Dissipation and persistence of thiacloprid in pepper fruits

SUMMARY This study gives insight into the behavior of the neonicotinoid insecticide thiacloprid in greenhouse production of pepper. Thiacloprid was applied at a concentration of 96 g a.i./ ha, as recommended by the manufacturer for aphid control. Degradation of thiacloprid in pepper fruits was evaluated over a ten-day period, starting from the moment of insecticide application. Sample preparation was performed using the QuEChERS method for liquid chromatography with diode array detection for identification and quantification of thiacloprid residues in extracts of pepper samples. The method was validated in accordance with the SANCO/12571/2013 document. The obtained mean recovery value was 83.69%, with RSD 5.05%. Intraday precision was 3.21%. Within a concentration range from 0.01-2.0 µg/ml, thiacloprid showed linear calibration with R 2 0.997%, while the quantification limit of the method was 0.01 mg/kg. The results of a field trial showed that thiacloprid dissipated rapidly from 1.136 mg/kg to 0.321 mg/kg with a loss of 72% in the first two days after application. Throughout the experimental period, thiacloprid residues in pepper fruits basically remained at a stable low level, and no residue exceeding 0.198 mg/kg was detected in the terminal residue experiment, which was below the MRL of 1.0 mg/kg. The half-life (DT 50 ) of thiacloprid in pepper fruits obtained in this study was 4.95 days. Finally, the pre-harvest interval (PHI) for thiacloprid prescribed by Serbian authorities was proved to be safe enough in greenhouse production of pepper.


INTRODUCTION
Pepper (Capsicum annum) is one of the most important vegetables with great economic significance. It is usually infested by a variety of rapidly reproducing pests, and growers usually apply a combination of several types of pesticides to control them (Lazić et al., 2014a).
Intensified production and inadequate application of cultural practices lead to increased insect infestation and diseases, especially under greenhouse conditions of pepper growth. The insecticide thiacloprid is widely used for control of economically highly important pests of pepper, such as aphids. Thiacloprid [(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2 ylidenecyanamide] is the chloronicotinyl insecticide that acts selectively on the insect nervous system by inhibiting nicotinic acetylcholine receptors (Li et al., 2016). The structure of thiacloprid is shown in Figure 1. Thiacloprid is the second member of Bayer's chloronicotinyl insecticide (CNI) family (Jeschke et al., 2001) with a rising usage trend as it has been registered for use in many crops. Previous studies had indicated that thiacloprid could produce delayed lethal and sub-lethal effects on freshwater arthropods at low concentrations (Beketov & Liess, 2008). However, very little information is available on the behavior of thiacloprid in plants and soils (Oliver et al., 2005;Wang et al., 2011).
Due to intensive use of pesticides in vegetable farming, residues may be accumulated at levels higher than those permitted by their respective international maximum residue levels (MRLs). Assessment of dissipation rate of a pesticide after application is a key process for determining the residual behavior of pesticides in agricultural crops and for detecting pre-harvest intervals (PHIs) (Abd-Alrahman & Almaz, 2012). Dissipation rate is one of the most important parameters in assessing the fate of pesticide residues (Li et al., 2016) and it can be used to estimate the time required for bringing residues down below MRLs (Ambrus & Lantos, 2002).
Residue dynamics of thiacloprid have been studied in different matrices (Omirou et al., 2009;Wang et al., 2011;Yu et al., 2007;Jovanov et al., 2013). However, information regarding the behavior of thiacloprid in pepper is still lacking (Li et al., 2016). The MRL for thiacloprid residues in pepper fruits, according to EC (Regulation, 2005) and Serbian legislation (Pravilnik, 2014), is 1.0 mg/kg. Numerous analytical methods have been described for determination of thiacloprid in different matrices (Ying & Kookana, 2004;Hengel, 2011). Currently, QuEChERS is a method which has been mainly applied for the extraction of different classes of pesticides (Lehotay, 2005;Lazić et al., 2014b). The method has the status of an Official Method of AOAC International (Lehotay, 2007).
In this work, a field study was performed to investigate thiacloprid dissipation in pepper fruits grown in greenhouses. The study presents the results of our examination of a commercial formulation of thiacloprid and its behavior in pepper, and the results of determination of this insecticide's half-life in pepper fruits.

Reagents and standard solutions
An analytical standard of thiacloprid (purity 99.5%) was obtained from Dr Ehrenstorfer GmbH (Augsburg, Germany). Acetonitrile (HPLC purity) and acetic acid were purchased from J.T. Baker (Darmstadt, Germany), while ultrapure water was obtained from TKA apparatus (Germany). The dispersive SP extraction (Cat. No. 5982-5650) and clean-up (Cat. No. 5982-5056) kits for QuEChERS sample preparation were purchased as ready-to-use from Agilent Technologies (USA). A stock solution of thiacloprid was prepared in acetonitrile at a concentration of 100 µg/ml. Working standard solutions for HPLC analysis were prepared by further dilution with acetonitrile, achieving concentrations from 0.01 to 2.0 µg/ml. All standard solutions were stored in a refrigerator (4 °C).

Field experiment and sampling
The plants were grown in a greenhouse in 2015. The trial was set up complying with the principles of good agricultural practice, and OEPP/EPPO methods were used for trial design and data processing (EPPO, 2012), as well as the efficacy evaluation of the insecticide in control of aphids in vegetables (EPPO, 2004). The experiments were conducted in three replications. The control plot was allocated some distance from the tested plots and such untreated pepper plants were the source of blank samples in our study of method validation and matrix-matched calibration. The insecticide was applied at the manufacturer's recommended rate of 96 g a.i./ha using a portable hand sprayer. The product was used in the phase of pepper fruits ripening (BBCH 81), at the beginning of aphids' colony formation.
Samples of about 0.5 kg were randomly collected immediately after drying of the spraying mixture and 2, 3, 4, 5, 6, 7 and 10 days after application. Pepper fruits were stored in individual polyethylene bags at -20 °C until extraction (Commission Directive 2002/63/EC).

Sample extraction
The samples were chopped, homogenized and extracted according to the QuEChERS method. In brief, 10.0 g of homogenized pepper sample was weighed into a 50 ml centrifuge tube and 10 ml of acetonitrile was added. The tube was sealed and shaken for 1 min, then votexed for 1 min. To induce the phase of separation and pesticide partitioning, 1000 mg sodium citrate, 500 mg sodium hydrogen citrate sesquihydrate, 4000 mg magnesium sulfate and 1000 mg sodium chloride were added. This was followed by immediate sealing of the tube and its shaking for 1 min, vortexing for 1 min and centrifugation for 5 min at 3000 rpm, and then the tube was sealed, shaken for 1 min, vortexed for 1 min and centrifuged for 5 min at 3000 rpm. For sample clean-up, 6.0 ml of the upper layer was transferred to a tube with primary-secondary amine sorbent (150 mg) and magnesium sulfate (900 mg). After closing the tube, it was vortexed for 1 min and centrifuged for 5 min at 3000 rpm. An aliquot of the upper layer (3 ml) was evaporated to dryness under gentle nitrogen stream, dissolved in 1 ml of acetonitrile, filtered through a 0.45 µm membrane filter and transferred into an autosampler vial (Figure 2).

LC analysis
Liquid chromatographic analysis was performed with an Agilent Technologies Liquid Chromatograph 1100 Series. Chromatographic data were collected and recorded using the Chemstation system software. Separation was carried out using a C 18 column (50 mm × 4.6 mm internal diameter, 1.8 µm particle size, Agilent Technologies) and isocratic elution of acetonitrile and water acidified with 1.5% CH 3 COOH (30/70). The flow rate of the mobile phase was 1 ml/min and column temperature was ambient. The DAD was set at 225 nm with a bandwidth of 4 nm. Each analytical sample was considered in triplicates.

Method validation
The linearity of the method was evaluated by spiking untreated pepper samples with thiacloprid analytical standard because more reliable calibration may be obtained with matrix-matched calibration (European Commission Health & Consumer Protection Directorate-General, 2013). The calibration curve of thiacloprid in pepper matrix in 0.01-2.0 µg/ml concentration range showed an excellent linearity and strong correlation between concentrations and area in the studied range (r 2 =0.997).
Blank analyses were also used for interference check from the matrix. Figure 3 shows the chromatograms of blank and matrix-match thiacloprid standard. Absence of any signal at the retention time of thiacloprid (4.601 min) indicated that no matrix compounds were present, which may give a false positive signal.
Recover y a ssays were performed at three concentration levels: 0.01, 1.0 and 2.0 mg/kg. The samples were processed using the described procedure. At each fortification level, three replicates were analyzed. Quantification of recovery was carried out with the standard dissolved in pepper matrix. The chromatogram of spiked pepper sample (1.0 mg/kg) is shown in Figure 4. In all cases, the recovery of thiacloprid was >70% (83.73-91.14), the average value being 83.69% and RSD=5.05%. These data are generally considered satisfactory for residue determination. Precision, expressed as RSD, was studied by performing repeatability studies (n=6) with the standard dissolved in pure solvent and pepper matrix. The results indicated satisfactory precision of 1.26% and 3.21%. Instrumental LOQ based on S/N of 10:1 was 0.01 mg/kg.

Results of dissipation experiment
Dissipation results are shown in Table 1. The experiment was conducted indoors, the average value of relative humidity was 39.52% and temperature 24.7 °C. Thiacloprid mean residue levels during the sampling period for each application were derived from three sub-samples. The highest residue levels were found in samples taken at the first sampling time 1 h after pesticide application, and the mean value was 1.136 mg/kg. Thiacloprid residue levels kept decreasing in the following period, finally reaching the level of 0.321 mg/kg with dissipation of 72% only 2 days after application, with almost unchanged values on the 3 rd day. Over the further three days, thiacloprid residue level was relatively stable, ranging from 0.276 mg/ kg on the 4 th day to 0.282 mg/kg on the 6 th day. At the end of the pre-harvest interval, dissipation of thiacloprid was 83%, and residue level in pepper fruits was 0.198 mg/kg. During the sampling time, thiacloprid content in pepper fruits was far below the MRL of 0.184 mg/kg. Similar results had been reported by Sharma and Perihar (2013). They detected an intensive thiacloprid dissipation (60.42%) on the 2 nd day after application. The relationship between residues and time is described by a first-order model, C t = C 0 e -kt , where C t (mg/kg) is the residue after time t (d), C 0 (mg/kg) is initial residue, and k is dissipation rate constant (d -1 ). This model was originally adopted to interpret relationships between residues and time and it is widely used to describe the fate of pesticides in soil and plants (Beulke & Brown, 2001).
Half-life (DT 50 ) is defined as the time required for disappearance of 50% of the pesticide (based on initial residue levels after application). For the first-order model, the DT 50 was calculated by the following equation: DT 50 =ln2/k, using dissipation rate constant value. Dissipation of thiacloprid residues was simulated by a first-order model (Table 2). Degradation monitoring lasted ten days, slightly longer than PHI. Under such conditions, thiacloprid DT 50 in pepper fruits was 4.95 days. In the study conducted by Li et al. (2016) in pepper plants, thiacloprid was applied at 1.5-fold recommended dose of 144 g a.i./ha. The trial was set up in the field. Samples were collected randomly from sampling plots at 0 (2 h after spraying), 1, 3, 7, 10, 14, 21 and 28 days after treatment. The initial residue level of thiacloprid in pepper was 0.503 mg/kg, which is much lower than the established EU MRL. The half-life was 0.81 days.
Considerating the differences in doses of thiacloprid residues and DT 50 value in our experiment and the trial reported by Li et al. (2016), they can be attributed to different temperature conditions, humidity, and possibly to the insolation level, but also to the plants' habitats. Previous studies had reported that dissipation of pesticide deposits was a complex process depending on various environmental factors, such as temperature, relative humidity and UV irradiation, metabolism and translocation (pesticide penetration and plant growth), application technique and pesticide formulation (Brouwer et al., 1997).
The results of this study indicate that thiacloprid dissipates relatively rapidly in pepper fruits grown in greenhouses as its half-life was 4.95 days. At the end of PHI, thiacloprid residues in pepper plants were far below the MRLs. Finally, the PHI for the thiacloprid insecticide prescribed by the Serbian authorities was proved to be safe enough in greenhouse production of pepper.