Morbidity/mortality following developmental exposure to BPA (A,B), E2 (C), or GSK4716 (D). (A) Percentage of chorion intact (dotted black line) versus chorion removed (solid grey line) larvae exhibiting abnormalities following static waterborne exposure to BPA (10 – 100 μM). Statistical significance determined by unpaired T-test for each concentration (p<0.05). Error bars depict SEM. (B-D) Percentage of larvae exhibiting abnormalities (grey circles) or percent mortality (black squares) following static waterborne exposure to BPA (10 – 70 μM; note: data points (x-axis) are plotted at the actual exposure concentrations, which were adjusted following quantification of stock concentration and were slightly higher than expected; e.g., “20 μM” exposure was actually 24.46 μM) (B), E2 (0.1 – 25 μM) (C), or GSK4716 (0.1 – 30 μM) (D) from 8-120 hpf. Plot shows % affected versus log(exposure concentration) ±SEM (BPA: N=6, 12 embryos per group; E2 and GSK4716: N=5, 8 embryos per group). Curves were fit by a nonlinear regression dose-response analysis. Note: controls for BPA were 12.8% (abnormality) and 0% (mortality); controls for E2 and GSK4716 were 2.5% (abnormality) and 0% (mortality). Statistical significance determined by one-way ANOVA on ranks (Kruskal-Wallis), Student-Newman-Keuls post-test compared to vehicle-exposed embryos, *p<0.05. Error bars depict SEM. (E) Representative photos of 5 dpf larvae showing physical effects of BPA, E2, or GSK4716 exposure. Numbers depict exposure concentrations (μM). Most common observed effects were yolk sac edema, pericardial edema, craniofacial abnormalities, or axis defects.

Tissue dose associated with waterborne BPA exposure. BPA concentration was derived from whole body homogenates (50 pooled embryos) by HPLC following 8-58 hpf static exposure. Black squares are measured values (mean ±SEM, n=6 or n=3 (1μM)); grey triangles were derived by fitting a linear regression curve to the measured values. Values are plotted as log(tissue dose) versus log(exposure concentration). Figure shows log-transformed data plotted on log-scale axes. Error bars (not visible due to data transformation to a log scale) depict SEM.

Locomotor activity of 5 dpf larvae following exposure to 0.1% DMSO (A), BPA (B), E2 or GSK4716 (C) during neurogenesis. (A) Comparison of individual control larvae on each of the days used for the BPA concentration-response (B) and BPA/E2/GSK4716 comparison (C) experiments. (A) Embryos were static exposed to 0.1% DMSO between the 8 -10 hpf start time and 58 hpf. Duration of burst activity (>5 pixels displaced during 1/30^th second between consecutive frames) per minute for each individual (n=12-16) was summed for 5 minutes in the dark following a 20 minute acclimation period in the light. Y-axis depicts average duration of burst activity per minute. (B) Embryos were static exposed to BPA (0.001 – 10 μM) between the 8 -10 hpf start time and 58 hpf, then duration of burst activity per minute for each individual (n=42-47) was summed as above. Statistical significance was determined by one-way ANOVA, Student-Newman-Keuls post-test compared to vehicle-exposed embryos (**p<0.01). (C) Activity following exposure to 0.1 μM of the classical estrogen receptor agonist E2 or the ERRγ agonist GSK4716 compared to 0.1% DMSO control or 0.1 μM BPA (n=53-57). Since this data did not pass a normality test (Shapiro-Wilk), significance was determined by one-way ANOVA on ranks (Kruskal-Wallis), Dunn's post-test, (p<0.05).

a: drainage chamber, b: holding chamber, c: manually operated entry gate to raceway, d: raceway, e: perforated shim, f: computer operated gate to arm, g: electrode plate

Number of trials for adults to learn to select the correct arm in a T-maze following exposure to BPA during development (8 – 120 hpf) (n=5). (A) Number of trials by sex to reach criterion in T-maze first reversal task. Zebrafish receive mild shock in first arm chosen in a T-maze. Criterion = 3 consecutive choices of opposite, non-shocking arm. Similar letters between exposure levels within same sex indicate no significant difference. Two-way ANOVA (p < 0.001). (B) Number of trials (sexes combined) to reach criterion in T-maze first reversal task. Zebrafish receive mild shock in first arm chosen in a T-maze. Criterion = 3 consecutive choices of opposite, non-shocking arm. One-way ANOVA (*p < 0.001). Error bars depict SEM.