This work developed and evaluated the toxicological effects of single nanoemulsions (NEs) of peppermint or palmarosa essential oils (EO) combined with β-cypermethrin (CP) using ultrasound technique. For NEs formulation ultrasound parameters were: ultrasound power = 65 W, sonication time = 2 min and cycles = 30 on/20 off. The optimized active ingredients:surfactant ratio was 1:2. The NEs of peppermint EO combined with CP showed two peaks at 12.77 (33.4% intensity) and 299.1 nm (66.6% intensity) and polydisperse index (PDI) values of 1, while the NEs of palmarosa EO combined with CP had a size of 104.5 nm and their PDI value, 0.220. Both NEs were transparent and they were stable for 2 months. The insecticidal effect of these NEs (active ingredients:surfactant ratio was 1:2) were evaluated against T. castaneum and S. oryzae adults and Cx p. pipiens larvae. The NEs of peppermint EO combined with CP showed an enhancement of bioactivity of β-CP between 4.22 and 16 folds, while NEs of palmarosa EO + CP between 3.90 and 10.6 folds in all these insects. Moreover, both NEs demonstrated high insecticidal activity in all insect for 2 months, although slightly increase of particle size was detected. In this regard, the NEs elaborated in this work can be considered as highly promising formulation for the development of new insecticides.
Research Article
β-cypermethrin and essential oils incorporated into a single nanoformulation: chemical and biological characterization
https://doi.org/10.21203/rs.3.rs-2004809/v1
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Nanoemulsions
stability
peppermint oil
palmarosa oil
β-cypermethrin
insecticidal activity
- A single nanoemulsion loaded with geranium or peppermint essential oil and β-cypermethrin.
- Nanoformulations were developed using an ultrasound probe.
- Nanoemulsions showed small size and were stable for 2 moths.
- Nanofomulations enhaced significantly the insecticidal activity of β-cypermethrin.
- These nanoinsecticides showed effects for 2 months.
Nowadays, many countries worldwide face problems with Mosquito-borne diseases, that puts the health of their population at risk and avoid their economic development (Kelly et al., 2022). One of the most common mosquito specie is Culex pipiens pipiens Say (Diptera: Culicidae), which can transmit several parasitosis and arborivus, specially in tropical and subtropical regions (Tabbabi et al., 2019). In most cases, these regions have another important issue, the insect pests of stored products. They can produce 10–15% losses during storage (Adak et al., 2020). According to Attia et al. (2020) these losses in developing countries ranges from $500 million to $1 billion. Sitophilus oryzae L. (Coleoptera, Curculionidae) and Tribolium castaneum Herbst (Coleoptera, Tenebrionidae) are the most prevalent species in storage facilities (Guru-Pirasanna-Pandi, et al. 2018). S. oryzae is primary pest insect of wheat, maize and rice in storage while T. castaneum, a secondary and generalist one. Both insects produce quantitative (grain weight) and qualitative (nutritional and industrial values) losses (Gad et al., 2020; Upadhyay et al., 2018).
Synthetic and natural pesticides are a key tool for management of sanitary and stored product insect pest (Stejskal et al., 2021). Pyrethroids, specially cypermethrin (CP), are the most widely used synthetic insecticides, since they had lower environmental impact than organochlorine, organophosphate and carbamates (Yadav et al., 2017). On the other hand, in the last 20 years, essential oils (EO) have received particular attention as novel biopesticides to replace or minimize the use of chemical insecticides (Subaharan et al., 2022). EO are effective against Cx. p. pipiens larvae and adults of S. oryzae and T. castaneum (Al-Sarar et al. 2020, El-Sabrout et. al, 2019, Teke and Mutlu, 2021, Upadhyay et al., 2018).
Over the last decade, research about nanopesticides have increased since they could be a positive solution of some problems associated with application of traditional pesticides such as harmful effects of them on the environment, humans, and other organisms or resistance development (Badawy et al., 2017). Kah et al. (2013) defined a nanopesticide as any formulation that intentionally includes materials in the nanometer size range and claims novel properties associated with these small size range. The application of nanotechnology plays a critical role in the reduction of the amount of pesticide needed to insect pest management. For example, nanoformulation can improve the solubility, bioactivity and stability of active ingredients (Kah et al., 2018). Nowadays, there are many different nanopesticides such as polymeric or metallic nanoparticles, nanotubes, microemulsions or nanoemulsions (NEs) (Hayles et al., 2017). NEs are colloidal delivery systems with droplet size in the range 20–200 nm and high thermodynamic stability. Moreover, they show higher solubility and bioavailability than bulk material (Badawy et al., 2022). NEs are elaborated with low and high-energy methods. Generally, high-energy methods (high-pressure homogenizer, high shear homogenizer or ultrasonicator) use lower concentration of surfactant compared with low-energy methods (Jesser et al., 2020). The ultrasonication provide the energy force to reduce the droplets from the macroemulsions.Furthermore, the ultrasonic technique is economically viable, energy efficient, and offer better control over formulation variables (Subaharan et al., 2022).
In the literature, there are few works about the combination of plant extract, EO or their major compounds and CP for arthropod pest management. Islam and Aktar (2013) demonstrated that plant extracts of Calotropis procera Aiton (Gentianales, Asclepiadaceae), Piper longum L. (Piperales, Piperaceae) and Polygonum hydropiper L. (Caryophyllales, Polygonaceae) + CP are more effective than the insecticide or plant extracts alone against Musca domestica L. (Diptera, Muscidae). Khalequzzaman et al. (2006) showed that extracts of Azadirachta indica A. Juss. (Sapindales, Meliaceae) + CP increased pyrethoid toxicity on T. castaneum adults. Melia azedarach L. (Sapindales, Meliaceae) and Jatrapha gossypifolia L. (Malpighiales Euphorbiaceae) EO synergize CP toxicity on larvae Spodoptera frugiperda Walker (Lepidoptera Nuctuidae) (Bullangpoti, et al., 2012). Ismail (2021) informed that CP activity was increased 2.81-fold by garlic oil on Spodoptera littoralis Boisduval (Lepidoptera, Noctuidae). Monoterpenes such as linalool and 1–8 cineol improved CP effects against Spodoptera litura Fabricius (Lepidoptera, Noctuidae) (Ruttanaphan et al. 2019). Tavares et al. (2022) demonstrated that the combination of thymol and CP increased 4-fold acaricidal efficacy of pyrethroid on Rhipicephalus microplus Canestrii (Ixodida, Ixodidae). Moreover, Jesser et al. (2020 a,b) evaluated the effect of geranium, peppermint and palmarosa NEs and post-application of β-cypermethrin (β-CP) against Plodia interpunctella (Lepidoptera, Pyralidae) and Cx p. pipiens. The authors demonstrated that NEs enhanced the insecticidal activity of β-CP between 3.78–12.25 times in P. interpunctella and 1.67–2.84 times in Cx p. pipiens.
In the attempt to develop effective and safer insect control tools, the aim of this work was to incorporate CP and EO into a single nanoformulation. Consequently, NEs loaded with peppermint or palmarosa oils and β-CP were developed using an ultrasound probe.
To the best of our knowledge, this is the first report about a new nanopesticide which combined EO and β-CP in a single NE. Taking into account that our goal is to minimize the use of conventional insecticides against insect pest, stable NEs were elaborated with minimal amount of β-CP. Moreover, surfactant:active ingredients ratio were optimized.
Materials
Peppermint and palmarosa EO were procured from Swiss-Just (manufactured under supervision and control of Ulrich Justrich AG, Walzenhausen, Switzerland). Tween 80 and β-CP were purchased from Sigma-Aldrich Commercial. Analytical grade Acetone (Dorwill, Argentina) were used as solvent.
Insect
Colonies of T. castaneum and S. oryzae were kept without exposure to any insecticide in a growth chamber at 27 ± 1°C, 45–50% r.h. (relative humidity) and 16:8 h L:D photoperiod. The insects grew in glass containers (7.5 cm diameter × 12 cm high) covered by a fine mesh cloth for ventilation. Colonies of T. castaneum contained a mixture of wheat flour and powdered milk (5:1 w/w) whereas colonies of S. oryzae, wheat grains.
The mosquito larvae were collected from a water stagnated area. The species identification was conducted at the Laboratorio de Zoología Invertebrados II, UNS, Argentina, and it was identified as Cx p. pipiens. The larvae were maintained under suitable temperature for acclimatization.
Preparation of nanoemulsions (NEs)
For NEs elaboration, different ratios of peppermint or palmarosa EO and β-CP were dissolved in acetone. After solvent evaporation, Tween 80 and 25 mL of double distilled water were added and mixed. The coarse emulsion was stirred at 700 rpm for 2 min and put into a plastic container (3 cm diameter × 50 cm high). Thereafter, it was sonicated using a Sonics Vibra cell, VCX 130 with a titanium probe tip (9.5mm diameter, 130W nominal power, 20 kHz frequency). According with Jesser et al. (2020 a,b), the ultrasound parameters were: power: 65 W, cycles: 30 on/20 off and sonication time: 2 min.
Droplets sizes
The average droplets sizes and polydispersity index (PDI) of NEs were measured by dynamic light scattering (DLS) using a Zetasizer nano instrument ZEN 3690 model (Malvern, UK). Three replicates of each measurement were carried out at room temperature.
Emulsion stability
To study the stability of the emulsion over time, changes in the droplet size and the visual appearance were observed. For two months, the samples were maintained at 25°C and were checked one time a week. Three replicates of each measurement were carried out.
Bioassays
T. castaneum and S. oryzae bioassays
To evaluate the combined effect of peppermint and palmarosa EO + β-CP or their NEs, aliquots of each product were topically applied to batches of 10 adults of T. castaneum and S. oryzae. For EO, a pre-treatment was carried out using doses at equal amount of oil in NEs. After a 2 h pre-treatment, acetonic solutions of β-CP were applied in doses ranging from 0.06 to 2 µg insect− 1. NEs were topically applied in doses ranging from 0.075 to 0.6 µg β-CP insect− 1. Acetone or surfactant alone (in the corresponding ratio of NEs) were topically applied as control. Moreover, the toxicity of NEs were evaluated for one and two months with the same assay. All experiments were performed in quadruplicate (n = 40) and the insects were maintained at 27 ± 2°C, 45–50% r.h. and 16:8h L:D photoperiod. Insect mortality was recorded after a 24 h exposure.
Cx. p. pipiens bioassays
Bioassays were performed according to WHO (2005) with fourth instar larvae of Cx p. pipiens. All experiments were performed in quadruplicate with 20 fourth instar larvae in each replicate (n = 80).
To evaluate the combined effect of peppermint and palmarosa EO + β-CP, these active ingredients where mixed in tap water containing Tween 80 (at the same NEs ratio). To study the toxic effect of single NEs, the nanoformulations were diluted in tap water alone. As positive control, β-CP was mixed in tap water containing Tween 80 (1% p/v). In all treatments, the concentrations of β-CP ranged from 0.010 to 1 ppm. Two negative controls were performed; the first one with tap water alone and the second one with tap water containing the surfactant alone (in the corresponding ratio). Moreover, the toxicity of NEs were evaluated for one and two months with the same assay. The biological assays were performed at 27 ± 2°C, 45–50% r.h. and 16:8h L:D photoperiod. After a 24 h exposure, the mortality level was recorded.
Statistical analysis
Mortality data were used to obtain to obtain LD50 and LC50 values (SPSS 25.0 statistical software). The LD50 and LC50 values were considered significantly different if their 95% confidence intervals did not overlap.
Moreover, potentiation effect of different products based on LD50 or LC50 values were calculatedas:
The potentiation effect was considered statistically significant when confident intervals of LD50 or LC50 values did not overlap.
β-cypermethrin optimization
The development of a new single NE, which combine a pyrethroid with EO, is a big step in the reduction of use of conventional insecticides and the implementation of novel pest management strategies.
Based on our previous works (Jesser et al. 2020 a, b) the ratio between Tween 80:active ingredients (EO + β-CP) was fixed at 2:1 to obtain the minimum possible droplet size (MPDS) of NEs. Then, nanosystems loaded with different amount of EO (from 0.230 to 0.275 g) and β-CP (from 0.015 to 0.060 g) were formulated and physiochemical parameters were analyzed (Table 1). It is known that cypermethrin is a water insoluble insecticide. Therefore, for de development of a stable NE, it is critical the study of the interactions between surfactant concentration, oil type and ultrasonic parameters (Badawy et al, 2019). Generally, NEs droplet size grew when the amount of β-CP increased, affecting their visual appearance. Wang et al. (2007) observed similar tendency in β-CP nanoemulsions developed with methyl decanoate as oil phase and polyoxyethylene lauryl ether as surfactant. In our work, when the amounts of β-CP were 0.030 and 0.060 g, the visual appearance of NEs were whitish and white, respectively, and the sizes were > 249 nm. Transparent NEs were obtained when the amount of EO was 0.275 g and β-CP was 0.015 g. The NEs loaded with peppermint EO + β-CP showed two peaks at 12.77 (33.4% intensity) and 299.1 nm (66.6% intensity) and a PDI value of 1. The size of palmarosa NEs was 104.5 nm and their PDI value, 0.220. Similar values of droplet size using CP alone and different surfactants were found in the literature. Du et al (2016) developed NEs of β-cypermethrin using methyl laurate as the oil phase, alkyl polyglycoside and polyoxyethylene 3-lauryl ether as surfactants. These NEs had a mean droplet size of 113.5 nm and a PDI value of 0.128. NEs with β-CP, methyl oleate (solvent) and polyoxyethylene castor oil ether (surfactant) showed a mean droplet size of 166 nm and the PDI value was 0.160 (Feng et al., 2016). Taktak et al. (2021) developed NEs of 0.5% CP, 44% solvent (DMSO), 15% surfactant (tween 80) and 40.5% water using ultrasound. These NEs had a mean droplet size of 90.3 nm, a PDI value of 0.377 and the visual appearance was transparent. There is a relation between visual appearance and sizes of NEs and its interaction with light. When droplet diameter size is reduced, the interaction with visible light is smaller. Consequently, there are a change in color of nanosystem from white to transparent (Salvia-Trujillo et al. 2015).
| Essential oils | Sample | Tween 80 (g) | Essential Oil (g) | β-Cypermethrin (g) | Water (g) | Size (nm) ± SE | PDI ± SE | Visual appareance |
|---|---|---|---|---|---|---|---|---|
| Peppermint | 1 | 0.580 | 0.275 | 0.015 | 25 | Peak 1: 12.77 ± 1.72 Peak 2: 299.1 ± 43.56 | 1 ± 0.0 | Transparent |
| 2 | 0.580 | 0.260 | 0.030 | 25 | 269.2 ± 33.50 | 0.481 ± 0.017 | whitish | |
| 3 | 0.580 | 0.230 | 0.060 | 25 | 319.8 ± 26.93 | 0.122 ± 0.023 | white | |
| Palmarosa | 1 | 0.580 | 0.275 | 0.015 | 25 | 104.5 ± 27.98 | 0.220 ± 0.011 | transparent |
| 2 | 0.580 | 0.260 | 0.030 | 25 | 249.0 ± 46.95 | 0.504 ± 0.016 | whitish | |
| 3 | 0.580 | 0.230 | 0.060 | 25 | 325.7 ± 20.76 | 0.224 ± 0.020 | white |
Stability of NEs
NEs stability were analyzed by monitoring the droplet size, the PDI and visual appearance at 1, 30 and 60 days of storage at 25°C. Figure 1A. showed the stability of peppermint NEs over time. At 1 day of storage, it observed in the NEs two peaks at 12.77 and 299.1 nm, a PDI value of 1 and visual appearance was transparent. At 30 days, two peaks were observed at 11.43 (8.3%) and 187.8 (91.7% intensity) nm, the PDI value decreased (0.390) and the visual appearance was translucent. At 60 days of storage, the NEs showed a droplet size of 299.1 nm, a PDI value of 0.571 and visual appearance was whitish. However, the NEs not showed a phase separation for 60 days. The increase in mean droplet size can be explained by the Ostwald ripening phenomenon. Owing to the better solubility of the minor droplets in the continuous phase, mean droplets size of NEs tend to grow over time by the coalescent of minor droplets to bigger ones (Falleh et al. 2021). Similar trend was observed in NEs elaborated with peppermint oil alone and Tween 80 using the same ultrasound parameters (Jesser et al., 2020b). Falleh et al. (2021) reported an increase in mean droplet size of NEs formulated with peppermint oil alone and Tween 20 using a high-pressure homogenizer during 22 days of storage at 4°C. An increase of 20 − 30 nm in mean droplet size of peppermint NEs formulated with succinylated waxy maize starch using a high-pressure homogenizer was informed by Liang et al. (2012). As in the present work, authors did not show phase separation when stability test was concluded.
Figure 1B. expressed the stability of palmarosa NEs for 60 days. Generally, it observed a slight increase in mean droplet size, demonstrating a good NEs stability. At 1 day of storage, the mean droplet size was 104.5 nm, a PDI value of 0.220 and visual appearance was transparent. At 30 days, NEs showed a mean droplet size of 153.5 nm, a PDI value of 0.274 and visual appearance was translucent. Finally, at 60 days, the mean droplet size was 180.1 nm, a PDI value of 0.294 and visual appearance was translucent. Jesser et al. (2020b) informed that nanosystems formulated with of palmarosa oil alone and Tween 80 were unstable. However, when linalyl acetate was incorporated to palmarosa NEs, they were stable for 120 days with a slowly increase in the mean droplet size. It is important to emphases that the β-CP could promote a similar effect in the stabilization of that nanoformulation as linalyl acetate.
It is known that precipitated pesticides have lower bioavailability and bioactivity (Wang et al. 2007). In present study, after 60 days no β-CP precipitation was observed. In literature, there is a lack of information about the stability of NEs containing CP. Moreover, the works about this topic performed the stability test for few days. In this regard, Feng et al. (2016) studied the stability of NEs prepared with β-CP and polyoxyethylene castor oil ether (surfactant) for 7 days and showed no substantial change in droplet size. Wang et al. (2007) developed NEs of β-CP and ethylene oxide as surfactant, which were stable for 24 h. In our study, the stability of a single NEs of EO + β-CP was better than those informed in previous cited works. Probably, some EO compounds could act as co-surfactant, improving the stability of NEs (Ziani et al., 2012).
Bioassays
Effect of NEs in the enhancement of cypermethrin bioactivity
The insecticidal activity of the NEs formulated in this work (ingredient active:surfactant ratio from 1:2 and ultrasound parameters: ultrasound power = 65 W, sonication time = 2 min and cycles = 30 on/20 off.) were tested on T. castaneum and S oryzae adults and Cx. p. pipiens larvae.
The results from the bioassay in T. castaneum showed that the LD50 values from β-CP alone and the combination of peppermint EO + β-CP were not significant different (P > 0.05). The values after 24 h of exposure were 0.767 and 0.412 µg insects − 1, respectively (Table 2). These LD50 values were significantly higher than those for NEs (0.176 µg insects − 1) at 24 h (P < 0.05). This result showed that NEs significantly potentiated the insecticidal activity of β-CP 4.35-folds (P < 0.05) (Table 3). The improved β-CP bioactivity produced by NEs was similar in S. oryzae (4.22-folds) (P < 0.05) (Table 3). In this insect, after 24 h exposure, the LD50 values from β-CP alone and the combination of peppermint EO + β-CP were 0.882 and 0.412 µg insects− 1, respectively (Table 2). The LD50 value of NEs at 24 h was significantly lower (0.209 µg insects − 1) (P < 0.05). Table 2 also showed the toxicity effect of β-CP alone or combine with peppermint EO and their NEs in Cx. p. pipiens after 24 h of exposure. The toxicity order based on LC50 values was NEs (0.018 ppm) > β-CP + peppermint EO (0.030 ppm) > β-CP alone (0.288 ppm) (P < 0.05). The NEs significantly potentiated the toxic effect of β-CP (16-folds) (P < 0.05). Briefly, except for T. castaneum, peppermint oil significantly increased the bioactivity of β-CP (P < 0.05). Moreover, for all insect pest, NEs toxicity was approximately two-fold higher than β-CP + peppermint EO treatment (P < 0.05).
| β-CP alone | β-CP + EO | Nanoemulsion (1 day) | Nanoemulsion (30 day) | Nanoemulsion (60 day) | |
|---|---|---|---|---|---|
| T. castaneum | 0.767 (0.567–0.946) a | 0.412 (0.306–0.572) a | 0.176 (0.152–0.206) b | 0.169 (0.141–0.201) b | 0.195 (0.165–0.232) b |
| S. oryzae | 0.882 (0.701–1.166) a | 0.472 (0.366–0.576) b | 0.209 (0.193–0.261) c | 0.257 (0.206–0.301) c | 0.314 (0.257–0.374) c |
| Cx. p. pipiens | 0.288 (0.254–0.328) a | 0.030 (0.026–0.040) b | 0.018 (0.016–0.021) c | 0.025 (0.021–0.029) bc | 0.036 (0.032–0.042) b |
| For each row, different letters indicate significant statistical differences (P < 0.05). | |||||
| T. castaneum | β-cypermethrin alone | β-cypermethrin + EO | Nanoemulsion |
|---|---|---|---|
| β-cypermethrin alone | - | NS | * |
| β-cypermethrin + EO | 1.86 | - | * |
| Nanoemulsion | 4.35 | 2.34 | - |
| S. oryzae | β-cypermethrin alone | β-cypermethrin + EO | Nanoemulsion |
|---|---|---|---|
| β-cypermethrin alone | - | * | * |
| β-cypermethrin + EO | 1.86 | - | * |
| Nanoemulsion | 4.22 | 2.25 | - |
| Cx. p. pipiens | β-cypermethrin alone | β-cypermethrin + EO | Nanoemulsion |
|---|---|---|---|
| β-cypermethrin alone | - | * | * |
| β-cypermethrin + EO | 9.6 | - | * |
| Nanoemulsion | 16 | 1.66 | - |
| “*” indicated significant differences (p < 0.05) and “NS” not significant differences (p > 0.05). | |||
Table 4 informed the biological activity of β-CP alone or combine with palmarosa EO and their NEs in T. castaneum after 24 h of exposure. The results showed that not significant different were found between the LD50 value of β-CP alone (0.767 µg insects− 1) and the combination of β-CP + palmarosa EO (0.436 µg insects− 1) (P > 0.05). The LD50 value of NEs was 0.089 µg insects− 1, which indicated that nanoformulation significantly potentiated the toxicity effect of β-CP 8.62 times (P < 0.05) (Table 5). In S. oryzae, after 24 h of exposure, the toxicity order based on LD50 values was NEs (0.226 µg insects− 1) > β-CP + palmarosa EO (0.313 µg insects− 1) > β-CP alone (0.882 µg insects− 1) (P < 0.05) (Table 4). It was observed that the NEs significantly enhanced 3.9-folds the toxic effect of β-CP (P < 0.05) (Table 5). In Cx. p. pipiens, after 24 h of exposure, the toxicity order based on LC50 values was NEs (0.027 ppm) > β-CP + palmarosa EO (0.156 ppm) > β-CP alone (0.288 ppm) (P < 0.05) (Table 4). NEs potentiated 10.6-folds the toxic effect of β-CP (P < 0.05) (Table 5). Summarizing, except for T. castaneum, palmarosa oil significantly increased the bioactivity of β-CP (P < 0.05). Moreover, for all insect pest, NEs enhanced the insecticidal activity of β-CP + palmarosa EO between 1.38 and 8.66 folds (P < 0.05).
Peppermint and palmarosa EO demonstrated insecticidal activity in different insect pest such as S. oryzae, T. castaneum, Plodia interpunctella (Hübner) (Lepidoptera, Pyralidae), Spodoptera frugiperda Walker (Lepidoptera, Noctuidae), Cx. p. pipiens, Musca domestica L. (Diptera, Muscidae) and Blatella germanica L. (Blattodea, Blattellidae) (Devi, et al. 2020; Jesser et al., 2020c; Mohafrash et al., 2020; Sinthusiri, J., & Soonwera, 2013; Sousa-Barbosa et al. 2018; Yeguerman et al., 2020; 2022). It is known that EO can enhance or synergize the insecticidal activity of synthetic insecticides (Ruttanaphan et al., 2019). It was probed that EO inhibit detoxifying enzymes and improve the overall toxicity of synthetic insecticides by increasing their bioavailability and their interaction with the target site in the insect (Norris et al., 2019). Particularly, some works informed about the use of EO or plant extract as synergists of CP against arthropods pest (Bullangpoti, et al., 2012; Islam and Aktar, 2013; Ismail, 2021; Jesser et al., 2020 a,b; Khalequzzaman et al., 2006; Ruttanaphan et al., 2019; Tavares et al., 2022). However, to the best of our knowledge, this is the first report about the elaboration of a single NEs which combine EO and CP in the same formulation using Tween 80 and ultrasound. In our work, the NEs showed an enhancement of bioactivity of CP. Probably, this phenomenon could be attributed to the interaction between EO and CP and the nanometric size of NEs. It is important to emphases that, at nanoscale, the materials have unique chemical, physical and biological properties that are not observed in their bulk forms (Chhipa, 2017). For example, it was demonstrated that nanometric size facilitate the adhesion of NEs to insect body part promoting irritation and serious damage of cuticle, increasing their penetration (Hashem et al., 2018). It is important to emphases that NEs reduce organic solvent content in conventional insecticides formulation.
Finally, when the insecticidal activity of β-CP enhanced by peppermint or palmarosa oil was compared, it was observed that just in Cx. p. pipiens the treatment β-CP + peppermint EO was significantly higher than β-CP + palmarosa EO (P < 0.05). In addition, just in T. castaneum the NEs of β-CP + palmarosa EO was significantly more effective than the other NEs. In this regard, several studies reported that insecticidal activity of EO compounds is species-dependent (Alkan, 2020; Benelli et al., 2018; Bett et al., 2016;2017; Saleem et al., 2014). Recently our research group reported that the major compounds of peppermint oil were menthol and isomenthone and for palmarosa EO, geraniol (Yeguerman et al. 2020). Probably, these compounds could be involved in the insecticidal activity of the oils.
| β-CP alone | β-CP + EO | Nanoemulsion (1 day) | Nanoemulsion (30 day) | Nanoemulsion (60 day) | |
|---|---|---|---|---|---|
| T. castaneum | 0.767 (0.567–0.946) a | 0.436 (0.236–0.626) a | 0.089 (0.073–0.111) b | 0.231 (0.122–0.322) a | 0.267 (0.195–0.341) a |
| S. oryzae | 0.882 (0.701–1.166) a | 0.313 (0.275–0.412) b | 0.226 (0.147–0.255) c | 0.323 (0.275–0.378) b | 0.403 (0.342–0.484) b |
| Cx. p. pipiens | 0.288 (0.254–0.328) a | 0.156 (0.132–0.184) b | 0.027 (0.017–0.043) c | 0.035 (0.032–0.038) c | 0.041 (0.037–0.046) c |
| For each row, different letters indicate significant statistical differences (P < 0.05). | |||||
| A | β-cypermethrin alone | β-cypermethrin + EO | Nanoemulsion |
|---|---|---|---|
| β-cypermethrin alone | - | NS | * |
| β-cypermethrin + EO | 1.76 | - | * |
| Nanoemulsion | 8.62 | 4.89 | - |
| B | β-cypermethrin alone | β-cypermethrin + EO | Nanoemulsion |
|---|---|---|---|
| β-cypermethrin alone | - | * | * |
| β-cypermethrin + EO | 2.81 | - | * |
| Nanoemulsion | 3.90 | 1.38 | - |
| C | β-cypermethrin alone | β-cypermethrin + EO | Nanoemulsion |
|---|---|---|---|
| β-cypermethrin alone | - | * | * |
| β-cypermethrin + EO | 1.84 | - | * |
| Nanoemulsion | 10.6 | 8.66 | - |
| “*” indicated significant differences and “NS” not significant differences. | |||
Insecticidal activity of NEs over time.
Table 2 showed the insecticidal activity of NEs containing peppermint EO + β-CP for 2 months in different insect pest. It was observed that in T. castaneum and S. oryzae, the NEs maintained the bioactivity for 2 months and no significant changes in LD50 values were detected (P > 0.05). However, in Cx p. pipiens, LC50 value at 2 months had a slightly increase compared to NEs at 1 day (P < 0.05). In addition, no significant differences were observed in LC50 value between 1 and 2 months (P > 0.05).
Table 4 presented de bioactivity of NEs elaborated with palmarosa EO + β-CP for 2 months in different insect pest. In T. castaneum and S. oryzae, the NEs at 1 month were slightly less toxic than NEs at 1 day (P < 0.05). However no significant differences were observed in LD50 values between 1 and 2 months (P > 0.05). In the other hand, in Cx p. pipiens, the NEs maintained the larvicidal activity for 2 months and no significant changes in LC50 values were observed (P > 0.05).
In literature, there is a lack of information about this topic. The NEs developed in this work showed a better insecticidal activity over time compared with other NEs elaborated with EO or synthetic insecticide. Almadiy et al. (2022) evaluated the residual activity of NEs formulated with Deverra tortuosa DC (Apiales, Apiaceae) EO and Tween 80 in treated grains at values LC95 against Callosobruchus maculatus Fabricius (Coleoptera, Chrysomelidae). These NEs showed a lethal time 50 (LT50) = 29.4 ± 2.4 days. Faustino et al. (2020) evaluated the toxicological effect of NEs of Protium heptaphyllum Marchand (Sapindales, Burseraceae) and Tween 80 against Aedes aegypti Meigen (Diptera, Culicidae). At 20 µg mL− 1 theses nanosystems caused mortality for 96 h. NEs elaborated with Cinnamomum zeylanicum J.Presl (Lauraceae) or Myrtus communis L. (Myrtales, Myrtaceae) EO (0.5% w/w) and Tween 80 and Span 20 showed larvicidal activity for 8 days on Anopheles stephensi Liston (Diptera, Culicidae) (Firooziyan et al., 2021; 2022). Feng et al. (2021) evaluated the residual activity of NEs with pyriproxyfen, methyl oleate (solvent) and polyoxyethylene castor oil ether (surfactant) in M. domestica. These NEs showed insecticidal activity for 14 days.
Despite the fact that both NEs of peppermint or palmarosa oils + β-CP had a slightly increase of particle size for 2 months, these nanosystems demonstrated an excellent insecticidal activity in all insect pest over time.
As is known, green chemistry tends to minimize human health and environmental risks at the time to maximize efficiency throughout the chemical production process (Mulvihill et al., 2011). NEs elaborated in our work could be thought an innovation in this area since they produce a high insecticidal activity with low synthetic insecticide amount, as a result of the potentiation improved by the incorporation of EO in the NEs. Moreover, organic solvents are commonly used when synthetic insecticides are applied at field. In this sense, our NEs eliminate the hazards of organic solvent due to they are aqueous systems that could be directly applied. Finally, it is important to note that ultrasonic technique is considered a green method attributable to its energy-efficiency, low production cost, ease of system manipulation and better control over formulation (Jesser et al., 2020b).
In this work, single NEs elaborated with peppermint or palmarosa EO, β-CP and tween 80 have been successfully formulated by ultrasound method. Our results indicated that both singles NEs were stable for 2 months and enhanced the insecticidal activity of β-CP against T. castaneum, S. oryzae and Cx p pipines. Based on these findings and in order to minimize the use of conventional insecticides and organic solvents, the current study represents an advancement in green pest management programs.
COMPETING INTERESTS
The authors declare no conflicts of interest.
AUTHOR CONTRIBUTION STATEMENT
EJ, CD and JW conceived and designed research. EJ, CY and RU conducted experiments. EJ, AP CD and JW analyzed data. EJ wrote and JW supervised the manuscript. All authors read, reviewed and approved the manuscript.
FUNDING SOURCES
This research was supported by Grants PGI 24/B277 from UNS; PICT- 2018- 03283 from ANPCyT (Agencia Nacional de Promoción Cientifica y Tecnológica).
ETHICAL APPROVAL
not applicable
AVAILABILITY OF DATA AND MATERIALS
not applicable
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No competing interests reported.