Dissipation rate of acetamiprid in sweet cherries

Sanja Lazić1, Dragana Šunjka1*, Srđan Panić1, Dušanka Inđić1, Nada Grahovac2, Valéria Guzsvány3 and Pavle Jovanov4 1 University of Novi Sad, Faculty of Agriculture, Department of Environmental and Plant Protection 2 Institute of Field and Vegetable Crops, Novi Sad 3 University of Novi Sad, Faculty of Sciences 4 University of Novi Sad, Institute of Food Technology *(draganas@polj.uns.ac.rs) Received: November 7, 2013 Accepted: December 27, 2013


INTRODUCTION
The use of pesticides in contemporary agricultural production helps to increase yields and improve the quality of products.However, their inadequate usage leads to accumulation of their residues in the environment and agricultural products.This is especially important for fruits and vegetables that are mostly consumed fresh.One of such fruit species is the sweet cherry as only 15% of its overall production is planned for processing (Commission of the European Communities, 2006).
Sweet cherry (Prunus avium L.) is a highly valued fruit species owing to its pleasant taste, but also because of its nutritive value.It is a significant species with considerable potentials in terms of export to the international market.As the acreage of this crop is increasing worldwide, the producers are facing a challenge to their efforts to produce quality fruit despite the increasing pressures from harmful agents.One of the most important pests of the sweet cherry is the cherry fly (Rhagoletis cerasi L.).Its attack results in a reduced market value of fruits, and they additionally become susceptible to saprophytes and frequently to rot, and fall prematurely.The control of this pest is a great challenge in integrated as well as conventional systems of production, and both in terms of ecotoxicology and pesticide residues (Kovanci and Kovanci, 2006).
Its control is successfully performed by insecticides of the group of organophosphates.However, their application time and toxicity, and especially the short period of fruit ripening and the violation of its prescribed harvest waiting period create preconditions for residues of these compounds to occur in sweet cherry fruits.Good agricultural practice therefore requires that protection be performed with products that have shorter harvest waiting periods and more convenient ecotoxicological properties than the insecticides used at earlier times (Lazić et al., 2012).
Acetamiprid, a neonicotinoid insecticide, has been introduced as an alternative to organosphosphate insecticides for control of major cherry pests (Table 1).It is an antagonist of the acetylcholine receptor in postsynapses of insects that has excellent systemic properties, relatively low toxicity to warm-blooded organisms and long lasting effect.The pre-harvest period for acetamiprid in sweet cherries is 14 days (Sekulić and Jeličić, 2013).
However, due to a growing use of insecticides from the family of neonicotinoids, their increased presence in the environment is evident.The European Commision has adopted a proposal for a two-year restriction on the use (as of 1 December 2013) of three pesticides belonging to the neonicotinoid family (clothianidin, imidacloprid and thiamethoxam) (Official website of the European Commission, 2013).
Besides its positive effects, acetamiprid also poses various health risks to consumers (Lazić et al., 2012a).Pesticide residues can be found even when products are applied in accordance with good agricultural practices.The European Union specified a maximum residue level (MRL) for acetamiprid in sweet cherries of 0.5 mg/kg [Commission Regulation (EU) No. 978/2011], while it is 1.5 mg/kg under the EU Reg.No. 500/2013.The maximum permissible level for acetamiprid in sweet cherries set by the Serbian legislation is 0.2 mg/kg (Pravilnik, 2010).
The dissipation rate of pesticides after application depends on many factors, including their chemical and photochemical degradation, climatic conditions, volatilization, cultivated species, formulation class and application method (Sur et al., 2000).
The aim of this study was to generate data showing the persistence and residue levels of acetamiprid in sweet cherry fruits under controlled conditions.The extraction of acetamiprid from sweet cherry samples was done using a QuEChERS-based method with insecticide determination and quantification performed by HPLC/DAD.

Field experiment
The study was conducted in a sweet cherry orchard located at Stepanovićevo village near Novi Sad.In order to protect the sweet cherry crop from Rhagoletis cerasi L., a commercial formulation (SP) with 200 g/kg acetamiprid active ingredient was used.The insecticide was applied by a portable hand sprayer and the solution was prepared at the recommended concentration of 0.025%, according to the manufacturer's instructions.and 2, 4, 6,  8, 10, 12 and 14 days after application.The samples were collected from different heights.The weight of each laboratory sample of sweet cherries was approximately 500 g.The samples were packed in plastic bags, hand delivered to a laboratory freezer and stored at -20 °C.Each sample was represented by triplicates used for calculation of the mean values of acetamiprid residue levels.

Chemicals and solutions
A certified standard of acetamiprid (purity 98.1%) was purchased from Dr Ehrenstorfer (Augsburg, Germany).The extraction solvent acetonitrile (ACN), of a suitable grade (HPLC) for pesticide residue analysis, and CH-3 COOH were purchased from J.T. Baker (Germany).
The dispersive SP extraction (Cat.No. 5982-5650) and clean-up (Cat.No. 5982-5056) kits for QuECh-ERS sample preparation were purchased as ready-to-use from Agilent Technologies (USA).The water was purified with a water purification system (TKA, Germany).
A stock solution of acetamiprid was prepared in acetonitrile at a concentration of 100 µg/ml and stored at -10 °C, in the dark.Calibration solutions for the HPLC analysis were prepared by further dilution with acetonitrile, achieving concentrations in a range from 0.05 to 2.5 µg/ml.

Validation of the analytical method
The extraction and determination procedures had been optimized in our previous study (Lazić et al., 2013).Insecticide determination and quantification were performed by HPLC with diode-array detection (Agilent 1100 Series LC system, United States) and Agilent Zorbax Eclipse C18 column (50 mm × 4.6 mm internal diameter, 1.8 µm particle size).The analysis was done under the conditions described below (Table 2).Under the selected conditions, the linearity of detector response was evaluated at a concentration range between 0.05 and 2.5 µg/ml using nine calibration solutions prepared in acetonitrile.Calculations were done using the peak areas and linear regression was used for the calibration curve.The linearity of calibration curves was expressed by regression equation and the correlation coefficient (R 2 ).
The limit of detection was determined at a signalto-noise ratio of three, whereas the limit of quantification (LOQ) was determined by considering a signalto-noise ratio of 10.
The accuracy and precision of the proposed method were evaluated by spiking blank sweet cherry samples to fulfill all the necessary requirements of SAN-CO/12495/2011 -the Method Validation and Quality Control Procedures for Pesticide Residues Analyses in Food and Feed (EU Commission Health and Consumer Protection Directorate-General, 2011).
Precision was evaluated through the repeatability of acetamiprid determination.Repeatability was checked by injecting 2.5 µl of acetamiprid standard in the matrix (0.75 and 1.5 µg/ml, respectively) five times.The samples were analyzed on the same day, using the same instrument by the same operator, and repeatability was calculated as a relative standard deviation (RSD%).
The accuracy of the proposed method was evaluated as the mean recovery (%) at three spiking levels.According to the EU validation guideline for pesticide residues, the mean recovery values should be within the range of 70-120% at each spiking level with RSDs≤20%.

Sample extraction
The extraction of acetamiprid from sweet cherry samples was performed using the QuEChERS-based method (Anastassiades et al., 2003).Each homogenized sweet cherry sample (10 g) was weighed into a polypropylene tube (50 ml volume), 10 ml volume of ACN was added as an extraction solvent and the tube was tightly capped and vigorously shaken for 1 min.A mix of buffered salts (1000 mg of sodium citrate, 500 mg of sodium hydrogen citrate sesquihydrate, 4000 mg magnesium sulphate and 1000 mg sodium chloride) from separate pouches was added to each tube and immediately mixed for 1 min.After that, the tube was centrifuged at 3000 rpm for 5 min (Sigma, Germany).An aliquot of 6 ml of the upper acetonitrile layer was transferred to each 15 ml centrifuge tube containing the sorbent, 150 mg of primary-secondary amine (PSA) and 900 mg of magnesium sulphate.The tubes were vigorously mixed for 1 min and then centrifuged at 3000 rpm for 5 min.An aliquot of the final upper layer was filtered through a 0.45 µm membrane filter and transferred into an autosampler vial for HPLC/DAD analyses.

Method validation
In some official methods, acetamiprid is determined by the GC/NPD (Ministry of Health and Welfare of Japan, 1997).However, GC determination can produce overestimated values of highly polar compounds such as acetamiprid because of the matrix-induced enhancement effect (Sasaki et al., 1998).For determination of other neonicotionoids, such as imidacloprid, the highperformance liquid chromatography is already commonly used (Mandić et al., 2005).In this study, acetamiprid was determined by HPLC.HPLC/DAD chromatograms of acetamiprid standard in acetonitrile and in spiked sweet cheery samples at a concentration of 0.75 µg/ml are shown in Figure 1.UV apex spectrums of acetamiprid in ACN and in matrix are illustrated in Figure 2.
In the studied range of mass concentrations of acetamiprid, a good linearity of detector response was achieved.The obtained values suggest that the increase in concentration of acetamiprid working solutions linearly followed the increase in peak area.The calibration curves were linear over the range with the correlation coefficient of 0.995.The precision of measurement of an analyte can be evaluated as repeatability or reproducibility.In this study, precision is expressed as repeatability.Precision was examined by analysing the same samples (n=5) at two different concentrations on the same day.The retention time of acetamiprid was 1.382 min.The RSD values were within the range of 0.06-0.25%for retention times and from 0.96 to 1.61% for peak area, fulfilling the mentioned criteria of RSD≤20% (EU Commission Health and Consumer Protection Directorate-General, 2011).
The calculated LOD, determined as an S/N ratio of three, and LOQ, determined by considering an S/N of 10 by using matrix-matched calibration curves, are 5 and 14 µg/kg, respectively.The presented LOQ is lower than the acetamiprid MRLs in sweet cheeries set by the Serbian legislation and the European Commission.
The accuracy of the proposed method was evaluated as recovery, using blank samples spiked with the solution of acetamipid insecticide at three levels (0.1, 0.2 and 0.3 mg/kg).A sweet cherry sample from a known locality without acetamiprid contamination was used as the blank sample.In this study acetamiprid was successfully extracted with acetonitrile, while in other studies neonicotinoid insecticides had been extracted with acetone and eluted from columns with dichloromethane (Tsumura et al., 1998, cit. Obana et al., 2002).Imidacloprid was extracted with acetone and transferred to a mixture of dichloromethane and petroleum ether to reduce interferences (Fernandez-Alba et al., 1996).The use of dichloromethane in these methods is not advisable because of environmental concerns (Obana et al., 2002).
The mean recovery achieved was 85.4% with an associated relative standard deviation (RSD) of 2.5%.The average recoveries and RSD of the analyzed samples complied with the EU Commission Health and Consumer Protection Directorate-General ( 2011) criteria (70-120%), and were also used for method validation.Having in mind that the maximum permissible level of acetamiprid in sweet cherries is 0.2 mg/kg (Pravilnik, 2010), the method is sensitive enough for determination of that pesticide at concentrations well below the permissible level.

Acetamiprid residues in treated sweet cherry samples
The validated method was applied to analyse acetamiprid residues in sweet cherry samples, to which acetamiprid was applied under controlled conditions.The detected pesticide was selected by a respective retention time and UV spectra comparison with the reference standard.To obtain quantitative data, a method of external standard with calibration on nine levels was used.
Considering the importance of fresh fruits in a healthy diet, the concentration of acetamiprid residues in agricultural products should be monitored.The identification of acetamiprid in this study was based on its retention time and the spectrum was obtained by comparing it with the Rt standard of acetamiprid and its spectrum.No residues were detected on sweet cherry samples collected immediately before acetamiprid application (Figure 3).
Acetamiprid residues in sweet cherry samples after its application are presented in Figure 4.The maximum residue levels of acetamiprid were detected in the samples collected immediately after its application (0) with an average concentration of 0.529 mg/kg.The samples collected on day 2 had a mean acetamiprid residue concentration of 0.359 mg/kg, which corresponds to 32% dissipation rate.The degradation of acetamiprid increased, and the content of acetamiprid was 0.313 mg/kg 4 days after treatment.The content of acetamiprid in sweet cherry samples collected 6 days after treatment was at the maximum permissible level in Serbia (0.209 mg/kg).Eight and 10 days after treatment, the concentrations of acetamiprid were 0.152 and 0.134, and the respective dissipation rates 71 and 75%.In sweet cherry samples collected on day 12, the content of acetamiprid residues was 0.139 mg/ kg (74%), a slightly higher concentration than it was on day 10, which may be due to an error in handling and/or analysis of the sample (Park et al., 2011).The pre-harvest interval for acetamiprid in sweet cherries in Serbia is 14 days (Sekulić and Jeličić, 2013).At that time interval, the concentration of acetamiprid in our sweet cherries samples was 0.111 mg/kg, with a dissipation rate of 79%.

Figure 3 .
Figure 3. HPLC/DAD chromatogram of a blank uncontaminated sweet cherry sample

Figure 4 .
Figure 4. Acetamiprid residues in sweet cherry samples at various time intervals following its application

Table 1 .
Physicochemical properties of acetamiprid (Tomlin, 2006)Field sampling was carried out during the technological maturity of a medium late sweet cherry variety.Sweet cherry samples were collected at eight intervals: immediately after acetamiprid application,

Table 2 .
Conditions for HPLC/DAD analysis of acetamiprid