Monitoring of Cu²⁺ release from controllably synthesized nano-copper pesticides

ABSTRACT

Copper-based nanopesticides hold significant promise for agricultural applications. However, a critical challenge that persists is the need for effective control over the release of Cu²⁺. To this end, this study systematically investigated the Cu²⁺ release characteristics of Cu-based NPs with varying morphologies, species, and coating agents. It is found that CuO NPs exhibited extremely low Cu²⁺ concentration, rendering them ineffective for plant disease control. Cu(OH)₂ nanowires cannot continuously release Cu²⁺ due to its larger size and enhanced stability. Conversely, Cu(OH)₂ NPs exhibited sustained Cu²⁺ release, albeit at a slower rate. Modification with surfactants could significantly affect the Cu²⁺ release behavior, particularly the coating of carboxymethyl starch, which notably promoted Cu²⁺ release from Cu(OH)₂ NPs, achieving sustained release for 27 days. Furthermore, carboxymethyl starch-coated Cu(OH)₂ particles could consistently release Cu²⁺ in the pH range of 5–7. This finding may offer the inspiration of long-lasting control of plant diseases.

KEYWORDS:

• Nanopesticides
• copper
• copper hydroxide
• Cu²⁺ release
• surfactant modification

1 Introduction

In recent years, the application of nanotechnology in agriculture has garnered significant attention, particularly in the development of nanopesticides [1,2]. Nanopesticides are pesticides made using nanomaterials, which offer higher activity and efficacy compared to traditional pesticides. The small size and unique surface properties of NPs enable better adsorption on plant surfaces, enhancing adhesion and persistence [3]. Additionally, nanopesticides play an active role in crop nutrient management by improving the utilization and absorption of nutrients through nano-sized soil applications [4]. This allows for controlled fertilizer release, reducing pollution and waste while improving crop yield and quality.

However, the development and application of nanopesticides still face challenges. Among them, Cu-based NPs have shown excellent antimicrobial and cost-effective properties [5,6]. Cu(OH)₂ and CuO NPs (NPs) are the primary components of these pesticides [7–9]. These small particles could gradually release Cu²⁺ which is a broad-spectrum antibacterial antibiotic [10]. Therefore, Cu-based NPs has been commonly used to protect crops from bacteria induced diseases [11]. Compared with traditional Cu²⁺ pesticide, Cu-based NPs require lower concentrations while exhibiting higher bactericidal activity, resulting in more efficient disease control [12]. Cu-based NPs also demonstrate superior adhesion and penetration properties, allowing for better attachment to crop surfaces and entry into plant tissues [3]. Furthermore, the release of Cu²⁺ is relative slow, providing long-term protection for corps.

The control of Cu²⁺ release from Cu-based NPs remains a challenge. On the one hand, the Cu²⁺ concentration cannot be too low as its lowest bactericidal concentration is 63.5 μg/L [13]. On the other hand, the high Cu²⁺ concentration could also result in toxicity to plants as Cu²⁺ could replace Mg²⁺ in chlorophyll [14]. Therefore, the concentration of Cu²⁺ should be controlled in an appropriate concentration range. From the cost and benefit perspective, controlling the Cu²⁺ releasing at an appropriate rate could reduce the number of pesticide sprays and thus reduce the labor costs. Thus, control of Cu²⁺ release from Cu-based NPs is of great significance. Nevertheless, the study of Cu²⁺ release from Cu-based nanopesticides is still scarce [15,16].

Modification of Cu-based NPs with organic surfactant may be a possible solution for Cu²⁺ release control. The control of active constituent release has been commonly studied in the field of drug delivery [17]. For example, encapsulating the active ingredient in polymers or liposomes and controlling the pore structure on nanoparticle surfaces allows for gradual release, prolonging its action time [18,19]. Learning from this pathway, chemical modification of metal oxide nanopesticides through covalent bonding with a polymer or other compound was adopted to can control release rates [20–22]. This method enables adjustable release rates and durations based on specific needs, while remaining controllable and cost-effective. Whereas, chemical modification methods often require the use of capping agents which may pose ecological risks [23]. In addition, environmental factors such as temperature, pH might affect the Cu²⁺ release in their practical application. Given that, the critical environment factor such as pH should be taken into consideration when modification of Cu-based NPs [24]. Nevertheless, research on the Cu²⁺ release control from Cu-based NPs nanopesticides remains limited, making it crucial to explore environmentally friendly release strategies for Cu-based NPs.

Herein, this study aims to systematically investigate the Cu²⁺ release from Cu-based NPs. To achieve the impact of particle component, morphology, and the species of modification surfactants, seven Cu-based NPs, i.e. CuO NPs, Cu(OH)₂ NPs, Cu(OH)₂ nanowires (NWs), and rhamnolipid (Rha), gelatin (Gel), carboxymethyl starch (CMS), and PEG (PEG) coated Cu(OH)₂ NPs, were prepared by sintering method and coordination precipitation method. Their Cu²⁺ release behavior in 27 days were quantitatively monitored to screen for long-lasting and effective Cu-based NPs. Additionally, the influence of pH on Cu²⁺ release was also investigated, considering real application processes. This work may contribute novel insights for the green and controlled release of Cu-based NPs.

2 Materials and methods

2.1 Reagents and materials

High-purity anhydrous copper sulfate, ammonia (NH₄OH, GR, 25–28%), sodium hydroxide (NaOH, AR, ≥96.0%), oxalic acid (AR, ≥99.8%), and ethanol (AR, ≥95%) were procured from Beijing Sinopharm Chemical Reagent Co. Ltd. Rhamnolipid (95%) was bought from Sigma-Aldrich Co. Ltd (Germany). Gelatin (from pigskin), sodium starch glycolate (AR) and PEG (AR) were sourced from J&K Scientific Co. Ltd. Ultra-pure water (18.2 MΩ) produced with a Milli-Q Gradient system (Millipore, Bedford, U.S.A.) was used throughout.

2.2 Synthesis of Cu-based NPs

Preparation of Cu(OH)₂ NPs and Cu(OH)₂ NWs. Cu-based NPs were prepared through coordination precipitation method based on previous study with moderate modification [25]. The coordination precipitation method entailed adding ammonia to a CuSO₄ solution to form intermediate Cu₂(OH)₂²⁺ species, which further reacted with excess NH₄OH to yield Cu₂(NH₃)₄²⁺ complexes. The gradual addition of NaOH then facilitated the release of Cu²⁺ from these complexes, leading to the formation of Cu(OH)₂ NPs. The morphology of these NPs, influenced by the nucleation and growth rates within the liquid-phase reaction, was modulated by varying the concentrations and addition rates of NH₄OH and NaOH. In this study, the morphology of Cu(OH)₂ NPs was tailored by controlling the NaOH addition rate. The Cu(OH)₂ NPs was synthesized by first mixing 30 mL of 3 mol/L ammonia solution into 0.05 mol/L CuSO₄ solution, under continuous magnetic stirring for 20 minutes to yield flocculent copper precursors. Subsequently, 10 mL of 0.5 mol/L NaOH solution was introduced into the mixture at a rate of 10 mL/min, followed by an additional 20 min of stirring. The resulting solution was washed three times with pure water until a neutral pH was achieved, centrifuged at 4500 rpm, and the precipitate was dried in a blast oven to obtain the Cu(OH)₂ NPs. The synthesis process of Cu(OH)₂ NWs is identical to that of Cu(OH)₂ NPs, except adjust the NaOH addition rate to 2 min/L.

Preparation of surfactant coated Cu(OH)₂ NPs. The flocculent copper precursors solution was obtained through above described method. Then, surfactants including Rha, Gel, CMS, and PEG, were added into above solutions with final concentration of 1 mM. After incubation for 1 hours under stirring, 10 mL of 0.5 mol/L NaOH solution was added at a rate of 2 mL/min. Post-stirring, washing, centrifuging, and drying steps were identical to the Cu(OH)₂ NPs preparation.

Preparation of CuO NPs. The precipitation roasting method used for creating CuO NPs involved reacting oxalic acid with Cu²⁺ ions to form copper oxalate precipitates, which were then converted into CuO NPs through high-temperature sintering. An ethanol/water (1:1, v:v) solution served as the solvent for preparing 0.10 mol/L solutions of CuSO₄ solution and oxalic acid. The oxalic acid solution was added dropwise to the CuSO₄ solution under magnetic stirring for 20 min. The precipitate obtained was copper oxalate, which was centrifuged, washed with ethanol 3–5 times, vacuum dried at 60°C for 3 h, and roasted at a ramp of 10°C/min to 270°C, held for 2 hours to yield black CuO NP powder.

2.3 Monitoring of Cu²⁺ release from Cu-based NPs

A 250 mg sample of the synthesized Cu-based NP powder was dispersed in 500 mL of deionized water and shaken at 150 rpm. Samples with volume of 10 mL were taken out at 6 h, 1, 2, 3, 6, 9, 14, 20, and 27 days. The samples were filtered using a 50 mL ultrafiltration tube (Millipore, Amicon Ultra), centrifuged at 7000 rpm for 15 min. The filtrate had 200 µL of 65% nitric acid solution added dropwise, mixed thoroughly, and analyzed for Cu²⁺ concentration using inductively coupled plasma mass spectrometry (ICP-MS). To evaluate the influence of pH on Cu²⁺ release, the pH of the water was adjusted to approximately 5, 6, and 7 using diluted NaOH and HNO₃ solutions, and the aforementioned steps were replicated. To ensure the accuracy of Cu analysis, experimental blank group, which used pure water to substitute the Cu-based NP suspension was set, and Cu did not to be detected, indicating interference would not be introduced in this procedure.

2.4 Characterization

Particle morphology, composition, and surface functional groups were characterized using different analytical instruments. A Quanta FEG 250 scanning electron microscope (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS) was utilized to examine particle morphology. The sample preparation involved dispersing a small quantity of Cu-based NPs in deionized water, ultrasonic dispersion, and then placing 25 µL of this dispersion onto a clean silicon wafer, which was subsequently vacuum dried for SEM analysis. Fourier transform infrared spectroscopy (FTIR, Thermo-Nicolet Nexus 6700) was employed to identify surface functional groups. The concentration of Cu²⁺ was quantified using an Agilent ICP-MS 8900.

3 Results and discussions

3.1 Preparation and characterization of Cu-based NPs

The experimental results are illustrated in Figure 1a–c, indicating that a rapid addition of NaOH solution (0.5 M, 10 mL/min) resulted in Cu(OH)₂ NPs with a lamellar granular morphology, due to the orthorhombic crystal system of Cu(OH)₂ favoring such a structure. These particles ranged in size from 47.0 to 232.7 nm and exhibited relative uniformity. Conversely, when the NaOH addition rate was reduced to 10 mL/min, the resulting Cu(OH)₂ NWs with morphologies of rod-like or wire structures, with lengths spanning 174.5 to 1595.2 nm and diameters between 26.7 and 81.2 nm. The CuO NPs also adopted a lamellar morphology but with more defined edges and a broader size range of 100.4 to 690.2 nm, compared to the Cu(OH)₂ NPs.

Figure 1. Characterization of copper pesticide NPs. SEM images of Cu(OH)₂ NPs (a), Cu(OH)₂ NWs (b), CuO NPs (c), rha-coated Cu(OH)₂ NPs (d), gel-coated Cu(OH)₂ NPs (e), CMS-coated Cu(OH)₂ NPs (f), and PEG-coated Cu(OH)₂ NPs (g), respectively.

The role of surfactants in nanoparticle preparation, particularly in stabilizing particles and altering their morphology, prompted the use of Rha, Gel, CMS, and PEG in this study. The synthesis of surfactant-capped Cu(OH)₂ particles followed a similar procedure to that of the Cu(OH)₂ NPs, with the addition of surfactants after the formation of the flocculent Cu₂(NH₃)₄SO₄ precipitate, followed by thorough mixing and subsequent NaOH addition to induce precipitation. The findings, depicted in Figures d-g, reveal that Rha-coated Cu(OH)₂ NPs maintained a lamellar structure akin to Cu(OH)₂ NPs but were significantly larger, ranging from 51.7 to 628.4 nm. This increase in size may be attributed to the tighter binding of the surfactant to the particle surfaces, which elevates the nucleation potential energy, reduces the number of crystalline nuclei, and consequently enlarges the particle size. Gel and CMS-coated Cu(OH)₂ NPs exhibited similar spike-like morphologies, with sizes of 138.2 to 409.8 nm and 418.6 to 1269.4 nm, respectively. When utilizing PEG as a surfactant, the Cu(OH)₂ NPs predominantly appeared as thick flakes, sized between 181.0 and 979.0 nm, accompanied by occasional fine needle-like structures possibly arising from localized high concentrations during NaOH addition.

In addition, the particles were characterized using X-ray scattering spectroscopy and FTIR spectroscopy to analyze the presence of surfactants on their surfaces. The EDS results, presented in Figure 2, indicated strong carbon signals for all surfactant-coated particles, which, when correlated with SEM imagery and Cu elemental distribution maps, suggested the presence of surfactants on the particle surfaces. Further characterization was conducted using FTIR spectroscopy, with findings shown in Figure 3. All particles, except for the CuO NPs, exhibited pronounced peaks at 3572 cm⁻¹ and 3314 cm⁻¹, corresponding to the stretching vibrations of free O-H groups and hydrogen-bonded O-H groups in Cu(OH)₂ particles, respectively. The surfactant-coated Cu(OH)₂ particles, including those coated with Rha, Gel, and CMS, displayed broad peaks between 2500 and 3500 cm⁻¹, aligning with the characteristic peaks of carboxylate O-H stretching vibrations. Notably, these peaks partially overlapped with the free O-H stretching vibration peaks of uncoated Cu(OH)₂ particles. However, the O-H stretching vibration peaks of the surfactant-coated particles were more pronounced and sharper compared to those without carboxyl groups, such as PEG, indicating that the surfactants were successfully attached to the Cu(OH)₂ particles using the modification method. Additionally, post-modification, the Cu(OH)₂ particles exhibited a distinct and sharp peak at 1641 cm⁻¹, likely due to the C=O stretching vibration and O-H bending vibration under hydrogen bonding. Therefore, the combined SEM and FTIR characterization results confirm the successful modification of Cu(OH)₂ particles with different surfactants.

Figure 2. SEM and EDS characterization of Cu(OH)₂ NPs coated with different surfactants.

Figure 3. FTIR characterization of Cu(OH)₂ NPs coated with different surfactants.

3.2 Monitoring of Cu²⁺ release from different Cu-based NPs

The sterilization effect of copper nanopesticides is primarily achieved through the release of Cu²⁺ ions, which are crucial for controlling plant diseases. Consequently, monitoring the release of Cu²⁺ is a vital aspect of evaluating nano-copper pesticides. According to existing research, the minimum bactericidal concentration of Cu²⁺ is 63.5 μg/L [13], making it necessary for the released Cu²⁺ concentration to exceed this threshold. However, excessive Cu²⁺ can displace Mg²⁺ from chlorophyll, disrupting plant photosynthesis and causing phytotoxicity; thus, the Cu²⁺ concentration must be carefully controlled. Ideally, Cu²⁺ should be released in low doses and at a sustained rate to ensure bactericidal efficiency while extending its effectiveness for long-term disease control.

In this study, various Cu-based NPs (500 mg/L) were added to water, sampled at different time points, and separated via ultrafiltration to determine the concentration of free Cu²⁺ ions and obtain their release kinetic curves. The results, depicted in Figure 4a, showed that the Cu²⁺ concentration released from CuO NPs was approximately 25 μg/L, falling short of the bactericide requirement. The concentration of Cu²⁺ from Cu(OH)₂ NWs was ~125 μg/L, but remained relatively constant over time, which may be attributed to the larger, more stable nature of the wire structure, hindering the release of Cu²⁺. The initial Cu²⁺ concentration in the Cu(OH)₂ NPs group was approximately 108.6 μg/L, gradually increasing over time to reach 214.9 μg/L by day 27. Thus, granular Cu(OH)₂ particles appear to be more suitable for use as nanopesticides due to their favorable release profile.

Figure 4. Cu²⁺ release kinetic profiles of different Cu-based NPs. (a) Cu²⁺ release profiles of Cu(OH)₂ NPs, Cu(OH)₂ NWs, and CuO NPs; (b) Cu²⁺ release profiles of rha, gel, CMS, and PEG-coated Cu(OH)₂ NPs.

The Cu²⁺ release kinetics of Cu(OH)₂ NPs modified with four different coating agents are presented in Figure 4b. The Rha-coated Cu(OH)₂ NPs exhibited a steady and gradual increase in Cu²⁺ concentration, reaching 349.9 μg/L by day 27. In contrast, the Gel-coated Cu(OH)₂ particles maintained a stable Cu²⁺ level for the first 15 days, suggesting minimal release during this period. Afterward, the concentration surged to 584.9 μg/L, which could be attributed to the initial strong encapsulation by Gel that prevented early release, followed by eventual dissolution and delayed release of Cu²⁺. The CMS-coated Cu(OH)₂ NPs showed a continuous increase in Cu²⁺ concentration, with a modest rise during the first 3 days and then a consistent upward trend, culminating in 653.7 μg/L on day 27. The pattern of Cu²⁺ concentration change in PEG-coated Cu(OH)₂ NPs group was notably distinct, characterized by an initial decrease, stabilization, and subsequent increase. While there is no definitive theory to explain this behavior, it is hypothesized that fine needle-like Cu(OH)₂ NPs dissolved quickly upon water addition as particles with smaller sizes are more likely to release counterpart ions due to its high surface energy [26]. This results in an initial high Cu²⁺ concentration of 354.6 μg/L. Subsequently, these ions may have been adsorbed by larger Cu(OH)₂ particles, causing a reduction in free Cu²⁺ levels and establishing an equilibrium. Over time, the dissolution of PEG from the surfaces of the larger particles could have facilitated a gradual increase in Cu²⁺ concentration. Among the seven Cu-based NPs assessed, the CMS-coated Cu(OH)₂ NPs demonstrated the highest initial Cu²⁺ concentration and sustained release capability, making them the most suitable candidate for copper nanopesticides.

3.3 Monitoring of Cu²⁺ release under different pH conditions

Given that the pH of the solvent for Cu(OH)₂ NPs or rainwater can vary between 5.0 and 7.0 during practical applications, the study investigated the Cu²⁺ release kinetics of each Cu-based NPs across this pH range. The results, depicted in Figure 5, showed that for all groups except the CuO NPs, the Cu²⁺ concentration at pH 5 was slightly higher than at pH 6 and pH 7, which both exhibited similar trends and concentrations. Notably, the Cu²⁺ concentration at the sixth hour was higher at lower pH values, likely due to smaller Cu(OH)₂ particles being more sensitive to pH and dissolving more rapidly within the initial 6 h. In the Cu(OH)₂ NWs, Rha-coated Cu(OH)₂ NPs, CMS-coated Cu(OH)₂ NPs, and PEG-coated Cu(OH)₂ NPs groups, there was an observed decrease in Cu²⁺ concentration initially, presumably because larger particles adsorbed the released Cu²⁺, as previously discussed. Upon comparative analysis of the seven copper-based NPs, the carboxymethyl starch-coated Cu(OH)₂ particles were found to release Cu²⁺ continuously, with a release concentration meeting sterilization requirements and demonstrating minimal pH sensitivity, thus offering broader applicability.

Figure 5. Cu²⁺ release monitoring of Cu-based NPs at different pH values. Characterization of copper pesticide NPs. Cu²⁺ release kinetic curves of Cu(OH)₂ NPs (a), Cu(OH)₂ NWs (b), CuO NPs (c), rha-coated Cu(OH)₂ NPs (d), gel-coated Cu(OH)₂ NPs (e), CMS-coated Cu(OH)₂ NPs (f), and PEG-coated Cu(OH)₂ NPs (g), respectively.

4 Conclusions

In this study, we conducted a comprehensive investigation into the release kinetics of Cu²⁺ from Cu-based NPs with varying shapes, compositions, and surfactant coatings. We also examined the impact of environmental pH levels on Cu²⁺ release. The findings revealed that CuO NPs exhibited minimal Cu²⁺ solubility in water, rendering them ineffective for plant disease control. The Cu(OH)₂ NWs were found to be large and stable, but they struggled to release Cu²⁺ in a sustained manner. In contrast, granular Cu(OH)₂ NPs were capable of a sustainable release, albeit at a slower rate. The application of surfactants notably influenced the release behavior of Cu²⁺. Among the surfactants tested, CMS coating significantly enhanced the release of Cu²⁺ from Cu(OH)₂ NPs, sustaining the release over a period of 27 days. The release kinetics across different pH values was investigated, it was observed that CMS-coated Cu(OH)₂ NPs could maintain a consistent release of Cu²⁺ and fulfill the bactericidal concentration requirements under real-world environmental pH conditions (pH 5–7). This suggests that such particles have the potential to provide long-term protection of plants from bacterial induced diseases, highlighting their applicability for agricultural use.

Acknowledgements

This work was kindly supported by the Yunnan (China) Province Enterprise Basic Research Application Basic Research Joint Project (202101BC070001-026, 202101BC070001-011), National Natural Science Foundation of China (22206181 and 22106028), and Scientific Research Funding of National Institute of Metrology of China (AKYCX2202).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the National Natural Science Foundation of China [22206181 and 22106028]; Yunnan (China) Province Enterprise Basic Research Application Basic Research Joint Project [202101BC070001-011]; Yunnan (China) Province Enterprise Basic Research Application Basic Research Joint Project [202101BC070001-026]; Scientific Research Funding of National Institute of Metrology of China [AKYCX2202].