CHITOSAN-STABILIZED COPPER NANOPARTICLES ENABLE CONTROLLED COPPER AVAILABILITY AND REDUCE PHYTOTOXICITY IN SOIL-GROWN SOYBEAN

Abstract

Chitosan-stabilized copper nanoparticles (CuNPs) have emerged as promising alternatives to conventional copper sources in agriculture due to their potential to improve micronutrient delivery while reducing metal-associated phytotoxicity. However, their biological effects and interactions with soil-plant systems remain insufficiently understood. In this study, CuNPs were synthesized by chemical reduction within a chitosan matrix and characterized by ultraviolet visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and transmission electron microscopy (TEM), revealing well-dispersed, predominantly spherical nanoparticles (ca. 6 nm) with strong polymer coordination. Soybean (Glycine max) seedlings were cultivated in soil amended with CuNPs (10-100 mg L^–1) or copper(II) sulfate (CuSO₄; 10-50 mg L^–1) to evaluate seedling vigor and anatomical responses. Ionic copper induced a significant, dose-dependent reduction in seedling vigor, with marked inhibition at 50 mg L^–1 CuSO₄ (p < 0.05), accompanied by severe anatomical damage, including cortical cell collapse and vascular disruption. In contrast, CuNP exposure did not significantly affect germination or seedling vigor and, at lower concentrations, produced responses comparable to the control. Anatomical analyses further revealed fewer structural abnormalities in roots and stem-root transition zones in CuNP-treated plants. Collectively, these results indicate a distinct copper availability profile for chitosan-stabilized CuNPs, consistent with reduced acute phytotoxicity while preserving the essential micronutrient function of copper. Overall, polymer-stabilized CuNPs represent a promising strategy for controlled micronutrient delivery in agriculture, although further studies addressing long-term environmental behavior and safety remain necessary.

Keywords:
nanoparticles; chitosan; copper; phytotoxicity; bioavailability

INTRODUCTION

Nanotechnology has emerged as a transformative tool for agriculture, enabling the development of advanced materials designed to improve nutrient use efficiency, reduce environmental losses, and enhance crop performance. Within this context, engineered nanoparticles offer unique opportunities for the controlled delivery of agrochemicals due to their tunable size, surface chemistry, and reactivity. Metallic nanoparticles, in particular, represent one of the most extensively produced classes of nanomaterials, accounting for approximately 37% of commercially available nanomaterials.¹ Their increasing incorporation into agricultural inputs highlights the need to understand not only their agronomic functionality but also their interactions with soil-plant systems and potential environmental implications.²

Copper-based nanoparticles have attracted growing interest in agricultural nanotechnology owing to their multifunctional roles as micronutrient sources, antimicrobial agents, and components of fungicidal formulations.³,⁴ Compared to conventional copper salts, nanoparticulate copper offers the potential for greater distribution efficiency and reduced application rates. However, agricultural soils represent complex and dynamic environments that can strongly influence nanoparticle stability, transformation, and bioavailability. Global production estimates indicate that nearly 200 tons of copper nanoparticles are produced annually, with at least 36 tons entering terrestrial environments,⁵-⁹ emphasizing the importance of evaluating their performance and safety under realistic soil-based conditions relevant to crop production.

From a plant nutrition perspective, copper is an essential micronutrient involved in key physiological processes, including photosynthesis, respiration, and redox homeostasis.¹⁰ The effectiveness of copper-based nanomaterials in agriculture therefore depends on their ability to supply bioavailable copper while avoiding phytotoxic effects associated with excessive ionic release. While the uptake and transport of ionic copper via membrane transporters are well understood, the mechanisms governing the interaction of copper nanoparticles with plant roots, their internalization pathways, and subsequent physiological availability remain insufficiently characterized. Experimental studies¹¹-¹⁵ suggest that nanoparticles can penetrate root tissues and be translocated through vascular systems, even when their size exceeds the nominal pore dimensions of plant cell walls. It is important to emphasize that most of these findings are derived from simplified experimental systems, whereas soil-grown crops experience a much more complex physico-chemical environment that can substantially alter nanoparticle behavior, aggregation, and dissolution.¹⁶,¹⁷

Recent studies¹⁸-²³ have demonstrated that metallic nanoparticles can exert both beneficial and adverse effects on crop plants, depending on nanoparticle composition, surface functionalization, and application rate. These dual responses highlight the importance of rational nanomaterial design for agricultural applications. Soybean (Glycine max), one of the most economically important crops worldwide and responsible for nearly 40% of global oilseed production,²⁴ represents an ideal model system for evaluating the agronomic performance of copper-based nanomaterials. Emerging evidence²⁵,²⁶ suggests that soybean exhibits heightened sensitivity to metallic nanoparticles. For example, CuO nanoparticles have been reported to be taken up by root tissues without impairing germination, yet to induce genotoxic effects,²⁷ while other studies²⁸,²⁹ have shown that copper-based nanomaterials can interfere with biological nitrogen fixation and nutrient balance. Despite these findings, critical gaps remain in our understanding of how copper nanoparticles influence early plant development, structural integrity, and copper availability under soil-based conditions relevant to agricultural practice.²⁸,²⁹

Despite the rapid growth of agricultural nanotechnology, comparative studies directly assessing nanoparticulate versus ionic copper as nutrient sources under soil-grown conditions remain scarce. In this context, the present study investigates chitosan-stabilized copper nanoparticles as a nano-enabled copper source for soybean cultivation. By comparing their effects with those of ionic copper on seed germination, seedling vigor, and anatomical characteristics of roots and stem-root transition zones, this work aims to elucidate how nanoparticle stabilization and controlled copper availability influence early plant development. The results provide insights into the design of copper-based nanomaterials that balance agronomic functionality with reduced phytotoxicity, contributing to the development of safe and effective nanotechnologies for sustainable agriculture.

EXPERIMENTAL

Reagents

Copper(II) sulfate (CuSO₄), chitosan, and ascorbic acid were purchased from Sigma-Aldrich and used as received. Soybean (Glycine max L., cultivar BRS 9080RR) seeds were obtained from Brazilian Agricultural Research Corporation (Embrapa). Ultrapure water (Milli-Q system, resistivity of 18.2 MΩ cm at 25 °C) was used throughout all experiments.

Synthesis of nanomaterials

A 1.0 wt.% solution of medium-molecular weight chitosan was prepared in 2.0 wt.% acetic acid (25 mL) and heated to boiling under continuous stirring. Subsequently, copper(II) sulfate (2.5 g) and ultrapure water (25 mL) were added to the reaction mixture. After the solution reached the boiling point again, ascorbic acid (11.0 g) was added as a reducing agent. The reaction was maintained at boiling temperature for 10 min. After cooling to room temperature, the resulting chitosan-stabilized copper nanoparticle dispersion was used as obtained without further purification.

Characterization of nanoparticles

The optical properties of the synthesized copper nanoparticles (CuNPs) were analyzed by ultraviolet-visible (UV-Vis) spectroscopy using an HP 8453 spectrophotometer. Spectra were recorded in the wavelength range of 300-900 nm at room temperature using quartz cuvettes with a 1.0 cm optical path length. Deionized water was used as the reference blank.

Fourier transform infrared (FTIR) spectroscopy was performed using an Agilent Cary 630 FTIR spectrometer equipped with an attenuated total reflectance (ATR) accessory. Spectra were collected over the range of 4000-650 cm^–1 with a spectral resolution of 4 cm^–1 and 32 scans per sample. All measurements were conducted at room temperature, and background spectra were recorded prior to each analysis.

The morphology and size distribution of the CuNPs were examined by transmission electron microscopy (TEM) using a JEOL JEM-2100F microscope operated at an accelerating voltage of 100 kV. TEM samples were prepared by depositing a drop of a diluted CuNP dispersion onto carbon-coated copper grids, followed by solvent evaporation under ambient conditions. Particle size distributions were determined by measuring the diameters of individual nanoparticles from TEM micrographs using the software ImageJ 1.54g (Wayne Rasband, National Institute of Health, USA, 2023), with at least 100 particles analyzed per sample.

Seedling vigor index

Seed viability (maximum germination potential) and early seedling vigor were evaluated using a standard germination test. For each treatment, twelve soybean seeds were sown, and germination was monitored under controlled conditions. The germination percentage (GP) was calculated based on the number of seeds that produced normal seedlings. After the germination period, root and shoot lengths were measured, and the mean root length (L_r) and mean shoot length (L_s) were determined.

Seedling vigor was quantified using the seedling vigor index (SVI), which integrates germination performance and early seedling growth, according to Equation 1.³⁰

(1) $\text{SVI}=\left({\text{L}}_{\text{r}}+{\text{L}}_{\text{s}}\right)\times \text{GP}$

where GP is the germination percentage (%), L_r is the mean root length (cm), and L_s is the mean shoot length (cm).

Germination percentage was determined independently in two planting experiments conducted one day apart, with twelve soybean seeds per treatment in each experiment. The mean germination percentage reported in this study corresponds to the average value obtained from these two independent biological replicates.

Soil preparation and planting

A 1:1 (v/v) mixture of agricultural soil and vermiculite was prepared three days prior to planting to allow for equilibration. The physicochemical properties of the soil are provided in Table 1. The equilibrated soil mixture was amended with chitosan-stabilized copper nanoparticles at nominal concentrations of 10, 20, 30, 40, and 100 mg L^–1. Prior to soil application, CuNPs were dispersed in deionized water by ultrasonication for 30 min to ensure homogeneous suspension. An equivalent volume of deionized water without nanoparticles was applied to the control treatment.

Table 1.
Physicochemical properties of the experimental soil used in this study

Soil properties	Value
Clay / %	35
Slit / %	28
Sand / %	37
Organic matter / %	2.5
pH	5.2
Cation exchangeable capability / (cmol kg–1)	8.5
Cu content / (mg kg–1)	25
Nitrogen content / (g kg–1)	1.8
Phosphorus content / (g kg–1)	0.012
Potassium content / (g kg–1)	0.18

Soybean (Glycine max L.) seeds were surface-sterilized by immersion in 70% (v/v) ethanol, followed by thorough rinsing with deionized water, and subsequently sown in the prepared soil. Plants were cultivated under controlled conditions at 25 °C under a 12 h light / 12 h dark photoperiod with a light intensity of 2450 lux until approximately 80% of the control seedlings reached the VC (unifoliate) developmental stage. At harvest, plants were carefully removed from the soil and separated into roots, stems, cotyledons, and leaves. Fresh length and biomass measurements were recorded immediately after harvest, and samples were subsequently stored at 5 °C for further analyses.

Anatomical analysis

Two independent planting experiments were conducted with a one-day interval between them, and each experiment included twelve soybean seeds per treatment. For the anatomical evaluation, four soybean seedlings were collected from each independent experiment for each treatment, including the control and the four copper-based treatments: chitosan-stabilized copper nanoparticles (CuNPs) at 10 and 100 mg L^–1, and copper(II) sulfate at 10 and 50 mg L^–1. Plant tissues were fixed in FAA 50 (formalin-acetic acid-ethanol) for 48 h and subsequently stored in 70% (v/v) ethanol until further processing.

Sample preparation and sectioning were conducted at the Plant Anatomy Laboratory of the University of Campinas (Unicamp). Two anatomically relevant regions were analyzed: (i) the stem-root transition zone and (ii) the region proximal to the root apex. Samples were embedded in methacrylate resin (Historesin®, Leica Instruments, Heidelberg, Germany), and transverse sections were obtained using a Leica RM2245 rotary microtome at a thickness of 6 μm. Sections were stained with toluidine blue,³¹ and micrographs were acquired using an Olympus DP71 digital camera coupled to an Olympus BX51 optical microscope.

Statistical analysis

Biological assays were conducted in two independent planting experiments performed with a one-day interval between them, with twelve soybean seeds used per treatment in each experiment. Germination percentage and seedling vigor index data were expressed as mean values. Statistical comparisons between each treatment and the control were performed using Student’s t-test, with p < 0.05 considered statistically significant.

RESULTS AND DISCUSSION

Copper nanoparticles

The UV-Vis spectrum of the synthesized CuNPs exhibited a broad absorption band centered at approximately 790 nm, corresponding to a pronounced bathochromic shift relative to the surface plasmon resonance (SPR) region typically reported for bare or weakly capped copper nanoparticles (550-650 nm).³² This optical behavior should not be interpreted solely in terms of particle size or aggregation state. The position, width, and intensity of the SPR band in copper nanoparticles are highly sensitive to the local dielectric environment, surface chemistry, and electronic damping effects, particularly in polymer-stabilized systems.³³

In the present synthesis, the high concentration of chitosan plays a dual role, acting both as a stabilizing agent and as a surrounding dielectric medium for the metallic core. Such polymeric environments are known to induce significant red shifts and band broadening due to increases in the local refractive index and strong plasmon-polymer interactions. Similar bathochromic shifts extending into the near-infrared region have been reported³⁴ for copper nanoparticles embedded in or coated with polymeric matrices, including chitosan and other high-molecular-weight stabilizers. In addition, copper nanoparticles are intrinsically susceptible to plasmon damping effects arising from surface oxidation and interband electronic transitions, which further contribute to SPR broadening and red shifting.³² Taken together, these effects indicate that the observed optical response is consistent with the formation of polymer-stabilized CuNPs rather than extensive particle aggregation, in agreement with the nanoscale dimensions observed by TEM.

FTIR spectroscopy provided further evidence for the interaction between CuNPs and the chitosan matrix. The spectrum of chitosan exhibited a broad band at 3306 cm^–1, attributed to overlapping O–H and N–H stretching vibrations. The band at 2865 cm^–1 corresponds to C–H stretching, while absorptions at 1648 and 1550 cm^–1 are assigned to N–H bending vibrations of amino groups. Additional bands at 1374 and 1309 cm^–1 arise from C–H bending modes, and the absorption at 1025 cm^–1 is associated with C–O skeletal stretching.

Upon formation of the CuNPs, a systematic decrease in band intensities was observed, accompanied by slight shifts toward higher wavenumbers, specifically from 3306 to 3241, 1648 to 1629, 1374 to 1337, and 1025 to 1081 cm^–1. These spectral changes indicate coordination interactions between copper species and the functional groups of chitosan. Notably, the appearance of a new absorption band at 663 cm^–1 is attributed to Cu–N and/or Cu–O vibrational modes, confirming direct interactions between the nanoparticle surface and the amino and hydroxyl groups of the biopolymer.³⁵,³⁶

These spectroscopic features demonstrate that chitosan effectively coats and stabilizes the CuNPs through coordination interactions, resulting in a polymer-bound nanostructure. From an agricultural nanotechnology perspective, such stabilization is particularly relevant, as it can modulate copper release kinetics, reduce acute phytotoxicity associated with ionic copper, and enhance the controlled delivery of micronutrients in soil-plant systems.

Figure 1.
(a) UV-Vis absorption spectrum of CuNPs, (b) FTIR spectra of chitosan and CuNPs

TEM analyses revealed key morphological and dimensional characteristics of the synthesized nanostructures. As shown in Figure 2a, the copper nanoparticles (CuNPs) predominantly exhibit a spherical morphology with a narrow and homogeneous size distribution. Particle size analysis (Figure 2b) yielded an average diameter of approximately 6 nm, with the size distribution well described by a normal profile.

Figure 2.
(a) TEM micrograph of CuNPs; (b) particle size distribution histogram obtained from TEM analysis (n = 107), and (c) TEM micrograph of a chitosan nanoparticle encapsulating CuNPs

Notably, TEM micrographs further indicate that the CuNPs are effectively embedded within the chitosan matrix, which itself adopts a quasi-spherical conformation (Figure 2c). This structural organization suggests a uniform encapsulation mechanism, in which metallic nanoparticles are homogeneously dispersed throughout the polymeric network. Such morphology is particularly relevant for agricultural nanotechnology applications, as homogeneous polymer encapsulation can enhance colloidal stability, modulate copper release, and mitigate aggregation under soil conditions. Overall, the observed morphological compatibility between the CuNPs and the chitosan matrix confirms the effectiveness of the synthesis and stabilization strategy and supports the use of these nanostructures as controlled copper delivery systems for soil-plant interfaces.

Analysis of soybean plants

Germination percentages of soybean seedlings exposed to the copper treatments are presented in Figure 3. Overall, germination remained high across all treatments, suggesting that neither ionic nor nanoparticulate copper caused severe inhibition of seed germination under the tested conditions. Although some variation among treatments was observed, the limited number of biological replicates does not allow robust conclusions regarding treatment-related differences in germination percentage. Therefore, the germination results should be interpreted as descriptive evidence that seed germination was generally maintained across the evaluated copper concentrations.

Figure 3.
Germination percentage of soybean (Glycine max L.) seedlings cultivated in soil and exposed to ultrapure water (control), CuSO₄, or CuNPs at different concentrations

It is important to note that the germination experiment was conducted using only two independent biological replicates. Consequently, the statistical power of the analysis is limited, and subtle treatment-related effects on germination may not have been detected. Therefore, although germination percentages remained generally high across all treatments, these findings should be considered preliminary and should be confirmed through larger-scale experiments including a greater number of biological replicates.

Treatments with copper(II) sulfate resulted in a significant, dose-dependent reduction in seedling vigor relative to the control (p < 0.05), with the most pronounced inhibition observed at 50 mg L^–1 (Figure 4). This response indicates physiological stress associated with excessive accumulation of Cu²⁺ ions and is consistent with the well-documented phytotoxicity of ionic copper, which disrupts cellular metabolism, membrane integrity, and redox homeostasis.³⁷ In contrast, seedlings exposed to CuNPs maintained vigor indices comparable to, or slightly higher than, those of the control, with no statistically significant differences across the tested concentration range. This distinct physiological response suggests that copper delivered in nanoparticulate form interacts differently with plant tissues than ionic copper, likely reflecting differences in copper availability, transport, and interaction dynamics under soil-based conditions.³⁸ Consequently, CuNPs appear to mitigate acute copper toxicity while preserving the essential micronutrient function of Cu.

Figure 4.
Seedling vigor index of soybean (Glycine max L.) seedlings cultivated in soil and exposed to ultrapure water (control), CuSO₄, or CuNPs at different concentrations. *Indicates values statistically different from the control according to a t-test (p < 0.05)

Colloidal stability under realistic exposure conditions is a critical parameter governing nanoparticle bioavailability and biological effects in agricultural systems. Although the colloidal stability of CuNPs in the soil-vermiculite cultivation medium was not directly quantified, several qualitative observations support their effective dispersion during the early exposure period. First, CuNP suspensions were ultrasonically dispersed prior to soil application, and no visible macroscopic precipitation was observed throughout the cultivation period. Second, CuNP-treated seedlings did not exhibit significant reductions in germination or vigor, even at higher nominal concentrations, suggesting the absence of acute toxicity typically associated with rapid Cu²⁺ release or localized nanoparticle aggregation. Third, anatomical analyses revealed preserved cellular organization in roots and stem-root transition zones of CuNP-exposed plants, in marked contrast to the pronounced structural damage observed following CuSO₄ exposure. Finally, the lack of a clear dose-dependent phytotoxic response under CuNP treatments further suggests that nanoparticle availability was not dominated by rapid sedimentation or aggregation during early development. Collectively, these observations are consistent with the absence of acute phytotoxic effects during the initial exposure window, although changes in nanoparticle behavior, aggregation, or transformation under longer exposure times or field-relevant conditions cannot be excluded.³⁹

Representative photographs of soybean seedlings cultivated under the different treatments are shown in Figure 5. The seedlings in the control group and those exposed to lower copper concentrations displayed morphological features consistent with this developmental stage, including developed hypocotyls, cotyledons, and root systems. Seedlings exposed to the highest copper concentrations showed visible morphological differences compared with the control, particularly in overall seedling appearance and root system development. However, because no morphometric measurements were obtained from these images, these observations should be regarded as qualitative and complementary to the physiological and anatomical analyses discussed below.

Figure 5.
Soybean (Glycine max) seedlings cultivated under different treatments and photographed when approximately 80% of the control plants reached the VC (unifoliate) developmental stage: (a) control; (b) chitosan-stabilized copper nanoparticles (CuNPs), 10 mg L^–1; (c) chitosan-stabilized copper nanoparticles (CuNPs), 100 mg L^–1; (d) copper(II) sulfate (CuSO₄), 10 mg L^–1; and (e) copper(II) sulfate (CuSO₄), 50 mg L^–1. hy: hypocotyl; mr: main root; tr: stem-root transition region. Scale bars (a–e) = 2 cm

Anatomical examination of the stem-root transition region revealed a single-layered epidermis and a cortex composed of parenchymatic cells across all treatments (Figures 6a-6e). However, exposure to higher concentrations of ionic copper and sulfate induced pronounced structural alterations, including epidermal deformation and collapse of outer cortical cells (Figures 6c and 6e). Within the vascular cylinder, high copper concentrations led to marked changes in both phloem and xylem tissues, including visual alterations consistent with reduced xylem vessel diameter and evidence of secondary wall thickening (Figure 6h, arrow). Sulfate treatments inhibited phloem fiber differentiation at both tested concentrations, and at the highest sulfate level, xylem vessels showing tylosis formation were observed and exhibited apparent reduced diameters relative to the control.

Figure 6.
Transverse sections of the stem-root transition region (a-j) and of the root in the primary structure (k-t) of soybean (Glycine max L.) seedlings subjected to different treatments. (a-e) Peripheral region of the stem-root transition zone showing the epidermis and parenchymatic cortex. (f-j) Details of the vascular cylinder. (h) Arrow indicates xylem vessel elements with thickened secondary cell walls. (j) Asterisks indicate xylem vessel elements containing tyloses. (k-o) Peripheral region of the root showing the epidermis and parenchymatic cortex. (l) Arrow indicates parenchyma cells with lignified cell walls. (m) Epidermal cells with thick outer and inner periclinal walls and degraded cortical parenchyma adjacent to the epidermis. (o) Detail of the epidermis and four to five subepidermal layers exhibiting cellular degradation. (p-t) Details of the root vascular cylinder. (r, t) Altered cortical parenchyma and primary phloem cells. (t) Protoxylem vessel elements showing tylosis formation (abbreviations: ep = epidermis; fi = fiber; pc = parenchyma cells; ph = phloem; px = protoxylem; xy = xylem vessel element. Scale bars = 200 µm)

Similar trends were observed in root sections near the shoot apex, which normally display a single-layered epidermis, parenchymatic cortex, and actively differentiating primary xylem with four protoxylem arms (Figures 6k-6t). High concentrations of ionic copper and sulfate were associated with pronounced anatomical alterations in epidermal and cortical parenchyma cells, as well as pronounced disruptions in phloem tissues. At the highest sulfate concentration, the conducting elements of the protoxylem were obstructed by tyloses, further indicating impaired vascular functionality (Figure 6t).

The dissolution behavior of copper nanoparticles and the subsequent release of Cu²⁺ ions represent key mechanisms underlying their biological effects. Although copper ion release and nanoparticle transformation were not directly quantified in this study, the sharply contrasting physiological and anatomical responses observed between CuNP- and CuSO₄-treated plants provide strong functional evidence of distinct copper availability profiles. Ionic copper induced pronounced, dose-dependent phytotoxicity, whereas CuNP treatments produced minimal adverse effects, even at higher nominal concentrations. This contrast is consistent with the hypothesis that chitosan-stabilized CuNPs act as a moderated or buffered copper source, suggesting that the polymeric coating may limit rapid Cu²⁺ dissolution and attenuates acute copper exposure.⁴⁰ Similar behavior has been reported⁴¹-⁴³ for polymer-coated metallic nanoparticles, where surface functionalization reduces dissolution rates and mitigates metal-induced oxidative stress in plants. Thus, the reduced phytotoxicity observed in CuNP-treated soybean seedlings is likely associated with differences in copper availability governed by nanoparticle stabilization and soil interactions.⁴⁴

Taken together, the physiological and anatomical responses of soybean seedlings exposed to nanoparticulate and ionic copper indicate fundamentally different interaction pathways during early development. While this study focused on early-stage responses under soil-based conditions, direct measurements of copper accumulation, dissolution kinetics, and nanoparticle transformation were beyond its scope. Accordingly, the mechanistic interpretations proposed herein should be regarded as indicative rather than conclusive, highlighting the need for future studies addressing long-term exposure, copper speciation, and nanoparticle fate in agricultural soils.

CONCLUSIONS

This study demonstrates that chitosan-stabilized copper nanoparticles (CuNPs) exhibit a predominantly spherical morphology and strong interactions with the polymeric matrix, features commonly associated with enhanced colloidal stability and moderated copper availability in polymer-stabilized nanomaterials. These physicochemical characteristics are particularly relevant at the soil-plant interface, where they are expected to influence nanoparticle mobility, transformation, and ultimately their biological effects in agricultural systems.

Comparative evaluation of nanoparticulate and ionic copper revealed markedly different physiological and anatomical responses during the early development of soybean seedlings. Exposure to copper(II) sulfate induced pronounced, dose-dependent phytotoxic effects, reflected by reduced seedling vigor and severe anatomical alterations, including cortical cell collapse and vascular obstruction. In contrast, seedlings exposed to CuNPs, particularly at lower concentrations, maintained vigor levels comparable to the control and exhibited substantially fewer structural abnormalities. These contrasting responses are consistent with a distinct copper availability profile for chitosan-stabilized CuNPs, supporting the hypothesis that the polymeric coating may moderate Cu²⁺ release and thereby attenuate acute toxicity while preserving the function of copper as an essential micronutrient.

Microscopic analyses further corroborated these findings, revealing better preservation of root tissues and stem-root transition zones in CuNP-treated plants compared with those exposed to ionic copper. Collectively, the results indicate that nanoparticulate and ionic forms of copper interact with soybean seedlings through fundamentally different pathways during early development, largely governed by differences in copper delivery and bioavailability. These findings reinforce the potential of polymer-stabilized copper nanomaterials as promising platforms for controlled micronutrient delivery in agriculture.

Overall, this work contributes to the growing body of evidence supporting the use of rationally engineered nanomaterials as alternative micronutrient sources for sustainable crop production. Although the present study focused on early developmental and anatomical responses, the findings provide an important foundation for future investigations addressing long-term exposure, copper accumulation, nanoparticle transformation, and environmental safety under field-relevant conditions, key aspects for the responsible integration of copper-based nanotechnologies into modern agricultural systems.

DATA AVAILABILITY STATEMENT

In this work, all data supporting the findings are fully available within the text of the manuscript.

ACKNOWLEDGMENTS

The authors acknowledge CNPq (303231/2020-3), INCTBio-LK (408338/2024-5), and FAPESP (2018/02507-0, 2019/24445-8, 2022/01418-8) for financial support; the Multi-User Equipment Center of the Institute of Chemistry-Institutional Mass Spectrometry Laboratory (EMU/FAPESP 2019/24445-8) for infrastructure; Unicamp Institute of Chemistry for institutional support; and LISpec (RRID:SCR_027391, CEMUIQ-UNICAMP) for technical support.

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Edited by

• Executive Editor handled this article:
  Júlio S. Rebouças

Publication Dates

• Publication in this collection
  03 Aug 2026
• Date of issue
  2026

History

• Received
  17 Apr 2026
• Accepted
  16 June 2026
• Published
  29 June 2026