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Fig. S15 Commitment to grade 3 phenotype occurs during the first 3 h post-exposure. (A) 7 dpf zebrafish larvae were exposed to 3 µM chlorpyrifos-oxon (CPO) and the prevalence of grade 3 phenotype was evaluated after 3, 6 and 24 h exposure. No differences en the grade 3 prevalence were found between larvae exposed to CPO for 6 and 24 h [n= 6 experimental groups with 8 larvae each; p=0.492, Student’s t-test (t(10) = 0.0199)]. (B-D) 7 dpf zebrafish larvae were exposed to 3 µM CPO for 1 h (B), 2 h (C) and 3 h (D) and then transferred to clean water for additional 23, 22 and 21 h. For each experimental group, prevalence of grade 3 phenotype was analysed at the end of the CPO exposure period and after the period in clean water. At 8 dpf, no differences in grade 3 prevalence were found between the larvae continuously exposed to 3 µM CPO for 24 h and those exposed for only 3 h and then transferred to clean water for additional 21 h [n= 6 experimental groups with 8 larvae each; p=0.212, Student’s t-test (t(10) = 0.836)]. (E) Lateral view of a single representative zebrafish grade 3 larva exposed to 3 uM CPO for 3 h (top) and then transferred to clean water for additional 21 h (bottom). At 3 h are already evident the changes in the morphology of the head typical of grade 3 larvae. No recovery of the phenotype was found after 21 h of depuration in clean water (bottom). By contrast, the phenotype of the larvae after the depuration period was indistinguishable of the larvae exposed continuously during 24 h. Data presented as mean ± s.e.m.