Kent Michael L, Buchner Cari, Barton Carrie, Tanguay Robert L
Department of Microbiology, and Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon, USA.
Dis Aquat Organ. 2014 Jan 16;107(3):235-40. doi: 10.3354/dao02683.
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 to 50 ppm unbuffered chlorine solution for 5 to 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, but is less effective against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbuffered and buffered chlorine solutions to embryos exposed at 6 or 24 h post-fertilization (hpf) to determine whether 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 pH, and chlorine causes elevated pH. Consistent with this, we found that unbuffered 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 embryos for 5 min with unbuffered chlorine solution at 100 ppm.
受精鱼卵的表面消毒在水产养殖中被广泛应用，以减少产卵期间亲鱼可能释放的卵外病原体，这在斑马鱼（Danio rerio）研究实验室中是常规操作。大多数实验室使用约25至50 ppm的无缓冲氯溶液处理5至10分钟。用氯处理胚胎对许多革兰氏阴性菌、病毒和原生动物的滋养体阶段具有显著的杀菌作用，但对原生动物的包囊或孢子阶段以及某些分枝杆菌属效果较差。因此，我们评估了无缓冲和缓冲氯溶液对受精后6小时或24小时（hpf）暴露的胚胎的毒性，以确定是否可以使用更高浓度来处理斑马鱼胚胎。我们的大多数实验使用的是远交系（5D），以死亡率和畸形作为终点指标。我们发现，6 hpf的胚胎对氯的毒性作用始终比24 hpf的胚胎更具抗性。氯在较低pH值时毒性和杀菌性更强，并且氯会导致pH值升高。与此一致的是，我们发现相应浓度下，无缓冲氯溶液（pH约为8 - 9）比缓冲至pH 7的溶液毒性更小。基于我们在此的研究结果，我们建议用100 ppm的无缓冲氯溶液处理6 hpf的胚胎10分钟，处理24 hpf的胚胎5分钟。
