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Titanium dioxide nanoparticles (TiO 2 -NPs) in aquatic environments, originating from urban run-off, product use and post-consumer degradation, interact with aquatic organisms through water and sediments. Thorough toxicity assessment requires comprehensive data across all ecosystem compartments especially the benthic zone, which is currently lacking. Moreover, a proper physicochemical characterization of the particles is needed before and during toxicity assessment. In the present work, we used the planarian Schmidtea mediterranea to investigate the effects of TiO 2 -NPs (5 mg/L and 50 mg/L). Planarians are benthic organisms that play an important role in the food chain as predators. Our study integrated particle characterization with toxicokinetic and toxicodynamic parameters and showed that the uptake of TiO 2 -NPs of 21 nm occurred through the epidermis and intestine. Epidermal irritation and mucus production occurred immediately after exposure, and TiO 2 -NPs induced stronger effects in regenerating organisms. More specifically, TiO 2 -NPs interfered with neuroregeneration, inducing behavioral effects. A delay in the formation of the anterior commissure between the two brain lobes after seven and nine days of exposure to 50 mg/L was observed, probably as a result of a decrease in stem cell proliferation. Our findings underscore the need to incorporate multiple exposure routes in toxicity screenings. Additionally, we highlight the vulnerability of developing organisms and recommend their inclusion in future risk assessment strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Karen Smeets reports financial support was provided by Hasselt University., (Copyright © 2024. Published by Elsevier B.V.)