Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce
extraovum pathogens that may be released from brood fish during spawning, and this is routinely
used in zebrafish (Danio rerio) research laboratories. Most laboratories use approximately 25-50 ppm unbuffered chlorine solution for 5-10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages
of protozoa, it has reduced efficacy against cyst or spore stages of protozoa and certain
Mycobacterium spp. Therefore, we evaluated the toxicity of unbufferred and buffered chlorine
solution to embryos exposed at 6 or 24 hours post-fertilization (hpf) to determine if higher
concentrations can be used for treating zebrafish embryos. Most of our experiments entailed
using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6
hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of
chlorine. Chlorine is more toxic and germicidal at lower pHs, and chlorine causes elevated pH.
Consistent with this, we found that unbufferred chlorine solutions (pH ca 8-9) were less toxic at
corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we
recommend treating 6 hpf embryos for 10 min and 24 hpf for 5 min with unbuffered chlorine
solution at 100 ppm. One trial indicated that AB fish, a popular outbred line, are more
susceptible to toxicity than 5Ds. This suggests that variability between zebrafish lines occurs, and researchers should evaluate each line or strain under their particular laboratory conditions for selection of the optimum chlorine treatment procedure.Keywords: Zebrafish, Danio rerio, Chlorine, Mortality, Malformation
