Journal of Entomological Research

Islamic Azad University, Arak Branch

ISSN 2008-4668
www.entomologicalresearch.ir

Volume 4, Issue 4, pages: 297-305

Insecticidal effect of silica and silver nanoparticles on the cowpea seed

beetle, Callosobruchus maculatus F. (Col.: Bruchidae)
M. Rouhani1*, M. A. Samih2, S. Kalantari3
1- Ph.D Student, Department of Plant Protection, Faculty of Agriculture, Razi University, Kermanshah, Iran
2- Assistant Professor, Department of Plant Protection, Faculty of Agriculture, Vali-e-Asr University, Rafsanjan, Iran
3- Ph.D Student, Department of Agronomy and Plant Breeding, Ferdowsi Univrsity, Mashhad, Iran

Abstract

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Nanotechnology has become one of the most promising new approaches for pest control in
recent years. In our screening program, laboratory trials were conducted to determine the
effectiveness of silica nanoparticles (SNP) and silver nanoparticles (AgNP) on larval stage and
adults of Callosobruchus maculatus on cowpea seed. Nanoparticles of silica and silver were
synthesized through a solvothermal method and different concentrations (1, 1.5, 2 and 2.5 g kg-1) of
them were tested on C. maculatus. In the experiments, the LC50 value for SiO2 and Ag
nanoparticles were calculated 0.68 and 2.06 g kg-1 cowpeas on adults and 1.03 and 1.00 g kg-1 on
larvae, respectively. Result showed that, the both nanoparticles (silica and silver) were highly
effective on adults and larvae with 100% and 83% mortality, respectively. The result also showed
that SiO2 nanoparticles can be used as a valuable tool in pest management programs of C.
maculatus.

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Key words: Callosobruchus maculatus, nanosilver, nanosilica, storage pest

Introduction

The cowpea seed beetle, Callosobruchus maculatus F. (Col: Bruchidae), is the most important
storage pest of cowpea throughout the tropics. The control of stored grain pests stands mostly on
broad action insecticides and fumigants. Unfortunately, this leads to contamination of food with
toxic pesticide residues (Debnath et al., 2011). Moreover, the main problem in controlling pests in
stored grain is the resistance to pesticides. Regarding the resistance of grain pests and pesticide
residues, it seems that chemical control is not an appropriate approach for controlling the
population of these pests.
One alternative is the use of Diatomaceous Earths (DEs), composed mainly of amorphous silica
and derived from fossilized phyto-plankton (Subramanyam & Roesli, 2000; Mewis & Ul-richs,
2001). DE becomes more effective against insects if it possesses high silica content with uniform
size distribution (Korunik, 1997) and a number of physical properties of materials change as their
size approach nanoscale (Debnath et al., 2011).
Nanoparticles represent a new generation of environmental remediation technologies that could
provide cost-effective solution to some of the most challenging environmental clean-up problems
(Chinnamuthu & Murugesa Boopathi, 2009). Nanoparticles help to produce new pesticides,
insecticides and insect repellants (Owolade et al., 2008). Also, researchers believe that
nanotechnology will revolutionize agriculture including pest management in the near future
(Bhattacharyya et al., 2010). Although there have been numerous studies on the toxicity effects of
nanoparticles on bacteria, fungi, and animal pathogens (Bragg & Rannie, 1974; Feng et al., 2000;
Samuel & Guggenbichler, 2004; Elchiguerra et al., 2005; Reddy et al., 2007). Little research has

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* Corresponding Author:, E mail: Rouhani_valiasr @yahoo.com

Received: 1 Jun. 2012 -Accepted: 19 Dec. 2012

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Rouhani et al: Insecticidal effect of silica and silver nanoparticles on

been carried out to investigate the toxicity effect of nanoparticles on insects specially storage pest.
Wan et al. (2005) studied the effect of action of mixture of two nanoparticles with two insecticides
to the pest mite (Epitrimerus pyri (Nalepa)). According to the authors cypermethrin and alpha
Terhienyl mixed with nano-particled zinc oxide and copper oxide was effective on the tested mite.
Yang et al. (2009) expressed that nanoparticles loaded with garlic essential oil is efficacious against
Tribolium castaneum (Herbst). Stadler et al. (2010) showed that nano alumina could be
successfully used to control stored grain pests.
These cues led us to investigate the entomotoxicity of silica and silver nanoparticles against
C. maculatus.

Material and Methods

Synthesis of silica and silver nanoparticles
To synthesis the silica and silver nanoparticles, Mathew & Narayanankutty (2010) and Aslani et
al., (2003) methods with some modification were used, respectively. Nanosilica was synthesized by
acid hydrolysis of sodium silicate using dilute hydrochloric acid 15%. Sodium silicate solution was
prepared by 1% polyvinyl alcohol solution. Then 0.5 NHCl, was added to it slowly with stirring at
the temperature of 60oC. The pH of the mixture was maintained between 1and 2. The solution was
stirred at 60oC for 30 minute to carry out acid hydrolysis of sodium silicate. The sol-gel mixture
was then washed well to remove all the sodium chloride formed. It was dried at 50oC and then
muffled at 600oC.
For synthesis nanosilver, sodium hydroxide solution (4 M, 10 ml) was added to a solution of
AgNO3 (2 mmol) in EtOH/H2O solvent (25 ml). To investigate the role of surfactants on the size
and morphology of nanoparticles, 0.5g of polyethylene glycol (PEG) was used in the reactions with
optimized conditions. The obtained mixtures were sonicated for 150 min with ultrasound powers
followed by centrifugation and separation of the solid and liquid phases. The morphology and size
of samples were characterized by Scanning Electron Microscope*.

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Bioassay
Adults of C. maculatus were reared at 301oC and 655% RH in the dark on cowpea seed.
Insects were obtained from cultures maintained in the Kerman Agricultural Organization laboratory
for at least three years, with no history of exposure to insecticides. Adults with less than 2 weeks
old and first instar larvae (Negahban & Moharramipour, 2007) were used for the experiments.
Effects of the nanosilica and nanosilver on adults and larvaes of C. maculatus were determined
by contact toxicity assay at three doses of 1, 1.5 and 2 g nanoparticle kg-1 cowpea seed. The
experiments were carried out in Completely Randomized Design with five replications each
consisted of 20 adults or larvae of C. maculatus in small plastic screw capped jars containing 20 g
of cowpea seed. Seeds in each jar were treated individually with nanoparticles. Then, the jars were
shaken manually for approximately 1 min to achieve equal distribution of nanoparticles on cowpea
seed (Subramanyam & Roesli, 2000). In one additional set no nanoparticle was mixed with cowpea
and this set served as control. After 24 h, 20 unmated adults of C. maculatus were introduced into
each jar. All bioassays were performed at 301oC and 655% RH. Insect mortality was checked
after 1, 2, 4, 7 and 14 days for adults and 48 hours for larvae stages.

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Statistical Analysis
The mortality data was analyzed with SPSS 16 software followed by one-way analysis of
variance (ANOVA) and Duncan's multiple range tests. The results were expressed as means (SE)
of untransformed data and considered significantly different at P<0.05. Probit analysis was
conducted to estimate LC50 values with their fiducially limits by Probit analysis software.

* Model Philips XL 30

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Results
Structural study of nanoparticles
The shape and size of the nanoparticles prepared in this study were checked by Scanning
Electron Microscope (SEM). Figure 1, indicates that the original morphology of the silica and
silver nanoparticles are approximately spherical with the diameter varying between 20 to 60 nm.

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Fig. 1- The SEM images of synthesized Sio2 (left) and Ag (right) nanoparticles

Contact toxicity bioassay

Mortality of adults treated with the nanoparticles started after 18 hours under the laboratory
conditions. The presence of nanosilica and nanosilver resulted 100 and 75% mortality at the highest
concentration of 2.5 g kg-1 on day 14, respectively (Table 1). The results showed that there was
significant interaction effects between dates and treatments. Based on this finding the effect of
treatments on different days were reported, separately. The analysis of variance showed that the
effect of nanosilica on adults on first day (F9,20 = 53.92, P = 0.0000), second day (F9,20 = 123.68, P =
0.0000), fourth day (F9,20 = 126.97, P = 0.0000), seventh day (F9,20 = 119.51, P = 0.0000),
fourteenth day (F9,20 = 115.56, P = 0.0000), SiO2 on larvae (F4,20= 309.75, P = 0.0000) and Ag on
larvae (F4,20= 106.52, P = 0.0000) had a significant difference at 1% level. In all treatments using
NP, percentage of mortality of adults and larvae (Tables 1 and 2) increased with increase in NP
concentrations.
Table 1 shows that on the first day, nanosilica was not at all proper effect on adults. Since the
mortality less than 50% is not efficient, the efficiency of nanosilica was observed on day 4 with
concentration of 1.5 g kg-1. At the dose of 2.5 g kg-1 more than 85% and 96% of beetles died in case
of nanosilica after 4th and 7 th days of exposure, respectively. Also these results showed that SNP at
2 and 2.5 g kg-1 could kill all insects after 14 days. The highest percent mortality of silver
nanoparticles (51.66) was obtained on day 14 with concentration of 2.5 g kg-1. However, at the
similar concentration, nanosilica killed 100% of C. maculatus adults.
Application of NPs on larval stage showed higher killing effect of AgNP in all rates of
applications (Table 2). Both nanoparticles had the highest mortalities at concentration of 2.5 g kg-1.

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Rouhani et al: Insecticidal effect of silica and silver nanoparticles on

Table 1- Percent mortality (mean SE) of C. maculatus adults treated with silica and silver nanoparticles

Concentration
(g kg-1)
1

1.5

2.5
0

Nanoparticles
SiO2

1
11.653.33d

2
28.34.40c

Ag

1.651.66e

3.31.66eg

SiO2

23.31.66c

Ag

Mortality (%)
Day
4
41.61.66c

7
61.66.00c

14
78.33.33c

8.31.66e

23.31.66g

33.33.33c

33.34.40c

58.34.40b

75.05.00b

91.64.40c

3.31.66e

6.651.66deg

13.33.33e

28.31.66g

43.31.66c

SiO2

33.33.33b

63.31.66b

802.88a

93.003.33a

100.00.00c

Ag

3.31.66e

8.31.66de

28.33.33d

41.61.66e

55.05.00c

SiO2

43.333.33a

71.61.66a

85.00.00a

Ag

5.00.00a

11.61.66d

40.62.88c

SiO2 - Ag

0.00.00e

0.00.00g

6.61.66e

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96.01.66a

100.00.00c

51.61.66d

75.02.88c

10.010.00h

15.02.88c

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* Means followed by same letter in column are not significantly different at 5% level

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Table 2- Percentage mortality (mean SE) of C. maculatus larvae treated with silica and silver nanoparticles

Concentration
(g kg-1 of cowpea)
1
1.5
2
2.5
0

Mortality (%)
SNP
AgNP
37.002.00c
33.001.22c
52.002.54b
58.001.22b
a
61.001.87
71.002.44a
a
65.002.23
73.002.00a
2.001.22d
2.001.22d

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* Means followed by same letter in column are not significantly different at 5% level

The LC50 value for SiO2 and Ag nanoparticles on larvae and adults of C. maculatus is shown in
Table 3 and 4. According to these values, adults of C. maculatus were more sensitive to SiO2 than
Ag nanoparticles. However, there was not any significant difference between LC50 of Ag and SiO2
nanoparticles. These results indicated that LC50 decreased with increasing in NP concentrations
(Table 1 and 3). However the mortality increased with increasing NP concentrations. Due to the
highest mortality in superlative concentration LC50 was compared after 14 days and 48 hours for
adults and larvae, respectively.
The LC50 values for SiO2 and Ag nanoparticles on adults and larvae after 14 days and 48 hours
were estimated 0.68, 1.03, 2.06 and 1.00 g kg-1 cowpea, respectively (Table 4). The LC50 value
comparison using the LC50 ratio (0.328) and their lower and upper 95% confidence limits (0.2110.511) showed that there was a significant difference between LC50 value for SiO2 (0.68 g kg-1) and
this value for Ag nanoparticles (2.06 g kg-1) on adults.
Comparision between SiO2 and Ag nanoparticles on larvae, the LC50 ratio (1.023) with their
lower and upper 95% confidence limits (0.0803-1.303) showed that there was no significant
difference between the LC50 value for SiO2 (1.03 g kg-1) and Ag nanoparticles (1.00 g kg-1). For the
all nanoparticles the statistic t ratio was >1.96, the g factor was <0.5 and the heterogeneity factor
was <1.
Because of no significant difference between the LC50 value for SiO2 and Ag nanoparticles on
larvae, silica nanoparticles could be more appropriate to control C. maculatus.

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Table 3- LC50 (g kg-1 Seed weight) value of silica and silver nanoparticles against adults of C. maculatus

Nanoparticles

1
2.91
84.12

SNP
AgNP

Day
4
1.27
2.95

2
1.67
15.00

7
0.92
2.80

14
0.68
2.06

Table 4- LC50 (g kg-1 Seed weight) value of silica and silver nanoparticles against larvae after 48 hours and adults of
C. maculatus after 14 days

Nanoparticles
SNP
AgNP

Stage

LC50

Slop ( SE)

Limits 95%

Adults
Larvae
Adults
Larvae

0.68
1.03
2.06
1.00

3.91 0.97
2.82 0.54
2.70 0.74
4.90 0.67

0.30 - 0.89
0.94 1.11
1.80 2.50
0.76 1.17

Chi square
(2)
0.49
1.73
0.24
0.91

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Doseresponse gradient was used to evaluate the relationship between pesticide and mortality.
The presence of a dose-response gradient is one of the key criteria for determining the effectiveness
of toxicity is causal (Robertson & Rappaport, 1979). According to Fig. 1 gradient of mortality in
higher doses was more than low doses in all treatments. The dosage-response gradient for SiO2 and
Ag on adults and larvae were estimated 3.91, 2.70, 2.93 and 4.90 based on calculated LC50,
respectively. These gradients also demonstrates that SiO2 (3.91) is more effective on adults than Ag
(4.90) is more effective on larvae than SiO2 (Fig. 1).

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7500
7000

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6500

Probit

6000
5500

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5000
4500

4000

AgNP Larvae
AgNP Adult
SNP Larvae
SNP Adult

3500

0,0

0,1

0,2

0,3

0,4

0,5

Log (Dose)
Fig. 1- Dose-response gradient of silica and silver nanoparticles against C. maculatus

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Rouhani et al: Insecticidal effect of silica and silver nanoparticles on

Discussion
Management of stored-grain pests stands traditionally on use of synthetic insecticides and long
term application of these chemicals develops resistance to pesticides. In recent years, nanoparticles
have received much attention for controlling of pathogens in agriculture (Eleka et al., 2010; Guan
et al., 2008; Sang Woo et al., 2009). The use of nanomaterials in agriculture is still at a rudimentary
stage. Stadler et al. (2010) applied successfully nano alumina against two stored grain pests.
However, Yang & Watts (2005) showed that nano alumina in ground water inhibits the growth of
carrot, cabbage, cucumber, corn, and soybean. This study showed that NPs could be applied to
facilitate pest control management (IPM) of stored grain pests as C. maculatus. Comparison of our
results with earlier investigations (Debnath et al., 2011) demonstrates that application of SNP could
significantly increase mortality effect of NPs with increasing the time after application. They reveal
that SNP has a high potential as pesticide. This may be one of the possible reasons for which there
is an age-old tradition of using silica dust as protective agent for stored seeds by different ethnic
races all over the world (Ebeling, 1971). On the basis of our results, mortality effect of silica was
far more effective on adults than larvae, which this mortality could be attributed to the impairment
of the digestive tract (Smith, 1969) or to surface enlargement of the integument as a consequence of
dehydration or blockage of spiracles and tracheas. Also it refers to their enormously increased
exposed surfaces which could interact with the insect cuticle. Damage occurs to the insects
protective wax coat on the cuticle, both by sorption and abrasion.
It is well obvious from our results that nano silver is not effective on adults; this may be due to
cuticle but it shows effective entomotoxic potential against larval stage. This reaction could refer to
various toxicity mechanisms of Ag, which have been reported. Examples of such mechanisms
include generation of reactive oxygen Types, oxidative stress, membrane disruption, protein
unfolding, and/or inflammation (Bragg & Rannie, 1974; Feng et al., 2000; Samuel &
Guggenbichler, 2004; Elchiguerra et al., 2005; Reddy et al., 2007; Meng et al., 2009; Donaldson et
al., 2009).
Insect mortality effect of SiO2 nanoparticles was slightly more than that of Ag nanoparticles on
adults. These results also showed that Ag nanoparticles was more than that of SiO2 nanoparticles on
larvae but there was no significant difference between LC50 of Ag and SiO2 against larvae.
According to these findings, it is well obvious from our results that Sio2 could be selected as a good
agent to control this pest. This should be done whether the nanoparticles can act as a good method
of control and bring stored grain pests populations under control following EIL or not. Many
researchers have studied the effect of nanoparticles, especially that of against pathogens (Debnath
et al., 2011; Feng et al., 2000; Reddy et al., 2007). Our finding is consistent with other researchers
reports (Debnath et al., 2011; Stadler et al. 2010; Rouhani et al. 2011). According to Debnath et al.
(2011), the mortality of Sitophilus oryzae (Linnaeus) was significantly affected by hydrophilic and
hydrophobic of Sio2. Finding of Stadler et al. (2010) is also consistent with our results. They
demonstrated insecticidal effect of alumina nanoparticles on S. oryzae and Rhyzopertha dominica
(Fabricius). In addition, Rouhani et al. (2011) reported that ZnO-TiO2-Ag nanoparticles has
insecticidal activity on Frankliniella occidentalis (Pergande) and showed the most mortality effect
pertained to 28% ZnO-70% TiO2-2% Ag (LC50=195.27 mg/L).
For the practical use of SiO2 nanoparticles and test compounds as novel pesticide to proceed,
further research is required on the safety issues of these materials for human health. Other areas
requiring attention are its mode of action and development of formulations to improve potency and
stability, as well as to reduce cost.

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Conclusion
Motivated by the fact that little is known regarding the effects of NPs on stored-grain pests. The
present study demonstrated strong toxicity of the tested NPs, nanosilica and nanosilver to
C. maculatus. Our findings suggest that the use of NPs cause a reduction in density of
C. maculatus, but among these, nanosilica have more toxic effects than nanosilver on adults and
nanosilver is more effective on larvae stage. The NPs can be removed by conventional milling

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process unlike sprayable formulations of conventional pesticides, leaving residues on the stored
grain (Debnath et al., 2011). Therefore, SNP and AgNP have an excellent potential as stored grain
as well as seed protecting agent if applied with proper safety measures.

Acknowledgements
We thank Dr. Alireza Aslani in Vali-e-Asr University, Iran and Dr. Mehdi Kalantari at
Maryland University, College Park USA for helpful discussions. The authors are also thankful to
Tarbiat Modares University, Tehran, Iran for providing access to Scanning Electron Microscope
(SEM).

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