the extent of arsenic and of metal uptake by aboveground ... · the extent of arsenic and of metal...
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The Extent of Arsenic and of Metal Uptake by AbovegroundTissues of Pteris vittata and Cyperus involucratus Growingin Copper- and Cobalt-Rich Tailings of the Zambian Copperbelt
Bohdan Krıbek • Martin Mihaljevic •
Ondra Sracek • Ilja Knesl • Vojtech Ettler •
Imasiku Nyambe
Received: 1 April 2010 / Accepted: 1 September 2010 / Published online: 15 October 2010
� Springer Science+Business Media, LLC 2010
Abstract The extent of arsenic (As) and metal accumula-
tion in fronds of the As hyperaccumulator Pteris vittata
(Chinese brake fern) and in leaves of Cyperus involucratus,
which grow on the surface of an old flotation tailings pond in
the Zambian Copperbelt province, was studied. The tailings
consist of two types of material with distinct chemical
composition: (1) reddish-brown tailings rich in As, iron (Fe),
and other metals, and (2) grey-green tailings with a lower
content of As, Fe, and other metals, apart from manganese
(Mn). P. vittata accumulates from 2350 to 5018 lg g-1 As
(total dry weight [dw]) in its fronds regardless of different
total and plant-available As concentrations in both types of
tailings. Concentrations of As in C. involucratus leaves are
much lower (0.24–30.3 lg g-1 dw). Contents of copper (Cu)
and cobalt (Co) in fronds of P. vittata (151–237 and
18–38 lg g-1 dw, respectively) and in leaves of C. invo-
lucratus (96–151 and 9–14 lg g-1 dw, respectively) are
high, whereas concentrations of other metals (Fe, Mn, and
zinc [Zn]) are low and comparable with contents of the given
metals in common plants. Despite great differences in metal
concentrations in the two types of deposited materials,
concentrations of most metals in plant tissues are very sim-
ilar. This indicates an exclusion or avoidance mechanism
operating when concentrations of the metals in substrate are
particularly high. The results of the investigation show that
Chinese brake fern is not only a hyperaccumulator of As but
has adapted itself to high concentrations of Cu and Co in
flotation tailings of the Zambian Copperbelt.
Although most metals and metalloids are toxic to plants, a
number of plants have been identified as metallophytes or
hyperaccumulators, which grow preferentially or exclu-
sively in soils containing high levels of toxic elements.
Hyperaccumulators (Salt et al. 1995; Brooks 1998; Boyd
2007) have attracted particular interest. Not only are they
able to grow in soils with high concentrations of toxic
elements, but they also accumulate these elements in their
stems and foliage in amounts that may be higher than the
levels lethal for other living organisms. This tolerance and
the capacity to accumulate metalloids and metals have
fueled the concept of remediating contaminated soils by
cultivating such plants and harvesting the aboveground
parts with their accumulated contaminants so that they can
be removed. This is the process of phytoremediation or,
more exactly, phytoextraction (Wenzel et al. 1999; Tu et al.
2002).
The recent discovery of an arsenic (As) hyperaccumu-
lator, the Chinese brake fern, Pteris vittata L., (Ma et al.
2001) has offered the hope that phytoextraction might be
B. Krıbek (&) � I. Knesl
Czech Geologic Survey, Klarov 3, 118 21 Praha 1,
Czech Republic
e-mail: [email protected]
M. Mihaljevic � V. Ettler
Institute of Geochemistry, Mineralogy and Mineral Resources,
Faculty of Science, Charles University, Albertov 6, 128 43 Praha
2, Czech Republic
O. Sracek
OPV s.r.o. (Protection of Groundwater Ltd.), Belohorska 31,
169 00 Praha 6, Czech Republic
O. Sracek
Department of Geology, Faculty of Science, Palacky University,
17. listopadu 12, 771 46 Olomouc, Czech Republic
I. Nyambe
Department of Geology, School of Mines, University of Zambia,
P.O. Box. 32 379, Lusaka, Zambia
123
Arch Environ Contam Toxicol (2011) 61:228–242
DOI 10.1007/s00244-010-9604-4
developed into an efficient, environmentally friendly, and
cost-effective technology for As decontamination (Tu et al.
2002). This fern can accumulate B22.6 g kg-1 As (dry
weight [dw]) in its fronds, that is, 2% As in the above-
ground biomass. The As in the aboveground tissues of this
fern is much higher than that found in most common plant
species (\10 mg kg-1; Matschullat 2000). The biocon-
centration factor (a ratio of frond As concentration to soil
As concentration) is[10 (Ma et al. 2001; Zhao et al. 2002).
Knowledge of the mechanisms of As uptake and
detoxification in Chinese brake fern may contribute to
optimization of phytoremediation processes (Clemens et al.
2002). Recent advances in understanding the mechanisms
of As absorption, translocation, and compartmentalization
within the vacuoles of P. vitatta cells have provided novel
insights into plant physiology and the molecular biology of
phytoremediation (Meharg and Hartley-Whitaker 2002;
Tripathi et al. 2007; Zhao et al. 2009; Xie et al. 2009).
Recently, many As hyperaccumulators have been iden-
tified in different regions of China, Southern Thailand,
Scotland, Australia and North America. The majority of
them are ferns of the genus Pteris (Meharg et al. 1994; Ma
et al. 2001; Chen et al. 2002a; Du et al. 2005; Srivastava
et al. 2006; Wang et al. 2007; Koller et al. 2007). Although
great progress in understanding the uptake, transport, and
detoxification of As in P. vittata has been made (Xie et al.
2009), data on the uptake of metals by this species, espe-
cially in heavily contaminated environment, are limited.
In the area of the Zambian Copperbelt, P. vittata grows
not only in contaminated soils but also in copper (Cu)- and
cobalt (Co)-rich tailings dumped in numerous tailings
ponds. Therefore, the main objectives of the current
investigation were as follows:
1. Collect information on the characteristics of the
tailings and conditions governing growth of P. vittata
in tailings deposited in the Copperbelt impoundments.
2. Determine the amount of As, Cu, Co, and other metals
in P. vittata relative to the contents of these metals in
two different types of tailings.
Although most studies have been focused on the mecha-
nism of As uptake by P. vittata, a few studies have directly
compared As uptake by this plant with As uptake by other
plants growing at the same sites. Therefore, the accumulation
of As and metals by P. vittata in Zambia was compared with
that by Cyperus involucratus Rottb. growing together with
P. vittata in tailings of the Copperbelt impoundments.
Site Description
Mining has shaped the landscape of the Zambian Copper-
belt during several centuries. The area hosts one of the
world’s highest-grade resources of Cu and Co ore and is
estimated to contain 34% and 10% of global Co and Cu
reserves, respectively (United States Geological Survey
[USGS] 2010). As a result, the Copperbelt supports one of
the highest densities of large-scale commercial mining
operations in the world (Krıbek et al. 2010). The Copper-
belt sediment-hosted strata-bound and stratiform deposits
are characterized by finely disseminated Cu, Co, and iron
(Fe) sulphides. The principal minerals are chalcopyrite,
Co-rich pyrite, and bornite ± carrolite. The host rocks
include quartzite (arkose), shale, and dolomite. The grades
average 3 wt% Cu and 0.18% Co in ore deposits from
which both metals are extracted. Trace amounts of gold
(Au), platinum (Pt), and silver (Ag) are recovered from the
Cu slimes during the refining process. Approximately 30
million metric tonnes of Cu metal have been produced
since large-scale mining operations began in 1930
(Kamona and Nyambe 2002). The extracted ore is pro-
cessed by flotation or by chemical treatment. The flotation
tailings are usually dumped in tailings ponds constructed
mostly in areas of wetland, locally called ‘‘dambos.’’ The
dambos are usually the source of streams and rivers. Such
systems are ubiquitous throughout the Copperbelt, forming
6–10% of the land surface (Mendelsohn 1961; Limpitlaw
2002). Active tailings ponds are usually entirely free of
vegetation. However, the surface areas of old tailings
ponds have gradually become partly or entirely colonised
by Cu- and Co-tolerant plants.
Field Survey
Numerous flotation tailings ponds can be found in the
Chambishi region of the Zambian Copperbelt (Fig. 1a, d).
One of these is an old tailings pond north of Chambishi
(‘‘Chambishi-North’’) and is described in this study. A field
survey on the old Chambishi-North pond (E 28�01034.000 to
28�01053.200, S 12�37032.700 to 12�37035.500, total area
352.5 ha), located approximately 1 km west of Chambishi
town in the Copperbelt province of Zambia, was under-
taken during June to July in 2002, 2008, and 2009. Based
on the results of this survey, the area of old impoundment
can be subdivided into four vegetation zones (Fig. 1d) as
follows:
1. Reed swamp zone: The central part of the tailings pond
around the lagoon is dominated by dense growth cover
of Phragmites mauritianus Kunth, but Typha doming-
ensis Pers. is a common associate. Individual plants of
Phragnites and Typha reach almost 2 m in height. In
some places, Phragmites and Typha monoculture is
fringed by stands of Eleocharis dulcis (Burm. F.)
Hensch.
Arch Environ Contam Toxicol (2011) 61:228–242 229
123
2. Tailings essentially without vegetation: A great part of
the tailings impoundment, between the reed swamp
and peripheral papyrus and fern zone, is essentially
free of vegetation apart from some isolated stands of
P. mauritianus. Individual plants of Phragmites reach up
to 1 m in height. This zone is fully exposed to sunlight.
3. Papyrus and fern zone: The peripheral zone of the
tailings pond is dominated by stands of C. involucratus
and P. vittata together with minor P. mauritianus
and T. domingensis. In some places, clusters of
C. involucratuss and P. vittata give way to patches
that are entirely free of vegetation. The surface of the
tailings in this zone is partly covered with efflores-
cences of gypsum, which indicate strong capillary
action of groundwater during the dry season. On many
stands, P. vittata is associated with the vigorous
perennial sedge Bulbostylis pseudoperenis Goetgh.
Toward the banks of the impoundment, the vegeta-
tion cover becomes denser, and C. involucratus and
P. vittata form thickets together with other common
species, such as C. imbricatus Retz., E. dulcis,
Lagarosophon ilicifolius Oberm. and Kotschya Afri-
cana Endl. Dense vegetation cover is usually clustered
around extensive stands of C. papyrus L., which partly
overshadows this zone. Individual plants of C. papyrus
reach up to 3 m in height.
4. Mixed semideciduous tree forest zone: The transition
from the papyrus and fern zone of the peripheral parts
of the tailings impoundment to the mixed semidecid-
uous tree forest zone at the banks of the impoundment
is gradual. The fern- and papyrus-dominated vegeta-
tion is within a distance of 5 m replaced with Acacias
COPPERBELT
CONGO DR
ZAMBIA
Chililabombwe
Chingola
Chambishi
Kalulushi
Kitwe
Mufulira
Luanshya
Ndola
Kafue river
Kafue river
50 km
Old tailings pondChambishi-North
South Africa
Lesotho
Swaziland
BotswanaNamibia
Zimbabwe
Zambia Malawi
Angola
DR CongoTanzania
Chambishi MineComplex
CHAMBISHI
0 km 10 km
Kafue
Old tailings pondChambishi-North
New tailings pondChambishi-North
Musakashi
Cham
beshi
Lukashi
New tailingspond CambishiSouth (TD6)
Chambishi-Southwetland
1 km
N
Old tailings pondChambishi-North
Lagoon
12
Dischargepoint
Reed swamp Tailings without vegetation
Papyrus and fern swamp
Tree mixed semi-deciduous forest
Road
plot1 Sampling
A
C D
B
Fig. 1 Position of Zambian Copperbelt within Central Africa (a),
synoptic map of the Copperbelt (b), lay of the old Chambishi-North
and other tailings ponds in the Chambishi region (c), and distribution
of vegetation zones with sampling sites within the old Chambishi-
North tailings pond (d)
230 Arch Environ Contam Toxicol (2011) 61:228–242
123
and shrubby species. The most common species
include Acacia polyacantha Willd. and A. sieberana
DC together with Albizia versicolor Welw. Ex Oliv.
and Manilkara obovata (Sabine & G.Don) J. H. Hemsl.
Among the most important shrubby species are
Craterispermum laurinum (Poir.) Benth., Garcinia
smeathmannii (Planch. & Triana) Oliv. and Gardenia
imperialis K. Schum. Within the forest zone, P. vittata
locally forms dense stands in shady places spoiled by
escapes of the slurry from pipelines used in the past for
transporting tailings material to the impoundment.
Four sampling plots of 1 m 9 1 m were located in the
papyrus and fern zone (Fig. 1d). For each plot, tailings
samples were taken randomly from the top 20 cm to deter-
mine their physical and chemical properties. At least three
individuals of selected plants, together with tailings samples
taken closely to the root system, were collected from indi-
vidual plots. Moreover, groundwater was collected from a
shallow trench (30-cm deep) excavated in the central part of
the papyrus and fern zone (sampling plot 1; Fig. 1d).
Water, Tailings, and Plant Analysis
A sample of groundwater was taken from a shallow trench
excavated in the papyrus and fern zone using a syringe
equipped with a disposable microfilter (pore size 0.45 lm).
After stabilization of pH, the water sample was split into
one subsample acidified with ultrapure HCl for determi-
nation of cations and metals plus a second unacidified
subsample. The alkalinity of the unacidified subsample was
determined by titration with HCl in a field laboratory using
a Gran plot to determine the end point. Cations in the
acidified water subsample were determined using induc-
tively coupled plasma–mass spectrometry in the Acme
Analytic Laboratories, Vancouver, Canada. Concentrations
of anions in the unacidified water subsample were deter-
mined using a high-pressure Shimadzu LC6A liquid
chromatograph in the accredited Central Laboratories of
the Czech Geologic Survey in Prague.
Tailings samples were air-dried, and after homogeniza-
tion, half of each sample was passed through a 2-mm mesh
screen using a USGS sieving set and pulverized in an agate
ball mill to \0.063 mm mesh. To determine the total
content of metals, tailings samples were digested with aqua
regia in accordance with the ISO 11466 procedure (Inter-
national Organization for Standardization 1995). For the
determination of plant-available metals, samples were
extracted with a solution of diethylentriaminepentaacetic
acid (DTPA) and triethanolamine (TEA) according to the
ISO/DIS 14870 method (International Organization for
Standardization 2001).
All reagents were declared pro analysi, and all solutions
were prepared with double-distilled water. Standard
working solutions were prepared from original certified
stock solutions (MERCK) concentration 1000 mg of
chemical element l-1 in 1% super pure HNO3. Co, Cu, Fe,
Mn, and Zn were determined using flame atomic absorp-
tion spectroscopy (FAAS; Perkin Elmer 4000 spectrome-
ter). As was determined using hydride-generation atomic
absorption spectrometry (HGAAS; Perkin Elmer 503).
All tailings samples were analyzed at the accredited
Central Laboratories of the Czech Geologic Survey. The
quality-control procedure involved analysis of reagent
blanks, duplicate samples, and several reference materials
(RMs). Analytic precision was determined by duplicate
analysis of 10% of randomly chosen samples and of ref-
erence samples as well. The coefficient of variation for all
investigated elements was \8%. Reliability of analyses
determined by RMs was ± 5% for Co, Cu, and Zn; ± 12%
for Fe and Mn; and ± 10% for As.
Sequential extraction for selected samples was per-
formed using the BCR procedure (Rauret et al. 1999) The
following sequence of steps was used: a 0.11 M acetic acid
(CH3COOH) step targeting exchangeable- and acid-soluble
fraction; a 0.5 M hydroxylamine–chloride (NH2OH�HCl)
step targeting reducible fraction (mostly poorly crystalline
Fe/Mn oxides); an oxidisable step (8.8 M H2O2/1 M
CH3COONH4 extractable) targeting organic matter and
sulphides; and an Aqua Regia step targeting the residual
fraction. The full details of the experimental procedure are
given in Rauret et al. (1999).
The amount of total carbon (Ctot) was determined using
an ELTRA CS 500 instrument. Samples were combusted at
1400�C, and Ctot was measured as carbon dioxide using an
infrared (IR) detector. The amount of carbonate carbon
(Ccarb) was established using another ELTRA CS 500
instrument. Samples were digested in a saturated solution
of H3PO4, and the amount of CO2 liberated was recalcu-
lated to that of Ccarb. The amount of organic carbon (Corg)
was determined by subtraction of Ccarb from Ctot content,
i.e., Corg = Ctot - Ccarb. Total sulphur (Stot) was deter-
mined using the ELTRA CS 500 equipment. Samples were
combusted at a temperature of 1400�C, and the Stot, mea-
sured as released SO2, was determined by an IR detector.
The variation coefficient for Ctot and Ccarb is \0.5%, and
for Stot it is \1%. Relative errors of Ctot, Ccarb, and Stot
determined using reference materials were ±2.5% for Ctot
and Stot and ±2% for Ccarb.
To establish the pH value of tailings leachates, 2.5 g
material, sieved through 2-mm sieve mesh, were leached in
a periodically shaken solution of 1 M KCl. The pH mea-
surements were made with a precision of 0.01 pH unit
using a pHC 2085 pH electrode connected to a PHM 201
pH meter after 24 h of leaching. Differences in water
Arch Environ Contam Toxicol (2011) 61:228–242 231
123
temperature were automatically compensated using a T 201
temperature compensator. Calibrations were performed
using two standard IUPAC (Radiometer A/S, Copenhagen,
Denmark) buffers with pH values of 4.01 and 7.00,
respectively. The measured pH value was recorded auto-
matically with a precision of 0.01 pH unit.
Fresh plant materials (200-g weight) were washed
thoroughly with tap water, cleaned with distilled water, and
then separated into roots and shoots. All plant parts were
then oven-dried (65�C for 48 h). The plant tissues were
then cut into small pieces and pulverized in an agate ball
mill to \0.063 mm mesh. Approximately 1-g aliquots of
the plant samples were burned down in a muffle oven.
A temperature programme from 25�C to 550�C was
employed, with a temperature increase of 2�C/min, and
then the temperature was kept at 550�C for 2 h. The
amount of resulting ash was weighed, and the metals were
analytically determined in HNO3 and HCl at 5:1 (v/v)
leachate. As and metals were analysed in leachate as
described previously. Due to the requirements for precision
in As and metals analyses, standard RMs (SRM 1575a pine
needles and SRM 1515 apple leaves) were used for plant
samples. Relative errors determined using reference
materials were ±9.9% for Cu, ±10.8% for Fe, ±7.3% for
Mn, and ±11.4% for Zn. The relative error for As was
higher (±28%) due to a low concentration of this element
in SRMs (0.038 and 0.039 lg g-1, respectively).
Because of the high volatility of As during combustion
of plant samples, selected samples of plants were there-
fore analyzed without combustion using x-ray fluores-
cence (XRF) spectrometry (XRFARL 9400 ADVANTXP;
Applied Research Laboratories, Switzerland). The results
of both methods, i.e., the analyses of burnt samples using
the FAAS method and those of unburnt samples using the
XRF method showed a significant correlation (r = 0.994)
on the probability level of p \ 0.01. However, As con-
centrations established by the XRF method turned out to be
on average 15% higher than values obtained by FAAS
method in plants ash, especially in case of low As con-
centrations in plant material (\1 lg g-1 dw).
The accumulation of Fe and aluminium (Al) by plant
roots in Chambishi-North was studied using an optical
microscope and CamScan 3200 electron microprobe in
scanning electron microscopy mode equipped with an
energy-dispersive analyzer LINK-ISIS.
Statistical Methods
The tailings at Chambishi-North consist of reddish-brown
and grey-green material. Seven samples of P. vittata fronds
and five samples of C. involucratus leaves were collected
from reddish-brown tailings, and five samples of P. vittata
and six samples of C. involucratus were collected from
grey-green tailings. Data are presented as arithmetic
mean ± SD for plants growing in reddish-brown and grey-
green tailings. Values were calculated using S-Plus
programme version 4.5 (MathSoft, Seattle, WA). Further
statistical analysis was not deemed appropriate due to the
small number of samples. The bioaccumulation factor (BF)
is defined as the ratio of As or metal concentration in
shoots (total dw) to that in tailings, which is a measure of
the ability of a plant to take up and transport metals to the
foliage (Caille et al. 2005). The translocation factor, indi-
cating preferential partitioning of metals and defined as the
metal concentration in the plant foliage divided by that in
the roots (McGrath and Zhao 2003), was not calculated
because it was not possible to separate completely the roots
of the plants from the Fe- and Al-rich particles of tailings.
Results
Groundwater
The groundwater sampled in the old Chambishi-North tail-
ings pond has a pH of 7.16 and high specific conductivity
(1860 lS cm-1). The dominant cation is Ca (654.7 mg l-1),
but the concentrations of Mg (99.8 mg l-1) and potassium
(51.7 mg l-1) are also significant. Sulphate (2149 mg l-1) is
the principal anion; the concentration of bicarbonate is much
lower (393.12 mg l-1); and the chlorine concentration
is negligible. Concentrations of Fe (60 lg l-1) and Al
(20 lg l-1) are low compared with Mn (2085 lg l-1).
Respective concentrations of Cu and Co are 687 and
637 lg l-1, respectively. The concentration of As is
54 lg l-1 and that of Zn is 22 lg l-1.
Tailings
The tailings consist of two types of material with distinct
chemical compositions: (1) reddish-brown tailings rich in
total As, Fe, and other metals, and (2) light grey-green
tailings with lower contents of total As, Fe, and other
metals, apart from Mn (Table 1).
Reddish-brown tailings occupy most of the fern and
papyrus zone, whereas grey-green tailings occur mostly in
the central part of the tailings pond (i.e., in the reed swamp
zone) close to the central lagoon. In many places, however,
grey-green tailings form a layer up to 0.5 m thick, which
overlies reddish-brown tailings, especially in the fern and
papyrus vegetation zone. The pH of the tailings leachate
ranges from near-neutral to slightly alkaline, with an
average pH of 8.19 in reddish-brown and 7.23 in light grey-
green tailings. The mean concentration of Corg and Ccarb
232 Arch Environ Contam Toxicol (2011) 61:228–242
123
are higher in reddish-brown (0.29% and 1.33%, respec-
tively) compared with grey-green tailings (0.04% and
0.75%, respectively). The mean concentration of Stot is
much higher in reddish-brown (0.91%) than in light grey-
green tailings (0.25%).
Concentrations of plant-available As and metals were
determined by extraction with DTPA and TEA (Table 1).
The amounts of plant-available As and metals (expressed
as % of the total concentration of As and metals in the
tailings) show roughly similar trends in both tailings types
(Fig. 2). For reddish-brown tailings, the amount of plant-
available As and metals increases in the following
order: Fe (0.06%), ? As (1.53%), ? Cu (3.62%), ? Co
(4.36%) ? Mn (6.72%), ? Zn (14.81%). In grey-green
tailings, the amount of plant-available As and metals is
slightly different but increases in a similar sequence:
Fe ? (0.61%), ? As (1.15%), ? Co (1.84%), ? Cu
(3.35%), ? Mn (4.41%), ? Zn (5.34%).
It is pointed out that the methods of determination
of plant-available metals extracted with DTPA, as well
as with other similar chelating agents, were developed
for establishment of cation forms of metals. Therefore,
for better evaluation of the plant-availability of As, a
sequential analysis was used, particularly its first step
consisting of extraction of samples by 0.11 M acetic acid.
The results of sequential analyses of reddish-brown and
Table 1 Average concentration (mean value) of Ccarb, Corg, and Stot (all in wt%), total (T), and plant-available (PA) As and metals (in lg g-1)
and average pH value of reddish-brown and grey-green tailings at the old Chambishi-North impoundment, Zambia
Element Reddish-brown tailings (n = 12) Grey-green tailings (n = 14)
Mean SD Max Min Mean SD Max Min
pHleachate 8.19 0.16 8.23 7.95 7.23 0.07 7.32 7.12
Corg 0.29 1.18 0.01 0.64 0.04 0.01 0.04 0.02
Ccarb 1.33 0.41 2.32 1.07 0.75 0.03 0.44 0.08
Stot 0.91 0.55 0.30 1.92 0.25 0.16 0.44 0.06
As
T 175.26 17.18 211.08 156.86 18.81 9.36 43.33 9.21
PA 2.68 0.68 3.79 1.67 0.28 0.32 0.79 9.07
Co
T 3088 1881 8710 1690 272 182 663 148
PA 132 73 328 67 7 9 25 1
Cu
T 11,294 2174 14,200 7900 1728 633 2790 750
PA 738 253 1005 94 393 125 530 200
Fe
T 253,500 20,851 280,000 200,000 28,975 19,801 71,500 15,400
PA 141 38 207 89 121 24 148 80
Mn
T 268 169 810 130 0.018 0.005 0.027 0.01
PA 16 4 20 12 1066 181 1290 680
Zn
T 57 49 209 32 47 15 65 20
PA 8 8 29 2 40 3 47 35
n Number of analysed samples
As Co Cu Fe Mn Zn
20
15
10
5
0
Chemical element
Pla
nt-a
vaila
ble
amou
nt o
f ele
men
ts
(in %
of t
otal
con
cent
ratio
n)
Fig. 2 Average concentration (mean value) of plant-available
(DTPA- and TEA-extracted) As and metals in reddish-brown tailings
(white column, n = 11) and in grey-green tailings (black column,
n = 9) in the Chambishi-North pond in % average total concentration
of elements in tailings. The bar on top of the column is the SD
Arch Environ Contam Toxicol (2011) 61:228–242 233
123
grey-green tailings (Fig. 3) generally correspond with the
results obtained from the determination of the amount of
DTPA- and TEA-extractable elements.
Almost all Fe ([95% of the total content of Fe in the
analysed samples) is retained in the residual fraction.
Together with Fe, most of the As (60–65% of the total As
content of analysed samples) and Zn (55–60%), as well as
high amounts of Co (40–55%), Cu (30–50%), and Mn
(30–40%), are confined to the residual fraction.
The content of metals in the oxidizable fraction, i.e., the
amount of metals confined to sulphides and organic matter
is relatively small (\3%), with the exception of Cu
(20–45%). Particularly As (20–25%), Co and Cu (15–20%)
and, to a lesser extent, Zn (10–15%) are confined to the
reducible fraction, i.e., to poorly crystallized oxides of Fe
and Mn. Relatively high contents of Mn (40–65%) and Co
and Zn (25–35%) are released into the acid-extractable
fraction. Contents of As and Cu in this fraction are lower
(10–20%), whereas contents of Fe are negligible (\2%).
Elements retained in acid-extractable fraction are believed
to be confined to carbonates, absorbed on the surface of
minerals by ion-exchange processes or present in the form
of water-soluble mineral species. The amount of As in
acid-extractable fraction of the sequential analysis is higher
(10–15% of total As) compared with the amount of plant-
bioavailable As determined by extraction with DTPA and
TEA (1–1.5% of total As) because of a weak capacity of
chelating agents used to extract anions.
Arsenic and Metals in Plants
The metal concentrations in fronds of P. vitatta and in
leaves of C. involucratus were investigated in plants
growing in reddish-brown as well as in grey-green tailings.
The results of this study and the BF values versus tailing
types are listed in Tables 2 and 3. Mean concentrations
of As in P. vittata fronds were found to be very high in
samples collected in reddish-brown tailings (mean
3390 lg g-1 dw; Table 2) and even higher in samples
collected in grey-green tailings (mean 5534 lg g-1 dw,
Table 3 and Fig. 4a). Because of the much higher con-
centrations of As in reddish-brown tailings, the BF of
samples collected in this type of tailings is substantially
lower (BF = 18; Table 2 and Fig. 4b) compared with that
of samples taken from grey-green tailings (BF = 184),
regardless of very similar concentrations of As in plants.
This indicates that the BF value does not necessarily reflect
the capacity of plants to accumulate As (and metals) from
substrates. In case of the Chambisi-North tailings, the
much higher BF of fern growing in grey-green tailings does
not indicate higher capacity of As bioaccumulation but
simply reflects lower concentration of As in such tailings.
Concentrations of metals in fronds of ferns growing in
both types of tailings decrease in the following sequence:
Fe ? Cu ? Mn ? Co, Zn, and the BFs decrease in the
following order: Zn, ? Mn, ? Cu, ? Co, ? Fe. With
an exception of Mn, the BFs of all metals are higher in
ferns growing in grey-green compared with those growing
in reddish-brown tailings.
Compared with P. vittata, the concentration of As in
samples of C. involucratus collected from the same sam-
pling plots is substantially lower. However, concentration
of As in C. involucratus, as well as the BF value, is one
order higher in the foliage of specimens growing in red-
dish-brown tailings (mean 25.3 lg g-1 dw, BF = 0.20)
compared with specimens growing in grey-green tailings
(mean 0.46 lg g-1, BF = 0.04).
The mean concentration of metals in the foliage of
C. involucratus decrease in the following order: Fe ?Cu ? Mn ? Co, Zn. Compared with P. vittata, concen-
trations of metals are slightly lower, with the exception of
Mn. The BFs decrease in the following order: Zn, ? Cu,
Co ? Fe. The BF for Cu is one order higher in the foliage
of plants growing in metal-poor tailings (BF = 0.11)
compared with those growing in metal-rich tailings
(BF = 0.01). Like in the case of As, this difference does
not reflect a different capacity of the plant to accumulate
0%
20%
40%
60%
80%
100%
Dis
trib
uti
on
(in
% o
f to
tal a
mo
un
t)D
istr
ibu
tio
n (
in %
of
tota
l am
ou
nt)
Co Cu Zn As Mn FeChemical element
Residual Oxidizable Reductible Acid extractable
Residual Oxidizable Reductible Acid extractable
0%
20%
40%
60%
80%
100%
Co Cu Zn As Mn Fe
Chemical element
A
B
Fig. 3 Distribution of As and metals extracted in individual steps of
sequential analysis of reddish-brown (a) and grey-green tailings (b) in
the old Chambishi-North tailings pond in Zambia
234 Arch Environ Contam Toxicol (2011) 61:228–242
123
Cu but simply the different Cu concentrations in deposited
tailings.
Discussion
Chemical Composition of Groundwater in Relation
to the Concentration of As and Metals in Plants
The pH value and concentrations of Ca and Mg in
groundwater collected from a shallow trench (30-cm depth)
in the old Chambishi-North tailings pond correspond with
data by Sracek et al. (2010), who reported pH value of 7.5
and concentrations of Ca and Mg corresponding to 568 and
67 mg l-1, respectively, in a groundwater sample collected
at the old Chambishi-North tailings pond from a depth of
approximately 3.0 m. In contrast, concentrations of bicar-
bonate and sulphate in our groundwater sample (SO4
2149 mg l-1, HCO3 393 mg l-1) are higher compared with
data reported by Sracek et al. in 2010 (SO4 1820 mg l-1;
HCO3 71.4 mg l-1), presumably due to evaporation of
groundwater in the shallow part of tailings. High concen-
tration of sulphate in groundwater from the shallow part of
Table 2 Average total concentration of chemical elements in aboveground tissues of plant species (in lg g-1 dw, mean value ± SD) in their
associated reddish-brown tailings (in lg g-1) and BF values
Element P. vittata (n = 7) C. involucratus (n = 5)
Frond ± SD Tailings ± SD BF Leaf ± SD Tailings ± SD BF
As 3389.9 ± 885.8 180.41 ± 18.2 19.28 25.3 ± 3.6 168.0 ± 9.3 0.20
Co 28.2 ± 7.2 3706.11 ± 2257.3 0.01 13.3 ± 1.1 2222.5 ± 285.3 0.01
Cu 164.1 ± 39.3 12287.11 ± 2210.4 0.01 112.2 ± 14.3 9902.3 ± 1 089.3 0.01
Fe 663.0 ± 43.2 250571.01 ± 24070.0 0.003 144.2 ± 112.6 257600.0 ± 14 264.0 0.002
Mn 35.2 ± 9.1 321.31 ± 202.1 0.14 43.3 ± 1.3 193.1 ± 36.2 0.22
Zn 20.2 ± 5.3 75.41 ± 59.4 0.39 10.1 ± 2.3 33.0 ± 1.0 0.32
n Number of analysed plants and tailings samples
Table 3 Concentrations of chemical elements in aboveground tissues of plant species (in lg g-1 dw, mean value ± SD) in their associated
grey-green tailings (in lg g-1) and BF values
Element P. vittata (n = 4) C. involucratus (n = 4)
Shoot ± SD Tailings ± SD BF Shoot ± SD Tailings ± SD BF
As 5533.8 ± 532.6 32.35 ± 7.5 184.45 0.46 ± 0.18 12.45 ± 3.2 0.04
Co 28.1 ± 3.2 551 ± 88.2 0.05 9.0 ± 1.1 163.2 ± 6.0 0.02
Cu 216.3 ± 10.3 2560 ± 395.2 0.01 112.3 ± 23.2 1114.3 ± 342.4 0.11
Fe 546.2 ± 31.1 59000.0 ± 10254.0 0.009 253.1 ± 35.2 15967.2 ± 475.3 0.016
Mn 45.2 ± 1.3 810.1 ± 125.2 0.06 41.2 ± 1.1 1135.2 ± 79.3 0.04
Zn 28.1 ± 4.1 42.2 ± 1.1 0.67 12.1 ± 4.0 42.3 ± 4.3 0.29
n Number of analysed plants and tailing samples
CuCoAs0.001
0.01
0.1
1
10
100
1000
Chemical element
Bio
accu
mul
atio
n fa
ctor
(B
F)
ZnMnFe
As Co Cu
10000
1000
100
10
1
0.1
Chemical element
Con
cent
ratio
n, p
lant
(uµ
g.g-1
DW
)
ZnMnFe
natattiv.P , reddish-brown tailings ( = 7) P. vittata n, grey-green tailings ( = 5)
nsutarculovnI.C , reddish-brown tailings ( =5) C. Involucratus n, grey-green tailings ( = 6)
Fig. 4 Average concentration (mean value) of As and metals in
P. vittata fronds and C. involucratus leaves (a) and mean BF values
(b) for both plants growing in reddish-brown and grey-green tailings
in the Chambishi-North pond. The bar on top of the column is the SD.
n = Number of analysed samples
Arch Environ Contam Toxicol (2011) 61:228–242 235
123
the deposited material at the old Chambishi-North tailings
pond resulted in formation of gypsum on the surface of
tailings. The concentration of dissolved manganese (Mn),
Cu, and Co (2085, 687, and 637 lg l-1, respectively) in
groundwater sample collected from a shallow trench is
high, whereas the contents of Al Fe, Zn, and As (20, 60, 22,
and 54 lg l-1, respectively) are lower. The contents of As
and metals in groundwater are believed to vary over the
whole of the tailings pond being governed by precipitation
of respective mineral phases, by their coprecipitation, and
by cations exchange processes or by chemisorption,
depending on physical properties of either type of dumped
materials. However, it is obvious that the accumulation
ratio (AR) for As for P. vittata calculated using the fol-
lowing equation:
AR
¼ Arsenic ðmetalsÞconcentration inplant ðmg kg�1; DWÞArsenic ðmetals) concentration ingroundwater ðmg 1�1Þ;
which is a few orders of magnitude higher compared with
C. involucratus (Table 4). The AR for Fe appears relatively
high due to its low concentration in groundwater. As
concerns the other elements, the AR values are of the same
order of magnitude, which indicates their passive uptake by
both plants.
Chemical Composition, Mineralogy, and Origin
of Tailings
The results of chemical analyses proved two types of
material to have been deposited in the old Chambishi-
North tailings pond. The reddish-brown tailings are rich in
hematite and/or poorly crystalline Fe(III) phases and gyp-
sum (Sracek et al. 2010), which corresponds to high con-
centration of total Fe (mean 25 wt%) and Stot (mean
1.3 wt%; Table 1). A small part of sulphur is confined to
residual sulphides (chalcopyrite, bornite, and pyrite; Sracek
et al. 2010). High concentrations of total Cu and Co reflect
geochemical composition of extracted ore, and high
amount of Ccarb (mean 1.33%) indicates an occurrence of
carbonates (calcite). The reddish-brown tailings were most
likely produced by in situ deep tropical weathering of
flotation wastes initially deposited in the tailings pond.
In contrast, the grey-green tailings exhibit lower content
of total Fe (2.8 wt%), Stot (0.25 wt%), and Ccarb
(0.04 wt%; Table 1). Compared with reddish-brown tail-
ings, they show higher contents of quartz and aluminosil-
icates (muscovite, amphibole, and orthoclase; Sracek et al.
2010) and are poor in sulphides. Consequently, the con-
centrations of total As, Cu, Co, and other metals are gen-
erally low. The only exception is a high concentration of
total Mn (mean 1066 mg g-1), which is likely to be linked
with carbonates or with poorly crystallised Mn oxides.
Nevertheless, the origin of grey-green tailings is not evi-
dent. It can only be assumed that these tailings represent a
product of redeposition and deferrification of reddish-
brown tailings under reducing conditions during periods of
inundation of the tailings pond during rainy seasons. An
increase in thickness of grey-green tailings from the
peripheral to central part of the pond with intermittent
lagoon supports this hypothesis.
Table 4 Accumulation ratio (average total concentration of elements in plant, mean value, mg kg-1 dw)/concentration of elements in
groundwater [mg l-1]) for P. vittata and C. involucratus growing in reddish-brown and grey-green tailings at Chambishi-North pond
Element P. vittata reddish-brown
tailings (n = 7)
P. vittata grey-green
tailings (n = 5)
C. ivolucratus reddish-brown
tailings (n = 5)
C. involucratus grey-green
tailings (n = 6)
AR.103
As 6275.9 10246.3 468.5 0.85
Co 0.04 0.04 0.02 0.01
Cu 0.24 0.31 0.24 0.16
Fe 11.1 9.11 2.41 4.21
Mn 0.051 0.022 0.021 0.019
Zn 0.92 1.31 0.46 0.55
n Number of analysed plant samples
Table 5 Correlation coefficients (r) between total and plant-available
(extraction with DTPA and TEA) concentration of elements in tail-
ings from the old Chambishi-North impoundment in Zambia
Element Reddish-brown
tailings (n = 12)
Grey-green
tailings (n = 14)
Astotal–Asplant-available 0.54 0.86
Cototal–Coplant-available 0.95 0.98
Cutotal–Cuplant-available 0.92 0.93
Fetotal–Feplant-available 0.37 –0.86
Mntotal–Mnplant-available 0.92 0.86
Zntotal–Znplant-available 0.87 0.65
Significant correlations on the probability level p \ 0.05 (95%) are
printed in bold
n Number of samples
236 Arch Environ Contam Toxicol (2011) 61:228–242
123
The concentrations of plant-available As, Co, Cu, Mn,
and Zn, as established by extraction with DTPA and TEA,
show significant correlation with total contents of the given
elements in both types of tailings (Table 5). Therefore, the
concentrations of plant-available As, Co, Cu, and Mn
increase proportionally with an increase of their total
concentrations in tailings. Fe is the only metal for which
the value of correlation coefficient is low (Table 5), indi-
cating extremely low plant-availability of this element
regardless of the total amount of Fe in tailings.
Results of the determination of plant-available elements
correspond with the results of sequential analyses, in which
almost all Fe ([95% of the overall content of Fe in ana-
lysed samples) is retained in the residual fraction. The
distribution of As and metals in individual fractions of
sequential analysis (Fig. 3) is similar in both reddish-
brown and grey-green tailings, which indicates that all of
the analysed elements are present in the same chemical
forms in either type of the substrate. The only exception is
Cu, which is more abundant in the oxidizable fraction of
reddish-brown tailings because of a higher amount of
sulphides and organic matter compared with grey-green
tailings. The most important thing, from the point of view
of plant availability, is the amount of chemical elements in
the acid-extractable fraction. However, the applied method
of sequential analysis does not allow us to distinguish to
what extent the chemical elements are confined to the
surface of individual mineral phases by ion-exchange
processes or to what degree they are confined to or con-
tained as isomorphic admixture in carbonates within the
given (acid-extractable) fraction. This particularly applies
to Mn, of which the contents in carbonates may be high. In
contrast, large amount of metals can be absorbed on the
surface of amorphous Fe- and/or Mn-hydrated oxides.
Metal affinity to amorphous Fe oxides in general increases
in the following range: Cu [ Zn [ Co [ Mn (McKenzie
1980), which corresponds to higher amount of Mn, Co, and
Zn and lower amount of Cu in acid-extractable fraction of
sequential analysis of tailings. At higher values of pH, even
anions such as AsO33- can be absorbed on the surface of
amorphous Fe oxides or hydroxides due to negatively
charged sites, which begin to prevail on the surface of the
solid phase (Drever 1997). For example, Otte et al. (1995)
reported high amount of As to be retained on Fe plaques on
the surface of wetland plant roots.
As in Plants and Relation to Tailings
Concentrations of As in fronds of P. vittata growing on the
surface of the old Chambishi-North tailings pond
(2350–6200 lg g-1) are comparable with or slightly lower
than those detected in the same plant by other investigators
(3000–22,000 lg g-1 dw; Matschullat 2000; Cao et al.
2004; Lombi et al. 2002; Ma et al. 2001; Tu and Ma 2003).
Despite much higher contents of total and plant-available
As in reddish-brown compared with grey-green tailings
at Chambisi-North, concentrations of As in fronds of
P. vittata growing in both tailings types are similar or even
higher in plants growing in grey-green tailings (Fig. 5a).
Many investigators have reported that contents of As in
fronds of P. vittata increase with the increase of As in the
substrate (for survey see Xie et al. 2009). However,
Gumaelius et al. (2004) concluded that the uptake of As in
gametophytes of P. vittata follows a linear relation at lower
concentrations of As in substrate and then levels off at high
concentrations. This indicates an exclusion or avoidance
mechanism operating at high concentrations of As in sub-
strate. The surface layer of both types of tailings at
Chambishi-North is deposited in the aerobic environment
as indicated by the occurrence of hematite, poorly crys-
tallized Fe(III) oxides, and gypsum. In an aerobic envi-
ronment, As occurs in its oxidized form, i.e., arsenate, and
has been reported to be taken by plants by way of phos-
phate-transport systems (Meharg & Hartley-Whitaker
2002; Wang et al. 2002). However, in fronds of P. vittata,
arsenite is the main storage form of As (Lombi et al. 2002;
Wang et al. 2002) and is mainly confined to the vacuoles
(Lombi et al. 2002). Zhao et al. (2002) assumed that
arsenate in P. vittata is likely to be reduced to arsenite in
roots because there was almost no competition between As
and phosphate during transport from roots to fronds. With
the information reported so far, it is expected that arsenate
is taken up by way of phosphate transporters and is then
reduced to arsenites in roots and then transported to fronds
by arsenite transporters. Experiments performed by Duan
et al. (2005) showed that like in other plants, the reaction
rate of arsenate reductase (AsR) in roots of P. vittata
decreased at high concentrations of arsenate ([20 mM) in
substrate. Therefore, it may be speculated that relatively
high arsenate concentrations in reddish-brown tailings
might inhibit the AsR reaction rate and subsequently inhibit
the rate of transport of arsenate by way of arsenate trans-
porter to fronds. The same investigators (Duan et al. 2005)
reported that the activity of AsR showed an optimum pH of
approximately 6.5 and sharply decreases at alkaline pH.
Therefore, the pH of reddish-brown tailings (pH = 8.19)
compared with near-neutral pH of greenish-grey tailings
(pH = 7.23) can decrease the AsR reaction rate. In addition
to internal mechanisms that may decrease translocation of
As from roots to aboveground tissues of plants, the chem-
istry of the substrate may be an important control of As
uptake. Liao et al. (2003), for example, reported that high
contents of Ca2? in the growth media negatively affect As
translocation in P. vittata. Therefore, higher concentrations
of Ccarb in reddish-brown tailings (mean 1.33 wt%), com-
pared with grey-green tailings (mean 0.75 wt%), may be one
Arch Environ Contam Toxicol (2011) 61:228–242 237
123
of the external factors that efficiently decrease the uptake of
As by plants growing in reddish-brown substrate. Previous
experiments have shown (Meharg and Macnair 1990, 1991,
1992; Meharg et al. 1994; Chen et al. 2002b; Meharg and
Hartley-Whitaker 2002; Wang et al. 2002; Huang et al.
2007) that arsenate competes with phosphate for uptake in
plants and that increased external phosphate leads to
decreased As in P. vittata. Our preliminary data indicate that
the concentration of available phosphorus (P) in reddish-
brown tailings (27–36 mg kg-1) is much higher than in
grey-green tailings (1–2 mg kg-1). This corresponds with
much higher content of Corg in reddish-brown tailings
compared with grey-green tailings (Table 1). Therefore, the
higher content of available P in reddish-brown tailings may
inhibit the uptake of As by plants growing in reddish-brown
tailings, whereas lower P content may increase its uptake by
plants growing in grey-green tailings.
Compared with P. vittata, concentrations of As in
C. involucratus leaves are much lower and differ in plants
growing in reddish-brown (21–30 lg g-1 dw) and grey-
green (0.2–0.6 lg g-1 dw) tailings as it is characteristic of
common plants in which uptake and translocation of As
reflect As concentrations in the substrate (Pickering et al.
2000; Meharg and Hartley-Whitaker 2002).
Metals in Plants and Relation to Tailings
Concentrations of Cu and Co in P. vittata and C. invo-
lucratus growing in the Chambishi-North tailings pond are
higher, whereas concentrations of other studied metals are
10000
1000
100
10
1
0.11 10 100 1000
40
35
3025
2015
10
50
0 1000 2000 3000 4000 5000 6000 7000
250
200
150
100
50
00 4000 8000 12000
35
30
25
20
15
10
5
00 10 20 30 40 50 60 70 80
60
50
40
30
20
10
00 500 1000 1500
natattivsiretP , Reddish-brown tailings ( = 7) Pteris vittata n, Grey-green tailings ( = 5)nsutarculovnisurepyC , Reddish-brown ailings ( = 6) Cyperus involucratus, Grey-green tailings (n = 6)
Mn, tailings (µg.g-1)
Zn, tailings (µg.g-1)
As, tailings (µg.g-1) Co, tailings (µg.g-1)
As,
pla
nt (
µg.g
-1 D
W)
Co,
pla
nt (
µg.g
-1 D
W)
Zn,
pla
nt (
µg.g
-1 D
W)
Mn,
pla
nt (
µg.g
-1 D
W)
Cu,
pla
nt (
µg.g
-1 D
W)
Fe,
pla
nt (
µg.g
-1 D
W)
A
800
700
600500
400300
200
100
00 100000 200000 300000
Fe, tailings (µg.g-1)
Cu, tailings (ppm)
B
C D
FE
Fig. 5 Average concentration (mean value) of As (a), Co (b), Cu (c),
Zn (d), Fe (e), and Mn (f) in plant species studied in relation to
average concentrations of the same elements in reddish-brown (full
symbols) and grey-green tailings (open symbols). The bars are SDs.
n = Number of analysed samples of plants and tailings
238 Arch Environ Contam Toxicol (2011) 61:228–242
123
comparable with those in common plants (Table 6). High
concentrations of Cu and Co in plants growing in tailings
ponds were reported by several investigators. For instance,
Brooks and Malaisse (1985) mentioned concentrations of
Cu and Co in several plants from the Copperbelt region
reaching 200 and 73 lg g-1 (dw), respectively. Fonkou
et al. (2002) reported B12,500 lg g-1 Cu in shoots of
C. papyrus growing in wetland contaminated by heavy
metals in Cameroon, and Osman (2001) demonstrated
substantial accumulation of Cu and Co by Typha sp. and
Cyperus sp. within the Copperbelt tailings impoundments.
In contrast, high contents of Mn in leaves of C. involuc-
ratus reported by Cheng et al. (2002) were not confirmed in
our study. However, it is possible that high contents of Cu
and Co in substrate of the Chambishi-North tailings pond
prevent the uptake of Mn or other metals (antagonism in
metals uptake [Brooks 1972; Ross 1994]).
The concentrations of metals accumulated in the
aboveground tissues of P. vittata and C. involucratus
growing on both types of tailings of the old Chambishi-
North tailings pond are similar regardless of different
values of total and plant-available amount of metals in both
types of deposited materials. This is similar to the findings
of MacFarlane et al. (2003), who established that Cu levels
in mangrove leaves were not associated with Cu levels in
the sediment. In case of P. vittata, the mean total con-
centration of Co (Fig. 5b) is similar regardless of its con-
tent in deposited materials. The mean concentrations of Cu
and Zn in fronds are even higher in plants growing on
flotation tailings exhibiting lower concentrations of the
respective metals (Fig. 5c, d), and the mean concentration
of Fe in P. vittata growing on different types of tailings is
similar relative to the great difference in concentration of
Fe in the substrate (Fig. 5e).
As for C. involucratus, the mean concentration of Co is
slightly enhanced in plants growing in reddish-brown
tailings, but the mean content of Zn, Cu, and Fe in this
plant is similar in both types of tailings. As for both plants,
the concentrations of Mn in their aboveground tissues are
similar regardless of very different concentrations of Mn in
tailings (Fig. 5f).
In common (‘‘index’’) plants growing on substrate with
low contents of metals, the concentrations of metals in
aboveground tissues reflect contents of metals in substrate,
but their tolerance to high concentrations of metals is
limited. However, some plants (metallophytes) can grow
under conditions of extremely high concentrations of
metals in the substrate. Metallophytes are incapable of
excluding completely potentially toxic elements, but they
may restrict their uptake and/or translocation within the
plant. The uptake of metals by metallophytes can be
restricted over a wide range of substrate metal concentra-
tion. Plant survival in environments heavily contaminated
with potentially toxic elements can be achieved either by
avoidance mechanisms, whereby a plant is protected from
the influence of metals stress, or by true tolerance mech-
anisms whereby a plant survives the effect of an internal
stress (Baker 1987; Ross and Kaye 1994). The mechanisms
are not mutually exclusive (Macnair 1990; Tomsett and
Thurman 1988), and they can operate in different ways for
different metals. Avoidance can be defined as an organ-
ism’s ability to prevent excessive metal uptake into its
body (Levitt 1980). Such avoidance or metal-exclusion
mechanisms include mycorrhiza formation, alteration of
membrane permeability, proliferation of roots in uncon-
taminated horizons, changes in the metal-binding capacity
of the cell wall, or increased exudation of metal-chelating
substances (for surveys see Verkley and Schat 1990;
Clemens 2001; Raab et al. 2004).
Unlike As, which is stored mostly in fronds of P. vittata,
many investigators (see surveys by Weis and Weis 2004;
Sheoran and Sheoran 2006) have reported that mostly roots
of wetland plants, for example, species of Cyperus, Typha,
and Phragnites, tend to trap metals. Cheng et al. (2002)
Table 6 Concentrations of selected chemical elements (lg g-1 dw) in common plants and in aboveground tissues of P vittata and C. invo-lucratus growing in Cu- and Co-rich tailings of the Chambishi-North impoundment in Zambia
Element Normal rangea Normal rangeb P. vittata Chambishi-
North tailings
C. involucratus Chambishi-
North tailings
As 0.12–0.25 NR 2350.2–6197.4 0.24–30.27
Co 0.25–4.15 0.03–2 18.3–38.6 9.2–14.8
Cu 5.2–14.8 5–25 123.2–237.4 96.3–151.8
Fe 272–470 NR 492–690 215–500
Mn 47.4–337 20–400 26.7–46.2 6.2–56.7
Zn 24–130 20–400 13–33 6–14
NR not reporteda Reported by Reimann and de Caritat (1998), bark data excludedb Reported by Reeves and Baker (2000) and Ma et al. (2001)
Arch Environ Contam Toxicol (2011) 61:228–242 239
123
reported B15,600 lg g-1 Cu, 4850 lg g-1 Mn, and
4579 lg g-1 Zn (dw) in lateral roots of C. involucratus
growing in artificial wetland after application of metals in
solution. Concentrations of Cu, Mn, and Zn in leaves of the
same plant were much lower: 7.1, 68.9, and 77.3 lg g-1
(dw), respectively.
Excess of essential as well as nonessential metals can be
sequestered in root cells by various mechanisms, e.g., (1)
metal sequestration in roots by specially produced organic
compounds, (phytochelatins; Cobbett 2000); (2) subcellulal
compartmenisation, particularly in vacuoles of the root
cell; or (3) organic ligand exudation (Clemens et al. 2002).
Moreover, organic molecules exuded by root cells, as well
as mucilage at root tips, can bind, or chelate, with metals in
the rhizosphere, thus rendering them unavailable or less
available for root uptake (Levitt 1980; Verkley and Schat
1990).
A striking feature of roots of wetland plants is the
presence of metal-rich rhizoconcretions or ‘‘plaques’’ on
the roots (Mendelssohn and Postek 1982; Vale et al. 1990;
Weis and Weis 2004). These structures are composed
mostly of Fe(III) hydroxides and other components, i.e.,
Mn-hydroxides and carbonates, that are precipitated on
the root’s surface. The presence of these accumulations
appeared to decrease the amount of Zn, Mn, Cu, and other
metals in plants (Otte et al. 1989; Batty et al. 2000). At
Chambishi-North, the formation of plaques on the surface
of plant roots (Fig. 6a), as well as the subcelullar com-
partmenisation of inorganic matrix in root cells of P. vittata
(Fig. 6b), was observed using an optical microscope and
electron microprobe studies. Compared with other inves-
tigators who reported the formation of Fe- or Mn-rich
rhizoconcretions on the roots in an acid environment, Al
predominates over Fe on the root surface and in root tissues
at Chambishi-North, which is probably due to the neutral to
alkaline environment of deposited tailings (pH of tailings
leachates 7.12–8.23). The concentration of Fe is close to
the detection limit of the microprobe (0.2 wt% Fe). The
solubility of Fe(III) in the surface layer of tailings under
aerobic, neutral to alkaline conditions is low, whereas Al
may be dissolved as Al(OH)4- and precipitated on the roots
or within the root tissues in the form of amorphous Al
hydroxide or as a crystalline phase (gibbsite). As shown by
Garcia-Sanchez et al. (2002), the absorption capacity of Al
hydroxides for metals and As is comparable with or even
higher than that of Fe(III) oxyhydroxides. Therefore, it can
be assumed that the formation of Al-rich accumulations on
root surfaces or in root cells, and the adsorption of As and
metals on these accumulations, may restrict their uptake
and/or translocation within the plants at the Chambishi-
North tailings pond. If so, then the concentrations of metals
in shoots of plants growing in metalliferous substrates at
Chambishi-North do not reflect the metal contents in the
substrate but rather the efficiency of metal-exclusion
mechanisms in plant roots.
Conclusion
The results of the investigation summarized in this article
show that the Chinese brake fern (P. vittata) is not only a
hyperaccumulator of As, but it has adapted itself to high
concentrations of Cu, Co, and other metals contained in
flotation tailings in mud-settling ponds of the Zambian
Copperbelt. This is an important conclusion because this
plant may possibly be used for phytoremediation. Con-
tamination of soils or ground waters by a single element,
such as As, is actually rare. Pollution by metallurgical and
industrial wastes usually introduces a range of heavy
metals so that any phytoremediation should be based on the
selection and cultivation of plant assemblages that can
accumulate the widest range of pollutants contained in the
B
A
B
Cortex
Cortical cellsCentralcylinder
Al (Fe)deposit
Al (Fe)deposit
Centralcylinder
50 µm
Fig. 6 Deposition of Al-rich and Fe-poor material on root surface
and in root cells of P. vittata growing in the Chambishi-North tailings
pond in Zambia. a Photomicrograph of polished section of degraded
root tissues with accumulation of Al-rich and Fe-poor material (rusty-brown color) on the surface of cortical cells and in the neighborhood
of the central cylinder. b Accumulation of Al-rich and Fe-poor
material (whitish color) close to the central cylinder of P. vittata root.
Back-scattered electron image (Color figure online)
240 Arch Environ Contam Toxicol (2011) 61:228–242
123
substrate. Consequently, it is useful to study plant assem-
blages growing in regions contaminated by high levels of
potentially toxic elements. The tailings ponds of the
Zambian Copperbelt have proven to be the ideal setting for
such a study. Although the results of this study are prom-
ising from the viewpoint of phytoremediation, it will be
necessary to carry out germination experiments and pot
trials to obtain more accurate information about vegetation
growth and the response of different accumulator plants to
As, Co, and Cu mineralization as well as the uptake of
these elements by the root systems and aboveground foli-
age. The natural revegetation of mine wastes contaminated
by combinations of metals and metalloids is crucial for
environmental remediation and human health in populated
areas affected by mining. The value of plant hyperaccu-
mulators in this process is of the utmost importance, and
further investigations should be given high priority.
Acknowledgments This work was supported by the Czech Grant
Agency (Project No. 205/08/0321). The authors are grateful to
F. Veselovsky (Czech Geologic Survey) for participation in the field
operations. The authors are also obliged to J. Macurova (Institute of
Soil Amelioration and Preservation, Czech Republic) for analyses of
available phosphorus in tailings and to J. Malec (Czech Geologic
Survey) for microprobe analyses of root tissues. We are thankful to
David Chuba of the Biological Sciences Department, University of
Zambia for the botanic names of the Zambian plants described in this
paper. The authors are indebted to Ales Soukup (Department of
Experimental Biology, Charles University) for valuable information
about the tolerance and the capacity of plants to accumulate metal-
loids and metals and to Jaroslav Hak and Chriss Halls (the British
Museum of Natural History, London) for English editing. Our thanks
are also directed to Daniel R. Doerge, the journal manager, and to
three anonymous reviewers for their effort to go carefully through the
text and for their reasonable and inspiring recommendations.
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