Microemulsion-based delivery of peppermint essential oil as a sustainable strategy for Aedes aegypti control

Arboviral diseases such as dengue, Zika, and chikungunya remain major public health challenges worldwide. In the absence of broadly effective antiviral therapies and considering the limitations of current vaccination strategies, vector control remains the primary approach for reducing disease transmission. Essential oils have emerged as promising alternatives to synthetic insecticides because of their renewable origin, biodegradability, and multiple mechanisms of action. Nevertheless, their practical application is often limited by poor water solubility and physicochemical instability. This study investigated the chemical composition, larvicidal activity, and microemulsion-based formulation of Mentha piperita L. essential oil against Aedes aegypti larvae. The chemical composition of the essential oil was determined by gas chromatography–mass spectrometry (GC–MS). Larvicidal activity against third-instar A. aegypti larvae was evaluated by determining mortality rates and the median lethal concentration (LC 50). A pseudoternary phase diagram was constructed to identify suitable microemulsion regions, and selected formulations were characterized according to droplet size, polydispersity index (PDI), and short-term physical stability. The larvicidal activity of the resulting microemulsions was subsequently evaluated at different essential-oil concentrations. A blank microemulsion containing the formulation components without M. piperita essential oil was also evaluated as a control. GC–MS analysis identified 14 compounds representing nearly 100% of the essential oil composition, with menthol (50.17%) and menthone (23.02%) as the predominant constituents. The free essential oil exhibited concentration-dependent larvicidal activity against A. aegypti, with an LC 50 of 175.7 μg/mL and complete larval mortality at 250 μg/mL. Among the tested formulations, PME-05 was selected as the most promising formulation for further development because it achieved complete larval mortality while containing only 5% peppermint essential oil and exhibited the lowest PDI (0.06 ± 0.007), indicating a highly homogeneous dispersed system. The results support the use of microemulsion technology as a suitable delivery system for M. piperita essential oil, facilitating its dispersion and application in aqueous environments while retaining its larvicidal activity. The developed formulation represents a promising platform for the application of botanical larvicides in mosquito control. Further studies are needed to evaluate long-term physicochemical stability, residual activity, effects on non-target organisms, and performance under semi-field and field conditions.

Arboviral diseases represent a growing challenge for public health systems worldwide. Over the last decades, dengue, Zika, and chikungunya have undergone remarkable geographic expansion, driven by urbanization, climate change, and increased human mobility, leading to recurrent outbreaks and placing an increasing proportion of the global population at risk of infection (Abbasi, 2026). Among these diseases, dengue remains the most prevalent mosquito-borne viral infection, causing millions of cases annually and imposing a substantial burden on healthcare systems, particularly in tropical and subtropical countries (Ferreira et al., 2016; Pourzangiabadi et al., 2025; Ferreira et al., 2026). Beyond their direct effects on morbidity and mortality, arboviral infections are associated with significant socioeconomic impacts, including healthcare expenditures, productivity losses, and long-term neurological and developmental complications, as observed during recent Zika virus epidemics (Abbasi, 2026; Ryan et al., 2019).

The transmission dynamics of these diseases are strongly influenced by environmental and anthropogenic factors that favor mosquito proliferation and dispersal. Rising temperatures, inadequate sanitation, rapid urbanization, and the widespread availability of breeding sites have facilitated the expansion of Aedes aegypti and Aedes albopictus, reinforcing the need for effective and sustainable vector control strategies (Abbasi, 2026; Ryan et al., 2019). In the absence of broadly effective antiviral therapies and considering the of current vaccination strategies, vector control remains the cornerstone of arboviral disease prevention (Wilson et al., 2020). However, the extensive use of synthetic insecticides has led to the emergence of resistant mosquito populations while raising concerns regarding environmental contamination and toxicity to non-target organisms. These challenges have intensified the search for safer and more sustainable alternatives for mosquito management (Wilson et al., 2020; Abbasi, 2026).

Among the alternatives under investigation, plant-derived products have attracted considerable attention as sources of bioactive compounds for vector control. Essential oils are complex mixtures of terpenes and other secondary metabolites capable of acting on multiple physiological targets in insects, thereby reducing the probability of resistance development. Their renewable origin, biodegradability, and broad spectrum of biological activities make them promising candidates for the development of environmentally friendly larvicidal agents (Hosseinpour et al., 2026; Santana et al., 2026). Supporting this potential, a comprehensive review by Luz et al. reported that more than 337 essential oils obtained from 225 plant species have been evaluated against A. aegypti, with over 60% demonstrating significant larvicidal activity (LC 50 < 100 μg/mL). Notably, Mentha piperita L (peppermint) was identified among the most active species, highlighting its potential as a source of natural larvicidal compounds (Luz et al., 2020).

Despite their biological potential, the practical application of essential oils remains limited by their hydrophobicity, volatility, and susceptibility to oxidation, which compromise their stability and persistence in aquatic breeding sites. Consequently, considerable efforts have focused on developing delivery systems capable of improving the stability, dispersion, and biological performance of these natural products (Chaudhary et al., 2024; Silva et al., 2025; Luz et al., 2020).

Among the available formulation approaches, microemulsions have emerged as particularly attractive delivery systems for essential oils and other lipophilic bioactive compounds. These thermodynamically stable and isotropic systems, composed of oil, water, surfactants, and often co-surfactants, enhance the aqueous dispersion and physicochemical stability of essential oils while facilitating their interaction with biological targets. In addition, microemulsions may improve the bioavailability and controlled release of incorporated compounds, making them promising platforms for developing sustainable larvicidal formulations (Chaudhary et al., 2024; Lawrence and Rees, 2012).

Although the larvicidal potential of M. piperita essential oil has been previously reported, studies integrating comprehensive chemical characterization, rational microemulsion development, physicochemical characterization, and standardized larvicidal evaluation against A. aegypti remain limited. Addressing this gap, the present study characterized the chemical composition of M. piperita essential oil, developed stable microemulsion formulations, and evaluated their larvicidal activity against A. aegypti larvae. By integrating phytochemical analysis, formulation science, and biological evaluation, this work provides new evidence supporting the development of sustainable natural product-based technologies for mosquito control and arboviral disease prevention.

Raw materials

Mentha piperita L (peppermint) essential oil was purchased from a certified commercial supplier (Phytoterpica®, Brazil) and stored in amber glass vials at 4 °C until use. Ethyl acetate (HPLC grade; Merck, Germany), Polysorbate 20 (Tween® 20; Sigma-Aldrich, United States), propylene glycol (Synth, Brazil), and ethyl oleate (Sigma-Aldrich, United States) were used for microemulsion preparation. Ultrapure water was obtained from a Milli-Q® purification system (Millipore, United States). A homologous series of n-alkanes (C7–C40 Saturated Alkanes Standard; Supelco, United States) was used for the calculation of linear retention indices during GC–MS analysis.

Aedes aegypti larvae used in this study were obtained from a laboratory colony established from eggs collected in ovitraps installed at Fundação Ezequiel Dias (FUNED), Belo Horizonte, Brazil. The colony has been maintained under controlled insectary conditions for more than 5 years through successive generations and, therefore, does not correspond to a reference strain. The research activities were registered in SisGen (registration no. A00AD3B), and mosquito blood feeding on mice was performed under CEUA approval (protocol no. 001/2026).

Chemical characterization by GC–MS

The chemical profile of peppermint essential oil was determined by gas chromatography coupled to mass spectrometry (GC–MS) using a GCMS-QP 2010 Ultra system (Shimadzu, Kyoto, Japan) equipped with a SH-RTx-5MS fused-silica capillary column (30 m × 0.25 mm i. d.; 0.25 μm film thickness; 5% diphenyl/95% dimethylpolysiloxane stationary phase). Prior to analysis, the essential oil was diluted in ethyl acetate (20 μL in 980 μL).

Chromatographic separation was performed with the oven initially maintained at 60 °C for 3 min, followed by a temperature ramp of 10 °C min -1 up to 290 °C, where it was held for 30 min. Helium was employed as the carrier gas at a constant flow rate of 0.92 mL min -1. The injector temperature was set at 220 °C, and samples were introduced using a split ratio of 1:5. Mass spectra were acquired under electron ionization (EI) at 70 eV within a scan range of m/z 20–500 (Ribeiro et al., 2025).

Compound identification was achieved through comparison of the obtained mass spectra with those available in the NIST 11 spectral library, together with the calculation of linear retention indices (LRI). For this purpose, a homologous series of n-alkanes (C7–C40) was analyzed under the same chromatographic conditions, using a 2% (v/v) solution in ethyl acetate. The calculated LRI values were subsequently compared with literature data to confirm compound assignments (Ribeiro et al., 2025; Luz et al., 2020). The essential oil composition was determined from three independent injections analyzed under the conditions described above. where: LRI - linear retention index; Tx - retention time of the compound of interest; Tn–retention time of the previous alkane; Tn+1 - retention time of the subsequent alkane; n - number of carbons from the previous alkane.

Development and physicochemical characterization of microemulsions

A pseudoternary phase diagram was constructed as an exploratory approach to identify composition regions capable of forming transparent and macroscopically homogeneous microemulsion systems. Different proportions of M. piperita essential oil, ultrapure water, and a surfactant/co-surfactant mixture (Smix) were evaluated. The Smix consisted of the selected surfactant (Polysorbate 20) and co-surfactant (propylene glycol) at different ratios, while the relative proportions of essential oil, Smix, and water were varied to map the phase behavior of the system.

Formulations were prepared by mixing the oil phase with the surfactant under magnetic stirring, followed by the gradual addition of the co-surfactant. After homogenization, the aqueous phase was slowly incorporated under continuous stirring until transparent and homogeneous systems were obtained. The resulting formulations were visually evaluated for transparency, homogeneity, phase separation, and precipitation (Ribeiro et al., 2025; Fialho and Da Silva-Cunha, 2004). The compositions investigated were subsequently plotted in a pseudoternary phase diagram, allowing the identification of regions corresponding to stable microemulsions. Selected formulations were stored for 7 days prior to physicochemical characterization (Kreilgaard, 2002; Lawrence and Rees, 2012).

The mean droplet size and polydispersity index (PdI) of the microemulsions were determined by dynamic light scattering using a Zetasizer Nano ZS (ZEN3600, Malvern Instruments) analyzer at 25 °C. Samples were diluted (1:10) with distilled water prior to analysis to avoid multiple scattering effects. Measurements were performed in triplicate, and results were expressed as mean ± standard deviation.

Larvicidal bioassay against Aedes aegypti

Aedes aegypti eggs were obtained from a laboratory colony maintained under controlled conditions. Larvae were reared in dechlorinated water at 25 °C ± 2 °C, with a photoperiod of 12 h light/12 h dark, and fed with a standardized diet. Third-instar larvae were used for all larvicidal bioassays.

Larvicidal activity was evaluated according to WHO guidelines and methodologies commonly employed in studies with essential oils against A. aegypti larvae (Brandão et al., 2021; Faustino et al., 2020; Organization, W. H. 2005). Groups of 10 third-instar A. aegypti larvae were exposed to different concentrations of M. piperita essential oil or microemulsion formulations in disposable containers containing dechlorinated water. Tested concentrations ranged from 91 to 912 μg/mL. Control groups consisted of larvae exposed to water and blank microemulsions (without peppermint oil). Larval mortality was recorded after 24 h of exposure. Larvae were considered dead if they showed no movement after gentle prodding. All experiments were performed in quadruplicate under controlled environmental conditions.

Statistical analysis

Statistical analysis was performed using RStudio version 2025.05.0. The relationship between concentration and larval mortality was analyzed using nonlinear dose–response models with a binomial distribution in R, using the drc package. Two-parameter sigmoidal models were fitted, including log-normal with Probit link, log-logistic with Logit link, and Weibull type I and II functions, with lower and upper response limits fixed at 0 and 1, respectively. Concentration was treated as a continuous predictor, and mortality proportion as the response variable. For logarithmic model fitting, observations from the same test substance with zero or near-zero concentration values were assigned a pseudoconcentration corresponding to one-tenth of the lowest positive concentration tested; this adjustment was applied only for modeling purposes and did not represent a separate control group. The best-fitting model was selected based on the Akaike Information Criterion (AIC), and the median lethal concentration (LC 50) with its 95% confidence interval was estimated. The association between concentration and mortality was assessed by Probit regression and likelihood ratio tests, adopting a significance level of 5%.

Chemical composition of Mentha piperita essential oil

Since the biological activity of essential oils is closely related to their chemical composition, the first step of this study consisted of characterizing the volatile constituents of M. piperita essential oil. GC–MS analysis resulted in the identification of 14 compounds, accounting for almost 100% of the total oil composition. The chromatographic profile and compound identification are summarized in Table 1, while the representative GC–MS chromatogram is provided as Supplementary Figure S1.

The major compounds were menthol and menthone, and together represented approximately 73% of the total composition, indicating a profile rich in oxygenated monoterpenes (Table 1). The predominance of these constituents is consistent with previous reports for peppermint essential oil, although variations in the relative abundance of individual compounds have been described depending on geographical origin, cultivation conditions, harvesting period, and extraction procedures. Importantly, the presence of menthol, menthone, and other monoterpenes is consistent with previous reports describing larvicidal activity of M. piperita essential oil against A. aegypti and other mosquito vectors (Soltanbeigi et al., 2021; Abbas et al., 2024; Luz et al., 2020). Thus, the chemical profile identified in the present study provides a plausible chemical basis for the biological activity reported for this essential oil in the literature.

The chemical composition of peppermint essential oil is characterized by the coexistence of major and minor constituents, which may contribute collectively to its biological properties. Monoterpenes such as menthol, menthone, and pulegone have been investigated for their potential effects on physiological processes in insects, including neuronal signaling and other cellular pathways (Santana et al., 2026). However, the contribution of individual constituents to the activity of a complex essential oil cannot be inferred solely from their relative abundance, since interactions among multiple components may also influence the overall biological response. Therefore, the chemical profile obtained by GC–MS was considered primarily as a basis for characterizing the essential oil and for contextualizing the larvicidal results presented subsequently.

Larvicidal activity of Mentha piperita essential oil against Aedes aegypti

Previous studies have reported significant larvicidal activity of peppermint essential oil against A. aegypti, reinforcing the relationship between its chemical composition and biological activity. In a comprehensive review of essential oils evaluated against mosquito larvae, M. piperita was identified among the most promising species, exhibiting pronounced larvicidal effects that have been largely attributed to its high content of oxygenated monoterpenes, particularly menthol and menthone (Luz et al., 2020; Pavela, 2015). Consistent with these previous reports, the essential oil evaluated in the present study exhibited concentration-dependent activity against A. aegypti larvae, achieving complete mortality at concentrations equal to or greater than 250 μg/mL (Figure 1).

The activity observed in the present study is also consistent with previous reports involving Mentha species. Mentha arvensis essential oil, rich in menthol and menthyl acetate, exhibited significant larvicidal activity against A. aegypti larvae (Manh and Tuyet, 2020). Similarly, the predominance of menthone, menthol, and, to a lesser extent, pulegone in the chemical profile of M. piperita essential oil provides a plausible basis for its observed larvicidal activity.

The concentration–mortality curve obtained for the essential oil (Figure 1) demonstrated a clear dose-dependent larvicidal effect against A. aegypti larvae. Using the classification commonly applied in studies of plant-derived mosquito larvicides, LC 50 values between 100 and 750 μg/mL are generally considered indicative of effective larvicidal activity (Trongtokit et al., 2005; Alyahya, 2023). Probit regression analysis performed in R software estimated a median lethal concentration (LC 50) of 175.7 μg/mL to peppermint oil, indicating effective larvicidal activity under the experimental conditions. This result is consistent with the chemical profile of the oil, which was characterized by compounds frequently associated with insecticidal activity. Furthermore, the observed efficacy reinforces the potential of M. piperita essential oil as a promising alternative to conventional synthetic larvicides, whose long-term use has contributed to the emergence of resistant mosquito populations and raised concerns regarding environmental contamination and toxicity to non-target organisms.

The larvicidal activity of essential oils is likely influenced by the combined action of multiple constituents rather than by a single compound. Monoterpenes have been reported to interfere with several physiological processes in insect larvae, including acetylcholinesterase activity, neuronal signaling, and mitochondrial function and energy metabolism, which may ultimately impair larval mobility and development (Santana et al., 2026). In complex essential oils, interactions among major and minor constituents may also contribute to the overall biological response. Additive or synergistic interactions among components have been proposed as important factors underlying the activity of multicomponent essential oils (Santana et al., 2026; Pavela, 2015).

In addition to the concentration-dependent mortality observed in the larvicidal assays, larvae exposed to peppermint oil exhibited noticeable behavioral and morphological alterations. A progressive reduction in mobility was observed, characterized by slow and uncoordinated movements that ultimately evolved to complete paralysis. Interestingly, some larvae remained alive for a short period after losing locomotor capacity, maintaining only respiratory movements before death. Morphological examination also revealed darkening of the larval body and, in some individuals, disruption of midgut structures and tissue integrity. These alterations suggest that the essential oil affects multiple physiological systems and provide visual evidence supporting its larvicidal activity (Santana et al., 2026).

Overall, the larvicidal activity demonstrated in the present study is consistent with previous reports for M. piperita essential oil and with the chemical profile identified by GC–MS. The predominance of menthol and menthone, together with the presence of other monoterpenes, provides a chemically plausible context for the observed activity, although the contribution of individual constituents and their potential interactions cannot be established from the present experimental design. These findings support the use of M. piperita essential oil as a candidate natural larvicidal agent and provide the basis for its subsequent incorporation into a microemulsion system.

Development and characterization of Mentha piperita microemulsions

The incorporation of peppermint essential oil into a microemulsion system was proposed as a strategy to improve the dispersibility and handling of this hydrophobic material in aqueous media. Microemulsions can provide suitable vehicles for incorporating poorly water-soluble compounds and essential oils, potentially enhancing their practical application in settings such as mosquito breeding sites. Their ability to reduce interfacial tension and promote the spontaneous formation of nanometric droplets makes them attractive systems for the formulation of natural larvicidal agents (Faustino et al., 2020; Luz et al., 2020; Lawrence and Rees, 2012). More recently, micro- and nanoemulsion systems containing essential oils have continued to be investigated as strategies for improving the dispersion and practical application of plant-derived insecticidal agents, particularly in mosquito control (Peniche et al., 2022; Sonter et al., 2024). In the present study, the microemulsion was developed primarily as a formulation strategy to improve the handling and aqueous applicability of M. piperita essential oil, rather than to increase its intrinsic larvicidal potency.

The formulation design was conducted as an exploratory screening of the composition space defined by M. piperita essential oil, Smix, and water (Table 2). Different component ratios were examined to identify the domain in which transparent and macroscopically homogeneous systems could be obtained. The surfactant-to-co-surfactant ratio was also varied during this screening, allowing different interfacial compositions to be assessed in combination with different proportions of essential oil and water. Within the identified domain, six representative compositions (PME-01 to PME-06) were selected to encompass different relative proportions of the oil, Smix, and aqueous phases, as well as distinct surfactant-to-co-surfactant ratios. The resulting formulations therefore differed in essential oil content (2%–20%), Smix content (34%–70%), and water content (10%–64%), allowing the influence of formulation composition on droplet size, PDI, and short-term physical stability to be assessed. The systems were subsequently screened for their suitability for larvicidal evaluation. This approach was exploratory and was intended to identify promising candidate compositions rather than to establish an optimized formulation through a formal statistical design. The use of pseudoternary phase diagrams as an exploratory tool for defining suitable composition domains has also been reported in recent studies involving essential-oil-based microemulsions (Yang et al., 2025).

The pseudoternary phase diagram obtained from the formulation screening is shown in Figure 2. The identified domain comprised compositions that produced transparent and macroscopically homogeneous systems, indicating compatibility among the selected oil, Smix, and aqueous phases within the composition range investigated. The location of the selected formulations within this domain also allowed different combinations of oil, Smix, and water to be examined. The observed results highlight the importance of component ratios in determining the physical properties of the resulting systems. In pseudoternary systems, phase behavior is governed by the balance among the oil phase, aqueous phase, and interfacial components, and changes in composition can modify the extent and characteristics of the single-phase region (Kreilgaard, 2002; Lawrence and Rees, 2012). Consistent with this principle, a recent study of a Forsythia essential oil microemulsion used pseudoternary phase diagrams to identify a suitable domain and subsequently characterized the selected systems in terms of droplet size and PDI (Yang et al., 2025). Thus, the phase diagram in the present study served primarily as a screening tool to identify compositions suitable for subsequent physicochemical and biological evaluation.

The selected formulations exhibited distinct phase behavior depending on their composition, highlighting the role of the relative proportions of essential oil, Smix, and water in determining their properties. A particularly relevant finding was the contrasting behavior of PME-02 and PME-06. PME-02 contained the highest Smix proportion evaluated (70%), together with 20% essential oil and only 10% water, and exhibited phase separation during the storage period. In contrast, PME-06 contained 34% total Smix, 2% essential oil, and 64% water, and maintained a homogeneous appearance throughout the 7-day evaluation period. These findings suggest that microemulsion stability was influenced not simply by the total amount of surfactant/co-surfactant mixture, but by the overall balance among the oil, aqueous, and interfacial components, including the surfactant-to-co-surfactant ratio. Excessive Smix content combined with limited water availability may alter interfacial organization and, depending on the composition, favor the formation of liquid-crystalline or other organized phases that can compromise the physical characteristics of the system (Kreilgaard, 2002; Lawrence and Rees, 2012). Conversely, appropriate combinations of oil, Smix, water, and interfacial components may favor the formation of homogeneous oil-in-water systems. Taken together, the contrasting behavior of PME-02 and PME-06 suggests that overall compositional balance, rather than total Smix content alone, plays an important role in determining the physical behavior of the formulations. This interpretation is consistent with recent work demonstrating that the physicochemical characteristics of essential-oil microemulsions depend strongly on the relative proportions and interactions among the oil, surfactant, co-surfactant, and aqueous phases (Yang et al., 2025).

The partial physicochemical characterization of the six formulations (PME-01 to PME-06) is summarized in Table 3. The systems were assessed in terms of droplet size, polydispersity index (PDI), and short-term physical stability. Most exhibited droplet sizes within the nanometric range (9.6 ± 0.4 to 15.6 ± 6.4 nm) and relatively low PDI values (0.06 ± 0.01 to 0.26 ± 0.27). These results support the formation of microemulsions, as the observed droplet sizes fall within the nanometric range typically reported for these systems (5–200 nm) (Lawrence and Rees, 2012). Differences in short-term physical stability were observed during the 7-day evaluation period. However, these findings should not be interpreted as evidence of long-term stability or shelf life. Additional studies are required to establish storage stability, formulation persistence, and behavior under environmentally relevant conditions, including variations in temperature, water composition, organic matter, and light exposure, which may affect both colloidal organization and the persistence of volatile essential-oil constituents. PME-02 was excluded from further evaluation because phase separation was observed during storage, whereas PME-01, PME-03, PME-04, PME-05, and PME-06 maintained their visual appearance and showed relatively consistent droplet sizes and PDI values throughout the evaluation period. Small droplet sizes and relatively low PDI values are desirable characteristics for essential-oil-based colloidal systems because they indicate a relatively homogeneous dispersed phase and can facilitate the dispersion of hydrophobic materials in aqueous media. Recent studies have demonstrated the feasibility of formulating essential oils as nanoscale dispersed systems while retaining larvicidal activity against mosquito larvae (Peniche et al., 2022; Sonter et al., 2024). The combination of nanometric droplet size, relatively low PDI, and absence of visible phase separation during the evaluation period therefore provided a physicochemical basis for selecting these systems for subsequent larvicidal assays. Recent literature on essential-oil-based nanostructured systems for A. aegypti control likewise highlights their potential while emphasizing the need for further investigation of formulation performance and environmental applicability (Sonter et al., 2024; Cruz et al., 2025).

Evaluation of the larvicidal activity of the selected microemulsion formulations

Following the physicochemical characterization and short-term stability assessment, the microemulsion systems that maintained their physical characteristics during the 7-day evaluation period were assessed for larvicidal activity against A. aegypti larvae. PME-01, PME-03, PME-04, PME-05, and PME-06 were therefore subjected to the larvicidal assay. These systems differed in the proportions of M. piperita essential oil, Smix, water, and surfactant-to-co-surfactant ratios, as shown in Table 2. The assay was performed under identical experimental conditions to compare the biological performance of the selected microemulsion systems.

Larvicidal activity varied considerably among the selected systems, despite all of them meeting the short-term physical stability criterion (Figure 3). PME-06, which contained the lowest proportion of peppermint essential oil (2%), did not produce larval mortality under the tested conditions. In contrast, PME-03, PME-04, and PME-05 resulted in 100% mortality, whereas PME-01 showed approximately 35% mortality. The Kruskal–Wallis test indicated a statistically significant difference in larval mortality among the evaluated systems (χ 2 = 18.85; df = 4; p = 0.00084), showing that at least one treatment differed from the others. Dunn-Holm post hoc comparisons revealed significant differences between PME-03 and PME-06 (adjusted p = 0.0113), PME-04 and PME-06 (adjusted p = 0.0113), and PME-05 and PME-06 (adjusted p = 0.0113). The remaining pairwise comparisons were not significant after adjustment for multiple testing, including the contrasts between PME-01 and PME-03, PME-04, and PME-05, which showed significant unadjusted p-values but did not remain significant after Holm correction. Thus, the statistically significant differences were primarily associated with the lower-performing PME-06. The variation in mortality among the systems suggests that their distinct component ratios and physicochemical characteristics may influence the dispersion and availability of the essential oil in the aqueous medium. Notably, PME-01 and PME-05 contained the same proportion of essential oil (5%), yet resulted in markedly different mortality rates (35% and 100%, respectively), further indicating that biological performance cannot be attributed solely to essential oil content. This interpretation is consistent with previous studies showing that the larvicidal performance of essential-oil-based nanoemulsions can vary according to both essential oil composition and formulation characteristics, including droplet size and polydispersity (Sonter et al., 2024; Cruz et al., 2025; Faustino et al., 2020).

Despite their compositional differences (Table 2), PME-03, PME-04, and PME-05 all produced 100% larval mortality, indicating that more than one microemulsion composition can provide effective larvicidal performance under the conditions evaluated. These systems differed in the proportions of essential oil and other components, as well as in their physicochemical characteristics, suggesting that distinct formulation strategies may be suitable for the aqueous delivery of M. piperita essential oil. A recent review of essential-oil-based nanoemulsions targeting A. aegypti similarly highlighted that larvicidal efficacy varies according to the botanical source and chemical composition of essential oils, as well as the composition and physicochemical characteristics of the resulting dispersed systems (Silva et al., 2025).

Among the systems that achieved complete mortality, PME-05 was particularly noteworthy because it combined a relatively low essential oil content (5%) with the lowest PDI value (0.06 ± 0.007). The low PDI indicates a highly homogeneous dispersed system and may contribute to a more uniform distribution of the essential oil in the aqueous medium. Studies of essential-oil nanoemulsions have likewise reported low PDI values in systems exhibiting larvicidal activity. For example, nanoemulsions containing Ocimum basilicum essential oil presented PDI values below 0.25 and demonstrated larvicidal activity against A. aegypti, illustrating the feasibility of obtaining homogeneous nanoscale dispersed systems while retaining biological activity (de Sousa dos Santos et al., 2024). However, the present results do not establish a direct relationship between PDI and larvicidal activity, since PME-03 and PME-04 also achieved 100% mortality despite presenting different PDI values. Rather, the results suggest that biological performance may arise from the combined effects of formulation composition and physicochemical characteristics.

From a formulation development perspective, PME-05 was considered particularly promising because it achieved 100% larval mortality while containing a relatively low proportion of M. piperita essential oil (5%) among the systems that achieved complete mortality. The use of a lower essential oil content may be advantageous from a formulation and production perspective, particularly considering the cost and volatility of essential oils and the need to minimize the amount of active material while maintaining biological efficacy. In addition, the low PDI observed for PME-05 (0.06 ± 0.007) indicates a highly homogeneous dispersed system, supporting its potential for further development. Therefore, PME-05 was selected as the most promising candidate for subsequent investigation. Nevertheless, PME-03 and PME-04 also demonstrated complete larvicidal activity and should be considered as alternative formulation candidates, as their distinct compositions may provide different approaches for the aqueous delivery of M. piperita essential oil for A. aegypti control. Further studies are warranted to compare the performance, stability, and environmental safety of these systems under conditions more representative of their intended application.

Similar approaches have been reported for other nanostructured formulations containing essential oils for mosquito control. Faustino et al. demonstrated that a nanoemulsion based on Protium heptaphyllum essential oil exhibited high larvicidal activity against A. aegypti, supporting the feasibility of colloidal systems for dispersing and handling volatile bioactive compounds (Faustino et al., 2020). Likewise, nanoliposomal formulations containing Artemisia essential oils showed effective larvicidal activity, further supporting the potential of nanostructured delivery systems for vector control applications (Sanei-Dehkordi et al., 2023). A nanoemulsion containing Lantana camara essential oil exhibited relevant larvicidal and pupicidal activity against Anopheles culicifacies, with LC 50 values of 50.35 and 54.82 ppm, respectively, and was reported to inhibit key detoxification and neurophysiological enzymes (Sonter et al., 2024). Collectively, these studies support the use of colloidal systems as delivery platforms for essential oils in vector control, particularly when improved dispersion and practical aqueous application are required.

Notably, the microemulsion formulation successfully preserved the biological activity of the essential oil while providing a suitable platform for its delivery. This finding is in agreement with previous studies demonstrating that emulsification can preserve biological activity while improving physicochemical stability and reproducibility, and greater reproducibility compared with non-formulated oils (Santos et al., 2024; Viana et al., 2025). Importantly, the present study does not demonstrate an increase in the intrinsic larvicidal potency of the essential oil; rather, the results indicate that microemulsion technology can provide a physically stable aqueous-based system while maintaining substantial biological activity. Among the evaluated formulations, PME-05 combined complete larval mortality with a relatively low essential oil content and a low PDI, supporting its selection as the most promising candidate. Further studies are necessary to determine whether this formulation provides advantages in terms of persistence, dispersion, application performance, or environmental behavior under conditions relevant to mosquito control.

Mentha piperita essential oil showed effective larvicidal activity against A. aegypti, while microemulsion technology enabled its formulation in an aqueous-compatible system without loss of biological activity. PME-03, PME-04, and PME-05 achieved 100% larval mortality, and PME-05 was identified as the most promising formulation, combining complete mortality with only 5% essential oil and a low PDI (0.06 ± 0.007). This formulation strategy provides a promising basis for further development of peppermint essential oil as a botanical tool for A. aegypti control.

These findings are limited to laboratory conditions and short-term physicochemical assessment. Further studies should investigate long-term stability, residual activity, and effects on non-target organisms, as well as validate the efficacy of PME-05 under semi-field and field conditions.