Comparative assessment of uptake and effects of TiO and CeO nanoparticles in Algae using advanced single-entity analytical techniques.

Despite significant progress in understanding the toxicity of engineered nanoparticles (NPs) in aquatic environments, key gaps remain in our understanding of their uptake and effects on algae. Specifically, it is unclear whether NPs must be internalized and cross biological membranes to induce toxicity, or if surface interactions alone are sufficient. This study aimed to explore the relationship between uptake and effects of TiO-NPs and CeO-NPs on the green alga Raphidocelis subcapitata using advanced single-entity analytical techniques. Flow cytometry was used to distinguish algal cells from NP aggregates and determine growth rates, while single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) quantified adsorbed and internalized metals, operationally discriminated by washing cycles with EDTA. Single-particle ICP-MS (SP-ICP-MS) characterized NP size distribution and dissolution. Results showed greater toxicity for CeO-NPs (72h-EC of 13.6 ± 0.57 mg L⁻¹) compared to TiO-NPs (72h-EC of 28.3 ± 1.16 mg L), with hormesis observed for TiO-NPs between 11 and 20 mg L⁻¹. CeO-NPs. induced a significantly higher level of ROS production, showing a 71.8 % increase compared to the unexposed control, whereas TiO-NPs induced only a 39.46 % increase at highest tested concentration of 50 mg L. SC-ICP-MS revealed both adsorption and internalization of NPs, with Ti accumulation exceeding Ce, despite that CeO-NPs induced stronger growth inhibition and oxidative stress. Hetero-aggregation between NPs and algae, along with changes in cell granularity, was observed at higher NP concentrations. These findings offer insights into TiO-NPs and CeO-NPs interactions with microalgae and highlight the importance of advanced analytical techniques in assessing nanoparticle behavior in aquatic ecosystems.