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Application of diethanolamide surfactant derived from palm oil to improve

the performance of biopesticide from neem oil
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International Conference on Biomass: Technology, Application, and Sustainable Development IOP Publishing
IOP Conf. Series: Earth and Environmental Science1234567890
65 (2017) 012005 doi:10.1088/1755-1315/65/1/012005

Application of diethanolamide surfactant derived from palm

oil to improve the performance of biopesticide from neem oil

F N Nisya1, D Prijono1,2 and A Nurkania1

1
Surfactant and Bioenergy Research Center (SBRC), Bogor Agricultural University,
Jl. Pajajaran No.1. Bogor, Indonesia
2
Department of Plant Protection, Bogor Agricultural University, Bogor ,Indonesia

Email : [email protected]

Abstract.The purpose of this research was to improve the performance of organic pesticide de-
rived from neem plant using diethanolamide surfactant (DEA) derived from palm oil in con-
trolling armyworms. The pesticide was made of neem oil. Neem oil is a neem plant product
containing several active components, i.e. azadirachtin, salanin, nimbin, and meliantriol which
act as a pesticide. DEA surfactant acts as a wetting, dispersing and spreading agent in neem oil
pesticide. The neem oil was obtained by pressing neem seeds using a screw press machine and
a hydraulic press machine. DEA surfactant was synthesized from methyl esters of palm oil ole-
in. Pesticide formulation was conducted by stirring the ingredients by using a homogenizer at
5,000 rpm for 30 minutes. Surfactant was added to the formulation by up to 5%. Glycerol, as
an emulsifier, was added in to pesticide formulations of neem oil. The efficacy of the pesti-
cides in controlling armyworms fed soybean leaves in laboratory was measured at six concen-
trations, i.e. 10, 13, 16, 19, 22, and 25 ml/L. Results showed that the neem oil used in this
study had a density of 0.91 g/cm3, viscosity of 58.94 cPoise, refractive index of 1.4695, surface
tension of 40.69 dyne/cm, azadirachtin content of 343.82–1.604 ppm. Meanwhile, the aza-
dirachtin content of neem seed cake was 242.20 ppm. It was also found that palmitic (31.4%)
and oleic (22.5%) acids were the main fatty acids contained in neem oil. As the additive mate-
rial used in neem oil in this study, diethanolamide surfactant had a pH of 10.6, density of
0.9930 g/cm3, viscosity of 708.20 cP, and surface tension of 25.37 dyne/cm. Results of CMC,
contact angle, and droplet size analyzes showed that diethanolamide surfactant could be added
into insecticide formulation by 5%. Results of LC tests showed that on Spodoptera litura the
LC50 and LC95 values were 13 and 22 ml/L, respectively. Neem oil was found to inhibit the
development of Spodoptera litura and its larval molting process.

1. Introduction
Surfactant is a compound which has an ability to reduce surface tension of a medium. It can be pro-
duced biochemically or in a synthetically chemical way. It is able to reduce surface tension because it
has both hydrophilic (like water) and hydrophobic (like fat/oil) groups. These polar and non-polar
parts of surfactant make it able to make water and oil mix well in a mixture. According to [1], in gen-
eral, the functions of a surfactant are to reduce surface and interfacial tension, to increase the stability
of dispersed globules, and to control the formation type of emulsion, such as oil in water (O/W) or wa-
ter in oil (W/O). Today, surfactant is used as a component of adhesive material, coagulating agent,
wetting agent, foaming agent, emulsifier, penetrating agent, and dispersing agent [2]. Diethanolamide
surfactant is a surfactant whose molecules have no charges and its hydrophilic and hydrophobic prop-

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65 (2017) 012005 doi:10.1088/1755-1315/65/1/012005

erties are resulted from the existence of ester oxygen and hydrocarbon ester groups. Hydrocarbon
groups consist of carbon-carbon and carbon-hydrogen bonds. In organic molecules, these bonds are
non-polar bonds and hydrophobic. As these parts get longer, the solubility of the molecules in water is
low. According to [3], alcohol and ester groups consist of polar molecules. This property makes ester
oxygen groups hydrophilic. Diethanolamide can be produced in two ways, namely through (1) the
reaction between methyl ester and diethanolamide and (2) reaction between fatty acid and diethanola-
mide.

Insecticide from neem oil has been long known and used by farmers to organically control pests in
plants. Among other plant insecticides available in the market today, neem oil is the one which has
low toxicity for beneficial organisms. This has made it a promising plant insecticide [4]. The target
pests of neem oil include A. culicifacies [5], Ceraeochrysa claveri [6,7] Diaphorina
ri [8], Mamestra brassicae [9] Nilaparvata lugens Stal [10], Pieris brassicae [11[, Limnoperna for-
tunei (Dunker 1857) [13], and Spodoptera frugiperda [13].

Neem oil is extracted from seeds of neem plants. Neem plants belong to the family of Meliaceae and
are found to be limited in Indonesia and the Philippines-Azadirachta excelsa Kack [14]. Today, neem
oil is well known all over the world as a source of phytochemicals for human health and pest control.
It contains at least 100 active components but triterpenoid, better known as limonoids, is the main
component and azadirachtin which has a pesticide effect is the most important component. Aza-
dirachtin has a molecular weight of 720 g/mol [4]. In Indonesia, neem plants are found alongside the
north coast of Java, stretching from Indramayu to Banyuwangi. In addition, they are also found in
Nganjuk, Jombang, Blitar, Ponorogo, Madiun, Bojonegoro, Bondowoso, Gianyar, Negara, and East
Lombok [15].

One of the disadvantages of neem oil is that it is less stable under the field condition as a result of its
high photodegradation level, short staying time, and slow killing power compared to non-plant pesti-
cides [16, 17, 18]. Environmental factor and method of extraction gave significant effects on the
composition of neem oil. As a result, there is no standard active material in the composition of this
plant insecticide and this restricts the use of it in agricultural pest control [19,20,21]. Therefore, in its
application, an additive material which can stick, wet, and disperse it to the all parts of leaves, is re-
quired to make pest control more effective. One of the additive materials that can be used for this pur-
pose is diethanolamide (DEA) surfactant. This study was aimed at improving the performance of or-
ganic pesticide made of neem seed oil by using DEA surfactant from palm oil in controlling Spodop-
tera litura pests.

2. Materials and Methods

2.1 The process of neem oil production and the analysis of its physicochemical properties
Dried neem seeds were pressed by using a screw press. The cake produced from this pressing process
was repressed to take the remaining oil in the cake out. The crude oil obtained from this pressing pro-
cess was left alone for 24 hours to separate the sediment which was still found in the crude oil. The
sediment was then pressed by using a hydraulic press to take the oil out from the sediment. This crude
oil was then analyzed for its azadirachtin content and physicochemical properties. Measurements were
taken on azadirachtin content, refractive index, optical rotation, density, viscosity, surface tension, sol-
ubility, and contact angle. The analysis of physicochemical properties of neem oil was done at the
laboratories of Surfactant and Bioenergy research Center of Bogor Agricultural University and Center
of Agro industries, Indonesian Ministry of Industry.

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65 (2017) 012005 doi:10.1088/1755-1315/65/1/012005

2.2 The process of DEA surfactant production and the analysis of its physicochemical properties
DEA surfactant was produced by using an amidation reactor of 25 L/batch scale. The surfactant was
synthesized by reacting methyl ester of palm oil with diethanolamide at the mole ratio of 1:2 by using
1% NaOH 30% as a catalyst at 140 oC and the speed of 100-200 rpm for 3-5 hours.

2.3 Determination of optimum concentration of DEA surfactant as an additive material in the

formulation of plan insecticide from neem oil.
This trial was done to obtain the concentration of DEA surfactant that could dissolve neem oil to form
a stable emulsion as indicated by its droplet size analyzed by using a ICC 50 HD microscope (1000 x
magnification), had small contact angle as analyzed by using a Contact Angle Analyzer Phoenix 300,
and had a small surface tension as analyzed by a Critical Micelle Concentration (CMC) method. The
common way of CMC determination is done by measuring the surface tension of surfactant solution as
the function of concentration. DEA surfactant was dissolved in neem oil in the concentration of 1, 2,
3, 4, 5, 6, 7, 8, 9, and 10%. The surface tension of these surfactant solutions was measured by using a
Spinning Drop Tensiometer at Surfactant and Bioenergy Research Center, Bogor Agricultural Univer-
sity.

2.4 Formulation of neem insecticide by using DEA surfactant

Insecticide formulation process was done by using a homogenizer. The formulation was made by mix-
ing 90-95% neem oil, 1-10% DEA surfactant (in accordance with DEA surfactant concentration test
results), and 1-5% supporting material to improve the stability of pesticide emulsion. The formulation
process was done for 30-60 minutes at the speed of 5,000-6,000 rpm. Glycerol as much as 3% was
used as the supporting material in this study.

2.5 Assessment of the effects of lethal concentration of neem formula on S. litura larvae
The resulted insecticide formula was tested at six concentration rates (10, 13, 16, 19, 22, and 25 ml/l)
expected to cause death of 15-95% tested insects. Each insecticide formula was diluted with distilled
water containing surfactant at the concentration similar to that of each neem oil formula. As a control,
distilled water containing surfactant at the concentration similar to that contained in each formula was
diluted to reach the concentration of 25 ml/l. For each treatment, 60 instar III larvae of Spodoptera
litura were put into 6 petri dishes (10 in each petri dish). The tissue paper put in the lower and upper
parts of each dish was wetted every day in order to maintain the moist of the leaves in the dishes. On
day-2 and 4, treatment or control leaves were added. Observation on the mortality of tested insects
was done on day-9.

3. Results

3.1 Neem oil production and the analysis of its physicochemical properties
The extraction process of neem oil was done by using screw and hydraulic pressing methods resulted
in an oil yield of 12.3%. This analysis was aimed at assessing the properties of neem oil produced.
The physicochemical properties of neem oil are listed in Table 1. It was shown that neem oil produced
in this study had 0.91 g/cm3 density, 58,94 cPoise viscosity, and 1.4695 refractive index. As a com-
parison, at 250C water and olive oil have viscosity of 0.890 and 81 cPoise, respectively.

Neem oil produced in this study was found to have a surface tension of 40.69 dyne/cm which was con-
sidered to be higher than those (25-35 dyne/cm) of commercial pesticides available in the market.
High level of surface tension of neem oil has made the direct application of this oil in the field was less
effective as neem oil could not dissolve in water nor spread evenly to the surface of leaves. High sur-
face tension also made neem oil applied to the plants easy to fall down or evaporate as it could not at-
tach to the leaves. This made the spraying of this oil, particularly during rainy season, a waste as the
oil was easily washed away by the rain.

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IOP Conf. Series: Earth and Environmental Science1234567890
65 (2017) 012005 doi:10.1088/1755-1315/65/1/012005

Table 1. Physicochemical properties of neem oil

No. Physicochemical properties Value
1 Density 0.91 g/cm3
2 Iodine number 74.5 g Iodine/100 g
3 Saponification number 189 mg KOH/g
2 Viscosity 58.94 cPoise
3 Surface tension 40.69 dyne/cm
4 Refractive index 1.4695
5 Optical rotation Not measurable/dark
6 Azadirachtin content of neem oil 343.82—1,604 ppm
7 Azadirachtin content of neem seed cake 242.20 ppm
8 Fatty acid composition (%)
Butyric (C4) 0.98
Lauric (C12) 7.29
Palmitic (C16-0) 31.4
Stearic (C18-0) 9.45
Oleic (C18-1) 22.5
Linoleic (C18-2) 10.2
Linolenic (C18-3) 1.94

Results of the HPLC analysis showed that the azadirachtin content of neem oil was 343.82–1,604 ppm
while that of neem seed cake resulted from the pressing process was 242.20 ppm. These results were
lower than the azadirachtin content found by [22], namely 0.1-0.5% of dried neem seed weight.
Meanwhile, palmitic (31.4%) and oleic (22.5%) acids were found as the main fatty acids of neem oil.

3.2 Physicochemical properties of DEA surfactant

DEA surfactant produced in this study was found to have pH of 10.6, density of 0.9930 g/cm3, viscosi-
ty of 708.20 cP, and surface tension of 25.37 dyne/cm. These were in line with what was found by
[23]. The pH level of diethanolamide used by Kirk-Othmer was 9-10. The value of surface tension of
DEA surfactant produced in this study indicated that this surfactant could lower the surface tension of
water by 61-65% from 72 dyne/cm.

3.3 Determination of optimum concentration of DEA surfactant as an additive material in the

formulation of plant insecticide from neem oil

a. CMC Analysis on surface tension

Determination of optimum concentration of DEA surfactant by using a CMC analysis in order to as-
sess the highest reduction in the surface tension of neem oil. It was shown that the addition of DEA
surfactant at the concentration of 5 and 6% could lower the surface tension of neem oil from 40.69 to
23.82 dyne/cm. The addition of DEA surfactant higher than 6% was found to make the surface tension
of neem oil increase back. According to [24], the addition of surfactant to a solution would lower the
surface tension of the solution. After a certain concentration was reached, the surface tension would
stay constant even though the concentration of surfactant was increased. The adding of surfactant to
the amount that exceeded this concentration would make the surfactant aggregate to form micelles.
The concentration when miselles start to form is known as the Critical Micelle Concentration (CMC).
Surface tension will keep lowering down until CMC is reached. Once CMC is reached, surface ten-

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IOP Conf. Series: Earth and Environmental Science1234567890
65 (2017) 012005 doi:10.1088/1755-1315/65/1/012005

sion will remain constant indicating that the interfacial is saturated and the formed micelles are in their
dynamic equilibrium state with their monomers.

45.00
40.00
35.00
30.00 37.29
Dyne/cm

33.77 32.91
25.00 29.84
26.73 25.14 28.21 28.44 28.38
20.00 24.66
15.00
10.00
5.00
0.00
Dea Dea Dea Dea Dea Dea Dea Dea Dea Dea
1% 2% 3% 4% 5% 6% 7% 8% 9% 10%
Concentration DEA surfactant in neem oil

Figure 1. Surface tension values of neem oil at various concentrations of DEA surfactant

CMC is a standard parameter in the characterization of surfactant solution [25] as, in general, it indi-
cates the minimum concentration needed to obtain surfactant association structure. A number of stud-
ies on the methods or techniques of CMC determination and factors affecting CMC of various types of
surfactants have been done. The common methods or techniques used to determine CMC are based on
surface tension [26], capillary electrophoresis [27] and optical density [28]. Theories have also been
developed to explain the occurrence of CMC [29,30,31].

b. Analysis of Contact Angle

The analysis of contact angle was done to determine the contact angle of formulation which was dilut-
ed to 1%. This analysis was necessary to assess the ability of DEA surfactant in spreading neem oil on
the surface of leaves. The graph of the decrease in contact angle of neem oil is depicted in Figure 2.
A contact angle is an angle formed between the surface of tested material (leaves) and neem oil
dropped onto the surface of the tested material. The mean contact angles resulted from the addition of
DEA surfactant to neem oil solution were
about 17.82 to 52.52°. It is shown in the above graph that the addition of DEA surfactant at a concen-
tration higher than 5% resulted in smaller contact angles. This indicated that the solution of neem oil
was able to spread well on the surface of the leaves resulting in very small contact angles. Insecticide
formulation solution was expected to have small contact angles as small as possible to make it able to
attach to and spread over the surface of targeted objects. A contact angle close to 0° shows that neem
oil droplets do not only attach to but also spread over well on the leaves. A contact angle close to 90°
indicates that neem oil droplets are able to attach to the leaves but they do not spread well on the
leaves. A contact angle close to 180° indicates that neem oil droplets do not attach to the leaves at all
and even they directly slip down from the leaves.

The mixture of neem oil and surfactant with small contact angle indicated that the mixture spread over
the leaves well when it is applied by farmers in the field. Therefore, DEA surfactant produced in this
study could be utilized as a spreading and wetting agent in insecticide formula. Solution containing
surfactant spreading all over the leaves could penetrate and spread over the fine hair on the surface
through a capillarity force, while the droplets of solution containing solution with no surfactant did not
spread but only attached to the surface of leaves [32].

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65 (2017) 012005 doi:10.1088/1755-1315/65/1/012005

50.00
45.00
40.00
35.00
30.00
(o)

25.00
20.00
15.00
10.00
5.00
0.00
DEA DEA DEA DEA DEA DEA DEA DEA DEA DEA
1% 2% 3% 4% 5% 6% 7% 8% 9% 10%
Concentration DEA surfactant in neem oil

Figure 2. Effects of DEA surfactant concentration in neem oil on contact angles

c. Analysis of droplet size

This analysis was done to determine the droplet size of the emulsion formed by the mixture of neem
oil and DEA surfactant diluted in water until the formula concentration was 1%. The analysis was
done to ensure that the emulsion system of formulation product had a micro size. Small-sized droplets
made the emulsion more kinetically stable so that sedimentation and creaming during storage could be
avoided [33]. The graph showing the decrease in droplet size as a result of the addition of DEA sur-
factant produced in this study is depicted in Figure 3.

Emulsion is a preparation containing two phases, usually water and oil, which are not mixed. In an
emulsion, a liquid is dispersed into droplets and the other liquid is stabilized with an emulsifier or a
suitable surfactant [34]. An emulsion system is generally easy to get damaged by the addition of ener-
gy or by the passing by of time. This problem can be overcome by making the size of droplets smaller
and by using a stabilizing agent such as surfactant. Small-sized droplets can be reach through the use
of appropriate ratio of surfactant and oil and right temperature. In this study, it was found that the
mixture of neem oil and DEA surfactant diluted in water to a concentration of 1% had mean droplet
size of 1.61 to 8,17 µm. Smaller size droplets were obtained when higher concentration of DEA sur-
factant was added into neem oil.

As stated by [35], the length of hydrocarbon chains of a surfactant also affects the performance of sur-
factant in emulsion formation. A surfactant with long hydrocarbon chains increases the protective role
of steric in minimizing the possibility of particle or droplet mergers. Olein, as a synthetic feedstock of
DEA surfactant in this study, is composed dominantly of long chain fatty acids. Therefore, it was
found in this study that the size of droplets formed was smaller as more DEA surfactant was added.

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IOP Conf. Series: Earth and Environmental Science1234567890
65 (2017) 012005 doi:10.1088/1755-1315/65/1/012005

10.00

8.00

6.00
µm
4.00

2.00

0.00
DEA DEA DEA DEA DEA DEA DEA DEA DEA DEA
1% 2% 3% 4% 5% 6% 7% 8% 9% 10%
Concentration of DEA surfactant

Figure 3. Effects of DEA surfactant concentration in neem oil on droplet size

3.3 Lethal Concentration Test of Selected Insecticide Formula

Results of determination of optimum concentration of DEA surfactant as an additive material in the
formulation of plant insecticide from neem oil showed that DEA surfactant concentration of 5% was
the most effective in lowering the surface tension and contact angle of neem oil. This determination
was made as no statistical differences were found in the effectiveness of DEA surfactant concentration
of 5 and 6% in lowering the surface tension and contact angle of neem oil.

Results of the application test of insecticide formula of neem oil by using 5% DEA surfactant showed
that the mortality rates of tested insects at 2, 4, 6, and 9 days after treatment (DAT) were about 0 -
1.8%, 5 - 15.2%, 6.8 - 55.8%, and 22.8 - 100%, respectively. The effect of neem formula treatment on
mortality rates significantly increased at 9 DAT. This was caused by the existence of active substanc-
es, particularly azadirachtin, which inhibited insect development and this effect was observable long
after the treatment was given. Larvae which fail to molt can live for significantly long time before
they finally die [36].

Based on the values of LC50 and LC95 at 9 DAT and results of probit analysis of mortality rates of test-
ed larvae, the neem formula by using DEA surfactant was y = -3.13 + 7.28 x, with LC50 and LC95 val-
ues of 13 and 22 ml/L, respectively. This result was lower than [13] who found that neem oil of Aza-
dirachta indica juss could kill Spodoptera frugiperda Smith at mortality rates of 83.33 +/- 0.83% and
89.58 +/- 0.90% at 4 and 6 DAT, respectively. Differences in efficacy might be caused by differences
in types of pests and methods and techniques of insecticide production. Most neem product used in
this study was neem oil which was obtained by pressing neem seeds. A solvent was then added to ex-
tract the active substances of the pesticide. Different methods and techniques applied in neem oil pro-
duction resulted in different concentration of active substances and biological efficacy [37].

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Table 2 Mortality of larva S. litura larvae treated with neem oil insecticide with dilution concentration
of 10-25 ml/l at 2, 4, 6, and 9 days after application
Formulation concentration Mortality rate of tested insects (%) (n)a
(ml/l) 2 days 4 days 6 days 9 days
10 1.7 (60) 5.1 (59) 10.5 (57) 22.8 (57)
13 1.7 (60) 12.3 (57) 17.5 (57) 43.9 (57)
16 0 (60) 5.0 (60) 6.8 (59) 55.9 (59)
19 0 (60) 11.9 (59) 22.4 (58) 79.3 (58)
22 1.7 (59) 6.9 (58) 29.3 (58) 94.8 (58)
25 1.8 (55) 15.2 (53) 55.8 (52) 100 (52)
a
Number of tested insects in each treatment was 60 instar III larvae of S. litura. Figures in bracket (n) show the
number of larvae which were found back at a certain observation time. Figures less than 60 indicate that there
are larvae which were not found back at the time of observation for unknown reasons.

Insecticide of neem oil is a systemic insecticide [38]. The work mechanisms of neem oil include in-
hibiting feeding, affecting hormone function in juvenile stages, reducing ecdysone, deregulating
growth, altering development and reproduction, suppressing fertility, sterilizing, repelling oviposition,
and disrupting molting processes [39]. Based on the complex active substances they contain, deriva-
tive products of neem oil can be used as antifeedants, growth regulators, sterilants, anti-oviposition
agents, and repellents [40].

4. Conclusions
Neem oil used in this study had a density of 0.91 g/cm3, viscosity of 58.94 cPoise, refractory index of
1.4695, surface tension of 40.69 dyne/cm, azadirachtin content of neem seed oil of 343.82 – 1,604
ppm, and azadirachtin content of neem seed cake of 242,20 ppm. The main fatty acid composition
consisted of palmitic (31.4%) and oleic (22.5%) acids. As an additive substance in neem oil insecti-
cide, the ethanolamide surfactant used in this study had a pH of 10.6, density of 0.9930 g/cm3, viscosi-
ty of 708.20 cP, and surface tension of 25.37 dyne/cm. based on the results of CMC analysis and the
values of contact angle and droplet size, the most effective concentration of DEA surfactant used as an
additive in neem oil insecticide was 5%. The LC50 and LC95 values on Spodoptera litura were 13 and
22 ml/L, respectively. Neem oil worked by inhibiting the development of Spodoptera litura and its
larval molting process.

Acknowledgement
Financial support from Ministry of Research, Technology, and Higher Education through the National
Strategic Excellence Research Scheme was acknowledged.

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