Intensification of levofloxacin sono-degradation in a US/H2O2 system with Fe3O4 magnetic nanoparticles☆

Hong Wei*,Da Hu 2,Jie Su Kebin Li

1 State Key Laboratory Base of Eco-Hydraulic Engineering in Arid Area,Xi'an University of Technology,Xi'an 710048,China

2 China Huadian Electric Power Research Institute,Hangzhou 310030,China

3 Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education,School of Chemistry and Material Science,Northwest University,Xi'an 710069,China

Keywords:Fe3O4 magnetic nanoparticles H2O2 Levofloxacin Sonolysis HPLC/MS/MS Degradation pathway

ABSTRACT Fe3O4 magnetic nanoparticles(MNPs)were synthesised,characterised,and used as a peroxidase mimetic to accelerate levofloxacin sono-degradation in an ultrasound(US)/H2O2 system.The Fe3O4 MNPs were in nanometre scale with an average diameter of approximately 12 to 18 nm.The introduction of Fe3O4 MNPs increased levofloxacin sono-degradation in the US/H2O2 system.Experimental parameters,such as Fe3O4 MNP dose,initial solution pH,and H2O2 concentration,were investigated by a one-factor-at-a-time approach.The results showed that Fe3O4 MNPs enhanced levofloxacin removal in the pH range from 4.0 to 9.0.Levofloxacin removal ratio increased with Fe3O4 MNP dose up to 1.0 g·L?1 and with H2O2 concentration until reaching the maximum.Moreover,three main intermediate compounds were identified by HPLC with electrospray ionisation tandem mass spectrometry,and a possible degradation pathway was proposed.This study suggests that combination of H2O2,Fe3O4 MNPs and US is a good way to improve the degradation efficiency of antibiotics.

1.Introduction

In recent years,the release of pharmaceutical compounds into the environment due to their extensive use and incomplete removal in wastewater treatment processes has become a significant concern,pharmaceutical substances have come to be considered as environmental contaminants[1].Fluoroquinolones(FQs)are a class of broad spectrum anti-bacterial agents,which are widely used for both human medicine and livestock operations.Because FQs are incompletely metabolised during human therapy,20%-80%of FQs are excreted in their pharmacologically active forms,subsequently discharging into municipal sewerage.As a result,FQs are frequently identified pharmaceuticals in domestic and hospital wastewaters[2,3].For example,four FQs(ciprofloxacin,norfloxacin,enrofloxacin, and sarafloxacin)have been reported with mean concentrations from non-detectable to 0.12 μg·L?1in surface waters in the United States[4].Increasing evidences of pharmaceuticals in the environment result in concerns about the safety of drinking water,reclaimed and reused wastewater,and aquatic ecosystems.Therefore,FQs must be either removed from water or degraded.

The elimination of FQs from drinking water and wastewater by direct photolysis[5],ozonation[6]and advanced oxidation processes(AOPs)[7]has been reported.Among the proposed FQ treatment technologies,AOPs show the potential to degrade recalcitrant organic compounds and have received considerable attention[7].AOPs are expected to degrade soluble biorefractory antibiotics effectively due to their formation of reactive hydroxyl radical(·OH)species.Hydroxyl radicals involved in AOPs can be generated by the combination of H2O2and/or O3with catalysts(transition metals,semiconductors),UV or ultrasonic irradiation.Among all of the AOPs,sonochemical technology is a relatively new technique for destroying non-biodegradable pollutants.

Two reaction mechanisms are presumably responsible for the sonodegradation of pollutants:one is pyrolysis in the cavitation of bubbles,which is assumed to be the major reaction path for the degradation of non-polar compounds;another is generation of·OH in cavitating bubbles,which subsequently oxidise those polar organic compounds[8].In regard to FQ removal,single-use ultrasound is limited[9].Therefore,effort has been devoted to increasing the sono-degradation efficiency by using hybrid techniques.H2O2,as a green oxidant,could act as a resource for reactive oxygen species and thus it is important for the oxidative degradation of organic pollutants.In our previous study,it has been observed that H2O2facilitates the ultrasonic degradation of levofloxacin,with the removal efficiency and intermediate compound distributions influenced by pH of solutions greatly[10].It is necessary to further explore approaches that could enhance the generation of reactive oxygen species and clarify those factors affecting sono-degradation efficiency.The introduction of catalysts is an important way to drive the decomposition of H2O2for the oxidation of organic contaminants.Recently,it has been reported that Fe3O4MNPs exhibitan enzyme mimetic activity for·OH degradation[11].Moreover,it is reported that the degradation of dye organic pollutants by sonolysis is greatly promoted by H2O2/Fe3O4MNPs[12].However,reports on sono-degradation of pharmaceutical compounds in an ultrasound(US)/H2O2/Fe3O4MNP system are scarce.

The objective of this study is to evaluate Fe3O4MNPs as used in the sono-degradation of levofloxacin in an US/H2O2system.We choose levofloxacin as a model compound because it is a commonly used FQ when treating life-threatening bacterial infections and often fails to respond to other antibiotic classes[13].Additionally,levofloxacin cannot be effectively removed by conventional wastewater treatment processes due to its biological resistance and stability.We perform sonolysis in an US/H2O2/Fe3O4MNP system and investigate the effects of experimental parameters,such as Fe3O4MNPdosage,initial pH of the solution,and H2O2concentration,on the sono-degradation of levofloxacin.The intermediate compounds are monitored by HPLC and HPLC/MS/MS methods.A preliminary degradation pathway is proposed.Results from this study may provide useful information for improving the treatment of FQ antibiotic-contaminated water.

2.Experimental

2.1.Materials and reagents

The levofloxacin was provided by Xinchang Pharmaceutical Co.,Ltd.(Zhejiang)and used as received without further treatment.The physico-chemical characteristics of levofloxacin are listed in Table 1.

Table 1 Physiochemical characteristics of levofloxacin

Hydrogen peroxide(H2O2,30%solution)was purchased from Tianjin Chemical Reagent Co.,Ltd.All other chemicals are of analytical reagent grade.The water used in all experiments was purified with a Milli-Q water system.

2.2.Preparation of Fe3O4 nanoparticles

Fe3O4nanoparticles were synthesised by the modified coprecipitation method[14].A certain dosage of FeCl2·4H2O and FeCl3·6H2O was dissolved in 50 ml purified water and heated to 70°C.The heated Fe(II)/Fe(III)solution was added drop-wise into 40 mlof3.0 mol·L?1ammonia solution at70 °C with magnetic stirring.After a one hour reaction time,the generated black Fe3O4nanoparticles were collected by magnetic separation,washed with water to neutral pH, filtered,dried for5 h at70°C,and ground for use.The phase composition of the resulting Fe3O4nanoparticles was determined by a Philips X-ray diffractometer with Cu Kαradiation(λ =0.154051 nm).Transmission electron microscopy(TEM,JEOL,JEM-2010,Japan)was used to measure the size and distribution of Fe3O4nanoparticles deposited at an acceleration voltage of 200 kV.

2.3.Degradation procedure

Sonication was performed under normal atmosphere with a 20 kHz ultrasound generator(Model JY92-IIN,NingBo Xinzhi-Technology Co.,China),which was equipped with a titanium probe(8 mm diameter).The tip of the titanium probe was placed approximately 10 mm below the surface of the solution during sonication.The ultrasound generator was operated in an on/off(1 s/1 s)pulse mode at the given power.Experiments were conducted in a 250 ml cylindrical water-jacketed glass reactor(80 mm internal diameter).Fe3O4MNPs were dispersed into 100 ml of levofloxacin aqueous solution,followed by shaking for 10 min to reach adsorption-desorption equilibrium.The degradation of levofloxacin was initiated by rapidly adding a given concentration of H2O2to the reactor and immediately turning on the ultrasound generator.The pH value of each reaction solution was adjusted to the desired level using appropriate concentrations of H2SO4and NaOH solutions.During sono-degradation,1.5 ml of the reaction solution was sampled at given time intervals and immediately centrifuged at 5000 r·min?1for 15 min to remove Fe3O4MNPs.The supernatant was further filtered through 0.45 μm membranes for HPLC analysis.

2.4.Chemical analysis

2.4.1.HPLC analysis

The concentration of levofloxacin in aqueous solutions was analysed on an Agilent 1200 series HPLC with an Eclipse XDB-C18column(5 μm,4.6 mm×150 mm;Agilent).The mobile phase consisted of acetonitrile and Milli-Q water containing 0.2%formic acid(3:1,volume ratio)at a flow rate of 0.2 ml·min?1.The wavelength of the UV detector was set to 290 nm for quantification.Solution pH value was measured using a pHs-25 digital pH meter.The removal ratio of levofloxacin was calculated by the following equation:

where R is the removal ratio of levofloxacin(%),and C0and Ctrepresent the initial and remaining levofloxacin concentrations,respectively.

2.4.2.LC/MS/MS analysis

LC/MS/MS analysis of levofloxacin degradation products was performed on a Bruker microTOF-QII.Chromatographic separations were performed using an Eclipse XDB-C18column(5 μm,4.6 mm × 150 mm)and peaks were detected at 290 nm.The LC system was operated isocratically.The composition of the acetonitrile/water/formic acid mobile phase was 75:24.98:0.02(volume ratio)and the flow rate was 0.2 ml·min?1.The MS condition used was positive electron ionisation,in full-scan mode with a mass range scan of 50 to 20000.

2.4.3.Hydroxyl radical(·OH)analysis

Hydroxylradicals(·OH)were measured as follows:a certain dosage of Fe3O4MNPs and H2O2or H2O2was added to 1.0 × 10?3mol·L?1coumarin solution and reacted in the degradation procedure.Since the reaction of coumarin with·OH to form 7-hydroxy coumarin is highly specific toward the·OH radical,a highly fluorescent molecule of 7-hydroxy coumarin forms[15](excitation,λEx=335 nm;emission,λEm=460 nm).

3.Results and Discussion

3.1.Characterisation of synthesised Fe3O4 nanoparticles

Fig.1.XRD pattern of Fe3O4 magnetic nanoparticles.

Fig.1 shows the XRD patterns of Fe3O4MNPs.The diffraction peaks can be indexed to a cubic phase ofFe3O4(JPCDS No.19-0629).The average size of the Fe3O4nanoparticles is determined from their XRD pattern according to the Scherrer equation:D=kλ/β cos θ,where k is a constant equal to 0.89,λ is the X-ray wavelength of 0.154 nm,β is the full width at half maximum,and θ is the diffraction half-angle.The calculated result indicates that the average size of Fe3O4particles is approximately 15.3 nm.Fig.2 shows the transmission electron microscopy describing the size and shape of Fe3O4MNPs,which reveals the Fe3O4MNP agglomeration to some extent.The size of Fe3O4particles has a nanometric scale with an average diameter of around 12 to 18 nm,which is in good agreement with the result obtained by XRD.

Fig.2.TEM images of Fe3O4 magnetic nanoparticles.

3.2.Sonocatalysis of levofloxacin

Fig.3 shows the sonolysis of levofloxacin under five different experimental conditions.With levofloxacin treated only by ultrasound the removal ratio is about 1.88%in 240 min.The low efficiency suggests that an insufficient amount of active radicals forms and thermal decomposition is little in the US process[16].With H2O2used to oxidise levofloxacin alone,the removal ratio remains low(6.74%).Thermodynamically,oxidation of levofloxacin by H2O2is feasible as its redox potential(Eh=1.02 V)is lower than that of H2O2(0.87 V≤Eh≤1.80 V,14.0≥pH≥0).The limited oxidation of levofloxacin by H2O2in this study may suggest that the reaction is controlled by dynamics to a large extent.With ultrasound introduced into H2O2oxidation process(US/H2O2),levofloxacin removal ratio increases from 6.74%to 64.96%in the same reaction time,because the thermal decomposition of H2O2in the cavitation bubbles forms reactive radicals such as·OH.In the simple catalytic system of H2O2/Fe3O4MNPs without ultrasonic irradiation,the removal ratio of levofloxacin is 71.46%,higher than that in the US/H2O2system.The higher degradation efficiency could be attributed to the intrinsic peroxidase mimetic activity of Fe3O4MNPs[Eqs.(2)and(3)].For degradation of levofloxacin in an US/H2O2/Fe3O4MNP system,it is exciting to find that 99%of the levofloxacin can be removed after 150 min treatment.The degradation of levofloxacin is significantly enhanced by the combination of US,H2O2,and Fe3O4MNPs.The significant enhancement effect of Fe3O4MNPs on the sonocatalytic degradation of levofloxacin can be further explained according to Eqs.(4)and(5).The production of active radicals in an US/H2O2/Fe3O4MNP system obeys two simultaneous mechanisms:homogeneous and heterogeneous catalyses.Nevertheless,the controlling mechanism should be heterogeneous catalysis of Fe3O4MNPs,as they provide more nucleation sites to form cavities[17].As a result,the ultrasonic efficiency of levofloxacin is increased.In addition,the intrinsic peroxidase mimetic activity for Fe3O4MNPs is also responsible for the better performance of the US/H2O2/Fe3O4MNP system.

The degradation of levofloxacin under each of the five test conditions follows pseudo- first-order kinetics[Fig.3(b)],and the corresponding fitted results are listed in Table 2.Comparing the k values under different experimental conditions,it is found that the degradation rate constants increase in the following order:US/H2O2/Fe3O4MNPs＞H2O2/Fe3O4＞US/H2O2＞H2O2＞US.

We determine·OH radical concentrations in US/H2O2and US/H2O2/Fe3O4MNP systems by a fluorescence probe technique.100 ml of 1.0 × 10?3mol·L?1coumarin solutions mixed with 5.0 mmol·L?1of H2O2,or 5.0 mmol·L?1of H2O2and 1.0 g·L?1of Fe3O4MNPs.After the mixtures were ultrasonic irradiated for 30 min at the output power of 260 W,5.0 ml of aliquot was taken out to determine·OH concentration and the results are shown in Fig.4.Compared to that in the US/H2O2system,much more·OH radicals formed and accumulated in the US/H2O2/Fe3O4MNP system.

Fig.3.Degradation of levofloxacin in different systems(a)and corresponding plot of pseudo- first-order kinetics(b)(Experimental conditions:levofloxacin initial concentration,20 mg·L?1;pH 7.14;Fe3O4 MNPs,1.0 g·L?1;H2O2,5.0 mmol·L?1;ultrasound power,195 W;mixing speed,150 r·min?1).★ US;▼ H2O2;● US/H2O2;▲ H2O2/Fe3O4 MNPs;■ US/H2O2/Fe3O4 MNPs.

Table 2 Kinetic parameters for levofloxacin degradation under different experimental conditions

A specific quenching diagnostic technique was further used to examine·OH radicals and its role in levofloxacin degradation by adding appropriate scavenger.Isopropanol is a known hydroxyl radical scavenger and its effect on levofloxacin degradation is shown in Fig.5.The levofloxacin degradation is effectively quenched by the addition of isopropanol.Because isopropanol molecules can pass through cavitation bubbles,they are able to scavenge·OH in the bubble and thus significantly reduced the levofloxacin degradation efficiency.It is consistent with the fluorescence analysis result.

Fig.4.Fluorescence spectra observed in US/H2O2/coumarin and US/H2O2/Fe3O4 MNP/coumarin systems after 30 min of ultrasonic irradiation.

3.3.Effect of experimental parameters on sono-degradation

3.3.1.Fe3O4 MNP dose

Fig.5.Effect of isopropanol on levofloxacin ultrasonic degradation in an US/H2O2/Fe3O4 MNP system(Experimental conditions:levo floxacin initial concentration,20 mg·L?1;pH 7.14;Fe3O4 MNPs,1.0 g·L?1;H2O2,5.0 mmol·L?1;ultrasound power,195 W;isopropanol concentration,100 mmol·L?1).■ US/H2O2/Fe3O4 MNPs;● US/H2O2/Fe3O4 MNPs/isopropanol.

Fig.6 illustrates the levofloxacin degradation in US/H2O2/Fe3O4MNP system with various doses of Fe3O4MNPs.Compared with the US/H2O2system(without Fe3O4MNPs),the addition of Fe3O4MNPs significantly enhances the degradation of levofloxacin.As Fe3O4MNP concentration increased from 0 to 1.0 g·L?1,the first-order rate constant k increased from 4.69× 10?3to 21.3× 10?3min?1(Fig.7).When the dose of Fe3O4MNPs was higher,higher removal ratio was observed.Besides the peroxidase-like catalytic activity of Fe3O4MNPs,it is because higher doses of Fe3O4MNPs could provide more nucleation sites for the formation of cavities[18].Therefore,the optimum dose of Fe3O4MNPs under these experimental conditions was set to 1.0 g·L?1for subsequent tests.

Fig.6.Influence ofFe3O4 MNPdose on levofloxacin degradation(Experimental conditions:levofloxacin concentration,20 mg·L?1;pH 7.14;H2O2 concentration,5.0 mmol·L?1;ultrasound power,195 W).■0.3 g·L?1;● 0.4 g·L?1;▲0.6 g·L?1;▼0.8 g·L?1;★1.0 g·L?1.

Fig.7.The relationship between Fe3O4 MNP dose and levofloxacin decay rate constant k.

3.3.2.Solution acidity

The initial pH of the medium is an important parameter for degradation of chemical pollutants under ultrasound irradiation.To avoid secondary pollution caused by the dissolution of iron ions and reflect the recycling advantage of Fe3O4MNPs,the effect of initial pH on ultrasonic degradation of levofloxacin by US/H2O2/Fe3O4MNPs is analysed in the range 4.0≤pH≤9.0.The relationship between first-order kinetic rate constant k and pH value is shown in Fig.8,indicating the degradation rate constant k remaining between 21.3× 10?3and 28.51× 10?3min?1at 4.0≤pH≤8.0(R2＞0.95)and being slower at pH 9.0,with a reaction rate constant k of 12.58×10?3min?1(R2=0.968).

Fig.8.The effect of solution pH on species distribution of levofloxacin and its degradation rate constants(Experimental conditions:levofloxacin concentration,20 mg·L?1;Fe3O4 dosage,1.0 g·L?1;H2O2 concentration,5.0 mmol·L?1;ultrasound power,195 W).

The effect of pH on levofloxacin ultrasonic degradation is complex.Firstly,levofloxacin is a non-volatile compound,so a direct pyrolysis of levofloxacin in cavitation bubbles is less important,as evidenced in Fig.3.Therefore,levofloxacin decomposition proceeds mostly at the liquid-vapour interface of cavitation bubbles or in the bulk of the solution.Levofloxacin exists as different species depending on the pH values of the medium.At 5.7≤pH≤7.9,levofloxacin mainly exists in its zwitterionic form in the solution;at pH＞7.9 or＜5.7,levofloxacin exists in cationic or anionic form in the solution,respectively.Therefore,the hydrophilicity and solubility of levofloxacin at different pH values may lead to different degradation rates.As shown in Fig.7,levofloxacin degradation in the US/H2O2/Fe3O4MNP system is related to its chemical structure formed in the range 5.0≤pH≤8.0.

Secondly,degradation of levofloxacin by US/H2O2/Fe3O4MNPs includes homogeneous and heterogeneous catalytic processes.Changes of solution pH value affect not only the mode of substrate adsorption on Fe3O4MNPs but also the heterogeneous Fenton-like reaction on the catalyst surface[19].For pH＞4.0,hydroxyl radicals facilitated by homogeneous catalytic processes of Fe3O4MNP dissolution could be partly ignored[20].The enhanced degradation of levofloxacin by US/H2O2/Fe3O4MNPs over the wide range of 4.0≤pH≤8.0,which is due to nucleation sites of Fe3O4MNPs for the formation of cavities[18],becomes slower atpH 9.0,which is partly due to the decrease ofH2O2adsorption on the catalyst covered with Fe(OH)63?[21].Anotherreason may be due to the self-decomposition of H2O2at alkaline pH levels as indicated by Eq.(6)[21].Consequently,it results in a low availability of H2O2in the solution and reduces the production of·OH.Reactions(7)to(10)may also lead to depletion of H2O2and/or·OH at the elevated pH levels[22].It is worth noting that the super oxide radical(·O2-)and HO2·exhibit weaker reactivity toward organic compounds compared with·OH radicals[22].All the aforementioned factors may cause different levofloxacin degradation rates at different pH values.

3.3.3.Concentration of H2O2

Fig.9 shows the degradation of levofloxacin in the US/H2O2/Fe3O4MNP system with different initial concentrations of H2O2.Without H2O2present initially,the degradation ratio of levofloxacin is approximately 30%,which to some extent is attributed to adsorption of levofloxacin on Fe3O4MNPs.The addition of H2O2obviously enhances degradation of levofloxacin.As H2O2concentration increases from 1.5 to 15.0 mmol·L?1,the levofloxacin degradation ratio increases until reaching its maximum.The results are mainly related to the adsorption amount of H2O2on Fe3O4MNPs[12].

Fig.9.Effect of H2O2 concentration on levofloxacin degradation in US/H2O2/Fe3O4 MNP system(Conditions:levofloxacin initial concentration,20 mg·L?1;Fe3O4 dose,1.0 g·L?1;ultrasound power,195 W). ★ 0.0 mmol·L?1; ☆ 1.5 mmol·L?1; ▼3.0 mmol·L?1;■ 5.0 mmol·L?1;●10.0 mmol·L?1;▲ 15.0 mmol·L?1.

Fig.10.Changes in HPLC spectra of levofloxacin in US/H2O2/Fe3O4 MNP system(Conditions:initial levofloxacin concentration,20 mg·L?1;pH,7.14;Fe3O4 dosage,1.0 g·L?1;H2O2,5.0 mmol·L?1;ultrasound power,195 W).

3.4.Sono-degradation products and degradation pathway

The HPLC spectra of levofloxacin solution in an US/H2O2/Fe3O4MNP system at different times are shown in Fig.10.The retention time(tR)of levofloxacin is 10.91 min(tR=6.73 min is the adsorption of H2O2).The peak area of levofloxacin decreases with reaction time,suggesting that its structure is destroyed,producing three main intermediate compounds.In 30 min,two intermediate compounds are produced,and their corresponding retention time is 9.76 min(P2),and 14.24 min(P3).After 60 min,tRof 8.61 min(P1)appears and P1 and P2 degrade completely in 240 min.

The MS spectra of three intermediate products(P1,P2,and P3)are shown in Fig.11,with the protonated molecular ions at m/z 338.1451,348.1325 and 378.1435.Combined with the theoretical molecular weight and the report in literature,P1 with 338.1451 is found to be anthranilic acid analogues,and similar degradation products are identified during levofloxacin ozonation[6].Formation of isatin and anthranilic acid analogues by means of hydroxyl radicals is possible through the formation of intermediate.As one of the main intermediates,P2 with 348.1325 m/z may be attributed to the demethylation of piperazinyl ring.MS-fragment m/z 304.1438 corresponds to CO2loss[6,23].P3(m/z 378.1435)has a net gain of one oxygen atom compared to levofloxacin.The presence of MS-fragment m/z 361.1409 and 317.1515 indicates the degradation at oxazinyl group instead of piperazinyl substituent[24].With the three products,a tentative partial degradation pathway for levofloxacin in US/H2O2/Fe3O4MNP system is postulated and shown in Fig.12.

4.Conclusions

Fe3O4magnetic nanoparticles(MNPs)were successfully synthesised by modified co-precipitation method.The results of XRD and TEM revealed that the size of Fe3O4MNPs has an average diameter of approximately 12 to 18 nm.The results demonstrate that Fe3O4MNPs can effectively enhance levofloxacin degradation over the range 4.0≤pH≤8.0 in an US/H2O2system.Higher Fe3O4MNP doses and H2O2concentration are favourable for levofloxacin degradation.According to the HPLC/MS/MS results,three possible intermediates are identified,and a partial tentative degradation pathway is postulated.

Fig.11.MS2 spectrum of three intermediate products P1,P2 and P3 of levofloxacin(Other conditions:initial levofloxacin concentration,20 mg·L?1;pH 7.14;Fe3O4 dosage,1.0 g·L?1;H2O2 concentration,5.0 mmol·L?1;ultrasound power,195 W).

Fig.12.A tentative partial degradation pathway of levofloxacin in US/H2O2/Fe3O4 MNP system.