Environmental risk assessment of chemical contaminants requires prioritizing of substances taken up by biota as it is a starting point for potential adverse effects. Although knowledge about the occurrence of known chemical pollutants in aquatic organisms has significantly improved during the last decade, there is still a poor understanding for a broad range of more polar compounds. To tackle this issue, we proposed an approach that identifies bioaccumulative and biomagnifiable polar chemicals using liquid chromatography coupled with electrospray ionization to high resolution tandem mass spectrometry (LC-HRMS/MS) and combine it with trend analysis using hierarchical clustering. As a proof-of-concept, this approach was implemented on various organisms and compartments (sediment, litter leaves, periphytic biofilm, invertebrates and fish) collected from a small urban river. HRMS/MS data measured via data-independent acquisition mode were retrospectively analysed using two analytical strategies: (1) retrospective target and (2) suspect/non-target screening. In the retrospective target analysis, 56 of 361 substances spanning a broad range of contaminant classes were detected (i.e. 26 in fish, 18 in macroinvertebrates, 28 in leaves, 29 in periphyton and 32 in sediments, with only 7 common to all compartments), among which 49 could be quantified using reference standards. The suspect screening approach based on two suspect lists (in-house, Norman SusDat) led to the confirmation of 5 compounds with standards (three xenobiotics at level 1 and two lipids at level 2) and tentative identification of seven industrial or natural chemicals at level 2 and 3 through a mass spectra library match. Overall, this proof-of-concept study provided a more comprehensive picture of the exposure of biota to emerging contaminants (i.e., the internal chemical exposome) and potential bioaccumulation or biomagnification of polar compounds along the trophic chain.