Bioresource Technology 98 (2007) 17881794

Phytoextraction capacity of the plants growing

on tannery sludge dumping sites
Amit K. Gupta, Sarita Sinha

Ecotoxicology and Bioremediation Group, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India
Received 7 March 2006; received in revised form 23 June 2006; accepted 30 June 2006
Available online 14 September 2006

Abstract
The study was undertaken to assess the phytoremediation potential of four plants (Sida acuta, Ricinus communis, Calotropis procera,
Cassia stula) growing at a tannery sludge disposal site. Results showed that maximum amount of K, Fe and Ni was associated with residual fraction, whereas, Zn, Mn, Cr, Pb, Cd and Co was found in FeMn oxide fraction. Cu and Na were mostly associated with organic
matter and carbonate fraction, respectively. The results demonstrated that the levels of accumulation of metals was found high in all the
studied plants and followed the order; C. procera > S. acuta > R. communis = C. stula. The principal components analysis (PCA)
revealed that translocation behavior of Cu, Zn and Mn, Cr were found similar in the plants. Correlation analysis between metal accumulation in the plants with DTPA extractable metals emphasized that S. acuta and C. stula provide better value of correlation for most of
the tested metals. The values of transfer factor were also found high for most of the tested metals in the plants of S. acuta. Overall, the
plants of S. acuta and C. stula were found suitable for the decontamination of most of the metals from tannery waste contaminated sites.
2006 Elsevier Ltd. All rights reserved.
Keywords: Tannery sludge; DTPA; Sequential extraction; Metals

1. Introduction
Heavy metals are considered as one of the major toxic
pollutant due to their long persistent in the environment.
The sludge is considered as a source of potentially toxic elements and its disposal is problematic due to the presence of
several heavy metals. The production of sludge is increasing
day by day as a result of wastewater treatment. It is generally bulky with high moisture contents and their composition may range from highly organic to minerals
depending on their origin. It is likely to be used as fertilizer
material in many countries. The trace elements bioavailability has been considered as one of the most crucial problem
in agricultural and environmental studies. There has been a
steady increase in the number of investigations related to
both understanding the process involved in the uptake of
an element (essential and non essential) by the plants and
*

Corresponding author. Tel.: +91 522 2205831 35x221.

E-mail address: sinha_sarita@redimail.com (S. Sinha).

0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.06.028

to nd out the most reliable method for the prediction of

availability of a given element to plants (Kabata-Pendias,
2004; Gupta and Sinha, 2006). The mobility of trace metals
and their bioavailability are related to eco-toxicity in the
plants which depends strongly on their specic chemical
forms or binding. Consequently, these are the parameters
that have to be determined rather than the total element
contents in order to assess the toxic eects and to study geochemical pathways (Adriano et al., 2004). A potential strategy to remediate these soils is the use of plants to remove
pollutants from the habitat or to render them harmless (Salt
et al., 1998; Lasat, 2002). Soil contaminated with multiple
heavy metals can present a dicult challenge for both the
approaches to phytoextraction. Although, some hyperaccumulator plants may appear to be capable of accumulating
elevated concentrations of several heavy metals. Simultaneously, there is still considerable specicity in metal hyperaccumulation (Chaney et al., 2005).
India is one of the largest producers of leather in the
world and about 700,000 tons of wet salted hides and skins

A.K. Gupta, S. Sinha / Bioresource Technology 98 (2007) 17881794

are processed in about 3000 tanneries. Uttar Pradesh is an

important state of India holding sizeable population of
livestock and prominent center for leather processing.
Proper disposal of chromium contaminated hazardous
sludge has become a major environmental problem. The
economically important plants including medicinal plants
growing at contaminated site is a matter of serious concern.
In recent times, the accumulation of pollutants particularly
metals in such plants have been reported which may alter
or aect their medicinal properties (Ajasa et al., 2004;
Rai et al., 2005; Sinha et al., 2006).
The present study was carried out to assess the translocation of metals in dierent parts of the plants growing at
tannery sludge dumping site. Further, the metals were
extracted (single and sequential) from tannery sludge and
its correlation was calculated between the extracted metals
from the tannery sludge and metal accumulation in to the
plants in order to assess the potential of these plants for
phytoextraction purposes.
2. Methods
Plants of Sida acuta (sida), Ricinus communis (castor),
Calotropis procera (madar), and Cassia stula (cassia) growing on the tannery sludge at the dumping site of the UASB
treatment plant at Jajmau, Kanpur (Uttar Pradesh, India)
were collected randomly, packed into polythene bags and
brought to the laboratory for the analysis. The plants were
washed with doubled distilled water to eliminate the adhering soil and other contaminants. The dry weights (g) of the
plants at the time of sampling were as follows: S. acuta
(29.07), R. communis (21.71), C. procera (29.13) and C. stula (19.13). The plants were separated, dried, crushed and
mixed thoroughly for the processing of metal analysis.
Sludge samples were oven dried, ground and sieved with
2 mm mesh size and used for further analysis. All the samples were analyzed in triplicates. Certied aqueous standards of the elements (SIGMA) were used to prepare
standard curve for Atomic Absorption Spectrophotometer.
2.1. Physico-chemical parameters of the tannery sludge
The physico-chemical parameters of sludge i.e., organic
carbon (OC), organic matter (OM) and cation exchange
capacity (CEC) were estimated using methods of Kalra
and Maynard (1991). pH of the sludge (1:2.5 sludge water
suspension) was measured using Orion pH meter (Model

1789

420). The salinity (Sal.) and electrical conductivity (EC)

was measured using Orion Conductivity Meter (Model
150) in the sludge (1:2.5 sludge water suspension).
2.2. Sequential extraction
The sludge was fractionated (ve steps) by the method
of Tessier et al. (1979) and chemical reagent, extraction
conditions and corresponding fractions are listed in Table
1.
2.3. Digestion of samples for metal estimation
After homogenization, a small portion (0.10 g) of plant
samples was digested in 5 ml of 70% HNO3 using Microwave Digestion System MDS 2000 in closed teon vessels.
For digestion of sludge (ca. 0.50 g), 10 ml double distilled
water + 5 ml HCl + 4 ml HF + 1 ml HNO3 was used.
The metal contents of digested samples were determined
with GBC Avanta R Atomic Absorption Spectrophotometer. DTPA extractable metal fraction of sludge samples
(4 g) was obtained by mechanical shaking with 40 ml of
0.5 M DTPA, 0.01 M CaCl2, 0.1 M TEA (triethanolamine)
buered at pH 7.3 for 2 h (Lindsay and Norvell, 1978).
2.4. Statistical analysis
Statistical analysis, including principal component analysis (PCA) was performed using SPSS statistical software.
In PCA, it was calculated based on the correlation matrix.
Varimax Normalization was used as the rotation method in
the analysis. The heavy metals, which showed a close correlation, were identied and grouped for further analysis.
2.5. Quality control and quality assurance
The standard reference material of metals (E-Merck,
Germany) was used for the calibration and quality assurance for each analytical batch. Analytical data quality of
metals (Cd, Cr, Cu, Pb) was ensured using EPA samples
(Lot TMA 989) in water and the results were found to be
within 4.8% of certied values. Method validation (accuracy and repeatability) was performed by analyzing the
certied materials reference solution (BND 1101.02) of
multi-elements (Zn, Fe, Cu) and single element, BND
102.03 (Pb); BND 402.02 (Cr) and BND 1001.02 (Ni) provided by National Physical Laboratory (NPL), New Delhi,

Table 1
Sequential extraction procedures and their corresponding fractions
Steps

Fractions

Extraction procedures

1
2
3
4

Exchangeable (EXC)
Bound to carbonate (CAR)
Bound to FeMn oxides (FeMn)
Bound to organic matter (OM)

Residual (RES)

1 g of soil sample, 8 ml 1 mol l 1 MgCl2, pH 7, shake 1 h, room temperature

8 ml 1 mol l 1 CH3COONa, adjusted pH to 5.0 with CH3COOH, shake 5 h, room temperature
20 ml 0.04 mol l 1 NH2OH HCl in 25% CH3COOH, pH 2.0, water bath, 96 C, 6 h, occasional shaking
3 ml 0.02 mol l 1 HNO3, 30% H2O2 (adjusted to pH 2.0), water bath, 85 C, 5 h, 3.2 mol l 1
CH3COONH4 in 20% (v/v) HNO3, shake 30 min
3 ml HNO3 + HClO4 + HF under high pressure, 170 C

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A.K. Gupta, S. Sinha / Bioresource Technology 98 (2007) 17881794

India and the results were found to be within 1.70% of

certied values (n = 10). For plants, recoveries of metals
from the plant tissues were found to be more than 98.5%
as determined by digesting three samples each from an
untreated plant with known amount of metals.
3. Results and discussion
3.1. Physico-chemical analysis
The analysis of physico-chemical properties of tannery
sludge (Table 2) showed high level of EC (3850 lS cm 1),
salinity (7.4&), CEC (184.77 cmol (p+) kg 1), OM
(10.63%) and OC (6.17%) as reported earlier (Gupta and
Sinha, 2006). The level of total metal content was found
signicantly high as compared to DTPA extractable metals
in the tannery sludge (Table 2). The total metal present in
the soil is not available to the plant grown therein and
DTPA-extractable metals can be used as an indicator of
bioavailability and toxicity of heavy metals. Lindsay and
Norvell (1978) selected DTPA for bioavailable metals to
identify near-neutral and calcareous soils with insucient
transition metals. The physico-chemical characteristics
(pH, CEC, quantity and quality of OM, oxides, clay minerals) of soil are known to regulate the fate of the metals
and largely inuence rhizospheric processes. The metals
bioavailability of soil also depends to a large extent on their
distribution between solid and solution phases, which, in
turn, dependent on the soil processes like cation exchange,

Table 2
Physico-chemical properties of tannery sludge collected from Jajmau,
Kanpur
Parameters

Tannery sludge

pH (1:2.5)
EC (lS cm 1)
Salinity (&)
CEC (cmol (p+) kg 1)
OC (%)
OM (%)

7.83 0.01
3850 10
7.4 0.00
184.77 11.91
6.17 0.03
10.63 0.09

Metals

DTPA extractable
metals (mg kg 1 dw)

Total metals
(mg kg 1 dw)

Na
K
Fe
Zn
Mn
Cu
Cr
Pb
Ni
Cd
Co

1039.48 10.96c
153.64 6.71b
94.05 5.66c
55.06 2.48c
8.61 1.40c
25.10 2.57c
19.59 0.75c
19.66 1.51b
4.63 0.17b
23.24 3.06b
12.35 2.11a

25876.35 1145
12305.09 1662
19401.44 61
340.69 11.10
294.19 15.50
188.09 5.81
7489.76 203
143.43 8.72
219.16 21.73
95.1 1.25
31.7 3.40

All the values are mean of three replicates SD. Student t-test (two tailed
as compared to control).
a
p < 0.02.
b
p < 0.01.
c
p < 0.001.

specic adsorption, precipitation, and complexation. Correlation analysis between metal accumulations in the plants
with DTPA extractable metals emphasized that S. acuta
and C. stula provide better value of correlation for most
of the tested metals. The eect of organic matter amendments on heavy metal solubility also depend greatly upon
the degree of humication of their OM and their eect
upon soil pH (Walker et al., 2003). The high level of
CEC in the tannery sludge is due to the presence of organic
matter, a factor that may aect both soluble and exchangeable metal levels (Gupta and Sinha, 2006).
3.2. Sequential extraction
The metals in tannery sludge were fractionated in to ve
dierent fractions (EXC, CAR, FeMn, ORG, RES)
(Table 3). The analysis of the data showed that maximum
level of K, Fe and Ni was bound with RES fraction and
Zn, Mn, Cr, Pb, Cd and Co was mostly associated with
FeMn oxide fraction. However, majority of Cu and Na
are associated with OM and CAR fraction, respectively.
Gupta and Sinha (2006) reported that Mn, Zn, Cr and
Cd were mostly associated with FeMn oxide fraction in
most of the amendments, K and Ni was found in RES fraction, Fe and Cu was bound with OM and RES fractions
and Na was associated with CAR fraction of tannery
sludge. Koleli (2004) reported that maximum level of Cr
was associated with RES fraction followed by the OM
form. The organic matter is one of the factors that may
reduce the ability of these metals to be phytotoxic in the
soil due to metal-organic complexation. Amir et al.
(2005) argued that copper strongly bound to OM and
would be released slowly over time as the OM of the sludge
is decomposed, whereas, Cd and Zn are not bound as
strongly to OM. Similar to the present study, Walter and
Cuevas (1999) reported that Cr and Cd was found maximum in FeMn oxide fraction of the soil amended with
sewage sludge. Fuentes et al. (2004) reported maximum
amount of Ni bound with RES fraction. The signicant
increase in the level of Ni with residual fraction may be
due to alkaline stabilization process. Metals conned in
Table 3
Fractionation of metals in tannery sludge among dierent phases obtained
by ve steps procedure
Metals

Na
K
Fe
Zn
Mn
Cu
Cr
Pb
Ni
Cd
Co

Extraction percentage (%)

EXC

CAR

FeMn

ORG

RES

1.95
0.25
0.04
0.77
1.37
0.71
0.06
0.34
1.88
5.88
10.93

78.09
2.46
0.06
11.61
14.35
1.48
0.15
1.37
1.47
23.67
26.65

0.26
4.32
39.27
69.32
52.42
3.70
62.10
50.96
29.55
65.67
39.86

0.15
5.73
7.06
12.02
5.77
82.69
10.21
35.50
14.60
4.78
13.84

19.55
87.24
53.57
6.28
26.10
11.42
27.48
11.82
52.51
0.00
8.72

A.K. Gupta, S. Sinha / Bioresource Technology 98 (2007) 17881794

1791

Fig. 1. Accumulation of metals (mg kg 1 dw) in dierent parts of plants growing at tannery sludge dumping sites. All the values are mean of three
replicates SD. Sc = S. acuta; Rc = R. communis; Cp = C. procera; Cf = C. stula.

the residual fractions are usually not expected to release

over short periods of time under the conditions usually
encountered in nature (Su and Wong, 2003).
3.3. Metal accumulation in plants
The analysis of the results of metal accumulation in the
plants grown on sludge dumping sites (Figs. 1 and 2)
showed that the magnitude and relative distribution of
metals in the plant was dierent. Overall, the total metal
accumulation was in the order K > Na > Fe > Cr > Mn >
Zn > Cu > Pb > Ni > Cd > Co, which varied from one part
to another. In most of the plants, the translocation of Cr
was better in upper parts of the plants. The accumulation
of toxic metals Pb and Cd was maximum in fruit parts of
the plants whereas, accumulation of essential micronutrients (Fe, Zn, Cu and Mn) was least. However, accumulation of macronutrients Na and K was recorded maximum
in upper part of the plants including leaves and fruits.
The transport of metals from roots to shoot includes
long distance translocation in the xylem and storage in
the vacuole of leaf cells and these processes aected by
many factors (Yang et al., 1997). The process of metal

uptake and accumulation by dierent plants depend on

the concentration of available metals in soils, solubility
sequences and the plant species growing on these soils.
Generally, the concentration of metals in shoots is lower
than in roots (Gupta and Sinha, 2006). Similarly, Sinha
et al. (2006) reported the accumulation and translocation
of metals in the vegetables/crops growing at Jajmau, Kanpur (India) where treated tannery wastewater is being used
for irrigation. In most of the plants, the accumulation of
metals was found less in upper part of the plants. In the
present study, the accumulation of essential micronutrients
was found higher in upper parts of the most of the tested
plants, whereas, toxic metals accumulation was restricted
in lower parts of the plants except Cr.
The accumulation and distribution of metals in the plant
tissue are important aspects to evaluate the role of plants in
remediation of contaminated sites (Salt et al., 1998; Garbisu and Alkorta, 2001). The success of a phytoremediaton
process depends on adequate plant yield and hyperaccumulator plants possess an ability to take up abnormally high
amounts of heavy metals in their shoots (Chaney et al.,
2005). The results of the present study also showed high
accumulation of metals in the upper part of the plant,

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A.K. Gupta, S. Sinha / Bioresource Technology 98 (2007) 17881794

Fig. 2. Accumulation of metals (mg kg 1 dw) in dierent parts of plants growing at tannery sludge dumping sites. All the values are mean of three
replicates SD. Sc = S. acuta; Rc = R. communis; Cp = C. procera; Cf = C. stula.

which showed usefulness of these plants to be used for phytoremediation of metals from contaminated sites. Khan
(2001) reported that plant cover (Dalbergia sissoo, Acacia
arabica and Populus euroamericana) reduced the level of
metals at tannery euent contaminated site, however,
roots of the plant showed no correlation between Cr content and DTPA extractable metals in soil. Similarly, Armienta et al. (2001) also reported high concentration of Cr in
the soil and plants irrigated with tannery wastewater in the
Leon valley, Mexico. The roots in all the plants mostly
retained chromium and no correlation seemed to exist
between total Cr content in the soils and plants. In contrast, Gupta and Sinha (2006) recently reported poor translocation of Cr, Fe, Mn, Cu, Pb, Cd and Ni in the plants of
Sesamum indicum grown on soil amended with dierent
percentage of tannery sludge.
3.4. Correlation coecient between total metals
accumulation and DTPA extractable metals
In the present study, DTPA extractant was used to
assess the bioavailability of metals. Correlation analysis

(r) was performed between total metal accumulation in

the plants and DTPA extractable metals. Correlation data
showed signicant positive correlation (P < 0.05) of DTPA
extractable metals with Zn in case of S. acuta (r = 0.856),
whereas, signicant negative correlation (P < 0.05) was
observed with Ni in case of C. procera (r = 0.862). It is
interesting to observe that the plants of C. stula showed
positive correlation with all toxic metals and negative
correlation with essential micronutrients and signicant
positive correlation with Ni (r = 0.974, P < 0.01) and Co
(r = 0.855, P < 0.05). Overall, the plants of S. acuta
showed positive correlation with most of the tested
metals.
The DTPA extraction method was found only suitable for neutral and near-calcareous soil with insucient transition metals (Zn, Fe, Mn, Cu) (Lindsay
and Norvell, 1978). Several reports (Walker et al.,
2003; Sinha and Gupta, 2005; Gupta and Sinha,
2006; Ortiz and Alcaniz, 2006) showed close relationship between concentrations of heavy metals extracted
with DTPA and the levels of metals in the plant
tissue.

A.K. Gupta, S. Sinha / Bioresource Technology 98 (2007) 17881794

Table 4
Transfer factor from soil (DTPA extractable metals) to plants
Metals

Na
K
Fe
Zn
Mn
Cu
Cr
Pb
Ni
Cd
Co

Transfer factor
S. acuta

R. communis

C. procera

C. stula

4.07
73.15
6.40
0.67
8.71
0.58
10.79
0.53
1.12
0.38
0.49

5.28
60.17
6.97
0.49
2.25
0.49
7.05
0.63
1.38
0.41
0.52

6.03
36.19
9.72
0.96
1.64
0.76
4.65
0.69
2.30
0.40
0.22

2.42
39.99
5.24
0.58
1.62
0.41
1.30
0.67
0.64
0.40
0.34

3.5. Transfer factor (TF) from soil (DTPA extractable

metals) to the plants
Soil-to-plant transfer ratio is one of the key components
of phytoextraction. The values of transfer factor (Table 4)
for all the tested metals vary from one plant to another. All
the plants showed maximum TF value for K followed by
Fe. Among all the plants, maximum TF value for Cr and
Mn was observed in S. acuta followed by R. communi > C.
procera > C. stula. Among all the plants, it was observed
that the plants of S. acuta removed maximum amount of
Cr from tannery waste contaminated sites on the basis of
high TF value. Recently, Gupta and Sinha (2007) reported
high TF values for all the tested metals in the plants of
Chenopodium album grown on soil amended with 10%
tannery sludge.
3.6. Principal component analysis
The results of PCA for the metal concentration in the
dierent parts of the plants demonstrated that four principal components were considered in the PCA analysis,
accounting for over 79.9% of the total variance in the
complete data set. Elements such as Zn, Cu was closely
associated in the rst principal component (PC1), which
explained over 30.7% of the total variance. However, Mn
and Cr were also signicantly closely associated with
PC3, which explained over 14.55% of the total variance.
The second factor with signicant loadings for K and Cd
contributed to 23.67% of total variance, and was considered to originate from sewage euent. However, signicant
loadings for Na contributed to 10.89% of total variance
and were considered to originate from tannery process
(PC4). This could indicate the inuence of similar translocation pattern of these elements in to the studied plants.
4. Conclusion
Correlation analysis between metal accumulation in the
plants and DTPA extractable metals showed better value
of correlation for most of the tested metals in S. acuta

1793

and C. stula. Further, the plants of S. acuta showed better

TF for most of the tested metals, especially Cr. Overall
analysis of the results revealed that the plants of S. acuta
and C. stula could be suitable for the decontamination
of most of the metals from tannery waste contaminated
sites.
Acknowledgements
We thank the Director, National Botanical Research
Institute, Lucknow (India) for providing required research
facilities. Amit K. Gupta is grateful to NRCD, Ministry of
Environment and Forests, Govt. of India, New Delhi for
providing nancial assistance.
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