Northern Wild Rice (Zizania palustris L.) as a Phytoremediation Species in Eutrophic Wetlands – Investigation of Root-Sediment Interactions A thesis presented to The Faculty of Graduate Studies of Lakehead University by Kimberly Jorgenson, B.Env.Sc. In partial fulfillment of requirements for the degree of Master of Science in Biology January 2013 © Kimberly Jorgenson, 2013 i ABSTRACT The causes of anthropogenic eutrophication in water bodies are multi-faceted and multi- generational, presenting an ever increasing need for effective and sustainable solutions. Phytoremediation presents a cost-effective strategy to improve water body nutrient retention and removal, contributing to eutrophication mitigation efforts. This thesis examines the potential for northern wild rice (Zizania palustris L.) to be used as a phytoremediation species in eutrophic wetlands. An investigation into root-sediment interactions was undertaken to determine how northern wild rice affects water and sediment pore water chemistry. Northern wild rice growth was found to alter sediment pore water chemistry, contributing directly to nutrient retention during the summer growing season through nutrient assimilation in its tissues, and indirectly through increasing pore water Fe and Mn in the fall. The majority of P and N within the plant was found to be contained in the stems and leaves (44-53%), followed by the inflorescence (22-28%). Harvesting northern wild rice vegetation (including the seeds) at the end of the growing season would present a permanent nutrient removal mechanism. Substantial iron plaque forms on the roots of northern wild rice, visible as an orange- brown coating that ranges structurally from <1 µm to 14 µm thick. Iron plaques were found to be composed mainly of Fe, O, Al and K, with Fe found within and on root epidermal cells. P was not found to be associated with iron root plaques. With proper harvesting and management techniques, northern wild rice grown in eutrophic water bodies could present a viable phytoremediation method for nutrient removal. ii LAY SUMMARY Faculty and students in the Department of Biology at Lakehead University are bound together by a common interest in explaining the diversity of life, the fit between form and function, and the distribution and abundance of organisms. Northern wild rice (Zizania palustris L.) is a valuable wetland plant with significant cultural importance for Canada’s Aboriginal people. The purpose of this research was to examine the root-sediment interactions of northern wild rice, contributing to “the fit between form and function” of this important aquatic plant. Nutrients (phosphorus and nitrogen) are required for plant growth; human activities have released excessive nutrients into freshwaters worldwide, resulting in nutrient pollution. This research explored how northern wild rice could help remediation efforts in nutrient-impacted water bodies. Northern wild rice growth was found to alter water chemistry, reducing the amount of available nutrients in the summer by incorporating nutrients in its tissues and in the fall by increasing iron and manganese availability. The majority of nutrients within the plant were found in the stems and leaves, thus harvesting the entire plant at the end of the growing season could permanently remove nutrients. Northern wild rice roots were found to be covered in an orange-brown coating known as iron root plaques. These plaques were composed mainly of iron, oxygen, aluminum and potassium; phosphorous was not found in the iron root plaques. The results of this research found that, with proper harvesting and management practices, northern wild rice grown in nutrient-rich water bodies could contribute to nutrient removal efforts. iii ACKNOWLEDGEMENTS I am grateful for the field assistance contributed by the students of the Lakehead University Orillia Campus, with special thanks to Sheri O’Connor and Michael Thorn for their ingenuity and commitment. Thank you to Jack Siegel for your hospitality in allowing us to conduct research in Marchmont Marsh. I am also grateful for the analytical assistance contributed by the LUEL and LUIL staff, with special thanks to Allan MacKenzie for his instrumentation expertise and advice, and to Johane Joncas for sharing her knowledge and laboratory space. Thank you to my supervisors, Dr. Peter Lee and Dr. Nanda Kanavillil for your knowledge, guidance, revisions and support. Thank you to my committee members, Dr. Kam Leung and Dr. Sree Kurissery, your comments have contributed significantly. I would also like to acknowledge Environment Canada’s Lake Simcoe Clean-up Fund who partially financed this research. Special thanks to my university colleagues, Christina, Susanne and Shannon, for sharing advice and a beverage when needed. To my parents, thank you for your advice, encouragement and free labour, I would not be where I am today without the foundation you provided me. This thesis is dedicated to my husband Kelly. Without your patience, love and support, I could not have reached this goal. Thank you. iv TABLE OF CONTENTS Abstract ............................................................................................................................... i Lay Summary .................................................................................................................... ii Acknowledgements .......................................................................................................... iii Table of Contents ............................................................................................................. iv List of Tables ................................................................................................................... vii List of Figures ................................................................................................................. viii List of Appendices ............................................................................................................ ix General Introduction .........................................................................................................1 Chapter 1: Influence of northern wild rice (Zizania palustris) on water chemistry in freshwater wetlands .....................................................................................................7 1.1 Introduction ........................................................................................................7 1.2 Materials and Methods .......................................................................................9 1.2.1 Field Experiment .................................................................................9 1.2.1.1 Study Area .........................................................................10 1.2.1.2 Site Preparation ..................................................................11 1.2.1.3 Pore Water Sampler ...........................................................13 1.2.1.4 Sample Collection ..............................................................15 1.2.2 Laboratory Procedures ......................................................................17 1.2.2.1 Water Analysis ...................................................................17 1.2.2.2 Sediment Analysis .............................................................18 1.2.2.3 Vegetation Analysis ...........................................................18 1.2.3 Data Analysis ....................................................................................19 1.3 Results ..............................................................................................................20 1.3.1 Water Chemistry ...............................................................................20 1.3.2 Water Chemistry Data Trends ..........................................................24 1.3.3 Sediment Chemistry ..........................................................................32 1.3.4 Vegetation Chemistry .......................................................................33 v 1.4 Discussion ........................................................................................................35 1.4.1 Water/Sediment Chemistry and Nutrient Retention .........................36 1.4.2 Vegetation Influence .........................................................................37 1.4.2.1 Northern Wild Rice Water Chemistry Influence ...............37 1.4.2.2 Northern Wild Rice Tissue Assimilation ...........................40 1.4.3 Northern Wild Rice and Nutrient Retention .....................................40 1.4.4 Conclusion ........................................................................................42 Chapter 2: Electron microscopy study of iron plaques on the roots of northern wild rice (Zizania palustris) .................................................................................43 2.1 Introduction ......................................................................................................43 2.2 Materials and Methods .....................................................................................47 2.2.1 Study Area ........................................................................................47 2.2.2 Sample Collection and Preparation Procedures ................................48 2.2.2.1 Northern Wild Rice ............................................................48 2.2.2.2 Surface Water and Sediment ..............................................49 2.2.3 Analytical Procedures .......................................................................50 2.2.3.1 Northern Wild Rice ............................................................50 2.2.3.2 Surface Water and Sediment ..............................................52 2.2.4 Data Analysis ....................................................................................52 2.2.4.1 Northern Wild Rice ............................................................52 2.2.4.2 Surface Water and Sediment ..............................................52 2.3 Results ..............................................................................................................52 2.3.1 Surface Water and Sediment .............................................................52 2.3.2 Northern Wild Rice ...........................................................................54 2.3.2.1 Root Anatomy ....................................................................54 2.3.2.2 Plaque Presence .................................................................55 2.3.2.3 Plaque Structure .................................................................56 2.3.2.4 Plaque Composition ...........................................................59 2.3.2.5 Plaque Deposition ..............................................................59 2.3.2.6 Plaque Anomalies ..............................................................62 vi 2.4 Discussion ........................................................................................................64 2.4.1 Surface Water and Sediment .............................................................64 2.4.2 Northern Wild Rice ...........................................................................64 2.4.2.1 Root Anatomy ....................................................................64 2.4.2.2 Plaque Distribution ............................................................65 2.4.2.3 Plaque Composition ...........................................................66 2.4.2.4 Plaque Morphology ............................................................67 2.4.2.5 Plaque Anomalies ..............................................................70 2.4.3 Conclusion ........................................................................................71 Summary and General Conclusions ...............................................................................72 References .........................................................................................................................74 Appendices ........................................................................................................................87 vii LIST OF TABLES Chapter 1 Table 1.1: Marchmont Marsh water chemistry .................................................................21 Table 1.2: Victoria Point water chemistry ........................................................................22 Table 1.3: Sediment chemistry data ..................................................................................32 Table 1.4: Marchmont Marsh wild rice vegetation chemistry data ..................................34 Table 1.5: Victoria Point wild rice vegetation chemistry data ..........................................34 Chapter 2 Table 2.1: Surface water chemistry, northern wild rice sample sites ...............................53 Table 2.2: Sediment chemistry, northern wild rice sample sites ......................................54 viii LIST OF FIGURES Chapter 1 Figure 1.1: Location of study sites near Orillia, Ontario (Canada) ..................................11 Figure 1.2: Plot delineation in study sites .........................................................................12 Figure 1.3: Sediment pore water sampler (peeper) design ...............................................14 Figure 1.4: Control apparatus ...........................................................................................15 Figure 1.5: VP and MM August and September P and N water profiles .........................25 Figure 1.6: VP and MM August and September pH, alkalinity and conductivity water profiles .........................................................................................................27 Figure 1.7: VP and MM August and September Ca, Fe, K and Mg water profiles ..........29 Figure 1.8: VP and MM August and September Mn, Na, S and Zn water profiles ..........30 Chapter 2 Figure 2.1: Location of sample sites in Ontario, Canada ..................................................48 Figure 2.2: Northern wild rice roots with orange-brown colouration ...............................49 Figure 2.3: Northern wild rice root anatomy ....................................................................55 Figure 2.4: SEM images of iron plaques on northern wild rice roots ...............................57 Figure 2.5: SEM images of northern wild rice roots with iron plaque casts ....................58 Figure 2.6: EDXA x-ray spectra of iron plaques on northern wild rice roots ..................60 Figure 2.7: SEM images and EDXA x-ray spectra of iron plaques on northern wild rice roots.................................................................................................................61 Figure 2.8: SEM image and EDXA element distribution maps of select elements in northern wild rice root cross-section with iron plaque ..........................................62 Figure 2.9: SEM images of northern wild rice roots with grooves present in thick crust iron plaque .....................................................................................................63 Figure 2.10: Hypothetical model of iron plaque development on northern wild rice roots.................................................................................................................69 ix LIST OF APPENDICES Appendix A: Marchmont Marsh Data ..............................................................................88 Appendix B: Victoria Point Data ....................................................................................132 Appendix C: Zizania palustris Root Plaque Examination Data .....................................177 1 GENERAL INTRODUCTION Clean freshwater is a vital resource that is rapidly becoming degraded (Carpenter et al. 1998). Of the vast water resources present on Earth, less than 0.5% is accessible as freshwater in ground and surface reservoirs (Wetzel 1992). This freshwater has been used to dispose of pollutants throughout human history, resulting in further stress to the crucial resource (Carpenter et al. 1998). Sources of human-induced surface water degradation include the direct release of toxic chemicals, increased erosion due to forestry and agriculture, and nutrient enrichment (leading to eutrophication) through fertilizer and wastewater discharge (Wetzel 1992). The cultural eutrophication of water bodies is caused by excessive inputs of nutrients (phosphorus and nitrogen) into aquatic systems from human sources (Carpenter et al. 1998). Aquatic organisms require nitrogen (N) and phosphorus (P) to survive (Wetzel 2001), however when these nutrients are available in excess they are considered contaminants to an aquatic system (Reddy et al. 1999). Symptoms of this artificial enrichment include an increase in primary productivity, turbid water and a loss of biodiversity (Wetzel 2001; Søndergaard et al. 2003). As additional stresses are placed on the worlds’ freshwater resources and individuals become aware of the health and ecological risks of nutrient pollution (Wetzel 1992; Galvez-Cloutier et al. 2006), the need for effective and sustainable solutions for cultural eutrophication increases. Eutrophication can be reversed through the reduction of nutrient sources to a water body, however the rate of recovery is highly variable and internal P loading (i.e. P release from the water body sediments) can cause a persistent eutrophic state (Carpenter et al. 1998; Søndergaard et al. 2003). P has a tendency to accumulate in sediments, resulting in an 2 internal load of P in eutrophic water bodies that can delay water quality improvements (Søndergaard et al. 2003; Hickey and Gibbs 2009). Effective remediation strategies must include a reduction in external nutrient loads and a mechanism for capturing nutrients from the water and sediment and rendering them unavailable (Søndergaard and Jeppesen 2007). External load reductions, accomplished through government regulations/enforcement and public education campaigns, must target point and non-point sources of pollution including the capture and treatment of nutrient-rich wastewater, municipal sewage, fertilizer runoff and industrial discharges (Carpenter et al. 1998; LWSB 2006). Remediation mechanisms to improve contaminant retention in water bodies involve the manipulation of biotic and abiotic processes such as settling and sedimentation, sorption, chemical oxidation/reduction, biodegradation and plant uptake (Lavrova and Koumanova 2006). Several strategies exist to reduce P release from sediments, including hypolimnetic aeration and the addition of flocculating agents/capping materials, however these methods are frequently cost-inhibitive and require high implementation and monitoring efforts (Reddy et al. 1999). Phytoremediation, remediating contaminants through the use of vegetation, presents a cost-effective strategy to improve nutrient retention within water bodies (Williams 2002). Wetland systems (constructed and natural) are considered a low-cost alternative for treating non-point source pollutants (Jiang et al. 2007; Lu et al. 2009). The retention and transformation of nutrients in wetlands decreases the quantity of available nutrients in the water column and sediment and thus lowering downstream export (Johnston 1991; Reddy et al. 1999; Mitsch and Gosselink 2007; En-Hua et al. 2010). Wetlands have been shown 3 to retain organic and inorganic nutrients from the waters that flow through them by physical, chemical and biological processes (Dunbabin and Bowmer 1992; Reddy et al. 1999; Mitsch and Gosselink 2007). Aquatic plants (macrophytes) are the main biological components of wetlands (Maine et al. 2005a) and have a major role in nutrient assimilation and storage (Reddy et al. 1999). Emergent macrophytes with extensive root systems and a high ratio of above to below-ground biomass are well-suited to the phytoremediation of nutrient contaminants (Reddy et al. 1999; En-Hua et al. 2010). Emergent macrophytes contribute to nutrient retention mainly through nutrient assimilation, denitrification and rhizosphere oxidation (Moore et al. 1994; Hoagland et al. 2001). Wetlands function as nutrient sources or sinks depending on the season (Kao et al. 2003), with wetlands typically functioning as net sinks for total P during the growing season (Reddy et al. 1999). Nutrient retention during the growing season, when the majority of problems associated with eutrophication are experienced, can significantly improve water quality and assist in the remediation of eutrophic water bodies (Reddy et al. 1999; Hoagland et al. 2001; Hupfer and Dollan 2003; Kao et al. 2003). Emergent macrophytes acquire the nutrients required for growth directly from wetland sediments and incorporate them into the living biomass (Wetzel 2001; En-Hua et al. 2010). This process influences the sediment pore water chemistry, reducing the quantity of available nutrients in the sediment and lowering nutrient transfer into the water column (En-Hua et al. 2010). After the growing season, nutrients are released from the senesced vegetation at a variable rate through decomposition and leaching (Mitsch and Gosselink 2007; Reddy et al. 1999). The aboveground portion of the macrophyte returns nutrients 4 to the water column, while the belowground biomass returns nutrients to the sediment pore water (Reddy et al. 1999; Shilla et al. 2006). Denitrification, the conversion of nitrate (NO3 - ) to gaseous nitrous oxide (N2O) and molecular nitrogen (N2) by microbes in anaerobic conditions (Mitsch and Gosselink 2007), can permanently remove N from a water body (McCarthy et al. 2007; Hickey and Gibbs 2009). Denitrification is promoted by decomposing vegetation providing a nutrient source and substrate for bacteria (Hoagland et al. 2001). Maine et al. (2005a) found that constructed wetlands efficiently removed both N and P from wastewater high in Fe and SO4 2- by promoting both denitrification and P precipitation and adsorption to Fe and CaCO3. Rhizosphere oxidation occurs when emergent macrophytes transport oxygen to their roots which subsequently “leaks” out (by radial oxygen loss) into the surrounding anaerobic rhizosphere (Armstrong 1964; Conlin and Crowder 1989; Mitsch and Gosselink 2007). Numerous biogeochemical processes occur within this oxidized rhizosphere, including the oxidation of iron which often results in the precipitation of ferric hydroxide onto root surfaces termed iron root plaque (Mendelssohn and Postek 1982; Crowder and MacFie 1986; St-Cyr et al. 1993; Christensen and Wigand 1998; Caetano and Vale 2002). Iron hydroxides have a high adsorption capacity, resulting in the adsorption or co-precipitation and incorporation of a number of elements including P into plaques (Chen et al. 1980a; Chambers and Odum 1990; Liu et al. 2011). The ability of a wetland to retain nutrients is strongly influenced by the aquatic species present (Kao et al. 2003). Macrophytes best suited for eutrophic wetland phytoremediation are fast-growing indigenous species with vigorous root systems and 5 high oxygenation ability (Dunbabin and Bowmer 1992; En-Hua et al. 2010). Many studies have shown constructed wetlands planted with emergent macrophytes including Typha, Schoenoplectus, Phragmites, Juncus and Cyperus species are successful in remediating polluted waters (Dunbabin and Bowmer 1992; Tanner 1996; Hoagland et al. 2001; Maine et al. 2005a; Jiang et al. 2007; Lu et al. 2009; En-Hua et al. 2010). Zizania latifolia (Griseb.) Turcz. ex Stapf, Manchurian wild rice, has been studied for its nutrient uptake and retention potential in Eastern Asia (Tanner 1996; Jiang et al. 2007; Lu et al. 2009; En-Hua et al. 2010). Tanner (1996) examined eight wetland species, concluding that Z. latifolia accumulated the largest amount of N, P, S, K, Zn and Fe due to its high growth rate, well-developed root system, high root-zone aeration and stress tolerance. Jiang et al. (2007), Lu et al. (2009) and En-Hua et al. (2010) all found that Z. latifolia had a high capacity for N and P uptake, concluding that plant harvest at the end of the growing season provided a permanent nutrient removal mechanism. Zizania palustris L., northern wild rice, is an emergent annual aquatic grass that grows in shallow lake waters and slow-moving rivers across eastern and north-central North America (Aiken et al. 1988; Painchaud and Archibold 1990). An important cereal crop, wild rice has been harvested for centuries by First Nations people (Aiken et al. 1988). Wild rice is the only wild grass in Canada that grows annually from seed (without being planted) with a grain of sufficient size for widespread human consumption (Aiken et al. 1988). Z. palustris has been cultivated in North America since the 1950s (Malvick and Percich 1993), while research into its cultivation potential in Eastern Asia has increased in recent years (Gemma et al. 1993; Jin et al. 2005). 6 Z. palustris grows in a wide range of water and sediment types and is known to oxidize the rhizosphere through its well-defined system of aerenchyma (Stover 1928; Aiken et al. 1988; Day and Lee 1989; Lee and McNaughton 2004). Stands of wild rice maintain wetland water quality through binding loose soil, retaining nutrients and reducing wind erosion (Bennett et al. 2000). The historic range of Z. palustris has been significantly reduced due to human disturbance including water pollution, boat turbulence and water level manipulations (Bennett et al. 2000; Meeker 2000). Since restoration is not a viable option on many historic waters, efforts have been made to seed wild rice into suitable habitats across its former range with variable success (David 2000). The objective of this thesis was to examine the potential of Z. palustris as a phytoremediation species in eutrophic wetlands. An investigation of root-sediment interactions was undertaken with the following aims: 1) To determine how the establishment of Z. palustris in a eutrophic wetland alters water and sediment pore water chemistry; 2) To study the deposition, composition and structure of iron plaques formed on the roots of Z. palustris. 7 CHAPTER 1 Influence of northern wild rice (Zizania palustris) on water chemistry in freshwater wetlands 1.1 Introduction Cultural eutrophication is caused by excessive inputs of the nutrients phosphorus (P) and nitrogen (N) into aquatic systems from human sources (Carpenter et al. 1998). When nutrients are available in excess, water bodies experience an increase in primary productivity, turbid water and a loss of biodiversity (Reddy et al. 1999; Wetzel 2001; Søndergaard et al. 2003). This condition can be reversed through the reduction of nutrient sources, however the rate of recovery for water bodies is highly variable since internal P loading from sediments often persists (Carpenter et al. 1998; Søndergaard et al. 2003). Effective remediation strategies include a reduction in external nutrient loads and a mechanism for capturing nutrients from the water and sediment (Søndergaard and Jeppesen 2007). Phytoremediation through wetland macrophytes presents a cost- effective strategy to improve nutrient retention (Williams 2002). Nutrient retention and transformation in wetlands decreases the load to downstream water bodies (Johnston 1991; Reddy et al. 1999; Mitsch and Gosselink 2007; En-Hua et al. 2010). Emergent macrophytes in wetlands contribute to nutrient dynamics mainly through nutrient assimilation, denitrification and rhizosphere oxidation (Moore et al. 1994; Hoagland et al. 2001). Nutrient retention by macrophytes during the growing season can significantly improve water quality and assist in the remediation of eutrophic water bodies (Reddy et al. 1999; Hoagland et al. 2001; Hupfer and Dollan 2003; Kao et al. 2003). 8 Zizania palustris L., northern wild rice, is an emergent annual aquatic grass that grows in shallow lake waters and slow-moving rivers across eastern and north-central North America (Aiken et al. 1988; Painchaud and Archibold 1990). In North America, Z. palustris has been harvested for centuries by First Nations people and has been cultivated commercially since the 1950s (Aiken et al. 1988; Malvick and Percich 1993). Z. palustris grows in a wide range of water and sediment types and is known to oxidize the rhizosphere through its well-defined system of aerenchyma (Stover 1928; Aiken et al. 1988; Day and Lee 1989; Lee and McNaughton 2004; Jorgenson et al. 2013 Botany - in press). The phytoremediation potential of Z. palustris in nutrient-impacted water bodies has not been examined to date, however the commercial value and physical attributes of this annual aquatic grass suggest that this species may be suitable in such initiatives. Sediment interstitial pore waters represent an important source of bioavailable nutrients and metals within water bodies (Teasdale et al. 1995). Pore water dynamics are useful in examining interactions between lake water and sediment, are more sensitive to seasonal variations and are a better indicator of the trophic status of a water body (Teasdale et al. 1995; Søndergaard 1990). Examining sediment pore water chemistry can help explain many chemical processes occurring within sediments, and in-situ methods of pore water collection are best for minimizing sampling artefacts (Azcue et al. 1996). An in-situ sampling field experiment was designed based on the research of Moore et al. (1994) to determine how water and sediment pore water chemistry are affected by northern wild rice. Dialysis pore water samplers (peepers) were used to examine the effect of northern wild rice on water chemistry and nutrient partitioning in adjacent vegetated and non- vegetated plots in two freshwater wetlands. 9 The objective of this study was to examine the potential of Z. palustris as a phytoremediation species for eutrophic water bodies. Lake Simcoe is a mesotrophic lake located in southern Ontario that has undergone numerous environmental changes due to anthropogenic P loading (Evans et al. 1996; Ginn 2011). P levels began to increase in Lake Simcoe in the 1930s, however P was not identified as a water quality problem until the 1970s when a decline in the coldwater fishery and excessive algal and aquatic macrophyte growth were observed (Eimers et al. 2005; Winter et al. 2007; Kilgour et al. 2008; Hawryshyn et al. 2012). Anthropogenic nutrient sources within the watershed include urban and agricultural run-off from 23 municipalities, livestock waste, effluent from 15 sewage treatment plants and atmospheric pollution (LSRCA 2009; Ontario Ministry of the Environment 2009a). Initiatives to reduce total P loading to Lake Simcoe, including the Lake Simcoe Protection Plan, have been ongoing since the 1990s with a substantial improvement in lake water quality observed in recent years (Winter et al. 2007; Ontario Ministry of the Environment 2009b; Ginn 2011; Winter et al. 2011; Hawryshyn et al. 2012). The goal of this study was to determine how water and sediment pore water chemistry are affected by the presence of Z. palustris, in an effort to determine the usefulness of Z. palustris as a phytoremediation species in eutrophic wetlands. 1.2 Materials and Methods 1.2.1 Field Experiment Two freshwater wetlands were selected near Orillia, Ontario, to examine the influence of Z. palustris growth on water chemistry in differing wetland environments. Surface water and sediment pore water samples were collected in-situ from plots containing wild rice vs. plots containing no vegetation and submitted for laboratory analysis. 10 1.2.1.1 Study Area Orillia, Ontario (44° 35’ 59” N, 79 24’ 59” W) is located on the northern shore of Lake Simcoe, adjacent to the Lake’s outflow to Lake Couchiching at Atherley Narrows (Winter et al. 2007). Both wetlands selected for this study (Figure 1.1) are part of the Lake Huron Drainage Basin, with Victoria Point located within the Lake Simcoe Watershed and Marchmont Marsh located within the Severn Sound Watershed (Environment Canada 2010; South Georgian Bay-Lake Simcoe Source Protection Committee 2011). Victoria Point (VP) wetland (44° 35’ 36” N, 79 22’ 58” W), adjacent to the southeast boundary of Orillia, is a shallow open water marsh within Lake Simcoe with dark brown flocculent organic sediment (> 2 metres (m) deep) and a seasonally fluctuating water level (0.1 - 2 m depth). Dominant macrophyte species within the study area included Nymphaea odorata Aiton, Nuphar variegata Engelm. ex Durand, Ceratophyllum demersum L. and Myriophyllum sp., with Typha angustifolia L. dominant along the shoreline. Marchmont Marsh (MM) (44° 38’ 03” N, 79 31’ 03” W), located approximately 5 km west of the City of Orillia, is an open water marsh along the shoreline of a slow-moving tributary of the North River with a stable water level (1 m depth). Within the study area, a firm light brown clay layer is located beneath the overlying brown organic sediments (0.3 – 0.5 m deep). Dominant macrophyte species within the study area included Z. palustris, N. variegata and C. demersum, bounded by a riparian forest dominated by Abies balsamea (L.) Mill. and Thuja occidentalis L. 11 Figure 1.1 – Location of study sites near Orillia, Ontario (Canada). Basemap imagery from ESRI (2012). 1.2.1.2 Site Preparation Victoria Point In early May 2010, VP wetland was observed to be lacking vegetation cover. A suitable plot location was selected for seeding Z. palustris based on accessibility, adequate water depth (0.5 - 1 m), flocculent sediment conditions, a lack of competitive species and shelter from wind (Aiken et al. 1988; Gemma et al. 1993; Painchaud and Archibold 1990). A fence (constructed of ABS pipe and plastic safety fencing) was erected to eliminate waterfowl disturbance, delineating a study area approximately 7.5 m by 11 m in size. Z. palustris seeds (collected and over-wintered in a pond near Kakabeka Falls, Ontario) were scattered throughout the fenced area, with excess seed distributed in an area just north of the fence. Growth of the seeded northern wild rice was monitored in June and July, with the majority of successful growth observed adjacent to the fence due 12 to the reduced turbidity from strong winds. Competitive species (N. odorata and C. demersum) were removed from within the fenced area as required to encourage growth. In late July 2010, six plots were delineated within the study area based on the observed vegetation growth (Figure 1.2): three vegetated plots containing only northern wild rice vegetation; two non-vegetated plots containing no vegetation; and, one large mixed- vegetation plot containing N. odorata (dominant), Myriophyllum sp., C. demersum and Z. palustris. Non-vegetated plots were maintained throughout the growing season with bi-weekly vegetation removal. Marchmont Marsh In late June 2010, Z. palustris was observed to be growing vigorously along the southern shore of MM. Six plots were delineated (Figure 1.2) within the study area: three vegetated plots containing only northern wild rice vegetation, and three non-vegetated plots containing no vegetation. Non-vegetated plots were maintained through monthly vegetation removal of C. demersum. Figure 1.2 – Plot delineation in study sites. 13 1.2.1.3 Pore Water Sampler Dialysis pore water samplers (peepers) were constructed to collect sediment pore water samples. Originally designed by Hesslein in 1976, peepers allow for the collection of discrete pore water samples from specified depths, through the equilibration of a contained quantity of water with the surrounding pore water (Hesslein 1976). Many modifications have been designed and used over the years to meet specific experimental requirements (Teasdale et al. 1995; Azcue et al. 1996; Jacobs 2002; Bally et al. 2005). In this study, peepers were designed to collect the large sample volumes required for a wide range of chemical analyses. Fisherbrand® 50 mL centrifuge tubes were modified to use as the individual dialysis water samplers (Figure 1.3 C). A 180 mm diameter hole was drilled in each cap and fitted with a 0.45 µm Millipore Durapore® membrane filter, adhered with silicone between two 500 µm Nitex® screens. ABS pipe and fittings were used to construct an apparatus for anchoring multiple dialysis water samplers (Figure 1.3 A). Three dialysis water samplers were inserted into each 10 cm interval of the sampling apparatus, collecting 150 mL of water per 10 cm interval (Figure 1.3 B). Collectively, the sampling apparatus and multiple dialysis water samplers are herein referred to as a peeper. 14 Figure 1.3 – Sediment pore water sampler (peeper) design. At the time of deployment, each dialysis water sampler was filled with degassed distilled deionized water (DDW), capped (zero headspace) and placed in the sampling apparatus. The compiled peeper was pushed vertically into the sediment at a random location within each plot, with one 10 cm depth interval visible above the sediment-water interface (SWI) (Figure 1.3 B). In total, 36 peepers were constructed, 30 peepers with five 10 cm depth intervals to sample up to 40 cm below the SWI, and six “deep peepers” with eight 10 cm depth intervals to sample up to 70 cm below the SWI. Deep peepers were designed to examine pore water trends below the rooting zone of Z. palustris. After deployment, all peepers remained undisturbed for approximately one month (26-33 days), allowing adequate equilibration time with the surrounding water (Teasdale et al. 1995). 15 1.2.1.4 Sample Collection At the time of sample collection, the peeper was carefully pulled vertically from the sediment and each individual dialysis water sampler was extracted and collected by 10 cm sampling depth. Compromised samples (e.g. from samplers with damaged caps) were discarded. The contents of all samplers from the same 10 cm depth interval (usually three samplers) were emptied into clean, pre-labelled 250 mL plastic bottles, resulting in one 150 mL water sample per 10 cm depth per peeper. Samples were placed in ice-filled coolers and transported to the Lakehead University Environmental Laboratory (LUEL) for analysis. After sample collection, the sampling apparatus was scrubbed clean, loaded with new degassed DDW-filled dialysis water samplers and re-deployed. A control apparatus was also constructed to determine if peeper components contributed to the analytical results. A short length of ABS pipe was capped at both ends and filled with degassed DDW and three filled dialysis water samplers (Figure 1.4) at each deployment event. Samples of the control apparatus water, degassed DDW (used to fill samplers at deployment) and site surface water were collected at each sampling event. Samples were collected directly into clean 250 mL plastic bottles, sealed, labelled and stored in a cooler along with the peeper samples for transport to the LUEL for analysis. Figure 1.4 – Control apparatus. 16 Sediment and vegetation samples were also collected at select intervals from each study site. Sediment samples were collected by pre-cleaned shovel into new 1 L plastic bags, sealed, labelled and transported in coolers to the LUEL for analysis. Vegetation samples of Z. palustris were removed by hand from the sediment and placed into new plastic bags, sealed, labelled and transported in coolers to the LUEL for analysis. Victoria Point In total, 20 peepers were deployed and collected monthly in VP from July to October 2010. Three peepers each were located in the three vegetated plots and two non- vegetated plots, and five peepers were located in the mixed vegetation plot. One deep peeper was included in each plot (six deep peepers total). Three sample collection events occurred (August, September and October) for a total of 333 samples collected. Water levels within VP dropped significantly throughout the summer so that only 0.1 to 0.3 m of water and little vegetation remained within the study area in October. Accordingly, only one deep peeper from each of the vegetated plots was collected and analyzed in October. One surface water sample was collected per month from July to October. One vegetation sample (10 plants) was collected in September, and one sediment sample was collected in October. Marchmont Marsh In total, 18 peepers were deployed and collected monthly in MM from June to October 2010. Three peepers each were located in the three vegetated plots and three non- vegetated plots. Firm clay impeded peeper deployment beyond 40 cm below the SWI, 17 thus no deep peepers were used. Four sample collection events occurred (July, August, September and October), for a total of 388 samples collected. Water level and vegetation cover within MM was consistent throughout the study. One surface water sample was collected per month from July to October. Two vegetation samples were collected, one in August (3 plants) and one in September (20 plants). Two sediment samples were collected, one each in June and October. 1.2.2 Laboratory Procedures All sample analyses were conducted at the LUEL, a Canadian Association of Laboratory Accreditation (CALA) ISO 17025 accredited laboratory. All analyses followed standard operating procedures and included the use of blanks, quality control samples and replicates. 1.2.2.1 Water Analysis As per the in-situ sample collection technique, water samples (surface and pore) were filtered in the field. The majority of samples contained a small portion of sediment, indicating that not all water passed through the 0.45 µm membrane (i.e. some water passed around the membrane and only through the 500 µm screen). In preparation for laboratory analysis, water samples were mixed, allowed to settle for 5 to 10 minutes and then decanted (eliminating the approximately 10 mL of water containing sediment particles from analysis). Water (surface and pore) samples were analyzed for: pH, conductivity, total alkalinity, P (total P and phosphate), N (nitrite and nitrate), and total Al, As, Ba, Ca, Fe, K, Mg, Mn, Na, S, Sr and Zn. Select samples in June and July were also analyzed for dissolved 18 organic carbon (DOC) and reactive silicates (MM only). For samples of insufficient quantity (i.e. less than 150 mL), analyses were prioritized as follows: 1) P and N; 2) Al, As, Ba, Ca, Fe, K, Mg, Mn, Na, S, Sr and Zn; 3) pH, conductivity and total alkalinity. Within 24 hours of reaching the laboratory, pH and conductivity were analyzed by a probe (with temperature correction) and alkalinity was analyzed by titration with H2SO4 to a pH of 4.5 (automated titration procedure). Al, As, Ba, Ca, Fe, K, Mg, Mn, Na, S, Sr and Zn analyses were carried out by ICP spectrometry subsequent to their digestion and concentration by microwave following the addition of HNO3. P and N were determined on filtered samples (0.45 µm) by ion chromatography on a Dionex DX-120. 1.2.2.2 Sediment Analysis Sediment samples were analyzed for total P, Al, As, Ba, Ca, Fe, K, Mg, Mn, Na, S, Si, Sr, Ti and Zn. Samples from MM were also analyzed for pH and conductivity. Sediment samples were air-dried and ground to pass through a 2 mm mesh. Total P, Al, As, Ba, Ca, Fe, K, Mg, Mn, Na, S, Si, Sr, Ti and Zn analyses were conducted by ICP spectrometry subsequent to their digestion and concentration by microwave following the addition of HCl and HNO3. Conductivity and pH were determined by probe (with temperature correction) on non-dried samples thoroughly mixed with DDW. 1.2.2.3 Vegetation Analysis Vegetation samples were separated into four parts (root, leaf, stem and inflorescence), oven-dried at 35°C and ground to pass through a 2 mm mesh. All samples were analyzed for total P, N, Al, Ba, Ca, Fe, K, Mg, Mn, Na, S, Si, Sr, Ti and Zn. Total P, Al, Ba, Ca, Fe, K, Mg, Mn, Na, S, Si, Sr, Ti and Zn analyses were conducted by ICP spectrometry 19 subsequent to their digestion and concentration by microwave following the addition of HNO3. Total N analyses were conducted by colourimetry through a SKALAR AutoAnalyzer® subsequent to their digestion and concentration by microwave following the addition of H2SO4 catalyzed with a metal sulphate. 1.2.3 Data Analysis All data was tabulated, with means and standard deviations calculated for each parameter, depth and plot type. Water data from depths ranging from 10-0 cm above the SWI to 30- 40 cm (MM) and 40-50 cm (VP) below the SWI were statistically analyzed and graphed using SigmaPlot™ 12.0 Graphing and Statistical Software to determine trends with depth between sampling plots (vegetated vs. non-vegetated) by month. Since a trend in the distribution of data with depth was observed (indicating additive data), a correction was applied to all depths per plot per month, in accordance with the methodology outlined by Lee and Stewart (1981). This involved calculating the overall mean for all samples (all plots) within the same depth interval, and then transforming the mean for all samples within individual plots to a percentage of the overall mean value: This approach allowed for the comparison of the concentrations of variables in one plot in relation to another, rather than comparing the actual concentration values (Lee and Stewart 1981). Paired t-Tests and ANOVAs were then conducted on the depth- independent water data for each site, comparing each plot to the other, both within and between months. If the plots were significantly different from each other within the same month or if individual plots were significantly different between months (P ≤ 0.05), a Tukey Post Hoc Test was conducted to determine where the data differed. 20 1.3 Results 1.3.1 Water Chemistry In MM, 108 surface water samples (from a depth range of 10 cm to 0 cm above the SWI) and 280 pore water samples (from a depth range of 0 cm to 50 cm below the SWI) were analyzed. Table 1.1 presents the concentration ranges (minimum value – maximum value), means and stand deviations for all surface and pore water samples analyzed (all depths, all months) from MM. The mean As concentrations were less than the MDL In VP, 39 surface water samples (from a depth range of 10 cm to 0 cm above the SWI) and 294 pore water samples (from a depth range of 0 cm to 80 cm below the SWI) were analyzed. Table 1.2 presents the concentration ranges (minimum value – maximum value), means and stand deviations for all surface and pore water samples analyzed (all depths, all months) from VP. The mean As concentrations were less than the MDL. At both sites, pH, total S and Zn concentrations were higher in the surface waters, while alkalinity, conductivity, and total P, N, Al, Ba, Ca, Fe and Mn concentrations were higher in the pore waters. Total K, Mg, Na and Sr showed opposite trends in VP vs. MM, with total Na concentrations higher in the surface waters of VP (pore waters of MM) and total K, Mg and Sr concentrations higher in pore waters of VP (surface waters of MM). The total P concentrations in the surface water of VP were indicative of a eutrophic state, while the surface waters of MM had P and N concentrations within the range of uncontaminated fresh waters (Wetzel 2001; Mitsch and Gosselink 2007). N concentrations in the surface water of VP were generally below the MDL, supporting the accepted theory that P is the nutrient of primary concern within Lake Simcoe (Winter et al. 2007; Palmer et al. 2011; Winter et al. 2011). Table 1.1 - Marchmont Marsh water chemistry. Data presented by plot type for all months. Units MDL Water Range Mean SD n Range Mean SD n Surface 0.003 - 0.238 0.026 0.034 60 0.003 - 0.128 0.031 0.029 48 Pore 0.006 - 0.355 0.072 0.068 133 0.003 - 0.263 0.081 0.053 147 Surface 0.013 - 0.013 0.013 0 60 0.013 - 0.013 0.013 0 48 Pore 0.013 - 0.013 0.013 0 133 0.013 - 0.078 0.014 0.006 147 Surface 0.003 - 0.003 0.003 0 60 0.003 - 0.098 0.008 0.019 48 Pore 0.003 - 2.410 0.039 0.236 133 0.003 - 0.003 0.003 0 147 Surface 0.005 - 0.044 0.008 0.007 60 0.005 - 0.171 0.018 0.031 48 Pore 0.005 - 0.166 0.015 0.028 133 0.005 - 0.329 0.030 0.064 147 Surface 7.23 - 7.93 7.67 0.16 42 6.87 - 7.88 7.59 0.23 31 Pore 6.60 - 7.51 7.03 0.19 107 6.48 - 7.87 6.88 0.22 126 Surface 124.4 - 167.4 130.0 8.0 42 123.2 - 163.6 130.6 8.3 31 Pore 91.3 - 260.9 145.4 26.3 107 101.0 - 205.2 140.8 20.0 126 Surface 256.40 - 338.00 278.18 14.51 42 261.60 - 333.30 278.72 14.63 31 Pore 188.00 - 504.00 314.76 46.96 107 223.10 - 391.10 303.03 32.31 126 Surface 0.006 - 0.066 0.024 0.016 52 0.006 - 0.071 0.019 0.013 40 Pore 0.008 - 3.080 0.128 0.329 128 0.007 - 0.727 0.050 0.081 138 Surface 0.054 - 0.089 0.059 0.006 52 0.055 - 0.092 0.061 0.008 40 Pore 0.042 - 0.154 0.073 0.018 128 0.057 - 0.119 0.085 0.013 138 Surface 30.500 - 41.940 33.842 2.677 52 30.740 - 44.752 34.308 3.185 40 Pore 26.972 - 67.820 40.380 6.061 128 29.100 - 58.220 39.960 4.624 138 Surface 0.011 - 1.839 0.128 0.339 52 0.008 - 1.754 0.270 0.462 40 Pore 0.077 - 4.558 1.280 0.835 128 0.009 - 3.472 1.735 0.647 138 Surface 1.01 - 1.47 1.20 0.16 52 0.50 - 1.45 1.15 0.18 40 Pore 0.15 - 2.46 0.86 0.43 128 0.11 - 4.42 0.75 0.51 138 Surface 8.84 - 11.24 10.47 0.44 52 7.12 - 11.09 10.11 0.92 40 Pore 4.60 - 13.31 7.44 1.73 128 5.26 - 11.82 7.91 1.32 138 Surface 0.0009 - 0.3736 0.0277 0.0763 52 0.0011 - 0.8448 0.0771 0.1499 40 Pore 0.0061 - 0.8744 0.1731 0.1319 128 0.0012 - 1.0704 0.3131 0.1888 138 Surface 4.54 - 6.07 5.05 0.50 52 4.21 - 5.99 4.96 0.44 40 Pore 1.89 - 21.54 7.89 4.31 128 2.27 - 13.24 5.16 2.02 138 Surface 0.36 - 2.12 1.81 0.35 52 0.11 - 2.06 1.68 0.43 40 Pore 0.06 - 1.81 0.39 0.26 128 0.10 - 1.97 0.40 0.31 138 Surface 0.097 - 0.118 0.103 0.005 52 0.089 - 0.133 0.104 0.007 40 Pore 0.063 - 0.179 0.101 0.016 128 0.074 - 0.144 0.104 0.013 138 Surface 0.001 - 0.012 0.006 0.004 52 0.001 - 0.015 0.007 0.004 40 Pore 0.001 - 0.013 0.005 0.003 128 0.001 - 0.012 0.005 0.003 138 Surface a 3.8 - 9.3 5.0 1.0 30 1.3 - 8.6 4.9 1.4 26 Pore b 4.1 - 15.6 7.5 2.8 63 1.4 - 10.1 6.3 2.0 72 Surface c 7.70 - 22.4 11.13 5.57 6 8.60 - 10.30 9.12 0.69 5 Pore d 15.20 - 29.60 22.31 4.88 9 16.80 - 28.80 22.95 4.29 11 Notes MDL = method detection limit; N/A = not applicable; SD = standard deviation; n = sample size; a = measured in June, July & August; b = measured in July & August; c = measured in June & July; d = measured in July. 1.5 µm) - = N/A Comments Site Root Colour SEM Observation Data ImageSample ZoomPlaque Present Primary Deposition Secondary Deposition Other Particulate Deposits Sample Data Image Location Appendix C Zizania palustris Root Plaque Examination 187 C.1.1 - Lake Tamblyn Table C.1.1.2 - Lake Tamblyn, Zizania palustris Root Plaque EDXA X-Ray Spectra Data Compilation 1 2 3 4 5 LT 1 orange-brown A Y root surface O C Fe Si Ca none LT 1 orange-brown B Y root surface O Fe C Si Al CaCO3 & soil LT 2 orange-brown A Y root surface Fe O C Si Al none LT 2 orange-brown B Y root surface Fe O Al Si C none LT 2 orange-brown C Y piece of plaque Fe Al O Si Ca none piece of root plaque on plaque-crusted surface LT 3 orange-brown A Y root surface Fe O C Ca Si CaCO3 & soil LT 3 orange-brown B Y root surface O C Fe Si Ca CaCO3 & soil LT 4 orange-brown B Y root surface Fe O C Si Ca(Fe) soil LT 4 orange-brown C.1 Y root surface Al C Fe O Si none LT 4 orange-brown C.2 N/A outer cortex C O Ca Fe Al none root interior, high Ca and Fe LT 4 orange-brown C.3 N/A cortex C O Fe Si Ca none root interior, high Fe LT 4 orange-brown D.1 Y root surface C O Fe Si(Fe) Ca none LT 4 orange-brown D.2 N/A epidermis cells C O Fe Si Ca(Fe) none cell interior, similar to cell surface LT 4 orange-brown D.3 N/A epidermis cells C O Fe Si Ca(Fe) none cell interior, similar to cell surface LT 4 orange-brown E N/A vascular cylinder C O - - - - other element peaks extremely low LT 5 orange-brown A.1 Y root surface Fe O C Si Ca(Fe) CaCO3 & soil plaque-packed cell LT 5 orange-brown A.2 Y root surface Fe C O Al Ca CaCO3 & soil thin plaqued cell LT 5 orange-brown B.3 Y epidermis cells C O Fe Si Ca CaCO3 & soil interior of hallow-plaque cell cast (Chen et al., 1980b) LT 5 orange-brown B.4 Y epidermis cells O Fe C Si Ca(Fe) CaCO3 & soil exterior of hallow-plaque cell cast (Chen et al., 1980b) Notes Relative Peak Height of Elements: Xx(Yy) means that element Xx had the next highest peak relative to element Yy, shown in brackets because it is the second peak for element Yy - = element peaks too low to determine relative height Sample Data Other Particulate Deposits X-Ray Spectra Data Comments Site Sample Root Colour Relative Peak Height of Elements Spectrum Plaque Present Location Appendix C Zizania palustris Root Plaque Examination 188 Appendix C Zizania palustris Root Plaque Examination 189 C.1.2 – Marchmont Marsh Figure C.1.2.1 – Marchmont Marsh, Sample 1 (longitudinal section: root surface orange- brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x85, root surface showing lateral roots and particulate deposits (likely soil) and plaqued cells (white); B. x600, root surface with one plaqued cell.     Appendix C Zizania palustris Root Plaque Examination 190 C.1.2 – Marchmont Marsh Figure C.1.2.2 – Marchmont Marsh, Sample 2 (longitudinal section: root surface orange- brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x600, root surface with particulate deposits; B. x1,200, root surface; C. x600, root surface.       Appendix C Zizania palustris Root Plaque Examination 191 C.1.2 – Marchmont Marsh Figure C.1.2.3 – Marchmont Marsh, Sample 3 (longitudinal section: root surface orange- brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x600, root surface with particulate deposits and little plaque present; B. x600, root surface with particulate deposits; C. x1,000, root surface with particulate deposits (likely CaCO3).       Appendix C Zizania palustris Root Plaque Examination 192 C.1.2 – Marchmont Marsh Figure C.1.2.4 – Marchmont Marsh, Sample 4 (longitudinal section: root surface orange- brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x110, root surface near cut edge; B. x900, root surface with particulate deposits (soil and plaque); C. x600, general root surface, showing white plaqued cells.       Appendix C Zizania palustris Root Plaque Examination 193 C.1.2 – Marchmont Marsh Figure C.1.2.5 (cont’d on next page)       1 2   Appendix C Zizania palustris Root Plaque Examination 194 C.1.2 – Marchmont Marsh Figure C.1.2.5 – Marchmont Marsh, Sample 8 (longitudinal section: oldest portion of root, root surface orange-brown), SEM images with associated EDXA x-ray spectra (x- ray location shown by box); A. x500, root surface with plaque-crusted cells (spectrum A.1) and plaqued cells (spectrum A.2); B. x1,000, root surface with plaque-crusted cells; C. x950, root surface, habit of plaque crust on cell edge; D. x300, unplaqued lateral root surface (spectrum D.1) and root surface with plaque-crusted cells (spectrum D.2).     1 2 Appendix C Zizania palustris Root Plaque Examination 195 C.1.2 – Marchmont Marsh Figure C.1.2.6 (cont’d on next page)     1 2     1 2 Appendix C Zizania palustris Root Plaque Examination 196 C.1.2 – Marchmont Marsh Figure C.1.2.6 – Marchmont Marsh, Sample 9 (longitudinal section: middle-aged portion of root, root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x650, root surface with plaque-packed cells (spectrum A.1) and surface-plaqued cells (spectrum A.2); B. x800, root surface with plaque-packed cells (spectrum B.1) and surface-plaqued cells (spectrum B.2); C. x1,000, plaqued root surface.   Appendix C Zizania palustris Root Plaque Examination 197 C.1.2 – Marchmont Marsh Figure C.1.2.7 – Marchmont Marsh, Sample 10 (longitudinal section: youngest portion of root, root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x1,000, plaqued root surface (spectrum A.1) and deposit between two cells (spectrum A.2); B. x900, unevenly plaqued root surface with light areas (spectrum B.1) and dark areas (spectrum B.2).     2 1     1 2 Appendix C Zizania palustris Root Plaque Examination 198 C.1.2 – Marchmont Marsh Figure C.1.2.8 (cont’d on next page)         Appendix C Zizania palustris Root Plaque Examination 199 C.1.2 – Marchmont Marsh Figure C.1.2.8 – Marchmont Marsh, Sample 11 (longitudinal section: youngest portion of root, root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x200, root surface with plaque-crusted areas; B. x2,200, plaque-crusted cells with cell cast; C. x800, plaque crust; D. x1,500, plaque crust; E. x1,000, root surface with plaque-crusted areas (spectrum E.1) and surface-plaqued areas (spectrum E.2).     1 2 C.1.2 - Marchmont Marsh Table C.1.2.1 - Marchmont Marsh, Zizania palustris Root Plaque SEM Image Data Compilation Type % Coverage Thickness Location Type % Coverage Thickness Location MM 1 orange-brown A whole section N/A 85 - - - - - - - - soil longitudinal section image for reference to subsequent image locations MM 1 orange-brown B root surface Y 600 thin 95 thin cell surface packed cells 5 unknown unknown soil single plaque-packed cell in image (white in image) MM 2 orange-brown A root surface Y 600 thin 100 thin cell surface - - - - KCl KCl appears evenly distributed with plaque MM 2 orange-brown B root surface Y 1,200 thin 100 thin cell surface - - - - KCl KCl appears evenly distributed with plaque MM 2 orange-brown C root surface Y 600 thin 75 thin cell surface crust 25 unknown cell surface KCl KCl appears evenly distributed with plaque MM 3 orange-brown A root surface N 600 - - - - - - - - present, unknown may be a very slight plaque present, slight peak in Fe shown in x-ray spectra MM 3 orange-brown B root surface N 600 - - - - - - - - present, unknown no plaque present MM 3 orange-brown C root surface N 1,000 - - - - - - - - present, unknown no plaque present MM 4 orange-brown A whole section N/A 110 - - - - - - - - soil longitudinal section image for reference to subsequent image locations MM 4 orange-brown B root surface Y 900 unknown - - - - - - - soil plaque may be thin, too much soil to confirm MM 4 orange-brown C root surface Y 600 thin 70 thin cell surface packed cells 30 unknown unknown soil white cells indicative of plaque-packed cells MM 8 orange-brown A root surface Y 500 crust 85 thick cell surface thin 15 thin cell surface soil oldest portion of root: crust follows shape of cells, could be on and within cells, difficult to determine MM 8 orange-brown B root surface Y 1,000 crust 50 1.5 - 3 µm cell surface thin 50 thin cell surface none oldest portion of root: crust could be on and within cells, difficult to determine MM 8 orange-brown C root surface Y 950 crust 60 2 µm cell surface thin 40 thin cell surface none oldest portion of root: crust appears to be on the surface of compacted cells (likely compacted from sample processing), deposition % based on observed root surface cell area MM 8 orange-brown D root surface Y 300 crust 60 thick cell surface thin 40 thin cell surface none oldest portion of root: plaque type and deposition % based on observed root surface area (minus lateral root area which has no plaque) MM 9 orange-brown A root surface Y 650 thin 50 thin cell surface packed cells 50 unknown on and within cells soil middle-aged portion of root: plaque appears to be within plaque- packed cells MM 9 orange-brown B root surface Y 800 thin 90 thin cell surface packed cells 10 unknown on and within cells soil middle-aged portion of root: plaque-packed and thin plaqued cells MM 9 orange-brown C root surface Y 1,000 thin 85 thin cell surface packed cells 15 unknown unknown none middle-aged portion of root MM 10 orange-brown A root surface Y 1,000 thin 100 thin cell surface - - - - S youngest portion of root: strip of particulate deposit along centre cell, composed of sulphur MM 10 orange-brown B root surface Y 900 thin 100 thin cell surface - - - - soil youngest portion of root: plaque unevenly distributed MM 11 orange-brown A whole section N/A 200 - - - - - - - - - youngest portion of root: longitudinal section image, plaque crust over lower portion of root and along middle-line MM 11 orange-brown B root surface Y 2,200 crust 100 2 to 7 µm on and within cells - - - - none youngest portion of root: plaque-crusted area with plaque cell cast (similar to that in Chen et al., 1980b) MM 11 orange-brown C root surface Y 800 crust 100 thick on and within cells - - - - none youngest portion of root: thick plaque crust MM 11 orange-brown D root surface Y 1,500 crust 100 4 µm on and within cells - - - - none youngest portion of root: thick plaque crust MM 11 orange-brown E root surface Y 1,000 thin 75 thick on and within cells crust 25 thin cell surface none youngest portion of root: crusted area, thin plaque on remainder of root Notes Plaque thickness = thin (too thin to measure, approximately <1 µm) Plaque thickness = thick (not able to measure, at least >1.5 µm) - = N/A Image Location Plaque Present Zoom Primary Deposition Secondary Deposition Other Particulate Deposits Sample Data SEM Observation Data Comments Site Sample Root Colour Image Appendix C Zizania palustris Root Plaque Examination 200 C.1.2 - Marchmont Marsh Table C.1.2.2 - Marchmont Marsh, Zizania palustris Root Plaque EDXA X-Ray Spectra Data Compilation 1 2 3 4 5 MM 1 orange-brown B Y root surface Fe C Al O Si soil spectra on single plaque-packed cell MM 2 orange-brown A Y root surface C O Cl K Fe KCl KCl present, evenly distributed MM 2 orange-brown B Y root surface C Fe Cl K O KCl KCl present, evenly distributed MM 2 orange-brown C Y root surface K Fe C Cl O KCl KCl present, evenly distributed MM 3 orange-brown A N root surface C O Cl Al Fe present, unknown may be a slight Fe plaque, not prominent MM 3 orange-brown B N root surface C O Al - - present, unknown particulate deposits visible, unsure of composition MM 3 orange-brown C N root surface C Al O Ca - present, unknown particulate deposits visible, unsure of composition MM 4 orange-brown B Y root surface C O Si Fe Al soil soil particulates and plaque present MM 4 orange-brown C Y root surface C O Fe Al K soil MM 8 orange-brown A.1 Y root surface O Fe C Si Al soil oldest portion of root: crust follows shape of cells, Fe peak stronger in crust MM 8 orange-brown A.2 Y root surface C O Fe Al Ca soil oldest portion of root: cells with thin plaque MM 8 orange-brown B Y root surface C Fe O Al Si none oldest portion of root: crust follows shape of cells MM 8 orange-brown D.1 N lateral root C O - - - none oldest portion of root: other element peaks extremely low MM 8 orange-brown D.2 Y root surface Fe O C Ca S(Fe) none oldest portion of root: crust MM 9 orange-brown A.1 Y root surface C O Fe Si(Fe) S soil middle-aged portion of root: plaque-packed cell MM 9 orange-brown A.2 Y root surface Fe C O Si Al soil middle-aged portion of root: thin-plaqued cell MM 9 orange-brown B.1 Y root surface Fe C Al Ca O(Fe) soil middle-aged portion of root: plaque-packed cell MM 9 orange-brown B.2 Y root surface Fe C Si O Al soil middle-aged portion of root: thin-plaqued cell MM 9 orange-brown C Y root surface Fe C Si O Al none middle-aged portion of root: dark area, soil particulates and plaque present MM 10 orange-brown A.1 Y root surface Fe C O(Fe) Ca Al none youngest portion of root: thin plaque MM 10 orange-brown A.2 N between-cell deposit S C Fe - - S youngest portion of root: particulate deposit between cells MM 10 orange-brown B.1 Y root surface Fe C O Si Al(Fe) soil youngest portion of root: uneven thin plaque distribution: light area MM 10 orange-brown B.2 Y root surface C Fe O Si Al soil youngest portion of root: uneven thin plaque distribution: dark area MM 11 orange-brown B Y root surface Fe O C Al(Fe) Si none youngest portion of root: plaque cast (Chen et al., 1980b) MM 11 orange-brown C Y root surface Fe O C Si(Fe) Al none youngest portion of root: plaque crust MM 11 orange-brown D Y root surface O C Fe Al(Fe) Si none youngest portion of root: plaque crust MM 11 orange-brown E.1 Y root surface C Fe O Si(Fe) Ca none youngest portion of root: plaque crust MM 11 orange-brown E.2 Y root surface C O Fe Al Ca none youngest portion of root: thin plaque Notes Relative Peak Height of Elements: Xx(Yy) means that element Xx had the next highest peak relative to element Yy, shown in brackets because it is the second peak for element Yy - = element peaks too low to determine relative height Relative Peak Height of Elements Other Particulate Deposits X-Ray Spectra DataSample Data Comments Site Sample Root Colour Spectrum Plaque Present Location Appendix C Zizania palustris Root Plaque Examination 201 Appendix C Zizania palustris Root Plaque Examination 202 C.1.3 – Partridge Crop Lake Figure C.1.3.1 – Partridge Crop Lake, Sample 1 (longitudinal section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x750, plaque-crusted root surface, note deposit follows contours of cells; B. x1,000, plaque-crusted root surface; C. x250, root surface with lateral root protruding; D. x600, plaque-crusted root surface.       Appendix C Zizania palustris Root Plaque Examination 203 C.1.3 – Partridge Crop Lake Figure C.1.3.2 – Partridge Crop Lake, Sample 2 (longitudinal section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x300, root surface with plaque; B. x750, root surface cell packed with plaque; C. x750, root surface with particulate deposits.       Appendix C Zizania palustris Root Plaque Examination 204 C.1.3 – Partridge Crop Lake Figure C.1.3.3 (cont’d on next page)         1 2 1 2 Appendix C Zizania palustris Root Plaque Examination 205 C.1.3 – Partridge Crop Lake Figure C.1.3.3 – Partridge Crop Lake, Sample 3 (carbon-coated cross section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x180, outer cortex (spectrum A.1) and cortex (spectrum A.2); B. x450, root surface (spectrum B.1) and cortex (spectrum B.2); C. x600, epidermis / root surface (spectrum C.1), epidermis cell interior (spectrum C.2) and outer cortex (spectrum C.3).     1 2 3 Appendix C Zizania palustris Root Plaque Examination 206 C.1.3 – Partridge Crop Lake Figure C.1.3.4 (cont’d on next page)     1 2 3   5 4 Appendix C Zizania palustris Root Plaque Examination 207 C.1.3 – Partridge Crop Lake Figure C.1.3.4 – Partridge Crop Lake, Sample 4 (carbon-coated cross section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x550, epidermis cell interior (spectrum A.1), plaque-packed epidermis cell (spectrum A.2), outer cortex (spectrum A.3), and cortex (spectra A.4 and A.5); B. x850, root surface (spectrum B.1) and outer cortex (spectrum B.2).     1 2 Appendix C Zizania palustris Root Plaque Examination 208 C.1.3 – Partridge Crop Lake Figure C.1.3.5 (cont’d on next page)     1 2 3 Appendix C Zizania palustris Root Plaque Examination 209 C.1.3 – Partridge Crop Lake Figure C.1.3.5 – Partridge Crop Lake, Sample 5 (carbon-coated cross section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x330, epidermis cell interior (spectrum A.1), outer cortex (spectrum A.2) and cortex (spectrum A.3); B. x700, root / epidermis surface (spectrum B.1), epidermis cell interior (spectrum B.2), outer cortex (spectrum B.3) and cortex (spectrum B.4).     1 2 3   4 Appendix C Zizania palustris Root Plaque Examination 210 C.1.3 – Partridge Crop Lake Figure C.1.3.6 (cont’d on next page)         1 2 Appendix C Zizania palustris Root Plaque Examination 211 C.1.3 – Partridge Crop Lake Figure C.1.3.6 – Partridge Crop Lake, Sample 6 (longitudinal section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x100, root surface with plaque crust; B. x600, plaque crust; C. x600, plaque crust; D. x1,500, plaque crust; E. x1,000, root surface with plaque crust broken away (spectrum E.1) and present (spectrum E.2); F. x500, root surface with plaque crust broken away (spectrum F.1) and present (spectrum F.2).     1 2 C.1.3 - Partridge Crop Lake Table C.1.3.1 - Partridge Crop Lake, Zizania palustris Root Plaque SEM Image Data Compilation Type % Coverage Thickness Location Type % Coverage Thickness Location SeR 1 orange-brown A root surface Y 750 crust 100 thick cell surface - - - - none crust appears to be of variable thickness SeR 1 orange-brown B root surface Y 1,000 crust 100 thick cell surface - - - - none crust appears to be of variable thickness SeR 1 orange-brown C root surface Y 250 crust 100 thick cell surface - - - - none crust appears to be of variable thickness; plaque appears present on lateral root SeR 1 orange-brown D root surface Y 600 crust 100 thick cell surface - - - - none consistent thickness in crust, some cracks SeR 2 orange-brown A root surface Y 300 thin 85 thin cell surface packed cells 15 unknown within cells none deposition % based on observed root surface area SeR 2 orange-brown B root surface Y 750 thin 90 thin cell surface packed cells 10 unknown within cells soil SeR 2 orange-brown C root surface Y 750 thin 100 thin cell surface - - - - soil SeR 3 orange-brown A cross section Y 180 thin 100 thin cell surface - - - - soil deposition % based on observed root surface area SeR 3 orange-brown B cross section Y 450 thin 100 thin cell surface - - - - none deposition % based on observed root surface area SeR 3 orange-brown C cross section N/A 600 - - - - - - - - - cross section, no root surface visible to assess plaque SeR 4 orange-brown A cross section N/A 550 crust 100 thick on and within cells - - - - none plaque deposits on root surface and within root surface cells, but not past epidermis SeR 4 orange-brown B cross section Y 850 crust 100 1.5 µm on and within cells - - - - soil plaque deposits on root surface and within root surface cells, but not past epidermis SeR 5 orange-brown A cross section N/A 330 - - - - - - - - - cross section, no root surface visible to assess plaque SeR 5 orange-brown B cross section Y 700 unknown - - - - - - - - cross section, not enough root surface visible to assess plaque thickness, can confirm presence SeR 6 orange-brown A root surface Y 100 crust 90 thick on and within cells broken away 10 unknown within cells none deposition % based on observed root surface area SeR 6 orange-brown B root surface Y 600 crust 100 thick on and within cells - - - - none thick plaque crust, follows contours of cells SeR 6 orange-brown C root surface Y 600 crust 90 thick on and within cells broken away 10 unknown within cells none where plaque crust has broken away, appears that the cell wall of the root surface cell is gone as well SeR 6 orange-brown D root surface Y 1,500 crust 95 3 µm within cells broken away 5 unknown within cells none plaque-crusted area with plaque cell cast (similar to that in Chen et al., 1980b) SeR 6 orange-brown E root surface Y 1,000 crust 80 thick on and within cells broken away 20 unknown within cells none where plaque crust has broken away, appears that the cell wall of the root surface cell is gone as well SeR 6 orange-brown F root surface Y 500 crust 60 thick unknown broken away 40 unknown unknown soil cannot tell if plaque is in or on cells Notes Plaque thickness = thin (too thin to measure, approximately <1 µm) Plaque thickness = thick (not able to measure, at least >1.5 µm) - = N/A Image Location Plaque Present Zoom Primary Deposition Secondary Deposition Other Particulate Deposits Sample Data SEM Observation Data Comments Site Sample Root Colour Image Appendix C Zizania palustris Root Plaque Examination 212 C.1.3 - Partridge Crop Lake Table C.1.3.2 - Partridge Crop Lake, Zizania palustris Root Plaque EDXA X-Ray Spectra Data Compilation 1 2 3 4 5 SeR 1 orange-brown A Y root surface Fe O C(Fe) K Si none plaque crust SeR 1 orange-brown D Y root surface Si Fe O Al K none visible soil may be incoporated into plaque crust SeR 2 orange-brown B Y root surface Fe K C O Al none plaque-packed cell SeR 2 orange-brown C Y root surface Fe O C K Al none SeR 3 orange-brown A.1 N/A outer cortex C K O Fe P N/A root interior, for comparison to root surface SeR 3 orange-brown A.2 N/A cortex Ca K C O Fe N/A root interior, for comparison to root surface SeR 3 orange-brown B.1 Y root surface Fe C Al K O none SeR 3 orange-brown B.2 N/A cortex Ca K Fe(Ca) - - N/A root interior, for comparison to root surface SeR 3 orange-brown C.1 Y root surface Al Fe O C K none SeR 3 orange-brown C.2 N/A epidermis cells Fe Al Ca K - N/A root interior, for comparison to root surface SeR 3 orange-brown C.3 N/A outer cortex C O K - - N/A root interior, for comparison to root surface SeR 4 orange-brown A.1 Y epidermis cells C O Fe Al Si none surface cell SeR 4 orange-brown A.2 Y epidermis cells Fe C Al O K none plaque-packed cell SeR 4 orange-brown A.3 N/A outer cortex C O - - - N/A root interior, for comparison to root surface SeR 4 orange-brown A.4 N/A cortex C K O Fe - N/A root interior, for comparison to root surface SeR 4 orange-brown A.5 N/A cortex C K Ca Fe O N/A root interior, for comparison to root surface SeR 4 orange-brown B.1 Y epidermis cells C O Fe Si Al none plaque crust SeR 4 orange-brown B.2 N/A outer cortex C O Si K - N/A root interior, for comparison to root surface SeR 5 orange-brown A.1 N/A epidermis cells C O Fe Al K N/A surface cell SeR 5 orange-brown A.2 N/A outer cortex C O K Ca Si N/A root interior, for comparison to root surface SeR 5 orange-brown A.3 N/A cortex C K Ca O - N/A root interior, for comparison to root surface SeR 5 orange-brown B.1 Y root surface Fe C O Al K none plaque visible SeR 5 orange-brown B.2 N/A epidermis cells C O Al Fe K N/A Fe spectrum greatly reduced compared to root surface SeR 5 orange-brown B.3 N/A outer cortex C O K Fe - N/A root interior, for comparison to root surface SeR 5 orange-brown B.4 N/A cortex C O K Ca Fe N/A root interior, for comparison to root surface SeR 6 orange-brown E.1 Y root surface Fe K Al C O none root surface, appears that crust is broken away SeR 6 orange-brown E.2 Y root surface Fe K Ca(Fe) Al O none plaque crust, extremely high Fe SeR 6 orange-brown F.1 Y root surface C O Fe K Al none root surface where plaque crust has broken away SeR 6 orange-brown F.2 Y root surface Fe O C Si K(Fe) soil plaque crust, extremely high Fe Notes Relative Peak Height of Elements: Xx(Yy) means that element Xx had the next highest peak relative to element Yy, shown in brackets because it is the second peak for element Yy - = element peaks too low to determine relative height Relative Peak Height of Elements Other Particulate Deposits X-Ray Spectra DataSample Data Comments Site Sample Root Colour Spectrum Plaque Present Location Appendix C Zizania palustris Root Plaque Examination 213 Appendix C Zizania palustris Root Plaque Examination 214 C.1.4 – Lower Steep Rock Lake Figure C.1.4.1 – Lower Steep Rock Lake, Sample 1 (longitudinal section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x1,000, plaque-crusted root surface; B. x600, root surface; C. x950, root surface with particulate deposits.       Appendix C Zizania palustris Root Plaque Examination 215 C.1.4 – Lower Steep Rock Lake Figure C.1.4.2 – Lower Steep Rock Lake, Sample 2 (longitudinal section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x600, root surface; B. x1,000, root surface with particulate deposits; C. x750, root surface with particulate deposits.       Appendix C Zizania palustris Root Plaque Examination 216 C.1.4 – Lower Steep Rock Lake Figure C.1.4.3 (cont’d on next page)         1 2 3 1 2 3 Appendix C Zizania palustris Root Plaque Examination 217 C.1.4 – Lower Steep Rock Lake Figure C.1.4.3 – Lower Steep Rock Lake, Sample 3 (cross section: root surface orange- brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x650, “crusted” root surface (spectrum A.1), outer cortex (spectrum A.2) and cortex (spectrum A.3); B. x650, root surface (spectrum B.1), outer cortex (spectrum B.2) and cortex (spectrum B.3); C. x1,000, root surface (spectrum C.1), outer cortex (spectrum C.2) and cortex (spectrum C.3).     1 2 3 Appendix C Zizania palustris Root Plaque Examination 218 C.1.4 – Lower Steep Rock Lake Figure C.1.4.4 (cont’d on next page)         1 2 3 Appendix C Zizania palustris Root Plaque Examination 219 Figure C.1.4.4 – Lower Steep Rock Lake, Sample 4 (carbon-coated cross section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x140, root cross section; B. x270, root vascular cylinder; C. x600, epidermis/root surface cells filled with plaque (spectrum C.1), outer cortex (spectrum C.2) and cortex (spectrum C.3); D. x600, epidermis cell interior (spectrum D.1), outer cortex (spectrum D.2) and cortex (spectrum D.3); E. x650, epidermis plaque-filled cell interior (spectrum E.1), epidermis empty cell interior (spec. E.2) and cortex (spec. E.3).     1 2 3     1 2 3 C.1.4 – Lower Steep Rock Lake Appendix C Zizania palustris Root Plaque Examination 220 C.1.4 – Lower Steep Rock Lake Figure C.1.4.5 – Lower Steep Rock Lake, Sample 5 (carbon-coated cross section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x220, root surface (spectrum A.1), outer cortex (spectrum A.2) and cortex (spectrum A.3); B. x350, root surface cells (spectrum B.1), outer cortex (spectrum B.2) and cortex (spectrum B.3).         1 2 3 1 2 3 Appendix C Zizania palustris Root Plaque Examination 221 C.1.4 – Lower Steep Rock Lake Figure C.1.4.6 (cont’d on next page)       1 2   Appendix C Zizania palustris Root Plaque Examination 222 C.1.4 – Lower Steep Rock Lake Figure C.1.4.6 – Lower Steep Rock Lake, Sample 6 (cross section: root surface orange- brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x85, root cross section; B. x1,000, root surface (spectrum B.1) and outer cortex (spectrum B.2); C. x450, lateral root, showing root surface plaque coverage; D. x950, root vascular cylinder; E. root surface with plaque, three zoomed images showing plaque thickness (area shown by dashed box), E.1 x1,000, E.2 x2,000, E.3 x7,000.       C.1.4 - Lower Steep Rock Lake Table C.1.4.1 - Lower Steep Rock Lake, Zizania palustris Root Plaque SEM Image Data Compilation Type % Coverage Thickness Location Type % Coverage Thickness Location StR 1 orange-brown A root surface Y 1,000 crust 100 1 µm cell surface - - - - none thin plaque crust, cell surface flaking up StR 1 orange-brown B root surface Y 600 crust 100 1 µm cell surface - - - - none even crust StR 1 orange-brown C root surface Y 950 crust 100 1 µm cell surface - - - - soil bits of soil and plaque on surface StR 2 orange-brown A root surface Y 600 thin with crust pieces 100 thin cell surface - - - - soil thin plaque with pieces of plaque on surface StR 2 orange-brown B root surface Y 1,000 thin with crust pieces 100 thin cell surface - - - - soil thin plaque with pieces of plaque (up to 20 µm in length) on surface StR 2 orange-brown C root surface Y 750 thin with crust pieces 100 thin cell surface - - - - soil thin plaque with pieces of plaque (up to 10 µm in length) on surface StR 3 orange-brown A cross section Y 650 unknown - - - - - - - soil cross section, not enough root surface visible to assess plaque thickness, can confirm presence StR 3 orange-brown B cross section Y 650 unknown - - - - - - - soil cross section, not enough root surface visible to assess plaque thickness, can confirm presence StR 3 orange-brown C cross section N/A 1,000 - - - - - - - - soil cross section, not enough root surface visible to assess plaque presence StR 4 orange-brown A cross section N/A 140 - - - - - - - - - cross section image for reference to subsequent image locations StR 4 orange-brown B cross section N/A 270 - - - - - - - - - cross section image for reference to subsequent image locations StR 4 orange-brown C cross section Y 600 crust/packed cells N/A - - - - - - none root surface cells filled with loosely-packed plaque, from root surface to epidermis cells, up to 40 µm in depth StR 4 orange-brown D cross section Y 600 crust/packed cells N/A 2 µm on and within cells - - - - none deposition % based on observed root surface area StR 4 orange-brown E cross section Y 650 crust/packed cells N/A 2 to 30 µm on and within cells - - - - none deposition % based on observed root surface area StR 5 orange-brown A cross section N 220 - - - - - - - - soil no plaque on root surface StR 5 orange-brown B cross section Y 350 thin N/A thin cell surface - - - - soil thin plaque on root surface StR 6 orange-brown A cross section N/A 85 - - - - - - - - - cross section image for reference to subsequent image locations StR 6 orange-brown B cross section Y 1,000 crust 100 1 µm cell surface - - - - soil flakey crust on root surface, deposition % based on observed root surface area StR 6 orange-brown C root surface Y 450 crust 100 thick cell surface - - - - soil plaque crust on root and lateral root surface StR 6 orange-brown D vascular cylinder N/A 95 - - - - - - - - - root interior StR 6 orange-brown E.1 root surface Y 1,000 crust 100 1 to 2 µm cell surface - - - - none plaque crust on root surface StR 6 orange-brown E.2 root surface Y 2,000 crust N/A 1 to 2 µm cell surface - - - - none zoom of above; plaque crust on root surface StR 6 orange-brown E.3 root surface Y 7,000 crust N/A 1 to 2 µm cell surface - - - - none zoom of above; plaque crust on two root surface cells Notes Plaque thickness = thin (too thin to measure, approximately <1 µm) Plaque thickness = thick (not able to measure, at least >1.5 µm) - = N/A Primary Deposition Secondary Deposition Other Particulate Deposits Sample Data SEM Observation Data Comments Site Sample Root Colour Image Image Location Plaque Present Zoom Appendix C Zizania palustris Root Plaque Examination 223 C.1.4 - Lower Steep Rock Lake Table C.1.4.2 - Lower Steep Rock Lake, Zizania palustris Root Plaque EDXA X-Ray Spectra Data Compilation 1 2 3 4 5 StR 1 orange-brown A Y root surface Fe O Al C(Fe) K(Fe) none flakey plaque crust StR 1 orange-brown B Y root surface Fe O Al(Fe) C K none even plaque crust StR 1 orange-brown C Y root surface Fe O Al C Si soil bits of soil and plaque on surface of crust StR 2 orange-brown A Y root surface C Fe O Na Al soil thin plaque with pieces StR 2 orange-brown B Y piece of plaque Fe O Si Al C soil large piece of plaque on root surface StR 2 orange-brown C Y root surface C Fe O Al Na/Si soil thin plaque with pieces StR 3 orange-brown A.1 Y root surface Fe C O Al Si soil unsure of plaque type StR 3 orange-brown A.2 Y outer cortex Fe C O Al Si N/A root interior, for comparison to root surface; plaque presence may be result of sample preparation StR 3 orange-brown A.3 N/A cortex C O Al Fe K N/A root interior, for comparison to root surface StR 3 orange-brown B.1 Y root surface C O Fe Al Si soil unsure of plaque type StR 3 orange-brown B.2 Y outer cortex Fe C K Al O N/A high Fe, plaque present, consistent with that of A.2 StR 3 orange-brown B.3 N/A cortex C O Si K Al N/A root interior, for comparison to root surface StR 3 orange-brown C.1 N root surface Si Al O Na Ca soil surface "crust" appears to be soil StR 3 orange-brown C.2 N/A outer cortex C O Fe K Cl N/A root interior, for comparison to root surface StR 3 orange-brown C.3 N/A cortex Fe K Cl C Ca N/A root interior, for comparison to root surface StR 4 orange-brown B N/A vascular cylinder C K Cl O S N/A root interior, for comparison to root surface StR 4 orange-brown C.1 Y epidermis cells C Fe O K - none root surface cells filled with plaque, collapsed down to epidermis StR 4 orange-brown C.2 Y outer cortex Fe K Cl Ca(Fe) - none epidermis with root surface cells collapsed in StR 4 orange-brown C.3 N/A cortex C O - - - N/A cortex intact, no plaque StR 4 orange-brown D.1 Y epidermis cells Fe K C O Cl none plaque-filled surface cells StR 4 orange-brown D.2 Y outer cortex Fe K Cl Ca C none epidermis cells with plaque StR 4 orange-brown D.3 N/A cortex C K Cl Fe O N/A root interior, for comparison to root surface StR 4 orange-brown E.1 Y epidermis cells Fe O C K(Fe) Si none plaque-packed surface cell StR 4 orange-brown E.2 Y epidermis cells Fe K Cl(Fe) - - none empty surface cell; still high Fe StR 4 orange-brown E.3 N/A cortex C Fe O K Si N/A root interior, for comparison to root surface; high Fe StR 5 orange-brown A.1 N root surface Si O C Mg Al soil no plaque StR 5 orange-brown A.2 N/A outer cortex C O - - - N/A root interior, for comparison to root surface StR 5 orange-brown A.3 N/A cortex C O K - - N/A root interior, for comparison to root surface StR 5 orange-brown B.1 Y epidermis cells C O Fe Al Si soil slight root surface plaque StR 5 orange-brown B.2 N/A outer cortex C O K - - N/A root interior, for comparison to root surface StR 5 orange-brown B.3 N/A cortex C K O P Ca N/A root interior, for comparison to root surface StR 6 orange-brown B.1 Y root surface Fe O Si C K/Al soil flakey plaque crust StR 6 orange-brown B.2 Y outer cortex C Fe O K Si none root interior, for comparison to root surface; plaque present StR 6 orange-brown C Y root surface C O Fe Si Al(Fe) soil plaque crust on root and lateral root surface StR 6 orange-brown D N/A vascular cylinder C O K Cl P N/A root interior, for comparison to root surface StR 6 orange-brown E.4 Y root surface Fe O C Si Al(Fe) none plaque crust Notes Relative Peak Height of Elements: Xx(Yy) means that element Xx had the next highest peak relative to element Yy, shown in brackets because it is the second peak for element Yy Relative Peak Height of Elements: Xx/Yy means that element Xx and Yy had equal peak heights - = element peaks too low to determine relative height Relative Peak Height of Elements Other Particulate Deposits X-Ray Spectra DataSample Data Comments Site Sample Root Colour Spectrum Plaque Present Location Appendix C Zizania palustris Root Plaque Examination 224 Appendix C Zizania palustris Root Plaque Examination 225 C.1.5 – Whitefish Lake Figure C.1.5.1 – Whitefish Lake, Sample 1 (longitudinal section: root surface orange- brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x450, root surface; B. x750, root surface, no plaque; C. x700, root surface with little plaque.       Appendix C Zizania palustris Root Plaque Examination 226 C.1.5 – Whitefish Lake Figure C.1.5.2 – Whitefish Lake, Sample 2 (longitudinal section: root surface orange- brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x1,100, root surface with plaque/particulate deposits; B. x850, root surface with plaque.     Appendix C Zizania palustris Root Plaque Examination 227 C.1.5 – Whitefish Lake Figure C.1.5.3 (cont’d on next page)         1 2 3 Appendix C Zizania palustris Root Plaque Examination 228 C.1.5 – Whitefish Lake Figure C.1.5.3 – Whitefish Lake, Sample 3 (carbon-coated cross section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x90, root cross section; B. x700, root surface with plaque crust; C. x750, epidermis / root surface (spectrum C.1), epidermis cell interior (spectrum C.2) and outer cortex (spectrum C.3); D. x1,000, root surface with plaque crust; E. x1,200, epidermis / root surface (spectrum E.1), outer cortex (spectrum E.2) and cortex (spectrum E.3).     1 2 3   Appendix C Zizania palustris Root Plaque Examination 229 C.1.5 – Whitefish Lake Figure C.1.5.4 (cont’d on next page)     1 2 3     1 2 Appendix C Zizania palustris Root Plaque Examination 230 C.1.5 – Whitefish Lake Figure C.1.5.4 – Whitefish Lake, Sample 4 (carbon-coated cross section: root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x110, lateral root within epidermis (spectrum A.1), lateral root outside of epidermis (spectrum A.2) and root surface (spectrum A.3); B. x370, root surface (spectrum B.1) and cortex (spectrum B.2); C. x650, lateral root surface within epidermis; D. x500, root surface (spectrum D.1) and cortex (spectrum D.2).     1 2   Appendix C Zizania palustris Root Plaque Examination 231 Figure C.1.5.5 – Whitefish Lake, Sample 8 (longitudinal section: oldest portion of root, root surface orange-brown), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x1,000, root surface, no plaque; B. x2,000, cell with CaCO3 deposit; C. x700, root surface, cell with CaCO3 deposit (spectrum C.1) and cell without deposit (spectrum C.2).     1 2     C.1.5 – Whitefish Lake Appendix C Zizania palustris Root Plaque Examination 232 C.1.5 – Whitefish Lake Figure C.1.5.6 (cont’d on next page)     1 2     Appendix C Zizania palustris Root Plaque Examination 233 C.1.5 – Whitefish Lake Figure C.1.5.6 – Whitefish Lake, Sample 9 (longitudinal section: middle-aged portion of root, root surface orange-brown), SEM images with associated EDXA x-ray spectra (x- ray location shown by box); A. x850, general root surface; B. x500, lateral root protruding through root surface; C. x1,500, lateral root surface, light coloured cell (spectrum C.1) and dark coloured cell (spectrum C.2); D. x450, root surface with Fe particulate matter deposit.   Appendix C Zizania palustris Root Plaque Examination 234 C.1.5 – Whitefish Lake Figure C.1.5.7 (cont’d on next page)     1 2     Appendix C Zizania palustris Root Plaque Examination 235 C.1.5 – Whitefish Lake Figure C.1.5.7 – Whitefish Lake, Sample 10 (longitudinal section: youngest portion of root, root surface orange-brown), SEM images with associated EDXA x-ray spectra (x- ray location shown by box); A. x200, general root surface showing lateral roots; B. x1,200, root surface, cell packed with plaque (spectrum B.1) and un-packed cell (spectrum B.2); C. x2,000, close-up of plaque-packed cell; D. x1,200, root surface with precipitate (spectrum D.1) and without precipitate (spectrum D.2).     1 2 C.1.5 - Whitefish Lake Table C.1.5.1 - Whitefish Lake, Zizania palustris Root Plaque SEM Image Data Compilation Type % Coverage Thickness Location Type % Coverage Thickness Location WF 1 orange-brown A root surface N 450 - - - - - - - - none may be some thin plaque, not visible WF 1 orange-brown B root surface N 750 - - - - - - - - none no plaque WF 1 orange-brown C root surface Y 700 thin 100 thin cell surface - - - - none very little thin plaque WF 2 orange-brown A root surface Y 1,100 crust 100 thin cell surface - - - - none flakey plaque crust WF 2 orange-brown B root surface Y 850 crust 100 thin cell surface - - - - none flakey plaque crust WF 3 orange-brown A cross section N/A 90 - - - - - - - - - cross section image for reference to subsequent image locations WF 3 orange-brown B root surface Y 700 crust 85 3 µm on and within cells broken away 15 unknown within soil crust follows contours of cells, where plaque crust broken away, appears that cell wall of root surface cell is gone WF 3 orange-brown C cross section Y 750 crust N/A 1 µm cell surface - - - - none cross section, not enough root surface visible to assess plaque thickness, can confirm presence WF 3 orange-brown D root surface Y 1,000 crust 85 1 to 14 µm cell surface broken away 15 unknown within soil thick crust, follows contours of cells WF 3 orange-brown E cross section Y 1,200 crust N/A 2 µm cell surface - - - - none thin plaque precipitate may also be present on root epidermis cells WF 4 orange-brown A cross section N 110 - - - - - - - - none cross section image for reference to subsequent image locations WF 4 orange-brown B cross section N 370 - - - - - - - - none cross section, no plaque on root surface WF 4 orange-brown C lateral root surface N 650 - - - - - - - - none lateral root within epidermis, has flakey surface, not likely plaque WF 4 orange-brown D cross section N 500 - - - - - - - - none cross section, no plaque on root surface WF 8 orange-brown A root surface N 1,000 - - - - - - - - - oldest portion of root: no plaque WF 8 orange-brown B root surface N 2,000 - - - - - - - - - oldest portion of root: no plaque WF 8 orange-brown C root surface N 700 - - - - - - - - CaCO3 oldest portion of root: no plaque, CaCO3 deposits WF 9 orange-brown A root surface Y 850 thin 100 thin cell surface - - - - none middle-aged portion of root: extremely thin plaque WF 9 orange-brown B root surface N 500 - - - - - - - - Fe deposit middle-aged portion of root: Fe/particulate matter deposit WF 9 orange-brown C lateral root surface N 1,500 - - - - - - - - none middle-aged portion of root: lateral root has flakey surface, not likely plaque WF 9 orange-brown D root surface N 450 - - - - - - - - Fe deposit middle-aged portion of root: Fe/particulate matter deposit WF 10 orange-brown A whole section N/A 200 - - - - - - - - - youngest portion of root: longitudinal section image for reference to subsequent image locations WF 10 orange-brown B root surface Y 1,200 thin 65 thin cell surface packed cells 35 unknown within none youngest portion of root: plaque-packed cells WF 10 orange-brown C root surface Y 2,000 packed cells 65 unknown within thin 35 thin cell surface none youngest portion of root: zoom of prior image of plaque-packed cells WF 10 orange-brown D root surface Y 1,200 thin 100 thin cell surface - - - - none youngest portion of root: plaque precipitate appears uneven, thicker on certain cells Notes Plaque thickness = thin (too thin to measure, approximately <1 µm) Plaque thickness = thick (not able to measure, at least >1.5 µm) - = N/A Primary Deposition Secondary Deposition Other Particulate Deposits Sample Data SEM Observation Data Comments Site Sample Root Colour Image Image Location Plaque Present Zoom Appendix C Zizania palustris Root Plaque Examination 236 C.1.5 - Whitefish Lake Table C.1.5.2 - Whitefish Lake, Zizania palustris Root Plaque EDXA X-Ray Spectra Data Compilation 1 2 3 4 5 WF 1 orange-brown B N root surface C Al O S - none no plaque WF 1 orange-brown C Y root surface C O S Fe - none very little plaque WF 2 orange-brown A Y root surface Fe Al C S O none flakey plaque crust WF 2 orange-brown B Y root surface Fe Al C O Cl none flakey plaque crust WF 3 orange-brown B Y root surface Fe O C S(Fe) - soil plaque crust WF 3 orange-brown C.1 Y root surface C O Fe Al S(Fe) none WF 3 orange-brown C.2 Y epidermis cells C O Fe S Al none plaque present within cells WF 3 orange-brown C.3 N outer cortex C O Fe S - N/A root interior, high Fe WF 3 orange-brown D Y root surface Fe Al - - - soil thick plaque crust WF 3 orange-brown E.1 Y root surface Fe Al C S O(Fe) none plaque crust WF 3 orange-brown E.2 Y outer cortex Fe C Al S O none plaque precipitates visible WF 3 orange-brown E.3 N/A cortex Fe C S K Ca N/A root interior, Fe elevated though not high WF 4 orange-brown A.1 N lateral root surface C O - - - none lateral root within epidermis, no plaque WF 4 orange-brown A.2 N lateral root surface Al C K O - none lateral root protruding outside of epidermis, no plaque WF 4 orange-brown A.3 N root surface Al C Fe S Si none no plaque WF 4 orange-brown B.1 N root surface Al C Fe Cl K none no plaque WF 4 orange-brown B.2 N cortex C O S Na K N/A root interior WF 4 orange-brown C N lateral root surface Fe K S Cl - none Fe elevated, though not high (only 1.7 counts per second) WF 4 orange-brown D.1 N root surface Al C Fe - - none no plaque WF 4 orange-brown D.2 N/A cortex Fe Al K C(Fe) - none root interior, Fe elevated though not high WF 8 orange-brown A N root surface C O Al S - none oldest portion of root: root surface with no plaque WF 8 orange-brown B N root surface C Ca O Al K none oldest portion of root: root surface cell with Ca deposit WF 8 orange-brown C.1 N root surface C O Ca Al - CaCO3 oldest portion of root: root surface cell with particulates WF 8 orange-brown C.2 N root surface C O - - - none oldest portion of root: root surface cell without particulates WF 9 orange-brown A Y root surface C O Fe K - none middle-aged portion of root: extremely thin plaque WF 9 orange-brown B N root surface C O Fe Al Si Fe deposit middle-aged portion of root: Fe/particulate matter deposit WF 9 orange-brown C.1 N lateral root surface C O Fe Ca - none middle-aged portion of root: lighter coloured cell WF 9 orange-brown C.2 N lateral root surface C O Al - - none middle-aged portion of root: darker coloured cell WF 9 orange-brown D N deposit on root surface Fe C Ca Al K Fe deposit middle-aged portion of root: Fe/particulate matter deposit WF 10 orange-brown B.1 Y root surface cells C Fe O Si S none youngest portion of root: plaque-packed cell WF 10 orange-brown B.2 Y root surface cells C O Fe Si Al none youngest portion of root: thin plaque WF 10 orange-brown C Y root surface cells C Fe O Si P none youngest portion of root: plaque-packed cell WF 10 orange-brown D.1 Y root surface cells C Fe O Si Ca(Fe) none youngest portion of root: thicker thin plaque WF 10 orange-brown D.2 Y root surface cells C O Fe Al Ca none youngest portion of root: thin plaque Notes Relative Peak Height of Elements: Xx(Yy) means that element Xx had the next highest peak relative to element Yy, shown in brackets because it is the second peak for element Yy - = element peaks too low to determine relative height Relative Peak Height of Elements Other Particulate Deposits X-Ray Spectra DataSample Data Comments Site Sample Root Colour Spectrum Plaque Present Location Appendix C Zizania palustris Root Plaque Examination 237 C.1.6 – Plaque Observation Summary Table C.1.6.1 - Plaque Structure, Zizania palustris Root Plaque SEM Image Data Summary Site # Images Observed with Plaque Crust Thin Thin & Packed cells Thin & Crust Crust & Packed cells LT 16 8 5 3 0 0 MM 18 3 4 5 6 0 SeR 16 11 3 2 0 0 StR 15 8 1 0 3 3 WF 11 6 3 2 0 0 Totals 76 36 16 12 9 3 47.4% 21.1% 15.8% 11.8% 3.9% Notes Site = Marchmont Marsh (MM), Partridge Crop Lake (SeR), Lower Steep Rock Lake (StR), Whitefish Lake (WF) "Broken away" plaque type observation not included; not a type, only a different form of crust plaque % Images with Plaque Type Sample Data Plaque Type Observed Appendix C Zizania palustris Root Plaque Examination 238 C.1.6 - Plaque Observation Summary Table C.1.6.2 - Plaque Composition, Zizania palustris Root Plaque EDXA X-Ray Spectra Data Summary Site # Plaque X-ray Spectra Collected Fe + O Fe + Al Fe + K Fe + Si Fe + Cl O + Si O + S O + Cl LT 14 12 2 0 0 0 0 0 0 MM 23 15 2 1 2 1 1 0 1 SeR 14 7 3 3 1 0 0 0 0 StR 21 16 0 5 0 0 0 0 0 WF 15 9 5 0 0 0 0 1 0 Totals 87 59 12 9 3 1 1 1 1 67.8% 13.8% 10.3% 3.4% 1.1% 1.1% 1.1% 1.1% Notes Site = Marchmont Marsh (MM), Partridge Crop Lake (SeR), Lower Steep Rock Lake (StR), Whitefish Lake (WF) Two most abundant elements based on relative peak height observations in x-ray spectrum. Sample Data X-ray Spectra - Two Most Abundant Elements % X-ray Spectra with Two Most Abundant Elements Appendix C Zizania palustris Root Plaque Examination 239 C.1.6 - Plaque Observation Summary Table C.1.6.3 - Plaque Deposition, Zizania palustris Root Plaque EDXA X-Ray Spectra Profile Data Summary # X-ray Spectra Iron Presence # X-ray Spectra Iron Presence # X-ray Spectra Iron Presence # X-ray Spectra Iron Presence # X-ray Spectra Iron Presence LT 1 8 3 3 2 2 1 0 1 1 1 0 MM 0 0 0 0 0 0 0 0 0 0 0 0 SeR 3 21 3 3 6 5 6 0 6 0 0 0 StR 4 24 5 5 5 5 6 5 6 1 2 0 WF 1 8 4 4 1 1 2 2 1 1 0 0 Totals 9 61 15 15 14 13 15 7 14 3 3 0 Notes Site = Marchmont Marsh (MM), Partridge Crop Lake (SeR), Lower Steep Rock Lake (StR), Whitefish Lake (WF) Data from cross section samples with plaque (none collected from MM samples) Outer Cortex = layer of small cells 2 - 3 cells thick, directly adjacent to the epidermis "Iron Presence" based on Fe being one of the two most abundant elements, based on relative peak height observations in x-ray spectrum. Iron presence in cortex may be due to x-ray disperson since cortex spectra collected in cells adjacent to outer cortex Vascular Cylinder 100% 92.9% 46.7% 21.4% 0% Sample Data Location and Fe Presence % Observations with Fe Present at Location Site # Samples with Profiles Collected Total # X-ray Spectra Profiles Epidermis - Surface Epidermis - Interior Outer Cortex Cortex Appendix C Zizania palustris Root Plaque Examination 240 Appendix C Zizania palustris Root Plaque Examination 241 C.2 – Element Maps Figure C.2.1 – Partridge Crop Lake, Sample 5 (carbon-coated cross section: root surface orange- brown), SEM image with associated EDXA element maps. A. x90, original image; B. coloured points indicate the presence and location of each of the elements Fe (red), O (green) and P (blue); C. light coloured points indicate the presence and location of each element (as labelled).           Appendix C Zizania palustris Root Plaque Examination 242 C.2 – Element Maps Figure C.2.2 – Marchmont Marsh, Sample 8 (longitudinal section: root surface orange-brown), SEM image with associated EDXA element maps. A. x100, original image; B. coloured points indicate the presence and location of each of the elements Fe (red), O (green) and P (blue); C. light coloured points indicate the presence and location of each element (as labelled).           Appendix C Zizania palustris Root Plaque Examination 243 C.2 – Element Maps Figure C.2.3 – Marchmont Marsh, Sample 8 (longitudinal section: root surface orange-brown), SEM image with associated EDXA element maps. A. x350, original image; B. coloured points indicate the presence and location of each of the elements Fe (red), O (green) and P (blue); C. light coloured points indicate the presence and location of each element (as labelled).           Appendix C Zizania palustris Root Plaque Examination 244 C.2 – Element Maps Figure C.2.4 – Marchmont Marsh, Sample 8 (longitudinal section: root surface orange-brown), SEM image with associated EDXA element maps. A. x1,000, original image; B. coloured points indicate the presence and location of each of the elements Fe (red), O (green) and P (blue); C. light coloured points indicate the presence and location of each element (as labelled).           Appendix C Zizania palustris Root Plaque Examination 245 C.2 – Element Maps Figure C.2.5 – Whitefish Lake, Sample 10 (longitudinal section: root surface orange-brown), SEM image with associated EDXA element maps. A. x150, original image; B. coloured points indicate the presence and location of each of the elements Fe (red), O (green) and P (blue); C. light coloured points indicate the presence and location of each element (as labelled).           Appendix C Zizania palustris Root Plaque Examination 246 C.3 – Plaque Anomalies Figure C.3.1 (cont’d on next page)       Appendix C Zizania palustris Root Plaque Examination 247 C.3 – Plaque Anomalies Figure C.3.1 – Marchmont Marsh, Sample 8 (longitudinal section: oldest portion of root, root surface orange-brown), SEM images; root surface with grooves present in plaque crust, A. three zoom images (area shown by dashed box), A.1 x1,200, A.2 x2,500, A.3 x5,000; B. x1,600, C. x1,000, and D. x950.     Appendix C Zizania palustris Root Plaque Examination 248 C.3 – Plaque Anomalies Figure C.3.2 (cont’d on next page)       Appendix C Zizania palustris Root Plaque Examination 249 C.3 – Plaque Anomalies Figure C.3.2 – Marchmont Marsh, Sample 8 (gold-coated longitudinal section: oldest portion of root, root surface orange-brown), SEM images from Hitachi SU-70 SEM; A. x12,000, holes in plaque crust; root surface with grooves present in plaque crust, B. x4,000, C. x6,000, D. x45,000, E. x18,000 and F. x10,000.   Appendix C Zizania palustris Root Plaque Examination 250 C.3 – Plaque Anomalies Figure C.3.3 – Whitefish Lake, Sample 3 (carbon-coated cross section: root surface orange-brown), SEM images; root surface with grooves present in plaque crust, A. x700 and B. x1,000 (note grooves extending through entire depth of plaque crust).     Appendix C Zizania palustris Root Plaque Examination 251 C.3 – Plaque Anomalies Figure C.3.4 (cont’d on next page)       Appendix C Zizania palustris Root Plaque Examination 252 C.3 – Plaque Anomalies Figure C.3.4 – Whitefish Lake, Sample 3 (gold-coated longitudinal section: root surface orange-brown), SEM images from Hitachi SU-70 SEM; A. x2,200, grooves present across cell surface in plaque crust; root surface with grooves present in plaque crust, B. x12,000, C x20,000, D x9,990, E x12,000 and F x10,000   Appendix C Zizania palustris Root Plaque Examination 253 C.4 – Base/Control Data C.4.1 – Control Samples, SEM Images and EDXA X-Ray Spectra Figure C.4.1.1 – Marchmont Marsh, Sample 5 (longitudinal section: control, root surface white), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x400, root surface; B. x700, root surface ; C. x600, root surface.       Appendix C Zizania palustris Root Plaque Examination 254 C.4.1 – Control Samples Figure C.4.1.2 (cont’d on next page)     1 2 3   5 4 Appendix C Zizania palustris Root Plaque Examination 255 C.4.1 – Control Samples Figure C.4.1.2 – Marchmont Marsh, Sample 6 (cross section: control, root surface white), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x400, epidermis / root surface (spectrum A.1), epidermis cell interior (spectrum A.2), outer cortex (spectrum A.3) and cortex (spectra A.4 and A.5); B. x800, epidermis / root surface (spectrum B.1) and outer cortex (spectrum B.2).     1 2 Appendix C Zizania palustris Root Plaque Examination 256 C.4.1 – Control Samples Figure C.4.1.3 – Marchmont Marsh, Sample 7 (longitudinal section: control, root surface white), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x700, root surface (spectrum A.1) and root surface with CaCO3 particulate deposits (spectrum A.2); B. x200, root surface (spectrum B.1) and root surface with CaCO3 particulate deposits (spectrum B.2).     1 2     1 2 Appendix C Zizania palustris Root Plaque Examination 257 C.4.1 – Control Samples Figure C.4.1.4 – Whitefish Lake, Sample 5 (longitudinal section: control, root surface light yellow), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x150, root surface (white particulates likely CaCO3); B. x500, root surface (spectrum B.1) and CaCO3 particulate deposits (spectrum B.2); C. x1,100, root surface.       1 2   Appendix C Zizania palustris Root Plaque Examination 258 C.4.1 – Control Samples Figure C.4.1.5 – Whitefish Lake, Sample 6 (longitudinal section: control, root surface light yellow), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x400, root surface with CaCO3 particulate deposits; B. x600, root surface; C. x1,000, root surface.       Appendix C Zizania palustris Root Plaque Examination 259 C.4.1 – Control Samples Figure C.4.1.6 – Whitefish Lake, Sample 7 (cross section: control, root surface light yellow), SEM images with associated EDXA x-ray spectra (x-ray location shown by box); A. x95, cross section showing cortex (spectrum A.1) and root surface (spectrum A.2); B. x450, epidermis / root surface (spectrum B.1), outer cortex (spectrum B.2) and cortex (spectrum B.3).     1 2 3     1 2 Appendix C Zizania palustris Root Plaque Examination 260 C.4.1 – Control Samples Figure C.4.1.7 – Blank stub, no sample EDXA x-ray spectra. X-ray spectra of carbon- tape at three locations (A, B and C).       C.4.1 - Control Samples Table C.4.1.1 - Control Samples, Zizania palustris Root Plaque SEM Image Data Compilation Type % Coverage Thickness Location Type % Coverage Thickness Location MM 5 white A root surface N 400 - - - - - - - - none no plaque MM 5 white B root surface N 700 - - - - - - - - none no plaque MM 5 white C root surface N 600 - - - - - - - - none no plaque MM 6 white A cross section N 400 - - - - - - - - none no plaque MM 6 white B cross section N 800 - - - - - - - - none no plaque MM 7 white A root surface N 700 - - - - - - - - CaCO3 CaCO3 deposits (white spots), no plaque MM 7 white B root surface N 200 - - - - - - - - CaCO3 CaCO3 deposits (white spots), no plaque WF 5 light yellow A root surface N 150 - - - - - - - - CaCO3 CaCO3 deposits (white spots), no plaque WF 5 light yellow B root surface N 500 - - - - - - - - CaCO3 and soil CaCO3 deposits (white spots) and soil, no plaque WF 5 light yellow C root surface N 1,100 - - - - - - - - none no plaque WF 6 light yellow A root surface N 400 - - - - - - - - CaCO3 CaCO3 deposits (white spots), no plaque WF 6 light yellow B root surface N 600 - - - - - - - - CaCO3 CaCO3 deposits (white spots), no plaque WF 6 light yellow C root surface N 1,000 - - - - - - - - CaCO3 CaCO3 deposits (white spots), no plaque WF 7 light yellow A cross section N 95 - - - - - - - - none no plaque WF 7 light yellow B cross section N 450 - - - - - - - - none no plaque Notes - = N/A Primary Deposition Secondary Deposition Other Particulate Deposits Sample Data SEM Observation Data Comments Site Sample Root Colour Image Image Location Plaque Present Zoom Appendix C Zizania palustris Root Plaque Examination 261 C.4.1 - Control Samples Table C.4.1.2 - Control Samples, Zizania palustris Root Plaque EDXA X-Ray Spectra Data Compilation 1 2 3 4 5 MM 5 white A N root surface C O - - - none no plaque MM 5 white B N root surface C O Al - - none no plaque MM 5 white C N root surface C Al O Ca - none no plaque MM 6 white A.1 N root surface C O Al - - none no plaque MM 6 white A.2 N epidermis cells C Al O - - none no plaque MM 6 white A.3 N outer cortex C O - - - none root interior MM 6 white A.4 N cortex C O K - - none root interior MM 6 white A.5 N cortex C O K - - none root interior MM 6 white B.1 N root surface C O Al - - none no plaque MM 6 white B.2 N outer cortex C O Al - - none root interior MM 7 white A.1 N root surface C O Al Cl K none no plaque MM 7 white A.2 N root surface C O Ca - - CaCO3 CaCO3 deposits, no plaque MM 7 white B.1 N root surface C O Al - - none no plaque MM 7 white B.2 N root surface C O Ca Al - CaCO3 CaCO3 deposits, no plaque WF 5 light yellow A N root surface C O S - - none no plaque WF 5 light yellow B.1 N root surface C O K Al S none no plaque WF 5 light yellow B.2 N root surface C Ca Si O Al soil and CaCO3 CaCO3 deposits and soil, no plaque WF 5 light yellow C N root surface C O Al - - none no plaque WF 6 light yellow A N root surface Ca C O Al(Ca) - CaCO3 CaCO3 deposits, no plaque WF 6 light yellow B N root surface C O - - - none no plaque WF 6 light yellow C N root surface C O K S - none no plaque WF 7 light yellow A.1 N cortex C O K S Cl none root interior WF 7 light yellow A.2 N root surface C O Al - - none no plaque WF 7 light yellow B.1 N root surface Al C K O Fe none no plaque WF 7 light yellow B.2 N outer cortex C O Al K - none root interior WF 7 light yellow B.3 N cortex C O Al K S none root interior N/A N/A N/A A N/A carbon tape C O - - - N/A blank stub with carbon tape, no sample N/A N/A N/A B N/A carbon tape C O - - - N/A blank stub with carbon tape, no sample N/A N/A N/A C N/A carbon tape C O - - - N/A blank stub with carbon tape, no sample Notes Relative Peak Height of Elements: Xx(Yy) means that element Xx had the next highest peak relative to element Yy, shown in brackets because it is the second peak for element Yy - = element peaks too low to determine relative height Relative Peak Height of Elements Other Particulate Deposits X-Ray Spectra DataSample Data Comments Site Sample Root Colour Spectrum Plaque Present Location Appendix C Zizania palustris Root Plaque Examination 262 Appendix C Zizania palustris Root Plaque Examination 263 C.4.2 – Control Samples, Element Maps Figure C.4.2.1 – Whitefish Lake, Sample 6 (longitudinal section: control, root surface light yellow), SEM image with associated EDXA element maps. A. x150, original image; B. coloured points indicate the presence and location of each of the elements Fe (red), O (green) and P (blue); C. light coloured points indicate the presence and location of each element (as labelled).           Appendix C Zizania palustris Root Plaque Examination 264 C.4.2 – Control Element Maps Figure C.4.2.2 – Marchmont Marsh, Sample 6 (cross section: control, root surface white), SEM image with associated EDXA element maps. A. x100, original image; B. coloured points indicate the presence and location of each of the elements Fe (red), O (green) and P (blue); C. light coloured points indicate the presence and location of each element (as labelled).           C.4.3 - Water Base Data C.4.3 – Water Analytical Base Data Table C.4.3.1 - Water Analytical Data, Collected Near Zizania palustris Sample Sites Al Ca Fe K Mg Mn Na S Zn (mg/L) (µS/cm) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 25-Sep-10 MM MM-Water Water Surface 133.6 292.3 7.715 0.010 35.912 0.061 1.37 10.63 0.0035 5.41 1.80 0.007 - 0.0045 B - 0.022 25-Sep-10 MM MM-V-1a-1 Water Surface 135.5 290.6 7.774 0.010 38.192 0.225 1.45 10.93 0.0397 5.97 1.76 0.015 - 0.0045 B - 0.027 25-Sep-10 MM MM-V-1a-2 Sediment Porewater 0-10 cm A 127.9 270.4 6.981 0.013 35.392 1.503 0.95 7.81 0.3492 4.30 0.23 0.007 - 0.0045 B - 0.210 25-Sep-10 MM MM-V-1a-3 Sediment Porewater 10-20 cm A 127.8 268.0 6.867 0.016 36.972 1.961 0.87 7.17 0.2998 4.42 0.20 0.008 - 0.0045 B - 0.145 25-Sep-10 MM MM-V-1a-4 Sediment Porewater 20-30 cm A 133.4 287.1 6.829 0.043 40.092 2.408 0.64 7.35 0.2928 5.00 0.14 0.008 - 0.0045 B - 0.085 25-Jul-11 SeR PCrop4 Water Surface 20.3 56.8 6.831 0.046 6.947 0.168 0.50 1.43 0.0131 1.87 0.77 0.002 0.015 0.030 0.406 0.016 25-Jul-11 SeR PCrop6 Water Surface 20.4 57.3 6.825 0.052 6.993 0.165 0.52 1.44 0.0129 1.90 0.78 0.004 0.025 0.037 0.423 0.017 06-Sep-11 SeR PCrEpi Water Surface 20.7 59.0 6.710 0.049 6.754 0.163 0.50 1.35 0.0137 1.76 0.77 0.004 0.005 B 0.023 0.304 0.005 06-Sep-11 SeR PCrOut Water Surface 21.2 59.1 6.709 0.051 6.642 0.164 0.50 1.33 0.0132 1.72 0.74 0.004 0.005 B 0.024 0.349 0.008 27-Jul-11 StR LSR01 Water Surface 36.6 97.8 7.136 0.055 12.695 0.308 0.54 2.10 0.0461 3.23 1.04 0.004 0.057 0.023 0.506 0.019 27-Jul-11 StR LSR02 Water Surface 36.8 99.6 7.233 0.014 12.436 0.156 0.54 2.07 0.0300 3.34 1.01 0.005 0.034 0.0045 B 0.450 0.016 07-Sep-11 StR LSRIn1 Water Surface 37.9 100.0 6.903 0.034 10.880 0.093 0.74 1.99 0.0341 2.65 1.14 0.012 0.053 0.195 0.560 0.013 07-Sep-11 StR LSREpi Water Surface 38.3 101.1 8.126 0.033 9.885 0.094 0.56 1.64 0.0446 2.83 0.90 0.004 0.005 B 0.0045 B 0.445 0.010 Summer 1997 C WF Wild Rice (30m) Water Surface - 103.0 6.840 0.074 144.100 0.239 2.57 38.81 0.1090 16.10 1.03 0.003 - - 0.430 0.008 Notes No water data available for Lake Tamblyn sample collection site All samples analyzed at Lakehead University Environmental Laboratory (LUEL) according to LUEL QA/QC protocols, CALA approved. - = parameter not analyzed A = Depth below sediment-water interface B = Results reported as "less than detection limit" are shown as half of the value of the detection limit C = Data presented are means of seven sampling dates from mid-June to mid-September 1997, data published by Lee & McNaughton, 2004 Site = Marchmont Marsh (MM), Partridge Crop Lake (SeR), Lower Steep Rock Lake (StR), Whitefish Lake (WF) Sample Data Analytical Parameters (Units) Sample Date Site Depth N-NH4+NH3 Nitrate NO3-N Total K. Nitrogen Total P pHSample ID Total MetalsTotal Alkalinity as CaCO3 Con-ductivity Matrix Appendix C Zizania palustris Root Plaque Examination 265 Appendix C Zizania palustris Root Plaque Examination 266 C.5 – Photos Figure C.5.1 – Lake Tamblyn wild rice root sample preparation prior to freeze-drying. Figure C.5.2 – Marchmont Marsh wild rice root sample. Appendix C Zizania palustris Root Plaque Examination 267 C.5 – Photos Figure C.5.3 – Prepared samples of Marchmont Marsh wild rice roots prior to freeze- drying. Figure C.5.4 – Partridge Crop Lake wild rice root sample preparation prior to freeze- drying. Appendix C Zizania palustris Root Plaque Examination 268 C.5 – Photos Figure C.5.5 – Lower Steep Rock Lake wild rice root sample preparation prior to freeze- drying. Figure C.5.6 – Prepared samples of Whitefish Lake wild rice roots prior to freeze-drying. Appendix C Zizania palustris Root Plaque Examination 269 C.5 – Photos Figure C.5.7 – Prepared samples for freeze-drying. Figure C.5.8 – LABCONCO Freeze Dry System (freeze-dryer) used to dry wild rice root samples. Located in the Lakehead University Instrument Laboratory (LUIL). Appendix C Zizania palustris Root Plaque Examination 270 C.5 – Photos Figure C.5.9 – Freeze-dried wild rice root samples mounted on stubs with carbon tape, in preparation for SEM and x-ray analysis. Sample 10 is gold-coated. Figure C.5.10 – JEOL JSM-5900LV Scanning Electron Microscope (SEM) fitted with EDXA to view and x-ray wild rice root samples. Located in the Lakehead University Instrument Laboratory (LUIL).