Article Figures & Data
Figures
- Figure 1.
Image of a zebrafish larva illustrating the area used for head size measurements. Dorsal view of a zebrafish larvae embedded in low melting point agarose. Red lines outline the area measured to determine the head size of larvae. Scale bar: 150 μm.
- Figure 2.
BPA exposure can increase mortality, deformity, and delays in hatching in zebrafish. A, Five days of exposure to BPA increased mortality measured at 6 dpf according to a one-way ANOVA (F(5,174) = 3.56; p < 0.01). Tukey’s HSD post hoc tests revealed that the group exposed to 50 μm BPA had significantly (p < 0.05) more mortality compared with the groups exposed to 0, 10, and 20 μm BPA. B, BPA exposure (5 d) increased the number of deformities measured at 6 dpf as indicated by a one-way ANOVA (F(5,126) = 63.41; p < 0.001). Subsequent Tukey’s HSD post hoc tests demonstrated that the 40 and 50 μm groups had significantly (p < 0.001) more deformities than did the groups exposed to 0, 10, 20, and 30 μm BPA. Furthermore, the 30 μm BPA group had significantly (p < 0.01) more deformities than did the 0 μm BPA group. C, BPA exposure (5 d) caused hatching delays as indicated by a one-way ANOVA (F(5,136) = 11.48; p < 0.001). Tukey’s HSD post hoc tests revealed that hatching in the group exposed to 50 μm BPA was significantly (p < 0.05) more likely to be delayed compared with hatching in the 0, 10, 20, 30, and 40 μm BPA groups. Furthermore, the fish in the 10 and 30 μm BPA groups demonstrated significantly (p < 0.01) more frequent delayed hatching than did those in the 0 μm BPA group. D, Zebrafish exposed to BPA (5 d), and with no visible deformities, did not exhibit deficits in the C-start reflex as shown by a one-way ANOVA (F(3,80) = 0.23; p = 0.87). This and subsequent figures present means ± SEM; * specifies a significant difference between groups in this and subsequent figures.
- Figure 3.
Extended exposure to DMSO does not increase mortality, delays in hatching, or abnormal sensorimotor responses. A, Exposure of zebrafish larvae to DMSO (0.05%) from 0 to 5 dpf did not change the mortality rate (DMSO group = 22.00 ± 5.92%, n = 50) compared with a control group exposed only to E3 (E3 group = 18.75 ± 4.39%, n = 80) according to an unpaired t test (t(128) = 0.45, p = 0.66). B, Extended exposure (5 d) to DMSO (0.05%) did not significantly delay hatching in zebrafish compared with fish exposed to E3 solution (DMSO group = 2.50 ± 2.50%, n = 40; E3 group = 7.58 ± 3.28%, n = 66; unpaired t test; t(104) = 1.09, p = 0.28). C, An unpaired t test (t(104) = 1.45, p = 0.15) revealed no significant differences in startle response probability between the DMSO group (88.33 ± 3.00%, n = 40) and the E3 group (93.18 ± 1.87%, n = 66).
- Figure 4.
Exposure to 25 μm BPA does not alter mortality, or startle probability in zebrafish larvae, but does delay hatching. A, Bathing fish in BPA for 5 d did not significantly increase mortality (BPA group, n = 30) compared with bathing them in the vehicle (DMSO group, n = 30; unpaired t test; t(58) = 1.00, p = 0.32). B, BPA treatment significantly delayed hatching (BPA group, n = 30; DMSO group, n = 30; unpaired t test, t(58) = 2.32, p = 0.02). C, The startle probability of BPA-treated larvae (BPA group, n = 29) did not differ from that of vehicle-treated larvae (DMSO group, n = 30; unpaired t test; t(57) = 0.45, p = 0.66).
- Figure 5.
BPA exposure disrupts PPI in zebrafish larvae. Effect on PPI of exposure to 25 μm BPA for 5 d (BPA group, n = 83) compared with that of exposure to the vehicle (DMSO group, n = 83; unpaired t test, t(164) = 2.31, p < 0.05).
- Figure 6.
BPA exposure reduces STH. A, Response rates during habituation training of BPA-exposed fish (n = 20) and DMSO-treated fish (n = 20). Data were binned as a running average of five consecutive auditory pulses. According to a two-way ANOVA, the number of responses during training and the probability of startle at 30 s posttest produced a significant interaction (F(1,76) = 27.24; p < 0.01). B, Results of a one-way ANOVA, subsequent to the two-way ANOVA in A, comparing the number of startle responses during habituation training by the BPA-treated group to the DMSO-treated group. This analysis revealed that the BPA group habituated less than did the DMSO group (F(1,38) = 30.58; p < 0.001). C, Results of a one-way ANOVA comparing the number of startle responses evoked on the 30 s posttest in the BPA-treated and DMSO-treated groups (F(1,38) = 8.10; p < 0.01).
- Figure 7.
Thigmotaxis was not increased in zebrafish exposed to BPA. The mean distance from the edge of the experimental dish during swimming in the BPA-exposed group (n = 14) and the DMSO-treated group (n = 15; t test, t(27) = 0.77, p = 0.45).
- Figure 8.
BPA exposure did not modify locomotion in zebrafish larvae. A, Mean distance traveled by BPA-exposed (n = 32) and DMSO-exposed (n = 31) fish (unpaired t test; t(61) = 0.30, p = 0.77). B, Number of circles made by larvae exposed to BPA (BPA group, n = 32) and the vehicle (DMSO group, n = 31; t(61) = 1.44, p = 0.15).
- Figure 9.
Exposure to BPA did not change head size in zebrafish larvae. Mean volume of the head in BPA-treated (BPA group, n = 30) and vehicle-treated (DMSO group, n = 30) fish (t(58) = 1.63, p = 0.11).
- Figure 10.
Brain volume was not changed in zebrafish larvae by BPA exposure. A, 3D reconstruction of a larval brain. Sample confocal images of optical sections of a brain exposed to DMSO. Scale bar: 150 μm. B, Mean volumes of the forebrain (FB), midbrain (MB), and hindbrain (HB) in BPA-exposed (BPA group, n = 15) fish compared with the vehicle-exposed fish (DMSO group, n = 15). A two-way ANOVA revealed no significant interaction or group effect (interaction, F(2,84) = 0.03; p = 0.75: group, F(1,84) = 0.10; p = 0.97. C, Mean volume of the whole brain in BPA-exposed fish (BPA group, n = 15) and vehicle-exposed fish (DMSO group, n = 15). The difference between the two groups was not significant (t(28) = 0.23, p = 0.82).
Tables
Data structure Type of test Power (α = 0.05) a(Fig. 2A) Non-normally distributed One-way ANOVA test 0.92 b(Fig. 2B) Non-normally distributed One-way ANOVA test 1.00 c(Fig. 2C) Non-normally distributed One-way ANOVA test 1.00 d(Fig. 2D) Normally distributed One-way ANOVA test 0.09 e(Fig. 3A) Normally distributed Unpaired t test 0.07 f(Fig. 3B) Non-normally distributed Unpaired t test 0.19 g(Fig. 3C) Normally distributed Unpaired t test 0.30 h(Fig. 4A) Non-normally distributed Unpaired t test 0.17 i(Fig. 4B) Non-normally distributed Unpaired t test 0.63 j(Fig. 4C) Normally distributed Unpaired t test 0.07 k(Fig. 5) Normally distributed Unpaired t test 0.63 l(Fig. 6A) Normally distributed Two-way ANOVA test (interaction) 1.00 m(Fig. 6B) Normally distributed One-way ANOVA test 1.00 n(Fig. 6C) Non-normally distributed One-way ANOVA test 0.79 o(Fig. 7) Normally distributed Unpaired t test 0.12 p(Fig. 8A) Normally distributed Unpaired t test 0.06 q(Fig. 8B) Normally distributed Unpaired t test 0.30 r(Fig. 9) Normally distributed Unpaired t test 0.36 s(Fig. 10B) Normally distributed Two-way ANOVA test (interaction) 0.05 t(Fig. 10B) Normally distributed Two-way ANOVA test (main effect) 0.06 u(Fig. 10C) Normally distributed Unpaired t test 0.06 - Table 2
Mortality, deformity, hatching delays, startle response in zebrafish larvae exposed to BPA
BPA Mortality; mean and SEM Deformity; mean and SEM Hatching delays; mean and SEM Startle response; mean and SEM 0 μm 16.67 ± 6.92%
n = 300.00 ± 0.00%
n = 254.00 ± 4.00%
n = 2588.67 ± 4.38%
n = 2510 μm 13.33 ± 6.31%
n = 3011.54 ± 6.39%
n = 2644.83 ± 9.40%
n = 2991.30 ± 3.61%
n = 2320 μm 20.00 ± 7.43%
n = 308.33 ± 5.76%
n = 2428.00 ± 9.17%
n = 2593.18 ± 4.74%
n = 2230 μm 33.33 ± 8.75%
n = 3030.00 ± 10.51%
n = 2055.00 ± 11.41%
n = 2092.86 ± 4.85%
n = 1440 μm 23.33 ± 7.85%
n = 30100.00 ± 0.00%
n = 2356.52 ± 10.57%
n = 2350 μm 53.33 ± 9.26%
n = 30100.00 ± 0.00%
n = 1495.00 ± 5.00%
n = 20
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