j o u r n a l o f s u s t a i n a b l e m i n i n g 1 4 ( 2 0 1 5 ) 1 1 5 e1 2 3

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Evaluation of metal uptake factors of native trees

colonizing an abandoned copper mine e a quest for
phytostabilization

Ramkrishna Nirola a,c,*, Mallavarapu Megharaj b, Thavamani Palanisami b,

Rupak Aryal c, Kadiyala Venkateswarlu d, Ravi Naidu b
a
Future Industries Institute, Division of Information Technology, Engineering and the Environment,
University of South Australia, SA 5095, Australia
b
Global Institute for Environmental Research (GIER), University of Newcastle, ATC Building, Callaghan, Newcastle,
NSW 2308, Australia
c
School of Natural and Built Environments, Division of Information Technology, Engineering and the Environment,
University of South Australia, SA 5095, Australia
d
Formerly Professor of Microbiology & Dean, Faculty of Life Sciences, Sri Krishnadevaraya University, Anantapur
515055, India

article info abstract

Article history: Accumulation and enrichment of heavy metals in the above ground parts of Australian native
Received 16 September 2015 Acacia pycnantha (Ap) and Eucalyptus camaldulensis (Ec) growing in an abandoned copper mine
Received in revised form located in Kapunda, South Australia have been studied. Cu and other metals (Na, Al, K, Ca, Fe,
23 October 2015 Zn, Cd and Pb) in plants and corresponding soils were analysed to evaluate plant interaction
Accepted 2 November 2015 with soils containing heavy metals. As per the total metal analysis of leaf and corresponding
Available online 18 November 2015 soil samples, Ap accumulated 93.6 mg kg
1 of Cu in leaf while the corresponding soil con-
centration was 1632 mg kg
1. The Ec accumulated 5341 mg kg
1 of Cu in leaf while the con-
Keywords: centration of this heavy metal in soil was 65 mg kg
1 in soil. The ESEM spectral analysis also
Abandoned mine site showed a high leaf concentration of Cu in Ec (7%) as against only 0.12% in Ap. The average
Heavy metals bioconcentration factor for Cu, Zn, Cd and Pb in Ec was much higher than that of Ap. Similarly,
Bioconcentration factor enrichment factor was more in Ec for Cu, Zn and Pb than in Ap. In contrast, translocation
Translocation factor factor for only Zn and Cd was high in Ap. This study points out that Ec and Ap have different
Enrichment factor stabilising potential in remediating heavy metals like Cu in mined soils.
Phytostabilisation © 2015 The Authors. Productioin and hosting by Elsevier B.V. on behalf of Central Mining
Institute in Katowice. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Ecological impacts in places such as AMLs can be controlled

1. Introduction effectively by evaluating the accumulators against the non-
accumulator plants prior to the phytoremediation processes
The effect of heavy metal toxicity on biota is severe and there is (Ma, Rajkumar, Luo, & Freitas, 2013). A right rehabilitation
an urgency to control the level of toxicity and impact on public practice is certainly a tool to control the heavy metal pollution
health, particularly from abandoned mine lands (AMLs). and one such practices have been by the use of

* Corresponding author. Future Industries Institute, Division of Information Technology, Engineering and the Environment, Building X,
Room X2-O3-EO3, University of South Australia, SA 5095, Australia. Tel.: þ61 883025170; fax: þ61 883023057.
E-mail address: [email protected] (R. Nirola).
Peer review under responsibility of Central Mining Institute in Katowice.
http://dx.doi.org/10.1016/j.jsm.2015.11.001
2300-3960/© 2015 The Authors. Productioin and hosting by Elsevier B.V. on behalf of Central Mining Institute in Katowice. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
116 j o u r n a l o f s u s t a i n a b l e m i n i n g 1 4 ( 2 0 1 5 ) 1 1 5 e1 2 3

phytoaccumulators (Ali, Khan, & Sajad, 2013). The phytor- based on translocation factor (TF) study. Therefore, it has been
emediation technology is employed over polluted sites and established that species of plants having high metal concen-
have served to be a boon to assist in pollution clean up efforts tration ratio (1) of roots to soil is called bioconcentration factor
(Nirola, Mallavarapu, Aryal, & Naidu, 2015). As such, copper is (BCF), a low shoots to roots metal concentration ratio (1)
one of those toxic metals that have a capacity to bind to known as the translocation factor (TF) and 1 ratio of metal
albumen and other small molecules in the human body as ‘free content in above ground plant parts over metal content in soil
copper’ to cause nerve damage (Brewer, 2010). An effective known as enrichment factor (EF) is considered to be ideal HM
phytoremediation would have saved the inhabitants near a stabilizers (Sagiroglu et al., 2006; Sinha, Herat & Tandon, 2007).
mine site in Mexico from elevated levels of lead and arsenic in Therefore, in the present context of the use of plant species for
their blood, and are more severe in children (Gonza  lez & mine site rehabilitation, phytoextraction or phytoaccumulation is
Gonza  lez-Cha
 vez, 2006). Identification of suitable remediating effectively based on EF since the metal accumulation in above
species of plants could reduce the ecological risks of heavy ground parts is exposed to biosphere (Baker, 1981).
metals such as Cu to contaminate water bodies and damage We consider two native tree species growing naturally over
gills causing premature death of fishes (Couillard, Courtenary & many years in mine site as per our earlier study (Nirola et al.,
Mcdonald, 2008). It is thought to be an important component for 2015). The leguminous Acacia pycnantha Benth (golden wat-
identifying the species positively responsible for bio- tle), and non-leguminous Eucalyptus camaldulensis Dehnh (river
accumulation of pollutants being transferred from plants into red-gum) were included with an aim to find answers to the
the food chain (Krumins, Goodey, & Gallagher, 2015). issues of bioavailability, bioaccumulation and biotransforma-
A typical practice during industrial revolution was to tion of metals. The Australian legume A. pycnantha (Ap) has
abandon mines once mineral extraction was conducted. The attributes of root nodule formation and an active microbial
estimated number of abandoned mines in the developed na- activity, and give upper hand in growing resilient at the AML.
tions was 630309 by the year 2000 (Van Zyl, Sassoon, Digby, Our earlier survey and screening at the mine site also indicated
Fleury, & Kyeyune, 2002). Wait (2012) reports 5858 derelict more Cu accumulation in the root zone for this species (Nirola
and ownerless mines in South Africa, and Mackasey (2004) et al., 2015). Further evidence for such resilience by legume
records 5500 derelict (abandoned) mines in Japan alone. Acacia has been documented for sites in the Sahara Desert
There are about 1800 final mine voids and 150 operational (Brockwell, Searle, Jeavons, & Waayers, 2005). An estimated
open cut mines in Western Australia (Doupe  & Lymbery, 2005). annual quantity of nitrogen fixed worldwide by legumes is
Moreover, approximately 2000 derelict mine site exists only in 70e100 million tonnes with acacias fixing a substantial
New South Wales, Australia that date back to the mid-1800s amount growing in around 5 million hectares of land world-
(Grant, Campbell, & Charnock, 2002). However, the exact wide (Brockwell et al., 2005). In terms of its drought tolerance
figure of abandoned and post operational mine differs mechanism, acacias have a morphological advantage to adapt
depending on the definition of what a mine site is. to arid or xeric conditions including AMLs (Brodribb & Hill,
The threshold references used for metals in the surface soils 1993). Their leaves are modified into phyllodes with an abun-
are Cd-3 mg kg
1, Cu-60 mg kg
1, Pb-300 mg kg
1, and Zn- dance of thick sclerenchyma in the outer palisade mesophyll
200 mg kg
1 with a level above 20 mg kg
1 for Cd and aiding its ability to survive drought conditions (Boughton, 1986;
300 mg kg
1 for Pb considered to be a health hazard (Ash & Midgley & Turnbull, 2003). The other native Australian species,
Truong, 2003). The threshold reference values are important E. camaldulensis (Ec) is widely distributed and exhibits a sub-
indicators to determine soil pollution level as these values help stantial drought tolerance capacity which is attributed to deep
to measure phytoremediation degree before and after the pro- roots and tough leaf cuticles as seen through the ESEM image
cess. The accumulation of metals in the foliar region could be (Boland et al., 2006). However, most of the species of eucalyptus
severe due to the reoccurring of soil pollution by metal accu- including E. camaldulensis have indicated an allelopathic effect
mulation in litter of mine site plants (Nouri et al., 2011). In a on other vegetation, hampering the germination of seed
study of plant behaviour with soil heavy metals in Turkey, (Ahmed, Hoque, & Hossain, 2008; Khan, Hussain & Khan, 2008;
species such as Euphorbia macroclada, Verbascum cheiranthifolium Yang, Liu, Ren, & Wang, 2009). In the current study site both of
and Astragalus gummifer were found to accumulate 3e4 times these species were found to be dominating the different
higher levels of heavy metals than those found in the soil landscapes of the polluted area. A curious thought to evaluate
(Sagiroglu, Sasmaz, & Sen, 2006). In another study, Reichman, two taxonomically different plants is expected to add impetus
Menzies, Asher, and Mulligan (2006) assert that plant soil to the issues of mine site rehabilitation with respect to avoid-
interaction explores its capacity to stabilise the heavy metal ing phytoextraction and encouraging phytostabilization.
(HM) using native plants in AMLs. Of all the available rehabili- Moreover, no earlier studies on metal uptake factors are
tation technologies, phytostabilisation is a plant's ability to available for widely used A. pycnantha in mine site
avoid exposure of pollutants to the environment by not trans- rehabilitation.
locating metals from soil to the above ground parts (Kurek &
Majewska, 2012; Yoon, Cao, Zhou, & Ma, 2006). This is to
contain heavy metal pollution by immobilizing the bioavailable 2. Materials and methods
components in the earth's biosphere through revegetation of
operational, derelict and abandoned mine sites. One analysis of 2.1. Site description and characterization
metal toxicity on copper in an abandoned mine in Florida (Yoon
et al., 2006) reported the native grass species Gentiana pennelli- The abandoned mine at Kapunda is one of the oldest settle-
ana that could stabilise heavy metals such as Pb, Cu and Zn ments in Australia that mined copper till 1879 AD. The mine
 j o u r n a l o f s u s t a i n a b l e m i n i n g 1 4 ( 2 0 1 5 ) 1 1 5 e1 2 3 117

site is located 79 km from Adelaide city. The site harbours arid 2 m and homogenized. Plant parts were collected from around
to semi-arid type of vegetation with a calcareous soil. A the canopy and bulked to form a composite sample to main-
thorough screening of nine prominent shrub and tree species tain a uniform representation. Fresh leaf samples were
were undertaken to check their metal enrichment factors collected in an ice filled thermo-col box (5  C) for ESEM studies
namely A. pycnantha, Racosperma pycnanthum, E. camaldulensis, to be analysed within 24 h. Inductively Coupled Plasma Mass
Rosa canina, Lycium ferocissimum, Olea europaea, Acacia acinacea, Spectrometer/Optical Emission Spectrometer (ICP-MS/OES) to
Schinus molle, Leptospermum lanigerum, and Pinus halepensis determine the total metal concentration in plant and soil
(Nirola et al., 2015). Among the nine species, the two species samples were employed (Thomas, 2013). The plant samples
emerged as an outstanding metallophytes growing in the for ICP-MS analysis were collected in the polypropylene con-
abandoned mine site. The golden wattle (Ap) was found tainers and stored in laboratory at room temperature. The
growing around the periphery of the open cut area whereas plant samples were washed three times in deionised water
the river red gum (Ec) was mostly concentrated towards the and oven dried at 60  C. The oven-dried plant samples for ICP-
clayey soil downstream. Currently, the local Light Regional MS analysis (leaf, stem and roots) were ground separately into
Council is responsible for managing the area as a heritage site 1 mm mesh sieve powder using an electric stainless steel
to control pollution and to promote tourism (http://www.light. grinder and were stored in the polypropylene containers. The
sa.gov.au). The plan of the regional council divides the mine soil samples were air-dried and sieved using a 2 mm plastic
site into cultural landscape (C), geological landscape (G) and mesh sieve and stored in polypropylene containers for ICP-
environmental (regenerative) landscape (E) within the area of OES analysis.
approximately 2 km (Fig. 1). Drainage from the site originates
mainly from the regenerative landscape and flows into the
Light River. The currently operated heritage mine track runs 2.3. Characterization of samples
about 1.5 km mainly through the geological landscape (G). The
geological landscape is a barren ground, fenced as a safety Soil characterization included texture analysis using hy-
measure to avoid human intrusion. Fresh leaf, stem and roots drometer method; standard tests were employed to measure
of Ap and Ec including the corresponding soils were collected pH, electrical conductivity, total dissolved carbon (TC), total
from G and E zones, respectively. Some of the sites having the dissolved nitrogen (TN) and total organic carbon (TOC)
unconfirmed indications of earlier land refilling by off-site (Me ndez, Go
 mez, Paz-Ferreiro, & Gasco , 2012). The potentio-
soils were avoided. metric measurement of the supernatant suspension of soil:
deionised water (w/v) at 1:5 ratio was used to measure the pH
2.2. Sampling and processing and EC (Sposito, 2008). A 5 gram soil was separately oven
dried to compare and record the moisture content of the soil.
The sampling plots were modelled to extend a maximum A microwave accelerated reaction system (CEM-MARS X®)
coverage (ISO, 2002; Hazelton & Murphy, 2007), starting from served to digest metals in aqua-regia for soils and HNO3 for
the geological landscape (G) towards the environmental plants separately following USEPA method 3015a (Agazzi &
landscape (E). Altogether each of 16 Ap and Ec plots were Pirola, 2000). The digested samples were diluted up to 50 ml
sampled including the associated soils as per the ecological using Mili-Q water and were passed through a 0.45 mm filter
quadrat principles (Krebs, 1999). The soil samples were using a syringe. The final test samples of 10 ml diluted sus-
collected from the rhizosphere at 15e20 cm depth using a pension were put into ICP tubes for analysis in the Agilent
plastic trowel from two points around the plant diameter of 7500c (Agilent Technologies, Tokyo, Japan).

Fig. 1 e Site map of abandoned copper mine at Kapunda in South Australia. The letter ‘C’ represents the Conservation zone,
‘G’ represents the Geological zone and ‘E’ represents the Environmental zone.
118 j o u r n a l o f s u s t a i n a b l e m i n i n g 1 4 ( 2 0 1 5 ) 1 1 5 e1 2 3

The TC, TN and TOC were measured using air-dried, 2 mm analysing the ESEM data, each picture was tagged so that it
mesh sieved soil dissolved in 1:5 (w/v) deionised (Mili-Q) water corresponded to the radiation outcome. The spectral data
(You, Dalal, Mulligan, & Huang, 2015). The soil solutions were were then compared with the corresponding ICP-MS data.
agitated in end-over shaker overnight and were centrifuged at The ESEM pictures were processed in Microsoft paint pro-
4000 rpm for 10 min. The supernatant was analysed in the gramme. The mean, range and average were calculated using
Formacsseries total organic carbon, total nitrogen analyser triplicate result of each sample. Pearson's correlation coeffi-
using the UV-promoted per-sulphate digestion methodology. cient determined the correlation between metals and soils,
Analyses of total C (%) and N (%) were done by introducing roots, stems and leaves.
0.5 g of air-dried 2 mm mesh sieved and ground sample in a
Trumac carbon-nitrogen-sulphur (CNS) analyser (Leco® Cor-
poration, Michigan, USA). The standard blank, controlled and
3. Results
cross-referencing samples were introduced to maintain
quality control after every 10 readings. The pH, EC, moisture
The physico-chemical parameters used in the present in-
content (H2O%), dissolved TC (mg kg
1), dissolved TN
vestigations include the EC, pH, TC, TN, TOC, N (%) and C (%)
(mg kg
1), dissolved TOC (mg kg
1), total N % and total C %
as presented in Table 1. The soil being tested in this study is
were recorded to average the duplicates of each sample.
from the copper and other metals contaminated dry mine
spoil. The Ap rhizosphere soil was a sandy loam comprising
2.4. Spectroscopic characterization of plant matter 8% clay whereas the Ec rhizosphere soil had 72% clay as per
the hydrometer readings (Me ndez et al., 2012). The lack of
The bioavailability of heavy metals like copper is based on
water stagnation and low water holding capacity of soil result
the influence of soil physical factors (Cerqueira, Arenas-Lago,
into low electrical conductivity and acidic nature of soil (Reuss
Andrade, & Vega, 2015). The localisation of the elemental
& Johnson, 2012). In our study, the soil pH at Ap rhizosphere
distribution in the leaf sample by ESEM is another appro-
ranged from 4.4 to 6.8 (acidic to slightly acidic) and Ec rhizo-
priate method to track the fate and status of accumulated
sphere ranged from 6.8 to 8.4 (neutral to basic). The average
metals in plant tissues (Stokes, Morris, & Groves, 2013). The
electrical conductivity of soils in Ap rhizosphere was
process for characterization began by selecting and washing
304 mS cm
1 and Ec rhizosphere was 7242 mS cm
1 (Table 1).
followed by the fresh leaves being air-dried. Leaf samples
Moreover, the leaf litter of Ec tends to have been responsible
were sliced using a surgical blade into the thinnest trans-
for basic soil and high salinity condition.
verse sections (TS) possible. The specimens were placed on a
Table 2 provides information on heavy metals present in
watch glass for sample sorting. The thinnest sections of both
soil, root, stem and leaf for plants Ap and Ec. Although the
the Ap and Ec were separately chosen using a light micro-
average soil concentration of Cu in E. camaldulensis rhizo-
scope Digitek QC 3199. The sections were mounted on the
sphere soil was lower with 64.9 mg kg
1, the plant and
aluminum specimen-mounts using double-sided tape and
particularly the stem (139.23 mg kg
1) and leaf
fitted onto the platform. The specimens were loaded into a
(5341.1 mg kg
1) recorded a higher accumulation ability
“FEI Quanta 450 FEG ESEM” with an attached “EDAX” Apollo X
compared to Ap (Baker & Brooks, 1989).
SDD Energy Dispersive X-ray (EDX) detector for multiple ex-
Ap showed a decreasing trend of Cu concentration as it
aminations. High voltage (HV), low voltage (LV) and ESEM
travels from soil to leaf, [1631.7(soil) /
modes were utilised to examine the three samples, respec-
90.4(root) / 152(stem) / 93.6(leaf) mg kg
1] whereas Ec
tively, namely: 1) coated dry sample with 5 nm chromium by
showed a much higher accumulation capacity in root and
Quorum Q150T coating unit; 2) uncoated dry sample; and 3)
stem [64.9(soil) / 185.7(root) / 139(stem) / 5341(leaf) mg
uncoated fresh wet sample. However, for the uncoated fresh
kg
1] and significantly high in its leaf (Fig. 2).
but wet specimen, the temperature of the sample holder was
The BCF, also referred to as root (below ground)-soil
reduced to maintain constant water inside the cells within
quotient of Ec is 1 for Cu (3.57) and Zn (2.81), are values
the optimum pressure as per standard procedure. Photo
regarded as a copper or zinc stabiliser. However, the BCF of
images and associated spectral data representing metal
Ap is 1 for Cu (0.43), Zn (0.84), Cd(0.11) and Pb (NA) as the
concentrations were obtained from leaves of Ap and Ec from
each sampling site. As a result, the anticlinal images (TS) of
the specimen were produced in the monitor along with cor-
responding spectral images that were saved for further Table 1 e The physico-chemical parameters of soils from
analysis. the abandoned mine site; ±se (range).
Average Plots of A. Plots of E.
2.5. Data analysis pycnantha (Ap) camaldulensis (EC)
pH 5.8 ± 0.41 (4.4e6.8) 7.6 ± 0.75 (6.8e8.4)
The data were analysed statistically using Microsoft Excel
EC mS 303.8 ± 121.9 (117e1276) 7242 ± 1929 (1382e13,240)
and IBM®SPSS®21 for Windows. Each sample had separate Moisture % 2.8 ± 0.52 (1.6e5.3) 4.8 ± 2 (2.8e6.8)
data arising from triplicate laboratory analysis results that TC mg kg
1 260.5 ± 63.6 (38.7e540.5) 203 ± 101.98 (91e315)
was subjected to ANOVA, including the descriptive statistics. TN mg kg
1 10.6 ± 3.7 (1e34.7) 35.8 ± 9.5 (26e45)
The data and pictures of ESEM anticlinal section of leaves TOC mg kg
1 180 ± 55.4 (27.1e497.8) 146 ± 77.6 (68e223)
corresponding to their graphical spectrum were carefully N% 0.07 ± 0.02 (0.01e0.18) 0.17 ± 0.13 (0.04e0.29)
C% 1.97 ± 0.4 (0.93e3.6) 2.7 ± 1.67 (1.1e4.4)
examined to compare with the data of ICP-MS. While
 j o u r n a l o f s u s t a i n a b l e m i n i n g 1 4 ( 2 0 1 5 ) 1 1 5 e1 2 3 119

Table 2 e The average concentration of metals in the soil, root, stem and leaf of samples from Kapunda abandoned mine site; ±se(range).
Metals Soil (mg kg
1) Root (mg kg
1) Stem (mg kg
1) Leaf (mg kg
1)
Ap Ec Ap Ec Ap Ec Ap Ec
Na 2425 ± 687 5557 ± 3225 4461 ± 8450 17,075 ± 9859 13,926 ± 4054 13,670 ± 969 34,823 ± 534 599 ± 174
(690e6554) (452e14,750) (1473e7749) (0.00e34,320) (2953e39,360) (11,930e15,740) (33,250e35,590) (203e1652)
Al 3160 ± 739 8155 ± 2824 293 ± 58 2414 ± 732 649 ± 7 946 ± 24 15,071 ± 2364 1913 ± 214
(539e6102) (1801e14,750) (157.5e667.2) (1132e3700) (367e1002) (906e1010) (4898e23,125) (1530e2297)
K 22,079 ± 4381 5034 ± 1563 39,353 ± 10,611 38,663 ± 4835 55,277 ± 12,106 57,088 ± 1560.9 102,450 ± 1680 3836 ± 654
(11,518e49,850) (2057e9017) (12,120e101,450) (30,140e47,840) (22,835e114,450) (52,870e59,750) (100,000e107,300) (1594e6591)
Ca 30,489 ± 6296 5707 ± 900 95,879 ± 20,433 128,075 ± 13,157 128,372 ± 19,345 167,325 ± 20,547 63,528 ± 730 14,334 ± 9205
(13,730e68,340) (4164e7823) (54,145e220,350) (104,100e153,700) (55,620e218,450) (127,900e203,500) (62,490e65,680) (244e72,300)
Fe 5755 ± 1195 28,200 ± 7240 595 ± 89 4059 ± 957 1288 ± 123 1355 ± 50 3134 ± 408 51,649 ± 7262
(835e11,010) (12,900e47,200) (412e1173) (2372e5819) (715e1836) (1268e1489) (2365e3919) (26,620e80,170)
Cu 1632 ± 241 65 ± 19 90 ± 22 186 ± 27 152 ± 30 139 ± 27 94 ± 9 5341 ± 1226
(458e2544) (25e117) (17e199) (137e233) (45e282) (90e195) (77e110) (946e11,710)
Zn 63 ± 17 40 ± 11 62 ± 15 107 ± 17 96 ± 16 71 ± 10 128 ± 4 80 ± 13
(11e169) (20e69) (19e126) (76e140) (35e165) (52e91) (119e136) (31e133)
Cd 0.28 ± 0.07 5 ± 1.45 0.28 ± 0.07 0.55 ± 0.01 0.19 ± 0.05 0.57 ± 0.10 0.27 ± 0.04 4.1 ± 1.0
(0.1e0.6) (2.3e9) (0.1e0.6) (0.5e0.6) (0.1e0.4) (0.39e0.7) (0.2e0.3) (1.1e9)
Pb 2.6 ± 0.6 10.9 ± 3.1 2.6 ± 0.6 2.4 ± 0.6 0.8 ± 0.2 10.3 ± 3.9 2.05 ± 0.4 21 ± 3.7
(0.0e4.8) (4.5e19.3) (0.0e4.8) (1.4e3.5) (0.1e1.6) (3.3e17.6) (1.4e2.7) (7.2e37)

value of BCF 1 an considered as inferior phytostabilizer

(Sagiroglu et al., 2006; Yoon et al., 2006). Root accumulation of 4. Discussion
HM (in Ap) is not as harmful as above ground parts accu-
mulation (in Ec) in terms of pollution and dispersion (Nouri 4.1. Soil characteristics and metal accumulation in plants
et al., 2011). The TF, also called shoot-root quotient, ex-
plains a plant's ability to translocate metal from its roots to An evaluation of metal uptake behavior of both the species Ap
shoots and to leaves where a value of 1 is ideal for phy- and Ec growing on mine site with respect to its adaptation and
tostabilization. The TF of Ec for Cd (1.04) and Pb (3.86) is 1, a soil interaction based on their interaction with heavy metal
good candidate for Pb and Cd phytoabsorption but less uptake behavior has been investigated. The extent of root and
preferred for phytostabilization (Yoon et al., 2006). The Ap the physico-chemical properties of soil influence the uptake
showed a negligible level of copper translocation (TF) along or accumulation values analysed currently using the top soil
metal concentrations (Zengin, Aka Sag  lıker, & Darıcı, 2008).
its tissue and such a quality is acknowledged as Cu tolerance
(not translocating) ability (Lamb, Ming, Megharaj, & Naidu, However, the correlation of soil PH to root copper concentra-
2010). An EF also called soil-leaf quotient with a value 1 is tion has R2 value of 0.58 (p  0.05), which is a less significant
considered to be an ideal stabilizer (Sagiroglu et al., 2006; result explains the feeble effect of soil pH on metal uptake to
Yoon et al., 2006). Therefore the EF of copper in Ec is 2.17, root zone of Ap (Fig. 2). This indicates plants ability to adapt
as Cu accumulator and 1.89 as Zn accumulator (Table 3). soil ionic impact on metal uptake as reflected with Ap where a

Fig. 2 e The accumulation pattern of underground and above ground parts of metallophyte (SD-%, N ¼ 3) a) Cu concentration
in Ap rhizosphere soil (bars) and A. pycnantha (Ap1, Ap 2, Ap3, Ap4 ¼ 4 £ 4) root, stem and leaf at 16 sampling locations.
b) Cu concentration in Ec rhizosphere soil (bars) and E. camaldulensis (Ec1, Ec2, Ec3, Ec4 ¼ 4 £ 4) root, stem and leaf at 16
sampling locations.
120 j o u r n a l o f s u s t a i n a b l e m i n i n g 1 4 ( 2 0 1 5 ) 1 1 5 e1 2 3

highest soil copper yielded less foliar concentration. In

Table 3 e The Translocation factor (TF), Bioconcentration factor (BCF) and Enrichment factor (EF) of A. pycnantha (Ap) in secondary axis and E. camaldulensis (Ec) in primary
axis against the metals under investigation; ±se(range). Note that TF of Ec for Pb and of Ap for Cd, BCF of Ec for Cu and Zn and of Ap for Zn and EF of Ec for Cu and Zn and of Ap for

(24.7e38.7)
(24.3e33.8)
(0.04e0.06)
(2.11e2.23)

(0.06e0.23)
(4.3e55.6)
(0.07e0.1)

(1.58e2.2)

(0.25e2.3)
contrast, there is a relatively higher concentration of metals in
Ec leaf and stem compared to soil and root concentration
growing on basic pH soil condition (Table 2). Therefore, this

EF

31.7 ± 7
29.9 ± 25.6
0.09 ± 0.02

29.1 ± 4.8
0.05 ± 0.01
2.2 ± 0.06
1.9 ± 0.31
0.15 ± 0.09
1.3 ± 1.01
result generally indicates that Ap while growing on acidic soils
translocate less metals than the Ec that is growing on basic
soil condition is a natural adaptation. This result could be also
attributed to the metal enrichment in Ec leaves through root
uptake which is called systemic uptake, and through bark
called dermal sorption, or by a combination of both (Chenery
Eucalyptus camaldulensis

et al., 2012; Reichman, Menzies, Asher, & Mulligan, 2006). Our

0.41 ± 0.19 (0.22e0.59)

0.67 ± 0.03 (0.64e0.70)

1.04 ± 0.4 (0.67e1.4)
3.9 ± 1.29 (2.6e5.2)
study shows that the accumulation level of Cu in Ap and Ec is
0.7 ± 0.0 (0.7e0.7)
0.6 ± 0.3 (0.3e0.9)
2.2 ± 0.5 (1.7e2.6)
1.4 ± 0.5 (0.9e1.9)

different in root, stem and leaf suggesting the independent

TF

behaviour of each species against copper (Fig. 2).

*NA

4.2. Soil e plant interaction

The ESEM spectral analysis shows a higher level of Ca depo-

sition in the vascular bundle of Ap with EF value 78.9. Thus, Ap
(0.08e0.42)
(14.7e14.9)
(17.6e27.8)
(0.07e0.27)
(1.58e5.56)
(2.48e3.14)
(0.09e0.17)
(0.10e0.44)

has an affinity to Ca salts as evident from the results shown in

Zn are high. BCF > 1, TF and EF < 1 means that it is a good stabiliser, NA- Not available due to calculation error.

(19e75)

Fig. 3. According to Maksymiec (1998) there is a physiological

BCF

effect on cellular processes of higher plants. One such effect is

47 ± 28

22.7 ± 5
0.3 ± 0.2
14.8 ± 0.13

0.17 ± 0.10
3.6 ± 1.99
2.81 ± 0.33
0.13 ± 0.04
0.27 ± 0.17

due to Cu2þ where enzymatic and leaf senescence is brought

about with Ca2þ moving into xylem through apoplast
pathway. Moreover, the study also points out about the
photosynthetic apparatus where Cu2þ and Ca2þ are involved
in the process of escaping metal toxicity. The Ap accumu-
lating more Ca in the vascular zone is a possible explanation
(0.01e0.04)

(0.01e0.02)
(0.01e0.04)
(0.17e1.77)
(0.02e1.27)
(0.00e0.04)

for its premature death after almost every 10 years due to the
(0.8e276)
(0.3e6.3)

(0.5e3.4)

Ca flakes clogging vascular bundles (Brockwell et al., 2005;

Gibson et al.; 2011; Miller, Murphy, Brown, Richardson, &
EF

Gonza  lez-Orozco, 2011). However, the accumulation of Ca in

3.9 ± 0.7
0.02 ± 0.00
1.5 ± 0.3
78.9 ± 31.5
0.02 ± 0.0
0.02 ± 0.00
0.90 ± 0.22
0.21 ± 0.15
0.02 ± 0.01

Ap vascular bundle leading to a mutualistic interaction be-

tween plant and microbe can be defined as “a necessary evil”.
Vadassery and Oelmüller (2009) assert a similar view
regarding the mutual relationship existing between the plant
and microbe based on Ca signalling. This relationship has an
advantage for Ap with its root nodules being able to survive in
1.42 ± 0.56 (0.42e5.23)
0.15 ± 0.04 (0.03e0.3)

1.20 ± 0.29 (0.33e2.8)

Acacia pycnantha

0.19 ± 0.1 (0.04e0.6)

contaminated soils due to the ability to draw moisture and

2.3 ± 0.5 (0.4e4.2)

0.9 ± 0.2 (0.3e1.7)

3.5 ± 0.6 (1.4e6.6)

nutrition such as nitrogen from the rhizosphere with the help

TF

of rhizobia present in root nodules and micorrhiza present in

root tissue (Gibson et al., 2011). Moreover, some species of
Acacia (A. nilotica and A. senegal) were found growing well in
*NA

*NA

calcareous soil supports Ca-philic and accumulating behav-

iour of this genera (Chaplot, 2013).
One study in Western Australia (Mikli, 2001) reported that
E. camaldulensis was found to grow well on riverine sites quiet
0.43 ± 0.14 (0.18e1.42)
0.84 ± 0.22 (0.20e2.21)
0.11 ± 0.05 (0.02e0.45)

similar to the habitat in the current study site. Mikli in his

0.24 ± 0.12 (0.03e1)
0.27 ± 0.1 (0.05e1)
1.8 ± 0.4 (0.9e4.5)
24 ± 9.5 (0.56e85)

thesis further reports that Eucalyptus robusta and E. camaldu-

2.4 ± 0.6 (1.2e6)

lensis struggled to grow in poor coal mine soils even though

BCF

they tolerated acidic soils. One of the possible reasons for

getting poor growth of Ec was due to sandy soil as found in
*NA

arid to semi-arid mine sites that prevented deeper root

penetration (Boland et al., 2006). In the stressed soil condi-
tion, the root hairs struggle to settle inside the nutrient
deficient soil, and preferentially, Ec is found to grow on grey
Metals

heavy clayey soils with basic pH soil condition is consistent to

our current finding as well (Brooker, Slee, Connors, & Duffy,
Na

Cu
Zn
Cd
Ca

Pb
Fe
Al
K

2005; Costermans, 1989; Haling et al., 2013). Generally, the

 j o u r n a l o f s u s t a i n a b l e m i n i n g 1 4 ( 2 0 1 5 ) 1 1 5 e1 2 3 121

Fig. 3 e ESEM spectral diagram with images in inset. a) the anticlinal section of Ap xylem bundle in the inset with spectral
diagram of highlighted area in red circle shows Ca flake deposition. b) the anticlinal section of Ap xylem vessel in inset with
spectral diagram of the highlighted area in red circle shows salts of chlorine and potassium respectively, at 2.4 and 3.4 keV.
c) the anticlinal section of Ec leaf in inset with spectral diagram of highlighted area in red circle shows Ca deposition in
mesophyll zone at 3.6 keV. d) the anticlinal cross-cut of Ec leaf in inset with spectral diagram of highlighted red circle shows
deposition of Cu and Zn in xylem vessel.

acacia species have been widely preferred to eucalyptus for accumulation) ratio (Masvodza, Dzomba, Mhandu, &
afforestation and greening programs because it can grow in Masamha, 2013). However, there is no convincing earlier
poor soils faster, can fix nitrogen, and revegetate even in data for both species regarding their interaction with heavy
nutrient deficient soil (Boyes, Gunton, Griffiths, & Lawes, metal, particularly in abandoned mine soils. According to
2011; Gawronski, Greger, & Gawronska, 2011). Therefore, in Baker and Brooks (1989), plants that accumulate
our study area containing acidic soil this leguminous Ap >1000 mg kg
1 of Cu, Co, Cr, Ni or Pb and >10,000 mg kg
1 of
species has been evaluated to address the phytotoxicity of Mn or Zn are called hyper-accumulators, and the present
metals on plants (Seigler, 2003; Zaets & Kozyrovska, 2012). Ap investigation implies to Ec leaf accumulation. So, the uptake
rhizosphere soil confirmed that this species grows well in factor values actually explain the qualitative aspect rather
acidic soil with low EC, low carbon and nitrogen content as than the quantitative one. The ESEM data of the leaf indicates
presented in Table 1. that Ap is an excluder of Cu because of its low spectroscopic
readings (Fig. 3). This is in contrast to the findings of
4.3. Accumulation factors of heavy metals on Ap and Ec Masvodza, Dzomba, Mhandu, and Masamha (2013) who re-
ported hyperaccumulation of Cu in Acacia saligna and Acacia
The observed metal concentration in leaf, stem and root were polycantha grown in mine slime dams in Harare, Zimbabwe.
used to measure BCF, TF and EF to determine whether a plant Baker (1981) suggests that it is the foliar concentration that
is an excluder, extractor or stabiliser (Baker, 1981). As per the disqualifies a plant to act as a stabiliser. In the present study,
current investigation, Ap has a better edge over Ec because of the foliar concentration of Cu in Ap was comparatively lower
its high BCF (high root accumulation) and low EF (lower leaf
122 j o u r n a l o f s u s t a i n a b l e m i n i n g 1 4 ( 2 0 1 5 ) 1 1 5 e1 2 3

than in Ec, indicating that AP a more suitable candidate for Brockwell, J., Searle, S. D., Jeavons, A. C., & Waayers, M. (2005).
phytoremediation. Nitrogen fixation in acacias: An untapped resource for sustainable
plantations, farm forestry and land reclamation (ACIAR).
Brodribb, T., & Hill, R. S. (1993). A physiological comparison of
leaves and phyllodes in Acacia melanoxylon. Australian Journal
5. Conclusions of Botany, 41(3), 293e305.
Brooker, M. I. H., Slee, A. V., Connors, J. R., & Duffy, S. M. (2005).
The present study of abandoned mine site at a semi-arid CD-ROM review EUCLID, Eucalypts of Southern Australia.
climate provides insight into the ability of native trees to South African Journal of Botany, 71(1), 130e131.
interact with metals present in soil. Our data suggest that E. Cerqueira, B., Arenas-Lago, D., Andrade, M. L., & Vega, F. A. (2015).
Using time of flight secondary ion mass spectrometry and field
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Chenery, S. R., Izquierdo, M., Marzouk, E., Klinck, B., Palumbo-
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Martin O'Leary, (DEWNR, SA) for plant identification, and perspective-interactions affecting vulnerability. Environmental
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