Evaluation of the utility of six measures for algal (Microcystis aeruginosa, Planktothrix agardhii and Pseudokirchneriella subcapitata) viability

Abstract

Standard algal toxicity tests are used to discern responses of algae to a variety of exposures including pesticides, personal care products and complex mixtures such as runoff and effluents. There are concerns regarding the accuracy, precision and utility of algal viability measures used as endpoints in algal toxicity test protocols. To definitively evaluate six algal viability measures, algae were heat-treated to produce known live:dead cell ratios. Cultures of two prokaryotic algae (Microcystis aeruginosa and Planktothrix agardhii) and a eukaryotic alga (Pseudokirchneriella subcapitata) were boiled for five minutes and mixed after cooling with untreated cultures to produce suspensions of 0%, 25%, 50%, 75% and 100% live algal cells. Optical microscopy was used to assess the viability of algae on a cell-by-cell basis by measuring cell density, uptake of a vital stain (neutral red) and exclusion of a mortal stain (erythrosin b). Aggregate measures of algal cell viability included chlorophyll a concentrations, pheophytin a concentrations and respiration (measured as 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium formazan absorbance (INT)). Cell densities, erythrosin b stained cells and chlorophyll a concentrations correlated with viable M. aeruginosa, P. agardhii and P. subcapitata cells (R(2)=0.97-0.78, 0.98-0.85 and 0.99-0.97 respectively). Pheophytin a concentrations and neutral red stained cells did not correlate with viable algae (R(2)=0.41-0.01 and 0.15-0.03 respectively). For INT formazan absorbance, 50%, 75% and 100% viable algae had greater variances and did not strongly correlate (R(2)=0.75-0.54). This result was likely confounded by respiration associated with resident bacteria. Three of the six methods provided accurate and precise information regarding the viability of both prokaryotic and eukaryotic algae. These methods also have a relatively low initial expense and can be used widely.

Keywords: Algae; Biomarkers; Cell density; Chlorophyll a; Endpoints; Pheophytin a.