Introduction 1 6 7 8 9 Xenopus laevis 10 14 15 17 18 21 22 23 24 X. laevis X. laevis X. laevis X. laevis −1 50 Material and methods Test organism X. laevis Rearing n  −1 −1 25 Feeding 26 27 −1 Study design −1 Replicates 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. Flow-through system 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). −1 Primary E2 solutions −1 E2 solutions −1 −1 −1 Analysis of E2 concentration Sampling −1 Sample analysis −1 −1 Environmental conditions Temperature 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. Light 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 28 29 Maintaining water quality 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. In-life assessment of tadpoles Tadpoles were monitored for changes in general health, swimming behaviour and morphological appearance daily. The numbers of dead or moribund tadpoles and tadpoles completing metamorphosis were recorded daily for each tank. Snout-to-vent length and weight −1 3 Gross pathology 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. Assessment of gonad morphology 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. 1 Table 1 X. laevis   Feature Definition 1. Adhesion (other tissue) Gonads joined to other abdominal tissue 2. Aplasia (agenesis) Complete lack of gonad development 3. Segmental Aplasia Gonad is longitudinally discontinuous   3-A. Tissue separation One or more areas along the length of the gonad is poorly developed, attenuated with an essentially complete lack of gonadal tissue   3-B. Extraneous gonadal tissue A small disjunct cluster of gonad-like tissue 4. Bifurcation Division of the gonad oriented longitudinally, along the cranial–caudal axis   4-A. Protuberance(s) Small projection(s) of the gonad along the margins   4-B. Symmetric A “Y” -shaped single gonad with relatively equal division 5. Angular deformity One or more bends in the gonad to an excessive degree   5-A. Gonad folded The gonad is bent over or doubled up so that one part lies on another 6. Displaced The gonad, or a section of it, is not located in the typical location   6-A. Displaced laterally The gonad, or a section of it, is not located in the typical location. Instead it is located further away from the frog’s midline   6-B. Displaced medially The gonad, or a section of it, is not located in the typical location. Instead it is located closer to or crosses over the frog’s midline   6-C. Displaced cranially The gonad is not located in the typical location. Instead it is located more towards the anterior   6-D. Displaced caudally The gonad is not located in the typical location. Instead it is located more towards the posterior 7. Fused Left and right gonads joined to varying degrees at one or more locations along cranial–caudal axis 8. Hypertrophy Size of the gonad is larger than typical   8-A. Wide Gonad appears broad. Lateral width is large relative to the length   8-B. Elongated Gonad appears long (cranial–caudal). Length is large relative to the length of the kidneys and the torso of the frog   8-C. Thick Gonad relatively increased in the distance between the dorsal and ventral surfaces   8-D. Enlarged A generalized increase in the size of the gonad along all axes 9. Segmental hypertrophy A gonad where one or more areas are excessively large   9-A. Enlargement (mass) A section of the gonad substantially bigger in width and/or thickness than the remaining tissue   9-B. Pearling Pronounced multiple segmental enlargement of the gonad   9-C. Partly thick A section or sections of the gonad is relatively increased in the distance between the dorsal and ventral surfaces 10. Hypoplasia Size of the gonad is decreased   10-A. Narrow Gonad appears strap-like. Lateral width is markedly reduced relative to the length   10-B. Slightly narrow Lateral width is somewhat reduced relative to the length   10-C. Truncated Gonad appears short (cranial–caudal). Length is small relative to the length of the kidneys and the torso of the frog   10-D. Slightly truncated Gonad appears somewhat short (rostal–caudal). Length is somewhat reduced relative to the length of the kidneys and the torso of the frog   10-E. Thin Gonad relatively reduced in distance between the dorsal and ventral surfaces   10-F. Margin entire All of the ovary or indeterminate gonad has a smooth edge; no scalloping or lobes 11. Segmental hypoplasia A gonad where one or more areas are excessively reduced, attenuated, or poorly developed but not separated   11-A. Partly narrow A section or sections of the gonad has a small lateral width relative to the length and the width of the other sections   11-B. Partly thin A section or sections of the gonad is relatively reduced in distance between the dorsal and ventral surfaces   11-C. Margin slightly sinuate Gonad has only shallow waved or scalloped edge (typically associated with a narrow or partly narrow ovary)   11-D. Margin partially entire A section or sections of the gonad has a smooth edge (typically associated with a narrow or partly narrow ovary)   11-E. Pearling Pronounced multiple segmental attenuation or narrowing of the gonad 12. Intersex Ovarian and testicular tissue present as separate structures (i.e. not contained in the same gonad) (left/right) 13. Mixed sex Ovarian and testicular tissue present in the same gonad 14. Translucent Gonad appears not so dense, light able to pass through diffusely   14-A. Tissue slightly translucent To a small extent the gonad appears not so dense, a small amount of light able to pass through diffusely 15. Segmental translucence A section or sections of the gonad appears not so dense, light able to pass through diffusely 16. Melanophores decreased Apparently fewer pigment-containing cells than typically seen. Applies only to females, mixed sex frogs, and frogs of undetermined sex   16-A. Apigmentation No melanophores in the internal tissue. Applies only to females, mixed sex frogs, and frogs of undetermined sex 17. Internal melanophores Pigment-containing cells within the gonadal tissue. Applies only to males, mixed sex frogs, and frogs of undetermined sex 18. Other Any finding not listed. Describe as needed. For any “other” finding that study personnel consider not incidental, notify the Study Director and other laboratories as soon as possible via email including a description and photograph Photography 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. Gonad measurement 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. Statistical analysis For both experiments, observations were collected for each animal. However, consistent with the experimental design, the tank is considered the primary experimental unit. When tank differences are present, statistical analysis should accommodate this experimental structure directly using nested “random effects” analyses or indirectly by analysing tank means or tank percentages. Neither approach is completely satisfactory for all endpoints. Nested models have specific distributional requirements and, in the case of incidence data, become unstable when frequencies are at or near zero. Analyses of tank means can also be problematic for these experiments. Estradiol-induced feminization causes the number of males per tank to be smaller for treated groups than for the control group. This, in turn, results in greater variation between estradiol-treated tanks than between control tanks. Most parametric and non-parametric statistical procedures, however, require equal variability between tanks within every group. When tank differences are small or absent, however, the individual data from all tanks within the same treatment group can be pooled and analysed more simply using the frog as the basic experimental unit. p p p p p Results X. laevis X. laevis 2 Table 2 X. laevis Parameter Characteristic Water-quality parameters   Supply water: filtered, contaminant free Filtered and UV sterilized; tested for chemical contaminants   Dissolved oxygen ≥   pH 7.9 to 8.3   Ammonia −1   Nitrate −1   Hardness 3 −1   Alkalinity 3 −1   Specific conductance −1   Supplemental aeration Initiated not later than dpf 36 Animal husbandry   Animal supply Xenopus, Dexter, MI, USA   Shipped from Supplier Day 3 post fertilization (dpf)   Acclimation dpf 4–7   Treatment period dpf 8–82   Feed: Sera Micron Sera Micron (contaminants and estrogenic potential evaluated) Contents: 50% protein, 8% fat; 4% fibre; 11% ash.   Feeding rate—acclimation dpf 4–5; per 300 larvae: 200 mg; if free swimming, dpf 6–8: 200 mg twice daily (based on the intake of food by the larvae)   Feeding rate—NF stages 46–66 −1   Exposure chamber cleaning Daily   Exposure chamber replacement As needed   Water-quality parameters Hardness, ammonia, nitrate, pH, specific conductivity twice a week; DO three times a week   Temperature 22 ± 1°C (verified twice daily, measured once a week)   Light intensity 100–500 lux (measured once weekly, at level of surface of water)   Light:dark cycle 12:12; 30-min transition (30% of full light) Experimental design   Performed in compliance with Good Laboratory Practice Standard Yes   Colour coded (blind) treatment Yes   Randomization of tadpoles Tadpoles distributed in rotation one or two at a time to transport vessels to contain 25, then vessels were randomly assigned to test tanks   Randomization of tank clusters Completely randomized placement; one cluster consists of four tanks   Negative control Water (eight replicates, two clusters)   Treatment 17β-estradiol concentrations −1   Tank volume 9 L (water volume 7 L), tanks covered   Tank flow rate −1   Number of animals 200 larvae per treatment group   Animal load density −1   HPLC–MS–MS verification dpf 8 all tanks; dpf 16–81 weekly (alternating tanks) Environmental conditions 2 3 −1 −1 −1 −1 E2 exposure verification −1 1 −1 −1 1 Fig. 1 a b −1 −1 −1  In-life assessment of tadpoles Appearance and behaviour 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. Survival −1 −1 Snout-to-vent length and weight −1 −1 2 Fig. 2 a b −1 −1 Neg Ctrl  −1 2 Time course of metamorphosis 3 −1 −1 −1 3 3 −1 −1 −1 Fig. 3 X. laevis n a b c −1 d Neg Ctrl  −1 Mean age at completion of metamorphosis −1 4 −1 −1 −1 −1 −1 4 Fig. 4 a b −1 −1 −1 Neg Ctrl asterisks p p p p p  p p   Appearance and examination of non-gonadal organs 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. Determination of sex, percent female, male, and mixed sex by gross examination −1 −1 5 50 50 Fig. 5 bars X. laevis n top of the bars 50 Neg Ctrl asterisks p p p  −1 −1 5 −1 −1 −1 −1 −1 Xenopus 6 6 X. laevis 1 Fig. 6 a X. laevis b c Arrows f m d p e segA  Gonadal measurement Because of the subjective nature of gross gonad morphological assessment, quantitative measurements of gonad area were performed on digital images. 2 2 2 2 −1 −1 2 2 2 2 −1 p −1 2 2 2 −1 2 2 2 2 p  p  Discussion 30 31 32 X. laevis 15 17 15 17 16 X. laevis 20 21 23 X. laevis 33 X. laevis 1 7 K ow 34 X. laevis Fig. 7 Correlation of changes in E2 concentration and the biomass of growing tadpoles during the exposure period in Experiments 1A and 1B  X. laevis X. laevis X. laevis 25 X. laevis 8 X. laevis Fig. 8 n  25 X. laevis −1 −1 50 −1 X. laevis −1 15 17 22 31 35 36 14 −1 X. laevis −1 −1 X. laevis −1 −1 37 38 50 X. laevis 1 39 −1 −1 −1 X. laevis X. laevis X. laevis