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Mount Polley mine tailings : uptake and distribution of metals in lodgepole pine and bluebunch wheatgrass… Leung, Anthony 2018

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MOUNT POLLEY MINE TAILINGS: UPTAKE AND DISTRIBUTION OF METALS IN LODGEPOLE PINE AND BLUEBUNCH WHEATGRASS UNDER CONTROLLED ENVIRONMENTS by  Anthony Leung  B.Sc., The University of British Columbia, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Soil Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2018  © Anthony Leung, 2018 ii  Abstract On August 4th, 2014, a partial breach of the Mount Polley Mine tailings storage facility (TSF) released over 4.6 million m3 of supernatant water, and 12.8 million m3 of slurry tailings (solids and interstitial water) into Polley lake and downstream of Hazeltine Creek into Quesnel Lake. The spill led to an enrichment of copper and selenium into the affected ecosystem. Concentrations of copper and vanadium in sandy and silty tailings exceeded provincial soil quality standards for the protection of parkland use. A study of potential toxicity of deposited tailings to plants was conducted to determine if the tailings material would support growth of plants representative of the biogeoclimatic zone; more specifically, the growth and uptake of metals in root and shoot tissue of lodgepole pine (Pinus contorta), and bluebunch wheatgrass (Pseudoroegneria spicata). The greenhouse study identified physical growth-limiting factors of hexagonal closed packing and surface crusting in silty tailings. The Environment Canada (2007)-compliant 42-day growth chamber study found seedling emergence and survival not affected in plants growing in tailings or in copper-spiked greenhouse soils. Copper uptake was significant in roots and shoots of both species grown in tailings. Neither species survived in 115 mg/kg of copper-spiked sandy and silty substrates as a result of osmotic burning from the high salt index of copper sulfate. A separate 100-day growth chamber study found proportionally greater copper uptake in roots of lodgepole pine and in shoots of bluebunch wheatgrass compared to the 42-day study. Lodgepole pine and bluebunch wheatgrass each showed various qualitative signs of phytotoxicity. The presence of metals and their respective associations to one another in tailings may have profound implications on species-specific phytotoxicity otherwise not found in the studies conducted. Results of this study contributed to the shaping of future terrestrial restoration at Hazeltine Creek.  iii  Lay Summary Following the Mount Polley Mine tailings storage facility breach on August 4th, 2014, sandy and silty tailings covered over 100 hectares of the forest floor along Hazeltine Creek. These tailings contain concentrations of copper and vanadium exceeding Canadian soil quality standards for the protection of parkland use. The objective of this thesis was to assess tailings as a growth medium and determine if physical and chemical characteristics inhibited emergence, survival, growth and development of lodgepole pine and bluebunch wheatgrass. Physical growth-limiting factors were identified in a greenhouse study. An Environment Canada (2007)-compliant growth chamber study found significant reductions in root growth and elevated concentrations of copper in roots of both species compared to controls. These findings were mirrored in a separate 100-day growth chamber study. Results of this study contributed to shaping future terrestrial restoration at Hazeltine Creek.    iv   Preface Chapter 2 provides an overview of the sandy and silty tailings collected by Mount Polley Mine staff in late 2015.   Chapter 3’s greenhouse study was conducted at the UBC Horticulture Greenhouse with consultation from Melina Boron. Experimental setup and laboratory work was conducted at UBC H. R. Macmillan Building. I supervised Crystal Chan and Pariya Torkaman in the setup of all substrate treatments. I was responsible for experimental setup, monitoring, extraction and all laboratory work for metal analysis. Inductive coupled plasma optical emissions spectroscopy (ICP-OES) analysis was also completed by Maureen Soon at UBC’s Earth Sciences Building.  Chapter 4 was conducted with ongoing consultation from Kerrie Serben and Trish Miller of Golder Associates Ltd. to ensure the experimental design was Environment Canada (2007) compliant. The growth chamber experiment took place at the UBC Forest Sciences Building with laboratory work conducted at the UBC H. R. Macmillan Building. I was responsible for experimental setup, monitoring, plant extraction and laboratory work for metal analysis. Inductively coupled plasma optical emissions spectroscopy (ICP-OES) analysis was also completed by Maureen Soon at UBC’s Earth Sciences Building. Kerrie Serben assisted with the compilation of data tables. I collaborated with Kerrie Serben and Dr. Suzanne Simard to complete a Summary of Key Findings Report in January 2017 to Golder Associates Ltd. as part of their ecological risk assessment at Hazeltine Creek. This report can be found in Appendix B   v  Table of Contents  Abstract .......................................................................................................................................... ii Lay Summary ............................................................................................................................... iii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................... ix List of Photos ................................................................................................................................ xi Acknowledgements ..................................................................................................................... xii Dedication ................................................................................................................................... xiii Chapter 1: Introduction ................................................................................................................1 Chapter 2: General Methods ........................................................................................................9 Chapter 3: Greenhouse Study.....................................................................................................17 3.1 Greenhouse Study Methods .......................................................................................... 17 3.2 Results and Discussion for Greenhouse Study ............................................................. 23 3.2.1 Lodgepole Pine ......................................................................................................... 24 3.2.2 Bluebunch Wheatgrass.............................................................................................. 29 3.2.3 Wild Rose.................................................................................................................. 34 3.2.4 Summary ................................................................................................................... 34 Chapter 4: Environment Canada Compliant Growth Chamber Study .................................37 4.1 Growth Chamber Study Methods ................................................................................. 37 4.2 Results and Discussion for Growth Chamber study ..................................................... 51 4.2.1 Lodgepole Pine ......................................................................................................... 51 4.2.2 Bluebunch Wheatgrass.............................................................................................. 66 4.2.3 Wild Willows ............................................................................................................ 80 Chapter 5: 100 Day Growth Chamber Study............................................................................81 5.1 Methods for 100 Day Growth Chamber Experiment .................................................... 81 5.2 Lodgepole pine results and Discussion for 100 Day Growth Chamber Study ............. 81 5.3 Bluebunch Wheatgrass.................................................................................................. 88 vi  Chapter 6: Summary and Further Study ..................................................................................95 References ...................................................................................................................................104 Appendices ..................................................................................................................................111 Appendix A Sandy and Silty Tailings: Certificate of Analysis .............................................. 111 Appendix B Summary of Key Findings Report Prepared for Golder Associates Ltd. ........... 117 Appendix C Chemical Analysis of Greenhouse Soils and Fertilizer ...................................... 152 C.1 Greenhouse Potting Soil Chemical Analysis: February 18, 2014........................... 152 C.2 Greenhouse Fertilizer Water Chemical Analysis February 26, 2014 ..................... 153 Appendix D Hexagonal Closed Packing/Squared Closed Packing ........................................ 154 Appendix E Statistical Analysis: Kruskal Wallis Test and Summary of p values .................. 155 E.1 Lodgepole Pine p values from 42-Day Growth Chamber Study ............................ 155 E.2 Bluebunch Wheatgrass p values from 42-Day Growth Chamber Study ................ 156 E.3 Lodgepole Pine p values from 100-Day Growth Chamber Study .......................... 158 E.4 Bluebunch Wheatgrass p values from 100-Day Growth Chamber Study .............. 159 Appendix F Principal Component Analysis Diagrams (PCA) ............................................... 160 F.1 Generic R Studio Codes for All Principal Component Analysis Diagrams ........... 160 F.2 Six Week Greenhouse Study: Bluebunch Wheatgrass ........................................... 161 F.3 42 Day Growth Chamber Study: Lodgepole Pine .................................................. 163 F.4 42 Day Growth Chamber Study: Bluebunch Wheatgrass....................................... 166 F.5 Tailings and Greenhouse Soils................................................................................ 169 F.6 100 Day Growth Chamber Study: Lodgepole Pine ................................................ 170 F.7 100 Day Growth Chamber Study: Bluebunch Wheatgrass..................................... 171 Appendix G Selenium and Vanadium Values (PPM)............................................................. 173 G.1 Lodgepole Pine 42-Day Growth Chamber Study ................................................... 173 G.2 Bluebunch Wheatgrass 42-Day Growth Chamber Study ....................................... 174 G.3 100-Day Growth Chamber Study ........................................................................... 175  vii  List of Tables  Table 1-1 Modified summary table of copper, selenium and vanadium concentrations found in soil samples (Golder Associates Ltd., 2016, p31) .......................................................................... 5 Table 2-1 Greenhouse soil bulk sample chemical analysis .......................................................... 11 Table 2-2 Plant species by experimental method, seed source and expected emergence rate ...... 12 Table 2-3 Environment Canada (2007) method terminology and definitions .............................. 13 Table 3-1 Experimental design of greenhouse study .................................................................... 17 Table 3-2 Substrate treatment concentrations of copper............................................................... 18 Table 3-3 Modified summary of chemical analysis of Lane Mountain #20-30 silica sand, the control substrate in the greenhouse study (Target Products Ltd., 2016) ...................................... 18 Table 3-4 Volumetric distribution of tailings and control substrates ........................................... 19 Table 3-5 Environmental conditions in greenhouse ..................................................................... 21 Table 3-6 Metal concentration in standards and blanks used for Inductively Coupled Plasma (ICP-OES) ..................................................................................................................................... 23 Table 3-7 Chemical analysis of copper spiked greenhouse soils before and after six weeks of growth ........................................................................................................................................... 34 Table 4-1 Experimental design of 42-day growth chamber study ................................................ 38 Table 4-2 Grain size distribution of control substrate .................................................................. 39 Table 4-3 Sandy tailings treatment by weight proportion ............................................................ 40 Table 4-4 Composition of silty tailings and control silica by dry weight (g) ............................... 41 Table 4-5 Copper sulfate application to sandy and silty substrates and greenhouse soils ............ 41 Table 4-6 Growth chamber test start and end dates by substrate .................................................. 43 Table 4-7 Environment Canada (2007) specified day and night growth chamber conditions ...... 44 Table 4-8 Elemental concentrations of blanks, experimental standards and standard reference material used for inductively coupled plasma optical emission spectrometry (ICP-OES) ........... 48 Table 4-9 Median relative percent difference in treatments after ICP-OES analysis of copper ... 49 Table 4-10 Expected and actual concentrations of copper in substrate by treatment ................... 49 Table 4-11 Significant difference in growth characteristics between substrates .......................... 60 Table 4-12 Median root to shoot copper translocation ratio ......................................................... 64 viii  Table 4-13 Chemical analysis significance between substrates ................................................... 65 Table 4-14 Bluebunch wheatgrass mean below to aboveground copper translocation ratio ........ 76 Table 4-15 Bluebunch wheatgrass significant shoot and root chemistry ..................................... 77 Table 5-1 Lodgepole pine 100 Day mean emergence and survival .............................................. 82 Table 5-2 Bluebunch wheatgrass 100 day mean emergence and survival in sandy tailings ........ 89 ix  List of Figures  Figure 2-1 Summary of physical and chemical characteristics of sandy and silty tailings .......... 10 Figure 3-1 Lodgepole pine emergence and survival in greenhouse after six weeks of in greenhouse .................................................................................................................................... 24 Figure 3-2 Bluebunch wheatgrass emergence and survival in greenhouse after six weeks ......... 30 Figure 3-3 Copper uptake in bluebunch wheatgrass in greenhouse after six weeks of growth .... 32 Figure 3-4 Principal Component Analysis of metals in bluebunch wheatgrass shoots in sandy tailings in greenhouse after six weeks .......................................................................................... 33 Figure 4-1 Lodgepole pine emergence in growth chamber after 42 days..................................... 52 Figure 4-2 Lodgepole pine survival in growth chamber after 42 days ......................................... 53 Figure 4-3 Lodgepole pine longest shoot and root lengths in growth chamber after 42 days ...... 56 Figure 4-4 Lodgepole pine total biomass in growth chamber study after 42 days ....................... 59 Figure 4-5 Particle size and water holding capacity in silty tailings treatments ........................... 62 Figure 4-6 Copper uptake in lodgepole pine seedlings in growth chamber after 42 days ............ 63 Figure 4-7 Principal component analysis of metals in lodgepole pine roots in silty tailings in growth chamber after 42 days ....................................................................................................... 66 Figure 4-8 Bluebunch wheatgrass emergence in growth chamber after 42 days ......................... 67 Figure 4-9 Bluebunch wheatgrass survival in growth chamber after 42 days .............................. 69 Figure 4-10 Bluebunch wheatgrass longest shoot and root lengths in 42-day growth chamber study .............................................................................................................................................. 72 Figure 4-11 Bluebunch wheatgrass total biomass in growth chamber after 42 days.................... 74 Figure 4-12 Bluebunch wheatgrass shoot and root tissue copper after 42 days ........................... 75 Figure 4-13 Principal component analysis of metals in bluebunch wheatgrass roots in sandy tailings after 42 days ..................................................................................................................... 78 Figure 4-14 Principal component analysis of metals in bluebunch wheatgrass roots in copper spiked greenhouse soils after 42 days ........................................................................................... 80 Figure 5-1 Lodgepole pine shoot and root lengths after 42 and 100 days .................................... 83 Figure 5-2 Lodgepole pine shoot and root biomass after 42 and 100 days .................................. 84 Figure 5-3 Lodgepole pine mean copper uptake after 42 and 100 days ....................................... 86 x  Figure 5-4 Principal component analysis of metals in lodgepole pine root in sandy tailings, 100 day growth chamber study ............................................................................................................ 88 Figure 5-5 Bluebunch wheatgrass longest shoot and root lengths after 42 and 100 days ............ 89 Figure 5-6 Bluebunch wheatgrass shoot and root biomass after 42 and 100 days ....................... 90 Figure 5-7 Bluebunch wheatgrass copper uptake in shoots and roots after 42 and 100 days....... 92 Figure 5-8 Principal component analysis of metals in bluebunch wheatgrass root in sandy tailings, 100 day growth chamber study ....................................................................................... 94  xi  List of Photos  Photo 1-1 Modified aerial photos of Mount Polley Mine before and after tailings dam breach .... 2 Photo 1-2 Distinct layers of sandy and silty tailings settled over native forest soil (Golder Associates Ltd., 2015)..................................................................................................................... 3 Photo 2-1 Determining longest shoot and root measurements (bluebunch wheatgrass) .............. 14 Photo 3-1 Sample preparation and mixing.................................................................................... 20 Photo 3-2 Greenhouse study at UBC Horticulture Greenhouse ................................................... 22 Photo 3-3 Lodgepole pine seedling health in four substrates ....................................................... 25 Photo 3-4 Dark greenish blue copper sulfate evaporites found on greenhouse soil surfaces ....... 26 Photo 3-5 Lodgepole pine desication of hypocotyl hooks in silty tailings ................................... 28 Photo 3-6 Bluebunch wheatgrass plant health comparison in sandy tailings ............................... 31 Photo 3-7 Variable survival between and within treatments ........................................................ 35 Photo 4-1 Pots in growth chambers at Day 1 ................................................................................ 43 Photo 4-2 Emergence scenarios of copper-spiked sandy and silty substrates .............................. 54 Photo 4-3 Lodgepole pine development in silty tailings .............................................................. 55 Photo 4-4 Lodgepole pine development in copper-spiked greenhouse soils ................................ 56 Photo 4-5 Lodgepole pine seedlings from greenhouse soil experimental control pot .................. 58 Photo 4-6 Nutrient competition from algal, mold and fungi ........................................................ 61 Photo 4-7 Common emergence scenarios in treatments of copper-spiked substrates .................. 68 Photo 4-8 Establishment challenges in loose substrates and algal growth ................................... 70 Photo 4-9 Shoots and roots of grown in 100 percent sandy and silty tailings .............................. 73 Photo 5-1 Lodgepole pine in sandy tailings after a 100 Days of growth ...................................... 82   xii  Acknowledgements I offer my greatest gratitude to the faculty, staff and fellow students in the Department of Soil Science. In particular, I would like to thank:  Dr. Les Lavkulich, my supervisor, for sharing his great technical knowledge on this project. His laughter and energy are limitless, and I am forever grateful to have had the opportunity to study with him. I cherish our friendship and will remember fondly our discussions over coffee.   Dr. Suzanne Simard, my co-supervisor, for her enthusiastic approach to research, to experimental design and to communicating science through TerreWEB initiatives.   Trish Miller, member of my supervisory committee, for her expertise in ecological risk assessment and in contaminated site studies.   Kerrie Serben from Golder Associates Ltd. for guidance in Environment Canada compliant toxicity testing.  Dr. Lyn Anglin, Chief Scientific Officer at Imperial Metals Ltd. for providing the opportunity to contribute to this important and interesting restoration project.  Dr. Gary Bradfield and Dr. Gabriela Cohen-Freue for offering statistical advice on data analysis.   Dr. Sandra Brown, for their technical skillset and encouragement during the toughest times.   Finally, I must thank my family and friends for their unconditional support and encouragement throughout my graduate studies and writing of this thesis. Dedication  xiii  Dedication    to Mark, Jessica and Charles family is forever, for always no matter what   1  Chapter 1: Introduction Mining in British Columbia began in the mid-1800s with placer gold and coal mines and has transformed the province into one of the world’s major mining districts following the discovery of the Canadian Cordillera. The mineral and coal rich mountainous belt has made the province an important producer and exporter of gold, silver, copper, lead, zinc, molybdenum, coal and industrial minerals (British Columbia Technical and Research Committee on Reclamation, 2018). Today, there are 19 major operating mines present in British Columbia (BC Mine Information, 2018). Among them is Mount Polley, located 56 kilometers northeast of Williams Lake in south-central British Columbia. The open pit copper-gold mine commenced operations in 1997 processing 18500 tons per day with an expected mine life to 2026 (Imperial Metals Ltd., 2018).   On August 4th, 2014, a partial breach of the Mount Polley mine tailings storage facility (TSF) released over 4.6 million cubic meters of supernatant water, and 12.8 million cubic meters of slurry tailings (solids and interstitial water) into Polley lake and downstream into Hazeltine Creek then Quesnel Lake (Golder Associates Ltd., 2015). The spilled tailings and water entered Polley Lake and Hazeltine Creek, scoured 1.2 million cubic meters of native soil and deposited 1.6 million cubic meters of mixed tailings and native soil downstream of Hazeltine Creek valley and into Quesnel Lake (Golder Associates Ltd., 2015). Overnight, the narrow 5 m wide 0.3 m deep channel of Hazeltine Creek was transformed into a 50 plus meter wide channel covered with various depths of tailings (Photo 1-1) (Golder Associates Ltd., 2015; NASA Earth Observatory, 2014). Water levels at Quesnel Lake rose 0.068 – 0.079 m following the breach (Golder Associates Ltd., 2015).   2   Photo 1-1 Modified aerial photos of Mount Polley Mine before and after tailings dam breach  NASA Earth Observation images taken on July 29th, 2014 (Left) and after the breach on August 5th, 2014 (Right) (NASA Earth Observatory, 2014). Red arrows indicate direction of flow entering Hazeltine Creek into Quesnel Lake.  On August 5, 2014, the British Columbia Ministry of Environment issued Pollution Abatement Order No. 107461 to Mount Polley Mining Corporation requiring documentation of the environmental response, progress and plans to prevent further erosion and sediment transport downstream (Golder Associates Ltd., 2016; Imperial Metals ltd., 2016). Golder Associates Ltd. was hired to conduct a detailed site investigation (DSI) and human health and ecological risk assessment (HHERA) compliant with contaminated site regulations (CSR).  Field observations of the 2015 Post-Event Environmental Impact Assessment Report (PEEIAR) identified two distinct types of tailings by physical and mineralogical characteristics layered on top of one another (Golder Associates Ltd., 2016; SRK Consulting (Canada) Inc., 2015). “Grey tailings” (or silty tailings as used in this thesis) were found to be most abundant in embankments and along upper creek benches. These tailings were grey in color with fine “silty” texture predominantly composed of potassium feldspar, plagioclase and minor biotite and quartz minerals (SRK Consulting (Canada) Inc., 2015). Beneath silty tailings or in low-lying zones near the creek, heavier orange black “magnetite sands” (or sandy tailings as used in this thesis) were found with dominant minerals of plagioclase feldspar, magnetite and trace copper-bearing sulfides (SRK Consulting (Canada) Inc., 2015). Deposition of tailings was thickest adjacent to 2 km 3  the initial site of breach (commonly referred to as the Polley Plug area) measuring over 3.5 m and ranges between 0.5 to 1.5 m along lower Hazeltine Creek (Golder Associates Ltd., 2015). Sandy and silty tailings were often found settled in distinct layers (due to difference in particle specific gravity) over native forest soil.    Photo 1-2 Distinct layers of sandy and silty tailings settled over native forest soil (Golder Associates Ltd., 2015)  Studies assessing the impacts of tailings deposition on human health, aquatic environment and terrestrial ecosystem along Hazeltine Creek have now been complete (Golder Associates Ltd., 2016; SNC Lavalin Inc., 2014; SRK Consulting (Canada) Inc., 2015). Golder Associates Ltd. (2015) described the physical changes in the environment as the primary impact on the terrestrial ecosystem as the tailings flow scoured and deposited a mixture of tailings and native soil into the lakes, creeks and terrestrial environment along the flow path (Golder Associates Ltd., 2016). The breach left adjacent forest floor along Hazeltine Creek smothered with varying thickness of sandy and silty tailings. Deposition of tailings on the forest floor impeded air exchange; these saturated anaerobic environments led to root decay and subsequent tree mortality in the spring of 2015 (Golder Associates Ltd., 2016). The PEEIAR (Golder Associates Ltd., 2015) did not find metal toxicity as the root cause nor was consistent with subsequent loss in standing plant communities. 4  Results from detailed geochemical characterization of sandy and silty tailings by SRK Consulting (Canada) Inc. (2015) indicated that tailings were not potentially acid generating (non-PAG) due to high pH and presence of carbonate minerals in tailings. These findings were consistent with historical understanding of low grade or low sulfur ore deposits (SRK Consulting (Canada) Inc., 2015). Following the breach, copper and selenium were the only elements found to be enriched in tailings compared to crustal basalt rocks (SRK Consulting (Canada) Inc., 2015). These results were in agreement with SNC-Lavalin Inc. (2014)’s terrestrial soil investigation of 18 transects oriented perpendicular to the course of the creek and spaced approximately 500 meters along the length of Hazeltine Creek.  The enrichment of copper and selenium was a result of two mineralogical forms of copper: 34 percent sulfide copper (ore material that bypassed mine extraction) and 66 percent non-sulfide copper found in silicate chloride (iron magnesium aluminum silicate mineral, of which 14 to 23 percent contained selenium) (SRK Consulting (Canada) Inc., 2015). Current and previous studies found non-sulfide copper to be relatively insoluble, considered non-reactive and at low risk of copper leaching under neutral pH (subaerial or subaqueous) conditions at Hazeltine Creek (Henry, 2009; SRK Consulting (Canada) Inc., 2015; Taplin, 2002). These results affirm the low potential of acid rock drainage of non-sulfide copper in sandy and/or silty tailings (SRK Consulting (Canada) Inc., 2015). The concern however, lies in the fact that these sulfide minerals containing copper and selenium remain leachable by natural oxidation processes (SRK Consulting (Canada) Inc., 2015).  Copper, selenium and vanadium were metals of particular interest in recent site studies (Golder Associates Ltd., 2015, 2016; SNC Lavalin Inc., 2014; SRK Consulting (Canada) Inc., 2015). Detailed site investigations by Golder Associates Ltd. (2016) found concentrations of copper exceeding British Columbia Regional Background Soil Quality standards and contaminated site regulations (CSR) in all 126 soil samples collected (Table 1-1). Concentrations of vanadium (expected to be associated with magnetite sands) also exceeded provincial soil quality standards in 37 of 126 samples by approximately 1.4 times the standard, but remain below CSR standard in soil (Golder Associates Ltd., 2016). Elevated levels of selenium have also been a focus in recent 5  studies as bioaccumulation of selenium in aquatic organisms impairs reproduction of important fish species and waterfowl populations (Barnhart, 1957; Ogle et al., 1988; Ohlendorf et al., 1986). Concentrations of selenium in soil samples were well below provincial soil quality standards and contaminated site regulations and thus not considered as a contaminant, only an enrichment (> 1 ppm) to regional crustal basalt rocks post breach. Thus, copper and vanadium were identified as the only metal contaminants entering Hazeltine Creek and adjacent areas (Golder Associates Ltd., 2016).  Table 1-1 Modified summary table of copper, selenium and vanadium concentrations found in soil samples (Golder Associates Ltd., 2016, p31) Concentrations of copper found exceeding both British Columbia Soil Quality standards and contaminated site regulations (CSR). Only 37 or the 126 samples had vanadium exceeding provincial soil quality standards by 1.4 times. Selenium was only found enriched compared to crustal basalts, did not exceed any standards.  Metal Samples exceeding CSR standards Maximum ppm Mean ppm 95% UCLM Local Reference BC Regional Background Soil Quality* CSR Standards Copper 126/126 1560 805 829.7 75 65 150 Selenium 0/126 1.7 1.0 1.03 0.61 4 3 Vanadium 37/126 289 186 189.2 113 100 200 Local reference is the 95th percentile of reference sample results (e.g. crustal basalt rocks); *(British Columbia Ministry of Environment, 2010)  UCLM = upper confidence limit of the mean; all concentrations shown in ppm.   The greatest emphasis of recent studies has been on aquatic ecosystems as the Pollution Abatement Order was focused on water turbidity, not terrestrial ecosystem. However, effective plant establishment and growth conditions between the (soil) medium and plant root, or rhizosphere, are critical. Adequate aeration, available nutrients and water must be available for seed germination and plant root growth and uptake (Miller & Donahue, 1995). Aeration is the balance of oxygen and carbon dioxide at root surface; most critical in uniform particle sized media such as tailings where hexagonal or cubic closed packing reduces substrate porosity and ultimately gas movement (Nawaz, Bourrié, & Trolard, 2013). Golder Associates Ltd. (2016) had noted that the deposition of tailings on the forest floor impeded air exchange. The saturated anaerobic environment resulted in root decay and increased tree mortality post breach.  6  There are seventeen most important nutrients for plant growth. Plants must obtain the following mineral and non-mineral nutrients from their growing medium: greater quantities of macronutrients of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), sulfur (S), magnesium (Mg); non-mineral nutrients of hydrogen (H), oxygen (O) and carbon (C); and in much less quantities of micronutrients (or trace minerals) of boron (B), chlorine (Cl), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo) nickel (Ni) and zinc (Zn) (Havlin, Tisdale, Nelson, & Beaton, 2013).  Plant nutrient uptake is generally attributed to three mechanisms of ion transport to plant roots: (1) mass flow of dissolved nutrients in soil water transported to roots by transpiration, (2) diffusion of nutrients from regions of high concentration to low concentration, and (3) root extension or physical contact from root growth (Foster & Miklavcic, 2016). Along root surfaces, nutrients are absorbed through selective, passive and active ion transport (Foster & Miklavcic, 2016).  However, to achieve desired outcomes of plant growth, it is not the availability of nutrients that is the only concern but the amount of each nutrient, or the balance among the various nutrients (Havlin et al., 2013). In addition, many micronutrients are trace nutrients required in relatively small amounts (compared to the macronutrients) and in large available concentrations can result in phytotoxicity or the degree in which a chemical or other compound is toxic to plants (Asati, Pichhode, & Nikhil, 2016). Environment Canada defines phytotoxicity as “unwanted detrimental deviations from the normal pattern of appearance, growth, and/or function of plants in response to test material. Phytotoxicity might occur during germination, growth differentiation, and/or maturation of plants” (2007, p xxiii).  At Hazeltine Creek, copper accumulation occurred relatively quickly in soils due to strong absorption to soil organic matter by cation exchange capacity (CEC) and only leached when copper concentrations exceed CEC sites (Alloway, 1990). Other soil factors affecting copper mobility include pH and presence of iron, manganese and aluminum oxides which were also found in tailings (Adriano, 1986; Slooff, Clevan, Janus, & Ros, 1989). Capacity of soil to absorb 7  copper is at its greatest at neutral to slightly alkaline conditions (pH 6.7-7.8) reflective of conditions at Hazeltine Creek (Adriano, 1986; SRK Consulting (Canada) Inc., 2015). Overexposure of copper to the terrestrial ecosystem can have serious adverse effects on soil microbial processes, terrestrial plants and invertebrates and ruminant wildlife through dietary uptake (Canadian Council of Ministers of Environment, 1999; Golder Associates Ltd., 2016). Cornfield (1977) reported that a concentration of 100 mg Cu/kg lead to a 25 percent decrease in carbon dioxide evolution in microbial populations. Phytotoxicity effects of copper on terrestrial plants are species-specific and vary with concentration. Typical symptoms of copper toxicity may include dark green leaves and reduction in radicle elongation (Canadian Council of Ministers of Environment, 1999).   Phytotoxicity and metal toxicity of tailings to the terrestrial ecosystem at Hazeltine Creek have not been extensively studied. In partnership with Mount Polley Mining Corporation, this thesis examined the edaphic effects of sandy and silty tailings on germination, growth, and metal uptake in roots and shoots in various plant species in two controlled environments. Primary focus was placed on copper given the severity of exceedance in provincial soil quality standards and contaminated site regulations and to a much lesser extent vanadium and selenium. The overall objective of this study was to determine the chemical and physical properties of sandy and silty tailings under controlled environmental conditions and to assess the establishment, survival and uptake of metals, notably copper, in lodgepole pine (Pinus contorta), bluebunch wheatgrass (Pseudoroegneria spicata), wild rose (Rosa nutkana) and wild willow (Salix scouleri). These results have assisted in the identification of growth limiting factors in tailings and the potential for food web transfer of metals into adjacent ecosystems.  A greenhouse study was conducted at the University of British Columbia to isolate several abiotic and biotic (herbivory) factors to provide favorable conditions for plant growth. The study focused on physical growth-limiting factors of sandy and silty tailings under greenhouse conditions using three species: lodgepole pine (Pinus contorta), bluebunch wheatgrass (Pseudoroegneria spicata) and wild rose (Rosa nutkana).   8  An Environment Canada (2007)-compliant study was then conducted following the Biological Test Method (2007) designed for measuring emergence and growth of terrestrial plants exposed to contaminants in soils. The 42-day growth chamber study incorporated critical physical findings from the greenhouse study to provide optimal growing conditions to further explore any limitations for plant growth and potential phytotoxicity in sandy and silty tailings. Substrate particle size and substrate copper concentrations were examined separately for three plant species: lodgepole pine (Pinus contorta), bluebunch wheatgrass (Pseudoroegneria spicata) and wild willows (Salix scouleri) with emergence, survival, longest shoot and root length, and metal uptake as indicators of species specific phytotoxicity. The 42-day study also assessed the concerns of applying a required universal procedure to test phytotoxicity on various substrates with limited knowledge of their respective physical characteristics.  An additional 100-day growth chamber study was designed to provide a form of comparison to trends and observations found in the 42-day study. This study was not intended to be Environment Canada (2007)-compliant, but rather to review substrate influences on lodgepole pine (Pinus contorta) and bluebunch wheatgrass (Pseudoroegneria spicata) growth after prolonged exposure in sandy tailings. Emphasis was placed on sandy tailings as it contains relatively high amounts of copper and had greater potential for seedling survival in environmental conditions found at Hazeltine Creek.  9  Chapter 2: General Methods This chapter provides a general overview of the common methods and materials used in all three studies. A single batch of sandy and silty tailings was collected in late 2015 and subsequently used in each study. Methods pertaining to data collection in all three studies were outlined by Environment Canada (2007) except for chemical extractions which were study specific. Statistical analysis was equally stringent across all studies. Details specific to each study are presented in their respective chapters.   Golder Associates Ltd. in conjunction with Mount Polley Mine staff collected a total of 50 (five gallon) buckets of sandy and silty tailings from Area 6 of the Mount Polley Plug Area (Easting 595421, Northing 5820734). This area was of particular interest as composition of tailings was least altered (free of debris), deposition was over 3.5 m thick adjacent to initial site of breach and undisturbed by mine reclamation traffic after the breach (Golder Associates Ltd., 2015). Silty and sandy tailings settled in distinct layers allowing samples to be collected from the same location. Detailed descriptions of collection methods can be found in Appendix B  : Summary of Key Findings Report Prepared for Golder Associates Ltd. Subsamples of sandy and silty tailings were directly sent to ALS Laboratory for chemical analysis (Figure 2-1; Appendix A  Sandy and Silty Tailings: Certificate of Analysis). Sandy and silty tailings were kept cool during transportation to HR MacMillian Building at the University of British Columbia on November 2015 and remained in their original containers until they were used for each study.  10   Figure 2-1 Summary of physical and chemical characteristics of sandy and silty tailings Tailings were collected by Golder Associates Ltd. and Mount Polley staff following the breach in November 2015.  ALS Laboratory Certificate of Analysis can be found in Appendix A  : Sandy and Silty Tailings: Certificate of Analysis.  As discussed in the previous chapter, metals of interest include copper, selenium and vanadium in sandy and silty tailings (Table 1-1). Concentrations of copper and vanadium were both found exceeding provincial soil quality standards and classified as contaminants at Hazeltine Creek (Golder Associates Ltd., 2016). Sandy and silty tailings used in this study contain 1130 and 805 mg/kg of copper, respectively; were well over the contaminated site regulation (CSR) limit of 150 mg/kg. Vanadium, however, was only 2 mg/kg over the CSR limit in silty tailings. Selenium was found to be enriched relative to local crustal basalt rocks, but did not exceed any pre-determined limit. Copper was the main focus of this study, to a much lesser extent, vanadium and selenium was reviewed.  To assess the effects of copper on plant emergence and growth, additional treatments involving copper-spiked greenhouse soils were included in both greenhouse and growth chamber studies. Equivalent to copper concentrations in tailings, copper spiked greenhouse soil trials ascertain the effects of copper on emergence and survival, plant growth and uptake in normal soil systems. Greenhouse soils were composed of peat potting mix with calcium carbonate grains (< 1 cm) to 11  ensure adequate soil aeration. The table below illustrates the major anions, cations and trace elements from bulk sampling of these soils (Appendix C). As with all growth experiments, fertilizer water was applied regularly to all substrates to ensure results were not affected by lack of macro, micro and essential nutrients. Fertilizer was obtained from the UBC Horticulture Greenhouse for both studies. The greenhouse study used an automated fertilizer mixer to mix fertilizer with a pH of 5.5 to 5.8 and electric conductivity of 1.6 to 1.7 S/m, whereas fertilizer used in the growth chamber studies was mixed manually and stockpiled for consistency (see details in respective chapters). Methods of enriching these soils with copper sulfides were specific to each experiment and discussed further in their respective chapters.   Table 2-1 Greenhouse soil bulk sample chemical analysis Values were converted from mmol/L to mg/L. See Appendix C  Chemical Analysis of Greenhouse Soils and Fertilizer analysis in 2014.  Sample Anions (mg/L) Cations (mg/L) Trace Elements (mg/L) NO3 S P NH4 K Na Ca Mg Si Fe Cu Potting Mix 142.61 76.96 11.77 <1.81 66.47 11.50 68.13 34.03 <2.81 0.09 <0.01  Three native plant species were selected by Dr. Suzanne Simard during preliminary stages of the greenhouse study. These were lodgepole pine (Pinus contorta), pinegrass (Calamagrostis rubescens) and willow (Salix scouleri). These species were selected for their abundance, growth rate, distribution, ecological importance and canopy level representation in the areas adjacent to Hazeltine Creek. Commencing the greenhouse experiment in January 2016 was a challenging time to search for viable seeds for pinegrass (which seeds infrequently between years) and willows (low viability, only produced in spring). As a result, it was advised by Dr. Suzanne Simard to test wild rose (Rosa nutkana) and bluebunch wheatgrass (Pseudoroegneria spicata, also known as Agropyron spicatum) in place of willow and pinegrass respectively for the greenhouse study (Table 2-2). Lodgepole pine seeds were obtained from the Ministry of Forests in 2016 from seedlots 31003, 08398, 14387, 31002 in the Kangaroo Creek area, located approximately 1200 m from Mount Polley Mine. Details on selection and seed sources can be found in Appendix B  Summary of Key Findings Report Prepared for Golder Associates Ltd. 12  Table 2-2 Plant species by experimental method, seed source and expected emergence rate Wild roses were replaced with wild willows in growth chamber study due to poor emergence. *Wild willows were not used in the 100-day growth chamber study. Plant Species Experimental Method Seed Source Expected Emergence Rate Greenhouse Growth Chamber Lodgepole Pine  (Pinus contorta) √ √ Ministry of Forest, British Columbia 94% (tested in 2013) Bluebunch Wheatgrass (Pseudoroegneria spicata) √ √ Premier Pacific Seeds Ltd 89% (tested 2016) Wild Rose  (Rosa nutkana) √  Linnaea Nurseries Field Collected, Unknown Wild Willow  (Salix scouleri)  √* Areas adjacent to Mount Polley Mine  Field Collected, Unknown  A similar combination of species was used for the growth chamber studies. Wild willows replaced wild roses due to poor emergence found in the greenhouse study. Wild willow seeds were hand-picked weeks before the commencement of the growth chamber study (July 2016). The key objective in choosing test species was their significance in the local ecosystem along Hazeltine Creek. Details regarding seed lots, seed origin, seed stratification and method to sow seeds are experiment-specific and discussed in their respective chapters. A new batch of seeds for each species was obtained for each experiment to ensure consistent seed viability.  Greenhouse and growth chamber studies each followed specific method development and applications from Environment Canada’s Biological Test Method: Test for Measuring Emergence and Growth of Terrestrial Plants Exposed to Contaminants in Soil published by Environment Canada (2007). A summary of relevant terms defined by Environment Canada was used to quantitatively and qualitatively assess toxicity of copper in Mount Polley Mine tailings (Table 2-3). Seedling emergence, survival, plant growth (shoot/root lengths), biomass (shoot/root mass) and metal uptake and translocation were assessed and discussed in their respective chapters.   13  Table 2-3 Environment Canada (2007) method terminology and definitions Definitions are specific to Environment Canada’s Biological Test Method: Test for Measuring Emergence and Growth of Terrestrial Plants Exposed to Contaminants in Soil published by Environment Canada (2007). Term Explanation Seedling Emergence Occurs following the germination of a plant, wherein the early growth of a seedling pushes the epicotyl through the soil surface. In this test method, emergence refers to the appearance of the seedling shoot 3 mm above the surface of the soil. Seedling Survival The mean percent survival is calculated from the percentage of emerged plants, in each test vessel containing negative control soil, that survived to the end of the test. For instance, if only four of the five emerged seedlings in a given vessel survived to the end of the test, the percent survival for the vessel was 80%. However, the mean percent survival is the average percent survival for emerged plants in all of the test vessels containing negative control soil. Plant Growth Live shoot and root length and shoot and root dry mass measured at the end of the test Shoot Above-ground portion of the plant such as the stems and leaves.  Root Below-ground portion of the plant Germination Refers to the process by which the plant embryo within the seed resumes growth after a period of dormancy and the seedling emerges from the seed. Biomass Total dry weight (mass) of a group of animals or plants. Desiccation When the plant, or portion of plant, is dried. Growth The increase in size or weight as the result of proliferation of new tissues.   The duration of greenhouse and two growth chamber studies were 6 weeks, 42 and 100 days, respectively. The 42-day growth chamber study was the only study fully compliant with Environment Canada (2007) standards. At the end of each study, seedlings were photographed and carefully extracted by hand to preserve fine roots and ensure accurate measurement of the extent of root growth. Following separation, roots were thoroughly rinsed with distilled water and cleaned by hand to remove all adhering substrates. Shoot lengths were measured from the base or transition point between the shoot and root to the tip of the longest grass blade or needle. Similarly, root lengths were measured from the same transition point to the furthest root extension. The photo below illustrates longest shoot length, root length and position of transition point determined for all surviving plant specimens at the end of each study.  14   Photo 2-1 Determining longest shoot and root measurements (bluebunch wheatgrass) Green line represents the transition point and reference point for measuring longest root (blue) and shoot (red). One by one centimeter graph paper as scale.  Shoots and roots were then dried separately to determine dry weight biomass. The greenhouse and growth chamber experiments used dry and wet ashing digestive methods respectively, to prepare samples for chemical analysis. Using inductively coupled plasma optical emissions 15  spectroscopy (ICP-OES), a series of eighteen elements (Cu, Se, V, Al, Ca, Cd, Cr, Co, Fe, K, Mg, Mn, Na, Ni, P, Pb, Si, Zn) were measured with particular attention to copper, selenium and vanadium as expressed in detailed investigations after the Mount Polley tailings dam failure (Golder Associates Ltd., 2016; SNC Lavalin Inc., 2014; SRK Consulting (Canada) Inc., 2015). Selenium and vanadium was not measured in the greenhouse study.   An ANOVA test was not suitable for statistical analysis as small sample sizes and assumptions of normality were not met. Statistical significance (p values) between treatments was determined using the Kruskal-Wallis test, a non-parametric equivalent of a one-way ANOVA in the SPSS software program. Pairwise comparisons between treatments and substrates were subsequently examined in SPSS with the Dunn’s post hoc test on each pair of pre-determined groups. SPSS automatically applies the Bonferroni adjustment, product of Dunn’s p values and total number of test carried between treatments, to p values of pairwise comparisons to account for error from multiple testing. Statistical analysis was conducted on emergence, survival, longest shoot and root, total biomass and chemical uptake of copper, selenium and vanadium in lodgepole pine and bluebunch wheatgrass. A summary table of statistically significant effects and corresponding p values is available in Appendix E  : Statistical Analysis: Kruskal Wallis Test and Summary of p values.  Principal component analysis (PCA) diagrams were generated for each plant species by growing medium and by shoot and root to graphically determine the correlations among the eighteen metals analyzed. PCA diagrams were created in RStudio (See 0for program codes). Each metal is a variable and represents one dimension; RStudio reduces the dimensionality of 18 dimensions to a two-dimensional diagram. “Dim 1” and “Dim 2” show the largest (principal direction) and second largest (second most important direction) of sample variation, respectively. The sum of Dim 1 and Dim 2 is the percentage of total data variance retained in each PCA diagram. RStudio uses R-values as coordinates to graphically represent metal correlations with relationship strengths (p values) shown by color and degree of shading. Correlation indicates redundancy in data; thus metals are correlated when arrows point in the same direction and no correlation when arrows are perpendicular to one another. Also included in each PCA diagram were replicates of 16  each treatment. Closely clustered treatment points indicate similar chemical composition (or high redundancy in metal data). The position of each replicate datapoint or treatment cluster also indicates their relevance to specific metals or principal components. Selected diagrams are found in respective chapters with the remainder of diagrams displayed in Appendix F  Principal Component Analysis Diagrams (PCA). 17  Chapter 3: Greenhouse Study Greenhouses have been the primary choice for controlled environmental testing as it provides a moderated environment reflective of diurnal changes. The simulated natural environment is an economically viable option for toxicity testing as facilities were capable of handling large replicate numbers. This study was conducted at the University of British Columbia Horticulture Greenhouse in spring of 2016.  3.1 Greenhouse Study Methods The objective of this greenhouse study was to determine the physical soil properties of tailings on plant emergence, survival, growth, copper toxicity and uptake under simulated natural conditions. Three common species (lodgepole pine, bluebunch wheatgrass and wild rose) were grown in various concentrations of sandy tailings, silty tailings and copper sulfate-spiked greenhouse soils over a period of four months. These concentrations began with coarse grained inert silica sand (0 percent) and progressed to 12.5, 50 and 100 percent of copper-bearing substrates. The partial factorial set of treatments shown in Table 3-1 were replicated six times for each plant species, comprising three-completely randomized experimental designs. This design resulted in a total of 66 one gallon pots for each of the three plant species or 198 pots all together.  Table 3-1 Experimental design of greenhouse study Sandy and silty tailings treatments shared experimental controls as only one type of sand was used to mix various treatment concentrations for both. Substrate Control 12.5% Treatment 50% Treatment 100% Treatment Sandy Tailings n = 6 pots each n = 6 pots each Silty Tailings n = 6 pots each Greenhouse Soil n = 6 pots each Total Treatments 66 treatments pots (4L) per plant species  Sandy and silty tailings contained 1130 and 805 mg/kg of copper, respectively. To examine the effects of copper on plant responses, this study used 1150 mg/kg of copper sulfate to artificially create soil equivalence of copper rich tailings. The following table illustrates the concentrations 18  of copper in each substrate by treatment. These values were obtained with the assumption that a given volume would only yield the respective chemical concentration in randomly selected homogenized samples of tailings, control substrate (inert neutral quartz sand) and bulk greenhouse soils. The preparation of treatment pots began with experimental control pots, followed by sandy and silty tailings with increasing concentration of tailings. Copper-spiked greenhouse soils were prepared last to minimize cross contamination of treatments due to usage of copper sulfate solution.  Table 3-2 Substrate treatment concentrations of copper Substrate Copper Concentration (mg/kg) Control 12.5% Treatment 50% Treatment 100% Treatment Sandy Tailings 0* mg/kg 141.3 mg/kg 565 mg/kg 1130 mg/kg Silty Tailings 0* mg/kg 100.6 mg/kg 402 mg/kg 805 mg/kg Greenhouse Soil 0* mg/kg 143.8 mg/kg 575 mg/kg 1150 mg/kg Calculated and measured concentrations of before and after six weeks of growth can be found in Table 3-7 *Control substrates may contain trace amounts of copper (<10 ppm) which were insignificant and irrelevant to the study. At a much lesser amount, the same control substrate was used to create other treatments.    Lane Mountain silica sand (#20-30) was used as the control substrate for diluting both sandy and silty tailings treatments in the greenhouse study. Appearing white to cream-white, the control substrate had an angular grain shape and was 99.56 percent inert quartz sand (or silica oxide) with 0.44 percent trace chemical properties listed in Table 3-3 (Target Products Ltd., 2016).   Table 3-3 Modified summary of chemical analysis of Lane Mountain #20-30 silica sand, the control substrate in the greenhouse study (Target Products Ltd., 2016) Property Typical Value (% weight) Silica (SiO2) 99.56 Aluminum Oxide (Al2O3) 0.24 Iron Oxide (Fe2O3) 0.042 Calcium Oxide (CaO) 0.0051 Magnesium Oxide (MgO) 0.0079 Titanium Dioxide (TiO2) 0.016 Loss on Ignition at 1180 oC     0.13  19  Bags of Lane Mountain silica sand (#20-30) were homogenized before use. A plastic mesh liner was placed under each of these pots to reduce sand spilling out from the pot’s drainage holes. Pots were filled in thirds with gentle compaction by hand in-between until two centimeters from the rim of the pot. This minimized air-pockets and overflowing of substrate during the first hydration. Beginning with 12.5 percent treatments, sandy tailings were measured by volume (12.5 percent) and added to the reciprocal volume of control substrate (87.5%) to achieve a full treatment pot. Volumetric measurements of both sandy and silty tailings are listed in the table below.  Table 3-4 Volumetric distribution of tailings and control substrates  Percent Distribution of Tailings and Control Substrate 0% (Control) 12.5% 50% 100% Sandy Tailings 0 12.5 50 100 Control Substrate LM #20-30 100 87.5 50 0 Sandy Tailings 0 12.5 50 100 Control Substrate LM #20-30 100 87.5 50 0  Silty tailings arrived at the University of British Columbia in various moisture conditions ranging from fully saturated and submerged, to saturated, to partly dry on the surface (Photo 3-1). To ensure the correct concentration of tailings was used in each treatment, each bucket was left to drain through Whatman 1 filter paper for twenty minutes, mixed with trowel and allowed to dry for 2 days before use. Fines captured through filter paper were then brushed back to their respective trays.     20   Photo 3-1 Sample preparation and mixing Top Left: varying moisture in silty tailings upon arrival to UBC (left to right: saturated and submerged in fluid, saturated, partly dry on surface); Top Right: silty tailings left to air try for 2 consecutive days; Bottom Left: 50% silty tailings treatment before mixing; Bottom Right: 50% silty tailings treatment after mixing.  Greenhouse soils were homogenized and filled in thirds, with gentle compaction by hand in between fillings, until a mean weight of 0.650 kg/pot was reached. Fertilizer water was added to each pot until saturation and allowed to drain naturally for two hours. Thereafter, a saucer was placed under each pot to catch any residue fluid, which was poured back into the respective pot.   As stated in Chapter 1, total copper concentration in sandy and silty tailings was found to be 1130 and 805 mg/kg respectively by ALS Laboratories. For ease of calculation and preparation, greenhouse soils received a maximum copper concentration of 1150 mg/kg (or 0.75 g copper sulfate) for the 100 percent treatment, with subsequent treatments 50 and 12.5 percent receiving 0.37 and 0.09 g of copper sulfate pentahydrate (Fisher Scientific C493-500; 100% CuSO4-5H2O; ACS Certified), respectively. Each pot was allotted 30.0 mL of the appropriate copper sulfate solution. Upon addition, pots were thoroughly mixed; excess fluids were poured back into the respective pot to ensure the correct concentration was maintained. Soil surfaces were then 21  levelled and seeds were sown. A total of five seeds were randomly selected and sown in a circular fashion in each pot. All seeds were sown in January 16-17, 2016. Each seed was gently covered by coarse sand (same material as control) to ensure seeds were held in place during watering.  Environmental conditions in the greenhouse (Table 3-5) were maintained throughout the experiment. However, ambient light and temperatures increased slightly entering mid spring. Unlike conventional greenhouse watering where benches/pots were flooded and drained several times a day, pots were kept moist as drainage from excess moisture could alter the pot’s original metal concentration. As a means of preventing any cross contamination, all pots were elevated six inches above the greenhouse bench.  Table 3-5 Environmental conditions in greenhouse Environmental Condition Day (7:00 am – 10:00 pm) Night (10:00 pm – 7:00 am) Air Temperature 24 ± 5°C* 15 ± 5°C Lighting 300 ± 100 µmol/(m2 s) to soil surface No light Humidity >50% Watering Hand water as required** None *Day and night temperatures depended on weather and became progressively warmer entering late Spring. **Pots were watered and kept moist but not saturated as excess drainage could flush away and alter original metal concentration.  Pots were uniquely identified and grouped by replicate number. Each replicate set was randomly re-arranged every two weeks and replicate sets shuffled left or right within the greenhouse bench biweekly. Even numbered replicate sets were shuffled towards the right end of the bench while odd numbered replicate sets were shuffled left (e.g. arrangement for first two weeks were 1-2-3-4-5-6 and re-arranged to 3-6-5-2-1-4 at the beginning of third week). The randomization within each replicate set and shuffling replicate sets biweekly was to ensure identical exposure to environmental conditions and to eliminate external greenhouse factors affecting plant growth (Photo 3-2).  22    Photo 3-2 Greenhouse study at UBC Horticulture Greenhouse  Pots were elevated to prevent cross contamination. Red bracket illustrates one full replica of entire experiment. There were a total of six replicates, each re-arranged biweekly along the bench.  Lodgepole pine seedlings grew at a slow rate and did not produce enough above and belowground biomass for tissue analysis during the time allotted for the trial. Test duration for lodgepole pine was extended to four months, however emergence and survival were fairly variable across treatments and particularly limited in tailings. As a result no chemical analysis was conducted on lodgepole pine seedlings. Wild rose seedlot was defective as seeds did not emerge (no data).   On February 29th and March 1st, all bluebunch wheatgrass pots were removed after emergence and survival data was collected. Shoot and root tissues of bluebunch wheatgrass were oven dried at 65 oC for 12 hours to reach constant weight and cooled in desiccators. Samples were then cut into pieces less than 2 cm in length and placed in crucibles in preparation for dry-ashing. Pots with an aggregated (above or belowground) sample weight of less than 0.050 grams of dry biomass would form a composite sample within the pot. If replicates in the same treatment did not meet the 0.050 gram criteria, the entire treatment was composited into a single sample. 23  Crucibles were placed in muffle furnaces at 350 oC for one hour with aeration (open door) before heating up to 500 oC (closed door) for a minimum of four hours or until no black ash remained, then allowed to cool in the furnace (Isaac, 1990; Lavkulich, 1981). Concentrated hydrochloric acid, 10 mL, of was added to each crucible and evaporated to dryness (hotplate at +100 oC) and baked for an hour to dehydrate the silica. The remaining ash was washed by 7 mL of 4 percent nitric acid and two drops of concentrated nitric acid through Whatman filter paper. The filtrate was collected, brought to volume in 25 mL volumetric flasks and sent for elemental analysis of metals by inductively coupled plasma optical emission spectrometer (ICP-OES). Table 3-6 illustrates the metal content of blanks and standards used for sample analysis. A recalibration of analyzing blank and standards 1 to 6 was conducted every 30-50 samples.  Table 3-6 Metal concentration in standards and blanks used for Inductively Coupled Plasma (ICP-OES)   Metal Concentration in (PPM) Al Ca Cd Co Cu Cr Fe K Mg Mn Na Ni P Pb Si Zn Blank 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 std 1 12.5 7.5 0.25 0.25 0.5 0.5 12.5 2.5 7.5 1 2.5 0.5 2.5 0.5 12.5 1 std 2 25 15 0.5 0.5 1 1 25 5 15 2 5 1 5 1 25 2 std 3 50 30 1 1 2 2 50 10 30 4 10 2 10 2 50 4 std 4 100 60 2 2 4 4 100 20 60 8 20 4 20 4 100 8 std 5 250 150 5 5 10 10 250 50 150 20 50 10 50 10 250 20 std 6 500 300 10 10 20 20 500 100 300 40 100 20 100 20 500 40  Available copper in artificial copper-spiked greenhouse soils was determined using the 1M hydrochloric acid method, a procedure designed for soils deficient in copper (Lavkulich, 1981). 10 grams of soil and 50.0 mL of 1M hydrochloric acid was held in a plastic bottle and shaken for one hour. The suspension was filtered through Whatman No. 2 filter paper and brought to volume in a 50 mL volumetric flask with 4 percent nitric acid and prepared for ICP-OES analysis.  3.2 Results and Discussion for Greenhouse Study The greenhouse study was an exploratory experiment designed to determine growth limiting properties of sandy and silty tailings under normal controlled growing conditions. The 24  experiment duration was reduced from four months to six weeks as a result of inconsistent emergence and poor survival across all treatments. Chemical analysis was only conducted on bluebunch wheatgrass seedlings as it was the only plant species with adequate biomass for further testing. In addition to copper, 15 other metals (Al, Ca, Cd, Co, Cr, Fe, K, Mg, Mn, Na, Ni, P, Si, Pb, and Zn) were analyzed. Principal component analysis graphs were generated by substrate for bluebunch wheatgrass. These graphs do not provide statistical measures of metal distribution, rather they assist in the generation of hypotheses for further study.   3.2.1 Lodgepole Pine Emergence of lodgepole pine was highly variable and inconsistent during the six week growing period (Figure 3-1). A concentration-response relationship was not observed. Experimental controls of greenhouse soils and tailings had least emergence while survival was most affected in 109 and 269 mg Cu/kg in greenhouse soils with the latter being equivalent to 25 percent tailings. There was no survival in silty tailings.    Figure 3-1 Lodgepole pine emergence and survival in greenhouse after six weeks of in greenhouse 0%10%20%30%40%50%60%70%80%90%100%Control 28 109 269 Control 12.5% 50% 100% 12.5% 50% 100%Copper-spiked Greenhouse Soil(mg Cu/kg)SilicaSandSandy Tailings Silty TailingsPercent Emergence and Survival Lodgepole Pine Emergence and Survival (6weeks) EmergenceSurvival25  Emergence (blue) and survival (red) is fairly variable with experimental controls under 50 percent in greenhouse soil and sandy substrate. No significance was found between and among treatments (n = 6); no seedlings emerged at 12.5 percent silty tailings. Median and standard error displayed.  Once established, plant health was visually similar across all treatments. Greenhouse soils were organic-based, which enhances growing conditions in warm and well hydrated environments. No sign of toxicity was found in surviving seedlings of copper-spiked soils after six weeks of growth. Healthy seedlings were also found in sandy and silty tailings, but survival was highly variable. Those that survived the first weeks in sandy and silty tailings continued to growth unaffected (Photo 3-3). In addition to poor emergence and survival, there was insufficient above and belowground biomass to determine copper uptake. Thus no chemical data were obtained from lodgepole pine seedlings.    Photo 3-3 Lodgepole pine seedling health in four substrates Control greenhouse soil (top left), 269 mg copper per kg greenhouse soil (top right), control silica sand (bottom left) and 100% sandy tailings (bottom right). 26  A number of factors could have led to poor emergence in lodgepole pine. The expected germination rates of lodgepole pine and bluebunch wheatgrass were 94 and 89 percent respectively. These values were also observed in subsequent germination tests conducted with the same package of seeds on a moist petri-dish for both species. In the experiment, seeds were sown on substrate surfaces and covered by a thin layer of silica sand and hydrated by spray bottle. One can speculate that for substrate surface conditions, variable and poor moisture retention of tailings may have hindered emergence or become inhabitable for early survival.  Greenhouse conditions were thought to be the greatest confounding factor to the experiment’s success. Under normal operating conditions, pots are placed in misting tents where high humidity is kept to allow seeds to germinate. Following germination, greenhouse benches are repeatedly flooded and drained to re-hydrate pots two to three times a day. This experimental setup was not suitable for testing tailings toxicity as neither simulate natural conditions nor prevent cross contamination between pots. Pots in the greenhouse study were carefully hand watered daily to keep substrates hydrated. Even so excess water may have flushed copper and tailings out of the pots, compromising the pot copper and tailings concentrations. This was often observed in silty tailings treatments where slow infiltration rates caused water to drain along pot walls and out of pots.   Photo 3-4 Dark greenish blue copper sulfate evaporites found on greenhouse soil surfaces 27  Substrate surface evaporation became more apparent as the experiment progressed. Dark greenish blue copper sulfate evaporites were found on soil surfaces of copper-spiked treatments (Photo 3-4). Hydration may have been adequate for plant growth, but could not suppress the constant evaporation or upward movement of soil water. This resulted to a concentrated zone of copper sulfate evaporites along soil surface. Evaporites were found sparingly and were variable within pots and across treatments. Thus it is plausible that not all plants within the pot were exposed to equal concentrations of copper sulfate.   Dryness was also a major factor affecting emergence in sandy and silty tailings. With organic matter, the cation exchange capacities (CEC) and anion exchange capacities (AEC) help retain toxic metals, nutrients and water while remaining readily available to root absorption. The tailings Mount Polley Mine staff collected was free of debris; this reduces complexity and variability within tailings and thus results direct represent plants grown in tailings. At Hazeltine Creek, scoured forest soil in tailings-soil mixtures could potentially act as a soil amendment to plants.  Irrespective of copper concentration, results indicated that sandy tailings were a more favorable growing medium than silty tailings. The size distribution of larger particles in sandy tailings would allow greater aeration, porosity for drainage and distribution of pore sizes for moisture retention. Substrates with greater particle distribution would eventually settle to “random closed packing” where void spaces between particles were minimized as particles rested closely to one another (Appendix D  ). As particles stacked, smaller particles in adjacent layers accommodate spaces between larger particles. This form of packing has a much greater void fraction and packing density should not be confused with “hexagonal or cubic closed packing” of equal sized particles (Dullien, 1992). Once settled, voids from several layers of particles become pore spaces.  Silty tailings treatments generally became more self-compacted after each hydration until water could no longer infiltrate and it seeped and drained along pot walls. Since moisture is no longer absorbed, a hard crust was formed along the surface. The effects of surface crusting is shown in 28  the photo below where the harden crust became an impermeable barrier for plant establishment in silty tailings treatments. Surface crusting was commonly found along Hazeltine Creek with some plant germinating along cracks where moisture was present between crusts.    Photo 3-5 Lodgepole pine desication of hypocotyl hooks in silty tailings Seedlings in 50 (left) and 100 (right) percent silty tailings , seedlings showed early signs of emergence but as soon as surface dried, an impenetratable crust formed. Crusting could not be resolved with hydration, and thus seedlings undergoing early stages of emergence were unable to survive due to dessication (see arrows).   Lodgepole pine seedlings undergo epigeal germination where the seed radicle extends beneath the surface to establish and secure the plant before a hypocotyl is developed (Owens, 2006). The hypocotyl then lifts the seed above substrate surface to complete germination. As the surface dried, crusting occurred and substrate mechanical resistance increases beyond the seedling’s potential to bore through the surface. At this point, seedling survival was strictly dependent on surface water content. In silty tailings, water infiltrated poorly and often drained directly along pot walls. The photos above illustrate the process where the hypocotyl hook developed on the hard surface (left) and desiccated over time (right).   5mm 5mm 29  3.2.2 Bluebunch Wheatgrass Bluebunch wheatgrass emergence and survival patterns were similar to those of lodgepole pine (Figure 3-2). First emergence was observed in control pots as early as 2 days after seeds were sown. Survival in copper-spiked greenhouse soils was generally above 50 percent with minimal growth and foliage toxicity observed. Survival in sandy tailings was largely attributed to the species’ robust growing habit and drought tolerance. Bluebunch wheatgrasses undergo rapid germination to develop shoots and roots to improve resistance to stress for successful establishment (Kitchen & Monsen, 1994; McKell, 1972). Both adventitious and seminal roots were often well developed in sandy tailings and similar to those grown in copper-spiked greenhouse soils. In sandy and silty tailings, roots were generally loose and comonly found near the surface.  Compaction in silty treatments became more pronounced after each watering session. Surface crusting was also present with increasing concentrations of sandy and silty tailings. Particle size separation became more noticable as the experiment progressed in treatments with sandy and silty tailings. Emergence and survival in tailings were signficiantly less than those of greenhouse soils (p < 0.05) and generally between 20 to 30 percent irrespective of treatment concentration. Survival was consistent in sandy tailings and not in greenhouse soils due to the source of copper and exposure in substrates. Greenhouse soils were spiked with copper sulfate which was much more mobile and available than copper held in minerals in sandy tailings. Coupled with high evaporation which concentrates copper along soil surfaces (see Photo 3-4), seedlings in greenhouse soils were more exposed to copper species. No ill-health affects were observed in copper-spiked soil treatments while plant growth and foliage were severely reduced in treatments of sandy tailings. Despite a higher copper concentration, seedling survival in sandy tailings was largely attributed to larger particle sizes which allow adequate aeration and effective infiltration not found in silty tailings.  30   Figure 3-2 Bluebunch wheatgrass emergence and survival in greenhouse after six weeks  Copper spiked soils generally had over 40 percent survival with minimal effect from copper. The tailings however, proved to be an unfavorable environment for growth. Compared to greenhouse soil, sandy and silty tailings effectively inhibited emergence (p = 0; p = 0) and thus survival was largely reduced (p = 0.04; p = 0). As with lodgepole pine, both species had poor emergence in silty tailings and no emergence in 12.5 percent treatment.  Median and standard error displayed (n = 6).  The images below show bluebunch wheatgrass exposed to 0, 50 and 100 percent concentrations of sandy tailings for a period of 6 weeks. A reduction in blade thickness and quantity, root thickness and number of adventitous roots were observed between control and 50 percent sandy tailings. In 50 percent tailings, grass blades were less developed with thinner and darker green in color. Seedlings in 100 percent tailings did not increase in size after first two weeks of study. It is uncertain as to whether root thickness and adventitious root development was associated with chemical properties of sandy tailings or a result of larger pore spaces in larger grain size distribution in control silica sand.  0%10%20%30%40%50%60%70%80%90%100%Control 28mg/kg109mg/kg269mg/kgControl 12.5% 50% 100% 12.5% 50% 100%Copper-spiked Greenhouse Soil CleanSandSandy Tailings Silty TailingsPercent Emergence and Survival Bluebunch Wheatgrass Emergence & Survival (6weeks) EmergenceSurvival31   Photo 3-6 Bluebunch wheatgrass plant health comparison in sandy tailings Comparison of plant vigor and growth in control silica sand (left), 50 % sandy tailings (middle) and 100 % sandy tailings (right). Note the difference in thickness of grass blades and color and adventitious root development and root thickness in control compared to other treatments with sandy tailings. Grids in each picture are one by one centimeter for scale.  Longest lengths of above and below ground biomass were not measured due to variable emergence and survival. Samples were chemically analyzed to determine the likelihood of copper uptake in root and shoot after a six week exposure (Figure 3-3). Copper uptake was substantially greater in roots than in shoots of greenhouse soils (p = 0.00) and sandy tailings (p = 0.024). Root copper was expected to be two to three times higher in tailings than greenhouse soils as a result of direct exposure to in tailings than indirect exposure in greenhouse soils. These results illustrate the importance of organic matter content in growth mediums. The cation exchange capacity in organic matter influences the soil’s ability to retain nutrients, effluents and to buffer pH. Despite being spiked with copper sulfate, copper is retained by organic matter in greenhouse soils and less available for plant uptake. This effect was observed in root copper between 12.5 percent sandy tailings and near equivalent 268 mg Cu/kg greenhouse soil. In sandy and silty tailings, roots were directly exposed to copper and various metal species allowing greater metal uptake and accumulation over a similar period of growth. These results could resemble metal uptake in plants emergening from Hazeltine Creek if tailings were not removed. 32   Figure 3-3 Copper uptake in bluebunch wheatgrass in greenhouse after six weeks of growth  Copper uptake was substantially greater in roots than shoots of greenhouse soil (p = 0) and sandy tailings (p = 0.024). Copper translocation was also greater (p = 0.031) in shoots of sandy tailings as a result of higher exposure. The distribution of n decreases with respect to increasing copper concentration in soil (n=18, 14, 9, 3), however no significant effects were found. Root copper in 12.5 percent sandy tailings and 269 mg Cu/kg illustrate the importance of organic matter in metal retention as well as direct and indirect exposure to copper, respectively. It should be noted that error bars in 269 mg Cu/kg (equivalent to about 25 percent of sandy tailings) was likely a result of one contaminated sample. Median and standard error displayed; error bars only available for treatments with n > 1.  A series of principal component analysis (PCA) were generated by substrate and by root and shoot to aid the interprations of metal associations with respect to each other. The first two dimensions (Dim1 and Dim2) captured 80 and 60 percent of total variabilty in tailings and greenhouse soil samples, respectively (0, Figure 3-4). Two strongly correlated clusters of metals in roots (Cu, Al, Ca, Mg, Fe, Mn and Ni, Cd, Co, Cr) were observed. Caution should be used in interpretating these results as the primary cluster of metals shown in PCA Diagrams (Cu, Al, Ca, Mg, Fe, Mn) were abundant in minerals of tailings and in essential nutrients in fertilizer given frequently throughout the study. Similarly, clusters in greenhouse soil were predominantly macro and essential nutrients from fertilizer as some metals within the cluster were weakly correlated. Each PCA diagram also shows variability of individual treatment points in respective colored dots. Individual treatment points in greenhouse soils were fairly scattered yet mostly cluster 0100200300400500600700800900Control 28 109 269 0% 12.5% 50.0% 100.0% 12.5% 50.0% 100.0%Greenhouse SoilPositive Cu Control (mg/kg)SilicaSandSandy Tailings Silty TailingsCopper (PPM) Treatments Bluebunch Wheatgrass: Copper Uptake Six Week Harvest Shoots Roots Shoots & Roots33  around the origin (no correlation) while clusters of each treatment were scattered across the plot in sandy/silty tailings. Although copper was the main focus of this study, these results appear to imply that growth inhibitation or copper toxicity in bluebunch wheatgrass could be a result of multiple factors (or metals) in tailings.   Figure 3-4 Principal Component Analysis of metals in bluebunch wheatgrass shoots in sandy tailings in greenhouse after six weeks Dimensions 1 and 2 which account for 73 percent of total variabilty in dataset. Scattered individual treatment points with the larger representing the center of confidence ecllipses of the respective treatment. Two main clusters of metals roots (Cu, Al, Ca, Fe, Mg, Na and, Cd, Co, Cr, Ni) correlated to one another were present in both tailings and greenhouse soils. Remaining graphs can be found in 0.  Greenhouse soil treatments were also chemically analyzed to determine the overall effectiveness of the copper-spiking technique (Table 3-7). A 20 percent error range was found between actual and expected concetrations. Large discrepancies after the 6 week growing period were 34  uncommon as the distribution of copper was highly dependent on the upward or downward movement of water within the pot. Greenhouse soils were thoroughly mixed before sampling as the presence or absence of copper evaporites would falsely escalate or de-escalate the actual copper concentration in substrate.    Table 3-7 Chemical analysis of copper spiked greenhouse soils before and after six weeks of growth Median treatment values presented in with standard error in parentesis. Copper Spiked Greenhouse Soil Treatments (PPM) Treatment (% tailings equivalent) Experiment Start Experiment End Control 9.95 (2.70) 5.85 (1.24) 28 mg Cu/kg (2.5 % tailings) 28.95 (8.85) 28.48 (5.97) 109 mg Cu/kg (9.5 % tailings) 148.85 (23.72) 90.51 (29.44) 269 mg Cu/kg Soil (25 % tailings) 217.25 (38.54) 240.63 (88.36) Tap Water: 0.28 (0.01) PPM  Fertilizer: 0.43 (0.02) PPM   3.2.3 Wild Rose There was no germination of wild roses in any treatment at the end of the six week period. It is believed that field collected seeds obtained from Linnaea Nurseries were defective.   3.2.4 Summary The greenhouse experiment was designed to examine the chemical growth limiting properties of tailings under simulated natural growing conditions. Instead, results have ascertain that physical soil properties must be addressed before toxicity can be examined. Further experimentation was required to improve emergence and survival to further understand the potential for copper biotranslocation. Survival of lodgepole pine seedlings, for example, were highly variable within (Photo 3-7, left) and among treatments. After germination, seedlings continued to be extremely sensitive to substrate conditions.  35   Photo 3-7 Variable survival between and within treatments  Sandy tailings 12.5 percent (left), 50 percent (middle) and 100 percent silty tailings (right). Results were inconclusive, however, it was hypothesized that unfavorable substrate conditions was the main cause of dessication in treatment containing sandy or silty tailings.  The primary reason for choosing a coarse variety of silica sand as the control substrate was to mitigate hexagonal or cubic closed packing in treatments containing tailings. Mixed with sandy and silty tailings, coarse silica sand increases the grain size distribution and effectively increases interparticle spaces for aeration, infiltration and drainage. The effectiveness was quickly lost in sandy tailings due to frequent irrigation which disturbed and resettled grains and in silty tailings where surface crusting led to poor substrate infiltration. The confounding factor that led to poor emergence and survival, surface crusting and high evaporation was that this study treated all three substrates (sandy tailings, silty tailings, greenhouse soils) of different water-holding capacities as one substrate with one watering schedule. Fluctuating relative humidity in the greenhouse was not accounted for and thus caused the constant upward flux in substrate evaporation. As a result, copper sulfate evaporites were found in greenhouse soils and inconsistent and inconclusive survival patterns in tailings.   Apart from the uncertainties described earlier, this exploratory study formed a basis for a more controlled study where environmental conditions (humidity, temperature, lighting) could be standardized by using growth chambers. Furthermore, improved emergence and survival, 36  especially in experimental controls, would provide adequate above and belowground biomass for a more comprehensive chemical analysis. As such the next phase of experiments followed a more rigorous experimental method largely outlined by Environment Canada’s Biological Testing Methods (2007): test for measuring emergence and growth of terrestrial plants exposed to contaminants in soil.    37  Chapter 4: Environment Canada Compliant Growth Chamber Study  Results from the greenhouse study found a need for a more controlled approach to improve emergence and survival in all treatments including experimental controls and silty tailings. The shift to using growth chambers and Environment Canada (2007)’s toxicity testing methods would address confounding factors of low humidity (causing surface crusting) and substrate moisture (high evaporation) in a rigorous manner. Consistent humidity, temperature and lighting in growth chambers enable optimal growing conditions allowing a more comprehensive experimental design. Both particle size and copper concentration were examined using similar particle sized sandy and silty substrates spiked with equivalent copper concentration in tailings. These optimal growing conditions enable seedlings to generate adequate biomass for chemical analysis.  4.1 Growth Chamber Study Methods The objective of this growth chamber study was to investigate the physical and chemical growth limiting factors of sandy and silty tailings following Environment Canada’s Biological Test Methods (2007). These methods are universal guidelines designed to “measure and assess the toxicity of samples of field-collected soil, biosolids, sludge, or similar particulate material; or of natural or artificial soil spiked with test chemical(s) or chemical product(s)” on terrestrial plants under controlled laboratory conditions (Environment Canada, 2007, p v).   The Environment Canada compliant experimental design examined growth response of three species (lodgepole pine, bluebunch wheatgrass, and willow) to various substrates (sand, silt, soil) and copper concentrations. From seed, each species was subjected to 0, 10, 25, 50, 100 percent concentration of sandy and silty tailings as well as corresponding concentrations of copper in artificially spiked inert control sand and greenhouse soil. Environment Canada (2007) requires 4-6 replicates per concentration per species. The 42-day experiment used three growth chambers, one for each plant species, for three separate experiments. Table 4-1 summarizes the treatments and replication (total of 107 pots per species) applied in a completely randomized design for each experiment.  38  Table 4-1 Experimental design of 42-day growth chamber study  Environment Canada (2007) provides specific potting procedures for substrate preparation, potting, seed placement and environmental conditions. Both sandy and silty tailings were prepared following Environment Canada’s protocol for substrate preparation before potting. From the same shipment received for the greenhouse study, remaining sandy and silty tailings were air-dried in a fume hood for a period of two weeks and prepared separately to eliminate cross contamination during treatment preparation.   The edaphic effects of grain sizes were examined using a control substrate of similar grain size specific to sandy and silty tailings. This experiment utilized inert silica sand of grades LM #70 and LM #125 from Lane Mountain Company (Valley, Washington, USA) for sandy and silty tailings, respectively (Target Products Ltd., 2016). Sandy tailings had a median cumulative diameter between 0.063 to 0.5 mm at 50 percent median diameter (d50) which is close to 0.103 to 0.300 mm in LM #70. Similarly, LM #125 was found to have a similar d50 as silty tailings (0.004 to 0.25 mm). The table below provides a detailed distribution of particle sizes in both control substrates used in growth chamber experiments.       Treatment Sandy Substrate Silty Substrate Cu-spiked Greenhouse Soil Tailings Cu-spiked Sand Tailings Cu-spiked Silt Control (inert silica or soil) n = 5 n = 5  n = 5 each   10% Tailings | 115 mg Cu/kg n = 5 each n = 4  each n = 5 each n = 4 each   25% Tailings | 288 mg Cu/kg   50% Tailings | 575 mg Cu/kg 100% Tailings |1150mg Cu/kg Total Pots 107 Pots per species (Growth Chamber Capacity = 109 pots) 39  Table 4-2 Grain size distribution of control substrate Grain size distribution of inert silica sand from Lane Mountain Company (Valley, Washington, USA) used in all sandy and silty treatments. Cells highlighted in gray indicate dominant grain sizes corresponding to the mean cumulative diameter (d50) of tailings (Target Products Ltd., 2016)  Sieve Size (mm) LM #70 (Control Substrate Sandy Tailings) LM #125 (Control substrate Silty Tailings) Cumulative Percent Passing (%) Individual Percent Retained (%) Cumulative Percent Passing (%) Individual Percent Retained (%) 0.425 100 0 100 0 0.3 95-100 0-5 98-100 0-2 0.212 50-80 20-45 95-100 0-5 0.15 20-40 20-45 85-95 0-10 0.103 8-18 10-25 50-80 15-25 0.075 0-10 5-15 20-40 30-50 Balance NA 0-10 NA 20-40  Sand and silt sized varieties of Lane Mountain inert silica sand were used as a control substrate to prepare treatment concentrations of 0 (control), 10, 25, 50 and 100 percent for sandy tailings, Cu-spiked sand as well as silty tailings and Cu-spiked silt, respectively. While different in particle size distribution (d50), Lane Mountain silica sand is 99.56 percent weight of SiO2 (chemically identical to those used in the greenhouse study) and does not contain copper (Target Products Ltd., 2016).   Copper concentrations were 1130 and 805 mg/kg in sandy and silty tailings, respectively (ALS Canada Ltd, 2015). For ease of calculation and treatment creation, all positive control treatments (particle size and greenhouse soils) had a maximum of 1150 mg Cu/kg comparable to sandy tailings. Thus treatments of 10, 25, 50 and 100 percent copper had 115, 287.5, 575 and 1150 mg Cu/kg (Table 4-3). This study also compared 100 percent sandy and silty tailings with different proportions of tailings with no adjustment to copper concentration. Preparation of sandy tailings treatments were created based on percentage dry weight of Lane Mountain (LM #70) sand and sandy tailings as indicated in the table below. For example, a 10 percent treatment was composed of 10 percent sandy tailings and 90 percent silica sand by dry weight.  40  Table 4-3 Sandy tailings treatment by weight proportion  Treatment total, 6550 g includes an additional 100 g for end-point chemical testing. Once hydrated with fertilizer water, pots were filled half way or with approximately 500 mL(Environment Canada, 2007).  Sandy Tailings Treatment by Dry Weight Proportion in Grams (mg Cu/kg) Control (0 mg/kg) 10% (115 mg/kg) 25% (287.5 mg/kg) 50% (575 mg/kg) 100% (1150 mg/kg) Control Silica 430 387 322.5 215 0 Sandy Tailings 0 43 107.5 215 430 Total per Replicate  430 430 430 430 430  Once thoroughly mixed, each treatment (6550 g) was hydrated with fertilizer water to field capacity or optimal water-holding capacity; “a homogeneous crumbly consistency with clumps [macro-aggregates] approximately 3-5 mm in diameter” as described by Environment Canada (2007, p57). Literature estimates of field capacities of sand, loamy sand and sandy loam were 10, 12 and 18 percent of dry weight, respectively (Dane & Toppe, 2002; Saxton & Rawls, 2006). All sandy substrates were able to hold 20 percent of its dry weight or 1310 mL (Becker-van Slooten, Campiche, & Tarradellas, 2003; ESG International Ltd. & Aquaterra Environmental Ltd., 2002).  Silty tailings treatments were prepared in a similar manner as sandy tailings. The composition of each treatment was determined on a dry weight basis and brought to volume of 500 mL or half the pot’s height (Environment Canada, 2007). Literature estimates of field capacities of silt and loam were 30 and 28 percent dry weight, respectively (Dane & Toppe, 2002; Saxton & Rawls, 2006). Following Environment Canada (2007) guidelines, a linear relationship between field capacity and substrate distribution was derived from testing 100 percent LM #125 (control), 10 and 100 percent silty tailings treatments,  as water-holding capacities change with respect to the volume of silty tailings. These initial laboratory findings, specific to Environment Canada (2007) guidelines, were used to determine the precise volume of hydration for the treatment composition. Table 4-4 presents substrate composition of each treatment with the corresponding volume of hydration required. Treatments containing silty tailings were generally heavier due to greater particle density.  41  Table 4-4 Composition of silty tailings and control silica by dry weight (g) Treatment total, 7925 g includes an additional 100 g for end-point chemical testing. Once hydrated with fertilizer water, pots are filled half way or approximately 500 mL (Environment Canada, 2007).  Dry Weight Treatment Composition in Grams (mg Cu/kg) Control (0 mg/kg) 10% (115 mg/kg) 25% (287.5 mg/kg) 50% (575 mg/kg) 100% (1150 mg/kg) Control Silica Silt 520 468 390 260 0 Silty Tailings 0 52 130 260 520 Total per Replicate  520 520 520 520 520 Hydration 2377 mL 2294 mL 2162 mL 1941 mL 1500 mL  Lane Mountain silica sand (LM #70) and silt (LM #125) was used to prepare corresponding copper concentrations as found in sandy and silty tailings, respectively (Table 4-5). Twelve 430 and 520 g (dw)  of sandy and silty substrates, respectively, were first hydrated with fertilizer water, then spiked with copper sulfate pentahydrate (Fisher Scientific C493-500; 100% CuSO4-5H2O; ACS Certified) dissolved in 500 mL fertilizer water and then mixed with the remaining volume of 1032 and 1903 mL fertilizer water. This process was then repeated for each treatment. Treatments in greenhouse soils were created similarly in batches of fifteen 80 g (dw) replicates with 3000 mL of fertilizer water to reach near field capacity. Table 4-5 provides the weight of copper sulfate required to match corresponding copper concentrations in sandy tailings.  Table 4-5 Copper sulfate application to sandy and silty substrates and greenhouse soils Copper sulfate application varies by substrate dry weight. The corresponding weight of copper sulfate dissolved in 500 mL fertilizer water for twelve replicates over three species. Copper Sulfate Application CuSO4-5H2O Treatments (g)  Total Fertilizer Used (mL) Control (0 mg/kg) 10% Cu (115 mg/kg) 25% Cu (287.5 mg/kg) 50% Cu (575 mg/kg) 100% Cu (1150 mg/kg) Sandy Substrate (LM # 70) 0.00 2.37 5.92 11.84 23.68 1032 Silty Substrate  (LM # 125) 0.00 2.87 7.16 14.32 28.65 1902 Greenhouse Soils 0.00 0.63 1.58 3.16 6.32 3000  Clear one liter polypropylene containers were used for each replicate. These new disposable containers were thoroughly rinsed with distilled water before use. As recommended by 42  Environment Canada (2007), lids were placed on top of each pot (or “test vessel”) to maintain consistent humidity within each pot. Given the growth rates expected in bluebunch wheatgrass and willow, all pots were covered by a second inverted pot (or “test vessel”) instead of lids to increase growing space (J. Princz and K. Serben, personal communication, May 3, 2016). Containers were held together by tape with a 1< mm gap for aeration.  Once thoroughly mixed, treatments were placed into polypropylene containers with identical wet weight equivalent to an approximate volume of 500 mL, or half-filled container. Pots were filled to the respective substrate weight, smoothed (not compressed) by spoon and gently tapped against laboratory benchtop to settle (Environment Canada, 2007). Since sandy and silty substrates were near field capacity, potting was gentle as excessive tapping or vibration (disturbance to substrate structure) could cause liquefaction (fertilizer water separating from substrate).  Three species were planted in the growth chamber experiment: lodgepole pine (Pinus contorta), bluebunch wheatgrass (Pseudoroegneria spicata, also known as Agropyron spicatum), and wild willow (Salix scouleri). Lodgepole pine seeds were stratified in distilled water for 24 hours, dried and refrigerated at least 2 weeks before use (S. Reitenbach, personal communication, May 3, 2016). Bluebunch wheatgrass did not require stratification. Wild willow seeds were kept in a freezer below -10 oC until use.  Five seeds were sown in each pot with four seeds equally spaced around one seed in the center (Environment Canada, 2007). Each seed was sown at a depth twice its diameter and gently covered with surrounding substrate (Environment Canada, 2007). Each pot was sprayed three times with fertilizer water before pots were sealed by tape with a 1< mm gap maintained for aeration (Photo 4-1). The start and end dates for each substrate is listed below (Table 4-6). All three plant species were sown on the day substrates were potted, or Day 1 of experiment. Sandy substrates, silty substrates and greenhouse soils were potted on July 18th, 21st and 26th of 2016, respectively.  43   Photo 4-1 Pots in growth chambers at Day 1  Table 4-6 Growth chamber test start and end dates by substrate Each substrate was potted and seeded on the same day or Day 1 of experiment. Experiment Start Date End Date Total Exposure Sandy Tailings July 18, 2016 August 29, 2016 42 days Copper Spiked Silica Sand July 18, 2016 August 29, 2016 42 days Silty Tailings July 21, 2016 September 1, 2016 42 days Copper Spiked Silica Silt July 21, 2016 September 1, 2016 42 days Copper Spiked Greenhouse Soil July 26, 2016 September 6, 2016 42 days  Three Conviron E-15 growth chambers were used for the duration of the 42 Day experiment. Each growth chamber housed one species irrespective of growth medium. Following manufacturer’s lighting recommendations, each chamber used 16 Philips fluorescent lights and 10 Valoya G2 LED bulbs. The Philips fluorescent tube lights, F72T8 TL841 HO, were 72 inches long, eighth-inch diameter each delivering a high output of 65 watts. These lights provided cool white light, Color Code TL841 with a Color Rendering Index (Nom) of 86 (Bulbs.com, 2016). Valoya G2 7 watt LEDs provided up to 10 µmol/m2/s of strong red, far red and additional wide blue spectrums enhanced plant growth (Valoya, 2014). The light fluence rate in each growth chamber was approximately 300 ± 100 µmol/m2/s adjacent to soil surface (P. Smets, personal communication, December 12, 2016) as specified by Environment Canada (2007). 44  Conditions in each chamber were set according to Environment Canada Biological Test Method standards (2007). Day time conditions were 24 oC with 300 ± 100 µmol/m2/s or full lighting from 7:00 am to 10:00 pm. At night, temperatures dropped to 15 oC with no light. After 6:00 am, chambers were programed to become progressively warmer and brighter to meet daytime conditions by 7:00 am. Humidity was monitored and kept above 50 % throughout the duration of study. Conditions of day and night are outlined in Table 4-7.  Table 4-7 Environment Canada (2007) specified day and night growth chamber conditions After 6:00 am, chambers become progressively warm and bright to meet day time conditions. After 10:00 pm, chamber darkens and allowed to cool to night time temperatures. *Hand watering is only conducted by day, as required. Environmental Condition Day (7:00 am – 10:00 pm) Night (10:00 pm – 7:00 am) Air Temperature 24 ± 3°C 15 ± 3°C Lighting 300 ± 100 µmol/m2/s to soil surface No light Humidity >50% Watering Hand water as required* None  Fertilizer concentrates, prepared from University of British Columbia’s Greenhouse Facility, were kept separately at room temperature until use. The first concentrate contained calcium nitrate, ammonium, potassium nitrate, calcium chloride and iron diethylenetriamine pentaacetic acid (DTPA; a chelating agent) (M. Biron, personal communication, January 19, 2016). The second concentrate contained magnesium sulfate, bicarbonate, micronutrients and nitrogen:phosphorus:potassium mixes (M. Biron, personal communication, January 19, 2016). Equal parts of concentrates (7 mL each) and bicarbonate (7 mL) were mixed into 1 L of distilled water in an alternating fashion of distilled water and concentrate to avoid co-precipitation of minerals such as calcium carbonate. A stock solution of fertilizer water was made before substrate preparation and re-made in 30 liter batches thereafter to maintain consistent pH at 5.5-5.8 and electric conductivity at 1.6-1.7 S/m throughout the experiment. A summary of analysis of anions, cations and trace elements in fertilizer water can be found in Appendix C   Chemical Analysis of Greenhouse Soils and Fertilizer.  45  Pots were inspected every other day for adequate substrate moisture throughout the 42-day growing period. Each pot was hand sprayed with fertilizer water to near saturation or when water was observed temporarily pooling at the bottom of the pot (Environment Canada, 2007). Pots were shuffled from center outwards such that each pot was exposed to slight variations (if present) of humidity, lighting, temperature and ventilation inside the growth chamber (Environment Canada, 2007). For example, when all sandy tailings and copper-spiked silica sand pots were taken out for hydration, the remaining pots were randomly placed along the chamber walls making room for the newly hydrated pots in the middle. This rotation was conducted throughout the 42-day growth period.  All pots were harvested on the 42nd growth day. Plants were carefully extracted and separated by hand. Extraction of lodgepole pine seedlings was relatively straight forward as roots were often short and not intertwined. Bluebunch wheatgrass, however, had a complex root system intertwined with other conspecific plants within the pot. Although a lengthy process, significant attention was required to avoid severing roots leading to incorrect root length representation. Once separated, each plant was individually washed with distilled water and dried with paper towel until all visible substrates were removed. This delicate process was conducted with precision to avoid loss of fine root hairs (the points of metal absorption). Several rinse cycles were needed for plants grown in tailings to remove all particles stuck at the transition point between shoots and roots and along the root itself. In artificial soils, roots would often penetrate though calcium carbonate and larger peat moss pieces, which required manual separation to remove all organics from root tissue. This was a critical step in preparation for metal analysis as any loose particle stuck in roots would cause an erroneous reading and a loss of that pot’s metal uptake data.   Longest shoot and root lengths of each surviving plant were measured, then separated by razor and compiled to their respective pot (as shown in Photo 2-1). Due to the small amount biomass present after 42 days of growth, composite samples were required to ensure adequate dry biomass available for analysis testing. Thus, each treatment would have five shoot replicates corresponding to five root replicates. 46  Samples were placed in 50 mL volumetric flasks and oven dried at 105 oC overnight (over 12 hours) to reach consistent dry weight before cooling back to room temperature in desiccators. Unlike the conventional drying method of 90 oC (Aquaterra Environmental Ltd. & ESG International Ltd., 2000), samples were oven dried at 105 oC as they were housed in glass volumetric flasks instead of paper bags. The lack of sufficient biomass for analysis in some samples led to the decision of directly using volumetric flasks to store each sample at the beginning of the experiment until samples were filtered into ICP-OES test vessels. This prevented the introduction of experimental error by loss of plant tissue and metal counts during each vessel transfer. Given enough time to aerate, the 50 mL volumetric flasks enabled adequate wet digestion compared with beakers.    Wet ashing or rapid wet digestion, is a procedure to achieve organic decomposition in relatively low temperatures to minimize volatile loss of elements (Government of B.C., 2015; Pequerul, Perez, Madero, Val, & Monge, 1993). Pequerul (1993)’s rapid wet digestion begins with 5 mL of concentrated nitric acid and 4 mL of (33%) hydrogen peroxide, gently stirred and allowed to oxidize at room temperature for one hour. Samples were heated on a hot plate at approximately 60 oC where strong effervescence of dense brown fumes was produced. At this time, amber fumes turned clear and volumetric flasks were left to cool back to room temperature. To ensure adequate digestion, a second round of 4 mL hydrogen peroxide was added and samples were reheated to 60 oC before final cooling. In absence of nitric acid, effervescence does occur; however, fumes are generally transparent to light amber in color. At room temperature, 5 mL of concentrated hydrochloric acid was added to each sample (to dissolve any remaining recalcitrant organics) and diluted up to 50 mL with 4 percent nitric acid as solution matrix for ICP-OES. Using Whatman 2 filter paper, the filtrate was collected and refrigerated until ICP-OES analysis.  Results from the greenhouse study have indicated significantly low concentrations of metals observed in samples after 6 weeks of growth. Thus ICP-OES standards were adjusted to fit the range of concentration in samples. In addition to the 16 metals analyzed in greenhouse study (Cu, Al, Ca, Cd, Cr, Co, Fe, K, Mg, Mn, Na, Ni, P, Pb, Si, Zn), selenium and vanadium were also analyzed. A full calibration or re-run of machine blank and standards 1-10 was conducted 47  every 50 samples with a reference standard, NBS tomato leaves 1573 (Gladney, 1987) and procedural or laboratory blanks analyzed every 25 samples to test instrument response and detection consistency. Concentrations of eighteen metals in each standard and reference standard used are listed in the Table 4-8.   48  Table 4-8 Elemental concentrations of blanks, experimental standards and standard reference material used for inductively coupled plasma optical emission spectrometry (ICP-OES) Standards were identical to those used in the greenhouse experiment; selenium and vanadium standards were made separately and thus solely present in Standards 6-10. NBS 1573 Tomato Leaves, USA H.S. Jun 1986 (Gladney, 1987), were used as reference material to ensure accuracy of machine detection particularly to copper. Values are shown in parts per million (PPM) with the exception of Ca, Co, K and Se. *values compiled from consensus data, but not NBS certified. Metal Concentration in Standards (PPM)  Al Ca Cd Co Cr Cu Fe K Mg Mn Na Ni P Pb Se Si V Zn Blank 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 std 1 2.5 1.5 0.05 0.05 0.1 0.1 2.5 0.5 1.5 0.2 0.5 0.1 0.5 0.1  2.5  0.2 std 2 6.25 3.75 0.125 0.125 0.25 0.25 6.25 1.25 3.75 0.5 1.25 0.25 1.25 0.25  6.25  0.5 std 3 12.5 7.5 0.25 0.25 0.5 0.5 12.5 2.5 7.5 1 2.5 0.5 2.5 0.5  12.5  1 std 4 25 15 0.5 0.5 1 1 25 5 15 2 5 1 5 1  25  2 std 5 50 30 1 1 2 2 50 10 30 4 10 2 10 2  50  4 std 6               0.01  0.1  std 7               0.05  0.5  std 8               0.1  1  std 9               0.5  5  std 10               1  10  Tomato Leaves NBS 1573 1200 3.00 ±0.03 % 3 600   ng/g 4.5  ±0.5 11  ± 1 690  ± 25 4.46  ± 0.03 % 7000 238 ± 7 *470 ± 110  *1.3 ± 0.2 3400 ± 200 6.3 ±0.3 *54  ± 6 ng/g *3000 *1.2 ± 0.2 62 ± 6 49  Replicates of each treatment were also randomly selected and re-analyzed as duplicates to ensure instrument consistency. Relative percent difference (RPD) is the percentage of differences between sample and duplicate result divided by the average of the two results. Median RPD in each treatment was calculated to determine the precision of machine detection in the table below.  Table 4-9 Median relative percent difference in treatments after ICP-OES analysis of copper  Relative Percent Difference Sandy Tailings Silty Tailings Greenhouse Soil Lodgepole Pine Shoot 11 % 12 % 21 % Lodgepole Pine Root 3 % 3 % 17 % Bluebunch Wheatgrass Shoot 3 % 2 % 2 % Bluebunch Wheatgrass Root 4 % 2 % 6 %  A replicate from each treatment was also randomly selected and re-analyzed (duplicate) to ensure instrument consistency. Samples containing less than 0.01 g of dry weight biomass were processed but removed from final results due a high level of experimental uncertainty. The analytical detection limit for copper and vanadium was 0.1 and selenium was 0.2 ppm (mg/kg dw) (M. Soon, personal communication, November 28, 2016).   The rapid wet digestion method was also used to determine available copper in all substrates (Table 4-10). Digestive methods should remain consistent such that the digestive power to release chemical species in plant material is equivalent to that used on substrates. Strong digestive methods such as ammonium oxalate or aqua regia was avoided as either method would provide total substrate copper and release additional chemical species otherwise not relevant to this study.   Table 4-10 Expected and actual concentrations of copper in substrate by treatment  Substrate –  Treatment Expected Copper Concentration (ppm) Actual Copper Concentration (ppm) Sandy Tailings – Control 0 % 0.0 2.61 Sandy Tailings – 10 % 113.0 119.25 Sandy Tailings – 25 % 282.5 278.50 Sandy Tailings – 50 % 565.0 662.87 50  Substrate –  Treatment Expected Copper Concentration (ppm) Actual Copper Concentration (ppm) Sandy Tailings – 100 % 1130.0 1252.71 Sandy Substrate – 10 % 115.0 121.54 Sandy Substrate – 25 % 287.5 275.49 Sandy Substrate – 50 % 575.0 574.72 Sandy Substrate – 100 % 1150.0 1198.85 Silty Tailings – Control 0 % 0.0 4.28 Silty Tailings – 10 % 80.5 85.95 Silty Tailings – 25 % 201.2 211.07 Silty Tailings – 50 % 402.5 447.58 Silty Tailings – 100 % 805.0 888.52 Silty Substrate – 10 % 115.0 123.94 Silty Substrate – 25 % 287.5 304.52 Silty Substrate – 50 % 575.0 575.55 Silty Substrate – 100 % 1150.0 1343.83 Greenhouse Soil - Control 0 % 0.0 33.83 Greenhouse Soil – 10 % 115.0 No sample  Greenhouse Soil – 25 % 287.5 549.79 Greenhouse Soil – 50 % 575.0 812.73 Greenhouse Soil – 100 % 1150.0 1740.22 Note: n=1 for all mineral samples, n=2 for all greenhouse soil samples  The Kruskal Wallis test in SPSS software program was used to determine statistical significance in data as outlined in Chapter 2. Medians and standard errors were presented in all figures. Principal component analysis (PCA) diagrams of eighteen metals analyzed (Cu, Se, V, Al, Ca, Cd, Cr, Co, Fe, K, Mg, Mn, Na, Ni, P, Pb, Si, Zn) were generated by plant species, by substrate and by shoot and root to determine metal correlation and trends for further study. In each of these diagrams, the R-values graphically represent metal correlation by color while metal relationship strengths (p values) were shown by degree of shading. See Appendix F   Principal Component Analysis Diagrams (PCA).  51  4.2 Results and Discussion for Growth Chamber study Following Environment Canada’s Biological Test Methods (2007), the growth chamber experiment was designed to further isolate and identify potential growth limiting factors in Mount Polley Mine’s tailings. This 42-day Environment Canada-compliant study tested three species (lodgepole pine, bluebunch wheatgrass and wild willows) potted on sandy tailings and sandy substrates, silty tailings and silty substrates, and greenhouse soils on July 18th, 21st and 26th of 2016, respectively (Table 4-6).   Environment Canada’s Biological Test Method (2007) requires 14 to 21 days of exposure to determine species emergence and survival. The data were collected on the 42nd day in this study as additional growing time was required to generate adequate biomass for chemical analysis. Environment Canada’s criterion for validating each test was only listed for specific species of common vegetable plants and cereal grasses to determine substrate toxicity since copper sulfate is a common effective herbicide in agriculture. None of these species listed were applicable to the goals and objectives of reclamation in the Cariboo Region of Central British Columbia. Furthermore, it would be neither realistic nor beneficial to use vegetable plants and cereal grasses to determine tailings toxicity and form the basis of an ecological risk assessment report. Thus test validation criteria were considered as guidelines for regional (lodgepole pine, wild willows) and reclamation (bluebunch wheatgrass) species. Results and discussion are presented by species in the following order: lodgepole pine, bluebunch wheatgrass and wild willows.  4.2.1 Lodgepole Pine Lodgepole pine emergence and survival were both over 95 percent across all treatments of tailings and greenhouse soils (Figure 4-1). In tailings, copper species were held tightly in sulfide and non-sulfide minerals due to low leachability of metals in tailings (SRK Consulting (Canada) Inc., 2015). In greenhouse soils, high electron affinity between copper and organic matter allows strong adsorption and thus limited mobility of copper species reduces seedling exposure to copper toxicity (Adriano, 1986).  Healthy foliage was observed in all other surviving seedlings; none of which showed signs of toxicity in foliage. Emergence and survival was significantly affected in sandy and silty substrates where the absence of organic matter results in direct 52  exposure to aqueous copper sulfate. Seedlings in sandy and silty substrates were prone all mechanisms of copper uptake (mass flow, diffusion, root extension).   Figure 4-1 Lodgepole pine emergence in growth chamber after 42 days Emergence was most affected when seeds were directly exposed to copper (spiked sandy, p = 0.004 and silty substrates, p = 0.001; n = 4). No significant effect was observed in remaining substrates (n = 5). Experimental controls for sandy and silty tailings were also shared with copper-spiked sandy and silty substrates. Median and standard error displayed.  For test validation, Environment Canada (2007) requires results to meet specific mean emergence and survival rates and exceed minimum root and shoot lengths in all negative soil controls. Criterion for validation is species specific; most of these were agricultural species not applicable or close to the genus Pinus and thus are expected to be considered as general guidelines. The only generic criterion applicable which this experiment met is mean survival of emerged seedlings exceeding 90 percent (at test end) in all negative control soils (Environment Canada, 2007). Thus the lodgepole pine experiment is Environment Canada compliant.   0%10%20%30%40%50%60%70%80%90%100%Control010%11525%287.550%575100%1150Emergence (%) Percent Tailings,Substrate Copper Concentration (mg/kg dw) Lodgepole Pine Seedling Emergence Sandy TailingsSilty TailingsSpiked Greenhouse SoilSpiked Sandy SubstrateSpiked Silty Substrate53   Figure 4-2 Lodgepole pine survival in growth chamber after 42 days Greatest reduction in copper-spiked sandy and silty substrates as a result of suspected osmotic burning from copper sulfate compared to controls (p = 0.002 and p = 0.001 respectively; n = 4). Median and standard error displayed.  Median seedling survival was 100 percent in sandy and silty tailings and copper-spiked greenhouse soils (Figure 4-2). The effective concentration (EC) of direct exposure to copper sulfate was found in copper-spiked sandy and silty substrates (not in tailings). Compared with the same controls as counterparts in sandy and silty tailings, emergence dropped to 20 and subsequently below 5 percent in spiked sandy and silty substrates; an indicator that lodgepole pine seedlings would not survive in direct exposure of 115 mg/kg of copper sulfate in substrate solution. This effect was not observed in any treatment containing tailings as the copper species were probably held tightly in crystal lattices of copper bearing minerals. Poor emergence and impaired growth were found in copper-spiked sandy and silty substrates. Critical values of copper sulfate stress, growth depression and toxicity could not be found in literature for lodgepole pine emergence and survival. However, forestry research on copper sulfate fertilizer foliar applications on lodgepole pine have consistently reported copper toxicity occurring when foliar copper concentrations exceed 17 parts per million (Lozano & Morrison, 1981; Majid, 1984; Majid & Ballard, 1990; Reuther & Labanauskas, 1966; Van Lear & Smith, 1972).  0%10%20%30%40%50%60%70%80%90%100%Control010%11525%287.550%575100%1150Survival (%) Percent Tailings, Substrate Copper Concentration (mg/kg dw) Lodgepole Pine Seedling Survival Sandy TailingsSilty TailingsSpiked Greenhouse SoilSpiked Sandy SubstrateSpiked Silty Substrate54  Two outcomes were found in both copper-spiked sandy and silty substrates: no emergence or emergence with visible growth stresses from copper toxicity (Photo 4-2). In the first scenario, seedlings began to emerge but were unable to generate adequate growth over the 42 days. Found across all treatments, seedlings either appeared unchanged or in some cases had developed seed radicle/hypocotyl hooks less than 3 millimeters in length. Copper sulfate evaporites were found on seed surfaces in higher treatments containing 575 and 1150 mg/kg copper sulfate. Emergence, exceeding 3 millimeters in growth, was commonly found in the lower 115 and 287.5 mg/kg treatments. In the second scenario, emerged seedlings grew up to 1 centimeter long, appeared dry (dark green color) and developmentally impaired compared to healthy counterparts. Hypocotyl hooks were stunted and often grew inverted (upwards) appearing to minimize substrate contact. Pre-mature needle development was evident, but the needles were often found remaining in the seed coat at the end of the 42-day period. No roots developed.   Photo 4-2 Emergence scenarios of copper-spiked sandy and silty substrates No emergence or emerged with visible growth stresses, likely from copper toxicity, in all treatments of coper-spiked sandy (left) and silty (right) substrates. Most growth was found in 10 percent treatments, as shown, with dark green stunted development of seed radicle/hypocotyl hooks (less than 3 mm length) and premature needle development compared to healthy seedlings in experimental controls. Osmotic burning from high salt index of copper sulfate may have impaired growth.  Both outcomes can be explained by the high salt index of copper sulfate (Tisdale & Nelson, 1975) which results in foliage scorching, also known as “osmotic burning” (Miller & Young, 55  1976). Osmotic burning occurs when cells rupture due to significant differences in osmotic pressure acting on cell walls from concentrated solutions such as copper sulfate outside the cell (Majid, 1984; Majid & Ballard, 1990). Under normal conditions, the cell wall exchanges fluids out as fertilizer solutes move in to equalize pressure. In this study, it is likely that differences in osmotic pressure occurred upon germination. A large flux of solutes moved into the cells more quickly than fluids exiting. This causes an imbalance in pressure and cells rupture. Osmotic burning may thus have led to considerable damage in the emergence and development of lodgepole pine seedlings in copper-spiked sandy and silty tailings. In lower concentrations, seedlings may have emerged stunted with less foliage scorching, whereas osmotic burning may have inhibited any form of emergence in higher copper sulfate treatments. Furthermore, Franke (1976) noted that urea, which occurred in trace volumes in fertilizer used throughout the experiment, can improve permeability of solutes into cells.  Surviving seedlings in sandy and silty tailings and copper-spiked greenhouse soils were extracted for measurements of longest above and belowground lengths, dry weight biomass and chemical analysis. Visually, foliage of surviving seedlings was healthy and did not differ between the control (left) and 1150 mg/kg (right) treatments for all three substrates (Error! Reference source not found.; Photo 4-4). Growth within each pot was generally consistent; however, seedlings developed less in treatments containing 1150 mg/kg copper or 100 percent tailings.   Photo 4-3 Lodgepole pine development in silty tailings Increasing concentration of silty tailings; from left to right, 0, 10, 25, 50, 100 percent tailings.  56   Photo 4-4 Lodgepole pine development in copper-spiked greenhouse soils Increasing concentration of copper-spiked greenhouse soils (bottom); from left to right, control, 115, 287.5, 575 and 1150 mg Cu/kg.  Longest length measurements, defined by Environment Canada (2007), were measured from the soil level to the furthest extent of the plant’s above and belowground development. Significant reductions in shoots and roots were found in sandy and silty tailings (p = 0.013, p = 0.012; p = 0.021, p = 0.034) respectively. Lodgepole pine roots and shoots in sandy and silty tailings declined slightly with increasing concentrations of tailings (Figure 4-3). Shoot and root lengths in copper-spiked greenhouse soils were not affected by the volume of copper in soil (p > 0.05).    Figure 4-3 Lodgepole pine longest shoot and root lengths in growth chamber after 42 days -15.0-12.5-10.0-7.5-5.0-2.50.02.55.0Control010%11525%287.550%575100%1150Lengths (cm), 0 = Soil Level Percent Tailings, SubstrateCopper Concentration (mg/kg dw) Lodgepole Pine Longest Shoot and Root Lengths Sandy-ShootSandy RootSilty-ShootSilty-RootSoil-ShootSoil-Root57  Significant reduction was found in shoots and roots of sandy and silty tailings (p = 0.013, p = 0.012; p = 0.021, p = 0.034) respectively. Between control and 100 percent tailings, shoots were shortened by 25 percent and roots by half in sandy tailings (p = 0.011). Seedlings were not affected in copper-spiked greenhouse soils. Median and standard deviations displayed. Detailed statistical values can be found in Appendix E.1  Belowground biomass followed a similar pattern where roots were longest in sandy tailings, followed by silty tailings and lastly greenhouse soils. Most significant was between control and 100 percent sandy tailings where shoots were shorted by a quarter and roots by half (p = 0.011). These results could be early signs of metal stresses. Although not specific to lodgepole pine, aluminum accumulation in roots has shown significant inhibitory effects on root growth in distantly related masson pine (Pinus massoniana) (Zhang et al., 2014). Foliar growth has been less effected by aluminum as it is maintained by reliance on a constant supply of fertilizer water to generate adequate foliage. Alternatively, copper concentrations may have hindered growth rates in lodgepole pine; however, severity was not observed due to the short period of exposure. Seedlings in spiked greenhouse soils were not different to control. Organic matter may have limited uptake because copper species are held tightly on exchange sites. Additionally, organic matter naturally retains more water, thus moderating pot conditions and improving aeration.  Longest length, defined by Environment Canada (2007), was measured from the soil level to the furthest extent of the plant’s above and belowground development. The concern with this method of comparison is that it ignores the lateral shoot or root development of each plant. While Environment Canada (2007) only requires a growing period of 14 to 21 days, not all plants can generate adequate biomass for chemical analysis. Doubling the growing period, as in this experiment, would require the five seedlings to develop different rooting strategies to compete for space and resources within the pot. Assuming survival was not affected; seedlings may develop lateral root systems causing a reduction in average root length per pot (Photo 4-5). For instance, the seedling on furthest left had lateral roots nearing the length of its tap root. Measured to the nearest millimeter, a minimal reduction changes the relative difference observed between concentrations and does not properly illustrate the effects, if any, from copper toxicity.  58   Photo 4-5 Lodgepole pine seedlings from greenhouse soil experimental control pot Most seedlings first developed long tap roots while others (left) developed complex lateral root systems in early stages of growth to mitigate resource and space competition. This reduced the average longest root length measured per pot.  A similar trend was found when comparing total biomass (dry weight) of surviving seedlings per pot (Figure 4-4). In this experiment, shoots and roots of mineral soils (sandy and silty tailings; p < 0.005) were more productive than greenhouse soils as nutrients were readily available. Unlike organic soils, mineral soils (sandy and silty tailings) have low cation exchange capacities and thus unable to retain either copper species or nutrients for root uptake. Notwithstanding a higher copper content and possibly copper uptake, total biomass production in sandy tailings was greater than silty tailings and ultimately greenhouse soils (p < 0.005). The larger distribution of grain sizes in sandy tailings resembles a hydroponic environment in most of its treatments than a normal soil system. In 100 percent sandy tailings, total biomass was substantially reduced (p < 0.008, compared to control) largely attributed to the concentration of copper. A detailed list of p values between and among treatment can be found in Appendix E  Lodgepole Pine p values from 42-Day Growth Chamber Study.  59   Figure 4-4 Lodgepole pine total biomass in growth chamber study after 42 days The effects of substrate physical characteristics were well captured in the growth and development of seedlings among the three substrates. As the study largely resembles a hydroponic system, organic soils naturally retain toxins, nutrients and water in substrate. Thus greater productivity is expected in tailings and particularly sandy tailings (p = 0.013, n = 5) as larger particle distribution further enhances root aeration, infiltration and drainage which were lacking in silty substrate. Significant differences were also found between control and 100 percent sandy tailings (p = 0.008, n = 5). Median and standard deviation displayed.  Unlike the greenhouse study which simulated natural conditions, the growth chamber study optimized growing conditions to foster growth in all substrates and alleviate the sowing factor of 2.18 seeds per successful seedling (Owens, 2006). This allowed us to study copper toxicity and particle size separately. Although tailings were diluted with inert sand matching their respective distribution of particle sizes (d50), the likelihood of “hexagonal close packing” were less prominent as pots were smaller and less disturbed between irrigation. Furthermore, constant humidity prevented surface crusting. Greater biomass was expected in sandy tailings as larger particle sizes improve infiltration and root aeration. Among the growth characteristics tested, significance between substrates was predominantly found in roots where seedlings were most influenced by physical and chemical differences of each substrate.   0.000.020.040.060.080.100.120.14Control010%11525%287.550%575100%1150Dry Weight (g) Percent Tailings, Substrate Copper Concentration (mg/kg dw) Lodgepole Pine Total Biomass Sandy TailingsSilty TailingsSpiked Greenhouse Soil60  Table 4-11 Significant difference in growth characteristics between substrates Significance Among Characteristics Treatment  Significance  (p < 0.05) Between Substrates (p < 0.05) Sandy Tailings - Silty Tailings Sandy Tailings - Greenhouse Soil Silty Tailings - Greenhouse Soil Shoot Biomass 0.000  0.000 0.004 Root Length 0.000 0.001 0.000  Root Biomass 0.000 0.003 0.000 0.009  Plant growth in fine particles (silty tailings) was far more complex as the physical characteristics render several growth limiting factors. In the greenhouse study, plants were unable to establish in higher concentrations of silty tailings due to surface crusting. As described in the previous chapter, surface crusting forms when humidity is lost and substrates harden, forming an impenetrable surface. Once crusted, substrates no longer return to initial moisture contents with watering as poor infiltration results in surface flow and drainage along pot walls and drainage holes at the bottom of the pot. The growth chamber study used inverted pots as lids to maintain consistent humidity and moisture in the climate controlled setting while providing adequate space for plant growth (J. Princz and K. Serben, personal communication, May 3, 2016).  Despite various root copper concentration, foliage appear to be healthy and did not show any sign of phytotoxicity. The effects of growth reduction did not occur until after 50 percent treatments of sandy and silty tailings (Figure 4-3; Figure 4-4) while no effects were found in greenhouse soils. Efficient and effective seedling growth in sandy and silty tailings is largely due to the ease of access to nutrients, water and compaction for stability (Blouin, Schmidt, Bulmer, & Krzic, 2008) resembling a hydroponics environment otherwise not possible in natural conditions (greenhouse study) or at Hazeltine Creek. The challenge in comparing seedling growth between two different substrates using a universal procedure is that substrates respond differently over time. In tailings, seedlings may face increased exposure to copper from fertilizer pH induced leaching and chalcopyrite oxidizing bacteria (Murr & Berry, 1976) while organic matter decomposition may increase bacteria, fungi, molds and protozoa competing against seedlings for nutrients. Although pots were frequently checked and cleaned, handful of seedlings were lost due to the smothering growth of algal and fungal (Photo 4-6).    61    Photo 4-6 Nutrient competition from algal, mold and fungi  Some seedlings became susceptible the smoothening growth of algal, mold and fungi in nutrient rich greenhouse soils as indicated in colored polygons.  In contrast, excess moisture in silty tailings can result in inadequate aeration or anaerobic environment as air exchange was limited due to closed packing. Unlike sandy tailings (that contain larger particle sizes and ultimately larger pore spaces), water holding capacities differed due to the distribution and proportion of particle sizes in each treatment. In excess moisture, the slightest vibration or changes in substrate (soil) structure lead to liquefaction. A significant linear relationship (R2 = 0.99) was found between water-holding capacity of three treatments 0 (silt sized sand control, LM #125), 10 and 100 percent silty tailings and the pot weight (Figure 4-5).  62   Figure 4-5 Particle size and water holding capacity in silty tailings treatments A linear model from water holding capacities of control, 10 and 100 percent tailings were used to determine water holding capacities of 25 (green) and 50 (purple) percent silty tailings treatments. The three data points generated a linear trendline (y = -0.1113x + 2382.3) with an R2 value of 0.9999.  Results from chemical analysis of copper were displayed in Figure 4-6. Several trends were observed in roots and copper transfers to shoots. In particular, uptake was most pronounced in sandy tailings (p = 0) with 1524 ppm at 50 percent treatment (p = 0.001) but decreased to 937 ppm in 100 percent treatment as a result of low biomass (p = 0.008) and shortened roots (p = 0.011) compared to control. This may have resulted in 67 ppm or twice the copper transferred to shoots than other treatments (p = 0.001). Silty tailings followed a similar trend with a third less copper in roots (p = 0.008) and similar in shoots compared to sandy tailings. No effects were observed in copper-spiked greenhouse soils, however shoot copper were similar to those of silty tailings. It should be noted that samples below 0.01 g dry weight were chemically analyzed, but excluded in calculations due to a high level of experimental uncertainty; in particular, 0 and 287.5 mg Cu/kg greenhouse soil treatments were based on 1 of 5 root sample replicates.  Control 10% Tailings 100% Tailings 0500100015002000250030000 1000 2000 3000 4000 5000 6000 7000 8000 9000Water (mL) Weight of Silty Tailings (g) Silty Tailings: Bulk Waterholding Capacity by Treatment  63   Figure 4-6 Copper uptake in lodgepole pine seedlings in growth chamber after 42 days Significant uptake observed in shoots and roots of sandy (p = 0.001; p = 0.0) and silty (p = 0.008; p = 0.008) tailings; no effects observed in greenhouse soils. Minimum replicate tissue weight of 0.01 g is required for adequate chemical analysis; only one of five root sample replicates in 0 and 287.5 mg Cu/kg greenhouse soil treatments. Note the scale changes in copper ppm from above ground (every 25 ppm) to belowground (every 200 ppm). Median and standard error displayed; error bars only available for treatments with n > 1. See Appendix E.1 for summary of all p values.  Copper translocation in tailings was generally under 30 ppm in all substrates. Root to shoot transfer ratios appear to be fairly consistent after 10 percent tailings or 115 mg Cu/kg in spiked -900-850-800-750-700-650-600-550-500-450-400-350-300-250-200-150-100-50050100-1800-1600-1400-1200-1000-800-600-400-2000200Control010%11525%287.550%575100%1150Copper PPM (negative values imply belowground) Percent Tailings & Substrate Copper Concentration (mg/kg) Lodgepole Pine Copper Conc. (PPM) Sandy Tailings - Root Silty Tailings - Root Greenhouse Soil - RootSandy Tailings - Shoot Silty Tailings - Shoot Greenhouse Soil - Shoot64  greenhouse soils (Table 4-12). These results are indicative of the following: copper transfer may not be associated to quicker growth rates as found in tailings; and high root copper tolerance. Since test duration was limited to 42 days, no sign of phytotoxicity was observed. Past studies have determined copper toxicity in lodgepole pine occurs when foliar concentrations exceed 17 ppm, however, this value was determined with full grown and established lodgepole pine trees and not from emerging seedlings nor copper rich substrate (Lozano & Morrison, 1981; Majid, 1984; Majid & Ballard, 1990; Reuther & Labanauskas, 1966; Van Lear & Smith, 1972). Furthermore, chemical analysis of shoots in this study includes all aboveground biomass. Thus foliar copper concentrations were not examined.   Table 4-12 Median root to shoot copper translocation ratio The lower the ratio the greater the transfer from root to shoot. Lodgepole Pine Copper Translocation: Median Root to Shoot Ratio Substrate Treatment (mg Cu/kg) Control 0 10% 115 25% 287.5 50% 575 100% 1150 Sandy Tailings 2.73 25.01 45.03 45.03 48.09 Silty Tailings 1.80 18.11 35.74 35.74 25.12 Greenhouse Soil 4.55 5.38 9.40 9.40 13.75  Golder Associates Ltd. (2016), reported that tailings were enriched in selenium (1.18; 1.03 ppm) and high in vanadium (187; 202 ppm) in both sandy and silty tailings, respectively (Appendix G). Selenium was highly variable across all substrates with each substrate exceeding the aquatic freshwater and marine life guideline of 2 ppm (Ministry of Environment, 2014). No significance was observed in roots and shoots of all three substrates. High variability could be a result of ICP-OES interference as detection was limited to one wavelength (196.026 nm) in chemical analysis. Experimental error may also be present as selenium was not detected in most roots replicates (0 ppm) but highly in shoots of those samples. Selenium speciation was not reviewed in this study and thus reasons for inconsistent selenium uptake could not be explained. Vanadium uptake did exceed the 50 ppm established by the British Columbia Water Guidelines (Ministry of Environment, 2017) for protection of marine aquatic life in roots of 50 (p = 0.028) to 100 (p = 0.002) percent sandy and silty tailings. Shoot transfer was generally below 5 ppm. Between substrates, chemical differences were mostly observed between sandy tailings (high abundance 65  of metals) and copper-spiked greenhouse soils where source of metals is only obtained from fertilizer (Table 4-13).   Table 4-13 Chemical analysis significance between substrates Chemical Analysis Between Substrates (Cu, Se, V) Treatment  Significance (p < 0.05) Between Substrates (p < 0.05) Sandy Tailings - Silty Tailings Sandy Tailings - Greenhouse Soil Silty Tailings - Greenhouse Soil Shoot Chemistry Selenium 0.000  0.000 0.000 Vanadium 0.000 0.000   Root Chemistry Copper 0.002  0.003  Vanadium 0.000  0.000 0.000  Principal component analyses were generated separately for roots and shoots of each substrate to summarize the associations between 18 elements analyzed in each sample (0, Figure 4-7). Dimensions 1 and 2 generally represent 50 to 60 percent of total variability. Individual treatment points were fairly scattered with no apparent pattern in all graphs except in roots of sandy and silty tailings where a weak progressive trend was observed with respect to metal correlation. In addition to the influx of metals from fertilizer water, metal influence in tailings were more pronounced as individual control data points were negatively and not correlated to most metals in roots and shoots of tailings respectively. This trend was less observable in copper spiked greenhouse soils as treatments only differed by copper sulfate. Positively correlated metals include Al, Ca, Cr, Cu, Fe, Mg, Na, Si in roots of sandy and silty tailings and predominantly Ca, Mg, Na, Ni (macro nutrients) in shoots for all substrates. Metals in roots and shoots of sandy and silty tailings do share a degree of similarity and generally divided between a secondary cluster or no correlation to the dominant cluster of metals. However, copper-spiked greenhouse soils show even more pronounced trends of Fe, Cr, Zn with no correlation to copper or aluminum in roots. In all charts, potassium was found to have no correlation with major clusters of metals.  66   Figure 4-7 Principal component analysis of metals in lodgepole pine roots in silty tailings in growth chamber after 42 days Although scattered, general clusters individual treatment points do show a progressive increase along dimension 1 where most metals are correlated. Larger individual treatment points indicate the center of treatment ellipse. Dimensions 1 and 2 represent 67 percent of total variability in dataset. Al, Ca, Cr, Cu, Mg, Na, Si were also positively correlated to one another in roots of sandy tailings and have no correlation to potassium.   4.2.2 Bluebunch Wheatgrass Bluebunch wheatgrass seedlings emerged within days after seeds were sowed in optimal germination temperatures of 15/25 oC (Kitchen & Monsen, 1994). Emergence exceeded 90 percent in most treatments, a value indicated in germination tests of seeds received Premier Pacific Seeds Ltd (2016) (Figure 4-8). Greatest variability was observed in sandy tailings and copper-spiked greenhouse soils where mean emergence was reduced to 80 percent in some 67  treatments. Median emergence in copper-spiked sandy (p = 0.002) and silty (p = 0.001) substrates was 5 and 20 percent respectively in 115 mg/kg of copper sulfate and no emergence onwards.   Figure 4-8 Bluebunch wheatgrass emergence in growth chamber after 42 days As with lodgepole pine, emergence was significantly affected by direct exposure to copper in spiked sandy (p = 0.002, n = 4) and silty substrates (p = 0.001, n = 4). Poor sowing methods may have led to variable emergence in sandy tailings and greenhouse soil; an 80 percent emergence is equivalent to 4 of 5 seedlings emerged. Experiment controls for sandy and silty tailings were also shared with copper-spiked sandy and silty substrates respectively. Median and standard error displayed.    Visible characteristics of emergence, less than 2 mm of growth, were often found in lower concentrations of copper-spiked substrates; although valuable as observations, Environment Canada (2007) defines emergence as greater than 3 mm of growth above surface (Photo 4-7). Lethal concentrations (LC) of copper toxicity for bluebunch wheatgrass were not found in the literature. As with lodgepole pine, the absence of emergence and growth in all copper-spiked substrates may largely be a result of osmotic burning from high salt index of direct exposure to copper sulfate. Similarly, seedling survival in copper-spiked substrates was rare, as with lodgepole pine, show obvious signs of developmental stress (emergence scenarios A and B in Photo 4-7). 0%10%20%30%40%50%60%70%80%90%100%Control010%11525%287.550%575100%1150Emergence (%) Percent Tailings or Substrate Copper Concentration (mg/kg dw) Bluebunch Wheatgrass Seedling Emergence Sandy TailingsSilty TailingsSpiked Greenhouse SoilSpiked Sandy SubstrateSpiked Silty Substrate68   Photo 4-7 Common emergence scenarios in treatments of copper-spiked substrates Regular emergence, poor seedling vigor and survival resistance at various stages of growth (A and B); signs of early emergence, up to 2 mm in growth (C); no sign of emergence (D). The pot shown was 115 mg/kg of copper-spiked silt (equivalent to 10 percent silty tailings), scenarios C and D were predominantly found in increased copper treatments.  In contrast to lodgepole pine, seedling survival was fairly uniform in silty tailings with some reductions in controls and treatments of sandy tailings and copper-spiked greenhouse soils (Figure 4-9). The cause of such occurrence should not be overlooked as traits associated to good seedling vigor, rapid germination, rapid root and shoot growth and stress resistance, have been carefully selected to form the current variety over the years (Jones, Nielson, & Carlson, 1991; Kitchen & Monsen, 1994; McKell, 1972; Ogle, 2010; Tilley & St. John, 2013). Thus the observed reduction would only suggest a response to the soil physical and chemical properties among the three substrates.   A D C B 69   Figure 4-9 Bluebunch wheatgrass survival in growth chamber after 42 days Survival was fairly consistent in silty tailings, but variable in treatments of sandy tailings and copper-spiked greenhouse soil treatments. Seedlings which emerged from spiked sandy and silty substrates (Figure 4-8) appear to have survived the 42-day test. Experiment controls for sandy and silty tailings were also shared with copper-spiked sandy and silty substrates respectively. Median and standard error displayed.    Silty tailings are far more susceptible to self-compaction (and crusting when dry) due to fine particles of similar sizes whereas loose particles in sandy tailings and greenhouse soils create a more stressful environment for plant establishment. Following Environment Canada (2007) protocol, seeds were sown at a depth twice their diameter and gently covered with surrounding substrates. This method may not be appropriate for bluebunch wheatgrass as seed depths were dependent on substrate texture for rapid germination, growth and establishment (Ogle, 2010). Even with good seedling vigor, seedlings become more susceptible to wilting or root separation due to poor plant stability if sown too shallow. Photo 4-8 shows a couple of instances where crown (and other roots) lay loosely above surface before roots penetrate the substrate. Substrate specific sowing depths could improve establishment and survival in sandy tailings and greenhouse soils treatments. Algal growth was commonly found along soil surfaces weeks after emergence; it would be beneficial to aid seedling establishment if seeds were sown deeper below to mitigate nutrient competition and aid root penetration. 0%10%20%30%40%50%60%70%80%90%100%Control010%11525%287.550%575100%1150Survival (%) Percent Tailings or Substrate Copper Concentration (mg/kg dw) Bluebunch Wheatgrass Seedling Survival Sandy TailingsSilty TailingsSpiked Greenhouse SoilSpiked Sandy SubstrateSpiked Silty Substrate70   Photo 4-8 Establishment challenges in loose substrates and algal growth Crown and roots (red arrow) of bluebunch wheatgrass seedlings appear loosely above surface before penetrating sandy tailings (left) and greenhouse soils (right). Seedlings become more susceptible to wilting or severe root separation due to poor plant stability (blue arrow). Seedlings were also subjected to competition against green algae on soil surfaces.  Bluebunch wheatgrass has been used extensively as forage and erosion control in reclamation due to its adaptability in a variety of soils in western half of North America. The United States Department of Agriculture Natural Resources Conservation Service (USDA-NRCS) recommends seed sowed at depths based on substrate texture: ¼ inch in fine textured soils, ½ inch or less in medium textured soils, and ¾ inch or less in coarse textured soils for adequate establishment (Ogle, 2010). In determining germination rate and emergence success, Kitchen (1994) used deep planting (4 cm in soil) to determine traits associated with good seedling vigor in bluebunch wheatgrass. Substrate texture/characteristics and species used should be considered when determining planting depth (Murphy & Arny, 1939).   For test validation, Environment Canada (2007) requires results to meet species specific mean emergence and survival rates and exceed minimum root and shoot lengths in all negative soil controls. The closest relatable species listed was northern wheatgrass (Elymus lanceolatus), a distant relative classified in the Family Poaceae of monocotyledonous flowering plants known as 71  grasses. Comparing among species is unrealistic even if results indicate a valid test. The only generic criterion applicable to this section of the experiment is mean survival of emerged seedlings exceeding 90 percent (at test end) in all negative control soils (Environment Canada, 2007). As the data suggest, mean survival for sandy and silty tailings and greenhouse soils were 96, 84 and 76 percent respectively. It is not appropriate to say this test is invalid as certain aspects of the protocol (such as sowing depth) were not species specific or tailored to specific growth media. A deeper seed depth is particularly important to tailings and greenhouse soils for improved establishment and access to adequate moisture. The consistency which Environment Canada (2007) aims to provide is important for standardization, however, the protocol should also allow a degree of flexibility to accept specific alternatives as indicated in peer-reviewed literature.  Several trends were observed in comparing longest shoot and root lengths (Figure 4-10). Both sandy (p = 0.001) and silty (p = 0.001) tailings showed a significant reduction in shoot (up to 10 cm) and root (10 cm) lengths in treatments after 115 mg Cu/kg or 10 percent tailings as well as between control and 50 and 100 percent tailings.  Shoots were generally longer in silty than sandy tailings while root lengths varied and weren’t definitive. Copper-spiked greenhouse soils were fairly consistent in treatments with no significant changes or trends observed. As with all greenhouse soil treatments, copper species would be retained by organic matter and thus seedlings were subjected to minimal exposure with no significant effect on plant growth. The reverse is observed in sandy and silty tailings where substrates do not retain/absorb water or copper species. Johnson and Aguirre (1991) examined the effects of daily water availability on bluebunch wheatgrass root lengths and concluded that altitude (longest root) and root magnitude (branching) were reduced when less water was applied. Thus it is expected that direct access to water in sandy and silty tailings would yield longer roots, however root lengths further reduced as copper concentrations exceeded 115 mg Cu/kg or 10 percent tailings or more.   72   Figure 4-10 Bluebunch wheatgrass longest shoot and root lengths in 42-day growth chamber study Growth and development was hindered in upper concentrations of sandy (p = 0) and silty (p = 0.005) tailings compared to consistent growth in greenhouse soils. Shoot and root lengths were significantly shorter in both tailings and pairwise (control to 50 and 100 percent treatment) with p values less than 0.015 (Appendix E  ). Median and standard error displayed (n = 5).  Although this study does not examine root morphology, Photo 4-9 shows similar observations of reduced root altitude and magnitude in 100 percent tailings as found in the greenhouse study (Photo 3-6). In some instances, shortened root pathways have led to an absence of primary root (Photo 4-9, second from left) were found in as early as 50 percent tailings. A reduction in shoot quality, color, quantity and robust growth became more pronounced from 25 percent tailings onwards.  -30-25-20-15-10-505101520253035Control010%11525%287.550%575100%1150Lengths (cm), 0 = Soil Level Percent Tailings, SubstrateCopper Concentration (mg/kg dw) Bluebunch Wheatgrass  Longest Shoot and Root Lengths Sandy-ShootSandy RootSilty-ShootSilty-RootSoil-ShootSoil-Root73   Photo 4-9 Shoots and roots of grown in 100 percent sandy and silty tailings Root pathways and longest lengths were shorten in sandy (left, p = 0.006) and silty (right, p = 0.009) as found in the greenhouse study. In some cases, the primary root was absent (e.g. second seedling in sandy tailings from left). Roots were commonly found within the top 2 cm of tailings.   As with shoot and root quality, total biomass was reduced to half in 10 percent sandy and silty tailings and significantly to one quarter at 50 (p = 0.06) and one sixth at 25 (p = 0.06) percent respectively (Figure 4-11). The significant reduction between treatments was largely attributed to direct exposure to substrate copper as no effects were observed in copper-spiked greenhouse soils where seedlings were indirectly exposed. The trade-off for protection against phytotoxicity in (greenhouse) soils has resulted in slower growth rates and productivity. Stand establishment, shoot/root quality and quantity were best in controls of silty tailings. This could be of several reasons. Fine-grained silty tailings were more compacted adding stability to seedling growth compared to loose sandy tailings and lighter greenhouse soils. Second, seeds were sown at the literature recommended depth of ¼ inch in fine textured soils (Ogle, 2010). The finer silt sized 74  particles may also provide a more preferred root surface area to volume ratio for nutrient absorption otherwise not found in other substrates.    Figure 4-11 Bluebunch wheatgrass total biomass in growth chamber after 42 days Compared to control, biomass was reduced to one quarter in 50, 100 percent sandy tailings (p = 0.004; p = 0.006) and one sixth in 25, 50, 100 percent silty tailings (p = 0.026; p = 0.026; p = 0.004). Greater shoot quality and stand establishment was found in silty tailings; an indication of preferred substrate. Median and standard error displayed (n = 5). See Appendix E.2 for summary of p values.  Root copper uptake was found significant in as early as 25 compared to control percent in sandy (p = 0.008) and silty tailings (p = 0.046) with a hard cap of under 800 ppm in both tailings (Figure 4-12). Root copper was different in all three substrates (p = 0.012), in particular between silty tailings and greenhouse soils (p = 0.016). Copper translocation was greatest in shoots of sandy tailings (p = 0.001); similar values found in willow staked along Hazeltine Creek (Golder Associates Ltd., 2016). In general, it is not surprising to see greater uptake in sandy than silty tailings; treatments were setup based on the percentage of tailings rather than actual concentration of copper which is less (325 mg Cu/k) in silty tailings. Copper uptake was unusually high in copper-spiked soils (p = 0.002). One may hypothesize that rapid growth rates lead to greater surface area of roots exposed to bio-available copper species. Through active transport, copper species were readily absorbed from organic matter. In greenhouse soils, mean 00.10.20.30.40.50.60.7Control010%11525%287.550%575100%1150Dry Weight (g) Percent Tailings, Substrate Copper Concentration (mg/kg dw) Bluebunch Wheatgrass Total Biomass Sandy TailingsSilty TailingsSpikedGreenhouse Soil75  shoot copper was consistent at 54 ppm as mean root copper concentration increased from 777 to 1945 ppm in 25 to 100 percent treatments. These sharp increases were also a result of lesser root biomass in greenhouse soils due to slower growth rates.   Figure 4-12 Bluebunch wheatgrass shoot and root tissue copper after 42 days Copper uptake in roots and transfer to shoots were significant in all treatments and substrates (see Appendix E.2). Greatest translocation was found in sandy tailings (p = 0.001), similar to trends observed in lodgepole pine seedlings. Shoot translocation in copper-spiked soils were consistent at 54 ppm while root uptake increased from 777 to 1945 ppm in 25 to 100 percent treatments; the cause of such occurrence may be attributed to experimental error due to low biomass for chemical analysis. Note the scale in copper ppm from aboveground (50 ppm) to belowground (500 ppm). Median and standard error displayed. -1350-1200-1050-900-750-600-450-300-1500150300-2250-2000-1750-1500-1250-1000-750-500-2500250500Control010%11525%287.550%575100%1150Copper PPM (negative values imply belowground) Percent Tailings & Substrate Copper Concentration (mg/kg) Bluebunch Wheatgrass Copper Uptake (PPM) Sandy Tailings - Root Silty Tailings - Root Greenhouse Soil - RootSandy Tailings - Shoot Silty Tailings - Shoot Greenhouse Soil - Shoot76  Copper translocation from root to shoot was greatest in both types of tailings where roots were directly exposed to copper species in substrate and substrate solution (Table 4-14). Mean copper root to shoot transfer rates were 3.7, 8.2 and 19.6 in sandy, silty and copper-spiked greenhouse soils, respectively (lower values indicate higher transfer). Greatest transfer was expected in sandy tailings as it contains higher concentration of copper per treatment than silty tailings. These results indicate that at similar root and shoot growth and biomass, higher substrate copper leads to greater root to shoot transfer (Figure 4-10; Figure 4-11).  Table 4-14 Bluebunch wheatgrass mean below to aboveground copper translocation ratio Lower values indicate greater the volume of copper translocated from root to shoot. Bluebunch Wheatgrass Copper Translocation: Mean Belowground/Aboveground Ratio Substrate Treatment (mg Cu/kg) Control 0 10% 115 25% 287.5 50% 575 100% 1150 Sandy Tailings 0.89 6.59 4.37 3.07 6.22 Silty Tailings 0.73 8.55 10.97 7.76 8.66 Greenhouse Soils 2.38 14.26 14.43 24.83 39.11  In contrast, seedlings grown in greenhouse soils undergo different growth strategies and response to copper. Median root and shoot lengths were 12.6 and 25.0 cm respectively with 0.14 g of dry weight biomass across all treatments. Growth strategies in greenhouse soils were predominantly shoot-focused whereas a balance was observed in tailings. Root copper increased significantly with respect to increasing copper-spiking (p = 0) while shoot copper remained capped at 54 ppm (Figure 4-12). These results indicate that substrates quality dictates growth responses and strategy and ultimately the quantity of copper transferred.  Bluebunch wheatgrass copper toxicity and root to shoot translocation information is fairly limited in literature. Redente et al. (2002) found similar responses of biomass reduction, copper uptake and root-shoot translocation in bluebunch wheatgrass from century long smelter-impacted copper-rich soils in southwest Montana, USA. Results from the 50 day greenhouse study found 7 and 5 times more copper in roots than in shoots of concentration gradient specific smelter-impacted and non-smelter-impacted soils respectively. These ratios were most applicable to sandy tailings by substrate texture with differences attributed to the lack of organic matter. 77  Golder Associates (2016)’s detailed site investigation noted enrichment of selenium (1.18; 1.03 ppm) and high vanadium (187; 202 ppm) in both sandy and silty tailings respectively (Appendix A  ; see Appendix G  for experimental data). Originated from silica chloride, an iron magnesium aluminum silicate mineral, selenium is considered highly mobile only in soluble forms. Due to interferences, ICP-OES detection was limited one wavelength (196.026 nm) in chemical analysis. Selenium speciation was not reviewed in this study and thus reasons for inconsistent selenium uptake could not be explained. In some instances, no data was found in roots while high concentrations were found in shoots. In general, selenium continued to exceed the aquatic freshwater and marine life guideline of 2 ppm in roots and shoots (Ministry of Environment, 2014). Vanadium uptake as well under the 50 ppm established by the British Columbia Water Guidelines (Ministry of Environment, 2017) for protection of marine aquatic life in roots and shoots. Significant translocation of vanadium was only found in shoots among treatments (Appendix E.2). Differences among shoot and root are summarized in the table below. As discussed earlier, significance effects were observed in as little as 25 percent tailings.   Table 4-15 Bluebunch wheatgrass significant shoot and root chemistry  Although mobile in soluble form, selenium was rarely found in roots (0 ppm) and thus significance in shoots could be a result of experimental error.  Chemical Analysis (Cu, Se, V) Treatment  Significance (p<0.05) Significance Among Treatment Significance Between Treatments Control to 10 Control to 25 Control to 50 Control to 100 Sandy Tailings Shoot Copper 0.001  0.006 0.003   Shoot Vanadium 0.011   0.023  10-50% p = 0.023 Root Copper 0.002  0.008 0.005   Silty Tailings Shoot Copper 0.000   0.004 0.001  Shoot Selenium 0.016      Shoot Vanadium 0.003   0.003 0.030  Root Copper 0.004  0.046 0.030 0.007  Root Vanadium 0.007    0.002  Cu-spiked Greenhouse Soil Shoot Copper 0.002  0.009 0.040 0.009  Shoot Selenium 0.001 0.008 0.008 0.008 0.008  Shoot Vanadium 0.014  0.007    Root Copper 0.000   0.038 0.000 10-100% p = 0.030 78  Principal component analysis of the 18 elements analyzed provided clear indication that most elements were positively correlated to one another (0, Figure 4-13). Separated by root and shoot and by substrate, each PCA from tailings showed clear indication of individual treatments and their association to the 18 elements. The first two dimensions were reviewed with each plot accounting for 58 to 70 percent of total sample variability. A dominant cluster of metals (Cu, Al, Ca, Mg, Fe, Na, Si and weakly Mn) was found positively correlated to one another closely along the first dimension of each plot. Metal correlation is mostly influenced by treatments of 50 and 100 percent where individuals were progressively scattered and mostly in the range and direction of correlated metals. Control (0) and 10 percent data points were closely clustered among treatment and negatively correlated to the major cluster of positively correlated elements while 25 percent treatments showed no correlation to metals. As with lodgepole pine, these results continue to indicate a synergistic effect or multiple factors causing phytotoxicity in bluebunch wheatgrass.   Figure 4-13 Principal component analysis of metals in bluebunch wheatgrass roots in sandy tailings after 42 days 79  Distinct individual treatment clusters were observed with fairly minimal influence compared to 50 and 100 percent treatments. A dominant cluster of metals (Cu, Al, Ca, Mg, Fe, Na, Si and weakly Mn) was observed in roots and shoots of sandy and silty tailings. Larger treatment points represent midpoints of confidence ellipse.   Unlike principal component analyses of sandy and silty tailings, individual treatments were thoroughly scattered across two dimensions in shoot and roots of copper-spiked greenhouse soils (Figure 4-14). In roots, copper was a major influence (almost parallel to dimension 1), but was positively correlated to micro nutrients (Fe, Mn, Cr, Si, Zn) and negatively correlated to macro nutrients (Ca, Mg, K, P) from fertilizer. These macro nutrients were mostly contributed from control, 10 and 25 percent treatments. A weaker resemblance of the dominant cluster of positively correlated metals was observed in shoots from copper-spiked greenhouse soils while no metal correlation was found in roots. In general, Se, Cd, Pb, Zn had no correlation in any bluebunch wheatgrass treatments; potassium and occasionally phosphorus were negatively correlated to most metals. Sandy and silty tailings had strong influence to positive correlation of metals in roots and weaker trends observed in shoots.  80   Figure 4-14 Principal component analysis of metals in bluebunch wheatgrass roots in copper spiked greenhouse soils after 42 days Individual treatment data points were fairly scattered. Copper is largely captured in dimension 1 while no dominant correlation trends were observed. Copper sulfate and metals from fertilizer were major influences. Larger treatment points represent midpoints of confidence ellipse. Note the low variability captured in dimensions 1 and 2.   4.2.3 Wild Willows Poor emergence and survival was observed from wild willow seeds in all five substrates. Only 10 percent emerged and far less survived. Seeds were collected in areas adjacent to Mount Polley Mine, however short seed life may have caused the seedlot to become defective. Willow seeds are non-dorminant and generally only viable for days without moisture (California Native Plant Society, 2014; Steele & Geier-Hayes, 1989) Cold storage was not effective to prolong seed viabilty. No data was collected for willows. 81  Chapter 5: 100 Day Growth Chamber Study Following the Environment Canada compliant growth chamber study, an additional study was conducted to identify changes in growth, metal uptake and translocation in lodgepole pine and bluebunch wheatgrass after prolonged exposure in sandy tailings. Emphasis was placed only on sandy tailings as it contains greater concentration of copper than silty tailings and that seedlings were unable to survive in copper-spiked sandy and silty substrates. Growing conditions were consistent in both growth chamber studies. An extended study was required to better understand potential toxicity of tailings and metal accumulation in established seedlings otherwise not present in the 42-day study.  5.1 Methods for 100 Day Growth Chamber Experiment This experiment only examines 0, 10, 25, 50, 100 percent concentration of sandy tailings on lodgepole pine seedlings. The experiment setup, substrate preparation, maintenance and extraction methods were identical to those written in the previous chapter aside from two main differences. This study is not Environment Canada compliant as pots were filled by weight but not to the required volume of 500 mL or half-filled container. Tailings have a higher specific gravity (abundance of iron oxide) than inert silica; weights remain consistent, but volume changes with increasing proportion of tailings. Second, test duration exceeds the specified 14 to 21 day duration in Environment Canada Biological Test Method Guidelines (2007). Two species, lodgepole pine and bluebunch wheatgrass were tested late June to late September of 2016. Extraction, chemical and statistical analysis were conducted as written in Chapters 2 and 4.  5.2 Lodgepole pine results and Discussion for 100 Day Growth Chamber Study The 100 day study was designed to provide a form of comparison in addition to the trends and observations found among substrates in 42 day study. This study was not intended to be Environment Canada (2007) compliant, rather to further study the changes in biomass production and copper uptake after a prolonged period of exposure to sandy tailings. Emphasis was placed on sandy tailings as it contains the greater concentration of copper and that seedlings were more likely to survive if grown in natural setting. The objective of the 100 Day study was to determine 82  if lodgepole pine and bluebunch wheatgrass were able to survive in prolong periods of sandy tailings and whether metal concentrations in plant tissues would change over time.  Emergence and survival in 100 Day growth period were similar to those of the 42 day study (Figure 4-1; Figure 4-2; Table 5-1). Foliage was generally healthy in all treatments. During the last three weeks, hydration cycles were reduced from biweekly to weekly due to lack of manpower. Seedlings were responsive to the accidental induction of stress which may have resulted in the slight browning of needle tips in some seedlings across all treatments (Photo 5-1). Alternatively, such observations indicate the sensitivity of seedlings to environmental change.   Table 5-1 Lodgepole pine 100 Day mean emergence and survival No significant found between and among treatments and to 42 Day Environment Canada (2007) compliant study. Median and standard error displayed. Lodgepole Pine Percent Sandy Tailings (mg Cu/kg) Control (0) 10% (115) 25% (287.5) 50% (575) 100% (1150) Emergence 100 ± 4% 100 ± 4% 100 ± 4% 80 ± 7.5% 100 ± 4% Survival 100 ± 4% 100 ± 4% 100 ± 4% 80 ± 7.5% 80 ± 7.5%   Photo 5-1 Lodgepole pine in sandy tailings after a 100 Days of growth Control (left) has developed extensive tap and lateral root development with mean treatment length of 28 cm. Seedlings in 100 percent sandy tailings (middle and right) have healthy shoots but poorly developed root systems which include thickened root tips, stunted tap roots, blackish root color and minimal lateral root growth (right).  83  Healthy shoots were observed in all treatments and significant (p = 0.039) to those of the 42 day experiment. In contrast, root development was minimal as concentration of tailings increased. Seedlings from control were able to develop both lateral and extensive tap roots; treatment average was 28 cm with the longest shown in Photo 5-1 (left, treatment control) at 39 cm. While longest lengths do not account for seedling competition and root morphology, it does provide a general quantitative comparison between treatments and experiments.   Root lengths were shortened (p = 0.001) in as little as 25 percent tailings while the 42 day study only found significant reduction in 100 percent tailings (Figure 5-1). Median root lengths between 25 to 100 percent tailings were nearly identical after 42 and 100 days of growth. Seedlings in 25 to 100 percent tailings of both 42 and 100 day study did show similar signs of stunted tap root and minimal lateral root growth. The extended exposure also revealed that seedlings, especially in 100 percent tailings, were unable to develop lateral roots or any root system at all (Photo 5-1, right). Blackening and thickening of root tips were more prominent in 50 to 100 percent tailings where root growth is stunted. These results indicate upper limits of seedling tolerance and growth habit response beyond 10 percent tailings and the importance of long term studies.    Figure 5-1 Lodgepole pine shoot and root lengths after 42 and 100 days -35-30-25-20-15-10-505Control(0)10%11525%287.550%575100%1150Lengths (cm), 0 = Soil Level Percent Tailings andSubstrate Copper Concentration (mg Cu/kg) Lodgepole Pine 42 and 100 Day: Shoot & Root Lengths 42 Day Shoot42 Day Root100 Day Shoot100 Day Root84  Significant reduction in root length (p = 0.001) was observed in as early as 25 percent tailings in the 100 day study compared to 100 percent in 42 day study respectively. Shoots were healthy and not affected by concentration of tailings, but differed between studies (p = 0.039). Negative values indicate belowground lengths. Median and standard error displayed; see Appendix E.3 for summary of all p values.  Alternatively, reviewing shoot and root biomass provides a clearer picture of how well seedlings were able to tolerate sandy tailings (Figure 5-2). The additional growing time increased shoot and root biomass, however productivity in 42 and 100 day study continue to decline with respect to concentration (p = 0.002 in shoot; p = 0.0 in root), in particular between control and 100 percent sandy tailings. These trends have led to the hypothesis that different growth strategies were used to counter various concentrations and seedling competition in sandy tailings. Seedlings in control and 10 percent treatments developed longer tap roots and fewer lateral roots while 25 to 50 percent treatments reduced tap root and focused on lateral root development for stability and nutrient uptake. Evidently hindered by tailings, seedlings in 100 percent sandy tailings appear to develop the bare minimum roots to survive and continued to remain competitive with adequate shoot development for photosynthesis.    Figure 5-2 Lodgepole pine shoot and root biomass after 42 and 100 days -0.15-0.10-0.050.000.050.100.15Control(0)10%11525%287.550%575100%1150Lengths (cm), 0 = Soil Level Percent Tailings orSubstrate Copper Concentration (mg /kg)  Lodgepole Pine 42 and 100 Day: Shoot and Root Dry Biomass 42 Day Shoot42 Day Root100 Day Shoot100 Day Root85  Shoots and roots steadily decline with respect to increasing concentration of tailings (p = 0.048 in shoot; p = 0.0 in root) especially in 100 percent treatment (see Appendix E   for list of p values). Different rooting strategies were observed as a means to counter concentration of tailings. Median and standard error displayed.  Root copper was expected to increase with concentration after the extended exposure to sandy tailings (p = 0.001). Compared to control, significant uptake occurred in as early as 25 (p = 0.021) percent tailings while the 42 day study only found significant uptake only in 100 (p = 0.001) treatment. Root to shoot transfers were found different compared to the 42 day study (p = 0.048); median values were below 30 ppm, an upper limit of shoot copper concentration as seen in Figure 4-6 yet remain unaffected by root copper concentration. Seedling shoots appear slightly affected by the unintentional change in hydration, however, phytotoxicity among treatments were not observed. As explained in the previous chapter, foliage was not isolated at the time of chemical analysis, thus foliar copper concentrations were unknown. The suggested literature value of foliar copper concentration exceeds 17 ppm (Lozano & Morrison, 1981; Majid, 1984; Majid & Ballard, 1990; Reuther & Labanauskas, 1966; Van Lear & Smith, 1972) would not be applicable as seedlings appear healthy with shoot quality indifferent among and between treatments of 42 and 100 day studies. These results may suggest that seedlings generally have high tolerance to tissue copper and can develop specific growth strategies to better adapt and survive in toxic environments. One particular case is the distribution of resources from root development to shoot development in 100 percent tailings.  86   Figure 5-3 Lodgepole pine mean copper uptake after 42 and 100 days Significant uptake observed in roots (p = 0.001) and shoots (p = 0.008) and between 42 and 100 day studies (p = 0.048 shoots; p = 0.005). Root to shoot copper transfers were generally capped at 30 ppm. Note the changes in scale from above ground (every 50 ppm) to belowground (every 500 ppm). Median and standard error displayed; see Appendix E.3 for summary of all p values.  Other metals of interest include selenium and vanadium (Appendix G). Although highly variable, selenium was only found significant in shoots (p = 0.017) and between 42 and 100 day studies. Due to the mobility of soluble selenium, detection was fairly limited which led to greater variability in metal detection. In general, the aquatic freshwater and marine life guideline of 2 ppm in roots and shoots was often exceeded (Beatty, J. M and Fusso, G. A., 2014). Vanadium however, was well below the 50 ppm established by the British Columbia Water Guidelines (Ministry of Environment, 2017) for protection of marine aquatic life in roots and shoots. No other significance results were found in these metals. -350-300-250-200-150-100-50050100-3500-3000-2500-2000-1500-1000-50005001000Control010%11525%287.550%575100%1150Copper PPM (negative values imply belowground) Percent Tailings & Substrate Copper Concentration (mg/kg) Lodgepole Pine 42 & 100 Day: Copper Conc. 42 Day Root 100 Day Root 42 Day Shoot 100 Day Shoot87  Principal component analysis of 100 day study samples showed strong correlations of metals in shoots and roots of sandy tailings with over 75 percent of total sample variability captured (0, Figure 5-4). Tight clusters of individual control data points were observed in shoot and root; positively correlated to potassium and negatively correlated to copper. In particular, roots showed an addition of Na, Ni, P, Se, V, Zn to the cluster of metals (Al, Ca, Cu, Cr, Fe, Mg, Si) previously observed in sandy and silty tailings of the 42 day study. Correlation between these metals varied in roots and became more pronounced in shoots where copper and magnesium showed no correlation to the group of strongly correlated metals (Cr, Fe, Na, Ni, P, Pb, Zn) in shoots. This observation is particularly unusual as copper was commonly associated to many other metals as seen in results of 42 day study. Two strong implications can be derived from 100 day growth. The first hypothesis is that seedlings have reached a phytotoxic maximum where copper uptake no longer occurs while uptake of other micro nutrients continues to maintain growth. Or that copper uptake became much slower due to prolonged exposure and uptake to copper rich substrate. The latter would also imply the importance of long term studies to determine phytotoxicity in lodgepole pine and effects affecting plant growth. At this point it remains uncertain as to whether copper and other metals form additive, potentiation, antagonism or synergistic effect on phytotoxicity.  88   Figure 5-4 Principal component analysis of metals in lodgepole pine root in sandy tailings, 100 day growth chamber study Strong cluster of positively correlated metals (Na, Ni, P, Se, V, Zn of which Al, Ca, Cu, Cr, Fe, Mg, Si were in sandy and silty tailings of the 42 day study) in root and also found in shoots. Larger treatment points represent midpoints of confidence ellipse. Dimensions 1 and 2 represent total variability in samples.  5.3 Bluebunch Wheatgrass  The 100 day study showed several differences in growth after seeds were subjected to longer periods of exposure. Changing hydration cycles from biweekly to weekly may have unintentionally induced substantial stress on the already stressed seedlings. Results generally indicate the need to dilute sandy tailings for adequate growth to take place.    Emergence and survival appear to be fairly steady with full emergence and survival in control and 10 percent sandy tailings (Table 5-2). Seedlings continue to have some difficulty emerging and surviving as concentration of tailings increase. In general, seedlings appear to be less vigorous and statistically indifferent to their counterparts in the 42 day study.  89  Table 5-2 Bluebunch wheatgrass 100 day mean emergence and survival in sandy tailings Seedlings appear to be similar (p > 0.05) to those of the 42 day study.  Bluebunch Wheatgrass Percent Sandy Tailings (mg Cu/kg) Control (0) 10% (115) 25% (287.5) 50% (575) 100% (1150) Emergence 100 ± 0% 100 ± 0% 100 ± 8% 80 ± 4.9% 100 ± 4.9% Survival 100 ± 0% 100 ± 0% 100 ± 8% 80 ± 4% 100 ± 8%  The quality of shoots and roots were considerably different that those of the 42 day study. Most grass blades were dark green, thin with early blades wilted at the time of harvest. Shoot lengths were significantly shorter (p = 0.004) than seedlings of the 42 day study (). Overall treatment growth appears to be slower as well. Although root growth was found significant in 50 percent tailings on wards (p = 0.004), they do not appear to differ much from the 42 study. The quality of growth, especially in greater proportions of tailings, could be attributed to the lack of fertilizer water present as pots were prepared by substrate weight. The specific gravity of tailings is greater than inert silica; thus pots with higher proportions of tailings will have less substrate and ultimately less fertilizer water to support adequate growth. Water stress corresponds to higher root to shoot ratio in length and biomass (Kiemned, Larson, & Garmmon, 2003; Klepper, 1991) not found in 50 to 100 percent sandy tailings treatments.   Figure 5-5 Bluebunch wheatgrass longest shoot and root lengths after 42 and 100 days  -35-30-25-20-15-10-50510152025Control(0)10%11525%287.550%575100%1150Lengths (cm), 0 = Soil Level Percent Tailings andSubstrate Copper Concentration (mg/kg) Bluebunch wheatgrass 42 and 100 Day:  Longest Shoot and Root Lengths 42 Day Shoot42 Day Root100 Day Shoot100 Day Root90  Roots (p = 0.003) and shoots (p = 0.016) were significantly different among treatments in shoots (p = 0.004) between 42 and 100 day study. Median and standard error displayed; see Appendix E.4 for list of p values.  Plant biomass is a strong indicator of productivity within each treatment. Shoot (p = 0.003) and root (p = 0.001) biomass were significantly reduced in treatments containing tailings and much less than values of the 42 day study (Figure 5-6). The 42 and 100 day study showed severe decrease in treatments especially in lower concentrations of tailings (0-25, p = 0.021 shoots; 0-25 p = 0.028 roots). These results were particularly important as the quick growing used for reclamation isn’t able to grow in sandy tailings over time. Seedling establishment was the primary concern in the 42 day experiment, however with extended growth period, seedlings seem to have poor vigour when subjected to sandy tailings. The lack of biomass produced is a clear indicator that bluebunch wheatgrass was unable to thrive in any concentration of tailings.    Figure 5-6 Bluebunch wheatgrass shoot and root biomass after 42 and 100 days Shoot (p = 0.003) and root (p = 0.001) biomass were significantly reduced in as little as 25 percent tailings while the 42 day study only showed significance in 100 percent tailings. Note the significant reduction between control and treatments of various sandy tailings. Median and standard error displayed.  -0.25-0.20-0.15-0.10-0.050.000.050.100.150.200.25Control(0)10%11525%287.550%575100%1150Lengths (cm), 0 = Soil Level Percent Tailings andSubstrate Copper Concentration (mg/kg) Bluebunch Wheatgrass 42 & 100 Day: Shoot & Root Biomass 42 Day Shoot42 Day Root100 Day Shoot100 Day Root91  The 100 day study found less copper uptake in roots (p = 0.001) and greater copper transfer to shoots in most treatments of the 42 day study (Figure 5-7). Root uptake was significantly less as a result of limited belowground biomass with only 1 to 2 times the copper in shoots compared to 3 to 6 or 5 to 7 times found in the 42 day and Redente et al. (2002)’s study, respectively. Shoot copper concentrations were above 230 ppm in 25 to 100 percent sandy tailings (p = 0.002). Lethal (LC) and effective (EC) concentrations of copper for bluebunch wheatgrass was not found in literature; however, one may hypothesize the high concentration of copper in shoots were toxic and inhabited further plant establishment and productivity.   As with lodgepole pine in the previous section, several indicators have suggested a change in seedling establishment to mitigate substrate toxicity from prolonged exposure to tailings. Under stressful environments, seedlings tolerate stress by increasing their root to shoot ratio (Kiemned et al., 2003; Klepper, 1991); instead, shoot biomass exceeds root biomass in 25 to 100 percent treatments. These results may suggest a survival scenario where seedlings produce enough roots for adequate nutrient and uptake and majority of developmental focus on aboveground biomass production. The redistribution of resources to increase shoot growth may accelerate phytotoxicity as copper accumulates in shoots over time.  92   Figure 5-7 Bluebunch wheatgrass copper uptake in shoots and roots after 42 and 100 days Significant root (p =0.002) and shoot (p =0.006) copper uptake found after 100 days of growth. The quantity of copper was only significant in roots of 42 and 100 day study (p =0.001). The lack of root biomass produced in 100 day study would have resulted in lower root copper collected. A list of p values is presented in Appendix G  . Median and standard error displayed; see Appendix E.3 for list of all p values.  Other metals of interest include selenium and vanadium (Appendix G). Higher concentrations of selenium was found in treatments of roots (p = 0) and shoots (p = 0.021) compared to the 42 day study. At low doses, selenium generally serves to protect plants from abiotic stresses such as metal stress, drought and desiccation (Gupta & Gupta, 2016) which was found in seedlings in the 100 day study. Literature on bluebunch wheatgrass and selenium phytotoxicity have not been found; in general high selenium uptake leads to induced oxidative stress and distorts protein structure and functions in plants (Gupta & Gupta, 2016). Selenium values were highly variable -1000-800-600-400-2000200400Control010%11525%287.550%575100%1150Copper PPM (negative values imply belowground) Percent Tailings & Substrate Copper Concentration (mg/kg) Bluebunch Wheatgrass 42 & 100 day: Copper Conc. 42 Day Root 42 Day Shoot 100 Day Root 100 Day Shoot93  and thus no trends/effects were observed. Root to shoot transfer ratios were 1 to 3 weakly corresponding to increase in tailings. Vanadium uptake closely resembled seedlings in the 42 day study (p = 0). Mean uptake in roots were 5 ppm in all tailings treatments with a 1:1 root to shoot transfer ratio in 50 and 100 percent sandy tailings (p < 0.05). Values were well below the established 50 ppm concentration for protection of marine aquatic life (Ministry of Environment, 2017).  Stronger metal correlations were observed in principal component analysis between 42 and 100 days of growth (0, Figure 5-8). The dominant cluster of metals in 42 day study (Cu, Al, Ca, Mg, Fe, Na, and weakly Si, Mn) now includes Ni, Co, P, Cr, Cd with dimensions 1 and 2 representing over 70 percent of total variability in dataset. In roots, the dominant cluster of metals were strongly and equally correlated along dimension 1 while shoots display a weaker correlation with Cu, Ca, Mg separating from the major cluster. The observed phenomenon in roots was driven by metals in tailings as correlations were based on 25, 50 and 100 percent treatments and less influenced by fertilizer (applied to all pots).  94   Figure 5-8 Principal component analysis of metals in bluebunch wheatgrass root in sandy tailings, 100 day growth chamber study  Major cluster of positively correlated metals include Ni, Co, P, Cr, Cd of which Cu, Al, Ca, Mg, Fe, Na, and weakly Si, Mn were in sandy and silty tailings of the 42 day study. Larger treatment points represent midpoints of confidence ellipse. Dimensions 1 and 2 represent total variability in samples.  Two strong implications can be derived from PCAs of 42 and 100 day studies. Results ascertain the hypothesis that phytotoxicity in sandy and silty tailings is synergistic and primarily evolve around 8 to 13 metals which have strong influences on bluebunch wheatgrass growth and development. Second, Environment Canada’s required test duration of 14 to 21 days is too short to provide critical information on metal uptake as metal correlations have appeared strong after an extended growth period. While these were preliminary and exploratory studies, results from lodgepole pine and bluebunch wheatgrass have demonstrated the importance of test duration as a critical assessment of substrate toxicity. 95  Chapter 6: Summary and Further Study The primary objective of this thesis was to assess Mount Polley Mine tailings as a growth medium and determine if the physical and chemical characteristics of tailings inhibit growth and generation of above and belowground biomass. Results of this exploratory study contributed to the restoration plan for impacted terrestrial environment along Hazeltine Creek downgradient from the Mount Polley Mine. Following field observations, a greenhouse study was prepared to assess the physical growth limiting factors of sandy and silty tailings under natural conditions. An Environment Canada (2007) compliant study was recommended by Golder Associates Ltd. using growth chambers to mitigate physical limitations of tailings and examine copper, selenium and vanadium uptake in below and aboveground biomass under optimal growing conditions. The growth chamber study indirectly examined the effectiveness of Environment Canada’s Biological Test Method (2007) which is commonly used to assess phytotoxicity in contaminated sites. These universal guidelines do not account for soil composition where physical growth limiting factors arise. An additional growth chamber study explored the possibility of plant establishment and changes in trends after prolonged period (100 days) of exposure to sandy tailings.  The greenhouse study primarily focused on identifying the effects of physical properties on plant growth and metal uptake in sandy and silty tailings. Coarse textured inert silica (Land Mountain #20-30) was used as control substrate to prevent “hexagonal close packing” or “cubic closed packing” which occurs when similar sized particles such as tailings become aligned and stacked horizontally in alternating layers over time. Field observations at Hazeltine Creek found areas where a thick layer of saturated silty tailings created an anaerobic environment and led to the decay of plant roots. Coarse grained silica was mixed into sandy and silty tailings to increase space between particles to increase substrate aeration and improve infiltration critical to plant growth. Although effective, results from the greenhouse study found other physical challenges of sandy and silty tailings affecting plant growth.   Lodgepole pine and bluebunch wheatgrass had poor emergence and limited survival as a result of low humidity and substrate dryness between hydration cycles. No emergence was found in wild 96  rose. Treatments, particularly with silty tailings formed hard surface crusts which became an impenetrable barrier for plant establishment; coupled with poor infiltration, surface flows occurred during hydration mimicked observations of exposed silty tailings along Hazeltine Creek (Miller, personal communication, 2016). Climatic variation and different water holding capabilities of sandy and silty tailings and greenhouse soils were not suited for a static hydration schedule. Pots were often dry with copper evaporites appearing on surfaces of greenhouse soils. Varying daily changes in humidity and temperature created inhabitable conditions causing seedling survival to plummet as susceptibility to desiccation were common in early stages of growth. Copper uptake was primarily found in bluebunch wheatgrass roots of sandy tailings (upwards of 500 to 650 ppm) or 5 to 6 times the concentration in shoots as found in other studies (Redente, Zadeh, & Paschke, 2002).  The growth chamber study applied Environment Canada’s Biological Test Method (2007) to measure any phytotoxic effects on terrestrial plants exposed to potentially toxic substances under a controlled setting. The Environmental Canada (2007) compliant study examined growth responses of lodgepole pine, bluebunch wheatgrass and wild willows by 0, 10, 25, 50, 100 percent concentration of sandy and silty tailings and respective copper equivalence in inert sandy and silty substrates and greenhouse soil. The growth chamber study mitigated abiotic factors to prevent surface crusting, but did not address the principal confounding physical, chemical and biological factors found in each substrate. Sandy and silty tailings in this study were collected in the Polley Plug area free of debris to determine plant growth and metal uptake. Tailings along Hazeltine Creek were well mixed with native forest soil containing various rock fragments, forest litter, soil fauna and flora, various soil organic matter, microbial communities and mycorrhizae fungi. These soil-tailings mixtures have phenomenal influence on plant-soil interactions, substrate texture and chemical processes not present in this study. The addition of slightly acidic fertilizer (pH 5.5) to alkaline tailings for example, would increase metal release and potential plant uptake as metals were not chelated by the biological community. Such limitation is a result of applying universal guidelines which defines “contaminated soils” as contamination entering a pre-existing system of developed heterogeneous agricultural or forest 97  soil. At Hazeltine Creek, however, the pre-existing system is no longer present after the scouring of native soil and deposition of sandy and silty tailings.   By definition, soil is a mixture of organic matter, minerals, liquids, gases and organisms that support life. Sandy and silty tailings (as well as inert sand and silt) should be considered as parent material as it neither contains any form of biological development nor the effective composition to support plant growth. Uniform particle sized substrates such as tailings self-compact resulting in hexagonal or cubic closed packing where inadequate porosity creates an anaerobic environment. The greenhouse study mixed coarse sand into tailings to improve substrate structure; it increased micro and macro pores for aeration, infiltration, drainage and prevention of hexagonal or cubic closed packing over time. While growth chambers maintain constant humidity to prevent surface crusting, pore spaces could be blocked by bacterial culture or algal growth as observed in well fertilized substrate.  Further to that, the Biological Test Method (2007) requires pots to be hydrated to “near saturation;” a condition only achieved in developed organic or mineral soils which contain high organic matter content. Near saturation in mineral substrates such as tailings could lead to liquefaction or anaerobic conditions inhibiting plant growth not resulting from substrate toxicity. These universal guidelines were “suitable for measuring and assessing toxicity of samples of field collected soil, biosolids, sludge, or similar particulate material; or of natural or artificial soil spiked […] with test chemicals” (Environment Canada, 2007, p. v). The limitations of each substrate should be identified and mitigated or isolated before testing such that results reflects method and substrate not phytotoxicity.  Environment Canada’s Biological Test Method (2007) was designed to measure emergence and growth of terrestrial plants exposed to contaminated soils. All compliant studies are required to meet minimal species-specific mean emergence and survival rates and exceed minimum root and shoot lengths in all negative soil controls for compliancy. For validation, the method provides a list of common vegetable plants and cereal grasses used to determine substrate toxicity such as copper sulfate herbicide applications in agriculture. The provided list of species specific criteria was neither applicable nor appropriate to validate lodgepole pine and bluebunch wheatgrass 98  results against distant relatives or by taxonomic Family. Phytotoxicity testing should use ecologically appropriate species native to the contaminated ecosystem or end objective of restoration work (Redente et al., 2002). Results of these studies should provide useful information to environmental risk assessments and future restoration activities much like how this study forms a basis for future restoration work at Mount Polley Mine and Hazeltine Creek. It would be impractical to use common vegetable plants and cereal grasses to assess mine tailings or even industrial contamination in order to achieve Environment Canada compliancy.   Results of the 42 day study were compliant to Environment Canada (2007) test standards. Mean survival in control substrates were 95, 100, 95 percent in lodgepole pine and 96, 84, 76 percent in bluebunch wheatgrass in sandy tailings, silty tailings and copper-spiked greenhouse soil, respectively. The only generic criterion applicable was mean survival of emerged seedlings exceeding 90 percent in all negative control soils at test end (Environment Canada, 2007). Bluebunch wheatgrass emergence and survival would improve if an option in the Biological Test Method (2007) was available to sow seeds at depths based on substrate texture, characteristics and test species recommended in peer reviewed literature. The United States Department of Agriculture Natural Resources Conservation Service (USDA-NRCS) recommends ¼ inch in fine textured soils, ½ inch or less in medium textured soils, and ¾ inch or less in coarse textured soils for adequate establishment (Ogle, 2010). Planting depths may improve seedling stability (and survival) as some crowns (and roots) were found lying loosely on substrate surfaces of sandy tailings and greenhouse soils. Even with good seedling vigor, method induced growth habit leading to increased susceptibility to desiccation should not be recorded as an effect from tailings. These changes may improve emergence and survival yet maintain stringent standardized methodology for all testing.  Lodgepole pine and bluebunch wheatgrass emergence and survival were not affected by concentration of sandy and silty tailings or copper-spiked greenhouse soils. Emergence and survival in copper-spiked sandy and silty substrates were approximately 20 percent, and less than 5 percent for both species in treatments containing 115 and 287.5 to 1150 mg Cu/kg respectively (equivalent to 10 and 25 to 100 percent tailings). Common characteristics of phytotoxicity 99  include stunted hypocotyl, pre-mature foliage development or no germination after 42 days of exposure. Seedlings become prone to foliage scorching or “osmotic burning” from solutes of high salt index such as copper sulfate, which causes cell walls to rupture due to an imbalance of water entering into cells (Miller & Young, 1976; Tisdale & Nelson, 1975). Similar effects were observed in bluebunch wheatgrasses in copper-spiked sandy and silty substrates. Low viability of wild seeds has resulted in a lack of success and limited emergence in the study. The effective concentrations (EC) of copper phytotoxicity in lodgepole pine and bluebunch wheatgrass were not found in literature.  The Environment Canada (2007) used longest lengths of shoots and roots to identify changes in growth habits across all treatments. Lodgepole pine shoots appeared healthy with similar lengths in all treatments. Roots were significantly shortened with limited lateral root development in 100 percent sandy and silty tailings. In contrast, significant shortening of shoot and roots were found in treatments greater than 575 mg Cu/kg or 50 percent sandy and silty tailings. Increased tailings have resulted in shorter branching root structures in bluebunch wheatgrass. Shoots were generally discolored and lacked robust growth in treatments greater than 287.5 mg Cu/kg or 25 percent sandy and silty tailings. Neither species had growth affected in greenhouse soil as copper species were largely retained in soil organic matter.   The drawback of comparing longest roots and shoots is that the approach is most suited for short term studies (14 or 21 days) where lateral root development, root thickness or competition induced survival strategies have the least effect on measurement. The Biological Test Method (2007) recommended test duration of 14 or 21 days while the growth chamber study was extended to 42 days to generate adequate biomass for chemical analysis. Studies requiring adequate plant biomass for chemical analysis, should review a combination of longest shoot and root lengths, above and belowground biomass and qualitative observations to assess any changes in rooting habits. Since fertilizer water was applied from the surface, developing lateral roots may increase seedling’s competitiveness.  100  Significant copper uptake was observed in shoots and roots of both species. Copper uptake was dominantly found in roots with steady transfers observed in shoots. Lodgepole pine root uptake increased with concentration (p < 0.05) while the copper concentrations in shoots, bulk aboveground biomass, remained under 30 ppm in most treatments and substrates except for 100 percent sandy tailings. Lodgepole pine copper phytotoxicity from root uptake were not found in literature, however, results from foliar copper sulfate fertilizer applications have indicated phytotoxicity occurring when foliar (needle) copper concentration exceeded 17 ppm (Lozano & Morrison, 1981; Majid, 1984; Majid & Ballard, 1990; Reuther & Labanauskas, 1966; Van Lear & Smith, 1972). Shoot copper concentrations in 42 and 100-day growth chamber studies were generally above 30 ppm in all treatments except controls. Shoot chemistry samples in this study includes both hypocotyl and foliar (needles) and thus, seedlings did not show any sign of phytotoxicity. This may indicate that majority of copper in shoots remain in hypocotyl.   In contrast, bluebunch wheatgrass had consistent root copper uptake of 385 to 800 ppm for all treatments containing sandy and silty tailings while copper-spiked greenhouse soils increased significantly from 400 to 1945 ppm with respect to treatments. Shoot copper in silty tailings and greenhouse soils were less than 85 and 54 ppm respectively; greatest transfer was found in sandy tailings (over 100 ppm) irrespective of root copper concentration. Likewise, root to shoot copper transfer was greatest in sandy tailings in both species as roots were directly exposed to copper-bearing minerals.  Copper and selenium were found to be enriched in tailings and contain the possibility of potential leaching as indicated in present geochemical studies (SRK Consulting (Canada) Inc., 2015). Two mineralogical forms of copper were present: 34 percent primary sulfide copper minerals (Bornite, Cu5FeS4 and chalcopyrite, Cu2FeS2) remaining after mine extraction and 66 percent non-sulfide copper (iron magnesium aluminum silicate mineral, of which 14 to 23 percent contain selenium) (SRK Consulting (Canada) Inc., 2015). High pH and presence of carbonate minerals in tailings prevents minerals from weathering and releasing copper while kept in tailings storage facility. Current and previous studies have found non-sulfide copper to be 101  relatively insoluble and low risk of copper leaching under neutral pH (subaerial or subaqueous) conditions at Hazeltine Creek (SRK Consulting (Canada) Inc., 2015).   The copper found in plant tissues was a result of several factors from aqueous copper species to weathering of exposed surfaces to bioweathering. Following the breach, the debris flow deposited supernatant water (containing aqueous copper species) and sandy and silty tailings on to the forest floor. Samples collected by Golder Associates Ltd. and Mount Polley Mine staff were fairly wet as a result of poor subsurface drainage. Tailings had to be air dried and thoroughly mixed before use to ensure all pots received tailings of similar nature. SRK Consulting (Canada) Inc. (2015) indicated that tailings had low leachability in under neutral conditions. However, slightly acidic conditions from fertilizer water, pH 5.5, may influence the release of copper, selenium and other metals from fragmented or exposed surfaces of sulfide and non-sulfide copper minerals throughout the study. Humid growth chamber conditions may further facilitate abiotic and biotic weathering of minerals. Biological tests were not conducted on tailings at the time of study; however, bacterial leaching or bacterial oxidation has been known to increase chalcopyrite weathering by 2 to 15 times (Murr & Berry, 1976).  The 100 day study of lodgepole pine and bluebunch wheatgrass in sandy tailings was a comparative study to identify trend and observation changes after prolonged period of exposure. Results were not Environment Canada (2007) compliant as pots were filled by weight (and not by volume); volume decreases slightly with increasing proportion of tailings due to greater specific gravity in sandy tailings.   Lodgepole pine shoot length and quality were similar in 42 day study. Results from the 100-day study found shoot copper below 30 ppm and significantly less than the 42-day counterparts (p = 0.048). Root lengths were significantly shorter than in the control and equivalent to the lengths of respective concentration of tailings in the 42 day study. The decreased root growth suggests that seedlings were solely relying on fertilizer to maintain survival; such uptake (p = 0.001 and 0.005 compared to 42 day study) is indicative of accumulation of copper belowground. Similarly, the quality of bluebunch wheatgrass roots and shoots were considerably different than 102  those of the 42 day study. Root to shoot transfer ratios were below 2 with shoot copper exceeding 200 ppm (p = 0.002) in 25 to 100 percent tailings. At the time of harvest, grass blades were thin and dark green indicative of severe stress. Although preliminary, the 100 day study found key differences which would not be known if experimentation and results were only limited to the Biological Test Method (2007)’s first 14 or 21 days of growth.   Both 42 and 100 day studies were exploratory studies focused on copper as it was initially suggested as the dominant cause of potential phytotoxicity at Hazeltine Creek and Mount Polley area.  An enrichment of selenium compared to regional crustal basalt rocks was also found in tailings and concentrations of vanadium exceeded the provincial soil standards (Golder Associates Ltd., 2016; SRK Consulting (Canada) Inc., 2015). Inductively coupled plasma (optical emission spectroscopy) was only able to detect selenium one wavelength (196.026 nm) with minimal interference and machine noise. The values, however, were fairly inconsistent between tailings and plant species. For instance, there was no selenium detected in roots of bluebunch wheatgrass while an excess of 85 ppm was found in shoots of 25 and 50 percent silty tailings. In contrast, weak trends of vanadium uptake were observed with respect to increasing sandy and silty tailings. Root to shoot ratios were in the range of 2 to 12 with mean vanadium transfer of less than 5 ppm in all studies.  Principal components analysis (PCA) were generated by substrate and by plant species to determine if correlations were present for all 18 common metals (Cu, Se, V, Al, Ca, Cd, Co, Cr, Fe, K, Mg, Mn, Na, Ni, P, Pb, Si, Zn). Definitive clusters of metals were not found in the first two dimensions (Dim 1 and 2) which account for 60 to 70 percent of total variability of samples analyzed. In sandy and silty tailings, one to two dominant metal clusters were often observed while no metal correlation was found in copper-spiked greenhouse soil treatments. It should be noted that greenhouse soils were essentially barren of metals until the addition of copper sulfate and fertilizer which contains nine of the eighteen metals (Cu, Ca, Fe, K, Mg, Mn, Ni, P, Zn) analyzed. PCA diagrams of greenhouse soils of both species did not show copper as the principal component as copper (along with other metals) were chelated by the abundance of cation exchange capacities in organic matter. In contrast, seedlings were prone all mechanisms of metal 103  uptake (mass flow, diffusion, root extension) in tailings. The proportion of PCA analyses influenced by metals uptaken as a nutrient rather than toxin in tailings and in greenhouse soils is beyond the extent of this study. Thus copper and other metals associated to phytotoxicity cannot be identified.  The duration of study as required by Environment Canada (2007) for toxicity testing was 14 to 21 days. The first growth chamber study found this timeframe to be unrealistic and too short for seedlings to produce adequate biomass for chemical analysis. The need for extended studies is critical as metal toxicity may not occur until plants reach maturity or full establishment. The 42 growth chamber studies have shown significant changes in growth and metal uptake in lodgepole pine and bluebunch wheatgrass compared to the 100-day study. Results of these studies do not affirm metal uptake as optimal growing conditions largely resemble a hydroponic system than normal soil systems and certainly not reflective of field conditions.   Comprehensive studies are required to accurately address the cause of phytotoxicity in lodgepole pine and bluebunch wheatgrass as well as many dominant species in the contaminated ecosystem at Hazeltine Creek. Restoration plans should not be based on 42 or 100 day growth chamber studies as these results were based on growth in optimal conditions to satisfy Environment Canada (2007) requirements for permitting. Rather the next step is to establish 1, 5 and 10 year long term field studies to better understand the potential for toxicity of tailings to plants and bioaccumulation of metals released from tailings.   Lodgepole pine and bluebunch wheatgrass each showed various qualitative signs of phytotoxicity. 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E., & Berry, V. K. (1976). Direct observations of selective attachment of bacteria on low-grade sulfide ores and other mineral surfaces. Hydrometallurgy, 2(1), 11-24. doi:10.1016/0304-386X(76)90010-4 NASA Earth Observatory. (2014). Dam breach at mount polley mine in british columbia. Retrieved from https://earthobservatory.nasa.gov/IOTD/view.php?id=84202&src=ve Nawaz, M. F., Bourrié, G., & Trolard, F. (2013). Soil compaction impact and modelling. A review. Agronomy for Sustainable Development, 33(2), 291-309. doi:10.1007/s13593-011-0071-8 Ogle, D. G., St John, L. and Jones, T. A. (2010). Plant guide for bluebunch wheatgrass (Pseudoroegneria spicata). USDA-Natural Resources Conservation Service. Idaho and Washington Plant Materials Program. Retrieved from https://www.fs.fed.us/rm/pubs_other/rmrs_2010_ogle_d001.pdf 109  Ogle, R. S., Maier, K. J., Kiffney, P., Williams, M. J., Brasher, A., Melton, L. A., & Knight, A. W. (1988). Bioaccumulation of Selenium in Aquatic Ecosystems. 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Chapman (Ed.), Diagnostic Criteria for Plants and Soils (pp. 157-179): University of California, Div. Agric. Sci. Saxton, K. E., & Rawls, W. J. (2006). Soil Water Characteristic Estimates by Texture and Organic Matter for Hydrologic Solutions. Soil Science Society of America Journal, 70(5). doi:10.2136/sssaj2005.0117 Slooff, W., Clevan, R. F. M. J., Janus, J. A., & Ros, J. P. M. (1989). Integrated criteria document copper. . National Institute of Public Health and Environmental Protection. Report No. 758474009, 147.  SNC Lavalin Inc. (2014). Comprehensive Environmental Impact Assessment and Action Plan, Mount Polley Mine Tailings Storage Facility Breach, Mount Polley Mining Corporation (MPMC). Retrieved from https://www2.gov.bc.ca/assets/gov/environment/air-land-water /spills-and-environmental-emergencies/docs/mt-polley/p-o-r/2014-0815_mpmc_ceia_and _action_plan.pdf SRK Consulting (Canada) Inc. (2015). Mount Polley Mine Tailings Dam Failure: Update on Geochemical Characterization of Spilled Tailings, Prepared for Mount Polley Mining 110  Corp. Retrieved from https://imperialmetals.com/assets/docs/mt-polley/02.16.16.SRK-consulting-mp-mine-tailings-dam-failure-up.pdf Steele, R., & Geier-Hayes, K. (1989). The douglas-fir/mountain maple habitat type in central Idaho: succession and management. In Preliminary draft on file at: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station, Fire Sciences Laboratory, Missoula, MT (pp. 8434). Super Quintet Chemistry I: Introduction to Chemistry. (2017). Structure of solids 10. Closed packing arrangements and corrdination numbers. Retrieved from http://library.tedankara .k12.tr/chemistry/vol2/close%20packing%20arrangements%20and%20coordination%20numbers/h47.htm Taplin, L. B. (2002). Recovery improvement process development for copper from oxide materials. Internal Imperial Metals report prepared with the National Research Council of Canada. Retrieved from Imperial Metals Ltd. Target Products Ltd. (2016). Lane Mountain Silica Sand. Retrieved from www.targetproducts .com/UserContent/SpecSheets/lmsilsnd.pdf Tilley, D., & St. John, L. (2013). Plant fact sheet for bluebunch wheatgrass (Pseudoroegneria spicata). Retrieved from USDA-Natural Resources Conservation Service, Arberdeen Plant Materials Center, Aberdeen, Idaho: https://www.nrcs.usda.gov/Internet/FSE _PLANTMATERIALS/publications/idpmcfs11626.pdf Tisdale, S. L., & Nelson, W. L. (1975). Soil fertility and fertilizers: 3ed: Macmillan Publ. Co., New York. Valoya. (2014). Professional grow lights. Retrieved from https://www.led-horticoles.eu/wp-content/uploads/2015/01/EN_All-products_2014.pdf Van Lear, D. H., & Smith, W. H. (1972). Relationships between macro- and micronutrient nutrition of slash pine on three coastal plain soils. Plant and Soil, 36(2), 331-347.  Zhang, H., Jiang, Z., Qin, R., Zhang, H., Zou, J., Jiang, W., & Liu, D. (2014). Accumulation and cellular toxicity of aluminum in seedling of pinus massoniana. BMC Plant Biol., 14, 264. doi:10.1186/s12870-014-0264-9  111  Appendices Appendix A  Sandy and Silty Tailings: Certificate of Analysis   112     ALS ENVIRONMENTAL ANALYTICAL REPORT L1698939 CONTD....  PAGE 2 of 5  20-NOV-15 18:36 (MT) Version: FINAL * Please refer to the Reference Information section for an explanation of any qualifiers detected. 113     ALS ENVIRONMENTAL ANALYTICAL REPORT L1698939 CONTD....  PAGE 3 of 5  20-NOV-15 18:36 (MT) Version: FINAL * Please refer to the Reference Information section for an explanation of any qualifiers detected. 114   115    116    117  Appendix B  Summary of Key Findings Report Prepared for Golder Associates Ltd.  Summary of Key Findings Report Prepared for Golder Associates Ltd.  Measuring Emergence, Growth and Copper Uptake of Terrestrial Plants Exposed to Mount Polley Mine Tailings  Prepared by Anthony Leung and Dr. Suzanne Simard University of British Columbia January 12, 2017             118  Table of Contents Chapter 1: Introduction ................................................................................................................4 Chapter 2: Methods .......................................................................................................................5 2.1 Experimental Design ....................................................................................................... 5 2.2 Test Species .................................................................................................................... 6 2.3 Test Media ...................................................................................................................... 7 2.3.1 Tailings ....................................................................................................................... 7 2.3.1.1 Preparation of Sandy Tailings ............................................................................. 8 2.3.1.2 Preparation of Silty Tailings ............................................................................... 8 2.3.2 Particle Size Control ................................................................................................. 10 2.3.3 Greenhouse Soil ........................................................................................................ 10 2.4 Test Vessels .................................................................................................................. 11 2.5 Nutrient Amendment .................................................................................................... 11 2.6 Test Initiation ................................................................................................................ 12 2.6.1 Hydration of Substrates............................................................................................. 12 2.6.2 Preparation of Dilution Series ................................................................................... 12 2.6.3 Preparation of Spiked Substrates .............................................................................. 12 2.6.4 Potting and Seeding .................................................................................................. 13 2.7 Test Maintenance .......................................................................................................... 14 2.7.1 Environmental Conditions ........................................................................................ 14 2.7.2 Watering .................................................................................................................... 14 2.8 Test Completion ............................................................................................................ 14 2.8.1 Seedling Emergence and Survival ............................................................................ 15 2.8.2 Biomass Measurements ............................................................................................ 15 2.8.3 Test Validity Criteria ................................................................................................ 16 2.8.4 Copper Concentrations in Substrates ........................................................................ 17 2.8.5 Copper Concentrations in Plant Tissue ..................................................................... 17 2.9 Statistical Analysis ........................................................................................................ 18 Chapter 3: Results........................................................................................................................18 3.1 Confirmatory Chemical Analysis ................................................................................. 18 3.2 Seedling Emergence and Survival ................................................................................ 19 3.3 Shoot and Root Length ................................................................................................. 22 3.3.1 Lodgepole Pine ......................................................................................................... 22 3.3.2 Bluebunch Wheatgrass.............................................................................................. 24 3.4 Shoot and Root Dry Biomass Weight ........................................................................... 25 3.4.1 Lodgepole Pine ......................................................................................................... 25 3.4.2 Bluebunch Wheatgrass.............................................................................................. 27 3.5 Plant Tissue Chemistry ................................................................................................. 28 119  Chapter 4: Discussion ..................................................................................................................30 Chapter 5: Conclusion .................................................................................................................31 5.1 Lodgepole Pine ............................................................................................................. 31 5.2 Bluebunch Wheatgrass.................................................................................................. 32 Chapter 6: Recommendations for Further Study .....................................................................34 References .....................................................................................................................................35                    120  Chapter 1: Introduction On August 4, 2014, a partial breach of the Mount Polley Mine tailings storage facility in British Columbia causing 4.6 million cubic meters of supernatant water, and 12.8 million cubic meters of slurry tailings (solids and interstitial water) into Polley Lake, Hazeltine Creek, and Quesnel Lake. The breach resulted in a cover of tailings and native soil mixed with tailings along Hazeltine Creek. Following the breach,  is characterized as either sandy or silty in texture, low in organic carbon and nutrients, and having higher concentrations of copper and vanadium (relative to provincial soil quality standards for the protection of soil invertebrates and plants and background soil).   Golder Associates Ltd. (2015, 2016) reported that the primary impact to the terrestrial environment appears to be the physical impact of the scouring and subsequent loss of plant and soil communities in the floodplain. The the soil/tailing mixture layer covering the forest floor in the halo area restricted air exchange and movement of water to the root zone. This smothering is believed to be the root cause of the change of the soil microbial community and tree mortality observed in spring 2015 where tailings settled on top of the forest floor (referred to as the halo area). The pattern of tree mortality is not consistent with metal toxicity. However, in order to determine if the metals in soil would inhibit restoration and to investigate if plants would accumulate potentially harmful concentrations of metals, additional investigation was needed.  To test the hypothesis that copper toxicity is not occurring, a laboratory toxicity testing study was initiated examining the effects of soil/tailings mixture on the germination, growth and foliar chemistry of three plant species: lodgepole pine (Pinus contorta), bluebunch wheatgrass (Pseudoroegneria spicata), and wild willow (Salix scouleri). This technical summary of key findings provides a description of the toxicity test methods and results.  1.1 Study Questions This study was designed to answer the following questions:  Do the copper concentrations found in the tailings inhibit seedling emergence or plant growth (above/belowground biomass generation)?  Do differences in particle size of the tailings affect seedling emergence or plant growth?  Do plants germinated in tailings take up more copper than plants germinated in copper-spiked greenhouse soil?  If the plants germinated in tailings take up more copper than plants germinated in greenhouse soil, do the resulting tissue concentrations correspond to toxicity to the plant (i.e., inhibition of seedling emergence or plant growth)?  121  Chapter 2: Methods The experimental methods used in this study were based on the guidance provided in the following standardized test protocols:  Environment Canada (2007) “Biological Test Method: Test for Measuring Emergence and Growth in Terrestrial Plants Exposed to Contaminants in Soil (EPS 1/RM/45)”.  Environment Canada (2013) “Biological Test Method: Test for Growth in Contaminated Soil Using Terrestrial Plants Native to the Boreal Region (EPS 1/RM/56)”.  ASTM (2014) “Standard Guide for Conducting Terrestrial Plant Toxicity Tests (E1963-09 [Re-approved 2014])”. The actual methods varied from these standardized protocols in the following ways:  Non-standard test species were selected because the chosen species represented plants that are on site or will be used as part of the remediation strategy. As such, test validity criteria as stated in the protocols were not directly applicable to the chosen test species.  Test duration was lengthened to allow sufficient time for plant growth so that shoots and roots could be analyzed for copper concentrations.   The study was primarily designed to answer the study questions and not to strictly adhere to the standardized test protocols. In addition, it was necessary to optimize the number of test vessels to fit the available space in the test chambers. Therefore, certain aspects of the protocol were adjusted such as reducing the number of test concentrations from nine to four, limiting the characterization of test soils to concentration of key contaminants of interest (i.e., copper, selenium, and vanadium), and testing copper sulfate as a positive control in greenhouse soil instead of performing a reference toxicant test with boric acid.  2.1 Experimental Design Five experiments were conducted with five different substrates (sandy tailings, sandy control substrate, silty tailings, silty control substrate, and greenhouse soil):   Sandy tailings, diluted with sand of similar particle size to achieve the following dilution series: 100%, 50%, 25%, 10%, 0%. Replicated five times in a completely randomized design.  Silty tailings, diluted with sand of similar particle size to achieve the following dilution series: 100%, 50%, 25%, 10%, 0%. Replicated five times in a completely randomized design. 122   Sandy control substrate, used as the negative control soil in the sandy tailing experiment, spiked with copper sulfate to achieve the same range of total copper concentrations as tested in the sandy tailings experiment (i.e., 1150, 575, 287.5, 115, and 0 mg/kg dw). Replicated four times in a completely randomized design.  Silty control substrate, used as the negative control soil in the silty tailing experiment, spiked with copper sulfate to achieve the same range of total copper concentrations as tested in the silty tailings experiment (i.e., 1150, 575, 287.5, 115, and 0 mg/kg dw). Replicated four times in a completely randomized design.  Greenhouse soil, spiked with copper sulfate to achieve the same range of total copper concentrations as tested in the tailings experiments (i.e., 1150, 575, 287.5, 115, and 0 mg/kg dw). Replicated five times in a completely randomized design. All substrates, irrespective of tailings composition, received nitrogen-phosphorus-potassium (N-P-K) liquid fertilizer amendments so that no major nutrient deficiencies were present. It was expected that the low nutrient content of the tailings would appreciably limit plant growth, and therefore, testing without nutrient amendment was considered unnecessary to answer the study questions.  2.2 Test Species Initially, three plant species were tested: lodgepole pine (Pinus contorta), bluebunch wheatgrass (Pseudoroegneria spicata, also known as Agropyron spicatum), and wild willow (Salix scouleri). Lodgepole pine seeds were obtained from the BC Ministry of Forest in May 2016 from seedlots 31003, 08398, 14387 and 31002 in the Kangaroo Creek area, which is located approximately 1200 m from Mount Polley Mine (Table 1). Lodgepole pine seeds were stratified under cold water for 24 hours, dried and kept in the refrigerator for approximately 2 weeks following BC Ministry of Forest procedures prior to use in the toxicity tests. Bluebunch wheatgrass was obtained from Premier Pacific Seeds in May 2016 and did not require stratification prior to testing. Willow seeds were collected by Mount Polley personnel and sowed as received.  Table 1: Source and Expected Emergence Rates for Seeds Notes: BC = British Columbia; % = percent.   Species Source Expected Emergence Lodgepole Pine BC Ministry of Forest 94% (tested in 2013) Bluebunch Wheatgrass Premier Pacific Seeds 89% (Premier Pacific Seeds) Wild Willows Mount Polley mine site Field Collected, Unknown 123  2.3 Test Media 2.3.1 Tailings Sandy and silty soil/tailings mixture (referred to as “sandy tailings or “silty tailings”) were collected by Mine staff from Upper Hazeltine Creek on November 5, 2015, specifically for the plant toxicity testing. The sampling site was within the floodplain near the Polley Plug in an area free of debris and with minimal disturbance by mine reclamation traffic. One representative sample of each type of tailings was collected, for a total of two samples. Both samples were collected from the same location; the silty layer was on top, and sandy layer was below. The sampled tailings were shipped to the University of British Columbia for use in toxicity testing. All tailings were shipped in waterproof sealed plastic pails and kept cool in coolers throughout the duration of shipment. Upon arrival, the tailings were kept in the original closed coolers and stored room temperature until use.  Sub-samples of the tailings were submitted to an accredited laboratory for physical and chemical characterization including: moisture content, pH, particle size distribution, nutrients, and metals.  Laboratory quality control (QC) procedures included analysis of laboratory duplicates, laboratory control samples, certified and internal reference materials, and method blanks. The QC results met the laboratory’s data quality objectives (DQOs) with the following exceptions. Sample holding times were met for all parameters except available nitrate, which has a holding time of three days, but was analyzed nine days after sample collection. Laboratory duplicate results for selenium were slightly outside of the relative percent difference limit of 30%, which the laboratory attributed to sample heterogeneity. All other QC results for selenium (certified reference materials, laboratory control sample, and method blank) were within laboratory DQOs. The results for select parameters are provided in Table 2. A copy of the analytical report is provided in Attachment 1.  Table 2: Physical and Chemical Characterization of Soil/Tailings Mixtures used in the Toxicity Tests Parameter Units Lowest Detection Limit Sandy Tailings Silty Tailings Physical Tests     Moisture % 0.25 15.2 16.5 pH (1:2 soil:water) pH 0.10 8.46 8.51 Particle Size     % Gravel (>2 mm) % 0.10 <0.10 <0.10 % Sand (1.00 to 2.00 mm) % 0.10 <0.10 <0.10 % Sand (0.50 to 1.00 mm) % 0.10 0.94 0.15 % Sand (0.25 to 0.50 mm) % 0.10 17.8 3.65 124  Parameter Units Lowest Detection Limit Sandy Tailings Silty Tailings % Sand (0.125 to 0.25 mm) % 0.10 48.5 18.8 % Sand (0.063 to 0.125 mm) % 0.10 17.6 23.9 % Silt (0.0312 to 0.063 mm) % 0.10 8.23 21.8 % Silt (0.004 to 0.0312 mm) % 0.10 5.38 24.9 % Clay (<4 µm) % 0.10 1.46 6.77 Nutrients     Total Nitrogen % 0.020 <0.020 <0.020 Total Carbon % 0.1 0.6 0.4 Available Ammonium-N mg/kg dw 1.0 2.8 1.3 Available Nitrate-N mg/kg dw 1.0 <1.0 <1.0 Available Phosphate-P mg/kg dw 2.0 <2.0 <2.0 Available Potassium mg/kg dw 20 52 127 Available Sulfate-S mg/kg dw 4.0 8.5 120 Metals     Copper (Cu) mg/kg dw 0.50 1130 805 Selenium (Se) mg/kg dw 0.20 1.18 1.03 Vanadium (V) mg/kg dw 0.20 187 202 Notes: % = percent; > = greater than; mm = millimetres; < = less than; N = nitrogen; P = phosphorus; S = sulfur; mg/kg dw = milligrams per kilogram dry weight.  2.3.1.1 Preparation of Sandy Tailings  Sandy tailings were extracted from shipment containers and thoroughly homogenized before potting. Treatments containing control substrate (50%, 25%, 10%, and 0%) were manually mixed by hand to ensure evenness and uniform distribution between sandy tailings and control substrate. Mixing was finished when homogeneous color and texture was observed. Sandy tailings were moist when extracted from containers. To thoroughly mix tailings with the control substrate, trowels and spatulas were used to compress and loosen up all colloids with minimal effort.   2.3.1.2 Preparation of Silty Tailings  Most silty tailings received were moist or saturated with approximately one third of the silty tailings completely dry (Photo 1). Containers of wet silty tailings were allowed to drain with fines captured with Whatman 1 filter paper for twenty minutes. All silty tailings, irrespective of their moisture content, were mixed by trowel and left to air dry for two days before use. Once 125  dried, the fines captured in Whatman 1 filter paper were brushed back into their respective lot of silty tailings. A mixture ratio of approximately 4 parts of saturated (upon arrival) silty tailings to 1 part dried (upon arrival) tailings were manually mixed together before potting individual treatments. In the 100% silt tailing treatments, colloids of 0.5 cm were commonly found; however, these colloids were readily dissolved upon addition of hydration water.    Photo 1: Silty Tailings – Physical Appearance Before and After Mixing for the Toxicity Tests. Top Left: Illustration of the varying moisture status of silty tailings as received (left to right: completely saturated with excess fluid, saturated/fluids present, no fluids present); Top Right: mixing and air drying silt tailings in trays for two days; Bottom Left: preparation of 50% silty tailings treatment before mixing; Bottom Right: finished product of 50% silt tailings treatment before potting. Once the respective portions of silty tailings were measured (Photo 1-Bottom Left), the mixing process began with manually grinding the colloids by rolling pin. Abundant colloids were found in silty tailings due to its ability to retain moisture; these were difficult to break apart when dry. During mixing, a small portion of silty tailings were first ground, and then mixed and loosened by hand into a small portion of control substrate. After a quick visual inspection, this process continued until all silty tailings had been crushed with the rolling pin. The entire mixture was then stirred and reground to ensure the silty tailings were thoroughly mixed into the control substrate. As indicated in Photo 1 (Bottom Left and Bottom Right), the rolling pin was an effective tool for loosening up all silty colloids and thus sieves were not required in this process. The larger grain size of the control substrate prevented from crushing of diluted silty tailings to smaller particles.     126  2.3.2 Particle Size Control  Silica sand from Lane Mountain Company (Valley, Washington, USA) was purchased from a local supplier and used as the control substrate for the tailings and spiked sand substrate experiments. Two sand grades were selected to best match the grain size distribution of the sandy and silty tailings:   Silica sand grade LM#70 with a general grain size range of >0.103 to <0.300 mm was used for sandy tailings, which mostly fell within a grain size of 0.063 to 0.5 mm (Table 2).   Silica sand grade LM#125 with a general grain size range of <0.075 to <0.150 mm was used for silty tailings, which mostly fell within a grain size of 0.004 to 0.25 mm (Table 2).  The typical gradations of these sand classes are presented in Table 3; the product sheet is provided in Attachment 2.   Table 3: Typical Gradations of Silica Sands used as Control Substrates Sieve Size (mm) LM#70 (Used as Control for Sandy Tailings) LM#125 (Used as Control for Silty Tailings) Cumulative Percent Passing (%) Individual Percent Retained (%) Cumulative Percent Passing (%) Individual Percent Retained (%) 0.425 100 0 100 0 0.300 95-100 0-5 98-100 0-2 0.212 50-80 20-45 95-100 0-5 0.150 20-40 20-45 85-95 0-10 0.103 8-18 10-25 50-80 15-25 0.075 0-10 5-15 20-40 30-50 Balance NA 0-10 NA 20-40 Notes: mm = millimetre; % = percent; NA = not applicable. 2.3.3 Greenhouse Soil  Regular potting mix was used for the greenhouse soil experiment. This greenhouse soil was a peat mixture with carbonate grains of <0.5 centimetres (cm). In 2014, chemical analysis was conducted from bulk sampling of these soils. A summary of anions, cations and trace elements is provided in the table below (Table 4; see Attachment 3 for the original laboratory report).   Table 4: Summary of Anions, Cations and Trace Elements in Greenhouse Potting Mix Sample Anions (mg/L) Cations (mg/L) Trace Elements (mg/L) NO3 S P NH4 K Na Ca Mg Si Fe Cu Potting Mix 142.61 76.96 11.77 <1.81 66.47 11.50 68.13 34.03 <2.81 0.09 <0.01 Notes: mg/L = milligrams per litre; NO3 = nitrate; S = sulfur; P = phosphorus; NH4 = ammonium; K = potassium; Na = sodium, Ca = calcium; Mg = magnesium; Si = Silicon; Fe = iron; Cu = copper. 127  2.4 Test Vessels  Each test vessel consist of two clear plastic 1 litre (L) polypropylene containers. The bottom container held substrate and experimental plants while the top container was inverted (to form a lid) to maintain adequate humidity within the vessel. Clear containers allowed for visual inspection of presence of moisture, especially when substrates were constantly hydrated to water holding capacity (or near saturation) Clear inverted 1-L containers were applied to retain substrate moisture while allowing light penetration. These containers were taped into place to minimized moisture loss from the test vessels.   2.5 Nutrient Amendment Each substrate (tailings, greenhouse soil, or silica sand control) were hydrated with nutrient-amended water. The nutrients were added to hydration water as a fertilizer amendment, which is a pre-mixed solution supplied by the Greenhouse facility at the University of British Columbia. In the Greenhouse, fertilizer chemicals are dissolved in cool clean tap water and held in separate tanks, and then these solutions are combined in relatively equal parts, in addition to sodium bicarbonate (to maintain pH), using a series of pipes. In this way, the two fertilizer solutions are kept separate until mixed immediately before use to avoid co-precipitation of minerals (e.g., calcium carbonate). One fertilizer solution contains calcium nitrate, ammonium, potassium nitrate, calcium chloride, and iron diethylenetriamine pentaacetic acid (DTPA; a chelating agent). The other fertilizer solution contains magnesium sulfate, bicarbonate, micronutrients, and nitrogen:phosphorus:potassium mixes. In 2014, a sample of this fertilizer-amended water was analyzed; a summary of the anions, cations and trace elements is provided in Table 5 (see Appendix C for the original laboratory report).  Occasionally the mixing of the two streams in the Greenhouse is not equal or consistent such that day to day colour changes are observed in the amended water used in the Greenhouse. To avoid issues with inconsistency in nutrient amendments from using water directly from the Greenhouse taps, batches of each fertilizer solution were collected and stored specifically for use in this study. These batches were mixed by the analyst prior to use in setting up the treatments or when hydrating the test vessels.  Table 5: Summary of Anions, Cations and Trace Elements in Greenhouse Fertilizer Sample Anions (mg/L) Cations (mg/L) Trace Elements (mg/L) NO3 S P NH4 K Na Ca Mg Si Fe Cu Fertilizer 570.45 9.62 10.22 9.05 148.57 11.50 156.30 19.44 <2.81 1.17 0.29 Notes: mg/L = milligrams per litre; NO3 = nitrate; S = sulfur; P = phosphorus; NH4 = ammonium; K = potassium; Na = sodium, Ca = calcium; Mg = magnesium; Si = Silicon; Fe = iron; Cu = copper.  128  2.6 Test Initiation  2.6.1 Hydration of Substrates  Each substrate (and treatment, in the case of the tailings experiments) was hydrated to 70% of its water holding capacity, which is consistent with Environment Canada (2007) protocol. Nutrient-amended water was used to hydrate the substrates (Appendix C).  2.6.2 Preparation of Dilution Series Treatments (10%, 25%, and 50% dilutions) for the tailings experiments were prepared individually. For the 50% treatment, an aliquot of either sandy or silty tailings was mixed with an equal aliquot of the appropriate control substrate. The amount of the aliquot was based on dry weight (i.e., measured using as-is substrate [wet weight] but taking into account the original dry weight volume). For the other two treatments, the ratio of tailings to control substrate was 10% tailings to 90% control substrate for the 10% treatment and 25% tailings to 75% control substrate for the 25% treatment. No mixing was required for the 0% and 100% treatment (100% control substrate and 100% tailings, respectively).   After mixing, the treatments were hydrated to 70% of the water holding capacity (Environment Canada, 2007). Hydration after mixing was necessary because the silty tailings and the control substrates had different water holding capacities, such that water holding capacity decreased with proportion of tailings content in the treatment.   2.6.3 Preparation of Spiked Substrates Toxicity of copper was tested with the greenhouse soil and particle size controls (sandy and silty control substrates) to provide information on toxicity to plants using a more bioavailable form of copper than that expected in the tailings experiments. Seedling emergence and growth in the tailings experiments were compared to these positive controls. Total copper concentrations in sandy and silty tailings were 1130 and 805 mg/kg dw, respectively. The copper concentration in sandy tailings was selected as the target highest test concentration. For ease of preparation, the study used copper concentration of 1150 mg/kg dw as its 100% treatment. To achieve a calculated dilution series of 100%, 50%, 25%, and 10%, the greenhouse soil and control substrates were mixed proportionally to achieve the targeted concentrations of 1150, 575, 287.5, and 115 mg/kg dw. Copper sulfate pentahydrate was obtained from Fisher Scientific (C493-500; 100% of CuSO4●5H2O; ACS Certified). Analytically measured copper concentration in each substrate can be found in the Master’s thesis.  129  Each treatment and substrate was spiked separately. Copper sulfate was weighed using a small glass beaker, dissolved in fertilizer-amended water, and sprinkled evenly over the surface of the batch of substrate. Then the substrate was thoroughly mixed before distribution. The substrate was hydrated prior to spiking, and the amount of spiking solution added was taken into account during the hydration process.  2.6.4 Potting and Seeding  Each test vessel was uniquely identified with waterproof labels. The naming convention was as follows: substrate – treatment – species – replicate #. Substrate labels were: TSA (sandy tailings), TSI (silty tailings), SOIL (greenhouse soil), CON-TSA (sandy control substrate), and CON-TSI (silty control substrate). Treatment concentration labels were: 10 (10%), 25 (25%), 50 (50%), and 100 (100%). For the 0% treatment, the labels did not include a treatment concentration (i.e., CON-TSA-x-x; CON-TSI-x-x; CON-SOIL-x-x). The species were abbreviated P (lodgepole pine), G (bluebunch wheatgrass), and W (wild willow). To distinguish the replicates, the last identification code was a numeric value of 1 to 5. There were five replicates for each treatment in the sandy and silty tailings experiments and in the greenhouse soil experiment, and four replicates for each copper-spiked treatment in the spiked sandy and silty control substrate experiments.  Each test vessel was filled approximately half with substrate (i.e., ~500 mL). The test vessels were seeded on the same day as potting. A total of five seeds were placed in a circular format approximately an inch away from the edge of the pot. Each seed was firmly pressed into substrates and covered with substrate to a depth approximately twice the diameter of the seed.  Due to the amount of time to prepare the substrates and treatments, the experiments were set up over several days, with all species set up on the same day (e.g., sandy tailings experiment – all three species seeded on the same day). The test initiation and termination dates are provided in Table 6. Each replicate was watered before they were placed randomly into the growth chamber. As the test progressed, each replicate was checked for adequate hydration every two to three days and randomly placed back into the growth chamber   Table 6: Test Initiation and Termination Dates Experiment Test Initiation Test Termination Total Exposure Period Sandy Tailings July 18, 2016 August 29, 2016 42 days Silty Tailings July 21, 2016 September 1, 2016 42 days Spiked Sandy Control Substrate July 18, 2016 August 29, 2016 42 days Spiked Silty Control Substrate July 21, 2016 September 1, 2016 42 days Spiked Greenhouse Soil July 26, 2016 September 6 2016 42 days  130  2.7 Test Maintenance 2.7.1 Environmental Conditions A preliminary study explored the possibility of using available greenhouse benches for a controlled setting. However, it was later found that seasonal temperature variation (spring to summer months) and exposure to sunlight in the greenhouse soil significantly changed the irrigation regimes and potentially affected the hydrological fluxes of substrates within each pot. Therefore, growth chambers were used instead, which allowed for greater controls on air temperature, humidity, and lighting as per Environment Canada’s requirements as follows:   Air temperature: daily range, 24 ± 3°C during the day, 15 ± 3°C at night.  Humidity: >50%.  Lighting: chamber lighting were a combination of 16 fluorescent and 10 LED lights. The Philips fluorescent tube lights, F72T8 TL841 HO, are 72 inches or 6 feet long, high output with a lamp diameter in eighth-inch increments. These lights are designed to provide cool white light with Color Rendering Index (Nom) of 86, Color Code TL841 delivering 65 watts each. The Valoya G2 LEDs cover red-far red spectrum. The combination of fluorescent and LEDs mimics the spectral composition of sunlight. Lights are approximately 300 ± 100 µmol/(m2 s) adjacent to the level of the soil surface; set to 16 hour light, 8 hour dark.  2.7.2 Watering All test vessels were partially enclosed with clear 1 litre (L) plastic polypropylene containers as lids. Containers are taped together to maintain a steady closure with one millimeter gap to allow gas exchange. The vessels were checked and hydrated frequently as necessary to maintain substrate hydration to a minimum of 70% water-holding capacity. Test vessels were hydrated with fertilizer-amended water.  2.8 Test Completion After 42 days of exposure, all experiments were terminated. Qualitative observation of plant health were made, and representative photos taken. Aboveground biomass (shoots) were removed, measured for length and dry weight, and analyzed for metal concentrations. Belowground biomass (roots) were carefully removed from the soil, washed in deionized water several times, dried on paper towels, measured for length and dry weight, and analyzed for metal concentrations. Plants from each test replicate were pooled for chemical analysis.   131  There was low seedling emergence in all of the willow experiments, including the control treatments, indicating that the batch of seeds had low viability. Although the willow experiments were maintained throughout the same exposure period as the other plants (i.e., monitored and hydrated as necessary), the emerged willows were insufficiently developed to measure for biomass nor were plant tissue samples collected for chemical analysis.  2.8.1 Seedling Emergence and Survival Seedling emergence was measured as per Environment Canada (2007), which defined emergence as the height of first shoot rising 3 mm above soil or when the hypocotyl hook are observed above the surface of the soil medium. At test termination, the total number of emerged seedlings was counted for each treatment and divided by the number of seeds planted to calculate percent emergence. Seedling survival (post-emergence) was recorded as the total number of surviving seedlings divided by the number of emerged seedlings.    2.8.2 Biomass Measurements Shoots and roots were harvested by hand. Each plant was separated manually from the substrate and individually washed. Excess substrates were first shaken off by hand, and flushed in clean distilled water and later dried by paper towel. The process was repeated until all visible substrates were removed. Although intensive care was taken to remove all substrate particles from the roots by hand, it is possible that some substrate remained attached. The alternative, using extensive vibrations from a sonicator to remove most substrate particles, was considered, but discarded because this process will also break off fine roots resulting in a loss of biomass. Once cleaned and dried, plants were individually photographed and shoots and roots separated using a razor (Photo 2). Nitrile gloves were used at all times and to prevent contamination, gloves were washed between test vessels of the same treatment and replaced between treatments.  The lengths of the longest shoot and root of each plant were measured to the nearest millimeter. The mean lengths of shoots and roots in each test replicate were calculated, and used to calculate the overall means and standard deviations of shoot and root lengths for that treatment.   Each plant within a test replicate were then pooled for dry weight analysis. The separated shoots and roots were placed into volumetric flasks and oven tried at 105oC overnight (over 12 hours) to reach consistent weight. Once removed from oven, the samples were placed in desiccators and allowed to cool back to room temperature. The means and standard deviations of shoot and root dry weights were calculated for each treatment.  132   Photo 2: Wash station where individual plants within each test replicate were identified and washed separately until roots were clear of visible substrate. Once dried, plants were individually photographed and separated by shoot and root for drying. 2.8.3 Test Validity Criteria There are no standard test validity criteria for the test species used in this study. Environment Canada (2007) has suggested requirements of test validity for similar species:   For jack pine, the Environment Canada test validity criteria are as follows: 1) ≥60% mean seedling emergence, 2) ≥62 mm mean root length, 3) ≥44 mm mean shoot length. These criteria could be reasonably applied to lodgepole pine.  For northern wheatgrass, the Environment Canada test validity criteria is as follows: 1) ≥70% mean seedling emergence, 2) ≥90% survival of emerged seedlings, 3) ≤10% of control seedlings exhibiting phytotoxicity or developmental abnormalities, 4) ≥110 mm mean root length, 5) ≥100 mm mean shoot length. These criteria could be reasonably applied to bluebunch wheatgrass.  There are no test validity criteria for willow (Salix spp.) in either Environment Canada test method or ASTM E1963. For the purposes of identifying that acceptable seedling emergence and growth can be achieved, it is proposed that these test validity criteria are applied to the greenhouse soil control. It is reasonable to assume that successful seedling emergence and growth should occur in the greenhouse soil. In contrast, we have no information about whether the silty or sandy control substrates would provide acceptable growing conditions for the test species. Therefore, applying the test validity criteria to the silty or sandy controls may not be appropriate.    133  2.8.4 Copper Concentrations in Substrates All treatments were prepared in large individual batches with sub-samples collected just before potting. To ensure that the calculated copper dilution values match those in the experiment, sub-samples were analyzed for copper concentration using the “rapid we digestion method” (Peguerul et al, 2015) as described below. Samples accidentally were not collected at test completion.   Substrate samples were prepared for “rapid wet digestion method (Pequerul et al., 2015) identical to the wet digestion method used for plant tissue analysis in this study. Since the objective of this study is to determine copper uptake by plants, a total copper digestion (such as Aqua Regia) would not be suitable as not all copper in tailings is available to plant roots. The objective of using a plant tissue specific digestion method on substrates is to provide experimental consistency in determining copper extracted in plant tissue. Using a different digestion method for plant tissues and substrates may not represent the precise copper exposed to plants.   Once dry weights are measured, each sample is given 5 mL of concentrated nitric acid and 4 mL of (33%) hydrogen peroxide, gently stirred and allowed to oxidize at room temperature for approximately one hour. The samples are then heated on a hot plant and a strong effervescence was produced followed by dense brown fumes. When fumes are less dense, the samples are cooled. To ensure adequate digestion of samples, another 4 mL of hydrogen peroxide is added, reheated and cooled again. Samples then receive 5 mL of concentrated hydrochloric acid, and diluted up to 50 mL with 4% nitric acid (as solution matrix) before collecting the filtrate.  Elemental analyses of metals was analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) in the Department of Geological, Atmospheric and Ocean Sciences at the University of British Columbia. Laboratory quality control samples included procedural blanks, laboratory blanks, and laboratory duplicates. With each set of 25 samples, a laboratory blank and one of the standard solutions were analyzed to track machine response. The analytical detection limit for copper was 0.1 mg/kg dw (M. Soon, personal communication, Nov. 28, 2016).  2.8.5 Copper Concentrations in Plant Tissue Plant tissues were dried in volumetric flasks and prepared for “rapid wet digestion (Peguerul et al. 2015). Metal analysis proceeded according to the same methods as for soil samples. There was insufficient plant tissue to analyze in the copper-spiked sandy and silty control substrate experiments; therefore, plant tissue chemistry data are limited to sandy and silty tailings experiments, and to the spiked greenhouse soil experiment.   134  2.9 Statistical Analysis  Seedling emergence and survival were summarized (mean ± SD); emergence was plotted; no further statistical analysis was conducted for these endpoints.  Shoot and root length and dry weight were summarized (mean ± SD), plotted, and statistically analyzed using one-way analysis of variance (ANOVA) and Dunnett’s post-hoc test (pairwise comparison to 0% control) to identify no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for each media type (i.e., sandy tailings, silty tailings, greenhouse soil). NOEC is the highest concentration that does not have a statistically significant effect compared to the control, and LOEC is the lowest concentration that has a statistically significant effect compared to the control. Assumptions of normality and equality of variances were confirmed using Shapiro-Wilk’s and Levene’s tests. Significance was determined using an alpha of 0.05.  To evaluate emergence, survival and growth effects related to particle size, each of the three experiments (un-spiked sandy and silty control substrates, and greenhouse soil) were statistically analyzed using one-way ANOVA and Tukey’s test.  Plant tissue chemistry was summarized (mean ± SD). Significant differences among treatments were evaluated with one-way ANOVA and Dunnett’s post-hoc test. Tissue concentrations were plotted against measured nominal substrate concentration to evaluate whether accumulation of copper increased with increasing proportion of tailings or copper sulfate in the greenhouse soil.   Chapter 3: Results 3.1 Confirmatory Chemical Analysis  The analytical chemistry report for fertilizer water, substrate chemistry at test initiation, and plant tissue chemistry at test completion is provided in Attachment 5.  Measured concentrations in treatment substrates were within 20% of nominal concentrations at Day 0 of experiment, with the exception of the copper-spiked greenhouse soil, which were greater (Table 7).        135  Table 7: Nominal vs Measured Copper Concentrations in the Substrates at Test Initiation Treatment Sandy Tailings Sandy Substrate Silty Tailings Silty Substrate Greenhouse Soil NOM* MEAS NOM* MEAS NOM* MEAS NOM* MEAS NOM* MEAS 0% (control) 2.6 2.6 2.6 2.6 4.3 4.3 4.3 4.3 34 34 10% 115 119 117 122 83 86 119 124 149 nd (b) 25% 284 279 289 275 203 211 291 305 321 550 50% 566 663 576 575 404 448 577 576 609 813 100% 1130 1253 1150 1199 805 889 1150 1344 1184 1740 Notes: Concentrations are mg/kg dw. * = Nominal copper concentration is based on previously measured (Table 2 for tailings) or spiked (for spiked substrates), with adjustment for the contribution of copper from the control substrates. For the control substrates (0%) it was assumed that measured = nominal as control substrates form the basis of the dilution series. (b) = Sample lost; no data available (nd). NOM = nominal; MEAS = measured; % = percent.  3.2 Seedling Emergence and Survival  Raw data for seedling emergence and survival in the five experiments are provided in Attachment 6.  Emergence:  Poor seedling emergence occurred in the willow control treatments; therefore, test not completed (i.e., no biomass data were collected from the willow experiments).  The adopted test validity criteria for seedling emergence (≥60% mean seedling emergence for pine, ≥70% mean seedling emergence for grass) was met in all control treatments (Table 8): Table 8: Seedling Emergence in Lodgepole Pine and Bluebunch Wheatgrass Experiments (Mean ± SD) Treatment Lodgepole Pine Sandy Tailings Copper-spiked Sandy Substrate Silty Tailings Copper-spiked Silty Substrate Copper-spiked Greenhouse Soil 0% (control) 96 ± 9% 96 ± 9% 100% 100% 100% 10% 100% 20 ± 16% 80 ± 28% 30 ± 26% 100% 25% 100% 5 ± 10% 96 ± 9% 0% 100% 50% 96 ± 9% 6 ± 10% 92 ± 11% 0% 96 ± 9% 100% 96 ± 9% 0% 100% 0% 100% 136   Treatment Bluebunch Wheatgrass Sandy Tailings Copper-spiked Sandy Substrate Silty Tailings Copper-spiked Silty Substrate Copper-spiked Greenhouse Soil 0% (control) 96 ± 9% 96 ± 9% 96 ± 9% 96 ± 9% 80 ± 14% 10% 84 ± 9% 5 ± 10% 88 ± 18% 20 ± 28% 80 ± 20% 25% 88 ± 11% 0% 96 ± 9% 0% 92 ± 11% 50% 96 ± 9% 0% 92 ± 11% 0% 96 ± 9% 100% 96 ± 9% 0% 96 ± 9% 0% 84 ± 17% Notes: The tailings and substrate experiments shared the same control (e.g., 0% sandy tailings = un-spiked sandy substrate).   All dilution treatments of the sandy and silty tailings mixtures, as well as all treatments of the spiked greenhouse soil, had similar, and high, percentage of seedling emergence (≥80%), indicating no effect of dilution on seedling emergence in any of these treatments (Figure 3, Figure 4).  All sandy and silty control substrates in the copper-spiked experiments also had high seedling emergence.   However, all dilutions above the control in the copper-spiked sandy and silty substrates had very low seedling emergence (79 to 95% effect size at the 10% treatment), indicating that the copper spiked into these substrates was likely highly toxic to the plants.  Figure 3: Seedling Emergence in Lodgepole Pine Experiments (Mean ± SD) 137     Survival:  The adopted test validity criterion for survival was met in un-spiked greenhouse soil control treatment, in that there was 96 ± 9% (mean ± SD) survival in both pine and grass trials.  Note that only grass had a survival test validity criterion of ≥90% mean survival.  In the particle size dilution experiments, survival in particle size controls were 100% for both pine and grass in silty control substrate, and 100% for pine and 88 ± 11% for grass in sandy control substrate.  Survival was greater than 80% in all treatments (sandy tailings, silty tailings and greenhouse soils). However, survival was significantly reduced in copper-spiked sand and silt treatments. This reduction was observed in 10% copper treatments and declined further in higher copper concentrations (Table 9). Table 9: Seedling Survival in Lodgepole Pine and Bluebunch Wheatgrass Experiments (Mean ± SD) Treatment Lodgepole Pine Sandy Tailings Copper-spiked Sandy Substrate Silty Tailings Copper-spiked Silty Substrate Copper-spiked Greenhouse Soil 0% (control) 100% 100% 100% 100% 96 ± 9% 10% 100% 75 ± 50% 93 ± 15% 75 ± 50% 100% 25% 100% 25 ± 50% 100% 0% 96 ± 9% 50% 100% 0% 100% 0% 100% 100% 100% 0% 100% 0% 84 ± 26%   Figure 4: Seedling Emergence in Bluebunch Wheatgrass Experiments (Mean ± SD) 138  Treatment Bluebunch Wheatgrass Sandy Tailings Copper-spiked Sandy Substrate Silty Tailings Copper-spiked Silty Substrate Copper-spiked Greenhouse Soil 0% (control) 88 ± 11% 88 ± 11% 100% 100% 96 ± 9% 10% 91 ± 12% 25 ± 50% 100% 50 ± 58% 96 ± 9% 25% 100% 0% 96 ± 9% 0% 92 ± 11% 50% 100% 0% 100% 0% 96 ± 9% 100% 95 ± 11% 0% 98 ± 4% 0% 90 ± 22% Notes: The tailings and substrate experiments shared the same control (e.g., 0% sandy tailings = un-spiked sandy substrate).  3.3 Shoot and Root Length  Raw data for shoot and root length and dry weight are provided in Attachment 7.  3.3.1 Lodgepole Pine  Average shoot lengths of lodgepole pine in the un-spiked control substrates and un-spiked greenhouse soil ranged from 38 mm (greenhouse soil control) to 41 mm (sandy control substrate), which were just under the test validity criterion for jack pine of 44 mm. While both jack pine and lodgepole pine seedlings may have similar growth rates in comparable substrates, it is irrelevant to compare species of different climatic zones (e.g. boreal forests vs sub-boreal and temperate forests). The difference observed in this study is relatively small and could be hindered by substrate texture. Thus literature values of jack pine should only be considered a reference, not ideal to determine the confidence of this study.    Average root lengths were greater than the test validity criterion of 62 mm for jack pine, and ranged from 63 mm in un-spiked greenhouse soil to 136 mm in un-spiked sandy control substrate.  Sandy tailings and silty tailings had good growth, but there was a slight trend of declining growth with increasing proportion of tailings content (Figure 5), and the highest treatment (100%) usually had significantly lower average shoot and root lengths (24% effect size for shoot length, and 62-66% effect size for root length; Table 10).   No significant reduction in shoot or root lengths compared to the control were observed in any of the copper-spiked greenhouse soil treatments, including the 100% spiked treatment, which had a measured copper concentration of 1,740 mg/kg dw.  There were no significant differences in shoot length among the un-spiked sandy or silty substrates or the un-spiked greenhouse soil. Root length was significantly lower in the 139  greenhouse soil control compared to the sandy control substrate, but neither were significantly different from the silty control substrate.  Length was not significantly lower in the un-spiked sandy or silty substrates compared to the un-spiked greenhouse soil (i.e., there was no obvious effect of particle size on plant growth).  Conclusion: reduction in shoot and root length was observed in 100% tailings, but the effect did not appear to be related to particle size.  Table 10: NOECs and LOECs for Lodgepole Pine Shoot and Root Length Dataset Statistical Test p values NOEC LOEC % Effect at LOEC Sandy Tailings - Shoot Length One-way ANOVA / Dunnett’s p < 0.05 50% 100% 24% Sandy Tailings - Root Length One-way ANOVA / Dunnett’s p < 0.05 50% 100% 66% Silty Tailings - Shoot Length One-way ANOVA / Dunnett’s p = 0.029 50% 100% 24% Silty Tailings - Root Length One-way ANOVA p = 0.093 100% >100% na Spiked Greenhouse Soil - Shoot Length One-way ANOVA p = 0.852 100% >100% na Spiked Greenhouse Soil - Root Length One-way ANOVA p = 0.941 100% >100% na Notes: NOEC = no observed effect concentration; LOEC = lowest observed effect concentration; % = percent; ANOVA = analysis of variance; < less than; na = not applicable.  3.3.2 Bluebunch Wheatgrass   Average shoot and root lengths of bluebunch wheatgrass in the un-spiked controls substrates and un-spiked greenhouse soil were greater than the test validity criteria for northern wheatgrass. Average shoot length ranged from 225 mm (un-spiked sandy control substrate) to 309 mm (un-spiked silty control substrate), which were greater than the criterion of 100 Figure 5 Shoot and Root Lengths of Lodgepole Pine Exposed to Sandy and Silty Tailings, Copper-spiked Greenhouse Soil, and Copper-Spiked Sandy and Silty Substrates 140  mm for northern wheatgrass. Average root lengths ranged from 138 mm in un-spiked greenhouse soil to 276 mm in un-spiked silty control substrate, which were greater than the criterion of 110 mm for northern wheatgrass.  There was a more pronounced declining trend in growth with increasing proportion of tailings content than that observed for pine length (Figure 6). In general, the lowest treatment that was significantly different from the control and had a >20% effect size was the 25% dilution treatment (Table 11).   In the copper-spiked greenhouse soil experiment, there was no significant difference in shoot length but a 32% significant reduction in root length in the 100% treatment, which had a measured copper concentration of 1,740 mg/kg dw.  Variable results were observed when comparing un-spiked sandy or silty substrates and un-spiked greenhouse soil. The lowest shoot growth was observed in the un-spiked sandy control substrate, which was also significantly different from the un-spiked silty control substrate. The un-spiked silty control substrate had the highest average shoot length and significantly different root length. In other words, growth was highest in the un-spiked silty control substrate, and lowest in the un-spiked greenhouse soil, with the growth in the un-spiked sandy control substrate in between the other two substrates. Therefore, no obvious negative effect of particle size related to the tailings was observed on grass growth.  Conclusion: Bluebunch wheatgrass was more sensitive than lodgepole pine with significant reduction in shoot and root length in 25% tailings, and this effect was not likely related to particle size.   Figure 6 Shoot and Root Lengths of Bluebunch Wheatgrass Exposed to Sandy and Silty Tailings, Copper-spiked Greenhouse Soil, and Copper-Spiked Sandy and Silty Substrates 141  Table 11: NOECs and LOECs for Bluebunch Wheatgrass Shoot and Root Lengths Dataset Statistical Test p values NOEC LOEC % Effect at LOEC Sandy Tailings - Shoot Length One-way ANOVA / Dunnett’s p < 0.05 10% 25% 31% Sandy Tailings - Root Length One-way ANOVA / Dunnett’s p < 0.05 10% 25% 46% Silty Tailings - Shoot Length One-way ANOVA / Dunnett’s p < 0.05 <10% 10% 15% Silty Tailings - Root Length One-way ANOVA / Dunnett’s p < 0.05 10% 25% 64% Spiked Greenhouse Soil - Shoot Length One-way ANOVA p = 0.458 100% >100% na Spiked Greenhouse Soil - Root Length One-way ANOVA / Dunnett’s p = 0.015 50% 100% 32% Notes: NOEC = no observed effect concentration; LOEC = lowest observed effect concentration; % = percent; ANOVA = analysis of variance; < less than; na = not applicable.  3.4 Shoot and Root Dry Biomass Weight 3.4.1 Lodgepole Pine  Treatments with sandy tailings and silty tailings had good growth, but there was a slight trend of declining growth with increasing proportion of tailings content in sandy tailings (Figure 7). The highest dilution treatment (100%) had significantly lower average shoot dry weight (50% effect) than the control and the 50% treatment had significantly lower average root dry weight (35% effect) than the control (Table 12).  No significant difference in shoot or root dry weights were observed in treatments containing 100% silty tailings (with a nominal copper concentration of 805 mg/kg dw) or in 100% copper-spiked greenhouse soil (nominal copper concentration of 1150 mg/kg dw) compared with their controls.  Average shoot and root dry weight was lowest in the un-spiked greenhouse soil, then the un-spiked silty control substrate. The highest average dry weight was observed in the un-spiked sandy control substrate. Shoot dry weight was significantly lower in un-spiked greenhouse soil compared to un-spiked sandy substrate, and shoot dry weight in un-spiked silty substrate was not significantly different from either un-spiked greenhouse soil or un-spiked sandy substrate. Root dry weight was significantly lower in un-spiked greenhouse soil compared to un-spiked sandy substrate, and the two un-spiked control substrates were significantly different from each other. These results suggested that particle size in the tailings did not negatively affect shoot dry weight relative to greenhouse soil, but that the silty substrate may have affected root dry weight compared to the sandy substrate.  Conclusion: reduction in shoot dry weight in in 50% and 100% treatments in sandy tailings, but, not silty tailings. Such effects do not appear to be related to particle size. 142    Table 12: NOECs and LOECs for Lodgepole Pine Shoot and Root Dry Weights Dataset Statistical Test Results NOEC LOEC % Effect at LOEC Sandy Tailings - Shoot Dry Weight One-way ANOVA / Dunnett’s p < 0.05 50% 100% 50% Sandy Tailings - Root Dry Weight One-way ANOVA / Dunnett’s p < 0.05 25% 50% 35% Silty Tailings - Shoot Dry Weight One-way ANOVA / Dunnett’s p = 0.421 100% >100% na Silty Tailings - Root Dry Weight One-way ANOVA p = 0.671 100% >100% na Spiked Greenhouse Soil - Shoot Dry Weight One-way ANOVA p = 0.760 100% >100% na Spiked Greenhouse Soil - Root Dry Weight One-way ANOVA p = 0.774 100% >100% na Notes: NOEC = no observed effect concentration; LOEC = lowest observed effect concentration; % = percent; ANOVA = analysis of variance; < less than; na = not applicable.  3.4.2 Bluebunch Wheatgrass  Shoot and root dry weight in the sandy and silty tailings were significantly reduced at 10% tailings, with effect sizes greater than or equal to 43% (Figure 8, Table 13).  No significant differences in shoot or root dry weights were observed in 100% copper-spiked greenhouse soil (nominal copper concentration of 1150 mg/kg dw).  Average shoot and root dry weight was lowest in the un-spiked greenhouse soil, then the un-spiked sandy control substrate. The highest average dry weight was observed in the un-spiked silty control substrate. Significant differences in shoot dry weight were observed between greenhouse soil control and silty control substrate, and between the two control substrates. Significant differences in root dry weight were observed between un-spiked greenhouse soil and both control substrates, but not between the control substrates. These Figure 7: Shoot and Root Dry Weights of Lodgepole Pine Exposed to Sandy and Silty Tailings, Copper-spiked Greenhouse Soil, and Copper-Spiked Sandy and Silty Substrates 143  results suggested that particle size in the tailings did not negatively affect root dry weight relative to greenhouse soil, but that the sandy substrate may have affected shoot dry weight compared to the silty substrate.  Conclusion: reduction in shoot and root dry weight in 10% tailings; effects do not appear to be related to particle size.   Table 13: NOECs and LOECs for Bluebunch Wheatgrass Shoot and Root Dry Weights Dataset Statistical Test Results NOEC LOEC % Effect at LOEC Sandy Tailings - Shoot Dry Weight One-way ANOVA / Dunnett’s p < 0.05 <10% 10% 45% Sandy Tailings - Root Dry Weight One-way ANOVA / Dunnett’s p < 0.05 <10% 10% 52% Silty Tailings - Shoot Dry Weight One-way ANOVA / Dunnett’s p < 0.05 <10% 10% 46% Silty Tailings - Root Dry Weight One-way ANOVA / Dunnett’s p < 0.05 <10% 10% 43% Spiked Greenhouse Soil - Shoot Dry Weight One-way ANOVA p = 0.622 100% >100% na Spiked Greenhouse Soil - Root Dry Weight One-way ANOVA p = 0.735 100% >100% na Notes: NOEC = no observed effect concentration; LOEC = lowest observed effect concentration; % = percent; ANOVA = analysis of variance; < less than; na = not applicable.  3.5 Plant Tissue Chemistry  Copper concentrations in shoot and root tissue were lower in the un-spiked control substrates than in the dilution treatments containing tailings or in the spiked greenhouse soil treatments Table 14). In general, treatments containing tailings had significantly higher copper tissue concentrations than in the un-spiked control substrates. Furthermore, copper concentrations in tissue increased proportionally to the concentration of tailings in substrate (Figure 9 for lodgepole pine and Figure 10 for bluebunch wheatgrass).  Figure 8 Shoot and Root Dry Weights of Bluebunch Wheatgrass Exposed to Sandy and Silty Tailings, Copper-spiked Greenhouse Soil, and Copper-Spiked Sandy and Silty Substrates 144  Table 14: Measured Copper Concentrations in Shoots and Roots of Lodgepole Pine and Bluebunch Wheatgrass at Test Completion Treatment Lodgepole Pine Sandy Tailings Silty Tailings Greenhouse Soil Shoots Roots Shoots Roots Shoots Roots 0% (control) 12.5 ± 3.4 32.6 ± 8.2 6.9 ± 4.0 21.2 ± 13.6 16.3 ± 12.9 58.1 ± 27.5 10% 41.6 ± 26.1* 719 ± 95.2* 25.3 ± 10.8* 597 ± 349* 21.7 ± 18.2 152 ± 149 25% 22.7 ± 3.8 1245 ± 536* 19.7 ± 4.0 680 ± 162* 23.9 ± 7.3 296 ± 137* 50% 30.2 ± 4.8* 1569 ± 323* 27.5 ± 8.3* 734 ± 69.3* 19.6 ± 4.4 290 ± 79.9* 100% 69.0 ± 27.8* 1028 ± 207* 27.8 ± 9.3* 870 ± 96.5* 59.3 ± 69.7 862 ± 1124*  Treatment Bluebunch Wheatgrass Sandy Tailings Silty Tailings Greenhouse Soil Shoots Roots Shoots Roots Shoots Roots 0% (control) 23.8 ± 5.0 21.2 ± 9.9 16.0 ± 4.4 13.2 ± 6.0 20.5 ± 3.5 48.4 ± 23.2 10% 94.2 ± 35.5* 510 ± 90.3* 44.5 ± 6.3* 445 ± 180* 33.2 ± 6.2 447 ± 124 25% 179 ± 8.6* 770 ± 174* 58.1 ± 7.3* 630 ± 119* 54.8 ± 13.2* 881 ± 222* 50% 219 ± 77.9* 811 ± 101* 79.7 ± 9.9* 672 ± 208* 54.0 ± 22.3* 1205 ± 363* 100% 123 ± 46.8* 838 ± 280* 94.0 ± 33.7* 696 ± 147* 54.1 ± 9.1* 1997 ± 297*    Notes: Concentrations are mg/kg dw. Mean ± standard deviation are presented (n=5). * = statistically significant from control. % = percent. Figure 9 Measured Copper Concentration in Shoots and Roots of Lodgepole Pine at Test Completion Compared to Measured Copper Concentration in the Substrate 145    Chapter 4: Discussion The purpose of the greenhouse soil experiment was to provide an indication of copper toxicity to plants in a media that plants were expected to survive and grow well in. Toxicity in greenhouse soils are expected to be suppressed due to the nature of high organic peat composition which naturally chelates copper in solution. Unlike mineral substrates which lack such chelation, plants in copper-spiked greenhouse soils are less exposed to copper. Thus toxicity was generally not observed in greenhouse soils until a measured copper concentration of 813mg/kg dw (corresponding to 50% treatment) where root lengths were reduced. In contrast, the copper-spiked control substrate experiments yielded high toxicity responses. These experiments were intended to generate concentration-response curves that could be compared to the tailings experiments. However, copper appeared to be more toxic in copper-spiked substrates compared to the tailings, with very pronounced effects on seedling emergence, survival, and growth observed at the lowest treatment tested (10%). In these experiments, appreciable toxicity was observed at the lowest measured concentration in media of 122 mg/kg dw.   In the tailings experiment, pine exhibited the highest tolerance with a toxicity threshold starting at 889 mg Cu/kg dw in the medium. As a naturally slow growing species, pines are largely less affected due to the length of exposure and slow response to toxins. In contrast, grasses with quicker growth rates develop finer root architectures resulting in greater surface area to volume ratio and ultimately leading to greater exposure to metals in substrate. This may explain the significant toxicity observed in grasses growing at concentrations above 86 mg/kg dw. While field observations by Golder Associates Ltd. have found grasses growing in areas assumed to contain copper concentrations up to 1130 mg/kg dw, there are a number of confounding factors to consider before comparing field and lab findings. Firstly, the use of plastic polypropylene experiment vessels (at 70% substrate moisture) could increase the exposure by simply limiting Figure 10: Measured Copper Concentration in Shoots and Roots of Bluebunch Wheatgrass at Test Completion Compared to Measured Copper Concentration in the Substrate 146  roots to grow within the vessel. Secondly, test vessels do not fully represent growth in a natural environment. Soil physical and biological properties such as aeration and microbial activity respectively are not present at the natural state. As indicated in Section 3.3, all substrates are dried, homogenized and later rehydrated. Factors such as spatial confinement and loss of microbial activity could largely affect fast growing species (i.e. grasses) in this study.   Chapter 5: Conclusion 5.1 Lodgepole Pine The results of the lodgepole pine experiment are summarized below relative to the study questions.   Do the copper concentrations found in the soil/tailings mixtures inhibit seedling emergence or plant growth (above/belowground biomass generation)? Seedling emergence was high across all treatment groups in the experiments with soil/tailings mixtures, indicating that concentrations of copper or other metals in the soil/tailings mixtures did not inhibit seedling emergence. In contrast, seedling emergence was significantly reduced in copper-spiked sandy and silty control substrates, which suggested that the copper sulfate spiked into these control substrates was highly bioavailable and toxic to the pine seeds. The highest copper concentration in tailings associated with no significant effect on seedling emergence was 1,253 mg/kg dw in the 100% treatment of the sandy tailings experiment.  Shoot and root length was significantly affected in the 100% tailings treatments with an effect size of greater than or equal to 24%, with the exception of root length in the silty tailings, which was not significantly different from control in the 100% treatment. The lowest copper concentration in tailings associated with a significant effect on length was 889 mg/kg dw in the 100% treatment of the silty tailings experiment.  Effects on shoot and root dry weights were observed only in the sandy tailings. Shoot dry weight was significantly reduced by 50% in the 100% treatment, and root dry weight was significantly reduced by 35% in the 50% treatment. The lowest copper concentration in tailings associated with a significant effect on dry weight was 663 mg/kg dw in the 50% treatment of the sandy tailings experiment. However, no significant effects on shoot or root dry weight were observed in the silty tailings experiment in the 100% treatment, which corresponds to a copper concentration in tailings of 889 mg/kg dw.  147   Do differences in particle size of the soil/tailings mixture affect seedling emergence or plant growth (above/belowground biomass generation)? Seedling emergence did not appear to be affected by particle size, as high emergence was observed in all media (sandy or silty control substrate, and greenhouse soil). Growth appeared to be highest in the un-spiked sandy control substrate, and lowest in the greenhouse soil. One plausible explanation is that greenhouse soils could contain inhibitory soil microflora absent in both tailings. Overall, the results do not suggest differences in particle size could explain the lower growth observed in the sandy tailings in the 50% and 100% treatments.   Do plants germinated in soil/tailings mixtures take up copper in a concentration-dependent manner (i.e., increasing copper concentrations in shoots and roots with increasing copper in the substrate)? No, although the copper concentrations in shoots and roots are higher in the treatments containing tailings than in the control treatments (un-spiked sandy and silty control substrates), there does not appear to be any patterns of increasing tissue concentrations with increasing copper concentration in the substrates.   If the plants germinated in soil/tailings mixtures take up more copper than plants germinated in control substrates, do the resulting tissue concentrations correspond to toxicity to the plant (i.e., inhibition of seedling emergence or plant growth)?  As stated above, seedling emergence was not affected in any tailings treatment. Therefore, the highest copper concentrations in tissue associated with no significant effects on seedling emergence was 69.0 mg/kg dw in shoots and 1,028 mg/kg dw in roots from the 100% treatment of the sandy tailings experiment.  Shoot and root length was significantly affected in the 100% tailings treatments. The lowest copper concentrations in tissue associated with significant effects on length was 27.8 mg/kg dw in shoots and 870 mg/kg dw in roots from the 100% treatment of the silty tailings experiment. Significant reduction in shoot dry weight was observed in the 100% treatment of the sandy tailings experiment; the corresponding shoot copper concentration was 69.0 mg/kg dw. Significant reduction in root dry weight was observed in the 50% treatment, with a corresponding root copper concentration of 1,569 mg/kg dw.  No significant effects on shoot or root dry weight was observed in the silty tailings experiment in the 100% treatment, which corresponds to copper concentrations of 27.8 mg/kg dw in shoots and 870 mg/kg dw in roots.    148  5.2 Bluebunch Wheatgrass The results of the bluebunch wheatgrass experiment are summarized below relative to the study questions.   Do the copper concentrations found in the soil/tailings mixtures inhibit seedling emergence or plant growth (above/belowground biomass generation)? As with lodgepole pine, seedling emergence was high across all treatment groups in the experiments with soil/tailings mixtures with at least 80% mean emergence, indicating that concentrations of copper or other metals in the soil/tailings mixtures did not inhibit seedling emergence to this plant species. In contrast, seedling emergence was significantly reduced in copper-spiked sandy and silty control substrates, which suggested that the copper sulfate spiked into these control substrates was highly bioavailable and toxic to the grass seeds. The highest copper concentration in tailings associated with no significant effect on seedling emergence was 1,253 mg/kg dw in the 100% treatment of the sandy tailings experiment.  Shoot and root length was significantly affected with a greater than 25% effect size in the 25% treatment. The lowest copper concentration in tailings associated with a significant effect on length was 211 mg/kg dw in the 25% treatment of the silty tailings experiment.  Shoot and root dry weight was a more sensitive endpoint than length, with significant effects observed in the 10% treatment. The lowest copper concentration in tailings associated with a significant effect on dry weight was 86 mg/kg dw in the 10% treatment of the silty tailings experiment.   Do differences in particle size of the soil/tailings mixture affect seedling emergence or plant growth (above/belowground biomass generation)? Seedling emergence did not appear to be affected by particle size, as high emergence was observed in all media (sandy or silty control substrate, and greenhouse soil). Shoot length appeared to be highest in the un-spiked silty control substrate, and lowest in the un-spiked sandy control substrate. This pattern was also observed for root length; however, whereas shoot length in un-spiked greenhouse soil was not significantly different from either sandy or silty control substrates, root length was markedly lower. For dry weight, shoot and root dry weight were highest in un-spiked silty control substrate and lowest in un-spiked greenhouse soil. These results suggest that the grass preferred the silty control substrate over the sandy control substrate or the greenhouse soil, which was predominantly peat. While greenhouse soil does naturally contain 30mg/kg dw of copper more than sandy or silty control substrates, the copper is long chelated from soil solution and should not have any affect on either species.Overall, the 149  results do not suggest differences in particle size could explain the lower growth in shoot and root length observed in the 25% treatments or in shoot and root dry weight in the 10% treatments.   Do plants germinated in soil/tailings mixtures take up copper in a concentration-dependent manner (i.e., increasing copper concentrations in shoots and roots with increasing copper in the substrate)? No, similar results were observed with grass as with pine. Although the copper concentrations in shoots and roots are higher in the treatments containing tailings than in the control treatments (un-spiked sandy and silty control substrates), there does not appear to be any patterns of increasing tissue concentrations with increasing copper concentration in the substrates.    If the plants germinated in soil/tailings mixtures take up more copper than plants germinated in control substrates, do the resulting tissue concentrations correspond to toxicity to the plant (i.e., inhibition of seedling emergence or plant growth)?  As stated above, seedling emergence of wheatgrass was not affected in any tailings treatment. Of surviving seedlings, the highest copper concentrations in tissue associated with no significant effects on seedling emergence was 123 mg/kg dw in shoots and 838 mg/kg dw in roots from the 100% treatment of the sandy tailings experiment.  Shoot and root length was significantly affected with a greater than 25% effect size in the 25% tailings treatments. The lowest copper concentrations in tissue associated with significant effects on length was 58.1 mg/kg dw in shoots and 630 mg/kg dw in roots from the 25% treatment of the silty tailings experiment.  Significant reduction in shoot and root dry weights were observed in the 10% tailings treatments. The lowest copper tissue concentrations were 44.5 mg/kg dw in shoots and 445 mg/kg dw in roots from the 10% treatment of the silty tailings experiment.    Chapter 6: Recommendations for Further Study This study was designed following Environment Canada’s Biological Test Methods, however, these methods were not tailored to account for the physical and chemical properties of tailings. The substrates collected for this experiment were pure/unmixed, debris-free sandy and silty tailings not present in Hazeltine Creek. Thus it would only be appropriate to interpret these 150  findings as indicators where field concentration of tailings closely matches treatment concentrations in this study.  Experimenting in growth chambers and vessels without drain holes could impose significant challenges to plant health, growth and exposure to tailings. Environment Canada has listed specific conditions for growth chambers, however these conditions do not account for seasonal temporal variations at Mount Polley Mine. In the field, survival rates of both pine and grasses are likely to decrease due to harsh winters and wet spring seasons during the first season of growth. The lack of canopy shielding, poor anchorage and substrate saturation could lead to higher mortality of pine seedlings unrelated to the presence of tailings. Field trials are recommended as it provides improved insight on regeneration and resilience to local climatic conditions.  Experiment vessels may also contribute to unfavorable exposure conditions within each treatment. Variability in hydrological regimes were observed among replicates of each sandy and silty treatments. Although partially enclosed to maintain moisture, surface crusting is commonly found in treatments of 50 to 100% silty tailings. Similar crusting was observed in the first 5 millimeters of sandy tailings, however with much less mechanical resistance. While surface drying is also observed in field investigations, the lack of drainage holes in experiment vessels could inhibit root growth. Field investigations from Golder Associates Ltd. have indicated that grasses are capable of growing well in tailings assumed to be of 1150 mg/kg dw. The concern here is that test vessels (as per Environment Canada’s Biological Test Methods) are far too shallow for fast growing species. In addition to competition between 5 plants within each replicate, grass roots were often found concentrated and circulating along the bottom of each test vessel where most of the moisture in each pot occurred.  Since largest growth effect is observed between 10-25%, it is recommended to repeat the experiment with one plant per pot to eliminate competition among plants as a potential cause of inhibiting growth.   This study has provided several indicators of pine and grass tolerance to the particle sizes and concentration of tailings at Mount Polley Mine. Trials in this study used pure and dried tailings and only accounted for the physical and chemical properties in these samples. The last confounding factors to consider are the biological activities/microbial communities present in field or reclaimed substrate mixes at Hazeltine Creek. It is recommended to repeat the experiment along sections of Hazeltine Creek to account for substrate heterogeneity and incorporation of microbial communities which may improve vegetation re-establishment and spatial root development. Alternatively, substrates could be collected and grown without drying to preserve biological processes in a growth chamber environment.    151  References ASTM. (2014). Standard Guide for Conducting Terrestrial Plant Toxicity Tests (E1963-09 [Re-approved 2014]). Environment Canada. (2013). Biological Test Method: Test for Growth in Contaminated Soil Using Terrestrial Plants Native to the Boreal Region. EPS 1/RM/56. Method Development and Applications Section, Environmental Technology Centre, Environment Canada, Ottawa, ON, Canada. Environment Canada. (2007). Biological Test Method: Test for Measuring Emergence and Growth of Terrestrial Plants Exposed to Contaminants in Soil. EPS 1/RM/45. Method Development and Applications Section, Environmental Technology Centre, Environment Canada, Ottawa, ON, Canada. Golder Associates Ltd. (2015). Post-event environmental impact assessment report - Key findings report. Prepared for Mount Polley Mining Corporation. Retrieved from https://www2.gov.bc.ca/assets/gov/environment/air-land-water/spills-and-environmental-emergencies/docs/mt-polley/p-o-r/2015-06-17_peeiar_summary_report_v1.pdf Golder Associates Ltd. (2016). Mount Polley Rehabilitation and Remediation Strategy: Detailed Site Investigation Mount Polley Tailings Dam Failure, Mount Polley, BC. Retrieved from https://www2.gov.bc.ca/assets/gov/environment/air-land-water/spills-and-environmental-emergencies/docs/mt-polley/p-o-r/2016-01-31_mpmc_detailed_site_investigation.pdf Pequerul, A., Perez, C., Madero, P. and Monge, E. (1993). A rapid wet digestion method for plant analysis. Optimization of Plant Nutrition. Development in Plant and Soil Sciences. Vol 53 pp 3-6          152  Appendix C  Chemical Analysis of Greenhouse Soils and Fertilizer C.1 Greenhouse Potting Soil Chemical Analysis: February 18, 2014  153  C.2 Greenhouse Fertilizer Water Chemical Analysis February 26, 2014  154  Appendix D  Hexagonal Closed Packing/Squared Closed Packing Substrates, namely silty tailings, with smaller distribution of grain sizes would yield a different packing orientation as particles settle. “Hexagonal closed packing” or “cubic closed packing” occurs when layers of similar sized particles are aligned and stacked in alternating layers (Figure X). Hexagonal closed packing operates in simple alternating A-B-A layers where particles of each layer are shifted slightly to cover the voids of the previous layer. Cubic closed packing follows an A-B-C-A pattern where layer C is directionally opposite to layer A.   Figure Above. Graphical explanation of closed packing. Hexagonal closed (top left) packing occurs when similar sized particles are aligned and stacked horizontally in alternating layers where each particle stacks directly on the voids of the previous layer forming a simple A-B-A pattern. In cubic closed packing (middle left), similar sized particles stack A-B-C-A pattern where layers A and C are directionally opposite to one another. Random closed packing (bottom left) occurs when various sized particles stack against one another. Void spaces are often uncovered due to large distribution of particle sizes. 3D imagery of hexagonal and cubic closed packing by layer, modified from(Super Quintet Chemistry I: Introduction to Chemistry, 2017).   155  Appendix E  Statistical Analysis: Kruskal Wallis Test and Summary of p values Median and standard error were displayed in all tables and figures due to the nature of low replicates (n =< 5) in all three studies. An ANOVA test was not suitable for statistical analysis as small sample sizes and assumptions of normality were not met. Statistical significance between treatments was determined using the Kruskal-Wallis test, a non-parametric equivalent of a one-way ANOVA in SPSS software program. Pairwise or among treatments comparisons was subsequently examined in SPSS with the Dunn’s post hoc test on each pair of pre-determined groups. The software provides p values for each statistical analysis between treatment and adjusted significant p values (using the Bonferroni error correction) among treatment to account for error from multiple testing. Statistical analysis was conducted on all results including emergence, survival, longest shoot and root, total biomass and chemical uptake of copper, selenium and vanadium in lodgepole pine and bluebunch wheatgrass. A summary of all relevant significant values (p < 0.05) are listed in the tables below by study, plant species and plant characteristic.   E.1 Lodgepole Pine p values from 42-Day Growth Chamber Study Emergence & Survival Treatment  Significance (p < 0.05) Significance Among Treatment Control to 10 % Control to 25 % Control to 50 % Control to 100 % Copper-Spiked Sandy Substrate Emergence 0.004  0.036 0.036 0.007 Survival 0.002  0.044 0.008 0.008 Copper-Spiked Silty Substrate Survival 0.001  0.009 0.009 0.009 Emergence 0.001  0.009 0.009 0.009  Growth Characteristics Treatment  Significance (p < 0.05) Significance Among Treatment Additional Notes Control to 10 % Control to 25 % Control to 50 % Control to 100 % Sandy Tailings Shoot Length 0.013    0.006  Shoot Weight 0.017    0.015  Root Length 0.012    0.011 10-100% p = 0.040 Root Weight 0.005    0.005 10-100% p = 0.026 Total weight 0.013    0.008  Silty Tailings Shoot Length 0.021    0.017  Root Length 0.034     25-100% p = 0.043 156   Significance Among Characteristics Treatment  Significance  (p < 0.05) Between Substrates Sandy Tailings - Silty Tailings Sandy Tailings - Greenhouse Soil Silty Tailings - Greenhouse Soil Shoot Biomass 0.000  0.000 0.004 Root Length 0.000 0.001 0.000  Root Biomass 0.000 0.003 0.000 0.009  Chemical Analysis  (Cu, Se, V) Treatment  Significance (p < 0.05) Significance Among Treatment Additional Notes Control to 10 % Control to 25 % Control to 50 % Control to 100 % Sandy Tailings Shoot Copper 0.001    0.001  Root Copper 0.000  0.023 0.001  10-50% p = 0.035 Root Vanadium 0.001   0.002 0.008  Silty Tailings Shoot Copper 0.008   0.020 0.017  Root Copper 0.008    0.004  Root Vanadium 0.003   0.050 0.003   Chemical Analysis Between Substrates (Cu, Se, V) Treatment  Significance (p < 0.05) Between Substrates Sandy Tailings - Silty Tailings Sandy Tailings - Greenhouse Soil Silty Tailings - Greenhouse Soil Shoot Chemistry Selenium 0.000  0.000 0.000 Vanadium 0.000 0.000   Root Chemistry Copper 0.002  0.003  Vanadium 0.000  0.000 0.000   E.2 Bluebunch Wheatgrass p values from 42-Day Growth Chamber Study Emergence & Survival Treatment  Significance (p < 0.05) Significance Among Treatment Control to 10 % Control to 25 % Control to 50 % Control to 100 % Copper-Spiked Sandy Substrate Emergence 0.001  0.009 0.009 0.009 Survival 0.001  0.009 0.009 0.009 Copper-Spiked Silty Substrate Survival 0.002  0.009 0.009 0.009 Emergence 0.002  0.009 0.009 0.009    157  Growth Characteristics Treatment  Significance (p < 0.05) Significance Among Treatment Additional Notes Control to 10 % Control to 25 % Control to 50 % Control to 100 % Sandy Tailings Shoot Length 0.001   0.009 0.003 10-100% p = 0.046 Shoot Weight 0.001   0.008 0.005  Root Length 0.000    0.006 10-100% p = 0.001 Root Weight 0.001   0.008 0.003  Total weight 0.001   0.006 0.004  Silty Tailings Shoot Length 0.000   0.013 0.000  Shoot Weight 0.001  0.020 0.035 0.003  Root Length 0.002   0.03 0.009  Root Weight 0.001  0.028 0.035 0.005  Total weight 0.001  0.026 0.026 0.004  Cu Greenhouse Soil  Root Length 0.027    0.046   Significance Among Characteristics Treatment Significance (p < 0.05) Between Substrates Sandy Tailings - Silty Tailings Sandy Tailings - Greenhouse Soil Silty Tailings - Greenhouse Soil Shoot Length 0.000  0.000 0.005 Root Biomass 0.004  0.020 0.008  Chemical Analysis (Cu, Se, V) Treatment  Significance (p < 0.05) Significance Among Treatment Additional Notes Control to 10 % Control to 25 % Control to 50 % Control to 100 % Sandy Tailings Shoot Copper 0.001  0.006 0.003   Shoot Vanadium 0.011   0.023  10-50% p = 0.023 Root Copper 0.002  0.008 0.005   Silty Tailings Shoot Copper 0.000   0.004 0.001  Shoot Selenium 0.016      Shoot Vanadium 0.003   0.003 0.030  Root Copper 0.004  0.046 0.030 0.007  Root Vanadium 0.007    0.002  Cu-spiked Greenhouse Shoot Copper 0.002  0.009 0.040 0.009  Shoot Selenium 0.001 0.008 0.008 0.008 0.008  158  Soil Shoot Vanadium 0.014  0.007    Root Copper 0.000   0.038 0.000 10-100% p = 0.030  Chemical Analysis Between Substrates (Cu, Se, V) Treatment  Significance (p < 0.05) Between Substrates Sandy Tailings - Silty Tailings Sandy Tailings - Greenhouse Soil Silty Tailings - Greenhouse Soil Shoot Chemistry Copper 0.000 0.026 0.000  Selenium 0.000 0.000  0.000 Vanadium 0.000  0.000 0.000 Root Chemistry Selenium 0.003   0.005 Vanadium 0.000 0.000  0.013   E.3  Lodgepole Pine p values from 100-Day Growth Chamber Study Growth Characteristics Treatment  Significance (p < 0.05) Significance Among Treatment Additional Notes Control to 10 % Control to 25 % Control to 50 % Control to 100 % Sandy Tailings (100 Day) Root Length 0.001    0.015 10-100% p = 0.001 Root Weight 0.002    0.001  42-Day Vs 100 Day Shoot Length 0.039   Shoot Weight 0.002 Root Weight 0.000  Chemical Analysis (Cu, Se, V) Treatment  Significance (p < 0.05) Significance Among Treatment Additional Notes Control to 10 % Control to 25 % Control to 50 % Control to 100 % Sandy Tailings (100 Day) Shoot Copper 0.008 0.037 0.013    Shoot Selenium 0.017      Root Copper 0.001  0.021 0.033 0.006  Root Vanadium 0.001   0.028 0.002 10-100% p = 0.032 42-Day Vs 100 Day Shoot Copper 0.048   Shoot Selenium 0.000 Root Copper 0.005 Root Selenium 0.000 Root Vanadium 0.000  159  E.4 Bluebunch Wheatgrass p values from 100-Day Growth Chamber Study Growth Characteristics Treatment  Significance (p < 0.05) Significance Among Treatment Control to  10 % Control to   25 % Control to   50 % Control to   100 % Sandy Tailings (100 Day) Shoot Length 0.016    0.011 Shoot Weight 0.003  0.021  0.005 Root Length 0.003   0.018 0.007 Root Weight 0.001  0.028 0.045 0.003 42-Day Vs 100 Day Shoot Length 0.004   Shoot Weight 0.003  Chemical Analysis (Cu, Se, V) Treatment  Significance (p < 0.05) Significance Among Treatment Additional Notes Control to 10 % Control to 25 % Control to 50 % Control to 100% Sandy Tailings (100 Day) Shoot Copper 0.002  0.003 0.032   Shoot Vanadium 0.040     10-50% p = 0.027 Root Copper 0.006  0.039 0.020   Root Vanadium 0.037   0.028   42-Day Vs 100 Day Shoot Selenium 0.021   Shoot Vanadium 0.000 Root Copper 0.001 Root Selenium 0.000 Root Vanadium 0.000         160  Appendix F  Principal Component Analysis Diagrams (PCA) F.1 Generic R Studio Codes for All Principal Component Analysis Diagrams     161  F.2 Six Week Greenhouse Study: Bluebunch Wheatgrass   162    163   F.3 42 Day Growth Chamber Study: Lodgepole Pine   164    165    166  F.4 42 Day Growth Chamber Study: Bluebunch Wheatgrass  167    168     169  F.5 Tailings and Greenhouse Soils PCA Diagrams based on substrate chemistry; no plant samples involved.   170  F.6 100 Day Growth Chamber Study: Lodgepole Pine   171  F.7 100 Day Growth Chamber Study: Bluebunch Wheatgrass   172    173  Appendix G  Selenium and Vanadium Values (PPM) G.1 Lodgepole Pine 42-Day Growth Chamber Study Lodgepole Pine Vanadium in Shoots (ppm) 42-Day Study Treatment Sandy Tailings Silty Tailings Copper-spiked Soils Control (0 mg/kg Cu) 1.13 ± 0.38 2.50 ± 2.45 3.85 ± 0.51 10% (115 mg/kg Cu) 0.00 ± 0.45 5.38 ± 1.35 5.01 ± 1.46 25% (287.5 mg/kg Cu) 0.32 ± 0.55 3.65 ± 1.87 0.00 ± 0.80 50% (575 mg/kg Cu) 1.48 ± 71.73 6.10 ± 1.55 4.96 ± 2.25 100% (1150 mg/kg Cu) 3.79 ± 1.13 4.73 ± 0.66 1.04 ± 1.56  Lodgepole Pine Vanadium in Roots (ppm) 42-Day Study Treatment Sandy Tailings Silty Tailings Copper-spiked Soils Control (0 mg/kg Cu) 15.20 ± 1.95 4.12 ± 4.69 0.00 ± 0.00 10% (115 mg/kg Cu) 45.47 ± 3.78 33.78 ± 8.47 0.00 ± 3.64 25% (287.5 mg/kg Cu) 56.97 ± 4.60 49.45 ± 6.04 10.13 ± 0.00 50% (575 mg/kg Cu) 69.18 ± 210.46 72.55 ± 2.14 16.36 ± 9.15 100% (1150 mg/kg Cu) 65.16 ± 16.39 115.79 ± 16.95 11.38 ± 2.62  Lodgepole Pine Selenium in Shoots (ppm) 42-Day Study Treatment Sandy Tailings Silty Tailings Copper-spiked Soils Control (0 mg/kg Cu) 0.00 ± 6.37 0.00 ± 0.00 86.42 ± 26.57 10% (115 mg/kg Cu) 6.65 ± 4.75 14.77 ± 9.54 80.84 ± 61.08 25% (287.5 mg/kg Cu) 0.00 ± 5.6 12.63 ± 12.86 99.46 ± 38.33 50% (575 mg/kg Cu) 0.00 ± 17.58 0.00 ± 4.42 128.72 ± 26.70 100% (1150 mg/kg Cu) 0.00 ± 8.47 0.00 ± 6.90 57.43 ± 10.03  Lodgepole Pine Selenium in Roots (ppm) 42-Day Study Treatment Sandy Tailings Silty Tailings Copper-spiked Soils Control (0 mg/kg Cu) 0.00 ± 15.34 0.00 ± 0.00 0.00 ± 0.00 10% (115 mg/kg Cu) 0.00 ± 9.86 0.00 ± 65.16 0.00 ± 0.00 25% (287.5 mg/kg Cu) 0.00 ± 28.46 158.58 ± 92.12 394.94 ± 0.00 50% (575 mg/kg Cu) 0.00 ± 23.37 84.97 ± 66.50 0.00 ± 10.20 100% (1150 mg/kg Cu) 0.00 ± 3.01 313.89 ± 231.96 335.34 ± 211.76          174  G.2 Bluebunch Wheatgrass 42-Day Growth Chamber Study Bluebunch Wheatgrass Vanadium in Shoots (ppm) 42-Day Study  Sandy Tailings Silty Tailings Copper-spiked Soils Control (0 mg/kg Cu) 0.92 ± 24.67 0.08 ± 0.35 1.94 ± 1.14 10% (115 mg/kg Cu) 2.29 ± 0.44 3.23 ± 0.78 0.00 ± 0.00 25% (287.5 mg/kg Cu) 8.57 ± 0.68 8.38 ± 1.79 0.00 ± 0.25 50% (575 mg/kg Cu) 17.46 ± 1.81 10.93 ± 0.45 0.00 ± 0.09 100% (1150 mg/kg Cu) 7.72 ± 1.66 4.87 ± 3.77 0.00 ± 0.57  Bluebunch Wheatgrass Vanadium in Roots (ppm) 42-Day Study  Sandy Tailings Silty Tailings Copper-spiked Soils Control (0 mg/kg Cu) 0.99 ± 29.35 0.18 ± 0.35 1.53 ± 3.93 10% (115 mg/kg Cu) 13.19 ± 3.47 19.03 ± 0.86 0.00 ± 2.30 25% (287.5 mg/kg Cu) 45.17 ± 65.52 16.56 ± 4.55 0.00 ± 1.04 50% (575 mg/kg Cu) 61.44 ± 4.87 25.09 ± 6.68 4.12 ± 0.95 100% (1150 mg/kg Cu) 43.44 ± 11.8 41.45 ± 6.61 6.23 ± 1.94  Bluebunch Wheatgrass Selenium in Shoots (ppm) 42-Day Study Treatment Sandy Tailings Silty Tailings Copper-spiked Soils Control (0 mg/kg Cu) 0.00 ± 1.92 19.74 ± 7.31 0.00 ± 0.00 10% (115 mg/kg Cu) 0.00 ± 13.01 56.46 ± 25.51 0.00 ± 0.00 25% (287.5 mg/kg Cu) 6.82 ± 6.51 97.57 ± 23.02 0.00 ± 0.00 50% (575 mg/kg Cu) 58.85 ± 43.94 67.89 ± 17.27 0.00 ± 0.00 100% (1150 mg/kg Cu) 8.53 ± 2.44 31.37 ± 16.87 0.00 ± 0.00  Bluebunch Wheatgrass Selenium in Roots (ppm) 42-Day Study Treatment Sandy Tailings Silty Tailings Copper-spiked Soils Control (0 mg/kg Cu) 0.00 ± 0.00 0.00 ± 0.00 52.15 ± 26.05 10% (115 mg/kg Cu) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 25% (287.5 mg/kg Cu) 0.00 ± 12.31 0.00 ± 0.00 0.00 ± 0.00 50% (575 mg/kg Cu) 0.00 ± 14.52 0.00 ± 0.00 0.00 ± 35.06 100% (1150 mg/kg Cu) 0.00 ± 1.46 0.00 ± 0.00 23.17 ± 25.12            175  G.3 100-Day Growth Chamber Study  100 Day Lodgepole Pine Shoot Treatment Mean Selenium (ppm) Mean Vanadium (ppm) Control (0 mg/kg Cu) 12.81 ± 5.95 1.2 ± 0.2 10% (115 mg/kg Cu) 33.3 ± 5.36 0.73 ± 0.65 25% (287.5 mg/kg Cu) 16.23 ± 11.38 1.37 ± 0.5 50% (575 mg/kg Cu) 27.8 ± 2.86 1.37 ± 0.42 100% (1150 mg/kg Cu) 74.53 ± 15.94 2.22 ± 1.35 100 Day Lodgepole Pine Root Treatment Mean Selenium (ppm) Mean Vanadium (ppm) Control (0 mg/kg Cu) 46.85 ± 6.08 11.09 ± 0.37 10% (115 mg/kg Cu) 26.91 ± 12.03 12.29 ± 0.99 25% (287.5 mg/kg Cu) 41.41 ± 13.88 20.27 ± 0.77 50% (575 mg/kg Cu) 73.59 ± 14.23 21.02 ± 1.11 100% (1150 mg/kg Cu) 98.73 ± 98.83 30.06 ± 7.60   100 Day Bluebunch Wheatgrass Shoot Treatment Mean Selenium (ppm) Mean Vanadium (ppm) Control (0 mg/kg Cu) 4.01 ± 3.03 0.43 ± 0.21 10% (115 mg/kg Cu) 27.39 ± 13.11 0.00 ± 0.28 25% (287.5 mg/kg Cu) 8.99 ± 17.83 0.33 ± 0.51 50% (575 mg/kg Cu) 36.19 ± 10.89 4.06 ± 1.16 100% (1150 mg/kg Cu) 146.78 ± 19.11 4.88 ± 4.71 100 Day Bluebunch Wheatgrass Root Treatment Mean Selenium (ppm) Mean Vanadium (ppm) Control (0 mg/kg Cu) 0.77 ± 7.14 0.00 ± 0.04 10% (115 mg/kg Cu) 80.74 ± 14.59 4.21 ± 1.08 25% (287.5 mg/kg Cu) 76.60 ± 25.82 4.30 ± 1.16 50% (575 mg/kg Cu) 25.39 ± 13.29 7.66 ± 2.26 100% (1150 mg/kg Cu) 42.82 ± 0.00 4.13 ± 0.00       

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