International Biodeterioration & Biodegradation 91 (2014) 119e127

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International Biodeterioration & Biodegradation

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Insecticidal activity of eucalyptus oil nanoemulsion with karanja and

jatropha aqueous filtrates
Megha Pant a,1, Saurabh Dubey a, *, P.K. Patanjali b, 2, S.N. Naik a, 3, Satyawati Sharma a, 4
a
Centre for Rural Development and Technology, Indian Institute of Technology, 110016 Delhi, India
b
Formulation Division, Institute of Pesticide and Formulation Technology, Gurgaon 122016, Haryana, India

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to utilize the aqueous filtrate of de-oiled karanja (Pongamia glabra) and
Received 16 July 2013 jatropha (Jatropha curcas) cakes left after extracting oil for preparing biodiesel to enhance the activity of
Received in revised form eucalyptus oil (Eucalyptus globulus) as a pesticide by making a nanoemulsion for the control of Tribolium
16 November 2013
castaneum, a secondary pest of stored grains. The aqueous filtrate obtained from the solid cake pos-
Accepted 22 November 2013
Available online 19 April 2014
sessing insecticidal properties was used in place of distilled water to enhance the shelf life of eucalyptus
oil for a longer period of time. The eucalyptus oil nanoemulsion containing karanja and jatropha aqueous
filtrate with concentrations of 300 and 1500 ppm gave 88e100% mortality rates against T. castaneum
Keywords:
Biopesticides
adults within 24 h. The LC50 values recorded for the nanoemulsions with and without the aqueous filtrate
Nanoemulsion were 0.1646 mg/l and 5.4872 mg l
1, indicating greater toxicity for the nanoemulsion containing aqueous
Eucalyptus oil filtrate. The GCeMS analysis also indicated the degradation of the marker compound, i.e., 1, 8-cineole, in
Solid biodiesel waste both types of nanoemulsions, i.e., with and without the aqueous filtrate. Analysis with ATR-FTIR also
Tribolium castaneum confirmed the presence of 1,8-cineole in the nanoemulsion containing the aqueous filtrate. The nano-
emulsion formulation with the lowest average particle size, 77 nm, was chosen for extensive efficacy
trials against T. castaneum. It was found that the loss of eucalyptus oil by volatilization was stabilised in
the nanoemulsion containing the aqueous filtrate.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction storage pests destroy approximately one-third of the world’s food

production, valued annually at more than $100 billion, and among
Food grain losses due to insect infestation during storage are a which the highest losses (43%) occur in the developing world
serious problem, particularly in developing countries. Accumula- (Jacobson, 1982; Ahmed and Grainge, 1986). Storage pests destroy
tion of high levels of insect detritus may result in infestation of approximately one third of the world’s food production in devel-
grain, making it unfit for human consumption, and in loss of food oping Asian and African countries (Talukder, 2006). In the USA and
commodities, both in terms of quality and quantity. Insect- Canada, 20e26% of stored wheat was infested by stored-product
infestation-induced changes in the storage environment may pests (White et al., 1985). In India, losses caused by insects
cause warm, moist “hotspots” that provide suitable conditions for accounted for 6.5% of stored grains (Raju, 1984). Food grain pro-
fungal growth, which causes further grain loss (Rajashekar et al., duction in India has reached 250 million tonnes/year in the year
2012). It is estimated that more than 20,000 species of field and 2010e2012; (Pugazhvendan et al., 2012); in which nearly 25% was
damaged by pests (Rajashekar et al., 2010). The global post-harvest
grain losses caused by insect damage and other bio-agents range
from 10 to 40% (Papachristos and Stamopoulos, 2002). Small scale
* Corresponding author. Centre for Rural Development & Technology (CRDT),
farmers may lose as much as 80% of their stock due to insects after
Indian Institute of Technology (IIT), Delhi 110016, India. Tel.: þ91 011 26591162, þ91
09873713209; fax: þ91 011 26591121.
storing for 6e8 months (Kitch et al., 1992; Nukenine et al., 2002). In
E-mail addresses: mpmegha04@gmail.com (M. Pant), drsaurabh_dubey@ the northern provinces of Cameroon, where cereals play an
rediffmail.com, mpmegha04@gmail.com (S. Dubey), pkpatanjali@yahoo.com essential role as human food, the maize weevil Sitophilus zeamais
(P.K. Patanjali), snn@iitd.ac.in (S.N. Naik), ssharma@hotmail.com (S. Sharma). Motschulsky (Curculionidae) is the main pest in granaries (Ngamo
1
Tel.: þ91 09899807318.
2 Tinkeu et al., 2004).
Tel.: þ91 09811221652.
3
Tel.: þ91 09818294067. Integrated pest management has now come up as a widely
4
Tel.: þ91 09873040455. accepted strategy in pest control; it includes post-harvest

http://dx.doi.org/10.1016/j.ibiod.2013.11.019
0964-8305/Ó 2014 Elsevier Ltd. All rights reserved.
120 M. Pant et al. / International Biodeterioration & Biodegradation 91 (2014) 119e127

infestation control, which involves the use of chemical (contact/ homogenizers, and ultrasound generators. Recently, a new low-
residual) insecticides along with fumigants (Shaaya et al., 1997). energy emulsification method has been developed by taking ad-
Although synthetic insecticides are commonly used to reduce the vantages of phase behaviour and properties to promote the for-
losses caused by insects, there is global concern about their nega- mation of ultra-small droplets with constant vigorous stirring, a
tive effects such as environmental pollution, pest resistance and “top-down” process (Ostertag et al., 2012). These low-energy
pesticide residues in food (Ogendo et al., 2008). The continuous techniques include self-emulsification, phase transition, and
application and excessive reliance on chemical pesticides have also phase inversion temperature (Izquierdo et al., 2005). In contrast,
resulted in toxicity hazards for non-target organisms and users solid nanoparticles are usually produced in a “bottom-up” process.
(Isman, 2006). The concept of “biopesticides” refers to products A recently developed process for generating nanometer-scale oil
obtained from natural sources such as animals, plants, and micro- droplets in water has been reported in the journal Angewandte
organisms, including ’’natural ingredient pesticides,’’ ’’microor- Chemie by Japanese researchers, who have developed a technique
ganism pesticides,’’ and ’’biochemical pesticides” that can help they named MAGIQ (monodisperse nanodroplet generation in
reduce pest populations and increase food production (Koul and quenched hydrothermal solution). This technique requires gener-
Walia, 2009). In recent years, studies have been focussed on the ally high temperature and pressure for the preparation of nano-
use of plant essential oils and their bioactive chemical constituents emulsions (Deguchi and Ifuku, 2013). In our nanoemulsion system,
as possible alternatives to synthetic insecticides (Rajendran and this technique is inapplicable because of the highly volatile nature
Sriranjini, 2008). Eucalyptus oil, with its wide range of desirable of the eucalyptus oil.
properties, can be used as a biopesticide for the control of various De-oiled seed cake of jatropha showed molluscicidal, insecti-
insect pests (Koul et al., 2008). cidal, fungicidal, and nematicidal activity. De-oiled seed cake of
Eucalyptus oil has been placed under the GRAS (generally karanja also possesses insecticidal and nematicidal activity (Kesari
regarded as safe) category by The U.S. Food and Drug Administra- et al., 2010; Sharma et al., 2011). Neem and karanja oil cakes in
tion and classified as nontoxic (USEPA, 1993) (Mahboubi et al., combination also showed insecticidal properties against mosquito
2013). Even the Council of Europe has approved use of eucalyptus vectors (Shanmugasundaram et al., 2008). Aqueous extract of kar-
oil as a flavoring agent in foods (>5 mg kg-1), candies, and con- anja and jatropha seed cakes had a significant effect on the termite
fectionary items (<15 mg kg
1) (Council of Europe, 1992). Vilela Odontotermes obesus (Sharma et al., 2011). But there is no relevant
et al. (2009) also studied the effect of eucalyptus essential oil by information regarding the use of these aqueous extracts to
contact and head space volatile assay on food-spoiling fungi. The formulate an essential oil to increase their shelf life and
antimicrobial effect of Eucalyptus globulus oil against bacteria effectiveness.
(gram-negative, gram-positive), fungi, and yeast in liquid as well as The LC50 value is the concentration of a pesticide that is lethal to
in the vapour phase was studied by Tyagi and Malik (2011). 50% of a population of test animals and is usually determined for a
The values of the oral and acute LD50 of eucalyptus oil and 1,8- specific exposure period. A pesticide with a lower LC50 is more toxic
cineole for rats is 4440 mg kg
1 body weight (BW) and than a pesticide with a higher number because it takes less of the
2840 mg kg
1 (BW), respectively (Regnault-Roger, 1997), rendering pesticide to kill half of the test animals (Mikhaiel, 2011).
it much less toxic than pyrethrins (with LD50 values of 350e This paper focuses on enhancing the bioefficacy of the highly
500 mg kg
1 BW [USEPA, 1993]) and even technical-grade pyre- volatile essential oil for a longer period of time by decreasing its
thrum (LD50 value e 1500 mg kg
1 BW [Batish et al., 2008]). volatility and also increasing its effectiveness using karanja and
The preparation of a submicron emulsion, called a nano- jatropha aqueous filtrate in the eucalyptus oil nanoemulsion
emulsion or miniemulsion, has emerged as a promising alterna- formulation.
tive for both intravenous and dermal application. Nanoemulsions
are fine oil-in-water dispersions having a droplet size ranging 2. Materials and methods
from 100 to 600 nm (Solans et al., 2003). Nanoemulsions are not
only kinetically stable but are also physically stable for a long Eucalyptus globulus essential oil was purchased from Gogia
time, with no apparent flocculation or coalescence (Bouchernal Chemicals, Delhi, India. Karanja and jatropha cakes were collected
et al., 2004). In addition to specific shelf life or stability depend- from the Centre for Rural Development Technology, I.I.T, Delhi; the
ing on the individual system or mixture of ingredients (active and butanol was from Merck; and Suprol T-60 (commercial
inert), low viscosity and droplet transparency make them an Polysorbate-80 or Tween-80) was supplied by Supreme Surfactant
attractive system for many industrial applications, such as drug Limited, Sonipat, Haryana, India.
delivery systems in the pharmaceutical field; they can also be
used in cosmetics and in pesticide delivery systems (Sonneville- 2.1. Test insect
Aubrun et al., 2004).
An emulsion is a thermodynamically unstable two-phase sys- Chosen as the test insect was the red flour beetle, Tribolium
tem consisting of at least two immiscible liquids, one of which is castaneum; it is a worldwide pest of stored products, particularly
dispersed in the form of small droplets throughout the other, and food grains, and a model organism for ethological research. The
an emulsifying agent. The dispersed liquid is known as the internal source of the insect was infested flour collected from the Depart-
or discontinuous phase, whereas the dispersion medium is known ment of Entomology, Indian Agricultural Research Institute, Delhi. A
as the external or continuous phase. If the aqueous phase consti- pure line culture of T. castaneum was maintained at 30e34  C and
tutes more than 45% of the total weight with a hydrophilic emul- 70% RH. The paper transfer method was used to transfer the beetles
sifier, oil/water emulsions are formed (Wang et al., 2007). Water/oil from the existing stock to the fresh jar. A clean paper strip was put
emulsions are generally formed if the aqueous phase constitutes in the glass jar containing beetles along with the flour. It was
less than 45% of the total weight and a lipophilic emulsifier is used inserted 1/3e1/4th of its length; adult beetles were collected from
(Abren and Massimo, 2008). the lower side of the strip. The strip was then withdrawn carefully
Nanoemulsions are metastable systems, and their stability de- and inserted into a jar of fresh flour. The strip was discarded and a
pends upon the method of synthesis or preparation (Gutiérrez new one was used to repeat the subculture. The method selects the
et al., 2008). The most common approach is high-energy emulsi- healthiest and most vigorous beetles and prevents any
fication, which involves high-shear stirring, high-pressure contamination.
 M. Pant et al. / International Biodeterioration & Biodegradation 91 (2014) 119e127 121

2.2. Preparation of karanja and jatropha aqueous filtrate Table 2

Amount of materials extracted from karanja and jatropha cakes using a mechanical
expeller reported in percentage values.
Five grams of jatropha and 5 g of karanja cakes in 100 ml cold
water were soaked for 24 h at room temperature (Sharma et al., S. no. Materials Amount Materials Amount
2011). After 24 h, the aqueous solution was filtered through extracted from extracted extracted from extracted
karanja cake (%) jatropha cake (%)
Whatman filter paper no. 1 and the filtrate was stored in an air-tight
bottle at 10  C and was used within a week’s time after filtration. 1. Nitrogen 4.0 Phorbol esters 0e0.8%
2. Phosphorous 0.9 Curcin protein 9.74%
After mechanical extraction of karanja and jatropha seeds on the
3. Potassium 1.3 Phytates 9.8%
expeller, the seed cakes were left as karanja and jatropha cakes. 4. Sulphur e Trypsin inhibitors 0.0084e0.00275
Estimation of material from the cakes after extraction is provided in 5. Karinjin 0.18 e
Table 2. 6. Oil content 5 e

2.3. Characterization of karanja and jatropha aqueous filtrate by

ATR-FTIR analysis 2.6. ATR-FTIR analysis of eucalyptus essential oil and their
nanoemulsion
Characterization of karanja and jatropha aqueous filtrate was
done using ATR-FTIR analysis. The carbonyl group in karanja and Infrared (IR) spectra were recorded on a Bruker alpha ATR-FTIR
jatropha filtrate was confirmed at 1737 and 1705 cm-1. A band at spectrophotometer using the attenuated total reflectance (ATR)
2923 cm-1 was due to axial deformation of CeH stretching vibra- technique, and values are expressed as gmax cm
1.
tion. The presence of a hydroxyl group (OH) in karanja and jatropha
aqueous filtrate was confirmed at 3300 cm-1, as shown in the
2.7. GCeMS analysis of eucalyptus oil after pre- and post-
spectra (Bar et al., 2009).
nanoemulsion

2.4. Preparation of eucalyptus oil nanoemulsion: low-energy Qualitative analysis of essential oil and its nanoemulsion was
emulsification method performed in triplicate on a GCeMS (Thermo Fisher, equipped with
Focus GC, DSQ II mass spectrometer and triplus auto sampler). A
Continuous phase of oil-in-water nanoemulsion was composed Thermo TR-5 MS capillary column (30 m  0.25 mm  0.25 mm) was
of distilled water and aqueous filtrate along with other ingredients used in Focus GC. The mass spectrometer was programmed in the
required for its preparation, as shown in Table 3. The active EI mode and ionization was done at 70 eV. Specifications were:
component (10% eucalyptus oil) was kept constant in the mass range, 40e250; carrier gas, helium; flow rate, 1.5 ml min
1;
formulation. split mode, 1:10; injection volume, 1 ml; injector port temperature,
The surfactant was melted at 45  C and was added to the 250  C; oven temperature, 50  C, increased to 100  C at the rate of
aqueous filtrate of karanja and jatropha to get a clear micro- 5  C min-1, then increased to 280  C at the rate of 25  C min-1 (hold
emulsion. Propylene glycol was also added afterwards. To this time 20 min). Quantification was done based on peak area.
microemulsion, eucalyptus oil was added slowly, with stirring at
750 rpm. The stirring was continued for 60 min to assure a stable
2.8. Insecticidal bioassay
and fine dispersion of oil particles in form of a nanoemulsion
(Ostertag et al., 2012). Samples of the nanoemulsion formulation
Insecticidal activity of the nanoemulsion formulation of euca-
prepared were observed after 24 h of their preparation to study the
lyptus oil with and without the karanja and jatropha aqueous
stability aspect. Also, the nanoemulsion samples prepared for bio-
filtrate was determined by direct contact application (Salunke et al.,
efficacy trials were evaluated in terms of droplet size, volatility, and
2005). A stock dilution (w/v) was prepared by dissolving the
stability.
desired quantity of nanoemulsion of eucalyptus oil in water. The
appropriate amount of each of the test materials containing 300,
2.5. Measurements of emulsion droplet size 600, 900, 1200, or 1500 ppm in 100 ml water was applied to filter
papers (Whatman no. 2, 4.25 cm diameter).
Measurements of droplet size and poly dispersity index of After complete drying at room temperature for 20 min, each
nanoemulsion prepared were done with a Beckman Coulter DelsaÔ filter paper was placed in the bottom of a petri dish (9 cm diam-
n
nano-particle analyzer. eter). Twenty unsexed adults (1 wk old) of Tribolium sp. were
released in each petri dish covered with a lid having holes for
proper aeration.
Table 1
Essential oil components of Eucalyptus globulus with highest percentage of 1,8-
cineole i.e. 66.28%. Table 3
Different concentrations of karanja and jatropha filtrate in different formulations.
Compound identified Percentage composition
S. Ingredients Formulation Formulation Formulation Formulation
cis-ocimen 21.33
no. (w/w) (in gm.) 1 (F1) 2 (F2) 3 (F3) 4 (F4)
Camphene 0.21
b-pinen 1.23 1. Distilled water 60 40 20 e
b-myrcene 1.00 2. Propylene glycol 12 12 12 12
1,8-cineole 66.28 3. Karanja and e 20 40 60
4-terpineol 0.52 jatropha
a-terpineol 1.73 filtrate
a-terpinol acetate 3.39 4. Surfactant 15 15 15 15
Aromadendrene 2.85 5. Eucalyptus oil 10 10 10 10
Globulol 1.43 6. Total 100 100 100 100
122 M. Pant et al. / International Biodeterioration & Biodegradation 91 (2014) 119e127

Table 4 3.2. ATR-FTIR analysis of eucalyptus oil nanoemulsion

Effect of karanja and jatropha aqueous filtrate on different properties viz. droplet
size, polydispersity index, percentage mortality and LC50 value.
The active component in eucalyptus oil is 1,8-cineole, which is
S. Properties Formulation Formulation Formulation Formulation responsible for its insecticidal activity (Yang et al., 2004). Confir-
no. F1* F2 F3 F4* mation of the active component in the nanoemulsion formulations
1. Aqueous karanja e 20 40 60 prepared was done by using an ATR-FTIR technique. The bicyclo-
and jatropha comphane compound was identified at 984 cm-1 due to CH2
filtrate (w/w)
wagging vibration (Sirvaityte et al., 2011).
in percentage
2. Distilled water 60 e e e Other characteristic but less intensive bands of 1,8-cineole
2. Droplet Size 569 233 103 77 (Fig. 2) that are seen in the ATR-FTIR spectrum can be attributed
(nm) at to CeOeC symmetrical (1079 cm-1) and asymmetrical (1214 cm-1)
product day
stretching, as well as to CH3 symmetrical deformation modes
3. Polydispersity 0.8 0.113 0.223 0.278
index
recognized at 1374 cm-1. The same bands appear in the nano-
4. Percentage (%) 80 88 90 88 emulsion formulation of eucalyptus oil due to incorporation of 1, 8-
mortality cineole as a main marker compound (Fig. 3). After two months of
(300 ppm) nanoemulsion preparation, there was no shifting of band in the
5. LC50 (mg/l) 5.4872 0.1256 0.1256 0.1646
ATR-FTIR spectra.
*
FI ¼ Distilled water based nanoemulsion of eucalyptus oil. Phorbol esters (Phasukarratchai et al., 2012) and karinjin are the
*F4 ¼ Karanja and jatropha aqueous filtrate based nanoemulsion of eucalyptus oil.
major constituents of jatropha and karanja cake (Table 2) that were
responsible for its biopesticidal properties (Baswa et al., 1990;
Verma et al., 2011). Their presence in the aqueous filtrate was
Control sets were made, in which the same numbers of adults confirmed by ATR-FTIR analysis (Fig. 1).
were released in petri dishes containing filter papers treated with
nanoemulsion. Treated and control sets were maintained at 3.3. Effect of karanja and jatropha aqueous filtrate on bioefficacy
30  2  C and 68  2 RH and in a 12:12 light: dark photoperiod. All
treatments were set up in three replicates and percentage mor- The effect of karanja and jatropha aqueous filtrate was studied
talities were determined 24 and 48 h post-treatment. Test insects by preparing nanoemulsion formulations with different concen-
were considered dead when, prodded with a fine brush (maximum trations and determining the LC50 of the samples. The nano-
three times), they showed no appendage movement. The LC50 value emulsions were divided into four groups, i.e., F1, F2, F3, and F4, with
was determined by log-probit regression using the SPSS 10.0 for 0, 20, 40, and 60% of aqueous filtrate of karanja and jatropha cake as
Windows/Microsoft Excel Programme. a continuous phase along with combination of 60, 40, 20, and 0% of
distilled water respectively (Table 3).
The maximum amount of karanja and jatropha aqueous
2.9. Probit analysis filtrate used for the preparation of nanoemulsion formulations
was restricted up to 60% and below that concentration there was
The Probit method is a parametric statistical procedure for no change in the LC50 value and hence the bioefficacy, as shown
estimating the LC50 value and the associated 95% confidence in- in Table 3. The aqueous filtrate of karanja and jatropha itself also
terval. The analysis consists of transforming the observed propor- showed some mortality, i.e., 31e60% (Table 5) for Tribolium sp.,
tion mortalities with a probit transformation, and transforming the but when it was used as a continuous phase in nanoemulsion
effluent concentrations to log10. This involves estimation of linear formulation of eucalyptus oil, the mortality results (Table 5) were
regression parameters, using an iterative approach. The estimated 88e100% at different concentrations of formulations prepared.
LC50 and associated confidence interval are calculated from the The variation of the toxicity of the nanoemulsion of eucalyptus oil
estimated linear regression parameters. Due to the intensive nature with and without the aqueous filtrate against T. castaneum is
of the calculations for the estimated LC50 and associated 95% con- shown in Fig. 4 (a). From the graph it can be seen that nano-
fidence interval using the probit method, it is recommended that emulsions of eucalyptus oil containing the aqueous filtrate were
the data be analyzed by a computer program, e.g., the SPSS Data more toxic and effective as compared to free eucalyptus essential
Programme (Rath et al., 2011). oil and its nanoemulsion formulation without using this filtrate
as a continuous phase. It is clear from Fig. 4a that there is not
3. Results and discussion much difference in the insecticidal activity of the two nano-
emulsion formulations containing distilled water and aqueous
3.1. GCeMS analysis of eucalyptus oil filtrate.
Fig. 4b shows the variation of insecticidal activity of the nano-
GCeMS analysis of Eucalyptus globulus essential oil showed the emulsion of eucalyptus oil with and without the aqueous filtrate
presence of 10 components that formed 99.97% of the total oil against T. castaneum after 2 months. From this figure, it is clear that
composition, with 1,8-cineole (66.28%), cis-ocimen (21.33%), and a- the toxicity of the nanoemulsion prepared using this filtrate as a
terpineol (3.39%) as major components (Table 1). The 1,8-cineole continuous phase remains the same, but there is considerable
content of Eucalyptus globulus oil reported in the literature varied decrease in insecticidal activity of the nanoemulsion formulations
between 18 and 66% (Cimanga et al., 2002). The wide variation in prepared using distilled water. The LC50 values of the formulations,
chemical composition of the oil could be attributed to environ- i.e., F1, F2, F3, and F4 (Table 4) indicate that F4 was more toxic than
mental and agronomic factors (Ahmadouch et al., 1985), as well as F2 and F3, but F1 was not as toxic as F4, F3, and F2 because of the
the extraction procedure (Jimenez-Carmona and Luque de Castro, absence of aqueous filtrate as a continuous phase in the formula-
1999). Since the insecticidal activity of essential oil is reportedly tion (Fig. 4a and b).
dependent upon its monoterpene content, its characterization may This decrease in insecticidal activity of the nanoemulsion pre-
help in deriving a general idea about the oil constituents respon- pared with distilled water may be due to the volatility of the active
sible for its bioactivity (Kumar et al., 2012). component, i.e., 1,8-cineole, in eucalyptus oil. This was confirmed
 M. Pant et al. / International Biodeterioration & Biodegradation 91 (2014) 119e127 123

Fig. 1. ATR-FTIR* spectra of karanja and jatropha (1:1) aqueous filtrate. * For analyzing the liquid samples by ATR-FTIR, a drop of sample is placed over the crystal (sample stage).
After the cleaning crystal properly, an infrared background is collected with a drop of water on the crystal so that water effect in the sample’s spectrum can be cleared, liquid is
simply poured onto the crystal. The whole crystal must be covered while performing qualitative analysis of the sample.

using GCeMS analysis. From this analysis, it was found that there The foregoing results indicate that biopesticidal constituents of
was a 5% degradation of active component, i.e., 1,8-cineole, after 2 eucalyptus oil (Table 1) and aqueous filtrate, i.e., 1,8 cineole, kar-
months in the formulation prepared using distilled water but there injin, and phorbol esters (Table 2), play a major role in increasing
was no change in concentration of the active component in the the efficacy and shelf life of the nanoemulsion (Fig. 5a, b) formu-
formulation (Fig. 5a and b) prepared using aqueous filtrate, as lation of eucalyptus oil against the stored grain pests (Solsoloy,
shown in the chromatogram. 1993; Silva et al., 2012).

Fig. 2. ATR-FTIR spectra profile of essential oil of Eucalyptus globulus.

124 M. Pant et al. / International Biodeterioration & Biodegradation 91 (2014) 119e127

Fig. 3. ATR-FTIR* spectra of nanoemulsion of eucalyptus oil containing aqueous filtrate. * For analyzing the liquid samples by ATR-FTIR, a drop of sample is placed over the crystal
(sample stage). After the cleaning crystal properly, an infrared background is collected with a drop of water on the crystal so that water effect in the sample’s spectrum can be
cleared, liquid is simply poured onto the crystal. The whole crystal must be covered while performing qualitative analysis of the sample. The eucalyptus oil nanoemulsion of
concentration 300 ppm was analysed using the technique.

3.4. Particle size

Nanoemulsions have been considered advantageous for opti-

mizing delivery of water-insoluble compounds or active in-
gredients (Wang et al., 2007). The small size of the droplets allows
them to deposit or spray uniformly on target areas (Solans et al.,
2003). Wetting, spreading, and penetration may also be enhanced
as a result of the low surface tension of the whole system and the
low interfacial tension of oil in water droplets due to nano-range
particle size (Tadros et al., 2004). The mean droplet size of the
nanoemulsion formulation prepared using distilled water was very
high, i.e., 569 nm, as compared to the nanoemulsion formulation
containing aqueous filtrate. Since dispersity is the ratio of standard

Table 5
Effect of karanja and jatropha filtrate alone on percentage mortality.

S. no. Ingredients Concentration Percentage LC50 (mg/l)

mortality

1. Aqueous filtrate 0.3 31.65 0.9844

0.6 40.00
0.9 48.30
1.2 51.65
1.5 60.00
Control 0.00
2. F1 0.3 80.00 5.4872
0.6 90.00
0.9 91.50
1.2 93.00
1.5 95.00
Control 0.00
Fig. 4. (a): Percentage mortality at different concentrations (ppm) of eucalyptus oil
3. F4 0.3 88.00 0.1646
nanoemulsion containing aqueous filtrate, nanoemulsion containing distilled water
0.6 91.50
and karanja and jatropha aqueous filtrate at zero month. A F4: Nanoemulsion þ60%
0.9 98.00
aqueous filtrate - F1: Nanoemulsion þ distilled water : Aqueous Filtrate: Filtrate of
1.2 98.00
karanja & jatropha before formulation. (b): Percentage mortality at different concen-
1.5 100.00
trations (ppm) of eucalyptus oil nanoemulsion containing aqueous filtrate and nano-
Control 0.00
emulsion containing distilled water after two months period. A F4:
FI ¼ Distilled water based nanoemulsion of eucalyptus oil. Nanoemulsion þ60% aqueous filtrate - F1: Nanoemulsion þ60% distilled water.
F4 ¼ Karanja and jatropha aqueous filtrate based nanoemulsion of eucalyptus oil. *Values are given as means  standard deviation (n ¼ 3).
Fig. 5. (a): Chromatogram of eucalyptus essential oil nanoemulsion with aqueous filtrate at zero month period. (b): Chromatogram of eucalyptus essential oil nanoemulsion with
aqueous filtrate after two months period.
126 M. Pant et al. / International Biodeterioration & Biodegradation 91 (2014) 119e127

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