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Journal of Environmental Management 133 (2014) 302e308

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Journal of Environmental Management

journal homepage: www.elsevier .com/locate/ jenvman

Degradation and antibiotic activity reduction of chloramphenicol inaqueous solution by UV/H2O2 process

Antonio Zuorro a, Marco Fidaleo b, Marcello Fidaleo c, Roberto Lavecchia a,*

aDepartment of Chemical Engineering, Materials & Environment, Sapienza University, Via Eudossiana 18, 00184 Roma, Italyb Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Roma, ItalycDepartment for Innovation in Biological, Agro-Food and Forest Systems, University of Tuscia, Viterbo, Italy

a r t i c l e i n f o

Article history:Received 24 September 2013Received in revised form26 November 2013Accepted 7 December 2013Available online

Keywords:ChloramphenicolAntibioticDegradationUV/H2O2

WastewaterResponse surface methodology

* Corresponding author. Tel.: þ39 (0)6 44585598;E-mail address: roberto.lavecchia@uniroma1.it (R.

0301-4797/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.jenvman.2013.12.012

a b s t r a c t

The efficacy of the UV/H2O2 process to degrade the antibiotic chloramphenicol (CHL) was investigated at20 �C using a low-pressure mercury lamp as UV source. A preliminary analysis of CHL degradationshowed that the process followed apparent first-order kinetics and that an optimum H2O2 concentrationexisted for the degradation rate. The first-order rate constant was used as the response variable and itsdependence on initial CHL and H2O2 concentrations, UV light intensity and reaction time was investi-gated by a central composite design based on the response surface methodology. Analysis of responsesurface plots revealed a large positive effect of radiation intensity, a negative effect of CHL concentrationand that there was a region of H2O2 concentration leading to maximum CHL degradation. CHL solutionssubmitted to the UV/H2O2 process were characterized by TOC and their activity against Escherichia coliand Staphylococcus aureus was assessed. No residual antibiotic activity was detected, even at CHL con-centrations higher than those used in the designed experiments. Overall, the obtained results stronglysupport the possibility of reducing the risks associated with the release of CHL into the environment,including the spread of antibiotic resistance, by the UV/H2O2 process.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Chloramphenicol (CHL) is a broad-spectrum antibiotic effectiveagainst many Gram-positive and Gram-negative bacteria, includingseveral anaerobic organisms (Balbi, 2004). CHL acts by binding tothe 50S ribosomal subunit of bacteria and inhibiting peptidyltransferase, the enzyme catalyzing the formation of peptide bondsbetween adjacent amino acids in the growing polypeptide chain(Schwarz et al., 2004). While topical application of CHL is consid-ered to be relatively safe, its systemic administration can haveserious side effects, such as bone marrow suppression and aplasticanemia, which are often fatal (Lam et al., 2002). For this reason, itsuse is currently restricted to ophthalmic applications or the treat-ment of meningitis, typhoid fever and other infections when saferalternatives are not available.

Because of its potential deleterious effects on human health, inthe 090s CHL has been banned from use in food-producing animalsin the European Union and the United States of America. However,CHL is still widely used in shrimp farming and other aquaculture

fax: þ39 (0)6 4827453.Lavecchia).

All rights reserved.

systems, particularly in Asian countries (Sapkota et al., 2008). Inaquaculture facilities, CHL is dissolved into water for pond disin-fection and/or added to feed for prophylactic purposes. A system-atic study on the use of antibiotics by the top 15 aquaculture-producing countries showed that CHL was the second mostcommonly used after oxytetracycline (FAO, 2005). For example, insurface waters in Singapore and Korea, CHL was found in concen-trations between 0.001 and 0.031 mg L�1, and in effluents of sewagetreatment plants in China average concentrations of 2.08 and26.6 mg L�1 were measured (Trovó et al., 2013).

In addition to toxic effects on aquatic and terrestrial organisms,the presence of CHL in water bodies may contribute to the emer-gence of resistant strains of pathogenic bacteria. Antibiotic resis-tance can result from mutations in genes encoding the moleculartargets for antibiotic compounds (Davies and Davies, 2010) or fromhorizontal gene transfer mechanisms such as conjugation, trans-duction and transformation (Andam et al., 2011). Several studieshave documented the prevalence of CHL-resistant bacteria inantibiotic-treated ponds and in aquaculture environments (Ruizet al., 1999; Sandaa et al., 2005; Huys et al., 2007; Gao et al.,2012). Furthermore, antibiotic resistance genes in bacteriacollected from aquaculture facilities were found to exhibit highsequence similarities with those present in clinical isolates (Rhodes

0

0.5

1

1.5

2

220 240 260 280 300 320 340 360 380

Abs

orba

nce

Wavelength (nm)

a

c

b

Fig. 1. e Absorption spectra of CHL before (a) and after 10-min (b) or 20-min (b)treatment with UV/H2O2 under the following conditions: ca0 ¼ 50 mg L�1,ch0 ¼ 40 mM, I ¼ 1000 mW cm�2.

A. Zuorro et al. / Journal of Environmental Management 133 (2014) 302e308 303

et al., 2000), thus supporting the hypothesis that the developmentof antibiotic resistance in aquaculture environments can contributeto the emergence and spread of antibiotic resistance among humanpopulations (Sapkota et al., 2008).

Due to the recalcitrant nature of antibiotics, their removal fromwastewater by conventional treatment processes is extremely low(Zhang and Li, 2011). Therefore, specifically designed treatmentsare necessary to degrade them and avoid their release into theenvironment (Elmolla and Chaudhuri, 2010). Studies on CHLremoval have investigated the possible use of photocatalyticmethods (Chatzitakis et al., 2008), Fenton process (Badawy et al.,2009), microwave irradiation (Lin et al., 2010), metallic nano-particles (Singh et al., 2012) and bioelectrochemical systems (Lianget al., 2013). Each of these technologies offers advantages overclassical methods, but their economic effectiveness and applica-bility to real situations have not yet been fully assessed. In addition,and more importantly, little or no information is available on thedegree of antibiotic activity reduction that could be achieved bythese methods.

The purpose of this study was to evaluate the efficacy of the UV/H2O2 process to degrade CHL in aqueous solution and to assess theresulting degree of antibiotic activity reduction. This technologybelongs to the so-called advanced oxidation processes (AOPs),which are considered among the most effective methods for thedegradation of refractory organic pollutants. AOPs are based on in-situ generation of highly reactive radical species that are capable ofdegrading the contaminants by the use of solar, chemical or otherforms of energy (Vogelpohl and Kim, 2004). In the UV/H2O2 pro-cess, the main species responsible for oxidation are the hydroxylradicals (HO�) generated from the photolysis of hydrogen peroxide(Wang and Xu, 2012). In order to assess the feasibility of thismethod for removing CHL from contaminated water, we investi-gated the influence of the main process variables (CHL and H2O2concentrations, reaction time and irradiance level) on the antibioticdegradation. To evaluate the antibiotic activity reduction achievedby this treatment, we determined the antibacterial activity of theUV/H2O2-treated CHL solutions against Escherichia coli and Staph-ylococcus aureus, two pathogenic bacteria that are highly sensitiveto CHL.

2. Materials and methods

2.1. Chemicals and bacterial strains

Chloramphenicol (C11H12Cl2N2O5, CAS Registry No. 56-75-7,purity >98%), hydrogen peroxide (CAS Registry No. 7722-84-1, 30%v/v aqueous solution), catalase (EC 1.11.1.6, CAS Registry No. 9001-05-2, activity 10,000 U/mg protein), MuellereHinton Agar 2 andMuellereHinton Broth were purchased from SigmaeAldrich(Milano, Italy). E. coli (ATCC 25922) and S. aureus (ATCC 25923)were supplied by KairoSafe (Duino Aurisina, Italy).

Working H2O2 solutions were prepared by diluting the stocksolution with double-distilled water. The resulting H2O2 concen-tration was checked spectrophotometrically at 240 nm using amolar extinction coefficient of 43.6 M�1 cm�1 (Noble and Gibson,1970).

2.2. Degradation experiments

Experiments to investigate the UV/H2O2 degradation of CHLwere carried out in a cabinet maintained at 20 � 2 �C and con-taining a low-pressure mercury lamp (ENF-260C/FE, SpectronicsCorp., USA) with a nominal power of 6Wand a maximum emissionat 254 nm. Rectangular quartz cells with an internal volume of 4 mLand an optical path length of 1 cm were inserted in the cabinet at

calibrated distances from the lamp, with the exposed surfaceperpendicular to the direction of light propagation. The irradiancelevels at the selected distances were periodically checked with a UVradiometer (XR-1000, Spectronics Corp., USA). All experimentswere made at pH 5.5 � 0.1.

Before starting the experiment, the lamp was warmed up for atleast 10 min to ensure a stable light emission. The cells were filledwith the antibiotic solution (with or without hydrogen peroxide)and irradiated for the prescribed time. Then, the CHL concentrationwas determined using a double-beam spectrophotometer (Lambda25, Perkin Elmer, USA). To avoid interferencewith H2O2, absorbancemeasurements weremade at 290 nm. At this wavelength, themolarextinction coefficient of CHL evaluated from a calibration curvewas7.4 � 103 Me1 cm�1. Representative absorption spectra of CHLbefore and after the treatment are shown in Fig. 1.

2.3. TOC measurements

Total organic carbon (TOC) measurements were made with aShimadzu analyzer (TOC-5000A, Shimadzu Corp., Japan) operatingat 680 �C furnace temperature. The analyzer was equipped with anIR detector and an autosampler.

2.4. Antibacterial activity assay

The antibacterial activity of the CHL solutions was determinedby the agar-well diffusion method, following the procedure re-ported by Fidaleo et al. (2013). Briefly, bacterial cells from anexponential-phase culture were first incubated overnight at 37 �Cusing an inoculum of approximately 1.5 � 106 CFU mL�1. Sub-cultures were then streaked on MuellereHinton agar plates andfour 9-mm wells were cut in the agar plate. Each well was filledwith 150 mL of the CHL solution. After 16-h incubation at 37 �C, thediameters of the inhibition zones around the wells were measured.

In order to remove the residual hydrogen peroxide that mightremain in the CHL solutions after the UV/H2O2 process, they weresubjected to a treatment with catalase before performing the ac-tivity assay (Brudzynski et al., 2011). This enzyme catalyzes thedecomposition of hydrogen peroxide to water and oxygen(H2O2 / H2O2 þ O2) and was applied to the CHL solutions using adose of 22 mU mL�1, a temperature of 25 �C and a reaction time of30 min.

0

0.2

0.4

0.6

0.8

1

0 15 30 45 60 75

c a/ca0

t (min)

H2O2

UV

UV + H2O2

Fig. 2. e Effect of UV, H2O2 and UV/H2O2 on the degradation of CHL. ca is the con-centration of CHL in solution calculated from the absorbance at 290 nm (other con-ditions: ca0 ¼ 100 mg L�1, ch0 ¼ 40 mM, I ¼ 1000 mW cm�2).

A. Zuorro et al. / Journal of Environmental Management 133 (2014) 302e308304

2.5. Experimental design

The effect of initial CHL (ca0) and H2O2 (ch0) concentrations, UVlight intensity (I) and reaction time (t) on the antibiotic degradationwas studied by using the response surface methodology (Zuorroet al., 2013). In particular, a central composite design consisting ofa full 24 factorial design (16 runs), axial points at distance a ¼ 2from the central point (8 runs) and a replicated central point (6runs) was selected. Factor levels were chosen by examining theresults of preliminary degradation experiments and are reported inTable 1. The original or natural values of factor levels were trans-formed into the coded values (xi) using the following linear trans-formations (Montgomery, 2005):

x1 ¼ ca0 � 6020

x2 ¼ ch0 � 3515

x3 ¼ I � 600200

x4 ¼ t � 30001200

(1)

Overall, the experimental design consisted of 16 þ 8 þ 6 ¼ 30runs, which were performed in random order to eliminate possiblebias (Table S1 in Supplementary Material). The CHL conversion (Xa)at the preset reaction time (t) was calculated as:

Xa ¼ 1� caca0

(2)

where ca is the antibiotic concentration at time t.The analysis of results was performed with the statistical soft-

ware JMP�, version 9.0.2 (SAS Institute Inc., Cary, NC, USA).

3. Results and discussion

3.1. Preliminary analysis of CHL degradation

A first series of experiments was performed to investigate theeffect of UV and H2O2, alone or in combination, on CHL degradation.As can be seen from Fig. 2, neither H2O2 nor UV alone had appre-ciable effects on the degradation of CHL. In contrast, a significantreduction in CHL concentration was observed when they wereapplied simultaneously. After approximately 1 h, the antibioticconversion reached values close to 100%. Furthermore, plotting theexperimental data points on a semi-log scale yielded straight lines,suggesting that the process followed apparent first-order kinetics(Fig. 3). Finally, the first-order rate constant calculated from theslope of these lines exhibited a bell-shaped dependence on theinitial H2O2 concentration (Fig. S1 in Supplementary Material).

From this preliminary analysis it can be concluded that: (a) thedegradation process requires both UV and H2O2 to occur; (b) thekinetics of the process is of the pseudo-first order with respect to

Table 1Natural and coded levels of the factors of the central composite design.

Factor Factor level Unit

�2 �1 0 þ1 þ2

CHL concentration (ca0) 20 40 60 80 100 mg L�1

Hydrogen peroxide (ch0) 5 20 35 50 65 mMUV light intensity (I) 200 400 600 800 1000 mW cm�2

Reaction time (t) 600 1800 3000 4200 5400 s

the antibiotic concentration; and (c) the antibiotic degradation ismaximum at H2O2 concentrations ranging approximately between25 and 40 mM.

The mass-balance for the degradation of antibiotic in theaqueous solution can be written as:

�dcadt

¼ kca (3)

where k is the first-order rate constant for the degradation process.Integrating this equation with the initial condition ca ¼ ca0 for t ¼ 0and accounting for Eq. (2) gives:

k ¼ lnð100� XaÞt

(4)

Eq. (4) can be used to determine k from conversion (Xa) and time(t) data. Accordingly, the quantity k can be regarded as a newresponse variable obtained by transformation of the original one(Xa) through the time factor.

3.2. Analysis of response surface

The first-order rate constants determined from conversion andtime data (Eq. (4)) were correlated in terms of the process variablesby the following 2nd-order polynomial equation, referred to as thefull model:

y ¼ b0 þX4

i¼1

bixi þX4

i¼1

biixi2 þ

X4

i¼1

X4

j¼1;i<j

bijxixj (5)

where y is the process response (k), b0 is the intercept, bi, bii and bijare the linear, pure quadratic and interaction regression co-efficients, respectively, and xi are the coded independent variables.Because of their good interpolation ability and ease of parameterestimation, polynomial models are commonly used to describe thebehavior of complex systems (Fidaleo et al., 2006; Kousha et al.,2012).

The coefficients of Eq. (5) were estimated by fitting the k data bythe least squares method. Many coefficients were not significant atthe 95% confidence level (a ¼ 5%) and were dropped from themodel to build a reduced model with better prediction capabilities.Examination of the non-significant coefficients indicated that the

0.01

0.1

1

10

0 10 20 30 40 50 60

c a/c

a0

t (min)

0.01

0.1

1

10

0 10 20 30 40 50 60

c a/c

a0

t (min)

a

b

Fig. 3. e Observed changes in CHL concentration (ca) as a function of time (t) on asemi-logarithmic plot for several treatments carried out in water with ca0 ¼ 50 mg L�1

(a) or 100 mg L�1 (b) in presence of both UV light (I ¼ 1000 mW cm�2) and H2O2

(ch0 ¼ 40 mM) (C); in presence of only H2O2 (-) or only UV light (:).

Table 2Estimates of the regression coefficients (bi, bij and bii) of the reduced polynomialmodel together with their corresponding standard errors (SE), t-statistics (t) and p-values (p).

Coefficient Effect Estimate SE t p

b0 e 2.04892 0.03586 56.856 0.000b1 ca0 �0.25879 0.02536 �10.206 0.000b2 ch0 0.06806 0.02536 2.684 0.013b3 I 0.70735 0.02536 27.895 0.000b12 t 0.07574 0.03106 2.439 0.023b11 ca0 � ca0 �0.10426 0.02329 �4.476 0.000b22 ch0 � ch0 �0.23355 0.02329 �10.027 0.000

A. Zuorro et al. / Journal of Environmental Management 133 (2014) 302e308 305

reaction time did not exert an appreciable effect on k, therebyconfirming that the assumed first-order kinetics for CHL degrada-tion was reasonable.

The reduced polynomial model proved to be adequate to fit allexperimental data, with coefficient of determination (R2), adjusted-R2, prediction-R2 and error standard deviation of 97.8%, 97.2%, 93.9%and 0.12 h�1, respectively. Analysis of residuals showed no apparentdepartures from basic ANOVA assumptions, i.e., normally distrib-uted errors with constant variance and independent of one another.Furthermore, lack of fit was not significant at a¼ 5%, indicating thatthe model was a good approximation to the mean structure in theregion of experimentation. The experimental and calculated kvalues were in fairly good agreement, with an average percentdifference of about 6% (Fig. S2 in Supplementary Material). Inaddition, the k values obtained for two factor level combinationscharacterized by all factors at the same levels except for time werereasonably close, further supporting the fact that the reaction timewas not a significant factor.

Table 2 reports the reduced model coefficients together withtheir standard errors, t statistics and corresponding p-values. Wenote that CHL degradation was mainly affected by UV light in-tensity, through a linear term, and to a lesser extent by initial CHLand H2O2 concentrations, through linear and quadratic terms. Aweak interaction between the latter two factors was also present.

Fig. 4 shows the response-surface plots of k as a function of twoindependent variables varying in the factorial part of the experi-mental space (between �1 and þ1), with the other independentvariable set at its central level. Examination of the plot of k as afunction of initial CHL and H2O2 concentrations (Fig. 4a) reveals anegative effect of CHL concentration, with a downward curvature,and that there is a region of H2O2 concentration in which k ismaximum. Since ch0 does not interact with I, it is possible todetermine, for each value of ca0 and independently of the value of I,a ch0 concentration that results in the maximum k. For ca0 varyingbetween 40 and 80 mg L�1, the optimum H2O2 concentrationranged from 34.1 to 39.3 mM, such a low variation being attributedto the weak interaction between ch0 and ca0. The large positive ef-fect of UV light intensity on k is apparent from Figs. 4b and c, wherek is plotted as a function of I and ca0 or ch0, respectively. Because I isnot involved in any interaction with the other factors, the obser-vations reported above about the effect of ca0 and ch0 are consistentalso with these plots.

The effects of the different factors on the kinetics of CHL removalcan be explained by considering the following reaction scheme forthe degradation process:

CHL þ hn / products (6)

H2O2 þ hn / 2 HO� (7)

CHL þ HO� / products (8)

which assumes that the antibiotic molecule is attacked by both UVphotons and the hydroxyl radicals generated from the photolysis ofH2O2 to give the final products.

The positive effect of UV light intensity can be ascribed to theincreased production of HO� (Eq. (7)), the main species responsiblefor the degradation of organic compounds in the UV/H2O2 process,and, to a lesser extent, to direct CHL photolysis (Eq. (6)).

The negative effect of initial CHL concentration can be due to theabsorption of UV radiation by the antibiotic molecule. At 254 nm,the wavelength of maximum emission of the UV lamp, themeasured molar extinction coefficient of CHL was 4.45 � 103 Me

1 cm�1, i.e., about a half of that at the peak maximum (see Fig. 1).Accordingly, higher CHL concentrations cause an increase in thefraction of UV photons absorbed and a decrease in the concentra-tion of HO� in the liquid. Another possible, but perhaps less sig-nificant cause of inhibition, could be the formation of reactionintermediates interfering with the degradation pathway (So et al.,2002).

The existence of an optimal concentration for hydrogenperoxide was also observed in other studies on the UV/H2O2 pro-cess (Morrison et al., 1996; Einschlag et al., 2002; Wu et al., 2007)and explained in terms of two opposing effects: the increasedproduction of HO� radicals at higher H2O2 concentrations,

Fig. 4. e Response surface and contour plots showing the effects on the first-order rate constant (k) of: (a) initial CHL (ca0) and H2O2 (ch0) concentrations; (b) initial CHL con-centration (ca0) and radiation intensity (I); (c) initial H2O2 concentration (ch0) and radiation intensity (I). For each surface plot the levels for the other factor are held at their centralvalues.

A. Zuorro et al. / Journal of Environmental Management 133 (2014) 302e308306

according to Eq. (7), and their quenching, according to the followingreactions:

H2O2 þ HO� / H2O þ HO2� (9)

2 HO2� / H2O2 þ O2 (10)

HO2� þ HO� / H2O þ O2 (11)

The combination of the two contributions can lead to theappearance of a maximum in the dependence of the degradationrate on initial H2O2 concentration. However, evidence indicates thatthe optimal H2O2 concentration is highly dependent on the char-acteristics of the degrading compound and the applied conditions.For this reason, the effects of H2O2 concentration should beexperimentally checked and analysed for each particular case.

The canonical analysis of the response surface, carried out byusing a model including all coefficients except the ones related tothe time factor, allowed the eigenvalues to be estimated as:l1 ¼ �0.0184, l2 ¼ �0.1059 and l3 ¼ �0.2507. Since l1 is muchsmaller than the others, the remaining ones are both negative andthe critical point falls outside the region of experimentation, thesurface can be classified as a rising ridge.

To validate the developed model, additional experiments werecarried out in the central composite design domain (xi varyingbetween �1 and þ1). By using the reduced polynomial modeltogether with the parameter estimates listed in Table 2, it waspossible to calculate the predicted values of k and the 95%-

prediction intervals for future observations. The predicted rateconstants were in good agreement with the experimental valuesand the majority of data points fell within the 95%-prediction band(Table S2 and Fig. S3 in Supplementary Material), further sup-porting the model presented above.

3.3. TOC reduction

Fig. 5 shows the TOC reduction for CHL solutions subjected toUV/H2O2 treatment under conditions close to optimal for CHLdegradation. In particular, the initial H2O2 concentration, the UVlight intensity and the reaction time were set at their center-pointvalues (ch0 ¼ 35 mM, I ¼ 600 mW cm�2 and t ¼ 50 min) and fourinitial antibiotic concentrations (20, 60, 100 and 150 mg L�1) wereexamined. As is known, TOC values are related to the concentrationof total organic compounds in the solution and the decrease of TOCreflects the degree of mineralization. Under the tested conditions,TOC removal decreased from about 50% (at ca0 ¼ 20 mg L�1) to41.5% (at ca0 ¼ 150 mg L�1). Similar results are reported by Lopez-Penalver et al. (2010), who studied the degradation of tetracy-clines in aqueous solution by the UV/H2O2 process and found amean mineralization ratio of 47.2% for antibiotic concentrationsranging from 10 to 100 mg L�1.

The above results indicate that CHL mineralization is slowerthan degradation and that degradation products or intermediatesstill contribute to the TOC of the solution. Of course, the crucialpoint is to assess whether these compounds also exhibit antibac-terial activity.

0

5

10

15

20

25

30D

(mm

)

E. coli Untreated

UV/H2O2 treated

0

10

20

30

40

50

60

20 60 100 150

TOC

(mg

L-1 )

ca0 (mg L-1)

Untreated

UV/H2O2 treated

Fig. 5. TOC removal in aqueous solutions at different initial CHL concentration (ca0).Treatment conditions: ch0 ¼ 35 mM, I ¼ 600 mW cm�2, t ¼ 50 min.

A. Zuorro et al. / Journal of Environmental Management 133 (2014) 302e308 307

3.4. Antibacterial activity of the treated CHL solutions

To assess the effectiveness of the process in terms of antibioticactivity removal, the residual activity of UV/H2O2-treated CHL so-lutions against E. coli and S. aureus was determined. These patho-gens are highly sensitive to CHL (Saxena and Gomber, 2008) andwere selected as representative of Gram-negative and Gram-positive bacteria, respectively.

Activity measurements were carried out on CHL solutions sub-mitted to UV/H2O2 treatment under conditions close to optimal forantibiotic degradation. In these experiments the initial H2O2 con-centration, the UV light intensity and the reaction time were set attheir center-point values (ch0 ¼ 35 mM, I ¼ 600 mW cm�2 andt ¼ 50 min) and four initial antibiotic concentrations (20, 60, 100and 150 mg L�1) were examined. Untreated antibiotic solutions atthe same CHL concentrations were also assayed. Typical results of

Fig. 6. e Inhibition zones on agar plate with S. aureus (ATCC 25923) produced byuntreated CHL at 150 mg L�1 and CHL treated with H2O2, UV (hv) or UV/H2O2

(hv þ H2O2). Treatment conditions: ca0 ¼ 150 mg L�1, ch0 ¼ 35 mM, I ¼ 600 mW cm�2,t ¼ 50 min.

agar-diffusion tests are presented in Fig. 6. The extent of the inhi-bition zone around each well can be regarded as an inverse mea-sure of the antibacterial activity of the solution in the well. Plottingthe diameters of the inhibition zones against the initial antibioticconcentrations gave the results reported in Fig. 7. For the untreatedsamples, a dose-dependent response was observed, with inhibitionzone diameters ranging from 10 to 23.5 mm (E. coli) and 9.5e24 mm (S. aureus). No residual antibacterial activity was detectedagainst both pathogens in CHL solutions subjected to UV/H2O2treatment. This clearly suggests that, under the applied processconditions, the antibacterial activity of CHL or its degradationproducts was completely lost, even at CHL concentrations higherthan those used in the response surface study.

It may be interesting to compare these results with those ob-tained in the few studies examining the drug activity reduction ofCHL solutions after treatment by other methods. Chatzitakis et al.(2008) investigated the photocatalytic degradation of CHL by TiO2particles and found that a reaction time of 90 min was needed tocompletely eliminate the antibiotic activity of CHL at 50 mg L�1

against E. coli. In the study of Liang et al. (2013) an innovativeelectrochemical system based on a microbial biocathode was usedto degrade CHL and compared with an abiotic cathode. The authorsreport a removal efficiency at 24 h of 96% for the bioelectrochemical

0

5

10

15

20

25

30

20 60 100 150

D (m

m)

ca0 (mg L-1)

S. aureus Untreated

UV/H2O2 treated

20 60 100 150

ca0 (mg L-1)

Fig. 7. e Antibiotic activity of untreated and UV/H2O2-treated CHL solutions againstE. coli (ATCC 25922) and S. aureus (ATCC 25923). D is the diameter of the inhibitionzone and ca0 the initial CHL concentration. The dashed lines indicate the size of theagar well. Treatment conditions: ch0 ¼ 35 mM, I ¼ 600 mW cm�2, t ¼ 50 min.

A. Zuorro et al. / Journal of Environmental Management 133 (2014) 302e308308

system and 73% for the abiotic control. They also found that verydifferent transformation products were formed in the two systemsand that those produced in the bioelectrochemical system lacked orhad low antibacterial activity against the tested strains (E. coli andStaphylococcus oneidensis), while the intermediates of CHL reduc-tion in the abiotic system were still toxic to microorganisms.

Turning to our results, we showed that the combined applica-tion of UV and H2O2, under appropriate conditions, allows completedrug activity removal at least up to initial CHL concentrations ashigh as 100 mg L�1, which demonstrates the efficacy of the pro-posed method.

4. Conclusions

In recent years, the spread of antibiotic resistance and its po-tential impact on human health have caused a growing concernworldwide. Among the factors contributing to this phenomenon,the extensive and unregulated use of antibiotics in aquacultureseems to play an important role, also because of the difficulty ofremoving these pollutants by conventional wastewater treatments.The results of this study demonstrate that CHL, a nitroaromaticantibiotic widely used in aquaculture facilities, can be effectivelydegraded by the UV/H2O2 treatment. We have also shown that theantibacterial activity of CHL solutions treated under optimal pro-cess conditions was completely lost, even at antibiotic concentra-tions higher than those commonly found in wastewaters.

From a methodological viewpoint, our results suggest that arigorous approach based on the response surface analysis and theuse of a transformed response variable can improve the interpre-tation and modeling of experimental results. In this regard, thesimplified model developed can provide useful indications on theoptimum set of operating conditions to be used in the practice andan easy evaluation of the influence of process parameters on anti-biotic degradation.

Acknowledgement

The authors gratefully thank Dr. Stefano Denetto for his valuableassistance in the experimental part of this work.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jenvman.2013.12.012

References

Andam, C.P., Fournier, G.P., Gogarten, J.P., 2011. Multilevel populations and theevolution of antibiotic resistance through horizontal gene transfer. FEMSMicrobiol. Rev. 35, 756e767.

Badawy, M.I., Wahaab, R.A., El-Kalliny, A.S., 2009. Fenton-biological treatmentprocesses for the removal of some pharmaceuticals from industrial wastewater.J. Hazard. Mat. 167, 567e574.

Balbi, H.J., 2004. Chloramphenicol: a review. Pediatr. Rev. 26, 284e288.Brudzynski, K., Abubaker, K., St-Martin, L., Castle, A., 2011. Re-examining the role of

hydrogen peroxide in bacteriostatic and bactericidal activities of honey. Front.Microbiol. 2, 213.

Chatzitakis, A., Berberidou, C., Paspaltsis, I., Kyriakou, G., Sklaviadis, T., Poulios, I.,2008. Photocatalytic degradation and drug activity reduction of chloram-phenicol. Water Res. 42, 386e394.

Davies, J., Davies, D., 2010. Origins and evolution of antibiotic resistance. Microbiol.Mol. Biol. Rev. 74, 417e433.

Einschlag, F.S.G., Lopez, J., Carlos, L., Capparelli, A.L., 2002. Evaluation of the effi-ciency of photodegradation of nitroaromatics applying the UV/H2O2 technique.Environ. Sci. Technol. 36, 3936e3944.

Elmolla, E.S., Chaudhuri, M., 2010. Comparison of different advanced oxidationprocesses for treatment of antibiotic aqueous solution. Desalination 256, 43e47.

FAO, 2005. Fishery Information Data and Statistics Unit. FISHSTAT Databases andStatistics, Roma, Italy.

Fidaleo, M., Esti, M., Moresi, M., 2006. Assessment of urea degradation rate in modelwine solutions by acid urease from Lactobacillus fermentum. J. Agric. Food Chem.54, 6226e6235.

Fidaleo, M., Zuorro, A., Lavecchia, R., 2013. Enhanced antibacterial and anti-quorumsensing activities of triclosan by complexation with modified b-cyclodextrins.World J. Microbiol. Biotechnol. 29, 1731e1736.

Gao, P., Mao, D., Luo, Y., Wang, L., Xu, B., Xu, L., 2012. Occurrence of sulfonamide andtetracycline-resistant bacteria and resistance genes in aquaculture environ-ment. Water Res. 46, 2355e2364.

Huys, G., Bartie, K., Cnockaert, M., Hoang Oanh, D.T., Phuong, N.T., Somsiri, T., 2007.Biodiversity of chloramphenicol-resistant mesophilic heterotrophs fromSoutheast Asian aquaculture environments. Res. Microbiol. 158, 228e235.

Kousha, M., Daneshvar, E., Dopeikar, H., Taghavi, D., Bhatnagar, A., 2012. Box-Behnken design optimization of Acid Black 1 dye biosorption by differentbrown macroalgae. Chem. Eng. J. 179, 158e168.

Lam, R.F., Lai, J.S., Ng, J.S., Rao, S.K., Law, R.W., Lam, D.S., 2002. Topical chloram-phenicol for eye infections. Hong Kong Med. J. 8, 44e47.

Liang, B., Cheng, H.Y., Kong, D.Y., Gao, S.H., Sun, F., Cui, D., Kong, F.Y., Zhou, A.J.,Liu, W.Z., Ren, N.Q., Wu, W.M., Wang, A.J., Lee, D.J., 2013. Accelerated reductionof chlorinated nitroaromatic antibiotic chloramphenicol by biocathode. Envi-ron. Sci. Technol. 47, 5353e5361.

Lin, L., Yuan, S., Chen, J., Wang, L., Wan, J., Lu, X., 2010. Treatment ofchloramphenicol-contaminated soil by microwave radiation. Chemosphere 78,66e71.

Lopez-Penalver, J.J., Sanchez-Polo, M., Gomez-Pacheco, C.V., Rivera-Utrilla, J., 2010.Photodegradation of tetracyclines in aqueous solution by using UV and UV/H2O2 oxidation processes. J. Chem. Technol. Biotechnol. 85, 1325e1333.

Montgomery, D.C., 2005. Design and Analysis of Experiments, sixth ed. John Wiley& Sons, New York.

Morrison, C., Bandara, J., Kiwi, J., 1996. Sunlight induced decoloration/degra-dation of non-biodegradable Orange II dye by advanced oxidation tech-nologies in homogeneous and heterogeneous media. J. Adv. Oxid. Technol.1, 160e169.

Noble, R.W., Gibson, Q.H., 1970. The reaction of ferrous horseradish peroxidase withhydrogen peroxide. J. Biol. Chem. 245, 2409e2413.

Rhodes, G., Huys, G., Swings, J., McGann, P., Hiney, M., Smith, P., Pickup, R.W., 2000.Distribution of oxytetracycline resistance plasmids between aeromonads inhospital and aquaculture environments: Implication of Tn1721 in disseminationof the tetracycline resistance determinant tet A. Appl. Environ. Microbiol. 66,3883e3890.

Ruiz, J., Capitano, L., Nunez, L., Castro, D., Sierra, J.M., Hatha, M., Borrego, J.J., Vila, J.,1999. Mechanisms of resistance to ampicillin, chloramphenicol and quinolonesin multiresistant Salmonella typhimuriumstrains isolated from fish.J. Antimicrob. Chemother. 43, 699e702.

Sandaa, R.A., Torsvik, V., Goksoyr, J., 2005. Transferable drug resistance in bacteriafrom fish-farm sediments. Can. J. Microbiol. 38, 1061e1065.

Sapkota, A., Sapkota, A.R., Kucharski, M., Burke, J., McKenzie, S., Walker, P.,Lawrence, R., 2008. Aquaculture practices and potential human health risks:current knowledge and future priorities. Environ. Int. 34, 1215e1226.

Saxena, S., Gomber, C., 2008. Comparative in vitro antimicrobial procedural efficacyfor susceptibility of Staphylococcus aureus, Escherichia coli and Pseudomonasspecies to chloramphenicol, ciprofloxacin and cefaclor. Brit. J. Biomed. Sci. 65,178e183.

Schwarz, S., Kehrenberg, C., Doublet, B., Cloeckaert, A., 2004. Molecular basis ofbacterial resistance to chloramphenicol and florfenicol. FEMS Microbiol. Rev.28, 519e542.

Singh, K.P., Singh, A.K., Gupta, S., Rai, P., 2012. Modeling and optimization ofreductive degradation of chloramphenicol in aqueous solution by zero-valentbimetallic nanoparticles. Environ. Sci. Pollut. Res. 19, 2063e2078.

So, M., Cheng, M.Y., Yu, J.C., Wong, P.K., 2002. Degradation of azo dye Procion RedMX-5B by photocatalytic oxidation. Chemosphere 46, 905e912.

Trovó, A.G., de Paiva, V.A.B., Machado, A.E.H., de Oliveira, C.A., Santos, R.O., 2013.Degradation of the antibiotic chloramphenicol by photo-Fenton process at lab-scale and solar pilot plant: kinetic, toxicity and inactivation assessment. Sol.Energ. 97, 596e604.

Vogelpohl, A., Kim, S.M., 2004. Advanced oxidation processes AOPs in wastewatertreatment. J. Ind. Eng. Chem. 10, 33e40.

Wang, J.L., Xu, L.J., 2012. Advanced oxidation processes for wastewater treatment:formation of hydroxyl radical and application. Crit. Rev. Environ. Sci. Technol.42, 251e325.

Wu, C., Shemer, H., Linden, K.G., 2007. Photodegradation of metolachlor applyingUV and UV/H2O2. J. Agric. Food Chem. 55, 4059e4065.

Zhang, T., Li, B., 2011. Occurrence, transformation, and fate of antibiotics inmunicipal wastewater treatment plants. Crit. Rev. Environ. Sci. Technol. 41,951e998.

Zuorro, A., Fidaleo, M., Lavecchia, R., 2013. Response surface methodology (RSM)analysis of photodegradation of sulfonated diazo dye Reactive Green 19 by UV/H2O2 process. J. Environ. Manage. 127, 28e35.