## 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
This pattern is in general agreement with previous studies with other cell lines, such as MCF-7 tested with PP concentrations above 3.604 \(\times\) 10\({}^{-3}\) mg/L [33], human breast carcinoma cells (MDA-kb2) at more than 0.0225 mg/L PP [34], and HepG2 at 4 \(\times\) 10\({}^{-4}\) to 4.6 \(\times\) 10\({}^{-3}\) mg/L PP [35]. However, [PERSON] et al. [36] reported that 0.0180 mg/L of PP slightly reduced cell viability, but in a non-cytotoxic manner, in human placenta (JEG-Tox) and HaCaT cell lines. These data are not fully comparable with our results since the concentrations of the untreated PP effluent affecting cell viability in the present work are higher (0.125 to 1 mg/L), with negative effects being noticed only at a concentration equivalent of 1 mg/L regardless of the exposure period (Figure 2 compared to Figure S1). The noticed negative effects could result from the increased production of peroxide anions, which causes a reduction in mitochondrial membrane potential and induces cytotoxicity [35]. The treated PP effluent showed significant toxicity only when tested at full-strength (after 48 h and 72 h). For 4-HBA, one of the identified by-products of the PP oxidation, no decrease in cell viability was reported in the immortalized human colorectal adenocarcinoma cell line (Caco-2) at 1, 3.5, 18, and 36 mg/L [37], or in HaCaT cells at 25, 50, and 100 mg/L [38]. Also, 3,4-DMBA, the other by-product identified, was not proved to induce cytotoxicity in this latter cell line at 10, 50, and 100 mg/L [39]. These data seem inconsistent with the results obtained herein after exposure to the treated PP effluent (Figure 2) after 72 h of exposure, unless unidentified by-products other than 4-HBA and 3,4-DMBA (Section 3.1) are present that induce cytotoxic effects. [PERSON] et al. [40] described that exposure to PCT at concentrations of 0.302 to 2.267 mg/L led to cell death in HepG2, probably due to damage to mitochondrial proteins, potentially reducing respiration and inducing oxidative stress, although the exact mechanism is not yet understood. This evidence is consistent with the results obtained herein, as cytotoxicity was identified at 1 mg/L of PCT in the untreated effluent after 72 h of exposure (Figure 2). In the treated PCT sample, the initial compound was degraded, so the toxicity evidenced following exposure to 50 and 100% treated effluent (Figure 2) should originate from the by-products produced, namely AO and HQ (Section 3.1). [PERSON] et al. [41] describe that AO exposure (\(\geq\)2 mg/L) causes cell death in human proximal tubule cells (HPT) and exhibits cytotoxicity in porcine renal tubule cells (LLC-PK1) and canine Madin-Darby kidney cells (MDCK). These toxic effects may reflect the accumulation of hydroxyl radicals [42]. [PERSON] et al. [43] showed that HQ decreased the cell viability of colon cancer cells (HCT116) and primary human fibroblasts. According to some authors, HQ may induce DNA damage by a combination of mitotic spindle damage, type II topoisomerase inhibition, and breaks in DNA chains through ROS generation [43]. There are not many studies evaluating the cytotoxicity associated with SMX as an isolated compound. However, [PERSON] [44] demonstrated that SMX (1 to 800 mg/L) significantly reduced cell viability in breast tumor (4T1), primary osteosarcoma (Saos-2), normal osteoblast (MC3T3), and fibroblast (3T3-L1) cell lines, like our results obtained following a 72-h exposure to 0.5 and 1 mg/L SMX in the untreated effluent for HaCaT (Figure 2). After ozonation, SMX was degraded, originating AMI as the main by-product (Section 3.1), and the cytotoxicity of the effluent appreciably increased: significantly decreased cell viability was found for treated effluents, undiluted and diluted by 50% and 75% (Figure 2). Although no studies concerning AMI cytotoxicity were found that can provide confirmation, it seems reasonable to postulate the link between the by-product formed and the observed cell viability decrease.