A simulation model (PostPLANT-Soil) for predicting pesticide concentrations in succeeding leafy vegetables: II. Validation with experimental data on plant uptake in a growth chamber.

We validated a simulation model (PostPLANT-Soil) for predicting pesticide concentrations in succeeding leafy vegetables reported in our first paper in this series, which includes the pesticide sorption process into plant roots. As a result of the model validation with the measured data from a plant uptake study in a growth chamber, the model successfully simulated the concentration changes of pesticides in a plant shoot. However, the simulated shoot concentrations for several pesticides were overestimated compared to the measured values. The leafy vegetable (Brassica rapa) used in this study probably has a high metabolic ability for the fungicide flutolanil from the result of the uptake study under a hydroponic condition.

A simulation model (PostPLANT-Soil) for predicting pesticide concentrations in succeeding leafy vegetables: I. Validation with experimental data in a Japanese Andosol field.

We developed a simulation model for predicting pesticide concentrations in succeeding leafy vegetables (PostPLANT-Soil), which includes the process of pesticide uptake from plant roots. To validate the model, we compared pesticide concentrations simulated by the model with values measured from field experiments in an upland Andosol. The model validation showed that pesticide concentrations in the plant shoot were correlated with the concentrations in the soil solution rather than those of the water-extracted pesticides. The model successfully simulated the concentration changes in plant shoots when the simulated concentrations of the pesticides in the soil solution were fitted to the measured values by considering the key parameter - the corrective coefficient for the soil adsorption coefficient. However, the simulated shoot concentrations at the appropriate harvest period exceeded the measured values. This indicates that the leafy vegetable used in this study may have some metabolic capacity for the pesticides.

Liquid Chromatographic Determination of Trace Bioavailable Neonicotinoids in Soil with Dispersive Liquid-Liquid Microextraction and Its Application for Experimental Monitoring.

Dispersive liquid-liquid microextraction (DLLME) was applied to extract bioavailable neonicotinoids involved in the uptake from soil through roots to plants. To quantitatively extract bioavailable neonicotinoids with the proposed DLLME, 3.5 mL of dichloromethane (extractant)/acetonitrile (dispersive solvent) (6:1, v/v) was injected into 5 mL of aqueous soil extracts in which 1 g of sodium chloride was previously dissolved. The separated dichloromethane phase after sonication and centrifugation was evaporated, reconstituted with a mobile phase, and determined with high-performance liquid chromatography. The established method showed sufficient analytical performance to quantify the amount remaining in soil in trace amounts. In a pilot trial conducted in the field, the changes in the concentrations of bioavailable neonicotinoids were confirmed using the method. After showing rapid degradation in soil, degradation of clothianidin and imidacloprid slowed after about 100 days of treatment, but it continued to be detected at around 0.02-0.05 µg/g-dried weight until 1097 days. This result suggests that once these neonicotinoids are treated in soil, they might remain for long periods, which supports the possibilities of crop contamination and exposure to pollinators.

Review of sample preparation methods for chromatographic analysis of neonicotinoids in agricultural and environmental matrices: From classical to state-of-the-art methods.

This review specifically examines the development of sample preparation methods for residue analyses of neonicotinoid insecticides in agricultural and environmental matrices. Pesticide residue analysis is fundamentally important to ensure the safety of foods and processed foods of plant and animal origin, and to preserve the environment, particularly soil and water. For the development of pesticide residue analysis, the sample preparation process is an important key to maximizing the analytical performance of highly sensitive and accurate chromatographic instruments and to acquiring reliable analytical results. This review outlines sample preparation methods that have been proposed to date for extraction of neonicotinoids that might remain in a complicated sample matrix in quantitatively trace amounts, and for cleaning up, to the greatest extent possible, the interfering components that coexist in the sample extract.

Detection of herbicide clopyralid at nanogram per gram level in agricultural products using easy-to-use micro liquid-liquid extraction followed by analysis with ultraperformance liquid chromatography-tandem mass spectrometry.

A new sample preparation method was developed for auxin-like herbicide clopyralid residue in agricultural products. The method uses extraction with sulfuric acid - acidified acetonitrile, with cleanup of sample extracts using solid-phase extraction (diatomaceous earth) and micro liquid-liquid extraction, followed by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This sample preparation method greatly reduces the influence on ionization during determination with UPLC-MS/MS. Quantification was achieved using external calibrators prepared in matrix-free 0.1% formic acid. The proposed analytical method supported good recovery of 73.7-91.4% with less than 8% relative standard deviation. Method quantification limits (MQL) of samples were 0.6-1.0 ng g-1. The method was applied to determine clopyralid residue in several crop samples. The herbicide was detected as near the MQLs in all samples.

Simultaneous Detection of Six Different Types of Pesticides by an Immunosensor Based on Surface Plasmon Resonance.

Six pesticides, azoxystrobin, boscalid, chlorfenapyr, imazalil, isoxathion, and nitenpyram, were simultaneously detected by using a surface plasmon resonance (SPR) immunosensor. The working ranges were 3.5 - 19 ng/mL for azoxystrobin, 4.5 - 50 ng/mL for boscalid, 2.5 - 25 ng/mL for chlorfenapyr, 5.5 - 50 ng/mL for imazalil, 3.5 - 50 ng/mL for isoxathion, and 8.5 - 110 ng/mL for nitenpyram. They showed adequate recovery results in tomato samples: 104 - 116% for azoxystrobin, 94 - 101% for boscalid, 90 - 112% for chlorfenapyr, 96 - 106% for imazalil, 107 - 119% for isoxathion, and 104 - 109% for nitenpyram. The correlation coefficient with liquid chromatography (HPLC or LC-MS/MS) using vegetable samples also agreed well: 0.91 - 0.99 as R2 without strong bias, except for nitenpyram for which the SPR immunosensor sensitivity was too low. The SPR immunosensor will have high applicability for pesticide residue analyses in vegetable samples.

Highly sensitive analytical method for herbicide clopyralid residue in cattle manure compost with ultraperformance liquid chromatography tandem mass spectrometry.

A micro liquid-liquid extraction has been applied to sample preparation in the current authorized method for clopyralid in compost. The method rendered matrix effects practically negligible during determination with ultraperformance liquid chromatography-electrospray ionization tandem mass spectrometry with an improved limit of quantification of 0.7 µg/kg dry weight. Moreover, it had good accuracy and reproducibility. Therefore, the method is proposed as a highly effective routine analytical technique for investigating the actual status of clopyralid residue in compost.

Development of a direct competitive enzyme-linked immunosorbent assay for determination of the fungicide mepanipyrim and its metabolite.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for determination of anilinopyrimidine fungicide mepanipyrim in vegetables. Two derivatives of mepanipyrim and mepanipyrim propanol type metabolite which carried carboxy acid were synthesized and conjugated with keyhole limpet hemocyanin. BALB/c mice were immunized to prepare anti-mepanipyrim monoclonal antibodies (MoAbs) by obtained conjugates. The dc-ELISAs based on the prepared MoAbs, MPP107 and MPP204, showed working ranges between 0.12 and 1.8 ng/mL with mepanipyrim for MPP107, 0.12 and 2.4 ng/mL with mepanipyrim for MPP204, and 0.2 ng/mL and 5.7 ng/mL with the mepanipyrim propanol type for MPP204. The dc-ELISAs showed the sufficient sensitivity to determine the mepanipyrim residues for the MRLs of 1-15 mg/kg among the majority of vegetables and fruits in Japan. Recovery and/or correlation results from HPLC suggested that the dc-ELISAs would be applicable to the residue analysis of mepanipyrim and its propanol type in vegetables.

Physiological disorder of plants depending on clopyralid concentration in the soil and plant.

The influence of clopyralid in soil on plant growth was investigated over time using three plants. The order of clopyralid sensitivity was as follows: Solanum lycopersicum>Solanum melongena>Momordica charantia, especially physiological disorder of S. lycopersicum were rapidly expressed as various serious symptoms with increasing concentration of clopyralid. In contrast, the clopyralid concentration of above-ground part was in the following order: M. charantia>S. lycopersicum, S. melongena, which differed from the order of sensitivity to clopyralid.

Direct determination of neonicotinoid insecticides in an analytically challenging crop such as Chinese chives using selective ELISAs.

Easy-to-use commercial kit-based enzyme-linked immunosorbent assays (ELISAs) have been used to detect neonicotinoid dinotefuran, clothianidin and imidacloprid in Chinese chives, which are considered a troublesome matrix for chromatographic techniques. Based on their high water solubility, water was used as an extractant. Matrix interference could be avoided substantially just diluting sample extracts. Average recoveries of insecticides from spiked samples were 85-113%, with relative standard deviation of <15%. The concentrations of insecticides detected from the spiked samples with the proposed ELISA methods correlated well with those by the reference high-performance liquid chromatography (HPLC) method. The residues analyzed by the ELISA methods were consistently 1.24 times that found by the HPLC method, attributable to loss of analyte during sample clean-up for HPLC analyses. It was revealed that the ELISA methods can be applied easily to pesticide residue analysis in troublesome matrix such as Chinese chives.

Potential application of immunoassays for simple, rapid and quantitative detections of phytoavailable neonicotinoid insecticides in cropland soils.

This study evaluated the applicability of commercially available kit-based enzyme-linked immunosorbent assay (ELISA) to simple, quick, and quantitative detection for three water-extractable (phytoavailable) neonicotinoid insecticides: dinotefuran, clothianidin, and imidacloprid in soils. ELISA showed excellent analytical sensitivity for determination, but with cross-reaction to structurally related neonicotinoid analogues, which might produce false positives. To analyze insecticides in soil samples of diverse physicochemical properties, they were extracted with water. The aqueous soil extracts were assayed directly with ELISA. No matrix interference was observed without additional dilution with water. Recovery experiments for the insecticides from aqueous soil extracts spiked at 2-10 ng/mL showed good accuracy (72-126%) and precision (<16%). Kit-based ELISAs were used to estimate soil-water distribution coefficients (Kd). Values estimated using this method showed positive correlation between organic carbon contents in soil and those for evaluated insecticides. Results indicate that the evaluated kit-based ELISA has applicability for simple, quick, and reliable detection of phytoavailable insecticides in soils and for estimating Kd values in soil.

Development of an Immunosensor for Determination of the Fungicide Chlorothalonil in Vegetables, Using Surface Plasmon Resonance.

An immunosensor based on surface plasmon resonance (SPR-sensor) was developed to analyze chlorothalonil residues and maximum residue limits (MRLs; 0.5-50 mg/kg) in vegetables in Japan. Conjugates of N-(pentachlorophenoxyacetyl)glycine and bovine serum albumin were covalently coated on the sensor chip. The SPR-sensor quantitatively determined chlorothalonil at concentrations ranging from 8.0 to 44 ng/mL, using TPN9A, a monoclonal antibody to chlorothalonil. The 50% inhibition concentration was 25 ng/mL. The reactivity was 10-fold lower than that of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). However, the SPR-sensor could determine chlorothalonil residues in vegetables at concentrations around the above MRLs. Chlorothalonil spiked in vegetables was recovered at 90-118% within 1 day and at 90-115% across 3 days, correlating with HPLC results. The sensor showed good performance for chlorothalonil residue analysis in vegetables with rapid determination, although the sensitivity and the cross-reactivity were less effective than with the ic-ELISA.

Highly sensitive quantification of pyrethroid insecticide etofenprox in vegetables with high-performance liquid chromatography and fluorescence detection.

This paper describes a highly sensitive analytical method using high-performance liquid chromatography and fluorescence detection (HPLC-FLD) capable of quantifying trace amounts of synthetic pyrethroid insecticide etofenprox residue in six vegetable samples: bell pepper, cucumber, eggplant, Japanese mustard spinach, spinach, and tomato. After extraction with acetonitrile, the crude sample extract was cleaned up with a solid-phase extraction cartridge. The matrix interference derived from the tested vegetable samples was evaluated. Quantification was conducted using external calibrators prepared in pure acetonitrile. The limits of quantification for etofenprox in each sample were 1.87-3.87 ng/g. Recoveries obtained by application of the proposed analytical method of vegetable samples spiked at the considerably low levels (5-100 ng/g) were 85-111% with relative standard deviations of less than 12%. The proposed method using the HPLC-FLD was applied for trace analysis of the insecticide residue in vegetable samples.

Development of direct competitive ELISA for residue analysis of fungicide chlorothalonil in vegetables.

A direct competitive (dc)-ELISA was developed for rapid and simple determination of chlorothalonil residue in vegetables. A carboxylic acid derivative of pentachlorophenol was used to prepare an anti-chlorothalonil monoclonal antibody (MoAb) that showed adequate reactivity for dc-ELISA. Before homogenization of vegetable samples, phosphoric acid was added (vegetable-10% phosphoric acid (2 : 1, w/v)) to block enzymatic decomposition of chlorothalonil. The use of phosphate buffer (100 mmol/L, pH 7.0) minimized the influence of phosphoric acid on competitive reaction in the dc-ELISA. Working range was 0.10 to 6.0 ng/mL in the optimized dc-ELISA. The recovery of chlorothalonil spiked in cucumber and eggplant was 97.1 to 125%. The results correlated well with those obtained by HPLC analysis. The dc-ELISA could rapidly determine chlorothalonil after a simple sample preparation procedure.

Chromatographic separation of arsenic species with pentafluorophenyl column and application to rice.

Arsenic species, including arsenous acid, arsenic acid, methylarsonic acid, and dimethylarsinic acid, were determined using HPLC-ICPMS. The species were separated with a Discovery HS F5 column and a simple, volatile, and isocratic mobile phase of 0.1% (v/v) formic acid and 1% (v/v) methanol. The Discovery HS F5 column with a pentafluorophenyl (PFP) stationary phase gave sharp peaks and full separation of the arsenic species in 5min, and other PFP columns showed lower performance. This separation method was applied to arsenic species analysis in rice. The extraction of arsenic from rice samples was performed using 0.15M nitric acid. The methodology was validated by use of certified reference materials, NMIJ CRM 7503-a and NIST SRM 1568a, and extremely low arsenic rice samples as blank samples.

Water-based extraction and liquid chromatography-tandem mass spectrometry analysis of neonicotinoid insecticides and their metabolites in green pepper/tomato samples.

This paper proposes an environmentally friendly method involving water-based extraction of the samples, cleanup of the extracts by solid-phase extraction, and subsequent liquid chromatography coupled with tandem mass spectrometry, which was used for simultaneous determination of seven hydrophilic neonicotinoid insecticides as well as their metabolites in agricultural samples. The effects of sample matrix on detection of the target compounds were negligibly small. Mean recoveries obtained at spiked concentrations between 0.01 and 1.00 mg/kg were 71.2-122.3% with relative standard deviations of ≤ 7.5%. When the method was applied to crop samples sprayed with commercial formulations of the target compounds, the residual concentrations of the compounds determined by the proposed method (0.015-0.27 mg/kg in green peppers and 0.017-0.31 mg/kg in tomatoes) were equivalent to those determined by the official Japanese method (0.017-0.26 mg/kg in green peppers and 0.013-0.30 mg/kg in tomatoes).

Aqueous acetonitrile extraction for pesticide residue analysis in agricultural products with HPLC-DAD.

To reduce hazardous organic solvent consumption during sample preparation procedures as much as possible, an extraction method of smallest feasible sample volume (5g) using aqueous acetonitrile (MeCN) was developed to extract pesticide residues from agricultural samples prior to HPLC-DAD determination. Extraction with MeCN/water (1:1, v/v), and adjustment of the MeCN concentration by diluting with water after extraction recovered successfully most pesticides showing various physicochemical properties. The matrix effects of tested samples on the proposed method developed herein were generally negligibly-small. The average recoveries were in the range 70-120% for all pesticides with the coefficient of variation values below 20%. The reduction rate of organic solvents used for the proposed sample preparation method was up to approximately 60% compared with the Japanese authorised official method for pesticide residue analyses. These results demonstrate the feasibility of the proposed method for pesticides with diverse properties.

Review of enzyme-linked immunosorbent assays (ELISAs) for analyses of neonicotinoid insecticides in agro-environments.

Immunoassay is a promising method that is suitable for rapid and simple analyses of pesticides, which are likely to persist at a trace level in agro-environments, including agricultural products, soil, and water. Particularly, enzyme-linked immunosorbent assay (ELISA) has wide application to development of analytical methods for pesticide residues because it can very sensitively and very accurately determine them in samples. This paper presents a review of the fundamental analytical performance, a device for the sample pretreatment methods before determination, and cases of applications to various samples on ELISA methods that have been developed for detection of neonicotinoid insecticides in food or environmental matrices. The reviewed ELISAs can be ranked as quantitative, rapid and simple analytical methods for single analytes. The recognition of ELISA as an analytical methodology for pesticide residues is expected to advance rapidly in the future.

Reduction of hazardous organic solvent in sample preparation for hydrophilic pesticide residues in agricultural products with conventional liquid chromatography.

An original extraction method using water as an extractant has been established for environmentally friendly sample preparation procedures for hydrophilic pesticides (acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, methomyl, pymetrozine, thiacloprid, and thiamethoxam) in agricultural samples with conventional HPLC. Water-based extraction and cleanup with two solid-phase extraction cartridges can recover target hydrophilic pesticides quantitatively. The matrix effects of tested samples on the proposed method developed herein were negligibly small. Under the optimized conditions, the recoveries of almost all tested pesticides were 70-120% with satisfactory precision (%CV < 20%). The analytical data are in good accordance with Japanese or European Union guidelines for pesticide residue analysis. The reduction rate of hazardous organic solvents used for the proposed method and by reducing the sample size for extraction was about 70% compared with the Japanese authorized reference method used in this work. The results demonstrate the feasibility of the proposed sample preparation procedures for hydrophilic pesticides.

Quantitative analysis of fungicide azoxystrobin in agricultural samples with rapid, simple and reliable monoclonal immunoassay.

This work presents analytical performance of a kit-based direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for azoxystrobin detection in agricultural products. The dc-ELISA was sufficiently sensitive for analysis of residue levels close to the maximum residue limits. The dc-ELISA did not show cross-reactivity to other strobilurin analogues. Absorbance decreased with the increase of methanol concentration in sample solution from 2% to 40%, while the standard curve became most linear when the sample solution contained 10% methanol. Agricultural samples were extracted with methanol, and the extracts were diluted with water to 10% methanol adequate. No significant matrix interference was observed. Satisfying recovery was found for all of spiked samples and the results were well agreed with the analysis with liquid chromatography. These results clearly indicate that the kit-based dc-ELISA is suitable for the rapid, simple, quantitative and reliable determination of the fungicide.