Cupressus sempervirens Essential Oil, Nanoemulsion, and Major Terpenes as Sustainable Green Pesticides against the Rice Weevil

Abstract

In order to find effective, biorational, and eco-friendly pest control tools, Cupressus sempervirens var. horizontalis essential oil (EO) was produced using hydrodistillation, before being analyzed with gas chromatography, specifically, using flame ionization detection. The monoterpene components α-pinene (46.3%), δ-3-carene (22.7%), and α-cedrol, a sesquiterpene hydrocarbon, (5.8%), were the main fractions. An oil-in-water nanoemulsion was obtained following a green protocol. The EO, its nanoemulsion, and its terpenes each exhibited both insecticidal and insect repellent activities against the rice weevil, Sitophilus oryzae. In a contact bioassay, the nanoemulsion induced a 100% adult mortality rate in a concentration of 10.0 µL/cm² after 4 days of treatment, whereas 40 µL/cm² of EO and α-cedrol was required to kill 100% of weevils. Using fumigation, nanoemulsion and EO at 10 µL/L air caused a 100% adult mortality rate after 4 days of treatment. The LC50 values of botanicals ranged between 5.8 and 53.4 µL/cm² for contact, and between 4.1 and 19.6 µL/L for fumigation. The phytochemicals strongly repelled the weevil at concentrations between 0.11 and 0.88 µL/cm², as well as considerably inhibiting AChE bioactivity. They were found to be safe for earthworms (Eisenia fetida) at 200 mg/kg, which also caused no significant alteration in wheat grain viability. This study provides evidence for the potential of using the EO of C. sempervirens and its nanoemulsion as natural, eco-friendly grain protectants against S. oryzae.

Full text

Citation: Almadiy, A.A.; Nenaah,
G.E.; Albogami, B.Z.; Shawer, D.M.;
Alasmari, S. Cupressus sempervirens
Essential Oil, Nanoemulsion, and
Major Terpenes as Sustainable Green
Pesticides against the Rice Weevil.
Sustainability 2023,15, 8021. https://
doi.org/10.3390/su15108021
Academic Editors: Barlin
Orlando Olivares Campos,
Miguel Araya-Alman and Edgloris
E. Marys
Received: 6 April 2023
Revised: 10 May 2023
Accepted: 11 May 2023
Published: 15 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
sustainability
Article
Cupressus sempervirens Essential Oil, Nanoemulsion, and Major
Terpenes as Sustainable Green Pesticides against the
Rice Weevil
Abdulrhman A. Almadiy 1, Gomah E. Nenaah 1, 2, * , Bader Z. Albogami 1, Dalia M. Shawer 3and Saeed Alasmari 1
1Biology Department, College of Science and Arts, Najran University, Najran 1988, Saudi Arabia;
aaalmady@nu.edu.sa (A.A.A.); bzalmarzoky@nu.edu.sa (B.Z.A.); smalasmari@nu.edu.sa (S.A.)
2Zoology Department, Faculty of Science, Kafrelsheikh University, Kafrelsheikh 33516, Egypt
3Economic Entomology Department, Faculty of Agriculture, Kafrelsheikh University,
Kafrelsheikh 33516, Egypt
*Correspondence: dr_nenaah1972@yahoo.com
Abstract:
In order to find effective, biorational, and eco-friendly pest control tools, Cupressus semper-
virens var. horizontalis essential oil (EO) was produced using hydrodistillation, before being analyzed
with gas chromatography, specifically, using flame ionization detection. The monoterpene compo-
nents
α
-pinene (46.3%),
δ
-3-carene (22.7%), and
α
-cedrol, a sesquiterpene hydrocarbon, (5.8%), were
the main fractions. An oil-in-water nanoemulsion was obtained following a green protocol. The
EO, its nanoemulsion, and its terpenes each exhibited both insecticidal and insect repellent activities
against the rice weevil, Sitophilus oryzae. In a contact bioassay, the nanoemulsion induced a 100%
adult mortality rate in a concentration of 10.0
µ
L/cm
2
after 4 days of treatment, whereas 40
µ
L/cm
2
of EO and
α
-cedrol was required to kill 100% of weevils. Using fumigation, nanoemulsion and EO
at 10
µ
L/L air caused a 100% adult mortality rate after 4 days of treatment. The LC
50
values of
botanicals ranged between 5.8 and 53.4
µ
L/cm
2
for contact, and between 4.1 and 19.6
µ
L/L for
fumigation. The phytochemicals strongly repelled the weevil at concentrations between 0.11 and
0.88
µ
L/cm
2
, as well as considerably inhibiting AChE bioactivity. They were found to be safe for
earthworms (Eisenia fetida) at 200 mg/kg, which also caused no significant alteration in wheat grain
viability. This study provides evidence for the potential of using the EO of C. sempervirens and its
nanoemulsion as natural, eco-friendly grain protectants against S. oryzae.
Keywords: cypress oil; nanoemulsion; terpenes; Sitophilus oryzae; bioactivity; biosafety
1. Introduction
The world’s population is expected to grow to more than 10 billion by 2050, which will
boost the global food demand; particularly, for food crops and cereal products. Therefore,
agricultural production should be doubled if we are to secure adequate food sources for
this huge number of people [
1
]. There is no doubt that many countries of the world will
face problems in this regard, especially those developing and underdeveloped countries
with poverty and inadequate technologies for modern agriculture. Furthermore, harmful
insects and other pathogens cause the loss of more than 30% of the world’s food production,
both in the field and during storage as well [
2
,
3
]. Coleopteran arthropod insect pests are
the major causal agents of grain loss during storage, worldwide. Grain weevils of the
genus Sitophilus are among the world’s most damaging and widespread pests for stored
grains. Infestations of grains by these weevils cause significant grain losses and promote
the growth of molds, including harmful toxigenic species, by increasing temperature and
moisture levels [3].
The rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae), is a serious primary
internal feeder pest, able to infest intact grains, causing both quantitative and qualitative
Sustainability 2023,15, 8021. https://doi.org/10.3390/su15108021 https://www.mdpi.com/journal/sustainability

Sustainability 2023,15, 8021 2 of 20
damage in the grains and altering seed viability [
4
]. Chemical insecticides have widely
been applied to prevent insect pests from attacking a variety of crop plants, both in the
field and in stores. The overuse of chemical insecticides negatively affects not only the
health of consumers and farmers, but also nontarget beneficial organisms and the quality
of foodstuffs, in addition to bringing environmental problems as well [
5
,
6
]. Populations
of S. oryzae have also developed resistance against many insecticides and fumigants, such
as pirimiphos-methyl and phosphine [
7
]. These problems make the intensive use of
chemical pesticides a point of renewed public debate, especially in light of increasing public
awareness of the risks of insecticides. This popular awareness of the adverse effects of
conventional pesticides has promoted researchers to seek novel agrochemical pesticides that
can meet the increasing grower, consumer, environmental, and regulatory requirements [
8
].
These new pesticides should be environmentally safe, with novel action mechanisms and
low negative impacts on the ecosystem. Pest control using phytochemicals, especially plant
essential oils (PEOs), seems a promising alternative strategy for decreasing the intensive
reliance on conventional insecticides [
6
,
9
]. Because of their wide spectrum bioactivity as
pest control tools against several pest insects (including those of stored grains), PEOs are
increasingly being considered a credible eco-friendly natural alternative to conventional
insecticides [
3
,
6
,
9
–
14
]. This is especially significant for the protection of stored products,
whose confinement promotes the action of molecules which are naturally highly volatile, in
order to avoid the toxic residue from chemical insecticides contaminating food products. In
this context, we can observe major shortcomings related to the procedures adopted for the
extraction, formulation, application, and performance of plant products (including EOs)
in pest control protocols. For strengthening and improving the inclusion of plant-based
products in pest control programs, nanotechnology has emerged as a promising field across
multidisciplinary research, opening up many application opportunities in such various
fields as medicine, electronics, drug delivery, and agriculture [
15
,
16
]. In agriculture, the
potential benefits of nanotechnology in pest control programs include the fabrication of
novel plant-based nanomaterial formulations with enhanced insecticidal activities, which
could diminish the need for repeated application of chemical pesticides [
6
,
15
–
18
]. Because
of their high reactivity and solubility, in addition to their novel physical and chemical
characteristics, nanopesticide materials show enhanced bioactivities as pest control tools
relative to their bulk counterparts [6,15,17].
The genus Cupressus (Cupressaceae) comprises about 12 species, spread across North
America, Mexico, the Mediterranean basin, southern Europe, and subtropical west Asian
countries, including the Kingdom of Saudi Arabia [
19
]. Members of Cupressaceae are com-
mon essential-oil-bearing plants, of which an important species is Cupressus sempervirens L.
var. horizontalis (The Mediterranean cypress). It is an aromatic evergreen tree, traditionally
used as an expectorant in anticough and antibronchitis medications; for stomach pain; as
an antidiabetic medicine; as an antiseptic; for antiulcer and anti-inflammatory purposes;
and for treatment of toothaches, flu, coughs, and laryngitis [
20
]. This plant species has
been screened for different bioactivities, including antimicrobial, antiviral, antihelmenthic,
antiseptic, cytotoxic, antioxidant, anti-inflammatory, antirheumatic, antihyperlipidemic,
anticancer, antispasmodic, antidiuretic, and hepatoprotective activities [
20
,
21
]. Insecticidal
bioactivities of the cypress tree have also been recorded [
9
,
22
,
23
]. However, there have
been no thorough investigations into the insecticidal bioactivity of the EO, nanoemulsion,
or bioactive terpenes of C. sempervirens against stored-grain insects. In this study, we aimed
to investigate not only the composition, but also the contact, fumigant, and repellence
bioactivities of the oil, nanoemulsion, and bioactive terpenes of cypress trees growing in
Saudi Arabia against S. oryzae. The effect of EO materials on acetylcholinesterase (AChE)
bioactivity—being a common target enzyme for insect control agents—was investigated.
The impact of EO products on earthworms (E.fetida), as well as their phytotoxic activity
on wheat plant in terms of the basic growth parameters (%germination, root, and shoot
growth) were evaluated.

Sustainability 2023,15, 8021 3 of 20
2. Materials and Methods
2.1. Chemicals
Analytical grade monoterpenes, sesquiterpenes, and oxygenated monoterpenes,
(
Sigma-Aldrich Co. Ltd., St Louis, MO, USA
; label purity 99.0–99.8%) were used for com-
parisons. To calculate retention indices, the series of hydrocarbons (C
5
–C
40
) known
as triacontane (Supelco, Bellefonte, CA, USA) was used. Dimethyl sulfoxide (DMSO)
and its solvents, all of an analytical grade (Carlo Erba Milan, Milan, Italy), were used
in experiments.
2.2. Test Insect
A laboratory strain of the rice weevil, the S. oryzae were reared in a pesticide-free
environment in our laboratory for more than twenty generations. Weevils were kept in 2 L
glass bottles, each containing 200–250 adults and 250 g sterilized wheat grains (moisture
content 14
±
2%). The jars were covered using muslin cloth that was held in place with
a rubber band, and then kept in laboratory conditions of 30
±
2
◦
C and 68.5% relative
humidity, in complete darkness, until the emergence of the adult specimens.
2.3. EO Extraction
On September 2020, the whole aerial parts of the cypress trees were collected from
random gardens in Abha, Kingdom of Saudi Arabia; latitude 18
◦
19
0
45.7824
00
(N), longitude
42
◦
45
0
33.7140
00
(E), and 718 m altitude. Specimens were identified by the botanists of
Najran University, Saudi Arabia. A plant specimen (No. Cs 02) was laboratory-deposited,
for reference. Plant leaves were air dried in the shade, powdered mechanically using a high-
speed blender, before being subjected to hydrodistillation using a Clevenger apparatus to
obtain the EO. In each run, 150 g powders were hydrodistilled for 3 h with 250 mL distilled
water, and in three replicates. The oil/water mixture was extracted with hexane, which
had been washed with anhydrous sodium sulfate before the oil had dried, before then
being concentrated under reduced pressure. For bioassays, the oil yield (% wt./wt.) was
calculated on a dry weight basis and stored at 4
◦
C, where triplicates were considered in
calculating the oil yield.
2.4. Analysis of EO and Identification of Constituents
Cypress oil was analyzed using an Agilent 6890 N gas chromatograph (Agilent
Technologies
, Palo Alto, CA, USA), coupled with a flame ionization detection (FID) and an
HP-5 capillary column (30 m 0.32 mm; thickness 0.25 m). The following conditions were
met by the GC–FID: one liter of EO; split mode; 50:1 split ratio; and an injector temperature
of 250
◦
C. Oven temperature was initially set at 40
◦
C for 3 min, then increased to 80
◦
C at a
rate of 5
◦
C/min, held at that temperature for 3 min, then increased to 250
◦
C at 10
◦
C/min,
which was held for 10 min. The injector and detector were set to 250
◦
C, and the carrier gas
was helium, at a 1.0 mL/min flow rate. The gas chromatograph was then connected to the
silica gel capillary column (HP-5 MS). At a split ratio of 1:100, 0.1
µ
L of EO was injected
onto the column; the carrier gas was helium (1.0 mL/min flow rate). Operation of the mass
detector was set at 70 eV ionization voltage. The mass range was taken at 45~550 AMU.
Temperatures of the ion source, transfer-line, and the quadrupole were 230, 250, and 150
◦
C,
respectively. Temperature of the oven was programmed as described for the GC. Retention
indices of terpenes were calculated depending on n-alkanes (C
5
–C
40
) co-injected into the
column, in accordance with Van Den Dool and Kratz’s equation. Identification of EO
profile was accomplished by comparing terpene retention indices and mass spectra to those
recorded by Adams [
24
] and the data stored in the database NIST Standard Reference
Database Number 69, [
25
]. The oil terpenes were quantified as percentages by integrating
their peak areas, calibrating, and comparing them to internal standards without the use of a
response factor correction. The remaining terpenes were likewise quantified as percentages,
calculated by integrating their peak areas, calibrating, and then comparing to standards
without using a response factor correction.

Sustainability 2023,15, 8021 4 of 20
2.5. Isolation of Main Terpenes
Ten milliliters of cypress oil was fractionated on a silica gel capillary column (Kieslgel
60, 230–400 mesh, Merck). Trials were conducted in order to determine the best eluent.
To accomplish this, several solutions of both n-hexane: ethyl acetate and toluene: ethyl
acetate were prepared, which were then tested using Thin-layer Chromatographic plates
(TLC), with toluene: ethyl acetate (90:10, then 93:7) being chosen as the best eluent [
26
].
According to TLC data, developed fractions were divided into 3 main fractions: Main
fraction 1 (fractions 8–14, 274.3 mg) was fractionated on a silica gel column, affording 41 mg
of
α
-pinene. The main fraction 2 (fractions 21–27, 193.1 mg) yielded 23 mg of
δ
-3-carene. By
contrast, when fraction 3 (fractions 31–37, 82.5 mg) was developed it provided 9.2 mg of
α
-cedrol. Terpene fractions were visualized using an UV lamp (254 and 365 nm), and the R
f
values of terpenes were calculated and then compared against standards. The structures of
the terpene fractions were elucidated using spectroscopic instruments.
2.6. Nanoemulsion Formulation and Characterization
An oil/water nanoemulsion was made following a low-energy emulsification protocol
at a constant temperature, with the following proportions: deionized H
2
O (90%), EO 5%
(wt./wt.), and Tween 80 (5%) as a nonionic surfactant [
17
]. The EO/emulsifier mixture was
stirred at 800 rpm for 30 min in a water bath at 35
±
5
◦
C. After reaching an oily phase,
deionized H
2
O was gradually added (2.5 mL/min). After stirring for 45 min at 800 rpm,
the temperature was gradually reduced to room temperature, and the nanoemulsion was
formed and then preserved in dark screw-capped vials at 24
±
2
◦
C. At 0, 1, 10, 20, 30, and
45 days following its preparation, the nanoemulsion was examined for thermodynamic
changes. Nanoemulsion was characterized using a polydispersity index (PDI), mean
droplet size, and thermodynamic stability measurements (centrifugation, heating, cooling,
freezing cycles, and viscosity) [
17
]. The oil emulsion was first centrifuged (5000 rpm
at 25
◦
C for 25 min), and checked for phase separation, turbidity, and cracking, if any.
Heating/cooling tests were performed on the stable formulations for 6 cycles (4–40
◦
C, each
cycle lasting 48 h). Emulsions that demonstrated stability were subjected to a freeze–thaw
stress test by alternately storing them at 2 different temperatures (20
◦
C and 20
◦
C, 24 h each).
Nanoemulsions that demonstrated stability were stored in dark, tightly closed vials at room
temperature for one month to observe any creaming, phase separation, or flocculation.
The emulsion’s pH was measured at 25
±
0.2
◦
C, and its viscosity (
µ
) was elucidated at
200 rpm. Samples were left to stand for about 2 min to reach an equilibrium; thus, readings
and experiments were each taken and performed in triplicate. The Z-average diameter of
droplets and PDI were measured using a nanoparticle analyzer apparatus (Zetasizer, Nano
ZS, Malvern Instruments, Worcestershire, UK) that operates on a dynamic light-scattering
basis. Prior to measurements, test formulations were diluted to 10% with deionized H
2
O
to avoid multiple scattering. The droplets’ size and their PDI were calculated using DLS
data. The measurements were taken at a scattering angle of 90
◦
, and trials were repeated
three times. A Scanning Electron Microscope, or SEM (JEOL, JFC-1600, Tokyo, Japan), was
used to determine the morphology of droplets; for this, 15
µ
L of emulsion that had been
dissolved in deionized H
2
O was placed onto a carbon-coated copper grid that had been
stained with 2% phosphotungstic acid (pH of 6.8). The test samples were dried at 26
◦
C
before being imaged at 80 kV.
2.7. Contact Insecticidal Activity
The contact bioactivity of the EO, nanoemulsion, and terpenes from cypress trees
against S. oryzae adults were determined using the dipping filter paper technique [
6
].
Test concentrations of 0.398, 0.795, 1.59 and 3.18 mL of EO, nanoemulsion,
δ
-3-carene,
α
-cedrol, and
α
-pinene were dissolved in 5 mL acetone to obtain the test solutions. Each test
concentration was uniformly dropped onto a filter paper (Whatman No. 1, 9 cm d, 63.6 cm
2
)
to achieve serial test concentrations of 5.0, 10.0, 20.0, and 40.0
µ
L/cm
2
, respectively. Acetone
was evaporated from the treated papers; then a treated paper was placed into the bottom

Sustainability 2023,15, 8021 5 of 20
of a Petri dish (9 cm d) and twenty adult weevils (of mixed sexes, and 15–20 days old) were
introduced. Control groups (adults exposed to acetone-treated filter papers) were included.
Treatment and control dishes were kept in the dark at 30
±
2
◦
C and 68
±
5% r.h. After
24 h, insects were placed into clean Petri dishes, enriched with wheat grains, and kept in
rearing conditions. Experiments were performed six times alongside control, and mortality
was recorded 1, 2, 4 and 7 days after the treatment, whereafter the end-point mortality was
reached, and the resulting contact toxicity was expressed in µL/cm2.
2.8. Fumigation
Insecticidal bioactivity using fumigation was investigated as follows: a filter paper
(7.0 cm diameter) was dipped in 25
µ
L of an appropriate concentration of each terpene
dissolved in acetone, and control sets were made using acetone, only [
6
]. Botanicals were
screened at 4 dose rates (2.5, 5.0, 10.0, and 20.0
µ
L/L air). After evaporating the acetone,
each treated paper was attached to the undersurface of the screw cap of 250 mL volume
glass bottles, which served as fumigant chambers. Twenty weevils were placed into each
bottle as adults (15–20 days old), and the bottle was covered with a tape-fixed fine gauze.
Experiments were undertaken in six replicates, alongside control groups. After 24 h, the
weevils were transferred back to food-enriched clean vials and kept in the rearing conditions
described before; whereupon mortality was measured after 1, 2, 4 and 7 days had elapsed
from treatment, and the resulting fumigant bioactivity was expressed in µL/L air.
2.9. Repellence Bioactivity
The repellence bioactivity of C. sempervirens oil materials against adult weevils was
studied by adopting a chosen (area preference) bioassay [
22
]. A piece of filter paper
(Whatman No. 1, 9 cm diameter) was divided into two halves. Test solutions of oil
materials were prepared, with 3.5, 7.0, 14.0, and 28
µ
L of each material dissolved in 0.5 mL
n-hexane. Each test concentration was uniformly dropped onto a half filter paper disc,
which served as a test area, to obtain bioassay concentrations of 0.11, 0.22, 0.44, and
0.88
µ
L/cm
2
. The second half was treated only with n-hexane, representing a control. The
treated and untreated paper discs were then air dried for 5 min, and thereafter attached to
their corresponding opposite surface with adhesive tape, and put in the bottom of a Petri
dish (9 cm). Twenty (15–20 days old) unsexed adult weevils of S. oryzae were released at the
center of each disc, then the lid was covered using a parafilm. Five replicates (100 adults)
were considered for each concentration, and the experiments were achieved in rearing
conditions. The number of weevils that were observed across both the treated and control
halves were counted after 2, 6, 12, and 24 h. The Repellency percentage (RP) was calculated
using the following formula: RP = (C
−
T)/(C + T)
×
100, where C is the No. of weevils on
untreated zone, and T is the No. of weevils on control zone.
2.10. AChE Inhibition and Estimation of IC50
Anticholinesterase (AChE) enzymatic activity was measured in accordance with Ell-
man et al. [
27
]. One gram’s worth of the adult weevils were homogenized in 20 mL of an
ice-cold phosphate buffer (50 mM and pH 7.4). Acetylthiocholine iodide (25
µ
L of 15 mM)
was dropped as a substrate. The inhibition in AChE activity was measured calorimetrically
using a supernatant as an enzyme source [
17
]. Botanicals were formulated initially in
acetone, then in Triton-X 100 (0.01%), and were then tested at 2.5~100 mM. Test and control
solutions were corrected using blanks for the nonenzymatic hydrolysis. Trials were per-
formed in triplicate. Absorbance of the solution reflecting AChE specific activity (
∆
OD/mg
protein/min) was monitored at a wavelength of 412 nm.
2.11. Phytotoxicity
The phytotoxic impact of the oil terpene components (as indicated by basic growth pa-
rameters (%germination, root, and shoot growth)) was evaluated on wheat plants (Triticum
aestivum L.). Wheat seeds were sterilized using a solution of sodium hypochlorite (15%) for

Sustainability 2023,15, 8021 6 of 20
about 40 s, followed by rinsing in sterile deionized water. The grains were placed in clean
9 cm diameter Petri plates, each containing five layers of Whatman filter paper, onto which
1 mL of each botanical (at concentrations of 50, 100, and 150
µ
L/mL) was dropped. 2 mL
of methanol was sprayed on the control. After the evaporation of methanol, ten healthy
grains (~0.3–0.36 g) were deposited in each dish. Dishes were maintained at 20
±
2
◦
C;
65 ±5% R.H.
, with a natural photoperiod (optimized environmental conditions for wheat
germination). Additionally, 10 mL of water was given daily. Each concentration had five
replicates, as well as a control. Germination and the seedling growth were noticed after
10 days of planting. The length of shoot and number of leaves were counted 2 weeks later,
and seed germination was indicated by the emergence of radicles.
2.12. Toxicity on Earthworm
The acute toxicity of the botanicals against E. fetida earthworms was investigated,
according to the guidelines of OECD (Organization for Economic Co-Operation and Devel-
opment [
28
]. The animals were reared on artificial diet, as detailed by Pavela [
29
]. Terpenes
were admixed with the soil at concentrations equaling 50, 100, and 200 mg kg
−1
. The
positive control was
α
-cypermethrin at 10 and 20 mg kg
−1
soil alongside, with deionized
water as a negative one. In 1 L glass pots containing either treated or untreated (control)
soil, the earthworms were confined as ten adults, and triplicates were made for each run.
Pots were covered with a fine gauze, then incubated at 22
±
2
◦
C, 75
±
5% R.H., and 16:8 h
light/dark photoperiod. Mortality was recorded after five and ten days of treatment.
2.13. Statistical Analysis
Data of mortality were adjusted for control mortality and corrected using Abbott’s
formula [
30
] when mortality in control exceeded (5%), and data were expressed as % means
(
±
S.E.). A one-way analysis of variance (ANOVA) at the probability level (=0.05) was
adopted on transformed data to compare significance differences between means in both
the treatment samples and the controls, followed by individual pairwise comparisons,
adopting Tukey’s HSD test. Dose–response mortality was analyzed using Finney’s Probit
analysis to estimate the LC
50
and LC
95
and their limits across 48 exposure periods [
31
].
Probit analysis was adopted to calculate the concentrations that inhibited AChE bioactivity
by 50% (IC
50
). The Statistical Package for Social Sciences was used for data analysis (version
23.0; SPSS, Chicago, IL, USA).
3. Results
3.1. Composition of EO
A pale yellowish EO with a strong odor (yield 0.74% w/w) was obtained from
C. sempervirens
var. horizontalis using hydrodistillation. A total of 62 terpenes amounting
99.7% (wt./wt.) were identified in the oil (Table 1and Figure 1a), and then listed accord-
ing to their retention indices. The main oil terpenes were (1S,5S)-2,6,6-trimethylbicyclo
[3.1.1] hept-2-ene (
α
-pinene, 46.3%), 3,7,7-trimethylbicyclo [4.1.0] hept-3-ene (
δ
-3-carene,
22.7%), and (1S,2R,5S,7R,8R)-2,6,6,8-tetramethyltricyclo [5.3.1.0] undecan-8-ol (
α
-cedrol,
5.8%) (
Figure 1b
). The structure of the terpenes was confirmed using physical and spectro-
scopic methods, which corroborates with published data:
α-pinene
Colorless, C
10
H
16
.
1
H NMR (CDCl
3
, 300 MHz):
δ
1.92 (m, 1H),
δ
1.4 (s, 3H),
δ
1.6 (s,
3H),
δ
1.8 (s, 3H),
δ
1.9 (m, 2H),
δ
2.3 (m, 1H),
δ
2.4 (m, 1H),
δ
4.2 (s, 1H),
δ
5.6 (t, 1H), 20.67,
22.35, 22.47, 26.36, 32.62, 36.08, 68.03, 94.15, 123.87, 134.38; 13C NMR (125 MHz, CHCl3): δ
46.99 (C-1), 144.6 (C-2), 116.0 (C-3),
δ
31.3 (C-4), 40.69 (C-5), 37.97 (C-6),31.5 (C-7),
δ
26.3
(C-8), 20.8 (C-9), 23.01 (C-10) [32,33].
δ-3-carene
Colorless, C
10
H
16
.
1
H-NMR (600 MHz, CDCl
3
)
δ
ppm: 5.23 (2H, t, H-2), 2.62 (2H, t,
H-6), 1.60 (4H, m, H-3, 5), 1.022 (1H, m, H-4), 0.761 (6H, s, 9, 10-CH
3
), 0.49 (3H, s, 7-CH
3
);

Sustainability 2023,15, 8021 7 of 20
13
C-NMR (125 MHz, CDCl
3
)
δ
ppm: 131.30 (C-6), 119.56 (C-1), 28.42 (C-7), 24.93 (C-5), 23.63
(C-4), 20.89 (C-2), 18.71 (CH3-8), 16.90 (C-3),16.78 (CH3-9), 13.20 (CH3-l0) [34,35].
α-cedrol
Colorless, C
15
H
26
O.
νmax
/cm
−1
3380, 1460, 1030, and 1000;
δH
(300 MH
z
) 0.74 (3H, s,
Me), 0.78 (3H, d, J7.1, Me), 0.85 (3H, s, Me), 0.88 (3H, s, Me), 0.85–1.61 (10H, m), 1.87–1.98
(1H, m), 2.1–2.2 (1H, m), and 3.94 (1H, ddd, J2.2, 5.6 and 9.7, CHOH); m/z 222 (M1, 44%), 206
(15), 178 (100), and 123 (40) (Found: M1, 222.1992. C15H26O requires M, 222.1985) [36–38].
Table 1. Chemical profile of Cupressus sempervirens essential oil.
a,b Components cRI exp. dRI lit. Concentration
(%)
2-Hexanal 860 862 0.2
Tricyclene 918 916 0.1
a-Thujene 920 921 0.4
α-Pinene 928 930 46.3
Camphene 930 932 1.2
a-Fenchene 941 942 0.1
Sabinene 966 967 0.6
ß-Pinene 980 980 0.9
ß-Myrcene 988 988 0.1
α-Phellandrene 1006 1008 0.2
δ-3-Carene 1010 1010 22.7
α-Terpinene 1016 1018 1.3
p-Cymene 1021 1020 0.6
Limonene 1032 1029 1.6
β-Phellandrene 1034 1032 0.2
Z-ß-Ocimene 1038 1037 0.2
E-ß-Ocimene 1044 1044 0.1
γ-Terpinene 1054 1055 0.3
cis-Sabinene hydrate 1067 1066 0.4
p-Cymenene 1070 1072 0.9
α-Terpinolene 1085 1086 1.3
Linalool 1096 1095 0.4
trans-Sabinene hydrate 1099 1097 1.1
Pinocarveol 1138 1140 0.1
Camphor 1142 1144 0.2
Pinocarvone 1158 1162 1.6
Borneol 1162 1165 0.2
Terpinen-4-ol 1176 1174 1.1
p-Cymen-8-ol 1180 1181 0.4
trans-Pinocarveol 1182 1184 0.6
α-Terpineol 1188 1186 0.2
Myrtenol 1192 1195 0.2
Pulegone 1235 1233 0.3
Carvacrol methyl ether 1241 1241 0,2
cis-Chrysanthenyl acetate 1244 1242 0.1
cis-Piperitone epoxide 1247 1248 0.4
trans-Piperitone epoxide 1251 1252 0.7
Carvone 1254 1258 1.1
Carvenone oxide 1260 1260 0.2
Bornyl acetate 1285 1186 0.4
Thymol 1289 1288 0.3
trans-Sabinyl acetate 1290 1292 0.2
Carvacrol 1296 1298 0.3
α-Cedrene 1295 1294 0.1
α-Copaene 1372 1374 0.2
β-Bourbonene 1382 1384 0.1
α-Gurjunene 1408 1408 0.3
β-Caryophyllene 1414 1417 0.1

Sustainability 2023,15, 8021 8 of 20
Table 1. Cont.
a,b Components cRI exp. dRI lit. Concentration
(%)
β-Gurjunene 1430 1432 0.2
α-Humulene 1450 1452 0.2
Alloaromadendrene 1472 1474 0.3
Germacrene D 1480 1478 0.3
Bicyclogermacrene 1496 1495 0.1
β-Bisabolene 1508 1510 0.2
cis-Calamenene 1544 1443 0.2
Spathulenol 1574 1576 0.6
α-Cedrol 1596 1591 5.8
α-Acorenol 1632 1630 0.3
β-Acorenol 1635 1637 0.2
γ-Cadinol 1648 1649 0.1
Cadalene 1674 1674 0.1
Manool 1990 1989 0.3
Grouped compounds (%) - -
Monoterpene hydrocarbons - - 77.1%
Oxygenated monoterpenes - - 12.9%
Sesquiterpene hydrocarbons - - 9.7%
% peaks identified - - 99.7
Total yield % (mL/100 g) - - 0.74
a
Compounds are listed in the order of their elution from a HP-5MS column.
b
Identification methods: a, based on
comparison of RT, RI, and MS with those of authentic compounds; b, based on comparison of mass spectrum
with those reported in Wiley, Adams [
24
], and NIST 69 MS libraries [
25
].
c
Linear retention index on the HP-5MS
column, experimentally determined using homologous (C
5
–C
40
)n-alkane series.
d
Linear retention index based
on Adams [24] or NIST 69 [25], and literature.
Sustainability 2023, 15, x FOR PEER REVIEW 9 of 21
(a)
α-pinene δ-3-carene 𝛼-cedrol
(b)
Figure 1. (a) GC–FID chromatogram of cypress EO. Major terpenes are highlighted; (b) Major ter-
pene components of cypress EO.
3.2. Nanoemulsion Characterization
The developed emulsion (droplet size 67.8 ± 3.1 nm) showed stability during ex-
treme conditions of centrifugation, temperature, heating–cooling cycle (4–40 °C), and a
freezing cycle at −4 °C. The optimum conditions of nanoemulsion preparation, droplet
size, and their PDI, are listed in Table 2 and illustrated in Figure 2. The SEM revealed that
a transparent nanoemulsion consisting of dispersed and spherical-shaped nanoparticles
had been developed (Figure 3).
Table 2. Characterization of Cupressus sempervirens oil nanoemulsion.
Storage Period (Days)
Viscosity (mPa·s)
pH
PDI
Size (nm ± S.E.)
0
4.1
6.1 ± 0.04 c
0.18 ± 0.03 a
67.8 ± 3.1 a
1
4.1
5.8 ± 0.06 b
0.20 ± 0.05 a
69.2 ± 3.6 a
10
4.4
5.6 ± 0.04 b
0.20 ± 0.02 a
73.4 ± 4.2 b
20
4.8
5.1 ± 0.08 a
0.23 ± 0.03 b
78.6 ± 5.7 bc
30
5.1
4.9 ± 0.16 a
0.25 ± 0.02 bc
86.1 ± 6.3 c
45
5.5
4.7 ± 0.14 a
0.28 ± 0.02 c
92.1 ± 6.1 d
Each experiment is the mean of three replicates. Within a column, means followed by same letter(s)
are not significantly different (p ≤ 0.05).
Figure 1.
(
a
) GC–FID chromatogram of cypress EO. Major terpenes are highlighted; (
b
) Major terpene
components of cypress EO.

Sustainability 2023,15, 8021 9 of 20
3.2. Nanoemulsion Characterization
The developed emulsion (droplet size 67.8
±
3.1 nm) showed stability during extreme
conditions of centrifugation, temperature, heating–cooling cycle (4–40
◦
C), and a freezing
cycle at
−
4
◦
C. The optimum conditions of nanoemulsion preparation, droplet size, and
their PDI, are listed in Table 2and illustrated in Figure 2. The SEM revealed that a transpar-
ent nanoemulsion consisting of dispersed and spherical-shaped nanoparticles had been
developed (Figure 3).
Table 2. Characterization of Cupressus sempervirens oil nanoemulsion.
Storage Period (Days) Viscosity (mPa·s) pH PDI Size (nm ±S.E.)
0 4.1 6.1 ±0.04 c0.18 ±0.03 a67.8 ±3.1 a
1 4.1 5.8 ±0.06 b0.20 ±0.05 a69.2 ±3.6 a
10 4.4 5.6 ±0.04 b0.20 ±0.02 a73.4 ±4.2 b
20 4.8 5.1 ±0.08 a0.23 ±0.03 b78.6 ±5.7 bc
30 5.1 4.9 ±0.16 a0.25 ±0.02 bc 86.1 ±6.3 c
45 5.5 4.7 ±0.14 a0.28 ±0.02 c92.1 ±6.1 d
Each experiment is the mean of three replicates. Within a column, means followed by same letter(s) are not
significantly different (p≤0.05).
Sustainability 2023, 15, x FOR PEER REVIEW 10 of 21
Figure 2. Particle size of nanoemulsion from cypress oil after: (a) 0 day, (b) 1 day, (c) 10 days, (d) 20
days, (e) 30 days, and (f) 45 days.
Figure 3. SEM of cypress oil nanoemulsion.
3.3. Contact Bioactivity
Contact bioactivity of EO materials was both dose- and time-dependent (Table 3).
The oil/water nanoemulsion caused the strongest activity, with which 100% adult mor-
tality of S. oryzae was reached at 10.0 µL/cm2 at 4 days’ following exposure. Under these
conditions, the percentage mortality was 71.1, 60.3, 37.1, and 33.6% for EO, α-cedrol,
δ-3-carene and α-pinene, respectively. After 7 days from exposing the weevils to 40
µL/cm2 of botanicals, the mortality of the adults ranged between 77.5 and 100%.
Table 3. Contact insecticidal bioactivity of S. oryzae exposed to essential oil, nanoemul-
sion, and terpene fractions of C. sempervirens.
Test Material
Concentration
(µL/cm2)
Mortality (% mean ± S.E.) after Exposure Period
Day1
Day 2
Day 4
Day 7
Crude oil
5.0
18.1 ± 2.3 ghi
25.3 ± 1.1 fg
44.3 ± 2.6 g
70.3 ± 2.1 d
10.0
27.6 ± 2.1 f
46.6 ± 2.3 e
71.1 ± 2.3 d
84.1 ± 2.2 c
20.0
39.9 ± 2.1 e
63.6 ± 3.1 d
88.0 ± 3.2 b
100.0 ± 0.0 a
Figure 2.
Particle size of nanoemulsion from cypress oil after: (
a
) 0 day, (
b
) 1 day, (
c
) 10 days,
(d) 20 days, (e) 30 days, and (f) 45 days.
Sustainability 2023, 15, x FOR PEER REVIEW 10 of 21
Figure 2. Particle size of nanoemulsion from cypress oil after: (a) 0 day, (b) 1 day, (c) 10 days, (d) 20
days, (e) 30 days, and (f) 45 days.
Figure 3. SEM of cypress oil nanoemulsion.
3.3. Contact Bioactivity
Contact bioactivity of EO materials was both dose- and time-dependent (Table 3).
The oil/water nanoemulsion caused the strongest activity, with which 100% adult mor-
tality of S. oryzae was reached at 10.0 µL/cm2 at 4 days’ following exposure. Under these
conditions, the percentage mortality was 71.1, 60.3, 37.1, and 33.6% for EO, α-cedrol,
δ-3-carene and α-pinene, respectively. After 7 days from exposing the weevils to 40
µL/cm2 of botanicals, the mortality of the adults ranged between 77.5 and 100%.
Table 3. Contact insecticidal bioactivity of S. oryzae exposed to essential oil, nanoemul-
sion, and terpene fractions of C. sempervirens.
Test Material
Concentration
(µL/cm2)
Mortality (% mean ± S.E.) after Exposure Period
Day1
Day 2
Day 4
Day 7
Crude oil
5.0
18.1 ± 2.3 ghi
25.3 ± 1.1 fg
44.3 ± 2.6 g
70.3 ± 2.1 d
10.0
27.6 ± 2.1 f
46.6 ± 2.3 e
71.1 ± 2.3 d
84.1 ± 2.2 c
20.0
39.9 ± 2.1 e
63.6 ± 3.1 d
88.0 ± 3.2 b
100.0 ± 0.0 a
Figure 3. SEM of cypress oil nanoemulsion.

Sustainability 2023,15, 8021 10 of 20
3.3. Contact Bioactivity
Contact bioactivity of EO materials was both dose- and time-dependent (Table 3). The
oil/water nanoemulsion caused the strongest activity, with which 100% adult mortality of
S. oryzae was reached at 10.0
µ
L/cm
2
at 4 days’ following exposure. Under these conditions,
the percentage mortality was 71.1, 60.3, 37.1, and 33.6% for EO,
α
-cedrol,
δ
-3-carene and
α
-pinene, respectively. After 7 days from exposing the weevils to 40
µ
L/cm
2
of botanicals,
the mortality of the adults ranged between 77.5 and 100%.
Table 3.
Contact insecticidal bioactivity of S. oryzae exposed to essential oil, nanoemulsion, and
terpene fractions of C. sempervirens.
Test Material Concentration
(µL/cm2)
Mortality (% Mean ±S.E.) after Exposure Period
Day1 Day 2 Day 4 Day 7
Crude oil 5.0 18.1 ±2.3 ghi 25.3 ±1.1 fg 44.3 ±2.6 g70.3 ±2.1 d
10.0 27.6 ±2.1 f46.6 ±2.3 e71.1 ±2.3 d84.1 ±2.2 c
20.0 39.9 ±2.1 e63.6 ±3.1 d88.0 ±3.2 b100.0 ±0.0 a
40.0 71.3 ±3.0 b93.8 ±2.6 a100.0 ±0.0 a100.0 ±0.0 a
Nanoemulsion 5.0 36.3 ±3.1 e48.1 ±2.1 e83.6 ±3.1 b c92.4 ±2.1 b b
10.0 54.4 ±3.1 d70.9 ±2.1 c100.0 ±0.0 a100.0 ±0.0 a
20.0 67.8 ±2.6 bc 81.3 ±1.9 b100.0 ±0.0 a100.0 ±0.0 a
40.0 92.1 ±3.3 a100.0 ±0.0 a100.0 ±0.0 a100.0 ±0.0 a
α-Cedrol 5.0 15.8 ±1.1 ijk 29.3 ±2.1 f36.1 ±2.1 h44.8 ±2.1 h
10.0 25.5 ±2.1 fg 41.9 ±2.3 e60.0 ±2.1 e71.0 ±2.1 d
20.0 36.0 ±2.3 e60.8 ±2.6 d70.9 ±1.9 d83.3 ±1.7 c
40.0 61.3 ±3.1 cd 83.1 ±3.1 b100.0 ±0.0 a100.0 ±0.0 a
δ-3-Carene 5.0 11.3 ±2.0 jk 16.6 ±2.1 h22.6 ±3.1 i29.3 ±4.2 j
10.0 19.0 ±3.0 ghi 30.3 ±1.9 f37.1 ±2.1 h46.7 ±2.4 h
20.0 27.3 ±2.3 f41.9 ±1.6 e54.3 ±2.6 ef 65.9 ±2.0 f
40.0 38.2 ±3.3 e60.4 ±3.1 d77.3 ±1.9 cd 84.3 ±1.9 c
α-Pinene 5.0 8.3 ±1.1 k14.3 ±1.1 h19.4 ±1.3 i25.4 ±2.1 k
10.0 13.1 ±1.1 jk 20.8 ±1.3 gh 33.6 ±1.3 h42.6 ±1.8 hi
20.0 19.6 ±1.6 ghi 28.5 ±2.3 f51.3 ±1.9 f56.3 ±1.7 g
40.0 23.0 ±2.3 fgh 44.0 ±2.9 e70.5 ±2.3 d77.5 ±2.0 e
*F-value - 167.90 278.00 323.96 436.45
Each result is the mean of 6 replicates, each made with 20 adults (n= 120). Means within a column followed by
same letters are not significantly different (p
≤
0.05) (Tukey’s HSD test). * All F-values are significant, at p
≤
0.001.
3.4. Fumigation Bioactivity
Results of fumigation bioactivity (Table 4) tests demonstrated that the nanoemulsion
and the crude oil exhibited the strongest fumigant bioactivity (10
µ
L/L air of both botanicals
caused 100% adult mortality after 4 days). At these conditions, (%) mortality was 68.6, 53.3,
and 51.6% for
α
-cedrol,
α
-pinene, and
δ
-3-carene, respectively. After 4 days of exposing
weevils to 20
µ
L/L air,
α
-cedrol caused 100% adult mortality. After 7 days of exposing
insects to 20
µ
L/L air of botanicals, the mortality of adult weevils ranged between 81.5
and 100%.
Table 4.
Fumigant insecticidal bioactivity of S. oryzae exposed to essential oil, nanoemulsion, and
terpene fractions of C. sempervirens.
Test Material Concentration
(µL/L Air)
Mortality (% Mean ±S.E.) after Exposure Period
Day1 Day 2 Day 4 Day 7
Crude oil 2.5 15.6 ±1.3 ijk 23.1 ±1.6 fg 39.3 ±2.4 e53.8 ±2.1 h
5.0 24.1 ±1.3 hi 36.0 ±2.3 e63.6 ±3.2 c72.7 ±2.4 e
10.0 33.6 ±2.1 efg 55.3 ±3.6 cd 100.0 ±0.0 a100.0 ±0.0 a
20.0 55.3 ±2.3 c94.6 ±2.2 a100.0 ±0.0 a100.0 ±0.0 a

Sustainability 2023,15, 8021 11 of 20
Table 4. Cont.
Test Material Concentration
(µL/L Air)
Mortality (% Mean ±S.E.) after Exposure Period
Day1 Day 2 Day 4 Day 7
Nanoemulsion 2.5 27.5 ±1.1 fgh 36.1 ±2.3 e66.6 ±2.1 c74.1 ±2.4 e
5.0 49.9 ±2.3 cd 60.3 ±3.6 c92.1 ±2.1 a100.0 ±0.0 a
10.0 68.4 ±3.1 b94.3 ±3.0 a100.0 ±0.0 a100.0 ±0.0 a
20.0 91.1 ±3.3 a100.0 ±0.0 a100.0 ±0.0 a100.0 ±0.0 a
α-Cedrol 2.5 13.3 ±1.6 jk 20.6 ±3.3 fg 34.3 ±4.5 ef 49.5 ±3.4 i
5.0 20.3 ±2.1 hij 32.3 ±3.3 e49.3 ±4.5 d58.9 ±2.7 g
10.0 34.8 ±2.1 ef 47.9 ±3.6 d68.6 ±3.3 c76.1 ±2.3 e
20.0 46.4 ±2.3 d70.3 ±4.1 b100.0 ±0.0 a100.0 ±0.0 a
δ-3-Carene 2.5 9.3 ±1.3 k15.3 ±3.3 fg 30.3 ±4.5 fg 40.8 ±3.4 j
5.0 14.3 ±2.3 jk 23.0 ±3.3 f40.0 ±4.5 e52.5 ±2.8 h
10.0 25.0 ±2.0 gh 37.3 ±3.6 e53.3 ±3.3 d64.0 ±3.2 f
20.0 37.3 ±2.9 e56.3 ±4.1 c77.0 ±3.9 b89.9 ±2.1 c
α-Pinene 2.5 8.9 ±1.1 k12.9 ±3.3 g26.0 ±3.2 g33.0 ±3.3 g
5.0 12.1 ±1.1 jk 19.1 ±1.8 fg 36.3 ±4.5 ef 47.4 ±3.6 ef
10.0 19.6 ±2.1 hij 30.9 ±1.5 e51.6 ±3.1 d59.7±3.1 g
20.0 33.1 ±2.3 efg 53.3 ±2.3 cd 70.1 ±2.9 c81.2 ±2.6 d
* F-value - 134.04 265.03 317.22 302.08
Each result is the mean of 6 replicates, each made with 20 adults (n= 120). Means within a column followed by
same letters are not significantly different (p
≤
0.05) (Tukey’s HSD test). * All F-values are significant, at p
≤
0.00.
3.5. The Dose-Response Mortality
The LC
50
and LC
90
and their confidence limits are illustrated in (Table 5). For the
contact bioassay, LC
50
values of the botanicals after 48 h of treatment were: Nanoemulsion
(LC
50
= 5.8
µ
L/cm
2
,
χ2
= 0.94, df = 4), crude oil (LC
50
= 13.3
µ
L/cm
2
,
χ2
= 1.33,
df = 4
),
α-cedrol
(LC
50
= 15.1
µ
L/cm
2
,
χ2
= 2.04, df = 4),
δ
-3-carene (LC
50
= 30.7
µ
L/cm
2
,
χ2= 2.77
,
df = 4
), and
α
-pinene (LC
50
= 53.4
µ
L/cm
2
,
χ2
= 3.12, df = 4). The LC
500s
of the phytochemi-
cals after 48 h fumigation were as follows: Nanoemulsion (LC
50
= 4.1
µ
L/L air,
χ2= 0.91
,
df = 54
), crude oil (LC
50
= 8.7
µ
L/L air,
χ2
= 1.08, df = 4),
α
-cedrol (LC
50
= 12.2
µ
L/L
air,
χ2= 2.16
,
df = 4
),
δ
-3-carene (LC
50
= 17.2
µ
L/L air,
χ2
= 3.06, df = 54), and
α
-pinene
(LC50 = 19.6 µL/L air, χ2= 3.22, df = 4).
Table 5.
* LC
50
and LC
95
and their fiducial limits of EO materials against S. oryzae 48 h post treatment.
Test Material Bioassay LC50
** (95% fl)
LC95
** (95% fl)
Slope
(±S.E.)
*** χ2
(df = 4)
Crude oil Contact (µL/cm2)13.3 (11.1–16.3) 25.9 (19.3–32.2) 2.1 ±0.20 1.33
Fumigation (
µ
L/L)
8.7 (7.5–10.1) 16.3 (13.8–21.3) 2.0 ±0.24 1.08
Nanoemulsion Contact (µL/cm2)5.8 (5.3–7.2) 10.2 (8.6–13.3) 1.5 ±0.18 0.94
Fumigation (
µ
L/L)
4.1 (3.7–4.9) 7.3 (6.1–8.6) 1.6 ±0.14 0.91
α-Cedrol Contact (µL/cm2)15.1 (13.4–18.9) 27.5 (22.9–35.3) 2.1 ±0.28 2.04
Fumigation (
µ
L/L)
12.2 (10.5–15.8) 22.9 (18.6–27.1) 2.6 ±0.26 2.18
δ-3-Carene Contact (µL/cm2)30.7 (27.6–36.6) 55.3 (48.4–64.8) 2.9 ±0.32 2.77
Fumigation (
µ
L/L)
17.2 (15.4–21.3) 39.6 (34.7–47.6) 2.8 ±0.40 3.06
α-Pinene Contact (µL/cm2)53.4 (46.3–63.1) 114.8 (101.8–119.1) 3.1 ±0.30 3.12
Fumigation (
µ
L/L)
19.6 (17.3–24.5) 42.2 (37.0–50.3) 2.7 ±0.41 3.22
Each result is the mean of 6 replicates, each including 20 individuals (n= 120). * LC
50
and LC
95
are considered
significantly different when the 95% fiducial limits (f.l.) fail to overlap. ** fl = fiducial limits. *** Chi-square value,
significant at p≤0.05 level; df = degree of freedom.
3.6. Repellence Bioactivity
As illustrated in Table 6and Figure 4, the EO materials strongly repelled the adult
weevils, and the repellent bioactivity was both time- and dose-dependent. The crude oil of
C. sempervirens was the strongest insect repellent, even at low concentrations, followed by

Sustainability 2023,15, 8021 12 of 20
nanoemulsion,
α
-cedrol, and
δ
-3-carene; by contrast, the monoterpene
α
-pinene showed a
weak-to-moderate repelling efficacy. The crude oil completely repelled the adult weevils at
0.44
µ
L/cm
2
after 12 h. The crude oil, nanoemulsion, and
α
-cedrol caused 100% repellency
when the weevils were treated with a concentration equaling 0.88
µ
L/cm
2
of these products
after 24 h. At this concentration, the remaining monoterpenes caused moderate repelling
activities. At the lowest concentration tested (0.22
µ
L/cm
2
), the percentage repellency was
73.9, 61.3, 43.9, 33.3, and 22.1% for the EO, the nanoemulsion,
α
-cedrol,
δ
-3-carene, and
α-pinene, respectively.
Sustainability 2023, 15, x FOR PEER REVIEW 13 of 21
Table 6. Repellence activity of EO, nanoemulsion, and terpenes of C. sempervirens against S. oryzae
adult weevils.
Test Material
Concentration
(µL/cm2)
Repellency (% mean ± S.E.) after Period (h)
2
6
12
24
Crude oil
0.11
29.6 ± 1.1 gh
51.3 ± 2.3 efg
62.3 ± 3.1 f
73.9 ± 2.6 e
0.22
44.9 ± 2.3 e
72.9 ± 2.3 bcd
80.1 ± 2.9 c
100.0 ± 0.0 a
0.44
63.3 ± 3.1 b
80.6 ± 1.9 abc
100.0 ± 0.0 a
100.0 ± 0.0 a
0.88
82.3 ± 3.1 a
91.1 ± 2.6 a
100.0 ± 0.0 a
100.0 ± 0.0 a
Nanoemulsion
0.11
20.1 ± 1.3 j
37.1 ± 1.3 gh
46.1 ± 2.3 h
61.3 ± 2.1 h
0.22
29.3 ± 1.6 gh
53.3 ± 2.1 ef
68.6 ± 2.6 e
80.1 ± 2.2 d
0.44
44.6 ± 2.1 e
66.1 ± 3.6 cde
80.9 ± 3.3 c
89.6± 0.0 b
0.88
59.3 ± 2.6 c
83.9 ± 2.6 ab
90.3 ± 3.1 b
100.0 ± 0.0 a
𝛼-Cedrol
0.11
15.6 ± 1.3 k
28.6 ± 2.1 hi
37.3 ± 2.3 i
43.9 ± 2.3 i
0.22
23.6 ± 2.1 i
44.6 ± 2.3 fg
60.3 ± 2.1 f
70.1 ± 2.1 f
0.44
37.9 ± 2.1 f
58.9 ± 2.6 def
72.9 ± 1.9 d
83.3 ± 1.6 c
0.88
48.3 ± 2.3 d
71.3 ± 3.1 bcd
83.6 ± 2.1 c
100.0 ± 0.0 a
δ-3-Carene
0.11
9.6 ± 1.3 lm
17.9 ± 1.9 ij
28.6 ± 3.1 k
33.3 ± 3.1 k
0.22
15.3 ± 2.1 k
27.6 ± 1.6 hij
36.1 ± 2.1 i
41.3 ± 2.6 j
0.44
27.6 ± 2.1 h
43.9 ± 2.6 fg
51.3 ± 2.6 g
60.9 ± 2.3 h
0.88
39.1 ± 2.3 f
51.3 ± 3.3 efg
62.3 ± 1.9 f
66.1 ± 1.9 g
α-Pinene
0.11
7.9 ± 1.1 m
12.6 ± 1.1 k
20.9 ± 1.6 m
22.1 ± 2.3 m
0.22
11.3 ± 1.3 l
16.1 ± 1.3 ij
24.6 ± 1.9 l
30.6 ± 1.9 l
0.44
18.9 ± 1.6 j
23.9 ± 2.3 hij
32.3 ± 1.9 j
39.3 ± 2.6 j
0.88
31.1 ± 2.1 g
37.9 ± 2.9 gh
44.1 ± 2.1 h
46.1 ± 2.3 i
Control
-
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
* F-value
-
961.75
61.65
1237.03
2644.87
Each result is the mean of 5 repeats, each including 20 individuals (n = 100). Means within a column
followed by same letter(s) are not significantly different. (p ≤ 0.05) (Tukey’s HSD test). * All
F-values are significant at p ≤ 0.001.
Figure 4.
Repellent activity of cypress EO products against S. oryzae at (
a
) 0.11
µ
L/cm
2
;
(b) 0.22 µL/cm2; (c) 0.44 µL/cm2; and (d) 0.88 µL/cm2. (%) (Repellency in control was nil).
Table 6.
Repellence activity of EO, nanoemulsion, and terpenes of C. sempervirens against S. oryzae
adult weevils.
Test Material Concentration
(µL/cm2)
Repellency (% Mean ±S.E.) after Period (h)
2 6 12 24
Crude oil 0.11 29.6 ±1.1 gh 51.3 ±2.3 efg 62.3 ±3.1 f73.9 ±2.6 e
0.22 44.9 ±2.3 e72.9 ±2.3 bcd 80.1 ±2.9 c100.0 ±0.0 a
0.44 63.3 ±3.1 b80.6 ±1.9 abc 100.0 ±0.0 a100.0 ±0.0 a
0.88 82.3 ±3.1 a91.1 ±2.6 a100.0 ±0.0 a100.0 ±0.0 a
Nanoemulsion 0.11 20.1 ±1.3 j37.1 ±1.3 gh 46.1 ±2.3 h61.3 ±2.1 h
0.22 29.3 ±1.6 gh 53.3 ±2.1 ef 68.6 ±2.6 e80.1 ±2.2 d
0.44 44.6 ±2.1 e66.1 ±3.6 cde 80.9 ±3.3 c89.6±0.0 b
0.88 59.3 ±2.6 c83.9 ±2.6 ab 90.3 ±3.1 b100.0 ±0.0 a
α-Cedrol 0.11 15.6 ±1.3 k28.6 ±2.1 hi 37.3 ±2.3 i43.9 ±2.3 i
0.22 23.6 ±2.1 i44.6 ±2.3 fg 60.3 ±2.1 f70.1 ±2.1 f
0.44 37.9 ±2.1 f58.9 ±2.6 def 72.9 ±1.9 d83.3 ±1.6 c
0.88 48.3 ±2.3 d71.3 ±3.1 bcd 83.6 ±2.1 c100.0 ±0.0 a

Sustainability 2023,15, 8021 13 of 20
Table 6. Cont.
Test Material Concentration
(µL/cm2)
Repellency (% Mean ±S.E.) after Period (h)
2 6 12 24
δ-3-Carene 0.11 9.6 ±1.3 lm 17.9 ±1.9 ij 28.6 ±3.1 k33.3 ±3.1 k
0.22 15.3 ±2.1 k27.6 ±1.6 hij 36.1 ±2.1 i41.3 ±2.6 j
0.44 27.6 ±2.1 h43.9 ±2.6 fg 51.3 ±2.6 g60.9 ±2.3 h
0.88 39.1 ±2.3 f51.3 ±3.3 efg 62.3 ±1.9 f66.1 ±1.9 g
α-Pinene 0.11 7.9 ±1.1 m12.6 ±1.1 k20.9 ±1.6 m22.1 ±2.3 m
0.22 11.3 ±1.3 l16.1 ±1.3 ij 24.6 ±1.9 l30.6 ±1.9 l
0.44 18.9 ±1.6 j23.9 ±2.3 hij 32.3 ±1.9 j39.3 ±2.6 j
0.88 31.1 ±2.1 g37.9 ±2.9 gh 44.1 ±2.1 h46.1 ±2.3 i
Control - 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0
*F-value - 961.75 61.65 1237.03 2644.87
Each result is the mean of 5 repeats, each including 20 individuals (n= 100). Means within a column followed
by same letter(s) are not significantly different. (p
≤
0.05) (Tukey’s HSD test). * All F-values are significant at
p≤0.001.
3.7. AChE Inhibition
All test EO materials caused a remarkable inhibition of AChE activity in S. oryzae
(Table 7). The nanoemulsion (IC
50
= 9.88 mM,
χ2
= 1.68, df = 5, p= 0.201) was the superior
AChE inhibitor, followed by EO (IC
50
= 14.03 mM,
χ2
= 2.41, df = 5, p= 0.243), and
α
-cedrol
(IC
50
= 17.21 mM,
χ2
= 2.88, df = 5, p= 0.311). By contrast,
δ
-3-carene and
α
-pinene caused
moderate effects. The IC50 of methomyl was 2.44 ×10−3mM.
Table 7.
Inhibition of acetylcholinesterase (AChE) of S. oryzae larvae by EO materials of
C. sempervirens.
Plant Material * IC50 (mM) (95% Fiducial Limits) Slope (±S.E.) ** χ2(df = 5) p
Crude oil 14.03 (12.21–16.28) 1.44 ±0.22 2.41 0.243
Nanoemulsion 9.88 (7.94–11.71) 1.09±0.14 2.68 0.201
α-Cedrol 17.21 (15.20–20.07) 1.30 ±0.19 2.88 0.311
δ-3-Carene 34.54 (30.00–39.33) 1.66 ±0.25 3.82 0.355
α-Pinene 39.83 (34.44–46.12) 2.08 ±0.34 4.05 0.512
Methomyl 2.17 ×10−3(1.73 ×10−3–3.66 ×10−3)1.03 ±0.18 2.62 0.377
* The concentration causing 50% enzyme inhibition. ** Chi-square value, not significant at p
≤
0.05 level;
df = degree of freedom.
3.8. Phytotoxicity Assessment
Phytotoxicity testing revealed that the botanicals were not phytotoxic to wheat plants,
where the agronomical parameters of wheat (%) germination, and the growth of radicals and
shoots were unaffected after treatment with botanicals at concentrations ranging between
50.0 and 150
µ
L/mL (Table 8and Figure 5). Percentage germination and growth of shoots are
slightly affected at 150
µ
L/mL, especially with EO, nanoemulsion, and
α-cedrol
. By contrast,
the remaining compounds were nonphytotoxic, even at high test concentrations.
Table 8.
* Phytotoxic activities of essential oil, nanoemulsion, and major fractions of C. sempervirens
against wheat plants.
Plant Material Concentration (µL/mL) Germination (%) RL SL
Crude oil 50 90.6 ±1.4 a9.08 ±0.31 a3.32 ±0.14 ab
100 80.3 ±1.5 ab 8.89 ±0.23 a3.12 ±0.11 ab
150 70.1 ±1.3 b8.07 ±0.18 a2.26 ±0.15 c

Sustainability 2023,15, 8021 14 of 20
Table 8. Cont.
Plant Material Concentration (µL/mL) Germination (%) RL SL
Nanoemulsion 50 88.9 ±1.3 a9.03 ±0.19 a3.16 ±0.12 ab
100 76.3 ±1.2 ab 8.70 ±0.20 a3.03 ±0.14 ab
150 65.2 ±1.2 bc 7.08 ±0.28 a2.05 ±0.12 c
α-Cedrol 50 88.2 ±1.3 a9.11 ±0.41 a3.28 ±0.15 ab
100 83.4 ±1.9 ab 9.02 ±0.26 a3.20 ±0.13 ab
150 74.2 ±1.4 b8.24 ±0.12 a2.59 ±0.17 bc
δ-3-Carene 50 90.6 ±1.7 a9.20 ±0.18 a3.39 ±0.16 a
100 88.7 ±1.4 a9.12 ±0.31 a3.34 ±0.11 a
150 88.3 ±1.9 a9.01 ±0.22 a3.09 ±0.11 ab
α-Pinene 50 91.8 ±1.1 a9.24 ±0.19 a3.41 ±0.13 a
100 91.0 ±1.3 a9.23 ±0.08 a3.31 ±0.11 ab
150 90.2 ±1.3 a9.19 ±0.22 a3.16 ±0.12 ab
Control - 91.7 ±1.6 a9.22 ±0.32 a3.43 ±0.18 a
F-value - 4.28 1.16 7.94
* Each value is the mean
±
S.E. of 4 trials; RL = Radicle growth (length of seeds, cm); SL = Shoot length (cm). In a
column, means followed by same letter (s) are not significantly different (p
≤
0.05). All F-values are significant at
p≤0.001.
Sustainability 2023, 15, x FOR PEER REVIEW 15 of 21
* Each value is the mean ± S.E. of 4 trials; RL = Radicle growth (length of seeds, cm); SL = Shoot
length (cm). In a column, means followed by same letter (s) are not significantly different (p ≤ 0.05).
All F-values are significant at p ≤ 0.001.
Figure 5. Viability of wheat grains as (%) germination treated with cypress EO products at (a) 50
µL/mL; (b) 100 µL/mL; and (c) 150 µL/mL.
3.9. Toxicity against Earthworms
The test botanicals showed relative safety toward E. fetida. Neither mortality nor
toxicity signs were recorded in treated animals, even at 200 mg per kg−1 soil. On the oth-
er hand, the chemical pesticide α-cypermethrin at 20.0 mg per kg−1 caused 100% mortal-
ity of earthworms after 10 days.
4. Discussion
The yield and composition of C. sempervirens var. horizontalis oil are in a good ac-
cordance with previous studies of the same Saudi species or those of other similar flora,
where α-pinene was the main component of cypress EO [21,39–42]. Variations both in the
yield and the abundant oil terpenes of plant oils have been recorded in previous reports
[22,43–45]. However, β-thujene was presented as the main terpene component (31.4%) in
the EO of Brazilian C. sempervirens [43]. These variations are mainly dependent on many
factors, including genetic and geographic factors. The soil status, the method of cultiva-
tion, water availability, seasonality, the extracted parts, and extraction techniques are
also of major influence [6,46–48].
According to our findings, the EO of cypress, its nanoemulsion, and individual ter-
penes exhibited remarkable insecticidal, repellent, and AChE effects against S. oryzae. To
our knowledge, bioactivity of the EO of cypress belonging to Saudi flora, particularly its
nanoemulsion and individual terpenes, had not been investigated against insects of
stored grain; hence our study is considered a first report. A remarkable fumigation bio-
0
10
20
30
40
50
60
70
80
90
100
Crude oil
Nanoemulsion
α-cedrol
δ-3-carene
α-pinene
Control
Crude oil
Nanoemulsion
α-cedrol
δ-3-carene
α-pinene
Control
Crude oil
Nanoemulsion
α-cedrol
δ-3-carene
α-pinene
Control
Germination (%)
(a)
(b)
(c)
Figure 5.
Viability of wheat grains as (%) germination treated with cypress EO products at
(a) 50 µL/mL; (b) 100 µL/mL; and (c) 150 µL/mL.
3.9. Toxicity against Earthworms
The test botanicals showed relative safety toward E. fetida. Neither mortality nor
toxicity signs were recorded in treated animals, even at 200 mg per kg
−1
soil. On the other
hand, the chemical pesticide
α
-cypermethrin at 20.0 mg per kg
−1
caused 100% mortality of
earthworms after 10 days.

Sustainability 2023,15, 8021 15 of 20
4. Discussion
The yield and composition of C. sempervirens var. horizontalis oil are in a good ac-
cordance with previous studies of the same Saudi species or those of other similar flora,
where
α
-pinene was the main component of cypress EO [
21
,
39
–
42
]. Variations both in
the yield and the abundant oil terpenes of plant oils have been recorded in previous re-
ports
[22,43–45]
. However,
β
-thujene was presented as the main terpene component (31.4%)
in the EO of Brazilian C.sempervirens [
43
]. These variations are mainly dependent on many
factors, including genetic and geographic factors. The soil status, the method of cultivation,
water availability, seasonality, the extracted parts, and extraction techniques are also of
major influence [6,46–48].
According to our findings, the EO of cypress, its nanoemulsion, and individual ter-
penes exhibited remarkable insecticidal, repellent, and AChE effects against S. oryzae. To
our knowledge, bioactivity of the EO of cypress belonging to Saudi flora, particularly its
nanoemulsion and individual terpenes, had not been investigated against insects of stored
grain; hence our study is considered a first report. A remarkable fumigation bioactivity
of the EO extracted from Egyptian cypress was recorded against adults of S. oryzae with
LC50 = 17.2 mg/L air.
The EO of C. sempervirens was reported to possess toxic and repellent bioactivities
against Sitophilus zeamais and Tribolium confusum using the impregnated filter paper bioassay,
as well as treated grains [
22
]; a remarkable repellent potential against the codling moth,
Cydia pomonella; and moderate toxicity and repellent activities against the mosquitoes, Aedes
albopictus and Ae. aegypti [
49
–
51
]. The oil materials tested herein strongly repelled
S. oryzae
adult weevils. There are many factors affecting the repellent bioactivity of the plant-based
products against harmful insects, which depend mainly on the nature of products under
investigation; the respiratory system upon which the plant bioactive substances act, and
the insect’s olfactory receptors are of a major influence [
52
]. The high volatile nature
of EOs play a main role in this phenomenon, where they can be inhaled, ingested, or
easily absorbed through the insect’s skin [
53
,
54
]. The repellence bioactivity of cypress EO
products toward S. oryzae was in accordance with studies and reports that investigated the
bioinsecticidal and repellant potential of plant EOs against insects of stored grain, including
S. oryzae [10,11,55–57].
In this study, a green approach was followed to prepare an oil-in-water nanoemulsion
(droplet size 67.8
±
3.1 nm) from cypress oil using fewer toxic chemicals at acceptable
concentrations, in the proportions 5:90:5% (EO:H
2
O:Tween 80 as an emulsifier). Tween
surfactants, especially Tween 80 and Tween 20, are frequently utilized as emulsifiers in
the preparations of oil/water nanoemulsions as they can produce stable formulations
without using cosurfactants [
58
]; However, Tween 80 was selected herein as a nonanionic
emulsifier, due to its miscibility with water and good solubility for EOs. It is characterized
by a high hydrophilic–lipophilic balance (HLB = 15); hence, decreasing the tension between
the oil and aqueous phases, and resulting in the formation of stable emulsions [
17
]. In
most cases, Tween 80 also appeared to perform better than Tween 20 in terms of droplet
size distribution and the stability of the nanoemulsion, which may be due to the structural
differences in the nonpolar tail of the two molecules [
59
,
60
]. In both micro- and nanoemul-
sion preparations, the surfactant functions to reduce the interfacial energy by providing a
mechanical barrier to coalescence [
17
]. The nanoemulsion of cypress oil exhibited a good
stability up to 45 days after preparation when exposed to stress conditions during storage.
Meanwhile, nonequilibrium emulsion formulations may undergo a breakdown, resulting
in sedimentation, flocculation, and coalescence, resulting in many shortcomings in their
biological activities. Alternatively, because of their novel properties, such as subcellular
size, nanoemulsions have good stability under extreme conditions [
17
,
61
]. The pH of the
nanoemulsion stabilized around 6.5 during storage. The pH of an emulsion is critical to
its stability because changes in pH affect the surface charge of the globules, disrupting
their stability. Furthermore, increases in the surface charge of globules cause electrostatic
repulsion, which reduces flocculation and leads to the dissolution of micro- and nanoemul-

Sustainability 2023,15, 8021 16 of 20
sions [
17
]. In a nanoemulsion formulation, the PDI determines droplet size stability and
uniformity; a low PDI ensures high droplet size uniformity. Over 30 days of storage, the
PDI of cypress oil nanoemulsion ranged between 0.18
±
0.03 and 0.24
±
0.02. Many authors
have reported that a PDI of less than 0.25 indicates a narrow distribution of particle size,
providing stability and homogeneity due to a reduced Ostwald ripening [17,61].
The nanoemulsion of cypress oil exhibited superior bioactivity against the target
weevil. When materials are formulated at the nanoscale, they acquire novel chemical
and physical properties, such as increased surface area, solubility, and high affinity to the
targeted biosystems, which promotes their biological activities [
17
]. Because of these novel
criteria, nanomaterials are promising candidates for developing effective eco-friendly insec-
ticides. To avoid the overuse of the toxic solvents or high-energy inputs that are commonly
used in pesticide synthesis, the “green synthesis” concept has been proposed, outlining the
potential use of animal, microbial, and plant-borne compounds as stabilizing agents for the
production of bioactive nanomaterials [
15
,
62
]. As a result, nanotechnology is being consid-
ered as an alternative strategy to improve the stability and bioactivity of pesticide materials
that rely on various nanocarriers, such as plant-oil-based nanoemulsions [
6
,
15
,
17
,
63
]. In
the literature, the reported insecticidal activity of plant-based nanopesticides, including
oil nanoemulsions against serious insects, such as those infesting stored grains, has been
reported [
6
,
15
,
16
,
18
]. Nenaah reported that nanoemulsions made from the EOs of three
Achillea species, A. biebersteinii, A. santolina, and A. millefolium, outperformed their bulk
counterparts in adulticidal activity against T. castaneum [
6
]. Similar results have been
reported for T. confusum and Cryptolestes ferrugineus [18,63].
The bioactivity of plant oils are attributed to several components, with demonstrable
insecticidal activity contained in the plant EO, especially in monoterpenes such as
α
-
pinene,
α
-terpinene, limonene, camphor, carvacrol, thymol,
δ
-3-carene,
α
-thujone, 1,8-cineol
(eucalyptol), eugenol, and ascaridole [
3
,
6
,
9
–
11
,
17
,
57
]. Although a synergism with other
minor constituents is common where each oil component participates in penetration,
fixation, and distribution into biomembranes [
6
,
9
,
17
], synergy between components of an
EO might be occur between several components contained in the same oil, or between
different essential oils with known biological activities [17,64,65].
Essential oils, particularly monoterpenes, are volatile and lipophilic, allowing them
to quickly penetrate the integument of insects, interfering with physiological parameters
and causing alteration in all vital functions. [
6
,
9
,
17
,
53
]. The EO materials caused a consid-
erable inhibition in the AChE bioactivity of S. oryzae, indicating a neurotoxic mechanism
of action. As mixtures, the toxicity of EOs is not yet fully understood. Nevertheless, the
rapid action against some pests is major evidence of a neurotoxic action, which is attributed
to AChE inhibition, as described herein [
9
–
12
,
17
]. Comparing our results with previous
reports, the dichloromethane, acetone, ethyl acetate, and methanol extracts of the cones and
leaves of Cupressus sempervirens var. horizantalis displayed a moderate inhibition of butyryl-
cholinesterase, AChE, and tyrosinase bioactivities at 200
µ
g/mL [
66
]. Aazza reported the
acetylcholinesterase inhibitory effect of cypress oil (where IC
50
was 0.2837 mg/mL) using
bovine acetylcholine [
67
]. Recently, Alimi et al. found that the EO of cypress displayed a sig-
nificant inhibition in AChE activity of Hyalomma scupense (Acari: Ixodidae) [
68
]. The plant
EOs can interfere with other protein targets, which disrupts the insect’s nervous system,
such as the nicotinic acetylcholine receptors (nAChR), and the octopamine or the neuro-
transmitter inhibitor
γ
-aminobutyric acid (GABA). EOs were found to inhibit enzymatic
biosystems (superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST),
and glutathione reductase (GR)), peroxidases (POx), and the nonenzymatic (glutathione
(GSH)) antioxidant defense biosystems [9,11].
According to our findings, the EO materials showed a relative safety within the limit
of the test concentrations when tested on E. fetida (a common earthworm) and wheat
plants. Plant products with pesticidal activities are often wrongly considered safe with
no negative effects on nontargets, including humans, without this being experimentally
verified. Nevertheless, many authors stated that EOs, nanoemulsion preparations, and oil

Sustainability 2023,15, 8021 17 of 20
terpenes were relatively safe when assessed against several nontarget species [
3
,
17
,
28
,
69
].
In that regard, most reports have focussed on assessing the acute toxicity, whereas both
subchronic and chronic evaluations have not been fully undertaken [
5
,
17
,
69
–
71
]. Regarding
cypress oil, health risks or side effects following administration of designated therapeutic
dosages are not recorded. Nevertheless, kidney irritation was recorded with the intake
of large doses [
72
]. However, not all natural products are free of risk, therefore deep
investigations are always required to explore the biosafety of the plant-based pesticides
before practical use in stored-product insect control programs. The authors should discuss
the findings and how they can be interpreted in light of previous research and the working
hypotheses. The findings and implications should be discussed in the broadest possible
context. Future research directions may be highlighted as well.
5. Conclusions
According to the results of the present study, cypress EO, the oil nanoemulsion, and its
individual terpenes showed remarkable insecticidal, repellence, and acetylcholinesterase
inhibitory bioactivities against the rice weevil, S. oryzae. There were no significant adverse
effects on the earthworms, nor the agronomical parameters of wheat plant. When properly
prepared, cypress oil, its nanoemulsion, and its main terpenes could be applied as novel
ecofriendly natural pest-control options against S. oryzae, being more appropriate than the
chemical insecticides. However, deep toxicological evaluations should be carried out to
substantiate the relevant concentrations and adverse effects of the test products against
mammals and other nontarget organisms.
Author Contributions:
Conceptualization, G.E.N.; methods, investigation, and validation, G.E.N.,
B.Z.A., A.A.A., D.M.S. and S.A.; analysis, G.E.N.; resources, curation, G.E.N., B.Z.A., S.A., D.M.S. and
A.A.A.; writing original draft, G.E.N.; review and editing, G.E.N. and A.A.A.; supervision, G.E.N. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Institutional Ethics Committee.
Informed Consent Statement: Not applicable.
Data Availability Statement:
Data supporting the conclusions of this article are presented in the
main manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
FAO. The Future of Food and Agriculture—Trends and Challenges; Food and Agriculture Organization of the United Nations: Rome,
Italy, 2017.
2.
Sparks, T.C.; Bryant, R.J. Impact of natural products on discovery of, and innovation in, crop protection compounds. Pest Manag.
Sci. 2021,78, 399–408. [CrossRef] [PubMed]
3.
Prakash, B.; Kumar, A.; Singh, P.; Das, S.; Dubey, N.K. Prospects of plant products in the management of insect pests of food
grains: Current status and future perspectives. In Natural Bioactive Compounds; Sinha, R.P., Häder, D.-P., Eds.; Academic Press:
Cambridge, MA, USA, 2021; pp. 317–335. [CrossRef]
4.
Hossain, F.; Boddupalli, P.M.; Sharma, R.K.; Pradyumn Kumar, P.; Bahadur Singh, B. Evaluation of quality protein maize
genotypes for resistance to stored grain weevil Sitophilus oryzae (Coleoptera: Curculionidae). Int. J. Trop. Insect Sci.
2007
,27,
114–121. [CrossRef]
5.
Desneux, N.; Decourtye, A.; Delpuech, J.M. The sublethal effects of pesticides on beneficial arthropods. Annu. Rev. Entomol.
2007
,
52, 81–106. [CrossRef] [PubMed]
6.
Nenaah, G.E. Chemical composition, toxicity and growth inhibitory activities of essential oils of three Achillea species and their
nano-emulsions against Tribolium castaneum (Herbst). Ind. Crops Prod. 2014,53, 252–260. [CrossRef]
7.
Okpile, C.; Zakka, U.; Nwosu, L.C. Susceptibility of ten rice brands to weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae),
and their influence on the insect and infestation rate. Bull. Natl. Res. Cent. 2021,45, 2. [CrossRef]
8.
Sparks, T.C.; Bryant, R.J. Innovation in insecticide discovery: Approaches to the discovery of new classes of insecticides. Pest
Manag. Sci. 2022,78, 3226–3247. [CrossRef]

Sustainability 2023,15, 8021 18 of 20
9. Isman, M.B. Bioinsecticides based on plant essential oils: A short overview. Z. Naturforsch. 2020,75, 179–182. [CrossRef]
10.
Abdelgaleil, S.A.M.; Mohamed, M.I.E.; Shawir, M.S.; Abou-Taleb, H.K. Chemical composition, insecticidal and biochemical effects
of essential oils of different plant species from Northern Egypt on the rice weevil, Sitophilus oryzae L. J. Pest Sci.
2016
,89, 219–229.
[CrossRef]
11.
Chaudhari, A.K.; Singh, V.K.; Kedia, A.; Das, S.; Dubey, N.K. Essential oils and their bioactive compounds as eco-friendly novel
green pesticides for management of storage insect pests: Prospects and retrospects. Environ. Sci. Pollut. Res.
2021
,28, 18918–18940.
[CrossRef]
12.
Pavela, R.; Benelli, G. Essential oils as ecofriendly biopesticides? Challenges and constraints. Trends Plant Sci.
2016
,21, 1000–1007.
[CrossRef]
13.
Spinozzi, E.; Pavela, R.; Bonacucina, G.; Perinelli, D.R.; Cespi, M.; Petrelli, R.; Cappellacci, L.; Fiorini, D.; Scortichini, S.; Garzoli,
S.; et al. Spilanthol-rich essential oil obtained by microwave-assisted extraction from Acmella oleracea (L.) R.K. Jansen and its
nanoemulsion: Insecticidal, cytotoxic and anti-inflammatory activities. Ind. Crops Prod. 2021,172, 114027. [CrossRef]
14.
Kavallieratos, N.G.; Bonacucina, G.; Nika, E.P.; Skourti, A.; Georgakopoulou, S.K.C.; Filintas, C.S.; Panariti, A.M.E.; Maggi,
F.; Petrelli, R.; Ferrati, M.; et al. The Type of Grain Counts: Effectiveness of Three Essential Oil-Based Nanoemulsions against
Sitophilus oryzae. Plants 2023,12, 813. [CrossRef] [PubMed]
15.
Benelli, G. Green synthesis of nanomaterials and their biological applications. Nanomaterials
2021
,11, 2842. [CrossRef] [PubMed]
16.
Srivastava, S.; Bhargava, A. Applications of biosynthesized nanoparticles. In Green Nanoparticles: The Future of Nanobiotechnology;
Springer: Singapore, 2022. [CrossRef]
17.
Nenaah, G.E.; Almadiy, A.A.; Al-Assiuty, B.A.; Mahnashi, M.H. The essential oil of Schinus terebinthifolius and its nanoemulsion
and isolated monoterpenes: Investigation of their activity against Culex pipiens with insights into the adverse effects on non-target
organisms. Pest Manag. Sci. 2022,78, 1035–1047. [CrossRef]
18.
Giunti, G.; Palermo, D.; Laudani, F.; Algeri, G.M.; Campolo, O.; Palmeri, V. Repellence and acute toxicity of a nano-emulsion of
sweet orange essential oil toward two major stored grain insect pests. Ind. Crops Prod. 2019,142, 111869. [CrossRef]
19.
Rawat, P.; Khan, M.F.; Kumar, M.; Tamarkar, A.K.; Srivastava, A.K.; Arya, K.R.; Maurya, R. Constituents from fruits of Cupressus
sempervirens.Fitoterapia 2010,81, 162–166. [CrossRef]
20.
Grunewald, J.; Brendler, T.; Jaenicke, C. PDR for Herbal Medicine, 4th ed.; Medical Economics Co.: Montreal, QC, Canada, 2007;
pp. 248–249.
21.
Galoviˇcová, L.; ˇ
Cmiková, N.; Schwarzová, M.; Vukic, M.D.; Vukovic, N.L.; Kowalczewski, P.Ł.; Bakay, L.; Kluz, M.I.; Puchalski, C.;
Obradovic, A.D.; et al. Biological Activity of Cupressus sempervirens Essential Oil. Plants 2023,12, 1097. [CrossRef]
22.
Tapondjou, A.L.; Adler, C.; Fontem, D.A.; Bouda, H.; Rechmuth, C. Bioactivities of cymol and essential oils of Cupressus
sempervirens and Eucalyptus saligna against Sitophilus zeamais Motschulsky and Tribolium confusum du Val. J. Stored Prod. Res.
2005
,
41, 91–102. [CrossRef]
23.
Hasnaoui, F.; Zouaoui, I.; Dallali, S.; Dibouba, F. Insecticidal effects of essential oils from six aromatic and medicinal plants on the
pine processionary caterpillar (Thaumetopoea pityocampa Schiff). Adv. Med. Plant Res. 2020,8, 81–88.
24.
Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Allured Publishing
Corporation: Carol Stream, IL, USA, 2007; ISBN 9781932633214.
25.
Linstorm, P. NIST Chemistry Webbook, NIST Standard Reference Database Number 69. J. Phys. Chem. Ref. Data Monogr.
1998
,9,
1–1951.
26.
Matysik, E.; Wofniak, A.; Paduch, R.; Rejdak, R.; Polak, B.; Donica, H. The new TLC method for separation and determination of
multicomponent mixtures of plant extracts. J. Anal. Methods Chem. 2016,6, 1813581. [CrossRef] [PubMed]
27.
Ellman, G.L.; Courtney, K.D.; Andres, V.; Featherstone, R.M., Jr. A new and rapid colorimetric determination of acetyl-
cholinesterase activity. Biochem. Pharmacol. 1961,7, 88–95. [CrossRef] [PubMed]
28.
Organization for Economic Development (OECD). Guideline for Testing of Chemicals No. 207. Earthworm, Acute Toxicity Tests;
OECD—Guideline for Testing Chemicals; OECD: Paris, France, 1984.
29.
Pavela, R. Essential oils from Foeniculum vulgare Miller as a safe environmental insecticide against the aphid Myzus persicae Sulzer.
Environ. Sci. Pollut. Res. 2018,25, 10904–10910. [CrossRef] [PubMed]
30. Abbott, W.S. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 1925,18, 265–267. [CrossRef]
31. Finney, D.J. Probit Analysis, 3rd ed.; Cambridge University: London, UK, 1971; pp. 68–78.
32. Lee, S.-G. α-Pinene and myrtenol: Complete 1H NMR assignment. Magn. Res. Chem. 2002,40, 311–312. [CrossRef]
33.
Matsuo, A.L.; Figueiredo, C.R.; Arruda, D.C.; Pereira, F.V.; Borin Scutti, J.A.; Massaoka, M.H.; Travassos, L.R.; Sartorelli, P.;
Largo, J.H.G.
α
-Pinene isolated from Schinus terebinthifolius Raddi (Anacardiaceae) induces apoptosis and confers antimetastatic
protection in a melanoma model. Biochem. Biophys. Res. Commun. 2011,411, 449–454. [CrossRef]
34.
Baldwin, S.J.; Clarke, S.E.; Chenery, R.J. Characterization of the cytochrome P
450
enzymes involved in the
in vitro
metabolism of
rosiglitazone. Br. J. Clin. Pharmacol. 1999,48, 424–432. [CrossRef]
35.
Dvorakova, M.; Valterova, I.; Saman, D.; Vanek, T. Biotransformation of (1S)-2-carene and (1S)-3-carene by Picea abies suspension
culture. Molecules 2011,16, 10541–10555. [CrossRef]
36.
Shanker, P.S.; Rao, G.S. Synthesis based on cyclohexadienes. Part 25. Total synthesis of (+/–)-allo-cedrol (khusiol). J. Chem. Soc.
Perkin Trans. 1998,1, 539–548.

Sustainability 2023,15, 8021 19 of 20
37.
Kim, K.H.; Moon, E.; Kim, S.Y.; Choi, S.U.; Son, M.W.; Choi, S.Z.; Lee, K.R. Bioactive sesquiterpenes from the essential oil of Thuja
orientalis.Planta Med. 2013,79, 1680–1684. [CrossRef]
38.
Perez-Hernandez, N.; Gordillo-Roman, B.; Arrieta-Baez, D.; Cerda-Garcia-Rojas, C.M.; Joseph-Nathan, P. Complete
1
H NMR
assignment of cedranolides. Magn. Res. Chem. 2017,55, 169–176. [CrossRef] [PubMed]
39.
Asgary, S.; Naderi, G.A.; Shams Ardekani, M.R.; Sahebkar, A.; Airin, A.; Aslani, S.; Kasher, T.; Emami, S.A. Chemical analysis and
biological activities of Cupressus sempervirens var. horizontalis essential oils. Pharm. Biol. 2013,51, 137–144. [CrossRef]
40.
Selim, S.A.; Adam, M.E.; Hassan, S.M.; Albalawi, A.R. Chemical composition, antimicrobial and antibiofilm activity of the
essential oil and methanol extract of the Mediterranean cypress (Cupressus sempervirens L.). BMC Complement. Altern. Med.
2014
,
14, 179. [CrossRef] [PubMed]
41.
Jahani, M.; Akaberi, M.; Khayyat, M.H.; Emami, S.A. Chemical Composition and Antioxidant Activity of Essential Oils from
Cupressus sempervirens. var. sempervirens, C. sempervirens. cv. Cereiformis and C. sempervirens var. horizentalis. J. Essent. Oil
Bear. Plants 2019,22, 917–931.
42.
Argui, H.; Ben Youchret-Zalleza, O.; Can Suner, S.; Periz, C.D.; Türker, G.; Ulusoy, S.; Ben-Attia, M.; Büyükkaya, F.; Oral, Y.; Said,
H. Isolation, Chemical Composition, Physicochemical Properties, and Antibacterial Activity of Cupressus sempervirens L. Essential
Oil. J. Essent. Oil Bear. Plants 2021,24, 439–452. [CrossRef]
43.
Pansera, M.R.; Silvestre, W.P.; Sartori, V.C. Bioactivity of Cupressus sempervirens and Cupressus lusitanica leaf essential oils on
Colletotrichum fructicola.J. Essent. Oil Res. 2023,35, 51–59. [CrossRef]
44.
M’barek, K. Chemical composition and phytotoxicity of Cupressus sempervirens leaves against crops. J. Essent. Oil Bear. Plants
2016
,
19, 1582–1599. [CrossRef]
45.
Fadel, H.; Benayache, F.; Chalchat, J.C.; Figueredo, G.; Chalard, P.; Hazmoune, H.; Benayache, S. Essential oil constituents of
Juniperus oxycedrus L. and Cupressus sempervirens L. (Cupressaceae) growing in Aures region of Algeria. Nat. Prod. Res.
2021
,35,
2616–2620. [CrossRef]
46.
Miloš, M.; Radoni´c, A.; Masteli´c, J. Seasonal variation in essential oil compositions of Cupressus sempervirens L. J. Essent. Oil Res.
2002,14, 222–223. [CrossRef]
47.
Nejia, H.; Séverine, C.; Jalloul, B.; Mehrez, R.; Stéphane, C.J. Extraction of essential oil from Cupressus sempervirens: Comparison
of global yields, chemical composition and antioxidant activity obtained by hydrodistillation and supercritical extraction. Nat.
Prod. Res. 2013,27, 1795–1799. [CrossRef]
48.
Jain, P.L.B.; Patel, S.R.; Desai, M.A. Patchouli oil: An overview on extraction method, composition and biological activities.
J. Essent. Oil Res. 2022,34, 1–11. [CrossRef]
49.
Landolt, P.J.; Hofstetter, R.W.; Biddick, L.L. Plant essential oils as arrestants and repellents for neonate larvae of the codling moth
(Lepidoptera: Tortricidae). Environ. Entomol. 1999,28, 954–960. [CrossRef]
50.
Giatropoulos, A.; Pitarokili, D.; Papaioannou, F.; Papachristos, D.P.; Koliopoulos, G.; Emmanouel, N.; Tzakou, O.; Michaelakis,
A. Essential oil composition, adult repellency and larvicidal activity of eight Cupressaceae species from Greece against Aedes
albopictus (Diptera: Culicidae). Parasitol. Res. 2013,112, 1113–1123. [CrossRef] [PubMed]
51.
Drapeau, J.; Fröhler, C.; Touraud, D.; Kröckel, U.; Geier, M.; Rose, A.; Kunz, W. Repellent studies with Aedes aegypti mosquitoes
and human olfactory tests on 19 essential oils from Corsica, France. Flavour Fragr. J. 2009,24, 160–169. [CrossRef]
52.
Moore, S.J. Plant-based insect repellents. In Insect Repellents Handbook, 2nd ed.; Debboun, M., Frances, S.P., Strickman, D., Eds.;
CRC Press: Boca Raton, FL, USA, 2014; pp. 179–213.
53.
Regnault-Roger, C. The potential of botanical essential oils for insect pest control. Integr. Pest Manag. Rev.
1997
,2, 25–34.
[CrossRef]
54.
Nerio, L.S.; Olivero-Verbel, J.; Stashenko, E. Repellent activity of essential oils: A review. Bioresour. Technol.
2010
,101, 372–378.
[CrossRef]
55.
Khani, M.; Marouf, A.; Amini, S.; Yazdani, D.; Farashiani, M.E.; Ahvazi, M.; Khalighi-Sigaroodi, F.; Hosseini-Gharalari, A. Efficacy
of three herbal essential oils against rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae). J. Essent. Oil Bear. Plants
2017
,20,
4937–4950. [CrossRef]
56.
Bincy, K.; Remesh, A.V.; Prabhakar, P.R.; Babu, C.S.V. Chemical composition and insecticidal activity of Ocimum basilicum
(Lamiaceae) essential oil and its major constituent, estragole against Sitophilus oryzae (Coleoptera: Curculionidae).J. Plant. Dis.
Prot. 2022,130, 529–541. [CrossRef]
57.
Fouad, H.A.; Tavares, W.; Zanuncio, J.C. Toxicity and repellent activity of monoterpene enantiomers to rice weevils (Sitophilus
oryzae). Pest. Manag. Sci. 2021,77, 3500–3507. [CrossRef]
58.
Balasubramani, S.; Rajendhiran, T.; Moola, A.K.; Kumari, R.; Diana, B. Development of nanoemulsion from Vitex negundo L.
essential oil and their efficacy of antioxidant, antimicrobial and larvicidal activities (Aedes aegypti L.). Environ. Sci. Pollut. Res.
2017,24, 15125–15133. [CrossRef]
59.
Dai, L.; Li, W.; Hou, X. Effect of the molecular structure of mixed non-ionic surfactants on the temperature of miniemulsion
formation. Colloids Surf. A Physicochem. Eng. Asp. 1997,125, 27–32. [CrossRef]
60.
Mondal, P.; Laishram, R.; Sarkar, P.; Kumar, R.; Karmakar, R.; Hazra, D.K.; Banerjee, K.; Pal, K.; Choudhury, A. Plant essential
oil-based nanoemulsions: A novel asset in the crop protection arsenal. In Agricultural Nanobiotechnology, Biogenic Nanoparticles,
Nanofertilizers and Nanoscale Biocontrol Agents; Ghosh, S., Thongmee, S., Kumar, A., Eds.; Woodhead Publishing Series in Food
Science, Technology and Nutrition; Woodhead Publishing: Sawston, UK, 2022; pp. 325–353. [CrossRef]

Sustainability 2023,15, 8021 20 of 20
61. Reddy, S.R.; Fogler, H.S. Emulsion stability: Determination from turbidity. J. Colloid Interface Sci. 1981,79, 101–104. [CrossRef]
62.
Hano, C.; Abbasi, B.H. Plant-based green synthesis of nanoparticles: Production, characterization and applications. Biomolecules
2022,12, 31. [CrossRef] [PubMed]
63.
Palermo, D.; Giunti, G.; Laudani, F.; Palmeri, V.; Campolo, O. Essential Oil-Based Nano-Biopesticides: Formulation and Bioactivity
Against the Confused Flour Beetle Tribolium confusum.Sustainability 2021,13, 9746. [CrossRef]
64.
Santana, A.S.; Baldin, E.L.L.; dos Santos, T.L.B.; Baptista, Y.A.; dos Santos, M.C.; Lima, A.P.S.; Tanajura, L.S.; Vieira, T.M.; Crotti,
A.E.M. Synergism between essential oils: A promising alternative to control Sitophilus zeamais (Coleoptera: Curculionidae). Crop
Prot. 2022,153, 105882. [CrossRef]
65.
Arokiyaraj, C.; Bhattacharyya, K.; Reddy, S. Toxicity and synergistic activity of compounds from essential oils and their effect on
detoxification enzymes against Planococcus lilacinus.Front. Plant Sci. 2022,13, 1016737. [CrossRef] [PubMed]
66.
Tumen, I.; Senol, F.S.; Orhan, I.E. Evaluation of possible
in vitro
neurobiological effects of two varieties of Cupressus sempervirens
(Mediterranean cypress) through their antioxidant and enzyme inhibition actions. Türk. Biyokimya. Dergisi. (Turk. J. Biochem.)
2012,37, 5–13. [CrossRef]
67.
Aazza, S.; Lyoussi, B.; Miguel, M.G. Antioxidant and antiacetylcholinesterase activities of some commercial essential oils and
their major compounds. Molecules 2011,16, 7672–7690. [CrossRef] [PubMed]
68.
Alimi, D.; Hajri, A.; Jallouli, S.; Sebai, H. Phytochemistry, anti-tick, repellency and anti-cholinesterase activities of Cupressus
sempervirens L. and Mentha pulegium L. combinations against Hyalomma scupense (Acari: Ixodidae). Vet. Parasitol.
2022
,303, 109665.
[CrossRef]
69.
Almadiy, A.A.; Nenaah, G.E. Bioactivity and safety evaluations of Cupressus sempervirens essential oil, its nanoemulsion and main
terpenes against Culex quinquefasciatus Say. Environ. Sci. Pollut. Res. 2022,29, 13417–13430. [CrossRef]
70.
Giunti, G.; Benelli, G.; Palmeri, V.; Laudani, F.; Ricupero, M.; Ricciardi, R.; Maggi, F.; Lucchi, A.; Guedes, R.N.C.; Desneux, N.;
et al. Non-target effects of essential oil-based biopesticides for crop protection: Impact on natural enemies, pollinators, and soil
invertebrates. Biol. Control 2022,176, 105071. [CrossRef]
71.
Song, S.Y.; Chang, H.J.; Kim, S.D.; Kwag, E.B.; Park, S.J.; Yoo, H.S. Acute and sub-chronic toxicological evaluation of the herbal
product HAD-B1 in Beagle dogs. Toxicol. Rep. 2021,8, 1819–1829. [CrossRef]
72.
IUCN Centre for Mediterranean Cooperation. A Guide to Medicinal Plants in North Africa. C. sempervirens; IUCN Centre for
Mediterranean Cooperation: Malaga, Spain, 2005; p. 106.
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.