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The determination of the real toxicity of sediments in aquatic ecosystems is challenging and necessary for an appropriate risk assessment. Different approaches have been developed and applied over the last several decades. Currently, the joint implementation of chemical, ecological and toxicological tools is recommended for an appropriate and successful toxicity risk assessment. We chose the combination of the toxic unit approach with acute pore water tests (Vibrio fischeri, Pseudokirchneriella subcapitata and Daphnia magna) and whole-sediment exposure tests (V. fischeri, Chironomus riparius), together with invertebrate community composition (multivariate analyses) to detect short and long-term responses of the organisms in four rivers of the Iberian Peninsula. High toxicity was detected in three sites (the downstream sites of the Llobregat and the Júcar, and the most upstream site of the Ebro). We identified organophosphate insecticides and metals as the main variables responsible for this toxicity, particularly in the whole-sediment tests. In particular, chlorpyrifos was mostly responsible for the toxicity (TUs) of D. magna, coinciding with the C. riparius mortality (long-term toxicity) in the mentioned sites, and copper was the main pollutant responsible for the short-term toxicity of P. subcapitata. The combination of the different approaches allowed us to detect ecotoxicological effects in organisms and identify the main contributors to the toxicity in these multi-stressed rivers.