Chemical, Antibacterial and Cytotoxic Properties of Abuos

or Weaver Ants (Oecophylla smaragdina)

Science Research Paper

Presented to 2014 National Science and Technology Fair

In Partial Fulfillment of the Requirements in

Science Research and Statistics II

Submitted to:

Ms. Elsa L. Cajucom

SRAS II Teacher / Project Adviser

Submitted by:

Samuel Heinrich L. Soliven

IV-Explorers

December 2014
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TABLE OF CONTENTS
Page
Title Page

Table of Contents

Introduction .

Statement of Null Hypotheses

Electrolytes and Chemistry Analyses .

Antibacterial Analysis

Cytotoxicity Assay

19

19

Research Environment

19

Experimental Designs

19

Data Analysis Procedure

21

24

24

24

Effect of Larvae Extraction Methods on Zones of Inhibition .

26

Effect of Pupae Extraction Methods on Zones of Inhibition .

28

Cytotoxic Effect of Abuos Larvae and Pupae on Brine Shrimp

29

Probit Analysis in Determining LC 50 for Larvae and Pupae.

31

Rationale

Statement of the Problem

Materials and Methods

Safety Precautions

Results

Electrolytes in Abuos Larvae and Pupae

Chemical Content of Abuos Larvae and Pupae

Discussion

Electrolytes in Abuos Larvae and Pupae

34

34

34

Chemical Content of Abuos Larvae and Pupae

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Effect of Larvae Extraction Methods on Zones of Inhibition .

38

Effect of Pupae Extraction Methods on Zones of Inhibition .

39

Cytotoxic Effect of Abuos Larvae and Pupae on Brine Shrimp

39

Conclusions .

42

Acknowledgement

40

Bibliography .

41

Appendix 1. Probit Analyses .

43

Appendix 2. Communication Letters .

49

Appendix 3. Laboratory Results

51

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INTRODUCTION
Rationale

Entomology is the study of insects. Insects come in various species and forms. Insects
can be harmful or beneficial. Ants are insects. They are social insects. There are ant species
that are beneficial to a certain community of people. One of them is locally known among
Ilocanos in Nueva Vizcaya as abuos. The ant eggs are known as itlog ti abuos. Scientifically,
they are known as Oecophylla smaragdina. They are called weaver ants, too.

In the unpublished autobiography of Samuel R. Soliven (2006) entitled A Tale of

Perseverance, Hope, Faith and Love as his entry to the UP Centennial Years Non-Fiction
Writing Contest, he narrated that he was a collector of ant eggs during his younger days. He also
claimed that these ant eggs are edible. Consider his account below:
Most of the ants that we normally see are red and black. Some are
small while others are bigger. I also enjoyed eating eggs of big red ants
that thrive and lay eggs in trees. Aside from that, I had tried selling them
also in the market.
Collecting ant eggs was difficult and challenging. The months of
April and May are considered the best time to collect them. I had to walk
more than a kilometer on hills where trees with big red ants were found.
With me were a bundle of dried cogon grass, match, a long and slender
dried bamboo with a basket tied on its tip and a bilao.
Once a tree with big red ants was found, I had to locate ants houses
where eggs were laid. Their houses consisted of leaves which they
especially assembled. If the leaves in the ants house are already brown or
dried, the eggs could already be collected.
I had to position the long bamboo with basket towards the ants
house with eggs. As soon as the house was disturbed many red ants would
surface with their tails lifted indicating anger, perhaps. Some also would
walk through the bamboo towards me. I would then create a hole through
their house by inserting the bamboo pointer with basket tied in it. I would
send a vibration through the bamboo and would eventually vibrate the ants
house. The eggs would fall then to the basket. If red ants would get
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through my hands, they would start biting me. I had to move very fast. I
would remove some ants on my hands and bring the bamboo with basket
away to a safe place. I would set a fire using the cogon grass and start
killing the red ants using the flame. The eggs in the basket would then be
transferred to the bilao. Flames would be passed over the eggs to kill the
remaining alive ants. After which, the eggs would be put in a safe
container.
I could collect several cups of eggs. Some were eaten while others
were sold to the market of Bayombong.

Pea, E. (2010) shared that there are several ways to cook abuos but the most common is
sauteed. The steps are quick and simple.

Other well-liked recipes are adobo, sinigang

and omelet. Other local chefs introduced a rather unique and exciting recipe which is roasted ant
eggs stuffed on red bell pepper with cheese. In Nueva Vizcaya, abuos-eating is a highlight in
their Ammungan Festival, held every May of the year in tune with the provinces celebration of
their founding anniversary. Abuos eggs are a common offering in public markets in the north
during the dry season.

These claims were provided with evidences by Raksakantong, et. al (2010) who studied
the fatty acid and proximate composition of Oecophylla smaragdina and other insects. It was
found out that O. smaragdina has good nutritional value for fat and protein.

Ants form part of the ecosystem. Definitely, they are important in the balance of nature
or homeostasis.

As such, their contribution to the world of living things cannot be

underestimated.

In fact, they come in different sizes and colors. They adapt to different

environments. Some live in the soil while others stay on trees. The kind of discipline that they

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have is admirable from finding foods to storing foods.

The trail that they create also

emphasizes cooperation and unity in their activities.

Prior to laying their eggs, they weave the young leaves of trees into a hollow sphere-like
or ellipsoid-like space. They cut some leaves to make the covering more secured. Inside it,
these ants lay their eggs. As days pass by, the cut leaves become dry and brown signaling that
the eggs have grown, become larvae, pupae and eventually as ants others with wings known
as their queens.

In the study of Das Priya, et. al (2013) published in the Journal of Entomological
Research, the gastric secretions of Oecophylla smaragdina were found to have antibacterial
property against Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis and four
gram negative bacteria: Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa,
Vibrio cholerae. Excellent antibacterial activity was exhibited against Vibrio cholerae, Bacillus
subtilis and Staphylococcus epidermidis.

This reveals that these secretions have medicinal

properties. In the research conducted by Keegan, et. al. (2013) it was found that the mandibular
glands of Oecophylla smargadina contain chiefly hexyl hexanoate, 3-decanone and octyl
hexanoate for major workers while minor workers contain nerol and 1-nonanol. Males contain
3-dodecanone, 3-decanone and 3-decanol.
These ants are like soldiers that fight for their territories. This characteristic makes them
best for controlling pests and other insects. Peng, et. al (2000) found out that Oecophylla
smaragdina has the ability to control insect pests of cashew and other trees. On the other hand,

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Hosseti and Rudresh (2012) proved that Oecophylla smargadina can act as a biocontrol agent
against bugs on trees.

Because these ants are sources of food too, Offenberg (2011) assessed cost-benefits of
Oecophylla smaragdina farming based on artificial feeding, food consumption and food
conversion. This farming was found to be highly profitable.

The studies cited above revealed that Oecophylla smaragdina ants are beneficial to
people. However, no studies were found to have delved on the chemical, antibacterial and
cytotoxic properties of Abuos eggs, larvae, and pupae. Thus, these became the foci of this
research.

Statement of the Problem

This research sought to determine the chemical, cytotoxic and antibacterial properties of
Oecophylla smaragdina or abuos.

Specifically, it answered the following questions:

1. What are the chemical constituents of (a) mixture of eggs and larvae and (b) pupae of
Oecophylla smaragdina or abuos?
2. What are the antibacterial properties of (a) mixture of eggs and larvae and (b) pupae of
Oecophylla smaragdina or abuos when fresh or when extracted using Methanol or
Chloroform using gram-positive bacteria and gram-negative bacteria?

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3. Is there a cytotoxic property of (a) mixture of eggs and larvae and (b) pupae of
Oecophylla smaragdina or abuos?

Statement of Null Hypotheses

The following null hypotheses were tested:
(1) There was no significant difference in the zones of inhibition when (a) mixture of Oecophylla
smaragdina or abuos eggs and larvae and (b) pupae,
(i) applied as fresh extract; or
(ii) applied as methanol-based extract; or
(iii)chloroform-based extract; or
(iv) applied with an antibiotic as control
using Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) and Gram-negative
bacteria (Escherichia coli Salmonella typhimurium)

(2) The mean percentage death of Artemia nauplii brine shrimp per concentration of Oecophylla
smaragdina or abuos extract is not significantly different from 50%.

This study on Oecophylla smaragdina or abuos would be helpful to chemists,

nutritionists and microbiologists who are in constant search for better ways to improve lives.
Indigenous peoples and other local consumers will also be informed of the food and medicinal
qualities of Oecophylla smaragdina or abuos.

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MATERIALS AND METHODS

Electrolytes and Clinical Chemistry Analyses
Materials used in these analyses were from the Veterans Regional Hospital (VRH)
Laboratory in Bayombong, Nueva Vizcaya. These were characterized by great diversity. They
were classified as non-biological and biological materials. The biological materials included the
Abuos eggs, larvae and pupae. These were weighed and processed well. The non-biological
materials included the various laboratory devices and equipment.
Two reports were generated, namely: Electrolytes Report and Clinical Chemistry Report.
The Electrolytes Report showed the Sodium ion, Potassium ion and Chloride Ion contents of
Abuos eggs and larvae and Abuos pupae. The Clinical Chemistry Report revealed the presence
or absence of glucose, triglycerides, AST/SGOT, ALT/SGPT, Urea, Creatinine, Uric Acid and
Cholesterol of the Abuos eggs larvae and Abuos pupae.

Anti-bacterial Analysis
Materials for the Anti-bacterial experiments included abuos extract, agar, peptone, meat extract,
petri dishes, filter paper discs, vernier caliper, beaker, test tubes, distilled water, inoculating loop

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Procedure
Preparation of Culture Media
Test tubes with 20 mm diameter were prepared. These were used for all pours: i.e., nutrient
agar, Sabourauds agar, EMB agar, etc. Pours were used for filling petri plates. The test tubes
were cleaned with warm water and detergent and using a test tube brush. These were rinsed
twice, first with tap water, and finally with distilled water to rid them of all traces of detergent.
These were placed in a test tube rack in an inverted position to allow draining.
Reagents needed were prepared as follows: 30 g agar/gulaman, 15 g peptone, 3 g meat/beef
extract, 1000 ml tap water. Dissolved the peptone, meat extract and agar in 1000 ml tap water
and heated to boiling by stirring constantly then transferred into tubes. Provided a closure for
each tube. Plastic (polypropylene) caps were used to close each tube. All caps that slipped over
the tube end have inside ridges that gripped the side of the tube and provided an air gap to allow
steam to escape during sterilization. The tubes were sterilized in the autoclave for 15 minutes at
121 C and 15 psi.

If the tubes are to be converted to slants, it is necessary to lay the tubes down in a nearhorizontal manner as soon as they are removed from the autoclave. Solidification should occur in
about 30-60 minutes. If the tubes are to be converted into tubes of broth, agar deeps, nutrient
gelatin, etc., the tubes should be allowed to cool to room temperature after removal from the
autoclave. Once they have cooled down, place them in a refrigerator or cold-storage room. If
tubes of media are not to be used immediately, they should be stored in a cool place. When
stored for long periods of time at room temperature media tend to lose moisture. At refrigerated
temperatures, media will keep for months.
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Dispensing Media in Agar Plates

The nutrient agar was liquefied, cooled to 50oC, and poured the medium into the bottom of
the plate. The tube was sterilized by flaming the neck of it. After pouring the medium into the
plate, the plate was gently rotated to evenly distribute it without any splash of the medium up
over the sides. There should be no moisture on the cover to prevent the drop of it on any colony
on the medium.

Inoculation Procedure
STREAK PLATE PROCEDURE. The culture tube was shaken from side to side to suspend
organisms. Do not moisten cap on tube. The loop and wire was heated to red-hot. The handle
was flamed slightly also. The cap was removed and the neck of the tube was flamed. Do not
place the cap down on the table. After allowing the loop to cool for at least 5 seconds, a loopful
of organisms was obtained. Avoid touching the sides of the tube. The mouth of the culture tube
was flamed again. Then, return the cap to the tube and placed the tube in a test-tube rack. The
plate was streaked. Do not gouge into the medium with the loop. The loop was flamed before
placing it down.

STREAKING PATTERNS. One loopful of organisms was streaked back and forth over area 1
near edge of the plate. The loop was applied lightly. Dont gouge into the medium. The loop
was flamed, cooled down in 5 seconds and made 5 to 6 streaks from Area 1 through Area 2
momentarily touching the loop to a sterile area of the medium before streaking insures a cool
loop. The loop was flamed again, cooled it, and made 6 or 7 streaks from area 2 through area 3.
The loop was flamed again and made as many streaks as possible from area 3 into area 4 using
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up the remainder of the plate surface. The loop was flamed before putting it aside. Filter paper
discs of 6 mm diameter were immersed in the extract and in a positive control for 24 hours. The
discs were removed from the vial and aseptically put 3 of the paper discs immersed in the extract
and one of the paper discs immersed in the control equidistantly on the surface of the assay
plates. The plates were incubated at 35oC for bacteria-containing plates. Then the zones of
inhibition were measure by a digital caliper and interpreted data as follows:
<10 mm=inactive
10-13 mm =partially active
14-19 mm=active
>19 mm =very active

In summary, the fresh extract of a) mixture of eggs and larvae and b) pupae in a blender
and was placed each on a beaker. 50 mL of the fresh extract of the mixture of eggs and larvae
was extracted using 10 mL chloroform. 50 mL of the fresh mixture of eggs and larvae extract
was extracted using 10 mL methanol. The same processes were repeated using fresh pupae
extract.

The extracts were covered using aluminum foil and left them for one day. At the same
time, the bacteria were cultured using an incubator at 35C and left them there for one day. Once
extracted, the fresh, methanol and chloroform extracts were filtered using gauze bandages and
placed them in separate beakers. Once filtered, around 12 paper discs were prepared and placed
them on each beaker including one for the control and waited for around 2 hours for it to absorb
the extract. While waiting for it, the incubated bacteria were removed and streaked it on the
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ready-made broth placed in a petri dish using an inoculating loop. The inoculating loop was
sanitized using the flame from an alcohol lamp. Once the streaking was done, placed 1 paper
disc from the fresh larvae-egg extract, 1 from the chloroform-extract, 1 from the methanolextract and 1 from the control in certain areas on each petri dish with corresponding bacteria.
Marked a certain part on the petri dish so that we may not forget which paper disc is which. Did
this thrice then leave the set-ups for a day.
The same process was followed for the pupae extract. After a day, the zones of inhibition
were measured using a digital caliper.

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ANTIBACTERIAL EXPERIMENTATION

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Cytotoxicity Assay

Abuos eggs, larvae, and pupae were screened. These were processed for the brine shrimp
bioassay test.
Cysts: For this experiment, resting Brine shrimp eggs were obtained from St. Marys
University, Biology Laboratory. Eggs in desiccator were stored in a refrigerator at 5 C.

Culture Medium: The artificial seawater (ASW) was used in the study. Although natural
seawater (about 38 g of salts per liter) is the medium of choice, it has been shown the hatching
rate and the viability of the nauplii in natural seawater (NSW) are similar to those in the artificial
seawater (ASW).
Hatching the shrimp. Brine shrimp eggs were hatched in a shallow rectangular dish filled
with artificial seawater. A plastic divider with several 2 mm holes was clamped in the dish to
make two unequal compartments. The eggs were sprinkled. After 48 h, the phototropic nauplii
were collected by pipette from the lighted side, having been separated by the divider from their
shells. Lamp positioned above the uncovered side attracted hatched shrimp. It took 2 days (48h)
for the eggs to hatch and mature as nauplii. The lamp provided direct light and warmth (about
25 C) throughout embryogenesis.

Preparation of test samples:

Samples were prepared by dissolving 20 mg of the ethanolic Abuos extract in 2 mL of a
suitable solvent (Stock solution # 1). Dilution of this stock solution gave the series of
concentrations required for testing. Four concentrations (replicates) were obtained for each series
of tests.
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Bioassay:
To each sample vial, a drop of DMSO solvent was added, ten shrimps were transferred
using a Pasteur pipette, and artificial seawater was added to make a total volume of 5 mL. The
nauplii was counted against a lighted background. Counting for the chronic LC50 began 24 hours
after initiation of tests. Nauplii was considered dead if they were immobile at the bottom of the
vials, and the percentage of deaths at each dose was determined.

In summary, the brine shrimp eggs were hatch in artificial saline solution (3.8 g rock
salt/100 mL distilled water) placed in 2 petri dishes within 48 days. While hatching the eggs, the
fresh extract from a) mixture of eggs and larvae and b) pupae were prepare for blending. Once
the eggs have been hatched, the egg shells from the brine shrimp were separated using a pipette
and placed the brine shrimp on a separate petri dish. For the larvae, a 6 mg extract/3 mL H2O
mixture and a 6 mg extract/6 mL H2O mixture were created. 500 L of the first mixture and 500
L of the brine with the hatched brine shrimp were measured using a micrometer to create a
1000 ppm concentration into a micro-centrifuge tube (MCT). This was done 3 times. The
MCTs according to concentration was labeled, time to be measured (6 hours, 12 hours or 24
hours) and type of weaver ant. The lid was kept open.

500 L of the second mixture and 500 L of the brine with the hatched brine shrimp were
measured using a micrometer to create a 500 ppm concentration into an MCT. This was done 3
times. The MCTs according to concentration was labeled, time to be measured and type of
weaver ant. The lid was kept open. 250 L of the second mixture and 750 L of the brine with
the hatched brine shrimp was measured using a micrometer to create a 250 ppm concentration
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into an MCT. This was done 3 times. The MCTs according to concentration was labeled, time to
be measured and type of weaver ant. The lid was kept open. 125 L of the second mixture and
875 L of the brine with the hatched brine shrimp was measured using a micrometer to create a
125 ppm concentration into an MCT. This was done 3 times. The MCTs according to
concentration was labeled, time to be measured and type of weaver ant. The lid was kept open.
During the process, the number of brine shrimp present was measured. The same processes were
used for the pupae. After 6 hours, the number of brine shrimps remaining of all of the MCTs
labeled with 6 hours was counted. This was done also after 12 hours and 24 hours. The
percentages of the dead per MCT were computed with the formula: [(OriginalAlive)/Alive]*100.
CYTOTOXICITY EXPERIMENTATION

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Safety Precautions in the Laboratories

Doing science experiments in standard laboratories requires a great deal of self-care and
understanding of the laboratory dos and donts. Safety rules were observed to ensure that the
experimental procedure is executed well with high precision and accuracy of data.
Research Environment
Mixture of Abuos eggs, larvae and pupae were collected in Bansing, Bayombong, Nueva
Vizcaya in April-May, 2014. The electrolytes and clinical chemistry analyses were done at the
Veterans Regional Hospital (VRH) Laboratory in Bayombong, Nueva Vizcaya on May 3-5,
2014. The anti-bacterial and cytotoxicity assay were conducted in April-May 2014 at the
Biology Laboratory of St. Marys University, Bayombong, Nueva Vizcaya.

Experimental Designs
Below are the experimental designs:
Antimicrobial Assay of Oecophylla smaragdina using Its Eggs, Larvae, and Pupae
Experimental Design 1
Title: The Effect of the Extraction Mechanism for Oecophylla smaragdinas (a) mixture of eggs
and larvae and (b) pupae on Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus)
and Gram-negative bacteria (Escherichia coli Salmonella typhimurium)

Hypothesis:
There is no significant difference in the zones of inhibition when (a) mixture of Oecophylla
smaragdina or abuos eggs and larvae and (b) pupae,

applied as fresh extract; or

applied as methanol-based extract; or

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chloroform-based extract; or

applied with an antibiotic as control

using Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) and Gram-negative

bacteria (Escherichia coli Salmonella typhimurium)
IV: Extraction Mechanism for Oecophylla smaragdinas (a) mixture of eggs and larvae and (b)
pupae
DV: Zones of Inhibition (mm) with the use of
Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus)
Gram-negative bacteria (Escherichia coli Salmonella typhimurium)
Positive
Fresh
Methanol Chloroform
Control
Extract
Extract
Gram-positive Bacteria
(Bacillus subtilis)
Disc 1
Disc 2
Disc 3
Gram-positive Bacteria
(Staphylococcus aureus)
Disc 1
Disc 2
Disc 3
<10 mm=inactive
14-19 mm=active

10-13 mm =partially active

>19 mm =very active

Constants: materials and laboratory factors, No. of repeated trials: 3

Cytotoxicity Assay of Oecophylla smaragdina using Its Eggs, Larvae, Pupae and Adults
Experimental Design 2
Title: The Effect of the Oecophylla smaragdinas (a) mixture of eggs and larvae and (b) pupae
on the Artemia nauplii brine shrimp
Hypothesis: The mean percentage of death of Artemia nauplii brine shrimp per Oecophylla
smaragdina or abuos (a) mixture of eggs and larvae and (b) pupae extract is not significantly
different from 50%

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IV: Oecophylla smaragdina (a) mixture of eggs and larvae and (b) pupae
DV: Death of Artemia nauplii brine shrimp
1000 ppm 500 ppm 250 ppm 125 ppm
D O % D O % D O % D O %
After 6 hours
After 12 hours
After 24 hours
D-number of dead brine shrimp
O original number of brine shrimp
Constants: materials and laboratory factors

Data Analysis Procedure

The following statistical procedures were used.
Descriptive statistics: Means, standard deviations and qualitative descriptions to describe
the data on zones of inhibitions. The qualitative descriptions of the means are as follows:
If the zone of inhibition is
0 mm, there is no inhibitory effect
<10 mm, there is inhibitory effect but inactive
10-13 mm, there is inhibitory effect but partially active
14-19 mm, there is inhibitory effect and is active
>19 mm, there is inhibitory effect and is very active
Inferential statistics (anti-bacterial assay)
F-test for One-way Analysis of Variance (ANOVA) was used for this null hypothesis.
There is no significant difference in the zones of inhibition when (a) mixture of Oecophylla
smaragdina or abuos eggs and larvae and (b) pupae,

applied as fresh extract; or

applied as methanol-based extract; or

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chloroform-based extract; or

applied with an antibiotic as control

using Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) and Gram-negative

bacteria (Escherichia coli Salmonella typhimurium)
Special values derived were the F-ratio and p-values.
If the p-value > 0.05, do not reject the null hypotheses.
p-value <0.05, reject the null hypothesis
When the null hypothesis was rejected, the Scheffe method was used to determine which
pair of means were statistically different.
Special values derived are the MD (Mean Difference) and p-values.
If the p-value >0.05, the mean difference is not significant. This reveals that the mean
zones of inhibition for the two extracts against a certain bacterium are not significantly different.
Meaning, the extracts have the same inhibitory effects.

Inferential statistics (cytotoxicity assay)

Probit analysis was used. According to Vincent (2013), probit analysis is a specialized
regression model of binomial response variable like death/no death of pests or brine shrimps in
toxicity studies. It is the preferred statistical model in understanding the death of brine shrimps
relative to solution concentration. It is the basis in determining the LC50 of the larvae and pupae
solution in this study. LC50 represents the concentration at which 50% of the brine shrimp
would die.

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The probit regression model is a logarithmic function of the form

Probit (p) = q(a + b log x)
Where a and b are characteristic constants
depending on the solution and extract
q - cumulative normal distribution function
x is the solution concentration
Using a statistical software, a table was generated to easily show the LC 50 based on the
probit regression model.

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RESULTS
This section presents the data, analyses and interpretation based on the actual
experiments conducted.
Electrolytes in Abuos Larvae and Pupae
Table 1
Electrolytes Content of Abuos Larvae and Pupae
TEST NAME
Larvae Pupae UNITS
Sodium (Na+)
37.5
39.5
mmol/L
Potassium (K+) 59.84
55.91
mmol/L
Chloride (Cl-)
47.0
43.8
mmol/L
The table 1 above reveals that Abuos larvae and pupae contain sodium (Na+) ions,
Potassium (K+) ions and Chloride (Cl-) ions. It was noted that as the larvae metamorphosed to
pupae, the Na+ ions content increased while the K+ and Cl- ions content decreased.

Chemical Content of Abuos Larvae and Pupae

Table 2
Chemical Content of Abuos Larvae and Pupae
Assay
Larvae Pupae
Units
Flags
Range
(for humans)
Glucose
3104
1514
mg/dl
HIGH
70-99
Triglycerides 3391
3773
mg/dl
HIGH
0-149
AST/SGOT 4052
2966
U/L
HIGH
5-34
ALT/SGPT
4241
1553
U/L
HIGH
0-55
Urea
<200
41
mg/dL
<
7-26
Creatinine
<20.00 1.8
mg/dL
<
0.57-1.25
Uric Acid
>33.1 >33.1
mg/dL
>
2.6-7.2
Cholesterol
170
138
mg/dL
Within
0-199
Notes: 1. Sample was diluted to 1:100 for convenient running
2. Reference Ranges are for human serum only.
The table 2 above reveals the following:

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Glucose
Relative to the normal range of glucose for humans, the Abuos larvae contains 3104
mg/dl while Abuos pupae contains 1514 mg/dl. These values are quite high compared to the
normal range for humans. It was noted too that as the larvae metamorphosed to pupae, the
glucose content decreased.
Triglycerides
Abuos larvae and Abuos pupae contain 3391 mg/dl and 3773 mg/dL, respectively. The
values are very high compared to the normal range for humans. Notably, the triglycerides
increased in the metamorphosis.
AST/SGOT
From Abuos larvae to Abuos pupae, the AST/SGOT decreased from 4052 U/L to 2966
U/L. These values are quite high compared to the normal range for humans.
ALT/SGPT
It is interesting to note that the Abuos larvae has 4241 U/L while Abuos pupae has 1553
U/L. As the larvae transforms to pupae, the ALT/SGPT decreased. These values are quite large
compared to the normal values.
Urea
The Abuos larvae to pupae urea content shows a decrease from 200 mg/dL to 41 mg/dL.
These information are higher in values compared to the normal values.
Creatinine
These is a decrease in the creatinine values of Abuos larvae to Abuos pupae from 20
mg/dL to 1.8 mg/dL. The values are greater than the normal values.

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Uric Acid
From Abuos larvae to Abuos pupae, the uric acid content is constant at 33.1 mg/dL yet
much higher than the normal range of values for humans.
Cholesterol
The cholesterol content of Abuos larvae was 170 mg/dL while that of pupae was 138
mg/dL. These values are within the normal values for humans.

Effect of Larvae Extraction Methods on Zones of Inhibition

Table 3
Effect on S. aureus
Larvae
Chloro
Fresh
Methanol

SAT1
12.6
10.7
NI

SAT2
21.8
19
8

SAT3
21.7
15.8
NI

+ control

26.9

31.1

28.2

<10 mm=inactive

N Mean SD
3 18.7 5.28
3 15.17 4.19
1
8
.
3

df

MS

446.203

148.734

8.914*

.012

100.113
546.316

6
9

16.686

28.73 2.15

10-13 mm =partially active

T1, T2, T3 trials

BG
WG
T

SS

14-19 mm=active

>19 mm =very active

* significant at 0.05

The larvae extract using chloroform as solvent and the fresh one were found active against
S. aureus with the means of 18.7mm and 15.17mm, respectively. The positive control was very active
against the said bacteria showing further that it was significantly the best compared to the Abuos extracts
as shown by the F value = 8.914 at p=0.012.
Table 4

Effect on B. subtilis
Larvae

BST1

BST2

BST3

Chloro

18.6

21

Fresh

19.4

Methanol
+ control

SS

df

MS

BG

238.167

119.083

80.341**

.000

1.11

WG

8.893

1.482

30.03

0.9

247.060
4>1,2,3

N Mean

SD

21.5

20.37

1.55

17.2

18

18.2

NI

NI

NI

29

30.5

30.6

<10 mm=inactive

T1, T2, T3 trials

10-13 mm =partially active

14-19 mm=active

>19 mm =very active

* significant at 0.05
26 | P a g e

The larvae extract using chloroform as solvent was very active against B. subtilis
(mean=20.37mm) while the fresh was active against it (mean=18.2mm). The positive control was very
active against the said bacteria showing further that it was significantly the best compared to the Abuos
extracts as shown by the F value = 80.341 at p<0.0005.

Table 5
Effect on S. typhimurium
Larvae

STT1

STT2

STT3

Chloro

25.2

27.6

Fresh

12.6

Methanol
+ control

SS

df

MS

BG

1340.949

446.983

106.870**

.000

0.61

WG

33.460

4.183

0.5

1374.409

11

N Mean

SD

28.7

27.17

1.79

13

13.8

13.13

9.4

8.4

8.9

8.9

32.8

33.4

39.3

<10 mm=inactive

10-13 mm =partially active

T1, T2, T3 trials

4>1,2,3

35.17 3.59

14-19 mm=active

>19 mm =very active

* significant at 0.05

The larvae extract using chloroform as solvent was very active against S. typhimurium
(mean=27.17mm). The positive control was very active against the said bacteria showing further that it
was significantly the best compared to the Abuos extracts as shown by the F value = 106.870 at
p<0.0005.
Table 6

Effect on E. coli
Larvae

ECT1 ECT2 ECT3 N Mean

SD

SS

df

MS

3.450

.072

Chloro

19.2

17.6

10.8

15.87

4.46

BG

72.749

24.250

Fresh

14.7

16.5

12.9

14.7

1.8

WG

56.233

7.029

Methanol

8.6

11.9

7.7

9.4

2.21

11

+ control

14.4

13.8

14.2

128.982
4=1,2>3

<10 mm=inactive

T1, T2, T3 trials

14.13 0.31

10-13 mm =partially active

14-19 mm=active

>19 mm =very active

* significant at 0.05

The larvae extract using chloroform as solvent was active against E. coli (mean=15.87mm).
Fresh extract is also active against it (mean=14.7mm). The positive control was also active against the
said bacteria showing that all three treatments were of the same effect as shown by the F value = 3.450 at
p=0.072.
27 | P a g e

Overall
In summary, the Abuos larvae extract with the use of chloroform was found very active
against B. subtilis, active against S. aureus, S. typhimurium and E. coli. The fresh Abuos extract
was generally active for these bacteria except on S. typhimurium.

Effect of Pupae Extraction Methods on Zones of Inhibition

Table 7
Effect on S. aureus
Pupae
Chloro

SAT1 SAT2 SAT3 N Mean

NI
NI
NI
0
.

SD
.

Fresh

NI

NI

NI

Methanol
+ control

7.2
27.6

6.8
29.8

8.3
31.6

3
3

7.43
29.67

0.78
2

<10 mm=inactive

10-13 mm =partially active

T1, T2, T3 trials

Methanol
+ control

1.15
4.75

T1, T2, T3 trials

10.6
27.5

NI
37

3
3

9.45
32.2

10-13 mm =partially active

T1, T2, T3 trials

BG
WG

741.482

741.482

321.219**

.000

9.233

2.308

750.715
4>1,2,3

5
>19 mm =very active

BG
WG
T

SS

df

MS

1115.987
48.765
1164.752
4>1,2,3

3
6
9

371.996
8.128

45.770**

.000

14-19 mm=active

>19 mm =very active

* significant at 0.05

S. typhimurium
Pupae
STT1 STT2 STT3 N Mean SD
Chloro
11.9
12.6
15.4 3 13.3 1.85
Fresh
9.4
9.1
16.8 3 11.77 4.36
Methanol
9.9
11.4
11.9 3 11.07 1.04
+ control
33
45
41.4 3 39.8 6.16
<10 mm=inactive

MS

14-19 mm=active

SD
0.7
.

<10 mm=inactive

df

* significant at 0.05

B. subtilis
Pupae
BST1 BST2 BST3 N Mean
Chloro
9.7
10.4
9
3
9.7
Fresh
7
NI
NI
1
7
8.3
32.1

SS

10-13 mm =partially active

BG
WG
T

SS

df

MS

1741.163
122.913

3
8

580.388
15.364

37.775**

.000

1864.077
4>1,2,3

11

14-19 mm=active

>19 mm =very active

* significant at 0.05

28 | P a g e

In summary, the Abuos pupae fresh and extracts using different extraction methods were
found inactive against the bacteria except on S. typhimurium where they were found partially
active.
Cytotoxic Effect of Abuos Larvae and Pupae on Brine Shrimp
Table 8
Cytotoxic Assay using the Crude LC 50
Abuos Larvae
1000 ppm
6 hrs.
12 hrs.
24 hrs.

19
21
17

23 82.6% 14
24 87.5% 12
19 89.5% 12

Abuos Pupae
1000 ppm
6 hrs.
12 hrs.
24 hrs.

12
26
16

22
31
19

250 ppm

500 ppm
25
20
22

56.0%
60.0%
54.6%

4
4
12

8
15
16

24
26
25

33.3%
57.7%
64.0%

24 16.7%
15 26.7%
25 48.0%

0
2
5

0%
17
18 11.1%
14 35.7%

250 ppm

500 ppm
54.6%
83.9%
84.2%

125 ppm

3
9
16

21
22
27

14.3%
40.9%
59.3%

125 ppm
0
4
7

12
14
17

0%
28.6%
41.7%

It can be inferred from the tables above that the LC50 in the larvae is located between
250 ppm; 24 hrs and 500 ppm; 24 hrs. The LC50 in the pupae is located between 250 ppm; 12
hrs and 500 ppm; 12 hrs, between 500 ppm; 6 hrs and 500 ppm; 12 hrs and between 250 ppm; 12
hrs and 250 ppm; 24 hrs. The highest percentage for the larvae is 89.5% located at 1000 ppm; 24
hrs. and the lowest is 0% located at 125 ppm; 6 hrs. The highest percentage for the larvae is
84.2% located at 1000 ppm; 24 hrs and the lowest is 0% located at 125 ppm; 6 hrs.

Considering these, in a relatively small concentration in terms of ppm and that it is indeed
seen in the data that it can kill cancer cells, Oecophylla smaragdina can kill 100% of cancer cells
29 | P a g e

(twice the LC50) and in 1000 ppm of larvae, 89.5% of cancer cells can be killed in 24 hrs. These
examples can be used when a patient is required to have his cancer cells eliminated within a
certain period.

30 | P a g e

Probit Analysis in Determining LC 50

Table 9
Cytotoxic Assay and the Probit Regression Models
Below are the regression models and the corresponding graphs of the transformed raw
data gathered to aid in determining the LC 50. To locate the LC 50, find the probit p = 0.50 in
the table and locate the corresponding concentration. This concentration is the LC 50. Refer to
the Appendices showing the tables.

Larvae Probit (p) Model where X = concentration (in ppm)

After 6 hours Probit (p) =-9.263 + 3.445logX Significant at 0.01
After 12 hours Probit (p) =-6.958 + 2.684logX

Significant at 0.01

After 24 hours Probit (p) =-3.810 + 1.566logX

Significant at 0.01

Probit (p) =-9.263 + 3.445logX

Probit (p) =-6.958 + 2.684logX

Probit (p) =-3.810 + 1.566logX

31 | P a g e

Pupae Probit (p) Model where X = concentration (in ppm)

After 6 hours Probit (p) =-6.555 + 2.245logX Significant at 0.01
After 12 hours Probit (p) =-4.386 + 1.750logX

Significant at 0.01

After 24 hours Probit (p) =-2.729 + 1.202logX

Significant at 0.01

Probit (p) =-6.555 + 2.245logX

Probit (p) =-4.386 + 1.750logX

Probit (p) =-2.729 + 1.202logX

32 | P a g e

From these models, the LC 50 was determined. The table 10 below shows the LC 50.

6 hrs.
12 hrs.
24 hrs.

Table 10
Cytotoxic Assay using the Probit Analysis to Determine LC 50
LC50
95% Confidence Limits LC50
95% Confidence Limits
Larvae
of the Concentration
Pupae
of the Concentration
(Larvae)
(Pupae)
488.61 ppm
392.014-619.371
831.64 ppm 595.265-1707.466
391.50 ppm
287.576-526.154
320.35 ppm 188.041-459.816
271.01 ppm
127.137-420.661
186.69 ppm 23.314-316.081

LC 50 for Abuos Larvae

It can be deduced that the Abuos larvae have anticancer potential. The desired Abuos
larvae concentration to kill 50% of the Brine Shrimp is 488.61 ppm after 6 hours, 391.50 ppm
after 12 hours, and 271.01 ppm after 24 hours.

LC 50 for Abuos Pupae

It can be deduced that the Abuos pupae have anticancer potential. The desired Abuos
pupae concentration to kill 50% of the Brine Shrimp is 831.64 ppm after 6 hours, 320.35 ppm
after 12 hours, and 186.69 ppm after 24 hours.
A comparison of the LC 50 for Abuos larvae and Abuos pupae revealed that the pupae
have better ability to kill Brine Shrimp than the larvae. After 24 hours, the LC 50 of pupae is
186.69 ppm of pupae.

33 | P a g e

DISCUSSION

This study determined the electrolytes and chemical contents of Abuos larvae and pupae
and their antibacterial and cytotoxic effects. These Abuos larvae and pupae were subjected to
anti-bacterial and cytotoxic laboratory procedures at St. Marys University, Bayombong, Nueva
Vizcaya while the determination of electrolytes and clinical chemical contents was done at the
VRH Laboratory in Bayombong, Nueva Vizcaya.
Results of the study revealed the following:

Electrolytes in Abuos Larvae and Pupae

These are tests usually grouped together for the purpose of determining either hydration
(presence of water) or dehydration (lack of water).

Abuos larvae and pupae contain sodium (Na+) ions, Potassium (K+) ions and Chloride
(Cl-) ions. It was noted that as the larvae metamorphosed to pupae, the Na+ ions content
increased while the K+ and Cl- ions content decreased.

Chemical Content of Abuos Larvae and Pupae

Glucose
A form of sugar or carbohydrate that is used as an immediate source of energy is glucose.
A certain amount is always maintained in the blood. Diabetics are unable to use glucose properly
and usually maintain high blood concentrations.
Exercise like jogging, stretching and doing something may influence the better utilization of
glucose by the tissues. This can be an important therapeutic benefit for diabetics because it may
34 | P a g e

under certain conditions lessen their dependence on insulin. Diabetics should work closely with
their medical doctor to monitor insulin therapy during any exercise program.

Abuos larvae contains 3104 mg/dl while Abuos pupae contains 1514 mg/dl. These
values are quite high compared to the normal range for humans. It was noted too that as the
larvae metamorphosed to pupae, the glucose content decreased.

Triglycerides
They are the main lipids of foods and mammalian fat storage depots (adipose tissue).
Triglycerides are neutral fats, totally insoluble in water (hydrophobic) which makes them ideal as
storage fuel. However, significant triglyceride increases reduce the fluidity and oxygen-carrying
capacity of the blood. Triglycerides in the blood originate from either dietary fat in the liver
which in turn comes about due to high sugar intake. The blood triglyceride level is considered an
essential lipid component, along with cholesterol, in the prediction of arterial suffocation.

Abuos larvae and Abuos pupae contain 3391 mg/dl and 3773 mg/dL, respectively. The
values are very high compared to the normal range for humans. Notably, the triglycerides
increased in the metamorphosis.

SGPT (ALT or Alanine Aminotransferase), SGOT (AST or Aspartic Transaminase)

These are enzymes. These enzymes are produced by all the cells in the body, but the
liver and muscle are the largest producers. If there is liver or muscle damage -- such as in
hepatitis, gall bladder disease, or heart attack -- levels of these enzymes are increased. Normally,
35 | P a g e

modest increases of any of these enzymes are not cause for concern, unless they remain elevated
on a repeated test.
AST/SGOT
From Abuos larvae to Abuos pupae, the AST/SGOT decreased from 4052 U/L to 2966
U/L. These values are quite high compared to the normal range for humans.
ALT/SGPT
It is interesting to note that the Abuos larvae has 4241 U/L while Abuos pupae has 1553
U/L. As the larvae transforms to pupae, the ALT/SGPT decreased. These values are quite large
compared to the normal values.
Urea / Blood Urea Nitrogen (BUN)
Urea nitrogen is a protein breakdown product circulating in the blood which is expelled
by the kidney. The kidney continually filters urea from the blood. It is excreted through the
urine. Many carry high levels of protein breakdown products in their blood primarily because of
their high protein diet particularly animal protein. Healthier low-protein nutrition
characteristically lowers the urea nitrogen circulating in the blood.

The Abuos larvae to pupae urea content shows a decrease from 200 mg/dL to 41 mg/dL.
These information are higher in values compared to the normal values.

Creatinine
Creatinine is also a protein breakdown product but not significantly changed by high
protein intakes. However, high protein intake will accelerate kidney damage, and if the kidney is
destroyed, the creatinine level will increase.
36 | P a g e

These is a decrease in the creatinine values of Abuos larvae to Abuos pupae from 20
mg/dL to 1.8 mg/dL. The values are greater than the normal values.

Uric Acid
Uric acid is a waste product of protein breakdown. It is normally filtered by the liver and
excreted by the kidney. In large amounts it collects in the joints causing inflammation and
"gouty" arthritis. Recently, elevated uric acid has been found to be a strong indicator of heart
disease. Like the blood cholesterol level, it should be kept at a low level within the normal range.
From Abuos larvae to Abuos pupae, the uric acid content is constant at 33.1 mg/dL yet
much higher than the normal range of values for humans.

Cholesterol
Cholesterol is the most infamous of the essential fat components. It is one of the most
common sterols found in human tissue. Cholesterol is used by the body for cell membrane
integrity, hormonal functions and digestive processes. The body manufactures all the cholesterol
normally needed to maintain these bodily functions. Excess cholesterol levels are generally
related to dietary patterns, exercise habits and gender.

The cholesterol content of Abuos larvae was 170 mg/dL while that of pupae was 138
mg/dL. These values are within the normal values for humans.

37 | P a g e

Effect of Larvae Extraction Methods on Zones of Inhibition

The larvae extract using chloroform as solvent and the fresh one were found active
against S. aureus with the means of 18.7mm and 15.17mm, respectively. The positive control
was very active against the said bacteria showing further that it was significantly the best
compared to the Abuos extracts as shown by the F value = 8.914 at p=0.012.

The larvae extract using chloroform as solvent was very active against B. subtilis
(mean=20.37mm) while the fresh was active against it (mean=18.2mm). The positive control
was very active against the said bacteria showing further that it was significantly the best
compared to the Abuos extracts as shown by the F value = 80.341 at p<0.0005.

The larvae extract using chloroform as solvent was very active against S.
typhimurium (mean=27.17mm). The positive control was very active against the said bacteria
showing further that it was significantly the best compared to the Abuos extracts as shown by
the F value = 106.870 at p<0.0005.

The larvae extract using chloroform as solvent was active against E. coli
(mean=15.87mm). Fresh extract is also active against it (mean=14.7mm). The positive control
was also active against the said bacteria showing that all three treatments were of the same
effect as shown by the F value = 3.450 at p=0.072.
Overall
In summary, the Abuos larvae extract with the use of chloroform was found very active
against B. subtilis, active against S. aureus, S. typhimurium and E. coli. The fresh Abuos extract
was generally active for these bacteria except on S. typhimurium.
38 | P a g e

Effect of Pupae Extraction Methods on Zones of Inhibition

In summary, the Abuos pupae fresh and extracts using different extraction methods were
found inactive against the bacteria except on S. typhimurium where they were found partially
active.
Cytotoxic Effect of Abuos Larvae and Pupae on Brine Shrimp
LC 50 for Abuos Larvae. It can be deduced that the Abuos larvae have anticancer potential.
The desired Abuos larvae concentration to kill 50% of the Brine Shrimp is 488.61 ppm after 6
hours, 391.50 ppm after 12 hours, and 271.01 ppm after 24 hours.

LC 50 for Abuos Pupae. It can be deduced that the Abuos pupae have anticancer potential.
The desired Abuos pupae concentration to kill 50% of the Brine Shrimp is 831.64 ppm after 6
hours, 320.35 ppm after 12 hours, and 186.69 ppm after 24 hours.
A comparison of the LC 50 for Abuos larvae and Abuos pupae revealed that the pupae
have better ability to kill Brine Shrimp than the larvae. After 24 hours, the LC 50 of pupae is
186.69 ppm of pupae.

39 | P a g e

CONCLUSIONS

Abuos larvae and pupae contain sodium ions, potassium ions and chloride ions. Relative to
human range of normal values, they have very high content of the following:
triglycerides, AST/SGOT, ALT/SGPT, Urea, Creatinine, and Uric acid.
cholesterol content.

glucose,

They have low

Thus, individuals with diabetes, with poor liver profile, and/or with

abnormal kidney must avoid eating Abuos (O. smaragdina) larvae and pupae.

The Abuos larvae extract with the use of chloroform was found very active against B.
subtilis, active against S. aureus, S. typhimurium and E. coli. The fresh Abuos extract was
generally active for these bacteria except on S. typhimurium. The Abuos pupae fresh and
extracts using different extraction methods were found inactive against the bacteria except on S.
typhimurium where they were found partially active. The positive control was best. However,
the antibacterial effect is the same against the E. coli. These results show that Abuos larvae have
better antibacterial properties than Abuos pupae. Thus, these Abuos larvae and pupae have
active components that can help cure illnesses due to these bacteria.

The cytotoxic potential of Abuos larvae and pupae is evident. After 24 hours, LC 50 for
larvae is 271.01 ppm and LC 50 for pupae is 186.69 ppm. These results revealed that Abuos
pupae have better cytotoxic property than the Abuos larvae. These also mean that Abuos larvae
and pupae have anticancer chemicals that must be isolated using high technology laboratory.

40 | P a g e

ACKNOWLEDGEMENT
The science researcher would like to express his gratitude to the following who helped
him along the various stages of the experimentation process:

Rev. Fr. Neil H. Sta. Ana, CICM, President of St. Marys University; Dr. John Octavios
Palina, Dr. Moises Alexander Asuncion, Mrs. Venica Acosta, Vice Presidents; Dr. Ma.
Theresa B. Tayaban, and Dr. Melfei E. Bungihan for their approval and assistance in using
the SMU Science laboratories for the conduct of the antibacterial and cytotoxic experiments.

Dr. Cirilo Galindez, Dr. Nathanael Vidad and Dr. Claire Dontogan of the Veterans Regional
Hospital (VRH) for the permission and assistance in conducting the chemical analyses of the
extracts.

Dr. Henry Navarro, Principal and Ms. Elsa L. Cajucom, Science, Technology and Research
Coordinator for the proper direction in conceptualizing the research topic and conducting the
experiments.

To his uncles and relatives in Bansing, Bayombong, Nueva Vizcaya, namely: Jay-Ar Cacayan
and Kag. Benny Soliven who harvested Oecophylla smaragdina.

To his parents, Samuel and Anivel Soliven for the love, inspiration and support in realizing this
research.
Samuel Heinrich L. Soliven
Science Researcher
41 | P a g e

BIBLIOGRAPHY
Das Priya, D. R., Anaswara, K., Achuthsankar, K. S. & Oommen, O. V. (2013). Antibacterial
action of gastric secretions from Oecophylla smaragdina, an Asian red weaver ant.
Journal of Entomological Research, 37 (4): 347-349. Retrieved October 14, 2013 from
http://anft.indianjournals.com/ijor.aspx?target=ijor:jer&volume=37&issue=4&article=01
2
Keegans, S. J., Billen, J. & Morgan, E. D. (2013). Volatile secretions of the green tree
ant Oecophylla smaragdina(Hymenoptera: Formicidae). Retrieved October 15, 2013
from http://www.sciencedirect.com/science/article/pii/030504919190273G
Offenberg J. (2011). Oecophylla smaragdina food conversion efficiency: prospects for ant
farming. Journal of Applied Entomology, 135(8): 575-581. Retrieved October 14, 2013
from http://europepmc.org/abstract/AGR/IND44611972
Peng, R. K., Christian, K & Gibb, K. (2000). The effect of the green ant, Oecophylla
smaragdina (Hymenoptera: Formicidae), on insect pests of cashew trees in Australia.
Australia: Northern Territory University Pub. Retrieved October 15, 2013 from
http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=2479200
Pea, E. (2010). Abuos: The Ilocanos version of caviar (Bayombong, Nueva Vizcaya)
Retrieved on September 22, 2013 http://loqal.ph/food-and-beverage/2010/04/14/abuosthe-ilocanos-version-of-caviar/
Raksakantong, P., Meeso, N., Kubola, J. & Siriamornpun, S. (2010). Fatty acids and proximate
composition of eight Thai edible terricolous insects. Food Research International,
43(1): 350-355. Retrieved October 14, 2013 from
http://europepmc.org/abstract/AGR/IND44328216
Soliven, Samuel R. (2008).
A Tale of Perseverance, Hope, Faith and Love (An
Autobiography). Unpublished Non-Fiction Story, Bayombong, Nueva Vizcaya

Tawaha, K. (2005). Cytotoxicity evaluation of Jordanian wild plantsusing brine shrimp

lethality test. Retrieved September 28, 2013, from
http://asu.edu.jo/TestWeb/userfiles/file/natural_pdf/Volume-8-2006/Number1/Cytotoxicity%20Evaluation%20of%20Jordanian%20Wild%20Plants%20using
%20Brine%20Shrimp%20Lethality%20Test.pdf
Vincent, K. (2013). Probit analysis. Retrieved May 5, 2014 from
http://userwww.sfsu.edu/efc/classes/biol710/probit/ProbitAnalysis.pdf

42 | P a g e

APPENDIX 1
PROBIT ANALYSES
Probit Analysis for Larvae: After Six hours

Probability

PROBIT

.010
.020
.030
.040
.050
.060
.070
.080
.090
.100
.150
.200
.250
.300
.350
.400
.450
.500

.550
.600
.650
.700
.750
.800
.850
.900
.910
.920
.930
.940
.950
.960
.970
.980
.990
a. Logarithm base = 10.

Confidence Limits
95% Confidence Limits for concen
Estimate Lower Bound Upper Bound
103.197
42.296
160.046
123.822
55.996
183.893
138.996
66.859
200.977
151.625
76.364
214.967
162.739
85.052
227.145
172.839
93.194
238.122
182.208
100.946
248.247
191.028
108.407
257.738
199.419
115.645
266.741
207.468
122.710
275.362
244.402
156.457
314.922
278.391
189.030
351.772
311.294
221.436
388.365
344.143
254.150
426.287
377.668
287.423
466.895
412.496
321.398
511.558
449.247
356.201
561.793
392.014
619.371
488.609
LC 50
531.421
429.154
686.466
578.767
468.152
765.912
632.139
509.832
861.661
693.722
555.449
979.641
766.926
606.948
1129.446
857.567
667.558
1328.075
976.829
743.310
1609.665
1150.725
847.806
2057.896
1197.174
874.767
2184.706
1249.761
904.880
2331.727
1310.254
939.018
2505.284
1381.283
978.473
2714.978
1467.004
1025.264
2976.326
1574.535
1082.807
3316.603
1717.592
1157.589
3790.023
1928.083
1264.424
4527.833
2313.428
1451.854
5998.457

95% Confidence Limits for log(concen)

Estimate
Lower Bound Upper Bound
2.014
1.626
2.204
2.093
1.748
2.265
2.143
1.825
2.303
2.181
1.883
2.332
2.211
1.930
2.356
2.238
1.969
2.377
2.261
2.004
2.395
2.281
2.035
2.411
2.300
2.063
2.426
2.317
2.089
2.440
2.388
2.194
2.498
2.445
2.277
2.546
2.493
2.345
2.589
2.537
2.405
2.630
2.577
2.459
2.669
2.615
2.507
2.709
2.652
2.552
2.750
2.689
2.593
2.792
2.725
2.763
2.801
2.841
2.885
2.933
2.990
3.061
3.078
3.097
3.117
3.140
3.166
3.197
3.235
3.285
3.364

2.633
2.670
2.707
2.745
2.783
2.824
2.871
2.928
2.942
2.957
2.973
2.991
3.011
3.035
3.064
3.102
3.162

43 | P a g e

2.837
2.884
2.935
2.991
3.053
3.123
3.207
3.313
3.339
3.368
3.399
3.434
3.474
3.521
3.579
3.656
3.778

Probit Analysis: Larvae After 12 Hours

Probability

PROBIT

.010
.020
.030
.040
.050
.060
.070
.080
.090
.100
.150
.200
.250
.300
.350
.400
.450
.500

.550
.600
.650
.700
.750
.800
.850
.900
.910
.920
.930
.940
.950
.960
.970
.980
.990
a. Logarithm base = 10.

Confidence Limits
95% Confidence Limits for concen12
Estimate
Lower Bound Upper Bound
53.197
13.972
98.495
67.215
20.358
117.254
77.967
25.829
131.070
87.175
30.879
142.598
95.461
35.692
152.779
103.132
40.363
162.066
110.363
44.946
170.720
117.267
49.477
178.904
123.920
53.981
186.731
130.377
58.476
194.278
160.893
81.213
229.535
190.163
104.996
263.170
219.484
130.312
297.213
249.649
157.463
333.094
281.290
186.648
372.180
315.014
218.002
416.019
351.486
251.614
466.524
287.576
526.154
391.500
LC 50
436.070
326.073
598.150
486.558
367.502
686.910
544.892
412.635
798.711
613.952
462.825
943.128
698.330
520.359
1135.996
806.004
589.213
1406.218
952.638
677.013
1814.096
1175.612
801.321
2514.918
1236.871
833.987
2723.437
1307.043
870.753
2970.398
1388.805
912.789
3268.814
1486.181
961.834
3638.828
1605.600
1020.628
4113.701
1758.218
1093.845
4753.401
1965.864
1190.453
5680.773
2280.342
1331.205
7204.931
2881.217
1585.427
10493.328

95% Confidence Limits for log(concen12)

Estimate
Lower Bound Upper Bound
1.726
1.145
1.993
1.827
1.309
2.069
1.892
1.412
2.118
1.940
1.490
2.154
1.980
1.553
2.184
2.013
1.606
2.210
2.043
1.653
2.232
2.069
1.694
2.253
2.093
1.732
2.271
2.115
1.767
2.288
2.207
1.910
2.361
2.279
2.021
2.420
2.341
2.115
2.473
2.397
2.197
2.523
2.449
2.271
2.571
2.498
2.338
2.619
2.546
2.401
2.669
2.593
2.459
2.721
2.640
2.687
2.736
2.788
2.844
2.906
2.979
3.070
3.092
3.116
3.143
3.172
3.206
3.245
3.294
3.358
3.460

2.513
2.565
2.616
2.665
2.716
2.770
2.831
2.904
2.921
2.940
2.960
2.983
3.009
3.039
3.076
3.124
3.200

44 | P a g e

2.777
2.837
2.902
2.975
3.055
3.148
3.259
3.401
3.435
3.473
3.514
3.561
3.614
3.677
3.754
3.858
4.021

Probit Analysis: Larvae After 24 Hours

Probability

PROBIT

.010
.020
.030
.040
.050
.060
.070
.080
.090
.100
.150
.200
.250
.300
.350
.400
.450
.500

.550
.600
.650
.700
.750
.800
.850
.900
.910
.920
.930
.940
.950
.960
.970
.980
.990
a. Logarithm base = 10.

Confidence Limits
95% Confidence Limits for concen12
Estimate
Lower Bound Upper Bound
8.858
.021
37.623
13.225
.060
48.409
17.055
.116
56.841
20.651
.192
64.166
24.129
.288
70.838
27.546
.408
77.082
30.939
.553
83.027
34.330
.725
88.758
37.735
.928
94.332
41.167
1.165
99.791
59.033
2.974
126.277
78.615
6.238
152.891
100.517
11.721
181.012
125.341
20.529
211.923
153.785
34.224
247.294
186.722
54.913
289.798
225.289
85.138
344.327
127.137
420.661
271.013
LC 50
326.018
181.060
538.884
393.356
243.560
737.955
477.604
310.603
1088.075
585.989
382.026
1720.943
730.703
462.001
2918.018
934.278
558.387
5371.297
1244.198
685.471
11112.886
1784.143
875.946
28097.708
1946.430
928.157
35200.281
2139.505
987.988
44984.125
2373.980
1057.770
58934.288
2666.354
1141.014
79723.602
3044.007
1243.343
112580.893
3556.589
1374.552
168967.930
4306.499
1553.886
278546.281
5553.658
1827.271
541874.057
8292.192
2355.190 1549210.259

95% Confidence Limits for log(concen12)

Estimate
Lower Bound Upper Bound
.947
-1.681
1.575
1.121
-1.224
1.685
1.232
-.935
1.755
1.315
-.717
1.807
1.383
-.540
1.850
1.440
-.389
1.887
1.491
-.258
1.919
1.536
-.140
1.948
1.577
-.032
1.975
1.615
.066
1.999
1.771
.473
2.101
1.896
.795
2.184
2.002
1.069
2.258
2.098
1.312
2.326
2.187
1.534
2.393
2.271
1.740
2.462
2.353
1.930
2.537
2.433
2.104
2.624
2.513
2.595
2.679
2.768
2.864
2.970
3.095
3.251
3.289
3.330
3.375
3.426
3.483
3.551
3.634
3.745
3.919

2.258
2.387
2.492
2.582
2.665
2.747
2.836
2.942
2.968
2.995
3.024
3.057
3.095
3.138
3.191
3.262
3.372

45 | P a g e

2.731
2.868
3.037
3.236
3.465
3.730
4.046
4.449
4.547
4.653
4.770
4.902
5.051
5.228
5.445
5.734
6.190

Probit Analysis: Pupae After 12 Hours

Probability

PROBIT

.010
.020
.030
.040
.050
.060
.070
.080
.090
.100
.150
.200
.250
.300
.350
.400
.450
.500

.550
.600
.650
.700
.750
.800
.850
.900
.910
.920
.930
.940
.950
.960
.970
.980
.990
a. Logarithm base = 10.

Confidence Limits
95% Confidence Limits for concen12
Estimate
Lower Bound Upper Bound
76.498
7.669
157.624
101.176
13.964
190.581
120.816
20.393
215.306
138.064
27.085
236.255
153.895
34.087
255.017
168.792
41.423
272.372
183.035
49.108
288.768
196.805
57.152
304.497
210.226
65.561
319.761
223.388
74.341
334.713
287.243
123.879
408.381
350.776
182.513
487.177
416.371
248.641
580.127
485.667
319.064
698.087
560.135
390.080
854.053
641.330
459.496
1062.366
731.072
527.386
1339.530
595.265
1707.466
831.642
LC 50
946.049
665.179
2198.387
1078.430
739.419
2862.055
1234.754
820.656
3778.542
1424.081
912.365
5083.553
1661.090
1019.640
7023.938
1971.711
1150.879
10095.193
2407.818
1321.990
15446.612
3096.089
1569.744
26448.064
3289.926
1635.705
30126.455
3514.280
1710.306
34708.640
3778.669
1796.037
40560.601
4097.531
1896.603
48276.933
4494.166
2017.859
58894.331
5009.483
2169.832
74402.974
5724.668
2371.836
99197.856
6835.882
2668.800
145448.583
9041.194
3211.952
266052.000

95% Confidence Limits for log(concen12)

Estimate
Lower Bound Upper Bound
1.884
.885
2.198
2.005
1.145
2.280
2.082
1.309
2.333
2.140
1.433
2.373
2.187
1.533
2.407
2.227
1.617
2.435
2.263
1.691
2.461
2.294
1.757
2.484
2.323
1.817
2.505
2.349
1.871
2.525
2.458
2.093
2.611
2.545
2.261
2.688
2.619
2.396
2.764
2.686
2.504
2.844
2.748
2.591
2.931
2.807
2.662
3.026
2.864
2.722
3.127
2.920
2.775
3.232
2.976
3.033
3.092
3.154
3.220
3.295
3.382
3.491
3.517
3.546
3.577
3.613
3.653
3.700
3.758
3.835
3.956

2.823
2.869
2.914
2.960
3.008
3.061
3.121
3.196
3.214
3.233
3.254
3.278
3.305
3.336
3.375
3.426
3.507

46 | P a g e

3.342
3.457
3.577
3.706
3.847
4.004
4.189
4.422
4.479
4.540
4.608
4.684
4.770
4.872
4.997
5.163
5.425

Probit Analysis: Pupae After 12 Hours

Probability

PROBIT

.010
.020
.030
.040
.050
.060
.070
.080
.090
.100
.150
.200
.250
.300
.350
.400
.450
.500

.550
.600
.650
.700
.750
.800
.850
.900
.910
.920
.930
.940
.950
.960
.970
.980
.990
a. Logarithm base = 10.

Confidence Limits
95% Confidence Limits for concen12
Estimate
Lower Bound Upper Bound
15.020
.508
47.262
21.497
1.040
60.271
26.989
1.638
70.367
32.027
2.304
79.092
36.811
3.040
87.008
41.441
3.847
94.390
45.978
4.729
101.398
50.461
5.688
108.135
54.916
6.727
114.670
59.363
7.848
121.055
81.949
14.828
151.836
105.885
24.498
182.447
131.920
37.537
214.435
160.713
54.802
249.113
192.976
77.334
288.025
229.559
106.321
333.373
271.542
142.944
388.664
188.041
459.816
320.349
LC 50
377.929
241.618
556.935
447.046
302.731
696.736
531.795
370.494
905.920
638.552
445.768
1228.489
777.924
532.267
1744.979
969.200
637.553
2623.584
1252.285
776.421
4276.817
1728.745
983.523
8000.966
1868.753
1040.043
9319.207
2033.732
1104.688
11002.988
2231.996
1179.920
13213.121
2476.358
1269.448
16217.457
2787.874
1379.198
20496.271
3204.289
1519.472
27001.843
3802.399
1710.454
37920.011
4773.767
2000.143
59609.172
6832.628
2555.430
121794.694

95% Confidence Limits for log(concen12)

Estimate
Lower Bound Upper Bound
1.177
-.294
1.675
1.332
.017
1.780
1.431
.214
1.847
1.506
.362
1.898
1.566
.483
1.940
1.617
.585
1.975
1.663
.675
2.006
1.703
.755
2.034
1.740
.828
2.059
1.774
.895
2.083
1.914
1.171
2.181
2.025
1.389
2.261
2.120
1.574
2.331
2.206
1.739
2.396
2.286
1.888
2.459
2.361
2.027
2.523
2.434
2.155
2.590
2.506
2.274
2.663
2.577
2.650
2.726
2.805
2.891
2.986
3.098
3.238
3.272
3.308
3.349
3.394
3.445
3.506
3.580
3.679
3.835

2.383
2.481
2.569
2.649
2.726
2.805
2.890
2.993
3.017
3.043
3.072
3.104
3.140
3.182
3.233
3.301
3.407

47 | P a g e

2.746
2.843
2.957
3.089
3.242
3.419
3.631
3.903
3.969
4.042
4.121
4.210
4.312
4.431
4.579
4.775
5.086

Probit Analysis: Pupae After 24 Hours

Probability

PROBIT

.010
.020
.030
.040
.050
.060
.070
.080
.090
.100
.150
.200
.250
.300
.350
.400
.450
.500

.550
.600
.650
.700
.750
.800
.850
.900
.910
.920
.930
.940
.950
.960
.970
.980
.990
a. Logarithm base = 10.

Confidence Limits
95% Confidence Limits for concen12
Estimate
Lower Bound
Upper Bound
2.164
.000
19.641
3.648
.000
26.561
5.081
.000
32.185
6.520
.000
37.200
7.986
.000
41.862
9.490
.000
46.297
11.041
.000
50.581
12.643
.000
54.761
14.301
.001
58.872
16.019
.001
62.937
25.623
.009
83.142
37.217
.040
104.063
51.265
.144
126.611
68.347
.457
151.665
89.220
1.324
180.365
114.891
3.604
214.500
146.738
9.359
257.351
23.314
316.081
186.687
LC 50
237.512
55.013
409.849
303.348
116.828
601.234
390.630
205.453
1106.500
509.923
299.664
2615.599
679.837
396.479
7517.548
936.455
508.476
25940.871
1360.204
656.496
113754.109
2175.628
884.928
747595.108
2436.981
949.110
1180532.537
2756.605
1023.473
1940436.500
3156.641
1111.273
3353389.985
3672.420
1217.460
6181810.078
4364.298
1350.057
12427258.416
5345.462
1523.216
28249152.767
6858.941
1765.192
77595653.690
9554.010
2144.775 297656480.201
16107.752
2909.553 2482324720.713

95% Confidence Limits for log(concen12)

Estimate
Lower Bound Upper Bound
.335
-6.417
1.293
.562
-5.495
1.424
.706
-4.909
1.508
.814
-4.469
1.571
.902
-4.112
1.622
.977
-3.807
1.666
1.043
-3.540
1.704
1.102
-3.302
1.738
1.155
-3.084
1.770
1.205
-2.885
1.799
1.409
-2.058
1.920
1.571
-1.403
2.017
1.710
-.842
2.102
1.835
-.340
2.181
1.950
.122
2.256
2.060
.557
2.331
2.167
.971
2.411
2.271
1.368
2.500
2.376
2.482
2.592
2.708
2.832
2.971
3.134
3.338
3.387
3.440
3.499
3.565
3.640
3.728
3.836
3.980
4.207

1.740
2.068
2.313
2.477
2.598
2.706
2.817
2.947
2.977
3.010
3.046
3.085
3.130
3.183
3.247
3.331
3.464

48 | P a g e

2.613
2.779
3.044
3.418
3.876
4.414
5.056
5.874
6.072
6.288
6.525
6.791
7.094
7.451
7.890
8.474
9.395

Appendix 2: Communication Letters

49 | P a g e

50 | P a g e

Appendix 3: Laboratory Results

51 | P a g e

52 | P a g e

53 | P a g e

54 | P a g e

55 | P a g e

56 | P a g e

57 | P a g e

ABSTRACT

58 | P a g e