Residual characteristics and safety assessment of the insecticides spiromesifen and chromafenozide in lettuce and perilla

Chemicals and materials

Analytical standard (> 99% purity) of spiromesifen, BSN2060-enol, and chromafenozide were purchased from AB Solution Co., Ltd., Hwaseong-si, Gyeonggi-do, Republic of Korea. HPLC grade water and acetonitrile were supplied by Merck, Darmstadt, Germany. QuEChERS kit (4.0 g magnesium sulfate, 1.0 g sodium chloride, 1.0 g sodium citrate tribasic dihydrate, 0.5 g disodium citrate sesquihydrate) were obtained from Phenomenex, California, USA. Individual stock solutions of the target compounds were prepared in acetonitrile and stored at − 20 °C before use.

Field experiments

The trials were carried out in a greenhouse farm during the season 2018 at two different sites (with approximately 24 km distance between both sites) located in Chuncheon and Hongcheon-gun, Gangwon-do, Republic of Korea following the method described by the Organization for Economic Co-operation and Development (OECD)³⁸. The field test of lettuce (Latuca sativa L.) crop was conducted in Chuncheon city, and perilla (Perilla frutescens (L.) Britton) crop in Hongcheon city. The area of each field was divided into two plots (treatment and control). The treatment plots were further divided into three replicates (subplots 33 m²). The control plot was separated by a buffer zone of 3 m² from the treated site. To minimize spray overlap, buffer zones (1 m) were set up between subplots. The commercial products of spiromesifen 20% SC diluted 2000 times and chromafenozide 5% EC diluted 1000 times were sprayed twice at 7-days intervals using an automatic sprayer. After the second spray samples (lettuce and perilla leaves) were collected from each subplot at 0 (2 h after spraying), 1, 3, 5, and 7 days according to the Korean RDA²³ method. Thirty samples 1.0 kg each from the collected crop were placed in polyethylene bag and labeled. After collection, the samples were transported to the laboratory, where they were chopped and homogenized. The homogenized samples were kept frozen at − 20 °C until analysis.

We confirm all plant samples used in the current work comply with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Samples pretreatment

A QuEChERS method was used for the extraction of the targeted compounds from lettuce and perilla leaves. A 10 g of previously homogenized samples were weighed into a 50 mL polypropylene centrifuge tube and mixed with 10 mL of water followed by 10 mL of acetonitrile. The samples were shaken at 1500 rpm in a shaker machine for 10 min. Then commercial QuEChERS kit was added, and the mixtures were shaken vigorously for 2 min in a shaker. Subsequently, the samples were centrifuged at 3584 g-force for 10 min. After centrifugation, the supernatant was filtered with a 0.22 μm membrane filter and transferred into the glass vial for instrumental analysis.

LC-MS/MS analysis

Quantitative determination of the tested compounds was carried out by using HPLC system Dionex Ultimate 3000 (Thermo Science, USA) coupled with tandem mass spectrometry (MS/MS) (TSQ Quantum Access Max (Thermo Science, USA). Water (solvent A) and acetonitrile (solvent B) containing 0.1% formic acid and 5 mM ammonium format were used as mobile phase at a flow rate of 0.4 mL/min and injection volume 1.0 µL. To obtain desirable chromatographic peaks, two different instrumental conditions were used. The chromatographic separation of spiromesifen was separated by Capcell core-C18 (2.1 mm I.D. × 150 mm × 2.7 μm, Shiseido Co., Ltd., Tokyo, Japan) and BSN2060-enol was performed by C18 column (Poroshell 120 SB-Ag, 2.1 mm I.D. × 100 mm × 2.7-μm, Agilent Technologies, Santa Clara, California, USA) with a gradient elution as follows (mobile phase B%): 0.0 min, 5.0%; 2.0 min 5%; 2.5 min, 95%; 6.0 min, 95%; 6.5 min, 5.0%; 10 min, 5.0%. Likewise, chromafenozide was separated by C18 column (Imtakt Unison UK-C18, 2.0 mm I.D. × 100 mm × 3.0-μm, Imtakt, Portland, USA) with a gradient elution as follows (mobile phase B%): 0.0 min, 5%; 1.0 min, 5.0%; 1.5 min, 90%; 5.0 min, 90%; 7.0 min, 5.0%; 10 min, 5.0%. An MS/MS system (TSQ quantum ultra, Thermo Science, USA) equipped with an electrospray ionization source operating in positive mode (ESI+) was used. The MS/MS parameters and selected product ions are shown in supplementary Tables S2 and S3.

The calculation of spiromesifen total residues

The total residues in lettuce and perilla samples were calculated using Eq. (1)²³.

Total residues of spiromesifen (mg/kg) = spiromesifen + (BSN2060 residue × 1.36). The conversion factor was calculated as follow;

where MW molecular weight.

Initial deposition calculation

The initial residues of spiromesifen and chromafenozide deposition in lettuce and perilla leaves were calculated from 0-day according to Eq. (2) described by Kang et al.¹² as follow;

A: Initial residue (mg/kg), B: Residues (mg/kg) on 0 day, C: active ingredients, E: dilution factor.

Method validation

The analytical method was validated in terms of different performance criteria such as linearity, accuracy, precision, and method limit of quantitation (MLOQ). Matrix-matched standards were used to construct the calibration curve by evaporating (0.01, 0.05, 0.1, 0.2, 0.5, 0.7 and 1.0 mg/kg) working solution (1 mL) and re-dissolved in the extract of control sample. The linearity of the matrix-matched calibration curve was evaluated by the values of the correlation coefficient (R²). The accuracy and precision were obtained in terms of recovery (70–120%) and repeatability (n = 3). The recoveries were determined by spiking the analytes at two concentrations levels (0.1 mg/L) and (0.5 mg/L) in 10 g control samples and were quantified by comparing the response of analytes in samples with response in calibration standard solutions prepared in matrix. The repeatability expressed as the relative standard deviation (RSD) of the analyzed samples was calculated from three repetitions. The MLOQ was calculated by Eq. (3) taking into consideration the following factors: the instrument limit of detection, volume of extraction solvent, injection volume, dilution factor, and sample amount^39,40.

where A: instrument limit of detection, B: volume of extraction solvent, C: injection volume, D: dilution factor, E: sample amount.

Half-life calculation

The dissipation patterns of spiromesifen and chromafenozide in lettuce and perilla leaves over time were found following the first-order kinetics model²⁸. The half-life was determined by the following equation:

where C_t is the concentration of the insecticide, C₀ represents the initial residue concentration of insecticide, t is the time (days) after insecticide application, and k is the constant rate.

Safety assessment

In this study, the safety assessments (percent acceptable daily intake; %ADI) of the target insecticides that are consumed with lettuce and perilla leaves were calculated by the ratio of estimated daily intake (EDI) to acceptable daily intake (ADI). The EDI was calculated using insecticide concentration and average consumption of food commodities per person per day. In addition, the theoretical maximum daily intakes (TMDIs) of both insecticides were calculated using the maximum residue limits (MRLs) and average body weight (60 kg) of adults in Republic of Korea. TMDIs were calculated following the equation described by Kim et al.⁴¹.

Source: Ecology - nature.com