phytoremediation of arsenic …...phytoremediation is the removal of contaminants from soil and...
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PHYTOREMEDIATION OF ARSENIC CONTAMINATED SOILS BY FAST GROWING EASTERN COTTONWOOD Populus deltoides (Bartr.) CLONES
By
RICHARD WILLIAM CARDELLINO
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2001
ACKNOWLEDGMENTS
I extend my gratitude to Dr. Donald Rockwood for the opportunity to participate
in bioenergy and remediation studies of Eucalyptus and Populus and also to my
committee members Drs. P. K. Nair and Thomas Crisman for additional mentoring.
This thesis was possible due to the work started by Dr. Gillian Alker, and I thank
her for the guidance and opportunity to study phytoremediation. I also thank Dr. Lena Ma
for advice on soil remediation.
The Phyto Lab staff and students have been essential for the research done, and I
am very thankful to them.
My special thanks go to my Family.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. ii
LIST OF TABLES...............................................................................................................v
LIST OF FIGURES .......................................................................................................... vii
ABSTRACT..................................................................................................................... viii
CHAPTERS
1 INTRODUCTION ..........................................................................................................1
Phytoremediation and Short Rotation Intensive Culture ........................................... 2 Cottonwood in Short Rotation Intensive Culture....................................................... 2 Hypotheses and Objectives ........................................................................................ 3
2 LITERATURE REVIEW ...............................................................................................5
As Contamination ...................................................................................................... 5 As and Soil Interactions ............................................................................................. 7
As and Plant Interactions ........................................................................................... 9 As Toxicity in Plants................................................................................................ 12 Phytoremediation ..................................................................................................... 14 Biomass vs Hyperaccumulation............................................................................... 17 Clonal Selection....................................................................................................... 19
Chelates in As Phytoremediation............................................................................. 22 3 MATERIALS AND METHODS..................................................................................28
Field Studies............................................................................................................. 28 Laboratory and Greenhouse Studies ........................................................................ 34
4 RESULTS AND DISCUSSION...................................................................................38
Field Studies............................................................................................................. 38 Laboratory and Greenhouse Studies ........................................................................ 50
5 CONCLUSIONS...........................................................................................................54
6 FUTURE RESEARCH .................................................................................................57
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APPENDICES
A TABLES.........................................................................................................................58
B FIGURES .......................................................................................................................62
C ANOVA .........................................................................................................................72
LITERATURE CITED ......................................................................................................77
BIOGRAPHICAL SKETCH .............................................................................................83
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LIST OF TABLES
Table page 1-1 Variables assessed in three field sites and/or one greenhouse experiment. ................. 4
3-1 Four treatments applied to clone ST 121 grown in Archer contaminated soil and receiving ammonium nitrate. …. ............................................................................... 35
4-1 Archer clones least squares means and standard errors in parentheses for height, diameter, and leaf number for November 2000 and May 2001. ............................... 38
4-2 Concentrations (mg/kg) of As, Cu, and Cr in leaf tissue of selected Archer clones in November 2000. .................................................................................................... 40
4-3 Concentrations (mg/kg) of As in leaf and stem tissue of 11 Archer clones in May 2001. .......................................................................................................................... 40
4-4 Plant tissue As concentration (mg/kg) in naturally occurring vegetation at Quincy in November 2000...................................................................................................... 41
4-5 Cluster means for height (cm), diameter (mm), leaf number, and/or weight (g) from Quincy cluster analysis for November 2000, February 2001, and May 2001. .42
4-6 Best performing clones by Quincy cluster analysis for height (H in cm), diameter (D in mm), leaf number (LN) and/or dry weight (W in g) in November 2000, February 2001, and May 2001. . ................................................................................ 42
4-7 Leaf As concentration (mg/kg) of selected clones at Quincy in 2000 and 2001. ..... 43
4-8 Concentrations (mg/kg) of As in Quincy soil samples taken at three depths in August and September 2000. .................................................................................... 44
4-9 Comparison of soil As at 0.15 m and tissue As concentration (mg/kg) for a given clone for September 2000. . ....................................................................................... 45
4-10 Cluster means for height (cm), diameter (mm), leaf number, and/or weight (g) from Kent cluster analysis for November 2000, February 2001, and May 2001. ..... 48
4-11 Best performing clones by cluster analysis at Kent for November 2000, February 2001, and May 2001. . ............................................................................................... 48
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4-12 Comparison of height between clones for Quincy (contaminated) and Kent (uncontaminated). ..................................................................................................... 49
4-13 Greenhouse experiment means and multiple comparisons for height and percent leaf mortality. …........................................................................................................ 50
4-14 Greenhouse experiment tissue and soil As analysis. …. ........................................... 51
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LIST OF FIGURES
Figure page 4-1 Clonal mean unaffected leaf area in in vitro leaf disc test for October 1999. .. ........ 53
4-2 Clonal mean unaffected leaf area in in vitro leaf disc test for May 2001. . ............... 53
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
PHYTOREMEDIATION OF ARSENIC CONTAMINATED SOILS BY FAST GROWING EASTERN COTTONWOOD (POPULUS DELTOIDES) CLONES
By
Richard William Cardellino
December, 2001
Chairman: Donald L. Rockwood Major Department: Forest Resources and Conservation
Florida has many arsenic (As) contaminated soils due to past agricultural
pesticide and chromium copper arsenate (CCA) wood preservative use. The cost of
conventional chemical and physical remediation is high, and therefore most contaminated
soils remain untreated. Phytoremediation, in combination with woody biomass
production, could be an economically feasible method.
Eighty-five cottonwood (Populus deltoides) clones were assessed at two CCA
contaminated sites and a clay-settling pond in Florida for their ability to grow and tolerate
or extract As metals from soils. Variables used were height, diameter at 1 cm above
ground, biomass, number of leaves, and leaf and stem concentration of As. Cluster
analysis was used to rank clones using all variables. The overall better performing clones
were ST 71, ST 201, ST 202, ST 259, and S13C1.
Where soil As levels ranged from 33 to 259 mg/kg, leaf As uptakes were 15 to 29
mg/kg in the first growing season, and 4.1 to 6.4 mg/kg in the second growing season.
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For clone ST1, leaf uptake was 5.5 mg/kg and stem uptake was 1.4 mg/kg, confirming
that stem uptake is around 1/3 of leaf uptake. Where soil As levels ranged from 1.6 to 64
mg/kg, and a hardpan was present at 2 ft, leaf As uptakes were BDL to 1.1 mg/kg. Most
plants sampled growing at lower levels of As at .5-ft, showed uptake at BDL. Plants
growing at higher soil As levels showed some As uptake.
Clone ST 201was used in the greenhouse to evaluate EDTA and histidine for
uptake enhancement of As. EDTA produced rapid leaf mortality. Histidine plus EDTA
helped the plants cope with the rapid uptake of As and permitted accumulation of up to
23 mg/kg As.
A leaf disk test for screening clones in vitro was performed for identifying clones
suitable for phytoremediation. Leaf segments were placed in a petri dish containing a
solution with CCA. Clones that showed the least amount of necrosis were considered
most appropriate for phytoremediation. ST 259 had a higher unaffected leaf area than
110804. This matched the performance in the field.
Cottonwood is potentially easy to establish, is fast growing, can produce 10
t/ha/yr of biomass, and achieve tissue concentrations of up to 10 mg/kg of As. The
removal of up to 100 g/ha of As per year is possible.
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CHAPTER 1 INTRODUCTION
Florida has many arsenic (As) contaminated soils due to past pesticide uses: cattle
dipping vats, and chromated copper arsenate (CCA) wood preservation plants. There is a
high cost associated with site remediation using conventional chemical and physical
methods, and therefore most contaminated soils remain untreated (Alker et al., 2000).
Phytoremediation is the removal of contaminants from soil and groundwater using plants.
The cost of phytoremediation for 0.4 hectares of a Pb contaminated site is
$60,000, compared to the $400,000 cost of excavating and landfilling (US EPA, 1998).
Phytoremediation in combination with woody biomass production could be an
economically feasible way of treating these sites. Phytoremediation with woody biomass
is most feasible in low to medium level As contaminated soils.
Phytoremediation uses plants to remove, transfer, stabilize, or destroy organic or
inorganic contaminants in soil, sediment, or water (US EPA, 1998). Plants accomplish
this task by three mechanisms: uptake of contaminants via roots and accumulation in
plant tissue, release of root exudates and enzymes that detoxify contaminants directly or
stimulate microbial detoxification, and enhancement of mineralization in the soil
(Schnoor et al., 1995). Soil amendments are sometimes necessary to make metal
contaminants bioavailable for uptake. Contaminants such as As will not break down, but
can be taken up and stored in plant biomass.
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Phytoremediation and Short Rotation Intensive Culture
Woody biomass can be produced while also providing for other soil remediation
needs. There is a need to identify effective tree genotypes for contaminant uptake and fast
growth and to develop guidelines for phytoremediation systems. Growth and remediation
potential is being assessed for cottonwood (Populus deltoides Bartr.), cypress (Taxodium
distichum L.), eucalyptus (Eucalyptus amplifolia Naudin, E. camaldulensis Dehnh., and
E. grandis Hill), and leucaena (Leucaena leucocephala L.), which are all potential
biomass species (Alker et al., 2000).
These species can be grown in Florida using short rotation intensive culture
(SRIC) methods by Ledin and Alriksson (1992) and weed and pest control. In this
system, trees are coppiced or cut back to the base of the stems, on a harvest rotation of 3
to 5 years. After harvest, the trees re-sprout from the cut stems. Stands can be as dense as
20,000 trees/ha, and they attain heights of up to 4 meters within 2 years of planting. This
method maximizes growth and extraction of contaminants due to the high stocking and
the high demand for water and nutrients of young trees. Under SRIC, cottonwood will
produce as much as 10 t/ha/yr of biomass. If tissue concentrations of 10 mg/kg of As are
achieved, it has the potential to remove around 100 g/ha of contamination per year (Alker
et al., 2000).
Cottonwood in Short Rotation Intensive Culture
The cottonwood tree used in this study, eastern cottonwood, is potentially easy to
establish using rooted cuttings and is also fast growing. It has a high transpiration rate, a
widespread root system, and is a native species to the Southeast. In an effluent treatment
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study at Winter Garden, Florida, cottonwood reached up to 3.5 m of height after 9 months
but was not effective in controlling weed competition (Alker et al., 2000).
Over 200 genetically improved cottonwood clones have been selected for high
biomass production, but their ability to tolerate or extract metals from soils is unknown.
The advantage of using suitable clones in plantings is to increase uniformity and
performance. Many clones should be used in a clonal plantation to decrease the risk of
forest diseases. It is important to screen the high biomass-producing clones for As
phytoremediation, because biomass is the most important factor affecting contaminant
uptake in non-hyperaccumulating plants.
With improved site preparation, propagation materials, selection of better
performing clones, and use of chelating agents, phytoremediation can be an effective, low
cost, less invasive, long-term alternative to physical removal of the soil.
Hypotheses and Objectives
The study’s six hypotheses were as follows:
1) Cottonwood will grow well in contaminated soil and accumulate As.
2) Cottonwood is tolerant to As contamination.
3) An in vitro leaf disc test pre-screens cottonwood clones for CCA tolerance.
4) Naturally occurring terrestrial vegetation at sites is not useful for As
phytoremediation.
5) Some clones are going to be suitable for As phytoremediation.
6) Histidine and EDTA will enhance As uptake to above ground biomass.
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These six hypotheses lead to the four objectives of this study: 1) Assess cottonwood clones at two CCA contaminated sites and a clay-settling area
in Florida for six variables (Table 1-1).
2) Compare an in vitro leaf disc test with field performance of clones for developing a
screening trial technique.
3) Assess clone ST 121 in the greenhouse with Archer, Florida contaminated soil and
chelates.
4) Rank clones according to performance by site and relative performance of clones
according to growth in contaminated site (Quincy) and uncontaminated site (Kent).
Table 1-1. Variables assessed in three field sites and/or one greenhouse experiment.
Variable Unit Height centimeters
Diameter millimeters Oven dry weight grams
Leaf Number - Leaf As mg/kg Stem As mg/kg
CHAPTER 2 LITERATURE REVIEW
As Contamination
As is a gray element with atomic number 33 and atomic weight 75. Common
forms are metallic arsenides and other organic and inorganic arsenides. As compounds
have no smell or taste (US DHHS, 1993). Arsenic is commonly found in soil at two
oxidation states: arsenate+5 in aerobic soils, and arsenite+3 in anaerobic (wetland) soils
(Alker et al., 2000). As is found in nature in compounds with oxygen, chlorine, and
sulfur. As bonds with carbon and hydrogen to form organic compounds in plants and
animals. These compounds are not as dangerous as inorganic As forms (US DHHS,
1993). Methylated and other organo-arsenicals exist in seafood, where As is present as
non-toxic arsenobetaine (Thomas et al., 1998).
Arsenic is classified as a “known human carcinogen” (US EPA, Group A), and is
carcinogenic by inhalation and ingestion. Chronic consumption of As contaminated water
at low level carries a risk of developing cancer of skin, bladder, lung, liver, kidney, and
prostate. As poisoning is associated with gastrointestinal, cardiovascular, and
neurological symptoms. This is a lesser concern than chronic exposure from the
contaminated groundwater and soil particles (Florida DEP, 1998b).
Arsenic does not vaporize at field temperatures, and most As compounds can
dissolve in water. Compounds can become airborne when contaminated material is
burned or carried with dust particles. Soil type, hydraulic conductivity of the soil,
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hydrology of the site, and the association with aluminum and iron oxides or hydroxides
determine the movement of As in the soil and the heterogeneous nature of As
concentration in the soil (Thomas et al., 1998).
The State of Florida has guidelines but no standard for minimum safe levels of As
for soils. Residential levels are low compared to industrial levels for direct exposure. The
cleanup standards for contaminated soil in Florida are 0.8 mg/kg for residential direct
exposure and 29 mg/kg for leachability based on groundwater criteria (A. E. H. Soils,
1997). The US EPA also sets limits on the amount of As industrial sources can release.
There is a limit of 0.05 mg/kg for As in drinking water. The federal Occupational Safety
and Health administration (OSHA) established a maximum permissible exposure limit for
airborne As of 10 µg/m3 (US DHHS, 1993).
Arsenic in US soils ranges from 2 to 200 mg/kg, with 5 mg/kg as the mean, and
the mean for Florida soils is 0.4 mg/kg. Baseline As concentration for Florida soils is
0.02 to 7.01 mg/kg. Metal concentration was determined by soil composition (Chen et al.,
1999). There is no limit set for As in soil, except as an upper limit of 5 mg/ for the US
EPA, Toxicity Characteristic Leaching Procedure (TCLP). The TCLP yields a number
that correlates to the amount of the As that would leach out of the soil (Thomas et al.,
1998). Concentrations of As in the soil media are reported as total As, regardless of
chemical form (Florida DEP, 1998a).
CCA is widely used in the United States for housing and general construction. In
Florida, CCA pressure-treated wood effectively controls wood decay and termites. The
State of Florida has imported 1,400 tons of metallic As for use in treated wood (Florida
DEP, 1998b). The Florida DEP is researching the potential of groundwater contamination
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due to the disposal of treated wood in unlined landfills. There is also concern about direct
exposure from wood leachate into surfaces and soils (Chandler, 1999). Soil
contamination of As from former industrial and agricultural activities also poses a risk for
groundwater contamination (Florida DEP, 1998b).
As and Soil Interactions
Inorganic insecticides such as lead arsenate, calcium arsenate, and copper arsenate
were used in the past and have accumulated in some agricultural sites. Organic arsenides
are presently used as herbicides. Organic forms are considered less toxic than inorganic
forms, but conversion from organic to inorganic forms will occur with time. These
processes are driven by adsorption and desorption to soils and sediments, and microbial
interactions. Arsenic is generally adsorbed by soil cations such as iron, aluminum and
calcium, forming insoluble salts. This immobilization occurs most frequently in clay soils
or organic soils. In sandy soils, bound As is prone to movement by erosion of soil
particles. It can be very persistent in soil, with an average residence time of 2,400 years
(Florida DEP, 1998a).
Arsenic and phosphorus have similar characteristics in the soil. Phosphate (P)
competes with As for binding sites with cations. In the presence of high concentration of
P, As may become more mobile and more available for plant uptake. Arsenate, As+5, is
the most prevalent form in soils (Florida DEP, 1998a).
P fertilizer additions to soils containing lead arsenate (LA) increased As
solubility. Creger and Peryea (1994) grew apricot (Prunus armeniaca L.) seedlings in
pots with loam soil. Adding LA reduced shoot and root mass and increased As
concentration in shoots and roots. P amendment had less effect on soil Pb
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phytoavailability, indicating that P displaced primarily As from the soil. In another study,
canola (Brassica napus L.) grown in high phosphate (P) concentrations in hydroponics
solution, and corresponding high P levels in the plant, showed lack of As toxicity. On the
other hand, canola grown on soil showed high toxicity at the same levels of As (Cox et
al., 1996). Mono-ammonium Phosphate (MAP) use on soils contaminated with Pb
arsenate also enhanced As solubility and phytoavailability. Leaf As was higher in the
MAP treated trees. This trend diminished over time (Pereyra, 1998).
pH soil reaction and microbial action also affect conversion of As from one state
to another. Adsorption of As+5 increases rapidly up to pH 5, starts to decrease rapidly
thereafter, and is very low above pH 7 (neutral). As+3 adsorption increases steadily up to
pH 6, and decreases steadily starting at neutral pH. Oxidation and reduction of As can
occur in the soil by bacteria, fungi, and algae. Processes such as methylation of inorganic
As and production of arsine gas, have been studied for applications in bioremediation
(Smith et al., 1998).
Pongratz (1998) measured contaminated soil from an industrial site for As
speciation. Arsenate was the major component. Through time, transformation from
arsenate to arsenite, and presence of methylarsenic and dimethylarsinic acid occurred.
This indicated activity of microorganisms and possible applications for phytoremediation.
Volatilization by microorganisms from Arsine gases is a mechanism of loss of both
inorganic and organic As in the soil. The process occurs under aerobic or anaerobic
conditions. Organo-arsenicals are more easily converted to arsine gas. In silt loam soil,
the rate of formation of Arsine gases depended on the degree of methylation and the level
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of aerobic activity. Suitable microbial populations and the iron/clay amount in the soil
also play a role in volatilization (Florida DEP, 1998a).
In anaerobic conditions such as that of salt marsh soil, As behaves differently. It
can be taken up by plants and therefore also be transferred through the food web. In the
rhizosphere, As may be immobilized through the oxidation of arsenite to less mobile
arsenate or adsorption to iron hydroxides may occur. In Aster tripolium, As and iron
accumulation occurred in the roots. There is oxidizing activity induced by plant roots or
microorganisms (Otte et al., 1991).
As and Plant Interactions
As is a natural element that is not essential for plants. It is not involved in
metabolism or enzyme systems, but will interfere with them. Due to its similar chemical
characteristics with P, it can be taken up by the plant, and therefore interfere with plant
growth (Miteva and Peycheva, 1999). Translocation of P in the plant can give some idea
of how As is transported. P is absorbed by the roots and transported in the xylem to the
younger leaves. Concentrations in the xylem range from 1 mM in P starved plants to 7
mM in plants growing in high solution P. There is also re-translocation of P from older
leaves to growing shoots, and from these to roots. Mycorrhizal fungi also seem to play an
important role in uptake of P, because available P is usually low in the soil (Schachman
et al., 1998). Arsenate was much less toxic at high P supply. At low P levels, more As
was taken up by plants, and under chronic exposure, there was toxicity (Sneller et al.,
1999).
Plants are able to tolerate elevated heavy metal concentrations through the activity
of certain enzymes, such as peroxidase. Peroxidase appears in tissues of plants grown in
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media with toxic levels of metals. Peroxidase induction in root and leaf tissue is strongly
correlated with tissue concentration of heavy metals and with growth (Miteva and
Peycheva, 1999).
Miteva and Peycheva (1999) investigated peroxide synthesis activity at different
stages of green bean (Phaseolus vulgaris) and tomato (Licopersicum esculentum)
development after treatment with As. Soluble As was applied to the roots, and plants
were harvested after formation of the fourth leaf. Leaf samples demonstrated that green
bean plants stored more As than tomato plants. Peroxidase synthesis was induced in the
green bean plant at all stages of development, whereas in tomato plants, peroxidase
synthesis only occurred at early growth stages.
Thyme and rosemary clones were grown in soil medium with azo dye, an organic
pollutant. Dye tolerance was also associated with enhanced peroxidase activity.
Peroxidase activity appears in lignification, wound healing, aromatic compound
degradation, pathogen defense, and stiffening (Zheng et al., 2000).
Carbonell et al. (1995) determined that As uptake in tomato plants grown under
greenhouse conditions was dependent on As availability in the nutrient solution. The
uptake was also limited by As toxicity to root tissue. Plants were grown for one growing
season at three sodium arsenite solution levels (2 mg/L, 5 mg/L, 10mg/L). There was
progressive accumulation of As in roots at all solution levels, up to 1000 mg/kg. At a
certain threshold, physiological damage occurred. The higher the As level in the nutrient
solution, the higher the rates of accumulation in the plant root. Concentrations in the root
were also proportional to concentrations in leaf tissue and were much higher than in leaf
tissue. At the high As concentration (10 mg/L), damage began to occur to root tissue,
11
resulting in reduction of transport and translocation to upper parts. Biomass accumulation
was inversely proportional to the amount of As in the nutrient solution, with the highest
biomass production at the lower 2-mg/l level.
In a study of five vegetable species by Tlustos et al. (1998), biomass production
was dependent on soil fertility and not by different levels of As in the soil. Plants were
grown on three different soils that contained 4.4, 16.7, and 49.5 mg/kg of As, for a three-
year period. As levels were low in general and did not reach toxic levels (Tlustos et al.,
1998).
Plant growth and heavy metal uptake were studied in a soil amendment with 10%
sawdust containing CCA. Beetroot, white clover, and lettuce were grown in pots for three
years. The As level in the soil was 32 mg/g. The CCA treatment had no adverse effect on
growth. High levels of As were detected in roots, especially in beetroot. Above ground
parts had lower levels of As, below animal toxicity levels (Speir et al., 1992).
In a study by Qian et al. (1999), uptake of As and other heavy metals was studied
for 12 wetland plants in hydroponics solutions. Water lettuce (Pistia stratiotes L.),
presented the highest shoot As uptake with 35 mg/kg. All other species presented less
than 5 mg/kg. For root As uptake, five species presented significant uptake. Iris-leaved
rush (Juncus xiphioides E. Mey.) contained around 80 mg/kg. Smooth cordgrass
(Spartina alterniflora L.) and smartweed (Polygonum hydropiperoides L.) contained
between 100 and 120 mg/kg As. Water lettuce and water zinnia (Wedelia trilobata H.)
contained the highest concentrations at 150 to 170 mg/kg. Water lettuce was also a high
accumulator of mercury.
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Uptake and translocation of As has not been widely studied for the Populus
genus. Transferring and cycling of heavy metals and As in poplar trees has been
evaluated in China by Yu et al. (1996) to measure purification efficiency. Populus
canadensis Moench. was treated with soil amendments that included 30 mg per gram of
As along with other heavy metals. Average concentrations in the root bark, root core,
trunk, branch, and leaves were 4.25, 10.51, 2.27, and 6.31 mg/kg, respectively (Yu et al.,
1996).
Heavy metals and As distribution of Populus nigra L. were studied in Bulgaria for
pollution bioremediation. Median, minimum, and maximum levels of As were reported as
0.30, < 0.2, and 20.80 mg/g dry weight respectively (Rumiana et al., 1995).
Other studies with Populus and heavy metals have been conducted in Poland and
Russia, near heavily contaminated industrial centers. The effects of copper, lead, zinc,
iron, and manganese pollution on the growth of resistant Populus marilandica Dosc.
versus sensitive Populus balsamifera L. were evaluated. Aboveground tissues of Populus
marilandica accumulated considerable amounts of metals, and it was recommended as a
tree for land reclamation (Lukaszewski et al., 1993; Rachwal et al., 1992; Giniyatullin et
al., 1998).
As Toxicity in Plants
Metals can be phytotoxic due to the triggering of physiological mechanisms.
Excess metals interact with the functional groups of amino acids to induce protein
misfunction and also permit production of many free-radicals that are toxic due to
oxidative capacity (Gonzalez, 1997).
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As toxicity depends on the concentration of soluble As, such as sodium arsenate
and As trioxide that were formerly used as herbicides. The mobility of As is inversely
proportional to Fe and Al content. Phytotoxicity of As is highly dependent on soil
properties. In heavy soils, growth reduction appears at 1000 mg/kg; in light soils the same
growth reduction appears in 100 mg/kg. Increasing the oxidation state limits As
bioavailability. The application of P decreases bioavailability of As that is already in
solution, but it could also displace adsorbed or fixed As. Concentration of As in lettuce
was proportional to total soluble soil As. The symptoms of As toxicity are wilting, violet
color (anthocyanin), discolored roots, and cell plasmolysis (Kabata-Pendias, 2001).
Dickinson et al. (1991) describes different effects of pollution and potential
responses of trees as: acute toxicity, chronic toxicity, evolution of tolerance, innate
tolerance, and facultative adaptation. Phenotypical plastic responses are more significant
than previously thought. In grasses, this responses can be carried over through vegetative
propagation.
Uptake of As by turnip in non-soil culture conditions was conducted with added
arsenite, arsenate, both inorganic, and methylarsonic acid, and dimethylarsinic acid, both
organics. The organic arsenicals had higher upward translocation and therefore more
phytotoxicity (Carbonell et al., 1999).
Toxicity studies have been conducted for other Populus species. Differences in
the accumulation of heavy metals in poplar clones were measured in Kornic, Poland.
Three clones growing in the vicinity of copper smelters were selected based on their
relative tolerance to air pollution. Cuttings were planted in 2.4-l pots with two kinds of
media: pure quartz sand and quartz sand with peat. Both substrates were mixed with
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smelter dust as 0,1,5, and 25 % supplement to the medium. After 16 weeks, clones were
selected as either sensitive, medium tolerance, or high tolerance (Rachwal et al., 1992).
Cottonwood clones could be also classified according to their tolerance to soil
contamination.
Growth of Acer pseudoplatanus L. callus tissue on liquid growth media
containing elevated copper (Cu) concentration was studied to provide an index of field
tolerance for the whole plant. Cell suspension cultures had different responses to Cu
according to their site of origin. In cultures from uncontaminated sites, growth was
inhibited at 15.0 mg/l Cu. In cultures from metal-contaminated sites, there was no toxic
effect. This indicates that there is genetic variation in trees for surviving metal
contamination (Turner and Dickinson, 1993).
Cu and zinc (Zn) tolerance for micropropagated birch (Betula pendula L.) clones
were studied in hydroponics. Tolerance indices were determined, based on the mean
growth rate of the longest root in 1 week. Clone 142A from a contaminated site showed
more tolerance to Cu and Zn than clone KL-2-M from an uncontaminated area. Roots of
both clones had equal amounts of Zn (Utriainen et al., 1997).
Phytoremediation
Phytoremediation is an organic, low input, and solar-energy powered remediation
technique that is very applicable to sites with surficial and low to medium levels of
contamination. It is very useful for treating a wide variety of environmental contaminants
at one time. Problems can arise if high levels of contaminants are toxic to plants. It has
the same mass transfer limitations as other bioremediation systems, and determining fate
or transformation of contaminants is complicated. Phytoremediation process might be
15
seasonal if plants slow down growth or become dormant at certain times of the year (US
EPA, 1998). Phytoremediation is appropriate for metal, pesticide, solvent, explosives,
and crude oil contamination. The uptake and translocation of metal contaminants in the
soil by plant roots into plant biomass is called phytoextraction or phytoaccumulation.
Plants that can grow under these conditions and contain high concentrations of metals are
hyperaccumulators (US EPA, 1998).
Phytoremediation systems such as vegetation covers on mine spoil can be a low-
cost alternatives for erosion control and dispersion of contaminant prevention. In the
Jales mine spoils in Portugal, conditions of high concentration As and heavy metals, low
pH, poor soil physical structure, nutrients, organic matter, and water level, made
colonization by plants difficult. The spoils were sparsely colonized by Holcus lanatus.
Compost was added to increase the organic matter content and improve plant
establishment (Koe et al., 1999).
The ideal plant for phytoremediation must be able to tolerate high levels of the
element in root and shoot cells. Tolerance is believed to result from vacuolar
compartmentalization and chelation. A plant must have the ability to translocate an
element from roots to shoots at high rates. Root concentrations are 10 or more times
higher than shoot concentrations, but in hyperaccumulators, shoot metal concentrations
can exceed root. There must be a rapid uptake rate for the element at levels that occur in
soil solution (Chaney et al., 1997).
Geranium (Pelargonium sp.) plants were compared to Indian mustard (Brassica
juncea) and sunflower (Helianthus annuus) for Pb uptake and tolerance under greenhouse
conditions. Pb exposure did not significantly affect the efficiency of photosystem II
16
activity in scented geraniums, but the ratios decreased significantly in Indian mustard and
sunflower plants. In geranium, Pb accumulation was observed in the apoplasm and in the
cytoplasm, vacuoles, and as distinct globules (Pb-lignin or Pb-P complexes) on the cell
membrane and cell wall. Geranium has a number of mechanisms for metal tolerance
(Krishna-Raj et al., 2000).
Two mechanisms exist for resistance to the toxicity of metal ions in plants: 1)
Preventing toxic ions from reaching their target sites and 2) tolerance to metal ions in
symplasm by complexation (synthesis of metal chelating peptides). This synthesis is
enzymatically mediated and requires a protein activated by the metal. Peptides bind metal
ions and form metal binding complexes that are transported and sequestered in plant
vacuoles. Poplar trees have been modified by introducing a gene coding for the bacterial
enzyme g-glutamylcysteine synthetase for phytochelates production (Prasad and Oliveira
Freitas, 1999).
Ion transport from the cytosol to the vacuole is important for plant metal
tolerance. The antiporter calcium exchanger 2 may be a key factor. Tobacco (Nicotiana
tabacum L.) plants expressing CAX2 accumulated more Ca2+, Cd2+ ,and Mn2+ and
increased ion transport in root tonoplast vesicles (Hirschi et al., 2000).
Mechanisms of metal accumulation involving extracellular and intracellular metal
chelation, precipitation, compartmentalization and translocation in the vascular system
are poorly understood. Metal contaminants in the soil are usually bound to organics or
clay, or are present as insoluble precipitates. Plants can secrete metal-chelating molecules
into the rhizosphere. Graminaceous species secrete mugeneic acid in response to metal
deficiencies. Roots can reduce these metals by root plasma membrane reductases and can
17
lower the pH of the soil by releasing protons. These actions are aided by soil
microorganisms (Raskin et al., 1994). Kluyvera ascorbata was used to inoculate tomato
and Indian mustard seeds, which were grown in soil with Ni, Pb, or Zn. The bacteria
protected plants against the inhibitory effects of high concentrations of Ni, most likely by
providing the plants with sufficient iron, and so contributed to phytoremediation (Burd et
al., 2000).
Biomass vs. Hyperaccumulation
Remediation of heavy metals and radionuclides requires efficient removal from
contaminated sites. Plants can extract inorganics, but effective phytoextraction requires
plants that produce high biomass and possess high uptake capacity (Ow et al., 1998).
Generally, phytoremediation systems use hyperaccumulators that tend to be slow
growing. This biomass is usually considered a hazardous waste that requires specialized
disposal. Commercially important fast growing hardwoods are generally not
hyperaccumulators, but there is a potential that they can match the extraction of metals by
hyperaccumulators in the same time span due to fast growth and high biomass
production. The concentration of the contaminant in the wood has to be dilute enough for
it to be used as a fuel or mulch source (Alker et al., 2000).
Betula pendula Roth., Alnus cordata Desf., Crataegus monogyna Jacq., and Salix
caprea L. were planted in the UK near industrial sites for land reclamation and assessed
for survival rate and uptake of heavy metals. Uptake patterns of metals into foliage and
woody tissues were variable, with substantial uptake in some species and clones. Trees
were considered for reclamation as a low-cost, ecologically sound, and sustainable
18
method. Many brownfields, which are currently prohibitively expensive to restore, could
be brought into productive use (Dickinson and Wong, 2000).
Tossel et al., (2000) found that Eucalyptus camaldulensis and Tamarix parviflora
DC. tolerated elevated concentrations of dissolved As and sodium in groundwater found
at Palo Alto, CA. Hydraulic control of the contaminant was assessed for use in
conjunction to a slurry wall, or as a substitute.
Trees of the genus Populus have been recognized as being important for
phytoremediation. Deep roots and high transpiration rates help with immobilization,
absorption, and accumulation of pollutants. Another advantage is the potential for
hydraulic control of contaminants. Trees act as hydraulic pumps when the roots reach
groundwater and transpire high amounts of water. Populus trees can transpire between
150 and 900 l per day due to fast growth. This water movement towards the surface can
help prevent both metal contamination and downward movement of metals from the soil
(US EPA, 1998).
Tolerance for aluminum and air pollution has been reported for several poplar
species (Dix et al., 1997). At Argonne National Laboratory, studies to determine the
potential of hybrid poplar trees to extract Zn from contaminated soils indicated root tissue
concentrations of up to 2000 mg/kg dry wt of Zn. Poplar trees have the potential for
attenuation of metal groundwater contamination (Nyer and Gatliff, 1996).
Phytoremediation studies with Populus have mainly been conducted with
hydroponics systems and organic contaminants. Burken et al. (1996) used Populus nigra
L. cuttings to evaluate atrazine phytoremediation in two types of soil media, silt loam and
silica sand. Individual 25-cm long cuttings were planted in bioreactors that had separate
19
compartments for soil and leaf surfaces. Bioreactors were placed (airtight) under growth
lights (soils were light shielded) and operated for a 16-hour photoperiod. Poplar trees
decreased atrazine in contaminated soils. Direct uptake was greater in sand than in silt
loam (Burken et al., 1996). Similar hydroponics studies should be conducted with As to
determine fate of contaminants and toxicity, factors that are important for
phytoremediation.
Clonal Selection
Clonal selection for phytoremediation has been performed with agricultural
plants. Alfalfa (Medicago sativa L.) growth has been tested in soil with organic
contaminants. Twenty clones of alfalfa were studied in a greenhouse trial (Wiltse et al.,
1998). Sandy loam soil and crude oil were used as growth media. Forty grams of crude
oil were spread in 2 kg of soil, then transferred to 15-cm plastic pots. The experimental
design was a randomized complete block design with four replications. Forage yield,
plant height, plant maturity, vigor, and leaf burn were measured every month.
Screening of fast growing Populus clones has been conducted for disease
resistance and adaptation to local conditions. Sixteen cottonwood clones raised from
cuttings in pots were investigated for 42 days under flooding conditions in China. Three
treatments were applied: watered (control), flooded to 3 cm above the soil surface, and
completely submerged. All plants survived the 42-day period, but flooding inhibited leaf
initiation, and chlorosis and abscission occurred. Leaf size, leaf area, and number of
leaves were reduced for all flooded plants relative to watered plants. Based on clustering
of all measured values, clones were divided into three types: Type 1 (resistant to
20
flooding), Type 2 (moderately tolerant to flooding), and Type (3-flood intolerant) (Cao
and Conner, 1999).
Growth, damage by insects, diseases, frost or herbicides, and survival were
compared for 54 hybrid Populus clones in New York. Cluster analysis and indices of
total growth and canker severity were used to select better performing clones. Seven of
the 54 clones were recommended for further tests on a larger scale (Abrahamson et al.,
1990).
In a similar study, 25 cottonwood clones were evaluated in Buenos Aires
Province, Argentina for growth rate, form, and canker, rust, and wind damage. A
hierarchical cluster procedure was used to group clones on a multivariate basis.
Clustering may be useful in selecting clones with similar morphology and growth
characteristics to avoid monoclonal plantations (Ares and Gutierrez, 1996).
Damage caused by A. germari was investigated in 34 Populus clones in 5-year-
old plantations in Shadong Province, China, by measuring: insect density and tree
infestation percentage, infestation index, and damage duration. Vague cluster analysis
(Euclidean distance as the group average for the cluster variable) divided the clones into
four groups. Clones that exhibited a tree infestation percentage of 7-25%, an infestation
index of at least 11 (from 1 to 29), and an insect density of less than 1 head/tree were
classified as resistant (XiNan et al., 1998). A similar study could be developed for
phytoremediation that includes variables for growth, concentration of contaminant, and
toxicity.
Punjab Agricultural University, India, tested 70 poplar clones in nursery
plantings. Cuttings 23 cm long were planted, fertilized, and irrigated. In India, widely
21
planted fast growing clone G-3 is becoming susceptible to pests and diseases. The need to
introduce new clones for mosaics of monoclonal plots or polyclonal plots is needed
(Sidhu, 1989). In the UK, Salix viminalis clones were established at 20,000 cuttings/ha,
and dry weights and susceptibility to rust were recorded. Six one-year and 7 two-year-old
clones had significantly higher yields than the reference clone 78183. The aim was to
select clones for clonal mixtures. These clones must have high yield biomass and
resistance to diseases (Dawson and McCracken, 1998).
Populus hybrids were grown at three spacings (0.5, 1.0, and 1.5 m) in monoclonal
plots and in polyclonal plots in Washington. After three years, differences among clones
in growth (height, diameter and biomass) and stem form were greater in polyclonal than
in monoclonal plots. Monoclonal stands had greater uniformity in tree size than
polyclonal stands. Total aboveground oven-dry woody yield averaged 48.0 t/ha in the
0.5-m spacing and decreased as spacing increased. Mean yield of monoclonal plots (44.3
t/ha) did not differ from the yield of polyclonal plots (43.1 t/ha) (DeBell and Harrington,
1997). Polyclonal plots might be a good option for phytoremediation systems to protect
plots further from pests and diseases.
Most poplar breeding programs have been conducted by intense clonal selection
to identify best clones out of thousands of seedlings. This is effective in producing
significant genetic gains in a short time. On the other hand, there is a lack of long-term
sustained breeding program, as for example multi-generation pedigrees. This limits
genetic studies, since there is lack of genetic mapping and identification of genes. Long-
term support for germplasm collection and maintenance such as clone banks is
22
recommended (Bradshaw et al., 2000). This should also benefit genetic improvement
programs for phytoremediation.
Chelates in As Phytoremediation
Metal chelates are synthetically produced for agriculture and research. Metals
chemically bond to organic compounds at multiple sites to make a ring structure
involving the metal and the agent. Metal chelates have been used for plant nutrition and
were used originally for correction of iron deficiencies in citrus in Florida. Metal chelates
resist soil fixation, are more available to plants, and have an important role in the
diffusion of micronutrient cations in the soil-root environment. Ethylenediamine
tetraacetic acid (EDTA) is a metal chelate that has the trade names NaFe and NaZn. It
can be blended into mixed dry fertilizers, slurry, and liquid fertilizers. EDTA is the most
widespread metal chelate (Wallace, 1971).
The chelate structure is a ring configuration that results when a metal ion
combines with one or two electron donor groups of a single molecule. Metals bound in a
chelating agent have lost their cationic characteristics. In the soil, water molecules are
coordinated with a metal ion, and are replaced with more stable bi-, tri-, or polydentate
groups resulting in a ring formation. Metals are therefore prevented from inactivation in
the soil and remain available to plants. Synthetic chelates have to be resistant to
decomposition by soil microorganisms. Binding is stronger for some metals than for
others, and may form hydroxy complexes that are difficult to be absorbed by plants.
Metals can be toxic to plants, but metal chelate treated plants are not harmful to
organisms. Chelates can also be fixed on clay surfaces (Wallace, 1971).
23
EDTA has two amine nitrogens and four carboxylic oxygens that wrap around a central
metal ion, such as Fe (III). It is only effective up to pH 6.5. The use of EDTA or citric
acid increases metal solubility in the soil, while maintaining parameters such as pH,
osmotic potential, ion availability, adequate of plant growth (Robinson, 1997).
Pb contaminated soils were studied to determine the factors that limit Pb
extractability. Pb needs to be in soluble form for plant uptake. It bound to oxides and
EDTA did not easily solubilize organics. Caprylic acid, a surfactant, in combination with
0.25 mM EDTA, was as effective as 0.50 mM EDTA alone. Surfactants are more
biodegradable and less expensive than EDTA (Elless and Blaylock, 2000).
Contaminants such as Pb have low bioavailability for plant uptake in certain soils.
Chelating agents were evaluated for solubilizing Pb in the soil and facilitating absorption
and translocation from the roots to the shoots in soils containing 600 mg/kg Pb and
amended with EDTA. Indian mustard was able to accumulate up to 1.5 % Pb in the
shoots. The accumulation of Cd, Cu, Ni, and Zn was also present. EDTA facilitated
biomass production as well (Blaylock et al., 1996).
Indian mustard was also evaluated by Vassil et al. (1998) for Pb accumulation in a
hydroponics experiment. Direct measurements of a complex of EDTA and Pb in xylem
exudates indicated that EDTA is responsible for Pb transport in the plant. The
coordination of Pb and EDTA enhanced mobility outside and within the plant of a
practically insoluble metal ion. After application of EDTA, plants absorbed higher levels
of Pb in the shoot tissue compared to untreated controls.
Cr (III) and Cr (VI) phytoremediation by fast growing sunflower (Helianthus
annuus) and Indian mustard was studied using soils contaminated with different rates of
24
Cr with EDTA, citric acid, and oxalic acid (Shahandeh and Hossner, 2000a). Cr
concentration in plant shoots and roots increased for all amendments, but toxicity was not
avoided. Cr (III) and Cr (VI) were equally toxic, and phytoaccumulation varied according
to soil type.
A number of agricultural crop plants were evaluated for chromium
phytoremediation. The variables measured were size, dry matter production, and
tolerance to heavy metals. There was also a difference between Cr (III) and Cr (VI)
uptake and translocation in the plants. Sunflower was the least tolerant but contained the
highest Cr accumulation; and Indian mustard, bermuda grass (Cynodon dactylon) and
switch grass (Panicum virgatum) were the most tolerant to soil Cr. Most of the chromium
was bioavailable in the form Cr (VI). EDTA addition enhanced plant uptake of Cr (III).
Limitations included high accumulation in root tissues and toxicity to shoot from metal
accumulation (Shahandeh and Hossner, 2000b).
Poplars and willows are being studied in France for decontamination of soils
polluted with Cd, such as pastures fertilized with Cd-rich super-phosphate fertilizer.
Poplar Kawa (Populus deltoides Bartr. x P. yunnanensis Dode), and willow Tangoio
(Salix matsudana Koidz.x S. alba L.) clones were planted in soils with 0.6 to 60 mg/kg
Cd. Chelating agents (0.5 g/kg and 2.0 g/kg EDTA, 0.5 g/kg DTPA and 0.5 g/kg NTA)
added to soil with poplar Kawa increased uptake of Cd. Two of the chelating agents, 2
g/kg EDTA and 0.5 g/kg NTA, caused reduction in growth and leaf abscission. Poplar
trees accumulated up to 209 mg/kg Cd (Robinson et al., 2000).
Phytochelates (PCs) are biological molecules and part of peptides and proteins.
The plant produces them to aid in the transport and accumulation of metals.
25
Hyperaccumulators are able to tolerate large quantities of metals that would otherwise be
toxic. The plant A. lesbiacum is able to absorb Ni from roots to shoots using the amino
acid histidine. Nitrogen atoms in histidine donate electrons to Ni, forming a strong bond.
The Ni is then bound inside the histidine molecular structure. Histidine is able to move
freely in the plant root (Kramer et al., 1996; Coghlan, 1996).
PCs formation was studied in cell cultures of Rauvolfia serpentina and
Arabidopsis plants. As anions uptake induced the biosynthesis of PCs such as glutamate
in vivo and in vitro. Enzyme preparation from Silene vulgaris was also able to produce
PCs in the presence of EDTA, arsenate, and arsenite, and also sequester the As
compounds (Schmoger et al., 2000). Induction of PCs and desglycyl peptides by heavy
metals and As was investigated in root cultures of Rubia tinctorum. All metals induced
PCs to some degree. However, only Ag, Cd and Cu were strongly bound to the PCs that
they induced. Cu was also bound to the PCs induced by Ag+, As+3 and Cd+2 (Maitani et
al., 1996).
To optimize the process of phytoremediation, Pickering et al. (2000) studied the
mechanisms of bioaccumulation of As in Indian mustard using x-ray absorption
spectrometry. Arsenate was absorbed by the roots via the P transport mechanism and
transported to shoots by xylem transport as arsenate and arsenite oxyanions. As was
stored in the roots and shoots as As (III)-this-thiolate complex, or As (III)-this-
glutathione. Thiolate donors such as glutathione are PCs. The addition of PCs (amino
acids) to the hydroponics medium increased As levels in leaves five times compared to
control. The total amount of As fixed increased very little, but before treatment, the
26
majority of the As was stored in the roots. The use of PCs is important for
phytoremediation systems in which aboveground biomass is harvested.
Kramer et al. (1996) investigated selective metal chelation and metal translocation
from roots to shoots. Xylem sap was sampled as exudates from cut surfaces of root
systems from plants growing in hydroponics. In A. lesbiacum, exposure to Ni in the roots
caused a slight increase in the amino acid content of shoots. Exposure of A. montanum to
the same treatment caused no increase. The amino acid responsible for the reaction was
L-histidine. A wide range of exposure to Ni showed a linear relationship between xylem
Ni and histidine concentration. Ni hyperaccumulator Alyssum had a limited capability to
take up and accumulate cobalt. Histidine response in this plant could be started with the
presence of cobalt but with limited results. This indicated that PCs could be specific to
certain elements. A. lesbiacum samples were analyzed using x-ray absorption
spectrometry. Spectra were collected from xylem sap. Results indicated that that Ni is
complexed with histidine in plant tissues. Histidine participated in the mechanism of Ni
tolerance and transport at the same time.
Vassil et al. (1998) showed that EDTA-chelated Pb outside the plant and formed
an EDTA-Pb complex that increased the transportation through the roots. In Kramer et al.
(1996), the non-tolerant A. montanum was supplied with histidine as a foliar spray, and
also Glutamine (amino acid with similar properties). At high Ni concentrations, histidine
more than doubled plant biomass production and halved the inhibitory effect of Ni on
root elongation compared to plants growing with Ni and no histidine. Histidine also
increased the flux of Ni through the xylem of A. montanum but had no effect on A.
lesbiacum (hyperaccumulator). As uptake to leaves will be expected to increase
27
somewhat in non-hyperaccumulating Populus with EDTA application, but will be
expected to increase the As uptake to leaves with EDTA and histidine.
Ni hyperaccumulating plants usually exceed concentrations of 0.1 percent of
aboveground biomass accumulation. The genus Alyssum (Brassicaceae) contains 48
hyperaccumulators. A. lesbiacum is very tolerant to Ni contamination. This species
accumulates metals two orders of magnitude higher than the non-hyperaccumulator A.
montanum of the same genus, although the metal concentration in the roots is the same
for both species. This confirms that root-to-shoot transport and capability of
accumulation in the shoots are the most important components in phytoremediation of
metals (Kramer et al., 1996).
CHAPTER 3
MATERIALS AND METHODS
Field Studies
Archer
This 0.2-hectare site in Archer, Florida is a former CCA wood preservative plant
used until 1962. Previous studies at this site include an assessment of the contamination
of the soil and groundwater and vegetation sampling. Contamination in the soil is present
due to lack of a disposal program such as a holding pond. Three samples of each selected
plant and tree species growing naturally on the site were taken for chemical analysis. One
species of brake fern (Pteris vittata) showed hyperaccumulation of As (3280 to 4980
mg/kg), but most plants were not hyperaccumulators (Komar, 1999).
The Archer soils are Entisols and belong to the Arredondo-Jonesville-Lake soil
association. Soils are well-drained, leveled, deep sandy soils underlined by limestone.
More specifically, the NE portion of the property has Arredondo fine sands, with 0 to 5 %
slopes, good drainage, and low organic content. Most of the property, including the
cottonwood plots, is in the Arredondo-Urban land complex. This classification is due to
urban lands in which buildings, streets, parking lots have reworked the soil to the point
where it is unrecognizable (Black and Veatch, 1998). Construction debris and clay from
bricks under the cottonwood plots have increased the available water capacity and slowed
permeability, improving conditions for cottonwood growth in some patches.
28
29
Previous soil samples conducted in 1995 at the site were randomly collected at
depths of 20 and 100 cm at a different location than the current cottonwood plot. Soils
were analyzed with EPA method 3051 for As, Cu, Cr, and other trace metals by atomic
absorption spectrophotometer. Total As was (156-184 mg/kg) at 20 cm, with a maximum
concentration of 690 mg/kg, far above the natural background levels of 0.1 to 6.1 mg/kg
for Florida soils. At 100 cm, total As ranged from 0.8 to 5 mg/kg. Soil samples taken at
20-cm depth had a higher As average than samples taken at 100 cm (Komar, 1999).
The study consists of two plantings in a randomized block design. The first
planting was installed February 20, 2000 with 10 clones and 10 replications of single tree
plots at 1 x 1-m spacing. Drought conditions in the following months, and use of
unrooted cuttings resulted in 20 % survival. For replanting and for a second planting, 20
unrooted cuttings of each of the 10 clones were started in the greenhouse in “super
stubby” containers with contaminated Archer soil and misted for eight weeks. This
provided a pre-screening of cuttings that did not grow under contaminated soil conditions
for replanting.
In early 2001, irrigation was started in both cottonwood plots, and 112 kg of
nitrogen per hectare was applied. The total amount applied was 2.7 kg of ammonium
nitrate. Periodically, weed control was performed because competition with herbaceous
and perennial plants was prevalent in the past. Roundup, Transline, or Goal was used
depending on site-specific problems and need to avoid damaging cottonwood.
Measurements of height, diameter, and leaf count were conducted in November
2000, and May 2001. In January 2001, trees were felled at a 10-cm stump height to
equalize growth; biomass (weight) and leaf samples were collected for the 25 1.0 to 2.0
30
m tall plants that survived establishment on February 20, 2000, and were overtopping the
new plants. Six leaves were collected from the top, center, and bottom of each tree. Soil
samples were collected under trees. Soil and leaf samples were prepared for laboratory
analysis of As. Soils were sieved to remove large objects, stored in a dry room at 60 °C,
and dried for seven days to constant weight. Leaf and biomass samples were rinsed with
de-ionized water, placed in a dry room at 60 °C, dried for seven days to constant weight,
weighed, chipped or milled depending on the size, and placed in laboratory containers.
Since the data were unbalanced due to differences in age and missing trees,
analysis of variance used General Linear Model (GLM) of Statistical Analysis System
(SAS). The effects of blocking and overall differences among clones were evaluated.
Multiple comparisons of clones were performed with GLM and pairwise t test (P-Diff)
for all least square means (Statistical Analysis System, 1990).
Quincy
This 7-hectare site is located 5.6 km east of Quincy, Florida. The facility pressure
treated posts and lumber with CCA and PCP until 1982, disposing of waste products in a
clay-lined pit in the northeast portion of the site. Waste occasionally overflowed into a
stormwater pond south of the site. Contamination assessments conducted by EPA and
FDEP after 1982 revealed PCP and As levels that exceeded state and federal guidelines.
Quincy soils are Ultisols and Udults in origin. These are deep soils with a thermic
temperature regime, udic moisture regime, a loam or sand surface, and a loam or clay
subsoil (Thomas et al., 1961). Soils are categorized as part of the Citronelle formation.
The first 4 to 5 m consist mostly of fine to medium grained sands with varying amounts
of clay, silt, and gravel. There is also a hardpan, a rust colored cemented fine-grained
sand present sporadically at 0.3 to 0.6 m below surface (Rust, 1998).
31
EPA conducted a contamination assessment in July 2000 using a previously
established 15 x 15-m sampling grid. Soil samples were collected from 179 of the grid
intersects to determine the extent of soil PCP, CCA and dioxin contamination. As and
PCP concentrations were initially determined for all samples at each location. Subsequent
samples were analyzed only if there was detection in the soil above. Sampling events
were in July and October 2000. The results established baseline concentrations of 0.97 to
18 mg/kg for PCP and 1.6 to 64 mg/kg for As. Groundwater samples from 14 monitoring
wells located throughout the site were collected. Three wells were sampled at every
sampling event, and these were located in close proximity to the phytoremediation
planted and control plots. As concentrations ranged from 1.0 to 5.0 mg/kg and remained
constant throughout the study (Rust, 1998).
A 0.48-hectare study was established using intensive SRIC methods in a
randomized block design in May 2000. It had three replications of 14-tree block plots
consisting of two rows spaced 0.75 m apart with seven trees at 1-m spacing. Around
4,800 unrooted cuttings from 67 different clones were used. The plot was cleared of
vegetation, herbicided, and disked for soil aeration before planting. This treatment did not
break up the hardpan. A control plot 0.25 hectares in size was given the same weed
control and irrigation treatment as the study. Unrooted 20- cm long cuttings were
prepared from clones obtained in Mississippi and Florida in January 2000. Each
replication had between 81 and 100 plots depending on the available area. Spacing
between plots was 1.25 m. The whole study was bordered with two rows of unrooted
cuttings.
32
A drip irrigation system was installed on both the control and planted plots. Drip
lines were 2 m apart, and in the study, were between the 0.75m spaced rows to encourage
root growth. Water was drawn from the surface aquifer using a pump. The site was
previously herbicided with Goal and Select, so no weeds were present. Height, diameter,
and leaf number were recorded in November 2000, February 2001, and May 2001. Six
leaves were collected from the top, center, and bottom of each tree. Leaves were then
rinsed with de-ionized water to remove surficial dust and contaminants and dried at 60 °C
for seven days to constant weight. Samples were later ground and collected in sample jars
for laboratory analysis of As concentration.
In November 2000, 10 leaf samples were collected for As analysis. A sampling of
stems and leaves of indigenous plants was also conducted to determine concentrations of
contaminants in the natural occurring vegetation. In February 2001, all surviving trees
were harvested by cutting the new stem growth back to the original propagation cutting.
Dry stem samples were weighed. Stem samples from the six plots that produced the
greatest woody biomass were analyzed. In May, locations of soil sampling points were
recorded to perform comparisons between localized contamination and contaminant
uptake. The numbers used for comparisons were from 0.15 m.
Since the data were unbalanced due to missing trees, GLM was performed along
with cluster analysis (FASTCLUS) in SAS using least square means for each variable
measured at the 3 sampling events (Statistical Analysis System, 1990).
Cluster analysis organized data from different variables into relatively homogeneous
groups, or clusters. The first step was establishment of the similarity or distance around
seed values using Euclidean. Since seed values were selected at random, the program was
33
run two or three times to determine if clones fall into the same groups (Statistical
Analysis System, 1990). Because cluster analysis is very sensitive to noise (sources of
variation or error not associated with the main effect – treatment: clone effect) – e.g.
position in the experimental design (block, rep, and site) and unbalancedness. Noise was
reduced using least square means, which correct observations means for sources of
variation other than clone and unbalancedness. Using the number of observations (n) of
each clone as another input for FASTCLUS, more importance (weight) was given to the
means with larger observations.
Kent
The Kent site near Lakeland, FL, is an unused clay-settling area from phosphate
mining with cogongrass (Imperata cylindrica L.) cover. This site compares clone
performance on contaminated soil with clone performance under non-As contaminated
soil. The Kent study contained most of the same clones used in Quincy and Archer and
was planted during the same period. Kent is located in a sub-tropical climate, while
Archer and Quincy are in a transition zone to temperate climate. This poses a challenge
for clonal comparisons.
Cottonwood clones obtained in Mississippi and Florida in January 2000 were also
used in the Kent site. Cuttings were rooted in the greenhouse three months prior to
planting in June 2000. Three cottonwood blocks were contained within a larger Latin
Square design. Each block contained 73 clones in a randomized design with four
replications.
Height, diameter, and leaf number were recorded in November 2000, February
2001, and May 2001. In February 2001, biomass was measured and collected. Re-sprouts
34
were measured during May 2001. Competition was observed in the past with herbaceous
and perennial plants, mostly exotic. A more advanced pre-emergent herbicide that does
not affect cottonwood was used around the cottonwood plots.
Since the data were unbalanced due to missing trees, GLM was performed along
with Cluster analysis (FASTCLUS) in SAS using least square means for each variable
measured at the three sampling events (Statistical Analysis System, 1990). For the cluster
analysis, all clones that had only one tree were dropped, but were still taken into account
in the total tree and clones count.
Clones were ranked according to performance per site, and study sites were
compared for results. By dividing the growth under contaminated conditions (Quincy) by
the growth under ideal conditions (Kent), a relative performance for clones was obtained.
Metal uptake measurements demonstrated the degree of effectiveness of clones for soil
remediation.
Laboratory and Greenhouse Studies
Chelates in As Phytoremediation
Fourteen cm cottonwood cuttings of the clone ST 121 were rooted during fall of
2000 in Miracle-Gro medium and yellow “supper stubbies”. After root and shoot
formation, propagules were taken out of stubbies, and roots were rinsed with water.
Thirty propagules of the same size were transferred to 1-kg Deepots containing 0.7 kg
of As contaminated soil. Soil from three locations of known contamination in Archer was
homogenized for 30 minutes using a small shovel and a plastic container. These plants
were grown for an additional two months so they would obtain leaves and sufficient
growth before application of amendments. Lights were used in the greenhouse to extend
35
the photoperiod (6 PM to 10 PM), and the temperature was maintained at 35 °C. Water
misters operated for one hour every morning. Soil was wetted above capacity each time.
After two months, 24 pots with plants of similar size were selected. Two samples were
taken from the soils and analyzed for As, nutrients, and pH. A one-time application of
ammonium nitrate fertilizer was added to all the pots to promote shoot formation at
0.201-g ammonium nitrate per kg soil (0.150 g N per kg).
Four plants were placed in each tray, and each plant was assigned one of four
treatments (Table 3-1) at random. The distance between plants within trays and between
trays was 10 cm to prevent shading. Six trays were placed in a design that assured that
they would get equal watering through sprinklers. Histidine solution (100 ml) was
applied as a foliar spray only once before plants were placed in the design; subsequent
applications were added to the soil to prevent inter-tray contamination. EDTA solution
was applied as a soil amendment (Komar, 1999).
Table 3-1. Four treatments applied to Clone ST 121 grown in Archer contaminated soil and receiving ammonium nitrate.
Treatment Formulation Method of Application Control - - Potassium EDTA 2.06 g/l solution in de-
ionized water 70 ml per pot was added once
Histidine 3.10 g/l solution in de-ionized water
100 ml on leaves initially, and 50 ml applied in the soil every two days
Potassium EDTA and Histidine
as above as above
Height and shoot diameter at 1 cm, leaf number, and leaf mortality were collected
24 hours after application of amendments and after 12 days (05/17/01 and 5/29/2001).
36
Above ground biomass uptake of metals (mg/kg of biomass) was determined for each
treatment at the end of the experiment.
Stems and leaves were harvested (from the top, center, and bottom of each tree)
and rinsed in de-ionized water. Soils were sieved to obtain only fine sands and particles.
Soils were placed in specialized paper bags, and plants were cut into smaller pieces. Soil
and plant samples were put in a drying room set at 60°C for seven days and dried to
constant weight. Once the plants dehydrated, they were milled and collected into vials.
Material from the same treatment was homogenized, and two samples of each treatment
were then sent to an analytical laboratory. Plants were considered dead when no green
tissue was present below the bark. Dead leaves were collected in bags and labeled for
later analysis. Data were analyzed using ANOVA and Tukey multiple comparisons
procedure in SAS.
A supplementary study was also conducted with 12 Eucalyptus grandis, 12 E.
amplifolia, and 10 Salix caroliniana Michx. to evaluate the potential of future As
phytoremediation research with these fast growing species. The same methodology
followed for cottonwood was applied.
Leaf Disc Test for Screening Clones in vitro
This test involved placing leaf segments of the plant into a petri dish containing a
solution with metals. Leaf samples of clones ST 1, ST 71, ST 72, ST 81, ST 107, ST 109,
ST 121, ST 148, ST 153, ST 197, ST 202, ST 213, ST 229, ST 238, ST 240, ST 244,
S13C115, S13C15, S4C2, S7C4, 112016, 112107, 112236, 112631, and 112910, were
obtained from a clone bank in Quincy. Two sets of five leaf discs were cut from each
clone. One set was suspended in a petri dish containing Cu, Cr, As, and CaNO3, and the
other set was suspended in a petri dish containing CaNO3 only. The solution was
37
prepared using the same average proportions of metals at the Archer site (26.6 mg Cu/l,
3.81 mg Cr/l, and 22.6 mg As/l). The leaf discs were removed from the petri dishes after
seven days of soaking at a constant temperature of 10 °C (Alker et al., 2000).
Each leaf was mounted on paper to use for scanning. Sheets of leaf discs were
scanned and saved on disc in the Scan Image Program that measures the necrotic area.
After scanning, each sheet was measured and analyzed for the area of necrosis on each
disc. Each sheet of discs was scanned under medium compression, inverted, and set at a
threshold of 167. After the area of necrosis was found, the area of the total disc was
established to determine the percent-unaffected area. Clones that showed the least amount
of necrosis were considered more appropriate for phytoremediation. The percentages
were compared to growth of the clones at Archer to determine if the leaf disc test was
effective in screening for contaminant tolerant clones.
CHAPTER 4
RESULTS AND DISCUSSION
Field Studies
Archer
For November 2000 height and diameter, variation among age (150 and 270 days)
and 11 clones were significant. For leaf number, age was significant but not clone. The
average size of 270-day trees was significantly higher than 150-day trees (Table 4.1). ST
202 was significantly different than: ST 1, ST 97, ST 244, and 112016. ST 71 was
significantly different than all the clones but ST 202. Significance among clones was
tested for each trait. Height was used as the standard for comparisons among clones.
Table 4-1. Archer clones least squares means and standard errors in parenthesis for height, diameter, and leaf number for November 2000 and May 2001. Nov 2000 May 2001 Clone n Height (cm) Diam (mm) Leaf N n Height (cm) Diam (mm) Leaf N 112016 18 87.3 (7.9) 9.5 (1.0) 24 (9) 7 121.7 (18.3) 12.4 (2.3) 112 (42) S7C1 17 102.1 (8.3) 10.1 (1.0) 27 (9) 12 161.8 (15.2) 18.1 (1.9) 195 (35) ST 202 8 124.7 (11.6) 14.4 (1.5) 38 (14) 5 183.7 (22.6) 21.8 (2.9) 133 (52) ST 1 15 98.2 (8.2) 11.4 (1.0) 48 (9) 8 142.7 (17.0) 15.5 (2.2) 166 (39) ST 121 17 91.5 (8.3) 10.0 (1.0) 26 (9) 11 137.2 (15.4) 15.5 (1.9) 105 (367) ST 153 9 81.7 (10.4) 8.7 (1.3) 15 (12) 9 126.6 (17.1) 14.0 (2.2) 87 (40) ST 197 14 97.6 (8.9) 11.6 (1.2) 30 (11) 9 151.3 (16.8) 17.2 (2.1) 112 (39) ST 229 11 84.0 (9.5) 8.8 (1.2) 20 (11) 7 136.3 (18.2) 15.3 (2.3) 74 (42) ST 240 12 103.6 (9.3) 11.0 (1.1) 28 (10) 6 123.8 (20.4) 12.8 (2.6) 112 (47) ST 244 11 96.2 (9.9) 10.6 (1.3) 26 (11) 8 130.6 (18.0) 14.4 (2.3) 113 (42) ST 71 12 127.8 (9.2) 13.9 (1.1) 35 (10) 10 144.4 (16.2) 17.0 (2.1) 135 (38) Average 13 99.5 (9.2) 10.9 (1.1) 29 (10) 8 141.8 (17.7) 15.8 (2.2) 122 (41)
38
39
For May 2001 height and diameter, variation among age (10 weeks and 14 weeks)
and 11 clones were significant. For leaf number, age was significant but not clone. The
average size of 14-week trees was significantly higher than 10-week trees. ST 202 was
significantly different than ST 153, ST 240, and 112016.
The overall best performing clones were ST 202, ST 71, and S7C1 (ST259); other
good clones were ST 1 and ST 197 (Table 4-1). With the harvesting of all trees in
February 2001 to a 10-cm stump, and the establishment of two months of irrigation, trees
stabilized their growth. In the May 2001 measurement, the clone factor was not
significant, but pair-wise differences were significant for the top and bottom of the list
clones. No clones performed badly, with the exception of 110804 that were replaced
early in the experiment. Most of the Archer clones were also good performers at Quincy
and Kent.
Leaf data for November 2000 (Table 4-2) showed As uptakes of 15 to 29 mg/kg
and a possible variation of uptake between clones ST 71 and ST 229. In May 2001, As
uptakes ranged from 4.1 to 6.4 mg/kg. ST 71 again had higher uptake, and ST 229 had
less. For ST 1, leaf uptake was 5.5 mg/kg and stem uptake was 1.4 mg/kg, confirming
that stem uptake is around 1/3 of leaf uptake (Table 4-3). Reduced uptake in the second
sampling event is possibly due to decreasing bioavailable As in the top layers of the soil
due to uptake and natural attenuation. There was still much As in the soil, but there might
be a need to make it more bioavailable using nitrogen or chelates.
Soil samples were taken at nine locations throughout two plantings. Three
locations with poor growth did not have higher contamination but might have had higher
soil compaction or less soil moisture. By the time irrigation was established in March
40
2001, they were too damaged to recover. As levels in soil ranged from 33 to 259 mg/kg.
Compared to previous years’ soil samples performed, there might have been an overall
reduction in As levels. Future testing of these five points is needed to confirm As
reduction.
Table 4-2. Concentrations (mg/kg) of As, Cu, and Cr in leaf tissue of selected Archer clones in November 2000.
Clone n As Cu Cr ST71 2 19.5 9.4 0.2 ST1 3 16.0 7.9 0.1 ST197 1 18.4 6.8 0.2 ST229 1 14.6 3.8 < 0.1
Table 4-3. Concentrations (mg/kg) of As in leaf and stem tissue of 11 Archer clones in May 2001.
Clone Matrix n StDev Mean 112016 Leaf 2 0.07 3.8 S7C1(ST 259) Leaf 2 0.28 4.2 ST 202 Leaf 1 - 4.1 ST 1 Leaf 2 0.07 5.5 ST 1 Stem 1 - 1.4 ST 121 Leaf 1 - 6.0 ST 153 Leaf 2 1.41 5.7 ST 197 (O R5 tree 17) Leaf 1 - 3.0 ST 197 (N R3 t tree 11) Leaf 1 - 5.8 ST 229 Leaf 2 0.28 5.1 ST 240 Leaf 2 0.49 5.2 ST 244 Leaf 1 - 4.4 ST 71 Leaf 2 0.35 6.4
Quincy
The indigenous species analysis (Table 4-4) indicated that none were As
hyperaccumulators. As concentrations were lower than 3.4 mg/kg, and plants had low
biomass production, making them unsuitable for As phytoremediation. S. caroliniana
41
accumulated 2.4 mg/kg and has potential to be a fast growing hardwood through
conventional tree breeding.
Table 4-4. Plant tissue As concentration (mg/kg) in naturally occurring vegetation at Quincy in November 2000.
Trees Tissue Concentration Red Maple (Acer rubrum) Stem 0.5 Cottonwood (Populus deltoides Bartr.) Stem 1.1 Live Oak (Quercus virginiana) Stem 0.7 Winged Sumac (Rhus copallina) Stem 0.8 Wax Myrtle (Myrica cerifera) Stem 0.5 Sweetgum (Liquidambar styraciflua) Stem 0.5 Coastal plain willow (Salix caroliniana) Stem 2.4 Water Oak (Quercus nigra L.) Stem 0.8
Herbs Beautyberry (Callicarpa americana) Plant 2.2 Tropical bush mint (Hyptis mutabilis) Plant 3.0 Goldenrod (Solidago sp.) Plant 2.3 Southern dewberry (Rubus trivialis) Plant 3.3 Showy rattlebox (Crotolaria spectabilis) Plant 2.3 Canebrake (Arundinaria gigantea) Plant 1.3 Eyebane (Chamaesyce nutans) Plant 1.5
In May 2001, clones that resprouted had better growth compared to the initial
establishment. The average height of all trees for the first growth season was 48 cm
(n=334) The average height for the trees four months after resprout was 58 cm (n=268).
This demonstrates that during the first growing season clones established their root
system. Eleven clones in clusters 1, 2, and 3 may have high performance for use in
phytoremediation (Table 4-5). The two best performing clones in terms of growth
(S13C11 and ST 201) were ranked in the first cluster. Other good clones were 110412,
112127, 21-6, ST 273, ST 67, KEN8, S13C20, S4C2, and ST 92 (Table 4-6). Surviving
42
clones may tolerate elevated levels of As in the soil and may be suitable to establish on
As contaminated sites.
Table 4-5. Cluster means for height (cm), diameter (mm), leaf number, and/or weight (g) from Quincy cluster analysis for November 2000, February 2001, and May 2001.
Cluster Height (cm) Diam (mm) Leaf N Weight (g) November 2000
One 89.3 10.6 15 - Two 67.7 7.7 12 -
Three 45.7 5.6 11 - Four 34.3 5 6 - Five 22.3 4.3 4 -
February 2001 One 94.8 10.6 - 17.3 Two 63.1 7.3 - 5.3
Three 49.6 6.4 - 3.2 Four 37.9 5.7 - 1.7 Five 26.5 5.2 - 0.4
May 2001 One 100.8 9.6 66 - Two 80.3 8.6 44 -
Three 60.6 6.6 50 - Four 39.9 5.5 23 - Five 59.8 6.6 31 -
Table 4-6. Best performing clones by Quincy cluster analysis for height (H in cm), diameter (D in mm), leaf number (LN) and/or dry weight (W in g) in November 2000, February 2001, and May 2001.
November 2000 February 2001 May 2001Clone Cluster n H D LN Clone Cluster n H D W Clone Cluster n H D LNST-201 First 4 89.3 10.6 15 ST-201 First 4 94.8 1.06 17.4 S13C11 First 4 110.8 9.2 65112016 Second 3 72.7 7.8 14 S13C11 Second 3 65.6 0.75 7.5 ST-201 First 4 90.8 10 67ST-67 Second 3 73.5 7.6 9 112016 Second 4 61.6 0.71 5.0 110412 Second 6 87.3 10 46110312 Second 5 60.3 7.5 12 110412 Second 6 59.8 0.72 4.6 112127 Second 4 75.2 8 39111014 Third 2 47.8 6.7 10 111014 Third 2 53.3 0.67 3.4 21-6 Second 4 82.7 9.2 44112614 Third 2 42.8 6.7 7 112614 Third 2 47.3 0.72 5.1 ST-273 Second 9 73.6 7.2 471358 Third 2 43.1 4.6 10 1358 Third 2 49.5 0.55 1.9 ST-67 Second 3 90.3 9.7 40ST-244 Third 2 43 3.8 13 ST-197 Third 2 51.1 0.64 3.5 Ken8 Third 56 60.9 6.7 51ST-261 Third 3 44.7 6 8 ST-244 Third 2 47.6 0.59 1.9 S13C20 Third 14 61.6 5.7 55ST-72 Third 3 54.2 6.1 10 ST-109 Third 3 48.5 0.6 2.2 S4C2 Third 9 57.7 7.2 44S13C11 Third 4 57 6.6 10 ST-261 Third 3 47.5 0.67 2.8 ST-92 Third 2 58.8 6.8 40S7C15 Third 5 50.2 6 5 ST-67 Third 3 53.1 0.76 5.5 1358 Fourth 2 63.4 7.2 27ST-275 Third 5 48 7.2 13 ST-72 Third 3 54.5 0.68 3.6 110312 Fourth 4 64.3 7.4 35110412 Third 7 53 7 11 S7C15 Third 5 55.4 0.71 2.7 110804 Fourth 4 68.2 8.6 28S4C2 Third 9 42.7 5.2 11 ST-240 Third 5 45.5 0.62 2.3 111510 Fourth 7 61.7 6.1 31111829 Third 10 43.8 5.4 11 ST-275 Third 5 52.4 0.71 4.2 111829 Fourth 9 64 7.2 29S13C20 Third 14 42.1 5 12 110312 Third 6 55.8 0.7 5.0 112016 Fourth 3 63.9 5.5 26Ken8 Third 55 44.8 5.4 11 S4C2 Third 8 47 0.64 4.1 112415 Fourth 3 53.6 5.3 26ST-197 Fourth 2 41.5 3.8 7 111829 Third 10 46 0.58 2.4 22-4 Fourth 8 55.7 6.2 35ST-240 Fourth 5 41.5 5.6 7 Ken8 Third 52 49.2 0.61 3.0 ST-109 Fourth 3 73.7 8.3 24
43
Arsenic was detected in 15 of the 47 leaf samples collected between August 2000
and June 2001 (Table 4-7). Of 26 clones sampled, As was detected in leaf tissue during
one or more sampling event in 12. Leaf As concentration ranged from less than 0.5
mg/kg to 2.5 mg/kg. All leaf samples collected in October and December 2000 contained
As at levels below the detection limit, including samples taken from clones where As was
detected in August 2000, suggesting that As is translocated from leaf tissues into stem
and root tissues prior to dormancy.
Table 4-7. Leaf As concentration (mg/kg) of selected clones at Quincy in 2000 and 2001.
Clone (Rep) Aug-00 Oct-00 Dec-00 Jun-01 11032 BDL 110412 BDL 0.61 111733 BDL 111829 0.69 BDL 0.56 112016 BDL BDL KEN8 (1) BDL BDL BDL 0.91 KEN8 (2) BDL BDL BDL BDL KEN8 (3) 2.5 BDL BDL BDL S13C20 BDL 1.1 S4C2 BDL 0.59 ST 12 BDL BDL ST 183 0.57 0.74 ST 197 BDL ST 201 0.61 0.7 ST 202 BDL ST 213 BDL ST 229 0.9 ST 238 BDL 0.61 ST 240 BDL 0.52 ST 244 BDL ST 261 BDL ST 273 1.3 BDL ST 63 BDL ST 67 BDL ST 72 BDL ST 92 BDL
44
In general, the average As contamination at 0.15 m in the soil throughout all the
sampling points. Contamination at 0.6 m remained relatively constant, but the
contamination at 0.9 to 1.2 m increased. Data suggest that the overall soil contamination
decreased in the planted plot. The unplanted plot remained relatively constant in
contamination (Table 4-8). US EPA Methods for Chemical Analysis of Water and
Wastes were followed for all contamination analyses. To assure the significance of the
data, duplicate digestions were done with randomly selected samples.
The increased leaching could be explained by removal of dense groundcover
(grasses and herbaceous plants) that was not followed by rapid cottonwood growth. Most
of the rows remained as bare soil.
Table 4-8. Concentrations (mg/kg) of As in Quincy soil samples taken at three depths in August and September 2000. Planted Plot Sample Points Sampled on 8/10/2000 Sampled on 10/27/2000
Row Tree L 0.15 m 0.6 m 0.9-1.2 m 0.15 m 0.6 m 0.9-1.2 m 11 21 25 22 75 28 2.9 70 100 11 38 24 22 170 140 6.7 100 120 28 21 21 2.4 63 45 4.6 19 68 28 65 23 12 63 43 4 69 69 41 21 18 42 60 46 6.5 69 80 41 38 17 43 75 57 9.6 88 100 41 65 16 21 69 19 2.5 35 90 57 38 13 7.1 57 96 2.6 45 74 57 65 14 45 50 - 37 78 - 57 21 15 23 80 - 1.5 43 -
Unplanted Plot 1 3.9 33 37 1.6 38 44
7 2.9 1 1.1 6 1 0.7
Ammonium nitrate and herbicides might have lowered pH by acidification, to
increase solubility and leaching. The total amount of As decreased in the planted plot,
suggesting volatilization of As by microorganisms, enhanced natural attenuation
45
encouraged by roots, nitrogen, and irrigation. As decline cannot be explained by the small
uptake due to abnormal slow growth. It is not known how much As is in the root biomass
of cottonwood. Groundwater levels remained the same, so leaching into the groundwater
is not apparent, but there is leaching into deeper soils. Future cottonwood root growth
could affect this contamination.
At the present height of plants, the majority of the roots are present within 0.3 m.
There is also a hardpan present at 0.6 m and roots are probably not yet reaching the
contamination at 0.6 and 0.9 m. At many sample points, most of the As contamination is
at 0.6 and 0.9 m. In four months of growth, plants took up from BDL to 1.1 mg/kg As.
Most plants sampled growing at lower levels of As at 0.15 m, showed uptake at BDL.
Plants growing at higher soil As levels showed some As uptake (Table 4-9).
Table 4-9. Comparison of soil As at 0.15 m and tissue As concentration (mg/kg) for a given clone for September 2000.
Clone Rep Row Tissue As Tissue As Soil ST12 1 5-6 Leaf BDL 16 112016 1 27-28 Leaf BDL 6 Ken 8 (tree 3 col 1) 2 33-34 Leaf BDL 7 Ken 8 (tree 7 col 1) 2 33-34 Leaf BDL 7 Ken 8 3 71-72 Leaf BDL 12 Ken 8 3 71-72 Stem BDL 12 ST 72 3 55-56 Leaf BDL 4 183 2 33-34 Leaf 0.74 42 S1C320 3 75-76 Leaf 1.1 23 110412 1 25-26 Leaf 0.61 42 S4c2 2 49-50 Leaf 0.59 43 Ken 8 1 9-10 Leaf 0.91 25 Detection Limit As: 0.5 mg/kg
46
Due to limitations on the number of samples analyzed and the high degree of
variability observed, it was not possible to identify the factors controlling leaf As
concentration. In a number of clones, As was detected at relatively high levels in one
sampling event, but was below detection limits in others. Factors that may affect tissue
As concentration include: season, clone, and tree growth and soil As concentration.
However, As was detected in two clones, ST 183 and ST 201, in both August 2000 and
June 2001 ranging from 0.57 to 0.74 mg/kg. Clone ST 201 was ranked in the highest
cluster in terms of growth performance, suggesting that this clone may have superior
growth and uptake for phytoremediation.
For November 2000 height and diameter, variation among 67 clones and three
blocks was significant. For leaf number, clone was not significant, and block was
significant. For February 2001 height, diameter, and biomass variation among 65 clones,
and three blocks was significant. Leaf number was not analyzed due to absence of leaves
in winter. For May 2001 height and diameter, variation among 62 clones and three blocks
was significant. For leaf number, clone was not significant, and block was significant.
Kent
For November 2000 height and diameter, variation among 73 clones and 10 reps
was significant. Leaf number was not performed due to high leaf numbers. Cluster
analysis was conducted using the two variables present. For February 2001, only Reps 1
to 4 were harvested and measured due to superior performance of this area and the high
amount of biomass present. For height and diameter, variation among 71 clones and four
Reps was significant. For biomass, clone was not significant, but Rep was. Leaf number
was not performed due to absence in winter. Cluster analysis was conducted using the
three variables present. For May 2001, Reps 1 to 4 were analyzed separately due both to
47
superior performance of this area and to harvesting being performed previously. For
height and leaf number, variation among 72 clones and four blocks was significant. For
diameter, clone was not significant, but Rep was. Cluster analysis was conducted using
the three variables present.
Constant weed infestation by Sesbania in plots 5 to 10 made data from Reps 1 to
4 more reliable for selecting the better performing clones: ST 107, ST 67, ST 240,
112127, 110226, ST 261, ST 265, ST 213, and ST 153 (Tables 4-10 and 4-11). The Kent
data were used to compare growth between Quincy (contaminated) and Kent
(uncontaminated) (Tables 4-12). ST 229 grew almost the same in both sites, indicating
that it is very resistant, but because it does not grow much, it is not recommended for use
in medium level contaminated sites such as Quincy or Archer. This clone is a candidate
for studies in highly contaminated areas. Clones such as ST 201 and 110412 also
performed well in both sites. Other clones that performed well at both sites (ST 67, ST
275, ST 273, and ST 261) are recommended for phytoremediation in As contaminated
sites.
FASTCLUS procedure of SAS was used because with large data sets it was
impractical to do pairwise comparisons. FASTCLUS is recommended if there are more
than 100 observations, and it permitted analysis of more than one variable at a time.
FASTCLUS is designed for disjoint clustering of very large data sets and can find good
clusters with only two or three passes over the data. The maximum number of clusters
was specified. Clones were separated into groups according to their potential for
phytoremediation (Statistical Analysis System, 1990).
48
Table 4-10. Cluster means for height (cm), diameter (mm), leaf number, and/or weight (g) from Kent cluster analysis for November 2000, February 2001, and May 2001.
Cluster Height (cm) Diam (mm) Leaf N Weight (g) November 2000
First 131.4 15.5 - - Second 108.2 13.5 - -
Third 95.3 10.8 - - Fourth 80.6 9.6 - - Fifth 56.9 7.4 - -
February 2001 First 160.9 16.5 - 82
Second 132.6 15.7 - 77.4 Third 129.5 13.9 - 47.7
Fourth 101.1 11.2 - 25.1 Fifth 69.6 8.7 - 3.4
May 2001 First 141.1 13.6 33 -
Second 130.7 12.6 46 - Third 112.4 12.1 32 -
Fourth 87.7 8.9 26 - Fifth 33.8 5.1 12 -
Table 4-11. Best performing clones by cluster analysis at Kent for November 2000, February 2001, and May 2001.
November 2000 February 2001 May 2001Clone Cluster n H D Clone Cluster n H D W Clone Cluster n H D LNST107 First 4 130.7 14.6 ST67 First 3 186.3 16.4 82.4 110226 First 2 148 13.2 30ST67 First 8 126.6 14.7 ST153 First 4 155 16.5 77.9 112127 First 3 146.2 13.6 39ST240 First 9 135.6 17.1 ST240 First 4 156.8 18.7 95.4 112620 First 4 141.3 15 24110226 Second 4 111.6 10.3 ST261 First 4 152 14.3 72.6 ST107 First 3 142.1 14.1 42ST265 Second 5 108.2 12.1 ST121 Second 2 145.2 16.2 83.8 ST213 First 1 128.4 9.8 27ST153 Second 6 115.6 14.9 112121 Second 3 144.6 21 67.9 ST240 First 4 154.8 16.6 42112016 Second 7 102.8 13.4 ST92 Second 3 127 14.1 99.9 ST265 First 3 134.2 16.8 36112121 Second 7 106.4 16.3 112620 Second 4 122.5 12.8 70.6 ST274 First 4 128.3 11.7 25ST121 Second 7 103.7 14.5 ST273 Second 4 131.8 18.6 70.9 ST275 First 4 135.3 13.3 33ST201 Second 7 102.8 12.4 ST72 Second 4 132.5 12.4 77.8 ST63 First 3 129.8 11.5 24110412 Second 8 116.7 13.1 112614 Third 2 136.3 14 46.7 ST67 First 3 161.5 13.6 35ST1 Second 8 110.9 12.2 ST1 Third 2 143.7 15.5 56.7 ST70 First 3 138.2 13.2 37ST275 Second 8 105.6 15.1 ST244 Third 2 146.3 13.6 46.6 ST72 First 4 146.5 14.2 34ST259 Second 9 106.7 12.9 110312 Third 3 129 13.4 41.1 110412 Second 4 142.3 12.7 48ST91 Second 9 107.4 11.9 ST201 Third 3 130.4 14 51.1 112740 Second 4 122.5 11.9 34ST202 Second 10 110 12.7 ST221 Third 3 108 12 52.3 ST1 Second 4 131.8 12.8 39ST239 Second 10 103.5 13.2 ST265 Third 3 152.9 14.6 57.2 ST153 Second 4 141.3 11.7 48ST261 Second 10 107.5 12.7 ST66 Third 3 109.5 12.6 46.4 ST183 Second 3 131.2 12.6 61ST273 Second 10 111.5 16.9 110412 Third 4 135.3 14.7 66 ST201 Second 3 114.2 10.9 50112614 Third 3 97.6 12.5 111733 Third 4 119.3 13 48.2 ST229 Second 2 137.5 15.8 48ST197 Third 5 89.4 8.4 112016 Third 4 137.5 15 35.6 ST239 Second 4 125 12.8 47ST244 Third 5 97.9 12.1 112127 Third 4 124.5 14.4 42.8 ST261 Second 4 130.3 12 37
49
Table 4-12. Comparison of growth between clones for Quincy (contaminated) and Kent (uncontaminated).
Clone Quincy Height (cm) Kent Height (cm) Ratio ST229 53.5 55.7 96 111829 64.0 68.8 93 ST75 53.9 73.3 74 112415 53.6 73.4 73 ST201 90.8 146.1 62 ST109 73.7 118.8 62 110412 87.3 151.3 58 110312 64.3 115.0 56 110702 38.2 68.8 56 112127 75.2 136.3 55 ST273 73.6 138.8 53 ST70 64.7 127.5 51 ST12 60.0 120.4 50 ST148 56.3 115.0 49 ST92 58.8 122.5 48 ST67 90.3 190.4 47 ST200 38.1 86.3 44 112016 63.9 147.5 43 ST91 54.7 140.0 39 ST183 47.6 122.5 39 111234 42.9 112.5 38 ST239 47.5 138.8 34 ST265 53.7 165.1 33 ST275 47.0 146.3 32 ST238 38.7 127.5 30 111733 38.7 131.3 29 ST261 46.4 161.3 29 111101 34.9 127.5 27 ST107 37.7 162.5 23 110319 22.9 112.8 20 ST240 29.4 175.0 17
50
Laboratory and Greenhouse Studies
Chelates for As Phytoremediation
Chelating treatments and Reps were significant for height and leaf mortality
(Table 4-13). The chelate study indicated that the histidine treatment produced more
growth in cottonwood than the addition of EDTA, or both histidine and EDTA. The
Control was not significantly different from treatment, but differed from the E treatment,
suggesting that EDTA alone had a negative influence on growth compared to the Control
(Table 4-13).
Table 4-13. Greenhouse experiment means and multiple comparisons for change of height and percent leaf mortality.
Treatment n Duncan Grouping Starting H (cm)
Change H (cm)
Std Dev Duncan Grouping
% Leaf Mortality
Std Dev
H 6 A 16.7 3.1 1.6 B 5.7 11.4 C 6 B A 18.5 2 1 B 3.3 8.2
HE 6 B C 22.8 0.6 1.7 B 18.2 14.4 E 6 C 19.3 -0.6 3.3 A 62 44.5
Means with same letter are not significantly different
The Control, histidine, and histidine and EDTA, treatments suffered significantly
less mortality than EDTA treatment alone. This demonstrates that the addition of
histidine to EDTA diminishes plant mortality. The addition of EDTA was supposed to
increase the concentration of As in aboveground biomass (leaf and stem), but the rapid
mortality produced by the rapid uptake of As induced by EDTA did not give time for
such uptake.
The addition of histidine to EDTA helped the plants cope with the rapid uptake of
As and permitted accumulation. Results indicate that EDTA with histidine produced the
most uptake (22.7 mg/kg) (Table 4-14). Histidine produced more uptake than the Control,
probably because it made the plant grow more, and therefore uptake more As. Average
51
soil contamination was 670 mg/kg. This level is very high, but As was probably
unavailable before addition of EDTA. Further research with E. grandis should be
conducted due to the tolerance and growth experienced with addition of EDTA and
histidine, and the As uptake of 6 mg/kg.
Metal chelates such as EDTA increase uptake potential and also participate in
shoot transport, although toxicity is present. Histidine is more efficient in binding metals
and facilitating storage without the effects of toxicity. The problem with chelates in the
soil is the danger to make currently soil-bound metals available to leaching. Metal
chelates must be applied in small amounts close to the root systems, and they may not be
applied were in close proximity to groundwater.
Table 4-14. Tissue and soil As concentration (mg/kg) in Greenhouse experiment.
Matrix Treatment n Mean StdDev Leaf and Stem Histidine 2 17.1 0.07 Leaf and Stem EDTA 2 14.4 1.63 Leaf and Stem Control 2 11.8 1.70 Leaf and Stem Histidine and EDTA 2 22.7 1.77
Greenhouse Soil Watered 2 months 2 670.5 30.41 Leaf and Stem Eucalyptus Grandis 1 5.9 -
In the supplemental study, E. grandis treated with EDTA (50 ml) and histidine
experienced average growth of 7 cm in two weeks. Immediately after application, one
plant lost 50 % of its leaves, 2 other plants lost 20 %, but no mortality was experienced
out of 12 plants. E. amplifolia suffered 20 % mortality and negligent growth. S.
caroliniana experienced 100% mortality, perhaps because these clones were from
unimproved individuals collected at Kent.
52
Leaf Disc Test for Screening Clones In Vitro
The leaf disc test (Figure 4-1) ranked clones from less tolerant (smaller unaffected
leaf area) to more tolerant (larger unaffected leaf area). Comparison of the October 1999
leaf test with the field performance of clones in Archer was inconclusive, as some clones
did not match their performance in the field. ST 1 and ST 71 had a small-unaffected leaf
area, indicating low performance in the field, which was not the case.
In the second leaf test (Figure 4-2), a small number of clones were used to avoid
deterioration from transport and high numbers. Some comparisons were also made with
clones available at Quincy, although the metal solution was customized for the
contamination in Archer. For Archer clones, S7C1 (ST 259) had a higher unaffected leaf
area than 110804. For Quincy clones, ST 201 had higher unaffected leaf area than
110312 and 111101, matching the performance in the field. This test has potential for
identifying clones suitable for remediation.
53
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
ST24
4
ST19
7
ST21
3
ST72
ST71
ST1
ST15
3
ST20
2
S4C
2
ST14
8
ST23
8
1129
10
ST10
9
ST81
ST12
1
1122
36
S13C
11
1126
31
1121
07
ST10
7
S13C
15
S7C
4
ST24
0
1120
16
ST22
9
Clone
Una
ffect
ed le
af a
rea
(% o
f tot
al le
af a
rea)
Figure 4-1. Clonal mean unaffected leaf area in in vitro leaf disc test for October 1999.
0
10
20
30
40
50
60
70
80
90
100
111101 110804 110312 ST201 S7C1
Clone
Una
ffect
ed le
af a
rea
(% o
f tot
al le
af a
rea)
Figure 4-2. Clonal mean unaffected leaf area in in vitro leaf disc test for May 2001.
CHAPTER 5 CONCLUSIONS
Cottonwood grew well under Archer As soil and contamination levels with the aid
of irrigation and herbiciding for a few weeks during establishment or resprout. All Archer
clones accumulated As principally because of the higher contamination throughout the
soil. Some clones had better growth in Quincy, and As accumulation was present in
places were there was significant contamination in the upper 0.3 m of soil. Overall, the
uptake for Quincy trees was less because this site had less surficial As contamination than
Archer, but more importantly because the trees did not grow well due to poor soil
conditions. There is a need to identify soil amendment needs by conducting soil tests for
structure and nutrients and to break the hardpan to permit root establishment.
Comparisons between sites are difficult due to the transition from sub-tropical to
temperate climate. Further research will indicate if clonal performances are similar
throughout Florida.
Stem As concentrations were often 1/3 lower than leaf sample concentrations and
therefore significantly lower than mean concentrations found in better US coal (15
mg/kg), suggesting that biomass produced on As contaminated sites is suitable for wood
products or bioenergy for cofiring with coal. There is also the potential to match
hyperaccumulators due to cottonwood’s higher biomass production (10 t/ha/yr) (Alker et
al., 2000). In comparison, CCA wood 0.25 PCF contains 1,000 mg/kg As. Background
levels in US soils average 5 mg/kg, in Florida 0.4 mg/kg. Drinking water contains 0.05
mg/l to 0.1 in the US, and up to 4 in Bangladesh.
54
55
The only evidence of cottonwood toxicity at high levels of contamination was in
the greenhouse experiment when high levels of As were made available through EDTA.
In the field, some trees grew in soils with up to 600 mg/kg, but most of this As is
probably bound and unavailable for plant uptake. The in vitro leaf disc experiment can be
used to pre-screen cottonwood clones for CCA tolerance if it is done at a small scale,
because it is labor intensive, time-consuming, and sensitive to leaf condition. It is
difficult to maintain fresh leaves; furthermore, the timing needs to be optimal for removal
from solution, proper drying, and scanning. Perhaps no more than five clones should be
used at a time for a test. More research is needed to determine if this test can replace field
trials.
Most naturally occurring terrestrial vegetation at Quincy was not useful for As
phytoremediation due to the limited uptake and growth. Some clones selected for
biomass production could also be used for As phytoremediation because they maintained
their fast growth characteristics and also absorbed As.
Histidine and EDTA enhanced As uptake in above ground biomass and increased
tolerance. Protecting the plant from metal toxicity is best done by phytochelates such as
histidine, while supplying of metals from soils to roots is best done by metal chelates
such as EDTA. The use of both types has the potential to maximize As phytoremediation
in non-hyperaccumulating plants such as cottonwood.
56
With improved site preparation, propagation material, selection of better
performing clones and possibly the use of chelating agents to improve uptake
concentrations, phytoremediation may be an effective, low cost, less invasive, long-term
alternative to physical removal of the soil.
CHAPTER 6 FUTURE RESEARCH
Future research needs to focus on planting density and harvesting
recommendations. There is also a need to conduct Toxic Characteristics Leaching
Potential (TCLP) for harvested material to identify its usefulness as a fuel source. Further
investigation in the areas of site preparation, propagation material, and selection of better
performing clones, is important.
There is a need to improve physical and chemical characteristics of the soil to
promote growth and uptake. The use of amendments and herbicides in combination with
irrigation and plowing may or may not increase leaching depending on the uptake of the
trees. The uses of amendments like phosphate and organic matter may hold some of the
contaminants. These questions have not yet been answered.
More studies are needed to study the role of microorganisms (bioremediation),
amendments, and leaching in As phytoremediation systems with fast growing trees. The
use of hyperaccumulating ferns in the understory, genetic improvements for uptake, and
combination with wetlands and engineering systems should also be considered. The use
of chelates for phytoremediation must be tested in the field, and more leaf tests must be
conducted for a higher number of clones to determine if it can replace field tests.
57
APPENDIX A
TABLES
Table A-1. Distribution of Cottonwood clones in Archer, Quincy, and Kent field sites.
Clone Archer Quincy Kent Clone Archer Quincy Kent1358 X ST1 X X2218 X ST12 X X110120 X X ST13 X X110226 X ST63 X X110312 X X ST66 X110319 X X ST67 X X110320 X ST70 X110412 X X ST71 X X110531 X ST72 X X110702 X X ST75 X X110804 X X ST81 X X110814 X ST91 X X110919 X ST92 X X111014 X X ST107 X X111032 X X ST109 X X111101 X X ST121 X X111234 X X ST124 X X111322 X X ST148 X X111510 X ST153 X X111733 X X ST163 X111829 X X ST165 X112016 X X X ST183 X X112107 X X ST197 X X X112121 X X ST200 X X112127 X ST201 X X112236 X X ST202 X X X112332 X ST213 X X112415 X X ST221 X X112614 X X ST229 X X X112620 X X ST238 X X112631 X ST239 X X112740 X ST240 X X XKen8 X ST244 X X XS13C20 X ST259 (S7C1) X XS4C2 X ST260 (S7C2) X XS7C15 X ST261 X X15-3 X ST264 X X21-6 X ST265 X X22-4 X ST272 (CL552) X X
ST273 (CL723) X X ST274 XST275 X XST276 XST278 X XST279 (114-2) X X
Total 11 67 73
58
59
Table A-2. Quincy Cluster Analysis Fall 2000, February and May 2001. November 2000 February 2001 May 2001
Clone Cluster nHeight(cm)
Diam(mm) Leaf N Cluster n
Height(cm)
Diam(mm) Weight (g) Cluster n
Height(cm)
Diam(mm) Leaf N
ST-92 Fourth 2 34.7 4.7 8.8 Fourth 2 36.5 0.86 5.75 Third 2 58.8 6.8 40ST-91 Fourth 9 34.5 4.7 6.2 Fourth 9 35.8 0.44 1.32 Fourth 4 54.7 6.4 35ST-75 Fifth 2 25.6 5.1 6.4 Fifth 2 30.5 0.5 0.37 Fourth 2 53.9 6.2 19ST-72 Third 3 54.2 6.1 9.5 Third 3 54.5 0.68 3.6 Fourth 3 64.7 7 37ST-67 Second 3 73.5 7.6 8.8 Third 3 53.1 0.76 5.53 Second 3 90.3 9.7 40ST-63 Fourth 6 34.8 4.6 5.4 Fourth 5 43.5 0.56 1.66ST-275 Third 5 48 7.2 13.4 Third 5 52.4 0.71 4.21 Fifth 2 47 6.8 31ST-273 Fourth 11 37.4 5.5 5.7 Fourth 12 39.8 0.58 1.64 Second 9 73.6 7.2 47ST-265 Fourth 4 35.1 6.1 8.1 Fourth 4 34.8 0.66 1.76 Fourth 2 53.7 6.6 18ST-264 Fifth 4 15.1 3.8 5.2 Fifth 3 18 0.45 0.18ST-261 Third 3 44.7 6 8.3 Third 3 47.5 0.67 2.81 Fifth 3 46.4 5.8 30ST-244 Third 2 43 3.8 13 Third 2 47.6 0.59 1.9ST-240 Fourth 5 41.5 5.6 6.9 Third 5 45.5 0.62 2.29 Fifth 3 29.4 5.6 24ST-239 Fourth 4 30 2.8 2 Fifth 2 47.5 5 27ST-238 Fourth 15 33.2 5.5 5.8 Fourth 15 34.8 0.59 1.47 Fifth 10 38.7 5.7 21ST-229 Fourth 3 31.7 4.1 9.4 Fourth 3 37.6 0.62 2.04 Fourth 2 53.5 6.2 37ST-221 Fifth 2 20 2.8 3ST-201 First 4 89.3 10.6 14.8 First 4 94.8 1.06 17.35 First 4 90.8 10 67ST-200 Fourth 3 37.7 5.7 4.6 Fourth 3 38.9 0.6 0.78 Fifth 3 38.1 6.4 17ST-197 Fourth 2 41.5 3.8 6.5 Third 2 51.1 0.64 3.5ST-183 Fourth 9 33.9 5.1 10.5 Fourth 9 36.8 0.57 1.31 Fifth 7 47.6 5.9 32ST-148 Fourth 6 39.1 5 6.9 Fourth 6 40.2 0.61 2.23 Fourth 4 56.3 6.6 37ST-12 Fourth 6 38.2 6.5 7.4 Fourth 6 42.6 0.65 4.1 Fourth 6 60 8.1 36ST-109 Fourth 3 33.4 5.3 10.3 Third 3 48.5 0.6 2.17 Fourth 3 73.7 8.3 24ST-107 Fourth 6 31 4.8 7.2 Fourth 6 32.2 0.52 0.01 Fifth 2 37.7 7.8 11S7C15 Third 5 50.2 6 5 Third 5 55.4 0.71 2.71 Fifth 3 41.5 6.2 19S4C2 Third 9 42.7 5.2 11.2 Third 8 47 0.64 4.09 Third 9 57.7 7.2 44S13C20 Third 14 42.1 5 11.7 Fourth 15 43.6 0.59 2.58 Third 14 61.6 5.7 55S13C11 Third 4 57 6.6 9.8 Second 3 65.6 0.75 7.48 First 4 110.8 9.2 65Ken8 Third 55 44.8 5.4 10.5 Third 52 49.2 0.61 2.98 Third 56 60.9 6.7 5122-4 Fourth 10 33.6 4.5 4.5 Fourth 9 42.9 0.58 1.64 Fourth 8 55.7 6.2 3521-6 Fourth 4 38.6 4 6.7 Fourth 4 41.1 0.6 2.18 Second 4 82.7 9.2 44112614 Third 2 42.8 6.7 6.7 Third 2 47.3 0.72 5.07112415 Fourth 9 38.8 4.8 7.4 Fourth 9 40.8 0.6 1.29 Fourth 3 53.6 5.3 26112127 Fourth 6 33.3 5.7 4.7 Fourth 6 37.9 0.62 2.01 Second 4 75.2 8 39112016 Second 3 72.7 7.8 13.7 Second 4 61.6 0.71 4.98 Fourth 3 63.9 5.5 26111829 Third 10 43.8 5.4 10.5 Third 10 46 0.58 2.4 Fourth 9 64 7.2 29111733 Fourth 7 31.4 4.8 6.9 Fourth 8 32.9 0.56 1.59 Fifth 6 38.7 4.1 25111510 Fourth 6 29.4 5.2 5.5 Fourth 5 36.6 0.57 1.32 Fourth 7 61.7 6.1 31111322 Fifth 2 21.6 5.1 3.9 Fifth 2 24.5 0.5 0.38 Fifth 3 42.9 4.9 21111234 Fourth 12 30.2 4.7 6.7 Fourth 12 33.2 0.48 1.13111101 Fifth 3 20.9 3.7 2.2 Fifth 3 26.7 0.54 0.41 Fifth 2 34.9 5.1 21111014 Third 2 47.8 6.7 10.2 Third 2 53.3 0.67 3.42110804 Fourth 6 41.1 5.4 8.3 Fourth 6 42.6 0.61 2.57 Fourth 4 68.2 8.6 28110702 Fifth 4 27.2 5.3 6.5 Fourth 3 32.3 0.56 0.66 Fifth 2 38.2 6.6 22110412 Third 7 53 7 10.5 Second 6 59.8 0.72 4.63 Second 6 87.3 10 46110319 Fourth 2 29.6 3.6 5.4 Fourth 2 32 0.4 0.02 Fifth 2 22.9 3.2 13110312 Second 5 60.3 7.5 11.7 Third 6 55.8 0.7 5.02 Fourth 4 64.3 7.4 35110120 Fifth 2 22.3 3.7 2.211032 Fourth 6 27.6 5.4 6.1 Fourth 6 31.5 0.53 1.512218 Fifth 4 22.7 5.1 3.8 Fifth 4 29.9 0.49 0.59 Fifth 3 37.7 4.8 171358 Third 2 43.1 4.6 9.9 Third 2 49.5 0.55 1.92 Fourth 2 63.4 7.2 27
60
Table A-3. Kent Cluster Analysis November 2000, February and May 2001 (Plots 1-4). November 2000 February 2001 May 2001
Clone Cluster nHeight(cm)
Diam(mm) Cluster n
Height(cm)
Diam(mm)
Weight(g) Cluster n
Height(cm)
Diam(mm) Leaf N
ST92 Third 10 99.5 12.4 Second 3 127 14.1 99.9 Third 4 100.5 8.6 35ST91 Second 9 107.4 11.9 Third 4 127 13.5 53.1 Third 4 111 10.3 33ST81 Fourth 6 83.2 8 Fourth 4 80.5 9.6 14.3 Fourth 3 94.8 9.3 24ST75 Fifth 7 57.5 10.1 Fourth 4 111.3 11.7 31.8 Third 4 116.5 10.6 33ST72 Third 8 96 10.8 Second 4 132.5 12.4 77.8 First 4 146.5 14.2 34ST71 Third 9 102.8 11.2 Third 4 131.3 12.8 35.1 Third 3 116.2 10.1 42ST70 Third 10 89 8.5 Fourth 4 111.3 9.6 19.5 First 3 138.2 13.2 37ST67 First 8 126.6 14.7 First 3 186.3 16.4 82.4 First 3 161.5 13.6 35ST66 Third 6 96.7 11.1 Third 3 109.5 12.6 46.4 Fourth 4 83.3 7 20ST63 Fourth 9 85.6 9.5 Fourth 4 106.3 11.1 18.4 First 3 129.8 11.5 24
ST279 Fifth 2 32.8 3 Fifth 3 23.1 3.9 7ST278 Fourth 10 81.5 9.5 Fourth 4 98 11.1 22.7 Third 3 116.5 10.4 26ST276 Fourth 6 67.2 8 Fourth 2 97.5 12.9 29.9 Third 3 114.8 12.2 36ST275 Second 8 105.6 15.1 Third 4 133.3 16.6 54.4 First 4 135.3 13.3 33ST274 Third 7 98 11 Third 4 125.5 12.6 39.2 First 4 128.3 11.7 25ST273 Second 10 111.5 16.9 Second 4 131.8 18.6 70.9 Fourth 4 96 8.1 38
Fifth 1 44.4 6.3 16ST265 Second 5 108.2 12.1 Third 3 152.9 14.6 57.2 First 3 134.2 16.8 36ST264 Fourth 4 78.4 8ST261 Second 10 107.5 12.7 First 4 152 14.3 72.6 Second 4 130.3 12 37ST260 Third 9 95 10.1 Third 4 123.3 11.7 34.9 Fourth 4 91.3 8.9 33ST259 Second 9 106.7 12.9 Third 4 133.8 14.5 61.4 Third 4 115.8 11.1 32ST244 Third 5 97.9 12.1 Third 2 146.3 13.6 46.6 Third 3 119.1 10.8 22ST240 First 9 135.6 17.1 First 4 156.8 18.7 95.4 First 4 154.8 16.6 42ST239 Second 10 103.5 13.2 Third 4 126.5 14.8 56.4 Second 4 125 12.8 47ST238 Third 7 96.7 11.5 Fourth 4 108.3 13.1 38.3 Fourth 3 95.2 11.5 46ST229 Fifth 5 49.8 10.4 Fifth 2 51.2 12.1 -4.3 Second 2 137.5 15.8 48ST221 Fourth 8 82.1 10.1 Third 3 108 12 52.3 Fourth 4 94 10.9 24ST213 Fourth 5 86.1 8.8 Fourth 2 110.2 9.7 4.7 First 1 128.4 9.8 27ST202 Second 10 110 12.7 Third 4 118.8 14.3 39.1 Third 3 102.8 36.7 34ST201 Second 7 102.8 12.4 Third 3 130.4 14 51.1 Second 3 114.2 10.9 50ST200 Fifth 9 66.1 6.9 Fifth 4 77.5 8.5 11.2 Fourth 4 76 7.6 31ST197 Third 5 89.4 8.4 Fourth 2 104.8 9.9 28.6 Fourth 2 95.5 9.8 25ST183 Fourth 9 85.2 10 Fourth 4 101.3 11.6 21.4 Second 3 131.2 12.6 61ST165 Fourth 8 81.5 7.8 Fourth 4 92.5 9.4 18.7 Third 4 112.8 10.4 26ST163 Fourth 7 86 11 Fourth 3 118.6 13.3 32.7 Fourth 4 94 9.3 21ST153 Second 6 115.6 14.9 First 4 155 16.5 77.9 Second 4 141.3 11.7 48ST148 Third 7 87.5 11.2 Fourth 4 95 12.8 27.7 Third 4 119 11.4 35ST13 Fourth 9 76.3 9.3 Fourth 4 107.5 10.9 26.7 Fourth 3 95.8 8 26
ST124 Fourth 9 81.4 8.8 Fifth 2 82.7 10.4 6.7 Third 3 107.5 12 25ST121 Second 7 103.7 14.5 Second 2 145.2 16.2 83.8 Fourth 2 86 9.1 18ST12 Fourth 7 76.2 10.4 Fourth 3 104.6 12.1 37 Fourth 4 78 9.4 25
ST109 Third 8 90.6 9.4 Fourth 4 95 11 20.7 Fourth 3 90.5 9.2 22ST107 First 4 130.7 14.6 Third 4 150 16.2 64.7 First 3 142.1 14.1 42
ST1 Second 8 110.9 12.2 Third 2 143.7 15.5 56.7 Second 4 131.8 12.8 39112740 Fourth 6 82.2 10.5 Third 4 122.5 12.1 32.6 Second 4 122.5 11.9 34112631 Fourth 9 68.7 9.4 Fourth 3 106.2 10.9 16.8 Fourth 3 77.2 7.7 22112620 Third 6 101.8 11.2 Second 4 122.5 12.8 70.6 First 4 141.3 15 24112614 Third 3 97.6 12.5 Third 2 136.3 14 46.7 Third 2 119.5 10.4 34112415 Fifth 7 62.2 5.5 Fifth 2 69 6.9 4.2 Fourth 3 67.5 7.1 24112332 Fourth 4 79.7 8.5 Fourth 3 83.7 10.1 26.3 Third 2 119 11.4 29112236 Fourth 7 86.2 11.2 Fourth 4 106 12.6 39.4 Third 4 117.3 12.8 26112127 Third 8 96.1 12.8 Third 4 124.5 14.4 42.8 First 3 146.2 13.6 39112121 Second 7 106.4 16.3 Second 3 144.6 21 67.9 Third 4 105.8 11 35112107 Fourth 7 75 9.5 Fourth 3 88 11.7 14.5 Third 4 105.5 10.5 32112016 Second 7 102.8 13.4 Third 4 137.5 15 35.6 Third 4 113.8 10.7 32111829 Fifth 8 56.9 5.3 Fifth 3 58 7 3.9 Fourth 3 99.8 10 24111733 Third 9 92.1 11.4 Third 4 119.3 13 48.2 Third 4 118.8 11.5 31111322 Fifth 6 56.6 7.5 Fifth 3 81.2 9.1 8.9 Fourth 4 81 7.7 27111234 Fourth 7 82.4 8.8 Fourth 4 101.3 10 18.4 Fourth 3 85.1 8.1 29111101 Fourth 8 85.8 10.9 Fourth 4 111.3 12.5 27 Fourth 4 95.5 9.4 27111032 Third 9 92.6 10.7 Fourth 3 114.6 12.4 26.2 Third 4 101.8 10.8 41111014 Fifth 5 49.5 7.1 Fourth 3 82.9 8.6 12 Fourth 3 61.9 6.4 24110919 Fifth 6 57.1 9.2 Fourth 2 78.8 11.1 29.8 Fourth 3 83.8 8.6 18110814 Fifth 9 59.3 7.3 Fifth 3 71.6 9 3.5 Fourth 3 94.2 8.7 20110804 Fourth 4 80.8 8.6 Fourth 3 97 10.2 29.5 Fourth 4 78.3 7.7 14110702 Fifth 8 63.5 6 Fifth 2 56.2 7.7 9.5 Fourth 4 91.8 11.4 34110531 Third 7 102.1 10 Fourth 3 103 11.6 38.6 Fourth 3 97.5 9.4 25110412 Second 8 116.7 13.1 Third 4 135.3 14.7 66 Second 4 142.3 12.7 48110319 Fourth 7 76.6 8.7 Fourth 3 110.4 10.3 31.9 Fourth 4 91.3 10.4 20110312 Fourth 9 79.6 11.2 Third 3 129 13.4 41.1 Fourth 3 93.8 9.3 42110226 Second 4 111.6 10.3 First 2 148 13.2 30
61
Table A-4. Greenhouse Experimental Design and Data.
HistidineEDTAHis-EDTANone
R1 R2 R4 R61 3 5 9 13 17 21 232 4 6 10 14 18 22 24
7 11 15 19R3 8 12 16 20 R5
5/17/2001 5/29/2001
Plant Tment Rep Height(cm) % Leaf
Necrosis Height(cm)
% Leaf Necrosis
1 H 1 14.8 0 18.4 0 2 HE 1 17.2 58 20.1 29 3 E 1 17.1 50 17.0 100 4 C 1 25.5 0 27.5 0 5 E 2 17.0 60 11.9 100 6 C 2 16.8 13 17.9 20 7 HE 2 26.5 9 25.1 19 8 E 2 15.9 0 14.5 50 9 HE 3 28.6 0 29.6 0 10 H 3 17.6 0 19.9 0 11 C 3 12.1 0 13.1 0 12 H 3 21.0 0 22.1 0 13 C 4 19.4 0 22.6 0 14 HE 4 22.5 0 21.9 8 15 HE 4 28.2 13 30.3 40 16 E 4 18.0 100 14.9 100 17 H 5 22.1 0 23.9 6 18 E 5 21.5 50 24.5 22 19 H 5 14.0 0 19.5 0 20 C 5 21.0 0 22.5 0 21 HE 6 13.8 0 13.1 14 22 H 6 26.7 33 30.8 29 23 C 6 16.3 0 19.6 0 24 E 6 10.9 22 14.0 0
APPENDIX B FIGURES
Figure B-1. Map of Archer site.
62
63
February 20, 2000 PlantingJuly 20, 2000 Planting Replanting (July 20, 2000)
Rep 3 Rep 11 2 3 4 5 1 2 3 4 5
ST240 ST229 ST121 ST71 ST 202 1 ST153 ST240 S7C1 112016 ST153ST 202 S7C1 ST229 ST197 ST71 2 ST1 ST229 ST121 ST121 ST71ST197 ST71 112016 ST244 ST240 3 ST71 ST244 ST197 ST71 ST240ST121 ST 202 S7C1 ST121 ST1 4 ST240 ST153 ST240 ST244 ST229
Flag ST1 ST244 ST1 ST 202 ST197 5 ST197 ST121 112016 S7C1 ST121Color 112016 ST240 ST244 S7C1 ST121 6 ST244 ST1 ST153 ST197 ST1
ST1 ST197 ST240 112016 ST244 7 ST229 S7C1 ST244 ST1 S7C1ST229 ST121 ST71 ST1 ST229 8 S7C1 ST71 ST229 ST240 ST244S7C1 ST1 ST 202 ST240 112016 9 112016 ST197 ST1 ST153 112016ST244 112016 ST197 ST229 S7C1 10 ST121 112016 ST71 ST229 ST197ST197 ST229 ST197 ST71 S7C1 11 112016 ST121 ST1 ST244 ST121ST71 S7C1 ST71 ST240 ST1 12 ST229 ST240 ST229 ST153 ST1
ST121 ST121 ST121 ST121 ST240 13 S7C1 112016 ST244 S7C1 S7C1ST240 ST244 ST240 ST1 ST197 14 ST153 ST71 ST197 ST1 ST153ST1 ST1 ST1 ST244 ST121 15 ST121 ST1 ST153 ST121 112016
112016 ST197 112016 ST 202 ST229 16 ST244 S7C1 ST240 112016 ST229ST 202 ST71 ST 202 ST197 ST244 17 ST71 ST153 S7C1 ST197 ST197ST229 ST 202 ST229 ST229 ST71 18 ST240 ST244 ST71 ST229 ST71S7C1 112016 S7C1 S7C1 ST 202 18 ST197 ST229 112016 ST71 ST240ST244 ST240 ST244 112016 112016 20 ST1 ST197 ST121 ST240 ST244
6 7 8 9 10 6 7 8 9 10Rep 4 Rep 2
1 x 1 m spacing February 20 planting
Warehouses
Road
Figure B-2. Archer Experimental Design and clonal allocation plots for February and July 2000.
New Plot Old PlotRow 1 2 3 4 5 1 2 3 4 5
1 21 41 61 81 101 121 141 161 181Tree 2 22 42 62 82 102 122 142 162 182Number 3 23 43 63 83 103 123 143 163 183
4 24 44 64 84 Rep 3 104 124 144 164 184 Rep15 25 45 65 85 105 125 145 165 1856 26 46 66 86 106 126 146 166 1867 27 47 67 87 107 127 147 167 1878 28 48 68 88 108 128 148 168 1889 29 49 69 89 109 129 149 169 18910 30 50 70 90 110 130 150 170 19011 31 51 71 91 111 131 151 171 19112 32 52 72 92 112 132 152 172 19213 33 53 73 93 113 133 153 173 19314 34 54 74 94 114 134 154 174 19415 35 55 75 95 115 135 155 175 19516 36 56 76 96 Rep 4 116 136 156 176 196 Rep 217 37 57 77 97 117 137 157 177 19718 38 58 78 98 118 138 158 178 19819 39 59 79 99 119 139 159 179 19920 40 60 80 100 120 140 160 180 200
6 7 8 9 10 6 7 8 9 10
Sample # As mg/kg43 5048 259
Suspected high contamination 54 49.459 76
143 42.6148 44.1154 57.5159 55.3196 33
Figure B-3. Archer Soil samples of As conducted in May 2001.
64
Figure B-4. Map of Quincy site.
65
Figure B-5. Quincy map soil samples in control plot and in planted plot.
66
Figure B-6. Map of Kent site. Latin square plot located in the NW corner.
67
Figure B-7. Quincy Map of Experimental design.
68
Figure B-8. Kent Map, location of cottonwood (CW) plots in Latin square.
69
Figure B-9. Kent, CW plots Experimental Design and clonal allocation.
70
Figure B-10. Leaf disc test scanning sheets for clones.
Figure B-11. Leaf disc test. Mortality area detected by scanning software for different clones.
71
Figure B-12. Quincy Map. Clonal allocation and location of soil and plant tissue samples.
APPENDIX C ANOVA
Table C-1. Archer November 2000. The SAS System The GLM Procedure Class Levels Values Age 2 150 270 days Clone 11 S7C1 ST 202 ST1 ST121 ST153 ST197 ST229 ST240 ST244 ST71 X112016 Number of observations 144 Height Dependent Variable: Height Source DF Type III SS Mean Square F Value Pr > F Age 1 165817.1118 165817.1118 171.26 <.0001 Clone 10 24157.5561 2415.7556 2.50 0.0089 Error 132 127804.7688 968.2179 Corrected Total 143 331238.8889 Dependent Variable: Diameter Source DF Type III SS Mean Square F Value Pr > F Age 1 1508.992179 1508.992179 103.57 <.0001 Clone 10 355.671612 35.567161 2.44 0.0108 Error 125 1821.166081 14.569329 Corrected Total 136 3784.657664 Dependent Variable: Leaf Source DF Type III SS Mean Square F Value Pr > F Age 1 19913.50978 19913.50978 16.50 <.0001 Clone 10 9732.48231 973.24823 0.81 0.6228 Error 126 152068.4177 1206.8922 Corrected Total 137 183026.8841
Table C-2. Archer May 2001. The SAS System The GLM Procedure Block 2 N O Clone 11 S7C1 ST 202 ST1 ST121 ST153 ST197 ST229 ST240 ST244 ST71 X112016 Age 2 10 14 weeks Number of observations 92 Dependent Variable: Height Source DF Type III SS Mean Square F Value Pr > F Block 1 23312.67715 23312.67715 10.14 0.0021 Clone 10 20914.41094 2091.44109 0.91 0.5290 Age 1 45740.97273 45740.97273 19.89 <.0001 Error 79 181716.8629 2300.2135 Corrected Total 91 324545.7391
72
73
Dependent Variable: Diam Source DF Type III SS Mean Square F Value Pr > F Block 1 61.7508481 61.7508481 1.67 0.1998 Clone 10 424.9241214 42.4924121 1.15 0.3368 Age 1 799.5001102 799.5001102 21.65 <.0001 Error 79 2917.858496 36.934918 Corrected Total 91 4558.944674 Dependent Variable: Leaf Source DF Type III SS Mean Square F Value Pr > F Block 1 12884.1256 12884.1256 1.04 0.3109 Clone 10 112562.7254 11256.2725 0.91 0.5296 Age 1 110568.3940 110568.3940 8.93 0.0037 Error 79 978683.994 12388.405 Corrected Total 91 1190621.859
Table C-3. Quincy November 2000. The SAS System The GLM Procedure Block 3 1 2 3 Clone 67 110120 110312 110319 11032 110412 110702 110804 110814 111014 111032 111101 111234 111322 111510 111733 111829 112016 112127 112236 112415 112614 112620 114-2 1358 15-3 21-6 22-4 2218 CL552 (ST272) CL723 (ST273) Ken8 S13C11 S13C20 S4C2 S7C15 ST 107 ST 109 ST 12 ST 124 ST 148 ST 183 ST 197 ST 200 ST 201 ST 202 ST 213 ST 221 ST 229 ST 238 ST 239 ST 240 ST 244 ST 260 ST 261 ST 264 ST 265 ST 273 ST 275 ST 278 ST 63 ST 67 ST 72 ST 75 ST 81 ST 91 ST 92 ST29 Number of observations 348 Dependent Variable: Height Source DF Type III SS Mean Square F Value Pr > F Block 2 3201.96223 1600.98112 5.62 0.0040 Clone 66 43202.67322 654.58596 2.30 <.0001 Error 279 79471.4523 284.8439 Corrected Total 347 124702.1695 Dependent Variable: Diameter Source DF Type III SS Mean Square F Value Pr > F Block 2 26.6683341 13.3341670 3.92 0.0210 Clone 66 465.6533443 7.0553537 2.07 <.0001 Error 279 949.279213 3.402434 Corrected Total 347 1452.068966 Dependent Variable: Leafcount Source DF Type III SS Mean Square F Value Pr > F Block 2 470.802012 235.401006 6.83 0.0013 Clone 66 2793.797608 42.330267 1.23 0.1305 Error 279 9612.62014 34.45384 Corrected Total 347 12651.00000
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Table C-4. Quincy February 2001. The SAS System The GLM Procedure Block 3 Clone 65 110120 110312 110319 11032 110412 110702 110804 110814 111014 111032 111101 111234 111322 111510 111733 111829 112016 112127 112236 112415 112614 112620 114-2 1358 15-3 21-6 22-4 2218 CL552 (ST272) CL723 (ST273) Ken8 S13C11 S13C20 S4C2 S7C15 ST 107 ST 109 ST 12 ST 124 ST 148 ST 183 ST 197 ST 200 ST 201 ST 202 ST 213 ST 229 ST 238 ST 239 ST 240 ST 244 ST 260 ST 261 ST 264 ST 265 ST 273 ST 275 ST 278 ST 63 ST 67 ST 72 ST 75 ST 91 ST 92 ST29 Number of observations 334 Dependent Variable: Height Source DF Type III SS Mean Square F Value Pr > F Block 2 1600.87816 800.43908 3.15 0.0444 Clone 64 36370.57347 568.29021 2.24 <.0001 Error 267 67841.3919 254.0876 Corrected Total 333 105044.9461 Dependent Variable: Diam Source DF Type III SS Mean Square F Value Pr > F Block 2 0.27332762 0.13666381 8.79 0.0002 Clone 64 2.65112502 0.04142383 2.66 <.0001 Error 264 4.10472244 0.01554819 Corrected Total 330 6.98525680 Dependent Variable: Weight Source DF Type III SS Mean Square F Value Pr > F Block 2 58.106882 29.053441 3.59 0.0290 Clone 64 1463.569987 22.868281 2.83 <.0001 Error 259 2096.437978 8.094355 Corrected Total 325 3575.767477
Table C-5. Quincy May 2001. The SAS System The GLM Procedure Block 3 1 2 3 Clone 62 110120 110312 110319 11032 110412 110702 110804 111014 111032 111101 111234 111510 111733 111829 112016 112127 112236 112415 112614 112620 114-2 1358 15-3 21-6 22-4 2218 CL552 (ST272) CL723 (ST273) Ken8 S13C11 S13C20 S4C2 S7C15 ST 107 ST 109 ST 12 ST 148 ST 183 ST 197 ST 200 ST 201 ST 202 ST 213 ST 221 ST 229 ST 238 ST 239 ST 240 ST 260 ST 261 ST 264 ST 265 ST 273 ST 275 ST 278 ST 63 ST 67 ST 72 ST 75 ST 91 ST 92 ST29 Number of observations 268 Dependent Variable: Height Source DF Type III SS Mean Square F Value Pr > F Block 2 13446.60085 6723.30043 10.61 <.0001 Clone 61 63903.19376 1047.59334 1.65 0.0051 Error 204 129328.6595 633.9640 Corrected Total 267 207916.9067
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Dependent Variable: Diam Source DF Type III SS Mean Square F Value Pr > F Block 2 56.5432592 28.2716296 5.18 0.0064 Clone 61 461.1909749 7.5605078 1.38 0.0489 Error 204 1113.917102 5.460378 Corrected Total 267 1673.264291 Dependent Variable: Leaf Source DF Type III SS Mean Square F Value Pr > F Block 2 15459.23930 7729.61965 12.03 <.0001 Clone 61 47497.62509 778.64959 1.21 0.1632 Error 204 131095.3940 642.6245 Corrected Total 267 197834.1194
Table C-6. Kent November 2000. The SAS System The GLM Procedure Rep 10 Clone 73 110120 110226 110312 110319 110412 110531 110702 110804 110814 110919 111014 111032 111101 111234 111322 111733 111829 112016 112107 112121 112127 112236 112332 112415 112614 112620 112631 112740 ST1 ST107 ST109 ST12 ST121 ST124 ST13 ST148 ST153 ST163 ST165 ST183 ST197 ST200 ST201 ST202 ST213 ST221 ST229 ST238 ST239 ST240 ST244 ST259 ST260 ST261 ST264 ST265 ST272 ST273 ST274 ST275 ST276 ST278 ST279 ST63 ST66 ST67 ST70 ST71 ST72 ST75 ST81 ST91 ST92 Number of observations: 527 The GLM Procedure Dependent Variable: Height Source DF Type III SS Mean Square F Value Pr > F Rep 9 609387.1577 67709.6842 64.03 <.0001 Clone 72 182112.6248 2529.3420 2.39 <.0001 Error 445 470608.164 1057.546 Corrected Total 526 1283170.638 Dependent Variable: Diam Diam Source DF Type III SS Mean Square F Value Pr > F Rep 5 2069.405967 413.881193 27.92 <.0001 Clone 70 1502.421024 21.463157 1.45 0.0280 Error 170 2519.790699 14.822298 Corrected Total 245 6251.594350
Table C-7. Kent February 2001. The SAS System The GLM Procedure Class Levels Values Rep 4 1 2 3 4 Clone 71 110120 110226 110312 110319 110412 110531 110702 110804 110814 110919 111014 111032 111101 111234 111322 111733 111829 112016 112107 112121 112127 112236 112332 112415 112614 112620 112631 112740 ST1 ST107 ST109 ST12 ST121 ST124 ST13 ST148 ST153 ST163 ST165 ST183 ST197 ST200 ST201 ST202 ST213 ST221 ST229 ST238 ST239 ST240 ST244 ST259 ST260 ST261 ST265 ST272 ST273 ST274 ST275 ST276 ST278 ST63 ST66 ST67 ST70 ST71 ST72 ST75 ST81 ST91 ST92 Number of observations 233
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Dependent Variable: Height Source DF Type III SS Mean Square F Value Pr > F Rep 3 185208.9772 61736.3257 44.16 <.0001 Clone 70 141831.0545 2026.1579 1.45 0.0293 Error 159 222291.5228 1398.0599 Corrected Total 232 564640.5665 Dependent Variable: Diam Source DF Type III SS Mean Square F Value Pr > F Rep 3 1684.655689 561.551896 38.25 <.0001 Clone 70 1586.248352 22.660691 1.54 0.0137 Error 156 2290.345145 14.681700 Corrected Total 229 5796.893957 Dependent Variable: Weight Source DF Type III SS Mean Square F Value Pr > F Rep 3 203975.3721 67991.7907 41.69 <.0001 Clone 70 128114.6609 1830.2094 1.12 0.2753 Error 159 259339.2945 1631.0647 Corrected Total 232 603043.5096
Table C-8. Kent May 2001. The SAS System The GLM Procedure Rep 4 Clone 72 110120 110226 110312 110319 110412 110531 110702 110804 110814 110919 111014 111032 111101 111234 111322 111733 111829 112016 112107 112121 112127 112236 112332 112415 112614 112620 112631 112740 ST1 ST107 ST109 ST12 ST121 ST124 ST13 ST148 ST153 ST163 ST165 ST183 ST197 ST200 ST201 ST202 ST213 ST221 ST229 ST238 ST239 ST240 ST244 ST259 ST260 ST261 ST265 ST272 ST273 ST274 ST275 ST276 ST278 ST279 ST63 ST66 ST67 ST70 ST71 ST72 ST75 ST81 ST91 ST92 Number of observations 241 Dependent Variable: Height Source DF Type III SS Mean Square F Value Pr > F Rep 3 84260.8165 28086.9388 21.18 <.0001 Clone 71 143537.9037 2021.6606 1.52 0.0147 Error 166 220155.6835 1326.2391 Corrected Total 240 450056.9378 Dependent Variable: Diam Source DF Type III SS Mean Square F Value Pr > F Rep 3 429.782271 143.260757 3.66 0.0138 Clone 71 3313.241553 46.665374 1.19 0.1824 Error 166 6504.21796 39.18204 Corrected Total 240 10225.25336 Dependent Variable: Leaf Source DF Type III SS Mean Square F Value Pr > F Rep 3 2141.54537 713.84846 3.51 0.0167 Clone 71 20262.42400 285.38625 1.40 0.0406 Error 166 33784.87130 203.52332 Corrected Total 240 56388.34025
LITERATURE CITED
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BIOGRAPHICAL SKETCH
Richard Cardellino was born on February 18, 1973, in Raleigh, NC. He attended
elementary school in Rio Grande do Sul, Brazil, until 1982, and in Bonn, Germany until
1984. He later attended elementary school, high school, and university in Salto, Uruguay.
Beginning 1993 he lived in East Lansing, Michigan and attended Lansing Community
College and Michigan State University while working at the Michigan Department of
Natural Resources. He obtained a Bachelor of Science degree in forestry in summer
1998, and then he worked at the Los Alamos National Lab in New Mexico. In the Fall of
1999, Richard began a Master of Science program in the School of Forest Resources and
Conservation at University of Florida in Gainesville, which will be completed in
December 2001.
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