ISSN 1062-3590, Biology Bulletin, 2023. © Pleiades Publishing, Inc., 2023.

ECOLOGY

Efficacy of Some Nano-Organic Formulations and Their Normal

Formulations against the Black Cutworm, Agrotis ipsilon (Hufnagel)
Al-Kazafy Hassan Sabrya (ORCID: 0000-0002-1301-0588)
and Mona Ahmed Husseina, * (ORCID: 0000-0002-1727-0491)
a Pests and Plant Protection Department, Agricultural and Biological Institute, National Research Centre,
Dokki, Cairo, 12622 Egypt
*e-mail: ma.hussein@nrc.sci.eg
Received September 24, 2022; revised October 3, 2022; accepted October 11, 2022

Abstract—Normal formulations of some organic chemicals were developed to nano-organic chemicals and
tested against the fifth instar larvae of the black cutworm, Agrotis ipsilon. These organic chemicals include
etofenprox, imidacloprid and indoxacarb. The nanosizes of these formulations ranged between 246 to
390 nm. The loading capacities were 60.7 ± 5.7, 60.7 ± 5 and 52.0 ± 6.3% for etofenprox, indoxacarb, and
imidacloprid, respectively. The results found that etofenprox was the most potent insecticide followed by
indoxacarb and imidacloprid in both formulations (normal and nano). The LC50s were 1.7, 2.5, and 13.6 ppm
for nanoetofenprox, nanoindoxacarb, and nanoimidacloprid, respectively. The corresponding results with
normal formulations were 9.1, 17.5, and 51.1 ppm, respectively. The results found that also the nanoformula-
tions with all tested compounds were more potent than the normal formulation with all tested insecticides.
Nanoetofenprox, nanoimidacloprid, and nanoindoxacarb were five, four, and seven times more effective
than normal formulations. The results confirmed that the nanoformulations can be used instead of the nor-
mal formulations against the larvae of A. ipsilon to reduce the hazards of insecticides to nontarget organisms,
the cost of insecticides application, insecticide residues in food and quantities of insecticides used.

Keywords: organic compounds, Agrotis ipsilon, normal formulations, nanoformulations, nontarget organisms
DOI: 10.1134/S1062359022700030

INTRODUCTION used silica nanoparticles in control of the 4th instar

The black cutworm, Agrotis ipsilon is considered
one of the most dangerous pests for crops and vegeta-
bles (Binning et al., 2105). This pest can eat over
400 cm2 of foliage during its developmental stages
(Amin, 2019). This means that one larva can damage
up to five plants during the larval duration. The crop
damage by A. ipsilon reached 80% of the yield.
Using synthetic chemical compounds is still one of
the most potent tools to suppress the population den-
sity of A. ipsilon in the fields. Due to the hazards of the
conventional insecticide formulations used it’s a dire
need to find a new strategy to overcome this pest dam-
age without any side effects. Nanopesticide formula-
tions may be achieving this purpose (Yan et al., 2021).
Sabry et al. (2021) used some nanopesticide formula-
tions against the cotton leafworm. Awad et al. (2022)
used some insecticide nanoformulations against the
larvae of A. ipsilon. The authors found that the
nanoparticles of these insecticides were more potent
than the normal formulations. Yan et al. (2021) tested
nanoformulation of thiamethoxam against the green
peach aphid, Myzus persicae. Mesbah et al. (2020)
larvae of A. ipsilon.
Etofenprox is a new insecticide used against wide
range of harmful insects. This insecticide belongs to
the pyrethroid ether group while other the pyrethroids
belong to pyrethroid ester. The main target of this
insecticide is the nerve cell membranes. This insecti-
cide is blocking of ion gates of the sodium channel
during the re-polarization process. Abd-El-Aziz et al.
(2020) used some pyrethroid insecticides against the
black cut worm.
Imidacloprid is related to the neonicotinoids pesti-
cides group that the nervous system is the main target
for this group. The specific target in the nervous sys-
tem is the post-synaptic nicotinic acetylcholine recep-
tors which cause paralysis and death to treated insects.
Rimpy and Verma (2018) used imidacloprid against
the larvae of A. ipsilon.
Indoxacarb is an organic insecticide related to a
methyl ester that belongs to the oxadiazine insecticide
group. This insecticide acts by blocking neuronal
sodium channels. Shakur et al. (2007) used indoxacarb

1
2 AL-KAZAFY HASSAN SABRY, MONA AHMED HUSSEIN
Table 1. The concentrations of tested compounds against the fourth instar larvae of Agrotis ipsilon
Normal formulations concentrations, ppm
Compounds the first the second the third
concentration* concentration** concentration*** concentration
Etofenprox 25 12.5
Imidacloprid 180 90
Indoxacarb 75 37.5
* The first concentration: the recommended field rate, ** the second; half of the recommended field rate, *** the third, the one–fourth
recommended field rate.
against A. ipsilon. Sabry et al. (2021) used nanoindox-
acarb against the cotton leafworm larvae.
This work aims to use a new strategy for pest con-
trol by developing nanopesticide formulations instead
of the normal formulations to reduce the side effect of
the normal formulations and increase the efficacy of
the new formulations against the fourth instar larvae of
A. ipsilon.
MATERIALS AND METHODS
The Organic Compounds
1. Etofenprox is the common name; the chemical
name is (2-(4-ethoxyphenyl)-2-methylpropyl 3-phe-
noxybenzyl ether); the trade name is Primo 10% SC).
Produced by Beta Chem., China. The recommended
concentration used in field is 200 mL/feddan (4200 m2).
2. Imidacloprid is the common name; the chemical
name is (1-(6-chloro-3-pyridylmethyl)-N-nitroimid-
azolidin-2-ylideneamine); the trade name is Sunclo-
prid 35% SC) produced by Imprimerie and Routage,
France. The recommended concentration used in
field is 250 mL/feddan.
3. Indoxacarb is the common name; the chemical
name is ((methyl(4aR)-7-chloro-2-[methoxycar-
bonyl-[4-(trif luoromethoxy)phenyl]carbamoyl]-3,5-
dihydroinde-no[1,2-e][1,3,4]oxadiazine-4a-carbox-
ylate); the tarade name is Avaunt 15% EC), the pro-
duction company is Du Pont De Nemours. This
insecticide belongs to a new group of insecticides
named oxadiazine. The main target of this insecticide
is sodium and potassium channel. The recommended
concentration used in field is 200 mL/feddan.
Each compound used has three concentrations, the
recommended field concentration and other two
lower concentrations (the half and one–fourth of rec-
ommended field rate) (Table 1).
The Tested Insect
Laboratory culture of the black cutworm, Agrotis
ipsilon was developed under laboratory conditions
Nanoformulations concentrations, ppm
the first the second the third
concentration concentration
6.3 5 2.5 1.25
45 36 18 9
18.7 15 7.5 3.7
(26 ± 2°C and 65 ± 5 RH). The adults were fed on
sugar solution until eggs put. After eggs hatching the
first instar larvae were fed on lettuce leaves until
reached the fourth instar. The fourth instar larvae were
tested against the selected compounds.
Preparation of Nanoformulations
The normal formulations of the tested compounds
(etofenprox, indoxacarb and imidacloprid) were
developed to nanoformulations by method described
by Vaezifar et al. (2013). This method depends on con-
verting of the carrier to nanoscales. In this method,
chitosan (a high molecular weight produced by Sigma
Aldrich Co., USA) was used as a polymer to the nano-
formulation produced. It was known that chitosan
doesn’t dissolve in the water, so acetic acid (2% v/v)
was used to dissolve the particles of chitosan. The
solution consists of 120 mL distilled water and 0.8 g
chitosan. The solution (acetic acid and chitosan) was
put on a magnetic stirrer for 25–30 min or until con-
verted to a transparent solution. On the other hand,
0.8 g of tripolyphosphate dissolved in 120 mL of con-
ductive water. One mL of tested insecticide was mixed
with tripolyphosphate in conductive water. The solu-
tion was dropwise by dropwise to the chitosan mixture
with continuous stirring for 20–30 min. After that, the
suspension obtained was centrifuged at 10000 rpm for
30 min. The obtained pellet was harvested and lyo-
philized to gain nanoparticle formulation. This work
was done with all tested compounds. The obtained
nanoparticles were photographed under a scan elec-
tronic microscope (SEM) (HRTEM, JEM2100, Jeol,
Japan) (Fig. 1).
The nanosize of these compounds under scan elec-
tronic microscope was not enough to sure these com-
pounds were converted to nanoformulations. So,
another test must be carried out. This test is the load-
ing capacity of these compounds on a carrier (chi-
tosan). This test was carried out according to He et al.
(2017). Thirty mL of the obtained nanoformulation
was dissolved in 50 mL of acetonitrile. This obtained
solution was put in a shaking tank for 12 h in a tem-
BIOLOGY BULLETIN 2023
 EFFICACY OF SOME NANO-ORGANIC FORMULATIONS...
270 nm
241 nm
870 nm
(а) (b)
HV mag spot WD det
20.00 kV 2000 × 4.0 11.1 mm BSED
Fig. 1. The nanoformulations of etofenprox (a), imidacloprid (b) and indoxacarb (c) under SEM.
perature room to dissolve the chitosan particles com-
pletely. The obtained mixture was filtered, the mass of
the active ingredient of all tested pesticides in acetoni-
trile was determined by HPLC (The HPLC system was
equipped with an XTerra RP18 column, 5 μm particle
size, 4.6 mm internal diameter × 250 mm length
(Waters®, USA) with the wavelength of 278 nm (Fig. 2).
The loading capacity was determined by dividing the
mass of loading pesticide (etofenprox) on the mass of
etofenprox
Loading Capacity (LC)
Mass of loaded pesticide
= × 100
Mass of pesticide nanoparticles
nanoparticles ×100.
According to this formula, the loading capacities
were 60.7 ± 5.7, 52.0 ± 6.3, and 60.7 ± 5.7% for
etofenprox, imidacloprid, and indoxacarb, respec-
tively.
The Bioassay
Two formulations were tested against the 4th instar
larvae of A. ipsilon, normal formulation and nanofor-
mulation for each tested organic compound. Three
concentrations were used for each tested compound as
cleared in Table 1. Three replicates for each concen-
tration were used each replicate has 10 healthy starved
larvae. The larvae in control replicates were treated by
water only. Lettuce leaves were used as natural food.
Dipping technique was used by dipping of the lettuce
leaves in each concentration and the tested larvae were
fed in. All replicated were inspected after 24 h, 2, 5,
and 9 days. The dead larvae were counted and
excluded. The percent of mortalities in all replicates
was calculated and the lethal concentration for 50% of
the treated population calculated by Proban Program
Software.
BIOLOGY BULLETIN 2023
3
450 nm
371 nm
621 nm
(c)
HFM 9/1/2021 50 Pm
207 Pm 2:13:06 PM NRC (QUANTA FEG250)
Statistical Analysis
Data were subjected to the analysis of variance test
(ANOVA) (F test) and analysis of variance (one-way
classification ANOVA) followed by a least significant
difference (LSD) at 5% (Costat, 1990).
RESULTS AND DISCUSSION
The toxicity of both nanoformulations and normal
formulations is determined by using of the 4th instar
larvae of Agrotis ipsilon.
Toxicity of the Normal Formulations of Tested
Compounds against A. ipsilon Larvae
The normal formulations of etofenprox, imidaclo-
prid and indoxacarb are evaluated by using of the 4th
instar larvae of A. ipsilon (Table 2). The obtained
results show that the percent of mortalities with the
first concentration (recommended field concentra-
tion) are 80, 93.3 and 90% for etofenprox, indoxacarb
and imidacloprid, respectively. This result clears that
indoxacarb is the most toxic compound followed by
imidacloprid and etofenprox. The statistical analysis
shows that there is no significant difference between
indoxacarb and imidacloprid but there is a significant
difference between indoxacarb and etofenprox. The
LSD value is 7.7 (Table 2). The same results have
existed with the second and third concentrations.
Indoxacarb is the most effective followed by imidaclo-
prid and etofenprox. In spite of etofenprox causing less
percent mortality, the mean efficacy of all tested
compounds and the LC50 values found that etofen-
prox is the most potent compound followed by
indoxacarb and imidacloprid. This is due to the dif-
ference in the percent of the active ingredients in all
tested compounds. The LC50s are 9.1, 17.5, and
4 AL-KAZAFY HASSAN SABRY, MONA AHMED HUSSEIN

mAU (a)

250
1750
1500
1250
1000
750
500
Etofenprox st
250
0

5 10 15 20
(b)
3.038
2.600

200

150 p r id
iclo
nd
le I
mp
100 Sa

50

0
20 40 60 80
(c)
3.038

40 b
car
oxa
nd
le I
30 mp
Sa

20

10

0
20 40 60
Fig. 2. The loading capacities of etofenprox (a), imidacloprid (b) and indoxacarb (c).

51.1 ppm for etofenprox, indoxacarb, and imidaclo-
prid, respectively.
Ismail (2021) used indoxacarb against the first,
third, and fifth instar larvae of A. ipsilon. The author
found that the LC50s of indoxacarb 0.259, 0.809, and
1.23 mg/L, respectively.
Toxicity of the Nanoformulations of Tested Compounds
against A. ipsilon Larvae
Table 3 shows that indoxacarb is the most toxic
compound against the 4th instar larvae followed by
etofenprox and imidacloprid with all tested concentra-
tions. The percentages of mortalities with the first
concentration are 86.7, 83.3, and 76.7%, respectively.
The statistical result shows that there are no significant
differences among all tested compounds with the first
concentration. The results show also there is a signifi-
cant difference between all tested compounds and
control.
The obtained results with the second concentration
(the half of recommended field rate) the percentages
of mortalities are 73, 60 and 66.7%, respectively. This
means that imidacloprid is more effective than etofen-
prox with the second concentration. The statistical
result shows that there is a significant difference
between etofenprox and other tested compounds.
With the third concentration the percentages of mor-

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 EFFICACY OF SOME NANO-ORGANIC FORMULATIONS... 5

Table 2. Toxicity of the normal formulations to the 4th instar larvae of A. ipsilon
The mortality percentages LC50 and fiducial
Tested insecticides Slope ± SE
C1 ± SE C2 ± SE C3 ± SE limits

9.1
Etofenprox 80.0 ± 0.0b 56.7 ± 5.8b 40.0 ± 0.0b 1.8 ± 0.3
(6.9–10.9)
51.1
Imidacloprid 90.0 ± 0.0a 66.7 ± 5.8ab 46.7 ± 5.8ab 2.2 ± 0.3
(38.9–61.4)
17.5
Indoxacarb 93.3 ± 5.8a 76.7 ± 5.8a 53.3 ± 5.8a 2.3 ± 0.4
(12.6–21.4)
Control 6.7 ± 5.8c 5.0 ± 5.0c 0.0c
F-values 301.8*** 96.3*** 103.3***
LSD 7.7 10.9 7.7
Means under each treatment sharing the same letter in a column are not significantly different at P = 0.05.

Table 3. Toxicity of the nanoformulations to the 4th instar larvae of A. ipsilon

The mortality percentages LC50 and fiducial
Tested insecticides Slope ± SE
C1 ± SE C2 ± SE C3 ± SE limits

1.7
Etofenprox 83.3 ± 5.8a 60.0 ± 0.0b 40.0 ± 0.0b 1.9 ± 0.3
(1.4–2.1)
13.6
Imidacloprid 76.7 ± 5.8a 66.7 ± 5.8ab 33.3 ± 5.8b 1.9 ± 0.3
(10.8–16.2)
2.5
Indoxacarb 86.7 ± 5.8a 73.3 ± 5.8a 60.0 ± 0.0a 1.4 ± 0.3
(1.1–3.7)
Control 3.3 ± 5.8b 3.3 ± 5.8c 6.7 ± 5.8c
F-values 141.6*** 123.9*** 87.3***
LSD 10.9 9.4 7.7
Means under each treatment sharing the same letter in a column are not significantly different at P = 0.05.

talities are 60, 40 and 33.3%, respectively. Although
the percent of mortality of etofenprox against the 4th
instar larvae is less than another tested compound, the
lethal concentration of 50% of the treated population
(LC50) more than other tested compounds. This is due
to the percent of the active ingredient of etofenprox
(10%) being less than other compounds (imidacloprid
35% and indoxacarb 15%). The LC50s are 1.7, 2.5, and
13.6 ppm for etofenprox, indoxacarb, and imidaclo-
prid, respectively. The most treated larvae are dying
after 24 h of treatment by all tested nanoformulations
(Fig. 3).
The same results were obtained by Sabry et al.
(2021). The results showed that imidacloprid and
indoxacarb nanoformulations were used with the 2nd
instar larvae of Spodoptera littoralis. The results also
showed that the indoxacarb nanoformulation was
more potent than imidacloprid nanoformulation. The
LC50s were 2.9 and 15.9 ppm for indoxacarb and imi-
dacloprid, respectively. Huang et al. (2022) used
nanoindoxacarb against the larvae of Spodoptera
litura. The results showed that the nanoindoxacarb
was effective against the larvae of S. litura. Bilal et al.
(2020) used the indoxacarb nanoformulation against
the larvae of Plutella xylostella. The results found that
the indoxacarb nanoformulation was very potent to
the tested pest. Sabry and Hussein (2022) evaluated
that the nanoetofenprox to the adults of chocolate
banded snail, Eobania vermiculata. The LC50 of nano-
etofenprox was 20.3 ppm.

BIOLOGY BULLETIN 2023

6 AL-KAZAFY HASSAN SABRY, MONA AHMED HUSSEIN
(а)
(c)
Fig. 3. The efficacy of etofenprox (a), imidacloprid (b) and indoxacarb (c) nanoformulation compared to untreated (d) against
the 4th instar larvae of A. ipsilon.
CONCLUSIONS
The nanoformulations of all tested compounds
have the same efficacy as the normal formulations.
Although the concentration of normal formulations
was five times more than the nanoformulations, the
efficacy of both formulations was the same. The
obtained results demonstrated that the recommended
field rate of etofenprox in normal formulation caused
80% mortality compared with 83.3% in nanoformula-
tion. The LC50 of nanoetofenprox and its normal for-
mulation were 1.7 and 9.1 ppm, respectively. This
result clears that the nanoetofenprox was 5-times
more potent than the normal formulation. The statis-
tical analysis showed that there is no significant differ-
ence between nanoetofenprox and normal etofenprox
with all tested concentrations (Table 4). The first con-
centration of imidacloprid in normal formulation
(90%) was more effective than the nanoformulation
(76.7) but the mean efficacy of all tested concentra-
tions cleared that the nanoimidacloprid was more
effective than the normal formulation. The LC50 of
nanoimidacloprid and normal formulations were 13.6
and 51.1 ppm. This means that the nanoimidacloprid
was 4-times effective than the normal formulation.
The same results were found with the nanoindox-
acarb. The nanoindoxacarb was 7-times more effec-
tive than the normal formulation.
This means that the nanoformulations can be used
instead of the normal formulations to reduce the
amount of pesticide treatment and the pesticide toxic-
ity on the nontarget organism.
ACKNOWLEDGMENTS
Great thanks to National Research Centre (NRC),
Cairo, Egypt for supporting this work through projects nos.
12050118 and 12050137.
(b)
(d)
FUNDING
All authors declared that there was no funding.
COMPLIANCE WITH ETHICAL STANDARDS
Conflict of interest. All authors declared that there is no
conflicts of interest.
Statement on the welfare of animals. All applicable inter-
national, national, and/or institutional guidelines for the
care and use of animals were followed.
AUTHOR CONTRIBUTIONS
All authors designed the research, conducted the exper-
iments, analysed the data, wrote, revised, and approved the
manuscript.
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