REMEDIATIVE ABILITIES OF COWPEA (Vigna unguiculata ...

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REMEDIATIVE ABILITIES OF COWPEA (Vigna

unguiculata), BAMBARA GROUNDNUT (Vigna

subterranean) AND MAIZE (Zea mays) ON SOILS

POLLUTED WITH LEAD AND ZINC

BY

OMITIRAN, ESTHER OLUWASAYO

B.Sc. (Ed) ILORIN; M.Sc., LAGOS

A THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE

STUDIES OF THE UNIVERSITY OF LAGOS, AKOKA, LAGOS,

NIGERIA FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D)

DEGREE IN CELL BIOLOGY AND GENETICS

NOVEMBER, 2012

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REMEDIATIVE ABILITIES OF COWPEA (Vigna

unguiculata), BAMBARA GROUNDNUT (Vigna

subterranean) AND MAIZE (Zea mays) ON SOILS


BY


B.Sc. (Ed) ILORIN; M.Sc., LAGOS

A THESIS SUBMITTED TO THE SCHOOL OF POST GRADUATE

STUDIES OF THE UNIVERSITY OF LAGOS, AKOKA, LAGOS,

NIGERIA FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D)

DEGREE IN CELL BIOLOGY AND GENETICS

3

NOVEMBER, 2012

SCHOOL OF POSTGRADUATE STUDIES

UNIVERSITY OF LAGOS

CERTIFICATION

This is to certify that the thesis:

REMEDIATIVE ABILITIES OF COWPEA (Vigna unguiculata), BAMBARA

GROUNDNUT (Vigna subterranean) AND MAIZE (Zea mays) ON SOILS


Submitted to the School of Postgraduate Studies

University of Lagos

For the award of the degree of

DOCTOR OF PHILOSOPHY (Ph. D)

is a record of original research carried out

By


in the Department of Cell Biology and Genetics

___________________ ___________________ ____________________

AUTHOR’S NAME SIGNATURE DATE

___________________ ____________________ ___________________

1st SUPERVISOR’S NAME SIGNATURE DATE

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2nd SUPERVISOR’S NAME SIGNATURE DATE

___________________ ____________________ ___________________

1st INTERNAL EXAMINER SIGNATURE DATE

___________________ ____________________ ___________________

2nd INTERNAL EXAMINER SIGNATURE DATE

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___________________ ____________________ ___________________

EXTERNAL EXAMINER’S NAME SIGNATURE DATE ___________________ ____________________ ___________________

SPGS REPRESENTATIVE SIGNATURE DATE

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DEDICATION

To the living God, by whose grace, this piece of work is completed and by whom we all have our being.

You are worthy to be praised and adored.

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ACKNOWLEDGEMENT

I wish to express my sincere gratitude to my supervisor, Prof. P.G.C. Odeigah for his tolerance,

fatherly advice and firm discipline which made this piece of work a success. May His countenance

always shine upon you. To my second supervisor, Dr. I. A. Taiwo for his support and valuable

comments, your encouragement and advice helped a great deal, thank you sir. My gratitude goes to

Professor M.O Akinola and Professor O.Oboh for their valuable instructions, great concern and

insightful comments, thank you and God bless. To Professor Egonmwan, for her motherly advice,

thank you ma. I also sincerely appreciate Professor Ilori and Professor Olowokudejo for their valuable

advice.

My sincere gratitude also goes to Dr. L.A. Ogunkanmi and Dr. K. L Njoku, for their support and great

help. My gratitude also goes to Mr. O. Adebesin, Mr. Begusa, Mr. Adelabu, Mr.Obu , Mr Sunday

Adenekan (Biochemistry Department, Unilag) and my colleague Mr. T. Yahaya for their help and

support.

My sincere appreciation also goes to my sweetheart Oluwasanmisirere; a rear gem (your push went a

long way) and my son, Ore-ofeoluwa; a great relief through the grace of God. My appreciation also

goes to my parents, Overseer and Deaconess G.S. Omitiran for their love, encouragement and support.

To my siblings, Rebecca, Bukky, Ife and Adeola you are all special jewels, I say thank you and God

bless.

And to all my loved ones who cared to ask about the progress of this work. I say thank you.

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TABLE OF CONTENTS

CONTENTS PAGE

Title page i-ii

Certification iii

Dedication iv

Acknowledgement v

Table of Contents vi-x

List of Tables xi-xiii

List of Figures xiv

List of Plates xv-xvi

Abstract xvii-xviii

CHAPTER ONE: INTRODUCTION

1.0 Introduction 1

1.1 Background to the study 1

1.2 Statement of problem 5

1.3 Aim of the Study 5

1.4 Objectives of Study 5

1.5 Significance of the study 6

1.6 Operational Definition of Terms 7

1.7 List of Abbreviations and Acronyms 8

CHAPTER TWO: LITERATURE REVIEW

2.0 Literature review 9

2.1 Phytoremediation as a technique in soil management 9

2.1.1 Merits of Phytoremediation 12

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2.1.2 Demerits of Phytoremediation 12

2.1.3 Limitation of Phytoremediation 13

2.1.4 Mechanisms of Metal Sequestration/ Uptake 13

2.2 Cowpea (Vigna unguiculata) 16

2.3 Bambara groundnut (Vigna subterranean) 16

2.4 Maize Plant (Zea mays) 17

2.5 Heavy metals in the Environment and Plant Growth 18

2.6 Genotoxicity of Heavy Metals 20

2.7 Oxidative stress/ Reactive oxygen species/ Free Radicals 22

2.7.1 Oxidative Stress 22

2.7.2 Chemical and Biological Effects of Oxidative stress 22

2.7.3 Free radicals 23

2.8 Enzymatic antioxidants and Metals Tolerance 23

2.8.1 Superoxide dismutases (SODs) 23

2.8.2 Catalases 23

2.8.3 Glutathione Synthetase (GSH) 24

2.9 RAPD-PCR Analysis 24

CHAPTER THREE: MATERIALS AND METHODS

3.0 Materials and Methods 26

3.1 Materials 26

3.1.1 Sources and Collection of Test plants 26

3.2 Study sites 30

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3.3 Methods 30

3.3.1 Determination of metal salt and chelant concentration 30

3.3.2 The Screening test 30

3.3.3 Soil Analysis before planting 31

3.3.3.1 Soil Characteristics determination 33

3.3.4 Cytological Study 36

3.3.4.1 Seed Germination for cytological procedure 36

3.3.5 Experimental Description 37

3.3.6 Macromorphometric Studies on Treated and Untreated Plant Samples 38

3.3.7 Determination of Chlorophyll content in Treated and Untreated Plant Samples 40

3.3.8 Biochemical Analysis 41

3.3.8.1 Determination of Superoxide Dismutase (SOD) Activity 41

3.3.8.2 Determination of Glutathione (GSH) Activity 42

3.3.8.3 Determination of Catalase (CAT) Activity 42

3.3.8.4 Determination of Product of Lipid Peroxidation (MDA) 43

3.3.9 Sample preparation and Determination of Heavy Metals in 44

Treated plant samples and soils

3.3.9.1 Plant Sample preparation and Digestion 45

3.3.9.2 Digestion of soil samples 45

3.3.9.3` Determination of Heavy Metals in the Digested Plant and Soil Samples 45

using Atomic Absorption Spectrophotometer (AAS)

3.3.9.4 Determination of the Translocation factor/ bioconcentration factor

and the Plant-soil coefficient. 47

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3.3.10 Genomic DNA Isolation for PCR Analysis 47

3.3.10.1 Reagents and Chemicals 47

3.3.10.2 DNA Extraction 47

3.3.10.3 DNA Quantification and Quality Analysis using Agarose Gel Electrophoresis 48

3.3.11 Statistical Analysis 49

CHAPTER FOUR: RESULTS

4.0 Results 50

4.1 The Screening Test 50

4.1.1 Percentage Germination 50

4.2 Macromorphometric parameters and Nutritional Evaluation following 56

Metal treatment and augmentation

4.2.1 Leaf Aea/Size, Stem length, Root length,Fresh and Dry weight characteristics 56

4.2.2 Chlorophyll content 74

4.3 Effects of Heavy Metals on the enzymatic activities and Lipid Peroxidation 78

of treated plants.

4.3.1 Effects of Heavy Metals on the Enzymatic activities and Lipid Peroxidation 78

of Treated and Untreated Cowpea plants.


of Treated and Untreated Bambara groundnut plants.


of Treated and Untreated Maize plants

4.4 Cytological Study 84

4.4.1 Micrographs of cells showing normal mitosis in the control plants and 88

aberrations in the heavy metal treated plants

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4.5 Phytoremediation Study 96

4.6 Soil sample analysis after planting 109

4.7 Influence of Heavy metals on plant’s DNA with and without EDTA and 111

Manure augmentation

4.7.1 RAPD (Random Amplified Polymorphic DNA) Analysis 111

4.7.2 Coefficient of Similarity among the Control and Treated Plants 117

CHAPTER FIVE: DISCUSSION

5.0 Discussion 119

5.1 Summary 130

5.2 Summary of Findings 131

5.3 Contribution to Knowledge 132

5.4 Recommendation 133

5.5 Future Research 134

References 135

Appendices 152

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LIST OF TABLES

Table Title Page

3.1 Physico-chemical characteristics of soil sample used for the experiment 32

4.1 Percentage Germination of Cowpea seeds (Vigna unguiculata) Accession Tvu 3788 and

Cowpea seeds (Vigna unguiculata) Accession IT99K-377-1 in different Concentrations of lead

and zinc nitrate. 53

4.2 Percentage Germination of Bambara groundnut seeds (Vigna subterranea) Accession

Tvsu 1685 and Bambara groundnut seeds (Vigna subterranea) Accession Tvsu 102 in different

Concentrations of lead and zinc nitrate. 54

4.3 Percentage Germination of Maize seeds (Zea mays) Accession DMR-LSRW and Maize seeds (Zea

mays) Accession ACR.91SUWANI-SRC1 in different concentrations

of lead and zinc nitrate. 55

4.4 Effects of Different Concentrations of Lead and Zinc Nitrate on

Leaf Area (cm2) of Cowpea (Vigna unguiculata) 58

4.5 Effect of Different concentrations of Lead and Zinc Nitrate on Leaf Area (cm2)

of Bambara groundnut (V.Subterranea ) 59

4.6 Effect of Different concentrations of Lead and Zinc Nitrates on Leaf Area (cm2)

of Maize (Zea mays) 60

4.7 Effect of Different concentrations of Lead and Zinc Nitrates on Stem Length (cm)

of Cowpea (Vigna unguiculata) 61

4.8 Effect of Different concentrations of Lead and Zinc Nitrates on Sem Length (cm)

of Bambara groundnut (V.subterranea ) 62

4.9 Effect of Different concentrations of Lead and Zinc Nitrates on Stem Length (cm)

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of Maize (Zea mays) 63

4.10 Effect of Different concentrations Lead and Zinc Nitrates on RootLength (cm) 64

of Cowpea (Vigna unguiculata)

4.11 Effect of Different concentrations of Lead and Zinc Nitrates on Root Length (cm) 65

of Bambara groundnut (V.subterranea )

4.12 Effect of Different concentrations of Lead and Zinc Nitrates on Root Length (cm) 66

of Maize (Zea mays)

4.13 Effect of lead concentrations on mean fresh weight (grams) of the three test plants 70

4.14 Effect of zinc concentrations on mean fresh weight (grams) of the three test plants 71

4.15 Effect of lead concentrations on mean dry weight (grams) of the three test plants 72

4.16 Effect of zinc concentrations on mean dry weight (grams) of the three test plants 73

4.17 Effect of lead concentrations on Total chlorophyll (mg/g) of the three test plants 76

4.18 Effect of zinc concentrations on Total chlorophyll (mg/g) of the three test plants 77

4.19 The Effect of different concentrations of lead and zinc nitrate on some 79

enzymes and Lipid peroxidation in Cowpea

4.20 The Effect of different concentrations of lead and zinc nitrate on some enzymes 81

and Lipid peroxidation in Bambara groundnut

4.21 The Effect of different concentrations of lead and zinc nitrate on some enzymes 83

and Lipid peroxidation in Maize

4.22 Chromosome Aberrations in Cowpea root tips of control and treated plants 85

in different concentrations of lead and zinc nitrate

4.23 Chromosome Aberrations in Bambara groundnut root tips of control and treated 86

plants in different concentrations of lead and zinc nitrate

4.24 Chromosome Aberrations in Maize root tips of control and treated plants in different 87

concentrations of lead and zinc nitrate

4.25 Concentration of lead (Pb) (mg/kg) in the roots and shoots of cowpea after metal treatment and

augmentation 97

4.26 Concentration of lead (Pb) (mg/kg) found in the roots and shoots of bambara groundnut after

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metal treatment and augmentation 98

4.27 Concentration of lead (Pb) (mg/kg) found in the roots and shoots of maize after metal treatment and

augmentation 99

4.28 Concentration of zinc (Zn) (mg/kg) found in the roots and shoots of cowpea after metal treatment

and augmentation 100

4.29 Concentration of zinc (Zn) (mg/kg) found in the roots and shoots of bambara groundnut after metal

treatment and augmentation 101

4.30 Concentration of zinc (Zn) (mg/kg) found in the roots and shoots of maize after metal treatment and

augmentation 102

4.31 Soil Parameters Analyzed after the Remediation Experiment 110

4.32 Primer Sequence for the Control and Treated Plant Samples 112

4.33 Number of Monomorphic, Polymorphic bands and Polymorphism Percentage

produced by each RAPD primer for the Control and some Treated Plant Samples 116

4.34 Total number of bands by all the Primers and the Coefficient of Similarity 118

among Control and Some Treated plants

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LIST OF FIGURES

Figure Title Pages

3.1 Experimental Layout 39

4.1 Percentage Pb accumulation within plants treated with 200mg/kg lead nitrate 103

4.2 Percentage Pb accumulation within plants treated with 200mg/kg lead nitrate and EDTA 104

4.3 Percentage Pb accumulation within plants treated with 200mg/kg lead nitrate and Manure 105

4.4 Perecntage Zn accumulation within plants treated with 200mg/kg zinc nitrate 106

4.5 Perecntage Zn accumulation within plants treated with 200mg/kg zinc nitrate and EDTA 107

4.6 Perecntage Zn accumulation within plants treated with 200mg/kg zinc nitrate and Manure 108

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LIST OF PLATES

Plate Title Page

3.1 Cowpea, Vigna unguiculata (Cultivar IT 99K-377-1) 27

3.2 Cowpea, Vigna unguiculata (Cultivar Tvu 3788) 27

3.3 Bambara groundnut, Vigna subterranea (Tvsu102) 28

3.4 Bambara groundnut, Vigna subterranea (Tvsu1685) 28

3.5 Maize, Zea mays (Cultivar DMR-LSRW) 29

3.6 Maize, Zea mays (ACR.91SUWANI-SRC1) 29

4.1 Anaphase observed in control Cowpea 88

4.2 Anaphase observed in control Bambara groundnut 88

4.3 Anaphase observed in control maize 89

4.4 Early telophase with observed in control maize 89

4.5 Vagrants in cowpea treated with 25mg/L lead nitrate 90

4.6 Stickiness at telophase observed in cowpea treated with 25mg/L lead nitrate 90

4.7 Anaphase bridge and laggard chromosome observed in cowpea treated 91

with 25mg/L lead nitrate

4.8 Vagrant observed in cowpea treated with 50mg/L zinc nitrate 91

4.9 Scattered chromosomes observed in bambara groundnut treated with 100mg/L

Lead nitrate 92

4.10 Vagrant chromosomes at metaphase observed in bambara groundnut treated with 25mg/L zinc

nitrate 92

4.11 Vagrant and bridged anaphase observed in maize treated with 25mg/L

lead nitrate 93

4.12 Sticky chromosomes at telophase observed in maize treated with 50mg/L

zinc nitrate 93

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4.13 Vagrant observed in maize treated with 50mg/L lead nitrate 94

4.14 Laggard at telophase observed in maize treated with 100mg/L zinc nitrate 94

4.15 Vagrant at metaphase observed in maize treated with 100mg/L zinc nitrate 95

4.16 Fragmented chromosomes at metaphase observed in maize treated with

25mg/L lead nitrate 95

4.17 RAPD-PCR Amplification based on the use of Primer OPJ-12 on the

Plant Samples Genotypes 113

4.18 RAPD-PCR Amplification based on the use of Primer OPJ-13 on the


4.19 RAPD-PCR Amplification based on the use of Primer OPO-08 on the


4.20 RAPD-PCR Amplification based on the use of Primer OPO-09 on the


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ABSTRACT

Accessions Tvu 3788 and IT 99K-377-1, Tvsu 102 and Tvsu 1685 and ACR.91SUWANI-SRC1 and

DMR-LSRW of Cowpea (Vigna unguiculata), Bambara groundnut (Vigna subterranean) and Maize

(Zea mays) respectively were subjected to lead (Pb) and zinc (Zn) treatment and screened for their

ability to tolerate these metals. Accessions Tvu 3788, Tvsu 102 and ACR.91SUWANI-SRC1 of

cowpea, bambara groundnut and maize respectively were chosen and used for the experiment. The

toxic effects of lead and zinc on the growth and development and biochemical activities of the three

crop plants were carried out. The genotoxic effect of the heavy metals (Pb) and (Zn) on cowpea,

bambara groundnut and the maize crop was investigated. The potential of cowpea, bambara groundnut

and maize in the remediation of lead and zinc from polluted soils as well as the effects of

Ethylenediamine tetra acetic acid (EDTA) and farmyard manure on the remediative abilities of the

crop plants were also investigated. The genetic variability for lead and zinc tolerance by the crop

plants were also determined.

Fresh and dry weights of plants were obtained using a weighing balance and by oven-drying. The

chlorophyll content was determined by spectrophotometry. Genotoxicity was determined by

chromosomal squash technique with lactic acetic orcein stain. The amount of metals contained in

plants’ tissues was determined by Atomic Absorption spectrophotometry. RAPD-PCR analysis for

determining genetic variation of plants was also carried out. One-way ANOVA and student’s t-test

using Microcal origin 5.0 software procedures were carried out on the morphological and biochemical

parameters.

A statistically significant difference (P<0.05) between control and treated plants was observed for the

morphological parameters, chlorophyll content and enzyme activity of the three test plants. Lead and

zinc caused a reduction in the mitotic index of treated plants. A statistically significant difference

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(P<0.05) between control and treated plants was observed. For instance, the mitotic index for cowpea

were 2.70 ± 0.83, 0.50 ± 0.33 and 1.20 ± 0.52 for the control, 50mg/L of Pb and 50mg/L of Zn

respectively. At lower concentrations of 25mg/L, bridges, vagrant and laggard chromosomes were

observed, whereas at higher concentrations of 100mg/L, sticky chromosomes were the most common

especially in plants treated with lead. Maize being able to translocate these metals especially lead

through the vascular system act as a phytoextractor while cowpea could be employed for

phytoextraction of zinc for translocating it through the vascular system probably by mechanisms such

as uptake and metal redistribution to various tissues in the shoot through phloem and xylem transport.

Bambara groundnut displayed the property of a metal excluder by immobilizing these metals at its root

zone, possibly through mechanisms such as adsorption and accumulation in roots by vacuole

sequestration, cell wall binding and complex formation by root exudates. When cowpea, bambara

groundnut and maize were treated with 100 mg/kg of Pb, the total percentage of Pb accumulated

within their tissues were 54.24%, 49.79% and 62.85% respectively. When treated with lead and

augmented with manure, the total percentages of Pb accumulated within plants’ parts were 49.24%,

30.57% and 82.14% for cowpea, bambara groundnut and maize respectively. However, when treated

with 100mg/kg of Pb and augmented with EDTA, 65.27%, 58.48% and 70.64 % of Pb was

accumulated for cowpea, bambara groundnut and maize respectively. It is therefore suggested that for

better removal of Pb, cowpea and bambara groundnut may be assisted with EDTA while maize be

assisted with manure. RAPD-PCR analysis, revealed decrease and increase in the total number of

bands of treated plants compared to their control. However, the close values obtained from their co-

efficient of similarity and regions of cluster showed the effects of metal treatment to be minimal on the

DNA of treated plants for these concentrations tested.

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It is suggested that metal treated plants be composted and the compost especially for zinc be applied to

Zn-deficient soil. The metal treated plants can also be incinerated.

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CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the Study

Phytoremediation has proven to be quite effective in the removal of metal ions from the soil in an

environmentally-friendly manner (Kalay et al., 1999). The major advantages of phytoremediation over

conventional treatment methods include low cost, high efficiency of metal removal from dilute

solution, minimization of chemicals, no additional nutrient requirement and the possibility of metal

recovery (Kalay et al., 1999). Phytoremediation (such as phytoextraction and phytostabilization) of

soils polluted by heavy metals has been accepted as a cost-effective and environmentally-friendly

cleanup technology (Kalay et al., 1999). Potentially useful approaches to phytoremediation of heavy

metal-polluted soils include (i) phytoextraction (phytoaccumulation and phytosequestration) – the use

of plants to remove metals from soils and to transport and concentrate them in above-ground biomass,

(ii) phytostabilization – the use of plants to minimize metal mobility in contaminated soil through

accumulation by roots or precipitation within the rhizosphere, (iii) phytofiltration – the use of plant

roots (rhizofiltration) or seedlings (blastofiltration) to absorb or adsorb pollutants, mainly metals, from

groundwater and aqueous-waste streams rather than the remediation of polluted soils, and (iv)

phytovolatilization – the use of plants to turn metals into gaseous forms ( McGrath et al, 2001;

Garbisu and Alkorta, 2001; Lasat, 2002 and Ghosh and Singh 2005).

Cowpea (Vigna unguiculata) is a popular leguminous staple food in Nigeria. It constitutes an

important source of dietary protein and secondary staple carbohydrate (Adelaja, 2000; Adaji et al.,

2007). Cowpea is grown mainly for its protein-rich grains and quality fodder for livestock. Minerals

like Sodium (Na), Potassium (K),Magnesium (Mg), Calcium (Ca), Phosphorus (P), Cobalt (Co), Iron

(Fe), Copper (Cu), Manganese (Mn), Cadmium (Cd), Zinc (Zn) and Chromium (Cr) have been found

in many soils where cowpea is cultivated (Sebetha et al., 2010). Islam et al., (2006) reported that

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cowpea is more tolerant to drought, infertile soils and acid stress than common beans. It is a semi-arid

crop adaptable to a wide range of geographical and environmental conditions including poor soil and

limited rainfall due to its well developed taproots, which can grow to a depth of about 1 m (Islam et

al., 2006).

Bambara groundnut (Vigna subterranea) is a leguminous seed crop grown in semi arid parts of Africa

on a small scale. The plant originated in West Africa. It can withstand drought, resist pests and

diseases and is able to thrive well in soils that are not fertile. The seeds can be eaten unripe or stored as

dried pulse for later consumption (National Research Council, 2006). The Bambara groundnut is a

member of the family Fabaceae. According to some authors it is Voandzeia subterranea, but others

place it in the genus Vigna. Bambara groundnut is a traditional food plant in Africa with the potential

to improve nutrition, boost food security, foster rural development and support sustainable land care

(National Research Council, 2006). According to Adu-Dapaah and Sangwan (2004), the seed is

considered as a balanced food because when compared to similar food legumes, it is rich in iron and

contains high lysine and methionine. Bambara groundnut is known to contain 63 % carbohydrates, 18

% oil and the fatty acid content is predominantly linoleic, palmitic and linolenic acids (Minka and

Bruneteau, 2000). Bambara groundnut is eaten in several ways and at different stages of maturation.

The young fresh seeds may be boiled and eaten as a snack in a manner similar to boiled peanuts and

could be made into pudding locally called “Moin Moin” or “Okpa” (bean porridge) in some parts of

Nigeria. Brough et al., (1993) reported that in Zambia, bambara groundnut is used to make bread

while Poulter and Caygill, (2006) also noted its use in milk making.

Maize (Zea mays) is the most important staple cereal in Nigeria after sorghum and millet and it has the

widest geographical spread in terms of production and utilization among cereals (Omoloye, 2009).

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Maize is grown in all parts of the country, though slightly more in the savannah belt of the country.

Maize exist in different colours, textures, shapes and sizes. The common types are white, yellow and

red. All parts of the crop can be used for food and non-food products (I.I.T.A, 2010). Maize or corn is

a versatile cereal crop that is grown widely throughout the world in a range of agro-ecological

environments. Maize particularly is a widely cropped annual cereal that grows rapidly, produces

extensive fibrous root system with large biomass, withstands adverse conditions and produces

abundant seeds with ease of cultivation under repeated cropping (Garbisu and Alkorta, 2001; Zhang et

al., 2007).

Heavy metals, according to Ademoroti (1996) are defined as metals with density greater than 5g/cm3.

These include transition metals and metals with higher atomic weights of groups III to V of the

periodic table. Metals get to plants from various sources such as the earth’s crust, soil erosion, mining,

industrial discharge, urban runoff, sewage effluents, air pollution fallout and pest or disease control

agents applied to them. These heavy metals are passed through the food chain and are toxic as a result

of bioaccumulation and bio-magnification. Heavy metals become pollutants when the quantity

present in living organism is greater than the tolerable quantity for good functioning of the system

(Ademoroti, 1996). Unlike many organic pollutants that can be eliminated or reduced by chemical

oxidation technique or microbial activity, heavy metals will not degrade (Cline and Reed, 1998).

Information relating to the rate and absolute quantity of heavy metals deposited within a living

organism is necessary for better understanding of their long-term effects.

Heavy metals are the most hazardous pollutants as they are non-degradable and get accumulated and

become toxic both to plants and animals (Ademoroti, 1996). Among heavy metals, lead and zinc are

the major contaminants found in soil, sediments, air and water. Lead can remain in the environment

for 150-5000 years. Once in water, it enters the food chain and adversely affects the flora and fauna.

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Tomar et al., (2000) reported that increased level of lead in soil caused significant reduction in plant

height, root-shoot ratio, dry weight, nodule per plant and chlorophyll content in Vigna radiata.

In animals, heavy metals most especially lead (Pb) and zinc (Zn) have being known to damage nerves,

liver, kidney and bones, and also block functional groups of vital enzymes (Wang, 2002). For over a

decade, crops grown in heavy metal-contaminated soils are an important route for these toxic

pollutants to enter the human food chain. Thus, researchers have been looking for cheaper and more

effective methods of remediating heavy metal-contaminated soils. Pollution by these metals in soils

has become an increasing environmental problem and this will continue unless remediation techniques

are developed by identifying the versatile crop plant which has the capacity to bioaccmulate /

biotransform or remediate the soil.

This study investigated the level of tolerance and removal of lead (Pb) and zinc (Zn) by three (3)

crops: Cowpea (Vigna unguiculata), Bambara groundnut (Vigna subterranea) and Maize (Zea mays).

The crops were exposed to three concentrations (100, 150 and 200 mg/kg) of each metal salt during

the study. Three approaches were used for phytoremediation of heavy metals in this study namely:

natural phytoextraction (without amendment), soil organic amendment using farm yard manure and

chemical enhancement using Ethylene diamine tetra-acetate acid (EDTA).

1.2 Statement of the Problem

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• Heavy metal pollution has caused damage to both animals and plants’ growth and

development.

• Weeds which are difficult to manage and are of little or no economic value have usually been

used in remediation. There is a need to investigate the usefulness of more economically

valuable plants such as legumes (cowpea and bambara groundnut) and cereals (maize) in

remediation.

• Over 400 taxa of plant hyperaccumulators of heavy metals have been identified; most are

exotic species and are low biomass producers. There is, thus, the need to add to the list of

plants available for phytoremediation. Generally, native species are preferred to exotic plants,

which can be invasive and endanger the balance of the ecosystem.

• Some remediation techniques (such as excavation) are not eco-friendly and are more laborious

to achieve, hence the need for phytoremediation.

1.3 Aim of the Study

The overall aim is to identify accessions of the crop plants studied that are more tolerant to heavy

metal pollution.

1.4 Objectives of this Study are to:

1. Investigate the effects of the Pb and Zn on the growth, development and enzymatic activities of

cowpea, bambara groundnut and the maize crop to be used for remediation.

2. Investigate the genotoxic effect of the heavy metals (Pb and Zn) on cowpea, bambara

groundnut and maize crop.

3. Evaluate the potential of cowpea, bambara groundnut and the maize crop when not supported

with the amendments- EDTA and farm yard manure as well as the impact of the amendments

on the crop plants in the remediation of heavy metals polluted soils.

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4. Determine the genetic tolerance for lead and zinc by the different crop plants and establish

molecular markers associated with heavy metal tolerance.

1.5 Significance of the Study

The research is initiated to determine the efficacy of three important food crops in heavy metal

remediation. The use of amendments such as farmyard manure and EDTA will enhance the ability of

the crop plants in removing heavy metals removal from the soil. Furthermore, there is the need to add

to the list of plants that are used for the removal of heavy metals from polluted soils.

1.6 Operational Definition of Terms

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Heavy metals: Are metals with density greater than 5g/cm3. They include transition metals and

metals with higher atomic weights of groups III to V of the periodic table of elements.

Genotoxicity: The degree to which something causes damage to or mutation of DNA

Vagrant: Random movement displayed by distorted chromosomes

Stickiness: The lumping together of chromosomes thereby preventing their migration to the poles

Bridge: Chromosomes linking up and not separating especially at anaphase.

Primer: A short sequence of RNA that is made before DNA formation can proceed.

Phytoremediation: The use of green plants to remove pollutants from the environment or render them

harmless.

Phytoextraction: Use of green plants to remove metals or organics from soils by concentrating them

in the harvestable parts.

Phytostabilization: The use of plants to reduce the bioavailability of pollutants in the environment.

Metal excluders: Green plants capable of retaining metals at their root zones or within the soils.

The Plant-Soil Coefficient (PSC): This is the ratio of metal (whole plant) / metal (soil).

The translocation factor (TF): The ratio of metal (shoot) / metal (root) within plants.

Bioconcentration factor (BCF): This is the ratio of metal (root) / metal (soil).

Bioaccumulation factor (BAF) : This is the ratio of metal (shoot) / metal (soil).

Bioconcentration: Building up of contaminants within the tissues of a living organism.

Mitotic index (M/I): The fraction of cells undergoing mitosis in a given sample.

30

Superoxide dismutases (SODs): A class of closely related enzymes that catalyze the breakdown of

the superoxide anion into oxygen and hydrogen peroxide.

Glutathione synthetase (GSH): An enzyme that plays several important roles in the defense of plants

against environmental threats.

Malondialdehyde (MDA): A naturally occurring product of lipid peroxidation and prostaglandin

biosynthesis that is mutagenic and carcinogenic. It reacts with DNA to form adducts to

deoxyguanosine and deoxyadenosine.

Chlorosis: Yellowing of leaves and other tissues due to loss of chlorophyll.

Synthetase: Enzyme that catalyses the biosynthesis of a compound requiring the concurrent breaking

of a disulphide bond in a triphosphate such as adenosine triphosphate.

1.7 List of Abbreviations and Acronyms

F.E.P.A: Federal Environmental Protection Agency

DPR-EGASPIN: Department of Petroleum resources-Environmental Guidelines and Standards for the

Petroleum industry, Nigeria

NJDEP: New Jersey Department of Environmental Protection

USEPA: United States Environmental Protection Agency

N.R.C: National Research council

I.I.T.A: International Institute of Tropical Agriculture

PCR: Polymerease chain reaction

RAPD: Random Amplified Polymorphic DNA

N.A.P.E.P: National Poverty Eradication Programme

N.D.E: National Directorate of Employment

EDTA: Ethylene diamine tetra-acetate

31

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Phytoremediation as a Technique in Soil Management

Heavy metals, unlike organic pollutants, cannot be chemically degraded or biodegraded by

microorganisms. An alternative biological approach to deal with this problem is phytoremediation,

that is, the use of plants to clean up polluted waters and soils (Salt et al., 1995). This cost-effective

plant-based approach to remediation takes advantage of the remarkable ability of plants to concentrate

elements and compounds from the environment and to metabolize various molecules in their tissues.

Toxic heavy metals and organic pollutants are the major targets for phytoremediation. Some

phytoremediation mechanisms such as rhizosphere accumulation, complexation (phytostabilization),

phytoextraction and volatilisation by leaves (phytovolatilization) have been described by Nascimento

et al., (2006). However, progress in this field is hindered by lack of understanding of the chemically-

assisted phytoextraction with cereals and legumes which increases metal bioavailability, translocation

and accumulation by plants (Lombi et al., 2001). Plants have long adapted the traits necessary to

survive in a wide variety of stressful environments-including areas of high salinity, extreme heat,

drought and freezing temperatures (Bentjen, 2002).

Plants are especially useful in the process of phytoremediation because they prevent erosion and

leaching which can spread the toxic pollutants to surrounding areas (USEPA, 2001). Removal of

heavy metals from polluted sites can be done by phytoextraction and phytostabilization (Kumar et al.,

1992). Phytoextraction of metals take place in stages. The basic mechanism of phytoextraction

involves mobilization of ions, uptake and sequestration in roots, xylem transport, redistribution to

various tissues in the shoot through phloem, trafficking and sequestration in the shoot (Hooda, 2007).

32

A good phytostabilizing plant should have low shoot metal accumulation to eliminate the necessity to

treat harvested shoot residues as hazardous wastes, tolerate high levels of heavy metals and

immobilize these metals in the soil through root uptake and precipitation. Hooda, (2007) also

described the basic mechanism of phytostabilization to include adsorption, absorption and

accumulation in roots by vacuole sequestration and cell wall binding, complex formation by organic

acids and root exudates, precipitation within the root zone by complex formation, no xylem loading

and translocation. According to Hooda (2007) for phytoremediation to be effective, the following

factors should be considered: the physical and chemical properties of the soil, plant and microbial

exudates, metals bioavailability, the ability of plants to take up, accumulate, translocate, sequester and

detoxify metals. A chelant such as Ethylenediaminetetraacetic acid (EDTA) needs to be added to the

soil as an amendment. The EDTA makes the lead available to the plant. Hyperaccumulator are usually

slow growing and of low biomass. Thlaspi caerulescens is known as the best metal hyper accumulator,

it can produce 2-5 mgha-1

(Scragg, 2006). Thlaspi caerulescens can remove up to 30,000 mgkg-1

of Zn

(Baker and Brooks, 1989). Hyperaccumulators contain more than 1,000 mgkg-1

Co, Cu, Cr, Pb and Ni

as well as 10,000mgkg-1

(1%) of Mn or Zn in dry matter (Baker and Brooks, 1989). Commonly, the

plant used for lead extraction is Indian mustard (Brassisa juncea) (Scragg, 2006). According to Lasat

et al., (2000), crops like Alpine penny cress (Thlaspi caerulescens), Ipomea alpine and Astragalus

racemosus have very high bioaccumulation potential for Cd/Zn, Cu and Se respectively.A leafy

vegetable Amaranthus dubius was found to tolerate and translocate up to 100 mgkg-1

of Arsenic to its

aerial parts and 25 mgkg-1

of Cr, Hg, As, Pb, Cu and Ni (Mellem, 2012). Hamizahh et al., (2011)

reported 99.6% and 97.3% copper removal from copper solutions of 1.5mg/L, 2.5mg/L and 5.5mg/L

by two aquatic plants: Centella usiatica and Eichhornia crassipes respectively. Betula removed up to

528mgkg-1

of Zn while grasses such as Pennisetum glaucum and Paspalum notatum removed 0.03

33

mgkg-1

of Zn (Scragg, 2006). Phytoremediation is less expensive compared to excavation and reburial

of soil (Cunningham and Ow, 1996) and it is suitable for treatment of large volumes of substrate with

low concentration of heavy metals. However, these metals contained in plants prevent plant growth

thereby hindering phytoremediation. As a result, the ability of plants to tolerate the toxic metals being

extracted from the soil is of great importance. Phytoextraction may reduce the levels of heavy metals

in sediments to acceptable levels with time, because metals are translocated to easily harvestable plant

parts. A few plants are able to survive and reproduce heavily on polluted soils or sediments with Pb,

Cd, Cu and Zn (Baker, 1981). Higher pH values (near neutrals) in sediments result in greater retention

and lower Heavy metal solubility. Huang et al., (1997) described four categories of heavy metals

accumulation (Plant-soil co-efficient (PSCF) / Bioaccumulation factor (BAF) by plants: (1) BAF <

0.01 (shows non-accumulator plant) (2) BAF between 0.01-0.1 (shows low accumulator plants) (3)

BAF between 0.1-1(shows moderate accumulator plants) and (4) BAF between 1-10 ( shows high

accumulator plants/ hyperaccumulator plants). Huang et al., (1997) also reported that the content value

of metal per plant is a better estimate of heavy metal extraction efficiency in a given plant species. Pb

has been detected in the transpiration fluid in chelated form (Tanton and crowdy, 1971). Increasing the

soil clay content, cation exchange capacity of the soil decreases Pb solubility: hence its availability. In

pot trials, using soils contaminated with Pb, Zn and Cd (690, 410 and 4.5 mg/kg soil respectively),

addition of CaHPO4 markedly lowered the accumulation of these metals in kohlrabi (cabbage plant),

Kale (cabbage plant) and Celeriac (root vegetable) (Leh, 1986). The difference between arsenate, Pb

and Zn is that arsenate is adsorbed and desorbed rather than precipitated and dissolved like Pb and Zn

(Qafoku et al., 1999). Roots can actively exude protons and other substance like organic acids and

nitrogenous compounds. This has to do with the nutrient acquisition of the plant, especially that of P,

Mn, Fe and Zn and is also influenced by other factors like Ph (Ryan et al., 2001). Organic acids like

34

malate, oxalate and citrate form metal ion complexes and their role as regards metallic elements may

relate to tolerance mechanism, although this function is yet to be clarified further (Jones, 1998a).

However, root exudated oxalate has been reported to enhance Pb tolerance in rice (Yang et al., 2000).

2.1.1 Merits of Phytoremediation

• It is less expensive compared to other bioremediation techniques and more environmentally

friendly than the traditional method of land escavation. The cost of phytoremediation could be

as much as 20 times less than any other types of methods (Lasat, 2002).

• It is important in protection of human health and lives because both Human and Livestock can

be exposed to toxic levels of contaminants from soil through ingestion (Lasat, 2002).

• Roots may be described as exploratory, liquid-phase extractors that can find, alter and/or

translocate elements and compounds against large chemical gradients. Therefore, plants can

also be a cost-effective alternative to physical remediation systems. (Lasat, 2002).

2.1.2 Demerits of Phytoremediation

• It is a slow process compared to mechanical methods like excavation.

• Climatic restrictions in growing some species of plants and problem of unknown long-term

environmental costs.

• Potential danger exists for animals that live in the areas in which phytoremediators are grown

especially if these animals typically feed on these species being used for phytoremediation.

• Potential for contaminants to move up the food chains quickly leading to toxicity (Hooda,

2007).

35

2.1.3 Limitation of Phytoremediation

Contaminants that are highly water-soluble may leach outside the root zone and require containment.

Phytoremediation is also frequently slower than physio-chemical process; may need to be considered

as long term remediation process.

2.1.4 Mechanisms of Metal Sequestration/Uptake and Tolerance

Metal uptake depends solely on metal bioavailability. Metal species existing in the soil is governed by

the physical, chemical and biological processes of the soil (Hooda, 2007). Bioavailability of soil

pollutants, a primary basis of remediation efficacy, refers to a fraction of the total pollutant mass in the

soil and sediment available to plants. The ability of plants to take up metals involves root interception

of metal ions, entry of metal ions into roots and translocation of these metals to the shoot through mass

flow and diffusion. It is characteristic of metals like As, Pb and Zn to remain in the upper layers of the

soil with high organic matter contents. (Matschullat, 2000). When free metal ions are at high

concentrations in soil solution, they tend to be more toxic to plants than corresponding concentrations

of other metals in soluble forms (Mc Bride, 1995). Biotic and Abiotic factors such as plant root’s

activity, soil microflora, chemical composition of the rhizosphere, soil pH and reduction/oxidation

potential are observed to influence the bioavailability of an element. (Mc cully, 1999). Effect of soil

pH, on the solubility and availability of Pb varies. Soil pH (range 4.6-6.2) among other factors in an

extensive study in England and Wales, influenced the uptake of Pb into Raphanus sativus (Davies,

1992). The solubility of soil Pb is influenced by both the organic and mineral fractions of the soil to a

greater extent than that of Zn (Alloway et al., 1998). Similarly, Pb also adheres readily onto soil

substrates such as clay and Fe/ Mn oxides, depending on the pH conditions (Mc Bride et al., 1997). It

also associates with organic matter and carbonates present in the soil. (Blaser et al., 2000).

36

According to Salt et al., ( 1995) this uptake is achieved by mobilizing metals bound to soil particles

through the metal-chelating molecules which are secreted into the rhizosphere, specific plasma-

membrane-bound metal reductase and the proton extrusion from roots. A type of plant exudate is

phytosiderophores produced by grasses, which binds Iron (Fe) and facilitate its uptake.

Phytosiderophores are biosynthesized from nicotinamide, which is composed of three methionines

along with non-peptide bonds. It is however possible to enhance the bioavailability of metal pollutants

by manipulating the root processes. Chelates are used to enhance phytoextraction of a number of metal

contaminants including Cd, Cu, Ni, Pb and Zn (Blaylock et al., 1997). The chelate-mediated

accumulation of toxic metals in a non-accumulator species is referred to as "chelate-assisted

hyperaccumulation". Metal-accumulation efficiency appears to be directly related to the affinity of the

applied chelating agent (Salt et al., 1995). Thus, synthetic chelating agents with a high affinity for the

metal of interest (e.g., EDTA for Pb, EGTA for Cd) are preferred (Blaylock et al., 1997).

Higher concentrations of Zinc found to be more toxic to P. sativum and Zea mays (Nobbe et al., 1884)

and Knop, (1885) than Pb. Pb reduced the dry matter produced more than Zn. According to Hevesy,

(1923) Pb accumulated mostly in the roots of Vicia faba and was suggested that one-third of it could

be removed with dilute HNO3, this therefore suggests that lead was attached to the cell wall’s

apoplastic space. However, Pb nitrate claimed to be a better fertilizer compared to others like Na

nitrate (Berrry, 1924). Hoagland et al., (1936) has demonstrated that Zn altered plant growth

metabolically and showed external toxicity.

According to Krupa et al., (2002), roots usually accumulate Pb and are translocated in the transpiration

stream. It moves in the apoplastic space of the root cortex and it can bypass the endodermis and gain

symplastic access in the young root zone. Pb can enter and move within the cytoplasm. Its uptake is

thought to be by passive absorption (Tung and Temple, 1996). Proteins such as channel type in the

37

root plasma membrane of tobacco have been identified to mediate cross-membrane movement of Pb

(Arazi et al., 1999). Zn is an essential micronutrient and is mobile in plants. Vacuolar zinc trafficking

in Silene vulgaris has been observed (Chardonnes et al., 1999).

Baker (1987) grouped plants into accumulators or excluders. Excluder plants reduces uptake of

elements (Baker, 1987). Exclusion property is poor or absent in higher plants. (Ernst,1976). Neither

tolerance nor toxicity mechanisms are fully understood yet. However, the mechanisms likely to be

involved in plant tolerance may include responses like altered membrane permeability, enhanced metal

binding capacity of the root apoplasm and root exudates (Hall, 2002).

Synthesis of phytochelatins (PCs) and metallothionens (MTs) is one of the responses of plants to

higher concentrations of a number of metals or metalloids. PCs (cysteine-rich polypeptides) primary

function in plant is detoxification while MTs help in translocation of some metallic elements. PCs are

enzymatically produced while a gene family is known to encode MTs. PC synthethase genes are not

yet identified in higher plants. However, the roles of PCs need more clarification (Clemens et al.,

2002). PCs are induced in plant’s response to Ag, Au, Cd, Cu, Hg, Ni, Pb, Sb, Sn and Zn (Grill et al.,

1987). In legumes, Pb is a strong inducer of PCs. Production of PCs therefore implies toxicity but not

necessarily tolerance (Ebbs et al., 2002). PCs also help in micronutrient homeostasis. Zn is however,

known as a weak inducer of PCs (Grill et al., 1987). Zn uptake is shown by response mechanisms such

as complexation with organic acids (root exudates). This is observed to be a more prevalent and

effective mechanism for inactivating Zn than are PCs (Wuang et al., 1992)

2.2 Cowpea (Vigna unguiculata)

Cowpea has been the subject of genetic research since the beginning of the 1900s. It is one of the most

important pulse crops in tropical Africa (Duke, 1990). In Nigeria, cowpea is utilized essentially for dry

38

seed consumption. The seed constitutes a main source of dietary protein since approximately 80% of

the protein in cowpea is seed storage protein (Oyenuga, 1967). It is useful for plant breeding and

genetic research because it is a diploid plant with a relatively short life cycle. Although, it has been

reported to undergo limited out crossing (Purseglove, 1985), it exhibits self-pollination under most

environmental conditions. The total seed protein, globulin and albumin fractions of 20 cowpea

accessions from IITA gene bank have been investigated by SDS-polyacrylamide gel electrophoresis

(Odeigah and Osanyinpeju, 1996). The cultivation of cowpea in recent times has increased

tremendously because of its nutritional value to man and livestock. It is drought tolerant with a great

agronomic interest as food and fodder. It can adapt to poor soil with limited rainfall. Cowpea has the

ability to improve its nutrient uptake by mycorrhizal associations between their roots and soil fungi

(IITA, 2010). Cowpea (Vigna sinensis) was reported to have a translocation factor less than one (TF <

1) when treated with Zn and Cd below the concentrations of 1000mgkg-1

and 100mgkg-1

respectively

in pot cultures (Saleh and Saleh, 2006).

2.3 Bambara Groundnut (Vigna subterranean)

In South Africa, Bambara groundnut is known as Jugo beans while in Zimbabwe, it is known as

Nyimo beans. It is referred to as an underutilized African legume in South Africa but widely cultivated

in sub-Saharan Africa (NRC, 2006). The origin of the bambara groundnut is thought to be Bambara in

Central Mali, West Africa. It is widely distributed in Asia, Australia, South and Central America. The

bambara groundnut can adapt to different harsh conditions, it can survive in hot, dry places with low

rainfall. Bambara groundnut is important because it fixes atmospheric nitrogen as nitrates into the soil

which improves soil fertility. It is high in methionine, an essential amino acid. The beans are eaten

fresh after harvest and can be dried and stored for later use and consumption (NRC, 2006). The great

genetic diversity potential of bambara groundnut has been described by Wazael (2004). Okpuzor et al.,

39

(2010) reported the nutritional and health value as well as the different types of protein in the seeds of

bambara groundnut obtained in Lagos, Nigeria. Bambara groundnut could be used to remediate heavy

metal contaminated soils owing to its unique characteristics viz: toughness to stress, good root system

and bunch growth. In a study by Nwaichi et al., (2012), bambara groundnut (Vigna subterranean)

achieved 53.62%, 55.20% and 42.26% PAH, BTEX and Oil/ Grease removal respectively without

nutrient amendments while these rates were improved by amendments as follows: Urea- 63.37%,

NPK- 65.99%, PM- 70.04,% for PAH; Urea- 78.80%, NPK- 79.80%, PM-87.90%, for BTEX; and

Urea- 71.23%, NPK- 71.39%, PM- 88.13%, for Oil and Grease. Generally, the performance was best

with poultry manure amendment.

2.4 Maize Plant (Zea mays)

Maize (Zea mays), or corn is a versatile cereal crop that is grown widely throughout the world in a

range of agro-ecological environments. Maize is the most important cereal crop in sub-Saharan Africa

(SSA) and an important staple food for more than 1.2 billion people in SSA and Latin America

(Nippon Foundation, 2002; IITA, 2010). Production and utilization of maize has increased recently in

Nigeria as a result of the introduction of high yielding, drought-tolerant, early and extra-early maturing

varieties (e.g. 80 – 90 days). The government’s encouragement through initiatives such as the National

Poverty Eradication Programme (NAPEP) and the National Directorate of Employment (NDE) has

also improved Maize production in Nigeria (Omoloye, 2009).

Maize forms 16 to 22 leaves per plant. The tassel forms at the top of the plant and provides the pollen

for fertilizing the ear (also known as a cob). Maize grains usually weigh around 25 – 40 g per 100

kernels (IKISAN, 2000). Maize needs bright sunny days for its accelerated photosynthetic activity and

rapid growth of plants. An intermittent sunlight and cloud of rain is the best for its growth, prolonged

cloudy period is demanding to the crop (IKISAN, 2000). In terms of soil requirements, deep fertile

40

soils rich in organic matter and well-drained are most preferred; however maize can be grown on a

variety of soils. The ideal soil types are loam or silt loam surface soil and brown silt clay loam that is

fairly permeable. The crop is very sensitive to water logging. The tolerable pH is 6.0 – 7.5. Maize, is a

widely cropped annual cereal that grows rapidly, produces extensive fibrous root system with large

biomass, withstands adverse conditions and produces abundant seeds with ease of cultivation under

repeated cropping (Garbisu and Alkorta, 2001; Zhang et al., 2007). Although maize is not listed as a

hyperaccumulator, the attributes mentioned above are sufficient to consider its use in phytoextraction

(Ebbs et al., 1997). Transfer coefficients in maize have been reported as Zn,1-2, Cd, Cu and Pb,0.01-

0.05 (Korentejar,1991). Wuana et al., (2010) also reported the transfer coefficients of maize as Zn,

0.23, Cd, 0.18, Cu, 0.15 and Pb,0.12.

2.5 Heavy metals in the Environment and Plant Growth

Heavy metals are metals with higher atomic weights (usually greater than 20) found within groups III

to V of the periodic table of the elements. They are not easily degraded. Heavy metals are a source of

environmental pollution in industrialized societies. Pollution by metals varies from air, water to soil,

because heavy metals stay much longer in soil than other parts of the biosphere (Lasat, 2002). Most

industrial operations release various toxic heavy metals in their effluents which eventually find their

way into water sources such as lakes, rivers and streams. The alarm of heavy metal pollution started

with the effects of Minamata disease caused by consumption of sea foods containing mercury (Gingell

et al., 1976). Industrial chemicals had been strongly implicated in metal pollution of water bodies.

High levels of copper (Cu), Zinc (Zn), lead (Pb), Cadmium (Cd), Nickel (Ni) and Iron (Fe) were found

in leafy vegetables grown in domestic gardens near a Copper smelter in Australia (Bearington, 1975).

Arsenic acid poison comes from white arsenic (arsenic trioxide), which is used in manufacturing

arsenical pesticides and herbicides. Large volumes of arsenicals come from leather industries (leather

41

pigments), landfills, mines, pit heaps, smelters, and antifungal wood preservatives. Sewage effluents

containing heavy metals such as cadmium, lead and zinc have been found to cause adverse effects on

soil properties and plant growth (Amin and Migahid, 2000). The Federal Environmental Protection

Agency (FEPA, 1991) gave the highest desirable level of substances in drinking water for humans;

children are mostly affected but adults also get affected. Lead is used as an industrial raw material for

storage battery manufacture, printing, fuels pigment, photographic materials, matches and explosives

manufacturing. It has been noted that lead decreases with depth down the soil profile, and the

concentration increases with increasing decomposition of litter (Lasat, 2002). Lead accumulation

within the leaf litters gets incorporated into the soils in a woodland ecosystem. (Gingell et al.,1976).

Studies of heavy metals distribution in a contaminated woodland showed that 60% of the top soil is

lead accumulation. Lead accumulation is not affected by liming and it is less translocated than zinc

(Bergquivist and Sundbom, 1980). The level of lead present in a plant varies according to the part of

the plants. Ademoroti (1996) has shown that lead occurs more in the stem than in the root tips of

Abelmoschus esculentus (Okro) and bitter leaf plants. Variation in lead contents in plants also depends

on whether the plants are grown in contaminated soil or not. Plants grown in contaminated soil have

higher levels of lead (as well as other heavy metals) than those grown in an uncontaminated soil

(Bearington, 1975, Gingell et al., 1976). Elevated Pb in soils may adversely affect soil productivity

and even low concentration can inhibit some vital plant processes, such as photosynthesis, mitosis and

water absorption. The toxic symptoms shown include: dark leaves, wilting of older leaves, stunted

foliage and brown short roots (Patra et al., 2004).

Zinc pollution occurs as a result of natural and anthropogenic processes and these can lead to the

deposition of zinc on land. Ademoroti (1996) and Odiete (1999) noted that zinc arises as industrial

effluents and industries that produce zinc as effluents include electroplating industries, metallurgical

42

industries, dye, paint and pigments, pharmaceuticals and mining industries. Water-soluble zinc that is

located in soils can contaminate ground water. Plants often have a zinc uptake that their systems

cannot handle, due to the accumulation of zinc in soils. On zinc-rich soils, only a limited number of

plants have a chance of survival. That is why there is not much plants diversity near zinc- disposing

factories (Heyman, 1991). According to Chaudri et al., (1993), soil microflora shows sensitivity to

zinc within the soil. Zn is known to be involved in protein synthesis, DNA replication and serve as

catalyst of many enzymes in plants. The potential of high toxicity of Zn lies in its role as a

micronutrient, high solubility and ready uptake by plants (Longnecker and Robson,1993). Seregin et

al., (2004); Stiborova et al., (1987); Vojtechova and Leblova, (1991) and Prassad and Prassad (1987)

reported that heavy metals such as lead inhibited seed germination and seedling growth. Early seedling

growth was also reported to be inhibited in rice (Verma and Dubey, 2003; Yang et al., 2000), Spruce

(Vodnik et al., 1999), Barley (Stiborova et al., 1987), tomato and egg plant (Khan and Khan, 1983).

Paivoke (2003) observed a decline in seedlings growth and reduced shoot yield of Pisum sativum

exposed to 300mg/kg of Zn and 500mg/kg of Pb. The effect of heavy metals on plants growth depends

on the type of metal ion, plant species and growth stage at which the metal is applied (Sheoran and

Singh, 1993).

2.6 Genotoxicity of Heavy Metals

The general principles of the mechanisms of mitosis are easily studied in the actively growing regions

of plants such as the root apex. Metals are one of the major groups of genotoxic environmental

pollutants causing serious threat to human as well as environmental well being (Panda and Panda,

2002). Excess heavy metal stress causes oxidative damage in plants. The elevated levels of heavy

metals in plants may suppress the metabolism and translocation of reserve material to the growing

regions (cell division) and their subsequent utilization (Panda and Panda, 2002).

43

Heavy metals at high level concentrations affect the morphological traits of plants (Sinha and Gupta,

2005). Rank and Nielsen (1998) analyzed wastewater sludges and found to contain heavy metals (Pb,

Ni, Cr, Zn and Cu ). The genotoxic effect of these wastewater sludges were investigated in Allium

cepa, and it was found that these heavy metals induced significant chromosome aberrations such as

stickiness and vagrants (Odeigah et al., 1997a).

Tannery effluent and Chromium caused a reduction in mitotic index and induced chromosomal

aberrations in Allium cepa (Gupta et al., 2012). Chromosomal and mitotic aberrations were observed

on exposing Vicia faba and Pisum sativum to copper (Souguir et al., 2008). Odeigah et al., (1997b)

reported the genotoxic potential of leachates from solid wastes on Allium cepa.

Olorunfemi et al., (2011) observed a rapid decrease in mitotic index, chromosomal aberration and root

length inhibition with increasing cassava effluent concentration on Allium cepa. Bakare (2001)

reported a reduction in the number of dividing cells of Allium cepa treated with different

concentrations of leachates containing metals. Sewage effluents containing Pb, Zn, Cr and Hg induced

chromosomal aberrations such as vagrants, stickiness and bridges on Allium cepa (Ukaegbu and

Odeigah, 2009). Kumar and Rai (2009), reported chromosomal aberrations such as stickiness, bridges,

laggards and scattering as genotoxic effects of Hg and Cd on six inbred lines of maize at the different

concentrations of 25mg/L, 75mg/L and 100mg/L.

Arsenic has been observed to interfere with cell division by disturbing organization of microtubule and

subsequent formation of mitotic spindle. It may also inhibit DNA repair enzyme (Panda and Panda,

2002). Steinkellner et al., (1998) found that at elevated concentrations, As, Pb, Cu and Zn disrupt cell

division in many plant species. Even at low concentrations (10-7

M) of organic or inorganic Pb, the

mitotic index was reduced. According to Liu et al., (1994), polynucleated cells and micro nuclei are

44

found as common effects of Pb on mitosis. However, Steinkellner et al., (1998) also found that

elevated concentrations of Zn are not strongly genotoxic.

2.7 Oxidative Stress/ Reactive Oxygen Species/ Free Radicals

2.7.1 Oxidative Stress

Oxidative stress represents an imbalance between the production of reactive oxygen species and a

biological system's ability to readily detoxify the reactive intermediates or to repair the resulting

damage (Chao et al., 1999). Disturbances in the normal redox state which can be due to the presence

of heavy metals can lead to toxic effects through the production of peroxides and free radicals that

destroy all components of the cell, including proteins, lipids, and DNA (Imlay, 2003).

2.7.2 Chemical and Biological effects of Oxidative stress

In chemical terms, oxidative stress is a large rise (becoming less negative) in the cellular reduction

potential, or a large decrease in the reducing capacity of the cellular redox couples, such as

glutathione. The effects of oxidative stress depend upon the size of these changes, with a cell being

able to overcome small perturbations and regain its original state. However, more severe oxidative

stress can cause cell death and even moderate oxidation can trigger apoptosis, while more intense

stresses may cause necrosis. A particularly destructive aspect of oxidative stress is the production of

reactive oxygen species, which include free radicals and peroxides. The major portion of long term

effects is caused by damage to DNA. Most of these oxygen-derived species are produced at a low

level by normal aerobic metabolism and the damage they cause to cells is constantly repaired.

However, under the severe levels of oxidative stress that cause necrosis, the damage causes ATP

depletion, preventing controlled apoptotic death and causing the cell to simply fall apart (Lee and

Shacter, 1999).

45

2.7.3 Free Radicals

Free radicals are atoms or groups of atoms with an odd (unpaired) number of electrons. They can be

formed when oxygen interacts with certain molecules. They can cause great damage in a living

organism when they react with important cellular components such as DNA, or the cell membrane.

Cells may function poorly or die if this occurs (Rattan, 2006).

2.8 Enzymatic Antioxidants and Metal Tolerance

2.8.1 Superoxide dismutases (SODs)

These are a class of closely related enzymes that catalyze the breakdown of the superoxide anion into

oxygen and hydrogen peroxide (Zelko et al., 2002), (reaction is shown below). SOD enzymes are

present in almost all aerobic cells and in extracellular fluids. Superoxide dismutase enzymes contain

metal ion cofactors that, depending on the isozyme, can be copper, zinc, manganese or iron. There also

exists a third form of SOD in extracellular fluids, which contains copper and zinc in its active sites. In

plants, SOD isozymes are present in the cytosol and mitochondria. An iron SOD is also found in

chloroplasts of plants but absent in vertebrates and yeast.

2.8.2 Catalases

These are enzymes that catalyse the conversion of hydrogen peroxide to water and oxygen, using

either an iron or manganese cofactor (Hiner et al., 2002). The reaction is shown below. Hydrogen

peroxide is the only substrate for catalase, it is therefore an unusual enzyme since and it follows a

ping-pong mechanism. Here, its cofactor is oxidised by one molecule of hydrogen peroxide and then

regenerated by transferring the bound oxygen to a second molecule of substrate (Mueller et al., 1997).

SOD

46

2.8.3 Glutathione Synthetase (GSH)

GSH plays several important roles in the defense of plants against environmental threats, oxidative

stress, heavy metals and xenobiotics. Glutathione is not only a substrate for glutathione S-transferases,

enabling neutralization of potentially toxic xenobiotics, but is also a reductant of dehydroascorbate

(Foyer and Haliwell, 1976). Moreover, GSH is the precursor for phytochelatins (PCs), heavy-metal-

binding peptides involved in heavy-metal tolerance and sequestration (Steffens, 1990). The

detoxification of heavy metals by plants is achieved by uptake and translocation, sequestration into the

vacuole and metabolization, including oxidation, reduction or hydrolysis and conjugation with

glucose, GSH or amino acids (Salt et al., 1995; Dietz and Schnoor, 2001).

2.9 RAPD-PCR Analysis

The PCR-based genetic assay known as randomly amplified polymorphic DNA (RAPD) was

developed by Welsh and McClelland (1990) and Williams et al., (1990). This procedure detects

nucleotide sequence polymorphisms in DNA by using a single primer of arbitrary nucleotide

sequence. The current method used in measuring genetic variation within germplasm collections is the

use of RAPD for identification of cultivars through DNA profiling (Williams et al., 1990 and

Hernendez et al., 2001). PCR-based RAPD markers are dominant markers that are used greatly in

genetic mapping (Chalmers et al., 2001) and identification of loci linked with different traits (Sun et

al., 2003). According to Demek et al., (1996), due to technical simplicity and speed, RAPD

methodology has been used for several analyses in different crops. Criteria for the estimation of

genetic diversity include: pedigree records, morphological traits or molecular markers (Heckenberger

47

et al., 2002). Molecular markers detect variation of the DNA sequences among cultivars and therefore

directly bypass problems connected with environmental effects (Maric et al., 1998). RAPD-PCR helps

to identify variations and discover the effects of heavy metals on the quality of plant DNA. PCR-based

RAPD markers linked to a gene governing metal stress have been identified in several studies. For

instance, cadmium stress in durum wheat (Penner et al., 1995), Aluminum tolerance in rice (Wu et al.,

2000) and manganese efficiency in barley (Pallotta et al., 2000 and Khabaz-Saberi et al., 2002).

48

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Materials

3.1.1 Sources and Collection of Test Plants/Materials

Dry seeds of Cowpea (Vigna unguiculata) accessions: Tvu 3788 and IT 99K-377-1, Bambara

groundnut (Vigna subterranean) accessions: Tvsu 102 and Tvsu 1685 and Maize (Zea mays)

accessions: ACR.91SUWANI-SRC1 and DMR-LSRW were collected from the International Institute

of Tropical Agriculture (I.I.T.A) Ibadan. The cowpea accessions were of a white seed coat, the

bambara groundnut a light brown seed coat and maize of a yellow seed coat (Plates 3.1 to 3.6). The

farmyard manure (cow dung) was obtained from a local abattoir along LASU Road, Igando, Lagos.

The metal salts used were lead nitrate and zinc nitrate. The chelant used was ethylene diamine acetate

(EDTA). The metal salts and chelant were purchased from Finlab, Nigeria Ltd, Anthony, Lagos and

Labio Scientific, Mushin, Lagos.

49

Plate 3.1: Cowpea, Vigna unguiculata (Accession IT 99K-377-1) x 100

Plate 3.2: Cowpea, Vigna unguiculata (Accession Tvu 3788) x 100

50

Plate 3.3: Bambara groundnut, Vigna subterranean (Accession Tvsu102) x 100

Plate 3.4: Bambara groundnut, Vigna subterranean (Accession Tvsu1685) x 100

51

Plate 3.5: Maize, Zea mays (Accession DMR-LSRW) x 100

Plate 3.6: Maize, Zea mays (Accession ACR.91SUWANI-SRC1) x 100

52

3.2 Study Sites

Genotoxicity study was carried out at the Cell Biology and Genetics Departmental laboratory in the

Biological garden, University of Lagos. The phytoremediation and phytotoxicity studies were carried

out at a garden along Lagos State University (LASU) Road, Akesan, Lagos. The biochemical analyses

were carried out at the Department of Biochemistry, College of Medicine, University of Lagos, Idi-

Araba. The analyses of metal contents in the plant samples were carried out at the Department of

Chemistry, University of Lagos, Akoka, Yaba. The DNA isolation and RAPD-PCR analyses were

carried out at the Cell Biology and Genetics Department (Laboratory) and the Biotechnology unit of

the Federal University of Agriculture, Abeokuta (FUNAAB), Ogun State.

3.3 Methods

3.3.1 Determination of Metal Salt and Chelant concentration

Before the start of the experiment, the concentrations of the metals (Pb and Zn) were determined by

the root growth inhibition test as described by Jorge and Arruda (1997) and considering the certified

reporting regulatory limits by US EPA (2001), DPR-EGASPIN (2002) FEPA (1991) and NJDEP

(1996) (see Appendix I, II and III a and b). The concentrations of the metals (100mg/kg, 150mg/kg

and 200 mg/kg) were of acceptable levels (NJDEP, 1996). The concentrations of Ethylene diamine

acetate (EDTA) were determined following the method by Nascimento et al., (2006).

3.3.2 The Screening Test

There were two accessions available for each crop plant under study. In order to have reliable and

valid results, there was a need to conduct a screening test for the accessions of each crop plant. The

percentage viability and germination were investigated under ten days. The percentage (%)

53

germination was obtained by dividing the number of germinated seeds by the total number of seeds

sown (Odoemena, 1988).

3.3.3 Soil Analysis before Planting

The following soil characteristics were determined: Soil type, Soil pH, Total Organic Matter, Total

Nitrogen, Total Organic Carbon, Available Phosphorus, Bulk density, Cation exchange capacity,

moisture content and the amount of lead and zinc present in the soil (Table 3.1). Some of these

parameters were also evaluated at the end of the experiment.

54

TABLE 3.1: PHYSICO-CHEMICAL CHARACTERISTICS OF SOIL SAMPLE USED FOR

THE EXPERIMENT.

Soil parameters Result/Values obtained

Soil type Sandy loam (silt 28.58%, clay 30.93% and sand

49.81%).

Soil pH 6.42

Organic matter 3.28 %

Total Organic carbon 3.2 %

Moisture content 8.18%

Total Nitrogen content 0.16mg/kg

Phosphorus content 0.026mg/kg

Lead content 1.4220mg/kg

Zinc content 0.7800mg/kg

55

3.3.3.1 Soil Characteristics Determination:

Soil type

This was determined following the method by White (2006).

Soil pH

The soil pH was determined following the method of Hendershot et al., (1993). Soil pH was estimated

by suspending 10 g of air dried soil in 20 ml of 0.01M CaCl2 solution. The air-dried soil was mixed

with 5 ml of distilled water and stirred. The mixture was allowed to stand for thirty minutes to settle.

The slurry was decanted into a test tube. The electrode of a pH meter was put into the slurry and the

pH read off.

Cation Exchange Capacity

The Cation exchange capacity was estimated according to the procedure of Anderson and Ingram

(1998a). The soil was air-dried and crushed with a mechanical device and screened to pass a 20-mesh

sieve. 2.5g of soil was weighed out in a 125-mL Erlenmeyer flask. Sample was placed in a shaker for

15 minutes. The solution was then filtered and analyzed by flame atomic absorption.

Total Organic Matter

Five grams (5g) of soil was put in a clean, dry and accurately weighed porcelain crucible and ignited

over a Bunsen burn flame or hot plate until smoking stopped. The crucible was transferred into a

muffle furnace and heated for 2 hours at 550oC.It was allowed to cool in a desiccator and weighed.

The mass of organic matter is difference between mass of dish and the moisture (White, 2006).

Total Nitrogen

Ten grams (10g) of soil was passed through a 20 mesh sieve. 150ml of sulphuric-salicyclic acid

mixture (1g salicyclic acid + 30ml conc. H2SO4) was added to the soil and shaken to obtain intimate

contact of the soil with the reagent. Five grams (5g) sodium thiosulphate was added and then heated

56

gently for 5minutes. Ten grams (10g) of the sulphate mixture was then added. The digestion continued

for an hour after which 100ml of concentrated sodium hydroxide (45%) and a large zinc granule were

added. Blanks were done and titration carried out using 10 drops of the Bromocresol green-methyl red

as indicator (Bremner, 1996).

Total Organic Carbon

The soil organic carbon was determined by the procedure described by (Anderson and Ingram, 1998b.)

Ten (10g) of soil sample was weighed into a separating funnel. The sample was extracted with 25ml

chloroform thrice. The extract collected was weighed and put in a dry beaker and evaporated to

dryness.

Total Organic Carbon = Weight of residue x 1000

Weight of sample

Available Phosphorus

Five grams (5g) of soil is placed in a clean glass test tube. 10ml distilled water and one drop of (1: 3)

acetic acid were added, shaken for few minutes and filtered. 10 ml of the filtrate was added to

phosphate powder pillow content. The reaction time was allowed to lapse and the concentration read

directly at a wavelength of 430nm. The value obtained was then converted to mg/l phosphorus (AOAC,

1990).

i.e. mg/l phosphorus = mg/L phosphate x 31

= mg/L phosphate x 0.3263

Moisture Content

10g of the soil sample was weighed into a re-weigh crucible. The crucible was placed in the oven at

105oC for 3 hours. A pair of crucible tongs was used to transfer the crucible into a dessicator to allow

57

cooling and weighing. The dish was returned to the oven for half an hour and the process repeated to

obtain a constant weight. The calculation is shown below:

% moisture content = W2- W3 × 100

W2-W1 1

W2 = mass of crucible + sample, W3 = mass of dried crucible to a constant weight ,W1 = mass of

crucible alone (AOAC, 1990).

Bulk Density (Density-can method)

Bulk density is the ratio of the weight of soil to its volume expressed in grammes per cubic centimeter.

This was determined following the procedure by Grossman and Reinsch (2002). A solid block sample

of of the soil was trimmed into a more or less regular shape avoiding re-entrant angles. It was then

coated with paraffin wax, allowed to dry and the coating repeated. The coated samples was weighed as

Wx, the weight of sample was given as Ws and then the weight of paraffin wax (Wp) was obtained as

Wp = Wx-Ws. Coated sample was totally immersed in a density can filled with water. The water

displaced by the sample was run into the measuring cylinder and the volume (Vw) recorded. The

coated sample is removed from the can and wiped dry on the outside. The paraffin wax coating was

removed and the moisture content determined as shown below:

Calculations: Vs =Vw-Wp/Gp (ml)

Where Vs= volume of soil and Gp = density of paraffin wax (usually 0.908 approximately).

r = 62.425 Ws lb per cu ft

Vs

Where r = wet bulk density.

rd = 100 Dw lb per cu ft

100 + m

Where rd = corresponding dry bulk density, Dw = dry weight of soil, m = moisture content.

After the remediation and phytotoxicity experiments, the soils were analyzed again to find out the

effects of the different metal treatments on the soil characteristics.

58

3.3.4 Cytological study

Viability Test for the Genotoxicity Study:

The seeds were subjected to viability test using floatation technique according to Agbogidi (2010).

This is a preliminary experiment carried out to ascertain the viability of the seeds under study. 50

seeds of each crop plant were soaked in petridishes containing tap water to trigger sprouting. After

thirty minutes for cowpea, two days for maize and three days for bambara groundnuts in order to

soften the hard seed coats especially for maize and bambara groundnut. This was also done to allow

water imbibition by these seeds. Ten seeds out of the soaked seeds were transferred to each of the

petridish containing cotton wool moistened with tap water for the control and treated groups. The

control group had tap water while the treated groups had 25mg/L, 50mg/L and 100mg/L of Pb and Zn

nitrates solutions respectively in three replicates. The seeds were moderately watered for three days

after which the percentage viability was calculated as:

Number of sprouted seeds X 100%

Total number of seeds sown

3.3.4.1 Seed Germination for Cytological Procedure

Seeds of the three crop plants were spread uniformly in Petri dishes lined with filter paper. The Petri

dishes were treated with equal volumes of the different concentrations of lead and zinc nitrates

solutions (25, 50 and 100 mg/L) in three replicates. The seeds were then allowed to germinate at room

temperature for 5 (five) days. Root tips which are brittle, translucent and gently tapering were selected

from the three seedlings grown in the different concentrations and from the seedlings grown in

ordinary water which serves as the control. About 2-3 mm terminal root tips were cut off using a

sharp blade and then placed on a clean glass slide and macerated with the aid of two dissecting needles

and the remaining portion discarded. A drop of 1N Hydrochloric acid (HCL) was added to the root tip

59

and left for 5 minutes; this softens the root tissue breaking up the middle lamellae. The excess acid

was sucked up with a filter paper and the softened tissue is further macerated with dissecting needles

so that the cells easily absorb the lactic acetic orcein stain and spread adequately for microscopic

observation. Then, a drop of lactic acetic orcein stain was placed on the macerated root tip and

allowed to stand for 20 minutes. Each slide was covered with a cover slip and pressed down to allow

the tissue spread out and also to allow the excess stain seep out at the edges of the cover slip. This was

removed by placing the slide between the folds of the filter paper and squashing was done with the

base of the dissecting set. All prepared slides were examined under the Wild M20 light microscope

with power magnification (x 40 objective): then slides that showed better contrast of root-tips

cytologically were preserved by sealing the edges of the cover slip with nail varnish to prevent the

stain from evaporating (Michelle-Frainer et al., 2006). The photomicrographs of good slides were then

taken under the oil immersion lens (x1000 objective) using a Wild M20 microscope with MPS 55

photoautomat attachment. For each plant sample, slides that showed better contrast of root-tips

cytologically were selected and examined for the mitotic and non mitotic cells. The mitotic index was

calculated using the formula by Balog (2002):

Mitotic Index (M/I) = Number of cells in mitosis per field X 100

Total Number of cells per field 1

3.3.5 Experimental Description

The soil was mixed thoroughly and then filled into 180 black cellophane bags. Each cellophane bag

has a radius 13 cm and height 17 cm. Three thousand grams (3kg) of soil were placed in each bag.

Within each bag, a depth of 4cm above the soil surface was maintained for the addition of manure and

water. Ninety (90) bags were for the phytotoxicity test and the other 90 bags were for the remedation

60

study. The bags were perforated at the sides and bases to avoid water logging and also to increase the

soil aeration. The bags were arranged in four (4) rows designated as Control, A, B and C of three

replicates each (Figure 3.1). For the remediation study, the EDTA (Chelant) and manure were added to

the soils in about 50 bags and left to stabilize for 5days before the introduction of metal salt (Wu et al.,

2000). The soils were watered during this period. The whole garden was divided into two parts. One

was for phytoremediation experiment and the other for phytotoxicity experiment. Ninety pots were

arranged in four major groups as follows: Control (with cowpea, bambara groundnut and maize seeds

irrigated with distilled water), treatment A (with cowpea, bambara groundnut and maize seeds treated

with 100,150 and 200 mg/kg concentration of lead and zinc nitrates respectively), treatment B (with

cowpea, bambara groundnut and maize seeds treated with 100,150 and 200 mg/kg concentrations of

lead and zinc nitrates and augmented with 0.08g, 0.12g, 0.16g EDTA respectively) and lastly

treatment C (with cowpea, bambara groundnut and maize seeds treated with 100,150 and 200 mg/kg

concentration of lead and zinc nitrates and augmented with 100g, 150g and 200g of manure

respectively).

3.3.6 Macromorphometric Studies on Treated and Untreated Plant Samples

The plant height (cm), root length (cm) and leaf area (cm2) were measured from the tenth day of

growth to the thirtieth day. The fresh weight (g) and dry weight (g) were also measured on the thirtieth

day. Three seedlings from each of the metal treated and untreated soil sample were measured after

uprooting. The fresh weights were obtained by uprooting the plant from each bag. The roots were

washed thoroughly with deionized water to remove the soil. The plants were then weighed using a

weighing balance (model PN 163) immediately after harvest to avoid water loss. The dry weights were

obtained by oven-drying the control and treated plants at 60oC for 48 hours to ensure a constant weight

(AOAC, 1990).

61

X1

X2

A1

A2

B1

B2

C1

C2

D1

D2

3.5m

3.0m

Phytotoxicity Experiment

Remediation Experiment

Figure 3.1 Experimental Layout

Key

(Phytotoxicity Experiment) (Remediation Experiment)

X1= Untreated soil without plant X2= Untreated soil without plant

A1=Untreated soil with plant (Control) A2= Untreated soil with plant (Control)

B1=Treated soil with lead and zinc nitrate B2= Treated soil with lead and zinc nitrate

C1=Treated soil with Pb and Zinc nitrate +EDTA C2= Treated soil with Pb and Zinc nitrate +EDTA

D1=Treated soil with Pb and Zinc nitrate +Manure D2=Treated soil with Pb and Zinc nitrate + Manure

62

3.3.7 Determination of Chlorophyll content in Treated and Untreated Plant Samples

The photosynthetic pigment: the leaf total chlorophyll content (%) was evaluated. The chlorophyll

content of the seedling was determined using the method of Arnon (1949). Leaves from three plants

from each treatment and the control were separately put in a clean mortar, 10ml of 80% acetone was

added and the leaf tissue was ground to a fine pulp for three minutes. The resulting green liquid was

carefully transferred into a buchner funnel containing a pad of Whatman No. 1 filter paper. After

filtering, the grinding of the pulp was repeated with 5ml of 80% acetone for another 3 minutes. The

second extract was filtered as before into the flask containing the first extract. The sides of the mortar

and the funnel were rinsed with 5ml of 80% acetone to ensure that all the chlorophyll was collected.

The optical density (O.D) of the chlorophyll extract was determined with a spectrophotometer at

663nm and 645nm respectively against a 100% acetone solvent blank. The amount of chlorophyll

present in the extract (milligram of chlorophyll per gram of leaf tissue extracted) was determined using

the following equations:

mg chlorophylla /g tissue = 12.7D663 - 2.69D645

mg chlorophyllb /g tissue = 22.9D645 - 4.68D663

mg total chlorophyll/g tissue = Chlorophyll a + chlorophyll b

= (12.7D663 – 2.69D645) + (22.9D645 - 4.68D663)

= (12.7D663 – 4.68D663 + 22.9D645 - 2.69D645)

= 8.02D663 + 20.21D645

mg total chlorophyll tissue = 8.02D663 + 20.21D645

63

3.3.8 Biochemical Analysis

The effects of Pb and Zn on the activity of enzymes involved in metal tolerance of test plants were

also investigated.

3.3.8.1 Determination of Superoxide Dismutase (SOD) Activity

The level of SOD activity in the control and treated plant samples were determined by the method of

Magwere et al., (1997). From each of the treated and untreated sample solution, 0.1ml was diluted in

0.9ml of distilled water to make a 1 in 10 dilution. An aliquot of 0.2ml of the diluted enzyme

preparation was added to 2.5ml of 0.05M carbonate buffer (pH 10.2) to equilibrate in the

spectrophotometer, and the reaction was started by adding 0.3ml of freshly prepared 0.3mM

adrenaline to the mixture which was quickly mixed by inversion. The reference cuvette contains 2.5ml

of 0.05M carbonate buffer, 0.3ml of adrenaline (substrate) and 0.2ml of distilled water. The increase in

absorbance at 480 nm was monitored every 30 seconds for 150 seconds.

Calculation

Increase in Absorbance per minute = A2.5 – Ao

t

Where, Ao = initial absorbance, A2.5 = final absorbance and t = total time taken (150 secs or 2.5mins)

% inhibition = increase in absorbance of sample / min x 100%

Increase in the absorbance of blank / min

1 unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the

oxidation of adrenaline to adrenochrome during 1 minute.

SOD activity (units) = % inhibition

50% Therefore,

Specific activity of SOD (units/mg protein) = SOD activity x dilution factor

mg protein

64

3.3.8.2 Determination of Glutathione (GSH) Activity

The total sulphydryl groups, protein – bound sulphydryl groups and free sulphydryl groups like GSH

in biological samples can be determined using Ellman’s reagent, 5,5'-dithio-bis-2-nitrobenzoic acid

(DTNB) according to the modified method by Jollow et al., (1974).

Estimation of GSH level in Test Sample

From the sample solution, 0.2ml was mixed with 1.8ml of distilled water to give 1 in 10 dilutions.

About 3ml of precipitating reagent (4% sulphosalicyclic acid) was added to the diluted sample and

then allowed to stand for 10minutes for a precipitate to occur. 0.5ml of supernatant withdrawn was

added to 4ml of phosphate buffer followed by addition of 0.5ml of Ellman’s reagent. The blank was

prepared with 4ml of 0.1M phosphate buffer pH 7.4, 1ml of diluted precipitating solution and 0.5ml of

Ellman’s reagent, 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB). The absorbance was read within 20

minutes of colour development at 412nm against blank using a spectrophotometer. Reduced

glutathione concentration was proportional to the absorbance at 412 nm.

3.3.8.3 Determination of Catalase (CAT) Activity

Catalase activity of the control and treated plant samples were determined according to the method of

Sinha (1972) but modified by Artenie et al., (2008). Different amounts of H2O2 ranging from 20 to

160 moles were taken in small test tubes and 2ml of dichromate/acetic acid was added to each.

Addition of the reagents instantaneously produces an unstable blue precipitate of perchromic acid.

Subsequent heating for 10mins in a boiling water bath changed the colour of the solution to stable

green due to formation of chromic acetate. After cooling at room temperature, the volume of the

reaction mixture was made to 3ml with distilled water and the absorbance measured with a

spectrophotometer at 570nm.

65

From the sample solution, 0.1ml was mixed with 4.9ml of distilled water to give a 1 in 5 dilution of

the sample. The assay mixture contained 4ml of 0.2M H2O2 and 5ml of 0.01M Phosphate buffer in a

10ml flat bottom flask. 1ml of properly diluted enzyme preparation (test sample) was rapidly mixed

with the reaction mixture by gentle swirling motion. The reaction was performed at room temperature.

A 1ml portion of the reaction mixture was withdrawn and put into a test tube containing 2ml of

dichromate/acetic acid reagent at 60 seconds intervals for 3minutes. The H2O2 contents of the

withdrawn sample were determined by the method described above.

Calculation:

The mononuclear velocity constant, K, for the decomposition of H2O2 by catalase was determined by

using the equation for a first – order reaction.

K = 1/t log So/S.

Where, So is the initial concentration of H2O2

S is the concentration of peroxide at t min (60 seconds interval)

t is time – interval (1minute).

The value of K is plotted against time in minute and the velocity constant of catalase K(o) at time zero

determined by extrapolating the catalase content of the enzyme preparation was expressed in terms of

katalase feiahigkeit or ‘Kat F’ Artenie et al., (2008).

Kat F = K (o)

mg protein / ml (U/ mg protein)

3.3.8.4 Determination of Product of Lipid Peroxidation (MDA)

This was assayed by measuring the TBA (Thiobarbituric acid), reactive products present in the treated

and untreated plant samples using the procedure of Vashney and Kale (2007) and expressed as

micromolar of malondialdehyde (MDA)/g tissue.

66

Method

An aliquot of 0.4ml of the test sample was mixed with 1.6ml of Tris KCl buffer (which was first

placed into the test tube before the test sample). Then 0.5ml of 30% TCA was added followed by

0.5ml of 0.75% TBA and the mixture was placed in a water bath for 1hour between 90 – 95oC. This

was then cooled in ice and centrifuged at 3000 r.p.m. for 15mins. The clear pink supernatant was

collected and absorbance measured against a reference blank of distilled water at 532 nm in a

spectrophotometer.

Calculation:

Lipid peroxidation product (MDA) was expressed in units / mg protein, where E532 is molar extinction

coefficient which is 1.56 x 105M

-1CM

-1.

Therefore, MDA (units/mg protein) = Absorbance of test X volume of mixture

E532 X volume of sample X mg protein

3.3.9 Sample Preparation and Determination of Heavy Metals in Treated Plant Samples and

Soils

3.3.9.1 Plant and Soil Sample Preparation and Digestion

After treatment, the plant and soil samples were taken to the laboratory for heavy metal analysis. Each

plant sample was separated into leaves, roots, and stems and then dried at 50ºC for 8 hours in an oven

(USEPA, 1995). The separated and dried plant parts were milled using a laboratory blender and kept

for digestion. The same procedure applies to the soil samples. The soil sample was further broken

down into finer particles using a laboratory mortar and pestle. The soil samples were then dried for 8

hours at 80ºC using an oven. (USEPA, 1995 and Lone et al., 2008).

Plant samples treated with lead and zinc nitrate with and without augmentation were digested before

metal analysis to reduce organic matter interference and allow for the conversion of the metal into a

form that can be analyzed by the Atomic Absorption Spectrophotometer (AAS). The treated plant

67

samples were digested following a modified method described by Lone et al., (2008). Three (3g) of

the milled plant sample were weighed into a conical flask using a digital weighing balance. Three

milliliters (3 ml) of 60% hydrochloric acid and 10 ml of 70% nitric acid were added to the weighed

milled plant sample. The conical flask was then placed on a laboratory hot plate for digestion until the

white fume evolving from the conical flask turned brown. The digest was allowed to cool and filtered

through a Whatman’s filter paper, leaving a whitish residue. The filtrate was then made up to 50 ml

using distilled water and kept for further analysis.

3.3.9.2 Digestion of Soil Samples

Treated soil samples were digested with 10ml of a mixture perchloric acid: nitric acid (HCLO4:HNO3-

1:5 v/v) Acid digestion was carried out on a hot plate at 70-100oC until yellow fumes of HNO3 and

white fumes of HCLO4 were observed. The digestion process continued until a clear solution remained

after volatilization of acids, and was stopped when the residue in the flask was clear and white. The

digested samples were dissolved in distilled water and then filtered (Lone et al., 2008).

3.3.9.3 Determination of Heavy Metals in the Digested Plant and Soil Samples using Atomic

Absorption Spectrophotometer (AAS)

The digested plant and soil samples were analyzed for lead (Pb) and Zinc (Zn) using Atomic

Absorption Spectrometer (AAS). Lead and zinc accumulation (mgkg-1

DW) in different parts of

Bambara groundnut, Cowpea and Maize (roots, stems and leaves) was determined by using Atomic

Absorption Spectrophotometer after samples were digested with concentrated HNO3 + HClO4

(USEPA, 1995 and Lone et al., 2008). The total concentration of heavy metals in stems, leaves and

roots of test plants were measured in the 3 trials. The different plant parts were washed thoroughly

with deionized water before drying. In order to remove cations slightly absorbed on the root surface,

the roots were washed 5 minutes with 20 mM Na ethylenediamine tetraacetic (EDTA) and then rinsed

68

quickly with deionized water. Roots and shoots were then dried at 80°C for a week till constant weight

was attained and weighed. Shoots were divided into leaves and stems that were then individually

ground in a tungsten Retsch mill (Haan, Germany) and hot-digested in HNO3 65% and HClO4 70%.

Lead and zinc concentrations in all extracts and digests were measured by inductively coupled plasma

atomic emission spectrometry (ICP-AES; Plasma 2000; Perkin Elmer, Wellesley, USA) and by

graphite furnace atomic absorption spectrometry (GF-AAS; 5100PC atomic absorption spectrometer

equipped with a HGA 600 graphite furnace, a Zeeman-corrected furnace module, and a AS-60

Furnace autosampler; PerkinElmer, Wellesley, USA). The readings were taken from the equipment

and the results were converted to actual concentration of metals in the samples. The concentration of

each element is determined by means of the calibration graph. The sample prepared is aspirated and

the concentration of each element is calculated from the measured absorbance. 5 % acetic acid used

for the extraction is submitted to the test procedure to provide a blank value to be deducted from the

extract value. Using atomic absorption spectrometer, 1mL of 30 % hydrogen peroxide is added to each

100 mL of standard solution or sample before aspirating. The calibration curve for the metals is based

on an original standard concentration before addition of hydrogen peroxide. The blank value is taken

into consideration in the evaluation. The result is calculated with a computer or graphically. The result

is expressed in mg/L and later converted to mg/kg.

69

3.3.9.4 Determination of the Translocation Factor/ Bioconcentration Factor and the Plant-Soil

Coefficient

Multiplication Coefficient (MC) /Bioconcentration Factor (BCF) according to Stoltz and Greger

(2002) were also calculated using the equation:

MC/BCF = Concentration of R (µg/g)

Concentration in S (µg/g)

Where concentration of heavy metal in R is the concentration of heavy metal in the roots and

concentration of heavy metal in S is the concentration of heavy metal in soil.

The Plant-Soil Coefficient (PSC): This is the ratio of metal (whole plant) / metal (soil) according to

Stoltz and Greger (2002) was also calculated.

The translocation factor (TF): The translocation factor for metals within a plant was expressed by the

ratio of metal (shoot) / metal (root) to show metal translocation properties from roots to shoots (Stoltz

and Greger, 2002).

3.3.10 Genomic DNA Isolation for PCR Analysis

3.3.10.1 Reagents and Chemicals:

The stock solution concentrations were: 1 M Tris- HCl (pH 8), 0.5 M EDTA (pH 8), 5 M NaCl,

absolute ethanol (AR grade), chloroform: Isoamylalcohol (24:1 [v/v]), polyvinylpyrrolidone (PVP) (40

000 mol wt) (Sigma), ß-mercaptoethanol. All the chemicals used in the experiments were of analytical

grade. The extraction buffer which included 100 mM Tris –HCl (pH 8), 25 mM EDTA (pH 8), and 2

M NaCl. The PVP and ß-mercaptoethanol were freshly prepared.

3.3.10.2 DNA Extraction

DNA was isolated from young seedlings grown using a modified CTAB method (Khan et al., 2007).

The young seedlings after 15 days of sowing were ground into extraction buffer. The suspension was

70

gently mixed and incubated at 65ºC for 20 minutes with occasional mixing. The suspension was then

cooled to room temperature and an equal volume of chloroform: isoamyl alcohol (24:1) was added.

The mixture was centrifuged at 12,000 rpm for 5 min. The clear upper aqueous phase was then

transferred to a new tube, ice-cooled isopropanol added and incubated at -20 ºC for 30 minutes. The

nucleic acid was collected by centrifugation at 10,000 rpm for 10 minutes. The resulting pellet was

washed twice with 80 % ethanol. The pellet was air-dried under a sterile laminar hood and the nucleic

acid was dissolved in TE (10 mM tris buffer pH 8, 1 mM EDTA) at room temperature and stored at

4ºC until used. The RNA from crude DNA was eliminated by treating the sample with RNase (10

mg/ml) for 30 min at 37 ºC. DNA concentration and purity were determined by measuring the

absorbance of diluted DNA solution at 260 nm and 280 nm. The quality of the DNA was determined

using agarose gel electrophoresis stained with ethidium bromide.

3.3.10.3 DNA Quantification and Quality Analysis using Agarose Gel Electrophoresis

1.4% agarose was prepared using 2.8g in 200mls, 1 x TAE (Tris Acetate EDTA), microwave to

dissolve the agarose and allowed to cool to room temperature before it was poured into the gel tray. 1

µl of 6 x gel loading dye was added to 2-3µl of each DNA sample before loading into the wells of the

gel. At least one or two wells were loaded with quality DNA samples (50ng and 100ng) as molecular

weight standards. The submarine electrophoretic gel was run at 70V for 2hours till the dye migrated

one-third of the distance in the gel. DNA was visualized using a UV transilluminator and qualified in

comparison with the florescent yield of the standards (Khan et al., 2007 and Ogundipe and

Ogunkanmi, 2010).

Staining

The gel was stained in 2.5mg/l Ethidium Bromide for 3minutes and further distained for 10 minutes in

sterile water and then viewed under UV light and the picture was taken.

71

Dilution of DNA for PCR

About 10 µl of each DNA was taken into eppendorf tube together with 90µl Sterile distilled water to

have 20-50ng/µl (1:10 dilution).

PCR Amplification

RAPD reactions were assembled and mixed to create a cocktail of 20 µl total volume per unit. The

sample was mixed thoroughly and centrifuged briefly to bring down the contents of the tube. They

were then placed in the thermo cycler machine for DNA amplification. The PCR was carried out with

the RAPD profile of 45 cycles preheated at 94oC for 3 min, 94

oC denaturation for 30 s, 37

oC for 40 s

annealing and 72oC final extension for 7 minutes.

Gel Scoring and Data Analysis

Fragments that were clearly resolved on gels were scored as 1 or 0 for present or absent, respectively,

while bands that could not be confidently scored were regarded as missing data. Pair wise distance

(similarity) matrices were computed using sequential, hierarchical and nested (SAHN) clustering

option of the NTSYS-pc version 2.02j software package (Rohlf, 2006). The program generated a

dendrogram, which grouped the tested plants into clusters.

3.3.11 Statistical Analysis

All the experiments were conducted in triplicates. All data collected were analyzed using standard

deviation, t-test and analysis of variance (ANOVA) for statistical significance at 95% confidence

interval. Analysis of variance was performed on each measured variable, means and standard errors

(SE) were calculated. Descriptive statistics were calculated using the Microcal origin 5.0 and

Microsoft Excel. Graphical illustrations were also carried out to get vivid representation of the data

obtained.

72

CHAPTER FOUR

4.0 RESULTS

4.1 THE SCREENING TEST

4.1.1 Percentage Germination

Tables 4.1 to 4.3 show the percentage germination of the six cultivars used for screening. Germination

of seeds began 3 (three) days after planting in the control and treated cowpea and maize, while seed

germination began on the sixth day for bambara groundnut in both control and treated. However, the

percentage of seeds that germinated varies according to the concentration of the heavy metals applied

and also with time (in days). The percentage germination in all treated seeds decreased generally with

increased metal concentrations and increased with the number of days till there was no more

germination. In cowpea that did not receive heavy metal treatment, by the fourth day, the germination

rate was 50.0% (12 seeds) and 75.0% (18 seeds), 37.5% (9 seeds) and 62.5 % (15 seeds) for accession

Tvu 3788 and IT 99K-377-1 respectively. Germination then increased till the sixth day to 100% (24

seeds) and 75% (18 seeds) for accession Tvu 3788 and IT 99K-377-1 respectively. For cowpea treated

with lead and zinc nitrate, germination decreased with increased heavy metal concentration but

increased with number of days. Cowpea treated with 100 mg/kg of lead nitrate and 200 mg/kg of lead

nitrate had the percentage germination of 37.5% and 25% respectively for accession Tvu 3788 while

accession IT 99K-377-1 had 25% and 12.5% respectively by the third day. The percentage

germination became 75.0 % and 62.5 % in cowpea treated with 100 mg/kg of lead nitrate and 200

mg/kg of lead nitrate respectively for accession Tvu 3788 by the fifth day. This percentage was

maintained till the tenth day. However, by the fifth day, accession IT 99K-377-1 had 50% germination

in all the different concentrations. This percentage was also maintained till the tenth day.

73

In bambara groundnut, by the sixth and seventh day, the control plant had 25.0% (6 seeds) and 50.0%

(12 seeds), 50.0% (12 seeds) and 75.0 % (18 seeds) germination for accessions Tvsu 1685 and Tvsu

102 respectively. Germination then increased till the eight day and maintained at 75% (18 seeds) for

accession Tvsu 1685 and 87.5 % (21 seeds) for accession Tvsu 102. These values were maintained till

the end of the experiment. For bambara groundnut treated with lead and zinc nitrate, germination

decreased with increased heavy metal concentration but increased with number of days. By the eight

day, bambara groundnut plant treated with100 mg/kg of lead nitrate and 200 mg/kg of lead nitrate, had

percentage germination of 62.5% and 50% respectively, for accession Tvsu 102 while accession Tvsu

1685 had 37.5% and 50% respectively. These values were stabilized till the end of the experiment. For

bambara groundnut plants treated with zinc nitrate, by the ninth day, percentage germination was

62.5% and 50% in 100 mg/kg of zinc nitrate and 200 mg/kg of zinc nitrate respectively for accession

Tvsu102. This percentage was maintained till the tenth day. However, by the ninth day, accession

Tvsu 1685 had 55.0% and 37.5% germination in 100 mg/kg of zinc nitrate and 200 mg/kg of zinc

nitrate respectively.

In maize, by the fourth day, the untreated seedlings had percentage germination of 62.5% (15 seeds)

and 75.0 % (18 seeds) for accessions DMR-LSRW and ACR.91SUWANI-SRC1 respectively.

Germination increased till the fifth day and was stabilized at 75% (18 seeds) for accession DMR-

LSRW and 100.0 % (24 seeds) for accession ACR.91SUWANI-SRC1. These values were maintained

till the end of the experiment. For maize treated with lead and zinc nitrate, germination decreased with

increased heavy metal concentration. The percentage germination was 75.0% and 62.5% in maize

plants treated with 100 mg/kg of zinc nitrate and 200 mg/kg of lead nitrate respectively for accession

DMR-LSRW while accession ACR.91SUWANI-SRC1 had 87.5% and 75% respectively by the fifth

day. These values were stabilized till the end of the experiment. For maize plants treated with lead

74

nitrate, by the seventh day, percentage germination was 62.5% and 62.5% for seeds treated with 100

mg/kg and 200 mg/kg of lead nitrate respectively for accession DMR-LSRW. This percentage was

maintained till the tenth day. Also, for accession ACR.91SUWANI-SRC1 treated with 100mg/kg of

lead nitrate and 200 mg/kg of lead nitrate, percentage germination was 87.5% and 75.0% respectively

by the seventh day. This percentage was also maintained.

75

Table 4.1: Percentage Germination of Cowpea seeds (Accession Tvu 3788) and (Accession IT 99K-377-1)

in different concentrations of Lead and Zinc Nitrate

Days after

Treatment

control

Accession Tvu 3788 control Accession IT 99K-377-1

Lead nitrate concentrations

(mg/kg)

Zinc nitrate

concentrations (mg/kg)

Lead nitrate


Zinc nitrate


100 150 200 100 150 200 100 150 200 100 150 200

3 50.0

37.5

37.5

25.0

62.5

50.0

37.5

37.5 25.0 25.0 12.5 50.0 37.5 25.0

4 75.0

50.0

50.0

50.0

62.5

50.0

50.0

62.5 37.5 37.5 37.5 50.0 37.5 37.5

5 87.5

75.0

75.0

62.5

75.0

62.5

62.5

75.0 50.0 50.0 50.0 62.5 50.0 50.0

6 100.0

75.0

75.0

62.5

87.5

75.0

62.5

75.0 50.0 50.0 50.0 62.5 50.0 50.0

7 100.0

75.0

75.0

62.5

87.5

75.0

62.5

75.0 50.0 50.0 50.0 62.5 50.0 50.0

8 100.0

75.0

75.0

62.5

87.5

75.0

62.5

75.0 50.0 50.0 50.0 62.5 50.0 50.0

9 100.0

75.0

75.0

62.5

87.5

75.0

62.5

75.0 50.0 50.0 50.0 62.5 50.0 50.0

10 100.0

75.0

75.0

62.5

87.5

75.0

62.5

75.0 50.0 50.0 50.0 62.5 50.0 50.0

76

Table 4.2: Percentage Germination of Bambara groundnut seeds (Accession Tvsu 1685) and (Accession

Tvsu 102) in different concentrations of Lead and Zinc Nitrate

Days after

Treatment

control

Accession Tvu 1685 control Accession Tvsu 102


(mg/kg)

Zinc nitrate


Lead nitrate


Zinc nitrate


100 150 200 100 150 200 100 150 200 100 150 200

6 25.0

25.0

25.0

25.0

25.0

25.0

25.0

50.0 37.5 37.5 37.5 50.0 37.5 37.5

7 50.0

37.5

37.5

25.0

25.0

25.0

25.0

75.0 50.0 50.0 50.0 50.0 37.5 37.5

8 75.0

50.0

37.5

37.5

25.0

37.5

37.5

87.5 62.5 62.5 50.0 62.5 50.0 50.0

9 75.0

50.0

37.5

37.5

50.0

37.5

37.5 87.5 62.5 62.5 50.0 62.5 50.0 50.0

10 75.0

62.5

37.5

37.5

50.0

50.0

50.0

87.5 75.0 62.5 62.5 75.0 62.5 62.5

11 75.0

62.5

37.5

37.5

50.0

50.0

50.0

87.5 75.0 62.5 62.5 75.0 62.5 62.5

12 75.0

62.5

37.5

37.5

50.0

50.0

50.0

87.5 75.0 62.5 62.5 75.0 62.5 62.5

13 75.0

62.5

37.5

37.5

50.0

50.0

50.0

87.5 75.0 62.5 62.5 75.0 62.5 62.5

77

Table 4.3: Percentage Germination of Maize seeds (Accession DMR-LSRW) and (Accession

ACR.91SUWANI-SRC1) in different concentrations of Lead and Zinc Nitrate

Days after

Treatment

control Accession DMR-LSRW control Accession ACR.91SUWANI-SRC1


(mg/kg)

Zinc nitrate


Lead nitrate


Zinc nitrate


100 150 200 100 150 200 100 150 200 100 150 200

3 50.0

37.5

25.0

25.0

50.0

37.5

37.5

50.0 25.0 25.0 25.0 62.5 50.0 50.0

4 62.5

37.5

25.0

25.0

62.5

50.0

50.0

75.0 50.0 37.5 37.5 75.0 62.5 62.5

5 75.0 50.0

37.5

50.0

75.0

62.5

62.5

87.5 75.0 62.5 62.5 87.5 87.5 75.0

6 75.0

62.5

62.5

50.0

75.0

62.5

62.5

100.0 87.5 75.0 75.0 87.5 87.5 87.5

7 75.0

62.5

62.5

62.5

75.0

62.5

62.5

100.0 87.5 75.0 75.0 87.5 87.5 87.5

8 75.0

62.5

62.5

62.5

75.0

62.5

62.5

100.0 87.5 75.0 75.0 87.5 87.5 87.5

9 75.0

62.5

62.5

62.5

75.0

62.5

62.5

100.0 87.5 75.0 75.0 87.5 87.5 87.5

10 75.0

62.5

62.5

62.5

75.0

62.5

62.5

100.0 87.5 75.0 75.0 87.5 87.5 87.5

78

4.2 MACROMORPHOMETRIC PARAMETERS AND CHLOROPHYLL CONTENT

FOLLOWING METAL TREATMENT AND AUGMENTATION

4.2.1 Morphometric Parameters (Leaf Area, Stem Height, Root length, Fresh and Dry weight

Characteristics)

Leaf Area/Size (cm2)

Tables 4.4 to 4.6 show the effect of different concentrations of lead and zinc nitrate on leaf-sizes

of experimental seedlings of cowpea, bambara groundnut and maize. Leaf-size increased with

time (in days) in each treatment concentration whereas it decreased as the concentration of the

heavy metals increased. Statistical analysis revealed that the treatment effects on the leaf sizes

were significantly different (P<0.05) from control for all treated plants, except for cowpea treated

with zinc nitrate which was not significantly different (P>0.05) from control. The largest leaf size

was observed on the 30th

day in the control (untreated) with a leaf size of 26.18cm2 ± 0.82 while

the least leaf size of 12.24cm2 ± 0.12 was observed for seedlings treated with 200mg/kg of Pb on

the 30th

day as well.

Stem Height (cm)

Tables 4.7 to 4.9 show the effects of different concentration of two heavy metals on stem heights of

experimental seedlings. The stem height increased with time (in days) in each treatment

concentration while it decreases as the treatment concentration increased. Statistical analysis

revealed that the treatment effects were highly significant (P<0.05) on stem heights for the three

plants. The seedlings of cowpea in the control experiment were observed to have the highest mean

stem height (cm) of 70.0±0.14 compared with the treated cowpea seedlings of 200mg/kg Pb with

28.0±0.32. The untreated seedlings of bambara groundnut were also observed to have the highest

mean stem height of 20.0±1.05 while seedlings treated with 200mg/kg of Pb had the least stem

79

height (cm) as 12.0±1.04 In the same manner, the untreated seedlings of maize were also observed

to have the highest mean stem height (cm) of 14.0 ±0.08 while seeds treated with 200mg/kg of Pb

had the least stem height (cm) as 4.0±0.05 at the end of thirty days.

Root length (cm)

Tables 4.10 to 4.12 show the effects of different concentration of two heavy metals on root lengths

of experimental seedlings. As observed, the root length increased with time (in days) in each

treatment concentration while it decreases as the treatment concentration increased. The untreated

seedlings of cowpea were observed to have the highest mean root length (cm) of 70.0±1.14

compared with the treated cowpea seedlings of 200mg/kg Pb with 28.0 ±0.32 (cm). The untreated

seedlings of bambara groundnut were also observed to have the highest mean root length of

8.0±0.26 while seeds treated with 200mg/kg of Pb had the least root length (cm) as 7.0±0.17. In

the same manner, the control seedlings of maize were also observed to have the highest mean root

length (cm) of 17.10±0.45 while seedlings treated with 200mg/kg of Pb had the least root length

(cm) as 7.0 ±0.48 at the end of thirty days. Statistical analysis revealed that the treatment effects

were highly significant (P<0.05) on root length for the three plants when compared to the plants

that did not receive heavy metal treatment. Generally, the growth rate of roots and shoots were

found to be retarded with increasing concentrations of lead and zinc nitrate for the three test plants.

80

Table 4.4: Effects of Different Concentrations of Lead and Zinc Nitrate on Leaf Area (cm2) of

Cowpea (Vigna unguiculata, Accession Tvu 3788)

Days Treatment Concentrations

Control 100mg/kg

Pb

150mg/kg Pb 200mg/kg Pb 100mg/kg

Zn

150mg/kg Zn 200mg/kg Zn

Mean Leaf

Area ± SEM

Mean Leaf

Area ± SEM Mean Leaf





Area ± SEM

10 10.40 ± 1.12 6.42±0.09* 7.21±1.03* 6.14± 1.10* 16.30±0.12 15.32±0.12 13.44±0.12

15 14.15± 1.47 8.46±0.80* 9.14± 1.24* 8.18± 0.61* 18.45±0.12 17.24±0.12 15.50±0.12

20 16.50± 1.14 10.24±0.08* 11.12±0.22* 9.30± 0.19* 20.25±0.12 19.60±0.12 17.50±0.12

25 18.20± 1.22 12.20±1.04* 13.24±0.37* 11.28± 0.52* 22.50±0.12 21.30±0.12 19.20±0.12

30 26.18± 0.82 14.38±1.10* 16.30±0.68* 12.24± 0.12* 26.40±0.12 23.15±0.12 20.65±0.12

• Data were expressed as Mean ± SEM

• When * P<0.05 = Significantly different from control and When P>0.05 = Not significantly

different from control

81

Table 4.5: Effect of Different concentrations of Lead and Zinc Nitrate on Leaf Area (cm2) of

Bambara groundnut (V.Subterranean, Accession Tvsu 102)


Control 100mg/kg

Pb

150mg/kg

Pb

200mg/kg

Pb

100mg/kg

Zn

150mg/kg

Zn

200mg/kg

Zn

Mean Leaf







Area ± SEM

10 6.18±0.10 2.16± 0.44* 3.52± 0.02* 3.70± 0.10* 5.30± 0.37* 5.10 ± 0.17* 4.40±0.12*

15 7.20±0.12 4.42 ±0.62* 4.42±0.50* 4.20± 0.05* 6.45± 0.67* 6.10 ± 0.12* 6.00±0.23*

20 9.30±1.08 5.66± 0.82* 5.12±0.02* 5.50± 1.01* 7.20± 0.30* 8.20 ± 0.34* 7.10±0.46*

25 11.44±0.61 7.40± 0.21* 6.28±0.16* 6.00± 0.02* 9.40± 0.34* 9.50 ± 1.23* 8.20±0.49*

30 13.32±1.04 8.00± 0.27* 7.07±0.70* 6.70± 0. 07* 11.00±0.62* 10.80 ±0.49* 8.50±0.38*

• Data were expressed as MEAN ± SEM



82

Table 4.6: Effect of Different concentrations of Lead and Zinc Nitrates on Leaf Area (cm2) of

Maize (Zea mays Accession, ACR.91SUWANI-SRC1)


Control 100mg/kg

Pb

150mg/kg

Pb

200mg/kg

Pb

100mg/kg

Zn

150mg/kg

Zn

200mg/kg

Zn

Mean Leaf







Area ± SEM

10 24.00 ±1.28 22.30±2.29* 20.20±2.03* 18.30±1.28* 20.40±1.22* 18.20±1.20* 12.10±0.21*

15 30.40±2.77 28.20±3.06* 24.00±3.10* 22.50±1.29* 22.10±1.07* 20.30±0.86* 14.20± 0.80*

20 34.50±2.09 32.50±2.17* 30.20±1.45* 26.45±2.17* 26.15±1.15* 22.40±1.10* 16.10±1.23*

25 38.20±1.03 38.30±1.19* 36.50±2.01* 32.35±1.24* 28.20±0.61* 24.35±0.07* 18.00±1.02*

30 43.00±1.11 44.60±2.32* 40.65±0.89* 36.00±1.19* 32.30±0.72* 26.50±0.04* 22.40±0.04*




83

Table 4.7: Effect of Different concentrations of Lead and Zinc Nitrates on Stem Length (cm) of



Control 100mg/kg

Pb

150mg/kg

Pb

200mg/kg

Pb

100mg/kg Zn 150mg/kg

Zn

200mg/kg

Zn

Mean Stem

Length ±

SEM

Mean Stem

Length ± SEM Mean Stem





Length ± SEM

10 20.00±0.02 15.00±2.08* 16.00±1.32* 15.00±1.13* 18.00± 2.08 15.00±3.72* 13.00±3.14*

15 28.00±0.04 18.00±2.14* 19.00±1.60* 18.00±1.32* 26.00±1.85 25.00±3.39* 20.00±4.21*

20 44.00±0.08 23.00±1.56* 21.50±1.67* 21.00±1.02* 40.00±3.64 36.00±3.21* 34.00±3.82*

25 56.00±0.04 28.00±2.01* 29.00±1.45* 25.00±1.44* 55.00±3.01 49.00±4.11* 44.00±2.62*

30 70.00±0.14 36.00±0.55* 32.00±0.72* 28.00±0.32* 75.00±2.44 62.00±4.26* 58.00±4.32*




84

Table 4.8: Effect of Different concentrations of Lead and Zinc Nitrates on Sem Length (cm) of

Bambara groundnut (V.subterranean, Accession Tvsu 102)


Control 100mg/kg

Pb

150mg/kg

Pb

200mg/kg

Pb

100mg/kg

Zn

150mg/kg

Zn

200mg/kg

Zn

Mean Stem


Length ±SEM Mean Stem





Length ±SEM

10 10.00±0.80 6.00±0.98* 5.00±0.08* 4.00±0.08* 9.00±0.38* 7.00±0.65* 5.00±0.36*

15 12.00±0.77 8.00±1.10* 7.00±0.44* 6.00±0.91* 12.00±0.72 9.00±0.72* 8.00±1.18*

20 14.00±1.20 10.00±1.23* 9.00±2.17* 8.00±0.07* 14.00±0.58 11.00±0.69* 11.00±0.12*

25 17.00±0.16 13.00±0.11* 11.00±0.30* 10.00±1.06* 16.00±1.01* 13.00±0.44* 13.00±0.93*

30 20.00±1.05 15.00±0.18* 13.00±0.17* 12.00±1.04 18.00±1.70* 16.00±1.27* 14.00±1.11*




85

Table 4.9: Effect of Different concentrations of Lead and Zinc Nitrates on Stem Length (cm) of

Maize (Zea mays, Accession ACR.91SUWANI-SRC1)


Control 100mg/kg

Pb

150mg/kg

Pb

200mg/kg

Pb

100mg/kg

Zn

150mg/kg

Zn

200mg/kg

Zn

Mean Stem







Length ± SEM

10 15.00±1.75 6.00±0.21* 5.00±0.54* 4.00±0.52* 14.00±1.20* 12.00±0.83* 6.00±0.48*

15 20.00±3.07 8.00±0.72* 7.00±0.87* 6.00±0.44* 18.00±1.16* 14.00±0.87* 8.00±0.50*

20 28.00±2.11 11.00±1.10* 9.00±0.56* 8.00±0.45* 24.00±0.89* 18.00±0.92* 9.00±0.41*

25 32.00±1.04 13.00±0.89* 12.00±0.71* 9.00±0.52* 26.00±1.56* 20.00±0.65* 10.00±0.39*

30 35.00±2.48 16.00±1.04* 13.00±0.67* 10.00±0.48* 28.00±1.02* 22.00±0.88* 11.00±0.14*




86

Table 4.10: Effect of Different concentrations of Lead and Zinc Nitrates on Root Length (cm) of



Control 100mg/kg

Pb

150mg/kg

Pb

200mg/kg

Pb

100mg/kg

Zn

150mg/kg

Zn

200mg/kg

Zn

Mean Root

Length ±

SEM

Mean Root

Length ± SEM Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

10 11.00±0.23 9.40±0.19* 7.50±0.22* 5.30±0.11* 7.00±0.37* 6.00±0.12* 4.50±0.23*

15 12.00±0.30 9.00±0.11* 7.00±0.12* 4.80±0.2* 7.20±0.04* 7.00±0.17* 5.00±0.19*

20 13.00±0.19 8.60±0.10* 6.80±0.08* 4.50±0.07* 7.50±0.67* 7.30±0.29* 5.50±0.21*

25 13.50±0.40 8.30±0.09* 6.30±0.09* 4.20±0.08* 8.00±0.30* 8.00±0.18* 6.00±0.14*

30 14.00±0.08 8.00±0.07* 6.00±0.08* 4.00±0.05* 9.00±0.16* 8.50±0.20* 6.50±0.02*




87


Bambara groundnut (V.subterranean, Accession Tvsu 102 )


Control 100mg/kg

Pb

150mg/kg

Pb

200mg/kg

Pb

100mg/kg

Zn

150mg/kg

Zn

200mg/kg

Zn

Mean Root

Length ±

SEM

Mean Root


Length ±

SEM

Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

10 6.00±0.16 5.00±0.25* 5.50±0.27* 5.40±0.26* 4.50±0.40* 5.60±0.13* 5.80±0.06*

15 7.00±0.19 5.50±0.26* 6.00±0.30* 6.50±0.11* 5.20±0.27* 6.20±0.08* 6.00±0.22*

20 7.20±0.09 6.50±0.17* 8.00±0.15* 6.80±0.09* 5.80±0.10* 6.50±0.13* 6.20±0.07*

25 7.50±0.04 7.20±0.30* 8.40±0.36* 7.50±0.15 6.20±0.07* 6.80±0.16* 6.60±0.12*

30 8.00±0.26 8.00±0.24 8.20±0.32 7.00±0.17* 6.80±0.05* 7.40±0.17* 7.20±0.09*




88


Maize (Zea mays, Accession ACR.91SUWANI-SRC1)


Control 100mg/kg

Pb

150mg/kg

Pb

200mg/kg

Pb

100mg/kg

Zn

150mg/kg

Zn

200mg/kg

Zn

Mean Root

Length ±

SEM

Mean Root


Length ±

SEM

Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

Mean Root

Length ±

SEM

10 14.00±0.30 3.50±0.97* 6.00±0.07* 5.00±0.09* 7.00± 0.18* 6.00±0.02* 5.00±0.30*

15 15.20±0.11 8.00±0.79* 7.00±0.32* 6.80±0.06* 7.50±0.27* 6.50±0.06* 6.00±0.02*

20 16.00±0.23 9.00±0.34* 8.20±0.45* 8.00±0.07* 8.00±0.03* 7.00±0.13* 7.00±0.12*

25 16.80±0.12 10.00±0.42* 8.50±0.16* 8.50±0.65* 9.00±0.04* 7.20±0.11* 7.50±0.17*

30 17.20±0.45 9.50±0.10* 8.00±0.03* 7.00±0.48* 9.80±0.62* 7.50±0.22* 8.00±0.59*




89

Fresh and Dry Weights of Plants

Tables 4.13 to 4.16 show the effects of different lead and zinc nitrate concentrations and augmentation

on the fresh and dry weights of cowpea, bambara groundnut and maize. The fresh weight of plants

increased significantly with increase in lead treatment except for bambara groundnut. The fresh

weights of all treated bambara groundnut plants were reduced significantly by all concentrations of

lead and zinc nitrate. The fresh weights of treated plants were significantly lower (P< 0.05) than the

untreated plants for each crop plant. However, addition of a chelator and manure assisted the growth

of these three plants by causing an increase in the fresh weights of treated plants compared to when no

manure or chelator was added. For instance, the untreated cowpea plants had a mean fresh weight (g)

of 6.05±1.0 while cowpea plants treated with 100mg/kg and 200mg/kg lead concentrations had mean

fresh weights (g) of 6.45±1.0 and 25.34±1.0 respectively. Cowpea plants with 200mg/kg of lead

augmented with EDTA gave a mean fresh weight (g) of 19.45±1.0, while the 200mg/kg of lead and

manure gave 28.10±1.0. The untreated bambara groundnut plant had a mean fresh weight (g) of

4.44±0.06, 7.56±0.04 at 100mg/kg of lead concentration and 4.47±0.02 at 200mg/kg of lead

concentration, indicating decrease in fresh weight with increased concentration. Bambara groundnut

plants with 200mg/kg of lead augmented with EDTA gave a mean fresh weight (g) of 2.14±0.03,

while the 200mg/kg of lead augmented with manure gave 4.12±0.04 (g). Maize control plants had

4.24±0.09 mean fresh weight (g), maize treated with 100mg/kg and 200mg/kg of lead concentration

had a mean fresh weight (g) of 5.93±0.02 and 28.54±0.04 respectively. Maize plants with 200mg/kg of

lead augmented with EDTA gave a mean fresh weight (g) of 11.30±1.0, while the 200mg/kg of lead

augmented with manure gave 10.18±0.03 (g). The same trend was observed for the different zinc

concentrations but the effect was at a lower magnitude.

90

The dry weight of plants decreased significantly with increased lead and zinc supply for all treated

plants (P< 0.05) when compared with the untreated plants for each crop plant. The dry weights of all

treated plants were affected significantly at all concentrations. However, addition of a chelator and

manure assisted and caused tremendous increase in the dry weights of plants treated with zinc nitrates.

Farmyard manure assisted all plants treated with lead nitrate more than the chelator (EDTA). The

untreated cowpea plants had a mean dry weight (g) of 3.14±0.12. Cowpea plant treated with 100mg/kg

lead nitrate had mean dry weights (g) of 1.36±0.03 while cowpea plants treated with 200mg/kg lead

nitrate concentration had a mean dry weight (g) of 1.07±0.06. Cowpea plants treated with 200mg/kg of

Pb augmented with EDTA gave a mean dry weight (g) of 1.79±0.02, while the plants treated with

200mg/kg of lead augmented with manure gave 2.04±0.10. Statistical analysis showed that higher

concentrations of the metals decreased dry weights of treated plants significantly (P< 0.05).

The untreated (control) bambara groundnut plant had a mean dry weight (g) of 2.16± 0.05, 1.14±0.03

at 100mg/kg of lead concentration and 0.41± 0.01 at 200mg/kg of lead concentration. Bambara

groundnut plants treated with 200mg/kg of lead augmented with EDTA gave a mean dry weight (g) of

0.82±0.03, while the 200mg/kg of lead augmented with manure gave 1.87±0.03. Untreated maize

plants had 3.08±0.03 mean dry weight (g), maize treated with 100mg/kg and 200mg/kg of lead

concentration had a mean dry weight (g) of 2.02±0.04 and 1.81±0.02 respectively. Maize plants

treated with 200mg/kg of lead augmented with EDTA gave a mean dry weight (g) of 1.90±0.13, while

the 200mg/kg of lead augmented with manure gave a mean dry weight (g) of 2.82±0.03.

Farmyard manure assisted all plants treated with zinc nitrate more than the chelator (EDTA). The

untreated cowpea plants had a mean dry weight (g) of 3.14±0.12. Cowpea plant treated with 100mg/kg

zinc nitrate had mean dry weights (g) of 1.87±0.12 while cowpea plants treated with 200mg/kg zinc

nitrate concentration had a mean dry weight (g) of 1.73±0.03. Cowpea plants treated with 200mg/kg of

91

Zn augmented with EDTA gave a mean dry weight (g) of 2.24±0.13, while the plants treated with

200mg/kg of Zn augmented with manure gave 2.93±0.02. Statistical analysis showed that higher

concentrations of the metals decreased dry weights of treated plants significantly (P< 0.05).

The untreated (control) bambara groundnut plant had a mean dry weight (g) of 2.16± 0.05, 0.84±0.03

at 100mg/kg of zinc concentration and 0.37± 0.41 at 200mg/kg of zinc concentration. Bambara

groundnut plants treated with 200mg/kg of Zn augmented with EDTA gave a mean dry weight (g) of

0.78±0.04, while the 200mg/kg of Zn augmented with manure gave 0.67±0.03. Untreated maize plants

had 3.08±0.03 mean dry weight (g), maize treated with 100mg/kg and 200mg/kg of Zn concentration

had a mean dry weight (g) of 3.10±0.03 and 2.22±0.03 respectively. Maize plants treated with

200mg/kg of Zn augmented with EDTA gave a mean dry weight (g) of 1.64±0.02, while the 200mg/kg

of Zn augmented with manure gave a mean dry weight (g) of 3.29±0.06.

92

Table 4.13: Effect of Lead concentrations on Fresh Weight (grams) of the three Test Plants

Plants/ Treatment

concentration

Cowpea Bambara

groundnut

Maize

Mean Fresh

Weight± SEM

Mean Fresh

Weight± SEM

Mean Fresh

Weight± SEM

Control 6.05±0.04

4.44±0.06

4.24±0.09

100mg/kg Pb 6.45±0.07*

7.56±0.04*

5.93±0.02*

150mg/kg Pb 10.48±0.01*

5.92±0.03*

10.21±0.03*

200mg/kg Pb 25.34±0.08*

4.47±0.02*

28.54±0.04*

100mg/kg Pb+ EDTA 6.88± 1.0*

2.89±0.04*

4.18±0.01*

150mg/kg Pb+ EDTA 14.62±1.0*

2.56±0.01*

6.88±0.02*

200mg/kg Pb + EDTA 19.45±1.0*

2.14±0.03*

11.30±0.02*

100mg/kg Pb+ manure 24.13±1.20*

5.94±0.02*

7.97±0.01*

150mg/kg Pb+ manure 26.20±0.06*

4.70±0.01*

8.83±0.04*

200mg/kg Pb+ manure 28.10±0.04*

4.12±0.04*

10.18±0.03*


• When * P<0.05 = Significantly different from control

• When P>0.05 = Not Significantly different from control

93

Table 4.14: Effect of Zinc concentrations on Mean Fresh Weight (grams) of the three Test Plants

Plants/ Treatment

concentration

Cowpea Bambara

groundnut

Maize

Mean Fresh

Weight± SEM

Mean Fresh

Weight± SEM

Mean Fresh

Weight± SEM

Control 6.05±1.0

4.44±0.06

4.24±0.09

100mg/kg Zn 7.20 ± 0.02*

4.44 ± 0.01

6.13 ± 0.08*

150mg/kg Zn 11.13±0.03* 5.98 ±0.02* 7.50 ± 0.03*

200mg/kg Zn 25.29± 0.05 * 9.56± 0.03* 11.13±0.04*

100mg/kg Zn + EDTA 8.34± 0.01* 7.96 ± 0.04* 6.31 ± 0.02*

150mg/kg Zn + EDTA 12.36±0.02* 8.27±0.08* 13.20±0.01 *

200mg/kg Zn + EDTA 19.94±0.03*

10.59±0.07*

15.90±0.03*

100mg/kg Zn+ manure 6.72 ± 0.04*

5.36 ± 0.01*

8.64 ±0.02*

150mg/kg Zn + manure 14.94± 0.03*

7.28 ± 0.02*

10.58±0.01*

200mg/kg Zn + manure 18.21±0.02*

10.18±0.02*

13.28±0.03*




94

Table 4.15: Effect of Lead concentrations on Mean Dry Weight (grams) of the three Test Plants

Plants/Treatment

concentration

Cowpea Bambara

groundnut

Maize

Mean Dry

Weight± SEM

Mean Dry

Weight± SEM

Mean Dry

Weight± SEM

Control 3.14±0.12

2.16±0.05

3.08±0.03

100mg/kg Pb 1.36±0.03*

1.14±0.03*

2.02±0.04*

150mg/kg Pb 1.10±0.07*

0.98±0.02*

1.92±0.05*

200mg/kg Pb 1.07±0.06*

0.41±0.01*

1.81±0.02*

100mg/kg Pb+ EDTA 1.96±0.03*

1.32±0.16*

2.14±0.04*

150mg/kg Pb+ EDTA 1.88±0.02*

1.10±0.08*

2.07±0.08*

200mg/kg Pb + EDTA 1.79±0.02*

0.82±0.03*

1.90±0.13*

100mg/kg Pb+ manure 1.07±0.06*

0.75±0.03*

2.62±0.12*


1.20±0.10*

2.71±0.04


1.87±0.03*

2.82±0.03




95

Table 4.16: Effect of Zinc concentrations on Mean Dry Weight (grams) of the three Test Plants

Plants/Treatment

concentration

Cowpea Bambara

groundnut

Maize

Mean Dry

Weight± SEM

Mean Dry

Weight± SEM

Mean Dry

Weight± SEM

Control 3.14±0.12

2.16 ±0.05

3.08±0.03

100mg/kg Zn 1.87±0.12*

0.84 ±0.03*

3.10±0.03

150mg/kg Zn 1.79±0.02*

0.63 ±0.02*

2.44±0.05*

200mg/kg Zn 1.73±0.03*

0.37±0.41*

2.22±0.03*

100mg/kg Zn + EDTA 2.10±0.11*

0.56±0.05*

0.98±0.06*

150mg/kg Zn + EDTA 2.18±0.08*

0.72±0.03*

1.21±0.02*

200mg/kg Zn + EDTA 2.24±0.13*

0.78±0.04*

1.64±0.02*

100mg/kg Zn + manure 2.77±0.06*

0.75±0.03* 2.86±0.03*

150mg/kg Zn + manure 2.81±0.02*

0.86±0.02*

3.12±0.03

200mg/kg Zn + manure 2.93±0.02*

0.67±0.03*

3.29±0.06*




96

4.2.2 Chlorophyll Content

Tables 4.17 and 4.18 show the effects of the different concentrations of the metals on the total

chlorophyll content of treated plants. The chlorophyll content (mg/g) of treated plants decreased

significantly with increased lead and zinc concentrations in all treated plants. The chlorophyll contents

of all treated plants were affected significantly at all concentrations. Higher concentration of metals

decreased the quantity of total chlorophyll of plants significantly (P<0.05) when compared to the

untreated (control) plants. For cowpea, addition of a chelator (EDTA) and manure led to an increase in

the chlorophyll contents of plants treated with zinc and lead nitrates. For bambara groundnut, with

increased Pb concentrations in the presence of manure and EDTA, the chlorophyll content reduced.

However, there was an increase in the chlorophyll content with increased zinc nitrate concentrations

augmented with EDTA. Maize plants treated with Pb augmented with farmyard manure showed an

increase in chlorophyll content as concentrations of Pb increased while those assisted with EDTA still

experienced a decrease rather than an increase in chlorophyll contents as the metal concentrations

increased. The untreated (control) cowpea plants had mean chlorophyll content (mg/g) of 2.931 ±

0.05. Cowpea plants treated with 200mg/kg of Pb and EDTA gave a mean chlorophyll content (mg/g)

of 5.825 ± 0.05, while the 200mg/kg of lead and manure gave 5.102 ± 0.01 (mg/g). Cowpea plants

treated with the 100mg/kg and 200mg/kg lead nitrate concentrations had mean chlorophyll content

(mg/g) of 0.987±0.20 and 0.802 ± 0.02 respectively. The untreated (control) bambara groundnut plant

had mean total chlorophyll (mg/g) of 1.598 ± 0.001, bambara groundnut treated with 100mg/kg of lead

nitrate had a mean total chlorophyll (mg/g) of 2.944 ± 0.952 and the plant treated with 200mg/kg of

lead concentration had mean total chlorophyll (mg/g) of 2.002±0.616. Bambara groundnut plants with

200mg/kg of Pb augmented with EDTA gave mean chlorophyll content (mg/g) of 2.193±0.117, while

the 200mg/kg of Pb augmented with manure gave 5.418 ± 0.145. Maize control plants had mean

97

chlorophyll content of 2.350 ± 0.03 (mg/g). Maize treated with 100mg/kg and 200mg/kg of Pb

concentration had mean chlorophyll content (mg/g) of 1.242±0.02 and 1.092±0.01 respectively. Maize

plants treated with 200mg/kg of Pb augmented with EDTA gave mean chlorophyll content (mg/g) of

1.876±0.02, while the plant treated with 200mg/kg of Pb augmented with manure gave 1.621±0.04

(mg/g). Maize plants treated with Zn augmented with farmyard manure showed an increase in

chlorophyll content as concentrations of Zn increased while those assisted with EDTA still

experienced a decrease rather than an increase in chlorophyll contents as the metal concentrations

increased. The untreated (control) cowpea plants had mean chlorophyll content (mg/g) of 2.931 ±

0.001. Cowpea plants treated with 200mg/kg of Zn and EDTA gave a mean chlorophyll content

(mg/g) of 4.212 ± 0.16, while the 200mg/kg of Zn and manure gave 5.911 ± 0.07 (mg/g). Cowpea

plants treated with the 100mg/kg and 200mg/kg zinc nitrate concentrations had mean chlorophyll

content (mg/g) of 1.521±0.07 and 0.815 ± 0.02 respectively. The untreated (control) bambara

groundnut plant had mean total chlorophyll (mg/g) of 1.598 ± 0.001, bambara groundnut treated with

100mg/kg of zinc nitrate had a mean total chlorophyll (mg/g) of 3.649 ± 0.13 and the plant treated

with 200mg/kg of Zn concentration had mean total chlorophyll (mg/g) of 3.112±0.06. Bambara

groundnut plants with 200mg/kg of Zn augmented with EDTA gave mean chlorophyll content (mg/g)

of 4.918±0.21, while the 200mg/kg of Zn augmented with manure gave 5.816 ± 0.16. Maize control

plants had mean chlorophyll content of 2.350 ± 0.03 (mg/g). Maize treated with 100mg/kg and

200mg/kg of Zn concentration had mean chlorophyll content (mg/g) of 1.565±0.04 and 1.352±0.03

respectively. Maize plants treated with 200mg/kg of Zn augmented with EDTA gave mean chlorophyll

content (mg/g) of 3.989±0.07, while the plant treated with 200mg/kg of Zn augmented with manure

gave 6.101±0.15 (mg/g).

98

Table 4.17: Effect of Lead concentrations on Total Chlorophyll (mg/g) of the three Test Plants

Plants/Concentration Cowpea Bambara

groungnut

Maize

Mean value± SEM Mean value± SEM Mean value± SEM

Control 2.931±0.05

1.598±0.001

2.350±0.03

100mg/kg Pb 0.987±0.20*

2.944±0.952*

1.242±0.02*

150mg/kg Pb 0.921±0.01*

2.418±0.202* 1.108±0.02*

200mg/kg Pb 0.802±0.02*

2.002±1.616* 1.092±0.01*

100mg/kg Pb+ EDTA 1.127±0.15*

2.872±0.618*

2.028±0.03*

150mg/kg Pb+ EDTA 5.179±0.17*

2.648±0.320*

1.912±0.12*

200mg/kg Pb + EDTA 5.825±0.05*

2.193±0.117*

1.876±0.02*

100mg/kg Pb+ manure 1.612±0.03*

5.712±0.100*

1.308±0.01*

150mg/kg Pb+ manure 4.311±0.06*

5.528±0.150*

1.475±0.03*

200mg/kg Pb+ manure 5.102±0.01*

5.418±0.145*

1.621±0.04*



• When P >0.05 = Not significantly different from control

99

Table 4.18: Effect of Zinc concentrations on Total Chlorophyll (mg/g) of the three Test Plants

Plants/Concentration Cowpea Bambara

groundnut

Maize

Mean value± SEM Mean value± SEM Mean value± SEM

Control 2.931±0.05

1.598±0.001

2.350±0.03

100mg/kg Zn 1.521±0.07*

3.649±0.13*

1.565±0.04*

150mg/kg Zn 1.311±0.05*

3.501±0.1*

1.486±0.05*

200mg/kg Zn 0.815±0.02*

3.112±0.06*

1.352±0.03*

100mg/kg Zn + EDTA 3.819±0.03*

3.712±0.09*

4.141±0.10*

150mg/kg Zn + EDTA 4.103± 0.06*

4.518±0.10*

4.102±0.11*

200mg/kg Zn + EDTA 4.212±0.16*

4.918±0.21*

3.989±0.07*

100mg/kg Zn + manure 2.312±0.57

4.612±0.09*

5.122±0.11*

150mg/kg Zn + manure 4.416±0.01*

5.622±0.18*

5.893±0.06*

200mg/kg Zn + manure 5.911±0.07*

5.816±0.16*

6.101±0.15*



• When P > 0.05 = Not significantly different from control

100

4.3 EFFECTS OF HEAVY METALS ON THE ENZYMATIC ACTIVITIES AND LIPID

PEROXIDATION OF TREATED PLANTS

4.3.1 Effects of Heavy Metals on the Enzymatic Activities and Lipid Peroxidation of Treated

and Untreated Cowpea Plants

Table 4.19 shows the effect of lead and zinc treatment on superoxide dismutase (SOD), peroxidase,

gluathione (GSH), catalase activities and the level of malondialdehyde (MDA) in cowpea. Cowpea

plants treated with zinc nitrate experienced significantly increased enzyme activity compared to

control (P < 0.05). Cowpea plants treated with lead nitrate experienced significantly increased enzyme

activity except for GSH when compared to untreated (control) plants (P < 0.05). Cowpea plants treated

with 150mg/kg and 200mg/kg lead nitrate as well as 100mg/kg zinc nitrate experienced no significant

difference in the activities of GSH when compared to the untreated (control) plants (P> 0.05). Cowpea

plants treated with 100mg/kg Zn did not experience a significant difference in the level of MDA

compared to the untreated (control) plants.

101

Table 4.19: The Effect of different concentrations of Lead and Zinc nitrate on some Enzymes

and Lipid Peroxidation in Cowpea

TREATMENT SOD (µ/mg)

PEROXIDASE

(µ /mg)

GSH (µ /mg) CAT (µ /mg)

MDA (µ /mg)

Control 0.61±0.03 16.10±0.15 0.12±0.03 16.28±0.18 0.004±0.002

100mg/kg Lead 0.66±0.02* 15.65±0.08* 0.07±0.01* 19.98±0.22* 0.074±0.013*

150mg/kg Lead 0.74±0.02* 18.65±0.24* 0.09±0.01 20.83±0.08* 0.095±0.002*

200mg/kg Lead 0.98±0.02* 21.86±0.05* 0.15±0.01 22.41±0.21* 0.135±0.005*

100mg/kg Zinc 0.71±0.06* 22.04±0.20* 0.17±0.04 20.09±0.05* 0.008±0.003

150mg/kg Zinc 2.45±0.04* 42.39±0.28* 0.20±0.02* 69.12±0.10* 0.169±0.008*

200mg/kg Zinc 2.87±0.04* 46.87±0.15* 0.22±0.03* 73.12±0.10* 0.175±0.009*

• The values are the Means + SEM (range)

• When * P< 0.05 = Significantly different from control


102

4.3.2 Effects of Heavy Metals on the Enzymatic Activities and Lipid perpoxidation of Treated

and Untreated Bambara groundnut Plants

Table 4.20 shows the effect of lead and zinc treatment on superoxide dismutase (SOD), peroxidase,

gluathione (GSH), catalase activities and the level of malondialdehyde (MDA) in Bambara groundnut.

The effect of Zn and Pb treatment significantly decreased enzymes activity compared to control (P <

0.05) except for the MDA in plants treated with 100mg/kg zinc nitrate. The SOD and Catalase

activities were significantly reduced, while the MDA increased.

103

Table 4.20: The Effect of Different concentrations of Lead and Zinc Nitrate on some Enzymes

and Lipid Peroxidation in Bambara groundnut


PEROXIDASE

(µ /mg)


MDA (µ /mg)

Control 2.57±0.05 53.42±0.83 0.45±0.03 72.42±0.39 0.033±0.007

100mg/kg Lead 1.00±0.03* 38.48±0.53* 0.12±0.03* 40.08±0.24* 0.068±0.005*

150mg/kg Lead 0.72±0.02* 20.86±0.14* 0.09±0.02* 20.35±0.35* 0.071±0.002*

200mg/kg Lead 0.68±0.05* 20.10±0.14* 0.06±0.01* 18.65±0.35* 0.092±0.002*

100mg/kg Zinc 1.86±0.05* 51.44±0.05* 0.41±0.02* 66.78±0.12* 0.029±0.011

150mgkg Zinc 1.69±0.05* 48.40±0.55* 0.38±0.02* 64.38±0.29* 0.038±0.002*

200mg/kg Zinc 1.22±0.04* 44.30±0.40* 0.29±0.03* 54.34±0.09* 0.082±0.002*




104

4.3.3 Effects of Heavy Metals on the Enzymatic Activities and Lipid Peroxidation of Treated

and Untreated Maize Plants

Table 4.21 show the effects of lead and zinc treatments on superoxide dismutase (SOD), peroxidase,

gluathione (GSH), catalase activity and the level of malondialdehyde (MDA) in maize. Zn and Pb

treatment significantly increased the activities of CAT, peroxidase enzymes and the level of MDA

compared to control (P< 0.05) in maize. With increased zinc concentration, the GSH activity increased

with significant difference from the untreated plants (P < 0.05). Also, at the highest Pb concentration

of 200mg/kg, the GSH activity reduced with significant difference compared to the untreated plants

(P< 0.05). The SOD activity was also raised with increased zinc concentration with significant

difference (P < 0.05) compared to control. However, at the lowest Pb and Zn concentrations of

100mg/kg there was no significant difference (P > 0.05) in the SOD level compared to control. At the

highest lead concentration, the SOD level observed was significantly higher than the control (P <

0.05).

105

Table 4.21: The Effect of Different concentrations of Lead and Zinc nitrates on some Enzymes

and Lipid Peroxidation in Maize


PEROXIDASE

(µ /mg)


MDA (µ /mg)

Control 0.75±0.03 18.20±0.15 0.26±0.08 16.54±0.44 0.008±0.002

100mg/kg Lead 0.78±0.02 17.58±0.10* 0.08±0.03* 24.43±0.37* 0.118±0.018*

150mg/kg Lead 0.98±0.22* 22.20±0.27* 0.12±0.02* 27.54±0.37* 0.310±0.02

200mg/kg Lead 1.10±0.11* 26.10±0.50* 0.17±0.02* 30.42±0.07* 0.430±0.04*

100mg/kg Zinc 0.82±0.06 23.40±0.25* 0.18±0.04* 22.21±0.30* 0.080±0.02*

150mg/kg Zinc 2.77±0.24* 46.40±0.15* 0.21±0.07* 72.10±0.12* 0.202±0.005*

200mg/kg Zinc 2.98±0.45* 52.20±0.44* 0.30±0.03* 76.22±0.15* 0.345±0.015*




106

4.4 CYTOLOGICAL STUDY

The results for genotoxicity assay of the control and treated cowpea, bambara groundnut and maize

plants are shown on Plates 4.1 to 4.16. For all the three plants, 25mg/l and 50mg/l of lead and zinc

nitrates resulted in anaphase bridges, scattered chromosomes and C-mitosis. The 75mg/l and 100mg/l

concentrations mostly resulted in stickiness, vagrants and fragmented chromosomes. Tables 4.22 to

4.24 show the total number of cells analyzed, mitotic index values and the number of chromosome

aberrations observed in cowpea, bambara groundnut and maize treated with different concentrations of

lead and zinc nitrates. There was a decrease in mitotic index with increase in treatment concentrations.

The aberrations observed generally include: anaphase bridges, stickiness, vagrants, laggards,

fragments, c-mitosis and multipolar Anaphase. The highest treatment concentration of 100mg/l of lead

was observed to cause mostly stickiness and vagrant in all the three crop plants.

107

Table 4.22: Chromosome Aberrations in Cowpea Root Tips of Control and Treated Plants in

Different concentrations of Lead and Zinc Nitrate

TCN= Total Cell Number, ND= Number of dividing cells, ST= Stickiness, CM = C-mitosis, BF= Bridges fragment, VG=

Vagrant, LG= Laggard, MA= Multipolar anaphase, TA= Total aberrations, SD= Standard deviation, MI = Mitotic index,

P= Prophase, M=Metaphase, A= Anaphase, T= Telophase

When * P<0.05 = Significantly different from control and When P>0.05 = Not significantly different from control

Treatment

Concentrations

TCN ND ST CM BF VG LG MA TA(%) MI MI ± SEM

Control 1000 27(P5M13A6T3) 0 0 0 0 0 0 - 2.70 2.70±0.83

25mg/l Pb 1000 23(P1M8A12T2) 0 0 4 1 0 0 21.74 2.30 2.30±0.80*

50mg/l Pb 1000 5 (P0M5A0T0) 0 0 0 0 0 0 - 0.50 0.50±0.33*

100mg/l Pb - - - - - - - - - - -

25mg/l Zn - - - - - - - - - - -

50mg/l Zn 1000 12(P1M6A1T4) 0 3 0 0 1 0 33.33 1.20 1.20±0.52*

100mg/l Zn 1000 2(P1M1A0T0) 0 0 0 0 0 0 - 0.20 0.20±0.13*

108

Table 4.23: Chromosome Aberrations in Bambara groundnut Root Tips of Control and Treated

Plants in Different concentrations of Lead and Zinc Nitrates



P= Prophase, M=Metaphase, A= Anaphase, T= Telophase


Treatment

Concentrations

TCN ND ST CM BF VG LG MA TA(%) MI MI±SEM

Control 1000 34(P3M16A12T4) 0 0 0 0 0 0 - 3.40 3.40±0.88

25mg/l Pb 1000 21(P2M8A5T6) 0 0 3 5 0 0 38.10 2.10 2.10±0.76*

50mg/l Pb 1000 15(P0M5A6T4) 0 0 2 0 0 0 13.3 1.50 1.50±0.63*

100mg/l Pb 1000 13(P6M2A2T3) 1 1 3 1 3 0 69.23 1.30 1.30±0.48*

25mg/l Zn 1000 26(P6M8A6T6) 0 0 5 1 1 0 26.92 2.60 2.60±0.81*

50mg/l Zn 1000 24(P0M0A0T24) 24 0 0 0 0 0 100.0 2.40 2.40±0.80*

100mg/l Zn 1000 20(P1M9A7T3) 0 2 3 0 1 0 30.0 2.00 2.00±0.73*

109

Table 4.24: Chromosome Aberrations in Maize Root Tips of Control and Treated plants in Different

concentrations of Lead and Zinc Nitrates



P= Prophase, M=Metaphase, A= Anaphase, T= Telophase.


Treatment

Concentrations

TCN ND ST CM BF VG LG MA TA(%) MI MI ±SEM

Control 1000 34(P2M15A5T12) 0 0 0 0 0 0 - 3.40 3.40±0.88

25mg/l Pb 1000 25(P3M9A4T9) 4 0 2 5 0 1 48.00 2.50 2.50±0.82*

50 mg/l Pb 1000 3(P0M1A1T1) 0 0 0 1 0 0 33.33 0.30 0.30±0.21*

100mg/l Pb - - - - - - - - - - -

25mg/l Zn 1000 28(P7M9A4T8) 1 1 2 5 0 1 35.71 2.80 2.80±0.84*

50mg/l Zn 1000 25(P2M12A4T7) 4 0 4 2 0 0 40.00 2.50 2.50±0.83*

100mg/l Zn 1000 18(P3M6A1T8) 1 0 1 1 1 0 22.22 1.80 1.80±0.70*

110

4.4.1 Micrographs of Cells Showing Normal Mitosis in the Control Plants and Aberrations in

the Heavy Metal Treated Plants

Plate 4.1: Anaphase observed in control cowpea x 40

Plate 4.2: Anaphase observed in control bambara groundnut x 40

111

Plate 4.3: Anaphase observed in control maize x 40

Plate 4.4: Early telophase observed in control maize x 40

Early telophase

Anaphase

112

Plate 4.5: Vagrants in cowpea treated with 25mg/L lead nitrate x 40

Plate 4.6: Stickiness at telophase observed in cowpea treated with 25mg/L lead nitrate x 40

Stickiness

Vagrant

113

Plate 4.7: Anaphase bridge and laggard chromosome observed in cowpea treated with

25mg/L lead nitrate x 40

Plate 4.8: Vagrant observed in cowpea treated with 50mg/L zinc nitrate x 40

Vagrant

Scattered

chromosomes

Anaphase bridge

114

Plate 4.9: Scattered chromosomes observed in bambara groundnut treated with 100mg/L

lead nitrate x 40

Plate 4.10: Vagrant chromosomes at metaphase observed in bambara groundnut

treated with 25mg/L zinc nitrate x 40

Vagrant

115

Plate 4.11: Vagrant and bridged anaphase observed in maize treated with 25mg/L

lead nitrate x 40

Plate 4.12: Sticky chromosomes at telophase observed in maize treated with 50mg/L zinc

nitrate x 40

Stickiness

Vagrant at

metaphase

Bridged anaphase

116

Plate 4.13: Vagrant observed in maize treated with 50mg/L lead nitrate x 40

Plate 4.14: Laggard at telophase observed in maize treated with 100mg/L zinc nitrate x 40

Laggard

117

Plate 4.15: Vagrant at metaphase observed in maize treated with 100mg/L zinc nitrate x 40

Plate 4.16: Fragmented chromosomes at metaphase observed in maize treated with 25mg/L lead

nitrate x 40

Vagrant

118

4.5 PHYTOREMEDIATION STUDY

Tables 4.25 to 4.30 show that without assistance, maize extracted more lead compared to others

having bioconcentration / bioaccumulation factor greater than one. Cowpea showed more zinc

tolerance and accumulated it whether assisted or not than either maize or bambara groundnut.

However, all the three crop plants had bioaccumulation factor/plant-soil co-efficient above 1. When

the plants were treated with lead nitrate at a concentration of 150mg/kg, the amount and percentage of

lead removed and accumulated within plants’ tissues were 65.68 mg/kg (44.79%), 25.84 mg/kg

(17.06%) and 78.93 mg/kg (53.0%) for cowpea, bambara groundnut and maize respectively (Tables

4.28 to 4.30 and figures 4.1 to 4.6). However, when the plants were assisted they had greater

bioconcentration factor. Farmyard manure (cow dung) enhanced metal uptake by cowpea, bambara

groundnut and maize significantly than EDTA. Maize extracted more lead into its roots and

translocated to shoots when assisted with EDTA than cowpea and bambara groundnut. Also, in plants

treated with 200mg/kg of Pb and assisted with EDTA, the amount and percentage of lead removed and

accumulated within plants’ tissues were 67.67mg/kg (34.0%), 124.85mg/kg (62.0%) and 107.03

mg/kg (53.14%) for cowpea, bambara groundnut and maize respectively. However, in the plants

treated with 100mg/kg of lead and augmented with farmyard manure, the amount and percentage of

lead removed and accumulated within plants’ tissues were 49.88mg/kg (49.24%), 31.0mg/kg (30.0%)

and 82.89mg/kg (82.14%) for cowpea, bambara groundnut and maize respectively. The same trend

was observed for zinc translocation and bioaccumulation by these three plants (Tables 4.31 to 4.33).

All treated plants showed the trend of metal accumulation in this order: Root > Stem > Leaf. The

amount of Pb removed by plants in soils treated with Pb and augmented with EDTA, was greater when

compared with the amount of Zn, removed by plants in soils treated with Zn and augmented with

EDTA. (Tables 4.28 to 4.33).

119

Table 4.25: Concentration of Lead (Pb) (mg/kg) in the Roots and Shoots of Cowpea after Metal

Treatment and Augmentation

Treatment

concentration

Pb concentration

(mg/kg) in plant parts,

Soil and

Bioconcentration factor

(BCF)

Pb concentration

(mg/kg) in plant

parts, Soil and

Translocation factor

(TF)

Pb concentration

(mg/kg) in plant parts, Soil

and Plant-Soil Coefficient

(PSCF)

100 mg/kg Pb Root = 31.58±0.03

Soil = 43.41±0.11

BCF = 0.73

Shoot=23.43±0.42

Root =31.58±0.03

TF = 0.74

Shoot=23.43±0.42

Root =31.58±0.03

Soil = 43.41±0.11

PSCF = 1.27

150mg/kg Pb Root = 36.58±0.03

Soil = 88.39±0.14 BCF = 0.41

Shoot = 29.1±0.09

Root = 36.58±0.03

TF = 0.80

Shoot = 29.1± 0.09

Root = 36.58±0.03 Soil = 88.39±0.14

PSCF = 0.74

200mg/kg Pb Root = 32.91± 0.30

Soil = 88.11± 0.21

BCF = 0.37

Shoot = 24.5±0.14

Root = 32.91±0.30

TF = 0.75

Shoot = 24.5±0.14

Root = 32.91±0.30

Soil = 88.11±0.21

PSCF = 0.65

100mg/kg Pb + EDTA

Root = 47.68±0.24 Soil = 34.22±0.13

BCF = 1.39

Shoot = 18.52± 0.24 Root = 47.68± 0.24

TF = 0.38

Shoot = 18.52± 0.24 Root = 47.68± 0.24

Soil = 34.22± 0.13

PSCF = 1.93

150mg/kg Pb +

EDTA

Root = 70.97±0.24

Soil = 63.71±0.24

BCF = 1.11

Shoot = 13.33±0.51

Root = 70.97±0.24

TF = 0.19

Shoot = 13.33± 0.51

Root = 70.97± 0.24

Soil = 63.71± 0.24

PSCF = 1.32

200mg/kg Pb +

EDTA

Root = 47.25± 0.08

Soil = 71.56± 0.09

BCF = 0.66

Shoot = 20.12±0.16

Root = 47.25±0.08

TF = 0.43

Shoot = 20.12±0.16

Root = 47.25±0.08

Soil = 71.56±0.09

PSCF = 1.0

100mg/kg Pb+

manure

Root = 24.30±0.22

Soil = 43.40±0.21

BCF = 0.56

Shoot = 25.59±0.08

Root = 24.30±0.22

TF = 1.06

Shoot = 25.59±0.08

Root = 24.30±0.22

Soil = 43.40±0.21 PSCF = 1.13

150mg/kg Pb +

manure

Root = 56.15±0.03

Soil = 54.33±0.04

BCF = 1.03

Shoot = 38.13±0.11

Root = 56.15±0.03

TF = 0.67

Shoot = 38.13±0.11

Root = 56.15±0.03

Soil = 54.33±0.04

PSCF = 1.74

200mg/kg Pb + manure

Root = 92.79±0.06 Soil = 67.21±0.25

BCF = 1.38

Shoot = 35.42±0.08 Root = 92.79±0.06

TF = 0.38

Shoot = 35.42±0.08 Root = 92.79±0.06

Soil = 67.21±0.25

PSCF = 1.91

120

Table 4.26: Concentration of Lead (Pb) (mg/kg) in the Roots and Shoots of Bambara groundnut

after Metal Treatment and Augmentation

Treatment

concentration

Pb concentration


Soil and


(BCF)

Pb concentration

(mg/kg) in plant

parts, Soil and


(TF)

Pb concentration



(PSCF)

100 mg/kg Pb Root = 48.67 ± 0.04

Soil = 49.32±0.05

BCF = 0.99

Shoot=1.33±0.04

Root =31.58±0.03

TF = 0.03

Shoot=1.33±0.04

Root = 48.67 ± 0.04

Soil = 49.32±0.05

PSCF = 1.01

150mg/kg Pb Root = 25.63± 0.02

Soil = 108.84±0.09

BCF = 0.24

Shoot = 0.204 ± 0.13

Root = 25.63± 0.02

TF = 0.01

Shoot = 0.204 ± 0.13

Root = 25.63± 0.02

Soil = 108.84±0.09

PSCF = 0.24

200mg/kg Pb Root = 87.91±0.07 Soil = 106.33 ±0.03

BCF = 0.83

Shoot = 3.28 ± 0.11 Root = 87.91±0.07

TF = 0.04

Shoot = 3.28 ± 0.11 Root = 87.91±0.07

Soil = 106.33 ±0.03

PSCF = 0.86

100mg/kg Pb +

EDTA

Root = 33.79 ± 0.02

Soil = 40.51 ± 0.15

BCF = 0.83

Shoot = 25.52± 0.17

Root = 33.79 ± 0.02

TF = 0.76

Shoot = 25.52± 0.17

Root = 33.79 ± 0.02

Soil = 40.51 ± 0.15

PSCF = 1.46

150mg/kg Pb +

EDTA

Root = 70.97± 0.14

Soil = 51.44± 0.17

BCF = 1.25

Shoot = 13.33± 0.02

Root = 70.97± 0.14

TF = 0.50

Shoot = 13.33± 0.02

Root = 70.97± 0.14

Soil = 51.44± 0.17

PSCF = 1.86

200mg/kg Pb +

EDTA

Root = 88.40±0.02

Soil = 56.90±0.03

BCF = 1.55

Shoot = 36.45±0.08

Root = 88.40±0.02

TF = 0.41

Shoot = 36.45±0.08

Root = 88.40±0.02

Soil = 56.90±0.03

PSCF = 2.19

100mg/kg Pb+

manure

Root = 30.72±0.01

Soil = 31.80±0.08

BCF = 0.97

Shoot = 0.28±0.18

Root = 30.72±0.01

TF = 0.001

Shoot = 0.28±0.18

Root = 30.72±0.01

Soil = 31.80±0.08

PSCF = 0.98

150mg/kg Pb +

manure

Root = 61.25±0.06

Soil = 56.12±0.03

BCF = 1.62

Shoot = 33.76±0.07

Root = 61.25±0.06

TF = 0.03

Shoot = 33.76±0.07

Root = 61.25±0.06 Soil = 56.12±0.03

PSCF = 1.67

200mg/kg Pb +

manure

Root = 92.79±0.04

Soil = 70.10 ±0.08

BCF = 1.65

Shoot = 35.42±0.16

Root = 92.79±0.04

TF = 0.04

Shoot = 35.42±0.16

Root = 92.79±0.04

Soil = 70.10 ±0.08

PSCF = 1.72

121

Table 4.27: Concentration of Lead (Pb) (mg/kg) in the Roots and Shoots of Maize after Metal


Treatment concentration

Pb concentration (mg/kg) in plant parts,

Soil and


(BCF)

Pb concentration (mg/kg) in plant

parts, Soil and


(TF)

Pb concentration (mg/kg) in plant parts, Soil


(PSCF)

100 mg/kg Pb Root = 34.43 ± 0.12

Soil = 35.96 ±0.11

BCF = 0.96

Shoot=28.68 ±0.15

Root = 34.43 ± 0.12

TF = 0.83

Shoot=28.68 ±0.15

Root = 34.43 ± 0.12

Soil = 35.96 ±0.11

PSCF = 1.76

150mg/kg Pb Root = 46.67± 0.04

Soil = 79.26±0.15

BCF = 0.59

Shoot = 32.26 ± 0.05

Root = 46.67± 0.04

TF = 0.69

Shoot = 32.26 ± 0.05

Root = 46.67± 0.04

Soil = 79.26±0.15

PSCF = 1.0

200mg/kg Pb Root = 63.85 ±0.02

Soil = 81.41 ±0.15

BCF = 0.78

Shoot = 53.26 ± 0.25

Root = 63.85 ±0.02

TF = 0.83

Shoot = 53.26 ± 0.25

Root = 63.85 ±0.02

Soil = 81.41 ±0.15 PSCF = 1.44

100mg/kg Pb +

EDTA

Root = 30.80 ± 0.13

Soil = 29.11 ± 0.21

BCF = 1.06

Shoot = 40.84± 0.18

Root = 30.80 ± 0.13

TF = 1.33

Shoot = 40.84± 0.18

Root = 30.80 ± 0.13

Soil = 29.11 ± 0.21

PSCF = 2.46

150mg/kg Pb + EDTA

Root = 58.61± 0.31 Soil = 42.53± 0.03

BCF = 1.38

Shoot = 45.62± 0.31 Root = 58.61± 0.31

TF = 0.78

Shoot = 45.62± 0.31 Root = 58.61± 0.31

Soil = 42.53± 0.03

PSCF = 2.50

200mg/kg Pb +

EDTA

Root = 49.22±0.12

Soil = 53.04±0.04

BCF = 0.93

Shoot = 57.80±0.16

Root = 49.22±0.12

TF = 1.17

Shoot = 57.80±0.16

Root = 49.22±0.12

Soil = 53.04±0.04

PSCF = 2.01

100mg/kg Pb+

manure

Root = 56.38±0.02

Soil = 15.28±0.09

BCF = 3.69

Shoot = 26.52±0.10

Root = 56.38±0.02

TF = 0.47

Shoot = 26.52±0.10

Root = 56.38±0.02

Soil = 15.28±0.09

PSCF = 5.43

150mg/kg Pb +

manure

Root = 70.38±0.03

Soil = 32.18±0.14

BCF = 2.19

Shoot = 45.82±0.07

Root = 70.38±0.03

TF = 0.65

Shoot = 45.82±0.07

Root = 70.38±0.03

Soil = 32.18±0.14

PSCF = 3.61

200mg/kg Pb +

manure

Root = 83.62±0.11

Soil = 56.45 ±0.18

BCF = 1.48

Shoot = 57.32±0.12

Root = 83.62±0.11

TF = 0.69

Shoot = 57.32±0.12

Root = 83.62±0.11

Soil = 56.45 ±0.18

PSCF = 2.50

122

Table 4.28: Concentration of Zinc (Zn) (mg/kg) in the Roots and Shoots of Cowpea after Metal


Treatment

concentration

Zn concentration


Soil and


(BCF)

Zn concentration

(mg/kg) in plant

parts, Soil and


(TF)

Zn concentration



(PSCF)

100 mg/kg Zn Root = 32.70± 0.03

Soil = 21.22±0.07

BCF = 1.54

Shoot=38.42±0.10

Root = 32.70± 0.03

TF = 1.18

Shoot=38.42±0.10

Root = 32.70± 0.03

Soil = 21.22±0.07

PSCF = 3.35

150mg/kg Zn

Root = 44.90± 0.01

Soil = 35.16±0.04

BCF = 1.28

Shoot = 59.21 ± 0.08

Root = 44.90± 0.01

TF = 1.34

Shoot = 59.21 ± 0.08

Root = 44.90± 0.01

Soil = 35.16±0.04

PSCF = 2.96

200mg/kg Zn Root = 64.13±0.02 Soil = 49.20 ±0.03

BCF = 1.30

Shoot = 57.22 ± 0.16 Root = 64.13±0.02

TF = 0.89

Shoot = 57.22 ± 0.16 Root = 64.13±0.02

Soil = 49.20 ±0.03

PSCF = 2.47

100mg/kg Zn +

EDTA Root = 21.72±0.12

Soil = 36.14±0.10

BCF = 0.60

Shoot = 38.42± 0.08

Root = 21.72±0.12

TF = 1.77

Shoot = 38.42± 0.08

Root = 21.72±0.12

Soil = 36.14±0.10

PSCF = 1.66

150mg/kg Zn +

EDTA Root = 85.79±0.02

Soil = 45.14± 0.04

BCF = 1.22

Shoot = 13.33± 0.12

Root = 85.79±0.02

TF = 0.81

Shoot = 13.33± 0.12

Root = 85.79±0.02

Soil = 45.14± 0.04

PSCF = 2.20

200mg/kg Zn +

EDTA

Root = 74.50 ±0.02

Soil = 55.98± 0.02

BCF = 1.33

Shoot = 44.24±0.05

Root = 74.50 ±0.02

TF = 0.59

Shoot = 44.24±0.05

Root = 74.50 ±0.02

Soil = 55.98± 0.02

PSCF = 2.12

100mg/kg Zn +

manure

Root = 37.53±0.10

Soil = 25.18±0.06

BCF = 1.49

Shoot = 35.02±0.05

Root = 37.53±0.10

TF = 0.93

Shoot = 35.02±0.05

Root = 37.53±0.10

Soil = 25.18±0.06

PSCF = 2.88

150mg/kg Zn +

manure

Root = 36.60±0.01

Soil = 49.24±0.07

BCF = 0.74

Shoot = 57.52±0.15

Root = 36.60±0.01

TF = 1.57

Shoot = 57.52±0.15

Root = 36.60±0.01

Soil = 49.24±0.07 PSCF = 1.91

200mg/kg Zn +

manure

Root = 71.79±0.04

Soil = 46.41 ±0.13

BCF = 1.55

Shoot = 66.62±0.07

Root = 71.79±0.04

TF = 0.93

Shoot = 66.62±0.07

Root = 71.79±0.04

Soil = 46.41 ±0.13

PSCF = 2.98

123

Table 4.29: Concentration of Zinc (Zn) (mg/kg) in the Roots and Shoots of Bambara

groundnut after Metal Treatment and Augmentation

Treatment

concentration

Zn concentration


Soil and Bioconcentration factor

(BCF)

Zn concentration

(mg/kg) in plant

parts, Soil and Translocation factor

(TF)

Zn concentration


and Plant-Soil Coefficient (PSCF)

100 mg/kg Zn Root = 55.92± 0.01

Soil = 39.10 ±0.02

BCF = 1.49

Shoot=2.20±0.03

Root = 55.92± 0.01

TF = 0.04

Shoot=2.20±0.03

Root = 55.92± 0.01

Soil = 39.10 ±0.02

PSCF = 1.49

150mg/kg Zn

Root = 94.94 ±0.01

Soil = 48.71±0.06

BCF = 1.95

Shoot = 1.39 ± 0.11

Root = 94.94 ±0.01

TF = 0.02

Shoot = 1.39 ± 0.11

Root = 94.94 ±0.01

Soil = 48.71±0.06

PSCF = 1.98

200mg/kg Zn Root = 84.88 ±0.05

Soil = 75.0±0.01

BCF = 1.13

Shoot = 1.03 ± 0.05

Root = 84.88 ±0.05

TF = 0.01

Shoot = 1.03 ± 0.05

Root = 84.88 ±0.05

Soil = 75.0±0.01

PSCF = 1.15

100mg/kg Zn +

EDTA Root = 43.17±0.01

Soil = 48.17±0.13

BCF = 0.90

Shoot = 1.04± 0.05

Root = 43.17±0.01

TF = 0.05

Shoot = 1.04± 0.05

Root = 43.17±0.01

Soil = 48.17±0.13

PSCF = 0.92

150mg/kg Zn +

EDTA Root = 87.21±0.01

Soil = 52.18± 0.08

BCF = 1.69

Shoot = 1.00± 0.11

Root = 87.21±0.01

TF = 0.01

Shoot = 1.00± 0.11

Root = 87.21±0.01

Soil = 52.18± 0.08

PSCF = 1.69

200mg/kg Zn +

EDTA

Root = 61.63 ±0.01

Soil = 93.12 ± 0.04

BCF = 0.66

Shoot = 0.83±0.07

Root = 61.63 ±0.01

TF = 0.01

Shoot = 0.83±0.07

Root = 61.63 ±0.01

Soil = 93.12 ± 0.04

PSCF = 0.67

100mg/kg Zn +

manure

Root = 40.62±0.06 Soil = 42.88±0.08

BCF = 0.95

Shoot = 5.00±0.04 Root = 40.62±0.06

TF = 0.12

Shoot = 5.00±0.04 Root = 40.62±0.06

Soil = 42.88±0.08

PSCF = 1.06

150mg/kg Zn +

manure

Root = 74.20±0.02

Soil = 58.44±0.05

BCF = 1.27

Shoot = 11.94±0.05

Root = 74.20±0.02

TF = 0.16

Shoot = 11.94±0.05

Root = 74.20±0.02

Soil = 58.44±0.05

PSCF = 1.47

200mg/kg Zn +

manure

Root = 71.79±0.04

Soil = 46.41 ±0.13

BCF = 1.86

Shoot = 66.62±0.07

Root = 71.79±0.04

TF = 0.15

Shoot = 66.62±0.07

Root = 71.79±0.04

Soil = 46.41 ±0.13

PSCF = 2.15

124

Table 4.30: Concentration of Zinc (Zn) (mg/kg) found in the Roots and Shoots of Maize after

Metal Treatment and Augmentation

Treatment

concentration

Zn concentration


Soil and

Bioconcentration factor (BCF)

Zn concentration

(mg/kg) in plant

parts, Soil and

Translocation factor (TF)

Zn concentration



(PSCF)

100 mg/kg Zn Root = 15.25±0.03

Soil = 33.11±0.04

BCF = 0.46

Shoot=49.31±0.08

Root = 15.25±0.03

TF = 1.95

Shoot=49.31±0.08

Root = 15.25±0.03

Soil = 33.11±0.04

PSCF = 3.23

150mg/kg Zn

Root = 42.03 ±0.01 Soil = 43.12±0.08

BCF = 0.97

Shoot = 59.41 ± 0.13 Root = 42.03 ±0.01

TF = 1.41

Shoot = 59.41 ± 0.13 Root = 42.03 ±0.01

Soil = 43.12±0.08

PSCF = 2.35

200mg/kg Zn Root = 65.43±0.07

Soil = 42.42±0.11

BCF = 1.54

Shoot = 54.33 ± 0.05

Root = 65.43±0.07

TF = 0.83

Shoot = 54.33 ± 0.05

Root = 65.43±0.07

Soil = 42.42±0.11

PSCF = 2.82

100mg/kg Zn +

EDTA Root = 24.69±0.04

Soil = 39.14±0.02

BCF = 0.63

Shoot = 32.03± 0.06

Root = 24.69±0.04

TF = 1.30

Shoot = 32.03± 0.06

Root = 24.69±0.04

Soil = 39.14±0.02

PSCF = 1.45

150mg/kg Zn +

EDTA Root = 62.47±0.09

Soil = 32.12±0.02

BCF = 1.95

Shoot = 37.95± 0.03

Root = 62.47±0.09

TF = 0.61

Shoot = 37.95± 0.03

Root = 62.47±0.09

Soil = 32.12±0.02

PSCF = 3.13

200mg/kg Zn +

EDTA

Root = 104.41 ±0.05

Soil = 40.08 ±0.06

BCF = 2.61

Shoot = 39.81±0.09

Root = 104.41 ±0.05

TF = 0.38

Shoot = 39.81±0.09

Root = 104.41 ±0.05

Soil = 40.08 ±0.06

PSCF = 3.59

100mg/kg Zn +

manure

Root = 35.39 ±0.03

Soil = 16.92±0.05

BCF = 2.09

Shoot = 45.48±0.07

Root = 35.39 ±0.03

TF = 1.29

Shoot = 45.48±0.07

Root = 35.39 ±0.03 Soil = 16.92±0.05

PSCF = 4.78

150mg/kg Zn +

manure

Root = 52.09±0.06

Soil = 29.35±0.08

BCF = 1.77

Shoot = 59.12±0.11

Root = 52.09±0.06

TF = 1.14

Shoot = 59.12±0.11

Root = 52.09±0.06

Soil = 29.35±0.08

PSCF = 3.79

200mg/kg Zn +

manure

Root = 70.12 ±0.04

Soil = 32.48±0.12

BCF = 2.16

Shoot = 80.31±0.15

Root = 70.12 ±0.04

TF = 1.15

Shoot = 80.31±0.15

Root = 70.12 ±0.04

Soil = 32.48±0.12

PSCF = 4.68

125

Cowpea Bambara groundnut Maize-10

0

10

20

30

40

50

60

70

80

90

100

110

Perc

en

tage

(%

) A

ccu

mula

tion

of P

b

Lead Accumulation in Plants Treated with 200mg/kg Lead nitrate

Root

Stem

Leaf

Figure 4.1: Percentage Pb Accumulation within Plants treated with 200mg/kg lead nitrate

126

Cowpea Bambara groundnut Maize0

10

20

30

40

50

Perc

en

tag

e (

%)

Ac

cu

mu

lati

on

of

Pb

Lead Accumulation in Plants treated with 200mg/kg Lead nitrate and EDTA

Root

Stem

Leaf

Figure 4.2: Percentage Pb Accumulation within Plants treated with 200mg/kg lead and

EDTA

127

Cowpea Bambara groundnut Maize

0

10

20

30

40

50

60P

erc

en

tag

e (

%)

Accu

mu

lati

on

of

Pb

Lead Accumulation in Plants treated with 200mg/kg lead nitrate and manure

Root

Stem

Leaf

Figure 4.3: Percentage Pb Accumulation within Plants treated with 200mg/kg lead nitrate

and manure.

128

Figure 4.4: Percentage Zn Accumulation within Plants treated with 200mg/kg zinc nitrate


0

10

20

30

40

50

Perc

en

tag

e (

%)

Accu

mu

lati

on

of

Zn

Zinc Accumulation in Plants treated with 200mg/kg zinc nitrate

Root

Stem

Leaf

129


0

10

20

30

40

50

60

Pe

rcenta

ge (

%)

Accum

ula

tion o

f Z

n

Zinc Accumulation in Plants treated with 200mg/kg zinc nitrate and EDTA

Root

Stem

Leaf

Figure 4.5: Percentage Zn Accumulation within Plants treated with 200mg/kg zinc nitrate

and EDTA

130


0

10

20

30

40

50

60

Perc

en

tag

e (

%)

Accu

mu

lati

on

of

Zn

Zinc Accumulation in Plants treated with 200mg/kg zinc nitrate and manure

Root

Stem

Leaf

Figure 4.6: Percentage Zn Accumulation within Plants treated with 200mg/kg zinc nitrate and

manure

131

4.6 SOIL SAMPLE ANALYSIS AFTER PLANTING

After planting, the organic matter, pH and moisture content of the soil without EDTA and manure as

well as when assisted with farmyard manure and the chelator were analyzed to investigate the impact

of the heavy metals (lead and zinc). From table 4.31, it was observed that there was an increase in soil

pH from the initial value of 6.42 (before treatment/planting) for the following treated soil samples:

Soil with bambara groundnut treated with 100 mg/kg Pb, had pH of 6.83, soil containing maize

seedlings and treated with 100mg/kg Pb had 6.80, soil containing cowpea and treated with 200mg/kg

Pb + EDTA had 6.67, soil containing cowpea and treated with 200mg/kg Zn + EDTA had 6.59 and

soil containing cowpea and treated with 100 mg/kg Zn + manure had 6.80. The others showed

decrease in soil pH especially those treated with the highest lead concentration compared to the initial

values in the untreated soils and soils that had neither plants nor treatment. For all treated and

untreated soils, the moisture contents increased except for soil with cowpea and treated with 200mg/kg

Zn + EDTA. Also, for the organic matter contents, all treated and untreated plants experienced an

increase except for soils containing bambara groundnut and treated with 200mg/kg Pb + Manure, soil

containing cowpea and treated with 150mg/kg Pb and soil containing maize and treated with

100mg/kg Pb. Generally, the organic matter contents decreased in all soils treated with Pb without

augmentation. The Pb and Zn content of untreated soils without any plants were 1.42mg/kg and 0.78

mg/kg respectively. The untreated soils with cowpea plant had 0.10mg/kg and 0.01 mg/kg of Pb and

Zn respectively. The untreated soils with bambara groundnut plant had 0.13mg/kg and 0.21 mg/kg of

Pb and Zn respectively while the untreated soils with cowpea plant contained 0.10mg/kg and 0.01

mg/kg of Pb and Zn respectively.

132

Table 4.31: Soil Parameters Analyzed after the Remediation Experiment

Untreated Soil

Samples

Soil pH Moisture Content (%) Organic Matter (%) Pb content

(mg/kg)

Zn content

(mg/kg)

Untreated soil sample

without plants

6.42 7.86 3.28 1.42 0.78

Untreated soil with

Cowpea (Control )

5.92 7.46 4.641 0.10 0.01

Untreated soil with

Bambara groundnut

(Control)

6.18 12.24 3.986 0.13 0.11

Untreated soil with

Maize (Control)

6.37 11.00 9.671 0.06 0.04

Treated Soil Samples Soil pH Moisture Content (%) Organic Matter (%) Pb content

(mg/kg)

Zn content

(mg/kg)

150mg/kg Pb +

Cowpea

6.05 5.63 1.216 88.39 -

200mg//kg Pb + EDTA

Cowpea

6.67 6.28 5.841 71.56 -

200mg/kg Pb + Manure

+ Cowpea

6.10 13.71 7.461 67.21 -

150mg/kg Zn +

Cowpea

5.95 6.50 5.001 - 35.16

200mg/kg Zn + EDTA

+ Cowpea

6.59 2.49 14.516 - 55.98

100mg/kg Zn +

Manure + Cowpea

6.80 6.60 6.884 - 25.18

100mg/kg Pb +

Bambara groundnut

6.83 19.71 4.046 49.32 -

200mg/kg Pb + Manure

+ Bambara groundnut

6.02 11.33 2.606 70.10 -

150mg/kg Zn +

Bambara groundnut

6.23 13.7 3.648 - 48.71

200mg/kg Zn + EDTA

+ Bambara groundnut

6.08 12.41 5.698 - 93.12

133

4.7 INFLUENCE OF HEAVY METALS ON PLANT’S DNA WITH AND WITHOUT

EDTA AND MANURE AUGMENTATION

4.7.1 RAPD (Random Amplified Polymorphic DNA) Analysis

For the DNA analysis, the primer sequence involving four primers (OPJ-12, OPJ-13, OPO-08 and

OPO-09) and the band range/sizes of the primers between 1500bp-200bp is shown on Table 4.32. The

banding patterns of the primers are shown on plates 4.17 to 4.20. The total number of amplified

fragments observed among the genotypes of the three crops based on RAPD Analysis with all primers

was 44 with an average of 11 easily detectable fragments per primer. The number of amplified

fragments produced per primer ranged from 9 to 13 and size of the products ranged from 200bp to

1500bp. The total number of polymorphic fragments and the percentage of polymorphism were 28 and

62.61% respectively (Table 4.33). The primer OPJ-12 presented the highest percentage of RAPD

polymorphism (83.33%). RAPD primer OPJ 12 produced the highest number of bands (13).

Fragments 200bp and 560bp for lanes 2, 3, 7, 8, 9,10,12,14 and 15 were visualized using primer OPO-

08. Fragment 300bp for lanes 1, 4,7,10, 11, 13 and 15 were visualized using primer OPJ-12. Fragment

405bp for lanes 1 to 15 were visualized using primer OPJ-12. As well as Fragment 580bp for lanes

3,4,7,10,11, 13 and 15 were visualized using primer OPJ-12. However, in all the maize plants’

genotypes, the three fragments (unique bands) 300bp, 405bp and 580bp were visualized using primer

OPJ-12. Fragments 620bp for lanes 6,8,9,12,13 and 15 were visualized using primer OPO-09.

Fragments 890bp for lanes 3, 4, 7,8,9,10,12 and 13 were also visualized using primer OPO-09.

However, only fragment 863bp for lanes 2, 3, 6 to 15 was visualized using primer OPJ-13.

134

Table 4.32: Primer Sequence for the Control and Treated Plant Samples

S/N Primer Name Sequence Band range/size

1 OPJ-12 GTCCCGTGGT 1000bp-200bp

2 OPJ-13

CCACACTACC

1500bp-250bp

3 OPO-08

CCTCCAGTGT

1000bp-200bp

4 OPO-09

TCCCACGCAA

1400bp-250bp

135

Plate 4.17: RAPD-PCR Amplification based on the use of Primer OPJ-12 on the Plant Samples

Genotypes.

Plate 4.18: RAPD-PCR Amplification based on the use of Primer OPJ-13 on the Plant Samples

Genotypes.

-200

-300

-405

-580

-610

-710

-1000 bp

LANES

LANES

-250

-425

-618

-863

-1010 -1125

-1500 bp

136

Plate 4.19: RAPD-PCR Amplification based on the use of Primer OPO-08 on the Plant Samples

Genotypes.

Plate 4.20: RAPD-PCR Amplification based on the use of Primer OPO-09 on the Plant Samples

Genotypes.

LANES

-200

-370

-418 -490 -610

-970

-1000 bp

-250

-450

-620

-1090

-888

-1400 bp

LANES

137

Note that: Lane M: DNA Ladder, Lane 4: Control Maize, Lane 7: 200 mg/kg Pb + Manure

Maize, Lane 11:100 mg/kg Pb Maize, Lane 13: 200 mg/kg Zn Maize. Lane 5: Control Cowpea,

Lane 2: Manure + 100mg/kg Zn Cowpea, Lane 8: 150 mg/kg Pb Cowpea, Lane 9: 200 mg/kg Pb

Manure Cowpea, Lane 12:150 mg/kg Zn + Edta Cowpea, Lane 14: 150 mg/kg Pb + Edta

Cowpea, Lane 1: Control Bambara groundnut. Lane 3: Manure + 200 mg/kg Pb Bambara

groundnut, Lane 6:Manure + 150 mg/kg Zn Bambara groundnut, Lane 10: 100 mg/kg Zn +

Edta Bambara groundnut, Lane 15:150 mg/kg Pb Bambara groundnut.

138

Table 4.33: Number of Monomorphic, Polymorphic bands and Polymorphism Percentage

produced by each RAPD primer for the Control and some Treated Plant Samples.

Primers Band

Range/Size

(Bp)

Sequence Total

number of

bands

Monomorphic

band

Polymorphic

band

Percentage

Polymorphism

OPJ 12 1000bp-200bp

GTCCCGTGGT 13 4 8 61.54

OPJ 13 1500bp-250bp CCACACTACC 12 2 10 83.33

OPO-08 1000bp-200bp CCTCCAGTGT 9 4 5 55.56

OPO-09 1400bp-250bp TCCCACGCAA 10 5 5 50.00

Total - - 44 15 28 -

Average - - 11 3.8 7.0 62.61

139

4.7.2 Coefficient of Similarity among the Control and Treated Plants

The treated plants experienced decrease in their total number of bands compared to their control.

When assisted with manure, the plants had their total number of bands to be more than the treated

plants. Infact, bambara groundnut treated with 200mg/kg of lead and assisted with manure had a total

of 31 bands while the control had 23. The samples also differ from one another in their migration

rates. For instance, for primer OPJ-12, the 200bp band had slower migration rate than the 710bp band.

Dendograms of treated plant samples and control reflecting their similarity coefficients is shown in

Appendix IV (a-c). Samples with the same value and same variations/deviations fall in the same

cluster. For instance, control cowpea, cowpea with 100mg/kg Zn and manure, cowpea with 200mg/kg

Pb and manure as well as cowpea with 150 mg/kg Zn and EDTA all fell in the same cluster. Only

cowpea treated with 150 mg/kg Pb and EDTA fell in a different cluster entirely. The same trend was

observed for bambara groundnut with only plant treated with 150mg/kg Pb found in a different cluster

as well. However, for maize only the control plant had a different cluster, other treated plants

(augmented or not) fell in the same cluster. This indicates a deviation/variation from the control plants.

The similarity coefficients ranged from 0.51 to 0.80 (Table 4.34 and Appendix IV a-c). Cowpea plants

treated with 150mg/kg Pb and 200mg/kg Pb with manure showed the highest similarity index (0.80)

and the lowest similarity index (0.51) was shown by bambara groundnut treated with 150 mg/kg Pb.

The similarity index of treated plant samples was found to vary differently from the control samples

(Table 4.34 and Appendix IV a-c).

140

Table 4.34: Total number of bands by all the Primers and the Coefficient of Similarity among Control

and Some Treated plants

Lanes Samples Number of

bands for all

primers

Coefficient

of Similarity

5 Control Cowpea 29 0.76

2 100mg/kg Zn + Manure

Cowpea

21 0.76

8 150mg/kg Pb Cowpea 22 0.80

9 200mg/kg Pb + Manure

Cowpea

26 0.80

12 150mg/kg Zn+ EDTA

Cowpea

19 0.70

14 150mg/kg Pb + EDTA

Cowpea

18 0.58

1 Control Bambara

groundnut

23 0.79

3 200 mg/kg Pb + Manure

Bambara groundnut

31 0.66

6 150 mg/kg Zn + Manure

Bambara groundnut

27 0.79

10 100 mg/kg Zn + EDTA

Bambara groundnut

22 0.66

15 150 mg/kg Pb Bambara

groundnut

19 0.51

4 Control Maize 24 0.53

7 200 mg/kg Pb + Manure

Maize

23 0.63

11 100 mg/kg Pb Maize 18 0.73

13 200 mg/kg Zn Maize 19 0.73

141

CHAPTER FIVE

5.0 DISCUSSION

Physico-chemical characteristics of the soil used for planting

The soil was observed to be a loamy soil, which is suitable for plant growth. However, the dark grey

colour of the soil and the low values obtained for the organic matter, phosphorus, nitrogen and

moisture content indicates a low amount of humus and insufficient nutrients for plants’ growth. The

slightly acidic pH 6.42 recorded for the soil sample used for the experiment is within the range of

agricultural soils. Soil pH plays a major role in the sorption of heavy metals as it directly controls the

solubility and hydrolysis of metal hydroxides, carbonates and phosphates. Soil pH (range 4.6-6.8)

among other factors in an extensive study in England and Wales, influenced the uptake of Pb into

Raphanus sativus (Davies, 1992). The solubility of soil Pb is influenced by both the organic and

mineral fractions of the soil to a greater extent than that of Zn (Alloway et al., 1998). Similarly, Pb

also adheres readily onto soil substrates such as clay and Fe/ Mn oxides, depending on the pH

conditions (Mc Bride et al., 1997). It also associates with organic matter and carbonates present in the

soil. (Blaser et al., 2000).

The Screening Test

The accessions Tvu 3788, Tvsu 102 and ACR.91SUWANI-SRC1 of Vigna unguiculata, Vigna

subterranea and Zea mays respectively had higher values from the percentage viability and percentage

germination compared to the other accessions. These accessions were chosen and used for the

experiment. Higher concentrations of the metals (lead and zinc), especially lead inhibited both seed

germination and seedling growth. However, roots showed higher degree of growth inhibition

compared to shoots. This could be due to the direct contact the roots had with the treatments as well as

an imbalance of nutrients in the soil. It was observed that the rate of germination was significantly

142

lower in the treated seeds than the untreated seeds (control). It was also observed that with increase in

concentration, the rate of germination reduced for the treated seeds and increased with increase in

number of days. At certain period (day) for each treatment and accession, percentage germination

stabilized till the last day of treatment. However, in seeds treated with 200mg/kg of lead and zinc

nitrate, percentage germination reduced greatly especially for lead. This is in agreement with the work

of Seregin et al., (2004) that heavy metals such as lead, inhibited seed germination and seedling

growth of maize.

Growth changes are the first most obvious reactions of plants under stress. The observed decrease in

germination/growth of Vigna unguiculata, Vigna subterranea and Zea mays may be explained on the

basis of these metals interference with cell division and cell enlargement, especially in roots; thereby

reducing its germination rate which in turn affects the uptake of water and nutrients, and this

influences growth of the entire plant.

Morphometric Evaluation (Leaf size, Stem height and Root length)

Higher concentrations of the metals (lead and zinc), especially Pb inhibited both seed germination and

seedling growth. However, roots showed higher degree of growth inhibition compared to shoots. This

was possibly due to the direct contact of the roots with the polluted soils. However, Zn had no

significant effect on the leaf area of treated cowpea plants compared to the control (untreated plants).

Cowpea plants tolerated Zn probably due to its role as a micronutrient and easy uptake. This was

supported by the earlier work of Longnecker and Robson (1993) that the potential of high toxicity of

Zn lies in its role as a micronutrient, high solubility and ready uptake by plants. Seregin et al., (2004);

Stiborova et al., (1987) and Prassad and Prassad (1987) reported that heavy metals such as lead

inhibited seed germination and seedling growth. Early seedling growth was also reported to be

143

inhibited in rice (Verma and Dubey, 2003; Yang et al., 2000), corn plants (Tung and Temple, 1996).

Spruce (Vodnik et al., 1999), Barley (Stiborova et al., 1987), tomato and egg plant (Khan and Khan,

1983). Root, stem and leaf elongation, were inhibited by Pb in Allium and Barley species (Juwarkar

and Shende, 1986). Different effects of Pb and Zn on plant growth have been observed by various

workers. Tomar et al., (2000) reported that increased level of lead in soil caused significant reduction

in plant height, root-shoot ratio, dry weight, nodule per plant and chlorophyll content in Vigna radiata.

Paivoke (2003) observed a decline in seedlings growth, reduced shoot yield of Pisum sativum exposed

to 300mg/kg of Zn and 500mg/kg of Pb. Growth changes are the first most obvious reactions of plants

under stress. This was also noted from the morphology of plants exposed to the metal treatment.

Stunted growth, chlorosis, necrosis, white lesions and wilting were all observed as direct implication

of metal treatment. Heavy metals uptake and accumulation in plants have been shown to result in

negative effects on plant growth (Breckle and Kahle, 1992).

This study also observed that the metals (Zn and Pb) reduced the fresh and dry weights of test plants.

This result is in line with the work of Kumar et al., (1992) who reported lead inhibition in fresh

weight, dry weight and length of root and shoot of Sesamum indicum Cv. HT-I and Iqbal and Mushta

(1987) who also observed the same trend in maize. The inhibitory effects of lead and zinc on the

biomass and growth are possibly a consequence of the metals effect on metabolic activities of the

plants. This observation was also noted by Thapa et al., (1988) and Van Assche and Clijsters, (1990).

This study established that the metals (Zn and Pb) decreased the fresh and dry weights of test plants.

However, when augmented with EDTA and manure the effect was minimal and the plants biomass

increased especially at the lowest concentration tested. The increased plants biomass following

augmentation was possibly due to increased metal bioavailability and subsequent uptake by the plants.

144

Chlorophyll content

The chlorophyll content of treated plants reduced significantly with increased metal concentration

especially for lead In all treated plants, as the concentration of metals increased, the quantity of total

chlorophyll in the treated plants decreased.

This led to reduction in photosynthetic activities and an indication of stress. This finding therefore

agrees with Chettri et al., (1998) who reported a decrease in chlorophyll levels for Cladonia

rangiformis after treating with Cu, Zn and Pb. They added that decrease of chlorophyll after Cu

application may be due to blocking of enzymes acting in chlorophyll synthesis or to degradation of

chlorophyll. They also said that chlorophyll content is usually measured in plants in order to assess the

impact of environmental stress, as changes in pigment content are linked to visual symptoms of plant

illness and photosynthetic productivity. Augmentation with manure especially improved the

chlorophyll contents of treated plants indicating no hindrance to photosynthesis. In the case of maize

plants assisted with EDTA, the chlorophyll content reduced with increased zinc nitrate concentrations,

while the treated plants assisted with manure experienced increased chlorophyll content with increased

zinc nitrate. This was probably due to the utilization of the nutrients in the manure by the plants for

sustained growth and increased chlorophyll content.

Biochemical Analysis

The enzymatic activities of bambara groundnut, cowpea and maize were adversely affected when the

plants were exposed to different concentrations of lead and zinc. This was probably attributed to

oxidative stress induced by these metals. For all the test plants, there were increase and decrease in the

level of the enzymes. All treated cowpea plants experienced an increase in the level of MDA with

significant difference from the control (P < 0.05) except for cowpea plant treated with the lowest

145

concentration of 100mg/kg of zinc nitrate which showed an increased level of MDA with no

significant difference (P > 0.05) when compared to the control. This was probably due to the low

concentration of Zn. The increased level of MDA (malanodialdehyde) is highly indicative of

oxidative stress for the plants. Increase in the level of MDA (product of lipid peroxidation) can lead to

inactivation of enzymes, DNA damage and interaction with vital plant cells. The levels of glutathione,

peroxidase, SOD and catalase for the treated plants were also raised. The increased level of

glutathione, peroxidase and catalase also signifies possible release of free radicals. The level of

superoxide dismutase, catalase, peroxidase and glutathione were lowered in bambara groundnut,

except for the MDA. The low level of superoxide dismutase signifies possible release of radicals

within the treated bambara groundnut due to no inhibition to oxidation. Also, this could be due to low

metal translocation potential displayed by the plants. These findings corroborated the earlier work by

Dietz and Schnoor (2001) that lead stress causes multiple direct and indirect effects on plant growth

and metabolism and also alter some physiological processes. Edwards et al., (2000) also observed that

high internal levels of glutathione can be beneficial as a first response towards some of the effects

exerted by pollutants. Cells are usually protected from reactive oxygen species by the combined action

of enzymatic antioxidant systems like catalase, peroxidase and non enzymatic antioxidant like

ascorbate, glutathione and phenolic compounds (Edwards et al., 2000). Glutathione (GSH); a

precursor for phytochelatins (PCs) which are heavy-metal binding peptides is known to be involved in

heavy-metal tolerance and sequestration. GSH plays a central role in defense against oxidative stress

and heavy metals. Lead compounds have been observed to bind less strongly to phytochelatins (PCS)

unlike other metals like zinc due to larger ion radius (Pb, octahedral) and high coordination number

(Pb, 6-8). This could be the reason for the greater Pb tolerance and uptake by maize with or without

146

augmentation and less Pb tolerance by bambara groundnut and cowpea. This could also be attributed

to the great tolerance of Zn exhibited by all the three plants.

Genotoxicity Study

A concentration-dependent increase in chromosomal abnormalities was observed in all the treated

plants. The plants treated with lead nitrate showed several effects of chromosomal toxicity. There was

a highly significant difference between both the metal-treated set and the control set. Scattering,

bridges, stickiness were the most frequent aberrations observed for the two heavy metals. Similar

observations were shown by other studies (Fiskesjo, 1997; Ukaegbu and Odeigah, 2009) to be useful

signs of toxicity. The untreated roots (controls) had high mitotic indices in all the mitotic phases. All

the concentrations were capable of inducing different types of chromosomal abnormalities and the

frequency of abnormalities increased, in most cases, as the concentration of the metals increased. This

provides a case for comparison of the deleterious effects of these metals on the concerned plant. The

induction of cytological disturbances in meiotic cells is of great value, as it results in genetic damage

that is passed on to the next generation. Several researchers performed similar studies on the genotoxic

effects of different chemicals on different plant materials (Kumar and Tripathi, 2007). Chromosomal

stickiness was observed in mostly higher concentrations. This probably was due to incomplete

replication of chromosomes by defective enzymes. This observation is corroborated by the findings of

(Amin,2002) that chromosomal stickiness can lead to DNA-protein cross linking due to defective

enzymes. Metal-induced chromosomal stickiness has also been reported (Kumar et al., 2006).

Stickiness accompanied by pyknosis and chromatin degeneration has been reported in maize (Caetano-

Pereira et al.1995), Allium cepa (Kumar and Tripathi, 2003), Helianthus annuus (Kumar and

Srivastava, 2006) and Lens culinaris (Kumar and Kesarwani, 2004). Odeigah et al., (1997b) reported

that sticky chromosomes are indicative of a highly toxic usually irreversible effect, leading to cell

147

death. Scattering, in which the chromosomes spread irregularly over the cell, may be due to

disturbance of the spindle apparatus. The characteristic behaviour of laggard chromosomes is that they

generally lead to micronucleus formation. Fragments at metaphase may be due to the failure of

chromosomes that have broken to recombine. Fragments might also have arisen due to the stickiness

of the chromosomes and the consequent failure of the arrival of chromatids at the poles. Fragments

may also be acentric chromosomes formed as a result of inversion (Agarwal and Ansari, 2001).

Anaphasic bridges were formed due to unequal exchange or dicentric chromosomes. The dicentric

chromosome is pulled equally towards both poles at anaphase and a bridge is formed. As more

abnormalities accumulate, gamete formation is affected and leads to non-viable gametes, which

considerably reduces plant fertility. Studies on different plant species have shown that declines in seed

production are correlated with meiotic irregularities (Kumar and Rai, 2007).

Phytoremediation Study

Cowpea translocated more lead than bambara groundnut while maize translocated and extracted more

lead into its tissues than cowpea and bambara groundnut whether on amended soil or not. For bambara

groundnut, lead translocation from roots to stems and leaves whether on amended soil or not were

minimal because most metals remained at the root zone. Cowpea showed more zinc tolerance and

uptake whether assisted or not than for maize or bambara groundnut following its high translocation

factor, found to be greater than 1 in most cases. However, maize extracted more lead into its roots and

translocated to shoots when assisted with EDTA and manure than cowpea, having its translocation

factors being more than1. The maximum bioaccumulation coefficient was observed in maize plant.

Farmyard manure (cow dung) enhanced metal uptake by cowpea and bambara groundnut significantly

more than EDTA. Bambara groundnut displayed the property of an “excluder” having a translocation

factor well below 1 and little or no metals actually got translocated to its stem and leaves. This is

148

confirmed by Kimenyu et al., (2009), that the soil-plant transfer factor or bioaccumulation factor, f,

expressed as the ratio of plant metal concentration divided by the total metal concentration in soil, may

be used as indicators of the plant accumulation behavior. According to Stoltz and Greger (2002) metal

excluder species have translocation factor to be typically lower than 1. Bambara groundnut was able to

retain these metals especially zinc at its root zone possibly with the help of some plant exudates and

manure which increased the soil organic matter. It was able to stabilize/immobilize these metals at the

root zones possibly through mechanisms such as accumulation in roots by vacuole sequestration, cell

wall binding, complex formation by root exudates, precipitation within root zone due to little or no

xylem loading and translocation. Maize and cowpea phytoextracted more lead and zinc respectively,

their plant-soil coefficient/bioaccumulation factors were higher than 1. This was probably through

mechanisms such as uptake, sequestration in roots and metal redistribution to various tissues in the

shoot through phloem and xylem transport. These plants being able to tolerate these metals might also

be due to accumulation within the cell wall without penetration into the protoplast; a mechanism

described by Abdul (2010). All treated plants showed the trend of metal accumulation in this order:

Root > Stem > Leaf. Nascimento et al., (2006) reported that maize is capable of continuous

phytoextraction of metals from contaminated soils by translocating them from roots to shoots. The

higher the f factor, the more effective is the phytoextractor. Based on its ability to uptake heavy metal

and sensitivity to high metal pollution, maize accumulated significant amounts of heavy metals when

induced through the addition of metal chelates like EDTA. This study therefore shows that EDTA

supported the plants in metal uptake especially maize. This agrees with the findings of Mark and

Ronald (2006) that exposing plants to EDTA for a longer period (40 to 70 days) could improve metal

translocation in plant tissue as well as the overall phytoextraction performance. EDTA was also

observed to remove lead more than zinc. Farmyard manure supported cowpea, bambara groundnut and

149

maize more in metal tolerance and uptake due to the fact that increase in organic matter content of the

soil increases metal availability in the soil. This observation disagreed with the study by Brown et al,

(1994) that organic amendments like compost, farmyard manure or biosolids may effectively reduce

the bioavailability of heavy metals in soils due to its high content of organic matter. However,

following metal treatment along with augmentation, the soil parameters analyzed especially organic

matter increased, possibly due to availability of nutrients from the amendment. Roots can actively

exude substances like organic acids and nitrogenous compounds which improve soil nutrients and

enhance metal bioavailability, tolerance and uptake. This has to do with the nutrient acquisition by the

study plants (cowpea, bambara groundnut and maize). This observation corroborates the report by

Jones (1998a) that root exudates such as organic acids which include: malate, oxalate and citrate form

metal ion complexes and play a role as regards metallic element tolerance by plants. Also, root

exudated oxalate has been reported to enhance Pb tolerance in rice (Yang et al., 2000).

Accumulation and exclusion are two basic strategies shown by these plants in responding to increased

concentrations of heavy metals. As a result, bambara groundnut plants could be good candidates for

the revegetation and phytostabilization of heavy metal (Pb and Zn) in polluted soils.

Physico-chemical characteristics of the soil after planting

The organic matter contents reduced due to lead and zinc treatment without augmentation in the entire

test plants but improved following augmentation especially with manure. This improvement was

probably due to the soil being enriched with nutrients from the manure. The decrease and increase in

soil pH of test plants was due to the treatment effects of these metals. For all treated and untreated

plants, the moisture contents increased, indicating no significant difference in the moisture contents of

treated soils with respect to control. Patra et al., (2004) reported alterations in the organic matter, soil

150

pH, moisture content, phosphorus content and organic carbon of the soil following metal treatment

with mercury, lead and zinc.

DNA Analysis

From the RAPD-PCR analysis, all treated plants experienced reduction in the quality of DNA

extracted by having decrease/increase in their total number of bands compared to their control.

However, the close values obtained from their co-efficient of similarity showed the effects of metal

treatment to be minimal for these concentrations tested. This may be due to the fact that the DNA of

leaves of young seedlings with few days of exposure to lead and zinc nitrate were used for the

investigation and little amount of metals were found within these leaves especially for bambara

groundnut. When amended with manure, the total number of bands of treated plants was found to be

more than when not amended. Infact, bambara groundnut treated with 200mg/kg of lead and

amendedwith manure had a total of 31 bands while the control had 23. This still confirms bambara

groundnut as an “excluder” and metal stabilizer especially when amended with manure. The decrease

in the total number of bands for all treated plants compared to their control are however a great

indication of toxicity on the DNA as well as the genetic makeup of the plants hence, its effect on

reproduction and genotypes/phenotypes of successful offspring. Samples augmented with manure

were found to show little or no deviation from their control groups probably because of increased

organic matter in the presence of manure. Those augmented with EDTA and those without

augmentation had similarity index showing great deviation from their control groups. This was

probably due to reduced organic matter contents of the soil and the ability of the plants to uptake the

metals. However, the metal treatment did not affect the quality of the DNA especially in cowpea and

bambara groundnut treated samples. This may also be due to the short period of exposing cowpea,

bambara groundnut and maize to lead and zinc required by the phytoremediation technique employed

151

in this experiment. This work agrees with the findings of De Wolf et al., (2004) that higher plants have

been reported to produce varied response to heavy metals in their environment and these metals may

interfere with the genetic constitution of plants. Similarly, the genotoxicity of heavy metals in kidney-

bean (Phaseolus vulgaris) seedlings was studied and subjected to RAPD (Random Amplified

Polymorphic DNA) analysis. Polymorphisms were evident in all treated plants as the presence and/or

absence of DNA fragments in treated samples. A high number of both missing bands and new

amplified fragment were observed. Enan, (2006) also found missing bands and new fragments

showing the mutagenic effect of heavy metals on P. vulgaris. The unique bands (300bp, 405bp and

580 bp) obtained from this study which was common to most treated plants’ genotypes especially

maize plant clearly revealed the tolerance abilities of these plants and would act as marker for

assessment of environmental doses of lead and zinc. These bands can be considered as potential

markers to identify metal tolerant genotypes or may even be useful when converted into a simple-

sequence PCR based marker that can be used for large-scale lead and zinc tolerance screening of

genotypes. As a result, many researchers exploited DNA markers and detected some markers of

abiotic stress. Pakniyat and Tavakol (2007) found markers related to drought tolerance in bread wheat

genotypes using RAPD markers. Youssef et al., (2010) found molecular markers for new promising

drought tolerant lines of rice under drought stress through RAPD-PCR and ISSR markers. The

identification of DNA markers diagnostic of Pb and Zn tolerance can accelerate the development of

cultivars that can remain productive even under Pb and Zn stresses, and may be the starting point for

identifying the specific genes responsible for differences in the response of plant genotypes to toxic

lead and zinc levels.

152

5.1 Summary

Farmyard manure assisted all the plants in metal uptake and tolerance. EDTA also assisted in lead

uptake than zinc especially for maize. Lead at the 100mg/kg concentration was found to be toxic with

observable symptoms for all treated plants while zinc toxicity was obvious at the highest concentration

of 200mg/kg tested. It can be said that enzymes such as glutathione were possibly involved in metal

tolerance by maize and cowpea as well as resistance (exclusion) to uptake of metals especially for

bambara groundnut.

Atomic absorption spectrophotometric studies confirmed the accumulation of these metals primarily in

the roots of bambara groundnut and this makes the plant useful for phytostabilization. Maize being

able to translocate these metals especially lead through the vascular system act as a phytoextractor

while cowpea could be employed for phytoextraction of zinc for translocating it through the vascular

system. Composting these plants and then applying the compost of the metals especially zinc to a Zn-

deficient soil could be an effective technique for remediation of contaminated soils and redistribution

of the zinc as a plant nutrient for Zn-deficient soils. The plants used for remediation can be

incinerated to prevent transfer of metals through the food chain. However, it has been suggested that

the metals (Pb and Zn) can be removed chemically and the plants be used as fodder for animals. Also,

after remediation, these plants can still be allowed to undergo recovery if replanted in clean (metal-

free) soils with high organic matter content (nutrients) after remediation.

The treatment effects of lead and zinc nitrates were minimal on the treated plants’ DNA. The patterns

obtained with primer OPJ-12 particularly for the genotypes of maize plant (accession

ACR.91SUWANI-SRC1) suggested that this primer possibly has the ability to produce heavy metal

tolerant markers.

153

5.2 SUMMARY OF FINDINGS

AIMS AND OBJECTIVES SUMMARY OF FINDINGS

Investigate the effects of the metals on the growth,

development and enzymatic activities of cowpea, bambara

groundnut and the maize crop to be used for remediation.

The different concentrations of Pb and Zn caused reduced

growth. Enzymes’ activities were altered. However, the

plants biomass improved following augmentation.

Investigate the genotoxic effect of the heavy metals (Pb and

Zn) on cowpea, bambara groundnut and the maize crop.

The most frequent chromosomal aberrations observed at

the highest concentration tested were vagrants and

stickiness while lower concentrations resulted in bridges

and scattering of the chromosomes.

Evaluate the potential of cowpea, bambara groundnut and the

maize crop when not supported with the amendments- EDTA

and farm yard manure as well as the impact of the

amendments on the crop plants in the remediation of heavy

metals polluted soils.

.

Cowpea (Accession Tvu 3788) can tolerate zinc and

accumulate it within its stem even at the highest

concentration tested. Bambara groundnut (Accession

Tvsu 102 ) can stabilize lead and zinc within its roots by

immobilizing them.

Maize plant (Accession ACR.91SUWANI-SRC1) can

naturally uptake Pb and Zn and withstand their toxic

effects in moderately polluted soils.

The use of farmyard manure and EDTA assisted in metal

uptake by these plants from the roots up to the stem to a

great extent.

Plants’ roots exudates, enzymes and antioxidants (such as

glutathione) probably assisted in the metal tolerance by

these plants.

The soil properties such as organic matter and pH

improved due to manure and EDTA augmentation.

Determine the genetic tolerance for lead and zinc by the

different crop plants and establish molecular markers

associated with heavy metal tolerance.

.

Treated (augmented or not) accessions of cowpea and

bambara groundnut showed little or no genetic variation

from the control plants. The treated maize accession

showed greater deviation from the control plants.

The patterns obtained with primer OPJ-12 for the plants’

genotypes especially maize plants suggested that this

primer possibly has the ability to produce heavy metal

tolerant markers.

154

5.3 Contribution to Knowledge

• Bambara groundnut (Accession Tvsu 102) can stabilize lead and zinc within its roots by

immobilizing them.

• The treatment effects of the metal salt (Pb and Zn) were minimal on plants’ genomic DNA.

• Maize plant (Accession ACR.91SUWANI-SRC1) uptakes lead and zinc without assistance

and withstand their toxic effects in polluted soils.

• Cowpea (Accession Tvu 3788) can tolerate zinc most and accumulate it within its stem

even at the highest concentration tested.

• The use of farmyard manure and EDTA assisted in Pb and Zn uptake by these plants from

the roots up to the stem to a great extent.

155

5.4 Recommendation

Genetic engineering will be essential in creating transgenic plants that will be able to combine the

natural agronomic benefits associated with plants such as ease of harvest and rapid expansive growth

with the remediation capabilities of bacteria used in bioremediation.

The systemic screening of plant species and genotypes for metal accumulation and resistance will

enhance the spectra of genetic material available for optimization of phytoremediation technology and

application on a commercial scale.

156

5.5 Future Research

• Identification of candidate plants with substances that may deter the herbivores from feeding

on them and the subsequent transformation of such plants with improved metal tolerance

capabilities.

• Discovery of an approach involving simultaneous transfer of several genes into suitable

candidate plants to remove contaminants of complex nature.

• To understand the mechanisms involved in mobilization and transfer of metals by rhizobacteria

in order to develop future strategies and optimize the phytoextraction process.

157

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APPENDICES

APPENDIX 1: Regulatory limits on heavy metals applied to soils (Adapted from U.S. EPA,

2001, EPA,1999).

Appendix II

Soil concentration ranges and regulatory guidelines for soil heavy metals (Adapted from

NJDEP,1996)

Metal Soil

concentration/Range(mg/kg)

Regulatory limit

(mg/kg)

Pb 1.00-69,000 600

Cd 0.10-345 100

Cr 0.05-3950 100

Hg < 0.01-1800 270

Zn 150-5000 1500

175

Appendix IIIa

Target and Intervention values for some metals for a standard soil (Adapted from DPR-

EGASPIN, 2002)

Appendix III b

The growth Inhibition test according to Jorge and Arruda (1997) was used to determine the lethal dose

concentration. This was indicated by the occurrence of wilting by the roots after plants were treated

with different metal concentrations of 50mg/kg, 100mg/kg, 150mg/kg, 200mg/kg, 250mg/kg,

300mg/kg and 350 mg/kg after 96 hrs. The lethal dose from the root growth curve was 250mg/kg.

There was no significant reduction in root growth at the 50mg/kg concentration.

Metal Target value

(mg/kg)

Intervention

value (mg/kg)

Ni 140 720

Cu 0.30 10.00

Zn - -

Cd 100 380

Pb 35 210

As 200 625

Cr 20 240

Hg 85 530

176

APPENDIX IVa-c

a) The Dendrogram of Coefficient of Similarities among the Control and Treated Cowpea Plants

based on band Polymorphisms generated by PCR after using the Primers.

b) The Dendrogram of Coefficient of Similarities among the Control and Treated Bambara

groundnut Plants based on band Polymorphisms generated by PCR after using the Primers.

177

c) The Dendrogram of Coefficient of Similarities among the Control and Treated Maize Plants

based on band Polymorphisms generated by PCR after using the Primers.

178

APPENDIX V: PROCEDURES FOR ENZYMES AND LIPID PEROXIDATION

DETERMINATION

Determination of Superoxide Dismutase (SOD) Activity.

The level of SOD activity was determined by the method of Magwere et al., (1997).

Principle

The ability of superoxide dismutase to inhibit the auto-oxidation of epinephrine at pH 10.2 makes the

reaction a basis for simple assay for SOD. Superoxide anion (•O2-) generate by the xanthine oxidase

reaction is known to cause the oxidation of epinephrine to adrenochrome. The yield of adrenochrome

produced by •O2- introduced increases with increasing pH (Valerino and McCormack, 1971) and also

does concentration of epinephrine. These led to the proposal that auto – oxidation of epinephrine

proceeds by at least two distinct pathways, one which is a free radical chain reaction involving

superoxide anion radical and hence inhibitable by SOD.

Reagents/materials:

1. 0.05M carbonate buffer (pH 10.2)

14.3g of Na2CO3.10H2O (Sigma Chemicals Ltd, USA) and 4.2g of NaHCO3 (Sigma Chemicals

Ltd, USA) were dissolved in distilled water and made up to 1000ml mark in a liter standard

flask, then the solution was adjusted to pH 10.2.

2. 0.3mM Adrenaline

0.0013g of epinephrine (molecular weight of 182.21gmol-1

) (Sigma Chemicals Ltd, USA) was

dissolved in 250ml of distilled water. The solution was prepared fresh just before use.

Procedure

0.1ml of sample was diluted in 0.9ml of distilled water to make a 1 in 10 dilution. An aliquot of 0.2ml

of the diluted enzyme preparation was added to 2.5ml of 0.05M carbonate buffer (pH 10.2) to

equilibrate in the spectrophotometer, and the reaction was started by adding 0.3ml of fleshly prepared

0.3mM adrenaline to the mixture which was quickly mixed by inversion. The reference cuvette

contains 2.5ml of 0.05M carbonate buffer, 0.3ml of adrenaline (substrate) and 0.2ml of distilled water.

The increase in absorbance at 480nm was monitored every 30seconds for 150 seconds.

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Calculation:

Increase in Abs per minute = A2.5 – Ao

T

Where, Ao = initial absorbance

A2.5 = final absorbance and t = total time taken (150 secs or 2.5mins)

% inhibition = increase in absorbance of sample/min X 100%

Increase in the absorbance of blank/min

1 unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the

oxidation of adrenaline to adrenochrome during 1 minute.

SOD activity (units) = % inhibition

50%

Therefore,

Specific activity of SOD (units/mg protein) = SOD activity X dilution factor

mg protein

Determination of Reduced Glutathione (GSH) Level

The total sulphydryl groups, protein – bound sulphydryl groups and free sulphydryl groups like GSH

in biological samples can be determined using Ellman’s reagent, 5,5'-dithio-bis-2-nitrobenzoic acid

(DTNB) as described by Jollow et al, 1974.

Principle

This method is based on the development of a relatively stable yellow complex formed as a result of

reaction between Ellman’s reagent and free sulphydryl groups. The reduced form of glutathione

(GSH) in most instances is the bulk of cellular non – protein sulphydryl groups. The chromophoric

product, 2 – nitro – 5 – thiobenzoic acid, resulting from the reaction of Ellman’s reagent with GSH

possesses a molar absorption at 412nm. The absorbance of this complex at 412nm is proportional in

the level of GSH in the sample.

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GSH + R-S-S-R’ R-S- + GSS

-

R-S- = Yellow complex.

Reagents

1. Reduced Glutathione (GSH) working standard

40mg of GSH (Sigma Chemicals Co, USA) was dissolved in 100ml of 0.1M phosphate buffer,

pH 7.4 and then stored at 4oC.

2. 0.1M phosphate buffer pH 7.4

a) 0.1M K2HPO4.3H2O (molar mass; 228.22gmol-1

): 22.87g of K2HPO4.3H2O was

dissolved in distilled water and made up to 1000ml.

b) 0.1M KH2PO4 (molar mass; 186.09gmol-1

): 3.4g of KH2PO4 was dissolved in distilled

water and the volume made up to 250ml.

c) 8 volumes of (a) was added to 2 volumes of (b) and pH adjusted to 7.4 with 1M HCl or

NaOH.

3. Ellman’s reagent (DTNB)

4.0mg of Ellman’s reagent, 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) (Sigma Chemical Co,

USA) was dissolved in little amount of 0.1M phosphate buffer, pH 7.4 and made up to 100ml

mark in a standard flask with the same buffer. This was prepared flesh.

4. 4% Sulphosalicylic acid (Precipitating reagent)

4.0g sulphosalicylic acid (BDH Chemicals Co, England) was dissolved in little quantity of

distilled water and made up to 100ml mark in a standard flask.

Procedure

Serial dilution of the stock GSH were prepared as shown in table below. To each tube, appropriate

volumes of phosphate buffer were added and then followed by the addition of 4.5ml Ellman’s reagent.

The absorbance of the yellow color formed upon the addition of Ellman’s reagent was read within

5mins at 412nm using spectrophotometer. A plot of absorbance Vs Concentration of reduced GSH was

then obtained.

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Assay mixture for Calibration of Glutathione Standard curve

Tube number 1 2 3 4 5 6 7 8

GSH Stock (ml) - 0.025 0.05 0.1 0.2 0.3 0.4 0.5

GSH Conc. (µg/ml) 0.0 8 20 40 80 120 160 200

Phosphate buffer pH

7.4 (ml)

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Ellman’s reagent

(ml)

4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5

Absorbance at

412nm

0.00 0.137 0.203 0.283 0.451 0.509 0.670 0.859

Glutathione Standard Curve

Estimation of GSH level in test sample

0.2ml of sample was mixed with 1.8ml of distilled water to give 1 in 10 dilution. About 3ml of

precipitating reagent (4% sulphosalicyclic acid) was added to the diluted sample and then allowed to

stand for 10minutes form precipitate to occur. 0.5ml of supernatant withdrawn was added to 4ml of

phosphate buffer followed by 0.5ml of Ellman’s reagent was added. The blank was prepared with 4ml

of 0.1M phosphate buffer pH 7.4, 1ml of diluted precipitating solution and 0.5ml of Ellman’s reagent

(DTNB). The absorbance was read within 20minutes of color development at 412nm against blank

using spectrophotometer. Reduced glutathione concentration was proportional to the absorbance at

412nm.

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Determination of Glutathione –S– transferase Activity.

Glutathione -S- transferase activity was determined by the method according to Habig et al., 1974.

Principle

This based on the fact that all known glutathione -S- transferase demonstrate a relatively high activity

with 1-Chloro -2,4 – dintrobenzene (CDNB) as the second substrate. Consequently, the conventional

assay for glutathione – S – transferase activity utilizes 1 – Chloro -2, 4-dintrobenzene as substrate.

When this substrate is conjugated with reduced glutathione (GSH), its absorption maximum shifts to a

longer wavelength. The absorption increase at the new wavelength of 340nm which provides a direct

measurement of the enzymatic reaction.

Reagents

a) 20mM 1-Chloro-2, 4-dintrobenzene (CDNB)

3.37mg of CDNB (Sigma Chemicals Co, London) was dissolved in 1ml of ethanol.

b) 0.1M Reduced Glutathione

30.73mg (0.0307g) of reduced glutathione powder (Sigma Chemical Co, London) was

dissolved 100ml of 0.1M phosphate buffer pH 6.5.

c) 0.1M Phosphate buffer (pH 6.5)

a) 0.1M K2HPO4.3H2O (molar mass; 228.22gmol-1

): 22.87g of K2HPO4.3H2O was

dissolved in distilled water and made up to 1000ml.

b) 0.1M KH2PO4 (molar mass; 186.09gmol-1

): 3.4g of KH2PO4 was dissolved in distilled

water and the volume made up to 250ml.

8 volumes of (a) was added to 2 volumes of (b) and pH adjusted to 6.5 with 1M HCl or NaOH.

Procedure

The medium for the estimation of GST activity was prepared as shown in table 2.4 below and the

reaction was allowed to run for 60 seconds each time before the absorbance was read against the blank

at 340nm. The temperature was maintained at approximately 31oC. The absorbance was measured

using spectrophotometer.

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Glutathione -S- transferase assay medium.

REAGENT BLANK TEST

0.1M Reduced glutathione

(GSH)

30µl 30µl

20mM CDNB 150µl 150µl

0.1M phosphate buffer pH 6.5 2.82ml 2.79ml

Cytosol/microsomes --- 30µl

Total mixture 3ml 3ml

Calculation

The extinction coefficient of CDNB = 9.6mm-1

Cm-1

Glutathione – S – transferase activity = O.D/min X 1

9.6 0.03ml X mg protein

= µmole/min/mg protein.

Determination of Catalase (CAT) Activity

Catalase activity was determined according to the method of Sinha (1972) and modified by Artenie et

al., (2008).

Principle

This method is based on the fact that dichromate in acetic acid is reduced to chromic acetate when

heated in the presence of H2O2 with the formation of perchromic acid as an unstable intermediate. The

chromic acetate produced is measured colorimeterically at 570 – 610nm. It should be noted that

dichromate has no absorbance at this wavelength and hence its presence in the assay mixture does not

interfere with the determination of chromic acetate. Catalase preparation (in samples) is allowed to

split H2O2 for different periods of time. The reaction was stopped at a particular time by the addition

of dichromate/acetic acid mixture and the remaining H2O2 is determined by measuring chromic acetate

colorimetrically after heating the reaction mixture.

Reagents

1. 5% Potassium heptaoxodichromate (5% K2Cr2O7)

5g of dichromate K2Cr2O7 (BDH Chemicals Co., England) was dissolved in some volume of

distilled water in a 100ml standard flask and made up to the mark.

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2. 0.2M Hydrogen Peroxide (H2O2)

This was prepared by adding 5.15ml of H2O2 stock (30% purity) to 250ml of distilled water.

3. Dichromate/acetic acid solution

This solution was prepared by mixing solution of 5% K2Cr2O7 with glacial acetic acid (1:3 v/v)

and stored in brown bottle at room temperature.

4. 0.01M Phosphate buffer pH 7.0

2.28g of K2HPO4.3H2O and 1.42g KH2PO4 were dissolved in 900ml of distilled water. The pH

adjusted to 7.0 distilled water was added to make up to 1litre.

Procedure

Different amounts of H2O2 ranging from 20 to 160 moles were taken in small test tubes and 2ml of

dichromate/acetic acid was added to each. Addition of the reagents instantaneously produces an

unstable blue precipitate of perchromic acid. Subsequent heating for 10mins in a boiling water bath

changed the colour of the solution to stable green due to formation of chromic acetate. After cooling at

room temperature, the volume of the reaction mixture was made to 3ml with distilled water and the

absorbance measured with a spectrophotometer at 570nm. The concentration of the standard was

plotted against absorbance.

Preparation of H2O2 standard curve

Tube number 1 2 3 4 5 6 7 8 9

H2O2 (ml) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Dichromate/acetic

acid (ml)

2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

Distilled water

(ml)

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2

H2O2 conc.

(mmoles)

0.0 20 40 60 80 100 120 140 160

Absorbance@

570nm

0.00 0.08 0.21 0.25 0.29 0.39 0.47 0.54 0.60

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Hydrogen Peroxide Standard Curve

Estimation of Catalase Activity on Test Samples

0.1ml of sample was mixed with 4.9ml of distilled water to give a 1 in 5 dilution of the sample. The

assay mixture contained 4ml of 0.2M H2O2 and 5ml of 0.01M Phosphate buffer in a 10ml flat bottom

flask. 1ml of properly diluted enzyme preparation (test sample) was rapidly mixed with the reaction

mixture by gentle swirling motion. The reaction was run at room temperature. A 1ml portion of the

reaction mixture was withdrawn and blown into a test tube containing 2ml of dichromate/acetic acid

reagent at 60 seconds intervals for 3minutes. The H2O2 contents of the withdrawn sample were

determined by the method described above.

Calculation:

The mononuclear velocity constant, K, for the decomposition of H2O2 by catalase was determined by

using the equation for a first – order reaction.

K = 1/t log So/S.

Where, So is the initial concentration of H2O2

S is the concentration of peroxide at t min (60 seconds interval)

T is time – interval (1minute).

The value of K is plotted against time in minute and the velocity constant of catalase K(o) at time zero

determine by extrapolating the catalase content of the enzyme preparation was expressed in terms of

katalase feiahigkeit or ‘Kat F’ according to Von Euler and Josephson, 1927.

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Kat F = K(o)

mg protein/ml (U/mg protein)

Assessment of Lipid Peroxidation

This was assayed by measuring the TBA reactive products present in the test sample using the

procedure of Vashney and Kale (2007) and expressed as micromolar of malondialdehyde (MDA)/g

tissue.

Principle

The assay was based on the reaction of chromogenic reagent (2 – TBA) with MDA (end product of

lipid peroxidation) under acidic condition to yield a stable pink chromophone with maximum

absorbance at 532nm. It is ready extractable into organic solvents like butanol-1-ol.

MDA – TBA complex (pink color).

Reagents

1. 30% Trichloroacetic acid (TCA) solution

9g of TCA powder was dissolved in distilled water and made up to 30ml.

2. 0.75% Thiobarbituric acid (TBA) solution in 0.1M HCl

a) 0.1M HCl was prepared by adding 0.4ml of conc. HCl to 99.6 of distilled water.

b) 0.225g of TBA powder was dissolved in 30ml of 0.1M HCl and dissolution was aided

by shaking in boiling water.

1. 0.15M Tris KCl buffer (pH 7.4)

a) 1.15g of potassium chloride (KCl) was dissolved in water and volume made up to

100ml mark of standard flask.

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b) 2.36g of Tris base was dissolved and made up to 100ml with distilled water.

c) Both solutions (a) and (b) were mixed together and the buffer was standardized at pH

7.4

Method

An aliquot of 0.4ml of the test sample was mixed with 1.6ml of Tris KCl buffer (which was first

placed into the test tube before the test sample). Then 0.5ml of 30% TCA was added followed by

0.5ml of 0.75% TBA and the mixture was placed in a water bath for 1hour between 90 – 95oC. this

was then cooled in ice and centrifuge at 3000r.p.m. for 15mins. The clear pink supernatant was

collected and absorbance measured against a reference blank of distilled water at 532nm in a

spectrophotometer. The MDA level was calculated according to the method of Adam – Vizi and

Seregi (1982).

Calculation:

Lipid peroxidation was expressed in units/mg protein

where E532 is molar extinction coefficient is 1.56 x 105M

-1CM

-1

Therefore, MDA (units/mg protein) = Absorbance of test X volume of mixture

E532 X volume of sample X mg protein

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