甲基异噻唑啉酮对斑马鱼胚胎的急性毒性和机制研究

Acute Toxicity and Toxicology of Methylisothiazolinone to Zebrafish Embryos

甲基异噻唑啉酮(methylisothiazolinone,MIT)作为防腐剂,广泛用于个人护理品、日用品和涂料中。MIT随着污水进入地表水循环,普遍存在于水体中,但目前关于MIT对水生生物毒性的研究还比较少。本文以模式生物斑马鱼的胚胎作为受试对象,评价MIT对斑马鱼胚胎的毒性。将受精后3 h的健康斑马鱼胚胎暴露于梯度浓度的MIT下,观察其对胚胎生长发育的影响,用吖啶橙(AO)染色检测细胞凋亡情况。结果发现,48 h暴露浓度大于1.0 mg·L-1的胚胎孵化被显著抑制,72 h浓度大于1.52 mg·L-1的幼鱼心率显著降低,统计96 h幼鱼死亡和畸形数,并重复验证和计算得到96 h半致死浓度(96 h 50%lethal concentration,96 h-LC50)为6.15 mg·L-1,96 h半致畸浓度(96 h 50%teratogenesis concentration,96 h-TC50)为3.89 mg·L-1,测量96 h胚胎体长,分析最小生长抑制浓度(minimum concentration to inhibit growth,MCIG)为2.31 mg·L-1,AO染色显示72 h胚胎的凋亡细胞主要集中在脑部和尾部。不同时期下镜检观察到,胚胎出现的畸形主要包括尾部发育不良,脊柱弯曲,卵黄囊水肿和心包水肿。此外,高浓度处理组24 h胚胎自主抽动次数增加,72 h和96 h活动能力减弱,触碰反应迟钝。因此,推断MIT对斑马鱼胚胎的发育有较大影响,同时有一定的神经毒性。根据《危险化学品鱼类急性毒性分级试验方法》,判定MIT对斑马鱼胚胎为高毒,该毒性实验结果可为MIT在工业生产和环境中的风险管理提供依据。

Personal care products, daily necessities and coatings are widely used in daily life, and in most of them methylisothiazolinone （MIT） is used as a preservative. As a widespread pollutant in surface water, however, few toxicologic studies on MIT to aquatic organisms are reported. Acute toxicity test was conducted to study the effects of MIT on zebrafish embryos. 3 hours post-fertilization （3 hpf） zebrafish embryos were exposed to a gradient of concentration of MIT solutions. Meanwhile we used acridine orange （AO） staining to detect cell apoptosis in vivo and observed embryonic morphologic change after 24 h, 48 h, 72 h and 96 h exposure to MIT under a stereoscopic microscope. Results show that the embryo hatching rate and heart rate was significantly decreased under the concentration higher than 1.0 mg·L^-1 after 48 h and 1.52 mg·L^-1 after 72 h respectively. We found that 96 h half lethal concentration （96 h-LC50） and 96 h half teratogenic concentration （96 h-TC50） were 6.15 mg·L^-1 and 3.89 mg·L^-1 respectively. The 96 h minimum growth inhibitory concentration （MCIG） was found to be 2.31 mg·L^-1. In addition, the results of acridine orange （AO） staining showed that in the concentration of 1.0 and 2.0 mg·L^-1 cell apoptosis appeared mainly in brain and tail after 72 h. Microscopic observation also found that the malformation included caudal dysplasia syndrome, spinal curvature, yolk edema and pericardial edema, most of which were body axis curving. Moreover, in the concentrations greater than 1.52 mg·L^-1 independent twitch in 24 h embryos treatment groups increased significantly compared to control group. Furthermore, the embryos locomotion decreased, and touch response became sluggish when exposed to MIT up to 72 h and 96 h. It is concluded that MIT has both developmental toxicity and neurotoxicity on zebrafish embryos.