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TOLERANCE OF ORNAMENTAL SUCCULENT PLANTCROWN OF THORNS (Euphorbia milli) TO CHROMIUMAND ITS REMEDIATIONSivakoti Ramana a , Ashis Kumar Biswas a , Amar Bahadur Singh a , Ajay a , Narendar KumarAhirwar a & Annangi Subba Rao aa Indian Institute of Soil Science , Nabi bagh, Berasia Road, Bhopal , 462 038 , MadhyaPradesh , IndiaAccepted author version posted online: 28 Jan 2014.Published online: 28 Jan 2014.
To cite this article: International Journal of Phytoremediation (2014): TOLERANCE OF ORNAMENTAL SUCCULENT PLANTCROWN OF THORNS (Euphorbia milli) TO CHROMIUM AND ITS REMEDIATION, International Journal of Phytoremediation, DOI:10.1080/15226514.2013.862203
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TITLE: TOLERANCE OF ORNAMENTAL SUCCULENT PLANT CROWN OF THORNS
(Euphorbia milli) TO CHROMIUM AND ITS REMEDIATION
Authors’ names: *Sivakoti Ramana, Ashis Kumar Biswas, Amar Bahadur Singh, Ajay, Narendar Kumar
Ahirwar and Annangi Subba Rao
Address: Indian Institute of Soil Science, Nabi bagh, Berasia Road,Bhopal- 462 038; Madhya Pradesh,
India Telephone: 0755-2730946(Ext:227), Fax: 0755-2733310 ; *E-
mail:[email protected]
Running Head : Phytoremediation of Cr by Crown of thorns
Abstract
The potential of an ornamental shrub Crown of thorns (Euphorbia milli) was evaluated for
remediation of soil contaminated with Cr. The plant is one of the rare succulent ornamental shrubs
with a slow to moderate growth rate and is capable of blooming almost year-round. The plant could
tolerate well up to 75 mg of applied Cr and beyond that there was mortality of plants. Though the plant
could not be classified as a hyperaccumulator, the plant was still very efficient in translocating Cr from
roots to shoots as evident from the data on uptake and translocation efficiency values. The
translocation efficiency of over 80 % in our study demonstrates that a large proportion of Cr has
been translocated to the harvestable biomass of the plant and therefore, this plant could be effectively
recommended for the remediation of soils contaminated with low to medium level of contamination
i.e., up to 50 mg/kg soil.
Key words
Phytoremediation, chromium, crown of thorns, translocation factor, bioconcentration factor,
translocation efficiency
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INTRODUCTION
Chromium is one of the most toxic heavy metals which deteriorate the environment. Chromium
is used on a large scale in many different industries, including metallurgical, electroplating,
production of paints and pigments, tanning, wood preservation, Cr chemicals production, and
pulp and paper production. The leather industry is the major cause for the high influx of Cr to the
biosphere, accounting for 40% of the total industrial use (Barnhart, 1997). Chandra et al. (1997)
estimated that in India alone about 2000 to 3200 tonnes of elemental Cr escape into the
environment annually from the tanning industries, with a Cr concentration ranging between 2000
and 5000 mg L−1 in the effluent compared to the recommended permissible limit of 2 mg L−1.
Typical concentrations in natural soils are 1-1000 mg /kg soil (Frink, 1996; Lindsay, 1979). The
introduction of treatment processes has considerably reduced the chromium content of the
effluent in many modern tanneries. However, many older tanneries still incorporate little or no
treatment of the effluent prior to discharge, which leads to substantial chromium contamination
of the receiving waters (Fuller et al. 1990). Restoration of soils contaminated with potentially
toxic metals and metalloids is of major global concern. Considerable efforts have been made to
develop suitable methods for the remediation of chromium-contaminated soils. Remediation of
heavy metals polluted soil could be carried out using physico-chemicals processes such as ion-
exchange, precipitation, reverse osmosis, evaporation and chemical reduction. However, the
measures require external man-made resources and therefore are very costly (Mangkoedihardjo
and Surahmaida 2008). Phytoremediation is an emerging technology that can be considered for
remediation of contaminated sites because of its cost effectiveness, aesthetic advantages, and
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long term applicability. For a country like India, phytoremediation is best suited as it requires
low investment, and relies on plants natural capability to take up metal ions from soil (Ghosh and
Singh 2005). Identification/selection of plant species for phytoremediation is a continuous
process and till date, many plants have been found as remediation plants but there are very few
reports about the use of ornamental plants for phytoremediation purpose (Liu et al. 2009, Lal et
al . 2008; Ramana et al 2008a, Ramana et al.2008b; Ramana et al. 2009).Especially, in urban
areas, ornamentals can beautify the environment, and also resolve heavy metal pollution( Wang
and Zhou 2005). In our previous studies, we have studied the feasibility of remediation of soils
contaminated with Cr using different floriculture plant species i.e., tuberose (Polianthes
tuberosa) (Ramana et al. 2012), chrysanthemum, calendula, aster and dahlia(Ramana et al.
2013a); and fibre yielding plant mestha(Hibiscus sabdarifa)( (Ramana et al. 2013b). From these
studies, it was found that majority of the plant species could tolerate at the most 10-15 mg Cr/kg
soil. However, the contaminated sites would have very high levels of Cr and at times would even
be unfit for cultivation of the crops. Therefore, looking at the very harsh environmental
conditions at the polluted areas, in the present study, we have evaluated the potential of Crown of
thorns (Euphorbia milli) for remediation of soil contaminated with Cr. Crown of Thorns is one
of the rare succulent ornamental shrubs with a slow to moderate growth rate and is capable of
blooming almost year-round. It grows to a height of 4-5 feet and spread of 2.5 feet and has
strong, upright and heavily branched spiny stems with 1 inch long straight thorns and obovate
leaves that are medium to dark green of variable sizes. Thus, the study focuses on the ability of
Crown of thorns (Euphorbia milli) to tolerate and extract Cr from contaminated soil.
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MATERIALS AND METHODS
Pot culture experiment
The experiment was carried out in a screen house at the Indian Institute of Soil
Science,Nabi Bagh, Bhopal, India. The soil for the experiment was collected from 0-15 cm depth
from the nearby agricultural field. The soil was clayey in texture and classified as Typic
Haplusterts. The soil was dried under shade, ground and passed through two mm sieve and was
then transferred to 5 kg plastic pots. The soil contamination was performed by adding a specific
amount of stock solution of potassium dichromate. A stock solution of 1000 ppm potassium
dichromate solution was prepared by dissolving 2.83 g K2Cr2O7 in 1000 ml distilled water. The
soil in the pot was then treated with aqueous solution of K2Cr2O7 so as get the desired level of
contamination. Overall, the experiment consisted of 7 treatments including soil without
chromium i.e., control(Cr0); 25 mg Cr/Kg soil, 50 mg Cr/Kg soil; 75 mg Cr/Kg soil; 100 mg
Cr/Kg soil 150 mg Cr/Kg soil and 200 mg Cr/Kg soil. The soil was then subjected to wetting and
drying cycles for one month to ensure soil equilibrium with chromium and later the soil was
taken out and mixed uniformly and the soil was analysed for pH, EC, DTPA extractable Cr and
dehydrogenase activity (Table 1). Soil pH and EC were measured on 1:1 extract (Soil:Water).
The soil pH was unaffected by applied Cr but the EC increased considerably at higher levels of
Cr (Table 1). The CEC, OC, available N,P, and K and were 46 C mol (p+)/kg soil; 4.75 g/kg soil;
112 mg/kg soil; 2.61 mg/kg soil and 227 mg/kg soil respectively. The total Cr in the pretreated
soil was in found to be in traces. Extractable Cr in soil samples was carried out by DTPA in
accordance the Standard Methods (APHA 1998). The % of available Cr increased from 20% at
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25 mg Cr /kg soil to 50% at 200 mg Cr/kg soil. The dehydrogenase enzyme activity is commonly
used as an indicator of biological activity in soils/direct measure of soil microbial activity. The
dehydrogenase activity was determined as per the procedure given by Casida et al. (1964). In the
present experiment, however, there was reduction in the dehydrogenase activity of the soil
indicating decreased microbial activity in the soil with the applied Cr.
Two months old seedlings of Crown of thorns (Euphorbia milli) were procured from
local nursery and one plant was transferred to each pot. The plants were watered whenever
necessary. No fertilizers were applied. The plants were grown for six months. After six months,
the plants were harvested and separated in to roots and shoots. The roots were first washed with
running tap water followed by distilled water. The samples were then dried in an oven at 70-
800C till constant weight was obtained and the dry weight was recorded. The dried plant samples
were ground, digested with 10 ml di-acid mixture (9 HNO3 : 4 HClO4) and the concentration of
Cr was determined with Atomic Absorption Spectrophotometer (Perkin Elmer) and expressed as
µg-1 g DW. Subsequently, the uptake of Cr was computed by multiplying the concentration of Cr
in the plant tissue and dry weight of the plant tissue and expressed as µg pot-1. Besides the
accumulated concentration, bioconcentration factor(BCF), translocation factor (TF) and
translocation efficiency (TE %) the three most important parameters which are used to evaluate
the accumulating capacity of HMs by plants were calculated.
Bioconcentration factor (BCF) was calculated as by following formula given by
(Zhuang et al. 2007).
BCF = Charvested tissue / C soil
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where Charvested tissue is concentration of the target metal in the plant harvested tissue
(roots, stem or leaves) and Csoil is concentration of the same metal in soil. Csoil represents the
total Cr applied to the soil.
Translocation factor (TF) was quantified using the formula proposed by
Padmavathiamma and Li, (2007); Adesodun et al. (2010).
TF == C shoots/ C roots
Translocation efficiency (TE %) was calculated as per formula proposed by Meers et
al.(2004).
T E (%) =Cr content in the shoots (µg g) /Cr content in the whole plant(µg g) X 100
The % Cr crop removal
(Total Cr uptake by plant / Total Cr applied to the soil) X 100
Determination of proline and electrical leakage
One week before harvest of the plants, proline content and electrolyte leakage were
determined. Proline was determined by the method developed by Bates et al. (1973). 0.5 g of
plant material (leaf) was homogenized using 10 ml of 3% aqueous sulphosalicylic acid. The
homogenate was filtered through Whatmann No.1 filter paper and mixed with 2 mL of acid
ninhydrin (1.25 g of ninhydrin + 30 mL of glacial acetic acid + 20 mL of 6 M phosphoric acid)
and 2 mL of glacial acetic acid. The sample was heated for one hour at 100 °C in a water bath
and followed by addition of 4 mL of toluene. This solution was mixed well and read at 520 nm in
a UV-visible spectrophotometer. The proline concentration was determined using a standard
curve.
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Electrolyte leakage was measured using an electrical conductivity meter as described by
Lutts et al. (1996). Leaves were excised and washed with deionized water. After drying with
filter paper, 1 g fresh weight of leaves were cut into small pieces (about 1 cm2) and then
immersed in 20 mL deionized water and incubated at 25°C. After 24 h, electrical conductivity
(EC1) of the bathing solution was recorded. These samples were then autoclaved at 120°C for
20 min to completely kill the tissues and release all electrolytes. Samples were then cooled to
25°C and the final electrical conductivity (EC2) was measured. The electrolyte leakage (EL) was
expressed following the formula EL=EC1/EC2×100.
Statistical analysis
The experiment was conducted in a completely randomized block design and there were
five replications per each treatment. The data was analyzed statistically and the treatment means
were compared using least significant difference technique (LSD) at 5% probability appropriate
for CRBD (Gomez and Gomez 1964).
RESULTS AND DISCUSSION
The success of phytoremediation depends on biomass of shoot and its metal
concentration bioconcentration and bioavailable fraction of the applied metal. Therefore in the
present study, the data on biomass of the plant, concentration of chromium in roots and shoots,
its uptake, bio concentration factor (BCF), translocation factor (TF) and translocation efficiency
(TE %) were recorded.
Tolerance of Euphorbia milli to graded levels of Cr
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The plant growth expressed as dry weight of shoots and roots, shoot height and leaf area
(Table 2) was adversely inhibited when exposed to Cr stress. In general, the application of Cr
resulted in reduction in growth of the plants compared to control (Table 2). However, the
differences between control, 25 mg Cr/Kg soil and 50 mg Cr/Kg soil were not significant.
Significant reduction was observed only at 75 mg Cr/Kg soil treatment. Furthermore, in the
present study, beyond 75 mg Cr/ kg soil, the applied Cr was highly toxic leading to wilting and
death of the plants. Immediate wilting leading to death of plants has also been observed in
previous studies(Ramana et al. 2012, Ramana et al. 2013). This finding has been corroborated by
Hara and Sonoda (1979); Parr, (1982).Decrease in root growth is a well-documented effect due
to heavy metals in trees and crops (Breckle, 1991; Tang et al. 2001). Reduction in root length
and dry weight of crops with applied Cr has been reported by Iqbal et al.( 2001); Chen et
al.(2001). The reduction in root growth could be due to the direct contact of seedlings roots with
Cr in the medium causing a collapse and subsequent inability of the roots to absorb water from
the medium (Barcelo et al. 1986).Adverse effects of Cr on plant height and shoot growth have
been reported (Shahandeh and Hossner 2000; Han et al. 2004; Joseph et al. 1995).The reduction
in plant height could be due to the reduced root growth and consequent lesser nutrients and
water to the above parts of the of the plant. Further, it has been reported that, Cr transport to the
aerial part of the plant can have a direct impact of cellular metabolism of shoots contributing to
the reduction in plant height (Shankar et al. 2005). Leaf growth, area development and total leaf
number determine the yield of crop crops (Shankar et al. 2005). Tripathi et al. 1999 found that
leaf area and biomass of Albizia lebbak was severely affected by Cr applied at the rate of 200
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ppm. In the present investigation also there was a drastic reduction in number of leaves/plant,
leaf size, leaf area and LAR at 75 mg Cr/kg soil treatment.
The electrolytes leakage (EL) constitutes an indicator of the membrane permeability and
it was measured in the leaves. Electrolytes are contained within the membranes of plant cells and
these membranes are sensitive to environmental stresses. Unstressed, undamaged plant cells
maintain electrolytes within the membrane. As the cells are subjected to stress, electrolytes leak
into surrounding tissues. An estimation of cell damage can be made by comparing the
conductivity of the leaked contents from injured and uninjured tissues in water (Mattsson 1996,
McNabb and Takahashi 2000). In the present study, the exposure of the plants to Cr stress
resulted in a significant increase (17%) in electrolytes leakage (Table 2) compared to the control.
However, the among the chromium levels, the differences were not significant.
Significantly higher proline content was found in Cr treated plants compared to control
indicating that the applied chromium had created stress in plants. The difference between 25 mg
Cr/kg soil and 50 mg Cr/kg soil was not significant. However, between 50 mg Cr/kg soil and
the highest level i.e, 75 mg Cr/kg soil, the difference was significant. Proline accumulation is a
common metabolic response of higher plants to stress. The accumulation of proline could be
attributed to the strategies adapted by the plants to cope up with chromium toxicity as proline has
multiple functions such as providing the plants protection against damage by ROS. Proline plays
important roles in osmoregulation (Ahmad and Hellebust, 1988), protection of enzymes
(Nikolopoulos and Manetas, 1991); stabilization of the machinery of protein synthesis (Kadpal
and Rao, 1985), regulation of cytosolic acidity (Venekemp, 1989), and scavenging of free
radicals (Smirnoff and Cumbes, 1989).
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Partitioning of Cr and its uptake, BCF, TF and TE(%)
The accumulation of Cr in roots and shoots of Euphorbia milli is shown in Figure 1. The
data revealed that both root and shoot concentrations were significantly affected by varying Cr
concentrations in the soil. The concentrations of Cr in roots and stems of Euphorbia milli were
significantly higher in Cr-treated plants than in control plants. The concentration increased
significantly in both roots and shoots with increase in the level of applied Cr. The data on the
partitioning of Cr indicated that, invariably the roots accumulated higher levels of Cr than shoots
indicating that Cr is not easily translocated within plants. The concentration of Cr ranged from
56 µg/g at 25 mg Cr/kg soil to 634 µg/g at 75 mg Cr/kg soil and that of shoot ranged from 41
µg/g at 25 mg Cr/kg soil to 231 µg/g at 75 mg Cr/kg soil. On an average, the roots accumulated
1.86 times higher chromium than shoots. The big difference between root and shoot
concentrations indicates an important restriction of the internal transport of Cr from roots to
shoots resulting in higher root concentrations rather than translocating to shoots. Golovatyj et al.
(1999) have shown that Cr distribution in crops had a stable character and the maximum
quantity of element contaminant was always contained in roots and a minimum in the vegetative
and reproductive organs. In bean, only a 0.1% of the Cr accumulated was found in seeds against
98% in the roots(Huffman and Allaway, 1973a). The reason for high accumulation in roots of
plants could be because Cr is immobilized in the vacuoles of the root cells, thus rendering it less
toxic which may be a natural toxicity response of the plant(Shankar et al. 2004 a). Lower
transport of Cr from root to aerial parts in Euphorbia milli is in accord with our previous
observations in tuberose (Ramana et al. 2012) and other floriculture plant species(Ramana et al.
2013). Further, it has been reported that the poor translocation of Cr from roots to shoots is a
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major hurdle in using plants and trees for phytoremediation. Pulford et al. (2001) in a study with
temperate trees confirmed that Cr was poorly taken up into the aerial tissues but was held
predominantly in the root.
Bio concentration factor (BCF) indicates the efficiency of a plant in up-taking heavy
metals from soil and accumulating them into its tissues. It is a ratio of the heavy metal
concentration in the plant tissue (root, stem or leaves) to that in soil (Zhuang et al. 2007).For a
plant to be efficient tool in the contaminated soil phytoremediation, the BCF have to be higher
than 1. In our experiment, BCF of Cr was found to >1suggesting Euphorbia milli a good plant
for phytoremediation of Cr-contaminated soils. The BCF increased considerably ( 2.23 at 25 mg
Cr/Kg soil 25to 8.46 at 75 mg Cr/Kg soil) with increasing Cr concentration of soil (Table
2).However, it has been reported that the BCF decreases with increasing soil heavy metal
concentrations (Zhao et al.2003; Mertens et al. 2005) that was in contrary to our result.
Translocation factor TF is defined as a ratio of the concentration of the heavy metal in
shoots (stem or leaves) to that in its roots (Padmavathiamma and Li, 2007; Adesodun et al.
2010). It indicates the efficiency of the plant in translocating the accumulated heavy metals
from roots to shoots. If TF >1, it shows that the accumulation of heavy metals in the shoots is
higher than roots. Moreover, the higher the TF value is, the stronger the phytoextraction ability
(Zhao et al. 2006). In the present study, the TF values were found to be <1 indicating restricted
movement of Cr from root to shoot. Further, a linear reduction in TF values was observed. The
TF values decreased from 0.73 at 25 mg Cr/kg soil to0.36 at 75 mg Cr/kg soil. This observation
is in accordance with the findings of Sun et al. (2009).
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From the results, it was evident that, Euphorbia milli could not be classified as a
hyperaccumulator Cr as it could not meet the criteria of TF value being greater than one and
concentration of Cr in the shoots being 500 µg/g as suggested by Baker and Brooks (1989), the
plant was still very efficient in translocating Cr from roots to shoots as evident from the data on
uptake (Fig 2)and translocation efficiency values (Table 2). The uptake of Cr increased with
increase in the level of applied Cr to the soil. The uptake of Cr and its translocation to shoots in
Euphorbia milli is far greater than the uptake recorded in our previous studies with tuberose
(Ramana et al. 2012) and other floriculture plant speices(Ramana et al.2013a). Although roots of
plants accumulated higher concentration of Cr compared to shoot, the total removal of Cr by
shoot was larger because of its larger biomass. Translocation efficiency (TE %) defined as the
ratio of the metal accumulated in the shoot to the amount of metal accumulated in the whole
plant increased from 80.5% at 25 mg Cr/Kg soil to 83%up to 50 mg Cr/Kg soil and declined
sharply (56 %) at 75 mg Cr/Kg soil. TE of over 80 % in our study demonstrates that a large
proportion of Cr has been translocated to the harvestable biomass of the plant. Further, when
the fraction of Cr removed by the crop from the soil from applied Cr was calculated, on an
average 0.6% of Cr applied was removed by the crop. This value is similar to mustard (0.4%),
another important crop advocated for phytoremediation of soils contaminated with heavy metals
(Wu et al. 2003) and aromatic grasses (Lal et al. 2007).
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CONCLUSIONS
Our results clearly demonstrated that Cr was highly toxic to plants beyond 75 mg Cr/kg
soil and grew well up to 50 mg Cr/kg soil. Though the plant could not be classified as a
hyperaccumulator, the plant was more tolerant to Cr and very efficient in the translocation of Cr
to the shoots compared to the other crops we have studied earlier i.e., tuberose (Ramana et al.
2012), chrysanthemum, calendula, aster and dahlia(Ramana et al. 2013a); and fibre yielding
plant mestha(Hibiscus sabdarifa)( (Ramana et al. 2013b). Though roots accumulated higher
concentration of Cr compared to shoot, but because larger biomass of shoots, the total removal
of Cr by shoot was higher. The translocation efficiency of over 80 % in our study demonstrates
that a large proportion of Cr has been translocated to the harvestable biomass of the plant.
Therefore, it was concluded that, Euphorbia milli could be effectively recommended for the
remediation of soils contaminated with Cr if the level of Cr is up to 50 mg/kg soil.
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Table 1 Effect of different levels of Cr on some physico-chemical properties of soil
Treatment
(mg/kg soil)
pH EC
µmhos cm-1
DTPA Cr
(mg/kg soil)
Dehydrogenase activity
µg TPF/Kg soil/24 h
0 8.03 226 0.03 7.21
25 8.07 301 5.12 7.18
50 8.09 312 17.24 6.37
75 8.08 323 28.01 5.39
100 8.06 348 47.24 4.72
150 8.09 355 69.50 3.70
200 8.11 383 98.03 3.46
CD(0.05) NS 43 4.78 1.59
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Table 2 Effect of Cr on some physiological and biochemical parameters in Euphorbia milli
Treatment
(mg
Cr/kg
soil)
Dry
weight(g/pot)
Root Shoot
No of
leaves
/plant
Single leaf
size(cm2/leaf)
Leaf
area
(cm2/
plant)
LAR
(cm2/g)
Plant
height
(cm) Proline
(µg
gFW)
Electrical
leakage(%)
0 3.96 17.73 69.0 26.9 1619 254 31.3 10.40 17.36
25 2.99 15.97 61.0 22.9 1441 244 29.3 15.60 20.31
50 2.72 11.68 55.3 20.3 1230 218 27.7 17.07 21.23
75 1.48 5.65 39.3 6.1 179 167 22.7 27.07 21.23
CD(0.05) 0.48 2.62 15.7 4.7 75 38 5.87 4.52 1.95
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Table 3 Effect of different levels of Cr on Bio Concentration Factor(BCF), Translocation Factor
(TF), Translocation efficiency (TE %)
Treatment
(mg/kg soil)
Bio Concentration
Factor(BCF)
Translocation
Factor (TF)
Translocation efficiency
(TE %)
% Cr crop
removal
25 2.23 0.73 80.54 0.686
50 3.14 0.68 83.20 0.689
75 8.46 0.36 55.94 0.569
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0
100
200
300
400
500
600
700
0 25 50 75
Levels of Cr(mg/Kg soil)
Con
c. o
f Cr(
µg/g
)
RootShoot
Figure 1.Partitioning of Cr in Euphorbia milli
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0
500
1000
1500
2000
2500
0 25 50 75
Levels of Cr(mg/kg soil)
Upt
ake(
µg/p
ot)
RootShootTotal
Figure 2.Uptake of Cr by Euphorbia milli
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