Photocatalytic difference of amoxicillin and cefotaxime under visible light by mesoporous g-C3N4: Mechanism, degradation pathway and DFT calculation

Journal Pre-proofs Photocatalytic difference of amoxicillin and cefotaxime under visible light by mesoporous g-C3N4: mechanism, degradation pathway and DFT calculation Mengmeng Dou, Jin Wang, Boru Gao, Ce Xu, Fan Yang PII: DOI: Reference:

S1385-8947(19)32546-X https://doi.org/10.1016/j.cej.2019.123134 CEJ 123134

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

23 July 2019 26 September 2019 10 October 2019

Please cite this article as: M. Dou, J. Wang, B. Gao, C. Xu, F. Yang, Photocatalytic difference of amoxicillin and cefotaxime under visible light by mesoporous g-C3N4: mechanism, degradation pathway and DFT calculation, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej.2019.123134

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier B.V.

Photocatalytic difference of amoxicillin and cefotaxime under visible light by mesoporous g-C3N4: mechanism, degradation pathway and DFT calculation Mengmeng Doua,b, Jin Wanga,b,*, Boru Gaoa,b, Ce Xua,b, Fan Yanga,b a. Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Haidian District, Beijing, China, 100044 b. Beijing Key Laboratory of Aqueous Typical Pollutants Control and Water Quality Safeguard, Beijing, China, 100044 *Corresponding

author. E-mail address: [email protected]. Tel.: +86 10 51685628.

Fax: +86 10 51683764.

1

Abstract β-lactam antibiotics are generally used for disease prevention in humans and animals. The antibiotics, which have been excreted into the surrounding environment, have caused serious concerns. In this study, mesoporous carbon nitride (MCN) was synthesized using a template-free method. The photocatalytic degradation of the two typical β-lactam antibiotics, amoxicillin (AMX) and cefotaxime (CFX), was performed using MCN. Considering the complexity of the actual environment, four factors that may influence the photocatalytic degradation of AMX and CFX were studied, containing the initial antibiotics concentration, catalyst dosage, pH and humic acid (HA). The possible mechanism of photocatalysis was presented using ESR spectroscopy and free radical trapping experiments. Furthermore, rational photocatalytic degradation pathways were proposed through the combination of LC-Q-TOF-MS/MS and density functional theory (DFT). Toxicity Estimation Software Tool (TEST) was used to predicate the toxicity of the byproducts. This study identified the catalytic activity difference between AMX and CFX firstly and elucidated their degradation mechanisms. The new findings are very meaningful for optimizing the conditions of photocatalytic degradation of β-lactam antibiotics. In addition, the results of wastewater test and stability experiments indicated that MCN has excellent performance. Therefore, MCN is a promising treatment material for the elimination of the antibiotic activity and mineralization of antibiotics under visible light. Keywords: Mesoporous g-C3N4; Photodegradation; Amoxicillin (AMX); Cefotaxime (CFX); DFT calculation

2

1. Introduction The presence of antibiotics in the environment has been reported as an emerging risk to the biological environment[1]. Amoxicillin (AMX) (Structure SⅠ) and cefotaxime (CFX) (Structure SⅡ), both of which are broad-spectrum and semisynthetic β-lactam antibiotics, have been widely used for disease prevention in humans and animals[2]. These antibiotics were introduced into various environments as excipients or by-products through excretion. Although the toxicity of AMX and CFX and their concentrations in the environment are relatively low, the emergence of resistant bacteria and resistance genes due to the presence of antibiotics is a more impending problem[3]. In addition, on account of the biodegradability of AMX and CFX, they cannot be effectively removed by conventional wastewater treatment processes[4]. Therefore, it is required to develop novel alternative ways to solve the antibiotics pollution problems. Up to now, various methods have been applied including physical, chemical and biological remediation strategies in the field of the removal of organic pollutants. Advanced oxidation as a common chemical treatment are widely applied, such as UV irradiation, Fenton, ozone and so on[5-7]. But these conventional treatments have problems with high energy consumption and secondary pollution. Therefore, the semiconductor photocatalysis has been considered as a promising technique to treat environmental pollutants due to its clean, cost-effective, and environmentally friendly conversion by utilizing solar energy[8]. Multifarious photocatalysts have been widely reported, such as ZnO, TiO2[9,10]. However, due to these catalysts have a large band gap, it can only be excited by ultraviolet light, which accounts for only 4% of the total solar energy. Therefore, developing a new narrow-band gap semiconductor catalyst are urgent problems to be solved. 3

Recently, graphite carbon nitride (g-C3N4), which is a promising metal-free polymeric n-type semiconductor with distinct two-dimensional structure, excellent chemical stability, small band gap (2.70 eV), easy synthesis, adjustable electronic structure and low cost, has received more and more attention[11]. Meanwhile, researchers are constantly improving the performance of g-C3N4 to overcome some of its shortcomings, which limit its photocatalytic activity, such as low utilization of visible light, rapid recombination of photogenerated electrons, and small specific surface area. Zeng et al aquired a sea-urchin-structure g-C3N4 with ∼2.0 eV bandgap using a hydrothermal strategy to improve the utilization rate of visible light[12]. Carbon quantum dots modified porous g-C3N4 was prepared to enhance separation of charge carriers which was used to degrade the diclofenac[13]. In addition, there have been many reports of improving the photocatalysis efficiency of g-C3N4 through metal doping and heterojunction[14,15]. Mirzaei et al demonstrated that magnetic fluorinated Fe3O4/g-C3N4 exhibited better photocatalytic activity for amoxicillin degradation than g-C3N4 itself[16]. And some other references on photocatalytic degradation of AMX and CFX are summarized in the Supporting Information (Table S1). But most of the previous reports have focused more on the characterization and analysis of materials, there were only a handful of studies focusing on the degradation mechanism of antibiotics. Both AMX and CFX have typical β-lactam rings in structure, but the stability and pharmacology of the molecule are different due to different side chains. The degradation mechanism based on photocatalysis and the analysis of difference of AMX and CFX during the g-C3N4 based photocatalytic process remain unknown. And the data relating to the toxicity of the byproducts of AMX and CFX during the process of photodegradation has not been obtained. Therefore, the elevant mechanism analysis of AMX and CFX via g-C3N4 based

4

photocatalysts are strongly required to be further explained. Herein, we successfully synthesized MCN using a template-free method and used it for the photocatalytic degradation of AMX and CFX. Some factors that may affect the photocatalytic process, including the environmental pH, natural organic matter, and catalyst’s dose were investigated. The degradation intermediates of AMX and CFX were analyzed with the combination of LC-Q-TOF-MS/MS and the density functional theory (DFT) calculations. Based upon the results, a reasonable degradation path was proposed and the toxicity of the byproducts was predicted by TEST from United States Environmental Protection Agency (USEPA). In addition, the catalytic activity differences between AMX and CFX were further explained based on MCN. The study is among the first to identify the differences in the photodegradation of AMX and CFX and elucidate the mechanisms. Moreover, four different water matrices were considered and the stability of MCN was made to test the practicability and recyclability of the photocatalyst. This work is instructive for optimizing the conditions for the photocatalytic degradation of antibiotics. Structure SⅠ/Ⅱ

5

2.

Experimental

2.1

Chemicals Melamine, hydrochloric acid (HCl), isopropyl alcohol (IPA), ammonium oxalate

monohydrate (AO), ascorbic acid (AA) and 5,5-dimethyl-1-pirroline-N-oxide (DMPO) were purchased from Aladdin Industrial Corporation. Amoxicillin (98%) and cefotaxime (97%) were provided by Shanghai Macklin Biochemical Co., Ltd. Methanol, acetonitrile, and formic acid were of HPLC grade and purchased from Thermo Fisher Scientific. All chemicals and drugs were used without further purification. 2.2 Photocatalyst preparation Mesoporous g-C3N4 was prepared according to the procedure reported in a previous work[17]. Typically, 3 grams of melamine was added to 80 ml of boiling water with continued stirring. After the melamine solution was cooled to 50 ℃, 2 ml of HCl (37%) (molar ratio of HCl to melamine was 1:1) was slowly added under magnetic stirring. After 30 minutes, the solution was transferred to an oven and dried at 80℃ to finally obtain melamine hydrochloride. The melamine hydrochloride and unacidified melamine were then placed in a covered crucible and heated to 500℃ (heating rate of 20℃/min) under nitrogen for 2 h, respectively. Then they were heated to 520℃ for 2 h at a heating rate of 4℃/min. The mesoporous g-C3N4 (labeled as MCN) and the bulk-g-C3N4 (labeled BCN) were obtained after the sample was naturally cooled. 2.3 Characterization Detailed characterization techniques are shown in Supporting Information.

6

2.4 Photodegradation of AMX and CFX The photocatalytic properties of BCN and MCN were evaluated based upon the degradation of two typical aqueous solutions of β-lactam antibiotics. The 300W xenon lamp (with filter >420 nm) was used to simulate the visible light. In a typical photocatalytic degradation experiment, 100 mg of photocatalyst was dispersed in the antibiotic aqueous solution (100 mL) under magnetic stirring, whereas dark reaction was continued for 30 min to establish the adsorption-desorption equilibrium. After light exposure, 3 mL of the solution was taken at a certain time interval for centrifugal filtration, and then, analyzed using HPLC. In order to more accurately explore the degree to which it is susceptible to the influence of substances in the real water body, a low-concentration solution (2 ppm) was adopted to study the influencing factors. In the process of intermediate product analysis, a relatively high concentration of solution (20 ppm) was used to analyze the degradation process. All of the experiments were undertaken in triplicate. 2.5 Analysis of the intermediate products AMX and CFX concentrations were determined using HPLC using a Shimadzu LC-16 system equipped with an auto-injector, a column oven, a binary pump, a degasser, and a UV detector. Further degradation mechanism was studied using LC-Q-TOF-MS/MS (Agilent 6540 USA). The detection conditions are given in the Supplementary Information (SI). 2.6 Methodology of chemical calculations The Gaussian 09 program was employed for density functional theory (DFT) at the B3LYP/6-311G level. The optimization of geometric structure and condensed Fukui function analysis were carried out. Yang et al provided a condensed Fukui function (Eqs. (1) - (3)) based on the atomic charges (Hirshfeld and natural 7

population analysis (NPA) charges) and related to the change of electron density, which is used to explain the reaction mechanisms as well as stereo-selectivity[18]. Additionally, the Multiwfn software was used to calculate the qN 1 , qN and qN 1 , which represents the electron density of the molecule lost an electron, the neutral molecule and the molecule getting an electron, respectively. Then, the electrophilic (f +), nucleophilic (f -) and radical attack (f 0)

were calculated and used to evaluate

the reactive sites on the molecule[19].

fi   qN 1  qN

(1)

fi   qN  qN 1

(2)

fi 0  ( fi   fi  ) / 2

(3)

8

3.

Results and discussion

3.1 Characterization of the photocatalyst The prepared samples were characterized using FE-SEM and BET. As shown in Fig. 1, BCN was superimposed using a two-dimensional sheet-like structure with a smooth surface. Compared to the structure of BCN, the structure of MCN became significantly fluffy. The volume of MCN was around 2 times higher than that of the BCN for the same mass in the bottles. It can be seen that the density of MCN was smaller than that of BCN, and therefore, it was fluffier than BCN. It is well known that the specific surface area of a photocatalyst is one of the factors affecting the photocatalytic performance. Semiconductor materials with larger specific surface area have more surface-active sites, which are beneficial to the adsorption of pollutants and improve the utilization of light. MCN exhibits a Type IV isotherm with an H3 hysteresis loop, which is a typical feature of mesoporous materials. According to the BET theory, the specific surface area of MCN was 3.2 times that of the BCN, which are 18.06 m2/g and 5.64 m2/g, respectively. These values showed that MCN, prepared using the template-free method, successfully formed porous structure. The prepared samples were subjected to pore size distribution analysis using the BJH method. It can be seen that BCN has almost no porous structure, and the main pore size distribution range of MCN was in the range of mesopores (2-50 nm), indicating that the porous structure was not destroyed during the preparation of catalyst. The results of XRD, FT-IR, PL, UV-vis, Mott-Schottky, XPS and TEM are shown in the Supporting Information (SI). Fig. 1

9

3.2 Photocatalytic performance of photocatalysts AMX and CFX were employed as a representative pollutant to evaluate the photocatalytic activities of the prepared samples. As shown in Fig. 2, under visible light irradiation, no obvious change was observed in AMX and CFX without the addition of any photocatalytic catalyst. When the photocatalytic material was added, BCN could degrade AMX to 40% and the removal rate of CFX was found to be 80% after 60 min. However, the photodegradation rate of AMX and CFX increased to 90% and 99% after the addition of MCN. The kinetic analysis showed that both the AMX and CFX photodegradations followed first-order kinetics, and the results for the kinetic constant (k) are shown in Fig. 2. For AMX and CFX, the conversion from BCN to MCN was 2.5 times and 4 times enhanced with regards to the degradation efficiency, respectively. Obviously, the photocatalytic activity of MCN was higher than that of the BCN, whereas the improved photocatalytic activity was mainly attributed to the formation of porous structure, which improved the photocatalytic efficiency of the catalyst[17]. Fig. 2 3.3 Factors affecting the photodegradation process 3.3.1 Effect of initial antibiotic concentration The effects of different initial concentrations of antibiotic solution on the photocatalytic activity of MCN were investigated. As shown in Fig. 3, when the solution concentration increased from 1 mg/L to 10 mg/L, the removal rate of AMX and CFX decreased from 99% to 40% and 69%, respectively. It was obvious that photocatalytic activity was inhibited when the concentration of antibiotic solution increased. This may be due to three reasons. Firstly, the number of free radicals produced by a certain amount of materials is fixed. As the concentration of solution

10

increases, the reactant in the system becomes more competitive with the limited reacting oxygen species (ROSs)[20]. Secondly, when the concentration of the solution increases, the transmittance of the solution will be affected, due to which, the absorption of photons by the material will be weakened, and the number of free radicals produced will be reduced[21]. Thirdly, when the concentration of the initial solution increases, the intermediate products produced will compete with the original substances for free radicals, which will also lead to the reduction of degradation rate[22]. Fig.3 3.3.2 Effect of catalyst dosage The catalyst dosage is also a key factor affecting the photodegradation efficiency. As shown in Fig. 3c, four different dosages were carried out in the photodegradation study of AMX (20 mg, 50 mg, 100 mg, and 150 mg). When the dosage was increased from 20 mg to 100 mg, the photocatalytic efficiency gradually increased from 63% to 96%. The phenomenon may be due to the prepared MCN, which cannot provide sufficient active sites for the photodegradation of AMX under the condition of low dosage. Higher the dosage, more are the produced ROSs within limits[23]. However, when the catalyst dosage was further increased to 150 mg, the reaction efficiency decreased. This was because the excessive dosage increased the turbidity of solution, due to which, light transmittance was reduced, and the absorption of photons was cut down for MCN[24]. In the degradation of CFX (Fig. 3d), the same pattern was presented. However, the optimum dosage for CFX was 150 mg, and not 100 mg as was the case for AMX. It was confirmed that the best dosage of the same catalyst for different substances may be different. Therefore, the optimum dosage of the catalyst

11

may depend on the relationship between the structural characters of the antibiotics and the material properties. Fig. 3 3.3.3 Effect of pH The effect of acidity and alkalinity of the solution on photocatalysis is important and cannot be ignored. Experiments were conducted by varying the pH within the range of 3-11 to study the effect of pH. The initial pH of the antibiotics’ aqueous solution was adjusted to a predetermined value using 0.01M HCl and 0.01M NaOH. As shown in the inset of Fig. 4(a, b), AMX and CFX were stable in the acidic environment in the absence of MCN. However, when the pH changed to 11, both the AMX and CFX underwent significant degradations of 14% and 27%, respectively. It is due to the fact that beta rings are prone to hydrolysis under alkaline conditions[25]. As shown in Fig. 4a, compared to the blank, the addition of MCN had the lowest degradation rate of AMX at the pH of 5, whereas the maximum stability of AMX has been reported at this pH[24]. The same is true for CFX in neutral environment, as shown in Fig. 4b[26]. Clearly, both Fig. 4a and 4b showed that the degradation rate of AMX and CFX are the highest at the pH of 3, and reached 80% and 99%, respectively. The phenomenon may be attributed the increasing of hydroxyl radicals in the acidic condition which is shown in Eqs. (4) – (6). Superoxide radicals (∙O2- ) generated by MCN combine with electrons to produce hydrogen peroxide (H2O2), then create hydroxyl radicals(∙OH) by the bonding of electrons and H2O2. Redox potential of ∙OH was higher than that of ∙O2-.

O2  e   O2 

(4)

O2   2 H   e _  H 2O2

(5)

12

H 2O2  e _  OH  OH 

(6)

Fig. 4 3.3.4 Effect of HA Humic acid (HA) is a macromolecular organic substance widely found in nature. The effect of HA on the photocatalytic degradation of AMX and CFX using MCN was investigated. Different HA concentrations of 2 mg/L, 5 mg/L, and 10 mg/L were tested, and the corresponding results are shown in Fig. 5. The results showed that the photodegradations of AMX and CFX were inhibited with the addition of HA. As the concentration of HA increased, the inhibitory effect gradually strengthened. This may be due to the following two reasons. On one hand, HA has photosensitivity and will compete with MCN for the absorption of photons. The generation of active species was inhibited, leading to the decline of degradation rate[27]. On the other hand, HA will compete with AMX and CFX to obtain free radicals in the solution, which could reduce the reaction rate[28]. Fig. 5 3.4 Mechanism analysis 3.4.1 Possible photocatalytic reaction mechanism Generally speaking, the active oxidants in the photocatalytic process are of great significance to infer the reaction process and electron transfer mechanism. In this study, IPA (1 mmol/L), AO (1 mmol/L), and AA (1 mmol/L) were used as scavengers for hydroxyl radical (∙OH), hole (h+), and superoxide radical (∙O2-), respectively. Fig. 6(a, b) shows the effect of MCN catalysts on the degradation of AMX and CFX in the presence of different free radical scavengers. Obviously, ∙O2- is the main active species in the photocatalytic process. In order to further demonstrate that ∙O2- is 13

present in the photocatalytic reaction system, ESR spectra were used. 5 mg of MCN was added to 5 mL of CH3OH, and stirred for 2 min. Then, 60 μL of the sample was added to 2 μL of DMPO with stirring for 2 min. The prepared sample was sucked using capillary and placed immediately into ESR (emxplus-10/12, Bruker, USA) chamber with the detailed process in the Supporting Information. The test results are shown in Fig. 6(c), indicating that ∙O2- does exist in the photocatalytic system. The probable mechanisms for the photodegradation of MCN can be deduced as following. The analysis consequence of PL, photocurrents and EIS demonstrated that the photogenerated electrons and holes separation efficiency of MCN were improved after the formation of the pore structure (Fig. S3). As shown in Fig. 6(d), MCN was driven by visible light to produce electrons and holes. The electrons can combine with oxygen to further generate superoxide radicals, which could promote the oxidation and decomposition of pollutants. As an oxidant, the holes can directly act on the pollutants. Moreover, both Fig. 6a and Fig. 6b indicated the presence of small amount of ∙ OH in the system, which is maybe produced by the combination of superoxide radicals and hydrogen ions in water as explained by Eqs. (7)-(9)[29,30].

g-C3 N 4  h  g-C3 N 4 (e  , h  ) O2  e   O2  O2   2 H   2  OH

(7) (8) (9) Fig. 6 and Fig. S3

3.4.2 Proposed degradation pathways and toxicity prediction In the photodegradation of AMX and CFX, the initial concentration was 20 ppm for a more accurate analysis of the decay mechanism. As shown in Fig. 7(a), with the increase in reaction time, the degradation rate of AMX was up to 60% in 120 min, while the rate of degradation of CFX was up to 99% in 60 min. However, in TOC 14

detection, as shown in Fig. 7(b), the degree of mineralization for AMX and CFX were 25% and 32%, respectively. Due to this reason, the antibiotics are not completely mineralized. Therefore, we carried out the next LC-QTOF-MS/MS catalysis and DFT calculation to analyze the products. In Fig. S5, AMX is a molecule with m/z value of 366.11 [C16H19N3O5S+H] with cleavages, whereas the corresponding fragment masses show lack of an amidogen with [M+H]=349.08[31]. Furthermore, the peak at [M+H]=456.09 corresponds to the protonated molecule of CFX. Secondly, the characteristics of AMX and CFX have also been studied in Fig. 10(a) after the Gaussian optimization, in which different angles of molecules could be observed. In addition, the results for the DFT calculations are presented in Table S2 and Table S3. The condensed Fukui index representing atomic free radical attack (f 0) on AMX and CFX were used to calculate the free radical attack reaction sites[18]. As shown in Table S2, the f 0 values of O10, N12, C14, O15, S18, C23, and O24 are all larger than others, and therefore, free radical attacks are more likely to occur. Combined with the detected intermediates and the Fukui index, the degradation pathway of AMX is shown in Fig. 8. In the process of the photocatalytic experiment, two ways of decomposition have been found. They are the lipidation of β-lactam ring and the fracture of AMX. As shown in the Pathway 1, the Fukui has shown that C14 had higher f

0,

producing A1 Amoxicillin penicilloic acid with m/z of 383.

Subsequently, decarboxylation reaction and deamination reaction were generated due to the loss of -COOH and -NH2. Therefore, the intermediates of A2 (m/z of 339) and A3 (m/z of 368) were produced, respectively[32]. This result is consistent with the result of the Fukui function, which shows that N12 and O15 have a larger Fukui index with values of 0.04220 and 0.07382, respectively. Both of the above molecules eventually become A4 (m/z 324)[33]. The Pathway Ⅱ begun based on the fact that

15

the O10 had a high f

0

and the location was easy to be attacked by ROSs. This

phenomenon could mean that the N11-C9 bond was affected and became easy to break in the central section of AMX, producing A6 (m/z 166) and A5 6-aminopenicillanic acid with m/z of 216, as shown in the work of Hirte et al[34]. Then, A5 with the addition of hydroxyl radical generated A8 (m/z of 219)[35]. After that, A9 (m/z of 256) may change from A5 due to the methyl carboxylation. The A10 with m/z of 241 was produced and resulted from the deamination reaction. Meanwhile A6 was oxidized to A11 with the m/z of 165. Both reactions are also based on the fact that N12 and C14 are attacked with very high values of ∙O2- and ∙OH. Although the f 0 of S atoms was relatively high, no related decomposition products were detected, which may be because the cracking reaction of the heterocyclic ring of S occurred in the next process. As the reaction went on, small carboxyl groups and aliphatic compounds are gradually formed, which slowly and progressively mineralized into CO2 and H2O after sufficient reaction time, for example the intermediates of m/z of 188, 176, 117 and so on[24,36]. In the molecule of CFX, the f 0 values of N1, S4, C6, N8, O12, C20, S22, and O24 have been found to be higher than other atoms. The degradation pathway of CFX is shown in Fig. 9 in combination with the detected intermediates and the Fukui index. Firstly, the O24 has a large f 0 (0.03387), due to which, the atoms around it are very unstable, leading to the formation of B2 (m/z of 411). At this time, the CFX de-esterification (hydrolysis of 3-acetoxy group) reaction occurred based on the reason that C20 has a high f 0(0.04685), which could be broken off by ROSs, producing B1 (m/z of 413)[37]. Both of the B1 and B2 were translated into B3 (m/z of 353). Then, the β-lactam rings cleaved (lactonization), producing B4 (m/z of 255) and B5 (m/z of 114)[38]. Thereafter, the N1 atom with a high f 0 (0.06548) may also

16

be directly attacked by ∙O2- and ∙OH, leading to the hydroxylation of the -NH2 producing B6 (m/z of 117). Finally, all the above-mentioned intermediates will be further attacked by ROSs and break into smaller molecules or mineralize into CO2 and H2O with extended time. Toxicity Estimation Software Tool (TEST) was used to calculate the toxicity of the compounds from molecular structure, basing on mathematical models of Quantitative Structure Activity Relationship (QSAR). The toxicity of AMX was considered as the lethal concentration of 50% (LC50) (96h) fathead minnow and Ames mutagenicity[39]. Shown as Table S4 and S5, as the reaction went on, the increase of LC50 (96h) of fathead minnow of byproducts indicated the decrease of toxicity, but the predicted results of Ames mutagenicity were positive at A6, A8, A11, B4 which manifested these byproducts had the possibility of mutagenesis. So high toxic intermediates were maybe generated in the process of photodegradation which were possibly attributed to the formation of carboxylic acids as the reports of Pintar et al and Trovó et al[40,41]. A longer reaction time may be needed to reduce toxicity to increase the mineralization of pollutants. The similar results has been reported in the previous literature[13,16]. Therefore, the evaluation of the toxicity of photocatalysis products is a necessary problem that cannot be ignored and deserves further discussion. Fig. 8, Fig. 9, Fig. 10 and Table S2, S3, S4, S5

3.4.3 Difference of the photodegradations of AMX and CFX As shown in Fig. 7(a), the degradation rates of AMX and CFX have a significant difference with the increase in reaction time. Additionally, the degradation rate constant (k) of CFX was 4 times that of the AMX. From the study of LC in Fig. 7(c, 17

d), this evident difference could also be observed with the increase in time. As shown TOC detection in Fig. 7(b), the mineralization rate was about 30% for AMX by MCN, which was also significantly lower than that of the CFX. The above results indicate the fact that: two antibiotics belonging to β-lactam antibiotics exhibit different degradation patterns and mechanism under MCN photocatalysis. It was a very obvious and interesting phenomenon. This may be due to the following two reasons. On one hand, it depends on the different structure of the material itself. As shown in Fig. 10, the structures of AMX and CFX are obviously different. For AMX, the parent molecule is fused by a four-membered β-lactam ring and hydrogenated thiazole ring. Additionally, the two rings are not in the same plane, as seen from Fig. 10(a, b). The N17 atom and C14 atom have electronegativity differences, which causes the length asymmetry between C-C and C-N bonds in the four-membered β-lactam ring, and due to which, the C-N bond of β-lactam ring became very unstable. The four-membered ring structure is prone to distortion because of the increase in tension. However, for CFX, the β-lactam ring and hydrogenated thiazine ring were not the same as AMX. Due to this reason, the difference of cracking modes of β-lactam ring occurred, which is shown in Fig. 10(c, d). The oximido of CFX is easy to photoisomerization and its 3-acetoxy group is very weak[42]. Additionally, the calculation results based on Fukui faction suggest that the two different molecules act differently at the vulnerable sites. The above characteristics of the structure show the differences in the subsequent degradation process. On the other hand, the catalyst also has some selectivity to the degradations of antibiotics. The attacking sites of superoxide radical hole and hydroxyl radical, produced are different in the photocatalytic system. And it was confirmed in Wang et al.'s previous research using the frontier electron densities theory. As shown in Fig. 7,

18

the degradation degree of AMX was much lower than that of the CFX under the production of a large number of ∙O2-, which may be due to the fact that CFX is more vulnerable to the attack of superoxide radicals that are maximum in the system. However, when higher energy reactive oxygen species are produced, the result may be different. In addition, the zeta potential values of the catalyst are also a key factor determining the catalytic activity, and leading to the difference in sorption properties between the antibiotics and catalyst, and which has also been mentioned in a previous report[43]. Fig. 7 and Fig. 10 3.5 Wastewater Test and Stability Four actual water matrices as DI (deionized water), filtered sea water (SW), the effluent of secondary treatment unit of hospital wastewater (HW) and municipal wastewater (MW) were considered for AMX and CFX as shown in Fig.11(a, b) respectively. And the characteristics of the water matrices were shown in Table S6. In three kinds of relatively complex water substrates, MCN also showed certain degradation efficiency for AMX and CFX. And the TOC and COD were effectively eliminated which were shown in Fig. S6, which indicated that MCN is a promising material in actual wastewater treatment. In addition, as shown in Fig.11(c, d), the stability of MCN was very well. Therefore, this photocatalyst has a good application prospect in the future. Fig. 11, Fig. S6 and Table S6

19

4. Conclusions In this work, MCN was successfully synthesized using a template-free method, whereas BET analysis showed that the specific surface area of MCN was three times higher than that of the BCN. The efficiencies of MCN photocatalytic degradation of AMX and CFX were 2.5 and 4 times higher than that of BCN, respectively, which is mainly due to the porous structure that improved the utilization of light. Four influencing factors were studied in the photocatalytic degradation based on MCN. These factors included the initial antibiotics concentration, the catalyst dosage, the pH and the addition of HA. The feasible photodegradation mechanism of MCN was proposed with free radical quenching experiments and ESR. Combined with LC-QTOF-MS/MS and DFT, the possible pathways were studied in detail. The degradation pathway of AMX included the lipidation of β-lactam ring and the direct fracture of the molecule. For CFX, the de-esterification (hydrolysis of the 3-acetoxy group) reaction happened first, and then, the decarboxylic reaction occurred. In addition, the toxicity of the byproducts predicted by TEST has decrease tendency as the reaction goes on. Moreover, the differences were analyzed in the degradation pathways which were due to two reasons: the antibiotics have different structures, leading to the difference in the sensitivity to active species; and the type and amount of active species produced by the catalyst as well as different radicals’ attack at different sites. This study provides a more comprehensive insight into the degradation mechanism for AMX and CFX, and aids an in-depth understanding of the differences of the catalytic activity on two different antibiotics under mesoporous g-C3N4. Moreover, the results of wastewater test and stability experiments indicated that MCN is a promising treatment photocatalyst for the elimination of the antibiotic activity and mineralization of antibiotics under visible light.

20

Acknowledgements This study was supported by the State Key Laboratory of Environmental Chemistry and Ecotoxicology, the Research Center for Eco-Environmental Sciences, the Chinese Academy of Sciences, the Beijing Key Laboratory of Aqueous Typical Pollutants Control and Water Quality Safeguard, and the Beijing Key Laboratory of Aqueous Typical Pollutants Control and Water Quality Safeguard. The authors are thankful to Prof. Jing Chuanyong, Associate Professor Yan Wei and Yan Li for their guidance during the research process.

21

References: [1] F. Yuan, C. Hu, X. Hu, D. Wei, Y. Chen, J. Qu, Photodegradation and toxicity changes of antibiotics in UV and UV/H2O2 process,J HAZARD MATER, 185(2011)1256-1263. [2] R. Gothwal, T. Shashidhar, Antibiotic Pollution in the Environment: A Review,CLEAN – Soil, Air, Water, 43(2015)479-489. [3] N.F.F. Moreira, J.M. Sousa, G. Macedo, A.R. Ribeiro, L. Barreiros, M. Pedrosa, J.L. Faria, M.F.R. Pereira, S. Castro-Silva, M.A. Segundo, C.M. Manaia, O.C. Nunes, A.M.T. Silva, Photocatalytic ozonation of urban wastewater and surface water using immobilized TiO2 with LEDs: Micropollutants, antibiotic resistance genes and estrogenic activity,WATER RES, 94(2016)10-22. [4] F. Wang, Y. Feng, P. Chen, Y. Wang, Y. Su, Q. Zhang, Y. Zeng, Z. Xie, H. Liu, Y. Liu, W. Lv, G. Liu, Photocatalytic degradation of fluoroquinolone antibiotics using ordered mesoporous g-C3 N4 under simulated sunlight irradiation: Kinetics, mechanism, and antibacterial activity elimination,Applied Catalysis B: Environmental, 227(2018)114-122. [5] G. Matafonova, V. Batoev, Recent advances in application of UV light-emitting diodes for degrading organic pollutants in water through advanced oxidation processes: A review,WATER RES, 132(2018)177-189. [6]

S.O.

Ganiyu,

M.

Zhou,

C.A.

Martínez-Huitle,

Heterogeneous

electro-Fenton

and

photoelectro-Fenton processes: A critical review of fundamental principles and application for water/wastewater treatment,Applied Catalysis B: Environmental, 235(2018)103-129. [7] A.M. Chávez, O. Gimeno, A. Rey, G. Pliego, A.L. Oropesa, P.M. Álvarez, F.J. Beltrán, Treatment of highly polluted industrial wastewater by means of sequential aerobic biological oxidation-ozone based AOPs,CHEM ENG J, 361(2019)89-98. [8] J. Luo, S. Zhang, M. Sun, L. Yang, S. Luo, J.C. Crittenden, A Critical Review on Energy Conversion and Environmental Remediation of Photocatalysts with Remodeling Crystal Lattice, Surface, and Interface,ACS NANO, 13(2019)9811-9840. [9] E.S. Elmolla, M. Chaudhuri, Degradation of amoxicillin, ampicillin and cloxacillin antibiotics in aqueous

solution

by

the

UV/ZnO

photocatalytic

process,J

HAZARD

MATER,

173(2010)445-449. [10] M.E. Borges, M. Sierra, E. Cuevas, R.D. García, P. Esparza, Photocatalysis with solar energy: Sunlight-responsive photocatalyst based on TiO2

loaded on a natural material for wastewater

treatment,SOL ENERGY, 135(2016)527-535. [11] X. Wang, W. Lu, Y. Chen, N. Li, Z. Zhu, G. Wang, W. Chen, Effective elimination of antibiotics over hot-melt adhesive sheath-core polyester fiber supported graphitic carbon nitride under solar irradiation,CHEM ENG J, 335(2018)82-93. [12] Y. Zeng, H. Li, J. Luo, J. Yuan, L. Wang, C. Liu, Y. Xia, M. Liu, S. Luo, T. Cai, S. Liu, J.C. Crittenden, Sea-urchin-structure g-C3N4 with narrow bandgap (˜2.0   eV) for efficient overall water splitting under visible light irradiation,Applied Catalysis B: Environmental, 249(2019)275-281. [13] W. Liu, Y. Li, F. Liu, W. Jiang, D. Zhang, J. Liang, Visible-light-driven photocatalytic degradation of diclofenac by carbon quantum dots modified porous g-C3N4: Mechanisms, degradation pathway and DFT calculation,WATER RES, 151(2019)8-19. [14] W. Oh, V.W.C. Chang, Z. Hu, R. Goei, T. Lim, Enhancing the catalytic activity of g-C3N4 22

through Me doping (Me = Cu, Co and Fe) for selective sulfathiazole degradation

via

redox-based advanced oxidation process,CHEM ENG J, 323(2017)260-269. [15] L. Wang, T. Huang, G. Yang, C. Lu, F. Dong, Y. Li, W. Guan, The precursor-guided hydrothermal synthesis of CuBi2O4/WO3 heterostructure with enhanced photoactivity under simulated

solar

light

irradiation

and

mechanism

insight,J

HAZARD

MATER,

381(2020)120956. [16] A. Mirzaei, Z. Chen, F. Haghighat, L. Yerushalmi, Magnetic fluorinated mesoporous g-C3N4 for photocatalytic

degradation

of

amoxicillin:

Transformation

mechanism

and

toxicity

assessment,Applied Catalysis B: Environmental, 242(2019)337-348. [17] G. Dong, L. Zhang. Porous structure dependent photoreactivity of graphitic carbon nitride under visible light[J]. J MATER CHEM, 2012, 22(3): 1160-1166. [18] J. Du, W. Guo, H. Wang, R. Yin, H. Zheng, X. Feng, D. Che, N. Ren, Hydroxyl radical dominated degradation of aquatic sulfamethoxazole by Fe0/bisulfite/O2 : Kinetics, mechanisms, and pathways,WATER RES, 138(2018)323-332. [19] T. Lu, F. Chen, Multiwfn: a multifunctional wavefunction analyzer,J COMPUT CHEM, 33(2012)580-592. [20] D. Dimitrakopoulou, I. Rethemiotaki, Z. Frontistis, N.P. Xekoukoulotakis, D. Venieri, D. Mantzavinos, Degradation, mineralization and antibiotic inactivation of amoxicillin by UV-A/TiO2 photocatalysis,J ENVIRON MANAGE, 98(2012)168-174. [21] Y. Chen, K. Liu, Preparation and characterization of nitrogen-doped TiO2/diatomite integrated photocatalytic pellet for the adsorption-degradation of tetracycline hydrochloride using visible light,CHEM ENG J, 302(2016)682-696. [22] Y. Deng, L. Tang, C. Feng, G. Zeng, J. Wang, Y. Zhou, Y. Liu, B. Peng, H. Feng, Construction of plasmonic Ag modified phosphorous-doped ultrathin g-C3N4 nanosheets/BiVO4 photocatalyst with enhanced visible-near-infrared response ability for ciprofloxacin degradation,J HAZARD MATER, 344(2018)758-769. [23] A. Kumar, A. Kumar, G. Sharma, A.H. Al-Muhtaseb, M. Naushad, A.A. Ghfar, F.J. Stadler, Quaternary magnetic BiOCl/g-C3N4/Cu2O/Fe3O4 nano-junction for visible light and solar powered degradation of sulfamethoxazole from aqueous environment,CHEM ENG J, 334(2018)462-478. [24] D. Kanakaraju, J. Kockler, C.A. Motti, B.D. Glass, M. Oelgemöller. Titanium dioxide/zeolite integrated photocatalytic adsorbents for the degradation of amoxicillin,Applied Catalysis B: Environmental, 166-167(2015)45-55. [25] D. Y. Padmashree. Degradation of β - lactam antibiotics. CURRENT SCIENCE, 87(2004)12-25. [26] I. A. Alsarra, M. O. Ahmed, M. El-Badry, et al. Effect of beta-cyclodextrin derivatives on the kinetics of degradation of cefotaxime sodium in solution state[J]. J DRUG DELIVERY SCI TECH, 2007, 17(5): 353. [27] Y. Song, J. Tian, S. Gao, P. Shao, J. Qi, F. Cui, Photodegradation of sulfonamides by g-C3N4 under visible light irradiation: Effectiveness, mechanism and pathways,Applied Catalysis B: Environmental, 210(2017)88-96. [28] Y. Yang, J. Jiang, X. Lu, J. Ma, Y. Liu, Production of Sulfate Radical and Hydroxyl Radical by Reaction of Ozone with Peroxymonosulfate: A Novel Advanced Oxidation Process,ENVIRON SCI TECHNOL, 49(2015)7330-7339. [29] Z. Cai, A.D. Dwivedi, W. Lee, X. Zhao, W. Liu, M. Sillanpää, D. Zhao, C. Huang, J. Fu, 23

Application of nanotechnologies for removing pharmaceutically active compounds from water: development and future trends,Environmental Science: Nano, 5(2018)27-47. [30] L. Tang, C. Feng, Y. Deng, G. Zeng, J. Wang, Y. Liu, H. Feng, J. Wang, Enhanced photocatalytic activity of ternary Ag/g-C3N4/NaTaO3 photocatalysts under wide spectrum light radiation: The high

potential

band

protection

mechanism,Applied

Catalysis

B:

Environmental,

230(2018)102-114. [31] E. Nägele, R. Moritz, Structure elucidation of degradation products of the antibiotic amoxicillin with ion trap MSn and accurate mass determination by ESI TOF,J AM SOC MASS SPECTR, 16(2005)1670-1676. [32] A.G. Trovó, R.F. Pupo Nogueira, A. Agüera, A.R. Fernandez-Alba, S. Malato, Degradation of the antibiotic

amoxicillin

by

photo-Fenton

process

–

Chemical

and

toxicological

assessment,WATER RES, 45(2011)1394-1402. [33] X. Weng, Q. Sun, S. Lin, Z. Chen, M. Megharaj, R. Naidu, Enhancement of catalytic degradation of

amoxicillin

in

aqueous

solution

using

clay

supported

bimetallic

Fe/Ni

nanoparticles,CHEMOSPHERE, 103(2014)80-85. [34] K. Hirte, B. Seiwert, G. Schüürmann, T. Reemtsma, New hydrolysis products of the beta-lactam antibiotic

amoxicillin,

their

pH-dependent

formation

and

search

in

municipal

wastewater,WATER RES, 88(2016)880-888. [35] S.O. Ganiyu, N. Oturan, S. Raffy, M. Cretin, R. Esmilaire, E. van Hullebusch, G. Esposito, M.A. Oturan, Sub-stoichiometric titanium oxide (Ti4O7) as a suitable ceramic anode for electrooxidation of organic pollutants: A case study of kinetics, mineralization and toxicity assessment of amoxicillin,WATER RES, 106(2016)171-182. [36] T. Shen, X. Li, Y. Tang, J. Wang, X. Yue, J. Cao, W. Zheng, D. Wang, G. Zeng, EDTA and electricity synergetic catalyzed Fe3+/H2O2 process for amoxicillin oxidation,WATER SCI TECHNOL, 60(2009)761-770. [37] H. Fabre, N. Hussam-Eddine, A stability-indicating assay for cefotaxime utilizing thin-layer chromatography with fluorescence detection,J PHARM PHARMACOL, 34(1982)425-428. [38] V.V. Kondalkar, S.S. Mali, R.M. Mane, P.B. Dandge, S. Choudhury, C.K. Hong, P.S. Patil, S.R. Patil, J.H. Kim, P.N. Bhosale, Photoelectrocatalysis of Cefotaxime Using Nanostructured TiO2 Photoanode: Identification of the Degradation Products and Determination of the Toxicity Level,IND ENG CHEM RES, 53(2014)18152-18162. [39] S. Li, J. Hu, Transformation products formation of ciprofloxacin in UVA/LED and UVA/LED/TiO2 systems: Impact of natural organic matter characteristics,WATER RES, 132(2018)320-330. [40] A. Pintar, M. Besson, P. Gallezot, J. Gibert, D. Martin, Toxicity to Daphnia magna and Vibrio fischeri of Kraft bleach plant effluents treated by catalytic wet-air oxidation,WATER RES, 38(2004)289-300. [41] A.G. Trovó, R.F. Pupo Nogueira, A. Agüera, A.R. Fernandez-Alba, S. Malato, Degradation of the antibiotic

amoxicillin

by

photo-Fenton

process

–

Chemical

and

toxicological

assessment,WATER RES, 45(2011)1394-1402. [42] D. A. Lerner , G. Bonnefond, H. Fabre, et al. Photodegradation paths of cefotaxime[J]. Journal of pharmaceutical sciences, 1988, 77(8): 699-703. [43] S. Barışçı, F. Ulu, M. Sillanpää, A. Dimoglo, The usage of different forms of ferrate (VI) ion for amoxicillin and ciprofloxacin removal: density functional theory based modelling of redox 24

decomposition,Journal of Chemical Technology & Biotechnology, 91(2016)257-266.

25

Figure captions Fig. 1 N2 adsorption-desorption isotherms and Barret-Joyner-Halenda (BJH) pore size distribution plots (inset) of BCN and MCN samples and volume comparison (inset) in same quality (m=1g). And SEM images of BCN and MCN. Fig. 2 Photocatalytic degradation and kinetics curves (inset) of (a) AMX and (b) CFX under visible light (λ>420 nm) in the presence of BCN and MCN( initial antibiotics solution concentration C0=2mg/L). Fig. 3 The effects of antibiotic solution concentration of (a) AMX and (b) CFX with initial photocatalyst dosage as 100mg. And the effect of photocatalyst dosage of (c) AMX and (d) CFX with initial antibiotic solution concentration as 2mg/L. Fig. 4 The effects of initial pH of (a) AMX and (b) CFX in the presence of MCN and in the absence of MCN (inset). Fig. 5 The Effect of HA on (a) AMX and (b) CFX photodegradation by MCN ( initial pH = 7). Fig. 6 The photocatalytic degradation plots of (a) AMX and (b) CFX over MCN with the addition of hole, ∙O2 − and ∙OH- radical scavenger under visible light irradiation; (c) ESR spectra in methanol dispersion for DMPO ∙O2- ； (d) Possible photocatalytic reaction mechanism. Fig. 7 (a) Photocatalytic degradation and kinetics curves (inset) of AMX and CFX under visible light (λ>420 nm) in the presence of MCN( initial antibiotics solution concentration C0=20mg/L); (b) TOC removal curves of AMX and CFX on MCN; Photocatalytic degradation of (c) AMX in 120min and (d) CFX in 60min by HPLC.

26

Fig.8 The proposed pathways for AMX decay. Fig.9 The proposed pathways for CFX decay. Fig.10 Optimized structure from different angels of (a,b) AMX (c,d) CFX and the numbering system by Gaussian 09. (gray, carbon; red, oxygen; blue, nitrogen; yellow, sulfur). Fig.11 The photodegradation of the AMX (a) and CFX (b) in the different water matrices as DI (deionized water), SW (sea water), HW(hospital water) and WWTP(waste water treatment plant); Recycling runs in the degradation of AMX (c) and CFX (d) over MCN.

Fig. 1

27

Fig. 1 N2 adsorption-desorption isotherms and Barret-Joyner-Halenda (BJH) pore size distribution plots (inset) of BCN and MCN samples and volume comparison (inset) in same quality (m=1g). And SEM images of BCN and MCN.

Fig. 2

28

Fig. 2 Photocatalytic degradation and kinetics curves (inset) of (a) AMX and (b) CFX under visible light (λ>420 nm) in the presence of BCN and MCN( initial antibiotics solution concentration C0=2mg/L).

Fig. 3

29

Fig. 3 The effects of antibiotic solution concentration of (a) AMX and (b) CFX with initial photocatalyst dosage as 100mg. And the effect of photocatalyst dosage of (c) AMX and (d) CFX with initial antibiotic solution concentration as 2mg/L.

Fig. 4

30

Fig. 4 The effects of initial pH of (a) AMX and (b) CFX in the presence of MCN and in the absence of MCN (inset).

Fig. 5

31

Fig. 5 The effects of HA on (a) AMX and (b) CFX photodegradation by MCN ( initial pH=7).

Fig. 6

32

Fig. 6 The photocatalytic degradation plots of (a) AMX and (b) CFX over MCN with the addition of hole, ∙O2 − and ∙OH- radical scavenger under visible light irradiation; (c) ESR spectra in methanol dispersion for DMPO ∙O2- ； (d) Possible photocatalytic reaction mechanism.

Fig. 7

33

Fig. 7 (a) Photocatalytic degradation and kinetics curves (inset) of AMX and CFX under visible light (λ>420 nm) in the presence of MCN( initial antibiotics solution concentration C0=20mg/L); (b) TOC removal curves of AMX and CFX on MCN; Photocatalytic degradation of (c) AMX in 120min and (d) CFX in 60min by HPLC.

Fig.8

34

Fig.8 The proposed pathways for AMX decay.

Fig.9

35

Fig.9 The proposed pathways for CFX decay.

Fig.10

36

Fig.10 Optimized structure from different angels of (a, b) AMX (c, d) CFX and the numbering system by Gaussian 09. (gray, carbon; red, oxygen; blue, nitrogen; yellow, sulfur).

Fig.11

37

Fig.11 The photodegradation of the AMX (a) and CFX (b) in the different water matrices as DI (deionized water), SW (sea water), HW (hospital wastewater) and MW(municipal watewater); Recycling runs in the degradation of AMX (c) and CFX (d) over MCN.

38

Highlights  Mesoporous g-C3N4 was propared for the photodegradation of AMX and CFX.  Influencing factors were studied including pH, HA, initial concentration and catalyst dose.  The degradation mechanisms of AMX and CFX were investigated.  Density functional theory was applied to predict the reactive sites and pathways.  Difference in the photocatalysis of AMX and CFX under MCN was proposed.

39