Bryant University Bryant Digital Repository
Master of Science in Global Environmental Studies Graduate Theses
6-2015
Assessment of Trace Metal Concentrations in Lythrum salicaria at Three Rhode Island Sites Using ICP-MS Analysis
Allison Rose Hubbard
Follow this and additional works at: https://digitalcommons.bryant.edu/msglobalenv
Recommended Citation Hubbard, Allison Rose, "Assessment of Trace Metal Concentrations in Lythrum salicaria at Three Rhode Island Sites Using ICP-MS Analysis" (2015). Master of Science in Global Environmental Studies. Paper 2. https://digitalcommons.bryant.edu/msglobalenv/2
This Thesis is brought to you for free and open access by the Graduate Theses at Bryant Digital Repository. It has been accepted for inclusion in Master of Science in Global Environmental Studies by an authorized administrator of Bryant Digital Repository. For more information, please contact [email protected].
Assessment of Trace Metal Concentrations in Lythrum
salicaria at Three Rhode Island Sites Using
ICP-MS Analysis
Presented to the Faculty of the Graduate School of Bryant University in
Partial Fulfillment of the Requirements for the Degree of
Master of Science, Global Environmental Studies (MSGES)
©Allison Rose Hubbard
Bryant University
June 2015
ii Allison Hubbard Bryant University
Acknowledgements
I would like to thank all of the individuals who helped me to complete my graduate thesis research. My thanks to Julia Crowley-Parmentier for her guidance in the instrumentation and input in the writing process, Jaqueline Kratch and other previous students who developed and tested select protocols, and Dan McNally as the chair of the department. I wish to thank Phyllis Schumacher and Alan Olinsky for their invaluable help with statistics analysis. I would also like to thank Brian Blais for his help in creating innovative visualization of the data. My warmest thanks to the graduate committee
(Gaytha Langlois, Qin Leng, Dan McNally, and Hong Yang) for their valued input and guidance throughout the editing process. My deepest gratitude to Gaytha Langlois for her continued guidance and mentorship throughout the thesis process.
iii Allison Hubbard Bryant University
Abstract
Throughout human history terrestrial environments have been disturbed and contaminated through improper waste practices and disposal methods. New technology has emerged for cleanup and mitigation, but may cause additional ecosystem perturbance. One way of reducing ecological stress is to utilize bioremediation, mainly phytoremediation. Due to its persistence in polluted environments Lythrum salicaria (Purple Loosestrife), an invasive plant species, was thought to have the potential to uptake pollutants (such as heavy trace metals) from
the environment and thus help to effectively clean up a polluted environment. Three Rhode Island
sites with large stands of L. salicaria were studied by analyzing plant and soil samples. All sites
had potentially been exposed to some contamination, as a result of being located near a roadway
or industrial operation, or in the path of storm water runoff. ICP-MS was used to analyze trace
metal levels of Zn, Mg, Ca, Cr, Ni, Mn, Cu, As, Se, Ag, Cd, Ba, and Pb in all samples. Trace
metal levels were compared between the whole plant and soils as differentiated by site, as well as among plant leaves, stems, roots, and at each site. None of the trace metal levels (in soils or plant tissues) were above soil and plant regulatory standards. The highest concentration of metals within plant tissue varied among organs (e.g., Zn, Mg, and Ca concentrated in the leaves while other metals, e.g., Cr, concentrated in the roots). While some trace metal levels were higher in plant tissue than the soils (e.g., Ca, Zn, and Cr) others were not. The high density of clustered populations of L. salicaria would enhance the effectiveness of the plant’s phytoremediation capacity even when the concentration of a particular metal is only slightly higher than ambient soil levels.
Keywords: Purple Loosestrife, Lythrum salicaria, ICP-MS, Trace Metal Uptake,
Bioremediation, Phytoremediation, In-situ Remediation
iv Allison Hubbard Bryant University
Table of Contents
Title Page and Statement of Use and Copyright……………………………………….i
Signature Page...……………………………………………………………………....ii
Acknowledgements…….…………………………………………………………….iii
Abstract………………...…………………………………………………………...... iv
Table of Contents…………………………………..……………………………………v
List of Tables………………………………………………………..…………..viii
List of Figures……………………………………………………………………ix
List of Abbreviations……………..…………………………..………..………….x
Chapter 1: Introduction and Literature Review…………………………………….1-1
Statement of Importance…………………………………………..…………….1-1
Invasive Species…………………………………………………….…………..1-1
Description of Lythrum salicaria.…………………………………………….1-3
Introduction to North America and Uses……………………….……………….1-5
Spread and Removal Efforts…………………………………………………….1-5
Conflicting Opinions and Evidence……………………………….……….……1-6
Indicator of Human Disturbance and Pollution……………………..……….….1-6
Bioremediation and Phytoremediation as In-Situ Remediation………..……….1-7
Possibility of Pollution Uptake………………………………………...………..1-8
Scope of Project…………………………………………………………………1-9
Chapter 2: Materials and Methods………...………………………..………………..2-1
Location and Site Description…………………………………………………..2-1
Description of Samples……………………………………………...…………..2-1
v Allison Hubbard Bryant University
Procedures and Instrumentation……………………………………..………….2-4
Field Sampling………………………………………………..…………2-4
Field Procedure (Repeated at Each Sample Site)…………….…………2-5
Preserving Samples………………………………………………………..…….2-8
Soil Storage………………………………………………………...……2-9
Sample Preparation……………………………………………………………2-9
Acid Digestion………………………………………………………..2-10
ICP-MS……………………………………………………………………..….2-13
Data Analysis Techniques………………………………………………….….2-16
Chapter 3: Results……………………………………………………..………………3-1
Site Characterization and Plant Ecology………………………………………3-1
ICP-MS Results…………………………………………………………………3-3
Chapter 4: Discussion of Results……………………………………………...………4-1
Plant Biology and Ecology……………………………………………….……..4-1
Trace Metal Discussion…………………………………………………………4-4
Regulatory Comparison………………………………………………………..4-13
Chapter 5: Conclusions and Recommendations………………………………..……5-1
Appendices……………………………………………………………………….……A-1
References………………………………………………………………………..…….R-1
vi Allison Hubbard Bryant University
List of Tables
Table 1-1……………………………………………………………………………..….1-3
Depicts the taxonomic organization of Lythrum salicaria
Table 4-1…………………………………………………...……………………………4-5
Depicts a comparison of trace metal levels found in this study to previously
published values
Table 4-2……………………………………………………………………………….4-14
Depicts a comparison of trace metal levels found in this study to regulatory
standards from several agencies
Table 4-3……………………………………………………………………………….4-16
Depicts a comparison per ug/g of trace metals to dry weight of plant tissue
vii Allison Hubbard Bryant University
List of Figures
Figure 1-1…………………..…………………………………….……………………..1-4 Photo of Lythrum salicaria in native environment
Figure 1-2……………………………………………………………………….………1-5 A diagram of physiological structures of Lythrum salicaria
Figure 3-1……………………………………………………………………….………3-1 Field photos of the three separate sample sites in Rhode Island
Figure 3-2……………………………………………………………………………….3-2 Concentrations of Zn in Lythrum salicaria and soil
Figure 3-3……………………………………………………………………………….3-3 Concentrations of Mg and Ca in Lythrum salicaria and soil
Figure 3-4……………………………………………………………………………….3-4 Concentrations of Levels of Cr and Ni in Lythrum salicaria and soil
Figure 3-5……………………………………………………………………………….3-5 Concentrations of Mn and Cu in Lythrum salicaria and soil
Figure 3-6……………………………………………………………………………….3-6 Concentrations of As and Se in Lythrum. salicaria and soil
Figure 3-7……………………………………………………………………………….3-7 Concentrations of and Ag and Cd in Lythrum salicaria and soil
Figure 3-8……………………………………………………………………………….3-8 Concentrations of Ba and Pb in Lythrum salicaria and soil
Figure 3-9…………………………………………………………………………...…..3-9 Concentrations of Ba, Mg, Ca, and Ni in Lythrum salicaria and soil
Figure 3-10…………………………………………………………………………….3-10 Concentrations of Cd and Cu in Lythrum salicaria and soil
viii Allison Hubbard Bryant University
List of Abbreviations
Maximum Contaminant Level……………………………………………..…….MCL
National Oceanic and Atmospheric Administration…………………….………NOAA
Rhode Island Department of Environmental Management………………..…….RI DEM
Parts per Billion…………...……………………………………………………………ppb
Inductively Coupled Plasma Mass Spectrometry……………….…………………ICP-MS
Inductively Coupled Plasma Atomic Emission Spectroscopy……………...…….ICP-AES
ix Allison Hubbard Bryant University
Chapter 1: Introduction and Literature Review
Statement of Importance
The purpose of this study is to examine the uptake and sequestration capabilities
of the invasive macrophyte Lythrum salicaria L. 1753 (Purple Loosestrife) in regard to
selected trace metals (1). The uptake and sequestration of trace metals could be an indicator that L. salicaria could potentially be viewed as a gauge of human disturbance in an ecosystem, and as a potential mode of phytoremediation. It was observed in a previous study that instead of a strict monoculture, as described in the literature, native loosestrife was routinely comingled with invasive L. salicaria in field samples (2). Many locations that had L. salicaria present appeared to be within zones that were known to have had previous human disturbance, or nearby current human disturbance (2). These earlier observations prompted the current investigation of the capacity of L. salicaria to uptake selected metals and thereby potentially provide beneficial attributes of phytoremediation of contaminated environments. Instead of a complete eradication, it might be more effective for programs to switch to a different model of managing L. salicaria, based on its location, particularly if the area in question had a history of human use with evidence of pollution.
Invasive Species
A species is classified as invasive if there is a sizeable ecological disturbance that causes damage to an ecosystem in a way that influences humans (3). Many invasive species (also known as exotic species) are notable only when they negatively influence human welfare. Invasive species are a result of a rearranging of biota across a geographic
1-1 Allison Hubbard Bryant University
area that was previously uninhabited by the species in question (4, 5). The distribution of humans in all regions of the globe, coupled with diversified vectors, such as their extensive travel patterns, increased commerce, communication, and business, and the extensive trans-shipment of goods, have provided diversified vectors for expanding the range of invasive species, called “hitchhikers” by some observers. While some invasive species are introduced on purpose or through neglect (e.g.., the Burmese python in
Florida, or the Chinese snakehead fish on the U.S. east coast) a large portion of introduced species are from the increased rate of people traveling and goods being exchanged (4). Seeds, small insects, mice, reptiles, etc. can inadvertently be transported on a plane or boat to new territory. Invasive species can be accidentally introduced because people may not realize that the imported or planted species could spread easily from a confined space to the surrounding habitat (i.e., English ivy). Invasive species will often maintain a small population in a minor area and then once the species has reached a critical mass, the population explodes spreading at an exponential rate (4). Consequently, the invasion process can take over a decade for any changes to be noticeable, thus showing a population lag time. Land use and human disturbance in an ecosystem can make certain habitats more susceptible to invasion by an invasive species, for example farmland, new roadways, or canals (6). These human interventions have broken down barriers that had previously contained species to their geographic region of origin (7, 8).
Invasive species can crowd out native species in a given area and result in niche displacement, as well as change the genetic landscape through hybridization and mixing of the invasive species with a native species (4, 9). The increase in competition can result
1-2 Allison Hubbard Bryant University in the extinction of native species. Consequently, extinction of native species can result in a destabilization of an entire ecosystem and potentially lead to ecosystem collapse (10).
There has been recent speculation as to whether exotic species moving into a new environment should be seen as an invasion or as an increase in the natural extension of that species’ range (7, 9). An additional consideration involves whether the incidence of invasive species has actually become more prevalent, or the means of detection simply better than in the past, or both (7). Darwin alluded to observations of invasive plants in the reports from his travels, with a direct observation of an invasive species of thistle in
Argentina (7, 11). The first formal international declaration of the intent to curb the spread of invasive species and to control and eradicate them when present was set forth in
1992 at the Earth Summit Convention called by the United Nations. This led to the
Global Invasive Species Programme (GISP) which became established in 1997 (7).
Efforts in the United States to control and eradicate all identified invasive species have already reached over $30 billion a year in combined costs and losses (7). Yet, the influx and impacts of invasive species continues.
Description of Lythrum salicaria
L. salicaria is a flowering plant of the family Lythraceae. It is one of several members of the family that are identified as various types of loosestrife (12, 13, 14).
Table 1-1. Taxonomic classification of Purple Loosestrife (Lythrum salicaria) (15, 16, 17, 18).
Taxonomy of L. salicaria
Kingdom Division Class Order Family Genus Species
Plantae Tracheophyta Magnoliopsida Myrtales Lythraceae Lythrum. Lythrum salicaria
1-3 Allison Hubbard Bryant University
L. salicaria is native to Europe and Asia, but its exact origins are not clearly known (19). Large populations of L. salicaria have been found to extend up to the northern 65th parallel. The plant has been observed in areas of the Middle East, Russia,
India, parts of China, and Japan. L. salicaria is known as an invasive species in other temperate and subtropical areas such as Eastern Africa, and Australia (12). Commonly found along waterways, roadside ditches, and wetlands, the plant can survive in a variety of environments and can withstand bouts of flooding (thus is known as a hydrophyte) and periods of drought (14, 20). An illustration of L. salicaria is shown in Figure 1.1.
Figure 1-1. A dense population of Lythrum salicari at Site 1 (taken on 25 July 2014 by a Nikon Coolpix S6200 Camera).
L. salicaria is a perennial, herbaceous plant that can stand over 2m tall (12, 13,
14). As the plant ages the stems can become woodier and have an increased number of stems originating from a single root crown, becoming a subshrub (21, 22). This perennial plant with a regenerating root crown and adventitious roots is able to grow and spread quickly from year to year. Leaves are a semi-darker green color on older plants and can
1-4 Allison Hubbard Bryant University
reach 14cm in length. Stems are topped with a purplish cone-shaped inflorescence (spike)
with many flowers and vary in size (12, 13). L. salicaria flowers through July and August and even into early September if conditions allow for it (10, 12, 13, 19). It reproduces via seed production in late August through to October (12, 13). One individual plant can produce over 2.5 million seeds, and if conditions are not conducive to growth, the seeds can remain dormant for up to 3 years (12). All of these features help to account for the difficulty in eradicating or even containing the spread of this invasive plant.
A detailed illustration (Figure 1-2) of the L. salicaria plant shows an extensive root stucture, rigid stems,opposite leaves, and a conical inflorescence. Mature plants can have multiple stems growing from a single root crown (23).
Figure 1-2. A detailed illustration of the typical structures of L. salicaria (23).
1-5 Allison Hubbard Bryant University
Introduction to North America and Uses
L. salicaria was introduced to North America in the early 19th Century from
Europe (24). Seeds of L. salicaria made their way across the Atlantic in the ballast of
ships and by traders bringing them for garden ornamentation. The plant was also popular
in medicine gardens; L. salicaria was sometimes used as a remedy for intestinal distress
(25). Birds such as the American Goldfinch regularly nest in L. salicaria, and with other
nesting spaces disappearing, the maintenance of nesting areas (i.e. L. salicaria) could be
vital to the preservation of certain bird species (26, 27, 28, 29).
Spread and Removal Efforts
Once introduced to North America, L. salicaria spread easily by exploiting
human disturbance and by not having any native predators to control the population (24,
30). Many different methods have been employed to control and/or eradicate L. salicaria
from specific habitats. These methods include herbicides, manual pulling and digging
removal, burning, and biocontrols. Beetles and weevils native to Europe, and primarily
eat L. salicaria, were introduced into select areas to mitigate the spread of L. salicaria without harming native vegetation (4, 25, 31). In some instances this was able to stop the spread of L. salicaria and effectively eradicate it after long term continuous applications.
Without having natural predators, L. salicaria can effectively proliferate out of control.
The efforts to remove L. salicaria have cost the United States an estimated $45 million a year (7). While long term ecosystem sustainability is the end goal of any eradication effort, many times the results are short-lived and it is rare that long-term impacts are witnessed (32).
1-6 Allison Hubbard Bryant University
Many scientists, researchers, and environmental managers strongly claim that L. salicaria must be eliminated to preserve the natural habitats (32). Some studies have shown that the presence of L. salicaria can reduce the biomass of native plants through pollinator competition (33, 34). Most studies fail to assess the potential damage that could be done to an environment during the removal process, especially if L. salicaria is fulfilling a vital role in the ecosystem (29). Unfortunately, studies usually focus on specific aspects of an ecosystem versus the big picture when it comes to L. salicaria.
Studies tend to focus on the elimination of invasive species without weighing the costs to the ecosystem (some that may be detrimental). It has been documented that in some habitats, once L. salicaria is removed, that other invasive species that were previously held in check by L. salicaria (e.g., Phragmites australis Trinius ex Steudel, 1840, common reed) proliferate and spread at rapid rates (32). L. salicaria is also used by beekeepers to supplement their honey stocks, and to provide their bees with a food source
(25, 32). The actual effect that L. salicaria may have on an environment varies upon location and other species present. Additionally, there is sparse data pertaining to how long L. salicaria may have been in a given habitat (9).
Indicator of Human Disturbance and Pollution
Macrophyte hydrophytes are the primary producers found within aquatic and semi-aquatic environments, representing crucial components of chemical pathways within an ecosystem (14, 35, 36). Macrophyte hydrophytes have been used in multiple studies on the potential of bioremediation and phytoremediation of contaminated wetland areas (14, 37). In general, macrophyte hydrophytes are considered good indicators of
1-7 Allison Hubbard Bryant University
heavy metal pollution due to their resiliency, and being able to act as filters (38, 39).
Therefore, they can still thrive in areas of human disturbance. It then makes sense that since L. salicaria is considered a macrophyte hydrophyte, that human environmental disturbance would be noted as one of the leading causes of invasion by L. salicaria (40).
Bioremediation and Phytoremediation as an Example of In-situ Remediation
Implementation of bioremediation (particularly phytoremediation) in areas with contaminated substrates is becoming more widely used throughout the world as a viable treatment for areas exposed to pollutants. These applications are both examples of in-situ site remediation (41). Bioremediation is the utilization of a living organism to remove pollutants from a contaminated area; essentially any removal process that relies on biological processes to remove pollutants (43). Phytoremediation is the use of plants as a means of environmental decontamination. This is feasible if plants are able to uptake and sequester the contaminants or to degrade the contaminants into a less volatile or harmful form (41). Factors that can affect how specific plant species can take up trace metals include the plant species, the root zone, and actual plant uptake (42). However, in order for an herbaceous plant to be an effective phytoremediator, some type of seasonal harvesting would probably be necessary.
In-situ remediation is a designed treatment plan for a contaminated area that does not involve the direct removal of contaminated material (41, 43, 44). This approach can reduce cleanup costs, as well as causing a smaller disturbance to the environment undergoing treatment. One approach to in-situ remediation would be the extraction of the pollutants through a technique called phytoextraction. Phytoextraction is the use of plants
1-8 Allison Hubbard Bryant University
that accumulate pollutants and thereby extract toxins from the environment, followed by harvesting the plants to remove the toxic material (44). Plants that can be used for phytoextraction either need to hyperaccumulate the pollutants or to have a great yearly yield (44, 45).
Hyperaccumulation, or plants that are hyperaccumulators of certain chemicals, would have a greater capacity for in-situ remediation. For example, hyperaccumulators for metals are most effective where a shoot to root ratio of metals can exceed 1:1 (42).
The growth patterns shown by L. salicaria and the relative ease of harvesting the plants would be features favoring the consideration for phytoremediation or phytoextraction in certain locations.
Possibility of Pollution Uptake by Lythrum salicaria
L. salicaria is a hardy persistent plant that can survive under a variety of extreme conditions ranging from nutrient deprivation to heavy metal toxicity (6, 19, 20, 46, 47).
It is known that many plants possess the ability to uptake trace levels of heavy metals and to store them in the plant tissue with few consequences to the plant in its entirety (48).
Trace metals are an excellent indicator of persistent pollutants that may be in the environment (49). The uptake levels of trace metals by an individual plant is not necessarily high, but if there is a large proliferation of the species in a polluted area, the effect could be significant and its presence can be very beneficial to the ecosystem and to those who are looking to clean up the contaminated area (37, 48). Given that L. salicaria grows in dense stands in locations prone to human disturbance, especially from petroleum products or industrial chemicals, e.g., along heavily used river banks and
1-9 Allison Hubbard Bryant University
wetlands bordering industrial centers, the plant should be considered for uptake of
environmentally significant trace metals. Other external factors that can affect the plant’s ability to uptake trace metals would include pH, redox reactions, organic content of the soil, fertilizer exposure, and temperature (48, 50). Accumulation in the roots is common in ecosystems because the toxicity is centralized instead of being transferred through the food chain. Seasonal impacts can affect the accumulation of trace metals in plants due to when and how the plant is storing nutrients (44, 48). Plants tend to have higher concentrations of chromium (Cr) in the spring, while manganese (Mn) and zinc (Zn) tend to accumulate in the fall and in different areas of the plant (i.e. levels tend to be higher in the extremities of the plant during the summer or spring, while levels tend to be higher in root structures in the fall) (44, 51). However, different phytoremediators accumulate metals in different plant tissues depending on their uptake processes, tissue structure, and bacteria in the root systems (42). If the trace metals concentrate in above ground plant tissue, then a harvesting technique could be employed to remove contaminated L. salicaria as a phytoremediation technique. Determining where the metal levels are concentrated in L. salicaria could enable a tailored management program to be made per type of trace metal (i.e., whole plant removal for root extraction or just above ground harvesting).
When exposed to heavy metal contaminants such as lead (Pb), L. salicaria has been reported as displaying a decrease in above ground biomass, but the root system and biomass underground remained relatively unaffected (6). Pb is one of the most common heavy metal contaminants (52). Even if individual plants appeared to be dead after exposure to high levels of toxins (specifically Pb and copper (Cu)), the plant was able to
1-10 Allison Hubbard Bryant University
regrow during the following growing cycle (47). L. salicaria has also been employed to
remediate areas affected by drug pollution with some success and the plant is considered an indicator for exotoxicity (20). Some evidence exists that L. salicaria may be able to
accumulate Zn better than other hydrophytes (53, 54).
Based on these initial studies of trace metal accumulation by L. salicaria this
study was designed to establish additional baseline data about the concentrations of trace
metals within plants sampled from three different locations, and thereby to be considered
as a phytoremediator species.
Scope of Project
This project involved the analysis of the trace metal concentrations of L. salicaria
sampled from three different sites in northern Rhode Island in the United States.. The
assessment was designed to evaluate the potential for sequestration of trace metals, and
determine whether the plant is capable of hyperaccumulation. The results of this study
were intended to expand baseline data needed for designing future treatment modalities
and management programs for L. salicaria, primarily whether eradication efforts should
be continued, or if treatment should shift to a management standpoint (i.e. a harvesting
program). Therefore, we specifically analyzed whether L. salicaria could uptake heavy
trace metals/trace metals of concern, if L. salicaria would uptake different trace metals at
different rates, and if the concentrations of trace metals would vary within the leaf, stem,
and root tissues in the plant.
1-11 Allison Hubbard Bryant University
Chapter 2: Materials and Methods
Due to numerous materials used, a description of all materials is subsequently
provided within the specific methodologies inside this section. Major equipment used
included a SYNTH Microwave Synthesis Labstation and an Agilent 4500 ICP-MS. The
SYNTH Microwave Synthesis Labstation was used instead of alternative digestion
methods due to the speed of wet digestion and ease of use (55). The Agilent ICP-MS
trace metal analysis was used due to the accuracy, precision, and resolution of the
instrument, which is available in the Science and Technology Department Analytical
Laboratory at Bryant University.
Location and Site Description
The sites were chosen through visual observation by the experimenters at different localities throughout the state of Rhode Island. Sample sites were chosen within the towns located near Bryant University. Site 1 in North Smithfield, RI is at 41.97067°N and 71.53653°W and was first sampled on 25 July 2014. A second sampling trip was conducted on 31 July 2014. Site 1 was a protected wetland area located underneath high tension power lines next to a power station. Samples at Site 2, also in North Smithfield,
RI, at 41.42273°N and 71.54717°W were taken on 31 July 2014. Site 2 was located on a farm, where the soil was loose around the base of the L. salicaria, but obtaining a whole plant was difficult due to many of the accessible plants being connected by adventitious roots. Site 3, located in Smithfield, RI, at 41.95370°N and 71.54730°W, was sampled on
07 August 2014. Site 3 was located off Douglas Pike near a hotel, a composting operation, and a sand/gravel/salt area. Sampling at Site 3 was difficult due to the soil layer only
2-1 Allison Hubbard Bryant University
being a few inches deep with a rock layer below; however, the soil was dark and loosely grained. The roots of L. salicaria sampled at Site 3 were shallower and spread out across a greater area.
Description of Samples
Sites 2 and 3 were suspected of past or present contamination, while Site 1 was chosen as a comparison because it was believed to be relatively uncontaminated. Site 1 has been relatively protected in recent years as a Wetland Conservation Area (there was a sign stating this onsite). Site 2 was located immediately adjacent to a farm with running equipment and various chemical applications for the growth of crops. Site 3 was thought to be contaminated as it was located alongside a ditch next to a busy roadway with human and automobile traffic. Three plant samples were taken from each site (9 plant samples total), and one surface soil sample was taken from each site (3 samples). Each plant sample was processed through the ICP-MS by segmenting the plant (leaves, stems, roots), with each segment being duplicated depending on the amount of biological material available.
History of Land Use Near Sample Sites
North Smithfield and Smithfield have a long standing history in Rhode Island starting from ~1666 (56). Geologically, the bedrock is primarily granite with overlaying layers of till (a mixture of rock and soil deposited by glacial movement over the land) (56,
57, 58, 59). Once formed, both towns have undergone repeated development and change.
These land use changes stem from industrialization, a growing human population, and the
2-2 Allison Hubbard Bryant University
spread of transportation corridors. In their early years, both towns consisted mainly of
farms resulting in much of the land being cleared, farmed, and used for raising livestock.
Intersecting highways changed the landscape, and industry in North Smithfield and
Smithfield was focused on sawmills and gristmills in the 1800s, and then to iron works later on. Granite quarries were made as the towns and their manufacturing grew. Many farms transitioned to cultivating fruit instead of livestock and crops such as corn. In the
1900s many of the roads were upgraded and expanded and more homes emerged on the landscape (56, 57).
All of these activities would have contaminated the soil substrate over the years in
North Smithfield, RI and Smithfield, RI in some fashion. Farms used pesticides and other fertilizers to enhance plant growth and these compounds in turn reside in the soil.
Farming practices could well have been a primary means of introduction of certain trace elements (e.g., Cr) (60). Human disturbance (e.g., constructing roads and buildings) would have introduced trace metal pollutants into the environment that can persist for long periods of time in the environment.
Procedures and Instrumentation
Field Sampling
Several techniques were employed in the field sampling and lab protocols in order to preserve the integrity of L. salicaria. Procedures were primarily taken from the
“Manual of Herbarium Specimen Preparation” authored by Qin Leng (with contributions from Joseph E. Liljegren (Class of 2012) and Jessica May Vickers (Class of 2014)). This technique (pressing and freezing) enables the plant structures to remain intact and the
2-3 Allison Hubbard Bryant University
plants to be preserved for further testing without decaying. This is to keep the plants from molding or decaying. Parts of the plants and whole plants can also be preserved using a
70% alcohol solution in order to preserve the structural integrity for study later.
Field Procedure (Repeated at Each Sample Site)
An overview photograph of each habitat was taken with a Nikon COOLPIX
S6200. The coordinate location was tagged using a GIS device (Magellan Trition). One plant was selected as a representative sample. Three separate L. salicaria plants were photographed with a scale marker, sampled, and pressed (61). The soil around the roots of the plant samples was loosened to extract as much of the root crown as possible. A surface soil sample was extracted at each site, and a live L. salicaria specimen was transferred back to the laboratory.
Preserving Samples - Drying and Sterilization
Drying and sterilization are important to the preservation of the plant samples for extended use. Bacteria, fungi, and insects can cause decay of dried plant samples if they are not properly sterilized. The plant samples were dried in a press for seven days, and then placed into a clear plastic bag, with excess air removed, and sealed. The bag was placed into the deep freezer (~80°C) for around 7 days to achieve sterilization, and then removed and allowed to reach room temperature on the lab bench. The bag was left sealed during the temperature change to prevent moisture from reentering the plant samples.
2-4 Allison Hubbard Bryant University
Soil Storage
The sealed bags containing the three surface soil samples were placed in the deep
freezer (~80°C) until they were needed for testing. The surface soil samples were
removed from the freezer and allowed to reach room temperature before use.
Sample Preparation
L. salicaria tissue samples and soil samples were ground using a mortar and pestle. The plant tissue samples were ground separately by type (roots, stems, leaves).
Approximately 1g of material was massed per sample based on the material available.
The sample was ground into a fine powder, removed using sterile plastic spatulas (so as not introduce trace metals into the sample), and placed into a small glass vial (that had previously been cleaned with 2% HNO3) and labeled. Surface soil samples followed the same steps as the plant samples when grinding, except several grams of the soil samples oven-dried at 60°C for 48 hours before they were ground to ensure extraction of all moisture (Julia Crowley-Parmentier, personal correspondence).
Acid Digestion
A microwave digester, SYNTH Microwave Synthesis Labstation, was used to accelerate the complete digestion of samples. Approximately 0.3g of sample was placed in each sample vessel, along with 6mL of trace metal grade HNO3 and 2mL of H2O2.
Once capped and placed in the containers, the tops were tightened using a specialized torque wrench, and the vessels were left to sit overnight. The following day the
MUD25o.mpr program was used for processing the samples in the digester, according to
2-5 Allison Hubbard Bryant University
the operating instructions provided for the equipment. The time for runs 1 and 2 was set to 20 minutes each; the temperature for run 1 (T 1) was set to 200°C, and the temperature for run 2 (T 2) was set to 110°C at 1000W. When the digestion was done and the vessels
were cool enough to handle (~80-100 minutes after completion) the vessels were
removed from the oven. Vapors were allowed to dissipate before the tops were
completely removed (Julia Crowley-Parmentier, personal correspondence).
Dilutions for use in the ICP-MS were a 1:10 dilution in the first sample set generating raw data, and then a 1:5 dilution in the second sample set generating raw data.
ICP-MS
Agilent 4500 ICP-MS protocols were used for assessing trace metal levels in the plant tissues and soil samples and a batch file was created in the ICP-MS software. The sample data were added to the sample list, along with control samples if needed. Using the manufacturer’s guidelines, with adaptations developed for the Department of Science and Technology Analytical Laboratory, the ICP-MS was set up to process ~100 plant samples and ~9 soil samples, along with necessary blanks for comparison (47).
Data Analysis Techniques
Data analysis was completed using a Minitab statistical analysis software.
ANOVAS were employed during the analysis of trace metal levels by site (overall and by soil), and by plant segments. Two Subject T-Tests were used to compare trace metal levels in the plants versus the soils. Visual representations of the collected data were created in Microsoft Excel.
2-6 Allison Hubbard Bryant University Chapter 3: Results
Site Characterization and Plant Ecology:
The three sample sites are illustrated in Figure 3-1, showing dense clustered
populations of Lythrum salicaria at each site. Mixed plant communities were observed at all three sites, including mixtures of native loosestrife, cattail (Typha sp.), and common reed (Phragmites sp.), with distributions associated with the amount of moisture present at the site. As noted, samples were taken in mid-summer from each site.
A. B.
C.
Figure 3-1. Dense stands of Lythrum. salicaria at three separate sample sites. All photos were taken with a Nikon Coolpix S6200 Camera. (A). The photo of L. salicaria at sample site 1 (41.97067°N, 71.53653°W) was taken on 25 July 2014 at 2:19 PM. The L. salicaria plants were clumped and numerously scattered among other vegetation. (B). The photo of L. salicaria at sample site 2 (41.42273°N, 71.54717°W) was taken on 31 July 2014 at 12:10 PM. The stands of L. salicaria were ringed around the small pond next to the corn fields on a farm. (C). The photo of L. salicaria at sample site 3 (41.95370°N, 71.54730°W) was taken on 07 August 2014 at 12:02 PM. The L. salicaria were found centered around a ditch along the side of a main road.
3-1 Allison Hubbard Bryant University
ICP-MS Results
Following the ICP-MS analysis, the data were analyzed to depict the variation in
the concentration of metals associated with each sample. The metals in the following
figures were selected from the raw data as many of them are classified as metals of
concern by various agencies such as the U.S. Environmental Protection Agency (EPA).
Some of the metals were chosen because they display interesting results. As seen in
Figures 3-2 and 3.3, which compare the levels of metals ( e.g., Zn, Mg, and calcium (Ca)) found in plant tissues with the levels found in soil, there are differences among the metals.
Some of this variation is related to basic plant physiology, but prior land use and present contamination sources may also be influencing these levels. For example, the levels of Zn were higher in the leaves than in the other plant tissues.
A. B.
Figure 3-2. A comparison of the levels of trace metals in plant tissue (Lythrum salicaria) and soil. (A). The figure displays the comparison of Zn levels that were obtained from the samples of L. salicaria tissue (across the whole plant), and from samples of soil. (B). This graph shows a comparison of trace levels of Zn found in different plant tissues and soil, grouped by sample site.
3-2 Allison Hubbard Bryant University
A. B.
C. D.
Figure 3-3. Depicts a comparison of the levels of trace metals in plant tissue (Lythrum salicaria) and soil, for Mg and Ca. These metals are commonly associated with plant tissues and soils, as well as possibly being elevated by contamination in the soil. For example, Mg is a component of the chlorophyll molecule, which is of obvious importance to plant tissue. (A). The graph displays the comparison of Mg levels that were obtained from the samples of L. salicaria, and from samples of soil. (B). A comparison of trace levels of Mg found in different plant tissues and soil, also differentiated by sample site. (C). The graph illustrates the comparison of levels of Ca found in plant tissue and soil by site. (D) Shows a comparison of Ca in different plant tissue segments, soil, and sample sites.
3-3 Allison Hubbard Bryant University
The ICP-MS analysis of the various trace metals also yielded variations among other metals that were analyzed. Figures 3-4 and 3-5 illustrate comparisons of Cr, nickel
(Ni), Mn, and Cu, where there are noticeable distinctions among metals, sites, and plant tissues. For example, the graphs show that the levels of Cr were significantly higher in plant tissue than in the soil substrate, while Ni and Cu were higher in the soil than in the plant material.
A. B.
C. D.
Figure 3-4 (A-D). These graphs depict a comparison of the levels of trace metals in plant tissue (Lythrum salicaria) and soil, for Cr and Ni. (A). The graph displays the comparison of Cr levels that were obtained from the samples of L. salicaria, and from samples of soil. (B). A comparison of trace levels of Cr found in different plant tissues and soil, also differentiated by sample site. (C). The graph illustrates the comparison of levels of Ni found in plant tissue and soil by site. (D) Is a comparison of Ni in different plant tissue segments, soil, and sample sites.
3-4 Allison Hubbard Bryant University
A. B.
C. D.
Figure 3-5. A comparison of the levels of trace metals in plant tissue (Lythrum salicaria) and soil for Mn and Cu. (A). The graph displays the comparison of Mn levels that were obtained from the samples of L. salicaria, and from samples of soil. (B). A comparison of trace levels of Mn found in different plant tissues and soil, also differentiated by sample site. (C). The graph illustrates the comparison of levels of Cu found in plant tissue and soil by site. (D) Is a comparison of Cu in different plant tissue segments, soil, and sample sites.
3-5 Allison Hubbard Bryant University
The ICP-MS data yielded additional information that demonstrated noticeable
differences in concentration among the metals, soils, and sites. Figures 3-6 and 3-7 display the analysis and comparison of arsenic (As), selenium (Se), silver (Ag), and cadmium (Cd). Levels of As were much higher in the soil than in the plant tissue, as was
Se, whereas, Ag was higher in the plant tissue only at sample Site 1.
A. B.
C. D.
Figure 3-6. A comparison of the levels of trace metals in plant tissue (Lythrum salicaria) and soil. (A) The graph displays the comparison of As levels that were obtained from the samples of L. salicaria, and from samples of soil. (B) A comparison of trace levels of As found in different plant tissues and soil, also differentiated by sample site. (C) The graph illustrates the comparison of levels of Se found in plant tissue and soil by site. (D) Is a comparison of Se in different plant tissue segments, soil, and sample sites.
3-6 Allison Hubbard Bryant University
A. B.
C. D.
Figure 3-7. A comparison of the levels of trace metals in plant tissue (Lythrum salicaria) and soil. (A). The graph displays the comparison of Ag levels that were obtained from the samples of L. salicaria, and from samples of soil. (B). A comparison of trace levels of Ag found in different plant tissues and soil, also differentiated by sample site. (C). The graph illustrates the comparison of levels of Cd found in plant tissue and soil by site. (D) Is a comparison of Cd in different plant tissue segments, soil, and sample sites.
3-7 Allison Hubbard Bryant University
Based on the apparent variations among the different metals, the ICP-MS data
were further analyzed for more noticeable differences in concentrations among levels in
plant tissues, sites, and soil. Figure 3-8 displays the comparison of barium (Ba) and Pb
by plant tissue, soil, and site. Levels of Ba were higher in plant tissue from Site 1, but
were higher in the soil at sample Sites 2 and 3. Ba and Pb are shown here to demonstrate
a sharp contrast in metal distribution through plant tissues.
A. B.
C. D.
Figure 3-8. A comparison of the levels of trace metals in plant tissue (Lythrum salicaria) and soil. (A). The graph displays the comparison of Ba levels that were obtained from the samples of L. salicaria, and from samples of soil. (B). A comparison of trace levels of Ba found in different plant tissues and soil, also differentiated by sample site. (C). The graph illustrates the comparison of levels of Pb found in plant tissue and soil by site. (D) Is a comparison of Pb in different plant tissue segments, soil, and sample sites.
3-8 Allison Hubbard Bryant University
The following graphs shown in Figure 3-9 and 3-10 display some of the previous data (concentrations of Ba, Mg, Ca, Ni, Cd, and Cu) in a different visualization, to show an overall comparison of concentration levels in plants and soils at different sites. If the plant concentrations fall below the 45° line, the concentration of trace metal is higher in the soil. If the plant concentrations fall above the 45° line, then the concentration of trace metals is higher in the plant tissue than in the soil. If the concentration in plant tissue falls on the 45° line, then the trace metal concentrations in plant and soil are equal.
A. B. Concentration Plantin [ppb ]
C. D.
Concentration Plantin [ppb] Concentration Plantin [ppb]
Figure 3-9. A comparison of trace metal concentrations among plant tissues, soil, and site for Lythrum salicaria at Sites 1, 2, and 3). These graphs illustrate the relative differences in metal concentrations in plants and soils across the entire dataset. (A) Ba concentrations; (B) Mg concentrations; (C) Ca concentrations; (D) Ni concentrations.
3-9 Allison Hubbard Bryant University
A. B.
Figure 3-10. A comparison of trace metal concentrations among plant tissues, soil, and site for Lythrum salicaria at Sites 1, 2, and 3). These graphs illustrate the relative differences in metal concentrations in plants and soils across the entire dataset. (A) Cd concentrations; (B) Cu concentrations.
Other metals were analyzed using the ICP-MS, and a complete copy of the raw data files can be found in Appendix B.
3-10 Allison Hubbard Bryant University
Chapter 4: Discussion
Site Variations
As noted earlier, the three sample sites were chosen based on visual observation
(Figure 3-1), present and former land use patterns, and the relative likelihood of metal pollutants at the site. However, the results of this study challenged some of the initial assumptions about the sites. For example, at Site 1, initially presumed to be the most undisturbed due to its location in a protected wetland conservation area near an electrical substation, showed higher arsenic (As) concentrations than at either of the other two sites
(Figures 3-7A and 3-7B). At Site 2, located on a farm property adjacent to a busy road, and across from a school parking lot, selected particularly because of the multiple potential pollution sources, including a roadway, parking lot, and farm fields, all activities in which there would be a high likelihood of ongoing chemical pollution, yielded higher levels of Cr (Figures 3-4A and 3-4B) than the other sites. At Site 3, located next to a major roadway with heavy traffic flow as well as a gravel/salt storage area, a hotel, and a composting/mulch area, representing a different set of potential pollution sources, was characterized by the highest levels of Cu (Figures 3-5C and 3-5D) and Pb (Figure 3-8C and 3-8D).
For Sites 2 and 3, considering most of the trace metals examined in this study, the data correlated with what was expected in high traffic areas (Figures 3-2 to 3-10); whereas, the high level of As found at Site 1 raised questions about past land use at this location. Although presently part a preserved conservation area, Site 1 is in fact located beneath high tension power lines. It is possible that trace metal contamination was introduced into the area via runoff from the power lines, or possibly during the installation of the facility and its associated power lines (47). These factors could have
4-1 Allison Hubbard Bryant University
also affected other levels of trace metals at Site 1 (e.g., Ag, which was high in the plants,
particularly in root tissue, as shown in Figures 3-7A and 3-7B ). Trace metal contaminants can be associated with human sources such as mining, metal smelting,
electroplating, gas exhaust, energy and fuel production, runoff from power lines,
agriculture, power transmissions, sludge dumping, and military operations (10, 12, 13).
Before discussing in more detail the trace metal data assembled in this study, the
biological and ecological aspects of L. salicaria will be examined.
Plant Biology and Ecology
While all of the plants were identified as being L. salicaria, there was a
differentiation in morphology by site. Site 1 plants appeared to vary greatly in size across
the site. Samples obtained from Site 1 were smaller and the stems were less woody,
indicating that younger stands of L. salicaria were sampled (39). It was observed that
there were other stands of L. salicaria at Site 1 that exceeded 1.6m in height. Stands of
taller L. salicaria were located further off the footpath and in damper soil among thick
vegetation. Site 2 samples that were obtained were larger than those at Sites 1 and 3.
There were stands of L. salicaria at sample Site 2, but the majority of the plants appeared
to be approximately the same age/height. It was hard to find complete L. salicaria plants
with a full root system that were not directly attached to adventitious roots. This could be
due to the plants being on a farm, and it was observed that certain areas around the fields
had been cleared to remove the vegetation. Sample Site 3 samples were intermediate in
size compared to Site 1 and Site 2, but most of the L. salicaria plants along the edges of
the ditch were shorter than those in the center, and appeared to be younger and possibly
new from that season. The maturity of the plants could be important because if they are
4-2 Allison Hubbard Bryant University
new stands of L. salicaria their capability of uptaking trace metals may be different from those of more matured stands..
Terrestrial plants in an active growth phase such as the sampled L. salicaria, can readily absorb trace metals through their roots, whereas, submerged plants can also absorb trace metals from water through other organs. It is during the active growth phase that both terrestrial and aquatic plants store larger amounts of the trace metals than other phases (39, 50, 61). Multiple factors can affect the bioavailability and mobility of trace metals in an ecosystem. Those factors include, but are not limited to; vegetation, climate, parent soil material, living organisms, topography, time, sediment pH, organic matter, plant genotypes, seasons, and water flow (50).Seasonal fluctuations can affect the redox reactions in the stratification of layers of soil. This directly affects what trace metals are available for uptake by plants (63). A wide variety of plants possess metal bonding phytochains, or metal-binding polypeptides, which could be the key to hyperaccumulation of trace metals (39, 47). In highly tolerant plants, exchanges of ions and cations draw the trace metals into the plant and plant processes, and can cause a greater portion of the heavy trace metals to be bonded to the cell walls (62).
Plants that accumulate trace metals have been found to have certain specific processes that allow them to be more tolerant of high levels of trace metal contamination.
The plants can have the trace metals bind to the cell wall through carboxyl groups, reduce the uptake of trace metals by stopping the trace metals from entering the cell membrane, detoxify trace metals in cell cytoplasm through the use of ligands, or store the trace metals in the large central vacuole (45). Other evidence suggests that there are three patterns observed in trace metal tolerant plants. These include the trace metals being restricted from entering the plant, the trace metals accumulating in the roots and then
4-3 Allison Hubbard Bryant University
being restricted from entering the other organs, and the trace metals accumulating in
specific plant segments (62). Future biochemical and physiological studies may help
understand the processes by which L. salicaria sequesters trace metals. Such studies would be able to show if the trace metals are accumulated through biosorption or bioaccumulation. Biosorption is a passive process where the plant has surface attachment of the trace metals, and the process does not require energy to be expended.
Bioaccumulation is an active transportation process where trace metals are transported into live cells through the expenditure of energy (47, 61).
Trace Metal Distribution by Site Location and in Plant Tissues
Although trace metals are found naturally in the environment, they can be introduced through human activities. The difference between natural levels and contamination levels is related to the concentration within the substrates and the levels within organisms in an ecosystem. Trace metals that regularly occur in varying amounts within certain plant tissues would include Zn, Cu, Mn, Mg, Se, and Cd (45,47,61). A
comparison between this study’s results and those from previously published studies can
be found in Table 4-1. Zn and Cu can be utilized as part of plant proteins and within
enzymes for plant growth and development (61). Ca can be found in higher
concentrations within plant tissue because Ca can facilitate the movement of substances
through cell membranes (61). Mn has been found to activate select enzymes, and Mg is
critical in the formation of the chlorophyll molecules, and can also activate enzymes (61).
Plants can sometimes have high levels of Se and Cd because the plant may not be able to
differentiate between heavy metals and the trace metals needed for plant growth and
development (44, 53, 54).
4-4 Allison Hubbard Bryant University
Previously published studies included varied environments such as a contaminated site in France, a restored wetland area, and a lake system in India exposed to large amounts of urban runoff (44, 53, 54). Unlike the current study which utilized
ICP-MS, the other studies used ICP-AES (inductively coupled plasma atomic emission spectroscopy) or ICP coupled with flame atomic absorption (53). Differences in sensitivity and resolution of the results between different equipment could partially explain the differences in results.
Table 4-1. A comparison of trace metals found in this study’s L. salicaria samples and soils, L. salicaria samples from a site analysis in France that was contaminated (53), L. salicaria samples from a restored wetland (44), and L. salicaria samples from a lake site in Armenia that was contaminated by runoff from several urban areas around the lake (54). The study that detailed the trace metal contaminants in the site in France only gave information on Zn and Cd levels for L. salicaria. All concentrations are in ppb (parts per billion). Trace Levels in L. Levels in Soil (ppb) Site Analysis in Restored Sevan Lake, Metals Salicaria (ppb) France, L. Wetland, L. Armenia, L. salicaria Tissue salicaria Tissue salicaria Tissue (ppb) (53) (44) (ppb) (54) (ppb) Zn Site 1: 178,487.7 Site 1: 42,748.7 Aerial: 46,900 Site 2: 87,773.6 Site 2: 56,218.0 Roots: 57,900 59,600 3,981,000 Site 3: 93,219.1 Site 3: 270,246.6 Mg Site 1: 3,469,593.0 Site 1: 2,780,496.0 Site 2: 4,741,095.0 Site 2: 3,135,246.6 N/A N/A 46,620,000 Site 3: 377,8441.7 Site 3: 5,382,706.6 Ca Site 1: 892,271.7 Site 1: 234,749.0 Site 2: 1,140,553.6 Site 2: 281,147.2 N/A N/A 156,000,000 Site 3: 1,246,813.0 Site 3: 1,700,273.1 Cr Site 1: 922.0 Site 1: 904.9 Site 2: 4,160.2 Site 2: 672.4 N/A 2,200 9,650 Site 3: 3,887.2 Site 3: 503.0 Ni Site 1: 3,567.4 Site 1: 9,894.1 Site 2: 1,469.7 Site 2: 10,819.0 N/A 489 1,710 Site 3: 814.0 Site 3: 13,225.1 Mn Site 1: 1,101,363.7 Site 1: 677,307.0 Site 2: 223,562.9 Site 2: 248,344.5 N/A 115,000 454,600 Site 3: 186,126.0 Site 3: 515,787.6 Cu Site 1: 11,487.8 Site 1: 19,425.6 Site 2: 10,073.5 Site 2: 21,363.9 N/A 6,810 52,830 Site 3: 14,435.7 Site 3: 33,933.9 As Site 1: 380.9 Site 1: 15,399.3 Site 2: 381.8 Site 2: 7,251.3 N/A 432 N/A Site 3: 567.8 Site 3: 3,897.1 Se Site 1: 264.1 Site 1: 1,347.0 Site 2: 262.4 Site 2: 2,906.3 N/A N/A N/A Site 3: 131.1 Site 3: 2,414.4 Ag Site 1: 400.6 Site 1: 39.6 Site 2: 44.6 Site 2: 126.9 N/A N/A N/A Site 3: 22.9 Site 3: 97.1 Cd Site 1: 327.2 Site 1: 106.6 Aerial: 300 Site 2: 249.5 Site 2: 282.0 Roots: 200 173 210 Site 3: 108.7 Site 3: 232.1 Ba Site 1: 64,831.6 Site 1: 32,581.5 Site 2: 27,086.7 Site 2: 47,770.7 N/A N/A 77,930 Site 3: 18,249.4 Site 3: 74,995.9 Pb Site 1: 2,191.7 Site 1: 9,850.0 Site 2: 4,542.7 Site 2: 59,510.6 N/A 1,020 10,390 Site 3: 3,887.1 Site 3: 69,145.9
4-5 Allison Hubbard Bryant University
The difference in results between this study’s data and other studies could also be
related to location differences, specific soil types, seasonal patterns, the age of the plant
populations, and sampling variations. However, it seems imperative to compare the
findings in order to establish the range of variation of trace metals in the plant tissues of L.
salicaria, which may indicate the extensive resilience of this species in terms of its
tolerance for trace metal contamination.
This study includes Zn, Mg, Ca, Cr, Ni, Mn, Cu, As, Se, Ag, Cd, Ba, and Pb.
Compared with previous studies, as seen in Table 4-1, the current study scrutinized more
trace metals. These metals are all found in varying amounts within ecosystems. EPA
standards do not include Fe because it was not deemed as important/detrimental to
ecosystems as heavier metals (64). Other metals such as Fe, though not included in the
Results section, or in Tables 4-1 and 4-2, are included in Appendices A and B in the raw
data files. We are particularly interested in the toxic metals on the EPA list.
The levels of Zn were compared between whole plant and soils as differentiated
by site (Figure 3-2A) and for comparison of soils to segments of plant tissue at each site
(Figure 3-2B). Site 1 has the highest concentration of Zn in L. salicaria tissue, but Site 3 had the highest level of Zn in the soil by a large margin, while having the lowest concentration of Zn in the plant tissue. Unlike previously published data shown in Table
4-1, the highest concentrations of Zn in this study were in the leaf tissue of the L. salicaria samples, whereas, another study stated that Zn was more concentrated in the roots of their L. salicaria samples (44, 54). This variation may have been related to the age of the plants, or the particular phase in the annual growing season. The levels of Zn in our samples were higher than the levels reported in L. salicaria by the study that processed samples from a restored wetland, and conversely, our samples had significantly
4-6 Allison Hubbard Bryant University
lower levels of Zn than reported by the study conducted at contaminated lakes in India,
where the level of pollution was very high (53). It was expected that there would be large amounts of Zn found in the samples due to L. salicaria being able to accumulate higher levels of Zn than other wetland plants, as well as the fact that Zn is needed for a plant to properly function (63). The levels of Zn may have been lower in the plant tissue from
Site 3 because of the plants being younger, as well as the reality that we do not know how long the stands of L. salicaria had been present on that site. The Zn could also have been newly introduced into the environment, without sufficient time for uptake by the plant.
Analyses of Mg addressed differences between whole plant and soils by site
(Figure 3-3A) and compared concentrations of this metal in soils to different plant tissues at each site (Figure 3-3B). The Mg Levels followed a similar pattern as the Zn levels.
Sites 1 and 2 had higher levels of Mg in plant tissue than soil, while at Site 3 the Mg levels were higher in the soil than in the plant tissue. The highest concentration of Mg was in the leaf tissue at all three sites. This is not surprising given that Mg, like Zn, is used in essential plant processes. Finding the highest concentrations of Mg in the L. salicaria leaves was expected due to it being a primary element in the formation of chlorophyll (54). The levels of Mg found in this study’s L. salicaria samples were much lower than those found in the Indian lake study, but that was to be expected due to much higher levels of constant contamination in the lakes (Table 4-1) (63). Site 3 could have had higher levels of Mg because of the location of the sampling site. It would be difficult to pinpoint one specific contributing factor because of the proximity to the compost/mulch, a parking lot/hotel, a busy roadway, and the salt/sand area.
The levels of Ca were compared between whole plant and soils as differentiated by site (Figure 3-3C) and for the comparison of segments of plant tissue to the soil
4-7 Allison Hubbard Bryant University
samples at each site (Figure 3-3D). The Ca resembled the pattern of the Zn and Mg levels.
The levels of Ca were higher in the plant tissue at all sites except for Site 3, and the
concentration of Ca was highest in the L. salicaria leaves. Having a large amount of Ca
present in the plant tissues was expected since Ca is essential to plant function,
particularly to facilitate the movement of substances across cell membranes (44, 54). The
plants were collected around mid-day in full sunshine during the active growth cycle, so
it would be reasonable to conclude that the Ca levels could be high in the leaf tissues due
to active photosynthesis and production of substances being moved back and forth
between cells. Site 3 could have had higher levels of Ca because of the location of the
sampling site influenced by its proximity to the compost/mulch, a parking lot/hotel, a busy roadway, and the salt/sand area.
Concentrations of Cr were compared between whole plant and soils differentiated by site (Figure 3-4A) as well as addressing the comparison of soils to segments of plant tissue at each site (Figure 3-4B). The levels of Cr were higher in plant tissue than in soils at all three sampling sites. The concentration of Cr in L. salicaria tissue was higher in this study at Sites 2 and 3 than the restored wetland site, but lower than the Indian lake system study (Table 4-1) (44, 54, 68). This would indicate that it is possible for L. salicaria to sequester potentially toxic trace metals such as Cr in varying amounts. Interestingly, the highest bioconcentration of Cr in plant tissue occurred at Site 2 which is currently a farm.
The levels could have been higher than expected because of the location of the farm. Cr is associated with metal working and cars, and the farm is located on the intersection of two roads (65). The sample location of the L. salicaria at the site was below street level and probably received runoff from a large school parking lot nearby. Therefore there would be enhanced traffic flow and possible exposure to sources of Cr. Depending on the
4-8 Allison Hubbard Bryant University
age of the farm, it is also possible that old vehicles, farm equipment, and substances
related to those were dumped in a “trash” area. This was common on older working
farms (44, 54). Cr could leach from the old equipment and eventually end up in the area of the small pond on the property (next to sampling location) because of the slope.
The concentrations of Ni were compared in a similar fashion (i.e. between whole plant and soils as differentiated by site (Figure 3-4C) and between soils and segments of plant tissue at each site (Figure 3-4D). The levels of Ni were significantly higher in the soils at each site than the plant tissue. However, the level of Ni at Site 1 in the plant tissue was roughly seven times higher than the levels found in L. salicaria tissue from the restored wetland and around two times as high as the levels found in L. salicaria tissue from the Indian lake system (54). As seen in Table 4-1, the level of Ni at Site 2 in plant tissue was three times as high as those found in the restored wetland tissues (44, 54). The level of Ni was highest in the soil in Site 3, but lowest in the plant tissue from Site 3. The level of Ni in Site 1 could have come from previous land use or runoff from the power lines. The Ni levels in Site 3 could be attributed to younger stands of L. salicaria on the site, as well as a constant means of contamination from the roadway. This is significant because the Ni levels demonstrate the possibility of factors working synergistically so that L. salicaria could uptake higher levels of select trace metals than previously reported.
Comparison of the levels of Mn between whole plant and soils differentiated by site (Figure 3-5A) and for soil concentrations to plant tissue at each site (Figure 3-5B).
Site 1 had much higher levels of Mn than the other three sampling sites, as well as being higher than the published literature sites (Table 4-1) (66). The second highest concentration of Mn in plant tissue occurred in Site 2 samples. The Mn in Site 1 could have come from previous land use of the area, especially if there was a metal processing
4-9 Allison Hubbard Bryant University
plant nearby, industrial exhaust depositing in the area, or from batteries if the area had a
trash site near it (66). At Site 2 the levels of Mn in the soil were similar to those in the
plant tissue, where levels in the soil at Site 3 were much higher than the plant tissue. The
levels at Site 2 could be explained by exposure to fertilizers, and the levels could have
been lower in the soil because the fertilizer was deposited on the fields with runoff
draining primarily into the area of the L. salicaria (44). The bulk of Mn may have been
taken up by the crops that the fertilizer was intended for.
Concentrations of Cu between whole plant and soils differentiated by site location
(Figure 3-5C) and between soils and plant tissues at each site (Figure 3-5D) showed that
while the levels of Cu were higher in the soil than in the plant tissue at all three sites, the
levels of Cu found were higher than those found at the restored wetland (Table 4-1). The
levels of Cu in the plant tissue are therefore notable even though lower than the levels
from the Indian lake system (47, 61). It is not surprising that there were significant levels
of Cu found because Cu can be used in plant growth processes (39). Cu can come from
wastewater runoff (such as from roadways), and Cu is more likely to settle into the
sediment and to form stronger bonds. This makes it less bioavailable for plant use
depending on the chemical reactions occurring in the soil (47). This could explain the higher levels of Cu in the soil at Site 3 since it was next to a busy roadway. However, higher levels of Cu in plant tissue can interfere with plant growth because it can cause displacement of K, Zn, Mg, and Fe (67).
The levels of As were assessed using whole plant concentrations and soils as differentiated by site (Figure 3-6A) and for comparison of soils to segments of plant tissue at each site (Figure 3-6B). The highest levels of As were found in Site 1 soil and
Site 2 soil and the highest levels of As in plant tissue were from Site 3 (Table 4-1). The
4-10 Allison Hubbard Bryant University
level of As found in Site 3 plant tissue was higher than the level reported from the
restored wetland study. Conversely, the lowest levels of As in soil came from Site 3. It is
troubling that the level of As in Site 1 soil was the highest, since we had initially assumed
Site 1 to be the least contaminated site, and viewed plants at that site to be “controls.”
This is most likely linked to previous land use especially if metals were used in the area.
It is possible that As was introduced through the power lines because As is used as a
semi-conductor (67). There could also be As naturally found in the area as part of the
natural geology (67). The higher level of As in the soil at Site 2 could have come from
agricultural applications of a compound containing As (67).
Concentrations of Se varied between whole plant and soils when considered by
site (Figure 3-6C) and as well as between soils and plant tissue at each site (Figure 3-6D).
The levels of Se were higher in the soil than the plant tissue, but the highest level of Se
was found in the soil at Site 2 (Table 4-1). The Se could have come from runoff from the
road, parking lot at the school, or petroleum contamination from farm equipment (68, 69).
The concentrations of the trace metal Ag were likened between the whole plant
and soils as well as by site (Figure 3-7A) and for comparison of soils to segments of plant
tissue at each site (Figure 3-7B). Remarkably, the highest level of Ag was found in the
Site 1 plant tissue, and then in the Site 2 soil (Table 4-1). The levels of Ag in the plant
tissue of Site 1 would point to previous contamination of the site, and not necessarily
present contamination of the site. Ag typically comes from ore processing, electric
equipment, and electroplating (53). Again, this raises the question of the previous land use in and around the area of Site 1.
The levels of the trace metal Cd were gauged between whole plant and soils as extricated by site (Figure 3-7C) and for evaluation of soils to segments of plant tissue at
4-11 Allison Hubbard Bryant University
each site (Figure 3-7D). The highest levels of Cd were found in Site 1 plant tissue, and
then in the Site 2 soil. Unlike the site analysis of the contaminated site in France, the Cd
was concentrated in the root tissue instead of the aerial plant tissue (Table 4-1) (44, 69,
70). The levels of Cd in Sites 1 and 2 plant tissues were higher than those found in the
restored wetland and Indian lake system (44, 54). The L. salicaria samples from Site 1 could have higher levels of Cd because plants can have higher levels of heavy metals than expected because the plant confuses them with metals needed for plant growth (71).
Cd contamination can come from runoff that contains petroleum, which would make sense in the case of Site 2 due to its location. Again, Cd like some of the other trace metals found at Site 1 is used in metal plating (71). Cd is also associated with batteries,
paint, plastics, electrical equipment, jewelry making, and brake lines in automotive
vehicles (54, 70, 71). It would be interesting to know if there is, or ever has been, battery
disposal associated with the operation of the utility operations near Site 1.
Assessment of Ba concentrations followed a similar pattern of comparing whole
plant and soils differentiated by site (Figure 3-8A) and soils to segments of plant tissue at
each site (Figure 3-8B). Ba levels were highest in the Site 1 plant tissues (which were in
turn closest to the level reported from the Indian lake system), but lowest in Site 3 plant
tissues (Table 4-1) (72). The highest level of Ba in soil was found in Site 3, while the
lowest was in Site 1. The plants at Site 1 appear to be much more efficient at sequestering
Ba. Ba usually comes from natural ores found in soils. However, they can be used in
metal alloys and electronic equipment (44, 54). It is possible that the Ba contamination
from Site 1 could come from previous land use, or the high tension power lines. It is
unclear as to how the contamination in Site 3 would have been introduced.
4-12 Allison Hubbard Bryant University
Finally, the levels of Pb were also compared between whole plant and soils by site location (Figure 3-C) and as well as between soils to segments of plant tissue at each site
(Figure 3-8D). In plant tissue the level of Pb was highest in Site 2, and in the soils the highest level of Pb was found in Site 3. The levels of Pb found in plant tissue in this study were all higher than those found at the restored wetland and lower than those found in the
Indian lake study (Table 4-1) (67). Pb commonly enters the environment through industrial applications such as petroleum products or old pipes (39, 47). These could be modes of introduction at any of the three sites depending on the previous land use of each area, and depending on the location and age of water lines. Current introduction of Pb into the environment would probably be from petroleum based products. It is not surprising that the levels of Pb are higher in the soils than in the plant tissues because Pb tends to make strong bonds within the sediments, which makes it less bioavailable to plants (73).
Regulatory Comparison
Government agencies have diverse regulations for allowable levels of trace metals in varying environments. This creates a complicated regulatory structure for trace metal limits, and levels can also be evaluated by local municipalities. The level of selected trace metals in L. salicaria tissue and site soil (from our study) is compiled in a table which compared those levels to the Maximum Contaminant Levels (MCLs) set forth by NOAA,
RI DEM, and the EPA (Table 4-2).
In Table 4-2 the MCLs (Maximum Contaminant Levels) for NOAA and RI refer mainly to trace metal concentrations in soil except for the levels labelled “P” under
NOAA. The concentrations labelled “P” are the trace metal concentrations expected in
4-13 Allison Hubbard Bryant University
plant tissues. The EPA standards listed are those for drinking water and groundwater
MCLs.
Table 4-2. A comparison of trace metal levels found in Lythrum salicaria by site and trace metal levels in the soils by site. Further comparison is exhibited which compares Maximum Contaminant Levels (MCLs) as stated by NOAA, MCLs for Residential and Industrial/Commercial areas as stated by the Rhode Island Department of Environmental Management (RI DEM), the MCLs for Drinking Water Standards as stated by the Environmental Protection Agency (EPA), and MCLs for Groundwater Standards as stated by the RI DEM. Trace Levels in L. Levels in Soil MCL NOAA MCL RI (74) MCL MCL Metals Salicaria (ppb) (ppb) (73) Target, Residential and Drinking Ground- Intervention, Industrial/ Water water Plants (ppb) Commercial Standards Standards (ppb) (75) (ppb) (74, 75) (ppb) Zn Site 1: 178,487.7 Site 1: 42,748.7 T:16,000L R: 6,000,000 Site 2: 87,773.6 Site 2: 56,218.0 I: 350,000L I/C: 10,000,000 5,000 N/A Site 3: 93,219.1 Site 3: 270,246.6 P: 50,000a
Mg Site 1: 3,469,593.0 Site 1: 2,780,496.0 T: N/A R: 390,000 Site 2: 4,741,095.0 Site 2: 3,135,246.6 I: N/A I/C: 10,000,000 N/A N/A Site 3: 377,8441.7 Site 3: 5,382,706.6 P: N/A Ca Site 1: 892,271.7 Site 1: 234,749.0 T: N/A R: N/A Site 2: 1,140,553.6 Site 2: 281,147.2 I: N/A I/C: N/A N/A N/A Site 3: 1,246,813.0 Site 3: 1,700,273.1 P: N/A Cr Site 1: 922.0 Site 1: 904.9 T: <380L R: 1,400,000 Site 2: 4,160.2 Site 2: 672.4 I: <220,000L I/C: 10,000,000 100 100 Site 3: 3,887.2 Site 3: 503.0 P: <1,000a Ni Site 1: 3,567.4 Site 1: 9,894.1 T: 260L R: 1,000,000 Site 2: 1,469.7 Site 2: 10,819.0 I: 100,000L I/C: 10,000,000 N/A 100 Site 3: 814.0 Site 3: 13,225.1 P: 30,000a Mn Site 1: 1,101,363.7 Site 1: 677,307.0 T: N/A R: 390,000 Site 2: 223,562.9 Site 2: 248,344.5 I: N/A I/C: 10,000,000 50 N/A Site 3: 186,126.0 Site 3: 515,787.6 P: 220,000 Cu Site 1: 11,487.8 Site 1: 19,425.6 T: 3,400L R: 3,100,000 Site 2: 10,073.5 Site 2: 21,363.9 I: 96,000L I/C: 10,000,000 1,000 N/A Site 3: 14,435.7 Site 3: 33,933.9 P: 70,000 As Site 1: 380.9 Site 1: 15,399.3 T: 900L R: 7,000 Site 2: 381.8 Site 2: 7,251.3 I: 55,000 I/C: 7,000 10 10 Site 3: 567.8 Site 3: 3,897.1 P: 18,000 Se Site 1: 264.1 Site 1: 1,347.0 T: 700L R: 390,000 Site 2: 262.4 Site 2: 2,906.3 I: 100,000S I/C: 10,000,000 50 50 Site 3: 131.1 Site 3: 2,414.4 P: 520 Ag Site 1: 400.6 Site 1: 39.6 T: N/A R: 200,000 Site 2: 44.6 Site 2: 126.9 I: 15,000S I/C: 10,000,000 100 N/A Site 3: 22.9 Site 3: 97.1 P: 2,000a Cd Site 1: 327.2 Site 1: 106.6 T: 800 R: 39,000 Site 2: 249.5 Site 2: 282.0 I: 12,000 I/C: 1,000,000 5 5 Site 3: 108.7 Site 3: 232.1 P: 4,000a Ba Site 1: 64,831.6 Site 1: 32,581.5 T: 160,000 R: 5,500,000 Site 2: 27,086.7 Site 2: 47,770.7 I: 625,000 I/C: 10,000,000 2,000 2,000 Site 3: 18,249.4 Site 3: 74,995.9 P: 500,000a Pb Site 1: 2,191.7 Site 1: 9,850.0 T: 55,000L R: 150,000 Site 2: 4,542.7 Site 2: 59,510.6 I: 530,000 I/C: 500,000 0 15 Site 3: 3,887.1 Site 3: 69,145.9 P: 50,000a
Due to the trace metal levels found in the L. salicaria tissue in this study (e.g., Cr,
Zn, Mg, Mn), it would be possible to speed the remediation of a contaminated area if a large enough population of L. salicaria was present to take up trace metals to meet the
4-14 Allison Hubbard Bryant University
various environmental standards. The important point is to not look at individual plant
uptake, or even the uptake of a few plants, but what the plants could do at a critical mass.
Even when the differential between soil and plant levels of a trace metal are not large, the
combined effect of a large L. salicaria population acting simultaneously, coupled with the ease of harvesting the plants, might enable the plant to act as a bioaccumulator.
The Mg levels were higher in the L. salicaria tissue than the soil at both Site 1 and Site 2, and those levels are higher than the acceptable level for residential areas in
Rhode Island (Table 4-2). These levels suggest that L. salicaria could be sequestering sizable amounts of Mg. The levels of Cr are particularly significant because not only were the levels of Cr in L. salicaria tissue higher than those found in the soil, two out of the three were also higher than what NOAA sets as the target ppb for plants (Table 4-2).
This suggests that L. salicaria is able to uptake higher levels of Cr and continue to thrive.
Levels of Cu, while higher in the soil than the plant tissue, can still be significant due to the fact that they were at least ten times higher than the MCL for drinking water standards. If L. salicaria was to grow along waterways that feed into a reservoir or along a reservoir it could potentially help to filter Cu contamination out of the water. L. salicaria has a large potential that should be explored in future studies.
Where the value of L. salicaria truly becomes apparent, is in its comparison to the uptake abilities of other comparable herbaceous plants. As seen in Table 4-3, L. salicaria stacks up well in performance for the uptake of Cu, Pb, Cr, As, Mn, and Cd against other plants. A snapshot of the various plant abilities is demonstrated between L. salicaria,
Urtica dioca L., Phragmites australis (Cav) Trin ex Steudel, Rumex obtusifolius L., and
Batrachium divaricatum (Shrank) Wimm.(44, 54), as shown in Table 4.3.
Table 4-3. Depicts a comparison per ug/g of trace metals to dry weight of plant tissue.
4-15 Allison Hubbard Bryant University
Trace Metal Levels in ug/g of Dry Weight Plant Tissue Trace L. salicaria L. salicaria Urtica dioica Phragmites australis Rumex obtusifolius Batrachium Metal (Our Study) (Restored (Restored (Restored Wetland) (Restored Wetland) divaricatum Wetland) (44) Wetland) (44) (44) (44) (Sevan Lake, Armenia) (54) Cu Site 1: 11.49 Site 2: 10.07 6.81 8.24 4.64 7.52 250.5 Site 3: 14.44
Pb Site 1: 2.19 Site 2: 4.54 1.02 2.38 0.592 2.37 24.77 Site 3: 3.89 Cr Site 1: 0.92 Site 2: 4.16 2.2 4.63 2.98 2.70 31.43 Site 3: 3.89 As Site 1: 0.38 Site 2: 0.38 0.43 0.688 0.339 0.77 N/A Site 3: 0.57 Mn Site 1: 1,101 Site 2: 223.6 115 44.6 110 73.4 5,454 Site 3: 186 Cd Site 1: 0.33 Site 2: 0.25 0.173 0.136 0.06 0.508 0.83 Site 3: 0.11
Comparatively, using ug/g of dry weight of plant tissue, the L. salicaria in our study was able to uptake more trace metals than other plants. One plant, B. divaricotum did outstrip L. salicaria in the uptake of trace metals from an environment. However,
Sevan Lake, Armenia is highly contaminated and would have a higher likelihood that trace metals would be bioavailable to the plants present (54, 65). While L. salicaria may perform differently than other plant species, it is important to note that it possesses a broad range of uptake capabilities.
4-16 Allison Hubbard Bryant University
Chapter 5: Conclusions and Recommendations
There are several conclusions that can be made from this study. L. salicaria can selectively uptake trace metals from soil/sediment, and while some of those trace metals are used for physiological purposes, others are linked to environmental pollution. Due to the broad spectrum of metals examined, it provides a wider view of what L. salicaria can bioaccumulate, and provides valuable baseline data for future studies. The uptake of the trace metals is related to site dynamics, age and maturity of plants, physiological needs, and developmental dynamics of the plants. The uptake of the trace metals, coupled with the sequestration of select metals, suggests that L. salicaria may be able to be utilized in phytoremediation efforts, especially because of the dense growth of the plant.
The ICP-MS, due to its precise high resolution was very useful in demonstrating trace levels of metals in soil and plant samples. Although there is evidence that L. salicaria accumulates metals in plant tissues, more testing in polluted environments and lab simulated environments should be done to further verify the findings of this study, including seasonal variation, age of plants, larger sample size for plant analyses, and deeper background study of former land use patterns. Going forward, it would be prudent to evaluate if L. salicaria is doing more harm than good in a specific ecosystem, especially if the environment is polluted before eradication. Future work should determine if L. salicaria could be easily harvested at a commercial scale, and if not harvested, to determine if the plant could transform the metals into a less harmful form.
Further studies would elucidate how management strategies for this invasive species should be altered in order to balance the positive and negative ecological impacts of L. salicaria.
5-1 Allison Hubbard Bryant University
Therefore, on the basis of the data that have been collected L. salicaria can uptake
heavy trace metals/trace metals of concern, L. salicaria does uptake different trace metals
at different rates, and the concentrations of trace metals vary within the leaf, stem, and
root tissues in the plant.
The study limitations included small processing errors that were able to be
corrected, such as the initial mislabeling of one sample that was able to be correctly
notated, and failure to include one blank in the ICP-MS analysis, although there was a
consistent pattern for the other blanks. It was suggested that there could have been a
possible transfer of trace metals from the ink in the newspaper used during the pressing
and drying process, and this concern about sample preparation protocols should be
addressed for future studies. Data may have been limited in a comparison standpoint
with the soil samples due to only one surface sample being taken from each site. The pool
of data would have been better if there had been more samples. Some biological tissue
(e.g., roots) was only able to be run in technical duplicates due to the amount of tissue available (some plants were smaller than others and it was difficult to determine how much dry material would be available once the plant dried out). Finally, during the last microwave digestion run, six out of the ten Teflon vessel inserts in the digester warped.
All procedures were carried out as they had been on previous run, and at this time it is unknown as to what caused the warping of the vessels, but it is clear that the processing protocols for the acid digestion should be re-examined.
Future Studies
It is recommended that future studies should continue assess the phytoremediation ability of L. salicaria. It would also be important to know how the binding of metals to
5-2 Allison Hubbard Bryant University
soils and plant tissues would affect the bioremediation capabilities of L. salicaria. This
would then lead to assessment as to the ecotoxicity to other organisms. Another avenue
that could be explored would be how the metals may be transformed before entering the
environment, or within the environment.
While the levels of trace metals found in this study fall below the intervention
limits set by the various agencies (See Table 4-2), some of the metals could still be of concern, especially on Site 2, since it is a farm. The As levels at Site 1 are of concern, particularly because Site 1 was considered to be the clean site located in a preserved habitat, and show the importance of tracking down past land use for phytoremediation sites. Also of concern is that the second highest Pb level was at Site 2. While the levels of
Pb are below what is considered actionable with intervention, we should be concerned about crops being grown in soils with trace metals near MCL levels due to the fact that some vegetable crops have the potential to bioconcentrate metals (76, 77). Higher levels of Mg and Ca could have come from liming of the soil.
Soil coring might yield additional information about the land use history of contaminated sites, as reflected by the concentration of heavy metals at various depths, but if sampled, investigators should use coring devices not made of metal.
While the data in this study lends significant credence to the goals of the study, it is clear that there is much work to be done in the field of plants and trace metals as a whole. The
data provided from this study can serve as a baseline for future studies in this field.
5-3 Allison Hubbard Bryant University
Appendix A: Raw Preliminary Data
A-1 Allison Hubbard Bryant University
A-2 Allison Hubbard Bryant University
A-3 Allison Hubbard Bryant University
A-4 Allison Hubbard Bryant University
A-5 Allison Hubbard Bryant University
A-6 Allison Hubbard Bryant University
A-7 Allison Hubbard Bryant University
A-8 Allison Hubbard Bryant University
A-9 Allison Hubbard Bryant University
A-10 Allison Hubbard Bryant University
Appendix B: Raw Main Data
A-11 Allison Hubbard Bryant University
A-12 Allison Hubbard Bryant University
A-13 Allison Hubbard Bryant University
A-14 Allison Hubbard Bryant University
A-15 Allison Hubbard Bryant University
A-16 Allison Hubbard Bryant University
A-17 Allison Hubbard Bryant University
A-18 Allison Hubbard Bryant University
A-19 Allison Hubbard Bryant University
A-20 Allison Hubbard Bryant University
A-21 Allison Hubbard Bryant University
A-22 Allison Hubbard Bryant University
A-23 Allison Hubbard Bryant University
A-24 Allison Hubbard Bryant University
A-25 Allison Hubbard Bryant University
A-26 Allison Hubbard Bryant University
A-27 Allison Hubbard Bryant University
A-28 Allison Hubbard Bryant University
A-29 Allison Hubbard Bryant University
A-30 Allison Hubbard Bryant University
A-31 Allison Hubbard Bryant University
A-32 Allison Hubbard Bryant University
A-33 Allison Hubbard Bryant University
A-34 Allison Hubbard Bryant University
A-35 Allison Hubbard Bryant University
A-36 Allison Hubbard Bryant University
A-37 Allison Hubbard Bryant University
A-38 Allison Hubbard Bryant University
A-39 Allison Hubbard Bryant University
A-40 Allison Hubbard Bryant University
References
1. USDA (2015) USDA, ARS, National Genetic Resources Program. Germplasm Resources Information Network - (GRIN) [Online Database]. National Germplasm Resources Laboratory, Beltsville, Maryland. URL: http://www.ars- grin.gov.4/cgi-bin/npgs/html/taxon.pl?23022.
2. Balogh, G.R. (1986) Ecology, Distribution, and Control of Purple Loosetrife (Lythrum salicaria) in Northwest Ohio THESIS. Ohio State University 1-111.
3. EPA United States Environmental Protection Agency. (2012) Invasive Species. http://water.epa.gov/type/oceb/habitat/invasive_species_index.cfm.
4. Mooney, H. A., and Cleland E. E. (2001) The evolutionary impact of invasive species. PNAS 98(10):5446-5451.www.pnas.org/cgi/doi/10.1073/pnas.091093398.
5. Arim, M., Abades, S.R., Neill, P.E., Lima, M., and Marquet, P.A. (2005) Spread dynamics of invasive species. PNAS 103(2): 374-378.
6. Uveges, J.L., Corbett, A.L., and Mal, T.K. (2002) Effects of lead contamination on the growth of Lythrum salicaria (purple loosestrife). Environmental Pollution 120(2): 319-323.
7. Henderson, S., Dawson, T.P., and Whittaker, R.J. (2006) Progress in invasive plants research. Progress in Physical Geography 30(1): 25-46. DOI: 10.1191/0309133306pp468ra.
8. Rachich, J., and Reader, R.J. (1999) An experimental study of wetland invasibility by purple loosestrife (Lythrum salicaria). Canadian Journal of Botany 77(10):1499-1503.
9. Farnsworth, E.J., and Ellis, D.R. (2001) Is purple loosestrife (Lythrum salicaria) an invasive threat to freshwater wetlands? Conflicting evidence from several ecological metrics. Wetlands 21(2):199-209.
10. Jacobs, J. (2008) Ecology and Management of Purple Loosestrife (Lythrum salicaria L.). US Department of Agriculture, Natural Resources Conservation Service (NRCS).
11. Darwin, C.R. 1845: Journal of researches into the natural history and geology of the countries visited during the voyage of the H.M.S. Beagle round the world under the command of Capt. Fitz Roy, R.N. John Murray
12. USGS. (2013) Spread, Impact, and Control of Purple Loosestrife (Lythrum salicaria) in North American Wetlands. http://www.npwrc.usgs.gov/resource/plants/loosstrf/biology.htm.
R-1 Allison Hubbard Bryant University
13. USDA Forest Service. (2015) Lythrum salicaria. http://www.fs.fed.us/database/feis/plants/forb/lytsal/all.html#BOTANICAL AND ECOLOGICAL CHARACTERISTICS.
14. Miranda, M.G., Galvan, A., and Romero, L. (2014) Nitrate removal efficiency with hydrophytes of Los Reyes Aztecas lake water, México. Journal of Water Resource and Protection 6(11): 945-950.
15. ITIS Report. Lythrum salicaria L. (2015) http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_valu e=27079.
16. ARCTOS. (2015) Collaborative Collection Management Solution. http://arctos.database.museum/name/Lythrum%20salicaria.
17. NCBI. (2010) Lythrum salicaria. http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=13129.
18. USDA (2015) Natural Resources Conservation Service, Plants Database, Lythrum salicaria L. http://plants.usda.gov/core/profile?symbol=lysa2.
19. Olsson, K. and Agren, J. (2002) Latitudinal population differentiation in phenology, life history and flower morphology in the perennial herb Lythrum salicaria. Journal of Evolutionary Biology 15(6): 983-996.
20. Migliore, L., Cozzolino, S. and Fiori, M. (2000) Phytotoxicity to and uptake of flumequine used in intensive aquaculture on the aquatic weed, Lythrum salicaria L. Chemosphere 40(7):741-750.
21. Katovich, E.J.S., Becker, R.L., and Ragsdale, D.W. (1999) Effect of Galerucella spp. on survival of purple loosestrife (Lythrum salicaria) roots and crowns. Weed Science 47(3):360-365.
22. Qin, Hai-Ning, Graham, Shirley (2007) (Qin, Hai-Ning; Graham, Shirley. 2007. 4. Lythrum Linnaeus, Sp. Pl. 1: 446. 1753, Flora of China, Vol. 13: 281-282 (Electronic version can be retrieved from http://flora.huh.harvard.edu/china/PDF/PDF13/Lythrum.pdf)).20
23. U.S. Department of Transportation, Federal Highway Administration: Purple Loosestrife, a “Poster Child” for Invasive Plants. (2015) http://www.environment.fhwa.dot.gov/ecosystems/greenerroadsides/gr_fall01p6.a sp (Accessed 25 April, 2015).
24. Albright, M.F., Harman, W.N., Fickbohm, S.S., Meehan, H., Groff, S., et al. (2004) Recovery of native flora and behavioral responses by Galerucella spp. following biocontrol of purple loosestrife. The American Midland Naturalist. 152:248-254.
R-2 Allison Hubbard Bryant University
25. Blossey, B., Schroeder, D., Hight, S.D., and Malecki, R.A. (1994) Host specificity and environmental impact of two leaf beetles (Galerucella calmariensis and G. pusilla) for biological control of purple loosestrife (Lythrum salicaria). Weed Science 42(1):134-140.
26. Kiviat, E. (1996) American goldfinch nests in purple loosestrife. The Wilson Bulletin. 182-186.
27. Maddox, J.D., and Wiedenmann, R.N. (2005) Nesting of birds in wetlands containing purple loosestrife (Lythrum salicaria) and cattail (Typha spp.). Natural Areas Journal 369-373.
28. Tavernia, B.G., and Reed, J.M. (2012) The impact of exotic purple loosestrife (Lythrum salicaria) on wetland bird abundances. American Midland Naturalist. 168(2):352-363.
29. Environmental Services of the City of Portland (2011) Terrestrial ecology enhancement strategy guidance: avoiding impacts on nesting birds during construction and revegetation projects. TEES Summary and Update. 1-67.
30. Hovivk, S.M., Bunker, D.E., Peterson, C.J., and Carson, W.P. (2011) Purple loosestrife suppresses plant species colonization far more than broad-leaved cattail: experimental evidence with plant community implications. Journal of Ecology 99(1):225-234.
31. Hunt-Joshi, T.R., Blossey, B., and Root, R.B. (2004) Roots and leaf herbivory on Lythrum salicaria: implications for plant performance and communities. Ecological Society of America 14:1574-1589.
32. Blossey, B., Skinner, L.C., and Taylor, J. (2001) Impact and management of purple loosestrife (Lythrum salicaria) in North America. Biodiversity and Conservation 10:1787-1807.
33. Brown, B.J., Mitchell, R.J., and Graham, S.A. (2002) Competition for pollination between an invasive species (purple loosestrife) and a native cogener. Ecology. 83:2328-2336.
34. Flanagan, R.J., Mitchell, R.J., and Karron, J.D. (2010) Increased relative abundance of an invasive competitor for pollination, Lythrum salicaria, reduces seed number in Mimulus ringens. Oecologia 164:445-454.
35. Templer, P., Findlay, S., and Wigand, C. (1995) Sediment chemistry associated with native and non-native emergent macrophytes of the Hudson River marsh ecosystem. Final Report of The Tibor T. Polgar Fellowship Program, Hudson River Foundation.
R-3 Allison Hubbard Bryant University
36. Mishra, V.K., and Tripathi, B.D. (2008) Concurrent removal and accumulation of heavy metals by the three aquatic macrophytes. Bioresource Technology 99(15):7091-7097. doi://10.1016/j.biortech. 2008.01.002.
37. J.L., N.K., Das, M., Mukherji, R., and Kumar, R.N. (2012) Trace metal contents in water, sediment, and hydrophytes at freshwater wetland in Central Gujarat, India. Bulletin of Environmental and Scientific Research 1(1):16-24.
38. Hoseinizzadeh, G.R., Azarpour, E., Ziaeidoustan, H., Moraadi, M., and Amiri, E. (2011) Phytoremediation of heavy metals by hydrophytes of Anzali Wetland (Iran). World Applied Sciences Journal. 1478-1481.
39. Sheoran, A.S.., and Sheoran, V. (2006) Heavy metal removal mechanism of acid mine drainage in wetlands: a critical review. Minerals Engineering 19(2):105-116.
40. Nagel, J.M., and Griffin, K.L. (2001) Construction cost and invasive potential: comparing Lythrum salicaria (Lythraceae) with co-occurring native species along pond banks. American Journal of Botany 88(12):2252-2258.
41. O’Niell, W.L., and Nzengung, V.A. (2004) In-situ bioremediation and phytoremediation of contaminated soils and water: three case studies. Environmental Research, Engineering and Management 4:49-54.
42. Tangahu, BV, Abdullah, SRS, Basri, H, Idris, M, Anuar, N, et al (2011) A Review on Heavy Metals (As, Pb, and Hg) Uptake by Plants through Phytoremediation. International Journal of Chemical Engineering: 21:1-31.
43. Bankston, J.L., Sola, D.L., Komor, A.T., and Dwyer, D.F. (2002) Degradation of trichloroethylene in wetland microcosms containing broad-leaved cattail and eastern cottonwood. Water Research 36(6):1539-1546. PII: S0043- 1354(01)00368-2.
44. Teuchies, J., Jacobs, A., Oosterlee, L., Bervoets, L., and Meire, P. (2013) Role of plants in metal cycling in a tidal wetlands: implications for phytoremediation. Science of Total Environment. 146-154. http://dx/doi/org/10/1016/j/scitotenv.2012.11.088.
45. Kamal, M., Ghaly, A.E., Mahmoud, N., and Côte, R. (2004) Phytoaccumulation of heavy metals by aquatic plants. Environment International 29(8):1029-1039.
46. Shamsi, S.R.A., and Whitehead, F.H. (1977) Comparative eco-physiology of Epilobium hirsutum L.and Lythrum salicaria L. IV Effects of temperature and inter-specific competition and concluding discussion. Journal of Ecology 65(1):71-84.
47. Nicholls, A.M., and Mal, T.K. (2003) Effects of lead and copper exposure on growth of an invasive weed, Lythrum salicaria L. (Purple Loosestrife). Ohio Journal of Science 103(5):129-133. http://hdl.handle.net/1811/23985.
R-4 Allison Hubbard Bryant University
48. Jung, M.C., and Thornton, I. (1996) Heavy metal contamination of soils and plants in the vicinity of a lead-zinc mine, Korea. Applied Geochemistry 11:53-99. 0883-2927/96.
49. Mishra, V.K., and Tripathi, B.D. (2008) Concurrent removal and accumulation of heavy metals by the three aquatic macrophytes. Bioresource Technology 99(15):7091-7097. Doi://10.1016/j.biortech.2008.01.002.
50. Kortesky, C.M., Haas, J.R., Miller, D., and Ndenga, N.T. (2006) Seasonal variations in pore water and sediment geochemistry of littoral lake sediments (Asylum Lake, MI, USA). Geochemical Transactions 7:11. doi:10.1186/1467- 4866-7-11. . 51. Marchand, L., Mench, M., Jacob, D.L., and Otte, M.L. (2010) Metal and metalloid removal in constructed wetlands, with emphasis on the importance of plants and standardized measurements: a review. Environmental Pollution 158(12):3447-3461.
52. Farooqi, Z., Iqbal, M.Z., Kabir, M., and Shafiq, M. (2011) Tolerance of Albizia lebbeck (L.) Benth to different levels of lead in natural field conditions. Pakistan Journal of Botany 43(1):445-452.
53. Capilla, X., Schwarts, C., Bedell, J.P., Sterckeman, T., Perrodin, Y., et al. (2006) Physicochemical and biological characterization of different dredged sediment deposit sites in France. Environmental Pollution 143(1):106-116. Doi: 10.1016/j.envpol.2005.11.007.
54. Vardanyan, L.G., and Ingole, B.S. (2006) Studies on heavy metal accumulation in aquatic macrophytes from Sevan (Armenia) and Carambolim (India) lake systems. Environment International 32(2):208-218.
55. Balcerzak, M. (2002) Sample digestion methods for the determination of traces of precious metals by spectrometric techniques. Analytical Sciences:International Journal of the Japan Society for analytical Chemistry 18(7):737-750.
56. Rhode Island Historical Preservation Commission. (1980) Historic and architectural resources of North Smithfield, Rhode Island: a preliminary report. Rhode Island Historical Preservation Commission. I-69.
57. Rhode Island Historical Preservation Commission. (1992) Historic and architectural resources of Smithfield, Rhode Island. Rhode Island Historical Preservation Commission. I-92.
58. Payne, M. (2012) Soil parent materials of Rhode Island. USDA Natural Resources Conservation Service Rhode Island. Available at: http://www.nrcs.usda.gov/wps/portal/nrcs/site/ri/home/.
R-5 Allison Hubbard Bryant University
59. NRCS, RIGIS. (2009) Soil parent materials of Rhode Island. USDA, RI USDA NRCS. http://www.nrcs.usda.gov/wps/portal/nrcs/site/ri/home/.
60. Munson, R.D. (1998) Principles of plant analysis. Handbook of Reference Methods for Plant Analysis. Ed. Kalra, Y.P.. 1-24.
61. Bidlack, JE, Jansky, SH (2008) Roots and Soils. In Stern’s Introductory Plant Biology, Eds. McGraw Hill, pp 64-79.
62. Olguín, EJ, Sánchez-Galván, G (2012) Heavy metal removal in phytofiltration and phycoremediation: the need to differentiate between bioadsorption and bioaccumulation. New Biotechnology 30(1): 3-8.
63. Bidlack, JE, Jansky, SH (2008) Water in Plants. Stern’s Introductory Plant Biology, Eds. McGraw Hill, pp 146-161.
64. EPA, Drinking Water Contaminants. http://water.epa.gov/drink/contaminants/index.cfm#List (accessed April 30, 2015).
65. Hubbard, HJ (2015) Personal Interview.
66. SEPA. Scottish Environment Protection Agency, Manganese. http://apps.sepa.org.uk/spripa/Pages/SubstanceInformation.aspx?pid=106 (Accessed May 11, 2015).
67. EPA. Basic Information about Arsenic in Drinking Water. http://water.epa.gov/drink/contaminants/basicinformation/arsenic.cfm (Accessed May 11, 2015).
68. EPA. Basic Information about Selenium in Drinking Water. http://water.epa.gov/drink/contaminants/basicinformation/selenium.cfm (Accessed May 11, 2015).
69. USGS. Contaminants Found in Groundwater. https://water.usgs.gov/edu/groundwater-contaminants.html (Accessed May 11, 2015).
70. EPA. Basic Information about Cadmium in Drinking Water. http://water.epa.gov/drink/contaminants/basicinformation/cadmium.cfm (Accessed May 11, 2015).
71. Industrial Automation Control Products and Systems (1IS) Section (2008) NEMA WHITE PAPER CADMIUM IN ELECTRICAL CONTACTS. Industrial Automation Control Products and Systems: 2-8.
72. EPA. Basic Information about Barium in Drinking Water. http://water.epa.gov/drink/contaminants/basicinformation/barium.cfm (Accessed May 11, 2015).
R-6 Allison Hubbard Bryant University
73. Buchman, MF (2008) NOAA screening quick reference tables, NOAA OR&R report 08-1. National Oceanic and Atmospheric Administration: 1-34.
74. State of Rhode Island and Providence Plantations Department of Environmental Management Office of Waste Management (2011) Rules and Regulations for the Investigation and Remediation of Hazardous Material Releases. DEM: 1-125.
75. EPA, Drinking Water Contaminants. http://water.epa.gov/drink/contaminants/index.cfm#List (accessed April 30, 2015).
76. Khan, S, Farooq, R, Shahbaz, S, Khan, MA, Sadique (2009) Health Risk Assessment of Heavy Metals for Population via Consumption of Vegetables. World Applied Science Journal 6(12):1602-1606.
77. Intawongse, M, Dean, JR (2006) Uptake of heavy metals by vegetable plants grown on contaminated soil and their bioavailability in the human gastrointestinal tract. Food Additives and Contaminants 31(1): 36-48.
R-7