Development of an Improved Rapid Enzyme Inhibition Bioassay with Marine and Freshwater Microalgae Using Flow Cytometry

Abstract

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 μg Cu L⁻¹ (95% confidence limits 88–143) and 51 μg Cu L⁻¹ (95% confidence limits 38–70) for S. capricornutum and 47 μg Cu L⁻¹ (95% confidence limits 43–51) and 9.1 μg 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 μg 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 μg 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.