phytoextraction of lead from contaminated soil by panicum virgatum l

PHYTOEXTRACTION OF LEAD FROM CONTAMINATED SOIL BY PANICUM VIRGATUM L. (SWITCHGRASS) AND ASSOCIATED GROWTH

RESPONSES

by

ANNE MAUREEN GLEESON

A Thesis submitted to the Department of Biology

In conformity with the requirements for

The degree of Master of Science

Queen’s University

Kingston, Ontario, Canada

July, 2007

Copyright © Anne M. Gleeson, 2007

ABSTRACT

Growing interest in biomass crops for energy production has focused attention on

Panicum virgatum L. (switchgrass) as a promising perennial feedstock native to much of

North America. Switchgrass may be processed into products such as pulp and paper,

ethanol, and fuel pellets. A robust C4 grass, switchgrass typically produces 10-12 dry

tonnes/ha on agricultural soils in average growing conditions; this study shows that

reduced but substantive yields are also possible on soils rendered unsuitable for food crop

production by lead contamination over the acceptable level of 70 ppm. Switchgrass offers

not only tolerance to lead in the soil environment, but also the potential for extraction of

lead contaminants. Integrating the growth of switchgrass for biomass with

phytoremediation could provide greater opportunity for biomass production, while

eventually increasing the suitability of such lands for agricultural production. This study

examines the potential for using switchgrass to remove lead from the soil and to

translocate the contaminant to the leafy portions of the plant, where it could be removed

from the site through biomass harvest. Roots of plants treated with 6000 and 10000 ppm

lead solution from the time of seeding displayed some morphological changes and growth

inhibition, yet, produced biomass comparable to expected yields and removed up to 0.1%

of the applied lead solution. Established switchgrass displayed few morphological

changes and no significant loss of biomass when treated with lead acetate solution while

extracting lead into harvestable tissues. As the primary root is the organ through which

water, solutes and heavy metals pass into the plant, the dense, fibrous root system of

switchgrass may aid in the ability of this plant to extract contaminants from soil without

significantly hindering above ground biomass production. Switchgrass grown on

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brownfield soil, with contamination levels of up to 23600 ppm translocated lead into

harvestable tissues without significant changes in biomass production or growth

characteristics. However, symptoms of phytotoxicity were observed in switchgrass

grown on soil containing 36100 ppm lead. Atomic absorption spectrometry and

inductively coupled plasma analyses indicate that lead accumulation in harvestable

tissues of switchgrass occurs at rates comparable to those in previous studies. The

presence of dark staining deposits, detected by light microscopy in the root and shoot

tissue of switchgrass treated with high concentrations of applied lead (13800, 10000),

suggests an adaptive response within the plant. Successful integration of

phytoremediation with growth of biomass crops such as switchgrass may provide

effective reclamation of contaminated soils, while contributing to a sustainable energy

economy.

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ACKNOWLEDGMENTS I would like to thank Dr. Susan Wood for the wonderful help and guidance she

provided over the course of this project. I would also like to thank Dr. William

Newcomb, Barry Weese, Annie Craig, Nikki Philbrook and all the members of the

Newcomb lab for their help, patience and good humour, all of which were invaluable to

me throughout this process.

Thanks to Dr. Barb Zeeb for her help in the initiation of the project, and for

providing the soil for the brownfield study; Allison Rutter, Kalam Mir and Mary

Andrews at Queen’s Analytical Services for their guidance and for the use of their

facilities; and Dave Kempson for his help with the electron microprobe analysis.

Special thanks to my family and friends for their support, especially Travis

Lusney who is always ready to provide encouragement, support and much needed

laughter.

This research was supported by Natural Sciences and Engineering Research

Council Discovery grants to Dr. Susan Wood and Dr. William Newcomb.

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TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGMENTS iv

TABLE OF CONTENTS v

ABBREVIATIONS vii

LIST OF FIGURES viii

LIST OF TABLES x

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1

1.1. Lead Contamination 2 1.2. Removal of Lead from Contaminated Soils 3 1.3. Hyperaccumulation of Metals 5 1.4. Bioavailability of Lead in Soil 6 1.5. Plant Response to Lead Exposure 10 1.6. Biomass Production and the Need for Alternative Energy Sources 12 1.7. Switchgrass as a Biomass Feedstock 14 1.8. Biomass and Phytoextraction 15 1.9. Switchgrass in Phytoextraction Applications 17

CHAPTER TWO: MATERIALS AND METHODS 182.1. Plant Culture 18 2.2. Lead Treatment 19 2.3. Plant Harvest 222.4. Analysis 22

2.4.1. Morphological 22 2.4.2. Fixation 22 2.4.3. Histochemical Lead Detection 23 2.4.5. Dehydration 23 2.4.6. Embedding 242.4.7. Microscopy of Preserved Tissues 24 2.4.8. Atomic Absorption Spectrometry 24 2.4.9. 30 Element Analysis 25 2.4.10. Electron Microprobe Analysis 26 2.4.11. Statistical and Numerical Analyses 26

2.5. Methodological Error 27

CHAPTER THREE: RESULTS 293.1. Qualitative Observations 29 3.2. Growth Trend Analysis 33 3.3. Biomass Production 36 3.4. Morphology 43 3.5. Phytoextraction of Lead 47

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3.5.1. Lead Accumulation in Plants Grown on Soil Treated with Lead acetate 47 3.5.2. Lead Accumulation in Switchgrass Grown on Brownfield Soil 53 3.5.3. Electron Microprobe Analysis 56

3.6. Light Microscopy 56

CHAPTER FOUR: DISCUSSION 674.1. Lead Treatment Affects Switchgrass Establishment 67 4.2. Switchgrass Biomass Decreases with High Lead Treatment Concentrations 68 4.3. Lead Treatment Affects Switchgrass Root Morphology 70 4.4. Lead Affects Switchgrass Shoot Height and Appearance 72 4.5. Effects of Brownfield Soil on Switchgrass Growth Patterns 74 4.6. Phytoextraction of Lead by Switchgrass 75 4.7. Effects of Lead on Shoot and Root Cell Structure 80 4.9. Conclusions 83 4.10. Future Directions 85

SUMMARY 88

LITERATURE CITED 90 APPENDIX 1 - Hoagland’s Nutrient Solution 98

APPENDIX 2 - Sample of Randomized Growth Chamber Layout 99

APPENDIX 3 - Study Summary 100

APPENDIX 4 - 30 Element Analysis of Brownfield Soil and Soil Dilutions 101

APPENDIX 5 - Recipes for Buffer, Fixative and Resin 103

APPENDIX 6 - Mean Pb Concentration in Growth Media, Nutrient Solution, Seed Stock and Control Plants 104

APPENDIX 7 - Mean Percentage of Elements Taken up by Switchgrass from Brownfield Soil 105

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ABBREVIATIONS

AAS – Atomic absorption spectrometry

DDSA – Dodecenyl succinic anhydride

DMP-30 – 2,4,6-Tri Dimethylaminomethyl Phenol

DTPA – Diethylene triamine pentaacetic acid

EDTA – Ethylenediamine tetraacetic acid

EDXS – Energy-dispersive X-ray spectroscopy

EGTA – Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid

FBC – Fluidized bed combustor

ICP-OES – Inductively coupled plasma – optical emission spectrometer

LHL – Low-high-low combustor

LM – Light micrograph

Pb – Lead

PEPC – Phosphoenolpyruvate carboxylase

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LIST OF FIGURES

Figure 1: Colour photograph of Pb-treated and control switchgrass roots 31

Figure 2: Colour photograph of switchgrass grown on brownfield soil 33

Figure 3: Switchgrass biomass accumulation over 8 weeks of growth 35

Figure 4: Mean dry weight of switchgrass root tissue 39

Figure 5: Mean dry weight of switchgrass shoot tissue 40

Figure 6: Mean fresh weight of switchgrass root tissue 41

Figure 7: Mean fresh weight of switchgrass shoot tissue 42

Figure 8: Mean root length and number of roots on switchgrass plants 44

Figure 9: Mean plant height (cm) and mean number of offshoots per plant 46

Figure 10: Mean measured root (a) and shoot (b) lead concentration per plant in study 1. 49

Figure 11: Mean measured root (a) and shoot (b) lead concentration per plant in

study 2 50 Figure 12: Mean measured root (a) and shoot (b) lead concentration per plant in

study 3 51 Figure 13: Mean root and shoot Pb concentration per plant in brownfield soil 55 Figure 14: LM of the node of a control shoot 58

Figure 15: LM of a Pb-treated shoot (a) and cells of the developing leaf tissue (b) 59

Figure 16: LM of a 6000 ppm, Pb-treated switchgrass shoot from Study 2 60

Figure 17: LM of a 10000 ppm, Pb-treated switchgrass shoot from Study 3 60

Figure 18: LM of a control switchgrass shoot 61

Figure 19: LM of a 10000 ppm, Pb-treated switchgrass shoot vascular bundle 61

Figure 20: LM of a 6000 ppm, Pb-treated switchgrass shoot vascular bundle 62

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Figure 21: LM of a 10000 ppm, Pb-treated switchgrass shoot vascular bundle from Study 2 62

Figure 22: LM of a shoot from switchgrass grown on brownfield soil containing

36100 ppm Pb 63 Figure 23: LM of a control switchgrass root cross section 64

Figure 24: LM of a 10000 ppm, Pb-treated switchgrass root cross section from Study 1 65 Figure 25: LM of a root cross section from switchgrass grown on brownfield soil

containing 13800 ppm Pb 65 Figure 26: LM of a root cross section from switchgrass grown on brownfield soil

containing 36100 ppm Pb 66

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LIST OF TABLES

Table 1: Days to switchgrass seed development 32

Table 2: Mean fresh and dry weights of study 1 switchgrass plants 36




Table 6: Mean measured Pb concentration (ppm) in switchgrass plants 52

Table 7: Mean percentage of Pb total applied Pb treatment present in switchgrass tissue in studies 1, 2 and 3 53

Table 8: Mean percentage of total soil Pb present in switchgrass tissue grown on

brownfield soil 56

x

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

Growing global population coupled with increased demands for food, fibre and

fuel is driving demand for clean, usable agricultural soil and clean, renewable alternatives

to petroleum based fuels. In order to meet the demand for these fuels, economically

feasible and environmentally conscious alternatives such as energy from biomass have

been proposed. Yet, biomass feedstocks have relatively low energy density when

compared to fossil fuels and vast areas would be required to grow enough biomass to

meet even a fraction of Canada’s energy demand. If a high yield biomass feedstock such

as Panicum virgatum L. (switchgrass) may be grown on contaminated land that would be

otherwise unsuited to agriculture, it may help to rejuvenate that land for future use

(Cunningham and Ow, 1996; Liebig et al., 2005), without compromising the available

land base for food production.

Many tracts of land in North America are considered contaminated due to the

presence of industrial pollutants. As the cost for restoration is very high, and

contaminants such as lead (Pb) may pose health and ecological hazards, a substantial

proportion of this land is unused for any purpose (Begonia et al., 1998; Päivöke, 2002;

Sharma and Dubey, 2005; Garza et al., 2006). Thus, integrating perennial bioenergy

feedstock with phytoremediation—the use of plants to extract, neutralize, stabilize or

destroy pollutants in the environment and render them harmless (Salt et al., 1998)—

would meet the goals of supporting “green” energy production and remediating much

needed agricultural land.

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1.1. Lead Contamination

Although global efforts to reduce lead (Pb) contamination and to prevent further

accumulation of Pb in the environment have been made through the implementation of

higher soil standards, Pb contamination in soil, streams and ground water continues to

pose a serious threat to human health and the environment (Begonia et al., 1998; Päivöke,

2002; Sharma and Dubey, 2005; Garza et al., 2006). In Canada alone there are currently

more than 1600 heavy metal contaminated sites (Federal Contaminated Sites Inventory,

2005) occupying tracts of land spanning the country.

Brownfield sites are plots of land that are currently unused or underutilized due

primarily to environmental contamination, caused by industrial developments or spills

(Licht and Isebrands, 2005). Various anthropogenic practices release Pb into the air

allowing for sedimentation and deposition of Pb onto soil. Some of these practices, such

as the use of Pb-treated sewage as a fertilizing agent in agriculture, may cause Pb to build

up in soil (Päivöke, 2002; Licht and Isebrands, 2005; Sharma and Dubey, 2005; Garza et

al., 2006), at concentrations exceeding the maximum allowable Pb threshold of 70 ppm

(Canadian Council of Ministers of the Environment, 2006). Other possible sources of

contamination include fertilizers and herbicides; mining; industrial activities, such as the

production of Pb paint, pipes, welding alloys, batteries and leaded fuels; and the leaching

of Pb from metal shell casings used in ammunition (Päivöke, 2002; Garza et al., 2006).

Removal of heavy metals such as Pb from soil is necessary for maintenance of

environmental and human health as well as for agricultural success. Research has shown

that Pb may cause memory loss; muscle paralysis; irritability; anaemia; damage to the

gastrointestinal tract, myocardium, liver, kidneys and reproductive system; and loss of

2

mental function in humans (Liu et al., 2006; Garza et al., 2006; Wilde et al., 2005).

Lead is able to mimic other cations such as calcium, magnesium and zinc, and to bind to

receptor sites, allowing the metal to be transported into cells and incorporated into bodily

tissues such as organs and bones (Garza et al., 2006). Lead may also act as a co-

carcinogen, interfering with zinc binding sites within cell nuclei and preventing genetic

repair and regulation, thus increasing the carcinogenic effects of other chemicals such as

methyl methanesulfonate and other alkylating agents (McNeill et al., 2004). Lead may

remain in the blood stream for approximately 35 hours, but can remain dormant in the

brain and bone tissue for periods of years or decades respectively, leading to prolonged

detrimental health effects (Garza et al., 2006).

The presence of Pb in the human body affects many metabolic processes as

mentioned above, and may also cause permanent damage to the nervous system (Garza et

al., 2006). The effects of Pb on nervous system development are particularly detrimental

to young children and may lead to decreased aptitude, limited motor and cognitive skills,

as well as behavioural disorders (Garza et al., 2006). Successful removal of Pb from

polluted soil may decrease the potential for human exposure to Pb and the onset of

associated toxic effects.

1.2. Removal of Lead from Contaminated Soils

Reclamation, the act of reclaiming land for cultivation, and remediation, the

process of using corrective measures to restore contaminated land, traditionally involve

excavation of soil, and thus are expensive, invasive and pose a threat to the nutritional

and microbial balance of soil (Huang and Cunningham, 1996; Begonia et al., 1998; Shen

et al., 2002; Garcia et al., 2004). Removing soil from a contaminated site may have

3

detrimental effects on the ecosystem. Microorganisms that enhance plant growth and

increase soil nutrient content, such as nitrogen-fixing bacteria and mycorrhizal fungi, are

likely to be removed in the excavation process (Fesquez et al., 1987; EPA, 1995; Citterio

et al., 2005). Microbes that aid in the immobilization of metals, (Barkay and Schaefer,

2001) and seed stock which is native to a brownfield site are also likely to be lost during

excavation and removal of contaminated soil (Fresquez et al., 1987; Koch et al., 1996).

The elimination of these seeds and organisms may change nutrient cycling patterns, water

holding capacity and the revegetation rate of the land (Lyle, 1987). In addition,

remediation by excavation and burial of the polluted soil in landfill, the most common

method used with heavy metal contaminants, can cost between 60-300 USD per m3,

while in situ remediation costs range between 10-100 USD per m3 (Cunningham et al.,

1995; Begonia et al., 1998). Consequently, clean up activities are likely to be limited to

areas where the exposure and health threats are severe or where land value is very high.

Bioaccumulation of metals by some plants was observed in the early days of

biogeochemical prospecting (Freeman et al., 2004), and is now called

phytoaccumulation. Using this characteristic to specifically remove contaminants from

the soil is called phytoextraction and it has the potential to provide cost effective,

renewable alternatives to previously used remediation techniques while preventing the

loss of topsoil which occurs through the excavation process (Blaylock et al., 1997). For

example, after only two years of study, a poplar stand reduced boron leachate levels to

measurable concentrations meeting drinking water standards in a study performed in New

Zealand (Mills et al., 2002).

4

Phytoremediation may be used as a filter for ground water and runoff, as a cap or

as a reactor to remove contaminants from the soil, particularly within the top 1-2 m of

soil, where the majority of plant root activity occurs (Licht and Isebrands, 2005; Wilde,

2005). Though phytoremediation is a process which generally requires more time to

complete than other methods, the limited disturbance to the soil chemistry and the costs

associated with phytoremediation, approximately 0.02-1.00 USD per m3/year, represent a

fraction of those of excavation and burial (Cunningham et al., 1995), making this

approach appealing in numerous situations. Phytoremediation also provides opportunities

for economic gain where land values are lower, through landscape reclamation,

employment opportunities and healthier air and water quality (Licht and Isebrands, 2005).

Phytoremediation includes a number of different processes. However, this study

will focus on phytoextraction, which involves the translocation of contaminants to shoot

tissues where it may be available for harvest and processing (Cunningham et al., 1995;

Begonia et al., 1998; Lasat, 2000; Lombi et al., 2001; Martin and Ruby, 2004; Sharma

and Dubey, 2005).

1.3. Hyperaccumulation of Metals

The success of a phytoremediation venture which attempts to couple

phytoremediation with agricultural production may be evaluated based on whether the

crop is eligible for traditional agricultural practices such as harvesting and replanting;

whether sufficient biomass is produced along with accumulation; and whether toxic

metals are preferentially accumulated (Blaylock et al., 1997). Hyperaccumulators, plants

which have the ability to extract high concentrations of metal into their above ground

tissue while maintaining average metabolic function, have traditionally been considered

5

primary candidates for use in phytoremediation due to their affinity for metal

accumulation (Robinson et al., 2006).

While over 400 plants have been identified as hyperaccumulators there are few

that are able to produce a large amount of biomass while taking up contaminants from the

soil (McGrath and Zhao, 2003; Robinson et al., 2006,). Hyperaccumulators are generally

known as low-biomass plants (McGrath and Zhao, 2003); a fact that limits the speed of

soil recovery and efficiency of contaminant removal. The ability of the plant to positively

respond to agricultural practices is as important in the phytoremediation process as the

extraction itself (Cunningham and Ow, 1996; Blaylock et al., 1997).

Hyperaccumulators have been identified for remediation of other heavy metals,

including Berkheya coddii for nickel (Robinson et al., 1997) and alpine pennycress

(Thlaspi caerulescens) for zinc and cadmium (Pence et al., 2000). While proposed lead

hyperaccumulating plants such as Armeria martima, Thlaspi rotundifolium, Thlaspi

alpestre, Alyssum wulfenianum and Polycarpaea synandra have been identified, these

have been found to be generally low yield or slow growing species and therefore are not

good candidates as phytoremediators (Baker and Brooks, 1989). Although Panicum

virgatum has not previously been studied with regard to heavy metal extraction from soil,

its high biomass productivity and tolerance of low quality soils make it an attractive

candidate.

1.4. Bioavailability of Lead in Soil

The movement of heavy metals such as Pb into plant tissue is dependent on a

number of factors including soil type, pH, the age of the contaminant, solubility, and

lipophilicity of the compound (Punz and Sieghardt, 1993; Cunningham et al., 1995). Due

6

to the water potential gradient between soil and air which acts as a driving force for

mineral uptake into primary root tissues (Punz and Sieghardt, 1993), the heavy metal

contaminant must be in a soluble, ionic form that may be transported into the root. Soil

composition plays a role in the degree of metal ion availability. Humus and clay

components, solute behaviour, climatic conditions and electrolyte content all affect the

rate of uptake by changing the availability of the metal in the soil matrix, while heavy

metal binding and dissolution are indicators of metal availability (Punz and Sieghardt,

1993).

Heavy metals are bound to organic and inorganic compounds in the soil, leaving

approximately 2% of the metal in an available form, either adsorbed onto soil colloids, or

dissolved in soil water (Punz and Sieghardt, 1993). As a result, the solubility and

mobility of heavy metals is highly influenced by their affinity to react with other ions or

compounds within the soil matrix (Punz and Sieghardt, 1993). Lead is classified as a

weak Lewis acid, meaning it forms strong bonds with the organic matter in the surface

layers of the soil (Begonia et al., 1998; Päivöke, 2002; Sharma and Dubey, 2005). Lead

forms complexes with sulfur, including lead sulfate, an insoluble complex, and lead

sulfide a slightly soluble complex (Xintaras, 1992), and Pb also readily precipitates as

carbonates, phosphates and hydroxides (McBride, 1994). The most highly soluble forms

of lead include lead acetate, lead nitrate and lead chloride, which are the forms of lead

most readily available for phytoextraction (Xintaras, 1992). Thus, lead acetate, the

chemical form of Pb used in this study, represents a highly available form that mimics

actual soil conditions.

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Although the ability of plant roots to extract Pb from soil has been observed in

previous studies (Pahlsson, 1989; Huang and Cunningham, 1996; Xiong, 1997;

Chantachon et al., 2004), Pb movement within the plant is less well characterized.

Translocation of Pb into harvestable shoot tissue may be limited by binding at the surface

of roots or in root cell walls (Pahlsson, 1989). Dissolved ions taken up by roots

commonly move through the apoplast along the water potential gradient (Punz and

Sieghardt, 1993). Water flowing into root hairs may carry heavy metals such as Pb into

the root cortex providing the metal ions with access to the interconnected system of cell

walls and intercellular spaces found there, thus allowing for apoplastic movement further

into the plant (Punz and Sieghardt, 1993).

Lead accumulation in the apoplast of cells has been described in many species

including Raphanus sativus (Lane and Martin, 1977), Zea mays (Tung and Temple,

1996), Allium cepa (Wierzbicka, 1998), Pisum sativum and Vicia faba (Piechalak et al.,

2002). In addition, the formation of lead carbonates deposited in the cell wall has been

previously described (Blaylock et al., 1997). Lead is thought to move radially through the

root apoplast across the cortex (Sharma and Dubey, 2005). As apoplastic passage into the

stele is blocked by the Casparian strip of the endodermis, transport into vasculature likely

occurs through symplastic transport with the movement of ions through the plasma

membrane and cytoplasm of the cell (Sharma and Dubey, 2005). There are also two

regions through which ions may pass apoplastically (Punz and Sieghardt, 1993). The

first is the apical zone where the endodermis has not finished developing; the second is

the lateral root development zone. The lateral root zone may be important for the

8

transport of Pb through the root tissue, into the stele, and further on into above ground

tissue (Punz and Sieghardt, 1993).

Once the ability of a plant to take up Pb has been determined, it may be necessary

to identify the extent to which that species has an affinity for a particular metal. Some

plants possess greater affinities toward elements or compounds of a certain size or ionic

charge. Thus the efficiency of targeted contaminant uptake may be hindered in the field

by the existence of a multi-contaminant soil matrix (Robinson et al., 2006). Polluted

soils rarely contain only one contaminating substance, and this trait may cause an

inconsistency in the availability of a targeted substance. Such multi-contaminant systems

may lead to discrepancies between the observed and expected uptake of a targeted

substance in the field (Robinson et al., 2006).

The presence of more than one source of contamination can not only decrease the

efficiency with which a species is able to remove the targeted contaminant but may also

decrease the biomass production of the plant, as pollutants may have antagonistic

interactions (Pahlsson, 1989; Robinson et al., 2006). Lead may bind to other elements in

the soil matrix making it less available for uptake or a plant may have a higher affinity to

other elemental contaminants. For instance, synergistic reactions were observed between

Pb and cadmium (Cd) in the soil matrix (Miller et al., 1977). The presence of Pb

increased Cd uptake in corn, while Cd had the opposite affect on Pb uptake. Reduction

of root, stem and leaf growth has also been observed in the presence of Pb and Cd

(Hassett et al., 1976).

Though there are few identified hyperaccumulators of lead, it is possible to induce

hyperaccumulation using chelating agents (Schmidt, 2003; Robinson et al., 2006). In a

9

previous study, the addition of chelators to Pb contaminated soil increased the

accumulation of Pb in shoots up to 140-fold in corn and pea (Blaylock et al., 1997;

Schmidt, 2003). Citric or malic acid as well as synthetic chelators such as EDTA, DTPA

and EGTA, form soluble complexes with metals in the soil and can increase the uptake

and translocation of heavy metals through the above ground tissues (Blaylock et al.,

1997). EDTA-metal complex formation, for example, is more efficient in increasing

metal availability of Pb and Cd, than for most other heavy metals as it binds to Pb and Cd

more readily (Schmidt, 2003). However, the use of chelating agents may also make metal

ions more readily available for runoff in ground water, enhancing the threat of further

contamination of the ground water and soil profile (Römkens et al., 2002).

1.5. Plant Response to Lead Exposure

Lead induces a variety of stress responses in plants including reduced biomass

production, lowered photosynthetic capacity and anatomical variations (Pahlsson, 1989;

Punz and Sieghardt, 1993; Huang and Cunningham, 1996; Xiong, 1997; Chantachon et

al., 2004; Kosobrukhov et al., 2004). Changes to plant morphology and plant

productivity have been observed following lead exposure (Hampp and Lendzian, 1974;

Pahlsson, 1989; Punz and Sieghardt, 1993; Huang and Cunningham, 1996; Xiong, 1997).

Reductions in root biomass; a decrease in the shoot/root ratio of biomass; enhanced

lateral root formation; compression of the root axis; reduced distance between root tip

and youngest lateral roots; changes in growth rate, inhibition of root elongation and

damage to root cell membranes are only a few of the morphological changes exhibited by

species exposed to heavy metal contamination (Pahlsson, 1989; Punz and Sieghardt,

1993; Huang and Cunningham, 1996; Xiong, 1997). Lead toxicity may also promote

10

chlorosis, changes to the pigmentation of roots and shoots, smaller leaves and stunted

growth (Pahlsson, 1989; Huang and Cunningham, 1996). A deficiency of nutrients such

as Mg, K Fe, Zn, Ca and Mn caused by Pb exposure as observed in corn and ragweed

(Huang and Cunningham, 1996; Xiong, 1997) may also contribute to a reduction in

photosynthetic capacity related to the inhibition of chlorophyll biosynthesis (Hampp and

Lendzian, 1974). This decrease in pigment formation has been coupled with increased

chloroplast number, which may indicate an attempt by the plant cell to maintain

photosynthetic working capacity during a period of reduced pigment number

(Kosobrukhov et al., 2004).

Plants may be placed into three primary categories based on the methods of metal

uptake: accumulators, indicators and excluders (Baker, 1981). Accumulators are able to

accrue concentrations of a contaminant in above-ground tissues; indicators regulate the

extent and speed of uptake and transport of metals, with internal concentrations mirroring

external concentrations; and excluders maintain constant concentrations of internal metal

contaminants independent of external levels up to a critical limit (Baker, 1981). The

most effective accumulators of Pb are able to accumulate this heavy metal at

concentrations greater than 0.1% of dry weight of above ground biomass without

significantly hindering plant growth (Piechalak et al., 2002).

Tolerant species are able to survive lead exposure by utilizing avoidance and

inactivation response mechanisms (Piechalak et al., 2002). While stress responses tend to

be species specific, sequestration of heavy metal ions from the protoplasm through the

binding of ions to the cell wall, as well as expulsion of the ions from the cell, are methods

of avoidance observed in herbaceous plants (Pahlsson, 1989; Punz and Sieghardt, 1993).

11

Deposition of Pb in root cell walls (Seregin et al., 2004), and the formation of Pb-

containing vesicles within the stele, which became larger with increased Pb exposure,

were observed in maize (Malone et al., 1974). Compartmentalization of ions in vacuoles

may be employed as a method of tolerance (Malone et al., 1974). Inactivation of lead

ions is achieved through detoxification, involving the formation of thiol peptides-

phytochelatins formed through enzymatic action activated by the presence of heavy metal

ions such as Pb (Piechalak et al., 2002).

1.6. Biomass Production and the Need for Alternative Energy Sources

The use of biomass feedstocks for energy production is a rapidly growing industry

(USDOE, 2005). As environmental and economic consequences of petroleum fuels

become more apparent and energy security is threatened by dwindling supply and

political instability, much attention has been directed toward biological feedstocks that

may be grown for ethanol and fuel pellet production. High output biomass cultivars such

as switchgrass are considered to have great potential for use in this area (Nelson et al.,

2006). Energy consumption is expected to increase by an average of 1.5% per year

between 2004 and 2025 in the United States of America (USDOE, 2004). The net carbon

dioxide (CO2) emissions associated with this energy consumption have been identified as

significant contributors to global climate change (Nelson et al., 2006). As the focus of

international energy production shifts from traditional petroleum based sources to carbon

neutral or carbon negative alternatives, renewable, high biomass output resources are

becoming more important.

There are a number of feedstocks that have been identified as high yield crops,

and Miscanthus x giganteus (miscanthus) and switchgrass are two of the major biomass

12

feedstocks currently being studied for energy production through processes such as

pelleting and ethanol production (Mehdi et al., 2000). These perennial rhizomatous, C4

grasses are highly productive, have high water use efficiency, high rates of solar energy

conversion and require low fertilizer inputs (Heaten et al., 2004). However, switchgrass

is considered to be better suited to growth in eastern Canada as it has a large available

seed stock and is native to North America, thus circumventing the potential ecological

disturbance and genetic drift associated with other non-native crops (Heaten et al., 2004).

Using herbaceous and woody crops in energy production can allow for a

rotational crop system able to provide wind and water erosion protection (Casler and Boe,

2003). The reduced need for nitrogen fertilizer may decrease runoff and water

contamination, while the nature of the perennial grass allows for minimal soil

disturbance, low demand for pesticide and enhanced soil organic carbon (Nelson et al.,

2006).

Research and development into using biomass feedstock in co-firing and pelleting

for energy production is increasing the economic potential of biomass for use in the

energy industry. Economic parity between biomass and fossil fuels is reached when a

barrel of oil costs approximately 70 USD (Layzell et al., 2006); with crude oil prices

projected to average 65 USD/barrel over the 2006-2008 period (EIA, 2007), using

biomass for energy production is already a viable option (Layzell et al., 2006).

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1.7. Switchgrass as a Biomass Feedstock

Panicum virgatum is a C4 grass native to much of central and eastern North

America (Mehdi, 2000; Casler and Boe, 2003); the multiple cultivars of which are

believed to have diverged evolutionarily (Parrish and Fike, 2005). The various cultivars

of switchgrass exhibit morphological factors that enable them to thrive in a wide range of

climates and soil conditions (Casler and Boe, 2003; Parrish and Fike, 2005).

Incorporated into the agricultural industry as a crop merely 50 years ago (Parrish and

Fike, 2005), switchgrass has proven to be successful on soil with low nutrient

availability, and with minimal nitrogen fertilizer (Iowa State University, 2003). Warm

season grasses such as switchgrass have been shown to be effective when grown as

biomass feedstock and also when used in the control of wind erosion and as filter strips

for reducing water and contaminant run-off (Byrd and May II, 2000; Sanderson et al.,

2001; Belden and Coats, 2004). In Kansas, switchgrass proved to be more effective at

preventing soil erosion and nutrient loss than all conventional crops across the state (King

et al., 1998) and a study of potential yield recorded a return of approximately 15

tonnes/ha/yr of biomass with the application of only 0.05 tonnes N/ha/yr (Parish and

Fike, 2005).

While different cultivars produce slightly varying annual yields, average annual

yields in the range of 10-12 dry tonnes/ha may be expected from switchgrass (Liebig et

al., 2005). The growth rate of switchgrass is linked with temperature, while the plant

reproductive development is linked to photoperiod (Parrish and Fike, 2005). That is, by

planting cultivars which are cold tolerant but native to lower latitudes in high latitude

regions, including Canada, one might increase the overall productivity and biomass yield

14

of a specific cultivar (Casler and Boe, 2003; Parrish and Fike, 2005). Optimization of

energy production may also be achieved through the selection of switchgrass for

increased fibre content, and decreased concentrations of ash, in order to allow for

efficient combustion and reduced fly ash during gasification or combustion (McLaughlin

et al., 1999; Casler and Boe, 2003). Switchgrass has the potential to sequester carbon

within its substantial root system (Casler and Boe, 2003) adding value to this species

where carbon credit systems are in place such as the EU and the USA.

1.8. Biomass and Phytoextraction

Greater importance is being given to the selection of an appropriate

phytoremediating plant species, as concern for the preservation and reclamation of

natural ecosystems increases (Licht and Isebrands, 2005). The rapid growth rate, high

biomass yield, vast fibrous root system and ease of harvest associated with switchgrass

make it a strong candidate for use in phytoremediation. As switchgrass is native to much

of North America, with a vast pool of available germ plasm, it may also be an ideal

candidate for remediation in areas in need of protection from invasive species.

Connecting biomass feedstock production with remediation techniques may be the

next generation of phytoremediation systems. By combining the remediation of

otherwise unusable land with biomass growth, competition between agricultural and

alternative energy groups for fertile agricultural land may be avoided (Man and Tolbert,

2000). Growing biomass feedstock on contaminated soil also provides opportunities for

carbon sequestration, improvements in soil quality and increased agricultural profit (Man

and Tolbert, 2000). This collaboration may make soil remediation, through long term

phytoextraction, a more economically competitive technology. Extraction of harvested

15

contaminants may also provide opportunities for economic gain through pollutant

recycling into saleable goods, and the creation of employment opportunities (Licht and

Isebrands, 2005).

Crops which are successful in extracting metals from soil are normally processed

through burning or condensation and stored as hazardous waste materials in order to

prevent any further risk to human or environmental health (Robinson et al., 2006).

Harvested tissue may be processed using microbial, thermal or chemical processing to

reclaim or to break down the extracted materials (Cunningham et al., 1995). The process

of extracting metal contaminants from plant ash to be condensed and sold is considered to

be a type of mining (Nicks and Chambers, 1994). This process, otherwise known as

phytomining has been patented and put into practice by Viridian Environmental

(Robinson et al., 2006). Potential applications of this venture include bioenergy such as

cofiring and pellet combustion; paper and feed stock production; and biofuel extraction

providing carbon negative or neutral fuels (Licht and Isebrands, 2005).

In order to prevent extracted contaminants from being re-released into the

environment through hazardous ash or gaseous emissions, specialized facilities may need

to be developed in which tissue may be burned or processed in controlled systems, thus

preventing environmental exposure to contaminants through processing wastes (Keller et

al., 2005). Alternative combustion systems utilizing low-high-low (LHL) temperature

ranges as opposed to traditional fluidized bed combustor (FBC) systems may solve the

problems associated with biomass-metal interactions during combustion (Eldabbagh et

al., 2005). The high temperature reactions in the LHL combustion system result in heavy

metal containment pooling in the centre of ash particles, decreasing metal leachability to

16

below hazardous levels, while the charge of the ash causes submicron particles (which

are difficult to seize in filter systems) to adhere to the surface of supermicron particles

allowing for capture in pollution control filters (Eldabbagh et al., 2005). Although

phytoextraction in a multi-contaminant setting requires greater lengths of time to reach

completion than single contaminant sites, by combining remediation with biomass

production using switchgrass and modified combustion systems, the economic gain of

biomass production may help to offset the costs associated with the time of extraction

(Robinson et al., 2006).

1.9. Switchgrass in Phytoextraction Applications

The series of experiments discussed in this paper examine the efficiency of Pb

extraction from soil by switchgrass. The amount of Pb extracted by the plant, the

location in which Pb is stored or processed by cellular activities as well the effects of Pb

on biomass production, growth trends and plant morphology will be discussed.

Successful phytoextraction by switchgrass will mean little to no loss of establishment and

biomass output by the plants in addition to successful translocation of lead into

harvestable tissues.

The goal of this project was to determine the extent to which switchgrass meets

the two criteria essential to a phytoremediation application. Thus, the major questions

addressed in this paper are:

To what extent does switchgrass extract the contaminant, Pb, from the soil into

harvestable, aerial plant tissues?

Does switchgrass maintain an average biomass yield when grown on Pb-

contaminated soil?

17

CHAPTER TWO

MATERIALS AND METHODS

2.1. Plant Culture

Switchgrass (Panicum virgatum L. cv. Shelter) seeds (Ontario Seed Company,

Waterloo, ON) were planted in Promix BX (Premier Horticulture, Riviere-du-Loup, QC)

or brownfield soil (from Kingston, Ontario, provided by Dr. Zeeb, RMC) for each of the

studies addressed in this paper. All plants that were grown in a growth chamber in the

Phytotron at Queen’s University (Conviron Environments Ltd, Winnipeg, Manitoba)

were exposed a to 16 h light: 8 h dark photoperiod. The temperature was 22o C during

the light periods and 17o C during the dark period, with an average of 70% relative

humidity. Glasshouse grown plants were grown at 26-30oC from 9:30 a.m.-4:30 p.m.

and 18-22oC for the remainder of the 24 hr period. Switchgrass plants grown in the

glasshouse were exposed to a 16 h photoperiod. All plants were watered as needed with

Hoagland’s solution (Appendix 1) for controls and brownfield soil, while the treatment

series were watered using various concentrations of lead acetate (Pb(C2H3O2)2·3H2O) in

Hoagland’s solution.

Control contamination was prevented by placing plastic catch plates under each

pot, preventing runoff from contaminated soil from becoming available to roots of

controls or other treatment groups. Excess runoff was removed prior to the next day of

watering to prevent overflow and water logging of roots. A random number table was

used to randomize the arrangement of pots in the glasshouse and growth chamber

(Appendix 2). Randomization of pot placement was used as a means of offsetting

18

different light intensities and air flow within growth environments. Six studies were

performed, each with varied growth time and parameters as described below.

2.2. Lead Treatment

All control and treatment plants were watered as needed through the treatment

period with Hoagland’s solution (controls) or lead acetate (Pb(C2H3O2)2·3H2O) in

Hoagland’s solution as described below and in Appendix 3. Prior to determining

appropriate treatment concentrations, three areas were considered: previous literature

were consulted to identify comparable experimental Pb concentrations, typical

concentrations present in Brownfield sites were identified, and the precipitation point of

Pb acetate in Hoagland’s solution was determined. A stock lead solution of 10000 ppm

Pb, as lead acetate in Hoagland’s solution, was used to prepare one treatment series

consisting of the concentrations 75.8, 1200 and 6000 ppm Pb and a second series of 6000,

7000, 8000 and 9000 ppm Pb. The stock solution was thoroughly mixed to prevent the

formation of Pb-precipitates in the treatment solution. To ensure saturation of the soil

with lead solution, treatments were added to the soil in liquid form until soil was

completely soaked through with solution.

Study 1

Switchgrass seeds were planted in Promix BX on March 31 2006, and watered as

needed with Hoagland’s solution for seven weeks. Ten, 25.4 cm pots were planted

allowing for two replications of each treatment group and two pots of control plants.

Seven weeks after seeding, treatment of the plants began with the concentrations 75.8,

1200, 6000 and 10 000 ppm Pb for an additional five weeks, as needed, and the plants

were harvested on June 23. This study was performed in order to re-evaluate previous

19

preliminary studies on established plants in which contamination of controls occurred, as

well as to evaluate any changes in the growth regime of established plants relative to

those exposed to a contaminant from the time of seeding.

Study 2

As a corollary to Study 1, switchgrass seeds were planted 8-14 plants per pot in

Promix BX on May 19 2006 and grown for 12 weeks in a growth chamber. These plants

were treated from the time of seeding. Ten, 25.4 cm pots were planted and four treatment

groups (75.8, 1200, 6000 and 10 000 ppm) and one control group were each assigned two

pots. All plants were watered as needed with lead solution or Hoagland’s solution until

the soil was completely saturated with solution.

Study 3

Switchgrass plants were planted 8-14 plants per pot in 10, 25.4 cm pots

containing Promix BX. Plants were grown in the glasshouse for five weeks, from June

28 until Aug 2, and were watered as needed with lead solution or Hoagland’s solution

(controls). The applied treatments consisted of the concentrations 75.8, 1200, 6000 and

10000 ppm Pb. The glasshouse temperature was between 26 and 30oC.

Study 4

Five treatment groups were grown over a period of eight weeks in order to study

biomass growth in lead treated Promix BX. Treatments of 6000, 7000, 8000, 9000 and

10 000 ppm Pb in Hoagland’s solution were applied to the plants as needed until the soil

was saturated. Randomly chosen pots were harvested weekly and fresh weights were

recorded. The plants were dried for three days at 60oC after which the dry weights were

recorded.

20

Study 5

Switchgrass plants were seeded in Promix BX and grown in the glasshouse from

June 28 until December 5 2006, at which time each treatment group included plants that

had flowered. Seeds were planted in 10, 25.4 cm pots and treated daily with the 75.8,

1200, 6000 and 10 000 ppm lead acetate solution series in addition to Hoagland’s

solution for control plants. Daily temperatures in the glasshouse ranged between 26 and

30oC during the light period.

Study 6

Lead-contaminated brownfield soil used in this study contained mean lead

concentrations of 36100 ppm. As the soil was known to contain numerous elemental

contaminants, a 30 element analysis was performed by the Analytical Services Unit at

Queen’s University in order to identify and quantify reportable contaminants within the

soil (Appendix 4). Switchgrass seeds were planted in 11, 25.4 cm pots at a density of 8-

12 seeds per pot and at a depth of 1-2 cm. Control plants were grown in two pots

containing Promix BX, allowing for experimental replications. The brownfield soil was

thoroughly mixed with Promix BX to obtain diluted concentrations of approximately

5900, 6875, 13800, 23600 ppm (Appendix 4), and switchgrass was grown in these

treatment groups, with two pots of each group except the 36100 ppm treatment. Plants

grown in Pb-contaminated soil at the above concentrations as well as the controls were

watered as needed with Hoagland’s solution and harvested on February 5, 2007, 12

weeks after sowing and were prepared for light microscopy and 30-element, ICP Analysis

as described below.

21

2.3. Plant Harvest

The plants were harvested after the respective treatment period for each study

series. Beginning with control groups, the plants and soil were gently removed from the

pots. The roots of the plants were carefully separated to prevent damage to fine root

structures. After being removed from the soil, each plant was gently dipped in deionised

water to remove any remaining soil particles. Three to four plants were randomly chosen

for transfer to fixative and further microscopic analysis. The remaining plants were used

to record morphological observations. For study 5, only seed parts were collected and

prepared for light microscopy.

2.4. Analysis

2.4.1. Morphological

In order to determine morphological effects of lead contamination, as well as the

effects Pb may have on biomass production, plants not used for microscopy were used for

qualitative analysis. Shoot colour, root colour, shoot height, and overall appearance of

the plants were examined. Shoot and root parts were separated by cutting at the

shoot/root interface and the fresh weight of each was recorded. The total number of

roots; number of developed roots, defined as the number of roots greater than 5 cm in

length; and the number of offshoot stems were recorded from a representative sample of

each treatment.

2.4.2. Fixation

After plants were removed from soil, 1-3 mm thick hand sections were cut using a

double-edge razor blade; sections were immediately immersed in modified Karnovsky’s

22

fixative as this is an effective primary fixative for use with plants high in lignin content.

Fixation proceeded for 2-4 h at room temperature in the Karnovsky’s fixative containing

2% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde (Appendix 5). Replicates of

each sample type were made using 0.025 M potassium phosphate buffer (pH 6.8) (Wood

et al., 2002) for half of the samples and 0.05 M sodium cacodylate buffer (pH 6.8) for the

remaining samples (Appendix 5). The use of sodium cacodylate buffer in addition to

potassium phosphate buffer aimed to offset potential lead precipitation which may have

occurred due to the presence of phosphate. After primary fixation, samples were washed

several times with the corresponding buffer over a 30 minute period, and post-fixation

was completed in 1% osmium tetroxide in buffer at room temperature followed by

several washes with buffer.

2.4.3. Histochemical Lead Detection

Prior to fixation, additional samples of fresh tissue in study 1 were immersed in a

freshly prepared solution of saturated sodium rhodizonate which is reported to stain Pb-

containing tissue bright scarlet (Tung and Temple, 1996). After 20 minutes, the samples

were removed from the stain and prepared for light microscopy following the procedure

described above.

2.4.5. Dehydration

Tissue samples were slowly dehydrated using a graded acetone series over several

hours. Using 10% increments, the tissue was brought to 100% acetone then treated with

a 50:50 solution of dehydrated acetone and propylene oxide. Finally, tissues were

23

submerged in 100% propylene oxide with several changes over two hours (Wood et al.,

2002).

2.4.6. Embedding

Tissues were infiltrated with Epon/Araldite resin (Appendix 4) using a 1:1 (v/v)

mixture of plastic resin and propylene oxide. The mixture was added over 4-5 hours, 4-6

drops at a time. The mixture was left in the fume hood over night, allowing the

propylene oxide to evaporate. The tissue was then added to fresh resin in peel-away

moulds (Canemco Inc., St Laurent, QC) and placed under vacuum in a dessicator for 24

hours, allowing for complete infiltration and the removal of air bubbles. After

infiltration, samples were polymerized overnight at 60oC.

2.4.7. Microscopy of Preserved Tissues

Embedded samples were individually selected for light microscopy based on

appearance and position within the mold, cut from hardened resin and mounted on

Epon/Araldite beam capsules. Blocks were trimmed by hand and thick sections were cut

on a Sorvall, Porter-Blum ultramicrotome (MT-1) (Newtown, CT) using glass knives.

Medial sections were heat affixed to glass slides and stained with 0.025% (w/v) toluidine

blue O in 1% (w/v) borax. Slides were observed using a Leitz Dialux 20 light

microscope (Leitz Wetzlar, Ottawa, ON). Black and white photographs were taken on

Ilford FP4 film (ASA 125) film and processed commercially.

2.4.8. Atomic Absorption Spectrometry

Atomic absorption spectrometry (AAS) analysis was performed on studies 1, 2, 3,

and 6. Three replicates of each treatment group were taken from root and shoot samples.

24

Two replications of reference material containing 47 ± 3 μg Pb (Bush branches and

leaves, Institute of Geophysical and Geochemical Exploration, Langfang, China) and two

blanks were also run. Dried plant material was cut and ground into uniform pieces in a

coffee grinder (Procter Silex, Fresh grind). Approximately 0.5 g of each sample was

weighed and placed into high temperature resistant crucibles. The samples were ashed

overnight in a muffle furnace (Fisher Scientific, Isotemp programmable muffle furnace)

at 500oC. Following ashing, each sample was soaked in double deionised water, covered

with a watch glass and acid digested using 1 mL 16M nitric acid and 3 mL of 12M

hydrochloric acid. Acid digestion occurred over 4 hours on a hot plate at 110oC. The

watch glass was removed and two drops of 29-32% hydrogen peroxide were added to

each crucible. Samples were transferred to 50 mL test tubes using double deionised

water to ensure full quantitative transfer. The test tubes were filled to a volume of 12.5

mL, mixed with a cyclone mixer, filtered and transferred to smaller test tubes for AAS

analysis. High concentration samples were diluted using double deionised water to make

analysis possible.

2.4.9. 30 Element Analysis

Root and shoot tissue samples from switchgrass grown on Pb contaminated

brownfield soil were acid digested as described above and prepared for analysis using a

Varian, AX-Vista Pro CCD Simultaneous Inductively Coupled Plasma - Optical

Emission Spectrometer (ICP-OES). Samples of switchgrass seed, Promix-BX, and

brownfield soil were also analyzed for 30 elements using ICP.

Soil samples were dried overnight, weighed into 50 mL test tubes and acid

digested overnight on a hot plate using 2 mL 16M nitric acid and 6 mL of 12M

25

hydrochloric acid. Following digestion, samples were brought to a volume of 25 mL and

filtered into smaller test tubes for ICP analysis. High concentration samples were diluted

using double deionised water to make analysis possible.

2.4.10. Electron Microprobe Analysis

Samples of root and shoot materials from study 1, which had been treated with

10000 ppm Pb were mounted onto beam capsules and carbon-coated for electron

microprobe analysis. Analysis was conducted using and ARL SEM Quantometer (Bruce

Wing, Queen’s University) at an accelerating potential of 20 keV, an emission current of

100 microamps and a beam current of 30 nanoamps (500X magnification). EDS spectra

were collected from 0-20keV for 3000 seconds, and peaks were identified using Tracor

Northern Software (Thermo Noran Instruments Inc., Massachusetts, USA). Vascular

tissues of both roots and shoots were examined; cortical tissues of the roots were also

considered.

2.4.11. Statistical and Numerical Analyses

Data were analyzed for all treatment groups, within and between studies. The

presence of significant differences between fresh weights, dry weights, average heights,

number of offshoots, number of roots, number of long roots and plant lead concentration

was determined using one-way ANOVA analyses. If the F value showed a significant

difference, means were compared using a t-test. P≤ 0.05 was considered to be

significant. All statistical analyses were performed with the JMP 6.0 program.

All percentages were calculated based on the total μg Pb in plant tissue, relative to

the total applied Pb (μg), or the total soil lead content (μg). Total applied Pb values were

26

determined based on the volume of applied Pb treatment over the course of the treatment

period, while the total soil Pb concentration for study 6 was determined based on the total

mass of soil.

2.5. Methodological Error

Lead was present in all control plants, in both shoot and root tissues. Analysis of

the control Hoagland’s solution used in experimentation, the seed stock obtained from

Ontario Seed Company (Waterloo, ON) and Promix growth medium indicated a presence

of lead. The concentration of lead in Hoagland’s solution was above reportable limits

(Appendix 6) and may be due to an accumulation of trace amounts of lead in the stock

chemicals. The combined lead concentration of the nutrient solution and the low levels

found in both Promix and the seed stock (Appendix 6), may account for the presence of

lead in control plants. In order to compensate for the presence of lead in controls, all

spectrometry readings have been adjusted to represent control values at 0 ppm Pb.

Measurements of the Pb content of control plants for studies 1, 2, 3 and 6 are listed in

Appendix 6.

Epon/Araldite resin failed to properly polymerize in some molds for studies 1-3 as

there was a problem with the available DDSA. As the polymerization reactions depend

upon the ratio of the epoxy (Araldite) to the hardening agent (DDSA), evaporation or

contamination of chemicals such as DDSA can lead to changes in the properties of the

polymer (O’Brien and McCully, 1981). Also, in tissues that are highly suberized, the

suberin can act as a filter preventing complete infiltration of the tissue, resulting in soft

cells which are difficult or impossible to section (O’Brien and McCully, 1981).

27

Contamination of osmium tetroxide (OsO4) may have caused variation in the

efficiency of secondary fixation of samples. Failure of some tissue samples to fix

completely may also have occurred due to the release of water by the outer tissues of a

sample as they were fixed, diluting the fixative and altering its action on the inner tissues,

after which the specimen may not be accurately preserved (O’Brien and McCully, 1981).

Also, a tissue becomes more resistant to the fixative the longer it is exposed, and by the

time it penetrates the innermost tissues of a sample, the tissue may not be responsive to

its effects, again compromising the integrity of the preserved sample (O’Brien and

McCully, 1981).

Due to the heterogeneity of Pb in soil, ICP analyses of the brownfield soil

indicated large standard deviations in soil Pb content, and suggested that soil dilutions

may not meet expected concentrations, based on dilutions by volume. Soil dilutions

presented in this paper are based on a percent-mass ratio as described in Appendix 4.

28

CHAPTER THREE

RESULTS

3.1. Qualitative Observations

Switchgrass plants grown for seven weeks before the onset of a five week Pb

treatment (Study 1) were similar in appearance to control plants at the end of the growth

period. However, as treatment concentration increased, morphological changes also

increased, as slight changes in pigmentation appeared on some leaves of the 6000 and

10000 ppm treatment plants (Data not shown). Leaf tips of some plants were purple in

colour and had yellowing on the edges of leaves (Data not shown). Plants treated with

6000 and 10000 ppm Pb acetate solution also displayed changes in root morphology;

there was an increase in the number of short, thick roots and long roots were thinner than

those of the control, 75.8 and 1200 ppm plants (Figure 1).

Changes in plant appearance were more apparent in plants that were treated with Pb

from the time of seeding than those given time to establish before the start of the

treatment period. In study 2, the 10000 ppm treatment group did not survive for the

duration of the 12 week growth period. Of the few plants that survived to 6 weeks, all

had highly stunted root systems and were unable to remain erect. The 6000 ppm plants

expressed purple pigmentation on shoots and leaves. Leaves were also yellow and wilted

with a rolled appearance on some plants treated with high concentrations of Pb (Data not

shown). Plants in the 6000 ppm treatment group also had stunted root growth with very

few well developed roots per plant when compared to the control and lower treatment

concentration groups. While there were some pigmentation changes observed in the 1200

29

ppm and 75.8 ppm plants the appearance of treatment groups did not vary substantially

from the controls.

Similar changes in growth pattern were observed in switchgrass that was treated from

seed and grown for 5 weeks (Study 3) to those described above, as variations in plant

appearance increased as treatment concentration increased. In this study, the 10000 ppm

treatment plants had hindered root development, as many plants produced only 1 or 2

long roots which were thin, while there were numerous thick, stunted or emerging roots

at the base of the plant, as shown in Figure 1. The 10000 ppm plants in this study had

fewer root hairs than were observed on control plants. Plants treated with 6000 ppm Pb

developed pale yellow leaves with purpling at the tips. Roots were smaller in diameter

than controls, and small stunted roots which were darker than those of the controls and

yellow near the root tip were also observed. The 1200 ppm treatment plants also had

yellow discolouration on leaves, and thin, brown roots. In this study the 75.8 ppm

treatment looked similar to control plants but some yellowing was present on leaves and

one plant developed very thick roots.

30

a b

Figure 1: Switchgrass treated with 10000 ppm Pb acetate in Hoagland’s solution over a 5 week growth period (a) and a control plant (b). Switchgrass treated with high concentrations of Pb underwent changes in root development and growth patterns including root thickening and stunting. Magnifications are 1.2x for a, and 0.8x for b.

In order to determine whether Pb treatment affected seed production and time to seed,

switchgrass was grown for 23 weeks and treated with lead acetate at concentrations of 0,

75.8, 1200, 6000 and 10000 ppm (Study 5). The time until the first plant produced seed

was shortest for the 6000 ppm and 10000 ppm treatments (56 days) while the control

plants did not ever flower in the 161 day study period (Table 1). Malformations of seed

were observed in the 10000 ppm plants; shoots were produced with a single seed on the

end and others germinated mid-shoot while the seed was still on the plant.

31

Table 1: Number of days from time of planting until the first plant from each treatment produced seeds for switchgrass treated with 0, 75.8, 1200, 6000 and 10000 ppm lead

study 6, plants grown on brownfield soil containing Pb at concentrations of

approx

acetate in Hoagland’s Solution.

Pb Treatment

(ppm) Time to seed

(d) Control >161

75.8 78 1200 63 6000 56

10000 56

In

imately 5900, 6875, 13800, 23600 and 36100 ppm were compared to controls and

the only treatment group that showed a prominent change in appearance was the 36100

ppm group (Figure 2). Plants grown on soil containing 36100 ppm Pb displayed

severely stunted growth of both root and shoot tissue compared to controls and the other

treatment groups. By the end of the 12 week growth period, the shoots of plants in the

highest concentration treatment group were yellow and only a small fraction of the height

of the plants in all other groups. All other treatments were similar in appearance to

controls, although there was some purple colouration of leaves in the 13800 and 23600

ppm plants.

32

Figure 2: Switchgrass grown on soil containing 36100 ppm Pb (left) and control plants (right) after 12 weeks of growth. Magnification is 0.12x.

3.2. Growth Trend Analysis

A decrease in root and shoot biomass was observed during the eight week long

growth trend study (Study 4). Root and shoot biomass decreased with increasing Pb

treatment concentrations of 6000, 7000, 8000. 9000 and 10000 ppm Pb acetate solution

when compared to control plants (Figure 3). While there were no significant differences

in biomass for shoot or root tissue between the 6000 ppm treatment and controls, there

was a significant difference in root weight between the 7000 ppm treatment and the

control group beginning in week eight (ANOVA, t-test, P<0.05, n=5-8). This trend

continued with the 8000 ppm treatment group where the treatment plants produced

significantly less biomass in both root and shoot parts (ANOVA, t-test, P<0.05, n=5-8)

33

in week eight. After seven weeks of growth, the 9000 ppm (ANOVA, t-test, P<0.05,

n=5-8), and 10000 ppm (ANOVA, t-test, P<0.05, n=5-8) treatment groups produced

significantly less root biomass than controls, while the shoot weight of both the 9000 and

10000 ppm treatments was significantly less than control shoot biomass plants after 8

weeks of growth (ANOVA, t-test, P<0.05, n=5-8).

34

Figure 3: Dry root and shoot weight values for switchgrass seedlings, harvested weekly for an 8 week period. Switchgrass root and shoot weight values were recorded for plants treated with 6000, 7000, 8000, 9000 and 10000 ppm lead acetate in Hoagland’s solution in addition to control plants. Symbols represent weekly pairings of treatment and control measurements that are significantly different (n=4-8). Vertical error bars represent standard deviation.

35

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 1 2 3 4 5 6 7 8 9

Week

Mea

n D

ry R

oot M

ass

(g)

Control 9000 ppm

*

*α

α

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)

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*

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(g)

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(g)

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*

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(g)

Control 8000 ppm

*

*

0

0.02

0.04

0.06

0.08

0.1

0.12

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0.16

0 1 2 3 4 5 6 7 8 9

Week

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n D

ry R

oot M

ass

(g)

Control 10 000 ppm

*

*α

α

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 1 2 3 4 5 6 7 8 9

Week

Mea

n D

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s (g

)

Control 6000 ppm

0

0.05

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0.2

0.25

0.3

0 1 2 3 4 5 6 7 8 9

Week

Mea

n D

ry S

hoot

Mas

s (g

)

Control 7000 ppm

0

0.05

0.1

0.15

0.2

0.25

0.3

0 1 2 3 4 5 6 7 8 9

Week

Mea

n D

ry S

hoot

Mas

s (g

)

Control 8000 ppm

*

*

0

0.05

0.1

0.15

0.2

0.25

0.3

0 1 2 3 4 5 6 7 8 9

Week

Mea

n D

ry S

hoot

Mas

s (g

)

Control 10 000 ppm

*

*

3.3. Biomass Production

Mean dry weight (Figures 4 & 5) and fresh plant weight (Figures 6 & 7) were

recorded for switchgrass in studies 1, 2, 3, and 6. There were no significant differences

in fresh weight or dry weight observed for either root or shoot tissues in study 1, where

plants were treated for 5 weeks after a 7 week establishment period (Table 2).

Table 2: Mean fresh and dry weights (mean ± standard deviation) of switchgrass plants treated with 0, 75.8, 1200, 6000 and 10000 ppm lead acetate in study 1. Means with the same superscript letter do not differ significantly (ANOVA, t-test, P<0.05, n=3-6).

Treatment (ppm) Root Shoot Root Shoot

Control 2.83±1.29 2.71±0.92 00.28±39 0.67±0.3375.8 3.72±3.09 3.44±3.14 0.57±0.51 0.87±0.791200 8.04±4.33 6.01±1.71 0.98±0.34 1.85±1..056000 8.01±3.62 6.03±2.55 0.74±0.55 1.78±0.3210000 7.07±5.27 6.98±5.37 1.34±0.87 2.95±2.51

Fresh weight (g) Dry weight (g)

Plants grown and treated with 6000 ppm Pb acetate in study 2 produced

significantly less fresh root and shoot biomass than plants in both the control and 75.8

ppm groups (ANOVA, t-test, P<0.05, n=8-15) (Table 3). The dry weight values indicated

that there were no significant differences between study 2 treatment and control groups

for shoot weight. However, the mean dry root weight of the 6000 ppm treatment plants

was significantly less than the other treatment groups ( ANOVA, t-test, P<0.05, n=8-15)

(Table 3).

36


Treatment (ppm) Root Shoot Root ShootControl 2.86±0.99a 3.83±0.15a 1.09±0.38a 1.57±0.47

75.8 3.28±1.65a 4.66±2.25a 1.19±054a 1.82±1.021200 2.49±1.30ab 3.59±2.08ab 0.94±0.47a 1.57±0.936000 1.08±1.00b 1.54±1.12b 0.34±0.24b 1.03±0.80


The fresh weight values for shoots of study 3 did not vary significantly (Table 4).

However, the 6000 ppm treatment mean dry weight value was significantly less than both

the control and the 75.8 ppm groups (ANOVA, t-test, P<0.05, n=20-25). Dry weight

measurements indicated that the mean root weight of the 6000 ppm treatment plants was

significantly less than control, 75.8 and 1200 ppm plants. The dry shoot weight of the

6000 ppm treatment was also significantly less than the mean dry shoot weight of the

75.8 ppm group (ANOVA, t-test, P<0.05, n=20-25).


Treatment (ppm) Root Shoot Root ShootControl 0.49±0.22a 0.92±0.41 0.03±0.02a 0.11±0.06ab

75.8 0.32±0.19ab 0.75±0.39 0.04±0.02a 0.13±0.07a

1200 0.23±0.16bc 0.78±0.42 0.03±0.02a 0.11±0.06ab

6000 0.12±0.05c 0.54±0.20 0.01±0.01b 0.07±0.02b

10000 0.16±0.14bc 0.61±0.41 0.02±0.01a 0.09±0.06ab


37

Switchgrass grown in brownfield soil containing approximately 36100 ppm Pb

produced significantly less root and shoot biomass than all other treatments in study 6 for

fresh and dry weight measurements (Table 5) (ANOVA, t-test, P<0.05, n=15). There

were no significant differences observed in root fresh weight between the control plants

and the plants grown on soil containing 5900, 6875, 13800 and 23600 ppm Pb. The

mean fresh shoot tissue of control plants did, however, weigh significantly more than that

of the other treatments (ANOVA, t-test, P<0.05, n=15). There was no significant

difference in dry biomass of the root or shoot tissue of controls and 5900, 6875, 13800

and 23600 ppm treatment groups.

Table 5: Mean fresh and dry weights (mean ± standard deviation) of switchgrass plants grown in soil containing approximately 5900, 6875, 13800, 23600 and 36100 ppm lead in study 6. Means with the same superscript letter do not differ significantly (ANOVA, t-test, P<0.05, n=15).

Treatment (ppm) Root Shoot Root Shoot

Control 0.50±0.27a 1.48±0.67a 0.18±0.11a 0.42±0.28a

5900 0.34±0.17ab 0.87±0.47b 0.13±0.08a 0.32±0.16a

6875 0.53±0.29a 0.68±0.20b 0.21±0.09a 0.29±0.08a

13800 0.38±0.15a 0.71±0.28b 0.13±0.05a 0.31±0.11a

23600 0.59±0.35a 0.95±0.50b 0.18±0.09a 0.36±0.2a

36100 0.02±0.01b 0.02±0.004c 0.003±0.002b 0.01±0.002b


38

Figure 4: Mean dry weight of root tissue for switchgrass grown in studies 1, 2, 3, and 6. Plants were treated with concentrations of 0, 75.8, 1200, 6000 and 10000 ppm lead acetate in Hoagland’s solution and grown for either 5 or 12 weeks (studies 1-3), or grown on brownfield soil containing lead at concentrations of 0, 5900, 6875, 13800, 23600 and 36100 ppm (study 6). Data points labelled with the same symbol do not differ significantly (ANOVA, t-test, P<0.05). Vertical error bars represent standard deviation (study 1, n=3-8; study 2, n=8-15; study 3, n=20-25; study 6, n=15).

00.

20.

40.

60.

811.

21.

41.

61.

822.

22.

4

Cont

rol

75.8

1200

6000

1000

0

Lead

Tre

atm

ent C

once

ntra

tion

(ppm

)

Mean Root Dry Mass (g)

Stud

y 1

0

0.2

0.4

0.6

0.81

1.2

1.4

1.6

1.82

Cont

rol

75.8

1200

Lead

Tre

atm

ent C

once

ntra

tion

(ppm


6000

)

Stud

y 2

α

α

α

β

0Co

ntro

l59

0068

7513

800

2360

036

100

Soil

Lead

Con

cent

ratio

n (p

pm)

0.050.1

0.150.2

0.250.3

0.35


Stud

y 6

α

β

α

α

α

α

0Co

ntro

l75

.812

0060

0010

000

Lead

Tre

atm

ent C

once

ntra

tion

(ppm

)

0.01

0.02

0.03

0.04

0.05

0.06

0.07


Stud

y 3

α

α

αβ

α

β

39

Figure 5: Mean dry weight of shoot tissue for switchgrass grown in studies 1, 2, 3, and 6. Plants were treated with concentrations of 0, 75.8, 1200, 6000 and 10000 ppm lead acetate in Hoagland’s solution and grown for either 5 or 12 weeks (Studies 1-3), or grown on brownfield soil containing lead at concentrations of 0, 5900, 6875, 13800, 23600 and 36100 ppm (Study 6). Data points labelled with the same symbol do not differ significantly (ANOVA, t-test, P<0.05). Vertical error bars represent standard deviation (study 1, n=3-8; study 2, n=8-15; study 3, n=20-25; study 6, n=15).

0123456

Cont

rol

75.8

1200

6000

1000

0

Lead

Tre

atm

ent C

once

ntra

tion

(ppm

)

Mean Shoot Dry Mass (g)

Stud

y 1

0

0.51

1.52

2.53

Cont

rol

75.8

1200

Lead

Tre

atm

ent C

once

ntra

tion

(p


6000

pm)

Stud

y 2

0Co

ntro

l59

0068

7513

800

2360

036

100

Soil

Lead

Con

cent

ratio

n (p

pm)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


α

αα

Stud

y 6

α

α

β0

Cont

rol

75.8

1200

6000

1000

0

Lead

Tre

atm

ent C

once

ntra

tion

(ppm

)

0.050.1

0.150.2

0.25


Stud

y 3

α

α

α

β

β

βαβ

40

Figure 6: Mean fresh weight of root tissue for switchgrass grown in studies 1, 2, 3, and 6. Plants were treated with concentrations of 0, 75.8, 1200, 6000 and 10000 ppm lead acetate in Hoagland’s solution and grown for either 5 or 12 weeks (Studies 1-3), or grown on brownfield soil containing lead at concentrations of 0, 5900, 6875, 13800, 23600 and 36100 ppm (Study 6). Data points labelled with the same symbol do not differ significantly (ANOVA, t-test, P<0.05). Vertical error bars represent standard deviation (study 1, n=3-8; study 2, n=8-15; study 3, n=20-25; study 6, n=15).

02468101214

Con

trol

75.8

1200

6000

1000

0

Lead

Tre

atm

ent C

once

ntra

tion

(ppm

)

Mean Root Wet Mass (g)

Stu

dy 1

0123456

Con

trol

75.8

1200

Lead

Tre

atm

ent C

once

ntra

tion

(


6000

ppm

)

Stu

dy 2

α

αβα

β

0C

ontro

l59

0068

7513

800

2360

036

100

Soi

l Lea

d C

once

ntra

tion

(ppm

)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.91


αα

α

αα

Stu

dy 6

β

0C

ontro

l75

.812

0060

0010

000

Lead

Tre

atm

ent C

once

ntra

tion

(ppm

)

0.1

0.2

0.3

0.4

0.5

0.6

0.7


Stu

dy 3

α

βΩ

Ω

αβ

βΩ

41

Figure 7: Mean fresh weight of shoot tissue for switchgrass grown in studies 1, 2, 3, and 6. Plants were treated with concentrations of 0, 75.8, 1200, 6000 and 10000 ppm lead acetate in Hoagland’s solution and grown for either 5 or 12 weeks (Studies 1-3), or grown on brownfield soil containing lead at concentrations of 0, 5900, 6875, 13800, 23600 and 36100 ppm (Study 6). Data points labelled with the same symbol do not differ significantly (ANOVA, t-test, P<0.05). Vertical error bars represent standard deviation (study 1, n=3-8; study 2, n=8-15; study 3, n=20-25; study 6, n=15).

02468101214

Con

trol

75.8

1200

6000

1000

0

Lead

Tre

atm

ent C

once

ntra

tion

(ppm

)

Mean Shoot Wet Mass (g)

Stu

dy 1

0123456

Con

trol

75.8

1200

Lead

Tre

atm

ent C

once

ntra

tion

(ppm


6000

)

Stu

dy 2

α

α

αβ

β

0

0.51

1.52

2.5

Mean Shoot Wet Mass (ppm)

Con

trol

5900

6875

1380

023

600

3610

0

Soil

Lead

Con

cent

ratio

n (p

pm)

Stu

dy 6

Ω

α

β

ββ

β

0

0.2

0.4

0.6

0.81

1.2

1.4


Con

trol

75.8

1200

6000

1000

0

Lead

Tre

atm

ent C

once

ntra

tion

(ppm

)

Stu

dy 3

α

α

α

αα

42

3.4. Morphology

The number of roots per plant did not vary significantly across treatment for

studies 1, 2 or 3. However, while the number of longer, developed roots with length

greater than 5 cm remained fairly consistent in study 1, the number of long roots did vary

in studies 2 and 3 (Figure 8). The number of roots greater than 5 cm long decreased with

increasing Pb treatment concentration (Figure 8). In study 2, the 6000 ppm treatment had

significantly fewer long roots than all other treatments (ANOVA, t-test, P<0.05, n=8-15).

In study 3, both the 6000 and 10000 ppm treatments had significantly reduced root

development, with fewer roots greater than 5 cm long compared to the 1200 and 75.8

ppm treatments and the control plants (ANOVA, t-test, P<0.05, n=20-25).

In study 6, switchgrass grown in brownfield soil containing 36100 ppm Pb had

significantly fewer total roots than any other treatment group or the controls (ANOVA, t-

test, P<0.05, n=15). Plants grown on soil containing 5900 ppm Pb also had significantly

fewer roots than the treatment with the most roots, 6875 ppm (ANOVA, t-test, P<0.05,

n=15). When comparing developed roots greater than 5 cm in length, switchgrass grown

on 36100 ppm soil had significantly fewer long roots than any other treatment and the

controls (ANOVA, t-test, P<0.05, n=15), while there were no significant differences

between the other treatments.

43

Figure 8: Mean number of roots per plant and mean number or roots greater than 5cm in length for switchgrass grown on soil treated with 75.8, 1200, 6000 and 10000 ppm Pb acetate in Hoagland’s solution in studies 1, 2 and 3, switchgrass grown on brownfield soil containing 5900, 6875, 13800, 23600 and 36100 ppm Pb in study 6, and control groups. Values with the same symbol are not significantly different (ANOVA, t-test, P<0.05). Vertical error bars represent standard deviation (study 1, n=3-8; study 2, n=8-15; study 3, n=20-25; study 6, n=15).

44

0

2

4

6

8

10

12

14

Control 75.8 1200 6000 10000

Lead Treatment Concentration (ppm)

Mea

n N

umbe

r of R

oots

per

pla

nt

Study 1

0

2

4

6

8

10

12

Control 75.8 1200 6000 10000


Mea

n N

umbe

r of R

oots

per

pla

nt

>5cm

Study 1

0

5

10

15

20

25

30

Control 75.8 1200 6000


Mea

n N

umbe

r of R

oots

per

pla

nt

Study 2

0

5

10

15

20

25

30

Control 75.8 1200 6000


Mea

n N

umbe

r of R

oots

per

pla

nt >

5 cm

Study 2

αα

α

β

0

1

2

3

4

5

6

7

8

9

10

Control 75.8 1200 6000 10000


Mea

n N

umbe

r of R

oots

per

pla

nt

Study 3

0

1

2

3

4

5

6

7

8

Control 75.8 1200 6000 10000


Mea

n N

umbe

r of R

oots

per

pla

nt

>5 c

m

Study 3

αα

ββ

α

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

Control 5900 6875 13800 23600 36100

Soil Lead Concentration (ppm)

Mea

n N

umbe

r of R

oots

per

pla

nt

Study 6

αβαβ

ααβ

β

Ω

0.00

2.00

4.00

6.00

8.00

10.00

12.00

Control 5900 6875 13800 23600 36100


Mea

n N

umbe

r of R

oots

per

pla

nt

>5cm

Study 6

α

αα

α

α

Ω

Mean height of switchgrass plants grown in each treatment group was recorded

(Figure 9). In study 1, there was no significant difference in plant height between the

control plants and those treated with Pb acetate in Hoagland’s solution at concentrations

of 75.8, 1200, 6000 and10000 ppm for 5 weeks after establishment.

Switchgrass grown and treated for 12 weeks with Pb acetate solution at a

concentration of 6000 ppm were significantly shorter than controls, and those treated with

75.8 and 1200 ppm Pb acetate (ANOVA, t-test, P<0.0.5, n=8-15).

Study 3 plants grown and treated for 5 weeks in the above concentrations of Pb

acetate in Hoagland’s solution followed similar growth patterns to those observed in

study 2, as plant height decreased with increasing Pb treatment concentration.

Switchgrass treated with concentrations of 6000 and 10000 ppm Pb were significantly

shorter than control plants (ANOVA, t-test, P<0.05, n=20-25), but did not differ

significantly from plants treated with 75.8 or 1200 ppm Pb.

Switchgrass plants grown for 12 weeks on brownfield soil were significantly

shorter than controls for all Pb concentrations (ANOVA, t-test, P<0.05, n=15). Mean

plant height decreased in the following order, control, 5900, 23600, 13800, 6875 and

36100 ppm with the 5900 and 23600 ppm treatment heights being significantly greater

than those corresponding to the 13800 and 6875 ppm treatments (ANOVA, t-test, P<0.05,

n=15).

Although the number of shoots per plant generally decreased with an increase in

treatment concentration, there were no significant differences in shoot number observed

in any study (Figure 9).

45

Figure 9: Mean plant height (cm) and mean number of offshoots per plant for switchgrass grown in soil treated with Pb acetate in Hoagland’s solution at concentrations of 0, 75.8, 1200, 6000 and 10000 ppm in studies 1, 2 and 3, as well as switchgrass grown on brownfield soil containing approximately 0, 5900, 6875, 13800, 23600 and 36100 ppm Pb in study 6. Values with the same symbol are not significantly different (ANOVA, t-test, P<0.05). Vertical error bars represent standard deviation (study 1, n=3-8; study 2, n=8-15; study 3, n=20-25; study 6, n=15).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Control 75.8 1200 6000 10000


Mea

n N

umbe

r of S

hoot

s pe

r pla

nt

Study 1

0

20

40

60

80

100

120

140

Control 75.8 1200 6000 10000


Mea

n P

lant

Hei

ght (

cm)

Study 1

0

0.5

1

1.5

2

2.5

3

3.5

Control 75.8 1200 6000

Lean Treatment Concentration (ppm)

Mea

n N

umbe

r of S

hoot

s pe

r pla

nt

Study 2

0

20

40

60

80

100

120

140

Control 75.8 1200 6000


Mea

n Pl

ant H

eigh

t (cm

)

Study 2

α

αα

β

0

10

20

30

40

50

60

70

Control 75.8 1200 6000 10000


Mea

n Pl

ant H

eigh

t (cm

)

Study 3

α αβαβ

ββ

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Control 5900 6875 13800 23600 36100


Mea

n N

umbe

r of S

hoot

s pe

r pla

nt

Study 6

0

10

20

30

40

50

60

70

80

90

Control 5900 6875 13800 23600 36100


Mea

n Pl

ant H

eigh

t (cm

)

Study 6

α

ββ

ΩΩ

φ

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Control 75.8 1200 6000 10000


Mea

n N

umbe

r of S

hoot

s pe

r pla

nt

Study 3

46

3.5. Phytoextraction of Lead

The amount of lead extracted by plants treated with concentrations of lead acetate

solution between 0 and 10000 ppm, as well as by plants grown in brownfield soil, was

measured using AAS and ICP-OES analyses.

3.5.1. Lead Accumulation in Plants Grown on Soil Treated with Lead acetate

Mean plant lead concentration increased with increasing treatment concentration

in studies 1, 2 and 3. Root tissue extracted more lead from the soil than shoot tissue in

the majority of Pb treatments and studies. Switchgrass grown for 12 weeks and treated

for the last 5 weeks of the growth period extracted more lead as the treatment

concentration increased (Figure 10). There were significant differences between the 6000

ppm treatment and the lower treatments as well as the 10000 ppm treatment and all other

treatments (ANOVA, t-test, P<0.05, n=1) for both root and shoot parts.

In switchgrass grown for 12 weeks and treated for the duration of the growth

period (Figure 11), there were significant increases in the plant Pb concentration in both

root and shoot tissues between all treatments (75.8-6000 ppm) (ANOVA, t-test, P<0.05,

n=1).

Switchgrass grown and treated for 5 weeks displayed similar trends to those

presented above, as the measured Pb concentration increased as the applied treatment

concentration increased (Figure 12). There were significant differences in Pb

concentration in the root tissue of plants treated with 75.8 and 10000 ppm Pb acetate

(ANOVA, t-test, P<0.05, n=1). In the shoot tissue there were significant differences in Pb

concentration between the 75.8 ppm treatment and the other treatments, as well as the

1200 ppm treatment and both the 6000 and 10000 ppm treatments (ANOVA, t-test,

47

P<0.05, n=1). There was not a significant difference between the shoot Pb concentration

of the 6000 and 10000 ppm treatment groups.

In study 1, the established switchgrass plants extracted more lead from the soil

than plants in studies 2 and 3 (Table 6). There was significantly more Pb in study 1 root

tissue than in the roots tissue of study 2 for treatment concentrations of 75.8 and 1200

ppm (ANOVA, t-test, P<0.05, n=1), while there were no significant differences between

the extracted Pb concentrations in the root tissue of plants treated with 6000 ppm in these

studies. Study 1 took up significantly more Pb than study 3 for all treatment

concentrations, 75.8-10000 ppm (ANOVA, t-test, P<0.05, n=3). Switchgrass plants in

study 1 also had significantly more Pb in shoot tissues than both study 2 (ANOVA, t-test,

P<0.05, n=1) and 3 (ANOVA, t-test, P<0.05, n=1) across all treatment concentrations.

There was no significant difference in the amount of Pb extracted into the shoot

tissue of the 12 and 5 week old plants in Studies 2 and 3 respectively. The Study 2 plants

which were treated and grown for 12 weeks did, however, extract significantly more Pb

into the root tissue than study 3 (ANOVA, t-test, P<0.05, n=1).

The mean percentage of root and shoot Pb concentration relative to the applied

treatment concentration was greatest in the established plants of study 1. Study 2 plants

extracted a greater percentage of the treatment than study 3 plants which were grown

over a shorter growth period (Table 7).

48

Figure 10: Mean measured root (a) and shoot (b) lead concentration per plant for switchgrass treated with 75.8, 1200, 6000 and 10000 ppm lead acetate in Hoagland’s solution in study 1. Switchgrass in this study were treated for 5 weeks after a 7 week growth period. Values labelled with the same symbol are not significantly different (ANOVA, t-test, P < 0.05). Vertical error bars represent standard deviation (n=1, 3 replications).

49

1

10

100

1000

10000

100000

75.8 1200 6000 10000


Mea

n M

easu

red

Roo

t Lea

d C

once

ntra

tion/

plan

t (pp

m)

a

Ωβ

α

α

1

10

100

1000

10000

100000

75.8 1200 6000 10000


Mea

n M

easu

red

Shoo

t Lea

d C

once

ntra

tion/

plan

t (pp

m)

Ωβ

αα

b

Figure 11: Mean measured root (a) and shoot (b) lead concentration per plant for switchgrass treated with 75.8, 1200, and 6000 ppm lead acetate in Hoagland’s solution. Study 2 plants were grown and treated over a 12 week growth period. Values labelled with the same symbol are not significantly different (ANOVA, t-test, P<0.05). Vertical error bars represent standard deviation (n=1, 3 replications).

50

1

10

100

1000

10000

100000

75.8 1200 6000


Mea

n M

easu

red

Roo

t Lea

d C

once

ntra

tion/

plan

t (pp

m)

α

β

Ω

a

1

10

100

1000

75.8 1200 6000


Mea

n M

easu

red

Shoo

t Lea

d C

once

ntra

tion/

plan

t (pp

m)

Ω

β

α

b

Figure 12: Mean measured root (a) and shoot (b) lead concentration per plant for switchgrass treated with 75.8, 1200, 6000 and 10000 ppm lead acetate in Hoagland’s solution. Study 3 plants were grown and treated for 5 weeks. Values labelled with the same symbol are not significantly different (ANOVA, t-test, P<0.05). Vertical error bars represent standard deviation (n=1, 3 replications).

1

10

100

1000

75.8 1200 6000 10000


Mea

n M

easu

red

Roo

t Lea

dC

once

ntra

tion/

plan

t (pp

m)

αβ

αβ

α

β

a

1

10

100

1000

75.8 1200 6000 10000


Mea

n M

easu

red

Shoo

t Lea

d C

once

ntra

tion/

plan

t (pp

m)

α

β

Ω

b

Ω

51

Table 6: Mean measured Pb concentration (ppm) for root and shoot tissue of switchgrass plants for all treatment groups, given as Pb per plant and Pb per gram of plant tissue. Means with the same superscript within treatment groups do not differ significantly (ANOVA, t-test, P<0.05, n=2-3).

75.8 1200 6000 10000

75.8 1200 6000 10000

75.8 1200 6000 10000

75.8 1200 6000 10000

Mean Pb Treatment Concnetration (ppm)

87.7 ± 7.48b 190 ± 44.1b 190 ± 30.2b

Study

Study

11.4 ± 3.10bStudy 2

Study 3 2.46 ± 0.75b

19310 ± 2150a

127 ± 39c 217 ± 89b 390 ± 202a

N/A850 ± 99b104 ± 21b

710 ± 85a 8555 ± 2055a

Mea

n M

easu

red

Roo

t Pb

Con

cent

ratio

n (p

pm) p

er p

lant

Mea

n M

easu

red

Shoo

t Pb

Con

cent

ratio

n (p

pm) p

er p

lant

Study 1 347 ± 54a

Study 3 12.4 ± 1.99c

Study 1 590 ± 137a

2070 ± 75a 14327 ± 3350a 37115 ± 8560b

Study 2 192 ± 11.4b 1372.90 ± 295.50b 13997.85 ± 286.62a N/A

Mean Pb Treatment Concnetration (ppm)

Mea

n M

easu

red

Roo

t Pb

Con

cent

ratio

n pe

r gra

m ti

ssue

(p

pm)

StudyMean Pb Treatment Concnetration (ppm)

Study 1 796 ± 91a 2320 ± 76a 19600 ± 4540a 27750 ± 6350a

Study 2 240 ± 8.13b 1126 ± 240b 24900 ± 512a N/A

Study 3 330 ± 42b 1435 ± 420b 16756 ± 6876a 12626 ± 6509b

Mea

n M

easu

red

Shoo

t Pb

Con

cent

ratio

n pe

r gra

m ti

ssue

(p

pm)

StudyMean Pb Treatment Concnetration (ppm)

Study 1 960 ± 157a 660 ± 48a 5076 ± 1160a 6830 ± 730a

Study 2 50 ± 2.9b 89 ± 12b 790 ± 85b N/A

1870 ± 290bStudy 3 91 ± 6.7b 340 ± 21c 2570 ± 610c

52

Table 7: Mean percentage of Pb treatment present in plant tissue for switchgrass grown on soil treated with concentrations of 75.8, 1200, 6000 and 10000 ppm Pb acetate in Hoagland’s solution. STUDY 1 STUDY 2 STUDY 3

% Uptake of Total

Applied Pb Treatment % Uptake of Total

Applied Pb Treatment % Uptake of Total

Applied Pb Treatment Pb

Treatment (ppm) Root Shoot Root Shoot Root Shoot 75.8 0.070 0.192 0.038 0.018 2.012E-04 5.867E-04 1200 0.037 0.038 0.013 0.003 2.059E-04 6.683E-04 6000 0.036 0.054 0.011 0.001 9.440E-06 5.405E-05 10000 0.101 0.119 N/A N/A 6.145E-04 1.053E-03

Note: Percent uptake was calculated based on the total mass of Pb added to the soil over the treatment period (for 10000 ppm treatment, 5 weeks: 50 g; 12 weeks: 120g).

3.5.2. Lead Accumulation in Switchgrass Grown on Brownfield Soil

Switchgrass grown on brownfield soil containing concentrations of Pb ranging

from 5900-36100 ppm translocated larger amounts of lead into shoot tissue as the

concentration of Pb in the soil increased, with the highest concentration of Pb measured

in the 36100 ppm treatment group (Figure 13). The 23600 ppm study group accumulated

the highest mean concentration of Pb in the root tissue per plant (Figure 13), with

significantly more Pb in root tissues than any other treatment group (ANOVA, t-test,

P<0.05, n=1-3). While the root Pb concentration was greater than the shoot concentration

of the 5900, 6875, 13800 and 23600 ppm treatments, the 36100 ppm treatment plants

translocated more Pb into shoot tissues than were present in root tissues.

The mean percentage of Pb uptake into plant tissues relative to the soil Pb

concentration was highest in the 6875 ppm treatment, as root tissues extracted 0.007% of

the total lead and shoot tissues contained 0.001% of the total soil Pb (Table 8).

53

ICP 30-element analysis was performed on all root and shoot tissue for this study

in order to determine the concentrations of other elements that were extracted from the

soil into the plant tissues. Mean percentage of the original soil elemental concentration

present in the root or shoot tissue for each treatment group is shown in Appendix 7.

Switchgrass extracted between 0.003 and 0.034% of arsenic and between 0.04 and 0.03%

of Cd from brownfield soil. Concentrations of zinc decreased with increasing soil

concentration in both root and shoot tissues.

54

Figure 13: Mean root and shoot Pb concentration per plant in switchgrass grown for 12 weeks on brownfield soil containing 5900, 6875, 13800, 23600 and 36100 ppm Pb. Values labelled with the same symbol are not significantly different (ANOVA, t-test, P<0.05). Vertical error bars represent standard deviation (n=1-3).

0

100

200

300

400

500

600

700

800

5900 6875 13800 23600 36100


Mea

n M

easu

red

Roo

t Lea

d C

once

ntra

tion/

plan

t (pp

m)

a

φ

β

βΩ

Ω α

α

0

50

100

150

200

250

300

350

5900 6875 13800 23600 36100


Mea

n M

easu

red

Shoo

t Lea

d C

once

ntra

tion

/pla

nt (p

pm)

b

α

Ωβ

ββ

Ω

55

Table 8: Mean percentage of Pb treatment present in plant tissue for switchgrass grown on brownfield soil containing 5900, 6875, 13800, 23600 and 36100 ppm Pb.

Treatment Root Shoot5900 1.288E-03 8.870E-046875 7.001E-03 1.458E-03

13800 7.832E-04 3.566E-0423600 8.330E-04 2.393E-0436100 1.723E-06 4.213E-06

% Uptake of Pb Treatment

Note: Percent uptake was calculated based on the total mass of Pb in the soil at the onset of the study period (36100 ppm: 39 g Pb).

3.5.3. Electron Microprobe Analysis

Analysis of root and shoot tissues which were fixed and embedded for electron

microscope analysis did not yield any significant results. While x-ray spectra indicated

the possibility of Pb presence in plant tissues, the Pb levels were close to the detection

limit of the equipment and the resultant EDS peaks were so closely associated with those

of osmium that no clear determination as to the location of Pb within the plant tissue

could be made (data not shown).

3.6. Light Microscopy

Variations in structure and development were observed in light micrographs of

switchgrass plants which received high concentration lead treatments (13800, 10000 or

36100 ppm) when compared with control root and shoot cross-sections. Cell walls

appeared thicker in both root and shoot tissues of plants treated with high concentrations

of Pb, particularly in the case of xylem cells. Vascular bundles appeared smaller in size

and more densely arranged at the nodes of treated plants when compared with controls

(Figures 14-17).

56

Dark-staining bodies, absent in control shoot tissues (Figures 14 & 18) were

observed in treated shoot tissues. Deposits were observed in developing leaf structures of

plants treated with 10000 ppm Pb in study 1 (Figures 15 &19), as well as around the

vascular bundles in the study 2, 6000 ppm treatment (Figure 20) and the study 3, 10000

ppm (Figure 21) treatment group. Micrographs of switchgrass grown on brownfield soil

containing approximately 36100 ppm Pb also showed similar dark deposits (Figure 22).

Dark staining deposits were absent in control root tissues (Figure 23). However,

dark staining bodies were observed within the stele of switchgrass roots treated with

10000 ppm Pb acetate in Hoagland’s solution in study 1 which extracted the highest

concentrations of Pb (Figure 24). Deposits were also observed in plants grown on

brownfield soil (Figure 25), but the deposits were not present to a significant extent in

plants from studies 2 and 3. Dark staining bodies were observed in high concentration

plants for which the fixation process was performed using potassium phosphate buffer as

well as that using sodium cacodylate buffer. Therefore, the dark-staining deposits are not

likely a result of Pb interactions with potassium phosphate buffer during the fixation.

There were no deposits observed in plant roots treated with 36100 ppm Pb; however, the

cell walls of root structures in this treatment group were highly suberized (Figure 26).

Though a subset of tissue was soaked in sodium rhodizonate for histochemical

analysis, any staining that occurred in Pb treated root and shoot tissues was not visible

following post-fixation of the tissue with osmium tetroxide and embedding of the tissues

in resin.

57

X

Figure 14: Light micrograph of a toluidine blue O (0.025%) stained, resin-embedded, cross section through the node of a control shoot which received no lead treatment, showing xylem (X), phloem (Ph), sclerenchyma (Scl), epidermis (Ep) Magnification is 230x.

Ph

Ep

Scl

58

X

b

DD

Ph

DD

a

X

Figure 15: Light micrographs of cross sections through the node of a shoot (a) and cells of the developing leaf tissue (b) treated with 10000 ppm lead in study 1, showing xylem (X), phloem (Ph) and dark staining deposits in Pb treated plant (DD). Magnifications are 140x for a, and 900x for b.

59

Figure 16: Light micrograph of a cross section through the node of a shoot treated 6000 ppm lead in study 2 showing xylem (X), phloem (Ph) and epidermis (Ep). Magnification is 140x. Figure 17: Light micrograph of a cross section through the node of a shoot treated 10000 ppm lead in study 3 showing xylem (X), phloem (Ph) and epidermis (Ep). Magnification is 140x.

XPh

Ep

X

Ph

Ep

60

Figure 18: Light micrograph of cross section through the vascular bundle of a control shoot which received no lead treatment, showing xylem (X) and phloem (Ph). Magnification is 570x. Figure 19: Light micrograph of a cross section through the vascular bundle of a shoot treated with 10000 ppm lead acetate in study 1 showing xylem (X) and phloem (Ph). Magnification is 570x.

XX

X

Ph

XX

X

Ph

XPh XPh

61

Figure 20: Light micrograph of a cross section through the vascular bundle of a shoot treated with 6000 ppm lead in study 2 showing xylem (X) and phloem (Ph). Magnification is 570x. Figure 21: Light micrograph of a cross section through the vascular bundle of a shoot treated with 10000 ppm lead in study 3, showing xylem (X) and phloem (Ph). Magnification is 570x.

62

Ph

XX

XX

Ph

Ph X

X

X

Figure 22: Light micrograph of a cross section through the vascular bundle of shoots grown on brownfield soil containing 36100 ppm lead in study 6, showing xylem (X) phloem (Ph). Magnification is 570x.

63

PhX

Scw

Pe X

X

Figure 23: Light micrograph of a cross section through the stele of a control root which received no lead treatment, showing xylem vessels (X) and phloem cells (Ph) within the stele, the highly suberized secondary cell walls of the endodermis (Scw) and the pericycle (Pe). Magnification is 230x.

64

Figure 24: Light micrograph of a cross section through the stele of a root treated with 10000 ppm lead acetate in study 1, showing xylem vessels (X) and dark-staining deposits (DD) within the stele in the Pb-treated root. Magnification is 230x. Figure 25: Light micrograph of a cross section through the stele of a root grown on brown field soil containing 13800 ppm Pb showing xylem vessels (X) and phloem cells (Ph) within the stele, the highly suberized secondary cell walls of the endodermis (Scw), and dark-staining deposits (DD) within the stele in the Pb-treated root. Magnification is 140x.

X

X

X

X

X

DD

Ph

65

X

X

X

Scw

Pe

Figure 26: Light micrographs of a cross section through the stele of a root grown on brown field soil 36100 ppm lead, showing xylem vessels (X) \ within the stele, the highly suberized secondary cell walls of the endodermis (Scw), the pericycle (Pe) in the Pb-treated root. Magnification is 570x.

66

CHAPTER FOUR

DISCUSSION

4.1. Lead Treatment Affects Switchgrass Establishment

Declines in switchgrass biomass production were observed in plants treated with

7000-10000 ppm Pb acetate in Hoagland’s solution over an eight week period. At the

lower concentration of 6000 ppm, effects were insignificant. The applied Pb treatment

resulted in a significant decrease in root biomass at the end of eight weeks for the 7000

and 8000 ppm groups while a similar decrease in root biomass was observed after seven

weeks in the 9000 and 10000 treatment groups. Shoot biomass was significantly less than

that of the controls after 8 weeks for the 8000, 9000 and 10000 ppm treatments. Delayed

maize germination and root growth inhibition were observed in seeds exposed to 10-2-10-3

M Pb(NO3) (330-3300 ppm) (Obroucheva et al., 1998). Decreased establishment

capability associated with hindered growth and germination may be related to nutrient

deficiencies, which often occur in metal-exposed plants (Sharma and Dubey, 2005).

Lead may block the uptake of certain essential elements such as phosphorous, calcium

and magnesium (Begonia et al., 1998; Wierzbicka, 1998) leading to changes in plant

growth patterns and appearance. Increased ion concentration in the rhizosphere may in

turn lead to a loss of turgor pressure in shoot cells as water may be reallocated to root

tissues to relieve salt stress (Hsiao, 1973). Lead-interference with nutrient uptake may

lead to decreased biomass output by switchgrass grown on highly contaminated soils,

thus limiting plant productivity in such a situation. Therefore, switchgrass may be most

successful in a phytoremediation role where the available Pb is less than 6000 ppm.

67

High Pb treatment concentrations may also be associated with decreased time to

seed development and plant maturity. The first appearance of flowering by switchgrass

plants treated with 10000 ppm Pb solution flowered more than 100 days before control

plants. Growth inhibition and water stress resulting from the presence of Pb in the soil

(Hsiao, 1973) and within plant tissues may incite stress responses, such as the early onset

of seed production as a means of ensuring reproductive success. A decline in the leafing

period for beech trees under Pb stress, with late emergence and early senescence relative

to controls has been observed (Breckle and Kahle, 1992). Early maturation may reduce

biomass yield of a crop, thus decreasing overall efficiency of remediation and total

harvestable tissue.

4.2. Switchgrass Biomass Decreases with High Lead Treatment Concentrations

Switchgrass is considered a strong candidate for use in phytoremediation because

of the high biomass output associated with this feedstock. Switchgrass may be expected

to produce 10-12 tonnes/ha of dry biomass per year (Liebig et al., 2005), which is

approximately triple that of tame hay, which produces an average yield of 4.1 tonnes/ha

(Statistics Canada, 2006). A fundamental qualification for use of switchgrass in

phytoremediation is that it be able to maintain an adequate level of biomass production.

It is important to note that variations in biomass, as determined by fresh and dry weight

readings are not always reliable for plants exposed to a contaminant, as dead or decaying

leaves as well as the metal contaminant itself can add to the mass of the plant, resulting in

varied and potentially unreliable results (Malkowski et al., 2002).

Switchgrass plants that were grown for seven weeks prior to treatment with Pb did

not undergo a reduction in dry root or shoot biomass after five weeks of Pb treatment at

68

concentrations ranging from 0-10000 ppm Pb. However, a slight increase in shoot and

root weight was observed with increased treatment concentration relative to controls in

this study group. The observed increase may be associated with the production of cell

wall polysaccharides and increased cell wall thickness due to Pb exposure and

accumulation (Malkowski et al., 2002).

Switchgrass root biomass production was inhibited at concentrations of 6000 and

10000 ppm in plants that were treated for both 5 and 12 weeks from the time of seeding.

Switchgrass treated with 10000 ppm Pb in study 2 did not survive for the duration of the

12 week growth period which may suggest that Pb is phytotoxic to switchgrass above a

specific threshold. Vetiveria nemoralis (vetiver grass) displayed a similar response to Pb

treatment in a previous study, in which all plants of this species died after one week of

treatment with 9000 and 11000 ppm Pb (Chantachon et al., 2004). Reduction in plant

biomass may be a result of nutrient deficiencies caused by Pb interference with root

access to essential macronutrients and micronutrients in the soil matrix (Sharma and

Dubey, 2005). Inactivation of peroxidase and hydrolytic enzymes may have also

accelerated the onset of senescence in lead-stressed plants (Pahlsson, 1989). PEPC is an

important regulatory enzyme involved in C4 photosynthesis, the decreased activity of

which may be linked to Pb exposure; while decreased peroxidase activity may alter cross-

linking of cell walls (Pahlsson, 1989). Changes in enzyme production and regulation

may have altered the ability of switchgrass to grow and respond to a stress such as Pb.

While there was no change in shoot biomass for switchgrass treated for 12 weeks

with Pb, there was some variation in biomass for switchgrass treated and grown for five

weeks. The 6000 ppm treatment group was significantly less productive than the 75.8

69

ppm treatment. This result may be due to a large standard deviation in the shoot weight,

may indicate an inhibition of early plant growth due to the presence of Pb in the growth

medium or may be due to changes in resource allocation in response to stress. Previous

studies have found as much as a 10% decrease in biomass yield in Pisum sativum when

treated with concentrations of Pb acetate between 2-9.4 mmol Pb acetate/kg dwt soil

(760-3600 ppm) (Päivöke, 2002) and 30-40% when eggplant was treated with 300 and

600 ppm Pb in soil (Khan and Khan, 1983). Abiotic stress, such as drought, can result in

a decrease in growth rate due to a loss in turgor pressure within plant cells as well as

changes in osmoregulation (Hsiao, 1973). Changes to solute content in growth media can

lead to reduced transpiration rates and changes in osmotic pressure in shoots (Hsiao,

1973), as the plant responds to salt stress in the root zone. The prevention of solute

uptake from the soil due to interference by Pb ions may lead to water and salt stress

responses (Hsiao, 1973; Breckle, 1991).

The results of these studies indicate that transplanted or established plants treated

with high Pb concentrations may be able to maintain the mean expected growth rate as

represented by the controls, while switchgrass grown from seed at moderate treatment

concentrations will also maintain a high level of success.

4.3. Lead Treatment Affects Switchgrass Root Morphology

Switchgrass root development and the number of developed roots per plant, were

inhibited as the applied Pb treatment concentration increased. While there was little

variation in total number of roots per plant in any of the study groups, the number of

roots that were greater than 5 cm long decreased significantly for switchgrass treated with

70

6000 and 10000 ppm Pb from seed. This stunting of root growth was accompanied by

decreased length of root axis and decreased number of root hairs. Root stunting has

previously been observed in corn and ragweed exposed to Pb, and this decrease may be

attributed to deficiencies in essential nutrients such as magnesium, potassium, calcium,

iron, manganese and zinc (Huang and Cunningham, 1996).

Uptake of various ions may be inhibited by the presence of Pb in the soil as the

metal cations may interfere with ion transport sites of root cells or block ion channels

(Sharma and Dubey, 2005). Lead ions may compete with uptake sites of Ca and Zn

making it possible for Pb to interfere with the normal metabolic processes associated with

these cations, thus disrupting intracellular functions (Garza et al., 2006).

Root growth inhibition has also been associated with reduced size of the root

meristem upon root emergence due to metal suppression of cytokinesis and cell

elongation mechanisms (Obroucheva et al., 1998). Interactions between Pb and nucleic

acids have also been linked with the inhibition of cell division, and though the majority of

affects attributed to these associations are reversible with time, they have the largest

effect within 1 day of exposure to Pb (Breckle, 1991). Inhibition upon emergence may

account for average root development observed in established switchgrass as elongation

and cell division of emerging roots had already taken place.

Increased root system density was coupled with a change in root colour. Root

colour change in the thick, stunted roots observed in plants treated with high

concentrations of Pb may be associated with Pb deposition and increased cell wall

suberification (Breckle, 1981). Seregin et al. (1998) reported brown coloured roots and

measured a decrease in root growth of up to 50% in maize treated with 10-4 M Pb nitrate.

71

A change in colour gradient from yellow to dark brown was also observed in Vicia faba

and Pisum sativum which were treated with 10-2 M Pb in Hoagland’s solution and the

colour change was associated with distinct changes in root structure, elongation and

shape (Piechelak et al., 2002).

4.4. Lead Effects Switchgrass Shoot Height and Appearance

Exposure to high concentrations of Pb that cause symptoms of toxicity in roots

can produce only minor effects on shoot growth and elongation; perhaps because of

sluggish translocation of Pb from root to shoot which is likely limited by the movement

of Pb into the xylem (Obroucheva et al., 1998).

Switchgrass treated with high concentrations of lead appeared to be suffering

slightly from water stress, as some plants displayed leaf rolling. The presence of Pb

within the plant and in the soil matrix may have prevented the uptake of phosphorus by

the root system through the formation of precipitates in and around root cells (Breckle,

1991). Phosphorous deficiencies have been linked to decreased water absorption

capacity, loss of turgidity and decreased transpiration rates in plants (Sharma and Dubey,

2005).

Loss of green pigment intensity (chlorosis) and purplish pigmentation on leaf

edges were observed in switchgrass treated with high concentrations of Pb acetate

solution. Pigmentation change or loss may be attributed to Pb toxicity (Hampp and

Lendzian, 1974; Sharma and Dubey, 2005). Purple pigmentation can be associated with

increased anthocyanin concentration in leaves of phosphorous deficient plants (Begonia

et al., 1998). Lead forms insoluble complexes with phosphorous (Begonia et al., 1998).

The build up of Pb-phosphorous complexes has been observed in ragweed roots exposed

72

to Pb (Huang and Cunningham, 1996), and such associations may have rendered

phosphorous unavailable in switchgrass leaves, leading to high levels of anthocyanin and

the associated pigmentation.

Chlorosis was previously observed in turfgrass varieties grown in lead solutions

as low as 150 mg/L Pb (150 ppm) (Qu et al., 2003) and may, therefore, indicate a loss of

photosynthetic pigments. Kosobrukhov et al., (2004) reported an increase in the number

of chloroplasts per cell when Plantago major (plantain) was treated with 2000 mg/kg Pb,

while the amount of chlorophylls a + b decreased by 26% and 25% respectively. Lead

toxicity has been associated with nutrient deficiencies including Mg and Fe deficits

(Sharma and Dubey, 2005). Decreased stomatal regulation of water diffusion and CO2

response may influence the rate of photosynthetic activity, which may have in turn

resulted in the loss of photosynthetic pigments (Kosobrukhov et al., 2004). The lack of

essential elements such as Mg and Fe due to Pb interference, in addition to CO2

deficiencies and decreased chlorophyll synthesis, may cause changes in chloroplast

ultrastructure (Rebechini and Hanzely, 1974), thus enhancing the chlorotic appearance of

leaves.

The Pb treatments did not inhibit shoot growth in the established plants of study

1, though significant reductions in shoot height relative to mean control height were

observed in switchgrass treated over the entire growth period with concentrations of 6000

and 10000 ppm. Reduced shoot height may be attributed to the more severe reduction in

root growth which was observed. The associated reduction in the ability of the roots to

take up essential nutrients may have resulted in the inhibition of cell division and

elongation in the shoot structures (Obroucheva et al., 1998). The reduction in

73

chlorophyll production and related nutrient deficiencies as discussed above have also

been associated with a decrease in shoot height (Xiong, 1997).

4.5. Effects of Brownfield Soil on Switchgrass Growth Patterns

While switchgrass root and shoot biomass production, root development and

shoot height were significantly affected when plants were grown on soil containing

36100 ppm Pb, only shoot height was affected by the other soil treatments (5900-23600

ppm). The soil type and amount of organic material within the brownfield soil may have

hindered plant growth at the 36100 ppm level. The addition of even small amounts of

Promix as a dilution medium may have provided enough added organic matter to allow

the plants to be much more successful in the lower level treatment groups (Punz and

Sieghardt, 1993).

The formation of Pb complexes within the soil matrix may have reduced the soil-

available Pb, thus reducing both the impact of Pb on the plant growth regime and the

uptake of Pb into the plant (Davies, 1990). Interactions between Pb and Cd have

previously been studied (Silviera and Sommers, 1977; Breckle and Kahle, 1992;

Fargašová, 1994; Miller et al., 1997; Seregin et al., 2004) in terms of the synergistic

relationship between them. Both lead and cadmium are considered non-essential

elements which are phytotoxic at high concentrations (Pahlsson, 1989).

Roots of switchgrass grown on brownfield soil extracted as much as 0.030% of

the total soil Cd, while the maximum concentration of Pb was 0.007% (Appendix 7), a

factor of 10 lower than switchgrass treated with Pb in solution in studies 1 and 2. Fagus

sylvatica saplings demonstrated a synergistic effect between Pb and Cd in favour of Cd

uptake; the presence of Pb results in increased Cd uptake, and the Cd results in decreased

74

Pb concentrations in roots (Breckle, 1991). The combined stress of Cd and Pb presence

also reduced shoot metal concentrations of both metals (Breckle, 1991). These findings

suggest that there may be a critical limit after which the presence of Cd may inhibit any

significant uptake of Pb by the plant. Further studies into the relationship between Pb

uptake and increased soil-Cd concentration may aid in determining the efficiency of Pb

uptake on Cd contaminated soil, and emphasize the need for advanced understanding of

contaminant interactions.

Interactions between soil elements irrespective of Pb may have also influenced the

growth responses of switchgrass grown in highly contaminated soil. At high

concentrations, Cd and Cu have synergistic interactions, greatly increasing the combined

toxicity on barley, and similar results were obtained when bean (Phaseolus vulgaris) was

exposed to high levels of Cd and Zn (Wallace et al., 1977). Copper, however, had little

additive effect on plant growth in combination with zinc or nickel (Beckett and Davis,

1978). Interactions between the numerous elements present in the soil used in this

experiment likely affected the growth patterns and uptake ability of switchgrass in

response to Pb stress. Insight into specific phytotoxic effects of combined metal

contaminants on switchgrass could be determined through the growth of switchgrass on

soils deliberately contaminated with the individual elements and Pb, as well as more

complex combinations and treatment regimes.

4.6. Phytoextraction of Lead by Switchgrass

Previous studies have reported the phytoextraction of Pb from contaminated

growth media and translocation into harvestable tissues in numerous species, such as

broad beans, peas, common beans (Piechalak et al., 2002), onion, ragweed, Indian

75

mustard, pennycress, alpine pennycress (Huang and Cunningham, 1996), vetiver grass

(Chantachon et al., 2004), corn (Miller et al., 1977) and smilo grass (Garcia et al., 2004).

While concentrations of lead in aerial parts varied with species and treatment

concentration, vetiver grass (Vetiveria zizanioides) had shoot concentrations of

approximately 800 ppm Pb when grown on soil containing 11000 ppm Pb (Chantachon et

al., 2004), and smilo grass treated with 900 ppm Pb extracted 153 ppm Pb into shoot

tissue (Garcia et al., 2004); values which are comparable to Pb extraction by switchgrass.

Mean Pb concentration per plant (ppm) increased as the treatment concentration

increased for both root and shoot parts for studies 1, 2 and 3 demonstrating a consistent

relationship between availability and uptake. Switchgrass that was grown prior to the

onset of treatment (study 1) extracted higher concentrations of Pb per plant in both root

and shoot tissue than switchgrass that was treated from the time of seeding (studies 2 &

3), probably because of better establishment of functional root tissues. Mean percentage

of soil lead measured in switchgrass shoots for established plants ranged from 0.04% to

0.2% of the total accumulated applied Pb across treatments. Switchgrass treated from

time of seeding translocated between 0.001% and 0.018% after 12 weeks of growth.

Sowthistle treated with 3200 ppm Pb resulted in 6.7% of Pb solution in shoots (Xiong,

1997), and smilo grass extracted 21.7%, 16.9% and 50% of soil lead in shoots when

treated with 300, 900 and 1500 ppm Pb respectively (Garcia et al., 2004). In addition,

vetiver grass, a species with a fibrous root system similar to that of switchgrass,

demonstrates high efficiency in taking up certain heavy metals, and reportedly extracted

up to 40% of Pb treatment into its roots (Chantachon et al., 2004). These results suggest

76

that switchgrass may be able to extract Pb at rates competitive with those displayed by

other species over an extended period of time.

Leaf Pb accumulation increases with age and increased size. Higher

concentrations of Pb have been measured in senescing leaves while comparatively

minimal values were recorded in young leaves (Sharma and Dubey, 2005). Switchgrass

that was grown for seven weeks prior to treatment may have benefited from having more

mature leaves and developed roots systems, allowing for a higher average ratio of the

treatment to be accumulated in plant tissues.

Though switchgrass treated with Pb solution at concentrations between 75.8 and

10000 ppm translocated Pb into harvestable tissues, the concentration of Pb in plant roots

was greater than in shoots for almost all treatments across studies 1, 2 and 3. Similarly,

Begonia et al. (1998) found that approximately 10 times as much lead accumulated in the

roots of Indian mustard (Brassica juncea L.) than in shoot tissues. Lead readily forms

complexes with phosphorous and carbon and is known to precipitate on the surface of

roots, in cell walls (Huang and Cunningham, 1996; Sharma and Dubey, 2005) and in the

intercellular spaces of root cells (Tung and Temple, 1996). The formation of insoluble

Pb precipitates in root tissue may explain the relatively low transfer of Pb into shoot

tissues when compared with root uptake (Begonia et al., 1998).

Metal interactions likely influenced the amount of Pb uptake by plants grown on

brownfield soil as well as the uptake of other soil nutrients. ICP analyses revealed that

switchgrass grown on brownfield soil was able to translocate Pb out of the roots, but did

so to a lesser extent than plants which received applied Pb treatments. Concentrations of

Pb in shoot tissue ranged from 0.001 to 0.007% of total soil-Pb content in switchgrass,

77

with the percentage of soil-lead translocation varying across treatment groups. While the

applied treatments in studies 1-3 in the form of Pb acetate allowed for a consistently high

availability of Pb in the growth medium, the fraction of Pb available in the brownfield

study would decrease as Pb was transferred into plant tissues.

Switchgrass grown on soil containing approximately 36100 ppm Pb translocated

a greater percentage of soil-lead into aerial parts per plant than was measured in root

tissue. This finding was contrary to most previous findings, in which a larger proportion

of Pb was found in the roots than in shoots. This variation in results may have been due

to the significant growth inhibition observed in the 36100 ppm treatment group, as plants

may have reached the maximum functioning capacity in root tissue, necessitating

continued transfer into shoot tissues.

While the concentration of Pb in shoot tissues increased with increasing soil-lead

concentration, root Pb concentration varied across brownfield treatments with the highest

concentration of Pb observed in the roots of plants grown on soil containing 23600 ppm

Pb. Interactions have been observed between Pb and various other elements, as Pb

readily forms synergistic relationships with elements, mimics essential elements such as

magnesium, calcium and zinc, and forms complexes with phosphorous and sulfides

(Silviera and Sommers, 1977; Breckle and Kahle, 1992; Fargašová, 1994; Huang and

Cunningham, 1996; Miller et al., 1997; Seregin et al., 2004; Sharma and Dubey, 2005).

Such interactions may lead to the formation of Pb-rich pockets within the soil, changing

the amount of plant-Pb exposure on the small scale, despite a measurable mean total Pb

concentration of the soil sample. While the effects of these interactions on plant uptake

78

should be considered species specific, they may have played a role in limiting the Pb

uptake by switchgrass.

Lead ions may compete for cation exchange at calcium uptake sites in roots

(Wierzbicka, 1998). Cation exchange results from the dissociation of pectin and

hemicellulose components which, once separated, may bind to cations allowing transport

into the cell (Wierzbicka, 1998). Dissolved ions entering plant roots generally move

apoplastically in a radial direction across the cortical cells of roots following the water

potential gradient (Punz and Sieghart, 1993; Sharma and Dubey, 2005). Transport

through the symplast is necessary for translocation through the vasculature and on, into

the above ground tissues (Sharma and Dubey, 2005). As apoplastic movement into the

stele is blocked by the suberized walls of the Casparian strip, failure to pass through the

symplast of the cell is likely to lead to the accumulation of Pb in the cortex of roots (Lane

and Martin, 1977; Sharma and Dubey, 2005).

Alternative methods of transport into vascular tissue through lateral roots, and in

the root tip apical zone where the endodermis has not fully developed, have also been

proposed (Punz and Sieghardt, 1993). High concentrations of Pb may also interfere with

the selective functioning of plasmalemma and tonoplast permeability (Sharma and

Dubey, 2005). While Pb ions move through the apoplast at low concentrations, the barrier

function of the plasmalemma may be disrupted at high concentrations allowing increased

Pb transport into cells (Sharma and Dubey, 2005).

The presence of Pb at much higher concentrations in roots than in shoots in these

studies suggests the presence of more than one form of Pb within the plant. The

occurrence of two chemical forms of Pb within the roots of corn has been suggested; an

79

immobile, insoluble form which is more likely to remain in plant roots, and a soluble

active form which, while able to translocate more readily through the plant, represents a

much smaller fraction of the total plant lead concentration (Malone et al., 1974).

While Pb uptake into switchgrass shoots may have been hindered by low Pb-

availability, structural barriers such as the Casparian strip and the formation of complexes

with other elements, switchgrass extracted Pb from soil at rates comparable to those

previously reported in the literature in both root and shoot tissues.

4.7. Effects of Lead on Shoot and Root Cell Structure

Changes in the appearance of cells within root and shoot tissue of switchgrass

treated with high concentrations of Pb (6000, 10000, 13800 and 36100 ppm) may be due

to the physical deposition of Pb within cells and cell walls, as well as the interference of

Pb with cellular metabolism (Malone et al., 1974; Lane and Martin, 1977; Wierzbicka,

1987; Punz and Sieghardt, 1993).

Dark deposits were observed within the stele of switchgrass roots treated with

10000 ppm Pb acetate solution (Figure 24). Precipitation of Pb or the formation of

crystalline structures may have occurred at high concentrations within the plant such as

those recorded in established switchgrass treated with 10000 ppm Pb. Malone et al.

(1974) reported the presence of lead crystals in corn roots, and vacuolization of heavy

metal contaminants has been identified as a method of stress tolerance and cell

detoxification (Piechalak et al., 2002; Sharma and Dubey, 2005). In corn roots,

crystalline lead deposits were enclosed in vesicles upon passage into the symplast. The

vesicles were then enclosed in cell wall material either through exudations by the vesicle

80

itself or through movement to the periphery of the cell followed by incorporation into the

cell wall (Malone et al., 1974).

Deposits similar in appearance to those observed in root tissues were observed in

the leaf cells of study 1, 10000 ppm switchgrass plants. Though there is limited literature

on the cellular effects of Pb on shoot tissues, the proximity of the deposits to the vascular

bundles suggests that it is likely in a form similar to that found in the roots. Malone et al.,

(1974) found crystals in the stems and leaves of corn in every cell type except phloem

sieve tube elements. Binding of Pb within vesicles or through incorporation into the cell

wall may immobilize Pb and prevent the onset of phytotoxicity (Seregin et al., 2004).

Further analysis using Transmission Electron Microscopy, or histochemical analysis

using fresh tissues, may provide a more clear depiction as to the encapsulation

mechanism taking place in switchgrass cells.

Transport of lead into the cell symplast is necessary in order for transport into the

vascular tissue and aerial plant parts to occur. Transfer into the cell may occur through

the disruption of plasmalemma function and the loss of the integrity of that barrier

(Seregin et al., 2004; Sharma and Dubey, 2005). High concentrations of Pb may cause

cell injury, increasing plasmalemma permeability and allowing Pb to be transported into

the cell with greater ease (Seregin et al., 2004). This form of cell damage has primarily

been observed at toxic levels of contamination, which may explain the limited presence

of visible deposits observed in lower treatment concentrations. Fixatives were made in

both a potassium phosphate buffer and sodium cacodylate buffer so as to identify the risk

of reactions between Pb and phosphorous. There did not appear to be any significant

precipitates formed using the potassium phosphate buffer. Lead transport across the

81

plasma membrane may occur through voltage-gated calcium channels in the plasma

membrane of root cells (Sharma and Dubey, 2005). Following transport into the stele, Pb

ions likely return to apoplastic transport so that the hydraulic gradient of the transpiration

stream may be used to facilitate transport into aerial tissues (Punz and Sieghardt, 1993).

Changes in vessel diameter were observed in switchgrass exposed to high lead

concentrations, relative to control roots. Shoot node and leaf tissues of switchgrass

treated with high Pb concentrations produced compact vascular bundles in which the

vessels themselves, particularly xylem, appeared smaller in diameter than those of control

plants. Decreased vessel diameter has been attributed to heavy metal toxicity in wheat

(Barcelo and Poschenreider, 1990) which may account for decreased vessel size observed

in switchgrass. Smaller vessel size has also been linked to decreased water carrying

capacity and a drop in transpiration rate under heavy metal stress (Kosobrukhov et al.,

2004). The vascular bundles were also more densely arranged in treated plants than in

control tissues which may be a secondary symptom of Pb deposition leading to the

observed smaller vessel size. Increased vessel thickness may have resulted in vessel

rigidity preventing the expansion of the cells (Seregin et al., 2004).

Xylem elements appeared to have thickened cell walls in all studies, which may

be due to a build up of polysaccharides in the cell wall (Wierzbicka, 1998) or the

deposition of additional cell wall material in order to accommodate the fusion of Pb with

the cell wall (Malone et al., 1974). The increased thickness through polysaccharide

deposition may be a defence mechanism allowing the plant to accumulate higher

concentrations of Pb without experiencing toxic effects (Wierzbicka, 1998). Further

analysis of the cell ultrastructure could be performed using Transmission electron

82

microscopy, while the identification of Pb deposition within cell walls may be identified

using radioisotope tagging.

4.9. Conclusions

The results of this study provide important insight into the feasibility of using

switchgrass in a phytoremediation role in combination with its growth as a biomass

feedstock. Switchgrass successfully translocated lead into harvestable tissues at all

treatment concentrations used in this investigation.

While symptoms of phytotoxicity were observed at high treatment concentrations,

there were few reportable changes in biomass output, shoot and root development, or

plant establishment in plants treated with lower concentrations of Pb solution.

Switchgrass grown on soil treated with low to moderate concentrations of Pb acetate in

Hoagland’s solution, extracted Pb from the soil and translocated it into harvestable tissues

without significant loss of biomass production.

Switchgrass grown on lead-contaminated brownfield soil displayed few toxic

symptoms in both root and shoot development relative to control plants for all

concentrations except those grown on soil containing 36100 ppm Pb. These findings

suggest that switchgrass could be grown successfully on soil containing moderate

concentrations of Pb contamination as part of a more complex mixture.

Under treatment conditions switchgrass was highly successful in accumulating

and transporting the contaminant to aerial tissues, with mean uptake percentages

comparable to previous studies. Established plants were most successful, and presented

the fewest symptoms of toxicity in comparison with those treated from seed. While plants

treated with low concentrations of Pb presented few visible changes to growth patterns, a

83

decrease in time required to complete the lifecycle was observed. Switchgrass grown on

brownfield soil translocated smaller percentages of Pb into shoots than the applied study

plants. Research into using a chelating agent to facilitate uptake may increase the

percentage of uptake in situ and decrease the time needed to perform significant site

remediation.

The presence of darkly staining bodies in the root and shoot of switchgrass treated

with 10000 ppm Pb indicates a specific ultrastructural response in the plant to Pb

exposure and may suggest that there is a critical limit up to which switchgrass is able to

accumulate Pb within the cell wall. Further studies aimed at identifying the

compartmentalization mechanisms of these deposits should be performed. The absence

of darkly staining bodies in switchgrass treated for the duration of the growth period may

indicate plant tolerance to Pb at lower internal concentrations. The thickening of cell

walls also suggests the deposition of Pb within the apoplast of plant tissues. The presence

of Pb in the apoplast may hinder the movement of macronutrients and micronutrients,

interfering with the energy metabolism of the plant while cell growth may be hampered

by the added rigidity of the cell walls.

84

4.10. Future Directions

As the biomass study and qualitative analysis of root growth have suggested that

high concentrations of Pb may lead to stunted roots or root death, problems may arise

when trying to establish a switchgrass crop over a number of years at high lead

concentrations, including applied treatments greater than 8000 ppm and brownfield

contaminations of approximately 30000 ppm. Growth of switchgrass in soil

contaminated with an increasing Pb concentration gradient could identify the critical limit

of exposure in heterogeneous contaminant situations.

Changes in development pattern, and biomass production time, warrant further

research into the factors affecting shoot development, the associated plant responses to

nutrient deficiencies, and the longevity of switchgrass on highly contaminated soil.

Identification of the fraction of various Pb compounds, such as lead phosphate and lead

carbonate, present in plant tissues may help to identify the specific sources of observed

nutrient deficiencies as well as the mobile forms of Pb within plant tissues. Further to

that, soil analysis to determine the fraction of available Pb in contaminated soil and the

chemical form in which the Pb is present, may aid in determining the form in which Pb is

most readily taken up by the plant.

Seed viability and the effects of Pb on early seeding and biomass output should

also be investigated further. Seed size, weight and viability may be used as indicators of

changes to the metabolic function of switchgrass exposed to Pb. Changes in growth and

reproductive patterns, such as decreased seed dormancy, may be indicative of changes to

nutrient assimilation, energy allocation, and hormone function. By determining the

85

metabolic cause of morphological variations, the effects of Pb toxicity may be more

clearly identified.

Histochemical studies using fresh tissues may provide insight into the specific

location of lead within the cell. Lead-staining using sodium rhodizonate was not visible

in tissues which were post-fixed in preparation for light microscopy in this investigation.

Further work using fresh tissue and confocal microscopy may yield results. In addition,

radioisotope tagging using 210Pb, could be used to investigate the location and chemical

form of Pb as it travels through plant tissues, and may provide a more clear understanding

of how switchgrass reacts to the presence of Pb. Electron Microprobe Analysis could be

performed using tissues containing higher concentrations of Pb which have not been post-

fixed using osmium tetroxide, in order to identify the plant tissues or structures in which

Pb is deposited. Metabolic changes as well as reasons for observed changes in cell

distribution and development should be considered. Transmission electron microscopy

could be used to determine quantitative measurements of cell wall thickness and vessel

diameter, as well as the compartmentalization mechanism of the observed dark staining

deposits.

Changes to chloroplast structure and number in plants exposed to high

concentrations of Pb may indicate plant response mechanisms, leading to the inhibition of

chlorophyll production due to Pb stress. Spectrophotometry analysis of pigments and

chloroplast content in addition to electron microscopy may be used to determine

structural changes as well as changes to chlorophyll content.

Comparative studies may be used to investigate the metal specific interactions

between Pb and other essential and non-essential elements. Identifying the species

86

specific responses to elemental interactions should provide an understanding of plant

responses in naturally occurring soil matrices.

Finally, variations in growth and biomass production of various switchgrass

cultivars should be considered when selecting an appropriate variety for use in

phytoremediation. Changes to growth patterns in response to Pb contamination may vary

between cultivars, and optimization of characteristics such as biomass output, ash

content, and susceptibility to stress may increase efficiency of phytoextraction while

ensuring the production of a valuable energy feedstock.

87

SUMMARY

1. Exposure to high concentrations of Pb was associated with reduction in plant

height, root stunting, chlorosis and pigmentation change in switchgrass. Changes

in plant appearance are thought to be the result of nutrient deficiencies and an

ionic imbalance resulting from Pb interference with ion uptake as well as Pb

accumulation.

2. Switchgrass growth regime is affected by high Pb concentrations. High Pb

concentrations induce early onset of maturation and seed production, indicating

stress responses to Pb uptake. This early senescence in lead-stressed plants may

be the result of the inactivation of enzymes such as PEPC and peroxidase.

3. Switchgrass is able to extract and translocate Pb into aerial tissues at rates

comparable to those reported in previous studies.

a. Switchgrass which is allowed to grow before Pb exposure presents fewer

signs of phytotoxicity after exposure relative to plants treated from seed.

Older, established plants are likely able to extract and withstand greater

concentrations of Pb since the older, developed fibrous root system would

have a greater capacity for both extraction of, and tolerance for the

contaminant. Higher concentrations of Pb have also been measured in

leaves approaching senescence than in young leaves.

b. Lower concentrations of Pb in shoots relative to roots may be due to the

formation of insoluble Pb precipitates in root tissues.

c. Metal uptake by switchgrass on brownfield soil was lower than in applied

treatments. Variation in plant Pb concentrations may be due to

88

antagonistic and/or synergistic interactions between the various

contaminants in the soil matrix.

4. Switchgrass treated with high concentrations of Pb (13800, 10000 ppm) contained

dark staining bodies in the stele. Dark deposits were also found in leaf tissues.

Encapsulation of Pb contaminant in vesicles may be a defence mechanism of the

plant at high internal concentrations of Pb.

5. Light microscopy of Pb treated plants revealed the thickening of cell walls,

smaller vessel diameter and increased density of vascular bundles relative to

control plants. Smaller cell size and cell wall thickening may be due to Pb

deposits in the cell wall as well as increased polysaccharide content.

89

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97

Appendix 1 - Hoagland’s Nutrient Solution

Mol weight Stock (g/L) Stock (mM) Macronutrients KH2PO4 136.09 70 0.5 K2HPO4 174.18 20 0.5 K2SO4 174.3 87.5 1 MgSO4·7H2o 246.48 120 0.5 MgCl2·H2O 203.22 100 0.5 CaCl2·2H2O 147 220 0.5 Fe Sequestrine 550 20.7 1 Micronutrients MnSO4·4H2O 169 3.870 0.5 CuSO4·5H2O 249.8 0.500 0.5 ZnSO4·7H2O 287.54 0.580 0.5 H3BO3 61.83 3.830 0.5 Na2MoO4·2H2O 241..95 0.242 0.5 CoSO4·7H2O 272.00 0.118 0.5 NO- Modifications [Final Solution] Stock (g/L) KNO3

- 5.0 mM 0.50555

98

Appendix 2 - Sample of Randomized Growth Chamber Layout

Study 1 Study 2

3 7

10 2 9

4

8 3

1

7

10 2 9

6 5 1 5 4 6

8

Plant number assignments:

Pot Number Treatment1 Control 2 Control 3 75.8 ppm 4 75.8 ppm 5 1200 ppm 6 1200 ppm 7 6000 ppm 8 6000 ppm 9 10000 ppm10 10000 ppm

NOTE: Arrangement taken from a random number table

99

Appendix 3 – Study Summary

•Sw

itchg

rass

gro

wn

on b

row

nfie

ld s

oil f

or 1

2 w

eeks

•Gro

wn

on s

oil c

onta

inin

g: 5

900,

687

5, 1

3800

, 236

00, 3

6100

•Inve

stig

atin

g re

spon

ses

to P

b co

ntam

inat

ion

in re

al s

oil,

and

inth

e pr

esen

ce o

f oth

er

cont

amin

ants

6

•Sw

itchg

rass

gro

wn

until

see

ds d

evel

oped

for e

ach

treat

men

t gro

up•T

reat

men

t Ser

ies:

75.

8, 1

200,

600

0, 1

0000

ppm

Pb

acet

ate

•Inve

stig

atin

g ef

fect

s of

Pb

on th

e le

ngth

of g

row

th c

ycle

and

see

d pr

oduc

tion

5

•Sw

itchg

rass

gro

wn

over

8 w

eeks

and

har

vest

ed w

eekl

y•T

reat

men

t Ser

ies:

600

0, 7

000,

800

0, 9

000,

100

00•A

imed

at d

eter

min

ing

effe

cts

of P

b on

ear

ly g

row

th a

nd b

iom

ass

prod

uctio

n4

•Sw

itchg

rass

gro

wn

for 5

wee

ks, t

reat

ed fo

r ent

ire 5

wee

k pe

riod

•Tre

atm

ent S

erie

s: 7

5.8,

120

0, 6

000,

100

00 p

pm P

b ac

etat

e•In

vest

igat

ing

grow

th o

f sw

itchg

rass

from

tim

e of

see

ding

, with

trea

tmen

t per

to s

tudy

1io

d eq

ual i

n le

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3

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itchg

rass

gro

wn

for 1

2 w

eeks

, tre

ated

for e

ntire

12

wee

k pe

riod

•Tre

atm

ent S

erie

s: 7

5.8,

120

0, 6

000,

100

00 p

pm P

b ac

etat

e (1

0000

ppm

di

•Inve

stig

atin

g gr

owth

of s

witc

hgra

ss fr

om ti

me

of s

eedi

ng, w

ith g

row

th p

erio

dst

udy

1

ed) equ

al in

leng

th to

2

•Sw

itchg

rass

gro

wn

for 1

2 w

eeks

, tre

ated

for l

ast 5

wee

ks•T

reat

men

t Ser

ies:

75.

8, 1

200,

600

0, 1

0000

ppm

Pb

acet

ate

•Inve

stig

atin

g gr

owth

of e

stab

lishe

d pl

ants

1

Met

hod

and

Purp

ose

Stud

y

•Sw

itchg

rass

gro

wn

on b

row

nfie

ld s

oil f

or 1

2 w

eeks

•Gro

wn

on s

oil c

onta

inin

g: 5

900,

687

5, 1

3800

, 236

00, 3

6100

•Inve

stig

atin

g re

spon

ses

to P

b co

ntam

inat

ion

in re

al s

oil,

and

inth

e pr

esen

ce o

f oth

er

cont

amin

ants

6

•Sw

itchg

rass

gro

wn

until

see

ds d

evel

oped

for e

ach

treat

men

t gro

up•T

reat

men

t Ser

ies:

75.

8, 1

200,

600

0, 1

0000

ppm

Pb

acet

ate

•Inve

stig

atin

g ef

fect

s of

Pb

on th

e le

ngth

of g

row

th c

ycle

and

see

d pr

oduc

tion

5

•Sw

itchg

rass

gro

wn

over

8 w

eeks

and

har

vest

ed w

eekl

y•T

reat

men

t Ser

ies:

600

0, 7

000,

800

0, 9

000,

100

00•A

imed

at d

eter

min

ing

effe

cts

of P

b on

ear

ly g

row

th a

nd b

iom

ass

prod

uctio

n4

•Sw

itchg

rass

gro

wn

for 5

wee

ks, t

reat

ed fo

r ent

ire 5

wee

k pe

riod

•Tre

atm

ent S

erie

s: 7

5.8,

120

0, 6

000,

100

00 p

pm P

b ac

etat

e•In

vest

igat

ing

grow

th o

f sw

itchg

rass

from

tim

e of

see

ding

, with

trea

tmen

t per

to s

tudy

1io

d eq

ual i

n le

ngth

3

•Sw

itchg

rass

gro

wn

for 1

2 w

eeks

, tre

ated

for e

ntire

12

wee

k pe

riod

•Tre

atm

ent S

erie

s: 7

5.8,

120

0, 6

000,

100

00 p

pm P

b ac

etat

e (1

0000

ppm

di

•Inve

stig

atin

g gr

owth

of s

witc

hgra

ss fr

om ti

me

of s

eedi

ng, w

ith g

row

th p

erio

dst

udy

1

ed) equ

al in

leng

th to

2

•Sw

itchg

rass

gro

wn

for 1

2 w

eeks

, tre

ated

for l

ast 5

wee

ks•T

reat

men

t Ser

ies:

75.

8, 1

200,

600

0, 1

0000

ppm

Pb

acet

ate

•Inve

stig

atin

g gr

owth

of e

stab

lishe

d pl

ants

1

Met

hod

and

Purp

ose

Stud

y

100

Appendix 4 - 30 Element Analysis of Brownfield Soil and Soil Dilutions Results of 30-element analysis for Brownfield soil used in Study 6, showing mean soil element concentrations (n=2-3).

[Ag] (ppm) [Al] (ppm) [As] (ppm) [B] (ppm)[Ba]

(ppm)[Be]

(ppm) [Ca] (ppm)16.86 9540.1 393.76 66.51 11.71 <4 11212

[Cd] (ppm) [Co] (ppm) [Cr] (ppm) [Cu] (ppm) [Fe] (ppm) [K] (ppm) [Mg] (ppm)35.69 47.74 324.99 6658.66 191664 1474.1 2375.5

[Mn] (ppm) [Mo] (ppm) [Na] (ppm) [Ni] (ppm) [P] (ppm)[Pb]

(ppm) [S] (ppm)1164.35 48.43 1369.3 664.98 641.72 36105 16375

[Sb] (ppm) [Se] (ppm) [Sn] (ppm) [Sr] (ppm) [Ti] (ppm) [Tl] (ppm) [U] (ppm)10829 <10 1631.70 106.49 311.18 1.21 26.33

[V] (ppm) [Zn] (ppm)36.21 2557.73

101

Appendix 4 - 30 Element Analysis of Brownfield Soil and Soil Dilutions

In order to produce dilutions of the brownfield soil, the soil which contained 36100 ± 2975 ppm Pb was mixed with Promix BX on a per volume basis, and the concentration of the resultant soil mixture was determined on a mass basis. The percent change in mass between the original soil and the Promix/brownfield soil mixture was determined and that value was used to calculate the mean soil concentration for the soil dilutions. For example, if a 50:50 mixture of soil and Promix was produced, while the soil alone had a mass of 21.63 g and an equal volume of soil/Promix 50:50 dilution had a mass of 14.139 g, the following equation,

[ ] [ ]soilm

mdilution

soil

dilution ⋅=

was used to obtain the mean Pb concentration of 23600 ppm Pb in the diluted soil mixture.

Mean soil [Pb] in Brownfield soil and dilution based on mass ratios

SampleSoil Pb

reading (ppm)Mean total soil

[Pb] (ppm)Standard Deviation

1 32800.002 38573.70 36104.63 2976.163 36940.20

Expected [Pb]% Brownfield

soil Sample Mass (g)

Mean Calculated

[Pb]Standard Deviation

36100 100 21.63 36100 297618050 50 14.14 23600 19459025 25 8.27 13800 11384513 12.5 4.12 6875 5672256 6.25 3.55 5926 489

102

Appendix 5 - Recipes for Buffer, Fixative and Resin

0.025M potassium phosphate buffer 1. Weigh out 4.35g of K2HPO4 and 3.85g of KH2PO4 into a II flask 2. Add double distilled water, and mix well at room temperature 3. Adjust the pH with 0.1N HCl to 6.8 for plant tissues 4. Store in the refrigerator for up to three weeks. Bring to room temperature before

using. 0.05M sodium cacodylate buffer 1. Weigh out 5.4g of sodium cacodylate into a 500mL flask. 2. Add 500mL distilled water and mix thoroughly at room temperature 3. Adjust the pH to 6.8 with 0.1N HCl for use with plant tissues. Modified Karnovsky’s fixative – full strength 1. Add 2g of paraformaldehyde to 25mL distilled water in a 125 mL Erlenmeyer flask. 2. Cover the flask with foil while heating and stirring. Add a few drops of 10% sodium

hydroxide. Heat until gently boiling, at which point the solution should become clear. 3. Cool and add 5mL of 50% glutaraldehyde. 4. Bring the volume up to 50 mL with buffer. Epon/Araldite Rein Araldite 6.04g Epon 8.17g DDSA 15.6g DMP-30 0.63g Literature Cited Newcomb W, Wood SM. 1985. Methods in Transmission Electron Microscopy Laboratory Manual. Queen’s University, Kingston, ON, pg 7.

103

Appendix 6 -Mean Pb Concentration in Growth Media, Nutrient Solution, Seed stock and Control Plants

Mean [Pb] (ppm)20.245.0

Promix BX 5.24Switchgrass seed stock 6.32

Mean [Pb] (ppm) per plantStudy 1 Control Root 132 ± 9.2

Control Shoot 186 ± 27Study 2 Control Root 107 ± 23

Control Shoot 84 ± 8.5Study 3 Control Root 2.9 ± 0.26

Control Shoot 11 ± 2.8Study 6 Control Root 5.3 ± 2.9

Control Shoot 9.5

Hoaglands Sample 1--taken on October 2 2006Hoaglands Sample 2--taken on October 11 2006

104

Appendix 7 - Mean Percentage of Elements Taken up by Switchgrass from Brownfield soil

Sample# Treatment [Ag] [Al] [As] [Cd] [Co] [Cr] [Cu] 3.00 5900 Shoot <RL <RL <RL <RL <RL <RL 0.0035%4.00 5900 Root <RL <RL <RL <RL <RL <RL 0.0018%5.00 6875 Shoot <RL <RL <RL <RL <RL <RL 0.0011%6.00 6875 Root <RL 0.007% 0.003% 0.030% <RL <RL 0.0034%7.00 13800 Shoot <RL <RL <RL <RL <RL <RL 0.0003%8.00 13800 Root <RL 0.004% 0.017% 0.005% <RL <RL 0.0004%9.00 23600 Shoot <RL <RL <RL <RL <RL <RL 0.0011%10.00 23600 Root <RL 0.000% 0.034% 0.004% <RL <RL 0.0003%11.00 36100 Shoot <RL <RL <RL <RL <RL <RL 0.0000%12.00 36100 Root <RL <RL <RL <RL <RL <RL 0.0000%

Sample# Treatment [Mo] [Ni] [Pb] [S] [Sb] [Se] [Si] 3.00 5900 Shoot <RL <RL 0.001% 0.118% <RL <RL <RL4.00 5900 Root <RL <RL 0.001% 0.034% <RL <RL <RL5.00 6875 Shoot <RL <RL 0.001% 0.032% <RL <RL <RL6.00 6875 Root <RL <RL 0.007% 0.032% 0.002% <RL <RL7.00 13800 Shoot <RL <RL 0.0004% 0.011% <RL <RL <RL8.00 13800 Root <RL <RL 0.001% 0.005% 0.000% <RL <RL9.00 23600 Shoot <RL <RL 0.0002% 0.004% <RL <RL <RL10.00 23600 Root <RL 0.001% 0.001% 0.002% 0.000% <RL <RL11.00 36100 Shoot <RL <RL 0.0000% 0.0000% <RL <RL <RL12.00 36100 Root <RL <RL 0.0000% 0.0000% <RL <RL <RL

Sample# Treatment [Sn] [Sr] [Ti] [Tl] [U] [V] [Zn]3.00 5900 Shoot <RL <RL <RL <RL <RL <RL 0.043%4.00 5900 Root <RL <RL <RL <RL <RL <RL 0.015%5.00 6875 Shoot <RL <RL <RL <RL <RL <RL 0.012%6.00 6875 Root 0.002% <RL <RL <RL <RL <RL 0.013%7.00 13800 Shoot <RL <RL <RL <RL <RL <RL 0.003%8.00 13800 Root 0.000% <RL <RL <RL <RL <RL 0.002%9.00 23600 Shoot <RL <RL <RL <RL <RL <RL 0.002%10.00 23600 Root 0.000% <RL <RL <RL <RL <RL 0.002%11.00 36100 Shoot <RL <RL <RL <RL <RL <RL <RL12.00 36100 Root <RL <RL <RL <RL <RL <RL <RL

NOTE: Values below the reportable limit are marked as <RL

105

phytoextraction of lead from contaminated soil by panicum virgatum l

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