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Bioresource Technology 99 (2008) 7091–7097

Concurrent removal and accumulation of heavy metals

by the three aquatic macrophytes
Virendra Kumar Mishra *, B.D. Tripathi
Centre for Advanced Studies in Botany, Banaras Hindu University, Varanasi 221005, India

Received 4 October 2007; received in revised form 23 December 2007; accepted 3 January 2008
Available online 4 March 2008

Abstract

Under the present investigation effectiveness of three aquatic macrophytes Pistia stratiotes L. (water lettuce), Spirodela polyrrhiza W.
Koch (duckweed) and Eichhornia crassipes were tested for the removal of five heavy metals (Fe, Zn, Cu, Cr and Cd). These plants were
grown at three different concentrations (1.0, 2.0 and 5.0 mg l 1) of metals in laboratory experiment. Result revealed high removal (>90%)
of different metals during 15 days experiment. Highest removal was observed on 12th day of experiment, thereafter it decreased. Results
revealed E. crassipes as the most efficient for the removal of selected heavy metals followed by P. stratiotes and S. polyrrhiza. Results
from analysis confirmed the accumulation of different metals within the plant and a corresponding decrease of metals in the water. Sig-
nificant correlations between metal concentration in final water and macrophytes were obtained. Plants have accumulated heavy metals
in its body without the production of any toxicity or reduction in growth. Selected plants shown a wide range of tolerance to all of the
selected metals and therefore can be used for large scale removal of heavy metals from waste water.
Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Heavy metals; Aquatic macrophytes; Phytoremediation; Hyperaccumulation; Toxicity

1. Introduction removal of pollutants by the use of plants offers a promis-

Contamination of the aquatic environment by the heavy
metals has become a serious concern in the developing
world. Heavy metals unlike organic pollutants are the per-
sistent in nature, therefore, tends to accumulate in the dif-
ferent components of the environment (Chandra et al.,
1997). These metals are released from a variety of sources
such as mining, urban sewage, smelters, tanneries, textile
industry and chemical industry. These heavy metals are
highly toxic to the aquatic plants and animals as well as
do not vanish easily from the environment. Their treatment
usually requires removal through some technology. The
technologies used for their treatment are reverse-osmosis,
ion-exchange, electrodialysis, adsorption, etc. Most of
these technologies are quite costly, energy intensive and
metal specific. Contrary to this phytoremediation, i.e.
*
Corresponding author.
E-mail address: virendra78@rediffmail.com (V.K. Mishra).
ing technology for heavy metal removal from waste water
(Singh et al., 1996; Miretzky et al., 2004). Aquatic macro-
phytes have great potential to accumulate heavy metals
inside their plant body. These plants can accumulate heavy
metals 100,000 times greater than in the associated water
(Mishra et al., in press). Therefore, these macrophytes have
been used for heavy metal removal from a variety of
sources (Axtell et al., 2003; Maine et al., 2001; Miretzky
et al., 2004; Hassan et al., 2007; Mishra et al., 2008). The
aquatic macrophytes are thought to remove metals by three
patterns (a) metals are restricted from entering the plant
and attaches to the cell wall (b) metals are accumulated
in the root, but translocation to the shoot is constrained
(c) hyperaccumulation, metals are concentrated in the
plant parts. The hyper accumulative capacities of the aqua-
tic macrophytes are beneficial for the removal of heavy
metals. Most of the studies regarding the phytoremediation
of heavy metals are confined to a few plants and metals.
Still, there are a few studies available regarding the removal

0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2008.01.002
7092 V.K. Mishra, B.D. Tripathi / Bioresource Technology 99 (2008) 7091–7097
efficiencies of different plant groups and removal of several
heavy metals at a time. Therefore, present study was per-
formed to harness the metal removal capacities of selected
macrophytes and to determine the suitability of these
plants for their large scale utilization.
2. Methods
2.1. Experimental plants
Three aquatic macrophytes Pistia stratiotes L. (water
lettuce), Spirodela polyrrhiza W. Koch (duckweed) and
Eichhornia crassipes (Water hyacinth), were selected to
asses their heavy metal removal capacities for five heavy
metals (Cu, Cd, Cr, Zn and Fe) from water under labora-
tory conditions. These macrophytes are perennial aquatic
weed spread all over the world, considered noxious and
extremely invasive for freshwater environments. These spe-
cies carry out their entire life cycle as free-floating plant,
only the root system is completely submerged. All of the
above species take up metals from water, producing an
internal concentration several folds greater than their sur-
roundings and showing much higher metal-accumulating
capacity than non-hyper accumulating terrestrial plants.
Higher biomass, greater removal efficiency, fibrous root
system and greater tolerance for metals make them excel-
lent choice for the phytoremediation process (Maine
et al., 2001).
2.2. Experimental set-up
Experimental plants were collected from Govind Bal-
labh Pant (GBP) Sagar (Mishra et al., in press). These
plants were grown in 15 l experimental tanks filled with
10 l of water. Five heavy metals i.e. Fe, Cu, Zn, Cd and
Cr were added together in an amount which makes their
concentration in the experimental tanks as 1 mg l 1, 2 mg
l 1, 5 mg l 1, respectively. Experimental plants were grown
in duplicate as following.
1. P. stratiotes 100 g (fresh weight) grown in 10 l of water.
Heavy metals were added simultaneously in the experi-
mental tanks to make their concentration in the all exper-
imental tank 1 mg l 1, 2 mg l 1, 5 mg l 1, respectively.
2. E. crassipes 100 g (fresh weight) grown in 10 l of water.
Heavy metals were added simultaneously in the experi-
mental tanks to make their concentration in the all exper-
imental tank 1 m l 1, 2 mg l 1, 5 mg l 1, respectively.
3. S. polyrrhiza 100 g (fresh weight) grown in 10 l of water.
Heavy metals were added simultaneously in the experi-
mental tanks to make their concentration in the all exper-
imental tank 1 mg l 1, 2 mg l 1, 5 mg l 1, respectively.
4. Metal control – Heavy metals Fe, Cu, Zn, Cd and Cr
were maintained at 1 mg l 1, 2 mg l 1, 5 mg l 1, respec-
tively in the experimental tank without any plants.
5. Plant control – Plants were grown in 10 l of water con-
taining no metals.
2.3. Heavy metal preparation
Five heavy metals Fe, Cu, Zn, Cd and Cr were added to
make their concentrations 1 mg l 1, 2 mg l 1, 5 mg l 1,
respectively in water. These heavy metals were added as
ferrous ammonium sulphate [Fe(NH4)2(SO4)2
6 H2O],
cupric nitrate [Cu(NO3)2
3H2O], zinc nitrate
Zn(NO3)2
3H2O, cadmium nitrate (Cd(NO3)
H2O) and
potassium dichromate (K2Cr2O7
5H2O). The reagents
were dissolved in distilled water to get the desired contam-
ination level.
2.4. Analysis of heavy metals in water and plant samples
Water samples of 40 ml were taken from all the experi-
mental sets at 24 h interval for heavy metal analysis. Phys-
ico-chemical analysis of water sample was done by
adopting Standard Methods for the Examination of Water
and Wastewater as prescribed by American Public Health
Association (APHA, 1998), American Water Works Asso-
ciation and Water Pollution Control Federation. The met-
als analyses were carried out by means of atomic
absorption spectrophotometry (AAS, Perkin Elmer). Plant
samples were analyzed for heavy metals on two occasions,
first on initiation date of removal experiment and second
after 15 days removal experiment. Plants samples (2.0 g)
were dried in a fan forced oven for 24 h at 80 °C. After
the drying the plant samples were grounded and digested
with HNO3–HClO4 (2:1 v/v) and diluted to 100 ml with
de-ionized distilled water. The digested plant samples were
analyzed for heavy metals by the same procedure as it was
done for water sample. Heavy metals were analyzed with
two procedures contained in ‘‘Standard Methods for the
Examination of Water and Wastewater” (APHA, 1998):
Extraction air acetylene flame method (3111C) and direct
air–acetylene flame method (3111B). The former was used
to measure metals present at low concentration and latter
to measure high metal concentration (above the linear
range of the first method). The accuracy of the results for
metal was evaluated with the certified reference material
(CRM) with the 3111C method (APHA, 1998). Two ali-
quots of the CRM were spiked with a known amount of
metal spike standard and one spike was analyzed according
to 3111C method and other with the 3111B (APHA, 1998).
The metal recoveries were in the 96–105% which was
acceptable.
2.5. Analysis of plants for biochemical parameters
Biochemical analyses of the plant tissues were per-
formed on two occasions, first on the initiation date of
removal experiment and second after 15 days removal
experiment. Initially after the collection plants were washed
thoroughly with water from their sampling sites. Two spec-
imens of the each species were collected as a whole plant
sample from each site. Plants were carefully washed using
tap water then distilled water to remove all the debris.
 V.K. Mishra, B.D. Tripathi / Bioresource Technology 99 (2008) 7091–7097
The washed samples were cut into small pieces and dried to
a constant mass into fan forced oven at 80 °C for 24 h. The
oven dried material was chopped finely then grounded to
facilitate organic matter digestion. The method of Lowry
et al. (1951) and phenol sulphuric acid method were used
for protein and sugar estimation, respectively. For the esti-
mation of photosynthetic pigment leaf samples were
homogenized with mortar and pestle in 3 ml of phosphate
buffer, at pH 7.2 with 10 mM KCl. The homogenized was
then extracted with cold acetone (80%). The absorbance of
pigment was measured at wavelength of 470, 626, 645, 663
and 730 nm with spectrophotometer and contents of chl a,
chl b were calculated.
2.6. Statistical analysis
Metal concentration in mining effluent and water were
reported in mg l 1 and metal concentrations of sediments
and plant material are reported in kg g 1 dry weight and
are means of three replicates. Correlation and regression
analysis were performed by using SPSS 12 package.
3. Results and discussion
3.1. Heavy metal removal from metal solution
Chemical composition of the water used for the removal
experiment was slightly alkaline with moderately high bio-
chemical oxygen demand (BOD) and chemical oxygen
demand (COD). All the parameters were maintained within
the same range except the heavy metal concentration which
was 1, 2 and 5 mg l 1 in three different experimental sets.
The ambient air and water temperature was maintained
between 25 and 27 °C. Results revealed increasing trend
of removal with the increasing incubation period. Analysis
of variance showed significant difference (p < 0.001)
between the removal and incubation period for all the
experimental sets. Analysis of metal concentration with
increasing time has suggested that the metal concentrations
decreased up to 12th day in the different experimental sets.
Further analysis showed a little increase, however it was
slightly decreased on some instances (Fig. 1). Accumula-
tion of heavy metals in macrophytes under present study
was higher (Table 1) and this is in agreement with Gupta
and Chandra (1998), Deng et al. (2004), Miretzky et al.
(2004), and Mishra et al. (2008). The results of the regres-
sion analysis confirm that the metal removal by the three
macrophytes were proportional to metal concentrations
(p < 0.01). The results obtained from the present study
indicated that metal removal percentages were highest at
2 mg l 1 for all the three macrophytes for most of the
times. Removal percentage decreases for the metals at
5.0 mg l 1 with few exceptions (Table 2). The heavy metals
removed by E. crassipes were very high and ranged between
77% and 95% in 12 days incubation period. P. stratiotes
proved equally efficient in the cleaning of heavy metals
from the different experimental sets. A small decrease was
7093
observed on 15th day in all the experimental sets that indi-
cated the release of metals from plants into the water. The
removal efficiencies varied for different heavy metals with
varying concentration. The metal removal efficiencies were
increased initially with increasing time and concentrations
as shown by greater removal efficiency at 2.0 mg l 1 in
comparison to the same at 1.0 mg l 1, 5.0 mg l 1, respec-
tively. The highest removal percentage at 1.0 mg l 1 was
shown by E. crassipes (92) followed by P. stratiotes (91)
for Zn where as this was increased at 2.0 mg l 1 and it
was maximum for E. crassipes and P. stratiotes as 95%
for Fe. Removal efficiencies reached highest at 2.0 mg l 1
for all the metals except Cu which has shown slightly
increased removal percentage at 5.0 mg l 1 (Table 2). The
experimental plants showed excellent performance in
removing the metals as they were able to remove up to
95% of heavy metals in 12 days incubation period. Varia-
tion in metals concentration with in water with time frame
showed >50% removal within the first three days of the
experiment. Although the removal percentages shown by
P. stratiotes and E. crassipes were very high for Fe, Zn
and Cu, nevertheless these percentages were lower for Cd
and Cr. Chromium is supposed to be one of the most dif-
ficult metals to remove from the water due to the fact that
macrophytes do not require this element for any physiolog-
ical purposes. S. polyrrhiza removed least percentage of
Cr(62) and Cd(65) at 2.0 and 5.0 mg l 1. Many plant spe-
cies have low mobility of Cr due to the fact that there
are barriers or lack of transport mechanism suitable for
Cr transport from roots to shoots (Kleiman and Cogliatti,
1998). Control experimental sets showed loss of 1.7–5.1
percent heavy metals from the water at all the three concen-
trations. This loss might be due to sedimentation, adsorp-
tion to clay particles and organic matter, co-precipitation
with secondary minerals. Among the different plant used
for the E. crassipes is highly efficient in heavy metal
removal especially at lower concentrations (Soltan and
Rashed, 2003). These macrophytes having high biomass
fibrous root and broad leaves due to which it absorb higher
concentrations of heavy metal. Study reveals significantly
higher (p < 0.001) accumulation of heavy metals in the
macrophytes. Higher removal of heavy metals by P. strati-
otes and S. polyrrhiza may be attributed to their luxuriant
growth in heavy metal and nutrient rich media, greater bio-
mass accumulation (Mant et al., 2007) increased diversity
and preferential absorption of metals from medium.
3.2. Accumulation of heavy metals in macrophytes
A comparison between initial and final metal concentra-
tions within the plant had shown that the final concentra-
tions were more than 10 times to the initial for S.
polyrrhiza, E. crassipes and P. stratiotes, but Fe showed a
smaller increase in total concentration most likely because
the macrophytes had previously accumulated the higher
concentrations in the plant (Table 1). Iron (Fe) had the
highest initial concentration within plants among all the
7094 V.K. Mishra, B.D. Tripathi / Bioresource Technology 99 (2008) 7091–7097

5
4.5
4.5
4
4
2
3.5 2
3.5 R = 0.9048 R = 0.8899
3
3
2.5
Zn (mg l )
-1

Cu (mg l )
2.5

-1
2 2
2 R = 0.951
2 1.5
1.5 R = 0.9246
2
R = 0.9493
1 1
2
R = 0.8319
0.5 0.5

0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
-0.5 -0.5
No. of days Time (No.of Days)

4.5
5 4
2
R = 0.9221
3.5
4 2
R = 0.8836 3
Cd (mg l )
-1

3 2.5
Fe(mg l )

2
-1

2
2 2 R = 0.9065
R = 0.8741
1.5

1 2
1 R = 0.929
2
R = 0.9124
0.5

0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16

-1 Time (Days) Time (No. of days)

4.5

3.5

2.5
Cr (mg l )
-1

2
R = 0.8074
2
2
1.5 R = 0.874

1 2
R = 0.9101
0.5

0
0 2 4 6 8 10 12 14 16
-0.5
Time (No. of days)

Fig. 1. Removal of heavy metals by Eichhorrnia cressipes.

metals used in the experiment. Accumulation of the Fe ran- tosynthesis and has been shown for plants cultivated at
ged between 15.335 and 25.5 g kg 1. Its highest value was enhanced concentrations.
observed in E. crassipes while S. polyrrhiza showed the low- The highest concentration of Cu was 2.75 g kg 1 dry
est value at 5.0 mg l 1. Accumulation of Fe increased with weight of the plant tissue in E. crassipes at 5 mg l 1and
increasing concentration. Higher concentration did not the lowest was 0.165 g kg 1 dry weight in S. polyrrhiza.
affect the growth of plants. Accumulation of Zn ranged Although Cu is an essential micronutrient for normal plant
between 0.96 and 6.5 g kg 1. Highest accumulation of Zn metabolism, playing an important role in a large number of
was shown by E. crassipes at 2.0 mg l 1 and lowest by S. metalloenzymes, photosynthesis related membrane struc-
polyrrhiza at 5.0 mg l 1. This often results in inhibited pho- ture; it has been reported to be among the toxic heavy met-
 V.K. Mishra, B.D. Tripathi / Bioresource Technology 99 (2008) 7091–7097 7095

Table 1 2.0 mg l 1. Cadmium has become an increasing problem

Initial and final concentrations of heavy metals because of its toxic effects on biological systems. Although
Heavy Concentrations of heavy metals not essential for plant growth, Cd2+ ions are readily taken
metal up by roots and translocated into the leaves in many
Initial Final Final Final
(mg g 1) (1.0 mg l 1) (2 mg l 1) (5 mg l 1) plants, depressing growth by affecting photosynthesis,
(mg g 1) (mg g 1) (mg g 1) chlorophyll fluorescence and nutrient uptake by plants.
Pistia stratoites In present study the toxicity of Cd was not up to the level
Fe 8.682 10.8 12.226 15.334 to affect the growth of plants.
Zn 0.1 0.62 0.75 0.98
Cu 0.03 0.652 0.782 0.875
Chromium is a non-essential element to the plants. Its
Cd 0.02 0.21 0.39 0.321 compounds are highly toxic to plants, detrimental to their
Cr 0.01 0.018 0.098 0.075 growth and development (Rai et al., 1995). It is reported to
Spirodela polyrrhiza be toxic to most of the higher plants at 100 g kg 1 dry
Fe 3.2 9.08 15.556 18.996 weight. Under present investigation E. crassipes showed
Zn 0.05 0.782 1.3 1.5 highest accumulation of Cd as 0.30 g kg 1 followed by P.
Cu 0.02 0.186 0.165 0.145 stratiotes as 0.096 g kg 1 at 2.0 mg l 1 concentration.
Cd 0.01 0.055 0.25 0.14
The maximum quantity of element contaminant was
Cr 0.001 0.08 0.071 0.065
always contained in roots and a minimum in the vegetative
Eichhornia crassipes and reproductive organs. Plants acquire essential metals as
Fe 12.5 21.2 24.56 25.5
micronutrients, but also to regulate their concentrations
Zn 0.7 2.31 6.512 5.52
Cu 0.05 0.85 2.55 2.76 and prevent intoxication plants have a wide variety of
Cd 0.015 0.175 0.31 0.27 metal influx as well as efflux transporters (Williams et al.,
Cr 0.012 0.142 0.306 0.276 2000; Hall and Williams, 2003). Previous studies on the
accumulation of various metal ions by aquatic plants have
shown that the deposition of most metals was higher in
Table 2
Removal of heavy metals through three aquatic macrophytes
roots than the other parts of plants (Satyakala and Jamil,
1992; Zaranyika and Ndapwadza, 1995; Chandra and
Heavy Concentrations % Removal
metals (mg l 1)
Kulshreshtha, 2004). This is in line with the findings of
Pistia Spirodela Eichhornia present study.
stratoites polyrrhiza cressipes
Fe 1 87 83.5 85.7 3.3. Heavy metal concentration in plant tissue and its impact
2 95 81 90.1
5 90 77.5 78.6
on biochemical parameters
Cu 1 96 91 95
Heavy metal accumulation in aquatic macrophytes is
2 87 83 89
5 88 76 86 known to produce significant physiological and biochemi-
cal responses towards the growth of roots, stems and leaves
Cd 1 78 63 81
2 82 71 85
(Satyakala and Jamil, 1992; Chandra and Kulshreshtha,
5 70 65 77 2004; Shankers et al., 2005). Biochemical parameters like
protein, sugar and chlorophyll content of plants before
Cr 1 81 83 85
2 75 75 89 and after treatment is cited in Table 3. The results revealed
5 70 62 81 that these parameters decreased in plant tissues after 15
Zn 1 90 90 92
days incubation period. Chlorophyll content decrease from
2 92 92 95 10.31 mg g 1 to 4.17 mg g 1 in E. crassipes, 2.67 mg g 1 to
5 82 82 85 1.6 in P. stratiotes and 7.12 to 3.06 mg g 1 in S. polyrrhiza.
The same decreasing trend was also observed for protein
and sugars (Table 3). Morphological symptoms such as yel-
als (Li and Xiong, 2004). Excessive Cu accumulated in lowing and chlorosis of leaves and root shedding due to
plant tissue can be toxic to plants, affecting several physio- metal toxicity were evident in the some of the macrophytes
logical and biochemical processes and growth. Copper exposed to 5.0 mg l 1 Cr and Cd. Other metals (Fe, Zn and
treatment brings changes in nitrogen metabolism with a Cu) did not show such symptoms at all the three concentra-
reduction of total nitrogen (Llorens et al., 2000). Copper tions of metals to which macrophytes were exposed.
exposure results in increase of free amino acids. Under Reduction in both protein and chlorophyll contents in
present investigation toxicity or growth reduction did not the test plants with increasing duration of exposure and
occur. concentrations is in agreement with Shankers et al.
Cadmium (Cd) is a non-essential toxic heavy metal, can (2005). The reduction of chlorophyll content in macro-
have significant effects on higher plants (Sanita de tappi phytes may be attributed to inhibition of chlorophyll syn-
and Gabbrielli, 1999). P. stratiotes showed highest accumu- thesis which results in the loss of photosynthetic activity
lation of Cd as 0.39 g kg 1 and E. crassipes 0.31 g kg 1 at (Chandra and Kulshreshtha, 2004). The decrease in sugar
7096 V.K. Mishra, B.D. Tripathi / Bioresource Technology 99 (2008) 7091–7097

Table 3 Acknowledgement
Chlorophyll, protein and sugar content in aquatic macrophytes at different
concentrations of heavy metals
Authors are thankful to the Council of Scientific and
Concentration Chlorophyll Protein Sugar Industrial Research, New Delhi, for financial assistance.
(mg/l) (mg g 1) (mg g 1) (mg g 1)
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