## 4 Conclusions  
Starting from synthetic effluents contaminated with single or mixed PPCPs (MP, PP, PCT, SMX, CBZ), ozonation fully degraded the initial compounds, either if they were occurring singly or in mixtures. Exposure to ozone-treated effluents induced adverse effects on HaCaT cells, comparable to those following exposure to parent compounds in counterpart effluents. The cytotoxicity of treated effluents, formerly contaminated with single PPCPs or their mixtures, is likely related to by-products formation during ozonation. The cytotoxicity of untreated and treated effluents tended to decrease with increasing complexity (number of contaminants) of the effluents, with the mixture of parabens showing the highest toxicity. For this mixture, there was also an evidently increased toxicity compared to single PPCPs, suggesting a synergistic interaction among the produced by-products. Our results suggest, for the first time, that ozonation is poorly efficient in rendering water contaminated with PPCPs non-toxic for human cells, flagging the need for optimization of the treatment processes towards a safe use of reclaimed water. However, the scarcity of comparable studies in the literature for validation of the present conclusions should be noted, claiming for further investment in understanding the potential human health toxicity of treated effluents beyond the successful degradation of parent contaminants. This naturally includes the follow-up on highly controlled studies with synthetic effluents such as ours by addressing more complex (real) matrices contaminated with PPCPs, individually or in mixture, and the by-products formed during ozonation and other advanced oxidation processes. Lastly, the experimental procedure developed in the present study should be highlighted, as it was proven effective to appropriately meet the sterilization conditions required and to test full-strength solutions. This is a new asset that promotes the much-needed knowledge enrichment concerning the safety of reclaimed water to human health. **Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app15147784/s1](https://www.mdpi.com/article/10.3390/app15147784/s1)]([https://www.mdpi.com/article/10.3390/app15147784/s1](https://www.mdpi.com/article/10.3390/app15147784/s1)), Table S1: Summary of the one-way ANOVA (F-test) or non-parametric equivalent (Kruskal-Wallis test; H) applied to the data obtained in the cell viability tests with the untreated and ozonated effluents under study, considering exposures of 24 h, 48 h and 72 h (all independent tests); Table S2: Summary of the one-way ANOVA (F-test) or non-parametric equivalent (Kruskal-Wallis test; H) applied to the data obtained in the cell viability tests with the untreated and ozonated effluents under study, considering exposures of 24 h, 48 h and 72 h (all independent tests); Figure S1: Cell viability recorded in MTT assays with the HaCaT cell line exposed for 24 and 48 h to a range of concentrations of the independent tests); Figure S1: Cell viability recorded in MTT assays with the HaCaT cell line exposed for 24 and 48 h to a range of concentrations of the untreated and ozone-treated Mir, PP, PCT, SMX and CBZ effluents. Bars represent the mean of three independent assays and error bars represent the mean of three independent assays and error bars represent the corresponding standard deviation. Asterisks indicate the groups the corresponding standard deviation. Asterisks indicate the groups that differ significantly from the control (Dunnett's test or non-parametric equivalent; \(p<0.05\)); Figure S2. Cell viability recorded in MTT assays with the HaCaT cell line exposed for 24 and 48 h to a range of concentrations of the untreated and ozone-treated Mir 2 (MP + PP), Mix 3 (MP + PP + PCT), Mix 4 (MP + PP + PCT + SMX) and Mix 5 (MP + PP + PCT + SMX + CBZ) effluents. Bars represent the mean of three independent assays and error bars represent the corresponding standard deviation.