Optimization of a QuEChERS method for 15 pesticides followed by determination the transport behavior in cowpea-soil system

Optimization of a QuEChERS method for 15 pesticides followed by determination the transport behavior in cowpea-soil system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimization of a QuEChERS method for 15 pesticides followed by determination the transport behavior in cowpea-soil system Jiazhen Wu, Yufei Li, Xingsheng Yue, Xingyue Li, Kai Guo, Ye Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5625297/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pesticide residues have always been one of the food safe issues troubling consumers. Agricultural production is usually accompanied by a mixture of pesticides, and pesticide residues are not only present on plants but also contaminate soil in the environment. In this study, 15 pesticides commonly found in cowpea and soil were analyzed by optimizing QuEChERS and combining GC-MS/MS and HPLC. Various extractants and different methanol water ratios were evaluated to extract the pesticides from cowpea and soil.C18, PSA and GCB were used to purify the cowpea extracts, while in soil the ratios of de-watering agent and NaCl were optimized. The average recoveries were 91.81-109.95% and 89.89-104.08% in cowpea and soil at spiked levels of 0.0-1.0 mg/kg and 0.1-10.0 mg/kg, respectively. This method is suitable for the detection of pesticides in different types of soil (red soil, yellow soil, sandy soil, sandy loam soil, paddy soil) and different cowpea tissues. In addition, pesticide residues were detected and analyzed in the cowpea- sandy loam soil system. This demonstrates that the developed method can be used to detect the multiple pesticide in various types of soils and crops, and provides the necessary technical support for agricultural product pesticide detection and safety supervision. QuEChERS Cowpea-soil system Multiple Pesticides Behavior Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Pesticides are crucial in agriculture, primarily used to control the proliferation of weeds and fungi (Kaur et al., 2019 ; Tudi et al., 2021 ). These pesticides may inadvertently cause harmful effects on human health while being toxic to their targets (Lushchak et al., 2018 ). As the variety of pesticides continues to increase, new active ingredients, adjuvants or co-formulations, and new formulation combinations are constantly being developed, leading to a gradual increase in the amounts of pesticides used in agriculture (Tudi et al., 2021 ). Furthermore, pesticides are often applied in mixtures to crops or persist in the environment, posing significant risks to ecological systems and environmental sustainability. Many pesticides have toxicity, bioaccumulation, and environmental persistence (Kaur et al., 2019 ). Once they enter the environment, their residues may persist prolonged periods, eventually being absorbed and accumulated in crops (Umetsu and Shirai, 2020 ). The overaccumulation of pesticides not only suppresses the normal growth of crops and compromises the quality and safety of agricultural products, but also potentially poses risks to human health (Chang et al., 2024 ; Ye et al., 2024 ). Although there has been extensive research on pesticide residues in crops and their impact on human health (Li et al., 2024 ; Yang et al., 2021 ; Zhang et al., 2023 ), continued attention is still needed on the behavior of pesticides within the plant-environment system, which will help provide theoretical basis for reducing pesticide residue. Soil is an important foundation for agriculture and food production, and the safety of soil environment is related to food security (Gomes et al., 2019 ). In agricultural production, and the application of excessive pesticides in agricultural production and irrigation sewage will bring organic pollutants into the soil (Tang et al., 2021 ). Organic pollutants in the soil can undergo migration and transformation, directly or indirectly affecting soil environmental quality and the growth of plants and animals. These organic pollutants may ultimately enter the human body through the food chain, and due to their high bioaccumulation potential, they can accumulate in fatty tissues, posing a threat to human health (Ye et al., 2024 ; Zhang et al., 2023 ). Soil particles have many charged sites on the surface that can interact with chemicals of different charges through electrostatic interaction (Amutova et al., 2022 ), thereby affecting the soil’s ability to adsorb pesticides. Additionally, the characteristics of the soil and pesticides, such as soil pH, organic matter content, particle size and the molecular structure of the pesticide, also significantly influence the strength of soil adsorption of pesticides (Diagboya and During, 2024 ). For example, the adsorption of benzovindiflupyr by eight soils was significantly correlated with the organic matter content, and the uptake of pesticides by soils exhibited a negative correlation with pH and a positive correlation with the organic matter content (Chang et al., 2024 ). The quantity of pesticides absorbed by crops from soil is mainly affected by the ability of soil to adsorb pesticides, which in turn affects the amount of pesticide migration in plants (Amutova et al., 2023; Wang et al., 2021 ; Yu et al., 2022 ). Therefore, studying the migration and transformation behavior of pesticides in the crop-soil system can help clarify the pattern of pesticide residue and accumulation in crops, which is beneficial to food safety and environmental protection. QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) is a widely used sample pretreatment method for the analysis of pesticide residues in food, environment and agricultural products. The method was proposed by Anastassiades et al. in 2003, and has several advantages. The QuEChERS method consists of two main steps, namely, sample extraction and purification (Bruzzoniti et al., 2014 ; Lehotay et al., 2010 ). Due to the differences in sample composition and impurities levels in the extraction process, the QuEChERS method can be optimized to improve the effectiveness of multi-residue pesticide detection. For example, optimization of the QuEChERS method for pesticide extraction in rice resulted in significant improvements in sensitivity and accuracy for four pesticides (Zheng et al., 2024 ). In addition, the improved QuEChERS method using citrate combined with acetonitrile for the extraction of 51 pesticides in food can be accurately used for residue detection in market samples and food evaluation (Su et al., 2024 ). Compared with other pretreatment methods, such as solid-phase extraction and liquid-liquid extraction, QuEChERS method is simpler, more efficient, and suitable for a variety of complex matrices, especially in the detection of multi-residue pesticides, which shows obvious advantages (Mastovska and Lehotay, 2003 ). Cowpea ( Vigna unguiculata L.) is the main species of winter vegetable in Hainan Province, with a planting area of 23077 hm 2 and an output of 582,100 tons in 2022, making it the second largest vegetable in Hainan Province (Hainan Provincial Statistical Yearbook). Cowpea is popular among consumers for its full pods, tender texture and high nutritional value (Avanza et al., 2013 ; Jayathilake et al., 2018 ). However, during the cultivation process, cowpea is often affected by various diseases and pests (Singh and Allen, 1979 ). Therefore, it is often necessary to apply pesticides to mitigate the impact of these pests and diseases on the yield and quality of cowpeas. The objective of this study was to establish QuEChERs for 15 pesticides (thiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole, cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, ifenoconazole, azoxystrobin, dimethomorph) that are used with high frequency in cowpea, thereby improving assay precision and pretreatment efficiency, and combining HPLC and GC-MS/MS for detection. In addition, the behaviors of 15 pesticides in cowpea-soil were investigated to clarify the effects of soil physicochemical and pesticide properties on the fate of pesticides in cowpea. 2. Materials and methods 2.1 Chemicals and reagents Thiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole, cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, ifenoconazole, azoxystrobin, dimethomorph standard was obtained from Tiankang Yunmu Technology Co Ltd. Acetonitrile and ethyl acetate were purchased from Xilong Chemical Co. Sodium chloride (NaCl), PSA, GCB, MWCNTs, MWCNTs-NH 2 , C18 were purchased from Shanghai Ampoule Experimental Technology Co. 2.2 Experimental material Cowpea ( Vigna unguiculata L.) was purchased from Longxi Seed Industry Co. Ltd. in Suqian, Jiangsu Province (Jiangsu, China). Five types of soil were used in this experiment, including red soil (Sanjia, Dongfang City), yellow soil (Jianfengling, Ledong County), sandy soil (Foluo, Ledong County), sandy loam soil (Sigeng, Dongfang City), and paddy soil (Yazhou, Sanya City). The topsoil was collected and air-dried in a cool and dry place. After removing roots, stones and other debris, the soil was crushed and sieved through a 2 mm mesh for further use. The phychemical properties of the soils are shown in Table S1 . 2.3 Sample pretreatment 2.3.1 Pre-treatment of cowpea samples Cowpea samples were weighed (5.0 g) and placed in centrifuge tube, extracted with 10 mL of acetonitrile solution and were mixed for 5 min. Then, added 5.0 g NaCl, vortexed for 2 min, and centrifuged at 5000 r/min for 5 min. Added supernatant (2 mL) to a centrifuge tube with varying additions of PSA, C18 and anhydrous MgSO 4 and vortexed for 5 min, then centrifuged (5000 r/min, 5 min). Supernatant (1 mL) was rotary evaporated to near dryness at 40°C, reconstituted with ethyl acetate or methanol, and filtered through a membrane (0.22 µm) and left to be measured. 2.3.2 Pre-treatment of soil samples Weighed 5.0 g of soil after air-drying in centrifuge tube, added 10 mL acetonitrile and 5 mL ultrapure water, vortexed for 2 min. Then put it into an ultrasonic cleaner and added 1 g NaCl + 4 g anhydrous MgSO 4 , continued to shake for 2 min and centrifuged (5000 rpm, 10 min). The supernatant was filtered. 2.4 Detection condition 2.4.1 HPLC analysis Thiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole were analyzed by HPLC system (Shimadzu LC-AD20, Japan). The separation was performed on a PICKERING C18 column (4.6 mm×250 mm, inner diameter). The mobile phase consists of methanol and water (60:40, v/v). Flow rate: 1.0 mL/min. Wavelength: 255 nm. Injection volume:10 µL. Column temperature: 30℃. The overall analysis duration was 19 min. 2.4.2 GC-MS/MS analysis The analysis of cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, difenoconazole, azoxystrobin, dimethomorph was performed by using the same equipment and configuration as that of Ren et al. ( 2023 ). The ramp-up procedure is shown in Table S2 and the total run time was 23 min. 2.5 Analytical method validation 15 pesticides standard solutions of 0.1, 0.2, 0.5, 1, 2, and 5 mg/L were prepared. The linear regression equation and the coefficient of determination (R 2 ) the 15 pesticides were obtained. Detailed data are shown in Table S3. The accuracy and precision of the method were verified according to the description of Ren et al. ( 2023 ), with a recovery of 70–120% and a standard deviation (RSD) of less than 20%. 2.6 Data analysis 2.6.1 Enrichment and migration of 15 pesticides in soil by cowpea plants The extent of pesticide uptake and accumulation from the soil by cowpea plants was expressed as bioconcentration factor (BCFs), with root bioconcentration factor (RCF), stem bioconcentration factor (SCF) and leaf bioconcentration factor (LCF) was calculated as follows: RCF = C R /C S SCF = C S /C S LCF = C L /C S The extent of pesticide translocation in cowpea plants is expressed as translocation factor (TF), and the root-to-stem (TF s/r ) and stem-to-leaf (TF l/s ) translocation factors were calculated as follows: TF s/r = C s/ C r TF l/s = C l/ C s 2.6.2 Data processing and analysis Excel 2016 software was used to summarize the experimental data; origin 8.5 software was used for graphing; SPSS 17.0 software was used for data correlation analysis, and a, b, c was used to indicate the significance of differences between treatment groups (p < 0.05). 3. Results and discussion 3.1 Optimization of pre-treatment for cowpea samples 3.1.1 Selection of extraction solvent for cowpea The extraction effects of the extractant on pesticides varies depending on the type of pesticide, polarity and differences in vegetable substrates. Acetonitrile, ethyl acetate and methanol are commonly used extractants for pesticide extraction. Acetonitrile is moderately polar, volatile, and widely used, especially in the QuEChERS method showing high purity and relatively clean extracts (Kecojević et al., 2021 ). Ethyl acetate has slightly lower polar than acetonitrile, better water solubility, easy to penetrate into plant cell, can effectively extract a wide range of pesticides, which is superior to acetonitrile in the extraction of some specific pesticides, but it may carry more lipid contaminants (Madej et al., 2018 ). Methanol is more polar and suitable for the extraction of pesticides with higher polarity (Li et al., 2017 ). The effect of acetonitrile, ethyl acetate and methanol extraction in cowpea spiked with pesticides at a level of 1.0 mg/ kg is shown in Fig. 1 . Methanol, as an extractant, produced recoveries outside the range of 70–120% for the remaining 12 pesticides, except for imidacloprid, acetamiprid, carbendazim, indicating that methanol is not suitable for the simultaneous extraction of 15 pesticides in cowpea. In addition, the recovery of some pesticides was too high when extracting them with methanol, and the recovery of thiamethoxam was even as high as 129.61%, which indicated that methanol introduced impurities that interfered with pesticide extraction during the extraction process (Rutkowska et al., 2017 ). Additionally, rotary evaporation of methanol extracts was inefficient for high-pigment content cowpea leaf samples. In contrast, acetonitrile was more effective in extracting the 15 pesticides, with recoveries ranging from 70–120%. When ethyl acetate was used as the extraction agent, ethyl acetate was more effective than acetonitrile for chlorpyrifos and pyrimethanil, this is because chlorpyrifos is a nonpolar pesticide, and ethyl acetate is able to efficiently penetrate and solubilize nonpolar pesticides (Yin et al., 2020 ). Whereas, ethyl acetate was less effective for imidacloprid, chlorantraniliprole, cyromazine and tebuconazole, with recoveries below 70%. In addition, the residual pigment content after ethyl acetate extraction was also higher. Therefore, acetonitrile, as a polar medium extractant (Madej et al., 2018 ), gave the most stable recovery of pesticide, and the extracted solution was clear with little pigment residue. 3.1.2 Selection of cowpea adsorbent The fruit and vegetable matrix are rich in pigments, water, polysaccharides, and acidic substances (Gao et al., 2023 ). Adsorbents such as MWCNTs, MWCNTs-NH 2 , GCB can purify fruit and vegetable matrices and are widely used in the pretreatment of pesticide residues in food (Chen et al., 2014 ; González-Curbelo et al., 2013 ). Adsorbents suitable for cowpea pesticide extraction were screened, and the results are shown in Fig. 2 . For GCB as the adsorbent, the recoveries were in the range of 70–100% for all pesticides except for cyromazine (39.83%) and chlorpyrifos (63.28%) which were not in the standard range. In contrast, when MWCNTs-NH 2 was used as the adsorbent, the recoveries of cyromazine (105.13%) and chlorpyrifos (74.72%) were within the standard range. It can also be observed from the Fig. 2 that under the same addition amount, MWCNTs have a stronger adsorption capacity than the other two adsorbents, resulting in the recovery of pesticides being less than 60%, which is attributed to MWCNTs having a larger specific surface area and strong adsorption capacity for interfering substances (Pallavi et al., 2021 ). It mainly relies on van der Waals forces, π-π interactions and hydrophobic interactions, so it is particularly suitable for the adsorption of nonpolar compounds, which resulted in the recoveries of less than 70% for all 15 pesticides (Dehghani et al., 2019 ). Therefore, MWCNTs-NH 2 and GCB were selected to be used for the remaining 13 pesticides except for cyromazine and chlorpyrifos which only used MWCNTs-NH 2 as adsorbent. 3.1.3 Optimization of cowpea purifier content Commonly used purifying agents include PSA (N-propyl ethylenediamine), and C18. PSA, with 2 amino groups, can remove carbohydrates, phenols, and fatty acids from the matrix through ion exchange. However, excessive use of PSA can lead to pesticide adsorption and reduced recovery due to hydrogen bonding with functional groups such as -NH, -SH, and -OH in some pesticides (Jin et al., 2021 ). C18 is a reverse-phase adsorbent that has strong adsorption capabilities for non-polar substances such as cholesterol, vitamins, and fats in the matrix (Háková et al., 2018 ). GCB exhibits strong adsorption for pigments but may also affect recovery by adsorbing certain planar-structured pesticides (Long et al., 2023 ). GCB is not required as cowpea roots do not contain chlorophyll, different amounts of C18 and PSA (50 mg, 100 mg, 150 mg, 200 mg, 250 mg) were selected along with 200 mg MgSO 4 to investigate their impact on pesticide recovery. The results are shown in Fig. 3 A, the recovery within the range of 70–110% for thiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole, cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, ifenoconazole, azoxystrobin and dimethomorph, after the addition of 100 mg and 150 mg of C18 and PSA. The pesticide recovery was too high when the addition amount of C18 and PSA was 50 mg, while the pesticide recovery was too low when the addition amount of C18 and PSA was 200 mg and 250 mg. The amount of GCB used in stems and leaves was subsequently optimized. In the stems of cowpea, 20, 30, 40, 50, 60 mg were selected for pesticide extraction The results showed that the recovery of several pesticides was more than 120% when GCB was used at 20 and 30 mg, while recovery of some pesticides was less than 70% when GCB was used at 60 mg (Fig. 3 B). This was attributed to the adsorption of pesticides by GCB, insufficient addition of GCB will result in the substrate not achieving the purification effect, while excessive addition will lead to the absorption of target pesticides, resulting in a decrease in recovery (Kim et al., 2018 ). When the amount of GCB was 40 and 50 mg, the recoveries of 15 pesticides were within the standard range, and the recovery range of 40 mg fluctuated less and the amount was less. Therefore, 40 mg of GCB was selected for the decontamination of cowpea stems. The higher chlorophyll content in cowpea leaves resulted in correspondingly higher levels of GCB used (30, 50, 80, 100 and 150 mg). Similar to the GCB additions to the stems, the lower doses of GCB (30 mg and 50 mg) were less effective in adsorbing impurities, resulting in excessive pesticide recovery (> 120%), whereas the higher dose of GCB (150 mg) excessively adsorbed pesticides, resulting in pesticide recoveries below 70%. The recoveries of all 15 pesticides ranged from 70–120% at GCB dosages of 80 mg and 100 mg (Fig. 3 C). Based on the unit of recovery and dosage considerations, we chose 80 mg of GCB for pesticide extraction from cowpea leaves to obtain the best extraction results. 3.2 Optimization of soil purification agent content The main function of a desiccant is to induce salting-out effect during the extraction process of the target analyte, thereby promoting phase separation. Commonly used desiccants include MgSO 4 and NaCl, etc. The combination of MgSO 4 and NaCl is widely applied in QuEChERS to improve the purity and recovery of extracts (Zhang et al., 2024 ). In order to find the optimal amount of NaCl and MgSO 4 in soil matrix, this study conducted experiments on five different combinations. The results showed that both 0.2 g + 0.5 g and 0.5 g + 1 g ratios of NaCl + MgSO 4 resulted in recovery within standard range with good stratification between water and organic phase. This is because insufficient addition of NaCl will lead to incomplete distribution of the organic and aqueous phases, the pesticide components cannot be completely transferred to the organic phase, especially for pesticides with high polarity, the recovery may be significantly reduced. And when the amount of anhydrous MgSO 4 is insufficient, the water in the sample is not sufficiently absorbed to remain in the extract. The excess water may dilute the extracted pesticide components, which especially has a negative effect on the detection of water-soluble pesticides (Hwang et al., 2024 ). Whereas, 2 g + 3 g and 3g + 4g NaCl + MgSO 4 would lead to salting-out, which makes it difficult for certain pesticide components with high polarity to be fully dissolved in the organic phase. While excessive use of anhydrous MgSO 4 can lead to an increase in solid particles in the organic phase, too many particles can be difficult to settle completely during centrifugal separation, affecting the purity of the extraction solution (Faraji et al., 2018 ). The ratio of 1g + 2 g of NaCl + MgSO 4 resulted in pesticide recoveries that were all within the standard range and well stratified between the aqueous and organic phases. 3.3 Validation of the QuEChERS method The precision and accuracy of the established method were validated using different cowpea tissues and five types of soil, the optimized method was used to conduct recovery experiments on blank cowpea samples and soil spiked with 0.01, 0.1, and 1.0 mg/kg. The recoveries of the 15 pesticides in cowpea were determined. As shown in Table S4, the recovery in cowpea matrix ranged from 74.15–118.63%, with a precision range of 0.3–18.36%. Similarly, the average recovery in soil matrix also ranged from 74.15–118.63%, with a precision range of 0.3–18.36%. In addition, the recoveries and RSDs of the 15 pesticides in the five different soils were also within the standard range (Table S5). Therefore, the developed method fully complies with the requirements of SANTE/11312/2021 guidance, where stipulate recovery is within the range of 70–120% and RSD is less than 20% (European Food Safety Authority (EFSA)). 3.4 Analysis of the fate of 15 kinds of pesticides in soil-vegetable system 3.4.1 Exposure experiment Pesticides that are not effectively utilized enter the environment and remain in the soil, where they are then taken up by plant roots and translocated to other sites (Zhang et al., 2023 ). Pesticide residues, accumulation and metabolism in crops not only contribute to the quality and safety of agricultural products, but are also toxic to non-targets (Xia et al., 2024 ; Zhang et al., 2022 ). The distribution of the 15 pesticides in the cowpea-soil system is shown in Fig. 5 . Initially, pesticide residue levels were measured in soils with and without cowpea seedlings (Fig. 5 ). After 21 days, soils with cowpea seedlings exhibited significantly lower pesticide residues, indicating active uptake or degradation soil without cowpea seedlings. The above results suggest that the presence of cowpea plants may accelerate pesticide residues in the soil by absorbing pesticides or promoting degradation. During the soil cultivation of cowpea, 15 pesticides were detected in different tissue, indicating that roots are able to uptake pesticides and transport them to other organs. Most of the pesticide uptake by roots reached the maximum on the 3–5 d, fluopyram reached the maximum on the 10th day, and tebuconazole reached the maximum on the 14 d, after which the levels gradually stabilized. The average concentrations of thiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole, cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, difenoconazole, azoxystrobin and dimethomorph in the roots were 1.173, 1.124, 1.140, 4.127, 2.916, 0.852, 1.970, 2.224, 2.226, 0.579, 0.962, 1.322, 1.312, 7.039 and 2.704 mg/kg, respectively. The transport of pesticides in the stems of cowpea plants mostly reached equilibrium after the 10th day and decreased after the 14th day. The concentration of metalaxyl in the stems peaked on the 3rd day and gradually declined thereafter. The concentrations of thiamethoxam, acetamiprid, cyromazine, and metalaxyl in the leaves gradually exceeded those in the stems and roots after 1–3 d (Fig. 5 A-D), whereas metalaxyl and fluopyram were required on 7 or 21 d (Fig. 5 E, 5 F), respectively. Cyromazine showed a distribution pattern of leaves > stems > roots on day 14 d, while metalaxyl showed leaves > stems ≈ roots, which indicated that these two pesticides had strong upward translocation capabilities. The accumulation of chlorpyrifos, chlorfenapyr, pyraclostrobin, difenoconazole, azoxystrobin, and dimethomorph in the roots increased over time, reaching a maximum on the 14th day before declining (Fig. 5 I-O). The distribution of these six pesticides in the cowpea plants followed the pattern of roots > stems ≈ leaves, suggesting that these pesticides are not easily translocated upwards and are mainly concentrated in the roots. Kow (octanol-water partition coefficient, Log Kow ) is a coefficient that measures the partition of pesticides between octanol and water, reflecting the lipophilicity and hydrophilicity of the compound (Miller et al., 2016 ). It is closely related to the absorption and transportation of pesticides. Kow directly affects the transmembrane diffusion of pesticides. Pesticides with high Kow values are more lipophilic and therefore more likely to cross lipid bilayers and enter the cells of plants or other organisms (Zhang et al., 2023 ). In contrast, pesticides with a low K ow are more hydrophilic, less absorbed, and have difficulty in crossing cell membranes, usually staying more in the aqueous phase or being blocked by the more polar cell wall or extracellular regions (Wang et al., 2021 ). The content of the six pesticides thiamethoxam, imidacloprid, acetamiprid, cyromazine, metalaxyl, and fluopyram was present at higher levels in leaves than roots after a period of stable exposure. This phenomenon occurs because the first five pesticides have a Log Kow less than 3, making them less likely to be significantly accumulated in the roots, leading to their upward transport and accumulation in the leaves. Although fluopyram has a Log Kow greater than 3, its high solubility results in a greater concentration in the leaves compared to the roots during the later stages of the exposure trial molecular weight (MW). In contrast, the pesticides carbendazim, chlorantraniliprole, chlorpyrifos, chlorfenapy, tebuconazole, pyraclostrobin, difenoconazole, pyraclostrobin and dimethomorph exhibit a Log Kow greater than 3 or a Log MW greater than 300 g/mol (Wang et al., 2021 ). Their higher hydrophobicity facilitates retention in the root lipids, and their relatively low water solubility hinders upward migration through transpiration. 3.4.2 Correlation analysis 3.4.2.1 Bioconcentration factor analysis The phychemical properties of pesticides play a crucial role in determining how they behave within plants. Among them, MW has a significant effect on their uptake and translocation in plants. In general, pesticides with smaller MW are more readily absorbed and can be extensively distributed throughout the plant, while pesticides with larger MW are usually difficult to diffuse through plant cell walls and membranes, which restricts their movement within the plant (Miller et al., 2016 ). Solubility in Water (S W ) can also determine the behavior of pesticides in the plant-environment, especially their uptake through plant roots and their ability to move within the plant. In addition, the Kow also serves as a significant indicator influencing the distribution of pesticides in plants (Miller et al., 2016 ). Therefore, the Kow, S W , and MW were used as the physicochemical properties of the pesticides, along with the average BCF of 15 pesticides in various parts of the cowpea, to conduct a linear regression analysis to investigate how these physicochemical properties affect the behavior of pesticides in the cowpea-soil system. The findings indicated that the logarithmic values of the BCF in the leaves had a negative correlation with Log Kow (Fig. 6 C) (R 2 = 0.8831, P = 0.0000), whereas the roots and stems exhibited a positive correlation with Log Kow (R 2 = 0.4529, 0.5513; P = 0.0060, 0.0015) (Fig. 6 A, B). No significant correlation was observed between Log RCF and Log MW (Fig. 6 D), while both Log SCF and Log LCF exhibited a weak negative linear correlation with Log MW (R 2 = 0.3724, 0.2840; P = 0.0157, 0.0408) (Fig. 6 E, F). These indicated that as the MW of the pesticides increases, the BCF in the roots and stems gradually decrease, while those in the stems and leaves also decreases. Additionally, there was no significant correlation between Log Sw and the BCF in the roots (Fig. 6 G), but a positive correlation was observed with the BCFs in the stems and leaves (R 2 = 0.5714, 0.6433; P = 0.0011, 0.0003) (Fig. 6 H, I). The above results suggested that with the S W of the pesticides increases, the BCF in the stems and leaves gradually decrease, resulting in a decrease in the accumulation of these pesticides in these parts. The results mentioned above align with the findings regarding pesticide accumulation in the same tissues of cowpea (Fig. 5 ). 3.4.2.2 Transfer factor analysis The TF can be used to indicate the ability of pesticides to be transferred in plant (Xia et al., 2024 ; Ye et al., 2024 ). To clarify how the physicochemical properties of pesticides influence their transfer capabilities, linear regression analysis was conducted on the three physicochemical properties that reveal accumulation patterns in relation to cowpea TF s/r and TF l/s . The results are shown in Fig. 7 . There was a strong linear negative correlation was observed between the TF s/r and Log Kow (R 2 = 0.7441, P = 0.0003), indicating that pesticide translocation within the plant may gradually increase with increasing hydrophobicity (Fig. 7 A). However, the TF l/s had no significant correlation with Log Kow (R 2 = 0.7441, P = 0.0003) (Fig. 7 B). Additionally, the TF showed a high linear negative correlation with Log MW (R 2 = 0.6738, P = 0.0001), indicating that as MW increases, the mobility of the pesticides within the plants gradually decreases (Fig. 7 C). No significant correlation was observed between the TF l/s and the MW of the pesticides (Fig. 7 D). Between TF s/r and Log Sw , a strong linear positive correlation was found (R 2 = 0.8021, P = 0.0000), which implied that pesticide accumulation in the roots might decrease with increasing Sw, thereby enhancing their translocation within the cowpea (Fig. 7 E). However, no significant correlation between TF l/s and Log Sw (Fig. 7 F). Other studies have shown that the transport of organic pollutants in vegetables is negatively correlated with log Kow as hydrophobicity increases (Ye et al., 2024 ; Zhang et al., 2023 ). And the smaller the MW and higher the Sw of the pesticide, the easier the pesticide is to be transported to the top of maize, cherry radish, wheat, etc. (Wang et al., 2021 ; Xia et al., 2024 ; Ye et al., 2024 ; Yu et al., 2022 ). However, pesticide physicochemical properties are not the only factors affecting pesticide uptake and transport in plants, which may be related to the structural organization, physicochemical properties (e.g., fats, proteins, etc.), and growing environment (hydroponic vs. soil) of vegetables (Chang et al., 2024 ; Chang et al., 2021 ). In addition, differences in soil physicochemical properties can influence a plant’s capacity to absorb pesticides from the soil and redistribute them to other locations. For example, soil pH significantly affects the translocation and accumulation of imidacloprid in corn, which can be toxic to corn (Chang et al., 2021 ). Similarly, the uptake of imidacloprid and propiconazole by wheat roots is influenced by soil physicochemical properties, which in turn affects their translocation in wheat (Ju et al., 2020 ). In addition, Pearson correlation analyses of cowpea enrichment factors, transcription factors, and physicochemical parameters were performed, and the results are shown in Fig. 8 . The correlation coefficient between log SCF and log LCF was 0.94, showing a strong positive correlation. This indicates a high degree of covariance between these two variables, suggesting a high positive correlation between the pesticide enrichment capacity of cowpea stems and leaves. TFs/r and log Sw both had a correlation coefficient of 0.86, which is also a strong positive correlation. It indicates that solubility (log Sw ) may be positively correlated with the migration efficiency of TF s/r under certain conditions, the greater the Sw of pesticides, the easier it is to migrate to the above-ground parts of cowpea. However, log Kow and log Sw showed a strong negative correlation of -0.76, suggesting that log Kow and log Sw are mutually exclusive under certain conditions. The strong negative correlation between TFs/r and log MW suggests that larger molecular weights are unfavorable for pesticide translocation from roots to cowpea above-ground parts. Pearson correlation analysis verified the results of the Fig. 7 . 4. Conclusions This study optimized the QuEChERS method combined with GC-MS/MS and HPLC for the analysis of 15 common pesticides in cowpea and soil. Different extraction solvents were compared for the extraction of pesticides from cowpea, ultimately selecting acetonitrile as the extraction solvent for cowpea. C18, PSA, and GCB were employed to purify the cowpea extracts, with different amounts of pesticide cleanup sorbents being optimal for different pesticides in cowpea. For soil, a final addition ratio of 1 g sodium chloride and 2 g MgSO 4 was selected. Ultimately, at spiking levels of 0.01-1.0 mg/kg and 0.1–10.0 mg/kg, the recoveries varied between 74.15% -118.63%, with precision between 0.3–18.36%. The average recovery in soil matrix also fell within the range of ranged from 74.15–118.63%, with precision ranging from 0 .3% to18.36%. At the same time, we studied the enrichment and interpretation patterns of 15 pesticides in cowpea and soil, and found that 6 pesticides were easily enriched in the above-ground part of cowpea, which was mainly affected by the phychemical properties of pesticides and soil. The developed detection method in this study can better monitor the residues of multiple pesticides in cowpea and various types of soil. Declarations Declaration of Competing Interest The authors declare no competing financial interests. This is our original work, which has not been published previously or considered for publication elsewhere, and has been approved by all authors. Funding This research was supported by National Natural Science Foundation of China Regional Joint Priority Project (U22A20484), Key Laboratory of Tropical Fruits and Vegetables Quality and Safety for State Market Regulation (No. KF-2023001), and Hainan University Research Initiation Fund (Science and Technology) (XJ2400005859). Author Contribution W. J: Writing-original draft; L.Y: Data curation, Software; Y.X, L.X and G.K: Experimental operation; Y.Y: Writing-original draft;W. M: Conceptualization, Investigation; Z.S:Investigation, Writing-original draft, L.X and Z.Y Writing-review & editing, Supervision. All authors reviewed the manuscript. 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extractants\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/ccd071297296394a9696ebec.png"},{"id":71900821,"identity":"92c63b53-9bc2-4cb1-9e29-22f1805f17a9","added_by":"auto","created_at":"2024-12-19 14:23:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":21129,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different adsorbents on the recovery of 15 pesticides\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/c2350db205466c137625c013.png"},{"id":71899934,"identity":"107bf388-54c5-49e5-8aa1-f134c83f27f1","added_by":"auto","created_at":"2024-12-19 14:15:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60413,"visible":true,"origin":"","legend":"\u003cp\u003eRecovery of 15 pesticides in cowpea roots (A), stems (B), and leaves (C) under different concentrations of purifiers\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/57dd0649c001b909d1990df5.png"},{"id":71899961,"identity":"dba80628-0f14-4ebc-b725-3e00ac72e03c","added_by":"auto","created_at":"2024-12-19 14:15:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":23976,"visible":true,"origin":"","legend":"\u003cp\u003eRecovery rates of 15 different pesticides in soil with the addition of various concentrations of dewatering agent\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/bca9457b28cfef05427b243a.png"},{"id":71900832,"identity":"d2edb789-3b2e-4ad9-9e89-87dd60b00592","added_by":"auto","created_at":"2024-12-19 14:23:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":128360,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the contents of thiamethoxam (A), imidacloprid (B), Acetamidine (C), Cyromazine (D), Metalaxyl (E), Fluopyram (F), Carbendazim (G), Chlorantraniliprole (H), Chlorpyrifos (I), Chlorfenapyr (J), Tebuconazole (K), Pyraclostrobin (L), Difenoconazole (M), Azoxystrobin (N), Dimethomorph (O) in cowpea plants and sandy loam soil\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/73fe34f4665185cdec7d69de.png"},{"id":71900914,"identity":"843d8809-ccaf-423b-a3c9-0bbaf7bb9a49","added_by":"auto","created_at":"2024-12-19 14:23:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":38504,"visible":true,"origin":"","legend":"\u003cp\u003eThe concentration factors of roots, stems and leaves of 15 pesticides were correlated with Log K\u003cem\u003eow \u003c/em\u003e(A-C), Log \u003cem\u003eMW\u003c/em\u003e (D-F), Log \u003cem\u003eSw\u003c/em\u003e(G-I)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/f232eceb665a2a7b71760c96.png"},{"id":71900967,"identity":"630f5abd-1cf7-4d0d-99f7-819af729a089","added_by":"auto","created_at":"2024-12-19 14:23:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":43085,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between transport factors of 15 pesticides and Log \u003cem\u003eKow\u003c/em\u003e (A-B), Log \u003cem\u003eMW\u003c/em\u003e (C-D), Log \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sub\u003e (E-F)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/ff42dc6c48bd46297ceb5656.png"},{"id":71900962,"identity":"3ec0bb88-bb73-4b61-b625-890e8d1cf62b","added_by":"auto","created_at":"2024-12-19 14:23:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":129607,"visible":true,"origin":"","legend":"\u003cp\u003eLog BCF and TF correlation coefficient matrix heat map\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/c2cfb6d48d7908db53f19639.png"},{"id":72343556,"identity":"c46e7d7b-ba19-43d0-997c-e26f134e6c51","added_by":"auto","created_at":"2024-12-25 22:16:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1003275,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/bc753a6b-31c4-47a7-a30b-cdfa9480b188.pdf"},{"id":71899970,"identity":"59b6d4dc-07d1-4e1a-b2a6-9a0e5f2fc632","added_by":"auto","created_at":"2024-12-19 14:15:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":42671,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5625297/v1/e35783143e948540ee994b2f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimization of a QuEChERS method for 15 pesticides followed by determination the transport behavior in cowpea-soil system","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePesticides are crucial in agriculture, primarily used to control the proliferation of weeds and fungi (Kaur et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tudi et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These pesticides may inadvertently cause harmful effects on human health while being toxic to their targets (Lushchak et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As the variety of pesticides continues to increase, new active ingredients, adjuvants or co-formulations, and new formulation combinations are constantly being developed, leading to a gradual increase in the amounts of pesticides used in agriculture (Tudi et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, pesticides are often applied in mixtures to crops or persist in the environment, posing significant risks to ecological systems and environmental sustainability. Many pesticides have toxicity, bioaccumulation, and environmental persistence (Kaur et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Once they enter the environment, their residues may persist prolonged periods, eventually being absorbed and accumulated in crops (Umetsu and Shirai, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The overaccumulation of pesticides not only suppresses the normal growth of crops and compromises the quality and safety of agricultural products, but also potentially poses risks to human health (Chang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ye et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although there has been extensive research on pesticide residues in crops and their impact on human health (Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), continued attention is still needed on the behavior of pesticides within the plant-environment system, which will help provide theoretical basis for reducing pesticide residue.\u003c/p\u003e \u003cp\u003eSoil is an important foundation for agriculture and food production, and the safety of soil environment is related to food security (Gomes et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In agricultural production, and the application of excessive pesticides in agricultural production and irrigation sewage will bring organic pollutants into the soil (Tang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Organic pollutants in the soil can undergo migration and transformation, directly or indirectly affecting soil environmental quality and the growth of plants and animals. These organic pollutants may ultimately enter the human body through the food chain, and due to their high bioaccumulation potential, they can accumulate in fatty tissues, posing a threat to human health (Ye et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Soil particles have many charged sites on the surface that can interact with chemicals of different charges through electrostatic interaction (Amutova et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), thereby affecting the soil\u0026rsquo;s ability to adsorb pesticides. Additionally, the characteristics of the soil and pesticides, such as soil pH, organic matter content, particle size and the molecular structure of the pesticide, also significantly influence the strength of soil adsorption of pesticides (Diagboya and During, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For example, the adsorption of benzovindiflupyr by eight soils was significantly correlated with the organic matter content, and the uptake of pesticides by soils exhibited a negative correlation with pH and a positive correlation with the organic matter content (Chang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The quantity of pesticides absorbed by crops from soil is mainly affected by the ability of soil to adsorb pesticides, which in turn affects the amount of pesticide migration in plants (Amutova et al., 2023; Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, studying the migration and transformation behavior of pesticides in the crop-soil system can help clarify the pattern of pesticide residue and accumulation in crops, which is beneficial to food safety and environmental protection.\u003c/p\u003e \u003cp\u003eQuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) is a widely used sample pretreatment method for the analysis of pesticide residues in food, environment and agricultural products. The method was proposed by Anastassiades et al. in 2003, and has several advantages. The QuEChERS method consists of two main steps, namely, sample extraction and purification (Bruzzoniti et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lehotay et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Due to the differences in sample composition and impurities levels in the extraction process, the QuEChERS method can be optimized to improve the effectiveness of multi-residue pesticide detection. For example, optimization of the QuEChERS method for pesticide extraction in rice resulted in significant improvements in sensitivity and accuracy for four pesticides (Zheng et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In addition, the improved QuEChERS method using citrate combined with acetonitrile for the extraction of 51 pesticides in food can be accurately used for residue detection in market samples and food evaluation (Su et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Compared with other pretreatment methods, such as solid-phase extraction and liquid-liquid extraction, QuEChERS method is simpler, more efficient, and suitable for a variety of complex matrices, especially in the detection of multi-residue pesticides, which shows obvious advantages (Mastovska and Lehotay, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCowpea (\u003cem\u003eVigna unguiculata\u003c/em\u003e L.) is the main species of winter vegetable in Hainan Province, with a planting area of 23077 hm\u003csup\u003e2\u003c/sup\u003e and an output of 582,100 tons in 2022, making it the second largest vegetable in Hainan Province (Hainan Provincial Statistical Yearbook). Cowpea is popular among consumers for its full pods, tender texture and high nutritional value (Avanza et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jayathilake et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, during the cultivation process, cowpea is often affected by various diseases and pests (Singh and Allen, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). Therefore, it is often necessary to apply pesticides to mitigate the impact of these pests and diseases on the yield and quality of cowpeas. The objective of this study was to establish QuEChERs for 15 pesticides (thiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole, cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, ifenoconazole, azoxystrobin, dimethomorph) that are used with high frequency in cowpea, thereby improving assay precision and pretreatment efficiency, and combining HPLC and GC-MS/MS for detection. In addition, the behaviors of 15 pesticides in cowpea-soil were investigated to clarify the effects of soil physicochemical and pesticide properties on the fate of pesticides in cowpea.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and reagents\u003c/h2\u003e \u003cp\u003eThiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole, cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, ifenoconazole, azoxystrobin, dimethomorph standard was obtained from Tiankang Yunmu Technology Co Ltd. Acetonitrile and ethyl acetate were purchased from Xilong Chemical Co. Sodium chloride (NaCl), PSA, GCB, MWCNTs, MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e, C18 were purchased from Shanghai Ampoule Experimental Technology Co.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental material\u003c/h2\u003e \u003cp\u003eCowpea (\u003cem\u003eVigna unguiculata\u003c/em\u003e L.) was purchased from Longxi Seed Industry Co. Ltd. in Suqian, Jiangsu Province (Jiangsu, China). Five types of soil were used in this experiment, including red soil (Sanjia, Dongfang City), yellow soil (Jianfengling, Ledong County), sandy soil (Foluo, Ledong County), sandy loam soil (Sigeng, Dongfang City), and paddy soil (Yazhou, Sanya City). The topsoil was collected and air-dried in a cool and dry place. After removing roots, stones and other debris, the soil was crushed and sieved through a 2 mm mesh for further use. The phychemical properties of the soils are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Sample pretreatment\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Pre-treatment of cowpea samples\u003c/h2\u003e \u003cp\u003eCowpea samples were weighed (5.0 g) and placed in centrifuge tube, extracted with 10 mL of acetonitrile solution and were mixed for 5 min. Then, added 5.0 g NaCl, vortexed for 2 min, and centrifuged at 5000 r/min for 5 min. Added supernatant (2 mL) to a centrifuge tube with varying additions of PSA, C18 and anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e and vortexed for 5 min, then centrifuged (5000 r/min, 5 min). Supernatant (1 mL) was rotary evaporated to near dryness at 40\u0026deg;C, reconstituted with ethyl acetate or methanol, and filtered through a membrane (0.22 \u0026micro;m) and left to be measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Pre-treatment of soil samples\u003c/h2\u003e \u003cp\u003eWeighed 5.0 g of soil after air-drying in centrifuge tube, added 10 mL acetonitrile and 5 mL ultrapure water, vortexed for 2 min. Then put it into an ultrasonic cleaner and added 1 g NaCl\u0026thinsp;+\u0026thinsp;4 g anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e, continued to shake for 2 min and centrifuged (5000 rpm, 10 min). The supernatant was filtered.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Detection condition\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 HPLC analysis\u003c/h2\u003e \u003cp\u003eThiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole were analyzed by HPLC system (Shimadzu LC-AD20, Japan). The separation was performed on a PICKERING C18 column (4.6 mm\u0026times;250 mm, inner diameter). The mobile phase consists of methanol and water (60:40, v/v). Flow rate: 1.0 mL/min. Wavelength: 255 nm. Injection volume:10 \u0026micro;L. Column temperature: 30℃. The overall analysis duration was 19 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 GC-MS/MS analysis\u003c/h2\u003e \u003cp\u003eThe analysis of cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, difenoconazole, azoxystrobin, dimethomorph was performed by using the same equipment and configuration as that of Ren et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The ramp-up procedure is shown in Table S2 and the total run time was 23 min.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Analytical method validation\u003c/h2\u003e \u003cp\u003e15 pesticides standard solutions of 0.1, 0.2, 0.5, 1, 2, and 5 mg/L were prepared. The linear regression equation and the coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) the 15 pesticides were obtained. Detailed data are shown in Table S3.\u003c/p\u003e \u003cp\u003eThe accuracy and precision of the method were verified according to the description of Ren et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), with a recovery of 70\u0026ndash;120% and a standard deviation (RSD) of less than 20%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Data analysis\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Enrichment and migration of 15 pesticides in soil by cowpea plants\u003c/h2\u003e \u003cp\u003eThe extent of pesticide uptake and accumulation from the soil by cowpea plants was expressed as bioconcentration factor (BCFs), with root bioconcentration factor (RCF), stem bioconcentration factor (SCF) and leaf bioconcentration factor (LCF) was calculated as follows:\u003c/p\u003e \u003cp\u003eRCF\u0026thinsp;=\u0026thinsp;C\u003csub\u003eR\u003c/sub\u003e/C\u003csub\u003eS\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eSCF\u0026thinsp;=\u0026thinsp;C\u003csub\u003eS\u003c/sub\u003e/C\u003csub\u003eS\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eLCF\u0026thinsp;=\u0026thinsp;C\u003csub\u003eL\u003c/sub\u003e/C\u003csub\u003eS\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eThe extent of pesticide translocation in cowpea plants is expressed as translocation factor (TF), and the root-to-stem (TF\u003csub\u003es/r\u003c/sub\u003e) and stem-to-leaf (TF\u003csub\u003el/s\u003c/sub\u003e) translocation factors were calculated as follows:\u003c/p\u003e \u003cp\u003eTF \u003csub\u003es/r\u003c/sub\u003e= C\u003csub\u003es/\u003c/sub\u003eC \u003csub\u003er\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eTF \u003csub\u003el/s\u003c/sub\u003e = C\u003csub\u003el/\u003c/sub\u003eC\u003csub\u003es\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Data processing and analysis\u003c/h2\u003e \u003cp\u003eExcel 2016 software was used to summarize the experimental data; origin 8.5 software was used for graphing; SPSS 17.0 software was used for data correlation analysis, and a, b, c was used to indicate the significance of differences between treatment groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Optimization of pre-treatment for cowpea samples\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Selection of extraction solvent for cowpea\u003c/h2\u003e \u003cp\u003eThe extraction effects of the extractant on pesticides varies depending on the type of pesticide, polarity and differences in vegetable substrates. Acetonitrile, ethyl acetate and methanol are commonly used extractants for pesticide extraction. Acetonitrile is moderately polar, volatile, and widely used, especially in the QuEChERS method showing high purity and relatively clean extracts (Kecojević et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ethyl acetate has slightly lower polar than acetonitrile, better water solubility, easy to penetrate into plant cell, can effectively extract a wide range of pesticides, which is superior to acetonitrile in the extraction of some specific pesticides, but it may carry more lipid contaminants (Madej et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Methanol is more polar and suitable for the extraction of pesticides with higher polarity (Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe effect of acetonitrile, ethyl acetate and methanol extraction in cowpea spiked with pesticides at a level of 1.0 mg/ kg is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Methanol, as an extractant, produced recoveries outside the range of 70\u0026ndash;120% for the remaining 12 pesticides, except for imidacloprid, acetamiprid, carbendazim, indicating that methanol is not suitable for the simultaneous extraction of 15 pesticides in cowpea. In addition, the recovery of some pesticides was too high when extracting them with methanol, and the recovery of thiamethoxam was even as high as 129.61%, which indicated that methanol introduced impurities that interfered with pesticide extraction during the extraction process (Rutkowska et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, rotary evaporation of methanol extracts was inefficient for high-pigment content cowpea leaf samples. In contrast, acetonitrile was more effective in extracting the 15 pesticides, with recoveries ranging from 70\u0026ndash;120%. When ethyl acetate was used as the extraction agent, ethyl acetate was more effective than acetonitrile for chlorpyrifos and pyrimethanil, this is because chlorpyrifos is a nonpolar pesticide, and ethyl acetate is able to efficiently penetrate and solubilize nonpolar pesticides (Yin et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Whereas, ethyl acetate was less effective for imidacloprid, chlorantraniliprole, cyromazine and tebuconazole, with recoveries below 70%. In addition, the residual pigment content after ethyl acetate extraction was also higher. Therefore, acetonitrile, as a polar medium extractant (Madej et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), gave the most stable recovery of pesticide, and the extracted solution was clear with little pigment residue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Selection of cowpea adsorbent\u003c/h2\u003e \u003cp\u003eThe fruit and vegetable matrix are rich in pigments, water, polysaccharides, and acidic substances (Gao et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Adsorbents such as MWCNTs, MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e, GCB can purify fruit and vegetable matrices and are widely used in the pretreatment of pesticide residues in food (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gonz\u0026aacute;lez-Curbelo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Adsorbents suitable for cowpea pesticide extraction were screened, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. For GCB as the adsorbent, the recoveries were in the range of 70\u0026ndash;100% for all pesticides except for cyromazine (39.83%) and chlorpyrifos (63.28%) which were not in the standard range. In contrast, when MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e was used as the adsorbent, the recoveries of cyromazine (105.13%) and chlorpyrifos (74.72%) were within the standard range. It can also be observed from the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e that under the same addition amount, MWCNTs have a stronger adsorption capacity than the other two adsorbents, resulting in the recovery of pesticides being less than 60%, which is attributed to MWCNTs having a larger specific surface area and strong adsorption capacity for interfering substances (Pallavi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It mainly relies on van der Waals forces, π-π interactions and hydrophobic interactions, so it is particularly suitable for the adsorption of nonpolar compounds, which resulted in the recoveries of less than 70% for all 15 pesticides (Dehghani et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e and GCB were selected to be used for the remaining 13 pesticides except for cyromazine and chlorpyrifos which only used MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e as adsorbent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 Optimization of cowpea purifier content\u003c/h2\u003e \u003cp\u003eCommonly used purifying agents include PSA (N-propyl ethylenediamine), and C18. PSA, with 2 amino groups, can remove carbohydrates, phenols, and fatty acids from the matrix through ion exchange. However, excessive use of PSA can lead to pesticide adsorption and reduced recovery due to hydrogen bonding with functional groups such as -NH, -SH, and -OH in some pesticides (Jin et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). C18 is a reverse-phase adsorbent that has strong adsorption capabilities for non-polar substances such as cholesterol, vitamins, and fats in the matrix (H\u0026aacute;kov\u0026aacute; et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). GCB exhibits strong adsorption for pigments but may also affect recovery by adsorbing certain planar-structured pesticides (Long et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGCB is not required as cowpea roots do not contain chlorophyll, different amounts of C18 and PSA (50 mg, 100 mg, 150 mg, 200 mg, 250 mg) were selected along with 200 mg MgSO\u003csub\u003e4\u003c/sub\u003e to investigate their impact on pesticide recovery. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the recovery within the range of 70\u0026ndash;110% for thiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole, cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, ifenoconazole, azoxystrobin and dimethomorph, after the addition of 100 mg and 150 mg of C18 and PSA. The pesticide recovery was too high when the addition amount of C18 and PSA was 50 mg, while the pesticide recovery was too low when the addition amount of C18 and PSA was 200 mg and 250 mg.\u003c/p\u003e \u003cp\u003eThe amount of GCB used in stems and leaves was subsequently optimized. In the stems of cowpea, 20, 30, 40, 50, 60 mg were selected for pesticide extraction The results showed that the recovery of several pesticides was more than 120% when GCB was used at 20 and 30 mg, while recovery of some pesticides was less than 70% when GCB was used at 60 mg (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This was attributed to the adsorption of pesticides by GCB, insufficient addition of GCB will result in the substrate not achieving the purification effect, while excessive addition will lead to the absorption of target pesticides, resulting in a decrease in recovery (Kim et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). When the amount of GCB was 40 and 50 mg, the recoveries of 15 pesticides were within the standard range, and the recovery range of 40 mg fluctuated less and the amount was less. Therefore, 40 mg of GCB was selected for the decontamination of cowpea stems.\u003c/p\u003e \u003cp\u003eThe higher chlorophyll content in cowpea leaves resulted in correspondingly higher levels of GCB used (30, 50, 80, 100 and 150 mg). Similar to the GCB additions to the stems, the lower doses of GCB (30 mg and 50 mg) were less effective in adsorbing impurities, resulting in excessive pesticide recovery (\u0026gt;\u0026thinsp;120%), whereas the higher dose of GCB (150 mg) excessively adsorbed pesticides, resulting in pesticide recoveries below 70%. The recoveries of all 15 pesticides ranged from 70\u0026ndash;120% at GCB dosages of 80 mg and 100 mg (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Based on the unit of recovery and dosage considerations, we chose 80 mg of GCB for pesticide extraction from cowpea leaves to obtain the best extraction results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Optimization of soil purification agent content\u003c/h2\u003e \u003cp\u003eThe main function of a desiccant is to induce salting-out effect during the extraction process of the target analyte, thereby promoting phase separation. Commonly used desiccants include MgSO\u003csub\u003e4\u003c/sub\u003e and NaCl, etc. The combination of MgSO\u003csub\u003e4\u003c/sub\u003e and NaCl is widely applied in QuEChERS to improve the purity and recovery of extracts (Zhang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In order to find the optimal amount of NaCl and MgSO\u003csub\u003e4\u003c/sub\u003e in soil matrix, this study conducted experiments on five different combinations. The results showed that both 0.2 g\u0026thinsp;+\u0026thinsp;0.5 g and 0.5 g\u0026thinsp;+\u0026thinsp;1 g ratios of NaCl\u0026thinsp;+\u0026thinsp;MgSO\u003csub\u003e4\u003c/sub\u003e resulted in recovery within standard range with good stratification between water and organic phase. This is because insufficient addition of NaCl will lead to incomplete distribution of the organic and aqueous phases, the pesticide components cannot be completely transferred to the organic phase, especially for pesticides with high polarity, the recovery may be significantly reduced. And when the amount of anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e is insufficient, the water in the sample is not sufficiently absorbed to remain in the extract. The excess water may dilute the extracted pesticide components, which especially has a negative effect on the detection of water-soluble pesticides (Hwang et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Whereas, 2 g\u0026thinsp;+\u0026thinsp;3 g and 3g\u0026thinsp;+\u0026thinsp;4g NaCl\u0026thinsp;+\u0026thinsp;MgSO\u003csub\u003e4\u003c/sub\u003e would lead to salting-out, which makes it difficult for certain pesticide components with high polarity to be fully dissolved in the organic phase. While excessive use of anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e can lead to an increase in solid particles in the organic phase, too many particles can be difficult to settle completely during centrifugal separation, affecting the purity of the extraction solution (Faraji et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The ratio of 1g\u0026thinsp;+\u0026thinsp;2 g of NaCl\u0026thinsp;+\u0026thinsp;MgSO\u003csub\u003e4\u003c/sub\u003e resulted in pesticide recoveries that were all within the standard range and well stratified between the aqueous and organic phases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Validation of the QuEChERS method\u003c/h2\u003e \u003cp\u003eThe precision and accuracy of the established method were validated using different cowpea tissues and five types of soil, the optimized method was used to conduct recovery experiments on blank cowpea samples and soil spiked with 0.01, 0.1, and 1.0 mg/kg. The recoveries of the 15 pesticides in cowpea were determined. As shown in Table S4, the recovery in cowpea matrix ranged from 74.15\u0026ndash;118.63%, with a precision range of 0.3\u0026ndash;18.36%. Similarly, the average recovery in soil matrix also ranged from 74.15\u0026ndash;118.63%, with a precision range of 0.3\u0026ndash;18.36%. In addition, the recoveries and RSDs of the 15 pesticides in the five different soils were also within the standard range (Table S5). Therefore, the developed method fully complies with the requirements of SANTE/11312/2021 guidance, where stipulate recovery is within the range of 70\u0026ndash;120% and RSD is less than 20% (European Food Safety Authority (EFSA)).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Analysis of the fate of 15 kinds of pesticides in soil-vegetable system\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Exposure experiment\u003c/h2\u003e \u003cp\u003ePesticides that are not effectively utilized enter the environment and remain in the soil, where they are then taken up by plant roots and translocated to other sites (Zhang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Pesticide residues, accumulation and metabolism in crops not only contribute to the quality and safety of agricultural products, but are also toxic to non-targets (Xia et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The distribution of the 15 pesticides in the cowpea-soil system is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Initially, pesticide residue levels were measured in soils with and without cowpea seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). After 21 days, soils with cowpea seedlings exhibited significantly lower pesticide residues, indicating active uptake or degradation soil without cowpea seedlings. The above results suggest that the presence of cowpea plants may accelerate pesticide residues in the soil by absorbing pesticides or promoting degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the soil cultivation of cowpea, 15 pesticides were detected in different tissue, indicating that roots are able to uptake pesticides and transport them to other organs. Most of the pesticide uptake by roots reached the maximum on the 3\u0026ndash;5 d, fluopyram reached the maximum on the 10th day, and tebuconazole reached the maximum on the 14 d, after which the levels gradually stabilized. The average concentrations of thiamethoxam, imidacloprid, acetamiprid, carbendazim, chlorantraniliprole, cyromazine, metalaxyl, chlorpyrifos, fluopyram, chlorfenapyr, tebuconazole, pyraclostrobin, difenoconazole, azoxystrobin and dimethomorph in the roots were 1.173, 1.124, 1.140, 4.127, 2.916, 0.852, 1.970, 2.224, 2.226, 0.579, 0.962, 1.322, 1.312, 7.039 and 2.704 mg/kg, respectively. The transport of pesticides in the stems of cowpea plants mostly reached equilibrium after the 10th day and decreased after the 14th day. The concentration of metalaxyl in the stems peaked on the 3rd day and gradually declined thereafter.\u003c/p\u003e \u003cp\u003eThe concentrations of thiamethoxam, acetamiprid, cyromazine, and metalaxyl in the leaves gradually exceeded those in the stems and roots after 1\u0026ndash;3 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D), whereas metalaxyl and fluopyram were required on 7 or 21 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), respectively.\u003c/p\u003e \u003cp\u003eCyromazine showed a distribution pattern of leaves\u0026thinsp;\u0026gt;\u0026thinsp;stems\u0026thinsp;\u0026gt;\u0026thinsp;roots on day 14 d, while metalaxyl showed leaves\u0026thinsp;\u0026gt;\u0026thinsp;stems\u0026thinsp;\u0026asymp;\u0026thinsp;roots, which indicated that these two pesticides had strong upward translocation capabilities. The accumulation of chlorpyrifos, chlorfenapyr, pyraclostrobin, difenoconazole, azoxystrobin, and dimethomorph in the roots increased over time, reaching a maximum on the 14th day before declining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eI-O). The distribution of these six pesticides in the cowpea plants followed the pattern of roots\u0026thinsp;\u0026gt;\u0026thinsp;stems\u0026thinsp;\u0026asymp;\u0026thinsp;leaves, suggesting that these pesticides are not easily translocated upwards and are mainly concentrated in the roots.\u003c/p\u003e \u003cp\u003eKow (octanol-water partition coefficient, Log \u003cem\u003eKow\u003c/em\u003e) is a coefficient that measures the partition of pesticides between octanol and water, reflecting the lipophilicity and hydrophilicity of the compound (Miller et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is closely related to the absorption and transportation of pesticides. Kow directly affects the transmembrane diffusion of pesticides. Pesticides with high Kow values are more lipophilic and therefore more likely to cross lipid bilayers and enter the cells of plants or other organisms (Zhang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast, pesticides with a low K\u003cem\u003eow\u003c/em\u003e are more hydrophilic, less absorbed, and have difficulty in crossing cell membranes, usually staying more in the aqueous phase or being blocked by the more polar cell wall or extracellular regions (Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The content of the six pesticides thiamethoxam, imidacloprid, acetamiprid, cyromazine, metalaxyl, and fluopyram was present at higher levels in leaves than roots after a period of stable exposure. This phenomenon occurs because the first five pesticides have a Log \u003cem\u003eKow\u003c/em\u003e less than 3, making them less likely to be significantly accumulated in the roots, leading to their upward transport and accumulation in the leaves. Although fluopyram has a Log \u003cem\u003eKow\u003c/em\u003e greater than 3, its high solubility results in a greater concentration in the leaves compared to the roots during the later stages of the exposure trial molecular weight (MW). In contrast, the pesticides carbendazim, chlorantraniliprole, chlorpyrifos, chlorfenapy, tebuconazole, pyraclostrobin, difenoconazole, pyraclostrobin and dimethomorph exhibit a Log \u003cem\u003eKow\u003c/em\u003e greater than 3 or a Log \u003cem\u003eMW\u003c/em\u003e greater than 300 g/mol (Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Their higher hydrophobicity facilitates retention in the root lipids, and their relatively low water solubility hinders upward migration through transpiration.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Correlation analysis\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section4\"\u003e \u003ch2\u003e3.4.2.1 Bioconcentration factor analysis\u003c/h2\u003e \u003cp\u003eThe phychemical properties of pesticides play a crucial role in determining how they behave within plants. Among them, MW has a significant effect on their uptake and translocation in plants. In general, pesticides with smaller MW are more readily absorbed and can be extensively distributed throughout the plant, while pesticides with larger MW are usually difficult to diffuse through plant cell walls and membranes, which restricts their movement within the plant (Miller et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Solubility in Water (S\u003csub\u003eW\u003c/sub\u003e) can also determine the behavior of pesticides in the plant-environment, especially their uptake through plant roots and their ability to move within the plant. In addition, the Kow also serves as a significant indicator influencing the distribution of pesticides in plants (Miller et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the Kow, S\u003csub\u003eW\u003c/sub\u003e, and MW were used as the physicochemical properties of the pesticides, along with the average BCF of 15 pesticides in various parts of the cowpea, to conduct a linear regression analysis to investigate how these physicochemical properties affect the behavior of pesticides in the cowpea-soil system.\u003c/p\u003e \u003cp\u003eThe findings indicated that the logarithmic values of the BCF in the leaves had a negative correlation with Log \u003cem\u003eKow\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.8831, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0000), whereas the roots and stems exhibited a positive correlation with Log \u003cem\u003eKow\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.4529, 0.5513; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0060, 0.0015) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). No significant correlation was observed between Log \u003cem\u003eRCF\u003c/em\u003e and Log \u003cem\u003eMW\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), while both Log \u003cem\u003eSCF\u003c/em\u003e and Log \u003cem\u003eLCF\u003c/em\u003e exhibited a weak negative linear correlation with Log \u003cem\u003eMW\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.3724, 0.2840; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0157, 0.0408) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). These indicated that as the MW of the pesticides increases, the BCF in the roots and stems gradually decrease, while those in the stems and leaves also decreases. Additionally, there was no significant correlation between Log \u003cem\u003eSw\u003c/em\u003e and the BCF in the roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), but a positive correlation was observed with the BCFs in the stems and leaves (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.5714, 0.6433; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0011, 0.0003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, I). The above results suggested that with the S\u003csub\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sub\u003e of the pesticides increases, the BCF in the stems and leaves gradually decrease, resulting in a decrease in the accumulation of these pesticides in these parts. The results mentioned above align with the findings regarding pesticide accumulation in the same tissues of cowpea (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003ch2\u003e3.4.2.2 Transfer factor analysis\u003c/h2\u003e \u003cp\u003eThe TF can be used to indicate the ability of pesticides to be transferred in plant (Xia et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ye et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To clarify how the physicochemical properties of pesticides influence their transfer capabilities, linear regression analysis was conducted on the three physicochemical properties that reveal accumulation patterns in relation to cowpea TF\u003csub\u003es/r\u003c/sub\u003e and TF\u003csub\u003el/s\u003c/sub\u003e. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e. There was a strong linear negative correlation was observed between the TF\u003csub\u003es/r\u003c/sub\u003e and Log \u003cem\u003eKow\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.7441, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003), indicating that pesticide translocation within the plant may gradually increase with increasing hydrophobicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). However, the TF\u003csub\u003el/s\u003c/sub\u003e had no significant correlation with Log \u003cem\u003eKow\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.7441, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Additionally, the TF showed a high linear negative correlation with Log \u003cem\u003eMW\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.6738, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001), indicating that as MW increases, the mobility of the pesticides within the plants gradually decreases (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). No significant correlation was observed between the TF\u003csub\u003el/s\u003c/sub\u003e and the MW of the pesticides (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Between TF\u003csub\u003es/r\u003c/sub\u003e and Log \u003cem\u003eSw\u003c/em\u003e, a strong linear positive correlation was found (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.8021, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0000), which implied that pesticide accumulation in the roots might decrease with increasing Sw, thereby enhancing their translocation within the cowpea (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). However, no significant correlation between TF\u003csub\u003el/s\u003c/sub\u003e and Log \u003cem\u003eSw\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOther studies have shown that the transport of organic pollutants in vegetables is negatively correlated with log \u003cem\u003eKow\u003c/em\u003e as hydrophobicity increases (Ye et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). And the smaller the MW and higher the Sw of the pesticide, the easier the pesticide is to be transported to the top of maize, cherry radish, wheat, etc. (Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xia et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ye et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, pesticide physicochemical properties are not the only factors affecting pesticide uptake and transport in plants, which may be related to the structural organization, physicochemical properties (e.g., fats, proteins, etc.), and growing environment (hydroponic vs. soil) of vegetables (Chang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Chang et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, differences in soil physicochemical properties can influence a plant\u0026rsquo;s capacity to absorb pesticides from the soil and redistribute them to other locations. For example, soil pH significantly affects the translocation and accumulation of imidacloprid in corn, which can be toxic to corn (Chang et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, the uptake of imidacloprid and propiconazole by wheat roots is influenced by soil physicochemical properties, which in turn affects their translocation in wheat (Ju et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, Pearson correlation analyses of cowpea enrichment factors, transcription factors, and physicochemical parameters were performed, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The correlation coefficient between log \u003cem\u003eSCF\u003c/em\u003e and log \u003cem\u003eLCF\u003c/em\u003e was 0.94, showing a strong positive correlation. This indicates a high degree of covariance between these two variables, suggesting a high positive correlation between the pesticide enrichment capacity of cowpea stems and leaves. TFs/r and log \u003cem\u003eSw\u003c/em\u003e both had a correlation coefficient of 0.86, which is also a strong positive correlation. It indicates that solubility (log \u003cem\u003eSw\u003c/em\u003e) may be positively correlated with the migration efficiency of TF s/r under certain conditions, the greater the \u003cem\u003eSw\u003c/em\u003e of pesticides, the easier it is to migrate to the above-ground parts of cowpea. However, log \u003cem\u003eKow\u003c/em\u003e and log \u003cem\u003eSw\u003c/em\u003e showed a strong negative correlation of -0.76, suggesting that log \u003cem\u003eKow\u003c/em\u003e and log \u003cem\u003eSw\u003c/em\u003e are mutually exclusive under certain conditions. The strong negative correlation between TFs/r and log \u003cem\u003eMW\u003c/em\u003e suggests that larger molecular weights are unfavorable for pesticide translocation from roots to cowpea above-ground parts. Pearson correlation analysis verified the results of the Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study optimized the QuEChERS method combined with GC-MS/MS and HPLC for the analysis of 15 common pesticides in cowpea and soil. Different extraction solvents were compared for the extraction of pesticides from cowpea, ultimately selecting acetonitrile as the extraction solvent for cowpea. C18, PSA, and GCB were employed to purify the cowpea extracts, with different amounts of pesticide cleanup sorbents being optimal for different pesticides in cowpea. For soil, a final addition ratio of 1 g sodium chloride and 2 g MgSO\u003csub\u003e4\u003c/sub\u003e was selected. Ultimately, at spiking levels of 0.01-1.0 mg/kg and 0.1\u0026ndash;10.0 mg/kg, the recoveries varied between 74.15% -118.63%, with precision between 0.3\u0026ndash;18.36%. The average recovery in soil matrix also fell within the range of ranged from 74.15\u0026ndash;118.63%, with precision ranging from 0 .3% to18.36%. At the same time, we studied the enrichment and interpretation patterns of 15 pesticides in cowpea and soil, and found that 6 pesticides were easily enriched in the above-ground part of cowpea, which was mainly affected by the phychemical properties of pesticides and soil. The developed detection method in this study can better monitor the residues of multiple pesticides in cowpea and various types of soil.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interests. This is our original work, which has not been published previously or considered for publication elsewhere, and has been approved by all authors.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by National Natural Science Foundation of China Regional Joint Priority Project (U22A20484), Key Laboratory of Tropical Fruits and Vegetables Quality and Safety for State Market Regulation (No. KF-2023001), and Hainan University Research Initiation Fund (Science and Technology) (XJ2400005859).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eW. J: Writing-original draft; L.Y: Data curation, Software; Y.X, L.X and G.K: Experimental operation; Y.Y: Writing-original draft;W. M: Conceptualization, Investigation; Z.S:Investigation, Writing-original draft, L.X and Z.Y Writing-review \u0026amp; editing, Supervision. 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J Food Compos Anal 133, 106396. https://doi.org/10.1016/j.jfca.2024.106396.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"QuEChERS, Cowpea-soil system, Multiple Pesticides, Behavior","lastPublishedDoi":"10.21203/rs.3.rs-5625297/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5625297/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePesticide residues have always been one of the food safe issues troubling consumers. Agricultural production is usually accompanied by a mixture of pesticides, and pesticide residues are not only present on plants but also contaminate soil in the environment. In this study, 15 pesticides commonly found in cowpea and soil were analyzed by optimizing QuEChERS and combining GC-MS/MS and HPLC. Various extractants and different methanol water ratios were evaluated to extract the pesticides from cowpea and soil.C18, PSA and GCB were used to purify the cowpea extracts, while in soil the ratios of de-watering agent and NaCl were optimized. The average recoveries were 91.81-109.95% and 89.89-104.08% in cowpea and soil at spiked levels of 0.0-1.0 mg/kg and 0.1-10.0 mg/kg, respectively. This method is suitable for the detection of pesticides in different types of soil (red soil, yellow soil, sandy soil, sandy loam soil, paddy soil) and different cowpea tissues. In addition, pesticide residues were detected and analyzed in the cowpea- sandy loam soil system. This demonstrates that the developed method can be used to detect the multiple pesticide in various types of soils and crops, and provides the necessary technical support for agricultural product pesticide detection and safety supervision.\u003c/p\u003e","manuscriptTitle":"Optimization of a QuEChERS method for 15 pesticides followed by determination the transport behavior in cowpea-soil system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 14:15:35","doi":"10.21203/rs.3.rs-5625297/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27d2db97-d390-4ea2-b2cf-27d30131c319","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-25T22:08:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-19 14:15:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5625297","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5625297","identity":"rs-5625297","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}