In this work, an optimized photo-Fenton process was applied to remove pollutants from tannery industrial effluent (TIE) with its final toxicity level being assessed by a lettuce-seed-based bioassay test. A full 33 factorial design was applied for the optimization of long-term photo-Fenton experiments. The optimum conditions of the photo-Fenton process were attained at concentration values of 0.3 g Fe2+ L-1 and 20 g H 2O2 L-1 and pH 3, for 120 min UV irradiation time. Reactor operating parameter (ROP) effects on the removal of chemical oxygen demand, colour, turbidity, total suspended solids and total volatile solids were evaluated, suggesting that a broad range of ROP values are also suitable to give results very near to those of the photo-Fenton experiments under optimal conditions. Based on the low calculated median lethal dose (LD50) values from a lettuce-seed-based bioassay test, we suggest that recalcitrant substances are present in treated TIE samples. A possible cause of the high toxicity level could partly be attributed to the nitrate concentration, which was not completely abated by the photo-Fenton process. Apart from this, the photo-Fenton process can be used as a part of an industrial effluent treatment system in order to abate high organic pollutant loads. © 2013 Copyright Taylor and Francis Group, LLC.