Optimised Preparation and Formula of Deltamethrin Nanoemulsion for Enhanced Insecticidal Efficacy and Superior Biosafety

Abstract

Stable nanoemulsions with fine droplets reduce organic solvent use and improve the dispersion of hydrophobic pesticide. However, current studies on deltamethrin nanoemulsion lack systematic formula optimization, performance evaluation and biosafety assessment. This study developed a stable deltamethrin nanoemulsion (Del@Ne) and tested its physicochemical properties, insecticidal activity and non-target safety. In 2025, the effects of surfactant ratio, dosage, preparation temperature and emulsification method on emulsion stability was systematically investigated. The optimal formula contained an active ingredient (2.5% deltamethrin), a surfactant ratio of 8:1 (#601:#500), a 6% surfactant dosage, a 17.25% oil phase (S-100:DMF = 20:3), and deionised water filled to 100%, prepared by adding deionised water to an oil phase containing deltamethrin and surfactants at 40 °C. Del@Ne exhibited small droplet size and good storage stability (TSI ≈ 1), which had better wettability on peach leaves with contact angle falling from 40.4° to 21.6° in 120 s. Del@Ne also gave higher toxicity against Myzus persicae (LC₅₀ = 66.85 mg L⁻¹) than Del@EC (80.69 mg L⁻¹), while showing lower toxicity to zebrafish, earthworms and Harmonia axyridis, as well as better biocompatibility with human L02 hepatocytes. These results provide references for rapid screening of nanoemulsion formulation parameters and also offer insights for the efficient utilization of hydrophobic pesticides.

1. Introduction

In contemporary agriculture, pesticides serve as indispensable agents that efficiently control harmful microorganisms, insect infestations and unwanted vegetation in cultivated areas, playing an important role in enhancing agricultural productivity [1,2]. Most active ingredients used as pesticides exhibit poor water solubility, making formulation processing essential for enabling effective field application through spraying [3,4,5]. However, traditional pesticide formulations, such as emulsifiable concentrate and wettable powder, often cause irreversible environmental pollution and resource wastage [6]. Given these challenges, it is necessary to design more effective and relatively environmentally friendly pesticide formulations to improve food security and reduce ecological hazards [7].

Nanoemulsions are isotropic colloidal dispersion systems with droplet sizes typically between 20 and 200 nm [8,9,10]. They are prepared by stabilising two immiscible liquid phases using surfactants and have demonstrated significant application potential across various industries, including pharmaceuticals, food processing, agrochemicals and personal care products [11,12,13]. As an emerging nanocarrier for pesticide delivery, nanoemulsions not only can reduce the usage of organic solvents, but also exhibit several advantageous physicochemical properties, such as high dispersibility, nanoscale particle size, improved surface wettability and excellent sustained-release performance [14,15]. These attributes contribute to enhanced pesticide adhesion, deposition and penetration on plant surfaces, ultimately improving pesticide use efficiency, reducing application frequency and decreasing the environmental burden imposed by conventional formulations [16,17,18]. Therefore, processing pesticides into nanoemulsion-based systems can address key limitations of traditional pesticide formulations by using fewer organic solvents, having better dispersion of active ingredients and reducing wash-off by rainfall, thereby providing an efficient and relatively safe pesticide delivery method [19,20].

Myzus persicae, a Hemipteran pest species, frequently congregates on branches, buds and leaves, where it feeds on plant sap, leading to morphological deformities, growth inhibition, leaf abscission and, in severe infestations, fruit drop and plant mortality [21,22,23]. This pest has caused substantial economic damage to forestry plantations across China. At present, the management of M. persicae infestation relies primarily on chemical and biological control and agricultural practices. Chemical control remains a predominant strategy owing to its rapid action and high efficacy. Deltamethrin (Del), a synthetic pyrethroid insecticide with broad-spectrum efficacy, exhibits strong stomach toxicity and excellent aphid control performance [24,25]. Given its low dissolution in most organic solvents, commercial Del formulations predominantly exist as either emulsifiable concentrates (Del@EC) or wettable powders [26]. However, these formulations have low utilisation rates, and the organic solvents used cause serious environmental pollution. Del can be processed into a stable nanoemulsion to obtain efficient and environmentally friendly to obtain an efficient and relatively safe delivery system [27,28].

However, the combined effects of formulation parameters on the stability, efficacy, and biosafety of deltamethrin nanoemulsions remain insufficiently understood. In this study, a deltamethrin nanoemulsion (Del@Ne) was formulated using a low-energy emulsification method. To determine the most effective formula, the effects of key parameters, including surfactant ratio, surfactant dosage, preparation temperature and emulsification method, on nanoemulsion stability were systematically examined. After an optimal formula was identified, its wettability, adhesion performance on peach leaf surfaces and insecticidal efficacy against M. persicae were compared with Del@EC. Furthermore, its toxicity to zebrafish, earthworms, Harmonia axyridis and L02 cells was analysed. Altogether, this study provides a promising approach for the development of highly effective and relatively safe pyrethroid-based pest management strategies.

2. Materials and Methods

2.1. Materials

Del technical (98%) was purchased from Jiangsu Chunjiang Run Tian Agricultural Chemical Co., Ltd. (Huaian, China). Del@EC (25 g L⁻¹) was obtained from and Bayer Co., Ltd. (Shanghai, China). An aromatic solvent oil (S-100, 98%), a high-boiling aromatic hydrocarbon solvent mainly composed of alkylbenzenes and characterized by low water solubility, was obtained from Jiangsu Hualun Chemical Co., Ltd. (Yangzhou, China). Phenvinyl phenyl polyoxyethylene ether (#601) and calcium dodecylbenzenesulfonate (#500) were purchased from Jiangsu Haian Petrochemical Co., Ltd. (Nantong, China), whereas N, N-dimethylformamide (DMF, 99.5%) was obtained from Aladdin Reagent Co., Ltd. (Shanghai, China).

2.2. Preparation of Nanoemulsions

S-100 (15%) and DMF (2.25%) were thoroughly mixed in a beaker, and Del (2.5%) was dissolved in this solvent under ultrasonication. The resulting solution was mixed with a surfactant under stirring to obtain an oil phase. Deionised water was added drop-wise into the oil phase under constant agitation (800 rpm) for 30 min to obtain a Del nanoemulsion. A single-factor experimental design was adopted to evaluate the effects of formulation and preparation parameters on nanoemulsion stability. During this process, the effects of the surfactant ratio (4:1, 8:1, 12:1), surfactant dosage (4%, 6%, 8%) and preparation temperature (25 °C, 40 °C, 55 °C) on nanoemulsion stability were investigated. Here, the surfactant ratio refers to the mass ratio between surfactants #601 and #500. When the surfactant ratio was evaluated, the total surfactant dosage was kept constant at 6% of the total formula, while the proportions of the oil phase and other components, the preparation temperature, and the emulsification method were kept unchanged. When the surfactant dosage was evaluated, only the total surfactant dosage was varied, whereas the ratio of surfactant was unchanged and the preparation temperature and emulsification method remained constant. When the preparation temperature was evaluated, the surfactant ratio, surfactant dosage, and emulsification method were kept constant. In addition, the effect of different emulsification methods on the stability of nanoemulsions was examined. Two emulsification methods were included in the experimental design:

aqueous phase (deionised water) → oil phase (Del + S-100 + DMF + surfactant);

(R1)

oil phase (Del + S-100 + DMF + surfactant) → aqueous phase (deionised water).

(R2)

2.3. Observation of the Appearance of Nanoemulsions

Freshly prepared nanoemulsions were aliquoted into 4 mL glass vials. These nanoemulsions were stored at (54 ± 2) °C for 14 d to assess their stability during hot storage and at (0 ± 2) °C for 7 d to assess their stability during cold storage. Subsequently, the physical appearance of the nanoemulsions was carefully examined.

2.4. Droplet Diameter Determination

After the cold and hot storage tests, the nanoemulsions were diluted with deionised water, and their mean droplet size and distribution were measured using dynamic light scattering (Zetasizer Nano ZS90, Malvern Instruments Co., Worcestershire, UK).

2.5. Stability Analysis

The stability of nanoemulsions was determined through backscattering profiling using a Turbiscan Lab Tower system (Formulaction Instrument Co., Raymondis, France) equipped with paired detectors and an 880 nm near-infrared laser [29]. Each nanoemulsion was scanned every 10 min over a 10 h period at a constant temperature of 25 °C. The Turbiscan Stability Index (TSI) was determined by analysing internal physicochemical variations. A lower TSI value indicated fewer variations in backscattering profiles over time, reflecting greater stability [30].

2.6. Wettability Analysis

The leaf surface wettability of Del@Ne was assessed by measuring the contact angle and adhesion using an SL200KB contact angle analyser (24 V, 3000 mA, Shanghai Soren Information Technology Co., Shanghai, China). Leaf samples were mounted on glass slides, and droplets were applied using a microsyringe. Three replicates were prepared for each treatment, with deionised water serving as the control [31,32]. Similarly, Del@Ne and Del@EC were diluted to a certain multiple, and their surface tensions were measured at ambient temperature using the hanging plate method. The instrument used is a JK99B surface tension meter (220 V, 50 Hz), manufactured by Shanghai Zhongchen Digital Technology Equipment Co., Ltd (Shanghai, China). The rainfastness of Del@Ne and Del@EC on peach leaves was evaluated using a simulated rainfall experiment. FITC-labeled formulations were prepared by replacing the active ingredient with FITC and then sprayed onto the surface of peach leaves using a hand-held spray bottle. After air-drying, the fluorescence on the leaf surface was recorded using a plant in vivo imaging system (Tanon-5200Multi, Shanghai Tianneng Technology Co., Ltd., Shanghai, China). The leaves were then rinsed with deionized water, and the fluorescence status of the leaf surface was recorded again using the same imaging system [33,34].

2.7. Bioassay

The bioactivity of Del@Ne against M. persicae was investigated using the leaf dipping method. Briefly, gradient solutions of Del@Ne or Del@EC were prepared at concentrations of 400, 200, 100, 50, and 25 mg L⁻¹. Peach leaves were soaked and air-dried in the dark. The leaves were immersed in the nanoemulsions in Petri dishes, and 20 M. persicae individuals were added. Three replicates were prepared for each concentration, with the deionised water serving as the control. All bioassays were conducted at 25 °C and 50% relative humidity in an growth chamber and a photoperiod of 14 h:10 h (light/dark).

2.8. Safety Evaluation of Del@Ne

2.8.1. Acute Toxicity of Del@Ne to Zebrafish

The pyrethroid insecticide deltamethrin shows remarkably high acute toxicity when exposed to aquatic organisms [35]. Therefore, evaluating its environmental impact and toxicity is necessary. Herein, zebrafish with similar body sizes were selected as the test organism. Del@Ne and Del@EC were added to water to prepare five concentrations. Three replicates were prepared for each concentration, with deionised water serving as the control. The mortality of zebrafish was observed after 24, 48, 72 and 96 h of exposure to Del nanoemulsions or Del@EC. Zebrafish were considered dead if they exhibited no reaction to tactile stimuli.

2.8.2. Acute Toxicity of Del@Ne to Earthworms

To evaluate the potential ecotoxicological effects of Del@Ne on terrestrial invertebrates, Eisenia fetida was selected as the test organism in accordance with the standard protocol outlined in GB/T 31270.15-2014 [36]. Equal concentrations of Del@Ne and Del@EC were thoroughly mixed with a defined quantity of soil, and approximately 30% soil moisture content was achieved by adding deionised water. The earthworms were placed in incubators for culture, and their mortality rates were recorded on days 7 and 14 to assess acute toxicity associated with each treatment.

2.8.3. Acute Toxicity of Del@Ne to Harmonia Axyridis

To access the contact toxicity, Del@EC and Del@Ne series test solutions were prepared at concentrations of 0.25, 0.5, 1, 2, and 4 mg L⁻¹. A 9 cm Petri dish with filter paper was uniformly moistened with 1 mL of solution and air dried for 30 min at 25 °C. Ten second-instar larvae of Harmonia axyridis were placed in a Petri dish and fed enough atoxic aphids, and each treatment was replicated three times. After touching the larva with a fine needle, if there is no reaction, it is judged to be dead. The mortality of Harmonia axyridis was observed after 24 and 48 h [37].

2.9. Effect of Del@Ne on L02 Cells

L02 hepatocytes were used to assess the potential toxic effects of nanoemulsion on human cells [38]. L02 cells were provided by the Medical College of Yangzhou University and cultured in Dulbecco’s modified Eagle’s medium supplemented with foetal bovine serum and incubated at 37 °C with 5% CO₂. Fifth-generation cells were used in the experiments. The cytotoxicity of Del@Ne and Del@EC was assessed using CCK-8 assay [39]. Briefly, L02 cells were cultured in 96-well plates and exposed to equal concentrations of Del@Ne or Del@EC. Subsequently, the cells were incubated with CCK-8 reagent (10 μL/well) at 37 °C for 24 and 48 h. Absorbance was measured at 450 nm, and cell viability was calculated using the formula shown below.

$$\mathrm{Cell} \mathrm{viability} (\%) ={\displaystyle \frac{\mathrm{OD}\left(\mathrm{experiment}\right)\mathbf{ }-\mathbf{ }\mathrm{OD}\left(\mathrm{blank}\right)}{\mathrm{OD}\left(\mathrm{control}\right)\mathbf{ }-\mathbf{ }\mathrm{OD}\left(\mathrm{blank}\right)}}\times 100\%$$

(1)

To evaluate cell apoptosis, L02 cells reached logarithmic growth and were seeded into a 6-well plate at a density of 1 × 10⁵ cells mL⁻¹. The cultured cells were treated with equal concentrations of Del@Ne or Del@EC, along with water controls, for 24 h. After a 5 min centrifugation (1000 r min⁻¹), cells were harvested from six plates, washed three times with prechilled PBS, subjected to trypsinization, and finally resuspended in 100 μL of mixed media buffer. The cell suspension was then stained with 5 μL Annexin V-FITC and 10 μL PI, followed by a further 15 min incubation [40]. All sample processing was performed with light protection. After cryogenic freezing, samples were analyzed by flow cytometry (BD-FACS-Calibur, Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and the measured data were analyzed using FlowJo v10 software.

2.10. Statistical Analysiss

Duncan’s multiple range test was used to analyse the differences among different treatment groups, and different letters were considered significant (p < 0.05). Statistical analyses were performed using the DPS software (v9.01). A probit regression model was applied to calculate the LC₅₀ values.

3. Results

3.1. Effects of Surfactant Ratio

Achieving stable emulsions with a sole surfactant remains challenging in practical pesticide formulation [41]. Therefore, the use of a compound surfactant may result in the formation of a stable nanoemulsion. In this process, the ratio of surfactants is an important factor that determines the stability of the nanoemulsion. Figure 1A shows the appearance of new nanoemulsions prepared at different surfactant ratios, those stored under cold conditions for 7 d and those stored under hot conditions for 14 d. Newly prepared nanoemulsions with surfactant ratios of 4:1 and 12:1 appeared milky white, whereas those with a surfactant ratio of 8:1 showed a light blue lustre. The transparency and glossiness of the 8:1 nanoemulsion were better than those of the 4:1 and 12:1 nanoemulsions. The appearances of the 4:1 and 12:1 nanoemulsions after cold and hot storage were notably different from those of the corresponding newly prepared nanoemulsions, whereas the appearance of the 8:1 nanoemulsion showed no substantial changes and was more stable.

Figure 1B and Figure S1 show the particle sizes and distribution of nanoemulsions under different storage conditions and durations. The 4:1 nanoemulsion exhibited the largest particle size after formation (approximately 160 nm), followed by the 12:1 (76 nm) and 8:1 (43 nm) nanoemulsions. After 7 d of cold storage, the particle size of the 4:1 nanoemulsion significantly decreased to approximately 61 nm, whereas that of the 12:1 nanoemulsion increased to 151 nm. On the contrary, the 8:1 nanoemulsion showed only a slight increase in particle size. After 14 d of hot storage, an overall decreasing trend was observed in the particle sizes of all three formulations, with the 8:1 nanoemulsion demonstrating the highest stability.

The Turbiscan Lab Tower is a tool for monitoring variations in droplet size and dispersed phase concentration throughout the storage period, and the TSI serves as a crucial parameter for evaluating the stability of dispersion systems [42]. In this study, the stability of each nanoemulsion was assessed by evaluating its corresponding TSI value, with smaller TSI values indicating better dispersion stability. To identify the optimal surfactant ratio for nanoemulsion preparation, the TSI values of nanoemulsions with different surfactant ratios were compared. As depicted in Figure 1C, the 8:1 nanoemulsion showed the lowest TSI value, indicating the best stability, followed by the 4:1 and 12:1 nanoemulsions.

Furthermore, consistent results from three characterisation techniques confirmed that the nanoemulsion prepared at a surfactant ratio of 8:1 exhibited superior stability. Comparable findings have been reported in previous studies. When two surfactants are combined, different types of surfactants exhibit synergistic adsorption at the oil–water interface, enabling the rapid formation of a dense and robust interfacial film that significantly reduces interfacial tension. Simultaneously, the mixed system can balance both electrostatic repulsion and steric hindrance effects, enhancing the repulsion between droplets and reducing coalescence and flocculation after collisions [43].

3.2. Effects of Surfactant Dosage

The successful preparation of pesticide nanoemulsions also depends on the appropriate surfactant dosage [44]. Herein, the nanoemulsion with 4% surfactant showed a milky white appearance, whereas those with 6% and 8% surfactant had a light blue lustre and were more transparent (Figure 2A). After cold and hot storage, the nanoemulsion with 4% surfactant appeared milky white, and those with 6% and 8% surfactant appeared blue and transparent. No precipitation or crystallisation was observed in any of the three nanoemulsions.

Figure 2B and Figure S2 show changes in the particle sizes and distribution of nanoemulsions formulated with varying surfactant concentrations at different storage temperatures. An increase in the surfactant concentration led to a notable reduction in the particle size of nanoemulsions. In particular, the nanoemulsion with 4% surfactant had the largest particle size (approximately 120 nm), whereas those with 6% and 8% surfactant had significantly smaller particle sizes (42.5 and 28.4 nm, respectively). After cold and hot storage, the nanoemulsion containing 4% surfactant showed substantial fluctuations in particle size, indicating poor stability. Conversely, the particle sizes of nanoemulsions with 6% and 8% surfactant showed minor variations during storage, demonstrating superior stability.

The TSI values of nanoemulsions formulated with varying surfactant concentrations are presented in Figure 2C. The nanoemulsion formulated with 6% surfactant had the smallest TSI value (approximately 1), which indicated superior stability. The nanoemulsion with 4% surfactant had a slightly higher TSI value (approximately 2), whereas the nanoemulsion with 8% surfactant had the highest TSI value (>2.3). These results indicate that increasing surfactant dosage did not continuously improve nanoemulsion stability. Similar results have been reported in previous studies. At low surfactant dosage, incomplete interfacial coverage provides insufficient protection against droplet coalescence. When the surfactant dosage increases to an optimal level, the interfacial tension is reduced and droplet stabilization is enhanced. However, further increasing the surfactant dosage leads to interfacial saturation, and the excess surfactant remains in the aqueous phase as micellar aggregates, which may induce oil-phase solubilization and weaken emulsion stability, thereby causing larger droplet size [45].

3.3. Effects of Preparation Temperature

The physical appearances of nanoemulsions at varying preparation temperatures are depicted in Figure 3A. Immediately after preparation, the nanoemulsion synthesised at 55 °C exhibited a milky white appearance, whereas those prepared at 25 °C and 40 °C showed a light blue lustre with translucence. After 7 d of cold storage and 14 d of hot storage, the appearances of the nanoemulsions prepared at 25 °C and 40 °C remained relatively stable with no evident changes. However, the nanoemulsion prepared at 55 °C gradually turned transparent during storage, indicating poor stability.

Figure 3B and Figure S3 show the particle sizes and distribution of nanoemulsions prepared at different temperatures at different time points. The nanoemulsion prepared at 55 °C had the largest particle size (85.1 nm), followed by the nanoemulsions prepared at 25 °C (56.5 nm) and 40 °C (43 nm). After cold and hot storage, the nanoemulsion prepared at 40 °C showed the smallest change in particle size.

Figure 3C presents the TSI values of nanoemulsions prepared at different temperatures. The nanoemulsion formulated at 55 °C had the highest TSI value (exceeding 10), demonstrating the poorest stability. The TSI value of the nanoemulsion prepared at 25 °C was intermediate (approximately 8), indicating moderate stability. Notably, the nanoemulsion formulated at 40 °C had the lowest TSI value (approximately 1), demonstrating superior dispersion stability. At moderate preparation temperatures, the reduced viscosity of the system facilitates droplet disruption during homogenization, leading to a more uniform dispersion and improved stability. However, when the temperature is further increased, the viscosity decreases further and Brownian motion becomes more intense, which increases droplet collision frequency and promotes aggregation, thereby reducing nanoemulsion stability [46].

3.4. Effects of the Emulsification Method

The quality and stability of nanoemulsions are profoundly affected by the emulsification method employed [47]. The appearances of nanoemulsions prepared using different emulsification methods are shown in Figure 4A. The newly prepared P1 nanoemulsion exhibited a clear appearance with a characteristic light blue lustre. On the contrary, the P2 nanoemulsion showed significant creaming immediately after preparation, with the upper layer appearing milky white and the lower layer appearing clear with a faint blue tint. After cold storage, the appearance of the P1 nanoemulsion remained largely unchanged, indicating good stability. However, in the P2 nanoemulsion, the lower transparent blue layer gradually turned milky white over time. After 14 d of hot storage, the P1 nanoemulsion continued to demonstrate excellent stability, whereas the initial creaming observed in the P2 nanoemulsion disappeared, and the entire liquid turned uniformly clear with a light blue lustre.

The particle sizes and distribution of nanoemulsions prepared using different emulsification methods are presented in Figure 4B and Figure S4. The P1 nanoemulsion showed excellent stability after new preparation (42.8 nm), 7 d cold storage (51.2 nm) and 14 d hot storage (37.8 nm). However, the P2 nanoemulsion had poor stability. Its particle size was larger (500.8 nm) after new preparation, reduced to 422.3 nm after cold storage and sharply decreased to 41.9 nm after hot storage.

Figure 4C shows the TSI values of nanoemulsions prepared using different emulsification methods. The P2 nanoemulsion had a significantly higher TSI value than the P1 nanoemulsion (>50). The poor stability of P2 was closely related to the difference in emulsification pathway caused by the mixing order. When water was gradually added into the oil phase (P1), surfactant molecules could more effectively adsorb at the newly generated oil–water interface, thereby facilitating the formation of relatively small and uniform droplets with improved interfacial stability. In contrast, when the oil phase was added into the aqueous phase (P2), the transient continuous phase and surfactant distribution during emulsification were substantially altered, resulting in an unfavorable phase inversion pathway for this formulation. Consequently, the interfacial film was not formed rapidly enough to prevent droplet coalescence, which led to severe creaming and phase separation. Similar results were also reported by Shi et al. in their study on the effect of emulsification methods on emulsion stability [48]. Therefore, P1 was selected as the suitable emulsification method for subsequent optimization.

3.5. Quality Indices of the Optimised Nanoemulsion

In accordance with the FAO standards, the quality of the optimised nanoemulsion was assessed using a Collaborative International Pesticides Analytical Council method [49]. The detailed quality parameters obtained from this evaluation are summarised in

Table 1. Quality indices of the optimised nanoemulsion. MT * (Miscellaneous Techniques) from CIPAC Handbook.

Quality Indices	CIPAC Method	Result
Content of Deltamethrin		≥2.5%
pH value	MT31 *	4.1
Emulsion Stability and Re-emulsification	MT36.1 *	Emulsified completely after dilution. No oil slick or emulsifying cream was found after dilution for 0.5 and 2 h. After dilution for 24 h, it was completely reemulsified. No oil slick or emulsifiable paste after dilution for 0.5 h.
Persistent foaming	MT47.2 *	≤2.5 mL after 1 min.
Storing stability Storage at 54 °C	MT46.3 *	Content: ≥2.5%, pH: 4.07, emulsion stability and re-emulsification: same as mentioned above, continue foam after 1 min: ≤2.0 mL.
Storage at 0 °C	MT39.3 *	No oily substances or macroscopic droplets.

. Results showed that the optimised nanoemulsion met the essential quality criteria specified by the FAO guidelines.

3.6. Wetting Property

As shown in Figure 5A, the contact angles of Del@EC and Del@Ne on peach leaves were very small and gradually decreased over 120 s, indicating excellent wettability of the two formulations. The contact angle of Del@Ne on peach leaves (40.4–21.6°) was smaller than that of Del@EC (43.6–32.1°), indicating that the formulated nanoemulsion exhibits better wetting property [50,51].

Smaller contact angles are correlated with a higher work of adhesion. As shown in Figure 5B,C, Del@Ne had the highest work of adhesion, followed by Del@EC and deionised water. Analysis of the surface tension validated these results. Del@Ne (31.0 mN m⁻¹) had lower surface tension than Del@EC (41.0 mN m⁻¹) and deionised water (73.1 mN m⁻¹). The enhanced wettability of Del@Ne can facilitate its more efficient absorption on peach leaves after spraying, thereby improving pest control efficacy [52]. The rainfastness performance was evaluated by fluorescence imaging of peach leaves treated with FITC-labeled formulations (Figure 5D). Before rinsing, strong fluorescence was observed on the leaf surfaces of both Del@EC and Del@Ne, indicating effective coverage of the formulations. After simulated washing, although the fluorescence in-tensity decreased, a substantial amount of fluorescence signal remained on the Del@Ne-treated leaves, while much less fluorescence was retained on the Del@EC-treated leaves. This result suggests that Del@Ne possesses better rainfastness than Del@EC.

3.7. Bioactivity Analysis

The bioactivities of Del@EC and Del@Ne against M. persicae at different concentrations are shown in Figure 6. The insecticidal activity of Del@Ne against M. persicae was consistently better than that of Del@EC. The lethal concentration 50% (LC₅₀) of the two formulations against M. persicae is shown in

Table 2. The LC₅₀ of Del@EC and Del@Ne against M. persicae.

Title 1	LC50 (mg L−1)	95% Confidence Interval	Linear Fitting Equation	R2
Del@EC	80.69	62.92~103.47	y = 1.819x + 1.531	0.977
Del@Ne	66.85	53.81~83.06	y = 1.949x + 1.441	0.984

. The LC₅₀ of Del@EC and Del@Ne against M. persicae was 80.69 and 66.85 mg L⁻¹, respectively, indicating that Del@Ne had better control effect than Del@EC. The high insecticidal activity of Del@Ne may be attributed to its reduced droplet size. Owing to its larger specific surface area, Del@Ne can facilitate more effective contact and permeation, accelerating pesticide delivery and increasing bioactivity [53,54].

3.8. Safety Evaluation

3.8.1. Acute Toxicity to Zebrafish

As a key experimental model, zebrafish are instrumental in assessing the acute toxicity of pesticides for environmental risk analysis. As shown in

Table 3. The LC₅₀ values of Del@EC and Del@Ne against zebrafish at different time points.

Exposure Time	LC50 (95% Confidence Interval) (mg L−1)
	Del@EC	Del@Ne
24 h	0.0026 (0.0014–0.0048)	0.0032 (0.0016–0.0063)
48 h	0.0021 (0.0012–0.0034)	0.0026 (0.0013–0.0049)
72 h	0.0015 (0.0010–0.0023)	0.0020 (0.0009–0.0045)
96 h	0.0012 (0.0006–0.0023)	0.0017 (0.0009–0.0031)

, the acute toxicity of Del@EC and Del@Ne was assessed in zebrafish at 24, 48, 72 and 96 h. Del@Ne influenced zebrafish mortality in a concentration dependent manner, with LC₅₀ values of 0.0032, 0.0026, 0.002 and 0.0017 mg L⁻¹ at 24, 48, 72 and 96 h, respectively. Compared with Del@EC, Del@Ne showed significantly reduced toxicity to zebrafish at all time points. These findings suggest that the Del nanoemulsion prepared in this study exhibits favourable biosafety [55].

3.8.2. Acute Toxicity to E. fetida

Owing to their sensitivity to soil pollutants such as pesticides, earthworms serve as key model organisms in soil ecotoxicology.

Table 4. The LC₅₀ values of Del@EC and Del@Ne against E. fetida after 7 and 14 d of exposure.

Exposure Time	Del@EC		Del@Ne
	Regression Equation	LC50 (mg kg−1) 95% Confidence Interval	Regression Equation	LC50 (mg kg−1) 95% Confidence Interval
7 d	y = 1.975x + 0.582	172.69 (164.55–181.24)	y = 1.463x + 1.359	307.69 (256.23–369.50)
14 d	y = 3.939x − 3.008	106.56 (45.08–251.88)	y = 1.638x + 1.185	212.68 (175.86–257.21)

presents the results of the acute toxicity assessment of Del@EC and Del@Ne in earthworms after 7 and 14 d of exposure. The 7 d LC₅₀ values of Del@EC and Del@Ne against E. fetida were 172.69 and 307.69 mg kg⁻¹, respectively. However, after 14 d of exposure, these values decreased to 106.56 and 212.68 mg kg⁻¹, respectively. Compared with Del@EC, Del@Ne had lower acute toxicity to earthworms, which can be attributed to its environmentally friendly formulation, as well as fewer solvents and surfactants [56,57].

3.8.3. Acute Toxicity to Harmonia Axyridis

The toxicity of Del@EC and Del@Ne against second-instar larvae of Harmonia axyridis was determined after 24 h and 48 h (

Table 5. The LC₅₀ values of Del@EC and Del@Ne against Harmonia axyridis after 24 h and 48 h.

Exposure Time	Del@EC		Del@Ne
	Regression Equation	LC50 (mg L−1) 95% Confidence Interval	Regression Equation	LC50 (mg L−1) 95% Confidence Interval
24 h	y = 1.713x + 5.574	0.46	y = 1.135x + 4.289	4.23
48 h	y = 1.680x + 5.626	0.42	y = 1.123x + 4.334	3.92

). The 24 h and 48 h LC₅₀ values of Del@EC against Harmonia axyridis were 0.46 mg L⁻¹ and 0.42 mg L⁻¹, respectively. In contrast, Del@Ne exhibited higher LC₅₀ values, recorded as 4.23 mg L⁻¹ at 24 h and 3.92 mg L⁻¹ at 48 h. These comparative findings highlight that the Del@Ne is safer than Del@EC for Harmonia axyridis. Compared with Del@EC, Del@Ne contained fewer organic solvents and surfactant [58].

3.9. Cytotoxicity Evaluation

Exposure to pesticides during application may result in their absorption into the applicator’s body. The toxic effects of Del@Ne on L02 cells were evaluated after 24 and 48 h of incubation using CCK-8 assay (Figure 7A,B). After exposure to 50 mg L⁻¹ Del@Ne for 24 h, cell viability was 88.09%. However, treatment with Del@EC at the same concentration resulted in a lower survival rate (80.03%). After 48 h, the survival rate of cells treated with 10 mg L⁻¹ Del@EC decreased to 52.6%, whereas that of Del@Ne remained higher (76.31%). This difference may be related to the reduced amount of organic solvent in the nanoemulsion, which could alleviate formulation-induced cellular stress [40].

Flow cytometry was used to determine the apoptosis rate of L02 cells to evaluate the potential cytotoxicity of Del@EC and Del@Ne to humans. In the quadrant distribution results, Q1 represented living cells; Q2 and Q3 represented early and late apoptotic cells, respectively; and Q4 represented necrotic cells. The degree of cellular damage observed varied according to the specific Del formulation applied. The apoptosis rates of the blank control, Del@Ne and DeL@EC groups were 4.35%, 7.96% and 11.04%, respectively. These findings indicated that the Del nanoemulsion induced less severe apoptosis than Del@EC. Therefore, Del@Ne exhibited lower toxicity to L02 cells than Del@EC. The stronger adverse effects of Del@EC on human cells may be attributed to the presence of more organic solvents and surfactants in this commercial formulation [59].

4. Conclusions

This study evaluated three key parameters (appearance, droplet size, and TSI) for Del nanoemulsions. Results indicated that surfactant ratio, surfactant dosage, preparation temperature, and emulsification method significantly affected nanoemulsion stability. The optimal formulation (P1) consisted of 2.5% Del, 6% surfactant (#601:#500 = 8:1), 17.25% oil phase, with deionised water added up to 100%. The prepared Del nanoemulsion exhibited favourable stability and superior wettability on peach leaves. Moreover, compared with Del@EC, Del@Ne showed enhanced insecticidal activity against M. persicae and lower toxicity to zebrafish, earthworms, Harmonia axyridis, and human L02 cells. Stable Del nanoemulsions are the fundamental guarantee for achieving excellent wettability, rainfastness, and high insecticidal activity. Meanwhile, the reduction of organic solvents and surfactants in nanoemulsions contributes to improved safety for non-target organisms. This work provides a reliable reference for the rational design and optimisation of pesticide nanoemulsions. Nevertheless, several challenges remain to be addressed: (1) the effects of salt ions and dilution ratios on nanoemulsion stability; (2) the protective and controlled-release functions of active ingredients; (3) the application of eco-friendly additives; (4) toxicity evaluation against more non-target organisms. Effective solutions to these issues will promote the extensive application of nanoemulsions in agriculture.