Incubation at 32.5 °C and above causes malformations in the zebrafish embryo

Zebrafish embryos are increasingly used for developmental toxicity screening of candidate drugs and are occasionally co-incubated with a metabolic activation system at 32 °C for 1, 2 or 4 h, depending on their developmental stage. As this temperature is higher than the optimal temperature for zebrafish embryonic development (26-28.5 °C), we investigated whether continuous incubation of zebrafish embryos from 2.5 until 96 h post fertilization (hpf) at high temperatures (30.5-36.5 °C) causes malformations. At 32.5 °C tail malformations were observed as early as 24 hpf, and these became even more prominent at 34.5 and 36.5 °C. Cardiovascular and head malformations, edema and blood accumulations throughout the body were present at 36.5 °C. Finally, temperatures higher than 28.5 °C accelerated embryonic development except for 36.5 °C, at which a lower hatching rate and hatching enzyme activity were observed. In conclusion, incubation of zebrafish embryos at 32.5 °C and above from 2.5 until 96 hpf causes malformations as early as 24 hpf.