Exposure design of iFEDT experiment and cross design experiment: Zebrafish (Danio rerio) parents and offspring were exposed to PTU (iFEDT, results published in Pannetier et al. 2023a and Goelz et al. 2023). For the present study, eggs from the non-exposed parental generation (0 mg PTU/L) were exposed to either 0 or 78 mg PTU /L. Likewise, eggs from the parental generation exposed to 78 mg PTU/L were exposed to either 0 or 78 mg PTU /L. Between 4 and 18 days of exposure of the parental generation, eggs were collected and directly exposed for 4.5 d for evaluation of photomotor response and gene expression, or for 5 d for measurement of area of swim bladder.

Residual effect (difference between observed surface and expected surface according to standard length) of parental and/or embryonic exposure of zebrafish (Danio rerio) to propylthiouracil (PTU) on the surface of the posterior swim bladder (mean ± 95 % confidence interval) of 5 d old zebrafish embryos. Control: 4 replicates, N = 81; embryonic exposure: 5 replicates, N = 117; parental exposure: 3 replicates, N = 76; combined parental and embryonic exposure: 4 replicates, N = 86. * Significant main effect of “parental exposure” (p < 0.05).

Distance moved per minute by 4.5 d old zebrafish (Danio rerio) embryos during the green light-dark locomotion test, after parental exposure to propylthiouracil, PTU (A: blue line, 4 replicates, N = 88), after embryonic exposure (B: red line, 6 replicates, N = 131) or after combined exposure of parents and embryos (C: green line, 5 replicates, N = 104) in comparison to the control (black lines in A-C, 5 replicates, N = 109). Areas shaded in grey represent 95% confidence intervals of the control group, and the bar on top of the figure represents the green colour light or the darkness of each of the 10 min phases of the test.

Total distance moved (mean ± 95 % confidence interval) by 4.5 d old zebrafish (Danio rerio) embryos in the green light-dark locomotion test, in response to exposure of parents and/or embryos to PTU during the a) dark phases (10 × 10 min.) or b) green light phases (10 × 10 min.). Replicates and individual observations: control: 5 replicates, N = 109; embryonic exposure: 6 replicates, N = 131; parental exposure: 4 replicates, N = 88; combined parental and embryonic exposure: 5 replicates, N = 104. Different letters: significant differences between groups by Post-hoc Tukey test; capital and lowercase letters: differences in the main effect of embryonic exposure. * Statistically significant effect of main effect parental exposure (p < 0.05) by LMM and Kenward-Roger F-statistics (see Table 2).

Expression ( ) of A) thyroid peroxidase (tpo) and B) phosphodiesterase (pde6h) genes (mean ± 95 % confidence interval) by 4.5 d old zebrafish (Danio rerio) embryos in response to exposure of parents and/or embryos to propylthiouracil PTU. Gene expression normalised to the reference gene 18S rRNA. Control: 6 replicates; embryonic exposure: 5 replicates; parental exposure: 5 replicates; combined parental and embryonic exposure: 5 replicates. Each replicate is a sample from ca 30 pooled embryos. Different letters: significant differences between groups by Post-hoc Tukey test; capital and lowercase letters: differences in the main effect of parental exposure. Statistically significant effects of embryonic exposure: ** p < 0.01; *** p < 0.001 (ANCOVA; see Table 3).