Study on Mechanism of Synergistic Effect of pH and Dissolved Organic Matter in Water on Photochemical Transformation Kinetics of Antibiotics

Significance: Antibiotics，as a class of emerging pollutants，have drawn a great deal of attention，and photochemical transformation represents a significant environmental transformation behavior of antibiotics in water^［3-4］. Photochemical transformation brings about irreversible alterations to antibiotic molecules，which governs the persistence of antibiotics within the aquatic environment. Consequently，it exerts an influence on the reduction and ultimate fate of antibiotics therein and may also lead to changes in the toxicity，bioavailability，and ecological risks associated with antibiotics. The increasing presence of antibiotics in aquatic environment poses significant risks to ecosystems and human health. pH affects the photochemical persistence of organic pollutants^［5-6］，and dissolved organic matter (DOM)，which is ubiquitous in water，is an important photosensitive substance that mediates the photochemical transformation of organic pollutants^［1-2］. Therefore，investigating the effect of different pH on the photochemical transformation of pollutants mediated by DOM and revealing the influencing mechanism from the DOM molecular level are of great significance for evaluating the environmental fate of organic pollutants.

　　Globally，research on the photochemical transformation of antibiotics has gained momentum，yet significant challenges remain. Many studies have focused on isolated factors affecting degradation rates，such as light intensity，temperature，pH and DOM and so on. ³DOM^* has been proved to be the main photochemically produced reactive intermediate (PPRI) mediating the photochemical transformation of quinolones^［7］，sulfonamides^［8］ and tetracyclines^［9］，and antibiotics can react quickly with ¹O₂ and •OH^［10-11］. However，the synergistic effect of pH and DOM in influencing the photochemical transformation of antibiotics has not been thoroughly explored. This gap in knowledge limits the ability to predict the environmental fate of these pollutants under varying conditions.

　　In the study，norfloxacin (NOR) and ciprofloxacin (CIP) commonly detected in aquatic environment were taken as representatives of antibiotic emerging contaminants，and DOM extracted from Songhua River (S-DOM) and Suwannee River Natural Organic Matter (SRNOM) purchased from International Humic Substances Society were regarded as representatives of DOM. Under simulated sunlight irradiation，through photochemical simulation experiments and high performance liquid chromatography (HPLC) detection methods，the synergistic effect of pH and DOM on the photochemical transformation kinetics of antibiotics was studied. The molecular mechanism was elucidated by exploring effects of different pH values on quantum yields of ³DOM^*，¹O₂ and •OH produced by photosensitization of DOM and its small molecular analogues.

　　The finding reveals that pH significantly affects the occurrence forms of NOR and CIP，leading to enhanced photochemical transformation rates at pH=9.0，where both of antibiotics exist in zwitterionic forms. The study quantifies the contributions of PPRIs generated by DOM，specifically ¹O₂ and •OH，demonstrating their pivotal roles in promoting antibiotic degradation. The result indicates that the contribution of these species diminishes as pH increases，highlighting the complex dynamics of antibiotic phototransformation.

Methods: S-DOM water sample was taken from Songhua River，and the sampling point was near the sand quarry beside the Wujintun Bridge Toll Station. S-DOM was obtained by elution and drying through the solid phase extraction method，representing DOM in Songhua River under natural conditions. The key step for extracting S-DOM by the solid phase extraction method were as follows: (1) Water sample pretreatment: Filtration was performed using a 0.45μm glass fiber membrane; (2) Determination of TOC in water sample: Each solid phase extraction cartridge (Agilent，1g) adsorbed DOC＜2mmol/L or ＜10L of water sample; (3) Sample acidification: The water sample was acidified to pH=2.0 with formic acid; (4) Column activation: The cartridge was rinsed with methanol of twice the column volume，and then the filler was rinsed with acidified water with a pH=2.0 of twice the column volume; (5) Sample loading: The water sample was extracted using a semi-automatic solid phase extraction instrument，and the flow rate was adjusted to 4.9mL/min; (6) Washing: The ionic components were removed with 20mL of formic acid water，and then the excess formic acid was removed with 20mL of ultrapure water; (7) Drying the cartridge: The cartridge was dried with nitrogen; (8) Elution: Elution was carried out with methanol，and the eluate was stored in a sample bottle for later use; (9) Drying the eluate: The eluate was dried with a nitrogen blowing instrument; (10) Preservation of DOM: It was dissolved with 10ml of ultrapure water and preserved after sonication.

　　The photochemical experiment was carried out on an XPA-7 rotary photochemical reactor equipped with a condenser. A 500W mercury lamp was selected，and 290nm filters were added to simulate UV-A and UV-B parts of sunlight. The condenser was used to maintain the temperature at 25±1°C. Phosphate buffer solutions with different pH values (5.0，7.0，9.0) were used to prepare the reaction solutions. In the experiment on the photochemical transformation kinetic of antibiotics，initial concentrations of NOR and CIP were 20μmol/L. Sorbic acid (SA)，sodium azide (NaN₃)，and isopropanol (IPA) were selected as quenchers for ³DOM^*，¹O₂，and •OH respectively，with initial concentrations of 0.1mmol/L，1mmol/L，and 1mmol/L respectively. In the experiment regarding the photosensitized generation of PPRIs by DOM and its small molecule analogues，2，4，6-trihydroxy phenol (TMP)，with an initial concentration of 0.1mmol/L，was chosen as a chemical probe for ³DOM^* for the purpose of capturing it. Meanwhile，furfuryl alcohol (FFA)，at an initial concentration of 50μmol/L，was selected as a probe for ¹O₂ to trap it. Additionally，benzene，with an initial concentration of 3mmol/L，was picked as a probe for •OH to catch it. Initial concentrations of SRNOM and S-DOM were 5.0mgC/L，and that of small molecule analogues in the experiment were all 10mg/L. At the selected time intervals，1mL of sample was taken from each quartz tube and injected into a brown liquid bottle. All photochemical experiments were carried out in triplicate. Then，the concentrations of the target compounds were analyzed using HPLC. The relevant parameters for detecting compound concentrations by HPLC are shown in Table 1.

　　During the photochemical transformation process，apparent photolysis rate constants (k_obs) of NOR and CIP were fitted using a first order kinetic model. The quantum yield coefficient of ³DOM^* (f_TMP)，the quantum yield of ¹O₂ (Φ_1O2)，and the quantum yield of •OH (Φ_•OH) were analyzed and processed based on formulas (1)-(9).

Data and Results: The synergistic effect of pH and DOM on the photochemical transformation kinetics of NOR and CIP was studied by using photochemical simulation experiments and HPLC. The photochemical transformation of NOR and CIP is pH-dependent，and DOM inhibits their photochemical transformation through the light screening effect，as shown in Fig.1. When pH=5.0，7.0 and 9.0，k_obs of NOR in pure water are 0.05，0.18 and 0.39min⁻¹respectively (Fig.1a); k_obs of CIP in pure water are 0.05，0.20 and 0.43min⁻¹ respectively (Fig.1b); k_obs of NOR in SRNOM (S-DOM) solutions are 0.04，0.14 and 0.31min⁻¹ (0.05，0.16 and 0.36min⁻¹) respectively; k_obs of CIP in SRNOM (S-DOM) solutions are 0.02，0.15 and 0.28min⁻¹(0.03，0.15 and 0.30min⁻¹) respectively. Through quenching experiments，contributions of PPRIs generated by DOM under different pH conditions to the photochemical transformation of NOR and CIP were studied，and contributions of ¹O₂ and •OH are the most significant，as shown in Fig.2.

　　Regarding the effect of pH on the photosensitized generation of ³DOM^*，¹O₂，and •OH by DOM，results are as follows: (1) Under the same pH value，the ability of S-DOM to generate PPRIs is higher than that of SRNOM，and pH can affect the ability of DOM to generate PPRIs and the steady state concentrations of PPRIs，as shown in Fig.3. (2) DOM small molecule analogues with a higher degree of dissociation have a higher f_TMP，while DOM small molecule analogues with a lower degree of dissociation have a higher Φ_1O2 and Φ_•OH，and pH affects the generation ability of PPRIs by controlling the dissociation forms of dissociable DOM small molecule analogues，as shown in Fig.4. (3) pH affects the generation ability of PPRIs by influencing the redox activity changes of the quinone groups of three non-dissociable DOM small molecule analogues，as shown in Fig.5.

　　Unlike previous studies that mostly focused on a single factor，this paper comprehensively considered the synergistic effects of pH and DOM on the photochemical transformation kinetics of the two antibiotics，NOR and CIP. Moreover，it further explored the ability of DOM，dissociable DOM small molecule analogues，and non dissociable DOM small molecule analogues to generate PPRIs and their influencing factors，providing a more comprehensive perspective for in depth understanding of the role of DOM in the photochemical transformation process.