H2O2 process

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 in aqueous solution by UV/H2O2 process Antonio Zuorro a, Marco Fidaleo b, Marcello Fidaleo c, Roberto Lavecchia a, * a

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

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 September 2013 Received in revised form 26 November 2013 Accepted 7 December 2013 Available online

The efficacy of the UV/H2O2 process to degrade the antibiotic chloramphenicol (CHL) was investigated at 20
C using a low-pressure mercury lamp as UV source. A preliminary analysis of CHL degradation showed that the process followed apparent first-order kinetics and that an optimum H2O2 concentration existed for the degradation rate. The first-order rate constant was used as the response variable and its dependence on initial CHL and H2O2 concentrations, UV light intensity and reaction time was investigated by a central composite design based on the response surface methodology. Analysis of response surface plots revealed a large positive effect of radiation intensity, a negative effect of CHL concentration and that there was a region of H2O2 concentration leading to maximum CHL degradation. CHL solutions submitted to the UV/H2O2 process were characterized by TOC and their activity against Escherichia coli and Staphylococcus aureus was assessed. No residual antibiotic activity was detected, even at CHL concentrations higher than those used in the designed experiments. Overall, the obtained results strongly support 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. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Chloramphenicol Antibiotic Degradation UV/H2O2 Wastewater Response surface methodology

1. Introduction Chloramphenicol (CHL) is a broad-spectrum antibiotic effective against many Gram-positive and Gram-negative bacteria, including several anaerobic organisms (Balbi, 2004). CHL acts by binding to the 50S ribosomal subunit of bacteria and inhibiting peptidyl transferase, the enzyme catalyzing the formation of peptide bonds between adjacent amino acids in the growing polypeptide chain (Schwarz et al., 2004). While topical application of CHL is considered to be relatively safe, its systemic administration can have serious side effects, such as bone marrow suppression and aplastic anemia, which are often fatal (Lam et al., 2002). For this reason, its use is currently restricted to ophthalmic applications or the treatment of meningitis, typhoid fever and other infections when safer alternatives are not available. Because of its potential deleterious effects on human health, in the 0 90s CHL has been banned from use in food-producing animals in the European Union and the United States of America. However, CHL is still widely used in shrimp farming and other aquaculture

* Corresponding author. Tel.: þ39 (0)6 44585598; fax: þ39 (0)6 4827453. E-mail address: [email protected] (R. Lavecchia). 0301-4797/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jenvman.2013.12.012

systems, particularly in Asian countries (Sapkota et al., 2008). In aquaculture facilities, CHL is dissolved into water for pond disinfection and/or added to feed for prophylactic purposes. A systematic study on the use of antibiotics by the top 15 aquacultureproducing countries showed that CHL was the second most commonly used after oxytetracycline (FAO, 2005). For example, in surface waters in Singapore and Korea, CHL was found in concentrations between 0.001 and 0.031 mg L1, and in effluents of sewage treatment plants in China average concentrations of 2.08 and 26.6 mg L1 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 emergence of resistant strains of pathogenic bacteria. Antibiotic resistance can result from mutations in genes encoding the molecular targets for antibiotic compounds (Davies and Davies, 2010) or from horizontal gene transfer mechanisms such as conjugation, transduction and transformation (Andam et al., 2011). Several studies have documented the prevalence of CHL-resistant bacteria in antibiotic-treated ponds and in aquaculture environments (Ruiz et al., 1999; Sandaa et al., 2005; Huys et al., 2007; Gao et al., 2012). Furthermore, antibiotic resistance genes in bacteria collected from aquaculture facilities were found to exhibit high sequence similarities with those present in clinical isolates (Rhodes

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

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. 900105-2, activity 10,000 U/mg protein), MuellereHinton Agar 2 and MuellereHinton 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 stock solution with double-distilled water. The resulting H2O2 concentration was checked spectrophotometrically at 240 nm using a molar extinction coefficient of 43.6 M1 cm1 (Noble and Gibson, 1970).

calibrated distances from the lamp, with the exposed surface perpendicular to the direction of light propagation. The irradiance levels at the selected distances were periodically checked with a UV radiometer (XR-1000, Spectronics Corp., USA). All experiments were made at pH 5.5  0.1. Before starting the experiment, the lamp was warmed up for at least 10 min to ensure a stable light emission. The cells were filled with the antibiotic solution (with or without hydrogen peroxide) and irradiated for the prescribed time. Then, the CHL concentration was determined using a double-beam spectrophotometer (Lambda 25, Perkin Elmer, USA). To avoid interference with H2O2, absorbance measurements were made at 290 nm. At this wavelength, the molar extinction coefficient of CHL evaluated from a calibration curve was 7.4  103 Me1 cm1. Representative absorption spectra of CHL before and after the treatment are shown in Fig. 1. 2.3. TOC measurements Total organic carbon (TOC) measurements were made with a Shimadzu analyzer (TOC-5000A, Shimadzu Corp., Japan) operating at 680
C furnace temperature. The analyzer was equipped with an IR detector and an autosampler. 2.4. Antibacterial activity assay The antibacterial activity of the CHL solutions was determined by the agar-well diffusion method, following the procedure reported by Fidaleo et al. (2013). Briefly, bacterial cells from an exponential-phase culture were first incubated overnight at 37
C using an inoculum of approximately 1.5  106 CFU mL1. Subcultures were then streaked on MuellereHinton agar plates and four 9-mm wells were cut in the agar plate. Each well was filled with 150 mL of the CHL solution. After 16-h incubation at 37
C, the diameters of the inhibition zones around the wells were measured. In order to remove the residual hydrogen peroxide that might remain in the CHL solutions after the UV/H2O2 process, they were subjected to a treatment with catalase before performing the activity assay (Brudzynski et al., 2011). This enzyme catalyzes the decomposition of hydrogen peroxide to water and oxygen (H2O2 / H2O2 þ O2) and was applied to the CHL solutions using a dose of 22 mU mL1, a temperature of 25
C and a reaction time of 30 min.

2

a

1.5 Absorbance

et al., 2000), thus supporting the hypothesis that the development of antibiotic resistance in aquaculture environments can contribute to the emergence and spread of antibiotic resistance among human populations (Sapkota et al., 2008). Due to the recalcitrant nature of antibiotics, their removal from wastewater by conventional treatment processes is extremely low (Zhang and Li, 2011). Therefore, specifically designed treatments are necessary to degrade them and avoid their release into the environment (Elmolla and Chaudhuri, 2010). Studies on CHL removal have investigated the possible use of photocatalytic methods (Chatzitakis et al., 2008), Fenton process (Badawy et al., 2009), microwave irradiation (Lin et al., 2010), metallic nanoparticles (Singh et al., 2012) and bioelectrochemical systems (Liang et al., 2013). Each of these technologies offers advantages over classical methods, but their economic effectiveness and applicability to real situations have not yet been fully assessed. In addition, and more importantly, little or no information is available on the degree of antibiotic activity reduction that could be achieved by these 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 the resulting degree of antibiotic activity reduction. This technology belongs to the so-called advanced oxidation processes (AOPs), which are considered among the most effective methods for the degradation of refractory organic pollutants. AOPs are based on insitu generation of highly reactive radical species that are capable of degrading the contaminants by the use of solar, chemical or other forms of energy (Vogelpohl and Kim, 2004). In the UV/H2O2 process, the main species responsible for oxidation are the hydroxyl radicals (HO) generated from the photolysis of hydrogen peroxide (Wang and Xu, 2012). In order to assess the feasibility of this method for removing CHL from contaminated water, we investigated the influence of the main process variables (CHL and H2O2 concentrations, reaction time and irradiance level) on the antibiotic degradation. To evaluate the antibiotic activity reduction achieved by this treatment, we determined the antibacterial activity of the UV/H2O2-treated CHL solutions against Escherichia coli and Staphylococcus aureus, two pathogenic bacteria that are highly sensitive to CHL.

303

1

b

0.5

c

2.2. Degradation experiments

0 Experiments to investigate the UV/H2O2 degradation of CHL were carried out in a cabinet maintained at 20  2
C and containing a low-pressure mercury lamp (ENF-260C/FE, Spectronics Corp., USA) with a nominal power of 6 W and a maximum emission at 254 nm. Rectangular quartz cells with an internal volume of 4 mL and an optical path length of 1 cm were inserted in the cabinet at

220

240

260

280

300

320

340

360

380

Wavelength (nm) 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 L1, ch0 ¼ 40 mM, I ¼ 1000 mW cm2.

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2.5. Experimental design

H2O2

1

UV

0.8

ca/ca0

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

0.6

0.4 0.2

UV + H2O2

0 0

ca0  60 20 ch0  35 x2 ¼ 15 I  600 x3 ¼ 200 t  3000 x4 ¼ 1200

15

30

x1 ¼

60

75

t (min)

(1)

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

Xa ¼ 1 

45

ca ca0

(2)

Fig. 2. e Effect of UV, H2O2 and UV/H2O2 on the degradation of CHL. ca is the concentration of CHL in solution calculated from the absorbance at 290 nm (other conditions: ca0 ¼ 100 mg L1, ch0 ¼ 40 mM, I ¼ 1000 mW cm2).

the antibiotic concentration; and (c) the antibiotic degradation is maximum at H2O2 concentrations ranging approximately between 25 and 40 mM. The mass-balance for the degradation of antibiotic in the aqueous solution can be written as:

dca ¼ kca  dt

(3)

where ca is the antibiotic concentration at time t. The analysis of results was performed with the statistical software JMPÒ, version 9.0.2 (SAS Institute Inc., Cary, NC, USA).

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 0 and accounting for Eq. (2) gives:

3. Results and discussion

k ¼

3.1. Preliminary analysis of CHL degradation

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

A first series of experiments was performed to investigate the effect of UV and H2O2, alone or in combination, on CHL degradation. As can be seen from Fig. 2, neither H2O2 nor UV alone had appreciable effects on the degradation of CHL. In contrast, a significant reduction in CHL concentration was observed when they were applied simultaneously. After approximately 1 h, the antibiotic conversion reached values close to 100%. Furthermore, plotting the experimental 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 the slope of these lines exhibited a bell-shaped dependence on the initial H2O2 concentration (Fig. S1 in Supplementary Material). From this preliminary analysis it can be concluded that: (a) the degradation process requires both UV and H2O2 to occur; (b) the kinetics of the process is of the pseudo-first order with respect to

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

CHL concentration (ca0) Hydrogen peroxide (ch0) UV light intensity (I) Reaction time (t)

Factor level

Unit

2

1

0

þ1

þ2

20 5 200 600

40 20 400 1800

60 35 600 3000

80 50 800 4200

100 65 1000 5400

mg L1 mM mW cm2 s

lnð100  Xa Þ t

(4)

3.2. Analysis of response surface The first-order rate constants determined from conversion and time data (Eq. (4)) were correlated in terms of the process variables by the following 2nd-order polynomial equation, referred to as the full model:

y ¼ b0 þ

4 X i¼1

b i xi þ

4 X i¼1

bii xi 2 þ

4 X

4 X

bij xi xj

(5)

i ¼ 1 j ¼ 1;i
where y is the process response (k), b0 is the intercept, bi, bii and bij are the linear, pure quadratic and interaction regression coefficients, respectively, and xi are the coded independent variables. Because of their good interpolation ability and ease of parameter estimation, polynomial models are commonly used to describe the behavior of complex systems (Fidaleo et al., 2006; Kousha et al., 2012). The coefficients of Eq. (5) were estimated by fitting the k data by the least squares method. Many coefficients were not significant at the 95% confidence level (a ¼ 5%) and were dropped from the model to build a reduced model with better prediction capabilities. Examination of the non-significant coefficients indicated that the

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

10

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

ca/ca0

a 1

0.1

0.01 0

10

20

30

40

50

60

t (min)

ca/ca0

10

b

1

0.1

0.01 0

10

20

30

40

50

305

60

t (min) Fig. 3. e Observed changes in CHL concentration (ca) as a function of time (t) on a semi-logarithmic plot for several treatments carried out in water with ca0 ¼ 50 mg L1 (a) or 100 mg L1 (b) in presence of both UV light (I ¼ 1000 mW cm2) and H2O2 (ch0 ¼ 40 mM) (C); in presence of only H2O2 (-) or only UV light (:).

reaction time did not exert an appreciable effect on k, thereby confirming that the assumed first-order kinetics for CHL degradation was reasonable. The reduced polynomial model proved to be adequate to fit all experimental data, with coefficient of determination (R2), adjustedR2, prediction-R2 and error standard deviation of 97.8%, 97.2%, 93.9% and 0.12 h1, respectively. Analysis of residuals showed no apparent departures from basic ANOVA assumptions, i.e., normally distributed errors with constant variance and independent of one another. Furthermore, lack of fit was not significant at a ¼ 5%, indicating that the model was a good approximation to the mean structure in the region of experimentation. The experimental and calculated k values were in fairly good agreement, with an average percent difference of about 6% (Fig. S2 in Supplementary Material). In addition, the k values obtained for two factor level combinations characterized by all factors at the same levels except for time were reasonably close, further supporting the fact that the reaction time was not a significant factor. Table 2 reports the reduced model coefficients together with their standard errors, t statistics and corresponding p-values. We note that CHL degradation was mainly affected by UV light intensity, through a linear term, and to a lesser extent by initial CHL and H2O2 concentrations, through linear and quadratic terms. A weak interaction between the latter two factors was also present.

Coefficient

Effect

Estimate

SE

t

p

b0 b1 b2 b3 b12 b11 b22

e ca0 ch0 I t ca0  ca0 ch0  ch0

2.04892 0.25879 0.06806 0.70735 0.07574 0.10426 0.23355

0.03586 0.02536 0.02536 0.02536 0.03106 0.02329 0.02329

56.856 10.206 2.684 27.895 2.439 4.476 10.027

0.000 0.000 0.013 0.000 0.023 0.000 0.000

Fig. 4 shows the response-surface plots of k as a function of two independent variables varying in the factorial part of the experimental space (between 1 and þ1), with the other independent variable set at its central level. Examination of the plot of k as a function of initial CHL and H2O2 concentrations (Fig. 4a) reveals a negative effect of CHL concentration, with a downward curvature, and that there is a region of H2O2 concentration in which k is maximum. Since ch0 does not interact with I, it is possible to determine, for each value of ca0 and independently of the value of I, a ch0 concentration that results in the maximum k. For ca0 varying between 40 and 80 mg L1, the optimum H2O2 concentration ranged from 34.1 to 39.3 mM, such a low variation being attributed to the weak interaction between ch0 and ca0. The large positive effect of UV light intensity on k is apparent from Figs. 4b and c, where k is plotted as a function of I and ca0 or ch0, respectively. Because I is not involved in any interaction with the other factors, the observations reported above about the effect of ca0 and ch0 are consistent also with these plots. The effects of the different factors on the kinetics of CHL removal can be explained by considering the following reaction scheme for the 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 UV photons and the hydroxyl radicals generated from the photolysis of H2O2 to give the final products. The positive effect of UV light intensity can be ascribed to the increased production of HO (Eq. (7)), the main species responsible for 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 the absorption of UV radiation by the antibiotic molecule. At 254 nm, the wavelength of maximum emission of the UV lamp, the measured molar extinction coefficient of CHL was 4.45  103 Me 1 cm1, i.e., about a half of that at the peak maximum (see Fig. 1). Accordingly, higher CHL concentrations cause an increase in the fraction of UV photons absorbed and a decrease in the concentration of HO in the liquid. Another possible, but perhaps less significant cause of inhibition, could be the formation of reaction intermediates interfering with the degradation pathway (So et al., 2002). The existence of an optimal concentration for hydrogen peroxide was also observed in other studies on the UV/H2O2 process (Morrison et al., 1996; Einschlag et al., 2002; Wu et al., 2007) and explained in terms of two opposing effects: the increased production of HO radicals at higher H2O2 concentrations,

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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 concentration (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 central values.

according to Eq. (7), and their quenching, according to the following reactions: H2O2 þ HO / H2O þ HO2

(9)

2 HO2 / H2O2 þ O2

(10)

HO2 þ HO / H2O þ O2

(11)

prediction intervals for future observations. The predicted rate constants were in good agreement with the experimental values and the majority of data points fell within the 95%-prediction band (Table S2 and Fig. S3 in Supplementary Material), further supporting the model presented above. 3.3. TOC reduction

The combination of the two contributions can lead to the appearance of a maximum in the dependence of the degradation rate on initial H2O2 concentration. However, evidence indicates that the optimal H2O2 concentration is highly dependent on the characteristics of the degrading compound and the applied conditions. For this reason, the effects of H2O2 concentration should be experimentally checked and analysed for each particular case. The canonical analysis of the response surface, carried out by using a model including all coefficients except the ones related to the 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 much smaller than the others, the remaining ones are both negative and the critical point falls outside the region of experimentation, the surface can be classified as a rising ridge. To validate the developed model, additional experiments were carried out in the central composite design domain (xi varying between 1 and þ1). By using the reduced polynomial model together with the parameter estimates listed in Table 2, it was possible to calculate the predicted values of k and the 95%-

Fig. 5 shows the TOC reduction for CHL solutions subjected to UV/H2O2 treatment under conditions close to optimal for CHL degradation. In particular, the initial H2O2 concentration, the UV light intensity and the reaction time were set at their center-point values (ch0 ¼ 35 mM, I ¼ 600 mW cm2 and t ¼ 50 min) and four initial antibiotic concentrations (20, 60, 100 and 150 mg L1) were examined. As is known, TOC values are related to the concentration of total organic compounds in the solution and the decrease of TOC reflects the degree of mineralization. Under the tested conditions, TOC removal decreased from about 50% (at ca0 ¼ 20 mg L1) to 41.5% (at ca0 ¼ 150 mg L1). Similar results are reported by LopezPenalver et al. (2010), who studied the degradation of tetracyclines in aqueous solution by the UV/H2O2 process and found a mean mineralization ratio of 47.2% for antibiotic concentrations ranging from 10 to 100 mg L1. The above results indicate that CHL mineralization is slower than degradation and that degradation products or intermediates still contribute to the TOC of the solution. Of course, the crucial point is to assess whether these compounds also exhibit antibacterial activity.

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

Untreated

TOC (mg L-1)

50

UV/H2O2 treated

40 30

20 10 0 20

60

100

150

ca0 (mg L-1) Fig. 5. TOC removal in aqueous solutions at different initial CHL concentration (ca0). Treatment conditions: ch0 ¼ 35 mM, I ¼ 600 mW cm2, t ¼ 50 min.

3.4. Antibacterial activity of the treated CHL solutions To assess the effectiveness of the process in terms of antibiotic activity removal, the residual activity of UV/H2O2-treated CHL solutions against E. coli and S. aureus was determined. These pathogens are highly sensitive to CHL (Saxena and Gomber, 2008) and were selected as representative of Gram-negative and Grampositive bacteria, respectively. Activity measurements were carried out on CHL solutions submitted to UV/H2O2 treatment under conditions close to optimal for antibiotic degradation. In these experiments the initial H2O2 concentration, the UV light intensity and the reaction time were set at their center-point values (ch0 ¼ 35 mM, I ¼ 600 mW cm2 and t ¼ 50 min) and four initial antibiotic concentrations (20, 60, 100 and 150 mg L1) were examined. Untreated antibiotic solutions at the same CHL concentrations were also assayed. Typical results of

agar-diffusion tests are presented in Fig. 6. The extent of the inhibition zone around each well can be regarded as an inverse measure of the antibacterial activity of the solution in the well. Plotting the diameters of the inhibition zones against the initial antibiotic concentrations gave the results reported in Fig. 7. For the untreated samples, a dose-dependent response was observed, with inhibition zone diameters ranging from 10 to 23.5 mm (E. coli) and 9.5e 24 mm (S. aureus). No residual antibacterial activity was detected against both pathogens in CHL solutions subjected to UV/H2O2 treatment. This clearly suggests that, under the applied process conditions, the antibacterial activity of CHL or its degradation products was completely lost, even at CHL concentrations higher than those used in the response surface study. It may be interesting to compare these results with those obtained in the few studies examining the drug activity reduction of CHL solutions after treatment by other methods. Chatzitakis et al. (2008) investigated the photocatalytic degradation of CHL by TiO2 particles and found that a reaction time of 90 min was needed to completely eliminate the antibiotic activity of CHL at 50 mg L1 against E. coli. In the study of Liang et al. (2013) an innovative electrochemical system based on a microbial biocathode was used to degrade CHL and compared with an abiotic cathode. The authors report a removal efficiency at 24 h of 96% for the bioelectrochemical

30

E. coli

Untreated

25

UV/H2O2 treated

D (mm)

60

307

20 15 10 5 0

20

60

100

150

ca0 (mg L-1) 30

S. aureus 25

Untreated

D (mm)

UV/H2O2 treated 20 15 10

5 0 20

60

100

150

ca0 (mg L-1) Fig. 6. e Inhibition zones on agar plate with S. aureus (ATCC 25923) produced by untreated CHL at 150 mg L1 and CHL treated with H2O2, UV (hv) or UV/H2O2 (hv þ H2O2). Treatment conditions: ca0 ¼ 150 mg L1, ch0 ¼ 35 mM, I ¼ 600 mW cm2, t ¼ 50 min.

Fig. 7. e Antibiotic activity of untreated and UV/H2O2-treated CHL solutions against E. coli (ATCC 25922) and S. aureus (ATCC 25923). D is the diameter of the inhibition zone and ca0 the initial CHL concentration. The dashed lines indicate the size of the agar well. Treatment conditions: ch0 ¼ 35 mM, I ¼ 600 mW cm2, t ¼ 50 min.

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

system and 73% for the abiotic control. They also found that very different transformation products were formed in the two systems and that those produced in the bioelectrochemical system lacked or had low antibacterial activity against the tested strains (E. coli and Staphylococcus oneidensis), while the intermediates of CHL reduction in the abiotic system were still toxic to microorganisms. Turning to our results, we showed that the combined application of UV and H2O2, under appropriate conditions, allows complete drug activity removal at least up to initial CHL concentrations as high as 100 mg L1, which demonstrates the efficacy of the proposed method. 4. Conclusions In recent years, the spread of antibiotic resistance and its potential impact on human health have caused a growing concern worldwide. Among the factors contributing to this phenomenon, the extensive and unregulated use of antibiotics in aquaculture seems to play an important role, also because of the difficulty of removing these pollutants by conventional wastewater treatments. The results of this study demonstrate that CHL, a nitroaromatic antibiotic widely used in aquaculture facilities, can be effectively degraded by the UV/H2O2 treatment. We have also shown that the antibacterial activity of CHL solutions treated under optimal process conditions was completely lost, even at antibiotic concentrations higher than those commonly found in wastewaters. From a methodological viewpoint, our results suggest that a rigorous approach based on the response surface analysis and the use of a transformed response variable can improve the interpretation and modeling of experimental results. In this regard, the simplified model developed can provide useful indications on the optimum set of operating conditions to be used in the practice and an easy evaluation of the influence of process parameters on antibiotic degradation. Acknowledgement The authors gratefully thank Dr. Stefano Denetto for his valuable assistance 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 the evolution of antibiotic resistance through horizontal gene transfer. FEMS Microbiol. Rev. 35, 756e767. Badawy, M.I., Wahaab, R.A., El-Kalliny, A.S., 2009. Fenton-biological treatment processes 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 chloramphenicol. 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 efficiency of photodegradation of nitroaromatics applying the UV/H2O2 technique. Environ. Sci. Technol. 36, 3936e3944. Elmolla, E.S., Chaudhuri, M., 2010. Comparison of different advanced oxidation processes for treatment of antibiotic aqueous solution. Desalination 256, 43e47.

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