In Europe REACH framework directive imposes data acquisition concerning toxicity on acquatic species before the commercialization of chemicals to assess environmental risks. According to official methods, exposure tests are performed under in vitro and standardized conditions: OECD’s guideline rules external variables such as water type, feeding conditions, and exposure time. As consequence, such obtained results could be different from effects observed in natural environments. This study collects effects within 24–96 h of exposure to nano metal-oxides (ZnO, TiO2) on D. magna obtained by the exposure under standard OECD conditions comparing them with results obtained by the exposure under more similar conditions to natural environment (i.e. mixture, feeding). High doses exposure determines gas-bubble disease. Animals exposed to LC10 actively ingest nanoparticles under both fasting and feeding conditions. Furthermore, body burial by a coat of nanoparticles thicker in mixtures than in single dispersions was recorded. Furthermore, results show that: (i) effects increase over time; (ii) n-ZnO results less effective than n-TiO2 in both single dispersion, and mixture; (iii) the presence of surfactant increases toxicity of nanoparticles; (iv) immobilization is a more sensitive endpoint than mortality; (v) feeding increases test sensitiveness improving differences among treated and controls till 96 h and allowing longer exposure times than standard OECD test. As general remark, this study provides evidence that in vitro ecotoxicological results obtained under standardized OECD conditions could be significant different to animals’ responses under natural (feeding and mixtures) exposure conditions.
