Optimization of a Microplate-Based Assay to Assess Esterase Activity in the Alga Pseudokirchneriella subcapitata Manuela D. Machado & Eduardo V. Soares Received: 11 May 2012 / Accepted: 6 November 2012 / Published online: 24 November 2012 # Springer Science+Business Media Dordrecht 2012 Abstract The present work describes the optimiza- tion 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 μmolL -1 fluorescein diac- etate (FDA) for 40 min allowed discrimination be- tween 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 subse- quently stained with FDA, using the optimized proce- dure. 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 concentra- tion 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 μgL -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 toxic- ity of a high number of environmental samples. Keywords Bioassay . Fluorescein diacetate (FDA) . Growth phase . Microplate assay . Metabolic activity . Selenastrum capricornutum 1 Introduction The worldwide industrialization that occurred in the last century has increased the ecological problems and triggered the need of developing acute and chronic laboratory toxicity tests for assessing the impact caused by many thousands of chemicals released in the environment. The present chronic (with microor- ganisms) and acute (using invertebrates or fishes) benchmark tests proposed by international regulatory agencies, such as the Organisation for Economic Co- operation and Development (OECD) and the United States Environmental Protection Agency (US EPA), give slow responses (within days) (OECD 1984, 1992; US EPA 2002a, b). The long time (days) required for these chronic and acute toxicity tests can alter the Water Air Soil Pollut (2013) 224:1358 DOI 10.1007/s11270-012-1358-3 M. D. Machado : E. 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 e-mail: evs@isep.ipp.pt M. D. Machado : E. V. Soares IBB-Institute for Biotechnology and Bioengineering, Centre for Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal