Wheat, which is cultivated throughout China, ranks as the third leading crop in the country following rice (Oryza sativa) and maize (Zea mays)[1]. There are many diseases in wheat, and the yield loss caused by those diseases are serious. Currently, fungicides have played a prominent role in cereal disease control.
Epoxiconazole (Scheme 1) is a highly efficient systemic triazole fungicide developed by BASF (Germany) in 1993[2-3]. It is used as a broad-spectrum fungicide with preventive and curative action and is extensively used worldwide to control diseases caused by Ascomycetes, Basidiomycetes, and Deuteromycetes in cereals, rice, bananas, coffee, soybean, sugar beets, peanut, oilseed rape, apple, and ornamentals[4]. It is also used to regulate plant growth by acting as an inhibitor of C-14-demethylase in sterol biosynthesis. Due to the extensive use of this fungicide on crops, there is a potential risk in coastal ecosystems located close to agricultural areas and the entrance into food chains for humans and wildlife[5-6].
To the best of our knowledge, the residue analysis of epoxiconazole has been reported in various matrix such as rice plant and polished rice[7-9], vegetables and mosquito fish[7], soil[7-16], water[7-8, 12, 16], banana[14], wheat[15, 17], tubifex[16], wine[18], apple[19], fruit[20] and tea[20-21], grape[22], spicy flavor vegetables[23].
Lin et al.[7] established the method for the determination of epoxiconazole residues in field water, rice plant, polished rice, vegetables, soil, and mosquito fish using gas liquid chromatography with an electron-capture detector. The assay employs processing of field water extracted by n-hexane, rice plant or vegetables extracted by aqueous methanol solution, and cleaned up by silica gel column. Rybár et al.[13] developed an analytical method for the fast trace microanalysis of the chiral pesticides epoxiconazole in soil samples using reversed-phase high performance liquid chromatography. Fan et al.[15] developed a method to determine the residues in wheat and soil based on ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-MS/MS). Liu et al.[16] developed a method to determine residues in tubifex and soil based on high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (HPLC-MS/MS).
The present study developed a method to determine of epoxiconazole based on gas chromatography using an electron capture detector (GC-ECD). The price of GC-ECD is cheap, which is also widely used in analytical labs. Although the epoxiconazole dissipation have been studied in different matrices, such as wheat plants[15], grape and soil[24], and banana[14], no research regarding the epoxiconazole residues in wheat grains, wheat plants and soil in China is reported. Based on the available literature, the dissipation dynamics and the behavior of terminal residues of epoxiconazole in wheat and soil under field conditions were investigated. In China, the MRL value in wheat is 0.05 mg/kg. The purpose of this study was to determine whether epoxiconazole usage is safe under the recommended application methods. These results will aid the government in providing guidance concerning the proper and safe use of epoxiconazole.
1 Materials and methods 1.1 MaterialsEpoxiconazole standard (99.2% purity) was purchased from the Sigma-Aldrich Corporation, USA. An epoxiconazole 125 g/L suspension concentrate (SC) was provided by Jiangsu Qizhoulu Chemical Co., Ltd., Jiangsu, China.
Acetonitrile and n-hexane (HPLC grade) for chromatography were purchased from Thermo Fisher Co., Ltd., USA. Analytical-grade acetone was purchased from Luoyang Chemical Reagent Factory, Henan, China. Analytical-grade sodium chloride was purchased from Beijing Reagent Company, Beijing, China.
The wheat variety in this investigation was Zhengmai 9023.
1.2 Experimental designThe field trails were conducted in a randomized block design in farmers' fields, which were previously investigated to be free of the pesticide. The supervised field trials were carried out in Henan Province and Zhejiang Province and in Beijing City, China, from 2009 to 2010. The field treatments included the dissipation experiment and final residue experiment. Each treatment consisted of 1 control and 3 replicates of treated samples with an area of 30 m2. 2 metres wide buffer area was used to separate different treatments in the field. None of the plots had been treated with epoxiconazole in the past. To ensure the reliability of the experimental results, field management was carried out in accordance with local methods. During the trial, the daily average temperatures in Henan, Zhejiang and Beijing in the year of 2009 were 18.5, 19.25 and 15.25 ℃, respectively; the daily average temperatures in Henan, Zhejiang and Beijing in the year of 2010 were 17, 19.5 and 17.5 ℃, respectively. The rainfalls in Henan, Zhejiang and Beijing in the year of 2009 were 118.37, 479.32 and 96.74 mm, respectively; the rainfalls in Henan, Zhejiang and Beijing in the year of 2010 were 84.07, 384.04 and 131.56 mm, respectively. Physicochemical characterizations of the three types of soils are presented in Table 1.
To investigate the dissipation of epoxiconazole in wheat plants and in soil, when the wheat plant was at beginning of tillering, epoxiconazole (SC, 125 g/L) was dissolved in water and sprayed onto the surfaces of wheat plants using a manual sprayer (PS16-7, 40.5 cm × 17 cm × 57 cm, 16 L volume, max. pressure 1.0 MPa) at a application rate of 225 g a.i./ha (2 times of the highest recommended application rate). A plot of the same size but with no epoxiconazole application was conducted simultaneously. For the soil treatments, soil with no plants was sprayed at a application rate of 1 000 g a.i./ha. At 2 h and 1, 3, 5, 7, 10, 14, 21, 28, 35, 42 or 45, and 60 days after the spraying, approximately 200 g of wheat plants was randomly collected from several points in each plot, and an approximately 1 000 g soil sample was randomly sampled (at a depth of 0-10 cm) in each plot using a soil sampling apparatus (soil-sampling drill, i.d. 25 mm × height 20 cm) at 20 different points. Control samples were obtained from the control plots.
To investigate the terminal residues of epoxiconazole in wheat plants, wheat grains and soil, when the wheat plant was at beginning of flowering, two application rates were used: a low application rate at 112.5 g a.i./ha (1 time of the highest recommended application rate) and a high application rate at 168.75 g a.i./ha (1.5 times of the highest recommended application rate). Both the low and high application rates were used in two or three applications (7 day intervals each). The wheat plants, wheat grains and soil were sampled at 7, 14 and 21 days after the last spraying, and the final residues were analyzed. For the control treatment, samples without pesticide were collected.
Immediately after collecting, the samples were put into polyethylene bags and transported to the laboratory. The wheat grains were separated from the wheat spikes and concentrated using the cone-and-quartering method, after which, 200 g of the grain sample was ground into a coarse powder using a vegetation disintegrator. The soil samples were sifted through a 40-mesh sieve, and 100 g of the soil was concentrated using the cone-and-quartering method. The wheat plants were cut into small pieces (approximately 0.5 cm × 0.5 cm) prior to analysis. All samples were stored at –20 ℃ until analyzed.
1.3 Analytical methodsSoil and wheat grains: 10 g subsample of homogeneous soil or wheat grains was put into a 50 mL centrifuge tube. 10 mL distilled water was added, followed by the addition of 20 mL acetonitrile. The centrifuge tube was then placed on an ultrasonic machine, and the sample was extracted for 30 min. After extraction, 3 g sodium chloride was added, and the sample was vortexed for 1 min at high speed on a vortexer. The mixture was centrifuged at 4 000 r/min for 5 min. The supernatant (20 mL of the acetonitrile layer) was transferred to a 50 mL glass beaker and then concentrated to less than 1 mL on a rotary evaporator at 40 ℃. The supernatant was further evaporated under a stream of nitrogen until dry. The residues were redissolved in 2 mL n-hexane for clean-up.
Wheat plants: 10 g subsample of homogeneous wheat plants was placed into a 250 mL glass bottle with a lid, and 10 mL of distilled water was added, followed by the addition of 40 mL acetonitrile. The glass bottle was placed on an ultrasonic machine, and the sample was extracted for 30 min. The extraction was then filtered into a mixing cylinder with a stopper along with 3 g sodium chloride, shaken for 1 min, and incubated for 1 h. The supernatant (20 mL of the acetonitrile layer) was transferred to a 100 mL glass beaker. The above method was identical to those described for the soil and wheat grain samples.
Clean-up was performed using a pre-activated Florisil® cartridge eluted with 5 mL acetone/n-hexane (2:8, V/V) and 5 mL n-hexane. The epoxiconazole residues were adsorbed onto the Florisil® cartridge and then eluted off the cartridge with 5 mL of acetone/n-hexane (2:8, V/V). The solvent in the eluate was evaporated to dryness in a nitrogen evaporator with a water bath at (50±1) ℃. The residues were redissolved in 2 mL of n-hexane and transferred into a 2 mL glass autosampler vial for GC-ECD analysis.
The samples were analyzed using a GC (Thermo Trace GC, America) equipped with an electron capture detector (ECD). The column used for epoxiconazole determination was a DB-608 column (0.25 mm film thickness, 30 m length, 0.32 mm i.d., Thermo Scientific, USA). The instrument conditions were as follows: the injector temperature was held at 230 ℃, the oven temperature was initially 180 ℃ for 1 min, then increased to 220 ℃ at a rate of 30 ℃/min and maintained for 1 min, finally increased to 270 ℃ at a rate of 5 ℃/min and maintained for 5 min. Ultra-pure nitrogen was used as the carrier gas, and injections were performed in splitless mode.
1.4 Statistical analysisThe dissipation of epoxiconazole was described using a first-order kinetic equation, ct = c0 × e–kt. The dissipation half-lives (t1/2) of epoxiconazole in each experiment was obtained using the function t1/2 = ln2/k, where ct is the concentration (mg/kg) at time t (days) after application, c0 is the initial concentration (mg/kg), and k is the first-order rate constant (days–1).
2 Results 2.1 Method validationA standard calibration curve of epoxiconazole was constructed by plotting the analyzed concentrations against the peak areas. Good linearity was achieved in the range of 0.01 to 10 mg/L, standard curve was y = 2.441 × 106x – 1.736 × 104, with a correlation coefficient of R2 = 0.999 4. The recovery study was conducted for different matrix at three different fortified levels (0.01, 0.1, 2 or 10 mg/kg). The average recoveries from fortified samples in five replicate experiments for each matrix ranged from 82% to 93%, with a relative standard deviation (RSD) from 3.0% to 9.7%. The limits of quantification (LOQ) were 0.01 mg/kg for all samples. The LOQ and recoveries obtained can meet Chinese national standards[25]. This result suggests that epoxiconazole could be detected with good precision and had good recoveries using the extraction procedure adopted.
2.2 Dissipation of epoxiconazole in wheat plants and soilThe half-lives and other statistical parameters of the epoxiconazole residue dissipation were calculated from the experimental data and were summarized in Table 2.
2.3 Terminal residue of epoxiconazole in wheat grains, wheat plants and soil
The residues of epoxiconazole in wheat grains, wheat plants and soil under different application rates, different frequencies, and different intervals to harvest are presented in Table 3. The residue levels in wheat grains were < 0.01-1.0 mg/kg at the 7th day, < 0.01-0.54 mg/kg at the 14th day, and < 0.01-0.075 mg/kg at the 21th day. The residue levels in wheat plants were < 0.01-1.5 mg/kg at the 7th day after spraying, < 0.01-1.1 mg/kg at the 14th day, and < 0.01-0.85 mg/kg at the 21th days. The residue levels in soil were < 0.01-0.62 mg/kg at the 7th day, 0.018-0.46 mg/kg at the 14th day, and < 0.01-0.42 mg/kg at the 21th day.
3 Discussion 3.1 Dissipation of epoxiconazole in plant and soil
The initial concentrations of epoxiconazole in wheat plants in Henan Province, Zhejiang Province and Beijing City was 2.1, 2.8, 2.6 mg/kg in the year of 2009, respectively and 2.1, 7.8, 9.2 mg/kg in the year of 2010, respectively. Based on the dissipation data obtained in 2009 and 2010 from the above-mentioned three sites, the different initial concentrations of epoxiconazole in wheat plants could attribute to the different plant sizes. The half-lives of epoxiconazole in wheat plants in Henan Province, Zhejiang Province and Beijing City in the year of 2009 and 2010 was 4.3-8.4, 3.5-5.4, and 4.4-5.5 d, respectively. Differences in the dissipation rate and half-lives of epoxiconazole in the wheat plants might caused by the weather conditions (such as temperature and rainfall) after the chemical application. Higher temperature and solar irradiance enhanced the dissipation rates and resulted in shorter half-lives of epoxiconazole in the crop[7]. Choi et al.[26] reported that the residue levels of fungicides rapidly decrease during the early stages of precipitation. After the early stages, the rate of decrease in the residue levels slowly. Other papers also reported the half-lives of epoxiconazole in banana and in soil were 7.2-9.9 and 8.0-10 d[14], respectively, and those in wheat plants and in soil were 8.1-12.6 and 16.9-21.3 d[15] respectively. Half-lives of epoxiconazole ranged from 2.5 to 6.0 months under aerobic conditions[27].The field measurements of behavior were desirable, as simulations based on laboratory measurements can overestimate persistence[10-11]. The half-lives of epoxiconazole in soil in Henan Province, Zhejiang Province and Beijing City in the year of 2009 and 2010 was 17-20, 10-17, and 20-30 d, respectively. Differences in the dissipation rate and half-lives of epoxiconazole in the soil might due to local soil characteristics, farming methods, microbial activities and weather conditions. The epoxiconazole half-lives estimated in sandy loam soil tend to be shorter than those in clay loam soil[2], and the half-lives are shorter in alkaline soils than those in acidic soils. This study demonstrated that the dissipation of epoxiconazole in wheat plants was faster than that in soil. A growth dilution factor might play an important role in the pesticide dissipation in the plant, in addition to the effect of physical and chemical factors such as light, heat, pH value and moisture[28]. The growth dilution factor is important in reducing epoxiconazole residue levels in wheat fields because the residue is expressed as a proportion of weight (mg/kg). The wheat plants were picked in different stages of growth in this study, and as the weight of wheat plant material increases, the proportion of residue decreases[24].
3.2 Terminal residues of epoxiconazoleAccording to the terminal residue results, the residue behavior of epoxiconazole in grain, straw and soil under different treatments almost followed a trend in which higher epoxiconazole doses and shorter harvest intervals led to more residual epoxiconazole.
The terminal residues at harvest in the wheat grains, wheat plants and soil were lower than 1.0, 1.5 and 0.62 mg/kg, respectively. In this study, the order of residue levels was wheat grains < wheat plants, which was similar with that in Europe[29]. These results could be explained by the following reasons: 1) in the residue experiments, the epoxiconazole was sprayed directly on the wheat plants and not on the soil. Although the dissipation of epoxiconazole in wheat plants was faster than that in the soil, the moisture content in the wheat plant was also greatly reduced at harvest, causing the epoxiconazole residues per unit weight to be higher than that in soil and wheat. 2) Epoxiconazole is a systemic pesticide, so when it is sprayed onto the wheat plants, it can be transferred to wheat grains. Pesticide residues in the soil raise several potential risks, such as an adverse effect on subsequent crops and contamination of the groundwater[30]. In the final residue experiments, epoxiconazole residues in the soil were detectable at three locations, but the potential risks of these residues remain undetermined.
4 ConclusionsA specific, sensitive and simple residue analytical method using GC-ECD for the detection and monitoring of epoxiconazole in wheat grain, wheat plant and soil was established. The half-lives of epoxiconazole in soil and wheat plant were 3.5-8.4 and 10-30 d, respectively. When the interval to harvest was 21 d and epoxiconazole was sprayed at a low application rate and frequency, the terminal residues at harvest in the wheat grain were lower than 0.05 mg/kg.
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