Impact of titanium dioxide and cerium dioxide nanoparticle mixtures on the green alga Raphidocelis subcapitata

Engineered nanoparticles (NPs) occur as mixtures in the environment, making the study of their combined toxicity more realistic than the assessment of individual NPs. Interactions between NPs can alter toxicity profiles, producing synergistic, antagonistic, or additive effects, yet current environmental assessments often overlook these cocktail effects. This study investigated the effects of titanium dioxide NPs (TiO₂-NPs) and cerium dioxide NPs (CeO₂-NPs) mixtures on the green alga Raphidocelis subcapitata compared individual NP exposures. Four mixtures with varying NP ratios were designed based on the toxicity units (TUs) concept and EC₅₀ growth-inhibition values for TiO₂-NPs of 28.3 ± 1.16 mg L^-1, and EC₅₀ for CeO₂-NPs of 13.6 ± 0.57 mg L^-1. Growth inhibition, reactive oxygen species (ROS) generation, and membrane damage were assessed using flow cytometry (FCM), while uptake was analyzed by single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Single-particle (SP) -ICP-MS distinguished NP size distribution and dissolution in mixtures. Results revealed antagonistic interactions in mixtures, with growth inhibition lower than expected based on individual treatments. The difference between predicted and measured TUs in the mixture treatments, used as a measure for the extent of antagonistic interaction ranged from 0.66 to 1.76. Cells with elevated ROS generation showed a two- to four-fold reduction in the mixtures versus single NP treatments. SC-ICP-MS indicated reduced bioavailability of NPs in mixtures: intracellular TiO₂-NP content decreased from 15.7 to 24.3 fg cell^-1 (single) to 12.9-15.9 fg cell^-1 (mixture), and CeO₂-NP content dropped from 3.6 to 8.6 fg cell^-1 to 2.7-3.8 fg cell^-1. This study demonstrates the importance of assessing NP mixture toxicity using advanced single-event techniques, such as FCM, SP- and SC-ICP-MS, to discriminate the fate and bioavailability of NPs present in mixtures and to better understand their environmental impacts.