EMERGENCE AND GROWTH OF SEVEN GRASS SPECIES
ACROSS A GRADIENT OF METALS AND ARSENIC IN
LIME-AMENDED CONTAMINATED SOILS
by
Tara Noel Martin
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Land Rehabilitation
MONTANA STATE UNIVERSITY
Bozeman, Montana
May 2009
©COPYRIGHT
by
Tara Noel Martin
2009
All Rights Reserved
ii
APPROVAL
of a thesis submitted by
Tara Noel Martin
This thesis has been read by each member of the thesis committee and has been
found to be satisfactory regarding content, English usage, format, citation, bibliographic
style, and consistency, and is ready for submission to the Division of Graduate Education.
Dennis Neuman & Cliff Montagne
Approved for the Department Land Resources and Environmental Sciences
Bruce Maxwell
Approved for the Division of Graduate Education
Dr. Carl A. Fox
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a
master’s degree at Montana State University, I agree that the Library shall make it
available to borrowers under rules of the Library.
If I have indicated my intention to copyright this thesis by including a
copyright notice page, copying is allowable only for scholarly purposes, consistent with
“fair use” as prescribed in the U.S. Copyright Law. Requests for permission for extended
quotation from or reproduction of this thesis in whole or in parts may be granted
only by the copyright holder.
Tara Noel Martin
May 2009
iv
ACKNOWLEDGEMENTS
I am forever grateful to my major advisor, Dennis Neuman, who wanted to do this
research and believed in me. He has been an expert in the field of phytostabilization and
degraded land reclamation, and I am so lucky to have coupled up with him for this
project. I am also thankful for such a great committee of people, Cliff Montagne (Cochair of committee), Cathy Zabinski and John Borkowski. Without their guidance and
support I might not have been successful. Dennis and all of my committee members have
been extremely generous with their time and I appreciate the value that they have placed
in mentoring me. Many other people at MSU assisted me in various ways throughout this
project, I would like to thank them here: David Baumbauer and the workers at the Plant
Growth Center; Stuart Jennings, Frank Munshower, John Goering, Greg Vandeberg, Pam
Blicker, Dawn Major, and Doug Dollhopf, Reclamation Research Unit; Harold
Armstrong and Lucy at the Seed Lab; Matt Lavin, Plant Sciences; Mike Giroux lab, Plant
Sciences; John Berardinelli lab, Animal Sciences; Clain Jones, Linda McDonald, Patty
Shea, Holly Jo Carpenter, & Bruce Maxwell, LRES; and Carol Johnson, Ecology. I
would also like to thank Mark Majerus & Joe Scianna of the Bridger Plant Materials
Center for sharing their experiences with native grass germination and growth. There are
many other people that I’ve probably forgotten. I would also like to thank my parents,
Mary Jo and David, my sister Danielle, and my close friends Marigold, Matt, Molly,
Kim, Ronnie, Ben and others for their support.
v
TABLE OF CONTENTS
1. INTRODUCTION ................................................................................................... 1
Statement of Problem............................................................................................... 1
Purpose of Research................................................................................................. 8
2. LITATURE REVIEW ........................................................................................... 11
World-wide Metals and Arsenic Contamination ................................................... 11
Remedial Strategies ............................................................................................... 12
Factors Affecting Phytostabilization Success ........................................................ 15
Soil pH ............................................................................................................... 16
Trace Elements and Phytotoxicity ..................................................................... 18
Contaminant of Concern: Lead .................................................................... 20
Contaminant of Concern: Copper ................................................................ 21
Contaminant of Concern: Cadmium ............................................................ 22
Contaminant of Concern: Zinc .................................................................... 22
Contaminant of Concern: Arsenic ............................................................... 23
Fertility............................................................................................................... 24
Nitrogen ........................................................................................................ 25
Phosphorus .................................................................................................... 25
Potassium ...................................................................................................... 26
Organic Matter ................................................................................................... 26
Soil Microorganisms .......................................................................................... 27
Electrical Conductivity ...................................................................................... 27
Plant Species Selection ...................................................................................... 28
Metal Tolerant Species .................................................................................. 28
Tests of Phytotoxicity ............................................................................................ 29
Greenhouse Phytotoxicity Tests Outside of the UCFRB................................... 31
Sand or Aqueous Culture Using Liquid Additions of Single
Contaminants ................................................................................................ 31
Soils Using Liquid Additions of Single Contaminants ................................. 33
Contaminated Field Soils Mixed with Uncontaminated Soils ...................... 33
Undiluted Contaminated Field Soils ............................................................. 35
History of Phytotoxicity Tests from the UCFRB .............................................. 36
Field Tests ..................................................................................................... 36
Greenhouse Tests .......................................................................................... 37
vi
TABLE OF CONTENTS – CONTINUED
Need for Further Research ................................................................................... 40
3. MATERIALS AND METHODS.......................................................................... 42
Summary .............................................................................................................. 42
Test Soils and Collection Sites Summary ............................................................ 42
ARTS Test Soil ................................................................................................. 42
Clark Fork Test Soil .......................................................................................... 44
Dilution Soils and Collection Sites Summary ..................................................... 44
German Gulch Dilution Soil ............................................................................. 45
Little Blackfoot River Dilution Soil ................................................................. 45
Soil Handling and Mixing to Create the Dilution Series ..................................... 45
Sampling the Mixed Soils and Methods of Analyses .......................................... 47
Experimental Design............................................................................................ 48
Preparation for Plant Toxicity Tests .................................................................... 48
Soil Filling ........................................................................................................ 48
Initial Watering ................................................................................................. 48
Seed Planting .................................................................................................... 49
Seed Pre-treatment ............................................................................................ 49
Greenhouse Facilities and Conditions ................................................................. 50
Watering During the Trials .................................................................................. 50
Plant Species and their Trials............................................................................... 52
Slender Wheatgrass........................................................................................... 52
Basin Wildrye ................................................................................................... 52
Bluebunch Wheatgrass ..................................................................................... 53
Big Bluegrass .................................................................................................... 53
Sheep Fescue..................................................................................................... 53
Redtop ............................................................................................................... 54
Tufted Hairgrass ............................................................................................... 54
Identification of Volunteer or Unintentionally Planted Species .......................... 54
ITS/5.8S DNA Sequencing .............................................................................. 55
Plant Harvest and Measurements ......................................................................... 56
Total Percent Emergence ................................................................................. 56
Shoot Measurements ........................................................................................ 56
Root Measurements and Observations ............................................................ 57
Statistical Analyses .............................................................................................. 57
ANOVA ........................................................................................................... 57
Interaction Plots ............................................................................................... 58
Correlation ....................................................................................................... 59
vii
TABLE OF CONTENTS – CONTINUED
4. RESULTS ............................................................................................................ 60
Results of Soil Mixing ......................................................................................... 60
Results of One-way ANOVA, Dunnett’s Method and Correlation by Species ... 63
Basin Wildyre ................................................................................................... 64
Bluebunch Wheatgrass ..................................................................................... 68
Big Bluegrass .................................................................................................... 72
Sheep Fescue..................................................................................................... 76
Redtop ............................................................................................................... 80
Slender Wheatgrass........................................................................................... 84
Tufted Hairgrass ............................................................................................... 90
Interaction Plots .................................................................................................. 94
Root Growth Observations and Descriptions ..................................................... 99
Seed Bank/Seed Source Species ....................................................................... 102
5. SUMMARY AND DISCUSSION.................................................................... 105
6. FUTURE DIRECTION .................................................................................... 121
REFERENCES CITED.......................................................................................... 124
APPENDICES ....................................................................................................... 139
APPENDIX A: Plant Response Raw Data ........................................................ 140
APPENDIX B: Statistical Output ...................................................................... 169
viii
LIST OF TABLES
Table
Page
1. Soil pH ranges .....................................................................................................16
2. Mean regional & global background concentrations of the COCs, as well as
literature-reported phytotoxicity ranges for and maximum tolerable dietary
limits of the COCs ..............................................................................................19
3. Soil salinity guidelines ........................................................................................27
4. Soil parameters tested and methods of analyses used.........................................47
5. Soil analytical results for ARTS and Clark Fork dilution series with a column
included (in bold) for sum of total metals and arsenic........................................61
6. Mean percent emergence (%) for basin wildrye in ARTS and Clark Fork ........65
7. Mean percent emergence (%) for bluebunch wheatgrass in ARTS and Clark
Fork ....................................................................................................................69
8. Mean percent emergence (%) for big bluegrass in ARTS and Clark Fork .........73
9. Mean percent emergence (%) for sheep fescue in ARTS and Clark Fork ..........77
10. Mean percent emergence (%) for redtop in ARTS and Clark Fork ....................80
11. Mean percent emergence (%) for slender wheatgrass in ARTS and Clark
Fork .....................................................................................................................85
12. Mean percent emergence (%) for tufted hairgrass in ARTS and Clark Fork .....91
13. Identifications of volunteers or unintentionally planted species, method of
identification and total metal and arsenic levels involved ................................104
14. Thresholds of total metal and arsenic concentrations (mg/kg) where first
significant differences occurred by species, response variable and dilution
series .................................................................................................................105
ix
LIST OF TABLES - CONTINUED
Table
Page
15. Ranking of species’ performance by dilution series and response variable
thresholds ..........................................................................................................106
16. Basin wildrye plant growth data – ARTS dilution series .................................141
17. Basin wildrye plant growth data – Clark Fork dilution series ..........................143
18. Bluebunch wheatgrass plant growth data – ARTS dilution series ....................145
19. Bluebunch wheatgrass plant growth data – Clark Fork dilution series ............147
20. Big bluegrass plant growth data – ARTS dilution series ..................................149
21. Big bluegrass plant growth data – Clark Fork dilution series ...........................151
22. Sheep fescue plant growth data – ARTS dilution series ...................................153
23. Sheep fescue plant growth data- Clark Fork dilution series .............................155
24. Redtop plant growth data – ARTS dilution series ............................................157
25. Redtop plant growth data – Clark Fork dilution series .....................................159
26. Slender wheatgrass plant growth data – ARTS dilution series .........................161
27. Slender wheatgrass plant growth data – Clark Fork dilution series..................163
28. Tufted hairgrass plant growth data – ARTS dilution series ..............................165
29. Tufted hairgrass plant growth data – Clark Fork dilution series ......................167
x
LIST OF FIGURES
Figure
Page
1. Map of the Upper Clark Fork River Basin Superfund sites ..................................2
2. UCFRB phytostabilized site near Warm Springs, MT displaying poor species
richness .................................................................................................................7
3. Mobility of elements as a function of soil pH, data for a light mineral soil .......17
4. Phytotoxins in the environment and their influence on ecosystems ...................30
5. An EPA model of the effects of total metals, arsenic and pH on species richness
and biomass .........................................................................................................38
6. Map of UCFRB showing soil collection locations .............................................43
7. Proportion of individual contaminants in each treatment of ARTS dilution
series ...................................................................................................................62
8. Proportion of individual contaminants in each treatment of Clark Fork dilution
series ...................................................................................................................63
9. Boxplots of Basin wildrye shoot height for ARTS and Clark Fork ....................66
10. Boxplots of Basin wildrye total biomass for ARTS and Clark Fork ..................67
11. Boxplots of Basin wildrye root mass ratio for ARTS and Clark Fork................68
12. Boxplots of Bluebunch wheatgrass shoot height for ARTS and Clark Fork ......70
13. Boxplots of Bluebunch wheatgrass total biomass in ARTS and Clark Fork ......71
14. Boxplots of Bluebunch wheatgrass root mass ratio in ARTS and Clark Fork ...72
15. Boxplots of Big bluegrass shoot height for ARTS and Clark Fork ....................74
16. Boxplots of Big bluegrass total biomass for ARTS and Clark Fork ..................75
xi
LIST OF FIGURES - CONTINUED
Figure
Page
17. Boxplots of Big bluegrass root mass ratio in ARTS and Clark Fork..................76
18. Boxplots of Sheep fescue shoot height in ARTS and Clark Fork.......................78
19. Boxplots of Sheep fescue total biomass in ARTS and Clark Fork .....................79
20. Boxplots of Sheep fescue root mass ratio in ARTS and Clark Fork ..................80
21. Boxplots of Redtop shoot height in ARTS and Clark Fork ................................81
22. Boxplots of Redtop total biomass in ARTS and Clark Fork ..............................82
23. Boxplots of Redtop root mass ratio in ARTS and Clark Fork ............................84
24. Boxplots of Slender wheatgrass shoot height in ARTS and Clark Fork.............87
25. Boxplots of Slender wheatgrass total biomass in ARTS and Clark Fork ...........88
26. Boxplots of Slender wheatgrass root mass ratio in ARTS and Clark Fork ........89
27. Boxplots of Tufted hairgrass shoot height in ARTS and Clark Fork .................92
28. Boxplots of Tufted hairgrass total biomass in ARTS and Clark Fork ................93
29. Boxplots of Tufted hairgrass root mass ratio in ARTS and Clark Fork .............94
30. Interaction plots showing mean shoot height separated by dilution series for all
7 species tested ....................................................................................................95
31. Interaction plots showing mean total biomass separated by dilution series for all
7 species tested ....................................................................................................97
32. Root quality by category of six species across total metals and arsenic (both
dilution series ....................................................................................................100
33. U.S. EPA model of plant community effects due to total COCs and pH, with
these parameters for the current study marked with a red box .........................120
xii
ABSTRACT
Montana’s Upper Clark Fork River Basin contains hundreds of square kilometers
of land impacted by mine wastes and/or smelter emissions from decades of copper
mining and related activities. Contaminated soils in the Basin are often acidic and highly
enriched with the trace elements cadmium, copper, arsenic, lead, zinc, and others.
Natural plant colonization is often impaired, as evidenced by barren areas that are so
phytotoxic that normal germination and establishment cannot occur. One reclamation
strategy being used is in-place treatment with soil amendments including lime and other
products. This provides a more hospitable substrate for plants by raising pH and
lowering the mobile and bioavailable fraction of metals. Since contaminants are not
removed with in-place treatment, short-term and long-term effectiveness of the soil
amendments and the vegetative cover continue to be debated. Several experimental plots
within the Basin have been treated in-place, but have developed plant communities of
limited diversity where some seeded species failed to establish or persist. A greenhouse
pot study was used to determine site-specific toxicity thresholds across a dilution of total
metals and arsenic that significantly reduced plant growth. Two sets of contaminated and
reference soils collected from the Basin were mixed to obtain metal and arsenic
concentration gradients from 244 to 5885 and 250 to 7521 mg/kg, respectively. Five
native and two non-native grasses were grown in separate trials. Percent emergence,
shoot height, total biomass and root mass ratio were analyzed. Sensitivity of the seven
grasses varied according to the response measured and dilution series. Most species
showed significant reductions in total biomass and shoot height when the sum of total
metals and arsenic was 559 to 1900 mg/kg. Redtop (Agrostis gigantea Roth) was the
most tolerant species, not displaying significant decreases in total biomass until the sum
of total metals and arsenic reached 5783 mg/kg. Because the study used contaminated
environmental samples and nonagricultural species, the results may better estimate sitespecific ecological risk and toxicity thresholds for in-place treated soils in the UCFRB
over studies performed in sand with inappropriate surrogate species.
1
INTRODUCTION
Statement of Problem
Metal- and arsenic-contaminated soils and waters represent a significant
environmental and human health problem that reaches to every continent. Within
Southwest Montana, over a century of copper extraction and processing near the towns of
Butte and Anaconda has resulted in one of the largest contiguous sets of Environmental
Protection Agency (EPA) Superfund sites in the United States. This massive area is
contained within the Upper Clark Fork River Basin (UCFRB), stretching 225 kilometers
from Butte to the Milltown Dam near Missoula (U.S. EPA & MT DEQ, 2004).
Superfund sites within the Basin include Silver Bow Creek/Butte (SBC/Butte), Milltown
Reservoir/Clark Fork River (CFR), and the Anaconda Smelter (AS) (Figure 1). This
cumulative area represents land primarily impacted by fluvial deposition of acidmetalliferous tailings mixing with surface and sub-surface soils and aerially-emitted
smelter fall-out. In some areas of the Basin, tailings and mine waste mixed with native
soils can reach over a meter in thickness. It is estimated that between 8.5 and 10.5
million cubic yards of contaminated mine tailings, buried tailing and contaminated soils
exist in the Clark Fork portion of the CFR (U.E. EPA & MDEQ, 2004). In the AS,
approximately 300 square miles have been impacted by smelter emissions (U.S. EPA &
MDEQ, 1998). Here forward, these contiguous Superfund sites will be referred to as the
UCFRB.
2
Milltown
Reservoir/Clark
Fork River Site
Silverbow
Creek/Butte Site
Anaconda Smelter
Site
Figure 1. Map of the Upper Clark Fork River Basin Superfund sites.
Sediment and basin fill in the UCFRB is characterized by granitic parent material
from the Boulder Batholith and andesite-rhyolite minerals from the Elkhorn Mountains
volcanic center on the East side of the valley (Alt & Hyndman, 1986; Woods et al.,
2002). Geology on the West side of the river valley (including the Anaconda area) is
composed of a variety of sedimentary, metamorphic and igneous parent material
including limestone and other carbonates, sandstone and granite (Alt & Hyndman, 1986;
Woods et al., 2002). The pyritic ore body originally mined at Butte contains the cooccurring trace elements, arsenic, cadmium, copper, lead and zinc. A large flood in 1908,
along with later flood events, distributed these trace elements and associated native
parent material, as sediments in the entire floodplain of the upper Clark Fork River, as
3
well as contaminating smaller tributaries of the river such as Warm Springs and Silver
Bow Creeks (U.S. EPA & MDEQ, 2004).
Arsenic, cadmium, copper, lead and zinc are often enriched in the contaminated
soils and waters in the UCFRB and are considered the “contaminants of concern” (COCs)
as defined by the U.S. EPA, and they must be mitigated to reduce the environmental and
human health risks (U.S. EPA, 2001). Mine wastes containing the COCs can affect
health of humans and wildlife (including fisheries and other aquatic resources) through
food chain transfer, and have been shown to be phytotoxic to plants (U.S. EPA, 2001;
Hettiarachchi and Pierzynski, 2002; Kapustka, Lipton, Galbraith, Cacela, & LeJeune,
1995; Kapustka, 2002).
Phytotoxicity in the UCFRB is apparent when viewing the alterations to plant
community structure and composition relative to reference areas (Galbraith, LeJeune &
Lipton, 1995; U.S. EPA, 2001). Natural plant colonization and establishment on
contaminated areas are often impaired, as evidenced by barren areas where phytotoxic
root zones preclude any vegetation establishment. In tailings and smelter-impacted areas
where plant establishment has occurred naturally, vegetation recovery is extremely slow
and species diversity is poor. Undesirable weedy species often dominate these harsh sites
and only the most resistant species can colonize the waste material (U.S. EPA, 2001).
Remediation of these lands is not only legally mandated, but imperative in order to
mitigate risk from the COCs.
Regulatory agencies, with the help of research consultants, have been assessing
remedial techniques available for clean-up of the UCFRB Superfund sites for over two
4
decades (ARCO, 2000; U.S. EPA & MT DEQ, 2004; RRU, 1996; RRU, 1997). One
major strategy being used is leaving the COCs on site (in situ) and treating the
contaminated soil with amendments, such as limestone and organic matter. This method
has been called “in situ treatment”, “in-place inactivation” or “phytostabilization” (Berti
& Cunningham, 2000). The use of plants in the treatment of contaminated soils has
gained international attention in recent decades (Raskin & Ensley, 2000), and the term
phytoremediation is defined in the scientific literature as the use of green plants to
remove, contain, or render harmless environmental contaminants (Cunningham & Berti,
1993). There are five major approaches to phytoremediation defined in the scientific
literature, and all involve treatment of the environmental contaminants in situ:
●Phytoextraction—the use of metal accumulating plants to transport and
concentrate metals from the soil into harvestable roots and above ground plant
shoots (Kumar, Dushenkov, Motto & Raskin, 1995).
●Phytodegradation—the use of plants and associated microflora to degrade
organic pollutants (Salt et al., 1995).
●Rhizofiltration—the use of plants to adsorb, precipitate, and concentrate toxic
metals from polluted effluents (Dushenkov, Kumar, Motto & Raskin, 1995).
●Phytovolatilization—the use of plants to volatilize pollutants (Salt et al., 1995).
●Phytostabilization—the use of metal tolerant plants to inhibit the mobility of
metals, thus reducing the risk of further environmental degradation by leaching
into groundwater or by airborne spread (Salt et al., 1995). Also defined as the use
of amendments to reduce the risk of soil contaminants by forming insoluble
5
contaminant species, after which plants are used to cover the surface of the soil
(Berti & Cunningham, 2000).
In the UCFRB, phytostabilization1 is conducted with application of limestone and
other amendments, then plant species are seeded directly into the amended waste.
Limestone raises the pH and renders the metals less mobile (hence “stabilization”), and
thus, less bioavailable and phytotoxic (Adriano, 1986; Bolan, Rowarth, de la Luz Mora,
Adriano & Curtin, 2008; Kabata-Pendias, 2001).
Because the COCs in the UCFRB are not physically excavated with
phytostabilization, skepticism exists about the permanence and effectiveness of this
practice. Contributing to this is the lack of long-term monitoring studies on
phytostabilized sites in the UCFRB. In 2003, six phytostabilized experimental plots of
varying ages in the UCFRB were re-visited to gather long-term data regarding the
effectiveness of this treatment (Munshower, Neuman & Jennings, 2003). These
investigators found evidence that root zones were remaining neutral or alkaline, and
several volunteer species were colonizing the sites, and suggested phytostabilization was
a valuable land reclamation technique where rapid successional changes in plant
composition could take place. However, the data showed several native species
originally seeded were often absent from the phytostabilized plots, such as slender and
western wheatgrasses. Plant cover was dominated by introduced 2 grasses. Weedy
species like spotted knapweed and thistle had colonized some plots (Munshower et al.,
1
Here forward, the term phytostabilization will refer to chemically amending the contaminated soil on site,
then using plants which may or may not have demonstrable metal tolerance, to “cover” the amended soil.
2
In this paper, introduced refers to any plant species that arrived to the Rocky Mountain west after
Columbus, invariably with human assistance (USDA & NRCS, 2009)
6
2003). While introduced species may stabilize compromised sites more quickly (most of
the monitored plots were purposefully seeded with introduced species), it is generally
understood that introduced species do not contribute the same genetic and ecological
soundness that native 3 plants confer to ecosystems and they often prevent establishment
of native plants on sites where they have been seeded (Richards, Chambers & Ross,
1998). At one phytostabilized site, introduced species seeded 19 years previously
accounted for over 96% of the 107.7% total cover, with very little percent cover from
colonizing native species (Munshower et al., 2003). Data from ARCO (cited by CH2M
HILL, 2001, p. 53) further corroborates these observations that introduced species often
dominate the plant cover on phytostabilized sites. Empirical observations of
phytostabilized sites in the UCFRB show stands of grasses dominated by putative metal
tolerant species such as the introduced grass Redtop (Agrostis gigantea Roth) or the
native grass Basin wildrye (Leymus cinereus (Scribn. & Merr.) A. Löve) (D. Neuman,
personal communication, October 2, 2004; Figure 2). Despite these observations, more
recent studies have shown persistence of seeded native species and evidence of
colonization onto a phytostabilized tailings pond in Montana (Neuman & Ford, 2006).
Why do certain grasses establish and persist on phytostabilized sites while others
do not? Why do introduced species seem to predominate on phytostabilized sites? The
answers may be the persistence of the originally seeded species, or natural successional
changes occurring very slowly, or the phytotoxic levels of COCs that remain in the
contaminated soils, despite the geochemical models which suggest mobility and
3
Native refers to any plant species that was present in the Rocky Mountain west at the time of Columbus
(USDA & NRCS, 2009)
7
Cover dominated
by two grass
species
Figure 2. UCFRB phytostabilized site near Warm Springs, MT
displaying poor species richness.
bioavailability of metal cations decrease with increasing pH (Kabata-Pendias, 2001;
Bolan et al., 2008). Much of the research in the last 25 years in the UCFRB has
focused on site characterization, i.e. quantifying the degree and perimeter of the soil
contamination (U.S. EPA, 2001). Greenhouse investigations with amended UCFRB soil
and related contaminated soils (Grant-Kohrs Ranch) have occurred, but the focus of those
studies was to determine the best amendments for improved plant growth (RRU, 1996) or
to rank the phytotoxicity of the soils using mostly agronomic species (Kapustka, 2002).
Co-located soil metal and plant data were analyzed and correlated from a phytostabilized
plot within the UCFRB, but this involved the agronomic species barley (Hordeum
vulgare; Neuman, Jennings & Reeves, 2002). Very little, if any, published data exists on
site-specific toxicity thresholds 4 for locally-important grass species known to colonize or
persist on lime-treated mine wastes in the UCFRB, native or not. As such, toxicity
4
In this paper, threshold is defined as “The point, level, or value above which something is true or will take
place and below which it is not or will not.” (Merriam-Webster online dictionary, 2009).
8
thresholds of COCs (both from the UCFRB and outside Montana), are largely unknown,
especially for native plants (Paschke, Redente & Levy, 2000).
In the last decade, there has been considerable effort to determine toxicity to
plants and other biota for a variety of chemicals, and to quantify toxicity of contaminants
for risk assessment purposes (Megharaj & Naidu, 2008; U.S. EPA, 2001). Unfortunately,
many of the controlled laboratory and greenhouse studies on metals contamination
represent unrealistic measures of toxicity in the field, because of the growth media or
plant species used. Aqueous or sand cultures with liquid additions of single contaminants
are often used to mimic or approximate contaminated field conditions (Paschke et al.,
2000; Paschke, Valdecantos & Redente, 2005; Ye, Shu, Zhang, Lan & Wong, 2002).
Agronomic or aquatic plant species are predominantly used (Kapustka et al., 1995;
Ireland et al., 1991; Brallier, Harrison, Henry & Dongsen, 1996; McBride & Martinez;
2000). When real environmental soils are used, the study’s focus is often to determine
the best amendment for reduced metal uptake as opposed to determining threshold
concentrations (Ye, Wong & Wong, 2000; Ruttens et al., 2006). Determining
phytotoxicity thresholds is the next logical step towards understanding why some plant
species do not establish and succeed on phytostabilized sites in the UCFRB.
Purpose of Research
Establishing vegetation on metalliferous sites is challenging, expensive and
cannot happen overnight. This is true whether the site has been treated with soil
amendments such as limestone or not. Further, field sampling in the UCFRB has
identified a high degree of spatial heterogeneity of COCs contamination at sampling
9
locations (Neuman et al., 2002). Greenhouse pot trials provide a controlled environment
for conducting phytotoxicity tests on individual grass species from UCFRB soils
contaminated with the COCs. Two dilution series-representing two substrate and habitat
types (riparian versus upland)-can be created by mixing uncontaminated reference area
soils with contaminated UCFRB soils to create gradations of total metals and arsenic for
exploring toxicity threshold concentrations that reduce plant emergence and growth. For
this project, the primary null and alternative hypotheses, and associated objectives, are as
follows:
1) Objective #1: Determine if each plant response (percent emergence, shoot
height, total biomass, and root mass ratio) for each species tested, is equally
sensitive to increasing COCs by determining a threshold of COCs
concentrations that produces a significant decline relative to the control.
H0: Responses measured for an individual species will have the same
COCs thresholds.
Ha: Responses measured for an individual species will have significantly
different COCs thresholds.
2) Objective #2: For each individual species, determine if the two different
dilution series (riparian versus upland), produce the same results for Objective
#1 above.
10
H0: The results of Objective #1 will be the same for both dilution series
used in the study.
Ha: The results of Objective #1 will be significantly different for the two
dilution series used in the study.
3) Objective #3: Determine the relative sensitivity of the grass species tested by
comparing thresholds of COCs for all response variables and both dilution
series amongst all species tested.
H0: The overall sensitivity will be the same amongst the species tested.
Ha: The overall sensitivity will be different amongst the species tested.
A better understanding of UCFRB phytotoxicity thresholds for individual grass
species used in reclamation may improve ecological risk assessments and help remedial
decision-makers and land managers more accurately evaluate end-land use goals, sitespecific vegetation performance standards, and appropriate soil amendments.
11
LITERATURE REVIEW
World-wide Metals and Arsenic Contamination
Metals and arsenic contamination from mining-related activities spans nearly
every continent on the globe. China, Poland, Spain, Australia, the United Kingdom,
Chile, South Africa, India and the United States represent far from an exhaustive list of
countries/nations impacted by the anthropogenic disturbances related to mining (Ye et al.,
2000; Stobrawa & Lorenc-Plucinska, 2007; Conesa, Schulin & Nowack, 2007; Jefferson,
2004; Bradshaw & Chadwick, 1980; Santibanez, Verdugo & Ginocchio, 2008; Brooks &
Malaisse, 1990; Mendez & Maier, 2008). Many contaminated sites represent active
mining and the active production of waste, while others represent defunct smelters,
abandoned tailings heaps and old mining ghost towns. Some countries require mining
companies to contain or remediate mine wastes, while others do not, allowing tailings
disposal in surface waters (Mendez & Maier, 2008). In the U.S., most agree it is critical
to reclaim these polluted areas and minimize the threat to human and animal exposure
and reduce phytotoxicity to plants (Pierzynski et al., 1994; Chaney et al., 2000). Left
unreclaimed, these sites could take hundreds to thousands of years to revegetate naturally
and the risk of contaminant spread is high (Mendez & Maier, 2008; Munshower, 1994).
Contamination from mine waste is widespread in the western United States and in
Montana (Mendez & Maier, 2008; RRU, 1993; Neuman & Ford, 2006). Many of these
sites are on the U.S. EPA National Priorities List (NPL), a way for the U.S. EPA to rank
which toxic sites require “further investigation” and potential remediation under the
12
jurisdiction of the Comprehensive Environmental Response, Compensation and Liability
Act (CERCLA) of 1980, better known as the Superfund program. The NPL is used to
characterize those sites that are in the greatest need of clean-up through a Hazard
Ranking System (HRS). In the state of Montana, 14 sites are listed on the NPL, of which
four are located in the watershed of the UCFRB (U.S. EPA, n.d.)
Remedial Strategies
Phytoremediation technologies are very promising, since complete excavation of
mine wastes is often cost and volume prohibitive (Pierzynski et al., 1994; Berti &
Cunningham, 2000). Berti and Cunningham (2000) estimated the net cost of excavation,
capping with cement and placement in a permanent landfill at $1.6 million/hectare (ha),
for a 30 cm deep excavation of 0.2% lead-contaminated soil. In comparison, this same
soil could be phytostabilized for an estimated $60,000/ha, which includes initial fertilizer
& lime treatments, and 30 years of mowing the site (Berti & Cunningham, 2000). At the
Bunker Hill Superfund NPL site in Idaho, phytostabilization was implemented for about
$3,000/acre (Argonne National Laboratory, n.d.). Phytostabilization costs will certainly
vary based on site conditions, such as depth of contamination to be amended.
In the U.S., several abandoned metalliferous mine waste sites have been treated
with some type of phytostabilization, though very little published data exists on the longterm vegetation development. The quality of the vegetation cover developing on
reclaimed mined lands is a frequent measure of successful rehabilitation, and vegetation
development is often measured by number and abundance of plant species (Flege, 2000).
Biomass and percent cover should compare to or exceed growth on uncontaminated
13
reference areas, and evidence of native species colonization should occur (Mendez &
Maier, 2008). Typically, research plots are phytostabilized and monitoring occurs for a
few growing seasons only (Mendez & Maier, 2008), this being the case with several NPL
sites in other states, like Bunker Hill, Idaho, Cherokee County 5 and Whitewood Creek 6,
South Dakota. Though some published works have come from phytostabilization data on
these sites, the focus has been on permanence and effectiveness of different amendments,
uptake of trace elements by plants, and whether the amended site can produce short-term
vegetative cover (Brown, Henry, Chaney, Compton & DeVolder, 2003; Pierzynski et al.,
1994, Pierzynski et al., 2002; Mench et al., 2006). Monitoring of other vegetative quality
measures like diversity and abundance may be occurring, but the data remain
unpublished. Or, the data may be difficult to locate or unavailable, perhaps falling under
the “Grey Literature.”
In Montana, measures of vegetative development on metalliferous,
phytostabilized sites seems to be better established, or at least more available in the
literature. Work by Munshower (2003) has already been reviewed (Introduction). The
McClaren Mine in the New World Mining District near Cooke City, MT and the
Keatings tailings site, in the Radersburg Mining District, MT are two such cases (Brown
et al., 2003; Neuman & Ford, 2006). At both sites, mine wastes were treated with lime,
organic matter, & fertilizer, and then planted. At the McClaren Mine, phytostabilization
first began in 1976 and monitoring continued for 22 years (Brown et al., 2003). An
5
Cherokee county is a Superfund site that is a part of the Tri-State Mining Region in parts of Kansas,
Missouri and Oklahoma.
6
Whitewood Creek was deleted from the NPL list in 1996 by the U.S. EPA, though the 2nd “Five Year
Review” of the Remedial Action in 2007 continued to identify deficiencies & concerns (U.S. EPA, 2007a).
14
assemblage of 18 local, mostly native grasses and forbs were growing on the
phytostabilized plots in year 19, with plant cover values exceeding that on two of the
three nearby reference areas (Brown et al., 2003). At the Keatings site, phytostabilization
was implemented in 2003 and species counts and plant cover have been taken each
growing season since. Plant cover exceeds that of plants growing in non-amended mine
tailings, but is not significantly different from plant cover on native soils (Neuman &
Ford, 2006). Though several introduced species have invaded the plots, introduced
species are known to exist on the reference areas. Four native grasses have established at
greater than 0.5% cover, and more than 10 other native species have established, though
cover is less than 0.5% (Neuman & Ford, 2006). In both of these studies, care was taken
to plant native species similar to reference areas.
Despite the success at the McClaren and Keating sites, skepticism exists in the
literature about the permanence of phytostabilization (Mendez & Maier, 2008). The main
criticisms come from a lack of confidence that phytostabilized sites will remain of neutral
or alkaline pH, and that a self-sustaining, weed-free, healthy plant community will
develop. Many of these issues were questioned by the U.S. EPA National Remedy
Review Board, and a document responding to these issues was prepared (CH2M HILL,
2001). Despite the continued dialogue regarding phytostabilization permanence, the
Record of Decision (ROD) for the Clark Fork River Operable Unit of the CFR was
released in 2004, outlining which lands will be treated with phytostabilization. In the
ROD, contaminated areas classified as “Impacted Soils and Vegetation” will have the
following remedy:
15
“Impacted soils and vegetation areas will generally be treated in-situ, unless the
tailings and impacted soils in a given area extend more than 2 feet below ground
surface. In that case, the tailings and impacted soils will be removed. Other
impacted soils and vegetation areas that are too wet for implementation of in-situ
treatment techniques will also be removed” (U.S. EPA & MT DEQ, 2004).
Contaminated areas classified as “Impacted Soil and Vegetation Areas” that will receive
phytostabilization treatment are estimated between 700 and 1760 acres, though these
figures are only for Reach A (the upper 43 miles of the river) of this Operable Unit (U.S.
EPA & MDEQ, 2004). Because phytostabilization is no longer just a proposed remedy,
and many more sites in the UCFRB will be selected to undergo phytostabilization in
future remedies, understanding mechanisms for successful plant establishment and
persistence on such sites is critical.
Factors Affecting Phytostabilization Success
A vegetative cover is considered a crucial step in reclamation efforts, both in the
short term (reducing erosion and spread of contaminants) and in the long-term, for
improving soil structure and function, including microbial processes (Tordoff et al, 2000;
Munshower, 1994; Osterkamp and Joseph, 2000; Rosario et al., 2007). Even so,
establishing vegetation on mine wastes can be difficult.
The chemical and physical properties of mine tailings and tailing-impacted soils
are notoriously poor and according to Brown et al., (2003) represent conditions which
“frequently exceed the physiological tolerance of virtually all vascular plants, and thus
inhibit or completely retard the establishment of plant seedlings and other natural
succession.” Richmond (2000) describes:
16
“…tailings are not soil. They represent time zero in soil formation and lack
typical soil features. Tailing have limited (or excessive) moisture holding
capacity, are deficient in essential nutrients, are devoid of organic matter, have
virtually no biological activity, and the sand fractions have low cation-exchange
capacity.”
The chemical and physical characteristics of the contaminated soils must be suitable or
made suitable for sustaining vegetation (Dickinson, 2002). The sub-headings that follow
are the most important factors for revegetation success on metalliferous phytostabilized
sites.
Soil pH
Vegetation performance is affected by soil pH (Table 1), maximum values
occurring when pH is near neutral, between 6.4 and 7.3, though slightly acid or slightly
alkaline conditions occur commonly in natural environments (Munshower, 1994).
Table 1. Soil pH ranges.
pH
Soil condition
<4.5
Extremely acid
4.5-5.5
Very strongly to strongly acid
5.6-6.5
Slightly to moderately acid
6.6-7.3
Neutral
7.4-7.8
Slightly alkaline
7.9-8.4
Moderately alkaline
8.5-9.0
Strongly alkaline
>9.0
Very strongly alkaline
(Adapted from Munshower, 1994)
Mine wastes often contain unoxidized forms of sulfur, such as the mineral pyrite (FeS2),
which when weathered by oxygen and water creates sulphuric acid (H2SO4), acidifying
soil conditions. Thiobacillus ferrooxidans, a bacteria in soils, catalyzes the production of
sulphuric acid, increasing acid generation up to a million times (Skousen, Sexstone &
Ziemkiewicz, 2000). Weathered pyritic mine tailings frequently have pH values between
17
2.2 and 3.5 (Mays, Sistani & Soileau, 2000). Because of the large reserve of “potential
acidity” of sulfide ore-containing wastes, limestone is typically used to neutralize the
acidity.
On metalliferous wastes, low pH increases mobility of trace metal elements
(Kabata-Pendias, 2001; Figure 3). Adriano (1986) describes the mobile fractions of trace
elements as those dissolved in soil solution, and those held onto exchange sites.
Cadmium, copper, lead and zinc are all known to have the highest mobility when pH is
less than 5.5 (Dickinson, 2002).
Figure 3. Mobility of elements as a function of soil pH, data for a light
mineral soil (Used with permission from Kabata-Pendias, 2001).
Removal of metals from the soil solution by formation of insoluble metal precipitates of
hydroxides, oxides, carbonates and phosphates occurs at alkaline pH (McLean &
Bledsoe, 1992). All cationic metals adsorb more strongly to exchange surfaces such as
Fe and Mn oxides, organic matter, and clay minerals as pH increases because of pHdependent charges on these surfaces (McLean & Bledsoe, 1992). Further, when pH
18
becomes acidic, metal cations face competition with Al3+ and H+ for charged sites on
surfaces (McLean & Bledsoe, 1992).
Trace Elements and Phytotoxicity
Different definitions of trace elements exist in the scientific literature. Adriano
(1986) defines trace elements as those that occur in natural and disturbed systems in very
small amounts, such that they are toxic to living organisms when present in “sufficient”
amounts. Generally, geologists and biologists agree that those elements that are not part
of the major rock forms (Si, Al, Fe, Ca, Na, Mg and K), or part of the major
biogeochemical cycles (C, O, H, N, K, P, Ca, Mg, Cl, Na, S) are considered trace
elements (Kabata-Pendias, 2001; Adriano, 1986). “Trace elements” is the term used in
biomedical and biochemical research to describe those elements making up less than
0.01% of an organism (Adriano, 1986). All of the COCs in the UCFRB are considered
“trace elements.”
Trace elements can be considered essential, or nonessential to plants. Essentiality
refers to those elements that cannot be substituted for in normal plant biochemistry, and if
missing, some aspect of plant growth or metabolism will not occur (Kabata-Pendias,
2001). Of the five COCs in the UCFRB (cadmium, copper, lead, zinc and arsenic), only
copper and zinc are known to be essential to plant growth and metabolism (KabataPendias, 2001). COCs in the UCFRB are excessive and although plants are known to
adapt to chemical stress readily, they represent concentrations that have been shown to be
phytotoxic (Kapustka et al., 1995; Kapustka, 2002).
19
Phytotoxicity is defined as a deadly or sub-lethal plant response to a toxic agent
(ASTM, 2003), or an adverse response of a plant to a chemical (U.S.EPA, 1996a), or a
chemical that reduces growth or alters normal development in plants (Fletcher, 1997).
Native soils throughout the world and within the UCFRB have background levels of the
COCs, but phytotoxic levels in the UCFRB are often an order of magnitude higher than
regional mean concentrations (Table 2).
Table 2. Mean regional & global background concentrations of the COCS, as well as literature-reported
phytotoxicity ranges for and maximum tolerable dietary limits of the COCs.
Regional Background
1
CoC
Level
(mg/kg)
Global Background
2
Level
(mg/kg)
Maximum Tolerable
3
Phytotoxicity
(mg/kg)
Dietary Limits4
(mg/kg)
Lead
35.7
22-44
100-1000
Copper
22.4
13-24
60-1636
Zinc
66.1
45-100
70-500
Cadmium
0.9
0.37-0.78
3-100
Arsenic
9.3
4.4-9.3
15-315
1
Data summarized from U.S. EPA, 1997
2
Data summarized from Kabata-Pendias, 2001
3
Data summarized from Adriano, 2001; U.S. EPA, 1997; CH2M Hill, 1987 a, b;
Kabata-Pendias and Pendias, 1984
4
Data for cattle from National Resource Council (NRC), 2005
100
40
500
10
30
In general, the most severe phytotoxicity symptoms in plants in the UCFRB have
been correlated with soils of low pH and high COCs (U.S. EPA, 2001; Kapustka et al.,
1995; Kapustka, 2002). Phytotoxicity is highly related to “phytoavailability” or
“bioavailability” (Morel, 1997), where these terms refer to the mobile and transportable
forms of elements that can be taken up into plant tissues (Bolan et al, 2008; Adriano,
1986; Morel, 1997). Phytoavailability depends on many factors such as the plant species,
age of plant, concentration of the COC, and soil conditions such as pH, organic matter
20
content, redox potential, clay content and concentration of oxide minerals (KabataPendias, 2001; Adriano, 1986; U.S. EPA, 2001).
Metals and arsenic chemistry in soils is complex and dynamic, involving
interactions between the soil solution and the solid phase of the soil (McLean & Bledsoe,
1992; Adriano, 1986). Trace elements exist in different “pools” in soils defined as:
1) dissolved species in the soil solution (free ions, or complexed with inorganic or
organic ligands); 2) solid precipitates; 3) adsorbed to organic and inorganic surfaces via
electrostatic or covalent bonds; and 4) bound in the structure of solid minerals (Adriano,
1986; Kabata-Pendias, 2001; McLean & Bledsoe, 1992). Cationic species of metals and
solid metal precipitates are the major species under most environmental conditions (pH 48.5; Naidu and Bolan, 2008), while arsenic (As) bonds with oxygen, forming anionic
species of As (V) under most environmental conditions (Kabata-Pendias, 2001).
Contaminant of Concern: Lead Of the heavy metals, lead (Pb) is the most
abundant in the Earth’s crust, and its primary forms in nature are in the minerals galena
(PbS), cerussite (PbCO3) and anglesite (PbSO4), which are very insoluble in natural
waters (Adriano, 1986; Kabata-Pendias, 2001). Lead mainly occurs as Pb2+, but
sometimes as Pb4+. Organic matter fixation of lead is generally accepted as the most
important factor for the immobilization of lead in soils, though Pb is also known to
adsorb to clay surfaces, and manganese (Mn) & iron (Fe) oxides (Adriano, 1986). Some
discrepancy exists in the literature on the mobility of Pb in soils, but most believe Pb is
the least mobile of the heavy metals, as evidenced by very low levels of Pb in soil
solutions (Kabata-Pendias, 2001). Elevated phosphate from fertilizers may reduce Pb
21
solubility by forming phosphate-Pb complexes at neutral to alkaline pH (Adriano, 1986;
Kabata-Pendias, 2001). Uptake of Pb from soil solution is well-documented, though it
appears Pb accumulation in plant tissue is mostly limited to roots because of poor
translocation to shoots (Kabata-Pendias, 2001; Pahlsson, 1989). Phytotoxic effects of Pb
are usually dark green, stunted, chlorotic or reddish leaves that wilt upon aging, stunted
leaves, stunted plant growth and stunted brown or black roots (Pahlsson, 1989; KabataPendias, 2001).
Contaminant of Concern: Copper Copper (Cu) occurs most frequently in the
minerals chalcopyrite (CuFeS2) and bornite (Cu5FeS4) and is the 26th most abundant
element in the Earth’s crust (Adriano, 1986). These minerals solubilize easily in
weathering processes and release free Cu2+ ions (Kabata-Pendias, 2001). Like Pb, Cu is
strongly fixed by organic matter, clay minerals and Fe, Mn and aluminum (Al) oxides in
soils, and is considered one of the least mobile trace elements (Adriano, 1986). Of all the
Cu species in solution, Cu-organic matter complexes form the majority especially at
higher pH values (Kabata-Pendias, 2001; Adriano 1986). Copper has been proposed as
the most extensively complexing divalent metal to organic matter, over Pb, Fe, nickel
(Ni) and zinc (Zn; Adriano, 1986). As such, plant Cu deficiencies have been reported
where organic matter is high (Adriano, 1986). Like Pb, Cu is translocated poorly from
roots to shoots by plants, and plants tend to concentrate Cu in their roots (KabataPendias, 2001). Copper can strongly sorb with phosphates in soils with high phosphate
levels (Kabata-Pendias, 2001; Adriano, 1986). Phytotoxic effects of Cu are usually
small, chlorotic or dark green leaves, with early abscission; shortened, thickened, poor
22
root development especially lateral roots; reduced tillering, and overall stunted growth
(Pahlsson, 1989; Kabata-Pendias, 2001).
Contaminant of Concern: Cadmium Cadmium (Cd) occurrence and pollution is
highly related to the natural occurrence and mining of zinc (Zn), as Cd is naturally
produced as a by-product of the Zn industry (Adriano, 1986). Cadmium ranks 64th in
Earth’s crust and is found mainly in Zn, Pb-Zn and Pb-Cu-Zn ores, the principal of which
is sphalerite (ZnS) (Adriano, 1986). Weathering of Cd-containing parent rock causes Cd
to go into solution easily and it occurs as Cd2+ or as ionic Cd species of various inorganic
ligands (Kabata-Pendias, 2001). Soluble chloride species of Cd form where high Cloccurs in soils, such as saline soils, and can be an affective predictor of phytoavailability
(Kabata-Pendias, 2001). Just as with Pb and Cu, Cd sorption on organic matter, clay and
Fe and Mn oxides surfaces occurs strongly, especially with increasing pH, although the
stability of complexation with organic matter is the weakest for Cd compared to Pb and
Cu (Adriano, 1986). Redox potential has also been shown to affect Cd solubility
(Adriano, 1986). Cadmium readily translocates within plants and depending on soil and,
species of plant, may readily accumulate in shoots (Adriano, 1986; Kabata-Pendias,
2001). Phytotoxicity of Cd presents as small, curled, chlorotic leaves with red-brown leaf
margins; chlorotic veins, reduced tiller number, and stunted, brown roots (KabataPendias, 2001; Pahlsson, 1989).
Contaminant of Concern: Zinc Zinc (Zn) is 24th in abundance in the Earth’s crust
and occurs in the highest concentrations in the minerals zincite (ZnO), sphalerite (ZnS),
and willemite (Zn2SiO4). Zinc minerals solubilize during weathering and produce mobile
23
Zn2+ (Kabata-Pendias, 2001). Zinc forms complexes with chlorides, sulfates, nitrates and
phosphates, many of which do not contribute significantly to soluble forms of Zn
(Adriano, 1986). Redox potential impacts Zn solubility by forming insoluble ZnS under
wet, reduced conditions (Adriano, 1986). As with the other metal cations, Zn is held
quite strongly by clay, organic matter and oxides & hydroxides of Al, Fe and Mn
(Kabata-Pendias, 2001). Zinc has been rated similarly to Cd in stability of metal-organic
matter complexes and adsorption processes; i.e., it is less stable than Pb or Cu (KabataPendias, 2001). At higher pH values, Zn-organo complexes and anionic Zn complexes
have been known to account for some of the Zn solubility (Kabata-Pendias, 2001). Zinc
may be immobilized by soils with excess calcium (Ca) and phosphorus (P), and in these
cases may be linked to Zn deficiencies (Kabata-Pendias, 2001; Adriano 1986).
Translocation of Zn within plants appears to occur fairly readily and concentration of Zn
in shoots occurs, especially when plants are young or there are “luxury” levels of soil Zn,
though the majority of Zn is concentrated in roots (Kabata-Pendias, 2001). Phytotoxicity
symptoms of excess Zn are retarded growth, stunted roots resembling barbed wire,
chlorotic leaf tips, chlorotic veins in new leaves and lignification of shoot & root cells
(Pahlsson, 1989; Kabata-Pendias, 2001).
Contaminant of Concern: Arsenic Arsenic (As) ranks 52nd in abundance in the
Earth’s crust and exists in over 240 minerals such as arsenides, sulfides and sulfosalts
(Adriano, 1986). The most common As-containing mineral is arsenopyrite (FeAsS).
Arsenic minerals and compounds are very soluble, but As migration can be limited due to
strong sorption by clays, organic matter and hydroxides (Kabata-Pendias, 2001). Arsenic
24
exists as -3, 0, +3 and +5 oxidation states in the environment. The +3 and +5 states of As
are the most common under most soil pH and redox conditions, and they form complex,
mobile anion complexes such as AsO2-, AsO43-, HAsO42- and H2AsO3- (Kabata-Pendias,
2001; Adriano, 2001). Arsenic behavior as arsenate (V; AsO43-) is similar to that of
phosphates (PO43-), and readily forms insoluble precipitates with Fe, Al and calcium
(Ca), with Fe controlling arsenate’s mobility the most (McLean & Bledsoe, 1992).
Arsenate is the most stable form of As in aerobic environments, while arsenite (III) is
more common in reducing conditions, as AsO2- (McLean & Bledsoe, 1992). Arsenite is
much more soluble and toxic than arsenate (Adriano, 1986). Both pH and redox potential
together determine the oxidation states of As in soils (Kabata-Pendias, 2001). Arsenate
has highest adsorption at pH 3-5, while arsenite has maximum adsorption on iron oxides
at pH 7 (McLean & Bledsoe, 1992). Arsenic is more mobile than Cd, Zn, Pb and Cu
through a clay matrix (McLean & Bledsoe, 1992). The effect of high soil P on As
availability is unclear; some studies showing As bioavailability goes down, while others
indicate it goes up (Adriano, 1986). Arsenic appears fairly mobile through plant tissue,
with several studies showing As concentrates in aerial parts as well as roots of plants
(Kabata-Pendias, 2001). Phytotoxicity symptoms of As include leaf wilting, reduced
tillering, browning or yellowing of roots and reddish-brown spots on old leaves (KabataPendias, 2001).
Fertility
Tailings are generally devoid of essential plant nutrients, particularly nitrogen
(N), and to a lesser degree P (Bradshaw & Chadwick, 1980; Brown et al, 2003;
25
Richmond, 2000), and require fertilization, at least initially. Organic matter provides a
slow release of nutrients (Richmond, 2000), and is a better choice over the long-term.
Nitrogen Nitrogen is the most important plant nutrient and is often deficient in
soils because it does not occur in Earth’s minerals (Bradshaw & Chadwick, 1980).
Nitrogen occurring in soils comes from decaying organic matter or is fixed from the
atmosphere by soil microorganisms, or nitrogen-fixing plant/bacterial symbioses.
Ammonium (NH4+) and nitrate (NO3-) are the plant available forms of N; NH4+ tends to
sorb to clays and organic matter, being slowly released, while NO3- is highly mobile and
leachable (Dickinson, 2002). Nitrogen requirements for temperate plants are about 100
kg /ha/year and a soil must supply about 1000 kg/ha from decomposing organic matter to
supply plants with these amounts (Dickinson, 2002). According to Munshower (1994),
30 kg/ha are adequate levels for native range plants.
Phosphorus Plants take up P as H2PO4- or HPO42- and P is released into soils by
the weathering of P-rich minerals like apatite (Bradshaw & Chadwick, 1980). Most of
the total P in a soil (96-99%) is unavailable for plant uptake, being complexed to organic
matter, trace metals or Al & Fe at low pH and Ca at high pH (Munshower, 1994;
Bradshaw & Chadwick, 1980). Mycorrhizae, important fungal species that form
symbiotic relationships with plant roots, are known to increase plant available P
(Bradshaw & Chadwick, 1980). Temperate plants require about 9 kg of P/ha/year
(Dickinson, 2002), and typical available P in western soils in the U.S. range from about
5-10 µg/g (Munshower, 1994).
26
Potassium Minerals such as orthoclase and biotite release potassium (K) upon
weathering and decay, and the available form of K to plants is the ion K+ (Munshower,
1994; Dickinson, 2002). Clay and organic matter in soils adsorb and retain K+, so a lack
of these materials can result in rapid leaching of K+ from soils (Bradshaw & Chadwick,
1980). Temperate plants require about 55 kg of K/ha/year and most soils contain 0.3 to
5% total K (Dickinson, 2002; Munshower, 1994).
Organic Matter
Organic matter improves soil structure, lowers bulk density, increases water
holding capacity and is a reservoir for plant nutrients and a source of beneficial soil
microorganisms (Dickinson, 2002; Johnson et al., 1994; Mendez & Maier, 2008).
Organic matter builds up in soils as a natural process of microbial break-down of dead
plants, and without vegetation growth, very little organic matter accumulates in the
surface layers. Since most degraded mine wastes have little organic matter, vegetation
establishment is limited. This situation represents a bottleneck to the revegetation
process.
Organic matter plays an important role in the cycling of trace elements by binding
them to form soluble or insoluble complexes, and can be considered an organic storage
and regulating medium for the cycling of these elements in soils (Adriano, 1986; KabataPendias, 2001). Phenolic and carboxyl functional groups on organic matter form stable
complexes with metals through the cation exchange property, or by chelating the metals
(Kabata-Pendias, 2001).
27
Soil Microorganisms
Microscopic organisms are integral parts of ecosystems via their role in nutrient
cycling, improving soil structure and possibly reducing metal toxicity, all which benefit
plants (Rosario et al., 2007). Established plants can provide a nutrient-rich source of
energy to stimulate microorganism growth (Rosario et al., 2007), therefore, plant growth
and microbes are interrelated in soils. Metals in soils are known to inhibit soil microbial
abundance, diversity and overall activity such as nitrification, denitrification and
decomposition of organic matter (Wong, 2003; Pierzynski et al., 1994) and untreated
tailings are known to affect bacterial & fungal counts and have decreased carbonutilization activity (Moynahan, Zabinski & Gannon, 2002). Drastically disturbed systems
often lack mycorrhizae, and as such, plant establishment on such sites is reduced (Wali,
1999).
Electrical Conductivity
Electrical conductivity (EC) is the best measure of total salt content or potential
salinity (Sobek, Skousen & Fisher, Jr., 2000). Tailings and mine wastes can have excess
salinity because of the natural weathering of halides and carbonates in the ore, from
evaporation during storage of tailings heaps or due to tailings being transported through
saline waters that are reused for milling (Richmond, 2000). In the UCFRB, salinity has
been linked to excess soluble salts from liming amendments, or poor drainage (CH2M
HILL, 2001). Soil salinity guidelines appear in Table 3 below.
Table 3. Soil salinity guidelines.
Parameter
Nonsaline
EC (µS/m)
<4,000
(Adapted from Munshower, 1994)
Slightly Saline
4,000-8000
Moderately Saline
8,000-16,000
Saline
>16,000
28
Soil values of EC greater than 4,000 µS/m are considered saline and unsuitable for
agronomic crops (Richmond, 2000; Munshower, 1994). Electrical conductivity values at
8,000 µS/m and higher are not tolerated by most plant species, though some native
species tolerate EC > 8,000 µS/m (Richmond, 2000). Some productive native grasslands
in the western U.S. are known to occur in soils that are saline (Munshower, 1994).
Plant Species Selection
The choice of plant species on metals-contaminated lands is a critical part of
successful, self-sustaining vegetation (Johnson et al, 1994; Wong, 2003). Plant species
selection should consider which species will be long-term competitors and survivors
under harsh and changing environmental conditions (Pivetz, 2001). Ultimately, it is the
end land use is that is the determining factor for species selection on sites to be reclaimed
(Macyk, 2000). Land use in the UCFRB is mostly agriculture (i.e., cultivated areas and
improved pasture; U.S. EPA, 1996; ARCO, 1998). This may mean using native species,
agricultural species, naturalized species or some combination (Johnson et al., 1994),
though it is generally agreed that using acid-tolerant, metal-tolerant and climatically
adapted species will increase success (Munshower, 2000).
Metal Tolerant Species Plants differ in their ability to tolerate excess trace metals
and arsenic in soils (Baker, 1987; Watkins & MacNair, 1991). Tolerant species have
been observed colonizing tailings heaps in Europe as early as the 1950s (Bradshaw,
1952). Metal-tolerant populations of Agrostis, Festuca, Deschampsia, and Thlaspi are
well-documented (Verkleij & Schat, 1990; Smith & Bradshaw, 1979; Ernst, 1990).
Several terms exist to describe metal tolerance in plants; only two will be used here.
29
“Sensitivity” is the injury or death of a plant from a stress (Baker, 1987). “Tolerance” is
a type of resistance, where a plant can still survive and reproduce (Baker, 1987). Debate
has been going on for years as to whether or not tolerant phenotypes are limited to a
particular family of plants, certain genera only, certain species within genera, or to
individuals within a species (Smith & Bradshaw, 1979; Ehinger & Parker, 1979; Marty,
2000). This has fueled programs like the “Development of Acid/Heavy Metal Tolerant
Releases” (DATR), a local effort by the Bridger Plant Materials Center (BPMC), Bridger,
Montana, to develop a reliable supply of indigenous seed material from species adapted
to metals and acid soils, sometimes referred to as ecotypes 7. The main objective of this
program is to identify superior-performing plant ecotypes for use on mine wastes, and to
release these ecotypes to the commercial market where seed production can occur for use
in reclamation (U.S. EPA, 2007b). To date, at least four DATR ecotypes have been
released to the commercial market, including two used in this study (Copperhead
Selected class germplasm slender wheatgrass and Washoe Selected germplasm basin
wildrye) (Majerus & Majerus, 2007; U.S. EPA, 2007b).
Tests of Phytotoxicity
Phytotoxicity testing using terrestrial plants is a burgeoning field involving plant
ecologists, risk assessors and regulatory agents, to name a few (Fletcher, 1997).
Objectives of tests include determining the maximum amount of chemical exposure that
7
In this paper, ecotype is defined as a “subgroup within a species, which is genetically adapted to a habitat
type that is different from the habitat type of other subgroups of that species” (Burns & Honkala, 1990).
30
has no adverse effect on natural and cultivated vegetation 8, and characterizing the effects
of soil contaminants on living biota and relating these to risk of further spread of
contaminants (Fletcher, 1997; Kapustka, 1997). Hundreds of different tests and protocols
exist, and thousands of phytotoxic agents have been identified (Fletcher, 1997). Many
measures of plant phytotoxicity can be evaluated such as emergence, root length,
biomass, evidence of reproduction, or plant tissue concentrations of the toxin (Kapustka,
1997; Morel, 1997). The ultimate goal of these laboratory/greenhouse tests is to predict
the potential negative effects of toxins on field ecosystems (Pedersen & Elmegaard,
2000; Fletcher, 1997; Figure 4).
Figure 4. Phytotoxins in the environment and their influence on ecosystems (Used with permission from
Fletcher, 1997)
Guidelines for standardized test protocols have been published by the American
Society of Testing and Materials (ASTM), the U.S.EPA, the Organization for Economic
8
This is a toxicity threshold, similar to the threshold definition used in the Introduction Chapter.
31
Cooperation and Development (OECD) and the Food and Drug Administration (FDA).
Unfortunately, these protocols have been established for limited target species, limited
test substrates or testing media, and often contain vague and non-uniform test protocols
(Fletcher, 1997; ASTM, 2003; U.S. EPA, 1996b). Therefore, the reliability and
usefulness of the tests is in question. This seems to be particularly true relative to the
duration of the test (e.g., 14 days), the media used (e.g., sand or solution culture when the
risk assessment is on soil), and the species used (most target species in test protocols are
agronomic crops like lettuce, wheat, radish, etc.). Despite the increase in testing in recent
years, data is still lacking on the majority of the world’s plants, especially native species,
and as such, risk assessments are based on threshold levels derived from agronomic
studies (Fletcher, Johnson & MacFarlane, 1988; Paschke et al., 2000).
Greenhouse Phytotoxicity Tests Outside of the UCFRB
Sand or Aqueous Culture Using Liquid Additions of Single Contaminants Tice
(1995) evaluated 75-day growth of four rangeland grasses (western wheatgrass, sheep
fescue, Kentucky bluegrass and basin wildrye) given four different concentrations of
As(III) and As (V). While critical loading concentrations of As in root and shoot tissues
were determined for each grass and correlated to growth reductions, the study used a
single contaminant, which is unlikely to occur in mine wastes, and employed sand.
Similarly, Surbrugg (1982) studied root growth of ecotypes of Agrostis and Deschampsia
to varying concentrations of liquid-applied copper and zinc, but thresholds of Cu and Zn
toxicity were not established, though regression equations were published. The main
goal of this study was to compare tolerance of the two ecotypes. Fourteen-day root
32
growth of three cultivars of Agrostis capillaris grown on beads floating in plastic beakers
were evaluated after spiking with metal salt solutions of Cd, Cu, Zn, Ni or Pb
(Symeonidis, McNeilly & Bradshaw, 1985). While actual threshold values were not
reported for each metal, regression equations were published. Ehinger and Parker (1979)
observed root growth of two ecotypes of the native grass Andropogon scoparius growing
in hydroponic cultures of varying concentrations of Cu or Zn for two days. Both
ecotypes developed root length reductions when concentrations were between 0 and 0.05
mg/L for Cu and 2.5 to 5.0 mg/L for Zn (Ehinger & Parker, 1979).
Even though tests in sand do not represent real soil conditions, major
contributions regarding Rocky Mountain rangeland plant species’ toxicity thresholds
have come from the Mark Paschke lab, Colorado State University. Greenhouse studies
were performed on the native species slender wheatgrass, tufted hairgrass, ‘Magnar’
basin wildrye, ‘Sherman’ big bluegrass and the introduced grass redtop, using varying
concentrations of Cu and Zn in 60 day tests (Pasche et al., 2000; Pashke & Redente,
2002). Concentrations that reduced total biomass by 50%, as well as caused mortality of
50% of seedlings, and the tissue concentrations of Cu & Zn that reduced biomass by 50%
after 50 or 60 days were determined for each species. For each of these measures, a
toxicity threshold was calculated, based on the best fit model to the data. Only some of
the thresholds are reported here: For 50% reductions in total plant biomass, water soluble
Cu and Zn toxicity thresholds ranged from 283 to 710 mg/L and 84 to 222 mg/L,
respectively. Shoot tissue Cu and Zn concentrations correlated to 50% reductions in
shoot biomass were between 737 and 10,792 mg/kg and 2,449 and 5,026 mg/kg for Cu
33
and Zn, respectively. These latter values were much higher than literature-reported
thresholds for agronomic species (Paschke et al, 2000; Paschke & Redente, 2002).
Because five different thresholds for each species were derived from the different
measurements of reduced growth and survival, overall threshold generalizations and
relative species’ tolerance to Cu and Zn are difficult to summarize. Continued work has
identified Zn threshold values for six reclamation forbs using sand culture (Pashke, Perry
& Redente, 2006).
Soils Using Liquid Additions of Single Contaminants A study using the weedy
species Fallopia convolvulus was performed in uncontaminated Danish soils, with liquid
additions of Cu, and evaluated after five weeks (Pedersen & Elmegaard, 2000).
Threshold values of 20 to 200 mg/kg were estimated based on both Cu tissue
concentrations and total soil Cu (Pedersen & Elmegaard, 2000). In Indiana, seven native
species were tested in uncontaminated soil with liquid additions of 0 to 100 mg/L Cd in
six-week trials (Miles & Parker, 1979). Germination, survival, height and biomass were
measured. While the threshold levels varied by species for germination, survival and
biomass declined significantly between 10 and 30 mg/L Cd for all species (Miles &
Parker, 1979).
Contaminated Field Soils Mixed with Uncontaminated Soils Very few studies in
the literature use actual contaminated soils mixed with uncontaminated soils to create a
gradient of contaminants for determination of threshold values of phytotoxins. In Spain,
neutral pH mine tailings containing high levels of Cd, Cu, Pb and Zn were mixed with
non-contaminated, neutral pH forest soil from Switzerland to create 25, 50, 75%
34
treatments, as well as 0 and 100% controls (Conesa et al., 2009). Growth and metal
uptake of two local species known to colonize metalliferous mine tailings, Piptatherum
miliaceum and Lygeum spartum were evaluated after 10-week trials. Tissue
concentrations in roots and shoots for each metal were analyzed for both species, and in
most cases, significant differences were observed in the first treatment up from the
control (25%), although the authors didn’t relate these values to a total soil metal level.
Summing the individual metals by treatment indicates significant reductions occurred in
tissue concentrations for roots and shoots when total soil metals were between <57 and
3505 mg/kg. Although the authors reported significant biomass reductions as the
concentration of tailings in each treatment increased, the data was not supplied in the
paper.
In Italy, the European grass Lolium perenne was grown for 90 days in two soils
contaminated with Pb, Zn, Cu, Ni and chromium (Cr) and mixtures of 3:1 contaminated
soil to non-contaminated agricultural soil collected nearby (Arienzo, Adamo &
Cozzolino, 2004). A negative control (pH 8.1-8.4) was also used. Although tissue
concentrations of Pb, Zn and Cu were determined for each treatment, as well as biomass
data reported, interpretations of threshold levels were difficult. Further, the gradation of
total metals was 419 mg/kg in control soil to 1574 mg/kg maximum in contaminated
soils, so threshold levels correlated to growth reductions would likely fall within this
range. In both of the previous studies, seed emergence was not evaluated across the
dilutions.
35
In Poland, the European tree species, Populus nigra was grown via root cuttings
in two dilution series of smelter-impacted, metalliferous soils mixed with unpolluted soils
(Stobrawa & Lorenc-Plucinska, 2008). Threshold values of chelate extractable (EDTA)
Pb and Cu were correlated with reduced shoot & root growth and cellular levels of
molecules involved in antioxidant mechanisms known to be involved in heavy metal
stress (Stobrawa & Lorenc-Plucinska, 2008). For all responses, these values ranged from
100 to > 650 mg/L for Cu and <30 to >200 mg/L for Pb, associated with treatments
where the contaminated soil made up at least 25% of the total mixture. Total soil levels
of Pb and Cu were not published in the paper.
Undiluted Contaminated Field Soils Kapustka (2002) performed greenhouse
trials on 45 contaminated soils collected from the Grant-Kohrs Ranch (GKR) National
Historic site, near Deer Lodge, Montana. Here, phytotoxicity was assessed for the native
shrubs, alder and dogwood, the native sedge, Carex utriculata, and the introduced forb,
alfalfa, in trials lasting 2-4 weeks (Kapustka, 2002). “Scores” of phytotoxicity were
assigned to each soil based on plant growth reductions from the control, then the mean
score was calculated based on averaging all four species. This data was used to develop
predictive models for phytotoxicity on other soils in the GKR. Although total metals were
analyzed and correlated to plant growth to determine the scores and predictive models,
threshold values of total COCs causing individual growth reductions for the four plant
species were not determined. Very similar work was performed by the U.S. EPA (Wall,
Hoff & Brattin, unpublished, date unknown), using contaminated soils collected in the
Arkansas river valley (Leadville, Colorado), part of which forms a U.S. EPA NPL site
36
very similar in contamination to the UCFRB. Growth of alfalfa, the introduced tall
wheatgrass and the native forb western yarrow was explored in greenhouse tests of 20
samples of contaminated soils from the floodplain. Phytotoxicity scores were assigned to
each soil based on the overall average across all three species, and although total metals
were analyzed, threshold levels of metals were not correlated to plant growth reductions
for each individual species.
History of Phytotoxicity Tests from the UCFRB
As early as 1957, field and greenhouse studies on the phytotoxicity of tailings
were performed in what is now the Anaconda Smelter (AS) Superfund site by Leonard
Eliason (RRU, 1993). During the 1970s and 80s, many graduate students produced
papers on plant growth on toxic waste materials from Anaconda and surrounding areas,
the majority focusing on plant responses to different amendments or pH (RRU, 1993).
During the 1980s and 1990s, extensive site characterization occurred in the CFR and AS
Superfund sites, mapping and quantifying the depth of the tailings, degree of mixing of
tailings with soils, degree of movement of COCs into uncontaminated over- or underlaying soils, and differences in pH, EC, and total & soluble metals on both
phytostabilized plots and unreclaimed areas (U.S. EPA, 2001; ARCO, 1998). Numerous
studies were also completed providing background data on plant communities naturally
colonizing tailings (U.S. EPA, 2001). What follows are the most critical results of the
phytotoxicity tests from the 1980s to today, in order to explain where phytotoxicity
testing has come and where it is going in the UCFRB.
37
Field Tests In the early 1990s, twelve sites in the CFR were sampled and
analyzed for pH & total metals, and correlated to plant production and canopy cover
(CH2M HILL, 1991). The results indicated that when pH was <5.5, the greatest decrease
in canopy cover and production occurred, while pH>6.5 produced the largest cover and
production measurements (CH2M HILL, 1991). Further work linked plant growth and
total soil metals data from forty-eight sampling sites in the CFR (PTI, 1994). Plant
biomass and species diversity (richness and evenness) were low at high total metal levels,
and highest at zero total metal levels, with an intermediate level of biomass and diversity
in between (PTI, 1994). These latter two studies established pH and COCs
concentrations as the biggest drivers for plant growth. Neuman et al. (2002) collected
and analyzed co-located total COCs and barley (Hordeum vulgare) biomass data at a
phytostabilized area. A model for phytotoxicity was developed where only total soil
metal concentrations predicted plant growth at neutral pH (the lowest pH value being
6.73). While it is valuable to correlate total COCs with plant biomass data, this
phytotoxicity assessment was performed from field samples, where tailings can be very
spatially heterogeneous and the species of interest was agronomic. Further, while total
COCs were known for each of the 20 sampling locations, threshold levels associated with
significant barley biomass reductions were not determined.
Greenhouse Tests Pivotal work by the RRU in the 1990s called the Anaconda
Revegetation Treatability Studies (ARTS) evaluated the growth of many grass, forb and
shrub species across a matrix of amendments like lime, lime + compost, lime + manure
and so on, in different combinations in laboratory and greenhouse conditions (RRU,
38
1996). These studies were hugely valuable for developing field protocols for
amendments, rates of applications and species recommendations for use in
phytostabilization in the UCFRB, though the studies did not correlate total soil COCs
with individual plant growth reductions (RRU, 1996). Further, very few native grass
species were evaluated in the study.
Kapustka et al. (1995) performed greenhouse experiments using 20 contaminated
upland soils from AS, measuring multiple plant responses versus growth in controls for
alfalfa and wheat. Each soil was ranked with a phytotoxicity score based on plant growth
reductions relative to growth in the control and these scores were correlated to total
COCs and pH for predictive modeling purposes. Although total COCs concentrations
were analyzed, individual threshold levels of plant growth declines for alfalfa and wheat
were not determined, and the trials lasted only 2 weeks (Kapustka et al., 1995). These
and previous field results were used by the U.S. EPA to develop a model for predicting
the effect of pH and total COCs on plant development on contaminated upland soils in
the UCFRB (Figure 5).
Figure 5. An EPA model of the effects of total metals, arsenic and pH on
species richness and biomass (Adapted from U.S. EPA, 2001).
39
As seen in the model, no effects to species richness or biomass are predicted when pH is
about 6.5 to 8, if total COCs remain below about 10,000 mg/kg
Kapustka and Lipton (1995) studied stem height, root length, and biomass of
hybrid poplar twigs placed in straight tailings from the CFR versus tailings mixed 50:50
with uncontaminated soil after 28 days. No significant reductions in growth or survival
occurred for the 50:50 mixtures, but straight tailings resulted in high mortality (Kapustka
& Lipton, 1995). It is unclear from the literature whether total COCs were analyzed for
these tailings.
Work by Rader, Nimmo & Chapman (1997) explored seed germination, root
length, and shoot height for lettuce, radish, redtop and the grass Echinochloa crusgalli in
tailings (total metals= 4851 mg/kg; pH=4.4) and tailings mixed with varying amounts
(2.5 -97%) of clean sand (pH 7.5). Results showed low to moderate plant growth
reductions for all four species when tailings concentration was low (2.5%), and severely
reduced growth and germination of all four species when tailings reached 20-25% (Rader
et al., 1997). Results also indicated that low pH alone cannot be used to predict
reductions in these plant responses, as Echinochloa grown in acidic, but nonmetalliferous soil did not produce reduced growth. Conversely, mean root length grown
in straight tailings adjusted to pH 6.6 was significantly reduced relative to
uncontaminated reference soil (pH 7.5; Rader et al., 1997). This study appears to be the
first study to address threshold levels of total COCs that significantly reduce plant
growth. This study also suggests limitations in the U.S. EPA model presented earlier
40
(Figure 5), as plant growth and germination were reduced at values of pH and total COCs
where the model states “No Effects” should occur. Although it was a major advance in
gaining threshold data, the work by Rader et al. (1997) used three agronomic species and
the weedy, introduced Echinocloa.
Redente et al. (2002) evaluated growth of the native grass bluebunch wheatgrass
and native forb western yarrow in 50 day greenhouse trials using contaminated AS soils
and uncontaminated German Gulch control soils treated with lime, fertilizer or nothing.
Fertilizer was the most significant amendment improving plant growth for both species,
irrespective of pH or which soil the plants were grown in. Although total soil COCs were
determined, threshold levels were not correlated to plant growth, however this study
suggested these species may have high tolerance to total COCs (Redente et al., 2002).
Keammerer & Redente (reviewed by Neuman, 2005) showed significant growth declines
of the native grasses bluebunch wheatgrass and big bluegrass, and the introduced grass
redtop, grown in contaminated soils collected in the CF representing a gradient of COCs
(Neuman, 2005). Threshold values of significant declines for shoot height, and shoot &
root biomass were determined by Neuman (2005), and were concluded to be at or below
1461 mg/kg.
Need for Further Research
For the last half century or longer, evidence of impaired plant growth and
colonization on both reclaimed and unreclaimed areas in the UCFRB, has driven
researchers to determine the factors involved in phytotoxicity. The edge of knowledge
seems to be in: 1) improving toxicity testing to determine site-specific threshold levels,
41
especially for native rangeland species; 2) quantifying COCs that are taken up by plant
tissues; 3) identifying and improving commercial-availability of metals-adapted ecotypes;
and 4) improving amendment combinations for optimum phytostabilization.
Toxicity thresholds of COCs can be used to estimate a species’ ability to establish
and survive on compromised sites (Paschke et al., 2005) and optimize selection of species
for phytostabilization. Determining toxicity thresholds from site-specific, lime-amended
UCFRB soils may help improve risk assessments and define site-specific exposure to
plants, and other members of the food chain.
42
MATERIALS AND METHODS
Summary
Soil collection & handling, and the plant greenhouse tests from planting to
harvesting were conducted with special care to the methods outlined by the ASTM
publication, “Standard guide for conducting terrestrial plant toxicity tests” (2003) and the
methods and guidelines reported by others (Kapustka, 1997; Redente et al., 2002; Conesa
et al., 2009).
Test Soils and Collection Sites Summary
Two lime-treated metalliferous test soils were collected from different sites in the
UCFRB (Figure 6, black stars). For both soils, selection of the area to be sampled was
guided by previous analytical data. Both soils were collected in the summer of 2004
from several places within a ten square meter area. Each soil collection area supported
between twenty-five and forty percent vegetation canopy cover. Soil samples were
collected from approximately the top 15 cm for both test substrates, composited into a
single sample, and transported to the RRU laboratory, Montana State University (MSU),
Bozeman, Montana, in plastic buckets.
ARTS Test Soil
The first test soil is an upland substrate referred to as “ARTS”, and was collected
from the designated AS site, northeast of the town of Anaconda, Montana (Figure 6).
43
The soil collection area was part of a series of field phytostabilization experiments
carried out in the mid 1990s (RRU, 1997). The ARTS test soil site was impacted by both
Figure 6. Map of the UCFRB showing soil collection locations. Black stars=test soil
collection sites. Red stars=dilution soil collection sites.
fluvial and aerial contamination from flood-deposited metals-enriched tailings and
smelter deposition, respectively. A lime product was not added to this site, however a
deep calcareous layer in the soil profile existed from limestone parent material, and in
1993 this layer was plowed and mixed to about the 60 cm (~23.5 in) depth, raising the pH
of the above materials. A fertilizer at a rate of 4.5-11.3-1.1-0.06 (N-P2O5-K2O-B)
kilograms/site was also added and incorporated to the 15 cm depth. The experimental
site was seeded with a variety of herbaceous species and mulched with straw at 2
44
tons/acre. Elevation at the site is approximately 1535 m with annual precipitation around
35 cm (WRCC, 2006).
Clark Fork Test Soil
The second test soil is a riparian substrate called “Clark Fork” and is named for its
proximity to the Clark Fork River near Warm Springs, MT (Figure 6). The site sits
within a large-scale phytostabilization demonstration in the floodplain of the Clark Fork
River. Soil was collected from easily-accessible private land on the demonstration area,
which has been sampled previously for COCs (Neuman et al., 2002). Incorporation of
CaO and CaCO3 took place in 1990 and was mixed to the 45-120 cm (~17-42 in) depth.
Wheatgrasses and alfalfa were then seeded following a 2240 kg/ha straw mulch
application which was crimped onto the soil surface. In June of 2000, the land owner
applied 16·16·16 (N·P·K) fertilizer to the plot and seeded with six-row barley (Hordeum
vulgare). Since then, the site has been converted to an alfalfa field. Approximate
elevation at the site is 1430 m and annual precipitation in the nearby Deer Lodge area
averages about 27 cm (WRCC, 2006).
Dilution Soils and Collection Sites Summary
Metals and arsenic contamination within the UCFRB is so widespread that noncontaminated dilution soil collection sites had to be carefully selected outside of the
Superfund areas. Thus, dilution soils were collected from areas that were still in the
greater UCFRB watershed, but were only minimally impacted by fluvial or aerial
processes. The two sites chosen for the dilution substrates were German Gulch and Little
45
Blackfoot River based on their proximity to the two test soil locations, ARTS and Clark
Fork, respectively (Figure 6, red stars). Dilution soils were collected at each area in
September of 2004 from several places within a ten square meter area. Soil samples were
collected from approximately the top 15 cm, composited into a single sample and
transported to the RRU laboratory in plastic buckets.
German Gulch Dilution Soil
The German Gulch drainage is a tributary of Silver Bow Creek near Butte, MT,
with approximate elevation of 1575 m. The gulch is located upstream from the
confluence of Silver Bow Creek and the Clark Fork River and has been used by others as
a non-impacted soil collection area for studies on impacts of smelter emissions (Redente
et al., 2002; Kapustka et al., 1995).
Little Blackfoot River Dilution Soil
Downstream from Deer Lodge, Montana, is the confluence of the Little Blackfoot
and Clark Fork Rivers near the town of Garrison. Upstream from Garrison is the town of
Avon, Montana where soil collection occurred approximately five to ten meters from the
river’s edge. Approximate elevation at this site is 1430 m. The Little Blackfoot River
has been selected as a reference area for characterization of soil and plant conditions in
past U.S. EPA risk assessments (U.S. EPA, 2001).
Soil Handling and Mixing to Create the Dilution Series
All four soils were air-dried, passed through a 2 mm sieve to remove rocks and
plant debris, and stored in bins. Several sub-samples of each soil were collected from
46
different locations within the bins and composited into single samples and analyzed for
total elemental copper by the Soil Analytical Lab (SAL), MSU. Copper levels were
analyzed as a substitute for analyzing all metals, in order to calculate the ideal dilution
factors to obtain at least a one order magnitude difference between the most concentrated
treatments and the controls (100% dilution soils). Most standardized tests using dilutions
obtain concentration gradients of one to two orders of magnitude (Kapustka, 1997). The
rationale for testing elemental copper as a measure of total metal and arsenic
concentration stems from analytical data from UCFRB tailings where a good correlation
between levels of one metal (e.g., copper) and total metal and arsenic levels were found
(U.S. EPA, 2001).
With the total copper data, the contaminated test soils and non-contaminated
dilution soils were mixed using the calculated masses of each, resulting in five dilution
factors (treatments) that were stored in separate bins. Test soils (undiluted ARTS and
Clark Fork test soils) and dilution soils representing the controls (native German Gulch
and Little Blackfoot River soils) were left alone and stored in each of their own storage
bins. The dilution factors were originally named according to the percent concentration
of test soil in each mixture. For example, “ARTS 6.25%” contained 6.25% contaminated
ARTS test soil and 93.75% uncontaminated German Gulch dilution soil by air-dried
weight.
47
Sampling the Mixed Soils and Methods of Analyses
Sub-samples of each of the dilution factors (including controls) were taken from
five randomly chosen locations within each of the storage bins, composited into a single
sample and characterized using standard procedures (Table 4) by the SAL.
Table 4. Soil parameters tested and methods of analyses used.
Parameter
Analytical Method
Total metals (Pb, Cu, Zn, Cd) and As
EPA method 3050, Standard EPA-CLP methods (SOW
787, U.S. EPA)
NO3-N
Method 84-3.1 (Bremner, 1965) and Method 4500-E
(APHA, 1998)
P
Olsen-P, Method 24-5.4.2 (ASA, 1982)
K
Method 13-3.5 (ASA, 1982)
Total Organic Carbon
Method 29-3.5.2 (ASA, 1982)
Additional volumes of soil from each treatment were collected and composited in the
same manner as above and used to make saturated pastes. Saturated paste extracts were
analyzed for pH using USDA Handbook 60, Method 3a, 21c (U.S. Salinity Lab, 1969) in
the laboratory of Dr. Michael Giroux, MSU, following calibration of the meter before
each sample with standard buffer solutions of pH 4.0, 7.0 and 10.0. Electrical
conductivity (EC) was analyzed from saturated paste extracts according to USDA
Handbook 60, Method 3a, 4b (U.S. Salinity Lab, 1969) in the RRU laboratory, using
standard calibration solutions of 447, 1500, 2764 and 8974 µS at the beginning and end
of day use. All temperatures during pH and EC measurements were recorded.
48
Experimental Design
The greenhouse study was organized in a randomized complete block design, with
five replications of each of the seven treatments (five dilution factors plus two controls)
for each of the two dilution series, hereafter referred to as simply ARTS and Clark Fork.
Replications were randomized every 2-3 days using a random number list generator.
Preparation for Plant Toxicity Tests
Soil Filling
Super Cell Cone-tainers (Stuewe & Sons, Inc., Tangent, Oregon, USA) measuring
3.8 cm diameter by 21 cm depth were color-coded and numbered according to the
treatment and replication number. Filter papers were rolled into a cone and placed in the
bottom of these tubes to prevent soil loss through the drainage holes. Soil was added to
within one to two cm from the tops of the tubes with the different soil treatments and
weighed. Mass of each replicate was recorded as “dry mass”.
Initial Watering
Treatment soils were initially watered with distilled water to bring them to field
capacity. Field capacity was the state of the tubes after slow, but steady, effluent dripped
from the filter paper cones at the base of the tubes initially, but stopped (Peters, 1965).
Effluent did not drain from the tubes at a fast rate, nor did excess effluent drain from the
tubes (5 mL or less). This was accomplished by slowly watering the tubes in 10 mL
portions in the beginning of wetting, and 5mL portions as each treatment got closer to
49
reaching field capacity. Each replicate was then weighed and the mass recorded as “wet
mass”.
Seed Planting
To standardize planting, a template was used to create holes in the soil surface for
each species’ planting. Five seeds per tube were planted to a depth of about 1.5 to 2
times the seed diameter for each species’ trial, totaling 25 seeds per treatment as
recommended by ASTM (2003). Only pure seed was planted, as evidenced by a
detectable embryo (H. Armstrong, personal communication, January 6, 2005). After the
seeds were placed in the holes, the displaced soil was covered over the top and lightly
tamped for good seed to soil contact.
Seed Pre-treatment
All greenhouse trials except slender wheatgrass were preceded by a cold
stratification (“pre-chill”) of 7 to 10 days to break primary dormancy that might exist and
to ensure the most uniform germination possible. In most cases, pre-chills were not
reported to be necessary for germination of the grasses used in the study (Baskin &
Baskin, 1990; AOSA, 2005). However, the author empirically observed more uniform
germination with basin wildrye (2nd trial) following a pre-chill than with slender
wheatgrass (1st trial) with no pre-chill. Thus, pre-chills were employed for each
subsequent trial, using the “cold-wet” room at the Plant Growth Center (PGC) at MSU.
This storage room is not actively humidified, but is maintained at about 5 ºC.
50
Greenhouse Facilities and Conditions
Greenhouse facilities at the PGC were used to grow all plant species.
Supplemental 420 Watt high pressure sodium lamps set at about two meters from the
plants were used to maintain a 16-hour photoperiod. Temperatures in the greenhouse
were maintained at about 22 ºC during the day and about 20 ºC during the night. Trials
occurred consecutively from July through December of 2005 and from May to June, and
August to October of 2006.
Watering During the Trials
Throughout the trials, tubes were re-watered with distilled water as needed to
achieve soil conditions that were conducive to plant emergence and growth, neither
saturated, nor dry. Because soil treatments were shown empirically (with the first trial on
slender wheatgrass) and analytically (with soil organic matter data once available) to hold
different amounts of water, it was important to record these differences. This was
accomplished by determining the soil water holding capacity as defined by the ASTM
guide (2003). The only modification to this method was that soils were air-dried for
several weeks and stored in bins versus oven-dried.
The different capacities for each soil to hold water can also be described as
“available water holding capacity”, a common measure of plant available water, usually
between field capacity and the “wilting point” (Troeh & Thompson, 2005). Because only
a portion of the water held in soils at field capacity is available and useful to plants
because of soil particles holding water so tightly (Troeh & Thompson, 2005), the term
51
available water holding capacity (AWHC) may be more accurate than soil water holding
capacity to describe water content held by soils. Therefore, although the wilting point
was not estimated in this study, AWHC will be used to describe the difference between
the water content held by the different soils at field capacity and that held in the soils
after air-drying in storage bins for several weeks.
Once the AWHC was determined as described above for each treatment,
calculations were made to generate target masses of the different treatments at 35%, 50%,
and 85% of AWHC. Tubes were maintained at or near field capacity during the
emergence stage (first 7-14 days). This was accomplished by weighing the tubes every
one to two days and adding distilled water back to the tubes according to the calculations
of “wet mass” described previously. As the seeds emerged and the plants grew bigger,
the tubes were allowed to dry down to between 35-50% AWHC and they were re-watered
to 85% AWHC. This was accomplished by weighing tubes every one to two days. This
method seemed the most reasonable, as keeping the tubes wetter than 85% AWHC
seemed too wet and seemed to be an excessive application of water. Available water
holding capacity of 35-50% maintained a moist substrate by touch (when tube was held
in hand), by sight (the way the surface of the soil looked) and by visual observation of
plant moisture stress (no leaf discoloration, loss of turgor, etc.; Small, 2009). Treatments
were watered back to 85% AWHC on different days because they did not dry down at the
same rate.
Even though the tubes were watered on different days, all treatments for each
species’ trial were given similar amounts of total water over each trial duration. This is
52
also true for the preliminary trial on slender wheatgrass where the protocol for weighing
tubes and re-watering was not yet established. For this trial, tubes were initially wetted to
just at field capacity, then re-watered in two ways: 1) same amount of water, same day
and 2) different amounts of water to bring back to field capacity, same day.
Nevertheless, the total water given in the slender trial was very similar for all treatments,
despite having a less standardized watering regime as the later plant trials.
Plant Species and their Trials
Slender Wheatgrass
Copperhead germplasm selected class (formally accession “9081620”) slender
wheatgrass (Elymus trachycaulus (Link) Gould ex Shinners) seed was obtained from the
BPMC. Original collection of the material to produce this seed occurred in 1995, three
miles east of Anaconda, MT, where plants were growing in mine waste material with a
surface soil pH of 4.3 (Majerus & Majerus, 2007). Copperhead slender wheatgrass was
grown in July and August of 2005. Harvest occurred after 31 days for ARTS and 35 days
for Clark Fork. The slender wheatgrass trial was a preliminary trial to develop test
protocols for the greenhouse trials and plant harvests before full implementation.
Basin Wildrye
Washoe germplasm (formerly accession “9081627”) basin wildrye (Leymus
cinereus (Scribn. & Merr.) A. Löve) seed was obtained from the BPMC after having been
originally collected from an area impacted by smelter stack fallout on Mount Haggin, one
mile southwest of the defunct Washoe copper smelter stack near Anaconda, MT with
53
surface soil pH ranging from 4.6 to 5.6 (Marty, 2003). Washoe basin wildrye was grown
in September and October of 2005. Harvest occurred after 33 days for both dilution
series.
Bluebunch Wheatgrass
‘Secar’ bluebunch wheatgrass (Pseudoroegneria spicata (Pursch) A. Löve) seed
was obtained from Wind River Seeds (WRS), Manderson, WY, a reclamation and native
seed company. The original collection site of this cultivar was from the Snake River
gorge area in Idaho (USDA & NRCS, 2009). Bluebunch wheatgrass was grown in
November and December of 2005. Harvest occurred after 27 days for both dilution
series.
Big Bluegrass
‘Sherman’ big bluegrass (Poa secunda J. Presl) seed was obtained from WRS
with origin listed as Oregon. Original collection of ‘Sherman’ occurred near the town of
Moro, Oregon in Sherman County (USDA & NRCS, 2009). ‘Sherman’ big bluegrass was
grown in May and June of 2006. Harvest occurred after 47 days for both dilution series.
Sheep Fescue
‘Covar’ sheep fescue (Festuca ovina L.) seed was obtained from WRS with origin
listed as Washington, but the original collection of seed to develop this material was from
Konya, Turkey (USDA & NRCS, 2009). ‘Covar’ sheep fescue was grown in late August,
September and early October of 2006. Harvest occurred after 49 days for both dilution
series.
54
Redtop
Redtop (Agrostis gigantea Roth.) accession “9081619” seed was obtained from
the BPMC, being originally collected in 1998 from an area adjacent to Anaconda’s Old
Works Golf Course. A soil pH sample was not taken at the time of collection, but pH
values on nearby Stucky Ridge range from 4.0-4.7 in the 0-15 cm depth (M. Majerus,
personal communication, November 27, 2006). “9081619” redtop was grown in late
August, September and early October of 2006. Harvest occurred after 45 days for both
dilution series.
Tufted Hairgrass
Tufted hairgrass (Deschampsia caespitosa (L.) Beauv) seed was obtained from
WRS with origin listed as Washington. The initial growing stock of this material most
likely came from Alaska or Canada (R. Holzhäuser, personal communication, March 3,
2009). Tufted hairgrass was grown in May and June of 2006. Harvest occurred after 48
days for both dilution series.
Identification of Volunteer or Unintentionally Planted Species
In a few cases throughout the greenhouse trials, unknown species were discovered
growing in some of the treatments and were set aside from the rest of the plants to grow
for many months in an attempt to produce flowers for taxonomic identification. These
unknown plants represented seed bank species or unintentionally planted species that
were in the seed source. Most of these species were identified by ITS/5.8 ribosomal
55
DNA sequencing (Results Chapter) by Dr. Matthew Lavin, Plant Scientist, MSU, because
flowers never formed.
ITS/5.8S DNA Sequencing
The techniques used by Dr. Lavin were as follows: DNA was isolated from a
small fragment of leaf tissue from each unknown species using the Qiagen DNeasy Plant
Mini Kit (Valencia, CA). The nuclear ribosomal internal transcribed spacers and the
intervening 5.8S regions (ITS/5.8S) were amplified using the 18S and 26S primers and
sequenced using the SSF and LSR primers, all of which are described in Beyra and Lavin
(1999). DNA sequencing, which was performed at Northwoods DNA (Solway, MN),
included the forward and reverse directions, and each pair reads were then contiged with
the program Sequencer 4.1 (Gene Codes Corp, Ann Arbor, MI). These final DNA
sequences where then submitted to a BLAST search at the National Institutes of Health
web site (http://blast.ncbi.nlm.nih.gov) for identification. This unequivocally identified
the genus of each unknown grass sample and furthermore narrowed down the possibility
of the species identity of each. The final species identity was determined by a final visual
inspection of the grass sample, using the knowledge of which common Montana grass
species was most closely related to the species that was the closest match from the
BLAST search.
56
Plant Harvest and Measurements
Total Percent Emergence
Seedling emergence was recorded once per week during the duration of each trial.
Plants were thinned to one individual per tube once 70% germination had occurred in the
control (100% dilution soil), leaving the tallest plant to grow in each tube. In the case
where two equally tall plants existed in a tube, the individual located closest to the center
of the tube was left to grow. For all trials this occurred between 10 and 14 days. In the
case of slender wheatgrass, thinning occurred after 70% emergence had occurred in a
potting mix control since the 100% dilution soil control had delayed emergence (Results
Chapter). Seedlings that emerged after thinning were removed upon counting unless they
represented the only seedling to emerge in that tube. Counts of emergence were
converted to percentages before statistical analysis.
Shoot Measurements
Shoot height was measured to the nearest mm, and the aerial parts of the plant
clipped at the soil level, after the trial ended (between 27 and 49 days depending on
species). Above-ground parts were put in separate paper bags and placed in the drying
oven (70 ºC) until constant weight was obtained. Once the shoots were fully dried, a
Denver Instruments DI-100 analytical balance was used to weigh shoots to the nearest
0.001 g as recommended by ASTM (2003), after initial calibration to within 0.002 g. The
balance was re-calibrated after every 10th sample.
57
Root Measurements and Observations
Tubes were cut longitudinally with a razor to expose the soil and roots. Soil
adhering to the roots was removed in a series of wash pans according to an established
protocol (C. Zabinski, personal communication, August, 15, 2005). After the roots were
washed clean, the length was measured to the nearest mm. A dissecting microscope set
between 10-30X was used to inspect the roots for the presence of fine roots and root
hairs. A qualitative rating system was used to describe the root systems during harvest,
after it was determined with the preliminary trial on slender wheatgrass that roots
growing in different treatments had different degrees of branching and densities.
Individual plant roots were then put into separate paper bags and placed in the drying
oven (70 ºC) until constant weight was obtained. The same analytical balance and
calibration protocol was followed for roots as with shoots.
Statistical Analyses
ANOVA
One-way analysis of variance (ANOVA) was used to analyze significant (p<0.05)
mean differences in percent emergence, shoot height, total biomass and root mass ratio
amongst the different treatments using Minitab (Minitab 15 Statistical Software, 2007).
Some data were normally distributed with equal variance while others were not. The
Anderson-Darling and Ryan-Joiner tests (Minitab 15 Statistical Software, 2007) were
used to check normality (α = 0.01). Equal variance was verified with the Levene’s test
(α = 0.01). Data whose residuals were not distributed normally or whose variance were
unequal were transformed using the rounded lambda value from a Box-Cox function.
58
After data transformation with the Box-Cox derived value, all data met normality and
equal variance assumptions except for total percent emergence with redtop & sheep
fescue, and root mass ratio with slender wheatgrass in ARTS. In the case of emergence
with redtop and sheep fescue, because the raw data (Appendix) show emergence was so
uniform and limited to only certain values (i.e., 100% or 80% emergence), the residuals
could not be transformed to agree with normality assumptions. Thus, this response
variable for these species was analyzed with the untransformed data. Root mass ratio
with slender wheatgrass was analyzed in two ways (Results Chapter). Following
ANOVA, the post hoc Dunnett’s test was used to compare each treatment mean with the
control mean (100% dilution soil) and determine where significant differences occur
along the gradient of total metals and arsenic (Minitab 15 Statistical Software, 2007).
Whether transformation occurred or not, boxplots of the raw, untransformed data were
used to display the ANOVA and Dunnett’s method results for ease in reading the graphs.
Interaction Plots
Because two different dilution series were studied, plant response could differ
across increasing total metals and arsenic as a function of the series used. Thus,
interaction plots were created to determine if differences in the main pattern of the
scatterplots of mean response (i.e., slopes of connect lines), varied by dilution series
(Minitab 15 Statistical Software, 2007). Interaction plots were created to observe these
differences graphically.
59
Correlation
The Pearson product moment correlation coefficient (r) was used to determine the
strength of the linear relationship between sum of total metals and arsenic and plant
responses, where significant correlation was designated at the p<0.05 level (Minitab 15
Statistical Software, 2007).
60
RESULTS
Results of Soil Mixing
Table 5 presents the soil analytical results after mixing ARTS test soil with the
German Gulch reference soil to create ARTS, and mixing Clark Fork test soil with the
Little Blackfoot River reference soil to create Clark Fork. For both dilution series, the
soil mixing was successful in creating a gradation of increasing total metals and arsenic
as the ratio of test soil in each treatment increased.
Soil pH for all treatments was between 7.2 and 7.9. Just as the individual
contaminants increase as the concentration of test soil increases, so do the values for soil
N, P, and EC for the treatments of both dilution series (Table 5). This is because the
concentration of these nutrients and EC in the full-strength test soils (i.e. ARTS 100%
and Clark Fork 100%) exceed the concentration in the dilution soils. When these test
soils were mixed with greater amounts of dilution soils to create the dilution series, it was
inevitable that the other parameters in the soils (such as N, P, etc.) would be diluted, as
were total metals and arsenic. This of course depended on the original ratio of the
concentration of each soil constituent in the test versus dilution soil.
61
Table 5. Soil analytical results for ARTS and Clark Fork dilution series with a column included (in bold) for sum of total metals and arsenic.
C indicates control soil (100% dilution soil). Soil sample names, such as “ARTS 6.25%” indicate the percentage of contaminated test soil (by weight)
in the treatment. For example, ARTS 6.25% was a mixture of 0.506 kg of ARTS soil and 7.594 kg German Gulch soil.
German GulchC
ARTS 6.25%
ARTS 12.5%
ARTS 25%
ARTS 50%
ARTS 75%
ARTS 100%
7.5
7.3
7.5
7.9
7.7
7.7
7.5
42
60
81
106
172
245
311
71
237
458
791
1497
2284
3043
92
184
304
482
862
1263
1678
2.6
2.9
3.5
4.5
6.9
9.5
12.2
36
75
127
201
371
558
739
244
559
974
1585
2908
4360
5783
15
16
17
17
18
19
19
4.2
3.9
3.9
3.8
3.4
3.5
2.8
27
31
28
38
42
44
52
552
568
560
522
502
504
452
493
563
605
757
839
1029
1096
Little BlackfootC
Clark Fork 6.25%
Clark Fork 12.5%
Clark Fork 25%
Clark Fork 50%
Clark Fork 75%
Clark Fork 100%
7.9
7.7
7.7
7.3
7.4
7.3
7.2
57
123
224
250
420
660
823
25
247
627
947
1912
3185
4314
151
200
299
370
553
968
1058
1.2
1.6
2.2
2.7
4.0
5.8
7.0
16
78
182
330
636
1066
1319
251
650
1334
1900
3525
5885
7521
18
19
23
25
35
49
57
2.9
3.0
3.9
3.9
5.0
5.5
7.7
26
30
41
45
46
49
50
290
328
348
418
522
606
756
917
1358
1667
2398
2962
3590
3708
Soil
Sample
Total Total Total Total Total metals
Cu
Zn
Cd
As
and As
NO3-N
mg/kg mg/kg mg/kg mg/kg
mg/kg
mg/kg
TOC
%
Olsen
P
mg/kg
K
mg/kg
EC
µS/m
61
pH
Total
Pb
mg/kg
62
As another example, in the Clark Fork dilution series only, K and total organic
carbon (TOC; a measure of soil organic matter) increase as the concentration of test soil
in each treatment in the series increases (Table 5). This is because the concentration of K
and TOC in the Clark Fork test soil exceeds that of the dilution soil and these constituents
naturally increase as more Clark Fork soil is added to create each treatment. As a further
example, K and TOC decrease in the ARTS dilution series as the concentration of ARTS
test soil in the series increases. This is because the concentration of K and TOC in
German Gulch dilution soil exceeds the concentration in the ARTS test soil.
Explanations of the effects of diluted soil constituents such as these are addressed in the
Discussion Chapter.
The proportion of each individual contaminant (i.e. Pb, Cu, Zn, Cd, As) in the
dilution series increased as the concentration of test soil increased. This is presented
graphically in Figures 7 and 8.
ARTS dilution series
Figure 7. Proportion of individual contaminants in each treatment of ARTS
dilution series.
63
Clark Fork dilution series
Figure 8. Proportion of individual contaminants in each treatment of
Clark Fork dilution series.
Results of One-Way ANOVA, Dunnett’s Method and Correlation by Species
Displayed in this section are results of one-way ANOVA and Dunnett’s method
for percent emergence, shoot height, total biomass and root mass ratio (root mass as a
fraction of total plant mass) for seven grass species grown in the greenhouse trials.
Under each species’ section, there are paired graphs for comparing the species’
performance in the two dilutions series. All p values first reported on tables/figures are
the result of one-way ANOVA. The Pearson correlation coefficient (r) for the strength of
the linear relationship between each response variable and a gradation of total metals and
arsenic is also presented on all figures/tables, with the associated p value listed in
64
parentheses. The range of assumed threshold concentrations of total metals and arsenic
are indicated with arrows on figures, based on the first significantly different treatment
mean versus the control (100% dilution soil) mean. On the figures of shoot height, total
biomass and root mass ratio, the percent reduction or increase in treatment plant response
versus the response in the control is listed with the threshold arrow. Asterices (*) on all
figures/tables indicate mean treatment response is significantly different (p<0.05) from
mean response in the control (100% dilution soil). On boxplots, + signs are mean
responses.
Basin Wildrye
Table 6 shows percent emergence (%) for basin wildrye grouped by dilution
series. For ARTS, no significant differences in mean percent emergence were observed,
even in 100% test soil, indicating threshold values of total metals and arsenic may exceed
the most concentrated treatment for this response. For Clark Fork, mean percent
emergence declined when total metal and arsenic concentration reached 7521 mg/kg, but
there was no significant decline at the 5885 mg/kg treatment. Thus, the assumed
threshold value for percent emergence is between 5885 and 7521 mg/kg for this species
in the Clark Fork series. The r value for ARTS was weak, 0.05 (p=0.77), but moderate
for Clark Fork (r=-0.64, p=0.00), indicating percent emergence and a gradation of total
metals and arsenic are moderately and negatively correlated in Clark Fork for this
species. However, in the ARTS series, there is not enough evidence to reject the null
hypothesis that a significant correlation other than zero exists between percent emergence
and total metals and arsenic.
65
Table 6. Mean percent emergence (%) for basin wildrye in ARTS (left) and Clark Fork (right). C
indicates control soil.
ARTS
p = 0.55
r = 0.05 (p=0.77)
Response
Total metals
and As
Number of
p = 0.00
r = -0.64 (p=0.00)
CLARK FORK
Response
Mean
Total metals
percent
Number of
replications (n)
percent
emergence (%)
Mean
Treatment
(mg/kg)
Treatment
and As
(mg/kg)
0%C
244
5
76
0%C
251
5
84
6.25%
559
5
72
6.25%
650
5
84
12.50%
974
5
68
12.50%
1334
5
76
25%
1585
5
76
25%
1900
5
92
50%
2908
5
84
50%
3525
5
64
75%
4360
5
84
75%
5885
5
56
100%
5783
5
68
100%
7521
5
32*
replications (n) emergence (%)
Boxplots of shoot height grouped by dilution series for basin wildrye are shown in
Figure 9. For ARTS, a significant decline in mean shoot height occurred when total
metal and arsenic concentration reached 1585 mg/kg, but there was no significant decline
at the 974 mg/kg treatment. Thus, the assumed threshold value for shoot height is
between 974 and 1585 mg/kg for this species in the ARTS series. For Clark Fork, a
significant decline in mean shoot height occurred when total metal and arsenic
concentration reached 1900 mg/kg, but there was no significant decline at the 1334
mg/kg treatment. Thus, the assumed threshold value for shoot height is between 1334
and 1900 mg/kg for this species in the Clark Fork series. Both series had strong r values
for the relationship between shoot height and a gradation of total metals and arsenic;
ARTS having an r value of -0.91 (p=0.00) and an r value in Clark Fork of
-0.87 (p=0.00). These values indicate that shoot height and a gradation of total metals
and arsenic are strongly and negatively correlated for both series for this species.
66
ARTS
35
25
*
20
15
Assumed threshold
occurs between
these concentrations
(26% reduction)
10
5
244
*
*
*
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
CLARK FORK
p = 0.00
r = -0.87 (p=0.00)
30
Shoot height (cm)
30
Shoot height (cm)
35
p = 0.00
r = -0.91 (p=0.00)
25
20
15
10
5
Assumed threshold
occurs between
these concentrations
(55% reduction)
*
*
*
*
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 9. Boxplots of basin wildrye shoot height in ARTS (left) and Clark Fork (right).
Figure 10 shows total biomass for basin wildrye grouped by dilution series. For
ARTS, a significant decline in mean total biomass occurred when total metal and arsenic
concentration reached 974 mg/kg, but there was no significant decline at the 559 mg/kg
treatment. Thus, the assumed threshold for total biomass is between 559 and 974 mg/kg
for this species in the ARTS series. For Clark Fork, a significant decline in mean total
biomass occurred when total metal and arsenic concentration reached 1334 mg/kg, but
there was no significant decline at the 650 mg/kg treatment. Thus, the assumed threshold
value for total biomass is between 650 and 1334 mg/kg for this species in the Clark Fork
series. Both series had strong r values for the relationship between total biomass and a
gradation of total metals and arsenic; ARTS having an r value of -0.90 (p=0.00) and an r
value in Clark Fork of -0.81 (p=0.00). These values indicate that total biomass and a
gradation of total metals and arsenic are strongly and negatively correlated for both series
for this species.
67
ARTS
CLARK FORK
p = 0.00
r = -0.90 (p=0.00)
100
*
80
*
60
Assumed threshold
occurs between
these concentrations
(22% reduction)
40
20
*
120
*
*
0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
p = 0.00
r = -0.81 (p=0.00)
100
Total biomass (mg)
Total biomass (mg)
120
80
*
60
Assumed threshold
40 occurs between
20 these concentrations
(35% reduction)
0
*
*
*
*
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 10. Boxplots of basin wildrye total biomass in ARTS (left) and Clark Fork (right).
Boxplots of root mass ratio grouped by dilution series for basin wildrye are shown
in Figure 11. For ARTS, a significant increase in this ratio occurred when total metal and
arsenic concentration reached 4360 mg/kg, but there was no significant increase at the
2908 mg/kg treatment. Thus, the assumed threshold value for this ratio is between 2908
and 4360 mg/kg for this species in the ARTS series. For Clark Fork, a significant
increase in this ratio occurred when total metal and arsenic concentration reached 7921
mg/kg, but there was no significant increase at the 5885 mg/kg treatment. Thus, the
assumed threshold value for this ratio is between 5885 and 7921 mg/kg for this species in
the Clark Fork series. These data indicate that basin wildrye increased the amount of
below ground biomass relative to the above ground biomass for both series as total metals
and arsenic increased. Rationale for this is addressed in the Discussion Chapter. Both
series had moderate r values for the relationship between root mass ratio and a gradation
of total metals and arsenic; ARTS having an r value of 0.62 (p=0.00) and an r value in
Clark Fork of 0.72 (p=0.00). These values indicate root mass ratio and a gradation of
total metals and arsenic are moderately and positively correlated for both series for this
species.
68
ARTS
0.8
0.6
Assumed threshold
occurs between
these concentrations
(40% increase)
CLARK FORK
p = 0.00
r = 0.62 (p=0.00)
*
*
0.4
0.8
0.6
0.4
0.2
0.2
0.0
0.0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
p = 0.00
*
r = 0.72 (p=0.00)
1.0
Root mass ratio
Root mass ratio
1.0
Assumed threshold occurs
between these concentrations
(73% increase)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 11. Boxplots of basin wildrye root mass ratio in ARTS (left) and Clark Fork (right).
Bluebunch Wheatgrass
Table 7 shows percent emergence (%) for bluebunch wheatgrass grouped by
dilution series. For ARTS, no significant differences in mean percent emergence were
observed, even in 100% test soil, indicating threshold values of total metals and arsenic
may exceed the most concentrated treatment for this response. For Clark Fork, mean
percent emergence declined when total metal and arsenic concentration reached 3525
mg/kg, but there was no significant decline at the 1900 mg/kg treatment. Thus, the
assumed threshold value for percent emergence is between 1900 and 3525 mg/kg for this
species in the Clark Fork series. The r value for ARTS was weak, -0.29 (p=0.01), but
strong in the Clark Fork (r=-0.91, p=0.00). These values indicate that percent emergence
and a gradation of total metals and arsenic are strongly and negatively correlated in Clark
Fork and weakly and negatively correlated for ARTS for this species.
69
Table 7. Mean percent emergence (%) for bluebunch wheatgrass in ARTS (left) and Clark Fork (right).
C indicates control soil.
ARTS
p = 0.44
r = -0.29 (p=0.01)
Response
Total metals
and As
Number of
p = 0.00
r = -0.91 (p=0.00)
CLARK FORK
Mean
Total metals
percent
Response
Mean
Treatment
(mg/kg)
Treatment
and As
(mg/kg)
0%C
244
5
84
0%C
251
5
6.25%
559
5
88
6.25%
650
5
92
12.50%
974
5
84
12.50%
1334
5
100
25%
1585
5
72
25%
1900
5
88
50%
2908
5
80
50%
3525
5
40*
75%
4360
5
84
75%
5885
5
12*
100%
5783
5
60
100%
7521
5
12*
replications (n) emergence (%)
Number of
percent
replications (n) emergence (%)
96
Boxplots of shoot height grouped by dilution series for bluebunch wheatgrass are
shown in Figure 12. For ARTS, a significant decline in mean shoot height occurred
when total metal and arsenic concentration reached 559 mg/kg, the first treatment with
elevated levels greater than the control (244 mg/kg). Thus, the assumed threshold value
for shoot height is between 244 and 559 mg/kg for this species in the ARTS series. For
Clark Fork, a significant decline in mean shoot height occurred when total metal and
arsenic concentration reached 1900 mg/kg, but there was no significant decline at the
1334 mg/kg treatment. Thus, the assumed threshold value for shoot height is between
1334 and 1900 mg/kg for this species in the Clark Fork series. Both series had moderate
to strong r values for the relationship between shoot height and a gradation of total metals
and arsenic; ARTS having an r value of -0.67 (p=0.00) and an r value in Clark Fork of
-0.90 (p=0.00). These values indicate that shoot height and a gradation of total metals
and arsenic are moderately and negatively correlated in ARTS and strongly and
negatively correlated in Clark Fork for this species.
70
ARTS
30
20
15
10
*
*
*
*
*
*
Assumed
5 threshold occurs between
these concentrations (34% reduction)
0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
p = 0.00
r = -0.90 (p=0.00)
25
Shoot height (cm)
Shoot height (cm)
25
CLARK FORK
30
p = 0.00
r = -0.67 (p=0.00)
20
*
15
Assumed threshold
occurs between
these concentrations
(40% reduction)
10
5
0
250
*
*
*
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 12. Boxplots of bluebunch wheatgrass shoot height in ARTS (left) and Clark Fork (right).
Figure 13 shows total biomass for bluebunch wheatgrass grouped by dilution
series. For ARTS, a significant decline in mean total biomass occurred when total metal
and arsenic concentration reached 559 mg/kg, the first treatment with elevated levels
greater than the control (244 mg/kg). Thus, the assumed threshold value for total
biomass is between 244 and 559 mg/kg for this species in the ARTS series. For Clark
Fork, a significant decline in mean total biomass occurred when total metal and arsenic
concentration reached 1900 mg/kg, but there was no significant decline at the 1334
mg/kg treatment. Thus, the assumed threshold value for total biomass is between 1334
and 1900 mg/kg for this species in the Clark Fork series. Both series had moderate to
strong r values for the relationship between total biomass and a gradation of total metals
and arsenic; ARTS having an r value of -0.58 (p=0.00) and an r value in Clark Fork of
-0.87 (p=0.00). These values indicate that total biomass and a gradation of total metals
and arsenic are moderately and negatively correlated in ARTS and strongly and
negatively correlated in Clark Fork for this species.
71
ARTS
30
20
10
CLARK FORK
40
Assumed threshold p = 0.00
occurs between
r = -0.58 (p=0.00)
these concentrations
(48% reduction)
*
*
*
*
*
*
0
Total biomass (mg)
Total biomass (mg)
40
p = 0.00
r = -0.87 (p=0.00)
30
20
10
Assumed threshold
occurs between
these concentrations
(56% reduction)
*
*
*
*
0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 13. Boxplots of bluebunch wheatgrass total biomass in ARTS (left) and Clark Fork (right).
Boxplots of root mass ratio grouped by dilution series for bluebunch wheatgrass
are shown in Figure 14. For ARTS, no significant differences were observed, even in
100% test soil, indicating threshold levels of total metals and arsenic may exceed the
most concentrated treatment for this response. For Clark Fork, a significant increase in
this ratio occurred when total metal and arsenic concentration reached 7921 mg/kg, but
there was no significant increase at the 5885 mg/kg treatment. Thus, the assumed
threshold value for this ratio is between 5885 and 7921 mg/kg for this species in the
Clark Fork series. These data indicate that bluebunch wheatgrass produced the same
amount of below ground biomass relative to above ground biomass for ARTS but
increased this ratio in Clark Fork as the gradient of total metals and arsenic increased.
Rationale for this is addressed in the Discussion Chapter. The r value for ARTS was
weak, 0.13 (p=0.48), but moderate for Clark Fork (r=0.67; p=0.00), indicating that root
mass ratio and a gradation total metals and arsenic are moderately and positively
correlated in Clark Fork for this species. However, in the ARTS series, there is not
enough evidence to reject the null hypothesis that a significant correlation other than zero
exists between root mass ratio and total metals and arsenic.
72
ARTS
1.0
CLARK FORK
1.0
p = 0.80
r = 0.13 (p=0.48)
0.8
Root mass ratio
Root mass ratio
0.8
0.6
0.4
0.4
0.0
0.0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
*
0.6
0.2
0.2
p = 0.00
r = 0.67 (p=0.00)
Assumed threshold occurs
between these concentrations
(119% increase)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 14. Boxplots of bluebunch wheatgrass root mass ratio in ARTS (left) and Clark Fork (right).
Big Bluegrass
Table 8 shows percent emergence (%) for big bluegrass grouped by dilution
series. No significant differences in mean percent emergence were observed for either
series, even in 100% test soil treatments, indicating threshold values of total metals and
arsenic may exceed the most concentrated treatments for this response. Both series had
weak r values for the relationship between mean percent emergence and a gradation of
total metals and arsenic, ARTS having an r value of 0.31 (p=0.07) and an r value in Clark
Fork of 0.28 (p=0.11). This indicates that for both series, there is not enough evidence to
reject the null hypothesis that a significant correlation other than zero exists between
percent emergence and total metals and arsenic.
73
Table 8. Mean percent emergence (%) for big bluegrass in ARTS (left) and Clark Fork (right). No
significant differences amongst treatment versus control means were found for either series. C
indicates control soil.
p = 0.54
r = 0.31 (p=0.07)
ARTS
Response
Total metals
Treatment
and As
(mg/kg)
p = 0.45
r = 0.28 (p=0.11)
Mean
Number of
percent
replications (n) emergence (%)
CLARK FORK
Response
Total metals
Treatment
and As
(mg/kg)
Mean
Number of
percent
replications (n) emergence (%)
0%C
244
5
84
0%C
251
5
6.25%
559
5
88
6.25%
650
5
76
12.50%
974
5
84
12.50%
1334
5
88
25%
1585
5
96
25%
1900
5
72
50%
2908
5
92
50%
3525
5
88
75%
4360
5
96
75%
5885
5
92
100%
5783
5
96
100%
7521
5
92
84
Boxplots of shoot height grouped by dilution series for big bluegrass are shown in
Figure 15. For ARTS, a significant decline in mean shoot height occurred when total
metal and arsenic concentration reached 559 mg/kg, the first treatment with elevated
levels greater than the control (244 mg/kg). Thus, the assumed threshold value for shoot
height is between 244 and 559 mg/kg for this species in the ARTS series. For Clark
Fork, a significant decline in mean shoot height occurred when total metal and arsenic
concentration reached 1900 mg/kg, but there was no significant decline at the 1334
mg/kg treatment. Thus, the assumed threshold value for shoot height is between 1334
and 1900 mg/kg for this species in the Clark Fork series. Both series had strong r values
for the relationship between shoot height and a gradation of total metals and arsenic;
ARTS having an r value of -0.81 (p=0.00) and an r value in Clark Fork of -0.93 (p=0.00).
These values indicate that shoot height and a gradation of total metals and arsenic are
strongly and negatively correlated for both series for this species.
74
ARTS
20
p = 0.00
r = -0.81 (p=0.00)
15
*
10
*
*
*
*
*
Assumed threshold occurs
5 between these concentrations
(28% reduction)
0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
Shoot height (cm)
Shoot height (cm)
20
CLARK FORK
p = 0.00
r = -0.93 (p=0.00)
15
10
5
0
*
Assumed threshold
occurs between
these concentrations
(37% reduction)
*
*
*
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 15. Boxplots of big bluegrass shoot height in ARTS (left) and Clark Fork (right).
Figure 16 shows total biomass for big bluegrass grouped by dilution series. For
ARTS, a significant decline in mean total biomass occurred when total metal and arsenic
concentration reached 1558 mg/kg, but there was no significant decline at the 974 mg/kg
treatment. Thus, the assumed threshold value for total biomass is between 974 and 1558
mg/kg for this species in the ARTS series. For Clark Fork, a significant decline in mean
total biomass occurred when total metal concentration reached 650 mg/kg, the first
treatment with elevated levels greater than the control (250 mg/kg). Thus, the assumed
threshold value for total biomass is between 250 and 650 mg/kg in the Clark Fork series.
Both series had strong r values for the relationship between total biomass and a gradation
of total metals and arsenic; ARTS having an r value of -0.87 (p=0.00) and an r value in
Clark Fork of -0.92 (p=0.00). These values indicate that total biomass and a gradation of
total metals and arsenic are strongly and negatively correlated in both series for this
species.
75
ARTS
CLARK FORK
p = 0.00
r = -0.87 (p=0.00)
100
80
Assumed threshold occurs
between these concentrations
(74% reduction)
60
40
*
20
0
*
*
*
244
559 974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
120
Total biomass (mg)
Total biomass (mg)
120
100
80
p = 0.00
r = -0.92 (p=0.00)
*
*
60
Assumed
*
40 threshold occurs
between these concentrations
20
*
(30% reduction)
*
*
0
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 16. Boxplots of big bluegrass total biomass in ARTS (left) and Clark Fork (right).
Boxplots of root mass ratio grouped by dilution series for big bluegrass are shown
in Figure 17. No significant differences were observed for either series, even in 100%
test soil treatments, indicating threshold values of total metals and arsenic may exceed the
most concentrated treatments for this response. These data indicate that big bluegrass
produced the same amount of below ground biomass relative to above ground biomass
for both series as the gradient of total metals and arsenic increased. Rationale for this is
addressed in the Discussion Chapter. Both series had weak r values for the relationship
between root mass ratio and a gradation of total metals and arsenic; ARTS having an r
value of -0.16 (p=0.37) and an r value in the Clark Fork of -0.32 (p=0.06). This indicates
that for both series, there is not enough evidence to reject the null hypothesis that a
significant correlation other than zero exists between root mass ratio and total metals and
arsenic.
76
ARTS
CLARK FORK
1.0
p = 0.63
r = -0.16 (p=0.37)
0.6
0.4
0.2
p = 0.38
r = -0.32 (p=0.06)
0.8
Root mass ratio
0.8
Root mass ratio
1.0
0.6
0.4
0.2
0.0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
0.0
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 17. Boxplots of big bluegrass root mass ratio in ARTS (left) and Clark Fork (right). No
significant differences amongst treatment versus control means were found for either series.
Sheep Fescue
Table 9 shows percent emergence (%) for sheep fescue grouped by dilution series.
No significant differences in mean percent emergence were observed for either series,
even in 100% test soil treatments, indicating threshold values of total metals and arsenic
may exceed the most concentrated treatments for this response. Both series had weak r
values for the relationship between percent emergence and a gradation of total metals and
arsenic; ARTS having an r value of 0.24 (p=0.16) and an r value in Clark Fork of -0.27
(p=0.12). This indicates that for both series, there is not enough evidence to reject the
null hypothesis that a significant correlation other than zero exists between percent
emergence and total metals and arsenic.
77
Table 9. Mean percent emergence (%) for sheep fescue in ARTS (left) and Clark Fork (right). No
significant differences amongst treatment versus control means were found for either series. C indicates
control soil.
p = 0.37
r = 0.24 (p=0.16)
ARTS
Response
Total metals
p = 0.22
r = -0.27 (p=0.12)
CLARK FORK
Response
Mean
Total metals
Treatment
and As
(mg/kg)
Number of
replications (n)
percent
emergence (%)
Treatment
and As
(mg/kg)
Number of
replications (n)
percent
emergence (%)
0%C
244
5
92
0%C
251
5
96
6.25%
559
5
96
6.25%
650
5
84
12.50%
974
5
96
12.50%
1334
5
96
25%
1585
5
100
25%
1900
5
100
50%
2908
5
92
50%
3525
5
88
75%
4360
5
100
75%
5885
5
88
100%
5783
5
100
100%
7521
5
84
Mean
Boxplots of shoot height grouped by dilution series for sheep fescue are shown in
Figure 18. For ARTS, a significant decline in mean shoot height occurred when total
metal and arsenic concentration reached 974 mg/kg, but there was no significant decline
at the 559 mg/kg treatment. Thus, the assumed threshold value for shoot height is
between 559 and 974 mg/kg for this species in the ARTS series. For Clark Fork, a
significant decline in mean shoot height occurred when total metal and arsenic
concentration reached 1900 mg/kg, but there was no significant decline at the 1334
mg/kg treatment. Thus, the assumed threshold value for shoot height is between 1334
and 1900 mg/kg for this species in the Clark Fork series. Both series had strong r values
for the relationship between shoot height and a gradation of total metals and arsenic;
ARTS having an r value of -0.80 (p=0.00) and an r value in Clark Fork of -0.84 (p=0.00).
These values indicate that shoot height and a gradation of total metals and arsenic are
strongly and negatively correlated for both series for this species.
78
ARTS
p = 0.00
r = -0.80 (p=0.00)
Assumed threshold occurs
between these concentrations
* (33%*reduction)
*
*
*
20
15
10
5
0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
25
Shoot height (cm)
Shoot height (cm)
25
CLARK FORK
p = 0.00
r = -0.84 (p=0.00)
20
15
*
10
*
Assumed threshold
*
*
5 occurs between these
concentrations (33% reduction)
0
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 18. Boxplots of sheep fescue shoot height in ARTS (left) and Clark Fork (right).
Figure 19 shows total biomass for sheep fescue grouped by dilution series. For
ARTS, a significant decline in mean total biomass occurred when total metal and arsenic
concentration reached 974 mg/kg, but there was no significant decline at the 559 mg/kg
treatment. Thus, the assumed threshold value for total biomass is between 559 and 974
mg/kg for this species in the ARTS series. For Clark Fork, a significant decline in mean
total biomass occurred when total metal and arsenic concentration reached 1900 mg/kg,
but there was no significant decline at the 1334 mg/kg treatment. Thus, the assumed
threshold value for total biomass is between 1334 and 1900 mg/kg for this species in the
Clark Fork series. Both series had moderate to strong r values for the relationship
between total biomass and a gradation of total metals and arsenic, ARTS having an r
value of -0.79 (p=0.00) and an r value in Clark Fork of -0.92 (p=0.00). These values
indicate that total biomass and a gradation of total metals and arsenic are moderately and
negatively correlated in ARTS and strongly and negatively correlated in Clark Fork for
this species.
79
CLARK FORK
ARTS
p = 0.00
r = -0.79 (p=0.00)
120
90
60
30
0
Assumed threshold
occurs between
these concentrations
(84% reduction)
*
*
*
*
*
244
559 974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
150
Total biomass (mg)
Total biomass (mg)
150
120
90
60
p = 0.00
r = -0.92 (p=0.00)
Assumed threshold
occurs between
these concentrations
* (71% reduction)
30
0
*
*
*
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 19. Boxplots of sheep fescue total biomass in ARTS (left) and Clark Fork (right).
Boxplots of root mass ratio grouped by dilution series for sheep fescue are shown
in Figure 20. For ARTS, no significant differences were observed, even in 100% test
soil, indicating threshold values of total metals and arsenic may exceed the most
concentrated treatment for this response. In Clark Fork, a significant decrease occurred
when total metals and arsenic reached 7521 mg/kg, but there was no significant decrease
at the 5885 mg/kg treatment. Thus, the assumed threshold value for root mass ratio is
between 5885 and 7521 mg/kg. These data indicate that sheep fescue produced the same
amount of below ground biomass relative to above ground biomass for ARTS but
decreased this ratio in the Clark Fork as the gradient of total metals and arsenic increased.
Rationale for this is addressed in the Discussion Chapter. Both series had weak r values
for the relationship between root mass ratio and a gradation of total metals and arsenic;
ARTS having an r value of 0.16 (p=0.39) and an r value in the Clark Fork of -0.43
(p=0.01). These values indicate that for the Clark Fork series, root mass ratio and a
gradation of total metals and arsenic is weakly and negatively correlated, and this result is
significant. With the ARTS series, there is not enough evidence to reject the null
80
hypothesis that a significant correlation other than zero exists between root mass ratio
and total metals and arsenic.
CLARK FORK
ARTS
1.0
p = 0.84
r = 0.16 (p=0.39)
0.8
Root mass ratio
Root mass ratio
1.0
0.6
0.4
0.2
0.0
p = 0.03
r = -0.43 (p=0.01)
0.8
0.6
*
0.4
0.2
0.0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
Assumed threshold occurs between
these concentrations (41% reduction)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 20. Boxplots of sheep fescue root mass ratio in ARTS (left) and Clark Fork (right).
Redtop
Table 10 shows percent emergence (%) for redtop grouped by dilution series. No
significant differences in mean percent emergence were observed for either series, even
in 100% test soil treatments, indicating threshold values of total metals and arsenic may
exceed the most concentrated treatments for this response.
Table 10. Mean percent emergence (%) for redtop in ARTS (left) and Clark Fork (right). No significant
differences amongst treatment versus control means were found for either series. C indicates control
soil.
p = 0.17
r = 0.17 (p=0.32)
ARTS
Response
Total metals
p = 0.02
r = 0.23 (p=0.18)
Mean
Number of
percent
replications (n) emergence (%)
CLARK FORK
Response
Total metals
Mean
Treatment
and As
(mg/kg)
Treatment
and As
(mg/kg)
0%C
244
5
84
0%C
251
5
80
6.25%
559
5
100
6.25%
650
5
96
12.50%
974
5
96
12.50%
1334
5
96
25%
1585
5
92
25%
1900
5
100
50%
2908
5
100
50%
3525
5
100
75%
4360
5
96
75%
5885
5
96
100%
5783
5
96
100%
7521
5
96
Number of
percent
replications (n) emergence (%)
81
Both series had weak r values for the relationship between percent emergence and a
gradation of total metals and arsenic; ARTS having an r value of 0.17 (p=0.32) and an r
value in Clark Fork of 0.23 (p=0.18). This indicates that for both series, there is not
enough evidence to reject the null hypothesis that a significant correlation other than zero
exists between percent emergence and total metals and arsenic.
Boxplots of shoot height grouped by dilution series for redtop are shown in Figure
21. No significant differences in mean shoot height were observed for either series, even
in 100% test soil treatments, indicating threshold values of total metals and arsenic may
exceed the most concentrated treatments for this response. Both series had weak r values
for the relationship between shoot height and a gradation of total metals and arsenic;
ARTS having an r value of -0.19 (p=0.27) and an r value in Clark Fork of -0.17 (p=0.34).
These values indicate that for both series, there is not enough evidence to reject the null
hypothesis that a significant correlation other than zero exists between shoot height and
total metals and arsenic.
CLARK FORK
ARTS
Shoot height (cm)
30
p = 0.91
r = -0.19 (p=0.27)
25
20
15
10
5
35
30
Shoot height (cm)
35
p = 0.43
r = -0.17 (p=0.34)
25
20
15
10
5
0
0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 21. Boxplots of redtop shoot height in ARTS (left) and Clark Fork (right). No significant
differences amongst treatment versus control means were found for either series.
82
Figure 22 shows total biomass for redtop grouped by dilution series. For ARTS, a
significant decline in mean total biomass occurred when total metal and arsenic
concentration reached 5783 mg/kg, but there was no significant decline at the 4360
mg/kg treatment. Thus, the assumed threshold value for total biomass is between 4360
and 5783 mg/kg for this species in the ARTS series. For Clark Fork, a significant decline
in mean total biomass occurred when total metal and arsenic concentration reached 5885
mg/kg, but there was no significant decline at the 3525 mg/kg treatment. Thus, the
assumed threshold value for total biomass is between 3525 and 5885 mg/kg for this
species in the Clark Fork series. Both series had moderate r values for the relationship
between total biomass and a gradation of total metals and arsenic; ARTS having an r
value of -0.58 (p=0.00) and an r value in Clark Fork of -0.77 (p=0.00). These values
indicate that total biomass and a gradation of total metals and arsenic are moderately and
negatively correlated for both series for this species.
ARTS
CLARK FORK
180
180
Total biomass (mg)
150
90
30
*
120
120
60
p = 0.00
r = -0.77 (p=0.00)
150
Total biomass (mg)
p = 0.01
r = -0.58 (p=0.00)
*
Assumed threshold occurs
between these concentrations
(53% reduction)
90
60
30
*
Assumed threshold
occurs between
these concentrations
(35% reduction)
0
0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 22. Boxplots of redtop total biomass in ARTS (left) and Clark Fork (right).
83
Boxplots of root mass ratio grouped by dilution series for redtop are shown in
Figure 23. For ARTS, the 4360 mg/kg treatment was the only treatment with a
significantly lower mean from the control. Thus, determining an assumed threshold
value of total metals and arsenic where significant differences occur is difficult because
the next treatment above 4360 mg/kg was not significant. In Clark Fork, no significant
differences were observed, even in 100% test soil, indicating threshold values of total
metals and arsenic may exceed the most concentrated treatment for this response. These
data indicate that redtop produced the same amount of below ground biomass relative to
above ground biomass for both series as the gradient of total metals and arsenic
increased, however this ratio was increased in the 4360 mg/kg treatment in ARTS.
Rationale for this is addressed in the Discussion chapter. Weak r values for the
relationship between root mass ratio and a gradation of total metals and arsenic were
found for both series; ARTS having an r value of -0.32 (p=0.06) and an r value in the
Clark Fork of -0.12 (p=0.51). These values indicate that for both series, there is not
enough evidence to reject the null hypothesis that a significant correlation other than zero
exists between root mass ratio and total metals and arsenic. Though there was a
significant increase in root mass ratio at the 4360 mg/kg treatment, significant differences
between treatment and control means above or below this concentration were not found.
84
CLARK FORK
ARTS
1.0
p = 0.01
r = -0.32 (p=0.06)
0.8
0.6
0.4
*
0.2
0.0
Root mass ratio
Root mass ratio
1.0
p = 0.25
r = -0.12 (p=0.51)
0.8
0.6
0.4
0.2
0.0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentation (mg/kg)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 23. Boxplots of redtop root mass ratio in ARTS (left) and Clark Fork (right).
Slender Wheatgrass
Table 11 shows percent emergence (%) for slender wheatgrass grouped by
dilution series. For ARTS, a significant decline in mean percent emergence occurred
when total metal and arsenic concentration reached 5783 mg/kg, but there was no
significant decline at the 4360 mg/kg treatment. Thus, one would deduce that the
assumed threshold value for percent emergence is between 4360 and 5783 mg/kg for this
species in the ARTS series. However, looking at Table 11 closely shows the control soil
had relatively poor overall emergence (36%) compared with the average emergence
across all treatments (72%, not displayed in Table 11). In fact, the next treatment in this
dilution series (559 mg/kg) had better percent emergence (76%) than the control. This
result was due to two tubes with very low emergence (20%) and a third tube with zero
emergence in the control.
85
Table 11. Mean percent emergence (%) for slender wheatgrass in ARTS (left) and Clark Fork
(right). C indicates control soil.
p = 0.08
r = 0.34 (p=0.05)
ARTS
CLARK FORK
p = 0.00
Response
Total metals
r = -0.75 (p=0.00)
Mean
Number of
percent
replications (n) emergence (%)
Response
Total metals
Mean
Treatment
and As
(mg/kg)
Treatment
(mg/kg)
0%C
244
5
36
0%C
251
5
92
6.25%
559
5
76
6.25%
650
5
96
12.50%
974
5
76
12.50%
1334
5
88
25%
1585
5
76
25%
1900
5
76
50%
2908
5
76
50%
3525
5
64
75%
4360
5
76
75%
5885
5
24*
100%
5783
5
88*
100%
7521
5
32*
and As
Number of
percent
replications (n) emergence (%)
Emergence in the control was not only poor, but delayed by about 7 days for this species
in the ARTS series. Thus, the germinants in the control were effectively 7 days younger
than those germinants in all other treatments. This is addressed in the Discussion
Chapter. (The effect of younger germinants is also evident in the results of total biomass
displayed in Figure 25). Because of this, it may make sense to perform ANOVA and
Dunnett’s method using the next closest “control-like” treatment (559 mg/kg) to detect
any significant differences amongst treatments. When the 559 mg/kg treatment is used as
this “pseudo-control”, the only significantly different mean is from the 244 mg/kg
treatment. Thus, the ANOVA results first reported above that the 5783 mg/kg
concentration may represent a threshold value is not likely. Rather, if the control soil had
produced emergence similar to all other trials in this research, slender wheatgrass would
have a likely assumed threshold value that exceeds the highest total metal concentration
for the ARTS series (5783 mg/kg).
86
There was no delayed or poor emergence observed in the control soil for this
species in the Clark Fork series. The first significant decline in mean percent emergence
occurred at 5885 mg/kg, but there was no significant decline at the 3525 mg/kg treatment.
Thus, the assumed threshold value for percent emergence is between 3525 and 5885
mg/kg for this species in the Clark Fork series. The r value for ARTS was weak, 0.34
(p=0.05) but strong for Clark Fork (r=-0.75, p=0.00), indicating percent emergence and a
gradation of total metals and arsenic is strongly and negatively correlated in Clark Fork
for this species. However, in the ARTS series, there is not enough evidence to reject the
null hypothesis that a significant correlation other than zero exists between percent
emergence and total metals and arsenic.
Boxplots of shoot height grouped by dilution series for slender wheatgrass are
shown in Figure 24. Despite the delayed and poor emergence in the control soil for the
ARTS series, shoot height was able to “catch up” to the next closest treatment (559
mg/kg). For ARTS, a significant decline in mean shoot height occurred when total metal
and arsenic concentration reached 1585 mg/kg, but there was no significant decline at the
974 mg/kg treatment. Thus, the assumed threshold value for shoot height is between 974
and 1585 mg/kg for this species in the ARTS series. For Clark Fork, a significant decline
in mean shoot height occurred when total metal and arsenic concentration reached 5885
mg/kg, but there was no significant decline at the 3525 mg/kg treatment. Thus, the
assumed threshold value for shoot height is between 3525 and 5885 mg/kg for this
species in the Clark Fork series. Both series had moderate to strong r values for the
relationship between shoot height and a gradation of total metals and arsenic; ARTS
87
having an r value of -0.70 (p=0.00) and an r value in Clark Fork of -0.87 (p=0.00). These
values indicate that shoot height and a gradation of total metals and arsenic are
moderately and negatively correlated in ARTS and strongly and negatively correlated in
Clark Fork for this species.
ARTS
Shoot height (cm)
25
*
20
p = 0.00
r = -0.70 (p=0.00)
*
*
*
15
Assumed threshold
occurs between
these concentrations
(28% reduction)
10
5
30
25
Shoot height (cm)
30
CLARK FORK
p = 0.00
r = -0.87 (p=0.00)
20
*
*
15
10
5
Assumed threshold
occurs between
these concentrations
(52% reduction)
0
0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 24. Boxplots of slender wheatgrass shoot height in ARTS (left) and Clark Fork (right).
Figure 25 shows total biomass for slender wheatgrass grouped by dilution series.
As mentioned above, because germination in the negative control was delayed by about
seven days, these germinants were not able to catch up to the ARTS 559 mg/kg treatment
by the conclusion of the 31 day trial, although they did catch up in height. Thus, the 559
mg/kg treatment showed a significant increase in mean total biomass versus the control,
and the 1585 mg/kg treatment on up also showed significant declines compared to the
control. No significant difference was detected with the 974 mg/kg treatment. Because
of this, it is difficult to assign a threshold value of total metals and arsenic where
significant differences occur. It may make sense to assign the threshold value near the
treatment level where continuous differences occur above that level (i.e., 1585 mg/kg).
Thus, the assumed threshold value for total biomass may be between 974 and 1585
88
mg/kg for the ARTS series. Interpretations of these results are addressed in the
Discussion chapter. For Clark Fork-where no delayed or poor emergence occurred-a
significant decline in mean total biomass occurred when total metal and arsenic
concentration reached 1900 mg/kg, but there was no significant decline at the 1334
mg/kg treatment. Thus, the assumed threshold value for total biomass is between 1334
and 1900 mg/kg for this species in the Clark Fork series. Both series had moderate to
strong r values for the relationship between total biomass and a gradation of total metals
and arsenic; ARTS having an r value of -0.63(p=0.00) and an r value in Clark Fork of
-0.87 (p=0.00). These values indicate that total biomass and a gradation of total metals
and arsenic are moderately and negatively correlated in ARTS and strongly and
negatively correlated in Clark Fork for this species.
80
60
ARTS
* Assumed threshold occurs between
CLARK FORK
100
these concentrations (58% reduction)
and significant result is continuous above
1585 mg/kg (559 mg/kg is
also significant)
40
*
*
*
*
p = 0.00
0 r = -0.63 (p=0.00)
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
20
Total biomass (mg)
Total biomass (mg)
100
p = 0.00
r = -0.87 (p=0.00)
80
60
40
20
0
*
Assumed threshold
occurs between
these concentrations
(44% reduction)
*
*
*
250 650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 25. Boxplots of slender wheatgrass total biomass in ARTS (left) and Clark Fork (right).
Boxplots of root mass ratio grouped by dilution series for slender wheatgrass are
shown in Figure 26. For ARTS, a significant increase in this ratio occurred when total
metals and arsenic reached 4360 mg/kg, but no increase was detected at 2908 or 5783
mg/kg. Thus, assigning a threshold value of total metals and arsenic where significant
89
results occur is difficult. A very low value (possible outlier) was observed in the 5783
mg/kg treatment which prevented normality assumptions to be met for one-way ANOVA.
With the outlier removed from the data set (liberal approach), normality is met and the
first significant increase occurs at the 2908 mg/kg treatment on up. With the outlier
preserved in the dataset (conservative approach), the only significant increase occurs with
the 4360 mg/kg treatment. For Clark Fork, a significant increase in root mass ratio
occurred when total metals and arsenic reached 5885 mg/kg, but there was no significant
increase at the 3525 mg/kg treatment. Thus, the assumed threshold value for root mass
ratio is between 3525 and 5885 mg/kg for this species in the Clark Fork series.
ARTS
Root mass ratio
p = 0.05
r = 0.49 (p=0.00)
*
0.4
0.2
0.8
Root mass ratio
Outlier is preserved
in this graph. If outlier
is removed, assumed
threshold occurs at
0.6
2908 mg/kg
0.8
CLARK FORK
1.0
1.0
244
559
974 1585 2908 4360 5783
Total metal and arsenic concentration (mg/kg)
*
*
0.6
0.4
0.2
0.0
0.0
p = 0.00
r = 0.75 (p=0.00)
Assumed threshold occurs
between these concentrations
(38% increase)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 26. Boxplots of slender wheatgrass root mass ratio for ARTS (left) and Clark Fork (right).
These data indicate that slender wheatgrass produced more below ground biomass
relative to above ground biomass in ARTS 4360 mg/kg if the outlier is preserved. If the
outlier is removed, root mass ratio increases continuously above 2908 mg/kg. Slender
wheatgrass produced more below ground biomass relative to above ground biomass for
the Clark Fork as the gradient of total metals and arsenic increased. Rationale for this is
addressed in the Discussion chapter. Both series had weak to moderate r values for the
90
relationship between root mass ratio and a gradation of total metals and arsenic; ARTS
having an r value of 0.49 (p=0.00) and an r value in Clark Fork of 0.75 (p=0.00). These
values indicate that root mass ratio and a gradation of total metals and arsenic are weakly
and positively correlated in ARTS and moderately and positively correlated in Clark Fork
for this species.
Tufted Hairgrass
Table 12 shows percent emergence (%) for tufted hairgrass grouped by dilution
series. The ARTS series had only the first 3 treatments (i.e. 559, 974, 1585 mg/kg) and
the control (244 mg/kg) analyzed because mortality occurred in the higher treatment
levels. This mortality was believed to be from a short absence of supervision of the
plants where water was limited. This affected the higher treatments which had less
organic matter (Table 5) to hold water when water was limiting. Rather than not report
any of the ARTS tufted hairgrass results, it is hoped that by including the first 3
treatments, one might still determine an assumed threshold level and/or evaluate the
performance of this species, possibly making inferences about how tufted hairgrass might
perform in higher treatments. The Clark Fork series did not experience mortality and all
7 treatments are shown in the response graphs that follow. No significant differences in
mean percent emergence were observed for either series, indicating threshold values of
total metals and arsenic may exceed the most concentrated treatments for this response.
Both series had weak r values; ARTS having an r value of 0.09 (p=0.72), and an r value
in the Clark Fork of 0.16 (p=0.36). This indicates that for both series, there is not enough
91
evidence to reject the null hypothesis that a significant correlation other than zero exists
between percent emergence and total metals and arsenic.
Table 12. Mean percent emergence (%) for tufted hairgrass in ARTS (left) and Clark Fork (right).
No significant differences amongst treatment versus control means were found for either series. C
indicates control soil.
p = 0.44
r = 0.09 (p=0.72)
ARTS
Response
Total metals
p = 0.51
r = 0.16 (p=0.36)
CLARK FORK
Mean
Total metals
percent
Response
Mean
Treatment
and As
(mg/kg)
Treatment
and As
(mg/kg)
0%C
244
5
96
0%C
251
5
88
6.25%
559
5
84
6.25%
650
5
88
12.50%
974
5
88
12.50%
1334
5
80
25%
1585
5
96
25%
1900
5
88
50%
3525
5
76
75%
5885
5
88
100%
7521
5
96
Number of
replications (n) emergence (%)
Number of
percent
replications (n) emergence (%)
Boxplots of shoot height grouped by dilution series for tufted hairgrass are shown
in Figure 27. For ARTS, a significant decline in mean shoot height occurred when total
metal and arsenic concentration reached 974 mg/kg, but there was no significant decline
at the 559 mg/kg treatment. Thus, the assumed threshold value for shoot height is
between 559 and 974 mg/kg for this species in the ARTS series. For Clark Fork, a
significant decline in mean shoot height occurred when total metal and arsenic
concentration reached 3525 mg/kg, but there was no significant decline at the 1900
mg/kg treatment. Thus, the assumed threshold value for shoot height is between 1900 and
3525 mg/kg for this species in the Clark Fork series. Both series had moderate to strong r
values for the relationship between shoot height and a gradation of total metals and
92
arsenic; ARTS having an r value of -0.71 (p=0.00) and an r value in Clark Fork of
-0.88 (p=0.00). These values indicate that shoot height and a gradation of total metals
and arsenic are moderately and negatively correlated in ARTS and strongly and
negatively correlated in Clark Fork for this species.
CLARK FORK
ARTS
p = 0.00
r = -0.71 (p=0.00)
*
*
Shoot height (cm)
12
10
8
6
4
2
Assumed threshold
occurs between
these concentrations
(44% reduction)
0
14
p = 0.00
r = -0.88 (p=0.00)
12
Shoot height (cm)
14
10
8
*
6
4
2
Assumed threshold occurs
between these concentrations
(59% reduction)
*
*
0
244
559
974
1585
Total metal and arsenic concentration (mg/kg)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 27. Boxplots of tufted hairgrass shoot height for ARTS (left) and Clark Fork (right).
Figure 28 shows total biomass for tufted hairgrass grouped by dilution series. For
ARTS, a significant decline in mean total biomass occurred when total metal and arsenic
concentration reached 974 mg/kg, but there was no significant decline at the 559 mg/kg
treatment. Thus, the assumed threshold value for total biomass is between 559 and 974
mg/kg for this species in the ARTS series. For Clark Fork, a significant decline in mean
total biomass occurred when total metal and arsenic concentration reached 1900 mg/kg,
but there was no significant decline at the 1334 mg/kg treatment. Thus, the assumed
threshold value for total biomass is between 1334 and 1900 mg/kg for this species in the
Clark Fork series. Both series had strong r values for the relationship between total
biomass and a gradation of total metals and arsenic; ARTS having an r value of -0.81
(p=0.00) and an r value in Clark Fork of -0.90 (p=0.00). These values indicate that total
93
biomass and a gradation of total metals and arsenic are strongly and negatively correlated
for both series for this species.
CLARK FORK
ARTS
100
75
50
25
125
p = 0.00
r = -0.81 (p=0.00)
Assumed threshold occurs
between these concentrations
(83% reduction)
*
*
Total biomass (mg)
Total biomass (mg)
125
100
75
50
p = 0.00
r = -0.90 (p=0.00)
Assumed threshold occurs
between these concentrations
(71% reduction)
*
25
*
*
0
0
244
559
974
1585
Total metal and arsenic concentration (mg/kg)
*
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 28. Boxplots of tufted hairgrass total biomass for ARTS (left) and Clark Fork (right).
Boxplots of root mass ratio grouped by dilution series for tufted hairgrass are
shown in Figure 29. No significant differences in this ratio were observed for either
series, indicating threshold values of total metals and arsenic may exceed the most
concentrated treatments for this response. These data indicate that tufted hairgrass
produced the same amount of below ground biomass relative to above ground biomass
for both series as the gradient of total metals and arsenic increased. Rationale for this is
addressed in the Discussion chapter. Both series had weak r values for the relationship
between root mass ratio and a gradation of total metals and arsenic; ARTS having an r
value of 0.41 (p=0.07) and an r value in Clark Fork of -0.46 (p=0.01). These values
indicate that root mass ratio and a gradation of total metals and arsenic are weakly and
negatively correlated for Clark Fork, and this result is significant. However, in the ARTS
series, there is not enough evidence to reject the null hypothesis that a significant
correlation other than zero exists between root mass ratio and total metals and arsenic.
94
ARTS
Root mass ratio
0.8
CLARK FORK
1.0
p = 0.04
r = 0.41 (p=0.07)
0.6
0.4
p = 0.17
r = -0.46 (p=0.01)
0.8
Root mass ratio
1.0
0.6
0.4
0.2
0.2
0.0
0.0
244
559
974
1585
Total metal and arsenic concentration (mg/kg)
250
650 1334 1900 3525 5885 7521
Total metal and arsenic concentration (mg/kg)
Figure 29. Boxplots of tufted hairgrass root mass ratio for ARTS (left) and Clark Fork (right). No
significant differences amongst treatment versus control means were found for either series.
Interaction Plots
Following the ANOVA, interaction plots were used to compare the two dilution
series on the same graph for shoot height and total biomass (the most significant
responses). On these plots, the thresholds determined previously are marked with arrows.
In interpreting the plots, one considers the relative change in mean response across the
main factor (total metals and arsenic), and compares this change for the two levels of the
2nd factor (ARTS or Clark Fork dilution series). If the change is similar for both series,
the lines in the interaction plot will be close to parallel and indicate no interaction
between the two factors. That is, the effect of total metals and arsenic on plant growth
does not depend on whether the plants are growing in the phytostabilized upland ARTS
or riparian Clark Fork substrate. This has important implications for determining if other
soil parameters (Table 5), could impact any of the plant responses, or if the type of
substrate or habitat (i.e., upland or riparian), impacts plant growth across the gradient of
COCs. For shoot height (Figure 30), all species had similar overall pattern of decreasing
shoot height as total metals and arsenic increased for both dilution series, except redtop.
95
BASIN WILDRYE
BLUEBUNCH WHEATGRASS
30
ARTS
1585 mg/kg
25
Mean shoot height (cm)
Mean shoot height (cm)
30
20
15
10
5
CLARK FORK
1900 mg/kg
0
0
1000
2000
25
CLARK FORK
1900 mg/kg
20
15
10
ARTS
559 mg/kg
5
0
3000
4000
5000
6000
7000
0
8000
1000
2000
BIG BLUEGRASS
Mean shoot height (cm)
Mean shoot height (cm)
5000
6000
7000
8000
7000
8000
30
25
20
CLARK FORK
1900 mg/kg
15
10
5 ARTS
559 mg/kg
0
1000
25
CLARK FORK
1900 mg/kg
20
15
10
ARTS
974 mg/kg
5
0
2000
3000
4000
5000
6000
7000
8000
0
1000
Total metals and arsenic (mg/kg)
2000
3000
4000
5000
6000
Total metals and arsenic (mg/kg)
REDTOP
30
SLENDER WHEATGRASS
30
Mean shoot height (cm)
Mean shoot height (cm)
4000
SHEEP FESCUE
30
0
3000
Total metals and arsenic (mg/kg)
Total metals and arsenic (mg/kg)
25
20
15
10 No significant
5 differences in
either series
0
25
CLARK FORK
5885 mg/kg
20
15
10
ARTS
5 1585 mg/kg
0
0
1000
2000
3000
4000
5000
6000
7000
8000
0
1000
Total metals and arsenic (mg/kg)
2000
3000
4000
5000
6000
7000
8000
Total metals and arsenic (mg/kg)
TUFTED HAIRGRASS
Mean shoot height (cm)
30
25
20
ARTS
974 mg/kg
15
10
CLARK FORK
3525 mg/kg
5
0
0
1000
2000
3000
4000
5000
6000
7000
8000
Total metals and arsenic (mg/kg)
Figure 30. Interaction plots showing mean shoot height separated by dilution series for all 7 species
tested. ARTS=black circles. Clark Fork=red squares.
96
The lines in the interaction plots for big bluegrass, bluebunch wheatgrass, sheep fescue
and redtop all appear approximately parallel. Though this is the overall pattern, there are
nonparallel areas from about 250-1900 mg/kg in big bluegrass, 250-2000 mg/kg in
bluebunch wheatgrass and 240-650 mg/kg in sheep fescue. These patterns indicate that
overall, there is not an interaction between the gradation of total metals and arsenic and
the dilution series so mean shoot height does not depend on which series the plants are
grown in, except where the pattern is nonparallel. With the other three species (basin
wildrye, slender wheatgrass and tufted hairgrass), the overall patterns appear to be mostly
nonparallel, indicating there may be dependence between total metals and arsenic and the
dilution series for mean shoot height. For basin wildrye, the slopes of the lines only
appear parallel between about 1000-1300 mg/kg and 2500-3500 mg/kg. For slender
wheatgrass, the plots appear parallel only from about 250-650 mg/kg, 1300-1900 mg/kg
and 3000-3500 mg/kg. For tufted hairgrass, only four data points exist for ARTS, and
though the values of height are similar, the slopes are not parallel in this range of metals
and arsenic. For the three species just listed, the overall nonparallel pattern indicates
mean shoot height depends on the dilution series the plants are grown in, as the gradient
of total metals and arsenic increases. With the dilution series plotted together, threshold
values can be easily compared.
For total biomass (Figure 31), all seven species have similar overall pattern of
decreasing mean total biomass as total metals and arsenic increased for both dilution
series. The only significant exception to this is slender wheatgrass where the overall
negative slope does not begin until after 559 mg/kg (ARTS only). For interactions,
97
BLUEBUNCH WHEATGRASS
BASIN WILDRYE
140
120
Mean total biomass (mg)
Mean total biomass (mg)
140
ARTS
974 mg/kg
100
80
CLARK FORK
1334 mg/kg
60
40
20
120
100
80
60
ARTS
559 mg/kg
CLARK FORK
1900 mg/kg
40
20
0
0
0
1000
2000
3000
4000
5000
6000
7000
0
8000
1000
2000
Total metals and arsenic (mg/kg)
BIG BLUEGRASS
Mean total biomass (mg)
Mean total biomass (mg/kg)
CLARK FORK
650 mg/kg
100
80
60
ARTS
1585 mg/kg
40
20
5000
6000
7000
8000
7000
8000
120
100
ARTS
974 mg/kg
80
60
CLARK FORK
1900 mg/kg
40
20
0
0
0
1000
2000 3000 4000 5000 6000
Total metal and arsenic (mg/kg)
7000
0
8000
1000
2000
3000
5000
6000
SLENDER WHEATGRASS
REDTOP
140
Mean total biomass (mg)
CLARK FORK
5783 mg/kg
120
100
80
60
40
ARTS
5885 mg/kg
20
4000
Total metals and arsenic (mg/kg)
140
Mean total biomass (mg)
4000
SHEEP FESCUE
140
140
120
3000
Total metals and arsenic (mg/kg)
0
120
100
ARTS
1585 mg/kg
80
For ARTS, the 559 mg/kg
treatment was also significant,
but not 974 mg/kg
CLARK FORK
1900 mg/kg
60
40
20
0
0
1000
2000
3000
4000
5000
6000
7000
8000
0
1000
Total metals and arsenic (mg/kg)
3000
4000
5000
6000
7000
8000
TUFTED HAIRGRASS
140
Mean total biomass (mg)
2000
Total metals and arsenic (mg/kg)
120
100
ARTS
974 mg/kg
80
60
CLARK FORK
1900 mg/kg
40
20
0
0
1000
2000
3000
4000
5000
6000
7000
8000
Total metals and arsenic (mg/kg)
Figure 31. Interaction plots showing mean total biomass separated by dilution series for all 7 species tested.
ARTS=black circles. Clark Fork=red squares.
98
basin wildrye and bluebunch wheatgrass appear to have approximately parallel lines,
indicating little interaction of total metals and arsenic with dilution series, though
bluebunch wheatgrass has nonparallel portions between about 250-650 & 1000-2000
mg/kg. The patterns are more complex for big bluegrass, sheep fescue, redtop, slender
wheatgrass and tufted hairgrass. Big bluegrass appears to have approximately parallel
slopes except about 1300-3500 mg/kg. For sheep fescue, the slopes of the lines are
approximately parallel between 650-1300 mg/kg, and above 3500 mg/kg. For redtop,
approximately parallel slopes exist from about 550 to 900 mg/kg, 1300-1600 mg/kg and
above 3000 mg/kg. For slender wheatgrass, approximately parallel slopes exist between
about 1300 to 3500 mg/kg, but above and below these levels the slopes of the lines do not
appear parallel. With tufted hairgrass, the plots appear approximately parallel below 650
mg/kg, but no comparative ARTS data exists above 1585 mg/kg. For the five species just
listed, the plots indicate mean total biomass across increasing total metals and arsenic
may depend on the dilution series.
These interaction plots show that assumed threshold values for shoot height and
total biomass always occur earlier on the metals and arsenic gradient for ARTS than for
Clark Fork except for total biomass with big bluegrass and redtop. Best-fit lines to the
data were plotted for a sub-set of the species (Analyses not shown). The slopes of the
lines for shoot height were similar and a linear model seemed appropriate. For total
biomass, best-fit models appeared to be functions other than linear models, but even then,
the patterns of these regressed models were very similar for both dilution series. This
99
indicates that shoot height and total biomass decrease similarly for each unit change in
total metals and arsenic, and this rate of growth decline does not depend on which site the
plants are growing in.
Root Growth Observations and Descriptions
At harvest, the author developed a qualitative rating system to describe the root
systems of the seven species growing in each of the treatments of the two dilution series,
however, this was not a blind categorical ranking. This descriptive system was
developed after the harvest of the first trial on slender wheatgrass in which it was
observed that differences in root bushiness appeared to occur as metal and arsenic
concentrations increased, especially compared to the control and potting mix soils. There
was a need to qualify the amount of lateral roots branching off the main first order roots,
and the presence and amount of root hairs that could be seen under a dissecting
microscope. Figure 32 below summarizes the root systems by category for all plant
species except slender wheatgrass. Replications within treatments had variability in root
quality, but assignment to a category was made based on the root quality exhibited by the
majority of the five replications. Lateral roots are defined here as fine, small roots that
grow off of any of the main first order roots that contribute to the look of the roots being
“bushy” and “branched”. These could also be described as secondary or tertiary roots.
Root hairs were defined as any root-like structure that could not be seen with the naked
eye, and needed a magnification of at least 10X on a dissecting microscope to be seen.
100
Redtop
Big bluegrass
Basin wildrye
Branched---
Bluebunch wheatgrass
Sheep fescue
Tufted hairgrass
100
Category
Moderately--branched
Linear---
Stunted---
244 A
250 CF
559 A
650 CF
974 A
1334 CF
1585 A
1900 CF
2908 A
3525 CF
4360 A
5783 CF
5885 A
7521 CF
Total metals and arsenic (mg/kg)
Figure 32. Root quality by category of six species across total metals and arsenic (both dilution series). Categories are Branched, Moderately Branched,
Linear and Stunted (defined in main text). Letters of A or CF following the metal and arsenic levels on the x axis refer to ARTS or Clark Fork dilution
series, respectively.
101
The categories and descriptions were as follows: 1) Branched: Long roots, very
branched and bushy throughout length, lateral roots numerous, root hairs long and
stringy, and easily viewed at 10X, roots near crown supple; 2) Moderately Branched:
Long roots, moderate branching and bushiness but not as much as “Branched” category,
lateral roots numerous but not as numerous as “Branched” category, root hairs shortened
from “Branched” category, but easily viewable at 10X, roots near crown supple; 3)
Linear: Mostly shorter roots than “Moderately Branched” category with very reduced
branching along length; if roots remained long, only one main root existed with
branching restricted to near crown, very few lateral roots except near soil surface, root
hairs short and sparse, and not easily viewable until 30X, roots near crown less supple
than “Branched” or “Moderately Branched” categories; and 4) Stunted: Very short roots
without lengthening to fill tube, branching was restricted to very near crown and soil
surface, roots appeared compact and dense, though branching appeared to be of
thickened, first order roots instead of fine lateral roots, root hairs short and sparse, and
not easily viewable until 30X, entire root system not supple (coarse).
In general, plants appeared more branched and bushy at lower total metal and
arsenic levels and more shortened, stunted and less bushy at higher levels. This pattern
was not evident with Redtop. Lower total metal and arsenic treatments had more fine
roots bearing numerous root hairs than was observed with the higher metal and arsenic
treatments. Fine roots and root hairs were very sparse and more difficult to view by
microscopy under the highest metal and arsenic treatments. Roots in the highest metal
and arsenic treatments felt coarse to the touch, and appeared coarse under the
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microscope, as opposed to supple and soft. Roots in the “Stunted” category were very
small, and appeared like a mass of thickened first order roots and were found right under
the soil surface. More generalizations about the root quality observations of Figure 32
are as follows: When total metal and arsenic levels were 244 to 250 mg/kg (control
values), there were no differences in root observations amongst the species. From 559 to
650 mg/kg, all species maintained at least the “Moderately Branched” category. At 974
mg/kg (ARTS series), the first of the “Linear” roots appeared in bluebunch wheatgrass
and tufted hairgrass. At 1585 mg/kg (ARTS series), the first of the “Stunted” roots are
observed with tufted hairgrass. At 1900 mg/kg (Clark Fork series), all species except
redtop and big bluegrass fall in the “Linear” category. At the 3525 mg/kg level (Clark
Fork series), all species are “Stunted” except for redtop, big bluegrass and sheep fescue.
When total metals and arsenic reached and exceeded 4360 mg/kg, all species except
redtop exhibited “Stunted” roots. It should be noted that in three cases (bluebunch
wheatgrass, tufted hairgrass and sheep fescue), a higher root quality rating was assigned
for the roots growing in the Clark Fork dilution series versus ARTS when there were
similar total metal and arsenic levels (e.g., sheep fescue in 2908 mg/kg ARTS treatment
versus 3525 mg/kg Clark Fork treatment). This result is addressed in the Discussion
Chapter.
Seed Bank/Seed Source Species
During some of the greenhouse trials, seeds germinated that were volunteers from
the seed bank or were unintentionally planted because the seed source could not
guarantee 100% purity. Recall that species were thinned to one plant/tube after 70%
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germination was reached in the 100% dilution soil controls. This usually occurred by day
10, and the plantlets at this stage were very young and appeared morphologically
identical. Because the young plantlets were so morphologically similar by leaf and
ligule, it was possible to accidentally thin out all the intentionally planted seeds, leaving
behind a volunteer or unintentionally planted species. Unfortunately, it wasn’t until the
plantlets were older and had developed morphological differences that it was realized the
wrong species had been thinned. Percent emergence was adjusted for tubes where this
occurred. In these few cases, these unknown species were set apart from the regular
greenhouse trials and grown for many months to produce flowers for taxonomic
identification. Some unknown species were grown for over two years in an attempt to
produce flowers. Only one species was identified with this method, cheatgrass (Bromus
tectorum L.). Tentative identifications of the other unknown species were made with ITS
ribosomal DNA sequencing. Table 13 below summarizes the species identified, the
method of identification and the total metal and arsenic levels that these species had
emerged and were growing in.
These identifications lend more information about species capable of germinating
and growing in lime-amended contaminated waste, and corroborate that of other authors
(Literature Review) and the findings of this study. That is, Redtop and other introduced
grasses like Bromes are known to colonize phytostabilized sites and can emerge and
grow in elevated COCs and the native grass Basin wildrye can do the same. Though two
of the four seedbank species below are introduced species, remedial decision-makers and
managers may find this seedbank information useful. For example, these species could
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add canopy cover and at least short-term stabilization to contaminated sites in the
UCFRB, and could be included in vegetation performance standards.
Table 13. Identifications of volunteers or unintentionally planted species, method of
identification and total metal and arsenic levels involved.
Plant species identified
Method of
Total metal and As levels
identification
(mg/kg)
Bromus tectorum L.
Flowers
1585
(Cheatgrass)
Bromus carinatus Hook & Arn.
(California brome)
ITS ribosomal
DNA sequencing
974
Elymus cinereus Scribn. & Merr.
(Basin wildrye)
ITS ribosomal
DNA sequencing
4360
Agrostis stolonifera L
(Creeping bentgrass)
ITS ribosomal
DNA sequencing
5783
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SUMMARY AND DISCUSSION
An important objective of this research was to determine the threshold
concentration of total metals and arsenic at which significant differences in responses
occurred for each species. A comparison of species’ performance across the two
different dilution series was also important, as the two dilution series represent different
types of reclaimed environments (i.e. riparian versus upland). A related objective was to
rank each species in terms of its relative sensitivity to increasing total metal and arsenic
concentrations in lime-amended waste and compare sensitivities amongst species. Table
14 below summarizes the results of the first two objectives. This table shows the
variability in thresholds between species, between response variables, and between
dilution series as total metals and arsenic increase.
Table 14. Thresholds of total metal and arsenic concentrations (mg/kg) where first significant
differences occurred by species, response variable and dilution series. T (for Tolerance)=No significant
difference detected. A=trial where only control plus next 3 treatments were used because of mortality in
upper treatments. B=Threshold value is questionable because of delayed germination in negative
control (see Results). C=Metal and arsenic level where all treatments above were also significant (see
Results). D=First significant metal and arsenic level, however, the next treatment level (5885 mg/kg)
was not significant.
Response variable by series
SPECIES
Sheep
Basin
Bluebunch
Big
wildrye
wheatgrass
bluegrass
fescue
Redtop
hairgrassA
Tufted
wheatgrass
Slender
B
Percent Emergence
ARTS
Clark Fork
T
7521
T
3525
T
T
T
T
T
T
T
T
5783
5885
Shoot height
ARTS
Clark Fork
1585
1900
559
1900
559
1900
974
1900
T
T
974
3525
1585
5885
Total biomass
ARTS
Clark Fork
974
1334
559
1900
1585
650
974
1900
5783
5885
974
1900
1585C
1900
Root mass ratio
ARTS
Clark Fork
4360
7521
T
7521
T
T
T
7521
4360
T
T
T
4360
5885
D
D
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Table 15 further summarizes the information in Table 10, in order to rank the
species by dilution series and by response variable to accomplish the final objective. It is
noted that ranking the seven species in an overall, cumulative way is impossible because
thresholds where significant differences occurred varied according to dilution series and
response variable measured.
Table 15. Ranking of species’ performance by dilution series and response variable thresholds. Species
to left of > symbol are more tolerant of total metals and arsenic than those to right of the symbol.
= between species indicates significant results occurred at the same total metal and arsenic threshold
for these species, and they rank the same (Grass names are shortened to first names only).
ARTS:
Percent emergence
Shoot height
Total biomass
Root: total biomass
Only significant difference was Slender at 5783 mg/kg
Redtop>Basin=Slender>Sheep=Tufted>Big=Bluebunch
Redtop>Big=Slender>Sheep=Basin=Tufted>Bluebunch
All non-significant except Slender=Basin=Redtop
Clark Fork:
Percent emergence
Shoot height
Total biomass
Root:total biomass
All non-significant except Basin>Slender>Bluebunch
Redtop>Slender>Tufted>Basin=Sheep=Big=Bluebunch
Redtop>Slender=Sheep=Tufted=Bluebunch>Basin>Big
All non-significant except Basin=Bluebunch=Sheep fescue>Slender
Overall, the following generalizations about the trials and species’ performances can be
made: 1) redtop outperformed all other species; 2) bluebunch wheatgrass consistently
performed most poorly or tied for poorest performance in several responses for both
series; 3) slender wheatgrass performed second best or tied for second best to redtop for
both series for shoot height and total biomass; 4) redtop and big bluegrass had the most
lateral root branching & root hairs (Figure 32) as metals and arsenic increased; 5) percent
emergence was the least significant response, often not showing significant differences
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between treatment and control means for several species; 6) root mass ratio was highly
variable amongst species and often not significant or significant only at high total metal
and arsenic levels; 7) significant differences in responses occurred mostly between 559
and 1900 mg/kg for shoot height and total biomass; 8) shoot height and total biomass for
all species (except shoot height for redtop) decreased as total metals and arsenic
increased; 9) interaction plots showed both dilution series on the same graph for shoot
height and total biomass; where threshold levels were higher for Clark Fork over ARTS
for most species, though the patterns of interactions between total COCs and dilution
series varied for each species as COCs increased; and 10) sensitivity of the response
variables appears to be percent emergence<root mass ratio <shoot height<total biomass
where percent emergence showed the fewest significant results and total biomass showed
the most significant results. Making further generalizations are too difficult because of
the varied nature of the responses according to species tested and dilution series used.
Difficultly in assigning tolerance or sensitivity has occurred before (Pashke &
Redente, 2002; Pashke et al., 2000). Here, the authors ranked species’ tolerance only for
the results of 50% biomass reductions, though many measurements were made and linked
to threshold concentrations. The species’ tolerance ranks were as follows for Cu
thresholds:
Redtop>big bluegrass>tufted hairgrass>basin wildrye>common wheat.
For Zn thresholds, species’ tolerance rankings followed the order:
Basin wildrye> slender wheatgrass>redtop>big bluegrass>tufted hairgrass.
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As seen above, it is clear that making clear-cut tolerance rankings amongst the species
varies according to contaminant. In mine wastes with mixed contaminants, such as the
UCFRB, the rankings of species’ tolerances may be even more complex. Despite this
difficulty, the native grasses slender wheatgrass, basin wildrye and big bluegrass may be
good candidates for revegetation on phytostabilized sites in the UCFRB based on this
study, and previous work by the RRU and Pashke. All three species have a DATR
ecotype available including one not used in this study, “Opportunity Germplasm Nevada
big bluegrass”. Redtop clearly is tolerant of elevated COCs, but is rarely used in
revegetation seed mixes in the UCFRB, as it is ubiquitous in the Basin and colonizes
many habitat types, including untreated mine waste.
Classic symptoms of metal toxicity are chlorosis, necrosis, and stunting of leaves,
roots & reductions in productivity (Pahlsson, 1989; Morel, 1997). Throughout this study,
no macroscopic symptoms of plant toxicity were observed except for visibly smaller
shoots, and after harvest, visibly smaller, less branched roots in treatments with higher
COCs. It is common to have no visual symptoms of plant toxicity, yet identify
significant growth reductions or high tissue concentrations of contaminants (Morel, 1997,
Conesa et al., 2009; Kapustka, 2002). Overall mortality was very low in the study,
though not for tufted hairgrass in the ARTS series, believed to be due to watering regime.
Low mortality in these rangeland grasses under increased soluble Cu and Zn
concentrations has been observed before (Paschke & Redente, 2002; Pashke et al., 2000).
Emergence was the least sensitive response and previous phytotoxicity studies
have shown this same result (RRU, 1996; Kapustka et al, 1995; Kapustka, 2002; Neuman
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review of ARCO, 2005). For slender wheatgrass, delayed emergence in the control is the
only reason the threshold of 5783 mg/kg was estimated, based on the one-way ANOVA.
If one can assume slender wheatgrass does not normally perform so poorly in straight
German Gulch soil (control), then no species in the ARTS series would have experienced
significant differences in emergence. It is unclear why the control for Slender wheatgrass
had such poor and slow emergence. This was the first trial performed and the first tubes
to be prepped for planting. Seeds in this treatment could have been planted too deeply or
with too much tamping of the soil surface. Emergence in the potting mix soil was 96%
and not delayed, indicating the poor results in the negative control may have been due to
human error. In unpublished work by Neuman, this same ecotype of slender wheatgrass
had 96% germination in control soils of a greenhouse experiment performed in 2008 (D.
Neuman, personal communication, February 26, 2009). Though significant results for
emergence were few, higher COCs treatments of Clark Fork produced significant results
more than ARTS treatments with similar COCs. This may be due to the higher salt
content (EC) in the upper treatments of the Clark Fork series. Salinity has been shown to
reduce germination in other studies (Neid & Biesboer, 2004; Fenner & Thompson, 2005).
Root growth is known to be a sensitive indicator of trace element phytotoxicity
and has been found to be the most sensitive endpoint in many phytotoxicity studies of
metals (Kapustka et al., 1995; Kapustka, 2002; Pashke & Redente, 2002; Rader et al.,
1997). Plants have the ability to change the size of their root system relative to the shoot
system (allocation of carbon to roots instead of shoots) when nutrients are limiting
(Lambers, Chapin & Pons, 1998). Conversely, decreasing the root system relative to the
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shoot system is known to occur under elevated concentrations of metals (Ryser &
Emerson, 2007). This has been called “allocation plasticity.” Root mass ratio is a
measure of this plasticity in plant growth. Root mass ratio is root biomass divided by
total biomass for the plant. Because values can only be between 0 and 1.0, it is an
interpretable measure of whether roots are making up less, equal to or more biomass than
shoots (e.g. 0.5 is equal root biomass to shoot biomass).
Results of root mass ratio in this study were highly variable. Significant
differences occurred between 4360 and 7521 mg/kg COCs, but the pattern of allocation
was not consistent. For example, basin wildrye and slender wheatgrass increased root
mass ratio in both series as COCs increased, but sheep fescue decreased the ratio in Clark
Fork series as COCs increased. Root mass ratio was expected to decrease as COCs
increased, as a way for plants to concentrate more energy in shoot growth and decrease
surface area of roots in response to COC stress. Because major macronutrients are not
believed to be limiting in this study (discussed later), an explanation for these results is
that they are an artifact of extremely small plant size in the higher COCs treatments. A
very small plant has less room to develop size differences in roots versus shoots, this
compared to a large plant. An alternate explanation for increased root mass ratio is that if
roots are avoiding one area inside a growth tube, they could increase their mass building
new roots to explore other parts of the tube. Roots are dynamic organs and have been
known to “avoid” contaminated parts of soil profiles (Kapustka, 2002; Hagemeyer &
Breckle, 2002)
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Root length (of the longest root) was also measured in this study, but it was nearly
impossible to not cut some of the roots during harvest, so the accuracy of this
measurement is questionable. Further, size of growth container is known to affect root
length (Bohm, 1979). Observations were instead made on root branching and root hairs.
Development of lateral roots and root hairs are ways that plants can increase surface area
to absorb or access more nutrients (Hagemeyer & Breckle, 2002) and lateral roots are
related to a plant’s adaptability in its environment, plant size and productivity (Lloret &
Casero, 2002). Root development depends on soil nutrients, temperature, precipitation
and the genetic make-up of the plant (Munshower, l994). In general, branching and
development of fine lateral roots and root hairs decreased as COCs increased, though in
the highest treatments, roots appeared thickened, and appeared dense and branched but
extremely small, right under the soil surface (“Stunted” category). This branching
appeared to be only thickened first order roots, as opposed to the fine, supple roots that
were second or third order observed in control or low COC soils. Studies have shown that
root hair density decreases under metals stress (Hagemeyer & Breckle, 2002). Lateral
root development has been found to increase (Hagemeyer & Breckle, 2002) or decrease
(Foy, Chaney & White, 1979; Hagemeyer & Breckle, 2002; Pahlsson, 1989; Ryser &
Emerson, 2007) under metals stress, depending on concentration and plant species.
When lateral root development increases under increasing metal levels, the overall root
system appears more compact and dense (Hagemeyer & Breckle, 2002). Root toxicity of
trace elements has been described as “coralloid” (coral-like) or resembling barbed wire
(Kabata-Pendias, 2001) and thickening of roots have been described (Ryser & Emerson,
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2007). Roots in the highest metal concentrations (“Stunted” category) of this study
appeared dense, with thickened branching, and were coralloid-shaped. Overall, roots
growing in lower concentrated treatments appeared to be more branched in Clark Fork
versus ARTS, when COCs concentrations were similar, though this only occurred in
three cases. This could be due to increased nitrates in the Clark Fork over ARTS, known
to increase lateral root branching (Lloret & Casero, 2002). This could also be a result of
only having four root description categories, and a lack of quantitative data like counts of
roots or root hairs making category assignment somewhat subjective.
The majority of thresholds of COCs for shoot height and total biomass fell
between 559 and 1900 mg/kg. This is consistent with the review of an ARCO report by
Keammerer & Redente, where Neuman (2005) found thresholds of total COCs that
reduced plant height and biomass were at or below 1461 mg/kg in lime-amended UCFRB
soils. For both shoot height and total biomass, significant reductions for all species
occurred at higher thresholds in Clark Fork versus ARTS, except for total biomass in Big
Bluegrass. This can be easily viewed on the interaction plots. The most likely
explanation for this is that the higher organic matter content in the Clark Fork soil helped
to adsorb and/or chelate metal cations, making them less bioavailable. Interaction plots
of these responses did not show any clear trends of how increasing total COCs depends
on dilution series, because many portions of the graphs showed a parallel pattern, while
other areas showed a non-parallel pattern. When a sub-set of shoot height and total
biomass data for the two dilution series were regressed to determine a best-fit line
(analysis not shown), overall patterns were very similar, indicating plant growth
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decreases in a similarly manner across a COC gradient irrespective of the dilution series.
Although the interaction plots often showed higher mean height or biomass in the first or
second treatment with elevated COCs up from the control, total biomass in ARTS 6.25%
for slender wheatgrass was the only significant increase. Although delayed emergence
occurred in the control for this species, it is possible that small amounts of metals and
arsenic can stimulate plant growth and this is why the 6.25% treatment was significantly
higher. Tice (1995) found that 7 mg/L arsenic (V) increased mean shoot biomass of
basin wildrye over control levels.
Though thresholds have been estimated for a variety of plant assays, the majority
of them are identified for soluble metals (mg/L). Few studies try to define total soil metal
thresholds, mostly because total soil metals have had poor correlation with plant metal
uptake and only a fraction of the total soil metals (that in solution and easily put into
solution) are considered bioavailable (Bolan et al., 2008; Gupkta, Vollmer & Krebs,
1996). However, positive relationships between total soil metals and soluble metals exist
(Krazklewski & Pietrozykowski, 2002; Conesa et al., 2009; Pedersen & Elmegaard,
2000). Further, elevated levels of soluble Zn were found in reclaimed field soils (pH 5.9
to 7.1) at the Comet Mine, Jefferson County, Montana, despite the geochemical models
which suggest Zn solubility should be low at this pH (Tafi, 2006). Warne et al., (2008, in
review of many papers) reports soluble or extractable metals were not useful predictors of
soil phytotoxicity, and added total metals to their models of phytotoxicity. Total soil
metals cannot be ignored, and should be included in phytotoxicity models, as they define
the extent of the contamination and indicate the potential release into the soil solution if
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the soil reaches its exchangeable capacity, or due to insoluble fractions of elements being
mobilized by biota (Gupta et al., 1996; Mench et al, 2006).
Levels of N, P and K in all treatments of both dilution series are considered
acceptable for range or agricultural production (C. Jones, personal communication,
October 19, 2005). Critical levels for P and K are 16 and 250 mg/kg, respectively (C.
Jones, 2005). According to a soil analysis guide, P values in both series are considered
“very high”, while K values are considered “high” to very high” in both series (Energy
Laboratories, n.d.). Values that could be detrimental to plant growth are 60 mg/kg and
above for P and 700-1000 mg/kg for K (C. Jones, 2005). Given this, the only treatment
in both dilution series that may contribute to reduced plant growth should be the Clark
Fork 100% which contains 756 mg/kg K, however the Ca deficiency that can occur when
K levels are very high would not likely occur at this concentration (C. Jones, 2005).
Critical levels for nitrogen are not defined values, but are based on different factors such
as plant species, and irrigation practices. For rangeland or agricultural species, the nitrate
levels in all treatments of the dilution series for this study are considered adequate (C.
Jones, 2005), though they fall in the “very low” category for ARTS and range from “very
low to low” for Clark Fork (Energy Laboratories, n.d.). For Clark Fork, though nitrogen
ranges from very low to low, the high values are in the 100% Clark Fork treatment, while
the lowest values are in the Little Blackfoot River control. Thus, reductions in growth
from low nitrogen would occur in the lowest nitrogen treatment (Little Blackfoot River
control), if they occur at all. If nitrogen was limiting in this study, then Clark Fork 6.25
or 12.5% treatments containing elevated nitrogen relative to the control should show
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increased growth. Significant increases in growth did not occur in the Clark Fork 6.25 or
12.5% versus the control. Therefore, any growth limitations due to nitrogen are unlikely.
EC increases as test soil concentration increases in both dilution series for reasons
mentioned in the Literature Review Chapter. This is particularly true for the Clark Fork
dilution series, though EC levels are not considered saline (Richmond, 2000; Munshower,
1994), and native grasses are known to tolerate soil conditions with much higher EC
values (Munshower, 1994). Higher EC values in upper treatments of the Clark Fork
series may account for significant emergence reductions where COC levels are similar
between ARTS and Clark Fork, as discussed previously.
Organic Matter is higher in Clark Fork test soil over Little Blackfoot River
reference soil and lower in ARTS test soil over German Gulch reference soil. High
organic matter content in the Clark Fork soil versus the Little Blackfoot soil may be due
to decreased microbial activity from poor soil structure and excess water holding capacity
or high C:N ratios of the organic matter. It could also be due to the cultivation of
nitrogen-fixing alfalfa at this soil collection site. Nitrogen-fixing plants are known to
load carbon into soils (Cole, Compton, Edmonds, Homann & Van Miegroet, 1995).
Lower organic matter content in ARTS soil versus German Gulch soil is likely due to
lower plant production on the ARTS site which is linked to organic matter accumulation.
Organic matter is known to be a strong sink for metal cations, therefore the increase in
organic matter is likely to lower metal availability in higher COC treatments of the Clark
Fork series. With the ARTS series, the reduction in organic matter in higher COCs
treatments could decrease adsorption capacity of the soil for cations and reduce plant
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performance, though even in 100% ARTS, organic matter approaches that normally
found in upper horizons of soils, 3-5% (Johnson et al., 1994).
Determining metal and arsenic phytotoxicity thresholds is difficult due to soil
physical and chemical factors such as clay and organic matter and biological factors, such
as rhizosphere activity due to microorganisms and plant root chemicals. Soil type
influences phytotoxicity and threshold determinations. A study of 12 Australian soils
with soluble Cu and Zn applications showed total soil thresholds varied by 7- to 330-fold
and 18- to 160-fold for Cu and Zn, respectively, after the metals solutions equilibrated for
8 weeks (Warne et al., 2008). It is no wonder that toxicity threshold studies use sand
(relatively inert chemically) or water and single contaminants in order to minimize these
confounding factors. However, these studies are unrealistic of environmental settings
where mixed contaminants are the norm and toxicity in co-contaminated sites can be
vastly different from single contaminants and often more severe (Megharaj & Naidu,
2008). Ecological risk assessments on co-contaminated sites in the UCFRB should not
be performed with greenhouse assays performed in sand, though admittedly, as Megharaj
& Naidu (2008) said: “No one single assay is adequate and a suite of tests are necessary
to accurately identify the risk caused by contaminants.” The current study is not without
its own drawbacks, and represents just one type of test that should be performed in
UCFRB phytostabilized settings to determine risk to plants and ecosystems. Some
drawbacks are the size of the tubes, short term growth of species and the sieving, airdrying & homogenization of the contaminated soils. Some benefits of this study design
are the use of real soils (known to mediate metal toxicity), use of dilution soils from
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within the UCFRB watershed with similar soil mineralogy, and the use of rangeland
grasses and metals-adapted ecotypes.
The current study is only an approximation of what could occur in lime-amended
field settings in the UCFRB. Ideally, this study would be replicated in the field, though it
may be difficult to perform large-scale field mixing to create a dilution of COCs. Several
authors have reported that heightened effects of contaminants have occurred in
greenhouse relative to field conditions (Fletcher, Johnson & McFarlane; 1988; Conesa,
Robinson, Schulin & Nowack, 2007). Whether this is true for these greenhouse studies,
estimates of phytotoxicity thresholds here represent conservative or “worst-case” values
that can be thought of as a safety margin as opposed to a liberal over-estimation of
thresholds where ecological risk could be underestimated. Further, field conditions
present a host of factors which could distort threshold determinations, such as
competition with other plants and environmental stress such as drought.
What is considered vegetation “success” on reclaimed sites varies widely,
depending on the type of disturbance, habitat (e.g., upland or riparian), post remedial land
use, the responsible party or regulatory agency, and who is making the judgment.
Different objectives guiding revegetation objectives are evident. Munshower (1994)
states that the objectives driving revegetation efforts on disturbed sites are erosion control
and forage for animals. Others cite goals such as “return the land to a productive use”
and “achieve an aesthetically pleasing landscape” (Richmond, 2000). Still others seem to
approach revegetation in more of an ecological restoration framework citing goals such
as “sustainable plant communities representative of the composition and diversity of the
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surrounding natural plant communities” (Jefferson, 2004) and the use of native reference
areas as a standard by which successful reclamation is evaluated (Chambers, Brown &
Williams, 1994). In an Operable Unit of the AS Superfund site and in the CF Operable
Unit of the CFR, the RODs define successful reclamation as the following:
“…establishment of self-perpetuating plant communities capable of stabilizing
contaminated soils against wind and water erosion, reducing COCs transport to
groundwater, reducing the risk to human health and the environment, and
compliance with ARARS 9, in perpetuity” (U.S. EPA & MDEQ, 1998; U.S.
EPA & MDEQ, 2004).
In the CF Operable Unit, vegetation performance standards to assess the
effectiveness of remedial actions are site-specific and defined by the post-remedial land
use (U.S. EPA & MDEQ, 2004). For example, agricultural cropland in this operable unit
meets vegetation performance standards if production is statistically equivalent to County
averages for the crop of interest. Conversely, agricultural areas designated as upland
grazing must have a minimum of 45% live cover, and support five species/100 m2.
“Invasive” species do not count towards species richness, and are defined in the ROD as
known invaders such as spotted knapweed and thistle, yet no mention seems to be made
of introduced grasses that are invasive like redtop or brome. In the riparian zone of the
CF operable unit, the ROD defines vegetation performance standards based on cover
alone.
It is understandable that the short term goals in the UCFRB are to stabilize soils
and reduce risk to humans and animals as opposed to goals of restoring a diverse
assemblage of native species similar to reference areas. It is also understandable that
9
ARARS stands for “Applicable or Relevant and Appropriate Requirements” and is terminology used in
the AS ROD. “Performance Standards” replaces ARARS in the definition of “successful reclamation” in
the CF Operable Unit ROD.
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there is a lack of long-term plant monitoring data because risk reduction takes precedence
over plant community development. This is a function of the different objectives of the
government agencies involved. For example, the U.S. EPA is a risk management agency,
while others such as the Bureau of Land Management, Montana Fish Wildlife and Parks,
National Park Service, U.S. Forest Service and U.S. Fish and Wildlife Service are land
management agencies. The lack of long-term plant community data is also a function of
the relatively small geologic time scale during which phytostabilization has been
implemented. All of this reduces our understanding of how vegetation develops on
phytostabilized sites. It is possible that these sites are simply too compromised with
elevated COCs to support the ecosystems that existed before they were disturbed.
(Though data at Keatings and McClaren do not support this—See Literature Review).
Evidence from this study supports the idea that reduced plant biomass of
rangeland grasses may occur on phytostabilized soils from the UCFRB, and therefore
impact these already compromised ecosystems, even when total COCs are well below
10,000 mg/kg. The U.S. EPA model for plant community development as a function of
pH and total COCs is given below with the results of the current study included (Table
33).
120
Figure 33. U.S. EPA model of plant community effects due to total COCs and pH,
with these parameters for the current study marked with a red box.
At pH and total COC levels in this study, the model states that “No effects” to biomass or
species richness should occur. The current study suggests that while this model may be
accurate for loss to species richness because of nonsignificant emergence results for most
species, loss of biomass is not accurate, and occurred for all seven grass species.
Therefore, as phytostabilization becomes more commonplace in the UCFRB, plant
community development should be monitored and continued impacts of elevated COCs
should be used to improve models of site-specific plant community development. As
phytostabilization is chosen as the remedial action for future acreages in the UCFRB,
knowledge of which plant species are capable of surviving, persisting and colonizing
these areas will be important for evaluating vegetation standards and post-remedial land
use goals.
121
FUTURE DIRECTION
During this study, several hypotheses came up as to why phytostabilized soils in
the UCFRB often support limited plant species, and why plant growth is affected by total
metals when pH is near neutral. Some of them are as follows:
1) Biogeochemistry trumps geochemistry, and provides a more complete explanation
for reduced plant growth. Plant roots and microbial activity modify the chemistry
in the rhizosphere, particularly root exudates that can chelate trace elements near
roots or acidify regions near the root, increasing metal solubility. Microbes can
impact redox potential and impact trace element chemistry.
2) Arsenic, known to be more bioavailable at higher pH values, is related to plant
growth declines, more than metal cations. Anion exchange capacities of soils are
generally lower than cation capacities, which may also affect arsenic
bioavailability. Therefore, reducing arsenic bioavailability on neutral or alkaline
phytostabilized sites needs to be addressed.
3) Dissolved organic matter--a known chelator of metal cations--is known to
increase at higher pH values, and in response to liming. These chelates are water
soluble, and if they reach the roots and if the molecules are small enough, they
can be taken up by plant roots, increasing bioavailability and potentially,
phytotoxicity.
122
4) Incorporation or mixing of the lime amendments was not adequate, so plant roots
encounter pockets in the soils that have high soluble levels of metals. Or, if
mixing is adequate, tiny microsites in soil remain acidic, and harm plant roots via
high soluble metals in these areas.
5) When cations are in high concentrations in soils, Ca can be an effective
competitor for exchange surfaces, releasing metal cations into soil solution, which
can increase trace element bioavailability and potential phytotoxicity.
6) In high contaminant soils, the cation and/or anion exchange sites reaches capacity,
causing trace elements to be pushed into solution, increasing bioavailability and
potential phytotoxicity.
7) Nutrient limitations, such as low nitrogen or high Cu or Zn-induced Fe
deficiencies are influencing plant growth in ways not encountered on other
degraded sites that are not metalliferous.
In addition to a large scale, long-term plant community development monitoring
program of phytostabilized sites in the U.S., experiments should be set up to address the
hypotheses given above. It is critical to understand why treating these sites with soil
amendments and lime and seeding does not always work, and why only certain plants
seem to succeed despite the suggestions of geochemical models. Focus should also be on
biological components that relate to improved microbial activity and assist plant
recovery. It is possible that this dilution series method could be used as a screening tool
for risk assessment and remedy selection in the UCFRB. Improving the “transfer factor”
123
from greenhouse to field settings for ecological risk assessments of field soils should
occur with mixed species studies, large non-root limiting containers and screening of
many site-specific contaminated soils. It is possible that the dilution series methods used
here could be an effective screening tool for risk assessment and remedy selection on
other sites in the UCFRB. Threshold testing for many more rangeland species should
also occur. Identifying native plant species that are desirable for foraging animals, are
non-invasive and accumulate low levels of trace elements in shoots would be ideal. In
addition to evaluating plant growth reductions, identifying tissue concentrations of trace
elements in plant shoots should occur for all site-specific threshold and risk assessment
studies.
124
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139
APPENDICES
140
APPENDIX A
PLANT RESPONSE RAW DATA
141
Table 16. Basin wildrye plant growth data - ARTS dilution series
▲=plant emerged but died during trial
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
244
244
244
244
244
559
559
559
559
559
974
974
974
974
974
1585
1585
1585
1585
1585
2908
2908
2908
2908
2908
4360
4360
4360
4360
4360
5783
5783
5783
5783
5783
Percent emergence
day 10 (%)
80
100
60
40
80
60
60
60
80
80
80
60
60
80
60
60
80
80
40
100
60
80
100
100
40
60
100
60
100
80
0
40
40
0
60
Percent emergence
day 20 (%)
80
100
80
40
80
60
80
60
80
80
80
60
60
80
60
60
80
80
60
100
80
80
100
100
60
60
100
80
100
80
60
80
40
80
80
Total percent
emergence (%)
80
100
80
40
80
60
80
60
80
80
80
60
60
80
60
60
80
80
60
100
80
80
100
100
60
60
100
80
100
80
60
80
40
80
80
142
Table 16. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
31.7
25.6
31.5
20.3
24.5
22.3
17.4
22.1
22.7
26.8
27.8
22.8
21.1
20.1
24.6
20.2
18.1
22.2
22.7
15.6
13.2
14.3
13.9
13.7
12.1
9.6
11.8
14.7
11.8
8.9
4.5
7.1
▲
6.5
6.4
Shoot
biomass (mg)
76.2
59.1
68.9
56.1
55.1
54.1
61.0
54.1
46.5
57.4
44.0
46.4
44.5
33.9
45.9
24.9
24.1
42.6
37.2
17.9
8.7
12.9
22.9
17.5
10.2
6.9
12.3
9.5
10.6
4.8
1.4
4.2
▲
2.9
2.8
Root
biomass (mg)
41.5
29.2
29.7
35.1
41.1
37.3
54.1
48.4
35.3
31.5
40.9
38.6
32.5
24.9
30.4
21.5
9.6
23.3
27.9
16.5
8.7
7.9
7.8
8.2
5.6
8.5
10.6
9.8
11.0
4.6
3.0
5.3
▲
4.6
2.8
Total
biomass (mg)
117.7
88.3
98.6
91.2
96.2
91.4
115.1
102.5
81.8
88.9
84.9
85.0
77.0
58.8
76.3
46.4
33.7
65.9
65.1
34.4
17.4
20.8
30.7
25.7
15.8
15.4
22.9
19.3
21.6
9.4
4.4
9.5
▲
7.5
5.6
Root mass
ratio
0.35
0.33
0.30
0.38
0.43
0.41
0.47
0.47
0.43
0.35
0.48
0.45
0.42
0.42
0.40
0.46
0.28
0.35
0.43
0.48
0.50
0.38
0.25
0.32
0.35
0.55
0.46
0.51
0.51
0.49
0.68
0.56
▲
0.61
0.50
143
Table 17. Basin wildrye plant growth data - Clark Fork dilution series
▲=plant emerged but died during trial
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
250
250
250
250
250
650
650
650
650
650
1334
1334
1334
1334
1334
1900
1900
1900
1900
1900
3525
3525
3525
3525
3525
5885
5885
5885
5885
5885
7521
7521
7521
7521
7521
Percent emergence
day 10 (%)
40
100
80
100
80
60
100
100
80
80
80
60
80
80
60
40
100
100
20
100
0
60
20
0
0
0
0
0
0
0
0
0
0
0
0
Percent emergence
day 20 (%)
60
100
80
100
80
60
100
100
80
80
100
60
80
80
60
60
100
100
100
100
40
100
60
40
80
80
100
60
0
40
40
20
20
20
60
Total percent
emergence (%)
60
100
80
100
80
60
100
100
80
80
100
60
80
80
60
60
100
100
100
100
40
100
60
40
80
80
100
60
0
40
40
20
20
20
60
144
Table 17. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
27.7
▲
21.2
30.2
22.7
26.5
25.7
26.5
25.8
25.3
28.6
21.4
20.3
23.5
19.9
9.3
13.2
11.6
10.8
12.4
7.0
7.3
8.8
4.2
4.5
6.0
6.5
2.9
0.0
7.4
2.2
0.3
1.7
3.5
4.6
Shoot
biomass (mg)
75.3
▲
54.9
68.9
55.7
72.7
54.9
53.9
69.1
61.6
52.8
34.6
49.7
32.9
46.1
6.7
17.3
11.9
10.6
24.8
4.3
2.5
7.6
3.4
1.5
3.6
4.3
1.6
0.0
3.7
1.0
0.0
0.4
0.6
1.8
Root
biomass (mg)
44.3
▲
46.3
48.3
41.3
46.9
30.1
30.7
31.5
37.6
31.3
21.1
30.3
24.1
31.1
8.2
9.1
9.3
5.4
13.8
3.1
1.7
4.5
2.5
2.8
4.7
2.9
1.1
0.0
3.6
1.4
0.9
0.7
1.5
3.5
Total
biomass (mg)
119.6
▲
101.2
117.2
97.0
119.6
85.0
84.6
100.6
99.2
84.1
55.7
80.0
57.0
77.2
14.9
26.4
21.2
16.0
38.6
7.4
4.2
12.1
5.9
4.3
8.3
7.2
2.7
0.0
7.3
2.4
0.9
1.1
2.1
5.3
Root mass
ratio
0.37
▲
0.46
0.41
0.43
0.39
0.35
0.36
0.31
0.38
0.37
0.38
0.38
0.42
0.40
0.55
0.34
0.44
0.34
0.36
0.42
0.40
0.37
0.42
0.65
0.57
0.40
0.41
0.00
0.49
0.58
1.00
0.64
0.71
0.66
145
Table 18. Bluebunch wheatgrass plant growth data - ARTS dilution series
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
244
244
244
244
244
559
559
559
559
559
974
974
974
974
974
1585
1585
1585
1585
1585
2908
2908
2908
2908
2908
4360
4360
4360
4360
4360
5783
5783
5783
5783
5783
Percent emergence
day 10 (%)
60
80
100
80
80
80
80
80
100
80
100
20
80
80
100
80
60
60
80
80
80
100
60
20
60
80
80
80
60
100
100
60
0
0
100
Percent emergence
day 20 (%)
80
80
100
80
80
100
80
80
100
80
100
40
100
80
100
80
60
60
80
80
80
100
100
60
60
80
100
80
60
100
100
60
20
20
100
Total percent
emergence (%)
80
80
100
80
80
100
80
80
100
80
100
40
100
80
100
80
60
60
80
80
80
100
100
60
60
80
100
80
60
100
100
60
20
20
100
146
Table 18. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
25.5
22.5
19.1
20.0
15.5
8.1
18.3
11.5
16.0
13.9
13.5
13.0
12.6
14.0
9.3
18.6
11.6
13.0
14.8
12.8
12.0
11.6
10.6
12.3
13.3
13.1
11.8
9.6
10.8
12.4
10.5
6.2
5.6
6.7
9.0
Shoot
biomass (mg)
18.3
16.2
20.2
15.6
10.0
5.2
11.4
3.1
9.2
9.1
8.8
5.4
5.1
6.6
5.0
12.3
6.7
5.8
9.5
7.1
7.6
6.2
6.5
5.2
6.4
6.7
6.0
6.7
4.3
8.8
6.7
2.0
3.4
0.9
6.8
Root
biomass (mg)
9.8
8.8
11.3
6.6
5.6
3.6
8.0
2.3
5.5
6.2
5.2
4.2
3.6
4.3
2.6
6.2
5.4
4.7
6.0
5.9
4.6
4.2
2.1
5.7
3.7
5.2
4.7
3.3
3.5
4.6
2.9
1.8
1.1
1.4
4.5
Total
biomass (mg)
28.1
25.0
31.5
22.2
15.6
8.8
19.4
5.4
14.7
15.3
14.0
9.6
8.7
10.9
7.6
18.5
12.1
10.5
15.5
13.0
12.2
10.4
8.6
10.9
10.1
11.9
10.7
10.0
7.8
13.4
9.6
3.8
4.5
2.3
11.3
Root mass
ratio
0.35
0.35
0.36
0.30
0.36
0.41
0.41
0.43
0.37
0.41
0.37
0.44
0.41
0.39
0.34
0.34
0.45
0.45
0.39
0.45
0.38
0.40
0.24
0.52
0.37
0.44
0.44
0.33
0.45
0.34
0.30
0.47
0.24
0.61
0.40
147
Table 19. Bluebunch wheatgrass plant growth data - Clark Fork dilution series
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
250
250
250
250
250
650
650
650
650
650
1334
1334
1334
1334
1334
1900
1900
1900
1900
1900
3525
3525
3525
3525
3525
5885
5885
5885
5885
5885
7521
7521
7521
7521
7521
Percent emergence
day 10 (%)
100
100
100
80
100
100
100
80
100
80
100
100
100
80
100
60
100
60
100
80
20
20
40
0
20
0
0
0
0
20
0
0
0
0
0
Percent emergence
day 20 (%)
100
100
100
80
100
100
100
80
100
80
100
100
100
100
100
80
100
60
100
100
40
40
60
0
60
0
0
20
20
20
0
20
0
20
20
Total percent
emergence (%)
100
100
100
80
100
100
100
80
100
80
100
100
100
100
100
80
100
60
100
100
40
40
60
0
60
0
0
20
20
20
0
20
0
20
20
148
Table 19. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
22.7
18.9
26.4
25.1
22.1
19.0
21.5
22.5
26.4
24.1
16.2
22.3
17.0
15.6
20.1
13.7
13.8
12.4
16.0
13.3
8.6
13.5
8.3
0.0
12.6
0.0
0.0
1.7
5.0
12.6
0.0
3.4
0.0
4.1
1.3
Shoot
biomass (mg)
18.6
16.6
23.1
22.6
20.0
11.8
16.1
15.4
23.1
19.0
13.4
17.9
14.7
12.2
12.6
9.8
9.9
5.6
10.6
7.0
4.1
10.9
3.4
0.0
7.2
0.0
0.0
0.3
1.2
6.0
0.0
0.5
0.0
0.3
0.4
Root
biomass (mg)
9.0
10.8
16.1
11.3
10.1
8.2
11.4
7.0
11.0
12.0
9.5
10.9
12.5
10.1
10.0
6.7
5.4
3.8
7.4
3.6
1.9
5.0
1.9
0.0
3.3
0.0
0.0
0.4
1.0
2.3
0.0
1.0
0.0
1.9
2.0
Total
biomass (mg)
27.6
27.4
39.2
33.9
30.1
20.0
27.5
22.4
34.1
31.0
22.9
28.8
27.2
22.3
22.6
16.5
15.3
9.4
18.0
10.6
6.0
15.9
5.3
0.0
10.5
0.0
0.0
0.7
2.2
8.3
0.0
1.5
0.0
2.2
2.4
Root mass
ratio
0.33
0.39
0.41
0.33
0.34
0.41
0.41
0.31
0.32
0.39
0.41
0.38
0.46
0.45
0.44
0.41
0.35
0.40
0.41
0.34
0.32
0.31
0.36
0.00
0.31
0.00
0.00
0.57
0.45
0.28
0.00
0.67
0.00
0.86
0.83
149
Table 20. Big bluegrass plant growth data - ARTS dilution series
w= Seedbank seed emerged, and wrong species was thinned. This is accounted for in
total percent emergence. Where seedbank caused emergence >100%, values were
adjusted back to 100%.
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
244
244
244
244
244
559
559
559
559
559
974
974
974
974
974
1585
1585
1585
1585
1585
2908
2908
2908
2908
2908
4360
4360
4360
4360
4360
5783
5783
5783
5783
5783
Percent emergence
day 10 (%)
100
80
80
80
20
80
80
100
80
60
60
100
100
60
80
80
100
100
100
100
100
100
100
100
60
80
100
100
100
100
100
80
100
100
100
Percent emergence
day 20 (%)
100
100
80
80
60
100
80
100
80
80
80
100
100
60
80
80
100
100
100
100
100
100
100
100
60
80
100
100
100
100
100
80
100
100
100
Total percent
emergence (%)
100
100
80
80
60
100
80
100
80
80
80
100
100
60
80
80
100
100
100
100
100
100
100
100
60
80
100
100
100
100
100
80
100
100
100
150
Table 20. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
16.1
14.3
16.6
12.5
13.7
8.5
11.2
9.2
13.1
11.1
7.0
10.5
9.5
w
12.0
11.5
9.1
8.8
10.1
8.0
5.5
6.7
6.0
8.7
5.6
4.2
6.6
w
3.6
4.4
w
5.8
6.3
5.2
5.2
Shoot
biomass (mg)
64.9
50.0
43.7
14.8
53.3
13.1
45.5
13.9
31.0
19.2
8.1
21.7
14.6
w
23.5
11.8
6.9
12.7
14.6
8.6
2.9
1.6
3.5
7.9
4.0
2.6
4.5
w
3.2
2.5
w
1.9
4.1
3.1
0.6
Root
biomass (mg)
40.9
31.4
31.2
7.0
30.8
10.0
31.9
7.7
25.8
13.9
6.4
17.8
9.3
w
19.0
9.4
5.5
7.1
6.6
11.6
1.3
1.8
1.6
7.6
1.9
0.6
3.4
w
1.6
1.5
w
1.8
2.1
1.1
0.6
Total
biomass (mg)
105.8
81.4
74.9
21.8
84.1
23.1
77.4
21.6
56.8
33.1
14.5
39.5
23.9
w
42.5
21.2
12.4
19.8
21.2
20.2
4.2
3.4
5.1
15.5
5.9
3.2
7.9
w
4.8
4.0
w
3.7
6.2
4.2
1.2
Root mass
ratio
0.39
0.39
0.42
0.32
0.37
0.43
0.41
0.36
0.45
0.42
0.44
0.45
0.39
w
0.45
0.44
0.44
0.36
0.31
0.57
0.31
0.53
0.31
0.49
0.32
0.19
0.43
w
0.33
0.38
w
0.49
0.34
0.26
0.50
151
Table 21. Big bluegrass plant growth data - Clark Fork dilution series
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
250
250
250
250
250
650
650
650
650
650
1334
1334
1334
1334
1334
1900
1900
1900
1900
1900
3525
3525
3525
3525
3525
5885
5885
5885
5885
5885
7521
7521
7521
7521
7521
Percent emergence
day 10 (%)
80
80
80
60
100
80
80
40
80
100
100
100
60
100
80
40
60
60
100
100
100
80
80
80
100
100
60
100
80
40
60
40
80
80
40
Percent emergence
day 20 (%)
100.0
80.0
80.0
60.0
100.0
80.0
80.0
40.0
80.0
100.0
100.0
100.0
60.0
100.0
80.0
40.0
60.0
60.0
100.0
100.0
100.0
80.0
80.0
80.0
100.0
100.0
80.0
100.0
80.0
100.0
100.0
80.0
80.0
100.0
100.0
Total percent
emergence (%)
100.0
80.0
80.0
60.0
100.0
80.0
80.0
40.0
80.0
100.0
100.0
100.0
60.0
100.0
80.0
40.0
60.0
60.0
100.0
100.0
100.0
80.0
80.0
80.0
100.0
100.0
80.0
100.0
80.0
100.0
100.0
80.0
80.0
100.0
100.0
152
Table 21. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
16.9
15.3
15.6
14.9
16.4
9.8
13.1
14.5
11.8
16.5
16.9
11.1
12.9
10.6
18.0
9.9
10.2
10.8
10.1
8.9
6.3
5.5
7.2
7.0
4.0
4.1
4.0
3.3
4.2
3.7
3.5
2.8
4.3
4.5
4.1
Shoot
biomass (mg)
64.6
62.7
62.8
61.8
67.9
41.3
44.0
49.2
34.8
51.4
45.2
24.8
25.3
42.2
41.2
23.6
25.4
19.5
23.3
18.7
4.8
2.6
8.5
7.8
4.9
3.7
3.1
0.7
1.4
1.5
1.5
1.0
2.5
4.5
1.4
Root
biomass (mg)
56.9
42.8
46.1
47.3
53.8
47.0
38.7
36.9
20.9
33.2
36.7
37.6
15.3
21.9
34.0
18.7
18.1
17.1
15.7
16.3
3.4
4.0
4.3
7.2
2.9
2.5
0.9
3.1
1.4
0.7
0.5
0.3
3.3
0.8
0.8
Total
biomass (mg)
121.5
105.5
108.9
109.1
121.7
88.3
82.7
86.1
55.7
84.6
81.9
62.4
40.6
64.1
75.2
42.3
43.5
36.6
39.0
35.0
8.2
6.6
12.8
15.0
7.8
6.2
4.0
3.8
2.8
2.2
2.0
1.3
5.8
5.3
2.2
Root mass
ratio
0.47
0.41
0.42
0.43
0.44
0.53
0.47
0.43
0.38
0.39
0.45
0.60
0.38
0.34
0.45
0.44
0.42
0.47
0.40
0.47
0.41
0.61
0.34
0.48
0.37
0.40
0.23
0.82
0.50
0.32
0.25
0.23
0.57
0.15
0.36
153
Table 22. Sheep Fescue plant growth data - ARTS dilution series
w= Seedbank seed emerged, and wrong species was thinned. This is accounted for in
total percent emergence. Where seedbank caused emergence >100%, values were
adjusted back to 100%.
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
244
244
244
244
244
559
559
559
559
559
974
974
974
974
974
1585
1585
1585
1585
1585
2908
2908
2908
2908
2908
4360
4360
4360
4360
4360
5783
5783
5783
5783
5783
Percent emergence
day 10 (%)
60
60
20
60
60
100
80
100
100
80
100
100
100
80
100
100
80
100
100
80
100
100
80
80
80
100
100
80
100
100
100
60
100
80
80
Percent emergence
day 20 (%)
80
100
80
100
100
100
100
100
100
80
100
100
100
80
100
100
100
100
100
100
100
100
80
100
80
100
100
100
100
100
100
100
100
100
100
Total percent
emergence (%)
80
100
80
100
100
100
100
100
100
80
100
100
100
80
100
100
100
100
100
100
100
100
80
100
80
100
100
100
100
100
100
100
100
100
100
154
Table 22. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
11.7
13.3
10.4
15.3
17.3
13.5
9.1
14.0
13.1
11.4
10.4
9.1
9.3
9.4
7.5
8.7
6.1
5.9
w
w
6.2
w
4.7
4.8
5.5
6.2
3.9
5.9
6.8
6.2
5.5
3.1
w
4.8
4.8
Shoot
biomass (mg)
29.5
38.7
22.6
100.3
55.1
10.2
9.3
46.3
26.7
15.4
12.0
8.0
5.4
4.6
4.4
3.9
3.2
3.7
w
w
0.8
w
0.1
1.5
4.7
3.2
2.6
0.6
2.3
1.8
1.7
0.6
w
0.8
1.9
Root
biomass (mg)
18.8
9.0
11.8
51.4
24.0
4.1
3.6
27.9
16.7
10.2
10.4
4.9
2.8
1.9
1.9
3.5
0.2
2.2
w
w
0.8
w
0.5
0.7
0.2
1.4
1.0
1.0
0.0
0.9
1.0
1.0
w
0.7
0.9
Total
biomass (mg)
48.3
47.7
34.4
151.7
79.1
14.3
12.9
74.2
43.4
25.6
22.4
12.9
8.2
6.5
6.3
7.4
3.4
5.9
w
w
1.6
w
0.6
2.2
4.9
4.6
3.6
1.6
2.3
2.7
2.7
1.6
w
1.5
2.8
Root mass
ratio
0.39
0.19
0.34
0.34
0.30
0.29
0.28
0.38
0.38
0.40
0.46
0.38
0.34
0.29
0.30
0.47
0.06
0.37
w
w
0.50
w
0.83
0.32
0.04
0.30
0.28
0.63
0.00
0.33
0.37
0.63
w
0.47
0.32
155
Table 23. Sheep Fescue plant growth data - Clark Fork dilution series
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
250
250
250
250
250
650
650
650
650
650
1334
1334
1334
1334
1334
1900
1900
1900
1900
1900
3525
3525
3525
3525
3525
5885
5885
5885
5885
5885
7521
7521
7521
7521
7521
Percent emergence
day 10 (%)
100
100
100
80
100
60
100
100
80
80
60
100
100
100
100
80
80
100
100
80
80
80
40
20
100
40
40
20
60
20
20
20
40
0
40
Percent emergence
day 20 (%)
100
100
100
80
100
60
80
100
80
100
80
100
100
100
100
100
100
100
100
100
100
80
80
80
100
80
80
100
80
100
100
80
100
60
80
Total percent
emergence (%)
100
100
100
80
100
60
80
100
80
100
80
100
100
100
100
100
100
100
100
100
100
80
80
80
100
80
80
100
80
100
100
80
100
60
80
156
Table 23. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
15.2
9.6
17.6
13.1
15.2
15.7
17.1
15.4
13.4
19.2
10.6
11.5
13.5
13.0
9.4
10.6
8.8
12.2
6.8
9.3
6.7
6.7
5.2
6.0
6.8
5.6
5.0
5.0
5.1
4.4
3.5
5.0
6.0
5.8
4.6
Shoot
biomass (mg)
51.0
17.4
74.2
33.9
75.0
61.7
70.3
48.0
37.1
43.8
35.8
31.3
38.0
32.1
27.3
19.7
13.0
16.2
9.7
32.2
6.4
5.0
1.6
2.0
4.3
2.6
1.4
3.4
1.8
3.1
0.8
2.3
2.4
2.5
2.0
Root
biomass (mg)
31.2
23.8
49.2
57.3
42.6
35.3
33.0
28.8
14.1
22.3
18.9
19.9
21.1
18.8
20.4
7.9
7.2
8.6
2.4
14.6
1.9
1.7
0.7
2.3
1.7
1.7
0.7
1.2
0.7
2.4
0.8
0.6
0.7
0.6
0.7
Total
biomass (mg)
82.2
41.2
123.4
91.2
117.6
97.0
103.3
76.8
51.2
66.1
54.7
51.2
59.1
50.9
47.7
27.6
20.2
24.8
12.1
46.8
8.3
6.7
2.3
4.3
6.0
4.3
2.1
4.6
2.5
5.5
1.6
2.9
3.1
3.1
2.7
Root mass
ratio
0.38
0.58
0.40
0.63
0.36
0.36
0.32
0.38
0.28
0.34
0.35
0.39
0.36
0.37
0.43
0.29
0.36
0.35
0.20
0.31
0.23
0.25
0.30
0.53
0.28
0.40
0.33
0.26
0.28
0.44
0.50
0.21
0.23
0.19
0.26
157
Table 24. Redtop plant growth data - ARTS dilution series
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
244
244
244
244
244
559
559
559
559
559
974
974
974
974
974
1585
1585
1585
1585
1585
2908
2908
2908
2908
2908
4360
4360
4360
4360
4360
5783
5783
5783
5783
5783
Percent emergence
day 10 (%)
100
80
80
80
80
100
100
100
100
100
100
80
100
100
100
100
100
100
100
60
100
100
100
100
100
100
100
100
80
100
80
100
100
100
100
Percent emergence
day 20 (%)
100
80
80
80
80
100
100
100
100
100
100
80
100
100
100
100
100
100
100
60
100
100
100
100
100
100
100
100
80
100
80
100
100
100
100
Total percent
emergence (%)
100
80
80
80
80
100
100
100
100
100
100
80
100
100
100
100
100
100
100
60
100
100
100
100
100
100
100
100
80
100
80
100
100
100
100
158
Table 24. (continued).
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
24.0
23.3
19.1
22.7
26.0
19.0
23.5
26.9
26.2
25.5
26.0
27.7
15.8
18.8
22.6
28.5
25.0
21.6
17.0
20.3
22.1
19.3
24.0
24.6
18.7
26.1
22.0
19.2
25.4
19.6
16.5
15.9
20.3
23.7
28.5
Shoot
biomass (mg)
79.9
68.7
36.9
82.0
99.6
48.1
52.7
62.7
59.6
66.2
91.3
72.0
63.4
72.8
64.1
64.0
60.1
69.5
38.0
78.7
61.7
61.7
27.2
51.7
73.4
68.9
52.4
64.4
61.1
39.8
25.7
29.2
35.6
49.8
51.8
Root
biomass (mg)
50.7
44.2
34.7
46.8
47.0
24.7
40.2
37.1
29.8
29.9
39.9
28.9
29.8
46.2
42.1
22.2
36.6
39.5
20.9
46.8
62.1
68.0
26.6
20.3
44.4
31.1
20.7
32.9
22.9
11.3
12.7
11.6
13.1
22.1
27.6
Total
biomass (mg)
130.6
112.9
71.6
128.8
146.6
72.8
92.9
99.8
89.4
96.1
131.2
100.9
93.2
119.0
106.2
86.2
96.7
109.0
58.9
125.5
123.8
129.7
53.8
72.0
117.8
100.0
73.1
97.3
84.0
51.1
38.4
40.8
48.7
71.9
79.4
Root mass
ratio
0.39
0.39
0.48
0.36
0.32
0.34
0.43
0.37
0.33
0.31
0.30
0.29
0.32
0.39
0.40
0.26
0.38
0.36
0.35
0.37
0.50
0.52
0.49
0.28
0.38
0.31
0.28
0.34
0.27
0.22
0.33
0.28
0.27
0.31
0.35
159
Table 25. Redtop plant growth data - Clark Fork dilution series
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
250
250
250
250
250
650
650
650
650
650
1334
1334
1334
1334
1334
1900
1900
1900
1900
1900
3525
3525
3525
3525
3525
5885
5885
5885
5885
5885
7521
7521
7521
7521
7521
Percent emergence
day 10 (%)
80
100
80
80
60
80
100
100
80
100
100
100
100
100
80
100
100
100
100
100
100
100
100
100
100
60
100
100
80
100
100
60
100
80
100
Percent emergence
day 20 (%)
80
100
80
80
60
100
100
100
80
100
100
100
100
100
80
100
100
100
100
100
100
100
100
100
100
80
100
100
100
100
100
80
100
100
100
Total percent
emergence (%)
80
100
80
80
60
100
100
100
80
100
100
100
100
100
80
100
100
100
100
100
100
100
100
100
100
80
100
100
100
100
100
80
100
100
100
160
Table 25. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
17.3
18.7
17.7
20.7
19.7
22.5
25.1
20.6
23.1
19.6
18.8
18.3
25.0
23.2
18.4
19.9
21.7
15.0
22.1
27.6
27.7
21.2
25.6
15.5
19.5
19.0
22.4
18.4
20.8
21.7
20.6
20.0
20.0
17.4
14.1
Shoot
biomass (mg)
67.9
63.7
73.3
68.0
62.7
69.4
66.6
96.0
94.2
96.3
71.1
68.5
85.8
78.1
102.0
71.5
81.5
81.4
94.5
84.1
59.4
69.4
59.8
80.8
71.1
40.5
62.2
47.7
25.4
33.6
41.8
50.2
44.2
41.8
24.4
Root
biomass (mg)
42.9
48.7
50.5
49.7
38.3
39.9
41.5
42.2
57.8
58.3
60.5
53.1
66.2
41.9
52.3
36.5
54.4
44.4
64.7
30.5
34.3
45.9
33.9
56.8
34.3
28.8
58.1
44.1
13.9
15.9
15.9
19.3
29.9
23.6
17.0
Total
biomass (mg)
110.8
112.4
123.8
117.7
101.0
109.3
108.1
138.2
152.0
154.6
131.6
121.6
152.0
120.0
154.3
108.0
135.9
125.8
159.2
114.6
93.7
115.3
93.7
137.6
105.4
69.3
120.3
91.8
39.3
49.5
57.7
69.5
74.1
65.4
41.4
Root mass
ratio
0.39
0.43
0.41
0.42
0.38
0.37
0.38
0.31
0.38
0.38
0.46
0.44
0.44
0.35
0.34
0.34
0.40
0.35
0.41
0.27
0.37
0.40
0.36
0.41
0.33
0.42
0.48
0.48
0.35
0.32
0.28
0.28
0.40
0.36
0.41
161
Table 26. Slender wheatgrass plant growth data - ARTS dilution series
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
244
244
244
244
244
559
559
559
559
559
974
974
974
974
974
1585
1585
1585
1585
1585
2908
2908
2908
2908
2908
4360
4360
4360
4360
4360
5783
5783
5783
5783
5783
Percent emergence
day 10 (%)
0
20
0
20
0
100
80
40
80
40
0
40
20
40
100
20
0
0
40
0
20
0
20
0
40
0
0
20
20
0
0
60
20
40
40
Percent emergence
day 20 (%)
0
20
20
20
20
100
80
40
80
40
20
40
20
40
100
20
0
0
40
20
20
20
20
20
40
20
0
20
20
20
0
60
20
40
40
Total percent
emergence (%)
0
60
80
20
20
100
80
60
80
60
40
60
80
100
100
80
60
80
100
60
100
60
80
40
100
40
60
100
100
80
40
100
100
100
100
162
Table 26. (continued).
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
0.0
26.8
22.3
28.2
24.5
26.9
23.3
23.9
27.5
25.3
20.1
20.4
23.1
23.8
24.7
20.3
17.9
13.8
18.7
21.5
16.4
17.7
11.2
19.7
14.3
13.8
13.5
18.3
10.6
17.3
6.3
21.4
9.8
16.3
22.3
Shoot
biomass (mg)
0.0
37.0
17.7
38.0
25.5
44.5
52.2
35.7
60.0
48.7
14.0
28.8
23.6
25.3
42.4
12.0
6.5
5.5
12.8
17.3
5.7
9.4
3.8
11.7
10.2
4.4
5.8
9.5
4.7
11.8
1.4
17.6
4.0
7.9
17.6
Root
Total
biomass (mg) biomass (mg)
0.0
0.0
15.5
52.5
9.6
27.3
15.8
53.8
10.1
35.6
20.6
65.1
21.4
73.6
17.2
52.9
34.8
94.8
22.7
71.4
6.9
20.9
16.3
45.1
14.3
37.9
13.4
38.7
21.1
63.5
7.4
19.4
2.7
9.2
2.6
8.1
12.1
24.9
10.8
28.1
5.5
11.2
6.2
15.6
3.3
7.1
6.7
18.4
7.2
17.4
3.5
7.9
5.2
11.0
8.5
18.0
5.0
9.7
8.8
20.6
0.2
1.6
12.2
29.8
4.7
8.7
10.4
18.3
11.6
29.2
Root mass
ratio
0.00
0.30
0.35
0.29
0.28
0.32
0.29
0.33
0.37
0.32
0.33
0.36
0.38
0.35
0.33
0.38
0.29
0.32
0.49
0.38
0.49
0.40
0.46
0.36
0.41
0.44
0.47
0.47
0.52
0.43
0.13
0.41
0.54
0.57
0.40
163
Table 27. Slender wheatgrass plant growth data - Clark Fork dilution series
▲=plant emerged but died during trial
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
250
250
250
250
250
650
650
650
650
650
1334
1334
1334
1334
1334
1900
1900
1900
1900
1900
3525
3525
3525
3525
3525
5885
5885
5885
5885
5885
7521
7521
7521
7521
7521
Percent emergence
day 10 (%)
100
100
100
40
80
100
100
100
100
60
60
80
80
100
60
20
40
0
40
40
40
20
20
20
20
0
0
0
40
0
0
0
0
0
20
Percent emergence
day 20 (%)
100
100
100
60
100
100
100
100
100
80
60
100
100
100
80
40
80
40
80
80
100
20
20
40
20
0
0
0
60
0
20
0
20
0
20
Total percent
emergence (%)
100
100
100
60
100
100
100
100
100
80
60
100
100
100
80
80
80
40
80
100
100
20
60
100
40
0
20
0
80
20
40
0
20
40
60
164
Table 27. (continued).
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
27.5
26.4
26.2
25.4
25.7
29.0
22.5
24.6
27.2
26.0
▲
23.2
24.9
28.8
▲
25.7
24.6
20.5
23.4
19.3
23.3
23.7
21.5
19.9
21.5
0.0
15.7
0.0
18.2
4.1
4.5
0.0
10.2
10.0
16.9
Shoot
biomass (mg)
44.6
29.8
36.6
28.9
42.4
47.4
47.1
37.3
48.7
43.6
▲
34.8
47.7
45.4
▲
26.3
32.5
18.1
23.7
9.5
25.5
24.1
18.6
15.3
20.3
0.0
5.9
0.0
9.9
0.4
1.0
0.0
5.4
2.5
8.1
Root
biomass (mg)
33.5
24.2
30.2
20.5
27.9
21.9
25.6
27.1
26.6
26.4
▲
22.1
21.1
17.4
▲
16.4
19.9
7.8
18.2
6.2
17.7
17.6
15.2
11.1
14.6
0.0
7.0
0.0
7.3
1.7
3.1
0.0
5.9
7.6
11.6
Total
biomass (mg)
78.1
54.0
66.8
49.4
70.3
69.3
72.7
64.4
75.3
70.0
▲
56.9
68.8
62.8
▲
42.7
52.4
25.9
41.9
15.7
43.2
41.7
33.8
26.4
34.9
0.0
12.9
0.0
17.2
2.1
4.1
0.0
11.3
10.1
19.7
Root mass
ratio
0.43
0.45
0.45
0.41
0.40
0.32
0.35
0.42
0.35
0.38
▲
0.39
0.31
0.28
▲
0.38
0.38
0.30
0.43
0.39
0.41
0.42
0.45
0.42
0.42
0.00
0.54
0.00
0.42
0.81
0.76
0.00
0.52
0.75
0.59
165
Table 28. Tufted hairgrass plant growth data - ARTS dilution series
▲=plant emerged but died during trial
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
244
244
244
244
244
559
559
559
559
559
974
974
974
974
974
1585
1585
1585
1585
1585
2908
2908
2908
2908
2908
4360
4360
4360
4360
4360
5783
5783
5783
5783
5783
Percent emergence
day 10 (%)
80
100
100
100
100
60
100
80
100
80
60
100
100
100
80
100
100
80
100
80
100
100
100
80
100
100
100
100
80
100
100
80
80
100
100
Percent emergence
day 20 (%)
80
100
100
100
100
60
100
80
100
80
60
100
100
100
80
100
100
100
100
80
100
100
100
100
100
100
100
100
100
100
100
80
100
100
100
Total percent
emergence (%)
80
100
100
100
100
60
100
80
100
80
60
100
100
100
80
100
100
100
100
80
100
100
100
100
100
100
100
100
100
100
100
80
100
100
100
166
Table 28. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
10.6
13.0
13.1
11.4
10.4
11.9
11.1
9.6
12.5
13.2
7.6
9.1
9.5
4.4
2.4
6.2
10.2
1.3
5.3
6.8
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
Shoot
biomass (mg)
41.4
44.7
45.8
79.9
48.4
28.0
41.5
23.7
25.0
37.6
11.7
14.5
15.3
3.9
0.4
4.7
21.5
0.1
2.9
7.2
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
Root
biomass (mg)
17.8
16.4
17.8
45.4
16.3
12.2
18.7
15.0
15.1
14.9
5.4
4.5
6.1
2.2
0.0
3.5
6.8
0.2
3.4
5.1
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
Total
biomass (mg)
59.2
61.1
63.6
125.3
64.7
40.2
60.2
38.7
40.1
52.5
17.1
19.0
21.4
6.1
0.4
8.2
28.3
0.3
6.3
12.3
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
Root mass
ratio
0.30
0.27
0.28
0.36
0.25
0.30
0.31
0.39
0.38
0.28
0.32
0.24
0.29
0.36
0.00
0.43
0.24
0.67
0.54
0.41
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
167
Table 29. Tufted hairgrass plant growth data - Clark Fork dilution series
▲=plant emerged but died during trial
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Percent test
soil (%)
0.00
0.00
0.00
0.00
0.00
6.25
6.25
6.25
6.25
6.25
12.50
12.50
12.50
12.50
12.50
25.00
25.00
25.00
25.00
25.00
50.00
50.00
50.00
50.00
50.00
75.00
75.00
75.00
75.00
75.00
100.00
100.00
100.00
100.00
100.00
Total metal and As
concentration (mg/kg)
250
250
250
250
250
650
650
650
650
650
1334
1334
1334
1334
1334
1900
1900
1900
1900
1900
3525
3525
3525
3525
3525
5885
5885
5885
5885
5885
7521
7521
7521
7521
7521
Percent emergence
day 10 (%)
80
100
100
60
100
100
100
80
80
80
60
100
100
80
60
80
80
100
80
100
60
100
80
60
80
100
60
100
100
80
100
100
100
80
100
Percent emergence
day 20 (%)
80
100
100
60
100
100
100
80
80
80
60
100
100
80
60
80
80
100
80
100
60
100
80
60
80
100
60
100
100
80
100
100
100
80
100
Total percent
emergence (%)
80
100
100
60
100
100
100
80
80
80
60
100
100
80
60
80
80
100
80
100
60
100
80
60
80
100
60
100
100
80
100
100
100
80
100
168
Table 29. (continued)
Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shoot
height (cm)
9.8
9.5
11.5
9.6
10.5
14.1
11.6
8.9
▲
12.6
9.0
8.1
7.1
6.4
8.5
11.3
7.9
7.2
10.4
5.6
3.0
5.5
7.0
2.5
3.0
2.4
1.5
1.3
2.5
2.9
2.6
2.6
2.2
1.3
2.3
Shoot
biomass (mg)
59.1
46.0
40.7
58.7
57.7
50.4
40.6
30.9
▲
26.0
32.8
15.6
5.1
38.9
47.5
22.6
11.9
7.6
19.3
12.9
1.9
4.0
5.0
0.7
0.6
2.3
0.7
1.8
1.6
1.9
1.0
2.7
0.3
0.5
0.1
Root
biomass (mg)
28.7
27.4
25.9
35.8
41.7
35.8
20.0
22.1
▲
12.4
21.1
12.4
4.8
27.9
32.5
15.2
5.9
4.8
13.0
10.3
3.4
3.1
1.8
0.2
0.3
0.8
0.3
0.3
1.4
0.8
0.1
0.2
0.0
0.4
0.1
Total
biomass (mg)
87.8
73.4
66.6
94.5
99.4
86.2
60.6
53.0
▲
38.4
53.9
28.0
9.9
66.8
80.0
37.8
17.8
12.4
32.3
23.2
5.3
7.1
6.8
0.9
0.9
3.1
1.0
2.1
3.0
2.7
1.1
2.9
0.3
0.9
0.2
Root mass
ratio
0.33
0.37
0.39
0.38
0.42
0.42
0.33
0.42
▲
0.32
0.39
0.44
0.48
0.42
0.41
0.40
0.33
0.39
0.40
0.44
0.64
0.44
0.26
0.22
0.33
0.26
0.30
0.14
0.47
0.30
0.09
0.07
0.00
0.44
0.50
169
APPENDIX B
STATISTICAL OUTPUT
170
Basin wildrye - ARTS dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 16.39
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 15.21%
SS
1349
7520
8869
Mean
76.00
72.00
68.00
76.00
84.00
84.00
68.00
StDev
21.91
10.95
10.95
16.73
16.73
16.73
17.89
MS
225
269
F
0.84
P
0.552
R-Sq(adj) = 0.00%
Individual 95% CIs For Mean Based on
Pooled StDev
------+---------+---------+---------+--(-----------*------------)
(------------*------------)
(------------*-----------)
(-----------*------------)
(------------*------------)
(------------*------------)
(------------*-----------)
------+---------+---------+---------+--60
72
84
96
Pooled StDev = 16.39
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-32.31
-36.31
-28.31
-20.31
-20.31
-36.31
Center
-4.00
-8.00
0.00
8.00
8.00
-8.00
Upper
24.31
20.31
28.31
36.31
36.31
20.31
--------+---------+---------+---------+(-------------*-------------)
(-------------*-------------)
(-------------*-------------)
(-------------*-------------)
(-------------*-------------)
(-------------*-------------)
--------+---------+---------+---------+-20
0
20
40
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent emergence (%) and Total metals and
arsenic = 0.052
P-Value = 0.767
171
Basin wildrye – ARTS dilution series continued
One-way ANOVA: Shoot height versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 2.975
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
4
R-Sq = 86.37%
DF
6
27
33
Mean
26.720
22.260
23.280
19.760
13.440
11.360
6.125
StDev
4.875
3.334
3.052
2.952
0.847
2.274
1.127
SS
1513.95
238.99
1752.94
MS
252.32
8.85
F
28.51
P
0.000
R-Sq(adj) = 83.34%
Individual 95% CIs For Mean Based on
Pooled StDev
------+---------+---------+---------+--(---*---)
(---*---)
(---*---)
(---*---)
(---*---)
(---*---)
(----*---)
------+---------+---------+---------+--7.0
14.0
21.0
28.0
Pooled StDev = 2.975
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0107
Critical value = 2.74
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-9.618
-8.598
-12.118
-18.438
-20.518
-26.066
Center
-4.460
-3.440
-6.960
-13.280
-15.360
-20.595
Upper
0.698
1.718
-1.802
-8.122
-10.202
-15.124
-------+---------+---------+---------+-(-------*------)
(------*------)
(------*------)
(------*------)
(------*------)
(-------*------)
-------+---------+---------+---------+--21.0
-14.0
-7.0
0.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.910
P-Value = 0.000
172
Basin wildrye – ARTS dilution series continued
One-way ANOVA: Total biomass (mg) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 10.45
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
4
DF
6
27
33
R-Sq = 93.42%
Mean
98.40
95.94
76.40
49.10
22.08
17.72
6.75
StDev
11.52
13.04
10.68
15.80
6.13
5.45
2.23
SS
41883
2951
44834
MS
6980
109
F
63.87
P
0.000
R-Sq(adj) = 91.96%
Individual 95% CIs For Mean Based on
Pooled StDev
-+---------+---------+---------+-------(--*--)
(--*--)
(--*---)
(--*---)
(--*---)
(--*--)
(--*---)
-+---------+---------+---------+-------0
30
60
90
Pooled StDev = 10.45
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0107
Critical value = 2.74
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-20.58
-40.12
-67.42
-94.44
-98.80
-110.87
Center
-2.46
-22.00
-49.30
-76.32
-80.68
-91.65
Upper
15.66
-3.88
-31.18
-58.20
-62.56
-72.43
--+---------+---------+---------+------(----*----)
(----*----)
(----*----)
(----*----)
(----*----)
(-----*----)
--+---------+---------+---------+-------105
-70
-35
0
Correlations: Total biomass (mg), Total metals and arsenic
Pearson correlation of Total biomass (mg) and Total metals and arsenic =
-0.901
P-Value = 0.000
173
Basin wildrye – ARTS dilution series continued
One-way ANOVA: Root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.06238
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
4
DF
6
27
33
SS
0.17934
0.10506
0.28440
R-Sq = 63.06%
Mean
0.35932
0.42724
0.43597
0.40200
0.36148
0.50424
0.58826
MS
0.02989
0.00389
F
7.68
P
0.000
R-Sq(adj) = 54.85%
Individual 95% CIs For Mean Based on Pooled StDev
+---------+---------+---------+--------(-----*-----)
(-----*----)
(-----*----)
(-----*-----)
(-----*-----)
(----*-----)
(------*-----)
+---------+---------+---------+--------0.30
0.40
0.50
0.60
StDev
0.04875
0.04887
0.03234
0.08150
0.09070
0.03258
0.07766
Pooled StDev = 0.06238
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0107
Critical value = 2.74
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-0.04023
-0.03150
-0.06546
-0.10599
0.03678
0.11424
Center
0.06792
0.07665
0.04268
0.00215
0.14492
0.22894
Upper
0.17606
0.18479
0.15083
0.11030
0.25307
0.34365
---------+---------+---------+---------+
(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
(--------*---------)
---------+---------+---------+---------+
0.00
0.12
0.24
0.36
Correlations: Root mass ratio, Total metals and arsenic
Pearson correlation of Root mass ratio and Total metals and arsenic
= 0.616
P-Value = 0.000
174
Basin wildrye – Clark Fork dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 22.80
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 47.05%
Mean
84.00
84.00
76.00
92.00
64.00
56.00
32.00
StDev
16.73
16.73
16.73
17.89
26.08
38.47
17.89
SS
12937
14560
27497
MS
2156
520
F
4.15
P
0.004
R-Sq(adj) = 35.70%
Individual 95% CIs For Mean Based on
Pooled StDev
------+---------+---------+---------+--(------*------)
(------*------)
(------*------)
(------*------)
(------*------)
(------*------)
(------*------)
------+---------+---------+---------+--30
60
90
120
Pooled StDev = 22.80
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-39.39
-47.39
-31.39
-59.39
-67.39
-91.39
Center
0.00
-8.00
8.00
-20.00
-28.00
-52.00
Upper
39.39
31.39
47.39
19.39
11.39
-12.61
------+---------+---------+---------+--(----------*----------)
(-----------*----------)
(----------*-----------)
(----------*-----------)
(----------*----------)
(----------*----------)
------+---------+---------+---------+---70
-35
0
35
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent germination (%) and Total metals and
arsenic = -0.637
P-Value = 0.000
175
Basin wildrye – Clark Fork dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 2.417
Level
250
650
1334
1900
3525
5885
7521
N
4
5
5
5
5
4
5
R-Sq = 95.00%
DF
6
26
32
Mean
25.450
25.960
22.740
11.460
6.360
5.700
2.460
StDev
4.213
0.527
3.561
1.503
1.960
1.954
1.656
SS
2885.38
151.93
3037.31
MS
480.90
5.84
F
82.30
P
0.000
R-Sq(adj) = 93.84%
Individual 95% CIs For Mean Based on Pooled StDev
+---------+---------+---------+--------(--*---)
(--*--)
(--*---)
(--*---)
(--*--)
(--*---)
(---*--)
+---------+---------+---------+--------0.0
7.0
14.0
21.0
Pooled StDev = 2.417
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0113
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-3.910
-7.130
-18.410
-23.510
-24.409
-27.410
Center
0.510
-2.710
-13.990
-19.090
-19.750
-22.990
Upper
4.930
1.710
-9.570
-14.670
-15.091
-18.570
-------+---------+---------+---------+-(----*---)
(---*----)
(---*---)
(----*---)
(---*----)
(---*---)
-------+---------+---------+---------+--20
-10
0
10
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.869
P-Value = 0.000
176
Basin wildrye – Clark Fork dilution series continued
One-way ANOVA: Total biomass (mg) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 9.555
Level
250
650
1334
1900
3525
5885
7521
N
4
5
5
5
5
4
5
R-Sq = 96.08%
DF
6
26
32
Mean
108.75
97.80
70.80
23.42
6.78
6.38
2.36
StDev
11.32
14.35
13.43
9.64
3.25
2.50
1.76
SS
58203.8
2373.6
60577.4
MS
9700.6
91.3
F
106.26
P
0.000
R-Sq(adj) = 95.18%
Individual 95% CIs For Mean Based on
Pooled StDev
--+---------+---------+---------+------(--*--)
(--*-)
(-*--)
(--*-)
(--*-)
(--*--)
(--*-)
--+---------+---------+---------+------0
35
70
105
Pooled StDev = 9.55
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0113
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-28.42
-55.42
-102.80
-119.44
-120.79
-123.86
Center
-10.95
-37.95
-85.33
-101.97
-102.37
-106.39
Upper
6.52
-20.48
-67.86
-84.50
-83.96
-88.92
-----+---------+---------+---------+---(----*----)
(----*----)
(----*----)
(----*----)
(-----*----)
(----*----)
-----+---------+---------+---------+----105
-70
-35
0
Correlations: Total biomass (mg), Total metals and arsenic
Pearson correlation of Total biomass (mg) and Total metal and arsenic =
-0.812
P-Value = 0.000
177
Basin wildrye – Clark Fork dilution series continued
One-way ANOVA: tran root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 1.490
Level
250
650
1334
1900
3525
5885
7521
N
4
5
5
5
5
4
5
DF
6
26
32
R-Sq = 63.16%
Mean
5.868
7.846
6.583
6.703
5.392
4.855
2.132
StDev
1.054
1.432
0.682
2.363
1.818
1.489
0.725
SS
98.94
57.70
156.64
MS
16.49
2.22
F
7.43
P
0.000
R-Sq(adj) = 54.66%
Individual 95% CIs For Mean Based on
Pooled StDev
-------+---------+---------+---------+-(-----*------)
(----*-----)
(----*-----)
(-----*----)
(-----*----)
(-----*------)
(-----*----)
-------+---------+---------+---------+-2.5
5.0
7.5
10.0
Pooled StDev = 1.490
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0113
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-0.745
-2.009
-1.888
-3.200
-3.884
-6.459
Center
1.979
0.715
0.836
-0.476
-1.013
-3.735
Upper
4.703
3.439
3.560
2.248
1.859
-1.011
--+---------+---------+---------+------(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
(---------*--------)
(---------*--------)
--+---------+---------+---------+-------6.0
-3.0
0.0
3.0
Correlations: tran root mass ratio, Total metals and arsenic
Pearson correlation of tran root mass ratio and Total metals and arsenic
= -0.715
P-Value = 0.000
178
Bluebunch wheatgrass – ARTS dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 21.65
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 17.77%
SS
2834
13120
15954
Mean
84.00
88.00
84.00
72.00
80.00
84.00
60.00
StDev
8.94
10.95
26.08
10.95
20.00
16.73
40.00
MS
472
469
F
1.01
P
0.440
R-Sq(adj) = 0.14%
Individual 95% CIs For Mean Based on
Pooled StDev
+---------+---------+---------+--------(---------*---------)
(---------*---------)
(---------*---------)
(---------*---------)
(---------*---------)
(---------*---------)
(---------*---------)
+---------+---------+---------+--------40
60
80
100
Pooled StDev = 21.65
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-33.39
-37.39
-49.39
-41.39
-37.39
-61.39
Center
4.00
0.00
-12.00
-4.00
0.00
-24.00
Upper
41.39
37.39
25.39
33.39
37.39
13.39
+---------+---------+---------+--------(-----------*------------)
(-----------*-----------)
(-----------*-----------)
(------------*-----------)
(-----------*-----------)
(-----------*-----------)
+---------+---------+---------+---------60
-30
0
30
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent germination (%) and Total metals and
arsenic = -0.286
P-Value = 0.096
179
Bluebunch wheatgrass – ARTS dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 2.612
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
R-Sq = 70.38%
DF
6
28
34
Mean
20.520
13.560
12.480
14.160
11.960
11.540
7.600
StDev
3.749
3.956
1.854
2.733
0.986
1.374
2.070
SS
453.79
191.00
644.79
MS
75.63
6.82
F
11.09
P
0.000
R-Sq(adj) = 64.03%
Individual 95% CIs For Mean Based on Pooled StDev
+---------+---------+---------+--------(----*----)
(----*----)
(----*----)
(---*----)
(----*----)
(----*----)
(----*----)
+---------+---------+---------+--------5.0
10.0
15.0
20.0
Pooled StDev = 2.612
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-11.471
-12.551
-10.871
-13.071
-13.491
-17.431
Center
-6.960
-8.040
-6.360
-8.560
-8.980
-12.920
Upper
-2.449
-3.529
-1.849
-4.049
-4.469
-8.409
----+---------+---------+---------+----(-----------*----------)
(----------*----------)
(----------*----------)
(-----------*----------)
(-----------*----------)
(-----------*----------)
----+---------+---------+---------+-----16.0
-12.0
-8.0
-4.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.671
P-Value = 0.000
180
Bluebunch wheatgrass – ARTS dilution series continued
One-way ANOVA: Total biomass (mg) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 3.874
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
R-Sq = 70.04%
DF
6
28
34
Mean
24.480
12.720
10.160
13.920
10.440
10.760
6.300
StDev
6.056
5.571
2.464
3.136
1.305
2.098
3.917
SS
982.7
420.3
1403.0
MS
163.8
15.0
F
10.91
P
0.000
R-Sq(adj) = 63.62%
Individual 95% CIs For Mean Based on
Pooled StDev
------+---------+---------+---------+--(----*----)
(----*----)
(-----*----)
(----*----)
(----*----)
(----*----)
(----*----)
------+---------+---------+---------+--7.0
14.0
21.0
28.0
Pooled StDev = 3.874
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-18.452
-21.012
-17.252
-20.732
-20.412
-24.872
Center
-11.760
-14.320
-10.560
-14.040
-13.720
-18.180
Upper
-5.068
-7.628
-3.868
-7.348
-7.028
-11.488
-+---------+---------+---------+-------(----------*-----------)
(----------*----------)
(----------*-----------)
(-----------*----------)
(----------*----------)
(----------*----------)
-+---------+---------+---------+--------24.0
-18.0
-12.0
-6.0
Correlations: Total biomass (mg), Total metals and arsenic
Pearson correlation of Total biomass (mg) and Total metals and arsenic =
-0.576
P-Value = 0.000
181
Bluebunch wheatgrass – ARTS dilution series continued
One-way ANOVA: root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.07463
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
R-Sq = 9.75%
DF
6
28
34
Mean
0.34315
0.40535
0.39186
0.41400
0.38287
0.39965
0.40543
SS
0.01685
0.15595
0.17280
MS
0.00281
0.00557
F
0.50
P
0.800
R-Sq(adj) = 0.00%
StDev
0.02601
0.01911
0.03696
0.05173
0.09950
0.05787
0.14372
Individual 95% CIs For Mean Based on
Pooled StDev
----+---------+---------+---------+----(----------*-----------)
(-----------*----------)
(----------*-----------)
(----------*----------)
(-----------*----------)
(-----------*----------)
(-----------*----------)
----+---------+---------+---------+----0.300
0.360
0.420
0.480
Pooled StDev = 0.07463
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-0.06670
-0.08019
-0.05806
-0.08918
-0.07241
-0.06663
Center
0.06220
0.04871
0.07084
0.03972
0.05649
0.06228
Upper
0.19110
0.17762
0.19975
0.16862
0.18540
0.19118
-+---------+---------+---------+-------(---------------*---------------)
(---------------*---------------)
(---------------*---------------)
(---------------*---------------)
(---------------*---------------)
(---------------*---------------)
-+---------+---------+---------+--------0.080
0.000
0.080
0.160
Correlations: root mass ratio, Total metals and arsenic
Pearson correlation of root mass ratio and Total metals and arsenic
= 0.125
P-Value = 0.476
182
Bluebunch wheatgrass – Clark Fork dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 13.94
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 89.87%
Mean
96.00
92.00
100.00
88.00
40.00
12.00
12.00
StDev
8.94
10.95
0.00
17.89
24.49
10.95
10.95
SS
48274
5440
53714
MS
8046
194
F
41.41
P
0.000
R-Sq(adj) = 87.70%
Individual 95% CIs For Mean Based on
Pooled StDev
+---------+---------+---------+--------(---*---)
(----*---)
(---*----)
(---*----)
(---*----)
(---*---)
(---*---)
+---------+---------+---------+--------0
30
60
90
Pooled StDev = 13.94
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-28.07
-20.07
-32.07
-80.07
-108.07
-108.07
Center
-4.00
4.00
-8.00
-56.00
-84.00
-84.00
Upper
20.07
28.07
16.07
-31.93
-59.93
-59.93
-+---------+---------+---------+-------(------*------)
(------*------)
(------*------)
(------*------)
(------*------)
(------*------)
-+---------+---------+---------+--------105
-70
-35
0
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent emergence (%) and Total metals and
arsenic = -0.906
P-Value = 0.000
183
Bluebunch wheatgrass – Clark Fork dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 2.893
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
4
3
3
R-Sq = 87.95%
DF
6
23
29
Mean
23.040
22.700
18.240
13.840
10.750
6.433
2.933
StDev
2.901
2.776
2.855
1.328
2.684
5.590
1.457
SS
1404.63
192.50
1597.13
MS
234.11
8.37
F
27.97
P
0.000
R-Sq(adj) = 84.80%
Individual 95% CIs For Mean Based on
Pooled StDev
-+---------+---------+---------+-------(---*---)
(--*---)
(---*---)
(---*---)
(---*----)
(----*----)
(----*----)
-+---------+---------+---------+-------0.0
7.0
14.0
21.0
Pooled StDev = 2.893
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0105
Critical value = 2.79
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-5.441
-9.901
-14.301
-17.701
-22.497
-25.997
Center
-0.340
-4.800
-9.200
-12.290
-16.607
-20.107
Upper
4.761
0.301
-4.099
-6.879
-10.716
-14.216
--+---------+---------+---------+------(------*-----)
(-----*-----)
(-----*------)
(------*-----)
(------*-------)
(------*------)
--+---------+---------+---------+-------24.0
-16.0
-8.0
0.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.902
P-Value = 0.000
184
Bluebunch wheatgrass – Clark Fork dilution series continued
One-way ANOVA: Total biomass (mg) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 4.342
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
4
3
3
R-Sq = 88.43%
DF
6
23
29
Mean
31.640
27.000
24.760
13.960
9.425
3.733
2.033
StDev
4.973
5.849
3.019
3.763
4.893
4.025
0.473
SS
3314.8
433.5
3748.3
MS
552.5
18.8
F
29.31
P
0.000
R-Sq(adj) = 85.42%
Individual 95% CIs For Mean Based on
Pooled StDev
---+---------+---------+---------+-----(---*---)
(---*---)
(---*---)
(---*---)
(---*----)
(----*----)
(----*----)
---+---------+---------+---------+-----0
10
20
30
Pooled StDev = 4.342
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0105
Critical value = 2.79
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-12.295
-14.535
-25.335
-30.335
-36.746
-38.446
Center
-4.640
-6.880
-17.680
-22.215
-27.907
-29.607
Upper
3.015
0.775
-10.025
-14.095
-19.067
-20.767
--+---------+---------+---------+------(-----*------)
(-----*------)
(-----*------)
(-----*------)
(-------*------)
(------*-------)
--+---------+---------+---------+-------36
-24
-12
0
Correlations: Total biomass (mg), Total metals and arsenic
Pearson correlation of Total biomass (mg) and Total metals and arsenic =
-0.870
P-Value = 0.000
185
Bluebunch wheatgrass – Clark Fork dilution series continued
One-way ANOVA: root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.06340
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
4
3
3
DF
6
23
29
SS
0.48001
0.09246
0.57247
R-Sq = 83.85%
Mean
0.35997
0.36934
0.42965
0.38280
0.32598
0.43436
0.78788
StDev
0.03936
0.04855
0.03331
0.03376
0.02170
0.14819
0.10606
MS
0.08000
0.00402
F
19.90
P
0.000
R-Sq(adj) = 79.64%
Individual 95% CIs For Mean Based on
Pooled StDev
----+---------+---------+---------+----(--*---)
(---*---)
(---*---)
(---*---)
(---*---)
(----*----)
(---*----)
----+---------+---------+---------+----0.32
0.48
0.64
0.80
Pooled StDev = 0.06340
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0105
Critical value = 2.79
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-0.10242
-0.04211
-0.08897
-0.15257
-0.05470
0.29882
Center
0.00938
0.06969
0.02283
-0.03399
0.07439
0.42791
Upper
0.12117
0.18148
0.13463
0.08459
0.20348
0.55700
--------+---------+---------+---------+(----*-----)
(----*-----)
(----*-----)
(-----*-----)
(------*-----)
(-----*------)
--------+---------+---------+---------+0.00
0.20
0.40
0.60
Correlations: root mass ratio, Total metals and arsenic
Pearson correlation of root mass ratio and Total metals and arsenic =
0.668
P-Value = 0.000
186
Big bluegrass – ARTS dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 13.31
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 15.56%
SS
914
4960
5874
Mean
84.00
88.00
84.00
96.00
92.00
96.00
96.00
StDev
16.73
10.95
16.73
8.94
17.89
8.94
8.94
MS
152
177
F
0.86
P
0.536
R-Sq(adj) = 0.00%
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
--------+---------+---------+---------+80
90
100
110
Pooled StDev = 13.31
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-18.99
-22.99
-10.99
-14.99
-10.99
-10.99
Center
4.00
0.00
12.00
8.00
12.00
12.00
Upper
26.99
22.99
34.99
30.99
34.99
34.99
-----+---------+---------+---------+---(---------------*--------------)
(--------------*--------------)
(--------------*--------------)
(--------------*---------------)
(--------------*--------------)
(--------------*--------------)
-----+---------+---------+---------+----15
0
15
30
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent emergence (%) and Total metals and
arsenic = 0.306
P-Value = 0.074
187
Big bluegrass – ARTS dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 1.526
Level
244
559
974
1585
2908
4360
5783
N
5
5
4
5
5
4
4
DF
6
25
31
R-Sq = 84.86%
Mean
14.640
10.620
9.750
9.500
6.500
4.700
5.625
StDev
1.699
1.819
2.102
1.347
1.317
1.311
0.532
SS
326.38
58.24
384.62
MS
54.40
2.33
F
23.35
P
0.000
R-Sq(adj) = 81.22%
Individual 95% CIs For Mean Based on
Pooled StDev
-+---------+---------+---------+-------(---*---)
(---*---)
(----*---)
(---*---)
(---*---)
(---*----)
(---*----)
-+---------+---------+---------+-------3.5
7.0
10.5
14.0
Pooled StDev = 1.526
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0106
Critical value = 2.76
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-6.687
-7.718
-7.807
-10.807
-12.768
-11.843
Center
-4.020
-4.890
-5.140
-8.140
-9.940
-9.015
Upper
-1.353
-2.062
-2.473
-5.473
-7.112
-6.187
---+---------+---------+---------+-----(--------*-------)
(---------*--------)
(--------*--------)
(--------*--------)
(---------*--------)
(--------*--------)
---+---------+---------+---------+------12.0
-9.0
-6.0
-3.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.806
P-Value = 0.000
188
Big bluegrass – ARTS dilution series continued
One-way ANOVA: trans total biomass versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.5325
Level
244
559
974
1585
2908
4360
5783
N
5
5
4
5
5
4
4
DF
6
25
31
R-Sq = 83.48%
Mean
4.1782
3.6201
3.3185
2.9234
1.7608
1.5462
1.1876
StDev
0.6261
0.5595
0.5000
0.2288
0.5858
0.3847
0.7052
SS
35.822
7.088
42.909
MS
5.970
0.284
F
21.06
P
0.000
R-Sq(adj) = 79.52%
Individual 95% CIs For Mean Based on
Pooled StDev
-----+---------+---------+---------+---(---*---)
(---*---)
(----*---)
(---*---)
(---*---)
(----*---)
(----*---)
-----+---------+---------+---------+---1.2
2.4
3.6
4.8
Pooled StDev = 0.5325
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0106
Critical value = 2.76
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-1.4884
-1.8464
-2.1851
-3.3477
-3.6187
-3.9773
Center
-0.5581
-0.8597
-1.2548
-2.4174
-2.6320
-2.9906
Upper
0.3722
0.1270
-0.3245
-1.4871
-1.6453
-2.0039
---+---------+---------+---------+-----(------*-------)
(-------*-------)
(-------*------)
(-------*-------)
(-------*-------)
(-------*-------)
---+---------+---------+---------+------3.6
-2.4
-1.2
0.0
Correlations: trans total biomass, Total metals and arsenic
Pearson correlation of trans total biomass and Total metals and arsenic =
-0.872
P-Value = 0.000
189
Big bluegrass – ARTS dilution series continued
One-way ANOVA: root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.08297
Level
244
559
974
1585
2908
4360
5783
N
5
5
4
5
5
4
4
DF
6
25
31
SS
0.03040
0.17210
0.20251
R-Sq = 15.01%
Mean
0.37524
0.41514
0.43205
0.42622
0.39300
0.33155
0.39678
StDev
0.03521
0.03646
0.02887
0.10036
0.10767
0.10394
0.11585
MS
0.00507
0.00688
F
0.74
P
0.625
R-Sq(adj) = 0.00%
Individual 95% CIs For Mean Based on
Pooled StDev
-----+---------+---------+---------+---(----------*----------)
(----------*----------)
(-----------*-----------)
(----------*----------)
(----------*----------)
(-----------*------------)
(------------*-----------)
-----+---------+---------+---------+---0.280
0.350
0.420
0.490
Pooled StDev = 0.08297
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0106
Critical value = 2.76
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-0.10507
-0.09695
-0.09398
-0.12720
-0.19745
-0.13222
Center
0.03990
0.05681
0.05098
0.01776
-0.04369
0.02153
Upper
0.18486
0.21056
0.19594
0.16272
0.11007
0.17529
------+---------+---------+---------+--(-----------*-----------)
(------------*------------)
(-----------*-----------)
(-----------*------------)
(-----------*------------)
(------------*------------)
------+---------+---------+---------+---0.12
0.00
0.12
0.24
Correlations: root mass ratio, Total metals and arsenic
Pearson correlation of root mass ratio and Total metals and arsenic
concent = -0.163
P-Value = 0.372
190
Big bluegrass – Clark Fork dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 17.57
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 17.47%
SS
1829
8640
10469
Mean
84.00
76.00
88.00
72.00
88.00
92.00
92.00
StDev
16.73
21.91
17.89
26.83
10.95
10.95
10.95
MS
305
309
F
0.99
P
0.452
R-Sq(adj) = 0.00%
Individual 95% CIs For Mean Based on
Pooled StDev
---+---------+---------+---------+-----(----------*----------)
(----------*---------)
(----------*---------)
(----------*----------)
(----------*---------)
(---------*----------)
(---------*----------)
---+---------+---------+---------+-----60
75
90
105
Pooled StDev = 17.57
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-38.34
-26.34
-42.34
-26.34
-22.34
-22.34
Center
-8.00
4.00
-12.00
4.00
8.00
8.00
Upper
22.34
34.34
18.34
34.34
38.34
38.34
-------+---------+---------+---------+-(-----------*-----------)
(------------*-----------)
(-----------*-----------)
(------------*-----------)
(-----------*-----------)
(-----------*-----------)
-------+---------+---------+---------+--25
0
25
50
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent emergence (%) and Total metals and
arsenic = 0.279
P-Value = 0.105
191
Big bluegrass – Clark Fork dilution series continued
One-way ANOVA: trans shoot height versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.1729
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 92.96%
Mean
2.7602
2.5601
2.6085
2.2986
1.7703
1.3469
1.3312
StDev
0.0514
0.1987
0.2408
0.0707
0.2393
0.0979
0.1934
SS
11.0621
0.8374
11.8995
MS
1.8437
0.0299
F
61.65
P
0.000
R-Sq(adj) = 91.45%
Individual 95% CIs For Mean Based on
Pooled StDev
-------+---------+---------+---------+-(--*--)
(--*--)
(--*--)
(--*--)
(--*---)
(--*--)
(---*--)
-------+---------+---------+---------+-1.50
2.00
2.50
3.00
Pooled StDev = 0.1729
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-0.4988
-0.4504
-0.7603
-1.2886
-1.7120
-1.7277
Center
-0.2001
-0.1517
-0.4616
-0.9899
-1.4133
-1.4290
Upper
0.0986
0.1470
-0.1629
-0.6912
-1.1146
-1.1303
-----+---------+---------+---------+---(-----*-----)
(-----*-----)
(-----*-----)
(-----*-----)
(-----*-----)
(-----*-----)
-----+---------+---------+---------+----1.50
-1.00
-0.50
0.00
Correlations: trans shoot height, Total metals and arsenic
Pearson correlation of trans shoot height and Total metals and arsenic =
-0.934
P-Value = 0.000
192
Big bluegrass – Clark Fork dilution series continued
One-way ANOVA: trans total biomass versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.6203
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 97.26%
Mean
10.641
8.886
8.000
6.262
3.135
1.919
1.750
StDev
0.359
0.803
1.026
0.289
0.559
0.382
0.569
SS
382.918
10.775
393.692
MS
63.820
0.385
F
165.85
P
0.000
R-Sq(adj) = 96.68%
Individual 95% CIs For Mean Based on
Pooled StDev
------+---------+---------+---------+--(*-)
(-*-)
(-*-)
(-*-)
(*-)
(*-)
(-*-)
------+---------+---------+---------+--3.0
6.0
9.0
12.0
Pooled StDev = 0.620
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-2.8265
-3.7126
-5.4507
-8.5775
-9.7935
-9.9631
Center
-1.7551
-2.6412
-4.3792
-7.5061
-8.7221
-8.8917
Upper
-0.6837
-1.5698
-3.3078
-6.4346
-7.6507
-7.8202
+---------+---------+---------+--------(---*---)
(---*----)
(---*----)
(---*---)
(---*---)
(---*----)
+---------+---------+---------+---------10.0
-7.5
-5.0
-2.5
Correlations: trans total biomass, Total metals and arsenic
Pearson correlation of trans total bio and Total metal and arsenic =
-0.924
P-Value = 0.000
193
Big bluegrass – Clark Fork dilution series continued
One-way ANOVA: trans root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.09284
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
SS
0.05716
0.24132
0.29848
R-Sq = 19.15%
Mean
0.65905
0.66146
0.66333
0.66208
0.66092
0.65675
0.54524
StDev
0.01756
0.04694
0.07331
0.02199
0.07804
0.16249
0.13952
MS
0.00953
0.00862
F
1.11
P
0.384
R-Sq(adj) = 1.83%
Individual 95% CIs For Mean Based on
Pooled StDev
--+---------+---------+---------+------(---------*----------)
(----------*---------)
(----------*----------)
(----------*---------)
(----------*---------)
(----------*----------)
(---------*----------)
--+---------+---------+---------+------0.480
0.560
0.640
0.720
Pooled StDev = 0.09284
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-0.15793
-0.15607
-0.15732
-0.15847
-0.16265
-0.27415
Center
0.00241
0.00428
0.00303
0.00187
-0.00230
-0.11380
Upper
0.16276
0.16463
0.16338
0.16222
0.15805
0.04654
---+---------+---------+---------+-----(------------*-------------)
(------------*-------------)
(------------*-------------)
(------------*-------------)
(-------------*------------)
(-------------*------------)
---+---------+---------+---------+------0.24
-0.12
0.00
0.12
Correlations: trans root mass, Total metals and arsenic
Pearson correlation of trans root mass ratio and Total metals and arsenic
= -0.323
P-Value = 0.059
194
Sheep fescue – ARTS dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 7.559
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 19.54%
Mean
92.00
96.00
96.00
100.00
92.00
100.00
100.00
StDev
10.95
8.94
8.94
0.00
10.95
0.00
0.00
SS
388.6
1600.0
1988.6
MS
64.8
57.1
F
1.13
P
0.369
R-Sq(adj) = 2.30%
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+(----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*----------)
(----------*-----------)
(-----------*----------)
(-----------*----------)
--------+---------+---------+---------+90.0
96.0
102.0
108.0
Pooled StDev = 7.56
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-9.056
-9.056
-5.056
-13.056
-5.056
-5.056
Center
4.000
4.000
8.000
0.000
8.000
8.000
Upper
17.056
17.056
21.056
13.056
21.056
21.056
---+---------+---------+---------+-----(------------*------------)
(------------*------------)
(------------*------------)
(------------*------------)
(------------*------------)
(------------*------------)
---+---------+---------+---------+------10
0
10
20
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent emergence (%) and Total metals and
arsenic = 0.244
P-Value = 0.158
195
Sheep fescue – ARTS dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 1.649
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
3
4
5
4
DF
6
24
30
R-Sq = 84.23%
Mean
13.600
12.220
9.140
6.900
5.300
5.800
4.550
StDev
2.762
1.999
1.045
1.562
0.698
1.111
1.021
SS
348.78
65.29
414.07
MS
58.13
2.72
F
21.37
P
0.000
R-Sq(adj) = 80.29%
Individual 95% CIs For Mean Based on
Pooled StDev
--+---------+---------+---------+------(---*---)
(---*---)
(---*---)
(-----*----)
(----*----)
(----*---)
(----*----)
--+---------+---------+---------+------3.5
7.0
10.5
14.0
Pooled StDev = 1.649
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0105
Critical value = 2.77
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-4.275
-7.355
-10.042
-11.370
-10.695
-12.120
Center
-1.380
-4.460
-6.700
-8.300
-7.800
-9.050
Upper
1.515
-1.565
-3.358
-5.230
-4.905
-5.980
-----+---------+---------+---------+---(-------*-------)
(-------*--------)
(---------*--------)
(-------*--------)
(--------*-------)
(--------*--------)
-----+---------+---------+---------+----10.5
-7.0
-3.5
0.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.798
P-Value = 0.000
196
Sheep fescue – ARTS dilution series continued
One-way ANOVA: trans total bio versus Total metal and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.5870
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
3
4
5
4
DF
6
24
30
R-Sq = 86.12%
Mean
4.1346
3.3075
2.2966
1.6667
0.5842
1.0206
0.7246
StDev
0.5783
0.7414
0.5370
0.4000
0.8687
0.4069
0.3326
SS
51.290
8.268
59.558
MS
8.548
0.345
F
24.81
P
0.000
R-Sq(adj) = 82.65%
Individual 95% CIs For Mean Based on Pooled StDev
+---------+---------+---------+--------(---*----)
(----*---)
(---*----)
(-----*-----)
(----*----)
(----*---)
(----*----)
+---------+---------+---------+--------0.0
1.2
2.4
3.6
Pooled StDev = 0.5870
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0105
Critical value = 2.77
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-1.8572
-2.8681
-3.6573
-4.6430
-4.1441
-4.5026
Center
-0.8271
-1.8381
-2.4679
-3.5504
-3.1140
-3.4100
Upper
0.2029
-0.8080
-1.2784
-2.4578
-2.0839
-2.3174
-+---------+---------+---------+-------(-----*------)
(------*------)
(-------*------)
(------*-------)
(------*------)
(------*-------)
-+---------+---------+---------+--------4.5
-3.0
-1.5
0.0
Correlations: trans total biomass, Total metals and arsenic
Pearson correlation of trans total bio and Total metals and arsenic =
-0.786
P-Value = 0.000
197
Sheep fescue – ARTS dilution series continued
One-way ANOVA: root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.1745
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
3
4
5
4
R-Sq = 9.93%
DF
6
24
30
Mean
0.3126
0.3450
0.3559
0.3016
0.4231
0.3081
0.4459
StDev
0.0757
0.0573
0.0699
0.2161
0.3323
0.2219
0.1338
SS
0.0806
0.7310
0.8116
MS
0.0134
0.0305
F
0.44
P
0.844
R-Sq(adj) = 0.00%
Individual 95% CIs For Mean Based on
Pooled StDev
----+---------+---------+---------+----(----------*----------)
(----------*----------)
(----------*---------)
(-------------*-------------)
(-----------*-----------)
(----------*---------)
(-----------*-----------)
----+---------+---------+---------+----0.15
0.30
0.45
0.60
Pooled StDev = 0.1745
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0105
Critical value = 2.77
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-0.2739
-0.2630
-0.3647
-0.2144
-0.3108
-0.1916
Center
0.0324
0.0433
-0.0111
0.1104
-0.0045
0.1332
Upper
0.3386
0.3495
0.3426
0.4353
0.3017
0.4581
-----+---------+---------+---------+---(-----------*------------)
(------------*-----------)
(--------------*-------------)
(------------*------------)
(-----------*-----------)
(------------*------------)
-----+---------+---------+---------+----0.25
0.00
0.25
0.50
Correlations: root mass ratio, Total metals and arsenic
Pearson correlation of root mass ratio and Total metals and arsenic =
0.161
P-Value = 0.385
198
Sheep fescue – Clark Fork dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 11.71
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 24.32%
SS
1234
3840
5074
Mean
96.00
84.00
96.00
100.00
88.00
88.00
84.00
MS
206
137
F
1.50
P
0.215
R-Sq(adj) = 8.11%
StDev
8.94
16.73
8.94
0.00
10.95
10.95
16.73
Individual 95% CIs For Mean Based on
Pooled StDev
-------+---------+---------+---------+-(----------*----------)
(----------*----------)
(----------*----------)
(----------*----------)
(----------*----------)
(----------*----------)
(----------*----------)
-------+---------+---------+---------+-80
90
100
110
Pooled StDev = 11.71
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-32.23
-20.23
-16.23
-28.23
-28.23
-32.23
Center
-12.00
0.00
4.00
-8.00
-8.00
-12.00
Upper
8.23
20.23
24.23
12.23
12.23
8.23
-+---------+---------+---------+-------(------------*------------)
(------------*------------)
(-------------*------------)
(-------------*------------)
(-------------*------------)
(------------*------------)
-+---------+---------+---------+--------30
-15
0
15
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent emergence (%) and Total metals and
arsenic = -0.265
P-Value = 0.123
199
Sheep fescue – Clark Fork dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 1.781
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 87.19%
Mean
14.140
16.160
11.600
9.540
6.280
5.020
4.980
StDev
2.996
2.152
1.690
2.019
0.683
0.427
1.006
SS
604.77
88.82
693.59
MS
100.79
3.17
F
31.77
P
0.000
R-Sq(adj) = 84.45%
Individual 95% CIs For Mean Based on
Pooled StDev
--+---------+---------+---------+------(---*---)
(---*---)
(---*---)
(---*---)
(---*---)
(----*---)
(---*----)
--+---------+---------+---------+------4.0
8.0
12.0
16.0
Pooled StDev = 1.781
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-1.056
-5.616
-7.676
-10.936
-12.196
-12.236
Center
2.020
-2.540
-4.600
-7.860
-9.120
-9.160
Upper
5.096
0.536
-1.524
-4.784
-6.044
-6.084
----+---------+---------+---------+----(-----*-----)
(-----*-----)
(-----*-----)
(-----*-----)
(-----*-----)
(-----*-----)
----+---------+---------+---------+-----10.0
-5.0
0.0
5.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.838
P-Value = 0.000
200
Sheep fescue – Clark Fork dilution series continued
One-way ANOVA: trans total biomass versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.3827
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 94.34%
Mean
4.4447
4.3361
3.9623
3.1747
1.6203
1.2695
0.9582
StDev
0.4401
0.2869
0.0814
0.4912
0.5002
0.4166
0.2788
SS
68.392
4.102
72.494
MS
11.399
0.146
F
77.81
P
0.000
R-Sq(adj) = 93.13%
Individual 95% CIs For Mean Based on
Pooled StDev
-----+---------+---------+---------+---(--*--)
(--*--)
(--*--)
(-*--)
(--*-)
(--*--)
(--*--)
-----+---------+---------+---------+---1.2
2.4
3.6
4.8
Pooled StDev = 0.3827
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-0.7697
-1.1434
-1.9311
-3.4854
-3.8362
-4.1476
Center
-0.1086
-0.4823
-1.2700
-2.8243
-3.1751
-3.4865
Upper
0.5525
0.1787
-0.6089
-2.1633
-2.5141
-2.8254
-----+---------+---------+---------+---(----*-----)
(-----*----)
(----*-----)
(----*-----)
(-----*----)
(-----*----)
-----+---------+---------+---------+----3.6
-2.4
-1.2
0.0
Correlations: trans total biomass, Total metals and arsenic
Pearson correlation of trans total bio and Total metal and arsenic =
-0.922
P-Value = 0.000
201
Sheep fescue – Clark Fork dilution series continued
One-way ANOVA: trans root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.2186
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 37.07%
Mean
1.4891
1.7374
1.6307
1.8556
1.8268
1.7368
1.9908
SS
0.7881
1.3379
2.1260
MS
0.1314
0.0478
F
2.75
P
0.031
R-Sq(adj) = 23.59%
StDev
0.1861
0.1082
0.0672
0.2312
0.2777
0.1894
0.3424
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+(-------*-------)
(-------*--------)
(-------*-------)
(-------*-------)
(-------*-------)
(-------*-------)
(-------*-------)
--------+---------+---------+---------+1.50
1.75
2.00
2.25
Pooled StDev = 0.2186
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-0.1292
-0.2360
-0.0111
-0.0399
-0.1299
0.1241
Center
0.2483
0.1416
0.3665
0.3377
0.2477
0.5017
Upper
0.6258
0.5191
0.7440
0.7152
0.6252
0.8792
--------+---------+---------+---------+(-----------*------------)
(------------*-----------)
(-----------*------------)
(-----------*------------)
(-----------*------------)
(------------*-----------)
--------+---------+---------+---------+0.00
0.30
0.60
0.90
Correlations: trans root mass ratio, Total metals and arsenic
Pearson correlation of trans root mass ratio and Total metals and arsenic =
0.434
P-Value = 0.009
202
Redtop – ARTS dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 9.562
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 26.32%
Mean
84.00
100.00
96.00
92.00
100.00
96.00
96.00
SS
914.3
2560.0
3474.3
MS
152.4
91.4
F
1.67
P
0.166
R-Sq(adj) = 10.53%
StDev
8.94
0.00
8.94
17.89
0.00
8.94
8.94
Individual 95% CIs For Mean Based on
Pooled StDev
-----+---------+---------+---------+---(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
-----+---------+---------+---------+---80
90
100
110
Pooled StDev = 9.56
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-0.515
-4.515
-8.515
-0.515
-4.515
-4.515
Center
16.000
12.000
8.000
16.000
12.000
12.000
Upper
32.515
28.515
24.515
32.515
28.515
28.515
-------+---------+---------+---------+-(------------*-------------)
(-------------*-------------)
(-------------*------------)
(------------*-------------)
(-------------*-------------)
(-------------*-------------)
-------+---------+---------+---------+-0
12
24
36
Correlations: Total percent emergence (%), Total metal and arsenic
Pearson correlation of Total percent emergence (%) and Total metals and
arsenic = 0.173
P-Value = 0.321
203
Redtop – ARTS dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 3.879
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 6.85%
Mean
23.020
24.220
22.180
22.480
21.740
22.460
20.980
SS
31.0
421.2
452.2
MS
5.2
15.0
F
0.34
P
0.908
R-Sq(adj) = 0.00%
StDev
2.519
3.182
4.934
4.420
2.675
3.198
5.251
Individual 95% CIs For Mean Based on
Pooled StDev
--+---------+---------+---------+------(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(----------*-----------)
(-----------*-----------)
(-----------*-----------)
--+---------+---------+---------+------18.0
21.0
24.0
27.0
Pooled StDev = 3.879
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-5.499
-7.539
-7.239
-7.979
-7.259
-8.739
Center
1.200
-0.840
-0.540
-1.280
-0.560
-2.040
Upper
7.899
5.859
6.159
5.419
6.139
4.659
-------+---------+---------+---------+-(------------*-------------)
(------------*-------------)
(------------*------------)
(------------*-------------)
(-------------*------------)
(------------*------------)
-------+---------+---------+---------+--5.0
0.0
5.0
10.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.194
P-Value = 0.265
204
Redtop – ARTS dilution series continued
One-way ANOVA: Total biomass (mg) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 22.99
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 45.78%
Mean
118.10
90.20
110.10
95.26
99.42
81.10
55.84
StDev
28.60
10.46
15.08
25.05
34.21
19.95
18.67
SS
12495
14798
27292
MS
2082
528
F
3.94
P
0.006
R-Sq(adj) = 34.16%
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+(------*------)
(------*------)
(------*------)
(------*------)
(------*------)
(------*------)
(------*------)
--------+---------+---------+---------+60
90
120
150
Pooled StDev = 22.99
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-67.61
-47.71
-62.55
-58.39
-76.71
-101.97
Center
-27.90
-8.00
-22.84
-18.68
-37.00
-62.26
Upper
11.81
31.71
16.87
21.03
2.71
-22.55
---------+---------+---------+---------+
(----------*----------)
(-----------*----------)
(----------*-----------)
(-----------*----------)
(----------*-----------)
(----------*-----------)
---------+---------+---------+---------+
-70
-35
0
35
Correlations: Total biomass (mg), Total metals and arsenic
Pearson correlation of Total biomass (mg) and Total metals and arsenic =
-0.584
P-Value = 0.000
205
Redtop – ARTS dilution series continued
One-way ANOVA: root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.05918
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
SS
0.07553
0.09808
0.17361
R-Sq = 43.51%
Mean
0.38966
0.35764
0.33899
0.34523
0.43584
0.28521
0.30780
StDev
0.06018
0.04724
0.05018
0.04987
0.10337
0.04397
0.03228
MS
0.01259
0.00350
F
3.59
P
0.009
R-Sq(adj) = 31.40%
Individual 95% CIs For Mean Based on
Pooled StDev
-------+---------+---------+---------+-(-------*------)
(-------*-------)
(------*-------)
(------*-------)
(------*-------)
(-------*------)
(-------*-------)
-------+---------+---------+---------+-0.280
0.350
0.420
0.490
Pooled StDev = 0.05918
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-0.13424
-0.15289
-0.14665
-0.05605
-0.20667
-0.18408
Center
-0.03202
-0.05067
-0.04443
0.04618
-0.10445
-0.08186
Upper
0.07021
0.05155
0.05780
0.14840
-0.00223
0.02037
-+---------+---------+---------+-------(---------*---------)
(---------*---------)
(----------*---------)
(----------*---------)
(----------*---------)
(---------*---------)
-+---------+---------+---------+--------0.20
-0.10
-0.00
0.10
Correlations: root mass ratio, Total metals and arsenic
Pearson correlation of root mass ratio and Total metals and arsenic =
-0.320
P-Value = 0.061
206
Redtop – Clark Fork dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 8.619
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 40.13%
SS
1394.3
2080.0
3474.3
Mean
80.00
96.00
96.00
100.00
100.00
96.00
96.00
StDev
14.14
8.94
8.94
0.00
0.00
8.94
8.94
MS
232.4
74.3
F
3.13
P
0.018
R-Sq(adj) = 27.30%
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+(-------*-------)
(-------*-------)
(-------*-------)
(-------*-------)
(-------*-------)
(-------*-------)
(-------*-------)
--------+---------+---------+---------+80
90
100
110
Pooled StDev = 8.62
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
1.113
1.113
5.113
5.113
1.113
1.113
Center
16.000
16.000
20.000
20.000
16.000
16.000
Upper
30.887
30.887
34.887
34.887
30.887
30.887
---------+---------+---------+---------+
(--------------*--------------)
(--------------*--------------)
(--------------*--------------)
(--------------*--------------)
(--------------*--------------)
(--------------*--------------)
---------+---------+---------+---------+
10
20
30
40
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent emergence (%) and Total metals and
arsenic = 0.233
P-Value = 0.177
207
Redtop – Clark Fork dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 3.184
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 18.11%
SS
62.8
283.8
346.6
Mean
18.820
22.180
20.740
21.260
21.900
20.460
18.420
MS
10.5
10.1
F
1.03
P
0.425
R-Sq(adj) = 0.57%
StDev
1.404
2.158
3.138
4.531
4.862
1.717
2.713
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+(----------*-----------)
(-----------*----------)
(-----------*-----------)
(-----------*-----------)
(-----------*----------)
(-----------*-----------)
(-----------*----------)
--------+---------+---------+---------+17.5
20.0
22.5
25.0
Pooled StDev = 3.184
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-2.139
-3.579
-3.059
-2.419
-3.859
-5.899
Center
3.360
1.920
2.440
3.080
1.640
-0.400
Upper
8.859
7.419
7.939
8.579
7.139
5.099
-----+---------+---------+---------+---(------------*-------------)
(-------------*-------------)
(-------------*-------------)
(-------------*------------)
(-------------*-------------)
(-------------*-------------)
-----+---------+---------+---------+----4.0
0.0
4.0
8.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.165
P-Value = 0.342
208
Redtop – Clark Fork dilution series continued
One-way ANOVA: Total biomass (mg) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 20.09
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 69.45%
Mean
113.14
132.44
135.90
128.70
109.14
74.04
61.62
SS
25681
11296
36977
MS
4280
403
F
10.61
P
0.000
R-Sq(adj) = 62.91%
StDev
8.48
22.55
16.38
20.11
18.29
32.74
12.81
Individual 95% CIs For Mean Based on
Pooled StDev
------+---------+---------+---------+--(-----*-----)
(-----*-----)
(-----*-----)
(-----*-----)
(-----*------)
(-----*-----)
(------*-----)
------+---------+---------+---------+--60
90
120
150
Pooled StDev = 20.09
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-15.39
-11.93
-19.13
-38.69
-73.79
-86.21
Center
19.30
22.76
15.56
-4.00
-39.10
-51.52
Upper
53.99
57.45
50.25
30.69
-4.41
-16.83
--+---------+---------+---------+------(--------*-------)
(--------*-------)
(--------*--------)
(--------*--------)
(-------*--------)
(--------*--------)
--+---------+---------+---------+-------80
-40
0
40
Correlations: Total biomass (mg), Total metals and arsenic
Pearson correlation of Total biomass (mg) and Total metals and arsenic =
-0.767
P-Value = 0.000
209
Redtop – Clark Fork dilution series continued
One-way ANOVA: root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.05174
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
SS
0.02239
0.07495
0.09734
R-Sq = 23.01%
Mean
0.40597
0.36233
0.40401
0.35275
0.37283
0.41077
0.34565
StDev
0.02282
0.03263
0.05569
0.05670
0.03409
0.07308
0.06582
MS
0.00373
0.00268
F
1.39
P
0.252
R-Sq(adj) = 6.51%
Individual 95% CIs For Mean Based on
Pooled StDev
-----+---------+---------+---------+---(----------*-----------)
(-----------*----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(----------*-----------)
-----+---------+---------+---------+---0.320
0.360
0.400
0.440
Pooled StDev = 0.05174
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-0.13299
-0.09132
-0.14258
-0.12250
-0.08456
-0.14968
Center
-0.04363
-0.00196
-0.05322
-0.03314
0.00480
-0.06032
Upper
0.04573
0.08740
0.03614
0.05622
0.09416
0.02904
-+---------+---------+---------+-------(------------*------------)
(------------*-----------)
(-----------*------------)
(-----------*------------)
(------------*-----------)
(-----------*------------)
-+---------+---------+---------+--------0.140
-0.070
0.000
0.070
Correlations: root mass ratio, Total metals and arsenic
Pearson correlation of root mass ratio and Total metals and arsenic =
-0.116
P-Value = 0.506
210
Slender wheatgrass – ARTS dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 25.07
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 31.68%
Mean
36.00
76.00
76.00
76.00
76.00
76.00
88.00
SS
8160
17600
25760
MS
1360
629
F
2.16
P
0.077
R-Sq(adj) = 17.04%
StDev
32.86
16.73
26.08
16.73
26.08
26.08
26.83
Individual 95% CIs For Mean Based on
Pooled StDev
-----+---------+---------+---------+---(--------*---------)
(--------*---------)
(--------*---------)
(--------*---------)
(--------*---------)
(--------*---------)
(--------*--------)
-----+---------+---------+---------+---25
50
75
100
Pooled StDev = 25.07
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-3.30
-3.30
-3.30
-3.30
-3.30
8.70
Center
40.00
40.00
40.00
40.00
40.00
52.00
Upper
83.30
83.30
83.30
83.30
83.30
95.30
-+---------+---------+---------+-------(----------------*----------------)
(----------------*----------------)
(----------------*----------------)
(----------------*----------------)
(----------------*----------------)
(-----------------*----------------)
-+---------+---------+---------+-------0
25
50
75
Correlations: Total percent emergence, Total metals and arsenic
Pearson correlation of Total percent emergence and Total metal and
arsenic = 0.341
P-Value = 0.045
211
Slender wheatgrass – ARTS dilution series continued
One-way ANOVA: Total percent emergence versus Total metals and arsenic
treating 559 treatment as control
Source
Total metal and arsenic
Error
Total
S = 25.07
Level
244
559
974
1585
2908
4360
5783
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 31.68%
SS
8160
17600
25760
Mean
36.00
76.00
76.00
76.00
76.00
76.00
88.00
StDev
32.86
16.73
26.08
16.73
26.08
26.08
26.83
MS
1360
629
F
2.16
P
0.077
R-Sq(adj) = 17.04%
Individual 95% CIs For Mean Based on
Pooled StDev
-----+---------+---------+---------+---(--------*---------)
(--------*---------)
(--------*---------)
(--------*---------)
(--------*---------)
(--------*---------)
(--------*--------)
-----+---------+---------+---------+---25
50
75
100
Pooled StDev = 25.07
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (559) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
244
974
1585
2908
4360
5783
Lower
-83.30
-43.30
-43.30
-43.30
-43.30
-31.30
Center
-40.00
0.00
0.00
0.00
0.00
12.00
Upper
3.30
43.30
43.30
43.30
43.30
55.30
----+---------+---------+---------+----(------------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*------------)
----+---------+---------+---------+-----70
-35
0
35
212
Slender wheatgrass – ARTS dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 3.677
Level
244
559
974
1585
2908
4360
5783
N
4
5
5
5
5
5
5
R-Sq = 63.52%
DF
6
27
33
Mean
25.450
25.380
22.420
18.440
15.860
14.700
15.220
StDev
2.596
1.825
2.063
2.946
3.262
3.114
7.043
SS
635.7
365.1
1000.8
MS
106.0
13.5
F
7.84
P
0.000
R-Sq(adj) = 55.42%
Individual 95% CIs For Mean Based on
Pooled StDev
-------+---------+---------+---------+-(-------*------)
(------*------)
(------*------)
(------*------)
(------*-----)
(-----*------)
(-----*------)
-------+---------+---------+---------+-15.0
20.0
25.0
30.0
Pooled StDev = 3.677
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0114
Critical value = 2.71
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-6.767
-9.727
-13.707
-16.287
-17.447
-16.927
Center
-0.070
-3.030
-7.010
-9.590
-10.750
-10.230
Upper
6.627
3.667
-0.313
-2.893
-4.053
-3.533
-----+---------+---------+---------+---(---------*--------)
(---------*--------)
(---------*---------)
(--------*---------)
(---------*--------)
(--------*---------)
-----+---------+---------+---------+----14.0
-7.0
0.0
7.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.704
P-Value = 0.000
213
Slender wheatgrass – ARTS dilution series continued
One-way ANOVA: Total biomass (mg) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 11.45
Level
244
559
974
1585
2908
4360
5783
N
4
5
5
5
5
5
5
DF
6
27
33
R-Sq = 79.84%
SS
14016
3540
17555
Mean
42.30
71.56
41.22
17.94
13.94
13.44
17.52
StDev
12.99
15.28
15.34
9.04
4.71
5.54
12.44
MS
2336
131
F
17.82
P
0.000
R-Sq(adj) = 75.36%
Individual 95% CIs For Mean Based on
Pooled StDev
---------+---------+---------+---------+
(-----*-----)
(----*----)
(-----*----)
(----*----)
(----*----)
(-----*----)
(----*----)
---------+---------+---------+---------+
20
40
60
80
Pooled StDev = 11.45
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0114
Critical value = 2.71
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
8.41
-21.93
-45.21
-49.21
-49.71
-45.63
Center
29.26
-1.08
-24.36
-28.36
-28.86
-24.78
Upper
50.11
19.77
-3.51
-7.51
-8.01
-3.93
+---------+---------+---------+--------(--------*-------)
(--------*-------)
(-------*--------)
(--------*-------)
(-------*--------)
(-------*-------)
+---------+---------+---------+---------50
-25
0
25
Correlations: Total biomass (mg), Total metals and arsenic
Pearson correlation of Total biomass (mg) and Total metals and arsenic =
-0.626
P-Value = 0.000
214
Slender wheatgrass – ARTS dilution series continued
One-way ANOVA: root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.07887
Level
244
559
974
1585
2908
4360
5783
N
4
5
5
5
5
5
5
DF
6
27
33
SS
0.09440
0.16797
0.26237
R-Sq = 35.98%
Mean
0.30607
0.32347
0.34948
0.37324
0.42624
0.46613
0.40804
MS
0.01573
0.00622
F
2.53
P
0.045
R-Sq(adj) = 21.75%
StDev
0.03081
0.02764
0.01998
0.07414
0.05131
0.03378
0.17563
Individual 95% CIs For Mean Based on
Pooled StDev
--+---------+---------+---------+------(---------*---------)
(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
(--------*--------)
--+---------+---------+---------+------0.240
0.320
0.400
0.480
Pooled StDev = 0.07887
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0114
Critical value = 2.71
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-0.12625
-0.10024
-0.07648
-0.02347
0.01641
-0.04168
Center
0.01740
0.04341
0.06717
0.12018
0.16006
0.10197
Upper
0.16105
0.18706
0.21082
0.26382
0.30371
0.24562
-+---------+---------+---------+-------(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(-----------*-----------)
(----------*-----------)
-+---------+---------+---------+--------0.12
0.00
0.12
0.24
Correlations: root mass ratio, Total metals and arsenic
Pearson correlation of root mass ratio and Total metals and arsenic
= 0.492
P-Value = 0.003
215
Slender wheatgrass – ARTS dilution series continued
One-way ANOVA: remove outlier root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.05108
Level
244
559
974
1585
2908
4360
5783
N
4
5
5
5
5
5
4
DF
6
26
32
SS
0.12696
0.06783
0.19480
R-Sq = 65.18%
Mean
0.30607
0.32347
0.34948
0.37324
0.42624
0.46613
0.47880
MS
0.02116
0.00261
F
8.11
P
0.000
R-Sq(adj) = 57.14%
StDev
0.03081
0.02764
0.01998
0.07414
0.05131
0.03378
0.08804
Individual 95% CIs For Mean Based on
Pooled StDev
----+---------+---------+---------+----(-------*------)
(-----*------)
(------*------)
(-----*------)
(------*------)
(------*-----)
(------*-------)
----+---------+---------+---------+----0.280
0.350
0.420
0.490
Pooled StDev = 0.05108
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0113
Critical value = 2.73
Control = level (244) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
559
974
1585
2908
4360
5783
Lower
-0.07599
-0.04998
-0.02622
0.02678
0.06666
0.07428
Center
0.01740
0.04341
0.06717
0.12018
0.16006
0.17273
Upper
0.11080
0.13681
0.16056
0.21357
0.25345
0.27118
--------+---------+---------+---------+(---------*--------)
(--------*---------)
(---------*--------)
(--------*--------)
(--------*--------)
(---------*---------)
--------+---------+---------+---------+0.00
0.10
0.20
0.30
Correlations: remove outlier root mass ratio, Total metals and arsenic
Pearson correlation of remove outlier root mass ratio and Total metals and
arsenic = 0.788
P-Value = 0.000
216
Slender wheatgrass – Clark Fork dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 24.14
Level
250
650
1334
1900
3525
5885
7521
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 60.83%
Mean
92.00
96.00
88.00
76.00
64.00
24.00
32.00
StDev
17.89
8.94
17.89
21.91
35.78
32.86
22.80
SS
25349
16320
41669
MS
4225
583
F
7.25
P
0.000
R-Sq(adj) = 52.44%
Individual 95% CIs For Mean Based on
Pooled StDev
---------+---------+---------+---------+
(-------*------)
(------*------)
(------*-------)
(------*-------)
(------*-------)
(------*------)
(-------*------)
---------+---------+---------+---------+
30
60
90
120
Pooled StDev = 24.14
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-37.70
-45.70
-57.70
-69.70
-109.70
-101.70
Center
4.00
-4.00
-16.00
-28.00
-68.00
-60.00
Upper
45.70
37.70
25.70
13.70
-26.30
-18.30
-------+---------+---------+---------+-(---------*---------)
(---------*---------)
(---------*---------)
(---------*---------)
(---------*---------)
(---------*---------)
-------+---------+---------+---------+--80
-40
0
40
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent germination (%) and Total metals and
arsenic = -0.751
P-Value = 0.000
217
Slender wheatgrass – Clark Fork dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 3.446
Level
250
650
1334
1900
3525
5885
7521
N
5
5
3
5
5
3
4
R-Sq = 78.43%
DF
6
23
29
Mean
26.240
25.860
25.633
22.700
21.980
12.667
10.400
StDev
0.808
2.477
2.871
2.716
1.540
7.524
5.075
SS
993.2
273.1
1266.3
MS
165.5
11.9
F
13.94
P
0.000
R-Sq(adj) = 72.81%
Individual 95% CIs For Mean Based on
Pooled StDev
---------+---------+---------+---------+
(-----*----)
(----*----)
(------*------)
(----*----)
(-----*----)
(------*------)
(-----*-----)
---------+---------+---------+---------+
12.0
18.0
24.0
30.0
Pooled StDev = 3.446
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0105
Critical value = 2.79
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-6.456
-7.623
-9.616
-10.336
-20.589
-22.285
Center
-0.380
-0.607
-3.540
-4.260
-13.573
-15.840
Upper
5.696
6.409
2.536
1.816
-6.557
-9.395
--------+---------+---------+---------+(-------*------)
(--------*--------)
(-------*------)
(-------*------)
(--------*--------)
(-------*-------)
--------+---------+---------+---------+-16.0
-8.0
0.0
8.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metal and arsenic =
-0.866
P-Value = 0.000
218
Slender wheatgrass – Clark Fork dilution series continued
One-way ANOVA: Total biomass (mg) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 9.293
Level
250
650
1334
1900
3525
5885
7521
N
5
5
3
5
5
3
4
R-Sq = 88.11%
DF
6
23
29
Mean
63.720
70.340
62.833
35.720
36.000
10.733
11.300
StDev
11.823
4.082
5.950
14.683
6.755
7.780
6.425
SS
14716.3
1986.4
16702.6
MS
2452.7
86.4
F
28.40
P
0.000
R-Sq(adj) = 85.01%
Individual 95% CIs For Mean Based on
Pooled StDev
+---------+---------+---------+--------(---*---)
(---*---)
(----*-----)
(---*---)
(---*---)
(----*-----)
(----*---)
+---------+---------+---------+--------0
20
40
60
Pooled StDev = 9.293
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0105
Critical value = 2.79
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-9.766
-19.808
-44.386
-44.106
-71.908
-69.800
Center
6.620
-0.887
-28.000
-27.720
-52.987
-52.420
Upper
23.006
18.035
-11.614
-11.334
-34.065
-35.040
---------+---------+---------+---------+
(------*-----)
(-------*------)
(------*-----)
(------*-----)
(-------*------)
(------*------)
---------+---------+---------+---------+
-50
-25
0
25
Correlations: Total biomass (mg), Total metals and arsenic
Pearson correlation of Total biomass (mg) and Total metals and arsenic =
-0.870
P-Value = 0.000
219
Slender wheatgrass – Clark Fork dilution series continued
One-way ANOVA: root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.07930
Level
250
650
1334
1900
3525
5885
7521
N
5
5
3
5
5
3
4
DF
6
23
29
SS
0.34347
0.14464
0.48812
R-Sq = 70.37%
Mean
0.42821
0.36387
0.32405
0.37886
0.42406
0.59219
0.65488
StDev
0.02305
0.03860
0.05766
0.04849
0.01511
0.19728
0.11798
MS
0.05725
0.00629
F
9.10
P
0.000
R-Sq(adj) = 62.64%
Individual 95% CIs For Mean Based on
Pooled StDev
-----+---------+---------+---------+---(----*---)
(----*----)
(------*-----)
(----*----)
(----*----)
(-----*------)
(-----*----)
-----+---------+---------+---------+---0.30
0.45
0.60
0.75
Pooled StDev = 0.07930
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0105
Critical value = 2.79
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650
1334
1900
3525
5885
7521
Lower
-0.20417
-0.26562
-0.18918
-0.14398
0.00252
0.07836
Center
-0.06434
-0.10415
-0.04935
-0.00415
0.16399
0.22668
Upper
0.07550
0.05731
0.09048
0.13568
0.32545
0.37499
---+---------+---------+---------+-----(------*------)
(-------*-------)
(------*------)
(------*------)
(-------*-------)
(------*-------)
---+---------+---------+---------+------0.20
0.00
0.20
0.40
Correlations: root mass ratio, Total metals and arsenic
Pearson correlation of root mass ratio and Total metals and arsenic
= 0.751
P-Value = 0.000
220
Tufted hairgrass – ARTS dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 13.78
Level
244
559
974
1585
N
5
5
5
5
DF
3
16
19
R-Sq = 15.08%
SS
540
3040
3580
Mean
96.00
84.00
88.00
96.00
StDev
8.94
16.73
17.89
8.94
MS
180
190
F
0.95
P
0.441
R-Sq(adj) = 0.00%
Individual 95% CIs For Mean Based on
Pooled StDev
---------+---------+---------+---------+
(------------*------------)
(------------*------------)
(------------*------------)
(------------*------------)
---------+---------+---------+---------+
80
90
100
110
Pooled StDev = 13.78
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0196
Critical value = 2.59
Control = level (244) of Total metal and arsenic conc.
Intervals for treatment mean minus control mean
Level
559
974
1585
Lower
-34.60
-30.60
-22.60
Center
-12.00
-8.00
0.00
Upper
10.60
14.60
22.60
---+---------+---------+---------+-----(--------------*--------------)
(--------------*--------------)
(--------------*--------------)
---+---------+---------+---------+------30
-15
0
15
Correlations: Total percent emergence (%), Total metals and arsenic
Pearson correlation of Total percent emergence (%) and Total metals and
= 0.086
P-Value = 0.718
221
Tufted hairgrass – ARTS dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 2.416
Level
244
559
974
1585
N
5
5
5
5
DF
3
16
19
R-Sq = 61.13%
Mean
11.700
11.660
6.600
5.960
SS
146.83
93.36
240.19
MS
48.94
5.84
F
8.39
P
0.001
R-Sq(adj) = 53.84%
StDev
1.288
1.387
3.088
3.197
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+(-------*-------)
(-------*-------)
(-------*-------)
(-------*-------)
--------+---------+---------+---------+6.0
9.0
12.0
15.0
Pooled StDev = 2.416
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0196
Critical value = 2.59
Control = level (244) of Total metal and arsenic conc.
Intervals for treatment mean minus control mean
Level
559
974
1585
Lower
-4.001
-9.061
-9.701
Center
-0.040
-5.100
-5.740
Upper
3.921
-1.139
-1.779
--------+---------+---------+---------+(----------*----------)
(----------*-----------)
(-----------*----------)
--------+---------+---------+---------+-7.0
-3.5
0.0
3.5
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.711
P-Value = 0.000
222
Tufted hairgrass – ARTS dilution series continued
One-way ANOVA: trans total biomass versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 1.458
Level
244
559
974
1585
N
5
5
5
5
DF
3
16
19
R-Sq = 76.75%
Mean
8.545
6.780
3.244
2.950
StDev
1.487
0.686
1.686
1.725
SS
112.21
33.99
146.20
MS
37.40
2.12
F
17.60
P
0.000
R-Sq(adj) = 72.39%
Individual 95% CIs For Mean Based on
Pooled StDev
----+---------+---------+---------+----(----*-----)
(----*-----)
(-----*-----)
(-----*----)
----+---------+---------+---------+----2.5
5.0
7.5
10.0
Pooled StDev = 1.458
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0196
Critical value = 2.59
Control = level (244) of Total metal and arsenic conc.
Intervals for treatment mean minus control mean
Level
559
974
1585
Lower
-4.155
-7.690
-7.985
Center
-1.765
-5.300
-5.595
Upper
0.625
-2.910
-3.205
--+---------+---------+---------+------(---------*--------)
(---------*--------)
(---------*--------)
--+---------+---------+---------+-------7.5
-5.0
-2.5
0.0
Correlations: trans total biomass, Total metals and arsenic
Pearson correlation of trans total bio and Total metal and arsenic conc. =
-0.806
P-Value = 0.000
223
Tufted hairgrass – ARTS dilution series continued
One-way ANOVA: Root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.1108
Level
244
559
974
1585
N
5
5
5
5
DF
3
16
19
R-Sq = 39.69%
Mean
0.2926
0.3324
0.2397
0.4576
StDev
0.0428
0.0466
0.1414
0.1585
SS
0.1292
0.1964
0.3256
MS
0.0431
0.0123
F
3.51
P
0.040
R-Sq(adj) = 28.38%
Individual 95% CIs For Mean Based on
Pooled StDev
---------+---------+---------+---------+
(-------*--------)
(--------*-------)
(--------*--------)
(--------*--------)
---------+---------+---------+---------+
0.24
0.36
0.48
0.60
Pooled StDev = 0.1108
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0196
Critical value = 2.59
Control = level (244) of Total metal and arsenic conc.
Intervals for treatment mean minus control mean
Level
559
974
1585
Lower
-0.1419
-0.2346
-0.0167
Center
0.0398
-0.0530
0.1650
Upper
0.2214
0.1287
0.3466
------+---------+---------+---------+--(-----------*-----------)
(-----------*------------)
(-----------*-----------)
------+---------+---------+---------+---0.15
0.00
0.15
0.30
Correlations: Root mass ratio, Total metals and arsenic
Pearson correlation of Root mass ratio and Total metals and arsenic =
0.408
P-Value = 0.074
224
Tufted hairgrass – Clark Fork dilution series
One-way ANOVA: Total percent emergence versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 15.31
Level
250.0
650.0
1334.0
1900.0
3525.0
5885.0
7521.0
N
5
5
5
5
5
5
5
DF
6
28
34
R-Sq = 16.08%
Mean
88.00
88.00
80.00
88.00
76.00
88.00
96.00
StDev
17.89
10.95
20.00
10.95
16.73
17.89
8.94
SS
1257
6560
7817
MS
210
234
F
0.89
P
0.513
R-Sq(adj) = 0.00%
Individual 95% CIs For Mean Based on
Pooled StDev
---------+---------+---------+---------+
(---------*--------)
(---------*--------)
(--------*---------)
(---------*--------)
(---------*--------)
(---------*--------)
(--------*--------)
---------+---------+---------+---------+
75
90
105
120
Pooled StDev = 15.31
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0108
Critical value = 2.73
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650.0
1334.0
1900.0
3525.0
5885.0
7521.0
Lower
-26.44
-34.44
-26.44
-38.44
-26.44
-18.44
Center
0.00
-8.00
0.00
-12.00
0.00
8.00
Upper
26.44
18.44
26.44
14.44
26.44
34.44
---------+---------+---------+---------+
(------------*------------)
(------------*------------)
(------------*------------)
(------------*------------)
(------------*------------)
(------------*------------)
---------+---------+---------+---------+
-20
0
20
40
Correlations: Total percent emergence, Total metals and arsenic
Pearson correlation of Total germination percentage (% and Total metals and
arsenic = 0.161
P-Value = 0.355
225
Tufted hairgrass – Clark Fork dilution series continued
One-way ANOVA: Shoot height (cm) versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 1.514
Level
250.0
650.0
1334.0
1900.0
3525.0
5885.0
7521.0
N
5
4
5
5
5
5
5
R-Sq = 87.24%
DF
6
27
33
Mean
10.180
11.800
7.820
8.480
4.200
2.120
2.203
StDev
0.835
2.189
1.057
2.340
1.956
0.687
0.535
SS
423.04
61.87
484.92
MS
70.51
2.29
F
30.77
P
0.000
R-Sq(adj) = 84.40%
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+(---*---)
(----*---)
(---*---)
(---*---)
(---*---)
(---*---)
(---*---)
--------+---------+---------+---------+3.5
7.0
10.5
14.0
Pooled StDev = 1.514
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0107
Critical value = 2.74
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650.0
1334.0
1900.0
3525.0
5885.0
7521.0
Lower
-1.164
-4.985
-4.325
-8.605
-10.685
-10.602
Center
1.620
-2.360
-1.700
-5.980
-8.060
-7.977
Upper
4.404
0.265
0.925
-3.355
-5.435
-5.353
-------+---------+---------+---------+-(------*------)
(-----*------)
(------*-----)
(------*------)
(------*-----)
(------*------)
-------+---------+---------+---------+--8.0
-4.0
0.0
4.0
Correlations: Shoot height (cm), Total metals and arsenic
Pearson correlation of Shoot height (cm) and Total metals and arsenic =
-0.875
P-Value = 0.000
226
Tufted hairgrass – Clark Fork dilution series continued
One-way ANOVA: trans total biomass versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.2444
Level
250.0
650.0
1334.0
1900.0
3525.0
5885.0
7521.0
N
5
4
5
5
5
5
5
R-Sq = 89.86%
DF
6
27
33
Mean
2.7130
2.4949
2.3039
2.0336
1.3012
1.2010
0.9455
StDev
0.1035
0.1887
0.4100
0.2039
0.2988
0.1201
0.2286
SS
14.2836
1.6122
15.8958
MS
2.3806
0.0597
F
39.87
P
0.000
R-Sq(adj) = 87.60%
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+(---*---)
(----*---)
(--*---)
(---*---)
(---*--)
(---*---)
(---*--)
--------+---------+---------+---------+1.20
1.80
2.40
3.00
Pooled StDev = 0.2444
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0107
Critical value = 2.74
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650.0
1334.0
1900.0
3525.0
5885.0
7521.0
Lower
-0.6675
-0.8328
-1.1031
-1.8355
-1.9357
-2.1912
Center
-0.2182
-0.4091
-0.6794
-1.4118
-1.5121
-1.7675
Upper
0.2312
0.0145
-0.2558
-0.9882
-1.0884
-1.3439
-+---------+---------+---------+-------(------*-----)
(-----*-----)
(-----*-----)
(-----*-----)
(-----*-----)
(-----*-----)
-+---------+---------+---------+--------2.10
-1.40
-0.70
0.00
Correlations: trans total biomass, Total metals and arsenic
Pearson correlation of trans total biomass and Total metals and arsenic =
-0.902
P-Value = 0.000
227
Tufted hairgrass – Clark Fork dilution series continued
One-way ANOVA: Root mass ratio versus Total metals and arsenic
Source
Total metal and arsenic
Error
Total
S = 0.1228
Level
250.0
650.0
1334.0
1900.0
3525.0
5885.0
7521.0
N
5
4
5
5
5
5
5
R-Sq = 26.93%
DF
6
27
33
Mean
0.3775
0.3713
0.4286
0.3934
0.3797
0.2928
0.2209
StDev
0.0335
0.0519
0.0366
0.0406
0.1673
0.1162
0.2327
SS
0.1500
0.4071
0.5571
MS
0.0250
0.0151
F
1.66
P
0.170
R-Sq(adj) = 10.69%
Individual 95% CIs For Mean Based on
Pooled StDev
-+---------+---------+---------+-------(--------*---------)
(----------*---------)
(---------*--------)
(---------*--------)
(---------*--------)
(--------*---------)
(--------*---------)
-+---------+---------+---------+-------0.12
0.24
0.36
0.48
Pooled StDev = 0.1228
Dunnett's comparisons with a control
Family error rate = 0.05
Individual error rate = 0.0107
Critical value = 2.74
Control = level (250) of Total metal and arsenic concent
Intervals for treatment mean minus control mean
Level
650.0
1334.0
1900.0
3525.0
5885.0
7521.0
Lower
-0.2320
-0.1617
-0.1969
-0.2107
-0.2976
-0.3695
Center
-0.0062
0.0511
0.0159
0.0022
-0.0847
-0.1566
Upper
0.2196
0.2640
0.2288
0.2151
0.1282
0.0563
---+---------+---------+---------+-----(-------------*-------------)
(------------*-------------)
(------------*------------)
(------------*------------)
(-------------*------------)
(------------*-------------)
---+---------+---------+---------+------0.32
-0.16
0.00
0.16
Correlations: Root mass ratio, Total metals and arsenic
Pearson correlation of Root mass ratio and Total metals and arsenic
= -0.460
P-Value = 0.006