(LATO) IN LOWERED SEAWATER pH - Central Visayas Campus ...

GROWTH PERFORMANCE OF Caulerpa lentillifera (LATO)
IN LOWERED SEAWATER pH
A Research Paper
Presented to the
Curriculum and Instruction Services Division
Philippine Science High School-Central Visayas Campus
Talaytay, Argao, Cebu
In Partial Fulfillment of the Requirements in Research II
CHRISTINE A. ILUSTRISIMO
ISABEL C. PALMITOS
RACHELDA D. SEÑAGAN
March 2013

Republic of the Philippines
Department of Science and Technology
PHILIPPINE SCIENCE HIGH SCHOOL – CENTRAL VISAYAS CAMPUS
Talaytay, Argao, Cebu
CERTIFICATE OF PANEL APPROVAL
The research attached hereto, entitled “GROWTH PERFORMANCE
OF Caulerpa lentillifera (LATO) IN LOWERED SEAWATER pH”,
prepared and submitted by CHRISTINE A. ILUSTRISIMO, ISABEL C.
PALMITOS and RACHELDA D. SEÑAGAN is hereby recommended for
approval.
ALCO KENNETH C. TOLENTINO _______________
Research Adviser Date
ELEAZAR B. GUIA __________________
Panel Member Date
REY GIOVANNI L.VILLAMORA __________________
Panel Member Date
This research paper is approved in fulfillment of the requirements in RESEARCH II.
RECOMMENDING APPROVAL:
SHERRY P. RAMAYLA JOSEPH P. HORTEZUELA
Unit Head, Research Unit CISD Chief
APPROVED BY:
FLORITA A. PONTILLAS, ED.D.
Campus Director
ii

BIOGRAPHICAL DATA
Researcher : Christine Albite Ilustrisimo
Age : 16 years old
Address : Lamacan, Argao, Cebu
Date of Birth : December 19, 1996
Mother : Teodora A. Ilustrisimo
Father : Estanislao P. Ilustrisimo Jr.
Siblings : Julie Ann A. Ilustrisimo
Carl John A. Ilustrisimo
Contact no. : 09295803297
Email Address : ilustrisimo_christine@yahoo.com
Researcher : Isabel Coritico Palmitos
Age : 16 years old
Address : Pig-ot, Loon, Bohol
Date of Birth : August 7, 1996
Mother : Milagros C. Palmitos
Father : Roy D. Palmitos
Sibling : Kaye Angel C. Palmitos
Contact no. : 09217386060
Email Address : isabelpalmitos@yahoo.com
iii

Researcher : Rachelda Dinauanao Señagan
Age : 16 years old
Address : Colase, Samboan, Cebu
Date of Birth : July 28, 1996
Mother : Celedonia D. Señagan
Father : Juanito F. Señagan
Siblings : Richard Horace D. Señagan
Rica Angela D. Señagan
Richell Marie D. Señagan
Ronald D. Señagan
Contact no. : 09334085887
Email Address : eldasenagan@yahoo.com
iv

ACKNOWLEDGEMENT
The researchers would like to thank the University of San Carlos-Talamban
Campus (USC -TC), Talamban, Cebu City for allowing them to conduct the
experimentation of their study there, and Philippine Science High School – Central
Visayas Campus (PSHS-CVisC) for the endless support they have given.
The researchers would also like to thank Dr. Anthony S. Ilano and Professor
Rolly V. Viesca from USC-TC for their help and guidance in the conduct of their
study.
The researchers would also like to express their deep appreciation and
gratitude to the residents of Brgy. Kalawisan, Mactan, Cebu most especially to Brgy.
Capt. Eduardo Binse, Brgy. Councilor Danilo Nuñez and Mr. Leopoldo Verame for
their full help in the collection of their samples. And to Mr. Estanislao Quillosa for
helping us get seawater.
The researchers would also like to thank Mr. Paul Isaac Dizon and the
Research II instructors including their adviser, Mr. Alco Kenneth Tolentino, and their
panel members, Mr. Eleazar B. Guia and Mr. Rey Giovanni Villamora, for teaching
them the concepts behind research and for sparing their time in editing and checking
their research paper.
The researchers would also like to express their deep appreciation to their
batchmates for supporting them and serving as inspirations to finish their study.
The researchers would also like to say thank you to their parents for
constantly supporting them in everything they encounter and to our Almighty God.
The researchers,
CHRISTINE ILUSTRISIMO ISABEL PALMITOS RACHELDA SEÑAGAN
v

Ilustrisimo, C. A., I. C. Palmitos, R. D. Señagan. 2013. “Growth Performance of
Caulerpa lentillifera in Lowered Seawater pH”. A Research Paper. Philippine Science
High School – Central Visayas Campus, Talaytay, Argao, Cebu.
ABSTRACT
Ocean acidification, as a result of increased atmospheric carbon dioxide (CO2)
concentrations, is predicted to lower the pH of seawater over the next 100 years
(Clark et al. 2009). The aim of the study was to determine the growth performed by
Caulerpa lentillifera as the test species in lowered seawater pH. To verify the growth
performance of the species, an acidic environment was made with a range of lower
seawater pH (pH 2.7 -3.3, 3.7-4.3, 5.7-6.3 and 7.7 to 8.3). Acidity was adjusted by
adding a reagent grade of hydrochloric (HCl) acid. Species treated with pH 7.7 to 8.3
were held as the controlled group. Experimental conditions (temperature, salinity,
dissolved oxygen, and pH) were observed daily as accounted by Battistoni et al.
(2007). Different physical changes (weight and color) were also observed. The
amount of chlorophyll a content of the seaweed after the treatment was examined by
reading its absorbance at 645 nm and 662 nm using UV-Vis spectrophotometer.
Lowering pH resulted in a decrease in survival of the species. Species treated in pH
7.7 to 8.3 contains 11.92 mg/L chlorophyll a and was the highest amount of
chlorophyll a content, followed by pH 5.7 to 6.3 which was 11.66 mg/L, pH 2.7 to 3.3
which was 0.1551 mg/L and lastly, pH 3.7 to 4.3 containing 0.1198 mg/L. The
statistical results proved that there was a significant difference on the weights of the
samples in the three trials among the four groups. The study concluded that lowering
seawater pH (ocean acidification) will result to a decrease in weight and chlorophyll a
content of C. lentillifera, implying a negative effect on the growth of the species.
vi

TABLE OF CONTENTS
Title PAGE
TITLE PAGE i
CERTIFICATE OF PANEL APPROVAL ii
BIOGRAPHICAL DATA iii
ACKNOWLEDGEMENT v
ABSTRACT vi
TABLE OF CONTENTS vii
LIST OF TABLES ix
LIST OF FIGURES x
CHAPTER I. INTRODUCTION 1
1.1 Background of the Study 1
1.2 Objective of the Study 2
1.3 Significance of the Study 3
1.4 Scope and Limitations of the Study 3
1.5 Definition of Terms 4
CHAPTER II. REVIEW OF RELATED LITERATURE 5
2.1 Ocean Acidification 5
2.2 Ocean Acidification’s Effects on Growth Performance of
Marine Organisms
7
2.3 Biological Classification of Caulerpa lentillifera 8
2.4 Chlorophyll Concentrations 9
2.5 Related Studies 10
2.6 Physico-Chemical Conditions that Affect Seawater pH 11
CHAPTER III. MATERIALS AND METHODS 13
3.1 Research Design 13
3.2 Collection of Species 13
3.3 Experimental Set-up 13
3.4 Growth Performance 14
3.4.1 Measurement of the Species’ Growth Rate 14
3.4.2 Color Changes of the Samples 15
3.4.3 Measurement of Chlorophyll a Concentration 15
3.4.3.1 Preparation of Sample 15
3.4.3.2 Spectrophotometric Determination 16
3.5 Statistical Analysis 16
CHAPTER IV. RESULTS AND DISCUSSION 17
4.1 Average Weight of Caulerpa lentillifera at Different pH 17
4.2 Growth Performance 19
4.3 Color Changes of the Samples 20
vii

4.4 Chlorophyll a Concentration 21
CHAPTER V. CONCLUSION AND RECOMMENDATIONS 23
5.1 Conclusion 23
5.2 Recommendations 23
BIBLIOGRAPHY 25
APPENDICES 29
Appendix A. Raw Data of the Initial and Final Weight of the Species 29
at Different pH
Appendix B. Raw Data of the Absorbance of Caulerpa lentillifera (Lato) 31
at 645 and 662 Nanometers (nm)
Appendix C. T-Test Statistics Results 32
Appendix D. Photos 33
viii

LIST OF TABLES
TABLE PAGE
1 t-Test Results 18
2 Growth Rate of C. lentillifera at different pH 19
3 Chlorophyll a Concentration of C. lentillifera 21
A.1 The weight of Caulerpa lentillifera (Lato) in grams 29
at pH 7.7 to 8.3
A.2 The weight of Caulerpa lentillifera (Lato) in grams 29
at pH 5.7 to 6.3
A.3 The weight of Caulerpa lentillifera (Lato) in grams 29
at pH 3.7 to 4.3
A.4 The weight of Caulerpa lentillifera (Lato) in grams 30
at pH 2.7 to 3.3
B.1 The absorbance of Caulerpa lentillifera (Lato) at 31
645 nanometers (nm)
B.2 The absorbance of Caulerpa lentillifera (Lato) at 31
662 nanometers (nm)
C.1 Statistical Analysis of Samples 32
C.2 Mass (in grams) of Caulerpa lentillifera at different pH 32
ix

LIST OF FIGURES
FIGURE PAGE
1 Planting of C. lentillifera in the tank 14
2 Mass (in grams) of C. lentillifera at different pH 17
D.1 Fresh samples of Caulerpa lentillifera 33
D.2 Samples after the treatment 33
D.3 Experimental Setup 33
x

1.1 Background of the Study
CHAPTER I
INTRODUCTION
Oceans act as important carbon sinks for anthropogenic emissions, reducing
atmospheric concentrations of carbon dioxide by up to one-third (Doney et al., 2009).
As oceans become saturated with CO2, the pH of seawater is predicted to drop from
8.1 to 7.7-7.8 by the end of this century (Le Quere et al., 2007). This increased acidity
may have dramatic effects on marine organisms, in part because it reduces the
concentration of carbonate ions and the solubility of aragonite, a form of calcium
carbonate used to create biological structures (Orr et al., 2005).
A dire prediction that has raised a serious concern in the climate-alarmist-
inspired campaign to force reductions in anthropogenic CO₂ emissions is the
contention that continued increases in the air's CO₂ content will lead to ever more
carbon dioxide dissolving in the surface waters of the world's oceans and lowering its
pH values. This phenomenon is claimed to make biological calcification more
difficult to occur in marine organisms. A pH reduction of the seawater is viewed by
many as a cause for great concern, as it has been postulated to harm marine organisms
most especially on calcifying marine life such as corals, not only by reducing their
calcification rates, but by negatively impacting their metabolism, fertility, growth and
survival. (http://www.co2science.org, 2011)
As carbon dioxide mixes with water, the two molecules combine to become
carbonic acid which makes the seawater slightly more acidic. As billions of
molecules combine and go through this process, the overall pH of the oceans begins
to change. This phenomenon is known to be ocean acidification, the lowering of sea-

water pH that can greatly alter the metabolism, fertility, growth and survival of
different marine organisms. Thus, the effect of lowering of the seawater pH on the
growth of Caulerpa lentillifera will be determined (http://www.co2science.org).
In this study, a species of green algae in the family Caulerpaceae, Caulerpa
lentillifera, was examined. This alga is stenohaline and cannot thrive in areas where
salinity is less than 25%. Salinities lower than 30% would already result in crop loss
(http://www.globinmed.com). Caulerpa lentillifera has become a source of food to
mankind. It has nutritional contents which can serve as dietary alternatives. Moreover,
this species is found out to be a potential source of food proteins ( Chirapart and
Ratana-arporn, 2006).
This study focused mainly on how lowered seawater pH affects the growth
performance of Caulerpa lentillifera. Lowered seawater pH has been a vital factor on
the destruction of marine life, most especially on the marine organisms.
1.2 Objectives of the Study
This study aimed to determine the growth performance of Caulerpa lentillifera
in lowered seawater pH. Specifically, this study aimed to:
determine the fresh weight of Caulerpa lentillifera (lato) in varying seawater
pH before and after the treatment;
compare the initial and final weight of C. lentillifera (lato) at the designated
seawater pH;
determine the color changes in C. lentillifera (lato) in the normal and lowered
seawater pH treatments; and
determine the amount of chlorophyll a present in the species treated with
lowered seawater pH.
2

1.3 Significance of the Study
This study is essential to environmentalists and people who get their income
from seaweeds such as Caulerpa lentillifera. By knowing the mechanism of such
species in reacting to climate change, those people will be able to create and innovate
ways on how to conserve and lessen the impact of climate change to the species.
In addition, this study will greatly make a contribution to the scientific
community. The results of this study will provide information and further knowledge
to scientists on how C. lentillifera will react to a change in the environment,
specifically, ocean acidification. Moreover, this study can be used as a reference for
possible researches using the test species in its response to climate change.
1.4 Scope and Limitations of the Study
This study included only the growth performance of Caulerpa lentillifera in
lowered seawater pH. A UV-Vis spectrophotometer was used to measure the level of
chlorophyll a in the algae. The samples used were obtained from Carbon Market,
Colon St., Cebu City. This experiment was only limited on the specified pH ranges
and due to the lack of time, the period of experiment was only seven (7) days for each
of the three trials. The pH ranges used were all below the normal seawater pH (about
pH 8.0) because the focus of this study was only about ocean acidification. Only the
optimal range of seawater pH and the three pH treatments were considered in this
study. Experimental conditions were monitored, but the study was only focused on the
seawater pH. The samples were exposed to sunlight during the experimentation. Each
of the tank only contained 20 L of seawater.
3

1.5 Definition of Terms
Carbon dioxide (CO 2) is one of the most abundant gases in the air. It is the most
important gas for plants to make food. For humans, carbon dioxide is a waste
product of respiration.
Caulerpa lentillifera is a stoloniferous plant that can grow up to 10 cm tall. It has
branched stolons, cylindrical in cross-section and measuring 1-1.5(-2) mm in
diameter. (Jenkins 2009)
Lowered seawater pH is also known as ocean acidification. This happens when the
normal seawater pH, which is about pH 8.0, decreases. This poses a dramatic
effect on marine organisms and the life beyond. (Fabry et al., 2008)
pH (potential of Hydrogen) is a measure of the acidity or alkalinity of a solution,
numerically equal to 7 for neutral solutions, increasing with increasing
alkalinity and decreasing with increasing acidity. The pH scale commonly in
use ranges from 0 to 14. (http://dl.clackamas.edu/ch105-05/definiti.htm, 2000)
Salinity is the saltiness or dissolved salt content of a body of water. It is a
dimensionless quantity with no units ( http://www.thefreedictionary.com,
2006).
4

2.1 Ocean Acidification
CHAPTER II
REVIEW OF RELATED LITERATURE
Over the last two centuries, atmospheric carbon dioxide has increased by more
than 30%, from 280 to 380 ppm (parts per million) by 2007 (Doney et al., 2007). This
causes more than 80% of the added heat to reside in the ocean, causing global
warming. This increased CO2 concentration then leads to ocean acidification. Ocean
acidification, the phenomenon of decreasing ocean pH due to the uptake of
anthropogenic carbon dioxide (CO2), has been identified as an important consequence
of rising CO2 emissions worldwide (Fabry et al., 2008).The magnitude and patterns of
these changes are consistent with an attribution to human activities and not explained
by natural variability alone (Doney et al., 2007).
Over half of human carbon dioxide emissions to the atmosphere are absorbed
by the ocean and land biospheres, and the excess carbon absorbed by the ocean results
in increased ocean acidity (Gruber & Sarmiento, 2002). Once carbon dioxide enters
the ocean, it combines with water to form carbonic acid and a series of acid-base
products, resulting in a lowering of pH values. A small decrease in pH affects the
chemical equilibrium of ocean water, reducing the availability of carbonate ions
needed by a wide range of organisms to build and maintain structures of calcium
carbonate (Science Daily, 2008).
For at least 650, 000 years prior to the Industrial Revolution, atmospheric CO2
concentrations varied between 180 and 300 ppm (Siegenthaler et al., 2005). As a
result of human activity, today’s atmospheric CO2 concentration reaches 380 ppm and
currently is rising at a rate of 0.5% to the year 2100 (Forster et al., 2007), which

is 100 times faster than any change during the past 650 000 years (Royal Society,
2005; Siegenthaler et al., 2005). Elevated partial pressure of CO2 in seawater (also
known as hypercapnia) can impact marine organisms both via decreased calcium
carbonate (CaCO3) saturation, which affects calcification rates, and via disturbance to
acid–base (metabolic) physiology. The oceanic uptake of anthropogenic CO 2 and the
concomitant changes in seawater chemistry have adverse consequences for many
calcifying organisms, and may result in changes to biodiversity, trophic interactions,
and other ecosystem processes (Royal Society, 2005; Fabry et al., 2006).
Climate change is a problem that is affecting people and the environment.
Climate-focused agencies have been designing and implementing programs to help
conserve the environment. Greater energy efficiency and new technologies hold
promise for reducing greenhouse gases and solving this global challenge
(http://www.epa.gov/climatechange, 2007).
All across the world, people are taking action because climate change has
serious impacts, locally and globally. For example, in 2007, scientists from the
International Panel on Climate Change (IPCC) predicted that warming oceans and
melting glaciers due to global warming and climate change could cause sea levels to
rise 7-23 inches by the year 2100. Worldwide, densely populated coastal communities
and infrastructure that supports them would be affected (such as city buildings and
homes, roads, ports and wastewater treatment plants). Some would be flooded or
more vulnerable to storm damage. In flat terrain, the shoreline could move many
miles inland (http://www.ecy.wa.gov/climatechange/whatis.htm, 2007).
6

2.2 Ocean Acidification’s Effects on Growth Performance of Aquatic Organisms
The growth rate of a microalgal population is a measure of the increase in
biomass over time and it is determined from the exponential phase. Growth rate, such
as the change in biomass, is one important way of expressing the relative ecological
success of a species or strain in adapting to its natural environment or the
experimental environment imposed upon it. The duration of exponential phase in
cultures depends upon the size of the innoculum, the growth rate and the capacity of
the medium and culturing conditions to support algal growth. Cell count and dry
weight are common units of biomass determination
(http://www.marine.csiro.au/microalgae/methods/Growth%20rate.htm, 2007).
The study conducted by Battistoni et al. in 2007 focused on the effects of
ocean acidification on a turtle grass meadow. The results showed that ocean
acidification negatively affected the health of turtle grass because of the decrease in
growth and an increase in leaf senescence of plants in acidified treatments. They had
also concluded that the acidic environment appeared to be detrimental to epiphytic
calcifying algae. Another study conducted by Vijayan and Diwan (1995) focused
on the influence of temperature, salinity, pH, and light on the molting and growth of
the Indian white prawn Penaeus indicus under laboratory conditions. The study
resulted in a depressed growth of the species. They also concluded that lowered
seawater pH is eventually harmful to crustaceans.
7

2.3 Caulerpa lentillifera
Kingdom: Plantae
Division: Chlorophyta
Class: Bryopsidophyceae
Order: Bryopsidales
Family: Caulerpaceae
Genus: Caulerpa
Species: lentillifera
The genus Caulerpa are fast-growing green algae with fronds (“leaves”) that
come in a variety of shapes. The fronds between 15-30 cm (6-12 inches) in length are
attached to long runners (“stems”) called rhizomes. Besides simply spreading
outwards, Caulerpa can also propagate themselves vegetatively through sections of
rhizome that break off the parent plant and become established elsewhere ( Jenkins,
2009).
Caulerpa lentillifera is one of the favored species of edible Caulerpa due to its
soft and succulent texture. It is also known as sea grapes, green caviar, "ar-arosep", or
"lato" in the Philippines. The pond cultivation of C. lentillifera has been very
successful on Mactan Island, Cebu, Philippines, with markets in Cebu and Manila.
About 400 hectares (ha) of ponds are under cultivation, producing 12-15 tonnes of
fresh seaweed per hectare per year (McGugh, 2003).
C. lentillifera is usually eaten raw with vinegar, as a snack or in a salad
(Jenkins, 2009). It was also found to be a potential source of plant food proteins
owing to its high protein levels and balanced amino acid profiles and mineral
supplements (Chirapart and Ratana-arporn, 2006). The study conducted by Chirapart
8

and Ratana-arporn in 2006 concluded that this seaweed can provide dietary
alternatives due to its nutritional values. It was also found to have reasonable
adsorption capacity for basic dyes, Astrazon ® Blue FGRL (AB), Astrazon ® Red
GTLN (AR), and Astrazon ® Golden Yellow GL-E (AY) (Pimol et al., 2007). The
biosorption of Cu(II), Cd(II), and Pb(II) by Caulerpa lentillifera was also investigated
by Apiratikul and Pavasant in 2007. Their study had showed that ion exchange was
one of the main sorption mechanisms of the species.
2.4 Chlorophyll Concentration
Chlorophyll is a green pigment found in cyanobacteria and the chloroplasts of
algae and plants. In chlorophyll the central ion is magnesium, and the large organic
molecule is a porphyrin. The porphyrin contains four nitrogen atoms that form bonds
to magnesium in a square planar arrangement. Chlorophyll is one of the most
important chelates in nature. It is capable of channelling the energy of sunlight into
chemical energy through the process of photosynthesis. In photosynthesis, the energy
absorbed by chlorophyll transforms carbon dioxide and water into carbohydrates and
oxygen. Since animals and humans obtain their food supply by eating plants,
photosynthesis can be said to be the source of our life.
It has been shown that chlorophyll degradation products may at times
constitute a significant fraction of the total green pigments present in seawater
(Menzel and Yentsch, 1963). On a study conducted by Fay and Wyman ( 1986), the
influence of light quantity on the underwater light climate and the growth and
pigmentation of planktonic blue-green algae (cyanobacteria) was investigated. In
light-limited cultures, the relative concentrations of chlorophyll and phycobiliproteins
per cell decline at a similar rate with increasing irradiance. These pigment synthesis
9

appear to be imposed by genotypic constraints on the maximum cell density and on
the size of photosynthetic units rather than by either light or nutrient limitation of
chlorophyll and phycobiliprotein synthesis.
Another study by Beer et al. in 1993 focused on the photosynthetic
performance and carboxylation capacity in a range of aquatic macrophytes of
different growth forms. The low chlorophyll content and high chlorophyll specific
photosynthesis of species with high carbon transport capability (i.e. particularly the
marine algae) determine a large section on the water quality and thus, to the life in
marine environment. This suggests that running costs associated with inorganic
carbon assimilation are reduced when a CO2 concentrating system operates.
According to the Australian Online Coastal Information, chlorophyll a
concentrations are an indicator of phytoplankton abundance and biomass in coastal
and estuarine waters. They can be an effective measure of trophic status, are potential
indicators of maximum photosynthetic rate (P -max) and are a commonly used
measure of water quality. High levels often indicate poor water quality and low levels
often suggest good conditions. However, elevated chlorophyll a concentrations are
not necessarily a bad thing in relation to water quality. It is the long-term persistence
of elevated levels that is a problem. For this reason, annual median chlorophyll a
concentrations in a waterway are an important indicator on the status of the
environment.
2.5 Related Studies
Clark et al. (2009) had conducted a research about the response of sea urchin
pluteus larvae (Echinodermata: Echinoidea ) to reduced seawater pH. The results
showed that lowered seawater pH had affected the survival of the species used.
10

Lowering pH resulted in a decrease in survival in all species, but only below pH 7.0.
The size of larvae was reduced at lowered pH, but the external morphology (shape)
was unaffected. Calcification of the larval skeleton was significantly reduced (13.8 –
36.9% lower) under lowered pH, with the exception of the Antarctic species, which
showed no significant difference.
In a similar study done by Kurihara and Ishimatsu (2008), exposure to
seawater equilibrated with high CO2 air did not significantly affect the survival rate of
the copepod Acartia tsuensis, as well as with marine algae. Consequently, Clark et al.
(2009) concluded that lowered pH levels will not have a direct effect on the survival
of marine species. They may, however, exhibit decreased growth and calcification
indices that could indirectly compromise their survival in the ocean.
Elevated temperature (28-34°C) has been hypothesized as the primary cause of
the loss of algal endo-symbionts in coral reef-associated invertebrates, a phenomenon
observed on a world-wide scale over the last decade. In a related study conducted by
Iglesias-Prieto et al. in 1992, elevated temperatures adversely affects photosynthesis
of Symbiodinium microadriaticum in culture. Growth of the cells at different oxygen
tensions did not also appear to have any direct influence on the temperature-related
decrease in photosynthesis.
2.6 Physico-Chemical Conditions that Affect Seawater pH
The pH of seawater responds to changes in: (1) the concentration of total
dissolved CO2; (2) alkalinity; (3) temperature; and for deep waters, (4) pressure. In
each case, the magnitude of change varies with salinity because the concentration of
salt influences the various equilibrium constants and because several components of
sea salt are involved in the acid-base reactions of seawater. The equilibrium pH of
11

surface seawater, for a given temperature and salinity, is the pH of seawater in
equilibrium with CO2 in the atmosphere (Skirrow and Riley, 1975).
The seawater has a good buffer system. This means that it would keep in track
the balance within its environment. The seawater’s buffer system equilibrates back
and forth the changes and stresses it encounters and thus, pH stays relatively constant
(http://www.livingreefs.com). However, when too much stress is encountered, the
seawater’s buffer system lessens its capabilities to maintain the balance within its
environment.
Dissolved carbon dioxide occurs as carbonic acid and its presence in water
lowers the pH. During the day when plants are illuminated and photosynthesis is
occurring, the dissolved CO2 is taken up from the water and the pH rises. At night
when no photosynthesis is taking place and the CO2 builds up thereby contributing to
a falling pH. On the other hand, high alkalinity promotes calcification, encouraging
the rapid growth of calcifying algae. High alkalinity combined with calcium dosing
promotes the precipitation of phosphate and this limits algae growth
(http://www.livingreefs.com). Temperature inversely controls the solubility of oxygen
in water; as temperature increases, oxygen is less soluble. In contrast, there is a direct
relationship between atmospheric pressure and DO; as the pressure increases due to
weather or elevation changes, oxygen solubility increases
(www.uwgb.edu/watershed). Low concentrations of dissolved oxygen leads to an
acidic environment, or lowered seawater pH (RiverTrends Manual, 2010).
12

3.1 Research Design
CHAPTER III
METHODOLOGY
This is an experimental study which aimed to collect data on the growth
performance of Caulerpa lentillifera in lowered seawater pH. The experiment was
conducted in three trials with nine replicates per trial. Measurement of the chlorophyll
a content with the use of the UV-VIS Spectrophotometer was done at the Department
of Biology-University of San Carlos-Talamban Campus, Cebu Laboratory and the rest
including pH treatment of the C. lentillifera were done in Philippine Science High
School-Central Visayas Campus, Talaytay, Argao, Cebu Laboratory.
3.2 Collection of Species
One kilogram of fresh species of Caulerpa lentillifera was bought from
Carbon Market, Colon St., Cebu City. The dark green samples were chosen as the
planting materials.
3.3 Experimental Set-up
An acidic environment was created by filling three (3) clear, square tank
which measures 18x12x12 inches of 45 L capacity filled with 20 L of seawater and
specific pH adjusted initially with 1.0 M Hydrochloric acid (HCl). The seawater pH
was lowered in order to create an environment similar to what ocean acidification is.
Each tank was provided with an aerator. The samples were selected and were held on
a plastic screen placed in the tank using glove-covered hands at the start of the
experimentation period (see Figure 1). After placing the species on the tanks, it was
left untreated for three days to stabilize the samples. After three days of stabilization,

a reagent grade Hydrochloric acid of different volumes was added daily to each tank
to obtain the following pH ranges: 2.7 to 3.3, 3.7 to 4.3 and 5.7 to 6.3. A GLX
Explorer pH meter was used to monitor the specified pH ranges.
Figure 1. Planting of C. lentillifera in the tank
The pH ranges were checked again two (2) hours after adjustment to ensure
that the pH was still within the desired ranges (Clark et al., 2009). A tank filled with
20 L of seawater (pH 7.7 – 8.3) was also created which served as control. All of the
tanks were placed strategically to expose it to sunlight.
3.4 Growth Performance
3.4.1 Measurement of the Species’ Growth Rate
The growth performed by the species was determined by measuring its
biomass using a digital weighing scale. The individual species’ weight was recorded
before and after the treatment. The data on the average weight of the samples were
plotted in a graph with the time on the x-axis and biomass on the y-axis. To determine
the growth performance of the species, two points, N1 and N2, which are the
extremes of the linear phase are taken and substituted into the equation below
(Levasseur et al., 1993).
14

where K = growth rate, N1 = initial weight and N2 = final weight at time1 (first day)
and time2 (seventh day), respectively.
3.4.2 Color Changes of the Samples
The color of the samples was observed before and after treating them in
lowered seawater pH. This was also true for the samples under the normal seawater
pH. The color changes were classified to be green, pale green, or pale white.
Photographs were used to compare the changes in the color of the samples.
3.4.3 Measurement of Chlorophyll a Concentration
The amount of chlorophyll a content of the seaweed was also determined
using a UV-Vis spectrophotometer. This was used as a guide in the growth
performance of the species. The methods used were based on the study conducted by
Humphrey and Jeffrey (1975), Porra et al. (1989), and Dere et al. (1997).
3.4.3.1 Preparation of Sample
All the samples in each tank were weighed using a digital weighing scale.
After weighing, ten (10) mL of 100% acetone was added to each sample. It was then
homogenized using a homogenizer at 1000 rpm for one minute. The homogenate was
filtered using filter paper, and was centrifuged at 2500 rpm for three minutes. The
supernatant was then separated from the precipitate. The precipitate was discarded.
This sample preparation was done at Philippine Science High School-Central Visayas
Campus Science Laboratory.
15

3.4.3.2 Spectrophotometric Determination
The supernatant for each treatment was analyzed for the determination of the
chlorophyll a content using a UV-Vis spectrophotometer. The chlorophyll a
concentration was determined by measuring the absorbance (optical density - OD) of
the extract at wavelengths 645 nanometer and 662 nanometer. The resulting
absorbance measurements were then applied to a standard equation (ESS Method
150.1: Chlorophyll-Spectrophotometric).
The formula below was used to calculate the amount of chlorophyll a:
C = (11.75) (A662) – (2.350) (A645)
where C = Chlorophyll a concentration,
3.5 Statistical Analysis
A662 = absorbance at 662 nanometer
A645 = absorbance at 645 nanometer.
Treatment effects on the weight of the species were statistically tested for
significance at the 5% level using t-test (Kurihara and Ishimatsu 2008). The initial and
final weights of the species for the three trials were used and compared statistically.
The program used was Past software in computing the t-test.
16

CHAPTER IV
RESULTS AND DISCUSSION
4.1 Average Weight of Caulerpa lentillifera at Different pH
Samples of Caulerpa lentillifera for four different pH levels were weighed
before and after treating them at varying pH. These were then calculated and the
average weight for the species in each tank were determined.
Figure 2. Mass (in grams) of C. lentillifera at different pH
Figure 1 shows the result of the average weight of the species. The graph of
pH ranging from 7.7 to 8.3 has showed an increase in the mass while the graph of pH
ranging from 5.7 to 6.3, pH 3.7 to 4.3 and pH 2.7 to 3.3 showed a decrease in the
mass. As the pH decreases compared to normal seawater pH, the final mass of C.
lentillifera also decreases. This coincides to the fact that C. lentillifera can thrive on
the normal seawater pH which is at around pH 8 (Battistoni et al., 2007).
Using t-test, the computed t-values of the trials with pH 8.0±0.3, pH 6.0±0.3, pH
4.0±0.3 and pH 3.0±0.3 have values that are lesser than the significance level of 0.05.

At 5% level of significance, the data showed a significant difference on the weights of
the samples in the three trials among the four groups. Table 1 shows the t-test
statistical analysis results.
Table 1. t-Test Results
Variables
F
Values
166.44
p(eq)
2.76E-10
t 3.7264 0.001173
Welch t 3.7264 0.003278
Permutation t test: 0.001
A related study conducted by Battistoni et al. in 2007 focused on the effects of
ocean acidification on a turtle grass meadow. The results showed that ocean
acidification negatively affected the health of turtle grass because of the decrease in
growth and an increase in leaf senescence of plants in acidified treatments. They had
also concluded that the acidic environment appeared to be detrimental to epiphytic
calcifying algae, as algal cover on turtle grass leaves was reduced in the acidified
tanks.
Another study by Hauton et al. (2008) showed that changes in the pH of
seawater will potentially cause a disruption to the internal acid/base balance in wider
range of marine species. To some extent, this can result to hypercapnia which had
been shown to cause metabolic suppression in protostome invertebrates, although it is
clear that differences do occur and some species have a better tolerance than others
(Michaelidis et al., 2004).
18

These studies correlate with the results shown in the study. Lowering the
seawater pH had brought a negative effect on the species’ weight. A negative effect
was shown when the samples treated in lowered seawater pH decreased compared to
its fresh weight.
4.2 Growth Performance
From the data in Table C.2 (see Appendix C), the growth performance of the
species was calculated following Levasseur et al. (1993).
Table 2. Growth Rate of C. lentillifera at different pH
pH Level Growth Rate (%)
7.7 to 8.3 0.0069
5.7 to 6.3 -0.039
3.7 to 4.3 -0.061
2.7 to 3.3 -0.089
Table 2 shows the growth rate of the species treated at pH 8.0±0.3, 6.0±0.3,
4.0±0.3 and 3.0±0.3. The species under the normal seawater pH had the highest
growth rate, implying a positive impact on the species. It was followed by the species
treated at pH 6.0±0.3, then pH 4.0±0.3 and pH 3.0±0.3. All of the species treated
under lowered pH had negative coefficient. The negative coefficient implies a
decreased growth on the species treated under their designated pH levels.
Hinga (2002) reported that an ion balance effect may have occurred within the
organism. At high or low pH, cells may have to spend energy maintaining an internal
19

pH necessary for cell function. In addition, Hinga (2002) also concluded that the low
concentrations of free CO2 may as well be the mechanism for reduced growth rate at
low pH. The pH also affects the rates of primary production of marine algae.
Accordingly, their growth rates change significantly with pH changes near the
equilibrium pH.
The results of the study showed that the growth rate of the species was
reduced in lowered pH, which also coincided with the results on the study by Hinga
(2002). At pH 8.0±0.3, the species are under their normal environment, enabling
them to perform its activities well.
4.3 Color Changes of the Samples
The samples treated at the acidic pH of 3.0±0.3 and 4.0±0.3 showed a change
in color, most specifically from green to pale white. Also, 100% of the sample at these
pH ranges did not survive the experimental period of 7 days. On the other hand, only
about 20% of the species treated at pH 6.0±0.3 showed a change in color (see
Appendix D). The species treated at pH 8.0±0.3 had shown an even darker green
color, implying an increase in growth (Atkinson and Marubini, 1999).
Chlorophyll is one of the most important part of a plant or algae. Chlorophyll
presence is indicated by a green-colored characteristic in plants mainly because
chlorophyll is green. The greener the algae, the more amount of chlorophyll it
contains (Iglesias-Prieto et al., 1992).
The results in the study by Iglesias-Prieto et al. in 1992 coincides with the
observations in the experiment conducted. The samples at pH 8.0±0.3 showed a
darker green color (see Appendix D), implying a higher chlorophyll a content
compared to the samples treated in lowered seawater pH. The samples under pH
20

6.0±0.3 changed from green to pale green. However, the samples under pH 3.0±0.3
and 4.0±0.3 changed their color to pale white. Since a greener color of the samples
implies more chlorophyll a content, the samples treated under lowered seawater pH
were not in their viable environment.
4.4 Chlorophyll a Concentration
The amount of chlorophyll a content of the sample was determined using a
UV-Vis spectrophotometer. This was also used to determine the growth performance
of the species.
Table 3. Chlorophyll a Concentration (in milligrams per liter) of C. lentillifera
pH Levels Chlorophyll a Concentration
7.7 to 8.3 10.97
5.7 to 6.3 2.723
3.7 to 4.3 1.721
2.7 to 3.3 1.023
Table 3 shows the chlorophyll a concentration of C. lentillifera at different pH
levels. The samples in the controlled setup contained the highest amount of
chlorophyll a, which was found out to be 10.97 mg/L. However, the samples treated
in lowered seawater pH showed a decreasing pattern on the chlorophyll a
concentration. It was found to be 2.723 mg/L, 1.721 mg/L and 1.023 mg/L at pH
6.0±0.3, 4.0±0.3, and 3.0±0.3, respectively.
21

A study conducted by Iglesias-Prieto et al. (1992) observed that
photosynthetic rates of algae increased between 20°C and 30°C and consequently, at
pH on or near the equilibrium pH in seawater, which is about pH 8. This observation
coincides with the results shown on Table 3 above.
Photosynthesis is a sensitive indicator of thermal stress in plants and has a
central role in the nutrition of symbiotic invertebrates. Through photosynthesis, plants
such as algae are capable to channel the energy of sunlight into chemical energy. As
algae engage in photosynthesis, more chlorophyll is present, and the energy absorbed
by chlorophyll transforms carbon dioxide and water into carbohydrates and oxygen.
In most plants, photosynthesis takes place mainly in the leaves. Like other
living things, plants are made up of tiny cells. The cells in a plant’s leaves contain
even smaller, disc-shaped parts called chloroplasts. Chloroplasts contain chemical
called chlorophyll, which is bright green. Chlorophyll gives plants their green color
and makes photosynthesis work with the aid of sunlight, air, and water. This, in turn,
produces beneficial products such as carbohydrates and oxygen.
Algae under normal seawater pH engage in photosynthesis more actively.
Hence, they can produce enough or even more of the products of photosynthesis.
However, those species treated under low pH may encounter physiological stress as
indicated in the study of Iglesias-Prieto et al. in 1992.
22

5.1 Conclusion
CHAPTER V
CONCLUSION AND RECOMMENDATIONS
Results showed in this study indicate that lowered seawater pH can negatively
affect the survival of Caulerpa lentillifera (lato) under the experimental conditions
used in the study. The species under pH 7.7 to 8.3 had shown a relatively positive
growth compared to the others treated in lowered seawater pH. As the pH of the
treatment decreased compared to the normal seawater pH, the final mass of the
species also decreased.
The chlorophyll a concentration of the species had also been a vital factor in
the species’ growth performance. The chlorophyll a concentration of the species
increased as the pH also increased. The highest concentration of the species was
found to be 10.97 mg/L treated under pH 7.7 to 8.3. The statistical results proved that
there was a significant difference on the weights of the samples in the three trials
among the four groups. Caulerpa lentillifera (lato) has shown a negative growth as
the pH of the seawater becomes lowered. Lowered seawater pH could therefore be
detrimental to the survival and growth of C. lentillifera (lato).
5.2 Recommendations
In consideration of the lack of resources during the conduct of the study, the
researchers recommend the following:
1. Optimization of experimental conditions (temperature, dissolved oxygen and
salinity) observed in the study.
2. Determine the effects of lowered seawater pH in a longer period of time.

3. Use of other marine species for comparative analysis of the effects of lowered
seawater pH.
4. Examine the effects of elevated seawater pH (basic environment) on Caulerpa
lentillifera.
5. Shorten the period of time in which the extract of Caulerpa lentillifera (Lato)
will be obtained and centrifuged before reading in the spectrophotometer.
6. Choose a site in which the resources (seawater and Caulerpa lentillifera (Lato))
needed in the study is near and abundant.
7. Use tanks that are larger in size.
24

BIBLIOGRAPHY
Apiratikul, R. & Pavasant, P. (2007). Biosorption of Cu2+, Cd2+, Pb2+, and Zn2+
using dried marine green macroalga Caulerpa lentillifera. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/16330209.
Atkinson, M. J. & Marubini, F. (1999). Effects of Lowered pH and Elevated Nitrate
on Coral Calcification. Marine Ecology Progress Series Volume 188: 117-
121.
Battistoni, M., Fitzgerald, K., Kelson, S. (2007). Effects of Ocean Acidification on a
Turtle Grass Meadow. Ecology Journal of Science Volume 7: 40-42.
Beer, S., Madsen, T. V., Sand-Jensen, K. (1993). Comparison of Photosynthetic
Performance and Carboxylation Capacity in A Range of Aquatic Macrophytes
of Different Growth Forms. Retrieved from
http://www.sciencedirect.com/science/article/pii/030437709390078B.
Caulerpa lentillifera. From http://www .globinmed.com/index.php? option=com
_content &view=article&id=79160:caulerpa-lentillifera-j-agardh &ca
tid=367:c.
Chirapart, A. & Ratana-arporn, P. (2006). Nutritional Evaluation of Tropical Green
Seaweeds Caulerpa lentillifera and Ulva reticulata. National Science Volume
40: 75-83.
Chlorophyll. From http://www.chm.bris.ac.uk/motm/chlorophyll/chlorophyll_h.htm.
Chlorophyll a concentrations. From http://www.ozcoasts. gov.au/indicators/ chloro
phyll_a.jsp.
Clark, D., Lamare, M., Barker, M. (2009). Response of Sea Urchin Pluteus Larvae
(Echinodermata: Echinoidea) to Reduced Seawater pH: A Comparison among
a Tropical, Temperate, and a Polar Species. Marine Biology Volume 156:
1125-1137.
Climate Change. From http://www.ecy.wa.gov/climatechange/whatis.htm.
Climate Change. From http://www.epa.gov/climatechange/.
Dere, S., Gunes, T., & Sivaci, R. (1997). Spectrophotometric Determination of
Chloro-phyll - A, B and Total Carotenoid Contents of Some Algae Species
Using Different Solvents. Turkish Journal of Botany Volume 22: 13-17.
Dissolved Oxygen. From www.uwgb.edu/watershed.

Diwan, A. D., Vijayan, K. K. (1995). Influence of Temperature, Salinity, pH, and
Light on Molting and Growth in the Indian White Prawn Penaeus indicus
(Crustacea: Decapoda: Penaeidae) Under Laboratory Conditions. Asian
Fisheries Science Volume 8: 63-72.
Doney, Fabry, Feely, Kleypas. (2009). Annual Reviews Marine Science, ©Elsevier,
Luhe, Germany.
Doney, S., Fabry, V., Feely, R., Kleypas, J. (2007). Ocean Acidification: The Other
CO2 Problem. Pacific Marine Environmental Journal Volume 28: 21-33.
Fabry, V., Feely, R., Orr, J., Seibel, B. (2008). Impacts of Ocean Acidification on
Marine Fauna and Ecosystem Processes. Convention on Biological Diversity
Volume 46: 3-23.
Fabry, V., Feely, R., Kleypas, J., Langdon, C., Robbins, L., Sabine, C. (2006).
Impacts of Ocean Acidification on Coral Reefs and Other Marine Calcifiers.
Fay, P. & Wyman, M. (1986). Underwater Light Climate and the Growth and
Pigmentation of Planktonic Blue-Green Algae (Cyanobacteria) I. The
Influence of Light Quantity. Retrieved from
http://adsabs.harvard.edu/abs/1986RSPSB.227..381W.
Forster, P., Ramaswamy, V., Artaxo, P., Berntsen, T., Betts, R., Fahey, D. W.,
Haywood, J., et al.( 2007). Changes in Atmospheric Constituents and In
Radiative Forcing. In Climate Change: the Physical Science Volume 8: 129–
234.
Growth Rate. From http:// www. marine. csiro.au /microalga e/methods /Growth% 20
rate.htm.
Gruber, N. & Sarmiento, J. (2002). Physics Today,© American Institute of Physics,
Maryland, West Virginia, USA.
Hauton, C., Tyrrell, T., & Williams, J. (2008). The Subtle Effects of Sea Water
Acidification on the Amphipod Gammarus locusta. Retrieved from
http://www.biogeosciences.net/6/1479/2009/bg-6-1479-2009.html.
Hinga, K. (2002). Effects of pH on Coastal Marine Phytoplankton. Marine Ecology
Progress Series Volume 238: 281-300.
Humphrey, G. F. & Jeffrey, S. W. (1975). New Spectrophotometric Equations for
Determining Chlorophylls a, b, c1 and c2 in Higher Plants, Algae, and Natural
Phytoplankton. Biochemistry & Physiology Volume 167: 191-194.
Iglesias-Prieto, R., Matta J., Robins, W., & Trench, R. (1992). Photosynthetic
Response to Elevated Temperature in the Symbiotic Dinoflagellate
Symbiodinium microadriaticum in Culture. Retrieved from
http://www.pnas.org/content/89/21/10302.abstract.
26

Jenkins, A. (2009). A Closer Look at Caulerpa – Common Aquarium Species and
Their Care. From http://www.wetwebmedia.com/ca/volume_6/volume_6_4
/caulerpa. html.
Kurihara, H. & Ishimatsu, A. (2008). Effects of High Carbon Dioxide Seawater on the
Copepod ( Acartia tsuensis) Through All Life Stages and Subsequent
Generations. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18455195.
Le Quere, J., Porra, K. & Stanley, J. (2007). Ocean Acidification. Science Volume
316: 1735-1737.
Levasseur, M., Harrison, P. & Thompson, P. (1993). Physiological Acclimation of
Marine Phytoplankton to Different Nitrogen Sources. Journal of Phycology
Volume 29: 587-595.
McGugh, D. (2003). A Guide to the Seaweed Industry. FAO Fisheries Volume 441:
73-98.
Menzel, D. & Yentsch, C. (1963). A Method for the Determination of Phytoplankton
Chlorophyll and Phaeophytin by Fluorescence. Retrieved from
http://www.sciencedirect.com/science/article/pii/0011747163903589.
Michaelidis, B., Ouzounis, C., Paleras, A., & Portner, H. (2004). Effects of Long -
Term Moderate Hypercapnia on Acid-Base Balance and Growth Rate in
Marine Mussels Mytilus galloprovincialis. Marine Ecology Progress Series
Volume 293: 109-118.
Ocean Acidification. From http:// www.co2s cience.org/ education/reports
/prudentpath /ch10.php.
Orr et. al. (2005). Anthropogenic Ocean Acidification Over the 21 st Century and Its
Impact on Calcifying Organisms. Nature Volume 437: 681-686.
Pattama Ratana-arporn and Anong Chirapart (2006). Nutritional Evaluation of
Tropical Green Seaweeds Caulerpa lentillifera and Ulva reticulata. National
Science Volume 40: 75-83.
pH. From http://dl.clackamas.edu/ch105-05/definition.htm.
Pimol, P., Khanidtha, M. & Prasert, P. (2007). Influence of particle size and salinity
on adsorption of basic dyes by agricultural waste: dried Seagrape ( Caulerpa
lentillifera). Environmental Science Volume 20: 760-768.
Porra, R. J., Thompson, W.A., and Kriedemann, P.E. (1989). Determination of
Accurate Extinction Coefficients and Simultaneous Equations for Assaying
Chlorophylls a and b Extracted with Four Different Solvents: Verification of
the Concentration of Chlorophyll Standards by Atomic Absorption
Spectroscopy. Biochimica et Biophysica Volume 975: 384-394.
Salinity. From http://www.thefreedictionary.com/.
27

Science Daily (2008). Cosmic Rays Do Not Explain Gl obal Warming, Study Finds.
From http://www.sciencedaily.com/releases/2008/12/081217075138.htm.
Seawater buffer system. From http://www.livingreefs.com/sea-water-buffer-systemt358.html.
Siegenthaler, U., Stocker, T. F., Monnin, E., Luethi, D., Schwander, J., Stauffer, B.,
Raynaud, D., et al. (2005). Stable Carbon Cycle-Climate Relationship During
the Late Pleistocene. Science Volume 310: 1313–1317.
Skirrow, G. & Riley, S. (1975). Chemical Oceanography. Limnology and
Oceanography Volume 21: 766.
Tortell, P., Giocoma, R., Sigman, D. & Morel, F. (2005). Ocean Acidification Due To
Increasing Atmospheric Carbon Dioxide. The Royal Society Volume 236: 37
-43.
Tukey Test Statistical Tool. Retrieved January 2013 from Past Software.
Vijayan, K. K. & Diwan, A. D. (2008). Influence of Temperature, Salinity, pH, and
Light on Molting and Growth in the Indian White Prawn Penaeus indicus
(Crustacea: Decapoda: Penaeidae) Under Laboratory Conditions. Asian
Fisheries Science Volume 8: 63-72.
Warming Effect. From http://timeforchange.org/co2-concentration-causingtemperature-increase.
Water Chemistry and Algae. From http://www.livingreefs.com.
Water Quality. RiverTrends Manual. Retrieved from http://allianceforthebay.org/wpcontent/uploads/2010/12/Volunteer-Water-Quality-Manual.pdf.
Wisconsin State Lab of Hygiene (1991). ESS Method 150.1: Chlorophyll-
Spectrophotometric. Environmental Sciences Section Volume 14: 357-363.
28

APPENDIX A
RAW DATA OF THE INITIAL AND FINAL WEIGHT OF THE SPECIES AT
DIFFERENT pH
Table A.1 The weight of Caulerpa lentillifera (Lato) in grams at pH 7.7 to 8.3
pH 7.7 to 8.3
Weight T1 T2 T3 Average
Initial 31.96 31.21 31.84 31.67
Final 37.37 33.61 28.71 33.23
Table A.2 The weight of Caulerpa lentillifera (Lato) in grams at pH 5.7 to 6.3
pH 5.7 to 6.3
Weight T1 T2 T3 Average
Initial 31.99 31.88 31.35 31.74
Final 33.36 20.47 18.57 24.13
Table A.3 The weight of Caulerpa lentillifera (Lato) in grams at pH 3.7 to 4.3
pH 3.7 to 4.3
Weight T1 T2 T3 Average
Initial 33.05 32.76 32.15 32.65
Final 21.82 23.02 19.14 21.33

Table A.4 The weight of Caulerpa lentillifera (Lato) in grams at pH 2.7 to 3.3
pH 2.7 to 3.3
Weight T1 T2 T3 Average
Initial 31.51 32.71 31.64 31.95
Final 20.66 15.98 14.93 17.19
30

APPENDIX B
RAW DATA OF THE ABSORBANCE OF Caulerpa lentillifera (Lato) AT 645
AND 662 NANOMETERS (nm)
Table B.1 The absorbance of Caulerpa lentillifera (Lato) at 645 nanometers (nm)
pH 8 pH 6 pH 4 pH 3
645 nm 0.09833 0.25033 0.032 0.01367
1.455 0.32833 0.264 0.11533
1.79733 0.09733 0.261 0.20667
Average 1.11689 0.22533 0.18567 0.11189
Table B.2 The absorbance of Caulerpa lentillifera (Lato) at 662 nanometers (nm)
pH 8 pH 6 pH 4 pH 3
662 nm 0.09833 0.25033 0.032 0.01367
1.45667 0.31267 0.26133 0.10833
1.82967 0.103 0.24767 0.20367
Average 1.15678 0.27678 0.18356 0.10944
31

Table C.1 Statistical Analysis of Samples
APPENDIX C
T-TEST STATISTICS RESULTS
Initial Weight Final Weight
N 12 12
Mean 32.004 23.97
Variance 0.33314 55.446
TABLE C.2 Mass (in grams) of Caulerpa lentillifera at different pH
pH Range Initial Weight Final Weight
7.7 to 8.3 31.67 33.23 A
5.7 to 6.3 31.74 24.13 B
3.7 to 4.3 32.65 21.33 C
2.7 to 3.3 31.95 17.19 D
Note: The superscript symbol indicates the t-test statistical analysis results of the weights of the
samples. Varying superscript symbol indicates a significant difference on the weights of the samples.
32

APPENDIX D
PHOTOS
Figure D.1 Fresh samples of Figure D.2 Samples after the treatment
Caulerpa lentillifera
Figure D.3 Experimental Setup
33