X-ray irradiation simultaneously mitigates chlorfenapyr and azoxystrobin residues and preserves postharvest quality of cowpea

Abstract

Cowpea faces a residue-quality trade off during postharvest storage, requiring effective pesticide dissipation without accelerating quality deterioration. This study evaluated the potential of X ray irradiation (0–350 Gy) to simultaneously dissipate chlorfenapyr and azoxystrobin residues, and preserve the postharvest quality of cowpea over 8 days. Pesticide dissipation was dose-dependent, influenced by pesticide properties and matrix effects, with azoxystrobin showing greater apparent degradability than chlorfenapyr. Transformation products were identified, with degradation pathways involving dealkylation, hydroxylation, hydrolysis or ether cleavage, and reductive dehalogenation, while ECOSAR screening indicated most products had potentially lower aquatic toxicity for most products. Irradiated samples also showed lower microbial growth and lipid peroxidation, together with higher antioxidant enzyme activities, phenolics, and flavonoids, suggesting an adaptive stress response. The optimal 300–350 Gy irradiation range balances residue reduction and short-term storage quality, providing a sustainable technical and theoretical foundation for safe postharvest commercial handling of fresh cowpea.

1. Introduction

Cowpea (Vigna unguiculata (L.) Walp.) is a major legume widely cultivated in tropical and subtropical regions. Valued for its high content of minerals, protein, vitamins and dietary fiber, it contributes to improve residents' dietary nutrition and support the development of regional agricultural economies (Zhang, Guo, et al., 2025; Zhang, Han, et al., 2025). Nevertheless, the production and commercial circulation of cowpea are currently restricted by two intractable bottlenecks. First, the tender pods exhibit high postharvest respiration rates, leading to rapid quality deterioration such as wilting, fiber accumulation and rust-like speckling. These issues limit shelf life of fresh cowpea to only 3–4 days under ambient conditions (Liu et al., 2024), thereby leading to substantial postharvest losses. Second, frequent pesticide applications are required to control pests and diseases during cowpea cultivation, while the harvest interval is extremely short (as brief as 1–2 days at the peak harvest period), making it difficult to strictly comply with the required preharvest interval. This significantly increases the risk of pesticide residue accumulation in cowpea, raising public concerns regarding food safety and potential health hazards (Zhang et al., 2022). Chronic low-dose exposure to pesticide residues is associated with adverse health effects, including neurotoxicity, endocrine disruption and carcinogenicity (Munir et al., 2024; Zhou et al., 2025). Meanwhile, frequent reports of pesticide residue exceedances in marketed cowpea have further amplified public anxiety about its edible safety (Li, Wu, et al., 2025). Accordingly, there is an urgent need for postharvest interventions that can simultaneously mitigate pesticide residues and inhibit quality deterioration of cowpea during storage.

Chlorfenapyr and azoxystrobin are the most widely used pesticides for cowpea cultivation. Chlorfenapyr is a halogenated pyrrole pro-insecticide with broad-spectrum activity, which exerts its insecticidal activity by uncoupling oxidative phosphorylation in mitochondrial membranes, disrupting ATP synthesis, and ultimately leading to the death of insects and mites at all developmental stages (Cheng et al., 2025). Azoxystrobin is a systemic methoxyacrylate strobilurin fungicide that inhibits fungal mitochondrial respiration, thereby suppressing fungal growth and spread (Wang, Chen, et al., 2024). While these pesticides help secure yield, their residues may persist on the crop. There is therefore an urgent need for postharvest interventions that can simultaneously reduce residues and extend shelf life.

To address the dual challenges of pesticide residue reduction and postharvest quality maintenance of fresh cowpea, conventional decontamination such as washing, peeling, blanching, and frying can partially remove surface pesticide residues (Yu et al., 2024). However, these approaches are generally unsuitable for preserving the freshness of vegetables during storage and may adversely affect color, texture and flavor, ultimately compromising overall quality (Jiang et al., 2024). To overcome these limitations, a series of eco-friendly non-thermal technologies have been developed, including electrolyzed water, cold plasma, pulsed electric fields, ozone, and irradiation (Bocker & Silva, 2024; Li et al., 2024; Pan et al., 2021). Among them, irradiation stands out for its superior performance in microbial inactivation, shelf-life extension, nutrient retention, and pesticide degradation over thermal treatments (Li, Zhang, et al., 2025). In particular, X-ray irradiation has unique advantages of deep penetration and uniform dose distribution, which enables effective degradation of internal contaminants and inactivation of endogenous microorganisms with minimal adverse effects on product quality (IAEA, 2021; Ye et al., 2024). Previous studies have verified that irradiation can degrade multiple pesticide classes (Khedr et al., 2019; Zhao et al., 2025), and maintain postharvest quality of various fruits and vegetables (Dong et al., 2022; Guo et al., 2022; Ye et al., 2023; Yoon, Ameer, et al., 2020). While irradiation shows potential to address the dual challenge of residue dissipation and quality maintenance, its effects are commodity- and condition-dependent. For cowpea, no systematic study has yet explored the coupled effects of X-ray irradiation dose on residue dissipation, transformation product ecotoxicity, and postharvest quality within a unified experimental system.

Here, it is hypothesized that an appropriate X-ray dose window can simultaneously enhance chlorfenapyr and azoxystrobin residue dissipation, while inhibiting storage quality deterioration of cowpea. To verify this hypothesis, key physiological and physicochemical quality indicators of postharvest cowpea and target pesticide dissipation dynamics were systematically determined, with correlation analysis conducted to investigate the effects of X-ray irradiation on pesticide degradation and core quality maintenance of cowpea. The specific objectives of this study were to: (i) quantify the degradation kinetics of chlorfenapyr and azoxystrobin under X-ray irradiation in aqueous solution and in cowpea to evaluate matrix effects; (ii) characterize transformation products and propose major transformation pathways, together with in silico ecotoxicity prediction (ECOSAR); and (iii) assess storage-related quality and physiological indicators during 8 days of storage, thereby identifying a condition-specific dose window for balancing residue reduction and postharvest storage quality in cowpea.

2. Materials and methods

2.1. Materials

Cowpea (Vigna unguiculata (L.) Walp.) fruit were harvested from a commercial field in Yacheng Town, Yazhou District, Sanya, Hainan, China. Experimental materials comprised only healthy pods exhibiting uniform length and thickness and no visible pest, disease or mechanical damage. Chlorfenapyr and azoxystrobin standards (purity >98%) were purchased from Tiankang Yunmu Technology Co., Ltd. (Haikou, Hainan, China). Ethyl acetate (purity >99.5%), anhydrous magnesium sulfate (anhyd. MgSO₄; purity >99.0%), and sodium chloride (NaCl; purity >99.5%) were obtained from Xilong Chemical Co., Ltd. (China). Primary secondary amine (PSA) and graphitized carbon black (GCB) sorbents were supplied by Ampoule Experimental Technology Co., Ltd. (China). Commercial assay kits for antioxidant enzyme activities were purchased from Nanjing Jiancheng Technology Co., Ltd. (Nanjing, China). All other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (China).

2.2. Sample preparation

Accurately weighed chlorfenapyr and azoxystrobin standards were dissolved in acetonitrile and brought to volume to prepare single-compound stock solutions at 1000 mg/L, which were stored at −20 °C under dark conditions. Working standard solutions (0.5, 1.0, 2.0, and 5.0 mg/L) were freshly prepared by diluting the stocks with ethyl acetate immediately before analysis for calibration and quantification. The immersion-spiking procedure was used to standardize initial residue loading and enable mechanistic comparison across doses; however, it may represent a worst-case scenario rather than typical field residues, which is noted as a limitation. Based on Chinese maximum residue limit (MRL, chlorfenapyr 1 mg/kg; azoxystrobin 3 mg/kg), spiking levels of 0.5–5 mg/kg were chosen to cover sub-MRL to above-MRL scenarios for conservative assessment.

Fresh cowpea pods were randomly divided into ∼2 kg groups. Each group was immersed for 1 h in a corresponding pesticide solution at a sample-to-solution ratio of 1:4 (w/v) with gentle stirring to ensure uniform coverage. After treatment, the pods were removed and air-dried at room temperature until no visible surface moisture remained, and then subjected to irradiation.

Irradiation was conducted at the Electron Accelerator Laboratory of Sanya Mingyao Germplasm Innovation Technology Co., Ltd. X-rays were generated using a linear electron accelerator with a tantalum conversion target (electron energy: 2.0 MeV; average power: 1 kW, YKFN-V1-BVE-FY2000). Dose levels (150, 200, 250, 300 and 350 Gy) were selected with reference to previous studies on low-dose irradiation for residue reduction and postharvest quality maintenance in fresh produce (Guo et al., 2022), combined with the practical operating parameters of this study.

Following irradiation, cowpea pods were stored for 8 d in an incubator under controlled conditions (25 ± 0.5 °C; 75 ± 5% RH) to simulate typical ambient conditions during retail display and wet-market marketing of cowpea, thereby providing a practical shelf-life scenario for evaluating treatment effects. Texture (hardness), soluble solids content (SSC), and total viable count were analyzed at 2-day intervals. The experiment was conducted with three biological replicates per treatment, each consisting of 12 cowpea pods, and statistical analyses were performed using replicate (batch-level) means (n = 3). Method validation was conducted using matrix-spiking recovery experiments in cowpea at 0.01, 0.5, and 10 mg/kg. Recoveries ranged from 87.61% to 105.84%, with relative standard deviation (RSD) of 0.81%–9.67%. The limits of quantitation (LOQ) for both chlorfenapyr and azoxystrobin were 0.01 mg/kg.

2.3. Extraction and determination of chlorfenapyr and azoxystrobin

Residues of chlorfenapyr and azoxystrobin in cowpea were extracted using a modified QuEChERS method (Zhang et al., 2023). Samples (5 g) were extracted with 10 mL acetonitrile (8000 rpm, 5 min), salted out with NaCl (5 g), vortexed 2 min and centrifuged (5000 rpm, 5 min). An aliquot (4 mL) was cleaned by d-SPE (PSA 100 mg, C18 100 mg, GCB 100 mg, MgSO₄ 1.5 g), vortexed 2 min, and centrifuged (5000 rpm, 10 min). The purified extract (1 mL) was evaporated to dryness, reconstituted in 5 mL LC/MS-grade ethyl acetate, filtered (0.22 μm PTFE), and transferred to vials. Determination was by GC–MS/MS (TRACE 1300-TSQ 9000; TG-5SilMS 30 m × 0.25 mm × 0.25 μm; inlet 250 °C, splitless; transfer 280 °C; source 300 °C). Oven: 120 °C (2 min) → 25 °C/min to 250 °C (8 min) → 30 °C/min to 300 °C (5 min). Detection used EI-SRM. Quantification employed external calibration (n = 3).

2.4. Metabolite analysis and toxicity prediction of chlorfenapyr and azoxystrobin

Metabolite profiling was performed by UHPLC-HRMS/MS on an AB Sciex TripleTOF 6600 with a BEH Amide HILIC column. Data processing/annotation used PeakView (AB Sciex), Compound Discoverer 3.0 (Thermo Fisher Scientific), and Xcalibur (Qual Browser, Thermo Fisher Scientific). Toxicity prediction employed ECOSAR v2.2 (U.S. EPA).

Sample preparation. Irradiated pods were ground to a fine powder in liquid nitrogen. Powder was transferred to centrifuge tubes and extracted with pre-chilled solvent (methanol/acetonitrile/water = 2:2:1, v/v/v). After vortexing, samples were sonicated at 4 °C, followed by protein precipitation at −20 °C. After centrifugation at 4 °C, the supernatant was collected, lyophilized under vacuum, and reconstituted in acetonitrile/water (1:1, v/v). The reconstituted solution was centrifuged, filtered through an organic-phase syringe filter, and transferred to LC vials.

Instrumental analysis. Metabolites were analyzed using ultra-high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) on an AB Sciex TripleTOF 6600 system. Chromatographic separation employed an ACQUITY UPLC BEH Amide HILIC column (1.7 μm, 2.1 mm × 100 mm). The mobile phases were A: water with 25 mM ammonium acetate and 0.1% ammonia and B: acetonitrile; a gradient elution program was used.

Data analysis. Using PeakView software (AB Sciex), the features detected under the applied UHPLC HRMS/MS conditions were tentatively annotated as metabolites of chlorfenapyr and azoxystrobin on the basis of accurate mass, isotopic pattern, and diagnostic fragment ions. Possible transformation pathways of chlorfenapyr and azoxystrobin were proposed based on the tentatively assigned transformation products and their fragmentation characteristics.

Toxicity prediction. The ecological toxicity of individual tentative metabolites was predicted using the ECOSAR (v2.2, U.S. EPA) platform, which estimates acute and chronic toxicity values (LC50, EC50, ChV) for aquatic organisms based on chemical structure.

2.5. Dietary risk assessment

For chlorfenapyr and azoxystrobin, chronic and acute dietary exposure was evaluated as percent acceptable daily intake (%ADI) and percent acute reference dose (%ARfD), respectively, over initial concentrations of 0.5–5.0 mg/kg and irradiation doses of 0–350 Gy. Calculations were stratified by sex and age category (2–7, 8–12, 13–19, 20–50, 51–65, >65 years). The equations were:

$$\%\mathrm{ADI}=\mathrm{EDI}/\mathrm{ADI}\times 100\%$$

$$\%\text{ARfD}=\text{ESTI}/\text{ARfD}\times 100\%$$

where EDI (mg/kg bw day⁻¹) is estimated daily intake, ADI (mg/kg bw day⁻¹) is acceptable daily intake, ESTI (mg/kg bw) is estimated short-term intake, ARfD (mg/kg bw) is acute reference dose, and bw denotes body weight.

2.6. Color measurements and cowpea rust-speckling index (CI)

Surface color (L*, a*, b*) was measured with a colorimeter (CR-410, Konica Minolta, Osaka, Japan). Six random positions per pod were recorded and averaged.

CI was scored by affected surface area: Grade 0 = 0%; 0% < Grade 1 < 25%; Grade 2, 25–50%; Grade 3, >50% (Fan et al., 2016). The CI was calculated as:

$$\mathrm{CI}\left(\%\right)=\sum \left(\mathrm{CL}\times \mathrm{NL}\right)/3\mathrm{TN}\times 100$$

where CL is the speckling grade (0–3), NL is the number of pods at that grade, and TN is the total number of pods in the treatment.

2.7. Total viable count (TVC), electrolyte leakage (EL) and malondialdehyde (MDA) content

TVC was determined using the plate count method (Tao et al., 2024). Briefly, 5 g of cowpea was homogenized with 45 mL of sterile 0.85% (w/v) NaCl and subjected to serial dilutions. Aliquots (0.1 mL) from appropriate dilutions were spread-plated onto nutrient agar and incubated at 37 °C for 48 h. Colonies were counted on plates containing 30–300 colonies, and results were expressed as log CFU/g.

EL was determined by the conductivity method. Cowpea samples (5 g) were immersed in 20 mL of distilled water and gently agitated at room temperature for 20 min to obtain the initial conductivity (C_i). The sample was boiled for 20 min and cooled, the final conductivity (C_f) was recorded. EL (%) was calculated as EL = (C_i/C_f) × 100.

The MDA content was quantified using the thiobarbituric acid reactive substances assay (Xu et al., 2023). The absorbance of the reaction product was measured at 532 nm for concentration calculation.

2.8. Ascorbic acid (VC), soluble solids content (SSC), chlorophyll (Chl) and soluble protein (SP)

VC was determined by the 2,6-dichlorophenolindophenol (DCPIP) assay. Frozen tissue (5.0 g) was homogenized with 10 mL oxalic acid (1%) and filtered. A 2 mL aliquot of the filtrate was mixed with 1 mL 0.025% DCPIP-Na for 1 min, and the absorbance was measured at 534 nm. The concentration was calculated using an ascorbic acid standard curve (0–0.5 mg/mL).

SSC was determined with a handheld refractometer (PAL⁻¹, Atago). Fresh cowpea samples were homogenized and the slurry was centrifuged at 8000 ×g for 10 min. The supernatant was directly used for SSC determination, expressed as % (w/w).

Chl content was determined by spectrophotometric method (Xu et al., 2023). Frozen tissue (1.0 g) was extracted with 80% (v/v) acetone, made up to a final volume of 20 mL, kept in the darkness for 60 min. The extract was then centrifuged at 11,000 g for 15 min at 4 °C, A₆₅₂ of the supernatant was recorded.

SP content was determined by the Bradford method using bovine serum albumin (0–100 μg/mL) as standard. Briefly, frozen tissue (1.0 g) was homogenized in phosphate buffer, centrifuged, and 0.1 mL of supernatant was mixed with 5 mL Bradford reagent. After 5 min of incubation, absorbance was measured at 595 nm. Results are expressed as mg protein per g fresh weight.

2.9. Total phenolics (TP) and total flavonoids (TF)

TP and TF levels were assessed using the method of Zhao et al. (2025).

TP content was determined using the Folin-Ciocalteu method and expressed as mg GAE per g FW. Briefly, tissue (1.0 g) was homogenized in 80% ethanol, centrifuged, and the supernatant was reacted with Folin-Ciocalteu reagent and 7.5% Na₂CO₃. After 60 min in the dark, absorbance was measured at 765 nm against a gallic acid standard curve (0–100 mg/L).

TF content was determined spectrophotometrically at 510 nm using a rutin standard curve (0–200 mg/L) and expressed as mg RE per g FW. Briefly, 1 mL of extract was sequentially reacted with 1 mL of 5% NaNO₂ for 6 min, 4 mL of 10% Al(NO₃)₃ for 6 min, and 4 mL of 4% NaOH, followed by dilution to 10 mL and a 15-min dark incubation before measurement.

2.10. Antioxidant enzyme activities

Activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer's instructions.

POD: guaiacol colorimetric assay; one unit corresponded to corresponds to an absorbance change of 0.01 min⁻¹ at 470 nm.

SOD: inhibition of nitroblue tetrazolium (NBT) photoreduction; one unit is the enzyme amount that inhibits NBT photoreduction by 50% under the assay conditions.

CAT: UV assay based on the decline in H₂O₂ absorbance at 240 nm; one unit catalyzes the decomposition of 1 μmol H₂O₂ per minute.

APX: UV assay based on the decrease in ascorbic acid absorbance at 290 nm; one unit oxidizes 1 μmol ascorbic acid per minute.

2.11. Statistical analysis

All experiments were conducted with three independent biological replicates, and data are presented as mean ± standard error (SE). Data visualization were prepared in Origin 2025. Statistical analyses were conducted in IBM SPSS Statistics 26.0. Group comparisons were performed using one-way ANOVA to compare mean differences among three or more independent groups; when significant (P < 0.05), Duncan's multiple range test was applied.

3. Results and discussion

3.1. Effects of X-ray irradiation on pesticide residues

The degradation of chlorfenapyr and azoxystrobin by X-ray irradiation was jointly governed by the irradiation dose, matrix type (aqueous solution vs. cowpea), pesticide physicochemical properties and initial concentration (Fig. 1). For both pesticides, the residual percentages generally decreased with increasing dose, although the extent of dissipation depended strongly on matrix and compound type. For example, in aqueous solution spiked with 0.5 mg/L azoxystrobin, the residual percentage decreased to 12.85% at 350 Gy. This dose-dependent degradation aligns with reported enhancement of glyphosate degradation in water at higher irradiation doses (Mendes et al., 2024). The underlying mechanism likely involves both direct bond cleavage and indirect reactions mediated by species generated during water radiolysis, such as hydrated electrons (e⁻_aq), hydroxyl radicals (·OH) and hydrogen atoms (H·). The reduced degradation efficiency in cowpea matrix was mainly due to the quenching effect of matrix organic components on active free radicals. Representative reactions include:

$$\mathrm{X}-\mathrm{ray}\text{photons}\left(\mathrm{keV}\text{range}\right)\text{cause ionization}:{\mathrm{H}}_2\mathrm{O}\to {\mathrm{H}}_2{\mathrm{O}}^{+}+{\mathrm{e}}^{-}$$

Fig. 1.

Effect of X-ray irradiation on pesticide residues. (A) chlorfenapyr in solution; (B) azoxystrobin in solution; (C) cowpea treated with chlorfenapyr; (D) cowpea treated with azoxystrobin.

$$\text{The ionized water molecule reacts to form}:{\mathrm{H}}_2{\mathrm{O}}^{+}+{\mathrm{H}}_2\mathrm{O}\to {\mathrm{H}}_3{\mathrm{O}}^{+}+·\mathrm{OH}$$

$$\text{Electrons become hydrated}:{\mathrm{e}}^{-}+{\mathrm{nH}}_2\mathrm{O}\to {{\mathrm{e}}^{-}}_{\mathrm{aq}}$$

$$\text{Excited water molecules dissociate}:{\mathrm{H}}_2{\mathrm{O}}^{\ast }\to \mathrm{H}·+·\mathrm{OH}$$

At higher doses, increased yields of reactive species such as ·OH, can more effectively attack key functional groups of target pesticides (e.g., the methoxyacrylate moiety of azoxystrobin and the pyrrole ring of chlorfenapyr), thereby accelerating pesticide degradation (Khan et al., 2023).

Besides irradiation dose, the degradation of chlorfenapyr and azoxystrobin by X-ray irradiation was co-regulated by matrix type, pesticide physicochemical properties and initial concentration. The cowpea matrix markedly suppressed degradation relative to aqueous solution, and this matrix dependent suppression was observed consistently all treatments, which aligns with the higher profenofos degradation in solution than vegetable matrices reported by Thihara Rodrigues et al. (2020). This matrix effect likely reflects several interacting factors. First, the physical structure of cowpea tissue may restrict the diffusion and local availability of reactive species generated during irradiation. Second, endogenous constituents such as sugars, proteins, phenolics, and pigments may compete with pesticide molecules for reactive species, thereby reducing the effective degradation of the target compounds. Third, pesticide partitioning behavior within the plant matrix may further influence accessibility to radiolytic attack, suggesting that degradation behavior under similar treatment conditions may differ among fruits and vegetables with different tissue structures and compositions.

Significant pesticide-dependent degradability was observed, with azoxystrobin showing consistently higher degradation efficiency than chlorfenapyr under identical conditions. For example, at 350 Gy and 1 mg/L, where azoxystrobin showed lower residual percentages than chlorfenapyr in both matrices (Fig. 1). Such pesticide-dependent differences align with findings from 37 compounds in dandelion (Zhao et al., 2025). The higher degradability of azoxystrobin is mainly driven by its molecular structure and physicochemical properties. The methoxyacrylate moiety of azoxystrobin is more susceptible to oxidative attack, while the conjugated aryl pyrrole structure of chlorfenapyr is more stable; additionally, the higher aqueous solubility of azoxystrobin facilitates contact with reactive species, whereas lipophilic chlorfenapyr tends to partition into matrix hydrophobic domains with limited radical availability (Armelim et al., 2018; Khedr et al., 2019).

Furthermore, a negative correlation was observed between the initial pesticide concentration and degradation efficiency. At a fixed dose, increasing the initial concentration generally reduced the apparent degradation efficiency of both pesticides. This trend can be explained by the fixed yield of radiolytic reactive species generated at a given irradiation dose (Khedr et al., 2019). As pesticide concentration increased, the ratio of reactive species to substrate decreased, resulting in lower degradation efficiency per molecule.

3.2. Transformation pathways of pesticide metabolites

The tentatively identified transformation products of chlorfenapyr (C1-C7) and azoxystrobin (A1-A9) identified in irradiated cowpea are summarized in Supplementary Table S1. X-ray irradiation induced the formation of seven chlorfenapyr-related and nine azoxystrobin-related metabolites. For chlorfenapyr, key reactions included O/N-dealkylation, hydroxylation, and reductive dehalogenation (Fig. 2A). Route 1 (C → C1) involved O-deethylation via α‑hydrogen abstraction and C—O scission, yielding a terminal alcohol (loss of C₂H₄). Route 2 (C → C2 → C3) featured N-dealkylation to remove the N-(2-ethoxyethyl) group (C2), followed by reductive debromination (C3) via e⁻_aq/H·. Route 3 (C2 → C4) featured radiolytic nucleophilic aromatic substitution, converting the p-chlorophenyl moiety to a phenol derivative. Route 4 (C2 → C5) substituted bromine with OH (Br → OH); Route 5 (C2 → C6) achieved reductive dechlorination (Cl → H) on the aryl ring. Route 6 (C → C7) was more complex, involving combined arene dechlorination, nitrile hydration (-C ≡ N → -CONH₂), N-dealkylation and N-formylation. For azoxystrobin, ·OH-dominated reactions included alkene hydration/oxidation, O-dealkylation, ester hydrolysis and ether cleavage (Fig. 2B). Route 1 (A → A1 → A2 → A3) involved vinylic hydroxylation, oxidation to carbonyl and further hydroxylation to a carboxylic acid. Route 2 (A1 → A4) featured hydrolytic dealkoxylation of the methoxycarbonyl to a carboxylic acid. Route 3 (A → A5) entailed cleavage of the vinylic C-O-C linkage. Route 4 (A → A6 → A7) included methyl ester hydrolysis to carboxylic acid, followed by hydration/methoxy-hydroxylation. Route 5 (A → A8) involved radical addition across the β-methoxyacrylate double bond. Route 6 (A → A9) resulted in cleavage of the heteroaryl-O-aryl linkage to a phenolic product.

Fig. 2.

Proposed metabolic reaction and pathway analysis for (A) chlorfenapyr, (B) azoxystrobin in cowpeas and (C) reactive species affecting pesticide structure and degradation.

The degradation of chlorfenapyr and azoxystrobin was primarily driven by reactive species generated during water radiolysis. Under X-ray irradiation, water radiolysis produces a series of reactive intermediates, including ·OH, e⁻_aq, H₂O₂, H₂, H₃O⁺/OH⁻and hydrogen atoms (H·), as illustrated in Fig. 2C. Among these, the highly oxidative ·OH mediates critical reactions such as O/N-dealkylation, addition to C C double bonds, and various bond cleavage reactions, while the reductive eₐq⁻ and H· dominate reductive degradation pathways including dehalogenation (Egerić et al., 2024).

3.3. Model based ecotoxicity assessment

The potential ecological risks of pesticide degradation products necessitate careful evaluation, as some metabolites may exhibit higher toxicity than their parent compounds (Yu et al., 2025). In this study, ECOSAR was used as an in silico screening tool to predict the acute and chronic toxicity of chlorfenapyr, azoxystrobin and their transformation products toward three aquatic species: fish, green algae and daphnids. As shown in Fig. 3, the parent compound chlorfenapyr was predicted to be “very toxic” (LC50/EC50 < 1 mg/L) to all three tested organisms in both acute and chronic exposure scenarios. Most of its degradation products showed lower predicted toxicity, with some metabolites shifting the acute toxicity classification for fish and daphnia from “very toxic” to “toxic” (1–10 mg/L). In contrast, azoxystrobin showed predicted “harmful” acute toxicity to fish (LC50/EC50 10–100 mg/L) and “toxic” chronic toxicity, with chronic toxicity rated as toxic (1 mg/L < LC50/EC50 < 10 mg/L), while its acute effects on Daphnia and green algae were predicted to be “non-harmful” (LC50/EC50 > 100 mg/L). With the exception of metabolite A5, all other degradation products of azoxystrobin exhibited lower predicted acute and chronic toxicity risks than the parent compound across all three aquatic species.

Fig. 3.

Predicted (A) acute and (B) chronic toxicity of chlorfenapyr, azoxystrobin, and their degradation products to aquatic species using ECOSAR.

3.4. Dietary risk assessment

Chronic and acute dietary risks were evaluated using %ADI and %ARfD as a scenario-based assessment under the spiked-residue design (0.5–5.0 mg/kg), which enables comparison of dose-dependent changes. As shown in Supplementary Table S2-S5, azoxystrobin presented low estimated chronic and acute dietary risk across the tested spiking scenarios, with all %ADI and %ARfD values remaining below 50%. For chlorfenapyr, the estimated chronic risk also remained at a low level across all scenarios, while its acute dietary risk required more attention, especially for younger age groups. In general, both residue levels and the corresponding %ARfD values decreased with increasing irradiation dose. Under the 0.5–2.0 mg/kg spiking scenarios, acute risk was below 100% for most population groups, although children aged 2–7 years remained comparatively more vulnerable.

At the highest spiking level (5.0 mg/kg), irradiation still reduced estimated acute risk, but this reduction was insufficient to eliminate concern in all subpopulations. Although the estimated risk for several groups fell below the 100% ARfD threshold after irradiation, children aged 2–7 years still exceeded this threshold even at the maximum irradiation dose of 350 Gy, indicating that the acute dietary risk remained unacceptable under this worst-case high-residue scenario. These findings suggest that X-ray irradiation can reduce estimated acute dietary risk under the tested spiking conditions, but the risk mitigation effect should not be overgeneralized, especially for samples with extremely high initial residue levels.

3.5. Effects of X-ray irradiation on color attributes, water loss and rust-speckling

Visual quality, a key indicator of freshness and marketability, was assessed in cowpea during 8-day storage at 25 °C under different X-ray irradiation doses of 0–350 Gy. The appearance changes are shown in Fig. 4A and B, and the color parameters are summarized in Table S6. Overall, color changed progressively during storage and storage time exerted a stronger effect on color changes than irradiation. Nevertheless, irradiated samples—particularly at 200–350 Gy—generally retained higher brightness and greener appearance in the mid-to-late storage than the control, as evidenced by higher L* values, more negative a* values and lower b* values. The total color difference ΔE increased in all treatments without significant between-group differences. Generally retained slightly higher brightness and a greener appearance than the control, especially during the middle and late stages of storage. For example, the a* values of 300–350 Gy treatments ranged from −12.27 to −11.98, compared with −9.98 in the control. These results indicate that X-ray irradiation moderately delayed color deterioration of cowpea, which is consistent with previous findings in irradiated jujube and mango (Guo et al., 2022; Truc et al., 2021).

Fig. 4.

(A) Representative images of cowpea fruits during 8 days of storage following different X-ray doses; (B) Enlarged local images of cowpea pods on day 6 of storage after different X-ray treatments; (C) Water loss in cowpea during storage; (D) changes in rust-speckling index over storage.

The water loss in cowpea increased over storage time (Fig. 4B), with no significant differences observed among treatment groups. The rust-speckling index rose significantly in all groups during storage, with the control consistently exhibiting higher speckling than irradiated samples. The anti-skeckling effect was dose-dependent, being most pronounced at 250–350 Gy. This observation is consistent with the significant suppression of browning in button mushrooms following X-ray exposure (Dong et al., 2022). The potential contributing factors include (i) inhibition of microbial growth—particularly pathogenic taxa—thereby diminishing pathogen-induced speckling, and (ii) enhancement of antioxidant enzyme activities, which mitigates oxidative stress-induced enzymatic browning.

3.6. Effects of X-ray irradiation on key nutritional components

The effects of X-ray irradiation on key nutritional and functional components in cowpea, including ascorbic acid, soluble solids content, chlorophyll, soluble protein, total phenolics and total flavonoids were monitored over 8 days of storage (Fig. 5).

Fig. 5.

Effects of X-ray irradiation on (A) ascorbic acid, (B) soluble solids content, (C) chlorophyll, (D) soluble protein, (E) total phenolics, and (F) total flavonoids in cowpea during storage.

Ascorbic acid is a major antioxidant that scavenges free radicals but is sensitive to storage conditions, decreased throughout storage in all treatments (Fig. 5A). No significant difference in initial ascorbic acid content was observed between the control and irradiated groups on day 0, despite slightly lower values in irradiated samples. However, during later storage, high-dose treatments generally retained more ascorbic acid than the control, suggesting that X-ray irradiation mitigated the net loss of this antioxidant in cowpea under the tested conditions.

The SSC of cowpea remained relatively stable during the early storage period and declined thereafter (Fig. 5B). From mid-storage onward, the higher-dose treatments, particularly 300–350 Gy, generally maintained higher soluble solids than the control, indicating better retention of soluble constituents during storage. This is attributed to the fact that irradiation inhibits polysaccharide-degrading enzymes (e.g., amylase) and the reduction of sugar consumption by spoilage microorganisms, which is consistent with previous findings in irradiated fresh produce (Jat et al., 2022).

Chlorophyll content showed a continuous downward trend in all groups during storage (Fig. 5C), which corresponded to the progressive senescence and greenness loss of cowpea pods. X-ray irradiation treatment, especially at higher doses, delayed the degradation of chlorophyll compared with the control, which agrees with the greener visual appearance described above. This preservation effect may be attributed to the combined suppression of microbial activity and protection of thylakoid membranes through enhanced redox buffering, as previously suggested (Ye et al., 2023).

Soluble protein showed a fluctuating pattern during storage (Fig. 5D), but no significant treatment effect was detected at either the beginning or the end of storage. This result suggests that the soluble protein fraction of cowpea remained comparatively stable under the X-ray irradiation doses applied in the present study.

Total phenolics and total flavonoids, the core functional antioxidant components in cowpea, showed a continuous upward trend in all groups during storage (Fig. 5E-F), with little difference among treatments at day 0 but clearer divergence at later stages. Irradiated samples, particularly at higher doses, tended to accumulate more phenolics and flavonoids than the control during storage. These results are consistent with reports of irradiation-induced phenolic accumulation in fresh produce (Surjadinata et al., 2021; Villavicencio et al., 2018; Yoon, Kim, et al., 2020). Overall, X-ray irradiation at appropriate doses (250–350 Gy) effectively maintained the key nutritional and functional components of postharvest cowpea during storage, providing a nutritional basis for the application of this technology in cowpea postharvest handling.

3.7. Effects of X-ray irradiation on TVC, electrolyte leakage and MDA

TVC (log CFU g⁻¹) reflects the overall microbial burden and sanitary status of fresh cowpea, with higher values indicating accelerated spoilage and increased safety risk. In parallel, electrolyte leakage and MDA content are indicators of membrane integrity and oxidative stress, reflecting secondary physiological responses associated with lipid peroxidation during senescence, rather than direct causal effects of irradiation (Wang, Jing, et al., 2024).

As shown in Fig. 6A, TVC increased continuously throughout storage in all treatments, reflecting progressive microbial proliferation. X-ray irradiation consistently suppressed the increase of TVC compared with the non-irradiated control, and this effect showed a clear dose-dependent pattern, with more pronounced effects observed at higher irradiation doses. Although the magnitude of the difference narrowed by day 8, irradiated samples still maintained a lower microbial burden than the non-irradiated control. This persistent microbial inhibition is consistent with radiation-induced DNA damage and impaired cellular repair in spoilage and pathogenic microorganisms.

Fig. 6.

Effects of X-ray irradiation on (A) total viable count, (B) electrolyte leakage, and (C) malondialdehyde content, and (D-G) the activities of POD, SOD, CAT, and APX, respectively, in cowpea during storage.

MDA content, a marker of lipid peroxidation, also increased over time in all groups (Fig. 6B). Irradiation treatment was associated with lower MDA accumulation, and this effect became more prominent at higher doses and in the mid-to-late storage period. MDA levels were comparable among treatments in the early storage stage, whereas from mid storage onward the irradiated samples, particularly those treated at 300–350 Gy, generally showed lower lipid peroxidation than the control. For example, on day 4 of storage, the MDA content in the 350 Gy treatment group was 1.11 nmol/g, which was lower than the 1.33 nmol/g observed in the control, and this difference became more apparent at later stages. Overall, the higher dose treatments, particularly 300–350 Gy, were more effective in mitigating oxidative deterioration of cowpea during storage.

Electrolyte leakage increased in all groups during storage (Fig. 6C), which was consistent with progressive membrane deterioration. No significant difference was observed at day 0, suggesting that irradiation itself did not cause immediate membrane injury. During mid-storage, however, irradiated samples—especially those treated at higher doses—generally showed lower electrolyte leakage than the control. This pattern was broadly consistent with the lower MDA levels observed in irradiated groups, implying that irradiation delays, rather than prevents, membrane deterioration by maintaining redox homeostasis and limiting lipid peroxidation.

These results are consistent with reports in jujube and other produce (Zheng et al., 2025). The beneficial effects of X-ray irradiation on cowpea postharvest quality are mainly mediated by two complementary pathways. First, radiation directly damages microbial DNA and generates reactive species through water radiolysis, thereby inhibiting the proliferation of spoilage and pathogenic microorganisms. Second, a transient oxidative signal induced by irradiation may activate antioxidant defense responses, including POD, SOD, CAT, and APX activities, and may also be associated with increased phenolics and flavonoids (Hou et al., 2025). This coordinated cellular response limits lipid peroxidation (as evidenced by lower MDA levels) and preserves cellular membrane integrity (Guo et al., 2022), which is further supported by the enzyme activity results shown in Fig. 8.

Fig. 8.

Time-resolved correlation heatmaps between antioxidant enzymes and Physiological/biochemical indices at (A) 0, (B) 2, (C) 4, (D)6 and (E) 8 days of storage under different irradiation doses.

3.8. Effects of X-ray irradiation on antioxidant enzyme activities

The activities of the major antioxidant enzymes (POD, SOD, CAT, and APX) in cowpea, key biomarkers of endogenous antioxidant defense capacity, were monitored during storage. As shown in Fig. 6D-G, X-ray irradiation was generally associated with higher antioxidant enzyme activities than the control, although the response magnitude and timing differed among enzymes.

POD activity increased progressively during storage (Fig. 6D), and irradiated samples generally exhibited higher values than the control from day 2 onward. This pattern suggests that irradiation was associated with a stronger peroxidative response during storage. SOD activity increased first and then decreased, with a peak occurring at the mid-storage stage (Fig. 6E). Compared with the control, irradiated groups, especially those treated with higher doses, showed higher SOD activity during the early and middle stages of storage. CAT activity increased over storage time in all groups (Fig. 6F). The irradiation effect of irradiation on CAT activity was most prominent during early storage, whereas differences became smaller at later stages. APX showed a pattern similar to that of SOD, with a mid-storage peak and generally higher activity in irradiated groups than in the control (Fig. 6G). Among all tested treatments, t high-dose irradiation treatments induced the strongest upregulation of APX activity.

These results are consistent with previous reports of irradiation-induced upregulation of antioxidant enzymes in fruits such as jujube and kiwifruit (Hou et al., 2025). These enhanced antioxidant enzyme activities may reflect an adaptive response associated with irradiation induced oxidative stress, in which radiolytically generated reactive oxygen species may act as signaling molecules to stimulate antioxidant defense responses (Wang et al., 2022). Combined with the reduced MDA content and electrolyte leakage in irradiated samples (Fig. 6B, C), these results indicate that X-ray irradiation triggers a coordinated antioxidant response to maintain redox homeostasis of cowpea during storage.

3.9. Principal component analysis (PCA) and variable correlations reveal irradiation-mediated regulation

PCA and correlation analysis were performed to decipher the multivariate relationship among X-ray irradiation dose, storage time, and physiological traits in cowpea. As shown in Fig. 7, the first two principal components collectively explained 82.8% of the total variance (PC1: 66.8%, PC2: 16.0%), which means the two PCs effectively captured the major variation characteristics of all detected indicators (Elhadef et al., 2024). The sample distribution in the PCA biplot revealed a clear temporal pattern along PC1, with samples shifting from left to right side with prolonged storage (Fig. 7B). This axis represented characteristic senescence changes: increased total viable count, electrolyte leakage, rust-speckling index, and MDA content. PC2 reflected the variation induced by irradiation treatment, with higher-dose samples showing higher PC2 scores at the same storage time.

Fig. 7.

PCA reveals the effects of storage time and irradiation dose on cowpea quality traits: (A) variable loading plot; (B) observation (score) plot.

Time stratified correlation heatmaps further revealed the dynamic regulatory effect of irradiation on the antioxidant-quality correlation network (Fig. 8). At day 0, no stable correlation patterns were observed, while from days 2–6, irradiated groups exhibited significant positive correlations between antioxidant enzymes (POD, SOD, CAT, APX) and quality parameters (TP, TF, SP, chlorophyll, SSC), alongside negative correlations with deterioration markers (MDA, rust-speckling, a* value). By day 8, although all samples underwent pronounced senescence, irradiated group, particularly those treated at higher doses, still maintained positive associations between antioxidant enzymes and key quality indices (TP, TF, Chl, SSC), alongside negative associations with deterioration markers (MDA, CI), suggesting that the coordinated regulatory effect of irradiation persisted into the late storage stage.

Collectively, storage time was the dominant source of variation in postharvest traits, while X-ray irradiation induced significant multivariate separation between treatments, via a coordinated multi-target effect including microbial inhibition, pesticide degradation promotion, and antioxidant defense activation. This integrated effect is consistent with previous reports in irradiated fresh produce (Dobón-Suárez et al., 2025; Jat et al., 2022), supporting the application potential of X-ray irradiation as a green postharvest treatment for fresh cowpea.

4. Conclusions

In this study, X-ray irradiation reduced chlorfenapyr and azoxystrobin residues in fresh cowpea in a dose dependent manner, with degradation efficiency regulated by pesticide identity, initial concentration and matrix conditions, and azoxystrobin showing higher degradability than chlorfenapyr. The main degradation pathways of the two pesticides included dealkylation, hydroxylation, hydrolysis, ether cleavage and reductive dehalogenation, and ECOSAR in silico screening indicated lower predicted aquatic toxicity for most transformation products. During 8 days of ambient storage, 300–350 Gy X-ray irradiation effectively maintained the core postharvest quality of cowpea, with suppressed microbial growth, reduced lipid peroxidation, enhanced antioxidant enzyme activities, higher phenolic and flavonoid contents, and better retention of key quality attributes. Several limitations of this study should be acknowledged: the 8-day storage trial only reflects short-term postharvest effects, no systematic sensory evaluation was performed, degradation pathways were only inferred from transformation products, and aquatic toxicity was assessed via in silico prediction without in vivo validation. Overall, this study provides fundamental data for the application of X-ray irradiation in cowpea postharvest handling, and pilot-scale industrial verification, long-term storage trials and systematic sensory assessment should be conducted in future research.

CRediT authorship contribution statement

Kai Guo: Writing – original draft, Methodology, Data curation. Meng Wang: Writing – original draft, Funding acquisition, Conceptualization. Chang Chen: Methodology, Investigation. Jie Ren: Methodology, Investigation. Xiumei Li: Methodology, Investigation. Xingsheng Yue: Software, Methodology, Formal analysis, Data curation. Xingyue Li: Software, Methodology, Funding acquisition, Data curation. Shanying Zhang: Writing – review & editing, Funding acquisition, Conceptualization. Chenghui Zhang: Writing – review & editing, Funding acquisition, Conceptualization.

Funding sources

This research was supported by the Key Research and Development Project of Hainan Province (ZDYF2023GXJS164), Key Laboratory of Tropical Fruits and Vegetables Quality and Safety for State Market Regulation (No. KF-2023001), Hainan University Research Initiation Fund (Science and Technology) (XJ2400005859), and 2024 Hainan Province Graduate Student Innovative Research Projects (Qhys2024–170).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.