Franklin N M, Adams M S, Stauber J L, Lim R P
Centre for Advanced Analytical Chemistry, CSIRO Energy Technology, Bangor, New South Wales, Australia.
Arch Environ Contam Toxicol. 2001 May;40(4):469-80. doi: 10.1007/s002440010199.
A rapid toxicity test based on inhibition of esterase activity in marine and freshwater microalgae (Selenastrum capricornutum, Chlorella sp., Dunaliella tertiolecta, Phaeodactylum tricornutum, Tetraselmis sp., Entomoneis cf. punctulata, Nitzschia cf. paleacea) was developed using flow cytometry. Uptake of fluorescein diacetate (FDA) was optimized for each species by varying the substrate concentration, incubation time, and media pH. Propidium iodide (PI) was utilized to assess membrane integrity. The optimized FDA/PI staining procedure was then used to assess the toxicity of copper in short-term exposures (1-24 h). Esterase activity was a sensitive indicator of copper toxicity in S. capricornutum and E. cf. punctulata. As copper concentrations increased, esterase activity decreased in a concentration-dependent manner. The 3- and 24-h EC50 values (based on mean activity states) were 112 microg Cu L(-1) (95% confidence limits 88-143) and 51 microg Cu L(-1) (95% confidence limits 38-70) for S. capricornutum and 47 microg Cu L(-1) (95% confidence limits 43-51) and 9.1 microg Cu L(-1) (95% confidence limits 7.6-11) for E. cf. punctulata, respectively. This enzyme inhibition endpoint showed similar sensitivity to chronic growth rate inhibition in E. cf. punctulata (48-h and 72-h EC50 values of 17 and 18 microg L(-1), respectively) but was less sensitive compared to growth for S. capricornutum (48-h and 72-h EC50 values of 4.9 and 7.5 microg L(-1), respectively). For the other five species tested, inhibition of FDA fluorescence was relatively insensitive to copper, even at copper concentrations that severely inhibited cell division rate. These short-term bioassays that detect sublethal endpoints may provide a more rapid and cost-effective way of monitoring contaminant impacts in natural waters.
基于海洋和淡水微藻（羊角月芽藻、小球藻、三角褐指藻、三角褐指藻、四爿藻、类点纹内丝藻、类苍白菱形藻）酯酶活性抑制的快速毒性试验采用流式细胞术开展。通过改变底物浓度、孵育时间和培养基pH值，对每种微藻的荧光素二乙酸酯（FDA）摄取进行了优化。碘化丙啶（PI）用于评估细胞膜完整性。然后，采用优化的FDA/PI染色程序评估短期暴露（1 - 24小时）中铜的毒性。酯酶活性是羊角月芽藻和类点纹内丝藻中铜毒性的敏感指标。随着铜浓度增加，酯酶活性以浓度依赖方式降低。羊角月芽藻的3小时和24小时EC50值（基于平均活性状态）分别为112微克铜/升（95%置信限88 - 143）和51微克铜/升（95%置信限38 - 70），类点纹内丝藻分别为47微克铜/升（95%置信限43 - 51）和9.1微克铜/升（95%置信限7.6 - 11）。该酶抑制终点在类点纹内丝藻中对慢性生长速率抑制显示出相似的敏感性（48小时和72小时EC50值分别为17和18微克/升），但与羊角月芽藻的生长相比敏感性较低（48小时和72小时EC50值分别为4.9和7.5微克/升）。对于测试的其他五个物种，即使在严重抑制细胞分裂速率的铜浓度下，FDA荧光抑制对铜也相对不敏感。这些检测亚致死终点的短期生物测定可能为监测天然水体中污染物影响提供一种更快速且经济高效的方法。
