The continuous ozonation of benzalkonium chloride (BAC) and nickel oxide nanoparticles (NiO-NPs) has been performed in a synthetic water matrix and in a sewage treatment plant influent. This study aims to assess ozonation as pre-treatment of an activated sludge process, with emphasis on the toxicity of treated water. BAC was completely removed in synthetic matrix independently of the presence of NiO-NPs, although the ozone dose was influenced by NPs co-occurrence. The extent of mineralization was limited and a number of intermediate transformation products (TPs) appeared, twelve of which could be identified. The degradation pathway was shown to initiate both on the hydrophobic (alkyl chain) and hydrophilic (benzyl and ammonium moiety) region of the surfactant. The reactions on the hydrophilic region were affected by the presence of NiO-NPs as a consequence of the adsorption of BAC onto NP surface via the aromatic group. Water matrix strongly influenced BAC depletion. The aquatic toxicity of treated mixtures was assessed using a biotest battery composed of single species (the bacteria Vibrio fischeri and Pseudomonas putida and the protozoan Tetrahymena thermophila), as well as a whole biological community assay using activated sludge. Although, BAC showed considerable aquatic toxicity in all bioassays, ozonation decreased the toxic effects of treated water samples at ozone dosages below those required for total BAC depletion. Further treatment would not be justified, neither for a significant increase in BAC abatement nor concerning the toxicity of treated wastewater, which increased as a result of nickel leaching from the NPs. (C) 2015 Elsevier B.V. All rights reserved.