**2. Experimental**

#### **2.1 Water samples, chemicals and solutions**

All chemicals were of reagent grade and were used without further purification. CLP, 99.4%, pestanal quality, was manufactured by Riedel-de Haën; 85% H3PO4 was obtained from Lachema (Neratovice, Czech Republic) and NaOH from ZorkaPharm (Šabac, Serbia). The other chemicals used, such as 30% H2O2, *cc* acetic acid and 96% ethanol, were obtained from Centrohem (Stara Pazova, Serbia), KBrO3, (NH4)2S2O8 and 60% HClO4, from Merck, while 99.8% acetonitrile (ACN) and HPLC gradient grade methanol (MeOH) were products of J. T. Baker and humic acids (HUM) technical, of Fluka. All solutions were made using DDW. The pH of the reaction mixture was adjusted using a dilute aqueous solution of HClO4 or NaOH. Aspirin was purchased from Bayer, doxorubicin (Doxorubicin-Teva) from Pharmachemie B.V. (Haarlem, Netherlands) and gemcitabine (Gemzar) from Lilly France S.A. (Fegersheim, France), fetal calf serum (FCS) from PAA Laboratories GmbH (Pashing, Austria), penicillin and streptomycin from Galenika (Belgrade, Serbia), trypsin from Serva (Heidelberg, Germany), and EDTA, trichloroacetic acid (TCA), mercury(II) chloride from Laphoma (Skopje), and tris(hydroxymethyl)amino methane (TRIS) from Sigma Aldrich.

Wackherr's ''Oxyde de titane standard'' (100% anatase form, surface area 8.5±1.0 m2 g–1, crystallite size 300 nm (Vione et al., 2005) hereafter ''TiO2 Wackherr'', and TiO2 Degussa P25 (75% anatase and 25% rutile form, 50 m2 g–1, about 20 nm, non-porous) were used as photocatalysts.

The tap water sample was taken from the local water supply network (Novi Sad, Serbia). River water, collected from the Danube (Novi Sad, Serbia) in May 2010, was filtered through Whatman filter paper 42 (diameter: 125 mm, pore size: 0.1 μm, ashless) before use. The physicochemical characteristics of the water samples, along with that of DDW are given in Table 1.


Table 1. The physicochemical characteristics of the analysed water types.

#### **2.2 Photodegradation procedures**

166 Herbicides – Properties, Synthesis and Control of Weeds

''Oxyde de titane standard'') are even more efficient than TiO2 Degussa P25 in the photodegradation of phenol (Rossatto et al., 2003; Vione et al., 2005) and herbicides with a

The aim of this work was to study the effect of water type (double distilled (DDW), tap and river water) on the efficiency of TiO2 Wackherr toward photocatalytic degradation of CLP. First of all, the study is concerned with the transformation kinetics and efficiency of photocatalytic degradation of CLP in DDW. The study encompasses the effects of a variety of experimental conditions such as the effect of the type of irradiation, catalyst loading, the initial concentration of CLP, temperature, pH, presence of electron acceptors, and hydroxyl

compared to the most often used TiO2 Degussa P25. An attempt has also been made to identify the reaction intermediates formed during the photo-oxidation process of CLP, using the LC–ESI–MS/MS method. The cell growth activity of CLP alone or in the mixture with its photocatalytic degradation intermediates was evaluated *in vitro* in rat hepatoma and human fetal lung cell line, using colorimetric Sulphorhodamine B assay. Finally, the matrix effect of

All chemicals were of reagent grade and were used without further purification. CLP, 99.4%, pestanal quality, was manufactured by Riedel-de Haën; 85% H3PO4 was obtained from Lachema (Neratovice, Czech Republic) and NaOH from ZorkaPharm (Šabac, Serbia). The other chemicals used, such as 30% H2O2, *cc* acetic acid and 96% ethanol, were obtained from Centrohem (Stara Pazova, Serbia), KBrO3, (NH4)2S2O8 and 60% HClO4, from Merck, while 99.8% acetonitrile (ACN) and HPLC gradient grade methanol (MeOH) were products of J. T. Baker and humic acids (HUM) technical, of Fluka. All solutions were made using DDW. The pH of the reaction mixture was adjusted using a dilute aqueous solution of HClO4 or NaOH. Aspirin was purchased from Bayer, doxorubicin (Doxorubicin-Teva) from Pharmachemie B.V. (Haarlem, Netherlands) and gemcitabine (Gemzar) from Lilly France S.A. (Fegersheim, France), fetal calf serum (FCS) from PAA Laboratories GmbH (Pashing, Austria), penicillin and streptomycin from Galenika (Belgrade, Serbia), trypsin from Serva (Heidelberg, Germany), and EDTA, trichloroacetic acid (TCA), mercury(II) chloride from Laphoma (Skopje), and tris(hydroxymethyl)amino methane (TRIS) from

Wackherr's ''Oxyde de titane standard'' (100% anatase form, surface area 8.5±1.0 m2 g–1, crystallite size 300 nm (Vione et al., 2005) hereafter ''TiO2 Wackherr'', and TiO2 Degussa P25 (75% anatase and 25% rutile form, 50 m2 g–1, about 20 nm, non-porous) were used as

The tap water sample was taken from the local water supply network (Novi Sad, Serbia). River water, collected from the Danube (Novi Sad, Serbia) in May 2010, was filtered through Whatman filter paper 42 (diameter: 125 mm, pore size: 0.1 μm, ashless) before use. The physicochemical characteristics of the water samples, along with that of DDW are given in

river and tap water on photocatalytic removal of CLP was also studied.

**2.1 Water samples, chemicals and solutions** 

OH) scavenger on the photodegradation kinetics in DDW. The results were

pyridine ring (Abramović et al., 2011).

radical (

**2. Experimental** 

Sigma Aldrich.

photocatalysts.

Table 1.

The photocatalytic degradation was carried out in a cell made of Pyrex glass (total volume of ca. 40 mL, liquid layer thickness 35 mm), with a plain window on which the light beam was focused. The cell was equipped with a magnetic stirring bar and a water circulating jacket. A 125 W high-pressure mercury lamp (Philips, HPL-N, emission bands in the UV region at 304, 314, 335 and 366 nm, with maximum emission at 366 nm), together with an appropriate concave mirror, was used as the radiation source. Irradiation in the visible spectral range was performed using a 50 W halogen lamp (Philips) and a 400 nm cut-off filter. The outputs for the mercury and halogen lamps were calculated to be ca. 8.8 × 10–9 Einstein mL–1 min–1 and 1.7 × 10–9 Einstein mL–1 min–1 (potassium ferrioxalate actinometry), respectively. In a typical experiment, and unless otherwise stated, the initial CLP concentrations were 1.0 mM, and the TiO2 Wackherr loading was 2.0 mg mL–1. The total suspension volume was 20 mL. The aqueous suspension of TiO2 Wackherr was sonicated (50 Hz) in the dark for 15 min before illumination, to uniformly disperse the photocatalyst particles and attain adsorption equilibrium. The suspension thus obtained was thermostated and then irradiated at a constant stream of O2 (3.0 mL min–1). During the irradiation, the mixture was stirred at a constant speed. All experiments were performed at the natural pH (~ 3.5), except when studying the influence of the pH on the photocatalytic degradation of the substrate. In the investigation of the influence of electron acceptors, apart from constant streaming of O2, H2O2, KBrO3 or (NH4)2S2O8 was added to the CLP solution to make a 3 mM concentration. Where applicable, ethanol (400 L) was added as a hydroxyl radical scavenger.

#### **2.3 Analytical procedures**

#### **2.3.1 Kinetic studies**

For the LC–DAD kinetic studies of the CLP photodegradation, samples of 0.50 mL of the reaction mixture were taken at the beginning of the experiment and at regular time intervals. Aliquot sampling caused a maximum volume variation of ca. 10% in the reaction mixture. Each aliquot was diluted to 10.00 mL with DDW. The obtained suspensions were filtered through a Millipore (Millex-GV, 0.22 µm) membrane filter. The absence of the CLP adsorption on the filters was preliminarily checked. After that, a 20 µL sample was injected and analysed on an Agilent Technologies 1100 Series liquid chromatograph, equipped with a UV/vis DAD set at 225 nm (absorption maximum for CLP), and a Zorbax Eclipse XDB-

Comparative Assessment of the Photocatalytic Efficiency

of TiO2 Wackherr in the Removal of Clopyralid from Various Types of Water 169

Table 2. MS/MS fragmentation data of CLP photodegradation intermediates (Part I).

C18 (150 mm × 4.6 mm i.d., particle size 5 µm, 25 oC) column. The mobile phase (flow rate 1 mL min−1, pH 2.56) was a mixture of ACN and water (3:7, v/v), the water being acidified with 0.1% H3PO4. Reproducibility of repeated runs was around 510%.

The total organic carbon (TOC) analysis was performed on an Elementar Liqui TOC II according to Standard US EPA Method 9060A. In studying the influence of the initial pH on the photocatalytic degradation use was made of a combined glass electrode (pH-Electrode SenTix 20, WTW) connected to a pH-meter (pH/Cond 340i, WTW).

#### **2.3.2 Identification of the reaction intermediates**

For the LC–ESI–MS/MS evaluation of intermediates, a 1.0 mM of CLP solution was prepared. Aliquots were taken at the beginning of the experiment and at regular time intervals during the irradiation. Then, a 20 µL sample was injected and analysed on an Agilent Technologies 1200 series liquid chromatograph with Agilent Technologies 6410A series electrospray ionisation triple-quadrupole MS/MS. The mobile phase (flow rate 1.0 mL min–1) consisted of 0.05% aqueous formic acid and MeOH (gradient: 0 min 30% MeOH, 10 min 100% MeOH, 12 min 100% MeOH, post time 3 min). Components were separated on an Agilent Technologies XDB-C18 column (50 mm × 4.6 mm i.d., particle size 1.8 µm) held at 50 °C; UV/vis signal of the eluate was monitored at 210 nm, 225 nm and 260 nm (bandwidth 16 nm for each); continuous spectrum in the range from 200 to 400 nm (2 nm step) was also recorded. The eluate was forwarded to the MS/MS instrument without flow splitting. Analytes were ionised using the electrospray ion source, with nitrogen as drying gas (temperature 350 °C, flow 10 L min–1) and nebuliser gas (45 psi), and a capillary voltage of 4.0 kV. High-purity nitrogen was used as the collision gas. Full-scan mode (*m/z* range 100– 800, scan time 100 ms, fragmentor voltage 100 V), using positive or negative polarity (depending on compound), was used to select precursor ion for CLP and each degradation product, as well as to examine isotopic peaks distribution (Table 2). Then, product ion scan MS2 mode (fragmentor voltage 100 V, scan time 100 ms, collision energy 0–40 V in 10 V increments) was used for structure elucidation of each degradation product.

#### **2.4 Cell growth activity**

**Cell lines.** For the estimation of cell growth effects, the cell lines H-4-II-E (rat hepatoma) and MRC-5 (human fetal lung) were grown in RPMI 1640 (H-4-II-E) and DMEM medium (MRC-5) with 4.5% glucose, supplemented with 10% heat inactivated FCS, 100 IU mL–1 of penicillin and 100 µg mL–1 of streptomycin. Investigated cell lines grew attached to the surface. They were cultured in 25 mL flasks (Corning, New York, USA) at 37 C in atmosphere of 5% CO2 and 100% humidity, sub-cultured twice a week and a single cell suspension was obtained using 0.1% trypsin with 0.04% EDTA.

**Samples and controls used in cell growth experiments.** For the analysis of cell growth effects, serial dilutions in distilled water were used. Samples were filtered through a 0.22 µm micro filters (Sartorius) to obtain sterility. The final concentrations of CLP before beginning the irradiation as well as in the CLP solution that was not irradiated were in the range from 6.25 to 100 µM, i.e. the dilution was from 10 to 160. Solution of CLP, filtered suspension of TiO2 Wackherr catalyst and Aspirin were used as negative controls, while cytotoxic drugs doxorubicin and gemcitabine, as well as HgCl2 were used as positive controls.


Table 2. MS/MS fragmentation data of CLP photodegradation intermediates (Part I).

C18 (150 mm × 4.6 mm i.d., particle size 5 µm, 25 oC) column. The mobile phase (flow rate 1 mL min−1, pH 2.56) was a mixture of ACN and water (3:7, v/v), the water being acidified

The total organic carbon (TOC) analysis was performed on an Elementar Liqui TOC II according to Standard US EPA Method 9060A. In studying the influence of the initial pH on the photocatalytic degradation use was made of a combined glass electrode (pH-Electrode

For the LC–ESI–MS/MS evaluation of intermediates, a 1.0 mM of CLP solution was prepared. Aliquots were taken at the beginning of the experiment and at regular time intervals during the irradiation. Then, a 20 µL sample was injected and analysed on an Agilent Technologies 1200 series liquid chromatograph with Agilent Technologies 6410A series electrospray ionisation triple-quadrupole MS/MS. The mobile phase (flow rate 1.0 mL min–1) consisted of 0.05% aqueous formic acid and MeOH (gradient: 0 min 30% MeOH, 10 min 100% MeOH, 12 min 100% MeOH, post time 3 min). Components were separated on an Agilent Technologies XDB-C18 column (50 mm × 4.6 mm i.d., particle size 1.8 µm) held at 50 °C; UV/vis signal of the eluate was monitored at 210 nm, 225 nm and 260 nm (bandwidth 16 nm for each); continuous spectrum in the range from 200 to 400 nm (2 nm step) was also recorded. The eluate was forwarded to the MS/MS instrument without flow splitting. Analytes were ionised using the electrospray ion source, with nitrogen as drying gas (temperature 350 °C, flow 10 L min–1) and nebuliser gas (45 psi), and a capillary voltage of 4.0 kV. High-purity nitrogen was used as the collision gas. Full-scan mode (*m/z* range 100– 800, scan time 100 ms, fragmentor voltage 100 V), using positive or negative polarity (depending on compound), was used to select precursor ion for CLP and each degradation product, as well as to examine isotopic peaks distribution (Table 2). Then, product ion scan MS2 mode (fragmentor voltage 100 V, scan time 100 ms, collision energy 0–40 V in 10 V

with 0.1% H3PO4. Reproducibility of repeated runs was around 510%.

SenTix 20, WTW) connected to a pH-meter (pH/Cond 340i, WTW).

increments) was used for structure elucidation of each degradation product.

**Cell lines.** For the estimation of cell growth effects, the cell lines H-4-II-E (rat hepatoma) and MRC-5 (human fetal lung) were grown in RPMI 1640 (H-4-II-E) and DMEM medium (MRC-5) with 4.5% glucose, supplemented with 10% heat inactivated FCS, 100 IU mL–1 of penicillin and 100 µg mL–1 of streptomycin. Investigated cell lines grew attached to the surface. They were cultured in 25 mL flasks (Corning, New York, USA) at 37 C in atmosphere of 5% CO2 and 100% humidity, sub-cultured twice a week and a single cell suspension was obtained

**Samples and controls used in cell growth experiments.** For the analysis of cell growth effects, serial dilutions in distilled water were used. Samples were filtered through a 0.22 µm micro filters (Sartorius) to obtain sterility. The final concentrations of CLP before beginning the irradiation as well as in the CLP solution that was not irradiated were in the range from 6.25 to 100 µM, i.e. the dilution was from 10 to 160. Solution of CLP, filtered suspension of TiO2 Wackherr catalyst and Aspirin were used as negative controls, while cytotoxic drugs

doxorubicin and gemcitabine, as well as HgCl2 were used as positive controls.

**2.3.2 Identification of the reaction intermediates** 

**2.4 Cell growth activity** 

using 0.1% trypsin with 0.04% EDTA.


\* previously identified using TiO2 Degussa P25

\*\* wide spectral band

*M*MI – monoisotopic weight

Table 2. MS/MS fragmentation data of CLP photodegradation intermediates (Part II).

Comparative Assessment of the Photocatalytic Efficiency

the control.

process.

CLP.

**3. Results and discussion 3.1 Effects of the type of TiO2** 

of TiO2 Wackherr in the Removal of Clopyralid from Various Types of Water 171

**Sulphorhodamine B (SRB) assay.** Cell lines were harvested and plated into 96–well microtiter plates (Sarstedt, Newton, USA) at a seeding density of 4 x 103 cells per well (Četojevic-Simin et al., 2011), in a volume of 180 µL, and preincubated in complete medium supplemented with 5% FCS, at 37 C for 24 h. Serial dilutions and solvent were added (20 µL/well) to achieve the required final concentrations and control. Microplates were then incubated at 37 C for additional 48 h. Cell growth was evaluated by the colorimetric SRB assay according to Skehan et al. (1990). Cells were fixed with 50% TCA (1 h, +4 C), washed with distilled water (Wellwash 4, Labsystems; Helsinki, Finland) and stained with 0.4% SRB (30 min, room temperature). The plates were then washed with 1% acetic acid to remove unbound dye. Protein-bound dye was extracted with 10 mM TRIS base. Absorbance was measured on a microplate reader (Multiscan Ascent, Labsystems) at 540/620 nm. The effect on cell growth was expressed as a percent of the control, and calculated as: % Control = (At/Ac) x 100 (%), where At is the absorbance of the test sample and Ac is the absorbance of

**Statistical analysis.** The results of cell growth activity were expressed as mean SD of two independent experiments, each performed in quadruplicate (*n* = 8). Differences between control and treated groups were evaluated using one-way analysis of variance at the significance level of p < 0.05 (Microsoft Office Excel 2003 software). IC50 values were

The photocatalytic activity of TiO2 Wackherr was compared to that of the most often used Degussa P25 under UV and visible irradiation. As can be seen from Figure 1, practically no degradation was observed under the visible light irradiation, either in the presence or absence of TiO2. The lack of CLP disappearance in the presence of TiO2 under these conditions also allows the exclusion of a significant adsorption of CLP on the catalyst surface during the course of the irradiation. In contrast, significant CLP removal could be observed under UV, and the process involving TiO2 Wackherr was slightly faster compared to that observed in the presence of Degussa P25. This insignificant acceleration of the degradation of CLP in the presence of TiO2 Wackherr is noteworthy, considering that this TiO2 specimen has much larger particles (average radii in solution are 3–4 times larger) than

The direct photolysis of CLP was also checked under the adopted irradiation conditions, in the absence of a catalyst (Figure 1). It appears that CLP can be degraded by direct photolysis in the near UV region, but at a significantly lower rate compared to the photocatalytic

Under the relevant experimental conditions, the reaction followed a pseudo-first order kinetics. On the basis of the kinetic curves ln*c* (substrate concentration) vs. *t*, the values of the pseudo-first order rate constant *k′* were calculated. The degradation rate of CLP was calculated for all the investigated as the product *k′ c*0, where *c*0 is the initial concentration of

Degussa P25 and a surface area that is almost six times lower (Vione et al., 2005).

calculated using Calcusyn for Windows (Version 1.1.0.0.; Biosoft).

**Sulphorhodamine B (SRB) assay.** Cell lines were harvested and plated into 96–well microtiter plates (Sarstedt, Newton, USA) at a seeding density of 4 x 103 cells per well (Četojevic-Simin et al., 2011), in a volume of 180 µL, and preincubated in complete medium supplemented with 5% FCS, at 37 C for 24 h. Serial dilutions and solvent were added (20 µL/well) to achieve the required final concentrations and control. Microplates were then incubated at 37 C for additional 48 h. Cell growth was evaluated by the colorimetric SRB assay according to Skehan et al. (1990). Cells were fixed with 50% TCA (1 h, +4 C), washed with distilled water (Wellwash 4, Labsystems; Helsinki, Finland) and stained with 0.4% SRB (30 min, room temperature). The plates were then washed with 1% acetic acid to remove unbound dye. Protein-bound dye was extracted with 10 mM TRIS base. Absorbance was measured on a microplate reader (Multiscan Ascent, Labsystems) at 540/620 nm. The effect on cell growth was expressed as a percent of the control, and calculated as: % Control = (At/Ac) x 100 (%), where At is the absorbance of the test sample and Ac is the absorbance of the control.

**Statistical analysis.** The results of cell growth activity were expressed as mean SD of two independent experiments, each performed in quadruplicate (*n* = 8). Differences between control and treated groups were evaluated using one-way analysis of variance at the significance level of p < 0.05 (Microsoft Office Excel 2003 software). IC50 values were calculated using Calcusyn for Windows (Version 1.1.0.0.; Biosoft).
