CHAPTER I

THE PROBLEM
Introduction

Rice is the staple food for more than 60 percent of the population globally (Gross

and Zhaob, 2014). The Philippines is the world’s eighth-largest rice producer and its

arable land totals 5.4 million hectares and it is the staple food for most of the Filipinos

(Rice Pedia, 2012). Reportedly, Philippines is experiencing vast amount of losses of crops

every year because of the different factors. One of which is the rice black bugs

(Caoyonan, 2014).

In Philippines, Scotinophara coarctata (rice black bug) was believed to originate

from Palawan then Mindanao. For many years, it has been a rice pest in Malaysia

(Catindig and Heong, 2005). Here in South Cotabato, rice black bug is one of the most

persistent pest among rice (Pablico, 2007).Adult one is just around 8-9 mm (OISAT, 2007)

but is very destructive that 10 adult rice black bugs per hill can cause losses of up to 35

percent (Tacio, 2014). Therefore, causes failure in harvest and losses of income.

Derris elliptica Benth, locally recognized as ‘Tubli’ is known to have pesticidal

activity. Its twigs and roots have been used as natural pesticides in artisanal fisheries and

aquaculture ponds in Nigeria. In the study, ethanolic extracts of dried roots elicited a toxic

reaction by fish with discouration, gulping of air, erratic swimming, loss of reflex, settling

at the bottom motionless (Flores and Santiago, 2013). The present study was undertaken

to test the secondary metabolites present and the insecticidal effect of Derris elliptica

Benth ethanolic root extract as applied to rice black bugs (Scotinophora coarctata).
Statement of the Problem

This study identified the phytochemical constituents and determined the insecticidal

activity of Derris elliptica Benth (Tubli) root extract. Specifically, this sought answers to

the following questions:

1. Which of the following phytochemical constituents, namely alkaloids, flavonoids,

glycoside, phenolic compounds, saponins, steroids, tannins, and terpenoids are

present in the ethanolic root extract of Derris elliptica Benth (Tubli)?

2. Can ethanolic root extract of D. elliptica (Tubli) kill the adult rice black bug?

3. Is there a significant difference in the insecticidal activity among the 50%, 75% and 100

% concentrations of D. elliptica (Tubli) ethanolic root extract?

Scope and Delimitation

This study was delimited to phytochemical screening and insecticidal activity of

Derris elliptica Benth (Tubli) root extract as applied against Scotinophara coarctata (rice

black bugs).

This study followed the standard procedures in conducting the insecticidal effect

of the plant extract against rice black bugs. To strengthen this study, the researcher

conducted Phytochemical screening to determine the secondary metabolites present in

the root extract.

The presence of secondary plant metabolites including alkaloids, flavonoids,

glycoside, phenolic compounds, saponins, steroids, tannins, and terpenoids were

determined using qualitative analysis and the insecticidal activity through median lethal
dose (LD50). Each treatment was subjected to three replicates (R1, R2, and R3) using film

residue method.

This study used 8-9 mm adult rice black bugs as experimental animals. The

phytochemical screening and insecticidal property test were performed at NDMU

chemistry laboratory. This study were conducted last July, 2017.

Significance of the Study

This study provided information regarding the possible insecticidal effect of D.

elliptica (Tubli) ethanolic root extract as applied to rice black bugs. Result of this study

could be a great help to the farmers. By this, they will be informed about the effectiveness

of Tubli root extract as an insecticide against rice black bugs. In this way, they can avail

an insecticide which is cheaper, safety, and environmental-friendly. Moreover, they will

be encouraged to plant more Tubli on their respective backyards and appreciate its

benefit in their living. In addition it can benefit the department of agriculture, they will be

guided and value the usefulness of Tubli as an organic insecticide. More to that, it can

make the community realize how this vine can be helpful to them and they will be

encouraged to make it as a part in their backyard. The university could get concerned

and promote conducting of thesis that contributes to the wellness of the society and is

very beneficial to everybody. The researcher can comply for his master’s degree but more

importantly this gives him a feeling of satisfaction and fulfilment as he helped for the

improvement of the community and his country. Lastly, the future researchers may have

proper and deeper understanding about insecticidal properties of plants and also this

research could serve as their guide or reference for future biological research.
 Chapter II

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter contains some related literatures and related studies having bearing

on the study. This includes information regarding the phytochemical screening of the plant

secondary metabolites and the insecticidal evaluation concerning Deris elliptica Benth

(Tubli) ethanolic root extract as applied to rice black bugs.

Phytochemical Screening

Ding (2017) defined Phytochemicals as non-nutritive plant chemicals that have

protective or disease preventive properties. It is also the natural chemical compounds

found in plants that make up its color.

Phytochemical Screening is the process of separation and isolation of active

principle from plant sources (Sunli, 2017). In addition, phytochemical screening is the first

thing to be done before major discoveries of molecules or drug entities are known. It is

used to provide concrete knowledge and research to what plant active constituents have

potential to benefit mankind (Tacio, 2012).

Alkaloids

Seneca (2017) referred alkaloids as a special group of secondary compounds and

are part of an organism's adaptation mechanism to its living environment. They are not

toxic when stored, but become toxic as a result of cell pH change. Also, these are

compounds needed for cell activity and gene code realization in the genotype. They are
biologically significant as active stimulators, inhibitors and terminators of growth, a part of

an endogenous security and regulation mechanism (Von Linne, 2007).

Lastly, Wanse and Kuete (2013) termed that alkaloids are one of the most diverse

groups of secondary metabolites found in plants, marine organisms, and microorganisms.

They have an array of structural types, biosynthetic pathways, and pharmacological

activities.

Flavonoids

Szalay (2015) indicated that flavonoids are diverse group of phytonutrients (plant

chemicals) found in almost all fruits and vegetables. Along with carotenoids, they are

responsible for the vivid colors in fruits and vegetables. Flavonoids are the largest group

of phytonutrients, with more than 6,000 types.

Mateljan (2017) reported that flavonoids are a quite remarkable group of

phytonutrients that fall into the chemical category of polyphenols. They're perhaps

most famous for their rich diversity of color-providing pigments. The name of these

phytonutrients actually derives from their color-related chemistry, with the Latin

word flavus meaning "yellow." As a group, however, flavonoids are highly bioactive

and play a wide variety of different roles in the health of plants, animals, and human

health.

Glycosides

Kumar (2016) stated that glycosides are natural substances that contain

carbohydrates where glycosidic part of the molecule (cyclic form of sugars) is connected
with organic radical that is not a sugar (aglycone or genin). Pharma Tutor (2016) added

that glycosides are define as organic compound from plants and animal source, which on

enzymatic hydrolysis gives one or more sugar moieties along with anon sugar moiety.

Moreover, glycosides are water soluble compounds and insoluble in the organic

solvents. The glycone part is water soluble, insoluble in the organic solvents. While the

aglycone part is water insoluble, soluble in the organic solvents. And some glycosides

are soluble in alcohol (Kharat, 2017).

Phenolic Compounds

Phenolic compounds from medicinal herbs and dietary plants include phenolic

acids, flavonoids, tannins, stilbenes, curcuminoids, coumarins, lignans, quinones, and

others. Various bioactivities of phenolic compounds are responsible for their

chemopreventive properties (e.g., antioxidant, anticarcinogenic, or antimutagenic and

anti-inflammatory effects) and also contribute to their inducing apoptosis by arresting cell

cycle, regulating carcinogen metabolism and ontogenesis expression, inhibiting DNA

binding and cell adhesion, migration, proliferation or differentiation, and blocking signaling

pathways (Huang, Cai, and Zhang, 2010).

Additionally, de Lourdes Rei Giada (2013) described phenolic compounds having

common characteristic, the presence of at least one aromatic ring hydroxyl-substituted.

Another characteristic of these substances is that they are presented commonly bound

to other molecules, frequently to sugars (glycosyl residue) and proteins. The existence of

phenolic compounds in free form also occurs in plant tissues. However, it is less common,
possibly because they are toxic when present in the free state and detoxified, at least in

part, when bound.

Saponins

Liwa and Nwokocha (2017) affirmed that saponins are naturally occurring

surface-active glycosides produced by plants, lower marine animals, and some bacteria.

They occur constitutively in a great many plant species, in both wild plants and cultivated

crops. Yang and Yu (2014) added that by expressing a large diversity of structures on

both sugar chains and aglycones, saponins exhibit a wide range of biological and

pharmacological properties and serve as major active principles in folk medicines,

especially in traditional Chinese medicines. Furthermore, the amphiphilic nature of the

saponins enables them to act as soaps and detergents as they can dissolve membranes;

however, since the saponins can hemolyze erythrocytes they are highly toxic if they reach

mammalian blood (Boysen and Hearn, 2010).

Steroids

Steroids are a group of cholesterol derived lipophilic, low-molecular weight

compounds found in / derived from a variety of different marine, terrestrial, and synthetic

sources. All steroid classes and their metabolites play important roles in the physiology

and biochemistry of living organisms in which these are found (Sultan and Raza, 2015).

Klodas (2016) defined steroids as one of a large group of chemical substances

classified by a specific carbon structure. Steroids include drugs used to relieve swelling

and inflammation, such as prednisone and cortisone; vitamin D; and some sex hormones,

such as testosterone and estradiol.

Tannins

The term tannin refers to the use of tannins in tanning animal hides into leather;

however, the term is widely applied to any large polyphenolic compound containing

sufficient hydroxyls and other suitable groups to form strong complexes with proteins and

other macromolecules (Ashok and Upaddhyaya, 2012). Bate-Smith and Swain (2007)

determined tannins as “water-soluble phenolic compounds having molecular weights

between 500 and 3000; besides giving the usual phenolic reactions, they possess special

properties such as the ability to precipitate alkaloids, gelatin and other proteins”.

In nature the tannins are found worldwide in many different families of the higher

plants such as in chestnut and oak wood, Divi-Divi, Sumach, Myrobalaen, Trillo, Valonea

or plant galls; depending on their origin, their chemistry varies widely, having a molar

mass of up to 20000 D (Khanbabaee and van Ree, 2001).

Terpenoids

Terpenoids form a group of naturally occurring compounds majority of which occur

in plants, a few of them have also been obtained from other sources. Terpenoids are

volatile substances which give plants and flowers their fragrance. They occur widely in

the leaves and fruits of higher plants, conifers, citrus and eucalyptus (Yadav, Yadav, and

Goyal, 2014).

Insecticidal Activity

Gilbert (2014) quantified that insecticides are pesticides that are used against

insects. Most modern insecticides act by poisoning the nervous system with a dose large

enough (relative to the insects' size) to be lethal. They can be of two types: organic and
organic. Organic are those that contains carbon while inorganic are those that do not

have (Bennett, 2012). Film Residue method (Amin et al, 2012) and LD50 determination

(Khaluquzzaman and Sultana, 2006; Overgaard, 2014) is usually the way used to

determine insecticidal activity of a plant extract.

Derris elliptica Benth (Tubli)

Flores and Santiago (2013) described Derris elliptica Benth (Tubli) as a rambling

climber, with branches covered with brown hairs. Leaves are pinnate and 30 to 50

centimeters long. Leaflets are usually oblong, 9 to 13, when matured smooth above, and

subglaucous and silky beneath, 10 to 15 centimeters long, and about half as broad.

Racemes are lax, 15 to 30 centimeters in length, with reddish flowers in stalked clusters.

Pods are 5 to 8 centimeters long and contain 1 to 3 seeds, flat and reniform, olive, brown

or black.

This plant is used as a pesticide. A compound obtained from the roots has been

used as a fish poison for quite some time, but more recently, it was discovered that it

could be an effective insecticide (Starr, Martz, and Loope, 2016).

Scotinophora coarctata (Rice Black Bugs)

Online Information Service for Non-Chemical Pest Management in the Tropics

(2005) described the eggs of rice black bugs as round, greenish or pinkish in color. The

eggs are laid in groups of 15 in parallel rows on the lower leaves near the water surface,

on stems, roots and on soil cracks.

Tacio (2014) asserted that a black bug is only as big as a "black bean" but it is very

destructive. It sucks the juice from the midrib of leaves and panicles at the milk stage. In
most cases, it feeds on the basal part of the tillers most often at night. In addition, Catindig

(2015) reported that the insect is common in rained and irrigated wetland environments.

Damages are observed more frequently in dry season rice crops and densely planted

fields. Staggered planting of the rice crop and excessive nitrogen also favor the build-up

of the pest.

RELATED STUDIES

This section presents the local and foreign related studies for the better

understanding of this research. It may also present findings that can be used to strengthen

this study.

Foreign Studies

A study entitled “Screening for Larvicidal Activity in Some Thai Plants against Four

Mosquito Vector Species” was conducted in Thailand by Narumon et al (2005). Ninety-

six ethanolic extracts from various parts of 84 Thai plant species were tested for their

larvicidal activity against Aedes aegypti mosquitoes. Extracts from Rhinacanthus

nasutus, Derris elliptica, Trigonostemon reidioides, Homalomena aromatica, Stemona

tuberosa and Acorus calamus possessed high larvicidal activity, with LC50 values

between 16.0 and 48.2 mg/l. Petroleum ether (PE) and methanol (MeOH) extracts were

tested for their larvicidal activity against 4 mosquito vector species. The PE extract of R.

nasutus exhibited larvicidal effects against Ae. aegypti, Culex quinquefasciatus,

Anopheles dirus and Mansonia uniformis with LC50 values between 3.9 and 11.5 mg/l,

while the MeOH extract gave LC50 values of between 8.1 and 14.7 mg/l. D. elliptica PE
extract showed LC50 values of between 11.2 and 18.84 mg/l and the MeOH extract

exhibited LC50 values between 13.2 and 45.2 mg/l.

Another study entitled “Effects of Some Herbal Plant Extracts against Pharaoh Ant,

Monomorium Pharaonis (Linnaeus) was conducted in China by Tangchitphinitkan (2007).

The research was to evaluate the efficiency of three Thai herbs, i.e., tuba root (Derris

elliptica Benth.), yam bean seeds (Pachyrhizus erosus L.) and tea seed cake (Camellia

sp.) against adult worker of the Pharaoh ant (Monomorium pharaonis L.) The results

showed that the tuba root extracts exhibited LC50 against adult worker ca. 0.22 % w/v

whereas yam bean seed extracts showed LC50 against adult worker ca. 0.35 % w/v and

tea seed cake extracts showed LC50 against adult worker ca. 0.55 % w/v after 24 hours

exposure, respectively.

Additional study was made by (Akinbulumo, Fagbenro and, Fasakin, 2010) entitled

“Acute Toxicity of Ethanolic Extract Of Derris elliptica Roots to Oreochromis Niloticus

Fingerlings” where six graded concentrations of 0 (Control), 93, 139.5, 186, 232.5 & 279

mg/litre of Derris elliptica were used as treatments. These were applied in the form of

root powder solution to O. niloticus fingerlings in glass tanks A, B, C, D, E and F (where

“A” served as the control). Ethanolic extracts of dried roots of D. elliptica were tested for

toxicity under laboratory conditions with a 24h LC10 of 139.5mg/10L (weight/volume).

Toxic reaction exhibited by the fish includes discouration, gulping for air, erratic

swimming, loss of reflex, slow opercular movement and settling at the bottom motionless.

Histological examination of O. niloticus fingerlings showed some pathological changes.

Damage became severe with increasing concentration of the plant extracts.

Local Study

A study was also conducted by Amistad et al (2013) entitled “The Effectivity of

Tubli plant (Derris elliptica Benth.) root extract as mosquito larvicide compared to a

commercial larvicide (Abate 10SG). The experimental group treated with 100% Tubli

(Derris elliptica Benth.) root extract showed a perfect number of deaths (30 wrigglers).

The positive control group treated with the commercial larvicide also showed a great

number of deaths (26-29 wrigglers). The negative control group with only rain water

showed no deaths of larvae. Based from the data gathered, the researchers concluded

that extract from Tubli (Derris elliptica Benth) was more effective than the commercial

larvicide.
THEORETICAL AND CONCEPTUAL FRAMEWORK

The study basically exhibited the important relationship between the two variables:

the independent and the dependent variable. The phytochemical screening result of

Derris elliptica Benth was correlated with the dependent variable which is its insecticidal

property. The graphical representation of the Conceptual Framework of the study is show

on figure 1.

INDEPENDENT VARIABLE DEPENDENT VARIABLE

Derris elliptica Benth Derris elliptica Benth

• Phytochemical • Insecticidal Activity

Screening

Figure 1. Conceptual Framework

Hypothesis

This study tested the hypothesis using 0.05 level of significance.

1. Derris elliptica Benth (Tubli) root extract does not exhibit insecticidal property.

Definition of terms

This section provides the significant terms that were used in the study provided

with their conceptual and operational meaning for the better understanding of the

research.

Phytochemical Screening –conceptually, it is the process of separation and

isolation of active principle from plant sources (Sunli, 2017). Operationally, it is the first

test in the study used to determine the secondary metabolites present in Tubli root extract.

Insecticidal Activity – conceptually, it is the ability of a substance to kill insects

or control their growth (Perveen, 2012). Operationally, it was the second test that was

conducted and determined the final goal of the study.

Derris elliptica Benth (Tubli) – Conceptually, it is a rambling climber, with

branches covered with brown hairs. Leaves are pinnate and 30 to 50 centimeters long.

Leaflets are usually oblong, 9 to 13, when matured smooth above, and subglaucous and

silky beneath, 10 to 15 centimeters long, and about half as broad. Racemes are lax, 15

to 30 centimeters in length, with reddish flowers in stalked clusters. Pods are 5 to 8

centimeters long and contain 1 to 3 seeds, flat and reniform, olive, brown or black.

Operationally, it was used as the sample plant and its insecticidal effect against rice black

bug was determined in the study.

 Crude Ethanolic Extract – conceptually, it is concentrated preparation of a drug

obtained by removing the active constituents of the drug using ethanol as solvents

(Farflex, 2012). Operationally, it was the form of the Tubli root extract used in the study.

Scotinophara coarctata (Rice black bug) – Conceptually, it is the common pests

among rice (Pablico, 2007). Operationally, it was used as experimental animal subjected

in the evaluation.
 Chapter III

METHODOLOGY

This chapter contains the procedures that were used in conducting the study. It

gave details on the progress of the study that were done in a step-by-step method. The

following were included: Research Design Materials and Methods, Experimentation and

the General Procedure.

Research Design

This section covers the design on how the procedures or the methods of the study

were done. The first test was phytochemical screening for the root extract’s existing

secondary metabolites. The second test which is the most important was the insecticidal

property evaluation that were tested in rice black bugs, the experiment animals.

The researcher used lethal dosage lethality test in the study for the evaluation of

the insecticidal activity of Derris elliptica Benth root extract. It was used in measuring the

results on the insecticidal experiment because it is suited to the experiment that contains

only small numbers of treatments.

 Plant Specimen

(Avocado Bark)
bbn

Plant Root
Extract

Phytochemical
Insecticidal Activity
Screening
Evaluation

Alkaloids R1
T1 R2

Flavonoids R3

R1
Glycosides T2 R2

R3
Phenolic Compounds
R1
T3 R2
Saponins
R3

Steroids

Tannins

Terpenoids

Figure 2. Research Design

 Figure 2 shows the two types of tests that were used in the study. The plant

specimen undergone rota evaporation and the crude extract was subjected to test the

presence of secondary metabolites and insecticidal property of the plant’s root extract.

First test was phytochemical screening. Alkaloids, flavonoids, glycosides, phenolic

compounds, saponins, steroids, tannins, and terpenoids were evaluated through

qualitative analysis.

Lethal dose (LD50) method was used for the evaluation of the second test, the

insecticidal property of Derris elliptica Benth (Tubli) root extract. Lethal dose (LD50)

method was also used in measuring the results because it is suited to an experiment

involving only small number of treatments. The insecticidal activity of Derris elliptica Benth

(Tubli) was studied against rice black bugs (Scotinophara coarctata) using residual film

method. There were five treatments for the test; T1= 50% concentration, T2= 75%

concentration, T3= 100% concentration root extract. Each treatment had three

replications.

Research Locale

Plant materials were gathered from Purok 7, Brgy. Kinilis, Polomolok, South

Cotabato. The phytochemical screening analysis and the insecticidal activity evaluation

will be conducted at Polomolok National High School laboratory.

Photo credit to: Google map Domolok, Alabel, Sarangani Province

Photo credit to: Google map

Notre Dame of Marbel University
Research Sample

The samples of this study were 150 adult rice black bugs (8-9mm taken from a rice

field situated at Brgy. Lumakil, Polomolok, South Cotabato. For the insecticidal activity

evaluation, the samples were separated in three groups with three replication each which

were randomly selected.

Photo credit to: Google map Brgy. Lumakil, Polomolok, South Cotabato
Research Procedures

This section shows the procedures followed by the researcher in conducting the

study. The flow chart was used to show how the study can be done and served as the

researcher’s guide in conducting the study. It demonstrated how the two tests were

directed, the following are: phytochemical screening and the insecticidal activity

evaluation.
Gathering of Plant
Samples

Washing and Air-

drying

Plant Extraction

Phytochemical Insecticidal
Screening Activity Test

Film Residue
Method

Calculation of %
Mortality

Probit Analysis
(LD50)

Figure 3. Flow Chart

Materials and Instrumentation

The materials that were used in this research are the; micropipettes, disposable

gloves, test tubes, beaker, stirring rod, filter paper, rotary evaporator, Erlenmeyer flask,

funnel, Petri dish, test tubes, cling wrap, rice black bugs.

Preliminary Procedures

This gives information about the equipment that were used in conducting the

experiments. It also include the processes on how the plant specimen were gathered and

prepared, which were used in the experiment. In addition, it contains standard methods

or protocols for the following tests of phytochemical screening and the insecticidal

property activity evaluation. This supported the researcher in conducting the study in an

organized manner.

Gathering of Derris elliptica Benth (Tubli)

Fresh roots of Derris elliptica Benth (Tubli) were collected from at Brgy. Domolok,

Alabel, Sarangani Province. The gathered roots were freed from any external stimuli like

insects.

Preparation of Derris elliptica Benth (Tubli) root for Phytochemical Screening

The collected roots were thoroughly washed with distilled water and weighed at

exactly 1 kg. After drying, the roots were pounded using mortar and pestle until powdered.

The powdered material was soaked in the 80% ethanol for 8 hours. The mixture was

filtered using the filter paper, funnel, and beaker to separate the solid residue from the

supernatant. The supernatant was placed in the beaker and was transferred in the rotary
evaporating flask and placed in the rotary evaporator to evaporate ethanol. The crude

ethanolic extracts obtained were used for the phytochemical analysis.

Phytochemical Screening

The procedures for the phytochemical screening were adopted from Chichioco-

Hernandez, Paguigan and Ramiro (2010).

Test for Alkaloids

The presence of alkaloids was tested by dissolving 5 mL of the extract in 2 mL

distilled water added with 3 drops of Wagner’s reagent (2 g iodine and 6 g potassium

iodide in 100 mL water). Formation of a blue black precipitate confirmed the presence of

alkaloids.

Test for Flavonoids

The presence of flavonoids was tested by dissolving 2 mL of the extract in diluted

NaOH, followed by the addition of HCl. A yellow to orange solution with NaOH that turned

colorless upon addition of HCl denotes the presence of flavonoids.

Test for Tannins

To test for tannins, about 2 mL of the extract was dissolved in 5 mL distilled water

followed by dropwise addition of 15% FeCl3 solution. Formation of a blue-black

precipitate indicates the presence of hydrolysable tannins while brownish-green

precipitate indicates the presence of condensed tannins.

Test for Saponins

The presence of saponin was tested by boiling 5 mL of the extract in 5 mL distilled

water. The mixture was then cooled and shaken vigorously. Frothing would indicate the

presence of saponins.

Test for Terpenoids

The presence of terpenoids was tested by treating 2 mL of the extract with 2 mL

CHCl3 followed by layering with H2SO4. The formation of a reddish brown coloration in

the interface would indicate the presence of terpenoids.

Test for Glycosides

The presence of glycoside was tested by conducting the Killiani-Keller

experiment. First, is the addition of one drop of 15% FeCl3 to 2 mL extract. Afterwhich

was the layering of the solution with 1mL concentrated H2SO4. Formation of a brown ring

at the interface indicates the presence of cardiac glycosides.

Test for Phenolic Compounds

The presence of phenolic compounds was tested by adding 1% FeCl3 to the 2mL

extract. Formation of a green, purple, blue or black solution implies the presence of

phenolic compounds.

Test for Steroids

The presence of steroids was tested by adding 2 mL diluted H2SO4 to 2 mL extract

dissolved in 2 mL acetic anhydride. The formation of a blue or green solution indicates

that steroid is present.

Insecticidal Activity Evaluation

This method was used to determine if the plant specimen Derris elliptica Benth

(Tubli) root extract is possible as an alternative insecticide. The researcher used the film

residue method for this test.

Preparation of the Experimental Animals

A total of 150 8-9mm adult rice black bugs were collected from the rice field of

Brgy Lumakil, Polomolok, South Cotabato. The rice black bugs were placed in the

sterilized glass jar and were covered with nylon mesh held in place with rubber bands.

Residual Film Method of Toxicity

Residual film method as described by Busvine (1971) was used. A preliminary

screening of different doses were performed on several instars of adults to obtain 0% to

100% mortalities. Then, Derris elliptica Benth root extract were dissolved in 5 ml of

corresponding solvent to get concentrations of 50%, 75% and 100%, respectively, which

were as stock solutions. 1 ml of various concentrations for each sample were applied on

Petri dishes (9 cm diameter) in such a way that it made a uniform film over the Petri

dishes. For solvent evaporation, the Petri dishes were air dried leaving the extract on it.

After drying, 10 rice black bugs were released in each Petri dish with three replications.

A control batch was also maintained with the same number of insects after preparing the

Petri dish by applying and evaporating the solvent only. The treated black bugs were

placed in an incubator at the same temperature as reared in stock cultures and the

mortality of the black bugs were counted after 2 hours post-exposure (Islam et al., 2004).
Statistical Treatment

Phytochemical screening was analysed qualitatively. Insecticidal activity was

analyzed using median lethal dose. LD50 were determined from the counts of different

doses in different time of exposure. With Probit analysis method also described by Finney

(1971). Mean percent mortality of rice black bugs was subjected to one way analysis of

variance (ANOVA) using SPSS version 23 at 0.05 level of significance to determine if

there is a significant difference among and between treatments in terms of the plant

extracts’ ability to repel and to kill the rice black bugs.