Remediation of Antiseptic Components in Wastewater by Photocatalysis Using TiO2 Nanoparticles

Remediation of Antiseptic Components in Wastewater byPhotocatalysis Using TiO2 NanoparticlesRanjana Das,† Santanu Sarkar,† Sudip Chakraborty,† Heechul Choi,‡ and Chiranjib Bhattacharjee*,†

†Chemical Engineering Department, Jadavpur University, Kolkata, 700032, India‡School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, 1Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea

ABSTRACT: Environmental awareness in both the public and regulatory sectors has necessitated proper treatment of medicinalcomponents-rich pharmaceutical effluents. Even the presence of trace antiseptic may cause adverse health effects includingdevelopment of “product resistant microbes” in the aquatic environment. The present study involves photomineralization ofchlorhexidine, which belongs to the class of antiseptic drug components. This study details investigations on photocatalyticdegradation of chlorhexidine in a slurry batch reactor using titanium dioxide photocatalyst. Emphases were given to study theeffects of operating parameters on the degradation behavior of the targeted compound and characterization of degraded products.About 68.14% removal of chlorhexidine digluconate (CHD) was found after 1 h at 25 °C with a substrate-to-catalyst ratio of2.5:1 under UV intensity of 50 μW·cm−2 at pH 10.5. Though the product profile illustrates several degraded products,toxicological analysis on Bacillus subtilis exhibited no inhibition zone, suggesting the eco-friendly nature of the degraded products.

1. INTRODUCTION

Pharmaceuticals (PhAs) constitute a large group of medicinalcompounds which have indispensable use worldwide for humanand veterinary applications. Though the quantity of thesepharmaceuticals in the aquatic environment is trace, for aquaticand terrestrial organisms, their continuous input may impose apotential risk and they are considered to be an emergingenvironmental concern. The primary route of pharmaceuticalsinto the environment is through their introduction into soil,surface waters, and groundwater after being excreted fromhumans or animals via urine or feces through the sewagesystem.1,2 In addition to metabolic excretion, disposal of PhAsfrom medical treatment organizations and common householdsalso contributes to their entry into fresh water resources. Usuallypharmaceuticals are designed in such a way that they exhibit aphysiological effect on humans and animals at low concentrationlevels, and they become persistent against biological degrada-tion, which is primarily responsible for their tagging as a“pollutant”.3,4 PhAs released in the environment may imposetoxicity, the extent of which depends on the specific compoundsand virtually on the biological structure. In addition to toxiceffects, some classes of the pharmaceuticals (antibiotics) maycause long-term and irreversible changes to the microorganism,making them resistant to biodegradation even at lowconcentration. Several studies have exemplified the directentering of PhAs in the sewage treatment process5−7 and theiradverse effect on aquatic life.8−10

With a broad spectrum of activity, chlorhexidine (CHD)compositions have been in use throughout the world for morethan 50 years as an antiseptic in various medicinal applications,including as the active component in toothpaste (0.1−0.2%);mouthwash (0.6−1%); veterinary application (1.5−2%);cosmetic preservatives (1−1.5%); and hand wash and scrubs(15−20%).11 It has been used as an antibacterial agent incommercial ophthalmic products as a replacement for

thimerosal (which is a mercury-containing bacteriostat). CHDsurpasses that of the similar preparations containing providone-iodine, triclosan, hexachlorophene.12 Studies on CHD show itstoxic effect on nerve tissue, and therefore its contact with brainand meninges is restricted. Intravenous administration exhibitedgreater toxicity because of the stromalytic effect on red bloodcells resulting from its surfactant activity. Systemic oraladministration of CHD formulations on rats have shown thatreceiving 50−200 mg/kg CHD in drinking water for 90 daysproduces evidence of histiocytosis of the mesenteric lymphnodes. In the bone marrow test of the mouse with dermalapplication of 0.2 mL of 0.5% CHD in distilled water twice dailyfor 28days (50 mg/kg of body mass), an increase inchromosomal aberrations was noted.13 It is also reported asenvironmentally hazardous, toxic to aquatic organisms andsewage microorganisms, and responsible for long-term adverseeffects in the aquatic environment with an acute oral toxicity(LD50) of 6.3g/kg (mouse, oral); 2g/kg (human, oral); 3 g/kg(rat, oral).11

Increasing human and livestock populations have raised theconcern of safe drinking water and nonpotable water supply. Inthe present context of potable water supply, the development ofadvanced, low-cost, and high efficiency water treatmenttechnologies are potentially desirable. To attain the stringentinternational environmental standards, technological develop-ments for removal of pharmaceuticals has become indispensableto the pharmaceutical industries. Conventional treatmenttechnologies like adsorption and coagulation only concentratesthe pollutants using phase change principle, but does notcompletely eliminate or destroy the components.14 To over-

Received: November 11, 2013Revised: January 12, 2014Accepted: January 29, 2014Published: January 29, 2014

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come the limitations of conventional wastewater treatmentmethodologies and effective utilization of available economicresources, various advanced treatment technologies have beenadopted, optimized, and have been applied.15−17 Amongmentioned technologies, membrane filtration, advanced oxida-tion processes (AOPs), and UV irradiation have been provenbeneficial in removal of a wide range of challengingcontaminants. Intensive studies on photocatalysis using nano-TiO2 have already been published relating the treatment of thepharmaceutical components in wastewater,18−30 but no studieshave yet been reported on the photocatalytic degradation ofCHD using nano-TiO2. The outstanding properties of nano-TiO2 such as high surface area, nontoxicity, photochemicalstability, light absorption, charge transport, superior excited-state lifetimes and favorable combination of electronic structuremakes it the “photocatalyst of choice”.31 Considering theadverse effects of CHD on the environment, and the highefficiency of the photocatalytic degradation process, the presentstudy aims at the photocatalytic treatment of the pharmaceuticalwaste to obtain an eco-friendly discharge alleviating the majorenvironmental concerns. The novelty of the study is in thechoice of the substrate as chlorhexidinde digluconate since nopublication still available attempts the use of this particular classof antiseptic component. The process efficiency has beenevaluated by varying the process controlling parameters using anantiseptic drug chlorhexidine digluconate (CHD) as a targetcomponent. This study also involves characterization of theprocess degraded products and assessment of related producttoxicity.

2. EXPERIMENTAL SECTION2.1. Chemicals. Two catalyst systems of titanium dioxide

photocatalyst nanopowder (pure anatase, 637254) of particlesize <25 nm and specific surface area 45−55 m2·g−1 (Cat1) andAeroxideP25 (mixture of rutile and anatase, 718467) of particlesize 21 nm with surface area (BET) 35−65 m2·g−1 (Cat2) fromSigma-Aldrich, were used as received. Chlorhexidine digluco-nate solution (20% w/v, from Sigma-Aldrich) was used toprepare the simulated solutions to standardize the analyticalprocess. All experiments were carried out with ultrapure waterfrom Arium Pro VF (Sartorius Stedim Biotech) of 18.2 MΩcmresistivity. Chloramphenicol (TC 204) from HiMedia was usedas standard antibiotic. Bacterium Bacillus subtilis (NCIM No.2655) was purchased from National Collection of IndustrialMicroorganisms (NCIM), Pune, India, to assess the toxicitypotential of CHD degraded products. All other chemicals, unlessmentioned were purchased from Sigma Aldrich Chemical Co.,USA.2.2. Sample Preparation. Chlorhexidine digluconate of

varying concentration (500 mg·L−1 to 1500 mg·L−1) wasdissolved in the ultrapure water to simulate the syntheticpharmaceutical wastewater. This work also forms an importantbasis for the future studies when, real pharmaceutical effluentsand various other components’ interference are attempted. Inthe current study, each experimental run was monitored andanalyzed in terms of residual chlorhexidine digluconateconcentration with reaction time.2.3. Setup of the TiO2 Photocatalytic Reactor System.

A batch slurry photoreactor (SP) (Figure 1) was used fortreatment of synthetic wastewater. The reactor comprises achamber (17 cm × 17 cm × 17 cm) with UVA lamp (10 W,Concept International, Kolkata, India) from the top of thereactor and a magnetic stirrer (Remi, India) to ascertain the

proper mixing of the reaction solution and even distribution ofUV irradiation to the catalyst system. The distance of samplesolution (upper surface) to the radiation source maintainedduring the photodegradation process was 7 cm, and the reactionsolution thickness was 3 cm. The UV light intensity wascontrolled with an external controller, and inside intensity wasmeasured using a solar UVA meter (TM 208, Tenmars,Taiwan).

2.4. Adsorption Study Chlorhexidine Digluconate onTiO2 Nanoparticle. Since adsorption of substrate (CHD) onthe catalyst systems restrain the extent of photodegradation,studies on the adsorption behavior of CHD onto a catalystsystem is quite relevant in the present context. To study theadsorption behavior of CHD on a catalyst system, a batch studywas conducted under dark (25 °C) for a time period of 1 h witha variation of catalyst loading weight and CHD concentration.

2.5. Batch Study of the Photocatalytic Degradation ofChlorhexidine Digluconate. To study the process ofphotocatalytic degradation of chlorhexidine digluconate, abatch study (100 mL) was conducted with varying concen-tration of the substrate (CHD) and the catalyst. For overallunderstanding of the process, the controlling parameters such asUV intensity and the pH of the reaction mixture were alsovaried. The batch studies were conducted for 1 h (at 400 rpm)with sample collection at 5 min interval. To ascertain thecomplete catalyst removal, an aliquot was centrifuged (4 °C,11000× g, 5 min), and supernatant was collected for furtheranalyses.After collection of the supernatant the remaining portion was

diluted with fresh ultrapure water (100 mL), and sedimentationof the catalyst was induced by adjusting the pH of the mediumto the isoelectric point (IEP) of the catalyst. Though both waterreuse and catalyst reuse is possible, in the present case only thecatalyst was reused, as with water reuse the degradation productsremain in the system, which probably will impart some productinhibition on the overall photodegradation process.

2.6. Analytical Procedures. Initially, chlorhexidine digluc-onate concentration of the reaction mixtures was determinedusing spectrophotometry at 275 nm.32 Quantification was donewith respect to the standard curve of pure chlorhexidinedigluconate dissolved in deionized water within the concen-

Figure 1. Operational set up of the batch slurry reactor forphotocatalytic degradation of CHD.

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tration range of 0.005−0.02 mg/mL with a correlationcoefficient (R2) of 0.99. To maintain the accuracy of themeasurements and to exemplify the product pattern, repeatconcentration measurements were done in a RP-HPLC system(Cyber Lab, Millbury, USA) with ‘Zorbax SB Phenyl column,(4.8 × 250 mm, 5 μm, Agilent, USA) at 25 °C with an eluentflow rate of 0.5 mL·min−1 and UV absorbance at 275 nm.33 Themobile phase used was methanol 60% and 40% water, and theinjection volume was 20 μL. Degraded product concentrationswere measured from the peak areas as obtained in the HPLCchromatogram (Figure 2). The chromatogram shows the

presence of the distinct peaks of the degraded products alongwith the unconverted chlorhexidine digluconate, which alsosuggests that the elution condition is appropriate for theseparation of the degraded products from chlorhexidinedigluconate.2.7. Characterization and Toxicological Analysis of

Chlorhexidine Digluconate Degraded Product. Tocharacterize the degraded products of CHD, mass spectraanalysis was done in Quadrapole-TOF Micromass Spectrometer(Waters Co., USA). Electrospray ionization (ESI) massspectrometry was used as ionization mode. Using ESI as anion source, the compound encompasses a proton, increasing themolecular mass by +1 so, the spectra was obtained in positiveion mode. The “time of flight” (TOF) detector was used with aflight tube length of 1.5 m. Table 1 presents the ion sourceparameters maintained during the ionization process. In thepresent study, the toxicity potential of CHD degraded productsis tested on Bacillus subtilis; here, a subculture of Bacillus subtilis,grown in nutrient broth (5 g peptone; 3 g beef extract; 3 g yeastextract, and 5 g of NaCl in 1 L of distilled water at pH 7.4). Thetest was performed by autoclaving the Muller Hinton agar

media, along with the petriplates at 121 °C at 66.68 Pa for 20min. The agar media was allowed to cool for some time suchthat the temperature might not be too high to kill the bacterialcells. About 5 mL of Bacillus subtilis culture broth suspensionwas poured into the Muller Hinton agar media. The bacterialculture suspension along with the media was mixed well suchthat a confluent lawn of growth would result on incubation.Whatman filter paper discs (5 mm diameter), dipped inchloramphenicol antibiotic solution (A), sample (S), and pureCHD (C), were placed on the agar surface. Plates were thenincubated for 20−24 h at 37 °C prior to observation of theinhibition zone.

3. RESULTS AND DISCUSSION

3.1. Adsorption Behavior of CHD on to the CatalystSystem. Extent of the adsorption for substrate molecules on tothe catalyst surface controls the rate of the photodegradationprocess. Adsorption behavior changes with the nature of thecatalyst system, respective surface properties, and availability ofthe substrate on to catalyst surface. Results show about 6.8%more adsorption of CHD for AeroxideP25 (Cat2) as comparedto pure anatase (Cat1) after the surface area is normalized, whichis probably due to the contribution of higher negative charges ofthe rutile phase compared to pure anatase in AeroxideP25.34 Inthe mentioned range of experimental process conditions forphotodegradation, maximum adsorption of CHD was observed,about 29.8% for AeroxideP25(Cat2) and 27.8 % for pure anatase(Cat1) at pH 10.5.

3.2. Effect of Substrate to Catalyst Ratio and SolutionpH on the CHD Degradation Behavior. The photo-mineralization of organics by semiconductor photocatalysts isan area of intensive research, as ideally the end products of theseprocesses should be carbon dioxide, water, and inorganicmineral salts, which have minimum environmental impact. Inthis study, the photocatalytic treatment of the pharmaceuticalCHD was carried out at a higher concentration level (∼1500mg·L−1) using the photocatalyst TiO2 in the presence of UVirradiation. Figure 3 illustrates the effect of the substrate tocatalyst ratio (S/C value) on the degradation behavior of theCHD for the Cat1 system. The figure shows the removal percent

Figure 2. Chromatographic pattern of chlorhexidine digluconatedegraded products.

Table 1. Ion Source Parameters Maintained during MassSpectroscopic Analysis

parameters value

capillary voltage (V) 3000sample cone (V) 45extraction cone (V) 1.0desolving temperature (°C) 125source temperature (°C) 80syringe flow (μL) 10

Figure 3. Effect of contaminating substrate to catalyst ratio (S/C) andsolution pH on percent removal of CHD.

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of CHD at different medium pH under UV irradiation of 50μW·cm−2. A decreasing trend of the removal efficiency withincreasing S/C value could be observed from the figure. Aregression analysis was done to correlate the percent removal ofCHD with the substrate to catalyst ratio (S/C value) whichhelps theoretically to predict the percent removal of CHD underan optimized set of reaction parameters for a chosen S/C value.In the pH range under investigation, the removal percent ofCHD followed a second-order fitting with R2 values rangingfrom 0.95 to 0.98.Physico-chemical properties of organic compounds in

wastewater differ greatly for several parameters, particularlysolubility in water and hydrophobicity, depending on the pH ofthe medium. On the basis of the associated pKa value of theorganic compounds, at pH below its pKa value the compoundexists as neutral state, whereas above this pKa value it attains anegative charge. This variation in molecular structuresignificantly affects the photocatalytic behavior of the targetedcompound. Thus, the pH of the reaction medium plays a crucialrole in controlling the efficiency of the process. Variation of thepH also determines the surface charge of the photocatalyst andthe size of aggregates it forms,35,36 and controls the electrostaticinteraction between a catalyst surface, target molecule, chargedradicals formed during photocatalytic oxidation, and solventmolecules. Stability of the organic compounds is also reported tobe influenced by the phenomenon of “protonation” and“deprotonation” of the molecule that happens in actual factwith changing pH of the reaction medium.37 Chlorhexidine(CHD) is a symmetrical cationic molecule containing two 4-chlorophenyl rings and two biguanide groups connected by acentral hexamethylene chain. It is a strongly basic dicationiccompound with pKa values of 10.3 and 2.2.33 In this study, thedegradation behavior was found to diminish with increase in pHwithin the range of 4−11. Removal percent was observed higherat alkaline pH region (pH 10.5) as compared to the acidic region(pH 4.0) (Figure 3). This observation could probably beexplained based on the unfavorable electrostatic repulsionsbetween the positively charged surface of the catalyst(TiOH2

+)37 and substrate CHD (cataionic), which lowers theefficiency of degradation at acidic pH. Moreover, thedegradation rate may also decline at lower pH values due tothe neutral state of CHD. Above pH 6.25 (the point of zerocharge for anataseTiO2),

38 the negative charge at the surface ofthe TiO2 increases with increasing medium pH. It is expectedthat at alkaline pH, the generation of OH• is higher due to thepresence of more available hydroxyl ions on the TiO2 surfaces.Thus, the extent of degradation is possibly enhanced at high pHmedium. The observed behavior can be attributed to thehydroxylation of catalyst surface due to the abundance of OH−

ions in alkaline conditions. Similar behavior was also reported byseveral groups of authors on various organic pollutants.39

3.3. Effect of the Catalyst Loading on the DegradationBehavior of CHD. In photocatalytic processes feasibility alsodepends on the behavior of the catalyst, specifically on thephysicochemical properties of the catalyst. In this study titaniumdioxide (pure anatase and AeroxideP25) was used as aphotocatalyst to treat CHD, considering respective broadapplication profiles and excellent performance. Results fromthis study showed an initial positive correlation of substratedegradation with catalyst loading for both catalyst systems,followed by a decline for further enhancement in catalystloading with fixed substrate (CHD) loading. At the lowestcatalyst loading of 100 mg·L−1, 31.42% removal was achieved for

an initial substrate (CHD) concentration of 500 mg·L−1 withinthe reaction period of 1 h at solution pH 4 (Figure 4) for Cat1

and 32.9% for Cat2. A further increase in the catalyst (Cat1)loading, under similar process conditions, exhibited a higherpercent removal value of 68.2% and 47.4% for catalyst loadingsof 200 mg·L−1 and 400 mg·L−1, respectively). The initialincrease is possibly due to the excess in the availability of activesites on the catalyst surface which generated excess of thereactive radicals for CHD degradation. Though it is expectedthat with increasing catalyst loading the active sites or effectivesurface area per unit volume of the reaction mixture increases,still in our observation the percent removal of CHD presents adecreasing trend at higher dosing value. This may be explainedin terms of increased “agglomeration” and “turbidity” whichreduces the “light penetration”. The tendency of agglomeration(particle−particle interaction) also increases at high solidsconcentration, resulting in a reduction in active catalyst surfacearea for light absorption and hence results in a fall in percentdegradation. In the reaction time scale, maximum degradationwas achieved at a faster rate for catalyst loading of 200 mg·L−1

(68.2% at 1 h), whereas for other loading conditions the percentremoval values obtained were 63.8% and 47.4% for 100 mg·L−1

and 400 mg·L−1, respectively. The inverse correlation betweenthe CHD degradation rate and catalyst loading, possibly is dueto the light scattering and screening effects. A similar trend wasobserved for the entire pH range of observation. In the neutralregion (pH 6.7), the rate of degradation was observed to bemuch less under similar process condition possibly due to theseparation of catalyst from the reaction mixture being close tothe point of zero charge. The effect of catalyst loading withvarying CHD concentration has been illustrated in Figure 5. Forvarying CHD concentration values, similar behavior wasobserved in the lower concentration region (500−1000 mg·L−1), but at high concentration region (∼CHD 1500 mg·L−1)the decreasing trend was prominent possibly due to the lack ofsufficient active radicals to react with the enhanced number oftarget molecules, which suggests that the observed loading rangeof catalyst needs to be widened to achieve more active sites andbetter removal efficiency for higher CHD loading ranges.

Figure 4. The decay performance of CHD as a function of catalystloading at different pH levels.

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3.4. Effect of the Initial Substrate Concentration. Aproficient photocatalytic oxidation process necessitates theinvestigation of the dependence of the photocatalytic degrada-tion rate on the initial CHD concentration (C0), which appearsto be the primary process controlling factor. The effect of theinitial substrate concentration (C0) and the nature of thetargeted compound also affect the photodegradation behavior toa great extent. Within the catalyst loading range of 100−400(mg·L−1), CHD removal behavior is shown in Figure 6 for

varying C0 values. The percent removal of CHD (after 1 hreaction period) decreased from 31.42% to 14.39% onincreasing the C0 from 500 to 1500 mg·L−1 at constant pH 4and catalyst loading of 100 mg·L−1. A similar trend was observedfor other catalyst loading values. In the other pH regions, theobservation was similar highlighting the fact that the C0predominates over the pH value of the medium. Similar trendswere observed for the photocatalytic degradation of triclopyrand daminozid by Qamar et al. (2006)40 and Parida et al.

(2006).41 The behavior may be elucidated in terms of increasingpopulation and probability of the target molecules to getadsorbed on the surface of the photocatalyst, hence involvingthe generation of more reactive species (OH− and O•

2) for thedegradation of targeted compound. Since, the formation ofreactive species remains constant for a given UV intensity andcatalyst amount, the available radicals become inadequate forCHD degradation at higher concentrations. Consequently therate of percent removal decreases as the CHD concentrationincreases.35 Another probability is the generation of moreintermediates; those consequently adsorb on the catalyst surfaceand cause partial deactivation of active sites as well as a slowerdiffusion rate onto the photocatalyst surface.The inset of the Figure 6 shows CHD removal on a reaction

time scale under varying initial CHD concentrations. It showsthat maximum removal can be achieved within the time range of5−10 min, after that the rate of removal shows insignificantchanges with further progress of the reaction time period up to60 min.

3.5. Effect of UV Intensity. At a given wavelength, UVintensity determines the extent of radiation absorbed by thesemiconductor catalyst. As reported in research publications, therate of initiation of photocatalysis, that is, the electron−holeformation in the photochemical reaction is strongly dependenton the UV intensity.42 The light intensity distribution within thereactor also was reported to invariably determine the overallsubstrate conversion and degradation efficiency.43 The effect ofUV intensity on the CHD removal percentage has beenhighlighted in Figure 7 under constant S/C value and medium

pH. In this study, CHD removal efficiency was found to increasewith increasing intensity from 50 to 80 μW·cm−2 which is likelybecause of enhanced formation of an electron−hole, facilitatingthe photocatalytic degradation. Under such a condition,electron−hole recombination is insignificant.44 (The initialincrease in degradation thus probably results from thegeneration of more electron−hole pairs, which ultimatelyproduce the reactive species.) The decreasing value of theCHD removal at 125 μW·cm−2 could probably be explained onthe basis of an increased rate of hole−electron recombinationrate under high UV-radiant flux.

Figure 5. Effect of catalyst loading on CHD removal at differentmedium pH and varying initial CHD concentration.

Figure 6. Effect of initial CHD concentration on overall CHD removalpercent and rate of removal (inset).

Figure 7. Effect of UV intensity on the removal efficiency of CHD atfixed pH and S/C value.

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3.6. Comparison of the Performance of AnataseTitanium Dioxide with AeroxideP25 Catalyst System.With an aim to compare the pure anatase titania (Cat1) withstandard photocatalyst AeroxideP25 (Cat2), a repeat study wasconducted at the condition of maximum degradation of CHD.Considering the accessibility and superior stability of the anatasephase, this study intended to assess the functionality of the pureanatase titania as a photocatalyst. For Cat2 the extent ofphotodegradation was found about 4.7−5% higher than for Cat1at pH 6.7 and above (Figure 8); in the acidic region (pH 4) the

extent of degradation was found to be much less compared tothat of Cat1 due to the presence of more positive charges of therutile phase in Cat2

34 (Barbour et al., 2007) which imparts anelectrostatic repulsive interaction between the cationic substrateand the catalyst surface. The overall removal percent of CHD atpH 4, S/C ratio of 2.5:1, and under UV intensity of 50 μW·cm−2

was 3.2% less than that of Cat1.3.7. Reuse of the Catalyst System. From the economical

point of view and to lower the environmental load, reuse of thecatalyst system is essential. In the context of heterogeneousphotocatalysis, several groups of authors have attempted toreuse the TiO2 catalyst system to enhance the process feasibilityand efficiency.45−48 The reuse of a catalyst system depends onthe substrate, specifically, on the nature of degraded productswhich limits its reutilization efficiency (Barbeni et al., 1987); andhence, necessitates typical studies for each substrate to beinvestigated. In present context, under UV irradiation of 50 μW·cm−2, four times catalyst reuse was achieved without significantvariation in photodegradation efficiency, after that the efficiencywas found diminishing (Figure 9) with increasing number ofrecycling which may be due to the inhibition imposed by thedegraded products that partially block the active sites and hencelowers the extent of active radicals generation and processefficiency. Cat2 shows comparatively rapid drop in recyclingefficiency than Cat1 which is possibly due to the availability ofmore active surface area. That no significant changes wereobserved in catalyst recycling efficiency with further increasingUV intensity value may be due to a profound product inhibitioneffect.

3.8. Characterization and Assay of the Toxicity of CHDDegraded Products. The choice behind the photocatalyticdegradation process over other conventional water treatmentprocesses lies on the ultimate fate of the pollutant molecules.Safe discharge from wastewater requires meeting a standard anddetailed toxicological reports. In this context, the degradedproduct profile was investigated by mass spectroscopic analysis.Figure 10 panels a and b illustrate the mass spectra analysis ofthe pure CHD and the degraded products. Electrosprayionization mass spectrometry for pure chlorhexidine isillustrated in Figure 10a. Spectra reflects the molecular ionsfor chlorhexidine (CHD) at 505 and 506, two peaks reflect thetwo Cl atoms present in the structure which have isotopes at 35(75% total abundance) and 37 (25% total abundance). Peakswith lower masses represent fragments of the molecule as itbreaks apart in the ion source, and are compound characteristic.The few extra small peaks (noise) reflect contamination. Spectrapresented in Figure 10b, show seven peaks with majorcontribution of a component of mass 234.95 g·mole−1.Identification of the masses is beyond the scope of this studyand hope to be reported in forthcoming research articles. Massspectral analysis confirms the incomplete degradation, and thusnecessitates the investigation of the toxicological parameters ofthe degraded products. The antimicrobial susceptibility study ofthe degraded products was done on Bacillus subtilis, known to bethe most prevalent micro-organism residing in human oralcavities.49 The method used for this purpose is commonlyknown as the agar disc-diffusion method.50

The plates were observed for their respective inhibition zones.The zones of growth inhibition around each of the discs weremeasured to the nearest millimeters. The diameter of the zone isrelated to the susceptibility of Bacillus subtilis and to thediffusion rates of samples and chloramphenicol through the agarmedium. The zone of inhibition around the pure sample ofCHD was found to be about 30 mm (diameter); whereas noinhibition zone was observed around the degraded productseven at a high concentration of 500 mg·L−1. For thechloramphenicol antibiotic, the inhibition zone was measuredto be 60 mm (diameter). The photographs of the petriplates areshown in Figure 11.

Figure 8. Comparison of the performance of anatase titanium dioxidewith AeroxideP25.

Figure 9. Effect of catalyst recycling on the overall removal percent ofCHD.

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The pattern of the inhibition zone obtained for all samplesillustrates the superior antibacterial activity of chemicalantiseptic CHD (pure) as compared to chloramphenicol. Thedegraded products even at a significantly high concentrationlevel (500 mg·L−1, as obtained from chromatogram) showed noinhibition zone, which may be due to no or insignificant toxicityof the related compounds. From the observations it can beforeseen that the products obtained after photocatalyticdegradation of CHD, upon release in the environment, aremost unlikely to cause any toxic effect. Since this preliminarytest is performed only with microorganisms, without preclinical

analysis it is not justified to assay the effectiveness of thisantiseptic. The normal microflora of the oral cavity of higherorganisms contains Bacillus sp., therefore susceptibility ofBacillus subtilis to the degraded products of CHD is tested topredict the nontoxicity of the photocatalyzed products on beingreleased in the environment.

4. CONCLUSION

This study highlights the feasible application of a TiO2

photocatalyst-based process for treatment of a pharmaceuticalcompound CHD, in pharmaceutical wastewater in a batch slurry

Figure 10. (a) TOF analysis and mass spectra of pure chlorhexidine digluconate. (b)TOF analysis and mass spectra of the degraded products underoptimal removal condition.

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photoreactor. The monitoring of the degradation profiles forCHD concentrations revealed that the proposed system iscapable of treating CHD and can reduce the toxicity level of thetreated wastewater being disposed. Under optimum reactionconditions, about 68.2% removal of CHD was achieved after 1 hreaction time. The antimicrobial susceptibility test was alsoperformed to assess the final toxicity level of the reactionproducts, which gave negative response suggesting a safe eco-friendly discharge to the atmosphere. The outcomes from thisstudy indicated that the TiO2-based system has a high potentialto be utilized as a sustainable treatment system forpharmaceutical wastewater containing CHD.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected], [email protected]. Tel.: +91 98364 02118. TeleFax: +91 33 2414 6203.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe work reported in this article is part of an Indo-Koreanproject (vide sanction letter no. INT/Korea/P-11 dated August23, 2011), funded in India by Department of Science &Technology (Government of India). The contribution of DST(India) is gratefully acknowledged.

■ ABBREVIATIONSAOP = advanced oxidation processesCHD = chlorhexidineLD50 = lethal dose 50PhAs = pharmaceuticalsTiO2 = titanium dioxide

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Figure 11. Microbiological susceptibility test of antibiotic (A), CHD(P), and degraded products (S) on Bacillus subtilis.

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