Uptake and translocation of metals in Spinacia oleracea L.

grown
on tannery sludge-amended and contaminated soils: Eect on lipid
peroxidation, morpho-anatomical changes and antioxidants
Sarita Sinha
*
, Shekhar Mallick, Rohit Kumar Misra, Sarita Singh, Ankita Basant,
Amit Kumar Gupta
Ecotoxicology and Bioremediation Group, National Botanical Research Institute, Lucknow 226 001, India
Received 9 May 2006; received in revised form 7 August 2006; accepted 23 August 2006
Available online 13 November 2006
Abstract
The plants of Spinacia oleracea L. grown on contaminated soil (CS) and dierent amendments of tannery sludge (TS) have shown
high accumulation of metals in its edible part. The accumulation of toxic metal (Cr) in the leaves of the plants grown on CS was recorded
as 40.67 lg g
1
dw. However, the leaves of the plants grown on 100% TS have accumulated about two times (70.80 lg g
1
dw) higher Cr
than the 10% TS (31.21 lg g
1
dw). Among growth parameters, the root length was more aected at 90 d than the shoot length, number
of leaves and leaf area. The study of scanning electron micrographs showed 29.31% increase in stomatal length in the leaves of the plants
grown on CS as compared to garden soil (GS), which served as control, however it decreased in the plants grown on higher amendments
of TS. The decrease in MDA content at initial period of exposure and lower amendment was recorded in the leaves, whereas, signicant
increase (>10% TS onward) was observed with increase in tannery sludge ratio at 90 d as compared to GS. A coordinated increase in all
the studied antioxidants (cysteine, non-protein thiol, ascorbic acid, carotenoid contents) was found up to 75 d of growth. At 90 d, most of
the antioxidant decreased as compared to 75 d causing oxidative stress as evidenced by increased level of lipid peroxidation and
decreased chlorophyll and protein contents. Maximum increase of 181.43% in MDA content and maximum decrease of 53.69% in total
chlorophyll content was recorded in the leaves of the plants grown on 100% TS after 90 d of growth. The plants grown on CS have shown
an increase in shoot length, number of leaves, leaf area, photosynthetic pigments and protein contents and in all the studied antioxidants.
Thus, these plants are able to combat stress involving defense mechanism, resulting in healthy growth of the plants. The results are well
coordinated as there is no change in the MDA content as compared to the plants grown on GS. In view of high Cr accumulation in edible
part of S. oleracea grown on CS after irrigation with tap water, it is not advisable to use these plants for edible purposes. Summing up, it
is recommended that the level of metals in the edible part should be checked instead of healthy growth as deciding parameter for con-
sumption. It is demonstrated through this study that metal enriched plants have detoxication mechanism and grow well on organic
matter enriched contaminated soil.
2006 Elsevier Ltd. All rights reserved.
Keywords: Spinacia oleracea; Tannery waste; Metals; Antioxidants; Lipid peroxidation; Scanning electron micrographs
1. Introduction
Heavy metal pollution in and around industrial sites of
any city in developing countries has become a common
phenomenon, due to the lack of proper waste disposal
practices. Among all the industries, the tannery industry
is one of the notorious in terms of heavy metal contamina-
tion to the soil. The leachate from the dried tannery sludge
cakes and sludge itself is posing a potential hazard of heavy
metals contamination, particularly hexavalent chromium
(Cr
+6
) into the soil, on which it is dumped. In addition,
in the peri-urban and rural areas of most of the developing
0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2006.08.026
*
Corresponding author. Tel.: +91 522 205831 35x221; fax: +91 522
205839, +91 522 205836.
E-mail address: sinha_sarita@redimail.com (S. Sinha).
www.elsevier.com/locate/chemosphere
Chemosphere 67 (2007) 176187
countries, the use of sewage and treated industrial waste-
water for irrigating the crops is a common practice (Nan
and Cheng, 2001; Wong et al., 2001; Sinha et al., 2006)
which leads to cumulative contamination of heavy metals
to the soil. Chromium is one of the major heavy metal,
which is predominantly being released through the waste
from the tannery industry, used for chrome tanning of
raw leather hides. Chromium merits a special reference
for its extreme toxicity due to interaction of its compounds
with living cells (Cieslak-Golonka, 1995; Costa, 1997)
Besides Cr, Fe is an another major element present in the
tannery sludge and comes from animals hides and ferrous
aluminum sulphate, which is used for precipitation of sus-
pended solids during wastewater treatment. However,
excessive concentrations of Fe have also been reported to
be toxic to the plants (Sinha et al., 1997; Sinha and Saxena,
2006). Metals in tannery waste occur in complex forms and
vary widely in their availability to the plants.
The generation of reactive oxygen species is stimulated
in the presence of metals which can seriously disturb
normal metabolism through oxidative damage to cellular
compartments.To counteract this damage, highly ecient
antioxidant defense mechanism in its cells can scavenge or
deactivate metal stress-generated by reactive oxygen radi-
cals. Antioxidant substances like cysteine, ascorbic acid,
non-protein thiol (sulfhydryl) and antioxidant enzymes
play a vital role in providing cellular defense towards oxi-
dative stress (Sinha et al., 1997, 2005a; Halliwell and Gut-
teridge, 2004; Sinha and Saxena, 2006). There are many
reports on metal-induced oxidative stress and response of
antioxidants in the plants grown on contaminated soil
(Singh et al., 2004a,b; Singh and Sinha, 2005).
Metal accumulation in the leaves of the plants grown on
contaminated soil has shown various morphological and
structural changes such as wider opening of stomata,
increase in the stomata size, thinning of wax deposition
and elevation of stomatal complex. Lesser wax deposition
has been attributed to increased lipid peroxidation induced
by metal toxicity (Rai et al., 2005).
Due to the crisis of the arable land compounded by the
contamination of fertile soil with improper disposal of tan-
nery waste, farmers are forced to grow food crops on lands
contaminated by heavy metals. Among all the parts of the
plant, maximum accumulation of metals was found in the
roots in most of the plants. However, some of the metals
are translocated to the edible part. Therefore, entry of met-
als into the food-chain through plants grown on contami-
nated soils is a matter of serious concern. Sinha et al.
(2006) reported metal accumulation in various parts of
the plants grown on soil receiving treated tannery wastewa-
ter, maximum being in leafy vegetables than fruits bearing
vegetables/crops. Further, the plants have shown better
growth in the plants grown on contaminated soil than nor-
mal soil. However, no work has been carried out to study
the toxicity and defense mechanism of leafy vegetables
grown on contaminated agricultural soil. Based on our ear-
lier work (Sinha et al., 2006), where high accumulation of
toxic metal (Cr) in the leaves of Spinacia oleracea was
observed, the present study is undertaken with a view (i)
to assess the accumulation of metals in the leaves of the
plants collected from Jajmau (Kanpur, India) irrigated
with treated tannery wastewater; (ii) to assess the transloca-
tion of metals in the plants grown on contaminated soil in
pots irrigated with tap water and to investigate the eects
on its physiological and biochemical parameters; and (iii)
to compare the translocation of metals in the plants grown
on various amendments of tannery sludge with the plants
grown on contaminated soil and physiological and bio-
chemical eects induced in these plants. The outcome of
the results should be helpful to elucidate metal detoxica-
tion mechanism in S. oleracea which have shown healthy
growth when grown on tannery waste contaminated soil.
2. Material and methods
In India, Jajmau (Kanpur) is a major industrial town
and lies in the Indo-Gangetic plains between the parallels
of 2628
0
N and 8024
0
E. It is one of the major centers to
process raw hides. The discharge from these industries is
treated in an Up-ow Anaerobic Sludge Blanket (UASB)
treatment plant before releasing. The treated wastewater
is being used by the local farmers for irrigation of edible
crops/vegetables in the adjacent agricultural elds (2100
acre).
2.1. Collection of plants from agricultural eld
Plants of Spinacia oleracea L. (spinach) were collected in
two consecutive years (June 2002, January 2003, June
2003) from the agricultural eld of Jajmau (Kanpur) and
brought to the laboratory to check the level of metals in
the edible part of the plant. The agricultural land is being
irrigated with treated tannery wastewater since last many
decades.
2.2. Experimental design
Air dried tannery sludge (TS) was collected from the
wastewater treatment plant at Jajmau, Kanpur (Uttar Pra-
desh, India) and were brought to the laboratory. Uncon-
taminated garden soil (GS) was collected from National
Botanical Research Institute (NBRI) and was used as a
control. The tannery sludge and the garden soil were nely
grounded and passed through 2 mm sieve to get a uniform
size, before lling up in terracotta earthen pots (35 cm in
diameter). Dierent amendments (10%, 20%, 35%, 50%
and 100%) of the TS were prepared using GS. In another
set, the contaminated soil (CS) was also collected from
Jajmau, Kanpur which is being irrigated with treated tan-
nery wastewater in the agricultural elds (Jajmau, Kanpur)
and sieved soil were lled in terracotta earthen pots (35 cm
in diameter). The experiment was carried out in these pots
for three months, with three harvests.
S. Sinha et al. / Chemosphere 67 (2007) 176187 177
Seeds of S. oleracea were sterilized in 3% formalin for
5 min followed by washing with double distilled water
and soaked in water overnight. These seeds were sown in
a nursery bed. After the seeds germinated and the saplings
grew to 10 cm, they were transplanted to earthen pots lled
with dierent amendments (10%, 25%, 35%, 50%, and
100%) of TS in triplicates along with one set GS and CS.
The plants were allowed to grow in the eld at National
Botanical Research Institute in a randomized block design,
at an average diurnal temperature of 2535 C. The plants
were watered by normal tap water and harvested after 60,
75 and 90 d of growth.
2.3. Metal accumulation
Harvested plants were washed thoroughly with distilled
water and blotted dry. Dierent parts were separated
manually, cut in small pieces and oven dried at 70 C till
constant weight. The dried samples were ground (> 2 mm)
and digested in HNO
3
(70%) in Microwave Digestion
System (MDS 2000) and analyzed for metals content
using Atomic Absorption Spectrophotometer (GBC,
AvantaR).
2.4. Estimation of various physiological and biochemical
parameters
Fresh weight, root length, shoot length, leaf area of the
plant were recorded immediately after harvesting. Fresh
leaves were used for the estimation of various parameters.
Chlorophyll content in the fresh leaves of the plant
(100 mg) was estimated following the method of Arnon
(1949). Protein content in the leaves and roots of the plants
were determined using BSA as standard protein (Lowry
et al., 1951). Lipid peroxidation in the plant tissue was
measured indirectly in terms of malondialdehyde (MDA)
content, determined by thio barbituric acid (TBA) reaction
(Heath and Packer, 1968). Cysteine content was estimated
by the method of Gaitonde (1967). Non-protein thiol (acid
soluble thiol) content was measured (Ellman, 1959) using
Ellmans reagent (5,5
0
-dithiobis 2-nitrobenzoic acid). Ascor-
bic acid content in the leaves was estimated as per method
of Keller and Schwager (1977).
2.5. Scanning electron microscopy
Central part of the leaves from each set of the treatment
was collected for studying the surface structure through
Scanning Electron Microscopy. The leaves were kept in
2.5% gluteraldehyde overnight for xation followed by
dehydration in ethanol series (30%, 50%, 70%, 90% and
100%). Further dehydration was done in BAL-TEC
CPD-030 critical point drier using liquid CO
2
as carrier
gas. The leaves were mounted on a stub and were coated
with 15 lm conductive gold, in an ion sputter coater
(TFC 1100). Coated specimens were mounted on Philips
XL-20 Scanning Electron Microscope.
2.6. Data analysis and interpretation
The experiment was performed in completely random-
ized block design involving six amendments of TS with
GS, in triplicates, for three time interval. All the dataset
obtained from the experiment, were subjected to two way
analysis of variance (ANOVA) using Microsoft Excel
2000 followed by least signicant dierence (LSD) calcula-
tion (Gomez and Gomez, 1984). Student t-test (two tailed)
was applied between the data obtained from CS as com-
pared to GS.
2.7. Quality control and quality assurance
The standard reference material of metals (E-Merck,
Germany) was used for calibration and quality assurance
for each analytical batch. The reference solution (BND
1101.02) of multi-elements (Zn, Fe, Cu) was also used
for calibration of analytical equipment and validation of
test methods provided by National Physical Laboratory
(NPL), New Delhi (India) and the results were found to
be within 1.50% of certied values. EPA quality control
samples (Lot TMA 989) for metals (Cd, Cr, Cu, Pb) was
used in order to ensure analytical data quality in water
and the results were found to be within 2.79% of certied
values. The 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. The blanks were run in triplicate to
check the precision of the method with each set of samples.
3. Results and discussion
3.1. Physico-chemical properties of dierent substrates
The results of physico-chemical analysis of GS and its
dierent amendments along with CS (Table 1) revealed that
pH, salinity, EC, CEC, OC and OM of both CS and TS
were signicantly higher than the level of respective param-
eters in GS. The level of metals (Cr, Zn, Mn and Cu) was
signicantly high in CS and TS as compared to the GS. In
contrast, the level of Fe was higher in GS as compared to
the CS and TS, which is due to the presence of Fe as one
of the major constituent in earth crust. The level of all
the physico-chemical parameters was found high in con-
taminated soil as it is receiving treated tannery wastewater
for irrigation except Fe (Sinha et al., 2006).
3.2. Level of metals in S. oleracea collected from
contaminated agricultural eld
The accumulation of metals in the leaves of S. oleracea
grown on contaminated soil was recorded for the period
(20022003), which was receiving treated tannery wastewa-
ter since last few decades at Jajmau, Kanpur (Table 2). The
level of toxic metal (Cr) was found high in the leaves of the
plants. Variation in metal accumulation in the plant from
178 S. Sinha et al. / Chemosphere 67 (2007) 176187
year to year can be attributed to the varying physico-chem-
ical parameters of the substrate and other edaphic factors.
Recently, Sinha et al. (2006) carried out extensive studies
on the accumulation of metals in the edible part of vegeta-
bles/crops growing on treated tannery wastewater contam-
inated soil. They reported that agricultural land is not
suitable for the cultivation of leafy vegetables due to high
accumulation of metals in the edible part. Although, the
plants grown on these soil have shown healthy growth pre-
sumably due to the presence of essential nutrients and
organic matter. Similarly, the use of metal contaminated
wastewater for irrigation of vegetables and crops may
results in elevated level of metals in the soil. Metals eventu-
ally get translocated to the plants which aect health and
agricultural and environmental quality (Singh et al.,
2004c). Plants take up metals via roots, which depend upon
the physico-chemical characteristics of the soil, concentra-
tion, solubility, species, cultivar age and organ of the plant.
In addition, strict measures have been taken in the area to
restrict the cultivation of leafy vegetables in the recent past.
Recently, Sinha et al. (2005b) reported accumulation of Cr
(3.048.55 lg g
1
dw) in the leaves of S. oleracea grown in
the area which is being irrigated with river water. As per
report of NIN (Anonymous, 1982), on metal levels in
Indian leafy vegetables, roots and tubers, metal content
(lg g
1
dw) in green leafy vegetables ranged from 16 to
95 for Zn, 8 to 96 for Mn, 1.9 to 18 for Cu and 0.52 to
4.37 for Cr. In general, Fe content ranged 50250
lg g
1
dw in the plants.
3.3. Metal accumulation
In pot experimental studies, the accumulation of metals
in the leaves and roots of the plant after 90 d of growth on
dierent substrates, exhibited partitioning of the metals in
both parts of the plant (Table 3). Overall metal accumula-
tion in the leaves was found to be in the order of
Fe > Zn > Mn > Cr > Cu. The accumulation of essential
metals (Zn, Mn, Cu) was found more in the leaves than
roots except in case of Zn and Cu in the plants grown on
10% TS. In contrast, the accumulation of toxic metal
(Cr) was found more in the roots than in the leaves except
for the plants grown on 10% TS. In case of Fe, the accumu-
lation in the leaves was recorded low at higher amendments
(35% TS onwards) than lower amendments. Leaves of
S. oleracea grown on 25% TS had the highest Fe content
and in roots grown on 10% TS after 90 d of growth.
Recently, Gupta and Sinha (2006) observed that Fe accu-
mulation by Sesamum indicum (L.) var. T55 decreased with
increase in tannery sludge amendments beyond 25% TS,
this was attributed to low level of metals in TS. They have
also reported that the level of Fe extracted with EDTA
(bioavailable) decreased with increase in TS amendments
(>25% TS). In the present study, low accumulation of Fe
Table 1
Physico-chemical properties of dierent substrates
Parameters Substrates
GS CS TS
pH (1:2 ratio) 6.63 0.04 7.86 0.005 7.84 0.005
Salinity (&) 0.2 1.3 7.5
EC (lS m
1
) 708 1.6 2506 5.47 3044 11.4
CEC (C mol (p+) kg
1
) 55.69 0.83 83.8 1.2 146.25 1.87
OC (%) 0.48 0.04 1.27 0.07 5.47 0.27
OM (%) 0.793 0.121 2.18 0.01 9.39 0.49
Total concentration of metals (lg g
1
dw)
Fe 38862 3684 22898 1740 19401.44 61
Cr 5.1 0.34 145.87 21.51 7489.76 203
Zn 45.15 0.86 217.03 22.81 340.69 11.10
Mn 238.93 15.3 310.76 68.8 294.19 15.50
Cu 18.67 0.85 18.67 0.85 188.09 5.81
All the values are mean of three replicates SD.
Table 2
Levels of metal content in the leaves of S. olarace collected from agricultural
a
eld of Jajmau, Kanpur for two consecutive years
Study periods Metals (lg g
1
dw)
Fe Cr Zn Mn Cu
June 2002 1646.15 232 35.19 6.34 67.74 9.86 43.67 4.34 21.74 18.0
January 2003 850.16 79.20 30.38 1.78 182.07 19.07 70.83 5.56 16.04 1.133
June 2003 1246.15 132 25.19 4.34 69.74 9.01 47.07 1.34 25.74 2.8
All values are mean of three replicates SD.
a
Tannery wastewater contaminated soil.
S. Sinha et al. / Chemosphere 67 (2007) 176187 179
at higher amendments of TS may be attributed due to low
level of EDTA extractable metal.
Pot experimental studies were also conducted on con-
taminated soil (CS) collected from the eld in order to
assess the metal accumulation potential of S. oleracea
under controlled conditions using tap water for irrigation
(Table 3). High accumulation of toxic metal (Cr) in the
leaves of the plants irrigated with tap water was observed,
however, it is almost same as recorded in the leaves of the
plants collected from Jajmau, Kanpur (Table 2) which is
being irrigated from treated tannery wastewater. In view
of high Cr accumulation in edible part of S. oleracea after
irrigation with tap water, it is not advisable to use such
plants for edible purposes. Further, the accumulation of
most of the metals in the leaves was recorded almost same
as in the plants grown on 25% TS.
The leaves to roots ratio of metal accumulation, were
mostly >1 except for Fe, Cr. Translocation of metals
(Cu, Zn, Mn) was found to be higher in the leaves than
its roots. The translocation of toxic metal chromium was
found less at higher amendments (25% TS onwards) of tan-
nery sludge, retaining most of the chromium in the roots of
the plant. Recently, Sinha et al. (2005a,b) reported that
most of the chromium in the plant, Pistia stratiotes was
found in the roots, which is probably due to binding of
metals to the ligands and thus reducing its mobility from
roots to aerial parts. It is a common strategy of the plants
to restrict metal translocation to the above ground parts as
reported recently in the plants grown on tannery sludge-
amended soil (Singh et al., 2004a,b; Singh and Sinha,
2005; Gupta and Sinha, 2006).
3.4. Morphological parameters
The eect on growth of the plants grown on dierent
substrates (tannery waste contaminated) expressed as root
length, shoot length, leaf area and number of leaves, are
shown in Fig. 1. The plants grown on all the TS amend-
ments have shown signicant increase in shoot length and
leaf area at all the exposure periods as compared to GS
(Fig. 1). At 75 and 90 d, the number of leaves of the plants
grown on all the amendments has shown an increase as
compared to their respective plants grown on GS. Thus,
maximum increase (p < 0.05) of 79.71% in shoot length,
165.4% in leaf area and 232.7% in number of leaves (com-
pared to GS) was observed in the plants grown on 35% TS
after 90 d of growth. On contrary to these growth param-
eters, the roots exhibited no denite trend, however, it
was recorded less in the plants grown on higher amend-
ments of TS. The root length signicantly (p < 0.05)
decreased in the plants grown on 50% and 100% TS at 75
and 90 d of exposure, respectively.
The growth parameters was also recorded in the plants
grown on contaminated soil (Fig. 1) collected from Jajmau,
Kanpur. The analysis of the results revealed that signicant
(p < 0.02) increase of 67.81% in shoot length, 123.29% in
number of leaves and 130.04% in leaf area of the plants
was recorded after 90 d of growth as compared to GS.
No marked dierence was observed in the roots of the
plants grown on contaminated soil.
The changes observed in the growth of S. oleracea were
consistent with the results reported recently by Gupta and
Sinha (2006). They also observed no change in root length,
however, shoot length increased at initial period of growth
and lower amendments of tannery sludge. In other studies,
it was reported that the shoot length of Brassica juncea var.
rohini and Helianthus annuus grown on 75% tannery sludge
amendment have shown an increase in its length as com-
pared to control, however, root length of the exposed
plants increased up to 35% tannery sludge as compared
to control. Other growth parameters increased in the plants
grown on 50% tannery sludge at 90 d. In the present study,
the analysis of the results of growth parameters have
shown reduced growth in the plants grown on 50% TS
Table 3
Levels of various metals (lg g
1
dw) in dierent parts of S. oleracea grown on dierent substrate types after 90 d of growth
Substrate types (%) Metals (lg g
1
dw)
Fe Cr Mn Zn Cu
10 1265.41 30.11 29.90 0.68 67.40 1.32 72.12 1.44 14.94 1.02
(1019.78 100.47) (31.21 3.49) (21.04 2.88) (34.13 3.66) (25.15 2.79)
25 921.40 35.05 63.12 5.78 51.74 3.08 65.17 7.49 9.06 1.68
(1411.86 147.8)9 (36.90 3.78) (70.20 7.89) (159.18 7.57) (15.40 0.07)
35 787.44 71.55 78.26 8.99 41.80 5.80 61.7 7.29 14.02 1.11
(393.08 19.30) (17.84 2.14) (58.36 6.01) (172.00 7.69) (26.73 1.07)
50 899.96 84.65 68.70 7.29 36.89 1.24 39.76 3.30 7.19 0.13
(419.32 16.02) (29.90 3.01) (56.52 5.54) (108.78 25.7) (15.42 0.03)
100 682.93 65.47 205.95 22.45 44.39 4.97 70.54 8.70 17.04 0.78
(395.38 30.92) (70.80 7.88) (50.24 0.78) (195.99 0.44) (32.88 1.14)
CS 850 49 34.21 19.26 49.83 23.65 60.51 6.30 11.09 2.68
(1511 152) (40.67 21.2) (138.6 41.8) (143.6 4.3) (21.74 18)
The values in the parenthesis are of leaves. All values are mean of three replicates SD.
180 S. Sinha et al. / Chemosphere 67 (2007) 176187
(Singh and Sinha, 2004a,b). The soil contaminated with
treated tannery wastewater oers the potential for recycling
of nutrients present in the organic matter which serve as
plant nutrients.
Scanning electron micrographs of the leaf surface of
S. oleracea (Fig. 2AJ) after 90 d of growth, showed an
increase in stomatal length (Fig. 3) in the plants grown
on 25% TS and decrease at 50% and 100% TS as compared
to GS. Further, the length of the stomata of the leaves of
the plants grown on CS has shown an increase of 29.31%
in stomatal length as compared to GS. Some of the sto-
mata were found closed in the leaves of the plants grown
on dierent TS amendments.
The metal ions seems to attack various cellular compo-
nents including cell wall and membranes resulting in
dierential alterations that ultimately lead to their disorga-
nization and mechanical injury i.e. necrosis. Stomatal
opening was thought to be due to either metal-induced
inhibition of an energy system or the alterations of K
+
channel activities in guard cells. Maurel (1997) reported
that aquaporins are present in guard cells and these toxins
interfere with the polymerization and depolymerization of
actin laments altering K
+
channel activities in guard cells
(Hwang et al., 1997). Singh and Sinha, 2004a reported an
increase in stomatal size in the plants of Brassica grown
on 50% amendment of tannery sludge. The decrease in sto-
matal aperture may be due to rapid and preferential
absorption of metals by subsidiary cells followed by
changes in membrane permeability causing decrease in cell
turgor as reported in Cd treated Phyllanthus amarus (Rai
et al., 2005). The closure of the stomata may also be due
to a strategy of the plants to prevent the water loss through
transpiration as the translocation of water and solutes get
disturbed in the presence of metals. Bondada and Oos-
terhuis (2000) reported that closed stomata of the leaf
result in a slower rate of diusion due to greater diusion
gradient of water vapour. The decrease in size of stomatal
aperture in the leaves is in the line with the hypothesis that
metals induce water stress (Singh and Sinha, 2004b).
Among other surface features, there is a marked increase
in surface roughness in the leaves of the plants grown on
dierent amendments of TS, as compared to control. In
the leaves of the plants grown on TS amended soil, stoma-
tal frequency increased in the leaves of exposed plants as
compared to GS. Slight elevation in the stomatal complex
is also observed in the plants growing on higher amend-
ment of tannery sludge. Guard cells were slightly swollen
in the leaves growing in higher amendment of TS.
3.5. Photosynthetic pigment
The total chlorophyll, chlorophyll a and b contents
(Table 4) increased in the plants grown at all the amend-
ments at 60 d and up to 25% TS after 75 d as compared to
the plants grown on GS. The plants grown on 100% TS
have shown signicant (p < 0.05) increase of 54.82% in
Fig. 1. All values are mean of three replicates SD. The eect on the levels of growth parameters in the plants of S. oleraceagrown on dierent substrates.
*
p < 0.05 as compared to GS based on LSD calculations. CS data not included in LSD calculations. t-test (two tailed as compared to GS)
a
p < 0.05,
b
p < 0.02,
c
p < 0.01.
S. Sinha et al. / Chemosphere 67 (2007) 176187 181
total chlorophyll and 61.94% in chlorophyll a contents at
60 d and signicant decrease (p < 0.05) at 75 and 90 d.
Maximum decrease of 53.69% in total chlorophyll and
50.54% in chlorophyll a content were observed at 90 d
in the plants grown on 100% TS. The increase (non-signif-
icant) in chlorophyll b content at 60 d and signicant
decrease of 70.17% in the leaves of the plant grown on
100% TS was found after 90 d as compared to GS. The
analysis of the results revealed that decrease in chloro-
phyll b was more than chlorophyll a content in the leaves
of the plants grown on 100% TS at the same exposure
period.
Fig. 2. SEM photographs of leaf surface of S. oleracea (AJ) after 90 d of growth: (A) leaf epidermis of control plant (500); (B) magnied view of
stomata in plate A (2000); (C) leaf epidermis of plant grown on 10% TS (500); (D) magnied view of stomata of plate C (2000); (E) leaf epidermis of
plant grown on 50% TS (500); (F) magnied view of stomata in plate E (2000); (G) view of leaf epidermis of plants grown on 100% TS(500); (H)
magnied view of stomata in plate G (2000); (I) view of leaf epidermis of plants grown on CS (500); (J) magnied view of stomata in plate I (2000).
182 S. Sinha et al. / Chemosphere 67 (2007) 176187
At initial period of growth (60 and 75 d), the carotenoid
content in the leaves of the plants grown on dierent
amendments of TS increased with increase in TS amend-
ments, however, increase was signicant (p < 0.05) at
higher amendments at 35% and 25% TS after 60 and
75 d, respectively as compared to GS. However, no change
in carotenoid content in the leaves of the plants grown on
35% TS was recorded followed by signicant decrease as
compared to GS at 90 d of growth. The maximum increase
of 62.22% at 75 d and maximum decrease of 34% at 90 d
was observed in carotenoid content in the leaves of the
plants grown on 100% TS.
The total chlorophyll, chlorophyll a, chlorophyll b and
carotenoid contents in the leaves of the plants grown on
CS has shown signicant (p < 0.05) increase at 60 d and
non-signicant increase at 90 d as compared to their
respective GS.
The initial increase in the chlorophyll content can be due
to the availability of essential elements in abundance in
comparison to GS. However, these metals,which were act-
ing as nutrients, crossed the threshold limit and became
toxic agent with the advance of growth. Therefore, the
decline in the chlorophyll concentration can be attributed
to the interference of heavy metals present in the substrate
in the formation of chlorophyll (Van Assche and Clijsters,
1990). The level of photosynthetic contents in the plants
grown on TS amended soil decreased which is considered
as one of the sensitive parameter in metal exposed
plants (Singh et al., 2004a,b). Besides, lipid peroxidation
also causes degradation of the photosynthetic pigments
0
5
10
15
20
GS 10% 25% 50% 100% CS
Substrate types
S
t
o
m
a
t
a
l

d
i
m
e
n
s
i
o
n

(

m
)
length
breath
Fig. 3. Graphical representation of the stomatal dimension (lm) of S.
oleracea grown on dierent amendment of TS and CS after 90 d of growth.
Table 4
Levels of plant pigments (chlorophylls and carotenoid) in the leaves of S. oleracea grown on dierent substrate types
Substrate types (%) Exposure periods (d) Photosynthetic pigments (mg g
1
fw)
60 75 90
GS 1.97 0.08 2.37 0.20 2.98 0.28 Total chlorophyll
1.34 0.13 1.49 0.15 1.84 0.06 Chlorophyll a
0.64 0.08 0.88 0.02 1.14 0.04 Chlorophyll b
0.41 0.07 0.45 0.02 0.50 0.03 Carotenoid
10 2.21 0.10
*
3.12 0.18
*
3.22 0.15
*
Total chlorophyll
1.46 0.08 1.95 0.21
*
2.06 0.06
*
Chlorophyll a
0.75 0.10
*
1.18 0.18
*
1.16 0.15
*
Chlorophyll b
0.47 0.02 0.50 0.01 0.53 0.02 Carotenoid
025 2.44 0.08
*
3.44 0.12
*
2.60 0.06
*
Total chlorophyll
1.63 0.04
*
2.20 0.08
*
1.47 0.09
*
Chlorophyll a
0.81 0.08
*
1.25 0.12
*
1.14 0.04 Chlorophyll b
0.52 0.10
*
0.57 0.04
*
0.53 0.01 Carotenoid
35 2.6 0.11
*
2.15 0.27 2.0 0.12
*
Total chlorophyll
1.75 0.09
*
1.40 0.29 1.11 0.12
*
Chlorophyll a
0.85 0.11
*
1.04 0.27
*
0.60 0.004
*
Chlorophyll b
0.63 0.02
*
0.67 0.01
*
0.52 0.12 Carotenoid
50 2.84 0.06
*
1.80 0.18
*
1.55 0.03
*
Total chlorophyll
1.91 0.07
*
1.18 0.14
*
1.06 0.03
*
Chlorophyll a
0.94 0.05
*
0.73 0.08
*
0.37 0.004
*
Chlorophyll b
0.67 0.01
*
0.70 0.01
*
0.34 0.02
*
Carotenoid
100 3.05 0.05
*
1.60 0.41
*
1.38 0.02
*
Total chlorophyll
2.17 0.02
*
1.04 0.30
*
0.91 0.05
*
Chlorophyll a
0.88 0.05
*
0.70 0.04
*
0.34 0.06
*
Chlorophyll b
0.71 0.04* 0.73 0.01
*
0.33 0.02* Carotenoid
CS 2.86 0.1
c
3.35 0.04
a
3.32 0.32 Total chlorophyll
1.93 0.05
b
2.26 0.21
a
2.1 0.15 Chlorophyll a
0.98 0.0.03
a
1.19 0.19 1.24 0.2 Chlorophyll b
0.75 0.02
b
0.61 0.02
b
0.65 0.06 Carotenoid
All values are mean of triplicates SD.
*
p < 0.05 as compared to GS based on LSD calculations. CS data not included in LSD calculations. t-test (two
tailed as compared to GS)
a
p < 0.05,
b
p < 0.02,
c
p < 0.01.
S. Sinha et al. / Chemosphere 67 (2007) 176187 183
(Somashekaraiah et al., 1992). In this study,increased lipid
peroxidation was observed in S. oleracea which might
cause degradation in photosynthetic pigments.
The plants of S. oleracea grown under stress condition
also shown increase in carotenoid content which served as
accessory pigments for photosynthesis. Carotenoids, which
are important constituents of chloroplast membranes,
quench singlet oxygen rapidly and can therefore, help to
protect chlorophyll and membrane against damage. Carote-
noids are also able to absorb energy from, and so diminish
the concentration of, those exited states of chlorophyll that
lead to singlet oxygen species formation. Hence they have a
dual role: decreasing the formation of singlet oxygen in vivo,
and helping to remove any singlet oxygen does happen to be
formed. They may also react directly with peroxy and alk-
oxy radicals, and so interfere with the chain reaction in lipid
peroxidation. (Halliwell and Gutteridge, 2004).Thus, carot-
enoid acts as a non-enzymatic antioxidant, and plays an
important role in protection of chlorophyll under stress
condition (Kenneth et al., 2000). Increase in the carotenoid
content is considered as a plants defense mechanism
towards metal stress; however, it is observed in the present
study that carotenoid content was found to decrease after
75 d of growth. Thus, it may be inferred that, excessive
accumulation of metals may cause toxicity.
It has been observed that, the eect on chlorophyll b
content was observed in the plants of S. oleracea by metal
toxicity in comparison to chlorophyll a as it can be inferred
from the increasing trend of chl a/b ratio at 90 d. There are
various reports where chl a/b ratio was recorded more in
metal treated plants (Larsen et al., 1998; Rai et al., 2005).
The carotenoid content increased signicantly in all the
amendment except at 10% TS at initial period of harvesting
(60 d, 75 d), however, at 90 d of growth, it decreased at
higher amendments (50% and 100% TS). This pattern
could be due to initial availability of all the metals and
organic matter in abundance in the substrate but prolonged
exposure to the substrate led to toxic eect and subsequent
decline in carotenoid levels.
3.6. Eect on protein content
The protein content (Table 5) in the roots and leaves of
the plants grown on various amendments of TS increased
at all the TS amendments at all the growth periods except
non-signicant decrease at higher amendments at 90 d in
both parts of the plant. In both parts, signicant increase
in protein content was recorded in the plants grown on
higher amendments (>10% TS) except non-signicant
increase in the leaves at 25% TS after 60 d. The maximum
increase was recorded as 132.58% in the roots 136.72% in
the leaves of the plants grown on 100% TS at 75 d. As com-
pared to control, the protein content increased in the roots
of the plants grown on 35% TS at 90 d, however, in the
leaves up to 50% TS.
Growth and development occur as a result of overall
balance between protein synthesis and biogenesis, and pro-
teolysis which is associated to oxidative stress promoted by
reactive oxygen species (Solomon et al., 1999; Palma et al.,
2002). The increase in protein content in this study may be
due to no induction in MDA content at 60 d and lower
amendments at 75 d, thus, the protein degradation is lower
down in the plants grown on tannery sludge amendments.
There are other reports (Singh et al., 2004a,b; Singh and
Sinha, 2005) showing an increase in protein content.
3.7. Malondialdehyde content
Malondialdehyde (MDA) is a major cytotoxic product
of lipid peroxidation and acts as indicator of free radical
production and therefore, formation of MDA is considered
as measure of lipid peroxidation (Singh and Sinha, 2005).
Malondialdehyde content in the leaves of S. oleracea was
observed to decrease with increase in sludge amendment
at 60 d, however, it was observed to increase signi-
cantly (p < 0.05) at 90 d of exposure except non-signicant
increase in the plants grown at 10% TS. The increase of
181.43% in MDA content was recorded in the leaves of
the plants grown on 100% TS as compared to GS after
90 d of growth (Table 6).
The role of redox metals in the onset of peroxidation
of membrane lipids in the plants have been realized due
to induction of toxic oxygen species. The formation of
malondialdehyde (MDA) was considered as a measure of
lipid peroxidation (Halliwell and Gutteridge, 2004). Similar
to these reports, excessive accumulation of metals was
recorded in the plants of S. oleracea at 90 d of growth
which resulted an increase in leaves MDA content. The role
of redox metal in the induction of MDA content was
reported in the plants treated with Zn (Chaoui et al.,
Table 5
Levels of protein content (mg g
1
fw) in roots and leaves of S. oleracea
grown on dierent substrate types
Substrate types
(%)
Exposure periods (d)
60 75 90
GS 18.71 0.70 19.15 0.65 20.05 0.71
(21.29 0.22) (22.11 2.14) (24.46 1.97)
10 21.33 1.37
*
23.56 0.66
*
27.10 1.22
*
(23.02 0.01) (25.81 1.33
*
) (26.19 2.39)
25 26.38 1.53
*
30.81 1.32
*
26.66 0.49
*
(25.88 1.94
*
) (31.34 2.97
*
) (39.20 3.74
*
)
35 30.58 0.82
*
38.31 0.95
*
24.29 1.05
*
(31.18 5.14
*
) (40.52 1.57
*
) (32.92 4.94
*
)
50 35.21 1.32
*
40.88 .59
*
19.86 .49
(36.88 0.83
*
) (42.38 1.62
*
) (30.60 3.56
*
)
100 40.70 2.44
*
44.54 2.45
*
16.73 1.01
*
(39.77 2.49
*
) (52.34 10.08
*
) (22.27 0.36
*
)
CS 25.35 2.46
a
31.01 1.25
b
25.56 0.39
b
All values are mean of three replicates SD. Values in parenthesis are
protein content in leaves.
*
p < 0.05 as compared to GS based on LSD
calculations. CS data not included in LSD calculations. t-test (two tailed
as compared to GS)
a
p < 0.05,
b
p < 0.01.
184 S. Sinha et al. / Chemosphere 67 (2007) 176187
1997), with Fe (Sinha et al., 1997; Sinha and Saxena, 2006),
with Cr (Sinha et al., 2005a), with Cu (Jouili and Ferjani,
2003). There are various reports (Singh et al., 2004a,b;
Singh and Sinha, 2005) which conferred similar conclu-
sions showing an increase in the level of MDA content in
the plants grown on treated tannery sludge amendments
due to the presence of redox metals.
3.8. Antioxidants
A coordinated increase was observed in all the studied
antioxidant parameters. At 60 and 75 d of growth, cys-
teine, non-protein thiol, ascorbic acid and proline content
in the leaves increased as compared to GS, however,
increased signicantly (p < 0.05) at higher amendments
(Fig. 4AD). At 60 d, cysteine, non-protein thiol, ascorbic
acid and proline contents in the leaves increased with
increase in TS amendments with maximum increase of
233.31%, 336.80%, 71.48% and 396.16%, respectively in
the plants grown at 100% TS. As compared to GS, these
antioxidants increased at all the amendments after 75 d
of growth. At 90 d of growth, cysteine and non-protein
thiol (Fig. 4A and B) contents responded in similar pattern
showing signicant increase in the leaves of the plants
grown up to 25% TS with maximum increase of 142.84%
and 128.65%, respectively. However, the level of ascorbic
acid and proline increased signicantly in the leaves of
the plants grown on 10% TS followed by decrease. Overall
analysis of the results revealed that, the non-protein
thiol and cysteine contents have shown increase in the
leaves of the plants grown on 100% TS as compared
to GS. Whereas, proline and ascorbic acid contents
increased in the leaves of the plants grown on 50% TS
and 25% TS, respectively after 90 d of growth followed
by decrease.
The plants grown on contaminated soil have shown sig-
nicant increase in all the antioxidant parameters studied.
The maximum increase of 134.70% (90 d), 145.3% (75 d),
61.82% (60 d) and 251.33% (75 d) in cysteine, non-protein
thiol, ascorbic acid and proline contents, respectively was
recorded as compared to the plants grown on GS
(Fig. 4AD).
Table 6
Levels of MDA content (lmol g
1
fw) in the leaves of S. oleracea grown
on dierent substrate types
Substrate types (%) Exposure periods (d)
60 75 90
GS 4.91 0.15 4.97 0.25 5.01 0.47
10 3.80 0.24
*
4.15 0.35
*
5.86 0.54
*
25 3.07 0.16
*
3.77 0.06
*
8.27 0.53
*
35 2.97 0.08
*
5.94 0.5
*
9.41 0.28
*
50 2.52 0.07
*
7.92 2.64
*
12.12 0.51
*
100 2.35 0.07
*
10.49 2.30
*
14.10 1.50
*
CS 4.76 0.17 5.00 0.81 5.11 0.59
All the values are means of triplicates SD.
*
p < 0.05 as compared to GS
based on LSD calculations. CS data not included in LSD calculations.
t-test (two tailed) as compared to GS.
Fig. 4. All values are mean of three replicates SD. The eect on the levels of cysteine (nmol g
1
fw) (A), non-protein thiol (lmol g
1
fw) (B), ascorbic
acid (lg g
1
fw) (C), free proline (lmol g
1
fw), (D) contents in the plants of S. oleracea grown on dierent substrates.
*
p < 0.05 as compared to GS based
on LSD calculations. CS data not included in LSD calculations. t-test (two tailed as compared to GS)
a
p < 0.05,
b
p < 0.01,
c
p < 0.001.
S. Sinha et al. / Chemosphere 67 (2007) 176187 185
As it is evident from r
2
values presented in Table 7 that
the accumulation of metals has shown positive correlation
with MDA content after 90 d of growth in the leaves of the
plants with all the metals except Fe. However, the accumu-
lation of metals has shown negative correlation with all the
growth parameters and antioxidants except Fe. There is a
positive correlation between Fe accumulation and antioxi-
dants in 90 d old plants. This is due to decreasing pattern of
both Fe and cysteine in the plant, as it has been discussed
earlier in this paper that Fe content in TS is lower than that
in GS, therefore, with increasing proportion of TS in the
substrate, Fe concentration is expected to fall. This could
be due to the consumption of all the antioxidants, against
providing defense mechanism to the plants, posed by oxi-
dative stress. The correlation calculation (data not shown)
was also performed between MDA content and various
antioxidants which showed negative correlation at 90 d.
Thus, with increase in MDA content, the level of antioxi-
dant was expected to fall.
The mechanism of metal detoxication adopted by the
plants to scavenge free radicals and peroxides include sev-
eral antioxidant substances. These non-enzymatic cellular
entities include cysteine, non-protein thiol, ascorbic acid,
carotenoid etc. which play an important role in inducing
resistance to metals by protecting macromolecules against
free radicals which are formed during various metabolic
reactions leading to oxidative stress. In the present study,
the enhanced level of cysteine and non-protein thiol sug-
gests, its active participation in detoxication of toxic oxy-
gen species. The stressed leaves of the plants showed
increase in the level of antioxidants in varying degree and
provide endogenous protection eects. Higher level of anti-
oxidants at 60 d of growth might be related with low levels
of MDA which may be due to ecient defense mechanism
against the stress-generated by metals. High thiol content
might enable metabolites to function in the detoxication
of reactive oxygen species, which are detoxied by oxida-
tion of sulfhydryl moieties to disulphides under metal
stress.
Ascorbic acid plays an important role in a-tocopherol
regeneration which has been reported to act as the primary
antioxidant. Besides this, ascorbate plays many other roles
in the antioxidant metabolism. In plants, the level of pro-
line enhanced in response to toxic metal exposures which
might be attributed to the strategies adapted to cope up
with toxicity as main function of metal-induced proline
accumulation may be associated with osmoregulation,
and enzyme protection against dehydration rather than
metal sequestration (Sharma et al., 1998). During the pres-
ent study, an increase in ascorbic acid and proline contents
in the leaves of S. oleracea at initial period of exposure
may be considered as defense strategy to combat metal
stress in the plant grown on tannery waste contaminated
soil as reported earlier (Singh et al., 2004b; Singh and
Sinha, 2005).
4. Conclusion
In conclusion, the morphological parameters of the
plants increased up to 50% TS showing that the tannery
sludge supports the growth of the mature plants at lower
amendments of sludge. Further, SEM micrograph revealed
that increased amendments of tannery sludge led to closure
of stomata, increase in their frequency and degeneration. A
coordinated increase in antioxidants was noted with
increase in metal concentrations in the leaves of the plants
at initial period of growth. This indicates that the plants
have a detoxication mechanism to cope with such a high
concentrations of metals and no eect on MDA content.
However, at higher amendment in mature plants, it experi-
enced stress conditions due to increase in MDA content
with increase in sludge amendments and the antioxidant
system of the plant was not sucient to revert the stress
of a prolonged period of metal exposition. The plants
grown on contaminated soil are able to combat stress
involving defense mechanism, resulting in healthy growth
of the plants as evidenced by the increase in growth param-
eters photosynthetic pigments and antioxidants. The
results are well coordinated as there is no change in the
MDA content as compared to the plants grown on GS
due to increase in the level of all the antioxidants studied.
Summing up it is recommended that the emphasis
should be given to the level of metals in the edible part
of the plants instead of healthy growth as metal enriched
plants have detoxication mechanism and grow well on
organic matter enriched contaminated soil. In view of high
Cr accumulation in edible part of S. oleracea grown on
contaminated soil after irrigation with tap water, it is not
advisable to use these plants for edible purposes; however,
these plants have shown healthy growth. Thus, it can be
concluded that the contaminated soil which is receiving
treated tannery wastewater should not be used for the cul-
tivation of leafy vegetables on the basis of these studies.
Acknowledgements
We thank the Director, National Botanical Research
Institute, Lucknow (India), for providing required research
Table 7
Correlation factors (r
2
) between metals and various parameters in the
leaves of in S. oleracea after 90 d of growth
Parameters Fe Cr Mn Zn Cu
Shoot length 0.21 0.90 0.34 0.03 0.39
Root length 0.93 0.14 0.24 0.34 0.06
No. of leaves 0.49 0.06 0.77 0.86 0.06
Leaf area 0.11 0.81 0.03 0.14 0.07
Total chl 0.78 0.41 0.49 0.68 0.04
Carotenoid 0.65 0.61 0.11 0.30 0.04
MDA 0.70 0.62 0.40 0.66 0.04
Cysteine 0.79 0.55 0.37 0.07 0.60
Proline 0.72 0.42 0.56 0.76 0.19
Non-protein thiol 0.75 0.71 0.07 0.35 0.54
Ascorbic acid 0.61 0.31 0.73 0.84 0.08
186 S. Sinha et al. / Chemosphere 67 (2007) 176187
facilities. We are also thankful to Sri V.K. Lal for helping
us in electron microscopy.
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