United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/R-99/069
August 1999
vvEPA
Potential Species for
Phytoremediation of
Perch I orate
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EPA/600/R-99/069
August 1999
POTENTIAL SPECIES FOR
PHYTOREMEDIATION OF PERCHLORATE
by
Sridhar Susarla1, Ph.D.
Sydney T. Bacchus, Ph.D.
Steven C. McCutcheon, Ph.D., P.E.
N. Lee Wolfe, Ph.D.
J(NRC Research Associate)
United States Environmental Protection Agency
National Exposure Research Laboratory
Ecosystems Research Division
960 College Station Road
Athens, GA 30605
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DISCLAIMER
The information in this document has been funded wholly or in part by the United States
Environmental Protection Agency. It has been subject to the Agency's peer and administrative
review, and it has been approved for publication as an EPA document. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
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FOREWORD
Perchlorate has contaminated surface and groundwater resources at various locations in the
United States. Concern over perchlorate contamination is due to the influence of this compound on
thyroid gland function. As a potential endocrine disrupter, perchlorate can threaten the health of
both human and wildlife populations.
Technologies commonly used to remove contaminants from ground and surface water either
are ineffective for remediation of perchlorate or too expensive for large-scale use. Microbial
transformation of perchlorate has not been accomplished for concentrations prevalent at
contaminated sites. This project was the first to evaluate the ability of vascular plants to remove
perchlorate from solution at field concentrations and transform this contaminant into an innocuous
end product. The results of these experiments suggest that vascular plants provide a rapid,
inexpensive option for remediation perchlorate-contaminated sites. Site-specific "prescriptions" will
be required for on-site remediation.
Rosemarie C. Russo
Director
Ecosystems Research Division
Athens, Georgia
m
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EXECUTIVE SUMMARY
Phytoremediation is the use of plants to cleanse soil and water contaminated with organic
or inorganic pollutants. This promising new field of research can be used for in situ clean up of
large volumes and expansive areas of contaminated soils or waters, including ground water. Three
laboratory-scale experiments were conducted to: 1) evaluate the ability of selected terrestrial,
wetland, and aquatic plants to remove perchlorate from an aqueous solution; 2) compare the
performance of different age classes of one plant species; 3) evaluate the role of nutrients on
perchlorate removal; 4) determine the fate of perchlorate removed from solution (e.g., plant tissue
distribution; accumulation vs. breakdown); 5) document external plant responses to perchlorate; and
6) predict field-scale performance of the plant species evaluated. Perchlorate concentrations of 0.2,
2.0, and 20 ppm were tested in aqueous and sand treatments for ten-day periods in each experiment.
Thirteen vascular plant species were selected for evaluation in these initial experiments.
Four were trees, one was an herbaceous upland species, four were herbaceous wetland species, and
four were herbaceous aquatic species. The species of trees included cabbage gum (Eucalyptus
amplifolid), sweetgum (Liquidambar styraciflua), eastern cottonwood (Populus deltoides), and
black willow (Salix nigrd). Tarragon (Artemesia dracuncularus saliva) was the herbaceous upland
species. The herbaceous wetland species were pickleweed (aka iodine bush, Allenrolfea
occidentalis), blue-hyssop (Bacopacaroliniana), smartweed (Polygonumpunctatuni), and perennial
glasswort (Salicornia virginica). Aquatic species were waterweed (Elodea canadensis),
parrot-feather (Myriophyllum aquaticum), fragrant white water-lily (Nymphaea odorata), and
duckmeat (Spirodelapolyrhiza).
A preliminary sorption experiment with unwashed sand and no plants revealed that 50-64%
of perchlorate in solution became adsorbed to the sand, displacing chloride. Consequently, for
treatments with unwashed sand and plants in the subsequent three experiments, the free chloride ions
in solution were available to be taken up by the plants. When perchlorate concentrations exceeded
2.0 ppm in unwashed sand treatments, an option was available for plants to take up excess
perchlorate, rather than chloride ions, from the solution.
Perchlorate was depleted from solution in the presence of all but two species (waterweed and
duckmeat). The mass of perchlorate depleted (g/kg wet weight) was classified into five general
categories (0 = no depletion; 1-99 = minimal depletion; 100-499 = moderate depletion; 500-999 =
moderately high depletion; and >1000 = high depletion). None of the tree species tested, nor the
herbaceous upland species tested were included in the highest category of performance. Wetland
and aquatic plants included in the highest category were blue-hyssop, perennial glasswort, and
parrot-feather. Results in the moderately-high category were obtained for one species of tree
(cabbage gum), the herbaceous upland species (tarragon), and three of the four herbaceous wetland
species evaluated (pickleweed, smartweed, and glasswort).
Depletion was calculated as a first-order kinetics reaction, with k values (day-1) in sand
treatments in the range of 0-0.22 for cabbage gum, sweetgum, rooted green-wood cuttings of
cottonwood, willow, pickleweed, smartweed, glasswort, and water-lily. Upper values for rooted
mature-wood cuttings of cottonwood, blue-hyssop, and parrot-feather were 0.31, 0.34, and 0.41,
respectively. The range for tarragon was 0.48-0.77. Plant tissues (e.g., roots, stems, leaves) were
analyzed from selected samples, based on maximum drop in perchlorate concentration, for each of
the 11 species for which perchlorate depletion was observed. Perchlorate, or transformation
iv
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metabolites (chlorate, chlorite, chloride) were observed in all tissues analyzed. Future work should
include: 1) a quantitative analysis of different plant tissues (e.g., roots, stems, leaves), and 2)
radiolabeled chloride to determine the amount and source of chloride contained within the plants.
Results suggested that significant influences on depletion of perchlorate include: 1) plant
species present, 2) concentration of perchlorate, 3) substrate (sand versus aqueous treatments), 4)
the presence or absence of nutrients, 5) stage of plant maturity, and 6) the presence of chloride ions.
Characteristics of cottonwood and willow cuttings obtained from a site with perchlorate in the
ground water, and incorporated into these experiments suggested that fungal pathogens may be
present in the donor plants on the site. Fungal pathogens, if present, may have influenced the
performance of these plants in the experiment. Conversely, exposure of plants to perchlorate may
create stresses that result in predispostion of the plant to infection by plant pathogens. Evaluation
of these factors was not within the scope of these initial experiments, but should be addressed in
future experiments. Another important aspect not evaluated in these short-term experiments was
the potential environmental hazard to wildlife that may consume plants used for phytoremediation,
that contain high concentrations of perchlorate. Future experiments of longer duration should
provide more information regarding the degree to which perchlorate is accumulated in plant tissue,
and any potential threat to wildlife. The experimental design of the final two experiments, and the
short duration of these experiments suggest that external microbes and algal contaminants were not
involved in the depletion of perchlorate observed in these experiments.
Based on the results of these experiments and ecological knowledge of the species evaluated,
the following species are recommended for initiating future research for phytoremediation of
perchlorate, and are grouped by the type of phytoremediation for which they appear to be suited.
Additional research using sweetgum, eastern cottonwood, and black willow is recommended for in
situ phytoremediation of contaminated soils in uplands, including areas with shallow ground water
accessible to plant roots, and if production of biomass for harvest is of interest. For in situ
phytoremediation of contaminated areas that are saturated or inundated periodically, or for wetlands
created for phytoremediation, additional research using blue-hyssop, smartweed, and perennial
glasswort is recommended. Additional research using parrot-feather and fragrant white water-lily
is recommended for in situ phytoremediation of contaminated water bodies, or for ponds created
artificially for phytoremediation of contaminated surface water or extracted ground water. Finally,
extracts from tarragon may be useful for injection into mechanized flow-through systems where
ground water is extracted, exposed to these compounds, then reinjected into the aquifer, or for
similar flow-through systems for contaminated surface water.
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TABLE OF CONTENTS
Page
Forward iii
Executive Summary iv
List of Figures vii
List of Tables viii
List of Appendices viii
Acknowlegements ix
1.0 Introduction 1
1.1 Background and Health Implications 1
1.2 Remediation Approaches 1
2.0 Objectives 2
3.0 Experimental Methods 3
3.1 Selection Criteria for Plant Species Evaluated 3
3.2 Sorption Experiment 5
3.3 Kinetics Experiments 6
3.4 Tissue Extraction 10
3.5 Analytical Methods 11
3.5.1 Analysis of Chloride, Perchlorate, and Metabolites 11
3.5.2 Tissue Analysis 11
3.5.3 Data Analysis 11
4.0 Results and Discussion 12
4.1 Adsorption of Perchlorate and Depletion Characteristics 12
4.2 Kinetics of Perchlorate Depletion from Solutions 21
4.3 General External Plant Responses and Transformation 21
4.3.1 Favorable Response 21
4.3.2 No Response 21
4.3.3 Moderate Adverse Response 25
4.3.4 Severe Adverse Response 27
4.4 Predicted Field-Scale Performance and
Possible Mechanisms for Depletion of Perchlorate 27
4.4.1 Trees 28
4.4.2 Upland Herbs 32
4.4.3 Wetland Herbs 34
4.4.4 Aquatic Herbs 35
5.0 Summary and Recommendations 36
References 38
VI
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LIST OF FIGURES
Page
Figure 1. First Set of Initial, Ten-Day Perchlorate/Nutrient
Kinetics Experiments Using Tarragon, Waterweed
and Parrot-feather
Figure 2. Second Set of Initial, Ten-Day Perchlorate/Nutrient
Kinetics Experiments Using Black Willow,
Pickleweed and Duckmeat
Figure 3. Third Set of Initial, Ten-Day Perchlorate/Nutrient
Kinetics Experiments Using Cabbage Gum,
Sweetgum, Eastern Cottonwood (Seedlings,
Mature-wood Cuttings, and Green-wood
Cuttings), Blue-Hyssop, Smartweed,
Perennial Glasswort, and Fragrant White
Water-lily 9
Figure 4. Depletion of Perchlorate (20 ppm) from Solution
in the Presence of Cabbage Gum: Unwashed Sand +
Deionized Water Treatment 13
Figure 5. Depletion of Perchlorate (20 ppm) from Solution
in the Presence of Sweetgum: Unwashed Sand +
Nutrient Solution Treatment 13
Figure 6. Depletion of Perchlorate (20 ppm) from Solution
in the Presence of Black Willow: Unwashed Sand +
Deionized Water Treatment 14
Figure 7. Depletion of Perchlorate (20 ppm) from Solution
in the Presence of Tarragon: Unwashed Sand +
Nutrient Solution Treatment 14
Figure 8. Depletion of Perchlorate (20 ppm) from Solution
in the Presence of Parrot-feather: Deionized
Water (No Sand) Treatment 15
Figure 9. Depletion of Perchlorate (20 ppm) from Solution
in the Presence of Pickleweed: Washed Sand +
Deionized Water Treatment 15
Figure 10. Potential Fate of Perchlorate in Plant Systems 29
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LIST OF TABLES
Page
Table 1. Plant Species Evaluated in Initial Perchlorate/
Nutrient Experiments 4
Table 2. Summary of Perchlorate Depletion from
Solution in the Presence of Plants 16
Table 3. Percent of Perchlorate Depletion from
Solution in the Presence of Plants 17
Table 4. Mass of Perchlorate Depletion from
Solution in the Presence of Plants 18
Table 5. First Order Kinetics of Perchlorate Depletion from
Solution in the Presence of Plants 19
Table 6. Summary of First Order Kinetics of Perchlorate
Depletion from Solution in the Presence of Plants 20
Table 7. Categorization of Plant Species Based on General
Responses to Perchlorate/Nutrient Treatments
During Ten-Day Experiments 22
Table 8. General External Responses of Plants with
Positive Results During Ten-Day Perchlorate/
Nutrient Experiments 23
Table 9. Possible Mechanisms for Depletion of Perchlorate
from Solution Based on Plant Responses During
Ten-Day Experiments 24
Table 10. Metabolites of Perchlorate Transformation
Identified in Plant Tissues 26
LIST OF APPENDICES
Appendix A. Sources of Plant Species Evaluated A-l
Appendix B. Contents of Peters Professional All Purpose Plant Food B-l
Appendix C. Related Peer-Reviewed Publications and Conference Presentations . . C-l
Vlll
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ACKNOWLEDGMENTS
The authors express their gratitude to Valentine Nzengung and Chuhua Wang, UGA Geology
Department, for comparative analysis of samples from the chloride displacement experiment, and
initial tissue analysis for perchlorate; and to Rupert Simon and George Yager for technical support
for the ion chromatography equipment. Greg Harvey, Wright Patterson Air Force Base Project
Manager for this research, supplied critical background information and arranged for cottonwood
and willow cuttings to be obtained from the Carswell Air Force Base site. Kerry Britton and Paula
Spaine, USDA Forest Service, identified the fungal pathogen in the cottonwood cuttings and
provided additional information about the pathogen. Ed Glenn and Jaqueline Garcia provided
speciments of pickleweed and guidance on acclimation of the plants prior to initiation of the
experiment. James Burnett and Red Gidden facilitated collection of a limited number of crucial
species for testing from the St. Marks National Wildlife Refuge. Andy Tull, Melanie Smith, and
Mark Zimmerman provided horticultural guidance and care for research plants maintained in the
UGA Botany greenhouses. Colleagues Amy Bergstedt, Stacy Lewis, Jane Overton, and Jason
Tucker provided assistance in collecting, maintaining or preparing some of the plants used in these
experiments, Chris Mazur provided initial plants for the duckmeat culture, and John Kennecke
contributed photographic expertise. Stacy Lewis also provided salient comments on general aspects
of phytoremediation for consideration. Candace Halbrook and Kay Millar with EPA's Region IV
Laboratory provided laboratory support during the early phase of the project. Computer guidance
and support was provided by OAO Corporation, and administrative support was provided by Vera
Madison and Brandy Manders.
IX
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1.0 INTRODUCTION
1.1 Background and Health Implications
Perchlorate, chlorate, chlorine dioxide and hypochlorite are produced on a large scale by the
chemical industry, for a wide range of applications. Ammonium perchlorate is an oxyanion that has
been used extensively as a strong oxidizing agent in solid rocket fuel (Ataway and Smith, 1993).
Perchlorate must be removed from inventory periodically and replaced with a fresh supply, because
of its shelf life. Contamination of ground water has occurred as the result of incidental discharges
of perchlorate fuel used in rockets and activities associated with World War II, Korea and Viet Nam.
The problem was compounded by liberal disposal practices during the 1950s through the 1970s,
prior to expanded knowledge of the impacts of these fuels on soils and water resources (California
Department of Health, 1998).
Wastewater generated from the manufacturing, maintenance, and testing of solid rocket
propellants can contain ammonium perchlorate concentrations in the range of grams per liter.
(Herman and Frankenberger, 1998). Ammonium perchlorate also is used in the production of
explosives, pyrotechnics and blasting formulations. Other perchlorate formulations are used in dry
batteries and oxygen-generating systems (Malmquist et a/., 1991). The large solid rocket motor
disposal inventory currently has 55 million pounds of propellant ready for treatment. Over the next
8-10 years this amount is expected to increase to 164 million pounds of solid propellant targeted for
disposal (Phillips Laboratory, 1997). Innovations in fuel handling methods have reduced current
contamination; however, the United States Air Force is attempting to clean-up past spills to attain
environmental quality goals in balance with national defense needs.
Perchlorate currently is not regulated under the safe drinking water act, although the
California Department of Health Services has established an action level for perchlorate in drinking
water of 18 micrograms per liter (California Department of Health, 1998). The primary human
health concern related to perchlorate is that chronic or longterm exposure can interfere with the
thyroid gland's ability to utilize iodine to produce thyroid hormones required for normal body
metabolism, as well as growth and development (Stanbury and Wyngaarden, 1952). Information
on the health effects and toxicology of perchlorate is limited. The majority of data available
regarding impacts of perchlorate on humans is from clinical reports of patients treated with
potassium perchlorate for hyperthyroidism resulting from an autoimmune condition known as
graves' disease (AWWARF, 1998). Potassium perchlorate continues to be used diagnostically to
test thyroid hormone production. The effect of perchlorate on thyroid hormone function is the
competitive inhibition of iodide anion uptake into the thyroid gland by perchlorate anion, resulting
in reduced thyroid hormone production. Iodine deficiencies in pregnant women are detrimental to
fetal development. Interference with the normal function of the thyroid gland suggests that
perchlorate is an endocrine disrupter (van Wijk and Hutchinson, 1995).
1.2 Remediation Approaches
A number of physical and chemical processes to treat perchlorate-contaminated sites are
under consideration. Some of the processes have been tested with limited success. The chlorine in
perchlorate is at an oxidation state of plus seven, which is much higher than the minus one oxidation
state of chloride, the most stable form of chlorine in water. This would seem to favor processes
based on chemical reduction. However, pechlorate has found to be resistant to chemical reduction
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processes. The factor that contributes to the chemical stability of dissolved perchlorate are the four
oxygen atoms surrounding each chlorine. This results in a completely filled outer electron shell and
tetrahedral packing of the four oxygen atoms. Consequently the charge is distributed evenly around
a relatively large surface area. Therefore, a large activation energy is required to disrupt the stable
structure of perchlorate to allow the very thermodynamically favorable reduction reaction to
proceed. Because of the stability of perchlorate under most environmental conditions, reduction
processes have failed in remediating perchlorate-contaminated waters (van Ginkle et a/., 1995).
Perchlorate salts dissociate in water to form perchlorate anions and are highly soluble (>200
g/L). Therefore, volatilization technologies such as air-stripping are ineffective. Activated carbon,
another common technology used for potable and wastewater treatment, also is ineffective in
treating perchlorate-contaminated waters (AWWARF, 1998). Ion-exchange technology has
potential for remediation, but is not used widely due to its high cost (Glass, 1998). Other advanced
processes for the removal of perchlorate, such as reverse osmosis and nanofiltration technologies,
also are expensive.
A biological process developed at Tyndall Air Force Base (AFB) consists of a two-step
process, an anaerobic reactor followed by an aerobic reactor (Wallace et a/., 1998). Development
of this process has progressed from laboratory scale, to bench scale, to a pilot scale facility. In this
microbial system, the perchlorate is reduced to chloride ions and oxygen. This process has been
applied successfully to input water with a 9000 ppm perchlorate concentration, reducing the
perchlorate to below 500 ppb (van Ginkle et a/., 1996 and Rikken et a/., 1996). However, the
system has not been evaluated at lower concentrations that are prevalent at contaminated sites.
Additionally, the current understanding of microbial reduction of perchlorate by pure cultures is
limited because the sequential reduction of perchlorate to chlorate, chlorite, and ultimately chloride
and oxygen, has not been studied in detail. In summary, no proven technology is available for the
treatment of large volumes of water or soil containing relatively low concentrations of perchlorate.
Consequently, the development of efficient and cost effective strategies for the remediation of
perchlorate contaminated sites is of immense interest.
Phytoremediation, using plants to cleanse soil and water contaminated with organic or
inorganic pollutants, is an alternative strategy. Plant-based systems provide an attractive
remediation strategy because complete transformation of the compound to the end products (chloride
ion and oxygen) occurs. This developmental process could be used for on-site and in situ clean-up
of large volumes and expansive areas of contaminated soils or waters, including ground water.
Initial experiments reported herein have been performed to elucidate the environmental behavior and
fate of perchlorate using selected vascular plants that may have potential for remediating sites
contaminated with perchlorate.
2.0 OBJECTIVES
Laboratory-scale experiments were conducted to: 1) evaluate the ability of selected upland,
wetland, and aquatic plants to remove perchlorate from solution; 2) compare the performance of
different age classes of single plant species; 3) evaluate the role of nutrients and chloride on
perchlorate removal; 4) determine the fate of perchlorate removed from solution (e.g., distribution
throughout the plant; accumulation vs. breakdown); 5) document external plant responses during
the experiments; and 6) predict field-scale performance of the plant species evaluated.
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3.0 EXPERIMENTAL METHODS
3.1 Selection Criteria for Plant Species Evaluated
A total of 13 plant species were selected for evaluation in the initial, laboratory-scale
experiments. Four of the species were trees, one was a herbaceous upland species, four were
herbaceous wetland species, and four were aquatic species (Table 1). The species of trees included
cabbage gum (Eucalyptus amplifolia), sweetgum (Liquidambar styraciflud), eastern cottonwood
(Populus deltoides), and black willow (Salix nigrd). The herbaceous upland species selected was
tarragon (Artemesia dracuncularus sativd). The four herbaceous wetland species included pickleweed
(aka iodine bush, Allenrolfea occidentalis), blue-hyssop (Bacopa caroliniana), smartweed (Polygonum
punctatum), and perennial glasswort (Salicornia virginica). Four aquatic species also were selected,
and included waterweed (Elodea canadensis), parrot-feather (Myriophyllum aquaticum\ fragrant white
water-lily (Nymphaea odorata), and duckmeat (Spirodelapolyrhizd).
The primary determinant in selection of plant species for these initial experiments was plants
of specific interest to the United States Air Force. Specifically, these included the four tree species
listed above, tarragon, and pickleweed. Two additional tree species, sycamore (Platanus occidentalis)
and hackberry (Celtis laevigata) also were of interest to the Air Force for inclusion in these initial
experiments. Unfortunately, commercially-grown seedlings of these species could not be located
during the time period that these experiments were being conducted and these species reportedly do
not root readily from cuttings. Seedlings of sycamore, sweetgum, and possibly hackberry can be
obtained from the Texas Forest Service for approximately $20/100, but may require a request a year
in advance for collection of seed. Orders are placed in early October, and seedlings that are ordered
are shipped, bare root, in January or February of the following year (Larry Schaapveld, Texas Forest
Service, pers. comm., 6/18/98). The germination rate for sycamore reportedly is approximately 10%,
consequently few growers carry sycamore (Helen Matthews, Rennerwood Nursery, pers. comm., 7/98).
The tree species referenced above were of interest to the Air Force for several reasons. First,
trees generally have the ability to develop extensive root systems that are capable of colonizing large
expanses of soil in areas where the water table may be contaminated with compounds such as
perchlorate. Second, the species of trees selected are relatively rapid-growing trees, which may have
higher rates of transpiration than trees that grow more slowly. If more water is transpired, theoretically
more ground water containing a given pollutant, such as perchlorate, can be removed from the system
and treated. If trees planted for phytoremediation grow rapidly, they provide a potential for
commercial biomass production, which means that they can be harvested and sold as wood, pulp, or
fuel products to help defray the cost of site-decontamination. Finally, the referenced species are
thought to be tolerant of a wide range of growing conditions, and might be adaptable to contaminated
sites at various locations throughout the United States.
Tarragon, one of the two herbaceous plants selected by the Air Force, was chosen based on the
ability of enzymes released from its crushed leaves to transform perchlorate (Nzengung, unpub. data).
Pickleweed, the other herbaceous species selected by the Air Force, is a succulent, high elevation
desert plant that occurs in coastal areas and playas (old land-locked lake beds) throughout the Great
Basin, from the southwestern U. S. (e.g., Nevada, New Mexico, Texas, Wilcox Playa in Arizona, the
Chihuahua Desert) to Mexico (e.g., Baja California, Sonora). This species may be the most
salt-tolerant plant in the country (Phil Jenkins, pers. comm. 6/11/98), and is capable of tolerating large
concentrations of salts (generally chlorides) in soils saturated with brackish or saline water. Plants with
these characteristics are known as halophytes. Halophytes may grow in areas of high salt content
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Table 1. Plant Species Evaluated in
Initial Perchlorate/Nutrient Experiments
Scientific Names
Eucalyptus amplifolia Naud.
Liquidambar styraciflua L.
Populus deltoides Bartr.ex Marsh.
seedlings
mature-wood cuttings **
green-wood cuttings **
Salix nigra L.
Trees
Common Names*
cabbage gum (3)
sweetgum (3)
eastern cottonwood (3)
black willow (2)
Upland Herbs
Artemisia dracunculus var. sativa L.
tarragon (1)
Wetland Herbs
Allenrolfea occidentalis (Watson) Kuntze.
Bacopa caroliniana (Walt.) Robins.
Polygonum punctatum Ell.
Salicornia virginica L.
pickleweed (2)
blue-hyssop (3)
smartweed (3)
perennial glasswort (3)
Elodea canadensis Rich, in Michx.
Myriophyllum aquaticum (Veil.) Verde.
Nymphaea odorata Ait.
Spirodelapolyrhiza (L.) Schleid.
Aquatic Herbs
waterweed (1)
parrot-feather (1)
fragrant white water-lily (3)
duckmeat (2)
* number in parenthesis designates experiment to which plant was assigned
** cuttings from Carswell Air Force Base, rooted in the laboratory
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by one of several means. They may have evolved mechanisms to exclude salts from entering through
their roots, or to extrude salts through their leaves or other organs. Some have developed a specialized
physiology to tolerate high salt content in their tissue. The latter mechanism of tolerance is the process
used by pickleweed. Unlike some halophytes, pickleweed requires high concentrations of ions such as
chloride to maintain its osmotic balance. This characteristic, and the presence of chloride with
perchlorate formed the basis for selection of this species.
Perennial glasswort is in a closely related genus of the same family (Chenopodiaceae) as
pickleweed. Perennial glasswort is a rhizomatous perennial, occurring in salt and brackish marshes and
flats along the Atlantic coast from New Hampshire to south Florida, westward along the Gulf coast from
Florida to Texas, from California to British Columbia, the West Indies, western Europe, and north
Africa (Godfrey and Wooten, 1981). It was selected for evaluation by the researchers because it has
the same physiological traits as pickleweed. It also has an extensive geographic range, appears to grow
more rapidly than pickleweed, and is more tolerant of transplanting and vegetative propagation.
The remaining two herbaceous wetland species, blue-hyssop and smartweed, were selected by the
researchers because of their widespread accessibility, ease of vegetative propagation, and apparent
robustness. Blue-hyssop occurs naturally in wetlands and open water systems throughout the Coastal
Plain, southeast from Virginia to south Florida, and westward to east Texas (Godfrey and Wooten,
1981). The species of smartweed selected by the researchers for this experiment is a vigorous perennial,
also occurring in wetlands and open water systems, but with a more extensive range than blue-hyssop.
Our test species of smartweed occurs throughout most of temperate and subtropical North America, and
tropical South America (Godfrey and Wooten, 1981).
Four aquatic species with different growth characteristics were selected by the researchers to test
the ability of aquatic plants to remove or transform perchlorate in the water column. The selected
species of aquatics all have potentially broad geographic ranges, are easily propagated, and are
relatively fast growing. Waterweed has dense leaves that can extend throughout the water column and
across the surface of the water. It has few roots, and does not need to be rooted in a substrate.
Parrot-feather has both submersed and emergent leaves, few roots, and also does not need to be rooted
in a substrate. Fragrant white water-lily has large floating leaves that extend from thick rhizomes
(horizontal stems) that creep along the substrate. Numerous large, spongy roots grow from the rhizome
to anchor the plant to the substrate. The hardiness, large leaves and rhizomes, and the ease of
reproducing this species contributed to its selection. The final species, duckmeat, was selected because
it is a floating-leaved aquatic that reproduces relatively rapidly, is easily transported, and is thought to
be adaptable to a wide geographic area. The working hypothesis for this species was that enzymes
released from dying or decomposing plants might transform perchlorate.
Taxonomic, range and habitat information can be obtained for cabbage gum from Mabberley
(1997), for the remaining tree species from Godfrey (1988), for tarragon from Bailey and Bailey (1976),
for pickleweed from Jaeger (1947), and for the remaining species from Godfrey and Wooten (1979 and
1981). A description of the sources of the plants, transport, and acclimation procedures is provided in
Appendix A.
3.2 Sorption Experiment
Sand was selected as the solid substrate for the perchlorate experiments. A small-scale sorption
experiment was conducted prior to initiation of the perchlorate/nutrient experiments to determine
whether chloride ions were associated with the sand. If chloride ions were present, these ions could be
displaced into the solution in the presence of perchlorate, as the perchlorate adsorbed to the sand grains.
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A series of test tubes containing 5 g of unwashed sand (All Purpose, Setcrete, Inc.) per test tube was
equilibrated withl 0 mL of sodium chloride solution at one of five test concentrations (0, 50,100,200,
400 mg/L) for 24 h. The test tubes were shaken on a rotary shaker overnight at room temperature. After
saturation with sodium chloride solution, the supernatant solution was replaced with 10 mL of 200 ppm
perchlorate solution and shaken overnight. The following day, 1 mL of solution was filtered and
analyzed for chloride and perchlorate concentrations using Dionex lon-chromatography. The
experiment was duplicated.
3.3 Kinetics Experiments
Laboratory-scale experiments were conducted for an initial determination of the kinetics of
perchlorate depletion from solution in the presence of selected species of trees, upland, wetland, and
aquatic herbs. Three concentrations of perchlorate, differing by an order of magnitude each (0.2,2.0,
and 20.0 ppm), were selected for evaluation (Figure 1). The highest concentration of perchlorate
evaluated was selected because it is equivalent to field concentrations of groundwater contamination
at sites of interest. The first run of these experiments contained three treatments with three
experimental units per treatment and one concentration of perchlorate per unit, as shown in Figure 1.
Run 1 also included two control treatments (Figure 1). Each experimental unit consisted of a 600-mL
beaker and an experimental plant (for duckmeat, 3 g wet weight of plants were used), in addition to the
other assigned treatment components. The experimental design for Run 1, shown in Figure 1, was used
to evaluate tarragon, waterweed, and parrot-feather. All purpose sand (Setcrete, Inc.) was used in all
treatments except the "no-sand" treatments. The influence of nutrients on perchlorate depletion was
evaluated by adding a dilute solution (0.1 g/L) of Peters Professional All Purpose Plant Food to some
of the treatments, with the paired treatments containing an equivalent volume of deionized (DI) water.
The contents of the nutrient source are provided in Appendix B.
For treatments containing sand, approximately 320 g of sand were weighed into each beaker and
300 mL of perchlorate solution at a known concentration (0.2-20.0 ppm) was added. Perchlorate
solutions were made either in Peters solution or in DI water prior to the run. Comparable specimens
of the three experimental species of plants for Run 1 were selected and numbered sequentially for each
species. Each plant then was assigned randomly to one of the treatments. The bare roots were rinsed
in DI water to free any remaining soil, and the roots were pressed lightly between paper toweling to
remove excess water. The fresh weight of each plant was determined and recorded prior to placement
of the plant in the beaker. For treatments with sand, the assigned plant was placed in the beaker in such
a manner that the surface of the sand was at the top of the root zone. For treatments without sand, the
assigned plant was maintained in an erect position by placing four strips of tape across the top of the
beaker in a cross-hatch pattern on four sides of the stem.
The top of each beaker was covered loosely with a plastic wrap to minimize loss of solution due
to evaporation. The experimental units were placed under Sylvania spot gro lights (150w, 120v), that
provided a full photo synthetic spectrum at 400 to 500 E m-2 s-1 at 20-30 cm above the plant. Lights
remained on continuously for all experiments. The experiments were conducted at ambient laboratory
temperature (20o C). The pH of the no-sand and washed-sand treatments was 7.0, while for
unwashed-sand it was at 8.5.
Based on the results of the sorption experiment and responses of some of the plants tested in the
initial perchlorate/nutrient experiments, some of the plant responses appeared to be due to chloride
toxicity. Consequently, the original experimental design was refined for the subsequent two
experiments to resolve these problems, and to incorporate other improvements. The modified designs
and plant species used for the two subsequent experiments are provided in Figures 2 and 3.
-------
1:
Treatment 2:
of
Ca = 0.2 ^ = 2 ppm C =20 ppm
o o
3;
o o o
4;
5;
KEY
Treatment 1: plant + ynwashed sand -j- Peters solution +perchlorate
Treatment 2: plant + Peters solution + perchlorate
Treatment 3: unwashed sand -+• Peters solution + psrchorate
Treatment 4: plant + unwashed sand + Peters solution
Treatment 5: plant + Peters solution
= 600 ml
Figure 1. First Set of Initial, Ten-Day Perchlorate/Nutrient Kinetics Experiments Using
Tarragon (n=l 1), Waterweed (n=l 1) and Parrot-feather (n=ll).
-------
Treatment 1:
Treatment 2:
Treatments 3 - 5:
Concentrations of Perchlorate
Ca= 0.2 ppm cb= 2 ppm C = 20 ppm
o o o
Black Willow, Pickleweed & Duckmeat
O O O
Black Willow & Duckmeat
O
Black Willow, Pickleweed & Duckmeat
Treatment 6:
Treatment 7:
Black Willow, Pickleweed & Duckmeat
Black Willow & Duckmeat
KEY
Treatment 1: plant + unwashed sand + Peters solution + perch I orate
Treatment 2: plant + Peters solution + perch I orate
Treatments: plant + washed sand + Peters solution + perchlorate
Treatment 4: plant + unwashed sand + Dl water + perchlorate
Treatments: plant + washed sand + Dl water + perchlorate
Treatments: plant + unwashed sand (control)
Treatment 7: plant + Peters solution (control)
O
= 600 ml beaker
Figure 2. Second Set of Initial, Ten-Day Perchlorate/Nutrient Kinetics Experiments Using
Black Willow (n=l 1), Pickleweed (n=7) and Duckmeat (n=l 1).
-------
Treatment 1:
Treatment 2:
Concentrations of Perchlorate
Ca= 0.2 ppm Cb= 2 ppm C = 20 ppm
o o o
Cabbage Gum, Sweetgum, Cottonwood seedlings, Cottonwood mature, Cottonwood green,
Blue-hyssop, Smartweed, Perennial Glasswort & Water-lily
O O O
Sweetgum, Cottonwood seedlings, Cottonwood mature,
Blue-hyssop, Smartweed & Water-lily
Treatments 3-5:
O
Cabbage Gum, Sweetgum, Cottonwood seedlings,
Blue-hyssop, Smartweed & Perennial Glasswort
Treatments: I00*0'
Cabbage Gum, Sweetgum, Cottonwood seedlings, Cottonwood mature, Cottonwood green,
Blue-hyssop, Smartweed, Perennial Glasswort & Water-lily
Treatment 7: ( Contr°l
Sweetgum, Cottonwood seedlings, Cottonwood mature,
Blue-hyssop, Smartweed & Water-lily
KEY
Treatment 1: plant + unwashed sand + Peters solution + perchlorate
Treatment 2: plant + Peters solution + perchlorate
Treatments: plant + washed sand + Peters solution + perchlorate
Treatment 4: plant + unwashed sand + Dl water + perchlorate
Treatment 5: plant + washed sand + Dl water + perchlorate
Treatment 6: plant + unwashed sand (control)
Treatment?: plant + Peters solution (control)
O
= 600 ml beaker
Figure 3. Third Set of Initial, Ten-Day Perchlorate/Nutrient Kinetics Experiments Using
Cabbage Gum (n=7), Sweetgum (n=ll), Eastern Cottonwood (Seedlings (n=ll)
Mature-wood Cuttings (n=8), and Green-wood Cuttings (n=4)), Blue-hyssop (n=l 1),
Smartweed (n=l 1), Perennial Glasswort (n=7), and Fragrant White Water-lily (n=8).
-------
In experiments 2 and 3, the sand to be used in Treatments 3 and 5 was washed to remove free
chloride. The initial wash was with tap water followed by distilled water. After draining excess
water, the sand was dried in an oven at 60o C overnight. Additionally, to eliminate the potential
influence of microorganisms, all solutions, unwashed-sand and washed-sand were autoclaved at
12 lo C for 30 min. In experiments 2 and 3, auto-timers provided a day length of 14 h, mimicking
the current seasonal photoperiod. Other modifications included extending the thin plastic sheet over
each test plant to minimize bias due to transpirational losses from plants extending above the top of
the beakers (e.g., trees) as compared with those contained within the beakers (e.g., submerged
aquatic species).
The species assigned to each run are listed in Table 1, with the designated run number in
parentheses following the common name. Plants were assigned to treatments randomly for all three
experiments, with roots rinsed and plants weighed, as described above. The solution from each
beaker was sampled daily at approximately 5 pm for the 10-day duration of each experimental run
to determine perchlorate depletion and to identify the formation of any metabolites. Prior to
sampling, contents of the beaker were mixed gently with a glass rod to collect a representative
sample. A 1- mL sample of solution then was collected using a disposable pipette tip. The sample
was filtered into a glass vial with a teflon-lined cap. Samples were refrigerated (5o C) in the dark
until analyzed.
After sample collection on the final day, all plant material was removed from each beaker,
rinsed in distilled water, and final wet weights determined for whole plants. Each plant then was
separated into individual organs (i.e., leaves, roots, and stems and, when present, rhizomes).
Exceptions included the following: pickleweed and perennial glasswort, which have scale-like
leaves incorporated with the stem; waterweed, which has small, dense, whorled leaves fused with
the stem at the base; and duckmeat, which was maintained intact because of the small size of the
entire plant. The plant material was placed in aluminum packets and dried at 45o C in a drying
oven for a minimum of 48 h to establish a constant weight. The dried plant material was weighed
by organ type, and the total dry weight for each plant was determined for use in future calculations.
The dried plant material was analyzed for perchlorate and its transformation products, as described
in 3.5.2. Controls without perchlorate were included in each run, for treatments with and without
sand. The controls were subjected to the same treatment as described above.
3.4 Tissue Extraction
To determine the perchlorate accumulation and formation of metabolites in plant tissues
qualitatively, plants were selected from a single treatment, based on the depletion of perchlorate
from solution. Dried plant organs (e.g., roots, leaves and stems) from the selected treatments were
ground separately into a powder using a mortar and pestle. Then the samples were transferred into
10 mL glass bottles containing 5 mL of 5-mM sodium hydroxide solution. The bottles were mixed
by shaking briefly at regular intervals of approximately 6 h. After 48 h, each sample was transferred
into a 2 mL vial and centrifuged at 7000 rpm for 10 min. Finally, the supernatant was transferred
into an auto-sampler vial for analysis.
10
-------
3.5 Analytical Methods
3.5.1 Analysis of Chloride, Perchlorate, and Metabolites
A Dionex Ion Chromatograph equipped with a gradient pump, UV detector, auto-sampler
and auto-injector was used for analysis of chloride ions from the sorption experiment. The same
equipment and analysis procedure were used to detect perchlorate in solution extracted during the
kinetics experiments and the perchlorate metabolites in organs from plants that had been included
in the kinetics experiments. No pretreatment of the samples was necessary with this method of
analysis.
Ion analysis was performed with an lonpac AS 11-HC (4-mm) analytical column. A guard
precedes the analytical column to prevent sample contaminants from eluting onto the analytical
column. The column was operated at 35° C, and the flow rate of solvent (sodium hydroxide
lOOmM) was 1.0 mL min-1. The injection loop volume was 25 1, and the runtime for perchlorate
analysis was 12 min. An anion self-regenerating suppressor (ASRS) was used for suppressed
conductivity detection. Water as a mobile phase was used for regeneration of the ASRS. The
detection limit of the analytical method was 10 ppb.
3.5.2 Tissue Analysis
Tissue samples were analyzed by ion chromatography using the procedure referenced above.
An lonpac AG9-HC analytical column with an AS9-HC guard column was used for the metabolites
such as chlorate, chlorite and chloride ions. The mobile phase was 9mM sodium carbonate. The
lonpac AG9-HC provided an improved separation over the AS-11 HC column for trace ion analysis.
3.5.3 Data Analysis
The total accumulation plus degradation of perchlorate by the whole plant was assumed to
be equal to the total depletion of perchlorate from solution at the end of the ten-day run (q). The
value for uptake plus transformation was normalized by taking the measured value of solution
depletion (q) and dividing by the wet weight of the plant at the end of the run. These normalized
values are the total perchlorate sink in mg/kg.
First-order rate constants were determined by plotting the solution phase concentration time
course-data as ln(C/C0) vs. t. A non-linear regression analysis was completed, and the resulting slope
of the line reported as the pseudo-first-order rate constant (K). The rate constants for each treatment
were determined using this procedure.
11
-------
4.0 RESULTS AND DISCUSSION
4.1 Adsorption of Perchlorate and Depletion Characteristics
The adsorption-desorption characteristics of perchlorate play an important role in the fate
and transport in natural systems. The fate of this chemical is dependent on its sorption, which is
thought to occur as a result of partitioning into soil or sediment, such as sand, by an ion-exchange
process. Our lab-scale sorption results showed that 50-64% of the perchlorate originally in solution
become adsorbed to sand, replacing an equivalent amount of the chloride ion formerly associated
with the sand. Chloride ions displaced were related directly to the amount of perchlorate removed
from solution. The displaced chloride poses a potential problem by inhibiting the perchlorate ion
uptake by plant systems, and by causing chloride toxicity to sensitive species. The results of this
preliminary adsorption experiment with perchlorate in unwashed-sand, and the apparent toxicity
responses by some plants in Run 1, led to the expanded experimental design used in the subsequent
two experiments specifically to evaluate the effects of washed and unwashed sand on perchlorate
depletion from the solution in the presence of various plant species.
Representative depletion curves are provided in Figures 4-9, and additional characteristics
of depletion of perchlorate from solution are provided in Tables 2-6. Total depletion of perchlorate
from solution occurred in the presence of cabbage gum within eight days, while perchlorate in the
"no-plant" control remained at 20.0 ppm for the unwashed-sand treatment without nutrients (Figure
4). The increase in concentration for the control at approximately 150 h was due to additional
solution added at this time. For the same concentration of perchlorate in the presence of sweetgum
for the unwashed sand treatment with nutrients, total depletion of perchlorate occurred after nine
days (Figure 5). Seedlings and mature-wood cuttings of eastern cottonwood exhibited responses
similar to the depletion curves of cabbage gum and sweetgum.
For the same treatment described in Figure 4, but with black willow, less than half of the
concentration of perchlorate was depleted at the termination of the experiment on day ten (Figure
6). Species with depletion curves similar to black willow included green-wood cuttings of
cottonwood and fragrant white water-lily. For the same treatment described in Figure 5, but with
tarragon, only trace amounts of perchlorate remained in solution on day five of this experiment
(Figure 7). Total depletion of perchlorate from the no-sand treatment without nutrients occurred by
day seven, in the presence of parrot-feather (Figure 8). Concentrations of perchlorate in the control
remained at 20.0 ppm. For the treatment with washed sand without nutrients at the same
concentration of perchlorate, and in the presence of pickleweed, less than half of the perchlorate was
depleted by day seven, when no solution remained for sampling (Figure 8). Smartweed and
perennial glasswort had depletion curves similar to pickleweed.
A summary of perchlorate depletion from solution in the presence of plants tested is provided
in Table 2. A summary of the percent of perchlorate depletion from solution in the presence of
tested plants with positive results is provided in Table 3. Mass of perchlorate depletion for the
species with positive results is summarized in Table 4. The values for the first-order kinetics are
provided in Table 5 by treatment, and are summarized in Table 6. Water weed and duck meat are
not included in Tables 3-5 because there was no depletion of perchlorate during the ten day
experiment for these species.
12
-------
=• 15-
O
50
100 150
Time (h)
200
250
Figure 4. Depletion of Perchlorate (20 ppm) from Solution in the Presence of Cabbage Gum:
Unwashed Sand + Deionized Water Treatment.
20i
D)
o
15-
10-
5-
50
100 150
Time (h)
200
250
Figure 5. Depletion of Perchlorate (20 ppm) from Solution in the Presence of Sweetgum:
Unwashed Sand + Nutrient Solution Treatment.
-------
20-
15-
O
10-
5-
50
100 150
Time (h)
200
250
Figure 6. Depletion of Perchlorate (20 ppm) from Solution in the Presence of Black Willow:
Unwashed Sand + Deionized Water Treatment.
o
50
100 150
Time (h)
200
250
Figure 7. Depletion of Perchlorate (20 ppm) from Solution in the Presence of Tarragon:
Unwashed Sand + Peters Solution Treatment.
-------
o
50
100 150
Time (h)
200
250
Figure 8. Depletion of Perchlorate (20 ppm) from Solution in the Presence of Parrot-feather:
Deionized Water (No Sand) Treatment.
201
O
15-
10-
5-
50
100 150
Time (h)
200
250
Figure 9. Depletion of Perchlorate (20 ppm) from Solution in the Presence of Pickleweed:
Washed Sand + Deionized Water Treatment.
-------
Table 2. Summary of Perchtorate Depletion from Solution In the Presence of Plants
Unwashed Sand
( w/ nutrients)
Plants 0,2 2.0 20,0
Trees
Cabbage gum *f
-------
Table 3. Percent of Perchlorate Depletion from Solution in the Presence of Plants
Unwashed Sand
(w/ nutrients)
Aqueous Solution
(w/ nutrients)
Washed Sand
(w/ nutrients) (w/o nutrients)
Unwashed Sand
fw/o nutrients)
Concentrations of Perchlorate (ppm)
Plants
Trees
Cabbage gum
Sweetgum
Cottonwood
(mature-wood)
(green-wood)
Willow
Upland Herbs
Tarragon
Wetland Herbs
Pickleweed
Blue-hyssop
Smartweed
Glasswort
Aquatic Herbs
Parrot-feather
Water-lily
0.2
100
100
100
0
78
100
0
100
100
0
100
0
2.0
100
45
52
0
58
100
0
36
65
0
100
100
20.0
55
45
33
15
57
100
29
55
47
0
97
0
0,2
NT
0
0
NT
68
100
NT
87
0
NT
100
0
2.0
NT
0
0
NT
68
100
NT
0
0
NT
100
44
20.0
NT
0
0
NT
69
100
NT
0
0
NT
100
13
20.0
100
40
NT
NT
98
NT
0
0
0
13
NT
NT
20.0
100
100
NT
NT
51
NT
43
29
54
68
NT
NT
20.0
0
0
NT
NT
50
NT
0
0 "
0
0
NT
NT
NT = not tested
-------
Table 4. Mass of Perchlorate Depletion from Solution In the Presence of Plants
Plants
Trees
Cabbage gum
Sweetgum
Cottonwood
(mature-wood)
(green-wood)
Willow
Upland Herbs
Tarragon
WetjandHerbs
Pickleweed
Blue-hyssop
Smart weed
Glasswort
Parrot-feather
Water-lily
Unwashed Sand
(w/ nutrients)
0.2 2.0 20.0
6,5
(9.2)
2.5
(23.2)
3.2
(18.6)
0
1.0
(15.1)
1.8
(11.0)
0
120
(0.5)
12.5
(4.8)
0
3.7
(5.4)
0
53.5
(11.2)
42.5
(14.1)
13.1
(23.9)
0
4.6
(24.8)
21.0
(9.1)
0
216
(1.0)
ISO
(2.6)
0
•46.5
(4,3)
127
(4.7)
402
(8.2)
145
(18.5)
44.5
(44.5)
250
(3,6)
5.8
(19.8)
162
(12.3)
305
(1.9)
6600
(0.5)
564
(5.0)
0
392
(5.2)
0
Aqueous Solution Washed Sand Unwashed Sand
(w/ nutrients) (w/ nutrients) (w/o nutrients) (w/o nutrients)
Concentrations of Perchlorate (ppm)
0.2 2.0 20.0 20.0 20.0 20.0
NT
0
0
NT
2.5
(16.6)
5.3
(11.4)
NT
74,1
(0.7)
0
NT
11.8
(5.1)
0
NT
0
0
NT
2.7
(14.3)
59.4
(10. 1)
NT
0
0
NT
117
(5,1)
91.3
(2.9)
NT
0
0
NT
3.0
(13.9)
504
(11.9)
NT
0
0
NT
1200
(5.0)
312
(2.5)
923
(6.5)
160
(6.5)
NT
NT
120
(16,3)
NT
0
0
0
780
(1.0)
NT
NT
674
(8.9)
340
(8.9)
NT
NT
73.1
(M.O)
NT
614
(1.4)
1933
(0.9)
981
(3.3)
3138
(1.3)
NT
NT
0
0
NT
NT
46.4
(21.8)
NT
0
0
0
0
NT
NT
All values are tng/kg wel weight of plants; numbers in parenthesis arc plant weight before initiation of experiments
NT = not tested
-------
Table 5, First-order Kinetics of Perchlorate Depletion from Solution
in the Presence of Plants*
Unwashed Sand
(w/ nutrients)
Aqueous Solution
(w/ nutrients)
Washed Sand
(w/ nutrients) (w/o nutrients)
Unwashed Sand
(w/o nutrients)
Plants
Trees
Cabbage gum
Sweetgum
Cottonwood
(mature-wood)
(green-wood)
Willow
Upland Herbs
Tarragon
Wetland Herbs
Pickleweed
Blue-hyssop
Smartweed
Glasswort
Aquatic Herbs
Parrot-feather
Water-lily
0.2
0.008
0.003
0.013
0
0.004
0.035
0
0.014
0,001
0
0.005
0
2.0
0.002
0.002
0.004
0
0.003
0.020
0
0.006
0.006
0
0.014
0.004
20.0
0.005
0.007
0.010
0.007
0.003
0.032
0.072
0.004
0.007
0
0.017
0
Concentrations of Perchlorate (ppm)
0.2 2.0 20.0 20.0
NT
0
0
NT
0.005
0.018
NT
0.009
0
NT
0.004
0
NT
0
0
NT
0.004
0.025
NT
0
0
NT
0.012
0.002
NT
0
0
NT
0.003
0.031
NT
0
0
NT
0.090
0.001
0.008
0.003
NT
NT
0.003
NT
0
0
0
0.216
NT
NT
20.0
0.007
0.007
NT
NT
0.001
NT
0.17
0.003
0.005
0.22
NT
NT
20.0
0
0
NT
NX
0.002
NT
0
0
0.002
0
NT
NT
*First-order rate constant (k) in hr"1
NT = not tested
-------
Table 6. Summary of First-order Kinetics of Perchlorate Depletion from Solution
in the Presence of Plants
Plants
Trees
Cabbage gum
Sweetgum
Cottonwood
(mature-wood)
(green-wood)
Willow
Upland Herbs
Tarragon
Wetland Herbs
Pickleweed
Blue-hyssop
Smartweed
Glasswort
Aquatic Herbs
Waterweed
Parrot-feather
Water-lily
Duckmeat
ND = no depletion
NT = not tested
Sand
k(day1)
0-0.19
0-0.17
0.09-0.31
0-0.17
0.02-0.09
0.48-0.77
0-0,17
0-0.34
0-0.14
0-0.22
ND
0.12-0.41
0-0.09
ND
Aqueous
k (day1)
NT
0
0
NT
0.10
0.43-0.74
NT
0.01
ND
NT
ND
'0.09-2.1
0.05
ND
-------
4.2 Kinetics of Perchlorate Depletion from Solutions
Depletion was calculated as a first-order kinetics reaction, with individual values reported for
each treatment in Table 5 for thel 1 species for which perchlorate depletion occurred. The values for
k, the pseudo-first-order depletion rate constants in Table 5 are given in hr"1. A summary of the
pseudo-first-order kinetics (day"1) for the depletion of perchlorate for all species is provided in Table
6. Values (day"1) for washed and unwashed-sand treatments were in the range of 0-0.22 for cabbage
gum, sweetgum, rooted green-wood cuttings of cottonwood, willow, pickleweed, smartweed, glasswort,
and water-lily. Upper values for rooted mature-wood cuttings of cottonwood, blue-hyssop, and
parrot-feather were 0.31, 0.34, and 0.41, respectively. The range for tarragon was 0.48-0.77.
4.3 General External Plant Responses and Transformation
The plant species evaluated in this study can be grouped into the following four categories,
based on external responses associated with experimental treatments: A) favorable response, B) no
response, C) moderate adverse response, D) severe adverse response (Table 7). Plants assigned to the
first category generally exhibited new growth in the presence of perchlorate. Plants assigned to
Category B generally exhibited no noticeable external change in the presence of perchlorate. Category
C plants generally exhibited some type of adverse response such as wilting, which may be reversible.
Plants assigned to the final category generally died, or exhibited some other severe response, such as
permanent wilting. The general external responses of plants with positive results during the experiments
are summarized in Table 8. Possible mechanisms for depletion of perchlorate from solution, based on
plant responses during these experiments are described in Table 9. Analysis of plant tissues verified
the presence of perchlorate and transformation metabolites, including chloride (Table 10). Radiolabeled
chloride and a quantitative analysis of plant tissues could be used in future studies to determine the
amount and source of chloride contained within the plants.
4.3.1 Favorable Response
Three species were included in Category A (Table 7). The halophyte, pickleweed produced new
growth in all treatments of perchlorate tested. These responses may have been mediated by
pretreatment acclimation with DI water that depleted internal concentrations of ions. Perennial
glasswort, the other halophyte tested, also appeared to have favorable responses in all perchlorate
treatments. The final species included in Category A was waterweed. Waterweed appeared to produce
new growth in the presence of perchlorate; however, no depletion of perchlorate was observed in the
presence of this species.
4.3.2 No Response
Duckweed also was excluded from Table 8, for the same reason as waterweed, but was included
in Category B (Table 7), because it exhibited no apparent external response to any of the treatments.
Other species assigned to this category included black willow, blue-hyssop, and fragrant white
water-lily. However, depletion of perchlorate occurred in the presence of these latter three species. The
lack of any response by blue-hyssop, fragrant white water-lily, and duckmeat suggests that these
species are tolerant of perchlorate and chloride, at least on a short-term basis. Blue-hyssop may be
sensitive to excess nutrients (Table 8). Although black willow was assigned to this category, it
exhibited severe adverse responses at lower concentrations of perchlorate in treatments with unwashed
sand. These responses were attributed to a sensitivity to chloride ions displaced from the unwashed
sand by perchlorate, and a complex interaction of higher concentrations of perchlorate in this species.
21
-------
Table 7. Categorization of Plant Species Based on
General External Responses to Perchlorate/Nutrient Treatments
During Ten-Day Experiments
Scientific Names
Allenrolfea occidentalis
Salicornia virginica
Elodea canadensis1
A - Favorable Response
Common Names
pickleweed
perennial glasswort
waterweed
Salix nigra1
Bacopa caroliniana1
Nymphaea odorata
Spirodela polyrhiza
B - No Response
black willow
blue-hyssop
fragrant white water-lily
duckmeat
Eucalyptus amplifolia 2
Populus deltoides
seedlings 2
mature-wood cuttings
green-wood cuttings J
Polygonum punctatum 4
Myriophyllum aquaticum 4
C - Moderate Adverse Response
cabbage gum
eastern cottonwood
smartweed
parrot-feather
Artemisia dracunculus sativa5
Liquidambar styraciflua'
Salix nigrd'
D - Severe Adverse Response
tarragon
sweetgum
black willow
possible favorable response to perchlorate
possible unrecoverable sensitivity to chloride ions/some sensitivity to perchlorate
unrecoverable sensitivity to chloride ions
recoverable sensitivity to chloride ions
at least partially related to experimental design
-------
Table S. General External Responses of Plants with Poslti¥e Results During Ten-Day
Percblorate/Nutrient Experiments
Plants External Responses
Unwashed Sand Aqueous Solution
(w/ nutr ientsj (w/ optrients)
Trees
Cabbage gum lower leaves dead at all cone.
Washed
(w/ nutrients)
lower leaves dead
Sweetgum wilted at all cone. leaves wilted/dead at 20 ppm;leaves wilted/dead
lower cone, OK
Cottonwood
seedling decline decreases w/conc, good at all cone,2
mature-wood' decline decreases w/conc. higher cone, ok
green-wood1 decline increases w/conc.
Willow dead at lower cone,1 good at all cone.
Herbs
Tarragon decline increases w/conc.4 shriveled, dead5
Pickleweed robust/new growth all cone.5
Blue-hyssop OK decaying at G. 2 ppm
Smartweed decline increases w/conc. decline decreases w/conc.
Glasswort OK
Parrot-feather wilted at lower cone.5 robust at all cone,
Water-lily OK OK
1 green-wood cuttings from Carswcll AFB, with possible i'ungal Infections
2 possible biochemical change w/nutrients only and nutrients with 20.0 ppm
3 wilted at 20.0 ppm pcrchloratc (chloride toxicity?)
4 black stain in sand (exndatcs?) for 2.0 and 20.0 ppm perchlorate
lower leaves dead
lower leaves dead
-
good
-
new growth
OK
lower leaves dead
OK
-
-
Sand
(w/o__nutirienj.s}
lower leaves dead
leaves epinastic
lower leaves dead
lower leaves dead
-
good
.
new growth
OK
lower leaves dead
OK
-
-
Unwashed Sand
(w/o nutrients)
lower leaves dead
leaves wilted/dead
lower leaves dead
lower leaves dead
-
dead
-
new growth
OK
leaves wilted/dead
OK
-
-
perchlorate attracting aphids
5 dark red stain (tissue/cellular degradation?) in solution for all concentrations of perchlorate
6 no influence on perchlorate at 0.2 or 2.0 ppm perehlorate
7 signs of recovery at Day 10
-------
Table 9. Possible Mechanisms for Depletion of Perchlorate from Solution
Based on Plant Responses During Ten-Day Experimentsl
Trees:
cabbage gum (Eucalyptus amplifolid)
sweetgum (Liquidambar styraciflua)
eastern cottonwood (Populus deltoides)
seedlings
mature-wood cuttings 4
green-wood cuttings 4
black willow (Salix nigra)
Possible Mechanisms:
B, C, D, E
B,C,D,E2
B, C, D, E 2
B,C,D,E2
B, C, D, E 2
A3,B,C,D,E2
Upland Herbs:
tarragon (Artemisia dracunculus sativa)
A3,B5,C,D,E
Wetland Herbs:
pickleweed (Allenrolfea occidentalis)
blue-hyssop (Bacopa caroliniand)
smartweed (Polygonum punctatuni)
perennial glasswort (Salicornia virginicd)
A3,B5,C,D,E
A, B, C, D, E 2
A,B,C,D,E2
B, C, D, E
Aquatic Herbs:
waterweed (Elodea canadensis)
parrot-feather (Myriophyllum aquaticum)
fragrant white water-lily (Nymphaea odoratd)
duckmeat (Spirodela polyrhizd)
no depletion
B,C,D,E2
B 5, C, D, E
no depletion
1 see Figure 10 for explanation of A-D; E = adsorption to sand; bold codes indicate observed
responses that suggest that mechanism
2 visible response of plant in unwashed sand at certain concentrations suggesting chloride ion
interaction
3 may be short-term only, as plant dies/decomposes
4 rooted cuttings from Carswell Air Force Base, possibly with fungal pathogens
dark staining observed
-------
Perchlorate, intermediate metabolites (chlorate, chlorite), and the end product chloride were
documented in black willow organs (Table 10), confirming uptake by this species. However, a
determination cannot be made from these data regarding whether the source of chloride was from
transformation of perchlorate, or from chloride ions displaced from the sand. Experiments with
labeled chloride are needed to resolve this question. If black willow is capable of transforming
perchlorate to the end product chloride, additional long-term experiments should be conducted to
evaluate the sensitivity of black willow to increasing concentrations of chloride ions. Additional
complications could arise if this species is used at a site where chloride is associated with the soils
at the site, and the chloride ions are displaced by perchlorate and taken up by the plant. Finally,
additional experiments should be conducted to determine if seedlings, saplings, and rooted cuttings
obtained from other sources respond similarly to the rooted cuttings from Carswell used in these
experiments, since the latter may have had additional factors involved (e.g., fungal pathogens), which
contributed to low rooting success.
4.3.3 Moderate Adverse Response
Species assigned to Category C, moderate adverse responses to treatments, included cabbage
gum, eastern cottonwood seedlings, mature-wood cuttings, and green-wood cuttings, smartweed, and
parrot-feather. In cabbage gum, the lower leaves of plants in all treatments died initially (Table 7).
Perchlorate and all metabolites were detected in cabbage gum roots. Stems contained perchlorate and
chlorate. Leaves contained only chlorate and chlorite (Table 10). Cabbage gum may be experiencing
mild chloride toxicity or may be responding adversely to the saturated conditions of the treatments.
Responses in cottonwood seedlings varied by treatment. No adverse response was observed
in the no-sand treatment with nutrients. However, the lower leaves died in all other treatments (Table
8). In the washed-sand treatment with nutrients, seedling decline decreased with increasing
concentration of perchlorate. These responses suggest that cottonwood seedlings are tolerant of
saturated soil conditions, sensitive to chloride ions, but may be able to take up perchlorate
preferentially, when both perchlorate and chloride are in solution. All of the tissue that was analyzed
(4c) from the cottonwood seedling organs (root, stem and leaf) contained chloride. Therefore, if the
source of the tissue chloride was from transformation of perchlorate, long-term exposure to
perchlorate could lead to chloride toxicity.
Rooted mature-wood cuttings of cottonwood responded similarly to cottonwood seedlings,
with adverse impacts declining with higher concentrations of perchlorate. Perchlorate and all
metabolites were found in roots and the woody stem of the rooted mature-wood cuttings of
cottonwood that were analyzed (Ic). Chlorate, chlorite, and chloride were found in leaf tissue of the
same sample (Table 10). Therefore, the same potential for chloride toxicity via accumulation from
perchlorate transformation exists for these cuttings. The presence of these metabolites in seedling
tissues suggests that perchlorate is being transformed within, or translocated through all organs. The
same conclusion cannot be made for cottonwood cuttings without analysis of control plants that were
not exposed to experimental concentrations of perchlorate, since the cuttings were obtained from a
site contaminated with perchlorate.
Rooted green-wood cuttings of cottonwood in washed-sand with nutrients responded
differently than cottonwood seedlings, with adverse impacts increasing with higher concentrations
of perchlorate. Other treatments were not included for green-wood cuttings because of the limited
plant material. Additional research is required to determine the reason for the differences in these
responses for different-aged tissue of cottonwood.
25
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Table 10. Metabolites of Perchlorate Transformation Identified
in Plant Tissues
Plants1
Cabbage gum (3c)
Sweetgum (3c)
Cottonwood
(seedlings)(4c)
(mature-wood)( 1 c)
Willow (5c)
Tarragon (Ic)
Pickleweed2 (4c)
Blue-hyssop (4c)
Smartweed(lb)
Glasswort2'3 (4c)
Parrot-feather (Ic)
Water-lily3 (2b)
Roots
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chloride
perchlorate
chlorate
chlorite
chlorate
chloride
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
chloride
Metabolites
Leaves
chlorate
chlorite
perchlorate
chlorite
chlorate
chlorite
chloride
chlorate
chlorite
chlorate
chlorite
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
chlorate
chlorite
chloride
perchlorate
chlorite
chloride
perchlorate
chlorate
chloride
Stems2
perchlorate
chlorate
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
chloride
perchlorate
chlorate
chlorite
perchlorate
chlorate
chlorite
perchlorate
chlorate
chlorite
perchlorate
chlorate
chlorite
perchlorate
chlorate
chlorite
chlorate
chlorite
chloride
1 treatment code for sample provided in parenthesis
2 leaves and stems fused, analyzed as a single tissue, and reported as leaves
3 metabolites in rhizomes are reported as stems
-------
Responses of smartweed also varied with treatment. Similar responses were observed in
unwashed-sand treatments without nutrients, and in washed-sand with, and without nutrients (Table 8).
In unwashed-sand treatments with nutrients, plant decline increased with increasing concentration of
perchlorate, similar to the response in green-wood cuttings of cottonwood. The reverse occurred in the
no-sand treatment with nutrients. Perchlorate and chlorate were found in roots, stems, and leaves of
smartweed, suggesting that perchlorate is being transformed within, or translocated through all organs
(Table 10). More research is required to analyze the complexity of responses observed in smartweed.
4.3.4 Severe Adverse Response
The three species included in Category D were sweetgum, black willow, and tarragon (Table
7). Sweetgum responded adversely to all treatments, except lower concentrations of perchlorate in
no-sand treatments (Table 8). No recovery from wilting was observed. The response in no-sand
treatments compared to treatments with sand suggest that chloride toxicity may be responsible, in part,
for the responses, and the treatments with washed-sand may have contained residual chloride. A
random test of sand from washed-sand treatments at the conclusion of the final experiment revealed
approximately 0.2 mg/L of residual chloride. The source of the chloride presumably was the sand,
although experiments with labeled chloride are required for an accurate determination. For the
sweetgum tissue selected for analysis (3c), perchlorate and all metabolites were found in roots and
stems, but leaves contained only perchlorate and chlorite (Table 10).
Black willow appeared to have an extreme sensitivity to chloride, as indicated by plants dying
in the unwashed-sand treatments (Table 8). Consequently, if perchlorate metabolites are accumulated
in black willow or if chloride is present at the treatment site, chloride toxicity could become a problem
with this species. Tissue analysis (5c) indicated that perchlorate and all metabolites were present in the
roots and woody stems of black willow. Leaves contained only chlorate and chlorite (Table 10).
Tarragon died by the end of the experiment in both no-sand and sand treatments, with decline
increasing with increasing concentrations of perchlorate (Table 8). Both perchlorate and chloride are
thought to be factors in tarragon's response. An analysis of plant tissue (2c) confirmed the presence of
perchlorate, chlorate, and chlorite in roots and stems of tarragon, while leaves contained only chlorate
and chlorite (Table 10).
4.4 Predicted Field-Scale Performance and Possible Mechanisms for Perchlorate Depletion
One important consideration for field performance of potential phytoremediation candidates is
the mass of perchlorate that can be removed by the selected plant. An arbitrary ranking system was
developed for general comparison of phytoremediation potential based on the mass of perchlorate
depleted from solution per mas of plant species tested (mg/kg wet weight). The five categories are as
follows: 0 = no depletion; 1-99 = minimal depletion; 100-499 = moderate depletion; 500-999 =
moderately high depletion; and >1000 = high depletion. None of the tree nor the herbaceous, upland
species tested were included in the highest category of performance (Table 4). Wetland and aquatic
plants included in the highest category were blue-hyssop for the 20.0 ppm perchlorate treatments with
unwashed-sand and nutrients (6600 mg/kg), and washed-sand without nutrients (193 3 mg/kg); perennial
glasswort for the 20.0 ppm perchlorate treatment with washed-sand without nutrients (3138 mg/kg); and
parrot-feather for the 20.0 ppm perchlorate no-sand treatment with nutrients (1200 mg/kg).
Results in the moderately-high category were obtained for one tree species, the herbaceous
upland species, and three of the four herbaceous wetland species evaluated, all at 20.0 ppm
27
-------
perchlorate (Table 4). For cabbage gum, the mass of perchlorate depleted was moderately high for the
washed-sand treatment with nutrients (923 mg/kg) and without nutrients (674 mg/kg). For tarragon,
the mass of perchlorate depleted was moderately high for the no-sand treatment with nutrients (504
mg/kg). For pickleweed, the mass of perchlorate depleted was moderately high for the washed-sand
treatment without nutrients (614 mg/kg). For smartweed, the mass of perchlorate depleted was
moderately high for the unwashed-sand treatment with nutrients (564 mg/kg) and the washed-sand
treatment without nutrients (981 mg/kg). Finally, for glasswort, the mass of perchlorate depleted was
moderately high for the washed-sand treatment with nutrients (780 mg/kg).
Results in the moderate category (100-499 mg/kg) also are shown in bold in Table 4. In
summary, depletion of perchlorate appears to be influenced by plant species, perchlorate concentration,
sand versus no-sand treatments, the presence or absence of nutrients, the age of plant tissue, and by
the presence of chloride ions. A discussion of the predicted field scale performance of each species
is provided in the following subsections.
Four possible mechanisms for the potential fate of perchlorate in plant systems are shown in
Figure 10. The first two mechanisms, (A and B) involve external degradation/transformation, while
the following two mechanisms (C and D) involve internal degradation/transformation after uptake of
perchlorate by the plant. Mechanism A occurs in dead or dying plants as tissues are degraded and cell
contents are released. MechanismB involves substances exuded from live plants (e.g., root exudates).
Mechanism C involves the internal transformation of perchlorate without accumulation of perchlorate
or metabolites, while mechanism D involves internal transformation with accumulation of perchlorate
or metabolites (e.g., chloride). A fifth mechanism that is physical rather than biological (E), also could
be responsible for depletion of perchlorate. This mechanism involves exchange of perchlorate for
chloride adsorbed to the sand. The possible mechanisms for perchlorate depletion from solutions are
provided in Table 9. Codes printed in bold indicate observed responses that suggest that mechanism.
Samples for analysis of tissue samples were selected based on the maximum reduction in
solution perchlorate concentration, for each of the 11 species for which perchlorate depletion was
observed. Tissues of individual plant organs (e.g., roots, stems, leaves) from the selected treatments
were analyzed. Perchlorate, or transformation metabolites (chlorate, chlorite, and chloride) were
observed in all tissues analyzed (Table 10). Future work should include: 1) a quantitative analysis of
individual plant organs (e.g., roots, stems, leaves), and 2) labeled chloride to determine the amount and
source of chloride contained within the plants.
4.4.1 Trees
4.4.1.1 Cabbage Gum
As indicated previously, one of the Air Force's considerations for plants to be used for
phytoremediation is wood biomass production to defray costs of remediation. Production of
Eucalyptus species was investigated in the 1960's and 1970's as a short-rotation woody crop for
Alabama, Florida, Georgia, Louisiana, North Carolina, and Texas, but the effort was abandoned
because of several problems. These problems included wood that was very brittle, leaves that
contained high concentrations of volatile oils (making the trees highly flammable), and sensitivity to
cold damage. Renewed interest in Eucalyptus species is due to improved genetic stock that is less
susceptible to cold damage. Frost hardiness reportedly has been developed inE. grandis, so that it may
survive at low temperatures (Don Rockwood, University of Florida, pers. comm., 7/13/98).
28
-------
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One of the primary concerns expressed by various sources in Florida is that Eucalyptus species, which
have been imported from Australia, have escaped from these early "production" sites, and have invaded
natural habitat. Reference to the invasive ability of these species is made by Wunderlin (1997) and
by the Florida Exotic Pest Plant Council. Wunderlin (1997) has documented invasion by E. grandis
and E. robusta in central and south Florida. The Council lists E. camaldulensis as a "Category II"
plant ("species that have shown a potential to invade and disrupt native plant communities"). From
a long-term perspective, consideration should be given to the liability associated with promoting
non-native species that can 1) increase fire hazards in commercial and residential areas, and 2) result
in additional costly, and possibly irreversible environmental damage in addition to the perchlorate
contamination problem. Cabbage gum is not recommended for additional experiments since
affected leaves did not recover during these experiments, new growth was not observed, and because
of the adverse factors referenced above.
4.4.1.2 Sweetgum
Generally, sweetgum is tolerant of a wide range of growing conditions, particularly soil
moisture content. This species occurs naturally in floodplain wetlands, and should be adapted to
phytoremediation sites requiring roots to be in contact with ground water. The relatively poor
performance of sweetgum in this project may have been due to the large root mass of the plants
(largest of the plants tested) and the small size of the experimental containers. On-site or in situ
experiments are recommended for further evaluation of the species.
4.4.1.3 Eastern Cottonwood
Four of the possible causal factors are discussed below to explain the small percentage of
cottonwoods that produced roots and leaves during our experiments. More extensive experiments
with cottonwood are required before the response of this species to perchlorate can be determined
and predictions can be made regarding the potential field performance of this species.
Light:
The rooting treatments for the cottonwood (and willow) were conducted under artificial
growth lights in the laboratory where the perchlorate exposure experiments were conducted. When
the first experiment was conducted, lights in the laboratory were on continuously. The study design
was modified for the second set of experiments to provide a more natural photoperiod (7 am to 9
pm). Either the initial, extended photoperiod, or the artificial light conditions, may have resulted
in some type of disruption of the normal rooting mechanism for the cuttings. However, root and
shoot growth under natural lighting (but different rooting conditions) in the greenhouse did not
produce more favorable results. Additionally, another researcher also received cuttings from the
same source and placed them outside in natural light to root (Valentine Nzengung, University of
Georgia, pers. comm. 7/17/98). None of those cuttings produced roots.
Diameter/Age of Woody Tissue:
The majority of the cottonwood cuttings were large-diameter branches. The diameter of the
branch tips and the appearance of the wood confirmed this plant material was older wood than
cuttings produced from the branch tips, representing the current year's growth. Wood from the
current year generally is thought to produce roots more readily. However, only approximately 15%
of the smaller-diameter, green-wood cuttings from the Carswell AFB site produced roots and leaves.
30
-------
Timing of Cuttings:
The cuttings for these experiments were taken in the middle of the summer. Timing of
cuttings reportedly can be a factor influencing success of rooting (Kerry Britton, USDA Forest
Service, pers. comm., 8/31/98). The lack of success in rooting the cuttings may have been due to
seasonal factors.
Fungal Pathogens:
One of the cottonwood cuttings (10B) that was assigned randomly to the sand bed treatment
in the EPA laboratory began exhibiting red-orange protuberances in the lenticels on July 11, 1998.
By July 13, 1998, the protuberances had expanded, and a fungal infection was suspected. The
cutting was removed from the rooting bed, enclosed in a protective wrap and transported to the
USDA Forestry Sciences Laboratory in Athens, Georgia. No similar signs were observed in any
other cuttings in the laboratory, or in the greenhouse at that time.
The next morning, Dr. Paula Spaine, Forest Pathologist with that laboratory, provided the
results of her evaluation of the cottonwood cutting and substance associated with the lenticels. She
indicated that the cutting had predisposing cankers (scar tissue from an earlier infection). She also
indicated that the red-orange substance was ascospores (fruiting bodies) of the opportunistic fungal
pathogen, Cytospora. She had not encountered this secondary pathogen prior to examining the plant
material from Carswell AFB; however, the taxonomic reference she was consulting stated that
Cytospora infects plants that are predisposed by stress (Sinclair etal., 1987). She also indicated that
pruning the trees while they are under stress, and in the presence of pathogens may increase
infection. She suggested that the remaining cuttings without roots and leaves be examined the
following week by another Forest Pathologist with the USDA Forest Service, Dr. Kerry Britton,
upon her return. Dr. Britton who conducted research on cottonwood, confirmed Dr. Spaine's
conclusions. She added that Cytospora probably was not the cause of the poor rooting success and
probably would not contribute to the decline of the trees if the trees were not predisposed by some
unidentified stressor.
Plants can experience stress if planted outside their naturally occurring range. Therefore,
an effort was made to determine the origin of the cottonwood trees that had been planted at Carswell
AFB. The original 240 cottonwood trees planted at the AFB are "Sioux Land" variety and were
supplied by Gandy Nursery in Ben Wheeler, Texas. The cottonwood trees were rooted from
greenwood (branch tip) cuttings obtained from a natural population in the vicinity of the nursery,
which is approximately two hours from the Carswell AFB site (Dennis Gandy, Gandy Nursery,
pers. comm., 6/18/98). The proximity of the parent material to the AFB site suggests that the trees
grown from the cuttings should be adapted to climatic conditions at Carswell AFB. However,
site-specific soil, or soil moisture conditions at the AFB may not be optimal for cottonwood growth.
After the trees were planted at the AFB, approximately 28 of the trees were gnawed-down by
beavers. However, all of these trees resprouted (Greg Harvey, Wright Patterson AFB, pers. comm.,
6/18/98). This is an additional factor that may have contributed to the poor rooting response if the
cuttings were taken from resprouted trees. The stress which is predisposing the trees may be a
natural phenomenon. Another possibility is that some site-specific condition at Carswell AFB, such
as components in the ground water, may be predisposing these trees to infection by opportunistic
fungal pathogens.
Several days after the cottonwood cuttings arrived and selected cuttings were placed in the
tanks with aerated DI water, a tan-colored gelatinous substance was observed exuding from the
31
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bases of some of the cuttings. A similar gelatinous substance, referred to as "gummosis"
(production of a thick, dark, gummy substance) has been reported in peach trees subjected to water
stress and infected by another opportunistic pathogen, Botryosphaeria (Brown and Britton, 1986).
The gelatinous substance associated with some of the cottonwood cuttings may have been related
to a fungal pathogen they were harboring. Some of the substance was collected for future analysis.
Similar gelatinous exudates were not observed associated with the cottonwood cuttings in
the sand bed in the laboratory. However, when the cuttings were removed from the sand bed in the
greenhouse on July 15, 1998, there were signs that some type of exudate was associated with many
of the cuttings. The signs included circular zones of dark green algae and a gelatinous sheen on the
surface of the sand around the base of many of the cottonwood cuttings. These zones extended
approximately 1 to 2 cm and the algal growth appeared to be supported by these exudates. The zone
were photographed for future analysis.
The cuttings that produced the most dramatic responses were small segments of mature-wood
cuttings (approximately 3 cm in length), that were the residuals from the cuttings selected for rooting
in the laboratory and for use in the final experiment. The diameter of these cuttings was
approximately 2 cm. These segments had been placed vertically into the sand, with the upper
surface approximately even with the surface of the sand. Similar exudate zones appeared around
both ends of the smaller diameter cuttings (1 cm, and 4 cm in length) from the same experimental
batch that were pushed into the sand horizontally. Similar stained zones also were observed around
the bases of some of the green-wood cuttings that were less than 0.5 cm in diameter.
Based on the evidence of past and possible current infection of the cottonwood trees at the
Carswell AFB site, and the long-term interest in cottonwood as a phytoremediation species and a
biomass producer, the following recommendations are made. No additional cuttings should be
taken from the Carswell AFB trees, based on recommendations of Forest Pathologists. The trees
should be evaluated by a Forest Pathologist knowledgeable about secondary fungal pathogens and
stressors in an attempt to identify whether the cottonwood (and willow) trees currently exhibit any
symptoms of stress and, if so, what causal factors can be identified. On-site research at Carswell
AFB should be initiated to investigate the degree to which perchlorate, or breakdown products
actually occur in the tissues of the cottonwood and willow trees on-site.
4.4.1.4 Black Willow
Typically, willows are propagated readily from cuttings, and grow vigorously, producing
considerable biomass in a short time. These characteristics make willow a prime candidate for
on-site and in situ phytoremediation. Unfortunately, rooting success was low for the willow
obtained for these experiments, and sensitivity to chloride ions was observed. The performance of
black willow may be enhanced in the field. However, the susceptibility of this species to chloride
toxicity and secondary pathogens should be investigated.
4.4.2 Upland Herbs
Total depletion of perchlorate from solution at all three concentrations tested occurred in the
presence of tarragon (Table 3). The depletion of perchlorate from solution with this species
occurred relatively rapidly in both unwashed sand with nutrients (Treatment 1) and no-sand with
nutrients (Treatment 2) in the first run of these experiments (Table 5). The pseudo-first-order kinetic
rates were comparable for the two treatments (Table 6), with total depletion occurring after
32
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approximately five days for the greatest concentration (20 ppm) of perchlorate (Figure 7).
Mass of perchlorate depleted in the presence of tarragon (Table 4) was in the moderate range
for Treatment 1(162 mg/kg wet weight), and the moderately high range for Treatment 2 (504 mg/kg
wet weight). When compared to the mass of perchlorate depleted for the same concentration in
Treatment 1, in the presence of trees, cabbage gum, sweetgum and green-wood cuttings of eastern
cottonwood also were in the moderate range. When compared to the mass of perchlorate depleted for
the same concentration in Treatment 1 in the presence of other herbaceous species, pickleweed and
parrot-feather also were in the moderate range, and blue-hyssop was ranked high. For the no-sand
treatment with nutrients at the same concentration (Treatment 2), water-lily also was ranked moderately
high, and parrot-feather was ranked high (Table 4).
Washed-sand treatments were not incorporated in the first run of these initial perchlorate
experiments when tarragon was tested in order to evaluate the degree to which exchange or adsorption
of perchlorate might be occurring with unwashed-sand. However, depletion of perchlorate was greater
in the no-sand treatment. These similar results in treatments with and without the unwashed-sand
suggest that actual transformation of perchlorate was occurring in addition to any adsorption of
perchlorate to the sand that may have occurred in Treatment 1 (Table 4).
Eventually, the bases of all of the stems of the tarragon that were tested, as well as those being
acclimated in no-sand treatments, decayed and the plants died. Because tarragon was used in Run 1,
washed-sand treatments were not included. The tarragon in the sand treatments may have been
responding to chloride ions displaced from the unwashed-sand. However, tarragon plants transplanted
into containers with the similar sand for use in future experiments, did not exhibit similar symptoms.
Consequently, it appears that the plants in the sand-based treatments may have been responding to
water-logged conditions. Some of the plants in the sand-based treatments had zones of black stain
around the roots, which may have been exudates in response to the treatments. However, they may have
been responding to water-stress by being contained in the undrained beakers.
The staining and tissue degradation are suggestive of external transformation processes A and
B (Figure 10). The fact that tarragon exhibited a severe adverse response to the treatments (Table 7),
and plant decline intensified as the concentration of perchlorate increased (Table 8) suggests that uptake
also may be occurring in the form of internal transformation (C) or, more probably, tissue accumulation
(D) that is debilitating or toxic to the plant. More detailed experimentation and tissue analysis are
required to identify which of these mechanisms is the primary factor responsible for the depletion of
perchlorate.
Whichever mechanism is responsible for perchlorate depletion in the presence of tarragon, this
species appears to have limited tolerance for high soil moisture and no tolerance for standing water.
Therefore, under field conditions the roots of tarragon probably would avoid contact with the water table
when possible, relying on infiltration from rain (in well-drained soil) as the preferred source of water.
The sensitivity of tarragon to water-logged soil may limit the usefulness of this species for
on-site phytoremediation of groundwater contaminants. It is possible that large stands of tarragon
plants placed in well-drained areas over contaminated sites might leach exudates into the soil, and the
exudates could be transported vertically via infiltration into the ground water. However, it appeared that
the exudates observed in the initial experiments were being produced in response to the water stress.
Consequently, production of the exudates may be limited if the plants are not forced, artificially, to be
in contact with water-logged soils.
Although tarragon may not be well-suited for on-site phytoremediation of ground water
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contaminated with perchlorate, treatment of the contaminated water may be possible using a
flow-through system. In this case the contaminated ground water might be pumped from the ground,
exposed to tarragon plants or extracts from tarragon tissue, then reinjected into the ground. This
approach is more energy-intensive than using plant species that can be planted on-site for
phytoremediation, but may be desirable under certain conditions.
Finally, several attempts were made to propagate tarragon vegetatively by rooting cuttings. This
would provide more uniform plants for future experiments. Tarragon cuttings were placed in a 50/50
mix of sand and perlite in metal rooting trays, saturated, then placed on the misting bench at the UGA
greenhouse on June 19,1998. None of the cuttings produced roots. This suggests that tarragon stock
would have to be grown from seed, introducing genetic variability into laboratory and field-scale
experiments, and increasing the time required to produce the necessary plant material for
phytoremediation.
4.4.3 Wetland Herbs
4.4.3.1 Pickleweed
When the selected pickleweed was transferred from the acclimation containers into the beakers
used for the Run 2 of the initial experiments, new roots were observed in the experimental plants.
However, because the condition of the above-ground portion of the majority of the plants was poor, the
watering regime for the remaining plants not incorporated in the experiment was changed to a 5% NaCl
solution made with DI water, with saturated, rather than moist soil conditions, as recommended by Dr.
Ed Glenn and his staff. Saturated conditions were achieved by placing the plastic flats, containing 15
containers each, into plastic tubs, then filling the tubs with the saline solution to within approximately
3.0 cm of the soil surface. The remaining containerized pickleweed for future experiments are being
maintained in a growth chamber. However, after being transferred to the growth chamber with the new
watering regime, the condition of the plants did not appear to improve. The saline water in
approximately one-third of the containers was replaced with freshwater again (saturated conditions) in
an effort to increase the probability of some of the plants surviving. The plants in nonsaline water did
not recover, but most of the remaining plants in the 5% NaCl solution stabilized after several weeks.
The sensitivity of pickleweed plants to transplanting and the uncertainty of success for
germinating seeds at locations where on-site phytoremediation is needed will be significant factors that
may influence field-scale performance of this species. In fact, future experiments of increased duration
using this species may require significant modifications even to maintain control plants through the
entire length of the experiment. The seed obtained at the initiation of these experiments is being
maintained in a sealed plastic bag under ambient conditions in the laboratory. Because of the sensitivity
of this species to transplant shock, the seed should be planted directly in containers to be used for
testing (Ed Glenn, University of Arizona, pers. comm., 6/13/98). The impacts on viability of extended
holding times, or attempted germination during periods other than the natural germination period are
unknown.
4.4.3.2 Blue-hyssop
Blue-hyssop was the top performer with respect to mass of perchlorate depleted (6600 mg/kg).
Additionally, this species did not exhibit any adverse responses to perchlorate or chloride in any
treatments during the duration of the experiment. This species can be propagated vegetatively, and can
cover large areas. Excellent field performance is predicted. However, blue-hyssop is a prime waterfowl
food (Red Gidden, SMNWR, pers. comm., 7/28/98). Consequently, the potential threat to wildlife must
be investigated.
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4.4.3.3 Smartweed
This species can spread rapidly, by rooting at nodes, and can become established vegetatively
in new areas. Although smartweed appears to be sensitive to chloride ions, it will take up perchlorate
preferentially when chloride is present. The field performance of this species should be investigated.
4.4.3.4 Perennial Glasswort
Perennial glasswort is similar to pickleweed, except that the former grows rapidly via rhizomes,
and is tolerant of transplanting. The smallest mass of perchlorate depleted in the presence of perennial
glasswort was greater than the greatest mass of perchlorate depleted in the presence of pickleweed. The
greatest mass of perchlorate depleted in the presence of perennial glasswort was the second largest mass
depletion recorded during these experiments. These traits, and its tolerance of chloride, suggest that
perennial glasswort will perform well in field trials.
4.4.4 Aquatic Herbs
4.4.4.1 Waterweed
This species has the potential for the greatest surface area of the aquatic species tested, and
reproduces rapidly and vegetatively. It can grow throughout the water column in shallow water and in
the upper zone of deeper water. Although no depletion of perchlorate occurred in the presence of this
species during the experiment, different experimental conditions may mediate transformation of
perchlorate by waterweed.
4.4.4.2 Parrot-feather
Parrot-feather also grows throughout the water column and reproduces rapidly and vegetatively.
Its apparent ability to recover from chloride shock is an advantage for performance in the field, as is its
adaptation to growth in both shallow and deeper water. Depletion of perchlorate in the presence of this
species was observed in all treatments tested, supporting its range of tolerance and performance.
4.4.4.3 Fragrant White Water-lily
Water-lilies have large leaves and rhizomes (horizontal, underground stems), with robust,
spongy roots that may be able to process contaminants in the sediment and water column. The rhizomes
are extensive and should be able to be propagated readily by cross-sectional segments. The large
biomass of this plant that extends throughout the water column should facilitate phytoremediation in
aquatic systems. Dark stains in the sand of acclimation tanks suggest that this species produces root
exudates even in the absence of perchlorate.
4.4.4. Duckmeat
This floating-leaved aquatic plant is small but spreads rapidly over the surface of the water via
vegetative reproduction, particularly in the presence of nutrients. Duckmeat may be tolerant of
chloride ions and perchlorate. Although no depletion occurred in the presence of Duckmeat during
this experiment, other conditions may result in Duckmeat depleting perchlorate. For example, as
individual plants die and decompose perchlorate may be transformed via Mechanism A (Figure 10).
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5.0 SUMMARY AND RECOMMENDATIONS
Contamination of soil, surface water, and ground water with perchlorate represents a
significant health risk in the United States. Remediation of contaminated sites using current
technology is difficult and expensive. Microbial approaches for remediation of perchlorate have
been successful at reducing perchlorate from 9000 ppm to approximately 500 ppm. However,
perchlorate concentrations in contaminated ground water generally is in the range of 20 ppm, which
may be below the range of efficiency for microbial applications.
The use of vascular plants for phytoremediation of soil and water contaminated with
perchlorate is an emerging field of research. Initial experiments to evaluate the potential for
phytoremediation of perchlorate by vascular plants included 13 species, and three concentrations
of perchlorate (0.2, 2.0, and 20.0 ppm) in treatments with and without sand. The plants included
four species of trees, one herbaceous upland species, four herbaceous wetland species, and four
herbaceous aquatic species. The tree species were cabbage gum (Eucalyptus amplifolid), sweetgum
(Liquidambar styraciflua), three age classes of eastern cottonwood (Populus deltoides), and black
willow (Salix nigrd). Tarragon (Artemisia dracunculus sativa) was the herbaceous species tested.
Pickleweed (Allenrolfea occidentalis), blue-hyssop (Bacopa caroliniand), smartweed (Polygonum
punctatuni), and perennial glasswort (Salicornia virginicd) were the four herbaceous wetland species
tested. Waterweed (Elodea canadensis), parrot-feather (Myriophyllum aquaticum), fragrant white
water-lily (Myriophyllum aquaticum), and duckmeat (Spirodelapolyrhiza) were the four herbaceous
aquatic species tested.
Results were favorable, but variable, suggesting that significant influences on depletion of
perchlorate include: 1) plant species present, 2) concentration of perchlorate, 3) substrate (sand
versus no-sand treatments), 4) the presence or absence of nutrients, 5) stage of plant maturity, and
6) the presence of chloride ions. For example, the presence of nutrients and other ions can inhibit
depletion of perchlorate (e.g., pickleweed, sweetgum); enhance depletion of perchlorate (e.g.,
cabbage gum); or have no influence on depletion of perchlorate (e.g., waterweed, duckmeat).
Results from the modified experimental design, and the short duration of these experiments,
supported the conclusion that depletion of perchlorate from solutions was not due to algal growth
(primarily green algae) present in some treatments, or external microbes.
A preliminary sorption experiment with unwashed-sand and no plants revealed that 50-64%
of perchlorate in solution became adsorbed to the sand, displacing chloride. Consequently, for
treatments with unwashed-sand and plants in the subsequent three experiments, the free chloride
ions in solution were available to be taken up by the plants. When perchlorate concentrations
exceeded 2.0 ppm in unwashed-sand treatments, an option was available for plants to take up excess
perchlorate, rather than chloride ions, from the solution.
Perchlorate was depleted from solution in the presence of all but two species tested
(waterweed and duckmeat). The mass of perchlorate depleted (mg/kg wet plant weight) was
classified into the following five general categories: 0 = no depletion; 1-99 = minimal depletion;
100-499 = moderate depletion; 500-999 = moderately high depletion; and >1000 = high depletion.
None of the tree species tested, nor the herbaceous upland species tested were included in the
highest category of performance. Wetland and aquatic plants included in the highest category were
blue-hyssop, perennial glasswort, and parrot-feather. Results in the moderately-high category were
obtained for one species of tree (cabbage gum), the herbaceous upland species (tarragon), and three
of the four herbaceous wetland species evaluated (pickleweed, smartweed, and perennial glasswort).
Depletion of perchlorate was calculated as a pseudo-first-order kinetics reaction, with k
36
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values (day-1) for sand treatments in the range of 0-0.22 for cabbage gum, sweetgum, rooted
green-wood cuttings of cottonwood, black willow, pickleweed, smartweed, perennial glasswort, and
fragrant white water-lily. Upper values for rooted mature-wood cuttings of cottonwood, blue-hyssop,
and parrot-feather were 0.31, 0.34, and 0.41, respectively. The range for tarragon was 0.48-0.77.
Tissues from plant organs (e.g., roots, stems, leaves) were analyzed from selected samples, based on
maximum drop in solution perchlorate concentration, for each of the 11 species for which perchlorate
depletion was observed. Perchlorate, or transformation metabolites (chlorate, chlorite, and chloride)
were observed in all tissue samples analyzed. Future work should include: 1) a quantitative analysis
of each plant organ (e.g., roots, stems, leaves), and 2) radiolabeled chloride to determine the amount and
source of chloride contained within the plants, to evaluate the potential for chloride toxicity.
Characteristics of eastern cottonwood and black willow cuttings obtained from a site with
perchlorate in the ground water, and incorporated into these experiments, suggested that fungal
pathogens may be present in the donor plants on that site. Fungal pathogens, if present, may have
influenced the performance of these plants in the experiment. Conversely, exposure of plants to
perchlorate may create stresses that result in predispostion of the plant to infection by plant pathogens.
Evaluation of these factors was not within the scope of these initial experiments, but should be
addressed in future experiments. Another important aspect not evaluated in these short-term
experiments was the potential environmental hazard to wildlife that may consume plants used for
phytoremediation that contain high concentrations of perchlorate and transformation products. Future
experiments of longer duration should provide more information regarding the degree to which
perchlorate is accumulated in plant tissue, and any potential threat to wildlife.
The multitude of influential factors identified in these preliminary experiments and unexplored
factors of concern necessitate additional research in the referenced areas to develop approaches for field
application of vascular plants for phytoremediation. Specifically, future research should simulate
specific site conditions to evaluate the role of factors such as nutrients and other ions (e.g., chloride) that
are present. Additionally, the influence of plant age and condition (e.g., relationship of stress and
predisposition to pathogens) on perchlorate uptake/transformation should be investigated. Experiments
of extended duration also can evaluate long-term decline and recovery of plants, and identify the active
period for enzymes in external transformation of perchlorate ("Type A" mechanisms).
Based on the results of these experiments and ecological knowledge of the species evaluated,
the following species are recommended for future research for phytoremediation of perchlorate. The
recommended plants are grouped by the type of phytoremediation for which they appear to be suited.
Additional research using sweetgum, eastern cottonwood, and black willow is recommended for on-site
and in situ phytoremediation of contaminated soils in uplands, including areas with shallow ground
water accessible to plant roots, and if production of biomass for harvest is of interest. For on-site or in
situ phytoremediation of contaminated areas that are saturated or inundated periodically, or for wetlands
created for phytoremediation, additional research using blue-hyssop, smartweed, and perennial
glasswort is recommended. Additional research using parrot-feather and fragrant white water-lily is
recommended for on-site and in situ phytoremediation of contaminated waterbodies, or for ponds
created artificially for phytoremediation of contaminated surface water or extracted ground water.
Finally, extracts from tarragon may be useful for injection into mechanized flow-through reactors or
plant systems where ground water is extracted, exposed to phytoremediation plants, then reinj ected into
the aquifer, or for similar flow-through systems for contaminated surface water. Related peer-reviewed
publications and conference presentations are listed in Appendix C.
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REFERENCES
Ataway, H. and Smith, M. (1993) Reduction of perchlorate by an anaerobic enrichment culture, J Ind.
Microbiol, 12,408-412.
AWWARF (1998) Perchlorate Issue Group Presentations,
http://www.awwarf.com/newprojects/
Bailey, L. H. and Bailey, E. Z. (1976) Hortus Third, A Concise Dictionary of Plants Cultivated in the United
States and Canada. MacMillan Publishers, New York, NY. 1290 pp.
Brown, E. A. and Britton, K. O. (1986) Botryosphaeria diseases of apple and peach in the Southeastern
United States. Plant Disease 70(5), 480-484.
California Department of Health (1998) Perchlorate in California drinking water.
http://www.dhs.cahwnet.gov/ps/dwem/chemicals/perchl/perchlindex.htm
Glass, D. J. (1998) The 1998 United States market for Phytoremediation.
http://www.channell.com/dglassassoc/infphytexec.htm
Godfrey, R. K. (1988) Trees, Shrubs, and Woody Vines of Northern Florida and Adjacent Georgia and
Alabama. The University of Georgia Press, Athens, Georgia. 734 pp.
Godfrey, R. K. and Wooten, J. W. (1979) Aquatic and Wetland Plants of the Southeastern United States:
Monocotyledons. The University of Georgia Press, Athens, Georgia. 712 pp.
Godfrey, R. K. and Wooten, J. W. (1981) Aquatic and Wetland Plants of the Southeastern United States:
Dicotyledons. The University of Georgia Press, Athens, Georgia. 933 pp.
Herman, D. C. and Frankenberger, W. T. Jr. (1998) Microbial-mediated reduction of perchlorate
in groundwater, J. Envrion. QuaL, 27, 750-754.
Jaeger, E. C. (1947) Desert Wild flowers, 4th Edition. Oxford University Press, London. 322pp.
Phillips Laboratory (1997) Reclaimed ammonium perchlorate characterization for rocket propellants.
h ftp ://www.plk. af.mil
Mabberley, D. J. (1997) The Plant Book, 2nd Edition. Cambridge University Press, Cambridge, MA. 858
pp.
Malmqvist, A., Welander, T. and Gunnarsson, L. (1991) Anaerobic growth of microorganisms with chlorate
as an electron acceptor, Appl. Environ. Microbiol., 57, 2229 -2232.
Rikken, G. B., Kroon, A. G. M. and van Ginkel, C. G. (1996) Transformation of (per)chlorate into chloride
by a newly isolated bacteriumn:reduction and disputation, Appl. Microbiol. Biotechnol., 45, 420-426.
Sinclair, W. A., Lyon, H. H., and Johnson, W. T. (1987) Diseases of Trees and Shrubs. Comstock Pub.,
Ithaca. N. Y. 574pp.
Stanbury, J. B. and Wyngaarden, J. B. (1952) Effect of perchlorate on the human thyroid gland, Metabolism,
1,533-539.
van Ginkel, C. G., Plugge, C. M. and Stroo, C. A. (1995) Reduction of perchlorate with various energy
substrates and inocula under anaerobic conditions, Chemosphere, 4057-4066.
van Wijk, D. J. and Hutchinson, T. H. (1995) The ecotoxicity of chlorate to aquatic organisms: a critical
review, Ecotoxicol. Environ. Safety, 32, 244-253.
van Ginkel, C. G., Rikken, G. B., Kroon, A. G. M. and Kengen, S. W. M. (1996) Purification and
characterization of chlorite dismutase: a novel oxygen-generating enzyme, Arch. Microbiol., 166,
321-326.
Wallace, W., Beshear, S., Williams, D., Hospadar, S. and Owens, M. (1998) Perchlorate reduction by a
mixed culture in an up-flow anaerobic fixed bed reactor, J. Industril Microbiol. Biotechnol., 20,
126-131.
Wunderlin, R. (1977) Guide to the Vascular Plants of Florida. University Press of Florida, Gainesville, FL.
806 pp.
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APPENDICES
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Appendix A.
Sources of Plant Species Evaluated and Pre-experimental Preparation
TREES:
Cabbage Gum (Eucalyptus amplifolia Naud.)
Only one source of Eucalyptus seedlings could be located at the time of these experiments.
Dr. Don Rockwood, School of Forest Resources, at the University of Florida in Gainesville grew
seedlings of three species, Eucalyptus amplifolia, E. camaldulensis, E. grandis, for potential
production of mulch, fuel, pulp, and for experimental phytoremediation of heavy metals at municipal
treatment sites. Unfortunately, only a limited number of one species (E. amplifolia) was available
at the time of these experiments because all of the other seedlings had been planted on various
research sites. These trees are evergreen in warmer areas, increasing the potential for "removal" of
water through transpiration (Don Rockwood, pers. comm 6/98).
All of the Eucalyptus species originated in Australia; however, the seedlings being grown
at the University of Florida were from many seed lots obtained from California. The seedlings of
cabbage gum used in our experiment were from seed lot 4823. A limited number of seedlings of this
species and the other two species may be available next summer; however, this is not a commercial
source, and those seedlings are being grown for ongoing research at that facility.
The seedlings were approximately 35 cm tall and were germinated and grown under
greenhouse conditions in 0.5 by 1.2 m plastic trays that hold 72 seedlings per tray. Seedlings were
approximately 10 weeks old, and ready for transplanting to the field, or to larger containers at the
initiation of the experiment. Seedlings were transported by automobile immediately prior to
initiation of the experiment. The roots were washed by dipping the root mass repeatedly in a bucket
filled with DI water, until all potting soil was removed.
Sweetgum (Liquidambar styraciflua L.)
The sweetgum seedlings were obtained from Rennerwood Nursery, Tennessee Colony,
Texas, and were grown from seed collected locally. The seedlings were grown in 10 cm
"root-makers" (custom-designed, inverted pyramid containers) and were approximately 60 cm tall.
Twenty seedlings were shipped, bare-root, via UPS ground delivery for arrival on July 31, 1998, to
be included in the final run of the experiment. The roots were washed as described above.
Eastern Cottonwood (Populus deltoides Bartr. ex Marsh.)
Mature-wood Cuttings:
Eastern cottonwood plant material was obtained as woody cuttings from mature trees
growing on the Carswell AFB, Texas. The cottonwood cuttings reportedly were taken from branches
of numerous two-year old trees that were planted on the AFB site and are now approximately 3.7
to 6 m tall (Glenn Rivers, USGS, pers. comm., 6/98). Consequently, the cottonwood cuttings are
not identical genetically. The cuttings were enclosed in plastic bags and shipped overnight to the
Athens, Georgia EPA facility in a plastic cooler containing block ice on June 16, 1998. The leaves
had been removed from the cuttings prior to shipping, to reduce water loss from transpiration. The
plant material was in excellent condition. Eighteen cuttings of similar diameter were selected to be
rooted and used in the screening experiments. Diameters of the selected cottonwood cuttings ranged
from approximately 1.0 to 2.5 cm. Minimum lengths of the selected cuttings were 40 cm.
The cuttings were placed in 3.5 L beakers with deionized (DI) water until June 19, 1998.
On that date, each branch was cut (diagonal end cuts) to provide two 20 cm mature-wood segments
A-l
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from each cottonwood branch. The newly-cut segments were labeled with consecutive numbers and
"A" or "B", to designate lower and upper segments, respectively. These paired segments were
selected randomly, for placement in either sand saturated with DI water or, seamless glass aquaria
with a 5 cm depth of DI water. The water in the aquaria was aerated with a Reva Air 200 aquarium
pump and 15 cm "aqua mist bar" to promote root growth. These plants were maintained in the
laboratory, under artificial light (120 w Plant Gro N Show). By June 23, 1998, some of the
cottonwood cuttings had produced new leaves approximately 2 cm in length.
The sand rooting bed and aerated water treatments were used because they coincided with
the experimental treatments with and without sand. Roots produced in water are different, both
structurally and physiologically, from roots produced in a solid substrate such as sand. Therefore,
the preferred approach is to initiate the type of roots that will be acclimated to the treatment being
used.
Unfortunately, only a small number (approximately 30%) of the similar-dimension cuttings
selected for the two treatments produced roots after 19 days in the rooting treatments. Production
of roots was most successful in the sand bed saturated with DI water, where 4 of 18 cuttings
produced roots. One of the four cottonwood cuttings that produced roots in the sand bed had not
produced leaves by July 14, 1998.
Results were less favorable in the other treatment (tank with aerated DI water), where only
1 of 18 cuttings produced roots. However, three cottonwood cuttings that did not produce roots, in
addition to the one that had rooted, produced leaves in the aqueous rooting treatment during the
same time span. On July 12, 1998, the unrooted cottonwood cuttings that had produced leaves in
the aerated water were removed, and transfered to the sand bed. This was a final effort to induce
root development in these mature-wood cuttings for the initial screening experiments.
The mature-wood cuttings that were not selected for these two rooting treatments and the
green-wood cuttings of variable dimensions, were transferred to a second sand bed and placed under
the mist system at the University of Georgia (UGA) greenhouse. The mist system routinely is used
to root cuttings for commercial and research use. For the mature-wood cuttings of cottonwood
under the UGA greenhouse mist system that were of similar-dimension to those selected for the
screening experiment, only one of those cuttings had produced roots and leaves after 14 days. None
of the remaining mature-wood cuttings of that size group had produced either roots or leaves during
that same time period.
Green-wood Cuttings:
The sand bed under the mist system also contained a limited number of branch tip cuttings
referred to as "green-wood" cuttings. These cuttings had not been selected to use in the Phase I
screening experiment because there were not enough similar-sized green-wood cuttings to allow for
natural attrition and still have enough cuttings for all treatments in the final experiment. In fact, only
8 of the 52 green-wood cuttings (15%) in the mist beds produced leaves and roots. A discussion of
possible causes for the low percentage of root production in the cuttings is included in Section 4.5.
Four of the green-wood cuttings were similar in size and tissue components. A decision was
made to include these four rooted cuttings in Run 3, for Treatments 1 and 6. This would provide a
comparison of responses from tissue of two different age groups. Information of this nature would
be useful in trying to predict field performance of cottonwood, and whether saplings might respond
differently to perchlorate than older trees.
A-2
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Seedlings:
No commercial sources for cottonwood seedlings were found in the vicinity of the Carswell
AFB site. However a single source for cottonwood seedlings was located in Florida. The source
was the same as the source for cabbage gum described above. The seedlings were approximately
15 cm tall, and were germinated in the same containers as the cabbage gum. The seed was collected
from a number of natural populations throughout the southeastern United States. Seedlings from
seed lot 4409 were selected because of the similar latitude and close proximity of that donor
population (Tupelo, Mississippi) to the Carswell AFB site. After delivery of the seedlings to the
laboratory, the roots were washed as described in cabbage gum, above, and the seedlings were
incorporated into the final experiment, without acclimation.
Black willow (Salix nigra L.)
Willow cuttings from the Carswell AFB also were supplied for evaluation in these
experiments. These cuttings reportedly were taken from a single tree approximately 4.6 to 6 m tall
that is growing at the margin of the creek on the AFB site. Therefore, all of these cuttings have the
same genetic composition. The bases of the willow cuttings ranged in diameter from approximately
0.7 to 1.0 cm. The minimum length of the cuttings were 30 cm. The willow cuttings were shipped
with the cottonwood cuttings, as described above, with the same acclimation and rooting procedures,
except that the eighteen comparable size willow cuttings were subdivided into two 15 cm segments
for the experiment. At the time the initial leaves appeared on the cottonwood cuttings, new leaves
approximately 2 mm also had appeared on the willow cuttings. This rooting response of the willow
cuttings was similar to that of the cottonwood cuttings. A sufficient number of willow cuttings from
each of the two rooting treatments produced roots and leaves so that this experiment could be
conducted (9 of 18 cuttings rooted in sand and 11 of 18 cuttings rooted in aerated DI water).
However, rooting success was low and insufficient for subsequent, large scale experiments.
UPLAND HERBS:
Tarragon (Artemesia dracunculus L,.)
Thirty tarragon plants in 10 cm square plastic containers were obtained from Charmar Flower
and Gift Shop in Athens, Georgia to be used in the first set of perchlorate/nutrient experiments.
These perennial plants reportedly were germinated the preceding winter, were growing in typical
potting soil, and had an extensive root system. Approximately half of the plants were removed from
the 10 cm plastic containers, the roots were washed in the manner described for cabbage gum. The
plants then were randomly assigned to either aerated DI water or sand for pre-treatment acclimation.
WETLAND HERBS:
Pickleweed (a.k.a. iodine bush, Allenrolfea occidentalis (Watson) Kuntze)
Three species in the genus Allenrolfea occur in the southwestern United States and Mexico.
However, Allenrolfea occidentalis is the only species that is indicated to occur in the southwestern
United States. The official common name assigned to this species is "pickleweed", although it also
is referred to as iodine bush and picklebush. Technically the common name "iodine bush" refers
only to the genus Allenrolfea (Phil Jenkins, University of Arizona, pers. comm., 6/11/98).
Pickleweed reportedly is fragile and does not transplant well (Ed Glenn, University of
Arizona, James Henrickson, California State University, pers. comm., 6/98). Consequently, a source
A-3
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for both plants and fresh seed was sought, so that plants would be available immediately for the
initial experiments and seedlings could be grown for use in future experiments. Approximately 100
mature pickleweed plants and fresh seed with chaff were collected on June 28, 1998 by University
of Arizona research personnel from a natural population at Rocky Point along the Sonoran Coast
in Mexico. This is a research site for Dr. Ed Glenn, Environmental Research Laboratory, University
of Arizona, Tucson, Arizona. The bare-root plants were wrapped in wet newspaper, and packed in
a styrofoam-lined boxed. The fresh seed and chaff were in plastic ziplock bags, included in the box
with the bare-root plants, and shipped to the Athens, Georgia EPA facility, via overnight Federal
Express service. The plants arrived in excellent condition on June 30.
Thirty pickleweed plants of similar size and weight were transplanted into 10 cm square
plastic containers in builder's sand. Prior to transplanting, the bare roots were rinsed in DI water,
as described for cabbage gum, to remove any remaining soil. The containerized plants remained in
the laboratory under grow lights to acclimate to conditions to be used in the screening experiment.
The remaining small to medium-sized plants were transplanted as described for the plants selected
for the second set of experiments.
Initially, all of the transplanted pickleweed remained in the laboratory, with soil moisture
supplemented every other day using DI water to maintain saturation. Use of non-saline water
during the acclimation period is recommended to promote the growth of new roots. Pickleweed
reportedly turns bluish as the ion concentration in their tissue decreases to detrimental levels (Ed
Glenn, University of Arizona, pers. comm., 6/98). This response did not occur. Instead, many of
the plants became chlorotic, with flaccid apexes.
On July 8, 1998, the second set of experiments was initiated, with pickelweed included as
one of the three test species. The seven most robust pickleweed were selected from the acclimation
set of 30 plants, then randomly assigned to the seven treatments for pickleweed. When the selected
pickleweed was transferred from the acclimation containers into the beakers used for the screening
experiment, new roots (white in color) were observed in the experimental plants. However, the
condition of the above-ground portion of the majority of the plants was poor.
Blue-hyssop (Bacopa caroHniana(\Valt.) Robbins.)
Approximately 20 comparable-sized blue-hyssop plants were collected from a natural
population located at the margin of the pondcypress wetland adjacent to Refuge Road 13, north of
the Aucilla Tram Road, in the St. Marks National Wildlife Refuge (SMNWR) in Wakulla County,
Florida (south of Tallahassee). The collected plants were wrapped in moistened paper towels and
transported overnight to the EPA laboratory in Athens. After arrival, the plants were rinsed
thoroughly with tap water, then DI water, and incorporated into the final set of experiments, without
an acclimation period.
Smartweed (Polygonum punctatum Ell.)
Approximately 20 comparable-sized smartweed plants were collected from a natural
population growing in the Headquarters Pond, 6.6 km (4. Imi.) south of Aucilla Tram Road 105, in
SMNWR. The collected plants were placed in a plastic tub with water from the pond, and
transported overnight to the EPA laboratory in Athens. After arrival, the plants were rinsed
thoroughly with tap water, then DI water, and incorporated into the final set of experiments,
without an acclimation period.
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Perennial Glasswort (Salicornia virginica L.)
Approximately 30 comparable-sized perennial glasswort plants were collected from a
natural population growing in a salt flat at the light house in the SMNWR. The sandy soil was
removed from the roots by gently shaking the collected plants. Then the roots were wrapped in
moist paper towels, and the plants transported overnight to the EPA laboratory in Athens. After
arrival, the plants were rinsed in DI water, and incorporated into the final set of experiments, without
an acclimation period.
AQUATIC HERBS:
Waterweed (Elodea canadensis Rich, in Michx.)
Waterweed was collected from an established population in the UGA Lake Herrick
impoundment, located in close proximity to the Athens EPA laboratory. After collection, the plants
were rinsed thoroughly with tap water, then DI water, and acclimated under plant growth lights in
the laboratory where the experiments were conducted.
Parrot-feather (Myriophyllum aquaticum (Veil.) Verde.)
Parrot-feather was obtained from an established population at Shaking Rock Park, in
Lexington, Georgia, in the vicinity of the Athens EPA laboratory. After collection, the plants were
cleaned and acclimated as described for waterweed.
Fragrant White Water-Lily (Nymphaea odorata Ait.)
Approximately 12 young specimens of fragrant white water-lily and 10 large, mature
rhizome segments with roots and leaves were collected from a naturally established population
growing at Mounds #1, in a ditch 2.9 km (1.8 mi.) north of Headquarters Pond, in SMNWR. The
collected plants were placed in a plastic tub with water from the collection site, and transported
overnight to the EPA laboratory in Athens. After arrival, the plants were rinsed thoroughly with tap
water, then DI water. Eight of the young plants with similar-sized rhizomes, and similar leaf area
were selected for incorporation into the final run of the experiment, without an acclimation period.
The rhizomes of the larger plants, with leaves and roots, were cut into segments approximately 5 cm
in length, and placed in a glass tank containing the same type of sand being used in the experiments,
and DI water. Aeration was provided as described in section 3.1.1.3, above, and the tank was placed
on a nursery bench in UGA Botany greenhouse #1, as a holding facility until the plants were needed
for subsequent experiments.
Duckmeat (Spirodela polyrhiza (L.) Schleid.)
Duckmeat was obtained from an established EPA stock that was being maintained under
plant growth lights in the laboratory. The plant material for this species used in the initial
experiments was grown from 10 g of the laboratory stock population. Conditions for growth
included placement in a shallow container filled with DI water, and with sufficient surface area to
prevent overlap of the leaves. These plants were maintained under plant growth lights until
initiation of the first experiment.
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Appendix B.
Contents of Peters Professional All Purpose Plant Food*
Chemical Component Percent
Total nitrogen (N) 20.00
Nitrate nitrogen 1.97
Urea nitrogen 18.03
Available phosphate (P205) 20.00
Soluble potash (K20) 20.00
Magnesium (Mg) (Total) 0.50
Magnesium (water soluble) 0.50
Boron (B) 0.02
Copper (Cu) 0.05
Chelated copper (Cu) 0.05
Iron(Fe) 0.10
Chelated iron (Fe) 0.10
Manganese (Mn) 0.05
Chelated manganese (Mn) 0.05
Molybdenum (Mo) 0.0005
Zinc (Zn) 0.05
Chelated zinc (Zn) 0.05
* Derived from: potassium nitrate, urea, potassium phosphate,
magnesium sulfate, boric acid, copper EDTA, iron EDTA,
manganese EDTA, sodium molybdate, zinc EDTA.
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Appendix C.
Related Peer-Reviewed Publications and Conference Presentations
Peer-ReviewedManuscripts Prepared for Publication:
1A. Phytotransformation of perchlorate and identification of metabolic products in
Myriophyllum aquaticum - International Journal of Phytoremediation (S. Susarla,
Bacchus, S. T., and McCutcheon, S. C.) Volume 1, pp. 97 - 107, 1999
IB. Phytoremediation of perchlorate \yyMyriophyllum aquaticum - Soil and Groundwater
Cleanup magazine (S. Susarla, Bacchus, S. T., and McCutcheon, S. C.- abstracted by
editors of Inter. J. Phytoremediation) Feb/Mar 1999
2. Uptake and transformation of perchlorate by vascular plants - Environmental Science
and Technology (S. Susarla, Bacchus, S. T., and McCutcheon, S. C.) in press
3. Phytotransformation of perchlorate contaminated waters - Water Research (S. Susarla,
Bacchus, S. T., Wolfe, N. L. and McCutcheon, S. C.) in press
4. Federal wetlands produce knights in shining armor for phytoremediation of perchlorate -
Science (S. T. Bacchus, Susarla, S., Wolfe, N. L. and McCutcheon, S. C.) in review
Proposed Peer-Reviewed Publications and Intended Journal:
1. Uptake and transformation of perchlorate by trees: Environmental factors - Environmental
Toxicology and Chemistry (Bacchus, S.T., Susarla, S., andMcCutcheon, S. C.)
2. Perchlorate and transformation products in vascular plant tissue: A mass balance approach -
Chemosphere (S. Susarla, Bacchus, S. T., and McCutcheon, S. C.)
Conference Presentations:
1. Potential Species for phytoremediation of perchlorate- Battelle Conference, San Diego,
CA, April 1999. (S. Susarla, Bacchus, S. T., and McCutcheon, S. C.)
2. Advantages of a multidisciplinary approach to on-site phytoremediation - Georgia Water
Resources Conference, Athens, GA, March 1999. (Bacchus, S. T., Susarla S., and
McCutcheon, S. C.)
3. Predicting field performance of herbaceous plants for phytoremediation of perchlorate -
ACS Conference, New Orleans, LA, August 1999. (Bacchus, S. T., Susarla S., Wolfe,
N. L., Harvey, G., and McCutcheon, S. C.)
4. Perchlorate uptake and transformation in aquatic plants - 15th Annual Contaminated
Soils Conference, Amherst, MA, October 1999. (S. Susarla, Bacchus, S. T., and
McCutcheon, S. C.)
5. Uptake and transformation of perchlorate by trees - 15th Annual Contaminated Soils
Conference, Amherst, MA, October 1999. (Bacchus, S. T., Susarla, S., Harvey, G.,
Wolfe, N. L., and McCutcheon, S. C.)
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