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EFFECT OF MIXED INDUSTRIAL WASTEWATER ON SOIL, TREE BIOMASS
PRODUCTION AND TRACE METAL
UPTAKE
By
Syed Fazal ur Rehman Shah M.Sc. (Forestry)
A thesis submitted to University of the Punjab in the
fulfillment of requirements for the degree of Doctor of
Philosophy
INSTITUTE OF GEOLOGY
UNIVERSITY OF THE PUNJAB, LAHORE-PAKISTAN 2009
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DEDICATED TO MY PARENTS
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CERTIFICATE
It is hereby certified that this thesis is based on the results
of experimental work carried out by Syed Fazal Ur Rehman Shah under
our supervision. We have personally gone through all the
data/results/materials reported in the manuscript and certify their
correctness/ authenticity. We further certify that the materials
included in this thesis have not been used in part or full in a
manuscript already submitted or in the process of submission in
partial/complete fulfillment for the award of any other degree from
any other institution. Mr. Shah has fulfilled all conditions
established by the University for the submission of this
dissertation and we endorse its evaluation for the award of PhD
degree through the official procedure of the University.
SUPERVISORS
Nasir Ahmad, PhD Professor Institute of Geology University of
the Punjab Lahore, Pakistan
Khan Rass Masood, PhD Professor Department of Botany University
of the Punjab Lahore, Pakistan D.M. Zahid, PhD Associate Professor
University College of Agriculture Bahaudin Zakaria University
Multan, Pakistan
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CONTENTS
Abstract i
Acknowledgments iii
List of Tables iv
List of Figures vi
List of Abbreviations vii
CHAPTER 1 INTRODUCTION 1
1.1 Background 1
1.2 Historical Purspective of Phytoremediation 2
1.3 Site Description 3
1.4 Objectives of the Study 5
1.5 Thesis Layout 5
CHAPTER 2 REVIEW OF LITERATURE 7
2.1 Origin and Occurrence 8
2.2 Effects of Heavy Metals on Human Health 9
2.3 Effect of Wastewater Application on Soil Properties 10
2.3.1 Factors influencing heavy metal availability and uptake by
plants
11
2.3.1.1 Soil pH 11
2.3.1.2 Cation Exchange Capacity (CEC) 11
2.3.1.3 Soil type 11
2.3.1.4 Plant associated factors 12
2.4 Heavy Metal Toxicity in Plants 12
2.4.1 Mobility, uptake and accumulation of heavy metals 13
2.4.2 Mechanism of metal tolerance 16
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2.5 Effect of Heavy Metals on Growth and Development 17
2.5.1 Germination 18
2.5.2 Root 19
2.5.3 Stem 20
2.5.4 Leaf 20
2.5.5 Dry biomass 21
2.6 Effect of Heavy Metals on Plant Physiology 22
2.6.1 Photosynthesis 22
2.6.2 Water relation 24
2.6.3 Essential nutrients 25
2.7 Effect of Heavy Metals on Enzymatic System 26
2.7.1 Root Fe III Reductase 27
2.7.2 Nitrate Reductase 28
2.7.3 Antioxidant Enzymes 28
2.8 Effect of Wastewater Application to Plantations 29
CHAPTER 3 MATERIALS AND METHODS 32
3.1 Wastewater Sampling and Analysis 32
3.2 Soil Analysis 33
3.3 Procurement and Raising of Seedlings 33
3.4 The Hudiara drain Wastewater Application 34
3.5 Synthetic Wastewater Application 34
3.6 Plant Growth Analysis 35
3.7 Plant Digestion and Analysis 35
3.8 Chlorophyll Determination 36
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3.9 Analytical Quality Assurance 36
3.10 Experimentation under Controlled Conditions 36
3.11 Phytosociological Survey. 37
3.11.1 Soil sampling 38
3.12 Statistical Analysis 38
CHAPTER 4 RESULTS AND DISCUSSION 39
4.1 The Influence of Heavy Metals on Dalbergia sissoo Seedlings
under Controlled Water and Climate Conditions.
39
4.1.1 Effects on the growth of seedlings 39
4.1.2 Effects on biomass production of seedlings 42
4.1.3 Effects on chlorophyll contents of seedlings 44
4.2 Effects of the Hudiara drain Wastewater on Growth and
Biomass Broduction of Eucalyptus camaldulensis and Dalbergia sissoo
Plants
46
4.2.1 Characterization of the wastewater 46
4.2.2 Changes in soil chemistry 46
4.2.3 Effects on plant growth 48
4.2.4 Effects on biomass production 53
4.2.5 Effects on chlorophyll contents 54
4.2.6 Effects on the uptake and accumulation of nutrient 56
4.2.6.1 Root 56
4.2.6.2 Shoot 57
4.2.6.3 Leaves 58
4.3 Effect of Synthetic Wastewater on Growth and Biomass
Production of Tree Species
61
4.3.1 Changes in soil chemistry 62
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4.3.2 Effects on plant growth 64
4.3.3 Effects on biomass production 65
4.3.4 Effects on chlorophyll contents 68
4.3.5 Effects on the uptake and accumulation of nutrients and
metal elements
70
4.3.5.1 Root 70
4.3.5.2 Shoot 70
4.3.5.3 Leaves 71
CHAPTER 5 PHYTOSOCIOLOGICAL SURVEY OF THE HUDIARA DRAIN
76
5.1 Vegetation Profile of the Hudiara Drain 76
5.2 Classification of Vegetation 78
5.3 Vegetation and Environmental Variables 80
CHAPTER 6 CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK
82
6.1 Conclusions 82
6.2 Suggestions for Future Work 83
REFERENCES 85
ANNEXURE-I PUBLICATIONS 115
ANNEXURE-II CURRAL FORMULA FOR PERCENTAGE COVER VALUES
CALCULATION
116
ANNEXURE-III VEGETATION DATA RECORDING SHEET 117
ANNEXURE-IV LIST OF SPECIES WITH RESPECTIVE FAMILIES FALLING
ALONG THE HUDIARA DRAIN
118
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i
ABSTRACT
Although the agricultural use of wastewater raises some
environmental and human
health concerns, irrigation with wastewater is usually carried
out by smallholders in dry
areas. The present study reports on the physiological effect of
several dilutions of the raw
wastewater of the Hudiara drain on Dalbergia sissoo and
Eucalyptus camaldulensis plants.
Six-month old seedlings were established in pots and irrigated
for 18 months with: tap water
(control, T0); 25% wastewater (T1); 50% wastewater (T2); 75%
wastewater (T3); and 100%
wastewater (T4). Results showed that the plant growth parameters
decreased as the percent
of wastewater increased. At T4 the shoot length, number of
leaves, leaf fresh weight, and leaf
oven dry weight were reduced by 17%, 72%, 72%, and 70% in
Dalbergia sisoo and 5%,
17%, 23%, and 29% in Eucalyptus camaldulensis plants
respectively, compared to the
control (T0).
The content of chlorophyll a, chlorophyll b and total
chlorophyll increased in
Dalbergia sissoo plants treated with wastewater at 25%, but
decreased in the T2, T3, and T4
treatments. Whereas chlorophyll a, chlorophyll b and total
chlorophyll increased up to T2 in
E. camaldulensis, in treated pots beyond that percentage, a
decline in chlorophyll was
observed.
As the percentage of wastewater in the treatments increased, the
accumulation of Na,
Cd and Cr in tissues increased, while the concentration of K, P,
Mg, and Fe decreased.
Similarly, Eucalyptus camaldulensis and Dalbergia sissoo plants
were irrigated with
synthetic wastewater containing Cd and Cr for 18 months.
Treatments were T0= Tap water
(control); T1= 0.05+1.0 mg L-1conc. of Cd(II)+ Cr(VI); T2=
0.10+2.0 mg L-1conc. of Cd(II)+
Cr(VI); T3= 0.20+4.0 mg L-1conc. of Cd(II)+ Cr(VI) and T4=
0.40+8.0 mg L-1conc. of
Cd(II)+ Cr(VI). Results showed that plants at T1 grew more
compared to the control, but
beyond that level, a gradual decline in growth was recorded with
a maximum reduction in T4 treated plants. Cd and Cr accumulation in
tissues increased (roots>shoot>leaves) as
external metal concentration increased, while nutrient
accumulation (K, P, Mg, Fe) and
chlorophyll content declined. However, the application of
synthetic wastewater containing
various concentrations (0, 10, 20, 40 and 80 mg L-1) of Cd and
Cr on the growth of
Dalbergia sissoo at the seedling stage for four weeks under
controlled conditions in a growth
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ii
chamber (300 µmol m-2s-1 of photosynthetically active radiation
with 16:8 hours
photoperiod) revealed a decline in growth after 10 mg L-1 and 40
mg L-1 for Cr and Cd,
respectively. A combined application of Cd and Cr wastewater
showed a growth reduction at
doses above 20 mg L-1. Results showed that Cr was more toxic to
Dalbergia sissoo plants at
the seedling stage than Cd. The present study suggests that
wastewater from the Hudiara
drain diluted to 25% and 50% with tap water is a feasible option
for the growth of D. sissoo
and E. camaldulensis plants in Lahore, Pakistan.
A phytosociological survey using the Braun-Blanquet’s approach
was undertaken to
investigate the influence of the Hudiara drain wastewater on the
surrounding vegetation.
Multivariate analysis of vegetation data classified the
vegetation into two major communities
including, Cynodon dactylon and Boerhaavia diffusa, and
Parthenium hysterphorus and
Xanthium strumarium groups. The fervent growth of these species
designated the area as
wasteland. The patterns of floral diversity exhibited
considerable variation. Canonical
Correspondence Analysis (CCA) revealed that the distribution of
vegetation correlates with
environmental variables, but their role in the grouping of
species was not significant.
However, soil EC played a role in the grouping of Stellaria
media and Fagonia cretic.
Similarly, some species, namely Riccinus communis, Boerhaavia
diffusa and Phragmites
karka showed a correlation with Fe and Cr respectively,
suggesting Phragmites karka as a
suitable candidate for chromium contaminated sites.
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iii
ACKNOWLEDGMENTS
All blessings are unto Almighty Allah, the Compassionate the
Merciful, Who
bestowed me strength to complete this uphill task. I consider it
my utmost duty to express
gratitude to the Prophet Muhammad (S.A.W.) who is a symbol of
guidance and source of
knowledge.
I feel pleasure to acknowledge my deep sense of gratitude to my
supervisors Prof. Dr.
Nasir Ahamd, Prof. Dr. Khan Rass Masood, and Dr. D.M. Zahid for
providing gracious
assistance, guidance, immaculate supervision and keen interest
throughout the study.
My deeply felt thanks go to my parents, brother and sisters who
taught me self-
reliance, determination and courage to face the challenges. I am
indebted to my wife who
always encouraged, supported and shared happiness even in
difficult times.
I am grateful to my colleagues and friends for their help and
encouragement during
this research work. Special thanks are due to Dr Jose R
Peralta-Videa, Department of
Chemistry, University of Texas at El-Paso, El-Paso, TX, USA, Dr
Amanda Stiles,
Department of Plant and Microbial Biology, University of
California, Berkeley, USA and Dr
Muhammad Zubair, University College of Agriculture, B.Z.
University for their insightful
comments on the thesis.
Last but not least, I would also like to acknowledge and thank
the Higher Education
Commission, Islamabad for the financial assistance to complete
this research project.
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iv
LIST OF TABLES
Table 4.1 Metal element dose effect on the number of leaves,
shoot length, root length and seedling length of Dalbergia sisso
under controlled conditions
41
Table 4.2 Effects of metal elements on the biomass production of
Dalbergia sissoo seedlings
43
Table 4.3 Effects of metal elements on the chlorophyll contents
of Dalbergia sissoo seedlings
45
Table 4.4 Characterization of the Hudiara drain wastewater
47
Table 4.5 Effects of industrial wastewater on soil chemistry
51
Table 4.6 Effects of industrial wastewater on the growth of tree
species 52
Table 4.7 Effects of industrial wastewater on the biomass
production of tree species
54
Table 4.8 Variation in chlorophyll contents of tree species in
response to industrial wastewater application
56
Table 4.9 Accumulation of mineral nutrients in the roots of tree
species in response to industrial wastewater application
59
Table 4.10 Accumulation of mineral nutrients and metal elements
in shoots of tree species in response to wastewater application
60
Table 4.11 Accumulation of mineral nutrients and metal elements
in leaves of tree species in response to wastewater application
61
Table 4.12 Effects of synthetic wastewater on soil chemistry
63
Table 4.13 Effects of synthetic wastewater on the growth of tree
species 66
Table 4.14 Effects of synthetic wastewater on the biomass
production of tree species
67
Table 4.15 Variation in chlorophyll contents of tree species in
response to synthetic wastewater application
69
Table 4.16 Accumulation of mineral nutrients and metal elements
in roots ree species in response to synthetic wastewater
application
73
Table 4.17 Accumulation of mineral nutrients and metal elements
in shoots 74
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v
of tree species in response to synthetic wastewater
application
Table 4.18 Accumulation of mineral nutrients and metal elements
in leaves of tree species in response to synthetic wastewater
application
75
Table 5.1 The most frequent species occurring along the Hudiara
drain (in order of decreasing frequency in 99 Quadrats)
77
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vi
LIST OF FIGURES
Figure 1.1 Location Map of the Hudiara drain 4
Figure 1.2 Monthly mean temperature and precipitation of the
study area 4
Figure 5.1 Location Map of Sampling Points along the Hudiara
Drain 76
Figure 5.2 Overall division of vegetation by TWINSPAN into major
and minor groups
79
Figure 5.3 Biplot diagram of species environmental variables as
demarked by CCA
81
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vii
LIST OF ABBREVIATIONS
ANOVA Analysis of Variance APX Ascorbate Peroxidase BOD
Biochemical Oxygen Demand CAT Catalase CEC Cation Exchange Capacity
COD Chemical Oxygen Demand DCA Detrended Correspondence Analysis DO
Dissolved Oxygen DTPA Diethylenetriaminepentaacetic Acid EC
Electrical Conductivity EDTA Ethylenediaminetetraacetic Acid FAAS
Flame Atomic Absorption Spectrophotometer NADP Nicotinamide Adenine
dinucleotide Phosphate NEQS National Environmental Quality
Standards NIST National Institute of Standards and Technology NR
Nitrate Reductase PC Phytochelatins POD Peroxidase ppm parts per
million SAR Sodium Adsorption Ratio PSI Photosystems I PSII
Photosystems II ROIs Reactive Oxygen Intermediates ROS Reactive
Oxygen Species SH-Group Sulfhydryl group SOD Superoxide Dismutase
SPSS Statistical Package for the Social Sciences SRWC Short
Rotation Willow Coppice TSS Total Suspended Solids TWINSPAN Two Way
Indictor Species Analysis UV Ultra Violet
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Chapter One Introduction 1
Chapter-1
INTRODUCTION
1.1 Background
Like many developing/under-developed countries, in Pakistan,
farmers are commonly
using untreated industrial and municipal wastewater for
irrigation, particularly in the suburbs
of large cities and in the vicinity of major industrial estates
(Ghafoor et al., 1994; Bose and
Bhattacharyya, 2008; Chandra et al., 2008). However, long-term
application of wastewater
may lead to the accumulation of heavy metal elements (Cd, Ni,
Cr, Pb, As, Zn etc) in soil
which may cause (i) yield loss and decline in soil microbial
activity, (ii) soil and groundwater
contamination, (iii) reduction in soil fertility and (iv)
contamination of the human food chain
(McGrath et al., 1995; Yadav et al., 2002). Among the heavy
metal elements, cadmium (Cd)
and chromium (Cr) are of special concern because of their
toxicity to the plant kingdom,
even at very low concentrations (Shukla et al., 2007). Cd is a
particularly dangerous pollutant
due to its high toxicity and high solubility in water (Pinto et
al., 2004). High contents of Cd
in soil retards plant growth, reduces biomass production (Rai et
al., 2005), adversely affects
mineral assimilation and induces changes in various
physiological and biochemical
characteristics of plants (Scebba et al., 2006). In some plant
species, the interactions of Cd
and metal nutrients have shown changes in the plant nutrient
concentration and composition
(Peralta-Videa et al., 2002). Similarly, elevated levels of Cr
in soil causes retardation of
growth, damages roots, reduces yield and hampers productivity
(Sharma et al., 2003).
This situation demands that wastewater be treated for safe
agricultural use. A wide
range of techniques (physico-chemical, biological and advanced
oxidation processes) are in
use to treat wastewater. These treatment methods are costly,
labor-intensive, time-consuming
and are associated with secondary disposal problems (Davies et
al., 2007). In addition, their
application is sometimes restricted due to technological or
economical constraints (Pino et
al., 2006). However, phytoremediation (the use of plants to
remove, reduce, or stabilize toxic
elements) may be a promising alternative (Zhu et al., 1999).
This study is aimed to investigate the effects of the Hudiara
drain wastewater and
synthetic wastewater (with special emphases on the heavy metal
elements) on the growth and
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Chapter One Introduction 2
biomass production of Dalbergia sissoo and Eucalyptus
camaldulensis plants. These plants
were selected due to their fast growing nature, adaptability to
wide range of soil types, high
biomass production (Abo-Hassan et al., 1988) and minimum role in
food chain
contamination.
1.2 Historical Perspective of Phytoremediation
The use of plants to clean wastewater is quite old; Hartman
(1975) and Baumann
(1885) reported plant species that accumulated high levels of
metals in their leaves.
According to Byers (1935), the genus Astragalus accumulated up
to 0.6 % selenium in dry
shoot biomass. Minguzzi and Vergnano (1948) reported plants that
accumulated Ni up to 1%
in shoots, followed by high Zn accumulation in shoots of Thlaspi
caerulescens (Rascio,
1977). The idea to use plants for cleaning heavy metal
contaminated soils was reintroduced
by Utsunamyia (1980) and Chaney (1983). Later, numerous
researchers used plants to
remediate soil and wastewater (Kisku et al., 2000; Kaushik et
al., 2005; Sawalha et al., 2009).
Phytoremediation is now a widely supported green technology
which may provide an
alternative to cleaning wastewater and contaminated soil because
of its cost-effectiveness,
environment-friendly and aesthetically pleasant nature, and
equal applicability for the
removal of both organic and inorganic pollutants present in
soil, water and air (Yu et al.,
2007).
Plants which can accumulate high concentration of metals in the
harvestable biomass
are termed hyperaccumulators. According to Baker et al. (2000),
the plants that can
accumulate >100 mg Cd kg-1 or >500 mg Cr kg-1in dry leaf
tissue are termed
hyperaccumulators. Reeves and Baker (2000) also identified
hyperaccumulator plants for
elements including Cd, Cr, Ni, Pb, Se and Zn. Gardea-Torresdey
et al. (2005) compiled a list
of hyperaccumulator plants from literature pertaining to the
period of 1997-2004. However,
such plants are typically slow-growing, small, and/or weedy
plants that produce only limited
amounts of biomass, and therefore, takes significant time (may
be several years) to
decontaminate polluted sites (Cherian and Oliveira 2005).
Therefore, fast-growing tree
species that guaranteeing high biomass yield, have a tendency
for higher heavy metal
accumulation, a deep root system and a strong
evapo-transpiration system are preferred for
phytoremediation over conventional hyperaccumulators
(Sebastiani, 2004). The application
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Chapter One Introduction 3
of trees in phytoremediation is also advantageous because trees
can survive and grow in soil
contaminated with exceptionally high levels of multiple metals
and phenotypically adjust to
metal stress (Vangronsveld and Cunningham, 1998; Pulford, et
al., 2002). Many researchers
(Cromer et al., 1983; Baker, 1995) have reported that an
application of nutrient rich
wastewater to tree plantations not only increased the
productivity but also helped sustain the
supply and beneficial use of nutrient rich wastewater resources
that could otherwise cause
eutrophication of water bodies with the attendant risk of toxic
algal blooms. The resultant
biomass may be converted into raw material for industrial
applications such as furniture
making, power generation, and fiber production. It also helps to
reduce processes of erosion
by wind and water, and reduces possible contaminations of lakes
and rivers (Pulford et al.
2002; Borghi et al., 2008). Woodlots may therefore, help to
reduce soil toxicity and
safeguard the environment (Stewart et al., 1990).
1.3 Site Description
The Hudiara drain constitutes a main tributary of the Ravi River
(Fig 1.1). The total
length of the drain is 98.6 km; 44.2 km of which is in India and
54.4 km in Pakistan. The
discharge of the Hudiara drain at its source in Pakistan R.D.
(138) is 73.3 m3s-1 and at outfall
R.D. (308) is 141.2 m3s-1. Originally, it was a storm drain,
which became a perennial drain
due to the discharge of untreated wastewater from over 120
different industrial units (textile,
dying, tanneries, pharmaceutics and others) and municipal
wastewater (Rashid and Majeed,
2002). The climate of the area is warm and semi-arid with a mean
annual rainfall and
temperature of 52.4 mm and 24.32 oC, respectively (Fig 2).
Extreme hot weather is observed
during the months of May, June, and July, while December,
January, and February are the
coldest months (Pakistan Metrological Department).
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Chapter One Introduction 4
N
Fig. 1.1 Location map of the Hudiara Drain.
0
5
10
15
20
25
30
35
40
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Tem
pera
ture
(o C
)
0
50
100
150
200
250
Prec
ipita
tion
(mm
)
Temperature Precipitation
Fig 1.2 Monthly mean temperature and precipitation of the study
area
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Chapter One Introduction 5
1.4 Objectives of the Study
The main objectives of the study are:
i. To characterize the Hudiara drain wastewater in terms of
physico-chemical
properties and heavy metal element contents.
ii. To determine the effect of various dilutions of the drain
wastewater and synthetic
wastewater containing Cd and Cr on the physiology of tree
species.
iii. To find an appropriate dilution of the Hudiara drain
wastewater for safe irrigation
of woodlots.
iv. To compare soil characteristics before (control) and after
the application of the
drain water.
v. To assess the effect of Hudiara drain wastewater on the
distribution of
surrounding native vegetation.
1.5 Thesis Layout This thesis is comprised of six chapters.
Chapter one gives the rational of the study. It
introduces the historical perspective of phytoremediation for
the decontamination of
wastewater and soil and briefly describes the study area and
climatic condtions of the area. It
also describes the main objectives of the study.
Chapter two reviews the relevant literature which mainly deals
with the toxic effects
of wastewater application on the soil, plants and human health.
It also describes the merits
for the possible utilization of industrial wastewater and its
significance for raising tree
plantations as is currently being practiced in different parts
of the world. It describes the
diversion of industrial wastewater to tree plantations from
their conventional disposal into
stream systems and to crop production.
Chapter three covers the methodology of the experimental field
research, laboratory
analysis and statistical tools for the data analysis. It
describes the various dilutions of the
Hudiara drain wastewater and synthetic wastewater applied for
the irrigation of Eucalyptus
camaldulensis and Dalbergia sissoo in a pot experiment. It also
describes the procedure for
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Chapter One Introduction 6
conducting the phytosociological survey of the area, the size
and location of the quadrats, and
the use of computer based programmes for floristic data
analysis.
Chapter four describes the results of synthetic wastewater
application on Dalbergia
sissoo and Eucalyptus camaldulensis at early growth stages under
controlled environmental
conditions. It also describes the effects of metal elements and
various dilutions of Hudiara
drain industrial wastewater on the soil, on the biomass mass
production of tree species, and
the accumulation of micro- and macro-nutrients in various plant
parts (roots, shoot and
leaves). Lastly, it discusses the effects of wastewater on the
chlorophyll content of the plants.
Chapter five discusses the results obtained from the
phytosociological survey of the
area and the influence of the Hudiara drain on the distribution
of adjacent herbaceous
vegetation and its effect on the soil properties.
Chapter six includes the conclusions of the research work and
some suggestions for
future work.
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Chapter Two Review of Literature 7
Chapter-2
REVIEW OF LITERATURE
Heavy metal elements are found in living cells but only in trace
amounts. Some
metals, namely Co, Cu, Fe, Mn, Mo and Zn, are considered to be
essential for plants, Cr, Ni
and Sn are essential for animals, and Cd, Hg and Pb have not
been found to be essential for
any living organism (Misra and Mani, 1991). When the
concentrations of these metal
elements exceed permissible limits, they pose serious threats to
both flora and fauna, and due
to their high reactivity, can directly influence growth,
senescence and energy generating
processes. Their concentration in soil beyond permissible limits
is toxic to plants, causing
oxidative stress through free radicals and/ or disrupting the
functions of enzymes by
replacing essential metals and nutrients (Henry, 2000). Although
changes in cell metabolism
permit plants to cope, the reduction in plant growth is the
primary symptom of metal toxicity.
However, the response of plants to the excess of metals depends
on their growth stage
(Skόrzyńska-Polit and Baszynski, 1997). For example, Maksymiec
and Baszyński (1996)
reported that beans (Dicotyledonous plants) and alfalfa
(Gardea-Torresdey et. al., 2004) were
more resistant to heavy metals at an early growth stage.
Conversely, the adaptation
mechanisms of older plants exposed to heavy metals are not so
flexible and efficient.
Due to industrialization hazardous wastewater containing non
essential metal
elements (Pb, Cd, Cr, As and Hg) is being discharged in city
drains which are toxic in their
combined and elemental form (Peralta-Videa et al., 2009). This
wastewater is being used for
the irrigation of crops and is contaminating the human food
chain. Keeping in view the
toxicity of Cr and Cd at low concentrations (Sharma et al.,
1995; Das et al., 1997; Shukla et
al., 2007) for living organisms and their availability in
Hudiara drain wastewater beyond
threshold limits (Yasir, 2005) for irrigation water, these two
metals are the focus of this
review. Chromium (Cr) toxicity in plants varies from the
inhibition of enzymatic activity to
mutagenesis (Barcelo et al., 1993). The visible symptoms of
chromium toxicity include leaf
chlorosis, stunting, and yield reduction (Das et al., 1997;
Boonyapookana et al., 2002).
Cadmium (Cd) is a particularly dangerous pollutant due to its
high toxicity and high
solubility in water (Pinto et al., 2004). Reports indicate that
in some plant species, Cd
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Chapter Two Review of Literature 8
interacts with the absorption of metal nutrients such as Fe, Zn,
Cu and Mn (Zhang et al.
2002; Wu and Zhang 2002), to induce lipid peroxidation and
chlorophyll breakdown in
plants, and resulting in the enhanced production of reactive
oxygen species (ROS) (Hegedüs
et al. 2004). Cadmium also inhibits the uptake of elements such
as K, Ca, Mg, Fe because it
uses the same transmembrane carriers (Rivetta et al., 1997). Its
accumulation in plants may
also pose a serious health hazard to human beings through the
food chain; however, it poses
an additional risk to children by the direct ingestion of
Cd-contaminated soil (Nordberg,
2003).
2.1 Origin and Occurrence
The main problem with heavy metals in soils is that, unlike
organic pollutants, they
cannot be biodegraded and therefore reside in the environment
for long periods of time. Their
presence in soils may be from natural or anthropogenic origins.
Natural sources include
atmospheric emissions from volcanoes, the transport of
continental dust and the weathering
of metal-enriched rocks (Ernst, 1998). However, the major source
of contamination is from
anthropogenic origin: the exploitation of mines and smelters,
the application of metal based
pesticides and metal-enriched sewage sludges in agriculture,
combustion of fossil fuels,
metallurgical industries and electronics (manufacture, use and
disposal), military training,
contribute to an increased input of heavy metals in soils
(Alloway, 1995).
Heavy metals exist in colloidal, ionic, particulate and
dissolved phases. The soluble
forms of metal elements are generally ionized or unionized
organometallic chelates. Among
chemical elements, Cr is considered to be the seventh most
abundant element on Earth and
constitutes 0.1 to 0.3 mg kg-1 of the crystal rocks (Cervantes
et al., 2001). About 60–70% of
its total world production is used in alloys and 15% in chemical
industrial processes, mainly
leather tanning, pigments, electroplating and wood preservation
(McGrath, 1995). Chromium
has several oxidation states ranging from Cr2- to Cr6+; however,
valences of I, II, IV and V
have also been shown to exist in a number of compounds
(Krishnamurthy and Wilkens,
1994). Additionally, Cr (VI) is considered to be the most toxic
form of chromium and is
usually associated with oxygen as chromate (CrO42_) or
dichromate (Cr2 O7 2_) oxyanions. Cr
(III) is less mobile, less toxic and is mainly found bound to
organic matter in soil and aquatic
environments (Becquer et al., 2003). Cr occurs mostly in the
form of Cr (III) in soil, and
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Chapter Two Review of Literature 9
within the mineral structures in the forms of mixed Cr (III) and
Fe (III) oxides (Adriano,
1986). Cr and Fe (OH)3 is a solid phase of Cr (III) having even
lower solubility than Cr
(OH)3 (Rai et al., 1987). Hence, in the environment, the total
soluble Cr (III) remains within
the permissible limits for drinking water for a wide pH range (4
to 12) due to precipitation of
(Cr, Fe)(OH)3 (Rai et al., 1989; Zayed and Terry, 2003).
Similarly, a major source of Cd is
the parental material, but the anthropogenic activities have
enhanced the amount of cadmium
in the soil (Kabata-Pendias and Pendias 2001). Heavy metals are
normally present at low
concentrations in freshwater (Le Faucheur et al., 2006).
However, the discharge of
wastewaters from a wide variety of industries such as
electroplating, metal finishing, leather
tanning, chrome preparation, production of batteries, phosphate
fertilizers, pigments,
stabilizers, and alloys had impacted aquatic environments
(El-Nady and Atta 1996; Booth,
2005; Stephens and Calder, 2005). In addition, large areas of
cultivated land have also been
reported to be contaminated with As and Cd due to agricultural
and industrial practices
(McGrath et al., 2001; Verma et al., 2007). Cadmium pollution is
also released onto streets
from rubber car tires, and after a rain, the Cd is washed into
sewage systems and collected in
the sludge.
2.2 Effects of Heavy Metal on Human Health
The increase in the use of heavy metals in industrial processes
and products has
resulted in a dramatic increase in human exposure to heavy
metals during in the last 50 years.
Heavy metals may enter the human body directly or indirectly
through food, water, air, or
absorption through the skin (Dokmeci et al, 2009). Now a days,
chronic cases of metal
toxicity has been testified for mercury amalgam dental fillings,
lead in paint and tap water,
chemical residues in processed foods, and personal care
products. In today’s industrial
society, there is no possibility to avoid exposure to toxic
chemicals and metals. Heavy metals
are associated with several detrimental effects viz-a-viz
damaged or reduced function of the
central nervous system, reduced availability of biological
energy, and damage to blood
composition, lungs, kidneys, liver, and other vital organs.
Whereas, Long-term exposure to
these metals reveal even further chronic physical, muscular, and
neurological degenerative
processes that mimic Alzheimer's disease, muscular dystrophy,
Parkinson's disease, and
multiple sclerosis. Further to that, toxic metals can increase
allergic reactions, cause genetic
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Chapter Two Review of Literature 10
mutation, compete with good trace metals for biochemical binding
sites, and act as broad
range antibiotics (Theophanides and Anastassopoulou, 2002;
Wasserman et al., 2004).
2.3 Effect of Wastewater Application on Soil Properties
Commercial fertilizers could better be replaced by Industrial
wastewater (Marecos do
Monte et al., 1989). Nonetheless, continued wastewater
irrigation can change soil properties,
e.g. pH, nutrient concentration (Russell et al., 1988). These
changes might be a threat to
sustainability of long term land use. On the other hand,
application of wastewater has been
reported significantly increase in crop yields, (Russell et al.,
1988) and forestry (Lowe,
1994). Use of wastewater also bring about changes the soil
moisture contents, adding
nutrients and organic matter, which may further induce favorable
changes in soil properties,
tree growth and tree nutrient uptake. Schipper et al. (1996)
revealed that wastewater
application was attributed with the addition of nutrients into
the soil rather than additional
water loading. Significant effects soil properties, tree biomass
production and nutrient uptake
has been reported when Eucalyptus short-rotation forests were
applied with slaughter house
wastewater irrigation (Guo, 1998). Contrary to that, increased
concentrations of heavy metals
through wastewater irrigation may also negatively influence crop
growth by interfering with
metabolic processes thereby inhibiting growth that may sometimes
leading to mortality In
plants. (Schaller and Diez, 1991).
It is widely known that soil physico-chemical properties for
instance pH, contents of
clay minerals and organic matter etc determine that
bioavailability and mobility of metals in
soils. Generally, sorption of soil particles reduces the
activity of metals in the system. Thus,
the higher the cation exchange capacity (CEC) of the soil, the
greater the sorption and
immobilization of the metals. In acidic soils, due to
competition of H+ for binding sites on the
colloidal components of the soil, metal desorption and release
into solution is stimulated.
Therefore, soil pH not only affects metal bioavailability but
also metal uptake into roots.
However, the effects on metal bioavailability appear to be
related to the properties of each
metal as well (Lasat, 2000).
http://jeq.scijournals.org/cgi/content/full/32/6/1939#BIB90#BIB90
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Chapter Two Review of Literature 11
2.3.1 Factors influencing heavy metal availability and uptake by
plants
2.3.1.1 Soil pH
Soil pH is a prime determining factor for element availability
to plants concerning
heavy metals (Krebs et al., 1998) and their chemical forms in
soil are influenced affected by
changing the soil pH. An increase in pH (basic range) results in
an increase in the adsorption
of Cu, Cd, Zn, to soi1 particles and reduces the uptake of Cd,
Zn, Pb by plants (Kuo et al.,
1985). On other contrary, at lower pH (acidic range) results in
an increases the metal
absorption by plants and a reduction of metal adsorption to soi1
(Brown et al., 1994). Thus,
soil pH affects not only on metal bioavailability but also
affects the process of metal uptake
by roots. This effect could be metal specific. For example, Zn
uptakes by roots of Thlaspi
caerulescens was not pH dependence. Whereas, in case of Mn and
Cd, the uptake by roots
was heavily dependent on soi1 acidity (Brown et al., 1995). The
solubility of metals like Cd
and Zn is sensitive to pH in a weakly acidic to neutral range,
which is pH range of most the
humid and temperate climate soils (Fotovat and Naidu, 1998).
2.3.1.2 Cation exchange capacity (CEC)
The CEC is the ability of soil to retain metal ions. The cation
exchange capacity
increases with increasing clay content of soi1, while the
availability of metal ions decreases
(Kabata - Pendias and Pendias, 1984). In acidic soils, metal
desorption from soi1 binding
sites into solution is stimulated due to H+competition for
binding sites.
2.3.1.3 Soil type
The bioavailability of heavy metals in soi1 is a function of
soil texture and varies with
soil type such as optimal availability in sandy loam soil
followed by clayey loam and finally
lowest in clayey loam. Similarly, heavy metal concentrations are
higher in Gleysols and
Luvisols followed by Brunisols and Podzols. However, this
observation can also be related to
soil texture because Gleysols and Luvisols have higher clay
content, compared to Brunisols
and Podzols (Webber and Singh, 1995). The complex of heavy
metals with organic matter,
humic acid in particular, has been well documented (Friedland,
1999). High organic matter
content also enhances the retention of the metals, drastically
reducing the metal availability.
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Chapter Two Review of Literature 12
2.3.1.4 Plant associated factors
The genetic make-up of a plant greatly influences its metal
uptake potential (Chen et
al., 1997). Irrespective of which approach of phytoremediation
is used, two major plant-
associated factors, including metal tolerance and metal
hyperaccumulation potential (Saxena
et al., 1999). The metal accumulation potential of different
plant species highly correlated to
their genotype.
2.4 Heavy Metal Toxicity in Plants
Although many metal elements are essential for the growth of
plants, they are toxic
when their concentration in soil exceeds a certain threshold
value and the toxic effect varies
with the nature of the element and plant species. Heavy metal
toxicity in plants depends on
the bioavailability of the element in a soil solution, which is
a function of pH, organic matter
and CEC of the soil (Kabata - Pendias and Pendias, 1984; Krebs
et al., 1998). The
bioaccumulation of heavy metals may replace essential metals in
pigments or enzymes either
by disrupting their function and/or causing oxidative stress.
Heavy metal toxicity hinders the
growth of both underground and aboveground plant parts and
inhibits the activity of the
photosynthetic apparatus. This is often correlated with
progressing senescence. Non-essential
metals/metalloids such as Hg, Cd, Cr, Pb, As, and Sb are toxic
both in their chemically
combined or elemental forms, and a plant’s response to these
elements varies across a broad
spectrum from tolerance to toxicity. Cd stress creates changes
in various physiological and
biochemical functions of plants (Talanova et al., 2001). In
general, Cd interferes with the
uptake, transport and use of several elements (Ca, Mg, P and K)
and water by plants.
Similarly, Cr toxicity depends on its oxidation state, with Cr
(VI) being more toxic and
mobile compared to Cr (III) Shanker et al., 2005. Both elements
interfere with the uptake of
Ca, Mg, and P. Since plants lack specific transporters for these
nonessential elements, they
are mobilized through the transport system as essential ions. To
avoid the toxicity, plants
have developed specific mechanisms by which toxic elements are
excluded, retained in the
roots, or transformed into physiologically tolerant forms.
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Chapter Two Review of Literature 13
2.4.1 Mobility, uptake and accumulation of heavy metals
Heavy metals supplied to the environment are transported by
water and air and
deposited in soil and sediments. However, the bonding process
may take a considerable
period of time. It has been noted that at the beginning of the
binding process, the bioavailable
fraction of metal elements in the soil is high, but then
decreases gradually with time (Martin
and Kaplan, 1998).
Metal solubility and bioavailability to plants is mainly
influenced by the chemical
properties of soil such as, soil pH, loading rate, CEC, redox
potential, soil texture, clay
content and organic matter (Williams et al., 1980; Verloo and
Eeckhout, 1990). Generally,
the higher the clay and/ or organic matter and soil pH, the
greater the metal adsorption
resulting in longer residence time and reduced bioavailability
to plants. Soil temperature is
also an important factor accounting for variations in metal
accumulation by crops (Chang et
al., 1987).
The bioavailability of metals is increased in soil through
several means. The most
common mechanism is the secretion of phytosiderophores into the
rhizosphere to solubilize
and chelate soil-bound metals (Kinnersely, 1993). Heavy metals
are captured by plant root
cells after their mobilization in the soil. Their movement in
the soil mainly depends on (i)
diffusion of metal elements along the concentration gradient
that is formed due to the uptake
of elements by the root, and subsequent depletion of the element
in the root vicinity (ii)
interception by roots, in which soil volume is displaced by root
volume and (iii) the flow of
metal elements from the bulk soil solution down the water
potential gradient (Marschner,
1995). Cell walls behave as an ion exchanger of comparatively
low affinity and low
selectivity when metals are first bound. From the cell wall, the
transport systems and
intracellular high-affinity binding sites mediate and drive the
uptake of these metals across
the plasma membrane. A strong driving force for the uptake of
metal elements through
secondary transporters is created due to the membrane potential,
which is negative on the
inside of the plasma membrane and may exceed –200 mV in root
epidermal cells (Hirsch et
al., 1998). However, the uptake of some heavy metals has been
reported to be passive,
metabolic, or partially metabolic and partially passive (Cataldo
et al., 1983; Bowen 1987).
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Chapter Two Review of Literature 14
The uptake of metals, both by roots and leaves, increases as
metal concentration in
the external medium increases. Nevertheless, the uptake has no
linear relation with increasing
concentration. This is mainly because the metals bound in the
tissue cause saturation that is
governed by the rate at which the metal is taken up. The uptake
efficiency of metals by the
plants (or accumulation factor) is highest when their
concentrations in the external medium
are low. This is true for Cd both in solution and in soil is
likely due to the low concentration
of metal per unit of absorption area, resulting in low
competition between ions at the uptake
sites (the situation is opposite at high concentrations) (Greger
et al., 1991; Greger 1997).
Both essential and non-essential metals can be taken up by
leaves. In the form of a gas, they
enter the leaves through the stomata, whereas in ionic form
metals mainly enter through the
leaf cuticle (Lindberg et al., 1992; Marschner 1995). Hgo in
gaseous form is taken up by the
stomata (Cavallini et al., 1999) and its uptake is reported to
be higher in C3 than C4 plants
(Du and Fang 1982). The uptake occurs mainly through
ectodesmata, non-plasmatic
“channels” (which are less dense parts of the cuticular layer),
that are situated foremost in the
epidermal cell wall/cuticular membrane system between guard
cells and subsidiary cells.
Furthermore, the cuticle covering guard cells is often different
from the cuticle covering
normal epidermal cells (Marschner 1995).
The majority of the metal elements are insoluble in the vascular
system of plant and
unable to move freely. They usually form sulphate, phosphate or
carbonate precipitates
which are immobilized in apoplastic (extracellular) and
symplastic (intracellular)
compartments (Raskin, 1997). High cation exchange capacity of
cell walls further limits the
apoplastic transport of metal ions unless the metal ion is
transported as a non-cationic metal
chelate (Raskin, 1997). The apoplast continuum of the root
epidermis and cortex is
permeable to the movement of solutes. In the apoplastic pathway,
water and solute particles
can flow and diffuse without crossing membranes. The cell wall
of the endodermal cell layer
acts as a barrier for apoplastic diffusion into the vascular
system.
Generally, prior to the entry of metal ions in the xylem,
solutes are taken up by the
root symplasm (Tester and Leigh, 2001), and their movement from
the root to the xylem is
mainly governed by three processes, including (i) metal
sequestration into the root symplasm
(ii) symplastic transport into the stele, and (iii) release of
metals into the xylem. Metal ion
transport into the xylem is generally mediated by membrane
transport proteins. Metal
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Chapter Two Review of Literature 15
elements which are not needed by the plants use the same
transmembrane carriers and
therefore compete with the essential heavy metals for their
transport.
Cr (III) uptake by the plant is mainly a passive process, while
Cr (VI) transport is
mediated by sulphate carrier (Skeffington et al., 1976). For
this reason, inhibitors such as
sodium azide and dinitrophenol inhibit the uptake of Cr (VI) but
not Cr (III) by barley
seedlings (Skeffington et al., 1976). Group VI anions (e.g.
SO4-2) also inhibit the uptake of
chromates whereas Ca 2+ stimulates its transport (Shewry and
Peterson, 1974). This
inhibition of chromate transport is due to competitive
inhibition based on the chemical
similarity, while the stimulated transport of Cr (VI) by Ca is
attributed to its essential role in
plants for the uptake and transport of metal elements. (Zayed
and Terry, 2003; Montes-
Holguin et al., 2006).
There is no correlation between Cr concentrations in plant
tissues and that in soils.
However, some plants such as Brassica species (e.g., Indian
mustard) have shown an unusual
ability to take up heavy metals from root substrates and
accumulate them in their tissues
(Kumar et al., 1995). Even though it is a common tendency of
plants to retain Cr in their
roots, there are quantitative differences (Zayed and Terry, 2003
and references therein).
Leafy vegetables (e.g., spinach, turnip leaves) that tend to
accumulate Fe appears to be the
most effective at translocating Cr to the plant top (Cary et
al., 1977). Those leafy vegetables
(e.g., lettuce, cabbage) that accumulate relatively low
concentrations of Fe in their leaves are
considerably less effective at translocating Cr to their leaves.
Some plant species are reported
to attain substantially higher shoot/root concentration ratios
than other species (Zayed and
Terry, 2003). However, reports show that a ‘Soil–Plant Barrier’
protects the food chain from
toxicity of heavy metals. It implies that levels of heavy metals
in edible plant tissues are
reduced to levels safe for animals and humans due to one or more
of the following processes
(i) prevention of uptake of metal element(s) due to their
insolubility in soil, (ii) prevention of
translocation of metal element(s) by making them immobile in
roots or (iii) lowering the
phytotoxicity of the metal element(s) to permissible level both
for animals and human beings
(Chaney, 1980).
Some elements (e.g. B, Cd, Mn, Mo, Se, Zn) are easily absorbed
and translocated
within plant tissues, while others (e.g., Al, Ag, Cr, Fe, Hg,
Pb) are less mobile due to their
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Chapter Two Review of Literature 16
strong binding with soil components or root cell walls (Chaney,
1983). However, beyond
certain concentrations, all of these elements are mobilized
within the transport system of the
plant, even against a concentration gradient. For example,
kinetic data demonstrate that
essential Cu2+, Ni2+and Zn2+ and nonessential Cd2+ compete for
the same transmembrane
carrier for their transport (Crowley, 1991). Metal chelate
complexes may also be transported
via specialized carriers across the plasma membrane as is the
case for Fe–phytosiderophore
transport in graminaceous species (Cunningham and
Berti.1993).
Of the factors influencing metal accumulation in plants, soil pH
is usually the most
important parameter (Ramos et al., 2002; Piechalak et al., 2002;
Kirkham, 2006; Deng et al.,
2006). At higher soil pH, metal elements in soil solutions form
low soluble compounds that
decrease their bioavailability, while metal bioavailability
increases at lower soil pH (Seregin
and Ivanov, 2001). However, Cr is reported to enhance Cd
accumulation in plants such as H.
verticillata and Chara corallina (Rai and Chandra, 1992; Rai et
al., 1995). But the
accumulation of Cr is found to be greater in comparison to Cd
when applied separately
(Shukla et al., 2005; Singh et al., 2006). It is probably due to
the fact that the properties of Cr
make this element more available for plant uptake.
2.4.2 Mechanisms of metal tolerance
Plants use complex processes to adapt their metabolism to a
rapidly changing
environment. These processes include perception, transduction,
and transmission of stress
stimuli (Turner et al., 2002; Xiong et al., 2002; Kopyra and
Gwόźdź 2004). The adaptation to
stress conditions includes mechanisms of resistance and
tolerance. Resistance involves the
immobilization of a metals in roots and in cell walls (Garbisu
and Alkorta, 2001). Tolerance
deals with the internal sequestration of the toxic element. In
addition, plants have developed
a series of mechanisms to avoid heavy metal toxicity that
include (i) production of reactive
oxygen species by auto-oxidation and the Fenton reaction, (ii)
main functional group
blocking and (iii) displacement of metal ions from biomolecules
(Clemens, 2006). All of
these mechanisms operate as strategies to allow survival in
contaminated soil. Plants are able
to growth in contaminated soils because (i) they prevent the
metal uptake into the aerial parts
and they maintain a low and constant metal concentration over a
broad range of metal
concentration in soil by holding metals in their roots. These
plants are called metal excluders
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Chapter Two Review of Literature 17
(Cunningham et al., 1995), (ii) plants termed metal indicators
actively accumulate metals in
their aerial tissues by producting metal binding compounds
(chelators) or altering their metal
compartmentalisation pattern by storing metals in non-sensitive
parts. Some plants can
concentrate metal in their aerial parts to levels far exceeding
than soil and are termed
hyperaccumulators (Raskin, 1994; Baker et al., 1994). The
mechanisms used for
hyperaccumulation are still unknown. The criteria to classify
plants as hyperaccumulators
are: (i) a plant that can accumulate either As, Cu, Cr, Ni, Pb,
or Co >1000 mg kg-1 or zinc
>10 000 mg kg-1 in their shoot dry matter (Baker et al.,
1994; Brown et al., 1994; Ma et al.,
2001; Brooks, 1998; Reeves and Baker, 2000) or Mo>1500 mg
kg_1 (Lombi et al., 2001), (ii)
a plant that concentrates in shoots 10-500 times as much as
those in a normal plant (Shen and
Liu, 1998), (iii) a plant that concentrates more of an element
in shoots than in roots (Baker et
al., 1994), and (iv) when an enrichment coefficient (element in
shoot/element in soil) >1 is
observed (Wei et al., 2002). A few of the higher plant species
have adaptations that enable
them to survive and to reproduce in soils heavily contaminated
with Zn, Cu, Pb, Cd, Ni, and
As (Dahmani-Muller et al., 2000; Pulford and Watson, 2003). Tree
roots of these plants can
actively grow towards less contaminated soil zones (Turner and
Dickinson, 1993) and, even
with highly reduced growth, they can “sit and wait” for
favorable growth conditions
(Watmough, 1994). Such species are divided into two main groups:
(i) pseudometallophytes
that grow on both contaminated and non-contaminated soils and
the (ii) absolute
metallophytes that grow only on metal contaminated and naturally
metal-rich soils.
2.5 Effect of Heavy Metals on Growth and Development
Heavy metals either retard the growth of whole plant or some
part of plant (Shafiq
and Iqbal 2005; Shanker et al., 2005). The plant parts which
have the direct contact with
contaminated soils, normally the roots, exhibit rapid changes in
their growth patterns (Baker
and Walker 1989). The significant effects of a number of metals
(Cu, Ni, Pb, Cd, Zn, Al, Hg,
Cr, Fe) on the growth of above ground plant parts is also well
documented (Wong and
Bradshaw, 1982). Heavy metals mainly affect plant growth through
the generation of free
radicals and reactive oxygen species (ROS), which pose constant
oxidative damage by
degenerating important cellular components (Pandey et al., 2005,
Qureshi et al., 2005). For
example, in cucumber plants, Cu limits K uptake by leaves and
inhibits photosynthesis via
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Chapter Two Review of Literature 18
sugar accumulation which results in the retardation of cell
expansion (Alaoui-Sosse´ et al.
(2004). Similarly, rice seedlings exposed to Cd or Ni (Moya et
al. 1993) and runner bean
plants treated with Cd (Skόrzyńska-Polit et al., 1998) and Cu
(Maksymiec and Baszyński
1998) have shown an increase in their carbohydrate content and a
decrease in photosynthesis,
resulting in growth inhibition. The typical symptoms of Cd
toxicity of rice plants are wilted
leaves, growth inhibition, progressive chlorosis in certain
leaves and leaf sheaths and
browned roots, especially root tips (Das et al., 1997; Chugh and
Sawhney, 1999). Moreover,
in maize (Zea mays), Cd also reduced plant growth (Talanova et
al., 2001; Liu et al.,
2003/2004). Tomato plants irrigated with polluted water also
showed some phenotypic
deformities like stunted growth, fewer branching and less
fruiting. However, accumulation of
heavy metals in fruit appeared to be extremely low compared to
the stems, roots, and leaves
(Gupta et al., 2008).
2.5.1 Germination
Seed germination and early seedling growth are quite sensitive
to changing
environmental conditions (Seregin and Ivanov, 2001). Thus,
properties such as germination
performance and seeding growth rate are often used to assess the
abilities of plant tolerance
to metal elements (Peralta-Videa et al., 2001). Higher
concentrations (1, 5 and 10 µM) of
heavy metals (Cu, Zn, Mg and Na) significantly inhibited seed
germination and early growth
of barley, rice and wheat seedlings compared to controls
(Mahmood et al., 2007). Since seed
germination is the first physiological process affected by toxic
elements, the ability of a seed
to germinate in a medium containing any metal element (i.e. Cr)
would be a direct indicater
of its level of tolerance to this metal (Peralta-Videa et al.,
2001). For instance, seed
germination of Echinochloa colona plants was reduced to 25% in
200 µM Cr (Rout et al.,
2000), and high levels (500 ppm) of Cr (VI) in soil reduced
germination of Phaseolus
vulgaris plants up to 48% (Parr and Taylor, 1982). Jain et al.
(2000) observed reductions up
to 32 and 57% in sugarcane bud germination at 20 and 80 ppm Cr
application respectively. In
another study (Peralta-Videa et al., 2001), lucerne (Medicago
sativa cv. Malone) germination
was reduced to 23% at 40 ppm Cr (VI). The reduced germination of
seeds under Cr stress
could either be a depressive effect of Cr on the activity of
amylases, transport of sugars to the
embryo axes or an increase in protease activity (Zeid,
2001).
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Chapter Two Review of Literature 19
2.5.2 Root
In plants, the root is the first organ to come in contact with
toxic elements and it
usually accumulates more metals than shoots (Salt et al., 1995;
Wójcik and Tukiendorf,
1999; Rout et al., 2001). The inhibition of root elongation
appears to be the first visible effect
of metal toxicity. Root elongation can be reduced either by the
inhibition of root cell division
and/or by a decrease in cell expansion in the elongation zone
(Fiskesjo, 1997). Since
inhibition of root elongation appears to be the first visible
effect of metal toxicity, the root
length can be used as an important tolerance index (Piechalak et
al., 2002; Belimov et al.,
2003; Odjegba and Fasidi, 2004; Han et al., 2007). It is
reported (Han et al., 2004) that Cr
(III) precipitates in the roots of Brassica juncea plants
avoiding translocation.
The response of roots to heavy metals has been extensively
studied in both
herbaceous plant species and trees (Khale 1993; Punz and
Sieghardt, 1993; Hagemeyer and
Breckle, 1996, 2002). After the work of numerous researchers
(Bracelo and Poschenrieder,
1990; Punz and Sieghardt 1993; Hagemeyer and Breckle 1996, 2002)
morphological and
structural effects caused by metal toxicity in roots can be
summarized as (i) decrease in root
elongation, biomass and vessel diameter, (ii) tip damage (iii)
root hair collapse or decrease in
number of roots, (iv) increase or decrease in lateral root
formation, (v) suberification and
lignifications enhancement, and (vi) alterations in the
structure of hypodermis and
endodermis.
Metal toxicity varies with the metal element type. Chromium
severely affects the root
length compared to the other heavy metals (Prasad et al., 2001).
Mokgalaka-Matlala et al.
(2008) observed that the root elongation decreased significantly
with increasing
concentrations of As (V) and As (III) in Prosopis juliflora
plants. It has been reported that
the root length in Salix viminalis plants is affected more by Cr
than by Cd and Pb (Prasad et
al., 2001). According to Fargaŝva´ (1994, 1998), the inhibition
effect of Cr in S. alba root
growth is similar to that of Hg, and stronger than that of Cd
and Pb, while Ni reduced root
length less than Cr. The order of metal toxicity to new root
primordia in S. viminalis plants is
reported to be Cd>Cr> Pb (Prasad et al., 2001).
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Chapter Two Review of Literature 20
2.5.3 Stem
It is reported that metal elements adversely affect plant height
and shoot growth (Rout
et al., 1997). The reduction in plant height might be mainly due
to reduced root growth and
decreased nutrient and water transport to the aerial parts of
the plant. In addition, Cr transport
to the aerial parts of a plant can have a direct impact on the
cellular metabolism of shoots,
thereby contributing to the reduction in plant height (Shanker
et al., 2005). Anderson et al.
(1972) observed a reduction of 11%, 22% and 41% compared to a
control in oat plants
treated with nutrient solutions containing 2, 10 and 25 ppm Cr.
Similar reduction in the
heights of Curcumas sativus, Lactuca sativa and Panicum
miliaceum plants due to Cr (VI)
was also observed by Joseph et al. (1995). Cr (III) inhibited
shoot growth in lucerne cultures
(Barton et al., 2000). Sharma and Sharma (1993) observed a
significant reduction in height of
wheat (cv. UP 2003) sown in sand and treated with 0.5 µM sodium
dichromate in a
greenhouse experiment after 32 and 96 days. A significant
reduction in height of Sinapsis
alba plants grown in soil with Cr contents of 200 or 400 mg kg_1
was reported by Hanus and
Tomas (1993). Very recently, a reduction in stem height at
various concentrations (10, 20, 40
and 80 ppm) of Cd and Cr was reported in Dalbergia sissoo Roxb.
seedlings compared to a
control (Shah et al., 2008).
2.5.4 Leaf
Healthy leaf growth, leaf area development and total leaf number
contributes to crop
yield (Shanker et al., 2005). Metal elements such as Cd and Cr,
however, induce
morphological changes such as the drying of older leaves,
chlorosis and necrosis of young
leaves. Datura innoxia plants grown in an environment
contaminated with Cr (VI) exhibited
toxic symptoms such as the fall of older leaves at 0.2 mM Cr
(VI) and wilting of leaves at 0.5
mM Cr (VI) in soil (Vernay et al., 2008). None of these symptoms
were, however, visible in
the medium with excess Cr (III). Sharma and Sharma (1993) and
Tripathi et al. (1999) found
that a high concentration (200 ppm) of Cr (VI) severely affected
the leaf area and biomass of
Albizzia lebbek seedlings. An addition of 100 ppm of Cr (VI) to
the soil showed up to a 45%
decrease in dry leaf yield in bush bean plants (Wallace et al.,
1976). There appears a
reduction in leaf area and leaf dry weight in Oryza sativa,
Acacia holosericea and Leucaena
leucocephala plants treated with tannery wastewater of different
concentrations (Karunyal et
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Chapter Two Review of Literature 21
al., 1994). In a study on the effect of Cr (III) and Cr (VI) on
spinach, Singh (2001) reported
that Cr applied to soil at the rate of 60 mg kg_1 and higher
levels reduced the leaf size causing
the burning of leaf tips or margins and a slow leaf growth rate.
According to Pedreno et al.
(1997), heavy metal contamination, especially Cr, preferably
affected the young leaves in
tomato plants.
2.5.5 Dry biomass
Plant biomass is an indicator of crop productivity in terms of
dry matter yield.
Increased photosynthetic process is considered a base for the
building up of organic matter,
which accounts for 80-90% of the total dry mass of plant
(Bishnoi et al., 1993a,b). However,
heavy metals like Cr and Cd reduced the biomass production in
Bacopa monnieri L. plants
(Tokalioglu and Kartal, 2006). According to another study (Arora
et al., 2006), fronds of
Azolla species showed toxicity symptoms in terms of increased
fragmentation, change in
color, development of necrosis and an overall decrease in
biomass production as compared to
controls when cultivated in an environment containing Cr.
Vallisneria spiralis plants treated
with Cr (VI) at a concentration above 2.5 µg mL_1 severely
affected dry matter production
(Vajpayee et al., 2001). According to Zurayk et al. (2001), the
combined effect of salinity
and Cr (VI) caused a significant decrease in the dry biomass
accumulation of Portulaca
oleracea. Cauliflower (cv. Maghi) treated with 0.5 mM Cr (VI)
showed decreased dry
biomass production (Chatterjee and Chatterjee, 2000). The effect
of Cr (VI) on biomass
production (Kocik and Ilavsky, 1994) in sunflower, maize and
Vicia faba plants grown in soil
with Cr (VI) concentration of 200 mg kg_1 was negligible.
However, the uptake of Cr by
plant tissue was positively correlated with the Cr (VI) content
in the soil. A distinct reduction
in dry biomass at flowering in S. alba was noted when Cr (VI)
was added to soil at rates of
200 or 400 mg kg_1 along with N, P, K and S fertilizers (Hanus
and Tomas, 1993). In pot
trials undertaken in soil duly amended with Cr at the levels of
100 or 300 mg kg_1, a
reduction in yield of barley and maize is reported as well
(Golovatyj et al., 1999). Dry matter
production decreased dramatically in tomato and corn plants with
increasing concentrations
of Cd , and a decrease in yield of both crops was observed at
0.1 mg L-1 Cd (reaching acute
toxicity at 2 mg L-1)(Yildiz, 2005).
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Chapter Two Review of Literature 22
2.6 Effect of Heavy Metals on Plant Physiology
Plants present morphological and metabolic changes in response
to metal stress that
are believed to be adaptive responses (Singh and Sinha, 2004).
For instance, Cd not only
inhibits growth (Lunáčková et al. 2003, Dong et al. 2005), but
also changes various
physiological and biochemical characteristics such as water
balance, nutrient uptake
(Vassilev et al., 1997, Dražić et al., 2006, Scebba et al.,
2006) and photosynthetic electron
transport photosystems PS 1 and PS II (Siedlecka and Baszynski
1993, Skόrzyńska-Polit and
Baszynski 1995, Vassilev et al., 2004). Similarly, Cr hinders
electron transport, reduces CO2
fixation, chloroplast disorganization (Zeid, 2001; Davies et
al., 2002; Shanker, 2003),
decreases water potential, increases transpiration rate, reduces
diffusive resistance, and
causes a reduction in tracheary vessel diameter (Vazques et al.,
1987).
2.6.1 Photosynthesis
The photosynthetic apparatus appears to be very sensitive to the
toxicity of heavy
metals. Heavy metals affect the photosynthetic functions either
directly or indirectly, inhibit
enzyme activities of the Calvin cycle and cause CO2 deficiency
due to stomatal closure
(Seregin and Ivanov, 2001; Linger et al., 2005; Bertrand and
Poirier, 2005). Negative impacts
of Cr on photosynthesis in terrestrial plants are also well
cited in the literature. According to
a study by Bishnoi et al. (1993a), the effect of Cr was more
pronounced on PS I than on PS II
activity in isolated chloroplasts of pea plants. Vernay et al.
(2007) observed photoinhibition
in leaves of Lolium perenne L plants treated with 250 µM Cr and
noted a decrease in the
maximal photochemical efficiency of PSII of plants at Cr levels
of 500 µM. Shanker et al.,
2005, argued that Cr caused oxidative stress in the plants
because Cr may enhance alternative
sinks for the electrons due to the reduction of molecular oxygen
(part of Mehler reaction).
According to Rocchetta et al. (2006), the overall effect of Cr
ions on photosynthesis and
excitation energy transfer could be due to Cr induced
abnormalities (widening of thylakoid
and decrease in number of grana) in the chloroplast
ultrastructure.
Although the effect of Cr on photosynthesis in higher plants has
been extensively
studied (Foy et al., 1978; Van Assche and Clijsters, 1983), it
is not well understood to what
extent Cr induces inhibition of photosynthesis due to disarray
of chloroplasts ultrastructure
and inhibition of electron transport or the influence of Cr on
the enzymes of the Calvin cycle
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Chapter Two Review of Literature 23
(Vazques et al., 1987). However, Krupa and Baszynski (1995)
attempted to explain some
hypotheses concerning the possible mechanisms of heavy metal
toxicity to photosynthesis
and presented a list of key enzymes of photosynthetic carbon
reduction, which were inhibited
in heavy-metal treated plants (mainly cereal and legume crops).
It has been noted that the
40% inhibition of whole plant photosynthesis in 52-day-old pea
plant (Pisum sativum L)
seedlings at 0.1 mM Cr(VI) was further enhanced to 65% and 95%
after 76 and 89 days of
growth, respectively (Bishnoi et al., 1993a). The
disorganization of the chloroplast
ultrastructure and inhibition of electron transport processes
due to Cr and a diversion of
electrons from the electron-donating side of PS I to Cr(VI) is a
possible explanation for a Cr-
induced decrease in photosynthetic rate. It is possible that
electrons produced by the
photochemical process were not necessarily used for carbon
fixation as is evident by the low
photosynthetic rate of the Cr stressed plants. Bioaccumulation
of Cr and its toxicity to
photosynthetic pigments in various crops and trees has been
extensively investigated
(Barcelo et al., 1986; Sharma and Sharma, 1996; Vajpayee et al.,
1999). Bera et al. (1999)
studied the effect of Cr in tannery wastewater on the
chloroplast pigment content in beans.
They reported that irrespective of Cr concentration, chlorophyll
a, chlorophyll b and total
chlorophyll decreased in 6-day-old seedlings as compared to the
control. Chatterjee and
Chatterjee (2000) reported that in cauliflower (cv. Maghi)
cultivated in sandy soil with Cr
and Cu levels of 0.5 mM, a drastic resulted in chlorophyll a and
b in leaves occured. The
order of stress was Co>Cu>Cr. Conversely, a study on the
Cr and Ni tolerance of E. colona
plants showed that the chlorophyll content was high in tolerant
calluses in terms of survival
under high Cr concentration (Samantaray et al., 2001). A
significant decrease in chlorophyll
a, b and carotenoids was reported in Salvinia minima plants
treated with Cr at concentration
of 1 and 2 mg L-1 (Nichols et al., 2000). The decrease in the
chlorophyll a/b ratio brought
about by Cr indicates that Cr toxicity possibly reduces the size
of the peripheral part of the
antenna complex (Shanker, 2003). It is assumed that a decrease
in chlorophyll b due to Cr
could be due to the destabilization and degradation of the
proteins (Shanker et al., 2005).
A significant decrease in the content of chlorophyll and
carotenoids was established
under the influence of Cd. This effect was a function of the Cd
concentration in the nutrient
solution (Šimonová et al., 2007). The decrease in chlorophyll
content was due to the
inactivation of PS II due to heavy metals like Cd (Siedlecka and
Baszynski 1993). Moreover,
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Chapter Two Review of Literature 24
PS II reaction centers and the PS II electron transport
mechanism are affected by the
impairment of enzyme activity and protein structure. The
interaction of heavy metals with the
functional SH-groups of proteins according to Van Assche and
Clijsters (1990) may be a
possible mechanism of action of heavy metals. However, an
earlier study by Haghiri (1974)
reported that higher Cd contents in the growing medium might
suppress Fe uptake by the
plants, while Root et al. (1975) stated that Cd-induced
chlorosis in corn leaves could possibly
be due to changes in Fe:Zn ratios. In others plant species, Cd
toxicity appeared to induce
phosphorus deficiency or reduced transport of manganese
(Goldbold and Huttermann, 1985).
2.6.2 Water relation
Water can be considered as a major factor to regulate the plant
growth since it affects,
directly or indirectly, the overall growth process (Kramer and
Boyer 1995). Plants raised in
metal contaminated soils often suffer drought stress mainly due
to poor physicochemical
properties of the soil and shallow root systems. Therefore,
researchers are interested in
investigating on plant/water relations under heavy metal stress.
Selection of drought
resistance species can be considered to be an important trait in
phytoremediation of heavy
metals polluted soils (Barcelo et al., 2001). Heavy metal stress
can induce in plants a series
of events leading to decreased water loss (i.e. enhanced water
conservation), including a
decrease in the number and size of leaves, stomatal size, number
and diameter of the xylem,
increased stomatal resistance, enhancement of leaf rolling and
leaf abscission, and a higher
degree of root suberization (Barcelo and Poschenrieder
1990).
Chatterjee and Chatterjee (2000) reported that excess Cr
decreased the water potential
and transpiration rates, and increased diffusive resistance and
relative water content in
cauliflower leaves. Barcelo et al. (1985) observed a decrease in
leaf water potential in bean
plants treated with Cr. Bush bean plants when treated with Cr
exhibited toxicity symptoms in
terms of decreased turgor and plasmolysis in the epidermal and
cortical cells and decreased
tracheary vessel diameter, which ultimately resulted in the
reduction of longitudinal water
movement (Vazques et al., 1987).
Turner and Rust (1971) reported the wilting of various crops and
plant species due to
Cr toxicity, but little information is available on the effect
of Cr on water potential of higher
plants. Impaired spatial distribution and reduced root surface
of Cr-stressed plants can lower
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Chapter Two Review of Literature 25
the capacity of plants to uptake water from the soil. A
significantly higher toxic effect of Cr
(VI) in declining the stomatal conductance could be instrumental
in damaging the cells and
membrane of stomatal guard cells. This could affect the water
relationship in all plant
species.
2.6.3 Essential nutrients
Heavy metals as micronutrients are essential for the biological
and physiological
functions of plants. These functions include biosynthesis of
proteins, nucleic acids, growth
substances, chlorophyll and secondary metabolities such as
metabolism of carbohydrates and
lipids, stress tolerance, structural and functional integrity of
various membranes and other
cellular compounds (Päivöke and Simola, 2001; Tu and Ma 2005).
However, heavy metals
like Cr and Cd can interfere with the functioning of
micronutrients. Reports have indicated
that higher concentrations of Cr in the soil reduced the N
content and increased the P
concentration in oat plant tissues, slashed micronutrient (Cu,
Zn, Mn, and Ni) uptake in
plants, decreased the levels of Fe and Zn (and increased Mn) in
bush bean, interfered with the
uptake of Ca, Cu, B, K, Pb and Mg in soybeans, diminished the
uptake of Fe, Zn and Mn in
maize and reduced the uptake of Fe, Ca, Cu, Mg, Mn and Zn in
sugar beet (Zayed and Terry,
2003 and references therein). Since Cr is a toxic and
non-essential element, plants may lack
any specific mechanism for its transport. Moreover, being
structurally similar to essential
nutrients and having competitive binding abilities to common
carriers of essential elements,
can affect uptake and transport of mineral nutrients in plants
in a complex way. For instance,
Cr may reduce S and Fe uptake. Similarly, P and Cr compete for
surface sites as well as Fe, S
and Mn. Thus, the competitive ability of Cr allows for its swift
entry into plant system.
Numerous studies on the effect of Cr on different plants are
available in the literature.
For example, Sujatha et al. (1996) observed that irrigation with
tannery wastewaters resulted
in micronutrient deficiencies in several agricultural crops.
Khan et al (2001) noted a decrease
in N, P and K contents in dried rice plant treated with water
having 0.5 ppm Cr. According to
Barcelo et al. (1985), there is a strong correlation between
chlorophyll pigments and Fe and
Zn uptake in Cr-stressed plants. In fruit plants, Cr hinders the
availability of nutrients like Fe,
Mn, Cu and Zn to plant parts such as roots, leaves and stems
(Sharma and Pant, 1994). The
N, P, K, Na, Ca and Mg content in stems and branches of tomato
plants treated with Cr at 50
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Chapter Two Review of Literature 26
and 100 mg L_1 were significantly reduced (Moral et al., 1995).
Likewise, Moral et al. (1996)
reported negative effects of Cr on Fe absorption in Lycopersicum
esculentum M plants.
Shanker (2003), however, explains that the impediment of
nutrient transport in heavy metal
stressed plants is due to the inhibition of the activity of the
plasma membrane H+ATPase.
Cadmium also influences the uptake and transport of essential
elements in plants,
either by reducing their availability in the soil or decreasing
the microbes in the soil (Moreno
et al., 1999). Cd toxicity causes the nutritional deficiency in
plants (Das et al., 1997) by
inhibiting chlorophyll synthesis and causing disorganization of
the chloroplast structure
(Clarkson and Luttage, 1989; Rivetta et al., 1997). Reports
indicate a reduction in the uptake
of Fe and an accumulation of Cd in the tissues of roots and
shoots of maize plants when the
Cd concentration was increased in the soil (Liu et al.,
2006).
2.7 Effect of Heavy Metals on the Enzymatic System
Enzymatic activity is indispensable in enhancing the stress
reaction response in plants
through biosynthesis of signaling molecules. It is reported that
heavy metals impede the
enzymes associated with the photosynthetic carbon reduction
cycle and all of three phases of
the Calvin cycle including; carboxylation, reduction and
regeneration in plants (Krupa and
Baszynski 1995: Prasad 1995, 1997).
In accordance with Sheoran et al. (1990), Cd and Ni may also
reduce photosynthetic
activity in plants by inhibiting various enzymes (Rubisco, 3-PGA
kinase, NADP, NAD
glyceraldehydes 3-P dehydrogenase, aldolase and FDPase) of the
photosynthetic carbon
reduction cycle. The toxicity of cadmium also damages cell
membrane and inactivates
enzymes, possibly by reacting with the SH-group of proteins
(Mathys, 1975: Fuhrer, 1988),
which reflects the inhibitory effects of Pb2+ Cd2+, Zn2+ and
Cu2+ on the activity of the
chloroplast enzymes (Stiborova et al., 1986; Assche and
Clijsters, 1990; Guliev et al, 1992).
However, many of the metal sensitive plant enzymes (rubisco,
nitrate reductase, alcohol
dehyrogenase, glycerol-3-phophate dehydrogenase and urease) are
reported to be Cd tolerant
in the form of a Cd-PC complex (Kneer and Zenk 1992). In an
investigation involving Zea
mays seedlings exposed to 50 uM Cd for 5 days, Cd enhanced
enzymatic activity involved in
sulfate reduction by acquiring more label from 35SO42- (Nussbaum
et al., 1988).
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Chapter Two Review of Literature 27
Many papers are available about hyperactivity of antioxidative
enzymes in various
plants under Cu, Pb, Zn stress (Ali et al., 2003; Assche and
Clijsters, 1990). Nevertheless,
literature on the role of enzymatic antioxidant system in
protecting plants from the toxic
effects of reactive oxygen species (ROS) under Cr stress is
scarce. The antioxidant system,
besides its function in detoxification, may also be a sensitive
target for Cr toxicity in plants.
Inside the cell, a reduction of Cr (VI) to Cr (III) is due to
the formation of free radicals by the
strong oxidative ability of Cr (McGrath, 1982; Cervantes et al.,
2001). Thus, plants growing
in a Cr (VI) stressed environment are prone to potential risk
induced by ROS. Therefore, in
response to Cr stress, the antioxidative defense system,
consisting of several non-enzymatic
and enzymatic mechanisms, is activated in the cell. One of the
protective mechanisms is the
enzymatic antioxidant system, which involves the sequential and
simultaneous action of a
number of enzymes including superoxide dismutase (SOD),
peroxidase (POD), catalase
(CAT), and ascorbate peroxidase (APX) (Clijsters et al., 1999).
Samantaray et al. (2001) and
Poschenrieder et al. (1991) observed that Cr toxicity increased
the CAT activity in bean
plants. However, Cr depressed the enzyme activity in Zea mays,
Triticum spp., and Brassica
chinensis (Ren et al., 2002; Sharma et al., 2003).
Montes-Holguin et al., (2006) suggested
that iron–porphyrin biomolecules (CAT) are able to interact with
Cr through their iron center,
affecting the availability of the active form of iron and
thereby suppressing the CAT activity.
2.7.1 Root Fe III Reductase
Heavy metal toxicity hinders Fe mobility and uptake by plants,
and restrains the
reduction of Fe (III) to Fe (II) and its availability.
Consequently, Fe deficiency causes
chlorosis in plants (Shanker and Pathmanabhan, 2004). Under
Fe-deficient conditions, an
enhancement of the root Fe (III) reductase activity increases
the capacity of the plant to
reduce Fe (III) to Fe (II); a form in which roots absorb Fe
(Alcantara et al., 1994). In a
similar manner, the application of Cr to iron-deficient Plantago
lanceolata roots enhanced
the activity of root-associated Fe (III) reductase. The
examination by Wolfgang (1996) in a
split root experiment applying Cr and iron-free treatments to
root medium exhibited
intermediate FeEDTA reductase activity as compared to non-split
control plants. Similarly,
under iron deficient conditions, addition of Cr (III) at 2 µM
restricted ferric chelate reductase
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Chapter Two Review of Literature 28
in roots of alfalfa plants, while at 10 µM it stimulated ferric
chelate reductase in media
containing cobalt, nickel, chromium, and copper (Barton et al.,
2000).
2.7.2 Nitrate Reductase
Various tree species are affected by higher concentrations of
heavy metals. In Cr (VI)
stressed Albezzia lebbek plants, the nitrate reductase (NR)
activity of leaves was substantially
enhanced as compared to control. However, the activity was
negatively correlated with other
parts including root and shoot length, leaf area and biomass of
the plants (Tripathi et al.,
1999). Similarly, Cr concentration up to 200 µM significantly
restrained the NR activity in
Nelumbo nucifera (Vajpayee et al., 1999) and Nymphaea alba
plants (Vajpayee et al., 2000).
However, low concentrations of Cr (1 µM) enhanced NR activity.
While higher
concentrations of Cr render it toxic, which significantly
inhibited enzymatic activity in wheat
(Panda and Patra, 2000). A heavy metal like Cd is instrumental
in reducing nitrate reductase
activity at higher concentrations and reducing the absorption
and transport of nitrate from
roots to shoots of plants (Hernández et al., 1996). Similar
reduction in the enzyme due to Cd
was also exhibited in Silene cucubalus plants (Mathys,
1975).
2.7.3 Antioxidant Enzymes
Oxygen affects the cell metabolism in two ways. On the one hand,
it provides energy
for enzymatic combustion of organic compounds. On the other
hand, it causes damage to
aerobic cells due to the formation of reactive oxygen
intermediates (Bartisz, 1997), which
may be excessively produced in various compartments or
organelles even under normal
conditions. However, living organisms possess a highly efficient
defense called antioxidative
or antioxidant systems against the toxicity of reactive oxygen
intermediates (ROIs). These
defense systems are comprised of both non-enzymatic and
enzymatic constituents.
Heavy metals, at low concentrations, also promote the
antioxidant activity of
enzymes. However, at higher metal contents, catalyse activity is
reduced and SOD activity
remains unaffected (Gwozdz et al., 1997). A study on the affect
of Cr (VI) on SOD activity
of root mitochondria in pea plants, revealed that SOD activity
was increased by 20% at 20
µM Cr, while higher Cr contents (200 µM) substantially reduced
SOD activity (Dixit et al.,
2002). A Cr dose ranging between 20-80 ppm inhibited the
specific activity of catalase in
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Chapter Two Review of Literature 29
sugarcane (Jain et al., 2000). According to Chatterjee and
Chatterjee (2000), an excess of Cr
(0.5 mM) restricted the activity of catalase in cauliflower
leaves. The activity of peroxidase
and catalase was reportedly increased in tolerant calluses
compared to non-tolerant ones in
Echinochloa colona (L) plants at a Cr treatment of 1.5 mg L_1
(Samantaray et al., 2001). The
application of Cr at a concentration of 15 µM showed an increase
in the catalase and
peroxidase activities in calli derived from Leucaena
leucocephala (K8) grown on Cr treated
as compared to untreated soil (Rout et al., 1999). Similarly,
cadmium adversely intervenes
the antioxidant enzymes.
2.8 Effect of Wastewater Applications on Plantations
Shahalam et al. (1998) raised alfalfa, radish and tomato in
plots irrigated with
wastewater and fresh water through a sprinkler along with a sub
treatment with or without
fertilizer to each crop. The physical and chemical properties of
soil, the crop yield and
subsurface drainage were monitored. In most of the cases, yield
remained unchanged. There
was no significant change in silty loam soil properties except
slight variation in soil porosity
and salinity.
Guo and Sims (2000) examined the effects of five different
irrigation rates of water
and slaughterhouse wastewater on the soil, tree biomass
production and nutrient uptake by
Eucalyptus globulus seedlings grown in three growth cabinets at
various temperatures (5°C,
15°C and 25°C) and seasons (winter, spring/autumn and summer).
Wastewater irrigation
influenced soil properties by reducing soil pH and increasing
soil nutrient concentrations. At
the same time, it enhanced tree leaf area, biomass production,
nutrient uptake and shoot:root
ratio. At 5°C, the seedlings showed no response to wastewater
irrigation rates, but the soil pH