Revegetation and Ecotoxicological Assessment of Abandoned Copper Mine for improved Remediation Utilizing Native Plant Species RAMKRISHNA NIROLA B.Sc (Botany Honours) North Bengal University Darjeeling India B.Ed Tribhuvan University Kathmandu Nepal M.Sc (Botany/Ecology and Environment) Tribhuvan University Kathmandu Nepal A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy Future Industries Institute (FII), School of Natural and Built Environments, Division of Information Technology, Engineering and the Environment University of South Australia July 2016 i TABLE OF CONTENTS ABBREVATIONS vii LIST OF FIGURES viii LIST OF TABLES xii LIST OF PHOTO PLATES xiv LIST OF PUBLICATIONS xv EXECUTIVE SUMMARY xvii i) Background problem xvii ii) Research gap xvii iii) Research approach xvii iv) Research activities xviii v) Research findings xx DECLARATION xxii ACKNOWLEDGEMENT xxiii Chapter 1 INTRODUCTION 1.1 Global scenario of abandoned mine pollution 1 1.2 The scope of study 3 1.3 Significance of the study 4 1.4 Objectives 6 1.5 Structure of research 8 ii 1.6 Layout of chapters 8 Chapter 2 LITERATURE REVIEW 2.1 The metal pollution by mines 11 2.2 Environmental sustainability in mine site rehabilitation 15 2.2.1 Microorganisms and non-vascular plants 16 2.2.2 Vascular-plants 18 2.2.3 Semi-arid and arid (SAA) plants 27 2.2.4 Revegetation challenges 29 2.3 Phylogenetic implications in semi-arid and arid (SAA) lands 31 2.3.1 Plant metal interaction in SAA zones 32 2.3.1.1 Order Caryophyllales 34 2.3.1.2 Order Poales 36 2.3.1.3 Order Fabales 37 2.4 Advances in remediation science 39 2.4.1 Plant biotechnology and tissue culture 40 2.4.2 Future of remediation technology 41 2.5 Conclusion 42 Chapter 3 SCREENING OF METAL UPTAKE BY PLANT COLONIZERS GROWING IN KAPUNDA COPPER MINE, SOUTH AUSTRALIA 3.1 Introduction 44 3.2 Objectives 46 3.3 Hypothesis 46 3.4 Experiments 46 3.5 Materials and methods 47 3.5.1 Site description 47 iii 3.5.2 Sampling and characterization 48 3.5.3 Data treatment 52 3.6 Result 52 3.6.1 Soil characters and metal uptake 52 3.6.2 Statistical interpretations 56 3.7 Discussion 57 3.8 Conclusion 61 Chapter 4 EVALUATION OF METAL UPTAKE FACTORS OF ACACIA PYCNANTHA AND EUCALYPTUS CAMALDULENSIS AT A COPPER MINE IN-SITU SOIL 4.1 Introduction 62 4.2 Objectives 65 4.3 Hypothesis 66 4.4 Experiments 66 4.5 Materials and methods 66 4.5.1 Site description 66 4.5.2 Sampling and processing 67 4.5.3 Sample characterization 68 4.5.4 Spectroscopic characterization of plant matter 69 4.5.5 Data analysis 71 4.6 Results 71 4.7 Discussion 75 4.7.1 Soil characteristics and metal accumulation in plants 75 4.7.2 Plant-soil interaction 77 4.7.3 Accumulation factors of heavy metals on Ap and Ec 80 4.8 Conclusion 81 iv Chapter 5 ASSESSMENT FOR METAL TOXICITY AND BIOAVAILABILITY IN METALLOPHYTE LITTERS AND METALLIFEROUS SOILS USING EISENIA FETIDA IN A MICROCOSM STUDY 5.1 Introduction 82 5.2 Objectives 84 5.3 Hypothesis 85 5.4 Experiments 85 5.5 Materials and methods 86 5.5.1 Soil and leaf litter samples 86 5.5.2 Metal analysis of soil and plant litter samples 88 5.5.3 Extraction of leaf lignin 90 5.5.4 Preparation of microcosm substratum 90 5.5.5 Earthworm toxicity and bioavailability test 92 5.5.6 Statistical analysis 94 5.6 Results 95 5.6.1 Metal bioavailability from soil and litter samples 96 5.6.2 Earthworm weight, survival and reproduction in microcosm 98 5.6.3 Metal bioavailability to earthworm 100 5.6.4 Statistical test 101 5.6.4.1 Earthworm body weight 101 5.6.4.2 Earthworm numbers and juveniles 101 5.6.4.3 Copper uptake by earthworms 103 5.7 Discussion 104 5.8 Conclusion 107 Chapter 6 METAL BIOAVAILABILITY TO EISENIA FETIDA THROUGH LEPUS TIMIDUS AND PLANT LITTER. 6.1 Introduction 109 6.2 Objectives 111 v 6.3 Hypothesis 112 6.4 Experiments 112 6.5 Materials and methods 113 6.5.1 Sampling and processing 113 6.5.2 Soil characterization 115 6.5.3 Earthworm essay 116 6.5.4 Microcosm analysis 116 6.5.5 ESEM analysis 118 6.5.6 Data analysis 118 6.6 Results 120 6.7 Discussion 124 6.7.1 Bioavailable metal and bioaccumulation 124 6.7.2 Population assessment in microcosm 125 6.7.3 Metal fraction and bioavailability 126 6.7.4 Microscopic examination and statistical linkages 129 6.7.5 Soil metal homogeneity and uptake relationship 130 6.8 Conclusion 132 Chapter 7 STRESS RESPONSES AND SPECIFIC METAL EXCLUSION ON MINE SOILS BASED ON GERMINATION AND GROWTH STUDIES BY AUSTRALIAN GOLDEN WATTLE 7.1 Introduction 133 7.2 Objectives 135 7.3 Hypothesis 136 7.4 Experiments 136 7.5 Materials and methods 137 7.5.1 Soil characterization 137 7.5.2 Seed germination 138 7.5.3 Plants cultivation 139 7.5.4 Pore-water sampling and analysis 141 7.5.5 Biochemical analysis of proline 142 vi 7.5.6 Spectrophotometric determination of chlorophyll a, b and carotenoid 142 7.5.7 Total metal analysis of plants 143 7.5.8 Statistical analysis 144 7.6 Results 145 7.6.1 Germination and biometry 146 7.6.2 Pore-water analysis and biochemical attributes 148 7.6.3 Metal accumulation and biochemical correlation 149 7.7 Discussion 151 7.8 Conclusion 158 Chapter 8 DISCUSSION AND RECOMMENDATION 8.1 Introduction 159 8.2 Metallophyte screening and ecotoxicity tests 160 8.3 Measure of metals from litter into organisms 161 8.4 Scope of this research 163 8.5 Recommendations for future work 165 REFERENCES 169 ABBREVATIONS AML: abandoned mined lands BCR: Bureau of Community Research BLM: Bureau of Land Management EAA: The European Environment Agency ESEM: Environment Scanning Electron Microscope ISO: International Organization of Standards vii PCA: Principal components analysis SAA: semi-arid and arid USEPA: United States Environmental Protection Agency LIST OF FIGURES Fig 1.1 Structure of research in a flow diagram 8 Fig 2.1 Landscape of an abandoned copper mine in Kapunda, South Australia showing revegetation using leguminous plants 14 Fig 2.2 A revegetation and phytoremediation dynamics in blue arrows either partially (horizontal arrows) or wholly (vertical arrows) involving different remediation processes listed in diamond blocks 19 Fig 2.3 The ecological and engineering challenges associated with revegetation of SAA abandoned mine sites such as the copper mine in Kapunda 30 Fig 2.4 Integrated remediation technology (IRT) for revegetation of metal contaminated sites designated to semi-arid and arid conditions 43 Fig 3.1 Location of Kapunda Cu mine site Mathews Quarry 49 Fig 3.2 Enrichment factor (EF) for Cu, Cd, Zn and Pb of the plant colonists. The EF of Cu for Ec is high compared to the other species 58 Fig 3.3 The component plots in rotated spaces showing three groups of metallophytes with similar adaptation based on soil character 60 Fig 4.1 Site map of abandoned copper mine at Kapunda in South Australia. The letter ‘C’ represents Conservation zone, ‘G’ represents the Geological zone and ‘E’ represents the Environmental zone 69 viii Fig 4.2 The accumulation pattern of underground and above ground parts of metallophyte (SD-%, N=3) a. Cu concentration in Ap rhizosphere soil (bars) and A. pycnantha (Ap1, Ap 2, Ap3, Ap4=4 x4) root, stem and leaf at 16 sampling locations. b. Cu concentration in Ec rhizosphere soil (bars) and E. camaldulensis (Ec1, Ec2, Ec3, Ec4= 4x4) root, stem and leaf at 16 sampling locations 74 Fig 4.3 ESEM spectral diagram with images inset. a. The anticlinal section of Ap xylem bundle in the inset with spectral diagram of highlighted area in red circle shows Ca flake deposition. b. The anticlinal section of Ap xylem vessel in inset with spectral diagram of the highlighted area in red circle shows salts of chlorine and potassium respectively, at 2.4 and 3.4 keV. c. The anticlinal section of Ec leaf in inset with spectral diagram of highlighted area in red circle shows Ca deposition in mesophyll zone at 3.6 keV d. The anticlinal cross-cut of Ec leaf in inset with spectral diagram of highlighted red circle shows deposition of Cu and Zn in xylem vessel 79 Fig 5.1 The metal cycle from litters to earthworm tissue during the process of bioaccumulation and bio magnification 85 Fig 5.2 Kapunda abandoned mine site in South Australia showing sampling locations 87 Fig 5.3 Bioaccumulation of Cu, Zn, Cd and Pb in E. fetida exposed to different treatments. Values are means ± SE (n=3) 100 Fig 5.4 a. The best fit with worm Cu content being a linear function of the logarithm of the substrate Cu concentration. b. The Q-Q plot of the residuals from an analysis of the logarithmically transformed juvenile count with an effectively s=a straight line relationship indicting that the residuals are normally distributed 102 Fig 5.5 Means and standard errors of total metal content of Cu (divided by 1000), Zn (Divided by 10), Cd and Pb by different treatments. Columns for each element sharing the same letter are not significantly different at P < .05 (Tukey’s test) 103 Fig 6.1 a. The earthworm microcosm health and population census with different metal concentration and substratum treatments, and b. Bioaccumulation factor of Eisenia fetida against the total metal (log) ix concentration in treatment samples 126 Fig 6.2 Log transformed percent expressions of metal bioavailability in BCR metal analysis a. Actual bioavailable percent of metals from BCR extracted water soluble metals in (log ) substratum. b. Actual bioaccumulation percent in worms from total BCR extracted metal in each (log) treatment pot 127 Fig 6.3 a. ESEM image and EDX spectrum of worm exposed to T3. b. ESEM image and EDX spectrum of worm exposed to T4 129 Fig.6. 4 Dendrogram using Ward Linkage with Rescaled Distance Cluster Combine of metal bioavailability and metal homogenisity. a. Metal bioavailability linkage in earthworms’ tissue b. Metal homogenisity linkage in sample treatment soils 131 Fig 7.1 Map of Kapunda mine site with soil and litter sampling spots (marked in numbers) in South Australia 137 Fig 7.2 Process of Acacia pycnantha seed germination, growth and nodulation, and pore-water collection in glass house experiment set up 141 Fig 7.3 Experimental process for analysis of leaf, phytoaccumulation of metals and pore water activity in mine soils. 143 Fig 7.4 Germination and biomass study a. Germination and survival pattern of heat treated and b. Non-heat treated seeds under different irrigation system c. Height gain by Acacia pycnantha over 16 weeks in different soils, and d. Leaf formation of Acacia pycnantha in different soils till 16 weeks after transplantation 147 Fig 7.5 Changes in pore water dynamics and biochemical attributes. a. The pore water metal dynamics showing prominent As, Pb and Cu activity. b. Response of proline and chlorophylls over time on metal polluted soil 149 Fig 7.6 Plant growth and metal relationship with biochemicals a. Plant biomass rates of potted plants on different soils harvested after 14 months. (above gr-above ground, below gr-below ground). x