Water Spinach (Ipomoea aquatica Forsskal) as Copper and

Zinc Accumulators through Phytoremediation

A Research Proposal presented to the

Faculty of Science, Technology and Mathematics
College of Teacher Development
Philippine Normal University
Taft Avenue, Manila

In partial fulfillment
of the academic requirements for
Science Research II (2S-SCI03C)

by

Medillo, Rae Anthony T.

Torio, Joan H.

JULY 16, 2022

 TABLE OF CONTENTS

LIST OF TABLES AND FIGURES .................................................................... 5

A. Figures ...................................................................................................... 5
Figure 1 - Conceptual Framework ........................................................... 5
Figure 2 – Set A Experimentation Set-Up ............................................... 5
Figure 3 – Set B Experimentation Set-Up ............................................... 5
B. Tables ....................................................................................................... 5
Table 1 – AAS Pre-Laboratory Result ..................................................... 5
Table 2 – AAS Post-Laboratory Results for Controlled Group ............. 5
Table 3 – AAS Post-Laboratory Results for 1 ppm Concentration ....... 5
Table 4 – AAS Post-Laboratory Results 3 ppm Concentration ............. 5
Table 5 – IBM SPSS (v.21) One-way ANOVA Analysis ........................... 5
Table 6 – IBM SPSS (v21) Table of Comparison for four different
means ........................................................................................................ 5
C. Graph ........................................................................................................ 5
Graph 1 - Scatter Graph for the comparison of average increase in
Copper and Zinc........................................................................................ 5
D. Pictures..................................................................................................... 5
Picture 1 – Map of the Research Locale ................................................. 5
LIST OF ACRONYMS AND ABBREVIATIONS ................................................ 6
ACKNOWLEDGEMENT .................................................................................... 7
ABSTRACT ....................................................................................................... 8
CHAPTER 1 ....................................................................................................... 9
INTRODUCTION ............................................................................................... 9
1.1 Background of the Study ....................................................................... 9
1.1.1 Heavy Metals .................................................................................... 9
1.1.2 Copper and Zinc as Heavy Metals ................................................ 10
1.1.3 Phytoremediation........................................................................... 11
1.2 Significance of the Study ..................................................................... 12
1.3 Statement of the Problem .................................................................... 13
1.4 Scope and Limitations of the Study.................................................... 13
1.5 Ethical Considerations ......................................................................... 14
1.6 Definition of Terms ............................................................................... 14
CHAPTER 2 ..................................................................................................... 16
REVIEW OF RELATED LITERATURE ........................................................... 16
2.1 Heavy Metals in the Environment ........................................................... 16
2.2 Copper and Zinc as Heavy Metals .......................................................... 17
2.2.1 Copper ............................................................................................ 18
2.2.2 Zinc ................................................................................................. 18
2.3 Phytoremediation ..................................................................................... 19
2.3.1 Phytoremediation and Aquatic Plants ......................................... 20
2.3.2 Efficiency of Aquatic Plants in Heavy Metal Removal and
Accumulation .......................................................................................... 21
2.3.3 Free-Floating Aquatic Plants ........................................................ 23
2.3.4 Water Spinach (Ipomoea aquatica Forsskal) as Heavy Metal
Accumulator ............................................................................................ 23
2.3.5 Requirements for Growing Water Spinach .................................. 24
2.4 Synthesis of the Review of Related Literature and Studies ................. 25
2.5 Conceptual Framework ........................................................................... 25
CHAPTER 3 ..................................................................................................... 27
METHODOLOGY ............................................................................................ 27
3.1 Materials and Equipment ..................................................................... 27
3.2 Research Design .................................................................................. 27
3.3 Research Locale ................................................................................... 28
3.4 Sample Preparation .............................................................................. 28
3.5 Preparation of Heavy Metals ............................................................... 30
3.6 Preparation of Fertilizer ....................................................................... 30
3.7 Heavy Metal Testing ............................................................................. 31
3.8 Waste Management and Disposal ....................................................... 31
3.9 Statistical Analysis and Interpretation of Results ............................. 31
CHAPTER 4 ..................................................................................................... 32
RESULTS AND DISCUSSION ........................................................................ 32
4.1 Pre-Experimentation of Water Spinach .............................................. 32
4.2 Atomic Absorption Spectrophotometer Test Result ......................... 32
4.3 Statistical Results................................................................................. 34
CHAPTER 5 ..................................................................................................... 38
CONCLUSION AND RECOMMENDATION .................................................... 38
5.1 Conclusion ............................................................................................ 38
5.2 Recommendation ................................................................................. 39
REFERENCES ................................................................................................ 40
GANTT CHART OF ACTIVITIES .................................................................... 48
RESEARCH BUDGET ..................................................................................... 49
APPENDICES .................................................................................................. 50
APPENDIX A: Calculations ............................................................................ 51
APPENDIX B: Water Spinach (Ipomoea aquatica Forsskal) ....................... 52
APPENDIX C: Map of the Research Locale .................................................. 53
APPENDIX D: Illustration of Experimentation Set-up ................................. 54
APPENDIX E: Actual Experimentation Set-Up ............................................. 55
APPENDIX F: Plant Identification ................................................................. 56
APPENDIX G: Experimentation ..................................................................... 57
APPENDIX H: AAS Initial Testing Result ..................................................... 58
APPENDIX I: Laboratory Results .................................................................. 59
APPENDIX J: Letters to Internal and External Experts ............................... 63
APPENDIX K: Meetings with Internal and External Experts ....................... 66
APPENDIX L: Summary of Comments and Suggestions ........................... 68
APPENDIX M: Curriculum Vitae of Researchers ........................................ 72
 LIST OF TABLES AND FIGURES

A. Figures

Figure 1 - Conceptual Framework

Figure 2 – Set A Experimentation Set-Up

Figure 3 – Set B Experimentation Set-Up

B. Tables

Table 1 – AAS Pre-Laboratory Result

Table 2 – AAS Post-Laboratory Results for Controlled Group

Table 3 – AAS Post-Laboratory Results for 1 ppm Concentration

Table 4 – AAS Post-Laboratory Results 3 ppm Concentration

Table 5 – IBM SPSS (v.21) One-way ANOVA Analysis

Table 6 – IBM SPSS (v21) Table of Comparison for four different means

C. Graph

Graph 1 - Scatter Graph for the comparison of average increase in Copper

and Zinc

D. Pictures

Picture 1 – Map of the Research Locale

 LIST OF ACRONYMS AND ABBREVIATIONS

AAS – Atomic Absorption Spectrophotometer

Cu – Copper

HM – Heavy Metal

NPK – Nitrogen, Phosphorus, Potassium

Ppm – Parts per million

Zn – Zinc
 ACKNOWLEDGEMENT

First and foremost, we would like to praise and thank our Almighty God,

for His countless blessings, strength, and opportunity throughout our research

work, so that we have accomplished the thesis.

Apart from our efforts, the success of this thesis is largely dependent on

the support and guidance of many people. We would like to take this opportunity

to thank everyone who helped to the success of this research.

Dr. Crist John Pastor, Ph.D., Research Adviser, thank you is not enough

for your unwavering support and assistance. Every time we attend the meeting,

we feel inspired and driven. This research would not have been possible without

your guidance.

Prof. Ruel Avilla, M.Sc., Internal Expert, and Prof. Gicel Christine Paclibar,

M.Sc., External Expert, we would like to show our deepest gratitude for your

substantial contributions to our research and generously spent time providing

advice, support, suggestions, and comments are highly appreciated.

Family and Friends, we are extremely grateful for your support, love,

prayers, and sacrifices for preparing us to build our future. Your assistance kept

us moving forward in our research.

To all who are not mentioned, we are sending our utmost gratitude to all

of you who stayed by our sides at a time when we are about to give up.
 ABSTRACT

Heavy metal pollution is a major source of contamination in the

environment. Technologies for removing pollutants from wastewater are typically

environmentally damaging, expensive, and inefficient. Phytoremediation is a low-

cost and environmentally friendly technology. The most important part of

successful phytoremediation is plant species selection. Aquatic plants have high

efficiency in the elimination of organic and inorganic contaminants. This study

examines the potentiality of Water Spinach (Ipomoea aquatica Forsskal) as

copper and zinc accumulators. The samples of water spinach were collected,

processed, and analyzed for heavy metals by Atomic Absorption

Spectrophotometry. One-Way Analysis of Variance (ANOVA) is used to observe

the difference between the final heavy metal concentration in contaminated water

treated with Water Spinach and the mean was compared using Tukey’s post hoc

test at 0.05 significance level. The findings revealed that Water Spinach did not

prove to be much effective for copper and zinc removal from water samples, the

data gathered about the absorbed copper and zinc did not even reach 1 mg/kg

where its highest is 0.14 in 3 ppm of zinc. Thus, it was suggested that the

phytoremediation potential of Water Spinach be further boosted by the use of

other common heavy metals and consider the bioaccumulation which will take

longer allotted time for experimentation.

Keywords: environmental pollution; heavy metal; phytoremediation; wastewater treatment; water

spinach
 CHAPTER 1

INTRODUCTION

1.1 Background of the Study

On a worldwide scale, the rise of industry has resulted in increased pollutant

issues. The contamination of water due to heavy metal is one the examples,

which is commonly seen as a severe problem to human health. Heavy metal

contamination is mostly caused by the wrong human activities, such as marine

dumping, mining, household, and domestic waste, sewage and wastewater, and

many more.

1.1.1 Heavy Metals

The most common heavy metals that are found in the bodies of water are

arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), nickel

(Ni), and zinc (Zn) (Jaishankar et al., 2014). These heavy metals in water can

be quickly transported through the trophic chain imposing a serious threat to

the organisms (Jaishankar et al., 2014; Azimi et al., 2017). As a result of the

intensification of human activities, the increase of the contaminated area in

bodies of water is due to the release of aqueous effluents containing heavy

metals (Azimi et al., 2017). Even though numerous methods are commonly

used to clean up contaminated water, many of these methods are technically

expensive, environmentally toxic, and ineffective (Ali, S. et al., 2020).

Heavy metal poisoning can occur as a result of drinking contaminated

water (e.g., lead pipes), excessive ambient air concentrations, emission

sources, or intake via the food chain (Gupta 2018). Heavy metals are

hazardous due to their proclivity for bioaccumulation (Ali et al., 2019). In

comparison to the chemical's concentration in the environment,

bioaccumulation refers to an increase in the concentration of a chemical in a

biological organism over time. When compounds are taken up and stored

quicker than they are broken down (metabolized) or expelled, they build up

in living things (Gupta 2018). Aside from the biological role of some heavy

metals in the body, the presence of these heavy metals still endangers the

health of organisms in their environment, unlike other wastes such as

nitrogenous organic wastes, heavy metals are not biodegradable and cannot

be broken down since they are already in the atomic level, however,

organisms find a way to deal with these metals. Organisms may detoxify the

metal cations by binding the atom in a protein and excreting in the body of

the organism as a farce (Briffa et al., 2020).

1.1.2 Copper and Zinc as Heavy Metals

Heavy metals are found naturally in the Earth's crust, they cannot be

ruined or degraded. They get into our bodies in little amounts from food and

drinking water. Some heavy metals such as copper (Cu) and zinc (Zn) are

required as trace elements to keep the human body's metabolism running

smoothly. However, they can cause toxicity in higher amounts.

Copper is an essential trace metal that has a biological role in the human

body, such as serving as a cofactor for proteins, particularly oxidase

enzymes. However, the cupric cation (Cu2+) is reduced to (Cu+) after

absorption. Overdosing (1 to 10 g) or high copper content in the body can

induce liver problems such as acute hepatotoxicity and gastrointestinal pain

due to stomach or small bowel erosions. Excessive copper consumption

would result in the circulatory system collapsing, a coma, and death within

hours (National Center for Biotechnology Information 2021).

Zinc is also an essential trace element that can be found in multivitamins

and mineral supplements over-the-counter. It can stop copper from being

absorbed. Zinc is needed as a cofactor for enzymes involved in immune

system function, protein synthesis, cell division, and DNA synthesis. Zinc has

an oral LD50 of three g/kg, and excessive intake will result in hepatotoxicity,

jaundice, and liver failure, similar to the effects of copper and iron overdose.

1.1.3 Phytoremediation

As the problem of heavy metal pollution continues to rise, new plant-

based technologies were made to address this environmental problem –

phytoremediation is an emerging technology with the use of plants. There is

a large number of plant species with a high potential to accumulate heavy

metals, making phytoremediation an eco-friendly, low-cost, and alternative

method (Ali, S. et al., 2020). As a result, using aquatic plants in contaminated

bodies of water to accumulate heavy metals has been proven to be cost-

effective for remediating large areas (Dhir 2013). Its great impact is
 determined by the qualities of the plants used, as well as other factors,

including high biomass yield and rapid growth capability.

1.2 Significance of the Study

This study determined the potentiality of Water Spinach as Copper and

Zinc accumulators through phytoremediation. This study would greatly

contribute to the following:

Community – The research findings would be able to inform the

community regarding their safety and health in relation to the condition of the

water spinach they buy and eat.

STEM Students – This study serves as a reference for STEM students

who are studying botany, environmental science, ecology, and even

biotechnology as the subject of research.

Municipal Environment and Natural Resources Office – The study is

intended to provide any local government from which the water spinach has

been harvested with the information regarding the safety of the people eating

water spinach and if farms contain high heavy metal concentration.

Future Researchers – This research serves as a basis for future research

that they conduct since it may be used as a framework for other related

studies, and it helps them have the courage to protect their communities and

educate them on proper waste management.

1.3 Statement of the Problem

This study determined the phytoremediation capabilities of Water Spinach

(Ipomoea aquatica Forsskal) on Zinc and Copper metals in water.

Specifically, this study answer the following questions:

1. Is there a difference in Zinc and Copper concentration before and after

using water spinach as heavy metals accumulator in:

1.1. 1.0 mg/L concentration?

1.2. 3.0 mg/L concentration?

2. Does Water Spinach (Ipomoea aquatica Forsskal) exhibit preferential

absorption of specific heavy metals?

3. Is there an effect of using Water Spinach (Ipomoea aquatica Forsskal)

in phytoremediation for Copper and Zinc metal absorption?

1.4 Scope and Limitations of the Study

The focus of the study was to determine the potential of Water Spinach as

Copper and Zinc accumulators through phytoremediation. The study used

two specific heavy metals, such as copper and zinc, and was conducted

within the span of four (4) to ten (10) weeks. The heavy metals that were used

were bought in Bambang Manila City, while the Water Spinach was

purchased in the farm in Tala Caloocan City and propagated by the

researchers. The experimentation was conducted in a controlled set-up.

Fourteen (14) controlled sets of aquariums were used, divided into three (3):

positive control (one each for copper and zinc), experimental (mixed copper
 and zinc), and negative control (no heavy metals). There was duplicate for

each amount of contamination – 1.0 mg/L (standard concentration) and 3.0

mg/L (high concentration). The study excludes the discussion of metabolic

transport of these heavy metals in the different tissues of the Water Spinach.

The study only test the leaves part of the water spinach and did not test

other heavy metals that could be present in the water and water spinach

sample. To ensure the heavy metals present in the water spinach sample, it

was tested before the experimentation through AAS. Also, underwent plant

identification. The experimentation process will last for two (2) weeks only.

1.5 Ethical Considerations

This study does not violate any ethical considerations and all data were

treated accordingly. The owner of the farm was informed that the water

spinach is used in the research and sold the product voluntarily, thus,

researchers considered the confidentiality and maintained the privacy of the

owner of the farm. Consequently, this study provides benefits to the local

government and community regarding the safety of the people buying and

eating water spinach and if farms contain high heavy metal concentration.

1.6 Definition of Terms

The following terms were used and defined operationally in this study:

a) Atomic Absorption Spectrophotometer - refers to the process of

spectroanalytical for determining the quantitative data of heavy metals.

b) Bioaccumulation - refers to the progressive accumulation of heavy

metals in the water.

c) Bioremediation - refers to the use of living organisms in removing

contaminants from the water.

d) Heavy metal - refers to the metallic chemical element with a high

density that is hazardous and lethal at low concentrations.

e) Phytoacculumation - refers to aquatic plants that are used to remove,

transport, and eliminate pollutants in the water.

f) Phytoremediation - refers to the clean-up of contaminants in a wide

range of organic and inorganic pollutants from the water.

g) Remediation - refers to the action of rectifying and protecting water

from a threat.
 CHAPTER 2

REVIEW OF RELATED LITERATURE

2.1 Heavy Metals in the Environment

Heavy metal contamination is mostly caused by the wrong human

activities, such as domestic and household waste, mining, sewage and

wastewater, marine dumping, and many more. There are also natural sources of

heavy metal contamination such as weathering, erosion, and volcanic eruption,

which also add to the concern of toxic heavy metals. This contamination in water

by organic and inorganic pollutants is a major concern around the world. The

pollutants are difficult to eliminate biologically and chemically, fortunately, they

can be converted from a hazardous toxic to a less harmful state (Jiang et al.,

2018; Zhang et al., 2017). When the aquatic environment becomes

contaminated, it will disrupt the entire water ecosystem, causing all aquatic

organisms to suffer. Water pollution has always had negative consequences on

the aquatic ecosystem, which further leads to detrimental effects on human life.

Heavy metals such as cadmium (Cd), chromium (Cr), copper (Cu), lead

(Pb), nickel (Ni), selenium (Se), vanadium (V), and zinc (Zn) are among the most

common and harmful contaminants that found in water. Due to the lack of

information and strict execution of laws, these heavy metals have become a

serious, consequential, and major issue around the world, which is still

unregulated. (Akpor & Muchie, 2010; Eid et al., 2020). According to the World

Health Organization, cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu),

lead (Pb), mercury (Hg), and zinc (Zn) have been highlighted into consideration
when it comes to the quality of drinking water. However, high doses above the

recommended 10 mg/kg standard of the World Health Organization may cause

major problems in human health. The standard concentration of Copper is 0.11

to 0.17 mg/L and Zinc is 0.35 to 1.19 mg/L in water (Kacholi & Sahu 2018).

2.2 Copper and Zinc as Heavy Metals

Elements that have a high atomic weight and a density that is at least five

times greater than the density of water is a common characteristic of heavy

metals. These metals include metalloids including arsenic (As) and tellurium (Te),

and metals such as lead (Pb) and tin (Sn), lanthanides, and actinides

(Hemenstine 2018). However, these metals tend to be toxic in living organisms,

but still, these atoms are useful in the agricultural, domestic, industrial, medical,

and technological fields. Due to their wide application, their presence in the

environment poses threat to human health and the environment (Tchounwou et

al., 2012).

The degree of their toxicity varies according to their interactions in

biological systems such as their route of exposure, other factors may include the

gender, age, genetics, and current nutrition status of individual exposure to these

metals. Heavy metals can damage multiple organs and cause cancer as it is also

classified as human carcinogenic according to the study by Tchounwou et al

2012. Some heavy metals are also classified as essential metals due to their

biological importance such as copper (Cu) and zinc (Zn). Bioaccumulation

explains how metals build up in the body of an organism faster and how they can
manage by the biological system such as broken down or excreting as a waste,

as we go higher in the food chain, the consumers tend to have more

concentration of heavy metals on their body (Azad 2019).

2.2.1 Copper

Copper is a heavy metal (Helmenstine 2018) with an atomic number of 29

and it belongs to the d-block transition metals, it has a density of 8.933 g/cm³

almost nine times denser than liquid water and it is solid in its standard state. Its

chemical symbol of Cu comes from the Latin word for cuprum. Copper is an

essential trace metal that has a biological role in the human body such as being

a co-factor of proteins, especially the oxidase enzymes but in the process of

absorption, the cupric cation (Cu2+) is reduced into (Cu+). However, the overdose

intake (1 to 10 g) or the concentration of copper in the body will cause liver

problems such as acute hepatotoxicity, and gastrointestinal distress resulting

from gastric or small bowel erosions. Excessive ingestion of Cu will lead to the

collapse of the circulatory system, coma, and death within hours (National Center

for Biotechnology Information 2021).

2.2.2 Zinc

The next heavy metal in the Periodic Table of Elements after Copper is

Zinc with an atomic number of 30, it is the last transition metal in the fourth period.

It is less dense than Copper with a density of 7.134 g/cm³. As also an essential

trace element, it is included in over-the-counter multivitamin and mineral

supplements. It has the capability of blocking the absorption of copper. Zinc is

required as a cofactor of enzymes that play an important function in the immune

system, protein synthesis, cell division, and DNA synthesis. The oral LD50 of

Zinc is three g/kg and the excessive intake of the zine will cause hepatotoxicity,

jaundice, and hepatic failure which is resembling the overdose ingestion of

copper and iron.

2.3 Phytoremediation

The word phytoremediation is made up of the Greek word “Phyto”, which

means plant, and the Latin word” remedium”, which means to repair or remove

an affliction (El-Gammal et al., 2014; Zarcinas et al., 2004). Phytoremediation

refers to an emerging technology that employs the utilization of specific plants to

clean the polluted surroundings and improve its quality by eliminating toxic

pollutants without additional excavation processes or modifications to soil. To

facilitate the phytoremediation, the root of the plant should be in contact with

contaminated water as the membrane of the root acts as the filter known as

rhizofiltration. After the first process, two other processes will determine how the

absorbed contaminant will be managed inside the system of the plant, first is the

photodegradation where the metabolic processes in the plant’s system break

down the contaminant, second is the phytoaccumulation common in inorganic

contaminants that are locked within the parts of the plant (Bruni & Mcleskey

2013).
 Phytoremediation is an environmentally plausible technology, in which

plants should contain credible characteristics, including high biomass yield, rapid

growth capability, can transfer of metals in aboveground parts of the plant, has

the ability to carry a large number of heavy metals, and a system to endure the

heavy metal hazardous effect (Ali, H. et al., 2013). In addition, other elements

have a significant impact on the plant's phytoremediation capability and growth,

such as the availability of its nutrient, pH, salinity, and solar radiation (Reeves et

al., 2017; Tewes et al., 2018). Phytoremediation is considered by Bruni &

Mcleskey (2013) as one of the most natural techniques that are available and

occasionally less expensive environmental treatment. As a result,

phytoremediation has become the preferred strategy of industry and academic

scientists because of its being sustainable technology that can apply both in

developed and developing countries.

2.3.1 Phytoremediation and Aquatic Plants

Phytoremediation is credible in clean-up and a profitable strategy for a

vast pollutant aquatic environment. Heavy metals and contaminants are naturally

absorbed by aquatic plants (Pratas et al., 2014). The most efficient and cost-

effective technique for removing any heavy metals and other toxins in water is to

use aquatic plants (Ali, S. et al., 2020; Guittonny-Philippe et al., 2015). Aquatic

plants have been widely used for wastewater treatment around the world (Gorito
et al., 2017). Finding the right aquatic plant for heavy metal accumulation is very

relevant for phytoremediation to be effective in its role (Galal et al., 2018).

Aquatic plants have built an enviable prominence over the years for their

capability to clean up contaminated areas worldwide (Gorito et al., 2017). Aquatic

plants produce a complex root system that aids and makes them the greatest

alternative pollutant accumulator through shoots and roots (Mays et al., 2001).

Aquatic plant development and cultivation take much time, which may limit the

growing need for phytoremediation (Said et al., 2015). Nonetheless, the number

of advantages that this method has for the treatment of pollutant areas

compensates for this disadvantage (Kozminska et al., 2018; Syukor et al., 2014).

2.3.2 Efficiency of Aquatic Plants in Heavy Metal Removal and

Accumulation

The study about Salvinia biloba (Emiliani et al., 2020) shows the

effectiveness of this aquatic plant to eliminate cadmium (Cd), copper (Cu), lead

(Pb), and zinc (Zn) from contaminated water with 50 ± 2 µM or 100 ± 1 µM of

heavy metals during 48 hours of the exposure in individual experiments. Based

on the metal and its concentration, the nature of the removal of metal from water

can be assessed with a first quick process in about 6 hours, and a slower phase

process from 10 hours onwards which reaches a plateau in 24–48 hours. Other

scientists have determined a similar order for removing heavy metals utilizing a

range of aquatic plants, which include water spinach (Suñe et al., 2007).
 Metal absorption by the aquatic plant roots is the rapid component of

heavy metal accumulation, and it can occur through a mix of chemical and

physical mechanisms, like an ionic exchange and ionic bonds (Tello Zevallos et

al., 2018). The presence of negatively charged groups in macromolecules such

as carbohydrates, lipids, and proteins in the plants is a critical component in

determining the process between biomass and cationic metals. This means that

aquatic plants have a large specific area of biomass that is rich in carbohydrates

and carboxyl groups, which explains that aquatic plants have heavy metal

accumulator capacity (Sánchez-Galván et al., 2008).

In the research of Tello Zevallos et al., 2018 about free-floating

macrophyte Salvinia biloba, they acquired the Fourier transform infrared

spectroscopy-Attenuated total reflectance (FTIR-ATR) spectrum in Salvinia

biloba and described the existence of functional groups, which includes the

amide (RCONH2), carboxyl (RCOOH), hydroxyl (ROH), and phosphate (PO 4-3)

implying that functional groups are involved in the lead (Pb) adsorption. On the

other side, the slower phase of the removal of heavy metals in Salvinia biloba is

due to biological mechanisms involving metal intracellular absorption by

transmembrane proteins or ionic channels and subsequent translocation to the

aerial regions of the plant (Suñe et al., 2007). In addition to this study, it shows

that the efficiency of Salvinia biloba in accumulating heavy metal is affected by

both metal and its concentration (Castillo Loria et al., 2019).

 2.3.3 Free-Floating Aquatic Plants

Free-floating aquatic plants, including water spinach (Ipomoea aquatica

Forsskal), are widely known for their ability to remove metals from contaminated

areas. Different research has looked into the ability of these free-floating aquatic

plants to eliminate heavy metals (Maine et al., 2001; Olguin et al., 2002;

Muthusaravanan et al., 2018). Heavy metals are actively transported in free-

floating aquatic plants through the roots, from where the heavy metals are

transmitted to other sections of the plant. The direct contact of the body plant

with the pollutant is the passive transport, heavy metals accumulate primarily in

the higher portions of the plant during passive transport.

2.3.4 Water Spinach (Ipomoea aquatica Forsskal) as Heavy Metal

Accumulator

With the fast economic development in Southeast Asia, more bodies of

water have become polluted. As a result, a hybrid culture emerges, in which

people attempt to reconcile traditional farming ways with the rapidly developing

metropolitan environment, this is why water spinach is frequently grown and

consumed in areas that are not suitable for food production due to the pollution.

Household and other types of wastes that are collected in the ditches, ponds,

and canals where water spinach grows serve as heavy metal accumulators

because these waters include not only nutrients but also a variety of
contaminants from human activities, which also turns in the risk of their health

(Gothberg et al., 2002).

In the Philippines, water spinach (Ipomoea aquatica Forsskal) is a

common aquatic vascular plant, which grows best in the tropics and subtropics

regions, and the temperature should be above 24 degrees Celsius. The hollow,

long, and viny stems grow recumbent or floating, with roots emerging from nodes

that penetrate mud or damp soil. Water spinach is abundant in vitamin A, vitamin

C, and iron, and has a high nutritional value.

According to Gothberg et al., 2002, the highest consumption of water

spinach was calculated based on the investigation of the consumption of this

plant in the local area. However, humans should be aware that plants accumulate

more heavy metals in leaves (highest mean value: 1,440 microg/kg dry wt) than

in stems (highest mean value: 422 microg/kg dry wt), so it is recommended to

avoid the above part of water spinach due to the accumulated heavy metals,

which possess serious threats, especially to children.

2.3.5 Requirements for Growing Water Spinach

Water spinach needs consistently damp, humid, and nutrient-rich soil with

a pH of 6-7 and needs full sun. It is essential to water spinach so that the soil is

regular watering to maintain the wetness. In the ground set-up, it doesn’t need to

fertilize the soil, however, in a container set-up, the soil needs to be fertilized with
a high concentration of nitrogen liquid fertilizer twice a month or use a water pump

(Sahira et.al., 2021).

2.4 Synthesis of the Review of Related Literature and Studies

Heavy metals in water are universally considered a major source of a

threat since they cannot be destroyed biologically or chemically, as a result,

contamination will have a long time in staying in waters, which cause detrimental

effects on human health. Phytoremediation is utilized by the use of plants to

remove or collect a wide range of organic and inorganic pollutants from the water,

which is one of the most widely used technologies in the world. To increase the

effectiveness and long-term viability of phytoremediation in water treatment

systems, it is important to use aquatic plants that are not only effective at

removing pollutants but also plentiful and easy to access in the target area (Azimi

et al., 2017).

2.5 Conceptual Framework

The conceptual framework depicts how the concepts of this study were

organized for researchers to reach their goals, which will primarily consist of

input, process, and output to serve as a guide for the methods to utilize.
 Input Process Output

Gathering of Propagation of Absorption of heavy

Materials: purchase aquatic metals concentration
plants in the aquatic plant
Water spinach/
Kangkong Experimentation

Copper Atomic Absorption

Spectrophotometry
Zinc

Figure 1 Conceptual Framework of the Study

Figure 1 shows the conceptual framework of the study, which begins with

input – these are necessary steps for the researchers to begin the experimental

procedure, which includes collecting aquatic plants and heavy metals. The

collected materials from the input were subjected to the procedure, which is the

second part of the framework. The plants that were propagated and tested using

an Atomic Absorption Spectrophotometer after two (2) weeks of experiment.

Lastly, after the testing procedure is completed, the data was statistically

evaluated, and the phytoremediation results were compiled and submitted to the

appropriate channel.
 CHAPTER 3

METHODOLOGY

In order to respond to the research questions, the samples obtained from

the environment location were subjected to a controlled set-up and proper

protocols to provide accurate results. This study also used a quantitative-

experimental design to acquire essential details in the study.

3.1 Materials and Equipment

The materials that were used for plant propagation: container and shear.

For experimentation set-up: fourteen (14) aquariums (144×mm×95mm×203mm),

distilled water, fourteen (14) water pump, wooden sticks, copper sulfate

pentahydrate (CuSO4 • 5H2O), zinc chloride (ZnCl2), and Water Spinach. For

plant analysis, atomic absorption spectrophotometer from the laboratory. For

optimization of plants during the experiment: 14-14-14 NPK (Nitrogen-

Phosphorous-Potassium) Agricultural Fertilizer.

3.2 Research Design

The researchers used a quantitative-experimental design. This research

method was appropriate for the research study in which one or more variables

were manipulated during the experimentation procedure. It is a research design

that provides the details required to meet the objectives of the study.
3.3 Research Locale

The study was conducted at the house of one of the researchers in Tala

Caloocan City during the 2nd term of the academic year 2021- 2022. The

researchers considered the house set-up to easily monitor and observe. Proper

protocols were done to conduct accurate results for the propagation of plants

and the remediation of plants using a controlled set-up.

TORIO’s House in
Tala Caloocan City

Picture 1. Map of the Research Locale

3.4 Sample Preparation

The water spinach (Ipomoea aquatica Forsskal) that was used in the study

was propagated in a controlled set-up at home only and applied requirements to

grow the plant. The researchers propagated using the cuttings of water spinach

that was purchase from a farm in Tala Caloocan City. When the roots developed,

they were transferred into a container with distilled water and loamy soil, and

tested using Atomic Absorption Spectrophotometer.

 The heavy metals, such as Copper and Zinc that were collected were

bought from Bambang Manila City.

The experimentation was conducted in a controlled setup with a span of

two weeks. Fourteen (14) set-up divided into two groups, set A consist of one

set-up for controlled group which has no heavy metals, one set-up for 1ppm Cu2+,

one set-up for 1ppm Zn2+, one set-up for mix of 1ppm Cu2+ and Zn2+, one set-up

for 3ppm Cu2+, one set-up for 3ppm Cu2+, and one set-up for mix of 3ppm Cu2+

and Zn2+. Set B is the duplication of the above mentioned.

Set A

Standard Concentration High Concentration

1 2 5

Water Only Copper Copper 7

3 4 6 Mixed

Zinc Mixed Zinc

Figure 3 Set A Experimentation Set-up

Set B (Duplication)

Standard Concentration High Concentration

1 2 5

Water Only Copper Copper 7

3 4 6 Mixed

Zinc Mixed Zinc

Figure 4 Set B Experimentation Set-up

3.5 Preparation of Heavy Metals

The preparation of heavy metals undergone in the two series of dilution to

achieve the parts per million concentrations. For 1ppm Cu 2+ 14.73 grams of

CuSO4 • 5H2O, for 3ppm of 44.20 grams of CuSO4 • 5H2O, for 1ppm of Zn2+ 7.82

grams of ZnCl2, and for 3ppm Zn2+ 23.45 grams of ZnCl2. All metal salt was

dissolved in a 500ml volumetric flask. Obtained 10ml in a 500ml volumetric flask

and diluted it in another 500ml volumetric flask. Another 10ml was obtained in

the second diluted solution and put it in an acetone bottle. In dimensional analysis

the unit of ppm is mg/L.

3.6 Preparation of Fertilizer

The fertilizer used in the experiment was intended for optimization of

Water Spinach before and during experimentation, it has a value of 14-14-14

NPK (Nitrogen-Phosphorus-Potassium). The numbers represent the percentage

of the substance in fertilizers.

For the preparation of a 10ppm concentration of potassium per dosage:

13.6 grams of the fertilizer dissolved in a 10mL graduated cylinder. Obtained 1mL

in the dissolved solution using a syringe and is diluted in a 10 mL graduated

cylinder. The researchers got 1 mL of diluted solution and dropped it in every set-

up.
3.7 Heavy Metal Testing

The testing for heavy metals in the collected plant samples were done in

Mach Union Laboratories Bldg 335 Real St. Alabang–Zapote Road, Las Pinas,

1740, Metro Manila.

3.8 Waste Management and Disposal

The copper and zinc waste collected and disposed of separately in a

labeled bottle under no circumstances may differ types of waste be mixed. The

collected waste was sent to the Science Resource Center - Philippine Normal

University (PNU-SRC).

3.9 Statistical Analysis and Interpretation of Results

The statistical analysis tools that were utilized in the study included

measures of central tendencies such as the mean of heavy metal concentration

in the sample plant. One-Way Analysis of Variance (ANOVA) is used to check

the difference between the final heavy metal concentrations in contaminated

water treated with Water Spinach and the mean was also compared using

Tukey’s post hoc test at 0.05 significance level.

Bioconcentration factor is also used to measure the accumulation of heavy

metals. It is calculated by dividing the trace element concentration in plant tissues

(ppm) at harvest by initial concentration of the element in the external nutrient

solution (ppm).
 CHAPTER 4

RESULTS AND DISCUSSION

4.1 Pre-Experimentation of Water Spinach

Cu concentration Zn concentration
Water Spinach 0.005 0.0450
Table 1. AAS Pre-Laboratory Result

Before the population of the sample was subjected to the experiment, the

researchers gathered leaves that were tested in AAS. The researchers assumed

that the result represents the concentration of heavy metals in the fourteen

samples, as it cannot test all the population. Table 1 shows the results of pre-

laboratory that the Water Spinach does contain a minimal concentration of the

two heavy metals.

4.2 Atomic Absorption Spectrophotometer Test Result

Sample Solution Metal Concentration Accumulated BCF

leaf Concentration (mg/kg) concentration
(mg/L) from pre-
experimentation
1 Controlled Copper 0.009 0.004 0.00
Group
Zinc 0.08 0.035 0.00

Table 2. AAS Post-Laboratory Results for Controlled Group

The table 2 shows the number of heavy metals in the water spinach

leaves that have been tested, the concentration is in parts per million (mg/kg).

For the control group, even if the plant is subjected to distilled water, it

accumulates 0.004 ppm which raises the concentration into 0.009, for the zinc

the plant absorbs 0.035 ppm that raises the concentration into 0.08. The
absorbed metals despite no added concentration of Cu and Zn may result from

copper and zinc that is attached in the root system and as the plant detects

scarcity of these metals in the environment, it will activate its metabolic system

to absorb the remaining metals it has (Hausman et. al. 2016). The researchers

assumed no contamination of heavy metals from the environment therefore

initial concentration of these metals for the bioconcentration factor is zero

therefore the product is also zero.

Sample Solution Metal Concentration Accumulated BCF

leaf Concentration (mg/kg) concentration
(mg/L) from pre-
experimentation
2 1.00 Copper 0.03 0.025 0.03
3 1.00 Zinc 0.11 0.065 0.11
Copper 0.036 0.031 0.036
4 1.00
Zinc 0.11 0.065 0.11
Table 3. AAS Post-Laboratory Results for 1 ppm Concentration

The sample leaf number 2 that has been put in 1 ppm concentration of

copper accumulated 0.025 and has a BCF of 0.03. The sample leaf number 3

absorbed 0.065 ppm from 1 ppm concentration of zinc, the BCF value is 0.11.

For sample leaf number 4, it gathered 0.031 ppm of copper and 0.065 ppm of

zinc from 1 ppm concentration of Cu and Zn, the bioconcentration factors for the

mixed Cu and Zn environment are 0.036 and 0.11.

Sample Solution Metal Concentration Accumulated BCF

leaf Concentration (mg/kg) concentration
(mg/L) from pre-
experimentation
5 3.00 Copper 0.053 0.048 0.018
 6 3.00 Zinc 0.14 0.095 0.047
Copper 0.044 0.039 0.017
7 3.00
Zinc 0.13 0.085 0.043
Table 4. AAS Post-Laboratory Results 3 ppm Concentration

The sample leaf number 5 in 3 ppm concentration of copper accumulated

0.048 ppm and a BCF of 0.018. Sample leaf number 6 that is put in 3 ppm

concentration of zinc absorbs 0.095 ppm, the BCF is 0.047. The last sample, the

sample leaf number 7 is in a mixed solution of 3 ppm copper and zinc, it

accumulated 0.039 ppm copper and 0.085 ppm zinc, the values of BCF for this

mixed solution is 0.017 for copper and 0.043 for zinc. There is no less than one

value in all bioconcentration therefore, it implies that the metals accumulated in

the plants are hydrophilic and the ratio of heavy metals in plants is lower to its

environment. The researchers suspect that if the experiment takes longer, it will

produce higher results.

4.3 Statistical Results

Concentration
Sum of Squares df Mean Square F Sig.
Between Groups .004 3 .001 53.473 .001
Within Groups .000 4 .000
Total .004 7
Table 5. IBM SPSS (v.21) One-way ANOVA Analysis

The analysis of the statistical software of IBM SPSS (v.21) shows 0.001

significance which is lower than Cronbach alpha 0.05 therefore that is a

significant difference between the means of four different concentrations of

copper and zinc.

 Multiple Comparisons
Dependent Variable: Concentration
Tukey HSD
(I) (J) Comparing Mean Std. Sig. 95%
Concentration Concentration Difference Error Confidence
of Metals (I-J) Interval
Lower Upper
Bound Bound
- -
1 ppm Zinc -.037000* .005208 .007
.05820 .01580
-
1 ppm Copper 3 ppm Copper -.015500 .005208 .127 .00570
.03670
- -
3 ppm Zinc -.062000* .005208 .001
.08320 .04080
1 ppm Copper .037000* .005208 .007 .01580 .05820
1 ppm Zinc 3 ppm Copper .021500* .005208 .048 .00030 .04270
- -
3 ppm Zinc -.025000* .005208 .029
.04620 .00380
-
1 ppm Copper .015500 .005208 .127 .03670
.00570
- -
3 ppm Copper 1 ppm Zinc -.021500* .005208 .048
.04270 .00030
- -
3 ppm Zinc -.046500* .005208 .003
.06770 .02530
1 ppm Copper .062000* .005208 .001 .04080 .08320
3 ppm Zinc 1 ppm Zinc .025000* .005208 .029 .00380 .04620
3 ppm Copper .046500*0 .005208 .003 .02530 .06770
* The mean difference is significant at the 0.05 level.

Table 6. IBM SPSS (v21) Table of Comparison for four different means

The table for multiple comparison tells that only the mean for 1 ppm

Copper and 3 ppm Copper has no significant difference showing p-value or

significance level of 0.127 that is higher than Cronbach’s alpha 0.05, hence, there

is no significant increase in the accumulated concentration of copper even the

concentration was raised in threefold. The second highest in significance level is

the p-value of 1 ppm Zn to 3 ppm Cu which is 0.048, though it is lower than 0.05

therefore there is a significant difference. The p-value of the third is 0.007, the

significant level of the mean of 1 ppm zinc and 1 ppm copper. The lowest p-value

and the highest significant difference are the mean of 3 ppm Zn and 1 ppm Cu.

The accumulated metals for the controlled group were not included in the

comparison for the significant difference, as they do not have a mean.

Graph 1. Scatter Graph for the comparison of average increase in Copper and

Zinc

The result exhibits that zinc has a higher accumulation than copper as the

graph shows a spike between Cu and Zn average of increase of concentration.

Therefore, the gathered data tells the preferential absorption of Ipomoea

aquatica towards Zn rather than Cu.

The data from the conducted study of Hausman et. al. (2016), the heavy

metals such as zinc enters from the root system of a dicot plant by the zinc-

regulated, iron-regulated transporter-like proteins (ZIP), these family of receptor

proteins also exhibit high affinity in the divalent cations such as cadmium, iron

and copper. Although the similarities of ionic charge that make Cu2+ competes

with Zn2+ in this cellular uptake, the Cu2+ is being expelled in H+/ Cu2+ antiporter

transport protein, the H+ will move inward to compensate the efflux of Cu2+

(Hausman et. al. 2016) a while there is no pathway how the Zn 2+ will release in

the environment. Data from the study suggest that plants prefer the monovalent

of cuprous (Cu+) than divalent cupric (Cu2+), the conducted experiment for this

study used copper sulfate pentahydrate where its cation is cupric. The reduction

of cupric into cuprous is being facilitated by the ferric reductase oxidase (FRO)

particularly in the root tips (Bernal 2012), the reduced copper is being absorb by

the copper transporter 1 and 2 (COPT 1 and 2) which contains sulfur-containing

residues such as cysteine and methionine, the Cu+ will bind in the lone pairs of

the sulfur (Hausman et. al. 2016). The researchers suggest that the discussed

mechanisms of metal absorption of the plants and the used copper salt for

experimentation are the reason why the Ipomoea aquatica exhibits higher

accumulated zinc than copper.

 CHAPTER 5

CONCLUSION AND RECOMMENDATION

In this study, naturally occurring Water Spinach (Ipomoea aquatica) were

analyzed to evaluate its efficiency to phytoremediate water artificially

contaminated with equal molar concentration of 1 ppm and 3 ppm of Copper and

Zinc.

5.1 Conclusion

This study for the potentiality for phytoremediation of Ipomoea aquatica

did not prove to be much effective for Cu and Zn removal from water samples,

especially for copper where it exhibits smaller accumulated concentration

compare to zinc, the data gathered about the absorbed Cu and Zn in I. aquatica

did not even reach 1 mg/kg where its highest is 0.14 in 3 ppm of zinc. The

researchers dare to suggest that the factors why the I. aquatica did not exhibit

satisfying results is because of lack of time allotted for the plant to absorb the

metals and the parameters that should be considered but it does not include due

to financial capabilities of the researchers. Still the conducted research provides

valuable data that can help other researchers who want to pursue the field of

phytoremediation.
5.2 Recommendation

The researchers of this study recommend for the colleagues in the field of

science who will pursue research in phytoremediation to look for the other

common heavy metals where possibly the Ipomoea aquatica would be effective

to remove. If possible, the future researchers should also consider the

bioaccumulation which will take longer allotted time for experimentation, the

cellular metabolic mechanism of I. Aquatica for metal uptake particularly in the

rate of reaction of ferric reductase and oxidase (FRO), copper transporter

(COPT) that affects the speed of copper uptake of plant. Other parameters such

as pH levels, temperature and presence of microorganisms are worthy of

consideration too.
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 GANTT CHART OF ACTIVITIES

Activities

Time (Months) (Weeks)

Duration: 4 months and 2 weeks

Task March April May June July

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2
Planning of
Chapter 4
Propagatio
n of Water
Spinach
Testing of
Water
Spinach
Consultatio
n with
Experts
Controlled
setup
experiment
ation
Testing of
Water
Spinach
Consultatio
n with
Experts
Writing of
Paper
Submission
of Draft
Chapters
4-5
Revision
Mock
Defense
Revision
Submission
of Final
Chapters
4-5
Final
Defense
Submission
 RESEARCH BUDGET

Quantity Item Amount (Php)

14 (pcs) Aquarium 900

(144×mm×95mm×203mm)

4 (pcs) Container/ Pot 100

25 (gal) Distilled Water 800

16 (pcs) Water Pump 1000

4 (bundles) Water Spinach 100

1 (time) Laboratory 10000

6 (times) Transportation 2000

100 (grams) Zinc chloride 200

100 (grams) Copper sulfate 200

pentahydrate

100 (grams) Fertilizer 200

Total Amount: Php 15,500.00

APPENDICES
 APPENDIX A: Calculations

Preparation of 1ppm Copper

1mg Cu2+ 1g Cu2+ 1 mol Cu2+ 1mol CuSO4 • 5H2 O

( )( )( )( )
𝐿 1000mg 63.55g 1mol Cu2+
249. 68 g CuSO4 • 5H2 O 500mL volumetric flask
( ) (1.5 L Aquarium) ( )
1mol 10mL
500mL volumetric flask
( ) = 14.77g CuSO4 • 5H2 O
10mL

Preparation of 3ppm Copper

3mg Cu2+ 1g Cu2+ 1 mol Cu2+ 1mol CuSO4 • 5H2 O

( )( )( )( )
𝐿 1000mg 63.55g 1mol Cu2+
249. 68 g CuSO4 • 5H2 O 500mL volumetric flask
( ) (1.5 L Aquarium) ( )
1mol 10mL
500mL volumetric flask
( ) = 44.20g CuSO4 • 5H2 O
10mL

Preparation of 1ppm Zinc

1mg Zn2+ 1g Zn2+ 1 mol Zn2+ 1mol ZnCl2

( )( )( )( )
𝐿 1000mg 63.55g 1mol Zn2+
136.29g ZnCl2 500mL volumetric flask
( ) (1.5 L Aquarium) ( )
1mol 10mL
500mL volumetric flask
( ) = 7.82g ZnCl2
10mL

Preparation of 3ppm Zinc

3mg Zn2+ 1g Zn2+ 1 mol Zn2+ 1mol ZnCl2

( )( )( )( )
𝐿 1000mg 63.55g 1mol Zn2+
136.29g ZnCl2 500mL volumetric flask
( ) (1.5 L Aquarium) ( )
1mol 10mL
500mL volumetric flask
( ) = 23.45g ZnCl2
10mL
Preparation of Fertilizer

10mg K + 1g K + 1 mol K + 1mol KCl 74.55 g 100 g fertilizer

( )( )( )( )( )( )
𝐿 1000mg 39.10g 1mol K + 1mol 14g KCl
10mL 10mL
( 1mL ) ( 1mL ) = 13.6 g of Fertilizer
 APPENDIX B: Water Spinach (Ipomoea aquatica Forsskal)

This picture was originally taken by Joan H. Torio on July 19, 2021. This
Water Spinach (Ipomoea aquatica Forsskal) or commonly known as Kangkong
was bought in moist soil at Caloocan City. Its stems are 5 to 10 inches long, the
leaves are 3 to 4 inches long and 1 to 2 inches broad.
 APPENDIX C: Map of the Research Locale

TORIO’s House in
Tala Caloocan City

This picture was taken from Google Map on May 6, 2022. This Map is at
Tala Caloocan City, where the experimentation was set-up.
 APPENDIX D: Illustration of Experimentation Set-up

Set A

Standard Concentration High Concentration

1 2 5

Water Only Copper Copper 7

3 4 6 Mixed

Zinc Mixed Zinc

Set B (Duplication)

Standard Concentration High Concentration

1 2 5

Water Only Copper Copper 7

3 4 6 Mixed

Zinc Mixed Zinc

APPENDIX E: Actual Experimentation Set-Up
APPENDIX F: Plant Identification
APPENDIX G: Experimentation
APPENDIX H: AAS Initial Testing Result
APPENDIX I: Laboratory Results
APPENDIX J: Letters to Internal and External Experts

Internal Expert
External Expert
APPENDIX K: Meetings with Internal and External Experts

Internal Expert
External Expert
APPENDIX L: Summary of Comments and Suggestions

Internal Expert
External Expert
APPENDIX M: Curriculum Vitae of Researchers