Description:    The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata , in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmol L −1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The short-term assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC 50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L −1 , respectively. P . subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC 50 values for Cu, for cells in the stationary phase, were 443 and 268 μg L −1 , respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P . subcapitata . The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples. Content Type Journal Article Pages 1-11 DOI 10.1007/s11270-012-1358-3 Authors Manuela D. Machado, Bioengineering Laboratory-CIETI, Chemical Engineering Department, ISEP-School of Engineering of the Polytechnic Institute of Porto, Rua Dr. António Bernardino de Almeida, 431, 4200-072 Porto, Portugal Eduardo V. Soares, Bioengineering Laboratory-CIETI, Chemical Engineering Department, ISEP-School of Engineering of the Polytechnic Institute of Porto, Rua Dr. António Bernardino de Almeida, 431, 4200-072 Porto, Portugal Journal Water, Air, & Soil Pollution Online ISSN 1573-2932 Print ISSN 0049-6979 Journal Volume Volume 224 Journal Issue Volume 224, Number 1