Test soil was collected from the surface layer (0–20 cm) of an agricultural field in Nanjing, Jiangsu Province, China, during the crop fallow period in June 2025. This site had no history of plastic film mulching. The collected soil was air-dried, ground, and sieved through a 2-mm mesh to remove coarse debris. The physicochemical properties of the soil were determined as follows: pH 6.8, organic matter content 15.2 g kg⁻¹, and a sandy loam texture comprising 65.3% sand, 22.1% silt, and 12.6% clay.

To simulate a realistic contamination scenario, waste polyethylene (PE) agricultural mulch films collected from local farmlands were used as the microplastic source. The films were first rinsed with tap water, followed by ultrasonic cleaning in anhydrous ethanol and deionized water for 30 min each to remove soil particles and potential agrochemical residues.

The cleaned films were air-dried, embrittled using liquid nitrogen, and pulverized using a cryogenic grinder. The resulting powder was fractionated into four distinct size classes using standard stainless-steel sieves via a wet-sieving method (95% ethanol). The fractions were defined as follows: 1–2 mm (T1), 0.2–1 mm (T2), 50–200 μm (T3), and < 50 μm (T4). The T4 fraction was obtained by centrifuging the filtrate that passed through the 50-μm sieve. All microplastic particles were dried at 50 °C and sterilized under UV light for 12 h prior to application.

Tomato seeds (Solanum lycopersicum L., cv. Zhongshu No. 4) were surface-sterilized with 5% sodium hypochlorite (NaClO) for 10 min and rinsed thoroughly with deionized water. Seeds were germinated on moist filter paper in the dark, and uniform seedlings with 3–4 true leaves were selected for transplantation.

A pot experiment was conducted in a climate-controlled greenhouse (25/18 °C day/night temperature, 16 h photoperiod). A randomized complete block design was employed with five treatments; each replicated four times: CK: Soil without microplastic addition (Control); T1: Soil spiked with 1–2 mm PE particles; T2: Soil spiked with 0.2–1 mm PE particles; T3: Soil spiked with 50–200 μm PE particles; T4: Soil spiked with < 50 μm PE particles.

PE particles were added to the soil at a concentration of 1% (w/w) (10 g kg⁻¹). To ensure homogeneity, a stepwise mixing procedure was used: 10 g of PE was first premixed with 100 g of soil, which was then thoroughly mixed with the remaining 890 g of soil. To validate the uniformity of PE distribution, soil samples were randomly collected from different depths of the pots prior to transplantation. The actual PE concentrations were quantified using the FTIR-ATR method described below (Section 2.7), confirming that the variation coefficient was less than 5%, indicating satisfactory homogeneity. Each pot contained 1 kg of soil and one tomato seedling. Soil moisture was maintained at 60% of field water holding capacity by daily gravimetric weighing (weighing pots daily and adding deionized water to reach the target weight) to minimize water potential variations caused by MP addition. The exposure period lasted for 45 days.

At harvest, plants were separated into shoots and roots. Roots were gently rinsed under running tap water to remove bulk soil, followed by three rinses in deionized water. Before scanning, roots were floated in water and cleaned using fine tweezers to remove any remaining particles to ensure high contrast for image analysis. Fresh weights (FW) were recorded immediately. Root morphological traits (total length and surface area) were analyzed using a root scanner (Epson Expression 12000XL) coupled with WinRHIZO software (Regent Instruments Inc., Canada).

In accordance with the method of Xu et al. (2023), Gas exchange parameters, including net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci), were measured on the third fully expanded leaf between 9:00 and 11:00 AM using a portable photosynthesis system (Li-Cor 6800).

Endogenous IAA was extracted and quantified following a modified protocol based on Xu et al. (2025). Briefly, fresh tissue samples (0.1 g) were ground in liquid nitrogen and extracted with 2 mL of cold 80% methanol (4 °C, 12 h). After centrifugation (12,000 rpm, 5 min, 4 °C), the supernatant was purified using a preconditioned C18 solid-phase extraction cartridge. Impurities were removed with 20% methanol containing 0.1% formic acid, and the target IAA fraction was retrieved using 1 mL of 80% methanol.

Chromatographic separation was performed on a Finnigan LC–MS/MS system (Thermo Electron, USA) equipped with a HiQ Sil C18 column (250 mm × 4.6 mm, 5 μm). An isocratic elution was applied using a mixture of methanol and water (80:20, v/v) containing 0.1% formic acid at a flow rate of 1.0 mL min⁻¹. The injection volume was 25 μL. Mass spectrometric detection was conducted using an electrospray ionization (ESI) source in positive ion mode. Key operating parameters were set as follows: spray voltage, 4 kV; capillary temperature, 350 °C; and capillary voltage, 70 V. Quantification was achieved in selected reaction monitoring (SRM) mode by monitoring the transition m/z 174 → 130, with a collision energy of 15 eV. The recovery rate and detection limit of the method were validated prior to analysis.

Microplastic was extracted and quantified following a modified protocol based on Rathore et al. (2023). The uptake and accumulation of PE microplastics in plant tissues were identified and quantified using Fourier Transform Infrared Spectroscopy equipped with an Attenuated Total Reflection accessory (FTIR-ATR). Prior to analysis, root and leaf samples were thoroughly washed, freeze-dried, and ground into a fine powder. To minimize experimental errors arising from heterogeneous distribution, the powdered samples were mixed thoroughly to ensure homogeneity before spectral acquisition.

Spectral measurements were performed in the range of 4000–400 cm⁻¹ using an FTIR spectrometer (Thermo Nicolet iS50). The characteristic absorption peak at 2850 cm⁻¹, corresponding to the symmetric stretching vibration of the methylene group (-CH₂-), was selected as the diagnostic marker for PE identification.

For quantitative analysis, the integrated peak area at 2850 cm⁻¹ was used as the quantitative index. A standard calibration curve was established by plotting the peak areas against a series of known PE concentrations mixed with the plant matrix. The method showed high linearity (R²≥0.99). The PE concentration in the tissue samples (mg kg⁻¹) was calculated by substituting the integrated peak areas of the samples into the established regression equation.

The Bioconcentration Factor (BCF) and Translocation Factor (TF) were calculated as follows: BCF=C_tissue/C_soil; TF= C_leaf/_Croot.

Where C_tissue, C_soil, C_leaf, and C_root represent the PE concentrations in the plant tissue, soil, leaves, and roots, respectively.

Following the method of Xu et al. (2023), lipid peroxidation was assessed by measuring malondialdehyde (MDA) content via the thiobarbituric acid (TBA) assay. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined spectrophotometrically at 560, 470, and 240 nm, respectively.

Data are expressed as mean ± standard deviation (SD) (n=4). Statistical differences were analyzed using one-way ANOVA followed by Duncan’s multiple range test (p < 0.05) using SPSS 26.0. Principal Component Analysis (PCA) was conducted to visualize the overall physiological responses. The relationship between particle size and tissue accumulation was fitted using the Freundlich isotherm model:

Where qe is the PE concentration in tissues, Ce represents the exposure availability (related to specific surface area or inverse of particle size), and the parameters KF and n represent the adsorption capacity and surface heterogeneity, respectively, as applied in plant uptake studies (Li et al., 2005; Liu et al., 2021). Variance Partitioning Analysis (VPA) was performed using the vegan package in R software (version 4.2.3) to quantify the relative contribution of IAA content and antioxidant enzyme activities to the observed variations in PE toxicity across different particle size groups.

Exposure to polyethylene (PE) microplastics resulted in a significant, particle size-dependent inhibition of tomato seedling growth. Phenotypic measurements showed a progressive decline in biomass and root development as particle size decreased from 1–2 mm (T1) to 1–50 μm (T4) (Figure 1).

Compared to the control (CK), PE treatments significantly reduced both shoot and root fresh weights (FW) (p < 0.05). The T4 treatment exhibited the lowest biomass, with reductions of 42.3% in shoots and 43.1% in roots relative to the CK (Figures 1A, B). Root morphological traits followed a similar downward trend. Total root length and root surface area in the T4 group were reduced by 55.1% and 56.4%, respectively, compared to the CK (Figures 1C, D), confirming that the inhibitory effect on root expansion was most pronounced in the smallest particle size treatment.

PE exposure significantly altered the photosynthetic parameters of tomato seedlings, with distinct response patterns observed across different particle sizes (Table 1). Net photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs) decreased progressively as particle size diminished (p < 0.05). The T4 treatment showed the lowest values, with Pn and Gs reduced by 52.8% and 68.8% respectively, compared to the CK.

Notably, the intercellular CO₂ concentration (Ci) exhibited a non-monotonic response. While Ci decreased slightly in the T1 and T2 treatments, it significantly increased in the T3 (290 μmol mol⁻¹) and T4 (320 μmol mol⁻¹) groups (p < 0.05). The observation of decreased Pn accompanied by increased Ci in the T3 and T4 treatments indicates that the photosynthetic inhibition in these groups was primarily attributed to non-stomatal limitations rather than stomatal closure.

The concentration of indole-3-acetic acid (IAA) in both leaves and roots was significantly affected by PE exposure (Figure 2). A consistent size-dependent decline was observed across tissues. In leaves, IAA levels dropped progressively, with the T4 treatment showing a 38.2% reduction compared to the CK (Figure 2A).

In roots, the decrease was similarly pronounced. Root IAA concentrations fell from 69.5 ng/g in the CK to 42.3 ng/g in the T4 treatment, representing a 39.1% reduction (Figure 2B). Statistical analysis confirmed significant differences among treatments (p < 0.05), indicating that the presence of smaller PE microplastics corresponded to lower levels of endogenous auxin in the plant tissues.

PE microplastics were detected in the root and leaf tissues of all treated plants, with uptake and distribution patterns varying significantly by particle size (Table 2). No PE was detected in the control group.

The concentration of PE in tissues increased as particle size decreased (p < 0.05). In the T4 treatment, PE concentrations reached 4.20 mg/kg in roots and 1.20 mg/kg in leaves, which were significantly higher than those in the T1 treatment. Consequently, the translocation factor (TF) increased from 0.125 in T1 to 0.286 in T4. Although all TF values were below 1.0, the higher TF in the T4 group indicates a relatively greater distribution of particles to the aerial parts in the 1–50 μm treatment compared to the larger size treatments.

PE exposure induced significant changes in oxidative stress markers and antioxidant enzyme activities (Figure 3). The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased significantly as the PE particle size decreased (p < 0.05). The highest enzymatic activities were recorded in the T4 treatment, where SOD, POD, and CAT levels exceeded the CK by 1.23-fold, 1.94-fold, and 1.79-fold, respectively.

Concurrently, the content of malondialdehyde (MDA), a marker of lipid peroxidation, rose significantly in the smaller particle treatments. The MDA content in the T4 group was 3.67 times higher than that of the CK (Figure 3D). Despite the significant upregulation of SOD and CAT activities, the MDA content in the T4 treatment surged by 263.4%, indicating that the rate of ROS generation induced by micro-sized particles exceeded the scavenging capacity of the activated antioxidant system.

Principal component analysis (PCA) revealed a distinct separation of treatment groups, with PC1 accounting for 76.6% of the total variance, illustrating a clear trajectory of physiological changes associated with decreasing particle size (Figure 4A). The PCA plot revealed distinct clustering: the Control, T1, and T2 treatments clustered closely, suggesting similar physiological states dominated by physical stress. In contrast, T3 and T4 formed a separate cluster along the PC1 axis, indicating a divergent physiological response characterized by oxidative stress.

The accumulation data for both roots and leaves fitted well to the Freundlich isotherm model (R² > 0.97), describing the non-linear relationship between particle size and tissue concentration (Figure 4B). Furthermore, Variance Partitioning Analysis (VPA) was used to quantify the relative contribution of different physiological factors to the observed variance (Figures 4C, D). In the large particle treatments (T1, T2), IAA content explained the largest proportion of variance (32.4%, p < 0.001). In contrast, in the small particle treatments (T3, T4), antioxidant enzyme activity became the dominant explanatory factor (38.6%, p < 0.001). This shift in explanatory variables indicates distinct physiological response patterns between the large-sized (T1, T2) and small-sized (T3, T4) PE treatments.

Correlation analysis demonstrated that BCF (including Root.BCF and Leaf.BCF) was positively correlated with PE accumulation (Root.PE and Leaf.PE), antioxidant enzyme activities (SOD, CAT, and POD), MDA content, TF, and Ci, but negatively correlated with biomass (Shoot.FW and Root.FW), root morphology (Root.length and Root.area), IAA content (Leaf.IAA and Root.IAA), and photosynthetic parameters (Pn, Tr, and Gs). IAA content was found to be positively correlated with biomass, root morphology, and gas exchange parameters (Pn, Tr, and Gs) and negatively correlated with PE accumulation, BCF, antioxidant activities, and MDA content. Furthermore, TF was positively correlated with Leaf.BCF and Leaf.PE, and Pn was positively correlated with Tr and Gs (Supplementary Figure S1).

Our findings challenge the sufficiency of current ecological risk assessments, which predominantly rely on mass-based concentrations (e.g., mg kg⁻¹) to define pollution thresholds. While the mass concentration in this study was constant (1% w/w) across all treatments, the ecological hazard varied dramatically, with the smallest particles (T4) causing exponentially higher phytotoxicity and bioaccumulation (Yang et al., 2024). This discrepancy underscores that mass concentration alone underestimates the risks posed by micro-sized fragments. Consequently, future regulatory frameworks and environmental monitoring standards must incorporate particle size distribution and particle number concentration as critical parameters to accurately predict the toxicity of microplastic pollution (Yu and Hu, 2022).

Furthermore, these results provide practical insights for plastic management in agro-ecosystems. The transition from macro- (T1, T2) to micro-sized (T3, T4) particles represents a critical threshold where the mode of toxicity shifts from physical soil alteration to severe cellular oxidative stress and hormonal disruption. This highlights the urgent necessity of removing agricultural mulch films before they degrade into the highly toxic < 50 μm size range (Sander, 2019). Effective management strategies should prioritize the retrieval of macro-plastic residues to prevent the formation of these vectors for contaminant transfer, thereby safeguarding crop productivity and minimizing the risk of trophic transfer to the human food chain (Macheca et al., 2024).