Tadpoles of X. laevis were obtained from the commercial supplier, Xenopus I, (Dexter, MI, USA). These larvae (approx. 1200 for each study) originated from ten pairs of adult breeders for each study. Xenopus I reared the eggs until day post fertilization (dpf) 3 and then shipped them to each laboratory.

Upon arrival on dpf 4, tadpoles were acclimated to water and temperature conditions (21 ± 2 °C) over a six-hour time period. During the acclimation period larvae were maintained in two 40-L glass aquaria containing 30 L well water (ca. 600 larvae/tank, Experiment 1A) and three 40-L glass aquaria containing 30 L dilution water (ca. 400 larvae/tank, Experiment 1B) derived from the flow-through system. At the initiation of treatment, twenty-five larvae were randomly assigned to each glass aquarium (n = 200 larvae per treatment group) so that the biological loading rate was always less than 1g L⁻¹ day⁻¹. In Experiment 1A exposures were initiated at dpf 6 NF stage 45–46 and in Experiment 1B at dpf 8 when tadpoles had reached NF stage 47–48 as identified by visible hind limb buds [25]. Exposures continued until completion of metamorphosis or dpf 82, whichever came first. Throughout the acclimation period, water temperature was recorded twice daily whereas dissolved oxygen (DO) concentration and pH were measured daily. Experiment 1B was conducted in compliance with the local animal protection committee (G0048/05).

Animals were fed Sera Micron (Lot No: 9698; Sera, Heinsberg, Germany), a commercially available food which was reported by the manufacturer to consist of 50.2% protein, 8.1% fat, 4.2% fibre, and 11.0% ash. A sample of Sera Micron was analysed by Lancaster Laboratories (Lancaster, PA, USA) for potential contaminants (metals and pesticides), and all tested contaminants were below their limits of quantification for analysis at that facility. A mixture of diet and dilution water was tested for estrogenicity and androgenicity using YES (yeast estrogen screen) [26] and YAS (yeast androgen screen) assays [27]. Based on these analyses no estrogenic or androgenic activity was detected.

During acclimation of larvae in Experiment 1A, 1 g Sera Micron was added to each tank daily. This amount of food was found to be excessive. In experiment 1B, after temperature acclimation, larvae were transferred to the acclimation tanks where they promptly attached to the glass aquaria walls. Therefore, no food was added on dpf 4. The following day some of the tadpoles were free swimming and 150 mg Sera Micron was added that evening by rinsing through a sieve. The amount of feed was chosen based on the intake of food by the larvae, and care was taken to ensure that feeding did not adversely affect water quality. From dpf 6 to 8 the free swimming tadpoles were fed 200 mg Sera Micron in each tank twice a day. Throughout the remaining course of both studies, tadpoles were fed a suspension of Sera Micron (77 mg L⁻¹) three times daily. The total quantity of food added per tank each day increased from 300 mg/tank on dpf 8 to 900 mg/tank on dpf 33. The quantity of food added to each tank was adjusted periodically in Experiment 1A and on a daily basis in Experiment 1B to account for mortality and removal of tadpoles as they completed metamorphosis.

Each study consisted of at least one negative control group plus three E2-treated groups, each comprising eight replicated glass aquaria/treatment groups. Eight glass aquaria were arranged in two clusters of four tanks each. The clusters of negative control tanks and E2 treated tanks were randomly distributed within the environmental chamber in Experiment 1A and within water baths in Experiment 1B. Each cluster was supplied water via the same water flow meter and mixing chamber. Experiment 1A included 16 control tanks that were initially divided into two 8-tank groups designated as negative control and reference control, respectively. The animals from all treatment groups were handled in a blinded fashion so that the biologists and histologists involved in the experiments did not know the treatment applied to frogs being observed. An additional non-blind reference control group was included to provide reference information on rates of spontaneous incidence of gonadal abnormalities for blind histological assessment of gonads from the E2 treatment groups. Histological analysis was conducted on all frogs of Experiment 1A. At the end of Experiment 1A, it was discovered that slightly elevated temperature (1 °C) in one cluster of four negative control tanks had resulted in accelerated growth of the frogs in the tanks. Therefore, measurements of weight, length, gonad image area, and age at metamorphosis from those tanks were dropped from analysis, and the four remaining negative control tanks were combined with the reference control group to provide these measurements.

Both studies were conducted using continuous flow-through systems operating at a flow rate of at least 50 L dilution water per tank per day. This was equivalent to a water exchange rate of approximately seven tank volumes per day. Test tanks consisted of 9-L glass aquaria (30 × 20 × 14.5 cm) containing 7 L water. Dilution water for Experiment 1A was obtained from a well of approximately 40 m depth located on the WLI site. In Experiment 1B the dilution water used was non-chlorinated municipal water. Dilution water samples from Experiment 1A were analysed for possible metal or pesticides by Lancaster Laboratories and water samples of Experiment 1B by Environmental Chemistry and Pharmanalytics (RCC Ltd., Itingen, Switzerland).

The flow-through systems in the two laboratories were essentially identical. Prior to use, the water was passed through several filters (5, 1, 0.5, and 0.45 μm) to remove particulate material, a UV-sterilizer, and a final particle filter (0.45 μm). After being filtered and temperature conditioned, the dilution water was divided into 16 streams. Each stream passed through a flow-control assembly, consisting of a rotameter flow gauge and a needle valve, to a mixing chamber. The flow from each mixing chamber was then split to supply a cluster of four tanks. Each tank received 140 mL min⁻¹ dilution water (±5%).

The primary E2 stock solutions were continuously delivered to each mixing chamber (0.140 mL min⁻¹), where it was vigorously mixed with the dilution water. Primary E2 stock solutions were metered into the mixing chambers by an eight-channel peristaltic pump (MasterflexL/S, Model 752455, Cole–Parmer, IL, USA). The flow-through system (temperature, water, filter pressure, flow-meter), and peristaltic pumps were calibrated four weeks prior to study initiation, and inspected visually twice daily throughout the study.

In Experiment 1A, water samples were collected from each tank in all treatment groups to confirm the operation of the diluter two days prior to study initiation. In Experiment 1B, E2 concentrations were monitored twice weekly for a two-week period prior to study initiation. After study initiation, water sampling was carried out twice weekly from alternating tanks in two tanks per cluster until study termination. Throughout the studies water samples were collected routinely two days after the freshly prepared E2 solution was connected to the flow-through system and a second time in the same week. At mid-depth from each test tank 190 mL water was collected using a glass pipette and 10 mL methanol (MeOH) was added. Of this solution 20 mL was decanted into a 20 mL amber scintillation vial for E2-enzyme-linked immunosorbent assay (ELISA) analysis. The remaining volume of 180 mL was stored as backup at 4 °C until E2 analysis was completed. Water samples from Experiment 1B were shipped to WLI for E2 analysis. The stability of E2 was confirmed by shipping samples of E2 (0.1 μg E2 L⁻¹) that were prepared and analysed in parallel with the water samples.

Rapid analysis was performed using ELISA kits (Ecologiena, Abraxis LLC, Warminster, PA, USA) to ensure the absence of the test substance in the negative control tanks and to verify the proper E2 concentration in each E2 tank. The ELISA method was based upon the E2 kit manual provided by the manufacturer. The method detection range of this assay was 0.05–0.5 μg E2 L⁻¹. Parallel analyses of water samples for E2 concentration were performed by direct aqueous injection using an Agilent Series 1100 High-Performance Liquid Chromatograph coupled with a MDS Sciex API 3000 tandem mass spectrometer (LC–MS–MS) (Applied Biosystems, Foster City, CA, USA) and MDS Sciex API heated nebulizer ion source (Applied Biosystems) operated in the multi-reaction mode (MRM). Chromatographic separations were achieved using a Keystone Betasil C-18 column (50 mm × 2 mm, 3 μm particle size) (Thermo Hypersil, Madison, WI, USA). The method limit of quantitation (LOQ) for HPLC–MS–MS estradiol analyses was defined as 0.005 μg L⁻¹.

In Experiment 1A, all glass aquaria were situated in a walk-in environmental chamber (21 ± 2 °C). In Experiment 1B, glass aquaria were placed in temperature controlled water baths (22 ± 1 °C). In both experiments water temperatures were continuously recorded, and temperatures in all glass aquaria were measured weekly using a liquid-in-glass thermometer.

The target light intensity at tank level was 100–500 lux and was measured weekly using a Sper Scientific (AZ, USA) light meter. A photoperiod of 16 h of light and 8 h of dark was used in Experiment 1A, and was adjusted to a 12 h light, 12 h dark cycle in Experiment 1B. A 30-min low light (around 30% intensity of full light) transition period was installed in both laboratories.

Water quality parameters were assessed according to ASTM (2003) [28] and US EPA guidelines. Water hardness, pH, conductivity, and the concentrations of nitrate, and ammonia were measured weekly in one tank from each cluster throughout the experiments. Hardness and alkalinity were measured by titration based on procedures described in “Standard Methods for the Examination of Water and Wastewater” [29]. Nitrate concentrations were analysed with a Hach DR/700 colorimeter (Hach Company, Colorado, USA) and specific conductance was detected using a YSI conductivity meter (WLI: Yellow Springs Instruments Model 33, IGB: 3200, Yellow Springs, OH, USA). pH and ammonia concentrations were measured with an Orion pH/ISE Meter 720Aplus (Thermo Electron, USA). DO concentrations were measured in alternate tanks per treatment three times a week with an Orion Model 850plus dissolved oxygen meter (Thermo Electron). Commencing at dpf 43 in Experiment 1A, and at dpf 36 in Experiment 1B, gentle external aeration was applied by a glass pipette in each test tank to ensure maintenance of an adequate DO concentration.

The tanks required daily cleaning to minimize microbial growth and to maintain adequate water quality. Bio-film that accumulated on the inner walls and bottom of the tanks was scraped off daily, and detritus was siphoned from the tanks. Daily cleaning proved inadequate to maintain appropriate water quality throughout the entire exposure period. Hence, in Experiment 1A all tanks were replaced with clean tanks weekly and in Experiment 1B twice throughout the experiment when siphoning did not lead to satisfactory results.

Each tadpole that had either completed metamorphosis by NF stage 66, or had failed to achieve metamorphosis by dpf 82, was removed from the test tank and euthanized by immersion in MS 222 (tricaine methanesulfonate) (WIL: Sigma Aldrich, Allentown, PA, USA; IGB: Sigma–Aldrich, Taufkirchen, Germany) (2 g L⁻¹ buffered to pH 7.5 with NaHCO₃). Immediately thereafter, the snout-to-vent length was measured to the nearest millimetre. Each frog was blotted dry and then weighed to the nearest milligram. A unique identifier was assigned to each frog that included: study number, in-life laboratory ID, colour code, replicate, and animal number.

The pleuroperitoneal cavity was opened to expose the visceral organs. The liver, stomach, and intestine were examined using a dissecting stereo microscope (WIL: Olympus SZ61-TR, Olympus America, Melville, NY, USA; IGB: Olympus, SZX7, Olympus, Hamburg, Germany), and unusual variations in size, colour, and/or abnormal structure were recorded. The gastrointestinal tract was excised to expose the ventral surfaces of the kidney and gonads, which were similarly assessed for gross abnormalities. Tadpoles that did not complete metamorphosis at study termination (Experiment 1A: 25, Experiment 1B: 7) were staged according to NF and, the gonad morphology was assessed (if possible), and the carcasses were archived in fixative. Data derived from such animals were excluded from further analysis.

Using a dissecting stereo microscope, gross evaluations of gonad morphology were performed on all animals that reached NF stage 66. To enhance visualization of the gonads, which presented as thin, pale-tan strips of tissue on the ventromedial margin of the kidney, several drops of Bouin’s solution (WIL: Sigma Aldrich, Allentown, PA, USA; IGB: Sigma–Aldrich, Taufkirchen, Germany) were applied to partially fix these organs. Based on gross observation, the gonads of each animal were identified as testes, ovaries, or malformed. Examples of observed malformations included mixed sex, intersex, pearling and segmental aplasia. Mixed sex was defined as the co-occurrence of both ovarian and testicular tissue in a single gonad. Intersex was assessed as ovarian and testicular tissue in the same individual as separate gonads (left/right). The term pearling was characterized by the presence of multiple, prominent segmental enlargements and/or attenuations along the length of one or both gonads, whereas gonads that presented as nodular islands of testicular or ovarian tissue, with either intervening membranous connections or no connections at all, were classified as segmental aplasia. Gross findings for the gonads of each frog were recorded separately for the left and right gonads. The gonadal findings for each frog were verified independently by a second biologist. If the two biologists differed in their interpretations, the disparate opinions were discussed and a consensus finding was recorded.

Prior to the initiation of the first E2 experiment a list of standardized terms for changes of potential morphological gonadal observations was developed. Subsequent to the completion of both experiments the applied terminology was refined in order to better characterize and group different types of gonad changes observed and which in turn could be analysed. For example, the subcategories narrow, slightly narrow, truncated, slightly truncated, and margin entire were integrated into the main category hypoplasia. This term describes all gonads which appear smaller than those characterised as “typical/normal”. Since the term “typical/normal” is rather general, further sub-categories allow the biological variability of gonadal morphology to be described. However, during the investigations it was revealed that too few observations in each sub-category ruled out reasonable statistical analyses. Therefore, a refined list of qualitative descriptive terminology for gonadal features was created as presented in Table 1. Table 1

Glossary of terms used to describe features observed during gross morphological examination of gonads in X. laevis NF stage 66

After gross gonad inspection was complete, each gonad was photographed in situ using a digital camera (WIL: Olympus DP12–2, Olympus America, Melville, NY, USA; IGB: Olympus DT5, Hamburg, Germany) attached to the stereo microscope. Each digitalized image included a millimetre measurement scale placed adjacent to the gonads to permit gonad size to be determined from the photograph. Following photography, each frog was placed in a labelled individual container of 30 mL of Bouin’s solution for approximately 48 h. At the end of the fixation period, each carcass was rinsed several times in 70% ethanol and was placed in 30 mL 10% neutral buffered formalin (WIL: VWR, Westchester, PA, USA; IGB: Histofix, Roth, Karlsruhe, Germany). A complete gonadal histological evaluation was performed on each frog of experiment 1A and results are presented in a separate paper (Wolf et al., in preparation). In some cases the growth and histological findings differ and a better understanding of results is gained by considering both types of observation.

The biological variability of morphological features of the gonads and the subjective nature of the assessment prompted us to develop a more quantitative metric of gonad size. Thus, individual measurements of gonad image area were derived from photographs of the gonads. Gonad image areas were obtained using image-processing software (Image Pro Plus, Version 5.1, Media Cybernetics, Silver Spring, MD, USA) that calculated the combined area of the left and right gonad whose outline had been manually traced around the digital image. The program was calibrated by the millimetre measurement scale prior to measurement for the gonad of each tadpole.

In both experiments experimental conditions generally met the criteria listed in Table 2. In Experiment 1B the mean and SD for hardness and alkalinity were 103 ± 5.8 mg and 162 ± 6.8 mg CaCO₃ L⁻¹, respectively. Specific conductance was 794 ± 18.0 μS cm⁻¹, pH 8.1 ± 0.09, nitrate 1.6 ± 0.5 mg L⁻¹ and ammonia 0.08 ± 0.09 mg L⁻¹. The measurements from negative control tanks were not significantly different from those from the E2 tanks. Technical problems in Experiment 1B prevented accurate determination of DO concentration, however, based on subsequent studies using the same procedures, DO was consistently above 60% saturation.

In each experiment, E2 was not present in any of the negative control tanks. Measured concentrations of E2 stock solutions ranged from 96.6 to 106% in Experiment 1A and between 90.5 and 108% in Experiment 1B. In Experiment 1A, the mean concentration of E2 for three treatment groups began to decline after experiment initiation and fell below 80% of nominal E2 concentration by dpf 29. Between exposure dpf 43 and 50 mean concentration of E2 decreased to minima of 0.4, 43, and 38% of nominal concentrations of E2 in the 0.2, 1.5, and 6.0 μg E2 L⁻¹ treatment groups, respectively. After dpf 50, concentrations of E2 began to increase in all treatment groups, and exceeded 60% of nominal concentration on dpf 64 (Fig. 1a). In Experiment 1B, concentrations of E2 declined after study initiation and dropped below 60% of nominal E2 concentration by dpf 23. On dpf 37 concentrations of E2 fell to 47, 46, and 50% in the 0.015, 0.2, and 1.5 μg E2 L⁻¹ treatment groups, respectively. The concentration of E2 increased to 60% of nominal concentration on dpf 65 (except the 1.5 μg E2 L⁻¹ with 44.4%) (Fig. 1b). Fig. 1

Comparison of measured mean E2 concentrations in water samples of E2 treatments in the flow-through system. Mean values represent the concentrations of E2 of weekly sampling intervals from dpf 6 or 8 through the last sampling interval on termination of the experiments on dpf 82. a, E2 concentrations of Experiment 1A; b, E2 concentrations of Experiment 1B. The limit of quantification was 0.00500 μg E2 L⁻¹, calculated as the product of the concentration of the lowest calibration standard (0.100 μg E2 L⁻¹) and the dilution factor of the matrix blank samples (0.0500 μg E2 L⁻¹)

No effects of E2 treatment on tadpoles condition, behaviour, or external appearance was observed in any of the treatment groups at any time throughout the entire course of Experiment 1A or Experiment 1B.

Excellent survival was achieved in both experiments in controls as well as in E2 treated groups. The total survival was 92% in Experiment 1A and 99% in Experiment B. In Experiment 1A, survival in the combined negative control, 0.2, 1.5, and 6.0 μg E2 L⁻¹ treatment groups was 90.8, 94.5, 95.0, and 88.0%, respectively. In Experiment 1B survival rates were 100% in the negative control and 97.5, 97.5, and 99.5% in the 0.015, 0.2, and 1.5 μg E2 L⁻¹ exposure groups, respectively.

In Experiment 1A, for females the average length values were, 22.2 ± 0.1, 22.1 ± 0.1, and 22.8 ± 0.1 mm in the 0.2, 1.5, and 6.0 μg E2 L⁻¹ treatment group compared with 22.0 ± 0.2 mm in the combined control group. Males treated with E2 had an average snout-to-vent length of 21.8 ± 0.1, 22.1 ± 0.6, and 23.0 ± 0.1 mm in the combined control, 1.5, and 6.0 μg E2 L⁻¹ treatment group, respectively. The increase in the mean weight of males was more pronounced than the increase in the mean weight of females (Fig. 2). Fig. 2

Body weight (means ± SD) of a, females, and b, males, after exposure to E2 from dpf 6 or 8 in a flow-through system until completion of metamorphosis or day 82 post-fertilization. 0.015 μg E2 L⁻¹ was not used in Experiment 1A and 6.0 μg E2 L⁻¹ was not used in Experiment 1B. Neg Ctrl, negative control group

On average, the process of metamorphosis for tadpoles took longer in Experiment 1A than in Experiment 1B. In Experiment 1A, the age at which the first frog in the experiment completed metamorphosis was delayed by 6 days (dpf 44) in each E2 treatment group compared with animals in Experiment 1B (dpf 38) (Fig. 3). In Experiment 1A exposure of females to 0.2, 1.5, and 6.0 μg E2 L⁻¹ resulted in a slight delay of metamorphosis. In Experiment 1B, the time course of metamorphosis for females in the 0.015 μg E2 L⁻¹ treatment group was similar to that for females in the negative control group but exposure to 0.2 and 1.5 μg E2 L⁻¹ caused a delay of completion of metamorphosis (Fig. 3a,b). A comparable picture for the cumulative time course of metamorphosis of males is shown in Fig. 3c,d. In Experiment 1A, males treated with 0.2 and 1.5 μg E2 L⁻¹ had a delay in completing metamorphosis of approximately ten days compared with males in the negative control group. In Experiment 1B metamorphosis of male frogs in the 0.2 μg E2 L⁻¹ treatment group was slightly postponed whereas for males treated with 1.5 μg E2 L⁻¹ completion of metamorphosis was delayed by three days. The lowest E2 concentration used obviously did not affect the mean time to complete metamorphosis. Fig. 3

Cumulative portion of X. laevis frogs completing metamorphosis after exposure to different concentrations of E2 from day 6 or 8 post-fertilization in a flow-through system until completion of metamorphosis or day 82 post-fertilization. The number of individuals per treatment group (n) is given in the legend. a, portion of females in Experiment 1A; b, portion of females in Experiment 1B; c, portion of males in Experiment 1A (just one male was observed in the 6.0 μg E2 L⁻¹ group); d, portion of males in Experiment 1B. Neg Ctrl, negative control group

The differences in the time course of metamorphosis described in the previous section are reflected in the mean ages of frogs at completion of metamorphosis in each of the treatment groups. In Experiment 1A, the mean ages at completion of metamorphosis of females treated with 0.2, 1.5, and 6.0 μg E2 L⁻¹ were dpf 64.2 ± 7.6, 62.9 ± 7.9, and 65.1 ± 9.2, respectively, and substantially greater than the mean age in the negative control group (dpf 61.4 ± 8.2) (Fig. 4). In contrast, in Experiment 1B females treated with the different E2 concentrations showed a dose response with a significant increase noted in the 0.2 μg E2 L⁻¹ group (dpf 50.7 ± 7.5) and a greater effect (dpf 51.5 ± 6.6) in the 1.5 μg E2 L⁻¹ treatment group compared with dpf 47.8 ± 5.6 for females in the negative control group. For males in Experiment 1A, the mean age at completion of metamorphosis was increased from dpf 60.6 ± 9.4 in the negative control to dpf 70.9 ± 9.3 and dpf 72.3 ± 9.0 in the 0.2 and 1.5 μg E2 L⁻¹ treatment groups, respectively. In Experiment 1B, the mean age at completion of metamorphosis of males was statistically significant greater in the 0.2 μg E2 L⁻¹ (dpf 51.2 ± 6.6) and 1.5 μg E2 L⁻¹ (dpf 58.0 ± 8.3) treatment groups than in the negative control group (dpf 48.5 ± 6.8) (Fig. 4). Fig. 4

Mean age at completion of metamorphosis (mean ± SD) by treatment group for female, a, and male, b, frogs at completion of metamorphosis or on day 82 post-fertilization. 0.015 μg E2 L⁻¹ was not used in Experiment 1A and 6.0 μg E2 L⁻¹ was not used in Experiment 1B. Only one male frog was observed in the 6.0 μg E2 L⁻¹ group in Experiment 1A. Neg Ctrl, negative control group. For Experiment 1B significant differences are marked by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001; two-sided Wilcoxon–Mann–Whitney test of equality with negative control group). A Kruskal–Wallis test of overall group differences was significant (females p < 0.0001, males p = 0.0009) and a Jonckheere–Terpstra test of trend was significant (females p < 0.0001, males p = 0.0062)

No externally visible morphologic abnormalities were observed in each treatment group in either experiment. Likewise, inspection of the liver, stomach, intestine, kidney for tumours, lesions or any other remarkable features had no effect.

The percentages of male, female, and mixed-sexed animals were calculated based on frogs that completed metamorphosis (NF stage 66) among all animals. In Experiment 1A, the percentage of female phenotype was increased in all E2 treated groups. In the 0.2, 1.5, and 6.0 μg E2 L⁻¹ treatment group 88.4, 95.8, and 98.3% of frogs showed female phenotype. In Experiment 1B, the incidence of female phenotype in the E2 treated groups displayed a statistically significant distinct dose-responsive increase with a high percentage of females observed for the 0.2 (70.5%) and 1.5 μg E2 L⁻¹ (92.3%) treatment groups, respectively (Fig. 5). The E2 concentration that results in an increase of female phenotype to 75% is defined as the effective concentration (EC₅₀). Based on the feminization effect observed in Experiment 1B the EC₅₀ was calculated to be 0.12 μg E2. Fig. 5

Individual bars represent frequencies of sex of X. laevis frogs among all animals according to treatment group based on gross morphological evaluation; the total number of frogs (n) that completed metamorphosis, is given on top of the bars. Tadpoles were exposed to E2, in a flow-through system, from day 6 or 8 post-fertilization through completion of metamorphosis or day 82 post-fertilization. The EC₅₀ was calculated from the results of Experiment 1B and was 0.12 μg E2. The numbers of frogs in the negative control in Experiment 1A was higher for evaluation of gender because the animals which were excluded from calculation of weight and length were included in calculation of the portion of males, females, and mixed sex. Neg Ctrl, negative control group. For Experiment 1B asterisks denote significant differences (**p < 0.01, ***p < 0.001; two-sided Fisher’s exact test for comparison to negative control). Both an overall exact chi-square test of equality of all experimental groups and a Cochran–Armitage trend test were statistically significant (p < 0.05) in every case

Because of the subjective nature of gross gonad morphological assessment, quantitative measurements of gonad area were performed on digital images.

In Experiment 1A, mean gonad areas of female and male frogs were approximately twice as large as in animals of the corresponding gender in Experiment 1B. The mean gonad image areas for females in Experiment 1A were 2.20 ± 0.82 mm², 2.21 ± 0.89 mm², 2.09 ± 0.92 mm², and 2.23 ± 0.92 mm² in the combined negative control, 0.2, 1.5, and 6.0 μg E2 L⁻¹ treatment groups, respectively. In Experiment 1B, for females treated with 0.015, 0.2, and 1.5 μg E2 L⁻¹ mean gonad image areas were 1.21 ± 0.41 mm², 1.27 ± 0.44 mm², and 1.32 ± 0.41 mm², respectively, compared with 1.23 ± 0.42 mm² in the negative control group. Mean gonad areas in females treated with 1.5 μg E2 L⁻¹ were significantly increased (p < 0.05). In Experiment 1A, the measured mean gonad areas of males in the combined control, 0.2 and 1.5 E2 L⁻¹ were 1.28 ± 0.2 mm², 1.39 ± 0.2 mm², and 1.70 ± 0.3 mm², respectively. In Experiment 1B, males treated with 0.015, 0.2, and 1.5 μg E2 L⁻¹ had a slight increase of mean gonad areas 0.82 ± 0.20 mm², 0.85 ± 0.19 mm², and 0.89 ± 0.14 mm², respectively, compared with the negative control (0.77 ± 0.22 mm²). A Kruskal–Wallis test of overall group differences was marginally non-significant (p = 0.079) but a Jonckheere–Terpstra test of trend was significant (p = 0.0092).