In this study, the presence of 23 pharmaceutically active compounds (PhACs) including antibiotics, analgesics, anti-inflammatories, psychiatric and cardiovascular drugs, antifungals and metabolites was investigated in surface waters. A total of 89 samples were collected during 3 years (2020, 2021 and 2022) from a European representative river basin (Tagus, Spain). To elucidate PhAC potential sources, sampling points located in areas with low, median and high anthropogenic influence were selected. The analytical method based on solid phase extraction (SPE) followed by UHPLC-MS/MS analysis was validated meeting SANTE/2020/12830 and SANTE/12682/2019 performance criteria. PhACs were quantified above limits of quantification (LOQs) in 96 % of water samples, being the antihypertensives valsartan (648 ng/L, 87 % quantification frequency) and irbesartan (390 ng/L, 75 %) and the antidepressant o-desmethylvenlafaxine (495 ng/L, 76 %) the predominant pollutants. The rest of the target PhACs showed median concentrations between 4 and 172 ng/L with quantification frequencies ranging from 35 to 75 %. ∑PhAC concentrations did not show temporal or seasonal trends. However, valsartan and naproxen presented lower levels in drier (spring and summer) compared to the wetter. Source identification revealed a clear anthropogenic origin since concentrations obtained in highly populated areas were statistically higher (p < 0.01) than those quantified in sparsely populated ones. This finding was also confirmed by calculating PhACs mass flow rates, which ranged between 1.4 and 235 kg/y. Finally, data generated were used to estimate the potential risk to the aquatic ecosystem for three trophic levels (phototrophic, invertebrate and vertebrate organisms). Risk quotient ratios (RQs) were calculated for all PhACs at the median (P50) and worst-case (max) scenarios. Up to 7 PhACs (acetaminophen, carbamazepine, gemfibrozil, ibuprofen, irbesartan, ketoprofen and venlafaxine) showed high risk for the highest trophic level (fish) in >45 % of investigated locations.
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