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Dissipation kinetics and residue of LH-2010A in cucumber and soil under greenhouse condition
Regulatory Toxicology and Pharmacology 73 (2015) 732e736
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Dissipation kinetics and residue of LH-2010A in cucumber and soil under greenhouse condition Ruijuan Li, Tongjin Liu, Guochun Song, Rumei Li, Jianlei Yu* Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, Shandong, 250100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 May 2015 Received in revised form 30 July 2015 Accepted 26 October 2015 Available online 30 October 2015
LH-2010A is a newly developed fungicide with novel mode of action in the treatment of cucurbit downy mildew. Dissipation kinetics and residue levels of LH-2010A in cucumber were investigated using a QuEChERS method with GC-ECD. Field trials were constructed at three different sites in China in 2013 and 2014. The average recoveries of LH-2010A in fortified samples were between 94.0 and 106.2% for cucumber and between 84.4% and 98.7% for soil, with relative standard deviations within 3%. The dissipation rate of LH-2010A residue was evaluated assuming a pseudo first-order kinetics. The half-lives of LH-2010A were 2.8e4.2 days and 6.3e9.4 days in cucumber and soil samples, respectively. The terminal residues in cucumber were 0.077e0.207 mg/kg and 0.109e0.307 mg/kg on the first day after spray at the recommended dosage and 1.5 times of the recommended dosage, respectively. Using this rapid and sensitive method, we determined the dissipation kinetics and residue level of HL-2010A in cucumber. The suggested MRL value of HL-2010A in cucumber is 0.5 mg/kg. The research would provide guidance for proper and safe use of this newly developed fungicide in cucumber in greenhouse ecosystems. © 2015 Elsevier Inc. All rights reserved.
Keywords: LH-2010A Fungicide Cucumber Residue Dissipation QuEChERS
1. Introduction Cucurbit downy mildew is a foliage disease on plants, such as pumpkin, watermelon and cucumber, caused by the pathogen Pseudoperonospora cubensis (Lebeda and Cohen, 2010; Palti and Cohen, 1980). The disease has re-emerged in the US in 2004 on pathogen resistant cucumber cultivars and was estimated to have reduced 40% of cucumber production (Holmes et al., 2014, 2006). In China, downy mildew is also one of the most important diseases on cucumber. Although forecasting the occurrence and movement of the pathogens may provide useful information for disease control (Yang et al., 2007), chemical control is still one of the most effective means. Over the years, a number of fungicides targeting P. cubensis with different action modes have been developed for the treatment of downy mildew (Hausbeck, 2014). LH-2010A (IUPAC name: N-(3chloro-5-trifluoromethyl- pyridin-2-ylmethyl-2,3,5,6-tetrafluoro4-methoxy-benzamide) is a novel fungicidal agent (Commercial name: Kanuozi. Jinan, China) developed by Shandong Sino-Agri
* Corresponding author. 202 Gongye North Road, Jinan, Shandong, 250100, China. E-mail address: [email protected] (J. Yu). http://dx.doi.org/10.1016/j.yrtph.2015.10.026 0273-2300/© 2015 Elsevier Inc. All rights reserved.
Union Biotechnology Co. Ltd, China in 2010 (Zhang et al., 2014). LH-2010A is a white powder with a solubility of 4.5 mg/l in water. The structure of LH-2010A is shown in Fig. 1. LH-2010A was derived from fluopicolide, which is an effective fungicide with novel mode of action targeting many pathogens such as Phytophthora infestans, Plasmopora viticola, and P. cubensis (Toquin et al., 2007). Since pathogens are constantly developing resistance to fungicides, novel or modified fungicides are highly desirable. Compared with fluopicolide, LH-2010A is more effective and has a broader spectrum targeting plant pathogens, such as Rhizoctonia solani and Botrytis cinere (Zhang et al., 2013). In previous study, its uptake-translocate behavior was studied in cucumber and the efficacy of LH-2010A against P. cubensis was determined, with an EC50 of 6.70 mg/ml, which is slightly lower than that of fluopicolide (Zhang et al., 2014). LH-2010A exhibits great efficacies in field trials against downy mildew in cucumber and sheath blight in rice (Zhang et al., 2013, 2014). Improper and extensive use can lead to high residue level of fungicides in human or animal consumption; it may accumulate and pollute soil and groundwater (Fatta et al., 2007; Yi and Lu, 2006; Yu and Zhou, 2005). Until today, no studies have been reported on LH-2010A dissipation and residue distribution in cucumber, soil and other matrixes. In the present study, we developed
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2.3. Sample preparation
Fig. 1. The structure of LH-2010A.
a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method with a GC-ECD detector to evaluate the dissipation kinetics of LH-2010A in cucumber and soil and suggested the MRL for LH2010A. The research would provide guidance for proper and safe use of this newly developed fungicide in cucumber in greenhouse ecosystems.
2. Materials and methods 2.1. Chemicals LH-2010A (purity, 98.0%) and its commercial formulation (Kanuozi, 50% water dispersible granule) were obtained from Shandong Sino-Agri Union Biotechnology Co., Ltd, Jinan, China. High-performance liquid chromatography (HPLC) grade acetonitrile was purchased from Dikma Technologies Inc., Jinan, China. Other reagents and chemicals are analytical grade and were purchased from Jinan Chemical Works, Jinan, China. Stock solution of HL-2010A (100 mg/L) was prepared in ethyl acetate and stored at 20 C, Standard solutions (0.01, 0.05, 0.1, 0.5, and 1 mg/mL) were prepared from the stock by serial dilution. All solutions were protected from direct light by wrapping the containers with aluminum foil and were stored at 4 C.
2.2. Field trials Field trials were carried out in research stations at three different locations, including Jinan of Shandong province, Fengyang of Anhui province and Xinji of Hebei province, in 2013 and 2014. The experiments were designed following the guideline on pesticide residue trials (NY/T 788-2004, the Ministry of Agriculture of China). Both dissipation kinetics and residue determination experiments were carried out. A randomized block design was employed to conduct experiments in three replicates under greenhouse condition. Each experiment was carried out in a plot of 30 m2 and was separated by a buffer zone. Seven treatments were applied including six formulation treatments and one control treatment. All areas were subjected to routine horticultural treatment. The dissipation rate of LH-2010A in cucumber and soil was evaluated with experiments where LH-2010A was sprayed in the form of 50% WDG (water dispersible granule) at a dose of 1.5 times of the recommended maximum dosage (101.25 g a,i./ha). As a control, untreated plots were sprayed with pure water. Representative cucumber and soil samples were collected on day 0 (2 h), 1, 3, 5, 7, 14 and 21 after application of the fungicide. The residue experiment was conducted with 50% WDG spayed at a dose of 67.5 g a,i./ha (the recommended maximum dosage) and 101.25 g a,i./ha (1.5 times of the recommended maximum dosage), respectively. The application interval was set at 7 days and the fungicide was applied 4 times in each experiment. On day 1, 2, 3 and 5, cucumber and soil samples were collected.
The collected cucumber samples were cut into small pieces with a stainless steel knife and thoroughly mixed in a stainless steel pot. The samples were divided into four equal portions by quartering technique and two portions (150 g, each) were used for the subsequent experiments. The collected soil samples were air-dried, pulverized in a hammer mill and sieved to obtain granules with the same size (2 mm sieve). All samples were stored at 20 C for further analysis. 2.4. Extraction and purification 2.4.1. Soil samples Soil samples were first thawed at room temperature. 10.0 g soil samples were placed into a 100-ml conical flask with stopper, and were extracted with 60 ml of dichloromethane by ultrasonic extraction for 15 min. The solution was filtered through a column packed with anhydrous magnesium sulfate. The remaining solid soil sample was undergone ultrasonic extraction with an additional 40 ml of dichloromethane for 15 min and filtered through the same column packed with anhydrous magnesium sulfate. The filtrates were combined and concentrated to 0.5 ml with a rotary evaporator (Heidolph LABOROTA 4001, German) at 45 C. The concentrated extract was dried with a nitrogen drier, re-dissolved in 5 ml of ethyl acetate, and transferred into 10 ml PTEF centrifuge tube containing 10 mg PSA and 120 mg MgSO4. The extract was vigorously vortexed for 1 min and was centrifuged at 4200 rpm for 3 min. The supernatant was transferred into an autosampler vial for GC-ECD analysis. 2.4.2. Cucumber samples 10.0 g homogenized cucumber samples were weighed and placed into a 50 ml centrifuge tube with 20 ml acetonitrile. The sample was homogenized again for 2 min. The homogenate was filtered through a Whatman No.1 filter paper into a 100-ml graduated cylinder with stopper packed with 3 g NaCl. The tube was capped tightly and shaken vigorously for 1e2 min. After standing for 20 min, 8.0 ml aliquot of upper acetonitrile partition was transferred into a pear-shaped evaporating flask and dried by a rotary evaporator (Heidolph LABOROTA 4001, German) at 45 C. The extract was further dried with a nitrogen dryer, re-dissolved in 2 ml ethyl acetate, and transferred into 10 ml PTEF centrifuge tube containing 10 mg PSA, 50 mg GCB and 120 mg MgSO4. The extract solution was vortexed for 1 min and was centrifuged at 4200 rpm for 3 min. The supernatant was transferred into an autosampler vial for GC-ECD analysis. 2.5. Instrumental analysis LH-2010A was determined by an Agilent 7890B GC System equipped with a 63Ni micro-electron capture detector (mECD) system (Agilent Technologies, Palo Alto, CA, USA). Chromatographic separation was achieved using a capillary column (DB-1701 30 m 0.32 mm I.D. 0.25 mm film thickness analytical column). Nitrogen (99.999% purity) was used as the carrier gas to maintain the constant flow rate at 1.5 ml/min 1.0 ml sample was injected at 260 C in the splitless mode. The temperature for detector was set at 300 C. The oven temperature was operated as follows: the initial temperature was set at 80 C for 1 min and increased gradually to 180 C at a rate of 10 C/min, and to 240 C at a rate of 20 C/min. The temperature was held at 240 C for 3 min, increased to 250 C and held for another 10 min. The retention time of LH-2010A was approximately 15.8 min under these experimental conditions.
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2.6. Recovery test Fortified cucumber and soil samples were prepared using standard solutions to final concentration of LH-2010A of 0.005, 0.05 and 0.25 mg/kg. Recovery studies were carried out using these samples for method validation and optimization. The extraction procedures and GC analysis for these samples were the same as for the collected samples from the experiment stations. Each experiment was conducted in five replicates. 3. Results and discussion 3.1. Calibration curve Matrix effects can lead to signal enhancement/suppression in a sample because of the interaction between analyte and matrix components (Kwon et al., 2012). To compensate bias/interferences, calibration was performed using external matrix-matched standards for fungicide quantification in cucumber, while quantification in soil was accomplished by using standard stock solution in ethyl acetate. The standard calibration curve of LH-2010A was constructed by plotting its concentration against peak area. A typical chromatogram of LH-2010A is shown in Fig. 2. The calibration curve showed linearity when the concentration of LH-2010A was in the range of 0.01e1 mg/ml. The data of LH-2010A in soil were fitted into y ¼ 94433x 1450.4 with R2 of 0.9958; the data of LH-2010A in cucumber were fitted into y ¼ 228268x þ 52.462 with R2 of 0.9991. 3.2. Limit of detection and limit of quantification Limit of detection (LOD) and limit of quantification (LOQ) were determined as described in the method section. In this study, LOD is defined as the minimum concentration of a fungicide when the peak signal of the fungicide is three times of the noise signal. Accordingly, LOD for LH-2010A was estimated to be 0.01 ug/ml. LOQ is defined as the lowest concentration of a fungicide that could be quantitatively and accurately determined. LOQ for LH-2010A was calculated to be 0.005 mg/kg. 3.3. Precision and accuracy The method performance was evaluated and validated by using untreated cucumber samples applied with LH-2010A at three fortification levels, 0.005, 0.05 and 0.25 mg/kg. Five samples were prepared at each level. All the batches met the criteria for acceptable quality control (Podhorniak et al., 2001). The average recoveries were in the range between 84.4% and 106.2% with a RSD in
the range between 1.0% and 2.9%, as shown in Table 1. These values are within the recommended recovery range (70%e110%) with RSDs below 20% set by the guideline on pesticide residue trials (NY/ T 788-2004, the Ministry of Agriculture of China), indicating the method is suitable for dissipation and residue determination of LH2010A. 3.4. QuEChERS method The QuEChERS method was developed for analysis of a large array of fungicides simultaneously and was gradually modified to improve its performance (Bruzzoniti et al., 2014; Kwon et al., 2012; Lehotay et al., 2010; Pay a et al., 2007; Wilkowska and Biziuk, 2011). A number of solid-phase sorbents were developed for the removal of interfering substances (Michelangelo Anastassiades, 2003). For example, C18 is used for the removal of cholesterol, sterols and lipids and GCB is used for the removal of acids and sugars. For removal of pigments, a weak ion exchange (PSA), GCB, or C18 can be used (Michelangelo Anastassiades, 2003). GCB is likely a better choice than PSA as shown in previous studies (Ge et al., 2011; Li et al., 2008). By using PSA for the cleanup of soil samples, we obtained satisfactory results. However, pigments were not completely removed in cucumber samples after cleanup with PSA. Hence, an additional step was applied to completely remove pigments, by applying 50 mg GCB as the d-SPE sorbent with PSA. The recoveries were acceptable even at very low concentration of the fungicide. 3.5. Dissipation kinetics of LH-2010A Residue of LH-2010A in cucumber and soil was detected according to the aforementioned method under greenhouse condition. Statistical analysis was performed in accordance with the method proposed elsewhere (Timme and Frehse, 1980). The dissipation curves of LH-2010A in cucumber are showed in Fig. 3. The initial concentrations of LH-2010A in cucumber were in the range between 0.101 and 0.246 mg/kg at the three locations in 2013 and 2014. On average, more than 79.0% of LH-2010A was degraded after 7 days under greenhouse conditions. Fig. 4 shows the dissipation kinetics of LH-2010A in soil. The initial concentrations of LH-2010A in soil were in the range between 0.283 and 0.422 mg/kg at the three locations in 2013 and 2014. Concentration of LH-2010A was decreased more than 68.0% after 14 days. The dissipation of LH2010A residue in cucumber and soil was fitted onto the experimental data assuming a pseudo first-order kinetics with R2 in the range between 0.8356 and 0.9862. The dissipation kinetics LH-2010A and half-lives of LH-2010A in samples from the three locations are summarized in Table 2. The half-lives of LH-2010A in cucumber were very similar in the three locations, ranging from 2.8 to 4.2 days. Because the field trials were carried out in greenhouse condition at these three different locations, local climate had little effect on the dissipation of LH-2010A in cucumber. However, the half-lives of LH-2010A in soil varied in these three locations. The half-lives of LH-2010A in soil from Anhui were the longest, with an average of 9.4 and 8.8 days for year 2013
Table 1 Recovery of LH-2010A from cucumber and soil samples (n ¼ 5).
Fig. 2. Typical chromatogram of LH-2010A. The concentration of LH2010A used for this chromatogram was 0.1 mg/ml. LOD and LOQ were calculate to be 0.01 mg/ml and 0.005 mg/kg, respectively.
Matrix
Fortification level (mg/kg)
Recovery (%)
RSD (%)
Soil
0.005 0.05 0.25 0.005 0.05 0.25
84.4 97.0 98.7 94.0 106.2 103.2
2.9 1.8 1.0 2.5 2.0 1.9
Cucumber
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Fig. 3. The dissipation kinetics of LH-2010A in cucumber samples at the three locations in 2013 (A) and 2014 (B).
Fig. 4. The dissipation kinetics of LH-2010A in soil samples at the three locations in 2013 (A) and 2014 (B).
Table 2 First order kinetic equations and half-lives of LH-2010A in soil and cucumber samples under greenhouse conditions. Matrix
Date
Locality
First-order kinetic equation
Cucumber
2013
Shandong Anhui Hebei Shandong Anhui Hebei Shandong Anhui Hebei Shandong Anhui Hebei
C C C C C C C C C C C C
2014
Soil
2013
2014
¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼
0.20820.231t 0.20030.224t 0.17040.244t 0.18960.165t 0.13460.192t 0.08720.202t 0.35070.11t 0.34170.079t 0.24830.101t 0.42090.107t 0.39120.074t 0.13460.094t
and 2014, respectively. On the contrary, the average half-life was 6.3 and 6.5 days for samples from Shandong for year 2013 and 2014. In Hebei, the average half-life was 6.9 and 7.4 days for year 2013 and 2014, respectively. These results indicate the characteristics of local soil, such as composition and pH, have impacts on the dissipation of LH-2010A. Another important observation of our studies is that the dissipation of LH-2010A in soil is generally two times slower than in the cucumber samples and the presence of LH-2010A can provide longer effects. This should be taken note when determining the interval between application and before harvest. Fluopicolide, which is the parent compound of the newly synthesized herbicide, has the same mode of action as LH-2010A and targets many pathogens such as P. infestans, P. viticola, and P. cubensis (Toquin et al., 2007). Dissipation of fluopicolide has been studied in grape and tomato (Jiang et al., 2014; Mohapatra et al., 2011). The half-life of fluopicolide dissipation in grape berries is 10.2 days (Mohapatra et al., 2011). Fluopicolide dissipates in soil with a half-life of 5.33 days (Jiang et al., 2014). In comparison, LH2010A has similar dissipation rates. In a laboratory setting,
Correlation coefficient (R2)
Half-lives (days)
0.9616 0.8356 0.978 0.9744 0.8703 0.9735 0.9818 0.9714 0.9564 0.9543 0.9839 0.9862
3.0 3.1 2.8 4.2 3.6 3.4 6.3 8.8 6.9 6.5 9.4 7.4
resistance to fluopicolide has been introduced by UV irradiation (Lu et al., 2011; Wang et al., 2014) in Phytophthora capsici, which can cause Crown, root, and fruit rot. LH-2010A could be a great addition with the same mode of action for treatment of downy mildew and sheath blight (Zhang et al., 2013, 2014). 3.6. Terminal residues of LH-2010A We determined the residue levels of LH-2010A after application of the recommended dosage and an elevated dosage (1.5 times of recommended dosage) in the three different locations in 2013 and 2014. The levels of LH-2010A were in the range of 0.077e0.307 mg/ kg in cucumber, and in the range of 0.0905e0.835 mg/kg in soil on day 1, 2, 3 and 5 after application. Under the recommended dose, the concentration of LH-2010A residue in cucumber on day 1 after application was 0.0770e0.207 mg/kg; at 1.5 times of recommended dosage, the concentration of LH-2010A residue in cucumber on day 1 after application was 0.109e0.307 mg/kg. Maximum residue limit (MRL) of LH-2010A has not been set in any country or organization.
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The MRL of fluopicolide, a fungicide with the same mode of action in cucumber is 0.5 mg/kg in China (Ministry of Agriculture, P. R. China and National Health and Family Planning Commission, P.R. China 2014), and the MRL of fluopyram in cucumber is 0.5 mg/kg suggested by EU (European commission 2005). Therefore, we suggest the MRL of LH-2010A in cucumber to be 0.5 mg/kg. Dosages of 45e67.5 g active Ingredient/ha should be applied at least one day before harvest to be considered safe for the health of human beings. 4. Conclusion LH-2010A is a newly developed fungicide with novel mode of action targeting a broad spectrum of pathogens. In this study, a rapid and sensitive method was successfully developed to evaluate dissipation kinetics and residue level of HL-2010A in cucumber and soil samples from three different locations in 2013 and 2014. Our results show that the dissipation of LH-2010A followed a pseudo first-order kinetics, with the half-lives in the range of 2.8e4.2 days and 6.3e9.4 days for LH-2010A residues in cucumber and soil samples, respectively. The suggested MRL value of cucumber is 0.5 mg/kg. The pre-harvest interval should be at least one day after the spray of LH-2010A at a 1.5 times of the recommended maximum dose (67.5 g (a.i.)/ha). Author contributions Jianlei Yu designed the experiments. Ruijuan Li, Tongjin Liu, Guochun Song, Rumei Li performed the experiments. Guochun Song and Ruijuan Li analyzed the data. Ruijuan Li wrote the paper. Acknowledgments This work is supported financially by funding (No: 2012F431) from the Ministry of Agricultural of China. We thank Shandong Sino-agri Union Biotechnology Co. Ltd for providing LH-2010A and its commercial formulation. Transparency document Transparency document related to this article can be found at http://dx.doi.org/10.1016/j.yrtph.2015.10.026. References Bruzzoniti, M.C., Checchini, L., Carlo, R.M.D., Orlandini, S., Rivoira, L., Bubba, M.D., 2014. QuEChERS sample preparation for the determination of pesticides and other organic residues in environmental matrices: a critical review. Anal. Bioanal. Chem. 406, 4089e4116. http://dx.doi.org/10.1007/s00216-014-7798-4. Fatta, D., Canna-Michaelidou, S., Michael, C., Demetriou Georgiou, E., Christodoulidou, M., Achilleos, A., Vasquez, M., 2007. Organochlorine and organophosphoric insecticides, herbicides and heavy metals residue in industrial wastewaters in Cyprus. J. Hazard. Mater. 145, 169e179. http://dx.doi.org/ 10.1016/j.jhazmat.2006.11.009. Ge, J., Zhao, L.W., Liu, C., Jiang, S., Lee, P.W., Liu, F., 2011. Rapid determination of melamine in soil and strawberry by liquid chromatographyetandem mass spectrometry. Food Control 22, 1629e1633. http://dx.doi.org/10.1016/ j.foodcont.2011.03.020. Hausbeck, M., 2014. Downy Mildew Watch: Fungicides Recommended for Cucumber Disease Control [WWW Document]. MSU Ext. URL. http://msue.anr. msu.edu/news/downy_mildew_watch_fungicides_recommended_for_ cucumber_disease_control (accessed 28.03.15.). Holmes, G., Ojiambo, P.S., Hausbeck, M., Quesada-Ocampo, L.M., Keinath, A.P., 2014. Resurgence of cucurbit downy mildew in the United States: a watershed event for research and extension. Plant Dis. http://dx.doi.org/10.1094/PDIS-09-140990-FE.
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Dissipation kinetics and residue of LH-2010A in cucumber and soil under greenhouse condition Ruijuan Li, Tongjin Liu, Guochun Song, Rumei Li, Jianlei Yu* Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, Shandong, 250100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 May 2015 Received in revised form 30 July 2015 Accepted 26 October 2015 Available online 30 October 2015
LH-2010A is a newly developed fungicide with novel mode of action in the treatment of cucurbit downy mildew. Dissipation kinetics and residue levels of LH-2010A in cucumber were investigated using a QuEChERS method with GC-ECD. Field trials were constructed at three different sites in China in 2013 and 2014. The average recoveries of LH-2010A in fortified samples were between 94.0 and 106.2% for cucumber and between 84.4% and 98.7% for soil, with relative standard deviations within 3%. The dissipation rate of LH-2010A residue was evaluated assuming a pseudo first-order kinetics. The half-lives of LH-2010A were 2.8e4.2 days and 6.3e9.4 days in cucumber and soil samples, respectively. The terminal residues in cucumber were 0.077e0.207 mg/kg and 0.109e0.307 mg/kg on the first day after spray at the recommended dosage and 1.5 times of the recommended dosage, respectively. Using this rapid and sensitive method, we determined the dissipation kinetics and residue level of HL-2010A in cucumber. The suggested MRL value of HL-2010A in cucumber is 0.5 mg/kg. The research would provide guidance for proper and safe use of this newly developed fungicide in cucumber in greenhouse ecosystems. © 2015 Elsevier Inc. All rights reserved.
Keywords: LH-2010A Fungicide Cucumber Residue Dissipation QuEChERS
1. Introduction Cucurbit downy mildew is a foliage disease on plants, such as pumpkin, watermelon and cucumber, caused by the pathogen Pseudoperonospora cubensis (Lebeda and Cohen, 2010; Palti and Cohen, 1980). The disease has re-emerged in the US in 2004 on pathogen resistant cucumber cultivars and was estimated to have reduced 40% of cucumber production (Holmes et al., 2014, 2006). In China, downy mildew is also one of the most important diseases on cucumber. Although forecasting the occurrence and movement of the pathogens may provide useful information for disease control (Yang et al., 2007), chemical control is still one of the most effective means. Over the years, a number of fungicides targeting P. cubensis with different action modes have been developed for the treatment of downy mildew (Hausbeck, 2014). LH-2010A (IUPAC name: N-(3chloro-5-trifluoromethyl- pyridin-2-ylmethyl-2,3,5,6-tetrafluoro4-methoxy-benzamide) is a novel fungicidal agent (Commercial name: Kanuozi. Jinan, China) developed by Shandong Sino-Agri
* Corresponding author. 202 Gongye North Road, Jinan, Shandong, 250100, China. E-mail address: [email protected] (J. Yu). http://dx.doi.org/10.1016/j.yrtph.2015.10.026 0273-2300/© 2015 Elsevier Inc. All rights reserved.
Union Biotechnology Co. Ltd, China in 2010 (Zhang et al., 2014). LH-2010A is a white powder with a solubility of 4.5 mg/l in water. The structure of LH-2010A is shown in Fig. 1. LH-2010A was derived from fluopicolide, which is an effective fungicide with novel mode of action targeting many pathogens such as Phytophthora infestans, Plasmopora viticola, and P. cubensis (Toquin et al., 2007). Since pathogens are constantly developing resistance to fungicides, novel or modified fungicides are highly desirable. Compared with fluopicolide, LH-2010A is more effective and has a broader spectrum targeting plant pathogens, such as Rhizoctonia solani and Botrytis cinere (Zhang et al., 2013). In previous study, its uptake-translocate behavior was studied in cucumber and the efficacy of LH-2010A against P. cubensis was determined, with an EC50 of 6.70 mg/ml, which is slightly lower than that of fluopicolide (Zhang et al., 2014). LH-2010A exhibits great efficacies in field trials against downy mildew in cucumber and sheath blight in rice (Zhang et al., 2013, 2014). Improper and extensive use can lead to high residue level of fungicides in human or animal consumption; it may accumulate and pollute soil and groundwater (Fatta et al., 2007; Yi and Lu, 2006; Yu and Zhou, 2005). Until today, no studies have been reported on LH-2010A dissipation and residue distribution in cucumber, soil and other matrixes. In the present study, we developed
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2.3. Sample preparation
Fig. 1. The structure of LH-2010A.
a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method with a GC-ECD detector to evaluate the dissipation kinetics of LH-2010A in cucumber and soil and suggested the MRL for LH2010A. The research would provide guidance for proper and safe use of this newly developed fungicide in cucumber in greenhouse ecosystems.
2. Materials and methods 2.1. Chemicals LH-2010A (purity, 98.0%) and its commercial formulation (Kanuozi, 50% water dispersible granule) were obtained from Shandong Sino-Agri Union Biotechnology Co., Ltd, Jinan, China. High-performance liquid chromatography (HPLC) grade acetonitrile was purchased from Dikma Technologies Inc., Jinan, China. Other reagents and chemicals are analytical grade and were purchased from Jinan Chemical Works, Jinan, China. Stock solution of HL-2010A (100 mg/L) was prepared in ethyl acetate and stored at 20 C, Standard solutions (0.01, 0.05, 0.1, 0.5, and 1 mg/mL) were prepared from the stock by serial dilution. All solutions were protected from direct light by wrapping the containers with aluminum foil and were stored at 4 C.
2.2. Field trials Field trials were carried out in research stations at three different locations, including Jinan of Shandong province, Fengyang of Anhui province and Xinji of Hebei province, in 2013 and 2014. The experiments were designed following the guideline on pesticide residue trials (NY/T 788-2004, the Ministry of Agriculture of China). Both dissipation kinetics and residue determination experiments were carried out. A randomized block design was employed to conduct experiments in three replicates under greenhouse condition. Each experiment was carried out in a plot of 30 m2 and was separated by a buffer zone. Seven treatments were applied including six formulation treatments and one control treatment. All areas were subjected to routine horticultural treatment. The dissipation rate of LH-2010A in cucumber and soil was evaluated with experiments where LH-2010A was sprayed in the form of 50% WDG (water dispersible granule) at a dose of 1.5 times of the recommended maximum dosage (101.25 g a,i./ha). As a control, untreated plots were sprayed with pure water. Representative cucumber and soil samples were collected on day 0 (2 h), 1, 3, 5, 7, 14 and 21 after application of the fungicide. The residue experiment was conducted with 50% WDG spayed at a dose of 67.5 g a,i./ha (the recommended maximum dosage) and 101.25 g a,i./ha (1.5 times of the recommended maximum dosage), respectively. The application interval was set at 7 days and the fungicide was applied 4 times in each experiment. On day 1, 2, 3 and 5, cucumber and soil samples were collected.
The collected cucumber samples were cut into small pieces with a stainless steel knife and thoroughly mixed in a stainless steel pot. The samples were divided into four equal portions by quartering technique and two portions (150 g, each) were used for the subsequent experiments. The collected soil samples were air-dried, pulverized in a hammer mill and sieved to obtain granules with the same size (2 mm sieve). All samples were stored at 20 C for further analysis. 2.4. Extraction and purification 2.4.1. Soil samples Soil samples were first thawed at room temperature. 10.0 g soil samples were placed into a 100-ml conical flask with stopper, and were extracted with 60 ml of dichloromethane by ultrasonic extraction for 15 min. The solution was filtered through a column packed with anhydrous magnesium sulfate. The remaining solid soil sample was undergone ultrasonic extraction with an additional 40 ml of dichloromethane for 15 min and filtered through the same column packed with anhydrous magnesium sulfate. The filtrates were combined and concentrated to 0.5 ml with a rotary evaporator (Heidolph LABOROTA 4001, German) at 45 C. The concentrated extract was dried with a nitrogen drier, re-dissolved in 5 ml of ethyl acetate, and transferred into 10 ml PTEF centrifuge tube containing 10 mg PSA and 120 mg MgSO4. The extract was vigorously vortexed for 1 min and was centrifuged at 4200 rpm for 3 min. The supernatant was transferred into an autosampler vial for GC-ECD analysis. 2.4.2. Cucumber samples 10.0 g homogenized cucumber samples were weighed and placed into a 50 ml centrifuge tube with 20 ml acetonitrile. The sample was homogenized again for 2 min. The homogenate was filtered through a Whatman No.1 filter paper into a 100-ml graduated cylinder with stopper packed with 3 g NaCl. The tube was capped tightly and shaken vigorously for 1e2 min. After standing for 20 min, 8.0 ml aliquot of upper acetonitrile partition was transferred into a pear-shaped evaporating flask and dried by a rotary evaporator (Heidolph LABOROTA 4001, German) at 45 C. The extract was further dried with a nitrogen dryer, re-dissolved in 2 ml ethyl acetate, and transferred into 10 ml PTEF centrifuge tube containing 10 mg PSA, 50 mg GCB and 120 mg MgSO4. The extract solution was vortexed for 1 min and was centrifuged at 4200 rpm for 3 min. The supernatant was transferred into an autosampler vial for GC-ECD analysis. 2.5. Instrumental analysis LH-2010A was determined by an Agilent 7890B GC System equipped with a 63Ni micro-electron capture detector (mECD) system (Agilent Technologies, Palo Alto, CA, USA). Chromatographic separation was achieved using a capillary column (DB-1701 30 m 0.32 mm I.D. 0.25 mm film thickness analytical column). Nitrogen (99.999% purity) was used as the carrier gas to maintain the constant flow rate at 1.5 ml/min 1.0 ml sample was injected at 260 C in the splitless mode. The temperature for detector was set at 300 C. The oven temperature was operated as follows: the initial temperature was set at 80 C for 1 min and increased gradually to 180 C at a rate of 10 C/min, and to 240 C at a rate of 20 C/min. The temperature was held at 240 C for 3 min, increased to 250 C and held for another 10 min. The retention time of LH-2010A was approximately 15.8 min under these experimental conditions.
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2.6. Recovery test Fortified cucumber and soil samples were prepared using standard solutions to final concentration of LH-2010A of 0.005, 0.05 and 0.25 mg/kg. Recovery studies were carried out using these samples for method validation and optimization. The extraction procedures and GC analysis for these samples were the same as for the collected samples from the experiment stations. Each experiment was conducted in five replicates. 3. Results and discussion 3.1. Calibration curve Matrix effects can lead to signal enhancement/suppression in a sample because of the interaction between analyte and matrix components (Kwon et al., 2012). To compensate bias/interferences, calibration was performed using external matrix-matched standards for fungicide quantification in cucumber, while quantification in soil was accomplished by using standard stock solution in ethyl acetate. The standard calibration curve of LH-2010A was constructed by plotting its concentration against peak area. A typical chromatogram of LH-2010A is shown in Fig. 2. The calibration curve showed linearity when the concentration of LH-2010A was in the range of 0.01e1 mg/ml. The data of LH-2010A in soil were fitted into y ¼ 94433x 1450.4 with R2 of 0.9958; the data of LH-2010A in cucumber were fitted into y ¼ 228268x þ 52.462 with R2 of 0.9991. 3.2. Limit of detection and limit of quantification Limit of detection (LOD) and limit of quantification (LOQ) were determined as described in the method section. In this study, LOD is defined as the minimum concentration of a fungicide when the peak signal of the fungicide is three times of the noise signal. Accordingly, LOD for LH-2010A was estimated to be 0.01 ug/ml. LOQ is defined as the lowest concentration of a fungicide that could be quantitatively and accurately determined. LOQ for LH-2010A was calculated to be 0.005 mg/kg. 3.3. Precision and accuracy The method performance was evaluated and validated by using untreated cucumber samples applied with LH-2010A at three fortification levels, 0.005, 0.05 and 0.25 mg/kg. Five samples were prepared at each level. All the batches met the criteria for acceptable quality control (Podhorniak et al., 2001). The average recoveries were in the range between 84.4% and 106.2% with a RSD in
the range between 1.0% and 2.9%, as shown in Table 1. These values are within the recommended recovery range (70%e110%) with RSDs below 20% set by the guideline on pesticide residue trials (NY/ T 788-2004, the Ministry of Agriculture of China), indicating the method is suitable for dissipation and residue determination of LH2010A. 3.4. QuEChERS method The QuEChERS method was developed for analysis of a large array of fungicides simultaneously and was gradually modified to improve its performance (Bruzzoniti et al., 2014; Kwon et al., 2012; Lehotay et al., 2010; Pay a et al., 2007; Wilkowska and Biziuk, 2011). A number of solid-phase sorbents were developed for the removal of interfering substances (Michelangelo Anastassiades, 2003). For example, C18 is used for the removal of cholesterol, sterols and lipids and GCB is used for the removal of acids and sugars. For removal of pigments, a weak ion exchange (PSA), GCB, or C18 can be used (Michelangelo Anastassiades, 2003). GCB is likely a better choice than PSA as shown in previous studies (Ge et al., 2011; Li et al., 2008). By using PSA for the cleanup of soil samples, we obtained satisfactory results. However, pigments were not completely removed in cucumber samples after cleanup with PSA. Hence, an additional step was applied to completely remove pigments, by applying 50 mg GCB as the d-SPE sorbent with PSA. The recoveries were acceptable even at very low concentration of the fungicide. 3.5. Dissipation kinetics of LH-2010A Residue of LH-2010A in cucumber and soil was detected according to the aforementioned method under greenhouse condition. Statistical analysis was performed in accordance with the method proposed elsewhere (Timme and Frehse, 1980). The dissipation curves of LH-2010A in cucumber are showed in Fig. 3. The initial concentrations of LH-2010A in cucumber were in the range between 0.101 and 0.246 mg/kg at the three locations in 2013 and 2014. On average, more than 79.0% of LH-2010A was degraded after 7 days under greenhouse conditions. Fig. 4 shows the dissipation kinetics of LH-2010A in soil. The initial concentrations of LH-2010A in soil were in the range between 0.283 and 0.422 mg/kg at the three locations in 2013 and 2014. Concentration of LH-2010A was decreased more than 68.0% after 14 days. The dissipation of LH2010A residue in cucumber and soil was fitted onto the experimental data assuming a pseudo first-order kinetics with R2 in the range between 0.8356 and 0.9862. The dissipation kinetics LH-2010A and half-lives of LH-2010A in samples from the three locations are summarized in Table 2. The half-lives of LH-2010A in cucumber were very similar in the three locations, ranging from 2.8 to 4.2 days. Because the field trials were carried out in greenhouse condition at these three different locations, local climate had little effect on the dissipation of LH-2010A in cucumber. However, the half-lives of LH-2010A in soil varied in these three locations. The half-lives of LH-2010A in soil from Anhui were the longest, with an average of 9.4 and 8.8 days for year 2013
Table 1 Recovery of LH-2010A from cucumber and soil samples (n ¼ 5).
Fig. 2. Typical chromatogram of LH-2010A. The concentration of LH2010A used for this chromatogram was 0.1 mg/ml. LOD and LOQ were calculate to be 0.01 mg/ml and 0.005 mg/kg, respectively.
Matrix
Fortification level (mg/kg)
Recovery (%)
RSD (%)
Soil
0.005 0.05 0.25 0.005 0.05 0.25
84.4 97.0 98.7 94.0 106.2 103.2
2.9 1.8 1.0 2.5 2.0 1.9
Cucumber
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Fig. 3. The dissipation kinetics of LH-2010A in cucumber samples at the three locations in 2013 (A) and 2014 (B).
Fig. 4. The dissipation kinetics of LH-2010A in soil samples at the three locations in 2013 (A) and 2014 (B).
Table 2 First order kinetic equations and half-lives of LH-2010A in soil and cucumber samples under greenhouse conditions. Matrix
Date
Locality
First-order kinetic equation
Cucumber
2013
Shandong Anhui Hebei Shandong Anhui Hebei Shandong Anhui Hebei Shandong Anhui Hebei
C C C C C C C C C C C C
2014
Soil
2013
2014
¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼
0.20820.231t 0.20030.224t 0.17040.244t 0.18960.165t 0.13460.192t 0.08720.202t 0.35070.11t 0.34170.079t 0.24830.101t 0.42090.107t 0.39120.074t 0.13460.094t
and 2014, respectively. On the contrary, the average half-life was 6.3 and 6.5 days for samples from Shandong for year 2013 and 2014. In Hebei, the average half-life was 6.9 and 7.4 days for year 2013 and 2014, respectively. These results indicate the characteristics of local soil, such as composition and pH, have impacts on the dissipation of LH-2010A. Another important observation of our studies is that the dissipation of LH-2010A in soil is generally two times slower than in the cucumber samples and the presence of LH-2010A can provide longer effects. This should be taken note when determining the interval between application and before harvest. Fluopicolide, which is the parent compound of the newly synthesized herbicide, has the same mode of action as LH-2010A and targets many pathogens such as P. infestans, P. viticola, and P. cubensis (Toquin et al., 2007). Dissipation of fluopicolide has been studied in grape and tomato (Jiang et al., 2014; Mohapatra et al., 2011). The half-life of fluopicolide dissipation in grape berries is 10.2 days (Mohapatra et al., 2011). Fluopicolide dissipates in soil with a half-life of 5.33 days (Jiang et al., 2014). In comparison, LH2010A has similar dissipation rates. In a laboratory setting,
Correlation coefficient (R2)
Half-lives (days)
0.9616 0.8356 0.978 0.9744 0.8703 0.9735 0.9818 0.9714 0.9564 0.9543 0.9839 0.9862
3.0 3.1 2.8 4.2 3.6 3.4 6.3 8.8 6.9 6.5 9.4 7.4
resistance to fluopicolide has been introduced by UV irradiation (Lu et al., 2011; Wang et al., 2014) in Phytophthora capsici, which can cause Crown, root, and fruit rot. LH-2010A could be a great addition with the same mode of action for treatment of downy mildew and sheath blight (Zhang et al., 2013, 2014). 3.6. Terminal residues of LH-2010A We determined the residue levels of LH-2010A after application of the recommended dosage and an elevated dosage (1.5 times of recommended dosage) in the three different locations in 2013 and 2014. The levels of LH-2010A were in the range of 0.077e0.307 mg/ kg in cucumber, and in the range of 0.0905e0.835 mg/kg in soil on day 1, 2, 3 and 5 after application. Under the recommended dose, the concentration of LH-2010A residue in cucumber on day 1 after application was 0.0770e0.207 mg/kg; at 1.5 times of recommended dosage, the concentration of LH-2010A residue in cucumber on day 1 after application was 0.109e0.307 mg/kg. Maximum residue limit (MRL) of LH-2010A has not been set in any country or organization.
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The MRL of fluopicolide, a fungicide with the same mode of action in cucumber is 0.5 mg/kg in China (Ministry of Agriculture, P. R. China and National Health and Family Planning Commission, P.R. China 2014), and the MRL of fluopyram in cucumber is 0.5 mg/kg suggested by EU (European commission 2005). Therefore, we suggest the MRL of LH-2010A in cucumber to be 0.5 mg/kg. Dosages of 45e67.5 g active Ingredient/ha should be applied at least one day before harvest to be considered safe for the health of human beings. 4. Conclusion LH-2010A is a newly developed fungicide with novel mode of action targeting a broad spectrum of pathogens. In this study, a rapid and sensitive method was successfully developed to evaluate dissipation kinetics and residue level of HL-2010A in cucumber and soil samples from three different locations in 2013 and 2014. Our results show that the dissipation of LH-2010A followed a pseudo first-order kinetics, with the half-lives in the range of 2.8e4.2 days and 6.3e9.4 days for LH-2010A residues in cucumber and soil samples, respectively. The suggested MRL value of cucumber is 0.5 mg/kg. The pre-harvest interval should be at least one day after the spray of LH-2010A at a 1.5 times of the recommended maximum dose (67.5 g (a.i.)/ha). Author contributions Jianlei Yu designed the experiments. Ruijuan Li, Tongjin Liu, Guochun Song, Rumei Li performed the experiments. Guochun Song and Ruijuan Li analyzed the data. Ruijuan Li wrote the paper. Acknowledgments This work is supported financially by funding (No: 2012F431) from the Ministry of Agricultural of China. We thank Shandong Sino-agri Union Biotechnology Co. Ltd for providing LH-2010A and its commercial formulation. Transparency document Transparency document related to this article can be found at http://dx.doi.org/10.1016/j.yrtph.2015.10.026. References Bruzzoniti, M.C., Checchini, L., Carlo, R.M.D., Orlandini, S., Rivoira, L., Bubba, M.D., 2014. QuEChERS sample preparation for the determination of pesticides and other organic residues in environmental matrices: a critical review. Anal. Bioanal. Chem. 406, 4089e4116. http://dx.doi.org/10.1007/s00216-014-7798-4. Fatta, D., Canna-Michaelidou, S., Michael, C., Demetriou Georgiou, E., Christodoulidou, M., Achilleos, A., Vasquez, M., 2007. Organochlorine and organophosphoric insecticides, herbicides and heavy metals residue in industrial wastewaters in Cyprus. J. Hazard. Mater. 145, 169e179. http://dx.doi.org/ 10.1016/j.jhazmat.2006.11.009. Ge, J., Zhao, L.W., Liu, C., Jiang, S., Lee, P.W., Liu, F., 2011. Rapid determination of melamine in soil and strawberry by liquid chromatographyetandem mass spectrometry. Food Control 22, 1629e1633. http://dx.doi.org/10.1016/ j.foodcont.2011.03.020. Hausbeck, M., 2014. Downy Mildew Watch: Fungicides Recommended for Cucumber Disease Control [WWW Document]. MSU Ext. URL. http://msue.anr. msu.edu/news/downy_mildew_watch_fungicides_recommended_for_ cucumber_disease_control (accessed 28.03.15.). Holmes, G., Ojiambo, P.S., Hausbeck, M., Quesada-Ocampo, L.M., Keinath, A.P., 2014. Resurgence of cucurbit downy mildew in the United States: a watershed event for research and extension. Plant Dis. http://dx.doi.org/10.1094/PDIS-09-140990-FE.
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