Journal of Hazardous Materials 296 (2015) 82–92 Contents lists available at ScienceDirect Journal of Hazardous Materials jo ur nal ho me p ag e: www.elsevier.com/locate/jhazmat Responses of the alga Pseudokirchneriella subcapitata to long-term exposure to metal stress Manuela D. Machado a,b , Ana R. Lopes c , Eduardo V. Soares a,b,∗ a Bioengineering Laboratory, Chemical Engineering Department, ISEP-School of Engineering of Polytechnic Institute of Porto, Porto, Portugal b CEB-Centre of Biological Engineering, University of Minho, Braga, Portugal c LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal h i g h l i g h t s • Heavy metals provoke a perturbation of the physiological status of Pseu- dokirchneriella subcapitata. • Cd(II), Cr(VI) and Cu(II), at high concentrations, cause the loss of membrane integrity. • Cd(II), Cr(VI), Cu(II) and Zn(II) inhibit esterase activity in a dose dependent manner. • Heavy metals affect mitochondrial function and photosynthetic activity. • Fluorescent probes are a useful tool in the identification of toxicity targets of the heavy metals. g r a p h i c a l a b s t r a c t a r t i c l e i n f o Article history: Received 30 July 2014 Received in revised form 7 March 2015 Accepted 8 April 2015 Available online 9 April 2015 Keywords: Esterase activity Heavy metals toxicity Membrane integrity Mitochondrial membrane potential Photosynthetic activity a b s t r a c t The green alga Pseudokirchneriella subcapitata has been widely used in ecological risk assessment, usually based on the impact of the toxicants in the alga growth. However, the physiological causes that lead algal growth inhibition are not completely understood. This work aimed to evaluate the biochemical and structural modifications in P. subcapitata after exposure, for 72 h, to three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II), corresponding approximately to 72 h-EC 10 and 72 h-EC 50 values and a high concentration (above 72 h-EC 90 values). The incubation of algal cells with the highest concentration of Cd(II), Cr(VI) or Cu(II) resulted in a loss of membrane integrity of 16, 38 and 55%, respectively. For all metals tested, an inhibition of esterase activity, in a dose-dependent manner, was observed. Reduction of chlorophyll a content, decrease of maximum quantum yield of photosystem II and modification of mitochondrial membrane potential was also verified. In conclusion, the exposure of P. subcapitata to metals resulted in a perturbation of the cell physiological status. Principal component analysis revealed that the impairment of esterase activity combined with the reduction of chlorophyll a content were related with the inhibition of growth caused by a prolonged exposure to the heavy metals. © 2015 Elsevier B.V. All rights reserved. ∗ Corresponding author at: Bioengineering Laboratory, Chemical Engineering Department, ISEP-School of Engineering of Polytechnic Institute of Porto, Rua Dr António Bernardino de Almeida, 431, 4200-072 Porto, Portugal. Tel.: +351 22 8340500; fax: +351 22 8321159. E-mail address: evs@isep.ipp.pt (E.V. Soares). http://dx.doi.org/10.1016/j.jhazmat.2015.04.022 0304-3894/© 2015 Elsevier B.V. All rights reserved.