# Bridging lab and field: Tracking environmentally relevant nanoplasics in crops using Py-GC/MS

Nataliia Ryzhenko, Guillaume Pecheul, Aline Dia, Lionel Dutruch, Maxime

Pattier, Mathieu Pédrot, Julien Gigault, Mélanie Davranche

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Nataliia Ryzhenko, Guillaume Pecheul, Aline Dia, Lionel Dutruch, Maxime Pattier, et al.. Bridging lab and field: Tracking environmentally relevant nanoplasics in crops using Py-GC/MS. Journal of Hazardous Materials, 2025, 947, pp.139693. 10.1016/j.jhazmat.2025.139693. insu-05237107

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Bridging lab and field: Tracking environmentally relevant nanoplastics in crops using Py-GC/MS

Nataliia Ryzhenko, Guillaume Pecheul, Aline Dia, Lionel Dutruch, Maxime Pattier, Mathieu Pédrot, Julien Gigault, Mélanie Davranche

<figure><img src="image_2.png" /></figure>

PII: S0304-3894(25)02612-3

DOI: https://doi.org/10.1016/j.jhazmat.2025.139693

Reference: HAZMAT139693

To appear in: *Journal of Hazardous Materials*

Received date: 25 May 2025

Revised date: 26 August 2025

Accepted date: 27 August 2025

Please cite this article as: Nataliia Ryzhenko, Guillaume Pecheul, Aline Dia, Lionel Dutruch, Maxime Pattier, Mathieu Pédrot, Julien Gigault and Mélanie Davranche, Bridging lab and field: Tracking environmentally relevant nanoplastics in crops using Py-GC/MS, *Journal of Hazardous Materials*, (2025) doi:https://doi.org/10.1016/j.jhazmat.2025.139693

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
# Bridging lab and field: Tracking environmentally relevant nanoplasics in crops using Py-GC/MS

Nataliia Ryzhenko<fnref n="1" />, Guillaume Pecheul<fnref n="1" />, Aline Dia<fnref n="1" />, Lionel Dutruch<fnref n="1" />, Maxime Pattier<fnref n="1" />, Mathieu Pédrot<fnref n="1" />, Julien Gigault<fnref n="1" />,<fnref n="2" />, and Mélanie Davranche<fnref n="1" />

## Abstract.

The increasing presence of nanoplasics (NPs) in terrestrial environments raises concerns about their bioavailability and potential impacts on crops. This study investigates the uptake and translocation of environmentally relevant polystyrene nanoplasics (eNPs-PS) in *Hordeum vulgare* L. via soil. eNPs-PS were produced from aged polystyrene (PS) foam using a top-down approach and characterized by Nanoparticle Tracking Analysis (NTA) and Scanning Electron Microscopy (SEM), revealing a polydisperse population with a mean size of 179.8 ± 2.8 nm. A novel acetone-assisted Pyrolysis-Gas Chromatography-Mass Spectrometry (Py-GC/MS) technique was developed for quantifying PS in plant matrices tissues. Barley cultivated in eNPs-PS -contaminated soil (228.9 mg kg⁻<fnref n="1" />), and results demonstrated substantial accumulation in roots (97.6 ± 39.4 µg g⁻<fnref n="1" />) and limited translocation to shoots (30.1 ± 10.1 µg g⁻<fnref n="1" />; translocation factor (TF) = 0.31). These findings provide the first experimental evidence of root uptake and systemic movement of environmentally relevant nanoplasics in plants. The observed restricted translocation highlights a potential shift in risk from food chain contamination to rhizospheric exposure, with possible implications for soil organisms and microbial symbioses. This work underscores the need to include realistic nanoplasics in environmental risk assessments and supports the development of adapted regulatory test protocols.

**Environmental implications.** This study confirms that environmentally realistic polystyrene nanoplasics are bioavailable to crop plants through soil exposure, accumulating mainly in roots due to their irregular shape and interactions with the soil matrix. Their presence in edible tissues, though limited, signals potential food safety risks. Unlike synthetic nanobeads, eNPs-PS reflect real-world behavior and persistence, underscoring the need to revise current assessment frameworks. This approach offers a robust basis for evaluating nanoplasics risks and guiding regulatory policies in agroecosystems.

## Highlights

* Aged polystyrene nanoplasics were prepared for environmentally relevant exposure

<footnote n="1">¹ Univ. Rennes, CNRS, Géosciences Rennes - UMR 6118, F-35000 Rennes, France</footnote>

<footnote n="2">² TAKUVIK Laboratoy, UMI3376 CNRS/Université Laval, Québec, Canada</footnote>
* New acetone-assisted protocol developed for quantifying nanoplastics in plants

* 228.9 µg g⁻¹ in soil leads to 97.6 µg g⁻¹ nanoplastic accumulation in barley roots

* Nanoplastics show roots uptake with a translocation factor of 0.31 to shoots

* Demonstrated method performance in realistic soil-plant systems

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**Key words:** environmentally relevant polystyrene nanoplastics (eNPs-PS), *Hordeum vulgare* L., bioaccumulation, translocation

# 1. Introduction.

With plastic pollution infiltrating even the most remote soils [1], concerns are mounting over the invisible threat posed by NPs to trophic chain and human health. NPs pollution has emerged as one of the most pressing environmental issues worldwide [2–4]. Among plastic-derived contaminants, NPs—typically defined as particles smaller than 1 µm—originate from the progressive degradation of larger plastic waste and microplastics [5,6]. Due to their small size and colloidal behavior, NPs can readily migrate into diverse environmental compartments, including aquatic systems, soils, and biota [7–9]. However, there is still a lack of data on the actual uptake of environmentally aged NPs by plants under realistic soil exposure scenarios.

The bioavailability and translocation of NPs in the soil-plant system are controlled by multiple factors, including polymer type, particle size and morphology, surface properties, soil pH, organic matter content, and plant species-specific physiology [6,10]. Experimental studies demonstrated that polystyrene NPs of 50 to 150 nm can be taken up by roots and translocated to the aerial parts of species such as *Daucus carota* L., *Arabidopsis thaliana* L., and *Hordeum vulgare* L. confirming their potential to accumulate within plant
biomass [2,8,11]. Once inside plant tissues, nanoplastics may bioaccumulate and exert toxic effects on living organisms, notably by disrupting physiological functions. Their transfer to herbivores through consumption introduces them into the trophic chain, thereby posing risks to ecosystem health and food safety [2,11,12].

Recent studies have increasingly demonstrated that nanoplastics pose a significant risk to human health and biological systems in general. Due to their small size, nanoplastics can cross biological barriers, accumulate in tissues, and interfere with cellular functions. Documented toxic effects include oxidative stress, mitochondrial dysfunction, inflammation, genotoxicity, reducing male fertility, and metabolic disturbances in human [13–17].

Once nanoplastics reach the rhizosphere, their interactions with plant roots are governed by complex physical–chemical and biological processes. Beyond size and surface charge, factors such as colloidal stability and root exudate composition influence their mobility and bioavailability. Uptake pathways include apoplastic transport, endocytosis, and possible penetration through lateral root junctions, enabling internalization and translocation [18]. The nanoscale dimension, large surface area, and high colloidal stability of nanoplastics enhance their ability to interact with biological membranes, cross physiological barriers, and trigger biological responses [19].

Hydrophobic nanoplastics such as PS and polyethylene (PE) tend to adsorb strongly to root surfaces and exhibit limited root-to-shoot translocation, primarily due to their low compatibility with aqueous vascular pathways [20–22]. In contrast, nanoplastics with more hydrophilic properties, such as polyamide (PA) and polypropylene (PP), show higher mobility within plant tissues, although their translocation remains substantially lower than that of inorganic nanoparticles. Inorganic nanoparticles such as silver, titanium dioxide, and cerium oxide etc. demonstrate significantly higher translocation rates owing to their ionic nature, solubility, and surface charge [23–26]. Additionally, recent studies emphasize that soil matrices further reduce nanoplastic mobility compared to hydroponic systems, especially for hydrophobic polymers, reinforcing the role of environmental conditions in determining nanoparticle fate [10,20,21,27–29]. This highlights the importance of considering polymer-specific properties in the environmental risk assessment of nanoplastics in soil–plant systems.

Amongst plastics, those PS, are widely used in packaging and construction materials, especially in the form of expanded PS (EPS) foams. In Europe, PS consumption reached 2,108 kt with a continued annual growth
[30]. It is thus expected that weathered PS will increasingly contribute to environmental NP pollution. Despite growing concern, the environmental behavior of PS-derived NPs in agroecosystems remains poorly understood, and no regulatory thresholds currently exist for their presence in plant tissues. A major limitation of current research lies in the use of commercial PS nanobeads—monodisperse, spherical, and highly idealized particles [5,9,12,31,32] —rather than environmental NPs derived from real plastic waste [8,33,34]. Environmental NPs are typically polydisperse, irregularly shaped, and carry surface functionalities acquired during environmental weathering (UV, oxidation, biofilm formation), affecting their interactions with soil and living organisms [31,35–37]. This lack of realism hinders our ability to predict actual NPs behavior and fate in soil-plant systems and ultimately limits the reliability of risk assessments. Furthermore, despite mounting evidence of NPs uptake by plants under hydroponic conditions, no study has yet quantified environmentally aged nanoplastics in crops exposed via soil—the most realistic exposure pathway in agriculture.

To narrow this knowledge gap, a novel acetone-based extraction technique was developed for plant tissues, coupled with Py-GC/MS. Plants were exposed to eNPs-PS in soil using the RHIZOtest system, a semi-controlled device allowing soil contact while avoiding root damage.

This study provides the first experimental quantification of environmentally relevant PS-NPs in plant tissues under soil exposure. Our results not only confirm the uptake and partial translocation of eNPs-PS in a crop plant but also, establish a methodological framework for future studies. These findings contribute significantly to closing the current knowledge gap regarding the environmental fate of realistic NPs in terrestrial food webs.

## 2. Experimental.

**Environmental eNPs-PS production.** The production of eNPs-PS was inspired from the protocol developed by Blancho et al. [38] (Fig. 1).

<figure><img src="image_4.png" /><figcaption>Fig. 1 Steps in the preparation of eNPs-PS</figcaption></figure>
Polystyrene insulation foam, approximately 15 years old, was collected from household waste in February 2024. A preliminary identification of the PS was conducted using Py-GC/MS based on Styrene detection (m/z 104). The PS foam was then crushed, and organic matter (OM) was removed using 3% H₂O₂ heated at 40°C for 1 h. The cleaned plastic fragments were then mixed with ultrapure water and ground using a blender (Waring commercial laboratory blender, 18000 tr min⁻¹) for 1h. The resulting suspension was exposed to UV-C for 1h using a RMR-600 Rayonet reactor. Nanoparticles were then isolated from macro and microparticles through a sequential filtration at 100 µm (metallic sieve, VWR), 3 µm and 1.2 µm (Versapor acrylic copolymer, PALL). The solution <1.2 µm containing eNPs-PS was then concentrated using an ultrafiltration device (Amicon Stirred Cell, 400 mL) equipped with 20 kDa membrane (PES, NADIR UP020 P). These steps were repeated until the eNPs-PS concentration reached a satisfactory level for further experiments. The exact concentration of produced eNPs-PS was quantified by Py-GC/MS (Styrene detection, m/z 104) using a calibration curve prepared with 200 nm PS-NPs standard (Polybead® Carboxylate Microspheres, Polyscience) at concentrations ranging from 1 to 50 mg L⁻¹. The number, size, and distribution of eNPs-PS were analyzed using Nanoparticle Tracking Analysis (NTA) (Nanosight LM10 device system, Malvern Panalytical) at Institut des Sciences Chimiques de Rennes (ISCR, France) (Table S1).

**Plant exposition experiments.** This experiment was conducted using RHIZOtest devices (Fig. 2) (https://rhizotest.cirad.fr/en). As described by the RHIZOtest developers [39] and demonstrated in previous studies [40–42], the system allows plant roots to maintain direct physical contact with soil particles, enabling sorption–desorption interactions at the root–soil interface. This setup minimizes confounding factors such as soil heterogeneity, fluctuating oxygen levels, and inconsistent water distribution while maintaining environmentally relevant root–soil interactions.
<figure><img src="image_5.png" /><figcaption>**Fig 2. Components of the device RHYZOtest**</figcaption></figure>

In a first stage, seeds were germinated in Petri dishes for 7 days. During the second stage, the germinated seeds were transferred to hydroponic boxes containing Hoagland solution for 5 D. In the final stage, the plants were placed directly into the RHIZOtest system for another 14 D. The experiment was conducted under controlled conditions in a climatic chamber (Phytotron, Weisstechnik) with a day/night temperature of 24/20°C and 70% humidity. Hordeum vulgare L.

was grown for 14 days.

After harvesting, the plants were dried in a drying oven (EU53 T+TS, Prolabo Astel, France) at a stable temperature of 40 °C with forced air circulation, under low-humidity conditions (≤30%), until constant weight was achieved. Strictly controlled conditions were upheld during all phases of the RHIZOtest procedure to minimize the impact of external variables on the bioavailability of eNPs-PS to plants.

Before application, to prevent NPs aggregation, eNPs-PS suspensions were sonicated for 10 min and subsequently divided into three aliquots for application in the experimental setup. Each RHIZOtest device contained a total of 6.8 g of soil. The soil is a Cambisol collected at Cléry-Saint-André (Loiret, France). Soil geochemical composition was performed by the Service d'Analyse des Roches et des Minéraux (SARM - Vandoeuvre-les-Nancy, France) [43]. The soil characteristics are provided in Table S2. Although Cambisol was selected as a representative agricultural soil, we recognize that variations in soil texture, organic matter content, and mineralogy may influence nanoplastics behavior and plant uptake. Future studies should explore a broader range of soil types to validate the generalizability of these findings and better reflect field heterogeneity.

To date, there are no quantitative data on the environmental concentrations of nanoplastics (<1µm) in soils. While several studies have reported their detection in soil [9], no validated concentration ranges are currently available. This lack of baseline information limits the design of environmentally realistic exposure scenarios and highlights the need for testing across a broader range of concentrations. Addressing this
challenge will require optimizing the current production process, which took nearly three months to generate sufficient material for a single exposure condition with three replicates. In such context, eNPs-PS were introduced into the soil surface at a concentration of 228.92 mg kg⁻¹ to simulate environmental NPs contamination. The concentration of 228.92 mg kg⁻¹ used in this study reflects the amount of eNPs-PS that could be generated through a multi-step production protocol. This high-exposure scenario, while lacking direct field-based parallels, allowed the detection and quantification of eNPs-PS nanoplastics in plant tissues. It also enabled comparison with previous experiments using synthetic nanobeads at similar concentrations [8], which exhibited high concentration in above-ground part of plants. The experimental design included both a control (uncontaminated) and a contaminated (eNPs-PS -contaminated soil) soils, with three biological replicates per modality. A total volume of eNPs-PS suspension was 300 mL with the concentration 15.5 ± 0.4 mg L⁻¹. The calculated eNPs-PS mass added per Rhizotest was 1.6 mg, namely 228.9 mg eNPs-PS kg⁻¹ of soil.

The translocation factor (TF) from roots to shoots was calculated as:

$$TF = \frac{Conc_{shoots}}{Conc_{roots}} \quad (1)$$

With Concₜhoots – concentration of eNPs-PS in shoots, µg g⁻¹ and Concₜoots – concentration of eNPs-PS in roots, µg g⁻¹.

**eNPs-PS detection and quantification in plants.** The eNPs analysis in roots and shoots was carried out by Py-GC/MS (Pyrolyzer 3030D Frontier Lab, Japan; gas chromatography-mass spectrometry 7890 B Agilent Technologies; MS 5977 Agilent) (Table S1). Py-GC/MS was selected as the analytical technique for the quantification of eNPs-PS due to its well-established reliability for mass-based polymer-specific detection in complex environmental and biological matrices. Unlike alternative detection approaches that rely on fluorescent, isotopic, or metallic labeling (e.g., hyperspectral imaging with deep learning) or single-particle ICP-MS with isotopic tagging [44,45], Py-GC/MS enables the quantification of eNPs-PS. This method is independent of particle morphology, size, or surface modifications, and provides matrix-tolerant, polymer-specific quantification without requiring extensive pre-treatment or particle labeling, which is impractical for real-world environmental samples. Samples were prepared by digesting 25 mg dried shoot or root matrix with acetone, followed by 30 min of sonication. Acetone was chosen as a mild solvent to protect GC-MS components (i.e., minimize wear or contamination of injector liners, columns, and detectors) and for its effectiveness in disrupting plant tissues and solubilizing polystyrene [13,46–48]. 160 µL of acetone extract
was dried at 45°C in a pyrolysis cup (Frontier Laboratories) and subsequently pyrolyzed at 600°C with a 20-min slope. Styrene peak areas were normalized to the dry plant mass introduced into the pyrolysis cup to ensure accurate comparison between samples. Pyrolysis products were separated via gas chromatography using the Rxi-5Sil MS column (60 m × 0.25 mm × 0.25 µm, RESTEK). Ionization was set at 70 eV with a m/z range of 40 to 350, and an analysis rate of 4.5 scans s⁻¹. Pyrolyzates were identified by comparing the mass spectra with the NIST Library using the MSD CHEMSTATION software.

The eNPs-PS quantification was performed using the m/Z=104 of Styrene (Fig S1, Table S3). However, Styrene (m/z 104) is also a pyrolyzate of OM and plant matrices [49,50] and then always present in roots and shoots matrices of Hordeum vulgare L of the control.

The calibration curves (Figure S2a for roots, S2b for shoots) were constructed by spiking uncontaminated Hordeum vulgare L. roots and shoots with 50 nm carboxylated PS nanobeads (Polysciences) standards at concentrations ranging from 0.1 to 2.5 µg per mg of dry tissue (Table S4). Spiked samples were processed using the same protocol as experimental samples, and acetone extraction. The Styrene peak increased proportionally with the PS nanobeads concentration, confirming the method sensitivity. To correct for background interference, the Styrene signal from control (non-spiked) plant material was subtracted from the total signal in spiked samples. Blank matrices were included in each analytical series and subjected to identical preparation and analysis, ensuring accurate baseline correction and minimizing matrix effects. This approach enabled robust, linear calibration and reliable quantification of PS-derived Styrene in plant tissues. The average PS recovery using this method was 91% (Table S5).

The relation between concentration of eNPs-PS (µg mg⁻¹) and Styrene peak area(m/Z=104) was calculated as:

$$Conc PS_{sample} = \frac{St_{sample} - St_{blank}}{a}, \quad (2)$$

With Conc PS sample, µg mg⁻¹ – concentration of eNPs-PS in sample (plant matrix); Stsample – Styrene peak area (kcps) in the sample; Stblank – Styrene peak area (kcps) in the control sample (blank); a-slope.

**Microscopic Observations.** The effective presence of eNPs-PS and nanobeads onto roots were observed by Scanning Electron Microscopy (SEM) (CMEBA, Rennes, France) using a JEOL JSM 7100 FEDS EBSD (Oxford) with resolution 2 nm at 1kV, 1.6 nm at 5 kV, 1.2 nm at 15 kV.

### 3. Results and discussion.
**eNPs-PS description.** To mimic field conditions, eNPs-PS were produced from aged PS foam using a modified version of the Blancho et al. protocol [38]. To prevent errors caused by particle aggregation, eNPs-PS were ultrasonicated (~10 min, 40 kHz, USC – TH, VWR) before each analysis. The e-NPs-PS are polydisperse with an average size of 179.8 ± 2.8 nm (Fig. 3; Table 1). 80% of eNPs-PS have a size between 125.1 ± 0.8 nm and 248.6 ± 6.7 nm. The concentration of eNPs-PS was estimated at 3.36 ± 0.06 × 10⁹ particles ml⁻¹. The size–intensity and concentration distribution demonstrate a unimodal size distribution with a peak centered around ~180 nm.

<figure><img src="image_6.png" /><figcaption>Fig 3. Particles distribution in the eNPs-PS standard prepared solution</figcaption></figure>

<table><caption>Table 1 Particles distribution and concentration in the eNPs-PS standard solution</caption><thead><tr><th></th><th>Merged Data</th></tr></thead><tbody><tr><td>Mean</td><td>179.8±2.8 nm</td></tr><tr><td>Mode</td><td>142.7±6.9 nm</td></tr><tr><td>SD</td><td>60.6±0.7 nm</td></tr><tr><td>D10</td><td>125.1±0.8 nm</td></tr><tr><td>D50</td><td>166.8±2.6</td></tr><tr><td>D90</td><td>248.6±6.7</td></tr><tr><td>Concentration</td><td>33.36e+09 +/- 6.54e+07 particles/ml</td></tr></tbody></table>
The scatter plot of light intensity versus particle size showed a broad and reproducible distribution across all replicates. Intensity values fell within the expected range for eNPs-PS, confirming the presence of individual particles with stable refractive properties and minimal aggregation.

The concentration of eNPs-PS in the prepared standard suspension was determined as 15.5 ± 0.4 mg L⁻¹ using Py-GC/MS with Styrene detection (m/z 104) (Table S3). Quantification was performed based on a calibration curve constructed with 200 nm PS-NPs polybeads (Polybead® Carboxylate Microspheres, Polysciences), selected due to their closer size similarity to the ePS-NPs, as confirmed by NTA. The calibration yielded a strong linear correlation (R² = 0.9628) between the polystyrene mass and the Styrene peak area. The resulting pyrograms confirmed the presence of polystyrene in the solution and demonstrated a chemical profile consistent with the standard PS nanobeads (Fig. S1b).

SEM imaging of eNPs-PS showed polydisperse particles with irregular shapes and rough, uneven surfaces, consistent with mechanical fragmentation processes (Fig. 4a). By contrast, commercial PS nanobeads exhibited uniform spherical morphology, smooth surfaces, and highly consistent diameters (Fig 4b). These differences underscore the structural heterogeneity and environmental realism of eNPs-PS as compared to synthetic nanobead standards.

<figure><img src="image_7.png" /><figcaption>Fig 4. Scanning Electron Microscopy (SEM) images of (a) environmentally relevant polystyrene nanoplastics (eNPs-PS) produced from aged foam, showing polydispersity and irregular morphology; and (b) synthetic PS nanobeads (50 nm), showing monodisperse spherical particles.</figcaption></figure>
**eNPs in plants: bioaccumulation and translocation.** SEM observation displayed presence of numerous nanoparticles of varying shapes, sizes, and surface textures on the root epidermis for barley exposed to eNPs-PS (Fig. 5 a). They seem to be adsorb onto or partially embedded within the root surface, suggesting possible interactions between eNPs-PS and the root tissue, likely influenced by root exudates or cell wall properties [2,22,51].

By contrast, the control root demonstrates a smooth and particle-free surface, with no observable
nanoplastic-like structures (Fig. 5 b). These micrographs provide visual evidence that eNPs-PS can attach to
and accumulate on root surfaces, supporting the quantitative findings of PS uptake. Furthermore, the
asymmetrical shape and size of the observed particles are consistent with the characteristics of eNPs-PS.

<figure><img src="image_8.png" /><figcaption>a)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>b)</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>c)</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>d)</figcaption></figure>
Fig. 5. SEM images of *Hordeum vulgare* L. tissues: Roots: (a) exposed to eNPs-PS; (b) control; Shoots: (c) exposed to eNPs-PS; (d) control. Red circles indicate eNPs-PS adsorbed on or embedded within the tissue surface

To investigate whether nanoplastics were also present in the aerial parts, SEM analysis was performed on freeze-dried leaf tissues. Although fewer particles were detected compared to roots, several nanosized objects consistent with eNPs-PS were observed on leaf surfaces in exposed samples (Fig. 5c), while control leaves showed no such features (Fig. 5d). Notably, the morphological characteristics of nanoplastics differed between roots and shoots. In roots, particles appeared with irregular geometries, including angular edges and sharp contours, reflecting the heterogeneous nature of environmentally aged plastics. In contrast, particles observed in leaves were predominantly rounded or near-spherical in shape.

This morphological distinction may suggest a selective translocation mechanism: only nanoplastics with smoother, more compact shapes may successfully pass through the plant's internal transport barriers, while irregular or angular particles are more likely retained in root tissues. This hypothesis aligns with the observed low translocation factor (TF = 0.31) and supports the idea that particle shape—alongside size and surface chemistry—may influence the internal mobility of nanoplastics in plants.

The concentration of eNPs-PS in the roots of *Hordeum vulgare* L. reached 97.6 ± 39.4 µg g⁻¹ for an initial spike of 229 mg kg⁻¹ in soil, namely 43% of the initial eNPs-PS concentration, indicating substantial uptake and mobility of eNPs-PS inside the plant (Table S6). The uptake by root occurs despite their asymmetric shape and polydispersity (Fig. 5a), but this shape may also explain this accumulation. According to NTA, NPs ranging from 50 to 60 nm represent 0.58% of the total eNPs-PS stock suspension. Given the applied dose of 228.9 mg kg⁻¹ of eNPs-PS in soil, 1.30 mg kg⁻¹ of soil is estimated to contain 50 to 60 nm eNPs-PS. Although this represents a minor proportion of the total eNPs-PS input, NPs (50-60nm) are widely recognized as the most bioavailable. The PS concentration in shoots reached 30 ± 10 µg g⁻¹, confirming root uptake and translocation. Assuming that only the 50–60 nm eNPs-PS is efficiently translocated to shoots; PS theoretical contribution would be limited to ~0.17 µg g⁻¹. This estimated value is over 2 300 times lower than PS concentrations reported in our previous work using 50-nm PS nanobeads [8] where shoot accumulation reached up to 400 µg g⁻¹ following exposure to 300 mg kg⁻¹ in soil. These results illustrate the markedly lower bioavailability of eNPs-PS as compared to idealized monodisperse nanobeads. Differences in bioavailability between PS
nanobeads and eNPs-PS likely arise from multiple factors, including size, shape, surface charge, chemical functionality, and degradation state—all of which influence aggregation, mobility in soil, and plant uptake.

PS nanobeads, with their monodisperse size, smooth spherical morphology, and manufacturer-defined surface properties, do not reflect the complexity of environmental NPs [38] and may interact more readily with plant tissues. By contrast, eNPs-PS display heterogeneous sizes and shapes, irregular surfaces, and may contain degradation by-products, additives, or environmental contaminants [5,35]. Their surfaces can also adsorb soil colloids and organic matter [35,52], potentially reducing their effective bioavailability to plant roots. Nevertheless, the successful detection of eNPs-PS in root tissues highlights their capacity to interact with and penetrate plant surfaces despite their structural complexity, reinforcing the ecological relevance of using environmentally realistic particles. Their internalization may involve several mechanisms, including apoplastic transport, endocytosis, or passage through discontinuities in the root epidermis [2,22,53,54]. Their successful internalization may be facilitated by their average size (~180 nm), which falls within the range compatible with apoplastic transport and vesicle-mediated uptake mechanisms [53,55]. Once inside root, translocation through the xylem can occur, facilitating movement into aerial parts. Moreover, the asymmetrical shape of eNPs-PS may allow for alternative adhesion or entrapment mechanisms at the rhizoplane that differ from monodisperse, spherical PS nanobeads [51]. By contrast, the interaction with root surface exudates, soil OM, and root-associated microbial communities could influence these uptake dynamics [2,22,51,56].

The concentration of eNPs-PS measured in *Hordeum vulgare* L. shoots was 30.1 ± 10.1 µg g⁻¹ (Table S6). Although this value falls slightly below the established limit of quantification (LOQ), it consistently exceeded background levels and was significantly lower than concentrations measured in roots, supporting the occurrence of actual uptake and partial translocation. The calculated translocation factor (TF) was 0.31±0.06 (Table S6), confirming a limited ability of eNPs-PS to move from root to shoot. This suggests that a substantial fraction of the eNPs-PS remains sequestered in the root system.

The limited translocation observed likely results from multiple interacting factors, including both physicochemical properties of eNPs-PS and biological barriers within plant tissues. According to recent literature [10, 20, 21, 28, 29], several mechanistic pathways have been proposed for nanoparticle uptake and translocation in plants [22]. Initially, nanoplastics may adsorb onto the root surface through hydrophobic
interactions, electrostatic forces, and entrapment within the mucilage or cell wall matrix. Entry into root tissues can occur via physical discontinuities such as lateral root emergence zones, mechanical cracks, or damaged epidermis, allowing passive penetration of larger particles. Additionally, active cellular uptake via endocytosis has been documented for nanoparticles ranging from 100 to 300 nm, which aligns with the average size of eNPs-PS (~180 nm) used in this study. Once internalized, further movement through the apoplastic pathway is likely constrained by the Casparian strip [57], a hydrophobic barrier in the endodermis, which redirects particles toward symplastic transport mechanisms requiring passage through plasmodesmata or further endocytic processes [20,56]. The inherently hydrophobic and irregularly shaped nature of eNPs-PS likely exacerbates retention within the root apoplast and limits their compatibility with aqueous vascular pathways such as the xylem.

Asymmetric and polydisperse eNPs-PS tend to accumulate at the root-soil interface and within root tissues, likely due to a combination of electrostatic interactions and physical entrapment within apoplastic barriers such as the Casparian strip [57]. By contrast to synthetic nanobeads—spherical and monodisperse as a result of manufacturing processes—environmental nanoplastics exhibit irregular shapes and heterogeneous surface properties that better reflect particles encountered in real-world conditions. This structural complexity may hinder their mobility within plant vascular tissues. Furthermore, plants may actively limit the upward translocation of such xenobiotics through defense mechanisms aimed at protecting photosynthetic tissues from potential damage [58,59].

Beyond uptake differences between nanobeads [8] and eNPs-PS, root and shoot concentrations showed notable variability across replicates, likely due to root architecture, microenvironmental heterogeneity, and uneven particle distribution despite homogenization. Although the translocation factor (TF) was low (0.31), the consistent detection of eNPs-PS in shoots confirms their ability to move through vascular tissues, albeit less efficiently when particles exhibit irregular shapes and complex surface chemistries. The Casparian strip—a hydrophobic barrier in the endodermis [60,61], —may limit apoplastic transport and favor symplastic pathways such as endocytosis, contributing to compartmental differences. Species-specific traits, including exudation profiles and endodermal permeability, may further modulate nanoplastic mobility [22,54]. Given the environmental relevance of the eNPs-PS used here, these findings highlight the need to investigate other plant species and exposure contexts to determine whether such physiological barriers consistently influence
nanoplastic uptake and translocation. Our observations are strongly supported by recent findings [62], which show that nanoplasics with more environmentally relevant properties—such as surface oxidation, roughness, and polydispersity—exhibit higher attachment efficiencies than synthetic nanobead models. This behavior promotes both homoaggregation and heteroaggregation with soil components and biological surfaces, significantly reducing nanoplastic mobility in the soil-plant continuum. The aggregation tendency is further enhanced under natural ionic conditions and in the presence of soil organic matter [9,63,64]. These processes explain the predominant accumulation of eNPs-PS in roots observed in this study and their limited translocation to aerial plant parts.

The results of this study may contribute to improving environmental risk assessments in agroecosystems contaminated by nanoplasics. Based on our findings, it would be advisable to apply more realistic approaches that reflect the complexity of environmental particles and exposure scenarios. In this context, the following considerations may help strengthen future risk evaluation strategies: (i) developing and using environmentally aged, polydisperse nanoplasics—including, but not limited to, polystyrene—as standardized test materials derived from real waste sources; (ii) implementing dose-response experimental designs to evaluate concentration-dependent effects on plant and soil systems; (iii) prioritizing soil-plant exposure pathways over hydroponic models to better simulate natural conditions; (iv) incorporating sublethal endpoints related to root function, microbial symbioses, and nutrient dynamics; and (v) employing analytical techniques such as Py-GC/MS that are compatible with complex biological matrices.

**Environmental implications.** This study demonstrates that environmentally realistic polystyrene nanoplasics (eNPs-PS) can be taken up by crop plants under soil exposure, confirming their bioavailability in agroecosystems. Unlike synthetic nanobeads, eNPs-PS exhibit asymmetrical shapes and complex surface chemistries that better reflect the diversity of nanoplasics present in contaminated soils. Their detection in edible plant tissues, even at low levels, highlights a potential pathway for entry into terrestrial food webs and raises concerns about food safety in the absence of regulatory thresholds. However, the limited translocation observed suggests that most eNPs-PS remain confined to the root system, likely due to shape irregularity, polydispersity, and interactions with the soil matrix. As a result, nanoplasics may act as localized vectors of co-contaminants (e.g., trace metals, organic pollutants) within the rhizosphere, rather than contributing to widespread aerial contamination. Ecologically, this retention shifts the focus of concern toward soil
organisms closely associated with roots—such as symbiotic fungi, rhizobacteria, and soil fauna—that may
experience chronic exposure to nanoplastics and their adsorbed contaminants. The rhizosphere thus
emerges as a key zone for nanoplastic-biota interactions, where further studies are needed on soil health,
microbial functions, and biogeochemical processes. In particular, the persistence of eNPs-PS in the
rhizosphere may alter nutrient bioavailability or disrupt microbial symbioses, with potential long-term
consequences for plant-soil interactions and ecosystem functioning.

Regulatory frameworks must now consider the behavior of environmentally derived nanoplastics, as synthetic nanobeads may overestimate plant uptake and underestimate soil persistence. Future research should address long-term accumulation in soils, potential remobilization, and their influence on the fate of associated contaminants under realistic exposure conditions.

Ultimately, the approach developed here—combining realistic particle production, soil exposure, and robust quantification—provides a solid foundation for improving environmental assessments and informing regulatory decisions on nanoplastic pollution in agroecosystems.

**4. Conclusions and perspectives.**

This study provides the first experimental evidence that environmentally aged polystyrene eNPs-PS,
produced from real insulation foam via top-down mechanical degradation, can be effectively extracted and
quantified in crop plants following soil exposure. The particles displayed irregular morphology, polydispersity,
and surface roughness, offering a realistic alternative to synthetic nanobeads for environmental risk
assessment.

To enable accurate quantification in biological matrices, we developed a new analytical approach combining
acetone-assisted extraction and Py-GC/MS, optimized for complex plant tissues. This method demonstrated
good analytical consistency across tested concentrations, supporting its potential use for trace analysis of
nanoplastics in environmental and food-related applications.

Our results show that eNPs-PS can accumulate substantially in barley roots (97.63 ± 39.40 µg g⁻¹) and, to a lesser extent, translocate to shoots (30.1 ± 10.1 µg g⁻¹), yielding a translocation factor of 0.31. This limited systemic movement suggests that root zones act as primary retention sites, particularly for particles mimicking environmentally relevant features. Although partial shoot uptake was observed, the low translocation efficiency reduces the direct risk of food chain contamination while raising concern for
rhizospheric exposure and potential impacts on soil organisms [21]. The observed accumulation of
nanoplastics in roots suggests potential interactions with rhizospheric microbes [65]. Although microbial
community responses were not directly assessed in this study, previous work has shown that nanoplastics
may alter microbial diversity, enzyme activity, and nutrient cycling [27,54,56]. Future studies should combine
molecular microbiology tools (e.g. enzymatic assays) with ecological endpoints to better understand how
environmentally realistic nanoplastics affect plant-microbe interactions and soil ecosystem functions.

By demonstrating the feasibility of using aged nanoplastics in plant exposure scenarios, this work bridges a
critical gap between laboratory models and field-relevant conditions. It underscores the need to include
realistic nanoplastics in regulatory testing and highlights their dual role—as both environmental pollutants
and potential vectors for co-contaminants—in agroecosystems.

While *Hordeum vulgare* L. was selected as a representative cereal species, we recognize that nanoplastics
uptake and transport may vary widely among plant types. Future efforts should extend to other crops and
wild plant species, soil types, and co-exposure contexts, and focus on developing methods for quantifying
nanoplastics in soil to better assess their accumulation, mobility, and long-term fate as well as characterize
the species-specific responses and improve ecological risk assessments at the agroecosystem level. Further
long-term field studies are essential to assess the accumulation dynamics of nanoplastics in plants and their
potential impacts on plant health, soil ecosystems, and agroecosystem functioning under realistic
environmental conditions.

Acknowledgments

This work was supported by (i) the PAUSE Program, Collège de France "How do nanoplastic and associated organic and inorganic pollutants affect plants: Focus on rhizospheric processes", Geoscience-Rennes 1, France (2 years funded), cofounded by the Foundation Rennes 1 and the Rennes Metropole, (ii) theANR-UKRAINE: "How do nanoplastic and inorganic pollutants affect plants?", Geosciences, University of Rennes, France, (iii) the Programme ANR non-thématique: PRC. "Environmental dissemination of NaNOplastics: Impact of pedoCLImatic conditions and Consequences on associated POLLUTANTS", NANOCLIPO and (iv) the Observatoire des Sciences de l'Environnement de Rennes Project, Nanoplastics in plants.

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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.

Environmental implications.

This study confirms that environmentally realistic polystyrene nanoplastics are bioavailable to crop plants through soil exposure, accumulating mainly in roots due to their irregular shape and interactions with the soil matrix. Their presence in edible tissues, though limited, signals potential food safety risks. Unlike synthetic nanobeads, eNPs-PS reflect real-world behavior and persistence, underscoring the need to revise
current assessment frameworks. This approach offers a robust basis for evaluating nanoplasics risks and guiding regulatory policies in agroecosystems.

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Highlights

* Aged polystyrene nanoplasics were prepared for environmentally relevant exposure

* New acetone-assisted protocol developed for quantifying nanoplasics in plants

* 228.9 µg g⁻¹ in soil leads to 97.6 µg g⁻¹ nanoplastic accumulation in barley roots

* Nanoplastics show roots uptake with a translocation factor of 0.31 to shoots

* Demonstrated method performance in realistic soil-plant systems