Applied Science, Faculty of
Mining Engineering, Keevil Institute of
DSpace
UBCV
Xie, Lina
Doctor of Philosophy - PhD
University of British Columbia
The reclamation of sulfidic mine tailings with the potential to release toxic metals into the surrounding environment, has been an urgent, yet difficult, task for a long period. Direct revegetation is a more cost-effective reclamation method than a multilayer covering system on tailings. Phytostabilization can stabilize toxic metals in the rhizosphere, which is an emerging revegetation method in mine reclamation. Since previous research has not looked into the inorganic geochemical processes in the rhizosphere, a greenhouse study was conducted to explore the geochemical processes affecting the mobility of As, Cu, Pb, and Zn through the establishment of phytostabilization on sulfidic mine tailings. The three-month greenhouse experiment selected candidates for phytostabilization from four grass species in North America: wheat, ryegrass, tall fescue, and sheep fescue. The effectiveness of amendments (bauxite residue, lime, and compost), on enhancing plant growth and metal stabilization were analyzed. Total metals in plants, tailings, and substrates after pot experiments were analyzed, the mineralogy of materials before and after experiments were quantified, and drainage from each pot was collected weekly for metal analysis. The results show wheat and ryegrass can stabilize more metals in roots than in aboveground tissues, so they are good candidates for phytostabilization. Three amendments can improve the tailing pH, grass survival, and plant growth, but compost should be carefully considered for the phytostabilization of Zn. During experiments, pH decreased and metal concentrations in the drainage from amended tailings started to increase from the 6th or 7th week, suggesting that sulfide oxidation, organic matter decomposition, and root exudates are three processes of soil acidification. The other geochemical processes affecting metal mobility include neutralization processes at such sequences: (1) dissolution of carbonates, (2) dissolution of Al- and Fe- (oxy)hydroxide, and (3) silicate weathering. The long-term reliability of phytostabilization is likely to be achieved by re-application of amendment to initiate neutralization in the rhizosphere. This research fills a gap of study on the geochemical processes controlling metal mobility during phytostabilization, and provides a reference for land reclamation and remediation practitioners considering the application of phytostabilization of sulfidic mine tailings.
A GREENHOUSE STUDY ON PHYTOSTABILIZATION OF SULFIDIC MINE TAILINGS by LINA XIE B.A.Sc., China University of Geosciences (Wuhan), 2013 M.A.Sc., China University of Geosciences (Wuhan), 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Mining Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2023 © Lina Xie, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: A greenhouse study on phytostabilization of sulfidic mine tailings submitted by Lina Xie in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Mining Engineering Examining Committee: Dirk van Zyl, Professor, Mining Engineering, UBC Supervisor Ulrich Mayer, Professor, Earth, Ocean and Atmospheric Sciences, UBC Supervisory Committee Member Maria E. Holuszko Associate Professor, Mining Engineering, UBC Supervisory Committee Member Scott Dunbar, Professor, Mining Engineering, UBC University Examiner Susan A. Baldwin, Professor, Chemical and Biological Engineering, UBC University Examiner iii Abstract The reclamation of sulfidic mine tailings with the potential to release toxic metals into the surrounding environment, has been an urgent, yet difficult, task for a long period. Direct revegetation is a more cost-effective reclamation method than a multilayer covering system on tailings. Phytostabilization can stabilize toxic metals in the rhizosphere, which is an emerging revegetation method in mine reclamation. Since previous research has not looked into the inorganic geochemical processes in the rhizosphere, a greenhouse study was conducted to explore the geochemical processes affecting the mobility of As, Cu, Pb, and Zn through the establishment of phytostabilization on sulfidic mine tailings. The three-month greenhouse experiment selected candidates for phytostabilization from four grass species in North America: wheat, ryegrass, tall fescue, and sheep fescue. The effectiveness of amendments (bauxite residue, lime, and compost), on enhancing plant growth and metal stabilization were analyzed. Total metals in plants, tailings, and substrates after pot experiments were analyzed, the mineralogy of materials before and after experiments were quantified, and drainage from each pot was collected weekly for metal analysis. The results show wheat and ryegrass can stabilize more metals in roots than in aboveground tissues, so they are good candidates for phytostabilization. Three amendments can improve the tailing pH, grass survival, and plant growth, but compost should be carefully considered for the phytostabilization of Zn. During experiments, pH decreased and metal concentrations in the drainage from amended tailings started to increase from the 6th or 7th week, suggesting that sulfide oxidation, organic matter decomposition, and root exudates are three processes of soil acidification. The other geochemical processes affecting metal mobility include neutralization iv processes at such sequences: (1) dissolution of carbonates, (2) dissolution of Al- and Fe- (oxy)hydroxide, and (3) silicate weathering. The long-term reliability of phytostabilization is likely to be achieved by re-application of amendment to initiate neutralization in the rhizosphere. This research fills a gap of study on the geochemical processes controlling metal mobility during phytostabilization, and provides a reference for land reclamation and remediation practitioners considering the application of phytostabilization of sulfidic mine tailings. v Lay Summary Sulfidic mine tailings are wastes produced from the mining industry and have potential to release high levels of toxic metals into the environment. To prevent toxic metal pollutions, phytostabilization can be used to stabilize the toxic metals in the area around roots via plant growth. Some materials need to be applied to improve the tailing conditions for plant growth and to stabilize metals in tailings. A greenhouse study was conducted to evaluate the efficiency of metal stabilization of various species and amendment materials, and the reactions affecting the metal stabilization in tailings were explored. It is concluded that phytostabilization is reliable for metal stabilization in the short-term period, and the re-application of amendments and re-seeding of plants may achieve long-term stabilization and successful phytostabilization. vi Preface This thesis contains a series of papers that have been either published in peer- reviewed scholarly journals or submitted for review and subsequent publication. The following is a statement of contributions made to the jointly authored papers contained in this thesis: 1. Xie, L. and van Zyl, D., 2020. Distinguishing reclamation, revegetation and phytoremediation, and the importance of geochemical processes in the reclamation of sulfidic mine tailings: A review. Chemosphere, 252, p.126446. DOI: https://doi.org/10.1016/j.chemosphere.2020.126446. Xie wrote the entire manuscript and van Zyl provided reviews and proofreading. This paper forms Chapter 2 of this thesis. 2. Xie, L. and van Zyl, D., 2023. Identification of grass species candidates for phytostabilization and enhanced metal(loid)s immobilisation using cost-effective amendments on sulfidic mine tailings. International Journal of Mining, Reclamation and Environment. DOI: 10.1080/17480930.2023.2200322. Xie performed all experiments and data analysis, and wrote the manuscript. van Zyl provided reviews and proofreading. This paper forms Chapter 4 of this thesis. 3. Xie, L. and van Zyl, D., 2023. The geochemical processes affecting metal mobility of phytostabilization on sulfidic mine tailings revealed by a greenhouse study. This paper has been submitted for peer review. Xie performed all experiments and data analysis, and wrote the manuscript. van Zyl provided reviews and proofreading. This paper forms Chapter 5 of this thesis. 4. Xie, L., van Zyl, D., and Mayer, U., 2023. The environmental reliability of phytostabilization when metal immobilization in the rhizosphere was impeded by soil acidification. This paper will be submitted for peer review and following publication. Xie vii performed all experiments and data analysis, and wrote the manuscript. van Zyl provided reviews and proofreading. Mayer provided idea and concept. This paper forms Chapter 6 of this thesis. Three papers or posters have been presented in international conferences, which are outcomes work during PhD study. 1. Xie, L. and van Zyl, D., 2022. Grass candidates for phytostabilization and cost-effective amendments for direct revegetation on acid-generating tailings. Poster and presentation, in Canadian Institute of Mining, Metallurgy and Petroleum 2022 Convention, Vancouver, BC, Canada. Xie performed all experiments and data analysis, and wrote the poster and presented at the conference in person. van Zyl provided reviews and proofreading. 2. Xie, L. and van Zyl, D., 2022. Geochemical processes controlling metals (Pb, Zn) mobility during amended phytostabilization on sulfidic mine tailings. Presentation, in 2022 Goldschmidt Conference, Honolulu, Hawaiʻi, USA. Xie performed all experiments and data analysis, and wrote the presentation and presented at the conference virtually. van Zyl provided reviews and proofreading. 3. Xie, L. and van Zyl, D., 2022. Phytostabilization efficiency on sulfidic mine tailings and geochemical changes: a greenhouse study. Presentation, in 2022 Tailings and Mine Waste Conference, Fort Collins, Colorado, USA. Xie performed all experiments and data analysis, wrote the conference paper and presented at the conference. van Zyl provided reviews and proofreading. viii Table of Contents Abstract .......................................................................................................................................... iii Lay Summary .................................................................................................................................. v Preface............................................................................................................................................ vi Table of Contents ......................................................................................................................... viii List of Tables ............................................................................................................................... xiii List of Figures ............................................................................................................................... xv List of Abbreviations ................................................................................................................. xviii Acknowledgements ...................................................................................................................... xix Dedication .................................................................................................................................... xxi Chapter 1: Introduction ................................................................................................................... 1 1.1 Environmental hazards of sulfidic mine tailings .................................................................. 1 1.2 Mine revegetation ................................................................................................................. 1 1.3 Phytostabilization ................................................................................................................. 3 1.4 Geochemical processes and greenhouse experiments .......................................................... 4 1.5 Innovations and significance ................................................................................................ 5 1.6 Dissertation structure ............................................................................................................ 6 1.6.1 Overarching research question ....................................................................................... 6 1.6.2 Research objectives ........................................................................................................ 6 1.6.3 Chapter organization ...................................................................................................... 6 ix Chapter 2: Literature Review .......................................................................................................... 8 2.1 Introduction .......................................................................................................................... 8 2.2 The Concept of Reclamation .............................................................................................. 10 2.3 Revegetation on Covers vs. Direct Revegetation ............................................................... 12 2.3.1 Revegetation on Covers ............................................................................................... 13 2.3.2 Direct Revegetation ...................................................................................................... 14 2.4 Direct Revegetation vs. Phytoremediation ......................................................................... 18 2.4.1 Potential of Phytostabilization ..................................................................................... 22 2.5 Interactions in the Rhizosphere .......................................................................................... 24 2.5.1 Geochemical Processes ................................................................................................ 25 2.5.2 Plant – Soil Interactions ............................................................................................... 28 2.6 Discussion ........................................................................................................................... 31 2.7 Conclusion .......................................................................................................................... 35 Chapter 3: Materials and Methods ................................................................................................ 37 3.1 Introduction ........................................................................................................................ 37 3.2 Materials acquisition........................................................................................................... 39 3.2.1 Tailings ......................................................................................................................... 39 3.2.2 Bauxite residue, compost, and lime ............................................................................. 41 3.2.3 Material Characterization and Testing ......................................................................... 42 3.2.4 Species Selection .......................................................................................................... 44 x 3.3 Greenhouse Experiments .................................................................................................... 45 3.3.1 Three stages of greenhouse experiments ...................................................................... 45 3.3.2 Monitoring and testing ................................................................................................. 53 3.3.3 Statistical analysis ........................................................................................................ 54 Chapter 4: Species Selection and Amendment Effectiveness for Phytostabilization ................... 56 4.1 Introduction ........................................................................................................................ 56 4.2 Material characterization .................................................................................................... 58 4.2.1 Metals in tailings .......................................................................................................... 58 4.2.2 The pH of materials ....................................................................................................... 59 4.3 Growth affected by different amendments ......................................................................... 61 4.3.1 Germination rate and shoots height .............................................................................. 61 4.3.2 Dry biomass ................................................................................................................. 62 4.4 Metals in plants affected by different amendments ............................................................ 64 4.4.1 As in species ................................................................................................................. 64 4.4.2 Cu in species ................................................................................................................ 65 4.4.3 Pb in species ................................................................................................................. 66 4.4.4 Zn in species ................................................................................................................. 68 4.4.5 Discussion .................................................................................................................... 69 4.5 Translocation factor and accumulation Factor ................................................................... 70 4.6 Principal component analysis (PCA) .................................................................................. 74 xi 4.7 Conclusions ........................................................................................................................ 75 Chapter 5: Geochemical Processes in the Rhizosphere ................................................................ 76 5.1 Introduction ........................................................................................................................ 76 5.2 Geochemistry changes after experiments ........................................................................... 79 5.2.1 Changes of pH in substrates ......................................................................................... 79 5.2.2 Changes of mineralogy ................................................................................................ 80 5.2.3 Changes of pH and metal concentrations in drainage .................................................. 83 5.3 Discussion ........................................................................................................................... 93 5.3.1 The sources of metals ................................................................................................... 93 5.3.2 Soil acidification processes .......................................................................................... 95 5.3.3 Neutralization processes ............................................................................................. 100 5.3.4 Geochemical processes in different amended tailings ............................................... 104 5.3.5 Amendment effectiveness on metal stabilization ....................................................... 107 5.4 Conclusions ...................................................................................................................... 108 Chapter 6: Environmental Reliability of Phytostabilization on Sulfidic Mine Tailings ............. 110 6.1 Introduction ...................................................................................................................... 110 6.2 Reason of grass death ....................................................................................................... 112 6.3 Soil quality ........................................................................................................................ 115 6.3.1 Metal concentrations in soil after experiments .......................................................... 115 6.3.2 Metal distribution ....................................................................................................... 118 xii 6.4 Drainage quality ............................................................................................................... 121 6.5 Conclusion ........................................................................................................................ 124 Chapter 7: Conclusions and Suggestions .................................................................................... 126 7.1 Concluding remarks .......................................................................................................... 126 7.1.1 Species selection and amendments effectiveness....................................................... 127 7.1.2 Geochemical processes in the rhizosphere ................................................................. 128 7.1.3 Environmental reliability ........................................................................................... 129 7.2 Major contributions and significance ............................................................................... 130 7.2.1 Theoretical contribution ............................................................................................. 130 7.2.2 Applied contribution .................................................................................................. 131 7.3 Limitations and suggestions ............................................................................................. 132 7.3.1 Experimental studies .................................................................................................. 132 7.3.2 Analytical methods ..................................................................................................... 133 7.3.3 Application considerations ......................................................................................... 135 Bibliography ............................................................................................................................... 136 Appendices .................................................................................................................................. 159 Appendix A: The parameters and testing methods .................................................................. 159 Appendix B: Refinement plots of quantitative X-ray powder diffraction and qualitative X-ray powder diffraction ................................................................................................................... 163 Appendix C: Results of two – way ANOVA .......................................................................... 176 xiii List of Tables Table 2-1 Main sulfide minerals may contain minor and trace metals constituents in the form of cation substitutes ........................................................................................................................... 10 Table 2-2 Amendment applications for successful direct revegetation on sulfidic tailings ......... 16 Table 2-3 Metals toxicity limits to soil, plant, and animal and the typical tailings metal concentrations ............................................................................................................................... 17 Table 2-4 Current approaches of phytoremediation for sulfidic mine tailings ............................. 21 Table 2-5 Potential candidates of plant species for successful phytostabilization on sulfidic mine tailings in arid and humid regions ................................................................................................ 24 Table 3-1. Rinse pH (solid:water =1:5) of tailings, bauxite residue, and compost before mixing 42 Table 3-2. Physical properties and chemical characteristics of tailings ....................................... 42 Table 3-3. The quantitative XRD results of tailings and bauxite residue ..................................... 43 Table 3-4. The composition of amendments................................................................................. 46 Table 3-5. The installed pots for six substrates and four grass species ........................................ 47 Table 3-6. Primary purposes for the experimental design ............................................................ 48 Table 3-7. Timeline of the 2nd stage of greenhouse experiments in 2021 ................................... 49 Table 3-8. Greenhouse environmental conditions, provided by the UBC Horticulture Greenhouse....................................................................................................................................................... 50 Table 3-9. Numbers of sample collected from the greenhouse experiments ................................ 53 Table 3-10. Tested indicators and purposes in this research ........................................................ 54 Table 4-1. Metal concentrations in tailings compared to different limits ..................................... 59 xiv Table 4-2. Rinse pH (solid: water = 1:5) of tailings, bauxite residue, compost and six substrates....................................................................................................................................................... 60 Table 4-3. TF and AF for four species on six substrates .............................................................. 73 Table 5-1. Rinsed pH of substrates (1:5 w/w soil: water) in pots before and after experiments .. 79 Table 5-2. Quantitative XRD results of samples before and after experiments ........................... 81 Table 5-3. The soil acidification processes in six substrates ........................................................ 98 Table 5-4. Minerals related to geochemical processes in the six substrates ............................... 106 Table C-1. Two – way ANOVA results of effects of amendments and species on plant growth..................................................................................................................................................... 176 Table C-2. Two – way ANOVA results of effects of amendments and species on the pH of mixed substrates before experiments ..................................................................................................... 176 Table C-3. Two – way ANOVA results of effects of amendments and species on the total metal uptake by plants .......................................................................................................................... 177 Table C-4. Two – way ANOVA results of effects of amendments and species on the pH and total metals remained in pot substrates after experiments .................................................................. 178 Table C-5. Two – way ANOVA results of effects of amendments and species on the total metals in drainage at the end of experiments ......................................................................................... 179 xv List of Figures Figure 2-1. Three potential outcomes of direct revegetation including the rhizosphere interactions related to metal release and root uptake ........................................................................................ 19 Figure 2-2. Relating reclamation, revegetation and phytoremediation. ........................................ 33 Figure 3-1. Highlights of this chapter in the research workflow .................................................. 38 Figure 3-2. Tailings sampling location (red triangle) at tailings storage ponds at Faro Mine ...... 40 Figure 3-3. Tailings, bauxite residue, lime and compost .............................................................. 41 Figure 3-4. Selected grass species ................................................................................................ 44 Figure 3-5. Photos of six substrates .............................................................................................. 46 Figure 3-6. Photos of the experiment ............................................................................................ 49 Figure 3-7. The growth of all plants after the last batch of drainage collection on May 14th, 2021....................................................................................................................................................... 52 Figure 3-8. The procedures for collecting the root samples ......................................................... 52 Figure 4-1. Highlights of this chapter in the research workflow .................................................. 58 Figure 4-2. The germination rates (a) and shoot heights (b) of four types of grass species on the six substrates ................................................................................................................................. 61 Figure 4-3. The dry biomass of shoots and roots of four species on six substrates. ..................... 63 Figure 4-4. As concentrations in roots and shoots (mg/kg) .......................................................... 65 Figure 4-5. Cu concentrations in roots and shoots (mg/kg) .......................................................... 66 Figure 4-6. Pb concentrations in roots and shoots (mg/kg) .......................................................... 67 Figure 4-7. Zn concentrations in roots and shoots (mg/kg) .......................................................... 68 Figure 4-8. Accumulation factor and translocation factor of As, Cu, Pb and Zn for the four species on six substrates................................................................................................................ 72 xvi Figure 4-9. PCA comparing measured parameters. ...................................................................... 74 Figure 5-1. Highlights of this chapter in the workflow ................................................................ 77 Figure 5-2. pH changes of drainage from pots with or without plants during the experiments ... 84 Figure 5-3. Cu concentration of drainage from pots with or without plants during the experiments........................................................................................................................................................ 86 Figure 5-4. Pb concentration of drainage from pots with or without plants during experiments. 87 Figure 5-5. Zn concentration of drainage from pots with or without plants during the experiments........................................................................................................................................................ 89 Figure 5-6. pH changes of drainage from different substrates ...................................................... 90 Figure 5-7. Cu concentration of drainage from different substrates ............................................. 92 Figure 5-8. Pb concentration of drainage from different substrates ............................................. 92 Figure 5-9. Zn concentration of drainage from different substrates ............................................. 92 Figure 6-1. The highlight of this chapter in the research workflow ........................................... 111 Figure 6-2. Seed production of wheat (a) and ryegrass (b) at the end of the experiments ......... 113 Figure 6-3. The relationships between total biomass and pH in soil, shoot heights, and pH in the soil after experiments. ................................................................................................................. 114 Figure 6-4. Metal concentrations in soil after experiments compared to their original concentrations in tailings. ........................................................................................................... 117 Figure 6-5. Metal distribution in plants, soil, and drainage at the end of experiments ............... 120 Figure 6-6. Changes in pH and metal concentrations in drainage during the first six weeks ..... 123 Figure B-1. Rietveld refinement plot of sample #1: Tailings (2-3m) (before the experiment, analyzed in June, 2021)............................................................................................................... 164 xvii Figure B-2. Rietveld refinement plot of sample: Bauxite Residue (before the experiment, analyzed in June, 2021)............................................................................................................... 165 Figure B-3. Rietveld refinement plot of sample #1: Tailings Blank (after the experiment, analyzed in April, 2022) ............................................................................................................. 166 Figure B-4. Rietveld refinement plot of sample #2: T+BR Blank (Tailings + Bauxite Residue) (after the experiment, analyzed in April, 2022) .......................................................................... 167 Figure B-5. Rietveld refinement plot of sample #3: T+L Blank (Tailings + Lime) (after the experiment, analyzed in April, 2022) ......................................................................................... 168 Figure B-6. Rietveld refinement plot of sample #4: T+C Blank (Tailings + Compost) (after the experiment, analyzed in April, 2022) ......................................................................................... 169 Figure B-7. Qualitative XRD plot of sample: T+BR+C Blank (after the experiment, analyzed in March, 2022) ............................................................................................................................... 170 Figure B-8. Qualitative XRD plot of sample: T+L+C Blank (after the experiment, analyzed in March, 2022) ............................................................................................................................... 171 Figure B-9.Qualitative XRD plot of sample: T-WG (after the experiment, analyzed in March, 2022) ........................................................................................................................................... 172 Figure B-10.Qualitative XRD plot of sample: T-RG (after the experiment, analyzed in March, 2022). .......................................................................................................................................... 173 Figure B-11.Qualitative XRD plot of sample: T-TF (after the experiment, analyzed in March, 2022) ........................................................................................................................................... 174 Figure B-12. Qualitative XRD plot of sample: T-SF (after the experiment, analyzed in March, 2022) ........................................................................................................................................... 175 xviii List of Abbreviations AF – Accumulation Factor AMD – Acid Mine Drainage ANOVA – Analysis of Variation AP – Acid Potential BR – Bauxite Residue C – Compost CEC – Cation Exchange Capacity ICP - MS – Inductively Coupled Plasma Mass Spectrometric L – Lime NAP – Net Acid Production NP – Neutralization Potential PCA – Principal Component Analysis RG – Ryegrass SF – Sheep Fescue T – Tailings TF – Tall Fescue TF – Translocation Factor TIC – Total Inorganic Carbon WG – Wheat XRD – X-Ray Diffraction xix Acknowledgements I would like to convey my sincere gratitude and appreciation to following people who have provided their enormous support during my studies at the University of British Columbia. This thesis represents many years of work, none of which I could have done without the help, encouragement and support of many people. First of all, I would like to express my heartfelt gratitude to my supervisor, Dirk van Zyl, for his encouragement, continued support and invaluable mentoring throughout my Ph.D. life. Your patience and indulgence provided me freedom and confidence to grow up as an independent researcher. You offered me freedom of scientific research, massive conference opportunities about mine wastes and mine closure, and abundant interpersonal interactions. I will carry the pride in my heart as a student of yours forever. I would like to extend my thankfulness to my committee members: Ulrich Mayer, for guiding me in research of geochemistry and answering my endless questions; Maria E. Holuszko, who generously let me use her XRD analyzer, and contributed her valuable comments on my proposal and research. I also appreciate all the faculty and staff of the Norman B. Keevil Institute of Mining Engineering at UBC. In particular, thanks to Anne Broženský and Carmen Jensen for their administrative support, to Marek Pawlik for his graduate advises, to Libin Tong for his technical assistance to my lab work at CMP building, and to Vinoth Kumar Kuppusamy and Frank Yan for their help to my lab work in room 503 of the Frank Forward building. The cooperation and support from other parties are greatly appreciated. Particularly, thanks to Faro Mine, Rio Tinto, and UBC Building Operations for generously providing buckets of tailings, xx bauxite residue, and compost, respectively, to the UBC Horticulture Greenhouse for their professional support for my greenhouse experiment, and to Prof. Harry Brumer from UBC Michael Smith Laboratories and Department of Chemistry for kindly letting me analyze water samples in his lab during the pandemic. This research would not be possible without the financial support by China Scholarship Council and the generous assistantship provided by Prof. Dirk van Zyl. I am also thankful for the financial assistance provided by UBC in the form of the Faculty of Applied Science Graduate Award, the International Partial Tuition Scholarship and the President's Academic Excellence Initiative PhD Award. Finally, my deepest gratitude goes to my family. I am grateful that my mom, Xiuzhen Li, came to Canada where is completely strange to her to help take care of my family. Thanks to my twin daughters, Junping Xiang and Junan Xiang, for coming to my life during this study and making me smile. You two are the strength of my everyday triumph, and you have made me stronger, better and more fulfilled than I could have ever imagined. Most of all, I am extremely grateful for all the sacrifices my beloved husband, Jinke Xiang, has made for me. The completion of this work would not have been possible without his everlasting love and unconditional support. xxi Dedication To my sweet twin daughters, Pingping and Anan. I love you to the moon and back. 愿你们一生平安， 岁月无忧。 1 Chapter 1: Introduction 1.1 Environmental hazards of sulfidic mine tailings In the mining industry, tailings are one of the most important mine waste types due to their large volume and the potential of metal release into the environment. In Canada, the domestic mining industry (metal and non-metal mineral extraction), produced 473 million tonnes of waste rock and tailings in 2008 (Canada, 2012), and this number has increased considerably since. Ritcey (1989) pointed out that the single most important factor affecting the quality of seepage and runoff in the environment is sulfidic minerals in the tailings that result in acid generation. Therefore, sulfidic mine tailings should be reclaimed properly during mine closure, to provide for a more aesthetic terrain, as well as to minimize the environmental impact of seepage and runoff. Most sulfidic tailings have a rather low pH and a large acid production potential, thus the heavy metals loading in the tailings can drain to groundwater, which is called AMD (acid mine drainage) (Blowes et al., 2014; Nordstrom & Southam, 1997). Contaminated groundwater may present a threat to drinking water and the toxic metals are susceptible to wind dispersal. Therefore, tailings management and after-mining reclamation are required in mine closure. This is the motivation for this research. 1.2 Mine revegetation Mine revegetation is the most widely used reclamation technique in tailings, since it can recover the landscape aesthetics. Physical and chemical covers can be applied to neutralize and stabilize the metals, reducing their potential environmental impact. In addition, the vegetation cover can act as a neutralizing and protective barrier where metals may be sequestered and precipitated by 2 the colloid materials in the topsoil layer (Leroy, 1973). In practical implementations, the physical, chemical and vegetation covers are applied together as a multilayer covering system on tailings, which is revegetation on covers (Xie & van Zyl, 2020). Compared to revegetation on covers, the direct revegetation of sulfidic mine tailings is implemented less frequently in the field. Direct revegetation on tailings entails growing plants on tailings without applying other physical or chemical covers, but some economical amendments for adjusting tailings conditions are necessary for successful vegetation on the sulfidic tailings. Tailings amendment methods are critical to create a suitable and nutrient-sufficient environment for revegetation (Alvarenga et al., 2008; Araujo & Costa, 2013; Phillips et al., 2016; Radziemska et al., 2017). Research has indicated that organic matter and fertilizer are necessary inputs to revegetation on tailings due to the lack of nutrients and microbial communities in tailings. Controlling acidity to increase the pH in sulfidic tailings is also important. Especially for sulfidic mine tailings, while revegetation on covers has been applied widely in many mine reclamation cases, it is more expensive due to the large requirement for earth crust materials. By contrast, direct revegetation can be more cost-effective. Examining cost-effective amendments to support and enhance the direct revegetation on sulfidic mine tailings is the first objective of this research. The second objective of the study is to seek proper plant species for direct revegetation on sulfidic mine tailings. Most plants cannot grow in extremely acid media, and heavy metals toxicity affects plant roots and shoots (Mulligan et al., 2006). Therefore, plants intended for direct revegetation should be selected such that the concentration of toxic metals does not exceed the tolerance of the species (Guittonny et al., 2016; Farahat & Galal, 2018). 3 1.3 Phytostabilization Phytostabilization is one of the five techniques of phytoremediation, which aims to immobilize the metals in the rhizosphere through the growth of plants on contaminated land. Phytovolatilization and phytoextraction (phytoaccumulation) which remove high concentrations of heavy metals or other toxic contaminants and translocate them to the aboveground tissues (Hine & Martin, 2016) (Figure 2-1). Compared to phytovolatilization and phytoextraction, the advantage of phytostabilization in the rhizosphere is that it can protect animals from contacting or eating the exposed plant tissues with elevated concentrations of toxic metals, which also prevents these toxic elements from entering food chains used by humans (Xie & van Zyl, 2020). As phytostabilization is a method of direct revegetation that can be aided with amendments (Martínez‐Martínez et al., 2019), this aspect of phytostabilization was explored in this research through greenhouse experiments. Previous research on the phytostabilization of mine tailings focuses on plant species selection and amendment material selection for improving the stabilization of metals in the rhizosphere. The species used for phytostabilization on sulfidic mine tailings should tolerate acidity and toxicity and the amendments must be able to adjust the tailings to suitable conditions for plant growth (Marques et al., 2009; Yang et al., 2016). Although species selection and amendment materials for phytostabilization has been the focus of other research, there are still many species and amendment types in need of being explored for their candidacy and effectiveness for phytostabilization, especially on sulfidic mine tailings. Chapter 4 focuses on candidate species and the effectiveness of amendment materials for phytostabilization on the sulfidic mine tailings. 4 1.4 Geochemical processes and greenhouse experiments Research on geochemical processes of metal stabilization within the rhizosphere in sulfidic mine tailings is limited. Roots – tailings interactions provide a better understanding of metal speciation and the migration fate of toxic metals, thus enhancing the successful establishment of phytostabilization on sulfidic mine tailings and protecting the groundwater from AMD pollution. The biogeochemical interactions in the rhizosphere include organic-related processes, such as microbial activity and root uptake, and inorganic-related processes. The success of direct revegetation depends on the nature of materials, the mineralogy, nutrients, and the organic matter contained in the soil. The mineral composition can be retained after revegetation, and the breakdown of primary minerals into secondary minerals and the formation of clay minerals can occur around plant roots (Justine et al., 2014). The inorganic geochemical processes controlling metal mobility in the rhizosphere mainly include three types (Dighton & Krumins, 2014): precipitation and dissolution of inorganic minerals; silicate minerals weathering process releasing metals; adsorption and desorption onto solid phase surfaces. The rhizosphere around plant roots acts as a biogeochemical barrier for stabilizing metals from transporting to groundwater. Currently, most research focuses on microbial activity and its role in immobilizing metals, whereas there exists a research gap in the inorganic geochemical processes taking place in the rhizosphere when establishing phytostabilization on the sulfidic mine tailings. The third objective of this study is to explore the inorganic geochemical processes controlling the metal immobilization in the rhizosphere through phytostabilization, which leads to a better understanding of metal release, migration, and transformation, as well as the long-term evolution of toxic metals during phytostabilization. Chapter 5 explores the geochemical processes affecting metal mobility in the rhizosphere during phytostabilization. 5 Finally, with regards to the method for this research, greenhouse experiments were chosen because the geochemical processes can be explored better when plants could survive and grow under optimized climate conditions. Numerous greenhouse experiments have been conducted in the selection of potential plant species and suitable growing conditions, while the processes underlying geochemical processes that affect the metal mobility in plants are not a focus in the existing literature. Due to the uncertainties of the climate conditions in the field, greenhouse experiments are considered valuable and suitable to reach the goals of this study. 1.5 Innovations and significance This research is important because sulfidic mine tailings generate AMD and release toxic metals. The study enhances the knowledge of phytostabilization at sulfidic mine tailings, a cost-effective and environmentally-friendly method of direct revegetation, as well as the understanding of geochemical processes for phytostabilization with regards to metals immobilization. This research provides a reference to land reclamation and remediation practitioners when considering the application of phytostabilization at sulfidic mine tailings. This research has three innovations: • Assessing species candidates for phytostabilization; • Evaluating cost-effective amendments for phytostabilization, including bauxite residue which is also a type of mine waste; • Exploring the chemical changes in drainage and geochemical processes in the rhizosphere during phytostabilization. 6 1.6 Dissertation structure 1.6.1 Overarching research question The below research question was investigated through this study: • What are the geochemical processes affecting metal immobilization in the rhizosphere following the establishment of phytostabilization on sulfidic mine tailings? 1.6.2 Research objectives The objectives are divided into three purposes parts: • To evaluate the candidacy of the four selected plant species for phytostabilization on sulfidic tailings; • To compare the effectiveness of different amendments (organic matter, lime, and bauxite residue), on enhancing plant growth and metal immobilization in phytostabilization; • To evaluate the geochemical processes and reactions controlling metal mobility in the rhizosphere during phytostabilization by analyzing drainage chemistry and soil minerals. 1.6.3 Chapter organization The body of this thesis consists of seven chapters that demonstrate how the research was conducted to answer the above question and meet the objectives. Chapter 1: Introduction Chapter 2: Literature Review Chapter 3: Materials and Methods 7 Chapter 4: Species Selection and Amendments Effectiveness for Phytostabilization Chapter 5: Geochemical Processes in the Rhizosphere Chapter 6: Environmental Reliability of Phytostabilization on Sulfidic Mine Tailings Chapter 7: Conclusions and Suggestions 8 Chapter 2: Literature Review1 2.1 Introduction Mine tailings, a significant type of mine wastes produced from mining activities, have the potential to threaten the environment. An estimated over 10 billion tonnes of mine tailings are produced per year all over the world (Adiansyah et al., 2015). Tailings management facilities are commonly constructed to store the millions of tonnes of tailings. Globally, approximately 4×105 km2 of land is disturbed by mining activities including the storage area of sulfidic tailings (Hooke et al., 2012). These figures will increase as more open pit mining with decreasing cut-off grade are developed (Asad et al., 2016). The existence of sulfides in tailings results from the processing of hard rock sulfidic bearing ores, especially metallic ore deposits (Cu, Pb, Zn, Au, Ni, U, Fe), phosphate ores, coal seams and oil sands that contain abundant sulfides, thus the sulfide-containing tailings are commonly considered as sulfidic mine tailings (Lottermoser, 2010). The long-term exposure of sulfidic tailings in the field can potentially cause environmental problems to a nearby community, including eolian dispersion by wind erosion that can contain toxic metals as well as polluted mine drainage by water erosion and seepage. Ritcey (1989) pointed out that the single most important factor affecting the quality of seepage and runoff with respect to the environment is 1 A version of this chapter has been published. Xie, L., & van Zyl, D., 2020. Distinguishing reclamation, revegetation and phytoremediation, and the importance of geochemical processes in the reclamation of sulfidic mine tailings: A review. Chemosphere, 252, 126446. DOI: https://doi.org/10.1016/j.chemosphere.2020.126446. 9 the presence and content of sulfides in the tailings, because the oxidation of sulfide minerals can produce acid and thus increase the mobility of toxic metals. For example, the general pyrite oxidation reaction can be simplified as below, producing sulfuric acid and metals (D. K. Nordstrom & Southam, 1997), which is commonly known as the formation of AMD from sulfidic tailings (Blowes et al., 2014): FeS2(s) + 154 O2(aq) + 72 H2O → Fe(OH)3(s) + 2SO42− + 4H(aq)+ Equation 2-1 Nieva et al. (2018) found that minerals recycling controls short-term release of Cu, Zn and Fe in sulfidic tailings, while sulfide oxidation, oxyhydroxide dissolution at acidic pH, and desorption control long term release of Cu, Zn, Fe, and Pb. In fact, the tailings mineralogy can contain various sulfide minerals that all have the potential to release associated toxic metals to the environment, as listed in Table 2-1. The high concentration of toxic metals that would be released largely from these sulfide minerals contained in the tailings under acid conditions could have deleterious effects on the ecosystem and on human health. Thus, the reclamation of sulfidic tailings is an urgent but difficult task during mine closure. The present review has been undertaken with the following objectives: • To distinguish the concepts of reclamation, revegetation and phytoremediation, and clarify their relationships; • To discuss and emphasize the importance of inorganic geochemical processes in the study on the direct revegetation of sulfidic mine tailings. 10 Table 2-1 Main sulfide minerals may contain minor and trace metals constituents in the form of cation substitutes Sulfide mineral Chemical formula Minor and trace elements may be contained in the Arsenopyrite FeAsS As, Sb, Fe, Cu Chalcocite Cu2S As, Sb, Fe, Co, Cu, Pb Chalcopyrite CuFeS2 Ag, As, Bi, Cd, Co, Cra, Mn, Mo, Ni, Pb, Sb, Se, Sn, V, Cinnabar HgS Co, Cd, Hg, As Galena PbS Ag, As, Bi, Cd, Cu, Fe, Hga, Mn, Ni, Sb, Se, Sn, Ti, Zn Marcasite FeS2 As, Hga, Se, Sn, Ti, Pb, V Molybdenite MoS2 Cu, Mo, Ni, Pb, Zn Pyrite FeS2 Ag, As, Au, Bi, Cd, Co, Ga, Ge, Hga, Mo, Ni, Pb, Sb, Se, Pyrrhotite Fe1-xS Ag, As, Co, Cra, Cu, Mo, Ni, Pb, Se, Sn, V, Zn Stibnite Sb2S3 Fe, Cu, Sb, As Sphalerite ZnS Ag, As, Sb, Cu, Cd, Co, Cra, Fe, Ga, Ge, Hga, Mn, Mo, a some elements may also be present as small inclusions in the host sulfides (Bernd G. Lottermoser, 2003). b cited from (Seal & Foley, 2002; Vaughan & Craig, 1978) 2.2 The Concept of Reclamation Reclamation aims to maintain environmental quality and enhance land management capacity, implying converting disturbed or damaged land to its former or other productive uses (Favas et al., 2018; Haigh, 2007; Naeth et al., 2021). This is typically essential to convert mining lands and surfaces of tailings ponds to a natural or economically usable state after mine closure or tailings facilities decommissioning. The objectives of sulfidic tailings reclamation include (Favas et al., 2018; Lima et al., 2016): • Stabilization of the surface to reduce eolian dispersion and surface water erosion • Prevention of the release of contaminants to the surrounding environment 11 • Development of an aesthetic appearance on the surface of the tailings facilities, usually by establishing vegetation In order to target these purposes, adding covers on top of the tailings has become the most common and direct method of tailings reclamation following mine closure, aiming to prevent the formation of AMD and metals releasing in sulfidic mine tailings. Former researchers have classified the reclamation measures into three independent types of cover: chemical cover, physical cover and vegetation cover (Wang et al., 2017). • Single or multi layered physical covers by means of enclosing with coarse rock, soil or other materials with low-permeability to form a cover that will result in the reduction of water infiltration, gas diffusion or capillary (Benson et al., 2001; Rogers et al., 1984; Waugh et al., 2015) • Chemical covers that allow interaction with the fine tailings to form a hardpan or that aim to convert the toxic components so that they are resistant to weathering and leaching, thus minimizing the environmental problems, including migration of AMD and transport of toxic metals (Ritcey, 1989; Hunter & Whiteman, 1975) • Vegetation cover through planting on the tailings resulting in a protective barrier from wind and water erosion and for the reduction of drainage downward and metals transport, and as well as an aesthetic landscape for the sites (Chen et al., 2018; Leroy, 1973) Equation 2-1 indicates that oxygen and water are two key factors affecting acid generation, and then the acidity would promote the metal mobility in tailings, so the above covers mainly aim to create barriers to prevent the oxygen and water from entering into tailings and the metals migration. However, tailings reclamation typically combines all of these covers to achieve the 12 reclamation goals rather than each being applied separately. In fact, as tailings facilities are usually quite large and occupy a large area of land resources, Ritcey (1989) pointed out that recovering the tailings surface to an aesthetic landscape for future land uses became the most significant and the ultimate purpose for tailings reclamation. Therefore, establishing revegetation on the upper layer of the tailings storage facilities has the potential to achieve the three objectives mentioned above and a long-term self-sustainable ecosystem (Farmer & Richardson, 1981). Potential problems associated with the establishment of vegetation directly on the surface of sulfidic tailings materials include there: (1) low organic contents; (2) low levels of plant nutrients such as nitrogen (N), phosphorus (P), and potassium (K); (3) the low pH and high toxicity (Yang et al., 2016; Hunter & Whiteman, 1975). Based on the various means to resolve the above obstacles, the revegetation technologies can further be divided into non-direct and direct revegetation. 2.3 Revegetation on Covers vs. Direct Revegetation The revegetation of mined lands in practice can largely be considered as ecosystem reconstruction and reestablishment of the originally ecological function, which should integrate goals and criteria in a systematic way in the planning stage, particularly for metallic mine wastes (Cooke & Johnson, 2002). Without considering the systematically complexity of the design of revegetation plan, this research is focusing on reviewing the concepts, purposes and approaches of revegetation only from the technical perspective when dealing with sulfidic mine tailings. Revegetation on covers refers to the combination of the different functional covers mentioned above, such as coarse rock and topsoil, and then building the vegetation cap as the final surface 13 cover. Direct revegetation aims to grow plants directly and sustainably on the tailings; the addition of several amendments to the tailings may be required in establishing the revegetation. 2.3.1 Revegetation on Covers In the early history of tailings reclamation, revegetation on covers, which is referred to as multilayer reclamation in Ritcey (1989), has achieved more success than the direct revegetation on tailings in practice. Ghassemi et al. (1983) recommended a multilayered cover system consisting of a vegetated top layer, a middle drainage rock layer, and a low permeability bottom clay layer. Korea Electric Power Corporation International Nuclear Graduate School (Vaysidin et al., 2016) designed a feasible cost-effective covering system which can provide a long period (100 years) of protection for the uranium tailings in the Republic of Tajikistan. It included a multilayer cover system with a 70 cm thick percolation layer, a 30 cm thick drainage layer, and a 60 cm thick barrier clay soil layer. The engineered soil layer under the top vegetation cap was formed by mixing varying rates of glacial till, stockpiled peat, and compost as an alternative to sourcing topsoil for sulfidic tailings revegetation (Smart et al., 2016), which is also an effective barrier against the upward migration of toxic metals to the upper vegetation (Callery & Courtney, 2015). Moreover, since the soil layer for planting have to maintain the hydraulic capacity and the physical structure for stable water supply to plants for better revegetation, the mixed engineered soil layer also provide the effective implementation method to create macropores for enhancing water infiltration, where coarse materials, such as glacial till, gravels and inert waste rocks, play a critical role in creating water-stable aggregates and hydraulic storage, and are usually applied in engineered soil for the revegetation on covers (Norland & Veith, 1995; Robson et al., 2018; Vaysidin et al., 2016; Wehr et al., 2005). 14 Extensive literature about revegetation on covers on acidic mine wastes can be found throughout past decades (Nason et al., 2013Waugh et al., 2015). It can be concluded from the previous applications of multilayer covers that the typical revegetation on covers system consists of the lower layer placed with gravel or coarse rocks to prevent upward capillary migration of acid and salts, a medium clay layer compacted as a gas diffusion barrier, and an upper topsoil layer to provide nutrients and favorable conditions for vegetation growth. Moreover, the layer of coarse rocks and clay are also considered as inert covering materials (Bussière & Aubertin, 2003; Tordoff et al., 2000) to sulfidic tailings to prevent the oxygen and water from interacting with sulfide minerals. As the engineering cover properties naturally change because of the evolution of the ecological system (DeJong et al., 2015), the performance of cover alternative and characteristics would be evaluated and assessed either in the field or in the laboratory (Benson et al., 2001; Bussière et al., 2004). However, there is not a general cover system that can be specified to fit all the types of tailings revegetation because of the various physical and geochemical characteristics of tailings, in addition to the particular site’s climate conditions and the numerous reclamation requirements at different mine sites. As the revegetation on covers technology requires significant amounts of materials such as rock, clay, and topsoil, which can be expensive in terms of practical implementation, direct revegetation has attracted increasing attention because of the potential advantage of its cost-efficiency (Dean et al., 1973; Mulligan et al., 2006). 2.3.2 Direct Revegetation The term of “direct revegetation” is described in the literature (Li et al., 2014; Luo et al., 2018; Luo et al., 2023; Tordoff et al., 2000). Compared to revegetation on covers, direct revegetation 15 on the surface of sulfidic tailings is implemented less in practice, but typically there are greenhouse studies on direct revegetation on sulfidic tailings to choose plant species and seek suitable growing conditions. In most situations, plants cannot grow normally even on the surface of a tailings pond that has already been abandoned for years (Macdonald et al., 2017). This is not only because the chemical conditions are deleterious to plant survival and growth (Yang et al., 2016; Hunter & Whiteman, 1975). The poor physical structure and properties of the tailings also contribute to the poor survival rate of vegetation (Guittonny et al., 2016). However, direct revegetation has been achieved successfully in several mine sites, especially on some sulfidic mine tailings from metallic mines where exists low to high levels of acid generation, and even the ecological restoration of degraded mine tailings can be achieved in practice (M. Li, 2006; Wong, 2003). Table 2-2 lists the main measures taken for direct revegetation are to select appropriate amendments for tailings conditions amelioration and to select suitable plant species. Appropriate amendments for tailings could be utilized before seeding and planting to establish self-sustainable vegetation directly on mine tailings. The common amendments to the sulfidic tailings include: lime addition (Dean et al., 1973; Z. Ye et al., 2000); fertilizer supply (Hunter & Whiteman, 1975; Mulligan et al., 2006; Roseby et al., 1998); organic matter application such as wood chips, straw, biosolids, compost, and manure (Alcantara et al., 2015; Asensio et al., 2013; Mummey et al., 2002; Munshower, 2018). These practical examples proved that typical amendments could successfully improve the quality of sulfidic tailings and enhance the establishment of plants: the amount and frequency of lime addition can be adjusted flexibly depending on the acid generation potential of sulfidic tailings; fertilizer is important to supply necessary nutrients to plants; and the application of organic matter is to establish microbial communities in tailings to enhance plant growth. 16 Table 2-2 Amendment applications for successful direct revegetation on sulfidic tailings Tailings type Amendment applications Plants Reference Cu/Pb/Zn fly ash, N/P fertilizer, Mixed grass species (Hunter & Whiteman, 1975) Gold mine N/P/K fertilizer Native trees, shrubs, (Roseby et al., 1998; Mulligan et al., 2006) Uranium mine biomass, bacteria addition Native species (Mummey et al., 2002) Pb/Zn mine lime, fertilizer Native grass, trees (Z. Ye et al., 2000) Copper mine sludge Maritime pine, trees (Asensio et al., 2013) Gold mine 75% biosolids India mustard, carrot (Alcantara et al., 2015) As mentioned in the previous review of the revegetation on covers, the physical structure and hydraulic capacity should be created as well in tailings of direct revegetation not only for water supply to plants during the dry season but also for water infiltration in the rhizosphere during the rainy reason (Huang et al., 2012; Woodard et al., 2008). In practical cases, the effective amendments for the formation of macropores in fine-grained tailings include application of organic matter or minerals, such as Fe-oxides (Barral et al., 1998). Organic matter, as one critical amendment to enhance biological function of sulfidic tailings, can also help improve the physical structure and hydraulic properties in fine tailings because of its positive effects on the formation of stable aggregation (Caesar-TonThat et al., 2008; L. Huang et al., 2012). For example, woodchip, as one type of the organic matter, has beneficial effects on direct revegetation establishment on fine-grained tailings, because it can decompose at lower and stable rates in the natural environment and then form organic aggregates in tailings to provide cation exchange capacity for regulating nutrient buffering and supply to plants (Baldock et al., 2004; Morgenthal & Van Rensburg, 2004). Therefore, knowledge of organic matter on biological, physical and 17 hydraulic improvements in sulfidic tailings will help to select suitable combinations of organic matter with other tailings amendments. Table 2-3 Metals toxicity limits to soil, plant, and animal and the typical tailings metal concentrations Type As Cd Cu Mn Ni Pb Zn mg/kg Soil plant toxicity levelsa 15 3 200 3000 90 100-500 400 Plant leaf tissue toxicity limitsb 5-20 5-30 2-20 400-1000 10-100 30-100 100- U.S. domestic animal toxicity 30 10 40 2000 100 100 500 Typical sulfidic tailings d 11-651 2.6- 15.5-520 215-2400 0.6-92 11-4350 29- a based on total metal(loid) concentrations commonly toxic to plant growth (Mendez & Maier, 2008) b based on mean values of toxic levels of metals accumulated in agricultural drops c based on maximum tolerable levels for cattle (NRC, 2006) d data collected from sulfidic mine tailings that have been successfully revegetated. (Alvarenga et al., 2008; Favas et al., 2014; Forján et al., 2018; ; Gil-Loaiza et al., 2016; R. C. González & González-Chávez, 2006; Padmavathiamma & Li, 2009) Besides, some metal-tolerant plant species need to be selected carefully so that they can still survive and grow in the toxic environment of sulfidic mine tailings (see Table 2-3). Direct revegetation implementation focuses on choosing appropriate plant species for successful vegetation more than considering the interactions between plant roots and tailings materials, and effects on the metals geochemistry changes in tailings (Guittonny et al., 2016; Alcantara et al., 2015; Hine & Martin, 2016). The rhizosphere of plants is the zone surrounding the actively growing plant roots where the biogeochemistry of the soil is influenced by the roots (Hine & Martin, 2016), which is an extremely active zone of microbial activities and geochemical interactions involving changes to metals levels in tailings (Dighton & Krumins, 2014). Some plant roots have a detoxification function by decreasing the mobile metal concentrations in the 18 rhizosphere as a result of the uptake by the roots, or some plant roots may promote the metals leaching from tailings due to acidification by dissolved organic acid excreted by the roots (Kabata-Pendias, 2010). Based on the function of some plants species that are to be able to reduce the concentration of some contaminants including metals and the purpose of reducing the metal mobility in sulfidic tailings, the technology of phytoremediation has been developed and studied further than that of direct revegetation in the past two decades (Pilon-Smits, 2005). 2.4 Direct Revegetation vs. Phytoremediation The definition of phytoremediation, published by Oxford University, is “the use of different plant species that are able to grow on contaminated sites and remove high concentrations of heavy metals, or other toxic substances to decontaminate polluted land, water, or sediments.” (Hine & Martin, 2016) (Figure 2-1). Certainly, sulfidic mine tailings with elevated metal contamination are included. The term “phytoremediation” firstly appeared two decades ago (Salt et al., 1995), but there is currently no distinguishing difference in the literature between direct revegetation and phytoremediation. However, from definitions of the two terms, it is clear that for tailings reclamation, phytoremediation refers to removal of metal contaminants from tailings through plant uptake (Marques et al., 2009; Wang et al., 2017), whereas direct revegetation aims for surface vegetation and aesthetic recovery, regardless whether metals are taken up by the roots or not. Therefore, the most important character of phytoremediation distinguishing from direct revegetation is that phytoremediation aims to reduce and stabilize the mobile metals in soil thus being able to control the soil contamination. 19 Figure 2-1. Three potential outcomes of direct revegetation including the rhizosphere interactions related to metal release and root uptake Another difference is that plant species selection for direct revegetation can have a wider scope when compared to phytoremediation. Some plants may not be able to uptake a great number of metals but are nevertheless able to grow normally in extreme environments, which makes them good candidates for direct revegetation but not necessarily for phytoremediation (Conesa & Faz, 2011; Mulligan et al., 2006). The plant species for phytoremediation on sulfidic mine tailings have to be not only acid-tolerant but also metal-tolerant. Depending on the plant properties of 20 accumulating metals or stabilizing metals, five types of phytotechnologies have been developed: phytovolatilization, phytoextraction (phytoaccumulation), phytostabilization, phytodegradation, and phytofiltration. Phytovolatilization is the process which contaminants are taken up by roots and transferred to the leaves where they may be volatized through leaves stomata as gas compounds (Vroblesky et al. 1999). It is a type of detoxification strategy that some plants can transfer special toxic elements, e.g. Hg and Se, into less toxic and volatile compounds (Marques et al., 2009). Phytoextraction, or phytoaccumulation, is based on the same process where contaminants are absorbed by root uptake and then stored in harvestable parts of plants for future disposal or metal value recovery (Varun et al., 2015). This method can be cost-effective for value recovery by extracting metals from mine tailings thereby also reducing the tailings toxicity for future land development (Nkrumah et al., 2018; Wilson-Corral et al. 2011). Phytostabilization is the establishment of vegetation that can immobilize the metals in the rhizosphere where metals can precipitate and stabilize as stable minerals, or be absorbed and accumulated by roots without transfer to aboveground parts (Mendez & Maier, 2008). Phytodegradation is the degradation of organic contaminants through the roots exudates or metabolic activities within plant tissues (Newman & Reynolds, 2004). Phytofiltration is a wastewater treatment technique involving plant roots (rhizofiltration), shoots (caulofiltration), or seedlings (blastofiltration) to remove contaminants from contaminated waters (Olguín & Sánchez-Galván, 2012). 21 Table 2-4 lists some applications of the three phytotechnologies and suggests that phytoremediation needs tailings amendments to some extent as well. Table 2-4 Current approaches of phytoremediation for sulfidic mine tailings Method Tailings type Amendments type Examples Phytovolatilization Se contaminated soil in California Hydrated lime and nutrients addition (Banuelos & Meek, 1990); Acid-generating tailings in Japan Neutralization method (Sakakibara, Watanabe, Inoue, Sano, & Kaise, 2010); Phytoaccumulation & Phytoextraction Gold/ Copper mine tailings N, P, K fertilization Wilson et al. 2011; Lead/Zinc mine tailings N, P, K fertilization (Bing Yang, Shu, Ye, Lan, & Wong, 2003); Lead/Zinc mine tailings Biochar addition (G Fellet, Marmiroli, & Marchiol, 2014) Phytostabilization Copper mine tailings Compost and transplant Rosario et al., 2007; Gold/Silver/Cu/Pb/Zn mine tailings Compost addition Gil-Loaiza et al., 2016; Copper mine aged tailings Compost and biochar Forján et al. 2018; For differentiating the plant species suitable for use in phytostabilization and phytoaccumulation, the metal concentrations in plant parts can be expressed as the Translocation Factor (TF) and the Accumulation Factor (AF). The Translocation Factor (TF) is a measure of the plant’s ability to translocate metal from roots through shoots and leaves (Nirola et al., 2015): translocation factor = total element concentration in shoot tissuetotal element concentration in the root tissue Equation 2-2 The Accumulation Factor (AF) can be used to express metals accumulation in plants (Nirola et al., 2015): accumulation factor = total element concentration in shoot tissuetotal element concentration in mine tailings Equation 2-3 22 Unlike the plants suitable for phytoextraction or phytoaccumulation that should have TF and AF values both > 1, plant species used in phytostabilization should have TF and AF values both << 1 (Mendez & Maier, 2008). 2.4.1 Potential of Phytostabilization Although there are three techniques of phytoremediation, phytostabilization has the evident advantage than phytovolatilization and phytoaccumulation. On the one hand, the fate of the gas released by plants in the phytovolatilization containing the high risk elements volatilized by the plants is questionable and unpredictable (Pilon-Smits, 2005), so it may be a new consideration for tailings reclamation but it still needs more exploration by scientists. On the other hand, when phytoremediating sulfidic mine tailings, phytoextraction usually focus on accumulating one or two specific metals, such as Au, Ag and Cu (González-Valdez et al., 2018; Wilson-Corral et al., 2011), other metals with elevated concentrations would still stay in tailings and migrate along groundwater flow. Moreover, an important risk that needs to be considered with regards to phytoextraction or phytoaccumulation is that plants in the field should be carefully protected from animals, because their exposure and contact with animals may increase the metals bioavailability and the metals could enter into the human food chain (Mendez & Maier, 2008). Compared to phytoaccumulation which stores metals in the shoot tissues, phytostabilization mainly aims at stabilizing or sequestrating the metals within the soil and in the rhizosphere. The plants will reduce the mobile metals in the tailings through some interactions, and the exudates from the roots will in turn affect the physiochemical environment in the rhizosphere and enhance the heterotrophic microbial community. Bravin et al. (2012) reported that root-induced alkalization considerably altered Cu dynamic speciation in the most acidic soils (pH < 5.5) by 23 decreasing Cu2+ concentration and Cu mobility, and the dissolved organic matter concentration induced by roots resulted in a decrease of Cu2+ activity. The function of metal stabilization of phytostabilization will decrease the risk of metals pollution and promote plant growth for a long-term self-sustainable ecosystem. The research on phytostabilization of mine tailings is mainly reported with respect to plant species selection. Some potential candidates of plant species for successful phytostabilization on sulfidic mine tailings reported in typical literature are listed in Table 2-5, where the elevated metals and the low pH of these tailings are also indicated. These plant species for phytostabilization must be both acid-tolerant and metal-tolerant, and must not accumulate in aboveground parts, but can absorb metals in the roots and stabilize metals in the rhizosphere (Yang et al., 2016). These plants do not need to have the capacity to produce large aboveground biomass, but must grow quickly to set up a ground cover and maintain a relatively long life under field conditions (Marques et al., 2009). However, research for metal stabilization processes within the rhizosphere in tailings phytoremediation is limited. The area of roots-tailings interactions requires further research, which will definitely provide a better understanding of metals speciation and the long-term fate of metals in phytostabilization, as well as enhance the successful revegetation establishment on the tailings. Therefore, one important direction of enhancing phytostabilization is to research the rhizosphere processes in sulfidic tailings by growing the plant species which are potential candidates referenced in previous research (Table 2-5). 24 Table 2-5 Potential candidates of plant species for successful phytostabilization on sulfidic mine tailings in arid and humid regions Climate Region Speciesa Metals Tailings pH Reference Arid & semiarid Mexico Dalea bicolor Humb. Cd, Cu, Mn, Pb, Zn 3.96 - 7.06 (Gonzalez et al., 2006) Euphorbia sparsiflora A. Heller Isocoma veneta (Kunth) Greene Schinus molle L. U.S. Atriplex canescens (Pursh) Nutt. As, Hg, Mn, Pb < 3 - 4 (Rosario et al., 2007) Atriplex lentiformis As, Pb, Zn, Cd, Cu, Ni 2.5 (Gil-Loaiza et al., 2016) Bouteloua dactyloides Cercocarpus ledifolius Nutt. Prosopis L. Festuca arizonica Vasey Senegalia greggii Ecuador Bidens humilis H.B.K. Ag, As, Cd, Cu, Pb, Zn acid (Bech et al. 2002) Humid Canada Lolium perenne L. Cu, Pb 4.8 - 5.3 (Padmavathiamma & Li, 2009) Festuca rubra L. Mn Poa pratensis L. Zn Portugal Lolium perenne L. Cu, Pb, Zn 3.9 ± 0.2 (Alvarenga et al., 2008) Spain Brassica juncea L. Cu, Pb, Ni, Zn 2.73 - 3.78 (Forján et al. 2018) a in which metals accumulation in aboveground part does not exceed the US domestic animal toxicity limits (NRC, 2006) 2.5 Interactions in the Rhizosphere The rhizosphere is an extremely active zone of soil-roots interactions, including inorganic geochemical reactions and microbial activities (Dighton & Krumins, 2014). Four main 25 biogeochemical processes related to metal mobility and sequestration happen in the rhizosphere (see Figure 2-1): • lon exchange or complexation involving exchangeable ions or metals adsorbed/desorbed onto solid phase surfaces; • Precipitation and dissolution involving inorganic minerals precipitating or dissolving; • Silicate minerals weathering process unbinding and releasing metals; • Microbial activities may release metals bound in soluble or insoluble organic matter (Marques et al., 2009). Based on the above description, one of the reclamation purposes of sulfidic mine tailings is to prevent metals or toxic contaminants from being released to surrounding environment, which means that the method used to stabilize the metals in the tailings and to mitigate the leachable metals in the drainage of tailings should be considered in both either revegetation on covers or direct revegetation. Metal immobilization could be achieved using chemical covers in revegetation on covers. However, as direct revegetation rarely focuses on metals reduction in tailings, phytoremediation should be the main strategy to reclaim sulfidic mine tailings. At the same time, phytostabilization has more potential to safely reduce and immobilize metals than other two phytoremediation techniques. During phytostabilization, the task of metal stabilization should take the effects of the plant roots into consideration, as well as any impact from geochemical processes resulting from tailings amendments. 2.5.1 Geochemical Processes The success of direct revegetation covering of tailings depends on the nature of the material, the mineralogy, nutrients, and the organic matter contained in the soil. Mineral composition can be 26 retained even after revegetation, and the breakdown of primary minerals into secondary minerals and the formation of clay minerals can be detected around plant roots (Justine et al., 2014). The inorganic geochemical processes controlling metal mobility in the rhizosphere mainly include three types (Dighton & Krumins, 2014), namely: • Precipitation & Dissolution of inorganic minerals The precipitation and dissolution of minerals around roots in an unsaturated soil environment can largely change the hydraulic properties of the soil and the dependency of these changes on water contents, thereby, affecting water uptake by roots and plant growth (Wissmeier & Barry, 2009). In the natural field conditions of an Iron mine site, precipitation of dissolved Fe (III)/Fe(II) ions as Fe(oxy)hydroxides (ferrihydrite, hematite, magnetite) was observed in aged tailings, which were amorphous secondary minerals with a higher surface area that contributed to the decrease and immobilization of metals (Blowes et al., 2014; Wu et al., 2019). The addition of lime or other carbonate minerals, such as CaMg(CO3), FeCO3, can neutralize the acidic pH of the sulfides tailings and precipitate metals as carbonates (Sherlock et al., 1995). The neutralization of sulfuric acid generated in tailings can result in the precipitation of metal sulfates and reactions that decrease metal solubility (Blowes et al., 2014; Radziemska, 2018). After the AMD generation (Equation 2-1), sulfide minerals are oxidized and secondary minerals could include sulfates, carbonates, and hydrated oxides, depending on the pH and geochemistry of tailings (Blowes et al., 2014). The precipitation/ dissolution of minerals around the roots can enhance the competitive cation exchange between roots and the soil, thereby increasing the nutrient availability for plant growth. • Silicate minerals weathering process unbinding and releasing metals 27 Silicate-bearing minerals make up approximately 90% of the earth’s crust, and clay minerals (Al-bearing silicates) are the main constituents of soil, even in fine tailings. The weathering process of primary rock minerals produces clay minerals containing a series of variable composition of Al-oxides and Al-hydroxides, which contributes greatly to soil properties. Al-(oxy) hydroxides, having positively charged surfaces, can largely enhance the adsorption and flocculation of anions and negatively charged particles, and Al- montmorillonite has a complex ability to immobilize some free cations, such as Cd and Zn (Kabata-Pendias, 2010). Wu et al. (2019) indicated that the lack of Al-Si bearing minerals resulted in a higher mobile Fe concentration, regardless of the age of tailings. In general, gangue minerals coexisting with sulfidic deposits, such as porphyry ores and metallic ores, include a group of silicate minerals (KAlSi3O8–NaAlSi3O8–CaAl2Si2O8) and carbonates (limestone, dolomite, etc.) (Dold, 2014; Smuda et al., 2008), and secondary minerals of silicate minerals weathering include clay minerals and quartz (Blowes et al., 1994). • Adsorption & Desorption onto solid phase surfaces The adsorption and desorption of metals onto solid phase surfaces are the most important geochemical process controlling metal mobility in soil, including ion exchange and complexation. They are closely controlled by other geochemical processes in soil as described above. As an example, in the soil-plant system, the mobility of silver is closely related to the sorption of Ag nanoparticles and Ag+ onto soil particles. Sorption of both Ag nanoparticles and Ag+ increased at higher pH and silver nanoparticles are ultimately unstable and transform to Ag+, thus silver uptake by plants may present a human health risk (Saleeb et al., 2019). A competitive cation exchange process can allow the low-demand cations accumulating against roots, such as Ca2+, Mg2+ and Na+, to desorb the nutrients substrates (NH4+ and K+) from soil grains, and simultaneously, the competitive ion exchange between accumulation and desorption to increase 28 with net roots water uptake (Espeleta et al., 2017). Zheng et al. (2016) identified that the ion exchange as the predominant processes of metal sorption by water hyacinth roots, where Cu(II) and Cd(II) were individually removed by the release of protons and cations such as Ca2+ and Mg2+, despite the existence of a competitive ion exchange between Cu(II) and Cd(II). Complexation depends on the availability of the Fe-Al-Si bearing minerals in tailings, which can form amorphous secondary minerals with large surface areas. Therefore, the success possibility of metal immobilization in the rhizosphere will be increased through the addition of suitable amendments that can promote these three main geochemical processes, thus enhancing the effectiveness of phytostabilization. 2.5.2 Plant – Soil Interactions • Metals Uptake Plant growth closely relies on root uptake in the rhizosphere, including water uptake and nutrient uptake, both of which are necessary for normal plant growth (Dobermann et al., 1998; Grewal, 2010; Xinxin et al., 2016). However, roots also uptake metals when absorbing water and nutrients from the rhizosphere, which is particularly an issue in direct revegetation of sulfidic mine tailings containing high metals contents. Metal uptake by plants is a critical process that can immobilize the metals and reduce the environmental impact during direct revegetation. More specifically, metal uptake is the passive absorption by roots, as most of metals are not the necessary nutrients for plants growth (Kramer & Boyer, 1995). Compared to those necessary nutrients, such as P, K, S, Mg, Ca, Fe, and Na, with typical concentrations in the order of 10-3 M, heavy metals such as Mn, Cu, Co, Zn, Mo are required in much lower concentrations, typically in the order of 10-6 M (Ritcey, 1989). In acidic 29 soil conditions with a pH value below 5, the heavy metals will be more toxic to soil microorganisms and plants, therefore sulfidic tailings may be more toxic to plants than mobile metallic ions, as sulfide oxidation processes produce a large amount of metals (Guittonny-Larchevêque et al., 2016; Ye et al., 2002). As introduced above, the phytoextraction or phytoaccumulation can immobilize metals through metals uptake. Evidence has shown that metals accumulation and translocation of metals in plants can be promoted by the presence of organics, such as organic acid (Simon et al., 2001), metal chelates forming (Mohsin et al., 2019), addition of marble sludge (González et al., 2017). • Microbial Activities The interactions between plant roots and a great diversity of soil organisms are also important for plant growth and metal mobility in the rhizosphere. The various microorganisms in soil play an important role in the decomposition of organic matter, which varies greatly according to its origin and nature, as well as the climate to which it is exposed. Organic matter substrates are necessary nutrient requirements in soil, and they help construct the heterotrophic microorganism environment that helps plants grow. In some cases, root exudates (Figure 2-1) that can promote bacteria growing in rhizosphere help roots tolerate high metals and acidic pH in soil (Benidire et al., 2016). Root exudates contain released ions (i.e. H+), inorganic acids, oxygen and water, but mainly consist of carbon-based compounds (Baetz & Martinoia, 2014). These organic compounds can often be separated into two classes: low-molecular weight compounds, which include amino acids, organic acids, sugars, phenolics and an array of secondary metabolites, and high-molecular weight compounds like mucilage and proteins (Badri & Vivanco, 2009). Root exudates could increase the solubility of metals and their subsequent uptake (Hinsinger et al., 2006), and may increase microbial decomposition process to cause metals leaching (Mayer, 30 1998). The uptake of nutrients by plant roots, mostly as cations, can lead to the release of H+ in exudates from roots to maintain electroneutrality in soil (Goulding, 2016; Haynes, 1983; Wang et al., 2016). Krishnamurti et al. (1997) investigated the effects of root exudates on the kinetics of Cd release from the soil, and found increasing amounts of Cd were released from soil to the soil solution with the renewal of root exudates every 2 h. Ullah et al. (2015) reviewed that plant growth promoting (PGP) bacteria have positive influences on the phytoremediation of heavy metals. With regards to sulfidic tailings revegetation, many applications has been explored to establish a high-quality rhizosphere containing enough nutrients and organic matter, including microbial community pathway (Zhang et al., 2017), early seeding establishment (Araujo & Costa, 2013; Macdonald et al., 2017), fertilizer application (Asma et al., 2011; Brown et al.,2007; Phillips et al., 2016), and mineral and humid substances application (Adhe & Hasan, 2015). Huang et al. (2012) discussed tailings properties that limit remediation effectiveness and concluded that sustainable phytostabilization of tailings requires the systematic consideration of hydro-geochemical interactions taking place within the rhizosphere, which supports the idea of this research that geochemical processes taking place in sulfidic tailings through direct revegetation are essential to be further studied. The microbial community’s positive effects on roots nutrients uptake and plant growth have been studied extensively and identified by biologists. Microorganisms are also known to have an impact on water-solid geochemical processes that control metal mobility by both minerals and organic matter (Kabata-Pendias, 2010), so the geochemical evolution during tailing revegetation resulting from microbial activities and organic matter decomposition is able to directly illustrate the fate of metals mobility. However, the complexity of how inorganic geochemical processes 31 influence the metal mobility through sulfidic tailings revegetation has been studied less. The future focus of sulfidic tailings revegetation is necessary to be placed on the roots-soil interactions with regards to the inorganic geochemistry. 2.6 Discussion For the reclamation of sulfidic mine tailings, although revegetation on covers is a technology commonly applied nowadays which has been being gained continuous success in many cases, it can inevitably be expensive because of the significant requirement for earth crust materials. In contrast, the direct revegetation method by which plants are directly growing on the tailings can be more cost-efficient in terms of practical implementation. Many disadvantages prevent plant establishment directly on the sulfidic tailings, such as the deficiency of nutrient and organic matter, an extremely acidic environment, and metals toxicity for plants. Therefore, the plant species selection and tailings amendments are essential and these two areas have been studied much so far. The suitable selection of plant species is necessary for the survival and normal growth of plants under the poor tailings conditions, while tailings amendments are needed to ameliorate the tailings deleterious conditions into a consistent growing environment for plants. These are the two main concerns in regard to direct revegetation as they can influence the success of the direct revegetation system on sulfidic tailings. Greenhouse experiments and field trials can both be conducted to select the dominant species and the preferred amendments for tailings. By comparison, in the recent two decades, phytoremediation has been a focus more than direct revegetation, as the objective of the latter is solely to grow plants successfully. Because phytoremediation aims to decrease or stabilize the leachable metal concentrations in mine 32 tailings, it is more critical for the metal stabilization in sulfidic tailings. For example, field revegetation has been established successfully through phytostabilization on the Iron King Mine where is adjacent to the town of Dewey-Humboldt, Arizona in United States, and the sulfidic tailings left behind are characterized by low pH and nutrient contents, and elevated concentrations of a range of metal(loids) including arsenic, lead, copper, cadmium, chromium, and zinc as well as pyrite (Gil-Loaiza et al., 2016). Table 2-5 actually shows several successful examples of phytostabilization which also achieved the purpose of metals reduction in sulfidic mine tailings. Figure 2-2 organizes and clarifies the relationship between reclamation, revegetation and phytoremediation. As discussed above and shown in Figure 2-2, reclamation is a general term for the rehabilitation of tailings, including sulfidic mine tailings. If the ultimate aesthetic recovery of the tailings surface is required, the revegetation should be implemented through either revegetation on covers or direct revegetation approach. Since the direct revegetation is more material-saving and cost-effective than revegetation on covers, the direct revegetation approach has become increasingly attractive in the reclamation of all tailing types. Regarding sulfidic mine tailings with elevated concentrations of toxic metals, the decrease of mobile metals in tailings is necessary and direct revegetation is considered as phytoremediation which aims to reduce the metal mobility in sulfidic tailings. At the end of Figure 2-2, the listed three techniques of phytoremediation can serve different purposes in the field implementation. According to Figure 2-2, land reclamation and remediation practitioners need to systematically distinguish objectives of the reclamation, thus choosing the appropriate strategy for the project when designing the reclamation plan for sulfidic tailings. 33 Figure 2-2. Relating reclamation, revegetation and phytoremediation. According to the definition of the three phytoremediation techniques, phytovolatilization and phytoextraction will accumulate metals in the aboveground part of plants while phytostabilization will stabilize the metals in the rhizosphere (Figure 2-1). The long-term protection of the plants from wildlife intake of phytovolatilization and phytoextraction on large surface area of tailings cannot be secured in practical implementations, so phytovolatilization and phytoextraction still have a long way before they would be widely applied in industry. The application of phytostabilization has the potential to be promoted in the area of sulfidic tailings revegetation in the future because phytostabilization can immobilize metals in the rhizosphere, and its effectiveness can be enhanced by applying some appropriate amendments. Reclamation Physical cover Chemical cover Vegetation cover Revegetation on Covers Direct Revegetation Revegetation Phytoremediation aiming to reduce the metal mobility in tailings Phytostabilization Phytoextraction Phytovolatilization Multilayered covers to reclaim sulfidic tailings Require ultimate aesthetic recovery Establishment of vegetation cap without considering reduction of metals mobility Cost-effective Sulfidic mine tailings 34 Figure 2-1 illustrates that the rhizosphere is an extremely active zone of inorganic geochemical reactions and microbial activities, implying that direct revegetation on sulfidic tailings through phytostabilization can be enhanced by promoting these geochemical reactions related to metals immobilization. For sulfidic tailings, the addition of lime or limestone can not only neutralize the acidity but also precipitate the heavy metals as carbonates; Fe/Mn materials such as red mud, which is an alkaline bauxite processing waste, can promote the adsorption and complexation of the metals with Fe/Mn oxides while silicate minerals weathering can release some binding metals. Microbial activities can sometimes enhance roots uptake. When the plant species for phytostabilization is selected appropriately, metals will be stored in plant roots and stabilized through various bio-geochemical reactions in the rhizosphere. Even though the decomposition of fallen leaves may release a small amount of metals into the surrounding soil (Silva et al., 1998), decomposing tissues of detritus can provide more adsorption sites for stabilizing metals (Antoniadis et al., 2017). Therefore, the rhizosphere of direct revegetation likely acts as an efficient biogeochemical barrier for controlling metals transport from tailings to groundwater, which means that phytostabilization that does not translocate metals to aboveground parts of plants can create a more environmentally friendly system of direct revegetation. Despite plant species selection and tailings amendments, a better understanding of metals geochemistry in the rhizosphere may help create a more efficient system of phytostabilization. Metal mobility in the rhizosphere of revegetation system on tailings is closely relevant to microbial activities, roots uptake and exudates, and geochemical reactions, but the geochemical evolution during tailings revegetation actually results from microbial and roots effects. 35 Current research about revegetation or phytoremediation is mostly related to plant species selection and tailings amendments, either in greenhouse experiments or in field applications. For the research in the rhizosphere, the study of microbial activities has also received much focus than other aspects. Since research on geochemical processes involving metal immobilization that take place in sulfidic tailings through direct revegetation or phytostabilization has received much less attention, this direction is still valuable to be studied further in the future for better understanding metals behaviors, thus enhancing metal immobilization in sulfidic tailings revegetation. The interactions between roots and tailings materials in the rhizosphere can lead to a better understanding of the metal release and transformation, which can then provide information about the long-term geochemical evolution of heavy metals. This can consequently enhance the effectiveness of phytostabilization that is permanently stabilizing metals and reducing environmental impacts caused by metals transport in sulfidic tailings. 2.7 Conclusion In this chapter, the literature of reclamation of sulfidic tailings is reviewed and the relationship of reclamation, revegetation and phytoremediation is proposed progressively. Reclamation is a general strategy of the rehabilitation of tailings. When it comes to reclaiming sulfidic mine tailings, three types of covers, including physical cover, chemical cover and vegetation cover, can be applied separately on the tailings or combined as multilayered covers. Revegetation is a strategy included in reclamation and it would be necessary when the ultimately aesthetic landscape recovery of the surface of tailings storage facility is required. Moreover, compared to revegetation on covers that can be considered as multilayered revegetation, direct revegetation is more material-saving and cost-effective. Two aspects, including plant species selection and effective amendments selection, should be implemented before the direct revegetation in the 36 field on the sulfidic mine tailings to improve the success. Phytoremediation is a concept included in the direct revegetation, aiming to reduce the metal mobility in tailings and minimize the potential environmental pollution. Roots-tailings interactions happening in the rhizosphere include organic-related microbial activities and inorganic geochemical processes. The key research of inorganic geochemical processes should be received more attention, and it is expected to be an important direction in the research area of sulfidic mine reclamation, especially in regards to the effects of direct revegetation on toxic metal mobility in sulfidic tailings. The study of geochemical processes in the rhizosphere can provide a reference to the amendments selection during phytoremediation; thus, the application of phytostabilization in the future can be more valuable and environmentally friendly with regards to the promoted metals immobilization. 37 Chapter 3: Materials and Methods 3.1 Introduction This chapter introduces the materials, the design of greenhouse experiments, sample collection, analytical indicators, and statistical analyses. Figure 3-1 shows the research workflow. This research started with the sulfidic mine site selection – a porphyry Pb-Zn mine with a high sulfidic mineral content in a tailings storage pond that poses a risk to the environment through the potential release of metals to the surrounding area. Tailings characterization is following samples collection. The greenhouse experiments were conducted after species and material acquisition. Data collection included the water chemistry of drainage, metal distribution in plants, and soil characterization. Species selection and amendment efficiency were analyzed based on the metal distribution in plants and the tailings characterization (Chapter 4), and the geochemical processes in the rhizosphere were analyzed based on drainage chemistry and soil characterization (Chapter 5). Chapter 6 shows the reliability of phytostabilization on sulfidic mine tailings based on the results observed from the greenhouse experiments. The conclusions and suggestions are presented in Chapter 7. 38 Figure 3-1. Highlights of this chapter in the research workflow Greenhouse conditions are thought to be the greatest confounding factor to guarantee experimental success. In the greenhouse experiments, the following were collected: • Drainage samples from each pot weekly; • Samples of plants and roots after one growing season for assessing the growth; • Soil samples in each pot. The data were used to examine: Mine Site Background Data Collection in Progress Greenhouse Experiments Native Species and Amendments Acquisition Tailings Acquisition Tailings Characterization Reliability of Phytostabilization Data Collection after experiments Drainage Chemistry Metals in Plants pH & Metals in Soil Geochemical Processes in the Rhizosphere Species Selection and Amendments Efficiency Conclusions and Suggestions Materials Characterization before experiments Pots Experiments Setup 39 • If the selected species can stabilize some heavy metals in the rhizosphere and be the potential species candidates for phytostabilization; • How concentrations of heavy metals change in the drainage resulting from various amendments to phytostabilization on sulfidic tailings; • How different amendments can enhance the efficiency of direct revegetation by changing tailings conditions; • How geochemical reactions control metal mobility and enhance the phytostabilization. 3.2 Materials acquisition 3.2.1 Tailings Tailings were collected from the Faro Mine Site, Yukon, Canada, which was a Zn and Pb mine located 14 km north of the town of Faro and 200 km north-northeast of Whitehorse. The deposit is lead-zinc-silver strati from pyritic massive sulfide deposits, where the dominant sulfide minerals are sphalerite, galena, and pyrite, along with locally abundant chalcopyrite, pyrrhotite, and marcasite. Gangue minerals include barite, magnetite, and Ba-Fe-Mg carbonates; however, carbonate minerals are not abundant. Inter-sample variability may exist as the tailings geochemistry report indicated that pyrite content of the tailings varies in the range of 25 – 41 % due to heterogeneity (SRK Consulting, 2019). The Faro Mine produced lead and zinc concentrate starting in 1992 and milling stopped in February 1998. Residue gangue and tailings were deposited in the Rose Creek Tailings Area between September 1969 and August 1992. Tailings were subsequently deposited in the Original Impoundment (1969 – 1975), the Secondary Impoundment (1975 – 1982), and the Intermediate Impoundment (1986 – 1992). The surface tailings in the Original and Secondary Impoundments 40 are generally free-draining and groundwater levels are typically in the range of 5 to 10 metres below the surface. Tailings above the groundwater table are unsaturated to partially saturated, and the surface of the Original and Secondary tailings has been exposed to oxidation for at least 43 and 36 years, respectively. Ponded water and/or finer and more saturated tailings resulted in more limited exposure of the tailings within the Intermediate Impoundment. Tailings samples were collected from a 2 – 3m depth in the Intermediate Impoundment, where the pH was around 5. The sampling location is shown in the layout of the tailings storage pond (Figure 3-2). Tailings (25 L) were collected with an excavator and then sealed in plastic buckets (lined with polyethylene bags) to prevent oxygen diffusion, water seepage, and external contamination. Figure 3-2. Tailings sampling location (red triangle) at tailings storage ponds at Faro Mine 41 3.2.2 Bauxite residue, compost, and lime Amendments were selected based on expected pH adjustment and organic matter addition for sulfidic tailings (Table 2-2 and Table 2-4), as well as the accessibility of the materials. Bauxite residue, lime, and compost were chosen for the greenhouse experiments. Bauxite residue, a by-product of alumina extraction from bauxite containing rich surface metals oxide phases, such as Fe/Al oxides, can immobilize metals (Lee et al., 2014). The sample of bauxite residue was obtained from Rio Tinto Alcan’s Arvida R&D Center in Jonquiere, Canada. Lime or limestone is usually applied in the revegetation of acid-generating tailings (Table 2-2 and Table 2-4). Lime (CaO) was added as one of the inorganic amendments, and it was purchased from Fisher Scientific, Canada. Organic matter is critical for microbial community establishment in the soil to promote plant growth. The influence of organic matter on metal mobility in the rhizosphere is also considered by adding compost. The material compost was obtained from UBC Building Operations. Tailings Bauxite residue Lime Compost Figure 3-3. Tailings, bauxite residue, lime and compost Table 3-1 lists the rinse pH of the original materials before mixing. Lime was not tested for pH because the pH of hydrated lime can be obtained from literature, which is 12.4 (Al-Mukhtar et al., 2010). Results show bauxite residue has the second highest pH with an average of 10.85, and compost is slightly alkaline with an average pH of 7.59. 42 Table 3-1. Rinse pH (solid:water =1:5) of tailings, bauxite residue, and compost before mixing Rinse pH Repetition 1 Repetition 2 Repetition 3 Average pH Tailings 4.71 4.60 4.77 4.69 Bauxite residue 10.85 10.79 10.92 10.85 Compost 7.54 7.58 7.56 7.59 3.2.3 Material Characterization and Testing Table 3-2 lists the physiochemical characteristics of tailings, and the details of testing methods are listed in Appendix A. The objective of acid-base accounting is to determine the inventory of potential acid generation and neutralizing materials, which is essential for requirement of liming amendment in the greenhouse experiment. Table 3-2. Physical properties and chemical characteristics of tailings Physical properties Particle size distribution Hydraulic conductivity (m/s) CEC (meq/100g) Porosity Bulk density (g/cm3) Clay (%) Silt (%) Sand (%) Gravel (%) 0.89 16.4 74.67 8 4.7E-06 32 0.14 1.619 Acid base accounting (kg/t CaCO3) Total S Sulfate S TIC AP NP NAP NP:AP 180 3.3 1.8 176.7 149.4 27.3 0.85 Total metals (mg/kg) Al As Cu Fe Mg Mn Pb Zn 4700 630 1000 150000 2000 1500 6000 14000 *NAP is net acid generation potential which is tested by an acid-base accounting static test. AP is the acid generation potential and NP is the acid neutralization. NAP = AP – NP. 43 Table 3-3. The quantitative XRD results of tailings and bauxite residue Mineral Ideal Formula Tailings (%) Bauxite Residue (%) Quartz SiO2 26.4 4.2 Anglesite PbSO4 0.5 Barite BaSO4 2.1 Celsian Ba(Al2Si2O8) 1.1 Chalcopyrite CuFeS2 0.2 Galena PbS 0.2 Gypsum CaSO4∙2H2O 0.9 Muscovite KAl2AlSi3O10(OH)2 2.5 Kaolinite Al2Si2O5(OH)4 1.9 Marcasite FeS2 8.9 Pyrite FeS2 43.2 Pyrrhotite Fe1-xS 2.2 Siderite Fe2CO3 7.6 Sphalerite ZnS 2.3 Anatase TiO2 4.6 Böhmite AlO(OH) 4.9 Calcite CaCO3 3.1 Gibbsite Al(OH)3 1.8 Goethite (Fe,Al)O(OH) 30.6 Hematite Fe2O3 39.4 Rutile TiO2 1.2 Sodalite Na4(Si3Al3)O12Cl 10.2 Total 100.0 100.0 The results of quantitative XRD showed the percentage of minerals of tailings and bauxite residues (Table 3-3). The dominant sulfide mineral in tailings was pyrite (FeS2, 39.2 %), followed by marcasite (FeS2, 8.9 %), pyrrhotite (Fe1-xS, 2.2 %), and sphalerite (ZnS, 2.3 %), and there was a small amount of chalcopyrite (CuFeS2, 0.2 %) and galena (PbS, 0.2 %). Siderite (Fe2CO3) was the only carbonate mineral (8.9 %). The secondary sulfate minerals include barite 44 (BaSO4, 2.1 %), anglesite (PbSO4, 1 %) and gypsum (CaSO4∙2H2O, 0.9 %). These results were slightly different from the geochemical report written by (SRK Consulting, 2019), likely because of the heterogeneity in Faro mine wastes (Bao et al., 2020). Bauxite residue (red mud) is the slurry-solid waste generated from aluminum production. The QXRD analysis of the bauxite residue sample from Rio Tinto Canada found that the hematite (Fe2O3, 39.4 %), goethite binding with aluminum ((Fe3+,Al)O(OH), 30.6 %), and sodalite (Na4(Si3Al3)O12Cl, 10.2%) are the main phases and the other minerals included böehmite (AlO(OH), 4.9 %), anatase (TiO2, 4.6 %), quartz (SiO2, 4.2 %), calcite (CaCO3, 3.1 %) and gibbsite (Al(OH)3, 1.8 %). The composition of minerals in bauxite residue is consistent with that reported previously by others (Anawati & Azimi, 2019; Klauber et al., 2011; Reid et al., 2017). 3.2.4 Species Selection On-site plant investigation typically precedes tailings revegetation, especially near waste dumps and tailings ponds. As a result, several dominant types of species were considered to improve germination and survival potential on tailings: wheat (Triticum aestivum), ryegrass (Lolium multiflorum.), tall fescue (Festuca arundinacea), and sheep fescue (Festuca ovina) (Table 3-4). Seeds of these species were purchased from West Coast Seeds, Canada. Wheat Ryegrass Tall fescue Sheep fescue Figure 3-4. Selected grass species 45 3.3 Greenhouse Experiments 3.3.1 Three stages of greenhouse experiments • 1st stage: substrate preparation and pot installation Six substrates, one of the pure tailings and five amendments, were employed (Table 3-4). The experiment with pure 100 % tailings without any amendment was designated as a control group and named substrate Ⅰ. The addition of 10 % bauxite residue and the addition of 1.25 × NAP of lime to tailings, the inorganic amendments, constitute substrate Ⅱ and substrate Ⅲ, respectively. The 20 % compost was applied as the organic amendment to support the microbial community in the rhizosphere and provide nutrients to plants. Substrate Ⅳ consisted of 20 % compost and 80 % tailings. Substrate Ⅴ and substrate Ⅵ were made by adding 20 % compost to substrate Ⅱ and substrate Ⅲ, in which tailings were already amended by inorganic amendments: bauxite residue and lime. The rationale of amendment composition in Table 3-4 is amending the tailings with one type of pH adjustment, one type of organic matter adjustment, or both of them. Lime and bauxite residue are both regarded as pH adjustments, and the compost is regarded as organic matter. Therefore, the substrates of tailings simultaneously amended with two types of pH adjustments were not designed, such as T+BR+L and T+BR +L+C. Net acid production provides a quantitative estimation of acidity that can be generated by the sulfide oxidation in mine wastes, is expressed as CaCO3 kg/t, and is determined by acid-base accounting analysis (Appendix A: The Parameters and Testing Methods). The amount of lime requirement as one of the tailing amendments is calculated as 1.25 × NAP (CaCO3 kg/t), and the additional 25 % of NAP accounts for the tailings heterogeneity and potential acidity in the organic materials (Boyter et al., 2009). When transferring the weight of NAP in CaCO3 to CaO 46 (lime), multiply by 0.56, which was about 0.1 g lime added into 5 tailings and it was 2 % lime mixed with tailings. Most lime amendments in previous reclamations were applied over limestone and this quantity was controlled in this study to lower costs. The controlled amount of lime also kept the pH of amended tailings (T+L, T+L+C) not too alkaline to allow the plant growth. Table 3-4. The composition of amendments. Substrates Tailings (w/w) Bauxite residue (w/w) Lime Compost (w/w) T* 100% T+BR 90% 10 % T+L 100% 1.25 × NAP T+C 80% 20 % T+BR+C 70% 10 % 20 % T+L+C 80% 1.25 × NAP 20 % *Name of substrates: T = 100 % tailings, T+BR = 90 % tailings +10 % bauxite residue, T+L = 100 % tailings + 1.25 NAP of lime, T+C = 80 % tailings +20 % compost, T+BR+C = 70 % tailings + 10 % bauxite residue + 20 % compost, T+L+C = 80 % tailings +1.25 NAP of lime +20 % compost Figure 3-5. Photos of six substrates T T+C T+BR T+BR+C T+L T+L+C 47 There were totally 50 pots installed: 5 for substrate Ⅰ and 9 for each substrate from substrate Ⅱ to Ⅵ. On each substrate, one pot is blank (no grass) and two pots were included for each grass species (Table 3-5). The pot size is 9 cm (top) × 6 cm (base) × 8.5 cm (height), so the volume of tailings needed for each pot is 1.5 L. After the installation of pots in the greenhouse, substrates in each pot were left undisturbed for five days to allow the mixed materials and the different particles to settle before the seeding. Table 3-5. The installed pots for six substrates and four grass species Substrates WG* RG TF SF Blank Ⅰ T ×1 ×1 ×1 ×1 ×1 Ⅱ T+BR ×2 ×2 ×2 ×2 ×1 Ⅲ T+L ×2 ×2 ×2 ×2 ×1 Ⅳ T+C ×2 ×2 ×2 ×2 ×1 Ⅴ T+BR+C ×2 ×2 ×2 ×2 ×1 Ⅵ T+L+C ×2 ×2 ×2 ×2 ×1 * Name of grass species: WG = Wheat, RG = Ryegrass, TF = Tall Fescue, SF = Sheep Fescue 48 Table 3-6. Primary purposes for the experimental design Design Purpose Four grass species Select potential grass species candidates for phytostabilization of sulfidic mine tailings. Grass growth vs. blank Assess the effects of planting on geochemistry and metal mobility in the rhizosphere. Ⅰ vs. Ⅱ Assess the effects of bauxite residue on the geochemical reactions of metals immobilization. Ⅰ vs. Ⅲ Assess the effects of lime on the geochemical reactions of metals immobilization. Ⅰ vs. Ⅳ Assess the effects of compost on metals immobilization, which includes organic reactions that are not intended in this research but can serve as the controls trial for outcome comparison and analysis. Ⅱ vs. Ⅴ Assess the effects of compost, including organic matter and some nutrients addition on bauxite residue amended tailings, in terms of metals immobilization, e.g. how do the additions promote or reduce metals stabilization? Ⅲ vs. Ⅵ Assess the effects of compost, including organic matter and some nutrients addition on lime amended tailings, in terms of metals immobilization, e.g. how do the additions promote or reduce metals stabilization? Comparisons of the five substrates Select effective amendments composition of enhancing grass growth and metals immobilization. Comparisons of all pots Conclude the geochemical processes controlling metal immobilization in the rhizosphere through phytostabilization on tailings • 2nd stage: plant growing and drainage collection Substrates were placed in pots on February 19th, 2021, let settle for five days, and the seeds were sowed on February 24th, 2021. In each pot, 16 seeds of each species were sowed at 3 cm intervals, and buried at depths of 0 to 2 cm below the surface, based on recommended seeding rates for mine reclamation (Guittonny-Larchevêque et al., 2016). 49 All pots were watered by misting once per day with tap water to keep the 1 – 2 cm depth moist 16 days, after which no seed emergence was observed. At this time, the numbers of germinated seeds in each pot were recorded and the first batch of drainage samples was taken on March 12th, 2021. Following germination, pots were carefully hand-watered every other day to keep substrates hydrated. Nine batches of drainage samples were taken each week in the following 9 weeks. Therefore, there are 10 batches of drainage being collected in total, and the last batch of samples was on May 14th, 2021. Table 3-7. Timeline of the 2nd stage of greenhouse experiments in 2021 Pots installed Seeds sowed 10 batches of drainage samples 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th Feb 19th Feb 24th Mar 12th Mar 19th Mar 26th Apr 2nd Apr 9th Apr 16th Apr 23th Apr 30th May 7th May 14th *The total time duration of experiments was 84 days. Figure 3-6. Photos of the experiment Greenhouse conditions were controlled to optimize plant and root growth. The total time of the experiments was 84 days. It was 79 days from sowing to harvesting, which is a typical growing season (Bélanger et al., 1992; Tadych et al., 2014; Tadych, Bergen, & White Jr, 2014). This duration is a reasonable period for greenhouse experiments in mine tailings revegetation, especially those dealing with grass species (Alcantara et al., 2015; Leung, 2018; Mendez et al., Pots installation Seeds sowing Drainage collection 50 2007). The minimum drainage volume of 100ml from each pot was needed weekly for analyzing the water chemistry. Table 3-8. Greenhouse environmental conditions, provided by the UBC Horticulture Greenhouse. Climate Conditions Day* (7:00am-10:00pm) Night (10:00 pm -7:00 am) Air Temperature 23 ± 3°C* 19 ± 2°C Growing Light 1120 ± 500 J/cm2 day CO2 440 ± 10 ppm Relative Humidity 50% ± 10% Watering Hand watering every second day** None *Day and night temperatures depend on weather and become progressively warmer entering the Summer. **Pots are watered to keep hydrated but not saturated as excess drainage could flush away and alter original metal concentrations. • 3rd stage: harvesting and soil sampling After the last collection of drainage samples, the heights of plants, from the soil surface to the tip of the longest tissue, were recorded (Figure 3-7). Then, the aboveground tissues were cut at the soil surface and harvested. T-WG T-RG T-SF T-TF 51 T+BR WG T+BR TF T+BR RG T+BR SF T+L WG T+L RG T+L TF T+L SF T+C WG T+C RG T+C TF T+C SF 52 Figure 3-7. The growth of all plants after the last batch of drainage collection on May 14th, 2021 Step 1: dry pot samples in oven at 30°C Step 2: separate the soil and roots Step 3: wash the roots and then oven dry at 80°C Figure 3-8. The procedures for collecting the root samples The soil in each pot was collected in large plastic bags and labeled, and then all the soil samples were sent to the lab for oven-drying at 30 °C for three days (Figure 3-9). The next step was separating the roots from the dry soil sample manually. After being washed with distilled water, T+BR+C WG T+BR+C RG T+BR+C TF T+BR+C SF T+L+C WG T+L+C RG T+L+C TF T+L+C SF 53 roots and aboveground tissues were oven-dried at 80 °C. The dry biomass of roots and tissue samples was recorded. Last, all the samples of soil, aboveground tissue, and roots were labeled and sent to the BC analytical lab in the BC Ministry of Environment, Victoria, BC, for metal analysis. 3.3.2 Monitoring and testing Table 3-9 lists the total number of samples for each type, including the characteristics of tailings and bauxite residue, water chemistry for collected drainage, trace metals analyses in plants shoots and roots, and characteristics of the soil after the experiments. Table 3-10. Numbers of sample collected from the greenhouse experiments Before the seeding Characteristics of tailings and bauxite residue Particle size distribution Quantitative XRD 1 2 During the process Chemistry for collected drainage pH Leachable trace metals Major cations and anions 280 280 280 After the harvest Trace metals analysis in plant shoots and roots 6 substrates × 4 species × 2 repetitions × 2 parts (shoots and roots) = 96 Characteristics of pot substrates Total metals pH Quantitative XRD Qualitative XRD 50 50 4 24 Table 3-10 lists the purpose of each indicator during different stages. The testing methods and instruments used for all parameters are listed in Appendix A. Patterns of qualitative XRD results of representative samples are listed in Appendix B. Charge balance of drainage samples was checked before analyzing changes in metal concentrations of drainage. 54 Table 3-11. Tested indicators and purposes in this research Indicator Purpose Before the experiments pH of materials Characterization of materials Particle size distribution of tailings Characterization of tailings Hydraulic conductivity of tailings Characterization of tailings Cations exchange capacity of tailings Characterization of tailings Porosity of tailings Characterization of tailings Bulk density of tailings Characterization of tailings Acid base accounting of tailings Characterization of tailings Quantitative XRD of tailings and bauxite residue Minerals percentages of tailings and amendments pH of mixed substrates Characterization of amended tailings During the experiments pH and metals in drainage Characterization of changing trends in drainage After the experiments Trace metals in plant shoots and roots Metal distribution in plant bodies pH of pot substrates Characterization of pot substrates at the end Metals in pot substrates Characterization of pot substrates at the end Quantitative XRD of blank group of T, T+L, T+BR, T+C Comparison with the QXRD data before experiments Qualitative XRD of the remain pot substrates Mineralogy of the remain pot substrates 3.3.3 Statistical analysis Two-way analysis of variance (two-way ANOVA) was used to compare the effects of amendments and species on plant growth, metal uptake by plants, pH and metals in pot substrates, and the total metals in drainage, where significance was assessed at p < 0.05 were obtained. The results of two – way ANOVA are attached in Appendix C: Results of two – way ANOVA. Principal Component Analysis (PCA) was used to compare changes in the amendments based on geochemical data. To reduce influences from outliers and variable data 55 ranges, data were square-root transformed before performing the PCA. All analyses were conducted using SPSS (IBM SPSS Statistics 28.0.0.0). 56 Chapter 4: Species Selection and Amendment Effectiveness for Phytostabilization2 4.1 Introduction Phytostabilization is the establishment of a vegetation cap that can immobilize heavy metals in the rhizosphere, where the metals can precipitate and stabilize, or be absorbed and accumulated by roots. An aboveground canopy can reduce eolian dispersion, and the roots prevent water erosion and metals mobilization (Mendez & Maier, 2008). Compared to phytoaccumulation, which stores metals in shoot tissues, phytostabilization aims at stabilizing or sequestrating the heavy metals within the rhizosphere. Phytostabilization can help prevent animals from being poisoned by eating aboveground parts with high levels of metal concentrations. For successful phytostabilization at mine sites, species and amendment material selection are two principal aspects explored by researchers. This is because most plants cannot grow in extremely acidic media and toxic heavy metal tailings negatively affects plants’ roots and shoots (Mulligan et al., 2006; Roseby et al., 1998; Xu et al., 2008; Ye et al., 2002). Plants intended for phytostabilization should be carefully selected to ensure that the concentration of toxic metals 2 A version of this chapter has been published. Xie, L. and van Zyl, D., 2023. Identification of grass species candidates for phytostabilization and enhanced metal(loid)s immobilisation using cost-effective amendments on sulfidic mine tailings. International Journal of Mining, Reclamation and Environment. DOI: 10.1080/17480930.2023.2200322. The section of Materials and Methods is not included in this chapter because the details of experiments and materials were introduced in Chapter 3. 57 does not exceed their tolerance (Bech et al., 2002; Farahat & Galal, 2018; Guittonny et al., 2016). Tailings amendment methods are also critical for creating a suitable and nutrient-sufficient environment for revegetation (Alvarenga et al., 2008; Araujo & Costa, 2013; Phillips et al., 2016; Radziemska et al., 2017). Organic matter and fertilizer are necessary inputs to revegetation on tailings due to the lack of nutrients and microbial communities in tailings (Ploughe et al., 2021; Juge, Cossette, Jeanne, & Hogue, 2021). Especially for sulfidic mine tailings, controlling acidity is also important. Conditions of sulfidic mine tailings can be too deleterious to plants, so the selection and the effectiveness of amendments should be evaluated. This chapter assesses the candidacy of four native grass species: wheat (Pascopyrum), ryegrass (Lolium), tall fescue (Festuca arundinacea), and sheep fescue (Festuca ovina) for phytostabilization, as well as the effectiveness of amendments in improving phytostabilization on sulfidic mine tailings. Figure 4-1 indicates the evaluation of species selection and amendments efficiency. Therefore, this chapter asks two sub-questions : • What species can be candidates for phytostabilization? • What amendments can promote growth as well as stabilize metals in the rhizosphere? The objectives of this chapter are: • To compare the effectiveness of different amendments in enhancing plant growth; • To evaluate the candidacy of the four selected plant species used in the greenhouse trials in terms of phytostabilization on sulfidic tailings. 58 This information is significant to provide a reference for land reclamation and remediation practitioners when considering the application of phytostabilization at sulfidic mine tailings. Figure 4-1. Highlights of this chapter in the research workflow 4.2 Material characterization 4.2.1 Metals in tailings The concentrations of four elements: As, Cu, Pb, and Zn were 630, 1000, 6000, and 14,000 mg/kg, respectively, which are above the level of industrial soil quality regulated by the Mine Site Background Data Collection in Progress Greenhouse Experiments Native Species and Amendments Acquisition Tailings Acquisition Tailings Characterization Reliability of Phytostabilization Data Collection after experiments Drainage Chemistry Metals in Plants pH & Metals in Soil Geochemical Processes in the Rhizosphere Species Selection and Amendments Efficiency Conclusions and Suggestions Materials Characterization before experiments Pots Experiments Setup 59 Canadian Council of Ministers of the Environment (CCME, 2014), the soil plant toxicity levels, and plant leaf tissue toxicity limits (Table 4-1). The Zn concentration (14,000 mg/kg) was 35 times the level of soil plant toxicity limits (400 mg/kg), and As concentration was 42 times the As toxicity limits. The concentrations of Pb and Cu in tailings were 5 times and 12 times the plant toxicity limits, respectively (Mendez & Maier, 2008). Phytotoxicity depends on the plant species and the stage of the plant, but it has been reported widely that high levels of heavy metals in soil are toxic to plants (Nagajyoti & Lee, 2010; Yadav, 2010). Therefore, species selection in the greenhouse or at a small scale is necessary before establishing the revegetation in the field. Table 4-1. Metal concentrations in tailings compared to different limits Type As Cu Pb Zn mg/kg Tailings 630 1000 6000 14000 Industrial Soil Quality (CCME, 2014) 12 91 600 360 Soil Plant Toxicity Levelsa 15 200 100-500 400 Plant Leaf Tissue Toxicity Limitsb 5-20 2-20 30-100 100-400 a, b Cited from (Mendez & Maier, 2008). 4.2.2 The pH of materials The pH is an important factor affecting the metal bioavailability and metal uptake by plants because soil with higher pH can bind more metals to make them less available to plants (Reijonen & Hartikainen, 2016). As heavy metals tend to be released from minerals under acidic conditions (Nieva et al., 2018; Hammarstrom & Smith, 2002), increasing the pH of tailings by application of amendments can reduce the possibility of metal mobility, thus promoting the survival and growth of plants through tailings revegetation (Xie & van Zyl, 2020). 60 Table 4-2 lists the rinse pH of materials before mixing and the substrates after mixing. The tailings are acidic with an average pH of 4.69, and the bauxite residue is alkaline with an average pH of 10.85. The average pH of compost is 7.59, slightly alkaline. The average pH of mixed substrates was: 8.43 in T+L, 7.97 in T+L+C, 6.55 in T+C, 6.09 in T+BR and 6.22 in T+BR+C. The pH results indicated that applying bauxite residue, lime, and compost into tailings can increase the pH and neutralize the acidic tailings. Mixing 20 % compost with tailings can increase pH to around 6, but only 10 % of bauxite residue was needed to raise pH to 6.1. A small amount of lime has the most effective and intermediate neutralization ability by increasing the tailings pH (~ 8.5). Therefore, the application of bauxite residue, lime, and compost can address the problem of low pH, and the range of pH neutralization depends on the type and amount of materials added. Table 4-2. Rinse pH (solid: water = 1:5) of tailings, bauxite residue, compost and six substrates Rinse pH Repetition 1 Repetition 2 Repetition 3 Average pH Tailings 4.71 4.60 4.77 4.69 Bauxite residue 10.85 10.79 10.92 10.85 Compost 7.54 7.58 7.56 7.59 T+BR 6.01 6.08 6.17 6.09 T+L 8.43 8.61 8.24 8.43 T+C 6.69 6.64 6.32 6.55 T+BR+C 6.49 6.23 5.93 6.22 T+L+C 7.91 7.98 8.01 7.97 61 4.3 Growth affected by different amendments 4.3.1 Germination rate and shoots height Plant growth can be revealed by germination rates and shoot heights (Figure 4-1). Sixteen seeds were sowed in each pot and then the germination rate (%) is calculated: 𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠 𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠 = 𝐠𝐠𝐧𝐧𝐠𝐠𝐧𝐧𝐠𝐠𝐠𝐠 𝐠𝐠𝐨𝐨 𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠 𝐬𝐬𝐠𝐠𝐠𝐠𝐠𝐠𝐬𝐬𝟏𝟏𝟏𝟏× 𝟏𝟏𝟏𝟏𝟏𝟏% Equation 4-1 The germination rates out of 16 seeds in each pot of four species all improved on amended tailings compared to those on pure tailings (Figure 4-2.a). Wheat had germination rates of 53.1 – 84.4 % on all amended tailings, which significantly increases from only 6.3 % on the unamended tailings. Similarly, ryegrass also had increased rates from 25 % on tailings to 62.5 – 93.8 % on other amended tailings. Although tall fescue and sheep fescue germinated on unamended tailings, they could not survive and did not produce usable data for biomass and metal concentrations. It is noticeable that tall fescue and sheep fescue also had increased germination rates of 50 – 90.6 % and 25 – 87.5 % on amended tailings, respectively. Figure 4-2. The germination rates (a) and shoot heights (b) of four types of grass species on the six substrates 62 The averages of longest shoot heights were higher on tailings with amendments except for sheep fescue which had the shortest (10.4 – 14.25 cm) shoots among the four species (Figure 4-2.b). The heights of tall fescue, ryegrass, and wheat increased at 51.35 – 82.5 cm, 39.65 – 51.65 cm, and 28.8 – 41 cm, respectively, in amended tailings. The germination rates of species were higher in T+L and T+L+C than those in other amendments, and most species grew higher on T+BR+C than in other amendments. Because germination happened at the beginning of experiments, lime, and compost can affect pH immediately and be beneficial for seed germination when applied to tailings. This can also be shown by the pH of tailings amended with lime, 8.43 of T+L and 7.97 of T+L+C, which is the highest pH among amended tailings (Table 4-2). Shoot heights were recorded at the end of experiments, so the highest heights on T+BR+C indicate bauxite residue and compost support plant growth until harvesting. Wheat had the longest shoots of the four species and relatively high germination rates; ryegrass had the highest germination rates and second longest shoots. Tall fescue and sheep fescue had good germination rates but did not grow high. Wheat and ryegrass grew more and better than tall fescue and sheep fescue on tailings with amendments. 4.3.2 Dry biomass Figure 4-3 indicates the dry biomass of roots on all amendments was less than those of shoots of the four species, even though all five types of amendments increased plant biomass. The biomasses of tall fescue and sheep fescue were null because the plants could not grow on unamended tailings. Plants gained the most biomass on T+C and T+BR+C, which is consistent with shoot heights data that bauxite residue and compost can improve plant growth. Tailings 63 without any amendments has lower germination rates, shoot heights and plant biomass, because toxic metals and acidic condition are deleterious to plants (Conesa et al., 2007a; Conesa et al., 2007b). Sheep fescue had the lowest biomass among the four species, and tall fescue grew higher than sheep fescue. Whereas the root biomass of ryegrass was more than those of wheat, the two species had similar and the highest shoot biomasses among the four species. The result matches that of germination rates and shoot heights, in which wheat and ryegrass can grow better than tall fescue and sheep fescue. Figure 4-3. The dry biomass of shoots and roots of four species on six substrates. 64 4.4 Metals in plants affected by different amendments As shown in Table 4-1, the concentrations of As, Cu, Zn, and Pb in tailings exceed soil plant toxicity and leaf tissue toxicity levels. Then the uptake of these four metals by plants and the influence of amendments are further studied here. Each graph consists of two diagrams of one metal(loid)’s concentrations in plant roots and shoots with the same y-axis scale for the convenience of comparison. As tall fescue and sheep fescue could not survive on tailings, there is no data on metal concentrations in these two species. 4.4.1 As in species It can be seen in Figure 4-4 that As concentrations in roots were higher than those in aboveground shoots, with the most accumulated As concentrations in roots of wheat and ryegrass on T+C. For wheat and ryegrass, when the total As concentrations in roots and shoots were lower on T+BR and T+L, the data increased on T+C, T+BR+C and T+L+C. Therefore, the application of compost can promote the total As uptake by plants. Roots store the most As because translocation to aboveground shoots is limited. This means As accumulated in roots and toxicity to the aboveground tissues were reduced when plants grow on tailings with compost. Bauxite residue and lime reduced the metal solubility and their uptake by plants due to increased pH and capacity of stabilizing metals. 65 Figure 4-4. As concentrations in roots and shoots (mg/kg) 4.4.2 Cu in species As can be seen in Figure 4-5, Cu concentrations in roots were significantly higher than those in shoots. The highest Cu accumulation was in wheat roots on unamended tailings, which is 750 mg/kg, and the concentrations were lower in roots on the other amended tailings. Also, amendments reduced total Cu accumulation in wheat because the higher pH after tailings was amended reduced the metal solubility. Cu concentrations in ryegrass roots and shoots were lower on T+BR and T+BR+C but higher on T+C and T+L+C. Meanwhile, Cu accumulation in 66 aboveground parts of ryegrass generally decreased on tailings with all amendments. This means the application of bauxite residue can reduce Cu accumulation by ryegrass, compost can increase Cu uptake, and the translocation to aboveground tissues are limited under all amendments. Figure 4-5. Cu concentrations in roots and shoots (mg/kg) 4.4.3 Pb in species Figure 4-6 shows roots accumulated much more Pb (1 – 2 orders of magnitudes) than aboveground tissues of the four species on all amended tailings. The highest Pb concentration was in the roots of wheat, and amendments in both shoots and roots of wheat due to the 67 decreased pH. Compared to tailings, Pb accumulation in shoots of ryegrass declined when any amendment was applied. On T+L+C, both wheat and ryegrass accumulated more Pb in roots and less Pb in aboveground shoots than other amended tailings. Tall fescue and sheep fescue accumulated the most Pb in roots on T+BR and the least on T+BR+C. Therefore, Pb, bauxite residue, lime, and compost all reduce accumulation and limit translocation to shoots, but the combination of lime and compost can achieve more effective phytostabilization. Figure 4-6. Pb concentrations in roots and shoots (mg/kg) 68 4.4.4 Zn in species The Zn concentrations in roots were the highest on tailings and the accumulation in shoots was only higher than those on T+BR+C (Figure 4-7). The largest decrease in total Zn concentrations compared to tailings appeared on T+BR+C, which means the combination of bauxite residue and compost may be not ideal for the phytostabilization of Zn. Nevertheless, Zn uptake by plants on T+L+C was highest among amended tailings and Zn concentrations in shoots were not low. Zn in roots of the four species was translocated to aboveground shoots more on amended tailings than unamended tailings. Therefore, the translocation factor to shoots needs to be studied further in the next section to see the effectiveness of amendments on enhancing phytostabilization. Figure 4-7. Zn concentrations in roots and shoots (mg/kg) 69 4.4.5 Discussion Previous research has shown that there are positive correlations between metals in soil and their concentrations in plants (Fang et al., 2012; Fernández et al., 2017; Puschenreiter et al., 2017), which is the reason why the metals accumulation of As, Cu, Pb and Zn in tailings is the highest among all series. As indicated in Table 4-2, applications of bauxite residue, lime, and compost neutralize pH to varying degrees. The pH is an important factor affecting metal bioavailability and uptake by plants, because soil with higher pH can bind more metals to make them less available to plants (Reijonen & Hartikainen, 2016). The uptake of As, Cu, Pb, and Zn by wheat all decreased from high accumulation to intermediate levels. One thing common for the four species is the application of bauxite residue can limit the metal uptake because of its capacity in neutralizing pH and reducing metal solubility. For evaluating the efficiency of amendments influencing phytostabilization, the roots should uptake and store metals, without transferring to aboveground shoots, as much as possible. The precondition is that the germination rates and plant growth be promoted by the amendments to enhance phytostabilization. For As, tailings with compost only are the most effective combination to store in roots. For Cu, Pb, Zn, the combination of T+L+C is the most effective amendment to promote the roots accumulation. The previous discussion of plant growth concluded compost can support plant growth consistently until harvest. Therefore, compost is generally an efficacious and cost-effective amendment to improve plant survival and enhance phytostabilization. From the above discussion, both wheat and ryegrass can accumulate and store high levels of metals on tailings amended with compost, no matter if the compost is mixed with bauxite residue and lime or not. This means wheat and ryegrass may be suitable candidates for amended 70 phytostabilization, which needs to be further confirmed with the translocation Factor (TF) and the accumulation factor (AF), as discussed below. 4.5 Translocation factor and accumulation Factor Plants species selected for phytostabilization must be acid- and metal-tolerant, must not accumulate too much metals in shoot tissues, and absorb metals in roots and stabilize them in the rhizosphere. The Translocation Factor (TF) and the Accumulation Factor (AF) are calculated to evaluate the suitability of plants, and species used in phytostabilization should have both TF and AF values << 1 (Mendez & Maier, 2008). The Translocation Factor (TF) is a measure of the ability of plants to translocate metals from roots through aboveground shoots and leaves (Nirola et al., 2015): translocation factor = total element concentration in shoot tissuetotal element concentration in the root tissue Equation 4-2 The Accumulation Factor (AF) can be used to express metal accumulation in plants (Nirola et al., 2015) as: accumulation factor = total element concentration in shoot tissuetotal element concentration in mine tailings Equation 4-3 Figure 4-8 indicates the accumulation factor and translocation factor of As, Cu, Pb, and Zn. The red line is a reference line with a value of 1. It can be seen from this figure that the AF and TF of four species were < 1 for As, Cu, and Pb, which means they are suitable for phytostabilization of As, Cu and Pb. The data of AF and TF are shown in Table 4-3. For Zn, the AF of tall fescue and sheep fescue on T+L+C and the TF of wheat and sheep fescue on T+C were larger than 1, which means compost has a negative influence on phytostabilization for Zn by wheat, sheep fescue and tall fescue. It should be mentioned that the AF and TF of Zn 71 concentrations on the combination of T+BR+C are both <<1 in the four species; however, T+BR+C is not ideal for phytostabilizing Zn because the Zn uptake in roots largely decreased on T+BR+C. Therefore, although compost has been found to generally provide a cost-effective amendment to improve plant survival and enhance phytostabilization, adding compost into tailings limits its candidacy for amended phytostabilization of Zn. 72 Figure 4-8. Accumulation factor and translocation factor of As, Cu, Pb and Zn for the four species on six substrates 73 Table 4-3. TF and AF for four species on six substrates Wheat Ryegrass Tall Fescue Sheep Fescue AF TF AF TF AF TF AF TF As T 0.007 0.008 0.033 0.124 - - - - T+BR 0.004±0.001 0.020±0.004 0.002 ±0.001 0.009± 0.002 0.006±0.002 0.036± 0.007 0.022± 0.018 0.128± 0.013 T+L 0.019±0.007 0.059± 0.012 0.010±0.005 0.023± 0.013 0.023±0.017 0.052± 0.034 0.009± 0.001 0.052± 0.002 T+C 0.032±0.002 0.023± 0.001 0.039±0.006 0.029± 0.007 0.050± 0.006 0.072± 0.039 0.026± 0.001 0.032± 0.007 T+BR+C 0.018±0.01 0.023± 0.008 0.014 ±0.001 0.017± 0.005 0.016±0.02 0.028± 0.016 0.006±0.003 0.014± 0.001 T+L+C 0.013±0.004 0.019± 0.007 0.010 ±0.005 0.012± 0.007 0.016± 0.001 0.027± 0.004 0.010± 0.001 0.020± 0.004 Cu T 0.027 0.036 0.084 0.205 - - - T+BR 0.021±0.002 0.129±0.040 0.035±0.001 0.156±0.008 0.021±0.006 0.102±0.017 0.036±0.023 0.102±0.069 T+L 0.087±0.008 0.356±0.063 0.091±0.013 0.251±0.060 0.079±0.018 0.181±0.053 0.071±0.001 0.181±0.017 T+C 0.061±0.015 0.154±0.040 0.056±0.008 0.100±0.005 0.071±0.015 0.190±0.035 0.045±0.011 0.190±0.052 T+BR+C 0.045±0.024 0.138±0.063 0.042±0.009 0.125±0.006 0.023±0.016 0.088±0.005 0.019±0.001 0.088±0.020 T+L+C 0.064±0.014 0.149±0.045 0.074±0.008 0.140±0.005 0.074±0.007 0.178±0.020 0.037±0.004 0.178±0.012 Pb T 0.022 0.025 0.038 0.164 - - - T+BR 0.009±0.002 0.043±0.026 0.008±0.002 0.019±0.004 0.015±0.004 0.031±0.007 0.039±0.034 0.199±0.256 T+L 0.009±0.002 0.030±0.020 0.006±0.001 0.041±0.020 0.014±0.002 0.031±0.016 0.028±0.008 0.229±0.067 T+C 0.006±0.003 0.019±0.009 0.004±0.001 0.012±0.002 0.0100.001 0.047±0.006 0.009±0.001 0.030±0.005 T+BR+C 0.003±0.001 0.013±0.003 0.002±0.001 0.014±0.004 0.003±0.002 0.018±0.003 0.003±0.001 0.025±0.009 T+L+C 0.011±0.006 0.030±0.019 0.008±0.002 0.016±0.005 0.011±0.003 0.034±0.005 0.014±0.002 0.035±0.004 Zn T 0.471 0.060 0.257 0.783 - - - T+BR 0.821±0.051 0.595±0.101 0.704±0.015 0.624±0.097 0.739±0.066 0.805±0.158 0.779±0.111 0.701±0.221 T+L 0.668±0.066 0.656±0.159 0.696±0.126 0.650±0.118 0.489±0.015 0.548±0.014 0.561±0.106 0.785±0.062 T+C 0.821±0.051 1.217±0.011 0.393±0.051 0.641±0.129 0.529±0.061 0.835±0.063 0.925±0.308 1.336±0.003 T+BR+C 0.250±0.081 0.962±0.429 0.207±0.001 0.624±0.009 0.125±0.127 0.763±0.618 0.179±0.020 0.766±0.078 T+L+C 0.929±0.101 0.742±0.051 0.929±0.202 1.000±0.218 1.214±0.101 1.007±0.167 1.143±0.202 0.727±0.129 *Values are means ± S.D. of two replicates. There is only one replicate for tailings. The biomass of tall fescue and sheep fescue is null because of no growth on tailings. 74 4.6 Principal component analysis (PCA) A PCA bi-plot (Figure 4-9) was illustrated to compare the plant growth parameters (height, germination rate, dry biomass), soil characteristics (pH), and total metals in plants (As, Cu, Pb, and Zn). The first two components of PCA explained 70.6% of the total variation. The first axis explained 42.3 % of the total variation and was positively correlated with height, germination rate, dry biomass, and soil pH. The second axis, which explained 28.3 % of the total variation, was positively correlated with the total concentrations of As, Cu, Pb, and Zn in plants. Sheep fescue (blue) and tailings (stars) are significantly negative with height, germination rate, and dry biomass, which indicate that sheep fescue did not grow well on all amendments, and tailings conditions are not conductive for plant growth. T+C (circles) and T+L+C (diamonds) show a low positive correlation with total metals in plants, meaning compost can slightly increase metal uptake. These conclusions are all consistent with the above discussion. Figure 4-9. PCA comparing measured parameters. 75 4.7 Conclusions First, it is concluded that wheat and ryegrass are candidates for phytostabilization as they grow better than tall fescue and sheep fescue on amended tailings. They accumulate more metals in roots than in aboveground shoots, which can answer the first sub-questions of this chapter, and answer the species selection part of the research question. Second, three amendments used in this study improve the deleterious conditions of tailings and enhance plant growth. The application of bauxite residue can limit the metal uptake because of its capacity to increase pH and reduce metal solubility. Lime can be effective immediately upon seed germination, but may not have a lasting influence on promoting metals uptake. Compost is found to be a great and cost-effective amendment to improve plant survival and enhance phytostabilization, but adding compost into tailings does not achieve effective phytostabilization of Zn. However, compost is still a more cost-effective amendment than lime and bauxite residue to improve plant survival and enhance phytostabilization of As, Cu, and Pb. The above can answer the second sub-question of this chapter and answer the part of amendments effectiveness of the research question. Overall, this chapter achieved the objectives of species selection and evaluation of amendment effectiveness, which provides a reference for design of phytostabilization on sulfidic mine tailings. 76 Chapter 5: Geochemical Processes in the Rhizosphere3 5.1 Introduction This chapter is exploring the geochemical processes in the rhizosphere during phytostabilization (Figure 5-1). Since phytostabilization aims to stabilize and sequester heavy metals within the rhizosphere, the roots – tailings interactions involving metal immobilization is critical. Roots – tailings interactions provide an understanding of metal speciation and the migration of toxic metals (Xie & van Zyl, 2020), These interactions happening in the rhizosphere include organic-related processes, such as microbial activity and root uptake, and inorganic-related processes. Much research focuses on organic-related processes in the rhizosphere, including the types of microbes that can promote plant growth (Khan et al., 2013; Lugtenberg et al., 2013), the establishment of microbial communities in the rhizosphere (Caesar-TonThat et al., 2008; Y. Li et al., 2016; Zhang et al., 2017), and microbial activity affecting metal mobility (Hagmann et al., 2015; Hinsinger et al., 2008). 3 A version of this chapter entitled “The geochemical processes affecting metal mobility of phytostabilization on sulfidic mine tailings revealed by a greenhouse study.” has been submitted for peer review. The section of Materials and Methods is not included in this chapter because details of experiments and materials were introduced in Chapter 3. 77 Figure 5-1. Highlights of this chapter in the workflow However, studies on geochemical processes of metal stabilization within the rhizosphere in sulfidic mine tailings are limited, because most research has discussed the geochemical results of revegetation instead of the processes controlling metals fate in the rhizosphere(Lee et al., 2014; Wu et al., 2019). Mineral composition can be retained even after revegetation, and the breakdown of primary minerals into secondary minerals can be detected around plant roots (Justine et al., 2014). A better understanding of the geochemical processes in the rhizosphere can help choose more suitable types and amounts of amendment materials, and then lead to the Mine Site Background Data Collection in Progress Greenhouse Experiments Native Species and Amendments Acquisition Tailings Acquisition Tailings Characterization Reliability of Phytostabilization Data Collection after experiments Drainage Chemistry Metals in Plants pH & Metals in Soil Geochemical Processes in the Rhizosphere Species Selection and Amendments Efficiency Conclusions and Suggestions Materials Characterization before experiments Pots Experiments Setup 78 success of phytostabilization on sulfidic mine tailings of metals immobilization. The inorganic geochemical processes controlling metal mobility in the rhizosphere mainly include: precipitation and dissolution of minerals; silicate mineral weathering processes releasing metals; adsorption and desorption onto solid phase surfaces (Dighton & Krumins, 2014). For this research, inorganic geochemical processes affecting metal immobilization in the rhizosphere were explored based on the successful establishment of phytostabilization in the greenhouse study. This helps to develop a better understanding of metal release, migration, and transformation, as well as the long-term fate of toxic metals. The geochemical changes include the changes in the mineralogy of pot samples, pH in the drainage, and metal concentrations in drainage during the experimental period. The focus of this study is on the geochemical processes in the rhizosphere, so organic geochemistry and microbial interactions are not included in this study. The study can fill the gap of the inorganic geochemical processes taking place in the rhizosphere when establishing phytostabilization in sulfidic mine tailings, which is meaningful and significant to provide a reference for the design of phytostabilization in sulfidic mine tailings in the field, and help guide mining companies, the government, and stakeholders to develop better mine closure plans. This chapter asks the following question: • What geochemical reactions can be identified that affect the mobility of heavy metals in the rhizosphere when direct revegetation with or without amendments is applied to sulfidic tailings? The objective of this chapter is: 79 • To evaluate the geochemical processes and reactions controlling metal mobility in the rhizosphere by analyzing drainage chemistry and soil minerals. 5.2 Geochemistry changes after experiments 5.2.1 Changes of pH in substrates Table 5-1 indicates the pH of substrates in pots after experiments in each type of amendment of each grass. The results show pH in all pots was lower compared to the original materials (Table 5-1). Sulfide mineral oxidation is one reason for soil acidification, in blank groups without planting, representing pure mineral oxidation, with a pH of ~4. However, it is noticeable that the pH in pots planted with grass species is lower than those in blank groups, which means plants are the reason for some acid-producing processes other than sulfide oxidation. According to previous studies, the release of H+ from plant roots due to excess uptake of cations over anions by plants is one of the causes of soil acidification, because the soil – root system needs to maintain electroneutrality (Gliński & Stępniewski, 1985; Ulrich, 1986). Table 5-1. Rinsed pH of substrates (1:5 w/w soil: water) in pots before and after experiments Before experiments After experiments Average pH Wheat Ryegrass Tall fescue Sheep fescue Blank T 4.69 3.38 3.72 3.92 3.54 4.77 T+BR 6.09 3.15 2.71 2.99 3.41 6.15 T+L 8.43 3.22 2.90 2.90 3.21 4.8 T+C 6.55 2.59 2.33 2.34 2.86 4.12 T+BR+C 6.22 3.77 2.97 2.97 3.19 4.01 T+L+C 7.97 2.88 2.35 2.51 3.69 3.95 80 5.2.2 Changes of mineralogy Mineralogy changes are one of the main factors in tailings evolution, especially for sulfide mine tailings. Table 5-2 indicates the quantitative XRD results of tailings and bauxite residue before the experiment, and of T blank, T+BR blank, T+L blank, and T+C blank after experiments. The original tailings comprise sulfides (57 %), silicates (31.9 %), carbonates in the form of siderite (7.6 %), and minor sulfates (3.5 %). The sulfide minerals include pyrite (43.2 %), marcasite (8.9 %), sphalerite (2.3 %), pyrrhotite (2.2 %), chalcopyrite (0.2 %), and galena (0.2 %). The original bauxite residue consists of hematite (39.4 %), goethite (30.6 %), silicates (10.2 % of sodalite and 4.2 % of quartz), aluminum hydroxides (4.9 % of böhmite and 1.8 % of gibbsite), calcite (3.1 %), and titanium oxide (5.8 %). The pot samples of blank groups without planting grass were analyzed for QXRD in unamended tailings, T+BR, T+L, and T+C after experiments. The qualitative XRD of other substrates was analyzed and the results of some samples are attached in Appendix B. It was found that there were no new minerals produced other than those listed in Table 5-2. The percentages of some minerals showed significant changes. The sample for QXRD analysis is 2g, which may cause heterogeneity and imbalance of Ba-minerals according to the size of mineral crystallization, e.g., the imbalance percentages between Barite and Celsian (Table 5-2). 81 Table 5-2. Quantitative XRD results of samples before and after experiments Mineral Ideal Formula Before experiments After experiments Tailings Bauxite Residue Tailings Blank T+BR Blank T+L Blank T+C Blank % Sulfides Pyrite FeS2 43.2 46.6 37.1 34.5 46.9 Marcasite FeS2 8.9 4.1 7.1 4.2 0.8 Sphalerite ZnS 2.3 0.9 1.2 0.4 0.6 Pyrrhotite Fe1-xS 2.2 1.4 0.4 Chalcopyrite CuFeS2 0.2 Galena PbS 0.2 Sulfates Anglesite PbSO4 0.5 0.4 0.5 0.2 0.2 Barite BaSO4 2.1 8.4 2.1 1.2 8.9 Gypsum CaSO4∙2H2O 0.9 2.5 1.5 2.4 6.5 Jarosite K2Fe6(SO4)4(OH)12 4.8 5.9 7.5 6.2 Silicates Muscovite KAl2AlSi3O10(OH)2 2.5 1.9 2.4 2 2.7 Kaolinite Al2Si2O5(OH)4 1.9 0.9 1.3 0.8 0.9 Celsian Ba(Al2Si2O8) 1.1 0.8 1.2 0.7 0.8 Sodalite Na4(Si3Al3)O12Cl 10.2 Carbonates Siderite FeCO3 7.6 3.6 7.1 0.9 0.9 Calcite CaCO3 3.1 Hydroxides Böhmite AlO(OH) 4.9 Gibbsite Al(OH)3 1.8 Goethite (Fe,Al)O(OH) 30.6 0.9 Iron oxides Magnetite Fe3O4 0.9 0.2 0.3 0.8 Hematite Fe2O3 39.4 0.4 Others Sulfur S8 0.7 1 1 0.7 Anatase TiO2 4.6 Rutile TiO2 1.2 Quartz SiO2 26.4 4.2 22.1 29.7 43.9 23.1 Total (%) 100.0 100.0 100.0 100.0 100.0 100.0 82 First, in either unamended or amended tailings, chalcopyrite and galena were not found after experiments, and the content of marcasite, pyrrhotite and sphalerite reduced, which means sulfide oxidation occurred during the experiments. Reference has shown the oxidation sequence of sulfide minerals in the variably saturated zone is galena → chalcopyrite → pyrite (Kalinnikov et al., 2001). At the same time, the total percentages of sulfates in these four substrates increased from 3.5 % in original tailings to 16.1 % in tailings, 10 % in T+BR, 11.3 % in T+L, and 21.8 % in T+C after experiments. The precipitation of sulfates was another sign of sulfide oxidation, with the increase of SO42- combined with multiple cations to precipitate sulfate minerals. In this study, they are gypsum (CaSO4), anglesite (PbSO4), barite (BaSO4), and jarosite (K2Fe6(SO4)4(OH)12) that were produced after sulfide oxidation. In addition, the contents of carbonates decreased from 7.6 % in original tailings to 3.6 % in tailings, 7.1 % in T+BR Blank, 0.9 % in T+L Blank, and 0.9 % in T+C Blank. Carbonates are the first type of minerals to neutralize the AMD, which contributes to the neutralization potential (NP) (Weber et al., 2005). However, siderite (FeCO3) does not contribute to NP, because the dissolution of siderite produces Fe2+ that can be oxidized to Fe3+, of which subsequent hydrolysis consume no acid or protons (Jambor et al., 2003; ; Paktunc, 1999). There was 3.1 % of calcite in the original bauxite residue, and lime is in the form of CaO that can produce calcite as well when exposed to air, which means the carbonate neutralization capacity (calcite) was consumed in T+BR Blank and T+L Blank. The total percentage of silicates reduced from 5.5 % in original tailings to 4.6 % in tailings, 4.9 % in T+BR, 3.5 % in T+BR, and 4.4 % in T+C after experiments. There was 10.2 % sodalite, which is also a silicate mineral, in original bauxite residues. Silicate mineral weathering can be accelerated in acidic environments (Lasaga, 2018; Oelkers, 2001), so the content of silicate 83 minerals declined in substrates of blank groups of tailings, T+BR, T+L, and T+C after experiments. Moreover, the Fe, Al hydroxides (böhmite, gibbsite, goethite) and Fe oxides (magnetite, hematite) in the blank groups of tailings, T+BR, T+L, and T+C after experiments were lower compared to those in the original bauxite residue. These are signals that the pH of pore water or drainage may drop to below 4 at the end of the experiment, because the (oxy)hydroxide dissolution happens across the zone with pH close to 4 in the neutralization sequence of AMD (Baleeiro et al., 2018; Gunsinger et al., 2006). 5.2.3 Changes of pH and metal concentrations in drainage 5.2.3.1 Compare the metal concentrations with regards to species According to the conclusions of Chapter 4, wheat and ryegrass are good candidates for revegetation on the sulfidic tailings in this study, so these two species were selected for geochemistry analyses. Another fact needs to be clarified is that As concentrations in most water samples could not be detected, so changes in As concentrations in drainage are not discussed further. First, Figure 5-2, Figure 5-3, Figure 5-4, and Figure 5-5 represent the changes in pH and Cu, Pb, and Zn concentrations of drainage from pots with or without plants, respectively. These four figures show the effects of plants on changes in pH and metal concentrations in drainage. Figure 5-2 indicates pH in all pots showed decreased, no matter whether there were plants or not. The pH of drainage from pots with wheat and ryegrass basically decreased similarly, both decreasing from the 6th week. Meanwhile, the trends of the pH of blank groups (blue dotted lines) on amended tailings without plants are flatter, and look similar to those in unamended tailings. On 84 unamended tailings, the pH decreased at similar, which means the acidification rates were not affected by planting and grass species. Figure 5-2. pH changes of drainage from pots with or without plants during the experiments 85 Figure 5-3 shows the trends of Cu concentrations in the drainage from all six substrates with or without grass during the ten weeks. Compared to the Cu concentrations in drainage from amended tailings (T+ C, T+BR, T+BR+C, T+L, T+L+C), Cu concentrations in drainage from pure tailings remained at a relatively low level with slightly decreasing trends, with grass or not. Blank groups without grass on all substrates (blue dotted lines) drained out water with low Cu concentrations as well. Cu concentrations started to increase from the 6th week on amended tailings with grass, and show a more significant increase than those of blank groups without plants. Regarding the grass species, amended tailings planted with ryegrass had the highest Cu concentrations release (red dotted lines), followed by the pots planted with wheat (black dotted lines). In addition, the Cu concentrations varied in the drainage from tailings with different amendments. The trends of Pb concentrations in the drainage from all six substrates with or without grass during the experiments are shown in Figure 5-4. Similar to Cu concentrations, Pb concentrations in drainage from unamended tailings were at low levels, when compared to those from the amended tailings. Blank groups (blue dotted lines) without grass on all substrates drained out water with low Pb concentrations. Pb concentrations started to increase from the 6th week on T+C and T+L+C with grass, and from the 8th week on T+BC, T+BR+C, and T+L with grass; these data show a more obvious increase than those of blank groups without plants. Amended tailings planted with ryegrass released with the highest Pb concentrations (red dotted lines), followed by the pots planted with wheat (black dotted lines). In addition, the Pb concentrations varied in the drainage from tailing with different amendments. Cu and Pb concentrations have similar trends in drainage. 86 Figure 5-3. Cu concentration of drainage from pots with or without plants during the experiments. 87 Figure 5-4. Pb concentration of drainage from pots with or without plants during experiments. 88 According to Figure 5-5, Zn concentrations in drainage show similar features to Cu and Pb concentrations. First, Zn concentrations in drainage from unamended tailings remained at a low level when compared to those from the amended tailings. Plus, blank groups without grass on all substrates (blue dotted lines) drained water with low Zn concentrations. As Cu, Pb and Zn concentrations were at lower levels on unamended tailing and blank groups, and the unamended tailings have about the same effect on these three metal concentrations in drainage as the blank groups without plants. This is because grass can hardly survive and grow well on pure tailings due to deleterious conditions. As such, pure tailings can be considered as blank groups without grass, which is supported by the plant growth in the greenhouse experiment (Figure 4-2 and Figure 4-3). Similar to Cu and Pb, Zn concentrations started to significantly increase from the 6th week on amended tailings with grass, with more obvious increases than those of blank groups without plants. Regarding the grass species, amended tailings planted with ryegrass released the highest Zn concentrations (red dotted lines), followed by the pots planted with wheat (black dotted lines). Last but not least, the Zn concentrations varied in the drainage from tailings with different amendments. 89 Figure 5-5. Zn concentration of drainage from pots with or without plants during the experiments. 90 5.2.3.2 Compare the metal concentrations with regards to amendments To compare the different pH trends affected by amended tailings and unamended tailings with grass, Figure 5-6 (a) and Figure 5-6 (b) display changes for wheat and ryegrass, respectively. In general, all pH values decreased from the first week to the experiment’s end, regardless of grass species, adding amendments or not, or the amendments types. The differences are their decreasing rates of various substrates, with the pH in drainage from pure tailings showing the least steep decreasing trend for both wheat and ryegrass. The pH in the first week for different amended tailings has the same order with planting either wheat or ryegrass, which is T+BR+C (purple dotted line) > T+L+C (yellow dotted line) > T+L (blue dotted line) > T+C (green dotted line) >T+BR (red dotted line) > T (black dotted line). The pH fluctuated in various ways with decreasing trends for six substrates. At the end of the experiments, the two species show different pH values in drainage at the 10th week, and the drainage in most amended tailings had a lower pH than that in unamended tailings (black dotted line). This indicates despite the neutralization capacity of the amendments, some processes decrease the pH of amended tailings via planting of grass. Figure 5-6. pH changes of drainage from different substrates 91 Figure 5-7, Figure 5-8 and Figure 5-9 show the changes of Cu, Pb, and Zn concentrations in drainage from unamended and five types of amended tailings with wheat and ryegrass, respectively. The figures of these three metals are discussed together in this paragraph, because they display quite similar features. First, at the beginning of experiments, Cu, Pb, and Zn concentrations in drainage from unamended tailings were higher than those from all types of amended tailings. This is because the amendments neutralized the acidic conditions of tailings at the start, resulting in the immobilization of soluble metals. During the ten weeks, metal concentrations in drainage from unamended tailings had different trends from those of amended tailings. On unamended tailings, Cu, Pb, and Zn concentrations in drainage decreased gradually throughout the experiment; conversely, metal concentrations in drainage from amended tailings fluctuated slightly before the 6th week and started to significantly increase after the 6th week. At the end of the experiment (10th week), it is noticeable that Cu, Pb, and Zn concentrations in drainage from the six substrates generally are released at similar elevated concentrations: T+C (green dotted line) > T+L+C (yellow dotted line) > T+BR+C (purple dotted line) > T+L (blue dotted line) > T+BR (red dotted line) > T (black dotted line). This may imply the geochemical processes in the rhizosphere are consistent across grass species and relate closely to the amendment types. The reason will be discussed following. 92 Figure 5-7. Cu concentration of drainage from different substrates Figure 5-8. Pb concentration of drainage from different substrates Figure 5-9. Zn concentration of drainage from different substrates 93 5.3 Discussion 5.3.1 The sources of metals This paragraph is discussing the sources of As, Cu, Pb, and Zn in the plants, soil and drainage. Cu, Pb, and Zn concentrations significantly exceeded the limits for plants (Table 4-1), soil (Table 4-1), and drainage (CCME, 1993), while As was not detected in most drainage samples and As concentrations in plants and soil had exceeded toxicity limits. The release of metals, such as Cu, Pb, Cu, and Fe are controlled by sulfide oxidation, oxyhydroxide dissolution at acidic pH levels, and desorption in sulfidic tailings. The tailings mineralogy contains various sulfide minerals that have the potential to release associated toxic metals to the environment, as listed in Table 2-1. The high concentration of toxic metals that are released largely from these sulfide minerals contained in the tailings under acid conditions could have deleterious effects on ecosystems and human health. According to the results of quantitative XRD (Table 5-2), pyrite is the most abundant sulfide mineral in tailings. Pyrite oxidation has been extensively studied due to its importance in environmental reclamation, and because it is the primary reason for the formation of AMD (Brown & Jurinak, 1989; ; Evangelou & Zhang, 1995; Lowson, 1982; Nordstrom & Alpers, 1999). Pyrite oxidation is complex, involving chemical, microbiological, and even electrochemical reactions (Nordstrom & Alpers, 1999; Rosso & Vaughan, 2006). In natural environments, oxidation occurs when exposed to an oxidant and water. The oxidant is normally oxygen and can be Fe3+ or mineral catalysts (e.g., MnO2) (Blowes et al., 2014; Nordstorm, 1982). The overall pyrite reaction is: FeS2(s) + 154 O2(aq) + 72 H2O → Fe(OH)3(s) + 2SO42− + 4H+ Equation 5-1 94 Fe(OH)3 is a substitute for ferrihydrite. Hematite (Fe2O3) and magnetite (Fe3O4) are also possible products from AMD formation (Shingwenyana et al., 2021), which were found in pots soil after experiments in this study as shown in Table 5-2. This reaction also indicates the AMD is acidic with high levels of SO42-, thus resulting in precipitation and increased sulfate minerals. In this study, they are gypsum (CaSO4), anglesite (PbSO4), barite (BaSO4) and jarosite (K2Fe6(SO4)4(OH)12) produced after sulfide oxidation (Table 5-2). Likewise, sphalerite is the only Zn-sulfide mineral in tailings (Table 5-2), so the oxidation of sphalerite, which also relies on the dissolved O2 and Fe3+, controls the release of Zn (Bobeck & Su, 1985; Heidel et al., 2011; Perez & Dutrizac, 1991): ZnS + 2O2(aq) → Zn2+ + SO42− Equation 5-2 ZnS + 8Fe3+ + 4H2O → Zn2+ + 8Fe2+ + SO42− + 8H+ Equation 5-3 Chalcopyrite is the only Cu-sulfide mineral in the tailings of this study. The oxidation of chalcopyrite releases Cu (Hackl et al., 1995; ; Prasad & Pandey, 1998; Baojun Yang et al., 2020): CuFeS2(s) + 154 O2(aq) → CuSO4 + 12 Fe2O3(s) + SO2 Equation 5-4 CuFeS2 + 4Fe3+ → Cu2+ + 5Fe2+ + 2S0 Equation 5-5 Pb comes from the oxidation of galena, which is similar to the oxidation of sphalerite because Pb and Zn have the same valence state. The oxidation is the cause of Pb release and anglesite (PbSO4) precipitation (da Silva, 2004; Heidel et al., 2013): PbS + 2O2(aq) → Pb2+ + SO42− Equation 5-6 PbS + 8Fe3+ + 4H2O → Pb2+ + 8Fe2+ + SO42− + 8H+ Equation 5-7 95 The source of soluble As in tailings, mainly comes from the oxidation of arsenopyrite (FeAsS). The process releases As to the surrounding environment (Silva et al., 2017): FeAsS(S) + 114 O2(aq) + 32 H2O → Fe2+ + SO42− + 3H+ + AsO33− Equation 5-8 However, the Rietveld quantitative XRD results in Table 5-2 of tailings did not include arsenopyrite, which may be because As does not exist in a mineral crystallized phase and was not detected (Flemming et al., 2005). As can also be included in other sulfide minerals as inclusion, resulting in the release of As when the sulfide minerals are oxidized. Table 2-1 lists most associated toxic metals that can be potentially released along with the oxidation of various sulfide minerals. In conclusion, the sources of As, Cu, Pb, and Zn are the sulfide minerals in tailings, and the oxidation of various sulfide minerals is the key process to release those metals into the surrounding environment. These processes are also acid-generating, which is one of the causes of soil acidification. However, Figure 5-2 and Figure 5-6 show pH of drainage from the most amended tailings decreased significantly at the end of experiments, even though some of the tailings were amended with alkaline materials, e.g. lime and bauxite residue. Therefore, there must be other processes of soil acidification to decrease the pH, which will be discussed in the next section. 5.3.2 Soil acidification processes This section is going to discuss the factors contributing to the acidity in soil as there are other processes causing soil acidification except for the reactions of sulfide oxidation. The experimental evidence is the pH of drainage from the most amended tailings decreases at the end 96 of experiments, even if the tailings were amended with alkaline materials, such as bauxite residue and lime (Figure 5-2 and Figure 5-6). Soil acidification has been a major environmental concern and a constraint to crop production worldwide (Sumner & Noble, 2003). A number of factors can lead to soil acidification, which has been identified in previous research (Goulding, 2016; Haynes, 1983): • The release of H+ ions: some reactions in the soil can generate acid, such as sulfide oxidation or nitrification (Khwaja & Husain, 1990). • Nutrient uptake by crops and root exudates: the uptake of nutrients, mostly as cations, can lead to the release of H+ in exudates from roots to maintain electro – neutrality in soil (Wang et al., 2016). • The accumulation of organic matter: organic matter decomposition produces CO2, forming carbonic acid (H2CO3) (Bolan & Hedley, 2003). • The inputs of acidifying substances from the atmosphere: the dissolution of carbon dioxide (CO2), sulfur dioxide (SO2), and ammonia (NH3) can generate carbonate acid (H2CO3), sulfuric acid (H2SO4), and nitric acid (HNO3) (Likens et al., 1972). • The application of acidifying fertilizers such as urea or ammonium salts and the planting of legumes such as clover: this is the main cause of soil acidification on agricultural land as ammonium salts can strongly acidify the soil through nitrification (Barak et at., 1997). In this study, sulfide oxidation is the main process of soil acidification in tailings. To describe other soil acidification processes, the pure sulfide oxidation needs to be quantified first. It is undoubted that the unamended tailings without amendments and plants can represent the contribution of sulfide oxidation. Figure 5-2 shows that pH in drainage from T-blank (tailings without plants) are similar to those from T-WG and T-RG (tailings with wheat and ryegrass). 97 This is because the groups of T-WG and T-RG can be considered blank groups due to the extremely low germination rates (Figure 4-2) and low growth (Figure 4-3) of wheat and ryegrass on pure tailings. Therefore, for several groups of unamended tailings, no matter if grasses were planted or not, pure sulfide oxidation is the only process of soil acidification because grass could not survive and grow. For other reasons of soil acidification, Figure 5-2 and Figure 5-6 show that pH in drainage from the most amended tailings started to decrease from the 6th week, and lower than pH levels from unamended tailings at the 10th week. Especially on T+BR and T+L, the tailings were only amended with alkaline materials not causing acidification: bauxite residue and lime. The pH around the 6th week started to decrease, which means the alkalinity in the soil was consumed and then the acidity started to increase. As discussed above, the tailings groups can be considered as blank groups without planting because of the extremely low germination rates and limited growth. Thus, the other major reason contributing to soil acidification on T+BR and T+L is grass. Root exudates could increase the solubility of metals and their subsequent uptake (Hinsinger et al., 2006), and may increase microbial decomposition process to cause metals leaching (Mayer, 1998). The uptake of nutrients by plant roots, mostly as cations, can lead to the release of H+ in exudates from roots to maintain electroneutrality in soil (Goulding, 2016; Haynes, 1983; Wang et al., 2016). In addition, the reaction rates of kinetic controlled sulfide oxidation may be promoted by root exudates because pH in groups (2-3) with planting was quite lower than those (4-6) in blank groups (Table 5-1). Sulfide oxidation can be enhanced by microbial metabolism (Kühl & Jørgensen, 1992; Lu & Wang, 2012; Vera et al., 2022), and root exudates contain organic matter for microbial communities and significantly support their colonization in the rhizosphere (Huang et al., 2014; Zhalnina et al., 2018). Pyrite oxidation was found to be greater 98 by increased plant activity in summer (Ferreira et al., 2007), and sulfide oxidation was greater in all crop rhizosphere soil than in non-rhizosphere soils because of the increased activity of sulfur-oxidizing microorganisms (Grayston & Germida, 1990). Moreover, comparing the data of blank groups of T and T+C, the reason that the pH at the end point from the T+C blank group was smaller than that from the T blank group was the addition of compost. Thus, compost is a third cause of soil acidification. This is because compost contains large amounts of organic matter, and the purpose of compost amendment in the experiments was to provide organic matter for plants growing. As presented above, the accumulation of organic matter in soil can cause soil acidification by producing CO2 that dissolves into carbonate acid (H2CO3) (Bolan & Hedley, 2003; Goulding, 2016) and other organic acids through decomposition (Guggenberger et al., 1994). The increased leachability of heavy metals in the phytostabilization of tailings was also found by Lee et al. (2014), which was because the dissolved organic carbon increases the solubility of metals through the formation of organic-metallic complexes after adding the organic-rich bone meal as one amendment into tailings. Table 5-3. The soil acidification processes in six substrates Six substrates Sulfide oxidation Compost decomposition Root exudates T √ √(very limited) T+BR √ √ T+L √ √ T+C √ √ √ T+BR+C √ √ √ T+L+C √ √ √ In conclusion, tailings, compost, and grass are three reasons causing soil acidification in this study, through the processes of sulfide oxidation, the decomposition of organic matter, and root 99 exudates, respectively. For the six substrates in the experiments, sulfide oxidation is the major soil acidification process in T; sulfide oxidation and root exudates are the two acidification processes in T+BR and T+L; soil acidification in T+C, T+BR+C, and T+L+C is caused by all three processes. All above is concluded in Table 5-3. Literature about the acidification of root exudates and organic matter during phytostabilization on tailings is limited, but root exudates and organic matter do have effects on metal mobility in soil. Krishnamurti et al. (1997) investigated the effects of root exudates on the kinetics of Cd release from the soil, and found increasing amounts of Cd were released from soil to the soil solution with the renewal of root exudates every 2 h. Root exudates could increase the solubility of metals and their subsequent uptake (Hinsinger et al., 2006), therefore, supporting the successful phytostabilization by increasing the bioavailability of specific metals (Tahir et al., 2022; Zeng et al., 2008). While the decomposition of organic matter increases the release of Cu, Cd, Ni, Pb, Zn, Fe, and Mn from sediments (Caplat et al., 2005; Gerringa, 1990), organic matter is considered a good amendment to enhance phytostabilization because it can increase N, P, and K content and alter physical properties of tailings (Martínez‐Martínez et al., 2019). There is limited data from former research about the quality of drainage or pore water on phytostabilization on sulfidic mine tailings for comparison, but there are findings about the effects of acidification on metal stabilization. Honeker et al. (2019) and Valentín-Vargas et al. (2018) found that the re-acidification of the rhizosphere inhibits phytostabilization of metalliferous tailings, so they suggest to re-application of amendments on tailings to maintain phytostabilization. Beauchemin et al. (2018) found the re-mobilization of Ni after 4 – 5 years of oxygen cover on acid-generating tailings. These studies indicate that the re-acidification in the 100 rhizosphere would cause the re-mobilization of metals during phytostabilization, of which the reason was revealed in the next section. 5.3.3 Neutralization processes This section is going to discuss the neutralization processes with regards to various minerals. As tailings condition is too acid for plants to grow on, amendments are necessary to neutralize the acidity and adjust the pH of tailings. In the experiments of this study, the pH amendments are conducted by bauxite residue and lime, which are both alkaline with neutralization capacity for pH adjustment. Various types of minerals have the ability to neutralize the pH, in other words, have the capacity to accept protons from the surrounding environment. Based on XRD analysis results of quantitative mineralogy in Table 5-2, the neutralization processes are analyzed, and found to be consistent with the sequence of pH buffering processes in sulfidic mine tailings that from previous research on the formation of AMD. Acidity in the soil is buffered sequentially against acidification by a series of neutralization processes: (1) the dissolution of carbonates; (2) the dissolution of aluminum or iron (oxy)hydroxides; (3) the dissolution of Al-Si bearing minerals (Goulding, 2016; Weber et al., 2005; Worrall & Pearson, 2001). These pH buffering processes happen at approximately 5 – 6, 4, and 3, respectively (Bain et al., 2000; Gunsinger et al., 2006). The pH in soil with the acidification and buffering processes happening at the same time decreases slowly in the first six weeks. This is followed by a relatively fast decrease in pH when one process ends or one type of mineral becomes exhausted, and then the next buffer process takes over. The pH rapidly drops to 2 – 3 when all buffer minerals are consumed (Dold, 2010). This is evident from Figure 5-2 and Figure 5-6 showing the pH in drainage from most pots started to decrease at the 6th week, which means all buffer 101 minerals had been consumed. The pH in drainage from all pots was ~3 at the end of the experiments. First, the neutralization is conducted by carbonates, which is typically the dissolution of calcite (CaCO3). This process consumes 2 mols of H+ by 1 mol of calcite (Sherlock et al., 1995): CaCO3(s) + 2H+ → Ca2+ + H2O + CO2(g) Equation 5-9 According to the quantitative XRD results, the bauxite residue used for the experiments contains 3.1 % calcite (Table 5-2). Another alkaline material for amendments is lime (CaO), and the depletion of lime was quick due to the application of small amount of lime (2 % (w/w)), causing the pH in drainage became acidic in T+L and T+L+C. The reasons of fast depletion of lime in pots may be because: (1) 2% (w/w) amount of lime is not enough for neutralization; (2) the frequent watering (every other day) flushed the lime away in the early weeks. Lime neutralizes acidity through below reaction (Kastyuchik et al., 2016): CaO(s) + 2H+ → Ca2+ + H2O Equation 5-10 After the calcite becomes depleted, other carbonates, such as dolomite (CaMgCO3) and siderite (FeCO3), will continue to dissolve. Table 5-2 indicates it is siderite (FeCO3) that would dissolve after the calcite dissolution. However, the presence of siderite does not contribute to consume H+, because the oxidation from Fe2+ to Fe3+ and the subsequent hydrolysis of Fe3+ following siderite dissolution results in no consumption of acid overall (Jambor et al., 2003). FeCO3(s) + 2H+ → Fe2+ + H2O + CO2(g) Equation 5-11 Fe2+ + 14O2 + H+ → Fe3+ + 12 H2O Equation 5-12 102 Fe3+ + 3H2O → Fe(OH)3(s) + 3H+ Equation 5-13 The siderite originally existed in the tailings sample (7.6%), and then decreased in blank groups of unamended tailings and amended tailings. The percentages of siderite were not depleted in most pots, which maybe because of the non-homogeneous distribution of mineral particles; the kinetic reaction may also limit its dissolution (Gotor et al., 2000). The soil acidification exceeded the neutralizing capacity available from carbonate minerals from amendments. Carbonate buffering is followed by the dissolution of secondary Al-hydroxide minerals (Gunsinger et al., 2006; Johnson etal., 2000). Böhmite (AlO(OH)) and gibbsite (Al(OH)3) were two Al- hydroxide minerals detected in bauxite residue (Table 5-2), and were depleted in T+BR and T+BR+C at the end of experiments. This means the two minerals had been neutralized with acid during the experiments. Al(OH)3(s) + 3H+ → Al3+ + 3H2O Equation 5-14 AlO(OH)(s) + 3H+ → Al3+ + 2H2O Equation 5-15 Similarly, the Fe (oxy)hydroxide neutralization follows the Al hydroxide mineral neutralization. In this study, goethite (FeO(OH)) was the most common Fe oxyhydroxide mineral (30.6%) in bauxite residue, which decreased to only 0.9% in T+BR at the end of experiments. The percentages of hematite(Fe2O3) in T+BR also decreased, because the minerals dissolved into Fe hydroxide and then were neutralized with acid. FeO(OH)(s) + 3H+ → Fe3+ + 2H2O Equation 5-16 Fe2O3(s) + 3H2O → Fe(OH)3(am) Equation 5-17 103 Fe(OH)3(s) + 3H+ → Fe3+ + 3H2O Equation 5-18 The next acid-neutralizing process is silicate mineral dissolution at low pH values. Although the dissolution of silicate minerals can occur over the whole pH range, the rate of dissolution increase under low pH conditions (Gunsinger et al., 2006; Bain et al., 2000). Under these low pH conditions, however, the dissolution of silicates is substantially slower than the dissolution of the more-soluble carbonate and Al- and Fe- oxyhydroxide minerals (Blowes et al., 2014; Jambor & Blowes, 1998). The acid neutralization capacity of silicates is particularly significant in that most rock types are dominated by silicate minerals, and they are thus the significant overall source of acid neutralization in the long term (Weber et al., 2005). In this study, the silicate weathering process was the dissolution of sodalite (Na4(Si3Al3)O12Cl) that was detected in bauxite residue. Similar to other neutralizing minerals, sodalite also became depleted in T+BR and T+BR+C at the end of the experiments. The weathering process is: Na4(Si3Al3)O12Cl(s) + 12H+ → 4Na+ + 3Al3+ + Cl− + 3H4SiO4 Equation 5-19 In addition, the original percentages of silicates in tailings also decreased when it came to the end of experiments in amended tailings, such as clay minerals muscovite(KAl2AlSi3O10(OH)2) kaolinite (Al2Si2O5(OH)4), and celsian (Ba(Al2Si2O8)). The precipitation of barite (BaSO4) is a sign of dissolution of celsian(Table 5-2). The neutralization of these silicates weathering is: KAl2AlSi3O10(OH)2(s) + 10H+ → K+ + 3Al3+ + 3H4SiO4 Equation 5-20 Al2Si2O5(OH)4(s) + 6H+ → 2Al3+ + 2H4SiO4 + H2O Equation 5-21 Ba(Al2Si2O8)(s) + 8H+ → Ba2+ + 2Al3+ + 2H4SiO4 Equation 5-22 104 In conclusion, the acid neutralization processes are in the sequences: (1) dissolution of carbonates; (2) dissolution of Al- and Fe- (oxy)hydroxide; (3) silicate weathering. When one process ends or one type of mineral is exhausted, the next neutralization process takes over. The pH drops to 2 – 3 when all pH buffer minerals are consumed. As introduced in Chapter 2, the above discussed reactions control the mobility of metals and the availability of nutrients/cations to plant roots, including sulfide oxidation, carbonate dissolution, neutralization of Al- and Fe- (oxy)hydroxide with acid, and silicate weathering. In fact, there are different set of reactions happening in unamended tailings and various amended tailings, which will be introduced and discussed in the next section. 5.3.4 Geochemical processes in different amended tailings First, sulfide oxidation is the main geochemical process in all substrates, which is one of the soil acidification processes. Other soil acidification processes caused by root exudates and the dissolution of organic matter are organic processes. The geochemical processes refer to various inorganic-related processes depending on the different amendments. On unamended tailings, the only carbonate mineral is siderite. Although the dissolution of siderite does not contribute to the net capacity of acid neutralization, siderite still dissolves. The silicate minerals in tailings, i.e., muscovite (KAl2AlSi3O10(OH)2), kaolinite (Al2Si2O5(OH)4), and celsian (Ba(Al2Si2O8), dissolve under the acidic conditions, and then produce secondary minerals such as SiO2(am) and jarosite (K2Fe6(SO4)4(OH)12). On tailings amended with bauxite residue, as calcite (CaCO3) was detected in bauxite residue, the calcite dissolution is the first acid neutralization geochemical process occurring in T+BR and T+BR+C, followed by the siderite dissolution. Besides, böhmite (AlO(OH)), gibbsite (Al(OH)3), 105 and goethite (FeO(OH)) were Al- and Fe- (oxy)hydroxide minerals detected in bauxite residue, then the dissolution of these (oxy)hydroxide minerals occurred in the tailings amended with bauxite residue, including T+BR and T+BR+C. The following geochemical process is the weathering of silicate minerals, which is specifically sodalite (Na4(Si3Al3)O12Cl) in bauxite residue, except for muscovite (KAl2AlSi3O10(OH)2), kaolinite (Al2Si2O5(OH)4), and celsian (Ba(Al2Si2O8) in the original tailings. On tailings amended with lime, the most significant change from tailings is that the addition of lime increases the amount of calcium hydroxide (Ca(OH)2, and sometimes calcite (CaCO3) when exposed to air (Bertos et al., 2004). Therefore, the geochemical processes occurring in tailings amended with lime (T+L and T+L+C) also included the dissolution of calcium hydroxide (Ca(OH)2) and calcite (CaCO3 ), except for the geochemical processes already existing in tailings: dissolution of siderite and silicate weathering of muscovite (KAl2AlSi3O10(OH)2), kaolinite (Al2Si2O5(OH)4), and celsian (Ba(Al2Si2O8). On tailings amended with compost that contains large amounts of organic matter, the obvious change to tailings is the decomposition of organic matter causing significant soil acidification. This resulted in the pH of drainage from tailings being amended with compost decreasing to a level below that of unamended tailings. As this research is figuring out inorganic-related geochemical processes occurring is the rhizosphere of direct revegetation, the decomposition of organic matter is organic-related which is out of scope of this study and will not be discussed further. There is another type of organic-related process in the rhizosphere of all amended tailings with grass growing on, which is the process of soil acidification caused by root exudates. All grass could grow well on all tailings with amendments, and root exudates were limited on 106 some tailings due to limitations of grass growth, resulting in trends of pH and metals from unamended tailings being similar to those from blank groups without grass. Up to now, the inorganic-related geochemical processes happening in the rhizosphere in different amended tailings are basically figured out. Although the organic-related processes are not further discussed in this study, they affect the results of pH of drainage, metal mobility in soil, and the reaction rates of geochemical processes in various ways, i.e., the soil acidification caused by root exudates and organic matter decomposition increases the mobility of metals and decreases the pH of the soil; the increased acid production results in the depletion of neutralization capacity provided by amendments; the increased mobility of metals also means the increased ion activities leading to the different reaction rates of other geochemical processes. Overall, all the minerals causing part of geochemical processes in the six substrates are summarized in Table 5-4. Table 5-4. Minerals related to geochemical processes in the six substrates Sulfide oxidation Carbonates dissolution (Oxy)hydroxide dissolution Silicates weathering T Pyrite, Galena, Chalcopyrite, Sphalerite Siderite / Muscovite, Kaolinite, Celsian T+BR Pyrite, Galena, Chalcopyrite, Sphalerite Calcite, Siderite Gibbsite, Böhmite, Goethite, Hematite Muscovite, Kaolinite, Celsian, Sodalite T+L Pyrite, Galena, Chalcopyrite, Sphalerite Calcite, Siderite Calcium hydroxide Muscovite, Kaolinite, Celsian T+C Pyrite, Galena, Chalcopyrite, Sphalerite Siderite / Muscovite, Kaolinite, Celsian T+BR+C Pyrite, Galena, Chalcopyrite, Sphalerite Calcite, Siderite Gibbsite, Böhmite, Goethite, Hematite Muscovite, Kaolinite, Celsian, Sodalite T+L+C Pyrite, Galena, Chalcopyrite, Sphalerite Calcite, Siderite Calcium hydroxide Muscovite, Kaolinite, Celsian 107 5.3.5 Amendment effectiveness on metal stabilization It is interesting that not only pH, and concentrations of Cu, Pb, and Zn in drainage have a similar pattern among species, but also the concentrations of each metal in drainage from the different amended tailings have the same sequence in the 10th week (Figure 5-6, Figure 5-7, Figure 5-8, Figure 5-9). In the 10th week, the substrates’ order of metal concentrations from the largest to the smallest with different grass species were: T+C > T+L+C > T+BR+C > T+L > T+BR > T. Metal concentrations in drainage in all amended tailings were higher than that from unamended tailings, which means the mobility of Cu, Pb, and Zn was increased at the end of experiments even when the tailings were amended with alkaline materials. As discussed above, the pure tailings represented only one process of soil acidification that is sulfide oxidation, and other soil acidification processes were caused by the addition of compost and the growth of grass. Therefore, on the tailings amended with alkaline materials such as bauxite residue and lime, the influence of other soil acidification processes was significant enough to decrease the pH and release the metals. When the alkalinity or neutralization capacity was consumed (Bain et al., 2000), pH decreased and metals started to leach out around the 6th week of experiments. Because the metal concentrations in drainage from the compost application (T+C) at the end of experiments were higher compared to those from other amended tailings, and the following is T+L+C, the effects of compost are the most significant on soil acidification. When comparing the effects of bauxite residue and lime, metal concentrations in drainage from lime were always larger than those from tailings amended with bauxite residue: T+L+C > T+BR+C, T+L > T+BR, this is because the bauxite residue contains more weak base minerals such as böhmite (AlO(OH)), gibbsite (Al(OH)3) and goethite (FeO(OH)) that can change pH at a relatively slow rate compared to strong base minerals such as calcium hydroxide (Ca(OH)2) (Aitken & Moody, 1994; 108 De Vries et al., 1989). Thus, metals are better immobilized in tailings amended with bauxite residue rather than in tailings amended with lime. In conclusion, the ability to stabilize metals of amendments is: bauxite residue > Lime > compost. The conclusion of ability to stabilize metals of amendments is concluded from the metal concentrations in drainage, which can be a consideration for helping land reclamation and remediation practitioners to select the amendments materials on tailings reclamation. 5.4 Conclusions Several conclusions can be summarized here. First, the sources of As, Cu, Pb, and Zn are the sulfide minerals in tailings, and the oxidation of various sulfide minerals is the key process to release those metals into the surrounding environment. These processes are also acid-generating, which is one of the causes of soil acidification. The other two processes of soil acidification are the decomposition of organic matter and root exudates. Second, tailings, compost, and grass are the three main reasons driving soil acidification in this study, which is from sulfide oxidation, the decomposition of organic matter, and root exudates, respectively. With regards to the six substrates in the experiments, sulfide oxidation is the major soil acidification process in T; sulfide oxidation and root exudates are the two main acidification processes in T+BR and T+L; soil acidification in T+C, T+BR+C, and T+L+C are caused by all three processes. The acidification by sulfide oxidation as the main acidification process may be accelerated by the microbial activities, since the weak acid produced by root exudates and organic matter decomposition cannot decrease the pH to below 4. The organic-related microbial activities are beyond the scope of this study, but this aspect should be studies further in the future. 109 Third, the acid neutralization processes are in the sequence: (1) dissolution of carbonates; (2) dissolution of Al- and Fe- (oxy)hydroxide; (3) silicate weathering. When one process ends or one type of mineral become exhausted, the next neutralization process begins. The pH decreased to 2 – 3 when the pH buffer minerals are consumed. There are different sets of reactions occurring in unamended tailings and amended tailings. The ability to reduce metal leachability of amendments is: bauxite residue > Lime > compost. In conclusion, the above can basically answer the question of this chapter and answer the part of geochemical processes of the research question. This chapter has achieved the objective of evaluation of geochemical processes and reactions potentially controlling metal mobility in the rhizosphere by analysing the change of drainage chemistry and soil minerals. The significance of this chapter is providing a reference for policymakers to select the more effective amendment materials for phytostabilization on sulfidic mine tailings. 110 Chapter 6: Environmental Reliability of Phytostabilization on Sulfidic Mine Tailings4 6.1 Introduction The former two chapters have basically answered the research question of the metal immobilization of phytostabilization with regards to species selection and amendments selection (chapter 4), and geochemical processes in the rhizosphere (chapter 5). However, in order to get more comprehensive insight of phytostabilization on sulfidic mine tailings, this chapter is going to discuss the environmental reliability and potential of phytostabilization with regards to the metals remained in plant, pot soil, and drainage at the end of experiments. Figure 6-1 highlights the contents in research workflow. Phytostabilization has been considered much more beneficial than other direct revegetation techniques by researchers, because it can prevent toxic metals from entering into the surrounding environment, such as groundwater, air, and aboveground tissues of plants that may be eaten by animals, through stabilizing metals in the rhizosphere (Bolan et al., 2011; Egendorf et al., 2020; Mendez & Maier, 2008). Since the purpose of phytostabilization is to immobilize metals in the rhizosphere, this research discusses metal immobilization from various perspectives herein (Figure 6-1), and the results of plant growth, species selection, and amendments application have 4 A version of this chapter entitled “The environmental reliability of phytostabilization when metal immobilization in the rhizosphere is impeded by soil acidification” will be submitted for peer review and following publication. The section of Materials and Methods is not included because details of experiments were introduced in Chapter 3. 111 confirmed that successful phytostabilization with metal immobilization can be achieved with specific species and amendments during the experiments. Figure 6-1. The highlight of this chapter in the research workflow However, it was found in the experiments of this dissertation that the metal concentrations (Cu, Pb, and Zn) in drainage from amended tailings started to increase around the 6th or 7th week and reached elevated levels at the end of experiments. This was caused by three acidification processes as discussed in Chapter 5, but the results are counter to the aim of metal immobilization of phytostabilization. Thus, the question remains of the reliability of Mine Site Background Data Collection in Progress Greenhouse Experiments Native Species and Amendments Acquisition Tailings Acquisition Tailings Characterization Reliability of Phytostabilization Data Collection after experiments Drainage Chemistry Metals in Plants pH & Metals in Soil Geochemical Processes in the Rhizosphere Species Selection and Amendments Efficiency Conclusions and Suggestions Materials Characterization before experiments Pots Experiments Setup 112 phytostabilization to keep the surrounding environment safe from metal contamination: is phytostabilization a reliable technique to keep metal immobilization when established successfully on sulfidic mine tailings? The objective of this chapter is to provide more insight into phytostabilization related to metal stabilization by analyzing the plant growth, soil quality, and drainage quality at the end of experiments. It is significant to conduct this discussion of the potential of phytostabilization and provide more useful and practical measures to enhance phytostabilization at sulfidic mine tailings in the field application afterwards. 6.2 Reason of grass death It was observed that grasses were dying at the end of the experiments. Since the pH of amended tailings dropped to 3 – 4 at the end of experiments, it is questionable if the grasses died from natural aging or the acidity of pot substrates. It has to be clarified that annual ryegrass is used in this work. Annual plants grow for one season and then die naturally, while the aboveground portion of perennial plants die off after one growing season and plant returns from the base and rootstock every spring (Garnier, 1992). Three reasons are considered here to think the natural aging mainly caused the death of grasses, while soil acidity has slight impacts. First, the experiment duration (84 days) was designed depending on the general lifespan of grass which is around three months (Bélanger et al., 1992). Bai et al. (2010) observed the root lifespan of Leymus chinensis (wheat or Chinese ryegrass) in a typical steppe of Inner Mongolia was 81 – 103 days. Van der Krift and Berendse (2002) found the average root life span of Lolium perenne L. (wheat) were 14 weeks. Therefore, the designed planting duration was a reasonable period for planting grass species in the greenhouse experiments. Especially for studies of mine tailings 113 revegetation (Alcantara et al., 2015; Leung, 2018; Mendez et al., 2007), grass species were expected to enter into the end of their life cycle. Figure 6-2. Seed production of wheat (a) and ryegrass (b) at the end of the experiments Second, an important sign of the grasses was dying because of aging instead of acidity is that the wheat and ryegrass produced seeds at the end of the experiments (Figure 6-2). Seed production is one of the most critical processes in the life history of plants, with the completion of the life cycle and regeneration of a population depending on reproduction (Li et al., 2021). Generally grass growth can be described by three stages: seed germination (1 – 2 weeks), growth stage (3 – 16 weeks), and reproductive stage (a few days), and the new life cycle of plants will begin when the seeds fall into the soil (Malaviya et al., 2013). Therefore, the seed production of wheat and ryegrass means the grasses had successfully entered into their end of life cycle and would die due a. wheat seeds b. ryegrass seeds 114 to aging. However, seed production of tall fescue and sheep fescue was not observed, but they were still green and alive at the end of experiments (Figure 3-7). Third, the relationships between grass growth and pH in pot substrates after the experiments are shown in Figure 6-3. The discrete points indicate the total biomass and shoot heights of four types of grass species are not significantly relevant to the pH in the soil at the end of experiments. Figure 6-3. The relationships between total biomass and pH in soil, shoot heights, and pH in the soil after experiments. Combined with the above discussion, acidity is not likely the cause of death of wheat and ryegrass. For tall fescue and sheep fescue, growth on all pot substrates was limited on either unamended or amended tailings (Figure 4-2 and Figure 4-3). This may be because the integrated physiochemical conditions of those pot substrates were not good for the growth of tall fescue and sheep fescue, such as the acidity, porosity, hydraulic conductivity, nutrient contents, and microbial communities, but the acidity is not likely the sole reason that limited their growth. This conclusion can be also supported by the fact that tall fescue and sheep fescue did not grow well even with amended tailings, indicating that these species are not good candidates for revegetation 115 or phytostabilization on sulfidic mine tailings. While in most cases grass yield can be improved by increasing pH (Fonseca et al., 2020; Hale et al., 2020), Edmeades et al. (1991) found increasing soil pH from 4.7 to 6.0 had no significant effect on the yield of ryegrass but significantly decreased yields of paspalum and veld grass. Therefore, for some acid-tolerant species, more factors should be considered rather than solely adjusting pH when establishing revegetation on tailings. In summary, experimental evidence suggests that the death of wheat and ryegrass was caused by aging instead of acidity. To establish sustainable phytostabilization on sulfidic mine tailings by using wheat and ryegrass, it is suggested that reseeding of these two species is necessary before they enter the end of their life spans. 6.3 Soil quality 6.3.1 Metal concentrations in soil after experiments Metal concentrations in soil after the greenhouse experiment are shown in Figure 6-4. The red dashed line in each graph represents the metal concentrations in the original tailings before experiments. When compared to the original concentrations in tailings, all metal concentrations (As, Cu, Pb, and Zn) in the substrate of unamended tailings without amendments did not change significantly at the end of experiments, which means plant uptake and drainage did not remove a significant amount of As, Cu, Pb, and Zn from pure tailings. This is because of the decrease of metal concentrations in the soil resulting from the uptake by plants and metal release along the drainage. The uptake by plants on tailings without amendments was low due to low germination rates (Figure 4-2) and plant biomass (Figure 4-3), and the Cu, Pb, and Zn in drainage (Figure 5-7, Figure 5-8, Figure 5-9) were minimal at the end of experiments, as compared to other 116 amendments. Thus, in unamended tailings, the metals in pure tailings at the end of experiments were still at similar levels as the original tailings. As was not detected in most drainage samples, which means the majority of soluble As was absorbed by plants or soil. This can also be proven by the distribution of metals in plants, soil, and drainage (Figure 4-3). The metal concentrations in other amended tailings can be compared to those in the original tailings (Figure 4-3). As and Cu concentrations in all amendments were reduced significantly lower compared to that in the original tailings. The Pb concentration was the lowest in T+BR, whereas in T+L, T+C, T+BR+C, and T+L+C concentrations were slightly less than that in the original tailings. Apparently, the different amendments have various effects on the metal concentrations in after experiments soil. In Figure 6-4, As, Cu, and Pb concentrations in different amendments show similar trends, whereas there are differences in Zn concentration. As, Cu, and Pb concentrations were the highest in T+C and the second highest in T+BR+C after experiments; conversely, Zn concentration was the lowest in T+BR+C and the second lowest in T+C. This means Zn was taken up by plants or was released more when tailings were amended by compost or bauxite residue and compost together than in other amended tailings. Besides, while As, Cu, and Pb concentrations in T+BR were significantly lower, the Zn concentration in T+BR was basically at the same level of as of the original tailings. Last, the remaining concentrations of As, Cu, Pb, and Zn represent the different behaviors of plant uptake and leachability in the drainage of these four metals, which is an integrated result of geochemical interactions affecting the mobility of metals. 117 *The red dash line represents metal concentrations in the original tailings before experiments. Figure 6-4. Metal concentrations in soil after experiments compared to their original concentrations in tailings. Compared to amendments that play a dominate role in metals stabilization, plants had slight effects on the remaining metal concentrations in soil after experiments. First, on either unamended tailings or amended tailings, the cluster of points representing the metal concentrations was similar in each type of amended substrates (Figure 6-4). This means the grass types had little influence on soil metal concentrations for either unamended or amended tailings, so geochemical processes in each pot substrate even with different grasses were generally similar. On five substrates of amended tailings, it can be seen that the blank groups without plants (purple dots) contained slightly higher metal concentrations than other groups with plants. 118 Also, the cluster of points of metal concentrations in soil indicates the grass types had little influence on soil metal concentrations for either unamended or amended tailings, so geochemical processes in each pot substrate, even with different grasses, may be generally similar. The metal concentrations in each component (plant, soil and drainage) are the results of root uptake, geochemical processes, and water-soil interactions. Thus, if there is no plant uptake, the remaining metal concentrations in the soil should be higher than those in planted soil. Therefore, blank groups without plants in Figure 6-4 contained slightly higher metal concentrations than other groups with plants, because no plant uptake of metals in blank groups resulted in slightly higher concentrations of remained metals than those in amended substrates. 6.3.2 Metal distribution The percentages of metal distribution in plants, soil, and drainage after experiments are illustrated in Figure 6-2. As it is concluded in Chapter 4 that wheat and ryegrass are suitable candidates for phytostabilization due to their good survival rates and higher metal accumulation in roots, wheat and ryegrass are selected for analyses the metal distribution in this part. It can be seen from Figure 6-2 that the metal distribution in plant, soil, and drainage for wheat and ryegrass on each substrate were generally similar, which means grass species types do not have significant effects on metals distribution. Figure 6-5 indicates that the upper bars representing metals in plants and the middle bars representing metals in soil were the majority of the total metals. The percentage of distribution at the end of experiments is calculated as below, assuming the sum of total metal concentration in drainage, plant, and soil is equal to the total metal concentration in original tailings in the 119 corresponding pot. The reason for not using the data of total metals in original tailings in Table 3-2 is because this number is only for one sample and ignores the heterogeneity among pots: 𝐠𝐠𝐠𝐠𝐬𝐬𝐠𝐠𝐠𝐠𝐠𝐠𝐧𝐧𝐧𝐧𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠 𝐩𝐩𝐠𝐠𝐠𝐠𝐩𝐩𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠 = 𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐭𝐭 𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐭𝐭 𝐩𝐩𝐠𝐠𝐠𝐠𝐩𝐩𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠 𝐠𝐠𝐠𝐠 𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠,𝐩𝐩𝐭𝐭𝐠𝐠𝐠𝐠𝐠𝐠,𝐬𝐬𝐠𝐠𝐠𝐠𝐭𝐭 𝐠𝐠𝐠𝐠 𝐠𝐠𝐠𝐠𝐠𝐠𝐬𝐬𝐧𝐧𝐠𝐠 𝐠𝐠𝐨𝐨 𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐭𝐭 𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐭𝐭 𝐩𝐩𝐠𝐠𝐠𝐠𝐩𝐩𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠 𝐠𝐠𝐠𝐠 𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠𝐠,𝐩𝐩𝐭𝐭𝐠𝐠𝐠𝐠𝐠𝐠,𝐬𝐬𝐠𝐠𝐠𝐠𝐭𝐭 𝐠𝐠𝐠𝐠 𝐠𝐠𝐠𝐠𝐠𝐠 Equation 6-1 The percentage of metals in drainage is extremely small with 0 – 2 % of As, 0 – 3 % of Cu, 0 % of Pb and 0 – 9 % of Zn. This means the majority of metals in the original tailings were stabilized in the rhizosphere either by plant uptake or stabilization in soil. On unamended tailings, it needs to be pointed out that although the influence of root exudates on soil acidification is not significant due to the poor survival of plants on tailings, the percentage of metal uptake by the roots was still significant. This is because the original tailings have the potential to release much more soluble metals that can promote passive adsorption by roots (Epstein, 1955), resulting in the metal uptake calculated in mg/kg unit of grass does not appear small when compared to the proportions of retained metals in soil. As and Cu concentrations were distributed in plants and soil nearly equally on all amendments, except As was higher in plants (64 – 66 %) and lower in soil (33 – 36 %) on tailings amended with compost. This may be because the applied compost contained more organic material that binds with arsenic, e.g., organic acid, which enhanced the arsenic chelation and uptake by plants (S. Wang & Mulligan, 2006). Compared to As and Cu, Pb was higher in soil and lower in plants, especially the percentages in soil for T+C and T+BR+C that were higher (70 – 80 %) among the amendments. This can explain why high Pb concentrations remained in T+C and T+BR+C after experiments (Figure 6-1) due to lower root uptake of Pb. Conversely, Zn concentrations were higher more in plants and lower in soil, and the percentages of Zn released in drainage were noticeable. Zn distributed only 16 – 19 % on T+C and 23 % on T+BR+C, which is consistent 120 with the results of the lowest Zn concentrations in T+C and T+BR+C in Figure 6-4. It was also found that Zn became more soluble and released more in drainage in amended tailings than in unamended tailings. Therefore, Zn tended to be absorbed by plants and released more on amended tailings. Figure 6-5. Metal distribution in plants, soil, and drainage at the end of experiments Overall, compared to the unamended tailings, amendments improved the percentages of As, Cu, and Pb that remained in soil and reduced uptake by plants. Amendments increased Zn concentrations in drainage which means the amendments will enhance the Zn leachability to the surrounding environment. According to the definition of direct revegetation and phytostabilization, while one of the revegetation purposes of sulfidic mine tailings is to grow 121 plants with prevention of metals or toxic contaminants from being released into the surrounding environment, phytostabilization targets metal immobilization in the rhizosphere where metals can precipitate and be retained in stable minerals, or be absorbed and accumulated by roots without being transferred to the aboveground tissues of plants. When comparing the metal concentrations in drainage in T+BR+C and T+L+C to those in T+BR and T+C, respectively, we can see the addition of compost releases more Zn in drainage, so compost is not ideal for the phytostabilization of Zn. Therefore, bauxite residue, lime, and compost can aid phytostabilization in stabilizing As, Cu, and Pb; however, compost and its combinations with other amendments are not ideal for the phytostabilization of Zn on sulfidic mine tailings. These conclusions are consistent with the discussion in Chapter 4. 6.4 Drainage quality It is noticeable that the metal concentrations in the drainage from amended tailings ended up increasing to high levels, which leads to a concern about the reliability of phytostabilization in metal stabilization. It can be seen from Figure 5-3, Figure 5-4 and Figure 5-5 that the Cu, Pb and Zn concentrations were maintained at extremely low levels before the 6th week, compared to those after the 6th week, which means the amendments likely had positive impacts on reducing the leachability of the metals and improving the drainage quality. In order to figure this out, Figure 6-6 shows the changes in pH and metal concentrations in drainage from pots with wheat (a) and ryegrass (b) during the first six weeks. The results are received under the situation of successful phytostabilization because wheat and ryegrass act effectively for metal stabilization in roots, and the metal percentages that remained in the rhizosphere are higher in amended tailings (Figure 6-5). During the first six weeks, it can be seen that compared to those from tailings without amendments, the concentrations of Cu, Pb, and Zn were lower in drainage from all 122 amended tailings, and the pH of drainage from amended tailings was higher. The mobility of Cu, Pb, and Zn was reduced by all amendments, and tailings amended with compost released more Pb (yellow line) and Zn (purple line). Therefore, amendments added to sulfidic mine tailings can effectively reduce the leachability of metals and enhance the environmental reliability of phytostabilization for a short-term period before the acidification processes overrides the neutralization processes. The effectiveness of amendments as immobilizing agents to reduce the leachability of metals has been indicated by a number of studies either in the greenhouse or in the field (Alvarenga et al., 2009; Fellet et al., 2011; Hashimoto, 2013; Houben et al., 2013). 123 Figure 6-6. Changes in pH and metal concentrations in drainage during the first six weeks 124 To achieve long-term metal immobilization and sustainability of phytostabilization, re-application of amendments may be necessary (Honeker et al., 2019; Valentín-Vargas et al., 2018), as well as re-seeding and monitoring throughout the stage (Cunningham & Berti, 2020). The species of grass, brush and trees can be re-seeded and the amendments can be re-applied until a sustainable ecosystem is established in the field, as long as their values have been proven for phytostabilization. Therefore, the reliability of phytostabilization should not be obscured due to elevated levels of metal concentrations in drainage. The long-term reliability of phytostabilization is likely to be achieved by various methods, such as applying different amounts of amendments, reapplication of amendments, applying new amendments to initiate neutralization processes, and re-seeding plants. Based on this study on the geochemical processes affecting the metal mobility in the rhizosphere, further research could explore new materials to amend the sulfidic mine tailings. 6.5 Conclusion Several conclusions regarding to the environmental reliability of phytostabilization can be summarized here. First, the acidity is not the main reason causing the death of species. The death of wheat and ryegrass was caused by aging instead of acidity. The acidity did not cause tall fescue and sheep fescue to die, but integrated factors limited the growth of these two species. This is consistent with the conclusions of Chapter 4: wheat and ryegrass are good candidates for phytostabilization on sulfidic mine tailings, but tall fescue and sheep fescue are not. Second, the metal stabilization in the rhizosphere was improved by amendments. The remaining concentrations of As, Cu, Pb, and Zn are due to the different behaviors of plant uptake and leachability, an integrated result of geochemical interactions affecting the mobility of metals. 125 While plants can improve metal uptake and reduce the remaining metals in pot substrates, and amendments can help improve metals remaining in soil and reduce their uptake by plants. The amendments used in this study can aid phytostabilization on stabilizing As, Cu, and Pb, whereas these amendments and even their combinations are not ideal for the phytostabilization of Zn on sulfidic mine tailings. These conclusions are consistent with the discussion in Chapter 4. Third, drainage quality can be maintained within a short-term period for the phytostabilization with amendments. The long-term reliability of phytostabilization may be achieved by various methods, such as applying different amounts of amendments, re-applying amendments, applying new amendments to initiate neutralization process, and re-seeding plants. In summary, the above discussion can answer the question of this chapter and answer the part of the environmental reliability of the research question. This chapter has achieved the objective of exploring more insight of phytostabilization regarding to metal stabilization by analyzing the plant growth, soil quality and drainage quality at the end of experiments. This chapter is significant to provide more insight into phytostabilization on sulfidic mine tailings, and can guide future application of the technique in practice. 126 Chapter 7: Conclusions and Suggestions This research addressed the question: what are the geochemical processes affecting metal immobilization in the rhizosphere following the establishment of phytostabilization on sulfidic mine tailings. A greenhouse study using sulfidic mine tailings plus amendments was sowed with four different plant species. Analytical work included geochemistry of tailings, chemical testing of the drainage as well as the planting media. It was found that the combined application of amendments can help more efficient establishment of phytostabilization. In addition, neutralizing geochemical processes, such as carbonate dissolution, (oxy)hydroxide dissolution, and silicate weathering, can be initiated and enhanced as much as possible by combined amendments in the rhizosphere. 7.1 Concluding remarks When establishing phytostabilization on sulfidic mine tailings which have large potential to generate acidity and release toxic metals, the phytostabilization’s purpose of metal stabilization in the rhizosphere needs to be maintained. This thesis is devoted to exploring metal stabilization of phytostabilization concerning species selection, amendment effectiveness, and geochemical processes. In order to reach this objective, updated studies on phytostabilization of sulfidic mine tailings and key factors affecting the effectiveness of phytostabilization have been reviewed (Chapter 2). Most cases of mine reclamation in practice use a multilayers covering method, while direct revegetation, which is more cost-effective and material-saving, has not been broadly applied at sulfidic mine sites due to the large potential of acid generation and metal release. Furthermore, studies of phytostabilization focus on species and amendment selection, and organic-related microbial activities to ensure the success of phytostabilization, whereas the 127 inorganic-related geochemical processes of metal immobilization in the rhizosphere in sulfidic mine tailings are less well-studied. Consequently, this research conducted greenhouse experiments using tailings from a lead-zinc mine (Chapter 3). Four grass species and three types of amendments were chosen for the three-month greenhouse trials to evaluate their effectiveness for phytostabilization. First, grass growth and metal uptake by plant shoots and roots were evaluated, and then two species were identified as suitable candidates for phytostabilization along with three amendments having various positive effects on plant growth (Chapter 4). In addition, changes in pH and metal concentrations in the drainage from the unamended and amended tailings were analyzed, the mineralogy of materials before and after the experiments was characterized, and the geochemical processes of metal stabilization in the rhizosphere were discussed (Chapter 5). Moreover, based on the results of Chapter 5, the reason for grass death, soil quality, and drainage quality were discussed to obtain insight to the environmental reliability of phytostabilization on sulfidic mine tailings (Chapter 6). Overall, this thesis has answered the research question and reached the research objectives. One conclusion of this work contributing to the industry is wheat and ryegrass can be the first choice of grass species to be planted for phytostabilization on sulfidic mine tailings, and the combination of amendments needs to be applied into tailings to initiate more possible neutralization processes and neutralize the acidification. 7.1.1 Species selection and amendments effectiveness First, it is concluded that wheat and ryegrass are good candidates for phytostabilization as they grow better than tall fescue and sheep fescue on amended tailings, and accumulate more metals in roots than that in aboveground shoots. Second, the three amendments used in this study can 128 improve the deleterious conditions of tailings and significantly enhance plant growth. The application of bauxite residue can limit the metal uptake because of its capacity to increase pH and reduce metal solubility. Lime can have an immediate effect on seed germination, but may not provide a lasting effect on promoting metal uptake. Compost is concluded to be a robust and cost-effective amendment to improve plant survival and enhance phytostabilization, but adding compost into tailings does not achieve effective phytostabilization of Zn. However, compost is still a more cost-effective amendment than lime and bauxite residue to improve plant survival and enhance the phytostabilization of As, Cu, and Pb. Chapter 4 has reached the objective of evaluation the candidacy of the four selected plant species to stabilize metals for phytostabilization on sulfidic tailings and comparison of the effectiveness of different amendments: organic matter, lime and bauxite residue on enhancing plant growth and metal immobilization for phytostabilization. 7.1.2 Geochemical processes in the rhizosphere First, the sources of As, Cu, Pb, and Zn are the sulfide minerals in tailings, and the oxidation of various sulfide minerals is the key process of metal release into the surrounding environment. These processes are also acid-generating, which is one of the causes of soil acidification. The two other processes of soil acidification are the decomposition of organic matter and root exudates. Second, tailings, compost, and grass are the three main reasons causing soil acidification, which are related to sulfide oxidation, the decomposition of organic matter, and the release of root exudates, respectively. For the six substrates in the experiments, sulfide oxidation is the major soil acidification process in T; sulfide oxidation and root exudates are the two main acidification 129 processes in T+BR and T+L; soil acidification in T+C, T+BR+C and T+L+C is caused by all three processes. Third, the acid neutralization processes occur in the following sequence: (1) dissolution of carbonates; (2) dissolution of Al- and Fe- (oxy)hydroxide; (3) silicate weathering. When one process ends or one type of mineral become exhausted, the next neutralization process takes over. The pH will dropped to 2 – 3 when all the pH buffer minerals were consumed. All the above-discussed reactions control the mobility of metals and the availability of nutrients/cations to plant roots, including sulfide oxidation, carbonate dissolution, neutralization of Al- and Fe- (oxy)hydroxide with acid, and silicate weathering. There are different sets of reactions occurring in amended tailings depending on the various minerals contained in amendments. The ability to reduce metals leachability of amendments is: bauxite residue > lime > compost. Chapter 5 has achieved the objective of evaluating geochemical processes and reactions potentially controlling metal mobility in the rhizosphere by analysing the change of drainage chemistry and soil minerals. The significance of this chapter is providing reference to land reclamation and remediation practitioners to select the more effective amendment materials when designing phytostabilization on sulfidic mine tailings. 7.1.3 Environmental reliability First, the acidity is not the main cause of the death of species. The death of wheat and ryegrass was caused by aging instead of acidity. The acidity did not cause tall fescue and sheep fescue to die, but integrated factors limited the growth of these two species. Second, the metal stabilization in the rhizosphere was improved by amendments. The remaining concentrations of As, Cu, Pb, and Zn are due to the different behaviors of plant uptake and 130 leachability, an integrated result of geochemical interactions affecting the mobility of metals. While plants can improve metal uptake and reduce the remaining metals in pot substrates, and amendments can help improve metals remaining in soil and reduce their uptake by plants. The amendments used in this study can aid phytostabilization on stabilizing As, Cu, and Pb, whereas these amendments and even their combinations are not ideal for the phytostabilization of Zn on sulfidic mine tailings. Third, drainage quality can be maintained within a short-term period for the phytostabilization with amendments. The long-term reliability of phytostabilization may be achieved by various methods, such as applying different amounts of amendments, re-applying amendments, applying new amendments to initiate neutralization process, and re-seeding plants. Chapter 6 has achieved the objective of exploring more insight of phytostabilization regarding to metal stabilization by analyzing the plant growth, soil quality and drainage quality at the end of experiments. This chapter is significant to provide more comprehensive insight of phytostabilization on sulfidic mine tailings, and giving confidence and reference to land reclamation and remediation practitioners to implement the technique of phytostabilization in practice. 7.2 Major contributions and significance 7.2.1 Theoretical contribution This work fills a research gap in inorganic-related geochemical processes affecting the metal stabilization of phytostabilization on sulfidic mine tailings. It has been shown that with phytostabilization on sulfidic mine tailings, except for the neutralization capacity and metal 131 immobilization improved by amendment materials, the acidification processes and metal release caused by the decomposition of organic matter and root exudates have significant effects in the rhizosphere. To successfully establish the phytostabilization on sulfidic mine tailings, the neutralization processes need to be promoted constantly throughout the stage of phytostabilization until a sustainable ecosystem is established. This work is highly significant, as sulfidic mine tailings have been being produced at metalliferous mine throughout the world. 7.2.2 Applied contribution It is significant to find two grass candidates for phytostabilization: wheat and ryegrass can be used as pioneer grass species in the field when establishing phytostabilization on sulfidic mine tailings. They are native and easily available in North America and can provide organic matter to the tailings after their life cycle and decomposition after the plant growing season. The evaluation of amendments has implications for reducing the traditional material requirements, such as rocks and topsoil, for mine reclamation. Fewer and more cost-effective materials can be used to reach efficient outcomes of phytostabilization, especially when wastes (compost and bauxite residue in this study) can be recycled to make this progress both environmentally and economically feasible. The findings of inorganic-related geochemical processes and reactions potentially controlling metal mobility in the rhizosphere have implied the importance of neutralization processes promoted by amendments. This provides land reclamation and remediation practitioners with information on more effective amendment materials for designing phytostabilization on sulfidic mine tailings. 132 This study also indicates that the short-term reduction of metals leachability during phytostabilization can be maintained by applying useful amendments. The long-term reliability of phytostabilization is most likely to be achieved by various methods, such as applying different amounts of amendments, re-applying amendments, applying new amendments to initiate neutralization process, and re-seeding plants. The research uses drainage from pots from greenhouse trials of phytostabilization and shows that drainage chemistry provides excellent insights into roots-tailings interactions. The methods can be used as a reference for larger-scale trials in the field, and then reveal more findings regarding to the processes of metals stabilization. This research also improves existing tools for direct revegetation on sulfidic mines, which is critical for metals control, landscape recovery, and reducing the impacts of mining activity on the environment. It provides information for land reclamation and remediation practitioners reference when designing phytostabilization on sulfidic mine tailings at the mine closure stage. 7.3 Limitations and suggestions Further investigation on the insight of phytostabilization should be expanded to more types of metalliferous mines, such as copper, antimony, gold, since tailings used in this study were from a lead-zinc mine. This study can be expanded upon in three key aspects: experiments, analytical methods, and application. 7.3.1 Experimental studies Further experiments and research on roots-tailings interactions are recommended. In particular, it would be useful to explore deeper of the coupling processes between the organic-related 133 processes and inorganic-related processes, seeking more efficient ways to stabilize metals in the rhizosphere. Continued research is suggested at larger spatial scales in greenhouses or the field and, extending the period of the experiments by choosing species with longer lifespans, and increasing the number of repetitions of the experimental and control groups. Further investigation should focus on the combined application of amendments instead of sole application to obtain integrated views of metal stabilization. Since each amendment material may only promote one or two types of metal stabilization, and organic residue can provide necessary organic matter, nutrients, and cause acidification at the meantime, comprehensive results of amendment effectiveness on metal immobilization should therefore be studied to ensure the sustainability of phytostabilization. The various effects from different organic amendment on acidification and metal behaviours should be studied further, because organic compound may have various binding processes affecting the speciation of different metals. 7.3.2 Analytical methods In general more samples should have been taken for analyses including QXRD of the heterogeneous tailings. A lack of analytical data from enough samples is one of the limitations that limited the ability to get a clearer understanding of geochemical processes. Sequential extraction of for analyzing the different portions of metals (e.g., exchangeable state, carbonated-bound state, Fe and Mn oxides-bound state, organic-bound state, and residual state) is recommended, which can help to better understand the processes of metal stabilization. Scanning electron microscopy is suggested to help understand the binding state of metals in the rhizosphere. Redox potential (Eh) of pot substrates and drainage is suggested to help understand 134 the redox environment and the extent of sulfide oxidation. Evaluating the organic matter in pot substrates is also recommended to understand the extent of organic matter decomposition. The types or colonization of microbes are recommended to be further investigated, as the changes in microbial communities before and after the vegetation establishment may have a big impact on the acidification processes and behaviours of different metals. Furthermore, the mass balance is a helpful interpretation method for this study but is limited by the lack of analytical data, so the further study should pay more attention to this aspect. There is data of total metals of just one tailings sample, which limits the possibility to do valuable mass balance of metals from the beginning to the end of experiments because of the heterogeneity among pots. The heterogeneous errors among pots can be mainly caused by special heterogeneity in tailings, while metals from compost and bauxite residue could be quite small and may cause further difficulties in interpretation. The data of total metals in each pot before sowing is suggested to be obtained to conduct more accurate mass balance and understand the fates of total metals. QXRD analyses of more tailings samples in each pot can also help to reduce the errors caused by heterogeneity, and help to evaluate the mass balance. Although the total metals in water, compost, and bauxite residue may be quite small, the tiny error from each may cause a large error together in mass balance. Therefore, the total metals in other materials besides tailings should be analyzed in future research. Water balance is also important for water management in phytostabilization. The volume of watering every time and the volume of drainage samples need to be recorded, which can help to calculate the water balance. 135 7.3.3 Application considerations Before applying the proposed species and amendments at mine sites, small-scale trials are always suggested to be conducted either in the greenhouse or in the field. Doing so would allow for adjustments based on variations of sulfides and climate conditions at different mine sites. When applying the phytostabilization for mine tailings in the field, the heterogeneity of tailings at different locations should be considered since the selected species and amendments may be only appropriate for specific species and amendments. Therefore, small-scale trials need to be conducted on tailings with different properties even on the surface of one tailings facility being decommissioned. The application of compost, a municipal waste, is cost-effective and can improve plant growth and stabilization of As, Cu, and Pb. 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Rinse pH: The pH of tailings, substrates, and soil was tested by a pH electrode by mixing with water by volume 1:5 (Thomas, 1996) in the Coal & Mineral Processing Laboratory (CPML) at the UBC Mining Engineering department. 2. Particle-size distribution: All tailings samples are to be air-dried at room temperature (20 – 30°C) and sieved from bulk (whole, unaltered sample) through a 2 mm (No.10) screen to separate gravel particles. The percentage of sand, silt, and clay was determined using a Malvern Mastersizer 2000 in the Coal & Mineral Processing Laboratory (CPML) at the UBC Mining Engineering Department. 3. Hydraulic conductivity: The falling head method was applied in acrylic columns (40 cm long, 10 cm in diameter, and 78.5 cm2 in area). Columns filled with tailings are weighed after draining to determine field capacity. The test was done in the Coal & Mineral Processing Laboratory (CPML) at UBC Mining Engineering department. 4. Bulk Density: The density of tailings was analyzed using the core method described by the Canadian Society of Soil Science (Gregorich & Carter, 2007). Soil bulk density is the ratio of the mass of oven-dried (105 °C) solids to the bulk volume of the soil, which includes the volume of the solids and the pore space between the soil particles. The test was done in the Coal & Mineral Processing Laboratory (CPML) at the UBC Mining Engineering department. 5. Porosity: The the measured volume of water is poured from the graduated cylinder slowly into the graduated beaker containing the same volume of tailings, stopping when 160 the level of water just reaches the top of the tailings, and then recording the amount of water left in the graduated cylinder (which is the pore space volume of the tailings). The porosity is equal to the pore space volume / total volume. The test was done in the Coal & Mineral Processing Laboratory (CMP) at the UBC Mining Engineering department. 6. Cation exchange capacity (CEC): Tailings samples were sent to the BC Analytical Laboratory for effective CEC analysis of BaCl2 exchangeable cations (Al, Ca, Fe, K, Mg, Mn, Na). They used the 0.1 N Barium Chloride extraction method according to the Canadian Society of Soil Science (Gregorich & Carter, 2007). 7. Quantitative XRD: Tailings and bauxite residue were grounded into powder material with a particle size < 420 µm (40 mesh), and then 10 g of each sample was sent to the Department of Earth, Ocean and Atmospheric Sciences at the UBC for quantitative X-ray diffraction (XRD) analysis. The equipment is Bruker D8 Advance with a LynxEye XE detector. 8. Acid-base accounting: The total S, sulfate S, and TIC (total inorganic carbon) were measured in the BC analytical lab. Total S was analyzed by ICP-MS after full digestion. Sulfate S was measured by ICP-MS after 500 ppm calcium phosphate extraction. TIC was tested by SKALAR Primacs after acidification and combustion at 150 °C. Then, NAP was calculated based on these results according to the modified Sobek Method. AP and NP typically are expressed in units of (Sobek, 1978): kgCaCO3equivalentsmetric ton(t)of sample/rock same as kg CaCO3eqt = g CaCO3kg = t CaCO3eq1000t AP = Sulfide S =Total S –Sulfate S 161 NPCalculated �kgCaCO3t �=%TIC×83, Net Acid Generation Potential (NAP) =AP – NP. 9. Drainage chemistry: Samples for metal analysis were acidified to pH < 2 by 2 % HNO3, and then filtered by 0.45 um filters before being sent to the Department of Earth, Ocean and Atmospheric Sciences at the UBC for ICP-OES analysis. Samples for anion (SO42-, Cl-, NO3-) analysis were filtered by 0.45 µm filters and sent to thr BC Analytical Laboratory for analysis. Alkalinity was titrated by an ABU93 TRIBURETTE located in Michael Smith Laboratories in the department of Chemistry at the UBC. 10. Total metals in soil: Soil samples were collected and dried in an oven at 30°C after plant harvesting, and then sent to the BC Analytical Laboratory for total metal analysis. Samples were fully digested in a closed vessel microwave by strong acid, and elements were measured by ICP-MS. 11. Total metals in plants: At the end of the experiments, all the plants were harvested as shoots and roots separately. Roots were separated from dried soil and then washed with tap water with deionized water three times. All plant samples were dried in an oven at 105 °C until their weights were consistent. The dry weights of samples were recorded as biomass before total metal analysis. The plant samples were sent to BC Analytical Laboratory and then determined by ICP-MS after full digestion in a closed vessel microwave by strong acid. 12. Qualitative XRD: Pot samples were oven-dried at 30°C, homogenized, grounded into particle size less than 45 µm, and then milled to the optimum grain-size range for XRD 162 analysis (<10 µm). XRD analysis was conducted by Rigaku MiniFlex600 in room 503 of Frank Forward Building at the UBC. 163 Appendix B: Refinement plots of quantitative X-ray powder diffraction and qualitative X-ray powder diffraction Before the experiments, two samples, tailings (2 – 3 m) and bauxite residue, were sent to the Department of Earth, Ocean & Atmospheric Sciences at the UBC for quantitative XRD analysis by using the Rietveld method and X-ray power diffraction data in June 2021. The Rietveld refinement plots are shown in Figure B-1 and Figure B-2. After the experiments, four-pot samples: Tailings Blank, T+BR Blank, T+L Blank, and T+C Blank, were sent to the Department of Earth, Ocean & Atmospheric Sciences at the UBC for quantitative XRD analysis using the Rietveld method and X-ray power diffraction data in April, 2022. The Rietveld refinement plots are shown in Figure B-3 to Figure B-6. All the remaining pot samples not sent for quantitative XRD analysis were conducted qualitative XRD analysis with a Rigaku MiniFlex600 in room 503 of Frank Forward Building at the UBC in March 2022. As it was found that there were no new minerals produced in pots with all grass species and all amendments after the experiment other than those results from the quantitative XRD, the patterns of qualitative XRD are not shown for all samples. There were 30 pot samples after the experiment, and the pot samples of the tailings group (T-WG, T-RG, T-TF, T-SF, and T-Blank) and the samples of the blank group unsent for QXRD analysis (T+BR+C Blank and T+L+C Blank) were selected to represent the patterns of qualitative XRD analysis. The patterns of qualitative XRD of selected samples are shown in Figure B-7 to Figure B-12. 164 * This sample is original tailings before mixing for the experiments. Blue line - observed intensity at each step; red line - calculated pattern; solid grey line below - difference between observed and calculated intensities; vertical bars - positions of all Bragg reflections. Coloured lines are individual diffraction patterns of all phases. Figure B-1. Rietveld refinement plot of sample #1: Tailings (2-3m) (before the experiment, analyzed in June, 2021) 165 * This sample is bauxite residue before mixing for the experiments. Blue line - observed intensity at each step; red line - calculated pattern; solid grey line below - difference between observed and calculated intensities; vertical bars - positions of all Bragg reflections). Coloured lines are individual diffraction patterns of all phases. Figure B-2. Rietveld refinement plot of sample: Bauxite Residue (before the experiment, analyzed in June, 2021) 166 * This sample is pure tailings without amendments and plants. Blue line - observed intensity at each step; red line - calculated pattern; solid grey line below - difference between observed and calculated intensities; vertical bars - positions of all Bragg reflections). Colored lines are individual diffraction patterns of all phases. Figure B-3. Rietveld refinement plot of sample #1: Tailings Blank (after the experiment, analyzed in April, 2022) 167 * This sample is mixed of tailings and bauxite residue, without plants. Blue line - observed intensity at each step; red line - calculated pattern; solid grey line below - difference between observed and calculated intensities; vertical bars - positions of all Bragg reflections. Coloured lines are individual diffraction patterns of all phases. Figure B-4. Rietveld refinement plot of sample #2: T+BR Blank (Tailings + Bauxite Residue) (after the experiment, analyzed in April, 2022) 168 * This sample is mixed of tailings and lime, without plants. Blue line - observed intensity at each step; red line - calculated pattern; solid grey line below - difference between observed and calculated intensities; vertical bars - positions of all Bragg reflections. Coloured lines are individual diffraction patterns of all phases. Figure B-5. Rietveld refinement plot of sample #3: T+L Blank (Tailings + Lime) (after the experiment, analyzed in April, 2022) 169 * This sample is mixed of tailings and compost, without plants. Blue line - observed intensity at each step; red line - calculated pattern; solid grey line below - difference between observed and calculated intensities; vertical bars - positions of all Bragg reflections. Coloured lines are individual diffraction patterns of all phases. Figure B-6. Rietveld refinement plot of sample #4: T+C Blank (Tailings + Compost) (after the experiment, analyzed in April, 2022) 170 *This sample is mixed of tailings, bauxite residue and compost, without plants. Red line - observed intensity at each step; blue line - calculated pattern; green line – background pattern. Figure B-7. Qualitative XRD plot of sample: T+BR+C Blank (after the experiment, analyzed in March, 2022) Meas. data:T+BR+C_BlankBG data:T+BR+C_BlankCalc. data:T+BR+C_Blank2-theta (deg)Intensity (cps)20 40 60 80 0.0e+000 2.0e+003 4.0e+003 6.0e+003 171 *This sample is mixed of tailings, lime and compost, without plants. Orange line - observed intensity at each step; purple line - calculated pattern; red line – background pattern. Figure B-8. Qualitative XRD plot of sample: T+L+C Blank (after the experiment, analyzed in March, 2022) Meas. data:T+L+C_BlankBG data:T+L+C_BlankCalc. data:T+L+C_Blank2-theta (deg)Intensity (cps)20 40 60 80 0.0e+000 2.0e+003 4.0e+003 6.0e+003 8.0e+003 172 *This sample is tailings planted with wheatgrass and without amendments. Red line - observed intensity at each step; blue line - calculated pattern; green line – background pattern. Figure B-9.Qualitative XRD plot of sample: T-WG (after the experiment, analyzed in March, 2022) Meas. data:T-WGBG data:T-WGCalc. data:T-WG2-theta (deg)Intensity (cps)20 40 60 80 0 1000 2000 3000 4000 5000 173 *This sample is tailings planted with ryegrass and without amendments. Red line - observed intensity at each step; blue line - calculated pattern; green line – background pattern. Figure B-10.Qualitative XRD plot of sample: T-RG (after the experiment, analyzed in March, 2022). Meas. data:T-RG-2BG data:T-RG-2Calc. data:T-RG-22-theta (deg)Intensity (cps)20 40 60 80 0 1000 2000 3000 4000 5000 174 * This sample is tailings planted with tall fescue and without amendments. Green line - observed intensity at each step; purple line - calculated pattern; orange line – background pattern. Figure B-11.Qualitative XRD plot of sample: T-TF (after the experiment, analyzed in March, 2022) Meas. data:T-TFBG data:T-TFCalc. data:T-TF2-theta (deg)Intensity (cps)20 40 60 80 0 1000 2000 3000 4000 5000 175 *This sample is tailings planted with sheep fescue and without amendments. Blue line - observed intensity at each step; orange line - calculated pattern; green line – background pattern. Figure B-12. Qualitative XRD plot of sample: T-SF (after the experiment, analyzed in March, 2022) Meas. data:T-SFBG data:T-SFCalc. data:T-SF2-theta (deg)Intensity (cps)20 40 60 80 0 1000 2000 3000 4000 5000 176 Appendix C: Results of two – way ANOVA Table C-1. Two – way ANOVA results of effects of amendments and species on plant growth DF Sum of Squares Mean Square F Value P Value Germination rate Amendments 5 1.5177 0.30354 22.30736 <0.0001 Species 3 0.14012 0.04671 3.43259 0.04433 Model 8 1.65782 0.20723 15.22932 <0.0001 Error 15 0.20411 0.01361 Corrected Total 23 1.86193 Shoot height Amendments 5 2185.47052 437.0941 6.85778 0.00162 Species 3 7225.50698 2408.50233 37.78814 <0.0001 Model 8 9410.9775 1176.37219 18.45666 <0.0001 Error 15 956.0549 63.73699 Corrected Total 23 10367.0324 Total dry biomass Amendments 5 874.90647 174.98129 6.33193 0.00237 Species 3 1574.30746 524.76915 18.98946 <0.0001 Model 8 2449.21393 306.15174 11.0785 <0.0001 Error 15 414.52129 27.63475 Corrected Total 23 2863.73522 At the 0.05 level, the amendments and species are both significant factors affecting the germination rate of seeds, shoot height and total dry biomass of plants. Table C-2. Two – way ANOVA results of effects of amendments and species on the pH of mixed substrates before experiments DF Sum of Squares Mean Square F Value P Value Amendments 5 42.07158 8.41432 237.98059 <0.0001 Species 3 0.28996 0.09665 2.73364 0.08043 Model 8 42.36154 5.29519 149.76298 <0.0001 Error 15 0.53036 0.03536 Corrected Total 23 42.8919 177 At the 0.05 level, the amendment is the significant factor affecting the pH of mixed substrates. The species is not significant factor affecting the pH of mixed substrates due to p > 0.05, because seeds were not sowed yet before experiments. Table C-3. Two – way ANOVA results of effects of amendments and species on the total metal uptake by plants DF Sum of Squares Mean Square F Value P Value Amendments 5 931141.62969 186228.32594 13.27771 <0.0001 Species 3 204615.36281 68205.12094 4.86289 0.01475 As Model 8 1135756.9925 141969.62406 10.12215 <0.0001 Error 15 210384.51156 14025.6341 Corrected Total 23 1346141.50406 Amendments 5 209142.58333 41828.51667 6.12069 0.00278 Species 3 133298.04167 44432.68056 7.01593 0.00145 Cu Model 8 342440.625 42805.07812 5.53531 0.00222 Error 15 409842.83333 27322.85556 Corrected Total 23 752283.45833 Amendments 5 6258917.38802 1251783.4776 3.43259 0.04433 Species 3 2550618.27865 850206.09288 4.30475 0.01129 Pb Model 8 8809535.66667 1101191.95833 2.40518 0.04902 Error 15 2.13659E7 1424394.58872 Corrected Total 23 3.01755E7 Amendments 5 1.55113E9 3.10226E8 18.6529 <0.0001 Species 3 1.03815E9 3.46049E8 3.83082 0.01807 Zn Model 8 2.58928E9 3.2366E8 12.05843 <0.0001 Error 15 3.86986E9 2.57991E8 Corrected Total 23 6.45914E9 At the 0.05 level, the amendments and species are both significant factors affecting the As, Cu, Pb and Zn uptake by plants. 178 Table C-4. Two – way ANOVA results of effects of amendments and species on the pH and total metals remained in pot substrates after experiments DF Sum of Squares Mean Square F Value P Value Amendments 5 3.51727 0.70345 3.48984 0.01981 Species 4 12.71157 3.17789 15.76557 <0.0001 pH Model 9 16.22883 1.8032 8.94572 <0.0001 Error 20 4.03143 0.20157 Corrected Total 29 20.26027 Amendments 5 583356.66667 116671.33333 66.92108 <0.0001 Species 4 11771.66667 2942.91667 1.68802 0.1922 As Model 9 595128.33333 66125.37037 37.92861 <0.0001 Error 20 34868.33333 1743.41667 Corrected Total 29 629996.66667 Amendments 5 2360766.66667 472153.33333 640.20791 <0.0001 Species 4 34900 8725 11.83051 <0.0001 Cu Model 9 2395666.66667 266185.18519 360.92906 <0.0001 Error 20 14750 737.5 Corrected Total 29 2410416.66667 Amendments 5 1.44104E7 2882083.33333 37.88061 <0.0001 Species 4 1445333.33333 361333.33333 4.74918 0.00739 Pb Model 9 1.58557E7 1761750 23.15553 <0.0001 Error 20 1521666.66667 76083.33333 Corrected Total 29 1.73774E7 Amendments 5 1.90984E8 3.81968E7 58.02707 <0.0001 Species 4 8572833.33333 2143208.33333 3.25588 0.03282 Zn Model 9 1.99557E8 2.2173E7 33.68432 <0.0001 Error 20 1.31652E7 658258.33333 Corrected Total 29 2.12722E8 At the 0.05 level, the amendments is not significantly affecting As remained in pot substrates, but species is. At the 0.05 level, amendments and species are both significant factors affecting the pH, Cu, Pb and Zn remained in pot substrates after experiments. 179 Table C-5. Two – way ANOVA results of effects of amendments and species on the total metals in drainage at the end of experiments DF Sum of Squares Mean Square F Value P Value Amendments 5 3.00526 0.60105 2.31103 0.12161 Species 2 11.07322 5.53661 21.28813 2.48915E-4 pH Model 7 14.07848 2.01121 7.73306 0.00229 Error 10 2.6008 0.26008 Corrected Total 17 16.67928 Amendments 5 473.61226 94.72245 3.49053 0.04381 Species 2 773.10978 386.55489 14.24457 0.00118 Cu Model 7 1246.72204 178.10315 6.56311 0.0043 Error 10 271.36997 27.137 Corrected Total 17 1518.09201 Amendments 5 2.9736 0.59472 3.64359 0.03886 Species 2 3.54623 1.77312 10.8631 0.00311 Pb Model 7 6.51983 0.9314 5.7063 0.00719 Error 10 1.63224 0.16322 Corrected Total 17 8.15207 Amendments 5 1.01262E7 2025238.13334 10.40977 0.00103 Species 2 3846776.86865 1923388.43433 9.88626 0.00427 Zn Model 7 1.3973E7 1996138.21933 10.26019 7.34139E-4 Error 10 1945517.50064 194551.75006 Corrected Total 17 1.59185E7 At the 0.05 level, the amendments is not significantly affecting pH of the drainage at the end of experiments, but species is. At the 0.05 level, amendments and species are both significant factors affecting the Cu, Pb and Zn of the drainage at the end of experiments.
Thesis/Dissertation
eng
Mining Engineering
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Van Zyl, Dirk J. A.
A greenhouse study on phytostabilization of sulfidic mine tailings
Text