Environmental Science and Pollution Research

https://doi.org/10.1007/s11356-019-07332-y

RESEARCH ARTICLE

Eco-friendly pesticide based on peppermint oil nanoemulsion:

preparation, physicochemical properties, and its aphicidal activity
against cotton aphid
Mojgan Heydari 1 & Amirmostafa Amirjani 2 & Mozhgan Bagheri 1 & Iman Sharifian 3 & Qodrat Sabahi 3

Received: 2 August 2019 / Accepted: 6 December 2019

# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Using organic insecticides including plant oils, it is possible to design a new perspective for the control of insect pests. In this
research, nanoemulsion formulations of Mentha piperita, wild-type essential oil (EO) were prepared utilizing high-energy
ultrasonication process. Physicochemical properties of nanoemulsions were precisely studied by measurement various parame-
ters including pH, viscosity, conductivity, and zeta potential. Experimental design by the aid of response surface methodology
(RSM) was used to highlight the physicochemical roles of EO percentage (1% to 5% (v/v)) and surfactant concentration (3% to
15% (v/v)) for achieving minimum droplet diameter with high physical stability. The nanoemulsion formulations were then
characterized using dynamic light scattering, transmission electron microscopy, and optical clarity. Afterward, an appropriate
model between the variable factors (EO percentage and surfactant concentration) and the response (hydrodynamic particle size)
was statistically developed. Under the optimum conditions, nanoemulsion with hydrodynamic particle size less than 10 nm with
high physical stability is obtainable. Bioassay experiments were carried out to elucidate the effects of nanoemulsion on the cotton
aphid. Synthesized nanoemulsion formulations showed relatively high contact toxicity (average value of LC50 was about 3879.5
± 16.2 μl a.i./L) against the pest. On the basis of the obtained results, prepared nanoemulsion using M. piperita is potentially
applicable as organic insecticides against cotton aphid.

Keywords Organic pesticide . Nanoemulsion . Ultrasonic . Aphis gossypii . Mentha piperita L. . Eco-friendly

Introduction multivoltine pests such as aphids are among the major agri-
cultural pests having significant yield loss every year in the
Crop losses arising from insect pests overcome 30% of crop world (Ebrahimi et al. 2013). The cotton aphid, Aphis gossypii
production all over the world (FAO 2011). Polyphagous Glover (Homoptera: Aphididae), is the main pest species
causing considerable losses to cotton and thus preventing sus-
Responsible editor: Giovanni Benelli tainable production of this crop (El Shafie and Basedow
Electronic supplementary material The online version of this article 2003). However, there is still a significant lack of an eco-
(https://doi.org/10.1007/s11356-019-07332-y) contains supplementary friendly and efficient method to overcome the pest-resistant
material, which is available to authorized users. issue. Essential oils (EOs) are mainly obtained from the steam
distillation of aromatic plants’ foliage that produces EO as
* Mojgan Heydari their secondary metabolites. Rapidly, EOs found their appli-
[email protected]
cations in foods, cosmetics, and pharmaceuticals due to their
antioxidant, antiradical, and antimicrobial properties
1
Department of Nanotechnology and Advanced Materials, Materials (Kalemba and Kunicka 2003; Burt 2004). EOs are widely
and Energy Research Center, P.O. Box 31787-316, Karaj, Iran
used as the natural protection agents and environmentally be-
2
Institute of Chemical Sciences and Engineering (ISIC), École nign pesticides to overcome the pest resistance issue (Tripathi
Polytechnique FÉdÉrale de Lausanne (EPFL),
CH-1015 Lausanne, Switzerland
et al. 2009; Balasubramani et al. 2017; Benelli et al. 2019;
3
Govindarajan et al. 2018a, b; Mossa et al. 2018; Osanloo
Department of Plant Protection, Faculty of Agriculture and Natural
Resources, University of Tehran, P.O. Box 14155-6619, Karaj, Iran
et al. 2018).
 Environ Sci Pollut Res

EOs can be used in crops protection, efficiently owing to Main difficulty for the application of EOs in industrial ag-
their wide range of activity which hinders the development of riculture is attributed to their more solubility in organic sol-
resistant pests (Isman 2000; Rajendran and Sriranjini 2008; vents rather than in water. Entrance of nanotechnology into
Figueiredo et al. 2008; González et al. 2017; Hashem et al. agricultural science not only has brought essential develop-
2018; Feng et al. 2019). The essential oil taken from industrial ments in crop protection, disease management, minimizing
hemp (Cannabis sativa L.) by-products was used as an effec- nutrient losses in fertilization, plant pathogen detection, and
tive tool for insect pest management in organic crops by plant germination and growth (Khot et al. 2012; Pavela and
Benelli et al. (2018c). Their results revealed the possibility Benelli 2016), but also helped to overcome the low water
of using crop residue of industrial hemp as a source of eco- solubility of EOs (Donsi et al. 2012). As an instance, encap-
friendly botanical insecticides in the integrated pest manage- sulation of EOs in the form of oil-in-water (O/W)
ment and organic agriculture, particularly in control of aphid nanoemulsions (oil droplet <100 nm) could be an effectively
and housefly populations (Benelli et al. 2018c). EOs of genus possible solution for the mentioned difficulty. Reduced toxic
Mentha spp. (family: Lamiaceae) were tested against various effects of synthetic pesticides along with the pest control are
insects (Pavlidou et al. 2004; Sharma and Vidyarthi 2010; the main advantages of nanoemulsions (Suresh-Kumar et al.
Khani and Asghari 2012; Motazedian et al. 2012). 2013).
Repellency and toxicity effects of Mentha longifolia L. Recently, utilization of EOs in nanoemulsion formulation
against Tetranychus urticae Koch have been reported by with the aim of efficiency increase and extend potential appli-
Motazedian et al. (2012). Mentha pulegium L. EO showed cations has been widely considered (Nuchuchua et al. 2009;
the highest insecticidal activity among the three plant EOs Anwer et al. 2014; Fernandes et al. 2014; Sugumar et al. 2014;
taken from Greek plants against two fruit flies (Pavlidou Nantarat et al. 2015). Reducing the size of natural oils from
et al. 2004). Also, an ethanolic extract of M. piperita leaves original to nano-size and their consequent usage in the form of
showed high toxicity against one of the most important agri- nanoemulsions is an effective key solution as larvicidal agents
cultural pests, A. gossypii (Sharma and Vidyarthi 2010). (Anjali et al. 2012).
Using organic insecticides including plant oils is a new out- In this research, a straightforward method has been pro-
look in the control of insect pests (Li et al. 2014; Pavela and posed for preparing highly stabilized nanoemulsion from
Benelli 2016). Benelli et al. (2018b) showed that EOs taken from M. piperita. In this method, a non-ionic surfactant (Tween
Cuminum cyminum and Pimpinella anisum had remarkable in- 80) and EO of M. piperita were homogenously mixed and
secticidal efficacy in terms of acute toxicity against insect species ultrasonicated. In order to precisely identify the effect of each
including the lymphatic filariasis and Zika virus vector of Culex factor in the synthesis procedure, as well as the final
quinquefasciatus, common housefly of Musca domestica, peach- nanoemulsion size, RSM technique was used. This method
potato aphid of Myzus persicae, and tobacco cutworm of presents reliable information about probable interactions be-
Spodoptera littoralis led to develop “green” botanical insecti- tween factors involved in a process, i.e. the optimum level of a
cides. It is worth mentioning that these EOs insecticides did not factor might be influenced by the other factors. In this regard,
affect any beneficial organisms, such as ladybugs and earth- the factorial design of experiments (DOE) was used which is
worms (Benelli et al. 2018b). capable to take into account several factors at different levels,
Despite the ability to control insect pests, many plant oils simultaneously. To the best of our knowledge, there is no
have poor water solubility which restricts their use as an in- report on preparation of M. piperita nanoemulsion with this
secticide at field conditions. Several researchers have focused size and through a simple approach and evaluation of it
on the evaluation of EOs as promising tools for pest control against one of the most important pests of vegetables,
through developing more efficient formulations (Oyedele A. gossypii. It should be noted that A. gossypii is found every-
et al. 2002; Sharifian et al. 2011; Saranya et al. 2012; where, sucks on nearly all of the green parts of its hosts, and
Sharifian et al. 2015; Hashem et al. 2018; Massoud et al. impairs photosynthesis. Hence, it can weaken the plant even
2018). Among the suggested formulations, nanoemulsions further and may render vegetables unmarketable. In addition,
based on plant oils having simultaneous biocompatibility like most aphids, A. gossypii is actually an important virus
and bioavailability properties have been recently sharpened vector, transmitting a number of economically important plant
(Saranya et al. 2012; Hashem et al. 2018; Massoud et al. viruses. It can transfer about 70 different types that some of
2018). Emulsion formulations are based on water-soluble them may cause more damage than the aphid itself. Thus, it is
forms of hydrophobic materials with different emulsifiers sim- extremely important to find a pesticide formulation against
plifying the use of insoluble pesticides (Fernandes et al. 2014). this pest and prevent any damage to agricultural crops and
EO-based nanoemulsions have been recognized as substan- products. The M. piperita nanoemulsion formulation in the
tially valuable products for mosquito control (Ghosh et al. present work is smaller in size and present higher stability
2013; Benelli and Pavela 2018; Benelli et al. 2018a; Pavela compared to those ones reported by other researchers (Liang
et al. 2019). et al. 2012; Barzegar et al. 2018; Massoud et al. 2018).
Environ Sci Pollut Res

Further, the parametrical study implemented in this work pro- order to ove rcome the heat and de naturation of
vides a useful range of experimental parameters for future nanoemulsions. The sonication time was fixed for 15 min in
studies in this field. Based on the obtained results, the opti- all cases. Although longer times were also considered, there
mized nanoemulsion could be used as an efficiently natural was not any meaningful difference between them. Then, the
pesticide against an important pest, A. gossypii, with accept- formulated nanoemulsions were characterized by different
able contact toxicity. techniques and their stability was also investigated.

Characterization of nanoemulsion
Materials and methods
Droplet size, morphology, and zeta potential analysis
Chemical reagents
The mean size of nanoemulsion droplets was measured using
Peppermint (M. piperita) EO was prepared from Behboo Co. a dynamic light scattering instrument (Zetasizer Nano ZS,
(Ghamsar Ltd., Kashan, Iran). EO of peppermint was extract- model 3000HAs, Malvern, UK). The samples were diluted
ed by hydrodistillation method using the leaves of the plant in using DI water to decrease multiple scattering effects, and
Ghamsar, Iran. A non-ionic surfactant (Tween 80) was pur- each data point was obtained from average of 24 individual
chased from MERCK Chemicals Ltd. (Darmstadt, Germany). runs. Zeta potential was measured by Zetasizer 3000HAs
Autoclaved double-distilled water was used for the prepara- (Malvern Instrument Ltd., Malvern, UK) equipped with a
tion of all aqueous solutions. Other chemicals were all of the He–Ne laser beam at 658 nm (scattering angle: 90°). The zeta
analytical grade. potential values were determined from electrophoretic mobil-
ity of the oil droplets. The measurements were carried out on
GC-MS analysis diluted emulsion formulations. The refractive index and vis-
cosity were kept at 1.33 and 1.0 cP, accordingly. The morphol-
The chemical composition of M. piperita oil was analyzed by ogy and size of the particles dispersed in the nanoemulsion
gas chromatograph-mass spectrophotometer (Agilent 6890N/ formulations were investigated using a transmission electron
5973N GC–MS, USA) with an HP-5 MS column (30 m microscope (TEM, Zeiss EM10C, 80 KV, Germany).
length, 0.25 mm ID, 0.25 μm film thickness and cross bond
of 5% diphenyl/ 95% dimethyl polysiloxane). Helium as the Physicochemical studies
carrier gas was applied at the flow rate of 1 ml/min. The
samples were injected at a temperature of 80 °C, while the The nanoemulsion formulations were characterized in terms
detector temperature was 280 °C. The oven was programmed of pH, viscosity, and conductivity. In the case of viscosity
from 50 to 250 °C at the rate of 8 °C/min. Mass spectra were measurement, as-prepared nanoemulsions were used without
recorded over 50–550 amu range with 70 eV of ionization any dilution. Viscosity was measured by the Brookfield
energy and ion source temperature of 230 °C. The split injec- Viscometer, model RV-DV III Ultra, USA. The conductivity
tion was performed. The oil constituents were identified based of nanoemulsion formulations was measured using 712
on the comparison of retention indices and mass spectra cor- Conductometer Metrohm, USA. The pH of the samples was
relations with NIST 2008 and Wiley 2007 mass spectrometry measured using a pH meter (Mettler Toledo, Switzerland). All
libraries as well as comparison with literature data (Adams characterizations were performed at ambient temperature.
2007; Schmidt et al. 2009; Babushok et al. 2011; Beigi et al.
2018; Benelli et al. 2018b); Buleandra et al. 2016; Bishr and Insect collection and rearing
Salama 2018; Chauhan et al. 2018).
Cotton aphid (A. gossypii) adults were collected from the cul-
Nanoemulsion preparation turing room in the Department of Plant Protection, University
of Tehran, Karaj, Iran. Cucumber (Cucumis sativus L.) leaves
Oil-in-water (O/W) nanoemulsions were prepared by mixing were used as the contact toxicity bioassay media. Collected
of M. Piperita oil, wild type as oil phase, non-ionic surfactant aphids were reared on the leaves of cucumber for three gen-
Tween 80, and water using magnetic stirrer according to a erations at 25 ± 2 °C, 60% R.H. (Relative Humidity), and a
slightly modified technique (Ghosh et al. 2013; Mishra et al. photoperiod of 16L:8D.
2018; Mossa et al. 2018). After emulsion preparation, samples
were sonicated using a 24-kHz ultrasonic processor (Labsonic Bioassay tests
P, Sartorius Stedim Biotech, Germany) with the maximum
power output of 400 W and probe diameter of 10 mm. The Twenty adult aphids (1 day old) were used in 24 h contact
ultrasonication process was performed under the ice bath in toxicity experiments. Bioassay test was performed on the
 Environ Sci Pollut Res

basis of leaf disc method in Petri dishes (7 cm in diameter) Table 1 Experimental ranges for the level of independent variables in
response surface study
with a 2-cm diameter hole on top to provide ventilation and
avoiding fumigation. The test was run at 25 ± 2 °C and 60 ± Factors Unit Notation Level
5% R.H. and under a L16:D8 photo period, in the germinator.
Nanoemulsions were prepared with different concentrations −1 0 +1
and were diluted by distilled water. Leaflets were soaked in EO percentage % EO 1 3 5
nanoemulsion solution for 30 s and let air-dried for about
Surfactant concentration % S 3 9 15
10 min. Preliminary tests were performed to select the precise
concentrations. The experiment was done in three replications
and mortality of different concentrations was recorded.
Through leaf dipping method, the surface of cucumber leaf the results are reported as mean values in a randomized ar-
was covered by nanoemulsions, while distilled water was used rangement to minimize the artifacts. To estimate the response
in control samples. In order to be completely homogenized, values through a range of input factors’ values, a quadratic
nanoemulsions were stirred before the experiment using a polynomial regression model was used (Eq. (1)):
magnetic stirrer. Mortality percent of aphids was counted
2 2 2
24 h after the treatment. Aphids not showing any movement Y ¼ b0 þ ∑ bi Xi þ ∑ bii X2i þ ∑ ∑ bij Xi X j ð1Þ
after touching with a fine brush were considered as died. i¼1 i¼1 i¼1 j¼iþ1

Mortality correction was done by the Abbott formula

(Abbott 1925). The LD50 values were obtained through
Probit analysis (Robertson et al. 2007) using Poloplus 2.0 In this equation, b0, bi, bii, and bij are the intercept term and
software (LeOra Software). Other data were compared by measures of the effect of variable Xi, Xi2, and XiXj, respec-
one-way ANOVA followed by the Tukey test to determine tively. Also, Y is the dependent response variable (i.e., mini-
which one has differed, significantly. The statistical program mum hydrodynamic particle diameter), and Xi, Xj, and XiXj
SPSS 22 was used for all bioassay analyses. represent the independent variables and first-order interaction
between Xi and Xj (i < j), respectively. The consistency of the
model was investigated using analysis of variance (ANOVA),
Experimental design sequential F-test, and lack-of-fit test at 10% confidence level
(P value < 0.1).
Multiple linear regression was used to estimate the model
coefficient of the selected factors, which is believed to influ-
ence the nanoemulsion hydrodynamic particle size. Each fac-
tor was set at its high level (+1), low level (−1) and medium Results and discussion
level (0). Two factors with three levels were involved in this
design, including EO percentage and surfactant concentration Statistical model fitting
along with the response (i.e., the particle size of the system).
The details of each factor level and their response values have Regarding the regression coefficients presented in
been collected in Tables 1 and 2. It is worth mentioning that Table 3, all of the terms (including linear, quadratic, and

Table 2 Composition of various

experiments in CCD design for Run order EO percentage (%) Surfactant concentration (%) Particle size
minimizing the hydrodynamic
particle size 1 1 3 11.98
2 5 3 25.12
3 1 15 9.85
4 5 15 20.98
5 1 9 10.27
6 5 9 23.71
7 3 3 13.22
8 3 15 11.87
9 3 9 10.77
10 3 9 10.53
11 3 9 10.64
Environ Sci Pollut Res

Table 3 Values of regression

coefficients calculated for the Independent Regression coefficient Regression coefficient (un- Standard T-value P value
synthesis’s factors factor (coded values) coded values) error

Constant 12.95 11.40 0.71 18.15 < 0.000

Linear
EO 12.94 0.05 0.56 22.80 < 0.000
S − 10.61 − 0.06 0.56 − 18.68 < 0.000
Quadratic
EO·EO 9.66 2.42 0.87 11.05 < 0.000
S·S 1.96 0.05 0.87 2.24 0.075
Interactive
EO·S − 10.76 − 0.90 0.69 − 15.46 0.063

interaction terms) are significant. Thus, a second-order The effect of physicochemical factors in ultrasonic
polynomial can be used to express the relevant data. preparation of nanoemulsions
Statistical analysis of regression coefficients represents a
polynomial regression model equation (Eq. (2)) based on The importance level of each factor can be examined with
uncoded coefficients with 99.6% accuracy: respect to the regression coefficient values, collected in
Table 3. As can be seen, EO% has the highest level of
Hydrodynamic particle size ¼ 11:40 þ 0:05EO−0:06S importance in ultrasonic preparation of nanoemulsions,
þ 2:42EO  EO þ 0:05S  S−0:90EO  S while the interaction term is in the second place. Surface
ð2Þ plots can be used to precisely predict the effect of factors
on the response. As can be observed in Fig. 2, at low
EO%, increase of the surfactant content (in the range of
3–15%) does not change the hydrodynamic particle size,
Regarding the low values of P, determined for the
significantly. On the other hand, at the high level of EO%,
regression (P < 0.000), as well as negligible unfitting
increase of the surfactant content (in the range of 3%–
of the model (0.005), the suitability of the model was
15%) leads to decrease in the hydrodynamic particle size
approved. In order to examine the linear distribution
(in the range of 20–60 nm). The contour plots for the
and the adequacy of this model, the hydrodynamic par-
hydrodynamic particle size with respect to EO% and S%
ticle size predicted values (from the Eq. (2)) are plotted
are depicted in Fig. 3. It can be deducted that the prepa-
versus experimental data, as shown in Fig. 1.
ration of nanoemulsions remarkably depends on the uti-
lized contents of surfactant and essential oils. This result
is in consistency with the previously reported data (Liang
et al. 2012; Barzegar et al. 2018; Massoud et al. 2018;
Mishra et al. 2018; Mossa et al. 2018).
These main two factors were optimized to achieve min-
imum hydrodynamic particle size. Optimization was

Fig. 1 The hydrodynamic particle size predicted values versus Fig. 2 Surface plots for hydrodynamic particle size with respect to EO
experimentally obtained values and surfactant percentile
 Environ Sci Pollut Res

Table 4 Chemical composition of M. piperita oil, wild type

a b
Peak No Compounds Area (%) RI exp RI lit

1 α-Pinene 1.05 928 924

2 4(10)-Thujene (Sabinene) 0.72 959 969
3 β-Pinene 1.42 962 974
4 β-Myrcene 0.40 972 988
5 3-Octanol 0.26 977 988
6 α-Terpinene 0.31 1012 1014
7 Limonene 6.75 1021 1024
8 1,8-Cineole 3.75 1024 1026
9 Trans-β-Ocimene 0.27 1028 1032
10 α-Ocimene 0.08 1036 1044
Fig. 3 Contour plots for hydrodynamic particle size with respect to EO 11 γ-Terpinene 0.57 1045 1054
and surfactant percentile 12 4-Thujanol 0.81 1053 1065
13 α-Terpinolene 0.18 1069 1086
carried out using the proposed second-order polynomial 14 Linalool 0.16 1076 1095
model (Eq. (2)). This exercise suggested that the hydro- 15 2-Methylbutyl 2-methylbutyrate 0.35 1102 1100
dynamic particle size would be very low under the fol- 16 3-Octyl acetate 0.33 1104 1120
lowing conditions: 7.5 < %S < 15 and 1 < %EO < 4. This 17 β-Linalool 0.10 1122 1131
can be further confirmed by close observation of Fig. 3, 18 P-Menthanone 12.23 1147 1148
i.e. there is a region where the particle size is lower than 19 P-Menthone 10.58 1150 1158
10 nm. Applying these optimal region seems to be more 20 Menthofuran 4.00 1152 1159
facile and better from an operational viewpoint since it 21 Neomenthol 5.09 1160 1165
allows the use of a range of factors to achieve uniform
22 Menthol 26.20 1162 1167
small in size nanoemulsions.
23 α-Terpineol 0.52 1167 1174
24 Dihydrocarvone 0.77 1173 1191
25 Pulegone 0.16 1237 1233
GC-MS analysis of wild M. piperita oil
26 Carvone 4.03 1242 1239
27 Hexenyl isovalerate 4.62 1243 1241
The composition of M. piperita oil was evaluated by GC-
28 Piperitone 0.50 1254 1249
MS technique. The percentage amount was obtained by
29 P-Menth-3-en-2-one 0.31 1261 1255
comparing retention time and standard addition using the
30 Carvone oxide 4.17 1268 1259
peak normalization method. Table 4 shows the constitu-
31 Neo-Menthyl acetate 0.21 1273 1271
ents of M. piperita oil, a wild type that contains 43 com-
32 Dihydro carveol acetate 0.08 1328 1306
ponents (99.58%). As can be seen in Table 4, M. piperita
33 β-Bourbonene 0.55 1380 1387
oil is mainly composed of menthol (26.20%) as a hydro-
34 β-Elemene 0.19 1384 1389
phobic and volatile compound followed by p-menthanone
35 Trans-Caryophyllene 3.08 1420 1417
(12.23%), p-menthone (10.58%), limonene (6.75%),
neomenthol (5.09%), and their precursors, respectively. 36 β-Arnesene 0.41 1437 1440
Among them, menthol is the most important component 37 α-Humulene 0.17 1459 1452
responsible for most of the pharmacological effects of the 38 Germacrene D 2.47 1474 1480
plant (Galeotti et al. 2002; Iscan et al. 2002; Shah and 39 α-Muurolene 0.51 1504 1500
Mello 2004). According to the previous studies (Schmidt 40 δ-Cadinene 0.11 1513 1513
et al. 2009; Buleandra et al. 2016; Beigi et al. 2018; 41 Caryophyllene oxide 0.19 1586 1582
Chauhan et al. 2018), menthol has also been identified 42 Viridiflorol 0.83 1590 1592
as the major component of M. piperita essential oil in 43 α-Cadinol 0.09 1637 1638
different percentages. The difference among the results Total identified compounds 99.58
reported in the literature about composition and quantity a
Experimentally determined retention index (RI) on HP-5 MS column
of peppermint oil could be attributed to the geographic b
Literature retention index (Adams 2007)
origin of the plant, seasonal variation, genetics, age, and
harvesting time as well as the extraction method (Beigi
et al. 2018).
Environ Sci Pollut Res

Interestingly, menthol acts as a strong antibacterial and an- widely used emulsifying technique for nanoemulsions
tifungal agent (Iscan et al. 2002; Abbaszadeh et al. 2014). The prepartion.
terpenes or terpenoids prove to be the largest class of second- For further characterization of nanoemulsions, their physi-
ary metabolites, and most of them are insoluble in water. cochemical properties such as pH, viscosity, conductivity, and
Some terpenes improve plant growth; therefore, they can be zeta potential (Table 5) were measured. As was expected, the
considered as primary metabolites rather than secondary me- viscosity of nanoemulsion formulations generally was very
tabolites. However, most of these terpenes are secondary me- low, which is in consistency with the characteristics of
tabolites, which can be necessitated in plant defenses (Wink nanoemulsion formulations (Ali et al. 2014; Kotta et al.
1988; Unsicker et al. 2009). The insecticidal activity of essen- 2015). Actually, by increase of oil concentration, water mole-
tial oils depends on the type and quantity of the monoterpenes cules are trapped in cross-linked chains of surfactants resulting
in essential oil. Therefore, the composition evaluation by GC- in increased viscosity of the nanoemulsions. The oil viscosity
MS helps to exactly identify the active constituents of essen- is a key role player in the disruption of droplets; the lower the
tial oil, being responsible for insecticidal activity (Chauhan viscosity, the shorter time needed to break a drop (Tadros
et al. 2018). 2005).
Although different surfactant concentrations were tested,
Optimizing ultrasonic preparation of nanoemulsions the surfactant concentration was fixed on 1:3 (v/v) ratios of
M. piperita oil: Tween 80 for all specimens. Since non-ionic
As described earlier, nanoemulsion formulations were pre- surfactant was used in this system, pH did not have any mean-
pared by stirring the oil in non-ionic surfactant Tween 80 ingful effect on nanoemulsion formulations confirming that
and water. Tween 80 was used owing to its uncharged inher- the formulation was not affected by the ionic strength. The
ence and having hydrophilic head groups. It has a high hydro- conductivity of nanoemulsions was increased by increase of
philic and lipophilic balance value of 15, which sterically oil phase percentage, which was mainly due to higher electro-
stabilizes nanoemulsion droplets. Besides, non-ionic surfac- static charges and absorption of electric charges by the
tants are less toxic than ionic ones (Tadros 2005; Aserin surrounded ions. This feature demonstrated fewer ions in
2007). In order to survey the optical clarity of nanoemulsions, low concentrated samples resulted in less conductivity
the concentration of M. piperita oil was changed in the range (Table 5). High steady state shows that water is the continuous
of (1–5) %v/v according to the optimal range predicted by phase. As demonstrated in Table 5, the zeta potential of dif-
DOE for the formulated emulsions. The optical clarity of ferent nanoemulsion formulations varied in the range of −
nanoemulsions was decreased with increase of oil concentra- 39.86 and − 9.53 for 1 to 5% samples, respectively. The zeta
tions (Fig. SM1, supplementary material). potential strongly depends on the surface properties of a par-
Figure SM2 (supplementary material) indicates the appear- ticulate material as well as on the pH and ionic concentration
ance of 4% (v/v) nanoemulsion formulation before and after of the dispersion liquid. The higher concentration of
sonication. Before ultrasonic emulsification, the opaque emul- nanoemulsion means the higher relevant zeta potential, which
sion consisted of micro-metered size droplets with turbid and in turn results in less stability of the nanoemulsion system.
milky white appearance. After the ultrasonic process, the This state is in appropriate agreement with the obtained results
droplet size was decreased to the nano-meter range and the and confirms the stability of lower concentrated or more di-
emulsion was more transparent and clear than the non- luted samples.
sonicated samples. It is important to supply the high power The droplet diameter of the nanoemulsion formulations of
energy in a homogeneous manner and in the shortest time to M. piperita oil was evaluated by means of dynamic light scat-
produce particles as small as possible (Tadros 2005; Mason tering method. Table 6 shows the droplet diameter of differ-
et al. 2006; Jafari et al. 2006, 2007; Lee and Norton 2013). On ently formulated nanoemulsions varying from 1 to 5% (v/v)
the other hand, ultrasonication generates intensive and disrup- ratio of oil. As shown, the droplet size of different formula-
tive forces that minimize nanoemulsion droplets (Tadros tions slightly increases by the increase of oil concentration.
2005; Goh et al. 2015). Therefore, ultrasonication is the most The intensity of scattered light will be changed, and the

Table 5 Physicochemical
properties of different Nanoemulsion (%v/v) pH Conductivity (μS/cm) Viscosity (c.p) Zeta potential (mV)
nanoemulsions
1% 5.01 ± 0.08 48.85 ± 0.31 14.23 ± 0.31 − 39.86 ± 0.87
2% 5.40 ± 0.09 57.20 ± 0.97 14.41 ± 0.42 − 33.87 ± 1.09
3% 5.43 ± 0.04 72.29 ± 0.72 15.72 ± 0.71 − 26.25 ± 1.98
4% 5.35 ± 0.06 78.76 ± 0.78 15.91 ± 1.46 − 11.74 ± 1.82
5% 5.33 ± 0.11 120.7 ± 0.68 16.46 ± 1.69 − 9.53 ± 1.57
 Environ Sci Pollut Res

Table 6 Droplet size distribution of different nanoemulsions at stabilization and prevent coagulation of emulsions. This could
sonication times of 15 min
be the result of strong Coulomb interactions between charged
Nanoemulsion (%v/v) Droplet size (nm) particles, which neutralize Van der Waals bonds (Zainol et al.
2012). An emulsion remains stable when the droplets diameter
1% 11.82 remain sufficiently small and also well separated. In this con-
2% 12.01 dition, Brownian motion of droplets occur and they could be
3% 12.21 evenly dispersed throughout the continuous phase (Tadros
4% 12.54 2005). In this case, the emulsion droplets are sufficiently
5% 13.11 small; therefore, the Brownian motion overcomes gravity
forces and prevents coagulation of the targeted emulsion.
The stability of nanoemulsions was evaluated over a year
droplets undergo Brownian motion in emulsion formulation time period. Figure 5 shows the nanoemulsion formulations
with changing in size (Mohankumar et al. 2014). after 12 months. As can be seen in Fig. 5, all the formulated
Morphology and size of nanoemulsion were monitored by nanoemulsions of M. piperita oil exhibit long-term stability
transmission electron microscopy (TEM). Figure 4 shows the with no phase separation, creaming, flocculation, coalescence,
TEM image of M. piperita oil nanoemulsion (5% (v/v) ratio of and sedimentation. This result is in accordance with the results
oil)). The droplets had sphere-like morphology and diameter of the zeta potential measurements. As previously described,
of about 12–13 nm. The droplet size data obtained by TEM the zeta potential of the optimized formulation (1%v/v EO and
analysis was compatible with the data provided by particle 3% Tween 80) was about − 39.86 mV that caused strong inter-
size analyzer (DLS). The size of M. piperita oil nanoemulsion particle repulsion, confirming the good stability of formulated
in the present work (12–13 nm) is smaller than the reported nanoemulsion (Table 5).
one by Barzegar et al. (2018) (50 nm) and Massoud et al.
(2018) (44 nm).
Bioassay

Stability study Utilizing nanoemulsion formulation significantly enhanced

insecticidal activity of Rosmarinus officinalis L. (family:
The optimized formulation (1%v/v EO and 3% Tween 80) of Lamiaceae) against Aedes aegypti L. (Dip.: Culicidae) in com-
nanoemulsion was tested over a period of 12 months. Then, parison with control (Duarte et al. 2015). In addition, compar-
the relevant nanoemulsions were evaluated in terms of particle ison of mortality after topical treatment of Dysdercus
size, optical clarity, and zeta potential. After 12 months, the peruvianus (Hemiptera: Pyrrhocoridae) with nanoemulsion
collected data represented almost the same values of particle taken from fruits of Manilkara subsericea Mart. (family:
size (12.05 nm) and zeta potential (− 35.42 ± 0.19 mV) for the Sapotaceae) and with blank nanoemulsion showed the consid-
optimized formulation of nanoemulsion (1%v/v EO and 3% erably higher insecticidal efficiency of synthesized EO
Tween 80). It should be noted that the zeta potential is a spe- nanoemulsion (Fernandes et al. 2014). Concerning this point,
cial stability parameter in colloidal systems (such as emul- the current study allowed achievement of a nanoemulsion
sions) due to electrostatic repulsion (Benita and Levy 1993; with potential activity against A. gossypii, as one of the major
Tamilvanan and Benita 2004; Tamilvanan 2009; Zainol et al. pests of cucurbit plants.
2012). It is expected that by increase of the oil percentage, the Insecticidal efficiency of nano-formulations can be varied
zeta potential would be increased. From Fig. 5, it can be con- with several parameters including insect species, exposure
cluded that by decreasing the percentage of oil, the optical
clarity of nanoemulsions have been increased. The effects of
surfactant along with the low addition rate can result in steric

Fig. 4 TEM image of M. piperita oil nanoemulsion (5% (v/v))

Fig. 5 Stability of different nanoemulsion formulations after a year
Environ Sci Pollut Res

Fig. 6 Mean mortality percent of

A. gossypii adults after contact
with different concentrations of
M. piperita nanoemulsion in 24 h.
Different letters over columns
indicate significant difference
between various concentrations
and formulated percents using
Tukey test (P ≤ 0.05)

time, the concentration of the formulations, and the method of 0.195, df = 4). Means of A. gossypii mortality exposed to dif-
application (Anjali et al. 2012; Duarte et al. 2015; ferent concentrations and synthesized formulation of
Balasubramani et al. 2017). On the basis of the obtained re- M. piperita nanoemulsion are shown in Fig. 6.
sults in the current research, a significant increase in aphid Bioassay results showed that 4% synthesized nanoemulsion is
mortality by increase of nanoemulsion concentrations (F = the most toxic one between various treatments, while no signif-
295.338, P = 0.00, df = 4) revealed the dose-dependent mor- icant difference was observed between the treatments. Results of
tality in aphids. It means that the increase in active ingredient the current research are compatible with Massoud et al. (2018),
cause more effectiveness, as proved in previous reports who studied the effect of peppermint EO nanoemulsion on
(Anjali et al. 2012; Duarte et al. 2015; Balasubramani et al. Sitophilus oryzae L. (Col.: Curculionidae) as a stored product
2017). pest. They showed that among the nanoemulsion formulations,
Secondary metabolites are responsible for major insecticid- the highest and fastest toxic effect was observed in the case of
al activities of EOs (Govindarajan et al. 2018a, b). Some M. piperita (4%) nanoemulsion against S. oryzae via thin film
chemical components like menthol, menthone, methyl acetate, residue method and treatment with wheat grains method. They
mentho-furan, and 1,8-cineole (found as major components of suggested that smaller particle size and increase in biological
peppermint oil) are responsible for the insecticidal activity) activity provide more opportunity of the formulation to come
Ikbal and Pavela 2019). Previous reports have shown that in contact with the pest (Massoud et al. 2018) by increased sur-
the insecticidal activity of M. piperita EO originate from the face area of emulsion droplets. As indicated in Table 7, the mor-
presence of major constituents such as menthol, menthone, tality of A. gossypii in 5% treatment was more dose-dependent
and menthofuran) Ikbal and Pavela 2019). The obtained re- than other treatments. Moreover, according to the LC50 ratio
sults of the present research also showed no significant differ- ranges, there was no significant difference between lethal con-
ence between nanoemulsion various percents (F = 1.56, P = centration 50 of treatments. On the other hand, the lower

Table 7 Probit analysis of results of mortality caused by different synthesized percent of Mentha piperita nanoemulsion

Percent LC50 (ppm) LC90 (ppm) Slope ± S.E. χ2(df) P LC50 ratio

1% 3880.058 4827.785 13.5 ± 1.765 2.5 (13) 0.99 –

(3772.665–3982.866)a (4602.091- 5212.491)
2% 3941.778 4927.587 13.22 ± 1.749 5.1 (13) 0.97 0.984
(3834.676–4050.207) (4681.647–5353.913) (0.948–1.022)
3% 3858.11 4841.269 13 ± 1.749 3.6 (13) 0.82 1.006
(3745.165–3963.509) (4606.31–5249.339) (0.968–1.045)
4% 3852.709 4772.136 13.78 ± 1.76 8.6 (13) 0.8 1.007
(3744.953–3952.525) (4561.781–5123.284) (0.970–1.046)
5% 3864.79 4710.335 14.915 ± 1.82 4.0 (13) 0.98 1.004
(3766.45–3958.507) (4519.028–5020.423) (0.968–1.041)
a
Confidence limit of 95% (using Poloplus 2.0)
 Environ Sci Pollut Res

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