Effect of resorcinol exposure on whole-body T4 levels at 120 hpf. Average T4 levels following continuous resorcinol exposure. Error bars show standard deviation and sample sizes are given in parentheses. Each sample consisted of 100 pooled eleutheroembryos. Different letters indicate significant differences. Treatment 9 µM was left out of the statistical analysis because only two replicates were included (see Section 2.9).

Effect of resorcinol exposure on swim bladder inflation. Percentage of non-inflated swim bladders (A) following resorcinol exposure at both 5 and 7 dpf at 28°C (experiment 5). Significant differences from the control (0 µM) are indicated with an asterisk. Effect of T4 supplementation on swim bladder inflation (B) from 5 up to 8 dpf at 26°C (experiment 6). Zebrafish eleutheroembryos were exposed to either T4, T4 in combination with 727 µM resorcinol or solely 727 µM resorcinol. Asterisks indicate significant difference in swim bladder inflation at a given day between either the control and 727 µM resorcinol exposed embryos or between 2.5, 5 or 10 nM T4 and 2.5 nM T4 + resorcinol, 5 nM T4 + resorcinol or 10 nM T4 + resorcinol exposed embryos, respectively.

Effect of resorcinol exposure on swimming activity at 5 dpf Effects of resorcinol on swimming speed (A), total time moved (B) and the total distance travelled (C). Error bars show standard deviation and sample sizes are given in parentheses. Different letters indicate significant differences. Capital and lowercase letters indicate the significant differences within inflated or uninflated swim bladders, respectively. Significant differences within a concentration (inflated vs non-inflated) are indicated with asterisks. Effect on turning angles in eleutheroembryos with inflated (D) and uninflated swim bladders (E) illustrated using radar plots. Each corner of the radar plot represents a range of different turning angles. The y-axis is represented by the concentric circles showing the average turning angle normalized to both the sum of the swimming distance and the controls.

Effect of resorcinol exposure on gene transcription of key genes of the thyroid hormone system. Relative gene transcript levels (relative to the control (0 µM)) of tpo (A), ttr (B), thraa (C), thrb (D), dio1 (E), dio2 (F), dio3a (G) and dio3b (H) following resorcinol exposure at 120 hpf. Significant differences are indicated with different letters. Inflation status of the posterior chamber of the swim bladder is indicated by either a filled bar (inflated) or an unfilled bar (uninflated). Five replicate samples where used per condition and each sample consisted of ten eleutheroembryos.

Hypothesis of how transthyretin (TTR)-binding inhibition and thyroid hormone receptor (TR) antagonism could be added into the existing fish adverse outcome pathway (AOP) network. The existing AOPs linking thyroperoxidase (TPO) inhibition to impaired anterior swim bladder inflation and altered retinal structure, AOPs 159 and 363 (https://aopwiki.org/), respectively, are indicated in grey/black. The two new candidate molecular initiating events (MIEs) that were identified in the current study as well as additional key events (KEs) related to these candidate MIEs are indicated in color (purple: TTR-binding inhibition, blue: TR antagonism). The dotted line indicates all the mechanisms that are important during embryonic development (above the line) and during larval development (below the line). Created with biorender.com.