CHAPTER III
METHODOLOGY
A. Research Design
The research design used for the study of the mortality of Hermit Crabs at
different bleach concentrations was Completely Randomized Design (CRD). Five basins
were designed to simulate the beach environment with bleach concentrations, control, 10
ppm, 100 ppm, 1,000 ppm and 10,000 ppm, respectively. The researchers monitored the
Hermit Crabs’ mortality rates until LC50 in at least one set-up had been reached and then
verified if there was a significant difference among the mortality rates of the Hermit
Crabs at different bleach concentrations.
B. Materials and Equipment
Materials:
Bleach
Beaker
Hermit Crabs
Feeds for the Hermit Crabs
Seawater
White Sand
Stones
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5 Basins (with diameter of 8 inches each)
C. General Procedure
Gathering of Materials
The researchers gathered fifty Hermit Crabs from the seashore of Hapitanan,
Initao, Misamis Oriental, regardless of their specie, weight, color and age. They also
purchased commercial bleach and five basins with diameter of 8 inches each.
Preparing of Bleached Water Samples
The researchers measured bleach concentrations for Set-up A, B, C, D, and E
which are control, 10 ppm, 100 ppm, 1,000 ppm and 10,000 ppm, respectively. This
is thoroughly explained in the Table of Concentrations in the Experimental Set-up. To
get the ppm concentration for each set-up, the solute, bleach, was divided by the
solvent, seawater, and then multiplied by 106. This was further explained in the
expression below.
amount of solute
ppm concentration =
x
106
amount of solvent
Preparing the Improvised Hermit Crab Habitat
The set-up for each concentration included a basin to simulate the beach
environment. The set-up is represented by Figure 3.1.
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Figure 3.1 Set-up for the Hermit Crabs to Simulate the Beach Environment
Figure 3.1 shows the set-up for the Hermit Crabs to simulate the beach
environment. The blue part of the figure represents the seawater and bleach
concentration. The brown part represents the sand and stones, and the red part represents
the feeds of the Hermit Crabs which were available for them twice a day. The basin is
elevated a little on the sand part so as to totally submerged it in seawater and prevent it
from being washed away. This serves as the land area where Hermit Crabs usually stay.
Monitoring the Hermit Crabs’ Mortality Rate
The researchers determined the Hermit Crabs’ mortality rate through observing it
everyday until one set-up had five dead Hermit Crabs. This is also known as LC50 or
Lethal Concentration 50% common to toxicity tests. Dead Hermit Crabs were
determined when they did not move any longer and their bodies lied limp out of their
shell. When LC50 had been reached, the experiment stopped. The mortality rate was
then calculated through the formula shown below.
# of dead hermit crabs
% mortality =
x 100
total # of hermit crabs
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Table 3.1 Statistical Design Layout
Set-up
Bleach Concentration (ppm)
A
0
B
10
C
100
D
1,000
E
10,000
# of Dead Hermit Crabs
Mortality Rate
Table 3.1 is the Statistical Design Layout which was used to show the number of
dead Hermit Crabs and their respective mortality rates at different bleach concentrations.
D. Experimental Set-up
Table 3.2 Components of Different Set-ups
Set-up
Amount of Sand
Amount of Feeds
A
B
C
D
E
Bleach (mL)
0
0.002 mL
0.02 mL
0.2 mL
2 mL
Seawater (mL)
200 mL
200 mL
200 mL
200 mL
200 mL
Table 3.2 shows the Components of Different Set-ups. For each set-up, the
amount of solute which is the bleach was divided by the amount of solvent which is the
seawater multiplied by 106 that then yield to their respective concentration in terms of
ppm. The amount of sand and feeds were also shown in this table.
E. Instrument Used in Data Gathering
F. Statistical Tools for Data Analysis
To verify if there is a significant difference among the mortality rates of the
Hermit Crabs at different bleach concentrations, the Analysis of Variance (ANOVA) was
conducted by the researchers.
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Table 3.3 ANOVA Table
Source of
Degrees of
Variation
Freedom
Treatment
k-1
SS
MS
F
SST
SST
k-1
MST =
Error
n-k
SSE
MSE =
Total
n-1
MST
MSE
SST
n-k
Total SS
The Megastat add-in from Microsoft Excel was also used to calculate the ANOVA.
Gathering of 50 Hermit Crabs,
seawater, white sand, rocks and
purchasing of bleach and 5 basins
Preparing of bleached water samples with 10
ppm, 100 ppm, 1,000 ppm and 10,000 ppm
Preparing of improvised Hermit Crab
Habitat to simulate the beach environment
Placing of bleach concentrations, sand,
stones and 10 Hermit Crabs for each set-up
Observation of the Hermit Crabs’ mortality rate when
at least 5 hermit crabs died at any set-up (LC50)
Data analysis
Conclusion
Figure 3.2 Flowchart of Procedures done in the experiment
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Figure 3.2 shows the Flowchart followed by the researchers during the
entire period of experimentation.