Flow cytometric analysis of the cadmium-exposed green alga Chlamydomonas reinhardtii (Chlorophyceae)

A flow-cytometric method was developed and evaluated as a rapid ecotoxicological tool using cultures of the microalga Chlamydomonas reinhardtii (Chlorophyceae) under cadmium exposure. Three staining protocols were developed to assess the toxicological impact of this trace metal on algal physiology. Algal cells were exposed to total nominal cadmium concentrations of 5 and 100 mM. After 48 and 72 h exposure the fluorescent probes, fluorescein diacetate (FDA), dihydrorhodamine 123 (DHR123) and tetramethylrhodamine methyl ester (TMRM), were used to assess esterase activity, presence of reactive oxygen species and membrane potential, respectively. Results indicated that cell size, cell granularity and internal complexity were influenced by cadmium, confirming earlier findings on ultrastructural changes in microalgae exposed to trace metals. An increase was observed in the percentage of DHR123 positive cells as well as in their mean fluorescence intensity, on increasing cadmium concentration, confirming that this metal exerts its toxicity through the generation of reactive oxygen species. Furthermore, cadmium exposure resulted in an increase in esterase activity, as reflected in fluorescein fluorescence. We suggest this observation was linked to possible detoxification activity and defence mechanisms. Measurements of control samples during protocol optimization for TMRM proved not to be reproducible, leading us to defer any judgment on results of exposed samples and to conclude that TMRM does not seem suitable for flow cytometric use in algae. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.