CHIRALITY 27:137–141 (2015)
Enantioselective Degradation of Chiral Insecticide Dinotefuran in Greenhouse Cucumber and Soil 1
XIU CHEN,1,2 FENGSHOU DONG,1* JUN XU,1 XINGANG LIU,1 YUNHAO WANG,1 AND YONGQUAN ZHENG1** State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, P.R. China 2 Shanghai Agriculture Technology Extension and Service Center, Shanghai, P.R. China
ABSTRACT The enantioselective degradation behavior of the chiral insecticide dinotefuran in cucumber and soil was investigated under greenhouse conditions based on the method established with a normal-phase high-performance chromatography (HPLC) on a ChromegaChiral CCA column (250 × 4.6 mm, 5 μm, ES Industries). The linearity range, matrix effect, precision, and accuracy of the method were evaluated and the method was then successfully applied for the enantioselective analysis of dinotefuran in cucumber and soil. Significant enantioselectivity of degradation was observed in soil according to the results. The (+)-dinotefuran was more persistent in soil with half-life of 21.7 d, which is much longer than that of (–)-dinotefuran (16.5 d). In cucumber, the (–)-dinotefuran also tended to be preferentially degraded both in foliar and douche treatment. However, the statistical analysis indicated the enantioselectivity of degradation in cucumber was not significant. The research provides the first report concerning the enantioselective degradation of dinotefuran enantiomers and the results can be used for understanding the insect-controlling effect and food safety evaluation. Chirality 27:137–141, 2015. © 2014 Wiley Periodicals, Inc. KEY WORDS: dinotefuran; enantioselective degradation; cucumber; soil; HPLC INTRODUCTION
The greenhouse vegetable system is a typical agricultural production mode in China, which is characterized by high planting density under high temperature and humidity, low light, and closed conditions. All these factors always lead to a high occurrence of pests and diseases, as well as more pesticides application in the greenhouse than in outdoor fields.1,2 The novel neonicotinoid chiral insecticide dinotefuran was recently approved for use in greenhouse vegetable growing in China, due to its effective control of piercing sucking agricultural insects, such as aphids and whiteflies (Figure 1).3 As is well known, enantiomers of chiral pesticides may differ in binding to structure-sensitive biological receptors or naturally occurring chiral molecules for various molecular configurations.4–6 As a consequence, enantiomers can have very different biological activities.7–11 Previous reports have shown that the (+)-dinotefuran enantiomer has much higher insecticidal activity than the (–)-dinotefuran enantiomer to target insects.12,13 Many studies indicated that chiral insecticide enantiomers always show different stereoselective degradation in plants and the environment,14–21 as well as different insecticidal activity.17,22 The unclear possible changes of high active (+)-dinotefuran enantiomers may affect the control efficiency of a pest, and even food safety. Therefore, it is essential to understand the specific environmental fate of the chiral insecticide dinotefuran enantiomers in greenhouse crops. However, to date, the environmental behavior studies of dinotefuran at the enantiomeric level have not been reported. In our previous study, an enantioselective analytical method for the separation and determination of dinotefuran enantiomers in rice, tomato, and apple was established.23 On this basis, minor modifications were done to make it applicable to cucumber and soil matrices. Consequently, a study about the degradation behavior of dinotefuran enantiomers was © 2014 Wiley Periodicals, Inc.
successfully carried out in cucumber and soil under greenhouse conditions after the application of the commercial formulation. The main object of the research was to investigate the enantioselective kinetics of dinotefuran in cucumber and soil under greenhouse conditions to provide more data for understanding the insect-controlling effect and evaluating food safety at the level of enantiomers. MATERIALS AND METHODS Reagents and Materials Racemic dinotefuran standard (purity: 99%) was purchased from Dr. Ehrenstorfer (Augsburg, Germany). The formulation applied in field trials was 20% dinotefuran soluble powder (Zhongnonglihua Agrochemical, Tianjin, China). Ethanol, methanol, and n-hexane (high-performance chromatography [HPLC] grade) were from Honeywell International (Morristown, NJ). Ultrapure water was obtained from a Milli-Q system (Bedford, MA). Analytical grade acetonitrile, acetone, anhydrous magnesium sulfate, and sodium chloride were purchased from Beijing Chemical (Beijing, China).
Degradation Experiment Plant care and application of dinotefuran formulation. Working areas for cucumber and soil were prepared in the greenhouse of the Chinese Academy of Agricultural Sciences (Hebei Province, China). The 2 fields were divided into 15-m -sized blocks and the buffer zone was set up between plots. Each treatment included three plots as three replicates. In addition, another plot was used as the control. The dinotefuran commercial product (20% dinotefuran soluble powder) was applied in two dif-
*Correspondence to: Fengshou Dong or Yongquan Zheng, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Key Laboratory of Pesticide Chemistry and Application, Ministry of Agriculture, Beijing 100193, China.
[email protected] or E-mail:
[email protected] Received for publication 15 July 2014; Accepted 14 September 2014 DOI: 10.1002/chir.22402 Published online 6 November 2014 in Wiley Online Library (wileyonlinelibrary.com).
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Pharmacokinetics and Calculation It was assumed that the degradation of the enantiomers in plant and soil accorded with first-order kinetics. The corresponding degradation rate constant, k, was calculated according to Eq. (1). The starting point (C0) was considered to be the maximum value of the concentration. The half-life (T1/2, day) was estimated from Eq. (2).
Fig. 1. The structure of dinotefuran.
ferent modes (foliar spraying and root douche treatment) for cucumber plant. In the foliar spraying treatment, the insecticide was sprayed at 3× 2 the recommended dosage of 225 g a.i/hm . For root douche treatment, each cucumber plant was treated with 1000 mL aqueous solution of commercial dinotefuran (about 100 mg/L). Representative examples of cucumber were sampled at different intervals after treatment. The soil was also treated by spraying the aqueous solution of commercial dinotefuran at 3× the recommended dosage and sampled to a depth of 0–10 cm in each plot using a tuber auger at different intervals after treatment. Stones and plant debris were manually removed. All samples were put into polyethylene bags and transported to the laboratory where they were prepared as subsamples. The subsamples were kept at –20 °C until analysis. Sample preparation. A portion of 15 g subsample was weighed into a 50 mL PTFE centrifuge tube with screw caps. Then 15 mL acetonitrile was added, while before 5 mL pure water was added to the soil samples. The mixture was vortexed for 3 min. Three g of NaCl and 5 g of anhydrous MgSO4 was added and vortexed for 1 min and then the tube was centrifuged for 5 min at relative centrifugal force (RCF) 2811 g. Ten mL of supernatant was transferred, concentrated to near dryness, and then dissolved in 2 mL of acetonitrile. Subsequently, the 2-mL extract sample was then purified by a Cleanert PestiCarb/PSA cartridge, which was preconditioned with 10 mL of acetonitrile. The analyte was eluted with 2 × 2 mL of acetonitrile, and the eluate was then concentrated to near dryness. After that, the residue was reconstituted in 1 mL of ethanol and filtered with 0.22-μm syringe filters for HPLC analysis.
Apparatus and Chromatographic Conditions The chromatographic determination was performed with an Agilent 1100 series HPLC (Agilent Technology, Palo Alto, CA) equipped with a G1322A degasser, G1311A pump, G1316A column oven, G1328A injector, a 50-μL sample loop, and G1315B diode array detector (DAD). The signal was received and processed by Agilent Chemstation software. The enantiomers of dinotefuran were separated on a ChromegaChiral CCA column (250 × 4.6 mm, 5 μm, ES Industries, West Berlin, NJ). The mobile phase was n-hexane-ethanol-methanol (85:5:10, v/v/v) with a flow rate of 1.0 mL/min at the detection wavelength of 270 nm. The injection volume was 20 μL. The column temperature was 30 °C.
C ¼ C0 ekt
(1)
T 1=2 ¼ lnð2Þ=k ¼ 0:693=k
(2)
The enantiomer fraction (EF) was used as a measure of the enantioselectivity of the degradation of dinotefuran enantiomers in the cucumber and soil samples, which provided a more meaningful representation in the field of environmental chemistry.20,24 The EF values defined a range from 0 to 1, with EF = 0.5 representing the racemic mixture. EF was defined as follows: EF ¼ peak areas of the ðþ Þ enantiomer= ½ðþ Þ enantiomer þð Þ enantiomer
RESULTS AND DISCUSSION Method Validation
Performance parameters including linearity, recovery, precision, limit of detection (LOD), and limit of quantiation (LOQ) were investigated to validate the developed method for determination of the dinotefuran enantiomers in cucumber and soil. Dinotefuran enantiomers were completely separated, and there were no interference peaks at the retention times under the developed conditions. Linear calibration curves were obtained from each dinotefuran enantiomer concentration ranging from 0.25 to 50 mg/L. The summary of calibration data in Table 1 showed excellent linearity for analysis of the two enantiomers. The matrix effect was investigated in cucumber and soil by comparing the standards in the solvent with the matrix-matched standards. The slope ratios of the matrix to the solvent ranged from 0.97–1.02, indicating the matrix effect could be ignored in the study. The mean recoveries of the two enantiomers in cucumber and soil were determined at three fortification levels of 0.1, 1, and 2.5 mg/kg for each enantiomer and the results were displayed in Table 2. Recoveries of both dinotefuran enantiomers ranged from 78.6% to 91.2% in cucumber and from 77.6% to 92.1% in soil, with the RSDs lower than 11.1%. The LODs for two enantiomers were about 0.025 mg/kg in cucumber and 0.02 mg/kg in soil, respectively. Correspondingly, the LOQs were estimated to be 0.08 mg/kg in cucumber and 0.07 mg/kg in soil for each enantiomer, respectively. Typical chromatograms of blank and fortified samples of the cucumber and soil were shown in Figure 2.
TABLE 1. Linear regression parameters of calibration curves of dinotefuran enantiomers in pure solvent and different matrices Standard calibration curve Enantiomers
Matrix
(+)-dinotefuran
Ethanol Cucumber Soil Ethanol Cucumber Soil
()-dinotefuran
Chirality DOI 10.1002/chir
Calibration range(mg/L)
Slope
Intercept
R2
LOD
0.25-50 0.25-50 0.25-50 0.25-50 0.25-50 0.25-50
96.293 93.328 98.323 96.245 93.511 98.200
-3.9700 -1.0544 -11.306 -4.4505 -0.8056 -10.192
0.9999 0.9999 0.9999 0.9999 0.9999 0.9999
/ 0.025 0.020 / 0.025 0.020
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TABLE 2. Intraday and interday accuracy and precision results Intraday (n = 5) (+)-dinotefuran
Matrix Cucumber
Soil
Spiked level, mg kg-1 a 0.1 1 2.5 0.1 1 2.5
Interday (n = 3)
()-dinotefuran
(+)-dinotefuran
()-dinotefuran
Recovery
RSDr
Recovery
RSDr
Recovery
RSDR
Recovery
RSDR
%
%
%
%
%
%
%
%
91.2 83.8 84.7 79.2 78.2 92.1
2.5 11.1 5.3 1.8 3.4 2.3
91.0 83.7 87.3 75.8 78.0 91.8
3.3 11.0 5.2 1.9 3.8 2.3
86.0 82.4 77.9 79.2 78.7 82.8
7.8 9.0 7.4 2.8 2.5 8.4
88.0 83.1 78.6 77.6 78.6 81.6
4.9 9.0 8.6 3.5 2.7 9.2
a
The spiked concentrations were on the level of enantiomers.
Fig. 2. Chromatograms of cucumber blank (A), cucumber spiked (B), soil blank (C), and soil spiked (D). The spiking level was 0.1 mg/kg for each enantiomer.
Degradation in Cucumber
There were two stages of changes of the dinotefuran enantiomers in cucumber under both application modes. At the first stage the concentration increased to the maximum, and after that the concentration decreased with time. In the foliar spraying treatment, the concentration of dinotefuran enantiomers both attained a maximum at the 3rd day. In the root douche treatment, the maximum concentration of the two enantiomers was acquired at the 10th day after the application of the dinotefuran formulation. The relatively long time to attain the maximum concentration of dinotefuran might be due to a slow distribution of dinotefuran in cucumber plant. Thus, the regressive degradation equations were established with the maximum concentration of the enantiomers in plant as the degradation starting point. Corresponding degradation kinetic parameters of the two enantiomers are displayed in Table 3. The data show that the dissipation of dinotefuran enantiomers in cucumber followed first-order kinetics under both foliar spaying and root douche treatment, with R2 from 0.9445 to 0.9982. The average half-lives of (+)dinotefuran and (–)-dinotefuran in cucumber were 8.3 d and 7.6 d under foliar spaying treatment, respectively, and
11.7 d and 10.8 d under root douche treatment. Although the half-life of (+)-individual was longer than that of (–)-individual, the difference was not significant between the two enantiomers (P > 0.05, one-way analysis of variance [ANOVA]). The degradation dynamics of dinotefuran enantiomers in cucumber are displayed in Figure 3A,B. The EF values of dinotefuran enantiomers in cucumber under the two treatments are shown in Figure. 3D. The EF value was nearly 0.50 at 2 h in the foliar spraying treatment, then slightly decreased below 0.5 within 14 d and finally increased to 0.57 at the 28th day. The (–)-dinotefuran could not be detected at the 35th day. The situation was quite different for the EF values in the root douche treatment. It was 0.56 at the beginning, which might be caused by the selective absorption of the root. Then the EF value finally increased to 0.60. The difference in enantiomer accumulation in cucumber resulting from the different application modes may have some implications on the proper use of chiral pesticides. Although the EF values were different under the two application modes, the (–)-dinotefuran was preferentially degraded in cucumber after a relatively long phase. Chirality DOI 10.1002/chir
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TABLE 3. First-order constants (k), half-lives (T1/2) and correlation coefficients (R2) for the degradation of dinotefuran enantiomers in cucumber and soil Matrix
Application mode
Cucumber
Foliar spray Root douche
Soil
/
Enantiomers
k (d-1)
R2
T1/2(d)*
(+)-dinotefuran ()-dinotefuran (+)-dinotefuran ()-dinotefuran (+)-dinotefuran ()-dinotefuran
0.084 0.091 0.059 0.064 0.032 0.042
0.9673 0.9445 0.9894 0.9982 0.9492 0.9668
8.3a 7.6 a 11.7 a 10.8 a 21.7 a 16.5 b
*Significant difference (P < 0.05, one-way ANOVA) is indicated with different letters in the same test material.
Fig. 3. The degradation kinetics of dinotefuran enantiomers in cucumber after foliar spraying (A) and root douche (B) treatment, in soil (C) and the changes of EF values (D).
Degradation in Soil
The degradation kinetics of dinotefuran enantiomers also followed first-order kinetics in soil. It was observed that both dinotefuran enantiomers degraded slower than in cucumber and it was also (–)-dinotefuran that was preferentially degraded in soil. The mean half-lives were 21.7 d and 16.5 d for (+)-dinotefuran and (–)-dinotefuran, respectively (Table 3). Moreover, the half-lives in soil were significantly different between the two enantiomers (P < 0.01, one-way ANOVA). The EF value in soil was about 0.5 within the first 3 d after the treatment, and then gradually increased to 0.57 (Figure 3D). The data show that enantioselectivity existed between the two enantiomers of dinotefuran in the soil dissipation process. CONCLUSIONS
In this study, an effective method was successfully established and the dissipation behaviors of dinotefuran enantiomers were investigated in cucumber and soil. For cucumber, (+)-dinotefuran proved to be more persistent under both foliar spraying and root douche treatments. In soil, the degradation was in accordance with that in cucumber, with (–)-dinotefuran preferentially degraded. Certainly, further studies of dinotefuran on enantioselective degradation on other crops, interaction with enzymes, as well as metabolic products should be Chirality DOI 10.1002/chir
carried out to supply more accurate information. The results of this study may help to understand the dissipation of dinotefuran in the environment at enantiomeric levels. ACKNOWLEDGMENTS
This work was supported financially by the foundation established by the Nature Science Foundation of China (NSFC, 31272071). The authors declare no conflicts of interest. LITERATURE CITED 1. Sanchez-Hermosilla J, Rincon VJ, Paez F, Aguera F, Carvajal F. Field evaluation of a self-propelled sprayer and effects of the application rate on spray deposition and losses to the ground in greenhouse tomato crops. Pest Manag Sci 2011;67:942–947. 2. Juraske R, Anton A, Castells F, Huijbregts MAJ. Human intake fractions of pesticides via greenhouse tomato consumption: Comparing model estimates with measurements for Captan. Chemosphere 2007;67:1102–1107. 3. US.EPA, Pesticide Fact Sheet. http://www.epa.gov/opp00001/chem_search/ reg_actions/registration/fs_PC-044312_01-Sep-04.pdf 2004. 4. Zhou Y, Li L, Lin K, Zhu X, Liu W. Enantiomer separation of triazole fungicides by high-performance liquid chromatography. Chirality 2009;21: 421–427. 5. Li X, Bao C, Yang D, Zheng M, Tao S. Toxicities of fipronil enantiomers to the honeybee Apis mellifera L. and enantiomeric compositions of fipronil in honey plant flowers. Environ Toxicol Chem 2010;29:127–132. 6. Li J, Dong F, Xu J, Liu X, Li Y, Shan W, Zheng Y. Enantioselective determination of triazole fungice tetraconazole by chiral high-performance
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Chirality DOI 10.1002/chir
