The Prestige oil spill caused severe effects on the coastal fauna and flora due to direct contact of organisms with the fuel oil. However, the water soluble fraction (WSF) of the fuel oil can also provoke deleterious effects in the long term and even in regions not directly affected by the spill. Our objective was to determine the toxicity of the WSF using a battery of laboratory toxicity tests. To obtain a WSF in the laboratory, a sample of the spilled fuel was mixed with adequate medium, sonicated, agitated and filtered. No cytotoxic effects were detected in RTG-2 cells exposed to the WSF. In an algae growth inhibition test (OECD test guideline 201) the WSF did not affect the growth of Chlorella vulgaris. Furthermore, acute and reproductive toxicity tests (OECD test guideline 202) carried out using Daphnia magna did not indicate any deleterious effect of the WSF. In a bioassay designed in our laboratory, D. magna were fed with algae previously exposed to the fuel, but no toxic effects were detected. However, the WSF was able to induce a dose-dependent increase of ethoxyresorufin-O-deethylase activity in RTG-2 cells, indicating the presence of chemicals that could cause sub-lethal effects to organisms. After chemical analyses it was established that the final total quantity of polyaromatic hydrocarbons dissolved in medium was approximately 70 ng/ml. These low concentrations explain the observed lack of toxicity.