Chapter III

Methodology

3.1 Research method

Research method is experimental, because it is a systematic and scientific approach to

research in which the researcher manipulates one or more variables, and controls and measures
any change in other variables (e.g. concentration, number of mortality).

3.2 Instrumentation and Materials

These are the instruments, materials, and chemicals that we will be using in making our
research:

a) Rotary vacuum Evaporator h) Stirring Rod

b) Beakers i) Diethyl ether
c) Acetone j) Dechlorinated water
d) Ethyl ether
e) Buchner Funnel
f) Filter paper
g) Electrical blender

3.3 Research Design

The research design that will be used is Statistical Package for the Social Sciences (SPSS) to
show how effective the water hyacinth (Eichhornia crassipes) extract against mosquito larvae
(Aedes aegypti). The treatments are mosquito larvae is to water hyacinth extract and labelled.
Replicate 1 is 30 larvae:80ppm, replicate 2 is 30 larvae:160ppm, replicate 3 is 30 larvae:240ppm,
replicate 4 is 30 larvae:320ppm and replicate 5 is 30 larvae:400ppm versus the commercial
larvicide. Each Treatment had at least (5) replicates.
 Number of Dead Larve at Various Cone. (g/L) in 24 hours
30 Mosquito Larvae 80 ppm 160 ppm 240 ppm 320 ppm 400 ppm w/ Clorox
Replicate 1
Replicate 2
Replicate 3
Replicate 4
Replicate 5
Total
Average
% Motality

(Table 3.1: Research Table showing Replicates)

3.4 Research locale

The experiment will be conducted at Arturo Eustaquio Memorial Science High School
particularly at the Chemistry Laboratory. Apparatuses will be borrowed from the Universidad de
Zamboanga Science Laboratory Services (SLS).

3.5 Data Gathering with Flowchart

The following flow chart below contains the methods of the research.

Maintenance of the
Collection of eggs of Blood feeding of
Aedes aegypti
the Aedes aegypti. adult Aedes aegypti.
larvae.

Statistical Analysis
Collection of
Eichhornia crassipes
Larval toxicity test
and preparation of
extract.
 (Figure 3.1: Flow of the Method)

a) Collection of eggs and maintenance of larvae

First, they shall make a starter out of bean sprouts by blending it until soft. Then they shall
sieve and add water to the starter. Once they have the starter we shall pour it onto a container
filled with water. Then they shall cover it until the bacterial bloom, which will take about 24
hours. Then they will open the lid and remove the biofilm from the container. Then they will
close it again, but this time they shall leave an opening so that the Aedes aegypti has an entrance.
After 24 hours they shall expect to find eggs on the surface of the container. These eggs will be
brought to the laboratory and transferred to 18 × 13 × 4-cm enamel trays containing 500 mL of
pond water for hatching. The mosquito larvae were fed pedigree dog biscuits and yeast at 3:1
ratio. The feeding was continued until the larvae transformed into the pupal stage. (See figure
3.1)

b) Maintenance of pupae and adults

The pupae will then be collected from the culture trays and transferred to plastic containers
(12 × 12 cm) containing 500-mL of water with the help of a dipper. The plastic jars will be kept
in a 90 × 90 × 90-cm mosquito cage for adult emergence. A 10% sugar solution was provided for
a period of 3 days before blood feeding. (See figure 3.1)

c) Blood feeding of adult A. aegypti

The adult female mosquitoes can feed on the blood of a laboratory rat (a lab rat per day,
exposed on the dorsal side) for 2 days, to ensure adequate blood feeding for 5 days. After blood
feeding, enamel trays with water from the culture trays were placed in the cage as oviposition
substrates. (See figure 3.1)

d) Collection of plant and preparation of extract

 Collection of Eichhornia crassipes and preparation of extract. The Eichhornia crassipes will
be first washed with water and then shade dried at room temperature. An electrical blender will
powder the dried plant. 100 g of the powder will be extracted using 300 ml of Diethyl ether
which will be left for 24 hours after which the extract will be filtered. Which then will evaporate
to dryness using the rotary vacuum evaporator. One gram of the plant residue will be dissolved
in 100 mL of acetone (stock solution) and considered as 1% stock solution. From this stock
solution, different concentrations were prepared ranging from 80, 160, 240, 320 and 400 ppm,
respectively. (See figure 3.1)

e) Larval toxicity test

A laboratory reared colony of A. aegypti larvae will be used for the larvicidal activity.
Twenty-five individuals of first, second, third, and fourth instars larvae will be kept in a 500-mL
glass beaker containing 249 mL of dechlorinated water and 1-mL of desired concentration of
Eichhornia crassipes were added. Larval food will be given for the test larvae. At each tested
concentration, two to five trials will be made, and each trial consists of five replicates. The
control was setup by mixing 1 mL of acetone with 249 mL of dechlorinated water. The larvae
will be exposed to dechlorinated water without acetone served as control. The control mortalities
were corrected by using Abbott’s formula (Abbott, 1925). The LC50 and LC90 will be
calculated from toxicity data by using Probit analysis (Finney, 1971). (See figure 3.1)

f) Statistical analysis

All data will have subjected to analysis of variance; the means will be separated using
Duncan’s multiple range tests by Alder and Rossler (1977). The average larval mortality data are
to be subjected to Probit analysis, for calculating LC50 and LC90, values will be calculated by
using the Finney (1971) method. SPSS (Statistical software package) 9.0 version was used.
Results with P < 0.05 were statistically significant. (See figure 3.1)