Influence of Polysorbate 80 on the Larvicidal and Ecotoxicological Profile of Essential Oil Nanoemulsion: Insights into Green Nanotechnology

Abstract

Essential oil (EO) nanoformulations are emerging as green nanotechnology strategies against and s mosquitoes, which are vectors of arboviruses such as dengue. For the first time, we investigated the larvicidal influence of polysorbate (PS₈₀) against mosquito using simplex centroid design (SCD) in the formulation of EODr nanoemulsions derived from the species (Plantaginaceae), a plant native to the Cerrado region of Maranhão, Brazil. In addition, we present the ecotoxicological profile of the formulation against microcrustaceans. EODr was extracted by hydrodistillation, and its chemical profile was analyzed by GC-MS-FID and NMR (¹H, ¹³C, DEPT-135, and 2D). The nanoformulations were characterized by DLS, PDI, ZP, and TEM. The chemical profile indicated the presence of compounds such as fenchol, fenchyl acetate, caryophyllene, and caryophyllene oxide. The SCD contributed to the development of nanostructured formulations with spheroidal morphology, demonstrating effective larvicidal action against mosquitoes with lower toxicity than commercial chemical larvicides.

Introduction

Emerging and reemerging arboviruses represent a global public health challenge. Dengue, chikungunya, and Zika viruses are primarily transmitted by and mosquitoes. , This is a global concern, but especially in Brazil and Maranhão, where vaccination coverage is still expanding and the recurrent increase in epidemic cases raises concerns and requires urgent measures to prevent and combat the mosquito vectors. The main control strategies are the use of larvicides and synthetic insecticides. However, the repeated use of these synthetic compounds has contributed to the selection of resistant populations of Aedes mosquito species.

In this way, the search for natural larvicides has proven to be a promising alternative strategy, with essential oils (EOs) standing out, since the complex mixture of bioactives in EOs includes classes of metabolites that give plants protection and reduce resistance against attacks by pathogens and insects.

The bioactivity of EOs can be attributed to their complex mixture mainly composed of monoterpenes, sesquiterpenes, and their oxygenated derivatives. , The biological activity of EOs against Aedes mosquitoes is associated with multiple mechanisms, depending on the target action, from digestive toxicity to enzyme inhibition and toxicity to the nervous system in the larval stage. They are therefore promising, effective, and potentially environmentally safe alternatives to current synthetic larvicides. −

However, the limited availability of raw materials, low yield, high volatility, and low miscibility of EOs in aqueous media make their application in natura unfeasible. In this context, the use of surfactants is necessary, with nonionic surfactants being the most viable for use with EOs, as they reduce changes in their properties and promote the formation of micellar and/or vesicular structures with greater efficiency on a micro and/or nanoemulsion scale. ,

In this context, the study focuses on the chemical composition of the EO of the plant species identified in the Cerrado biome region in the state of Maranhão, with two floral morphotypes, lilac and white, registered as Scatigna and Colletta (Plantaginaceae), characterized the chemical profile of the EO of (EODr) as terpenic and evaluated the larvicidal action against larvae of the mosquito, with this action being attributed to the EO matrix consisting of the major chemotypes, fenchyl acetate and fenchol.

In the above-mentioned study, polysorbate 80 (PS₈₀), a nonionic surfactant, was used in the composition of the emulsified system. Although PS₈₀ is widely used in the preparation of emulsions, microemulsions and nanoemulsions, optimal concentration for formulations of mixtures with EOs is not well-defined in the literature. −

In addition to the search for emulsified systems and promising strategies to enhance the action of the bioactives present in EODr, the use of design of experiments (DOE), such as simplex centroid design (SCD), are of paramount importance to understand the interaction between the constituents of emulsified systems, in addition to increasing target-biocidal action and decreasing biological resistance.

The SCD is a mathematical-statistical approach to mixture design aimed at developing and optimizing analytical responses. The adoption of this approach has proven to be methodologically effective for modeling the combined biological activities of different secondary metabolites. ,

Furthermore, this type of experimental design is of great practical interest, as it involves a minimum number of experiments, the development of improved and/or innovative formulations that provide targeted responses and highlight general aspects of the interactions between independent factors. −

The article presents the formulation and characterization of EODr nanoemulsion systems and the investigation of the influence of PS₈₀ on the larvicidal and ecotoxicological activity of emulsified EODr mixtures, using the SCD methodology, as well as their EODr-PS₈₀ interactions.

Experimental Section

Collection of Plant Material and Essential Oil Extraction

Plant material from the lilac morphotype of (Plantaginaceae) was collected in São Benedito do Rio Preto, Maranhão, Brazil (03°19′27.9’“S; 43°31′02.6”’W). A voucher specimen (SLUI 8656) was deposited in the Rosa Mochel Herbarium and registered with SISGEN (A5E5CD0).

The EODr was extracted by hydrodistillation (Ultrathermostatic bath; SSDu 10 L) using green solvent, water in the recycling system. ,, Detailed methodology is provided in the Supporting Information.

Chemical Composition: CG-FID and GC-MS

The GC-FID analysis was conducted using a Shimadzu GC-17A. The GC-MS analysis was performed using a Thermo Scientific Trace Ultra GC coupled to an ISQ MSQ. , Detailed methodology is provided in the Supporting Information.

Chemical Composition: NMR ¹H and ¹³C

The NMR spectra of the EODr sample and the chemical standards of the main compounds, including 1D (¹H, ¹³C, DEPT-135) and 2D (COSY, HSQC, HMBC), were recorded using a BRUKER AVANCE III HD spectrometer (Billerica, MA, USA), operating at 11.75 T (500.13 MHz for ¹H NMR and 125.76 MHz for ¹³C NMR). Detailed methodology is provided in the Supporting Information.

Nanoemulsions Formulation

The nanoemulsions were obtained by the low-energy oil–water emulsification method (Figure ) without the use of specialized equipment. The steps of preparation of the mixtures involved some of the 12 principles of green chemistry.

1.

General scheme for the preparation of EODr-PS₈₀ nanoformulations.

To determine the role of PS₈₀ in the EODr-PS₈₀ interaction, the concentration of EODr in the mixtures was kept constant (Figure ).

2.

Distribution of EODr-PS₈₀ interaction points in the SCD. Vertex points (1, 2, and 3); edge points (4, 5, and 6); center point (7); and face points (8, 9, and 10).

The proportions for emulsification were: 1EODr-1PS₈₀ (X₁); 1EODr-5PS₈₀ (X₂) and 1EODr-25PS₈₀ (X₃) g·L^–1. Detailed methodology is provided in the Supporting Information.

Hydrodynamic Diameter, PDI, and ZP

The hydrodynamic diameter, polydispersity index (PDI) were evaluated using the dynamic light scattering technique and zeta potential (ZP) was measured using the electrophoretic light scattering technique, both on a Zetasizer Nano ZS90 (Malvern). Detailed methodology is provided in the Supporting Information.

Morphology and Size by TEM

The mean diameter and morphology were analyzed using a Transmission Electron Microscope (TEM), JEM-2100 (JEOL, Tokyo, Japan) equipped with EDS, Thermo Scientific; Lanthanum hexaboride filament electron beam (LaB₆); Acceleration voltage of 200 kV, with a resolution of 2.5 Å resolution; ORIUS SC 1000 CCD camera, Gatan brand; Digital Micrograph software; Energy dispersive spectroscopy (EDS) with Thermo Scientific NSS Spectral Imaging detector for elemental identification. Detailed methodology is provided in the Supporting Information.

Larvicidal Bioassays

The larvicidal bioassays of the EODr-PS₈₀ emulsified systems against mosquito larvae followed the methodology recommended by the WHO with adaptations. ,− Detailed methodology is provided in the Supporting Information.

Ecotoxicity Tests

Aquatic ecotoxicology tests followed the protocols for toxicity tests standardized by the Brazilian Association of Technical Standards (ABNT), NBR 16530:2016 against microcrustaceans, meeting the requirements for the competence of chemical testing laboratories, based on ABNT, NBR ISO IEC 17025:2017. Detailed methodology is provided in the Supporting Information.

Statistical Analysis

The results were expressed as mean, standard deviation and mortality percentage values. Data analysis was performed using Origin software (version 8.5) and R software (version 4.2.3) to determine lethal concentrations (LC₅₀ and LC₉₀) and the adjusted coefficient of determination (R² _Adj) with a 95% confidence interval (p < 0.05).

The larvicidal efficacy of EODr-PS₈₀ against s larvae was determined by obtaining contour plots. The LC₅₀ and LC₉₀ values were obtained from the replicates in the SCD, the replicates being necessary for the analysis of variance (ANOVA). , Detailed methodology is provided in the Supporting Information.

Results and Discussion

Chemical Profile: GC-MS and CG-FID

The chemical profiling of the EODr matrix confirmed the plant species’ chemical composition and allowed us to associate the interactions between its constituents with the observed biological responses.

Table shows the chemical composition via GC-MS and CG-FID of the EODr matrix, lilac floral morphotype, as well as the analysis of the chemical patterns (Sigma-Aldrich) of the four chemotypes in relative percentage (Figures S1–S13).

1. Chemical Composition of EODr, Lilac Morphotype, and Patterns of the Major Chemotypes .

peaks	EODr compounds	RTmin	RICalc	RILit	%	MF	TC
1	α-pinene	10.70	928	932	0.08	C10H16	M
2	α-fenchene	11.29	944	945	0.08	C10H16	M
3	sabinene	12.32	969	969	0.02	C10H16	M
4	α-terpinene	14.11	1014	1014	0.02	C10H16	M
5	p-cymene	14.41	1022	1020	0.13	C10H14	M
6	limonene	14.59	1026	1024	0.12	C10H16	M
7	1,8-cineole	14.70	1029	1026	0.11	C10H18O	OM
8	γ-terpinene	15.77	1056	1054	0.07	C10H16	M
9	fenchone	16.93	1085	1083	0.14	C10H16O	OM
10	fenchol <endo>	18.23	1119	1114	42.97	C10H18O	OM
11	α-terpineol	21.01	1194	1186	0.07	C10H18O	OM
12	fenchyl acetate <endo>	21.86	1217	1218	46.03	C12H20O2	OM
13	(E)-caryophyllene	28.61	1416	1417	4.81	C15H24	S
14	α-humulene	29.72	1452	1452	0.38	C15H24	S
15	caryophyllene oxide	33.56	1579	1582	1.94	C15H24O	OS
16	humulene epoxide II	34.36	1606	1608	0.27	C15H24O	OS
17	caryophylla-4(12),8(13)-dien-5α-ol	35.05	1631	1639	0.29	C15H24O	OS
18	caryophylla-4(12),8(13)-dien-5β-ol	35.14	1634	1639	1.00	C15H24O	OS
19	pogostol	35.71	1654	1639	0.24	C15H26O	OS
	Σ monoterpenes (M)				0.52
	Σ oxygenated monoterpenes (OM)				89.32
	Σ sesquiterpenes (S)				5.19
	Σ oxygenated sesquiterpenes (OS)				3.74
	Σ Total identified				98.77
Standard compounds
1	fenchol <endo>	18.42	1124	1114	94.82	C10H18O	OM
2	fenchyl acetate <endo>	21.97	1221	1218	98.87	C12H20O2	OM
3	(E)-caryophyllene	28.87	1425	1417	97.98	C15H24	S
4	caryophyllene oxide	33.77	1586	1582	98.26	C15H24O	OS

^aP = chromatogram peaks (Figures S1, S6, S8, S10, and S12); RT_min = retention time; RI_Calc. = calculated retention indices (on TR-5MS capillary column 30 m × 0.25 mm × 0.25 μm) according to van Den Dool and Kratz, based on a homologous series of normal alkanes; RI_Lit. = literature retention indices, Adams; % = relative percentage; MF = molecular formula; TC = terpene classes.

The chemical matrix of EODr shows a terpene profile, displaying chemical compound classes of monoterpenes, oxygenated monoterpenes, sesquiterpenes and oxygenated sesquiterpenes, totaling 98.77% of the relative percentage of chemical constituents. The predominance of oxygenated monoterpenes fenchol and fenchyl acetate is significant, totaling 89.0% of the EODr matrix.

The chemical profile observed in the present study is in line with the results reported by Brandão and collaborators, as well as Galvão and associates who observed the terpene pattern of the EO of the same plant species, with emphasis on the chemotypes of the species, oxygenated monoterpenes, endo fenchol, 42.97%; endo fenchyl acetate, 46.03%; sesquiterpenes; (E)-caryophyllene, 4.81% and oxygenated sesquiterpenes, caryophyllene oxide, 1.94%.

The percentage variations observed in the chemical composition compared to previously published studies are due to circadian, seasonal and edaphoclimatic conditions, as the yield and chemical composition of EOs are associated with rainfall, relative humidity, solar radiation, atmospheric constitution and variations in temperature range, wind, soil and relief. −

Several studies suggest that the terpenic pattern of various chemical matrices of EOs extracted from different botanical families is correlated with high larvicidal activity against and larvae. , In this respect, studies indicate that the bioactives in the essential oil and crude extracts of the lilac morphotype of the species from the Maranhão Cerrado, , have effective larvicidal activity, respectively, against the species and . ,

Chemical Profile: ¹H NMR and ¹³C NMR

The ¹H NMR data (Table ) provide crucial insights into the differentiation of the four major compounds in the EODr sample: fenchol, fenchyl acetate, caryophyllene, and caryophyllene oxide (Figure ).

2. NMR Data for ¹H (125 MHz; CDCl₃) of the Sample Presented as Chemical Shifts (δ).

Position	Fenchol	Fenchyl acetate	Caryophyllene	Caryophyllene oxide
1	-	-
2	3.25 (d)	4.35 (d)
3	-	-
4	1.68–1.67 (m)	1.71–1.70 (m)
5	1.59–1.56 (m); 1.05–1.00 (m)	1.47–1.41 (m); 1.69–1.66 (m)	5.43–529
6	1.66–1.60 (m); 1.42–1.38 (m)	1.77–1.72 (m); 1.07–1.04 (m)
7	1.47–1.43 (m); 1.19–1.69 (dd)	1.59–1.57 (q); 1.13–1.11 (dd)
8	1.08 (s)	1.09 (s)
9	0.98 (s)	1.03 (s)
10	0.86 (s)	0.77 (s)
11	-	-
12	-	2.07 (s)
13	-	-	4.94; 4.82	4.97; 4.86
14	-	-
15	-	-

3.

Structures of the main compounds, chemical markers of the species .

Fenchol and fenchyl acetate exhibit characteristic singlets at δ 1.08 and 1.09 for C8, along with methyl signals at δ 0.98–1.03 (C9) and δ 0.86–0.77 (C10), which confirm their bicyclic structures. The presence of a doublet at δ 3.25 in fenchol and at δ 4.35 in fenchyl acetate further differentiates the hydroxyl and acetate functional groups at C2, respectively.

Caryophyllene is distinguished by its olefinic protons at δ 5.43–5.29 (H5), indicative of its bicyclo[3.3.0]­octane system, whereas caryophyllene oxide exhibits shifts at δ 4.97 and 4.86 (H13), confirming the presence of an epoxide moiety. Additionally, the overlapping multiplets in the aliphatic region (δ 1.05–1.77) for all four compounds complicate signal attribution, especially due to spectral congestion from fenchol and fenchyl acetate.

However, the combined use of ¹³C NMR, DEPT-135, and two-dimensional NMR techniques enabled the complete assignment of all proton resonances, ensuring accurate structural characterization (Figures S14–S19).

The NMR data (Table ) clearly distinguish the four major compounds in the EODr sample. Fenchol and fenchyl acetate exhibit similar chemical shifts, particularly at δ 49.1 and δ 48.1 (C1) and in the C3–C10 region. The key differentiating feature is the ester carbonyl signal at δ 171.6 in fenchyl acetate, absent in fenchol. Both compounds also display an oxygenated carbon at δ 85.1 and δ 86.1 (C2), indicative of a tertiary alcohol or ester functionality.

3. NMR Data for ¹³C (500 MHz; CDCl₃) of the Sample Presented as Chemical Shifts (δ).

Position	Fenchol	Fenchyl acetate	Caryophyllene	Caryophyllene oxide
1	49.1	48.1	53.5
2	85.1	86.1	29.3	27.2
3	39.0	39.4	28.3	39.1
4	47.9	48.3	135.5
5	25.1	25.8	124.3	63.7
6	26.1	26.5	39.9	30.2
7	40.9	41.3	34.8	29.7
8	19.4	29.7	155.0	151.8
9	30.7	19.3	48.4	48.7
10	20.1	20.0	40.3	39.7
11	-	171.6	33.0	34.0
12	-	20.9	16.2	17.0
13	-	-	111.6	112.7
14	-	-	30.0	29.8
15	-	-	22.6	21.6

Caryophyllene is characterized by sp² -hybridized carbons at δ 135.5 (C4) and δ 124.3 (C5), which are not observed in fenchol or fenchyl acetate. Upon oxidation to caryophyllene oxide, notable shifts occur at δ 63.7 (C5) and δ 151.8 (C8), confirming epoxide formation. Additionally, C13 signals at δ 111.6 and δ 112.7 further differentiate these sesquiterpenes.

The assignments of NMR signals were corroborated by comparison with reference spectra from standard samples of fenchol, fenchyl acetate, caryophyllene, and caryophyllene oxide, ensuring the accuracy of the structural characterization (Figures S20–S43). The corresponding chemical shifts are provided in Tables S1 and S2.

The larvicidal activity demonstrated by the essential oil of EODr can be attributed, in large part, to its major constituents, fenchol and fenchyl acetate, whose efficacy has been previously validated against . These findings corroborate the results of the present study, highlighting the relevance of these monoterpenes as principal bioactive agents. However, the presence of sesquiterpenes such as caryophyllene and caryophyllene oxide, even in lower concentrations, may contribute to the observed biological effect through synergistic interactions, as previously reported in studies involving essential oils of , where similar constituents exhibited larvicidal activity against both and . Such synergism between major and minor components is well-documented in essential oil research and underscores the importance of evaluating the complete phytochemical profile rather than isolated compounds alone. These results suggest that the larvicidal potential of EODr is a multifactorial phenomenon, in which both individual and combinatorial effects play critical roles.

DLS, PDI, ZP, and TEM Droplet Sizes

The EODr-PS₈₀ mixtures presented a homogeneous appearance with a clear and transparent visual appearance, pH (value ∼ 6), without visible precipitation or phase separation after 24 h and throughout the monitoring period of the experiments, over six months (Figure ).

4.

EODr-PS₈₀ mixtures X₁; X₂; X₃; and X_OP.

The formulation of lipid nanosystems with safe, stable and efficient accumulation in target tissues for in vitro and/or in vivo applications depends on their physicochemical properties, such as size, size distribution and zeta potential.

Table shows the values of the mean droplet size diameter via DLS and TEM techniques, as well as the polydispersity index (PDI) values for the pure components (X₁, X₂, X₃ and X_OP) of the EODr-PS₈₀ nanoemulsions.

4. Droplet Size, PDI, ZP, and TEM of EODr-PS₈₀ Interactions .

Components	Diameter (nm)	PDI	Zeta Potential (mV)	TEM (nm)
	MV ± SD	MV ± SD	MV ± SD	MV ± SD
X1	933.8 ± 43.1	0.691 ± 0.045	–18.1 ± 0.9	132.0 ± 103.0
X2	615.6 ± 39.7	0.660 ± 0.050	–17.9 ± 2.8	86.0 ± 100.0
X3	239.8 ± 3.9	0.122 ± 0.025	–36.2 ± 2.0	100.0 ± 72.0
XOP	253.4 ± 50.0	0.524 ± 0.031	–24.1 ± 1.1	110.7 ± 60.9

^aX₁: 1EODr-1PS₈₀; X₂: 1EODr-5PS₈₀; X₃: 1EOD-25PS₈₀; X_OP: 1EODr-19.4PS₈₀; MV ± SD: mean value and standard deviation; replicates: 3.

Droplet Size, PDI, and ZP

The mean hydrodynamic diameter (Table ) showed a decreasing trend with an increase in the proportion of nonionic surfactant (PS₈₀) in the EODr-PS₈₀ mixtures (X₃ < X₂ < X₁). Although increasing surfactant concentration helps reduce droplet size, excessive amounts may affect biological systems. Being observed for the mixture under optimality conditions (1EODr-19.4PS₈₀) average size (253.4 ± 50.0 nm) with nanoformulated system profile.

In line with the study described in this paper, research by Pascual-Mathey and collaborators indicated that the optimum conditions for producing rosemary essential oil nanoemulsions with nonionic surfactant were found to be a higher proportion of surfactant than EO in the formulation matrix. The average particle sizes shown in the study were in the 50 nm range, so the authors suggest that the droplets are completely covered by the surfactant and the excess surfactant in the continuous phase.

Regarding the use of the dynamic light scattering (DLS) technique, the heterogeneity or polydispersity index (PDI) is a dimensionless parameter that indicates that indicates the width of the size distribution of molecules, particles, droplets or nanovesicles in the population within a sample. It provides insights into system homogeneity, in which nanosystems are classified as highly monodisperse (0.0 < PDI ≤ 0.1), moderately polydisperse (0.1 < PDI ≤ 0.4) and highly polydisperse (PDI > 0.4). ,

The PDI values (Table ) in the EODr-PS₈₀ mixtures follow the same trend as the particle size of the mixtures, in which the pure components with a higher amount of PS₈₀ present a lower polydispersity index (X₃ < X₂ < X₁).

The mixtures X₁ (0.691 ± 0.045), X₂ (0.660 ± 0.050) and X_OP (0.524 ± 0.031) presented a highly polydisperse profile, while X₃ (0.122 ± 0.025) exhibited a moderately polydisperse profile.

In general, the aggregation or decantation phenomena in emulsified systems tend to be less evident when they exhibit PDI values (0.0 < PDI ≤ 0.1) presenting profiles classified as monodisperse systems. , In contrast, the EODr-PS₈₀ mixtures (X₁, X₂, X₃ and X_OP) did not exhibit visible phase separation or precipitation (Figure ).

5.

MET micrographs and histograms of the emulsified EODr-PS₈₀ nanoemulsions.

In general, ZP values between ± 0–10 mV indicate high instability due to weak electrostatic repulsion, while values in the range ± 10–20 mV suggest limited stability. Systems with ZP ± 20–30 mV are considered moderately stable and ZP ≥± 30 mV tend to be more electrostatically stable.

For the EODr-PS₈₀ mixtures, the negatively charged ZP values (Table ) can be attributed to the presence of functional groups of the nonionic surfactant (PS₈₀) that are associated with the differences in the degree of dissociation and dipole moments presented by the monoterpene compounds in the matrices of EOs, water and the ethylene oxide groups of the polysorbate. −

In general, values observed for the pure components, X₁ and X₂, are indicative of relative electrical stability (−18.1 and −17.9 mV). For the mixture under optimal conditions X_OP, moderate stability was observed (−24.1 ± 1.1 mV) and X₃, high electrical stability (−36.2 mV).

The evaluated EODr-PS₈₀ mixtures show a tendency to increase stability through repulsive electrostatic interactions and steric effect promoted by the long chain of polysorbate 80 responsible for the reduction of the droplet aggregation phenomenon with the increase of PS₈₀ in the EODr-PS₈₀ mixtures.

Morphology and Size by TEM

The size and shape of droplets in emulsified essential oil-surfactant–water systems depend on the molecular geometry and dimensions of the surfactant head and tail groups, because droplets tend to adopt a spherical shape as it minimizes the interfacial free energy. The TEM electron micrographs of EODr-PS₈₀ (X₁; X₂; X₃ and X_OP) exhibit the formation of nanodroplets with a spheroidal morphology (Figure ).

The electron micrographs and the distribution of size values in the histograms (Table ) indicate nanodroplets with average sizes of 132.0 ± 103.0 nm (Figure a), 86.0 ± 100.0 nm (Figure b) and 100.0 ± 72.0 nm (Figure c) with marked standard deviation values indicated by the polydisperse profile of the mixtures EODr-PS₈₀. Figure d shows the profile of EODr-PS₈₀ interactions under optimality conditions (X_OP) with value (110.7 ± 60.9 nm) showing characteristics of a nanostructured system.

In general, polydispersity phenomena were observed in the TEM micrographs of the mixtures (X₁, X₂, X₃ and X_OP), displayed by the asymmetric frequency of the normal distribution in the histograms and corroborated by the results of the polydispersity indices (Table ).

The relative trend of decreasing nanodroplet size increasing was observed with increasing PS₈₀ concentration in the pure components, EODr-PS₈₀ (X₃ < X₂ < X₁), up to a critical surfactant concentration. A similar trend was observed in the dynamic light scattering technique of the analyzed samples.

From the analysis of the micrographs and the evaluation of the average size distribution observed in the histograms, there are indications of the action of PS₈₀ in the formation, reduction and stabilization of the EO nanodroplets observed in the EODr-PS₈₀ mixtures, since PS₈₀ contributes to the stabilization of the EO nanodroplets by reducing the interfacial tension and steric and electrostatic stabilization.

Larvicidal Evaluation of PS₈₀

The use of surfactants is necessary to form a water-miscible system and, consequently, to reduce the volatility of EO emulsified systems. The interaction of EO-H₂O via a nonionic surfactant depends on its size, micro and/or nanoemulsified to the detriment of macro-emulsified systems. Size affects characteristics such as physical stability, water solubility, protection against degradation agents, controlled release, attenuation of loss of control due to evaporation and increased bioactivity, such as larvicidal activity.

The percentage of larval mortality against the mosquito was analyzed at different concentrations of PS₈₀, as shown in Table . Tests performed at the listed concentrations of PS₈₀ indicate a limitation to the use of PS₈₀ in the preparation of emulsified systems in EODr-PS₈₀ mixtures.

5. Bioactivity of PS₈₀ against Larvae in 24 h .

Concentrations (%)	n, Aa	Dead MV ± SD	%Mortality MV ± SD
		MV ± SD	MV ± SD
0.10	50	0.0 ± 0.0	0.0 ± 0.0
0.50	50	0.0 ± 0.0	0.0 ± 0.0
1.94	50	0.0 ± 0.0	0.0 ± 0.0
2.50	50	0.8± 0.4	8.0 ± 4.5
5.00	50	2.4± 1.5	24.0 ± 15.2
10.00	50	4.4 ± 1.7	44.0 ± 16.7

^aMV ± SE: mean value and standard deviation; replicates: 5; n: number of larvae used.

The concentrations equivalent to 0.10, 0.50 and 2.50% PS₈₀ showed mortality percentages ranging from 0 to 8%, thus indicating that PS₈₀ has no direct influence on the larvicidal action of the EODr-PS₈₀ mixture. Meanwhile, concentrations equivalent to 5 and 10% of PS₈₀ showed mortality in the larval populations tested of between 22 and 44%, respectively. In this case, the preparation of EODr-PS₈₀ mixtures with these levels of PS₈₀ has a direct influence on the larvicidal activity analyzed.

Concentrations of less than 2.50% PS₈₀ are necessary for the formulation of emulsified systems. Highlighting the optimal conditions (1.94% PS₈₀) that did not influence larval mortality, being an action inherent to the bioactivity of the EODr matrix. In general, it is suggested that the concentration of nonionic surfactants and their proportions used in emulsified EOs formulations be better described in the negative control tests of biolarvicidal formulations.

Larvicidal Evaluation via SCD

The use of SCD instead of conventional pseudoternary phase diagrams provided savings in time and financial resources, reducing the number of experiments and optimizing the development of the formulations. − The larvicidal evaluation against field larvae of the mosquito for the lethal concentrations (LC₅₀ and LC₉₀) of the EODr-PS₈₀ interactions.

These were analyzed at the primary mixture corresponding to the vertex points (1, 2 and 3), in the binary mixtures corresponding to the edge points (4, 5 and 6), the ternary mixture points (7, 8, 9 and 10) corresponding to the central and face points. The mortality percentages and larvicidal activity profile of EODr-PS₈₀ for the determination of LC₅₀ and LC₉₀ against in SCD are shown in Table S3; Figure S44 and Table .

6. LC₅₀ and LC₉₀ Values Obtained by Sigmoidal Adjustment for the Two Replicates and Their Mean and Standard Deviation .

SCD	LC50			LC90			Sigmoidal Fit
Points	R1 (mg·L–1)	R2 (mg·L–1)	MV ± SD (mg·L–1)	R1 (mg·L–1)	R2 (mg·L–1)	MV ± SD (mg·L–1)	R2 Adj,1	R2 Adj,2	R2 Adj,M
1	437.9	471.9	482.3 ± 56.4	640.4	548.5	529.5 ± 98.0	0.993	1.000	1.000
2	467.4	453.3	586.6 ± 34.5	715.2	587.5	535.7 ± 96.1	0.997	1.000	1.000
3	361.1	319.8	340.8 ± 44.8	589.8	428.3	528.1 ± 133.7	0.946	0.991	0.980
4	410.0	321.3	365.3 ± 26.9	603.1	596.1	615.5 ± 41.5	0.951	0.994	1.000
5	402.8	276.9	353.6 ± 6.9	772.1	297.7	509.5 ± 18.6	0.991	0.995	1.000
6	356.4	295.4	337.8 ± 18.8	514.4	349.6	456.4 ± 51.0	0.988	1.000	1.000
7	392.6	336.2	353.1 ± 28.8	562.0	532.6	501.1 ± 1.4	0.853	0.999	1.000
8	413.7	353.6	390.2 ± 12.9	694.8	499.8	609.4 ± 40.3	0.998	1.000	1.000
9	350.4	253.4	289.1 ± 36.0	670.7	463.5	507.8 ± 5.2	0.873	0.996	1.000
10	296.9	313.1	311.4 ± 61.3	643.7	392.0	520.4 ± 196.6	0.976	1.000	1.000

^aThe adjusted coefficient of determination (R² _Adj) was used to assess the adjustment.

^bR: replicate; MV: mean value and SD: standard deviation.

Table shows the LC₅₀ and LC₉₀ values obtained from the EODr-PS₈₀ interaction.

The data obtained indicate that the higher the concentration of PS₈₀, the greater the lethality of the emulsified system. We can visualize this logic when we compare the vertex points (1, 2 and 3) in the replicates and the average LC₅₀ values, where we see that increasing the concentration of PS₈₀ in relation to EODr reduces the size of the dispersed droplets and consequently increases the larvicidal potential.

It should also be noted that the lowest average LC₅₀ observed in the ternary mixtures corresponds to the face point 9 (289.1 mg·L^–1), while the lowest average LC₉₀ value in the binary mixtures refers to the edge point 6 (456.4 mg·L^–1).

With regard to values CL₅₀ and CL₉₀, the WHO does not establish concentration parameters to indicate larvicidal efficiency for the mosquito.

On the other hand, they have indicated that compounds and/or bioproducts with values CL₅₀ < 50 mg·L^–1 are highly active; 50 < CL 50 < 100 mg·L^–1 active; 100 < CL₅₀ < 750 mg·L^–1 effective and CL₅₀ > 750 mg·L^–1 inactive, ,,, the classification being adopted in general for mosquitoes, and . It should also be noted that some of these studies do not present data on the characterization of the formulations used in the selected bioassays, nor the LC₉₀ values.

It should be noted that the LC₅₀ values must be associated with the LC₉₀ values, since the lower and closer the LC₅₀ and LC₉₀ values, the more information is provided about the maximum larvicidal action analyzed, since low LC₅₀ values may not mean concentrations with maximum efficiency (LC₉₀) on the larval populations evaluated. In this respect, this logic can be observed in the ternary mixtures corresponding to edge point 6, which shows average values LC₅₀ (337.8 ± 18.8 mg·L^–1) and LC₉₀ (456.4 ± 51 mg·L^–1).

Therefore, the interaction between EODr and PS₈₀ was determined by analysis of variance (ANOVA) of the replicates (Table S4).

For LC₅₀, the coefficients of the components X₁, X₂ and X₃, of very high probabilistic significance (99.9%), X₁X₂, significance of 95%, and X₂X₃, statistical significance (90%), were defined to represent the EODr-PS₈₀ interaction via LC₅₀. Equation describes this interaction:

$${\mathrm{LC}}_{50}=461.3\mathrm{EO}\mathit{Dr}‐{\mathrm{PS}}_{80}+445.3\mathrm{EO}\mathit{Dr}‐5{\mathrm{PS}}_{80}+339.5\mathrm{EO}\mathit{Dr}‐25{\mathrm{PS}}_{80}-385.2\mathrm{EO}\mathit{Dr}‐{\mathrm{PS}}_{80}\mathrm{:}\mathrm{EO}\mathit{Dr}‐5{\mathrm{PS}}_{80}-330.1\mathrm{EO}\mathit{Dr}‐5{\mathrm{PS}}_{80}\mathrm{:}\mathrm{EO}\mathit{Dr}‐25{\mathrm{PS}}_{80}$$ 1

The contour plot (Figure ) presents the EODr-PS₈₀ interactions and the larvicidal profile against larvae, based on the LC₅₀ and LC₉₀.

6.

Contour plot of the EODr-PS₈₀ interaction in relation to LC₅₀ (a) and LC₉₀ (b).

The EODr-PS₈₀ interaction via LC₅₀ follows a reduced quadratic polynomial model (R² _Adj 0.981), illustrates according to the contour surface for this model (Figure a), indicating a region of optimality for the EODr-PS₈₀ interaction, around point 6. The optimal value found with the Nlcoptim package for the pure components was 28% of EODr-5PS₈₀ (X₂) and 72% of EODr-25PS₈₀ (X₃) was observed with no influence of PS₈₀ on larval mortality.

For LC₉₀ only the pure components (X₁, X₂ and X₃) were significant, in which case the model followed is linear (R² _Adj 0.946), shows according to the contour surface for the EODr-PS₈₀ interaction, indicating the vertex points as the best condition (Figure b).

This indicates that the higher the concentration of PS₈₀ the lower the LC₉₀ and, consequently, the greater the larvicidal action. Although the LC₉₀ (Figure b) signaled the vertex point (X₃ mixtures) as the best condition, the increase of PS₈₀ in the EODr-PS₈₀ interactions presents a limitation of the use of PS₈₀ (Table ) in the mixtures due to its influence on the larvicidal activity. Equation describes this interaction:

$${\mathrm{LC}}_{90}=596.7\mathrm{EO}\mathit{Dr}‐{\mathrm{PS}}_{80}+649.6\mathrm{EO}\mathit{Dr}‐5{\mathrm{PS}}_{80}+512.7\mathrm{EO}\mathit{Dr}‐25{\mathrm{PS}}_{80}$$ 2

The data obtained correlates with the EODr matrix, which has a majority profile of monoterpene and oxygenated monoterpene chemotypes. , The interaction of PS₈₀ should present variations with the types of chemical constituents present in the different EO matrices.

Larvicidal Evaluation under Optimal Conditions

The larvicidal profile against mosquito larvae was evaluated based on the LC₅₀ under experimentally determined optimal conditions, as observed in the contour diagram (Figure a).

Table shows the percentage of mortality observed in interactions under optimal conditions (1EODr-19.4PS₈₀).

7. Mortality Profile against under Optimal Conditions in the SCD over a 24 h Period .

EODrPS80 (mg·L–1)	n, Aa	Dead MV ± SD	%Mortality MV ± SD	LC50 MV ± SD	LC90 MV ± SD	R2 Aj
		MV ± SD	MV ± SD	MV ± SD	MV ± SD
100	50	0.8 ± 0.4	8.0 ± 4.5	214.5 ± 11.6	503.8 ± 12.1	0.997
250	50	6.6 ± 1.9	66.0 ± 19.5
500	50	8.0 ± 1.4	80.0 ± 14.1
750	50	9.4 ± 0.9	94.0 ± 8.9
1000	50	10.0± 0.0	100.0 ± 0.0
BC	50	0.0 ± 0.0	0.0 ± 0.0	-	-	-
NC	50	00.0 ± 0.0	0.0 ± 0.0
NP	50	10.0 ± 0.0	100.0 ± 0.0

^aMV: mean value; SD: standard deviation; BC: blank control (hatching system mineral water).

^bNC: negative control (19.4 × 10³ mg·L^–1); NP: positive control (fersol 1 G/temephos 100 mg·L^–1).

^cReplicates: 5; n: number of larvae used.

The bioassays conducted following the optimality parameters at concentrations of 100 to 1000 mg·L^–1 showed mortality rates ranging from 8% (100 mg·L^–1) to 100% (1000 mg·L^–1), with a percentage above 50% (at 250 mg·L^–1), exhibiting a sigmoidal profile with increasing mortality following a dose–response relationship (Figure S45).

This suggests a larvicidal effect of the EODr-PS₈₀ formulation attributed to the bioactive compounds in EODr. It is noteworthy that the concentration of PS₈₀ (NC) under optimality conditions did not influence the larvicidal activity of the nanoformulation (0.0 ± 0.0).

The bioassays larvicidal (Figure S45) conducted according to these parameters showed a high correlation coefficient (R² _Adj 0.997), indicating a strong sigmoidal fit and reliable biological response. The values LC₅₀ (214.5 ± 11.6 mg·L^–1) and LC₉₀ (503.8 ± 12.1 mg·L^–1) indicate effective bioactivity (100 < CL₅₀ < 750 mg·L^–1) against the field larvae of . ,,

In general, when comparing the values LC₅₀ (296.5 mg·L^–1) and LC₉₀ (895.4 mg·L^–1) from the study carried out by Brandão and collaborators, of the emulsified system of EODr-PS₈₀ against larvae, the LC₅₀ and LC₉₀ values obtained under the optimal conditions of the SCD applied in this study show effective target-action activity against larval populations of this species, originating from eggs collected in a real field environment that are susceptible to synthetic chemical larvicides.

In contrast, the use of chemical larvicides such as temephos (an organophosphate larvicide) has been associated with increased resistance in Aedes mosquitoes, including and and, consequently, to the increase in arboviruses. Furthermore, even low-dose use causes toxic effects in aquatic and terrestrial nontarget organisms (insects, plants, animals and humans) due to inhibition of the enzyme acetylcholinesterase with adverse effects on the respiratory, reproductive, nervous, hepatic and renal systems of nontarget organisms. ,

In general, the statistical parameters of SCD presented reliability above 90% for the biological assays, contributing with new insights in nanobiotechnology. The results support the use of SCD as an effective approach to optimize nanoemulsions, indicating the appropriate proportion of PS₈₀ in the formulation with effective bioactivity of EODr against arbovirus-transmitting agents, such as the mosquito.

Ecotoxicological Evaluation under Optimal Conditions

Ecotoxicological bioassays were performed on microcrustaceans in aquatic environments at EODr-PS₈₀. It should be noted that lethality tests against are rapid, low-cost, and reproducible for the preliminary evaluation of natural and/or synthetic products against a variety of substances. Furthermore, microcrustaceans are considered halophilic organisms that thrive in environments with high salt concentrations, thus playing an important role in aquatic and marine ecosystems as ecological and ecotoxicological bioindicators.

Table presents the results of the bioassays with microcrustaceans of from EODr-PS₈₀, based on the optimality parameters in the SCD.

8. Percentage of Mortality against under Optimal Conditions in the SCD in the 48 h Period .

EODr -PS80, mg L–1	n, As	Dead	%Mortality	LC50	LC50	R2 Aj
		MV ± SD	MV ± SD	MV ± SD	MV ± SD
100	100	1.8 ± 1.2	18.0 ± 11.4	378.8 ± 27.2	716.2 ± 102.7	0.979
250	100	2.6 ± 1.1	26.0 ± 10.8
500	100	6.7 ± 1.8	67.0 ± 18.3
750	100	8.4 ± 1.3	84.0 ± 13.5
1000	100	9.0 ± 1.4	90.0 ± 14.1
BC	100	0.4 ± 0.9	4.0 ± 9.7	-	-	-
NC	100	0.1 ± 0.3	1.0 ± 3.2
NP	100	10.0 ± 0.0	100.0 ± 0.0

^aMV: mean value; SD: standard deviation; BC: blank control (hatching system saline solution).

^bNC: negative control (PS₈₀ 19.4 × 10³ mg·L^–1); NP: positive control (K₂Cr₂O₇ 0.1%); replicates: 10.

^cn: Number of used.

The EODr-PS₈₀ nanoformulation under optimal conditions showed mortality rates below 50% at 100 mg·L^–1 (18.0 ± 11.4) and 250 mg·L^–1 (26.0 ± 10.8) against microcrustaceans. For concentrations from 500 to 1000 mg·L^–1, percentages above 50% were observed, displaying an acute toxicity profile (Table , Figure S46) of the nanoformulation under optimal SCD conditions against nontarget organisms ( microcrustaceans) with increasing dose tested.

The influence of PS₈₀ (NC) on the toxicity of the nanoformulation was negligible, as the mortality rate (1.0 ± 3.2) was below the limit percentage (10%) established as ecotoxicological against microcrustaceans .

The ecotoxicological profile (Figure S46) against of the EODr-PS₈₀ under optimal conditions in the SCD, indicates LC₅₀ (378.8 ± 27.2 mg·L^–1) and LC₉₀ (716.2 ± 102.7 mg·L^–1) values, with a correlation coefficient (R² _Aj 0.979), showing reliability of the tests carried out, above 90%. The LC₅₀ suggests moderate toxicity (100 ≤ LC₅₀ ≤ 500 mg·L^–1) against microcrustaceans. −

Considering that the microcrustacean plays a significant role in the dynamics of aquatic ecosystems, the ecotoxicological profile of EODr-PS₈₀ nanoformulations indicates that the terpenoid constituents have potential for use in habitats where arbovirus vector larvae reproduce. ,

The results obtained offer promising perspectives for the application of EODr-PS₈₀ nanoformulations, and further tests with nontarget organisms are needed to more fully evaluate the toxicity profile of the developed formulations. The EODr-PS₈₀ presented a toxicity profile against nontarget organisms with lower toxicity than the chemical larvicides currently used.

The formulation strategy adopted for the EODr-PS₈₀ nanoemulsion was deliberately guided by the principles of green chemistry and environmental sustainability. The system was developed using a low-energy oil-in-water emulsification method, relying exclusively on water and polysorbate 80-both nontoxic, biodegradable, and widely accepted as environmentally benign solvents. No hazardous organic solvents or energy-intensive procedures were employed at any stage. Moreover, the implementation of a Simplex Centroid Design (SCD), in lieu of conventional pseudoternary phase diagrams, enabled a substantial reduction in experimental workload, raw material use, and waste generation. Collectively, these features underscore the eco-friendly and resource-efficient character of the formulation, highlighting its potential as a sustainable alternative for larvicidal applications.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.5c04690.

• Additional experimental details, including figures and tables (PDF)

The Article Processing Charge for the publication of this research was funded by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Brazil (ROR identifier: 00x0ma614). The authors would like to thank the Instituto Federal de Educação, Ciência e Tecnologia do Maranhão (PRPGI/IFMA grant: 89/2021; 65/2023); Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA/MS-Decit/CNPq/SES/PPSUS grant: 02086/20); Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES/PROAP/PDG-Legal Amazon) for the financial support. E.V.C. thanks the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/INCT grant: 465357/2014) for the financial support.

The authors declare no competing financial interest.