Single cell level microalgal ecotoxicity assessment by confocal microscopy and digital image analysis

In ecotoxicological studies involving environmental contaminants, rapid and multi-parametric optical detection based methods have definite advantages over traditional growth inhibition assays. In this context, a confocal laser scanning microscopy (CLSM) based method to assess ecotoxicity arising out of biocide insult to marine microalgae is reported. Using this technique, the effect of in-use concentrations of chlorine (an oxidizing biocide) on a marine diatom (Cocconeis scutellum Ehrenb) was determined based on inhibition of chlorophyll autofluorescence and esterase activity (probed by fluorescein diacetate (FDA) staining). Determination of mean fluorescence intensity (MFI) per cell by collecting auto-fluorescence from single cells in x, y and z dimensions permitted reproducible toxicity evaluation at single-cell level. Chlorine-induced inhibition of autofluorescence in laboratory cultures was dose-dependent. Additional data on metabolic activity of the diatom cells following chlorine exposure was collected by FDA staining. Our results demonstrate that chlorine, an antifouling biocide commonly used in cooling water systems, causes significant reduction in chlorophyll autofluorescence and esterase activity in diatoms in short-term exposure experiments. Tests employing multiple organisms and multiple toxicity endpoints are superior to standard algal growth inhibition assays for they provide a better understanding of algal-algal interactions and real impact in the environment. The combined autofluorescence-FDA technique described here is rapid and has clear advantages in terms of using environmentally relevant toxicant and cell concentrations. Additional microalgal species and toxicity end points can be employed in order to develop multi-species and multi-parameter bioassay using confocal microscopy.