Petroleum coke and plants: Impact on growth and physiology

By:

Colin Keiji Nakata

A Thesis Submitted to the Faculty of Graduate Studies of

The University of Manitoba in partial fulfillment of the requirements for the degree of;

MASTER OF SCIENCE

Department of Botany University of Manitoba Winnipeg, MB., Canada

March 14th,2007

Copyright A 2007 by Colin Keiji Nakata THE TJNIVERSITY OF MANITOBA

FACULTY OF GRADUATE STT]DIES ****:* COPYRIGHT PERMISSION

Petroleum coke and Plants: Impact on growth and PhYsiolog¡r

BY

Colin Keiji Nakata

A Thesis/Practicum submitted to the Faculty of Graduate Studies of The University of

Manitoba in partial fulfillment of the requirement of the degree

MASTER OF SCIENCE

Colin Keiji Nakata @2007

permission has been granted to the Library of the University of Manitoba to lend or sell copies of this thesigpracticum,io the National Library of Canada to microfîlm this thesis and to lend or sell copies of túe film, aná to University Microfilms rnc. to publish an abstract of this thesis/practicum.

This reproduction or copy of this thesis has been made available by authority of the copyright owner solóty for the purpose of private study and research, and may only be reproduced and copied owner. as permitied by copyright laws or with express written authorization from the copyright l1

Ansrnacr:

Greenhouse studies were conducted to determine the effects of coke, a by-product

of the oil sand industry, on the emergence, growth and physiology of Triticum aestivum,

Deschampsia caespitosa, Calamagr-ostis canadensis, Agropyron trachycaulum,

Oryzopsis hymenoides, Fragaria virginiana and Cornus set"icea. Accumulation of

potentially toxic elements in plant tissues was also determined. Plants were grown in

peat-mineral mix (control) or coke produced by Slncrude Canada Ltd. or Suncor Energy

Inc. Coke had little effect on the emergence of grasses. In most cases, biomass,

chlorophyll a and chlorophyll b were lower in coke treated plants than in controls.

Transpiration rates of plants grown in coke either decreased or remained unchanged when compared with controls. In some cases, nickel, vanadium, boron and molybdenum were found at higher concentration in coke treated plants than in controls. The results suggested that plants growing in coke suffered from water stress, nutrient deficiency and potentially metal toxicity. Recommendations for revegetation of coke storage sites and future studies are made based on the results of these studies. ill

AcKNowLEDGMENTS:

First and foremost, I would like to thank my advisor Dr. Sylvie Renault. Thank

you for the time and effort that you invested in me to help me grow as a researcher. I

appreciate the experience gained from the conferences, workshops and meetings which

you made available to me during the past few years. I would also like to thank you for the

technical and morale support you provided when times were rough. J'ai beaucoup appris je et me sens bien preparé pour la vie dans le marché du travail. Encore une fois, merci

Sylvie.

Thank you to my committee members Dr. Mike Sumner and Dr. Tee Boon Goh as

well as ClataQualizza, Dr. Mike Mackiruron and Wayne Tedder for their input and

contributions towards this study and the completion of this thesis.

I would also like to extend a special thanks to Karen Kivinen, Carl Szczerski,

Scott Green, and Maha Afifi for moral and technical support. I am indebted to you for

your help during harvesting, which extended far too late into the night. I also appreciate

the time spent by Scott Boorman and the summer students at Syncrude who did their best

to keep my grasses alive in the hot, dry coke.

Thanks to Eric Hoffman and Greg Morden for their advice regarding the element

analysis of tissues.

Finally I would like to acknowledge Syncrude Canada Ltd., Suncor Energy Inc.,

Canadian Natural Resources Ltd. and NSERC for funding which made this project possible. 1V

We will be known forever by the tracks we leave -Dakota v

TABLE OF CONTENT

ACKNOWLEDGMENTS: vi

3.3.4. Transpiration and Stomatal Conductance ...... 39

3.3.6. Element Content: ...... 39

3.4.1. Seed Emergence in Coke...... 59 3.4.2. Water Status of Grasses Grown in Coke...... 60 3.4.3. Arsenic, Vanadium, Boron, Iron, Molybdenum and Nickel in Coke Treated Grasses...... 62 3.4.4. Species Comparison...... 70

CHAPTER 4 - GROWTH, PI{YSIOLOGY AND ELEMENT ACCUMULATION OF CONTVUS SERICEA AI.ID FRAGAKIA VIRGINIANI EXPOSED TO COKE ...... 72

4.1. INTRoDUCTToN: ...... 72 4.2. METHoDS ANDMATERTALS:...... 74 4.2.1. P\ant Material and Treatments ...... 74

4.2.3. Chlorophyll and Pheophytin...... 77 4.2.4. Transpiration ...... 78

4.2.6. Uptake of Elements ...... 7g 4.2.7. Data Analysis...... 79

4.3.1. Injury and Growth...... 79 4.3.2. Chlorophyll and Pheoplzytin...... 80 4.3.3. Transpiration ...... d1

4.3.5. Element Content: ...... 81

4.4.1. Nutrient Status ...... 94

4.4.1 .3. Macronutrients, Micronutrients Concentrations...... gg 4.4.2. Arsenic and VanadiumAccumulatton...... 102 4.4.3. l4tøter 5fatus...... 103 4.4.4. Species Comparisott...... 105

CHAPTER 5 _ CONCLUSIONS AND RECOMMENDATIONS...... 106 vii

Lrsr op T¿slns

(mean + Table 3.1: Root dry weight, shoot dry weight and roolshoot ratio SE) of Triticym aestivum (aftet 2 months), Deschampsia caespitosa, Calamagrostis (after 3 canadensis, Agropyron trachycaulutn, and oryzopsis hymenoides coke months) of grõ*th in peat-mineral mix (control), uncapped or capped the treatments. No signifiãant differences exist between means followed by 49 same letter (n : 5l u,: 0'05). """"""'

Table 3.2: chlorophyll a (chl a), chlorophyll b (chl b), chlorophyll a to + chlorophylt U rátio (Chl a/b) and carotenoid content (mean SE) of Triticum o s ti s c anadens is, A gr opyr o n ae s tivum, D e s c hamp s i a c a esp it o s a, C al amagr trachycaulum and oryzopsis hymenoides grown in peat-mineral mix (control), Syncrude .ãt", Syn.rude coke + cap, Suncor coke or Suncor coke + cap. Ño significant differences exist between means followed by the same 51 letter 1n : 5, o : 0.05). """"""""""" + root Table 3.3: Arsenic (As) and vanadium (v) concentrations (mean sE) in o s al o s t i s tissues of Triti cum a es tivum, D es champ s i a c a espit a, C amagr growing in canadensis, Agropyron trachycaulum and oryzopsis hymenoides peat-mineral mir(control), syncrude coke, syncrude coke * cap, Suncor coke or Suncor coke * cap. No significant differences exist between means followed by the same lettãr (n: 5, o: 0'05)' ""' 53 + shoot Table 3.4: Arsenic (As) and vanadium (V) concentrations (mean SE) in s t is tissues of Triti cum aes tivum, D es champ s ia c a esp it o s a, C al amagro growing in canadensis, Agropyron trachycaulum and Oryzopsis hymenoides peat-mineral mirlcontrol), SlTrcrude coke, Syncrude coke * cap' Suncor coke or Suncor coke t cap. No significant differences exist between means 54 followed by the same lettãr (n: 5' cr: 0'05)' ""'

Table 3.5: Element content (mean + SE) in root tissues of Triticum aestivum, is, A gropyro n D es champ s ia c a e spit o s a, c al amagro s tis c an a dens trachycaulum and-oryzopsis hymenoides growing in peat-mineral mix (contiol), Syncrude coke, Syncrude coke ¡ caP, Suncor coke or Suncor coke ì Ño significant differences exist between means followed by the same lettei"up. (n:5. o:0.05). --- denotes no available data"""" """"""""""" 55

Table 3.6: Element content (mean * SE) in shoot tissues of Trítícum aestivum, Deschampsia caespitosa, calamagrostis canadensis, Agropyron trachycaulum and-Oryzopsis hymenoides gfowing in peat-mineral mix + (contiol), Syncrude coke, Syncruds * cap, Suncor coke or Suncor coke same òap. No'significant differences exist between means followed by the 57 letìer (n : 5, o : 0.05). """"""""""" vll1

Table 4.1: chlorophyll a, chlorophyll b, pheophytin a andYo pheophytin (mean + SE) in Fragaria virginíana and Cornus sericea after 8 weeks of treatment. No significant differences exist between means followed by the same letter (a: 0.05, n: 5)...... 87

Table 4.2: Transpiration rates (mean + SE) of Cornus set"ícea and Fragaría virginiana growing in peat-mineral mix (control), syncrude coke or suncor coke. No significant differences exist between means followed by the same letter (a : 0.05, n: 5). .... 88

Table 4.3: Proline content (mean + SE) of F. virgíniana and C. sericea growing in peat-mineral mix (control), syncrude coke and Suncor coke at the time of harvest. No significant differences exist between means followed by the same letter (cr: 0.05, n: 5)...... 88

Table 4.4: Arsenic (As) and vanadium (V) concentrations (mean + SE) in root and shoot tissues of F. virginiana and C. sericea growing in peat-mineral mix (control), Syncrude coke and Suncor coke. No significant differences exist between means followed by the same letter (n: 5)...... 89

Table 4.5: Macro and micro nutrient concentrations (mean + SE) in root tissues of F. virginiana and C. sericea growing in peat-mineral mix (control), Syncrude coke or suncor coke. No significant differences exist between means followed by the same letter (o:0.05, n: 5)...... 90

Table 4.6: Macro and micro nutrient content (mean + SE) in shoot tissues ofF. virginíana and c. serÌcea growing in peat-mineral mix (control), Syncrude coke or suncor coke. No significant differences exist between means followed by the same letter (o : 0.05, n : 5)...... 92 ix

Lrsr oF FrcrrRES

Figure 3.1: Percent emergence (mean + SE) of Triticum aestiyam (4, B), Deschampsia caespitosa (C, D) and Calamagrostis canaderzsls (E, F) grown for 17 days in peat-mineral mix (control), Syncrude coke, Syncrude coke + cap, Suncor coke or Suncor coke + cap. * denotes significant difference from control at a.:0.05 (n : 5)...... 43

Figure 3.2: Percent emergence (mean t SE) of Agropyron trachycaulum (A,B) and Oryzopsis hymenoides (C, D) grown for l7 days in peat-mineral mix (control), Syncrude coke, Syncrude coke * câp, Suncor coke or Suncor coke + cap. * denotes significant difference from control at s : 0.05 (n : 5)...... 44

Figure 3.3: Shoot height (mean + SE) of Tritícum aestivum (4, B), Deschampsia caespitosa (C, D) and Calamagrostis canadensis (E, F) grown for 7 weeks (7. aestivum) or 12 weeks (D. caespitosa and C. canadensrs) in peat-mineral mix (control), Syncrude coke, Syncrude coke * cap, Suncor coke or Suncor coke * cap. All means are statistically different unless otherwise noted. Means followed by different letters represent significant difference at o, : 0.05 (n:5)...... 45

Figure 3.4: Shoot height (mean + SE) of Agropyron trachycaulum (A, B) and Oryzopsis hymenoides (C, D) grown for 12 weeks in peat-mineral mix (control), Syncrude coke, Syncrude coke * cap, Suncor coke or Suncor coke + cap. All means are statistically different unless otherwise noted. Means followed by different letters represent significant difference at ü:0.05 1n: s)...... 46

Figure 3.5: Transpiration rates and stomatal conductance (mean * SE) of Triticum aestit'am (4, B), Calamagrostis canadensis (C, D) and Agropyron trachycaulum (E,F) growing in peat-mineral mix (control), Syncrude coke, Syncrude coke * cap, Suncor coke or Suncor coke * cap. No significant differences exist between means followed by the same letter (n: 5, û, : o.os)...... 47

Figure 3.6: Proline content (mean + SE) of A) Triticum aestivum,B) Deschampsia caespitosa and C) Agropyron trachycaulum growing in peat- mineral mix (control), Syncrude coke, Syncrude coke + cap, Suncor coke or Suncor coke + cap at the time of harvest. No significant differences exist between means followed by the same letter (n: 5, a:0.05)...... 48

Figure 4. 1 : Shoot height (mean + SE) of Cornus sericea after 3 months of growth in peat-mineral mix (control), Syncrude coke or Suncor coke. Means followed by different letters represent significant difference (o : 0.05, n : 5)...... 85 Figure 4.2: Root dry weight, shoot dry weight and the roolshoot ratio (mean t SE) of ,Fra garia virginiana (4, C, E) and Cornus sericea (8, D, F) grown for 3 months in peat-mineral mix (control), Syncrude coke or Suncor coke. No significant differences exist between means followed by the same letter (o: 0.05, n : 5)...... 86 X1

Llsr or AppnNDrcES

AppENux A: LIsr or TaeL¡s

Table A.l: Aluminium (Al), arsenic (As), boron (B), barium (Ba), beryllium (Be), bismuth (Bi), calcium (ca), cerium (ce), cobalt (co), chromium (c.¡, (Cu), cesium (Cs), dysprosium (Dy), erbium (Er), europium (Eu), iron "opp",(Ée), gallium (Ga), gadolinium (Gd), hafnium (Hf), holmium (Ho), poiassiuÀ 1r¡, lanthanum (La), Lithium (Li), lutetium (Lu), magnesium Wfg), (Mn), -ungun.r" molybdenum (Mo), sodium (Na), niobir* (Nu), ana neoaymiim (Nd) concentrations in peat-mineral mix, Syncrude coke and Suncor ...... "ãt". 135 Table 4.2: Gravimetric water content, and volumetric water content of peat- mineral mix, S¡mcrude coke and Suncor coke...... l3l Table 4.3: Range of detection rimits for elements analyzedby ICp-oES...... r37

Table 4.4: Detection limits and method of analysis for macronutrients, micronutrients, arsenic and vanadium in plant tissues by inductively coupled plasma mass spectroscopy (ICP-MS), Inductively coupied plasma ôpticåt Emissions spectroscopy (ICp-oES), combustion chrómatography (òc) and Combustion Infrared (IR)...... l3g Table 4.5: As, B, Fe, K, Mo, Ni, s, Ti, and v concentrations uncorrected for peat- mineral mix/ coke contamination...... 139 Table 4.6: Element content data uncorrected for peat-mineral mix/ coke contamination...... 144 x1l

AppENorx A: Llsr op FlcuRps

Figure A.l: Storage of coke in a) coke cell and b) coke beach at the Mildred Lake mine site, Alberta, Canada. Diagram and photo courtesy of Syncrude Canada Lrd...... 127

Figure A.2: Triticum aestiwtm grown in peat-mineral mix (control), Syncrude coke, Syncrude coke * cap, Suncor coke or Suncor coke + V days "íp after seeding...... 12g

Figure A3 : Deschampsia caespitosa grown in peat-mineral mix (control), syncrude coke, syncrude coke + câp, suncor coke or suncor coke +'c¿p after 3 months...... 12g

Figure A.4: Calamagrostis canadensis gro\Ä/n in peat-mineral mix (control), Syncrude coke, Slmcrude coke + câp, Suncor coke or Suncor coke + óáp after 3 months...... 130

Figure A.5: Agropyron trachycaulum grown in peat-mineral mix (control), Syncrude coke, Syncrude coke + câp, Suncor coke or Suncor coke +'ç¿p after 3 months...... l3l

Figure A.6: oryzopsis hymenoides grownin peat-mineral mix (control), syncrude coke, Syncrude coke + câp, Suncor coke or Suncor coke + cap after j months' ...... 132 Figure A.7: Fragaria virginiana grownin peat-mineral mix (control), Syncrude coke or Suncor coke after 3 months...... 133 Figure A.8 : cornus sericea grown in peat-mineral mix (control), syncrude coke or Suncor coke after 3 months. xllr

AppENux B: Llsr or FlcuREs

Figure 8.1 : Setup of experimental plots at the coke beach, Mildred Lake Basemine, AB., Canada...... l5l

Figure B'2: Emergence of D¿s champsia caespitosa seeded direcly in coke at the coke beach, Mildred Lake Basemine, AÈ., Canada...... 152 CHAPTER 1 - INTnonUcTIoN

The oil sands in northern Alberta, Canada are one of the largest hydrocarbon

reserves in the world. Three major hydrocarbon deposits are found in northern Alberta,

namely the Athabasca, Cold Lake and Peace River deposits. Current estimates suggest

that northern Alberta contains over 300 billion barrels of oil, exceeding even the

conventional oil reserves of Saudi Arabia (George 1998). Petroleum companies such as

Syncrude Canada Ltd., Suncor Energy Inc., Canadian Natural Resources Inc. (CNRL)

and shell canada Ltd. are exploiting or planning on exploìting the Athabasca

hydrocarbon deposit (Jardine 1974, CNRL 2006, shell canada Ltd. 2006, eualizza

personal communication, Tedder personal communication).

Oil sands can be defined as sand in which the inter-granular spaces are filled by

an assemblage of clay, water, and bitumen (FTFC 1995). Bitumen is a high molecular

weight hydrocarbon which can be upgraded into synthetic crude oil. The bitumen present

in oil sands originates from the burial and decay of long-dead aquatic organisms. High temperatures and pressure alter the organic materials thereby decreasing the ratios of

oxygen-to-carbon and hydrogen-to-carbon (Tatsch lg74). once produced, bifumen can migrate from its origin by flowing laterally along slopes and/orbe pushed vertically by pressure produced by tectonic movements. In the case of the Alberta oil sands, the bitumen migrated upwards into overlying, porous sediments, namely sand (Hitchon

r97 t).

Separation of bitumen from the other oil sand components occurs via treatment with hot water, steam and sodium hydroxide (FTFC 1995). Bitumen must be upgraded prior to refining as its density exceeds 950 kg m-3 and therefore cannot be processed by 2

standard pipelines and refineries (National Energy Board 2004). Two methods, known as

hydrogen addition and carbon rejection, are used to upgrade bitumen. Carbon rejection

methods such as coking are predominantly used onsite to upgrade bitumen. Coking

utilizes heat to break C-C bonds in the hydrocarbon chains of heavy oil. This results in

the formation of shorter hydrocarbon chains and thus a lighter crude oil (Burnham l9g 1 , Hazletf et al. 1981). As the chemical composition of the heavy oils change, the fractions

resistant to thermal degradation precipitate as coke. Although coking is a safe, reliable,

and inexpensive method to upgrade bitumen, coking produces a considerable amount of a

waste by-product known as "coke" or "petroleum coke". Depending on the coking

system used, different types of coke are produced. For example, Syncrude Canada Ltd.

uses a fluid coker which produces a sandy textured coke whereas Suncor Energy Inc.

uses a delayed coker which produces a solid mass of coke. Heterogeneous shapes and

size of coke are produced during the mechanical decoking of a delayed coker.

It is estimated that over I billion m3 of coke will be produced over the life of

Syncrude Canada Ltd. Mildred Lake mine site. Coke has a potential value as a carbon

source and is currently being stockpiled until technologies or markets develop allowing

for economical use or sale of coke. Coke geochemistry, oxidation and potential toxicity to animals have been previously studied (Scott and Fedorak2004). However, the effects of coke on plants are still relatively unknown, even though many industries such as the petroleum industry, aluminium industry, some ammonia producers, some refineries, and even certain cement industries use or produce coke (Gary and Handwerk 19g4, Swain

1991). A research program aimed at determining the ecology of coke has been established to determine if stockpiled coke should be considered an environmental Ja

hazatd' Although current reclamation practices cap coke with a peat-mineral mix, the cap

depth is not homogenous and generally varies from 0.35 m to I m in depth (eualizza

personal communication). It is likely that plants will come into contact with coke as roots

can grow to depths exceeding that of the cap. Plants growing in coke may suffer from drought conditions due to the rapid drainage of water from coke through macropores. Furthermore, elements found in coke such as arsenic (As), boron (B), molybdenum (Mo),

nickel (Ni) and vanadium (V) are known to have ph¡otoxic effects on plants and

depending on the degree of accumulation, may be problematic to plants growing in coke

(Aller et al. 1990, FargaSovà 1998, Mitev a2002, Parida et al. 2003). The overall goal of this study was to determine the effects of coke on plants. The objectives were to:

determine 1) the emergence and survival of plants grown in coke

2) determine growth and physiological responses of plants grown in coke

3) determine the accumulation of As, B, Fe, Mo, Ni and V in root and shoot tissues

of plants grown in coke

A first experiment with 5 grass speci es (Triticum aestivum, Deschampsía caespitosa, calamagrostis canadensis, Agropyron trachycaulum, and oryzopsis hymenoides) and a second experiment with a forb (Fragaría virginiana) anda shrub species (Cornus sericea) were designed. A field experiment was also conducted at the

Syncrude Canada Ltd' mine site to determine if D. caespitosa, Koeleria macrantha, Elymus ínnovatus and Festuca saximontana couldbe established directly in coke. An outline of the field experiment is presented in Appendix B. These experiments were designed to provide information to make recommendations for reclamation of coke impoundment sites. CHAPTER 2 - LITnRaTURE Rrvrpw

2.1. INTnoDUCTIoN

With the increasing importance of oil sands as a source of fossil fuels, it can be

expected that the production of coke, a by-product of oil sands processing, will also increase. Currently, coke stockpiles are capped with soil and stabilized by plants;

however, environmental concerns arise as the ecology of coke is relatively unknown.

Coke can potentially release metals and hydrocarbons into the environment through

abiotic and biotic degradation. Understanding how plants respond to coke exposure is

important as it is likely that plant roots will penetrate the soil cap and grow into coke. The

focus of this review is to provide background information on potential problems related

to growth and establishment of plants in coke. This review addresses the production, use

and current research on coke followed by the chemical/physical effects coke may have on plants.

2.2. CoI

Coking is a pyrolytic process which converts low grade petroleum products into valuable petroleum distillate products (Hatch and Matar l98l). Thermal cracking or pyrolysis of "heavy" feedstock is the central reaction of the coking process. Coke feedstock consist of a variety of high morecular weight compounds including hydrocarbons' thermal tars, coal tar pitch, and/or vacuum residuals. pyrolysis of heavy feedstock involves dehydrogenation, dealkylation, condensation, hydrogen transfer and isomerization reactions which transform heavy hydrocarbons into shorter, more volatile 5

and hence more economically valuable products (Hatch and Matar 1981, Gary and

Handwerk 1984). Highly polar compounds such as asphaltenes are maintained in solution

by compounds of intermediate polarity such as resins. Thermal cracking of feedstock

depletes the resin fraction through cracking and volatili zationwhich results in the

precipitation of the asphaltene fraction. Furthermore, the coking process produces high

molecular weight free radicals which polymerize or condense into insoluble compounds.

The combination of asphaltenes and other insoluble components resistant to thermal

cracking produce a solid, carbonaceous by-product called coke or petroleum coke

(McNaught and Wilkinson 1998).

The first production of coke on an industrial scale was not from oil sands

processing but from primitive oil refineries distilling kerosene from crude oil (Ellis and

Paul 1998). As the heat distribution of the wood and coal powered stills was uneven, oil from the bottom of the still would overheat and undergo thermal cracking, leading to

coke production (Ellìs and Paul 1998). These early refineries required periodical de-

coking to retnove coke build-up from the stills. As the market demand for lighter

petroleum products (i.e. gasoline) increased, coking became a necessary procedure as it

allowed for production of desired distillates from otherwise undesired hydrocarbons.

Several coking methods are currently employed by various petroleum companies. A

coking method known as delayed-coking functions much like the aforementioned primitive stills and suffers from the down time associated with manual decoking. A newer method known as fluid coking removes coke as it is produced and can therefore be used continuously. 2.2.1. Dnr,ayno CoxrNc:

Delayed coking is currently used by Suncor Energy Inc. to upgrade bitumen into

synthetic crude oil. The basic operation of a delayed coker begins with feedstock entering

a fractionation column where the lighter fractions are removed. The "heavy,, feedstock is

pumped through a heater where it is partiall y vaponzed The hot liquid-vapour mixture is

emptied into the coke drum where the unvapourized portion of the feedstock settles to the

bottom. The combination of high temperature and long retention time pyrolyses the

hydrocarbons, thereby producing coke and lighter petroleum fraction such as gas,

naphtha and gas oils. The lighter fractions are removed from the top of the coke drum and

returned to the fractionation column where the desired fractions are collected (Gary and

Handwerk 1984). The "heavy ends" re-condense upon cooling and are recycled with

fresh feedstock into the coke drum. Once the coke drum is full, the solid mass of coke is

broken using a mechanical or a hydro drill and subsequently removed from the drum.

To circumvent the down-time associated with de-coking, delayed cokers utilize a

minimum of 2 coking drums which allows for one coke drum to be cooled and emptied

while others are in use (Ellis and Paul 1998). A major problem associated with primitive

cokers was the premature formation of coke in the feedstock supplylheater tubing which

would hinder the flow of heated feedstock into the coke drum. This was resolved by gradually heating feedstock and allowing it to enter the coke drum immediately after

attaining coking temperature thereby "delaying" coke formation until the appropriate time (Fletcher 1983). 2.2.2. Fluro CoxrNc:

Fluid coking is used by Syncrude Canada Ltd. to upgrade bitumen into s1'nthetic

crude oil. Feedstock is sprayed into the coker through nozzles in the coke drum wall

(Hammond et aL.2003). Thermal cracking occurs when sprayed feedstock comes into

contact with red-hot coke particles recycled from a previous cycle. Vaporized products

are removed and filtered while coke particles are retumed to the reactor. Accumulated

coke is removed from the reactor and a fraction of it is reheated and recycled to the reactor to maintain the reaction. Some of the coke produced is combined with air and combusted to produce heat for the thermal process decreasing the need for oil or gas heaters. Fluid coking burns approximately 20-25o/o of the coke produced in the reactor to produce the heat required for thermal cracking of heavy feedstock. Excess coke is removed from the system.

2.3. CoxB Usns aNo Sronacp

Depending on the quality, coke can be sold for use in the production of graphite or as a source of carbon for carbides and carbon graphite electrodes (anode). Coke with higher concentrations of impurities but low in sulphur can be used as fuels for refineries, the cement industry or ammonia production (Swain I 991 , Rossi 1993). At the Syncrude

Canada Ltd. Mildred Lake mine site, some coke is used as a fuel source to produce electricity, steam and hydrogen for the processing plant. Due to the geographical isolation of the cokers, elevated sulphur content and current market demands, sale of coke 8

produced by Syncrude Canada Ltd. and Suncor Energy Inc. was deemed unprofitable

(Scott and Fedorak2004). Coke is therefore stored onsite in coke cells or coke beaches (Figure A.l ) until future markets or technologies develop to increase the marketability of

coke.

2.4. Cor

Storage of coke in the landscape requires an understanding of how coke interacts

with the and biotic abiotic components of the environment. A few studies have been

undertaken to gain a better understanding of the ecology of coke in order to prevent

environmental damage. A report by Golder Associates Ltd. (1998) summ anzed, the findings of 4 independent studies which assessed the leachability of organics and

inorganic components from Syncrude and Suncor coke. Findings suggest that although

elements extracted from Syrcrude and Suncor coke through leaching was low, coke

leachate can negatively impact the quality of receiving waters. Water collected in and

around existing coke stockpiles contained chlorine (Cl), aluminium (Al), boron (B),

chromium (cr), copper (cu), arsenic (As), cadmium (cd), iron (Fe), manganese (Mn), and possibly lead (Pb) at concentrations which exceeded one or more regulatory guidelines (protection of aquatic life acuteichronic guidelines, human health carcinogen guidelines, and human health non-carcinogen guidelines) (Golder Associates Ltd. l99g).

Furthermore, a study by Squires and Liber (2003) reported that overall leachability of metals from Syncrude coke was low but that concentrations of Cu, manganese (Mn), molybdenum (Mo), and vanadium (V) exceeded the Canadian'Water euality Guidelines. A greenhouse revegetation study showed that Suncor coke was a poor growth medium for 22 grasses and 11 forb species (Scott and Fedorak 2004). An experiment growing barley (Hordettm wlgare) in coke has shown that coke could support plant growth if

supplemented with nutrients (Scott and Fedorak2004). Neither of these studies

emphasized the physiological effects of coke on plants. Prasad and Rao (l 981) tested the

effects of coke dust produced from a coke-calcination unit of the Barauni petroleum

Refinery (lndia) on the growth of Phaseolus aureus. The authors found that dusting

plants over 40 days with coke at arate of 2 gpetroleum --td-' resulted in increased leaf

temperature, increased transpiration over the short term, a decrease in transpiration over

the long term, decreased photosynthetic rates, and decreased N, p, K, s and ca

accumulation. Furthermore, plants dusted with coke exhibited lower protein and

carbohydrate content than undusted plants. It is suspected that heating of the soils and

leaves, pafüal occlusion of stomata, and shading of leaves due to the deposition of coke

was responsible for the altered growth and physiology of coke dusted plants (prasad and

Rao l98l). Current reclamation practices cap coke with 35 - 100 cm of soil; however,

due to the use of heavy machinery, the peat-mineral mix cap is not homogenous in depth

over reclaimed site. Furthermore, as plant roots can grow deeper than the reclamation

cap, they are likely to come into contact with stockpiled coke, particularly in locations

where the reclamation cap is thinner (Shaffer et aL. 1994). The effects of coke on plant

growth and physiology as well as the accumulation of elements from coke into root and

shoot tissues is largely unknown. Therefore, the responses of plants to petroleum coke

require further study.

2.5. CnrlucAr, EFFECTS oF Coxn oN PLANTS

coke is approximately 80% carbon (Kessler and Hendry personal communication). Depending on the bioavailability of the carbon, stockpiled coke has the l0

potential for a very elevated C/N ratio. Microorganisms can use the excess carbon for

growth but in doing so also sequester N. If the C/lr{ ratio exceeds 30, then any N released

from mineralization is immediately immobllizedby the micro-organisms (Adam and

Duncan 2002). As a result, the micro-organisms remove the N from the soil, leaving the

soil deficient in N. However, once the c/N ratio drops below 15, the rate of

mineralization exceeds the rate of immobilizationresulting in nitrogen being available for plant growth (USDA 1955). The principle of the C/lrtr ratio can be applied to any of the elements required for micro-organism growth and may pose special concerns for plant essential elements (8, ca, cl, cu, Fe, K, P, Mg, Mn, Mo, s and Zn). sulphur is another dominant component of coke, accounting for approximately 6.5Yo of coke. Thiophene sulphur, a carbon bonded sulphur, is the predominant form of sulphur in coke. Carbon bonded sulphur is not readily plant available, and must be mineralized to inorganic sulphate prior to plant absorption (Goh and Pamidi 2003). Although mostly carbon and sulphur, coke does contain other elements at concentrations which may be of concem to water quality, plant and animal life. Chung et aI. (1996) analyzed coke and found that As

(10 l8 pg g-'), B (r0 g8 - - rSpg g-'), Mo (31 - pg g-'), Ni (304 - 517 ¡tgg-r), and v (824

- ll66 pg g-') were present in coke at relatively high concentrations. Furthermore, polycyclic aromatic hydrocarbons (PAHs), many of which are carcinogenic or mutagenic, have been detected in coke in trace amounts (Suncor coke MSD S 2002). The potential for toxicity caused by the aforementioned elements will be discussed in the following sections. ll

2.5.1. ElnunNrs rN coKE

2.5.1.1. AnsnNlc (As)

The mean concentration of As in uncontaminated soils is generally below l0

Pg g-t (Leermakers et al. 2006). Arsenic is a relatively common contaminant in soil due

to its numerous commercial, industrial, mining and agricultural applications (Livesey and

Huang 1981, Schmögeret aL.2000, Leermakers et al. 2006). Several oxidation states

exist for As such as As*3 (arsenite) and As*5 (arsenate) which allow for a variety of , organic and inorganic forms (Leermakers et al. 2006). Under normal soil conditions As*s will be negatively charged (as H2AsO+- or HAsOo'-¡ while As*3 will be present as the uncharged H3AsO3 (Marin et al. 1993). Although both forms are present, arsenate is the predominant species of As in aerobic soils and arsenite dominating in soils subjected to reducing conditions (Takamatsu et al. 1982, Smith et al. 1998). The toxicity of As is highly dependent on its chemical form where elemental As is a non toxic form, arsenate is a form of intermediate toxicity and arsenite is the most toxic form (Miteva 1998,

Leermakers et al. 2006).

The mean As concentration in tissues of most plants species ranges from 0 to 3 pg g-t but is typically less than I pg g-' (Adriano 19g6, Munshower lgg4).Although arsenic is not essential for plant growth and development, plant exposure to low concentrations (<25 pg g-') has been found to increase root length, root and stem biomass, stem height, and photosynthetic pigment content (chlorophyll a, b, and carotenoids) in tomatoes (Lycopersicon esculentum). However, at higher concentrations

(>25 ltgg-'¡ ull the aforementioned physiological parameters were depressed (Miteva

1998, Miteva2002). Phyotoxicity of As is relatively well documented. Depending on the 12

plant species, exposure to 5 pg g-' to 50pg g-r of As will result in the occurrence of As

toxicity synnptoms (Munshower 1994, Miteva 2002). The s5rmptoms include leaf wilting,

development of necrotic spots on older leaves, leaf tips and margins, reduced chlorophyll

a and b content, increased anthocyanin production, root discoloration, reduction in root

and shoot growth and plant death (Aller et al. 1990, Miteva 1998, Miteva2002).

Arsenite has been shown to inhibit the activity of vacuolar ATPase (Dschida and

Bowman 1995). Arsenate is a phosphate analogue which reacts with a substrate to form an unstable organic-arsenate compound which spontaneously decomposes. This process is known as arsenolysis (Slocum and Varner 1960, Ter Welle and Slater 1967). For example, during oxidative phosphorylation, arsenate can replace inorganic phosphate (p¡) in the final step where adenosine diphosphate is typically phosphorylated by means of a proton gradient and ATP-synthase (Ter Welle and Slater 1967, Nelson and Cox 2000).

The resulting organic-arsenate compound is unstable and spontaneously decomposes thereby wasting an entire cycle of oxidative phosphorylation.

2.5.1.2. TrraNruvr (Tr)

Typical concentrations of Ti in soils range from 1,000 to 5,000 pg g-' (Adriano

1986). Anthropogenic enrichment of soils by Ti can originate from degradation of roof fittings, oil-combusting power plants, motor vehicles, sewage, mining and smelting industries (Kurkjian 2000, Rocher et a|.2004). Under normal soil conditions, Ti is relatively unavailable to plant roots as it precipitates as TiO2*H2O (Aller et al.. 1990).

Due to its low solubility, Ti can be used to determine the degree of contamination from soil during element analysis of plant tissues (Van Buren et al. 1989, Cary and Kubota

1990, Sheppard and Evenden 1995). l3

Certain discrepancies occur in the literature pertaining to Ti uptake in plants. As

previously stated, certain researchers claim that Ti is not biologically available and

attribute the Ti concentrations measured in tissues to contamination by dust or soil (Van

Buren et al. l989,cary and Kubota 1990, Sheppard and Evenden 1995). other

researchers claim that Ti concentrations in plant tissues typically range from 0. 15 to 4

-l pg g-r with concentrations in Eqttísetttm and (Jrtica species reaching up to l5 - 1500 pg

(Aller et al. 1990, Kabata-Pendias and Pendias 1992). As Ti is expected to be relatively

unavailable to plants, the accumulation of high concentrations of Ti in Equisetttm and

Urtica pose some interesting questions such as whether plants are increasing the

solubility of Ti. It is possìble that soil or dust contamination contributed to the high Ti

accumulation in the aforementioned plant tissues. Further research is required to elucidate

the bioavailability of Ti to planrs.

Although Ti is not essential to plant nutrition, studies have reported stimulatory

effects in yield, Fe and magnesium (Mg) uptake, enzyme activity, production of organic

acids and chlorophyll content in plants treated with a foliar spray containing 0.042mM to

0.047 mM of chelated Ti (Pais 1983, Adriano 1986, Alcaraz-López et al. 2004). A recent

study by Hrubf et al. (2002) reported positive changes in growth and physiological

parameters in Ti treated (0.04 - 0.36 mM Ti) Avena sativa grown hydroponically with

nitrate as the sole source of nitrogen. The growth stimulation was attributed to an

increased in nitrate reductase activity.

cawajal et al. ( t 994) found no phytotoxic effects in red pepper (capsicum annuum) treated with solutions of 0.042 mM Ti (IV) ascorbate or Ti (IV) chloride whereas Wallace et al. (1977) found that bush bean (Phaseolus rulgarrs) exposed to 4.5 14

pg g-r of Ti suffered from a reduction in yield. In a study by Hrubf et al. (2002), the

benefits of the increased nitrate metabolism masked the negative effects of Ti toxicity;

however, when ammonium was substituted for nitrate, plants suffered a severe reduction

in root and shoot dry weight, chlorophyll a and b content, plant height and root length.

Toxicity caused by the excessive uptake of Ti is unlikely to occur due to low solubility of

Ti under normal field conditions.

2.5.1.3. VaNauuvl (V)

Average vanadium concentrations in soil range from 3 to 500 pg g-l with a mean

of approximately 90 pg g-r (National Academy of sciences 1974, Adnano l9g6).

Combustion of fossil fuels is the primary cause of anthropogenic V enrichment; however,

residues from fly ash, slag and spent vanadium catalysts used in producing sulphuric acid

also contributes to vanadium enrichment of the soil (National Academy of Scienc e 1974,

Morrell et al. 1986, Poledniok and Buhl 2003).In the lithosphere, the oxidation state of V

varies from V*2 to V*s. In soil, V is typically fully oxidized to its pentavalent state (V*5)

and present as an oxyanion known as vanadate (vort-, vo+3-, or v2o7a-) (Morrell et al.

1986). Vanadium availability is highest at neutral or low pH; however; availability is

oftentimes reduced by chelation with organic ligands such as ketones, aldehydes,

catechols, amino compounds, and phenols (Morrell et al. 19g6, Aller et al. 1990).

The concentration of V in plant tissues typically ranges from 0.21 to 4.2 pg g-t with a mean of approximately I pg g-r (Aller et al. 1990). Basiouny (1984) derermined that V in tomato (Lycopersicon esculentum) accumulated mainly in roots (96-4%), followed by leaves (2%), petiole (0.9%), and stems (0.7%). This strong retention of vanadium in roots can be partially explained by the presence of aldehydes, ketones, 15

catechols, olefins (alkenes) or compounds containing sulphydryl groups in cell walls

which can reduce V*5 into unavailable V*a complexes (Basiouny 1984,Morrell et al.

l 986).

Although V is not considered an essential element for plant growth, Zea mays and

Chlorellafuscahave shown favourable results when treated with low doses of V.

Treatment of the aforementioned species with 0.250 pg ml-r to 20 pg g-r of V resulted in

increased biomass, chlorophyll content, carotenoid content, iron metabolism, cytochrome

f content and an acceleration of the Hill reaction (Singh 1971, Wilhelm and Wild 19g0,

Basiouny 1984, Wilhelm and Wild 1984). Meish and Bauer (1978) suggested thar V acts

as a catalyst accelerating the conversion of 4,5-dioxovaleric acid into ô- aminolevulinic

acid, a precursor to porphyrins such as chlorophylls (Meisch and Bauer 1978). The

authors suggested that stimulation of the photosynthetic apparatus leads to increased

photosynthetic capacity and hence biomass production (Meisch and Bauer 1978, Wilhelm

and Wild 1984)

Although V has beneficial effects in plants at low concentrations, it has shown

phytotoxic effects at higher concentrations. It has been suggested that V and cadmium

(Cd) are two of the most phytotoxic trace elements to vascular plants (Aller et al. 1990).

Vanadium toxicity results in chlorosis and/or stunting of plant growth, decreased leaf

area, decreased leaf number and depressed grain yield (Singh lg7l, Aller et al. 1990).

Jacobs andTaiz (1980) showed that exposing plants to solutions containing lmM

Na3VOa would inhibit cell wall elongation by inhibiting H*-ATPase pumps located on the plasma membrane. These pumps are responsible for producing and maintaining an H* gradient across the plasma membrane. This results in the acidification of the cell wall l6

environment which is required for cell wall expansion and for the transport of many

organic molecules via H*cotransport systems (Jacobs and,Taiz 1980,Tajz and, Zeiger

1998). Other toxic effects of V on plants include the inhibition of nitrate reductase

activity in plants treated with 0.08 ¡rM VOSOa + I pM NaVO¡ and the inhibition of

glycolipid and phospholipid production in plants treated with l0 pg g-' V (Mudd and

Garcia 1975, Ramadoss 1979, Khan and Malhortral9g7).

2.5.1.4.IRON (FE)

Iron accounts for 0.02 o/o to l0o/o of soils and is the most abundant plant

micronutrient (Barber 1995). Iron naturally originates from weathered minerals, such as

goethite, hematite, and pyrite (Guerinot and Yi 1994,Barber lgg5,Bartakova et al.

2001). Approximately 25o/o of all mined Fe is lost, on a yearly basis, due to corrosion,

which is reported as being the most important anthropogenic source of Fe input into soils

(Huebers l99l). Furthermore, the oxidation of Fe-sulphides produced by certain mining

operations also contributes to elevated Fe concentrations in the environment (Bartakova

et al. 2001).

Although abundant, under aerobic conditions and near neutral pHs, most Fe in

soils is oxidized into insoluble Fe*3 oxides and is therefore biologically unavailable

(Guerinot and Yi 1994, Schmidt and Fühner 1998). It has been well established that Fe*3 must be reduced to Fe*2 prior to absorption by plant roots (Chaney et al. 1972, Römheld

1987, Barber 1995). Plants can increase Fe availability through rhizosphere acidification, exudation of chelators or Fe reduction via a transmembrane redox system (Chaney et al.

1912, Römheld et al. 1984, Buckhout et al. 1989). Graminaceous monocotyledons can also secrete phytosiderophores (mugineic acid and avenic acid) which selectively binds to t7

Fe*3 and solubilize it (Rörnheld 1987). It has been speculated that the Fe*3-

phytosiderophores complexes are transported across the plasma membrane by a transport

protein which would function much like a system observed in micro-organisms (Römheld

and Marschner 1986).

Iron concentrations in plants ranges from 50 fo 7 5 p,g g-l and can reach up to

1,000 pg g-¡ in shoot tissues (Jones 1998). The ferrous form (F.*t) is the metabolically active form of Fe even though the majority of Fe in plants is under the ferric lFe*3) form

(Jones 1998). The importance of Fe in plants stems from its electron transfer properties vital to photosynthesis, respiration, nitrogen fixation, DNA synthesis, and hormone production (Briat and Lobréaux 1997).Iron is also a component of certain antioxidants such as catalases, peroxidases and Fe-superoxide dismutases (Becana et al. 1998).

Bronzing or yellowing of shoot tissues and the development of brown spots on leaves is typically associated with Fe toxicity. Concentrations of Fe leading to toxicity are undefined and vary from plant to plants (Jones 1998). Biologically active Fe*2 is highly reactive and must be stored in the apoplast, vacuoles or in specialized iron storing proteins called ferritin to prevent cellular damage.(f{arrison and Arosio 1996, Briat and

Lobréaux 1997). Under conditions of excess, Fe*2 is oxidized into Fe*3 while calabolizing the decomposition of H2O2 and producing .OH (hydroxyl radical) (Becana et al. 1998).

The Fe*3 is reduced back into Fe*2 by the superoxide radical (oz') by means of superoxide dismutase. These free radicals species are responsible for the peroxidation of membrane lipids and degradation of proteins (Halliwell 1982). 18

2.5.1.5. MoLystrNUM CMo)

Molybdenum is present at concentrations ranging from 1 to 3.3 pg g-r in the soils

(Adriano 1986, Reddy et al. 1997). Weathered shale and granite are reported as important

natural sources of Mo in soils (Adriano 1986). Molybdenum concentrations ranging from

2 to ll pg g-' have been reported in soils contaminated by industrial and/or agricultural

pollution (Reddy et al. 1997).

In soil solution with pH > 4.23, Mo is usually found as rnolybdate (Moo42-)

(Kaiser et al. 2005). Molybdate accumulates in the soil solution of poorly drained soils

and is readily plant available (Kubota et al.196l). Due to its anionic state, the solubility

of Mo increases with an increase in pH (Jones 1998).

In plants, Mo is biologically inactive unless complexed to an apoprotein. The Mo-

apoprotein complex is a pterin compound known as molybdenum-cofactor (Moco). In

plants, Moco combines with holoenzymes to form 4 types of enzymes; nitrate reductase,

aldehyde oxidase, xanthine dehydrogenase and sulfite oxidase (Mendel and Hänsch

2002). Biologically, the Mo-enzyme facilitates redox reaction involving the transfer of electrons from a donor to a final electron acceptor (Hale et al. 2001).

Instances of Mo related phytotoxicity in the literature are rare. A study by Choi et al. (1996) reported the development of an orange pigmentation on leaves and roots marigold (Tagetes patula) and petunia (Petunia hybrida) treated with a Mo solutions gteater than I mM. An other study by FargaSovà ( 1998) found that concentrations 1,000 to 2,866 times the expected concentrations in plant tissues would cause reductions in root elongation of Sínapis alba. Although accounts of Mo toxicity in plants are ÍaÍe, it is well established that Mo is quite toxic to ruminants at relatively low concentrations. Ingestion t9

of plants containing Mo concentrations in excess of 10 ¡rg g-l has been known to cause

molybdenosis in ruminants (Neunhäuserer et al. 2001). Molybdenosis is a potentially

fatal affliction caused by a copper deficiency resulting from reduced Cu absorption from

the gut (Smith and White 1997, Neunhäuserer er al. 2001).

2.5.1.6. NTcKEL (Nr)

Nickel is ubiquitous in the Earth's crust and is present in sandy and loamy soils at

concentrations not exceeding I pg g-'. In serpentine soils from India, Ni concentrations

exceeding 8,000 pg g-r have been reported (Greger l999,Pal et al. 2005). The naturally

elevated nickel cottent of soils is generally sourced from weathered bedrock with high Ni

contents. Many nickel contaminated sites are the result of anthropogenic activity such as

disposal of sewage sludge, industrial effluents and particulate emissions from nickel

mines and refineries (Kukier and Chaney 2001, Parida et al. 2003). The ionic form of Ni

(Nit*) is the predominant form of Ni under most normal soil conditions.

The concentration of nickel in plants varies among species and cultivars but

typically ranges between 0.I and 5 pg g-' (Mishra and Kar lg74). Certain species from

the Boraginaceae, Cruciferae, Myrtaceae, Leguminosae, Caryophyllaceae and Violaceae

can hyperaccumulate Ni to concentrations reaching 2 to 3o/o of the shoot dry weight

(Kovalevskiy 1979, Morrison et al. 1980).

Nickel is an essential component of urease, an essential enzyme in legumes

(Dixon et al. 1975, Brown et al. 1987). Urea is formed in legumes from the catabolism of ureides, the products of nitrogen assimilation. Urea is hydrolyzed by urease into

ammonium and COz. In nickel starved plants, urea accumulates in leaf tips leading to chlorosis and necrosis (Eskew et al. 1983, Walker et al. 1985, Checkai et al. 1986). 20

Although essential in legumes, the requirement of urease for nitrogen metabolism in all

higher plants is still unknown and certain authors still do not classify it as an essential

micronutrient (Jones I 998).

Although nickel is generally considered an essential element, it is quite phytotoxic

at high concentrations. Multiple studies have reported depression of yield and disorders

in metabolisms in plants treated with soil nickel concentrations ranging from 7pg g-r to

5,070 pg g-' frank et al l982.,Yanget al. 1996). Symptoms of Ni toxicity include weak

and stunted growth of roots and shoot, reduced branching of shoots and interveinal

chlorosis (Yang et al. 1996, Parida et al. 2003). As nickel is known to inhibit

translocation of Fe from the roots to shoots, it is speculated that the interveinal chlorosis

observed in nickel treated plants stems from an iron deficiency (Sunderman 1989).

Crooke (1955) and Khalid and Tinsley (1980) demonstrated that rhe ratio of nickel to iron

(Ni: Fe) was important in determining the degree of toxicity expressed by the plants.

Nickel toxicity was associated with Ni: Fe greater than l, where the toxicity symptoms

were aggravated with increases in the Ni: Fe.

2.5.1.7. Sulrnun (S)

In soils, S concentrations are quite variable but tend to fall between 100 and 500

pg g-l with concentrations in organic soils sometimes reaching excesses of 5,000 pg g-l

(Munshower 1994). Natural inputs of sulphur originates from weathering of sulphur bearing mineral, such as metal sulphides, which then oxidizes into sulphate (Barber

I 995). Atmospheric deposition (acid rain) of S and the decomposition of organic 21

materials are important sources of natural S deposition (Jones 1998). Fertilizer

application, pesticide use and industrial air pollution accounts for the majority of

anthropogenic S inputs.

The primary form of sulphur in the soil solution is the divalent sulphate anions

SO42- lsalisbury and Ross lgg2). Although inorganic sulphate is the most important form of sulphur for plants, it generally makes up less than 5o/o of the sulphur present in soils

(Kertesz and Mirleau 2004). Over 90o/o of S in soils exists in the soil organic matter

(Barber 1995, Jones 1998). The mineralization of sulphur is mediated and is dependent on the size and type of microbial community in the soil, soil characteristics and the ease of degradation of sulphur containing organic compounds (Ghani et al. 1992).

sulphur content in leaves ranges from 1,500 to 5,000 pg g-l (Jones l99g). The final metabolic step of S assimilation is the incorporation of sulphide into the amino acid cysteine (Bonner et al. 2005). Cysteine is produced when sulphides are joined to alanine

(Saito 2000, Kertesz and Mirleau 2004). Cysteine plays an important biochemical role as it is the metabolic sulphide donor for all cellular components containing reduced sulphur, and is the precursor for methionine, glutathione, phytochelatins, vitamin cofactors, and various other secondary metabolites (Bonner et al. 2006).

Sulphur is an essential macronutrient for plant growth. Reports of toxicity due to sulphur uptake by roots are rare. Jones ( 1998) states that excess of S may lead to premature leaf senescence. A study by Papadopoulos (1984) showed a decrease in shoot biomass and leld of tomato (Lycopersicon esculentum) exposed to SOa-2 concentrations of l6 mEq SOa-2 L-r. Drost et al. (1997) found a decrease in bean (Phaseolus vulgaris) and broccoli (Brassica oleracea) leaf area and shoot biomass with increasing SOa-2 22

concentrations. Furtherrnore, red-osier dogwood (Cornus sericea) treated with 50 mM

Na2SOa showed a greater reduction in growth than plants treated with NaCl at equimolar

Na2* concentrations suggesting that SO¿2- inhibits growth more than the Cl- anion

(Renault et al. 2001). Another form of S toxicity occurs via foliar exposure of sulphur dioxide (SOz), a common air pollutant released from combustion of fossil fuels and other hydrocarbons. At concentrations as low as 0.5 ml L-l SOz has been shown to inhibit plant growth (Taiz and Zeiger 1998). When SO2 enters the stomata, it dissolves in water and produces sulphite or bisulphite which leads to the inhibition of photosynthesis and chlorophyll degradation (Salisbury and Ross 1992, Taiz and zeiger 1998).

2.5.2. HynRocnnsoN

Hydrocarbons can range from simple methane (CH¿) to the complex mixtures such as diesel fuel (Adam and Duncan 2002). Coke contains hydrocarbons with high carbon-to-hydrogen ratio and trace amounts of polycyclic aromatic hydrocarbons (Suncor coke MSDS 2002).

Adam and Duncan (2002) demonstrated that soaking seeds in diesel fuel delayed gennination of grasses, herbs, legumes and commercial crops but had no overall effect on final germination. The authors suggest that diesel fuels form a hydrophobic film around seeds and roots which reduce the availability of water and oxygen to the seed during germination.

Hydrocarbons, especially oils, have phytotoxic properties which were historically exploited for agricultural purposes. Prior to modern day chemicals, oils were a popular active ingredient in many pesticides, herbicides fungicides and defoliants (Gauvrit and

Cabanne 1993). As a general rule, phytotoxicity of oils is proportional to their weight 23

where unsaturated hydrocarbons have higher phytotoxicity than saturated hydrocarbons

(Crafts and Reiber 1948, Adam and Duncan2002). The toxicity of oils is greatest in

aromatics followed by naphthenics and olefins while paraffins seemed the least toxic

(Ifuight et al. l929,Gauvnt and Cabanne 1993).

Polycyclic aromatic hydrocarbons (PAHs) are molecules composed exclusively of

C and H and have a molecular weight ranging from 178 to 300. PAHs are consist of three

or more fused benzene rings arranged in a linear, angular or cluster arrangement

(Edwards 1983). The pyrolysis of organic molecules during forest fires, prairie fires and

volcanic activity are the major source of naturally occurring PAHs (Blumer lg76).

Furthermore, certain plants and bacteria have been found to produce PAHs and are

thought to contribute to the environmental background concentrations. Anthropogenic

production of PAHs occurs principally from the combustion of fossil fuels for power and

heat generation, transportation, incineration of waste materials and agricultural burning

(suess 197 6, Edw ards I 983, Maliszewska-Kordybach and smreczak 2003).

As hydrocarbons are hydrophobic, their primary site of action is the cellular membranes. Certain PAHs have been found to partition in cellular membranes inhibiting the activity of ion pumps and increasing membrane permeability to protons and other larget molecules (Sikkema et al. 1992). A review by Edwards (1983) showed that over a quarter of all known PAHs were strongly mutagenic or carcinogenic and another quarter possessed weak mutagenic and carcinogenic properties. Certain PAHs such as benzofa]pyrene are harmless on their own but can be biologically activated by enzymes to form epoxides which are carcinogenic and mutagenic (Edwards l9g3). 24

2.6. Prryslcar, Enrncrs oF Coxn oN pr,aNrs

Although coke cannot be classified as soil, its physical properties can be

compared to soils where Syncrude coke could be compared to sand and Suncor coke to

cobble/gravel/sand mixture. Many of the soil physical properties are interrelated and

therefore the mechanism affecting plant growth is often the same for several soil physical

properties' The major physical properties of coke which may affect plant growth include

structure, texture and porosity.

Soil structure relates to the arrangelnents of soil particles into larger units of

variable sizes called aggregates or peds (White 2006). Syncrude and Suncor coke

particles do not aggregate into peds and are therefore comparable to sand and

gravel/cobble respectively suggesting that the coke is structureless (single grain). The

interaction between coke particles is weak and is therefore expected to provide poor

anchorage to plants. Large particles which are solid and closely distributed may inhibit

root growth resulting in stubby and gnarled roots. Greacen and Oh (lgl2) suggest that

root growth into hard, unyielding soil aggregates creates pressure on the root, thereby

inhibiting water uptake and cell elongation during the cell growth. A second hypothesis

(Russell and Goss 197 4) suggests that the physical pressure of soils is insufficient to inhibit growth and that an ethylene mediated hormonal signal, triggered by the pressure on the growing root, is responsible for the inhibition of root growth. Ethylene production in fava bean (Viciafob") roots increased 6 fold upon encountering a physical obstacle

(Kays et al. 1974). Furthermore, Veen (1982) observed that cellulose microfibrils in root parenchyma cell walls were deposited axially when treated with ethylene. The axial deposition limits longitudinal cellular expansion and promotes the radialgrowth of roots. 25

Porosity is the percentage of pore spaces (space filled with non-soil cornponents)

of a total volume of soil. Macropores (>75 prn diameter) and micropores (<30 ¡rm

diameter) are represented in different proportions depending on the soil type; sandy soils

are dominated by macropores and clay soils by micropores (White 2006). Syncrude coke

has a sandy texfure and is likely dominated mainly by macropores. Similarly, the large

size of Suncor coke aggregates lends itself to a high proportion of large macropores. The

predominance of many large macropores in soils may incur problems with water and

nutrient absorption. A common issue associated with alargeproportion of macropores is

the rapid drainage of water from the soil (Weiler and Naef 2003). This may lead to

conditions of drought even with frequent watering as the water quickly drains from the

root zone rnaking it unavailable to plants.

In soils, roots grow in close contact with the soil/liquid phase allowing for uptake

of water and nutrients. Root will preferentially grow in soil pores exceeding 500 pm in

diameter as opposed to growing through bulk soil, particularly when soil peds/particles

are large and solid (Volkmar 1996). When roots are clumped in macropores, exploration

by the root is limited to the region surrounding the pore. Furthermore, the uptake of water

and nutrients is severely limited by the lack of close root-to-soil contact (passioura l99l).

2.7. Suuuany

With the current market for fossil fuels, large volumes of coke will be produced.

It is imperative that the ecology of stockpiled coke be determined as large volumes will be incorporated into the landscape. Certain constituents of coke such as heavy metals are known to be problematic to plant growth and development. Furthermore, physical properties such as drainage may also affect the flora growing on revegetated coke storage 26 sites. Although several studies have been conducted, major gaps still exist in understanding the effects of coke on the components of the environment. As it is likely that roots will penetrate the reclamation cap and come into contact with coke, studies pertaining to the effect of coke on herbaceous and woody plants are required to assess potential problems/risks to flora associated with the integration of coke into the landscape for storage. 27

CHAPTER 3 - EnrnRcENCE, GRowttt, Pnyslolocy, AND THE AccuMULATroN oF As,

B, Nr, Mo ¿,No V lN Flvn Gnass Spnclns Exposrt ro Coxn

3.1. INTnoDUCTIoN:

coke consists mainly of carbon (approx. 80-90%), surphur (< gyo), oxygen,

nitrogen and hydrogen, but it also contains considerable amounts of iron (5,722 þLg g-'),

boron (13 pg g-r¡, nickel (501 ¡rg g-r), arsenic (r3 pg g-r), molybdenum (7g vgg-r) and

vanadium (1,616 pg g-') (Chung et al. 1996, Suncor coke MSDS 2002). Trace amounts of

polycyclic aromatic hydrocarbons (PAHs) have also been detected in coke (Coke MSDS

2002). Through abiotic and biotic action, coke may release the aforementioned elements

into various components of the environment. Sulphur, Fe, B and Mo are essential to plant

life in low concentrations but can become phytotoxic at higher concentrations (Aller et al.

1990, Carvajal and Alcaraz 1998). In addition, plants may absorb arsenic and vanadium

which have known ph¡otoxic properties at relatively low concentrations (Aller et al.

1990, Miteva2002). Furthermore, the sandy texture and coarse aggregates characterizing

different types of coke may induce other problems to plants related to the rapid drainage

of water, low root-to-soil contact and poor anchorage.

A greenhouse study was designed to observe the effects of coke on plants. The

first objective of the study was to determine the emergence and survival of 5 grass

species in coke. Sandy soils generally have a lesser amount of available water than loamy

or clay soils (USD A 1957). As coke had a very coarse and sandy texture, the likelihood that coke treated plants would suffer from water stress was greater than for plants growing in the clay textured controls. It was hypothesized that the lower available water in coke would hinder the imbibition of water into the seed thereby retarding germination 28

and emergence of plants growing in coke. The second objective was to determine the

effects of coke on plant growth and physiology. It was hypothesized that the

aforementioned water stress and toxicity caused by the uptake of elements from coke

would cause reduced growth and decreased chlorophyll content. It was also expected that

proline concentrations would be higher and transpiration/stomatal conductance rates

lower in coke treated plants as these are typical symptoms of water stress. The final

objective of this study was to determine the accumulation of elements such as As, Ni, Mo

andlor V into plant tissues. As coke contains elevated concentrations of the

aforementioned elements, it was hypothesized that grasses growing in coke would

contain higher concentrations of the elements than controls.

3.2. MnrnoDS AND Marnruals:

3.2.1. Pr,nNr Marrnrar, AND TREATMENTS

Plant species were selected from the Manual of Plant Species Suitability for

Reclamation in Alberta based on their natural distribution and application to land

reclamation (Hardy BBT Ltd. 1989).

Agropyron trachycaulum / Elymus trachycaulus - (slender wheatgrass)

o Natural range extends over Athabasca oil sand deposit in northern Alberta

(Scoggan 1957, Rydb erg t965, Hardy BBT Ltd. l9g9)

' The species has proven successful in colonizing disturbed sites such as gravel

road beds, abandoned coal mines, bitumen contaminated sites, textured

overburden and tailings sand dikes in Northern Alberta (Hardy BBT Ltd. l9g9) 29

o It is suggested that the species is a moderately good soil stabilizer and has good

soil building potential due to its high biomass production (Hardy BBT Ltd. l9g9)

Calamagrostis canadensrs (bluejoint, marsh reed grass)

o Natural range extends over Athabasca oil sand deposit in northern Alberta

(Scoggan 1957, Rydberg 1965)

¡ This species has demonstrated rapid invasion of oil spills sites in the Northwest

Territories (Hardy BBT Lrd. 1989, Farrell et al. 2000)

Deschampsia caespitosa (Tufted Hair Grass)

o Natural range extends over Athabasca oil sand deposit in northern Alberta

(Scoggan i957, Rydberg 1965)

c An observed pioneer of soils containing elevated concentrations of As, Cd, Cu,

Co, Ni, Pb, and Zn (Kuha and Hutchinson 1979)

Oryzopsis hymenoides (Indian rice grass, silkgrass, Indian millet)

¡ Natural range extends over Athabasca oil sand deposit in northern Alberta

(Scoggan 1957)

' The fibrous root system and moderate spreading ability of this species has been

exploited for soil stabilization and soil building (Hardy BBT Lrd. 1989, plummer

te70)

Trit i cum a e s t ivum (Wheat)

. Species used in several phytoxicology studies (Kukier and chaney 2001,

Maliszewska-Kordybach and Smreczak 2003)

o I species used by Organisation for Economic Co-operation and Development

(OECD) for toxicity testing (Fletcher l99l) 30

Commercial varieties of Agropyron trachycaulum, Calamagros tis canadensis,

Deschampsia caespitosa, Oryzopsis hymenoides and Triticum aestiwtm seeds were

purchased from Brett Young seeds (Winnipeg, MB., Canada).

Seeds were sown in 6" pots containing either; a) peat-mineral mix (control); b) coke produced by Syncrude Canada Ltd. (Syncrude coke); c) coke produced by Suncor

Energy Inc. (Suncor coke), d) Syncrude coke capped with 3-5 cm of peat-mineral mix, or e) Suncor coke capped with 3-5 cm of peat-mineral mix at predetermined seeding rates in order to yield l0 plants per pot. Uncapped treatments represented the worst case scenario for coke exposure to plants. Capping treatments represent a less stressful environment and have been shown to increase survival and growth of plants in tailings (Renault et al.

2003, Szczerski and Renault personal communication). The peat-mineral mix used for the controls and caps was collected from the Mildred Lake Basemine (north of Fort

McMurray, AB) by Syncrude Canada Ltd. Coke was supplied by Syncrude Canada Ltd. and Suncor Energy Inc. in 2004. Coke from Syncrude Canadaltd. was weathered and collected from the coke beach (Figure 4.1) whereas fresh coke was obtained from Suncor

Energy Inc. The concentrations of elements in the peat-mineral mix, Syncrude coke and

Suncor coke are outlined in Table A.l. The gravimetric and volumetric water content of all three growing medium are outlined in Table A.2.

Pots were watered with 50 ml of distilled water at the time of seeding. Plants were watered twice daily with approximately 50 rnl using water from the subtending trays whenever possible, otherwise, distilled water was used. Water soluble fertilizer was applied at 0, 35 and 56 days after seeding for a final application rate of 500 kg ha-r 12-36- 3l

12-4 (N-P-K-S). Plants were grown under an l8 hour photoperiod of natural light

supplemented with 400 W high pressure sodium vapour lights (P.L. Light Systems,

Beamsville, Ontario, Canada). Greenhouse temperature was maintained at24 + 3oC. The

experimental design was completely randomized and each treatment replicated 5 times to

ensure statistical validitv.

3.2.2. EnrnncsNcn AND Gnowru

Germination was measured as the percent emergence and was monitored over a

17 day period. Plant height was recorded on a weekly basis by measuring the distance between the soil surface and the plant apex. After 3 months (2 months for T. aestivam due to its rapid growth and maturation) grasses \¡/ere harvested, washed twice in distilled water and rinsed in deionized water. Roots and shoots were separated and their fresh weights recorded. The separated tissues were frozen in liquid nitrogen and lyophilized using afreeze dry system (Model 77520, Labconco co. Kansas city, Mo, uSA) for determination of dry biomass.

3.2.3. PuorosyNTHETrc PrcunNrs

Chlorophyll a, chlorophyll b and carotenoids content was assessed 5 weeks after seeding for T. aestivum and 8 weeks after seedin g for A. traclrycaulum, D. caespitosa, C. canadensis, and O. hymenoides. For each sample, the distal portion of a mature leaf was discarded as several plants suffered from tip necrosis. The following two centimetres

(length) by the full width of the leaf was collected, weighed and used for the extraction and quantification of chlorophyll a, chlorophyll b and carotenoids. Two leaves were used for D. caespitosa, C. canadenszs, and O. hymenoÌdes to ensure adequate amount of tissue

pigment for quantification. Pigments were extracted with 3 incubations of 2 ml B0o/o 32

acetone. The absorbance of the combined pigment extract was read at 480 nm, 645 nm,

and 663 nm using a UV/visible spectrophotometer (Ultraspec 2000 Uv/Visible lìght

spectrophotometer, Pharmacia Biotech, cambridge, England). chlorophyll and

carotenoid concentrations were calculated using the following equations (MacKinney

l94l,Davies 1976).

Chlorophyll a : 12.7 2* (,\bs. 66) - 2.58x(Abs.o¿s)

Chlorophyll b : 22.87 * (Abs.o¿s) - 4.67 x(4bs.663)

Carotenoids : Abs.aso + 0. I l4*(Abs.oo:) - 0.63 8*(Abs.6a5)

Values were expressed as mg pigment g-l fresh tissue.

3.2.4. Tn¡Nspln¡TroN AND Srouarar, CoNoucraxcn

Transpiration rates, which are the rate of water loss from stomata per unit area,

were measured for T. aestivum (42 days after seedin g), A. trachycaulum and C.

canadensis (63 days after seeding). Stomatal conductance, which is the rate of movement

of gases (e.g. Oz, H2O and CO2) from the leaf to the atmosphere via the stomates, was

measured at the same time as transpiration. Measurements were taken from the first

mature leaf from the top between l0:00 am and 12:00 pm. A total of l0 plants per

treatment were measured using a steady state porometer (LI-1600, LI-COR, Inc., Lincoln,

Nebraska, USA) and the area of the tissue used in measuring transpiration and stomatal

conductance was determined using aleaf areameter (LI-2100 Area meter, LI-COR, Inc.,

Lincoln, Nebraska, USA). Results were expressed in mmol m-' sec-I. Due to small leaf

area, transpiration rates and stomatal conductance could not be measured for D. caespitosa and O. hymenoides. JJ

3.2.5. PRorrNB

Proline extraction was conducted using a protocol adapted from Sofo et al.

(2004)- All steps of the protocol were conducted on ice unless otherwise noted. proline

was extracted by homogenizing 100 mg of lyophilized plant tissues in 3% sulfosalicylic

acid. Extracts were centrifuged at 30009 for l5 minutes to remove the insoluble fraction.

One millilitre of the supernatant was added to a new tube containing2ml of acid

ninhydrin (0.5 mg ninhydrin, 30 ml glacial acetic acid,20 ml DiH2O). The mixture was

incubated in a l00oC water-bath for I hour then transferred on ice to terminate the

reaction. Three ml of toluene were added to each tube and vortexed. The absorbance of

the chromophore containing toluene was read at 520 nm using toluene as a blank. proline

concentrations were obtained by comparison with a standard curve produced using L-

proline (Sigma Aldrich). Results are expressed as mg proline g-r tissue (dw).

3.2.6. Uprax¡ oF ELEMENTS

Inductively coupled plasma optical emissions spectroscopy (ICP-OES) was used

to quantify the concentrations of arsenic (As), boron (B), iron (Fe), molybdenum (Mo),

nickel (Ni), sulphur (S), titanium (Ti), and vanadium (V) in the root and shoot tissues of

plants growing in coke (Renault et a|.2002). Samples were prepared following the

protocol for wet digestion using nitric and perchloric acid as described in Richards

(1993)- Analysis of the sample was conducted by ICP-OES on a Varian ICp-Emission

Spectrometer Liberty 200 (warrington, cheshire, uK) by the Dept. of Geology,

University of Manitoba. No values are available for B concentrations in A. trachycaulum,

C. canadenszs, and O. hymenoides andMo concentrations inA. trachycauþtm, C. canadensis, and O. hymenoides due to technical problems. Detection limits for ICp-OES 34

are given in Table 4.3. Contamination of plant samples by soil or coke particles was

corrected for by assuming no uptake of Ti by plant roots as it precipitates as TiOz under

normal soil conditions (Cary and Kubota 1990, Sheppard and Evenden 1995). Thus the %

of soil contaminating plant samples could be estimated by comparing the soil-to-Ti ratio

in plant tissues and soil. The data was then corrected accordingly. The uncorrected data

can be found in Table A.5.

3.2.7. Gn¡vInrnrRIC AND Vor,un,rnrRJc WATER CoNTENT

Approximately 50 g of peat-mineral mix, Syncrude coke, and Suncor coke were

mixed with enough warm water to form a thin suspension of soil. Samples were mixed

for 2 minutes, left to settle for 30 minutes and mixed for another 2 minutes to ensure

complete water infiltration in the soil and/or coke. Suspensions were then filtered through

Whatman's #l filter paper and left to drain until a constant weight is attained. A

subsample of the drained peat-mineral mix/coke soil was then weigh ed, freezedried, and

reweighed. Gravimetric and volumetric water content were calculated using the following

equations (Topp 1993):

v/eight of water: weight of drained soil - weight of freeze dried soil

Gravimetric water content: weight of water / weight of freeze dried soil * 100

Volumetric water content: weight of water / volume of freeze dried soil * 100

3.2.8. Dnra ANar,ysrs

A one-way, nested Analysis of Variance (ANOVA) was used for analysis of height, biomass, pigment content, transpiration and proline content. A one way ANOVA was used to compare means of emergence and element uptake. Duncan's multiple range test 35

was used to compare differences between means. When comparing the concentrations of

elements in root and shoot tissues, concentrations which were below detection limit were

considered as 0. All statistical analyses were conducted using the SPSS statistical

software (Version I 1.0.0, SPSS Inc., Chicago, USA).

3.3. Rnsurrs

3.3.1. Eir,rnncBNcn

Emergence of coke treated T. aestivum did not differ from control plants throughout the 17 day monitoring period (Figure 3.1). Descharnpsia caespitosa, C. canadensis and A. trachycaulum growing in uncapped Syncrude and Suncor coke showed an increase in early emergence over the controls between the 4th and 8th day after seeding

(Figure 3.1 and Figure 3.2). Oryzopsis hyry¿nsides growing in uncapped Syncrude coke also showed an increase in the rate of emergence between days 4 and 8; however, no differences were observed between plants grown in uncapped Suncor coke and controls

(Figure 3.2). Despite early increases in emergence, final o/o emergence did not show any significant differences between treatments in D. caespitosa growing in Suncor coke or l. traclzycaulum and O. hymenoides grownin both types of coke (Figure 3.1 and Figure

3.2)- Deschampsia caespitosa grown in uncapped Syncrude coke was the only case where emergence decreased in response to coke exposure (Figure 3.1) where as Calamagrostis canadensis grown in all Suncor coke and capped Syncrude coke were the only cases where a significant increase in emergence was observed after 17 days (Figure 3.1). 36

3.3.2. IN¡uRy aNo GRowrn

Although all species survived the coke treatments, T. aestivum grown in uncapped

Syncrude and uncapped Suncor coke showed visual signs of stress in the form of

chlorosis, leaf tip necrosis and stunted growth (Figure A.2). In capped coke treatments,

these symptoms were still present, but the extent of the damage seemed to be alleviated by the presence of the peat-mineral mix cap (Figure 4.2). Although no visual injury was evident in D. caespitosa, C. canadensis, A. trachycaulum and O. hymenoide.s, a reduction in height and biomass was apparent (Figure A.3, Figure A.4, Figure A.5, Figure A.6).

A reduction in plant height was observed in Z. aestivum grown in capped and uncapped coke after I week (Figure 3.3). By the second week, differences were observed between plants grown in control, capped and uncapped treatments (Figure 3.3). Between weeks 3-5 a significant reduction from the control in plant height became apparent in all other species growing in all coke treatments (Figure 3.3 and Figure 3.4). Capping of coke generally led to a statistically significant increase in plant height over plants grown in uncapped coke (Figure 3.3 and Figure 3.4). These differences first occurred prior to the

6th week and with the exception of C. canadens¿s at 9 and 10 weeks and O. hymenoides at l l week, were preserved until the termination of the experiment (Figure 3.3 and Figure

3.4).

With the exception of T. aestivurn grownin capped Syncrude coke and uncapped

Suncor coke, root biomass of all coke treated plants were significantly lower than the controls (Table 3.1). Roots grown in capped coke treatments produced more biomass than roots grown in uncapped coke treatments with the exception of T. aestivum (Table 3.1).

In all species, a significant inhibition in shoot biomass was observed in all capped and 37

uncapped coke treatments when compared with controls (Table 3.1). As in roots, a trend

can be observed where most plants grown in uncapped coke treatments produced less

shoot biomass than plants grown in capped coke treatments (Table 3.1). The root/shoot ratio of T. aestivum, D. caespitosa and A. trachycaulum were significantly higher in all coke treatments than in the control plants (Table 3.1). Similarly, C. canadensis growing in all Suncor treatments and uncapped Syncrude coke, as well as O. hyrnenoides growing in uncapped Slmcrude coke had a significantly larger roolshoot ratio than controls (Table

3.1).

3.3.3. PrcunNrs

Chlorophyll a content was significantly less in T. aestivum, D. caespitosa and C. canadensis growing in both Suncor and Syncrude coke than in the controls (Table 3.2).

Chlorophyll a content of A. trachycaulum gro\¡/n in capped and uncapped Syncrude coke also followed this trend, while A. trachycaulum grown in both Suncor coke treatments showed no significant difference from the controls (Table 3.2). Oryzopsis hymenoides grown in uncapped Syncrude coke had significantly lower chlorophyll a content when compared to the controls while no differences were observed between plants growing in the remaining coke treatments and the controls (Table 3.2).lnall species, capping of coke did not significantly increase chlorophyll a concentrations over the uncapped treatments (Table 3.2). Chlorophyll b content was lower in all coke treated T. aestitum and D. caespitosa than in controls (Table 3.2). Catamagrostis canadensis grown in

Syncrude coke and capped Suncor coke showed a reduction in chlorophyll b while no differences in chlorophyll b were observed between plants grown in uncapped Suncor coke and controls (Table 3.2). Less chlorophyll b was observed inA. trachycaulum 38

grown in Syncrude coke than in controls; however, no differences were observed

between plants gïown in Suncor coke and controls (Table 3.2). Oryzopsis hymenoides

had lower chlorophyll b concentrations in Syncrude treated plants than in controls but

showed no other differences between treatments (Table 3.2). Capping of Suncor coke

lead to an increase in chlorophyll b in Triticum aestiwtmwhereas C. canadensrs grown in

capped Syncrude and Suncor coke showed a reduction in chlorophyll b when compared

to uncapped coke. Deschampsia caespitosa, A. trachycaulttm and O. hymenoid¿s showed

no significant differences between the chlorophyll b content of plants grown in capped

and uncapped coke (Table 3.2). The chlorophyll alb ratio showed no significant

differences between all coke treatments and the controls. Furthermore, no significant

differences were observed between the capped and uncapped treatments (Table 3.2).

A significant decrease was observed in the carotenoid concentrations of Z.

aestiwtm and caespitosa D- grown in capped and uncapped coke when compared to the

controls (Table 3.2). Similar trends were observed in C. canadensis grown in the capped

Syncrude treatments and both Suncor treatments as well as A. trachycaulumgrown in all

S1'ncrude treatments. No differences were observed in carotenoid concentrations of controls and coke treated o. hymenoides. Significant differences were observed between the carotenoid content of T. aestivum grownin capped and uncapped Suncor coke as well as c. canadenszs grown in capped and uncapped syncrude coke. No significant differences were observed between the capped and uncapped coke treatments ofD. caespitosa, A. trachycaulum and O. hymenoides (Table 3.2). 39

3.3.4. TnaNsplnarroN AND Srouarar, CoNoucraNcn

Triticum aestivum and A. trachycauh¿rn showed a decrease in transpiration and

stomatal conductance when gro\¡/n in uncapped coke (Figure 3.5). A trend was observed

where capping of coke limited the decrease observed in transpiration of T. aestivum, A.

trachycaulum and C. canadens¿s,'however, this trend was only significant in T. aestiwtm

and A- trachycaultun grown in capped Suncor coke. Stomatal conductance in T. aestiram

was lower in plants growing in all coke treatments than in control plants. With the

exception of plants grown in capped Suncor coke, the same trend as in Z. aesti,vum was

observed for A. traclrycaulum. No differences were observed between treatments in C.

canadensis (Figure 3.5). Capping of coke limited the decrease observed in stomatal

conductance for T. aestivum, A. trachycaulum and C. canadensrs; however, the trend was

only significant in A. trachycaulum grown in capped Syncrude coke and capped Suncor

coke (Figure 3.5).

3.3.5. Pnor,lNn

No significant differences in proline content were observed between treatments in

T. aestivutn (Figure 3.6), while proline content of A. n.achycaulum and D. caespitosa was significantly lower in all coke treated plants than in controls (Figure 3.6). Furtherïnore, higher proline concentrations were found in A. trachycaulum and. D. caespitosa grown in capped Suncor coke than in uncapped Suncor coke (Figure 3.6).

3.3.6. ElrunNr CoNrnNr:

Arsenic concentrations in root and shoot tissues of T. aestiwtnt and D. caespitosa were below detection limits in all treatments (Table 3.3, Table 3.4 and, Table 4.3). In roots, vanadium concentrations were significantly higher in T. aestivum, D. caespitosa, 40

C. canadens¡s and A. tt"achycaulum grown in uncapped Syncrude coke than in all other

treatments (Table 3.3). The accumulation of V in roots grown in Syncrude coke was

below detection limits when coke was capped (Table 4.3 and Table 3.3). In shoots, there

was not any significant accumulation of vanadium in the coke treated plants when

compared to the controls (Table 3.4).

No differences in B concentrations of T. aestiwtm roots were observed between

controls and all coke treatments (Table 3.5). However, there was an increase in B

concentrations in T. qestivam shoots grown in uncapped Suncor coke when compared to

controls (Table 3.6). Deschampsia caespitosa showed no differences in root B

concentrations while shoots growing in uncapped Syncrude coke had higher B

concentrations than all other treatments (Table 3.5 and Table 3.6).

Concentrations of Fe in root were higher in T. aestivum and C. canadensis grown

in uncapped Syncrude coke, D. caespitosa growing in capped and uncapped Syncrude

coke and O. hymenoides growing in both Syncrude treatments and capped Suncor coke

than in controls (Table 3.5 and Table 3.6). FurtherTnore, T. aestivum shoots growing in

capped and uncapped Syncrude coke and all coke treated shoots of D. caespitosa, C.

canadensis, A. trachycaulum and O. hymenoides had higher Fe concentrations than

controls (Table 3.6). Capping of coke resulted in lower Fe concentrations in roots ofD.

caespitosa and A. trachycaulum glown in Syncrude coke (Table 3.5). An increase in Fe

concentrations was observed in roots of O. hymenoides grown in capped Suncor coke and

shoots of T. aestiwtm, D. caespitosa and C. canadensis growing in capped Suncor coke

(Table 3.5 and Table 3.6). Concentrations of Fe in controls were oftentimes below detection limits (Table 4.3). 41

With the exception of shoots grown in capped Suncor coke, Triticum aestivam

grown in uncapped Syncrude coke had higher Mo concentrations than all other treatments

in root and shoot tissues (Table 3.5 and Table 3.6). Root and shoot tissues of D.

caespitosa grown in uncapped Suncor and both Syncrude coke treatments had

significantly more Mo than controls (Table 3.5 and Table 3.6). capping of coke

decreased Mo concentrations of roots and shoots of T. aestiwtm grownin Syncrude coke.

Furthermore, roots and shoots of D. caespitosa grown in both Syncrude and Suncor coke

had lower Mo concentrations in capped treatments than in uncapped treatments (Table

3.5 and Table 3.6). Molybdenum in certain capped and uncapped Suncor treatments were

below derection limits (Table A.3).

No statistically significant differences in Ni concentrations exist between

treatments in T. aestivum root tissues (Table 3.5). In many cases, Ni concentrations were

below detection limits (Table 4.3). Despite the higher level of Ni in T. aestivurz shoot

tissues, no statistically significant increases were observed due to the high variation in the

data (Table 3.6). Deschampsia caespitosa roots and shoots gro\¡/n in uncapped Suncor

coke contained higher Ni concentrations than controls (Table 3.5 and Table 3.6).

Calamagrostis canadensis roots grown in uncapped Syncrude coke and shoots grown in

uncapped Suncor coke had higher Ni concentrations than controls (Table 3.5 and Table

3.6). Higher Ni concentrations were observed in roots of A. trachycaulum grown in

uncapped and capped Syncrude coke when compared with controls whereas Ni

concentrations in shoots did not vary between treatments (Table 3.5 and Table 3.6). With the exception of plants grown in capped Suncor coke, all O. hymenoides shoots grown in coke had higher Ni concentrations than controls (Table 3.6). 42

ln T. aestivum roots,less sulphur was found in capped treatments than in the control plants and uncapped coke treatments (Table 3.5). No differences in sulphur concentrations in T. aestivum shoots were observed between coke treated plants and controls (Table 3.6). Descltatnpsia caespitosa roots and shoots also had less sulphur in capped treatments than in controls and uncapped coke treatments (Table 3.5 and Table

3.6). The S content in C. canaderzs¿s roots were similar in controls and plants growing in uncapped coke (Table 3.5). ln C. canade¡¿s¡s shoots, plants growing in uncapped coke had more S than control plants (Table 3.6). In both roots and shoots, plants grown in uncapped coke treatments had more S than plants grown in capped treatments (Table 3.5 and Table 3.6). Sulphur concentrations in C. canadersls grown in capped treatments were below the detection limit (Table A.3). Agropyron trachycaulwn had higher sulphur concentrations in roots of plants grown in uncapped Suncor coke and in shoots of plants glown in uncapped Syncrude and Suncor coke than in controls (Table 3.5 and Table 3.6).

Capping of coke lead to an overall decrease in the sulphur content of A. n"achycaulum tissues (Table 3.5 and Table 3.6). Oryzopsis hymenoldes shows no differences in S concentrations between controls and coke treatments in both root and in shoot tissues

(Table 3.5 and Table 3.6). Furthennore, capping had no effect on the sulphur concentrations in O. hvmenoides tissue. 43

100 B ¿__¿-+-ç__¿-+-p-_¿-+-*_-{,+-+ ': B0 à< : Èra +ai +ti ùH : È/ ãuoc o lt g, 40 !t o Control E --_ ß I -- control tu 20 - Syncrude ll I suncor * Syncrude + cap -- I I Suncor + cap 0 -- 10 12 14 16 1B

100 * Control -_r- Control o\í;BO o Syncrude _*- Suncor + Syncrude+cap Suncor + cap ãuoc -- c) Boo E tu 20

0

100 ----_o- Control G'. 80 o Syncrude o\ * Syncrude + cap ãuo C o * * * P40 !p)'Vlt{* o E ru 20

0

Days after seeding

Figure 3.1 : Percent emergence (mean + SE) of Triticum aestitam (4, B), Deschampsia caespitosa (c, D) and calamagrostis canadensís (E, F) grown for 17 days in peat- mineral mix (control), Syncrude coke, Syncrude coke + cap, Suncor coke or Suncor coke * + cap. denotes significant difference from control at o,: 0.05 (n: 5). 44

100

BO

\oo\ ()o60 C o P¿n (I) E uJ 20

0 16 180

----¡- Control ----+-- Syncrude + + s Syncrude cap ()o60 C C) Boo uE 20

0 16 180

Days after Seeding

Figure 3.2: Percent emergence (mean + sE) of Agropyron trachycaulnm (A,B) and Otyzopsis hymenoides (C, D) grown for 17 days in peat-minerai mix (control), Syncrude coke, Syncrude coke + cap, Suncor coke or Suncor coke * cap. * denotes significant difference from control at c:0.05 (n: 5). 45

60 A -¡- Control ] ---_o- Control --v- Suncot î+o + Suîcor+ cap J) Ë30 .9, Il920 10

+ Control -----¡- Control * Syncrude + Suncor * Syncrude + cap + Suncor+ cap 9zoE E, .o) c) ï10 a b a a a a

10

E _È-----¡- Control l _¡- Control Syncrude ì ---v- Suncor + Slncrude + cap + Suncor+Cap l 930E b6 -c .9, 20 Q) b b b - a a a a b a a a a a 10 a a a a a âa a a a a a a

6 t0 12 6 10 12 Weeks Weeks

Figure 3.3: Shoot height (mean + sE) of Tríticum aestivum (A, B), Deschampsia caespítosa (C, D) and Calamagrostis canadensis (8, F) grown for 7 week s (7. aestivam) or l2 weeks (D. caespitosa and C. canadensrs) in peat-mineral mix (control), Syncrude coke, Syncrude coke -| Cap, Suncor coke or Suncor coke + cap. All means are statistically different unless otherwise noted. Means followed by different letters represent significant difference at a:0.05 (n:5). 46

) + Control ---o- Control 60 r o Syncrude ---{- Suncor Syncrude + cap + Suncor+cap - Ê J) ¿o -c. .o) 0) :E 20

10 12

100 c -¡- Control -----r- Control + Syncrude + SunCoI * Syncrude + cap + SUncor+Cap b60c

-co) 'o 40 I

a a a a a a

6 l0 12 6

Weeks Weeks

Figure 3.4: Shoot height (mean + SE) of Agropyron trachycaulnm (4, B) and. oryzopsis hymenoídes (C, D) grown for 12 weeks in peat-mineral mix (control), Syncrude coke, Syncrude coke * cap, Suncor coke or Suncor coke * cap. All means are statistically different unless otherwise noted. Means followed by different letters represent significant difference at ü":0.05 (n: 5). qq s R 31 Transpiration Transpiration Transpiration )VJ¿\l!1 ,^O c;oo -2 -1 -' -1 ^tãÈ.i Ë'ù.rt (mmol m sec (mmol r-t lmmol m-2 sec -5 S p ""a (D98ñ '-1 Ë'^ (¡r - ^ I r .. aõ.oP-¡,2iO (Þ tÂJÞ ¡ril +vÊD --r' H Control LATTU) "p öa Ë's'Ë'Þ (ll5 ilp õ ñts Syncrude Þ' ezÊLì Syncrude + cap .vH.8: . È xËÈ;l,^'È u g- "È.a 3. Suncor Íto q e Suncor + cap P-rñC,$+Så o.St N- 9 PES Ë ã ã ã.P- Stomatal conductance Stomatal conductance Stomatal conductance 5Þ'1^ô -1¡ -1¡ (Dô o--'-c ôä x (mmol r-2 r"c (mmol r-2 se" -1¡ (mmol *-2 se. Ø (D NÀO)@ NÀo)co ÀcoNo)o l\) I ÊYl-X ooooo O ooooo OOOOOO 3âÞÈ;-. :- ô: )í r; i ã Control ãErã E êS ß Syncrude äð ? Þo*ca=õoSrr Syncrude + cap ã FÑ¡! P Suncor H^\¿+)ã'' +.:i T"U R :' &rSõl-" s s. Suncor + cap

Eã:SrOFrl CÞoFIlov ,f o {è 48

à EÈ \4 o) E3OJ ctc) õ L1L

^53 -;4þ C') 53ct) ct0) õ o_1L

E B =;4E o) E3OJ 0) Ca õ z rL1L

c) o- o E o o_ (Jñ o o C :f c o o O + + O C (¡) U)= E o U) f cC) O f, c U) U)

Figure 3.6: Proline content (mean + SE) of A) Triticum aestivum,B) Deschampsia caespitosa and C) Agropyron trachycaulum growingin peat-mineral mix (conirol), Syncrude coke, Syncrude coke + cap, Suncor coke or Suncor coke + ufthe time of "up harvest. No significant differences exist between means followed by thå same letter (n: 5, 0:0.05). Table 3.1: Root dry weight, shoot dry weight and root/shoot ratio (mean + SE) of Triticum aestivum(after 2 rnonths), Deschampsia caespitosa, Calamagrostis canadensis, Agropyron trachycaulttm, and, Otyzopsis hymenoides (after 3 months) of growth in peat-mineral mix (control), uncapped or capped toke treatments. No signiircant differences exist between rteans followed by the same letter (n : 5, o:-0.0S;.

Species Treatment

T. aestivum Control 0.727 t 0.039 c 0.046 + 0.003 c 0.063 r 0.003 , Syncrude 0.127 r 0.010 a 0.035 + 0.002 0.310 + 0.029" Syncrude + "u câp 0.235 + 0.019 u 0.043 r 0.003 . 0.195 r 0.011 b Suncor 0.200 + r 0.013 b 0.040 0.003 uc 0.211 ! 0.015b Suncor + câp 0.186 r 0.016 b 0.033 + 0.003 0.198 r 0.021 " b D. caespitosa Control 0.670 + 0.050 0.131 r 0.013 0.191 t 0.013 " " Syncrude 0.074 + 0.010 0.030 + 0.004 0.441 t0.017 " Syncrude + " " câp 0.176 r 0.014 b 0.064 " r 0.006 b 0.386 r 0.020 b Suncor 0.067 + 0.007 u 0.029 t 0.003 a 0.564 + 0.047 Suncor + ¿ câp 0.235 + 0.021 o 0.075 + 0.007 ¡ 0.340 t 0.019 b C. canadensis Control 0.557 r 0.063 b 0.131 r 0.026. 0.294 + 0.046 Syncrude 0.072 + 0,009 " u 0.038r0.004ab 0.619 + 0.069 . Syncrude + câp 0.133 r 0.013 a 0.041 10,005"6 0.349+0.037"u Suncor 0.083 r 0.009 a 0.035 r 0.004 a 0.443 r 0.034 b Suncor + ca 0.153 t 0.019 0.070 r 0.017 0.480 t 0.062 ¡, Table continued onfollowing page

\oÞ Table 3-l: Continued

Species Treatment

A. trachycaulum Control 0.620 + 0.060 . 0.130 t 0.010 d 0.520 r 0.290 Syncrude 0.073 + 0.009 0.037 + 0.003 0.595 r 0.038 " Syncrude + cap 0.159 r 0.014 " " " ¿6 0.085 10.005 c 0.611 I 0.032 . Suncor 0.12710.008"u 0.059 r 0.004 u 0.491 ! 0.019 b Suncor + cap 0.195 + 0.017 u 0.083 + 0.009 . 0.475 r 0.032 b O, hymenoides Control 0.621 ! 0.06b d + 0.075 0,009 c 0.118 t 0.007 a Syncrude 0.063 r 0.008 0.024 r 0.003 a 0.469 + 0.093 u Syncrude + câp 0.172 0.026 " r b 0.054 r 0.009 b 0.324 + 0.026 Suncor 0.144 + 0.010 u + "u 0.032 0.004 u 0.217 t 0.015 a Suncor + 0.278 ! 0.041 0.050 r 0.004 0.242 r 0.030

(^ Table 3.2: Chlorophyll a (Chl a), chlorophylt b (Chl b), chlorophyll a to chlorophyll b ratio (Chl a/b) and carotenoid content (mean * SE) of Triticum aestivum, Deschampsia caespitoro., Colo*ogrostis canadensis, Agropyron trachycaulum and Oryzopsis hymenoides grown in peat-mineral mix (controlj, Slmcrude coke, Syncrude coke * cap, Suncor coke or Suncor coke + cap. No significant differences exist between means followed by the same letter (n = 5, a, = 0.05).

Chl a cht b Carotenoids Chl a/b Species Treatment (mg g-' rw) (mg g-1 rw) (mg g-1 rW)

T. aestivum Control 3.05 r 0.23 6 0.73 r 0.05. 4.16t0.04"b 0.14 ! 0.01 . Syncrude 0.98 J 0,18" 0.24 !0.07 4.70 ! 0.40 b 0.07 r 0.01 Syncrude + cap 1.43 0.21 ^ " ! 0.36 10.06"b 4.11 ! 0.19"b 0.08 t 0.01 u6 Suncor ^ 0.99 r 0.19" 0.27 ! 0.05 3.41 t 0.25 0.07 r 0.01 . Suncor + cap 1.49 0.21 " ^ ! a 0.50 i 0.08 b 3.54 t 0.47 0.09 f 0.00 b " D. caespitosa Control 4.30 r 0.85 1.25 b ! 0.24 b 3.47 ! 0,06 0.21 ! 0.04 b " Syncrude 2.57 !0.21a 0.73 r 0.06, 3.51 r 0.05 0.13 r 0.01 Syncrude + " " câp 2.40 ! 0.25 a 0.71! 0.09" 3.41 ! 0.06 0.13 r 0.01 " a Suncor 2.73 ! 0.29, 0.78 r 0.09 3.56 r 0.11 0.14 ! 0.02 Suncor + cap 2.41 0.17 " ^ ^ ! 0.72 ! 0.05 u 3.37 ! 0.11 u 0.13 r 0.01 ^ " C. canadensis Control 3,12!0.16b 0.83 r 0.04 d 3.74 ! 0.11 0.16 r 0.01 Syncrude 2.50 " " ! 0.16 0.69 r 0.04b. 3.62 ! 0.09 0.14 ! 0.01 b" + " " Syncrude cap 2.09 ! 0.13u 0.56 r 0.05 3.97 t 0.36 0.11 r 0.01 " " Suncor 2.51 t 0.17 0.73 r 0.06.6 3.47 ! 0.07 0.1310.01 " + " ^ "6 Suncor ca 2.14 ! 0.12 0.5910.04 3.67 ! 0.14 0.12 r 0.00 Table continued onfollowing page

(.¡¡ Table 3-2: Continued Chl a cht b Carotenoids Chl a/b Species Treatment (mg g-' rw) (mg g-1 Fw¡ (mg g-' rw)

A. trachycaulum Control 2.06 ! 0.13 b 0.54 t 0.04 b 3.8810.01 ¿6 0,11r 0.146 Syncrude 1 .34 0.19 ! 0.35 I 0.06 4.02 ! 0.01 b 0.08 r 0.31 Syncrude + cap 1.34 " " " ! 0.20 0.36 r 0.06 3.80r0.01 ab 0.08 r 0.14 ^ " Suncor 1.62!0.15"6 0.49t0.05¡5 3.35 r 0.01 0.09 r 0.15"6^ Suncor + câp 2.01 0.13 " ! b 0.53 r 0.05 b 3.89 r 0.00"6 0.10 r 0.18b

O. hymenoides Control 2.40 ! 0.226" 0.63 t 0.06 bc 3.86 r 0.14 a 0.14!0.00"b Syncrude 1 .52 ! 0.15 , 0.35 t 0.05 4.73 ! 0.53 0.10 t 0.01 + " " " Syncrude câp 1.81 10.40"6 0.46 r 0.10ub 5.08 r 1.49 0.12! 0.01 Suncor 2.47 0.426ç " "6 ! 0.63 * 0.13 6. 4.45 ! 0.64 a 0.14!0.02ab Suncor + câp Ì 2.66 r 0.36. 0.68 r 0.09. 3.89 t 0.17 a 0.15 r 0.02 b

(rt N) 53

Table 3.3: Arsenic (As) and vanadium (v) concentrations (mean + sE) in root tissues of Triticum aestivum, Deschampsia caespitosa, a I am a gro s t i c s c ana den s i s, A gropyro n tr a c hy c artlum and oryz op s is hymenoides growing in peat-mineral mix (control), Syncrude coke, Syncrude coke + cap, Suncor coke or Suncor coke * cap. No significant differences exist between means followed by the same letter tr:5, g: 0.05

As (tig g-t) V (pg g-') T. aestivum Control 0.87 r 0.10, Syncrude 18.32 r 5.09 b Syncrude + câp 0.00 r 0.00 " Suncor 0.78 r 0.48. Suncor + cap 0.00 r 0.00 " D. caespitosa Control 4.64 ! 0.79 Syncrude ^ 27 .78 t 5.76 b Syncrude + cap 0.00 I 0.00 " Suncor 3.98 r 1.73 . Suncor + câp 0.00 t 0.00 " C. canadensis Control 6.98 r 1.22 Syncrude ^ 28.47 + 4.66 ¡ Syncrude + cap 0.00 r 0.00" Suncor 9.75 r 9.75 " Suncor + câp 0.00 r 0.00 " A. trachycaulum Control 3.43 r 1.00 " Syncrude 15.35 !3.24b Syncrude + cap 0.00 r 0.00 " Suncor 3.17 ! 0.61" Suncor + cap 0.00 r 0.00 " O. hymenoides Control 2.95 t 1.19 ^ Syncrude 3.64 I 2.66 ^ Syncrude + cap 0.00 r 0.00 " Suncor 0.02 ! 0.02. Suncor + 0.00 r 0.00 * below detection limit --- datanot available 54

Table 3.4: Arsenic (As) and vanadium (v) concentrations (mean + sE) in shoot tissues of Triîicum aestitam, Deschampsia caespitosa, Calamagrostis canadensis, Agropyron trachycaulum and oryzopsis hymenoides growing in peat-mineral mix (control), syncrude coke, syncrude coke + cap, Suncor coke or Suncor coke * cap. No significant differences exist between means followed by the same letter (n : 5, a: 0.05).

As (pg g') V (pg g-')

T. aestivum Control 6.39 r 0.51 b Syncrude 5.27 ! 0.30"0 Syncrude + cap 4.70!0.93"b Suncor 5.04 t 0.67 ^b Suncor + cap 4.13 !.0.40 " D. caespitosa Control 9.86 t 1.09 c Syncrude 5.44 ! 1.37 ,D Syncrude + cap 0.00 r 0.00 " Suncor 1.46 ! 0.66 " Suncor + cap 0.00 r 0.00 " C. canadensis Control 20.68 t 1.15 b Syncrude 18.68 + 0.05u Syncrude + cap 4.34 ! 1.60 Suncor " 17.64 t 3.43 b Suncor + cap 14.53 + 2.85 o

A. trachycaulum Control 17.95 + 0.96 " Syncrude 12.83 r 1.33b Syncrude + cap 4.91 x 1.07 " Suncor 14.56 + 1.25¿" + Suncor câp 14.71 ! 1.36 b"

O. hymenoides Control 13.95 r 1.13b Syncrude 15.24 + 1.Bgu Syncrude + câp 3.58 t 2.25 " Suncor 16.03 r 1.19b Suncor + c 5.40 r 3.40 " x below detection limit --- datanot available Table 3'5: Element content (rnean + SE) in root tissues of Triticum aestivum, Deschampsia caespitosa, Calamagrostis canadensis, Agropyron trachycaulttm and Oryzopsis hymenoide.r growing in peat-mineral mix (åontrol), Syncruãe coke, Syncrude coke +- cap, Suncor coke or Suncor coke *+ cap.s¿p. No significant differences exist between tneans followed by the same letter (n = 5. o: 0.05).0.05 --- denotes no available data

B (t¡g g-') Fe (ug g-') Mo (pg g-') Ni (pg g-') s (pg g-') T. aestivum Control + 11.37 3.33 0 + 0" 1.13 r 0.25" 5.48 3.60 " r 1505 r 168 b Syncrude 10.31 t 1.53" 560 r 285 5.75 " b ! 1.66 b 0.11 r 0.11 a 1221 !342b Syncrude + cap 18.99 t6.92a 192 + 45 0.31 f 0.19" 9.56 4.39 ^b t a 369 t 190" Suncor 13.54 + 3.19" 209+38"0 1.54 0.29 ! , 0,1910.19" 1508 r 96 b Suncor + cap + 16.26 3.66 282 ! 47 a,o 0.00 0.00 r 9.60 r 5.93 419 ! 291 a " " " D. caespitosa Control 11 .32 ! 1.76 0 + 0" 0.09 r 0.04 0.00 ^ t 0.00 1557 !71b Syncrude 17.67 + 2.50 " " 823 !221 c 17.46 ! 4.14 c 0.00 0.00 ^ r 2186!240 6 Syncrude + câp 12.87 2.17 " t a 380 r 73b 2.84!0.39"6 0.00 r 0.00 34!34u Suncor 14.46 1.96 " t a 315 r 107 6.20 ! 1.03 b 6.71 0.99 ^b ! b 1974 t 385b Suncor + cap 11.24 + 1.50 332 + 43 u 0.00 t 0.00 a 0.00 r 0.00 12+ 12" ^¿ " C. canadensis Control 0 + 0" 1.89 t 0.76 1504 ! 170 6 Syncrude 668 J 323 + ^ b 36.29 6.96 u 1260 !295 Syncrude + câp b 352139"b 1.82 ! 1.92 0+0 Suncor 84 !27 ^ " 4.27 ! 2.19 1275 t 316 b Suncor + ^ ^ ca 268 !71 0.00 r 0.00 0t0

Table continued onþllowing page (^(,¡r Table 3-5: Continued BFeMoNi (pg g-') (pg ,) g-,) (rrg ö 'l tus s

Control 0J0u 1.36 10.07, 1110r43b Syncrude 1371 t268b + Z2.gT 2.98" 1331 r 197 6ç Syncrude + câp 335 + 80 13.66 2.36 " t u 157 r 99a Suncor 200 ! 29 a S.3B r 0.44 a 1629 ! 121 Suncor + cap 255 ! 37 " a o.0o t 0.oo a 265 ! 146 a - O. hymenoides Control 0t0a 19.46+g.73¡" 1507 r 89" Syncrude 264 t 1126ç 20.36 + 9.09 990 r 373 Syncrude + cap 245 69 " " ! bc 15.41 t 4.go 6 535 + 195. Suncor 73 131 " " ab 1.g3t0.g5uu 1378 ! 573 a Suncor + ca 320 r 65 0.00 r 0.00 318 r 318

(Jl o\ Table 3'6: Element content (mean + SE) in shoot tissues of Triticttm aestivum, Deschampsia caespitosa, Calartagrostis canadensis, Agropyron trachycaulum and Oryzopsis hymenoides growing in peat-minerãl rnix (cóntrol), Syncrud-e coke, Syncrude * câP, Suncor coke or Suncor coke r cap. No significant differénces exist between means foilowed by the saÁe letter n: ).5, a:G: 0.05u.

g-') B (pg Fe (pg g-') Mo (pg g-') Ni (pg g-') s (pg g-') T. aestivum Control 10.51 1.28 + r 12.7 5.4 1.21 ! 0.31 0.00 r 0.00 1698 191 ^ ^ " r Syncrude 12.92r 1.15¿6 45.816.60" 7.58 2.74 " "b r ø 46.84 ! 32.57 a 1540 r 166"6 Syncrude + cap 16,83 r 3.93ab 42.5 ! 3.5 o" 1.17 ! 0.49 0.00 r 0.00 1350 t 169"u Suncor 20.38 + 2.99 " " u 26.5!1.6"u 0.00 r 0.00 68.51 r 43.62a 1745 ! 213,o Suncor + cap 10.49 1.04 " ! a 65.0 r 15.8. 3.99 r 2.01 ab 0.00 t 0.00 1171 !79 " a D. caespitosa Control 20.51 ! 1.32 0+0.0" 1.27 0.80 ! 0.00 r 0.00 2552 ! 216 b Syncrude ^ " " 45.86 r 5.64 b 100 + 4.5 27.66 r 0.98 c 1.68 t 1.22u 2890 I 351 b Syncrude + câp 26.84 r 1.66a 95+4.2"" + 12.60 1.16 o 1.78 ! 1.13, 1617 ! 172a Suncor 29.59 + 1.93 +5.4u 61 13.98 r 0.51 b 7.52 1.69 " ! b 2831 ! 177 6 Suncor + câp 26.90 ! 3.21 111 ! 12.9 3.62 ! 0.33 0.00 r 0.00 1147 !265a ^ " " " C. canadensis Control 0.9 t 0.ga 0.53 r 0.08 1619 t 42 b Syncrude gg.4 + 4.4 " c 15.96 + 1.07 ao 294 t 543 Syncrude + câp 107.0 + " 13,8" 3.42 ! 0.41 , 1304 ! 111 Suncor ,6 59.1 t 3.1 b 57.5 r 31.13 b 2676 t 174 c Suncor + ca 1 10.8 ! 13.7 0.00 t 0.00 853 r 160 L¡I Table continued onfollowing page \ì Table 3.6: Continued B Fe Mo Ni S (ug g-') (ug g-') (pg g-') (þrg g-t) (ug g-') A. trøchycøulum Control + 10.9 5.5 a 0.80 r 0.29 1527 Syncrude a !61 a 80.4 r 9.6. 7.23 ! 1.15 2177 !367 b Syncrude + cap 79.9 r 9.3 ^ c 2.67 ! 2.40 u 960 134a Suncor r 52.1 t 3.6 b 4,85 t 0.8S u 2176 ! 86,D Suncor + cap 71.9 ! 6.4¡. 14.14 + 14.14 1170 + 96a ^ O. hymenoides Control 20.6 r 6.9 0.17 !0.11 " 2271 !215 Syncrude 52.1 r 3.0 ^ ^b b 9.96 r 1.96 2020 ! 216 ab Syncrude + cap 51,6 3.0 " r b 6.05 t 1.04b 1756 ! 200 Suncor ^ 46.5 ! 2.9 6 4.64 ! 0.47 6 2413 ! 86 6 Suncor * ca 53.1 r 6.3 0.84 r 0.56 2030 !74 --- data not available

L'I oo s9

3.4. DrscussroN:

3.4.1. SBso EnlnRcnNcn rN Coxn

It was originally thought that emergence of seeds sown in coke would be inhibited by the low water holding capacity, rapid drainage of water from coke and the potential toxic effects of metals. However, the data shows an enhanced rate of emergence in D. caespitosa, A. trachycaulum, C. canadens¡s and O. hymenoides grown in uncapped coke between the days 4 through 7. Furthermore, in almost all cases, the final emergence in coke treated plants did not suffer from coke exposure (Figure 3.1 and Figure 3.2). Three key environmental factors contribute to seed germination and emergence; water

'Water availability, temperature, and air quality (oxygen) (Forcella et al. 2000). was not limiting emergence as coke treatments had higher emergence rates between days 4 to 7 despite holding less water than controls (Table 4.2). Although no data was collected to test this hypothesis, it could be argued that the black colouration of coke would convert more light energy into heat thereby accelerating the germination and emergence process

(Blackshaw 1990, Blackshaw et aI.2002).It is also likely that oxygen (air quality) is responsible, in part, for the observed differences in seedling emergence between treatments. As the peat-mineral mix used for controls contains a high proportion of clay, the size distribution of soil pores is skewed towards smaller pores, namely micropores.

Yanful (1993) has shown that gas exchange between the atmosphere and micropores in clay soil is hindered which would effectively limit the supply of oxygen to seeds. Bello et al. (2000) demonstrated that seeds exposed to oxygen concentrations lower than ambient 60

atmospheric conditions suffered from decreased rates of emergence and survival. This

could explain the reduced rate of emergence observed in control plants and the lower

emergence observed in control D. caespitosa.

Although commonly used, germination/emergence trials are insufficient for

assessing potential phytotoxic effects of a compound. Stored nutrients and biomolecules

in seeds may mask any effects caused by nutrient deficiencies and/or inhibition of

biochemical pathways required for physiological processes such as photosynthesis or

protein synthesis. For these reasons plants were grown and monitored over several

months so that longer term effects of coke exposure on gïasses could be observed.

3.4.2. Warpn Srarus oF GRÄssES GRowN rN CoKE

Drought conditions could occur in coke as its texture and strucfure is conducive to

rapid drainage of water. Decreased transpiration and stomatal conductance rates suggest

that T. aestivum and A. trachycaulum aÍe suffering from drought stress despite a regular

watering regime (Figure 3.5). Decreased transpiration is often associated with closure of

stomates, a strategy which prevents the loss of water by transpiration (Liu and Luan

1998). The decrease in stomatal conductance observed in coke treated T. aestiyam and D.

caespitosa suggest that stomates are partially closed providing further evidence that the

plants are suffering from drought stress (Figure 3.5). Flexas and Medrano (2002)

suggested that plants with stomatal conductance rates between 50 and 100 mmol H,,O m-2

-l' ^^ s are sufiêring fiom drought stress. According to these authors, even control l. tracltycaulum and C. canadens¿s would be suffering from drought stress. Calamagrostis canadensis has shown some drought tolerance (Hardy BBT Ltd. 19g9) which may explain why transpiration rates and stomatal conductance \ilere not affected by coke 6l

treatment. Drought conditions could explain, at least in part, the reduction in growth

observed in coke treated grasses. Growth inhibition in drought stressed plants can be related directly to a reduction in available water as low cellular turgor, which can be

caused by drought conditions, is known to limit cellular elongation (Hale and Orcutt

1987). However, limited CO2 supply caused by the closure of stomates is thought to be the principle cause of growth inhibition in plants suffering from drought stress (Flexas and Medrano 2002). Lawlor (1979) observed that closure of stomates caused an increase in Oz-to-CO2ratio in leaf tissues which resulted in an increase in photorespiration. Under conditions of high 02 conditions, the enzyme ribulose bisphosphate carboxylase/oxygenase (RUBISCO), which typically fixes atmospheric co2 for carbohydrate production, acts as an oxygenase and binds oxygen instead of COz. This process decreases the rate of carbohydrate synthesis and has been known to inhibit the production of biomass by up fo 50o/o (Nelson and Cox 2000). Furthermore, the activity nitrate reductase and sucrose phosphate synthase have been shown to be inhibited under drought conditions; however, enzpe activity can be restored by watering which suggests that the enzymes are not permanently damaged (Flexas and Medrano 2002). Under more severe drought conditions, damage to the photosynthetic apparatus does occur as it has been demonstrated that photosynthetic pigment content decreases under conditions of drought stress (Jagtap et al. 1998, Bartoli et al.2004). Energy absorbed by photosynthetic cells under stress conditions can be transferred from the photosynthetic pigments to oxygen thereby forming free radicals/reactive oxygen species (Smirnoff 1993). These free radicals can damage cellular membranes and bleach photosynthetic pigments

(Halliwell 1982). Under water stress, the rate of loss of chlorophyll a is not proportional 62

to the loss of chlorophyll b which often results in an increase in the chlorophyll alb ratio

(Bracher and Murtha 1993). No differences were detected in the chlorophyll a./b ratio

between controls and coke treated plants. It is therefore reasonable to suggest that

drought stress may be responsible for lower concentrations of photosynthetic pigments in

coke treated plants; however, it is very likely that other stresses are also responsible for

the reduction in growth and lower concentrations of photosynthetic pigments.

The root: shoot ratios in some coke treated plants were larger than controls (Table

3 ' I ). This is an expected response to water stress as many studies report that shoot growth

is more sensitive to water stress than root growth (Westgate and Boyer 1985, Creelmann

et al. 1990). It has been suggested that this change is initiated by the plant to allow for

continued exploration and water uptake during the drought period (Glass 2002).

3.4.3. AnsnNrc, vnNluuvr, BoRoN, InoN, MolvnoBNUM AND Nrcxnr, IN coKE

Tnr¡.rnp Gn¿.ssns

Coke contains elements such as arsenic (As), vanadium (V), and micronutrients

such as boron (B), iron (Fe), molybdenum (Mo) and nickel (Ni) at concentrations which

may pose a risk to plant health (Chung et al. 1996). A common problem when assessing

the elemental composition of plant tissues is contamination of tissues with growing

medium. Certain authors have circumvented this problem using a correction method

which compared the ratio of Tiro¡¡-to- elementro¡¡ to the ratio of Tio¡on1-to-elem€rtprant (Cary

and Kubota 1990, Sheppard and Evenden 1995). Despite its use, the aforementioned correction fails to differentiate between elements precipitated in the apoplast or elements bound to cell wall from the physiologically available elements present within the living OJ

portions of cells (Greger 1999). Therefore, all data related to uptake of elements should

be considered carefully as concentrations of elements found in tissues, particularly roots,

may not reflect biologically active concentrations of the element in living tissues.

Despite the presence of arsenic in coke, accumulation of As in root and shoots

were below detection limits. The sample detection limits were determined by multiplying

the machine detection lirnits by a dilution factor (volume of digest / weight of sample).

However, as a small amount of ground tissue were used for the ICP-OES analysis, the

detection limit for As was inflated and far exceed the literature values (0 to 3 pg g-') for

expected As concentrations in plant tissues (Table 4.3). Therefore it is possible that As

has accumulated in grasses at concentrations which may induce phytotoxic effects (Aller

et al. 1990)' A more sensitive analysis was used for the quantification of As in root and

shoot tissues in chapter 4 to elucidate the risk posed by As accumulation.

Literature values for V concentrations in plant tissues range between 0.27 ¡tg g-l

and 4.2 pg g-r (Aller et al. 1990). Concentrations of V in roots of coke treated plants

ranged from 0 to 28 pg g-r in roots and from 0 to 19 pg g-r in shoots. Partitioning of V was not consistently higher in roots than in shoots as noted by Basiouny (l9ga).

Vanadium concentrations which exceeded both the literature values and the concentrations in the controls were only found in roots of T. aestiwtm (136 x more than controls), D. caespitosa (6 x more than controls), A. trachycaulum (5 x more than controls) and C. canadensis (4 x more than controls) grown in uncapped Syncrude coke

(Table 3.4). Davies et al. (1978) showed that V concentrations as low as Z pg g-'of V in dried tissue caused chlorosis and stunting in barley (Hordeum vulgare). Other symptoms ofV toxicity such as decreasedleaf area, decreased leafnurnber and depressed grain 64

leld were observed in Zea mays (Singh 191l). A srudy by Jacobs andTajz (19g0)

showed that vanadate can inhibit cell wall elongation by inhibiting H*-ATpase pumps in

the plasrna membrane (P-type ATPase). These pumps are important for cell growth as

they acidify the cell wall environment which leads to breaking of cell wall bonds by

activating cell-wall-loosening enzymes (Jacobs and Taiz 1980). Furthermore, the p-type

ATPase pumps are responsible for producing and maintaining a transmembrane H*

gradient which is critical for the transport of anions, sugars and amino acids across the

membranes. Vanadium has also been shown to inhibit the function of the molybdo-

protein nitrate reductase (Ramadoss I979). Vanadium concentrations in shoots of

controls are equal to or greater than concentrations in plants grown in coke and therefore

are not expected to be solely responsible for the stress symptoms observed in coke treated

plants. Concentrations in roots of plants growing in uncapped Syncrude coke do exceed

concentrations in controls and could potentially explain, in part, some of the visual stress

symptoms observed in T. aestivurn as well as lower biomass and pigment content

observed in the other experimental species growing in Syncrude coke. The elevated

concentrations of V in roots of plants growing in Syncrude coke may be attributed to the

reduction of soluble v*s compounds into non-soluble v*a by aldehydes, ketones,

catechols, olefins (alkenes) and sulphydryl containing compounds present in the cell wall

(Basiouny 1984, Morrell et al. 1986). Further work is needed to determine the specific role of the aforementioned V concentrations in the experimental species grown in petroleum coke. 65

Many of the elements present at higher concentrations in coke are micronutrients,

which are required in low concentrations for plant growth and development. However, at

higher concentrations many micronutrients are known to have phytotoxic properties

(Aller et al. 1990). Boron concentrations in monocotyledons typically range from I to 6

¡rg g-r (Jones 1998). The concentrations of B in controls and coke treated plants of Z

aestivum and D. caespitosa exceed the higher range of the required concentrations and

are therefore not likely to be deficient (Table 3.5 and Table 3.6). Boron toxicity is

difficult to diagnose as critical concentrations for B toxicity spans over a wide range (10-

130 pg g-'¡ both within and between species (Nable et al. I 997).Inthe present study, it is

possible that plants from all treatments were suffering frorn B toxicity as concent¡ations

of l3 pg g-r in barley (Hordeurn vulgare) shoot tissue have been reported to cause leaf

injury typically associated to B toxicity (Brennan and Adcock2004). The primary visual

symptom of B toxicity is leaf burn, particul arly at the tips and margins of older leaves.

Syrnptoms of B toxicity bear a resemblance to those suffered by T. aestivum growing in

coke (Figure 4.2). However, Triticum aestivam shoots grown in uncapped Suncor coke

and D. caespitosa shoots grown in Slmcrude coke are the only cases where exposure to

coke is responsible for an increase in B concentrations in plants. Severe B toxicity caused

by exposure to coke is not likely to have occurred in the aforementioned plants as none of

the visible symptoms associated with B toxicity, such as chlorotic and necrotic patches at margins and tips of older leaves, reduction in height and reduction in dry weights were

exclusive to the treatments with higher boron concentrations. 66

The expected concentrations of Fe in plants ranges from 50 to 7 5 ¡tg g-l with

shoot concentrations reaching up to 1,000 pg g-r (Jones 1998). Although many coke

treated plants have higher iron concentrations than the controls, no negative effects

caused by iron overload stress are expected as the concentrations are quite low and would be considered deficient, particularly in controls. Common slrnptoms of Fe deficiency include interveinal chlorosis, decreased chlorophyll content, depressed photosynthetic rate, and lowerprotein contents (lturbe-Ormaetxe et al. 1995, Jones 1998). The decrease in chlorophyll observed in coke treated plants is not likely explained by Fe deficiency as controls have lower Fe concentrations but higher chlorophyll concentrations than coke treated plants.

Molybdenum concentrations in plant tissues typically range from 0.34 to 1.5 -r pg g (Jones 1998). Molybdenum concentrations in T. aestivum and D. caespitosa grown in coke was quite variable and ranged from below detection limit (Table 4.3) to almost 19 times the expected values (27.6 ¡tg g-'¡ ltabte 3.5 and Table 3.6). Reported cases of Mo induced ph¡otoxicity are rare and it is generally accepted to be relatively non-toxic to plants (Jones 1998). However, FargaSovà (1998) found that root length of

Sinapis alba was decreased when treated with Mo resulting in concentrations greater than

4,300 and 1,500 pg g-r of Mooa3- in root and shoots respectively. Although Mo concentrations in our experiment are not likely to cause toxic effects in plants, they may be problematic to grazingruminants. A review of the literature by Neunhäuserer et al.

(2001) found that ruminants grazing on feed containing over 10 pg g-t of Mo or having a 67

Cu: Mo ratio of less than 2 developed molybdenosis. Molybdenosis is a potentially fatal

disease in ruminants induced by a Cu deficiency caused by the binding of Cu to a

rnolybdenum-sulphur complex called thiomolybdate (Neunhäuserer et al. 2001).

Notwithstanding Ni hyperaccumulators, Ni concentrations in plant tissues

typically range from trace amounts to 8 pg g-' lAdriano 1986). Nickel deficiency is not

likely due to its relatively low requirement for plant growth. Coke treate d. T. aestiwun, A.

trachycaulum, C. canadensis and O. hymenoides accumulated between 2 (16 ¡tgg-'¡ to 9

times (68.5 pg g-r) more Ni than expected based on literature values. Nickel

concentrations in coke treated plants fall within the range of toxic concentrations (10 to

300 pg g-') (Davies et al. 1978, Khalid and Tinsley 1980, Aller er al. 1990, parida et al.

2003). Nickel toxicity has been reported to depress yield, cause metabolism disorders and

decrease photosynthetic pigment concentrations in plants treated with 7 ¡tgg-' - S070 pg

g-r lvergano and Hunter 1953, Frank et al. r 982,yanget al. 1996). However, the

severity of the interveinal chlorosis and tissue necrosis, both symptoms of Ni toxicity, are

dependent on the ratio of Ni-to-Fe in both the soil and plant tissues (Vergano and Hunter

1953, Crooke et al. 1954, Crooke 1955, Crooke and Inkson 1955). Crooke et al. (1954)

and Crooke (1955) demonstrated that the severity of the phytotoxic syrnptoms was

negligible when the Ni+o-Fe ratio in shoot tissues was below l. However, Ni toxicity

augmented in severity as the ratio ìncreased. These data agree with studies which suggest

that excess Ni induces sy'rnptoms related to Fe deficiency. It is speculated that an increase in the Fe relative to Ni decreases the likelihood of Ni displacing Fe in enzymes and other biological molecules (Crooke et al. 1954, Crooke 1955). Based on arguments by Crooke

(1954, 1955) it would seem that nickel toxicity is not a cause for concern in coke treated 68

plants as the Ni-to-Fe ratios in our experimental species were all below l. Care should be

taken when interpreting this conclusion as the results from Crooke's group have not been

confìrmed by current studies. At an ecological level, several studies have shown that

although Ni can be transferred from plant tissues into higher trophic levels it will not

bioconcentrate in the food web (HeliövaaÍaand Väisän en 7987,Torres and Johnson

2001, Peterson et aL.2003, Boyd et al.2006).

A deficiency in other nutrients required for plant growth may be responsible for

the slnnptoms observed in coke treated grasses. Analysis of coke by Chung et al. ( I 996)

indicates that coke does contain macronutrients and micronutrients required for proper

plant growth (18,000 pùg g-¡ N, 97 pg g-r p,l,O47 pg g-' K, 1,400 pg g-l Ca,576 F.g g-r

Mg, 66,000 pg g-t S, 4,578 pg g-r Fe, g-r 79 pg Mo, 136 pg g-' Mn, l3 pg g-' B, 12 ¡tg g- I Cu, 500 pg g-l Ni, and 17 ftg g-' Zn);however, little is known about the geochemistry of

coke and the form/availability of the abovementioned elements is unknown. Furthermore,

it is suspected that the elements are imbedded in a carbon matrix thereby limiting

bioavailability. Nutrient deficiency can also arise from high C-to-nutrient ratio in the

growing medium. As carbon accounts for approximately 80% of cok e can, depending on

the availability of the carbon, increase the carbon-to-nitrogen ratio. Under these

conditions, micro-organisms utilize carbon while sequestering (immobilization) other nutrients such as nitrogen, thereby inducing a deficiency (Uhart and Andrade 1995). This concept is not unique to nitrogen, and can be applied to any nutrient required for both plant and micro-organism growth. Although fefülizer containing nitrogen, phosphorus and potassium was added in all treatments, it is probable that micronutrient were limiting.

It is even likely that macronutrients may become limiting due to the potentially elevated 69

carbon-to-nutrient ratio. Studies describing the micro-organism community present in

coke and the bioavailability of coke components could determine the probability of an

induced nutrient deficiency caused by elevated carbon contents.

Plants growing in coke for 3 months (7. aestiwtm) or 4 months (D. caespitosa, A.

trachycaultnn, C. canadensis, O. hymenoides) were shorter, had less root and shoot

biomass and had an increased root-to-shoot ratio when compared to the controls (Table

3.1, Figure 3.3 and Figure 3.4). It is well documented that macronutrient andlor

micronutrient deficiencies yields stunted plants with lower biomass. For example,

deficiencies in Ca, which is important in the production of the middle lamellae and cell

wall components in new cells produced in meristematic regions, have been known to

result in abnormal development and necrosis of meristematic tissues thereby limiting

growth (Jones 1998, Kakegawa et al. 2005). Furthermore, an increase in root to shoot

ratio, as observed in most coke treated grasses, coupled with an overall decrease in root

biomass is a common response by plants to nutrient deprivation stress (Harris I 914, Glass

2002)- The reduction in photosynthetic pigments observed in plants grown in coke provides further evidence to support a nutrient defìciency (Table 3.2). Nitrogen and Mg

are components of the chlorophyll molecule (Jones 1998). Deficiencies in N and Mg have been associated with a decrease in the amount of thylakoids per grana in the chloroplasts as well as a decrease in the photosynthetic pigments (Guller and Krucka 1993,Laza et al.

1993, Kutík et al. 1995, Jones 1998). In coke treated plants, we observed a decrease or no change from the proline concentrations in the controls. These results were unexpected as proline is known to increase in certain plants as a mechanism for water stress tolerance

(McMichael and Elmore 1977, Taylor 1996). A study by Elmore and McMichael (1981) 70

demonstrated that proline does not accumulate under conditions of nutrient deficiency,

despite being grown under drought conditions. Therefore the decreased proline content

observed in coke treated D. caespítosa and A. trachycartlmn provides further evidence

supporting nutrient deficiency as an important factor limiting growth of coke treated

plants.

Unfortunately, no data pertaining to content of the macro and micro nutrients N,

P, K, ca, Mg, Mn, B, cu, andzn of gtasses gïown in coke or peat-mineral mix was

available as plant nutrition was not a primary objective of the study. Determination of

nutrient concentrations in tissues is required to elucidate the role of the plants nutrient

status on growth, pigment content, and proline production in coke treated plants. These

questions will be addressed in chapter 4.

3.4.4. Spncrrs Couparusox

Agronomic species, such as T. aestivum, do not perform as well as non agronomic

species in stressfull environments (Brown 1969). Triticttm aestivum, used as the test

species, was the only species to show visual signs of stress other than stunted growth in

plants grown in petroleum coke. Furthermore, a comparison of transpiration rates

between plants grown in coke and the controls reveals that transpiration rates in Z.

aestiwtm decreased by 45%to glYo whereas C. canadens¿s and A. trachycaulttm

decreased by -22% to 58o/o and -6.9%o to 80o/o respectively. Furthermore, chlorophyll a

and chlorophyll b content in T. aestivum grown in capped and uncapped coke were only

32o/o to 48 Yo and 33o/o to 680lo respectively of the chlorophyll found in controls. The chlorophyll content of non agronomic species gïown in coke treatments ranged from 560/o 7l

to lllo/o (Chl a) and 560/o to 108% (Chl b) of the concentrations found in controls. The results confìrm that the non-agronomic species performed better than the agronomic

species for land reclamation purposes.

Despite decreases in transpiration, stomatal conductance and photosynthetic pigment contents, T. aestiwtm growth did not suffer from a greater reduction relative to the non-agronomic species. Shoot biomass of coke treated T. aestivum, D. caespitosa, C. canadensis, A. trachycaulum, and O. hymenoides accounted for l8%o to 32o/o, l0o/o to

35o/o, l3o/o to 27o/o, ll%o to 31o/o and l0o/o to 45o/o of the control shoot biomass. Coke treated T. aestitntm accounted for 72o/o - 95% of the control root biomass where D. caespitosa, C. canadensis, A. trachycaulum and O. hymenoides accounted for 22%o to

57o/o,27yo to 54o/o,29o/o to 660/o and 32Yo to 72o/o of the control roots biomass respectively. Although the root dry weight data seem counterintuitive, it should be noted that a notable amount of T. aestivum roof biomass was lost during the root washing process which would express itself as less inhibition in root growth than otherwise expected.

With sufficient amendment in the form of fertilizer and capping, all species would likely survive reclaimed coke sites. Based on its apparent resistance to drought stress and moderate capability for ground cover, C. canadenszs appears to be better suited for revegetation of sites at higher risk of water stress. However, C. canadens¿s did accumulate higher concentrations of elements from coke than other speci es. Agropyron trachycaulum and O. lrymenoides produced tall plants and tillering was relatively low compared to other species suggesting that these species are poor candidates for use as a ground cover. 72

CHAPTER 4 - Gnowru, PttvslolocyAND Er,nlrpNr Accutlur,ATroN op Contvus sERrcEA tNo Fn¿c¿RrA vrRcrNrAiv,< Expospo ro Corn

4.1. INTnoDUCTIoN:

The previous chapter described the responses of grasses to coke. Based on trends in physiological measurements, growth, and elemental analysis of tissues, it was determined that grasses grown in coke were likely suffering from drought stress, nutrient deficiency andlor toxicity. Low transpiration rates, stomatal conductance, growth, pigment content and high roolshoot ratio provides evidence that plants are suffering from drought stress. Nickel, V, B and possibly As were found in root and shoot tissues at concentrations which may be phytotoxic. Evidence also suggested that nutrient deficiency could be problematic for plant growth in coke but the nutritional status of plants growing in coke could not be assessed as nutrient concentrations were not measured.

In the previous chapter monocotyledonous plants were tested. Despite the many similarities between monocotyledons and eudicotyledons, they do differ with respect to their nutritional requirements and some of their stress responses. Therefore, a second study using the forb Fragaria virginiana and the shrub species Cornus sericea was undertaken.

Although similar to the study outlined in chapter 3, the current sfudy emphasized the accumulation of elements in plant tissues in order to elucidate the risk of toxicity caused by As, B, Ni, and V. Furthermo¡e, the concentrations of nutrients in root and IJ

shoot tissues were measured to clarify whether inadequate nutrition is responsible for

slrnptoms observed in plants grown in coke. Additionally, an updated protocol with different extinction coeff,rcients was used to quantify chlorophyll concentrations.

Fragaria virginiana and Cornus sericea were gro\¡/n in coke under greenhouse conditions to measure the effects of coke on growth, physiology and nutrition of a forb and shrub species. The first objective of the current study was to determine the effects of coke on the growth and physiology of F. virginiana and C. set"icea. Growth and chlorophyll were measured as a general measure of plant stress. It was thought that transpiration rates would decrease as a response to water stress. The second objective was to determine the role of nutrient status in explaining the reduction in growth and physiology of coke treated plants. It was suspected that coke treated plants could potentially suffer from symptoms of micronutrient deficiency as coke contains low concentrations of micronutrients. Furthermore, the potential for an elevated C/ nutrient ratio in coke could potentially induce a deficiency in any/all nutrients in plants. symptoms of nutrient deficiency would be expressed as decreased growth, photosynthetic pigment (magnesium/ iron deficiency), increased roolshoot ratio and increased chlorophyll alb ratio. The third objective was to determine whether plants growing in coke were accumulating concentrations of non-essential elements, namely arsenic (As) and vanadium (V) in root and shoot tissues at concentrations which could induce phytotoxic effects in coke treated plants. It was thought that coke treated plants would contain elevated concentrations of the aforementioned elements as they are found at elevated concentrations in coke. 74

4.2. l4nrøoDs AND MarnRlru,s:

4.2.1. Pr,aNr M¿.rnnral aNn TnTaTMENTS

Upon mine closure, the oil sand industries are responsible for reconstructing a

landscape with similar composition, diversity, and productivity as the previous or

surrounding landscapes (Oil Sands Vegetation Reclamation Committee 1998). Fragaría

virginiana and Cornus sericea were listed during an inventory of boreal forest

communities conducted to aid in designing the species composition and distribution of

reclaimed sites (Purdy et al. 2005) . Fragaria virginiana and Cornus sericea were selected

based on their natural distribution, potential for use in land reclamation and tolerances to

stresses which may occur on reclamation sites.

corntts serÌcea (synonym cornus stolonifera) - Red osier dogwood

o Natural range extends over Athabasca oil sand deposit in northern Alberta

(Rydberg 1956, Scog gan 1957 , Hardy BBT Lrd. l9g9)

o Lack of literature pertaining to woody plants growing in coke

' Plummer (1970) recommends C. sericea for stabilization of disturbed

areas

' Can grow relatively well on saline consolidated tailings (CT) compared to

other reclamation species (Renault et al. 1999,2001)

Fragaria virginiana - Wild strawberry

o Natural range extends over Athabasca oil sand deposit in northem Alberta

(Scoggan 1957,Hardy BBT Ltd. 1989).

o Lack of studies looking at forb species and coke exposure 75

one of the most important herbaceous species colonizing surface coal

mine (Brenner et al. 1984)

. Found growing in sandy, nutrient poor soils of Sable Island, nova Scotia

(Catling et al. 1984)

Cornus sericea seeds were provided by Syncrude Canada Ltd. and originated

from wild populations near the mine area north of Fort McMurray, Alberta, Canada.

Seeds were stratified following the method outlined in Mustard (2002) and planted in a

3: I (V/V) mixture of peat moss and sand. Fragaria virginiana runners were collected from wild populations in south-eastern Manitoba, Canada. In this second experiment, C. sericea seedlings and F. virginiana runners were planted in coke instead of seeds as these species would be planted as seedlings in a reclamation site. The initial step in the experimental setup consisted of washing the peat and sand from roots of the experimental species and transplanting them into 700 ml deepots containing a) peat-mineral mix

(control); b) coke produced by syncrude canada Ltd.; or c) coke produced by suncor

Energy Inc. As past results demonstrated that plants could survive and grow in coke, the capped treatments were removed from the current study. The sources for peat-mineral mix and coke were the same as in chapter 3. The concentrations of elements in peat- mineral mix and coke are summarized in Table A.I and the gravimetric/volurnetric water holding capacity in Table 4.2.

High density polyethylene containers (37.9 L) subtended the deepots to ensure that water was not lost during watering. Plants were watered with approximately 50 ml of recycled water collected from the subtending trays. Distilled water was substituted in cases where inadequate volumes of recycled water were available. This was done to 76

prevent the loss of elements through leaching and/or the accumulation of salts at the

surface of the growing medium. Water soluble fertilizer was applied at 0, 35, and 56 days

after seeding for a final application rate of 500 kg ha-r of 12-36-12-4 (N-P-K-S). Plants

were grown under a 16 hour photoperiod of natural light supplemented with 400 W high

pressure sodium vapour lights (P.L. Light Systems, Beamsville, Ontario, Canada).

Greenhouse temperature was maintained at24 + 3oC. The experimental design was

completely randomized and each treatment replicated 5 times to ensure statistical validity.

4.2.2. Gnowrn

At the onset of the experiment, extra C. sericea and F. virginiana plants were harvested and their root biomass measured to provide a baseline for determination of new root growth. At the time of transplanting, C. sericea shoots were pruned back to the lowest 2 buds while F. virginianø shoots were cut back to the crown. This ensured that all shoot growth used in measurements was produced during exposure to coke. After th¡ee months of treatment, plants were harvested, washed twice in distilled water and rinsed once with deionised water. Root and shoot new growth was separated, shoot height determined (C. sericea only) and the fresh weight recorded. The tissues were frozen in liquid nitrogen and lyophilized for determination of dry weight. New root growth dry weight was estimated by subtracting the baseline values from the recorded values. The new shoot and new root growth will be referred to as shoot and root growth throughout the remainder of the chapter. 71

4.2.3. CHr,onopnyI,I, AND PnnopuyrrN

Chlorophyll a, chlorophyll b and pheophytin a content were assessed after 8

weeks of treatment. A different protocol than the one used in chapter 3 was used as it

allowed for the determination of pheophytin a, a degradation product of chlorophyll a.

Determination of pheophytin could clarify whether lower chlorophyll a content was

attributable to an inhibition of slmthesis or an increased rate of degradation.

Extraction and quantification of chlorophyll a, pheoph¡in a, and chlorophyll b was conducted using a modified protocol by Jeffrey and Humphrey (1975) and Axler and

Owen (1994). Two leaf discs (l cm diameter) were collected per plant and their fresh weights were recorded. The discs were then frozen with liquid nitrogen and finely ground using a mortar and pestle. Pigments were extracted 3 times with2 ml 90 o/o acetone. The

6 ml of pigment extract were centrifuged at 1000 g for 5 minutes and the absorbance of the supematant measured at 664 nm,64l nrn and 750 nm using a uv/visible spectrophotometer (Ultraspec 2000 UVfVisible light spectrophotometer, Pharmacia

Biotech, Cambridge, England). The extract was then acidified with 0.12N HCI (100 pL per 4 ml of extract). The absorbance of the acidified extract was read after 2 minutes at wavelengths 665 nm and 750 nm. Chlorophyll and pheophytin concentrations were determined using the following equations (Jeffrey and Humphrey 1975, Axler and Owen

19e4):

Chlorophylla={[(11.93*(Abs.66a-4bs.756)]-[1.g3.(Abs.o¿z-Abs.zso)]].V/Wt./1000

* * Pheophytin a - (Pheo. âr Chl. arl Chl. ar) V /Wt. / 1000

% pheophytin = pheophytin a / (pheophytin a + chlorophyll a)

Chlorophyll b = (20. 36.(Abs.e+z - Abs. 756))-( 5. 5*(4bs.664 - Abs.zso))*V^/Vt./1 000 78

Where:

Pheo. at 26.7*l(1.7"Abs = 665u.¡¿ - Abs.Tsoacid) - (Abs.66a - Abs.756)l

Chl. a1 = [1 1.93.(Abs.oo¿ - Abs.75s)]-[1.g3*(Abs.6a7 - Abs.756)]

Chl. a2 = 26.7*l( Abs.66a - Abs.75e)-(Abs.665".¡6 - Abs.zso""io)]

V = Volume in ml

Wt. = Fresh weight of tissue weight in g

The subscript "acid" refers to an absorbance measured after acidification

Values were expressed as mg pigment g-l fresh tissue.

4.2.4. Tn¡Nsprn¡rrou

Transpiration rates were measured for C. sericea and F. virginiana after 7 weeks

of treatment. Measurements were taken from the newest set of mature leaves as described

in chapter 3.

4.2.5. PnoI,l¡cn

Proline was quantified using the modified Sofo et al. (2004) protocol outlined in

chapter 3.

4.2.6. Upraxr oF ELEMENTs

Ground root and shoot tissues of F. virginiqna and C. sericeawere sent for analysis of As, v, K, ca, B, cu, Fe, Mn, Mo, Ni and znby Inductively coupled plasma

Mass Spectrometry (ICP-MS), p and Mg by Inductively coupled plasma optical

Emissions spectroscopy (ICP-OES), N by combustion infrared analysis and S by combustion chromatography (Activation Laboratories Ltd. Ancaster, ON, Canada).

Detection limits of the elements by the aforementioned methods are summarized on 79

Table 4.4. Contamination of plant samples by soil or coke particles was corected by

assuming no uptake of Ti. Thus the % soil in samples could be estimated by comparing

the soil-to-Tiro;l ratio to the ratio in plant tissues and correcting accordingly (Cary and

Kubota 1990, Sheppard and Evenden 1995). The uncorrected data can be found in Table

4.6.

4.2.7. Dara ANALysrs

A one-way, nested Analysis of Variance (ANOVA) was used to analyzebiomass,

pigment content, transpiration and proline content. A one way ANOVA was used to

compare means of emergence and element uptake. Duncan's multiple range test was used

to compare differences between means. When comparing the concentrations of elements

in root and shoot tissues, concentrations which were below detection limit were

considered as Yz the detection limit as recommended by the USEPA for statistical analysis

including censored data (USEP A2006). All statistical analyses were conducted using the

SPSS statistical software (Version I 1.0.0, SPSS Inc., Chicago, USA).

4.3. R¡sulr

4.3.I. IN¡uny AND GRowTH

Visual signs of stress in coke treated C. sericea and F. virginiana wasmanifested

as stunted shoot growth and red coloration in leaves (Figure A.7 and Figure A.g).

Fragaria virginíana grown in coke also suffered from chlorosis and necrosis at leaf margins of some leaves (Fìgure 4.7). 80

At the time of harvest, C. sericea gro\¡/n in coke were shorter than control plants

(Figure 4.1). Furtherrnore, plants growing in Suncor coke were significantly shorter than

plants growing in Syncrude coke (Figure 4.1). Plant height was not measured for,F.

virginiana due to its rosette growth pattern. The estimated amount of new root growth was signifìcantly lower in C. sericea grown in both types of coke than in controls while no differences were observed between treatments in F. virginiana (Figure 4.2).

Furthermore, roots of C. sericed grown in Suncor coke were larger than roots grown in

Syncrude coke whereas no differences were observed between the root dry weights of coke treated F. virginiana.ln both species, the dry weight of shoots was significantly lower in coke treated plants than controls with no differences between coke treatments

(Figure 4.2). The root/shoot ratio was significantly higher in C. sericea growing in

Suncor coke and lower in Syncrude treated plants. Fragaria virginiana growing in

Syncrude and Suncor coke had higher roolshoot ratio than controls (Figure 4.2).

4.3.2. Cur,onopnyr,r, AND PHBopHyrrN

Chlorophyll a content in both species was significantly lower in coke treated plants than in controls (Table 4.1). Fragaría virginiana grownin Suncor coke had higher concentrations of chlorophyll a than plants grown in Syncrude coke, whereas C. set"icea showed no differences in chlorophyll a content between coke treatments (Table 4.1).

Pheophytin a concentrations were significantly lower than controls in F. virginiana grown in coke while no differences were observed between treatments in C. sericea.

o/o When the results are expressed as pheophytin a, F. virginiana showed a higher proportion of chlorophyll a which had been degraded into pheophytin a, whereas no differences were observed between treatments in C. serícea (Table4.1). Chlorophyll b 81

content was significantly lower in F. virginiana growing in Syncrude coke and, C. sericea

growing in Suncor coke (Tabl e 4.1). Fragaria virginiana grown in Syncrude coke had

higher chlorophyll b concentrations than plants grown in Suncor coke whereas no

differences were observed between coke treatments for C. serícea (Table 4.1).

4.3.3. Tn¡NsprnauoN

Transpiration in,F. virginiana grown in coke was lower than transpiration in control plants and no differences were observed between coke treatments (Table 4.2).

The transpiration rates were not significantly different between all treatments of C. sericea (Table 4.2).

4.3.4. PRor,rnB

Proline concentrations in shoot tissues showed no differences between F. vírginiana grown in control and coke treatments while C. sericea grown in coke had higher proline content than controls (Table 4.3). Additionally, no differences in proline content were observed between F. vir"giniana and C. sericea grown in Syncrude and

Suncor coke (Table 4.3).

4.3.5. ELnÀ,rsNr Coxrexr:

Arsenic concentrations in coke treated F. vírginiana and C. sericea roots were approximately 50%o to 72o/o lower than concentrations found in the controls whereas shoot As concentrations were only 5Yo to 32o/o lower than in controls (Table 4.4).

Vanadium concentrations in roots of-F. vìrginiana and C. sericea gro\Mn in Syncrude coke were higher than controls and reached concentrations of 93.56 pg g-'. Vanadium concentrations in roots of Suncor treated plants were below detection limits in F. 82

virginiana and 2.55 pg g-l in C. set'ícea. With the exception of F. virginiana grown in

Syncrude coke the concentrations of V in shoot were below detection limits (Table 4.4).

There was no difference in N concentrations between treatments in F. virginiana roots whereas C. sericea roots grown in Syncrude coke had higher N concentrations than controls and plants grown in Suncor coke (Tabl e 4.5).In shoot tissues, F. virginiana grown in both types of coke had less N than in controls while C. sericea grown in both types of coke had more N than controls (Table 4.6). The root P concentrations of .F. virginiana showed no change from the control while C. sericea roots showed an increase in P concentrations in coke treated plants (Table 4.5). Shoots of .F. virginiana grown in both types of coke and C. sericea grown in Suncor coke had higher concentrations of P in tissues than the controls. No differences in root K concentrations were observed between treatments of F. virginiana whereas C. sericeø roots grown in Suncor coke had less K than controls and plants grown in Syncrude coke (Table 4.5). Shoot K concentrations in

F. virginianawere higher in plants grown in both types of coke than in controls while no differences were observed between controls and coke treated C. set"icea. Potassium concentrations were similar in F. virginiana roofs and shoots grown in both types of coke. Cornus sericea growing in Suncor coke had lower K concentrations than Syncrude treated plants in roots and higher K concentrations than Syncrude treated plants in shoots

(Table 4.6). Calcium was lower in root and shoot tissues of F. virginiana and. C. sericea grown in both types of coke than in controls (Table 4.5 and Table 4.6). No significant differences were observed in root Ca concentrations between the two coke treatments in both species; however, shoots of both species grown in Suncor coke had higher concentrations of Ca than plants grown in Syncrude coke (Table 4.5 and Table 4.6). 83

Roots and shoots of coke treated C. sericea and .F. virginiana contained less Mg than the

control plants (Table 4.5 and Table 4.6). Furtherïnore, no statistical differences were

detected between Mg concentrations in F. virginiana roofs and shoots grown in both

types of coke while C. sericea grown in Suncor coke had more Mg in roots and shoots

than plants grown in Syncrude coke (Table 4.5 and Table 4.6). Concentrations of S in

root tissues of F. virginiana and C. sericea showed no differences between treatments

whereas concentrations in shoots were higher in both species growing in Suncor coke

(Table 4.5 and Table 4.6). Furtherrnore, C. sericea shoots grown in Suncor coke had

accumulated more s than plants growing in syncrude coke (Table 4.6).

With the exception of C. serìcea roots grown in Syncrude coke, less B was found

in roots and shoots of coke treated plants than in controls (Table 4.5 andTable 4.6).

Additionally, F. virginiana root and shoots as well as C. sericea roots had lower root B

concentrations in suncor than in Syncrude treated plants (Table 4.5). Copper

concentrations were higher than controls in root tissues of plants grown in coke, while

plants grown in Suncor coke had higher Cu concentrations than controls (Table 4.5 and

Table 4.6). With the exception of C. sericea shoots, no differences were observed in the

Cu content between both coke treatments (Table 4.5 and Table 4.6). No differences in Fe

concentrations were observed between treatments in F. virginíana, whereas Fe concentrations in C. serícea plants growing in Syncrude coke were higher than all other treatments (Table 4.5 and Table 4.6).Manganese concentrations in coke treated F. virginíana roots were lower than concentrations in controls while Mn concentrations in

C. sericea were higher in Syncrude treated plants (Table 4.5 andTable 4.6). With the exception of F. virginiana grown in Suncor coke, shoot tissues of plants grown in coke 84

had significantly higher Mn concentrations than controls. Additionally, no differences in

Mn concentrations were measured between coke treatments in F. virginiana roots and

shoots whereas Syncrude treated C. sericea had signifìcantly more Mn in roots and

shoots than Suncor treated plants (Table 4.5 and Table 4.6). Roots and shoots of C.

sericea and F. virginiana grown in Syncrude coke had higher Mo concentrations than

control plants and plants grown in Suncor coke (Tabl e 4.5 and Table 4.6). Roots and

shoots of F. virginiana and C. sericea grown in Syncrude coke had significantly more Ni

than all other treatments. Fragaria virginiarza shoots and roots and shoots of C. sericea

also had higher Ni concentrations in Suncor treated plants than in controls (Table 4.5 and

Table 4.6). Furtherrnore, higher Ni concentrations were observed in roots and shoots of

F. virginiana and C. sericea roots and shoots gïown in Syncrude coke than in plants

grown in Suncor coke (Table 4.5 and Table 4.6). Zinc was higher in roots ofF.

virginiana growing in Syncrude coke than in controls while no differences were observed

in shoot Zn concentrations between controls and coke treated plants (Table 4.5 and,Table

4.6). Furtherrnore, no differences in F. virginiana Zn concentrations were found between

both coke treatments (Table 4.5 and Table 4.6). Zinc was higher in C. sericea root and

shoots grown in both coke treatments than in controls (Table 4.5 andTable 4.6). Cornus sericea roots grown in Syncrude coke accumulated more Znthanroots of plants grown in

Suncor coke while no differences were observed between coke treatments in shoots

(Table 4.5 andTable 4.6). 85

E {¿

=30.o) ro

Control Syncrude Suncor

Figure 4.1: Shoot height (mean + SE) of Corruts sericea after 3 months of growth in peat- mineral mix (control), Syncrude coke or Suncor coke. Means followed by different letters represent significant difference (a,: 0.05, n : 5). 86

o) -c .o', c) È Þ¿. o o É.

Control Syncrude Suncor Control Syncrude Suncor

o)

_c .C)) ìo) Ð ! o o -c ct)

Control Syncrude Suncor

0.5

0.4 Èo o 6 0.3 3 0-2 É. 0.1

0.0 Control Syncrude Suncor Control Syncrude Suncor

Figure 4.2: Root dry weight, shoot dry weight and the root/shoot ratio (mean + SE) of Fragaria virginiana (,A, C, E) and Cornus sericea (8, D, F) grown for 3 months in peat- mineral mix (control), Syncrude coke or Suncor coke. No significant differences exist between means followed by the same letter (o,: 0.05, n: 5). Table 4.1 : Chlorophyll a, chlorophyll b, pheophytin a and %o pheophytin (mean + SE) in Fragaria virginiana and Cornus sericea after 8 weeks of treatment. No significant differences exist between means followed by the same letter (a:a:0.05.n=5). 0.05. n = 5 o/o Chlorophyll a Pheophytin a Pheophytin Chlorophyll b Chlorophyll Treatment alb

F. virginiana Control 2.00 r 0.17 0.26 r 0.02 b 0.11 r 0.00" 1.57 0.13b 1 .41 " ! t 0,19 b Syncrude 0.89 0.11 t u 0.16 r 0.02. 0.16 r 0.01 b 0.99r0.11" 0.92 ! 0.06 1.21 0.1 1 " Suncor t b 0.18 r 0.01 0.14 ! 0.06 b 1.39 r 0.08 b 0.90 + 0.10" "

C. sericea Control 1.22 0.08 0.16 0.01 t b r a 0.13 r 0.01 0.74 ! 0.05 b 1.77 t 0.24 " ^ Syncrude 0.80 r 0.09 0,14 x0.02a 0.161 0.02" 0.69 0.13"¡ 1 " t .26 ! 0.13 , Suncor 0.70 r 0.05 0.13 J 0.01 0.16 r 0.01 0.58 t 0.07 1.55 r 0.34

{oo 88

Table 4.2: Transpiration rates (mean + SE) of Cornus set'icea and Fragaria virginianø growing in peat- mineral mix (control), Syncrude coke or Suncor coke. No significant differences exist between means followed by the same letter (o, : 0.05, n : 5).

Species Treatment ïranspirationrate (mmol m-'sec-t¡

F. virginiana Control 0.75 t 0.07 b Syncrude 0.35 r 0.08 Suncor 0.30 r 0.03 " " C. sericea Control 0.72 t 0.09 Syncrude 0.65 r 0.13a" Suncor 0.53 r 0.05 "

Table 4.3: Proline content (mean + SE) of F. virginiana and C. sericea growing in peat-mineral mix (control), Syncrude coke and Suncor coke at the time of harvest. No significant differences exist between means followed by the same letter (cr: 0.05, n: 5).

Species Treatment Proline (t,g g-t dw)

F. virginiana Control 993 t714 a Syncrude 719 + 202a Suncor 516 + 140 " C. sericea Control 60+21a Syncrude 128+36u Suncor 117 !44 89

Table 4.4: Arsenic (As) and vanadiurn (V) concentrations (mean r sE) in root and shoot tissues of F. vir"giniana and C. sericea growing in peat-mineral mix (control), syncrude coke and suncor coke. No significant differences exist between means followed by the same letter (n : 5 As V pggl pg g-1 F. virginiana Root Control 1.22 t 0.14,o 5.Bg r 0.43 u Syncrude 0.34 r 0.03 16.91 r 5.78 " b Suncor 0.35 I 0.04 0.05 t 0.00 ^ "* Shoot Control 0.38 r 0.05 0.09 r 0.01 " " Syncrude 0.19 r 0.04. 0.65 r 0.22,0 Suncor 0.26 ! 0.09" 0.05 r 0.00 "

C. sericea Control 1.07 t0.29," 15.92!0.21" Syncrude 0.41 t 0.07" 93.56 + 13.11u Suncor 0.45 t 0.14 2.55 ! 0.69 ^ " Shoot Control 0.46 r 0.04 0.05 t 0.00 " "* Syncrude 0.29 ! 0.09a 0.05 I 0.00 "* Suncor 0.35 r 0.10 0.05 0.00.* * r below detection limit Table 4.5: Macro and micro nutrient concentrations (mean + SE) in root tissues of F. vit giniana and, C. sericea growing in peat- mineral mix (control), Syncrude coke or Suncor coke. No significant differences exist between rteans followed by the same letter u,:0.05,n:5

F. virginiana Control 9033 463 '17995 t , 1163+49" 1813 r 155 r 515 b 2772 !72 h Syncrude 8927 t265 1487 " g6 a !166 a 1832 ! 170 a 8472 !397 a 1272 t a Suncor 8032 ! 552 1424 !63 2113 ! 148 9213 ! 452 1214!80 ^ ^ ^ ^ ^ C. sericea Control 4136 59 1239 + r 69 5389 r 297 b 6242 !79 b 3526 ! 124 " " " Syncrude 6434 285 ,D 2697 ! !329 h 5798 r 397 b 3733 ! 152 a 1696 t 75 " Suncor 3861 t 67 2177 !62 2561 r 189 3620 ! 145 2062 ! 107

Cu Fe Mn

F. Control virginiana 1901 r 96 a 23.78 t 0.68 c 27.48 t 5.86 1409 t 116 69+6u Syncrude 1500 254 " ^ t 11.81 ! 0.78 b 66.50 r 4.86 b 1410 ! 193 40 15" Suncor 1677 " ^ !423 a 10.82 r 0.57 u 63.43 * 4.64 6 1024 ! 173 , 26+3"

C. Control 1745 + + sericea 50 21.23 r 1.50 b 10.50 2.15 1007 !40 61 t1a Syncrude 1641 33 ^ ^ ^ r a 15.46 r 1.09 b 40.81 !7.17 ,, 6180 r 905 b 345r9. Suncor 1704 8.18 + !37 r 0.34 28.54 2.29 a 286!1 a 152 r 6 b ^ ^ Table continued onfollowing page

\o O Table 4-5: Continued

F. virginiana Control 1.89 r 0.09 3.27 ! 0.32 60.98 t 4.72 a Syncrude " ^ 12.80 r 0.84 b 14.40 r 5.97 b 99.03 r 9.32 b + Suncor 1,7 0.61 1.57 t 1.57 80.54 r 8.97 ¿6 ^ ^ C. sericea Control 2.58 t 0.18 4.13 ! 0.75 24.34 t2.26 a Syncrude " ^ 52.10 r 8.25 b 50,10 r 4.10 c 174.03 ! 20.44 c Suncor 2.98 ! 0.23 20.91 !6.44 70.98 !2.22

\o Table 4.6: Macro and micro nutrient content (mean + SE) in shoot tissues of F. virginiana and C. sericea growing in peat- mineral mix (control), Syncrude coke or Suncor coke. No significant differences exist between means followed by the same letter(o:0.05.n:5). Ca Mg

F. virginiana Control 1 1953 t778 b 2175 ! 117 8160 t 130" 15106 t 505c 5639 t 234,0 ^ Syncrude 8604r115a 3614 !92," 10091 + 4490 6255 t 117 3111 r93" ^ Suncor 9888 I 181 4786 ! 147 c 9443 r 309 b 7863 ! 227 b 3494 111 " ! u C. sericea Control 4457 !60 1182 ! 33 4861 !205"6 13225 ! 212 c 2351 !27 c ^ ^ Syncrude 5993 t 329 b 1402 t 146 4384 r 588 4233 !240 1598 r 68" ^ " ^ Suncor 5767 ! 194 2596 !76 5996 t 299 8558 t 273 1866 + 60 h ^

Cu Fe Mn

F. virginiana Control 1316 r 66" 74.37 r 3.10c 7.06 r 0.18 84.09 r 7.66 69.14 + 4.61 Syncrude 1605+52"u " " ^ 35,07 r 1.97 b 8.52!0.63"6 85.03 ! 12.81 a 85.53 r 6.71 b Suncor 1501 84b + r 23.42 0.62 u 9.58 r 0.55 b 83.13 r 15.61 a 71.49 t 3.14"6

C. sericea Control 1938 t 75b 32.49 ! 2.35 6 3.37 t 0.14 26.93 + 1,49 7.41 ! 0.20 Syncrude 1136 + ^ " ^ r 28" 19.66 1.34^ 3.41 ! 0.52 189.80 t 18.40 b 31.46 r 3.84 c ^ Suncor 3063 r 161 19.86 r 1.13" 6.82 r 0.27," 47.66 ! 17.61 16.90 ! 1.026 " " Table continued onfollowing page

\o l..J Table 4-6: Continued

F. virginiana Control 0,14 t 0.01 0,97 ! 0.24 30.91 r 2.11 " ^ ^ Syncrude 1.22 t 0.17 ,o 5.40 r 0.62,, 34.92 + 1.51 Suncor 0.11 " r 0.04^ 1.60 t 0.41 38.50 t 5,66 a ^ C. sericea Control 0.50 0.05 t u 1.25 ! 0.20 11 .87 r 1.15 a Syncrude 3.76 0.33 ^ t b 6.76 ! 1.44 17.04 + 2.30 u Suncor 0.74 0.06 " f 3.94 ! 0.12 17.09 t 0.67 r.

\o (¿) 94

4.4. DlscussroN:

4.4.1. NurnrnNr Srtrus

4.4.1.1. EFFECTS oN GRowru

No differences were detected in root growth among treatments of F. vir"giniana while coke treated C. sericeahad lower biomass than controls. Although the root biomass data were adjusted to account for new root growth only, care should be taken when interpreting the following data as the presence of an established root system may have masked any growth altering effects of coke. In plants with an established root system, the exploratory capacity of the plant is greater and may grant the plant increased access to water and nutrients. The shoot dry weight of F. vir"giniana and C. sericea were lower in coke treated plants than in controls. The increase in the roolshoot ratio observed in F. virginíana grown in both coke treatments and C. sericea grown in Suncor coke suggests that plants are expending energy for exploration of the growing medium. This response is often attributed to nutrient deficiencies, particularly when coupled with a decrease in overall root biomass (Harris 1914, Glass 2002). Based on the abovementioned evidence, it is likely that C. sericea and potentially F. virginiana plants gïown in coke are suffering from a defrciency in macro- and/or micro-nutrient defìciency. The validity of these results is strengthened by the similarities observed in the growth of grasses in coke observed in chapter 3.

A major drawback of the study outlined in chapter 3 was that nutrient concentrations in root and shoot tissues was not available to assess the nutrient status of plant growing on coke. This problem was addressed in the current study. Calcium is 9s

typically found in plants at concentrations accounting for 0.2 Yo to 5 % of plant dry weight (Jones 1998). In controls, Ca content ranged from 0.6 o/oto 18 o/o whereas the Ca

content of plants grown in coke was generally lower and ranged from 0.4 %o to 0.9 o/o. As the concentrations given by Jones ( 199S) are general guidelines, it is reasonable to suggest that C. sericea and F. virginiana grown in coke may be Ca deficient. Calcium is an integral component to the structure and development of the cell wall and middle lamellae during cell division. Deformations and breaks in cell walls have been observed in cells where Ca was defìcient or made unavailable with chelators (Skobeleva et al.

1996, Suzuki et al. 2003). The structural importance of Ca was described by Moris et al.

(1982) who suggested that Ca strengthens cell walls by networking pectin into "egg-box" linkages. Calcium is also important in maintaining the integrity of biological membranes"

The principle role of Ca in maintaining biomembrane integrity is to bridge the phosphate groups of phospholipids with the carboxylate groups of membrane proteins (Legge et al.

1982, Kirkby and Pilbeam 1984). Van Steveninck (1965) and Hecht-Bucholz (1919) observed the breakdown of cellular compartmentalisation in Ca starved cells thereby demonstrating the importance of Ca in maintaining membrane integrity. Calcium also plays an important role in signal transduction, the process of converting an extemal stimulus into a biochemical response (Raven et al. 2005). Cytosolic Ca is actively pumped and stored into the vacuole, endoplasmic reticulum and the apoplast. Upon reception of a signal, calcium channels are opened and Ca2* ions rapidly enter the cytosol and complexes with calmodulin. This complex is capable of affecting the activity of 96

certain proteins implicated in plant metabolism, motor protein and cytoskeleton

functions, ion transport, protein folding, protein phosphorylation and phospholipids metabolism (Snedden and Fromm 2001).

Boron concentrations in eudicotyledons typically fall between 20 pgg-r and 70

pg g-r. Coke treated plants generally had significantly less B than controls (Table 4.5 and

Table 4.6). Boron concentrations in roots of plants grown in coke would be mostly

considered deficient while concentrations in shoots were found towards the lower end of the expected range (Jones 1998).It is therefore plausible that F. virginiana and C. sericea were suffering from B deficiencies during the experiment. Over 90% of all plant

sequestered B is located ìn the pectin fraction of the cell wall (Matoh et al. 1993). Boron deficiencies are known to alter the anatomy, physiology and biochemistry of plants thereby inhibiting growth and causing abnormal development (Kakegawa et al. 2005).

Furthermore, under conditions of B deficiency, phenolics and auxins accumulate in growing points. As phenolics are known to inhibit IAA oxidase, IAA (indolyl-3-acetic acid) accumulates thereby inducing a hormonal imbalance which can lead to a disruption of normal plant development (Coke and Whittington 1968, Pilbeam and Kirkby 1983).

The conclusions drawn for B nutrition vary between monocotyledonous and eudicotyledonous plants as their requirement for B are quite different. In the previous study, it was shown that B had accumulated in T. aestiram and D. caespitosa at concentrations which could potentially be phytotoxic. Boron concentrations in the current study suggest that F. virginiana and C. sericea could be deficient in B despite having 97

higher concentrations than the monocotyledonous species from the f,rrst study. This

further demonstrates the importance of studying the effects of coke on a wide range of

plants species.

4.4.1.2. Erpncrs oN PTcMENTS

Chlorophyll a content in coke treated C. sericea and F. virginiana were lower

than controls as were the chlorophyll b concentrations in C. sericea grown in Slrncrude

coke and F. virginiar¿a in Suncor coke. Quantification of photosynthetic pigments by the

spectrophotometric method used in the study outlined in chapter 3 failed to explain whether the lower chlorophyll content in coke treated plants were caused by an inhibition in chlorophyll sl.nthesis or to accelerated degradation of chlorophylls brought on by plant stress. For the abovementioned reasons, an updated protocol was utilized in the current study to quantify chlorophyll a, chlorophyll b and pheophytin a. Pheophytin a is a degradation product of chlorophyll a and can be measured spectrophotometrically at the same time as the chlorophylls (Axler and Owen 1994). Total pheophytin concentrations in coke treated plants did not exceed the concentrations in controls. However, the proportion of degraded pigment relative to the sum of chlorophyll a and pheophytin a

(expressed as % pheophytin) is a better indicator of differences in chlorophyll degradation between treatments as the total amounts of chlorophyll a may vary between treatments. In F. vírginiana, an increase in % pheophytin a was associated with a decrease in chlorophyll a suggesting that lower chlorophyll a contents in plants grown in coke could be associated with pigment degradation. Various stresses such as drought stress or heavy metal stress are known to cause the degradation of photosynthetic pigment content (Vergano and Hunte¡ 1953, Yang et al. 1996, Jaglap et al. 1998, Bartoli 98

eI aL.2004). As the lower chlorophyll a content in C. sericea is not associated with a

significant change in the % pheophytin, it can be speculated that lower chlorophyll concentrations observed in coke treated C. sericea was caused by an inhibition in the synthesis of photosynthetic pigments. Nutrient deficiencies may be responsible for the inhibition in chlorophyll synthesis. Magnesium is the central atom in a chlorophyll molecule and synthesis of the photosynthetic pigment is therefore hindered under conditions of Mg deficiency (Jones 1998). A study on ultrastructural changes in Mg starved gapes (Vitis viniferu), observed that chloroplast within Mg starved plants suffered from very poor differentiation of thylakoids into grana, an internal membrane system required for the light reaction of photosynthesis (Guller and Krucka 1993, Raven et al. 1999). Furthermore, Thomson and Weier (1962) showed that grana and thylakoids in cells starved of Mg suffered a loss of compartmentalization, increased variation in granum shape and size and a reduction in the number of grana. Magnesium concentrations were lower in coke treated C. sericea and F. virginiana than in controls and ranged from 0.1% to 0.3o/o whereas concentrations in controls ranged from 0.2o/o -

0.6%. As the concentrations of Mg in coke treated plants were found at the lower end of the expected range (0.15% - l%) it is not urneasonable to suggest that coke treated plants may be deficient in Mg (Jones 1998). The inhibition of synthesis and the degradation of older thylakoid membranes, which contain the hydrophobic chlorophylls, suffered by Mg deficient plants, could potentially explain the lower concentrations in photosynthetic pigments observed in coke treated plants (Guller and Krucka 1993,Laza et al. 7993, 99

Kutík et al. 1995, Jones 1998). This data agrees well with the photosynthetic pigment and

pheophytin data for C. sericea which suggest that the lower pigments observed in coke

treated plants are caused by an inhibition of synthesis.

4.4.1.3. MIcRoNUTRIENTS, MrcnoNutRIENTS CoNcrNrnarroNs

Calcium, Mg and B concentrations in C. sericea and F. virginiana were lower in

coke treated plants than in controls and have been discussed in the previous sections.

Notwithstanding the aforementioned nutrienis, plants require N, p, K, S, and trace

amounts of the micronutrients Fe, zn, Cu, Mn, Mo, Cl, and Ni for growth. plant N

requirements typically fall within 1.5 % to 6 Yo of dry plant biomass (Jones 1998).

Nitrogen concentrations measured in C. sericea and.F. virginiana shoot tissues ranged

o/o o/o, from 0.4 to L2 which were below the generally excepted sufficiency values of 2.5

o/o to 3.5 % (Jones 1998). Despite addition of N containing fertilizer, it seems that all

plants are suffering from nitrogen deficiency. However, as C. sericea root and shoots and

F' virginiana roots growing in coke either showed no difference or had more nitrogen

than control plants it is not likely that the N deficiency is attributable to coke exposure.

Sufficient concentrations of P and K in plants range from 0.2 Yo to lo/o and l.5o/o to 5yo

respectively, with concentrations in shoot tissues ranging from 0. lo/o to 05% and 0.4yo

to lo/o respectively and in roots from 0.1% - 0.26% and 0. lg% - 0.58% respectively

(Jones 1998). Although seemingly deficient in P, no differences in P concentrations were

detected in F. vírginiana roots and C. sericea shoots between plants growing in coke and

controls. Potassium concentrations were deficient in all treatments, including controls.

Plants grown in coke had equal or greater K concentrations in their root and shoot tissues than controls suggesting that coke was not responsible for the defìcient K concentrations. 100

Sulphur deficiency and/or toxicity are not expected as S concentrations were within the

expected range of 1,500 to 5,000 pg g-' in all cases but control F. virginiarza shoots and

C. sericea roots grown in Syncrude coke. Furthermore, S concentrations were the same or

higher in coke treated plants than in controls (with the exception of C. sericea shoots

grown in Syncrude coke) suggesting that coke treatments does not induce a S imbalance

in plants.

Iron is typically found at concentrations ranging from l0 to 1,000 pg g-l with

suffìciency concentrations typically greater than 50 p.g g-r (Jones 1998). As the only

possibility for severe deficiency is in shoots of control C. sericea, Fe def,rciency can be

ruled out as a factor causing decreased growth and pigment content in plants grown in

coke. Iron concentrations leading to toxicity varies between species but usually exceeds

several hundred pg g-1. Although high Fe concentrations were observed in root tissues, care must be taken when interpreting these results as roots generally accumulate higher concentrations of elements than shoots by means of ion precipitation in the apoplast or binding to cell walls (Greger 1999). Furthermore, unless under strongly acidic or highly reducing conditions, Fe is generally unavailable in soils and does not typically accumulate to phytotoxic concentrations (Guerinot and Yi 1984, Römheld 1987, Schmidt and Fühner 1998). Copper concentrations were significantly higher in C. sericea and F. virginiana roots grown in coke and in shoots of plants grown in Suncor coke. Copper is required in minute amounts (5 - 20 pg g-t) and is suggested to be deficient at concentrations less than 4 pg g-l (Jones 1998). Although Cu concentrations in C. sericea shoots are less fhan 4 pg g-' in plants grown in Syncrude coke, the concentrations are similar to the controls and therefore the potentially deficient conditions is not likely 101

attributable to coke exposure. Molybdenum is only required for plant growth in trace

amounts and is considered deficient at concentrations typically less than 0.01 pg g-r

(Jones 1998). Deficiency is not likely to occur as concentrations in tissues ranged from

0.l4pg g-t to 52 pg g-r. In all cases, Mo was found at significantly higher concentrations in roots and shoots of plants grown in Syncrude coke. Molybdenum is generally thought to be relatively non-toxic to plants; however, FargaSovà (i998) did report a decrease in

Sinapis alba (white mustard) root elongation in plants with MoO¿ 3- concentrations of

4,300 and 1,500 pg g-r in roots and shoots respectively. The maximum Mo concentrations in coke treated plants are 82 tirnes smaller in roots and 399 times smaller in shoots when compared to the aforementioned study. Based on the low phytotoxicity of

Mo coupled with low Mo concentrations in tissues, it is unlikely that Mo phytotoxicity is a concem to F. virginíana and C. sericea. Unlike the grasses in chapter 3, Mo did not accumulate in the edible (shoots) parts of ,F. virgÌniana and C. sericea and the shoot tissues would therefore be considered safe for consumption by ruminants (Neunhäuserer et al. 2001). Nickel in coke treated plants was not significantly lower than in controls and is therefore not at risk of a coke induced def,rciency. Although required in low concentrations, Ni can be quite phytotoxic at higher concentrations (Khalid and Tinsley

1980, Parida et al.2003). Chlorophyll reduction is a common symptom of Ni toxicity. As discussed in chapter 3, Ni toxicity apparently increases with an increase in Ni to Fe ratio

(Crooke et al. 1954, Crooke 1955). The Ni-to-Fe ratios in our experimental species are well below I and based on arguments by Crooke's group, would suggest that Ni uptake in plant tissues is not likely to be responsible for the decrease in biomass and pigments observed in coke treated plants. These results agree with pheophytin data for C. sericea 102

which suggests that the lower chlorophyll concentrations in shoots are caused by the

inhibition of synthesis as opposed to the degradation of chlorophyll induced by toxic

components of coke. It is important to keep in mind that the study by Crooke is dated and that the experiments have not been currently confirmed, therefore, Ni should not be ruled

out as a potential cause for toxicity without having conducted specific toxicity studies.

With the exception of control C. sericea, shoot Zn concentrations fell within the expected range of l5 to 50 pg g-l and no deficiencies or toxicities are expected at these concentrations (Jones I 998).

4.4.2. AnsrNrc AND VANADIUM Accuiuur,ATloN

In the previous chapter, As concentrations were below detection limits. However, due to the limitations of the anal¡ical equipment, the concentrations of As in root and shoot tissues could potentially be as high as 31.25 pg g-r (Table A.3). At these phytotoxic concentrations As would be a serious cause for concern in coke treated plants. The current study used more sensitive analytical equipment with lower detection limits. It was determined that As concentrations in F. virginiana and C. sericea were at concentrations expected in plant tissues and did not accumulate in coke treated plants (Aller et al. 1990).

Furthermore, concentrations in coke treated plants were oftentimes lower than in controls; therefore it is not likely that symptoms observed in coke treated plants are caused by As uptake.

Typical V concentrations in plants approximate 1 pg g-r with toxicity recorded at concentrations exceeding2 - 10 pg g-' 1Alle. et al. 1990). Vanadium concentrations exceeded toxicity ranges in roots of C. sericea and F. virginiana grown in Syncrude coke. Furtherrnore, V concentrations in controls roots also exceed the expected 103 concentrations which could cause toxicity. Potential problems associated with V toxicity are discussed in chapter 3. It is important to note, however, that as previously explained for Fe, it is possible that the V in roots is precipitated in the apoplast or bound to components of the cell wall (pectins) making it biochemicaly inert (Greger 1999).

4.4.3. Warnn Srnrus

Closure of stomates reduces transpiration thereby conserving water under drought or osmotic stress conditions (Pei and Kuchitsu 2005). Fra.þaria virginiana grown in

Syncrude and Suncor coke did show a decrease in transpiration rates relative to the controls while C. serícea grown in coke showed no differences. Despite lower transpiration rates, coke treated F. vir"gíniana did not show an increase in the root to shoot ratio. Alberte et al. (1917) found that chlorophyll b would degrade at a faster rate than chlorophyll a in water stressed Zea mays resulting in an increase in the chlorophyll alb ratio; however, this was not observed in coke treated F. virginiana or C. serícea

Finally, plants are known to increase their root growth relative to their shoot growth under conditions of drought stress, leading to an increase in the root/shoot ratio (Westgate and Boyer 1985, Creelman et al. 1990). It is speculated that the increased root growth allows for continued exploration and water uptake during periods of drought (Glass

2002). Fragaria virginiana grown in coke had a higher roolshoot ratio than controls providing evidence that growth in coke may induce drought stress. The decreased transpiration and the increase in the roolshoot ratio suggest that F. virginiarza is suffering from drought stress despite a regular watering regime whereas C. serícea was not.

Although C. sericea is oftentimes associated with moist environments, it is suspected to have some drought tolerance as studies have shown C. sericea growing in the mine area 104 to be moderately salt tolerant (Renault et al. 2001). Salinity stress may induce water stress by decreasing the soil osmotic potential thereby reducing the availability of water.

Water stress may also be induced through the lack of water, rapid drainage of water from the root zone and/or low matric potential.

Proline is an amino acid which accumulates under conditions of drought stress and acts as a compatible osmolyte. The role of compatible osmolytes includes osmotic adjustment, cellular protection and/or stress recovery (Hare et al. 1998, Ain-Lhout et al.

2001, Sofo et al. 2004). Concentrations of proline in water stressed plants increases between 3 times to 320 times more than concentrations measured in unstressed plants

(Delauney and Verma 1993). Pisum sativa showed a 4 fold increase in proline concentrations when osmotically stressed; however, it was determined that the amino acid was only responsible for 0.5 % of the leaf s solute potential and was therefore deemed hardly significant as an osmotic adjustment response (Sánchez et al. 1998). As proline content in C. sericea was only twice as much proline in coke treated plants relative to the controls, it is not likely that proline is playing a substantial role in osmoregulation. Based on the abovementioned studies, it seems counterintuitive that increases in proline associated with no change in transpiration rates and vice versa, as both are responses to drought stress. One possible explanation is that C. sericea produced other osmol¡es such as sugar alcohols or glycine betaine. These compatible osmolytes aid in the osmotic adjustment of plants and could allow C. sericea to extract water from the growing medium thereby alleviating the stress (Taiz and Zeiger 1998). 105

4.4.4. Spncrrs CotrpaRrsoN

The root biomass of F. vir"giniana grown in coke was 4o/o-l5olo lower than controls as opposed to 63%o-83%o lower as observed in C. sericea. Similarly, the shoots of the F. virginiana species suffered less reduction (67%-68%) in biomass than the shoots of the shrub species (13%-83%). Although both species showed a decrease in chlorophyll a in plants grown in coke, the F. virginiana showed a higher reduction (40%-56%) than did

C. sericea (34%-43%). Furthermore, Fragaria virginia,?a seems to have suffered a degradation of chlorophyll whereas chlorophyll a synthesis seems to have been inhibited in C. sericea, possibly by a nutrient (Mg) deficiency. This may reflect differences in tolerances to nutrient deficiencies. Fragaria virginiana can tolerate some nutrient deficiency as it has been recorded growing in the nutrient poor soils of Sable Island,

Nova Scotia (Catling et al. 1984). Cornus sericea is generally associated with nutrient rich soils and no references to any tolerances to nutrient deficient conditions were found in the literature (Hardy BBT Ltd. 1989). Fragaria virginiana growing in coke showed a reduction of llYo-37% in chlorophyll b while C. sericea showed a7o/o-22o/o reduction in chlorophyll b concentrations. This resulted in decreases in the chl alb ratio of 2Yo-35o/o in

F. virginiana and 12%-24% in C. set"icea. Transpiration rates in F. virginiana grownin coke were lower than controls. The increase in the roolshoot ratio coupled with the decrease in transpiration rates suggest that ,F. virginiana is suffering from drought stress.

As C. sericea is not suffering from these symptoms, it is likely that C. sericea is more tolerant to the low water conditions present in present in the coke treatments than F. virginiana. Furthermore, C. sericea did show an increase in proline in plants growing in coke, a stress response to drought stress exhibited by some species. 106

CHAPTER 5 _ CoNcI,USIoNS a¡¡I RBcoMMENDATIoNS

Emergence of seeds sown in coke was faster during the early parts of the monitoring period than seeds sown in treatments containing peat-mineral mix. Early emergence in coke was likely caused by increased aeration and possibly slightly higher temperatures. Overall, coke did not seem to have any significant effect on the final emergence of seedlings suggestingthat coke has no immediate toxic effects on the developing seedling. However, over the long tenn, plants grown in petroleum coke oftentimes suffered from a number of symptoms which included reduced gtowth, increased root to shoot ratio, lower pigment content, lower transpiration and lower stomatal conductance. Based on the results, particularly the root to shoot ratio, transpiration and stomatal conductance rates, it does appear that plants growing in petroleum coke are suffering from water stress. The rapid drainage of water from coke coupled with the relatively low water holding capacity is likely to explain the source of water stress.

Despite the addition of fertilizer, the decreased root biomass coupled with increased root to shoot ratio and the reduction in pigment contents suggested that plants are also suffering from nutrient deficiencies. This problem was elucidated in the second experiment which showed lower B (in dicotyledons), Ca and Mg in coke treated plants than in controls. Deficiencies in B and Ca can readily explain reductions in growth while

Mg deficiencies have been associated with decreases in pigment content. Potential nutrient deficiencies are also present in controls where concentrations of N, K Fe (in monocotyledons) and sometimes P are below the expected concentrations in plant tissues.

This suggests that fertilizer application rates may need to be modified. t07

Concentrations of Ni, V, B (in monocotyledons) and Mo in tissues of plants grown in coke oftentimes exceed concentrations in control plants. Despite the relatively low concentrations, these elements may still induce phytotoxicity andlor affect the health of primary consufiìers. Studies are required to further understand the effects of Ni, B (in monocotyledons), Mo and V on the growth and physiology of coke treated plants.

Although not often associated with phytotoxicity, plants containing Mo concentrations exceeding l0 pg g-' or a Cu: Mo ratio of less than 2 can cause molybdenosis in ruminants. Results frorn the second experiment suggested that concentrations of As are low and well within expected concentrations. However, As concentrations in monocotyledons should be checked as results may be different from dicotyledons due to physiological and morphological differences between these groups.

Capping of coke with peat-mineral mix seemed to alleviate the stresses in coke treated plants. The plants are presumably responding to the increased water and nutrient holding capacity offered by the organic matter and clay fractions of the peat-mineral mix.

A cap l0 - 33 times larger than the caps used in our experiment are used for reclamation purposes by Syncrude Canada Ltd. The thicker cap will likely circumvent many problems associated with plant growth directly in petroleum coke. It is probable that all tested species would survive during the reclamation of coke capped with a peat-mineral mix cap. Under practical field conditions, other problems may arise which were not accounted for in our controlled greenhouse conditions. For example, the watering regime under field conditions is irregular and depends exclusively on precipitation events unless irrigated.

Furthermore, wind may increase water loss from plants which could exacerbate the effects of water stress on coke treated plants. 108

Although differences in responses to coke were observed between species, trends could be seen throughout the data sets. Based on the aforementioned research, the following recommendation for reclamation of coke impoundment sites and further studies can be made:

. Dose response curves could be carried out to fully understand the effects of B

(monocotyledons), Ni and V on plant growth and physiology. Furthermore, long-

term/field experiments would be required to determine the extent of element

accumulation from coke over time as well as their potential chronic effects in

plants. Due to the risk of molybdenosis, Mo concentrations of vegetation should

be monitored if ruminants are kept on revegetated sites.

. Monocotyledon tissues growing in coke could be analyzed to determine if

concentrations of As are similar to concentrations observed in F. virginiana and

C. sericea.

. Coke treated plants fertilized with 500 kg ha-r 12-36-12-4 (N-P-K-S) had

concentrations of N, K, Ca, Mg and sometimes P which were lower than controls

and were oftentimes bordering on deficiency. Certain control plants showed lower

concentrations of N, K, Fe (in monocotyledons) and P than expected. Monitoring

of peat-mineral mix nutrient availability and concentrations of nutrients in plants

could allow for the correction of the fertllizer regime which would maximize

plant establishment and growth.

o Although the symptoms and problems associated with growing in coke are

thought to be similar throughout all plants, the response of other plant species 109

such as conifers, ericaceous shrubs, deciduous trees etc. to coke are still unknown

and could be screened to ensure similar results.

¡ This study simulated a worst case scenario in coke revegetation. A fìeld

experiment aimed at determining the growth and partitioning of roots between the

peat-mineral mix cap and the underlying coke would complete this study.

Furthermore, fìeld trials could suggest species which were less likely to penetrate

the cap and hence be better suited to revegetation of coke impoundment sites.

o As coke drains quickly and has a relatively poor water holding capacity, it is

important to ensure that the cap is thick enough to hold the necessary moisture for

plant growth throughout the growing season. o Trees susceptible to windthrow may not be good candidates for reclamation of

these sites due to poor strucfure of coke which would provide poor anchorage. 110

CHAPTER 6 - RNTNNBNCNS

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Appendix A 127

reclamat¡on mater¡al

Figure 4.1: Storage of coke in a) coke cell and b) coke beach at the Mildred Lake mine site, Alberta, Canada. Diagram and photo courtesy of Syncrude Canada Ltd. 128

Control Uncapped Syncrude Capped Syncrude

Uncapped Suncor Capped Suncor

Figure A.2: Triticum aestivum grown in peat-mineral mix (control), syncrude coke, Syncrude coke + cap, suncor coke or suncor coke + cap 14 days after seeding. t29

A) Control, B) Syncrude, C) Syncrude + cap

A) Control, B) Suncor, C) Suncor + cap

Figure A.3 : Deschampsia caespitosa grown in peat-mineral mix (control), Syncrude coke, Syncrude coke + càp, Suncor coke or Suncor coke + cap after 3 months. 130

A) Control, B) Syncrude, C) Syncrude + cap

A) Control, B) Suncor, C) Suncor + câp

Figure A.4: Calamagrostis canadensis grown in peat-mineral mix (control), Syncrude coke, Syncrude coke + cap, Suncor coke or Suncor coke + cap after 3 months. 131

A) Control, B) Syncrude, C) Syncrude + cap

A) Control, B) Suncor, C) Suncor + câp

Figure A.5: Agropyron trachycaulum grown in peat-mineral mix (control), Syncrude coke, Syncrude coke + cap, Suncor coke or Suncor coke + cap after 3 months. 132

A) Control, B) Syncrude, C) Syncrude + cap

A) Control, B) Suncor, C) Suncor + cap Figure A.6: Oryzopsís hymenoides grown in peat-mineral mix (control), Syncrude coke, Syncrude coke * cap, Suncor coke or Suncor coke + cap after 3 months. 133

Figure A.7: Fragaria virginiana grown in peat-mineral mix (control), Syncrude coke or Suncor coke after 3 months. 134

Figure A.8: Cornus sericea grown in peat-mineral mix (control), Syncrude coke or Suncor coke after 3 months. Table A.l: Aluminium (Al), arsenic (As), boron (B), barium (Ba), beryllium (Be), bismuth (Bi), calciurn (Ca), cerium (Ce), cobalt (Co), chromium (Cr), copper (Cu), cesium (Cs), dysprosium (Dy), erbiurn (Er), europium (Eu), iron (Fe), galliurn (Ga), gadolinium (Gd), hafnium (Hf), holmium (Ho), potassium (K), lanthanum (La), Lithiurn (Li), lutetium (Lu), rnagnesium (Mg), manganese (Mn), molybdenum (Mo), sodium (Na), niobium (Nb), and neodymium (Nd) concentrations in peat-mineral mix, Slncrude coke and Suncor coke.

AI As B Ba Be Bi Detection limit 0.1% 0.5 ug g-' 2 vg g-' 1 ug g-' 0.1 ug g-' 0.1 ug g-' Control 2.4 t 0.1 3.6 r 0.1 62.7 ! 1.3 324 !9 0.9 r 0.1 0.3 I 0.0 Syncrude 0.7 r 0.0 7.4!1.7 18.3 I 1 .5 69t4 0.5 r 0.0 0.3 t 0.0 Suncor 0.4 r 0.0 4.4 ! 0.6 1 1.3 r 0.3 18119 0.2 J 0.0 0.21 0.0

Ca Ce Co Cr Cu Cs Detection limit 100 pg g-1 0.1 pg g-t 0.1 pg g-' 1 pg g-' 0.2 ug g-' 0.05 pg g-1 Control 11433 I 3530 48.9 t 1.2 6.0 I 0.3 150.7 ! 110.7 11.83 r 2.0 2.1 ! 0.1 Syncrude 1233 I 33 37.5 ! 0.12 13.3 r 1.5 32.3 È 8.3 9.4 ! 0.4 0.5 r 0.0 Suncor 367 r 33 16.5 r 0.6 6.7 t 0.1 13.7 !2.9 5.9 10.15 0.2 r 0.0

Dy Er Eu Fe Ga Gd Detection limit 0.1 ug g-t 0.1 ug g-t 0.05 pg g-1 100 ug g-1 0.1 pg g-t 0.1 pg g-t Control 2.5 ! 0.1 1.4 ! 0.1 0.8 r 0.0 18567 È1330 8.710.3 3.2 ! 0.1 Syncrude 2.2 t 0.0 1,3 r 0.0 0.7 r 0.0 8'133 r 537 5.2 ! 0.2 2.8 r 0.1 Suncor 1.2 ! 0.1 0,7 r 0.0 0.3 r 0.0 3067 r 260 3.1 r 0.'1 1.3 t 0.0

Hf Ho K La Li Lu _1 Detection limit 0.1 Lrg g-' 0.1 pg g-' 100 ug g-1 0.1 ug g-' u.5 trg g 0.1 pg g-t Control 3.5 t 0.2 0.5 J 0.0 10067 r 367 25.1 r 0.5 38.3 r 1.9 0.3 r 0.0 Syncrude 1.3 ! 0.2 0.5 r 0.0 1200 r 153 18.2 ! 0.1 11.4 ! 0.1 0.2 r 0.0 Suncor 1 t 0.2 0.2 r 0.0 467 !33 7.8 r 0.3 5.5 r 0.2 0.1 r 0.0 Table continued onfollowing page (,) (^ Table A.l: Continued

Mg Mn Mo Na Nb Nd Detection limit 100 Ug g-1 1 pg g-t 1 ug g-' 100 pg g-1 0.1 ug g-' 0.1 pg g-t Control 4767 !219 249 ! 1.5 1.3 t 0.3 3767 r 33 710.3 20.5 ! 0.7 Syncrude 700r0 203.3 r 1.3 72.3 ! 0.3 1267 !219 6.9 t 0.1 16.2 t 0.1 Suncor 267 !33 65.3 r 2.0 54 ! 1.5 633 t 88 3r0.1 7.2 ! 0.3

N¡ P Pb Pr Rb Re -1 _1 Detection limit u.b trg g ' 1o pg g-' u.5pgg' 0.1 pg g-' 0.2 pg g-' 0.001 pg g-1 Control 15.9 r 0.8 250 r 10 10.9 r 0.6 5.7 !0.2 45.9 r 0,5 0.003 r 0.000 Syncrude 655.3 ! 4.7 130 r 6 13.9 r 0.1 4.4 ! 0.0 7.0 I 0.3 0.100 r 0.030 Suncor 484.0 !6.2 40r6 7.3 ! 0.1 2.0 !0.1 3.1 J 0.1 0.100 I 0.020

S Sb Se Sm Sr Ta Detection limit 0.01% 0.1 pg g-' 0.1 ug g-' 0.1 ug g-' 0.2 pg g-' o.'1 pg s-' Control 0.13 r0.0 0.3 r 0,1 0.7 !0.2 3.9 r 0.1 120.3 ! 1.3 0.4 t 0.0 Syncrude 5.610.5 0.810.1 1.4 r 0.5 3.'1 r 0.0 59.8 t 0.4 0.310.1 Suncor 4.3 r 0.6 0.5 r 0.1 0.7 !0.2 1.5 r 0.0 12.7 t 0.5 0.2J0

Tb Th Ti TI U V Detection limit 0.1 pg g-' 0.1 pg g-' 100 Ug g-1 0.05 Ug g-1 o.'1 pg g-' 2 vg g-' Control 0.4 r 0.0 7,8!0.4 2070 !70 0.3 r 0,0 3.1 t 0.5 50.7 !2.2 Syncrude 0.4 r 0.0 5.8 r 0.1 2200 !0 0.1 r 0.0 1.6 t 0.1 1603.3 r 6.7 Suncor 0.2 t 0.0 2.2 ! 0.3 970 I 30 0.1 r 0.0 1.2 ! 0.2 1200 ! 20

Yb Zn Zr _a Detection limit 0.1 pg g-' 0.1 pg g-' u.bpgg' 1 pg g-' Control 12.6 r 0.5 1.3 r 0.1 29.9 r 1.5 135 t 4.5 Syncrude 11.3 r 0.1 1.1 r 0.0 14.8 r 0.5 55.3 t 6.8 UJ Suncor 5.9 r 0.2 0.6 r 0.0 6.4 t 0.3 41,3 !7.3 o\ t37

Table 4.2: Gravimetric water content, and volumetric water content of peat-mineral mix, Symcrude coke and Suncor coke Control Syncrude Suncor (%) (%) (%)

Gravimetric water content 59 t2 30+0 4+0 Volumetric water content 55+2 30r1 5+0

Table 4.3: Range of detection limits for elements analyzed by ICP-OES. Element Detection limit (pg g-') ICP-OES Range in sample As 0.s0 I 1.63 - 3t.25 B 0.0s l.r6 - 3.13 Mo 0.02 0.47 - 1.25 Ni 0.1 2.33 - 6.25 Ti 0.01 0.23 - 0.63 V 0.01 0.23 - 0.63 Fe 0.01 l.l6 - 0.63 K 0.05 16.5 - 3.r3 S 2 46.51 - 125 P 0.2 4.65 - 12.s 138

Table 4.4: Detection limits and method of an al ysi s for m acronutri ents, mi cronutri ents, arsenic and vanadium in plant tissues by inductively coupled plasma mass spectroscopy (lCP-MS), Inductively coupled Plasma Optical Emissions Spectroscopy (ICP-OES), Combustion chromatography (CC) and Combustion Infrared (IR) Element Method Detection limit

As ICP-MS 0.03 pg L-' V ICP-MS 0.1 pg L-l N CC 0.0t % P ICP-OES 0.4 pg g-' K ICP-MS 30 pg L-' Ca ICP-MS 700 pg L-' Mg ICP-OES I pg L-' S IR 0.01% B ICP-MS 0.2 ptg g'' Cu ICP-MS 0.2 ¡tgL'l Fe ICP-MS l0 pg L-' Mn ICP-MS 0.1 pg L-' Mo ICP-MS 0.1 pg L-' Ni ICP-MS 0.3 pg L-' Zn ICP-MS Table 4.5: As, B, Fe, K, Mo, Ni, S, Ti, and V concentrations uncorrected for peat-mineral mix/ coke contamination. T. aestivum Treatment As Fe K Mo

Shoot Control 0.0 t 0.0 10.5 r 1.3 43.3 ! 4.0 11026 ! 1236 1.21 t 0.31 Syncrude 0.0 r 0.0 12.9 t 1.2 50.8 f 7.6 1 0920 t 1054 7 .62 ! 2.75 Syncrude + cap 0.0 r 0.0 20.4 ! 3.0 32.4 ! 1.5 r338s ! 1114 0.00 t 0.00 Suncor 0.0 t 0.0 16.9 r 3.9 49.4 ! 3.4 7997 !825 1.23 ! 0.52 Suncor + câp 0.0 r 0.0 10.5 r 3.3 71.s r 53.0 8787 1398 4.08 r 0.25

Root Control 0.0 r 0.0 I1.8 r 3.3 464.2 t 53.0 2668 r 398 1.13 r 0.25 Syncrude 0.0 J 0.0 l0.s r 1.5 675.2 r 253.6 5828 !797 6.46 ! 1.43 Syncrude + câp 0.01 0.0 13.6 ! 3.2 232.6 r 36.9 7435 r 3BB t.97 !0.34 Suncor 0.0 r 0.0 19.3 16.9 261.1 r 55.0 4446 ! 474 0.99 t 0.41 Suncor + cap 0.0 r 0.0 r6.6 r 3.6 389.1 r 55.2 4718 !229 0.47 ! 0.44

Ni S Ti V Shoot Control 0.00 J 0.00 1702 ! 180 0.3s 1 0.07 6.4s r 0.50 Syncrude s0.0 r 33.3 1584 r 163 0.31 r 0.09 6.50 r 0.28 Syncrude + câp 68.9 t 43.8 t840 !211 0.33 r 0.02 7.ls t 0.65 Suncor 0.0 r 0.0 146r ! 170 0.38 10.03 7.t6 t 0.97 Suncor + câp 0.00 r 3.79 1277 ! 169 0.37 f 0.59 6.48 r 0.13

Root Control s.78 f 3.79 1607 t 169 8.17 r 0.59 2.34 t 0.13 Syncrude 30.74 r 0.83 1792 ! 149 2.3t ! 0.21 26.95 f 5.86 Syncrude + câp r.37 ! 1.37 1888 r 128 1.32 r 0.16 9.00 ! 1.17 Suncor 16.29 ! 5.22 1464 ! 151 3.89 r 0.58 7.46 ! 0.25 Suncor + ca t7.86 t 8.29 1706 r 135 s.16 r 1 .08 6.t3 t 1.70 Table continued onfollowing page UJ \o Table A.5: Continued D. caespitosa Treatment As Fe Mo

Shoot Control 0.0 r 0.0 20.7 t 1.3 198 r 18 t2r62 r 695 1.27 r 0.80 Syncrude 0.01 0.0 45.9 r 5.6 113t6 20689 !2402 27.81 r 0.96 Syncrude + cap 0.0 t 0.0 29.8 ! 1.8 891 9 18532 !420 14.70 t 0.57 Suncor 0.0 r 0.0 27.0 ! 1.6 t37+6 16010 I 584 t3.37 ! 1.11 Suncor + cap 0.0 J 0.0 27.r ! 3.2 168 t 24 8204 r 689 4.70 t 0.37

Root Control 0.0 r 0.0 I 1.7 r 1.8 577 t92 6t43 !275 0.09 r 0.04 Syncrude 0.01 0.0 t7.9 ! 2.5 878 ! 221 ts974 r 1881 18.r3 ! 4.12 Syncrude + câp 0.0 r 0.0 14.8 r 1.9 386 t 114 t3544 t 806 7.51 ! 1.29 Suncor 0.0 r 0.0 13.3 t2.2 49t !75 90t2 ! 937 4.87 r 0.36 Suncor + câp 0.0 r 0.0 12.0 I 1.5 512 160 4174 ! 420 Lt1r0.5'1

Ni S Ti V Shoot Control 0.00 r 0.00 2594 ! 211 3.36 r 0.66 10.46 J 1 .06 Syncrude r2.70 t 1.35 3006 + 341 0.82 r 0.13 8.78 t 1.70 Syncrude + cap 12.99 !2.82 3462 ! 250 2.20 t 0.64 r3.36 r 2.38 Suncor s.86 r 1.69 2297 ! 170 2.37 ! 0.26 8.32 r 0.80 Suncor + cap 0.00 r 0.00 2079 ! 166 3.24 ! 0.81 12.23 + 1.53

Root Control 0.00 r 0.00 1664 t 68 8.s9 r 1.00 6.18 r 0.71 Syncrude 33.ss t 6.44 2682 ! 258 3.52 r 0.38 42.07 ! 6.14 Syncrude + cap 15.40 !2j5 3126 ! 359 4.01 r 0.81 26.66 r 5.58 Suncor 25.07 r 1.90 1452 ! 125 6.23 r 0.39 s.32 ! 0.92 Suncor + câp 1s.28 r 2.68 20s7 !243 10.13 r 1.59 1s.29 ! 4.96 Table continued onfollowíng page À Table A.5: Continued C. canadensis Treatment As B Fe Mo

Shoot Control 132 !20 14040 r 709 Syncrude 10216 2s6s9 ! 2299 Syncrude + câp t52 + 19 11936 t 1253 Suncor 77 !6 16916 t 2091 Suncor + cap 382 t 91 1t279 r 580

Root Control 906 t1 60 5350 r 538 Syncrude 706 ! 329 13800 !2311 Syncrude + cap 492 ! 53 s654 ! 451 Suncor 47t !247 9s9r r 1669 Suncor + câp 382 t 91 390t !674

Ni Ti V Shoot Control 0.68 r 0.06 1750 ! 117 r.67 ! 0.31 20.99 r 1 .18 Syncrude 22.48 r 5.19 3070 J 536 0.86 r 0.12 21.6s !2.81 Syncrude + cap 9.69 ! 0.62 2027 ! 94 2.52 t 0.39 19.99 ! 1.07 Suncor 60.09 r 30.89 2969 ! 136 r.02 ! 0.43 24.t5 r 0.75 Suncor + câp 4.49 !0.41 t654 ! 111 2J9 ! 0.35 32.35 r 1.59

Root Control 2.82 r 0.83 1648 r 195 11.54 ! 2.43 9.t3 t 1.13 Syncrude 169.08 r 128.33 1880 r 263 2.16 t 0.44 42.28 ! 4.71 Syncrude + cap 17.38 r 1.85 1167 r 100 7.86 ! 0.87 1.98 r 1.63 Suncor 20.6r ! 2.03 3277 t283 6.96 ! 1.64 38.78 ! 10.57 Suncor + câp 6.10 ! 1.34 t324 ! 126 6.81 r 1.07 8.s8 r 4.31 Table continued onþllowing page 5 Table A.5: Continued A. trachycaulum Treatment As Fe Mo

Shoot Control 8s.8 15.0 10603 !47 Syncrude 85.8 r 9.9 22477 !2652 Syncrude + cap 98.1 ! 11.2 12996 ! 457 Suncor 60.7 ! 5.4 21492 t 1 185 Suncor + câp 90.6 t 10.2 7880 r '1500

Root Control s65 ! 122 3544 ! 519 Syncrude 1393 I 268 t0448 ! 1178 Syncrude + câp 401 r 83 637s t,373 Suncor 247 !30 10032 r 536 Suncor + câp 344 + 44 477 t ! 341

Ni Ti V Shoot Control 0.8691 0.296 1536 ! 62 0.7391 0.099 L9.l t2.1 Syncrude 7.772 ! 1.172 2225 ! 360 0.340 r 0.096 r2.9 ! 1.3 Syncrude + câp 4.527 ! 2.736 t254 ! 121 1.019 r 0.240 11.41 1.3 Suncor 32.357 r 26.33'l 23ls t 108 0.486 t 0.145 t7 .7 ! 0.6 Suncor + cap 15.785 ! 14.911 1.472 ! 58 r.0sl t 0.219 21.4 + 1.0

Root Control r.968 r 0.111 1506 t 334 6.891 r 1.835 4.7 ! 1.2 Syncrude 3s.997 r 0.028 1680 ! 171 t.214 ! 0.320 23.r ! 3.4 Syncrude + cap 22.87t ! 1.215 1102186 3.697 r 0.516 2.r r 0.9 Suncor 11.946 r 1.859 2387 !335 2.63s t 0,867 20.0 r 5.8

Suncor + cap 6.787 r 1.396 1628 !76 4.99810.613 3.9 r 1.8 5 Table continued onþllowing page N) Table A..5: Continued O. hymenoides Treatment As B Fe Mo

Shoot Control 82.t !7.5 tt279 t 542 Syncrude 55.5 !2.1 13625 r 1600 Syncrude + cap 56.6 ! 3.2 10829 r 1016 Suncor 49.7 !2.4 1458 r 2190 Suncor + câp 67.t ! 11.2 t0230 ! 527

Root Control 686.6 r 1 1B 4996 ! 442 Syncrude 391 r 98 9288 ! 1484 Syncrude + câp 367 ! 51.6 7632 ! 1133 Suncor 147.1 ! 16.6 9149 x 619 Suncor + câp s16.3 ! 117.3 s086 t 178

Ni S Ti V Shoot Control 2.ir 11.88 2279 t 216 0.662 ! 0.182 14.08 11 .15 Syncrude 26.02 r 15.81 20s0 !232 0.2r4 ! 0.137 15.28 r 1.89 Syncrude + câp 6.75 r 0.99 1836 r 191 0.277 t 0.03 4.29 !2.67 Suncor s.09 r 0.46 2464 t 87 0.179 r 0.088 17.18 r 1 .38 Suncor + cap 1.s9 t 0.67 2256 ! 82 0.786 ! 0.282 9.23 !3.92

Root Control 19.34 r 8.55 I63t ! 107 9.98 ! 2.14 4.7r!1.19 Syncrude 32.56 r 9.64 t944 ! 340 2.17 ! 0.44 18.28 r 1.86 Syncrude + cap 27.87 r.5.27 t66t ! 216 3.4s r 0.81 3.52 ! 2.04 Suncor 6.s5 r 0.20 2699 ! 133 t.40 !0.2v 4.7t !0.47 Suncor + cap 8.58 ! 2.49 2694 ! 200 11.05 r 3.05 8.12 t 1 .93 À UJ Table 4.6: Element content data uncorrected for peat-mineral rnix/ coke contamination As Ca Fe F. virginianø Shoot Control 386.5 ! 54.1 74.4 t 3.1 15140 r 504.6 122.6 ! 12.5 Syncrude 259.5 !20.4 35.1 ! 2.0 6256.0 r 1 16.6 91.5 t 12.4 Suncor 389.3 1111.3 23.4 ! 0.6 7866 r 227.1 97.6 110.1

F. virginiana Root Control 1381.4 ! 147.0 23.8 ! 0.7 18500 r 548.6 1973.9 r 163.5 Syncrude 395.4 r 26.8 1 1.8 r 0.8 8490 r 399.1 1498 r 198.1 Suncor 591.71101.1 10.810.6 9252 ! 456.4 1226.5 !224.8

C. sericea Shoot Control 461.2 t 39.2 32.5 ! 2.4 13240 t211.2 43.2 ! 2.1 Syncrude 410.8 r 96.4 19.7 ! 1.3 4234 !240.2 192.7 I 18.1 Suncor 421.2 r 107.5 19.9 r 1.13 8558 r 272,6 49.7 ! 1.1

C. sericea Root Control 1166.2 r 331.6 21.2 ! 1.5 7948 ! 1540.0 1492 ! 302.7 Syncrude 434.0 t 68.7 15.5 ! 1.1 3740 ! 151 .6 621 ! 904.3 Suncor 558,0 r 148.8 8.2 r 0.3 3624 ! 145.3 305.1 r 16.8 Table continued onfollowing page

è Table A.6: Continued K Mg Mn Mo F. virginiana Shoot Control 8182 ! 131 .5 5652 !234.6 69.7 r 4.6 0.1510.01 Syncrude 10092 ! 448.8 31 12 r 93.0 85.7 ! 6.7 1.29 ! 0.17 Suncor 9446.0 r 309.3 3494.1 t 111.4 71.9 ! 3.1 0.25 r 0.01

F. vir"giniana Root Control 2138 ! 160.8 2956.1r 76.9 77.6 ! 5.5 1.96 r 0.08 Syncrude 1846 r 170.8 1282 ! 87.3 42.4 ! 5.4 13.74 r 0.85 Suncor 2150 ! 156.7 1213 ! 80.1 31.8 t 4.6 5.72 ! 0.52

C. sericea Shoot Control 4870.1r 205.5 2356 !26.6 7.6 ! 0.2 0.50 r 0.05 S1'ncrude 4384 ! 587,9 1598 !67.7 31.6 r 3.9 3.80 r 0.34 Suncor 5996 r 298.8 1866 t 59,9 17.0 ! 1.0 0.78 t 0.06

C. sericea Root Control 5584 r 224.0 4272 ! 691.9 66.3 r 3.7 2.62 ! 0.17 Syncrude 5804 r 397.4 1700 r 75.3 345.9 r 8.9 52.48 ! 8.25 Suncor 2564 ! 189.5 2062 ! 107.4 152.0 r 6.3 3.37 !0.29 Table continued onþllowing page

(r¡s Table A.6: Contiru.ted Ni Ti F. virginiana Shoot Control 28.6 !27.6 4.9 ! 0.7 0.21 !0.02 0.13 t 0.01 Slmcrude 2.2 t 0.3 2.31 !0.32 0.16 r 0.01 Suncor 3.1 r 0.3 5.7 r 3.5 1.32 t 0.29 0.'16 r 0.00

F. virginiøna Root Control 3.9 r 0.3 71.5 !7.8 7.63 r 0.59 0,20 r 0.01 Syncrude 23.3 t 5.9 30.4 r 5.4 39.5 r 5.21 0.20 r 0,02 Suncor 24,6 ! 3.1 79.7 !20.7 50.8 t 9.20 0.33 r 0.03

C. sericea Shoot Control 1.3 ! 0.2 2.1 !0.2 0.08 r 0.01 0.19 r 0.01 Syncrude 31.5 ! 24.4 1.0 r 0.1 0.56 r 0.13 0.13 r 0.02 Suncor 4.7 !0.4 0.8 r 0.2 0.4210.05 0.31 r 0.02

C. sericea Root Control 4.5 r 0,8 42.8 ! 19.4 16.4 ! 0.25 0.18 r 0.00 Syncrude 53.7 ! 4.0 12.3 ! 1.4 102.7 ! 13.45 0.19 r 0.01 Suncor 24.6 ! 6.4 7.4 ! 1.9 11.87 ! 2.18 0.2 t 0.02 Table continued on.following page

Þ o\ Table A.6: Continued N Zn Cu F. virginiana Shoot Control 1.20 r 0.08 2176 ! 117.2 31.0 ! 2.1 7.1 !0.2 Syncrude 0.8610.01 3614 ! 92.1 62.3 ! 4.8 27.9 ! 5.9 Suncor 1.00 r 0.02 4786 ! 146.9 34.8 r 1.5 8,5 t 0.6

F. virginiana Root Control 0.91 10,05 1174 ! 48.4 99.2 r 9.3 66.6 r 4.9 Syncrude 0.91 10,03 1490 t 165:9 38.5 r 5.7 9.6 r 0.6 Suncor 0.9310.05 1430 ! 64 81.2 r 8.9 63.8 ! 4.7

C. sericea Shoot Control 0.45 r 0.01 1182 ! 33.1 11.9 ! 1.2 3.4 !0.1 Syncrude 0.60 r 0.03 1402 ! 145.8 25.1 !2.0 10.7 ! 2,3 Suncor 0.58 r 0.02 2596 !76.1 17.1 ! 2.3 3.4 r 0.5

C. sericea Root Control 0.42 ! 0.01 1246 !67.0 174.1 t 20.4 40.9 !7.2 Syncrude 0.6510.03 2698 !329.1 17.1 ! 0.7 6.8 r 0.3 Suncor 0.40 r 0.01 2178 !62.1 71.0 ! 2.2 28.6 ! 2.3

\ìÀ 148

Appendix B 149

FInlo ExppRrvrnNr

A field experiment was designed to study the responses of grasses growing on a reclaimed coke impoundment site and to determine the degree of root partitioning between the peat-mineral mix and the subtending coke. However, at the time of our arrival to the Syncrude Canada Ltd. field site, the reclamation material þeat-mineral mix) had not yet been spread. We then altered our plans and designed an experiment which aimed at mimicking the worst case scenario in revegetating coke. The objective of this study was to determine whether establishments of plants directly in coke would be feasible under field conditions. It was suspected that the rapid drainage of water from coke and high temperature associated with the black surface of coke would severely limit germination and/or survival of seeds and seedlings.

The field experiment was conducted on the coke beach (Figure A.1 and B.l ) at the Syncrude Canada Ltd. Mildred Lake mine site. One replicate consisted of a 3 m x 3 m square plot subdivided into 1 m x 1 m subplots. Each corner 1 m x I m sublot was randomly assigned an experimental species: Deschampsia caespitosø (Hairgrass),

Koeleria macrantha (Junegrass), Elymus innovatus (Hairy wildrye) or Festuca saximontanø (Rocky mountain fescue) (Figure 8.1). One hundred seeds, arranged in four rows of 25 seeds, were planted per sublot. Plots were irrigated daily by the summer staff of Syncrude Canada Ltd.

Surface temperature of coke could exceed 5loC on sunny days. At a depth of

10 cm, the temperature was still over 30oC (Boorman personal communication). Despite daily inigation, only I D. caespftosa seedling emerged (Figure 8.2). By 30 days after seeding survival was 0o/o. Excavated seeds showed evidence of germination manifested 150 by shoot and root growth. The sandy texture of the coke coupled with elevated temperatures created an extremely xeric environment for seedling establishment and was the likely cause for the 00lo survival. As expected, it does not appear that grasses can be seeded directly into unamended coke with any success for revegetation. 151

Figure 8.1: Setup of experimental plots at the coke beach, Mildred Lake Basemine, AB., Canada. t52

Figure 8.2: Emergence of Deschampsia caespitosa seeded direcly in coke at the coke beach, Mildred Lake Basemine, AB., Canada.