Ultrasound-assisted nanoemulsion of Trachyspermum ammi essential oil and its constituent thymol on toxicity and biochemical aspect of Aedes aegypti

Abstract

Aedes aegypti is the main vector of yellow fever, chikungunya, Zika, and dengue worldwide and is managed by using chemical insecticides. Though effective, their indiscriminate use brings in associated problems on safety to non-target and the environment. This supports the use of plant-based essential oil (EO) formulations as they are safe to use with limited effect on non-target organisms. Quick volatility and degradation of EO are a hurdle in its use; the present study attempts to develop nanoemulsions (NE) of Trachyspermum ammi EO and its constituent thymol using Tween 80 as surfactant by ultrasonication method. The NE of EO had droplet size ranging from 65 ± 0.7 to 83 ± 0.09 nm and a poly dispersity index (PDI) value of 0.18 ± 0.003 to 0.20 ± 0.07 from 1 to 60 days of storage. The NE of thymol showed a droplet size ranging from 167 ± 1 to 230 ± 1 nm and PDI value of 0.30 ± 0.03 to 0.40 ± 0.008 from 1 to 60 days of storage. The droplet shape of both NEs appeared spherical under a transmission electron microscope (TEM). The larvicidal effect of NEs of EO and thymol was better than BEs (Bulk emulsion) of EO and thymol against Ae. aegypti. Among the NEs, thymol (LC 50 34.89 ppm) had better larvicidal action than EO (LC 50 46.73 ppm). Exposure to NEs of EO and thymol causes the shrinkage of the larval cuticle and inhibited the acetylcholinesterase (AChE) activity in Ae. aegypti. Our findings show the enhanced effect of NEs over BEs which facilitate its use as an alternative control measure for Ae. aegypti.

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Environmental Science and Pollution Research
https://doi.org/10.1007/s11356-022-20870-2
RESEARCH ARTICLE
Ultrasound‑assisted nanoemulsion ofTrachyspermum ammi essential
oil andits constituent thymol ontoxicity andbiochemical aspect
ofAedes aegypti
KesavanSubaharan1 · PeriyasamySenthamaraiSelvan1· ThagareManjunathaSubramanya2·
RajendranSenthoorraja1· SowmyaManjunath1· TaniaDas1· VppalayamShanmugamPragadheesh3·
NandagopalBakthavatsalam1· MuthuGounderMohan1· SengottayanSenthil‑Nathan4· SreehariUragayala5·
PaulrajPhilipSamuel6· RenuGovindarajan6· MuthuswamyEswaramoorthy2
Received: 18 August 2021 / Accepted: 12 May 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Aedes aegypti is the main vector of yellow fever, chikungunya, Zika, and dengue worldwide and is managed by using
chemical insecticides. Though effective, their indiscriminate use brings in associated problems on safety to non-target and
the environment. This supports the use of plant-based essential oil (EO) formulations as they are safe to use with limited
effect on non-target organisms. Quick volatility and degradation of EO are a hurdle in its use; the present study attempts
to develop nanoemulsions (NE) of Trachyspermum ammi EO and its constituent thymol using Tween 80 as surfactant by
ultrasonication method. The NE of EO had droplet size ranging from 65 ± 0.7 to 83 ± 0.09nm and a poly dispersity index
(PDI) value of 0.18 ± 0.003 to 0.20 ± 0.07 from 1 to 60days of storage. The NE of thymol showed a droplet size ranging from
167 ± 1 to 230 ± 1nm and PDI value of 0.30 ± 0.03 to 0.40 ± 0.008 from 1 to 60days of storage. The droplet shape of both
NEs appeared spherical under a transmission electron microscope (TEM). The larvicidal effect of NEs of EO and thymol
was better than BEs (Bulk emulsion) of EO and thymol against Ae. aegypti. Among the NEs, thymol (LC50 34.89ppm) had
better larvicidal action than EO (LC50 46.73ppm). Exposure to NEs of EO and thymol causes the shrinkage of the larval
cuticle and inhibited the acetylcholinesterase (AChE) activity in Ae. aegypti. Our findings show the enhanced effect of NEs
over BEs which facilitate its use as an alternative control measure for Ae. aegypti.
Keywords Aedes aegypti· Nanoemulsion· Droplet size· Thymol· Trachyspermum ammi· Biochemical effect
Introduction
Mosquitoes (Diptera: Culicidae) have a significant role
in transmitting vector-borne diseases that cause mortality
and morbidity in humans (Benelli etal. 2018; Pavela etal.
2019). Among them, the yellow fever mosquito, Aedes
aegypti (L.) (Diptera Culicidae) is of concern as it transmits
Zika, dengue chikungunya, and yellow fever (Weaver etal.
2016; Ferreira-de-Brito etal. 2016; Benelli etal. 2019). The
annual economic loss caused due to dengue is estimated to
be US$ 9 billion (Shepard etal. 2016).
Responsible Editor: Philippe Garrigues
* Kesavan Subaharan
Kesavan.Subaharan@icar.gov.in; subaharan_70@yahoo.com
1 Division ofGermplasm Conservation andUtilization,
ICAR-National Bureau ofAgricultural Insect Resources,
Bengaluru, India560024
2 CPMU, Jawaharlal Nehru Centre forAdvanced Scientific
Research, Bangalore, India560065
3 CSIR-Central Institute ofMedicinal andAromatic Plants,
Regional Centre, Bengaluru, India560065
4 Division ofBiopesticides andEnvironmental Toxicology,
Sri Paramakalyani Centre forExcellence inEnvironmental
Sciences, Manonmaniam Sundaranar University,
Alwarkurichi, 627412Tirunelveli, TamilNadu, India
5 ICMR, National Institute forMalaria Research FU,
Bangalore, India562110
6 ICMR – Vector Control Research Centre, Field Station,
Madurai, India625002

Environmental Science and Pollution Research
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The Ae. aegypti adult females are anthropophilic and
use man-made receptacles in households for breeding
(Benelli and Mehlhorn 2016). Hence, the primary level
of management depends on source reduction by involving
community participation (Alvarado-Castro etal. 2017).
When the population is in excess, the synthetic insecti-
cides belonging to pyrethroids, organophosphates, carba-
mates and insect growth regulators are widely used for
Ae. aegypti management (Chen etal. 2009; Marcombe
etal. 2012). However, the continued use of synthetic
insecticides led to the development of insecticide resist-
ance among mosquito species (Thanigaivel etal. 2017)
and negative effects on humans and the environment (Roiz
etal. 2018). In recent years, the resurgence of dengue, chi-
kungunya, the rising incidence of Zika virus, and yellow
fever exposes the limitations in Ae. aegypti management
(Wilder-Smith etal. 2017). This will increase the necessity
to search and develop low-risk and environmentally safe
methods for Ae. aegypti management.
Natural products like essential oil (EO) obtained
from plant parts are an alternative source for Ae. aegypti
management (Silva etal. 2017). Essential oils (EO) are
a complex mixture of compounds that are hydrophobic,
volatile, and of low molecular weight (Rai etal. 2017).
EOs have a wide spectrum of biological action on pests
of agriculture and public health (Kavallieratos etal. 2020)
coupled with safety to the environment and non-target
organisms (Benelli 2018; Chellappandian etal. 2018). This
attribute qualifies them as “low-risk pesticides” (Kalita
etal. 2013) to replace or minimize the use of chemical
insecticides (Isman and Grieneisen 2014).
EOs from Tagetes spp. (Dharmagadda etal. 2005),
Trachyspermum ammi (Pandey et al. 2009) Ocimum
basilicum (Govindarajan et al. 2013), Sphaeranthus
amaranthoides (Thanigaivel et al. 2019) Kaempferia
galanga (AlSalhi etal. 2020) Thymus vulgaris (Pavela
etal. 2009), Eucalyptus sp. (Lucia etal. 2007), clove bud
oil (Kalaiselvi etal. 2019), and Zanthoxylum monophylum
(Pavela and Govindarajan 2016) have a significant effect on
mosquitoes as a larvicide, adulticide, and repellents.
The EO derived from Apiaceae plants has drawn inter-
est among research groups, as they have antimicrobial and
insecticidal properties (Evergetis etal. 2013; Singh etal.
2014). Notably, the EO derived from schizocarps of T. ammi
possesses larvicidal activity against Ae. aegypti (Seo etal.
2012), oviposition deterrence, fumigant toxicity, and repel-
lence activity against Anopheles stephensi (Pandey etal.
2009), and Musca domestica (Chantawee and Soonwera
2018). Thymol, a major constituent of T. ammi EO (Pandi-
yan etal. 2019) has larvicidal action on Ae. aegypti larvae
(Govindarajan etal. 2013; Silva etal. 2017; Junkum etal.
2021) and is 1.65 times more toxic than T. ammi EO when
exposed to An. stephensi larvae (Pandey etal. 2009). This
confirms the potential bioaction of T. ammi EO and thymol
formulations against the mosquitoes.
Though the EO formulations are effective in insect man-
agement, they have limitations in use due to their volatility,
water-solubility, physical destabilization caused by gravi-
tational separation, flocculation, and coalescence (Pavela
and Benelli 2016). The existing gap can be addressed by
developing oil-in-water (o/w) emulsion which will facilitate
improving the dispersion, availability, and scale down the
volatile loss of compounds (Pavoni etal. 2020). Utilizing the
nanotechnological approaches, the EO formulations could be
developed with better physical stability, smaller size, bio-
availability, and lower risk to non-targets (Nenaah 2014).
The nanoformulations like microemulsions (MEs) and
nanoemulsions (NEs) are a recent development in pest
management (Pavela etal. 2019; Jesser etal. 2020). The
MEs are thermodynamically stable, but sensitive to changes
in temperature and required a high amount of surfactants
(Forgiarini etal. 2000). Whereas the NEs are colloidal
delivery matrix prepared by low-energy and high-energy
methods (Fryd and Mason, 2012). NEs have robust stability,
high surface area, and tunable rheology (Gupta etal. 2016).
The high-energy method of NE preparation employs high-
pressure homogenization and ultrasonication (Homs etal.
2018). The ultrasonication provides energy to produce
turbulence that splits the oil and water phase to produce
small oil droplets (Delmas etal. 2011). It is a preferred
method to prepare NE as it is economically viable, energy-
efficient, and amenable to control the formulation variables
(Periasamy etal. 2016).
The NE of Rosmarinus officinalis L. and O. basilicum L.,
Vitex negundo EO and thymol caused higher larval mortality
to Ae. aegypti over the free form of EO (Ghosh etal. 2013;
Duarte etal. 2015; Balasubramani etal. 2017; Lucia etal.
2020). There is scanty work on assessing the efficacy of T.
ammi EO NE to manage Ae. aegypti, except for an attempt
to use β-cyclodextrin formulation of T. ammi to improve its
larvicidal effect on Ae. aegypti (Pandiyan etal. 2019). Previ-
ous studies on emulsion are more focusing on the efficacy
of EO with limited information on the effects of compounds
present in the EO. To develop a novel EO based formulation
for mosquito management, herein we present the methods
to develop NE of T. ammi EO and its constituent thymol by
ultrasonication method and assess their stability, larvicidal,
and biochemical effect on Ae. aegypti.
Materials andmethods
Chemicals
Polysorbate 80 (Tween 80) was purchased from Alpha Aesar.
Ethylenediaminetetraacetic acid (EDTA), phenyl thiourea,

Environmental Science and Pollution Research
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Coomassie brilliant blue (CBB), and bovine serum albumin
(BSA) were procured from Himedia Laboratories, Banga-
lore. Acetylcholineesterase kit and acetone were purchased
from Sigma Aldrich (St. Louis, MO, US).
Extraction andcharacterization ofessential oil
The seeds of T. ammi, sourced from Unjha, 23°48′07.6″N
72°23′03.9″E in Gujarat, India were shade dried for
(25 ± 2°C with RH 65 ± 5%) for 3days and cleaned for
physical and biological debris. The coarse ground seeds of
T. ammi (300g) was loaded into a 1000-ml round bottom
flask. To this, 500ml of distilled water was added, and the
temperature of the flask with contents was raised to 100°C
by placing over a heating mantle. The EO was extracted in
the Clevenger apparatus by hydro-distillation for 5h. The oil
collected in the receiver tube was separated from the aque-
ous layer using a separating funnel. The oil yield was 1.8%
(w/w). The EO was dehydrated by passing over anhydrous
sodium sulfate and stored at 4°C until use.
The extracted T. ammi EO was characterized by using
GC–MS (Agilent GC- 7890A and MS-5975) as previously
suggested by Subaharan etal. (2021). The analytical
conditions were maintained the same as those reported
by Senthoorraja et al. (2021). The components in EO
were identified by retention time and comparing the mass
fragmentation pattern using the National Institute of
Standards and Technology (NIST) library.
Preparation ofT. ammi EO andthymol inoil/water
nanoemulsion
The T. ammi EO and its constituent thymol oil-in-water
(O/W) nanoemulsion was prepared by mixing EO/Thymol
(5%) and the surfactant Tween 80 (5%) (v/v) at 400rpm for
20min under constant stirring at room temperature. Then,
water (90% v/v) was added dropwise into the mixtures of
EO/Tween 80 with continuous stirring for another 20min.
The obtained turbid mixtures of macroemulsions were sub-
jected to ultrasonic homogenization using a probe sonica-
tor (Model-VCX 750, Power—750W, Frequency—0kHz,
USA) for 10min at 50°C. The probe with a stepped microtip
(thickness about 3mm) was placed 25mm above the bot-
tom of the reservoir/container containing the liquid disper-
sion. The NE was stored at room temperature for further
characterization.
Nanoemulsion characterization
Droplet size analysis
The average droplet size distribution of NE was determined
using the dynamic light scattering technique (DLS) by
Zetasizer Nano ZS (Malvern Instruments Ltd., India) instru-
ment equipped with a 633-nm laser. The scattering intensity
was recorded with a detector kept at a backscattering angle
(173°). Measurement of NE was done on 1, 3, 5, 7, 15, 30,
and 60days after preparation. The oil droplet size (nm) was
characterized by distribution curves in intensity (%), average
droplet size, and polydispersity index (PDI). Droplet size
was expressed as mean diameter + SE (n = 3).
Transmission electron microscopy
The droplet shape in NE was observed by transmission elec-
tron microscope (TEM) JEOL—010 transmission electron
microscope operating at a voltage of 200kV. TEM samples
were prepared by mixing 100 μL of the sample with 200 μL
of 2 wt% phosphotungstic acids. A drop of the mixture was
placed on a copper grid and dried in a desiccator for removal
of water and then observed under the TEM.
Acute toxicity onAe. aegypti larvae
Aedes aegypti eggs obtained from the Indian Council of
Medical Research (ICMR)—Vector Control Research
Centre—Field station, Madurai was reared at the Veterinary
Entomology laboratory, ICAR—National Bureau of
Agricultural Insect Resources, Bengaluru, India in the
method suggested by Balasubramani etal. (2017). Briefly,
the eggs were seeded in an enamel trap having tap water
free of chlorine. On hatching, the larvae were fed with a
diet consisting of dog biscuit + yeast (3:1 ratio). Third instar
larvae were used to evaluate the efficacy of T. ammi EO,
thymol NE, and BE following the WHO method (WHO
2005). Based on the range-finding test, the NE and BE
were diluted in distilled water to obtain the concentration
ranging from 5 to 100ppm (relative to T. ammi EO and
thymol). The experiments were carried out with a group of
25 larvae of uniform size per replicate. Four replicates were
maintained. A control with surfactant alone was maintained
as a negative control. Imidacloprid tested between 0.2 and
2ppm was maintained as a positive control. The bioassays
were performed at 25 + 2°C, 60 + 5% RH and 16:8h (L:D).
Larval mortality was observed 24h after exposure. When
the mortality rate in control was above 20%, the mortality
was subjected to correction using Abbott’s formula (Abbott
1925). The lethal concentrations (LC50 and LC90) were
calculated by probit analysis (Finney 1971).
Scanning electron microscopy (SEM) ofAe. aegypti
larvae
The third instar Ae. aegypti larvae exposed to EO, thy-
mol, and control were used for SEM imaging in a method
reported by Subaharan etal. (2021). Briefly, the dead larvae

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exposed to LC50 dose of EO, thymol NE, and control were
separated and washed with double distilled water 5 × times to
detach the debris attached to the body surface. The primary
fixation of the samples was done with glutaraldehyde 2.5%
in distilled water for 1–2h. The samples were then rinsed
in distilled water for 10–0min, and the second fixation was
done for 1–2h in 1–% osmium tetroxide in distilled water.
The samples rinsed with distilled water (10–20min) were
serially dehydrated with 25, 50, 75, 90, and 100% ethanol
for 10min each. Ethanol was used for critical point drying
(CPD) of the samples. Samples were mounted on an alu-
minum stub and spluttered with gold particles by cathodic
spraying. External structures of Ae. aegypti larva was
observed on a scanning electronic microscope (Carl Zeiss
MERLIN VP Compact) for structural changes. Sample prep-
aration for SEM imaging was done at room temperature.
AChE inhibition
The surviving third instar Ae. aegypti larvae treated with
NE and BE of EO at LC50 (46.7 and 57.5ppm) and thymol
at LC50 (34.8 and 43.9ppm) from bioassay were used to
estimate the acetylcholineesterase (AChE). Larvae exposed
to tap water alone were used as a negative control and those
exposed to imidacloprid (LC50 dose 0.96ppm) was used as a
positive control. Ae. aegypti larvae exposed to the treatments
were homogenized in ice-cold 0.1-M phosphate buffer (pH
8.0), and the contents were centrifuged at 4°C for 20min
at 10,000rpm. The supernatant was used to estimate acetyl-
cholineesterase inhibition activity. The protein content was
estimated as suggested by Bradford (1976). Acetylcholinest-
erase (MAK119, Sigma Aldrich, US) inhibition activity was
measured following the modified method of Ellman etal.
(1961). The reaction mixture consisting of 10µl of enzyme
homogenate and 190µl of reagent (consisting of 5,5-dithio-
bis-2-nitrobenzoic acid, (DTNB) and acetyl thiocholine was
dissolved in Tris–HCl buffer (100Mm, pH 8.0 assay buffer).
Control had 10µl of distilled water and 190µl of reagent,
reaction mixture. The reaction mixture was incubated for
10min at room temperature. The rate of change in absorb-
ance was measured at 415 for 10min in a microplate reader
(iMark, BioRad). The percent inhibition was calculated as
suggested previously (Kim etal. 2013).
Statistical analysis
In toxicity assay when the control mortality was above
20%, it was subjected to correction using Abbott’s formu-
lae (Abbott 1925). The data on Ae. aegypti larval mortality
were subjected to probit analysis (Finney 1971) to determine
the median lethal dose LC50, and their 95% CI values and
chi-square test were calculated using the SPSS software ver-
sion 14.0. The variations in particle droplet size, PDI, and
variation in AChE enzyme activity were compared using
one-way analysis of variance (ANOVA) followed by Tukey’s
post hoc test (P < 0.05) using SPSS.
Results anddiscussion
Characterization ofEO
The constituents of T. ammi EO characterized by GC–MS
are shown in Table1. Twenty compounds were identified in
T. ammi EO. The major constituents were thymol (54.22%),
p-cymene (15.04%), and γ-terpinene (10.46%). The minor
compounds present between 0.5 and 1% include carvac-
rol (1.86%), p-cymenene (1.1%), carane, 4,5-epoxy-, trans
(0.85%), terpinen-4-ol (0.79%) 8,9-dehydrothymol (0.79%),
α-terpineol (0.76), p-cymene-2,5-diol (0.74), p-cymen-8-ol
(0.62), α-pinene (0.58), and camphene (0.51%). The con-
stituents in the EO in our studies agree with the previous
report (Benelli etal. 2017).
Characterization ofnanoemulsion
EOs have a potential effect on insects, but the hydrophobic-
ity and low solubility in water are a limitation for further use
as a larvicide in vector management. An effective mosquito
Table 1 Chemical composition of the T. ammi essential oil
Compounds Reported RI Percentage
composition
α-Pinene 933 0.58
Camphene 951 0.51
β-Pinene 980 0.24
β-Myrcene 993 0.3
α-Phellandrene 1005 0.08
δ-3-Carene 1011 0.03
α-Terpinene 1017 0.16
p-Cymene 1034 15.04
γ-Terpinene 1060 10.89
Terpinolene 1088 0.24
p-Cymenene 1090 1.1
Trans-2-caren-4-ol 1178 0.36
Terpinen-4-ol 1177 0.79
Carane, 4,5-epoxy-, trans 1179 0.85
p-Cymen-8-ol 1183 0.62
α-Terpineol 1190 0.76
8,9-Dehydrothymol 1221 0.79
Thymol 1291 55.84
Carvacrol 1299 1.86
p-Cymene-2,5-diol 1561 0.74
Total 91.78

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larvicide formulation needs to have a good dispersion in
water and stability. The O/W nanoemulsion of T. ammi EO
and its constituent thymol is an improved formulation to
overcome the limitations of dispersibility with enhanced
efficacy over free EO formulation.
Droplet size andpolydispersity index
The emulsion stability of EO and thymol NE was estimated
by recording the droplet size and the polydispersity index
(PDI) value on 1, 3, 5, 7, 15, 30, and 60days after stor-
age at 26°C. The droplet size and the PDI of NEs over a
period of time are shown in Table2. There was a signifi-
cant difference in the droplet size and PDI values between
NEs of EO and thymol, with the latter showing larger drop-
let size (F13,28 = 8411.39 P < 0.001) and higher PDI value
(F13,28 = 58.10 P < 0.001). Between the formulations, EO NE
showed smaller droplet size (65 ± 0.7 to 83 ± 0.09nm from 1
and 60days of storage) and lower PDI values (0.18 ± 0.003
to 0.20 ± 0.07 from 1 to 60days of storage). The slow desta-
bilization in EO NE until the 60th day of storage may be due
to the low PDI values (< 0.22) that reflects on the narrow
distribution of the particle size (Table1) which in turn attrib-
utes to the robust physical stability of NE due to reduced
Ostwald’s ripening of the droplets (Jesser etal. 2020). The
droplet size and stability of the EO NE maintained below
90nm on 60days of storage may be due to the steric effect
(Burt 2004).
Correlograms obtained during the droplet size meas-
urement of NEs of EO and thymol present a normal single
exponential decay of the intensities of scattered light over
time owing to the Brownian motion of the droplets (Figs.1
and 2), i.e., the correlogram curves are much steeper and
exhibit flat baseline at the end of the decay, suggesting that
the NEs remained monodispersed without any aggregation
throughout 60days of storage. These agree with the PDI
values of NEs of EO and thymol reported in Table2.
The mean droplet size of thymol NE during stor-
age ranged from 167 ± 1 on day 1 to 185 ± 0.6, 201 ± 1,
228 ± 0.03, and 230 ± 1nm on 5, 15, 30, and 60days after
storage, respectively. The PDI values for thymol NE ranged
from 0.30 ± 0.03 to 0.40 ± 0.008. The increase in droplet size
of thymol NE (230 ± 1nm after 60days of storage) may
be attributed to the droplet aggregation that resulted from
emulsion instability. Thymol has weak dispersibility as it has
a higher PDI value (Kumari etal. 2018), but when mixed
with eugenol, its dispersion in an aqueous medium increased
resulting in monodisperse droplets (Lucia etal. 2020).
Though the droplet size and PDI value of thymol NE
were greater than EO NE, the rate of increase in droplet
size during 60days of storage was 1.37 and 1.27-fold,
respectively. NE of Rosemarinus officinalis oil that had
a larvicidal effect on Ae. aegypti had a 3.6-fold rise in
droplet size 30days after storage (Duarte etal. 2015).
Smaller PDI values indicate that nanodroplets are more
homogeneous and the PDI of less than 0.6 is an aggrega-
ble homogeneity (Suyal etal. 2018). In our studies, the
PDI values of thymol NE was 0.40 ± 0.008 and droplet
size was 230 ± 1.0nm on 60days of storage. The narrow
range of increase in droplet size did not alter the concept
of it being termed a nanoemulsion, as previous studies
have reported that average droplet size with a diameter
lower than 300nm is to be considered as a nanoemul-
sion (Hashem etal. 2018). The EO and thymol NE did
not show any phase separation after 60days of storage.
TEM
Droplet shape of NEs of EO and thymol, when viewed
by transmission electron microscopy appeared well dis-
persed, spherical, and retained the overall size range
within < 250 nm (Fig.3). The transmission electron
microscopy (TEM) images of Tarragon, clove, and black
seed oil nanoemulsion revealed the spherical structure
of the droplets (Mossa etal. 2021). The spherical struc-
ture is due to the reduction of the interfacial area arising
due to a small radius and increased interfacial tension
(Pavoni etal. 2020).
Acute toxicity of nanoemulsion and bulk emulsion of T.
ammi EO and thymol to Ae. aegypti larvae.
The toxicity of NE and bulk emulsion BE of T. ammi
EO and thymol (referred to as EO BE and thymol BE)
on Ae. aegypti larvae is shown in Table3. Irrespective
of emulsion type, both EO and thymol showed larvicidal
Table 2 Nanoemulsion droplet size and polydispersity index
Means within a column followed by the same letter are not signifi-
cantly different by Tukey’s test (P < 0.05)
Nanoemulsion type Days
after
storage
Size (nm) + SE PDI + SE
T. ammi EO + Tween 80 165 ± 0.7a0.18 ± 0.003ab
366 ± 0.73ab 0.17 ± 0.005a
567 ± 0.89ab 0.17 ± 0.012a
769 ± 0.29bc 0.17 ± 0.002a
15 72 ± 0.46cd 0.17 ± 0.04a
30 74 ± 0.39d0.22 ± 0.001ab
60 83 ± 0.09e0.20 ± 0.07ab
Thymol + Tween 80 1167 ± 1.0f0.30 ± 0.03c
3177 ± 1.0g 0.30 ± 0.05c
5185 ± 0.60h 0.36 ± 0.11d
7196 ± 0.70i0.36 ± 0.12d
15 201 ± 1.0 j0.24 ± 0.02bc
30 228 ± 0.03k 0.40 ± 0.02d
60 230 ± 1.0k 0.40 ± 0.008d

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1 3
activity against Ae. aegypti. The toxicity of T. ammi EO
to Ae. aegypti (Silva etal. 2017; Seo etal. 2012) and
An. stephensi larvae (Pandey etal. 2009) was reported
earlier. Among the formulations tested, NE caused higher
toxicity than BE (Table3). Enhanced larvicidal activ-
ity of eucalyptus oil nanoemulsion than its bulk emul-
sion to Culex quinquefasciatus was reported by Sugu-
mar etal. (2014). Between the NEs tested, thymol NE
(LC50 34.89ppm) was more toxic than EO NE (LC50
46.73ppm). A similar trend of thymol (LC50 52.82ppm)
being more toxic than EO (59.67ppm) was observed in
BEs too. The order of toxicity was thymol NE > T. ammi
EO NE > thymol BE > T. ammi BE. Our observations
are in line with findings by Pandey etal. (2009) who
reported thymol to be 1.65 times toxic to An. stephensi
larvae as compared to T. ammi whole extract.
The previous studies revealed that the nanoemulsion
of thymol showed higher toxicity to Ae. aegypti lar-
vae (LC50 11.1ppm), but the addition of eugenol to it
declined the toxicity (Lucia etal. 2020). EOs constitute
20–60 compounds belonging to phenols, alkaloids, and
terpenes, among them the bioactivity is mainly attributed
to few compounds that are in higher concentration (Pavela
2015); nevertheless, the compounds present in lower con-
centrations also add to its efficacy (Benelli etal. 2017).
Artificial mixtures of T. ammi EO prepared and tested
without the major constituent, viz., thymol, p-cymene and
γ-terpinene caused a decrease in toxicity to Ae aegypti as
compared to their presence in the mixture that enhanced
the toxicity (Seo etal. 2012). Thymol present in EO is a
major contributor in causing toxicity to Ae. aegypti (Park
etal. 2011). In our study, thymol is a major constitu-
ent in T. ammi EO along with p-cymene (15.04%) and
γ-terpinene (10.46%); this explains the enhanced larvi-
cidal activity of NEs of thymol and EO.
The droplet size and PDI are important physical
characteristics to determine the interaction of nanofor-
mulations and insects (Benelli 2018). In our studies,
the droplet size of EO and thymol ranged from 90 to
230nm. Previous studies confirm that droplet size in
nanoemulsion with a size ranging from 100 to 400nm
impute benefits likes dispersion, thermal stability, per-
meability (Bordes etal. 2009). Though there was a dif-
ference in NE droplet size of EO (< 90nm) and thymol
Fig. 1 Size distribution by
intensity and correlogram of T.
ammi EO NE

Environmental Science and Pollution Research
1 3
(230nm) the LC50 values were lower than 100ppm, and
this qualifies both as potent mosquito larvicide, as sug-
gested by Pavela (2015). In such a condition, it permits
the use of NEs of thymol and EO as larvicide against Ae.
aegypti. Though thymol as a single component is effec-
tive larvicide than EO as a whole, the advantage of other
constituents in the EO will add to their efficacy, and the
possibility of developing insecticide resistance can be
scaled down. Added to this the fractionation of thymol
from EO and use would add to cost than use of EO as
Fig. 2 Size distribution by
intensity and correlogram of
thymol NE
Fig. 3 TEM images of a EO NE
and b thymol NE
Table 3 Acute toxicity of emulsions of T. ammi EO and its constitu-
ent thymol
95% CL confidence interval at 95% confidence level
Test sample LC50 (ppm) 95% CL df Chi-square P value
EO NE 46.73 35.78–63.20 4 9.25 0.05
EO BE 59.67 45.98–82.68 4 9.20 0.06
Thymol NE 34.89 26.79–45.87 4 9.0 0.05
Thymol BE 52.82 40.94–71.27 4 8.99 0.05
Imidacloprid 0.96 0.76–1.27 3 7.2 0.06

Environmental Science and Pollution Research
1 3
such. The NEs are economical over Bes as in nanoemul-
sions; the requirement of thymol and EO can be reduced
by 1.51 and 1.28 times over the BE using EO and thymol
as a whole. Downsizing the droplet in nanoemulsion of
eucalyptus oil enhanced the contact of the toxicity to
Tribolium castaneum (Adak etal. 2020).
The synthetic insecticide imidacloprid was more toxic
(LC50 0.96ppm) than NE and BE formulations of EO and
thymol (Table3). Though chemical insecticides are effec-
tive, their indiscriminate use adds to problems like insecti-
cide resistance and harm to humans and the environment.
Scanning electron microscopy
The impact of EO and thymol on III instar larvae of Ae.
aegypti by contact exposure is shown in Fig.4a. EO and
thymol at LC50 dose distorted the cuticle by shrinkage, as
shown in Fig.4b, c. The control larvae had smooth and nor-
mal skin texture. One of the mechanisms for an effective
insecticide is its ability to cross over the cuticle (Kasai etal.
2014). The droplet size of the particles in nm facilitates bet-
ter penetration coupled with lipophilicity. The wider surface
area of the nanoemulsion would have been a possible reason
for better action of the EO and thymol by causing shrinkage
in larval cuticle in Ae. aegypti larvae. A decrease in droplet
size in nanoemulsion enhanced the uptake and penetration
of bioactive compounds in EO into the insect body; thereby,
improving the efficacy of NE over the BE was reported ear-
lier (Pascual-Villalobos etal. 2019).
AChE inhibition assay
The effects of NEs of EO and thymol on larval acetylcho-
lineesterase activity were studied. The inhibition of AChE
activity of NEs of EO and thymol is shown in Fig.5. The
NE of thymol was a potent AChE inhibitor as it caused
the significant inhibition activity (83.48 ± 1.21%), fol-
lowed by imidacloprid that caused 78.78 ± 5.48% inhibi-
tion that was at par. EO NE caused an inhibition activity
(53.62 ± 3.77%) (F2,6 = 18.96 P < 0.003).
Acetylcholineesterase is an enzyme in an insect that
hydrolyzes acetylcholine (Ach). Inhibition of AChE
causes paralysis in insects that leads to death (Huang etal.
2020). Natural products having phenols, monoterpenoids,
and mixtures of compounds inhibit the activity of AChE
(Wu etal. 2020) and block insect octopamine receptors
(Almadiy 2020), thereby causing an insecticidal effect.
Our studies showed that thymol NE caused the high-
est inhibition of AChE (83.48%) followed by EO NE
(53.62 ± 3.775). The insecticidal effect of nanoemulsion
of EO is facilitated through enzymatic inhibition which
has an impact on neural signal transduction (Seo etal.
2015). Thymol inhibited AChE in the third instar larvae
of Ae. albopictus and interacted with GABA-A and octo-
pamine receptors in Culex pipiens (Youssefi etal. 2019).
Pterodon emarginatus nanoemulsion showed anti-acetyl-
cholinesterase activity in Ae. aegypti (Oliveira etal. 2016).
Monoterpenes in essential form hydrophobic interaction
with the catalytic subunit of AChE thereby reducing its
Fig. 4 SEM micrograph of Ae.
aegypti larvae intersegmental
region. a Control (without any
oil), b T. ammi EO, c thymol

Environmental Science and Pollution Research
1 3
activity (Oyedeji etal. 2020). This may be the mechanism
of insecticidal activity of T. ammi EO and thymol.
Conclusion
The NEs of T. ammi EO and thymol optimized in this study
facilitated the development of eco-friendly and effective
nanoformulation for mosquito management. The NEs
stability and enhanced bioactivity of EO and thymol against
Ae. aegypti larvae as compared to BE confirm the effect of
NE in causing acute larval toxicity, impact on the larval
cuticle and its ability to inhibit the acetylcholineesterase
activity. These properties ascribe NEs of EO and thymol
as potential candidates for Ae. aegypti management.
Considering the improved formulation characters of NE,
it is effective and economical as compared to BE which
uses EO as a whole. Though the synthetic insecticide had
better larvicidal activity than the NE, they also bring in
ill effects due to their indiscriminate use like insecticide
resistance and harm environment. The EO with multiple
modes of action prevents or delays the development
of insecticide resistance in mosquitoes. Hence, the
developed NE formulation has the benefit to minimize the
dependence of harmful chemical pesticides used for Ae.
aegypti management. Further research on the impact of
nanoemulsion on human health and the environment will
strengthen its use in area-wide management.
Acknowledgements The work was supported by ICAR–NBAIR as an
institutionally funded project. Imaging services were provided by Dr
Geetha; NICE Lab NCBS is acknowledged
Author contribution Kesavan Subaharan: conceptualization, writ-
ing — original draft, and data curation. Periyasamy Senthamarai
Selvan, Tania Das, and Rajendran Senthoorraja: investigation. T. M.
Subramanya: investigation, Sowmya Manjunath: investigation on bio-
chemical aspects. Muthu Gounder Mohan: resources. Vppalayam Shan-
mugam Pragadheesh: investigation and validation. Nandagopal Bak-
thavatsalam: writing — review and editing. M. Eswaramoorthy: writing
— review and editing, and supervision. Paulraj Philip Samueland Renu
Govindaraju: resources. Sreehari U: writing — review and editing.
Sengottayan Senthil-Nathan: writing — original draft and editing.
All the authors read and approved the final manuscript.
Funding The work was supported by ICAR–NBAIR as an institutional
project.
Data availability Data is available by request to the corresponding
author.
Declarations
Ethics approval and consent to participate Not applicable.
Consent for publication Not applicable
Competing interests The authors declare no competing interests.
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