## 3 Results and Discussion
### Potential Cytotoxicity Associated with the Use of Ozonation to Degrade PPCPs
#### 3.2.1 Effects of Single-Chemical Effluents on Cell Viability  
In general, exposure to the single-chemical effluent samples resulted in toxic effects on HaCaT cells, significantly decreasing cell viability, mostly when the full-strength effluent (100%) or its half dilution (50%) were tested (Figure 2; Table S1). Figure 3: Cell viability recorded in MTT assays with the HaCaT cell line exposed for 72 h to a range of concentrations of the untreated and ozone-treated Mix 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. Asterisks indicate the groups that differ significantly from the control ([PERSON]’s test; \(p<0.05\)). The detailed statistical summaries are available in Supplementary Materials (Table S2). Figure 2: Cell viability recorded in MTT assays with the HaCaT cell line exposed for 72 h to a range of concentrations of the untreated and ozone-treated MP, PP, PCT, SMX, and CBZ effluents. Bars represent the mean of three independent assays and error bars represent the corresponding standard deviation. Asterisks indicate the groups that differ significantly from the control ([PERSON]’s test; \(p<0.05\)). The detailed statistical summaries are available in Supplementary Materials (Table S1). The untreated MP effluent was ineffective towards cell viability after 24 h (Figure S1) and 72 h of exposure (Figure 2), while significantly decreased cell viability was found after 48 h of exposure to 25-100% effluent strength (Figure S1). This apparent inconsistency in toxicity records at 48 h is likely a result of the lower variability within treatment, exposing a potential negative effect of MP in cell viability. MP has already been reported to accelerate the cellular aging of human normal primary epidermal keratinocytes (NHEK) at concentrations ranging within 7.6 \(\times\) 10\({}^{-4}\)-0.152 mg/L [28], which is below the ranges expected for MP concentration in effluent concentrations of 25-100%. A possible explanation for the toxicity of parabens considers their mode of action at the cellular level: alteration of the mitochondrial permeability transition pore, causing an increase in intracellular calcium and an inhibition in electron transport, which may induce apoptosis [29]. On the contrary, the treated MP effluent at 50 and 100% significantly decreased HaCaT viability after 72 h of exposure (after 24 h no cytotoxicity was found, and after 48 h, the same pattern arose as after 72 h; Figure S1), suggesting that the formed by-products, 2,4-DHBA and 3,4-DHBA (see Section 3.1), should be cytotoxic upon prolonged exposure. In fact, 3,4-DHBA decreased the viability of breast cancer (MCF-7), lung cancer (A549), liver cancer (HepG2), cervical cancer (HeLa), and prostate cancer (LNCaP) cell lines [30], while 2,4-DHBA decreased viability in MCF-7 cell line and breast cancer cells (MDA-MB-231) [31]. There is some controversy about the mechanism of toxicity of these by-products, specifically concerning their interaction with reactive oxygen species (ROS). The antioxidant efficiency of hydroxybenzoic acids (the class to which some of these degradation products belong) is argued to depend on several factors, namely the number and position of the hydroxyl group attached to the aromatic ring [32], but overall, they would not be expected to present toxicity as observed herein (Figure 2). Although by-product concentrations could not be determined, the results obtained are consistent with the quoted works, showing toxicity by 2,4-DHBA and 3,4-DHBA to HaCaT cells rather than any protection derived from their claimed primary function as antioxidants, depending on molecular structure, but rather due to mechanisms not yet unveiled. The untreated PP effluent significantly decreased cell viability in HaCaT only at full-strength (1 mg/L PP), regardless of the exposure period (Figures 2 and S1).