Fig. 4

Sleeping pattern in 8 dpf larvae prenatally exposed. A. Average movement of control larvae across the sleep test protocol. B. Average Log2 fold change relative to controls during the night. M-exposed larvae showed a significant increased in movement. E- and EM-exposed larvae followed this same trend, although not reaching significance. C. Smoothing representation of the average movement during the night for each condition, this was analyzed using Linear mixed-modelling analysis of the group activity during the first 30 min of the night. E-, M- and EM-exposed larvae showed an abrupt increase in activity and N- and EMN- animals mostly showed a decrease in movement. D. Average number of time points with a log2 fold change greater than 1 during the night. M and EM-exposed groups had a significantly higher number of time point compare with controls. E. The probability of a larva being hyperactive (log2 fold change >1) was calculated across the night, then compared to control animals. The data shown here represent the percentage change in probability relative to the control. Animals from the M-group were almost 20% more likely to be hyperactive than controls. On the opposite N-exposed larvae showed a reduced probability of being hyperactive. B-D: * = p < 0.05 compared to controls. Evaluated using mixed-effects models, including Batch as a random effect, followed by Benjamini–Hochberg correction for multiple comparisons. C: * p < 0.05 compared to control animals. P-values were obtained using a linear mixed-effects model with condition over time as a fixed effect and subject (larva ID) and Batch as random effects. E: * = p < 0.05 compared to controls. Points show model-estimated probabilities (estimated marginal means; EMMs) on the response scale with 95% confidence intervals. Statistical significance was assessed using EMM contrasts comparing each treatment to the control (treatment-vs-control), reported as differences in predicted probability (Δp). P-values were adjusted for multiple comparisons using the Benjamini–Hochberg procedure. n ≥ 12 per condit