The potential ecotoxicological impact of ZnO nanostructured films was investigated using common Anabaena flos-aquae, Calothrix pulvinata cyanobacteria and Euglena gracilis euglenoid microalgae. The pre-formed ZnO nanoparticles were synthesized in di(ethylene glycol) medium by forced hydrolysis of ionic Zn(2+) salts. Particle size and shape were controlled by addition of protective agents such as tri-n-octylphosphine oxide and polyoxyethylene stearyl ether. ZnO nanostructured films were directly prepared by spray deposition of pre-formed polyol-based ZnO nanoparticles (ZnO, ZnO-TOPO and ZnO-Brij-76) at 250 degrees C heated glass substrate. Another sample was prepared from zinc acetate in di(ethylene glycol) diluted in ethanol medium without nanoparticles (ZnO-SOL). In this case, ZnO nanostructured film was formed directly on the glass substrate at the same temperature. Water contact angle on ZnO-based films showed that nanostructured ZnO films containing ZnO nanoparticles prepared without protective agent or with tri-n-octylphosphine oxide present a hydrophobic character while the ones containing nanoparticles prepared using polyoxyethylene stearyl ether or the control sample are hydrophilic. Here we showed that (i) the use of protective agents, (ii) the surface properties of the films and (iii) the nature of the biological system can strongly influence the ecotoxicological studies. Epifluorescence microscopy analyses and Live/Dead tests showed that all films are toxic for Euglena gracilis. In the case of Anabaena flos-aquae and Calothrix pulvinata, tri-n-octylphosphine oxide and polyoxyethylene stearyl ether molecules can prevent ZnO nanoparticle toxicity. (C) 2011 Elsevier B.V. All rights reserved.