning the day before exposures began (D-1) and extending to day 56 (D56). Concentrations are presented as ng/l and as % of nominal (in parentheses) Nominal concn. (ng/l) Measured concn. (ng/l, %) D-1 D7 D16 D21 D28 D35 D2 D9 D56 Mean Control Solvent control a.22 (112) 1.79 (91) 18.1 (92) 19 (55) 328 (82) 783 (98).22 (113) 1.93 (98) 18.1 (92) 128 (6) 268 (67) 85 (61).21 (19) 1. (73) 19.3 (98) 128 (6) 35 (89) 778 (97).28 (11) 2.3 (13) 2.3 (13) 13 (52) 315 (79) 693 (87).22 (11) 1.85 (9) 18.1 (92) 11 (71) 321 (8) 72 (93).23 (119) 1.56 (79) 17.1 (87) 91 (5) 17 (1) 75 (9).29 (18) 1.5 (76) 16.7 (85) 86 (3) 29 (7) 822 (13).26 (133) 1.73 (88) 17.5 (89) 121 (61) 329 (82) 731 (91).26 (135) 1.71 (87) 17.3 (88) 15 (73).2 (12) 1.73 (88) 18.1 (92) 117 (59) 328 (82) 723 (9) a not detected. insufficient numbers to include these treatments in the reproductive trials (beginning on day 3). Therefore, the remaining fish in these treatments were sacrificed for histological evaluation. In all other treatments (2-ng/L nominal and lower), no significant differences were observed in the survival of fish in EE2 treatments when compared to control and solvent controls (Fig. 2). Fish in the - and 8-ng/L nominal treatments showed evidence of distended abdomens prior to death; this was tentatively described as generalized edema. This observation was confirmed later by histological analysis. Data on the lengths and weights of minnows sacrificed at days 3, 57, and 73 of the F phase indicated that the fish increased in length and weight over this period. However, no statistically significant treatment differences were observed in length, weight, or length:weight ratios in fish in the.2- through -ng/l exposure treatments as compared to the controls and solvent controls (pooled). Fish from the 8-ng/L treatments were not measured because of high mortality and insufficient numbers for analysis. Histological analysis. All fish from treatments of 1,6- and 3,2-ng/L nominal EE2 concentrations, sacrificed at day 17 of exposure, showed massive generalized edema of the visceral cavity. Heavy eosinophilia of the edemic fluid obliterated cellular structures and made histopathological observations of the visceral organs and the gonads impossible. Cystlike structures were observed on the secondary gill lamellae, indicating a toxicopathic response within the gill epithelium. Fish exposed to EE2 at nominal concentrations of and 8 ng/l were sacrificed at days 3 and 2 of exposure, respectively; both treatments were terminated at this time because of excessive mortality. Fish exposed to 8-ng/L nominal EE2 concentration also showed eosinophilic edemic fluid in the visceral cavity and in extracellular spaces of all tissues, indicating generalized edema. Blood-filled cysts on the secondary gill lamellae and hyperplasia of gill epithelia (Fig. 3) were also observed on fish from this treatment. Hepatocytes were generally hyperbasophilic with little vacuolization, which may indicate loss of intracellular glycogen. Pronounced effects were observed in the gonads of male fish. Three male fish had testis ova, an Fig. 2. Survival (%) of sheepshead minnows in duplicate trials at test termination (day 73 for control [C] and solvent control [SC] and for treatments with.2, 2, 2, and 2 ng/l and days 3 and 2, respectively, for and 8 ng/l of EE2). Fig. 3. Blood-filled cysts (arrow) on the secondary gill lamellae of a sheepshead minnow exposed to 8 ng/l of EE2 hematoxylin and eosin (H&E), 1.

6 Estuarine fish model for assessing estrogenic effects Environ. Toxicol. Chem. 2, Table 2. Incidence of histological changes in the testis of sheepshead minnows sampled at day 57 and day 73 of the F phase of the experiment. Testicular changes included testicular fibrosis (F) and testis ova (TO). Fish were taken from exposure chambers for control and solvent control treatments and for treatments with EE2 at concentrations of.2, 2, 2, 2,, and 8 ng/l Treatment Number males scored Day 57 Day 73 Testicular changes Day 57 Day 73 Table 3. Incidence of histological changes in the ovaries of female sheepshead minnows sampled at day 57 and day 73 of the F phase of the experiment. Ovarian changes included evidence of sex-reversals (R) and atresia of oocytes (A). Fish were taken from controls and solvent controls and EE2 treatments at concentrations of.2, 2, 2, 2,, and 8 ng/l. Putative sex reversals were identified by the presence of spermatogonia in the ovary Treatment Number females scored (ovarian changes) Day 57 Day 73 Day 57 Day 73 Control Solvent control Estradiol.2 ng/l 2 ng/l 2 ng/l 2 ng/l ng/l b 8 ng/l b (F) 2(F) 1(F), 1(TO) 1(F), 3(TO) 3(F), 3(TO) 1(F) 1(F) 1(F) 6(F) 3(F), 1(TO) 1(F) a, 6(TO) Control Solvent control Estradiol.2 ng/l 2 ng/l 2 ng/l 2 ng/l ng/l a 8 ng/l a (A) 1(A) (R) 2(R) 1(R), 3(A) a Complete lack of spermatocytes (sterile male). b Because of mortalities, fish from - and 8-ng/L treatments were sacrificed at days 3 and 2 of exposure, respectively, rather than day 57. intersex condition characterized by small numbers of previtellogenic ovarian follicles distributed among the spermatocytic tissue. In all remaining male fish in this treatment, the testis were highly fibrotic. All female fish from this treatment had oocytes in advanced stages of oogenesis (postvitellogenic stage) and showed atresia of some postvitellogenic oocytes. Fish exposed to -ng/l nominal EE2 concentrations showed toxicopathic responses in extragonadal tissues, including pericardial edema, kidney pathology (such as dilation of Bowman s capsule), and blood-filled cysts on the secondary gill lamellae. Among treatments at lower concentrations of EE2, these effects were seen only in fish exposed to 2-ng/ L nominal EE2 concentrations; however, most fish from this treatment survived through the reproductive trials to the termination of the experiment at day 73. Fish from the.2-, 2.-, 2-, and 2-ng/L treatments, as well as control and solvent control fish, were sampled for histological analysis at day 3 (beginning of first reproductive trial), day 57 (2 d prior to end of exposure and beginning of second reproductive trial), and day 73 (termination of experiment). At day 57, fish from the control and solvent control and the three lowest EE2 nominal concentrations (.2, 2, and 2 ng/l) did not exhibit any toxicopathic lesions in extragonadal tissues (gill, liver, and kidney), except for one fish in the solvent control that had a small number of blood-filled cysts on the secondary gill lamellae. On day 3, fish from the control, solvent control, and the.2-ng/l EE2 treatment exhibited normal architecture and development of the gonad. Oocytes in the ovaries of these fish were often in the previtellogenic and vitellogenic stages of oogenesis, but with some fish in postvitellogenic stages. Germinal cysts in the testicular lobules of the testes of fish from the.2-ng/l treatment and controls were in all stages of spermatogenesis; however, no spermatozoa were observed. These observations are consistent with fish that are approaching breeding condition. Tables 2 and 3 summarize histological observations of sheepshead minnow testis and ova, respectively. Alterations to the architecture of the testis were observed in male fish exposed to 2, 2, 2,, and 8 ng/l of EE2. In two male a Because of mortalities, fish from - and 8-ng/L treatments were sacrificed at days 3 and 2 of exposure, respectively, rather than day 57. fish from the 2-ng/L treatment, mild fibrosis of the lobules of the testis was observed at day 57 and in an additional six fish at day 73. The severity of testicular fibrosis in males generally increased with EE2 concentration. Figure illustrates the highly fibrotic testis of a male fish from the 2-ng/L treatment. Fig.. Testis of sheepshead minnows (a) exposed to 2 ng/l of EE2 showing extensive fibrosis and (b) from a control chamber showing normal germ cells in various stages of development hematoxylin and eosin (H&E),.

7 197 Environ. Toxicol. Chem. 2, 21 E.J. Zillioux et al. Fig. 5. Testis of a sheepshead minnow exposed to 2 ng/l of EE2 showing testis ova hematoxylin and eosin (H&E), 1. However, very mild fibrosis of the testis was also observed in a single fish from each of the control and solvent control sampled at day 73. Several previtellogenic ovarian follicles were observed in one male fish exposed to 2 ng/l of EE2 at day 57, although only two were scored from this treatment. These ovarian follicles were distributed among the spermatocytic tissue. Six males of the seven scored on day 73 in the 2-ng/L treatment had ovarian follicles. Although the numbers of ovarian follicles present in the testes were generally low, this condition was classified as testis ova. In addition, there were three male fish with similar testis ova at days 3 and 2, respectively, in each of the - and 8-ng/L treatments (out of four and six scored, respectively). A small number of fish exposed to 2,, and 8 ng/ L of EE2 were identified as females by the presence of developing oocytes in the gonad. However, the sex of these fish was considered ambiguous because of the presence of clusters of spermatogonia in the ovary that were also often accompanied by ova in the previtellogenic stage of oogenesis. This condition was putatively identified as evidence of sex reversal in these fish (Table 3). Evidence of atresia of postvitellogenic ova was seen in females from the 2-, 2-, and 2-ng/L treatments (Table 3). The group of fish sampled at day 73 of the experiment included fish from the control and solvent control and fish from treatments with EE2 at concentrations of.2, 2, 2, and 2 ng/l. Only fish from the 2-ng/L treatment exhibited toxicopathic lesions in extragonadal tissues. This included mild dilation of the Bowman s capsule in the kidneys of most fish from this treatment and blood-filled cysts on the gill lamellae of two fish. Pericardial edema was noted in one fish from this treatment. Fatty liver and foci of vacuolated hepatocytes were observed in the liver of most fish from all treatments, but this was considered normal because of the presence of this condition in fish from controls. Oocytes were in the postvitellogenic stage of oogenesis in the control, solvent control, and all EE2 treatments. These observations are consistent with female fish in breeding condition. No alterations to the development or the architecture of the ovaries were observed in females from controls or the.2-, 2-, and 2-ng/L treatments sampled at day 73 (Table 3). Atretic postvitellogenic ova were seen in females from the 2-, 2-, and 2-ng/L EE2 treatments as well as evidence of sex reversal in females from the 2-ng/L treatment (sampled at day 73) and in females from the - and 8-ng/L EE2 treatments (sampled at days 3 and 2; Table 3). In most male fish from the control, solvent control, and.2-ng/l EE2 treatment, the condition of the testis was consistent with normal male fish that are in breeding condition. However, in each of the control, solvent control, and.2-ng/ L EE2 treatments, one male fish with mildly fibrotic testis was observed (Table 2). No obvious explanation exists for these results. Fibrosis of the testis was observed in many male fish exposed to 2, 2, and 2 ng/l of EE2 (Table 2). However, the examined severity of fibrosis in males increased with EE2 concentration, with sterile males observed at high exposure concentrations. For example, very severe fibrosis (highly vascularized, fibrotic testis with reduced numbers of spermatogonia) was observed in one of the males from the 2-ng/L treatment at day 73 (Fig. ). One male fish exposed to 2 ng/l of EE2 had a testis ova characterized by large numbers of previtellogenic ovarian follicles distributed among the spermatocytic tissue (Fig. 5). In the 2-ng/L treatment, the six male fish with obvious testis ova appeared to be almost completely feminized (Table 2). Female fish exposed to 2,, and 8 ng/l EE2 were identified as individuals that had undergone sex reversal because of the presence of clusters of spermatogonia cells in the ovary (Table 3). Spawning trials During the first reproductive trial, observations were made on duplicate spawning groups from the controls and solvent controls and treatments with.2-, 2.-, 2-, and 2-ng/L nominal EE2 concentrations. Not enough surviving fish were available to conduct spawning trials with fish from the - or 8- ng/l treatments. As shown in Figure 6, the number of eggs per female reproductive day was significantly reduced in the 2-ng/L treatment in both spawning groups in comparison to controls and solvent controls (pooled). In the second reproductive trial, where only one spawning group was available per treatment, there appeared to be reduced eggs per female reproductive day in both the 2- and 2-ng/L treatments (Fig. 6). Fig. 6. Eggs per female reproductive day (EPFRD) for spawning groups of sheepshead minnows previously exposed to EE2 in duplicate spawning groups in reproductive trial 1 and in a single spawning group in reproductive trial 2. Fish were taken from exposure chambers for control (C) and solvent control (SC) and for treatments with.2, 2, 2, and 2 ng/l of EE2.

8 Estuarine fish model for assessing estrogenic effects Environ. Toxicol. Chem. 2, Fig. 7. Percentage hatch for eggs produced by spawning groups of sheepshead minnows previously exposed to EE2 in duplicate spawning groups in reproductive trial 1 and in a single spawning group in reproductive trial 2. Fish that produced the eggs were taken from exposure chambers for control (C) and solvent control (SC) and for treatments with.2, 2, 2, and 2 ng/l of EE2. Poor hatching success in.2-ng/l treatment of trial 1a, control of trial 1b, and solvent control of trial 2 believed due to observed fungal infections. F 1 phase Hatching success (Fig. 7) in the controls (n 228) and solvent controls (n 18) of the first reproductive trial was 6 and 8%, respectively. Hatching success of eggs from females exposed to the three lowest treatments (.2-, 2.-, and 2-ng/L EE2 concentrations) was greater than 65% for the combined duplicate spawning groups in each treatment. In the 2-ng/L EE2 treatment spawning groups, hatching success was less than 1%. Moderate reduction in hatching success in the control and.2-ng/l EE2 treatments was probably due to fungal infections of eggs noted in these replicates. Among progeny from the second reproductive trial, no successful hatches occurred in the 2- and 2-ng/L treatments. Hatching success in the solvent control was only 2% (Fig. 7). Large numbers of unfertilized eggs were found in the solvent control treatment and in the treatments at higher EE2 concentrations in this trial. However, eggs from the nonsolvent control and the.2- and 2-ng/L treatments in the second reproductive trial had high rates of hatching success (77, 88, and 96%, respectively). The survival of fry 7 d after hatching was good in all treatments and controls. For the first reproductive trial, the pooled survival of fry was 99% for the control and solvent control treatments and 9, 95, 99, and 89% in the.2-, 2-, 2-, and 2-ng EE2/L treatments, respectively. For the second trial, the survival of fry was 1% in control treatments and 88 and 98% in the.2- and 2-ng EE2/L treatments. Fry survival in the 2- and 2-ng EE2/L treatments could not be evaluated for the second trial since embryos in these treatments did not hatch. DISCUSSION Survival was significantly reduced in subadult sheepshead minnows exposed to EE2 at, 8, 1,6, and 3,2 ng/l. Only limited histological analysis was possible of fish at the 1,6- and 3,2-ng EE2/L treatments because eosinophilic edemic fluid obliterated most cellular structures. Analysis of fish in the - and 8-ng EE2/L treatments indicated that these fish experienced generalized edema and damage to the gill epithelium and hepatic tissues. Similar pathologic effects have been noted in rainbow trout (Oncorhynchus mykiss) exposed to excessive doses of 17 -estradiol (E2) [23]. Histological observations of kidney tissues in the trout indicated tubular necrosis and deposits of eosinophilic material. The hepatocytes in the liver showed evidence of hyperplasia, and eosinophilic edemic fluid was present in various organs. These researchers attributed the pathological responses in trout to estradiol-induced secretion of vitellogenin, which accumulated because of inadequate deposition into the oocytes. While it cannot be stated with certainty that this was the mechanism for the toxic responses observed in sheepshead minnows, it is clear that these fish were under severe stress as a result of edema and damage to the gill and kidney. The fish from the 2-ng/L treatment were also stressed by the toxic effects of EE2, as indicated by dilation of the Bowman s capsule in the kidney, pericardial edema, and gill lesions noted in several fish. However, these effects did not affect the survival and growth of the fish relative to control treatments. Fish from EE2 test concentrations of.2, 2, and 2 ng/l showed no toxicopathic stress and displayed survival similar to the controls. Effects were noted on the development and differentiation of the gonads in sheepshead minnows exposed to concentrations of EE2. The stages of oogenesis in female sheepshead minnows used in this study were classified according to a scheme developed for Japanese medaka [22] since both fish species are asynchronous spawners and similarities exist in the size, appearance, and staining characteristics of the oocytes in both species. Among the female fish examined in this study, a suggestion exists of concentration-dependent responses in the ovaries that may be attributed to EE2 exposure. Atresia of pre- and postvitellogenic oocytes were noted in several female fish from the 2-ng/L treatment with EE2, and atresia also occurred, albeit at a lower frequency, in fish from the 2- and 2-ng/L treatments. Atresia of follicles is a natural feature of the fish ovary, and rates of ovarian atresia vary seasonally, showing a peak near the end of spawning season [2]. However, studies with mammalian test species have shown that estrogens and androgens may alter the rates of ovarian atresia [25], so it is possible that estrogenic compounds may cause enhanced ovarian atresia in fish species. In male fish, the testis is compartmentalized into fibrouswalled lobules that accommodate large numbers of developing germinal cysts. These lobules eventually link up with the efferent duct. In sheepshead minnows, the lobules appear to be loosely organized into zones of cystic development with lobules in more advanced stages of spermatogenesis located more centrally in the testis (surrounding the efferent duct). Moderate to severe fibrosis of the testicular lobules, involving a thickening of the lobule walls with increased amounts of fibrous tissue, was observed in many of the male fish exposed to 2,, and 8 ng/l of EE2, and this condition was also observed in some males from the 2-ng/L treatment. Mild fibrosis of the testes was observed in six of seven fish from the 2-ng/L treatment and in a small number of fish from the.2-ng/l treatment. Similar fibrosis of the testis was noted in male Japanese medaka exposed to an alkylphenol compound [26]. In a hermaphroditic fish (the white bream, Diplodus sargus), the histological events associated with sex inversion from male to female were characterized by spermatogonial degeneration and proliferation of connective cells and fibers in the testicular region of the gonad [27]. It is possible that the similar fibrosis

9 1976 Environ. Toxicol. Chem. 2, 21 E.J. Zillioux et al. observed in the sheepshead minnows was due to EE2-induced degeneration of spermatocytic tissue. Clusters of cells that appeared to be undifferentiated spermatogonia were observed in several female fish from the 2-, -, and 8-ng EE2/L treatments, and these were tentatively interpreted as evidence of sex reversal in these fish. Similar clusters of male germinal tissue were noted in the ovaries of phenotypic female sea bass (Dicentrarchus labrax) exposed to EE2 and 17 -estradiol [5]. In a review of sex manipulation in fish, Yamazaki [28] documented sex reversals from the male to the female phenotype induced with estrogenic compounds including EE2, E2, estrone, diethylstilbestrol, and estradiol butyryl acetate in experiments with a variety of gonochoristic fish exposed to estrogens in the diet. Sex reversals were detected through changes to the sex ratios of the fish relative to controls. Sex reversal has also been accomplished through aqueous immersion of alevins of masu salmon (Oncorhynchus masu) for 18 d to E2 at concentrations of.5 to 5 g/l [29] which is similar to the upper range of concentrations of EE2 used in this study (.8 g/l). Feminization of coho salmon, Oncorhynchus kisutch, was accomplished by exposing eggs just before hatch to g/l of E2 for only 2 h [3]. Medaka showed evidence of sex reversal and intersex when exposed to g/l of E2 after hatch for a period of only 8 h [31]. It appears that feminization takes place when fish are exposed to estrogens around the time of sexual differentiation of the gonad, which is a period that varies among fish species [5,22,3,32]. Testis ova were observed in several male sheepshead minnows exposed to EE2 from the 2-, -, and 8-ng/L treatments; this condition was also noted in one male from the 2- ng/l treatment. Testis ova have been induced in Japanese medaka by both dietary and aqueous exposures to E2 [31,33] and also have been induced in medaka by exposure to xenoestrogens [2,3,26]. However, testis ova induced in medaka generally show a gradation in differentiation in which oogonia are generally confined to the anterior part of the gonad [2,3]. Spermatogonia and oogonia in the testis ova induced in sheepshead minnows in this study were both distributed throughout the gonad. Komen et al. [35] induced testis ova in common carp, Cyprinus carpio, by dietary exposure to E2. Little work has been done to characterize the conditions under which fish undergo development of testis ova versus full sex reversals, but it is probable that the timing and dose of estrogen exposure are the variables that determine whether testis ova or complete feminization are induced in exposed fish [5]. In reproductive trials, only fish from the 2-ng EE2/L treatment showed any reduction in reproductive success in trial 1 as indicated by the number of eggs produced per female reproductive day. Some evidence was seen of reduced reproductive success in the 2-ng EE2/L treatment of trial 2, but it was not possible to conduct statistical analysis of these data because the spawning groups were not replicated. Histological evaluation indicates that these effects may have occurred as a result of reproductive impairment in the male fish since the males showed evidence of testicular fibrosis and intersex at these concentrations. However, atresia of oocytes and putative evidence of sex reversal in females also may have contributed to poor reproductive success in EE2 treatments at some concentrations (e.g., 2 ng/l). Hatching success also appeared to be lower among the progeny of fish treated with 2 ng EE2/L, although fungal infections of embryos may have affected these observations. The Table. No-observed-effect concentration (NOEC), lowest-observedeffect concentration (LOEC), and maximum acceptable toxicant concentration (MATC) (ng/l) determined for various toxicological and developmental endpoints for sheepshead minnows exposed to EE2 and their progeny, including survival of exposed fish, alterations to testicular development in exposed male fish, reproductive success of exposed fish in spawning trials, and hatching success of the progeny from exposed fish. Nominal concentrations are presented with average measured concentrations over the exposure period in parentheses (see Table 1). Because of the greater uncertainty associated with the lower concentrations, the actual values may be substantially lower (see text discussion) Endpoint (ng/l) NOEC LOEC MATC Fish survival Fibrosis of testis (males) Testis ova (males) Reproductive success Hatching success 2 (117).2 (.2) 2 (1.7) 2 (18) 2 (18) (328) 2 (1.7) 2 (18) 2 (117) 2 (117) 283 (196).6 (.6) 6.3 (5.5) 63 (6) 63 (6) numbers of unfertilized eggs in these treatments were not counted, but there appeared to be large numbers of unfertilized eggs in the 2-ng/L treatment. It is likely that poor fertilization of eggs was a consequence of the highly vascularized, fibrotic testis and reduced numbers of spermatogonia observed in all males at this concentration. Once eggs hatched, good survival of fry over the next 7 d occurred (88% or better). The LOEC and NOEC data summarized for various toxicological endpoints in Table indicate that the LOEC for reduced reproductive success was a nominal concentration of 2 ng/l, which corresponds to an average measured EE2 concentration of 117 ng/l over the exposure period. However, some evidence was seen of reduced reproductive success in the second reproductive trial at 2 ng EE2/L. It is not possible to confirm the actual concentration in this latter treatment since treatment concentrations at the lower concentrations of EE2 (.2, 2, and 2 ng/l) were below analytical sensitivity and thus could not be directly monitored with time. Kramer et al. [36] calculated a median effective concentration (EC5) for inhibition of egg production in fathead minnows (Pimephales promelas) exposed to E2 of 12 ng/l (measured concentration); this is slightly higher than the MATC for reproductive success in sheepshead minnows exposed to EE2 (63 ng/l; Table ). The LOECs for increased fibrosis and induction of intersex (testis ova) in the testis of male fish exposed to EE2 were 2 and 2 ng/l (nominal concentrations), respectively. Uncertainty is inherent in all LOEC and NOEC estimates (and large variability between studies); they are presented here only as relative indicators. In this study, however, the uncertainty in these estimates is greater for the lowest three concentrations since measurements were from stock solutions rather than from actual test chambers. We believe the resulting error was minimized by preconditioning the exposure systems with EE2 before introducing fish, although some additional loss in the test chambers no doubt occurred. We suggest that a worst-case correction might be applied to measured stock solution concentrations of the.2-, 2-, and 2-ng/L treatments by assuming the total loss of EE2 in their respective test chambers was equal to the average loss in the higher dose treatment having the lowest average percentage recovery based on nominal (59% for the 2-ng/L treatment; Table 1). If this were done, the estimated NOEC for reproductive success (Table ), for example, would change from 18 to 11.8 ng/l. The 17 -ethynylestradiol has been detected in STPs in Europe, the United Kingdom, Brazil, and Canada at concentra-