Researchers at the Institute of Plant Protection – National Research Institute Branch Sośnicowice, Gliwicka, Poland, and the Institute of Food Safety, Animal Health, and Environment “BIOR,” Riga, Latvia, along with the Department of Inorganic Chemistry, Analytical Chemistry, and Electrochemistry, Silesian University of Technology, Gliwice, Poland, have conducted research to determine the effect of pesticides and their metabolites on wheat metabolism.
Wheat (Triticum aestivum L.) is the second most widely cultivated crop worldwide after rice and provides dietary carbohydrates and proteins for around 40% of the global population. The widespread use of pesticides to protect wheat cultivation can result in pesticide residues within the grains, and it can affect plant metabolic processes during growth, causing developmental disruptions and lowering the nutritional value of wheat. Investigating plant metabolic profile changes under pesticide influence provides information on detoxification mechanisms and data about general plant conditions and their nutritional value.
A comprehensive approach was applied to evaluate the effects of pesticides on the metabolism of wheat. The application of commercially available pesticide formulations under field cultivation conditions provided a source of metabolic data unlimited by model conditions, representing a novel approach to studying the effects of pesticides on edible plants.
The applied pesticides: prothioconazole, tebuconazole, fluoxastrobin, diflufenican, florasulam, and penoxulam were detected at concentrations ranging from 0.0070–25.20 mg/kg and 0.0020–2.2 mg/kg in the wheat roots and shoots, respectively. Pesticide metabolites were detected in shoots: prothioconazole-desthio (prothioconazole metabolite), 5-(4-Chlorophenyl)-2,2-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)-1,3-pentanediol (tebuconazole metabolite), and N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)-2,4-dihydroxy-6-(trifluoromethyl)benzene sulfonamide (penoxulam metabolite). The 32 metabolic fingerprints and profiles changed during the experiment, reflecting the cumulative response of wheat to both its growth environment and pesticides and their metabolites. Approximately 15 days after the herbicide treatment, no further changes in the plant metabolic profiles were observed, despite pesticides and their metabolites in both roots and shoots. This study is the first to combine the determination of pesticides and their metabolites in plant tissues with the evaluation of plant metabolic responses under field conditions.
Gas and liquid chromatography coupled with tandem mass spectrometry was employed for targeted and non-targeted analysis of wheat roots and shoots sampled six times during the six-week experiment.
Plant tissues were extracted by using the QuEChERS approach.
Plant tissue sample preparation for pesticide determination
QuEChERS method EN 15662:2018 was used to extract the pesticides. Homogenized plant tissues (about 5 ± 0.05 g and 0.8 ± 0.05 g for shoots and roots, respectively) were weighed in 50 mL polypropylene (PP) centrifuge tubes, and water was added (5 mL and 2 mL, respectively). Samples were fortified with IS. 10 mL acetonitrile was added to each tube, and the samples were mechanically agitated for 1 min at 450 rpm. Mylar packets containing 4000 mg MgSO4, 1000 mg NaCl, 500 mg Sodium Citrate Dibasic Sesquihydrate, and 1000 mg Sodium Citrate Tribasic Dihydrate were poured into the tubes. The extraction tubes were shaken vigorously for 1 min and then centrifuged for 5 min at 4500 rpm. 6 mL of the supernatant was transferred into dispersive solid phase extraction (d-SPE) tubes containing 900 mg MgSO4, 300 mg C18, and 300 mg ChloroFiltr for shoots and 900mg MgSO4 and 150 mg PSA for roots. The tubes were mechanically agitated for 1 min at 450 rpm and then centrifuged for 5 min at 4500 rpm. After the cleaning step, 1 mL of the extract was evaporated under a gentle nitrogen stream until dry and then redissolved with 1 mL of 190 acetone (GC-EI-MS/MS) or acetonitrile: water (1:1, v/v) for (LC-ESI-MS-MS). Finally, the sample extracts were filtered through a 0.22 μm PTFE syringe filter into a 2 mL vial and analyzed. Three independent replicates were prepared for each sample.
Sample preparation for pesticide metabolite identification
Grind 10 g of plant shoots. Add aliquots of homogenized sample to 50 mL PP centrifuge tubes, followed by 20 mL of acetonitrile: water (1:1, v/v). Shake vigorously for 1 minute, and add the QuEChERS 4000 mg MgSO4, 1000 mg NaCl, 500 mg Sodium Citrate Dibasic Sesquihydrate, and 1000 mg Sodium Citrate Tribasic Dihydrate salt mixture. Place in a mechanical shaker and ultrasonic bath for 15 and 10 min, respectively. Then, centrifuge for 10 min at 3500 rpm. Transfer 8 mL of the supernatant to a 15 mL PP tube and place in a -80°C freezer for 10 min. Immediately after removing the sample from the freezer, centrifuge for 10 min at 3500 rpm. Take a 6 mL aliquot of the supernatant and transfer it to a 15 mL PP centrifuge tube containing 900 mg MgSO4, 150 mg PSA, and 150 mg C18. Vortex for 1 minute and centrifuge for 10 min at 3500 rpm. Take a 100 μL aliquot of the extract and transfer it to an autosampler vial and dilute in a (1:1 v/v) proportion of water with mobile phase A, 10 mM ammonium formate with 0.01% acetic acid (AA) (v/v), and analyze.
In the study, analyte recoveries ranged from 78.3–114.3% for shoots and 76.3–117.5% for roots and were consistent with the SANTE/12682/2019 requirements. RSD ranged from 0.65–13.8% for shoots and 1.1–11.4% for roots and satisfied the requirements. Shoot pesticide LOQs were 0.001 mg/kg, except for DFF, which had a LOQ of 0.005 mg/kg.
Pesticide accumulation in plant tissues was influenced more by the polarity of the pesticide molecules than by the mechanism of action and application type. Fungicides PCL, TBC, and FXS, as well as herbicides DFF, FSM, and PXM applied for crop protection, undergo metabolism while being translocated within the plant (from roots to shoots). However, from the extensive potential metabolite database, only PCL, TBC, and DFF metabolites were identified. Based on plant metabolic fingerprints and profiles, the applied pesticides influenced plant metabolism. Plant metabolic profiles changed during the experiment, likely due to the combined effects of biochemical transformations, the influence of external conditions, and the action of pesticides and their metabolites. However, agrochemical influence had a critical effect. The metabolic responses in roots and shoots obtained during samplings 5 and 6 did not significantly differ. The material had a high degree of pesticide metabolism relative to samples collected during the earlier stages of the study. The factors influencing plant metabolism at this stage of the experiment were naturally occurring biochemical transformations within the plants. Approximately two weeks after the herbicide application, the plant has metabolized the pesticides to such an extent that they do not affect its metabolism.
To download the original paper, visit https://doi.org/10.1016/j.envpol.2022.120468
Pszczolińska, K., Perkons, I., Bartkevics, V., Drzewiecki, S., Płonka, J., Shakeel, N., & Barchanska, H. (2023). Targeted and non-targeted analysis for the investigation of pesticides influence on wheat cultivated under field conditions. Environmental Pollution, 316, 120468.