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The distribution of pesticides in dissolved aqueous phase (DAP), suspended particulate matter (SPM) and Scrobicularia plana soft tissues from the Tagus River estuary was determined to evaluate the chemicals pollution status and their hazard potential in this area. Samples were collected in 6 campaigns (December 2012–October 2013), from 3 strategical sites, and analysed via different extraction procedures followed by gas chromatography tandem mass spectrometry (GC–MS/MS) determination. The contamination profile among matrices (DAP, SPM, and soft tissue from bivalves (STB)) was marked by average concentrations of 345 ng/L, 0.51 mg/kg, and 0.02 mg/kg, respectively, with several samples above the 2013/39/EU Directive of environmental quality standards (EQS); no differences were observed between sex. A wider range of pesticides was present in STB ( n = 53) than in SPM ( n = 36) and DAP ( n = 19) matrices. Sediment–water partition coefficient, bioaccumulation factor in both DAP and SPM fraction were estimated ranging between 2.5 and 4.4 and 0.008–2799, respectively. The spatial distribution of most pesticides and physicochemical parameters were consistent, indicating a pollution pattern primarily near the Trancão River mouth. Due to the presence of the target compounds, calculated risk quotients pointed out potential hazards for aquatic organisms, mainly to invertebrates. The estimated average daily intake, theoretical maximum daily intake, and hazard quotient of the studied pesticides–via bivalve ingestion–indicated no risk for human health, although it is important to note possible biomagnification processes that may happen along the estuarine food-chain. [ABSTRACT FROM AUTHOR]