## 3 Results and Discussion
### CAF Degradation by Fenton Process
This limitation is evident in the HPLC chromatograms depicted in Figure 3. The reaction intermediates were detected in the first 5 min of the reaction and their amount in the solution increased until the end of the 60 min of the reaction. This result shows that caffeine degradation generates persistent organic intermediates resistant to oxidation. Similar chromatograms to those found in this study were observed by [PERSON] [55]. Figure 3 illustrates that under the investigated conditions, there is degradation of the contaminant, resulting in the formation of smaller-chain molecules with lower molecular weights and retention times of less than 7 min. To prevent the creation of potentially hazardous secondary waste, pursuing mineralization becomes crucial. Byproducts formed in this process might possess greater toxicity compared to the original molecules [39]. The rate at which hydroxyl radicals are generated relies on the oxidative potential of the procedure, directly impacted by the reaction rate outlined in Equation (1). Figure 3: Caffeine chromatograms of the initial solution of 20 mg L\({}^{-1}\) (**a**) and after 5 min (**b**) and 60 min (**c**) of the Fenton reaction (H\({}_{2}\)O\({}_{2}\)/FeSO\({}_{4}\).7H\({}_{2}\)O = 150/4 mg L\({}^{-1}\)). Identifying the byproducts formed during caffeine degradation is a complex process requiring focused research using analytical chemistry techniques like chromatography coupled with mass spectrometry. In this study, previous works by [PERSON] et al. [36], [PERSON] et al. [52], [PERSON] et al. [37], [PERSON] et al. [56], and ZIYLAN-YAVAS et al. [57] provided insights into potential caffeine degradation pathways. [PERSON] et al. [36] reported reaction intermediates from caffeine oxidation under Fenton reaction conditions (H\({}_{2}\)O\({}_{2}\)/FeSO\({}_{4}\cdot\)7H\({}_{2}\)O = 1/15 mol L\({}^{-1}\)). [PERSON] et al. [37] proposed a theoretical computational mechanism for caffeine degradation by \(\bullet\)OH which closely resembled the pathway described by [PERSON] et al. [36]. Overall, these studies indicate that caffeine oxidation by the Fenton reaction generates persistent organic intermediates more resistant to oxidation compared to caffeine itself. Figure 4 depicts the theoretical degradation pathway of caffeine developed by [PERSON] et al. [37], specific to degradation by the hydroxyl radical. According to this figure, caffeine degradation by \(\bullet\)OH begins at the imidazole portion, with the radical primarily targeting the C=C (degradation route \(\bullet\)R(OH)-5) of caffeine and, to a lesser extent, the C8 (degradation route \(\bullet\)R(OH)-8) [36; 37; 56]. Two pathways are illustrated for caffeine degradation. The first pathway (\(\bullet\)R(OH)-5) demonstrates the hydroxyl radical transforming into P\({}_{1}\), the 5,6-dihydroxy of caffeine, which further reacts to form the intermediate IM\({}_{2}\) and results in the production of dimethylparabanic acid (P\({}_{2}\)) and di(N-hydroxymethyl) parabanic acid (P\({}_{5}\)). The second pathway (\(\bullet\)R(OH)-8) shows reactions yielding 1,3,7-trimethylamine acid (P\({}_{6}\)), 6-amino-5-(N-formylmethylamino)-1,3-dimethyluracil (P\({}_{7}\)), and 8-oxocaffeine (P\({}_{11}\)). These products are formed through distinct reactions involving hydrogen atom abstraction, displacement, and bond cleavage. Studies by [PERSON] et al. [37] highlighted the ecotoxicity of caffeine and its degradation products, indicating chronic toxicity to fish, acute and chronic harm to daphnia, severe acute and chronic toxicity to green algae, and non-hazardous effects on rats. [PERSON] et al. [52] found that lower concentrations of the solution obtained from Fenton-treated caffeine showed no chronic toxicity to zooplankton, but higher concentrations exhibited chronic toxicity. Figure 4: Theoretical degradation route of caffeine by hydroxyl radicals. Despite this, the hydroxyl radicals formed in the study were inefficient in degrading the byproducts as well, hindering effective solution mineralization.