Journal of Integrative Agriculture Advanced Online Publication: 2013

doi: 10.1016/S2095-3119(13)60506-7

Hydrolysis and Photolysis of Herbicide Clomazone in Aqueous Solutions and Natural Water under Abiotic Conditions CAO Jia 1,2, DIAO Xiao-ping1* and HU Ji-ye2* 1

School of Agriculture, Hainan University, Haidian Island, Haikou 570228, P.R.China School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, P.R.China

2

Abstract The hydrolysis and photolysis of clomazone in aqueous solutions and natural water were assessed under natural and controlled conditions in this work. The kinetics of hydrolysis and photolysis of clomazone was determined using HPLC-DAD and the identification of photoproducts was carried out with HPLC-MS. Hydrolysis experimental results showed that no noticeable hydrolysis occurred in aqueous buffer solutions (25 ± 2℃, pH 4.5 ± 0.1, pH 7.4 ± 0.1, pH 9.0 ± 0.1; 50 ± 2℃, pH 4.5 ± 0.1, pH 7.4 ± 0.1) or in natural water up to 90 d, and the half-life of clomazone in pH 9.0 ± 0.1 buffer solutions at 50 ± 2

℃ The was rate 50.2 of photodecomposition d. of

clomazone in aqueous solutions followed first-order kinetics both in UV radiation and natural sunlight. The degradation rates were faster under UV light than sunlight, with the half-lives of 51.4-58.8 min and 86.7-135.9 d, respectively. Under UV light, four major photoproducts were detected and tentatively identified according to HPLC-MS

spectral

information

as

2-[2-phenol]-4,4-dimethyl-3-isoxazolidinone

2-Chlorobenzamide, and

N-hydroxy-(2-benzyl)-2-methylpropan-amide, 2-[(4,6-dihydroxyl-2-

chlorine

phenol)]-4,4-dimethyl-3-isoxazolidinone. These results suggested that the photodegradation of clomazone proceeds via a number of reaction pathways: (1) dehalogenation, (2) substitution of chlorine group by hydroxyl, (3) cleavage of the side chain. Photosensitizers, such as H 2 O 2 and riboflavin, could enhance photolysis of clomazone in natural sunlight. The results obtained indicated that photoreaction was an important dissipation pathway of clomazone in natural water systems.

Key words: clomazone, hydrolysis, photolysis, photoproducts, abiotic

INTRODUCTION Pesticides are of great environmental concern due to their widespread use in the past several decades and their potential toxic effects. Determination of degradation products (DPs) of organic compounds is one of the major challenges in analytical chemistry of environmental pollutants. DPs are often more toxic and/or persistent in environmental matrices than their parent (Moran et al., 2008). Therefore, to fully understand pesticides’ impact on the environment, there is a need to

CAO Jia, E-mail: [email protected]; Correspondence HU Ji-ye, Tel: +86-10-82376002, E-mail:

[email protected]

investigate both the parent pesticides’ and their corresponding metabolites’ impact. Thus, the degradation study of pesticides has become increasingly important in recent years (Lin et al., 2011). Residual pesticides are known to be decomposed by various natural conditions in which hydrolysis and photodegradation are the most important factors involved in the decomposition of pesticides in the environment (Mandal et al., 2011; Chamberlain et al., 2012). In the surface layers of aquatic systems, photochemical reactions can play an important role in the degradation of herbicides，so photochemical studies in aqueous solution to assess the fate of herbicides over a wide range of environmental conditions should be taken into account (Da Silva et al., 2003). Clomazone (2-[(2-chlorophenyl) methyl]-4, 4-dimethyl-3-isoxazolidinone) is a herbicide produced by FMC Corp for use against species of annual broadleaf weeds and grass. Clomazone is currently used for weed control in the cultivation of soybeans, cotton, tobacco, and various vegetable crops under the trademark command. In plants, its mode of action is thought to occur via cytochrome P450 activation followed by carotenoid synthesis inhibition(Brown and Masiunas 2002; Ferhatoglu and Barrett 2006). Clomazone is highly water-soluble (1100 mg L-1), minimally volatile (P v = 1.44 × 10-4 mm Hg), resistant to hydrolysis under a wide range of pH values, and weakly sorptive to soil (k D = 0.47-5.30) with sorption dependent upon organic carbon content (k oc = 300 mL g-1). These physicochemical characteristics indicate that clomazone is likely to persist primarily in the water column (Liu et al., 1996). It is highly effective, but causes groundwater contamination due to its water solubility. Clomazone residues were detected in 90% of water samples collected from rivers in rice cultivation regions (Patrick et al., 2010; Crestani et al., 2007).

Some studies of clomazone fate in soils under controlled laboratory conditions have been published in the per-reviewed literature (Li et al., 2004; Mervosh et al., 1995; Quayle et al., 2006). Clomazone degradation was affected by soil mositure, temperature, microorganisms and pH etc., and sorption was influenced by organic matter, clay content and cation-exchange content (Li et al., 2004). Mervosh et al. (1995) examined clomazone microbial degradation in an aerobic soil, finding slow degradation (t 1/2 = 49.0-58.0 d). Clomazone degradation was biologically dependent. Quayle et al. (2006) conducted a field dissipation study of clomazone in water and soil in treated rice plots, finding the compound to degrade rapidly, with half-lives 7.2 d (water) and 14.2 d (soil), respectively. The Environmental Protection Agency (EPA) postulates anaerobic degradation to proceed faster than aerobic with only one metabolite (N-[2-(2-chlorophenyl) methyl] 3-hydroxy-2, 2-dimethyl propanamide, ring-open clomazone) reaching concentrations of > 10% of initial application.Little metabolite formation is believed to occur in aerobic soil, but anaerobic degradation in rice fields is likely to significantly contribute to the dissipation of clomazone. The herbicide degraded rapidly to ring-pen clomazone under anaerobic conditions mostly forming soil sorbed residues which was fairly slow under aerobically conditions. In rice fields, as soil redox potential decreases, clomazone degradation and ring-open clomazone

formation rates are expected to increase (Patrick et al., 2010).

However, very limited data have been reported concerning the hydrolysis and photolysis of clomazone and its degradation products in solutions. Relevant study (Zanella et al., 2008) has been conducted concerning the degradation of clomazone in distilled and surface water under UV irradiation, but the data obtained were not comprehensive without the hydrolysis experiment and potential effect of photosensitizers on degradation. To understand the fate of clomazone after its release into environmental water, it is necessary to characterize the degradation kinetics, the potential degradation intermediates, and the degradation pathways in water. The main purpose of this study is (1) to comprehensively investigate the stability of clomazone in aqueous solutions under different hydrolytic conditions including different pH values and temperatures, (2) to characterize the photodegradation kinetics in controlled and natural conditions using the high-performance liquid chromatography (HPLC-DAD), (3) to identify the main photoproducts in distilled water with HPLC-MS and propose plausible photodegradation pathways of clomazone, (4) to assess the potential effect of photosensitizers on the degradation of clomazone under sunlight.

RESULTS Analytical method Standard calibration curve of clomazone was constructed by plotting the analyte concentration against peak areas under the proposed chromatographic conditions. At 230 nm, a good linearity was achieved from 0.1 to 20 mg L-1 with correlation coefficients(γ) = 0.9999. The standard curve equation was y = 1238.75x+1849.88, where y was peak area, and x was clomazone concentration. The mean recoveries of clomazone at spiking levels (0.01, 0.1 and 0.5 mg L-1) were 96.35% -104.33% in buffer solutions and nature water samples, and the coefficient variations (CV%) of the method ranged from 2.37% to 5.56%. All recoveries and CV values were within the permissible range as for pesticide residue analysis.

Effect of pH and temperature on hydrolysis The degradation of clomazone in aqueous solution in the absence of light at ambient temperatures (25 ± 2 or 50 ± 2℃) was monitored at different pH values. The results showed no noticeable hydrolysis occurred at 25 ± 2℃ in queous buffer solutions (pH 4.5 ± 0.1, pH 7.4 ± 0.1, pH 9.0 ± 0.1) or in natural water Over a period of 90 days, except for pH 9.0 at 50 ± 2℃with half-life 50.2 d. Data observed at different pH and temperatures are presented in Table 1. No degradation

occurred with respect to pH at 25 ± 2℃. This means that the hydrolytic processes of clomazone during the course of the photolysis experiment can be ignored. Table 1 Dissipation of clomazone at 25 ± 2℃ and 50 ± 2℃ in aqueous buffer solutions

Residue (mg L-1) (n = 3)a 25 ± 2 ℃ Days 0

50 ± 2 ℃

pH 4.5

pH7.4

pH9.0

0.94±0.01

0.94±0.01

0.93±0.03

Distilled

Natural

water

water

0.91±0.02

0.93±0.01

pH 4.5

pH7.4

pH9.0

0.94±0.01

0.95±0.01

0.93±0.02

Distilled water 0.93±0.03

5

0.92±0.01

0.90±0.01

0.90±0.01

0.91±0.01

0.92±0.01

0.94±0.01

0.94±0.01

0.83±0.01

0.93±0.01

10

0.90±0.01

0.80±0.03

0.92±0.01

0.91±0.01

0.92±0.02

0.93±0.01

0.94±0.01

0.75±0.01

0.93±0.01

20

0.86±002

0.92±0.01

0.90±0.01

0.89±0.01

0.89±0.01

0.92±0.03

0.93±0.01

0.73±0.02

0.91±0.01

30

0.90±0.01

0.90±0.01

0.91±0.01

0.89±0.01

0.89±0.01

0.93±0.01

0.97±0.01

0.61±0.01

0.90±0.01

50

0.91±0.02

0.93±0.01

0.85±0.02

0.89±0.01

0.91±0.01

0.93±0.01

0.93±0.01

0.45±0.01

0.92±0.01

70

0.87±0.01

0.91±0.01

0.89±0.01

0.88±0.01

0.91±0.01

0.92±0.02

0.92±0.02

0.35±0.01

0.93±0.01

90

0.91±0.01

0.89±0.02

0.90±0.01

0.89±0.01

0.91±0.01

0.93±0.03

0.93±0.01

0.26±0.01

0.92±0.02

aclomazone

concentrations are denoted as the means ± standard deviation (for n = 3). The statistical difference is indicated at the significance level of p 9.0±0.1 > 7.4±0.1.

Photolytic degradation by sunlight The photodecomposition parameters of clomazone exposed to sunlight are shown in Table 3. The data suggested that at the wavelengths and intensity of light emitted by the sun, degradation of clomazone is relatively slow. The indirect photolysis of pesticide residues was obtained by means of riboflavin and H 2 O 2 additions into the sample solutions. Indirect photolysis (sunlight with H 2 O 2 or riboflavin) of clomazone also follows first-order kinetics. Riboflavin and H 2 O 2 accelerated clomazone photolysis in sunlight, with half-lives ranging from 1.1 to 74.5 days. The values of the rate constants, regression coefficients, and half-lives are listed in Table 3.

Characterization and evolution of clomazone intermediates During the irradiation of clomazone, different photoproducts were detected by HPLC-DAD. On the basis of the HPLC-MS analyses carried out at different degradation times, the structures of four detected products are suggested. The total ion current (TIC) chromatogram obtained after 80 min and 160 min of clomazone irradiation in pure water are presented in Fig. 1. The peak at retention time 4.25 min was clomazone. The other four peaks on the chromatogram were photoproducts: B, C, D, and E, at retention times 0.64, 3.06, 3.34, and 3.90 min, respectively. Concerning the molecular peaks and fragments detected by HPLC-MS, different structures for the photoproducts can be envisaged. All of the results are summarized in Table 4, where each compound is referenced by its structure, retention time, m/z, and molecular weight. Fig. 2a showed a characteristic chromatogram of an irradiated clomazone solution. Fig. 2b is the HPLC-MS spectrum of parent clomazone with a molecular ion peak at m/z 240 [M + H]+. Compond B was tentatively assigned as 2-Chlorobenzamide with a molecular ion peak at m/z 156 [M + H]+(Fig. 2c) and the ion at m/z 140 was formed by the loss of -O. Compond C was N-hydroxy-(2-benzyl) -2-methylpropana-mide with the molecular ion peak at m/z 194 [M + H]+ and

m/z

216

[M

+

Na]+

(Fig.

2d).

Compond

D

was

identified

as

2-[2-phenol]-4,4-dimethyl-3-isoxazolidinone, according to its mass spectrum with a molecular ion peak at m/z 222 [M + H]+ (Fig. 2e) and the ions at m/z 244 and 266 were the sodium-adducted ions.

From

Fig.

2f,

compond

E

was

identified

as

2-[(4,

6-dihydroxyl-2-Chlorine

phenol)]-4,4-di-methyl-3-isoxazolidinone, according to its mass spectrum, with the ion peak at m/z 272 [M + H]+, m/z 294 [M + Na]+, and m/z 316 [M +2 Na]+, and the ion at m/z 240 was formed by the loss of two -OH. The photocatalytic degradation process of clomazone corresponds to four major reaction pathways (Fig. 3). The structures of the four transformation products (TPs) were all derived from the parent compound, and their structures were initially confirmed by the LC-MS. Actually, the further study using pure material by 1H , 13C NMR, UV, and IR will improve more reliable results.

Table 2 Photodegradation kinetic parameters: rate constants (k), correlation coefficients (γ), and half-lives (t 1/2 ) of clomazone in distilled water and buffer solutions under UV light

k (×10-2)(min-1)

Distilled water

51.4

0.98

58.8

0.99

1.29

53.7

0.99

1.20

57.7

0.97

1.18

pH 7.4±0.1

solutions

γ

1.35

pH 4.5±0.1 Buffer

t 1/2 (min)

pH 9.0±0.1

Table 3 Photodegradation kinetic parameters: rate constants (k), correlation coefficients (γ), and half-lives (t 1/2 ) of clomazone in distilled water and buffer solutions under under sunlight k (×10-2)(day-1) H2O2

Riboflavin

Blank

H2O2

Riboflavin

Blank

H2O2

Riboflavin

Blank

Natural water

17.23

2.26

0.74

4.0

30.7

93.7

0.95

0.98

0.94

Distilled water

19.13

1.07

0.58

3.6

64.8

119.5

0.99

0.96

0.93

pH 4.5±0.1

9.92

0.93

0.51

7.0

74.5

135.9

0.99

0.95

0.97

pH 7.4±0.1

15.22

1.04

0.6

4.6

66.7

115.5

0.96

0.97

0.95

pH 9.0±0.1

62.3

4.32

0.8

1.1

16.1

86.7

0.98

0.99

0.93

Buffer solutions

H 2 O 2 , 1 mg

L-1;

γ

t 1/2 (day)

riboflavin, 1 mg

L-1.

Table 4 HPLC-MS data of clomazone and photoproducts Clomazone and photoproducts

Retention time （min）

m/z Mass

MW

A(Clomazone)

4.25

240[M+H]+

239

B

0.64

156[M+H]+

155

3.06

194[M+H]+ 216[M+Na]+

193

3.34

222[M+H]+

221

3.90

272[M+H]+ 294[M+Na]+ 317[M+2Na]+

271

C

Compound

D

E

DISCUSSION Photochemical treatment was an important mechanism for the degradation of environmental pollutants such as pesticides/herbicides (Chan and Chu 2009). Most of the organic substances did not strongly absorb the light energy or wavelength above 300 nm (Hawari et al., 1992). Outdoor photolysis produced much lower rate constants due to the lower light intensity and higher wavelengths of UV light. Riboflavin and H 2 O 2 accelerated clomazone photolysis in sunlight .The reason for this behavior is a radical mechanism. New reactions occur as a consequence of the presence of radicals (especially •OH and HO 2 •) from the decomposition of hydrogen peroxide by radiation (Prevot et al., 1999). These photosensitizers have broad absorption bands in the UV-VIS range and can photogenerate hydroxyl radicals (•OH), which can quickly react with aromatic and unsaturated bonds of organic compounds. They are the main responsible agents that attack the pesticide molecules and start their breakage (Evgenidou et al., 2002; Hu et al., 2008). From the

data obtained in this project, photolysis may be the main degradation pathway in the aqueous medium of the environment. Moreover, because the photosensitizers exist extensively in the aqueous environment, clomazone photolysis can be accelerated.

The photodegradation of clomazone proceeds via a number of reaction pathways. The PathⅠinvolves the attack of hydroxy radicals to the benzene ring leading to the hydroxylation of the benzenic ring. Hydroxyl radicals usually attack the aromatic substrates to form the corresponding •OH adducts still adsorbed onto the oxide particles, and photooxidation reaction with hydroxy radical had been found as the most probable pathway of decomposition. Hydroxylated aromatics are more easily oxidized than parent compounds and the reaction proceeds rapidly with further hydroxylation and ring opening (Konstantinou and Albanis, 2003). The PathⅡbased on the dechlorination through positive holes or solvated electrons to form the hydroxy derivative. Dechlorination and hydroxylation reactions, previously assessed during the transformation of chlorobenzenes and chlorophenols (Prevot et al., 1999). Some experiments showed that dehalogenation was found the main degradation pathway of atrazine resulting in the formation of hydroxylated compound (Kiss et al., 2007). The Path Ⅲ corresponds to the dechlorination and the opening of the isoxazolidinone ring and the loss of the -CH 3 to give photoproduct C. The Path Ⅳ includes the rupture of the N-C and N-O bonds in the isoxazolidinone to form compound B. The four degradation products were more polar than active substance that could make them easily leach to groundwater and potentially contaminate drinking water sources.

CONCLUSIONS The hydrolysis and photolysis studies carried out on clomazone in aqueous solutions have enabled us to better understand the behavior of this herbicide in the environment. The hydrolysis and photolysis rate of clomazone were characterized by first-order kinetics. Clomazone is relatively stable in aqueous solutions in dark, with no significant variations being observed in degradation at 25 ± 2℃ up to 90 d.

Photosensitizers ( H 2 O 2 , riboflavin) could enhance photolysis of

clomazone in natural sunlight. Four major photodegradation products were detected and tentatively identified. From the data obtained in this project, photoreaction may be the main dissipation pathway of clomzone in water systems.

MATERIALS AND METHODS Chemicals , reagents and solutions Residue analysis grade clomazone was supplied by Shandong Cynda Chemical (Group) Co., Ltd.

(Shandong, China) at purity higher than 97.3%. Water for HPLC was double-distilled. HPLC grade methanol was procured from Dikma Limited (China). Riboflavin and H 2 O 2 used as photosensitizers in the experiment were analytical-grade. KCl, HCl, KH 2 PO 4 , Na 2 HPO 4 , NH 3 , H 2 O, and NaOH of analytical grade were used for buffer preparation. Three buffer solutions of pH at 4.5 ± 0.1, 7.4 ± 0.1, and 9.0 ± 0.1 were used to study the aqueous degradation of clomazone, and HCl, NaOH and phosphate buffer pH 7 were used to adjust the pH when necessary. To avoid microbial degradation, buffer solutions were sterilized and all glass apparatuses were sterilized by autoclaving for 20 min at 121℃. Aseptic techniques were adopted throughout the study to maintain sterility.

To study the influence of natural water constituents on photodegradation, lakewater was collected from the Weiming Lake of Beijing university (China). Natural water was sampled by dipping a clean stainless-steel can into the top 1 m of water until the can was full. Samples were subsampled prior to beginning the experiment for measurements of dissolved total organic carbon (TOC), total suspended solids (TSS), pH, and electrical conductivity (EC). Data are as follows: TOC, 11.6 mg L-1; TSS, 18.3 mg mL-1; pH, 7.2; EC, 1.15 mS cm-1. A stock solution of 532 mg L-1 of clomazone was prepared in methanol. Working standard solutions of 0.1, 0.5, 1, 5, 10, 20 mg L-1 were obtained by volumetric serial dilutions. All solutions were protected against light with brown containers and stored in a refrigerator at 4℃.

Hydrolysis experiments Triplicate 50.0 mL samples, each containing 1.0 mg L-1 of clomazone, were obtained by adding appropriate volume of the stock solution into the buffer solutions. The treated buffer solutions were stored in the dark at ambient temperature (25 ± 2℃) in erlenmeyer flasks. Another set of triplicate 50.0 mL buffer solution samples containing 1.0 mg L-1 of the pesticide, was stored in the dark at 50 ± 2℃ to test the effect of temperature on hydrolysis. In all trials, the pH of each sample was periodically measured and did not vary by > 0.1 unit. Experimental bottles were left static in the laboratory and shaken every 8-12 h. Initially, the starting concentration was determined. Samples (2 mL) were drawn out from each bottle at day 5, 10, 20, 30, 40, 60 and 90. Samples were treated followed the sample preparation procedure below prior to HPLC-DAD or HPLC-MS analysis.

Photolysis experiments For UV light, kinetic studies of photodegradation were performed with a high-pressure mercury

lamp (HPK 150 W) in water-cooled quartz housing. The concentration of the pesticide clomazone in aqueous solutions (buffer solutions and distilled water) was 20 mg L-1. At specific time intervals (10 min), samples of 2 mL were withdrawn from the reactor for HPLC-DAD. Each series of photodegradation experiments was conducted in three replicates and accompanied by dark reaction controls.

For the sunlight experiment, the initial concentration of clomazone in aqueous solutions (buffer solutions and natural water) was 1 mg L-1, being close to the natural environmental conditions. An irradiation experiment in the sunlight was performed at Beijing. Photolysis under sunlight was conducted from March to May, 2012, in Beijing, China. The sunlight intensity at 300-400 nm wavelength was 400, 2009, and 365 mW cm-1 at the beginning, middle, and end of the day, respectively. Samples (2 mL) were drawn out from each bottle at 0, 1, 3, 5, 7, 10, 20, 30, 40, 50, and 60 days.

Different kinds of photosensitizers, such as riboflavin and H 2 O 2 , were added to samples to evaluate their behavior as possible photocatalysts and their effect on the photolysis of pesticides under natural sunlight. These compounds were used in the concentration of 1 mg L-1 with the initial concentration of the pesticides fixed at 1 mg L-1. Dark controls were conducted under an identical experimental setup, to ensure that no loss of the clomazone occurred via processes other than photolysis (i.e., hydrolysis or evaporation).

Photoproducts identification A solution of clomazone (20 mg L-1) in solvents was irradiated in quartz tubes under UV light to facilitate the identification of intermediate products. Samples were withdrawn at intervals of 0, 10, 30, 60, 90, and 120 min from quartz tubes and HPLC-MS analysis was used to identify clomazone photoproducts. With each set of samples collected, a 2 mL control sample without ultraviolet irradiation blank was also detected.

Pretreatment of the samples 2 mL aqueous solution sample was transferred to a 250 mL separatory funnel in the presence of 30 mL 2% Na 2 SO 4 aqueous solutions, and the analyte was extracted by liquid-liquid extractions with dichloromethane (20 mL, 20 mL, 20 mL) for three times. The organic phase was dehydrated on anhydrous sodium sulfate and collected in a 250 mL flask, then evaporated to near dryness using a vacuum rotary evaporator at 40℃. After the extract was made to dryness under a gentle nitrogen stream, 2 mL of methanol was added to dissolve the sample for HPLC-DAD or HPLC-MS analysis.

Analytical determinations A HPLC (Shimadzu LC-20A) equipped with an analytical column (250 × 4.6 mm inner diameter, 5 μm ODS) was attached to a DAD. The chromatographic conditions used for the analysis of clomazone residues were as follows: the mobile phase was methanol/water (70 : 30, v/v) with a total flow of 1.0 mL min-1. The injection volume was 20 μL. Detection was performed at 230 nm. Under these conditions, the retention time of clomazone was about 10.3 min. All measurements were carried out at room temperature. For HPLC-MS analysis, UPLC-MS/MS (Waters, Acquity-TQD) analysis was performed in positive mode (ESI+). Acquisition parameters were as follows: column, Acquity UPLC BEH Shield C18 (2.1 mm × 100 mm，1.7 μm); mobile phase, methanol/water (65 : 35, v/v) with a flow rate of 0.3 mL min-1; capillary voltage, 3 kV; source temperature, 120℃; and desolvation temperature, 350℃. The cone and desolvation gas (N 2 ) flows were 50 and 500 L h-1, respectively; ESI+ full-scan mode in the rangs of m/z 80-300.

Acknowledgements This study was sponsored by Shandong Cynda Chemical (Group) Co., Ltd. (Shandong, China) and the Institute for the Control of Agrochemicals, Ministry of Agriculture of the People’s Republic of China.

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Fig. 1 HPLC-MS TIC chromatogram of clomazone in aqueous solutions after 80 min (a) and 160 min (b) of irradiation.

Fig. 2 HPLC chromatogram and LC-MS spectrum of clomazone (A) and photoproducts (B, C, D, and E) .

Fig. 3 Proposition of clomazone photodegradation pathway in aqueous medium under UV irradiation