Figure 1. The toxicity effects of As₂O₃ on zebrafish (Danio rerio) embryos. (A) Exposure to As₂O₃ increased embryo mortality in a dose-dependent manner. (B) LC₅₀ for As₂O₃. (C) Exposure to 30 µM and 40 µM As₂O₃ significantly decreased the incidence of tail coiling in 24 hpf embryos. (D) Exposure to 40 µM As₂O₃ significantly decreased the heartbeat of embryos examined at 48 hpf. (E) Exposure to increased As₂O₃ concentrations significantly delayed hatching between 48 hpf and 72 hpf. Data are presented as mean ± SEM of triplicate wells (n = 90 embryos per exposure group), with significant differences relative to the control group. ∗ (p ≤ 0.05); ^a no tail coiling, and heartbeats were recorded for 50 µM As₂O₃ exposed embryos, as all embryos were dead at 24 hpf.

Figure 2. Effects of embryonic As₂O₃ exposure in 6 dpf larvae and adult zebrafish. At 6 dpf, As₂O₃ exposure increased swim bladder inflation/volume (B), although its anterior–posterior length was shortened (A). (C) With change in swim bladder volume, As₂O₃ exposure also affected swimming behavior, with reduced down preference. (D) In adults, embryonic As₂O₃ exposure resulted in a reduced percentage of survivability in As₂O₃ exposed larvae (x), which was mainly detected at 13–23 dpf, compared to the control group (□). Data are presented as mean ± SEM, (∗ p ≤ 0.05) n = 30–90 per group), with significant differences relative to the control group ∗ (p ≤ 0.05).

Figure 3. Effects of long-term impairment of 30 µM As₂O₃ exposure on exploratory activity. Exploratory activity was persistently decreased in larvae from 6 dpf (A) and 11 dpf (B) to adult stage (C). Data are presented as mean ± SEM, (∗ p ≤ 0.005), n = 30 larvae per group, n = 22 adults per group), with significant differences relative to the control group, ∗ (p ≤ 0.05).

Figure 4. Effects of embryonic As₂O₃ exposure on the color preference of larval (11 dpf, A) and adult zebrafish (B). (A) In the 11 dpf larval test, a cross maze with four different-colored cambers was used. Reduction in color preference for blue in As₂O₃-exposed larvae compared to the control group. No significant differences were observed in color preference for red and green in control or As₂O₃-exposed larvae. However, MK-801-exposed larvae showed no clear color preference (B) Five-minute video tracking of color preference in adult fish after associative learning. Adult fish were acclimated to the three-chamber maze for 6 days with red food association before the test on the 7th day. As₂O₃-exposed zebrafish showed no significant preference for either green or red color Data are presented as mean ± SEM, (∗ p ≤ 0.005). n = 30 larvae per group, n = 22 adults per group. ∗ Significance at p ≤ 0.05 between left/center/right and between two color arms for each test, n.s: not significant.

Figure 5. OPLS−DA score plot and loading scatter plot (A,B) of negative and positive (C,D) modes based on the zebrafish larvae normalized data exposed to 30 µM As₂O₃ in comparison to control larvae. (E) Differential expression of metabolites in As₂O₃-exposed larvae produced by hierarchical clustering of the most significantly upregulated (red) and downregulated (blue) metabolites obtained from in negative- and positive-ion modes compared to the control group based on the log2 fold change value. (F) Metabolic set enrichment analysis of lipid metabolites in 6 dpf zebrafish showed the biosynthesis of unsaturated fatty acids, arachidonic metabolism, and sphingolipid were dysregulated after embryonic exposure to As₂O₃. Color intensity (yellow–to–orange/red) represents increasing statistical significance, whereas circular diameter is related to pathway impact. The graph was obtained by plotting−log of p-values from pathway enrichment analysis on the y-axis and the pathway impact values derived from pathway topology analysis on the x-axis.

Figure 6. Embryonic exposure to As₂O₃ induced alterations in transcriptional regulation of ASD-associated genes. As₂O₃ exposure resulted in significant overexpression of adsl and shank3a, whereas tsc1b was downregulated. Data are presented as the mean ± SEM, (* p < 0.05) (n = 90 per group).