Phytoremediation potential of Bermuda grass (Cynodon dactylon )

and Carabao grass ( Paspalum cojugatum ) in lead deposition :

A comparative study

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An Undergraduate Thesis

Presented to the Chemistry and Physics Department

College of Arts and Sciences,Cebu Normal University

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In partial fulfillment of the Course Requirements

For the degree Bachelor of Science in Chemistry and Physics

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Submitted by:

Anoc, Hannie Lou F.

Rom,Sherlice Q.

March 2013

APPROVAL SHEET

This research paper entitled “Phytoremediation potential of Bermuda grass (Cynodon dactylon) and Carabao grass (Paspalum conjugatum) in lead deposition: A comparative study” prepared by Hannie Lou F. Anoc and Sherlice Q. Rom. In partial fulfillment for the subject ChemPhys 114: Research in Chemistry and Physics has been examined and is recommended for acceptance and approval for Oral Examination.

The Technical Panel

JOYCE R. CALUMBA, MAST-Chemistry

Chair, Chemistry and Physics Department

GIBSON T. MAGLASANG, MS- PhysicsProfessor, Chemistry and Physics Department

KARL PATRICK R. CASAS, MS-PhysicsProfessor, Chemistry and Physics Department

DR. STELLA THERESE R. AVILAChair, Biology Laboratory

NIMFA PANSIT, MS Envi. BiologyProfessor, Biology Department

ALLAN ROY ELNARProfessor, Chemistry and Physics Department

Adviser Accepted and Approved in partial fulfillment of the requirements for

the subject ChemPhys 114: Research in Chemistry and Physics.

FLORIZA N LAPLAP, Ed. D.Dean, College of Arts and Sciences

Cebu Normal Universityi

PANEL OF EXAMINEES

This research paper entitled “Phytoremediation potential of Bermuda grass (Cynodon dactylon) and Carabao grass (Paspalum conjugatum) in lead

deposition: A comparative study” prepared by Hannie Lou F. Anoc and Sherlice Q. Rom. In partial fulfillment for the subject ChemPhys 114: Research in Chemistry and Physics passed on the Oral Examination and is approved by the committee of publishing.

JOYCE R. CALUMBA, MAST-ChemistryChair, Chemistry and Physics Department

GIBSON T. MAGLASANG, MS- PhysicsProfessor, Chemistry and Physics Department

KARL PATRICK R. CASAS, MS-PhysicsProfessor, Chemistry and Physics Department

NIMFA PANSIT, MS Envi. BiologyProfessor, Biology Department

ALLAN ROY ELNARProfessor, Chemistry and Physics Department

Adviser

Accepted and Approved in partial fulfillment of the requirements for the subject ChemPhys 114: Research in Chemistry and Physics.

FLORIZA N. LAPLAP, Ed. D.Dean, College of Arts and Sciences

Cebu Normal Universityii

ACKNOWLEDGEMENTThe accomplishment of this research won’t be possible without the

unconditional support of our families, friends, panelists and those individuals

that have shared their time with us while our research is on progress despite

of their busy schedule. We would like to thank the following:

To Mr. Allan Roy Elnar, our adviser, mentor, and a good father-like

image to us, for his patience in checking and correcting our works to comply

a much better and effective research paper;

Mrs. Luzviminda Bato, for her heartfelt help to us, for being

approachable to our concerns regarding our experiments; for keeping us

motivated in our research and never stop believing in ourselves;

Laboratory Custodians, Mr. Joenard Algones and Ms. Flor Marie

Flores with their laboratory assistants, who willingly entrust to us their

laboratories for us to use and for extending their laboratory duties in order

for us to perform our experiment;

Kuya Lester Jan R. Bato and Alden Deniega with the Technolab

Analytic Group, for giving us lesser fees for the reading of our samples and

analyzing them with precision;

Kuya Tonyo, for offering his services and facilities open-hand while

preparing and unloading some materials to be used in our experiment;

Mr. Adonis Atuel, agriculturist, for sharing his experiences on

planting grasses and advices that could help us in growing them;

iii

Science Faculty, for sharing experiences in their respected field as a

member of the Panel during our Thesis Proposal Hearing;

Our beloved parents- Mama Alice and Papa Allan (Sherlice), and

Mama Haydee and Papa Ronie (Hannie Lou), for their immeasurable

support, emotionally and financially; from the start until the end of our study.

Last but definitely not the least but the greatest, to God, for being with

us in the ups and downs of our research, HE who has given us the

perseverance on our research and the accomplishment of everything and

finishing what we had started.

Heartfelt grateful thanks to all of them who has been with us in our

journey to our goal.

Hannie Lou and Sherlice

Cebu Normal University

Cebu City

March 2013

ivABSTRACT

The emergence of interests in phytoremediation studies were brought

about by the increasing deposition of heavy metals and pollutants in the

soil.In this study,Bermuda grass ( Cynodon dactylon ) and Carabao grass

( Paspalum conjugatum ) are compared based on their potential as lead

phytoaccumulator at 3000 ppm and 6000 ppm concentrations.Grasses were

grown for one month,prepared using acid-digestion and read by Flame-

AAS.The concentration of lead present in the amended samples shows that

at 3000 ppm.It accumulate 5.6 mg/Kg of lead absorbed at a rejection

percentage of 0.19%.These implied that carabao grass is a potential

phytoremediating agent of lead while Bermuda grass does not exhibit the

same potentiality.

Keywords: Bermuda grass,Carabao grass,Phytoremediation

v

Table of Contents

Title Page

Approval Sheet……….

………………………………………………………………………………………….i

Panel of Examinees………..

…………………………………………………………………………………ii

Acknowledgement…………..

……………………………………………………………………………….iii

Abstract…………………………….

……………………………………………………………………………..iv

Table of Contents…………….

……………………………………………………………………………..vii

List of

Tables……………………………………………………………………………………………

…….viii

List of Figures…………………..

……………………………………………………………………………ix

List of Appendices…………….

…………………………………………………………………………….x

-------------------------------------------------------------------------------------

Chapter 1

Introduction…………………………………………………………………………………..1

Rationale…………………………………………………………………………………………

…….1

Statement of the

Problem……………………………………………………………........3

Theoretical

Background…………………………………………………………………………3

Scope and

Limitations…………………………………………………………………………..9

Significance of the

Study………………………………………………………………………9

Definition of

Terms………………………………………………………………………………10

Chapter 2 Review of Related

Literature…………………………………………………………12

vi

Chapter 3

Methodology…………………………………………………............................16

Research

Design………………………………………………………………………...........16

Research

Environment…………………………………………………………………………16

Research

Procedure……………………………………………………………………………..16

Chapter 4 Results and

Discussions……………………………………………………………….19

Results……………………………………………………………………………………………

…….19

Discussions………………………………………………………………………………………

…..21

Chapter 5 Summary, Conclusion and

Recommendations…………………………..25

Summary…………………………………………………………………………………………

……25

Conclusion………………………………………………………………………………………

…….25

Recommendations……………………………………………………………………………

…..26

Bibliography……………………………………………………………………………………

………………27

Appendices………………………………………………………………………………………

…………….37

Curriculum

Vitae…………………………………………………………………………………………….5

2

viiLIST OF TABLES

Tables Page

1.0 Analysis of Variance One-Factor ANOVA result of

Plant Growth……………………………………………………………………

20

2.0 Absorption of Lead through AAS

(Atomic Absorption Spectrometer)…………………………………..

20

3.0 Analysis of Variance One-Factor ANOVA result of

Lead Absorption……………………………………………………………….

21

viii

List of Figures

Figures Page

1.0 Average Leaf Size (Carabao grass) and

Diameter (Bermuda grass)………………………

19

ix

List of Appendices

Appendix Page

A Gannt

Chart………………………………………………………………………………37

B Plant

Profile………………………………………………………………………………38

B.1 Bermuda

grass……………………………………………………………….38

B.2 Carabao

grass…………………………………………………………………39

C Lead Concentration Accumulated using the Mean AAS (Atomic

Absorption Spectrometer)

reading………………….....40

D Lead Accumulation and Rejection

Percentage………………………..41

E Preparation of 3000 and 6000 ppm Lead nitrate……………………

42

F Flame Atomic Absorption

Spectrometer………………………………….43

G

Documentation…………………………………………………………………………

45

H Thesis

Expenditures…………………………………………………………………48

I Raw Data of Growth Rate ( Carabao and Bermuda Grass)

……………………………………………………………………49

J Result of Analysis through AAS(Carabao grass)

……………………………………………………………..50

K Result of Analysis through AAS(Bermuda grass)

…………………………………………………………...51

x

CHAPTER 1

Introduction

RATIONALE

One of the many problems existing in the environment introduced in

the soil is lead (Pb) (Ona et al., 2006 ).The main problem coexist with human

activities such as mining (Liu et al.,2010),wet battery leaks (US

EPA,2003 ),lead- waste water from industries (Ona et al., 2006 ),as well as

heavy metal (e.g., lead (Pb), mercury (Hg),cadmium (Cd) ).While these

problems continue to exist adverse health effects of heavy metal intake were

caused through food intake from plants ( Nasreddine and Parent-

Massin,2002 ) and animals (Lead Poisoning in Livestock,2012).

The accumulation of lead (Pb) in soil greatly affects the ecosystem.

Since soils are considered as a major sink for lead it might be absorbed and

bioaccumulated by plants and animals that may be available for human

consumption in significant amounts (Effects of Lead in Plant Growth and

Photosynthetic Activity, 2003).The general effect of lead (Pb) in plants is that

it affects the physiological processes such as the slowing rate of

photosynthetic activity and may lead to plant death. On the other hand, its

effect on domestic animals took its effect in the central nervous system and

inhibits the ability to synthesize blood cells according to US EPA (1986).And

on a report it generalizes that when animals are on a regular diet of 2-8 mg

of lead per kilogram of body weight per day, over an extended period of time

can cause death to most animals (US EPA, 1996).

An alternative way of reducing lead (Pb) contamination is through

phytoremediation. It is an alternative method that uses plants in cleaning up

lead (Pb) contaminated areas (Uera et al., 2007). It is an easy to implement,

cost-effective and an environmentally-friendly process (Berti, 1997).

However, the success of phytoremediation depends on the choice of plant

species, which can adapt and be relatively tolerant to the high concentration

of heavy metals in soil (Uera et al., 2007).The study uses grass belonging to

the Poaceae family.

Grasses belongs to the plant family Poaceae in which according to

recent studies is one of the 101 families which is known to be effective in

metal hyperaccumulation (Kramer,2010).They are thought to be an excellent

candidate in phytoremediation, because of their fibrous rooting system that

can stabilize the soil and provide a large surface area for root-soil contact

(Kulakow et al.,2000).Hence, the study is aimed to determine the potential of

grasses as phytoremediating plants to lead (Pb).This further determines the

level of concentration accumulated in the leaves. Consequently, it will

benefit the process of taking lead from contaminated soils, particularly in

dumping sites.

2

STATEMENT OF THE PROBLEM

The study aims to compare the lead accumulation of grasses namely:

Carabao grass (Paspalum conjugatum) and Bermuda grass (Cynodon

dactylon). Specifically it determines the following:

1. The level of lead (Pb) absorbance in grasses for the following

treatment:

0 ppm of Pb (NO3)2

3000 ppm of Pb (NO3)2

6000 ppm of Pb (NO3)2

2. The effect of lead accumulation to growth rate of Carabao grass

(Paspalum conjugatum) and Bermuda grass (Cynodon dactylon ).

Leaf size (Carabao grass)

Diameter (Bermuda grass)

3. The Rejection percentage (RejP) of the concentration of lead on the

grasses being analyzed (see Theoretical Background for the

formula).

THEORETICAL BACKGROUND

The removal of heavy metals can be related to the following theory

such as the Reverse Osmotic Theory. Here is the following explanation of the

theory in the removal of heavy metals.

3

1. Phytoremediation

Phytoremediation is the cleaning up of heavy metals in soil. Here

are the mechanisms of phytoremediation.

There are several types of phytoremediation processes that

cover a large number of different organic and inorganic compounds.

Only three are relevant to the phytoremediation of Lead (Pb).These

three are termed (1) Phytoextraction – The uptake of contaminants

by roots and translocation within the plants (2) Rhizofiltration – the

adsorption or precipitation onto plant roots, or absorption into the

roots of contaminants that are in solution surrounding the root zone,

due to biotic or abiotic processes and (3) Phytostabilization- the

immobilization of a contaminant in soil through absorption and

accumulation by roots, adsorption onto roots, or precipitation within

the root zone of plants, and the use of plants and plant roots to

prevent contaminant migration via wind and water leaching, and

soil dispersion.

4

Source:http://www.scribd.com/doc/37203060/Phytoremediation-Technology-Hyper-Accumulation-Metals-in-Plants

Figure 1.0 Schematic representation of the accumulation of pollutants during

phytoremediation

(1)Phytoextraction. The uptake of contaminants by plant roots and

translocation within the plants. It is primarily used in the treatment of soils,

Source:http://www.scribd.com/doc/37203060/Phytoremediation-Technology-Hyper-Accumulation-Metals-in-Plants

sediments and sludges. Constituents amenable to phytoextraction include :

Metals – Ag, Cd, Co, Cr, Cu, Hg, Mn, Mo, Pb, Zn; Metalloids – As, Se;

Radionuclides -90 Sr,137 Cs,239 Pu ,238 U,234 U.

5

Fodor’s model of the accumulation of heavy metals in plants

The step-by-step process of the uptake of heavy metals on plants

According to Fodor (2002) suggestion, the accumulation of heavy

metal in plant is a stepwise process. Initially, is the interaction with other

ionic components taking place at the locus entry into the plant rhizosphere

that consequently have consequences for the metabolism. This is followed

by an impact on the formation of reactive oxygen species (ROS) in the cell

wall and an influence on the plasmalemma membrane system (stage 1).At

stage 2,the metal ion reacts with all the possible interaction partners within

the cytoplasm, including proteins, other macromolecules and metabolites.

Stage 3 is mainly related to the factors that influence homeostatic events,

which include water uptake, transport and transpiration. At this stage,

symptoms start to develop, and they become visible at stage 4.For instance,

the chlorophyll and usually to a smaller degree, carotenoid content decrease,

which have obvious consequences for photosynthesis and plant growth

(Barcelo and Poschenrieder 2004).The death of plant cell occurs at stage

5.This model of Fodor has the advantage that visible effects are linked to

metabolic events that are influenced by any metal ion.

II. Reverse Osmosis Theory

Reverse osmosis (RO) is a membrane process which initially was

developed to produce potable water from saline and brackish water (Sirkar

6

et al., 1994).However, through the recent years of improvisation of its

performance researchers begun to find out that it is not only applied in the

treatment of water but as well as the recovery of organic and inorganic

materials from chemical processes. Moreover, it can also remove organics,

colour, nitrates and low total dissolved solids (TDS) concentrations (Sirkar et

al., 1992).In like manner, removal of inorganic materials from soil requires

grasses to have relatively high permeable membrane .Their efficiency can be

described in the process of reverse osmosis (RO) (See Fig.3.0 ).

SOURCE: http://ph.images.search.yahoo.com/imags/view

Fig.3.0.Reverse Osmosis

Moreover, these physical characteristics implied the potential of

plants in general as phytoremediating agents. Quantitatively, characteristics

can be defined in terms of the plants rejection percentage (RejP) and

recovery percentage (RecP).

7

Rejection Percentage is determined accordingly:

CF -CP% RejP= ------------------ X 100

CF

Where: CF – concentration of a specific component in the feed solution

to the membrane process

CP – concentration of the same specific component in the

product stream leaving the membrane system

On the other hand, recovery percentage (RecP) can be determined

accordingly:

CP % RecP = -------- X 100 CF

Where: CF – concentration of a specific component in the feed solution

to the membrane process.

CP – concentration of the same specific component in the

product stream leaving the membrane system.

In the case of concentration of lead, these percentages (RejP and

RecP) can be translated to:

Input concentration of lead in plants – output concentration of lead in plants

%RejP =------------------------------------------------------------------- X 100 Input concentration of lead in soil

8

%RecP is measured from the input and output concentration of lead as

follows:

Output concentration of lead in plants % Rec P = ---------------------------------------------- x 100

Input concentration of lead in soil

SCOPE AND LIMITATIONS

The study covers the comparative analysis among two kinds of

grasses: Carabao grass (Paspalum conjugatum) and Bermuda grass

(Cynodon dactylon ) and there efficiency in phytoremediation in the

absorbance of Lead (II) nitrate Pb ( NO3)2.It also focuses on the effect of Lead

(II) nitrate Pb ( NO3)2 to these grasses.

The delimination of the study is only two kinds of grasses will be

analyzed: Carabao grass (Paspalum conjugatum) and Bermuda grass

(Cynodon dactylon) and the contaminant used is Lead (II) nitrate Pb (NO3)2 .

The grasses to be used are young seedlings due to the unavailability of the

seeds of grasses in the Philippines. The study limits on the comparison of the

percentage of absorbance in Lead (II) nitrate Pb (NO3)2 among the two

grasses using Atomic Absorption Spectrometry (AAS).

SIGNIFICANCE OF THE STUDY

This study will beneficial to the following:

(a) PUBLIC – provide information about the phytoremediation potential

9

of selected grasses common in Cebu Provice such as Bermuda grass

(Cynodon dactylon ) and Carabao grass (Paspalum conjugatum)

accumulating Lead Nitrate (PbNO3)2.

(b) COMPANIES – a tool in enhancing their waste management

programs; provides an innovative, economical, and environmentally-

friendly alternative in removing toxic metals specifically Lead.

(c) LOCAL GOVERNMENT – for the sustainable protection of the

environment through strictly implementing the segregation of waste

materials, proper disposal through having garbage cans and

relocation sites for people living in dump sites for their safety.

(d) DENR – for the proper storage and disposal of the waste materials

being handed to them for the safety of soil, plants and human health.

(e) GRASS BREEDERS- for the awareness that these grasses: Carabao

grass (Paspalum conjugatum) and Bermuda grass (Cynodon dactylon)

can help in the cleaning of the soil used in breeding plants.

(f) DEPARTMENT OF AGRICULTURE- to breed more grasses especially

for the benefit of the soil they will be using.

DEFINITION OF TERMS

Grasses – belongs to the Poaceae family which is known to be

effective in phytoremediation.

10

Reverse osmosis theory- a process in which it requires grasses to have

relatively high permeable membrane to remove the inorganic materials

found in the soil.

Potentiality of grasses – being adaptive and highly tolerant of

grasses in the high concentrations of lead treated in the soil.

Phytoremediation – the use of plants in removing heavy metals in

soil. The study focuses on the use of grasses as phytoremediating

agent.

Heavy metals – hazardous in soil which is derived either naturally or

chemically that maybe absorbed and bioaccumulated by plants and

animals.

11CHAPTER II

Review of related literature and related studies

The emergence of interests in phytoremediation studies were brought

about by the increasing deposition of heavy metals and pollutants in the soil

( Caussy et al.,2003; Cui et al.,2004;Dudka et al.,1996;Muller and Anke,

1994;Sanchez – Camazano et al.,1994 ).In effect, ways to remove heavy

metals brought hope for a cleaner environment .However ,the

unprecedented urbanization and other anthropogenic human activities make

this likely impossible ( Dean et al.,1972;Dorsey,2003;Nriagu,1996;Mage et

al.,1996;Pauleit et al.,2005;Ona et al., 2006;Randolph,2004;Widinarko et

al.,2005 ) and always a challenge for sustainable development

(Cleverland,2003;Rees,1992).The many studies on removing heavy metals in

the soil had been also a challenge because of the unbalanced rate of

deposition and rate of removal (Singh et al.,2012).Also ,the methods used

were costly (Cunningham et al.,1996;Singh et al.,2012 ).

Moreover ,the deposition of lead (Pb) persisted for over 5000 years

(Friedland,1990) and become the most common heavy metal contaminant in

the soil ( Alloway,1995;EPA,1993; Wanatabe,1997 ).These contaminants

were considered toxic to humans even when taken in minute amounts

(Brinkmann, 1994; Sheppard, 1998; Thornton, 1991).In addition,

leadcontamination prevailed due to existing mining and smelting activities

(Bridge,2004;Kodom et al.,2010;Lacatusu et al., 2009;Nakayama et

al.,2010;Nriagu,1996) as well as the use of paints ,gasoline, explosives, and

the disposal of municipal sewage sludge and industrial wastes (FAO and

WHO,2000;Reichman,2002;Zakrzewski,1991).These activities introduced

lead to the food chain and further into animals and human metabolism

(ATSDR,2000;Sauve et al.,1997;Wang et al.,2001).

The fatal effect of lead (Pb) intake into human metabolism includes

seizures, mental retardation (Canfield et al.,2003;Gosh and

Singh,2005;Goyer,1993),behavioral disorders ( Gosh and Singh,2005) as well

as brain and kidney damage ( Voroney,2006 ) and vomiting and appetite loss

(FAO and WHO,2000;Mushak,1993).In like manner, lead (Pb) can lead to

human genetic disorder, such as cancer (Beyersmann & Hartwig,2008;EPA

Toxic Release Inventory ,2000 ).Consequently, the effects are irreversible

( Bellinger and Dietrich ,1994 ) that includes inhibited photosynthetic

activities in plants and animals resulting from deficient mineral intake and

water imbalance (Adriano,1986;Afzal et al.,2006;Alloway,1990;Hao et al.,

2004;Schmidt,2003;Sadiq,1992;Sharma et al.,2005;Wahla &

Kirkham ,2008;WHO,1989).

Lead contaminated soil must be remediated to decrease the

environmental risk. Many remediation techniques have been employed to

address the rising number of heavy metal contaminated soils (Cholpecka et

13

al.,1996;Cunningham,1996;Cunningham et al.,1995 ). Most of the traditional

methods such as incineration, vitrification, electrokinetics and land filing are

extremely expensive (Danh et al., 2009;Mulligan et al.,2001;Pulford and

Watson,2003). Due to these problems the emergence of an environmentally

friendly (Ranskin and Ensley, 2000) technology called Phytoremediation is

widely accepted.

Plants are attractive, economic and non-invasive alternatives to

remove heavy metals (phytoextraction) from polluted soils as pointed out by

Blaylock and Huang (2000) and Salt et al. (1998).However, the plant species

being used must grow well in toxic levels of heavy metal conditions and can

produce high biomass (Berti, 2007).The success of phytoremediation is

greatly dependent upon the choice of plant species to be used.

The use of plants as agents to remove heavy metals includes spinach

that can uptake a maximum of 192 µg g-1 Cadmium (Cd) at 50 µg g-1

treatment (Salaskar et al., 2011 ), radish according to Dean and Intawongse

(2006) can accumulate Copper (Cu) – 62.5%,Cadmium (Cd) –

54.9%,Manganese (Mn) – 45.8%, duckweed hyperaccumulates Cadmium

(Cd),Copper (Cu) and Selenium (Se) (Lone et al.,2008 ) and as pointed out by

Singh et al., (2012) it can remove up to 90% of soluble Lead (Pb) from

water; and recently the work of Estrera and Banzon (2012) on yardlong

beans used in Pb accumulation.

14

In similar manner the use of grasses as phytoremediating plants were

studied by Sigua et al., (2007) and Xia (2003).They found out that the

Vetiver grass had the potential in removing Pb ( Sigua et al.,2007;Xia ,2003)

and Cadmium (Xia,2003).Accordingly, Vetiver grass are known for its

effectiveness in erosion and sediment control ( Greenfield,1995 ),and highly

tolerant to soil extreme condition ( Roongtanakiat and Chairoj,2001;Truong

and Baker ,1996,1998;Truong,1999).These characteristics are an immediate

requirement in removing heavy metals in soil. Therefore, the use of these

plants and perhaps variant grasses can be use as phytoremediation agent

because of their relative tolerance to high concentration of heavy metals

(Uera et al., 2007).

Phytoremediation due to its low cost compared to the conventional

cleaning-up technologies ( Chaney et al., 1997 ;Cunningham et al.,

1996.1997;January,2006;Salt et al.,1995;Sarma 2011 ) and being

environmentally friendly ( Chen & Cutright,2002;Fayiaga et al.,

2004;Pivertz,2001) is a very interesting topic for many researchers. Its great

impact to our lives serves as a tool for a greener and healthier environment.

15

CHAPTER III

METHODOLOGY

RESEARCH DESIGN

The study is experimental by nature. The amount of lead absorbed by

the Bermuda grass and Carabao grass will be analyzed through AAS (Atomic

Absorption Spectrometry). A 2X3 factorial experiment with two replications

(Bermuda grass, Carabao grass ) per treatments ( 0 ppm,3000 ppm,6000

ppm ).The concentration of Lead accumulated by the two grasses is

determined through the Rejection Percentage.

RESEARCH ENVIRONMENT

The study was performed at Cebu Normal University Chemistry

Laboratory where the young grasses were being grown for one month. The

grasses were then placed in aluminum foil pan and were arranged in blocks.

After one month, the grasses were harvested and digested, and then the

digested samples were forwarded at Technolab Analytical Group Inc. for the

reading of the samples.

RESEARCH PROCEDURE

1) Sample Germination and Collection

The young grasses of Bermuda grass and Carabao grass that was

grown for one month were labeled as (Control, T1 and T2), in which the

control samples has 0 ppm lead amendment, while T1 and

T2 has the following amendment; 3000 ppm and 6000 ppm of lead,

respectively. The grasses are allowed to grow for one month and the

corresponding measurement of leaf size (Carabao grass) and diameter

(Bermuda grass) were measured weekly. During the growth of the

grasses, all treatment was watered with 2000 ml of distilled water

(Ahmad et. Al., 2008) avoiding contamination aside from lead. The

grasses were then air-dried for one week after one month of

germination.

2) Sample Preparation and Analysis

a.) 0.20 g of each samples were placed in porcelain crucibles and

were heated for 3 hours at 300 ˚C and an additional 2 hours at 500 ˚C

inside a muffle furnace. Then 3 ml of 5 N Nitric acid was added to the

samples and was heated at 200 ˚C for 15 minutes to remove traces of

organic matter. Then, the samples were placed on the hot plate for

drying followed by the addition of 5 ml 2 N Nitric acid to dissolve the

residue of salts. The mixtures was filtered to catch its filtrate through a

Whattman # 42 in a 250 ml volumetric flasks and then transferred to

vials and was stored in the refrigerator ready for forwarding of analysis

at Technolab Analytical Group Inc.

b.) Analysis using Flame- AAS is based on the APHA AWWA- WEF,

Standard Methods for the Examination of Water and Wastewater,

17

21st Edition(American Public Association, 2005).

3) Statistical Analysis

Statistical analysis employed in this study uses the software

SPSS V16.

18

CHAPTER IV

RESULTS AND DISCUSSION

RESULTS

I. GROWTH RATE

The Bermuda and Carabao grass was being observed in terms of its

diameter and leaf size of the latter in which they were being measured

weekly as shown in Figure 4.1.

CARABAO(Leaf

size)

TRIAL 1

TRIAL 2

TRIAL 3

BERMUDA(diam

eter)

TRIAL 1

TRIAL 2

TRIAL 3

0

50

100

150

200

250

300

CONTROL(in cm)3000 ppm6000 ppm

Figure 1.0. Average Leaf size (Carabao) and Diameter (Bermuda)

In reference to Figure 4.1, the control samples of carabao grass exhibit

almost the same results in growth rate. The 3000 ppm amended sample

show that the leaf size of the carabao grass grows increasingly. On the other

hand, the 6000 ppm, however does not show the same result as with the

first treatment, its leaf size grows increasingly but not as tall as the first

treatment. On the other hand, Bermuda grass shows an almost the same

diameter in both concentrations.

TABLE 1.0. Analysis of Variance: One- Factor ANOVA result of plant growth

Source of

Variation

SS df MS F P-value F crit

Between groups 3611.444 2 1805.722 0.344678 0.713914 3.68232

Within groups 78583 15 5238.867

Total 82194.444 17

F-value<Fcrit (0.345<3.682) = Not Significant

II. ABSORPTION AND ACCUMULATION OF LEAD

The absorption of lead in these grasses through AAS (Atomic

Absorption Spectrometer) shows the following results:

CARABAO GRASS BERMUDA GRASS

Control(mg/kg)

3000 ppm

(mg/kg)

6000 ppm

(mg/kg)

Control(mg/kg)

3000 ppm

(mg/kg)

6000 ppm

(mg/kg)<0.03 5.6 <0.03 <0.03 <0.03 <0.03

%RejP <0.03 0.19% <0.03 <0.03 <0.03

Table 2.0. Absorption of Lead through AAS(Atomic Absorption Spectrophotometer)

In the control samples of each grass it shows that it is <0.03 of lead

concentration absorbed in which it the absorption value of the grass is less

and nearly negligible. The concentration of lead present in the amended

20

samples of Carabao grass show that at 3000 ppm it accumulates 5.6 mg/kg

of lead absorbed at a Rejection percentage of 0.19%(see Theoretical

Background for the formula). In other concentration of lead, Carabao grass

and Bermuda grass shows the same results as to its controlled sample with

<0.03 absorption.

TABLE 3.0. Analysis of Variance: One- Factor ANOVA result of Lead

Absorption

Source of

Variation

SS df MS F P-value F-crit

Between

groups

31.36 2 15.68 5 0.021684 3.68232

Within groups 47.04 15 3.136

Total 78.4 17

F value>F-crit (5>3.68232) = Significant

DISCUSSION

I. GROWTH RATE

The result as shown in Figure 1.0 represents the growth rate of each

grass in a lead contaminant at different parameters in which there is a slight

difference in their leaf size and diameter. According to Hasnain et al. (1995)

and Prodgers and Inskeep (1981), with the increase of concentration and

toxicity of heavy metals the growth of a plant gradually slows down its

21

growth rate which means it is concentration dependent (Miller and Koeppe,

1971). The slight difference of the plants’ growth is due to the limited test

measuring in which only the selected parts of the plants are only measured

(Banzon and Estrera, 2012).

The plant growth varies insignificantly, which implies that these

grasses were tolerant at a certain concentration. Carabao grass easily adapts

the presence of stress and developed resistance to lead without any harm to

its growth and development implying that it is phytotolerant on an elevated

Pb (Environmental Science Pollution Research, 2007). Though carabao

grasses possess this kind of characteristic, but Cunningham and Ow(1996)

exemplifies that heavy-metal stress can also be an attribution to the

inhibition of plant’s growth and mechanisms.

II. ABSORPTION AND ACCUMULATION OF LEAD

The absorption of lead in grasses differs through its rooting system,

since the Carabao grass has vigorous roots so it can absorb more Pb unlike

Bermuda grass. Through this, it emphasizes the relationship between Pb

accumulation and absorption and plant biomass in Pb extraction (Gray

2000). It has a direct relationship on its biomass and accumulation of

metal;which means the more biomass the plant has the more metal it can

accumulate, since the metal uptake is a function of the overall plant biomass

claimed by Gray (2000) and as stated by the Environmental Science

22

Pollution Research (2007).

Once lead has entered the root system it may accumulate there or

translocated to other parts of the plants. For most plant species, the majority

of absorbed lead is accumulated in the roots and only a fraction is

translocated to other parts of the plants as been reported to the studies of

Piechalak et al.2002; Małecka et al.2008; Shahid et al.2011; Kopittke et al.

2007; Gichner et al.2008; Brunet et al. 2009; Gupta et al. 2009; Yan et

al.2010; Gupta et al. 2010; Jiang and Liu 2010. The absorption of lead is less

at 3000 ppm and other concentration is almost negligible, is due to the

translocation of lead in the system in which the part of the grass being

tested is where lead absorption is less.

The uptake of nutrients and beneficial metals of plants is through its

channels, pores, and transporters in its roots. Through it, plants

characteristically have the capacity to absorb what they need and do not.

However, most of vascular plants absorb toxic metals through their roots in

varying degrees from negligible to substantial and sometimes there is

absorption because of the chemical similarity between the beneficial and

toxic chemicals. Some plants utilize exclusion mechanisms, in which there is

a reduced uptake of roots or a restricted transport of the metal from roots to

shoots (Baker 1981).

The characteristic of being a phytoremediating agent is calculated

23

quantitatively through the rejection percentage (%RejP) and recovery

percentage (%RecP), the study only limits on the former percentage. The

percentage of 0.19% of lead in carabao grass at a concentration of 3000

ppm implies on how much lead is being absorbed by the grasses. In some

cases, in which Bermuda grass has 0 rejection percentage, this does not

mean that no lead (Pb) is absorbed but of negligible amount. This implies

that carabao grass is a potential phytoaccumulator of lead at 3000 ppm

concentration while Bermuda grass does not possess the same characteristic

with the latter, based on the result presented.

24

Chapter V

SUMMARY, CONCLUSION AND RECOMMENDATIONS

SUMMARY

This study was conducted to compare the lead accumulation of

Bermuda grass ( Cynodon dactylon ) and Carabao grass ( Paspalum

conjugatum) in two different concentrations. After grown for one month, the

plant where harvested; air dried and prepared using the Acid Digestion

Method. The samples were tested for lead absorption usig the Flame Atomic

Absorption Spectrophotometer (FAAS) and were statistically analyzed using

One Factor ANOVA.

Results showed that only Carabao grass at 3000 ppm accumulates

lead with the amount of 5.6 mg/kg. This result implies that the carabao grass

is an accumulator of lead, due to its vigorous rooting compared to Bermuda

grass.

CONCLUSION

The amount of lead accumulated may have been very negligible due to

the rooting system of each grass. Carabao grass is a potential

phytoremediating agent of lead at a concentration of 3000 ppm while

Bermuda grass does not exhibit the same potentiality.

RECOMMENDATIONS

The researcher’s scope of study limits the duration of experiment

weight of sample used and the equipment used for the analysis and hence,

would suggest the following improvements of this study.

One of the first considerations in this study is its environment; there

must be a consistency in the location to avoid other aspects that can affect

the study. Second, is to prolong the duration of experiment for about 3

months. This is to ensure that there is a clear difference between the amount

concentrations absorbed by these grasses. Second, is to increase the dry

weight of the samples used for analysis since the minimum amount needed

for accurate reading in Flame-AAS is 0.5 g. Making the reading of

accumulated lead increase and not on a negligible amount. Third, is the

equipment used for more accurate reading, for a better reading of the

samples we suggested to use a Graphite Atomic Absorption

Spectrophotometer that can determine metals in quantities as low as 10 -12 g.

Fourth, is the proper disposal of lead with the help of Department of

Environment and Natural Resources (DENR).

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36APPENDIX A

GANNT CHART

MONTH

2012 2013

ACTIVITY

JUN

E

JULY

AU

GU

ST

SEP

TEM

BER

OC

TO

BER

NO

VEM

BER

DEC

EM

BER

JAN

UA

RY

FEB

RU

AR

Y

MA

RC

H

Topic searchingTitle/proposal

makingWrite-ups ( Chapter

1-III )Thesis proposal

hearingPreparation for

experimentPlanting and height

measurement Harvesting and

drying Sample

preparation/AAS Analysis

Analysis of Results Thesis writing

Proof reading and Final Defense

37

APPENDIX B

APPENDIX B.1

PLANT PROFILE

Common Name: Bermuda grass

Scientific Name: Cynodon dactylon

Classification:

Kingdom   Plantae – Plants

Subkingdom   Tracheobionta – Vascular plants

Superdivision   Spermatophyta – Seed plants

Division   Magnoliophyta – Flowering plants

Class   Liliopsida – Monocotyledons

Subclass   Commelinidae

Order   Cyperales

Family   Poaceae – Grass family

Genus   Cynodon Rich.

Species   Cynodon dactylon

38

APPENDIX B.1

PLANT PROFILE

Common Name: Carabao grass

Scientific Name: Paspalum conjugatum

Classification:

Kingdom   Plantae – Plants

Subkingdom   Tracheobionta – Vascular plants

Superdivision   Spermatophyta – Seed plants

Division   Magnoliophyta – Flowering plants

Class   Liliopsida – Monocotyledons

Subclass   Commelinidae

Order   Cyperales

Family   Poaceae – Grass family

Genus  Paspalum L. – crowngrass 

Species   Paspalum conjugatum

39

APPENDIX C

Lead Concentration Accumulated using the Mean of AAS Reading

Formula :

Lead concentration = AAS reading x Volume of Prepared Sample used kg dry weight dry weight used

Control

0 μg x 50 mlLead concentration = mL________ = 0 μg/g = 0 mg/kg Kg dry weight 0.20 g

Amended

0.024 μg x 50 mlLead concentration = mL________ __= 5.6 μg/g = 5.6 mg/kg Kg dry weight 0.20 g

40APPENDIX D

Lead Accumulation & Rejection Percentage

SAMPLESBermuda Grass

Total leadConcentration

Present insoil (ppm )

AccumulatedConcentratio

nin ppm

RejectionPercentage

(%)

CONTROL( 0 ppm )

<0.03 <0.03 <0.03

AMENDED( 3000 ppm )

3000 <0.03 <0.03

AMENDED( 6000 ppm )

6000 <0.03 <0.03

Carabao Grass

CONTROL( 0 ppm )

<0.03 <0.03 <0.03

AMENDED( 3000 ppm )

3000 5.6 0.19

AMENDED( 6000 ppm )

6000 <0.03 <0.03

Input concentration of lead in soil – output concentration of lead in plants

%RejP =------------------------------------------------------------------- X 100Input concentration of lead in soil

41

APPENDIX E

Preparation of 3000 ppm and 6000 ppm Lead nitrate

“Parts per million” – usually abbreviated as “ppm” – means “out of a

million” (ppm) commonly used in measuring small levels of the amount of

pollutants in air, water and body fluids etc.

1 ppm= 1 mg/L =1 mg/kg

Parts per million is the mass

ratio between the pollutant

component and the solution and

ppm is defined as

ppm = 1,000,000 mc / ms        

where

mc = mass of component (kg, lbm)

ms = mass of solution (kg, lbm)

In the metric system ppm can be expressed in terms of milligram versus kg where

1 mg/kg = 1 part per million

42

APPENDIX F

Flame Atomic Absorption Spectrometry (FAAS)

http://www.etslabs.com/images/methods/6.gif

Atomic Absorption Spectroscopy is a technique for determining the

concentration of a particular metal element within a sample. Atomic

absorption spectroscopy can be used to analyze the concentration of over 62

different metals in a solution. (See Table 6 for the Approximate Sensitivity of

some metals in AAS)

43

Element Approximate

Sensitivity (ppm)

Element Approximate

Sensitivity (ppm)

As

Ca

0.50

0.10

Mg

Ni

0.01

0.15

Cd

Co

Cr

Cu

Fe

Pb

0.05

0.20

0.25

0.10

0.15

0.50

K

Ag

Na

Sn

Zn

0.05

0.10

0.05

5.00

0.05

Table 6. Approximate sensitivity, expressed in ppm, for several elements that may be analyzed by flame

44

APPENDIX G

DOCUMENTATION

Watering of grasses with distilled water

Carabao and Bermuda grass(ongoing experiment)

45

CONTROL: Bermuda grass

Amended Samples: Bermuda and Carabao grass

46

Preparation and Filtering of the Samples ready for Reading

47

APPENDIX H

Thesis Expenditures

Distilled water 400

Grasses (Bermuda & Carabao) 250

Gloves 50

Spray 50

Transportation 300

Lead Analysis 5,400

Printing & Bookbinding 500

______________

TOTAL EXPENSES Php 6,950

48

APPENDIX I

RAW DATA of GROWTH RATE

CARABAO GRASS (Leaf size)

CARABAO GRASS1st week 2nd week 3rd week 4th week

TRIAL 1 (in cm)Control 55 55 55 553000 ppm 55 68 98 1056000 ppm 55 65 76 85TRIAL 2 (in cm)Control 60 60 60 603000 ppm 60 79 98.5 1256000 ppm 60 68.9 79 89TRIAL 3 (in cm)Control 55 55 55 553000 ppm 55 76.6 99.7 1156000 ppm 55 58.4 67.4 75

BERMUDA GRASS (Diameter)

BERMUDA GRASS1st week 2nd week 3rd week 4th week

TRIAL 1 (in cm)Control 190 190 190 1903000 ppm 190 160 144 1466000 ppm 190 160 144 145TRIAL 2 (in cm)Control 198 198 198 1983000 ppm 198 210.4 236 2506000 ppm 198 219.6 243 250TRIAL 3 (in cm)Control 197 197 197 1973000 ppm 197 210.8 200.3 2206000 ppm 197 200.9 220 240

49

APPENDIX J

RESULT of ANALYSIS through AAS(Carabao Grass)

50

APPENDIX K

RESULT of ANALYSIS through AAS

(Bermuda Grass)

51

Hannie Lou Faisan AnocTabok,Mandaue City,Cebuhannieloua@yahoo.com

09423483079/09106429083

PERSONAL INFORMATION

Date of Birth : March 7, 1993Civil Status : SingleNationality : FilipinoReligion : Roman Catholic

EDUCATIONAL ATTAINMENT

College : BS Chemistry-Physics Cebu Normal University Osmeña Blvd.,Cebu City

Secondary : Mandaue City Comprehensive National High School Plaridel St.,Reclamation Area,Mandaue City ,Cebu

JOB EXPERIENCES

Student Assistant Physics Laboratory-Cebu Normal University June 2011-March 2013

On-the-Job Trainee Department of Agriculture May 2011, Mandaue Experiment Station

o Soils Laboratoryo Pesticide Analytical Laboratory

May 2012, M.Velez, Cebu Cityo Regional Feeds Laboratory

POSITIONS HELD

Class Secretary, BS Chemistry-Physics – Cebu Normal University Batch 2009-2013

Member,Association of Student Assistants – Cebu Normal University 2011-2013

52ORGANIZATIONS INVOLVED

Council of Liberal Arts & Sciences (C.L.A.S.S.) – Cebu Normal University

Junior Physics and Chemistry Society (JPACS) – Cebu Normal University

Lector’s Ministry – National Shrine of St.Joseph

53Sherlice Quiros Rom

Cogon,Maslog,Danao City,Cebusherlyalicerom@gmail.com

09106488759/09323013004

PERSONAL INFORMATION

Date of Birth : December 2, 1993Civil Status : Single

Nationality : FilipinoReligion : Roman Catholic

EDUCATIONAL ATTAINMENT

College : BS Chemistry-Physics Cebu Normal University Osmeña Blvd.,Cebu City

Secondary : Compostela National High School Poblacion,Compostela,Cebu

JOB EXPERIENCES

Student Assistant,Physics Laboratory-Cebu Normal University November 2009-October 2011

Student Assistant,Chemistry Laboratory-Cebu Normal University November 2011-March 2013

On-the-Job Trainee –Cebu Provincial Capitol Laboratories May 2011

o Provincial Engineering’s Laboratoryo Provincial Agriculture’s Laboratoryo Provincial Veterinary’s Laboratoryo Provincial Water Analysis Laboratory

On-the-Job Trainee- Regional Feeds Laboratory May 2012

54

POSITIONS HELD

Alumni President,Batch 2009 - Compostela National High School 2011-Present

Class Mayor – Cebu Normal University 2009-2011

Treasurer,Junior Physics and Chemistry Society- Cebu Normal University 2011-2013

Member,Association of Student Assistants – Cebu Normal University 2009-2013

ORGANIZATIONS INVOLVED

Council of Liberal Arts & Sciences (C.L.A.S.S.) – Cebu Normal University

Junior Physics and Chemistry Society (JPACS) – Cebu Normal University

Parish Pastoral Youth Council (PPYC) – St. Francis of Assisi Parish

55