This document describes a study investigating the potential of plant species found in areas of historic gold mining in Bendigo, Victoria to remediate arsenic contaminated soils through phytoremediation. The study included sampling vegetation and soils from contaminated and uncontaminated sites in Bendigo and growing three early colonizing plant species (C. arcuata, E. leucoxylon, A. pycnantha) in collected contaminated soil and spiked/unspiked potting mix in a greenhouse. Results found that none of the species accumulated significant arsenic in above-ground biomass, making them unsuitable for phytoextraction. However, C. arcuata and E. leucoxylon

1. 1
Arsenic Phytoremediation Potential of Early Colonising Plant Species
found in an Area of Historic Gold Mining Activity in the Bendigo
Region
Brett McGregor
School of Applied Sciences
RMIT University
Submitted in partial fulfilment of the requirements of BIOL2231
Research Project 2
June 2015

2. 2
This thesis is submitted in accordance with the regulations of RMIT University in atrial
fulfilment of the requirements of BIOL2231 within the degree of Masters of Environmental
Science and Technology (MC191)
Declaration and statement of Authorship
1. I have not impersonated, or allowed myself to be impersonated by any person for the
purposes of this assessment.
2. This assessment is my original work and no part of it has been copied from any other
source except where due acknowledgement is made.
3. No part of this assessment has been written for me by any other person, except where
such collaboration has been authorised by the course coordinator.
4. I have not previously submitted this work for any other course.
5. I give permission for my assessment response to be reproduced, communicated, compared
and archived for the purposes of detecting plagiarism.
6. I give permission for a copy of my assessment to be retained by the university for review
and comparison, including review by external examiners.
I understand that:
Plagiarism is the presentation of the work, idea or creation of another person as though is your
own. It is a form of cheating and is a very serious academic offence that may lead to exclusion of
the University. Plagiarised material can be drawn from, and presented in, written, graphic and
visual form, including electronic data and oral presentations. Plagiarism occurs when the origin
of the material is not appropriately cited.
Plagiarism includes the act of assisting or allowing another person to plagiarise or to copy my
work. I agree and acknowledge that:
1. I have read and understood the Declaration and Statement of Authorship above.

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2. I accept that the use of my RMIT account to electronically submit this assessment
constitutes my agreement the Declaration and Statement of Authorship.
3. If I do not agree to Declaration and Statement of Authorship in this context, the
assessment outcome is not valid for assessment purposes and cannot be included in my
aggregate score for this course.
Signature of candidate:............................................................................ Date:.................................
This project DID NOT involve the use of invertebrates, vertebrates or human subjects. I, Brett
McGregor, hereby certify that the Project did not require RMIT University ethics committee
clearance, nor a permit from any external organisation.
Signature of candidate:............................................................................ Date:.................................

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Abstract
Arsenic contaminated soils are a human health concern in many parts of the world including
areas of historic gold mining activity in Australia. Recent attention has been given to the use of
plants for the remediation of such sites, known as ‘phytoremediation’. While much research has
been done elsewhere around the world on the potential of some species to remediate arsenic
contaminated areas, little work has been done investigating native Australian species for this
purpose. This project aims to investigate the phytoremediation potential of plant species found
colonising areas of historic gold mining in Bendigo, Victoria. This project consists of two
experiments. One involves the sampling and analysis of vegetation and soils from two sites in the
Bendigo region: one contaminated and one uncontaminated. The second is a greenhouse
experiment growing three early colonising species (C. arcuata, E. leucoxylon, A. pycnantha) in
collected contaminated soil as well as spiked and non-spiked potting mix. From these
experiments it was found that no species accumulated significant concentrations of arsenic in
their above-ground biomass, thus making them unsuitable for the extraction of arsenic via the
harvesting of plant parts (‘phytoextraction’). However, C. arcuata and E. leucoxylon did show
some characteristics that may make them suitable for site remediation, including the apparent
ability to concentrate arsenic in the soil around their roots. This study highlights the lack of
knowledge regarding the suitability of native Australian plant species for the purpose of
remediating arsenic contaminated soils. Future research should continue investigating the
remediation potential of plants found colonising arsenic-contaminated sites as well as
investigating native fern species for their potential to accumulate arsenic in their above-ground
biomass for the purpose of harvesting and thus removing arsenic.

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Acknowledgements
I would like to thank all those people who made this project possible and whose assistance was
invaluable in helping me accomplish this task. In particular I would like to thank:
 Associate Professor Nichola Porter, who provided me with the idea for this project and
has helped me every step along the way.
 Simon Harrison of Bendigo City Council, who took the time out of his day to assist us
and organise staff to help us sample vegetation.
 Mark Toohey, also of Bendigo City Council, who escorted us around Bendigo and
showed us the sites in which we could sample and whose expertise in plant recognition
were invaluable.
 Karl Lang who, despite the innumerous demands on his time, was still able to help me
purchase my equipment and set up the greenhouse.
 Susan Holden, who helped me organise my time in the soil lab.
 Paul Morrison, who ran my samples through ICP-MS.
 Zahra Homan and the prep room staff, who helped me set up my experiments.
 Nihal Albuquerque, whose suggestions on experimental techniques saved me countless
hours of extra work.
 Angela Whiffin of Newport Lakes Native Nursery, who provided me with the plants for
the greenhouse experiment as well as advice on how to grow them.

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Table of Contents
1 Introduction.....................................................................................................................................8
2 Literature Review........................................................................................................................... 10
2.1 Arsenic: an overview................................................................................................................ 10
2.2 Arsenic as a toxin..................................................................................................................... 11
2.3 Arsenic in soils in westernVictoria............................................................................................ 12
2.4 Phytoremediation.................................................................................................................... 13
2.5 Metal hyperaccumulators ........................................................................................................ 14
2.6 Plant uptake of arsenic............................................................................................................. 16
3 Experimental design, materials and methods................................................................................... 18
3.1 Experimental design ................................................................................................................ 18
3.2 Sampling of vegetation and soil at Bendigo............................................................................... 18
3.3 Greenhouse setup ................................................................................................................... 21
3.4 Preparation of samples............................................................................................................ 24
3.4.1 Preparation of soil............................................................................................................. 24
3.4.2 Preparation of vegetation samples..................................................................................... 24
3.5 Digestion of samples................................................................................................................ 25
3.5.1 Digestion of plant samples................................................................................................. 25
3.5.2 Digestion of soil samples.................................................................................................... 26
3.6 Instrumental analysis............................................................................................................... 27
4 Results........................................................................................................................................... 28
4.1 Bendigo site sampling.............................................................................................................. 28
4.1.1 Leaves .............................................................................................................................. 29
4.1.2 Seeds/fruit........................................................................................................................ 30
4.1.3 Stems............................................................................................................................... 31
4.1.4 Roots................................................................................................................................ 32
4.1.5 Soil................................................................................................................................... 33
4.2 Greenhouse experiment .......................................................................................................... 34
4.2.1 Bendigo soil treatment...................................................................................................... 34
4.2.2 Spiked and control treatments........................................................................................... 35
4.3 Confirmation of results ............................................................................................................ 37
5 Discussion...................................................................................................................................... 38

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5.1 Bendigofield samples.............................................................................................................. 38
5.2 Greenhouse experiment .......................................................................................................... 39
5.3 Comparison betweenfield and greenhouse samples ................................................................. 40
5.4 Comparisonwith existing literature.......................................................................................... 41
6 Conclusion..................................................................................................................................... 42
8 Recommendations......................................................................................................................... 42
9 References..................................................................................................................................... 43
Table of Tables
Table 1: Numberof plantssurvivinginthirdbatchby endof experiment..................................................23
Table 2: ArsenicconcentrationsforsamplescollectedatBendigo.............................................................28
Table of Figures
Figure 1 Map of Bendigo showinglocation of two sampling sites......................................................... 19
Figure 2 Photo taken of Site 1 showing light grey soil/mine tailings with lack of vegetation................... 20
Figure 3 Image taken from Site 2 showing more natural landscape...................................................... 21
Figure 4 Greenhouse set-up. ............................................................................................................. 23
Figure 5 Cleaning process for vegetation samples............................................................................... 25
Figure 6 Digestion of samples on heating block................................................................................... 27
Figure 7 ArsenicconcentrationsinleavesfromvegetationsamplescollectedatBendigo.........................29
Figure 8 Arsenicconcentrationsin seed-like structures fromvegetationsamplescollectedatBendigo...30
Figure 9 Arsenicconcentrationsin stemsfromvegetationsamplescollectedatBendigo.........................31
Figure 10 Arsenicconcentrationsin rootsfromvegetationsamplescollectedatBendigo........................32
Figure 11 Arsenicconcentrationsforsoil samplestakenwithplantsamples.............................................33
Figure 12 Arsenicconcentrationsinplantpartsandsoil fortwo plants(E.leucoxylon andA. pycnantha)
growinginsoil takenfromSite 1 at Bendigo...............................................................................................34
Figure 13 Arsenicconcentrationsfromplantsamplesgrowningreenhouse esperiment..........................36
Figure 14 Final arsenicconcentrationsinspikedandun-spiked(control) pottingmix...............................36
Figure 15 Arsenicconcentrationsfoundinmulti-elementstandard...........................................................37

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1 Introduction
Arsenic is a metalloid element which is the 20th most abundant element in the Earth’s crust. It
exists primarily in one of four oxidation states: arsenic (As0), arsenite (AsIII), arsenate (AsV) and
arsine (As-III). Whilst arsenic is a naturally occurring element it is also toxic to biota, including
humans (Singh et al. 2015). Ordinarily, arsenic is bound to other elements within arsenic-bearing
minerals, such as arsenopyrite (FeAsS). However, it may be released into the environment by
either natural or anthropogenic means, including the weathering of rock, volcanic or biological
activity, ore smelting, waste combustion, or the use of arsenic-based herbicides (Smith et al.
2003; Zhang et al. 2002).
The toxic effects of arsenic are well known, and in humans it is known to cause birth defects,
immune disorders, developmental and neurological defects as well as various cancers including
lung, bladder, kidney, liver, prostate and skin cancers (Hinwood et al. 1999). While many of
these arsenic-induced health impacts are due to the consumption of arsenic-contaminated
drinking water, much of the arsenic absorbed by humans is from soil (Martin et al. 2013).
Uncontaminated soil usually contains arsenic concentrations below 10 mg/kg, with
concentrations above 40 mg/kg believed to pose a health risk to humans, especially young
children (Ko et al. 2008; Srivastava et al. 2006; Zhang et al. 2002).
Due to the health concerns surrounding arsenic-contaminated soils, attempts are often made to
remedy these sites. Traditional methods include physically removing the contaminated soil to
another area or restricting access to contaminated site (Vangronsveld et al. 2009). These methods
do not actually solve the problem of arsenic contamination and either simply move the site of
contamination from one area to another or result in an site becoming prohibited and unusable. In
recent decades there has grown increasing interest in a technique called ‘phytoremediation’ in
which plants are used to remediate contaminated areas (Singh et al. 2015). With regards to the
remediation of areas contaminated with metals or metalloids there exists three types of
phytoremediation. The first of these is ‘phytoextraction’, which involves the use of plants, known
as ‘hyperaccumulators’, which accumulate high levels of the target metal/metalloid in their above
ground parts, so that these parts may be harvested and removed thus removing the contamination.
The second is ‘phytostabilisation’, which involves the use of plants that decrease the mobility and

9. 9
bioavailability of pollutants in the soil. The third is ‘phytovolatilisation’, which involves the use
of plants that volatilise pollutants into the atmosphere (Fitz & Wenzel 2002; Salt et al. 1998;
Singh et al. 2015).
Soil arsenic contamination is an issue in many areas of historic gold mining in central and
western Victoria, Australia, where concentrations have been recorded at up to 15,000 mg/kg.
(Arne at al. 1999; Smith et al. 2003). It has been found that the level of arsenic in the toenail
clippings of children in these areas correlate with soil arsenic concentrations of areas in which
they play (Pearce et al. 2010). As such, dealing with soil arsenic contamination is an issue of
immediate importance in these areas and phytoremediation may be a possible solution. Whilst
there exist several plant species known to hyperaccumulate arsenic, such as the Chinese Brake
Fern Pteris vittata (Zhang et al. 2002), which may be used for phytoremediation, little research
has been done into the suitability of native Australian species for this purpose.
This lack of knowledge provides the impetus for this project, which aims to investigate if there
exist plant species native to south-eastern Australia that are suitable for remediating areas
contaminated with high levels of soil arsenic. Specifically, this project has the objective of
ascertaining their suitability for remediating sites of historic gold mining in central and western
Victoria. This study is divided into two parts. The first part involves the sampling of vegetation
from two sites in the Bendigo area; one contaminated and one uncontaminated. The purpose of
this it to determine if there are any plant species that take up high levels of arsenic into their
above-ground biomass, thus making them suitable for phytoextraction. The second part involves
the growing of three species, known to be early colonisers of disturbed land around Bendigo, in a
greenhouse environment. In this experiment plant samples growing in an arsenic-spiked medium
are compared with those growing in an un-spiked medium and those growing in contaminated
soil taken from Bendigo. This experiment aims to discover if these colonising species may also
be suitable for phytoextraction. If any of these results do confirm the suitability of one or more
species for phytoremediation then these may be candidates for the future remediation of areas
known to be contaminated with high levels of arsenic in the Bendigo and surrounding region.

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2 Literature Review
2.1 Arsenic: an overview
Arsenic is a crystalline metalloid element, and is the 20th most abundant element in the Earth’s
crust. It is also the 14th most abundant element in seawater and 12th most abundant in the human
body. Arsenic exists mainly in four oxidation states; arsenic (As0), arsenite (AsIII), arsenate (AsV)
and arsine (As-III), and its solubility is dependent on the pH and ionic environment in which it
exists (Singh et al. 2015). Arsenic is also a toxin, with inorganic species being more toxic than
organic ones, and AsIII being more toxic than AsV. The primary source of arsenic in the
environment is its release from As-enriched minerals found naturally in the Earth’s crust (Singh
et al. 2015). This release can be done by natural or anthropogenic means. Natural pathways for
arsenic to enter the environment include the weathering of rock, known as pedogenisis, as well as
biological activity and volcanic activity (Smith et al. 2003; Zhang et al. 2002). Anthropogenic
pathways include solid waste combustion, fossil fuel combustion, ore smelting, wood treatments
and the use of arsenic-based herbicides and pesticides (Smith et al. 2003; Zhang et al. 2002).
Arsenic is present in the environment in more than 200 minerals, including arsenates, arsenites,
arsenides, sulphides, oxides and as elemental arsenic. The most abundant arsenic-bearing mineral
is arsenopyrite (FeAsS) (Smith et al. 2003). Arsenic is usually present in the ores of gold (Au),
tin (Sn), zinc (Zn), copper (Cu), and lead (Pb). Arsenic is also found naturally in soils, albeit
usually at small concentrations, with the concentration in uncontaminated soils rarely exceeding
10mg/kg. The availability of arsenic in soil is determined by a number of factors, including
mineral composition, organic matter content, pH, redox potential, and phosphate content (Ko et
al. 2008). In soils that are contaminated with high concentration of arsenic, inorganic forms (AsV
and AsIII) usually dominate. In aerobic soils the oxic conditions favour the presence of As(V)
over As(III), the latter of which is oxidised to As(V) (Smith et al. 2010).

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2.2 Arsenic as a toxin
Arsenic is one of the most toxic elements found in soils and water, despite it being a naturally
occurring element. Arsenic is toxic to all biota. In animals, arsenic is known to be a teratogen
(causes birth defects), a carcinogen, a mutagen, as well as affecting the immune system
(Srivastava et al. 2006). In plants, arsenic is known to have a phytotoxicity of roughly 3-10 µg
As g-1, depending on species (King et al. 2008). This toxicity has been exploited historically
through the use of organoarsenical agents as pesticides (Salt et al. 1998). Arsenic concentrations
of greater than 40 mg/kg may pose a health risk to humans, especially small children (Ko et al.
2008). Long-term exposure can lead to arsenicosis, a term referring to health effects resulting
from arsenic poisoning (Singh et al. 2015). The toxicity of arsenic is solubility- and species-
dependent. Generally, arsenite is more toxic than arsenate. (Ng 2005).
Arsenic has long been associated with various types of cancer, including lung, bladder, kidney,
bone, skin, liver and prostate cancers, with specific cancers correlated with specific routes of
exposure, such as inhalation for lung cancers or via drinking water for bladder cancers (Hinwood
et al. 1999). The epidemiological evidence for arsenic-induced lung, skin, and bladder cancers is
currently strong (Ng 2005). Arsenic exposure is also linked to other adverse health effects,
including peripheral and cardiovascular disease, hypertension, diabetes mellitus, hearing loss,
anaemia, hematologic, gastrointestinal, renal, and respiratory disorders as well as reproductive,
developmental, immunological and neurological defects (Martin et al. 2013; Zhang et al. 2002).
These effects are mainly associated with contaminated drinking water as opposed to
contaminated soil or biota. However, it is estimated that between 30% and 80% of arsenic
absorbed by humans is from soil, and soil ingestion is an important route of exposure for young
children, especially those living or playing in arsenic contaminated areas. The risk factor for
children is also increased to due to children’s decreased capacity to detoxify inorganic arsenic
into its metabolites, thus resulting in an increased exposure time after ingestion (Martin et al.
2013).
Environmental Protection Authority (EPA) Victoria guidelines currently classify soil containing
>20 mg/kg of inorganic arsenic as being contaminated (EPA Vic 2009). Soil arsenic
concentrations in residential areas of rural Victoria have been found to range up to 16,000 mg/kg

12. 12
and drinking water concentrations have been found as high as 0.4mg/L. While the correlation
between arsenic-contaminated drinking water and arsenic absorption in humans is well
documented, the correlation between arsenic absorption and arsenic-contaminated soil is less well
known (Hinwood et al. 1999). Hinwood et al. (1999) conducted a study of cancer incidences in
western rural Victoria and their correlation with arsenic-contaminated drinking water and soil.
While this study did find a significant relationship between incidences of prostate cancer and
areas of both high water and high soil arsenic contamination there was found to be no
relationship between cancers and high soil contamination only. Studies assessing a potential
relationship between arsenic and cancer are often confounded by other potential cancer-causing
factors including pesticide use, cigarette smoking or other carcinogenic contaminants that are
often present alongside arsenic (Hinwood et al. 1999). However, as arsenic-contaminated soil is
one of the major sources of arsenic in contaminated drinking water (Zhang et al. 2002) dealing
with the issue of arsenic-contaminated soil is still a priority for public health protection. Indeed,
Ng (2005) states that chronic arsenic poisoning from both natural and anthropogenic sources is
“one of the most serious mass poisonings by a chemical in recent history”.
2.3 Arsenic in soils in western Victoria
Arsenic is often associated with gold mineralisation, and as such, elevated levels of arsenic have
been observed in areas of historic gold mining in Australia (Pearce et al. 2010). In Victoria, gold
mining has been taking place since the ‘gold rush’ of the 1850’s with much of this activity taking
place in the State’s centre and west encompassing towns such as Ballarat, Bendigo and
Castlemaine (Earth and Energy Resources 2015). The area surrounding Bendigo accounts for
over 75% of all of Victoria’s historic gold production and thus many current and former mining
sites exist in this area. Given that gold-bearing quartz reefs in western and central Victoria are
often accompanied by the formation of arsenopyrite, it is not unexpected that high concentrations
of arsenic have been discovered around historic mine sites in the Bendigo area. Within mullock
heaps and other mine waste sites around Victoria arsenic concentrations have been recorded
ranging from 280 to 15,000 mg/kg (Arne at al. 1999; Smith et al. 2003). These sites represent a
source of arsenic contamination that may be a hazard to human health (Ashley & Lottermoser
1999). Pearce et al. (2010), in a study analyzing arsenic concentrations in the toenails of primary

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school students at two schools in an historic gold mining location in Victoria (specific
schools/location not given) using Instrumental Neutron Activation Analysis (INAA), found that
there existed a moderate positive correlation between arsenic concentrations in children’s toenail
clippings and soil samples taken from locations where the children were known to play
frequently. Thus, Pearce et al. (2010) concluded that arsenic in soil in this area of historical gold
mining contributed to arsenic uptake by children living in this area and recommended that future
epidemiological studies be conducted to ascertain if adverse health impacts are correlated with
arsenic exposure in the study area.
2.4 Phytoremediation
Whilst there are many existing techniques available to remediate contaminated soils, few are
applicable to soils contaminated with trace elements, especially trace metals and metalloids.
Because trace elements are immutable and relatively immobile, existing techniques such as
thermal volatilisation, biodegradation and phytodegradation are not feasible. Generally there are
two options available for site remediation: removing the contamination (decontamination) or
reducing the risks associated by the contamination (site stabilisation) (Vangronsveld et al. 2009).
Lately, there has been increasing interest in a new technique that allows sites contaminated with
trace metals and metalloids, such as arsenic, to be remediated, either through decontamination or
site stabilisation. This technique is known as ‘phytoremediation’ and refers to the use of plants to
remediate contaminated sites (Salt et al. 1998; Singh et al. 2015). There are several methods of
phytoremediation applicable to the removal of trace metals and metalloids, these being:
 Phyotextraction, which refers to the use of plants known to extract pollutants from the
soil and translocate these pollutants to the above-ground parts of the plant. Once the
contaminants have been taken up by the plant these above-ground parts can be harvested,
thus removing the contamination from the site;
 Phytostabilisation, also known as Phytoimmobilisation, which refers to the use of plants
that decrease the mobility and bioavailability of pollutants via the formation of
precipitates and insoluble compounds on the roots; and

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 Phytovolatilisation, which involves the use of plants that volatilise pollutants into the
atmosphere (Fitz & Wenzel 2002; Vangronsveld et al. 2009).
Each method has positive and negative aspects and may not be suitable for all scenarios. For
example, the main advantages of phytoextraction compared to existing techniques such as soil
excavation and removal include reduced cost, reduced possibility of spreading contamination by
dumping waste, and the possibility of recycling plant biomass for use as fertilisers due to the fact
that toxin-accumulating plants, known as ‘hyperaccumulators’, often consist of significant
amounts of trace elements. The primary disadvantages of phytoextraction include its limitation to
sites that are moderately contaminated, its restriction to sites that can adequately support known
hyperaccumulating plant species, and the ability to only decontaminate surface soil in the root
zone, typically less than 50 cm. Time is also another key disadvantage to this method, as
remediation to safe contaminant levels may take years or even decades (Vangronsveld et al.
2009). Phytoextraction can also be a liability in some circumstances as it directly transfers toxic
elements into the biosphere and thus may result in the transfer of toxic elements up the food
chain via herbivory. In these cases phytostabilisation is the preferred option, although this
method does not remove the contamination from the site but rather reduces the risks associated
with it (Craw et 2007; King et al. 2008).
2.5 Metal hyperaccumulators
Metal hyperaccumulaters are plants which are able to accumulate >1000mg/kg of metal in their
above-ground tissues and have a higher metal concentration in their above-ground biomass than
in their roots or the surrounding soil. In addition, its transfer factor (ratio of element
concentration in above ground biomass compared to roots) and its bioconcentration factor (ratio
of element concentration in plant tissues compared to soil) should be greater than one (Srivastava
et al. 2006). As such, metal hyperaccumulators are ideally suited to the task of phytoextracting
toxic metals and metalloids. Plants that are suitable for the extraction and removal of a variety of
metals including cadmium, chromium, copper, mercury, lead, nickel, selenium and zinc have
already been found (Zhang et al 2002, Vangronsveld et al. 2009). In order for a plant species to
be suitable for the phytoextraction of metals and metalloids it should possess the following

15. 15
characteristics: the ability to accumulate high levels of the target metal in its harvestable parts;
tolerance to high levels of the target metal; have a rapid growth rate; have the potential to
produce a high biomass in the field; and have a deep and profuse root system (Ko et al. 2008).
The discovery of plants that are able to hyperaccumulate arsenic are a more recent phenomenon,
with discoveries being made only in the past decade-and-a-half (Salt et al. 1998). These plants,
known as ‘arsenophytes’, have the potential to be used for the phytoextraction of arsenic from
contaminated sites (Zhang et al 2002). This may be done via the direct planting of arsenophytes
onto the contaminated land or onto capping soil. (Craw et al. 2007).
One such arsenophyte is the Chinese Brake Fern Pteris vittata. Brake ferns have been found to
grow healthily on mine tailings containing 23,400 mg/kg of arsenic and plant arsenic
concentrations have been found to reach as high as 2.3% (Wei et al. 2006; Zhang et al 2002).
Zhang et al. (2002) found that P. vittata accumulated arsenic mostly in its above-ground biomass,
specifically fiddle heads and fronds, with higher arsenic levels in older fronds compared to new
fronds. They speculate that the transport of arsenic from roots to fronds is via xylem sap. They
also speculate that the accumulation of arsenic in older fronds may be a detoxification technique
that allows the plant to rid itself of arsenic when older fronds are shed. Zhang et al. (2002) found
that As(III) was the major species in P. vittata fronds at 60-74%, compared to just 8.3% in the
roots. They postulate that the reduction of As(V) to As(III) is an essential process for arsenic
detoxification in P. vittata, although As(III) is believed to be more toxic to organisms than
As(V). The reason for this hyperaccumulation of arsenic in above-ground biomass in not yet
clear, although Salt et al. (1998) speculate that metal hyperaccumulation may be a method of
defence against fungal and herbivorous attack. Indeed, studies have found that arsenic-
accumulated P. vittata could deter herbivorous predators such as grasshoppers and scale insects
(Rathinasabapathi 2011). There are several other species of the Pteris genus known to
hyperaccumulate arsenic, including P. cretica, P. longifolia, P. umbrosa, P. biaurita, P.
wahlenbergii, P. quadriaurita Retz, P. ryukyuensis, P. multifida and P. oshimensis (Craw et al.
2007; King et al. 2008; Srivastava et al. 2006; Wang et al. 2007). Other non-pteris species have
also been found to hyperaccumulate arsenic, including Agrostis tenuis, and Pityrogramma
calomelanos (Craw et al. 2007; King et al. 2008; Srivastava et al. 2006; Wang et al. 2007). So

16. 16
far, it appears that arsenic hyperaccumulation in plants is unique to ferns (Rathinasabapathi
2011).
Despite the success of P. vittata overseas this species may not be practical in the Australian
environment. Niazi et al. (2012) ran a 27-month trial in northern New South Wales comparing the
efficacy of P. vittata and the Gold Dust Fern Pityrogramma calomelanos var. austroamericana
in remediating an arsenic-contaminated site in Australian conditions. They found that P. vittata
was less efficient at competing with weeds as it had a less dense canopy compared to P.
calomelanos. They also found that P. calomelanos re-established more successfully that P. vittata
and had more fiddle-heads and fronds after each frond harvest (conducted at 10, 22 and 27
months). P. calomelanos was also found to have a significantly larger biomass yield that P.
vittata, with frond dry biomass being 130, 151 and 68 g per plant compared to 81, 39 and 16 g
per plant for P. vittata at the 10-, 22- and 27-month harvest, respectively. With respect to arsenic
extraction, P. calomelanos had a significantly higher arsenic concentration in fronds, with
concentrations of 887, 423 and 581 mg/kg dry weight after harvest compared to 674, 292 and 401
mg/kg for P. vittata. P. calomelanos also had significantly higher arsenic uptake into fronds, with
uptakes of 124, 64 and 40 mg per plant after harvest compared to 27, 14 and 7 mg per plant for P.
vittata. P. calomelanos was able to remove 8,053 mg of arsenic from the soil over the 27-month
period (approx. 25.4 kg As ha−1), an amount 2.65 times greater than P. vittata, which removed
3,042 mg (approx. 9.7 kg As ha-1) from the soil over the trial period. In addition to this lack of
effectiveness there is a risk that P. vittata may become an invasive weed in its own right in some
environments (Ko et al. 2008).
2.6 Plant uptake of arsenic
Under aerobic conditions the main arsenic species found in soils is arsenate (AsV). This is mostly
bound to clay minerals as well as iron and manganese –oxi/hydroxides and organic substances
(Tu & Ma 2004). However, analysis has shown that within plant tissues arsenic exists mainly as
arsenite (AsIII). This has been shown to be the case even when plants have been exposed
primarily to arsenate (Zhao et al. 2009). This indicates that, following uptake, arsenate is reduced
efficiently to arsenite in plant cells, which is most likely done enzymatically via arsenic

17. 17
reductases (Smith et. al. 2010). The reduction of As(V) to As(III) takes place mostly in plant
roots, as As(III) is the dominant species found in xylem sap which transports arsenic from roots
to shoots. Also, As(III) is extruded from plant roots into the surrounding medium following
reduction from As(V), with Zhao et al. (2009) discovering that tomato roots preloaded with
arsenate extruded both arsenate and arsenite when transferred to an arsenic-free medium. While
this phenomenon has been observed in other species, it has not been observed P. vittata, where
there is minimal efflux of arsenite from roots to the surrounding medium. In aerobic soils As(III)
is rapidly oxidised to As(V) either via chemical reactions with manganese oxide or by arsenite-
oxidising microbes. Thus, plant roots, soil and microbes are likely to be constantly engaged in the
reduction-oxydation cycle of arsenate-arsenite.
Arsenic shares the same transport pathways in plants as phosphate, with these pathways having a
higher affinity for phosphate. Thus, phosphate inhibits arsenate uptake (Zhao et al. 2009). Tu &
Ma (2004) conducted a growth experiment comparing arsenic uptake in P. vittata with the non-
hyperaccumulating fern Nephrolepis exaltata. In both cases increases in levels of phosphorous in
the growth medium reduced the uptake levels of arsenic. According to the authors this result was
expected as arsenic and phosphorous are chemical analogues and the suppression of arsenic
uptake by phosphorous through competitive inhibition is a common phenomenon in many plant
species, with known examples including barley, Indian Mustard, and tolerant and non-tolerant
genotypes of the Holcus lanatus species. As such, Tu & Ma (2004) recommended that
phosphorus be limited in growth medium for arsenic hyperaccumulating plants. These findings
are at odds with Fitz & Wenzel (2002), who found that plant arsenic uptake increased upon the
application of phosphorous in both potted and field experiments, which they speculate is due to
increased plant growth triggered by the addition of phosphorous as well as the mobilization of
exchangeable arsenic allowing increased arsenic uptake.
In some instances the addition of an arsenic-mobilising agent can increase the plant uptake of
arsenic. Ko et al. (2008) added 0.2M ammonium oxalate (NH4)2C2O4 to arsenic-contaminated
soil in Fiji growing Indian Mustard Brassica juncea and found that arsenic concentrations in
plant biomass increased from 1.66 to 21.3 mg/kg.

18. 18
3 Experimental design, materials and methods
3.1 Experimental design
This experiment aims to investigate the uptake of arsenic in plant species local to the Bendigo
region to ascertain their potential usefulness in the phytoremediation of arsenic contaminated
sites. This investigation is broken into two distinct parts. The first part involves the collection of
plant samples from two sites in the Bendigo area; one known to be contaminated with high levels
of arsenic, and another believed to be uncontaminated. The second part involves the growing of
three native species, known to be early colonisers of disturbed sites in the Bendigo area, in a
controlled greenhouse environment, with each species divided between three treatments. These
experiments are detailed below.
3.2 Sampling of vegetation and soil at Bendigo
Plant and soil samples were collected on February 19th 2014 under the guidance of Bendigo City
Council ranger Mark Toohey. These samples were collected from two contrasting sites (see map
Figure 1). The first site (Site 1) was located at the Nell Gwynne Head Frame Reserve between
Dare Street and Ernest Street in the Bendigo suburb of Ironbark (lat. 36.755667°S, long.
144.25116E). This site was used as a dumping ground for arsenic-contaminated soil and mine
tailings taken from other sites around the Bendigo area and therefore the site itself was presumed
to be heavily contaminated (Mark Toohey 2014, pers. comm.), although no audit of contaminants
had been previously conducted (City of Greater Bendigo 2007). The soil at this site was
dominated by a light grey sandy soil which was notable for the lack of vegetation growing on it
(Figure 2). This was the soil/mine tailings that had been transported from off-site, dumped and
bulldozed. There was, however, vegetation growing in the area surrounding this soil, which was
sampled with the assistance of Mark Toohey, who was able to identify plant species and provided
cuttings from various species including leaves and stems/branches and, in cases where small
plants/shrubs with little conservation value were present, whole plants were taken, thus allowing

19. 19
the analysis of roots and attached soil for that species to be performed. In addition, two large
masses of soil were taken from two different locations at Site 1 to measure soil arsenic levels and
for use in the greenhouse experiment. The first mass was taken from the centre of the dumped
tailings while the second was taken from the bottom of a small gully where spoil had washed
down and was hypothesised to contain even greater concentrations of arsenic.
Figure 1 Map of Bendigoshowinglocationof two samplingsites (source: Google Maps).

20. 20
Figure 2 Photo takenof Site 1 showing light greysoil/mine tailings withlackof vegetation (Brett McGregor 19/2/2014).
The second site (Site 2) was located within the Crusoe Reservoir and Number 7 Reserve (lat.
36.825539°S, long. 144.230443°E). This site was undisturbed parkland (Figure 3) and was
presumed to be uncontaminated with high levels of arsenic. Again, Mark Toohey provided
clippings or whole plants where possible. Due to the variation in species available at each site not
every species was able to be sampled at both sites for a direct comparison.

21. 21
Figure 3 Image taken fromSite 2 showingmore natural landscape (Brett McGregor 19/2/2014).
3.3 Greenhouse setup
To test arsenic uptake levels of a sample of Australian native species within controlled conditions
a greenhouse experiment was set up within the grounds of the RMIT University’s City Campus.
This experiment utilised three native plant species – Eucalyptus leucoxylon (Yellow Gum),
Acacia pycnantha (Golden Wattle) and Cassinia arcuata (Chinese Scrub). These species were
known to be early colonisers of disturbed land in the Bendigo area and had been observed
growing on the edge of tailings dams (Marilyn Sprague 2013, pers. comm.). For use in this
experiment a Naturallife™ 200x200x220cm polyethylene greenhouse was purchased from
Bunnings Warehouse and established on the roof level of Building 15 at the city campus of
RMIT University (long. 37.808031°S, lat. 144.964944°E).

22. 22
For this experiment a total of 27 plants were purchased from Newport Lakes Native Nursery in
Newport, Melbourne – nine each of E. leucoxylon, A. pycnantha and C. arcuata. These plants
were divided into three treatments: one containing contaminated soil taken from Site 1 at
Bendigo; one containing potting mix spiked with arsenic to a concentration equal to that of the
Site 1 soil; and one control containing potting mix only, with three plants being used per species
per treatment. Plants were approximately one month old at the time of purchase and came in
small punnets. These plants were transferred to 125mm diameter plastic pots with each pot filled
with approximately 600g of soil or potting mix depending on treatment. Fifteen grams of Scott’s®
Ozmocote® for Native Gardens fertiliser was added to each pot before the potted plants were
transferred to the greenhouse.
The plants were first placed in the greenhouse on the 23rd of April 2014 where it was planned
they would grow for 12 weeks before sampling. However, after several weeks, plants potted in
the contaminated Bendigo soil began to perish, and before the planned growing period had
expired, all plants grown in the Bendigo soil had died. As such, a second batch was potted and
placed in the greenhouse on the 5th of November 2014. To increase chances of plant survival,
each plant was given Seasol® seaweed-based fertiliser on the recommendation of Angela
Whiffin, operator of Newport Lakes Native Nursery. This was applied one week after planting as
a 1:360 dilution in water as per packaging recommendations with each plant given 100mL of
dilute solution. A second application was made after a further week. Despite this, those plants
potted in the Bendigo soil again began to die. Advice was sought from Paul Ter who, upon
inspecting the dying plants, suggested the soil was hydrophobic and recommended soil wetter be
applied. As such, Seasol® Super Soil Wetter and Conditioner was applied as a 1:180 dilution in
water with each plant in the Bendigo soil treatment given 100mL of dilute solution. With this
application two plants, one A. pycnantha and one E. leucoxylon, were able to be recovered. All
other plants in the treatment died.
A third batch of plants were potted on the 12th of January with the aim of successfully growing all
plants in all treatments with the early application of soil wetter. Therefore, Seasol® soil wetter
was applied as above to all plants immediately after planting and one week following. One week
after planting those plants in the spiked treatment were given arsenic applied as sodium arsenate
dibasic heptahydrate (Na2HAsO4.7H2O). This was applied in five daily treatment of 79mg

23. 23
Na2HAsO4.7H2O diluted in 100mL of water per plant to bring the soil arsenic level to 260 mg/kg
of elemental arsenic, equal to that found in the contaminated Bendigo soil. To protect plant
samples against whitefly basil was purchased on the recommendation of Paul Ter and planted in
five pots which were placed in the greenhouse. The two surviving A. pycnantha and E.
leucoxylon in the Bendigo soil treatment from the previous batch were grown alongside the plants
in the third batch. Plants were watered with 100mL of water daily or as required. Pots were
covered in white tape to reduce heating due to sun exposure which may damage root systems.
The final setup for the third batch can be seen in Figure 4. A summary of plant survival is
detailed in Table 1.
Figure 4 Greenhouse set-up (Brett McGregor 4/2/2015).
Table 1 Number of plants surviving in third batch by end of experiment. Numbers in parenthesis represent
plants held over from second batch.
Species Control Spiked Bendigo soil
E. leucoxylon 2 0 0 (1)
A. pycnantha 3 0 0 (1)
C. arcuarta 3 0 0

24. 24
3.4 Preparation of samples
3.4.1 Preparation of soil
Soil samples, including those taken from Site 1 at Bendigo and soil taken from around the roots
of collected plant samples, as well as soil and potting mix used in the greenhouse experiment,
were placed in cardboard boxes lined with newspaper and shelved in milk creates in an
underground soil laboratory where they were allowed to air dry for at least two weeks. A portion
of soil was then oven dried for 48 hours at 70°C before being ground into a fine powder using a
ring-mill grinder. It was then oven dried at 105°C for 24 hours before being digested (see below).
3.4.2 Preparation of vegetation samples
Vegetation samples, including those sampled at Bendigo as well as plants grown in the
greenhouse, were in the first instance cleaned using the following multi-step process: 1) samples
were washed in tap water to remove surface dirt as well as soil that was adhering to roots; 2)
samples were washed in 0.2% Extran® phosphate-free detergent; 3) a second wash in tap water
was used to remove detergent suds; 4) samples were soaked in 0.1M hydrochloric acid (HCl) for
roughly 30 seconds to remove surface metal contamination; 5) samples were washed clean with
MilliQ® ultra-pure water.
Following cleaning samples were dried with paper towel before being stored in paper bags and
left to air dry for at least two weeks. This process can be seen in Figure 5. Samples were then
divided into leaves, stems and roots before being oven dried for 48 hours at 70°C before being
ground into a fine powder in a ring-mill grinder. They were then oven dried at 105°C for 24
hours before being digested (see below).

25. 25
Figure 5 Cleaningprocessfor vegetationsamples involving 1. removing ofplant from soil ifnecessary;2. multi-stage washing
process;3. drying, and;4. placing inbags for storage (Brett McGregor 6/2/2015).
3.5 Digestion of samples
3.5.1 Digestion of plant samples
Digestion of plant samples is based on Abedin et al. (2002) with minor alterations.
Approximately 200mg of dried ground vegetation sample was weighed into a 75mL digestion
tube that had been placed inside an analytical balance and the delivered weight recorded to the
nearest 0.0001g. Duplicates of each sample were created and two method blanks were also
created in which no sample was delivered but all other process steps were applied. A 5 mL
aliquot of concentrated (70%) HNO3 was added to each digest tube and watchglasses added to
avoid sample contamination and evaporative losses. The contents were shaken by hand to ensure
the sample was fully mixed with the acid and then allowed to sit overnight. The next day the
digest tubes were placed on a heating block and 1mL H2O2 added without warming to avoid
excessively vigorous reactions. Once reactions has settled the temperature was raised to 40°C
with a further 1mL H2O2 subsequently added. Again, once reactions had settled the temperature
was raised to 60°C and a final 1mL H2O2 aliquot added. The temperature was then raised
gradually in 20°C increments to 120°C. The samples were then digested at this temperature for
three hours before being cooled to room temperature (Figure 6).
Once cooled the watchglasses were washed with 3mL 2% HNO3 into the digestion tubes, the
contents of which were subsequently filtered through 15µm hardened ashless filter paper placed

26. 26
in glass funnels and into 100mL volumetric flasks. The digest tubes were then washed with three
10mL portions of 2% HNO3 with the washings filtered into the volumetric flasks. The filter paper
was then washed with three 10mL portions of 2% HNO3 into the volumetric flasks before the
samples were made up to volume with 2% HNO3 before being transferred to plastic bottles for
storage. All glassware and bottles were previously cleaned in a multi-stage process involving
rinsing with deionised water, scrubbing with Brighton Professional™ phosphate-free dishwashing
detergent, acid-washing in 10% HNO3 for at least two hours and then rinsing with MilliQ™ ultra-
pure water before drying and use.
3.5.2 Digestion of soil samples
Digestion of soil samples is based on the Australian Standard AS 4479.2-1997: Analysis of soils -
Extraction of heavy metals and metalloids from soil by aqua regia - Hotplate digestion method
with some minor modifications. Approximately 1g of dried ground soil sample was weighed into
a 75mL digestion tube placed inside an analytical balance and the delivered weight recorded to
the nearest 0.0001g. Duplicates of each sample were created and two method blanks were created
in which no sample was delivered but all other process steps were applied. 10mL of reverse aqua
regia (7.5mL HNO3 and 2.5mL HCl) was added to each digest tube and watchglasses added to
avoid sample contamination and evaporative losses. The contents were shaken by hand to ensure
the sample was fully mixed with the acid and then allowed to sit overnight. The next day the
digest tubes were placed on a heating block and the temperature raised to 70°C. After 1 hour at
70°C the temperature was raised to 80°C. After a further 1 hour the digests were allowed to cool
to room temperature. Once cooled the soil samples were prepared as for plant samples above with
filtering into a 100mL volumetric flask before being poured into a plastic bottle for storage.
Glassware and bottles were washed as per the process described above.

27. 27
Figure 6 Digestion ofsamples onheating block (Brett McGregor 3/10/2014).
3.6 Instrumental analysis
Both soil and plant samples were analysed for arsenic concentration using an Agilent
Technologies 7700X series Inductively Coupled Plasma – Mass Spectrometer running Mass
Hunter Workstation software and utilising an ASX 500 series autosampler. Arsenic standards of
100ppb, 50ppb, 10ppb and 5ppb were created from a stock multi-element standard in addition to
a blank standard. Where samples were predicted or found to have an arsenic concentration over
100ppb the samples were given a 1-in-100 dilution before analysis.

28. 28
4 Results
4.1 Bendigo site sampling
Soil taken from the centre of Site 1 contained an arsenic concentration of 255mg/kg while soil
taken from the small gully at Site 1 contained a concentration of 219mg/kg, indicating both soils
would be classified as Category C contaminated soils under EPA Vic. guidelines. Arsenic
concentrations in vegetation and soil samples collected at Site 1 and Site 2 are summarised in
Table 2.
Table 2. Arsenic concentrations (inmg/kg) for samples collectedat Bendigo. Where novalue is givennosample was taken.
Species leaves seeds/fruit stems roots soil
site 1 site 2 site 1 site 2 site 1 site 2 site 1 site 2 site 1 site 2
Acacia pycnantha 2.55 0.21 3.07 0.20 0.65 0.04 1.53
Eucalyptus sideroxylon 1.26 0.70 0.19 0.21 0.20
Melaleuca decussata 3.85 0.20 0.47 1.16 3.75 463.49
Eucalyptus polyanthemos 2.09 0.22 2.23 0.07 0.28 0.05
Acacia genistafolia 3.64 0.45 11.96 0.27 3.30 0.17 236.01
Cassinia arcuata 33.81 2.36 27.27 3.20 0.18 90.21 4.46 1184.00
Dianella admixta 6.20 0.86 49.81 159.16 5.91 41.31
Helianthus sp. 22.32 0.82 4.11 32.20
Casuarina sp. 0.23 0.07
Allocasuarina verticillata 2.84 0.51 0.04
Austrostypa sp. 5.09 1.88 156.87 28.22 243.54 40.44

29. 29
4.1.1 Leaves
Of leaf samples analysed two species, C. arcuata and Helianthus sp. (sunflower), both from Site
1, contained significantly elevated levels of arsenic (Figure 7), with C. arcuata having the highest
concentration at 34 mg/kg compared to 22 mg/kg for Helianthus sp., despite Helianthus growing
within the tailings as opposed to C. arcuata which was growing in the soil that surrounded the
tailings. C. arcuata also contained the highest arsenic concentration for all samples taken from
site 2 at 2.36 mg/kg, although this was lower than most concentrations found at site 1 (Table 1).
Figure 7 Arsenic concentrations inleaves from vegetation samples collectedat Bendigo. Error bars indicate variability between
sample replicates. † = no sample taken from site 1. ‡ = no sample taken from site 2.
0
5
10
15
20
25
30
35
40
Conc.(mgAs/kgplantmaterial)
Site 1
Site 2

30. 30
4.1.2 Seeds/fruit
This section contains any buds or segments that protruded from the leaf structure including seeds,
seed pods, fruit buds, flower buds, etc. Due to the difficulty accurately identifying the exact
nature of these plant parts for each species they are collectively presented here. As such, direct
comparisons between species cannot be made. Of the plant parts sampled C. arcuata (site 1)
contained the highest arsenic concentration at 27 mg/kg, followed by A. genistafolia (site 1) at 12
mg/kg (Figure 8). All other sampled parts contained low (<4 mg/kg) concentrations of arsenic.
Figure 8 Arsenic concentrations inseed pod-like structures from vegetation samples collectedat Bendigo. Error bars indicate
variabilitybetween sample replicates. † = no sample taken from site 1. ‡ = no sample taken from site 2. * = no sample taken
from site 1 or 2.
0
5
10
15
20
25
30
Conc.(mgAs/kgplantmaterial)
Site 1
Site 2

31. 31
4.1.3 Stems
Most stem samples contained low (<5 mg/kg) concentrations of arsenic (Figure 9). The exception
to this was D. admixta (site 1) which had an arsenic concentration of 50 mg/kg (Table 1). Notable
in these results is that the concentration for M. decasata is higher for the sample taken from Site
2 than the sample taken from Site 1. However, there is considerable error for the sample taken
from Site 2. This relatively high result is due to one of the replicates returning a result of 7.4
mg/kg, a result higher than any replicate for any sample with the exception of D. admixta (site 1).
Given the other replicate for M. decasata (site 2) returned a result of 0.09 mg/kg the averaged
result for this sample should be treated with scepticism and is likely due to sample contamination
in the higher-reading replicate.
Figure 9 Arsenic concentrations instems fromvegetationsamples collected at Bendigo. Error bars indicate variability between
sample replicates. † = no sample takenfrom site 1. ‡ = no sample taken from site 2. * = no sample taken from site 1 or 2.
0
10
20
30
40
50
60
70
Conc.(mgAs/kgplantmaterial)
Site 1
Site 2

32. 32
4.1.4 Roots
Root samples were available for less than half of vegetation samples collected (Figure 10). Two
samples contained high (>100 mg/kg) concentrations of arsenic, these being D. admixta (site 1) at
159 mg/kg and Austrostypa sp. (site 1) at 157 mg/kg (Table 1). C. arcuarta (site 1) also contained
a relatively high concentration of 90 mg/kg. For samples collected at site 2 only Austrostypa sp.
contained a moderate concentration of arsenic at 28 mg/kg (Table 1).
Figure 10 Arsenic concentrations inroots from vegetation samplescollectedat Bendigo. Error bars indicate variability between
sample replicates. † = no sample takenfrom site 1. ‡ = no sample taken from site 2. * = no sample taken from site 1 or 2.
0
20
40
60
80
100
120
140
160
180
Conc.(mgAs/kgplantmaterial)
Site 1
Site 2

33. 33
4.1.5 Soil
Of the soil samples collected along with plant samples the soil surrounding the roots of C.
arcuata taken from Site 1 was highest at 1184 mg/kg (Figure 11). This was significantly higher
than the next highest result, being M. decasata (site 1) at 463 mg/kg. Austrostypa sp. was the
only species to have soil samples collected from both sites, with the sample from Site 1 having a
concentration six time higher (244 mg/kg) than that from site 2 (40 mg/kg) (Table 1). D. admixta,
the only other species to have a soil sample taken from site 2, had a similar concentration to
Austrostypa sp. (site 2) at 41 mg/kg. A. genistafolia (Site 1) returned a result of 236 mg/kg,
similar to the level found in the contaminated tailings.
Figure 11 Arsenic concentrations for soilsamples takenwith plant samples. Error bars indicate variabilitybetweensample
replicates. † = no sample takenfrom site 1. ‡ = no sample taken from site 2. * = no sample taken fromsite 1 or 2.
0
200
400
600
800
1000
1200
1400
Conc.(mgAs/kgsoil)
Site 1
Site 2

34. 34
4.2 Greenhouse experiment
4.2.1 Bendigo soil treatment
The two plants from the second batch in the Bendigo soil treatment that had survived (A.
pycnantha and E. leucoxylon) were grown for 12 weeks before being analysed. Arsenic
concentrations from these samples were low in leaves and stems for both plants, with E.
leucoxylon having concentrations of 0.81 mg/kg in leaves and 0.98 mg/kg in stems while A.
pycnantha had concentrations of 0.72 mg/kg in leaves and 1.30 mg/kg in stems. For root samples,
E. leucoxylon had a concentration of 39.83 mg/kg compared to A. pycnantha, which had a root
arsenic concentration of 165.65 mg/kg (Figure 12). Soil taken from the pots after the growing
period showed that the soil in which E. leucoxylon was growing had a final arsenic concentration
of 253 mg/kg compared to the soil in which A. pycnantha was growing which had a final arsenic
concentration of 374 mg/kg.
Figure 12 Arsenic concentrations inplant parts and soil for twoplants (E. leucoxylon and A. pycnantha) growing in soil taken
from Site 1 at Bendigo. Error bars indicate variability between sample replicates.
Plants in the third batch growing in this medium all died before the fourth week of the
experiment and were not analysed.
0
50
100
150
200
250
300
350
400
450
Leaves Stems Roots Soil
Conc.(mgAs/kgplantmaterial)
E. leucoxylon
A. pycnantha

35. 35
4.2.2 Spiked and control treatments
Whilst the greenhouse experiment was planned to be conducted for 12 weeks, due to premature
plant deaths the experiment was concluded early. Two of the spiked E. leucoxylon had died by
the fourth week of the experiment, as had all spiked A. pycnantha by the sixth week. By the
eighth week of the experiment the remaining spiked E. leucoxylon had died as had one of the
control E. leucoxylon. By the end of the ninth week all spiked C. arcuata had died and as such
the experiment was concluded on the 16th of March 2015 after nine full weeks.
Arsenic concentrations in leaves, stems and roots for spiked and control plants are presented in
Figure 13. All leaf samples from the control treatment returned concentrations of <1 mg/kg while
of those from the spiked treatment C. arcuata had the highest mean arsenic concentration of 46
mg/kg followed by E. leucoxylon with 24 mg/kg and A. pycnantha with 19 mg/kg. For stem
samples again all control samples returned concentrations of <1 mg/kg. Stems from spiked plants
all returned higher arsenic concentrations compared to leaves from spiked samples with
concentrations of 62, 57 and 62 mg/kg for E. leucoxylon, A. pycnantha and C. arcuarta,
respectively. Roots from spiked plants returned even higher concentrations with E. leucoxylon
having a mean arsenic concentration of 222 mg/kg among replicates. A. pycnantha and C.
arcuarta returned lower mean root arsenic concentrations in spiked plants at 195 and 150 mg/kg,
respectively. However there was significant variation between replicate plants for these species
with A. pycnantha recording an upper concentration of 344 mg/kg and a lower concentration of
88 mg/kg while C. arcuata had an upper concentration of 240 mg/kg and a lower concentration
of 102 mg/kg. Arsenic concentrations in roots from plants in the control treatment were higher
than in other plant parts in the same treatment but still low with mean concentrations of 3.4, 7.2
and 3.7 mg/kg for E. leucoxylon, A. pycnantha and C. arcuata, respectively.

36. 36
Figure 13 Arsenic concentrations inplant parts fromplant samples grown in greenhouse experiment. Error bars represent
variability among species replicates within treatments.
Spiked and un-spiked (control) potting mix arsenic concentrations after the conclusion of the
growing period are shown in Figure 14. Control samples all showed low (<2 mg/kg) mean
arsenic concentrations indicating no evident contamination. For spiked samples those in which E.
leucoxylon were growing recorded the lowest mean arsenic concentration of 136 mg/kg
indicating it removed the largest amount of total arsenic from the potting mix over the growing
period. A. pycnantha and C. arcuarta both recorded similar final mean arsenic concentrations of
190 and 192 mg/kg, respectively, although C. arcurta had a higher variation between replicate
plants.
Figure 14 Final arsenic concentrations in spikedandun-spiked (control) potting mix for three species used in greenhouse
experiment. Error bars represent variability among species replicates within treatments.
0
50
100
150
200
250
300
350
400
E.leucoxylon
A.pycnantha
C.arcuarta
E.leucoxylon
A.pycnantha
C.arcuarta
E.leucoxylon
A.pycnantha
C.arcuarta
leaves stems roots
Conc.(mgAs/kgplantmaterial)
Control
Spiked
0
50
100
150
200
250
300
E. leucoxylon A. pycnantha C. arcuarta
Conc.(mg/kgAs.)
Control
Spiked

37. 37
4.3 Confirmation of results
To confirm that the results obtained from ICP-MS were accurate, the standards taken from the
multi-element standard (0 ppb, 5 ppb, 10 ppb, 50 ppb, 100 ppb) were compared with the
standards internal to the ICP-MS. It was found that the arsenic standards from the multi-element
standard were approximately 15% higher than expected, with values of 0 ppb, 5.5 ppb, 11.9 ppb,
59.4 ppb and 116.9 ppb (Figure 15). Give this discrepancy the internal standards were used to
measure the arsenic levels in the samples analysed. While the multi-element standard was more
concentrated than expected, the results are fairly linear, giving confidence in the accuracy of
dilutions used for ICP-MS analysis.
Figure 15 Arsenic concentrations found in multi -element standard compared to the expected concentrations .
0
20
40
60
80
100
120
0 20 40 60 80 100 120
Actualconcentration(ppbAs.)
Expected concentration (ppb As.)

38. 38
5 Discussion
5.1 Bendigo field samples
For all vegetation samples collected at Bendigo the arsenic concentration was higher in samples
taken from Site 1 than those taken from Site 2, the sole exception being the stem of M. decasata,
although this was likely due to a contaminated digest replicate. C. arcuata (Site 1) contained
elevated concentrations in all plant parts except in stems. The reason for this is unclear, but may
be indicative of a plant mechanism in which arsenic is taken up and stored in the leaves and seeds
rather than throughout the plant. What is most interesting is that the soil taken from surrounding
the roots of C. arcuata (Site 1) was exceptionally high at 1184 mg/kg, many times higher than
the concentration found in the centre of the contaminated tailings at Site 1 of 255 mg/kg. It is
possible that this may be due to the plant drawing arsenic towards it, in which case it would make
C. arcuata a possible candidate for phytostabilisation as it would draw arsenic from the
surrounding soil and localise it. In order to test this hypothesis a new field experiment in which a
transect radiating out from a single C. arcuata located in a similarly contaminated area would
measure arsenic concentrations at various distances from the plant roots. If this hypothesis is true,
arsenic concentrations should decrease with distance from the plant. If this is not found to be the
case, then the results found in this present study may be due to a random concentration of arsenic
near the C. arcuarta sampled. M. decasata (Site 1) also had a high arsenic concentration in the
soil surrounding it, although less than half that of C. arcuata at 463 mg/kg, but still higher than
the contaminated tailings. This species, however, did not have high arsenic concentrations in its
biomass. This could potentially make M. decasata suitable for phytostabilisation.
D. admixta (Site 1) was the only species to have an elevated arsenic concentration in its stems (50
mg/kg), with all other specimens taken from Site 1 having low (<5 mg/kg) concentrations.
Interestingly, this specimen only had a moderate concentration in its leaves (6 mg/kg). It did,
however, have a high concentration in its roots at 159 mg/kg. Austrostypa sp. had a similar
arsenic concentration in its roots at 157 mg/kg, but still less than the soil that surrounded it (244
mg/kg). All other species (A. pycnantha, E. sideroxylon, E. polyanthemos, A. genistafolia,
Helianthus sp., Casurina sp., A. verticilata) showed little uptake of arsenic into their biomass and
therefore little potential for phytoremediation.

39. 39
5.2 Greenhouse experiment
The high mortality rate among plants growing in the contaminated tailings taken from Site 1 is
consistent with observations in the field, in which little vegetation was growing on the
contaminated tailings with vegetation instead growing in the soil surrounding it. The implication
of this is that the potential for phytoremediation of this site is limited as any plants introduced to
the site would quickly perish. A possible solution to this would be to cover the site in a layer of
fertile capping soil before planting, although this endeavour would likely be expensive. For the
two plants that were able to be recovered from the second batch, neither accumulated significant
levels of arsenic in their above-ground biomass (<2 mg/kg). A. pycnantha was able to accumulate
much higher levels of arsenic in its roots than E. leucoxylon, with concentrations of 166 mg/kg
and 40 mg/kg, respectively. Strangely, A. pycnantha also had a higher final soil arsenic
concentration compared to E. leucoxylon. This is contrary to what would be expected, as a higher
root concentration would indicate more arsenic is being taken out of the soil, which should then
have a lower arsenic concentration. It may be possible that the arsenic concentrations measured in
the roots were, in part, due to residual soil adhering to root samples after cleaning, hence the
correlating root and soil concentrations. Most confounding is that the soil arsenic concentration
for A. pycnantha is over 100 mg/kg higher than the original concentration of 255 mg/kg. The
reason for this is unknown.
Plants potted in the spiked potting mix also had a very high mortality rate, with all plants dying
by the ninth week. Given that the arsenic concentration of the spiking solution was roughly equal
to the concentration found in the contaminated tailings from Site 1 (260 mg/kg), this may indicate
that all three growing species would be unsuitable for phytoremediation in such areas. However,
as the species or compound of arsenic found within the contaminated tailings was not researched,
the use of sodium arsenate dibasic heptahydrate (Na2HAsO4.7H2O) in the spiking solution may
not be representative of the arsenic found on site.
With regards to plant uptake of arsenic in the spiked treatment C. arcuarta contained the highest
concentration in leaves at 46 mg/kg, almost double that of the next highest, E. leucoxylon at 24
mg/kg. Concentrations in stems were roughly equal for all species at approximately 60 mg/kg,
and higher than in leaves. Concentrations in roots were much higher again, with E. leucoxylons’
average concentration of 222 mg/kg the highest for all three species but still lower than the

40. 40
spiking solution. This regression in arsenic concentration from spiking solution>roots>stems>
leaves is a likely indicator of arsenic progressively being taken up by the roots and translocated
through the stems and into the leaves.
The most important factor with regards to phytoremediation is the total amount of arsenic
removed from the soil. Both C. arcuata and A. pycnantha removed some arsenic from the spiked
growing medium, with final arsenic concentrations in the potting mix approximately 190 mg/kg
for both species, 70 mg/kg less than the starting concentration. E. leucoxylon, however, removed
a significant amount more arsenic from the potting mix, with a final mean concentration of 136
mg/kg. This would indicate that E. leucoxylon is the most suitable species for phytoremediation.
What is most interesting is that the E. leucoxylon replicates were the first to perish in the spiked
treatment, with two of the three potted replicates dying within two weeks of the final application
of spiking solution being applied, and the third plant dying four weeks subsequently without any
noticeable growth. Given this, it is likely that, had these plants grown for longer like C. arcuata,
the amount of arsenic removed from the potting mix may have been significantly greater.
5.3 Comparison between field and greenhouse samples
E. leucoxylon was not among the species sampled from either site at Bendigo, therefore no
comparison between samples taken from the field and those in the greenhouse experiment can be
made. A comparison can be made, however, between the sole E. leucoxylon recovered from the
Bendigo soil treatment growing in the second greenhouse batch and the examples growing in the
spiked sodium arsenate treatment in the third batch. In the spiked treatment E. leucoxylon had a
mean arsenic concentration of 24 mg/kg in leaves and 62 mg/kg in stems, yet in the sole example
growing in the Bendigo soil both leaves and stems had concentrations of below 1 mg/kg, despite
it growing for a full 12 weeks compared to less than 9 weeks for the spiked examples. This
indicates that results from the spiked greenhouse treatment may not be representative of actual
arsenic uptake if plants were planted in the field, and thus may not reflect the actual
phytoremediation potential of these species. This discrepancy between treatments may be due to
a number of factors such as arsenic species, bioavailability, and soil composition. There was a
similar discrepancy between the A. pycnantha example recovered in the Bendigo soil treatment

41. 41
and those in the spiked treatment, with leaves and stems in the spiked treatment having mean
arsenic concentrations of 19 mg/kg and 57 mg/kg, respectively, while leaves and stems from the
example in the Bendigo soil had concentrations of <2 mg/kg.
Examples of both C. arcuata and A. pycnantha were found at both Site 1 and Site 2 at Bendigo,
thus making a comparison with plants in the greenhouse experiment possible. For C. arcuata,
leaf samples had a higher mean concentration in the spiked greenhouse treatment than in the
sample taken from Site 1 (46mg/kg against 34 mg/kg) while for stems the difference was far
more significant, with the stems in the spiked treatment having a mean arsenic concentration of
62 mg/kg compared to just 3.2 mg/kg for the sample taken from Site 1. For A. pycnantha,
concentrations were again higher in the spiked greenhouse plants, with mean concentrations in
leaves and stems of 19 mg/kg and 57 mg/kg compared to the sample taken from Site 1 which
returned readings of 2.55 mg/kg for leaves and just 0.65 mg/kg for stems. These results again
show that results from the spiked treatment within the greenhouse experiment may not translate
to similar results if these species were planted in contaminated areas in the field and therefore
may not indicate their phytoremediation potential.
5.4 Comparison with existing literature
Srivastava et al. (2006) states that a hyperaccumulator is a plant that can accumulate metal
concentrations of above 1000 mg/kg in their above-ground parts. Given this definition, none of
the species sampled for this project can be classified as hyperaccumulators. This finding is not
unexpected as all known arsenic hyperaccumulating plants are ferns (Rathinasabapathi 2011).
Existing literature on arsenic accumulation on native Australian species is limited. Wilson et al.
(2013) conducted a greenhouse experiment growing Couch (Cynodon dactylon), Tussock Poa
(Poa sieberiana), Acacia ingramii and Eucalyptus michaeliana in two soils, taken from a historic
mining area in New South Wales currently undergoing rehabilitation, which contained arsenic
concentrations of 826 mg/kg and 1606 mg/kg. In their study, in which plants were grown for 14
weeks, final arsenic concentrations in leaves and stems did not exceed 40 mg/kg for any plants
growing in soil without any added nutrients and root arsenic concentrations did not exceed 140
mg/kg for plants in this same treatment. These finding generally reflect those from samples taken

42. 42
from Site 1 at Bendigo, although some of the results are slightly higher in the present study.
Wilson et al. (2013) state that these levels of arsenic uptake are similar to previous findings in
grasses and woody species outside Australia. Given these findings it appears unlikely that woody
species or grasses would be suitable for phytoextraction.
6 Conclusion
This study did not find any species that are able to hyperaccumulate arsenic in its above-ground
biomass and therefore no species sampled would be suitable for phytoextraction. Given that all
known arsenic hyperaccumulators are ferns this result is not surprising. However, there may be
potential for some species to be used for phytostabilisation. C.arcuata’s apparent ability to
concentrate arsenic in soil around its roots may be a way to localise soil arsenic. Also, E.
leucoxylon’s ability to remove significant amounts of arsenic from the soil in the spiked
greenhouse treatment despite its relatively low biomass concentrations may indicate its potential
for phytoremediation, although the sampling of examples in the field would be needed to
corroborate these results. The discrepancy between samples collected in the field and those
growing in the greenhouse demonstrate that spiking potted plants may not produce results that are
applicable to actual contaminated sites. To most accurately test the phytoremediation potential of
native species in contaminated soils, future experiments should conduct direct plantings of
candidate species into these soils. This is likely the only way to accurately assess which species
are suitable for the purpose of phytoremediation of arsenic contaminated soil.
8 Recommendations
One of the limitations of this study is that root and soil samples were not taken from all sampled
species at Bendigo. This was mostly due to mature trees and shrubs being sampled. Any future
study should aim to sample roots and soil where possible, which, by necessity due to plant size,
would mostly come from juvenile plants. Also, the species of arsenic found in the contaminated
soil should have been investigated so that the spiking solution could most accurately reflect the

43. 43
arsenic present in the contaminated soil. Lastly, a representative sample of soil from Site 1 taken
from outside the dumped tailings where most of the sampled plants were growing should have
been collected. This would have been informative of the arsenic levels in which the plants were
growing at Site 1 which may have been different from the concentration found within the tailings.
This could then be juxtaposed with the soil taken from around the plant roots and therefore better
gauge if the plants are concentrating arsenic around their roots.
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