Toxicity of chlorine to zebrafish embryos

This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Inter-Research and can be found at: http://www.int-res.com/journals/dao/dao-home/.Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce\ud extraovum pathogens that may be released from brood fish during spawning, and this is routinely\ud 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\ud of protozoa, it has reduced efficacy against cyst or spore stages of protozoa and certain\ud Mycobacterium spp. Therefore, we evaluated the toxicity of unbufferred and buffered chlorine\ud solution to embryos exposed at 6 or 24 hours post-fertilization (hpf) to determine if higher\ud concentrations can be used for treating zebrafish embryos. Most of our experiments entailed\ud using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6\ud hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of\ud chlorine. Chlorine is more toxic and germicidal at lower pHs, and chlorine causes elevated pH.\ud Consistent with this, we found that unbufferred chlorine solutions (pH ca 8-9) were less toxic at\ud corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we\ud recommend treating 6 hpf embryos for 10 min and 24 hpf for 5 min with unbuffered chlorine\ud solution at 100 ppm. One trial indicated that AB fish, a popular outbred line, are more\ud 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