Article pubs.acs.org/JAFC
Promoting Photosensitized Reductive Dechlorination of Chlorothalonil Using Epigallocatechin Gallate in Water Yongqiang Tan,†,§ Qinghua Huang,†,§ Taozhong Shi,† Laijia Jin,† Rimao Hua,*,† Xiangwei Wu,† Xiangqiong Li,† Xuede Li,† Haiqun Cao,† Jun Tang,† and Qing X. Li‡ †
Key Laboratory of Agri-food Safety of Anhui Province, School of Resource & Environment, Anhui Agricultural University, Hefei, Anhui 230036, China ‡ Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Road, Honolulu, Hawaii 957822, United States ABSTRACT: Chlorothalonil (CTL) is a broad-spectrum fungicide. Photodegradation is a main degradation pathway of CTL in water. Because of the high aquatic toxicity of CTL and its metabolite 4-hydroxy CTL (CTL−OH), it is significant to develop an effective method to degrade CTL but without formation of CTL−OH. Epigallocatechin gallate (EGCG) is an abundant tea byproduct and has more than 100-fold reducing power than vitamin C. The present study reports photosensitization effects of EGCG on CTL photodegradation in water under sunlight and artificial lights. The results indicated that EGCG significantly photosensitizes CTL photodegradation. Under high-pressure mercury light illumination, CTL underwent primarily reductive dechlorination. CTL−OH, a main CTL photolytic product, was not detected when EGCG was added in the water. We concluded that EGCG not only significantly enhances CTL photodegradation rate but also alters the photodegradation pathways, avoiding the production of the highly toxic CTL−OH. The results indicated high potential of using EGCG to minimize CTL aquatic toxicity and pollution. KEYWORDS: chlorothalonil, epigallocatechin gallate, photodegradation, photodechlorination, water its parent compound CTL.11 Therefore, it is necessary to develop innovative approaches to enhance CTL degradation and generate less toxic products in the environment. In our previous study,12 TiO2 nanoparticles were used to photosensitize photodegradation of chlorothalonil in aqueous solution and on the plant surface. TiO2 nanoparticles exhibited a strong photosensitizing effect on the degradation of chlorothalonil both in aqueous solution and on the surface of green pepper.12 In the present study, we focused on natural products as a photosensitizer to accelerate pesticide degradation in water. Polyphenols extracted from tea leaves include flavanols, anthocyanins, flavones, flavonols, and phenolic acids. Among tea polyphenols, flavanols (catechins) are the major ones, accounting for 60−80%. Catechins include mainly epigallocatechin gallate (EGCG), epicatechin gallate (ECG), gallocatechin (GC), epigallocatechin (EGC), and epicatechin (EC), among which EGCG is the most abundant, accounting for about 50−80% of catechins. The EGCG molecule contains eight hydroxyl groups and, thus, has a reduction power 100 times greater than vitamin C.13 Catechins have a strong reducibility and redox potential for degradation of pesticides in the environment.14 However, to our knowledge, the role of catechins in the photodegradation of organic pollutants has not been well studied. Tea seed cakes are an economical source of catechins which are not toxic to
1. INTRODUCTION Chlorothalonil (2,4,5,6-tetrachloro-1,3-dicyanobenzene, CTL) is a broad-spectrum, nonsystemic fungicide for control of fungal diseases in vegetables, fruit trees, wheat, rice, and beans. CTL is degraded mainly through photodegradation and biodegradation in water, soil, plant surface, and other environments. CTL in temperate soils can be degraded to 4-hydroxychlorothalonil (CTL−OH). The degradation half-life of CTL in temperate soils is approximately 2.5−3 months.1 CTL−OH may remain in soil for one year after repeated applications.2 Owing to its wide use and persistence in soil and water, CTL is commonly detected in vegetables and fruits,3,4 soil and surface water,5 groundwater, and greenhouse air.6 Therefore, pollution due to CTL is of concern, especially in agricultural aquatic systems since it is highly toxic to fish and invertebrates, with the 96-h LC50 values for rainbow trout (Oncorhnchus mykiss), common jollytail (Galaxias rnaculatus), spotted galaxias (Galaxias trutaceus), and golden galaxies (Galaxias auratus) ranged from 10.5 to 29.2 μg/L, depending upon the conditions of the bioassay. Chaves et al. 5 detected 11 photoproducts of CTL. Monadjemi et al.7 reported that CTL photodegradation mainly proceeded through reductive dechlorination. In soil and water, the main metabolites include CTL−OH, 1-carbamoyl-3-cyano4- hydroxy-2,5,6-trichlorobenzene, and 1,3-dicarbamoyl-2,4,5,6tetrachlorobenzene,8 which were considered to be derived from dechlorination9 and oxidation/hydration of the cyano (−CN) groups in CTL.8,10 The water-soluble polar metabolite, CTL− OH, is the major metabolite of CTL and was reported to have greater stability and persistence than CTL in natural water. CTL−OH is the most toxic, around 30 times more toxic than © 2014 American Chemical Society
Received: Revised: Accepted: Published: 12090
September 21, 2014 November 23, 2014 November 25, 2014 November 25, 2014 dx.doi.org/10.1021/jf504565b | J. Agric. Food Chem. 2014, 62, 12090−12095
Journal of Agricultural and Food Chemistry
Article
generated by H2O2 in aqueous solutions under the HPML irradiation. PNDA standard stock solution was transferred to a 100 mL flask, followed by addition of EGCG and H2O2 at a mole ratio of 1:1.64:1324 and 1:3.28:1324, respectively. The mixture solution was diluted with double-distilled water and subjected to ultrasonic solubilization. A control was prepared without adding EGCG. The reaction solution (10 mL) was transferred to a quartz tube with a stopper and placed under a HPML for the illumination treatment. Samples were taken at different time intervals to measure absorbance at 440 nm using a UV spectrophotometer (UV-1800, Shimadzu Corp.). The results were used to calculate the residual concentration of PNDA at each time interval to reflect •OH changes. 2.5. Analysis of CTL, CTL−OH, and EGCG. CTL was analyzed on an Agilent 1200 HPLC system equipped with an Agilent HC-C18 column (4.6 mm × 250 mm, 5 μm) at a detection wavelength of 236 nm (Agilent Technologies, USA). The mobile phase was 80% aqueous acetonitrile at a flow rate of 1.0 mL/min. The column temperature was 30 °C. The injection volume was 10 μL. The limit of detection (LOD) for CTL was 0.01 mg/L. The HPLC conditions for CTL−OH and EGCG were the same as CTL except the following differences: CTL−OH, detection at 248 nm, acetonitrile/0.5% phosphoric acid mobile phase (60:40 v/v), and injection volume of 20 μL. EGCG, detection at 280 nm, acetonitrile/ 0.1% citric acid mobile phase (20:80 v/v), and injection volume of 20 μL. The LOD for CTL−OH and EGCG was 0.01 and 0.05 mg/L, respectively. 2.6. Analysis of Photolytic Products of CTL and Photopolymerization Products of EGCG. Photolytic products of CTL and photopolymerization products of EGCG were analyzed on an Waters Xevo TQ MS ultra performance liquid chromatograph triple quadrupole tandem mass spectrometer (UPLC-MS/MS; Waters, USA) with an Acquity BEH C18 column (1.7 μm × 2.1 mm × 100 mm). The mobile phases were a mixture of water, methanol, and formic acid (98:2:0.1 v/v/v, A) and methanol and formic acid (100:0.1 v/v, B). The gradient elution program was 0−0.25 min 90% A+10% B, 0.25−6 min 0% A+100% B, 6−11 min 0% A+100% B, 11−12 min 90% A+10% B, and 12−15 min 90% A+10% B. The flow rate was 0.35 mL/ min. The column temperature was 40 °C. The MS was operated at electrospray ionization (ESI) mode with simultaneous detection of positive and negative ions, cone voltage of 30 V, mass range of 150− 1000 (m/z), ion source temperature of 150 °C, capillary voltage of 1.5 kV, desolvation gas flow of 800 L/h (N2), and desolvation temperature of 400 °C. 2.7. Analysis of Chloride Ion (Cl−). Chloride ion was analyzed on a Dionex ICS 1100 ion chromatograph system equipped with an isocratic pump, an AS-DV automatic injector, a Dionex ASRS-300 suppressor, and a DS6 heated conductivity cell. A mixture of 4.5 mM Na2CO3 and 0.8 mM NaHCO3 aqueous solution was used as mobile phase at a constant flow rate of 0.5 mL/min. An IonPac AG23 (50 mm × 4 mm) guard column and an IonPac AS23 (250 mm × 4 mm) analytical column (Dionex, Sunnyvale, CA, USA) were employed for separation of target ions. The LOD for Cl− was 0.2 μg/L. 2.8. Calculation Methods. Photodegradation rate was calculated with eq 1
humans. The present study investigated effects of natural product EGCG on photodegradation of CTL in aqueous solutions and photodegradation pathways and mechanisms.
2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. All chemicals and reagents were used as received. Chlorothalonil (99.1%), CTL−OH (99%), EGCG (98%), and N,N-dimethyl-p-nitrosoaniline (PNDA, 98%) were purchased from J&K Chemical Ltd. (Shanghai, China). Acetonitrile (Assay ⩾99.9%, HPLC/Spectro) and methanol (Assay ⩾99.9%, HPLC/Spectro) were purchased from Tedia Co. (Fairfield, USA). 2.2. Photodegradation Experiments. CTL standard stock solution was 1.0 g/L (3.76 mmol/L) in acetonitrile. To determine the effect of EGCG on CTL photodegradation in aqueous solution, CTL standard stock solution was transferred into 100 mL flasks followed by addition of EGCG aqueous solution at a mole to mole ratio (CTL: EGCG) of 1:0.6, 1:2.9, 1:5.8, 1:11.6, 1:29.0, and 1:58.1 (CTL is 0.00376 mmol/L). The mixture was then diluted with doubledistilled water and subjected to brief ultrasonication. An aliquot of a 10 mL reaction solution of CTL and EGCG was transferred to a quartz cuvette, which was covered with a stopper, and placed under different light sources for photodegradation experiments. The light intensity was 10000−11000 lx for a high-pressure mercury lamp (HPML) (emission line spectrum at 365 nm) and 100000−120000 lx for direct sunlight (N31°52′, E117°17′). The distance between the quartz tube and the HPML was 15 cm. The temperature of the reaction system was maintained at 25 ± 1 °C except for the sunlight treatment which temperatures varied between 25 and 30 °C. A rotary water bath was used to maintain the temperature for the mercury lamp illumination, and the quartz tube was tilted at a 30° angle with the ground to face the sun directly. Paddy, reservoir, and pond waters were used to determine the effect of EGCG on CTL photodegradation in natural water. The paddy water had turbidity 1.8 FTU, conductivity 0.29 ms/cm, pH 7.42, hardness 264 mg/L, DO 4 mg/L, COD 342 mg/L, BOD5 2.4 mg/L, TOC 15.08 mg/L, F−1 1.49 mg/L, Cl1− 46.4 mg/L, NO2−1 0.59 mg/L, SO42− 75.98 mg/L. The reservoir water had turbidity 1.4 FTU, conductivity 0.15 ms/cm, pH 7.09, hardness 124 mg/L, DO 15.2 mg/ L, COD 8 mg/L, BOD5 6.4 mg/L, TOC 9.62 mg/L, F−1 0.49 mg/L, Cl1− 14.61 mg/L, NO2−1 0.53 mg/L, SO42− 27.99 mg/L. The pond water had turbidity 2.3 FTU, conductivity 0.32 ms/cm, pH 7.38, hardness 140 mg/L, DO 4.8 mg/L, COD 72 mg/L, BOD5 2.4 mg/L, TOC 27.82 mg/L, F−1 1.15 mg/L, Cl1− 52.1 mg/L, NO2−1 0.57 mg/L, SO42− 72.54 mg/L. An aliquot of a 10 mL reaction solution of CTL and EGCG (CTL: EGCG of 1:2.9, CTL is 0.00376 mmol/L) was transferred to a quartz cuvette, which was covered with a stopper, and placed under sunlight for photodegradation experiments. The quartz tube was tilted at a 30° angle with the ground to face the sun directly. The light intensity was 82000−88000 lx for direct sunlight (N31°52′, E117°17′), and the temperature of the water solution varied between 40 and 45 °C. 2.3. Analysis of CTL and Chloride Ion in Photolysis Mixtures. An appropriate amount of CTL standard stock solution was transferred to a 100 mL volumetric flask, and EGCG was added at a mole to mole ratio of 1:5.8 (CTL is 0.00188 mmol/L). The mixture solution was diluted with double-distilled water and subjected to brief ultrasonication. Controls contained no EGCG. Twenty mL of the mixture was transferred to a quartz tube with a stopper and exposed to HPML. Afterward, samples were taken at different times to measure the concentrations of CTL and chloride ion. A dark control was the flasks completely covered with aluminum foil, while the other conditions remained the same as the treatments. The samples were taken at different time intervals, and each treatment was repeated twice. 2.4. Detection of Hydroxyl Free Radicals (•OH). PNDA has a yellow colored sensitive absorption band at 440 nm, and its adsorption decreases when it is reacted with •OH.15 The reduction of PNDA by • OH thus reflects the amount of •OH it captures. PNDA was used as the •OH scavenger to indicate the changes in the amount of •OH
photodegradation rate (%) = [(a − b)/a] × 100
(1)
where a was the residual concentration of CTL in the dark control, and b was the residual concentration of CTL in the light treatment. Photodegradation half-life (T1/2) was calculated with eq 2
T1/2 = ln 2/k
(2)
where k was the photodegradation rate constant and was calculated with eq 3 Ct = C0·e−kt
(3)
where C0 and Ct were residual concentrations at time zero and time t after the light treatment, respectively. Photosensitization efficiency was calculated with eq 4 12091
dx.doi.org/10.1021/jf504565b | J. Agric. Food Chem. 2014, 62, 12090−12095
Journal of Agricultural and Food Chemistry
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photosensitization efficiency (%) = [(k1 − k 0)/ k 0] × 100
Table 3. Quenching of Hydroxyl Free Radicals by EGCG as Determined with PNDA in Water Solution under HPML Irradiation
(4)
where k1 was the reaction rate constant of CTL with EGCG, and k0 was the reaction rate constant of CTL without EGCG.
concns of PNDA (mg/L) at different irradiation times (min)
3. RESULTS AND DISCUSSION 3.1. Photosensitization Effects of EGCG on Chlorothalonil Degradation in Water. The photolysis rate
experimental group PNDA PNDA/H2O2 PNDA/EGCG/H2O2 PNDA/EGCG/H2O2
Table 1. Effects of EGCG on Photodegradation of Chlorothalonil in Water under HPML and Sunlight Irradiation
mole ratio
0
10
20
30
1:1324 1:1.64:1324 1:3.28:1324
1.02 1.01 1.01 1.01
1.01 0.59 1.01 1.01
1.01 0.45 1.00 0.99
0.99 0.36 0.99 0.98
kinetic equations light sources
mole ratio (CTL/EGCG)
k (min−1)
R2
T1/2 (min)
HPML
1:0 1:0.6 1:2.9 1:5.8 1:11.6 1:29.0 1:58.1 1:0 1:0.6 1:2.9 1:5.8 1:11.6 1:29.0 1:58.1
0.0187 0.0252 0.04 0.0575 0.0755 0.0515 0.0326 0.0117 0.0288 0.077 0.1022 0.1265 0.1585 0.1469
0.9796 0.9949 0.997 0.9975 0.9918 0.9950 0.9977 0.9690 0.9747 0.9967 0.9948 0.9987 0.9940 1.0000
37.1a 27.5b 17.3c 12.1d 9.2d 13.5d 21.3c 59.2a 30.4b 9c 6.8c 5.5c 4.4c 4.7c
sunlight
Figure 1. Concentrations of CTL and Cl− in water under HPML illumination. △ The residual concentration of CTL in the control (5 mg/L CTL). ▲ The residual concentration of Cl− in the control (5 mg/L CTL). □ The residual concentration of CTL in treatment A mixture of CTL and EGCG at a mole ratio of 1:5.8. ■ The residual concentration of Cl− in treatment A mixture of CTL and EGCG at a mole ratio of 1:5.8. Vertical bars represent the standard errors of the means.
a-d
Different letters indicate significant difference at 0.05 level (P < 0.05).
constant of CTL without EGCG under HPML illumination (0.0187 min−1) was 1.6-fold greater than that (0.0117 min−1) under sunlight irradiation (Table 1). The maximum absorption wavelength of CTL was 233 nm. The HPML may contain shortwave light that could be adsorbed by chlorothalonil to facilitate its photodegradation, although the maximum emission spectrum wavelength of HPML was 365 nm.12 However, sunlight consisted primarily of visible light,16 consequently, CTL was degraded more slowly than that under HPML illumination. Ferrioxalate is a visible light-responsive photocatalyst. Ferrioxalate/H2O2 could photosensitize degradation of chlorothalonil in aqueous solution by solar irradiation.17 EGCG strongly photosensitized CTL photodegradation under both sunlight and HPML light. The photosensitization efficiency under sunlight was much greater than that under HPML illumination. As the concentrations of EGCG increased,
the photosensitization efficiency also increased. The rate constant increased 4-fold under HPML illumination at a molar ratio of CTL to EGCG of 1:11.6, whereas it increased approximately 12-fold under sunlight at a molar ratio of CTL to EGCG of 1:29.0. A maximum rate constant occurred at a ratio of CTL:EGCG 1:11.6 and 1:29.0 under HPML illumination and sunlight irradiation, respectively (Table 1). When the concentration of EGCG further increased, the rate constants decreased. It is noteworthy that EGCG can readily undergo oxidative polymerization and form polymers under irradiation.13,18In general, polymers could block light and slowdown
Table 2. Enhancement Effects of CTL on Degradation of EGCG in Water under HPML and Sunlight Irradiation initial concns (mg/L) EGCG
CTL
0
15
30
45
60
HPML
2 2 5 5 2 2 5 5
0 1 0 1 0 1 0 1
1.97 1.96 4.88 4.90 1.97 1.96 4.87 4.90
1.81 0.83 4.56 2.03 1.71 0.64 4.33 1.76
1.67 0.37 4.35 1.42 1.50 0.27 4.26 1.24
1.44 0.28 4.07 1.16 1.35 0.18 3.94 1.08
1.30 0.21 3.77 0.85 1.19 0.11 3.63 0.56
sunlight
a
concns of EGCG (mg/L) after different irradiation times (min)
light sources
decrease of EGCG after 60 min (%)a 33.8 89.4 22.6 82.6 39.3 94.3 25.4 88.5
± ± ± ± ± ± ± ±
3.2 7.7 1.9 6.8 3.1 9.2 3.1 8.3
Data were expressed as the mean ± SE of triplicate determinations. 12092
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Figure 2. HPLC chromatograms of CTL with no EGCG in the dark (A) and under HPML (B), with EGCG (CTL: EGCG of 1:11.6) in the dark (C) and with EGCG under HPML (D) for 60 min and proposed step-wide photosensitized degradation pathways of CTL in the presence of and without EGCG (bottom).
gradually declined as irradiation time increased (Table 2), which is probably due to oxidative polymerization of EGCG.13 Concentrations of •OH in natural waters are approximately 1.5 × 10−18−5 × 10−16 mol/L.19 Hydroxyl free radicals (•OH) can degrade organics via free radical reactions. To further confirm the reduction ability of EGCG, PNDA was used as a free radical trapping agent to detect •OH. The residual concentrations of PNDA decreased apparently when H2O2 was added, indicating generation of •OH from H2O2 (Table 3). However, when EGCG was added into the mixture of PNDA and H2O2, the residual concentrations of PNDA did not change, suggesting that EGCG quenched •OH. The results indicate EGCG photosensitized reductive photolysis of CTL rather than photooxidation by •OH free radicals. 3.3. Effects of EGCG on CTL Photodegradation Pathways and Minimizing Toxic Metabolite Formation. Concentrations of both CTL and chloride ion in the reaction mixture were monitored to further confirm the role of EGCG on CTL photodegradation through reductive dechlorination. Upon illumination for 120 min under HPML, 65% of CTL (5
Table 4. Inhibitory Effects of EGCG on Generation of CTL− OH from CTL (2 mg/L) in Water under HPML Irradiation concns (mg/L) at different irradiation times (min) concns of EGCG (mg/L) 0 20
test compds
0
30
60
90
CTL CTL−OH CTL CTL−OH
1.80 0 1.78
