Chemosphere 52 (2003) 1559–1570
www.elsevier.com/locate/chemosphere
Physiological aspects of vetiver grass for rehabilitation
in abandoned metalliferous mine wastes
J. Pang a, G.S.Y. Chan b, J. Zhang c, J. Liang
a,*
, M.H. Wong
c
a
b
College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, PR China
Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Hom, Hong Kong
c
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong
Abstract
Physiological aspects of why vetiver grass (Vetiveria zizanioides L.) can be tolerant to heavy metals and be used as an
alternative method for rehabilitation of abandoned metalliferous mine wastelands have been investigated. The results
showed that high proportions of lead and zinc (Pb/Zn) tailing greatly inhibited the leaf growth, dry matter accumulation, and photosynthesis of leaves, but stimulated the accumulation of proline and abscisic acid (ABA), and enhanced
the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), implying that different mechanisms to detoxify active oxygen species (AOS) existed in different parts of plants. Physiological responses to heavy metal
treatments differed greatly between roots and shoots. Nitrogen fertilizer application could greatly alleviate the adverse
effects of high proportions of Pb/Zn tailing on vetiver grass growth.
2003 Elsevier Ltd. All rights reserved.
Keywords: Pb/Zn tailing; Physiological responses; Rehabilitation of abandoned metalliferous mine wastes; Vetiver grass
1. Introduction
The heavy metals contamination of the environment
by soil erosion in agricultural lands, urban wastes and
by-products of rural, industrial and mining industries
attracts world-wide concern, especially in developing
countries (Tordoff et al., 2000; Mejare and B€
ulow, 2001).
In China, there are many abandoned metalliferous mine
wastelands and the areas become larger and larger
(Young, 1988). Economically there is an urgency to
decontaminate or re-vegetate the mine wastelands in
order to improve environment. Although there are many
methods used to treat them, most of them are either
expensive or impossible to carry out, as the volume of
contaminated material is very large, such as the coal
mine tailings (Salomons et al., 1995). Therefore, a more
*
Corresponding author. Tel.: +86-514-797-9320, fax: +86514-799-1747.
E-mail address: [email protected] (J. Liang).
economical and practical approach is urgently needed at
present, especially for the developing countries.
Vegetative methods are thought to be the most
practical and economical method for rehabilitation of
the mine wastelands (Flathman and Lanza, 1998).
However, re-vegetation of these sites is often difficult
and slow due to the hostile growing conditions, which
include toxic levels of heavy metals. Therefore, selection
or screening of plant species which are tolerant to toxic
levels of heavy metals has attracted much attention in
the treatment of the abandoned mine wastelands (Chaney et al., 1997; Salt et al., 1998). There are a wealthy of
evidence to show that vetiver grass is highly tolerant to
the hostile soil conditions and widely used as a natural,
effective, and low-cost alternative mean to vegetate the
heavy metal-contaminated lands (Truomg, 1996).
The aim of this paper is to investigate the physiological responses of vetiver grass to heavy metals. The
experiments were carried out in greenhouse, where vetiver plants were grown in different proportions of lead/
zinc (Pb/Zn) tailings collected from the abandoned mine
0045-6535/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0045-6535(03)00496-X
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J. Pang et al. / Chemosphere 52 (2003) 1559–1570
near Guangdong province, China. The results showed
that vetiver plants grown well in suitable proportions of
tailings-contained soil medium, and there exited doseand time-effects of the responses of vetiver plants to Pb/
Zn-tailings, and when being grew in the high proportion
of tailings-contained soil and/or for a extended period of
treatment, the growth of vetiver plants was significantly
influenced. The growth of vetiver plants when grew in
high tailing-contained soil could be greatly improved if
nitrogen fertilizer is applied.
2. Materials and methods
were measured with a gas exchange system (CIRAA-1,
PP System, Hitchin, Herts, UK) at ambient CO2 concentration and light intensity 400 lmol quanta m2 s1 .
Leaf fluorescence emission was measured on darkadapted leaves with a plant efficiency analyzer (PEA
System Hansatech, Norfolk, UK). The results of the
measurement were computed by the equipment as Fv =Fm ,
a ratio of maximal variable fluorescence out of fully
light-saturated peak fluorescence. Variable fluorescence
is subtracted from peak fluorescence with a constant
fluorescence of dark-adapted leaves. Dark-adaptation
was achieved with specially designed, light-proof clips
attached on leaves for at least 30 min (Liang et al.,
1997).
2.1. Plant materials and treatments
Vetiver plants (Vetiveria zizanioides L.) were provided by Zhongshan University, Guangzhou and precultivated in the John Innes No. 2 soil compost for
about 30 days when the vetiver grasses grown well. One
hundred and fifty uniform-sized plants (30 plants per
treatment) were selected and five treatments were designed (30 plants for each treatment). Plants were
transplanted into the plastic pots (18 cm in diameter, 25
cm in height; one plant per pot) containing different
proportions of Pb/Zn tailing (by weight): 100% of John
Innes No. 2 soil compost (JI-2) (Liang et al., 1996)
(treatment 1), 25% Pb/Zn + 75% JI-2 (treatment 2), 50%
Pb/Zn tailing + 50% JI-2 (treatment 3), 100% Pb/Zn
tailing (treatment 4), and 100% Pb/Zn tailing + 0.1 g
nitrogen fertilizer ((NH4 )2 SO4 ) (treatment 5), respectively. Plants were grown in greenhouse at the temperature of 25–28 C, 16/8 h (D/N) photoperiod and a
photosynthetic photo-flux density of 300 lmol m2 s1
and watered daily. The numbers of the tillers per plant
were recorded.
2.2. Measurements of leaf growth and biomass of the
plants
The growing leaves with the same leaf-age were labeled from each plant and the leaf length was measured
at an interval of 10 days with a ruler and the leaf growth
rate was calculated accordingly. At day 30 and 50 after
treatment, five plants were harvested and washed with
tap water thoroughly. The shoots and roots were separated and oven-dried to a constant dry weight for the
measurements of biomass and heavy metals.
2.3. Measurements of photosynthetic rate and chlorophyll
fluorescence
Twelve fully expanded leaves were selected from each
treatment for the measurements of leaf photosynthetic
rate, chlorophyll fluorescence, enzyme activities, etc.
Photosynthetic rate (A) of the fully expanded leaves
2.4. Measurements of water potential and electric conductivity of leaves
Leaf water potential was measured with a pressure
chamber (Model 3000, Soil Moisture Equipment Co.,
USA). Excised leaves were immediately placed into the
chamber lined with a moisture filter paper around the
inner wall to minimize evaporative water loss and
pressurized to the balance pressure.
For measurement of electric conductivity of leaves,
leaves were excised from plants and cut into 0.5 cm
segments and weighed. After being washed with distilled
water for at least three times, the leaf segments were
washed with deionized water for three times, and then
transferred into 25 ml beakers containing 10 ml deionized water. The leaf segments were vacuum-treated for
15 min leaf segments to be immersed fully into water.
The electric conductivity was measured with an electric
conductivity meter after 4 h under room temperature
(EC1). Thereafter, the beakers containing leaf segments
were placed into a boiling water bath for 15 min and
then cooled to room temperature, second measurement
was made as above (EC2). The percentage of electrolyte
leakage was calculated as (EC1/EC2) · 100.
2.5. Enzyme extraction and assays
Leaf samples were harvested at a given sampling time
and plunged into liquid nitrogen and stored at )80 C
pending enzyme assay. For enzyme extraction, frozen
leaves (0.5 g) were grinded in liquid nitrogen in a precold mortar. Ten ml of 50 mM pre-cold PBS buffer (pH
7.2) containing 1 mM EDTA and 1% (w/v) PVP was
added to the mortar and homogenized. The homogenate
was then filtered through four layers of cheesecloth and
centrifuged for 20 min at 15 000 g and 4 C. The supernatant was used for enzyme assays without further
purification.
Superoxide dismutase (SOD) (EC 1.15.1.1) activity
was analyzed based on the procedure described by
Beauchamp and Fridovich (1971) with minor modifica-
J. Pang et al. / Chemosphere 52 (2003) 1559–1570
tion. In brief, the reaction mixture contained 50 mM
PBS (pH 7.8), 8.6 lmol/l methionine, 42 lmol/l NBT,
1.3 lmol/l riboflavin, and 0.1 mmol/l EDTA, 50 ll crude
enzyme extract. The reaction was initiated by placing the
test tubes containing reaction mixture under 100 lmol
quanta m2 s1 of light intensity for 15 min later and the
color formed was determined spectrophotometrically
at 560 nm. One unit of SOD activity is defined as the
amount of enzyme required to inhibit 50% of the color
formation.
Analyses of peroxidase (POD) (EC. 1.11.1.7) and
catalase (CAT) (EC. 1.11.1.6) activities were performed
using guaiacol and H2 O2 substrates, respectively, as
described by Chance and Maehly (1995).
2.6. Quantification of leaf ABA
The concentration of abscisic acid (ABA) in the
leaves was determined by methods of the radio immu-
1561
noassay (RIA) according to the procedure described by
Liang et al. (1996).
2.7. Chlorophyll determination
The chlorophyll content was determined in 80% acetone extract of 0.2 g of dry weight of leaves based on
the procedure described by Arnon (1949).
3. Results
Pb/Zn mine tailing contains high levels of heavy
metals, especially Pb and Zn (Table 1), which are toxic
to plants or inhibitory to plant growth. Experiment was
conducted with different proportions of tailings to determine the tolerance of vetiver grass to high levels of
heavy metals. Vetiver plants were transplanted into
pots containing different proportions of tailings, and
Table 1
Heavy metal contents of different proportions of mine tailings (lg g1 dry weight)
Heavy metals
Treatment 1 (100% tailing)
Treatment 2
(50% tailing + 50% soil)
Treatment 3
(25% tailing + 75% soil)
Treatment 4
(100% soil)
Pb
Zn
Fe
Cu
1980
1700
36 870
30
1110
1000
28 280
20
530
550
17 450
10
100
150
13 550
10
Tailings collected from the abandoned mine near Guangdong province, China.
Fig. 1. Effects of different proportions of Pb/Zn tailings on tillering capacity of vetiver grass. Filled circles indicated control treatment
(100% soil), empty circles: 75% soil plus 25% Pb/Zn tailing, filled triangles: 50% soil plus 50% Pb/Zn tailing, empty triangles: 100%
Pb/Zn tailing, and filled squares: 100% Pb/Zn tailing plus nitrogen fertilizer. Data shown are the means of five plants ± SD.
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J. Pang et al. / Chemosphere 52 (2003) 1559–1570
Fig. 2. Effects of different proportions of Pb/Zn tailings on leaf
growth of vetiver grass. The treatments were similar to Fig. 1.
Each point is mean of 12 measurements ± SD.
physiological parameters were measured. Fig. 1 showed
the effects of different proportions of tailings on the tillering capacity of vetiver grass. As compared with the
control treatment (100% soil), there is no influence of
25% tailing treatment on the tillering capacity of vetiver
grass, and the numbers of tillers is about 8 per plant
after 50 days of treatment. However, the tillering capacity is significantly reduced as the tailing content increased and the tiller numbers decreased to only 60% of
those of control treatment when vetiver was treated with
100% tailing for 48 days. However, if nitrogen fertilizer
was applied to the 100% tailing treatment, the tillering
capacity was greatly improved and the tiller numbers
Fig. 3. Effects of different proportions of Pb/Zn tailings on dry
matter accumulation in shoots and roots of vetiver grass. Each
point is mean of five plants ± SD.
Fig. 4. Effects of different proportions of Pb/Zn tailings on leaf water potential of vetiver grass. The symbols are the same to those in
Fig. 1. Each data is mean of five measurements ± SD.
J. Pang et al. / Chemosphere 52 (2003) 1559–1570
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Fig. 5. Effects of different proportions of Pb/Zn tailings on chlorophyll content, photosynthetic rate and photochemical activity of
vetiver leaves. The symbols are the same to those in Fig. 1. Each data is mean of 12 measurements ± SD.
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J. Pang et al. / Chemosphere 52 (2003) 1559–1570
Fig. 6. Effects of different proportions of Pb/Zn tailings on electric leakage of vetiver leaves and roots. The symbols are the same to
those in Fig. 1. Each data is mean of 5 measurements ± SD.
was about 3 more than that of 100% tailing treatment
per plant (Fig. 1).
The leaf growth rate and dry matter accumulation of
both shoots and roots of vetiver grass were also inhibited when grown in the tailing-contained soil and the
degrees of inhibition increased with the increment of
tailing content and prolongation of treatment (Figs. 2
and 3). The effect was more pronounced on roots than
on shoots, and also application of nitrogen fertilizer
could significantly alleviate the inhibitory effects of
heavy metals (Fig. 3).
When plants were grown in a heavy metal-contaminated soil, the roots were the primary site of heavy metal
accumulation. Many reports showed that root growth
was severely inhibited by heavy metal treatment. The
inhibition of roots may lead to the decrease in water and
nutrient absorption. Fig. 4 showed the effects of different
Pb/Zn tailing treatments on leaf water potential. The
results indicated that the leaf water potential was almost
not significantly influenced when plants were grown in
the relatively low contents of tailing and short time of
treatments. However, obvious decrease in leaf water
potential was observed when plants were grown in 100%
tailing medium and the treatment time was longer than
13 days when the roots displayed an obvious injury
(data not shown).
J. Pang et al. / Chemosphere 52 (2003) 1559–1570
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Fig. 7. Effects of different proportions of Pb/Zn tailings on SOD activity of vetiver leaves and roots. The symbols are the same to those
in Fig. 1. Each data is mean of 3 measurements ± SD.
No significant inhibitory effects of tailing treatment
on chlorophyll content, photosynthetic rate and photosynthetic photochemical activity of leaves were observed, except for 50% and 100% tailing treatments (Fig.
5). In fact, nitrogen fertilizer treatment can greatly improve the photosynthetic characteristic of leaves of vetiver plants when grown in the tailing-containing soil
medium.
One of the most important effects of heavy metals at
the cellular level are the alteration of membrane integrity
and the formation of active oxygen species (AOS) (Dietz
et al., 1999). In order to determine whether high tolerance of vetiver grass to heavy metal is related to its
effective protective mechanisms to eliminate or reduce
AOS caused damages, the dose- and time-responses of
electric conductivity and the enzymatic antioxidant
system of leaves were investigated (Figs. 6–9). The results of Fig. 6 shown that electrolyte leakage of both
roots and leaves increased with the increment of tailing
contents and the progress of the treatment time, but a
significant increase was observed only for the 50% and
100% tailing treatments and the increase in the relative
electric conductivity was more pronounced in roots than
in shoots, implying that the injury of roots was severer
than that of shoots (Fig. 6). There were great differences
in the activities of SOD, POD and CAT between roots
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J. Pang et al. / Chemosphere 52 (2003) 1559–1570
Fig. 8. Effects of different proportions of Pb/Zn tailings on POD activity of vetiver leaves and roots. The symbols are the same to those
in Fig. 1. Each data is mean of 3 measurements ± SD.
and shoots in response to tailing treatments. Increases in
activities of all three enzymes were observed in roots and
in shoots after treatment of tailings. However, the increases in the activities of SOD and CAT were much
more distinct in shoots than in roots, whereas the activity of POD increased more greatly in shoots than that
in roots (Figs. 7–9), suggesting that the mechanisms to
scavenge reactive oxygen species used were different
between various parts of plants.
Abscisic acid (ABA), well known as a plant stress
hormones, plays an important role in the improvement of plant tolerance to adverse environmental
conditions. As shown in Fig. 10, ABA concentrations
in leaves and roots increased with the increment of
proportions of tailing and the progress of treatments,
but significant increase in ABA concentration was
observed only in leaves of 100% tailing treatment.
Nitrogen fertilizer treatment had no significant influence on ABA concentrations in leaves and roots, as
compared with 100% tailing treatment without nitrogen fertilization.
Similar increases in proline concentrations were observed in both leaves and roots (Fig. 11). However, the
proline concentration was much higher in roots than in
leaves after 50 days of treatments, especially in 100%
tailing-treated roots. Fertilizer treatment could stimulate
the accumulation of proline in both leaves and roots
(Fig. 11).
J. Pang et al. / Chemosphere 52 (2003) 1559–1570
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Fig. 9. Effects of different proportions of Pb/Zn tailings on CAT activity of vetiver leaves and roots. The symbols are the same to those
in Fig. 1. Each data is mean of 3 measurements ± SD.
4. Discussion
The areas of heavy metal-polluted soils increased
significantly throughout the world during past several
decades, as the results of industry development, mining
activity, irrigation of waste water, etc. (Smith et al.,
1996; Herawati et al., 2000; Tordoff et al., 2000),
which has become a global problem because of its
deterious influences not only on plant growth (yield
and quality) and environmental quality, but also on
the health of human beings. Therefore, much effort has
been made to decontaminate the polluted soil by using
either chemical, physical or biological methods (Salt
et al., 1998; Valls et al., 2000). For metalliferous mine
wastelands, both the physi-chemical methods and biological methods are impossible to be used to decontaminate the heavy metal polluted wasteland as a
consequence of the large amount of waste products of
mining and ore-processing operations. Use of a vegetation cover gives a cost-effective and environmentally
sustainable method of stabilizing and reclaiming
wastes such as mine-spoils and tailings (Tordoff et al.,
2000). Thus, screening of plant species which are high
tolerance to high-level heavy metals is urgently needed
in this aspect. Much progress has been made at the
levels of physiology, biochemistry and molecular
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J. Pang et al. / Chemosphere 52 (2003) 1559–1570
Fig. 10. Effects of different proportions of Pb/Zn tailings on ABA contents of vetiver leaves and roots. The symbols are the same to
those in Fig. 1. Each data is mean of 3 measurements ± SD.
biology of plant tolerance to heavy metals in past
decades (Chaney et al., 1997).
Vetiver grass (V. zizanioides), due to its unique
morphological and physiological characteristics, has
been widely known for its effectiveness in erosion and
sediment control, and has also been found to be highly
tolerant to extreme soil conditions including heavy metal
contamination (Truong and Baker, 1998). Nowadays
vetiver grass has been widely used as an alternative
method for rehabilitation of mine tailings in several
countries, including in China.
This paper highlights the physiological aspects of
vetiver grass in responses to heavy metal treatment. The
results showed that great physiolgical changes have oc-
curred when vetiver grass grown in heavy metal-containing soil medium. The adverse effects were more
pronounced in roots than in shoots (Figs. 3 and 6),
which might be related to high heavy metal accumulation within the root cells as compared with that in the
shoots.
As a visible symptom in the leaves, significant decrease in chlorophyll content was observed, especially
for the high proportions of tailing treatments (Fig. 5).
The decrease in chlorophyll content may be due to the
inhibition of chlorophyll biosynthesis (Stobart et al.,
1985; Stiborov
a et al., 1986) or accelerated degradation
of chlorophyll (Luna et al., 1994). The decrease in
chlorophyll content leads to the decrease of photosyn-
J. Pang et al. / Chemosphere 52 (2003) 1559–1570
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Fig. 11. Effects of different proportions of Pb/Zn tailings on proline contents of vetiver leaves and roots. The symbols are the same to
those in Fig. 1. Each data is mean of 3 measurements ± SD.
thesis of leaves, and thus growth of plants (Figs. 2, 3 and
5). Of course, the inhibition of photosynthesis may also
be due to inhibition of other photosynthesis-related
factors, such as Rubisco and photochemical activity.
However, the photochemical activity, indicated as Fv =Fm ,
decreased only when plants exposed to high level of
heavy metals (Fig. 5). Application of nitrogen fertilizer
combined with tailing treatment could greatly alleviate
the decreases in chlorophyll content and photochemical
activity, thus no influence on photosynthetic rate of
leaves was observed, as compared with the corresponding treatments (Fig. 5).
It is well known that exposure of plants to heavy
metals induces the generation of AOS, which is harmful
to plants (Zenk, 1996). The injury of plant cells caused
by heavy metals is, to a great extent, related to the destruction of the balance between the generation and
detoxification of AOS. Plants possess the protective
mechanisms to scavenge the toxic AOS, but the ability
to the balance between the generation and detoxification
of AOS varies greatly among different plant species. The
results shown in this paper suggest that tailing treatments could enhance the activities of POD, SOD and
CAT, which are the major enzymes involving in scavenge of AOS (Figs. 7–9). However, great differences
were observed in the changes of activities of these enzymes between shoots and roots, implying that different
mechanisms were used to scavenge AOS in different
parts of vetiver plants, which is wealthy of further study.
Similar results were observed for protective substances,
such ABA and proline (Figs. 10 and 11).
In conclusion, the results shown in this paper can
provide the guidelines in screening of plants high tolerant to heavy metals. Some physiological parameters
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J. Pang et al. / Chemosphere 52 (2003) 1559–1570
could be used in this aspect. Furthermore, it is very
useful to apply nitrogen fertilizer when vetiver grass is
used to re-vegetate the metalliferous mine wastelands.
Acknowledgements
This project is supported by the National Programme
on Key Basic Study and Development (G1999011700).
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