Oxytetracycline Mineralization inside a UV/H2O2 System of Advanced Oxidation Processes: Inorganic By-Product

Oxytetracycline (OTC) was widely used antibiotic in agricultural industry. However, most of them were secreted from the body and entered the water stream, due to low absorption. The occurrence of the antibiotics in water stream may led to serious health hazards. Hence, finding the effective method that capable to achieve total mineralization of antibiotic-contaminated wastewater, followed by the production of benign inorganic and organic byproduct, was necessarily deemed. Photochemical degradation method, such as: UV/H2O2 system, was capable to achieve total mineralization of OTC at its optimized condition. In this paper, inorganic by-products of OTC mineralization inside a UV/H2O2 system at its optimum condition were analyzed. The presence of nitrate, ammonium, chloride ions, and chlorine were detected at the sample solution after mineralization. The presence of these inorganic by-product has proven that the experimental setup chosen was capable to achieve total mineralization. In addition, possible routes of the inorganic by-products detachment from the OTC’s structure, were also presented. Copyright © 2021 by Authors, Published by BCREC Group. This is an open access article under the CC BY-SA License (https://creativecommons.org/licenses/by-sa/4.0).


Introduction
In livestock production, beside acting as a microbial agent, antibiotics are also used as a growth promoter, steroid, insecticides, and tranquilizing agent. Living body of organism only One distinct character of antibiotics that it is biologically active, hence it could cause the antibiotics to be resistant against biological degradation process. This is caused by the capability of antibiotics to reduce or even eliminate the microbial growth in the biodegradation tank of conventional treatment. Therefore, there is a need to remove antibiotic traces effectively from the water stream. In this regard, a non-biological method, such as Advanced Oxidation Processes (AOPs) could become alternatives. Generally, to identify the efficient process for removing certain type of organic contaminant, the term of organic degradation and mineralization are used. These two terminologies can be found in numerous studies and measured with different instruments [3][4][5]. Typically, to evaluate the degradation efficiency, the concentration of the parent compound of the organic contaminant is directly measured using chromatography or spectroscopy technique. Therefore, once the organic parent compound peak is not detected inside the chromatogram or spectrum, the organic degradation is considered completed. About 100% reduction of oxytetracycline concentration was achieved, after 20 min of ozonation treatment. However, its toxicity by products (after 5-30 min of treatment) were highly increased [5]. Similar trend was also observed for degradation of tetracycline using UV/H2O2/Fenton system, where tetracycline concentration was removed after 1 min of treatment [4]. Whereas, 72% Total Organic Carbon (TOC) removal was achieved after 60 min of treatment. Liu et al. studied the tetracycline decomposition by using photo electron Fenton [6]. Based on their study, after 120 min of treatment, 98.3% of tetracycline was removed, whilst 84.3% of total organic carbon reduction was observed after 10 h of treatment. Lower degree of mineralization compared to degradation of parent compound also shown for UV photolysis of Oxytetracycline (OTC) [7]. About 100% of OTC was removed after 7.5 h, while only 5.37% of OTC was mineralized. In addition, 80% increment of inhibition rate was observed after 2 h of UV photolysis, which indicates toxicity increment in the partially degraded solution.
Mineralization is the transformation of the organic parent compound into final product of inorganic compound, such as: CO2, H2O, NH3, or even diatomic gases like Cl2 and N2. Whereas the mineralization progress is commonly measured by evaluating the TOC value. Meanwhile, an UV/H2O2 process could destruct the antibiotics mainly by non-selective attack of hydroxyl radical. Hence, complex reaction path-ways may be triggered and various by-products along with the organic intermediates were formed [8]. These by-products also can be categorized as low molecular weight organic acids and inorganic ions. Hence, reduction of the TOC value implies the progress of the mineralization process. To date, most published works were mainly investigating the OTC parent compound removal and TOC reduction. However, no works have been published on investigating the inorganic by-product of OTC mineralization, especially inside a UV/H2O2 system.
In this paper, mineralization of OTC inside a UV/H2O2 system were conducted under the optimum condition. The inorganic by-products were analyzed to prove the complete mineralization of the optimized parameter. Possible released routes of inorganic by-product from the main structure of OTC were also presented. From this by-product analysis, possible pathways for OTC transformations can be established. Hence, additional insight on the OTC's transformation pathways could contribute for evaluating the treatment efficiency and possible changes and its interactions in the environment.

Materials
OTC hydrochloride, C22H24N2O9HCl (Merck) and hydrogen peroxide (30%, Merck) were used as the source of organic contaminant and the source of hydroxyl radical, respectively. H2SO4 (98%, Merck) and NaOH (Merck) were used for pH adjustment. All chemicals were used without further purification.

UV/H2O2 system
In this study, the optimized experimental parameters were applied, as described elsewhere [9]. All the present experiments were carried out in a glass reactor (400 mL working volume) with a provision for irradiation using a low-pressure Hg lamp (8 W power GPH295T5L; Serial no. EC90277, USA) to produce UV light at 254 nm. The schematic diagram of the reactor is shown in Figure 1. During irradiation, the solution was stirred using magnetic stirrer at which speed. The temperature was maintained using water circulation through the reactor jacket. After adjusting the OTC solution to the optimum pH, desired amount of H2O2 were then added into the reactor. To monitor the progress of mineralization, 3 mL of liquid samples were drawn from the reactor at scheduled time intervals. Since the total amount of samples withdrawn are less than 5% (<5%) of the total volume, for calculation purpose the total sample volume was neglected. About 180 min of irradiation time were applied for all experiments. All the experiments were conducted at the same UV lamp power which emits the light intensity of 12.06 mW/cm 2 .

Ion Chromatography
The concentrations of nitrate, nitrite, and chloride ions were analyzed using Ion Chromatography (Metro Ohm-761, Switzerland), equipped with Metrosep-A Supp 5-150 columns, and eluted using 3.2 mM Na2CO3/1 mM NaHCO3 as the mobile phase. The ion chromatography was using a conductivity meter as the ion detector and deionized water as the blank solution. Three sets of standard solutions of chloride, nitrate, and nitrite ions were used to construct the standard calibration curve as listed in Table 1.

Nessler Ammonia
Concentration of ammonia in the solution sample was estimated by using Nessler reagent (K2HgI4) and method No. 8038 (HACH, USA). The reaction occurs in alkaline condition produce complex compound with yellow colour. The intensities were proportional to ammonia concentration in the solution. In addition, Ness-ler's reagent, mineral stabilizer and polyvinyl alcohol were also added into the sample. These additions were carried out to minimize the interference from the water matrix. The ammonia concentration was measured using spectrophotometer DR 2000 (Hach, USA).

Total Chlorine
This method measures the value of total chlorine, which is equal to the summation of free chlorine and combined chlorine. The measurement was carried out based on method No. 10070 (HACH, USA) which is in accordance w i th US EPA D PD (N , N -d i eth yl -pphenylenediamine) method. The present total chlorine in the solution will oxidize iodide in the reagent to iodine. Then the iodine reacts with DPD along with the free chlorine present in the sample to form a pink color which is proportional to the total chlorine concentration. The absorbance value of pink solution was measured at 530 nm using spectrophotometer 2000 (HACH, USA).

Concentration of CO2
To monitor mineralization progress of OTC, the TOC measurements were used. The values of TOC were measured using a TOC-VCSH analyzer (Shimadzu, Japan). About 2 mL of samples were drawn from the sample port and diluted with distilled water until the final volume of 20 mL. As shown in Equation (1), the value of TOC was determined from the difference between the total carbon (TC) with total inorganic carbon (TIC). During TOC analysis, the sample solution was acidified using H3PO4 to ensure that all the carbonate and bicarbonate were transformed into CO2. In water solution, CO2 could transform into carbonate and bicarbonate ions which their transformation depends on the solution's pH. In this research work, after UV/H2O2 treatment, the pH final solution is expected to be in the range below the initial pH, and most probably lower than the pKa of carbonate (pKa = 10.3) and bicarbonate (pKa = 6.3). This is due to the concentration of organic acids and other acidic by-

Ion
Calibration  products will be higher compared to the basic by-product. Therefore, the concentration of CO2 can be approximated into the concentration of inorganic carbon as measured by the TOC analyzer.
where TC is total carbon and IC is inorganic carbon.

Results and Discussion
In the present study, OTC hydrochloride was used as the model component. Therefore, the OTC solution contains C, H, O and N elements and Cl ions. Inorganic compounds such as NO2 − , NO3 − , and Cl − were determined using ion chromatography. The nitrogen containing compound such as ammonia was identified using Nessler reagent, in the form of ammonium ion. Whereas the by-product analyses as well as the degradation mechanism development were carried out at the optimum condition. During the optimization, total mineralization was chosen to be as the optimized response. Therefore, at the end of the reaction, CO2, H2O, NH3, NH4 + , NO2 − , NO3 − , N2, Cl, Cl2, and fractions of organic acid are expected to be present in the solution.
As it mentioned earlier, the ratio of inorganic by-product containing nitrogen moieties was depend on the character of N-containing structure [10]. Based on Figure 2, OTC structure has two types of nitrogen bond configuration, namely the tertiary and primary amine. Both structures contain hydrogen extractable character. As shown in Figure 3, there were changes in concentration by time for the NO3 − ion and NH4 + ions throughout the mineralization process. By introducing the UV/H2O2 into the OTC solution, instead of by ionization, the re-lease of ammonia group was mediated by indirect oxidation of hydroxyl radical [11]. Therefore, there was an increase in NH4 + ions concentration at t = 10 min of irradiation which was followed by the formation of NO3 − and observed similarly at t = 10 min. During this first 10 min, the hydroxyl radical generation was initiated by the photon from the UV lamp and started to attack the OTC compound. The generations of NO3 − and NH4 + occurred until t = 180 min. Moreover, starting at t = 10 min, there was a sharp decline in the NH4 + ions concentrations indicating their conversion into NO3. However, at the end of the reaction, not all NH4 + was transformed into NO3 − . This was due to the reason that no H2O2 remained in the solution thus stopping the generation of hydroxyl radical. The presence of NO3 − and NH4 + were also detected for mineralization of levofloxacin which resulted from the released of nitrogen content from the antibiotic main structure [12]. Mineralization of tetracycline using electro Fenton process also produced NO3 − and NH4 + ions [13].
Basically, there are two steps of NO3 − formation [14]. In the first step, the conversion of nitro group into NO2 − ion and followed by the oxidation into the NO3 − ion. It is then followed by the oxidation of NH4 + into hydroxylamine and followed by the formation of NO3 − . However, in the present study, the most probable mechanism of OTC mineralization would be the second step, since no NO2 − ion was observed throughout the experiment. Moreover, the nitrogen attached to the heterocyclic aromatic rings could be transformed into NH4 + and NO3 − , whereas the secondary, tertiary, and quaternary nitrogen atom will be transformed into NH4 + ions [10]. Ammonium ions were formed directly from the release of nitrogen as ammonia when the oxidation state of the  bound nitrogen is −3. In addition, an organic compound with a weak C−H bond at amine nitrogen position also often experiences fragmentations via hydrogen transfer or electronproton transfer. The possible transformation pathways of nitrogen atom of OTC molecule are presented in Figure 4, which is derived based on Pelizetti et al. proposed pathways for formamide [15]. Pelizetti et al. [15] have studied the photocatalytic degradation of formamide which has similar primary amine position of with the of OTC (position b, Figure 2). They postulated that the degradation mechanism was affected by the carbon atom oxidation state rather than the nitrogen atom. In other word, the final product of degradation is determined by the amount of the carbon atom substituent (i.e. 1 = primary carbon, 2 = secondary carbon, etc.) rather than the nitrogen atom substituent. Moreover, the hydroxyl radical attack on nitrogen atom was also occurred with bicarbonate and nitrate ions as their final product, with lesser extent. The proposed OTC intermediate structure after the primary amine release is presented in Figure 5. Similar mechanism was also presented by Jeong et al. [16] that describe the primary amine fragmentation of OTC structure which was initiated through ketoenol tautomerization. Beside the primary nitrogen, OTC molecule also has another nitrogen atom which belong to the tertiary amine group (position a, Figure 2). Figure 6 shows the proposed transformation pathways of tertiary amine of OTC molecule which was proposed based on the study performed by Klare et al. [17]. The radical attack on −CH was carried out based on hydrogen abstraction mechanism. Further hydroxyl radical attack would drive the occurrence of hydroxyl radical addition and followed by the C−C bond cleavage, producing carbonylic compound and an amine. In accordance with Figure 4 and Figure 6, at the earlier stage the amide and tertiary amine of OTC molecules may contribute to the formation of ammonium. Meanwhile, the generation of nitrate ion come from the transformation of amide group of OTC. Hence nitrate ion was detected in smaller concentration, compared to ammonium. Moreo-  ver, longer irradiation time would cause the reduction of ammonium to nitrate ions, hence lead to increased nitrate ion concentration. Similar results were also observed for the photocatalytic degradation of n-penthylamine [18]. Figure 7 shows the pH profile during OTC mineralization. After 10 min of irradiation, the solution pH decreased from 6.5 to 3.8 which continues until 90 min of irradiation. This pH reduction most probably due to the higher concentration of acidic intermediates (organic acid) and CO2 formed, rather than ammonia. Similar results were also obtained for photocatalytic degradation of piperidine, after 10 min of irradiation, the piperidine solution pH decreased rapidly from 7.1 to 5.9 [18]. However, after 120 mins irradiation, pH of the solution started to increase until 180 min of irradiation. This increase was due to the higher concentration of amine intermediate (i.e. ammonia) in the solution compared to acidic intermediate (i.e. organic acid and CO2). In contrast with OTC, npentylamine oxidation experienced acidic pH at the end of irradiation time due to the excess of CO2 being produced compared to ammonia [18].
During TOC analysis, the sample solution was acidified using H3PO4 to ensure that all the carbonate and bicarbonate were transformed into CO2. However, since the partiallydegraded OTC sample had pH below the pKa  of carbonate (pKa = 10.3) and bicarbonate (pKa = 6.3), the value of inorganic carbon could be approximated as the concentration of CO2. Based on Figure 8, the CO2 concentration profile increased along the irradiation time which proved the occurrence of the OTC mineralization. Similar CO2 profiles were also shown during the piperidine and chloramphenicol removal [17][18].
Since the OTC hydrochloride was used as the antibiotic sample, the chloride ion was detected inside the solution sample due to the ionization of OTC compound. Inorganic chloride ion was also observed during the photocatalytic oxidation of cyclophosphamide and atrazine [10]. After irradiation took place, the hydroxyl radicals were started to form. Since the reduction potential of hydroxyl radical is higher (E°= +2.8 V) compared to chloride ion (E°= +2.2 V), the hydroxyl radical is capable to oxidize the chloride ions (Equation 2). The HOCl• − radical could decompose into radical (Equation 3) that could recombine into to form chlorine atom [19].
The oxidation of chloride ions into Cl2 was progressing as the mineralization of OTC took place. In the present study, the presence of chlorine atom was represented by the value of total chlorine total chlorine is equal to total amount of free chlorine plus chlorine attached to other components. Their profiles are shown in Figure 9. As the reaction proceed (t = 30 min), a certain amount of chloride ion was started to dissociate from the OTC parent structure hence increased in chloride ion concentration was observed. At the same time in-terval (t = 30 min), some of chloride ion were oxidized which led to the formation of chlorine atom. Thus, an increasing concentration of chlorine was observed. However, after 180 min of irradiation, the chloride ions were not oxidized completely into chlorine.

Conclusions
Mineralization of OTC have been conducted at its optimum TOC mineralization. Several inorganic by-products were detected, namely, nitrate, ammonium, chloride ion, chlorine. The detected by-products have proven the efficacy of the mineralization process. Based on the generated by-products, several reaction sequences have been proposed.