The production of ROS was studied on white blood cells as a model to screen the effect on eukaryotic cells after being exposed to samples and solar simulated irradiation (according to the level of penetration under the skin). For that purpose, the leukocytes were separated from anticoagulated fresh blood using the Ficoll-Hypaque reactive in a well-known technique [33]. Then, 50 μL of suspensions of P25TiO₂NPs (0.2 mg/mL and 0.02 mg/mL), vitaminB2@P25TiO₂NPs (0.2 mg/mL and 0.02 mg/mL) and vitamin B2 (0.2 mg/mL and 0.02 mg/mL) were prepared and mixed with 50 μL of white blood cells suspension. A solution of 3% H₂O₂ was used as positive control and PBS as negative control. Then, the samples were irradiated using the LED panel for 3 and 6 h to simulate the light penetration into the skin. Also, a set of samples was kept in the dark as control. Finally, the ROS were detected through the colorimetric assay employing the nitroblue tetrazolium salt (NBT salt) and the absorbance at 650 nm was measured. The experiment was reproduced twice; the standard deviation was calculated and p-value < 0.05 were considered significant.

Nano-sized TiO₂ generally shows low or no acute toxicity in both invertebrates and vertebrates. However, exposure of Daphnia magna to 20 ppm TiO₂ for 8 consecutive days was found to cause 40 % mortality. Zhu et al. showed minimal toxicity to D. magna after 48 h exposure, while upon chronic exposure for 21 days, D. magna suffered severe growth retardation and mortality. A significant amount of nano-sized TiO₂ was found also accumulated in the body of the animals. Similar findings with coated nano-sized TiO₂ (T-Lite™ SF, T-Lite™ SF-S and T-Lite™ MAX; BASF SE) were reported by Wiench et al. Biochemical measurements showed that exposure to TiO₂ NPs induces significant concentration-dependent antioxidant enzyme activities in D. magna. Lee et al. showed that 7 and 20 nm-sized TiO₂ induced no genotoxic effect in D. magna and in the larva of the aquatic midge Chironomus riparius.