## 3 Results and Discussion
### Potential Cytotoxicity Associated with the Use of Ozonation to Degrade PPCPs
#### 3.2.2 Effects of Effluents with Mixed PPCPs on Cell Viability  
There was a progressive and significant decrease in cell viability with increasing concentration of the effluents (both untreated and treated), at least from 25 to 100%, with, in many cases, a significant decrease being noticed also at 12.5% (Figure 3 for 72 h and Figure S2 for 24 and 48 h; Table S2 for statistical summaries). This decrease was more pronounced for the mixture of parabens (Mix 2), with average cell viability reaching values as low as 48.69% and 42.66% (untreated and ozone-treated effluent, respectively) upon exposure to the 100% effluent concentration (72 h exposure; Figure 3). As observed for single PPCPs, treated effluents yielded effects in cells that are similar across concentrations to their untreated counterparts, with differences in the cell viability between untreated and ozone-treated samples in the range 0.78-8.46%. Remarkably, the cell viability of the treated samples was commonly lower than that of the untreated samples. The narrower range observed for the mixtures suggests that ozonation is less efficient (concerning toxicity removal) towards mixtures than towards single PPCP contaminants. For instance, Mix 2 caused higher cytotoxicity (Figure 3) than each of the components alone (Figure 2), once cell viability inhibition higher than 20% was found from 12.5% effluent onwards, while these records were found only at full strength for single paraben effluents. Moreover, taking the treated 100% effluent concentration as an example, viability inhibition was \(\leq\)20% for MP and PP, individually, but reached 60% for the mixture. The same pattern was observed for untreated effluents. This suggests that the parabens or their by-products exhibit more-than-additivity regarding cytotoxicity to human keratinocyte cells. Interestingly, a synergistic interaction among the components of an ozone-treated mixture of MP and PP has also been observed for aquatic species, namely _Daplinia magna_ and _Raphidocelis subcapitata_[18], suggesting that the increased toxicity of this mixture following ozonation might be transversal to humans and aquatic species. Mixture 3, adding parabens and PCT, followed a similar trend, despite being less cytotoxic than Mix 2. The untreated and treated Mix 4 and 5 effluents presented lower cytotoxicity than Mix 2 and Mix 3 (Figure 3), as interpreted from the lower magnitude of the effects promoted (slightly more than 20% cell viability inhibition following exposure to full-strength effluents) compared to the effects noted in simpler mixtures (40-60% cell viability inhibition following exposure to full-strength effluents). This apparent decrease in relative toxicity with the increase in effluent mixture complexity, which is common to both the untreated and ozone-treated effluents, suggests that as one adds more compounds, there is a trend for reversing the interactive behavior of either the parent compounds or the by-products generated after treatment. Regarding the treated effluents, this might be partially explained by the increased reaction time (and TOD) with increasing the mixture complexity (Table 1). In fact, the increase in the ozonation time could enhance the by-products oxidation (e.g., [18]), which can result in less cytotoxic solutions. However, the similarity of the cell viability between untreated and ozone-treated samples suggests that this effect might be independent of the reaction time, being mainly determined by the number and type of contaminants in the mixture. Assessing the toxicity of treated mixtures of PPCPs to human cell lines is crucial to reliably conclude on the safety of reclaimed water. In the present study, it was shown that ozonation of synthetic effluents containing parabens, PCT, SMX, and CBZ had no pronounced beneficial effect on their cytotoxicity. Interestingly, these results agree, in general, with the conclusions of a previous study addressing the toxicity of these samples to aquatic species [18].