effect of mixed industrial effluents on soil, tree...

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

DEDICATED TO MY PARENTS

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

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

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

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

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

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

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.

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.

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

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

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

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

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


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


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).


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


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


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.

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


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


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


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


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.


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.


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).


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


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


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


(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


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).


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).


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


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).


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


(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,


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


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


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).


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


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


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

effect of mixed industrial effluents on soil, tree...

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