The Degradation of Aqueous Oxytetracycline by an O3/CaO2 System in the Presence of HCO3−: Performance, Mechanism, Degradation Pathways, and Toxicity Evaluation

This research constructed a novel O3/CaO2/HCO3− system to degrade antibiotic oxytetracycline (OTC) in water. The results indicated that CaO2 and HCO3− addition could promote OTC degradation in an O3 system. There is an optimal dosage of CaO2 (0.05 g/L) and HCO3− (2.25 mmol/L) that promotes OTC degradation. After 30 min of treatment, approximately 91.5% of the OTC molecules were eliminated in the O3/CaO2/HCO3− system. A higher O3 concentration, alkaline condition, and lower OTC concentration were conducive to OTC decomposition. Active substances including ·OH, 1O2, ·O2−, and ·HCO3− play certain roles in OTC degradation. The production of ·OH followed the order: O3/CaO2/HCO3− > O3/CaO2 > O3. Compared to the sole O3 system, TOC and COD were easier to remove in the O3/CaO2/HCO3− system. Based on DFT and LC-MS, active species dominant in the degradation pathways of OTC were proposed. Then, an evaluation of the toxic changes in intermediates during OTC degradation was carried out. The feasibility of O3/CaO2/HCO3− for the treatment of other substances, such as bisphenol A, tetracycline, and actual wastewater, was investigated. Finally, the energy efficiency of the O3/CaO2/HCO3− system was calculated and compared with other mainstream processes of OTC degradation. The O3/CaO2/HCO3− system may be considered as an efficient and economical approach for antibiotic destruction.


Introduction
In recent decades, antibiotics have been widely used in the pharmaceutical and animal husbandry industries.While providing substantial social benefits, they inevitably cause serious environmental pollution [1,2].Oxytetracycline (OTC) is a kind of tetracycline antibiotic [3] with broad-spectrum bacteriostasis and a low price.As a veterinary drug, it is widely used in animal husbandry worldwide [4].However, excessive use of OTC cannot be fully absorbed by animals, and there will be a large amount of OTC in the environment [5,6].A residual concentration of OTC in water is still present after treatment by sewage treatment plants.A high concentration of OTC in water will slow down the development of fish [7] and inhibit photosynthesis in plants [8].The naphthalene ring structure has antibacterial properties and chemical stability, which makes it difficult to remove OTC through traditional water treatment processes [9].Therefore, it is imperative to seek an effective method to remove OTC in water.
Oxidation is an essential reaction from an industrial and academic point of view.In general, selecting oxidants is significant for heterogeneous reaction systems.An advanced oxidation process (AOP S ) is recognized as an effective method for treating wastewater with potential environmental risks.One of the most common active species in AOP S is hydroxyl radicals (•OH), which have strong oxidation properties and can convert pollutants into carbon dioxide and water.A simple and commonly used substance in AOPs is O 3 [10].O 3 is considered to be a clean oxidant in which organic pollutants are oxidized directly or indirectly with free radicals generated by O 3 to achieve the degradation of organic pollutants [11].The direct oxidation reaction time is longer, the oxidation rate is slow, and it has a specific selectivity.Indirect oxidation refers to the solid oxidizing free radicals (mainly •OH) generated by the catalytic O 3 reaction, which can quickly decompose most organic pollutants in water.In the indirect oxidation method, the use of a catalyst to activate O 3 increases the oxidation capacity of O 3 and promotes the degradation of organic pollutants.Recently, the couple of O 3 and other oxidation processes has become very popular, such as H 2 O 2 /O 3 [12], ultrasonic treatment ozonation [13], and photocatalytic ozonation [14].Among them, the most effective method of O 3 indirect oxidation technology is the O 3 /H 2 O 2 method, which is also the simplest method for •OH [15].However, H 2 O 2 is relatively dangerous, with the characteristic of being flammable and explosive [16].
So far, many studies have confirmed that a combination of several treatment technologies can improve the degradation and mineralization of organic pollutants in wastewater [17].Some catalysts can promote the better degradation of pollutants by O 3 .As a substitute for liquid hydrogen peroxide, calcium peroxide (CaO 2 ), a new solid oxidant, has attracted a lot of attention in the environmental field due to its remarkable advantages, such as a high efficiency, safety, stability, controllability, and low cost [18,19].The process of releasing H 2 O 2 from calcium peroxide is slow, which can be controlled by adjusting the pH, which avoids the disadvantage of wasting oxygen and the short action time caused by the rapid decomposition of liquid H 2 O 2 .For some pollutants (such as PPCP and PAH), the removal effect of solid calcium peroxide will exceed that of liquid H 2 O 2 [20,21].The O 3 /H 2 O 2 system created by O 2 /CaO 2 has a low oxidation rate and poor selectivity in the absence of an activator.Sodium bicarbonate was added to the system as an auxiliary catalyst [22].On one hand, HCO − 3 reacts with H 2 O 2 to generate peroxymonocarbonate (HCO − 4 ), which continuously activates H 2 O 2 and converts it into highly active free radicals such as •O 2 − , singlet oxygen ( 1 O 2 ), and so on [23].On the other hand, HCO −  3 is a recognized promoter of O 3 decomposition, promoting the efficient oxidation of O 3 into •CO 3 − and the conversion of O 3 into indirect oxidation [24].O 3 /CaO 2 /HCO − 3 innovatively proposes a safe and fast O 3 /H 2 O 2 system.Meanwhile, the addition of HCO − 3 could activate H 2 O 2 and O 3 and significantly improve the oxidation capacity of the system [25].As a whole, the advantages of the O 3 /CaO 2 /HCO − 3 system can be listed as: firstly, the synergy effect between O 3 and CaO 2 can promote the generation of •OH.Secondly, the OH − released by CaO 2 will form an alkaline environment, which will further promote the transformation of O 3 into •OH.Thirdly, HCO − 3 activates O 3 radicals and catalyzes the production of •OH radicals.Finally, the decomposition products of CaO 2 /HCO − 3 are non-toxic and harmless.However, as far as is known, there have been few reports that O 3 activates CaO 2 /HCO − 3 for antibiotic OTC degradation, and its synergistic mechanism is still unclear.
Therefore, the O 3 /CaO 2 /HCO − 3 system was constructed for the efficient degradation of OTC in this paper.The influence of the effect of CaO 2 dosage, HCO − 3 dosage, O 3 concentration, initial pH value, and OTC initial concentration on OTC degradation was investigated.The role of active substances in the OTC decomposition system was inspected via the capture agent experiment.The generated active species were characterized using chemical methods and electron spin resonance (ESR).The degradation mechanism and process were examined.The toxicity of OTC and intermediates was analyzed, as was the feasibility of the O 3 /CaO 2 /HCO − 3 system for the treatment of other antibiotics and actual wastewater.Finally, the energy efficiency of the system was compared with that of other technology.

Effect of CaO 2
Figure 1a,b reveal the changes in the OTC degradation efficiency under different CaO 2 additions.The lowercase letters in the picture represent the difference between the data, and the star symbol represents the value of lnK obs .Experiments were carried out with fixed OTC concentrations at different CaO 2 concentrations.It was observed that the O 3 /CaO 2 system had a significant effect on the degradation of OTC within 30 min.When the dosage of CaO 2 was 0.050 g/L, the degradation efficiency of OTC could reach 85.3%, which was 23.2% higher than that of the sole O 3 system.The degradation efficiency (slope of degradation curve) of OTC increased with an increase in the CaO 2 concentration, which indicates that CaO 2 is conducive to the degradation of OTC.It is worth noting that, when the dosage of CaO 2 was higher than the optimal dosage (0.050 g/L), the degradation efficiency gradually slowed down.The first-order kinetic constant in the upper right corner of Figure 1a can also reflect this trend.3)-( 13)) [29][30][31][32][33].This •OH is a strong oxidant, which can effectively promote the degradation of OTC.However, when the concentration of CaO 2 is too high, too much H 2 O 2 will be produced [34].Excessive H 2 O 2 will decompose by itself and need to consume active free radicals such as •OH (Equations ( 14)-( 16)), which will adversely affect the degradation of OTC [35].The oxidation potentials of different free radicals are displayed in Table S1.

Effect of HCO - 3 Dosage
The degradation of OTC with time under the conditions of different HCO − 3 additions is shown in Figure 1c,d.To explore the CaO 2 /HCO − 3 combined process, with the addition of HCO − 3 as a variable, the experiment was carried out with the optimal CaO 2 concentration and fixed OTC concentration.It was observed that the O 3 /CaO 2 /HCO − 3 system had a further improvement in the degradation of OTC compared to O 3 /CaO 2 within the reaction time of 30 min.When the dosage of HCO − 3 was 2.25 mmol/L, the degradation efficiency of OTC could reach 91.5%, which was 7.3% higher than that of the O 3 /CaO 2 system and 32.1% higher than that of the sole O 3 system.The degradation efficiency of OTC increased with an increase in the HCO − 3 concentration.Further increasing the concentration of HCO − 3 based on the optimal dosage of HCO − 3 will inhibit the degradation of OTC.Similarly, the first-order kinetic constant in the upper right corner of Figure 1c 17)-( 22)) [36,37].The consumption of •CO − 3 for activating H 2 O 2 can be regenerated into •CO − 3 through in-termediate HCO − 3 to form a cycle, which is the synergistic effect of HCO − 3 and CaO 2 ((Equation ( 19)) [23,38].
The synergistic effect between HCO  23)-( 31)) [39][40][41].There is a balance point between the generation of •O − 2 and the decomposition of O 3 , which also explains the phenomenon that the degradation efficiency of OTC decreases when the HCO − 3 dosing amount is higher than the optimal amount [42]. •HO

Effect of O 3 Dosage
The degradation of OTC under the conditions of different O 3 concentrations is presented in Figure 1e,f.As the amount of O 3 increased from 0.125 g/h to 0.75 g/h, the OTC degradation efficiency increased significantly from 91.5% to 97.2%, and the reaction time when the OTC degradation efficiency reached 90% was shortened from 30 min to 5 min.The reasons were as follows: firstly, the larger O 3 flux increased the contact area between the gas phase and the liquid phase, and the number of effective molecular collisions increased, which improved the reaction rate.Secondly, according to Henry's Law, the more significant the O 3 concentration, that is, the increase in X Z per unit volume, the more increased the pressure of the O 3 gas, thus promoting the mass transfer rate of O 3 between the two phases [43].Third, the O 3 concentration will affect the concentrations of H 2 O 2 and •OH in the reaction system.A higher concentration will promote the generation of more active free radicals and affect the degradation effect [44].However, a high-concentration O 3 treatment process means a high cost, which will affect the economy of this process.

Effect pH Value
The degradation of OTC with time under the conditions of different initial pHs is shown in Figure 2a.With an increase in the initial pH of the solution, the environment changed from acidic to neutral and then to alkaline.The OTC degradation efficiency was enhanced from 71.4% to 94.4%.This phenomenon was mainly due to the direct oxidation of O 3 molecules with pollutants in an acidic environment.In an alkaline environment, the concentration of dissolved O 3 decreases [45], forcing O 3 molecules to be activated into active free radicals such as •OH and •O − 2 (Equations ( 24)-( 32)) to react with OTC [46].Because the indirect reaction is dominant, the oxidation potential of •OH is higher than that of O 3 molecules, and the oxidation ability is stronger, which is more conducive to the degradation of OTC.On the other hand, an alkaline environment is more conducive to the release of H 2 O 2 from CaO 2 and the self-decomposition of H 2 O 2 , which will produce more active free radicals and promote the degradation of pollutants [47].Similarly, the O 3 treatment process under alkaline conditions also means an increase in cost, and the natural water environment is mainly neutral or weakly alkaline, so neutral environmental conditions will continue to be used in subsequent studies.

Effect OTC Concentration
The degradation of OTC with time under the conditions of different OTC concentrations is shown in Figure 2c.As the initial OTC concentration was raised from 40 mg/L to 200 mg/L, the OTC degradation efficiency decreased significantly, the degradation efficiency decreased from 94.4% to 60.6%, and the kinetic constant decreased from 0.114 min −1 to 0.031 min −1 (inset Figure 2c).It is unreasonable to judge the OTC degradation only by the initial OTC concentration.We calculated the total removal of the four initial OTC concentrations in Figure 2c, which were 15.1 mg, 29.0 mg, 45.8 mg, and 48.5 mg, respectively.Comparing the kinetic constant and total removal amount of OTC, with an increase in the concentration, the kinetic constant decreased and the total removal amount increased.Therefore, it is speculated that the initial OTC treatment concentration is 40 mg/L, which is close to the limit of the O 3 /CaO 2 /HCO − 3 system.As shown in Figures 1b,d,e and 2b,d, good linear correlations between the ln k and different parameters were obtained.Therefore, the establishment of an O 3 /CaO 2 /HCO − 3 system can degrade OTC effectively.3a, it can be seen that the degradation efficiency of OTC decreased with increasing amounts of methanol.When the methanol concentration changed from 0 mmol/L to 10 mmol/L, the degradation efficiency of OTC decreased from 94.0% to 66.9%.Meanwhile, the kinetic constant declined from 0.114 min −1 to 0.039 min −1 (Figure S1a).These results indicate that •OH played an essential role in the degradation of OTC.•O − 2 can react to generate 1 O 2 (Equations ( 32)-( 34)), which has a strong oxidation potential.1,4-Diazabicyclooctane triethylenediamine (DABCO) is a highly efficient 1 O 2 -trapping agent that can capture the 1 O 2 existing in the solution.Therefore, this section intends to use DABCO to study the role of 1 O 2 in the O 3 /CaO 2 /HCO − 3 system.
•HO 2 Molecules 2024, 29, x FOR PEER REVIEW 8 of 19 (Figure S1d).This result shows that •CO 3 -is very involved in the degradation of OTC.The reaction rates of p-benzoquinone with •OH and •O2 − were 1.2 × 10 9 and 8.3 × 10 8 M −1 s −1 , respectively, which are comparable.The reaction rate of indole with •OH was 7.9 × 10 −14 M −1 s −1 , which can be ignored.Because of their low reaction rates, P-benzoquinone and Indole can be regarded as not reacting with •OH [48,49].As shown in Figure S2, compared with different active substances, •O2 made the greatest contribution to the degradation process of OTC, with 41.2% of the OTC being degraded by •O2.

Formation of Active Species
To further explore the synergetic mechanism, the formation of •OH under different systems was compared.The results are depicted in Figure 4a.Compared to the sole O3 system, the •OH concentration was seriously increased when CaO2 was added.These phenomena verified that O3 could react with the H2O2 released by CaO2 and promote the formation of •OH [50].As desired, the •OH concentration was further enhanced when HCO 3 was added.The production of •OH followed this law: O3/CaO2/NaHCO3 > O3/CaO2 > O3.Therefore, it can be explained that the higher degradation efficiency may have come from the higher production of •OH.It is worth noting that the generation of •OH in the O3/CaO2 system and the O3/CaO2/HCO 3 -system showed a trend of first rising, then decreasing, and lastly increasing over time.It was speculated that the conversion of H2O2 into •OH was violent, and a large amount of •OH was generated within 5 min.•OH could react with H2O2, and a certain amount of •OH production was consumed from 5 to 20 min.After the two reactions reached equilibrium, •OH production increased and tended to stabilize.Figure 3b shows the effect of various DABCO dosages on OTC elimination.Higher DABCO dosages will inhibit the degradation effect more obviously.When the addition of DABCO changed from 0 mmol/L to 10 mmol/L, the degradation efficiency of OTC decreased from 94.0% to 79.6%, with the kinetic constant varying from 0.114 min −1 to 0.060 min −1 (Figure S1b).DABCO has a significant inhibitory effect on OTC elimination.It is further inferred that 1 O 2 is actively involved in OTC degradation.
P-benzoquinone is a highly efficient  6)), which has a significant effect on OTC elimination.Figure 3c shows the effect of p-benzoquinone addition on OTC elimination.The degradation efficiency of OTC decreased with the p-benzoquinone addition.When the addition of p-benzoquinone was 0 mmol/L 0.5 mmol/L, 1.0 mmol/L, and 1.5 mmol/L, the degradation efficiency of OTC decreased from 94.0% to 53.2%, and the corresponding kinetic constant varied from 0.114 min −1 to 0.059 min −1 , 0.039 min −1 , and 0.033 min −1 (Figure S1c).This result showed that •O − 2 played a key role in the degradation of OTC.Indole is an efficient •CO − 3 capture agent, which can capture the HCO − 3 existing in a solution.After adding the catalyst NaHCO 3 to the solution, NaHCO 3 and H 2 O will produce part of •CO − 3 (Equation ( 19)).When the indole concentration changed from 0 mmol/L to 0.2 mmol/L, the degradation efficiency of OTC decreased from 94.0% to 59.5% (Figure 3d), and the corresponding kinetic constant varied from 0.114 min −1 to 0.033 min −1 (Figure S1d).This result shows that •CO −  3 is very involved in the degradation of OTC.The reaction rates of p-benzoquinone with •OH and •O 2 − were 1.2 × 10 9 and 8.3 × 10 8 M −1 s −1 , respectively, which are comparable.The reaction rate of indole with •OH was 7.9 × 10 −14 M −1 s −1 , which can be ignored.Because of their low reaction rates, P-benzoquinone and Indole can be regarded as not reacting with •OH [48,49].As shown in Figure S2, compared with different active substances, •O 2 made the greatest contribution to the degradation process of OTC, with 41.2% of the OTC being degraded by •O 2 .

Formation of Active Species
To further explore the synergetic mechanism, the formation of •OH under different systems was compared.The results are depicted in Figure 4a.Compared to the sole O 3 system, the •OH concentration was seriously increased when CaO 2 was added.These phenomena verified that O 3 could react with the H 2 O 2 released by CaO 2 and promote the formation of •OH [50].As desired, the •OH concentration was further enhanced when HCO − 3 was added.The production of •OH followed this law: O 3 /CaO 2 /NaHCO 3 > O 3 /CaO 2 > O 3 .Therefore, it can be explained that the higher degradation efficiency may have come from the higher production of •OH.It is worth noting that the generation of •OH in the O 3 /CaO 2 system and the O 3 /CaO 2 /HCO − 3 system showed a trend of first rising, then decreasing, and lastly increasing over time.It was speculated that the conversion of H 2 O 2 into •OH was violent, and a large amount of •OH was generated within 5 min.•OH could react with H 2 O 2 , and a certain amount of •OH production was consumed from 5 to 20 min.After the two reactions reached equilibrium, •OH production increased and tended to stabilize.ESR analyses further revealed that there were obvious signals of the •OH peak in various systems (Figure 4b).It can be seen that the characterized peaks of •OH followed as: O 3 /CaO 2 /HCO − 3 > O 3 /CaO 2 > O 3 , which illustrated that •OH was beneficial to generate in O 3 /CaO 2 /HCO − 3 system.Figure 4c presents the characteristic peaks of 1 O 2 in various systems.It can be seen that 1 O 2 can be generated in O 3 , O 3 /CaO 2 , and O 3 /CaO 2 /HCO − 3 systems.Compared to sole O 3 , the characteristic peaks of 1 O 2 were enhanced with the addition of CaO 2 and HCO − 3 , which indicates that the addition of CaO 2 and HCO − 3 effectively increased the production of 1 O 2 .

OTC Degradation Process 2.7.1. UV-Vis Spectra
Figure S3 shows the UV-Vis spectrum results of the OTC solution in the sole O 3 system and O 3 /CaO 2 /HCO − 3 system.The OTC spectrum shows two absorption bands near 276 nm and 353 nm, and the absorption peak near 353 nm is the characteristic peak of OTC.The common point of the two systems is that, after 30 min of processing, the characteristic peaks of OTC were significantly reduced, and the OTC molecules were weakened.The π-π * transition of its heteroaromatic ring makes the main contribution to this [51,52].Compared with Figure S3a, the characteristic peak of OTC in Figure S3b has a more rapid decline, which indicates that O 3 /CaO 2 /HCO − 3 had a better OTC degradation ability.

3D EEMFS
To study the degradation process of OTC more deeply, 3D EEMFs were used for characterization.The emission wavelength and the excitation wavelength were all set as 200-600 nm, whose sampling intervals were 5 nm and 10 nm, respectively.Figure 5a is taken from the original OTC sample, and Figure 5b,c are OTC samples processed for 10 min, 20 min, and 30 min, respectively.In the original OTC solution, there was a prominent fluorescence peak at E x /E m = 300-400/400-600 nm, which is located in the humic acid-like fluorescence region [53].With the passage of time, the characteristic peak intensity gradually weakened and the fluorescence intensity dropped from 2000.23 to 220.18, indicating that the OTC molecule was destroyed and may have been mineralized into some intermediates or decomposed into CO 2 and H 2 O (Figure 5d).The 3D EEMFs results show that the O 3 /CaO 2 /HCO − 3 system can degrade OTC, which is consistent with the UV-Vis spectrum results.and 353 nm, and the absorption peak near 353 nm is the characteristic peak of OTC.The common point of the two systems is that, after 30 min of processing, the characteristic peaks of OTC were significantly reduced, and the OTC molecules were weakened.The π-π * transition of its heteroaromatic ring makes the main contribution to this [51,52].Compared with Figure S3a, the characteristic peak of OTC in Figure S3b has a more rapid decline, which indicates that O3/CaO2/HCO 3 -had a better OTC degradation ability.

3D EEMFS
To study the degradation process of OTC more deeply, 3D EEMFs were used for characterization.The emission wavelength and the excitation wavelength were all set as 200-600 nm, whose sampling intervals were 5 nm and 10 nm, respectively.Figure 5a is taken from the original OTC sample, and Figure 5b and c are OTC samples processed for 10 min, 20 min, and 30 min, respectively.In the original OTC solution, there was a prominent fluorescence peak at Ex/Em = 300-400/400-600 nm, which is located in the humic acidlike fluorescence region [53].With the passage of time, the characteristic peak intensity gradually weakened and the fluorescence intensity dropped from 2000.23 to 220.18, indicating that the OTC molecule was destroyed and may have been mineralized into some intermediates or decomposed into CO2 and H2O (Figure 5d).The 3D EEMFs results show that the O3/CaO2/HCO 3 -system can degrade OTC, which is consistent with the UV-Vis spectrum results.

Variation of TOC and COD
TOC is an important parameter for evaluating the mineralization ability of oxidation systems.Figure 6a shows the degradation of OTC in terms of TOC removal, with or without two catalysts in the O 3 oxidation system.The carbon-containing organic matter in the OTC solution decreased as the treatment time increased.After the two catalysts were added, the TOC concentration decreased.When the treatment time was 30 min, the TOC degradation efficiency could reach 14.7%.Figure 6b depicts the COD concentration in the two systems.The COD concentration of the sole O 3 system underwent a slight drop, the speed was plodding, and it tended to be stable.The O 3 /CaO 2 /HCO − 3 system had a very high COD concentration at 0 min, with a significant drop and a fast speed, and a COD degradation efficiency of 48.8% was obtained within 30 min of the processing time.This O 3 /CaO 2 /HCO − 3 system produced a large number of active free radicals, which actively participated in the degradation process, which supports the derivation of the mechanism in the previous chapter.The O 3 /CaO 2 /HCO − 3 system has an excellent OTC degradation ability, but the degradation efficiency of TOC is far below 100%.Combining the results of the UV-Vis spectroscopy and 3D EEMFs, we speculated that a great deal of intermediates were formed during OTC degradation.degradation efficiency could reach 14.7%.Figure 6b depicts the COD concentration in the two systems.The COD concentration of the sole O3 system underwent a slight drop, the speed was plodding, and it tended to be stable.The O3/CaO2/HCO 3 -system had a very high COD concentration at 0 min, with a significant drop and a fast speed, and a COD degradation efficiency of 48.8% was obtained within 30 min of the processing time.This O3/CaO2/HCO 3 -system produced a large number of active free radicals, which actively participated in the degradation process, which supports the derivation of the mechanism in the previous chapter.The O3/CaO2/HCO 3 -system has an excellent OTC degradation ability, but the degradation efficiency of TOC is far below 100%.Combining the results of the UV-Vis spectroscopy and 3D EEMFs, we speculated that a great deal of intermediates were formed during OTC degradation.

Variation of pH and Conductivity
Figure S4a shows the pH change over time during OTC degradation.The O3/CaO2/HCO 3 -system presented a slightly alkaline environment, while the sole O3 system presented a neutral environment.As the treatment time increased, the pH values in both systems showed a gentle downward trend.Figure S4b shows the change in conductivity during OTC degradation.The conductivity of the sole O3 system was 21.2 μS/cm at 0 min, and after 30 min of treatment, the conductivity increased slightly to 30.3 μS/cm.The conductivity of the O3/CaO2/HCO 3 -system was 308 μS/cm at 0 min and dropped to 262 μS/cm after 30 min of treatment.We speculate that the addition of catalysts produced more inorganic ions, and acidic free radicals were generated during the degradation of OTC.In the process of degradation, HCO 3 -reacted with H2O2 to form HCO 4 -on the one hand and •OH to form •CO 3 -on the other, so HCO 3 -itself was consumed massively.The decrease in conductivity may have been due to the consumption of HCO 3 -.

Variation of pH and Conductivity
Figure S4a shows the pH change over time during OTC degradation.The O 3 /CaO 2 / HCO − 3 system presented a slightly environment, while the sole O 3 system presented a neutral environment.As the treatment time increased, the pH values in both systems showed a gentle downward trend.Figure S4b shows the change in conductivity during OTC degradation.The conductivity of the sole O 3 system was 21.2 µS/cm at 0 min, and after 30 min of treatment, the conductivity increased slightly to 30.3 µS/cm.The conductivity of the O 3 /CaO 2 /HCO − 3 system was 308 µS/cm at 0 min and dropped to 262 µS/cm after 30 min of treatment.We speculate that the addition of catalysts produced more inorganic ions, and acidic free radicals were generated during the degradation of OTC.In the process of degradation, HCO − 3 reacted with H 2 O 2 to form HCO − 4 on the one hand and •OH to form •CO − 3 on the other, so HCO − 3 itself was consumed massively.The decrease in conductivity may have been due to the consumption of HCO − 3 .

DFT Analysis and Degradation Pathway
The Fukui functions calculated with density functional and electron orbitals provide good evidence for predicting reaction sites.The molecular structure of OTC was calculated with the Gaussian 09 program.The Fukui function of OTC mapped the electron density isosurface and the calculation results are provided in Figure 7a and Table S2.Now, it is generally accepted that, the higher the f + function of the atom is, the easier will it be nucleophilically attacked; the higher the f − function of the atom is, the easier will it be electrophilically attacked; and the higher the f 0 function of the atom is, the easier will it be radically attacked.To indicate the possibility of the site being attacked clearly based on the above mechanism, electron cloud diagrams were drawn.It can be seen that the highlight is above C21 and O26, which means that these two sites would be easy to electrophilically attack.For the same reason, C4, O12, and C20 were easier to nucleophilically attack.C21 and O26 were easier to radically attack.Through a comprehensive comparison of the Fukui function table, it is inferred that OTC molecules may be decomposed into A (pathway 1) and B (pathway 2) due to electron and free radical attacks on C21 and O26.

Toxicity Evaluation
The U.S. Environmental Protection Agency's toxicity evaluation software tool (US−EPA−TEST, version 5.1.2) program of this study predicts the acute and chronic toxicity of OTC and its intermediates [55,57].The European Union criteria standard is adopted for acute toxicity, and the Chinese guidelines for the evaluation of hazardous chemicals (HJ/TI 154-2004) are adopted for chronic toxicity.The test results are shown in Table S4 and Figure 8.The daphnid LC50 of OTC is 9.34 mg/L, belonging to "toxic".After 30 min of treatment in this system, the toxicity of intermediates was significantly reduced, and all intermediates except F were located in "harmless" or "harmful" areas.A, B, and C were located in the "Harmless" area.Therefore, it can be speculated that the toxicity of OTC will decline significantly after O3/CaO2/HCO 3 -treatment, and detoxification may be achieved.S3.Combined with the Density Functional Theory (DFT) calculation and LC-MS detection results, the inferred degradation path of OTC is shown in Figure 7b. Figure 7c shows a free energy ladder diagram of these reactions.The initial energy of OTC was 0 kcal/mol, and the major degradation pathways are represented by negative energy levels.These reactions may occur, in theory, by different radicals.In the OTC molecular structure formula, weak chemical bonds between atoms are easy to destroy.C21 in pathway I is prone to enol ketone isomerization, and OTC is transformed into more stable isomers.The ketone/enol portion of C21-C25 is vulnerable to •OH attack, and further hydroxylation by-product A is obtained.O 3 in the system is an electrophilic reagent, which tends to attack the electron-rich region in the OTC structure [54].The double-bond structure of C21-C25 in pathway II and pathway III is broken by free radicals such as O 3 , and the main product is B. Fragile C5a breaks, causing B molecules to decompose into C and F. F is further dehydrated to obtain by-product G. C is demethylated to obtain by-product D, and D is further dehydrated to obtain small molecule by-product E. These intermediates could be further oxidized to small molecular organic acids, and ultimately mineralized to CO 2 and H 2 O [55,56].

Toxicity Evaluation
The U.S. Environmental Protection Agency's toxicity evaluation software tool (US-EPA-TEST, version 5.1.2) program of this study predicts the acute and chronic toxicity of OTC and its intermediates [55,57].The European Union criteria standard is adopted for acute toxicity, and the Chinese guidelines for the evaluation of hazardous chemicals (HJ/TI 154-2004) are adopted for chronic toxicity.The test results are shown in Table S4 and Figure 8.The daphnid LC50 of OTC is 9.34 mg/L, belonging to "toxic".After 30 min of treatment in this system, the toxicity of intermediates was significantly reduced, and all intermediates except F were located in "harmless" or "harmful" areas.A, B, and C were located in the "Harmless" area.Therefore, it can be speculated that the toxicity of OTC will decline significantly after O 3 /CaO 2 /HCO − 3 treatment, and detoxification may be achieved.

Feasible for Treatment of Other Antibiotics and Actual Wastewater
The feasibility of O3/CaO2/HCO 3 -for the treatment of other antibiotics and actual wastewater was investigated.The degradation of bisphenol A (BPA) and tetracycline (TC) under different systems is presented in Figure 9a,b.No matter the aspect of BPA or TC, the highest degradation efficiency was obtained in the O3/CaO2/ HCO 3 -system.As a whole, it can be seen that the degradation efficiency could reach above 80%, illustrating that the O3/CaO2/HCO 3 -system is also adapted to other antibiotic degradations.Figure 9c depicts the degradation of OTC in various water bodies.Over time, OTC molecules are gradually degraded in different water bodies such as deionized water, lake water, river water, and tap water.The degradation effect has not undergone significant changes, indicating that the system is also suitable for the treatment of actual wastewater.

Feasible for Treatment of Other Antibiotics and Actual Wastewater
The feasibility of O /CaO 2 /HCO − 3 for the treatment of other antibiotics and actual wastewater was investigated.The degradation of bisphenol A (BPA) and tetracycline (TC) under different systems is presented in Figure 9a,b.No matter the aspect of BPA or TC, the highest degradation efficiency was obtained in the O 3 /CaO 2 /HCO − 3 system.As a whole, it can be seen that the degradation efficiency could reach above 80%, illustrating that the O 3 /CaO 2 /HCO − 3 system is also adapted to other antibiotic degradations.Figure 9c depicts the degradation of OTC in various water bodies.Over time, OTC molecules are gradually degraded in different water bodies such as deionized water, lake water, river water, and tap water.The degradation effect has not undergone significant changes, indicating that the system is also suitable for the treatment of actual wastewater.

Feasible for Treatment of Other Antibiotics and Actual Wastewater
The feasibility of O3/CaO2/HCO 3 -for the treatment of other antibiotics and actual wastewater was investigated.The degradation of bisphenol A (BPA) and tetracycline (TC) under different systems is presented in Figure 9a,b.No matter the aspect of BPA or TC, the highest degradation efficiency was obtained in the O3/CaO2/ HCO 3 -system.As a whole, it can be seen that the degradation efficiency could reach above 80%, illustrating that the O3/CaO2/HCO 3 -system is also adapted to other antibiotic degradations.Figure 9c depicts the degradation of OTC in various water bodies.Over time, OTC molecules are gradually degraded in different water bodies such as deionized water, lake water, river water, and tap water.The degradation effect has not undergone significant changes, indicating that the system is also suitable for the treatment of actual wastewater.

Energy Efficiency Evaluation
Energy efficiency assessment is a part that cannot be underestimated.It is closely related to whether a new system can be promoted and applied.Therefore, we calculated the energy efficiency of the sole O3 system, O3/CaO2 system, and O3/CaO2/HCO 3 -system in different program doses, and the results are shown in Table S5.In the sole O3 system, the energy efficiency reached 1.55 g/kWh.When 0.050 g/L of CaO2 was added, the energy efficiency increased to 1.82 g/kWh.On this basis, when adding 2.25 mmol/L of HCO 3 -, the optimal energy efficiency could be increased to 1.91 g/kWh.The energy efficiency of the optimal O3/CaO2/HCO 3 -system could reach 1.91 g/kWh, which is an impressive result.
Table S6 lists the comparison results of the kinetic constants and energy efficiencies of other degradation OTC processes and this process.The degradation efficiency of the Al0-Gr-Fe0/O2 Fenton process for OTC was higher than that of other systems, close to 100%,

Energy Efficiency Evaluation
Energy efficiency assessment is a part that cannot be underestimated.It is closely related to whether a new system can be promoted and applied.Therefore, we calculated the energy efficiency of the sole O 3 system, O 3 /CaO 2 system, and O 3 /CaO 2 /HCO − 3 system in different program doses, and the results are shown in Table S5.In the sole O 3 system, the energy efficiency reached 1.55 g/kWh.When 0.050 g/L of CaO 2 was added, the energy efficiency increased to 1.82 g/kWh.On this basis, when adding 2.25 mmol/L of HCO − 3 , the optimal energy efficiency could be increased to 1.91 g/kWh.The energy efficiency of the optimal O 3 /CaO 2 /HCO − 3 system could reach 1.91 g/kWh, which is an impressive result.Table S6 lists the comparison results of the kinetic constants and energy efficiencies of other degradation OTC processes and this process.The degradation efficiency of the Al 0 -Gr-Fe 0 /O 2 Fenton process for OTC was higher than that of other systems, close to 100%, but the energy efficiency was extremely low [40].CO 3 O 4 /CNT catalyzed the degradation of OTC, and its degradation efficiency was close to O 3 /CaO 2 /HCO − 3 , but required a longer processing time and a relatively small kinetic constant [43].Among the many degradation OTC processes, O 3 /CaO 2 /HCO − 3 is undoubtedly the most economical and has the most potential [41,42,44,45].

Experimental Process
A reactor schematic diagram is presented in Figure S6.The O 3 was generated by plasma discharge, which was named as plasma-O 3 generator (CQ, Changqing-802S).The amount of O 3 used was extracted and adjusted using an atmospheric sampler.The active species were characterized using an electron paramagnetic resonance spectrometer (BRUKER, MESRV30077, Brooke Technology (Beijing) Co., Ltd., Beijing, China).To monitor the OTC degradation process, 3 mL of the aqueous solution was taken out of the reaction mixture every 5 min, and the experiment was repeated several times to take the average value for the subsequent analysis.The best degradation conditions were as follows: the concentration of CaO 2 was 0.050 g/L, the dosage of O 3 was 0.75 g/h, the dosage of HCO − 3 was 2.25 mmol/L, the pH was 2.4, and the initial concentration of OTC was 40 mg/L.

Analysis
The OTC calculation and analysis process is provided in Text S1.For the detection of O 3 , •OH, and H 2 O 2 in the OTC degradation process, the indigo method [58], the terephthalic acid probe method [59], and the titanyl sulfate method [60] were used, respectively.The data were analyzed and plotted with the origin (OriginLab Inc., version 9.8.0.200,Northampton, MA, USA) software.

DFT Analysis and Toxicity Evaluation
The molecular structures of the organic compounds studied were built using the Gauss View 6.0 software (Gaussian Inc., Wallingford, CT, USA).The built molecular structures were then optimized using chemical DFT methods (B3LYP/6-311++G (d, p)), employing Gaussian 16 (Gaussian Inc., Wallingford, CT, USA).Detailed information is shown in Text S2.US-EPA-TEST (US EPA, version 5.1.2,USA) was adopted to assess the toxicity of OTC and its intermediates based on the bioaccumulation factor and LC50 [57].

Conclusions
This study constructed O 3 /CaO 2 /HCO − 3 oxidation technology to degrade OTC wastewater.The introduction of CaO 2 /HCO − 3 significantly promoted the degradation of OTC

Molecules 2024 ,
29, x FOR PEER REVIEW 9 of 19tems.Compared to sole O3, the characteristic peaks of 1 O2 were enhanced with the addition of CaO2 and HCO 3 -, which indicates that the addition of CaO2 and HCO 3 -effectively increased the production of 1 O2.

Figure 6 .
Figure 6.Degradation efficiency of (a) TOC and (b) COD during OTC degradation.

Figure 6 .
Figure 6.Degradation efficiency of (a) TOC and (b) COD during OTC degradation.

Figure 7 .
Figure 7. (a) Fukui function of OTC mapped electron density isosurface: f − ; f + ; and f 0 , (b) pathways for OTC degradation and (c) energy barrier diagram.(Gray atoms represent C, white atoms represent H, red atoms represent N, and blue atoms represent O.).

Figure 7 .
Figure 7. (a) Fukui function of OTC mapped electron density isosurface: f − ; f + ; and f 0 , (b) pathways for OTC degradation and (c) energy barrier diagram.(Gray atoms represent C, white atoms represent H, red atoms represent N, and blue atoms represent O).To deeply explore the OTC degradation process in the O 3 /CaO 2 /HCO − 3 system, a Liquid Chromatograph Mass Spectrometer (LC-MS) (ES+, ES−) was adopted and the results are illustrated in Figure S5.According to the results of LC-MS, it can be inferred that there were eight main intermediates: A C 22 H 24 N 2 O 10 (m/z = 477), B C 15 H 13 NO 7 (m/z = 296), C C 4 H 7 NO 4 (m/z = 132), D C 3 H 7 NO 4 (m/z = 118), E C 3 H 7 NO 3 (m/z = 106), F C 6 H 10 O 2 (m/z = 113), and G C 6 H 12 O (m/z = 101).The molecular structures of the eight intermediates are shown in TableS3.Combined with the Density Functional Theory (DFT) calculation and LC-MS detection results, the inferred degradation path of OTC is shown in Figure7b.Figure7cshows a free energy ladder diagram of these reactions.The initial energy of OTC was 0 kcal/mol, and the major degradation pathways are represented by negative energy levels.These reactions may occur, in theory, by different radicals.In the OTC molecular structure formula, weak chemical bonds between atoms are easy to destroy.C21 in pathway I is prone to enol ketone isomerization, and OTC is transformed into more stable isomers.The ketone/enol portion of C21-C25 is vulnerable to •OH attack, and further hydroxylation by-product A is obtained.O 3 in the system is an electrophilic reagent, which tends to attack the electron-rich region in the OTC structure[54].The double-bond