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The aim of the present work was the optimisation and application of a simple laboratory test, using the phototactic behaviour of Balanus amphitrite nauplii as an index of physiological stress at sub-lethal level. The method consisted on counting the individuals attracted by a light source, and the percent of positive responding individuals (phototactic response, %R+) was calculated. The role of some cultural parameters which are likely to affect the entity of light-response (photoperiod, temperature, oxygen, developmental stage, presence of food), the sensibility of the different naupliar stages, and the effect of different potential antifouling substances and CdCl2 were investigated. The cultural parameters producing larvae with a higher phototactic response have been determined and fixed: 20°C, no aeration, no feeding and a 12:12 L:D photoperiod, with the phototactic test performed before 2:00 pm. The most sensible naupliar stage, in relation to toxic stress, resulted to be the II, both for acute toxicity and phototactic test. After 48-hours of exposure, the reduction in phototactic response for treated samples was significant for all the tested substances, while acute toxicity test showed no detectable difference compared to the controls.