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

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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 western Victoria...................................................................................................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 Bendigo field samples.......................................................................................................................38

5.2 Greenhouse experiment...................................................................................................................39

5.3 Comparison between field and greenhouse samples.......................................................................40

5.4 Comparison with existing literature.................................................................................................41

6 Conclusion...............................................................................................................................................42

8 Recommendations..................................................................................................................................42

9 References...............................................................................................................................................43

Table of Tables

Table 1: Number of plants surviving in third batch by end of experiment..................................................23

Table 2: Arsenic concentrations for samples collected at Bendigo.............................................................28

Table of Figures

Figure 1 Map of Bendigo showing location of two sampling sites.............................................................19Figure 2 Photo taken of Site 1 showing light grey soil/mine tailings with lack of vegetation.....................20Figure 3 Image taken from Site 2 showing more natural landscape..........................................................21Figure 4 Greenhouse set-up.......................................................................................................................23Figure 5 Cleaning process for vegetation samples.....................................................................................25Figure 6 Digestion of samples on heating block.........................................................................................27Figure 7 Arsenic concentrations in leaves from vegetation samples collected at Bendigo.........................29Figure 8 Arsenic concentrations in seed-like structures from vegetation samples collected at Bendigo...30

Figure 9 Arsenic concentrations in stems from vegetation samples collected at Bendigo.........................31

Figure 10 Arsenic concentrations in roots from vegetation samples collected at Bendigo........................32

Figure 11 Arsenic concentrations for soil samples taken with plant samples.............................................33

Figure 12 Arsenic concentrations in plant parts and soil for two plants (E. leucoxylon and A. pycnantha) growing in soil taken from Site 1 at Bendigo...............................................................................................34

Figure 13 Arsenic concentrations from plant samples grown in greenhouse esperiment..........................36

Figure 14 Final arsenic concentrations in spiked and un-spiked (control) potting mix...............................36

Figure 15 Arsenic concentrations found in multi-element standard...........................................................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

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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

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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

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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

15

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

16

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.

17

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

18

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 Bendigo showing location of two sampling sites (source: Google Maps).

19

Figure 2 Photo taken of Site 1 showing light grey soil/mine tailings with lack of 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.

20

Figure 3 Image taken from Site 2 showing more 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).

21

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

22

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 soilE. leucoxylon 2 0 0 (1)A. pycnantha 3 0 0 (1)C. arcuarta 3 0 0

23

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).

24

Figure 5 Cleaning process for vegetation samples involving 1. removing of plant from soil if necessary; 2. multi-stage washing process; 3. drying, and; 4. placing in bags 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

25

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.

26

Figure 6 Digestion of samples on heating 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.

27

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 (in mg/kg) for samples collected at Bendigo. Where no value is given no sample 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

28

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).

A. pycn

antha

E. sideroxyl

on

M. deca

sata

E. polya

nthemos

A. genist

afolia

C. arcu

arta

D. admixt

a

Helianthus s

p. ‡

Casurin

a sp. †

A. verti

cillata ‡

Austrosty

pa sp.

0

0.5

1

1.5

2

2.5

Site 1Site 2

Conc

. (m

g As

/kg

plan

t mat

eria

l)

Figure 7 Arsenic concentrations in leaves from vegetation samples collected at Bendigo. Error bars indicate variability between sample replicates. † = no sample taken from site 1. ‡ = no sample taken from site 2.

29

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.

A. pycn

antha

E. sideroxyl

on †

M. deca

sata ‡

E. polya

nthemos

A. genist

afolia

C. arcu

arta ‡

D. admixt

a *

Helianthus s

p.

Casurin

a sp. *

A. verti

cillata *

Austrosty

pa sp. *

0

0.05

0.1

0.15

0.2

0.25

0.3

Site 1Site 2

Conc

. (m

g As

/kg

plan

t mat

eria

l)

Figure 8 Arsenic concentrations in seed pod-like structures from vegetation samples collected at Bendigo. Error bars indicate variability between sample replicates. † = no sample taken from site 1. ‡ = no sample taken from site 2. * = no sample taken

from site 1 or 2.

30

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.

A. pycn

antha

E. sideroxyl

on

M. deca

sata

E. polya

nthemos

A. genist

afolia

C. arcu

arta

D. admixt

a ‡

Helianthus s

p. ‡

Casurin

a sp. †

A. verti

cillata

Austrosty

pa sp. *

0

0.5

1

1.5

2

2.5

3

3.5

4

Site 1Site 2

Conc

. (m

g As

/kg

plan

t mat

eria

l)

Figure 9 Arsenic concentrations in stems from vegetation samples collected at Bendigo. Error bars indicate variability between sample replicates. † = no sample taken from site 1. ‡ = no sample taken from site 2. * = no sample taken from site 1 or 2.

31

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).

A. pycn

antha ‡

E. sideroxyl

on *

M. deca

sata *

E. polya

nthemos *

A. genist

afolia *

C. arcu

arta

D. admixt

a

Helianthus s

p. ‡

Casurin

a sp. *

A. verti

cillata *

Austrosty

pa sp.

0

5

10

15

20

25

30

Site 1Site 2

Conc

. (m

g As

/kg

plan

t mat

eria

l)

Figure 10 Arsenic concentrations in roots from vegetation samples collected at Bendigo. Error bars indicate variability between sample replicates. † = no sample taken from site 1. ‡ = no sample taken from site 2. * = no sample taken from site 1 or 2.

32

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.

A. pycn

antha *

E. sideroxyl

on *

M. deca

sata ‡

E. polya

nthemos *

A. genist

afolia ‡

C. arcu

arta ‡

D. admixt

a †

Helianthus s

p. *

Casurin

a sp. *

A. verti

cillata *

Austrosty

pa sp.

40

40.2

40.4

40.6

40.8

41

41.2

41.4

Site 1Site 2

Conc

. (m

g As

/kg

soil)

Figure 11 Arsenic concentrations for soil samples taken with plant samples. Error bars indicate variability between sample replicates. † = no sample taken from site 1. ‡ = no sample taken from site 2. * = no sample taken from site 1 or 2.

33

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.

Leaves Stems Roots Soil0

50

100

150

200

250

300

350

400

E. leucoxylonA. pycnantha

Conc

. (m

g As

/kg

plan

t mat

eria

l)

Figure 12 Arsenic concentrations in plant parts and soil for two plants (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.

34

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.

35

E. le

ucox

ylon

A. p

ycna

ntha

C. a

rcua

rta

E. le

ucox

ylon

A. p

ycna

ntha

C. a

rcua

rta

E. le

ucox

ylon

A. p

ycna

ntha

C. a

rcua

rta

leaves stems roots

0

50

100

150

200

250

Control Spiked

Conc

. (m

g As

/kg

plan

t mat

eria

l)

Figure 13 Arsenic concentrations in plant parts from plant 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.

E. leucoxylon A. pycnantha C. arcuarta0

50

100

150

200

250

Control Spiked

Conc

. (m

g/kg

As.)

Figure 14 Final arsenic concentrations in spiked and un-spiked (control) potting mix for three species used in greenhouse experiment. Error bars represent variability among species replicates within treatments.

36

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.

0 20 40 60 80 100 1200

20

40

60

80

100

120

Expected concentration (ppb As.)

Actu

al co

ncen

trati

on (p

pb A

s.)

Figure 15 Arsenic concentrations found in multi-element standard compared to the expected concentrations.

37

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.

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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

39

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

40

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

41

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

42

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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