Forestry, Faculty of
DSpace
UBCV
Milla-Moreno, Estefanía
Doctor of Philosophy - PhD
University of British Columbia
The need to divest fossil fuels sets copper (Cu) in higher demand for use in clean energy systems. Chile’s Cu production has resulted in more than 750 tailing deposits across the northern and central regions, an area that also includes a biodiversity hotspot and the driest desert in the world. The quantity of tailings in Chile is expected to increase, and phytoremediation could help assist their management. To guide such efforts, I assessed the field performance and physiology of ten native woody plant species used for phytoremediation at a copper (Cu) mine in Chile, conducted a controlled environment experiment with a subset of these species, and undertook a ‘UBC public scholars initiative’ to discover professional stakeholders’ views on the use of phytoremediation in Chile. In the field, I assessed survival and hare damage in all species; and growth, vigor, chlorophyll content index and chlorophyll fluorescence in seven. Metal and metalloid concentrations in roots and shoots were measured in five species. Copper was most concentrated in the roots of Prosopis chilensis (Molina) Stuntz (algarrobo) and Quillaja saponaria Molina (quillay). Due to lower uptake overall and reduced mobilization to shoots, quillay is suggested for stabilizing tailings. In a controlled environment experiment with quillay and espino (Vachellia caven (Molina) Seigler and Ebinger) grown under three increasing Cu levels for 6 months in a greenhouse setting, I measured growth, photosynthetic performance and elemental concentrations of leaves and roots to further evaluate their potential for phytoremediation. Growth of quillay was unaffected by Cu increases but growth of espino was enhanced, as was its photosynthetic performance, indicating that espino may have an unusually high requirement for copper. Excess Cu was mostly restricted to the roots of both species, where X-ray fluorescence mapping indicated some tendency for Cu to accumulate in tissues outside the periderm. Finally, through an online questionnaire (n =43), I asked stakeholders linked to tailings management about the likelihood of using phytoremediation as an additional stabilization method. They were of the opinion that Chile should implement phytoremediation, but highlighted a need for more demonstrably successful phytoremediation projects, funding and access to suitable plant material.
AN ASSESSMENT OF NATIVE CHILEAN WOODY PLANTS FOR PHYTOREMEDIATION OF COPPER-CONTAMINATED SITES by Estefanía Milla-Moreno B.Sc., University of Chile, 2009 M.Sc., University of British Columbia, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Forestry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2023 © Estefanía Milla-Moreno, 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: An assessment of native Chilean woody plants for phytoremediation of copper contaminated sites submitted by Estefanía Milla-Moreno in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Forestry Examining Committee: Dr. Robert Guy, Professor, Faculty of Forestry, UBC Supervisor Dr. Cindy Prescott, Professor, Forestry, UBC Supervisory Committee Member Dr. Lacey Samuels, Professor, Botany, UBC Supervisory Committee Member Dr. Janusz Zwiazek, Professor, Agricultural, Life and Environmental Sciences, UAlberta Supervisory Committee Member Dr. Sean Smukler, Professor, Land and Food Systems, UBC University Examiner Dr. Christopher Chanway, Professor, Forestry, UBC University Examiner iii Abstract The need to divest fossil fuels sets copper (Cu) in higher demand for use in clean energy systems. Chile’s Cu production has resulted in more than 750 tailing deposits across the northern and central regions, an area that also includes a biodiversity hotspot and the driest desert in the world. The quantity of tailings in Chile is expected to increase, and phytoremediation could help assist their management. To guide such efforts, I assessed the field performance and physiology of ten native woody plant species used for phytoremediation at a copper (Cu) mine in Chile, conducted a controlled environment experiment with a subset of these species, and undertook a ‘UBC public scholars initiative’ to discover professional stakeholders’ views on the use of phytoremediation in Chile. In the field, I assessed survival and hare damage in all species; and growth, vigor, chlorophyll content index and chlorophyll fluorescence in seven. Metal and metalloid concentrations in roots and shoots were measured in five species. Copper was most concentrated in the roots of Prosopis chilensis (Molina) Stuntz (algarrobo) and Quillaja saponaria Molina (quillay). Due to lower uptake overall and reduced mobilization to shoots, quillay is suggested for stabilizing tailings. In a controlled environment experiment with quillay and espino (Vachellia caven (Molina) Seigler and Ebinger) grown under three increasing Cu levels for 6 months in a greenhouse setting, I measured growth, photosynthetic performance and elemental concentrations of leaves and roots to further evaluate their potential for phytoremediation. Growth of quillay was unaffected by Cu increases but growth of espino was enhanced, as was its photosynthetic performance, indicating that espino may have an unusually high requirement for copper. Excess Cu was mostly restricted to the roots of both species, where X-ray fluorescence mapping indicated some tendency for Cu to accumulate in tissues outside the periderm. Finally, through an online questionnaire (n =43), I asked stakeholders linked to tailings iv management about the likelihood of using phytoremediation as an additional stabilization method. They were of the opinion that Chile should implement phytoremediation, but highlighted a need for more demonstrably successful phytoremediation projects, funding and access to suitable plant material. v Lay Summary Tailings are the waste produced by mining companies. With this research, I hoped to learn if trees could be used to stabilize tailings or to remove heavy metals from soil. A mine site in Chile was used to study 10 species of woody plants that had been growing on tailings for six years. I assessed survival and stress status, and measured metal concentrations in five species. Quillay is suggested for stabilizing tailings because copper (Cu) uptake was restricted to its roots. I measured growth, photosynthesis and elementconcentrations in a greenhouse experiment using quillay and espino. Both species were resistant to Cu stress and Cu was mostly restricted to the roots. I then surveyed professional stakeholders in Chile about the use of native plants as an additional stabilization method for tailings. There was consensus in the openness to adopt this practice, if more studies with native plants are developed, properly communicated, and funded. vi Preface This dissertation is an original and independent work by the author Estefania Milla-Moreno. Throughout the Ph.D. program, the author received guidance and feedback from her Ph.D. supervisor, Dr. Robert Guy, and committee members, Dr. Cindy Prescott, Dr. Lacey Samuels, and Dr. Janusz Zwiazek. The supervisor and committee members helped shape the doctoral research purpose and questions and provided feedback for the dissertation manuscript. A version of Chapter 2 was published as Milla-Moreno E, Guy RD (2020) Growth response, uptake and mobilization of metals in native plant species on tailings at a Chilean copper mine. Int. J. Phytoremediation. 2020, 23:539–547; DOI: 10.1080/15226514.2020.1838435. I identified the research objectives, collected the data, performed analysis, and prepared the manuscript. R Guy provided advice concerning the data and analysis and recommendations and inputs for the manuscript. C Prescott provided editorial advice. A version of Chapter 3 was published as Milla-Moreno E, Guy RD, Soolanayakanahally RY (2022) Enlightening the pathway of phytoremediation: Ecophysiology and X-ray fluorescence visualization of two Chilean hardwoods exposed to excess copper. Toxics.10:237; DOI: 10.3390/toxics10050237. (Open Access; Editor’s Choice Article). In this publication, the conceptualization, formal analysis, X-ray visualization, and project administration were done by E Milla-Moreno and R Guy; investigation and writing the original draft by E Milla-Moreno; data curation and supervision by R Guy; funding acquisition by E Milla-Moreno, R Guy, and R Soolanayakanahally. Images related to this work are freely accessible through the Canadian Dataverse Repository Borealis (doi: https://doi.org/10.5683/SP3/OQSF3X). vii A version of Chapter 4 has been submitted for peer-review publication as: Likelihood of using of phytoremediation for mine tailings management in Chile. This section of the dissertation received research ethics permission from UBC’s Behavioral Research Ethics Board (ID: H21-00650) on July 9th, 2021. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ................................................................................................................................ v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations .................................................................................................................. xv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................... xix Chapter 1: Introduction ............................................................................................................... 1 1.1 Mining and the climate emergency ....................................................................................... 1 1.2. Tailings ................................................................................................................................ 3 1.3. Phytoremediation ................................................................................................................. 6 1.4. Metal stress and response in plants ...................................................................................... 8 1.4.1. Metal stress ................................................................................................................... 8 1.4.2 Metal response in plants............................................................................................... 10 1.5. Amendments ...................................................................................................................... 14 1.6. Research objectives ............................................................................................................ 15 ix Chapter 2: Growth response, uptake and mobilization of metals in native plant species on tailings at a Chilean copper mine .............................................................................................. 17 2.1. Summary ............................................................................................................................ 17 2.2. Introduction ........................................................................................................................ 17 2.3. Materials and methods ....................................................................................................... 21 2.3.1. Heavy metal analyzes of plant tissue and tailings ...................................................... 23 2.3.2. Statistical analyses ...................................................................................................... 25 2.4. Results and Discussion ...................................................................................................... 25 2.4.1. Tailing properties ........................................................................................................ 25 2.4.2. Survival ....................................................................................................................... 27 2.4.3. Growth, physiology and anatomy-related traits .......................................................... 28 2.4.4. Heavy metals in plant tissue ....................................................................................... 31 2.4.5. Bioaccumulation and Translocation Factors ............................................................... 33 2.4.6. Phytotoxicity ............................................................................................................... 34 2.5. Conclusions ........................................................................................................................ 36 Chapter 3: Enlightening the pathway of phytoremediation: ecophysiology and X-ray fluorescence visualization of two Chilean hardwoods exposed to excess copper .................. 37 3.1. Summary ............................................................................................................................ 37 3.2. Introduction ........................................................................................................................ 38 3.3. Materials and methods ....................................................................................................... 42 3.3.1. Plant Material and Treatments .................................................................................... 42 3.3.2. Physiological Measurements ...................................................................................... 43 x 3.3.3. Element Visualization ................................................................................................. 44 3.3.4. Statistics ...................................................................................................................... 45 3.4. Results ................................................................................................................................ 45 3.4.1. Soil Mix ...................................................................................................................... 45 3.4.2. Growth ........................................................................................................................ 47 3.4.3. Photosynthesis............................................................................................................. 47 3.4.4. Elemental Analysis ..................................................................................................... 48 3.4.5. X-ray Fluorescence Microimaging ............................................................................. 53 3.5. Discussion .......................................................................................................................... 59 Chapter 4: Likelihood of using phytoremediation for mine-tailing management in Chile . 66 4.1. Summary ............................................................................................................................ 66 4.2. Introduction ........................................................................................................................ 66 4.3. Materials and methods ....................................................................................................... 71 4.4. Results ................................................................................................................................ 71 4.5. Discussion .......................................................................................................................... 84 4.5.1. Demographic Composition ......................................................................................... 84 4.5.2. New Tailings ............................................................................................................... 86 4.5.3. Barriers ........................................................................................................................ 87 4.5.4. Social Context ............................................................................................................. 90 Chapter 5: General Discussion .................................................................................................. 92 5.1. Limitations of the study ..................................................................................................... 96 xi 5.2. Future Research and Recommendations ............................................................................ 98 References .................................................................................................................................... 99 Appendices ................................................................................................................................. 129 Appendix A ............................................................................................................................. 129 Appendix B ............................................................................................................................. 142 Appendix C ............................................................................................................................. 152 xii List of Tables Table 2.1 Information about species and measurements used in the study. ................................. 22 Table 2.2 Descriptive statistics of tailings properties. .................................................................. 26 Table 2.3 Percent survival of species, overall and by treatment ................................................... 28 Table 2.4 Descriptive statistics of growth and physiology related traits (Mean, ± SD) ............... 29 Table 2.5 Toxicity thresholds of heavy metals in leaves, animal feed and agricultural soils (mg kg-1). .............................................................................................................................................. 35 Table 3.1. Descriptive statistics for heavy metals and elemental content in leaves and roots of espino and quillay in control, 50 and 100 μM Cu treatments. ...................................................... 51 Table 4.1. Tailings volume (m3) and current tonnage by region built with information from the National Geology and Mining Service Registry collected in 2020 (decreasing order). ............... 68 xiii List of Figures Figure 1.1 Illustration of upstream, centreline and downstream tailing dam ................................. 4 Figure 3.1. Characteristics of soil mix from pots containing espino or quillay after 6 months growth ........................................................................................................................................... 46 Figure 3.2. Growth parameters of espino and quillay at time of harvest ...................................... 47 Figure 3.3. Effects of Cu treatments on photosynthetic traits and stomatal conductance in espino and quillay ..................................................................................................................................... 48 Figure 3.4. Multi-element spectra in plant tissues; (a) espino leaf, (b) quillay leaf, (c) espino root, (d) quillay root .............................................................................................................................. 54 Figure 3.5. X-ray fluorescence images of leaflets of espino grown at 100 μM Cu ...................... 55 Figure 3.6. X-ray fluorescence images of a leaf of quillay grown at 100 μM Cu ........................ 56 Figure 3.7. X-ray fluorescence images of a cross-section of an espino root grown at 100 μM Cu....................................................................................................................................................... 57 Figure 3.8. X-ray fluorescence images of a cross-section of a quillay root grown at 100 μM Cu........................................................................................................................................................ 57 Figure 3.9. X-ray fluorescence images of Ca and Ni, and their combined distributions, in leaflets of espino (first row) and a cross-section of a quillay root (second row), both grown at 100 μM Cu....................................................................................................................................................... 58 Figure 4.1. Is Chile doing a good job with the management of its tailing facilities? ................... 72 Figure 4.2. How important are the following stabilization methods?........................................... 73 xiv Figure 4.3. Ranking of the key aspects to be considered when approving more tailing facilities.74 Figure 4.4. How strongly do you think these barriers affect the use of Phytoremediation in Chile?....................................................................................................................................................... 75 Figure 4.5. Preferred stage in the life cycle of a tailing facility to (a) pursue phytoremediation, and (b) plant type to be used for phytoremediation. ..................................................................... 76 Figure 4.6. When can a restoration project using phytoremediation be considered successful? .. 77 Figure 4.7. Evaluation on regulations intended to address impacts. ............................................. 78 Figure 4.8. In your opinion, what compensatory practices to address environmental impacts are not well established in the management of tailing facilities? ....................................................... 81 Figure 4.9. To what extent do you think the International Labour Organization-convention 169 is enforced? ....................................................................................................................................... 82 Figure 4.10. What percentage of the Chilean mine royalty do you think should be diverted to fund ecological restoration? .......................................................................................................... 83 xv List of Abbreviations Amax: maximum photosynthetic rate (μmol CO2 m−2 s−1) Bafsoil mix: ratio of the mean Cu concentrations in the leaves to that of the soil mix Bafsolution: ratio of the mean Cu concentrations in the leaves to that of the fertigation solution in mg L−1 Bcfsoil mix: ratio of the mean Cu concentrations in the roots to that of the soil mix Bcfsolution: ratio of the mean Cu concentrations in the roots to that of the fertigation solution in mg L−1 Ci: intercellular CO2 concentration CCI: chlorophyll content index CR: crown radii ECe: extract electrical conductivity ECs: suspension electrical conductivity Fv/Fm: maximum quantum efficiency of PSII FW: fresh weight (g) gs: stomatal conductance (mol H2O m−2 s−1) H: height HD: hare damage xvi Kav: available potassium OM: organic matter content T: tailings alone T+C: tailings with planting spots amended with compost T+M: tailings with plants pre-inoculated with Rhizophagus irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler, formerly Glomus intraradices Tf: translocation factor (ratio of Cu concentration in the leaves relative to the roots) V: vigor WUEi: instantaneous Water Use Efficiency (μmol CO2 mmol H2O−1) xvii Acknowledgements I am extremely grateful to my supervisor, Dr. Rob Guy, for his patience and attention to detail, greatly improving my manuscripts. I also thank him for teaching me to take longer to produce more reflective and meaningful collaborations, a task that was surely challenging at times, but very impactful in my career. I am very thankful for the guidance and constructive feedback my committee members Dr. Lacey Samuels, Dr. Cindy Prescott, and Dr. Janusz Zwiazek provided over the course of my PhD. I am grateful to the Chilean National Agency for Research and Development (former CONICYT), as I had no other means to accomplish this research. I am very grateful to Felipe Munizaga and Cristobal Binvignat, whose help was crucial to translate my proposal into reality. I am forever grateful to Omar González, for assisting in the field and sharing his vast plant knowledge. I also thank the mine staff for allowing me to conduct my fieldwork at their facility. I’d like to thank the UBC Public Scholars Initiative. Dr. Susan Porter’s initiative allowed me to create a website to promote my research and travel to Chile to meet with members of environmental NGOs, industry, and academia, to discuss their impressions on the management of tailings. The funding also allowed me to host an exhibition of native plants with potential for phytoremediation. I am also thankful to Dr. Sergio Donoso, Dr. Karen Peña, and Laura Ugalde, for their help assisting with that meeting and the workshop that followed. During setup and harvesting for the greenhouse experiment, I received help and support from Shannon Bell, Rayen Tastets-Milla, José Arias-Bustamante, and Claudia Paez-Varas. The UBC greenhouse staff Melina Biron, Jimmy Sung, and Taylor Scriber, were very helpful. I’m very grateful to Dr. Soolanayakanahally (Agriculture and Agri-Food Canada) for his professional xviii support and collaboration, facilitating my access to the Canadian Light Source. I also want to thank the Canadian Light Source bioXAS team, particularly Dr. Biorica Bondici, for her pedagogical help and support during imaging. I am very grateful to Albina Gibadullina for the many sessions trying to troubleshoot my R code. I also thank Maria Larisheva, Norman Hodges, Evan Thornberry, and Eugene Barsky (Borealis) for their support. I also want to thank my support network, comprised of family, friends, colleagues and initiatives that made this period a memorable one: among them, my counsellor Andrea Jonsson, Jose Andres Murillo and Para la confianza team, Redicec, Vancouver Latin American Cultural Centre, Redes Chilenas, Instruments Beyond Borders, Diversity Crew, UBC JEDI, Dr. Sarah Gergel, Dr. Hisham Zeriffi, UBC Salsa Rueda, Jacky Yenga, chosen family in Acadia Park, veci Sarah, Silvi, Angela, Claudia Paez, Kristy, Jerry, Ale, Richie, JuanDi, Diana, Eve and Hugo, my lab members: Mina, Yi, Shalom, Lee and Athena, among others. Of course, my biggest gratitude goes to my dear family, my beloved compañero Jose Arias-Bustamante, and our precious daughters Rayen, Amancay and Antümalen – you made this journey much more enjoyable and meaningful, and I am grateful to live this life with all of you. Thanks to my dear mom, for her unconditional support and company through so many challenges. I know why I chose you <3. I also thank my dear brother Carlo Saavedra Moreno. I am grateful to three powerful role models, whose strength, resiliency, and wisdom helped me in the hardest moments of this doctorate, Dr. Wangari Maathai, Naomi Klein and Dr. Elisa Loncon. Finally, I want to thank Elhmapun, my ancestors, the Mapu, the Itxofill Mogenh with its genh, newen and pullü, for their company, guidance and Kimün. xix Dedication To my daughters Rayen, Amancay and Antümalen, my biggest inspiration and motivation for a better world. 1 Chapter 1: Introduction 1.1 Mining and the climate emergency There is disagreement in how the advancement of societies should be prioritized, either by promoting economic growth or protecting the health of the environment and its inhabitants. These priorities are often at odds with each other. For example, in la Higuera, Chile, north of the Coquimbo Region and just 30 km away from the Humboldt Penguin National Reserve, the Dominga venture takes place. This megaproject comprises two open pit mines, a commercial port, and a desalinization plant, and was approved by the Chilean Government in August 2021. The area is known to be home to one of the richest and most fragile marine ecosystems in the world (Urbina et al., 2021). Around 60-88% of the species in that ecosystem are endemic, and several are included in the International Union for Conservation of Nature Red List (Urbina et al., 2021). This area has been previously affected by industrial activity (Oceana, 2022). For example, there was a collapse of part of the infrastructure of the Los Pelambres Mine desalinization plant in August 2022, which subsequently produced hydrocarbon contamination in the Conchalí Bay in Los Vilos (SMA, 2022). Additionally, the iron (Fe) and steel port of the Pacific Mining Company, built between 1952 and 1954, is located at La Herradura and Coquimbo bays (CMP, 2018). Because of pressures from local communities, environmental advocates, and academics, demanding a balance between economic progress and social imperatives of environmental justice, Dominga was recently halted by the government. More than 100 projects from various sectors, which, like Dominga, are predominantly related to energy and mining (e.g., Alto Maipo, Pascua Lama, HydroAysén), are characterized by similar conflicts between economic development and socio-environmental protection (INDH, 2022). For example, about 40 mining projects have been identified within or in the proximity to 16 parks 2 and national reserves in Chile (Mongabay, 2022). There is a need to use native plants to mitigate environmental damage done by large extraction projects. This could be useful locally as well as in the fight against climate change. Evidence shows that human influence has warmed the atmosphere, ocean, and land. The last United Nations Climate Change conference confirmed the Paris Agreement's goal to prevent global temperatures from rising more than 2°C above pre-industrial levels and that carbon emissions should be reduced by 45% to achieve carbon neutrality around 2050 (United Nations, 2022). Such a large decrease in carbon emissions will depend in part on moving away from fossil fuels towards alternative energy sources. The last IPCC Physical Science Basis report indicated that changing temperatures have already led to shifts in climates zones towards the poles and an average two-day extension of the growing season, significantly affecting vegetation around the globe (IPCC 2021). Therefore, areas with high biodiversity should be protected. Current regulations lack requirements for a buffer zone around parks and reserves, resulting, for example, in up to eight mining projects surrounding a single protected area, the Nevado Tres Cruces National Park, located in the Atacama Region, in the north of Chile (Mongabay, 2022). Central Chile is one of 36 hotspots of biodiversity in the world, and is one of 25 hotspots that together account for only 1.4% of the land surface of the planet but contain 44% of all vascular plant species (Myers et al., 2000). Conservation priorities should ensure protection of these highly biodiverse and threatened ecosystems, but climate change may force their displacement. Central Chile’s flora overlaps with the Chilean mining sector, due to the extensive amount of copper ores present in that region. Moreover, Chile is included in the Pacific Rim, one of the five mining hotspots identified in the world that is coincident with a hotspot of biodiversity (Ginocchio and Baker, 2004). The rich quality of these ores paved the way for Chile’s historic 3 role as the main Cu producer globally. However, the modest level of innovation in the industry (e.g., lack of renewable energies across facilities, recycling of Cu from slags, poor tailings management), and a relaxation of the required mean ore quality required to extract the mineral from 0.87% in 2007 to 0.65% in 2016 (DatosGob, 2021), has increased the number of tailings in the country. The need to divest from fossil fuels has increased the pressure on mining, because the transition to clean energy systems relies heavily on at least 30 minerals and metals (Owen et al., 2022), including Cu and lithium (Li). The demand for Cu is predicted to increase 275% by 2050, and Li demand is expected to grow 40 times by 2040 (Owen et al., 2022). The demand for these elements has many impacts associated with increased ore extraction, transport and refining. One of the most prevalent impacts will be an increased volume of tailings, which must grow along with the demand. 1.2. Tailings The tailings deposits (hereafter referred to as tailings) are the waste produced in mineral extraction and generally comprise a mixture of crushed rock, small amounts of valuable minerals and metals, processing chemicals and processed water (Adiansyah et al., 2015; Kossoff et al., 2014; Schoenberger, 2016). The slurry can be disposed in many ways, but it is preferred to be stored behind dams made of mine waste, in a progressive fashion, as mine production progresses (Schoenberger, 2016). Chile has more than 750 tailings in various states (e.g., active, inactive, in construction, and abandoned). The oversight of the tailings has been assigned to the National Service of Geology and Mining (SERNAGEOMIN), created in 1980. However, this institution is only legally 4 equipped to supervise, control or request remediation of tailings with known owners and does not deal with tailings that have been declared abandoned. There are three types of dam configurations as illustrated in Fig. 1.1 (Kossoff et al., 2014): upstream (i.e., builds towards the interior of the storage facility), centerline (i.e., builds along the middle of the storage facility), and downstream (i.e., builds on the outside of the embankment). Figure 1.1 Illustration of upstream, centreline and downstream tailing dam (redrawn after Vick, 1983 in Kossoff et al., 2014). 5 The upstream configuration is the cheapest but also the most vulnerable to failure (Schoenberger, 2016). After the Supreme Decree 86 was enacted in 1970 by the Chilean Ministry of Mining, tailings could not be built upstream. Surprisingly, it appears that 36 tailings were still constructed in this way after this law was passed (El Andacollino, 2021). If such a tailing pond wall were to collapse, the heavy metals and chemical materials captive in the pond would be rapidly released, contaminating rivers and creeks downstream. This is even more concerning when considering the Chilean seismic activity, with the additional threat of liquefaction (i.e., loss of solid properties in response to a stress resulting in the material reacting in a liquid-like way) (Kossoff et al., 2014). The tailings registry of SERNAGEOMIN reports 213 tailings built upstream, from 1970 to 2014, with 161 identified as inactive, 49 abandoned and only three as active. Just two of these upstream tailing dams, Barahona 1 and 2, from the El Teniente facility, the largest underground Cu deposit in the world (Codelco, 2022), account for more than 63% of the total authorized volume of tailings in the country. The main cause of tailing dam facilities failure is water, either causing overtopping of embankment or a failure of the foundation (Gowan, 2012 cited by Schoenberger, 2016). To avoid dam failures, its crucial to have independent technical revisions, as well as maintenance by experienced professionals, at least once a year (Schoenberger, 2016). In order to reduce water content of tailings, companies have adopted vacuum- or pressure-driven elimination of water, to get to about 50-80% solids, compared to the 30-50% solids achieved in conventional storage (Schoenberger, 2016). This dry stacking of tailings reduces chemical reactions and the need for impoundment dams and long-term management (Adiansyah et al., 2015; Schoenberger, 2016). Once the tailings are closed, remediation technologies such as containment (e.g., surface capping, encapsulation, and landfilling), extraction (e.g., soil washing, soil flushing, 6 electrokinetics), and immobilization (solidification, vitrification, chemical stabilization) can be applied (Liu, et al., 2018). Biological approaches using either plants or microbes may also be employed. For example, in situ soil remediation is more cost-effective, but current practise leans towards traditional and less affordable methods of contaminant removal rather than immobilization and containment, because they permit restoration of agricultural uses (Liu, et al., 2018; Padmavathiamma and Li, 2007). The type of remediation to be used is selected based on the geography of site, degree of contamination, type of contaminants), remediation goal, budget, public acceptability, and maintenance. Several types of remediation can be used synergistically, like chemical stabilization to diminish bioavailability and overall toxicity prior to employing biological methods (Liu, et al., 2018). 1.3. Phytoremediation Some metal-tolerant plants (metallophytes) can sequester metals or otherwise reduce their mobilization for environmental cleanup, a technology coined “phytoremediation” (Pulford and Watson, 2003) and more recently also referred to as “phytotechnologies” (Conesa et al., 2011). Depending on the species, metallophytes may take up heavy metals from the soil into aerial tissues that can be harvested and disposed or processed for their metal concentrations (phytoextraction); their roots may precipitate and concentrate heavy metals from solution (rhizofiltration); and/or they may reduce the belowground mobility of heavy metals (phytostabilization) (Salt et al., 1995; Suman et al., 2018). Phytoremediation can also help restore degraded areas, fostering ecosystem conservation and land restoration efforts, which can reduce the vulnerability of biodiversity to climate change (IPCC, 2022). 7 Several studies have evaluated the Cu tolerance of native Chilean plant species growing in (or in proximity to) sites with a high level of Cu and other metals (Cárcamo et al., 2012; Ginocchio et al., 2002; Ginocchio et al., 2017; Meza-Ramirez et al., 2021; Milla-Moreno and Guy, 2020; Orchard et al., 2009; Ortiz-Calderón et al., 2008). These studies show a wide range of options for phytoremediation, but the hope is to restore those sites to a level of complexity that includes a great variety of microorganisms, flora and fauna, aiming to reflect complex interactions between diversity, structure and ecological processes (Ruiz-Jaen and Aide, 2005). Since the aim of ecological restoration is to assist the recovery of an ecosystem, species known to exist in those sites prior to the disturbance are likely to have better possibilities for survival, growth and reproduction than species that were not part of the pre-mining landscape. Given that invasive species can reduce biodiversity and alter ecosystem functions, native plants have been advised (Kettenring and Adams 2011), particularly if they are identified as vulnerable or endangered (Orchard et al., 2009). Moreover, native plants that have added value (e.g., are medicinal, produce nectar for honey, are good for forage) should be prioritized (Orchard et al., 2009), due to their greater benefit to local communities. As natural succession can take several years to decades, the early establishment of native metallophyte trees is also desirable (Munford et al., 2022). Research is needed to aid decision-makers in the mining sector in selecting the best plant candidates for phytoremediation. A major challenge to using the many native and endemic plant species that are potentially available is the lack of information regarding their propagation, basic physiological requirements, tolerance levels, metal uptake and sequestration patterns. Assessment of metal distribution in tissues is crucial to understanding environmental impacts, tolerance mechanisms, and interactions between multi-metal and other stressors in plants. In this 8 regard, finding plants that can manage several stresses (e.g., drought, compaction, lack of macronutrients) or build soil (e.g., N fixers) is also desirable. 1.4. Metal stress and response in plants 1.4.1. Metal stress Copper, cadmium (Cd), zinc (Zn) and lead (Pb) are the main heavy metals of concern in affected ecosystems (Adriano, 2001) and may originate from various sources, such as sewage sludge applications as fertilizers, irrigation water, pesticides, natural minerals and industrial wastes (Badilla-Ohlbaum et al., 2001; Ginocchio et al., 2009). Viticulture (Cruz-Aguayo, 2004) and the production of other vegetable and fruit crops (Aguilar et al., 2011) rely on Cu-based fungicides, because they are inexpensive and effective for controlling a variety of diseases. For this reason, the accumulation of Cu in agricultural soils and crops, especially for Latin-American countries, can be quite high (Adriano, 2001). For example, a range of 70-155 mg kg-1 was found as the background soil Cu concentration in areas free of mining impacts but affected by Cu-based fungicides in Chile (Aguilar et al., 2011). Mines are also a major source of heavy metal contamination, with Cu being the main contaminant in the soils of copper- mining areas in Chile (Goecke et al., 2011). Metal contamination from mining operations can enter ecosystems by way of atmospheric and particulate emissions as well as dispersal of liquid and solid wastes, with Cu smelting and mining being the greatest sources of soil Cu contamination (Ginocchio et al., 2009). One study of metal phytoxicity in soils due to mine wastes reported levels of total Cu up to ~220,000 mg kg-1 on smelter dust and up ~2,500 mg kg-1 on oxidized tailings (Ginocchio et al., 2009). Ventanas, a Cu smelter located at the Puchuncaví Valley (coastal central Chile), has not only affected adult plant 9 abundance but also soil seed bank abundance and micro-distribution, thereby affecting the regeneration and resilience of the system (Ginocchio, 2000). Either applied or deposited, Cu will remain in soils because it is highly complexed or sorbed by organic matter (OM), oxides of Fe and manganese (Mn), and (although at a lower rate) by clay minerals (Adriano, 2001; Ginocchio et al., 2009). Even though oxides of Mn, organic matter, oxides of Fe and clay minerals (descending order) are the most important soil constituents, the abundance of Fe oxide and clay minerals can override the others (Adriano, 2001). The ecological risk Cu may present is defined by its bioavailability related to soil pH and organic matter content (Adriano, 2001; Delorme et al., 2001). Copper bioavailability is severely reduced over pH 7 and high below pH 6 (Locascio, 1978; Lucas and Knezek, 1972). The buffering effect of OM is as important as pH in terms of immobilizing metals (Adriano, 2001; Delorme et al., 2001). Organic matter has a high sorption capacity for Cu, and it may be as important as the oxides of Mn and Fe (Adriano, 2001). Copper forms its most stable complexes with humic and fulvic acids (Adriano, 2001). Adsorption of Cu to organic matter increases with pH (Cavallaro and McBride, 1984). Since Cu uptake and bioavailable Cu have a negative relationship with higher pH, Cu uptake by plants may be decreased by adding organic matter of near-neutral pH, such as sewage sludge (Adriano, 2001). Consequently, the pH of sludge-treated soils should be kept above a certain level (> 6.5) to safeguard against excessive uptake of toxic metals (Adriano, 2001). In fact, keeping the pH of the soil above 5.5 improves plant performance due to the elimination of pH-dependent Cu/Zn solubility (Ginocchio et al., 2009). Bioavailability is a crucial determinant of Cu toxicity. The cupric ion (i.e., Cu2+) is the most bioavailable species of Cu and therefore the best indicator for Cu phytotoxicity (Sauvé et al., 10 1998). Though total Cu in soils varies over a large range, the range for bioavailable Cu tends to be narrower – around 0.1 to 10 ppm, but estimates also vary depending on the type of extractant used (e.g., water soluble and exchangeable; dilute acid and chelating agents) (Baker, 1974). Bioavailability is also connected to the depth of the profile, since the distribution of Cu in the profile declines rapidly with depth (Adriano, 2001; McBride, 1994). In a soil profile of central Chile, most of the Cu was concentrated within 20 cm of the surface (Badilla-Ohlbaum et al., 2001). Currently Chile lacks legislation limiting the maximum permissible concentration of metals in soils. If legislation is developed in the future, it must consider soil properties that affect metal bioavailability like soil pH and organic matter content. 1.4.2 Metal response in plants In addition to macronutrients, plant nutrition depends on the availability of seven traditional micronutrients: Zn, Cu, Mn, Fe, boron, molybdenum and chlorine (Adriano, 2001). Depending on their abundance, any one of these micronutrients may be deficient or available in excess, to the point of being phytotoxic (Adriano, 2001; McBride, 1994). In the case of Cu, concentrations in soil higher than 60 to 125 mg kg-1 are considered to be toxic to most plants (Kabata-Pendias and Pendias, 2001). The importance of Cu in plants is related to the Cu constituency of several plant enzymes (Adriano, 2001). If Cu is deficient in plants, several physiological processes are affected, such as carbohydrate metabolism, nitrogen (N) metabolism, cell wall metabolism, seed production, water relations and disease resistance (Bussler, 1981; Romheld and Marschner, 1991). Copper deficiencies tend to affect reproductive growth (seeds and fruit formation) more deeply than vegetative growth (Adriano, 2001). 11 Symptoms of Cu toxicity (and other heavy metals) include chlorosis (Chibuike and Obiora, 2014) and defoliation (Alva et al., 1999), inhibition of root elongation and damage of root cell membranes (Azeez et al., 2015). Metal stress can affect photosynthesis both directly and indirectly. Large reductions in stomatal conductance (gs) resulting from metal treatment or environmental exposure have been reported by Bernardini (2016) in Salix clones under Zn stress. Stomatal conductance is also likely to reflect the negative effects of heavy metals on xylem conductance to water movement (de Silva et al., 2012) and their interference with the plant’s ability to respond to improved soil moisture conditions (Santala and Ryser, 2009). Reductions in gs limit photosynthesis by decreasing the intercellular CO2 concentration (Ci), whereas direct effects on the photosynthetic apparatus may result in increased Ci (Parkhurst, 1994). In addition to metal stress, the growth of plants on mine tailings may be limited by poor access to water, nutrient resources, and the content of fine particles that may lead to soil compaction (Ginocchio et al., 2006; Ginocchio et al., 2017). Stomatal closure is the first response to drought that reduces transpiration, decreases Ci, and improves water-use efficiency (WUE). In contrast, declines in photosynthesis caused by nutrient deficiency (Livingston et al., 1999) and/or toxic metal stress (Ahmad et al., 2009; Hermle et al., 2007) at the biochemical level result in reduced WUE. Although measurements of gs and other gas exchange variables, as well as chlorophyll content, can provide valuable information about what may be happening to photosynthesis, more informative assessment is obtainable with a chlorophyll fluorometer (Baker, 2008; Murchie and Lawson, 2013). Any environmental factor that affects photosynthesis will likely affect chlorophyll fluorescence. For example, the ratio of the variable (Fv) over maximum (Fm) fluorescence indicates the potential maximum efficiency of photosystem II (PSII). Since Fv/Fm 12 and other fluorescence parameters can be determined in seconds, they are popular measures of stress in plants, including heavy metal stress (Bernardini et al., 2016; Evlard et al., 2014; Han et al., 2013; Pinheiro et al., 2013). Several studies have reported effects of heavy metal contamination on foliar N content. The activity of nitrate reductase (NR), the enzyme catalyzing the first step in NO3- assimilation, is repressed in many metal-stressed plants (Gomes et al., 2012; Llorens et al., 2000; Yadav, 2010). Less is known about the sensitivity of glutamine synthetase (GS), catalyzing the first step in ammonium (NH4+) assimilation, but similar effects are widely indicated. For example, several heavy metals are known to severely impact NH4+ uptake in Typha latifolia (Lim et al., 2003). Reduced N assimilation can reduce photosynthetic capacity and WUE. Plant strategies to overcome high concentrations of heavy metals can be grouped into mechanisms of tolerance or avoidance (Hall, 2002; Jabeen et al., 2009; Ali et al., 2013; Pulford and Watson, 2003), and the exact nature of these mechanisms can affect the utility of different species for phytoremediation. Plants that accumulate extremely high levels of heavy metals in their aerial tissues are called hyperaccumulators, whereas those that exclude metals from the aerial tissues, are called excluders (Baker, 1981). If the goal is phytoextraction, hyperaccumulator plants that concentrate heavy metals in accessible tissues and are the best candidates. Furthermore, if a hyperaccumulator also produces high biomass, it can yield a metal ‘crop’ (Brooks et al., 1998). There are about 721 known species identified in the Global Database of hyperaccumulator plants, with most of them (523) being nickel (Ni)-hyperaccumulators, and the second largest group (53) corresponding to Cu- hyperaccumulators (Reeves et al., 2018). Some examples of hyperaccumulator plants include the Ni-hyperaccumulator Sebertia acuminata Pierre ex Baill. (Padmavathiamma and Li, 2007) and other 13 species from ultramafic soils in Indonesia (van der Ent et al., 2013); cadmium (Cd)-hyperaccumulator Nicotiana tabacum L., Zn-accumulating Arabidopsis halleri L., Thlaspi goesingense Halácsy, and Sedum alfredii Hance (Liu et al., 2018); and Cu-hyperaccumulators Eleocharis acicularis (L.) Roem. & Schult. (Sakakibara et al., 2011) and Haumaniastrum katangense (S.Moore) P.A.Duvign. & Plancke (Brooks et al., 1998). Ortiz-Calderón et al. (2008) found that huingán (Schinus polygama (Cav.) Cabrera) and Atriplex desertícola Phil. growing in northern Chile are capable of accumulating over 1.2 mg g-1 Cu in their leaves, which, to my knowledge, makes these two species the only known Cu-hyperaccumulators native to Chile. On the other hand, for purposes of phytostabilization it is better if plants restrict accumulation to their roots, or avoid uptake altogether and otherwise promote the retention of heavy metals in the soil. Examples of excluders (of Ni) are Silene vulgaris L. and Rumex acetosella L. (Wenzel et al., 2003). Some excluders may be used to safely produce food in contaminated soils, such as with Cd-excluding brown rice in Shenyang Region of China (Zhan et al., 2012). After Cu uptake, tolerant plants may bind Cu to the cell wall, compartmentalize it in vacuoles or form complexes with proteins, oligopeptides, phenolic compounds or amino and organic acids (Goecke et al., 2011; Meharg, 2005). Metallothioneins are a group of cysteine-rich metal-binding proteins (Sytar et al., 2021). For example, Cu-tolerant Silene vulgaris (Moench) Garcke (Van Hoof et al., 2001) and Silene paradoxa L. (Mengoni et al., 2003) populations have higher constitutive levels of expression of the metallothionein gene MT2b, suggesting that this locus has been a major target of natural selection. Oligopeptides such as glutathione, a tripeptide, and the related phytochelatins may also bind heavy metals. For example, Brassica juncea (L.) Czern plants overexpressing ATP sulfurylase are more tolerant of Cu and other heavy metals because they have higher levels of glutathione, which not only forms glutathione-metal complexes but 14 also improves active scavenging of oxygen radicals (LeDuc and Terry, 2005). However, it has been reported that phytochelatins have no major role in detoxification of Cu in Arabidopsis thaliana (L.) Heynh. and Schizosaccharomyces pombe Lindner (1893) (Ha et al., 1999). Constituted by an aromatic ring with one or more hydroxyl groups, phenolic compounds scavenge reactive oxygen species generated during heavy-metal stress but also act as metal chelators (Balasundram et al., 2006). For example, phenolic compounds in tannin-containing idioblasts chelate Cu ions in Armeria maritima (Mill.) Willd. growing on Cu-polluted soils (Neumann et al., 1995). Amino (e.g., O-acetylserine, histidine) and organic acids (e.g., malate, citrate) can also form ligands with metal ions for detoxification, sequestration or mobilization (Shah and Nongkynrih, 2007). It has been widely reported that arbuscular mycorrhizal fungi (AMF) improve plant establishment in metal-contaminated soils (Curaqueo et al., 2014; Meier et al., 2015; Meier et al., 2012) due to their positive effect on plant nutrition and soil structure by external mycelium and the production of glomalin, a glycoprotein that supports sequestering of potential heavy metals (González et al., 2008). 1.5. Amendments A common practice to improve the ‘soil quality’ of tailings and influence plant uptake of heavy metals in soils is to add amendments, especially given the added cost advantage and efficiencies of recycling materials such as biosolids or agricultural wastes from nearby industries or communities. The type of amendment incorporated will affect the mobility of metals in the substrate (Neaman and Yañez, 2021; Paz-Ferreiro et al., 2014). For example, organic carbon from biochar helped immobilize Cd for Triticum aestivum L. (Yousaf et al., 2016), whereas lime 15 and fly ash reduced shoot concentration of Cu in Lolium perenne L. (Goeke et al., 2011). Addition of amendments translates into chemical and microbiological improvements that can increase plant yield even after only three years of application (Santibañez et al., 2012). Near a Cu smelter in central Chile, the incorporation of compost and the cumulative effect of compost and lime significantly increased the organic matter content, as well as the available N, phosphorous, potassium, calcium, and magnesium concentrations and the water holding capacity of the soil (Ulriksen et al., 2012). Furthermore, lime and compost application decreased the Cu2+ form in the soil solution by immobilizing Cu in the topsoil (Ulriksen et al., 2012). 1.6. Research objectives Clearly, more research is needed to develop sustainable solutions for mine site management, weighing technical aspects and balancing progress and timely action for the climate emergency with safety for local communities and ecosystems. Phytoremediation appears to be a promising part of the solution, particularly with Chile having a wide range of plants that are naturally adapted to growing in metalliferous soils (Ginocchio and Baker, 2004; Orchard et al., 2009). However, filling gaps in research related to expanding the list of useful plants, as well as understanding the existence of other barriers to phytoremediation is crucial. Towards these goals, the research objectives driving my research, were as follows: 1. Evaluate the phytoremediation potential of native woody plant species established on a copper mine tailing in Chile based on their physiological traits and heavy metal mobilization. 2. Examine copper uptake and sequestration patterns and effects on growth and photosynthesis of selected species in a controlled experiment. 16 3. Evaluate the likelihood of using phytoremediation as an additional stabilization method for mine-tailing management in Chile. To address the first objective, I assessed the performance of 10 native woody plant species growing in an established trial on a tailing site in Chile (Chapter 2). Based on my results, I chose a subset of species for a more detailed evaluation of the specific effects of Cu in a greenhouse study (Chapter 3), in which I used XRF imaging to study sequestration patterns within roots and leaves. Finally, I conducted a survey of professional stakeholders to gauge their views on the use of phytoremediation in practice (Chapter 4). This last chapter was facilitated by my participation in the UBC Public Scholars Initiative (PSI). This initiative supports scholarly work that entails ‘mutually beneficial partnership of university knowledge and resources with those of the public and private sector, to contribute to public good’. The policy paper presented in Chapter 4, and additional activities that I conducted on a trip to Chile in November 2018 were designed with this in mind. The activities comprised my participation as guest speaker at Gecamin, a Global Mine Conference, where I shared findings from Chapter 2; a Seminar about ‘Forest Sciences in Mining and Ecological Restoration’ with the participation of two representatives of Anglo American (Industry); one professor (Academia); and representatives of Oceana, FIMA, and the Association of Forest Engineers pro native Forest (Environmental NGOs). The seminar was free to the public and held in the Faculty of Forestry at the University of Chile. The event was followed by a workshop where attendees and presenters could brainstorm and reflect on the Seminar theme. Finally, there was an inauguration of a photo showcase of five native species with potential for phytoremediation, with two copies given to the Faculty of Forestry (UChile) and the Chilean Forestry Engineers Association for the Native Forest. More information about this event can be found in Alvarado (2019). 17 Chapter 2: Growth response, uptake and mobilization of metals in native plant species on tailings at a Chilean copper mine 2.1. Summary Chile has many mine-tailing deposits available for phytoremediation through the establishment of metal-tolerant plants. To guide such efforts, it is necessary to know whether roots exclude or take up metals, or if metals are mobilized to shoots. We evaluated a polyculture of ten native species 6 years after they were planted directly into tailings, amended with mycorrhiza before planting or planted with compost. All species were assessed for survival and hare damage. Given a limited number of plants available for some species, along with technical considerations (time and capacity), growth, vigor, chlorophyll content index and chlorophyll fluorescence were measured in seven species, and root and shoot concentrations of 11 metals and As were measured in five. All species had some level of stress, but there was no clear pattern related to foliar metal concentrations or amendments, the latter also having little effect on growth or survival. Copper (Cu), iron (Fe) and molybdenum (Mo) concentrations exceeded threshold toxicity levels for leaves and for animal feed in several species. Copper was most concentrated in the roots, and readily mobilized to leaves in three of the five species tested, but not in Prosopis chilensis (algarrobo) and Quillaja saponaria (quillay). Because of lower uptake overall, quillay is recommended for stabilizing tailings while also reducing transport to aerial tissues. 2.2. Introduction Chile is a major producer of Cu, rhenium, Mo, natural nitrates, iodine, lithium (Li) and other mining products (Matich, 2016; SONAMI, 2020). The Cu industry alone provides 10% of the gross domestic product and about 50% of exports (Orellana, 2019). However, this welfare is 18 linked with Chile’s holding of about 741 tailings (SERNAGEOMIN, 2018). A study performed in the Coquimbo region, which borders a biodiversity hotspot, discovered a remarkable 420 plant species (28% of the native regional flora) able to spontaneously colonize mine tailings (Orchard et al., 2009). The Coquimbo flora represents 30% of the total Chilean flora, with 54% of species being endemic to Chile (León-Lobos et al., 2011). The use of native species for restoration efforts can ease problems with seed/seedling supply and acclimation of plants, avoids potential introduction of exotic invasive species, restores ecosystem functionality and reduces costs due to lower maintenance. Several herbaceous Chilean species, and a sub-shrub, are reported to accumulate Cu (reviewed by Ginocchio and Baker, 2004) but work on woody plants is limited. Ginocchio et al. (2017) found two native tree species: pimiento (Schinus molle L.), and huingán (Schinus polygama (Cav.) Cabrera), along with five native shrubs, including romero (Baccharis linearis (Ruiz and Pav.) Pers.), spontaneously colonizing abandoned tailings dumps in the Coquimbo Region. Lam et al. (2017) found that the exotic shrub atriplex Atriplex nummularia Lindl., pimiento , and the endemic tree tamarugo Prosopis tamarugo Phil. were able to grow in Cu mine tailings in the Atacama Desert in the Antofagasta Region. In addition to macronutrients, proper plant nutrition requires seven primary micronutrients, including Cu and other metals (Adriano, 2001). Depending on abundance, any one of the micronutrients may result in a deficiency or an excess, to the point of being phytotoxic (Adriano, 2001). In the case of Cu in agricultural soils, if the concentration is higher than 100 mg kg-1 (DW) it is considered phytotoxic (Kabata-Pendias and Pendias, 2001). Symptoms of Cu toxicity include inhibition of root elongation and damage to cell membranes, and direct and indirect effects on photosynthesis and growth, including chlorosis, necrosis, defoliation and death (Alva et al., 1999; Azeez et al., 2015; Bouazizi et al., 2010; Panou-Filotheou et al., 2001). Although 19 measurements of gas exchange variables, as well as chlorophyll content, can provide information about what may be happening to photosynthesis, a more rapid assessment is obtainable with a chlorophyll fluorometer (Baker, 2008). Plant strategies to overcome high concentrations of heavy metals can be characterized as mechanisms of either avoidance or tolerance (Hall, 2002; Pulford and Watson, 2003) and these differences can be exploited to accomplish different goals in phytoremediation. For example, Eucalyptus spp. have been used for phytostabilization at an arsenical gold mine (King et al., 2008) and Ammania baccifera L. has been reported to have similar potential (Das and Maiti, 2007; Maiti and Jaiswal, 2007), because they have limited uptake of this metalloid and low mobilization to aerial parts (i.e., avoidance). This is a preferred strategy for tailings management if the ambition is to stabilize the site while also limiting the movement of metals into aboveground parts that may be subject to animal browse or lead to the offsite transfer of contaminants in litter. By contrast, Pteris vittate L., an arsenic (As) hyperaccumulator (i.e., tolerance) has been used for phytoextraction (Kertulis-Tartar et al., 2006). Hyperaccumulators have the ability to concentrate >10 mg g−1 (1%, DW) manganese (Mn) or Zn, >1mg g−1 (0.1%, DW) As, chromium (Cr), Cu, nickel (Ni), lead (Pb) or selenium (Se), and >0.1mg g−1 (0.01%, DW) cadmium (Cd) in their shoots, without suffering damage (Verbruggen et al., 2009). Other species may show both avoidance and tolerance, depending on the metal. Lam et al. (2018) have proposed using Adesmia atacamensis Phil., endemic to northern Chile, for phytostabilization of iron (Fe), Pb and Zn because of the limited uptake of these metals and low mobilization to aerial parts, but also for Cu phytoextraction because this same species actually hyperaccumulates Cu. Given that several factors, such as pH, nutrients, soil compaction, organic matter content, electrical conductivity and metal speciation, among others, can also limit plant survival and 20 growth in tailings (Adriano, 2001; Alva et al., 1999; Chibuike and Obiora, 2014; Kabata-Pendias and Pendias, 2001), several studies have reported on the use of amendments to promote growth and establishment (e.g., Ulriksen et al., 2012; Verdugo et al., 2011; Santibáñez et al., 2008). In A. nummularia and P. tamarugo, metal removal efficiency was higher when using calcium carbonate with compost as an amendment (Lam et al., 2017). Menares et al. (2017) found that in order to use the Cu-tolerant romero (Baccharis linearis (Ruiz and Pav.) Pers.), tailings should be improved by adding organic and inorganic colloidal particles. Chicken and soil manure amendments to Cu mine tailings significantly reduced uptake and accumulation of Cu and Ni in Cymbopogon citratus (DC.) Stapf and Vetiveria zizanioides (L.) Nash (Das and Maiti, 2009). Amendments with mycorrhizae fungi can also reduce heavy metal toxicity (Marschner, 1995; Jentschke and Godbold, 2000). Suillus bovinus (L.) Roussel (1806) and Tribulus terrestris L. have been found to protect Pinus sylvestris L. against Cu toxicity (Van Tichelen et al., 2001). Alleviation of growth depression caused by Ni (Jones and Hutchinson, 1986), Zn (Brown and Wilkins, 1985) and Cd (Jentschke et al., 1999) has been associated with the presence of ectomycorrhizal fungi. For example, Paxillus involutus (Batsch) Fr. can diminish the toxicity of Cd (Jentschke et al., 1999). We set out to explore the survival and performance of a set of 10 native plant species with high potential for remediating tailings and possibly other metal-containing sites. To do so we sampled an experimental polyculture of 25,700 plants after 6 years of growth on a mine tailing site, established with and without amendments. We hypothesized that 1) some species are better suited to the stresses of the site, 2) growth and survival were affected by the amendments, and 3) root vs. shoot metal sequestration patterns differ between species. Our objectives were to assess 21 growth related traits and survival, estimate photoinhibitory stress, and measure tissue metal concentrations. 2.3. Materials and methods We retrospectively sampled a study site located in Salamanca, Choapa province, in the Coquimbo Region. The original purpose of the trial was to assess growth, survival and metal accumulation in locally available and mostly native plants established on tailings, with or without additional treatments. The study design (Figure A1) comprised 30 ha containing three treatments: tailings only (T), tailings with planting spots amended with compost (T+C) and tailings with plants pre-inoculated with Rhizophagus irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler, formerly Glomus intraradices, (T+M). These treatments were organized in three blocks, each containing six plots. Seedlings (~20 cm tall) were planted in 2011 at a spacing of 2 m, and were drip irrigated with 4 L of water per plant per day throughout the first and all subsequent growing seasons. Ultrasol 15:30:15 (N:P:K) fertilizer was added to the irrigation water at a rate of 3.75 g per plant per week. The compost treatment was a 50:50 mixture of compost (pigeon manure, green manure and wood chips) and tailings added at planting (~8 L by volume per hole). The entire site was fenced, but not the individual blocks or seedlings. The following species, chosen for their local abundance and tendency to occur naturally on similar sites without tailings, were planted (Table 2.1): algarrobo (Prosopis chilensis (Mol.) Stunz.), espino (Acacia caven (Molina) Molina), litre (Lithraea caustica Hook. & Arn.), maitén (Maytenus boaria Molina), pimiento, quillay (Quillaja saponaria Molina), huigán, romero, talhuén (Trevoa quinquenervia Gillies & Hook) and quilo (Muehlenbeckia hastulata (Sm.) I.M. 22 Johnst.). The trial was non-homogenous in terms of the number of individuals planted per species: huingán (7084) and romero (6092) were the species with the largest representation. There were also relatively large numbers of algarrobo (2489), espino (1785), talhuén (1766), quillay (1732) and pimiento (1593). Litre, maitén and quilo were sparsely planted (71 individuals altogether). Table 2.1 Information about species and measurements used in the study. Common name Scientific name / Family Listed as Phenology Survival G & Ph HM Algarrobo Prosopis chilensis / Fabaceae Native tree X X X Espino Acacia caven / Fabaceae Native S-D tree X X X Litre Lithraea caustica / Anacardiaceae Endemic evergreen tree X Maitén Maytenus boaria / Celastraceae Native evergreen tree X Pimiento Schinus molle / Anacardiaceae Native evergreen tree X X Quillay Quillaja saponaria / Quillajaceae Endemic evergreen tree X X X Huingán Schinus polygama / Anacardiaceae Native evergreen shrub X X Romero Baccharis linearis / Asteraceae Native evergreen shrub X X X Talhuén Trevoa quinquenervia / Rhamnaceae Endemic evergreen shrub X X X Quilo Muehlenbeckia hastulata/ Polygalaceae Native evergreen shrub X G & Ph: Growth & Physiology, HM: Heavy Metals, S-D: semi-deciduous. Fieldwork was conducted in October-December 2017. Survival of all individuals originally planted on the site was assessed. Growth, physiological and anatomical traits were measured on a 23 subset of 210 plants (7 species; 10 individuals per species per treatment; 3 treatments). Their locations within each block were chosen randomly. Chlorophyll Content Index (CCI) was estimated on the youngest mature leaf of a fully sun-exposed branch/twig using a CCM-200 Plus (Opti-Sciences Inc, Hudson, NH). This variable is strongly indicative of leaf N content (Ali et al., 2017). Leaves were then dark-adapted for 20 minutes and the maximum quantum efficiency of PSII photochemistry (Fv/Fm) was obtained after a saturating flash (>7000 μmol m-2s-1) as per Genty et al. (1989), from 10:00 to 14:00 hrs with a FluorPen FP 100 (Photon Systems Instruments, Drasov, CZ). About 500 g FW of leaves were harvested from each plant and kept in paper bags inside a cooler until heavy metal analysis. Similarly, about 500 g of fine roots close to the taproot were harvested after digging a 10-20 cm deep hole at the immediate base of each plant. Height (H) and crown radii (CR) were measured with a tape, and neck diameter with an Absolute digimatic caliper (Mitutoyo, Sao Pablo, BR). Vigor (V) and hare damage (HD) were rated on a scale from 1 to 4; V was described as the proportion of crown with live foliage (1<25%, 2 = 25-50%, 3 = 50-75%, 4 =>75%; modified from Lakatos et al., 2014), and HD was defined as the proportion of near-ground-level bark removed (1<25%, 2 = 25-50%, 3 = 50-75%, 4 =>75%; modified from Eichhorn et al., 2016). 2.3.1. Elemental analyses of plant tissue and tailings A total of 45 plants (five species of the better performing species, from nine locations in one block) and nine tailing samples (2 kg combined from each location where plants were sampled) were analysed at the Agroanálisis at the Catholic University, Santiago. We intended to include huingán in this assessment but the samples were lost unexpectedly. Plant tissue was washed with distilled water to remove surface impurities. Dry, ground tissue was calcinated at 500°C and 24 dissolved in concentrated HCl (37%) for analysis of Cu, Fe, K, Mn, and Zn, according to Sadzawka et al. (2007). The reference material was IPE 990 (Wageningen Evaluating Programs for Analytical Laboratories). Copper, Fe, Mn, mercury (Hg), Zn and Se were detected using a Varian AA Spectra 220FS flame atomic absorption spectrometer with an Arquimed VGA 77 Vapor generation accessory. Arsenic, Cd, Cr, molybdenum (Mo), Ni, and Pb levels were analysed by Inductively Coupled Plasma Optical Emission Spectrometry (Varian Agilent 720-ES Axial). Consensus values (IPE, %) were: 22.5 As, 20.8 Cd, 16.6 Cr, 6.6 Cu, 11.2 Fe, 14.5 Pb, 9.6 Mn, 13.4 Mo, 16.8 Ni, 6.5 Zn. The bioaccumulation factor (Bf) was calculated following Eid et al. (2019), and the root-to-shoot translocation factor (Tf) was calculated following Maiti and Jaiswal (2008). Tailing samples were kept in plastic bags in a cooler until analysis (also by Agroanálisis) of suspension pH, suspension electrical conductivity (ECs), extract electrical conductivity (ECe), organic matter content (OM), available potassium (Kav), phosphorus (P), Fe, Cu, Zn, Mn, boron (B), sulfur (S), exchangeable K (Ke), calcium (Ca), magnesium (Mg), sodium (Na) and texture (e.g., clay, lime and sand). To obtain OM, samples were oxidized with a mixture of dichromate and sulfuric acid, and the reduced chromate was measured colorimetrically following Sadzawka et al. (2006). The microwave extraction method 3051A (United States Environmental Protection Agency, 2007) using conventional heating with nitric acid (HNO3) and then atomic aborption spectrophotometry was used to obtain Fe, Cu, Zn, Mn, B, Ca, Mg, and Na concentrations following Sadzawka et al. (2007). 25 2.3.2. Statistical analyses For most traits, the effects of species and treatment were tested by two-way analysis of variance (ANOVA) in Jamovi 0.8.1.18 (The Jamovi Project, 2019). Where necessary and possible, data were transformed to meet assumptions of normality and homogeneity of variance; we performed ANOVA on ranks where data could not be transformed. For per cent survival, the effects of species, treatment and block had to be tested separately using one-way ANOVA on ranks in Sigmaplot 12.5 (Systat Software Inc.). Relationships between plant physiological and anatomical traits were assessed using Pearson product-moment correlations. For statistical purposes, metal and metalloid concentrations that were under the detection limit (0.001 mg kg-1) were taken to equal half the detection limit (i.e., 0.0005 mg kg-1) as advised by the United States Environmental Protection Agency (1991). 2.4. Results and discussion 2.4.1. Tailing properties In general, Cu tailings typically have high metal concentration, low fertility and low water-holding capacity (Das and Maiti, 2007, 2008). Various amendments have been used to mitigate these challenges. In the present study, however, there were no significant treatment differences in terms of properties and mineral nutrient and metal/metalloid concentrations of the tailings (not shown). This might be explained by the variable condition of the tailings (standard deviations for some metals were high), or by the time since planting (6 years) and the amendments being limited in extent to only the planting holes (compost) or the roots (Rhizophagus) of each seedling. In all treatments, the tailings had a silt loam texture (Table 2.2), were acidic (pH 4.2-4.3) and slightly saline with ECe ranging from 2.88 to 2.97 mS/cm. Concentrations of OM (0.46-0.52%), 26 P (10-15 mg kg-1), and K (45-61 mg kg-1) were low (Penn State College of Agricultural Sciences, 2002; Beegle, 2015), which may be related to the acidic pH. Soil pH is important in determining the solubility and availability of mineral elements, including P (Jentschke and Godbold, 2000). In contrast, the tailings were high in S (784-845 mg kg-1), exceeding the mean global S level of 260 mg kg-1 found in continental crust (Sager, 2012). Table 2.2 Descriptive statistics of tailings properties. Treatment T T+C T+M pH ± SD 4.20 ± 0.22 4.30 ± 0.03 4.20 ± 0.22 ECe ± SD (mS cm-1) 2.93 ± 0.31 2.88 ± 0.15 2.97 ± 0.51 ECs ± SD (mS cm-1) 3.06 ± 0.07 3.03 ± 0.02 3.06 ± 0.11 OM ± SD (%) 0.48 ± 0.06 0.46 ± 0.16 0.52 ± 0.15 P ± SD (mg kg-1) 15.33 ± 11.93 9.67 ± 3.79 13.00 ± 6.08 Kav ± SD (mg kg-1) 61.00 ± 14.2 48.00 ± 32.90 45.00 ± 13.00 Cu ± SD (mg kg-1) 13.78 ± 10.55 3.86 ± 0.382 4.05 ± 1.81 Fe ± SD (mg kg-1) 65.50 ± 28.82 50.50 ± 23.95 49.60 ± 7.15 Mn ± SD (mg kg-1) 1.24 ± 0.35 0.94 ± 0.38 1.38 ± 1.03 Zn ± SD (mg kg-1) 0.35 ± 0.14 0.29 ± 0.02 0.28 ± 0.08 B ± SD (mg kg-1) 0.71 ± 0.17 0.94 ± 0.26 0.92 ± 0.52 S ± SD (mg kg-1) 845.00 ± 40.50 804.00 ± 51.40 784.00 ± 23.10 Ca ± SD (meq 100g-1) 40.90 ± 8.75 38.90 ± 14.27 41.60 ± 5.25 Mg ± SD (meq 100g-1) 0.23 ± 0.05 0.20 ± 0.03 0.34 ± 0.32 Ke ± SD (meq 100g-1) 0.16 ± 0.03 0.12 ± 0.09 0.11 ± 0.04 Na ± SD (meq 100g-1) 0.08 ± 0.01 0.09 ± 0.03 0.09 ± 0.01 Clay ± SD (%) 6.00 ± 0.00 5.70 ± 0.58 5.70 ± 1.53 Lime ± SD (%) 61.30 ± 17.01 51.10 ± 15.01 68.30 ± 6.03 Sand ± SD (%) 31.70 ± 17.01 42.40 ± 15.66 25.10 ± 7.09 27 N=3, T: Tailings only, T+C: Tailings with planting spots amended with Compost, T+M: Tailings with plants pre-inoculated with R. irregularis, SD: Standard Deviation, av (subscript): available, e (subscript): exchangeable, ECe: Electrical Conductivity (extract), ECs: Electrical Conductivity (suspended), OM: Organic Matter, Pav: Phosphorus, Kav: Potassium, Cuav: Copper, Feav: Iron, Mnav: Manganese, Znav: Zinc, Bav: Boron, Sav: Extract Sulfur, Cae: Calcium, Mge: Magnesium, Ke: Exchangeable potassium, Nae: Sodium. Mean available Fe concentrations ranged from 50 to 66 mg kg-1, which is higher than reported by Lam et al. (2017) for another Cu mine tailings site. The concentrations of available Cu (3.9-13.4 mg kg-1) and Zn (0.3-0.4 mg kg-1) were low compared to maximum allowable concentrations in agricultural soils (Table 2.5). Concentrations of these two metals were strongly positively correlated (r = 0.86, p = 0.003; data not shown). Manganese varied from 0.9 to 1.4 mg kg-1, which is much lower than the 352.5 mg kg-1 reported by Lam et al. (2017). Although acidic pH favours Cu solubility (Adriano, 2001; Ginocchio et al., 2009; Kabata-Pendias and Pendias, 2001), and OM content reduces bioavailability (Adriano, 2001), neither OM (r = 0.14, p = 0.73; data not shown) nor pH (r = -0.42, p = 0.26; data not shown) were significantly correlated with Cu. 2.4.2. Survival There were significant differences between the 10 species in which survival was assessed (p<0.001), being high in seven species, but below 50% in litre, maitén and quilo (Table 2.3). Algarrobo, romero and huingán had the highest survival, followed by espino. We did not detect treatment or block effects on survival. 28 Table 2.3 Percent survival of species, overall and by treatment Species Overall T T+C T+M Algarrobo 97.8 97.9 98.6 97.0 Espino 94.3 93.0 95.1 95.1 Huingán 96.0 96.4 96.6 94.9 Litre 5.9 0.0 8.3 9.1 Maitén 18.4 15.7 20.2 19.5 Pimiento 85.9 85.8 89.9 82.2 Quillay 82.7 81.5 87.9 78.7 Quilo 48.6 63.6 50.0 33.3 Romero 98.5 99.4 98.1 98.1 Talhuén 62.1 64.4 52.0 69.2 T: Tailings only, T+C: Tailings with planting spots amended with Compost, T+M: Tailings with plants pre-inoculated with R. irregularis. 2.4.3. Growth, physiology and anatomy-related traits As expected, there were highly significant species differences in all growth-related traits (Table A1). Algarrobo and romero were the largest, with mean heights of 189±53 and 147±19 cm, and mean crown radii of 338±106 and 168±43 cm, respectively (Table 2.4). There were no treatment differences or species×treatment interactions in H or CR, but there were significant treatment differences in neck diameter (p = 0.024), HD (p = 0.042) and V (p = 0.022). The growth-related traits were strongly positively correlated across species and, often, within species. Height and V were correlated with neck diameter in most cases (Table A2). 29 Table 2.4 Descriptive statistics of growth and physiology related traits (Mean, ± SD) 1 Species Treatment Height (cm) CR (cm) ND (cm) Vigour (adm) CCI (adm) Fv/Fm(adm) Hare (adm) Algarrobo T 164.7 347.4 31.9 3.0 3.3 0.7 1.0 ± 71.8 ± 114.3 ± 14.9 ± 0.8 ± 2.9 ± 0.1 ± 0.8 T+C 183.2 346.0 31.4 3.4 2.3 0.7 1.3 ± 36.2 ± 68.9 ± 9.9 ± 0.5 ± 0.7 ± 0.1 ± 0.7 T+M 219.0 319.5 41.3 3.6 2.9 0.7 1.6 ± 50.7 ± 134.2 ± 11.1 ± 0.5 ± 1.2 ± 0.1 ± 0.7 Espino T 122.2 124.8 33.3 3.3 1.9 0.6 0.5 ± 19.2 ± 44.3 ± 10.1 ± 0.7 ± 0.6 ± 0.1 ± 0.5 T+C 134.8 130.1 34.6 3.3 2.4 0.6 0.3 ± 35.1 ± 57.5 ± 16.0 ± 0.8 ± 0.7 ± 0.2 ± 0.5 T+M 130.4 147.3 36.8 3.2 2.0 0.7 0.6 ± 30.8 ± 35.8 ± 10.2 ± 0.4 ± 0.5 ± 0.1 ± 0.7 Huingán T 90.0 108.4 26.0 2.7 18.2 0.6 0.0 ± 34.8 ± 36.4 ± 9.7 ± 0.7 ± 14.6 ± 0.1 ± 0.0 T+C 100.1 128.8 29.6 2.9 19.1 0.7 0.0 ± 31.0 ± 27.5 ± 6.4 ± 0.9 ± 13.5 ± 0.0 ± 0.0 T+M 99.4 109.7 31.1 2.9 24.5 0.6 0.0 ± 22.8 ± 39.5 ± 9.9 ± 0.3 ± 21.8 ± 0.1 ± 0.0 Pimiento T 30.4 35.9 19.8 1.3 5.9 0.6 0.0 ± 18.2 ± 14.9 ± 11.1 ± 0.5 ± 6.2 ± 0.2 ± 0.0 T+C 49.3 72.3 19.2 1.9 12.5 0.7 0.0 ± 23.3 ± 23.9 ± 7.3 ± 0.7 ± 18.2 ± 0.1 ± 0.0 30 Species Treatment Height (cm) CR (cm) ND (cm) Vigour (adm) CCI (adm) Fv/Fm(adm) Hare (adm) T+M 39.3 49.5 24.5 2.3 13.7 0.7 0.0 ± 22.0 ± 24.9 ± 13.6 ± 0.8 ± 9.0 ± 0.0 ± 0.0 Quillay T 47.8 47.6 10.8 2.2 29.7 0.6 0.0 ± 36.5 ± 17.3 ± 4.0 ± 0.9 ± 8.0 ± 0.1 ± 0.0 T+C 38.3 63.1 14.1 2.1 44.7 0.6 0.0 ± 14.3 ± 26.4 ± 4.8 ± 0.3 ± 12.4 ± 0.1 ± 0.0 T+M 31.8 53.5 15.2 2.2 36.9 0.6 0.0 ± 21.4 ± 13.4 ± 3.1 ± 0.4 ± 15.2 ± 0.1 ± 0.0 Romero T 137.6 162.8 30.9 2.7 2.3 0.7 0.0 ± 24.2 ± 38.2 ± 9.5 ± 0.5 ± 1.2 ± 0.1 ± 0.0 T+C 149.2 177.8 38.1 2.8 2.2 0.6 0.0 ± 20.6 ± 27.9 ± 17.6 ± 0.4 ± 0.8 ± 0.1 ± 0.0 T+M 153.4 164.3 36.9 3.0 2.4 0.7 0.0 ± 12.4 ± 61.9 ± 10.4 ± 0.0 ± 0.8 ± 0.1 ± 0.0 Talhuén T 99.4 68.6 15.1 2.8 15.4 0.6 0.4 ± 47.1 ± 18.5 ± 5.5 ± 0.6 ± 21.6 ± 0.1 ± 0.5 T+C 76.7 71.8 13.0 2.4 15.1 0.7 0.4 ± 36.0 ± 35.7 ± 3.0 ± 0.7 ± 21.2 ± 0.1 ± 0.7 T+M 77.0 70.4 14.7 2.9 11.4 0.6 0.9 ± 47.5 ± 27.6 ± 5.7 ± 1.0 ± 16.9 ± 0.2 ± 0.9 N=10, T: Tailings only, T+C: Tailings with planting spots amended with Compost, T+M: Tailings with plants pre-inoculated with R. 2 irregularis, SD: standard deviation, CR: crown radius, ND: neck diameter, CCI: Chlorophyll Content Index, Fv/Fm: maximum 3 quantum efficiency of PSII photochemistry, Hare: damage by hares, adm: adimensional. 4 31 There were significant species (p<0.001) in HD. Most of the espino, algarrobo and talhuén, three of the four largest species present at the field site, were damaged by European hares (Lepus europaeus Pallas). For reasons that are unclear, damage to these species was more severe on the plots with seedlings pre-treated with mycorrhizae (p = 0.042). Romero, however, was very hare-resistant despite its large size. In algarrobo, H and HD were strongly positively correlated (r = 0.59, p<0.001; Table A2). In contrast, there was no relationship in talhuén (r = 0.04, p = 0.85), while in espino H and HD were negatively correlated (r = -0.45, p = 0.01). Stem damage in espino may have affected the allocation of resources, restricting growth in height. There were significant species differences in CCI (p<0.001, Table A1). The species with the thickest leaves, quillay, huingán and talhuén, had the highest mean values. There were no significant treatment differences in CCI. Although we found no consistent species or treatment effect on Fv/Fm, there was significant interaction (p = 0.033; Table A1). Most means were well below 0.83, suggesting some photoinhibitory stress (Björkman and Demmig, 1987). Severe stress, indicated by Fv/Fm values <0.5, occurred sporadically across treatments and species. Despite irrigation, the site was dry and sun-exposed, and we cannot ascribe this stress to the presence of heavy metals per se, but metal stress may be a contributing factor, along with others such as nutrient deficiency, substrate acidity and or salinity. 2.4.4. Heavy metals in plant tissue Tables A4 and A5 present mean heavy metal and metalloid concentrations in leaves and roots, respectively, of the five species assessed. Selenium and mercury were below detection limits in all cases and not included. Cadmium was also often below the detection limit, particularly in leaves. Foliar As and Cr levels were occasionally below detection limits. There were highly 32 significant species differences for metal concentrations in leaves (Cd, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Zn; Table A3) and roots (As, Cd, Cr, Fe, Mn, Mo, Zn). There were also some treatment effects (Mo in leaves and Mn, Mo, Ni, and Zn in roots). None of the species accumulated enough metals or metalloids to indicate hyperaccumulation. Although there were no significant species differences in foliar As concentrations, there was a significant species×treatment interaction (p = 0.009). We only found significant species differences for roots, where it ranged from 0.03±0.03 mg kg-1 in algarrobo to 1.26±0.56 mg kg-1 in talhuén. Romero was the only species to accumulate detectable amounts of Cd in leaves (0.57±0.28 mg kg-1) and had the highest root Cd levels (0.46±0.31 mg kg-1). Romero symbiosis with arbuscular mycorrhizal fungi has been shown to contribute to the sequestration of Cu and Zn in soil, as well as stabilizing pH and soil organic carbon (Cornejo et al., 2008). In comparison to Menares et al. (2017), we found higher foliar Cu and lower foliar Fe and Zn. Cadmium was undetectable in roots of algarrobo and, usually, espino. Chromium was mostly undetectable in leaves of quillay and occasionally in algarrobo, reaching maximum levels in talhuén (overall mean was 0.40±0.11 mg kg-1). In sharp contrast, Cr in roots tended to reach higher concentrations in algarrobo. Foliar Cu concentrations ranged from 5.67±3.8 mg kg-1 in quillay to 44.7±9.9 mg kg-1 in espino, averaged across treatments. Talhuén and espino had the highest foliar Fe concentrations, averaging 417±155 mg kg-1 and 372±49 mg kg-1, respectively. There was a marginally significant species×treatment effect for roots (p = 0.046) where Fe ranged from 270±158 mg kg-1 in talhuén on T to 1524±589 mg kg-1 in algarrobo on T+C. There was also a marginally significant species×treatment interaction for Pb in leaves (p = 0.044) where concentrations ranged from 0.1±0.1 mg kg-1 in quillay on T+M to 0.7±0.4 mg kg-1 in talhuén on T. 33 In addition to Cr, Cu and Fe, talhuén and espino also had the highest foliar Mn, Mo and Ni levels. Molybdenum was highest in leaves from plants growing on T. Mean foliar concentrations of Mn ranged from 51.3±16.9 mg kg-1 in quillay to 186±112 mg kg-1 in talhuén, while Mo ranged from 1.84±0.09 mg kg-1 in quillay on T+M to 17.0±2.7 mg kg-1 in talhuén on T, and Ni from 0.71±0.34 mg kg-1 in quillay to 2.75±1.01 mg kg-1 in espino (Table S4). In roots, Mn ranged from 24.3±11.0 mg kg-1 in talhuén on T to 82.3±21.6 mg kg-1 in romero on T+M, Mo from 3.23±1.18 mg kg-1 in algarrobo on T+C to 37.9±13.6 mg kg-1 in quillay on T, and Ni from 0.78±0.32 mg kg-1 on T+M to 1.22±0.50 mg kg-1 on T+C (Table A5). Talhuén and quillay had the lowest foliar Zn levels overall (11.6±1.94 mg kg-1 and 12.1±3.26 mg kg-1, respectively), while romero had the highest (48.4±15.0 mg kg-1). In contrast, quillay had the highest overall Zn concentration in roots (16.4±8.78 mg kg-1), whereas talhuén and algarrobo had the lowest (9.89±4.60 mg kg-1 and 8.44±2.01 mg kg-1, respectively). The treatment effect on root Zn levels indicated lower concentrations in plants on T. 2.4.5. Bioaccumulation and Translocation factors Because the amendments did not affect the tailing properties, species differences in Bf (not shown) directly reflect differences in tissue metal levels. Consequently, espino and talhuén had the highest foliar Bf for Cu and Fe, at 6.1±1.8 and 6.7±0.8 for espino and 5.8±2.4 and 7.6±2.6 for talhuén, respectively. For Zn, foliar Bf reached up to 100±36 in algarrobo and 157±51 in romero. The foliar Bf for Mn was highest in talhuén (137±74), espino (108±35) and algarrobo (100±34). Because of the way Bf is calculated, values greater than unity for Cu, Mn and Zn may result from the relatively low metal concentrations of the tailings and not necessarily reflect the capacity of these plants for hyperaccumulation. On the other hand, as noted above, tailing Fe concentrations 34 were high. Quillay had the lowest foliar Bf for Cu (1.0±0.5), Fe (0.9±0.4), and Mn (62±14), whereas for Zn (39±9) it was similar to talhuén (37±7). Similar foliar Bf values for Cu were reported for other plants (Chang et al., 2018; Gascó et al., 2019); in contrast, the same studies reported much smaller Bf values for Zn. Table A6 presents heavy metal and metalloid Tf values. There were highly significant species differences in Tf for Cd, Cu, Fe, Mo and Zn (p<0.001; Table A3). Significant treatment (p = 0.003) and species×treatment interactions (p = 0.013) were also found in Tf for Fe. As noted above, quillay had relatively high levels of Mo in its roots, but this species also had the lowest foliar Mo levels resulting in a very low Tf (0.10-0.18; Table A6). Similarly, the consistently low foliar Cu content in quillay compared to roots resulted in a very low Tf for this element as well (0.09-0.20). In addition, quillay had low Tf values for Cd (up to 0.04), Cr (0.01-0.03) and Fe (0.08-0.13). Algarrobo also had relatively low Tf values for Cu (0.16-0.61), Fe (0.06-0.20) and Cr (0.04-0.10), but overall uptake of these metals was higher than in quillay. Other studies have found Tf values <1 for Cu in water hyacinth (Eid et al., 2019) and Brassica napus (Gascó et al., 2019). 2.4.6. Phytotoxicity Table 2.5 presents foliar concentrations of heavy metals considered to be excessive or toxic to most plants (i.e., causing >10% reduction in growth, excluding very sensitive and tolerant species). The average foliar concentrations of most metals we measured were below minimum toxicity levels; including As, Cd, Cr, Pb, Mn, Ni and Zn. However, mean Mo concentration exceeded the toxicity threshold in leaves of talhuén on T and on T+C. Mean Fe concentrations were consistently above the toxicity threshold (300 mg kg-1) in leaves of espino and talhuén. 35 Similarly, although tailing Cu levels were relatively low, mean Cu concentrations entered the foliar toxicity threshold range (20-100 mg kg-1 DW) in algarrobo, espino, romero and talhuén. Mean concentrations of Cu, Fe and Mo were in some cases also above acceptable levels for animal feed (Table 2.5). Toxic Cu concentrations (>40 mg kg-1 DW) occurred in algarrobo, espino and talhuén. In the case of Fe, concentrations considered toxic for animal feed (>500 mg kg-1 DW) occurred only in talhuén. Table 2.5 Toxicity thresholds of heavy metals in leaves, animal feed and agricultural soils (mg kg-1). The reference for toxic concentration in mature leaf tissue is Kabata-Pendias and Pendias (2001); with the exception of iron (Tanaka et al. 1966). The reference for maximum tolerable limit allowed for animal feed is Weiss (2008). The reference for maximum allowable concentrations in agricultural soils is Canadian Council of Ministers of the Environment (2019). Metal Toxic concentration in mature leaf tissue Maximum tolerable limit allowed for animal feed Maximum allowable total concentrations in agricultural soils As 5-20 30 12 Cd 5-30 10 1.4 Cr 5-30 100 64 Cu 20-100 40 63 Fe 300 500 NIA Pb 30-300 100 70 Mn 400-1000 2000 NIA Hg 1-3 2 6.6 Mo 10-50 5 5 Ni 10-100 100 45 Se 5-30 5 1 Zn 100-400 500 250 NIA: no information available, As: Arsenic, Cd: Cadmium, Cr: Chromium, Cu: Copper, Fe: Iron, Pb: Lead, Manganese (Mn), Mercury (Hg), Molybdenum (Mo), Nickel (Ni), Selenium (Se), Zinc (Zn) 36 Toxicity thresholds specific to roots are not available in the literature, but mean root Fe and Cu concentrations exceeded foliar toxicity thresholds in all species. Molybdenum concentrations in roots of espino, quillay and talhuén were also above the minimum toxicity threshold. Interestingly, and despite low foliar concentrations, roots of quillay had relatively high Mo levels across all treatments. 2.5. Conclusions The amendments were not extensively applied and, six years after application, appeared not to have strong effects on plant growth and survival. In contrast, there were many clear differences between the plant species. Algarrobo, romero and espino had higher survival, and were the tallest and largest. The greenest species were quillay, huingán and pimiento. Evidence of photoinhibition was found in all species. Talhuén and espino had relatively high Bf and high Tf for Cu and Fe and might therefore be useful for phytoextraction. In contrast, quillay and algarrobo had low Tf for Cu and several other metals. The Tf for Cu in algarrobo was, however, roughly three times higher than in quillay and it had more consistently high root Cu concentrations; consequently, algarrobo still exceeded toxicity levels in the leaves despite its low Tf. Root uptake of Cu in quillay was comparable to the other species but, because of its very low Tf, its mean foliar Cu levels remained low. Quillay was also resistant to hare damage. We recommend employing quillay for the phytostabilization of the site used in this study and encourage its further evaluation on other tailings. 37 Chapter 3: Enlightening the pathway of phytoremediation: ecophysiology and X-ray fluorescence visualization of two Chilean hardwoods exposed to excess copper 3.1. Summary In the present climate emergency due to global warming, we are urged to move away from fossil fuels and pursue a speedy conversion to renewable energy systems. Consequently, copper (Cu) will remain in high demand because it is a highly efficient conductor used in clean energy systems to generate power from solar, hydro, thermal and wind energy across the world. Chile is the global leader in Cu production, but this position has resulted in Chile having several hundred tailing deposits. We grew two Chilean native hardwood species, quillay (Quillaja saponaria Molina) and espino (Vachellia caven (Molina) Seigler & Ebinger, under three increasing Cu levels (0, 50, and 100 µM) for 6 months in a greenhouse setting. We measured growth, photosynthetic performance and elemental concentrations of leaves and roots to further evaluate their potential for phytoremediation. Growth of quillay was unaffected by Cu treatment but growth of espino was enhanced, as was its photosynthetic performance, indicating that espino may have an unusually high requirement for Cu. Excess Cu was mostly restricted to the roots of both species, where X-ray fluorescence (XRF) mapping indicated some tendency for Cu to accumulate in tissues outside the periderm. Calcium oxalate crystals were prominently visible in XRF images of both species. Nickel (but not Cu) showed a concurrent distribution pattern with these crystals. 38 3.2. Introduction Evidence shows that human influence has warmed the atmosphere, ocean, and land. The last IPCC report indicated that, among other things, changing temperatures have already led to shifts in climates zones towards the poles and an average two days extension of the growing season, significantly affecting vegetation around the globe (IPCC, 2021). Therefore, areas with high biodiversity should be protected. Central Chile is one of 36 hotspots of biodiversity in the world, and is one of 25 hotspots that together account for only 1.4% of the land surface of the planet but in which 44% of all vascular plant species are confined (Myers et al., 2000). Conservation priorities should ensure protection of these highly biodiverse and threatened ecosystems, but climate change may force their displacement. For example, Muñoz-Sáez et al. (2021) reported that sclerophyllous forest in Chile is at a higher level of climate risk under global circulation models by 2080, with 65% of high risk in non-protected areas (13,675 km2) and 30% in protected areas (238 km2). Central Chile’s flora overlaps with the Chilean mining sector, due to the extensive amount of Cu ores present in that region. The rich quality of these ores paved the way for Chile’s historic role as the main Cu producer globally. However, because of incomplete extraction, about 450 thousand USD are lost daily in the Cu industry as slag resulting from the smelting process (PUCV, 2016). The modest level innovation of the industry (e.g., lack of renewable energies across facilities, recycling of Cu from slags, tailings management), and a relaxation of the required mean ore quality required to extract the mineral from 0.87% in 2007 to 0.65% in 2016 (DatosGob, 2021), has increased the number of tailings in the country. 39 Copper mines will continue to exist because Cu is a crucial element in clean energy systems and its use could balance out the cost from declining ore quality (Haas et al., 2020), particularly in high solar radiation and Cu-rich locations like Chile and Peru, when using renewable energy for Cu mining. Copper mining countries (e.g., Chile, Peru, United States) should aim for a fully renewable energy supply by 2030. Zaldívar is the first such fully renewable mine in the world, located in Antofagasta, Chile (Haas et al., 2020). To further minimize environmental impact, restoration of mining sites (e.g., tailings) must also be a priority. In this context, the use of plants and their associated microbes for in situ site remediation, a concept coined phytoremediation (Salt et al., 1998), has been used around the world with great success, particularly when using native species. Broadly defined, phytoremediation may involve one or more of phytoextraction, phytodegradation, rhizofiltration, phytostabilization, phytovolatilization and even the use of plants to filter air pollutants (Paz-Ferreiro et al., 2014; Rascio and Navari-Izzo, 2011). Plants deal with high concentrations of heavy metals, via mechanisms of tolerance, avoidance, or both (Hall 2002; Pulford 2003). The best species choice for any given phytoremediation application will depend on the strategy of the plant. For example, a study on metallophytes growing on a Cu mine tailing in northern Chile (Ortiz-Calderón et al.,2008) found that Schinus polygamus (Cav.) Cabrera and Atriplex deserticola Phil., both Cu-hyperaccumulators, accumulated more than 0.1% Cu in their shoots, showing good potential for phytoextraction; whereas Casuarina equisetifolia L. accumulated even more Cu, 2.9 g kg−1, but in the roots, making it an appropriate candidate for phytostabilization. In cases where phytostabilization rather than phytoextraction is the goal, the preferred plants should avoid metal mobilization to aerial parts where they may be subject to animal browse and/or result in the unwanted dispersal of heavy metals in the leaf litter. 40 There have been several studies that evaluated the Cu tolerance of native Chilean species growing in (or in proximity to) sites with a high level of Cu and other metals (Cárcamo et al. 2012; Ginocchio et al. 2002; Meza-Ramirez et al. 2021; Milla-Moreno and Guy 2020; Orchard et al., 2009; Ortiz-Calderón et al., 2008). These studies show a wide range of options for phytoremediation, but the hope is to restore those sites to a level of complexity that includes a great variety of microorganisms, flora and fauna, aiming to reflect complex interactions between diversity, structure and ecological processes, as advised by Ruiz-Jaen and Aide (2005). Since the aim of ecological restoration is to assist the recovery of an ecosystem, species known to exist in those sites prior to the disturbance are likely to have better possibilities for survival, growth and reproduction, over species that were not part of that landscape. In this context, and considering that invasive species reduce biodiversity and alter ecosystem functions, measures to promote native plants have been advised (Kettenring and Adams, 2011), particularly if they are identified as vulnerable or endangered (Orchard et al., 2009). Moreover, native plants that have added value (e.g., are medicinal, bee-friendly, good for forage, etc.) should be a priority (Orchard et al., 2009), due to their increased benefit to local communities. Research is needed to aid decision-makers in the mining sector in selecting the best candidates for phytoremediation. A major challenge to using the many native and endemic plant species that are potentially available is the lack of information regarding their propagation, basic physiological requirements, tolerance levels, metal uptake and sequestration patterns, etc. Assessment of metal distribution in tissues is crucial to understanding environmental impacts, tolerance mechanisms, and interactions between multi-metal and other stressors in plants. One relatively fast and non-destructive way to survey multi-element distributions in plant tissues is via X-ray fluorescence (XRF) as described by Vijayan (2015). This technique has been proven 41 versatile for visualizing Cu bioaccumulation in algae (Cervantes-Garcia et al., 2014), studying Cu tolerance of willow in flooded soils (Cao et al., 2019), and monitoring zinc distribution in drought-stressed leaves of wheat (Triticum aestivum L.) under drought stress (Willick et al., 2018). We set out to explore the ecophysiology and metal sequestration patterns of two species native to sclerophyllous forests in Chile, both with great potential for tailings restoration; namely, quillay (Quillaja saponaria Molina) and espino (Vachellia caven (Molina) Seigler & Ebinger, formerly assigned to Acacia). Espino (‘Kawen’: paddle in Mapuzugun), a member of the Fabaceae, is a nitrogen-fixing, semi-deciduous shrub or small tree (up to 6 m) with rigged thick bark, spinescent stipules, and bipinnately compound leaves that are ultimately dissected into tiny leaflets about 1–2 mm long and 0.5 mm wide (INFOR, 2012). Espino has been intensively used for charcoal production, due to its high caloric content, and its protein-rich seeds and pods are valued as forage (INFOR, 2012). In contrast, quillay (‘Küllay’: a certain tree in Mapuzugun), a member of the Quillaceae, is an evergreen tree (to 20 m) with thick rounded leaves that are 3–4 cm long and 1.5–3 cm wide (INFOR, 2011). Quillay provides good cover for foraging animals and is a rich source of saponins with a variety of industrial applications (INFOR, 2011). We compared seedling growth of these two species in a greenhouse experiment under two levels of Cu stress and, after 6 months, we measured photosynthesis, chlorophyll fluorescence (as a stress indicator) and elemental concentrations, and performed XRF mapping of metal distributions. In a previous field study (Milla-Moreno and Guy, 2020), we found that several native species, espino and quillay included, tolerated the stress of various metals in the tailings for six years with no clear advantage from amendments (i.e., mycorrhiza and compost). Moreover, Cu was prominent in quillay roots but not leaves, whereas in espino there was less Cu uptake overall and it was 42 more evenly distributed. We hypothesized that root versus shoot Cu distribution patterns would differ in espino and quillay under controlled environment conditions, possibly leading to differential effects on photosynthesis in these two species. 3.3. Materials and methods 3.3.1. Plant material and treatments Seeds of espino (Vachellia caven (Molina) Seigler & Ebinger) and quillay (Quillaja saponaria Molina) were obtained commercially (Vivero Antumapu, Chile). The seeds were germinated under a mist tent and transplanted after two weeks into 1-gallon pots (one seedling per pot) containing a 70% peat moss and 30% perlite mixture in a completely randomized design at the University of British Columbia greenhouse between December 2018 and June 2019. Espino seeds needed chemical scarification with sulfuric acid to break dormancy (INFOR, 2012). Quillay seeds were stratified by soaking in cold water for 72 h (INFOR, 2011). A fertigation system delivered 100 mL of liquid media (Table B1) to each pot every other day for the first 3 months, and then 150–300 mL each day for the remaining three months, a sufficient quantity to ensure that nutrient solution drained freely from the pots on every watering. Analyzes of the leachate using an ICP-MS confirmed an available Cu concentration of 377 ppb of Cu in the 50 µM Cu treatment and 620 ppb of Cu in the 100 µM Cu treatment. Dripper outlets were repositioned bi-weekly. After 2 months to allow plant establishment, CuSO4 was added to the fertigation solution to achieve Cu treatment concentrations of 0, 50 and 100 µM above background (0.0033 µM Cu in the unamended fertigation solution). There were five pots per species per treatment. Natural light provided a photosynthetic photon flux density (PPFD) of 470 μmol m−2 s−1 averaged over the entire 6 months. Supplemental lighting to ensure a minimum 43 PPFD of 240 μmol m−2 s−1 and a 12 h photoperiod was applied. Temperatures averaged 26 °C during the day and 19 °C at night. 3.3.2. Physiological measurements A LI-COR 6400 XT portable infrared system (LI-COR, Biosciences, Lincoln, NE, USA) was used to measure gas exchange traits on the youngest mature sun-exposed leaves (three times for each plant) over a 5-week period beginning 13 weeks after treatments began, including maximum photosynthetic rate (Amax; μmol CO2 m−2 s−1), stomatal conductance (gs; mol H2O m−2 s−1) and instantaneous WUE as determined by Amax over transpiration (WUEi; μmol CO2 mmol H2O−1). Leaves of quillay covered the full window of the cuvette, but the feathery leaves of espino only covered about 22% of the frame (calculated with a leaf area meter and three measurements of an espino leaf on a 6 cm2 frame to match the LI-COR). We applied a factor of 4.587 to account for this difference. After gas exchange measurements were completed, the same leaves were then dark-adapted for 20 min and the maximum quantum efficiency of PSII photochemistry (Fv/Fm) was obtained after a saturating flash (>7000 μmol m−2 s−1) as per Genty et al. (1989), from 10:00 to 14:00 hrs with a FluorPen FP 100 (Photon Systems Instruments, Drasov, CZ). Plants were harvested 18 weeks after treatment initiation, at which time the chlorophyll content index (CCI) was estimated on the youngest mature leaf using a CCM-200 Plus (Opti-Sciences Inc, Hudson, NH, USA). Two types of samples were taken from each plant after washing the potting soil mix from the roots and making basic growth measurements (shoot length, root length and fresh weight). Leafy twigs were pressed and air-dried, and root segments were placed into open vials and dried over silica gel in a desiccator. After further drying at 70 °C, followed by 44 microwave digestion, root and leaf Al (mg kg−1), Ca (mg kg−1), carbon (%), Cu (mg kg−1), Fe (mg kg−1), K (mg kg−1), Mn (mg kg−1), Mo (mg kg−1), Na (mg kg−1), nitrogen (%), P (mg kg−1) and Zn (mg kg−1) concentrations were determined by ICP-MS at the Analytical Chemistry Research Laboratory of the British Columbia Ministry of Environment. There was limited material for these analyses for espino; hence, for this species, composite samples consisting of an equal quantity of tissue from all five plants per treatment were constructed. After harvesting, about 300 g of fresh soil mix from each pot was also shipped to the same laboratory for immediate analysis of pH, soil mix organic matter (by loss on ignition), and concentrations of oxalic and citric acid (via water extraction, LC Analysis). Soil mix Ca (mg kg−1), Cu (mg kg−1), Fe (mg kg−1), K (mg kg−1), Mn (mg kg−1), Mo (mg kg−1) and Zn (mg kg−1) were determined by ICP-MS as above. The Cu bioconcentration and bioaccumulation factors were calculated in two ways: first as the ratios of the mean Cu concentrations in the roots (Bcfsoil mix) and leaves (Bafsoil mix) to that of the soil mix, respectively, and second as the ratios of the mean Cu concentrations in the roots (Bcfsolution) and leaves (Bafsolution) in mg kg−1 to that of the fertigation solution in mg L−1 (Eid et al., 2019), respectively, while the translocation factor (Tf) was calculated as the ratio of Cu concentration in the leaves relative to the roots (Yoon et al. 2006). 3.3.3. Element visualization X-ray fluorescence microimaging of single leaf and root subsamples from each species grown for 18 weeks with (100 μM) and without (0 μM) added Cu was performed at the Canadian Light Source Synchrotron facility (Saskatoon, SK). Dried leaves were placed directly on Kapton tape for imaging (Figure B1). Roots were placed in centrifuge tubes and immersed in millipore water overnight for rehydration. Samples were then taken out of the water and positioned in molds containing cryogel for freezing with liquid nitrogen. After this, radial cross-sections of 80 μm 45 thickness were obtained with a microtome (Leica CM1950, Leica Microsystems), re-dried, then placed on Kapton tape. Images were collected on the BioXAS-Imaging beamline (Canadian Light Source, 2021) configured as follows: incident energy of 15 keV, micro-mode, stage set at 90 degrees to the incident beam, detector at 45 degrees to the incident beam, beam focus of 5 µm for leaves and 12 µm for roots, with a dwell time of ~100 min. The data processing and mapping were done using PyMCA (Solé et al., 2007). 3.3.4. Statistics Soil mix, growth and physiological statistics were analyzed using 2-way ANOVAs in R (R Core Team, 2020). One-way ANOVA was used to explore situations where there was a significant interaction in the absence of a major treatment effect. We employed the Bonferroni correction when we analyzed 2-way ANOVA in growth and photosynthesis using R. For leaf and root elemental concentrations and heavy metals, and because tissues were pooled for espino, t-tests were conducted to evaluate differences between species and 1-way ANOVA was used to examine treatment effects in quillay. 3.4. Results 3.4.1. Soil mix We analyzed the soil mix in three pots without plants. Copper concentration averaged 15, 203 and 354 mg kg−1 in the control (hereafter referred to as 0 µM) and 50 µM and 100 µM Cu treatments, respectively. The soil mix was slightly acidic (5.7 to 6.0), with about 50% organic matter content and average oxalic and citric acid concentrations of 267 and 19 mg kg−1, respectively. Calcium, K and P accounted for 5–9%, 0.7–1% and 0.2–0.3% of the soil mix dry 46 mass, while concentrations of Fe, Mn, Mo and Zn ranged from 3–5 g kg−1, 0.13–0.15 g kg−1, 7–10 mg kg−1 and 30–34 mg kg−1, respectively. Soil mix pH, Cu, Fe, K, Mn, Mo and organic acid contents were modified by the presence of plants, depending on species and/or treatment (Figure 3.1, Table B2). Soil mix with plants rooted in it had somewhat less Cu, Mn and Mo, approximately doubled citric acid and slightly higher K and pH. Soil mix oxalic acid content was increased about 2.4-fold by the presence of quillay but not espino. We found a significant species effect for Cu, Fe, Mo, pH, and organic acids, and a significant treatment effect for Cu, as expected, but also for Mn, Mo, and pH (Table B3). There was a species by treatment interaction for Cu and pH. Soil mix Mo was reduced in the presence of additional Cu. Espino reduced the soil mix Cu and Mo concentrations more than quillay, but caused a greater rise in pH in the Cu treatments. Pots with espino also tended to have slightly higher Fe concentration. With both espino and quillay, soil mix in the 50 μM Cu treatment had significantly lower Mn than either of the other treatments. Figure 3.1. Characteristics of soil mix from pots containing espino or quillay after 6 months growth; (a) calcium, (b) Total copper, (c) iron, (d) potassium, (e) Total manganese, (f) molybdenum, (g) organic matter, (h) pH, (i) Total zinc, (j) citric acid, (k) oxalic acid. Whiskers in boxplots show the data range excluding outliers. 47 3.4.2. Growth Biomass (FW) of quillay exceeded espino by approximately 4-fold when averaged across treatments (Figure 3.2, Table B4), but shoot lengths were more comparable. Shoot lengths of espino and quillay, increased significantly as Cu was increased (Table B5). Similarly, the Cu treatments resulted in a slight increase in FW of espino (p < 0.009; Table B6) and the opposite in quillay (not significant). There were no significant treatment or species effects on root length. Figure 3.2. Growth parameters of espino and quillay at time of harvest; (a) biomass, (b) shoot length, (c) root length, and (d) CCI. 3.4.3. Photosynthetic performance Chlorophyll content index was much higher, by roughly nine times, in quillay than in espino (Figure 3.2d, Table B4). Two-way ANOVA (Table B5) indicated no significant effect of 48 treatment, but there was significant interaction with species and, when considered alone, CCI in espino increased significantly with Cu treatment (p < 0.001, Table S6), whereas it trended in the opposite direction in quillay (p = 0.080). Likewise, although Amax was unchanged with Cu treatment in quillay (Figure 3.3a, Table B7), in espino at 100 μM it was nearly tripled over the control level (p < 0.001). Overall, and in contrast to CCI, both Amax and gs (Figure 3a,b) were considerably higher in espino than quillay. There were no significant effects of treatment on gs or WUEi in either species, but quillay had a significantly higher WUEi than espino (Table B8, Figure 3.3c). Two-way ANOVA indicated no effect of species or treatment on QY, but again there was a significant interaction and, when considered alone, one-way ANOVA (Table B9) showed a significant increase in QY for espino (p < 0.003) but not quillay. Figure 3.3. Effects of Cu treatments on photosynthetic traits and stomatal conductance in espino and quillay; (a) maximum photosynthetic rate, (b) instantaneous water use efficiency, (c) stomatal conductance, (d) quantum yield. 3.4.4. Elemental analysis Root and leaf elemental concentrations are presented in Table 3.1. The pooling of tissue samples for espino precluded a 2-way ANOVA, but Cu was clearly increased in both leaves and, more so, 49 roots in rough proportion to the Cu supplied. In quillay, foliar Cu rose from 2.2 mg kg−1 in controls to ~10 mg kg−1 in Cu-treated plants (p = 0.007; Table B10), and from 12 mg kg−1 to 67 and 97 mg kg−1 in roots at 50 and 100 μM Cu, respectively (p < 0.001). No other elements in leaves were affected by the provision of additional Cu, but Ca (p < 0.004) and P (p < 0.003) were lower in roots at 50 μM than at 0 or 100 μM Cu. Although foliar Cu concentrations and bioaccumulation factors tended to be somewhat lower in quillay than in espino, they were not significantly different. Bioaccumulation factors relative to the soil mix (Bafsoil mix) fell considerably when the Cu was increased above the control (Table B12). Given the very low Cu concentration in the fertigation solution at 0 μM Cu, calculation of the bioaccumulation and bioconcentration factors relative to the fertigation solution (Bafsolution and Bcfsolution) is not relevant for this treatment. In espino at 50 and 100 μM Cu, the Bafsoil mix was 0.080 and 0.058 whereas the Bafsolution was 2.74 and 2.05, respectively. The respective bioconcentration factors were 0.860 and 0.752 (Bcfsoil mix) and 29.3 and 26.8 (Bcfsolution). In quillay at 50 and 100 μM Cu, respectively, the Bafsoil mix was 0.051 and 0.035 and the Bafsolution was 3.18 and 1.48. Quillay, however, accumulated significantly less Cu in roots than did espino at both 50 and 100 μM Cu (p < 0.02; Table B11) and consequently also had lower bioconcentration factors in these treatments, with the Bcfsoil mix being 0.336 and 0.360 at 50 and 100 μM Cu, while the Bcfsolution was 21.1 and 15.2, respectively. Mean translocation factors were similar in quillay and espino, dropping from 0.177–0.254 at 0 μM Cu, to 0.076–0.097 at 100 μM Cu. At all treatment levels, quillay had only about half as much foliar N as espino (p < 0.001), and about 30% less N in the roots. Quillay also always had lower concentrations of Fe in both leaves and roots (p < 0.001). Molybdenum too, was lower in quillay than espino in leaves (p < 50 0.001) and in roots (p < 0.02), except in roots at 100 μM Cu (p = 0.496). In contrast, quillay accumulated more Na in roots than espino (p < 0.02), and more K in leaves (p < 0.004). 51 Table 3.1. Descriptive statistics for elemental concentrations in dry leaves and roots of espino and quillay in control, 50 and 100 μM Cu treatments. Tissue Species Treatment Ca (g/kg) Cu (mg/kg) Fe (mg/kg) K (g/kg) Mn (mg/kg) Mo (mg/kg) N (%) Na (mg/kg) P (g/kg) Zn (mg/kg) Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean [SD] [SD] [SD] [SD] [SD] [SD] [SD] [SD] [SD] [SD] Leaves Espino Control 19 3.3 49 20 44 1.6 3.7 25 3 15 -- -- -- -- -- -- -- -- -- -- 50 µM Cu 14 8.7 53 11 34 2.9 3.5 29 1 18 -- -- -- -- -- -- -- -- -- -- 100 µM Cu 14 13 51 12 55 1 3.2 20 2 16 -- -- -- -- -- -- -- -- -- -- Quillay Control 10 2.2 26 47 30 0.54 1.7 570 2 16 [2.5] [1.2] [1.8] [9.8] [13.0] [0.2] [0.1] [697] [0.2] [2.2] 50 µM Cu 13 10.1 25 43 49 1.2 1.6 320 2 18 [1.5] [3.7] [6.3] [2.6] [20.5] [0.9] [0.1] [143] [0.3] [2.0] 100 µM Cu 10 9.4 21 37 52 0.9 1.6 119 2 15 [3.7] [4.8] [4.9] [6.2] [30.9] [0.5] [0.2] [40] [0.6] [2.5] Roots Espino Control 49 13 640 19 75 21 2.2 880 24 67 -- -- -- -- -- -- -- -- -- -- 50 µM Cu 17 93 420 18 24 15 2.7 550 4 25 -- -- -- -- -- -- -- -- -- -- 100 µM Cu 30 170 410 15 79 21 2.3 610 13 40 -- -- -- -- -- -- -- -- -- -- 52 Tissue Species Treatment Ca (g/kg) Cu (mg/kg) Fe (mg/kg) K (g/kg) Mn (mg/kg) Mo (mg/kg) N (%) Na (mg/kg) P (g/kg) Zn (mg/kg) Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean [SD] [SD] [SD] [SD] [SD] [SD] [SD] [SD] [SD] [SD] Quillay Control 16 12.2 158 20 53 7 1.6 1,780 8 43 [1.8] [2.9] [40.9] [1.8] [14.0] [2.2] [0.3] [497] [1.2] [7.4] 50 µM Cu 11 67.2 172 18 30 4.6 1.6 1,840 4 33 [2.0] [14.5] [59] [3.9] [7.0] [1.0] [0.3] [385] [1.1] [7.4] 100 µM Cu 15 96.6 126 21 63 4.7 1.5 1,820 6 38 [1.3] [23.1] [34.3] [2.7] [24.3] [1.7] [0.3] [536] [1.4] [8.1] N = 5 for quillay and espino (composite), SD: Standard Deviation, Ca: calcium, Cu: copper, Fe: iron, K: potassium, Mn: manganese, Mo: molybdenum, N: nitrogen, Na: sodium, P: phosphorus, Zn: zinc. 53 3.4.5. X-ray fluorescence microimaging X-ray fluorescence analysis was restricted to just eight specimens, including single leaf and root samples from each of four plants (one of each species at 0 and 100 μM Cu). The emission spectra from these samples are compared in Figure 3.4, where in all four panels the total counts are normalized to the control (0 μM) to account for signal strength variation due to sample size and/or total elemental concentration. Roots have a quite different general profile relative to leaves, suggesting that mobile elements were in large part leached from the roots when they were hydrated for purposes of sample preparation. Hence, peaks for P, S, Cl, K and Mn (at 2.014, 2.308, 2.622, 3.314 and 5.899 keV, respectively) are lower than expected based on our elemental analysis of tissues. The same is also very likely true for bromine, which accounts for the large peak at 11.924 keV in leaf tissues, but which is much smaller in roots. As expected, and noting that counts are plotted on a log scale, the Cu peak is much higher in leaves of both species when they were exposed to elevated Cu in the fertigation solution (Figure 3.4a,b). The Cu peak is also more pronounced in the root section of the Cu-treated quillay (Figure 3.4d), but not so much in espino (Figure 3.4c). In espino, the leaf sample from the Cu-treated plant shows markedly reduced fluorescence signals from silicon and nickel (arrows in Figure 3.4a). The root sample of Cu-treated espino shows a similar effect on peaks for vanadium and chromium, and there may be a similar effect of excess Cu on iron and zinc in the root of quillay. 54 Figure 3.4. Multi-element spectra in plant tissues; (a) espino leaf, (b) quillay leaf, (c) espino root, (d) quillay root. In every panel, the spectrum for Cu-treated tissue (100 μM treatment) is normalized relative to the spectrum for the control (0 μM treatment) by adjusting the total counts upwards or downwards, as appropriate to the sample. This normalization accounts for the lower signal-to-noise ratio for the Cu-treated quillay in panel b. Images for select elements associated with these spectra, in plants receiving 100 μM Cu, are presented in Figures 3.5 and 3.6 (leaves), and Figures 3.7 and 3.8 (roots). Calcium is shown in all four figures and we include K in the leaf images but not the roots, where instead we show the distribution of Mn. Copper was not restricted to a particular tissue in the leaves of either species, other than a bright spot in the espino sample that might represent contamination. Calcium was also found in concentrated form as calcium oxalate crystals, particularly near the veins of both 55 species. In espino, Ca also accumulated in the pulvinus at the base of each of the two secondary leaflets partially visible in the image. Figure 3.5. X-ray fluorescence images of leaflets of espino grown at 100 μM Cu. Scale bar of 100 μm; arrow in the lower left panel showcases the presence of calcium oxalate crystals in the secondary rachis; asterisk indicates a pulvinus at the base of the lower leaflet, and another pulvinus is visible on the uppermost leaflet; calcium oxalate crystals are also seen in the mid-rib of the lower leaflet. 56 Figure 3.6. X-ray fluorescence images of a leaf of quillay grown at 100 μM Cu. Scale bar of 100 μm; arrow in lower left panel points to calcium oxalate crystals at the mid-rib. View is perpendicular to the upper leaf surface. In roots of espino (Figure 3.7), Cu was distributed throughout but was particularly concentrated in tissues external to the phloem. Manganese had a similar distribution but was more evenly distributed across the inner (living phloem) and outer bark (periderm and remnant cortical and epidermal tissues), as was Ca. In quillay (Figure 3.8), the distribution of Cu clearly shows the structure of the xylem, with some increased presence near the cambium, but it is particularly abundant in the outer bark, presumably outside the periderm. Manganese had a similar pattern of localization. In contrast, Ca was most prominent in the phloem tissue near the cambium. 57 Figure 3.7. X-ray fluorescence images of a cross-section of an espino root grown at 100 μM Cu. Scale bar of 100 μm. Figure 3.8. X-ray fluorescence images of a cross-section of a quillay root grown at 100 μM Cu. Scale bar of 100 μm. 58 Nickel was easily visualized in the leaf of espino and the root of quillay (Figure 3.9) and in both cases its distribution was similar to Ca. Co-occurrence of Ni with the pattern of cells containing calcium oxalate crystals was evident in the espino leaf. There may be a similar association between Ni and Ca in the roots of quillay, but the image resolution is insufficient to clearly indicate the presence of calcium oxalate crystals. Figure 3.9. X-ray fluorescence images of Ca and Ni, and their combined distributions, in leaflets of espino (first row) and a cross-section of a quillay root (second row), both grown at 100 μM Cu. Scale bar of 100 μm. The samples imaged are the same as in Fig. 3.5 and 3.8, respectively. 59 3.5. Discussion Many studies have shown that excess Cu negatively impacts the growth of most plants, often at concentrations of less than 50 μM Cu (Kumar et al., 2021). For example, exposure of common bean (Phaseolus vulgaris L.) to 50 μM Cu resulted in chlorosis, necrosis and a severe reduction in dry weight after a mere 3 days (Bouazizi et al., 2010). In contrast, the growth of quillay was not affected after 4 months growth in our 50 and 100 μM Cu treatments, whereas shoot growth in espino was even enhanced. These observations are consistent with the ecologies of these two species and their excellent potential for use in phytoremediation. Total copper in Cu-affected native soil (in Central Chile) ranges from 30 to 200 mg kg−1 (Ginocchio et al., 2006) and 70–155 mg kg−1 in agricultural soils of north-central Chile not affected by mining (Aguilar et al., 2011). In Canada, the maximum allowable total Cu concentration in agricultural soils is 63 mg kg−1 (Canadian Council of Ministers of the Environment, 2019). These ranges were bracketed by the conditions we imposed, which resulted in final average Cu concentrations in the soil mix of 15, 203 and 354 mg kg−1 in the control (0 μM), 50 and 100 μM Cu treatments, respectively. Total Cu in tailings, however, can be much higher; e.g., from 360 to 4000 mg kg−1 in tailings of Central Chile (De La Iglesia et al., 2006). Further, our soil mix was high in organic matter (about 50%) relative to most tailings, which should have the effect of ameliorating metal stress. Clearly, much of the Cu provided in the fertigation solution was bound by the soil mix and its Cu concentration must have increased over the course of the experiment. Organic matter and pH are known to regulate Cu bioavailability and mobilization to aerial parts (Alloway, 1995; Kabata-Pendias and Pendias, 2001). Low molecular weight organic acids also have a profound effect on metal availability and toxicity (Adeleke et al., 2017). Analysis of our soil mix revealed no effect of the Cu treatments on citric or oxalic acid levels, but oxalic acid 60 was consistently higher in pots occupied by quillay, while citric acid tended to be higher in pots with espino. Although not detected here, plant roots often show increased exudation of organic acids when exposed to heavy metals. For example, Montiel-Rozas et al. (2016), found that Malva sylvestris exuded oxalic acid in response to a combined Cu, Cd and Zn stress, and Qin et al. (2007) reported a similar response to Cu stress in Populus tremula L. The binding of metals by organic acids exuded from roots may help in mobilization and increase metal uptake (Kim et al., 2010) or, contrastingly, limit uptake (Meier et al., 2012). Although several species have been defined as Cu-accumulators such as Ammania baccifera L. (Das and Maiti 2007), Oenothera affinis Cambess. (González et al., 2008), Mimulus luteus L. var. variegatus (Lodd.) Hook., Cenchrus echinatus L., Erigeron L., Mullinum spinosum (Cav.) Pers., Nolana divaricate (Lindl.) I.M. Johnst., and Dactylium sp. (Ginocchio and Baker, 2004), and Adesmia atacamensis Phil. (Lam et al., 2016), studies on quillay and espino are scarce (León-Lobos et al. 2011, Milla-Moreno and Guy, 2020; Pizarro et al., 2016). As in our previous field study (Milla-Moreno and Guy, 2020), we found that Cu accumulation in quillay was more pronounced in roots than in leaves. The translocation factors were similar between the two studies, averaging 0.07 in the field and 0.10 in the present study at 100 μM Cu, underscoring the potential of this species for phytostabilization. Although we reached Cu concentrations in our soil mix that greatly exceeded those of the tailings, Cu concentrations in roots and leaves of quillay were comparable between the two studies. In the greenhouse, however, quillay did not accumulate Cu from the fertigation solution any faster than it was bound by the peat-based soil mix and the Bcfsoil mix remained below 1.0 regardless of the treatment (Table B12). Consequently, from this perspective, the bioconcentration factor was much higher on the tailings than in the greenhouse, by as much as 36-fold. Likewise, the bioaccumulation factor was also considerably 61 higher on the tailings as compared to the Bafsoil mix that we found here. It is likely that the measurement of total Cu in the tailings and the soil mix (as opposed to bioavailable Cu) is behind these differences. We don’t know the bioavailability of the Cu in the tailings but the Cu concentration of the fertigation solution, though mitigated by the soil mix, provides a benchmark for the greenhouse study. From this other perspective, the field and greenhouse results are more comparable, with the Bcfsolution and Bafsolution averaging 15.2 and 1.48, respectively, for quillay at 100 μM Cu. Regardless, the bioconcentration and bioaccumulation factors from the field are more relevant for applied purposes, but unlike tissue concentrations and elemental distributions they provide little physiological information. In contrast to quillay, the distribution of Cu between the roots and leaves of espino was quite different here as compared to the field. In the greenhouse espino behaved like quillay and restricted Cu translocation to the leaves (Tf = 0.08 at 100 μM Cu), but in the field Cu was more evenly distributed between roots and leaves (mean Tf = 0.94). This inconsistency prevents us from recommending espino for purposes of phytostabilization without further study. We noted a few other differences in elemental composition between espino and quillay, independent of the treatments imposed. For example, Mo was higher in espino than quillay, which might be related to a greater need for this element in support of symbiotic nitrogen fixation via Rhizobium bacteria. Molybdenum acts as a cofactor for nitrogenase during the conversion of N2 into ammonium (Mendel and Hänsch, 2002). In contrast, across treatments and tissues the Na content of quillay was much higher than in espino (Table 1). There were large differences in Amax and gs between espino and quillay. These differences may in part relate to Amax and gs being over-estimated in espino because of difficulty in determining leaf area within the cuvette of the gas exchange system. The leaves of espino are finely dissected 62 and sensitive to touch, tending to fold when they are disturbed. In contrast, the CCI was considerably higher in quillay than in espino. This difference may be related to the evergreen nature of quillay leaves, but also likely again reflects the difference in leaf anatomy causing espino leaves to not fully cover the device aperture. Espino seemed to be lacking Cu for optimal photosynthetic performance, with Amax peaking in the 100 μM Cu treatment, whereas in quillay Amax remained stable across treatments. Although foliar nitrogen, Amax and gs were higher in espino than in quillay across all treatments, quillay accumulated a greater fresh mass. Unlike Amax and gs, calculations for WUEi and QY are independent of leaf area. The WUEi of espino seemed to increase with Cu while in quillay it was more stable and significantly higher. Similarly, in espino, QY, CCI, shoot length and biomass (FW) were all significantly higher when Cu was added. We conclude that espino has a requirement for Cu that is much higher than typical and suggest that it may be an obligate metallophyte (Baker et al., 2010). Nonetheless, regardless of treatment, the foliar Cu in both species remained below the typical toxicity threshold for leaves of average plants (i.e., 20 mg kg−1 (Kabata-Pendias and Pendias, 2001)) and the allowable threshold for animal feed (i.e., 40 mg kg−1 (Weiss, 2008)). X-ray elemental analysis and heavy metal mapping can shed light on the ecophysiology of metal accumulators and their tolerance and/or avoidance strategies (Van der Ent et al., 2018). This technique allowed us to analyze the distribution of many elements, several of which are not shown here. As noted in the results, however, the rehydration protocol necessary for making root sections appears to have leached some mobile constituents, leaving fixed or bound fractions behind. This effect will depend on the element. For example, K and Ca are normally abundant in plant tissues, given that K+ is the major free cation in plant cells (Ragel et al., 2019), whereas Ca is structurally important in cell walls and for membrane stability through its interactions with 63 pectin and phospholipids, respectively (Thor, 2019). Hence, rehydration is expected to affect the distribution of K more than Ca. We used K and Ca to highlight leaf structure in Figures 3.5 and 3.6, and Ca and Mn for roots in Figures 3.7 and 3.8. Another element with a strong XRF signal in leaves, but not roots, was bromine (Figure 3.4). Bromine was not supplied in the fertigation solution but is commonly found in peaty soils and readily taken up by plants (Shtangeeva, 2017). Emission spectra suggested that excess Cu may have affected levels of Si and Ni in the espino leaf, and other elements in the roots (V and Cr in espino, and Fe and Zn in quillay), but further work is needed to follow-up on these observations. Copper was shown to competitively inhibit Ni uptake and reduce transport to the shoot in Glycine max (L.) Merr. (Cataldo et al., 1978). Copper was widely distributed across both leaf and root tissues, with some tendency to reach higher levels (with Mn) in what we presume to be dead cortical tissue and/or the periderm of roots (Figures 3.7 and 3.8). Copper is an essential plant micronutrient, acting as a catalyst in the photosynthetic and respiratory electron transport chains, and as a cofactor for numerous other enzymes. It is substantially involved in the control of the cellular redox state (e.g., via the activity of Cu/Zn superoxide dismutase) and in the formation of lignin in cell walls (Printz et al., 2016). This latter role may account for the thin band of Cu in the vicinity of the cambium in the root of quillay (Figure 3.8). The root sections of both species also show the presence of Cu in the xylem. It is most clearly defined in quillay where it appears to outline the pattern of the cell walls, except in the wood rays. Cell walls are major sites of accumulation for heavy metals where they interact with -COOH, -OH and -SH groups, such as the carboxyl-groups of homogalacturonans (Krzesłowska, 2011). Mechanistically, the differences between the species in Cu concentration and distribution may relate to differences in transporter proteins that limit uptake and/or affect the redistribution of Cu 64 to shoots or, for example, root cortical tissue. There could be a role for the root cortex and/or epidermis in preventing the entry of excess Cu into the stele. Preferential localization of heavy metals in root epidermal and cortical tissues has been noted in other species (Shi et al., 2019; Hirano et al., 2012). These tissues die soon after the initiation of secondary growth and the development of the periderm, thus ridding the plant of the sequestered metals. Further studies are needed at different stages of root growth to investigate this as a possible stress avoidance strategy in espino and quillay. Plants may also deal with heavy metals by sequestering them within the vacuole or cell wall, often with the help of chelators such as phytochelatins, metallothioneins, and organic and amino acids (Cui et al., 2020; Hall 2002; Krzeslowska, 2010; Printz et al., 2016; Van Hoof et al., 2001). Further work is required to elucidate if and where such mechanisms may operate in quillay and espino. Crystals of calcium oxalate, which is minimally soluble in water (6.1 mg L−1 at 20 °C (Haynes, 2014)), were strikingly obvious by XRF imaging in both species. Their presence in quillay has been previously reported (Lersten and Horner, 2005), but, to the best of our knowledge, not in espino. Nevertheless, they are present in many plants and can take various forms (Raman et al., 2014). In leaves of quillay, spherical aggregates of crystals (druses) occur throughout the spongy mesophyll but more frequently beneath veins (Lersten and Horner, 2005), consistent with the pattern seen in Figure 3.6. Although druses also occur in the cortical parenchyma and pith of quillay stems, elongated column-shaped crystals (styloids) are abundantly interspersed with fibers in the phloem (Lersten and Horner, 2005) and are likely what we observed in the root section shown in Figure 3.8. Calcium oxalate crystals are hidden players in Ca cycling, and a possible heavy metal detoxification strategy (Franceschi and Nakata, 2005). Nickel was not deliberately supplied to 65 the plants but may have been naturally present in the potting mixture or as a trace contaminant of the fertigation solution (Alloway, 1995). In this context, and although we found no clear concurrence with Cu, the distribution of Ni in the espino leaf and quillay root suggested an association with these crystals. Nickel was seen to accumulate near abundant calcium oxalate crystals in the seed coats of Grevillea rubiginosa Brongniart & Gris when germinating in the presence of various Ni salts (Léon et al., 2005). In conclusion, both quillay and espino demonstrated high tolerance to the provision of excess Cu. Indeed, espino may require considerably more Cu than normal to support optimal growth, as also evidenced by its improved photosynthetic performance with increased Cu in the media. Excess Cu was mostly restricted to the roots of both species and showed some tendency to accumulate in tissue layers outside the periderm. Foliar Cu concentrations remained below toxicity levels in both species, in contrast to our previous study of older plants growing on tailings in the field (Milla-Moreno and Guy, 2020), where this threshold was exceeded in espino. These observations suggest that differences in age and/or other soil conditions may affect experimental outcomes. The possible role of calcium oxalate crystals in sequestering heavy metals other than Cu is an interesting avenue for further investigation. 66 Chapter 4: Likelihood of using phytoremediation for mine-tailing management in Chile 4.1. Summary Chile has more than 750 tailings across the country, mainly distributed in the northern region, which also includes a biodiversity hotspot and the driest desert in the world. So far, tailing management has included chemical and physical stabilization, exclusively. Through an online questionnaire (n =43), we examined the attitudes of stakeholders (Academia, Industry, environmental Non-Governmental Organizations, and Government officials), towards the management of tailings. We also explored their views towards the inclusion of plants and their associated microbes, as an additional stabilization alternative. The consensus opinion across sectors is that there is general lack of proper tailings management in Chile. There is a critical willingness to incorporate phytoremediation, with a preference for using native plants, as an additional stabilization method. An absence of demonstrably successful phytoremediation projects, funding and access to suitable plants are perceived to be significant limitations. Local community involvement is considered key to successful restoration of tailings. 4.2. Introduction The last United Nations Climate Change conference confirmed the Paris Agreement's goal to prevent global temperatures from rising more than 2°C above pre-industrial levels and that carbon emissions should be reduced by 45% to achieve carbon neutrality around 2050 (United Nations, 2022). Such a large decrease in carbon emissions will depend in part on moving away from fossil fuels towards alternative energy sources. This energy transition will be accompanied by a greater reliance on electricity and the necessary raw materials to support electrification (e.g., 67 metals such as Cu, and rare earth elements such as lithium). In the last 50 years, the global Cu demand has tripled (Jorratt, 2021) and it is anticipated to increase by 3% annually over the next several years, mainly due to the electrification of the transportation sector (Jorratt, 2021), which currently accounts for about one quarter of total greenhouse gas emissions (Agusdinata et al., 2018). Thus, addressing climate change will require increased mining activity, particularly in countries like Chile, Perú, and Mexico, respectively comprising 23, 10, and 6% of global Cu reserves (Bertram, 2021). Stakeholders comprising community, government, unions, non-governmental organizations (NGOs) and media have indicated that while mining has economic benefits it also has negative impacts on society (Viveros, 2016). Copper mines cause significant degradation of the environment, affecting ecosystems via acidification, pollution, and loss of biodiversity. Impacts include mechanical damage to the landscape, acid mine drainage, the creation of tailings deposits, and smelter emissions containing about 0.11 mg of S per mg of Cu produced (Dudka and Adriano, 1997). Presently, an average of 537 million tonnes of tailings are deposited in Chile every year (SERNAGEOMIN, 2022). There are more than 750 tailing deposits across the landscape, mainly concentrated in the country's north, from Antofagasta to the Metropolitan Region (SERNAGEOMIN, 2022). As of 2020, 112 tailings were active, 173 abandoned and 467 inactive, while five were under construction (Table 4.1). Furthermore, five municipalities (i.e., the smallest administrative unit) hold more than half of the total tonnage of tailings present in Chile (e.g., Antofagasta, Calama, Alhué, and Pica). The quantity of abandoned tailings (over 11 billion tonnes), mainly concentrated in Antofagasta, the Metropolitan region and Coquimbo, is a call to action to Chile’s environmental and social future. 68 Table 4.1. Tailings volume (m3) and current tonnage by region built with information from the National Geology and Mining Service Registry collected in 2020 (decreasing order). Generally, tonnage is obtained from volume by assuming a tailings density of 1.5. Numbers of tailings deposits that were active, abandoned, inactive or under construction are shown. Region Current volume (hm3) Total volume (%) Active Abandoned Inactive UC II 2942.47 34.8 12 16 24 0 MR 1748.37 20.7 6 6 14 0 III 1606.45 19.0 31 23 109 5 IV 861.71 10.2 39 106 244 0 I 706.22 8.36 2 6 0 0 VI 327.74 3.88 3 1 15 0 V 246.71 2.92 15 11 54 0 XI 9.78 0.12 0 4 5 0 VII 0.65 0.01 4 0 2 0 Total 8450.08 100 112 173 467 5 I: Tarapacá, II: Antofagasta, III: Atacama, IV: Coquimbo, V: Valparaíso, MR: Metropolitan Region, VI: Libertador General Bernardo O’Higgins, VII: Maule, IX: Araucanía, UC: under construction. In Chile, tailings are overseen by the National Geology and Mining Service (SERNAGEOMIN, 2022). Current local regulations governing tailings are framed by a complex set of overlapping laws and decrees. Law 19.300 of Environmental Bases (1994) mandates environmental accountability in the form of prevention, mitigation and/or compensation of environmental impacts, along with Supreme Decree 95 (2001) which enacted the System of Evaluation of Environmental Impacts. Supreme Decree 248 (2007) sets requirements to approve tailings based on their design, construction, operation and closure. Law 20.551 (2012) regulates environmental impacts during the closure phase. Supreme Decree 41 (2012) gave further clarification and unification of the pre-existing regulations on mine closures and mining facilities (i.e., Law 20.551, Law 19.300, and Supreme Decree 95). Finally, Law 20.819 (2015) amended the definition of a tailing’s life cycle. 69 The Cu mining industry has seen little advancement in the decontamination of soils (Pulford and Watson, 2003). Furthermore, it is concerning that Chile’s carbon neutrality relies on Cu extraction (Moreno-Leiva et al., 2020), a process that is still energetically and water intensive, whose total volume of tailings seems to be as strikingly high as its reserves. High lithium extraction in Chile would add even more pressure to water sources and fragile ecosystems, such as salt waters in the country's north (Agusdinata et al., 2018). In addition, the mining sector formerly benefited from favorable regulations and a mixed model of state- and free-market-driven water management (Prieto et al., 2022). However, the recently enacted Law 21.435 (Ministerio Obras Públicas, 2022) defined water access as a human right and indicated that its main use should be to sustain human consumption, subsistence and sanitation. International standards (i.e., not binding nor mandatory) such as the Global Industry Standard on Tailings Management prepared by the United Nations Environment Programme, Principles for Responsible Investment, and the International Council on Mining and Metals, have a stronger emphasis on social and environmental matters from the earliest phases of project conception (i.e., planning, design) until post-closure. Furthermore, corporate social responsibility has been described as greenwashing marketing without addressing social or environmental impacts (Viveros, 2016). Solutions to decrease erosion and leaching of barren soils, and to decontaminate and restore ecosystems are urgently needed. The mining industry generates a lot of tailings waste because only 1-3% of the ore is metal concentrate (Adiansyah et al., 2015). Tailings are either discharged directly into rivers, oceans, and lakes or more commonly, to an impoundment pond (Adiansyah et al., 2015). Tailings in Chile are maintained with chemical and physical stabilization methods to prevent contaminants entering into neighboring ecosystems and/or local communities via air 70 or water (SERNAGEOMIN, 2022). While chemical methods are meant to limit acidification and the resultant solubilization and release of metals and metalloids, physical barriers act to contain contaminated materials. Closed tailings facilities require ongoing maintenance. One additional solution to tailings management could be establishing metal-tolerant plants with the ability to sequester metals or otherwise reduce their mobilization (Salt et al., 1995). This technology, called “phytoremediation”, uses plants for environmental cleanup, either by (1) using hyperaccumulating species that take up heavy metals from the soil into aerial tissues that can be harvested and disposed or processed for their metal concentrations (phytoextraction), (2) using roots of metallophytes to precipitate and concentrate heavy metals (rhizofiltration), and (3) using metallophytes that reduce the mobility of heavy metals (phytostabilization) (Salt et al., 1995; Suman et al., 2018). Phytoremediation can help restore ecological functions, and may also contribute to local jobs and livelihoods, improvements in air quality, and a stronger sense of community attachment to the landscape. Chile has a wide range of plants proven to be suitable for phytoremediation (Ginocchio and Baker, 2004; Milla-Moreno et al., 2022), and many more that are yet to be assessed (Lam et al., 2018; Pizarro et al., 2016; Santibáñez et al., 2008). Knowing there are successful experiences with phytoremediation is crucial, but is there a willingness to advance its application to tailings management in Chile? To that end, we sought to assess stakeholder opinions on the management of tailings in Chile to date, analyze the likelihood of using phytoremediation for mining closure, and identify the main barriers tailings managers face in choosing phytoremediation. 71 4.3. Materials and methods The qualitative data were collected via an anonymous online survey (Appendix C1) to elucidate the likelihood of using phytoremediation for mine-tailing management in Chile by that stakeholder (i.e., Academia, Industry, environmental Non-Governmental Organizations, and Government officials). The survey helped us assess stakeholders’ opinions about barriers to implementing phytoremediation, as well as their evaluation of current regulations. The questionnaire consisted of 35 questions that included multiple choice, rating (from strongly agree to strongly disagree), ranking (from 1-6 where 1 is highest priority) and open questions. We defined technical terms when needed to clarify and standardize the information provided to respondents. We advertised our survey weekly for 11 months (i.e., July 2021 to May 2022) on Twitter, Facebook, and LinkedIn. In addition, we specifically reached out to Chilean Deputies and Senators whose work was related to the environment via their institutional emails. We received 43 completed surveys with optional open-ended questions. Descriptive statistics, ANOVA, Tukey Honest Significant Difference and Pairwise comparisons were performed in R (RStudio v3). We analyzed the open-ended responses using NVIVO (2022.07.01 Build 554) to construct tree word maps to identify the two most prominent themes by finding the 100 most frequent words with a minimum length of three letters. Comments selected in this way are provided in the Results to illustrate the respondents’ reflections (in Spanish with English translation). 4.4. Results We received 49 responses and analyzed 43 completed surveys (Table C2 and Table C3). Most respondents lived and worked in the same region (81%), with the majority residing in the capitol 72 Metropolitan Region (58%). About 84% identified as Latina/o (47%) or Mestiza/o (37%), with professionals, between 35 and 44 years old (44%) and 45-54 years old (21%) overly represented, particularly those with industrial or academic affiliations, the two categories with the highest numbers of participants. Men were overly represented (72% overall, 79% in Industry and 67% in Academia). Most academics held a graduate degree, whereas in Industry some respondents held superior or professional education as their highest qualification. About 65% of the respondents held a graduate degree, and 35% held professional/superior education. Even though Forestry (9.3%) and Mining (42%) comprised most of the sector representation, other categories such as environmental education, consulting, and community-based projects were mentioned by 49% of the respondents. When respondents were asked their opinions regarding the current state of tailings management in Chile, there was general discontent (Fig. 4.1). Figure 4.1. Is Chile doing a good job with the management of its tailing facilities? Responses from a multiple-choice list with “yes”, “no”, and “I don’t know” options (N = 43). 73 When respondents were asked about their preferred approach for tailings stabilization, there was a clear preference for both physical and chemical stabilization, with 95 % and 97 % ranking them as either extremely or very important, respectively. However, 74 % of respondents also designated phytoremediation as being extremely (44 %) or very (30 %) important, indicating that many respondents ranked it equal to the other two more traditional approaches (Fig. 4.2). Figure 4.2. How important are the following stabilization methods? Note: Stakeholders could select one or more of Physical, Chemical, and Phytoremediation. Proximity to local communities, hydrology, and seismic nature of the site were chosen from a provided list as the most important aspects to be considered prior to the approval of new tailing facilities (Fig. 4.3). Other factors prioritized included total amount of tailings of the region, the geology of the site, and the tailing facility lifecycle. Respondents provided other, more specific aspects to consider, such as proximity to abandoned tailings (i.e., many of them are not included in current regulations and therefore not properly managed), agricultural lands, water bodies, and 74 archeological sites. The biodiversity on site and the type of tailings to be managed were flagged as less relevant factors to consider. Figure 4.3. Ranking of the key aspects to be considered when approving more tailing facilities. Note: The list of these potential key aspects was provided to respondents, and they could add three more key aspects as an optional follow-up response. We asked respondents about their views regarding possible major factors precluding the use of phytoremediation for tailings remediation in Chile (Fig. 4.4). Lack of funds (i.e., to pay for the phytoremediation implementation) and scientific support were the most frequently selected responses, followed closely by the lack of nurseries, the biodiversity on site, and the proximity to water bodies. Fewer than half of the respondents agreed that the barriers to using phytoremediation relate to proximity to sacrifice zones, emergency response plans, proximity to local communities and protected areas, or total regional mass of tailings. Some respondents opted to comment further on this matter and mentioned that not knowing the regulations relevant to tailings management also plays a part. Water scarcity was pointed out as another relevant 75 factor. Insufficient information about suitable plants for phytoremediation and demonstrably successful phytoremediation projects were also mentioned as barriers. Figure 4.4. How strongly do you think these barriers affect the use of Phytoremediation in Chile? Note: The list of these potential barriers was provided to respondents, and they could add three more barriers as an optional follow-up response. Prox. = proximity. Even though phytoremediation is mainly implemented as a closure or post-closure decision, the implementation (e.g., design, planning, the establishment of nurseries) could be executed from the early stages of a mine. The respondents from this study preferred to incorporate phytoremediation at the closure (84%), post-closure (79%) and operation (56%) stages (Fig. 4.5a) rather than the earlier stages of planning, maintenance, design and construction. When asked about the plant choice, there was a clear preference for the type of plant to be used, with natives (and particularly endemic species) unanimously being the preferred option. Thirty percent of the respondents also considered exotic plants to be acceptable (Fig. 4.5b). 76 Figure 4.5. Preferred stage in the life cycle of a tailing facility to (a) pursue phytoremediation, and (b) plant type to be used for phytoremediation. Note: For (a), “None of the above” was also an option, only selected by one person along with “Operation”, so we only kept the latter. 77 There was consensus around when a phytoremediation project would be considered successful, based on the high agreement about the characteristics that would help for that evaluation, such as the establishment of permanent green cover, restoration of the water cycle, and the return of native animals and plants (Fig. 4.6). Additionally, active and meaningful participation of local communities, effective containment of the heavy metals from the tailings, and the re-establishment of an autonomous and resilient ecosystem were chosen as essential indicators of successful restoration. Figure 4.6. When can a restoration project using phytoremediation be considered successful? Note: The list of these potential barriers was provided to respondents, and they could add three more barriers as an optional follow-up response. Nationally, regulations are focused on the closure phase and environmental impacts (e.g., Law 19.300, 20.551, 20.819, and Supreme Decrees 95, 248, and 41). Most respondents considered this legal framework insufficient to address environmental (Fig. 4.7a) and social (Fig. 4.7b) impacts. 78 Figure 4.7. Evaluation on regulations intended to address impacts. For panel (a), respondents were asked if current regulations around environmental impacts were enough. For panel (b) respondents were asked if current regulations around social impacts were enough. Note: For both (a) and (b) respondents could only select one choice, but they could supplement their selections with optional text. Furthermore, respondents indicated that the framework is not binding nor effective, mainly because it does not target small to medium-scale mines, nor the more than 170 abandoned tailings to date. Others mentioned that it does not consider the climate emergency impacts, particularly in the central area: “[La normativa internacional no es obligatorio su uso y está limitado a la gran minería, dejando de lado la mediana y pequeña minería que son los depósitos de relaves más abundantes en Chile. En cuanto a la ley de cierre, solo toma aspectos cuando termina la vida útil del depósito]” Resp#1 79 “International regulations are not mandatory, and they are limited to large-scale mining, leaving aside medium and small-scale mining, which are the most abundant [sources of] tailings deposits in Chile. Regarding the closure Law, it only takes aspects once the lifecycle of the deposit finishes” Resp#1 “[Las exigencias para el cierre son altas, se cubre la estabilidad física y química, y coberturas de material para prevenir arrastre eólico, entre otros. En ese sentido actualmente la ley es exigente, sin embargo, no es una ley retroactiva, y no aplica a relaves en abandono.”] Resp#2 “The demands for closure are high, they include physical and chemical stability, and [also] covering of material so it is not dispersed by air, among others. In this sense, the law is currently demanding, however, it is not a retroactive law, and it does not apply to abandoned tailings.” Resp#2 “[El diseño de relaves en Chile hasta ahora ha sido exitoso. La ley de cierres sube el estandar en ese sentido. Sin embargo, la incerteza que implica el cambio climatico podrían llegar a afectar los relaves de la zona central. Los relaves del norte tiene menores dificultades.]” Resp#3 “[The] Tailings design in Chile has been successful so far. The closure Law raises the standard in that regard. However, the uncertainty implied by climate change could affect tailings in the central zone. The northern tailings have fewer difficulties.” Resp#3 The social impacts were also not considered to be properly addressed. Some respondents saw evidence of this failure in the many communities that today live in the proximity or even on top of tailings, along with administrative decisions that prioritize industry and politicians: 80 “[Porque vemos los impactos de los relaves activos, abandonados y cerrados en las comunidades, y nadie se hace cargo de ellos. Las comunidades tienen que soportar la contaminación, enfermedades y riesgos asociados con colapsos por lluvias o terremotos.]” Resp#4 “Because we see the impacts of active, abandoned and closed tailings on communities, and no one is responsible for them. Communities must endure pollution, diseases, and risks associated with collapse [of tailings] from rain or earthquakes.” Resp#4 “[Ni siquiera está del todo determinado el impacto sobre comunidades. Hay mucho desconocimiento de los riesgos en la población. En Atacama, hay gente que vive en los relaves.]” Resp#5 “The impact on communities is not even fully determined. There is much ignorance of the risks in the population. In Atacama, people are living on the tailings.” Resp#5 “[La ley actual castiga en impactos ambientales o sociales, u obliga a negociar con las comunidades. Además permite anular [Resoluciones de Calificación Ambiental] RCAs si la autoridad [municipal] lo considera conveniente. Se ha visto contra [mención de dos compañías].]” Resp#6 “The current law punishes via environmental or social impacts or obliges to negotiate with the communities. It also allows you to cancel RCAs [i.e., resolution of environmental qualification] if the [municipal] authority deems it convenient. It has been seen against [two companies are mentioned].” Resp#6 When we asked about compensatory practices to address environmental impacts, respondents indicated that promoting biodiversity, implementing environmental education, and promoting 81 carbon sequestration are not well addressed (Fig. 4.8). Some proffered that mitigation of atmospheric pollution, fair water management and the provision of clean public water supplies, are also not well-established compensatory practices. In addition, it was mentioned that support to local communities needs improvement, particularly in terms of supporting circular economy projects led by the community and effectively communicating local environmental impacts. Figure 4.8. In your opinion, what compensatory practices to address environmental impacts are not well established in the management of tailing facilities? Note: the choices provided were “Promoting carbon sequestration”, “Promoting biodiversity conservation”, and “Implementing environmental education”. Multiple responses were allowed. The International Labour Organization Convention, also known as ILO-convention 169, aims to protect the right of Indigenous and Tribal Peoples to be consulted before the exploration of potential sites for mining. Respondents from NGOs were the most critical about the extent of enforcement and, in this respect, differed significantly from respondents in Academia (p<0.02) and Industry (p<0.01) (Fig. 4.9). Respondents from Academia and Industry also tended to report that the percentage of ILO enforcement was low, and although there was more agreement 82 amongst academics, their median responses were similar (around 25%) and not significantly different from each other or from the remaining groups (around 50%). Figure 4.9. To what extent do you think the International Labour Organization-convention 169 is enforced? Note: A sliding scale from 0%-100% was provided. The boxplot displays the first quartile, median and third quartile (box lines), and minimum and maximum values. The Chilean mine royalty, implemented and amended by Laws 20.026 (2005) and 20.848 (2015), is a tribute applied to the utility margin of a mine. In most cases, this royalty amounts to 4.5% to 9% of the operating profit. For example, Escondida, Collahuasi, Pelambres and Los Bronces pay a royalty of about 5–6%. However, if the production of a mine exceeds 50,000 tonnes, the royalty can be as high as 14%. When we asked the respondents their opinion on what percentage of the royalty should be diverted to fund ecological restoration, those affiliated with NGOs and Industry did not favour allocating more than 25% for this purpose, whereas those in Academia, Government and Other did (Fig. 4.10). 83 Figure 4.10. What percentage of the Chilean mine royalty do you think should be diverted to fund ecological restoration? Note: a sliding scale from 0%-100% was provided. The boxplot displays the first quartile, median and third quartile (box lines), and minimum and maximum values. Some of the respondents mentioned that restoration efforts should be considered operational costs, so the royalty could be used to improve the quality of life of the communities where such mines are placed. Some of the responses were: “[Creo que cada empresa debiera tener incluído este gasto en sus inversiones y ser parte de lo que se les exige para poder abrir una faena.]” Resp#7 “I believe that each company should include this expense in its investments and be part of what is required of them in order to open a mine.” Resp#7 “[Royalty debería aplicarse en su mayor porcentaje en las necesidades de la region. La restauración debe ser parte de la empresa y no por parte del estado]” Resp#8 “Royalty should be applied in its greatest percentage to the region's needs. Restoration must be part of the company and not by the state” Resp#8 84 “[Debiera exigirse ese gasto a las empresas no al país. El royalty debe invertirse en I[Investigación]+D[Desarrollo].]” Resp#9 “This expense should be demanded of companies, not the state. The royalty must be invested in R&D [i.e., Research and Development"]” Resp#9 4.5. Discussion 4.5.1. Demographic composition Our survey solicited stakeholder opinions from academia, industry, environmental NGOs, and government officials, about the inclusion of phytoremediation as an additional stabilization alternative for the management of Chile’s tailings. Although these respondents are well qualified to comment on the technical aspect of the management of tailings, they are not representative of the general population, particularly the local communities that are most directly affected by tailing deposits. Most of the respondents live and work in the Metropolitan Region (57%), are professionals between 35 and 44 years old (46 %) and 45-54 years old (24 %) with industrial or academic affiliations, and are predominantly male (70 % overall, 79% in Industry and 62% in Academia). Furthermore, none identified as Indigenous, even though slightly more than 12% of the Chilean population is Indigenous according to the last census (Instituto Nacional de Estadística, 2017). Around 83% of the respondents identified as Latina/o (46 %) or Mestiza/o (37 %). Caution, however, must be taken when interpreting our survey data because concepts of indigeneity have been affected by a history of cultural erasure and/or racial combination (Silva et al., 2022). In Chile, around 4% of the population has not attended school, and only 35% have completed a college degree or obtained a professional education (census). An even smaller proportion, just 85 1.5%, have higher degrees. On a salary basis (i.e., without considering governmental aid), four of every ten people in Chile live below the poverty line (Durán and Kremerman, 2021). Among respondents, however, about 70% were graduates and mainly worked in academia, while 30% held professional/superior education and worked for industry. Unsurprisingly, forestry (11%) and mining (39%) were highly represented industrial sectors among respondents. Currently, about 3.6% of the population of Chile (i.e., around 319,000 people) and 3.2% (i.e., 285,000 people) are working in fields related to professional, scientific and technical activities or mining, respectively (Observatorio Laboral Nacional, 2022). Despite a clear affiliation of many of the respondents to the mining industry, the great majority are of the opinion that Chile is not doing a good job when it comes to the management of its tailings (Fig. 4.1). Their knowledge of the large volume of abandoned tailings could be behind this evaluation. Most of the respondents, regardless of sector, consider the current legal framework to be insufficient to address either environmental (Fig. 4.7a) or social (Fig. 4.7b) impacts. However, relative to respondents from Academia and Industry, those from NGOs attached a significantly lower score to their perceived extent of enforcement of ILO-convention 169 (Fig. 4.9). A similar pattern was found when respondents were asked about what percentage of the royalty should be directed to restoration efforts, but in this case the NGO group differed significantly only from Academia (Fig. 4.10). These differences could be related to NGOs generally working with local communities impacted by the mining industry (e.g., ONG Fima, Oceana, Greenpeace), while academics are perhaps more likely to work with industry. 86 4.5.2. New tailings When it comes to the approval of new tailing facilities, the proximity to local communities, whose residents often work in less qualified jobs for the mine and would be the most affected by its installations, was highly ranked (97% of respondents assigned it a priority level of 1-3, out of 6). Even though the impacts of the mine will primarily affect local communities, the fate of mining projects tends to be decided in regions geographically distant from those projects, lacking what Riofrancos (2017) defines as democratic justice and what the IPCC (2022) indicates as a principle of Climate (in)Justice. Furthermore, the cost of living, demographic growth, family, health, and Indigenous issues have been indicated to be negatively affected by mining, with education as the only socially favourable aspect perceived by local communities close to mines (Viveros, 2016). Our respondents indicated that site hydrology is another critical consideration when approving more tailings (78% gave it a 1-3 priority level). In this regard, Chile has been known for rather extreme circumstances when it comes to water regulations (Campero et al., 2021), from public regulation of water rights before 1981, passage of the Water Code in 1981 that privatized water rights (in perpetuity), to the recently enacted Water Code (Biblioteca del Congreso Nacional, 2022) that grants access to clean water as a human right and is managed by the state. Seismicity was also given high priority in approving more tailings, with 63% ranking it 1-3 out of 6. Chile is very prone to earthquakes, as recently evidenced by events in 2010 (Maule, central Chile, Mw 8.8), 2014 (Iquique, northern Chile, Mw 8.2), 2015 (Illapel, north-central Chile, Mw 8.3), and 2016 (Chiloe, south-central Chile, Mw 7.6) (Ruiz and Madariaga, 2018). Indeed, one of the primary causes for the failure or collapse of Chilean tailing dams is earthquakes (Ghorbani 87 and Kuan, 2017), and Chile is predicted to have at least two very large tsunamigenic events per century (Ruiz and Madariaga, 2018). 4.5.3. Barriers General discontent among respondents with the state of tailings management suggests that there could be opportunities to implement alternative strategies, such as phytoremediation, to improve current practices. Respondents were open to phytoremediation as an additional stabilization method, but cited lack of funds as the main reason precluding implementation (Figure 4.4). Chile accounts for 28% of global Cu production (International Trade Administration, 2022) and mining contributes 11% to the Chilean gross domestic product (GDP). Exports from the mining sector totalled US$15,378 million in 2022 (Banco Central de Chile, 2022). Given that phytoremediation is generally regarded to be quite cost-effective (Witters et al., 2012) – to the point of off-setting project costs in less than 7 years (Xiaoming et al., 2016) – the direct expense of phytoremediation to industry should be easily affordable. Another way to fund phytoremediation (especially of abandoned tailings) could be via the royalty. However, few respondents thought that this tax should be used to fund ecological restoration, and most favoured allocating no more than 25% to such purposes (Fig. 4.10). The main reasoning provided was that the royalty should be used to improve the quality of life of the communities and not be diverted to restoration efforts, because polluting companies should be accountable for their impacts; this, however, is only possible if clean-up costs are expended or collected before companies dissolve. History has shown that prevention, mitigation, and timely compensation for damage by extractive industries is cheaper than waiting to ameliorate impacts after companies have declared 88 themselves insolvent. Even though Law 20.551 establishes monetary guarantees by mining companies to the state to ensure funding for proposed closure plans, considering future insolvency of companies, ultimately proper management will depend on the capacity of SERNAGEOMIN. Restoration can be a costly item for the state. For example, in 2020 Canada spent about $1.56 billion cleaning Alberta's un-reclaimed oil and gas wells (Boychuk et al., 2021). Although this is a substantial sum, it represents just a fraction of the estimated total cost of reclamation of $55 billion for Alberta’s 300,000 unreclaimed oil and gas wells. Boychuk et al. (2021) suggested the establishment of a non-profit “Restoration Trust” to safeguard against failed companies, along with an industry-wide tax to cover clean-up costs not captured by other means. A similar paradigm change within the Chilean mining industry could translate into several full-time jobs for local communities, bringing job security to those zones impacted by mining, effectively stabilizing tailings, adding innovation to the sector, and ultimately contributing to Chile's GDP. Lack of scientific support was the second most important barrier to phytoremediation selected by the respondents (Fig. 4.4). Some indicated insufficient information about suitable plants for phytoremediation or cited a lack of demonstrably successful phytoremediation projects. Although there are many studies investigating the capacity of plants to accumulate or stabilize metals (Ginocchio et al., 2017; Lam et al., 2018; León-Lobos et al., 2011, Ortiz-Calderón et al., 2008; Santibáñez et al., 2008), large numbers of species remain understudied. Reports on operational phytoremediation projects, successful or not, are indeed rare. One positive example, outside Chile, involved the use of a hybrid poplar clone to degrade trichloroethylene in groundwater in Maryland, USA (CLU, 1998). We are unaware of similar success stories in Chile or anywhere else in South America, though they may exist. Scientific support that enhances 89 innovation in the Chilean mining sector needs to be expanded and better communicated to the public. The majority of respondents indicated that biodiversity on site and proximity to water bodies are additional barriers to phytoremediation. Water is in short supply in much of Chile, and the most significant environmental consequences of mining have been linked to contamination and overuse of water resources (Ghorbani and Kuan, 2017). Lack of nurseries to supply plants is another formidable barrier to using phytoremediation. Nursery establishment could be an opportunity for local communities bringing job security while helping to conserve local biodiversity, particularly in the Central-Northern part of Chile which is a biodiversity “hot spot”; in fact, half of the 4,000 vascular plants found there are endemic (CEPF, 2022). Respondents indicated that phytoremediation should be implemented at the closure (83%), post-closure (76%) and operation (57%) stages (Fig. 4.6a), unanimously favouring the use of native species, including endemics (Fig. 6b). Thirty percent also allowed for the use of exotic plants. The advantage of using native flora is related to the conservation of endemic species highly adapted to local abiotic and biotic site conditions, with potentially lower water requirements, maintenance costs, and emission of allergenic pollen (Arcos-LeBert et al., 2021; Fiedler et al., 2008; Muñoz-Sáez et al., 2021). In contrast, exotic species may become invasive and are not as likely to provide suitable habitat for local fauna (Souza-Alonso et al., 2017). Irrespective of the lack of industrial models, respondents generally agreed that in addition to effective containment/removal of heavy metals, successful phytoremediation entails the establishment of a permanent green cover, restoration of the water cycle, and the return of native plants and animals (Fig. 4.5). In addition to re-establishing a resilient and sustainable ecosystem, respondents mentioned that successful projects should promote active and meaningful participation of local 90 communities in tailings management, and company compliance with environmental regulations and standards. 4.5.4. Social context Mining companies are required to have an approved Environmental Impact Assessment in order to open new projects. However, concerns have been raised about deficiencies in assessments with regard to public participation, mitigation and compensation plans, as well as water-energy management (Campero et al., 2021). Phytoremediation could help articulate some of the preferred compensatory practices yet to be addressed (Fig. 4.8), such as promoting biodiversity (74%), implementing environmental education (70%), and promoting carbon sequestration (63%). Additionally, this approach could promote human health and well-being. Even though more research is needed, exposure to nature, such as in “forest bathing”, has mental health benefits and has been linked to improved quality of life (Hansen et al., 2017; Oh et al., 2017). Access to greater green areas, and less air pollution, can reduce the occurrence of attention-deficit/hyperactivity disorder (ADHD) in children and youth by 62% (Yuchi et al., 2022). The most recent report of the Plenary of the Intergovernmental Science – Policy Platform on Biodiversity and Ecosystem Services, expands on the value of nature to incorporate goals of justice and sustainability (IPBES, 2022). If the phtytoremediation of a site is paired with ‘commercial attractiveness’, such as through timber, bioenergy, biofortification, and/or biochar production (Conesa et al., 2011), or the production of a ‘crop’of a certain metal produced by a high-biomass hyperaccumulator (Brooks et al., 1998), the succesful implementation could be more easily achieved. In conclusion, we found a cross-sector consensus on the need to do better when it comes to tailings management. Currently, the damage caused by mining is not fully reversible and, with 91 increased activity following on the nascent energy transition, more innovation is required. Though more research is desirable, professionals clearly consider phytoremediation to be a feasible supplementary approach to tackle this situation. Phytoremediation with native plants may be particularly useful for restoration of abandoned sites, some of which are many decades old, and projects that meaningfully involve local communities are more likely to lead to success. 92 Chapter 5: General discussion The conversation around the economic, social and environmental fate of countries has ultimately been a debate on power distribution, with the majority of countries having a preference for neoclassical economics (Hickel et al., 2022). Even though there is consensus around the need to prevent global temperatures from rising more than 2°C above pre-industrial levels and reducing carbon emissions to achieve carbon neutrality around 2050 (United Nations, 2022), countries are delaying the profound transformations needed if such targets are to be reached. Major economic, environmental and climate forums have agreed that one of the critical transformations needed in this scenario is fossil fuels divestment, towards alternative energy sources. This energy transition, needed worldwide, will be accompanied by a greater reliance on electricity and the necessary raw materials to support electrification (e.g., metals such as Cu). Countries such as Chile, which exports most of its Cu and alone accounts for 28% of global production (International Trade Administration, 2022), are disproportionately affected by the waste produced by the industry. An average of 537 million tonnes of tailings are deposited in Chile every year, and at least 915 million tonnes are projected for 2026 (FCH, 2023). Most of the 750 tailings across the landscape today are mainly concentrated in the country's north, from Antofagasta to the Metropolitan Region (SERNAGEOMIN, 2022). As of 2020, 112 tailings were active, 173 abandoned and 467 inactive, while five were under construction. However, the volume of tailings cannot be accounted for by a mere tally of sites, as these installations vary in size (many are massive) and other risk factors. Four municipalities (i.e., the smallest administrative unit) hold more than half of the total tonnage of tailings present in Chile (Antofagasta, Calama, Alhué, and Pica). The reported quantity of abandoned tailings (over 11 billion tonnes), mainly concentrated in 93 Antofagasta, the Metropolitan region and Coquimbo, is a call to action to Chile’s environmental and social future. Companies have also tried to solve this problem, like in the case of Carmen de Andacollo, when a company removed 257,000 tonnes of abandoned tailings where they had active facilities (Teck, 2013). More recently, the Ministry of Mining has tried to improve the monitoring and risk communications with local communities with the Global System of Tailings Monitoring, part of the National Plan of Tailing Facilities for Sustainable Mining (DDS, 2020; FCH, 2023), and the infamously titled program ‘Adopt a Tailing’ (i.e., companies that have active operations in the area will deal with an abandoned tailing in their proximity, by properly disposing them or by finding a profitable alternative - such as turning them into bricks (Romo-Álvarez, 2018)). As mentioned in Chapter 4, the maintenance of tailings in Chile has been conducted exclusively with chemical and physical stabilization methods (SERNAGEOMIN, 2022). However, one additional solution to tailings management is establishing metal-tolerant plants with the ability to sequester metals, to either reduce mobilization through the soil or allow the harvesting of aboveground tissues to remove metals from the site (Salt et al., 1995). Phytoremediation can not only help restore ecological functions, but may also contribute to local jobs and livelihoods, improvements in air quality, and a stronger sense of community attachment to the landscape. Tailings management still has a long way to go. Phytoremediation appears to be a promising technique in this quest, particularly for the large and growing volume of tailings in Chile. The country has a wide range of plants proven to be suitable for phytoremediation (Ginocchio and Baker, 2004; Ginocchio et al., 2017; Milla-Moreno et al., 2022), and many more that are yet to be assessed (Lam et al., 2018; Pizarro et al., 2016; Santibáñez et al., 2008). For this and other reasons, Chile has the potential to be a pioneering country in tailings restoration. Chile’s 94 experiences can inform the design and maintenance of new tailing facilities worldwide and, with proper funding for research and implementation (either from industry and/or government), it can become a leader for environmental and social restoration. This doctoral study was able to shed light on the application of phytoremediation as an additional stabilization technique for tailings in three ways. In Chapter 2, I was able to assess growth, survival and metal accumulation in locally available and native woody plants established on tailings, with or without additional treatments. This phytoremediation project was a result of a long dispute between a local community and the mine. There were several benefits to working on this site: the establishment was already in place and comprised several native woody tree species, the company accepted my research placement in their facilities and provided support (accommodation, transport, food, assistance, and stipend), the entire site was fenced and the plants were drip fertigated. Some plants appeared to be better equipped than others in dealing with the site conditions. Although all species showed signs of photoinhibition, algarrobo, romero and espino had the highest survival, and were the tallest and largest. Although quillay was not one of the most productive plants on the site, it was one of the greenest species on the site, and may do better if soil is provided. Algarrrobo and espino are N-fixers, which can account for their better growth in the field (i.e., differences in growth were not significant in the greenhouse, where resources were not limiting). Quillay accumulated similar amounts of Cu in roots as other species but its translocation factor was very low (0.09-0.20), maintaining very low foliar concentrations of Cu. Furthermore, quillay was resistant to hare damage. Even though quillay is an endemic species, it was very abundant in the surrounding area of the phytostabilization site. Usually, quillay grows up to 15 m height and 1 m in diameter, but specimens have been found with up to 30 m height and 1.5 m in diameter (de la Vega, 2020). Quillay has other benefits (e.g., 95 is a good source of nectar for honey) and has been used as a source of saponins in vaccines to fight shingles, malaria and more recently COVID-19 (Laing and Martell, 2021). For Chapter 3, I followed up on my field study in a controlled environment experiment. Two Chilean native hardwood species, quillay and espino, were grown under three increasing Cu levels for 6 months in a greenhouse setting. I measured growth, photosynthetic performance and elemental concentrations of leaves and roots to further evaluate their potential for phytoremediation. Both quillay and espino demonstrated high tolerance to the provision of excess Cu. Indeed, espino may require considerably more Cu than normal to support optimal growth, as also evidenced by its improved photosynthetic performance with increased Cu in the media. Excess Cu was mostly restricted to the roots of both species and showed some tendency to accumulate in tissue layers outside the periderm. Foliar Cu concentrations remained below toxicity levels in both species, in contrast to our previous study of older plants growing on tailings in the field (i.e., Chapter 2), where this threshold was exceeded in espino. These observations suggest that differences in age and/or other soil conditions (i.e., multi-metal stress) may affect experimental outcomes. This study allowed us to confirm the potential of quillay in phytostabilization projects and provided a pilot experience in the use of XRF imaging for the screening of plants with potential for phytoremediation. This technique allows a rapid and fine screening of elemental distributions in plants, that can help understand differences and similarities in the accumulation of heavy metals as well as the roles that other elements may play in metal tolerance (e.g., the apparent association of Ni with calcium oxalate crystals). I have created a Borealis Dataverse Repository where images related to this work can be downloaded for educational purposes with proper copyright use (doi: https://doi.org/10.5683/SP3/OQSF3X). 96 In Chapter 4, I found a cross-sector consensus on the need to do better when it comes to tailings management in Chile. Currently, the damage caused by mining is not reversible and, with increased activity following on the nascent energy transition, more innovation is required. Though more research is desirable, professionals clearly consider phytoremediation to be a feasible supplementary approach to tackle this situation. Phytoremediation with native plants may be particularly useful for restoration of abandoned sites, some of which are many decades old, and projects that meaningfully involve local communities are more likely to lead to success. Throughout this doctoral thesis, but particularly when analyzing the data from the survey, I realized that the challenge of tailing management in Chile should not be siloed within either industry, non-governmental organizations, government or academia. A problem with so many implications must include an interdisciplinary approach and science-based solutions that account for power imbalances and social and environmental disparities. 5.1. Limitations of the study This research, based in three components (e.g., fieldwork, greenhouse, social), cannot provide a comprehensive understanding of tailing management in Chile, but can provide insight into the state of the art of phytoremediation in Chile and flag research gaps. The first couple of years of my research were used to get funding and permission to travel to the phytostabilization site in Chile to evaluate a phytoremediation pilot at a Cu mine. The second Chapter was not only challenging because it was conducted on the other side of the world, but also because it was pricey and required a lot of negotiation with the mine (and help from advocates), to be allowed to not only access to the site, but to actually stay there and measure plants for several months. This was only possible after we established a confidentiality 97 agreement that has framed how much I could analyze and share from that experience (i.e., I cannot provide identifiable information of the site or names of staff). The design of the phytostabilization site was done without the inclusion of a control (i.e., a site with the same plants growing on natural soil). Moreover, the amendments were not extensively applied and, six years after application, appeared not to have strong effects on plant growth and survival. We only examined a few species because a limited number were planted in the first place. There is much more that could be done. For example, we didn’t assess whether the phytoremediation of the site was successful, if ecological functions were re-established, if it was cost effective, if there was metal movement in the trophic chain, if plant reproductive organs contained metals, etc. In Chapter 3 we reduced the sample size even more than the previous chapter due to technical considerations (time and capacity). At the greenhouse, the slow process of measurements with the LI-COR and fluorometer (i.e., leaves were dark-adapted for 20 min), reduced the number of plants I was able to sample. In espino, the touch-sensitive feathery leaves only covered about 22% of the frame, so we accounted for the difference using a correction factor (calculated with a leaf area meter and three measurements of an espino leaf on a 6 cm2 frame to match the LI-COR), which is not ideal. The XRF imaging was costly (time at Canadian Light Source, accommodation, transportation), limiting the number of samples we could analyze. In the last research chapter, and despite multiple reminders via emails and social media platforms, I could only get 43 completed surveys, mostly representing industry and academia. 98 5.2. Future research and recommendations In Chapter 2, espino showed some potential for Cu and Fe phytoextraction, while quillay appeared to be a promising candidate for phytostabilization of Cu and other metals. I recommend further evaluation of those species. It would be interesting to also analyze heavy metal concentrations in other tissues, such as bark, wood and reproductive organs. As revealed in Chapter 4, professional stakeholders want to see more demonstration of plants suitable for phytoremediation. Chapters 2 and 3 made some small headway in this regard, but a much larger selection of native plants should be studied. 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Environment International 161:107120. Zhan J, Wei S, Niu R (2012). Selection of Cd-excluder rice in Shenyang region of China for food safety. Advanced Materials Research 518:1033–1039. 129 Appendices Appendix A Figure A1 Map of the study design, comprising 30 ha containing three treatments: tailings only (T), tailings with planting spots amended with compost (T+C) and tailings with plants pre-inoculated with Rhizophagus irregularis (Błaszk., Wubet, Renker and Buscot) C. Walker and A. Schüßler, formerly Glomus intraradices, (T+M). The treatments were organized in three blocks, each containing six plots. Points indicate the location of plants used for heavy metal analyzes. 130 Table A1 Results (p values) of two-way ANOVAs on growth-related traits. Trait Species Treatment Species × Treatment CCI < 0.001 0.271 0.598 CR < 0.001 0.273 0.967 Hare < 0.001 0.042 0.339 Height < 0.001 0.342 0.104 Neck Diameter < 0.001 0.024 0.898 QY 0.093 0.816 0.033 Vigour < 0.001 0.022 0.282 N=10, SD: Standard deviation, CR: crown radii, CCI: Chlorophyll Content Index, QY: Quantum Yield, Hare: Damage by Hare 131 Table A2 Pearson’s product-moment correlation coefficients (r). Colors from light to dark indicate significance levels of 0.05, 0.01 and 0.001. Height Height All 1 Algarrobo 1 Espino 1 Huingan 1 Pimiento 1 Quillay 1 Romero 1 Talhuen 1 Height Crown radii Crown radii All All 0.668 1 Algarrobo Algarrobo -0.083 1 Espino Espino 0.267 1 Huingan Huingan -0.014 1 132 Pimiento Pimiento 0.196 1 Quillay Quillay -0.028 1 Romero Romero -0.029 1 Talhuen Talhuen -0.037 1 Height Crown radii ND ND All All All 0.684 0.455 1 Algarrobo Algarrobo Algarrobo 0.755 -0.083 1 Espino Espino Espino 0.766 0.435 1 Huingan Huingan Huingan 0.544 0.345 1 Pimiento Pimiento Pimiento 0.417 -0.309 1 Quillay Quillay Quillay 0.403 0.318 1 Romero Romero Romero 0.207 0.24 1 Talhuen Talhuen Talhuen 0.672 -0.126 1 Height Crown radii ND Vigour Vigour All All All All 133 0.756 0.426 0.543 1 Algarrobo Algarrobo Algarrobo Algarrobo 0.87 -0.072 0.633 1 Espino Espino Espino Espino 0.738 0.301 0.702 1 Huingan Huingan Huingan Huingan 0.605 0.084 0.219 1 Pimiento Pimiento Pimiento Pimiento 0.619 0.077 0.406 1 Quillay Quillay Quillay Quillay 0.711 0.039 0.421 1 Romero Romero Romero Romero 0.363 0.119 0.166 1 Talhuen Talhuen Talhuen Talhuen 0.741 0.205 0.512 1 Height Crown radii ND Vigour CCI CCI All All All All All -0.414 -0.335 -0.356 -0.217 1 Algarrobo Algarrobo Algarrobo Algarrobo Algarrobo 0.372 -0.136 0.271 0.234 1 Espino Espino Espino Espino Espino -0.206 -0.249 -0.183 -0.06 1 Huingan Huingan Huingan Huingan Huingan 0.105 -0.028 0.125 0.104 1 Pimiento Pimiento Pimiento Pimiento Pimiento -0.041 0.122 -0.015 -0.013 1 Quillay Quillay Quillay Quillay Quillay -0.212 -0.027 0.018 -0.227 1 134 Romero Romero Romero Romero Romero 0.404 -0.124 -0.287 0.408 1 Talhuen Talhuen Talhuen Talhuen Talhuen 0.122 0.528 -0.06 0.235 1 Height Crown radii ND Vigour CCI QY QY All All All All All All 0.09 0.254 0.057 0.089 -0.015 1 Algarrobo Algarrobo Algarrobo Algarrobo Algarrobo Algarrobo 0.324 0.309 0.265 0.296 -0.173 1 Espino Espino Espino Espino Espino Espino -0.191 0.099 -0.044 0.052 -0.006 1 Huingan Huingan Huingan Huingan Huingan Huingan -0.196 0.312 0.036 -0.002 -0.163 1 Pimiento Pimiento Pimiento Pimiento Pimiento Pimiento 0.028 0.28 -0.118 0.343 0.129 1 Quillay Quillay Quillay Quillay Quillay Quillay -0.322 -0.06 -0.367 -0.232 0.067 1 Romero Romero Romero Romero Romero Romero -0.352 0.191 -0.234 0.059 -0.101 1 Talhuen Talhuen Talhuen Talhuen Talhuen Talhuen -0.273 0.341 -0.217 -0.269 0.342 1 Height Crown radii ND Vigour CCI QY Hare All All All All All All All Hare 0.478 0.479 0.217 0.36 -0.248 0.049 1 Algarrobo Algarrobo Algarrobo Algarrobo Algarrobo Algarrobo Algarrobo 0.59 0.037 0.543 0.487 0.337 0.207 1 Espino Espino Espino Espino Espino Espino Espino 135 -0.445 -0.168 -0.226 -0.258 0.131 0.12 1 Huingan Huingan Huingan Huingan Huingan Huingan Huingan ➖ ➖ ➖ ➖ ➖ ➖ 1 Pimiento Pimiento Pimiento Pimiento Pimiento Pimiento Pimiento ➖ ➖ ➖ ➖ ➖ ➖ 1 Quillay Quillay Quillay Quillay Quillay Quillay Quillay ➖ ➖ ➖ ➖ ➖ ➖ 1 Romero Romero Romero Romero Romero Romero Romero ➖ ➖ ➖ ➖ ➖ ➖ 1 Talhuen Talhuen Talhuen Talhuen Talhuen Talhuen Talhuen 0.037 -0.238 0.205 0.185 -0.115 -0.325 1 ND: Neck diameter, CCI: Chlorophyll Content Index, QY: quantum yield. 136 Table A3 Results (p values) of two-way ANOVAs on heavy metal concentrations in leaves and roots, and translocation factors (Tf). Tissue Metal Species Treatment Species × Treatment Leaves Arsenic (As) 0.108 0.372 0.017 Cadmium (Cd) <0.001 0.464 0.578 Chromium (Cr) <0.001 0.056 0.62 Copper (Cu) <0.001 0.858 0.742 Iron (Fe) <0.001 0.317 0.819 Lead (Pb) 0.003 0.964 0.044 Manganese (Mn) 0.021 0.156 0.484 Molybdenum (Mo) <0.001 0.015 0.384 Nickel (Ni) 0.001 0.736 0.893 Zinc (Zn) <0.001 0.492 0.806 Roots Arsenic (As) <0.001 0.926 0.697 Cadmium (Cd) <0.001 0.608 0.821 Chromium (Cr) <0.001 0.064 0.137 Copper (Cu) 0.384 0.109 0.905 Iron (Fe) 0.005 0.061 0.046 Lead (Pb) 0.090 0.469 0.327 Manganese (Mn) 0.013 0.001 0.053 Molybdenum (Mo) <0.001 0.015 0.656 Nickel (Ni) 0.146 0.011 0.435 Zinc (Zn) 0.005 0.005 0.349 Tf Arsenic (As) 0.001 0.164 0.257 Cadmium (Cd) <0.001 0.943 0.746 Chromium (Cr) 0.002 0.112 0.243 Copper (Cu) <0.001 0.133 0.289 Iron (Fe) <0.001 0.003 0.013 137 Tissue Metal Species Treatment Species × Treatment Lead (Pb) 0.601 0.779 0.343 Manganese (Mn) 0.016 0.105 0.259 Molybdenum (Mo) <0.001 0.364 0.928 Nickel (Ni) 0.117 0.704 0.871 Zinc (Zn) <0.001 0.301 0.738 138 Table A4 Descriptive statistics for heavy metal and metalloid accumulation in leaves (mg kg-1; Mean, ± SD). Species Treatment As Cd Cr Cu Fe Pb Mn Mo Ni Zn Algarrobo T 0.29 ND 0.11 30.67 87.00 0.28 167.70 8.06 0.92 25.30 ± 0.2 ± 0.1 ± 6.7 ± 6.2 ± 0.4 ± 53.5 ± 3.6 ± 0.2 ± 12.0 T+C 0.11 ND 0.10 41.33 80.30 0.62 80.30 5.37 1.62 34.70 ± 0.1 ± 0.1 ± 10.7 ± 11.9 ± 0.1 ± 27.2 ± 2.4 ± 0.8 ± 6.7 T+M 0.30 0.01 0.10 32.67 100.30 0.62 107.70 4.95 0.93 32.70 ± 0.3 ± 0.0 ± 0.1 ± 13.0 ± 28.3 ± 0.3 ± 38.4 ± 3.2 ± 0.6 ± 14.4 Espino T ND ND 0.26 37.33 373.30 0.22 137.00 8.24 2.75 21.70 ± 0.0 ± 8.5 ± 8.1 ± 0.0 ± 43.0 ± 4.7 ± 1.0 ± 3.8 T+C 0.18 ND 0.26 50.00 390.30 0.34 144.00 9.21 2.17 28.30 ± 0.2 ± 0.0 ± 21.4 ± 80.1 ± 0.1 ± 55.6 ± 6.1 ± 0.7 ± 3.2 T+M 0.01 ND 0.25 44.67 351.70 0.32 100.70 6.58 2.09 26.30 ± 0.0 ± 0.1 ± 9.9 ± 42.5 ± 0.1 ± 27.3 ± 4.6 ± 1.3 ± 2.9 Quillay T 0.99 ND 0.01 7.67 68.70 0.18 51.30 5.04 0.71 14.00 ± 0.4 ± 0.0 ± 1.2 ± 35.5 ± 0.2 ± 16.9 ± 4.4 ± 0.3 ± 5.0 T+C 0.18 ND ND 5.67 34.30 0.09 67.30 3.07 0.96 12.00 ± 0.3 ± 3.8 ± 4.0 ± 0.1 ± 17.0 ± 0.8 ± 1.0 ± 2.6 T+M 0.11 ND 0.01 8.33 46.30 0.07 103.30 1.84 0.90 10.30 ± 0.1 ± 0.0 ± 6.1 ± 20.6 ± 0.1 ± 16.9 ± 0.1 ± 0.7 ± 0.6 Romero T 0.24 0.71 0.26 31.33 234.70 0.22 135.00 6.66 0.76 50.00 ± 0.2 ± 0.3 ± 0.1 ± 4.7 ± 34.7 ± 0.0 ± 9.9 ± 2.0 ± 0.3 ± 14.7 T+C 0.16 0.60 0.16 32.67 220.00 0.26 62.70 4.38 1.05 54.00 ± 0.2 ± 0.3 ± 0.0 ± 11.9 ± 60.8 ± 0.2 ± 5.1 ± 0.9 ± 0.4 ± 20.0 T+M 0.75 0.41 0.13 30.67 179.70 0.37 113.00 5.58 0.89 41.30 ± 0.2 ± 0.2 ± 0.0 ± 3.5 ± 34.2 ± 0.0 ± 62.9 ± 1.4 ± 0.2 ± 12.5 Talhuen T 0.24 ND 0.49 51.67 503.00 0.65 185.70 17.02 1.48 12.30 ± 0.4 ± 0.0 ± 31.0 ± 248.0 ± 0.4 ± 111.6 ± 2.7 ± 0.7 ± 2.5 T+C 0.58 ND 0.36 37.00 374.00 0.21 131.00 13.94 1.31 11.00 ± 0.8 ± 0.1 ± 7.9 ± 49.7 ± 0.1 ± 109.5 ± 4.0 ± 0.8 ± 1.0 139 Species Treatment As Cd Cr Cu Fe Pb Mn Mo Ni Zn T+M 0.37 ND 0.35 37.67 372.70 0.23 171.00 6.99 1.28 11.30 ± 0.3 ± 0.1 ± 14.1 ± 125.9 ± 0.2 ± 41.9 ± 3.0 ± 0.5 ± 2.5 N=3, T: Tailings only, T+C: Tailings with planting spots amended with Compost, T+M: Tailings with plants pre-inoculated with R. irregularis, SD: Standard deviation, ND: non detected, As: Arsenic, Cd: Cadmium, Cr: Chromium, Cu: Copper, Fe: Iron, Pb: Lead, Manganese (Mn), Mercury (Hg), Molybdenum (Mo), Nickel (Ni), Selenium (Se), Zinc (Zn) 140 Table A5 Descriptive statistics for heavy metal and metalloid accumulation in roots (mg kg-1; Mean, ± SD). Species Treatment As Cd Cr Cu Fe Pb Mn Mo Ni Zn Algarrobo T 0.03 ND 1.50 217.70 466.00 0.39 33.70 7.98 0.79 6.67 ± 0.03 ± 0.60 ± 81.03 ± 123.00 ± 0.11 ± 12.42 ± 0.95 ± 0.28 ± 2.08 T+C 0.17 ND 2.30 100.00 1524.00 1.06 51.70 3.23 1.78 8.67 ± 0.09 ± 0.83 ± 72.38 ± 589.00 ± 0.84 ± 9.50 ± 1.18 ± 0.38 ± 1.53 T+M 0.23 ND 1.02 248.30 1118.00 0.50 74.30 5.78 0.82 10.00 ± 0.21 ± 0.41 ± 319.43 ± 548.00 ± 0.41 ± 33.01 ± 3.51 ± 0.50 ± 1.00 Espino T 0.40 0.09 1.12 45.70 929.00 0.99 47.30 23.04 1.18 8.33 ± 0.38 ± 0.12 ± 0.63 ± 17.10 ± 600.00 ± 0.35 ± 1.15 ± 10.39 ± 0.64 ± 2.52 T+C 0.16 ND 1.11 43.00 548.00 0.48 33.30 11.99 1.48 15.33 ± 0.14 ± 0.52 ± 14.93 ± 193.00 ± 0.17 ± 15.37 ± 5.14 ± 0.60 ± 1.53 T+M 0.48 ND 1.05 53.00 729.00 0.63 39.30 16.23 0.83 11.33 ± 0.55 ± 0.50 ± 68.43 ± 183.00 ± 0.25 ± 14.01 ± 18.26 ± 0.41 ± 3.22 Quillay T 0.41 0.18 0.42 264.30 527.00 0.29 36.00 37.86 0.74 12.67 ± 0.19 ± 0.14 ± 0.17 ± 395.85 ± 165.00 ± 0.04 ± 8.19 ± 13.58 ± 0.19 ± 3.06 T+C 0.77 0.08 0.31 59.30 487.00 0.24 32.70 19.06 0.79 23.00 ± 0.61 ± 0.10 ± 0.18 ± 65.68 ± 144.00 ± 0.25 ± 19.14 ± 6.83 ± 0.38 ± 14.18 T+M 0.55 0.14 0.21 91.70 478.00 0.35 41.30 21.32 0.90 13.67 ± 0.32 ± 0.03 ± 0.07 ± 9.81 ± 211.00 ± 0.17 ± 19.30 ± 9.73 ± 0.27 ± 0.58 Romero T 0.22 0.58 0.69 223.00 496.00 0.65 40.30 7.30 0.71 12.33 ± 0.06 ± 0.45 ± 0.42 ± 152.45 ± 380.00 ± 0.46 ± 6.81 ± 2.41 ± 0.27 ± 1.53 T+C 0.39 0.38 0.89 40.00 838.00 0.92 29.00 4.14 0.99 13.00 ± 0.11 ± 0.02 ± 0.49 ± 17.78 ± 432.00 ± 0.43 ± 12.17 ± 1.94 ± 0.31 ± 4.58 T+M 0.44 0.43 1.19 117.00 1133.00 0.83 82.30 7.64 0.71 16.33 ± 0.30 ± 0.38 ± 0.14 ± 116.05 ± 140.00 ± 0.42 ± 21.55 ± 3.12 ± 0.29 ± 2.52 Talhuen T 1.26 0.10 0.58 167.00 270.00 0.29 24.30 29.74 0.83 6.00 ± 0.56 ± 0.08 ± 0.53 ± 139.52 ± 158.00 ± 0.19 ± 10.97 ± 7.96 ± 0.67 ± 1.73 T+C 1.09 0.25 1.22 76.30 620.00 0.79 26.00 24.67 1.06 14.67 ± 0.60 ± 0.02 ± 0.20 ± 70.94 ± 281.00 ± 0.58 ± 7.55 ± 4.72 ± 0.11 ± 1.53 T+M 0.83 0.11 0.48 46.30 507.00 0.64 38.70 21.74 0.62 9.00 ± 0.50 ± 0.06 ± 0.19 ± 25.97 ± 150.00 ± 0.36 ± 4.04 ± 4.20 ± 0.24 ± 4.58 N=3, T: Tailings only, T+C: Tailings with planting spots amended with Compost, T+M: Tailings with plants pre-inoculated with R. irregularis, SD: Standard deviation, ND: non detected, As: Arsenic, Cd: Cadmium, Cr: Chromium, Cu: Copper, Fe: Iron, Pb: Lead, Manganese (Mn), Mercury (Hg), Molybdenum (Mo), Nickel (Ni), Selenium (Se), Zinc (Zn) 141 Table A6 Descriptive statistics about the translocation factor (mg kg-1; Mean, ± SD). N=3, T: Tailings only, T+C: Tailings with planting spots amended with Compost, T+M: Tailings with plants pre-inoculated with R. irregularis, SD: Standard deviation, ND: non detected, As: Arsenic, Cd: Cadmium, Cr: Chromium, Cu: Copper, Fe: Iron, Pb: Lead, Manganese (Mn), Mercury (Hg), Molybdenum (Mo), Nickel (Ni), Selenium (Se), Zinc (Zn). Species Treatment As Cd Cr Cu Fe Pb Mn Mo Ni Zn Algarrobo T 10.556 ND 0.093 0.155 0.196 0.652 5.690 0.993 1.250 4.230 ± 3.91 ± 0.07 ± 0.07 ± 0.05 ± 0.78 ± 3.10 ± 0.38 ± 0.40 ± 2.44 T+C 1.350 ND 0.037 0.610 0.058 0.915 1.640 1.722 1.010 4.189 ± 1.88 ± 0.04 ± 0.50 ± 0.02 ± 0.74 ± 0.84 ± 0.64 ± 0.72 ± 1.61 T+M 7.872 0.733 0.096 0.315 0.097 7.020 1.580 1.386 1.230 3.200 ± 11.44 ± 1.10 ± 0.09 ± 0.22 ± 0.03 ± 10.82 ± 0.69 ± 1.44 ± 0.74 ± 1.09 Espino T 0.034 0.042 0.335 0.928 0.627 0.249 2.900 0.344 2.470 2.770 ± 0.06 ± 0.05 ± 0.27 ± 0.46 ± 0.55 ± 0.11 ± 0.90 ± 0.06 ± 0.46 ± 0.90 T+C 0.800 ND 0.273 1.141 0.748 0.814 5.110 0.726 1.830 1.859 ± 0.90 ± 0.14 ± 0.20 ± 0.16 ± 0.45 ± 3.45 ± 0.25 ± 1.40 ± 0.27 T+M 0.059 ND 0.277 2.451 0.495 0.594 2.640 0.571 2.590 2.408 ± 0.05 ± 0.14 ± 1.88 ± 0.09 ± 0.37 ± 0.59 ± 0.35 ± 0.95 ± 0.48 Quillay T 2.477 0.009 0.025 0.183 0.133 0.584 1.480 0.144 1.060 1.119 ± 0.38 ± 0.01 ± 0.04 ± 0.18 ± 0.06 ± 0.58 ± 0.54 ± 0.12 ± 0.73 ± 0.40 T+C 0.418 0.042 0.005 0.197 0.076 6.382 2.450 0.182 1.820 0.649 ± 0.67 ± 0.05 ± 0.00 ± 0.18 ± 0.03 ± 10.93 ± 1.25 ± 0.09 ± 2.40 ± 0.32 T+M 0.220 ND 0.027 0.095 0.098 0.199 2.740 0.102 1.090 0.758 ± 0.21 ± 0.04 ± 0.07 ± 0.03 ± 0.28 ± 0.8 ± 0.06 ± 0.85 ± 0.08 Romero T 0.966 1.888 0.416 0.257 0.747 0.416 3.380 1.064 1.120 4.191 ± 0.85 ± 1.29 ± 0.26 ± 0.26 ± 0.58 ± 0.18 ± 0.30 ± 0.69 ± 0.53 ± 1.66 T+C 0.343 1.574 0.231 0.848 0.336 0.313 2.430 1.137 1.240 5.047 ± 0.39 ± 0.72 ± 0.18 ± 0.13 ± 0.22 ± 0.18 ± 0.96 ± 0.27 ± 0.87 ± 3.97 T+M 2.837 1.276 0.108 0.534 0.161 0.540 1.370 0.884 1.380 2.610 ± 2.46 ± 0.78 ± 0.03 ± 0.50 ± 0.04 ± 0.33 ± 0.56 ± 0.57 ± 0.60 ± 0.96 Talhuen T 0.187 0.016 1.765 0.602 2.218 2.352 9.060 0.587 2.240 2.298 ± 0.32 ± 0.01 ± 1.75 ± 0.61 ± 1.10 ± 0.64 ± 7.38 ± 0.08 ± 1.36 ± 1.27 T+C 0.222 ND 0.312 0.790 0.695 0.408 4.660 0.569 1.200 0.757 ± 0.38 ± 0.13 ± 0.50 ± 0.31 ± 0.29 ± 2.65 ± 0.16 ± 0.59 ± 0.12 T+M 0.838 0.012 0.750 0.941 0.739 0.280 4.420 0.317 2.420 1.392 ± 1.09 ± 0.01 ± 0.09 ± 0.36 ± 0.12 ± 0.25 ± 1.00 ± 0.09 ± 1.34 ± 0.40 142 Appendix B Figure B1 Reference pictures of sections used in XRF maps. Left picture shows an espino branch highlighting a leaflet. On the upper right side, the selection of a quillay leaf is shown (including the rachis). Below the quillay leaf, a typical selection of the roots mapped are shown. Table B1 Composition of unamended fertigation solution Ingredient Concentration (g per 100 L) Ca(NO3)2 + NH4OH 225 CaCl2 171 FeDTPA 3.8% Liquid 15 MgSO4 225 KH2PO4 57 KNO3 102 CuSO4 0.105 Mo 0.045 Na2B8O13·4H2O 0.9 MnSO4 0.9 ZnSO4 0.45 143 Table B2.1 Descriptive statistic for soil mix characteristics from pots containing espino or quillay in control, 50 and 100 µM Cu treatments. Species Espino Treatment Control 50 µM Cu 100 µM Cu Ca (%) Mean (SD) 7.68 (3.23) 6.86 (2.17) 6.72 (1.65) Median [Min, Max] 6.60 [5.10, 13.0] 5.80 [4.80, 10.0] 6.50 [4.50, 8.90] Cu (mg/kg) Mean (SD) 9.98 (1.60) 108 (21.2) 226 (50.3) Median [Min, Max] 9.70 [7.80, 12.0] 120 [83.0, 130] 230 [150, 280] Fe (mg/kg) Mean (SD) 3680 (286) 3800 (604) 3760 (279) Median [Min, Max] 3700 [3300, 4000] 3700 [3200, 4800] 3700 [3500, 4100] K (%) Mean (SD) 1.10 (0.143) 1.36 (0.182) 1.25 (0.298) Median [Min, Max] 1.20 [0.900, 1.20] 1.40 [1.10, 1.60] 1.20 [0.870, 1.60] Mn (mg/kg) Mean (SD) 129 (22.7) 109 (8.17) 120 (7.07) Median [Min, Max] 130 [96.0, 150] 110 [97.0, 120] 120 [110, 130] Mo (mg/kg) Mean (SD) 6.26 (1.09) 5.08 (0.798) 6.06 (0.961) Median [Min, Max] 6.00 [5.20, 7.40] 5.00 [4.20, 6.30] 5.70 [4.90, 7.40] OM (LOI; %) Mean (SD) 49.4 (1.95) 49.4 (3.21) 48.6 (5.68) Median [Min, Max] 50.0 [47.0, 52.0] 49.0 [45.0, 54.0] 51.0 [42.0, 54.0] pH (1:1, soil:water) Mean (SD) 6.00 (0.0853) 6.70 (0.104) 6.30 (0.129) Median [Min, Max] 6.02 [5.89, 6.09] 6.70 [6.55, 6.81] 6.30 [6.13, 6.44] Zn (mg/kg) Mean (SD) 27.6 (3.21) 26.6 (4.72) 25.0 (0.707) Median [Min, Max] 26.0 [24.0, 31.0] 27.0 [22.0, 34.0] 25.0 [24.0, 26.0] Citric Acid (mg/kg) Mean (SD) 60.4 (29.0) 57.4 (21.4) 55.8 (10.6) Median [Min, Max] 51.0 [34.0, 110] 49.0 [34.0, 86.0] 59.0 [43.0, 69.0] Oxalic Acid (mg/kg) Mean (SD) 170 (84.6) 338 (156) 237 (151) Median [Min, Max] 130 [120, 320] 400 [69.0, 440] 230 [73.0, 450] N=5, SD: Standard Deviation, Min: Minimum, Max: Maximum, Ca: calcium, Cu: copper, Fe: iron, K: potassium, Mn: manganese, Mo: molybdenum, OM: Organic Matter, Zn: zinc. 144 Table B2.2 Descriptive statistics for soil mix characteristics from pots containing espino or quillay in control, 50 and 100 µM Cu treatments. Species Quillay Treatment Control 50 µM Cu 100 µM Cu Ca (%) Mean (SD) 6.58 (1.98) 5.80 (0.748) 9.02 (1.88) Median [Min, Max] 6.20 [5.10, 10.0] 6.10 [4.90, 6.50] 8.30 [6.90, 11.0] Cu (mg/kg) Mean (SD) 13.6 (4.61) 200 (35.4) 268 (56.7) Median [Min, Max] 13.0 [8.80, 21.0] 200 [160, 240] 270 [210, 350] Fe (mg/kg) Mean (SD) 3040 (365) 2720 (130) 3400 (625) Median [Min, Max] 3200 [2400, 3300] 2700 [2600, 2900] 3100 [3000, 4500] K (%) Mean (SD) 1.32 (0.0447) 1.40 (0.0707) 1.24 (0.182) Median [Min, Max] 1.30 [1.30, 1.40] 1.40 [1.30, 1.50] 1.30 [1.00, 1.40] Mn (mg/kg) Mean (SD) 129 (28.2) 102 (7.82) 112 (9.10) Median [Min, Max] 120 [94.0, 170] 100 [94.0, 110] 110 [98.0, 120] Mo (mg/kg) Mean (SD) 8.30 (1.32) 6.68 (0.683) 6.70 (0.975) Median [Min, Max] 8.10 [6.30, 9.60] 6.80 [5.60, 7.30] 6.60 [5.40, 7.80] OM (LOI; %) Mean (SD) 48.4 (2.30) 50.0 (2.45) 51.0 (3.39) Median [Min, Max] 48.0 [46.0, 52.0] 50.0 [46.0, 52.0] 51.0 [47.0, 56.0] pH (1:1, soil:water) Mean (SD) 5.90 (0.113) 6.17 (0.250) 6.14 (0.236) Median [Min, Max] 5.89 [5.74, 6.02] 6.20 [5.91, 6.50] 6.16 [5.85, 6.48] Zn (mg/kg) Mean (SD) 44.6 (36.8) 24.6 (5.22) 25.2 (5.02) Median [Min, Max] 30.0 [22.0, 110] 23.0 [20.0, 33.0] 23.0 [20.0, 33.0] Citric Acid (mg/kg) Mean (SD) 28.0 (35.5) 45.6 (32.6) 41.2 (17.6) Median [Min, Max] 12.0 [5.00, 89.0] 60.0 [5.00, 79.0] 49.0 [15.0, 59.0] Oxalic Acid (mg/kg) Mean (SD) 764 (138) 674 (191) 568 (282) Median [Min, Max] 790 [580, 950] 750 [380, 840] 420 [330, 940] N=5, SD: Standard Deviation, Min: Minimum, Max: Maximum, Ca: calcium, Cu: copper, Fe: iron, K: potassium, Mn: manganese, Mo: molybdenum, OM: Organic Matter, Zn: zinc. 145 Table B2.3 Descriptive statistics for soil mix characteristics from pots containing espino or quillay in control, 50 and 100 µM Cu treatments. Species Overall Treatment Control 50 µM Cu 100 µM Cu Ca (%) Mean (SD) 7.13 (2.59) 6.33 (1.63) 7.87 (2.06) Median [Min, Max] 6.25 [5.10, 13.0] 5.95 [4.80, 10.0] 7.75 [4.50, 11.0] Cu (mg/kg) Mean (SD) 11.8 (3.76) 154 (55.6) 247 (55.2) Median [Min, Max] 11.0 [7.80, 21.0] 145 [83.0, 240] 245 [150, 350] Fe (mg/kg) Mean (SD) 3360 (458) 3260 (703) 3580 (494) Median [Min, Max] 3300 [2400, 4000] 3050 [2600, 4800] 3500 [3000, 4500] K (%) Mean (SD) 1.21 (0.154) 1.38 (0.132) 1.25 (0.233) Median [Min, Max] 1.25 [0.900, 1.40] 1.40 [1.10, 1.60] 1.25 [0.870, 1.60] Mn (mg/kg) Mean (SD) 129 (24.1) 106 (8.54) 116 (8.87) Median [Min, Max] 125 [94.0, 170] 110 [94.0, 120] 120 [98.0, 130] Mo (mg/kg) Mean (SD) 7.28 (1.57) 5.88 (1.10) 6.38 (0.973) Median [Min, Max] 7.40 [5.20, 9.60] 5.95 [4.20, 7.30] 6.40 [4.90, 7.80] OM (LOI; %) Mean (SD) 48.9 (2.08) 49.7 (2.71) 49.8 (4.59) Median [Min, Max] 48.5 [46.0, 52.0] 50.0 [45.0, 54.0] 51.0 [42.0, 56.0] pH (1:1, soil:water) Mean (SD) 5.95 (0.108) 6.43 (0.333) 6.22 (0.199) Median [Min, Max] 5.97 [5.74, 6.09] 6.53 [5.91, 6.81] 6.21 [5.85, 6.48] Zn (mg/kg) Mean (SD) 36.1 (26.2) 25.6 (4.81) 25.1 (3.38) Median [Min, Max] 28.5 [22.0, 110] 24.5 [20.0, 34.0] 25.0 [20.0, 33.0] Citric Acid (mg/kg) Mean (SD) 44.2 (35.0) 51.5 (26.7) 48.5 (15.7) Median [Min, Max] 42.5 [5.00, 110] 54.5 [5.00, 86.0] 50.0 [15.0, 69.0] Oxalic Acid (mg/kg) Mean (SD) 467 (331) 506 (242) 402 (276) Median [Min, Max] 450 [120, 950] 440 [69.0, 840] 340 [73.0, 940] N=5, SD: Standard Deviation, Min: Minimum, Max: Maximum, Ca: calcium, Cu: copper, Fe: iron, K: potassium, Mn: manganese, Mo: molybdenum, OM: Organic Matter, Zn: zinc. 146 Table B3 2-way ANOVA for soil mix of espino and quillay. Traits Species Treatment Species × Treatment Ca 0.951 0.272 0.132 Cu 0.002 < 0.001 0.033 Fe < 0.001 0.241 0.178 K 0.206 0.091 0.302 Mn 0.364 0.013 0.832 Mo < 0.001 0.014 0.290 OM 0.596 0.811 0.543 pH < 0.001 < 0.001 0.016 Zn 0.380 0.222 0.341 Citric Acid 0.050 0.820 0.635 Oxalic Acid < 0.001 0.433 0.189 N=5 147 Table B4 Descriptive statistics for CCI and growth of espino and quillay in control, 50 and 100 µM Cu treatments. Species Treatment CCI FW Shoot Length Root length Mean (SD) Median [Min, Max] Mean (SD) Median [Min, Max] Mean (SD) Median [Min, Max] Mean (SD) Median [Min, Max] Espino Control 1.88 (0.462) 1.80 [1.37, 2.80] 30.0 (17.9) 29.4 [11.6, 64.1] 60.5 (22.4) 57.2 [39.3, 90.6] 39.0 (8.57) 37.5 [25.3, 52.0] 50 µM Cu 2.69 (0.738) 2.50 [1.77, 3.77] 70.8 (9.45) 65.7 [60.7, 85.2] 107 (9.33) 107 [95.7, 120] 45.8 (18.7) 41.4 [21.5, 83.2] 100 µM Cu 3.40 (0.910) 3.73 [2.07, 4.67] 67.4 (31.9) 82.0 [10.4, 93.4] 113 (23.4) 118 [71.5, 136] 36.7 (3.35) 35.2 [33.4, 41.4] Quillay Control 31.2 (6.39) 28.2 [23.0, 38.8] 213 (49.1) 211 [157, 279] 45.0 (8.11) 43.7 [33.5, 55.8] 32.9 (5.51) 35.2 [25.5, 41.3] 50 µM Cu 20.4 (8.96) 17.4 [10.4, 31.5] 207 (39.1) 212 [144, 266] 70.6 (20.5) 65.1 [48.2, 102] 31.4 (8.46) 31.2 [16.3, 43.0] 100 µM Cu 22.5 (9.67) 20.1 [12.4, 37.5] 197 (68.9) 176 [105, 314] 58.4 (20.0) 57.2 [26.1, 91.5] 38.7 (9.48) 39.0 [23.5, 53.5] N=7, SD: Standard Deviation, Min: Minimum, Max: Maximum, FW: Fresh Weight 148 Table B5 2-way ANOVA for CCI and growth of espino and quillay at harvest. Traits Species Treatment Species × Treatment CCI < 0.001 0.091 0.030 Shoot length < 0.001 < 0.001 0.030 Root length 0.057 0.788 0.116 FW < 0.001 0.526 0.192 N=7 Table B6 1-way ANOVA for CCI and growth of espino and quillay at harvest. Traits Espino Quillay CCI < 0.001 0.080 Shoot length < 0.001 0.205 FW <0.009 0.586 N=7 149 Table B7 Descriptive statistics for gas exchange and chlorophyll fluorescence in espino and quillay in control, 50 and 100 µM Cu treatments. Species Treatment Amax (µmol CO2 m-2 s-1) gs (mol H2O m -2 s-1) WUEi (µmol CO2 mmol-1 H2O) QY Mean (SD) Median [Min, Max] Mean (SD) Median [Min, Max] Mean (SD) Median [Min, Max] Mean (SD) Median [Min, Max] Espino Control 9.67 (3.77) 8.91 [4.39, 13.8] 0.346 (0.190) 0.273 [0.138, 0.611] 41.7 (31.3) 24.3 [18.9, 92.5] 0.772 (0.0155) 0.770 [0.750, 0.793] 50 µM Cu 17.5 (2.50) 17.8 [14.1, 20.7] 0.333 (0.0209) 0.320 [0.317, 0.363] 52.3 (10.2) 48.5 [42.6, 67.3] 0.809 (0.0158) 0.810 [0.783, 0.823] 100 µM Cu 28.2 (7.22) 24.0 [22.1, 37.0] 0.463 (0.158) 0.410 [0.274, 0.637] 67.3 (11.6) 64.2 [58.3, 87.1] 0.804 (0.0104) 0.800 [0.793, 0.817] Quillay Control 4.86 (1.85) 4.39 [2.29, 6.90] 0.0748 (0.0407) 0.0620 [0.0260, 0.129] 72.9 (11.8) 68.3 [59.1, 86.5] 0.805 (0.0127) 0.810 [0.783, 0.813] 50 µM Cu 4.78 (1.36) 4.75 [3.02, 6.76] 0.0670 (0.0204) 0.0620 [0.0450, 0.0930] 76.5 (22.0) 71.2 [56.4, 114] 0.795 (0.0143) 0.800 [0.773, 0.810] 100 µM Cu 4.84 (1.80) 4.47 [2.99, 7.22] 0.0710 (0.0393) 0.0660 [0.0340, 0.137] 70.9 (17.2) 67.9 [53.2, 91.5] 0.795 (0.0105) 0.800 [0.780, 0.803] N=15, SD: Standard Deviation, Min: Minimum, Max: Maximum, Amax: maximum photosynthetic rate, gs: stomatal conductance, WUEi: intrinsic Water Use Efficiency, QY: Quantum Yield 150 Table B8 2-way ANOVAs for gas exchange variables and quantum yield. Traits Species Treatment Species × Treatment Amax < 0.001 < 0.001 < 0.001 gs < 0.001 0.318 0.326 WUEi 0.009 0.387 0.255 QY 0.510 0.076 < 0.001 N=15 Table B9 1-way ANOVAs for maximum photosynthetic rate (Amax) and quantum yield (QY). Traits Espino Quillay Amax < 0.001 0.997 QY <0.003 0.359 N=5 Table B10 Treatment effect 1-way ANOVAs for elemental and heavy metal analyses of quillay. Tissue Ca Cu Fe Mo N Na P Zn Leaves 0.197 0.007 0.237 0.23 0.558 0.261 0.779 0.202 Roots 0.003 <0.001 0.297 0.078 0.921 0.98 0.002 0.169 N=5 Table B11 t-tests for elemental and heavy metal analyses of quillay vs. espino in control, 50 and 100 µM Cu treatments. Tissue Treatment Cu Fe Mo N Na K Leaves 0 0.097 < 0.001 < 0.001 < 0.001 0.155 <0.004 50 0.429 < 0.001 0.011 < 0.001 0.010 < 0.001 100 0.168 < 0.001 0.496 < 0.001 0.005 < 0.001 Roots 0 0.546 < 0.001 < 0.001 0.010 0.015 0.501 50 0.016 < 0.001 < 0.001 <0.002 <0.002 0.914 100 0.002 < 0.001 < 0.001 0.006 0.007 0.008 N=5 for quillay; composite samples for espino. 151 Table B12 Bioconcentration, bioaccumulation and translocation factors of espino and quillay in control, 50 and 100 µM Cu treatments. Species Treatment Bcfsoil mix Bafsoil mix Bcfsolution Bafsolution Tf Espino 0 1.30 0.331 NR NR 0.254 50 0.860 0.080 29.3 2.74 0.094 100 0.752 0.058 26.8 2.05 0.076 Quillay 0 0.900 0.159 NR NR 0.177 50 0.336 0.051 21.1 3.18 0.150 100 0.360 0.035 15.2 1.48 0.097 NR: not relevant 152 Appendix C Survey C1. Survey provided to respondants (Spanish) Formulario de Consentimiento USO DE PLANTAS PARA LA RESTAURACIÓN ECOLÓGICA EN INSTALACIONES DE RELAVES EN CHILE: PROBABILIDAD DE SU USO POR PARTE DE TOMADORES DE DECISIONES BREB ID # H21-00650 Investigador Principal Dr. Robert Guy, Profesor del Departamento de Ciencias Forestales y de la Conservación, Facultad de Ciencias Forestales, Universidad de British Columbia. Teléfono: +1 604-822-6023. Correo electrónico: rob.guy@ubc.ca. Co-Investigadora Estefanía Milla-Moreno, Candidata a Doctorado del Departamento de Ciencias Forestales y de la Conservación, Facultad de Ciencias Forestales, Universidad de la Columbia Británica. Correo electrónico: emilla@alumni.ubc.ca. Objetivo Nuestro objetivo es recabar información para obtener una mejor comprensión sobre las barreras y la probabilidad del uso de plantas para la restauración ecológica de las instalaciones de relaves en Chile. Con ello, se podrá contar con mejores políticas públicas en torno a la gestión de los relaves. Procedimiento del estudio Se le invita a participar en un estudio de investigación a nivel nacional dirigido por Estefanía Milla-Moreno. Los resultados de esta investigación serán publicados como parte de la Tesis Doctoral de Estefanía Milla-Moreno y podrán ser publicados en revistas profesionales o presentados en conferencias científicas. En dichas presentaciones o publicaciones, se eliminarán los identificadores personales de los datos y se informará únicamente sobre la base de resultados agregados. Si es necesario, se seleccionarán cuidadosamente las citas anónimas para no violar la confidencialidad individual. Como participante, se le invitará a responder a un cuestionario en línea, que tardará aproximadamente 15 minutos en completarse. En la primera pregunta de la encuesta se le pedirá su consentimiento para analizar y difundir los resultados a partir de las respuestas agregadas. Puede retirarse en cualquier momento mientras responde a la encuesta, sin consecuencias. Las encuestas incompletas se descartarán de forma segura. No se registrará su identidad y, por tanto, se protegerá su anonimato. Eliminaremos la asociación de información personal de los participantes. Para garantizar la confidencialidad, le rogamos que no ponga su nombre en ninguna respuesta. Este cuestionario no está diseñado para poner a prueba sus conocimientos, sino para recoger su opinión. No hay respuestas correctas o incorrectas. Para garantizar la calidad de los resultados, le rogamos que responda a las preguntas de la forma más completa y honesta posible. Aunque le agradeceríamos que respondiera a todo el material con la mayor franqueza posible, no debe sentirse obligado a responder a ningún material que le resulte desagradable o incómodo. 153 En concreto, los objetivos de esta encuesta son: 1. Evaluar el conocimiento general de tomadores de decisiones potencialmente involucrados en la restauración ecológica de las instalaciones de relaves en Chile Analizar la probabilidad de que tomadores de decisiones utilicen la Fitorremediación para el cierre de minas. 3. Analizar la probabilidad de que tomadores de decisiones utilicen especies nativas al aplicar la Fitorremediación para el cierre de minas. 4. Identificar las barreras que enfrentan tomadores de decisiones en minería que los disuaden de utilizar la Fitorremediación. 5. Identificar las condiciones que limitan la aplicación de la Fitorremediación. Riesgos potenciales No hay riesgos conocidos asociados a este estudio, su anonimato estará garantizado. No habrá consecuencias adversas si decide no participar en esta encuesta. Posibles beneficios Este estudio le proporcionará la oportunidad de compartir sus historias y hacer que sus voces sean escuchadas, no sólo en los círculos académicos, sino también a nivel gubernamental y en la sociedad en general. La información recopilada puede mejorar las políticas actuales relativas a la gestión de relaves. Confidencialidad Los datos con los identificadores eliminados, serán accesibles para el Investigador Principal, Dr. Robert Guy, y la Co-Investigadora, Estefanía Milla-Moreno. El Investigador Principal será responsable de los datos. Los datos se conservarán durante 5 años después de la publicación de los resultados y se almacenarán en el ordenador de la oficina del Investigador Principal, Dr. Robert Guy, y en el ordenador personal de la Co-Investigadora, Estefanía Milla-Moreno. Los datos se destruirán posteriormente. Remuneración/Compensación Este estudio de investigación no contempla ninguna remuneración o compensación por su participación. Información de contacto sobre el estudio Si tiene alguna pregunta o desea más información con respecto a este estudio, puede ponerse en contacto con Estefanía Milla-Moreno en emilla@alumni.ubc.ca y con Dr. Robert Guy en rob.guy@ubc.ca. Si está interesado/a/e en recibir una copia de este estudio, titulado: Uso de plantas para la restauración ecológica en instalaciones de relaves en Chile: Probabilidad de su uso por parte de tomadores de decisiones, puede ponerse en contacto con Estefanía Milla-Moreno en emilla@alumni.ubc.ca. Inquietudes y quejas Si tiene alguna inquietud o queja sobre sus derechos como participante en la investigación y/o su experiencia durante su participación en este estudio, póngase en contacto con la Línea de Reclamaciones de Participantes en la Investigación de la Oficina de Ética de la Investigación de la UBC en el 604-822-8598 o, si es a larga distancia, envíe un correo electrónico a RSIL@ors.ubc.ca o llame gratis al 1-877-822-8598. De nuevo, gracias por su interés en participar en este estudio de investigación. Consentimiento Al seleccionar "Acepto participar" usted confirma que: 154 - Comprende lo que se requiere a partir de la lectura del Formulario de Consentimiento - Entiende que su participación es voluntaria y que es libre de retirarse en cualquier momento - Comprender las disposiciones de confidencialidad Opciones Acepto participar (si se selecciona se dirige a la encuesta) No acepto participar (si se selecciona se dirige a este mensaje: Le agradecemos su visita a esta encuesta y reconocemos que no participará en este estudio). PÁGINA 2 DE LA ENCUESTA SECCIÓN 1. En esta sección nos interesa conocer su opinión sobre la gestión de las instalaciones de relaves en Chile, particularmente por parte de la industria del cobre. ¿Está haciendo Chile un buen trabajo con la gestión de sus instalaciones de relaves? Sí No No sé ¿Qué importancia tienen los siguientes métodos de estabilización? Extremadamente Importante Muy importante Moderadamente importante Poco importante Nada importante No sé Estabilización química de los relaves (es decir, la prevención de la contaminación de los ecosistemas vecinos y/o de las comunidades locales a través del aire, o de las fuentes de agua) Estabilización física de los relaves (es decir, que los relaves estén debidamente contenidos por barreras físicas) La Fitorremediación (es decir, el uso de plantas y sus microbios asociados para estabilizar y, en última instancia, restaurar el terreno donde se encuentran los residuos). Priorice los aspectos clave que cree que deberían tenerse en cuenta a la hora de aprobar más relaves. [Ordene de mayor a menor importancia, arrastrando las opciones con su mouse. Deje espacio en blanco si no se le ocurre otro aspecto clave] 155 Cantidad total de relaves en la región Naturaleza sísmica en la ubicación de los relaves Proximidad a las comunidades locales Ciclo de vida de la mina Hidrología (ciclos y recursos hídricos) Geología (tipo de mineral) Otro Otro Otro ¿En qué medida está de acuerdo/desacuerdo con las siguientes afirmaciones? Las barreras para el uso de la Fitorremediación en Chile tienen que ver con Totalmente de acuerdo De acuerdo Ni de acuerdo ni en desacuerdo En desacuerdo Totalmente en desacuerdo No sé Proximidad a áreas protegidas Proximidad a cuerpos de agua Proximidad a zonas de sacrificio: definidas como áreas geográficas permanentemente deterioradas por daño ambiental y/o desinversión económica Proximidad a comunidades locales Masa total de relaves en la región Biodiversidad local Planes de respuesta a emergencias Falta de apoyo científico Falta de financiamiento Falta de viveros (para el suministro de plantas) Si gusta, describa otras barreras que inhiban el uso de Fitorremediación, de mayor a menor importancia Barrera 1 156 Barrera 2 Barrera 3 ENCUESTA PÁGINA 3 SECCIÓN 2. En esta sección nos interesa conocer su opinión sobre la Fitorremediación ¿En qué fase del ciclo de vida de la mina debería llevarse a cabo la Fitorremediación? [Elija todas las que correspondan] Planificación Diseño Construcción Operación Mantenimiento Cierre Post-cierre Ninguna de las anteriores La Fitorremediación debería incluir: [Elija todas las que correspondan] Especies exóticas: especies introducidas desde fuera del país Especies nativas: especies locales que se encuentran de forma natural en el país Especies endémicas: especies nativas cuya distribución está restringida a una zona concreta, a menudo pequeña ¿Cuándo se puede considerar que un proyecto de restauración con Fitorremediación ha sido exitoso? Totalmente de acuerdo De acuerdo Ni de acuerdo ni en desacuerdo En desacuerdo Totalmente en desacuerdo No sé Cuando se consigue revegetar permanentemente Cuando vuelven las plantas nativas Cuando vuelvan animales silvestres nativos Cuando se restablece el ciclo del agua Si gusta, describa otros factores, de mayor a menor importancia, que definirían una restauración con Fitorremediación exitosa 157 Factor 1 Factor 2 Factor 3 ENCUESTA PÁGINA 4 SECCIÓN 3. En esta sección nos interesa conocer su opinión sobre la gestión de relaves a nivel de políticas públicas. A nivel mundial, existen muchas normativas para el cierre de relaves. Aunque no es jurídicamente vinculante, la más actualizada y completa es la Norma Global de la Industria sobre Gestión de Relaves (2020), que establece el objetivo de "cero daños a las personas y al medio ambiente desde las primeras fases de concepción del proyecto". A nivel nacional, la normativa chilena se centra en la fase de cierre, e incluye las leyes 19.300 (1994), 20.551 (2012) y 20.819 (2015). La primera ordena la responsabilidad ambiental, en forma de prevención, mitigación y/o compensación de los impactos ambientales. La segunda regula el cierre de minas e instalaciones mineras y se centra en la evaluación de riesgos y los compromisos ambientales. Y la tercera modifica la Ley 20.551 en lo que respecta a los ciclos de vida de las minas y las garantías, especialmente, para la pequeña y mediana minería. ¿Considera que las regulaciones mencionadas en torno a los impactos ambientales son suficientes? Sí Sí, pero sólo las internacionales Sí, pero sólo las nacionales No No sé En un texto breve, ¿podría describir por qué? ¿Considera que la normativa mencionada sobre impactos sociales es suficiente? Sí Sí, pero sólo las internacionales Sí, pero sólo las nacionales No No sé En un texto breve, ¿podría describir por qué? 158 ¿Qué prácticas compensatorias en respuesta al impacto en el medio ambiente cree que no están bien establecidas en el manejo de relaves? [Marque todas las que correspondan] Promover el secuestro de carbono (por ejemplo, plantar y proteger suficientes árboles para secuestrar los gases de efecto invernadero producidos por el funcionamiento de la mina) Promover la conservación de la biodiversidad (por ejemplo, llevar la flora y la fauna nativas y endémicas a una zona bien establecida, bien diseñada y protegida dentro de la instalación) Implementar la educación ambiental (por ejemplo, en las escuelas primarias y secundarias de la zona que rodea a la mina) No sé Si gusta, describa de mayor a menor importancia, otras prácticas compensatorias que falten en la pregunta anterior Otra 1 Otra 2 Otra 3 El Convenio de la Organización Internacional del Trabajo, también conocido como Convenio 169 de la OIT, tiene como objetivo proteger el derecho de los pueblos indígenas y tribales a ser consultados antes de la exploración de posibles lugares para la minería. ¿En qué medida cree que se cumple la OIT en Chile? [Escala móvil] 0%-100% En un texto breve, describa sus razones para asignar esta puntuación El royalty chileno, implementado y modificado por las leyes 20.026 (2005) y 20.848 (2015), es un tributo que se aplica al margen de utilidades de una mina. En la mayoría de los casos, este royalty asciende a entre el 4,5% y el 9% del beneficio de explotación. Por ejemplo, Escondida, Collahuasi y Pelambres y Los Bronces, pagan un royalty de aproximadamente 5% y 6%. Sin embargo, si la producción de una mina supera las 50.000 toneladas, el royalty puede llegar al 14%. ¿Qué porcentaje del royalty cree que debería destinarse a financiar la restauración ecológica? [Escala móvil] 0% - 100% En un texto breve, describa sus razones para asignar esta puntuación 159 ENCUESTA PÁGINA 5 ¡Se está acercando al final de la encuesta! SECCIÓN 4. En esta sección nos interesa saber más sobre Usted. Recuerde que sus respuestas serán anónimas. ¿Cuál es su género? Femenino Masculino No binario Otro _______ Prefiero no contestar ¿Cuál es su edad? Menos de 24 años 25-34 35-44 45-54 55-64 65 años o más Prefiero no contestar ¿Cuál es el mayor grado o nivel de educación que ha recibido? Sin grado Educación Primaria (1° a 8° básico) Educación Secundaria/Media (1° a 4° medio) Educación Superior/Profesional (Centro de Formación Técnica, Institutos Profesionales, Universidades) Postgrado (Magíster, Doctorado) Otro ____ Cuál es la naturaleza de su experiencia según las áreas de investigación de la OCDE CIENCIAS NATURALES ----------------------------- Informática y Ciencias de la Información ----------------------------- Ciencias Físicas ----------------------------- Ciencias Químicas ----------------------------- Ciencias de la Tierra y del Medio Ambiente ----------------------------- Ciencias Biológicas ----------------------------- Otras Ciencias Naturales INGENIERÍA Y TECNOLOGÍA ----------------------------- Ingeniería Civil ----------------------------- Ingeniería Mecánica ----------------------------- Ingeniería Química ----------------------------- Ingeniería Ambiental ----------------------------- Biotecnología Industrial o Medioambiental ----------------------------- Otras Ingenierías y Tecnologías CIENCIAS AGRÍCOLAS ----------------------------- Agricultura ----------------------------- Ciencias Forestales ----------------------------- Pesquería ----------------------------- Ciencia Animal 160 ----------------------------- Otras Ciencias Agrícolas CIENCIAS SOCIALES ----------------------------- Psicología ----------------------------- Economía y Negocios ----------------------------- Sociología ----------------------------- Leyes ----------------------------- Ciencias Políticas ----------------------------- Geografía Social y Económica ----------------------------- Otras Ciencias Sociales HUMANIDADES ----------------------------- Historia ----------------------------- Lenguas y Literatura ----------------------------- Filosofía ----------------------------- Ética ----------------------------- Antropología ----------------------------- Otras Humanidades ¿Cuál de las siguientes categorías identifica mejor su afiliación principal? Gobierno Industria ONG Academia Ninguna Otra__________ ¿En qué región reside? Tarapacá (I) Antofagasta (II) Atacama (III) Coquimbo (IV) Valparaíso (V) Región Metropolitana (RM) O'Higgins (VI) Maule (VII) Araucanía (IX) 161 ¿Trabaja en la misma región en la que reside? Sí No Prefiero no contestar ¿Cuál es su situación laboral? [Marque todo lo que corresponda] Estudiante Trabajo a medio tiempo Trabajo a tiempo completo Independiente Jubilado/a/e Sin empleo Prefiero no contestar Otros _________ ¿En qué sector industrial trabaja? Minería Forestal Agricultura Turismo Otro_________ ¿Cómo se auto identifica Ud. respecto de las siguientes categorías? Indígena Mapuche Aymara Rapa Nui Atacameña 162 Quechua Colla Diaguita Kawashkar Yagán Chango Mestizo/a/e Negro/a/e Blanco/a/e Asiático/a/e Latino/a/e Otro___ Si ha seleccionado "Otro", describa a continuación ________________ ENCUESTA PÁGINA 6 Muchas gracias por su participación! Si tiene algún comentario adicional, escríbalo en el cuadro de texto de abajo. Si desea recibir una copia de los resultados de esta investigación, añada su correo electrónico a continuación: _______________________ 163 Table C2. Total counts of demographic characteristics and educational level in Academia, Government, Industry, NGO and Other. Academia Government Industry NGO Other M (N=8) F (N=4) M (N=2) F (N=1) M (N=15) F (N=4) M (N=2) F (N=2) M (N=4) F (N=1) Identity Latino/a 3 1 2 1 10 1 0 0 2 0 Mestizo/a 4 2 0 0 3 1 2 2 1 1 White 1 1 0 0 1 2 0 0 1 0 Other 0 0 0 0 1 0 0 0 0 0 Age 25-34 0 1 0 0 2 2 0 1 0 0 35-44 5 1 0 1 9 0 0 1 1 1 45-54 2 1 2 0 2 2 0 0 0 0 55-64 0 1 0 0 2 0 1 0 1 0 Under 24 1 0 0 0 0 0 0 0 0 0 65 or above 0 0 0 0 0 0 1 0 2 0 Degree Graduate 7 4 1 0 10 2 1 1 1 1 Superior/Professional education 1 0 1 1 5 2 1 1 3 0 Industry sector Forestry 2 1 0 0 0 0 1 0 0 0 Other 5 3 1 1 3 2 1 1 3 1 Mining 1 0 1 0 12 2 0 1 1 0 Region Antofagasta 0 0 0 0 2 1 0 0 0 0 Araucanía 1 0 0 0 0 0 0 0 1 0 Atacama 0 0 0 1 0 1 0 0 0 0 Biobío 0 0 0 0 0 0 0 0 0 1 Coquimbo 0 1 1 0 0 0 0 0 0 0 Libertador General Bernardo O’Higgins 0 1 0 0 1 0 0 0 0 0 Maule 1 0 0 0 0 0 0 0 0 0 Metropolitana de Santiago 6 1 1 0 10 2 1 2 2 0 Valparaíso 0 1 0 0 2 0 1 0 1 0 164 Table C3. Demographic characteristics based on Identity and Region. Latino/a Mestizo/a Other White Overall Male (N=17) Female (N=3) Male (N=10) Female (N=6) Male (N=1) Male (N=3) Female (N=3) Male (N=31) Female (N=12) Region Antofagasta 1.00 (5.9%) 0 (0%) 1.00 (10.0%) 1.00 (16.7%) 0 (0%) 0 (0%) 0 (0%) 2.00 (6.5%) 1.00 (8.3%) Araucanía 1.00 (5.9%) 0 (0%) 1.00 (10.0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2.00 (6.5%) 0 (0%) Atacama 0 (0%) 2.00 (66.7%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2.00 (16.7%) Biobío 0 (0%) 0 (0%) 0 (0%) 1.00 (16.7%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1.00 (8.3%) Coquimbo 1.00 (5.9%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1.00 (33.3%) 1.00 (3.2%) 1.00 (8.3%) LGBOH 0 (0%) 0 (0%) 0 (0%) 1.00 (16.7%) 0 (0%) 1.00 (33.3%) 0 (0%) 1.00 (3.2%) 1.00 (8.3%) Maule 0 (0%) 0 (0%) 1.00 (10.0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1.00 (3.2%) 0 (0%) MS 11.0 (64.7%) 0 (0%) 6.00 (60.0%) 3.00 (50.0%) 1.00 (100%) 2.00 (66.7%) 2.00 (66.7%) 20.0 (64.5%) 5.00 (41.7%) Valparaíso 3.00 (17.6%) 1.00 (33.3%) 1.00 (10.0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 4.00 (12.9%) 1.00 (8.3%) LGBOH: Libertador General Bernardo O’Higgins, MS: Metropolitana de Santiago.
Thesis/Dissertation
eng
Forestry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Guy, Robert D.
An assessment of native Chilean woody plants for phytoremediation of copper-contaminated sites
Text