Uptake, Translocation, and Metabolism of Pydiflumetofen in Hydroponic Cucumber and Tomato Planting Systems

Abstract

As a novel succinate dehydrogenase inhibitor (SDHI), pydiflumetofen (PYD) exhibits broad-spectrum bactericidal activity in various crops; however, little is yet known about its absorption, translocation, and metabolic behavior within plants. Cucumber and tomato plants were cultured in hydroponic conditions spiked at 0.5 mg/L of PYD, and samples were collected at certain intervals to investigate the residual fate of PYD within the plants. The results demonstrated that PYD was readily absorbed by the roots of both plants, with mean root concentration factors (RCFs) of 5.6–12.3 for cucumber and 5.0–12.4 for tomato. PYD exhibited higher translocation ability from stems to leaves and limited from roots to stems in cucumber, while comparably weak root-to-stem and stem-to-leaf translocation were observed in tomato. By the end of the exposure period, a mass loss of 51.55% and 56.67% was observed, and six and three metabolites were found to be generated in the cucumber and tomato systems, respectively. This study provides a foundation for comprehending the uptake and translocation of PYD and offers novel insights into its potential risks to agricultural products and food safety.

1. Introduction

In order to improve crop production and satisfy consumer demands, pesticides are essential in agriculture for the control of pests, diseases, and weeds [1]. However, there will always be pesticide residues in the environment due to irrational use [2,3], which may put human health and food safety at risk [4]. The main way residues enter crops is by root absorption, which is followed by plant internal translocation and accumulation [5]. Researches have demonstrated that many factors, such as plant features [6], physicochemical properties of the pesticides [7,8], and environmental circumstances [9,10], may affect the pesticide absorption and translocation within plants. Specifically, plants with high lipid and protein levels are likely to accumulate hydrophobic pesticides [9,11]. Pesticides with a higher octanol–water partition coefficient (logKow) have a greater tendency to accumulate in the roots, whereas pesticides with logKow ranging from 1 to 3 tend to be translocated to the aboveground parts of the plant after root uptake [10,12].

Following the uptake and accumulation of pesticides in plants, various biochemical reactions result in the generation of different metabolites. Certain metabolites may possess greater polarity and mobility, and even greater potential for toxicity than their parent compounds, which could significantly impede the safety of agricultural products and the surrounding environment [13,14,15]. Hence, it is of extremely great importance to monitor the metabolites generated by pesticides in plants, which could enhance our understanding of their environmental fate and metabolic mechanism.

Pydiflumetofen (PYD) (3-(difluoromethyl)-N-methyl-N-[(RS)-1-methyl-2-(2,4,6-trichloro phenyl) ethyl]-1H-pyrazole-4-carboxamide), a novel succinate dehydrogenase inhibitor (SDHI), was developed and marketed by Syngenta in 2017 [16]. Being highly effective and broad-spectrum, PYD is used to control fungal pathogens, including Fusarium spp., Alternaria spp., Cercospora spp. and Botrytis spp. etc. [17,18,19]. The action mechanism involves obstructing energy synthesis by interfering with the respiratory chain complex II, thereby inhibiting the growth of pathogens and achieving sterilization [20]. Currently, PYD is registered for use on various crops in China, including wheat, oilseed rape, peanuts, strawberries, grapes, and tomatoes for the control of fusarium head blight and leaf spot. To date, several studies have been conducted on its biological activity [16], adsorption–desorption in soil [21], dissipation dynamics in paddy fields [22], and comprehensive evaluation of chiral characteristics [23,24,25]. Furthermore, a previous study by Wang et al. demonstrated that several metabolites of PYD in zebrafish are more toxic than their parent compound [26]. However, the uptake and translocation, metabolic behavior, and underlying mechanisms of PYD in plants remain unclear.

The utilization of hydroponic systems in this study was based on their capacity to provide a more stable growth condition to plants than that afforded by field experiments [27]. Two common vegetables, cucumber (Cucumis sativus L.) and tomato (Lycopersicon esculentum Mill.), were selected as the model crops. The residual levels of PYD in the nutrient solution, roots, stems, and leaves were determined based on a validated analytical method. The extent of root, stem, and leaf uptake of PYD was evaluated using the bioconcentration factor (BCF), while the transport capacity of PYD within plants was assessed by the translocation factor (TF). Additionally, an analytical method employing high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF) was validated to determine the metabolic rules of PYD in two hydroponic vegetable–nutrient systems. These findings provide a valuable theoretical foundation and data for the risk assessment and food safety of PYD.

2. Material and Methods

2.1. Reagents and Materials

Standard PYD (99.4% purity, log Kow 3.8) was purchased from Badische Anilin-und-Soda-Fabrik (Beijing, China). Chromatographic-grade methanol and acetonitrile were purchased from Sigma-Aldrich (Steinheim, Germany). Formic acid (purity 98.0%) was purchased from Macklin Reagent (Shanghai, China), and analytical-grade sodium chloride was provided by Guangdahengyi Technology Co., Ltd. (Beijing, China). The nutrient solution (components are shown in Table S1) purchased from Rufeng was soilless cultivation nutrient solution, and ultrapure water was obtained from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China). The seeds of cucumber (Cucumis sativus L., “Jingyan xiamei No.2 F1”) and tomato (Lycopersicon esculentum Mill., “Yingfen No.8”) were obtained from Jingyan Yinong Seed Sci-Tech Co., Ltd. (Beijing, China).

2.2. Plant Cultivation and Exposure Experiments

The experiment was conducted based on a previously described procedure with slight modifications [14]. Briefly, the cucumber seeds underwent a germination period of 24 h at room temperature (25 °C), and tomato 72 h; after the formation of root hairs and young shoots, the seedlings with similar growth conditions were transplanted into hydroponic tanks containing nutrient solution, which were maintained at a concentration at 1200–1500 μS/cm and pH 7.0. PYD was added to the nutrient solution until the seedlings grew to have 3–4 leaves (around 10–12 cm tall). The environmental conditions for plant growth were maintained at a temperature of 25 °C during the day and 20 °C at night, with a humidity of 65%. Multiple batches of seedlings exhibiting consistent growth patterns were prepared for experiments.

In the treatment groups, seedlings of similar sizes were selected for the exposure experiments. Cucumber and tomato seedlings were transplanted into 12-well hydroponic buckets containing 600 mL nutrient solution, which were spiked with a standard solution at 0.5 mg/L. There were two types of control groups in this study. The first control group involved an equal volume of nutrient solution without PYD application but with plant growth, to reduce errors caused by interference from other environmental factors. The second group was the unplanted control in which PYD was applied in the nutrient solution at the same concentration, 0.5 mg/L, to observe the loss of PYD. A reduction in the volume of the nutrient solution was observed on a daily basis and documented, with replenishment performed by the addition of fresh nutrient solution to restore the initial volume.

At designated exposure intervals of 1, 3, 7, 10, and 14 days, three plants were randomly selected for analysis. The roots, stems, and leaves were separated, and roots were thoroughly rinsed twice with ultrapure water, and then dried using filter paper. A certain aliquot of corresponding nutrient solution was randomly collected into a centrifuge tube for processing and analysis. All plant tissue samples, roots, stems, and leaves were weighed, crushed with liquid nitrogen, and then stored at −20 °C before analysis. Unplanted and untreated controls were sampled on day 14.

2.3. Sample Extraction

An aliquot of 2.0 g of homogenized plant samples was extracted with 2.0 mL acetonitrile, and vortexed for 5 min. Subsequently, 0.4 g of sodium chloride was added, and the samples were vortexed for 5 min. Following this, centrifugation at 4000 rpm for 5 min was performed. The supernatant (1.5 mL) was clarified by filtration through a 0.22 μm organic filter for subsequent analysis by UPLC-MS/MS. The nutrient solution samples (2.0 mL) were extracted with 2.0 mL acetonitrile, and the subsequent steps were identical to those employed for the tissue samples. All sample extracts were prepared in duplicate for the quantitative and qualitative analyses of PYD and its potential metabolites.

2.4. UPLC-MS/MS and HPLC-Q-TOF Analyses

The separation, qualitative, and quantitative analyses of PYD were performed on a Waters Xevo TQD system coupled with a triple-quadrupole tandem mass spectrometer (Waters Corp, Milford, MA, USA), in positive electrospray ionization (ESI+) mode and using an ACQUITY UPLC BEH C18 analytical column (2.1 × 100 mm inner diameter, 1.7 μm) (Waters Corp, Milford, MA, USA). The injection volume was 1 μL, and the column was kept at 40 °C, while the sample temperature was set as 20 °C. A mixture of acetonitrile (A) and 0.1% formic acid aqueous solution (B) was used as the mobile phase at a flow rate of 0.3 mL/min for 6 min. The gradient elution procedure used for the mobile phase is presented in Table S2. The MS conditions are shown in the Supplementary Materials. The retention time of PYD was 2.59 min.

The metabolites of PYD in water–plant systems were analyzed using HPLC-Q-TOF (6546 Q-TOF-MS, Agilent Corp, Milford, MA, USA). A Waters ACQUITY HSS T3 analytical column (100 × 2.1 mm inner diameter, 1.8 μm) was used for chromatographic separation of the target analytes. A mixture of acetonitrile (A) and 0.1% formic acid aqueous solution (B) was used as the mobile phase at a flow rate of 0.3 mL/min for 11 min, and the injection volume was 5 μL. Gradient elution of the mobile phase is shown in Table S3. The samples were scanned in the entire scan mode (MS scan) and data analysis was performed using Metabolite ID 4.0 (Agilent). Potential metabolites were identified by molecular characterization analysis, binary alignment, isotope distribution, and mass deficit filtering. Targeted MS/MS analysis was then performed using Q-TOF, and the metabolism was further determined. An electrospray ionization source (Dual AJS ESI) was used for accurate mass correction, with mass-to-charge ratios (m/z) of 121.05 and 922.01. The scan ranges of MS and MS/MS were both 100–1000 m/z, and the scan rates were 1 sp/s and 5 sp/s. The secondary mass spectrometry scan range was 60–1000 m/z with a scan rate of 2 sp/s. The drying gas flow rates were 7.0 L/min and 12.0 L/min, and the drying gas temperatures were 280 °C and 370 °C, respectively. The atomization gas pressure was 30 psi, and the capillary and fragment voltages were 3000 V and 120 V, respectively.

2.5. Method Validation

A sequence of standard working solutions was prepared in the solvent or blank samples to obtain linearity and matrix effects. The spiked experiments for PYD in plant tissues and nutrient solution involved three spiked levels of 0.01, 0.10, and 0.50 mg/kg, with five replicates at each level. The limit of quantitation (LOQ) was defined as the lowest spiked level.

2.6. Data Calculation and Analysis

The bioconcentration factors (BCFs), including the root concentration factor (RCF), stem concentration factor (SCF), and leaf concentration factor (LCF), were used to evaluate the ability of plants to absorb PYD by roots, stems, and leaves [28] The translocation factor (TF) was used to evaluate the ability of plants to transport PYD, which involved TF_stem/root and TF_leaf/stem [29] These factors were calculated using the following formulas:

RCF = C_root/C_solution

SCF = C_stem/C_solution

LCF = C_leaf/C_solution

TF_stem/root = C_stem/C_root

TF_leaf/stem = C_leaf/C_stem

where C_root, C_stem, and C_leaf are the residue levels in plant roots, stems, and leaves (μg/g) and C_solution is the residual level in the nutrient solution (mg/L). The higher the RCF values are, the stronger the absorption or accumulation ability in root [28]. The TF value is ≥1, indicating the ease of translocation from root to stem, or from stem to leaf in plants [29].

3. Results and Discussion

3.1. QA/QC Validation

The developed method was validated in accordance with the Guideline for the testing of pesticide residues in crops (NY/T 788-2018), which stipulates that it should be tested for linearity, matrix effect (ME), spiked recovery, relative standard deviation (RSD), and limit of quantitation (LOQ). There was no interference in the blank samples at the retention time of PYD, indicating that this analytical method exhibited enhanced selectivity and specificity. As illustrated in Table S2, the solvent and matrix-matched calibration curves were constructed within the concentration range of 0.005–0.5 mg/L, and excellent linear relationships were obtained with coefficients (R²) > 0.99. The ME values of PYD in the nutrient solution, roots, stems, and leaves were found to range from −11.74% to −6.09%, −5.19% to 4.40%, −32.88% to −1.53%, and −74.38% to 4.91%, respectively, indicating that different extents of suppression or enhancement effects were observed in all matrices. Consequently, matrix-matched standard curves were selected for the quantitative analysis of the target compounds to avoid measurement errors caused by matrix effects for all samples. The recoveries of PYD ranged from 89.28% to 107.22% (RSDs ≤ 6.22%) in the nutrient solution, 84.64% to 114.26% (RSDs ≤ 7.51%) in cucumber roots, stems, and leaves, and 89.45% to 117.34% (RSDs ≤ 8.41%) in various tomato matrices. LOQs, which were defined as the lowest spiked concentration observed with satisfactory recoveries and RSDs, were assessed to be 0.01 mg/kg for PYD in the present study.

The parameters of the developed method comply with the requirements and can be used to analyze PYD residues in plant hydroponic systems.

3.2. Dissipation of PYD in Different Nutrient Solutions

The concentration variations of PYD in unplanted and planted nutrient solutions, and the dissipation trend, are shown in Figure 1A,B. The results showed no significant difference in concentration compared to the initial concentration, indicating that negligible evaporation or microbial degradation may have occurred in the nutrient solution. However, residual levels in cucumber and tomato nutrient solutions exhibited a significant decline over time. In the nutrient solution for cucumber, residues of PYD diminished from 0.47 mg/L to 0.11 mg/L over the period from day 1 to day 14. The dissipation followed the first-order kinetics equations Ct = 0.546e^−0.111t with the R² > 0.99, and the half-life was 6.2 day. In the tomato nutrient solution, its concentrations were from 0.38 mg/L on day 1 to 0.16 mg/L on day 14, and the dissipation equation was C_t = 0.413e^−0.065t with a half-life of 10.7 day. The consistent trend of residues in both nutrient solutions may be due to the continuous uptake of PYD by the roots of cucumber and tomato plants. Notably, the half-life of PYD in the cucumber solution was 1.73 times higher than that in tomato. This may be attributed to the different biological growth rates of the plant species. Similar results were observed for the dissipation pattern of phenamacril in lettuce and radish [14], which indicated that the high growth rate of lettuce could accelerate the absorption of pesticides.

3.3. Uptake and Translocation of PYD within Plants

The residual levels of PYD in cucumber and tomato plants at various exposure times are shown in Figure 2A,D, RCF, SCF, and LCF values in Figure 2B,E, while TF_stem/root and TF_leaf/stem are shown in Figure 2C,F. No cross-contamination of PYD was observed in unplanted controls throughout the experimental period. There were significant differences in residue behavior between the two crops.

3.3.1. Cucumber System

As shown in Figure 2A, PYD could be rapidly absorbed by the cucumber roots and reached the peak level of 2.65 µg/g on the day 1. Subsequently, the residual level gradually decreased to a minimum value of 1.39 µg/g on the day 14, representing 52.45% of the peak values. Similar with roots, the stem concentrations continued to decrease throughout the exposure period from 1.05 µg/g to 0.22 µg/g. Different from roots, there was an initial increase followed by a gradual decline in the leaves over time. At 1 day of exposure, the concentration of PYD in cucumber leaves was 0.9 µg/g, while the maximum was observed on the 3rd day, reaching 1.76 µg/g. Afterwards, the concentration gradually decreased to a minimum value of 0.50 µg/g at the end of the exposure, which was 28.4% of the peak concentration. This trend can be explained by the root absorption of PYD, subsequently translocated to the aboveground parts, resulting in an increase over a short period. As it reached a certain level, PYD may have translocated within the plant, causing it to be transmitted through the stem to reach the underground part. Another reason may be that as the leaf area continuously expanded, the growth dilution effect become one of the main factors affecting its residual level. During the exposure period, the average concentration in the roots was significantly higher than in the stems and leaves, ranging from 2.5 to 6.5 times that in stems, and 1.5 to 2.8 times that in leaves. Compared with residual levels in stems, PYD in leaves were comparably higher after 3 d exposure.

As illustrated in Figure 2B, the average RCF of the cucumber plants showed an increased trend throughout the experimental time, from 5.59 at 1 day to 12.3 at 14 day. This is significantly higher than SCF and LCF, indicating that PYD was easily absorbed by the roots compared with the stems and leaves. Furthermore, the TF_leaf/stem for PYD in cucumber plants was 2.74 to 3.32, which were >1 after 3 d exposure. As well as this, TF_stem/root ranged from 0.15 to 0.40, suggesting that PYD had higher translocation ability from stems to leaves, and limited from roots to stems (Figure 2C).

3.3.2. Tomato System

As shown in Figure 2D, PYD residues in tomato roots exhibited an initial increase followed by a decrease over the exposure period. During the 14 days of exposure, the lowest residue level of 1.89 µg/g was observed on day 1, representing 70.3% of the maximum level, which was observed as 2.69 µg/g on day 10. Subsequently, the residues of PYD in the tomato roots decreased, reaching a concentration of 1.92 µg/g at day 14. The translocation of PYD occurred within tomato plants, as evidenced by an increase in its concentration in tomato stems from 0.74 µg/g on day 1 to 1.56 µg/g on day 3, and a gradual decrease to 0.86 µg/g on day 14. Meanwhile, the residual levels in tomato leaves were from 0.51 μg/g at 1 day to 1.64 μg/g at 7 day as the highest, then followed by a decrease to 0.75 μg/g after the experiment. This may be due to the fact that the tomato root system continuously absorbed PYD in the nutrient solution following contact with the nutrient solution until it was gradually saturated. As the concentration of PYD in the nutrient solution gradually decreased, it is possible that the PYD in the root system was translocated to other parts of the plant, resulting in a gradual increase in the levels of PYD in the stems and leaves, and a decrease in the root system.

The RCF, BCF, and LCF of PYD in tomato plants are shown in Figure 2E, indicating a higher absorption capacity of tomato roots compared to stems and leaves. Correspondingly, TF_leaf/stem and TF_stem/root (Figure 2F) for PYD were from 0.69 to 1.45 and from 0.39 to 0.80, respectively. The TF values were almost <1, indicating a weak root-to-stem and stem-to-leaf translocation in tomato.

In cucumber systems, PYD residues in leaves were significantly higher than those in stems, while opposite results were obtained in tomato systems, which may be due to the difference in the characteristics of the plants themselves.

3.4. Factors Affecting PYD Absorption and Translocation

The process of organic pollutant absorption by plants can be viewed as a continuous distribution process occurring between water–root and root–leaf [6]. In this study, cucumber and tomato plants were exposed to a nutrient solution containing PYD, which prioritized absorption of the compound by the roots. The maximum levels of PYD observed in cucumber and tomato roots were recorded on 1 day and 10 days, respectively. The lipid content of the plant roots is a significant factor that influences the absorption of organic pollutants [9,30]. It can be postulated that the distinct morphological structures of cucumber and tomato roots may explain the disparity in PYD absorption. The lipid contents of the root of cucumber and tomato were 0.3 g per 100 g and 0.6 g per 100 g, respectively. It is proposed that the higher lipid content in tomato roots may contribute to their greater absorption capacity compared to cucumber roots. This finding aligns with the data of Schroll et al., which demonstrated that an elevation in root lipid content led to an increase in residual liposoluble hexachlorobenzene [31].

The physicochemical properties of compounds play a pivotal role in determining the uptake and translocation of plants. The results of the research conducted indicated that RCF demonstrated a positive correlation with logKow and molecular weight, while exhibiting a negative correlation with water solubility. In contrast, TF exhibited the inverse correlation with these three parameters when investigating the pesticide behavior of imidacloprid and difenoconazole [10,30,32]. Furthermore, it can be observed that non-ionic pesticides enter plant roots mainly through passive uptake [10]. In the present study, the results could be attributed to PYD’s relatively lower water solubility and logKow value of 3.8, which resulted in its accumulation in cucumber root. Similar findings were observed in difenoconazole (logKow 3.2) in rice and Chinese cabbage [33,34]. Additionally, it was postulated that the translocation situation of parent compounds could be attributed to the plant metabolic difference, which might explain the observed different phenomenon between cucumber and tomato plants. Consequently, further research is required to elucidate this hypothesis.

3.5. Distribution of PYD in the Plant–Water Systems

In this study, plant biomass was recorded accurately, and the PYD content in the different plant parts was calculated separately. The percentage of the PYD in the root, stem, leaf, and nutrient solution, as well as the percentage lost from the system at different exposure times, are presented in Figure 3.

3.5.1. Cucumber–Water System

As illustrated in Figure 3A, the concentration of PYD in the nutrient solution exhibited a gradual decline over time, accompanied by a corresponding increase in mass loss. Additionally, the proportion of PYD in the root, stem, and leaf showed different varying degrees of change. The content of PYD at day 1 in the nutrient solution, cucumber root, stem and leaf accounted for 89.00%, 5.33%, 2.17% and 2.33% of the initial value, respectively. As the exposure time increased, the PYD content in the nutrient solution gradually decreased, while the content in the cucumber plants gradually increased. At the end of the exposure period (day 14), the contents of PYD in the nutrient solution and cucumber plants were 23% and 25%, respectively. This indicated that the uptake of PYD in the cucumber is a gradual process that occurs over time.

The mass balance principle was employed to ascertain the mass loss of PYD in the nutrient solution–cucumber system, which was around 1.16% at 1 day exposure. At the last exposure of 14 day, the mass loss increased to 51.55%, indicating that PYD had undergone metabolism in the cucumber system and generated metabolites.

3.5.2. Tomato-Water System

As can be seen in Figure 3B, similar to cucumber plants, the concentration of PYD in the tomato nutrient solution decreased gradually with time, culminating in a maximum mass loss at 14 days. The initial concentration of PYD in the nutrient solution was recorded as 100%. After 1 day of exposure, the residual levels of PYD in the nutrient solution, tomato roots, stems, and leaves accounted for 75.63%, 2.37%, 2.10%, and 0.96%, respectively. The mass loss of PYD increased from 16% to 56% between 1 and 14 days of exposure. In contrast to the cucumber system, a greater proportion of PYD was found in the nutrient solution of the tomato systems. This discrepancy may be attributed to inherent differences between the plants themselves.

Phytoremediation represents a green strategy for the remediation of pesticide-contaminated water and soil. To date, there have been some studies investigating the potential of plants to remediate soil and water contaminated by organic micropollutants. Proportions of 96.1% and 99.8% of imazalil and tebuconazole in hydroponic nutrients were effectively uptaken and transported by reed [35], and the removal pathways of tebuconazole by five wetland plants were primarily involved in internal degradation [36]. Given that PYD is constantly degraded in the nutrient solution, resulting in a lower concentration, it is possible that PYD may also be absorbed by plants in order to achieve the purpose of soil and water resource remediation. This provides a novel approach to phytoremediation.

3.6. Identification of Metabolites

The most prevalent metabolic pathway for pesticides in plants is the I-phase metabolism, which is a catalytic metabolic reaction involving enzymes that encompasses reduction, hydrolysis, and oxidation reactions [37]. Cytochromes P450 is the most studied metabolic enzyme, known as “a universal biological catalyst”, which can participate in various metabolic reactions, including hydroxylation, dealkylation, and oxidation [37]. The metabolism of PYD in plants in this study was dominated by enzymes of the I-phase metabolism.

In this study, the metabolic pathways of PYD in a plant–nutritional solution system were analyzed using HPLC-Q-TOF. Six metabolites, namely M411, M441, M395, M455, M361, and M391, were identified in cucumber. The retention times of these metabolites were found to be 8.13, 6.84, 7.90, 8.47, 7.50, and 8.07 min, respectively, as shown in Table S3. The retention times of the metabolites were shorter than that of the parent compound (8.59 min), indicating that the metabolites exhibited a stronger polarity than the parent compound.

In comparison to PYD (m/z 426.03), M411 (m/z 412.02) exhibited a mass deviation of 14 Da. A multitude of fragments exhibited identical mass deviations to the parent fragment, providing unambiguous evidence that this transformation was unequivocally caused by the demethylation reaction, resulting in the loss of the -CH₂ group. Furthermore, it was observed that the fragment with m/z 192.94 was presented in both M411 and PYD, which suggested that methyl loss had occurred on the pyrazole ring. In contrast, M441 exhibited the same fragment m/z 206.07 as the parent compound, indicating that the pyrazole ring remained unaltered, while the benzene ring to which it was attached underwent modification. The mass difference of 16 Da between M441 and the parent, along with the presence of the fragment m/z 236.96 and its parent fragment m/z 220.97, as well as the complementary ions m/z 237 and 206, collectively provide compelling evidence that M441 was formed through hydroxylation. M395, which has a mass difference of 30 Da compared to PYD (m/z 406.03), showed significant fragmentation at m/z 376.02. The fragmentation of m/z 376.02 was broken into m/z 156.06 and m/z 220.97, a reaction also observed in PYD, implying modifications to the m/z 156.06 fragment. This alteration is likely attributed to demethoxylation, involving the elimination of a methoxyl group from the nitrogen atom of the amide moiety. Figure 4 illustrates the proposed metabolic reaction and pathway analysis for PYD in hydroponic systems. M455, which differs in mass by 30 Da from PYD, shares fragments m/z 206.07 and m/z 186.07 with the parent compound, suggesting that the benzene ring is the site of predominant metabolism. The initial analysis indicates that M455 is a hydroxymethoxylated derivative, with -OH and -CH₂ groups located at the meso-position of the benzene ring. The metabolite M361 exhibits a 64 Da mass difference from PYD, with corresponding fragments m/z 342.06 and m/z 406.03. The breakdown of m/z 342.06 at the nitrogen atom of the amide bond results in the formation of fragments 158.98 and 156.06. Many metabolic changes are reflected in these fragments, one of which occurred at the benzene ring. According to our hypothesis, M455 is formed by the removal of methoxyl groups and the substitution of benzene rings for the nitrogen atoms in the amide bond. It is noteworthy that M391 exhibits similarities with PYD, sharing fragments with m/z 186.07 and m/z 166.06. This indicated a 34 Da mass difference with major fragments at m/z 158.98. These similarities suggest that a loss may have occurred on the benzene ring, specifically the detachment of HCl.

In the tomato nutrient, PYD yielded three metabolites (M411, M441, and M395) with formation mechanisms consistent with those observed in cucumber. These metabolites were characterized by hydroxylation, demethoxylation, and demethylation as predominant processes.

The metabolic behavior of the same pesticide exhibits variability in different plants. In wheat, eight metabolites (M-412, M-408, M-374, M-330, M-186, M-396, M-392, and M-358) and one conjugate (C-570) were identified from PYD through hydroxylation, hydrolysis of acid amides, dechlorination, N-demethylation, demethoxylation, and glycosylation [4] In the case of Brassica napus, carbaryl buthionate was metabolized to carbaryl, 3-hydroxy-carbaryl, and 3-keto-carbaryl, whereas in cucumber it was metabolized to carbaryl and 3-hydroxy-carbaryl only [38]. Imidacloprid was found to have five metabolites in celery leaves, three metabolites in lettuce leaves, and two metabolites in radish leaves [39]. The findings indicated that different metabolites would be generated in different crops, even within the same crop, potentially due to the involvement of different enzymes participating in the biotransformation of organic compounds.

4. Conclusions

This research showed that PYD levels were found to be significantly higher in roots in both cucumber and tomato plants, which suggested that the roots have a higher ability to concentrate PYD. Additionally, TF_leaf/stem was observed to be 2.15–22 times higher in cucumber compared to TF_stem/root, indicating a greater ability transferring PYD from stems to leaves occurred in cucumber. However, weak root-to-stem and stem-to-leaf translocations (TFs < 1) were presented in tomato. The different degrees in proportion of mass loss implied that the generation of multiple metabolites within the two plants was different, and it is speculated that the main reason is the characteristics of plants themselves. Investigations on the accurate mechanism of the movement of PYD in plants by omics and biological techniques will be the focus of our future research.