Applicability of Gobiocypris rarus in Fish Embryo Acute Toxicity Test
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摘要: 为了验证我国本土鱼种稀有鮈鲫（Gobiocypris rarus）在鱼类胚胎急性毒性试验的适用性，评价其在鱼类替代试验中的应用潜力，选取3，4-二氯苯胺和五水硫酸铜，按照《OECD化学品测试准则No.236鱼类胚胎急性毒性试验》，分别开展6次稀有鮈鲫胚胎急性毒性试验，通过评价试验结果的重复性，验证稀有鮈鲫是否适用于鱼类胚胎急性毒性试验.结果表明：3，4-二氯苯胺和五水硫酸铜对稀有鮈鲫胚胎的96 h LC50（96 h半数致死浓度）平均值（x）分别为12.8和1.76 mg/L，标准差（s）分别为1.70和0.197 mg/L；变异系数（CV）分别为13.3%和11.2%，均小于30%；两种化学品6次试验的96 h LC50均在各自x±2s范围内.研究显示，稀有鮈鲫胚胎的形态特征、发育过程及孵化时间等生物学特征均与斑马鱼类似，3，4-二氯苯胺和五水硫酸铜这两种化学品的胚胎急性毒性试验结果具有良好的重复性，其敏感性也与成鱼类似.因此，稀有鮈鲫作为一种我国本土的标准试验鱼种，具有鱼类胚胎急性毒性试验的应用潜力.Abstract: The study is aimed to validate whether Chinese rare minnow (Gobiocypris rarus) is applicable for fish embryo acute toxicity test, and also to evaluate the possibility of using its embryo as an alternative to test fish in acute toxicity tests, which lay a foundation for producing the related national standard of chemical testing. According to OECD Guidelines for the Testing of Chemicals No.236:Fish Embryo Acute Toxicity Test, six independent embryo tests were performed using newly fertilized eggs of G. rarus that were exposed to two reference substances, i.e. 3, 4-dichloroaniline and copper sulfate pentahydrate, respectively. The repeatability of the test results was evaluated to validate whether G. rarus was applicable for fish embryo acute toxicity test. The mean estimates of the concentration causing 50% mortality (LC50, x) of G. rarus embryos for a period of 96 h were 12.8 mg/L for 3, 4-dichloroaniline and 1.76 mg/L for copper sulfate pentahydrate, with their corresponding standard deviations of 1.70 and 0.197 mg/L, respectively; and their coefficient of variation (CV) of LC50 values were 13.3% and 11.2%, which was less than 30%. The 96 h LC50 values of six runs for each chemical fallen into the range of (x±2s). The characteristics of G. rarus embryos regarding morphology, stages of development and hatch duration were quite similar to those of Danio rerio, and toxicity tests for two chemicals using G. rarus eggs presented a promising result, too. The sensitivity in G. rarus embryos' response to the two chemicals was comparable to that of adult fish. Therefore, the endemic fish species, G. rarus could serve as a candidate organism for fish embryo acute toxicity test.
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Key words:
- Gobiocypris rarus /
- embryo /
- acute toxicity /
- repeatability
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图 1 稀有鮈鲫与斑马鱼胚胎卵壳膜比较(原肠期) (体视镜放大40倍)
Figure 1. Comparison of the chorion between G. rarus and D. rerio at gastrula stage (×40 under stereoscope)
图 2 稀有鮈鲫胚胎在3, 4-二氯苯胺中暴露96 h后的异常表征(体视镜放大20倍)
Figure 2. Abnormalities on G. rarus embryos exposed to 3, 4-dichloroaniline for 96 h (×20 under stereoscope)
图 3 稀有鮈鲫胚胎在五水硫酸铜中暴露96 h后的异常表征(体视镜放大20倍)
Figure 3. Abnormalities on G. rarus embryos exposed to copper(Ⅱ) sulfate pentahydrate for 96 h (×20 under stereoscope)
表 1 胚胎急性毒性试验死亡表征及观察现象[7]
Table 1. The indicators of lethality and four apical observations in fish embryo acute toxicity test[7]
死亡表征 观察现象 卵凝结 卵黄凝结的胚胎肉眼观察呈乳白色，在显微镜下呈黑色.可分别在24、48、72和96 h观察 体节未形成 在(26±1)℃下，正常发育的胚胎在24 h后会形成大约20节体节.体节发育正常的胚胎会有自主运动(来回收缩)，自主运动的出现可表明胚胎体节发育成型.体节缺失可分别在24、48、72和96 h观察.如果暴露24 h后体节未形成，可归因于发育迟缓；如果48 h后仍未形成，可判定死亡 尾部未分离 正常发育的胚胎，在其身体后部伸长后可观察到尾部与卵黄囊分离的现象.尾部未分离可分别在24、48、72和96 h观察 失去心跳 在(26±1)℃下，正常发育的胚胎在48 h后会有心跳.心率不齐或有心跳但腹部无血流循环均不能判定胚胎死亡.判定胚胎无心跳，至少在放大80倍下观察持续1 min或以上.无心跳可分别在48、72和96 h观察 
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表 2 3, 4-二氯苯胺和五水硫酸铜对稀有鮈鲫胚胎的96 h LC50
Table 2. The 96 h LC50 values of 3, 4-dichloroaniline and copper(Ⅱ) sulfate pentahydrate in G. rarus embryo acute toxicity test
试验序号 96 h LC50/(mg/L) 3, 4-二氯苯胺 五水硫酸铜 D-1 14.6 2.07 D-2 12.9 1.91 D-3 10.4 1.73 D-4 12.0 1.57 D-5 14.8 1.60 D-6 11.9 1.65
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表 3 3, 4-二氯苯胺和五水硫酸铜对稀有鮈鲫的FET和AFT试验结果与预测结果
Table 3. Both tested and predicted results of 3, 4-dichloroaniline and copper(Ⅱ) sulfate pentahydrate in FET and AFT for G. rarus mg/L
化学品 FET LC50试验值 AFT LC50预测值 AFT LC50试验值 3, 4-二氯苯胺 12.8 20.4 6.59 五水硫酸铜 1.76 2.79 1.29
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