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Cobalt (Co) nanoparticles (NPs) may be diffusely dispersed into natural ecosystems from various anthropogenic sources such as traffic settings and eventually end up in aquatic systems. As environmentally dispersed Co NPs may be transferred through an aquatic food web, this study investigated this transfer from algae (Scendesmus sp.) to zooplankton (Daphnia magna) to fish (Crucian carp, Carassius carassius). Effects of interactions between naturally excreted biomolecules from D. magna and Co NPs were investigated from an environmental fate perspective. ATR-FTIR measurements showed the adsorption of both algae constituents and excreted biomolecules onto the Co NPs. Less than 5% of the Co NPs formed heteroagglomerates with algae, partly an effect of both agglomeration and settling of the Co NPs. The presence of excreted biomolecules in the solution did not affect the extent of heteroagglomeration. Despite the low extent of heteroagglomeration between Co NPs and algae, the Co NPs were transferred to the next trophic level (D. magna). The Co uptake in D. magna was 300 times larger than the control samples (without Co NP), which were not influenced by the addition of excreted biomolecules to the solution. Significant uptake of Co was observed in the intestine of the fish feeding on D. magna containing Co NPs. No bioaccumulation of Co was observed in the fish. Moreover, 10–20% of the transferred Co NP mass was dissolved after 24 h in the simulated gut solution of the zooplankton (pH 7), and 50–60% was dissolved in the simulated gut solution of the fish (pH 4). The results elucidate that Co NPs gain different properties upon trophic transfer in the food web. Risk assessments should hence be conducted on transformed and weathered NPs rather than on pristine particles.