With questions emerging on the presence and risks associated with metabolites and transformation products (TPs) of organic contaminants in the aquatic environment, progress has been made in terms of monitoring and regulation of pesticide metabolites. However, less interest is shown for pharmaceutical residues, although their pseudo-persistence and adverse effects on non-target organisms are proven. This study provides original knowledge about the contamination of ten sites located along three French rivers (water, sediments, biofilms, clams) by pharmaceutical metabolites and TPs, as well as a preliminary environmental risk assessment. Studied compounds included carbamazepine with five metabolites and TPs, and diclofenac with three metabolites and TPs. Results show that metabolites and TPs are present in all studied compartments, with mean concentrations up to 0.52 µg L -1 in water, 229 ng g -1 in sediments, 2153 ng g -1 in biofilms, and 1149 ng g -1 in clams. QSAR estimations (OECD toolbox) were involved to predict the studied compounds ecotoxicities. QSAR models showed that diclofenac and its metabolites and TPs could be more toxic than carbamazepine and its metabolites and TPs to three aquatic species representing green algae, invertebrates, and fish. However, real ecotoxicological effects are still to be determined. The environmental risk assessment showed that hydroxydiclofenac, 2-[(2-chlorophenyl)-amino]-benzaldehyde and 2 dibenzazepine could present a greater risk than other studied compounds for aquatic organisms. In addition, the risk associated with a mixture of diclofenac and its related metabolites and TPs has been found to be greater than that of the compounds considered individually.

A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was established for simultaneous quantification of eight pharmaceutical molecules (2-hydroxyibuprofen, diclofenac, ibuprofen, propranolol, ofloxacin, oxazepam, sulfamethoxazole, carbamazepine) and caffeine in environmental matrices. Analysis was performed by ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS-MS). Quantification was performed by using the 13 C internal standard method for each molecule. Two methods were firstly optimized on freezedried waste activated sludge and then applied and validated on real complex matrices, which have contrasted physicochemical properties, i.e., clarified wastewater and primary sludge. The combination of acetate buffer with MgSO 4 (protocol A) and citrate buffer with Na 2 SO 4 (protocol B) was found necessary to recover the nine targeted compounds. Adding a higher salts quantity of Na 2 SO 4 (protocol B) compared to MgSO 4 (protocol A) is crucial to increase the ionic strength of the aqueous solution and to obtain comparable extraction recoveries of the targeted molecules. Adding two times solvent volume to the aqueous phase leads to increased absolute recovery for all molecules and both protocols. After demonstration of the final protocol's performance on the control matrix, its robustness was tested on the matrices of interest. As a result, the two proposed detection methods exhibit good reproducibility, high sensitivity, and high reliability.