Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

Summary

Presented here is a protocol for the determination of the absorption, translocation, and distribution of imidacloprid in wheat under hydroponic conditions using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results showed that imidacloprid can be absorbed by wheat, and imidacloprid was detected in both the wheat roots and leaves.

Abstract

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. Imidacloprid, the most widely used insecticide worldwide, is a representative first-generation neonicotinoid and is used in pest control for crops, vegetables, and fruit trees. With such a broad application of imidacloprid, its residue in crops has attracted increasing scrutiny. In the present study, 15 wheat seedlings were placed in a culture medium containing 0.5 mg/L or 5 mg/L imidacloprid for hydroculture. The content of imidacloprid in the wheat roots and leaves was determined after 1 day, 2 days, and 3 days of hydroculture to explore the migration and distribution of imidacloprid in wheat. The results showed that imidacloprid was detected both in the roots and leaves of the wheat plant, and the content of imidacloprid in the roots was higher than that in the leaves. Furthermore, the imidacloprid concentration in the wheat increased with increasing exposure time. After 3 days of exposure, the roots and leaves of the wheat in the 0.5 mg/L treatment group contained 4.55 mg/kg ± 1.45 mg/kg and 1.30 mg/kg ± 0.08 mg/kg imidacloprid, respectively, while the roots and leaves of the 5 mg/L treatment group contained 42.5 mg/kg ± 0.62 mg/kg and 8.71 mg/kg ± 0.14 mg/kg imidacloprid, respectively. The results from the present study allow for a better understanding of pesticide residues in crops and provide a data reference for the environmental risk assessment of pesticides.

Introduction

In present day agronomy, the use of pesticides is essential to increase crop yield. Neonicotinoid insecticides alter the membrane potential balance by controlling nicotinic acetylcholine receptors in the insect nervous system, thereby inhibiting the normal conduction of the insect central nervous system, leading to the paralysis and death of the insects¹. Compared with traditional insecticides, neonicotinoids have advantages such as novel modes of action, high insecticidal activity, and strong root absorption, making them highly successful in the pesticide market^2,3. The sales volume of neonicotinoids was reported to account for 27% of the world pesticide market in 2014. The average annual growth rate of neonicotinoids was 11.4% from 2005 to 2010, of which about 7% was registered in China^4,5,6. From the end of 2016 to the first half of 2017, the sales of pesticides in China began to rebound after falling, and the prices of pesticides continued to rise, among which neonicotinoid insecticides showed a significant price increase⁷. So far, three generations of neonicotinoid insecticides have been developed, each containing pyridine chloride, thiazolyl, and tetrahydrofuran groups of nicotine, respectively⁸.

Imidacloprid represents the first generation of neonicotinoid insecticides, whose molecular formula is C₉H₁₀ClN₅O₂, and is a colorless crystal. Imidacloprid is used mainly to control pests, such as aphids, planthoppers, mealworms, and thrips⁹ and can be applied to crops such as rice, wheat, corn, cotton, and vegetables such as potatoes, as well as fruit trees. Due to the long-term, substantial, and continual application of pesticides, both beneficial insects and the natural enemies of pests have been rapidly reduced, and some agricultural pests have become resistant to pesticides, resulting in a vicious circle of applying continual and increasing amounts of pesticides¹⁰. In addition, the extensive application of pesticides has led to the deterioration of soil quality, persistent pesticide residues in agricultural products, and other ecological problems, which not only cause significant damage to the agricultural ecological environment¹¹ but also pose a serious threat to human health¹². Pesticide spraying severely impacts the growth and quality of soil microbes and soil animals¹³. The unreasonable or excessive use of pesticides has caused significant security risks to the soil and water environment, animals and plants, and even human life¹⁴. In recent years, the problem of excessive pesticide residues in crops has become more severe with the extensive application of pesticides. When imidacloprid was used to increase vegetable yield, the absorption rate of imidacloprid in the vegetables increased with the increase in the amount and residue of imidacloprid¹⁵. As a major food crop, both the production and safety of wheat are critical. Therefore, the residue and distribution policies of pesticides used for wheat need to be clarified.

In recent years, many methods have been developed to extract imidacloprid residues from water, soil, and plants. The QuEChERS method (quick, easy, cheap, effective, rugged, and safe) is a new method that combines solid-phase microextraction technology and dispersed solid-phase extraction technology and involves the use of acetonitrile as the extraction solvent and the removal of mixed impurities and water in the sample using NaCl and anhydrous MgSO₄, respectively¹⁶. The QuEChERS method requires minimal glassware and has simple experimental steps, making it one of the most popular pesticide extraction methods¹⁷. For the detection of imidacloprid, a detection limit as low as 1 × 10⁻⁹ g¹⁸ has been achieved with liquid chromatography (LC), and 1 × 10⁻¹¹ g¹⁹ has been achieved with gas chromatography (GC). Due to their high resolution and sensitivity, LC-MS and GC-MS have shown even lower imidacloprid detection limits of 1 × 10^-13 to 1 × 10^-14 g^20,21; these techniques are, therefore, well suited for the analysis of trace imidacloprid residues.

In the present study, imidacloprid was chosen as the target pollutant, and wheat was selected as the test crop to study the distribution of imidacloprid residues in wheat. This protocol details a method for the comprehensive analysis of the enrichment and transfer of the pesticide imidacloprid in wheat by exploring the absorption and storage of imidacloprid in different parts of wheat plants grown under hydroponic conditions. The present study aims to provide a theoretical basis for the risk assessment of pesticide residues in wheat, guide the rational application of pesticides in agricultural production activities to reduce pesticide residues, and improve the safety of crop production.

Protocol

1. Germination of wheat seeds

1. Select 1,000 wheat seeds (Jimai 20) with complete granules, intact embryos, and uniform size (length: 6 mm ± 0.5 mm).
2. Transfer 333.3 mL of 30% H₂O₂ solution to a 1 L volumetric flask and dilute with deionized water to prepare 1 L of 10% H₂O₂ solution. Immerse the wheat seeds in 10% H₂O₂ solution for 15 min to disinfect the seed surface (Figure 1).
3. Rinse the wheat seeds 5x with running sterile water for 10 s each time.
4. Spread the wheat seeds evenly with the embryos pointing up in a glass Petri dish containing moist sterile filter paper (Figure 2). Place the Petri dish in an artificial climate incubator at 30 °C and 80% relative humidity. Culture the wheat seeds in the dark for 3 days until they germinate and take root.

Figure 1: Disinfection of the wheat seeds. The wheat seeds were soaked in 10% H₂O₂ solution (in a beaker) for 15 min to disinfect the seed surface. Please click here to view a larger version of this figure.

Figure 2: Germinating the wheat seeds. The wheat seeds were spread evenly in a glass Petri dish containing moist sterile filter paper. The Petri dish was placed in an artificial climate incubator to germinate the wheat seeds. Please click here to view a larger version of this figure.

2. Cultivation of wheat seedlings

1. Dissolve 551 mg of Hoagland's basal salt mixture in 1 L of deionized water to prepare 1/2 Hoagland nutrient solution (containing 0.75 mmol/L K₂SO₄, 0.1 mmol/L KCl, 0.6 mmol/L MgSO₄, 4.0 × 10⁻² mmol/L FeEDTA, 1.0 × 10⁻³ mmol/L H₃BO₃, 1.0 × 10⁻³ mmol/L MnSO₄, 1.0 × 10⁻³ mmol/L ZnSO₄, 1.0 × 10⁻⁴ mmol/L CuSO₄, and 5.0 × 10⁻⁶ mmol/L Na₂ MoO₄).
2. After the wheat seeds (step 1.4) have germinated, place 15 wheat seedlings in hydroponic equipment (see Table of Materials) containing 100 mL of 1/2 Hoagland nutrient solution for hydroponics (Figure 3). Place the entire hydroponic apparatus in an artificial climate incubator (see Table of Materials) and incubate for 7 days at 25 °C and 80% relative humidity with a 16 h light/8 h dark photoperiod.

Figure 3: Hydroponic cultivation of the wheat seedlings. The wheat seedlings were hydroponically cultivated for 0 days, 3 days, and 7 days in 100 mL of 1/2 Hoagland nutrient solution. Please click here to view a larger version of this figure.

3. Experiment exposing the wheat plants to imidacloprid solution

1. After a 7 day hydroponic period, transplant the wheat plants into 1/2 Hoagland nutrient solution containing 0.5 mg/L or 5 mg/L imidacloprid to conduct the imidacloprid exposure experiments. Grow 15 wheat plants in each hydroponic device. Set up 15 hydroponic devices for each imidacloprid concentration group to ensure that adequate samples are taken during sampling.
2. Place the entire hydroponic equipment in an artificial climate incubator for 3 days at 25 °C and 80% relative humidity with a 16 h light/8 h dark photoperiod.
3. Throughout the exposure period, collect wheat roots (0.2 g per wheat plant) and leaves (0.5 g per wheat plant) daily. Integrate the wheat samples from every fifth hydroponic device as a parallel group and determine the imidacloprid content of the samples.

4. Procedure for extracting imidacloprid from wheat

1. Extraction of imidacloprid from wheat roots
   1. To avoid experimental errors, wash the wheat roots 4x with running sterile water for 10 s each time to remove any imidacloprid adsorbed on the root surface.
   2. Shred the wheat roots into approximately 1 cm pieces with scissors (Figure 4). Weigh 10.00 g of the shredded wheat roots and place in a 50 mL centrifuge tube.
   3. Add 10 mL of acetonitrile to the centrifuge tube and vortex the tube on a vortexer for 1 min. Then, add 4 g of anhydrous MgSO₄ and 1.5 g of NaCl to the centrifuge tube and vortex the tube immediately for 30 s. Centrifuge the tube for 5 min at 6,000 x g.
   4. Aspirate the supernatant with a disposable syringe and pass it through a syringe filter (0.22 µm pore size) to obtain the sample.
2. Extraction of imidacloprid from wheat leaves (Figure 5)
   1. Shred the fresh wheat leaves into approximately 1 cm pieces with scissors (Figure 4). Weigh 10.00 g of the shredded wheat leaves and place in a 50 mL centrifuge tube.
   2. Add 10 mL acetonitrile to the centrifuge tube and vortex the tube on a vortexer for 1 min.
   3. Add 4 g of anhydrous MgSO₄ and 1.5 g of NaCl to the centrifuge tube and vortex the tube immediately for 30 s.
   4. Centrifuge the tube for 5 min at 6,000 x g.
   5. After centrifugation, add 2 mL of the supernatant to a 5 mL centrifuge tube containing 50 mg of graphitized carbon black (GCB) and 150 mg of anhydrous MgSO₄ (to remove pigment and moisture from the sample), and vortex the centrifuge tube for 30 s (Figure 6). Centrifuge the tube for 5 min at 6,000 x g.
   6. Aspirate the supernatant with a disposable syringe and pass it through a syringe filter (0.22 µm pore size) to obtain the sample.

Figure 4: Shredded wheat roots and leaves. Fresh wheat roots and leaves were shredded using scissors into approximately 1 cm pieces. Please click here to view a larger version of this figure.

Figure 5: Extraction of imidacloprid in the wheat leaves. Imidacloprid in the samples was extracted using the QuEChERS method (steps 4.2.1-4.2.4 of the protocol). Please click here to view a larger version of this figure.

Figure 6: Purification of imidacloprid in the wheat leaves. The decontaminant was 50 mg GCB + 150 mg MgSO₄. Please click here to view a larger version of this figure.

5. Quantification of imidacloprid

1. Quantify the imidacloprid in the sample using liquid chromatography-tandem mass spectrometry (LC-MS-MS),based on a standard curve (y = 696.61x + 56.411, R=1) obtained from 0.2-250 µg/L imidacloprid concentrations. (Figure 7). The mass spectrometer was equipped with a C₁₈ column (100 mm x 2.1 mm, 3 µm) and an electrospray ionization source (ESI⁺). The elution program and ion source parameters are shown in Table 1.

Figure 7: Chromatogram and mass spectrogram of imidacloprid in the wheat leaves. The upper panel shows a chromatogram of imidacloprid (retention time = 0.93 min). The lower panel shows the mass spectrogram of imidacloprid at 0.93 min, showing the response intensity of the production (m/z = 208.8) of imidacloprid. Please click here to view a larger version of this figure.

Column temperature	40 °C
Solvent A	99.9% water/0.1% formic acid (v/v)
Solvent B	acetonitrile
Elution program	0–0.5 min, A = 20%
	0.5–2 min, A = 20%–50%
	2–3 min, A = 50%
	3–3.1 min, A = 50%–20%
	3.1–5 min, A=20%
Flow rate (mL/min)	0.3
Injection volume (μL)	5
Capillary temperature (°C)	330
Vaporizer temperature (°C)	350
Sheath gas flow rate (Arb)	40
Aux gas flow rate (Arb)	20
Spray voltage (V)	3900
Collision gas pressure (mTorr)	1.5
Precursor ion	256.1
Product ion/Collision energy (eV)	208.8/16

Table 1: Elution program and ion source parameters of the liquid chromatography-mass spectrometry method.

Results

The instrument limit of detection (LOD) of imidacloprid was 5.76 × 10⁻¹⁴ g, and the method's LOD of imidacloprid in the wheat root or leaf was 0.01 µg/kg; no matrix effect was observed. The recovery yields of imidacloprid in wheat are shown in Table 2. The recovery yields of imidacloprid from the wheat roots exposed to imidacloprid concentrations of 0.5 mg/L and 5 mg/L were 94.0%-97.6% and 98.8%-99.2%, respectively; the coefficients of variation were 1.92% and 0.20%, respectively. The recovery...

Discussion

In recent years, methods for the pretreatment and detection of residues of the pesticide imidacloprid have been frequently reported. Badawy et al.²³ used high-performance liquid chromatography to determine the content of imidacloprid in tomato fruit grown under greenhouse conditions and reported good linearity for imidacloprid in the range 0.0125-0.15 µg/mL. Zhai et al.²⁴ used LC-MS-MS to study the residue of imidacloprid in Chinese chives. In the present study, the Qu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetonitrile	Sigma-Aldrich (Shanghai) Trading Co. Ltd.	01-06-1995	Suitable for HPLC, gradient grade, >99.9%
Analytical balance	Sartorius Lab Instruments Co.Ltd.	GL124-1SCN
Artificial climate incubator	Shanghai Badian Instrument Equipment Co. Ltd.	HK320
Centrifuge	Eppendorf China Co. Ltd.	Centrifuge5804
Disposable syringe	Sigma-Aldrich (Shanghai) Trading Co. Ltd.	Z116866	Capacity 5 mL, graduated 0.2 mL, non-sterile
Formic acid	Sigma-Aldrich (Shanghai) Trading Co. Ltd.	Y0001970	European pharmacopoeia reference standard
Graphitized carbon black (GCB)	Sigma-Aldrich (Shanghai) Trading Co. Ltd.	V900058	45 μm
H2O2	Sigma-Aldrich (Shanghai) Trading Co.Ltd.	31642	30% (w/w)
Hoagland’s Basal Salt Mixture	Shanghai Yu Bo Biotech Co. Ltd.	NS1011	Anhydrous, reagent grade
Hydroponic equipment	Jiangsu Rongcheng Agricultural Science and Technology Development Co.Ltd.	SDZ04BD
Hypersil BDS C18 column	Thermo Fisher Scientific (China) Co. Ltd.	28103-102130
Imidacloprid	Sigma-Aldrich (Shanghai) Trading Co. Ltd.	Y0002028	European pharmacopoeia reference standard
MgSO4	Sigma-Aldrich (Shanghai) Trading Co. Ltd.	208094	Anhydrous, reagent grade, >97%
NaCl	Sigma-Aldrich (Shanghai) Trading Co.Ltd.	S9888	Reagent grade, 99%
pH meter	Shanghai Thunder Magnetic Instrument Factory	PHSJ-3F
Phytotron box	Harbin Donglian Electronic Technology Co. Ltd.	HPG-280B
Pipettes	Eppendorf China Co. Ltd.	Research plus
Syringe filter	Sigma-Aldrich (Shanghai) Trading Co.Ltd.	SLGV033N	Nylon, 0.22 µm pore size, 33 mm, non-sterile
Ultra performance liquid chromatography tandem triple quadrupole mass spectrometry	Thermo Fisher Scientific (China) Co. Ltd.	UltiMate 3000
		TSQ Quantum Access MAX
Vortex mixer	Shanghai Yetuo Technology Co. Ltd.	Vortex-2
Wheat seed	LuKe seed industry		Jimai 20