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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,[email protected]
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,[email protected]
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