prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/7611/1/... · v Table of Content Chapters Page No. Table of Content V List of Schematic Diagrams X List of Tables Xi List of

i

Effect of Chelating Agents, Fungi and Native

Plants on Remediation of Metals Contaminated

Soils

Shazia Akhtar

Department of Environmental Sciences

Fatima Jinnah Women University

The Mall Rawalpindi, Pakistan

2015

ii

Effect of Chelating Agents, Fungi and Native

Plants on Remediation of Metals Contaminated

Soils

By

Shazia Akhtar

A thesis submitted to Fatima Jinnah Women University,

Rawalpindi, in partial fulfillment of the requirement for the

Degree of

Doctor of Philosophy

In

Environmental Sciences

Department of Environmental Sciences

Fatima Jinnah Women University The Mall,

Rawalpindi, Pakistan

iii

2015

Dedicated to my dear teacher

Dr. Shazia Iftikhar

For her generous help and

kind cooperation

iv

Declaration

It is certify that this dissertation entitled “Effect of Chelating Agents,

Fungi and Native Plants on Remediation of Metals Contaminated Soils”

submitted by Shazia Akhtar, on accepted in its present form by the

Department of Environmental Sciences, Fatima Jinnah Women

University, Rawalpindi, Pakistan, as satisfying the dissertation

requirements for the degree of Ph.D in Environmental Sciences.

Supervisor: _____________________

External Examiner: _____________________

Chairperson: _____________________

Dated: -------------------

v

Table of Content Chapters Page No.

Table of Content

V

List of Schematic Diagrams X

List of Tables Xi

List of Figures Xiii

Abstract Xvi

Chapter 1

Introduction

1

1.1 Sources of Soil Contamination 2 1.2 Effects of Heavy Metals on Environment 4 1.3 Phytoremediation Technology 5 1.4 Hyper Accumulator Plants in Pakistan 6 1.5 Enhanced Phytoremediation 7

1.5.1 Chemically enhanced phytoremediation 7 1.5.2 Biologically enhanced phytoremediation 9

1.6 Significance of the Study 10 1.7 Goal of Study 13 1.8 Aims and Objectives 14

Chapter 2

Review of Literature

15

2.1 Metal Pollution 15 2.2 Phytoremediation 17 2.3 Native Plant Species of Pakistan for phytoremediation 19 2.4 Environmental Concerns Associated with Phytoremediation 22 2.5 Advantages of Phytoremediation 23 2.6 Disadvantages and Limitations of Phytoremediation 24 2.7 How to Make Effective Phytoremediation 25

Chapter 3

Materials and Methods

26

3.1 Phase 1: To conduct surveys for the assessment of peri-urban

agricultural areas (Multan, Kasur, Lahore and

Gujranwala) heavy metals contamination load in soils,

plants and untreated waste water.

24

3.1.1 Soil Samples Collection 28 3.1.2 Physiochemical Analysis of Soil Samples 37

3.1.2.1 Soil Texture 37 3.1.2.2 Soil pH 39 3.1.2.3 Electrical Conductivity (EC) 39 3.1.2.4 Lime Content/Calcium Carbonate (CaCO3) 39 3.1.2.5 Organic Matter Analysis 39 3.1.2.6 Heavy Metal Analysis of Soil Samples 40

3.1.3 Waste Water Samples Collection 39

vi

3.1.4 Waste Water Samples Physiochemical Analysis 41 3.1.4.1 Electrical Conductivity 45 3.1.4.2 pH 45 3.1.4.3 Soluble Na+ and K+ 45 3.1.4.4 Carbonate (CO3

-2) and Bicarbonate (HCO3-) 46

3.1.4.5 Chloride 46 3.1.4.6 Heavy Metals Analysis of Waste Water 46

3.1.5 Native Plants (Crop/Vegetable) Sampling and Analysis 46 3.1.5.1 Native Plants Heavy Metal Analysis 47

3.1.6 Isolation of Fungal Strains from Soil and Waste Water 47 3.1.6.1 Sterilization of Apparatus 45 3.1.6.2 Media Preparation 47 3.1.6.3 Preparation of Plates 468 3.1.6.4 Serial Dilution Method 48 3.1.6.5 Preservation and Identification of Fungi 48 3.1.6.6 Preparation of Slants 49 3.1.6.7 Analysis of Data 49

3.2 Phase 2: To test the various chelates and tolerant fungal strains

for their metal extraction and solubilizing efficiency in

laboratory batch experiments (shacking and incubation).

49

3.2.1 Selection of the Contaminated Soils 52 3.2.2 Preparation of Soil 52 3.2.3 Shaking Experiment (Efficiency of different chelating agents) 53 3.2.4 Incubation Experiments 53 3.2.5 Mycoremediation Experiments 54 3.2.6 Analysis of Data 54

3.3 Phase 3: To evaluate the natural, biological and chemically

enhanced phytoextraction potential of native plant

species through in-vitro experiments.

55

3.3.1 Soil Selection and Collection 56 3.3.2 Soil Preparation 56 3.3.3 Selection of Native Crops 56 3.3.4 In-Vitro Experiments 56

3.3.4.1 Seed Germination 56 3.3.4.2 Plant Growth 57 3.3.4.3 Harvesting of Plantlets and Digestion 57 3.3.4.4 Heavy Metals Analysis 58 3.3.4.5 Data Analysis 58

3.4 Phase 4: To predict the interactions between plant, contaminants

and soil characteristics through greenhouse pot

experiments

59

3.4.1 Soil Sampling 60 3.4.2 Soil Preparation 60 3.4.3 Green House Pot Experiment 60 3.4.4 Analysis of Data 61

3.5 Development of Phytoremediation Model 62

vii

Chapter 4 Results 63

4.1 Phase 1: To conduct surveys for the assessment of peri-urban

agricultural areas (Multan, Kasur, Lahore and

Gujranwala) heavy metals contamination load in soils,

plants and untreated waste water.

65

4.1.1 Physiochemical Analysis of Contaminated Soil Samples 65 4.1.2 Heavy Metal Analysis of Soil Samples 66 4.1.3 Physiochemical Analysis of Collected Waste Water Samples 70 4.1.4 Heavy Metals Analysis of Waste Water 72 4.1.5 Heavy Metals Analysis of Collected Plants (Crops/vegetables) Samples 76 4.1.6 Isolation of Fungi from Collected Soil Samples 80

4.1.6.1 Relative Dominance (d). 81 4.1.6.2 Diversity Index 84

4.1.7 Isolation of Fungi from Waste Water Samples 5

4.2 Phase 2: To test the various chelates and tolerant fungal strains

for their metal extraction and solubilizing efficiency in

laboratory batch experiments (shaking and incubation).

88

4.2.1 Shaking Experiments 88 4.2.1.1 Effect of Shaking Time and Concentration of EDTA on Metals

Solubilization 88

4.2.1.2 Effect of Shaking Time and Concentration of DTPA on Metals

Solubilization. 93

4.2.1.3 Effect of Shaking Time and Concentration of NTA on Metals

Solubilization 95

4.2.2 Incubation Experiments 97 4.2.2.1 Effect of Incubation Time and EDTA Concentrations on Metals

Extraction 98

4.2.2.2 Effect of Incubation Time and DTPA Concentrations on Metals

Extraction 101

4.2.2.3 Effect of Incubation Time and NTA Concentrations on Metals

Extraction 104

4.2.3 Analysis of Soluble Metal Contents in Fungal Treated Soil Sample 107 4.2.3.1 Penecillium sp. 107 4.2.3.2 Aspergiilus niger 110 4.2.3.3 Aspergillus flavus 113 4.2.3.4 Curvularia sp. 116 4.2.3.5 Aspergillus terrus 119 4.2.3.6 Aspergillus fumigatus 122 4.2.3.7 Aspergillus sp. 125 4.2.3.8 Fusarium sp. 128




131

4.3.1 Plants Growth Parameters 132 4.3.1.1 Shoot length of native crops 132

viii

4.3.1.2 Root length of native crops 137 4.3.1.3 Shoot fresh and dry weight 141 4.3.1.4 Root fresh and dry weight 145

4.3.2 Heavy Metals Accumulation in Plants Tissues 149 4.3.2.1 Metals accumulation in wheat 149 4.3.2.2 Metals accumulation in barley 150 4.3.2.3 Metals accumulation in maize 150 4.3.2.4 Metals accumulation in sunflower 151 4.3.2.5 Metals accumulation in bajra or pearl millet 152 4.3.2.6 Metals accumulation in barley 152 4.3.2.7 Metals accumulation in mustard 152

4.4 Phase 4: Greenhouse pot experiments to predict the interactions

between plant, contaminants and soil characteristics.

155

4.4.1 Green House Experiment of Maize 155 4.4.1.1 Maize shoot biomass 155 4.4.1.2 Maize root biomass 156 4.4.1.3 Residual soil metal contents 158 4.4.1.4 Metal analysis in maize shoots tissues 160 4.4.1.5 Metal analysis in maize roots tissues 160

4.4.2 Green house experiment of mustard 163 4.4.2.1 Mustard shoot biomass 163 4.4.2.2 Mustard root biomass 163 4.4.2.3 Residual soil metal contents 166 4.4.2.4 Metal analysis in mustard shoots tissues 167 4.4.2.5 Metal analysis in mustard roots tissues 168

4.4.3 Bioconcentration Factor (BCF) 171 4.4.4 Phytoextraction Rate (PR) 173 4.4.5 Extraction Factor 175 4.4.6 Phyto-remediation Model 177

4.4.6.1 Model assumptions 177 4.4.6.2 Parameters used in the model 178 4.4.6.3 Input parameters (Known parameter) 178 4.4.6.4 Required parameter (Unknown parameters) 178

Chapter 5

Discussion

180

5.1 Phase 1: Surveys for the assessment of heavy metals

contamination load in soils, plants and untreated waste

water of peri urban agricultural areas (Multan, Kasur,

Lahore and Gujranwala)

180

5.1.1 Use of Waste Water in Agricultural Land 181 5.1.2 Contaminated of Soil with Heavy Metals 182 5.1.3 Heavy Metals in Crops/Vegetables Samples 183 5.1.4 Fungal Diversity in Metals Contaminated Soils 184

5.2

Phase 2: To test the various chelates and tolerant fungal strains for

their metal extraction and solubilizing efficiency in

laboratory batch experiments (shacking and incubation).

186

ix

5.2.1 Shaking experiments 187 5.2.2 Incubation experiments 189 5.2.3 Heavy metals solubilization by fungi 190




191

5.4 Phase 4: Greenhouse pot experiments to predict the interactions

between plant, contaminants and soil characteristics. 193

Chapter 6

Conclusion

197

Phase 1 197

Phase 2 198

Phase 3 199

Phase 4 200

Chapter 7

Recommendations

201

Chapter 8

References

203

Appendix I I

Appendix II Ii

Appendix III Iv

Acknowledgement Xxxiv

Publications

A. Published papers B. Accepted Papers C. Conference Proceeding D. Abstract E. Paper Manuscript Ready for Submission F. Paper Presentation in Conferences G. Poster Presentation in Conferences

Reviewers Reports

Plagearism Certificate

Xxxv

x

List of Schematic Diagrams

Sr. No. Schematic Diagrams Page No.

1 Surveys and assessment of heavy metals contamination load in

soil, wastewater and plant samples of peri urban agricultural

areas of Punjab from April 2012-April 2013.

172

2 Laboratory batch experiments (Shacking and Incubation) for

the testing of various chelates and tolerant fungal strains for

their metal extraction and solubilizing efficiency.

178

3 In-vitro experiments to explore the natural and chemically

enhanced phytoextraction potential of native plant species. 182

4 To predict the interactions between plants, fungi and chelates

under greenhouse pot experiments. 184

xi

List of Tables

Table No. Titles Page No.

2.1 Metal ion hyper-accumulating plants. 18 3.1 Date of collection and total soil, plants, vegetables and effluents

samples from peri-urban agricultural areas of Multan, Kasur, Lahore

and Gujranwala.

26

3.2 Detail of soils and plant sampling sites of Multan and their global

positioning system coordinates. 31

3.3 Detail of soils and plant sampling sites of Kasur and their global

positioning system coordinate. 32

3.4 Detail of soils and plant sampling sites of Lahore and their global


3.5 Detail of soils and plant sampling sites of Lahore and their global


3.6 Detail of waste water sampling sites of Multan and their global

positioning system coordinates 40

3.7 Detail of waste water sampling sites of Kasur and their global


3.8 Detail of waste water sampling sites of Lahore and their GPS (global

positioning system) coordinates. 42

3.9 Detail of waste water sampling sites of Gujranwala and their global


3.10 Characteristics of Gujranwala soils used in shacking and incubation

experiments 49

3.11 Native crops used in in-vitro assessment. 53 4.1 Physiochemical analysis of soil samples of of peri-urban agricultural

areas of Multan, Kasur, Lahore and Gujranwala. 63

4.2 Total heavy metal concentrations (mg kg-1) of per-urban agricultural

soils of Multan, Kasur Lahore and Gujranwala irrigated with

municipal/industrial effluents.

66

4.3 Physiochemical analysis of municipal/industrial waste water of peri-

urban agricultural areas of Multan, Kasur, Lahore and Gujranwala. 68

4.4 Metal concentrations of municipal/industrial effluent used for

irrigation (mg L-1) in the study areas. 72

4.5 Metal concentrations (mg Kg-1) of crops/vegetable samples grown in

peri-urban agricultural area of Gujranwala irrigated with

municipal/industrial effluent.

73


peri-urban agricultural area of Kasur irrigated with


74


peri-urban agricultural area of Lahore irrigated with


74

xii


peri-urban agricultural area of Multan irrigated with


75

4.9 Diversity analysis of peri-urban agricultural areas of studied area. 80 4.10 Micro-fungi isolated from waste water samples of peri-urban

agricultural areas of Gujranwala. 83

4.11 Micro-fungi isolated from waste water samples of peri-urban

agricultural areas of Kasur. 83


agricultural areas of Lahore 84


agricultural areas of Multan. 84

4.14 Cadmium (Cd), Lead (Pb) Chromium (Cr) and Copper (Cu)

phytoextraction potential (mg g-1) of maize shoot grown on

Gujranwala and Lahore soils amended DTPA and fungal strains.

153


phytoextraction potential (mg g-1) of maize root grown on


154


phytoextraction potential (mg g-1) of mustard shoot grown on


161


phytoextraction potential (mg g-1) of mustard root grown on


162

4.18 Bioconcentration factors of Cadmium (Cd), Lead (Pb) Copper (Cu)

amd Chromium (Cr) of maize and mustard crops in Gujranwala and

Lahore soils amended with DTPA and Aspergillus species.

164

4.19 Phytoextraction rates (%) of Cd and Pb of maize and mustard grown

on Gujranwala and Lahore soils amended with fungi and different

DTPA rates.

166

4.20 Extraction factor (EF) percentages of Pb and Cu of maize and

mustard grown on Gujranwala and Lahore soils amended with fungi

and various rates of DTPA.

168

4.21 List of variables used in the model. 170

xiii

List of Figures

Figure No. Titles Page No.

3.1 Locations of sampling sites of peri-urban agricultural areas of Multan. 27 3.2 Locations of sampling sites of peri-urban agricultural areas of of Kasur. 28 3.3 Locations of sampling sites of peri-urban agricultural areas of Lahore. 29 3.4 Locations of sampling sites of of peri-urban agricultural areas of

Gujranwala. 30

3.5 The United State Department of Agricultur (USDA) Soil Textural

Triangle. 36

4.1 The Berger-Parker Dominance Index of fungal diversity in peri-urban

agricultural soils of Gujranwala. 78


agricultural soils of Kasur. 79


agricultural soils of Lahore. 80


agricultural soils of Multan. 80

4.5 Effect of time (hours) and concentration of EDTA on Cu solubilization

from metals contaminated soil of Gujranwala. 85

4.6 Effect of time (hours) and concentration of EDTA on Pb solubilization


4.7 Effect of time (hours) and concentration of EDTA on Cd solubilization

from metals contaminated soil. 87

4.8 Effect of time (hours) and concentration of EDTA on Cr solubilization


4.9 Effect of time (hours) and concentration of DTPA on Cu solubilization


4.10 Effect of time (hours) and concentration of DTPA on Pb solubilization


4.11 Effect of time (hours) and concentration of DTPA on Cd solubilization


4.12 Effect of time (hours) and concentration of DTPA on Cr solubilization


4.13 Effect of time (hours) and concentration of NTA on Cu solubilization


4.14 Effect of time (hours) and concentration of NTA on Pb solubilization


4.15 Effect of time (hours) and concentration of NTA on Cd solubilization


4.16 Effect of time (hours) and concentration of NTA on Cr solubilization

from 93

4.17 Solubilized Cu at different EDTA concentrations and incubation time. 94 4.18 Solubilized Pb at different EDTA concentrations and incubation time. 94

xiv

4.19 Solubilized Cd at different EDTA concentrations and incubation time. 95 4.20 Solubilized Cr at different EDTA concentrations and incubation time. 95 4.21 Solubilized Cu at different DTPA concentrations and incubation time. 96 4.22 Solubilized Pb at different DTPA concentrations and incubation time. 97 4.23 Solubilized Cd at different DTPA concentrations and incubation time. 98 4.24 Solubilized Cr at different DTPA concentrations and incubation time. 99 4.25 Solubilized Cr at different NTA concentrations and incubation time. 99 4.26 Solubilized Pb at different NTA concentrations and incubation tim 100 4.27 Solubilized Cd at different NTA concentrations and incubation time. 100 4.28 Solubilized Cr at different NTA concentrations and incubation time. 101 4.29a Solubilized Cu by Penicillium sp. in contaminated soil. 102 4.29b Solubilized Pb by Penicillium sp. in contaminated soil. 102 4.29c Solubilized Cd by Penicillium sp. in contaminated soil. 103 4.29d Solubilized Cr by Penicillium sp. in contaminated soil. 103 4.30a Solubilized Cu by Aspergillus niger. in contaminated soil. 104 4.30b Solubilized Pb by Aspergillus niger. in contaminated soil. 105 4.30c Solubilized Cd by Aspergillus niger. in contaminated soil. 105 4.30d Solubilized Cr by Aspergillus niger. in contaminated soil. 106 4.31a Solubilized Cu by Aspergillus flavus in contaminated soil. 107 4.31b Solubilized Pb by Aspergillus flavus in contaminated soil. 108 4.31c Solubilized Cd by Aspergillus flavus in contaminated soil. 108 4.31d Solubilized Cr by Aspergillus flavus in contaminated soil. 109 4.32a Solubilized Cu by Curvularia sp. in contaminated soil. 110 4.32b Solubilized Pb by Curvularia sp. in contaminated soil. 111 4.32c Solubilized Cd by Curvularia sp. in contaminated soil. 111 4.32d Solubilized Cr by Curvularia sp. in contaminated soil. 112 4.33a Solubilized Cu by Aspergillus terrus in contaminated soil. 113 4.33b Solubilized Pb by Aspergillus terrus in contaminated soil. 113 4.33c Solubilized Cd by Aspergillus terrus in contaminated soil. 114 4.33d Solubilized Cr by Aspergillus terrus in contaminated soil. 115 4.34a Solubilized Cu by Aspergillus fumigatus in contaminated soil. 116 4.34b Solubilized Pb by Aspergillus fumigatus in contaminated soil. 116 4.34c Solubilized Cd by Aspergillus fumigatus in contaminated soil. 117 4.34d Solubilized Cr by Aspergillus fumigatus in contaminated soil. 118 4.35a Solubilized Cu by Aspergillus sp. in contaminated soil. 119 4.35b Solubilized Pb by Aspergillus sp. in contaminated soil. 119 4.35c Solubilized Cd by Aspergillus sp. in contaminated soil. 120 4.35d Solubilized Cr by Aspergillus sp. in contaminated soil. 121 4.36a Solubilized Cu by Fusarium sp. in contaminated soil. 122 4.36b Solubilized Pb by Fusarium sp. in contaminated soil. 122

xv

4.36c Solubilized Cd by Fusarium sp. in contaminated soil. 123 4.36d Solubilized Cr by Fusarium sp. in contaminated soil. 124 4.37 Effect of different concentrations of metals on native plants shoots

length. 129

4.38 Effect of different concentrations of metals on native plants roots length.

Effect of different concentrations of metals on native plants roots length. 132

4.39a Effect of different concentrations of metals on native plants roots

length. 135

4.39b Effect of different concentrations of metals on native plants shoots dry

weight. 136

4.40a Effect of different concentrations of metals on native plants roots fresh

weight. 139

4.40b Effect of different concentrations of metals on native plants roots dry

weight. 140

4.41 Heavy metals accumulation in shoot and root of Wheat. 141 4.42 Heavy metals accumulation in shoot and root of Barley. 142 4.43 Heavy metals accumulation in shoot and root of Maize. 143 4.44 Heavy metals accumulation in shoot and root of Sunflower. 144 4.45 Heavy metals accumulation in shoot and root of Bajra 145 4.46 Heavy metals accumulation in shoot and root of Mustard. 145 4.47 Heavy metals accumulation in shoot and root of Soybean. 146 4.48 Maize shoot fresh biomass production in Gujranwala and Lahore soils in

the presence of fungi and DTPA concentrations. 148

4.49 Maize shoot dry biomass production in Gujranwala and Lahore soils in

the presence of fungi and DTPA concentrations 148

4.50 Maize root fresh biomass production in Gujranwala and Lahore soils in


4.51 Maize root dry biomass production in Gujranwala and Lahore soils in the

presence of fungi and DTPA concentrations. 150

4.52 Post harvest Copper (Cu) and Lead (Pb) concentrations in soils. 151 4.53 Post harvest Cadmium (Cd) and Chromium (Cr) concentrations in soils. 151 4.54 Mustard shoots fresh biomass production in Gujranwala and Lahore soils

in the presence of fungi and DTPA concentrations. 156

4.55 Mustard shoot dry biomass production in Gujranwala and Lahore soils in


4.56 Mustard root fresh biomass production in Gujranwala and Lahore soils in


4.57 Mustard root dry biomass production in Gujranwala and Lahore soils in


4.58 Post harvest Copper (Cu) and Lead (Pb) concentrations in soils. 158 4.59 Post harvest Cadmium (Cd) and Chromium (Cr) concentrations in soils. 158

xvi

Effect of Chelating Agents, Fungi and Native Plants on Remediation of

Metals Contaminated Soils

Abstract- In present research work four different peri-urban agricultural areas of Punjab

(Gujranwala, Kasur, Lahore and Multan) were surveyed in the first phase (2012-2013).

Total 138 contaminated soil samples, 131 waste water samples and 131 native plant

samples were collected and analyzed. Physiochemical analysis of soil and waste water

samples was done and also processed for fungal isolation. Native plants samples were

analyzed for heavy metal contents (Cu, Cd, Pb and Cr). In overall assessment Pb, Cd and

Cr were noticed above recommended permissible values in soil samples of all the study

areas. Whereas Cu was found above the recommended permissible limits only in samples

of Multan and Gujranwala as compared to Kasur and Lahore soil samples.

Physiochemical analysis of wastewater samples also showed high EC, bicarbonates,

chlorides and sodium in collected samples of Multan however, Kasur samples showed

salinity problems. In case of heavy metals in waste water samples Pb and Cd

contamination was found in all the four areas. But Cr contamination was found more in

Multan and Lahore waste water samples. Native plants of Multan were found

contaminated with Cd. In case of Kasur Cr was found maximum in native plant samples.

Plants samples of Lahore were also showing Pb, Cu, Cr and Cd contamination. In case of

fungal diversity maximum number of fungi were isolated from heavy metal contaminated

samples of Multan, Kasur, Lahore and Gujranwala.

In second phase of study (2013-2014) soil shaking and incubation experiments

were carried out to check the solubilization of heavy metals (Cu, Cd, Cr and Pb) in the

most polluted soils of Gujranwala by adding different levels of EDTA (Ethylene Dinitrilo

Tetra Acetic acid), DTPA (Diethylene Triamine Penta Acetic acid), NTA (Nitrilo Tri

Acetic acid) and fungal spore suspensions of three metals tolerant species. In shaking

experiments, it was noticed that with increasing doses of chelating agents heavy metals

solubilization also increased and 5.0 mM level of DTPA, EDTA, NTA and 120 hours

shaking time was noticed the best optimum value for further experiments. Whereas in

incubation studies more Cd and Cu were solubilized by DTPA and EDTA was noticed

best solubilizer for Pb. While NTA had solubilized maximum Cr and time period of 20

xvii

and 30 days was more suitable for solubilization of metals. Chelating agents have

capacity for the remediation of heavy metal contaminated soils of peri-urban agricultural

areas. In mycoremediation experiments (experiments in which fungi is used for metals

solubilization from contaminated soils) maximum Pb and Cu were solubilized by

Curvularia sp. and highest solubilization of Cr and Cd was observed by Aspergillus

niger.

In third phase of study (2014-2015) in-vitro experiments were conducted growing

different seed varieties of local crops wheat, maize, barley, bajra, sunflower, soybean and

mustard. Metals contaminated soil of Gujranwala was used for experiments. Seeds were

germinated and grown in laboratory in ambient environment and irrigated with different

concentrations (50,150, 250 mgkg-1 of soil) of each toxic metal (Cu, Cd, Cr and Pb).

Plants were harvested on 4th, 8th, 16th and 20th day. Root as well as shoot biomass was

recorded and heavy metal concentrations were also analyzed in roots and shoots of plants

using Flame Atomic Absorption Spectrophotometer. Evidences provided by in-vitro

experiments indicated that maize and mustard were more biomass producing crops.

Maximum root biomass was produced by barley and bajra crops. While maximum shoot

and root length was also produced by barley crop only. However, sunflower and barley

were noticed more effective accumulators of Cu. Similarly for Cd as well as Cr maize,

wheat and barley were proved effective accumulators. The heavy metal uptake analysis

showed that sunflower had the highest potential to uptake Cu and Pb in their shoot parts.

In phase four (2014-2015), based on all the experiments chemical and biological

enhanced phytoremediation potentials of maize (Zea mays) and mustard (Brassica

campstrus) were evaluated by cropping them on different contaminated soils of Lahore

and Gujranwala for 75 days. Soils were treated with varying amounts of DTPA (1.25,

2.5, and 5.0 mM kg−1 soil) and three fungal species (Aspergillus niger, A. fumigatus and

A. flavus) facilitated the metal uptake by plants. In pot experiment under green house,

addition of fungi and DTPA chelate significantly increased the Cu, Pb, Cr and Cd

concentrations in roots and shoots. Maximum maize shoot biomass was obtained in the

presence of A.s fumigatus and maximum root biomass was produced in the presence of A.

niger. In case of mustard crop both shoot and root biomass was produced maximum in

xviii

the presence of A.s fumigatus. Overall A. fumigatus presented good results in increasing

biomass production of maize and mustard plants in both Gujranwala and Lahore soils.

Post harvest metals contents were analyzed in maize growing soils and it was found that

maximum Cd and Cu were solubilized in A. flavus inoculated soils while Pb and Cr were

solubilized maximum in A. fumigatus inoculated soils. In case of mustard growing soils

post harvest metals contents were analyzed and results demonstrated that maximum Cd

and Cu were solubilized in A. niger inoculated soils while Pb and Cr were solubilized

maximum in A. flavus inoculated soils. In case of metal uptake efficiency of tested

plants, it was noticed that both chemical and biological treatments were proved effective.

Increases of metals uptake, bioconcentration factor, phytoextraction rate and

phytoextraction efficiency were noticed in both fungal and DTPA amended soils.

In present research on the basis of green house experiment conducted by using

maize crop a phytoremediation model was developed.

xix

Chapter 1

Introduction The agriculture sustainability largely depends on two natural resources; water and

land, agricultural production is affected badly if one of them is limited. On earth for

existence and survival of life water is very important. It is being used for agricultural,

domestic, industrial and recreational purposes, though agricultural sector is using 90% of

water (Dara, 1993).

There is shortage of irrigation water in Pakistan because surface water does not

fulfill the water requirements for crops. This water shortage is being fulfilled by

combined use of ground water and waste water (domestic and industrial) of urban

areas.This mixed water is being used for vegetable and crop growth in peri-urban

agricultural areas (Lone, 1995). In Pakistan it is estimated that in big cities like Lahore,

Karachi, Multan, Peshawar, Faisalabad, Hyderabad, Kasur, Quetta, Sukhur and

Islamabad/Rawalpindi sewage is being produced 116590 million gallons per day and

32000 hectares of land is being irrigated with this water (Saleem, 2005). Big cities have

no proper disposal and management systems but producing constantly huge volumes of

waste water (Ghosh & Singh, 2005). In Pakistan only 2% cities are using waste water

treatment plants (Clemett & Ensink, 2006). So, 90% of untreated waste water is being

used in agricultural activities in more than 80% cities of Pakistan (Ensink et al., 2004).

Currently 0.3 million hectares agricultural land is irrigated with waste water. The

use of waste water and its disposal ultimately boosts agricultural production and

minimizes the threats of environmental contamination (Saleem, 2009). This waste water

is used for irrigation as it is a rich source of nutrients which is beneficial for plant growth.

There are various types of industries situated in and around industrial cities. These

industries discharge their untreated effluent which ultimately mixes with urban waste

water and contain heavy metals such as chromium, cadmium, copper and lead etc. This

industrial waste water may be poured directly into water courses without pretreatment

(Malik et al., 2009) while on the other hand farmers use this contaminated water in their

fields (Rattan et al., 2005). Soil acts as filter of toxic chemicals it may adsorb and retains

heavy metals from waste water (Rattan et al., 2005) but when the capacity of soil to

retain toxic metals is reduced then these toxic metals are released into groundwater.

2

These toxic metals may enter the plants and whole food chain making it poisonous for

human beings. Thus environmental and human life quality simultaneously is under threat

by increasing soil pollution (Zia et al., 2008).

1.1 Sources of Soil Contamination

It is estimated that about 40,000 hectars agricultural land is irrigated with

municipal waste water. It is mainly used due to deficiency of canal water for irrigation.

This waste water contains nutrients for the plants and farmers can easily access this water

throughout the year. This waste water is mainly used for fodder crops and vegetables

cultivation. Lasa (2000) estimated that sweage water application to 40 cm depth of soil

may add 100 to 200 kg of nitrogen, 100 to 250 kg of potassium and 6 to 20 kg of

phosphorus. So, these waste water applications remove the fertilizer usage.This practice

is also dangerous because a big threat of utilizing raw city effluent is contamination of

food and this also poses threats of pathogens and water born diseases (Aziz, 2006).

Gujranwala has set up “Gujranwala Business Centre” for promotion of industries

situated in Gujranwala by Government of Pakistan. Different industries like master tiles

and ceramic industries, polymer industries, textiles, power and generators, steel mills,

mining and food industries. These industries discharge their waste water containing toxic

metals in the drainages. Local farmers across these drainages use that waste water to

irrigate their agricultural fields because they cannot use costly fertilizers (Mapanda et al.,

2005). Different contaminants are also added up in the soil from different anthropogenic

activities carried out in these cities such as emissions from industrial plants, vehicle

exhausts, and thermal power stations and dumping of different wastes.

Kasur city is well known for leather industries. About 600 tannaries are located

in the big cities (Karachi, Sialkot and Kasur) of Pakistan. Tanneries waste water is

discharged in the nearby water channels and soil which causes serious health hazards.

The most threatened and affected area is Kasur because it has higher number of tannery

industries. About nine millions liter of contaminated effluent is released daily. Here safty

measure for the exposure and work place protection is not adopted. Presently

environmental contamination is enhanced and affecting the health of nearby population.

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3

Diseases of different types like respiratory diseases infections of lungs,

dysentery/diarrhea and fever are reported more frequently, in native people of Kasur

(Tariq et al., 2005; Syed et al., 2010). Malik et al. (2009) reported that a larg area in the

vicinity of tannery industries in Kasur has become unusable due to heavy metals

pollution.

Lahore is a metropolitan city and 1121 industries are located which include 36

textile industries, 642 steel factories and foundries of discharging iron scrap, cadmium,

lead, and hazardous/ toxic chemicals and about 295 other industries like electroplating

miles, leather tanneries and pigment factories etc. (Saleem, 1990). These all industries

discharge their effluent in the twelve drainages of the city which contain hazardeous

chemical and heavy metals and these are managed by Water and Sanitation Authority

(WASA). Vegetable farming with this polluted water is a common practice in Lahore

(Mahmood & Malik, 2013).

Multan is a big producer of leather and leather related products. It also has

tradition of tanning. A large number of industries present in the city of Multan have no

approach to treatment plants so industries release waste water to agricultural fields and

nearby landfills. The toxic chemicals enter in the soil system and leach down to the

ground water. Therefore, soil and ground water quality is being affected adversely. This

method of discharging tannery effluents polluted the soil and nearby water and provide

pathway for the heavy metals to contaminate food chain (Raju & Tandon, 1999). It is

reported by the Parikh et al. (1995) that residents of theses areas are being suffering

serius types of diseases. Therefore, these areas are considerd for the heavy metals

contamination assessment and remediation purposes.

Rapid industrialization and urbanization also resulted in continuous loading of

toxic effluents in the soil. Hence soil pollution is a serious issue in these cities.

Continuous use of untreated city effluents for growing of crops and vegetables like

wheat, grams, barley, rice and maize may result in heavy metals accomulation such as Cr,

Cd, Pb and Cu in soils in phyto-toxic concentrations (Gratao et al., 2005). Soils may also

be contaminated by different environmental impacts such as sludge or urban composts,

4

pesticides and fertilizers, emissions from municipal waste incinerators, car exhausts,

residues from mining and metal smelting. Heavy metal content in soil has greater

importance because soil effectively acts as a reservoir which after temporary storage of

metals can act as a source under certain conditions. Therefore, soils act as both source

and sink for different types of environmental pollutants. Overall heavy metals

accumulation and their translocation and other form of soil pollutants are now important

challenges for these cities.

1.2 Effects of Heavy Metals on Environment

Soil contamination with heavy metals is a common practice and difficult to treat

because soil is source and also sink for the heavy metals. With concerned to human

health, heavy metals assessment in the soil is very important issue. Because they are toxic

in nature and their degredation is difficult. Therefore, these heavy metals remain

persistant in the soil and in ecosystem. Bioconcentrate in different level of food chain

threats all the living beings (Aragay et al., 2011). Soil contaminated with metals is a

primary route of toxic metals exposure to humans and causing cytotoxic, mutagenic and

carcinogenic effects in animals. Heavy metals could enter into the body of human beings

when they consumed food contaminated with heavy metals. From the literature, it is

reported that more than 70% of dietary intake of cadmium is contributed via food chain.

It has been reported by Zia et al. (2008) that dietry crops and vegetables grown in heavy

metals contaminated soils pose serious health problems. Heavy metals pollution increases

in biological and ecological systems and exert harmful effects. Even a very low level of

these heavy metals causes serious health disorders. Heavy metals can persist in the soil

for many thousands of years and dangerous for the higher organisms. These heavy metals

also affect the plants growth and soil microbiota. It is well known fact that heavy metals

cannot be degraded chemically, so they are physically removed and can be transformed

into less toxic and non toxic forms (Ghani, 2010). Contamination of soil with heavy

metals poses serouse effects on living organisms and ecosystem (Vaxevanidou et al.,

2008). Prolonged human exposure to heavy metals causes renal dysfunction disease-

tubular protein. Similarly inhaling dust and fumes having high metal concentrations can

cause destructive lungs disease that is pneumonitis. Cadmium pneumonitis can be

5

identified as pain in chest, reddish sputum and ultimately destroyed the inner layer of

tissues of the lungs due to excessive watery fluid accumulation. Excessive metal

exposure may cause pulmonary odema which lead death. Contamination of soil with

heavy metals needs the implementation of suitable remedial techniques (Vaxevanidou et

al., 2008). Thus there is need for the maintenance of soil quality, which is not a one-time

event but rather a continuing process (Wang et al., 2003).

1.3 Phytoremediation Technology

A variety of treatment technologies has been developed for the remediation of

metals contaminated soils, and phytoextraction is an economically feasible technique. It

is more acceptable publically because overall it increases the aesthetic beauty of the

contaminated area and also has potential to clean the environment (Chen & Cutright

2002). Phytoremediation is a plant based remediation strategy which uses the plants for

the environmental remdiation (Rauf et al., 2009).

Phytoextraction of heavy metals is a technique under phytoremediation in which

plants act as a solar energy driven pumps and extract and accumulate different elements

from the soil and environment (Luo et al., 2005). Heavy metals like Cd, Zn, Ni, Pb and

Cu can be extracted through this technique. Among the phytoremediation categories

phytoextraction is used to extract heavy metals from the soil system by plants because

some metals like Mn, Fe, Mg, Mo, Ni and Zn etc. are essential plant nutrients. But for Pb

removal it is commercially available technique. Phytoremdiation efficiency depends on

different factors like climate, time period, soil type and root depth. Vassilev et al. (2002)

reported metal phytoextraction protocol whice consists of the following strategies:-

(1) Cultivation of plants on metals contaminated soil.

(2) Harvesting of metals rich biomass.

(3) Post harvest treatments and subsequent disposal of the plants biomass as a

hazardous/toxic waste.

(4) Recovery of metals from metals loaded plants biomass.

Phytoremediation is an affordable and effective technical solution for removal of

heavy metals from the soil. Phytoremediation is an economical and envirmental friendly

6

technique. Plant roots ability of uptake of heavy metals is being used in this process of

phytoremediation along with the transformation, accumulation and biodegradation

capability of the whole plant body (Tangahu et al., 2011). Efficiency of remediation

depends on different factors such as soil type, nutrient status of soil and plants tolerance

against heavy metals.

For the management of contaminated agricultural land use of heavy metals

tolerant crops for remediation of heavy metals is new emerging technique. It is indicated

from the recent studies that different high biomass producing crop varieties have potential

for heavy metals accumulation such as oat (Avena sativa), Indian mustard (Brassica

juncea), sunflower (Helianthus annuus), maize (Zea mays), ryegrass (Lolium perenne)

and barley (Hordeum vulgare) ( Salt et al., 1998; Shen et al., 2002 and Meers et al.,

2005).

In this technique by using high biomass producing crops, with better management

of soil and improvement of plant husbandry an alternative strategy could be develop for

remediation of heavy metals polluted soils (Evangelou et al., 2007).

1.4 Hyperaccumulator Plants in Pakistan

Phytoremediation is a new and emerging technique in Pakistan and a lot of

research work has been done at the laboratory and field level (Mushtaq, 2010). Plants

with exceptional metal-accumulating capacity are known as hyperaccumulator plants

used in phytoremediation. More than 6000 higher plants species are found in Pakistan

(Chehregani & Malayer, 2007).

Different types of plants found in Pakistan have potential to concentrate different

environmental pollutants. In Pakistan different plants species are proved effective in Cd,

Cr, Pb and As absorption. Fifty plants species were tested for their heavy metals

accumulation potential in treatment of soil and contaminated water. In addition to this

plants ability to uptake a metal also affects the uptake of another metal ion. Nazir et al.

(2011) has reported that in Abbottabad specie like Arundo donax was used to

phytoremediate soil contaminated with chromium. In 2011 in Rawalpindi and Islamabad

23 species of plants were used for the phytoremediation of Cu, Zn, Pb contaminated soil.

7

In Taxila a hydroponic study was done which involved the remediation of Ni by

Eichhornia crassipes. In Gujranwala in 2010 Arundo donax was used effectively for

phytoremediation of soil contaminated with As and Hg while in Lahore in 2011

Plectranthus rugosus, Rumex hastatus, Fimbristylis dichotoma, Heteropogon were used

to phytoremediate the soil contaminated with Ni, Co, Cr, Cu,Cd, Zn and Pb. For

phytoremediation purpose use of native plants is more preferable because they have more

survival chances in the native environment, they can also continue growth under stressed

conditions as compared to the plants of different environment. There is a continuous

interest of scienctists to search the native plants for the phytoremediation purposes. Some

previous researches have demonstrated potential of native plsnts for phytoremediation in

field conditions (McGrath et al., 2001). Rapidly growing and producing more biomass

plants are more recommended for the phytoremediation purposes.

1.5 Enhanced Phytoremediation

Many chemical and biological treatments, such as inoculums of fungi and

bacteria, EDTA, DTPA, NTA and other organic compounds have been used in pot and

field experiments to facilitae the heavy metals extraction and to acquire the higher

phytoextraction efficiency (Blaylock et al., 1997; Huang et al., 1997; Kayser et al., 2000;

Ke et al., 2006 and Wu et al., 2006). It is known that microbial populations affect the

solubilization of heavy metals and their availability to plants, through acidification,

releasing chelatora and reduction-oxidation changes (Peer et al., 2006). It is reported that

presence of microbes in the rhizosphere increases the levels of Zn, Cu, Pb, Ni and Cd in

plants. Heavy metals tolerance and production of biomass could be enhanced by

improved interaction among the plants and rhizosphere microbes. It is also considerd as

an important phytoremediation technology factor (Whiting et. al., 2003).

1.5.1 Chemically Enhanced Phytoremediation

The phytoremediation effectiveness becomes limited oftenly because of low

solubility of metals and their sorption on surfaces of soil particales; however metals

solubilization could be increased by adding complexing/chelating agents with the time

(Pivetz, 2001). In the literature several chelating agents has been reported which enhance

the rate of phytoextraction. However EDTA and DTPA has been investigated widely and

8

they have high chelating ability towards most of the metals, like Cd, Cu, Cr and Pb,

which ultimately leads to increased translocation of metals from soil to plant (Barlow et

al., 2000; Wong et al., 2004 and Begonia et al., 2005).

From the literature it is seen that chelating agents may pose potential risk when

they are applied in-situ, because during extending period of time chelating agents leach

down to the ground water. However some studies showed that ammonium application to

soil might promote the heavy metals phytoavailability from polluted soils (Lasa et al.,

2000 and Zaccheo et al., 2006)

A lot of researhes on phytoextraction are based on green house experiments, few

tested the hyperaccomulators plants in the field and actually determined their heavy

metals accumulation potential (Hammer & Keller, 2003; McGrath et al., 2006 and

Zhuang et al., 2007).

Different chelates such as DTPA, EDTA and EDDS have shown enhancement in

the uptake of heavy metals by solubilizing them from the soil solid phase because of the

formation of water soluble complexes (Lestan et al., 2008). Primarily, heavy metals are

distributed mainly in two phases which are reversible and irreversible. The chelate try to

extract metals ion from reversible phase first and then fom irreversible phase (Ganguly et

al., 1998). Based on this criteria ability of chelates is analyzed. If a chelate dissolves

metals more from irreversible phase it is more efficient. Rescently NTA and EDDS are

used because of their ability to be biodegraded as compared to the EDTA (Evangelou et

al., 2007).

Synthetic chelants, such as EDDS, EDTA and NTA have been used in facilitating

the heavy metals solubility from the soil system and their uptake and translocation in the

shoot parts of plants (Blaylock et al., 1997; Huang et al., 1997; Cooper et al., 1999; Wu

et al., 1999; Shen et al., 2002 and Kos & Lestan, 2003a). For example Cu metal could be

toxic for many plant species (Pahlsson, 1989). The recommended threshold limit of Cu

metal for the plants is 30 mg per kg of plant dry matter (Marschner, 1995). In multimetals

polluted soil system Cu toxicity for the plants might be a constraint in the

9

phytoremediation process (Lombi et al., 2001).

Blaylock et al. (1997) reported that concentration of Cu in Brassica juncea shoots

in Cu contaminated soil containing 200 mg kg_1 of Cu reached 1000 mg kg_1 dry matter

one week after 2.5 mM application of EDTA. Chelant concentration is also an important

factor to develop an effective model for remediation of metals polluted soils. As reported

(Greman et al., 2003) that EDDS enhanced the phytoremediation and increased Cd, Pb

and Cu solubilization. Tandy et al., (2004) reported that chelates application rate affects

the metals extracton efficiency. Howere a single conclusion can not be drawn from their

concentrations; it varies in different conditions (Nowack, 2002). Chelants have high

affinity for differen metals, chelate metal ratio is important. Concequently, concentration

of chelating agent should be higher than metals for optimum extraction (Kim et al.,

2003). For soil conservation, lower chelates concentration was more favorable (Lim et

al., 2005).

Another important factor governing the solubilization of metal ions in the soil is

shaking time. Stability of the complex in soil determins the ability of chelate to susutain

the metals in soluble form. As reported by the Kim et al. (2003), a continuous steady state

condition between Pb and EDTA was not achieved within one day in Pb contaminated

soil. So by monitoring the time factor we can predict the persistant and availability of the

chelating agent in the soil matrix.

Incubation time is another important factor, which should be considerd while

evaluating the solubilization efficiency of chelating agent. Chaney (1988) demonstrated

that oxidation of metal ion and formation of metal-chelate complex may take different

time period for different heavy metals. Therefore, incubation period should not be

ignored while evaluating other environmental factors. In addition to this, incubation

period helps in determining the biodegradation period of chelants.

1.5.2 Biologically Enhanced Phytoremediation

Soil upper layer is interface of plant and soil and process of phytoremediation

takes place in this layer. Microorganisms in soil horizons release different organic

compounds and make the contaminated metals present in the rhizosphere available for the

10

plants through phytoremediation (Lasat, 2002). Soil microorganisms play very effective

roles in different processes and have effects on human beings (Doyle & Lee, 1986). Soil

microbes involve in different nutrient availability for the plants and develop symbiotic

relationships with plant roots; however these processes are needed to fully explored

(Khan, 2002).

Fungi play a very important role in solubilization and fixation of heavy metal ions

and change the availability of these ions for the plants (Birch & Bachofen 1990).

Different soil and plant factors affect the phytoremediation process and these also include

the soil fungi. There is a need of information about symbiotic relationships between soil

microbes (bacteria and fungi) and roots of plants. Heavy metals are also in compound

forms in soil which also affects the metals behavior in their solubilization and uptake

processes (Boruvka & Drabek, 2004). Different types of components present in the

fungal cell walls like carboxyl, hydroxyl, amino and other functional groups. Through

these functional groups fungi can bind with toxic heavy metals such as Pb, Cu, Cd and Ni

etc. (Kapoor & Viraraghavan, 1995).

A larg number of filamentous fungi may absorb heavy metal ions and used

commercially (Morley & Gadd, 1995). Protien present in the fungal cell wall has

potential to sorb the heavy metal ions this is in accordance with those fungi can tolerat

with the toxic heavy metals. Gonzalez-Chavez et al. (2004) reported that hyphae of the

arbscular micorhizae fungi consist of glomalin which can sequester the heavy metal ions.

Fungi play a very important role in the phytostabilization of toxic heavy metals in the

contaminated soils by sequestration and ultimately help mycorrhizal plants survive in

contaminated soils.

The phytoremdiation efficiency depends mainly on plants characteristics like

growth rate, biomass production, harvesting easiness, metals resistance and shoots ability

of heavy metal accumulation (He et al., 2005). Due to some constraints like large scal

cropping technique defeciency, less biomass and slow rate of plant growth researches are

directed to using amendments like addition of chelates which enhanced biomass

production of agronomic crops and their yield also increased (Luo et al., 2005, Meers et

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11

al., 2005 and Neugschwandtner et al., 2008).

The present study was planned by keeping in view the heavy metals exposure

directly to the water and agriculture soils. The proposed study was done in laboratory and

under green house conditions with the objectives to evaluate the efficacy of a synthetic

chelator and isolated fungi in the uptake and translocation of heavy metals as well as on

the total biomass of plants. The proposed research work was aimed to make an in-depth

investigation about the remediation of contaminated sites from various metal

contaminants using specific crops which have an experimental record as

phytoremediation tools. This work also helped to understand and develop the research

and development strategies for phytoremediation. In Multan, Kasur, Gujranwala and

Lahore soil contamination by heavy metals was becoming a serious environmental issue

and faced significant difficulties for large scale implementation of bioremediation due to

high cost technology, lack of awareness, limited resources and negligence of authorities.

In such situation small scale experiments could be recommended as the first step. Hence,

in this study assessment of heavy metals in plants, waste water and agricultural soil was

done. After that shaking and incubation experiments were conducted and in-vitro

assessment of contaminated soil of Gujranwala and Lahore was done by growing local

plants through uptake and accumulation of toxic metals like Cu, Cr, Pb and Cd. Based on

all these experiments ultimately a phytoremediation study was conducted in green house.

In future field testing of green house tested crops will be conducted to accelerate

implementation of phytoremediation technologies and enhancing biofuel production.

Much research has been carried out and a few plant species were discovered that have

been used for phytoremediation or phytodegradation. These few higher plants use their

rhizosphere microorganisms like fungi to remediate soils contaminated with toxic metals

by industrial activities. Crops used in the laboratory experiments and green house

experiments (maize, sorghum, wheat, mustard, canola and sunflower etc.) could be used

for multi-tasking like for the management of heavy metals polluted soils

(phytoremediation) as well as for biomass production which would be ultimately used for

biogas and biofuel production. Based on preliminary data, inoculums of best fungal

species would be used to enhance the process of metal uptake by the pre-tested plants in

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12

the field and in addition to these pre-tested fungal inoculums would be prepared and their

effect on biomass production of tested crops may evaluated.

1.6 Significance of the Study

Pakistan is a developing country and its population is more as compared to

resources. Most of the area is used for agriculture and urbanization purpose. In present

time for rapid progress industries are keys for achieving development. In major cities of

Pakistan industrial estates are established. Besides contributing major share of economy

to country GDP these industries are creating pollution problem. The increase level of

contamination is making land useless for better yield production. Contamination of lands

with toxic heavy metals is a widespread environmental issue. Conventional techniques

for reclamation of such soils are expensive and environmental non-friendly.

Phytoremediation is an emerging group of technologies utilizing green plants to clean up

the environment from contaminants and has been offered as a cost-effective and non-

invasive alternative to the conventional engineering-based remediation methods. The use

of plants and tolerant fungal strains to remove, contain, inactivate, or degrade harmful

environmental contaminants and to revitalize contaminated sites is gaining more and

more attention in the world. The main purpose of proposed research is to provide the

experience of the use of plants, chelates and fungi for the remediation of contaminated

soils.

In this study soils contaminated with heavy metals was considered and

remediation with fungi and plants was carried out because these technologies were

untested in Pakistan and were beneficial in the economical aspects, uses, and processing

of the biomass. Furthermore development of a model of plant-contaminants soil

interaction was used in remediation technology for rapid and successful remediation of

polluted peri-urban arable soils. The present study helped in developing new cost

effective strategy for heavy metals contaminated agricultural lands by selection of

appropriate local plants to remove metals and to harvest the valuable biomass. The

generated biomass could be either subjected to biomethanation or composting to reduce

the volume and then processed for recycling of heavy metals. Ethanol would be then

extracted and used as a biofuel in future. Use of plants on different sites would serve to

13

restore wetlands and other habitats, create natural parks and other green areas and resolve

the pollution problems. This research would help the farmers in selection of best

germinated seeds on contaminated agricultural lands. This further promoted the research

and development in future about implementing phytoremediation which makes use of

local plants to extract, transfer and stabilize potentially toxic metals from contaminated

soil. This study also advocated the development of a model of plant-contaminants soil

interaction and future remediation programs for rapid and successful remediation of

polluted peri-urban soil.

1.7 Goal of Study

In Pakistan soil, water and food are heavily polluted by heavy metals and the

overall goal of the present study was to use plants, chelates and fungi to de-contaminate

the contaminated soils. This was an effective technique in pollution control. The main

purpose of present research was to evaluate the accumulation of heavy metals in soils and

plants in contaminated soils and assess the role of hyperaccumulators plants, chelates and

fungi in removing pollutants and this study would made possible to understand the

implementation of phytoremediation, which made use of plants to extract, transfer and

stabilize heavy metals from soil and water.

The increase level of contamination is making land useless for better yield

production. This study was carried out on heavily contaminated areas of Multan, Kasur,

Gujranwala and Lahore in which major focus was on the areas having major threat to

food contamination and health hazards. Because irrigating the fields with heavy metal

contaminated water is a common practice and shortage of financial resources have always

put limitations on using advance remediation technologies

The goal of this research was to use the native vegetables of Punjab area so this

approach can be made accessible to local farmers and other entities interested in land

remediation. This study provided an economical and green approach to remediate the soil

that will be highly applicable in Pakistan. This study has opened the way to explore more

local plants having higher potential of heavy metal accumulation by which metals can be

recovered using technologies and also biofuel production can be made feasible at low

14

level.

Through this way, we can treat contaminated sites with local plants and utilize

them for biofuel, bioenegy and biomass production. The native crops which are

contaminated are possible to use them for eating purposes but possible to use for the

recovery of metals. Metal pollution is the problem of Pakistan and the ultimate goal of

this research was to develop a phytoremediation model to decontaminate the metals

contaminated soil.

1.8 Aims and Objectives

The specific research objectives of present study were as follows:

To conduct surveys for the assessment of heavy metals contamination load in

soils, plants and untreated waste water in peri-urban agricultural areas

(Multan, Kasur, Lahore and Gujranwala).

To test the various chelates and tolerant fungal strains for their metal

extraction and solubilizing efficiency in laboratory batch experiments

(shaking and incubation).

To evaluate the natural, biological and chemically enhanced phytoextraction

potential of native plant species through in-vitro experiments.

To predict the interactions between plants, contaminants and soil

characteristics through greenhouse pot experiments.

Chapter 2

Literature Review

15

Introduction

The present research was conducted to study the effect of chelating agents, fungi

and native plants species on remediation of metal contaminated calcareous soils of

Punjab. The selected cities of the Punjab were Multan, Kasur, Lahore and Gujranwala.

Metals pollution is an ecological and global concern and it is caused by different

anthropogenic activities (Adriano, 2001). Heavy metals accumulation in soil may pose

threats to the ecosystem and also human beings by contaminating food chain (Kabata-

Pendias & Pendias, 1992).

Metals do not destroy or degrade biologically and exist in the environment for

long time because of their affinity to bind with iron oxides, organic matter, clay minerals,

phosphates and carbonates (McBride et al., 1997). There are many physical and chemical

remediation techniques which limit the soil fertility and ultimately pose negative impacts

on the environment. However, new low cost and environment friendly techniques like

phytoremediation are publically acceptable. Plants are used to remove the metals from

contaminated soils to bring the metals concentration under safe recommended limits

(Garbisu & Alkorta, 2001).

2.1 Metal pollution

Heavy metals are natural constituents of earth crust and they are generally found

in low concentration. Anthropogenic activities have also increased metals concentration

in the environment (Ansari et al., 2004).

Due to rapid growth of population, industrialization, urbanization and increased

agricultural practices environmental pollution is also raising. Since 1900 heavy metals

pollution has increased sharply (Ensley, 2000). These heavy metals are difficult to

destroy biologically however they can be transformed from highly toxic form to less

toxic form (Garbisu & Alkortal, 2001). Some heavy metals are essential for global

ecosystem. These metals exist in the environment with different oxidation states and their

oxidation number is related to their toxicity. These pollutants are ultimately derived from

different sources affecting the whole ecosystem (Macfarlane & Burchett, 2001).

16

In Pakistan like other developing countries laws enforcement regarding industrial

waste disposal is ineffective so a large number of industries dump their waste into fresh

water, which in turn is responsible for soil pollution (Khan, 1997). Industrial waste

contain high concentrations of heavy metals, when added to water bodies and dispersed

in irrigation water caused serious type of environmental pollution, causing bad effects on

whole biota and ultimately causing a threat to the human beings (Ross, 1994). To avoid

these problems it is necessary to treat waste water before being discharged to the

environment (Naqvi, 1995).

Study areas Multan, Kasur, Lahore and Gujranwala are also receiving industrial

and domestic waste water as an irrigation source. Anonymous reported (1996) tannery

industries like Kasur, during tanning process, Cr is not attached to collagen and

discharged as effluent in the surrounding environment, preferably water and soil. It has

been estimated that with traditional method of tanning about 4 to 9.5 kg Cr per ton of

animal skin is not fixed during the process and if not properly carried out with proper

high tanning system, two third is poured in the liquid waste.

Heavy metals entered in the environment through different sources, especially

through water to soil and their concentrations build up in the soil. Such high

concentrations cause plant diseases like lesions in different crops and also cause

mutational and epigenetic changes in plants populations (Sharma et al., 1996).

Parsad & Parsad (1987) reported that heavy metals accumulation in nutrient

solution and soil results in impaired metabolism and growth retardation in plants.

Pakistan peri-urban agricultural soils often received contaminated waste water, polluted

or loaded with toxic heavy metals (Ghafoor et al., 1995).

Some heavy metals are biologically essential but in excess they become toxic

strongly, such type of metals pollution causes inhibition of plants growth and it is highly

toxic to plants cells and cause death (Lanaras et al., 1995). McGrath et al. (1995) reported

that high metals concentration in soil can decrease microbial activities, fertility of soil

and also crop yield. Steffens (1990) described that near mining operations, landfill or

waste disposal sites, on some agricultural sites and natural soil sites plant toxic and lethal

17

metals levels were experienced.

2.2 Phytoremediation

In term “Phytoremediation” phyto means plant and remediation means to restore

or to clean. It actually means a plant based technique that used natural or genetically

modified plants for environmental remediation (Cunningham et al., 1997). The main

focuse on developing phytoremediation technique is to develop a low-cost remediation

technique (Ensley, 2000).

Phytoremdiation is not a recent invention but also an old practice (Cunningham et

al., 1997). In Russia, at the dawn of nuclear era, for the treatment of radionuclide

contaminated water semi aquatic plants were used (Salt et al., 1995). Some plants grown

on metal contaminated soils have developed the potential to accumulate high amount of

metals in their tissues without inhibiting toxicity symptoms (Reeves & Brooks, 1983 and

Baker et al., 1989)

Pollutants of different types like metals, pesticides, organic compounds and

xenobiotics can be effectively removed by the plants. Plants hairy roots, cell culture and

algae have been used for their contaminant degradation ability (Suresh & Ravishankar,

2004).

A great progress in the field of phytoremediation was made by heavy metals (Salt

et al., 1995a and Blaylock & Huang, 2000). This technique is especially suitable for

moderate to low level of contamination. Whereas for heavily contaminated sites

enhanced phytoremediation technologies could be used.

Effectivness of phytoremediation depends on root zone of the plants. This may be

from few centimeters to many meters (Schnoor et al., 1995). This phytoremediation

technique is a long term strategy and it is more beneficial than other physical and

chemical technologies.

Concentration of heavy metals present in the contaminated soils affects their

uptake by plants. Heavy metals predominatly removed by the roots of plants. However

18

accumulation of heavy metals in the different parts of plants varies from plant to plant

specie (Kramer, 2010).

Hyperaccumulators are those plants which have 50-500 times greater capability to

absorb metals than average plants (Lasat, 2000). Hyperaccumulators have greater than

one bio-concentration factor, sometime it reaches 50-100 (McGrath & Zhao, 2003 and

Reeves & Baker, 2000). Hyperaccumulator plants are ideal model organisms for

scientists and have acquired attention all over the world for their use in phytoremdiation

technology.

Rhoades in 1999 has suggested mustard (Brassica juncea L.) as an effective

species capable of accumulating substantial amounts of Se in its shoots. Several studies

have shown that Thlaspi caerulescens is an efficient accumulator of cadmium (Cd) in its

above-ground parts (Brooks et al., 1998 and Whiting et al., 2000). Robinson et al. (1997)

have found Berkheya coddii as an excellent nickel (Ni) hyperaccumulator with the ability

to remediate moderately contaminated soils with only two crops.

However, hyperaccumulators often accumulate only a specific element and are, as

a rule, slow growing, low biomass-producing plants with little known agronomic

characteristics (Cunningham et al., 1995). With time scientists have developed

chemically enhanced phytoextraction techniques with fast growing and high biomass

crops (Zea mays, Helianthus annuus, Sorghum vulgare, Amaranthus spp., Sesbania

aculeata etc.) to overcome limitations due to low metal solubility and bioavailability

(Tandy et al., 2006; Komárek et al. 2007a and Melo et al., 2008).

According to the study of Wierzbicka (1999), barley and maize were species with

high constitutional tolerance towards Pb, whereas wheat and oats belonged to the group

of cultures capable of weak heavy metals accumulation (Bojinova et al., 1994). Opposite

results however were obtained by Vincenc et al. (1996), which indicated that heavy

metals accumulation was most apparent in cereals (maize, oats, rye, wheat and barley).

The studies connected with cultivation of sorghum crops in heavy metals-rich

soils are often contradictory. According to Youn’ study (2004) sorghum may be used as

an indicator plant to determine soil contamination due to presence of cadmium. Madejon

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3X-49BSC9D-1&_user=3419734&_coverDate=10%2F30%2F2003&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_searchStrId=1658262137&_rerunOrigin=scholar.google&_acct=C000060522&_version=1&_urlVersion=0&_userid=3419734&md5=e68126d893c32ca6258807d8fc6a54ee&searchtype=a#bib56#bib56http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3X-49BSC9D-1&_user=3419734&_coverDate=10%2F30%2F2003&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_searchStrId=1658262137&_rerunOrigin=scholar.google&_acct=C000060522&_version=1&_urlVersion=0&_userid=3419734&md5=e68126d893c32ca6258807d8fc6a54ee&searchtype=a#bib16#bib16http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3X-49BSC9D-1&_user=3419734&_coverDate=10%2F30%2F2003&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_searchStrId=1658262137&_rerunOrigin=scholar.google&_acct=C000060522&_version=1&_urlVersion=0&_userid=3419734&md5=e68126d893c32ca6258807d8fc6a54ee&searchtype=a#bib81#bib81http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3X-49BSC9D-1&_user=3419734&_coverDate=10%2F30%2F2003&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_searchStrId=1658262137&_rerunOrigin=scholar.google&_acct=C000060522&_version=1&_urlVersion=0&_userid=3419734&md5=e68126d893c32ca6258807d8fc6a54ee&searchtype=a#bib58#bib58

19

et al. (2002) established that sorghum was tolerant to soil contaminated with heavy

metals and was suitable for phytostabilization.

Nehnevajova et al. (2005) reported that sunflower can be used for the remediation

of metal-contaminated soils. Its high biomass production makes this plant species

interesting for phytoextraction and using sunflower oil for a technical purpose may

improve the economic balance of phytoremediation.

Sunflower (Helianthus annuus) a fast-growing crop has a reasonable tolerance to

heavy metals. It has been used for rhizofiltration because it has a high root uptake of

metals but shows low efficiency in their translocation from root to shoot (Saxena et al.,

1999; Kamnev & Van der Lelie, 2000 and Lin et al., 2003). Few reports on cadmium

accumulation by H. annuus are available (Madejón et al., 2003 and Pena et al., 2006).

Indian mustard (Brassica juncea) is also a suitable target species for

phytoremediation which has a large biomass production and a relatively high trace

element accumulation capacity. Most importantly, it can easily be genetically engineered

(Zhu et al., 1999b).

2.3 Native Plant Species of Pakistan for phytoremediation

Escalating heavy metals contamination in the environment is reported in many

developing countries including Pakistan (Hardoy et al., 1992; Jamali et al., 2007; Qadir

et al., 2001 and Kausar et al., 2012). In Pakistan tremendously higher concentration of

mercury (Hg) has noticed in marine and reverine ecosystems (Tariq et al. 1994; Tehseen

et al. 1994 and Mubeen et al. 2010). From literature it is concluded that soil and water in

Pakistan is most probably direct use of herbicides, pesticides and fertilizers in agricultural

sector, spills of hazardous substances and industrial and urban waste discarding methods.

Since soil is most important part of the environment. It is responsible for

supporting life and also provides habitat and other natural resources, so cleanup of soil

from heavy metals pollution is complicated task, especially when applied on large scale.

Soil has organic and inorganic solid components, water, and a combination of different

gases present in various proportions. The mineral constituents of the soil depend upon the

20

weathering process of rock which gives rise to particular type of soil, climatic conditions

have also great role to play in the formation of soil from rocks through weathering.

Consequently soils vary enormously in physical, chemical, and biological properties.

Water movement in soil is controlled by physical properties, such as soil structure and

texture. The soil moisture has great bearing on the controlling solute movement, salt

solubility, chemical reactions, and microbiological activities and ultimately, the

bioavailability of the metal ions.

Table 2.1: Metal ion hyperaccumulating plant species (Modified from Brooks et al.,

1998).

Heavy metal contamination in the soils has become a serious environmental issue

in Pakistan (Hussain et al., 1996). The rapid increase in population together with the

disposal of untreated effluents from tanneries and textile industries has increased the

threat of soil pollution (Khan, 2001). In Pakistan, the use of plant species to

decontaminate and remediate polluted soils with heavy metals is very scarce and limited.

Various hyperaccumulative plant species have been also broadly explored that

lead to the considerable advancement in this field. Plant species whose bioconcentration

factor (BCF) is greater than 1,000 are called hyperaccumulators while plants having BCF

are greater than 1 but less than 1,000 are called accumulators except plants whose BCF

Element Plant species above-

ground plant parts (µg g-1

dry matter)

Concentration in

ground

biomass (Mg ha-1)

Annual above-

ground biomass

(Mg ha-1)

Cadmium Thlaspi caerulescens 3000 (1) 4

Cobalt Haumaniastrun robertii 10200(1) 4

Copper Haumaniastrun katangense 8356(1) 5

Lead Thlaspi rotundifolium subsp. 8200 (5) 4

Manganese Macadamia neuraphylla 55000 (400) 30

Nickel Alyssum bertolonii 13400 (2) 9

Nickel Berkheya coddii 17000 (2) 18

Selenium Astragalus pattersoni 6000 (1) 5

Thallium Iberis intermedia 307() (1) 8

Uranium Atriplexconfertifolia 100 (0.5) 10

Zinc Thlaspi calaminate 10000 (100) 4

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21

are less than 1. Bioconcentration factor points out the efficiency of a plant in taking up

heavy metals from soil and accumulating. There are total 17 biomes in the world while

Pakistan has 9 out of them so it shows the unique geographical landscape of Pakistan and

subsequently Pakistan has more than 6000 species of higher plants (Ali & Qaiser, 1986).

Presently, more than 400 plant species among angiosperms have been examined and

identified as hyperaccumulators all over the world (Freeman et al., 2004). However, in

Pakistan, only 50 species have been examined and identified as metal accumulators to

remediate contaminated soil and water.

A study conducted by Qadir et al. (2001) remediated saline sodic contaminated

water from Faisalabad from the roots of cultivated plant species such as rice (Oryza

sativa L.) and wheat (Triticum aestivum L.) along with the application of manure

treatments. Active desalinization and desodication processes were found in all the

treatments (gypsum and manure) and they reported that the application of manure

treatments, efficiency of rice and wheat roots was increased which can help to enhance

the remediation ability of the above mentioned plants species. Application of sewage

sludge to agricultural land has become a common practice over the past several decades.

Findings of Mubeen et al. (2010) reported the utilization of locally available wild

plant material for the remediation of Cu from industrial waste water from Lahore city.

They have used Calotropis procera for the treatment of copper wastes. Roots of

Calotropis procera were used as biosorbent to remove copper from known concentration

of copper solutions. These experiments were carried out in order to determine some

operational parameters of Cu sorption such as the time required for the metal-absorbent

equilibrium, the effect of change in biomass quantity, and effect of contact time on

percentage removal of copper. At the end, it was concluded that adsorbent prepared from

Calotropis procera roots can be used for the treatment of heavy metals in waste waters.

The use of synthetic chelator increased the uptake and translocation of heavy metals in

plant biomass that could enhance the phytoremediation of Ni and Pb.

Substantial progress has been made in field of phytoremediation of metals,

various plant species have been extensively investigated in all over the world to date.

22

However, in context of the indigenous flora of Pakistan, inadequate information is

available. About 400 plant species among angiosperms have been inspected and

identified as hyperaccumulators from all over the world. Although the limited data

available in terms of the use of the flora from Pakistan (about only 30 studies have been

conducted to investigate the potential of flora from Pakistan for phytoremediation), still a

large number of plant species present in different localities of the country should be

tested for the phytoremediation purposes. Among those unfamiliar plant species, many of

them were used for remediation purposes in all over the world, i.e., Brassica juncea were

used by many researchers to remediate lead (Pd), zinc (Zn), and copper (Cu) from soil

samples (Ebbs & Kochian 1997; Salido et al., 2003; Belimov et al., 2005 and Zaidi et al.,

2006). This species is found in different locations in Pakistan (Islamabad, Rawalpindi,

Quetta, Lahore, and Karachi). Many studies reported the assessment of heavy metals

contamination in soil and water samples from Pakistan. Younas et al. (1998) and Malik et

al. (2010) investigated that soils from industrial zones of Lahore, Rawalpindi, and

Islamabad have been highly contaminated by heavy metals such as Cd, Ni, Cu, Zn, and

Pb. Thus, it could be useful to remediate the above mentioned contaminated locality

testing, for example, Brassica juncea, a plant species commonly found in these areas.

Similarly, Eichhornia crassipes has been found in many industrial and urban areas such

as Gujranwala, Lahore, and Rawalpindi from Punjab Province. Although this plant

species is not commonly used for remediation purposes but it has been reported as a

heavy metal accumulator (Cr, Zn, Cd, Cu, Ni, Pb, Hg, P, Pesticides) from different parts

of the world (Verma et al., 2005; Xia & Ma 2006; Odjegba & Fasidi 2007 and Mishra &

Tripathi 2009).

2.4 Environmental Concerns Associated with Phytoremediation

There are a number of environmental concerns pertaining to the use of

phytoremediation; one of the most significant of theses involves human health. There are

a number of different routes of exposure that must be taken into consideration. The

ingestion of heavy metals through contaminated soil by humans or animals, ingestion of

vegetation grown on the metal contaminated soil, ingestion of animals that have ingested

plants grown in the metal contaminated soil, and the leaching of metals into the water

23

supply are all concerns (Chaney et al., 2000).

Some studies have found that certain animals and insects will not consume plants

being used for phytoremediation because they merely taste bad (Chaney et al., 2000).

Field observations of livestock in areas where naturally occurring metal

hyperaccumulators have been found, have shown that cattle, sheep, and goats avoid the

metal rich vegetation like Alyssum and Thalspi. The seeds of hyperaccumulators are

generally small and lack nutritional and food value. Thus, large mammals and birds are

highly unlikely to have a diet limited to metal contaminated vegetation because of their

requirement for large habita

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