1. Introduction
Rice (
Oryza sativa L.) is the most important grain and widely cultivated in China [
1,
2,
3,
4]. Thus, the quality and safety of rice are highly related to human health and national strategies. Due to the long growth period of rice, weed problems appear frequently in paddy fields, which compete with rice for space, light, water, and nutrients, leading to a loss of about half of rice production [
5,
6]. With higher yield and quality requirements for rice, herbicides have been extensively used in paddy fields for weed control. Furthermore, the sales of herbicides have become the largest in the pesticide industry, and the wide use of herbicides in agriculture inevitably leads to higher residues in the environment [
7]. Studies indicate that herbicide residues could migrate into soil and water after application, which poses a great threat to the quality and safety of rice, as well as human health [
8,
9].
Pyrazosulfuron-ethyl ethyl 5-[(4,6-dimethoxypyrimidin-2-yl)carbamoylsulfamoyl]-1-methylpyrazole-4-carboxylate, and bensulfuron-methyl methyl 2-[(4,6-dimethoxypyrimidin-2-yl)carbamoylsulfamoylmethyl]benzoate are two commonly used rice herbicides belonging to the sulfonylurea group, which are generally used to control sedges and broadleaf weeds (
Figure 1) [
10,
11]. The action mechanism of sulfonylurea herbicides is to inhibit acetolactate synthase (ALS) activity and block the biosynthesis of branched-chain amino acids of weeds [
12]. Acetochlor 2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)acetamide and butachlor N-(butoxymethyl)-2-chloro-N-(2,6-diethylphenyl)acetamide are two chloroacetanilide herbicides generally used in rice fields for pre-emergent control of annual grasses and broadleaf weeds [
13,
14]. The four herbicides inhibit the growth of young shoots and roots, stimulate root-like deformities, and lead to the death of weeds after application in rice cropping systems [
15,
16,
17,
18]. Although the half lethal doses(LD
50) of four herbicides in rats are all over 5000 mg/kg according to the Pesticide Manual [
19], the four herbicides are extensively used in paddy fields in consideration of good selectivity, high efficiency, and low toxicity. Previous studies primarily concentrated on herbicidal activity and residue levels of four herbicides in rice cropping systems [
10,
20,
21]. The results indicated that pyrazosulfuron-ethyl, bensulfuron-methyl, acetochlor and butachlor were safe and the residues in rice were lower than the maximum residue limit (MRL) at the recommended dosage [
19,
20]. However, weed resistance to these four herbicides increased gradually due to widespread use in paddy fields, which inevitably increased the dosage of these four paddy herbicides [
22,
23,
24,
25]. Such phenomenon constitutes a serious threat to cropping ecosystem assessment and herbicide residues in rice.
Considering the complexity of matrices in paddy fields, a sensitive, rapid, and reliable sample preparation method is necessary, which should facilitate extraction, enhance enrichment of the target compound, and reduce interferences as much as possible. Solid-phase extraction, supercritical-fluid extraction, liquid-liquid extraction and QuEChERS are generally adopted in paddy sample preparation, and QuEChERS is the most frequently used method [
26]. Analytical methods for pyrazosulfuron-ethyl and bensulfuron-methyl have been mainly focused on high performance liquid chromatography (HPLC) [
27], capillary electrophoresis (CE) [
28], gas chromatography-tandem mass spectrometry (GC-MS) [
29], immunoassay [
30], and liquid chromatography-tandem mass spectrometry (LC-MS) [
31]. Acetochlor and butachlor analysis have been primarily performed on GC [
32], HPLC [
21], and GC-MS [
33]. HPLC-MS/MS and GC-MS have higher sensitivity and precision and lower detection limits than traditional methods at trace levels in various matrices [
34,
35]. In addition, the combination of QuEChERS and mass spectrometry has been considered as the most sensitive, rapid, and reliable method for pesticide residue analysis in different matrices.
In this study, a simple and reliable QuEChERS method coupled with HPLC-MS/MS and GC-MS methods was established to determine pyrazosulfuron-ethyl, bensulfuron-methyl, acetochlor, and butachlor residues in rice cropping systems. The dissipation dynamics of the four herbicides in water and soil, as well as the final residues in rice hull and husked rice, were carefully investigated under Good Agricultural Practices (GAP) use. Such results highlight the dissipation dynamics and residue of the four herbicides in rice cropping systems and contribute to risk assessment of herbicide residues in rice, as well as providing scientific guidance on the proper and safe application of rice herbicides in paddy fields.
2. Materials and Methods
2.1. Chemicals and Equipment
Pyrazosulfuron-ethyl (purity = 95%), bensulfuron-methyl (purity = 97%), acetochlor (purity = 92%), and butachlor (purity = 92.5%) were obtained from the Institute for Control of Agrochemicals (Beijing, China). Pyrazosulfuron-ethyl WP (10%), bensulfuron-methyl WP (10%), acetochlor WP (10%), and butachlor EC (900 g/L) were purchased from commercial sources. Acetonitrile, methanol, and formic acid were HPLC grade and purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ethyl acetate, acetone, sodium chloride, and anhydrous magnesium sulfate were analytical grade and bought from J&K Scientific Co., Ltd. (Beijing, China). Graphitized carbon black (GCB) and primary secondary amine (PSA) were purchased from Agela Technologies (Tianjing, China). Ultra-pure water was generated using a Milli-Q purification system from Millipore (USA). Pyrazosulfuron-ethyl, bensulfuron-methyl, acetochlor, and butachlor stock standard solutions were prepared with corresponding organic solvent and stored at −20 °C.
Pyrazosulfuron-ethyl and bensulfuron-methyl were analyzed on an Agilent 6410 high performance liquid chromatography-tandem triple quadrupole mass spectrometry equipped with electrospray ionization source (Agilent Technologies, Santa Clara, CA, USA). An Agilent 7890-5977B gas chromatography-tandem mass spectrometry (Agilent Technologies, USA) was used to determine acetochlor and butachlor. A Sigma 3K15 microcentrifuge (St. Louis, MO, USA), Xiangyi L550 centrifuge (Hunan, China), ME204 analytical balance (Sartorius, Germany), IKA T18 grinder (IKA, Germany), and ZWFR-200 shaker (Zhicheng, China) were adopted in sample preparation.
2.2. Field Experiment Design
Field experiments including the degradation dynamics and final residues in supervised field trials were conducted in Chongqing municipality in 2017 (
Table 1). All the experiments were designed based on the “Guidelines on pesticide residue trials (NY/T 788-2004)” published by the Institute for the Control of Agrochemicals, Ministry of Agriculture and Rural Affairs of the People’s Republic of China [
36]. The area of the field experiment plot was 30 m
2 and each treatment had three replicated plots. Furthermore, a buffer area was designed to isolate the experiment plots.
The degradation dynamics experiments were conducted with two dosage levels, pyrazosulfuron-ethyl (22.5 g a.i.ha−1, the recommended dosage and 45 g a.i.ha−1, double of the recommended dosage), bensulfuron-methyl (26.2 g a.i.ha−1, the recommended dosage and 52.4 g a.i.ha−1, double of the recommended dosage), acetochlor (52.5 g a.i.ha−1, the recommended dosage and 105 g a.i.ha−1, double of the recommended dosage) and butachlor (112.4 g a.i.ha−1, the recommended dosage and 224.8 g a.i.ha−1, double of the recommended dosage), respectively. All the four herbicides were sprayed one time after rice transplanting. Representative 2 kg paddy soil and 500 mL water samples were collected randomly in each plot at 0 (2 h post-treatment), 5, 10, 20, 30, 40, 80 days and pre-harvest interval (PHI) of 7 days after herbicides application. The representative 2 kg rice samples were randomly collected at pre-harvest interval (PHI) of 7 days. All the collected paddy soil, water, and rice samples were stored at −20 °C, respectively.
2.3. Analytical Procedure
2.3.1. Sample Preparation
All the samples were thawed at room temperature. 5 g of soil, 5 g of husked rice, 2 g of rice hull and 5 mL of water were weighed into a 50 mL polypropylene centrifuge tube, respectively. 5 mL of purified water with 1% formic acid were added to the rice hull and husked rice sample. 10 mL of acetonitrile was added in all samples for extraction. All the samples were shaken vigorously for 1 min, then 3 g of sodium chloride was added, and samples were oscillated for 30 min in an air bath oscillator at 300 rpm. After that, sample tubes were exposed to ultrasonic vibration for 10 min, and then centrifuged at 3500 rpm for 5 min.
For soil and water samples, 1 mL of the upper layer was placed into a 2 mL centrifuge tube including 20 mg of PSA and 100 mg of anhydrous magnesium sulfate. The samples were vortexed again for 1 min and then centrifuged at 10,000 rpm for 5 min. The upper extract was filtered through a 0.22 μm filter and transferred into a 2 mL autosampler vial for HPLC-MS/MS or GC-MS analysis, individually.
For husked rice and rice hull samples, 10 mL of the upper layer was transferred to a 100 mL conical flask, evaporated to dryness at 35 °C on a rotary vacuum evaporator, reconstituted with 1 mL of acetonitrile and transferred into a 2 mL single-use centrifuge tube including 50 mg of PSA, 10 mg of GCB, and 150 mg of anhydrous magnesium sulfate. The sample was vortexed vigorously for 1 min and centrifuged on a microcentrifuge at 10,000 rpm for 5 min. The resulting supernatant was filtered through a 0.22 μm filter and transferred into a 2 mL autosampler vial for HPLC-MS/MS or GC-MS analysis, respectively.
2.3.2. HPLC-MS/MS Analysis
The mobile phase was solvent A (methanol) and solvent B (0.1% formic acid in water) (
v/v = 90:10) with the flow rate of 0.3 mL/min. A sample of 5 μL was injected and the herbicides were separated on an Agilent ZORBAX SB-C
18 reverse-phase column (50 mm × 2.1 mm, 3 μm). Nitrogen was used as both nebulizer and collision gas in HPLC-MS/MS analysis. The electrospray ionization source (ESI) parameters were as follows: drying gas temperature, 350 °C; gas flow, 8.0 mL/min; nebulizer gas, 35 psi; and capillary voltage, 3000 V. The positive multiple reaction monitoring (MRM) mode was used for monitoring ions transitions. An Agilent Mass Hunter software package was used for method development and data acquisition. Under the above condition, the retention time of pyrazosulfuron-ethyl and bensulfuron-methyl were 7.08 and 6.12 min, individually. The MS parameters and representative chromatograms of pyrazosulfuron-ethyl and bensulfuron-methyl are shown in
Table 2 and
Figure 2.
2.3.3. GC-MS Analysis
Acetochlor and butachlor were analyzed on an Agilent 7890-5977B GC-MS system (Agilent, USA). The injector and detector temperature were set at 260 °C and 280 °C, respectively. Helium was served as the carrier gas at a constant flow rate of 1.0 mL/min and a sample of 2 μL was injected into the GC-MS system. The separations of two herbicides were performed on a HP-5 capillary column (30 m × 0.25 mm inner diameter and 0.25 μm film thickness). Oven temperature program was as follows: the column was held initially at 100 °C for 1 min, then ramped at 20 °C/min to 220 °C, ramped at 1 °C/min to 230 °C, further ramped 20 °C/min to 260 °C, and held at 260 °C for 3 min. The MS parameters were as follows: source temperature of 230 °C, emission current of 35 μA, and energy of −70 eV. The ions transitions were operated in the selective ion monitored (SIM) mode. The retention times of acetochlor and butachlor were 8.58 and 11.22 min, individually. The MS parameters and GC-MS chromatograms of acetochlor and butachlor are listed in
Table 3 and
Figure 3.
2.3.4. Data Analysis
The degradation dynamics of the four herbicides in paddy fields appeared to follow the first-order kinetic reaction and were calculated according to the following equation:
, where C
t and C
0 are the concentrations of herbicides at time t and time 0 after spraying (mg/kg), respectively, and k is the degradation rate constant [
37,
38]. The half-life (t
1/2) of each herbicide was calculated using the equation:
[
39,
40].
5. Conclusions
In the context of this study, a quick, easy, cheap, rugged, safe (QuEChERS) extraction method, coupled with HPLC-MS/MS and GC-MS, was developed to determine the dissipation dynamics and residue of pyrazosulfuron-ethyl, bensulfuron-methyl, acetochlor, and butachlor in rice cropping systems. The average recoveries of the four herbicides ranged from 78.9–108% with relative standard deviations (RSDs) less than 15% at three different fortified levels for soil, rice hull, and husked rice. The dissipation results indicate that the average half-lives of the four herbicides in soil are in the range of 3.5–17.8 days, and more than 95% of all herbicides dissipated within 5 days in water. Furthermore, the final residues of four herbicides were all below LOQ at harvest time. Such results highlight the dissipation dynamics and residue of four herbicides in rice cropping systems and contribute to risk assessment as well as scientific guidance on the proper and safe application of rice herbicides in paddy fields.