Aquatic organisms are often exposed to mixtures of low levels of pollutants whose presence and effects can pass easily unnoticed if only traditional monitoring strategies are employed. The main aim of this work was to assess the presence and effects of trace levels of pollutants in a scarcely affected area through the combination of chemical and biological approaches. Sediments were collected along a river with little anthropogenic pressure and assayed for cytochrome P450 (Cyp1a)-dependent ethoxyresorufin- O -deethylase (EROD) activity with the rainbow trout gonadal cell line RTG-2. Chemical analyses were performed in these sediments using two-dimensional gas chromatography-time-of-flight mass spectrometry. Sediment samples induced EROD activity, and chemical analyses evidenced the presence of a wide variety of contaminants in the range of nanograms per gram of dry weight. Correlation analysis between EROD induction and chemical analyses data showed an r value of 0.840 ( p   <  0.05). In addition, fish from a fish farm located downstream of the sampling points exhibited high hepatic EROD levels as well as an induced expression of cyp1a and cyp3a . In conclusion, only an appropriate combination of biological and chemical techniques allowed the detection of the presence of trace levels of contaminants in a theoretically nonaffected river.