chapter three study on entomotoxic efficacy of silica...

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CHAPTER THREE

Study on entomotoxic efficacy of silica nanoparticle in comparison with

other oxide nanoparticles: Value addition to these silica nanoparticles

3.1 INTRODUCTION

Infestation of crops by insects remains a major problem all over the world. There is no

specific insecticide for stored grains available in the market. Contact insecticides like some

organophosphates and pyrethroides or fumigants are used to protect stored grain.

Unfortunately this leads to contamination of food with toxic pesticide residues. Prolonged

exposure to these chemicals may lead to neuronal and hormonal disorders (Haviland et al.

2009; Bouchard et al. 2010; Harari et al. 2010) and may also lead to environmental

contamination. One such commonly used fumigant methyl bromide is directly implicated in

depletion of ozone layer. The Montreal Protocol has banned its use in developed countries

and its use is restricted in developing countries (USDA, Agriculture Information Bulletin No.

756). Moreover, there are numerous reports from around the globe on widespread resistance

in several insect pests to common insecticides including synthetic pyrethroids,

organophosphates, carbamates, chlorinated hydrocarbons, Bacillus thuringiensis, botanicals

and fumigants (Subramanyam et al. 1995).

The development of resistance in both stored product and field insects to conventional

insecticides, coupled with increased consumer awareness of the consequences of their

residual toxicity and environmental contamination have lead agro-chemical researchers to

reappraise the use of inert dusts as alternative insecticide for crop protection. Actually

‘bathing in sand’ is a well known phenomenon exhibited by birds to rid themselves of mites

and other parasites. Nearly four thousand years ago observations of such natural phenomena

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led the Chinese to use diatomaceous earth (DE), naturally occurring micron sized silica

derived from fossil phytoplanktons (Subramanyam et al. 2000; Mewis et al. 2001), to control

pests (Allen 1972). In 1880, it was noticed in the USA that road dust killed caterpillars of the

cotton moth (Stelle 1880). Until 1950s use of clay dusts, sand or silica gels were more

popular than DE. In the early 1950s, DE was used to fight fruit moths, cucumber beetles,

stored product pests and cockroaches (Barlett 1951). Earlier formulations of DE were not

widely accepted because of their adverse effects on bulk density (volume: weight ratio) of

grains, which is a very important physical property of grain mass (Korunic 1998). Moreover,

their insecticidal property depended on several other factors like temperature, humidity, silica

content, geological origin, access to food for the pest etc. (Subramanyam et al. 2000; Fields

et al. 2000; Vayias et al. 2004). In the last decade several improved DE formulations have

been successfully evaluated against several stored-product pest species (Athanassiou et al.

2004, 2006, 2007; Kavallieratos et al. 2007a; Vassilakos et al. 2006; Iatrou et al. 2010).

We stared our insecticidal assays with modified DE, “Fossil Shield 90.0s (FS90.0s®)”

and nanoporous alumino silicate nanoparticle (NP). Earlier researchers have shown that DE

was more effective against insects when it possessed high amorphous silica content with

uniform size distribution (Korunic 1997). This cue led us to investigate the effect of

amorphous silica nanoparticle (SNP) as a novel entomotoxic agent. Nanoparticles (NPs) are

more reactive than their bulk counterpart because of their increased surface to volume ratio

(Park et al. 2009) as in any physical or chemical interaction only the surface exposed to the

reaction condition participates in the process. So it is expected that nanocides would be

needed in smaller quantities to achieve crop protection.

Efficacy of SNPs was compared with other oxide NPs like Zinc oxide NP (ZNP),

Titanium dioxide NP (TNP) and Aluminium oxide NP (ANP). According to the International

Agency for the Research of Cancer (IARC), amorphous silica belongs to group 3; it is

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classified as not carcinogenic. United States Department of Agriculture (USDA) has already

approved the use of amorphous silica as safe (Stathers 2004). Zinc oxide and titanium oxide

have been used in cosmetics for years (Zvyagin et al. 2008; Newman et al. 2008). ANP was

used as positive control.

Insecticidal assays were performed on Sitophilus oryzae (L.) (Coleoptera:

Curculionidae), Lipaphis pseudobrassicae (Kaltenbach) (Hemiptera: Aphididae), Spodptera

litura (F.) (Lepidoptera: Noctuidae) and Epilachna vigintioctopunctata (F.) (Coleoptera,

Coccinellidae). S. oryzae is a very important insect pest that causes immense damage to

stored grains worldwide (Aitken 1975). It is classified as a primary pest which means that it

is capable of infesting unbroken grain kernels. Larvae of S. oryzae develop in the kernel and

therefore are protected from grain protectant residues on the exterior of the kernel. L.

pseudobrassicae is a common species on several Crucifereae plants and responsible for

serious losses in various crops such as Brassica juncea (L.) Czern (Brassicales:

Brassicaceae), Brassica napus (L.) (Brassicales: Brassicaceae) and Brassica rapa var. rapa

(Brassicales: Brassicaceae) (Kennedy et al. 1979; Kavallieratos et al. 2004, 2007b; Rana

2006) Both species are resistant to several conventional insecticides such as organochlorines

or pyrethroids (Heather 1985; Gaurav et al. 2004). The cutworm, S. litura, is another

economically important insect pest in many countries including India. This is a polyphagous

pest that has nearly 150 host species like cotton, jute, corn, tea, tobacco as well as many

vegetables such as eggplants, bean and a number of Cruciferous and Cucurbitaceae plants

(Rao et al. 1993). This pest has also developed resistance against numerous pesticides

(Armes et al. 1997). E. vigintioctopunctata is a major pest that attacks potato (Rajagopal et

al. 1989) and feeds on other plants of family Solanaceae and Cucerbitaceae.

The use of SNPs will increase if value addition is carried out. So the possible

fungitoxic property of SNPs was also studied together with entomotoxic efficacy. Anti fungal

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assays were performed on two fungi, having medical and agricultural importance: Aspergillus

niger and Fusarium oxysporum. A. niger is a causative agent of post harvest black mold

disease in important crops like onions, potatoes etc. It is an opportunistic pathogen in

immuno compromised humans, causing aspergillosis (Person et al. 2010). F. oxysporum is a

fungal plant pathogen that causes Panama disease of banana (Baayen et al. 2000). Panama

disease is one of the most destructive plant diseases of modern times, affecting a wide range

of banana cultivars.

3.2 MATERIALS & METHODS

3.2.1 DE and nanoparticles

FS90.0s® was obtained from Fossil Shield Company (Fig. 3.1a). Ten formulations of

nanoporous alumino silicate NPs of “AL” series (AL-06-101 – AL-06-110) were developed

in Humboldt University, Berlin. Lab scale synthesis of SNPs was performed in our own

laboratory. Moreover, surface functionalized SNPs designed by our group, were synthesized

on mass scale by M. K. Implex, Canada. SNPs were prepared by the vapor phase method

(Swihart 2003) and had size range of 15 – 20 nm (Fig. 3.1b-d). According to their surface

modification they were named as hydrophilic, lipophilic or hydrophobic SNP. Some other

custom synthesized oxide NPs were procured from M K Implex, Canada. They were

hydrophilic, lipophilic and hydrophobic ZNP, hydrophilic TNP (Anatase), hydrophilic and

hydrophobic TNP (Rutile), hydrophilic ANP-α and ANP-γ. ZNP was synthesized by the

plasma vapor method (Chou et al. 1992) and had the size range of 50 – 350 nm (Fig. 3.1e).

TNP, having size range of 45 – 60 nm (Fig. 3.1f), was made by the sol-gel method (Tao et al.

2008). The aluminium isopropoxide method (Park et al. 2005) was used for the production of

ANP. ANP-α had size range of 35-45 nm (Fig. 3.1g), and ANP-γ had size range of 13-20 nm

(Fig. 3.1h). As crystalline impurity is hazardous to human beings, X-ray diffraction (XRD)

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pattern of all NPs was studied by Bruker AXS Analytical Instruments to check the presence

of crystalline impurity in NP sample (Fig. 3.2).

Fig. 3.1 Electron micrograph of nanoparticle formulations. (a) FE-SEM image of FS90.0s®,

(b) TEM image of hydrophilic SNP, (c) TEM image of lipophilic SNP, (d) TEM image of

hydrophobic SNP, (e) FE-SEM image hydrophilic ZNP, (f) TEM image of hydrophobic TNP

(Rutile), (g) TEM image of hydrophilic ANP-α, (h) TEM image of hydrophilic ANP-γ.

20 30 40 50 60 70 80

100

200

300

400

500

600

700

800

900

1000

A

Inte

nsity

2 Theta

a b dc

e ff g h

Fig. 3.2 XRD of nanoparticles. (a) FS90.0s®, (b) hydrophilic SNP, (c) lipophilic SNP, (d)

hydrophobic SNP, (e) hydrophilic ZNP, (f) hydrophobic TNP (Rutile), (g) hydrophilic ANP-

α, (h) hydrophilic ANP-γ.

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3.2.2 Insects

S. oryzae were reared on whole rice grain (IR64) at 30°C ± 1°C, 75 ± 5% R.H. in continuous

darkness (insects were inbred in our laboratory by sib-mating for 20 generations). The R.H.

was maintained by using saturated solution of sodium chloride (Winston et al. 1960). Adults

less than 2 weeks were used for the experiments. Parts of the shoot infested with nymphs of

L. pseudobrassicae were collected from Brassica napus plants from the agricultural farm of

Indian Statistical Institute, Giridih, Jharkhand, India. 2nd instar nymphs (in which wing pads

were yet to be developed) were selected under stereo microscope in the laboratory. These

nymphs were used within 12 hours of collection from the field. Both adults and larvae of E.

vigintioctopunctata were collected from bitter gourd plants (Momordica charantia L.) from a

farm at Ranaghat, a village in West Bengal, India. S. litura were cultured on leaves of the

castor oil plant (Ricinus communis L.) in Bidhan Chandra Krishi Viswavidyalaya, an

agricultural university in West Bengal, India.

3.2.3 Studying insecticidal efficacy of “FS90.0s®” and “AL”

Bioassays were performed in plastic containers having surface area of 28 cm2 to test the

pesticidal efficacy of “FS90.0s®” and all ten “AL” formulations (“AL-06-101” – AL-06-

110”) on E. vigintioctopunctata. All formulations were sprinkled on the surface of plastic

screw capped boxes (diameter: 6 cm, height: 6.5 cm) uniformly at a concentration of 8 mg

cm-2. The caps were perforated for aeration. Untreated containers were used as controls.

Subsequently 10 adult and larvae of E. vigintioctopunctata were introduced into control and

treated boxes. After 24 and 48 hours, the morality data was taken.

2 g of M. charantia leaf was given for E. vigintioctopunctata in each box. The leaves

were changed daily. All bioassays were performed in three replicates.

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3.2.4 Assessment of entomotoxic efficacy of surface functionalized silica nanoparticles

The insecticidal assay on S. oryzae was performed in plastic screw capped boxes (diameter:

6cm, height: 6.5cm). The caps were perforated for aeration. 20g of rice (IR64) was placed in

each box. Rice in each box was treated individually with the SNPs and bulk sized silica at

three dose rates - 0.5, 1 and 2g NP kg-1 rice. Then the boxes were shaken manually for

approximately 1 minute to achieve equal distribution of NPs on rice (Subramanyam et al.,

2000). For each dose, there were five replicates. In one additional set no NP was mixed with

rice and this set served as control. Then 20 adults of S. oryzae were introduced into each box.

All bioassays were performed at 30°C ± 1°C, 75 ± 5% R.H. Insect mortality was checked

after 1, 2, 4, 7 and 14 days.

The entomotoxicity assays on L. pseudobrassicae were carried out in plastic Petri

dishes (Tarson, diameter 9.5cm). All SNPs were thoroughly sprayed on the Petri dishes at 3

dose rates – 0.025, 0.05 and 0.1 mg NP cm-2. For every case there were five replicates. In the

control set, no NP was given. 20 second instar nymphs of L. pseudobrassicae were added in

each Petri dish. All assays were performed at 27°C ± 1°C, 70 ± 5% R.H. Mortality data were

taken after 12th hour of the experiment.

The bioassays on S. litura were carried out in plastic boxes (diameter: 10 cm, height: 12.5

cm). Surface functionalized SNPs were dusted uniformly on the bottom surface of plastic

containers at four dose rates 0.125, 0.25, 0.5 and 1 mg cm-2. No NP was dusted in the control

set. The containers were covered with muslin cloth to allow aeration and larvae were fed with

equal amount of Ricinus communis leaves. The leaves were changed daily. For each dose

there were five replicates. 10 second instar larvae of S. litura were introduced in each box

after dusting. All bioassays were performed at 30°C ± 1°C and 75 ± 5% R.H. Insect mortality

was checked after 24 hours.

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3.2.5 Assessment of entomotoxic efficacy of surface functionalized zinc oxide, titanium

dioxide and aluminium oxide nanoparticles

These surface funtionalized oxide NPs were tested for their efficacy on S. oryzae and L.

pseobrassicae. As these NPs were tested to compare their pesicidal potency with SNPs, assay

protocol was similar to previous bioassays on these two insects with SNPs.

3.2.6 Antifungal assay of silica nanoparticles on A. niger and F. oxysporum

Antifungal assay of NPs was carried out on one strain of A. niger and F. oxysporum. A wild

strain of A. niger (MTCC-10180) was identified by phenotypic features and partial gene

sequences in Microbial Type Culture Collection (MTCC, India). The MTCC – 1043 strain of

F. oxysporum was purchased from National Collection of Industrial Microorganisms (NCIM,

India). Fungitoxic efficacy of SNPs was studied on both of these fungi in terms of their effect

on radial growth of isolated fungus. Radial growth (zone diameter) was evaluated after 2 and

3 days in case of A. niger and after 5 days in case of F. Oxysporum using modified poison

food technique (Nene et al. 2002). In case of A. niger, spores were harvested with a sterile

0.8% tween-80 solution and the number of spores per ml was enumerated with a

haemocytometer. Aliquots of a spore suspension at the concentration of ~104 spores / ml

were used as inoculums. Antifungal media was prepared by mixing ethanolic suspension of

SNPs with potato dextrose agar (PDA) at the concentration of 500 ppm, 1000 ppm and 2000

ppm. Ethanol containing PDA plates were used as controls throughout the study. 5 µl of

spore suspension (containing ~50 spores) was then inoculated in triplicate per plate and

incubated at at 30°C for 72 hrs. Circular mycelia discs of uniform size made with cork borer

from F. oxysporum culture were inoculated in control and SNP mixed PDA in disposable

Petri plates. Radial growth of the fungus was measured manually by zone diameter measuring

scale.

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3.2.7 Data analysis

The data were analyzed by using a two-way or one-way ANOVA with R 2.14.2 software.

Means were separated by using the Tukey-Kramer (HSD) test, at P = 0.05.

3.3 RESULTS & DISCUSSION

3.3.1 Insecticidal effect of “FS90.0s®” and “AL” on E. vigintioctopunctata

“FS90.0s®” is an amorphous micron sized particle, derived from diatom fossils, but its

surface was synthetically made hydrophobic. The size range of “FS90.0s®” was 5 to 30 µm.

60 to 80% of this formulation was amorphous silicon dioxide and 12 to 16% was aluminium

oxide. Other components were potassium oxide, calcium oxide, magnesium oxide and iron

oxide. “AL-06” was also hydrophobic in nature and was made from amorphous silica by top

down approach. All ten “AL-06” formulations had nanopore and had an average surface area

of 800 m2 g-1.

Under natural laboratory condition (temperature 30ºC and 60% RH) 100% mortality

was obtained for adult E. vigintioctopunctata in case of “AL-06-103”. “AL-06-102”, “-104”

and “-105” were also highly effective (Fig. 3.3). In contrast, “AL-06-106” showed mortality

of 70% after 48 hours. This difference in effectiveness among the “AL-06” formulations was

due to the difference in hydrophobicity of different “AL-06” particles. According to Völk et

al. (2004), Weishaupt et al. (2004) and Faulde et al. (2006) hydrophobicity plays a major role

in effectiveness of the particles in high relative humidity. Particle size was also important for

their effectiveness. However, this is not true for this particular experiment because all the

“AL-06” formulations were of comparable size.

50

0

20

40

60

80

100

120

Control AL-101 AL-102 AL-103 AL-104 AL-105 AL-106 AL-107 AL-108 AL-109 AL-110 FS 90.0s

Mort

ality

Treatment

A

a

B

bb

b

bb

b

b

b

b

b

B

B

BBB

B

BB

BBB

Fig. 3.3 Mean mortality (± S.E.) of adult E. vigintioctopunctata exposed for 24 and 48 hour

with “FS90.0s®” and “AL-06” formulations at of 8 mg cm-2 dose. (24 hour mortality

followed by the same lower case letter is not significantly different, 48 hour mortality

followed by the same upper case letter is not significantly different; Tukey – Kramer HSD

test; P < 0.001).

“AL-06” formulations were not so much effective on the larvae. This result was not

unusual as the cuticular composition of adult and larval beetle is different. Only “Al-06-102”

and “AL-06-104” showed higher mortality in comparison with adults (Fig. 3.4).

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Fig. 3.4 Mean mortality (± S.E.) of E. vigintioctopunctata larvae exposed for 24 and 48 hour

with “FS90.0s®” and “AL-06” formulations at of 8 mg cm-2 dose. (24 hour mortality

followed by the same lower case letter is not significantly different, 48 hour mortality

followed by the same upper case letter is not significantly different; Tukey – Kramer HSD

test; P < 0.001)

3.3.2 Entomotoxic effect of surface functionalized silica nanoparticles on S. oryzae, L.

pseudobrassicae and S. litura

Insecticidal assay on S. oryzae

All main effects and their associated interactions were significant at P < 0.01 level (Table

3.1).

Table 3.2 shows that on day 1, 0.5 g kg-1 dose was not at all effective on the insects.

But hydrophilic SNPs showed considerable insecticidal property at 1 g kg-1 dose or above. At

0.5 g kg-1, lipophilic SNP was most effective causing mortality of almost 50% insects on day

2. Application of hydrophilic SNP at 1 g kg-1 could kill more than 80% of the insects. Greater

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than 90% mortality was obtained with all the SNPs when dosage rate was 2 g kg-1 (Table

3.3). Bulk sized silica caused only 23% mortality at this dose. After day 4, more than 90% S.

oryzae died when hydrophilic SNP was applied at the dose of 1 g kg-1. 70% insect mortality

was found with application of lipophilic and hydrophobic SNP at this dose. Hydrophilic

SNPs were not very effective at the lowest dose (Table 3.4). After 7 days of exposure, 95%

and 86% mortality were obtained with hydrophilic and hydrophobic SNPs at 1 g kg-1

respectively. Nearly 70% of the insects were killed when the rice was treated with lipophilic

SNP at 1 g kg-1 (Table 3.5). Almost all insects were killed when SNPs were applied at a dose

of 2 g kg-1. Bulk sized silica caused only 34% insect mortality even at the highest dose. At

the end of 2 weeks, nearly 90% of the adults died when hydrophobic and lipophilic SNP were

applied at the rate of 1 g kg-1 (in the case of hydrophilic SNP mortality was 96%).

Hydrophobic and lipophilic SNP could kill 70% of the insects at the lowest dose.

Approximately 40% insects were killed when bulk silica was applied at a dose of 2 g kg-1

(Table 3.6).

Table 3.1 ANOVA parameters for main effects and their associated interactions Source df Day 1 Day 2 Day 4 Day 7 Day 14 F P F P F P F P F P

Treatment 3 148.97 <0.001 223.51 <0.001 228.66 <0.001 210.24 <0.001 285.85 <0.001 Dose 3 233.27 <0.001 642.22 <0.001 592.36 <0.001 616.96 <0.001 879.08 <0.001 Treatment 9 40.23 <0.001 59.38 <0.001 40.31 <0.001 37.13 <0.001 46.23 <0.001 x dose

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Table 3.2 Mean mortality (± S.E.) of S. oryzae adults exposed for 1 day on rice treated with

bulk and nano silica at 3 dose rates with control

Nanoparticle 0 g kg-1 0.5 g kg-1 1 g kg-1 2 g kg-1

SiO2 – hydrophilic 0.0 ± 0.0 Aa 14.2 ± 6.6 Ba 67.0 ± 8.4 Ca 86.0 ± 7.4 Da SiO2 – hydrophobic 0.0 ± 0.0 Aa 6.0 ± 8.2 Aa 7.0 ± 7.6 Ab 42.0 ± 7.6 Bb SiO2 – lipophilic 0.0 ± 0.0 Aa 7.9 ± 4.2 Aa 7.0 ± 5.7 Ab 34.0 ± 8.2 Bb SiO2 - bulk 0.0 ± 0.0 Aa 3.0 ± 4.5 Aa 7.0 ± 5.7 Ab 17.0 ± 2.7 Bc within each column, means followed by the same lower case letter are not significantly

different, within each row means followed by the same upper case letter are not significantly

different; Tukey – Kramer HSD test; P = 0.05.

Table 3.3 Mean mortality (± S.E.) of S. oryzae adults exposed for 2 days on rice treated with



SiO2 – hydrophilic 1.0 ± 2.3 Aa 23.3 ± 7.7 Ba 89.0 ± 4.2 Ca 95.0 ± 3.5 Da SiO2 – hydrophobic 1.0 ± 2.3 Aa 34.0 ± 6.5 Ba 49.0 ± 9.6 Cb 97.0 ± 2.7 Da SiO2 – lipophilic 1.0 ± 2.3 Aa 49.5 ± 8.4 Bb 48.0 ± 5.7 Bb 97.0 ± 4.5 Ca SiO2 - bulk 1.0 ± 2.3 Aa 7.0 ± 4.5 Ac 11.0 ± 4.2 Ac 23.0 ± 4.5 Bb Within each column, means followed by the same lower case letter are not significantly






SiO2 – hydrophilic 2.0 ± 2.7 Aa 33.4 ± 7.7 Ba 94.0 ± 6.5 Ca 97.0 ± 2.7 Ca SiO2 – hydrophobic 2.0 ± 2.7 Aa 55.0 ± 9.4 Bb 74.0 ± 9.6 Cb 100.0 ± 0.0 Da SiO2 – lipophilic 2.0 ± 2.7 Aa 61.4 ± 7.4 Bb 71.0 ± 8.9 Bb 100.0 ± 0.0 Ca SiO2 - bulk 2.0 ± 2.7 Aa 10.0 ± 5.0 ABc 15.0 ± 3.5 Bc 31.0 ± 4.2 Cb Within each column, means followed by the same lower case letter are not significantly



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SiO2 – hydrophilic 2.0 ± 2.7 Aa 35.4 ± 8.3 Ba 95.0 ± 5.0 Ca 97.0 ± 2.7 Ca SiO2 – hydrophobic 2.0 ± 2.7 Aa 62.0 ± 9.1 Bb 86.0 ± 8.2 Ca 100.0 ± 0.0 Da SiO2 – lipophilic 2.0 ± 2.7 Aa 62.4 ± 5.6 Bb 71.0 ± 8.9 Bb 100.0 ± 0.0 Da SiO2 - bulk 2.0 ± 2.7 Aa 16.0 ± 5.5 Bc 21.9 ± 6.5 Bc 34.0 ± 5.5 Cb Within each column, means followed by the same lower case letter are not significantly



Table 3.6 Mean mortality (± S.E.) of S. oryzae adults exposed for 14 days on rice treated

with bulk and nano silica at 3 dose rates with control


SiO2 – hydrophilic 4.1 ± 2.3Aa 42.5 ± 9.1 Ba 96.0 ± 4.2 Ca 100.0 ± 0.0 Ca SiO2 – hydrophobic 4.1 ± 2.3Aa 69.0 ± 9.6 Bb 92.0 ± 6.7 Ca 100.0 ± 0.0 Ca SiO2 – lipophilic 4.1 ± 2.3Aa 69.2 ± 5.8 Bb 89.0 ± 2.2 Ca 100.0 ± 0.0 Ca SiO2 - bulk 4.1 ± 2.3Aa 23.0 ± 5.7 Bc 25.0 ± 6.1 Cb 40.0 ± 6.1 Db Within each column, means followed by the same lower case letter are not significantly



One exciting finding from these experiments was that no fresh insect infestation is

found in the SNP treated stored rice even after 2 months of treatment. The nanocides could be

removed completely by conventional milling process unlike sprayable formulations of

conventional pesticides, which leave residues on the stored grain.

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Insecticidal assay on L. pseudobrassicae

In the laboratory condition, all L. pseudobrassicae were active and healthy till 12 hours. So

mortality data was taken after this time period. When SNPs were applied at 0.025mg cm-2

dosage more than 60% aphids were dead, whereas nearly 70% aphids died when surface

functionalized SNPs were applied at 0.05mg cm-2. Nearly 80% of the aphids were killed at

0.1mg cm-2 dosage (Table 3.7).

Table 3.7 Mean mortality (± S.E.) of L. pseudobrassicae nymphs exposed for 12 hours to

nano silica at 3 dose rates with control

Nanoparticle 0 mg cm-2 0.25 mg cm-2 0.05 mg cm-2 0.1 mg cm-2

SiO2 – hydrophilic 0Aa 63 ± 8.36 Ba 75 ± 6.12 Ca 76 ± 7.41 Ca SiO2 – hydrophobic 0Aa 67 ± 8.36 Ba 72 ± 7.58 BCa 80 ± 10 Ca SiO2 – lipophilic 0Aa 62 ± 5.7 Ba 68 ± 8.36 BCa 78 ± 6.7 Ca (Two way ANOVA: nanoparticle F = 0.85, P ≤ 0.44; dose F = 435.66, P < 0.001;

nanoparticle x dose F = 0.59, P ≤ 0.74). Within each column, means followed by the same

lower case letter are not significantly different, within each row means followed by the same

upper case letter are not significantly different; Tukey – Kramer HSD test; P = 0.05.

Insecticidal assay on S. litura

Table 3.8 shows all the SNPs had considerable insecticidal effect on S. litura. Especially

hydrophilic SNP could kill more than 90% larvae at 0.125 mg cm-2 dosage and at 0.25 mg

cm-2 dose or above all the larvae were dead within 24 hours of the treatment. Within this time

period, lipophilic and hydrophobic SNP caused 78% insect mortality at 0.125 mg cm-2

dosage. Application of lipophilic SNP at 0.25 mg cm-2 could kill 90% larvae. At this dose

hydrophobic SNP killed 86% S. litura. 90% or more insect mortality was obtained when

dosage rate was 0.5 or 1 mg cm-2 in case of lipophilic and hydrophobic SNP.

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Table 3.8 Mean mortality (± S.E.) of S. litura adults exposed for 24 hours to nano silica at 4

dose rates with control

Nanoparticle 0 mg cm-2 0.125 mg cm-2 0.25 mg cm-2 0.5 mg cm-2 1 mg cm-2

SiO2 – hydrophilic 0Aa 94 ± 8.94Ba 100 ± 0.0Ba 100 ± 0.0Ba 100 ± 0.0 Ba SiO2 – hydrophobic 0Aa 78 ± 8.36Bb 86 ± 8.94BCb 90 ± 7.07BCb 96 ± 5.47 Ca SiO2 – lipophilic 0Aa 78 ± 8.36Bb 90 ± 7.07BCab 94 ± 5.47Cab 94 ± 8.94 Ca (Two way ANOVA: nanoparticle F = 14.48, P < 0.001; dose F = 650.76, P < 0.001;

nanoparticle x dose F = 1.85, P ≤ 0.86). Within each column, means followed by the same

lower case letter are not significantly different, within each row means followed by the same

upper case letter are not significantly different; Tukey – Kramer HSD test; P = 0.05.

3.3.3 Assessment of entomotoxic efficacy of surface functionalized zinc oxide, titanium

dioxide and aluminium oxide nanoparticles on S. oryzae and L. pseudobrassicae


All main effects and their associated interactions were significant at P < 0.01 level (Table

3.9). Table 3.10 shows that at 2g kg-1 ANP-γ was much more effective than other NPs after 1

day of treatment. On day 2, more than 90% insect mortality was obtained with ANP, when

dosage rate was 2g kg-1. In fact at this dosage all the insects were killed in ANP-γ treatment.

Nearly 40% insects were dead when hydrophilic TNP (Anatase), hydrophobic TNP (Rutile)

and lipophilic ZNP were applied at 2g kg-1 dosage (Table 3.11). After day 4, more than 90%

S. oryzae died when ANP was applied at the dose of 1g kg-1. More than 45% of the insects

died in the application of ZNP at 2g kg-1. 60% insect mortality was obtained when

hydrophilic TNP (Anatase) and hydrophobic TNP (Rutile) were applied at this dose (Table

3.12). After 7 days of exposure, both the ANPs could kill almost all the insects with 1g kg-1

dose (Table 3.13). In this time period hydrophobic TNP (Rutile) killed 93% insects at 2g kg-1

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dose, whereas at this dose all ZNPs caused nearly 60% insect mortality. After 2 weeks, 60 -

75% mortality was found in all three types of ZNP and TNP at 1g kg-1. When the dosage was

2g kg-1 all TNPs and ZNPs caused more than 90% and more than 80% insect mortality

respectively (Table 3.14).

Table 3.9 ANOVA parameters for main effects and their associated interactions Source df Day 1 Day 2 Day 4 Day 7 Day 14 F P F P F P F P F P

Treatment 10 64.80 <0.001 303.88 <0.001 201.72 <0.001 188.96 <0.001 208.72 <0.001 Dose 3 170.19 <0.001 121.87 <0.001 563.41 <0.001 814.68 <0.001 1360.45 <0.001 Treatment 30 31.85 <0.001 18.89 <0.001 29.01 <0.001 31.87 <0.001 34.85 <0.001 x dose

Table 3.10 Mean mortality (± S.E.) of Sitophilus oryzae adults exposed for 1 day on rice

treated with surface functionalized oxide nanoparticles together with their bulk counterpart at

3 dose rates

Nanoparticle 0 g kg-1 0.5 g kg-1 1 g kg-1 2 g kg-1 Al2O3 (α) – hydrophilic 0 Aa 10 ± 6.12 Aab 18 ± 7.5 Abc 30 ± 10.6 Bc Al2O3 (γ) – hydrophilic 0 Aa 8 ± 2.73 ABab 17 ± 5.7 ABb 89 ± 8.94 Ac Al2O3 – bulk 0 Aa 7 ± 2.73 Aab 16 ± 4.18 Abc 25 ± 3.53 Bc TiO2 (Anatase) – hydrophilic 0 Aa 4 ± 4.18 ABa 7 ± 7.58 BCa 5 ± 8.66 Ca TiO2 (Rutile) – hydrophilic 0 Aa 3 ± 2.73 ABa 2 ± 2.73 Ca 4 ± 6.51 Ca TiO2 (Rutile) – hydrophobic 0 Aa 3 ± 4.47 ABa 3 ± 2.73 Ca 27 ± 5.7 Bb TiO2 - bulk 0 Aa 7 ± 2.73 Ba 16 ± 4.18 Ca 25 ± 3.53 Ca ZnO – hydrophilic 0 Aa 1 ± 2.23 Ba 3 ± 4.7 Ca 6 ± 6.51 Ca ZnO – lipophilic 0 Aa 1 ± 2.23 Ba 1 ± 2.3 Ca 7 ± 4.47 Cb ZnO – hydrophobic 0 Aa 1 ± 2.23 Ba 2 ± 2.73 Cab 7 ± 4.47 Cb ZnO – bulk 0 Aa 2 ± 4.47 BCa 4 ± 4.18 Ca 3 ± 4.47 Ca

Within each column, means followed by the same lower case letter are not significantly



58

Table 3.11 Mean mortality (± S.E.) of Sitophilus oryzae adults exposed for 2 days on rice


3 dose rates

Nanoparticle 0 g kg-1 0.5g kg-1 1g kg-1 2g kg-1 Al2O3 (α) – hydrophilic 0 Aa 44 ± 10.8 ABb 63 ± 10.36 Bc 91 ± 10.83 Ad Al2O3 (γ) – hydrophilic 0 Aa 57 ± 21.67 Ab 87 ± 7.58 Ac 100 Ac Al2O3 – bulk 0 Aa 11 ± 4.18 CDb 22 ± 4.47 Cc 32 ± 4.7 Bcd TiO2 (Anatase) – hydrophilic 0 Aa 14 ± 8.2 CDbc 25 ± 6.12 Cc 43 ± 10.36 Bd TiO2 (Rutile) – hydrophilic 0 Aa 19 ± 6.51 CDb 18 ± 13.5 CDb 20 ± 5 CDb TiO2 (Rutile) – hydrophobic 0 Aa 8 ± 7.58 CDa 12 ± 7.58 CDa 41 ± 8.94 Bb TiO2 - bulk 0 Aa 3 ± 4.47 Da 5 ± 3.53 Da 6 ± 4.18 Da ZnO – hydrophilic 0 Aa 25 ± 6.12 BCb 28 ± 10.95 Cb 32 ± 8.36 BCb ZnO – lipophilic 0 Aa 16 ± 7.41 CDb 20 ± 6.2 CDb 41 ± 8.94 Bc ZnO – hydrophobic 0 Aa 16 ± 6.51 CDb 17 ± 5.7 CDb 36 ± 7.41 BCc ZnO – bulk 0 Aa 2 ± 4.47 Da 4 ± 4.18 Da 5 ± 6.12 Da






3 dose rates

Nanoparticle 0 g kg-1 0.5g kg-1 1g kg-1 2g kg-1 Al2O3 (α) – hydrophilic 1 ± 2.23 Aa 61 ± 6.51 Bb 91 ± 12.44 Ac 100 Ac Al2O3 (γ) – hydrophilic 1 ± 2.23 Aa 94 ± 6.51 Ab 95 ± 6.12 Ab 100 Ab Al2O3 – bulk 1 ± 2.23 Aa 18 ± 7.58 CDEFb 30 ± 3.53 Bc 43 ± 5.7 CDd TiO2 (Anatase) – hydrophilic 2 ± 2.73 Aa 23 ± 9.08 CDb 37 ± 8.36 Bb 60 ± 10 Bc TiO2 (Rutile) – hydrophilic 2 ± 2.73 Aa 34 ± 7.41 Cb 29 ± 9.61 Bb 32 ± 8.36 Db TiO2 (Rutile) – hydrophobic 2 ± 2.73 Aa 13 ± 7.58 DEFab 25 ± 10.6 Bb 59 ± 6.51Bc TiO2 - bulk 2 ± 2.73 Aa 6 ± 4.18 EFa 6 ± 4.18 Ca 11 ± 4.18 Ea ZnO – hydrophilic 1 ± 2.23 Aa 33 ± 11.51 Cb 39 ± 8.94 Bb 46 ± 10.83 BDb ZnO – lipophilic 1 ± 2.23 Aa 27 ± 9.08 CDb 30 ± 5 Bb 47 ± 9.08 BDc ZnO – hydrophobic 1 ± 2.23 Aa 21 ± 5.47 CDEb 25 ± 5 Bb 51 ± 4.18 BCc ZnO – bulk 1 ± 2.23 Aa 3 ± 4.47 Fa 7 ± 2.73 Ca 8 ± 7.58 Ea Within each column, means followed by the same lower case letter are not significantly



59



3 dose rates

Nanoparticle 0 g kg-1 0.5g kg-1 1g kg-1 2g kg-1 Al2O3 (α) – hydrophilic 2 ± 2.73 Aa 72 ± 9.08 Bb 100 Ac 100 Ac Al2O3 (γ) – hydrophilic 2 ± 2.73 Aa 94 ± 6.51 Ab 97 ± 6.7 Ab 100 Ab

Al2O3 – bulk 2 ± 2.73 Aa 24 ± 8.94 CDEb 38 ± 7.58 BCc 58 ± 5.7 Bd TiO2 (Anatase) – hydrophilic 3 ± 2.73 Aa 27 ± 10.36 CDb 45 ± 6.12 Bb 65 ± 9.35 Bc TiO2 (Rutile) – hydrophilic 3 ± 2.73 Aa 39 ± 4.18 Cb 36 ± 9.61BCb 56 ± 9.61 Bb TiO2 (Rutile) – hydrophobic 3 ± 2.73 Aa 16 ± 10.83 DEb 29 ± 6.51 Cb 93 ± 4.47 Ac TiO2 - bulk 3 ± 2.73 Aa 8 ± 2.73 Eb 8 ± 6.7 Db 16 ± 2.23 Cb ZnO – hydrophilic 2 ± 2.73 Aa 40 ± 7.9 Cb 47 ± 12.54 Bb 59 ± 9.61 Bb ZnO – lipophilic 2 ± 2.73 Aa 33 ± 8.36 Cb 40 ± 5 BCb 65 ± 12.74 Bc ZnO – hydrophobic 2 ± 2.73 Aa 33 ± 7.58 Cb 36 ± 4.18 BCb 62 ± 7.58 Bc ZnO – bulk 2 ± 2.73 Aa 8 ± 5.7 Ea 11 ± 4.18 Da 11 ± 6.51 Ca Within each column, means followed by the same lower case letter are not significantly





3 dose rates

Nanoparticle 0 g kg-1 0.5g kg-1 1g kg-1 2g kg-1 Al2O3 (α) – hydrophilic 3 ± 2.73 Aa 89 ± 9.61 Ab 100 Ac 100 Ad Al2O3 (γ) – hydrophilic 3 ± 2.73 Aa 100 Ab 99 ± 2.23 Ab 100 Ab Al2O3 – bulk 3 ± 2.73 Aa 31 ± 5.47 Cb 44 ± 6.51 Cc 70 ± 5 Dd TiO2 (Anatase) – hydrophilic 3 ± 2.73 Aa 47 ± 10.36 Bb 69 ± 9.61 Bc 94 ± 8.21ABCd TiO2 (Rutile) – hydrophilic 3 ± 2.73 Aa 51 ± 7.41 Bb 58 ± 12.04 BCb 97 ± 4.47 ABc TiO2 (Rutile) – hydrophobic 3 ± 2.73 Aa 25 ± 7.07 CDb 75 ± 10.6 Bc 99 ± 2.23 Ad TiO2 - bulk 3 ± 2.73 Aa 10 ± 3.53 Db 12 ± 2.73 Db 23 ± 4.47 Ac ZnO – hydrophilic 4 ± 2.23 Aa 62 ± 9.08 Bb 74 ± 10.83 Bbc 83 ± 11.51 CDc ZnO – lipophilic 4 ± 2.23 Aa 54 ± 10.83 Bb 62 ± 6.7 Bb 84 ± 6.51 BCc ZnO – hydrophobic 4 ± 2.23 Aa 54 ± 6.51 Bb 62 ± 10.36 Bb 82 ± 7.58 CDc ZnO – bulk 4 ± 2.23 Aa 10 ± 3.53 Dab 16 ± 4.18 Db 18 ± 6.7 Eb




60


All main effects and their associated interaction were significant (NP F = 571.95, P < 0.001;

dose F = 738.88, P < 0.001; NP x dose F = 65.64, P < 0.001).

ZNP was not at all effective against nymphs of mustard aphids even at the highest

dose (0.1mg cm-2). According to Tukey’s HSD test at 95% level of significance ANP was

most effective in killing them followed by hydrophilic Anatase and Rutile TNP. At the dose

rate of 0.025mg cm-2 more than 75% aphids were dead in case of ANP treatment. Both ANPs

caused more than 90% aphid mortality at 0.05mg cm-2 dosage. At 0.1mg cm-2 72% of the

aphids died when treated with TNP (Anatase), whereas 66% and 61% insect mortality was

observed in hydrophilic and hydrophobic TNP (Rutile) treatment respectively. Almost all the

aphids died in the case of ANP application at this dosage (Table 3.15).

Table 3.15 Mean mortality (± S.E.) of L. pseudobrassicae nymphs exposed for 12 hours to

oxide nanoparticles at 3 dose rates with control

Nanoparticle 0 mg cm-2 0.25 mg cm-2 0.05 mg cm-2 0.1 mg cm-2 Al2O3 (α) – hydrophilic 0 Aa 79 ± 4.18 Ba 96 ± 4.18 Ca 99 ± 2.23 Ca Al2O3 (γ) – hydrophilic 0 Aa 76 ± 6.51 Ba 93 ± 10.36 Ca 97 ± 4.47 Ca TiO2 (Anatase) – hydrophilic 0 Aa 60 ± 9.35 Bb 66 ± 8.94 Bb 72 ± 8.36 Bb TiO2 (Rutile) – hydrophilic 0 Aa 51 ± 7.41 Bb 62 ± 7.58 BCb 66 ± 8.21 Cb TiO2 (Rutile) – hydrophobic 0 Aa 49 ± 10.83 Bb 57 ± 8.36 Bb 61 ± 6.51 Bb ZnO – hydrophilic 0 Aa 0 ± 0.0 Ac 3 ± 4.47 Ac 2 ± 4.47 Ac ZnO – lipophilic 0 Aa 0 ± 0.0 Ac 1 ± 2.3 Ac 2 ± 2.73Ac ZnO – hydrophobic 0 Aa 1 ± 2.23 Ac 2 ± 2.73 Ac 3 ± 4.47 Ac




61

3.3.4 A comparative analysis of entomotoxic efficacy of silica and other oxide

nanoparticles on S. oryzae and L. pseudobrassicae


For a comparative analysis on the entomotoxic efficacy of SNP with other oxide NP

formulations only 1 g kg-1 dose was selected. Figure 3.5 shows that on day 1, hydrophilic

SNP was far more effective than all other nanocides. On day 2, nearly 90% insect mortality

was obtained with hydrophilic SNP and ANP-γ (Fig. 3.6). After day 4, more than 90% S.

oryzae died when hydrophilic SNP and both ANPs were applied. 70% insect mortality was

found with application of lipophilic and hydrophobic SNP (Fig. 3.7). After 7 days of

exposure, 95% and 86% mortality were obtained with hydrophilic and hydrophobic SNP

treatment respectively. Nearly 70% of the insects were killed when the rice was treated with

lipophilic SNP at this dosage. ANP could kill almost all the insects with 0.1g kg-1 dose (Fig.

3.8). At this dose all SNPs (except lipophilic SNP) and ANPs were equally effective (Tukey’s

HSD test). Hydrophilic ZNP and TNP (Anatase) killed nearly 45% insects, whereas 29 – 40%

insect mortality was obtained for other ZNP and TNP formulations (Fig. 3.8). After 2 weeks,

nearly 90% of the insects died when they were exposed to hydrophobic and lipophilic SNP.

In case of hydrophilic SNP mortality was 96%. At this dose rate almost all the insects were

dead when treated with ANP. 60 - 75% mortality was found in all three types of ZNP and

TNP treatment (Fig. 3.9).

62

0102030405060708090

100

Con

trol

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

ZnO

-hyd

roph

ilic

ZnO

-lipo

phili

c

ZnO

-hyd

roph

obic

TiO

2 (A

nata

se)-

hydr

ophi

lic

TiO

2 (R

utile

)-hy

drop

hilic

TiO

2 (R

utile

)-hy

drop

hobi

c

Al2

O3

(Alp

ha)

Al2

O3

(Gam

ma)

Mort

ality

Nanoparticle

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

TiO

2(A

nata

se)-

hydr

ophi

lic

TiO

2(R

utile

)-hy

drop

hilic

TiO

2(A

nata

se)-

hydr

opho

bic

Al 2

O3

(Alp

ha)

Al 2

O3

(Gam

ma)

Fig. 3.5 Mean mortality (± S.E.) of Sitophilus oryzae adults exposed for 1 day on rice treated

with surface functionalized silica and other oxide nanoparticles at 1 g kg-1 dosage.

0102030405060708090

100

Con

trol

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

ZnO

-hyd

roph

ilic

ZnO

-lipo

phili

c

ZnO

-hyd

roph

obic

TiO

2 (A

nata

se)-

hydr

ophi

lic

TiO

2 (R

utile

)-hy

drop

hilic

TiO

2 (R

utile

)-hy

drop

hobi

c

Al2

O3

(Alp

ha)

Al2

O3

(Gam

ma)

Mo

rta

lity

Nanoparticle

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

TiO

2(A

nata

se)-

hydr

ophi

lic

TiO

2(R

utile

)-hy

drop

hilic

TiO

2(A

nata

se)-

hydr

opho

bic

Al 2

O3

(Alp

ha)

Al 2

O3

(Gam

ma)

Fig. 3.6 Mean mortality (± S.E.) of Sitophilus oryzae adults exposed for 2 days on rice treated


63

0102030405060708090

100110

Con

trol

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

ZnO

-hyd

roph

ilic

ZnO

-lipo

phili

c

ZnO

-hyd

roph

obic

TiO

2 (A

nata

se)-

hydr

ophi

lic

TiO

2 (R

utile

)-hy

drop

hilic

TiO

2 (R

utile

)-hy

drop

hobi

c

Al2

O3

(Alp

ha)

Al2

O3

(Gam

ma)

Mo

rtali

ty

Nanoparticle

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

TiO

2(A

nata

se)-

hydr

ophi

lic

TiO

2(R

utile

)-hy

drop

hilic

TiO

2(A

nata

se)-

hydr

opho

bic

Al 2

O3

(Alp

ha)

Al 2

O3

(Gam

ma)



0102030405060708090

100

Con

trol

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

ZnO

-hyd

roph

ilic

ZnO

-lipo

phili

c

ZnO

-hyd

roph

obic

TiO

2 (A

nata

se)-

hydr

ophi

lic

TiO

2 (R

utile

)-hy

drop

hilic

TiO

2 (R

utile

)-hy

drop

hobi

c

Al2

O3(α

)

Al2

O3(γ)

Mo

rtali

ty

Nanoparticle

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

TiO

2(A

nata

se)-

hydr

ophi

lic

TiO

2(R

utile

)-hy

drop

hilic

TiO

2(A

nata

se)-

hydr

opho

bic

Al 2

O3

(Alp

ha)

Al 2

O3

(Gam

ma)



64

0102030405060708090

100

Con

trol

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

ZnO

-hyd

roph

ilic

ZnO

-lipo

phili

c

ZnO

-hyd

roph

obic

TiO

2 (A

nata

se)-

hydr

ophi

lic

TiO

2 (R

utile

)-hy

drop

hilic

TiO

2 (R

utile

)-hy

drop

hobi

c

Al2

O3(α

)

Al2

O3(γ)

Mo

rtali

ty

Nanoparticle

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

TiO

2(A

nata

se)-

hydr

ophi

lic

TiO

2(R

utile

)-hy

drop

hilic

TiO

2(A

nata

se)-

hydr

opho

bic

Al 2

O3

(Alp

ha)

Al 2

O3

(Gam

ma)

Fig. 3.9 Mean mortality (± S.E.) of Sitophilus oryzae adults exposed for 14 days on rice

treated with surface functionalized silica and other oxide nanoparticles at 1 g kg-1 dosage.

ZNP showed moderate toxicity to S. oryzae. Both Anatase and Rutile TNP could kill

substantial number of S. oryzae only after 7 days. The “speed of kill” is very important for

any insecticide (Subramanyam et al. 2000). Because if treated with “slow-acting” insecticidal

agent, half-dead insects can migrate from the treated substrate, lay eggs before death, and

continue to cause grain damage. Considering all these facts the most effective among the

NPs were ANP followed by SNP. Yet SNP has some advantages over ANP. Yang et al.

(2005) have shown that ANP in ground water inhibits the growth of carrot, cabbage,

cucumber, corn and soybean. SNP has no such adverse effect on plant health; rather silica

enhances structural rigidity and strength of plant (Epstein 1994). 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).

65


ZNP could not kill the mustard aphid even with the highest dose 0.1 mg cm-2. SNP and ANP

were highly effective in killing them, while TNP was moderately effective (Fig. 3.10 - 12).

At the dose rate of 0.025 mg cm-2 more than 60% and 75% aphids were dead in case of SNP

and ANP treatment respectively (Fig. 3.10). At 0.1 mg cm-2 nearly 80% aphids died when

treated with SNP and almost all the aphids became dead in case of ANP treatment. At this

dosage TNP caused 60 -70% insect mortality (Fig. 3.12).

0102030405060708090

100

Con

trol

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

ZnO

-hyd

roph

ilic

ZnO

-lipo

phili

c

ZnO

-hyd

roph

obic

TiO

2 (A

nata

se)-

hydr

ophi

lic

TiO

2 (R

utile

)-hy

drop

hilic

TiO

2 (R

utile

)-hy

drop

hobi

c

Al2

O3

(Alp

ha)

Al2

O3

(Gam

ma)

Mort

ality

Nanoparticle

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

TiO

2(A

nata

se)-

hydr

ophi

lic

TiO

2(R

utile

)-hy

drop

hilic

TiO

2(A

nata

se)-

hydr

opho

bic

Al 2

O3

(Alp

ha)

Al 2

O3

(Gam

ma)

Fig. 3.10 Mean mortality (± S.E.) of L. pseudobrassicae nymphs exposed to surface

functionalized silica and other oxide nanoparticles at 0.025 mg cm-2 dosage after 12 hours.

66

0102030405060708090

100

Con

trol

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

ZnO

-hyd

roph

ilic

ZnO

-lipo

phili

c

ZnO

-hyd

roph

obic

TiO

2 (A

nata

se)-

hydr

ophi

lic

TiO

2 (R

utile

)-hy

drop

hilic

TiO

2 (R

utile

)-hy

drop

hobi

c

Al2

O3

(Alp

ha)

Al2

O3

(Gam

ma)

Mo

rtali

ty

Nanoparticle

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

TiO

2(A

nata

se)-

hydr

ophi

lic

TiO

2(R

utile

)-hy

drop

hilic

TiO

2(A

nata

se)-

hydr

opho

bic

Al 2

O3

(Alp

ha)

Al 2

O3

(Gam

ma)



0102030405060708090

100

Con

trol

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

ZnO

-hyd

roph

ilic

ZnO

-lipo

phili

c

ZnO

-hyd

roph

obic

TiO

2 (A

nata

se)-

hydr

ophi

lic

TiO

2 (R

utile

)-hy

drop

hilic

TiO

2 (R

utile

)-hy

drop

hobi

c

Al2

O3

(Alp

ha)

Al2

O3

(Gam

ma)

Mo

rta

lity

Nanoparticle

SiO

2-hy

drop

hilic

SiO

2-lip

ophi

lic

SiO

2-hy

drop

hobi

c

TiO

2(A

nata

se)-

hydr

ophi

lic

TiO

2(R

utile

)-hy

drop

hilic

TiO

2(A

nata

se)-

hydr

opho

bic

Al 2

O3

(Alp

ha)

Al 2

O3

(Gam

ma)



67

3.3.5 Antifungal assay of SNPs on A. niger and F. oxysporum

None of the SNP formulation could inhibit the growth of MTCC - 10180 strain of A. niger.

Though it seemed that hydrophilic SNP at 2000 ppm was slightly effective on this fungus

after 48 hours (Fig. 3.12), no significant inhibition of zone diameter with respect to control

was noticed after 72 hours (Fig. 3.13). Except hydrophilic SNP at highest dosage (2000 ppm)

no other SNP had fungicidal effect on MTCC – 1043 strain of F. oxysporum (Fig. 3.14).

Figure 3.15 shows the photograph of radial growth of control and SNP treated A. niger after

72 hours and F. oxysporum after 5 days.

10.00

15.00

20.00

25.00

30.00

35.00

Zon

e di

amet

er (m

m)

Treatment

*

Fig. 3.13 Radial growth (± S. E.) of control and SNP treated A. niger after 48 hours [A =

hydrophilic, B = lipophilic and C = hydrophobic SNP].

*Significant difference vs. control, P < 0.05.

68

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

Zon

e d

iam

eter

(m

m)

Treatment

Fig. 3.14 Radial growth (± S. E.) of control and SNP treated A. niger after 72 hours [A =


10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

Zon

e d

iam

eter

(m

m)

Treatment

Fig. 3.15 Radial growth (± S. E.) of control and SNP treated F. oxysporum after 5 days [A =


*Significant difference vs. control, P < 0.05.

69

Fig. 3.16 Photograph of radial growth of control and SNP treated [A] A. niger (MTCC –

10180) after 72 hours, [B] F. oxysporum (MTCC – 1043) after 5 days [a = control, b =

hydrophilic, c = lipophilic and d = hydrophobic SNP treatment].

3.4 CONCLUSION

The use of inert dusts, particularly those based on silica, has found increasing use as stored

grain protectants (Golob 1997). Among them DE, composed mainly of amorphous micron

sized silica, has become most popular (Athanassiou et al. 2007, 2008) as an alternative to the

conventional pesticides which have mammalian toxicity. The use of nanomaterials in

agriculture is still at a rudimentary stage. Stadler et al. (2010) successfully applied nano

alumina against two stored grain pests S. oryzae and Rhyzopertha dominica (F.). But Yang et

al. (2005) showed that nano alumina in ground water inhibits the growth of carrot, cabbage,

cucumber, corn and soybean. Our study presents the entomotoxic potential of SNP which has

no adverse effect on plant growth; rather silica enhances structural rigidity and strength of

plant (Epstein 1994). Insect mortality due to SNP treatment was obtained at dose rates almost

comparable with those of commercially available DE formulations ranging from 500 to 5000

mg kg-1 (Subramanyam et al. 2000; Vardeman et al. 2007). SNP does not affect the looseness

(a)

(d)

(c)

(b)

500 ppm 1000 ppm 2000 ppm 500 ppm 1000 ppm 2000 ppm

(a) (c)

(b)

(d)

A B

70

and bulk density of grain mass like DE (Korunic 1997) even with the highest dose used in our

bioassay.

Now it is known that inhalation of crystalline silica causes silicosis (Zaidi 1970).

Even the DE formulation (“FS90.0s®”) used in our experiments had crystalline impurity

(Fig. 2a). Though SNPs used in our experiments are amorphous in nature and silica is a less

reactive material, it will be premature to comment on its toxicity in living system. It should

be applied in controlled environment until detailed toxicity study of NPs is performed.

One exciting finding from these experiments was that no fresh insect infestation was

found in the SNP treated stored rice even after 2 months of treatment. The nanocides can be

removed by conventional milling process unlike sprayable formulations of conventional

pesticides, leaving residues on the stored grain. So SNP has an excellent potential as stored

grain as well as seed protecting agent if applied with proper safety measures.

Though SNP possibly does not have any fungicidal effect, it could reduce the Bombyx

mori nuclear polyhedrosis virus (BmNPV) induced 100% lethality in silk worm larvae to

70% (Rahman et al. 2009). Our research group has also showed that hydrophobic SNP can

effectively kill Anopheles stephensi larvae (Goswami et al. unpublished data).

This study could lead to open up newer pathways of using silica nanomaterial based

technology in pesticide industry.

chapter three study on entomotoxic efficacy of silica...

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