Development of flow cytometry-based algal bioassays for assessing toxicity of copper in natural waters

Copper toxicity to the freshwater algae Selenastrum capricornutum and Chlorella sp. and the marine algae Phaeodactylum tricornutum and Dunaliella tertiolecta was investigated using different parameters measured by flow cytometry: cell division rate inhibition, chlorophyll a fluorescence, cell size (i.e., light-scattering), and enzyme activity. These parameters were assessed regarding their usefulness as alternative endpoints for acute (1-24 h) and chronic (48-72 h) toxicity tests. At copper concentrations of 10 micrograms/L or less, significant inhibition (50%) of the cell division rate was observed after 48- and 72-h exposures for Chlorella sp., S. capricornutum, and P. tricornutum. Bioassays based on increases in algal cell size were also sensitive for Chlorella sp. and P. tricornutum. Copper caused both chlorophyll a fluorescence stimulation (48-h EC50 of 10 +/- 1 micrograms Cu/L for P. tricornutum) and inhibition (48-h EC50 of 14 +/- 6 micrograms Cu/L for S. capricornutum). For acute toxicity over short exposure periods, esterase activity in S. capricornutum using fluorescein diacetate offered a rapid alternative (3-h EC50 of 90 +/- 40 micrograms Cu/L) to growth inhibition tests for monitoring copper toxicity in mine-impacted waters. For all the effect parameters measured, D. tertiolecta was tolerant to copper at concentrations up to its solubility limit in seawater. These results demonstrate that flow cytometry is a useful technique for toxicity testing with microalgae and provide additional information regarding the general mode of action of copper (II) to algal species.