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Eco-Physiological responses of Sunflower (Helianthus annuus L.) against various

levels of Rhizospheric Arsenic (As) and Associated Metals in the Soil

Submitted by

Muhammad Asif Imran

A thesis submitted to the University of the Punjab in fulfillment of the requirement for

the degree of

Doctor of Philosophy

Supervisor

Prof. Dr. Muhammad Nawaz Chaudhry

Co-Supervisor

Dr. Khan Rass Masood

College of Earth and Environmental Sciences

University of the Punjab, Quaid-i-Azam campus,

Lahore – Pakistan

(2015)

DECLARATION

The thesis which is being submitted for the degree of Ph.D. in the University of the Punjab does not contain any material which has been submitted for the award of Ph.D. degree in any University and to the best of my knowledge and belief, neither does this thesis contain any material published or written previously by another person, except when due reference is made to the source in the text of the thesis.

Muhammad Asif Imran

Ph.D. ScholarCollege of Earth and Environmental

SciencesUniversity of the Punjab

Lahore - Pakistan

ACKNOWLEDGEMENTS

All the acclamations and appreciation are for Almighty Allah, the compassionate and

benevolent, the only creator who bestowed me with potential and ability to make

some contribution to the already existing ocean of knowledge. I offer my humblest

thanks from deepest core of my heart to the Holy Prophet Hazrat Muhammad

(SallAllaho alaihe wasallam) whose moral and spiritual teaching enlightened my heart

and mind and flourished my thoughts towards achieving high ideals of light.

With humble, profound and deep sense of devotion, I wish to report my sincerest

appreciation and gratitude to my supervisor Prof. Dr. Muhammad Nawaz Ch.

(Professor Emeritus) and co-supervisor Prof. Dr. Khan Rass Masood (Chairman

Department of Botany) for their valuable suggestions, kind and erudite guidance.

I acknowledge the profound gratitude for enthusiastic guidance, continuous support

and encouragement of Prof. Dr. Firdous-e-bareen, Chairman College of Earth and

Environmental Sciences for her sympathetic attitude.

I would also like to express my heartfelt thanks to Dr. Zahoor Ahmad Sajid,

Department of Botany for his cordial cooperation in each and every aspect of this

research work. My sincere thanks are also extended to my class fellows Shoaib,

Kamran Qamar, Farrukh, Ali Kamran and all others for their indescribable help and

cooperation. I am also thankful to Mr. Muhammad Imran Ch., Dr. Muzaffar Majeed

Ch. and all the laboratory staff serving in CEES especially Safdar Shehzad for their

cooperation in completion of this research work.

Last but by no means the least, I avail this opportunity to express my fervent thanks

and heartiest compliments towards my all family members for their affection,

amicable attitude and for remembering me in their prayers. I am cordially thankful to

HEC for their financial assistance during all this experimentation under 5000

Indigenous Fellowship scheme.

Muhammad Asif Imran

I dedicate this piece of research work to my beloved Late father (Muhammad Shaban Ch.)

and my sweet motherwho always encouraged and cooperated me during each and every field of my life.

“Tusi great o Abu g”

ABSTRACT

A series of four experiments was performed to evaluate effect of different

levels of two inorganic arsenicals on seed germination, vegetative and reproductive

growth or yield of sunflower cultivars. First experiment conducted in climatic room

using seeds of four sunflower cultivars revealed adverse effects of higher levels of

arsenic (6, 8 and 10 mg As/L) on seed germination. Reduction in germination

percentage, increased mean germination time, more days to 50 % germination and

stunted plumule and radicle growth with poor seedling vigour index depicted stressful

effects of arsenic for sunflower seedlings while lower levels of arsenic (2 and 4 mg

As/L) proved to be a little bit promoting for seeds to germinate. In further three pot

experiments arsenic was applied through soil, irrigation water as well as in

combination to two sunflower cultivars and different morphological, physiological

and plant water relation parameters were recorded. Arsenic bio-accumulative potential

as well as concentrations of 26 different micro, trace and heavy metal ions were also

determined in root, shoot, leaves and seeds of sunflower cultivars at crop maturity

using ICP-OES. As observed during the experimentation and concluded from physio-

chemical analysis of the plant organs, roots were found substantial sink for arsenic in

sunflower and least accumulation was recorded in seeds or achenes. Arsenic

application in soil proved more stressful than irrigation water alone and overall

arsenic application in combination was proved most deterrent for sunflower growth

and development. Plant accumulated arsenic according to its levels in rooting medium

and availability to plant because in aqueous rhizospheric environment it transforms

into various complex compounds and just a fraction is phytoextracted by sunflower

roots. Yield was also affected due to deleterious effects of higher concentrations of

arsenicals (80 and 100 mg As/kg soil) either as arsenate or arsenite with lower

capitulum diameter and reduced hundred achene weight. Both, cultivars or hybrids of

sunflower showed similar behavior towards its ability to cope with arsenic levels

more than 100 mg As/kg soil with very little phytoextraction potential and

accumulation in above ground biomass.

Table of Contents

CHAPTER 1. INTRODUCTION............................................................1

CHAPTER 2. REVIEW OF LITERATURE...........................................................6

2.1 Heavy metals...................................................................................................6

2.2 Arsenic (As).....................................................................................................6

2.3 Historical background.....................................................................................6

2.4 Characteristics of arsenic.................................................................................6

2.5 Arsenic minerals..............................................................................................7

2.6 Arsenic speciation...........................................................................................8

2.7 Sources..........................................................................................................10

2.7.1 Natural sources of Arsenic.....................................................................10

2.7.2 Unnatural or Anthropogenic pollution by arsenic..................................11

2.8 Products and uses of arsenic..........................................................................11

2.9 Arsenic pollution in Air, Water and Soil.......................................................12

2.10 Arsenic and coal burning...............................................................................13

2.11 Compatibility of arsenic with other elements................................................14

2.11.1 Arsenic (As) and iron (Fe):....................................................................14

2.11.2 Arsenic and Phosphorus:........................................................................14

2.12 Arsenic pollution in Asia...............................................................................15

2.13 Arsenic contamination in Pakistan................................................................17

2.14 Toxicity of arsenicals....................................................................................18

2.14.1 In Bacteria and Fungi:............................................................................18

2.14.2 Toxicity of arsenic to plants:..................................................................18

2.15 Adaptive mechanism in plants.......................................................................20

2.15.1 Metal excluders:........................................................................................21

2.15.2 Metal indicators:........................................................................................21

2.15.3 Accumulators:...........................................................................................21

2.15.4 Oxygen donor ligands............................................................................22

2.15.5 Sulfur donor ligands...............................................................................22

2.15.6 Nitrogen donor ligands...........................................................................23

2.16 Intracellular compartmentalization................................................................23

2.17 As contents of some edible crops and vegetables.........................................23

2.18 Sunflower......................................................................................................24

2.19 Uses of sunflower..........................................................................................26

CHAPTER 3. MATERIALS AND METHODS....................................................28

3.1 Seed Material:................................................................................................28

3.2 Metal and Treatment plan:.............................................................................29

3.3 Germination percentage (GP):.......................................................................30

3.4 Mean Germination Time (MGT):..................................................................30

3.5 Seedling Vigour Index (SVI):.......................................................................30

3.6 Days to 50% germination (T50):...................................................................31

3.7 Pots................................................................................................................32

3.8 Soil.................................................................................................................32

3.9 Physiochemical Characteristics of experimental soil....................................32

3.9.1 Determination of soil texture.................................................................32

3.9.2 Reagents.................................................................................................32

3.9.3 Method...................................................................................................33

3.9.4 Determination of Blank..........................................................................33

3.9.5 Determination of Silt and Clay..............................................................33

3.10 Determination of Sand...................................................................................34

3.11 Saturation percentage....................................................................................35

3.12 Moisture contents (%)...................................................................................35

3.13 Soil pH...........................................................................................................35

3.14 Application of arsenical treatments...............................................................36

3.15 Meteorological Data......................................................................................37

3.16 Plant measurements and arsenic determination.............................................37

3.17 Plant morphological observations.................................................................37

3.18 Plant physiological or water relation parameters..........................................39

3.19 Yield components..........................................................................................39

3.20 Preparation of plant samples for analysis:.....................................................40

3.20.1 Digestion of plant samples.....................................................................40

3.20.2 Reagents:................................................................................................40

3.20.3 Procedure................................................................................................40

3.20.4 Preparation of sample test solution........................................................40

3.21 Macro and Micro-nutrient ions......................................................................41

3.22 Heavy metal and other ions detected in plants..............................................41

3.23 Arsenic accumulation in root, stem and leaves.............................................42

3.24 Arsenic bioaccumulation coefficient.............................................................42

3.25 Arsenic contents of fruit or achenes..............................................................43

3.26 Left over arsenic............................................................................................43

3.27 Statistical analysis:........................................................................................43

CHAPTER 4. RESULT AND DISCUSSION.......................................................44

4.1 EXPERIMENT 1........................................................................................44

4.1.1 Germination percentage (G %age).........................................................44

4.1.2 Mean Germination Time (MGT)...........................................................45

4.1.3 Time to 50% germination (T50)..............................................................45

4.1.4 Plumule Length (mm)............................................................................45

4.1.5 Radicle Length (mm).............................................................................46

4.1.6 Seedling Vigour Index (SVI).................................................................46

4.2 RESULT & DISCUSSION EXPERIMENT NO. 2......................................53

4.2.1 First harvest (at vegetative stage):..........................................................53

4.2.1.1 Agronomic attributes......................................................................53

4.2.1.2 Physiological and plant water relation parameters.........................58

4.2.2 Final harvest (at maturity):.....................................................................62

4.2.2.1 Agronomic and yield parameters....................................................62

4.2.3 Arsenic accumulation in various plant tissues and left over arsenic......65

4.2.4 Bioaccumulation coefficient (BC) of arsenic.........................................66

4.2.5 Comparisons of ratios among arsenic concentrations in root, shoot,

leaves and seed.....................................................................................................68

4.2.6 Phosphorus (P), calcium (Ca) and magnesium (Mg) contents in root,

shoot, leaves and seeds of sunflower cultivars grown in arsenic contaminated soil

…………………………………………………………………………69

4.2.7 Potassium (K), boron (B) and copper (Cu) contents in root, shoot, leaves

and seeds of sunflower cultivars grown in arsenic contaminated soil.................72

4.2.8 Iron (Fe), manganese (Mn) and zinc (Zn) contents in root, shoot, leaves

and seeds of sunflower cultivars grown in arsenic contaminated soil.................75

4.2.9 Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in root,


…………………………………………………………………………78

4.2.10 Contents of barium (Ba), bismuth (Bi) and cadmium (Cd) found in root,


…………………………………………………………………………82

4.2.11 Cobalt (Co), chromium (Cr) and lithium (Li) contents in root, shoot,

leaves and seeds or achenes of sunflower cultivars grown in arsenic

contaminated soil.................................................................................................86

4.2.12 Nickel (Ni), lead (Pb) and antimony (Sb) contents found in root, shoot,

leaves and achenes of sunflower cultivars grown in arsenic contaminated soil. .89

4.2.13 Selenium (Se), strontium (Sr) and titanium (Ti) contents in root, shoot,

leaves and seeds of sunflower cultivars grown in arsenic contaminated soil......93

4.2.14 Thallium (Tl) and vanadium (v) contents in root, shoot, leaves and seeds

of sunflower cultivars grown in arsenic contaminated soil..................................96

4.2.15 Conclusion (Experiment 2)....................................................................99

4.3 RESULTS AND DISCUSSION EXPERIMENT NO. 3............................101

4.3.1 First harvest (at vegetative stage):........................................................101

4.3.2 Agronomic parameters.........................................................................101

4.3.3 Physiological and water relation parameters........................................105

4.3.4 Final harvest (at maturity):...................................................................108

4.3.5 Morphological and yield parameters....................................................108

4.3.6 Arsenic (As) contents accumulated in different sunflower organs and

left over arsenic, applied through irrigation water.............................................111

4.3.7 Arsenic bioaccumulation coefficient (BC) of sunflower cultivars......112

4.3.8 Arsenic concentration [As] ratios among various organs of sunflower

cultivars irrigated through As contaminated water............................................114


shoot, leaves and seeds of sunflower cultivars irrigated through arsenic

contaminated water............................................................................................116


and seeds of sunflower cultivars irrigated through arsenic contaminated water.

……………………………………………………………………….119


and seeds of sunflower cultivars irrigated through arsenic contaminated water.

………………………………………………………………………..123

4.3.12 Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in root,


contaminated water............................................................................................126

4.3.13 Barium (Ba), bismuth (Bi) and cadmium (Cd) contents in root, shoot,

leaves and seeds of sunflower cultivars irrigated through arsenic contaminated

water. ……………………………………………………………………….129


leaves and seeds of sunflower irrigated through arsenic contaminated water.. .133

4.3.15 Nickel (Ni), lead (Pb) and antimony (Sb) contents in root, shoot, leaves

and seeds of sunflower irrigated through arsenic contaminated water..............136

4.3.16 Selenium (Se) and strontium (Sr) contents in root, shoot, leaves and

seeds of sunflower irrigated through As contaminated water............................139

4.3.17 Titanium (Ti), thallium (Tl) and vanadium (V) contents in sunflower

cultivars irrigated through As contaminated water............................................142

4.3.18 Conclusion (Experiment 3):.................................................................146

4.4 RESULTS AND DISCUSSION EXPERIMENT NO. 4............................147

4.4.1 First harvest (at vegetative stage):........................................................147

4.4.2 Agronomic parameters:........................................................................147

4.4.3 Physiological and water relation parameters:......................................151

4.4.4 Final harvest (at maturity):...................................................................154

4.4.4.1 Agronomic and yield parameters:.................................................154

4.4.4.2 Arsenic contents in different sunflower organs:...........................156

4.4.4.3 Arsenic bioaccumulative coefficient of sunflower cultivars:.......158

4.4.4.4 Phosphorus (P), calcium (Ca) and magnesium (Mg) contents in

root, shoot, leaf and seed of sunflower cultivars under different arsenic

concentrations in soil as well as irrigation water...........................................160

4.4.4.5 Potassium (K), boron (B) and copper (Cu) contents of sunflower

under different As levels in rooting medium.................................................164

4.4.4.6 Iron (Fe), manganese (Mn) and zinc (Zn) contents in sunflower

under different arsenic salts and levels through soil plus irrigation water.....168

4.4.4.7 Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in

sunflower under different As conditions........................................................172

4.4.4.8 Barium (Ba) and bismuth (Bi) contents in sunflower under different

arsenic conditions...........................................................................................175

Figure 4.14(c): Barium (Ba) and bismuth (Bi) contents in sunflower under

different As conditions...................................................................................178

4.4.4.9 Cadmium (Cd), cobalt (Co) and chromium (Cr) contents in

sunflower under different arsenic conditions.................................................178

4.4.4.10 Lithium (Li), nickel (Ni) and lead (Pb) contents in sunflower under

different arsenic conditions in rooting medium.............................................182

4.4.4.11 Antimony (Sb), selenium (Se) and strontium (Sr) contents in

sunflower under different As conditions........................................................186

4.4.4.12 Titanium (Ti), thallium (Tl) and vanadium (V) contents in

sunflower under different conditions of arsenic.............................................190

4.4.5 Conclusion (Experiment 4)..................................................................194

4.5 Future prospects...........................................................................................195

4.6 Abbreviations..............................................................................................196

CHAPTER 5. REFERENCES...........................................................................198

List of Tables

Table 1.1: Total estimates of sunflower crop in Punjab, Pakistan, from 2000-2014.....4

Table 2.1: Common arsenic minerals, their composition and occurrence....................8

Table 2.2: Arsenic species commonly found in environment and living organisms.. . .9

Table 2.3: Normal ranges in plants and lower limits for hyperaccumulation of some

heavy metals.................................................................................................................21

Table 2.4: Arsenic concentrations in some common edible crops:.............................24

Table 3.1: Composition of Hoagland’s nutrient solution:...........................................29

Table 3.2: Chemical composition of soil....................................................................34

Table 3.3: Treatments plan..........................................................................................36

Table 3.4: Meteorological data recorded during the course of experimentation........37

Table 4.1: Two way analysis of variance (ANOVA) for germination percentage (G

%age), mean germination time (MGT), time to 50% germination (T50), plumule

length (Pl L), radicle length (Rl L) and seedling vigour index (SVI) of sunflower

cultivars........................................................................................................................47

Table 4.2: Means of arsenical (sodium arsenate and sodium arsenite) treatments for

germination %age, mean germination time, days to 50% germination, plumule length,

radicle length and seedling vigour index.....................................................................48

Table 4.3: Means of sunflower cultivars for Germination %age, Mean Germination

Time, days to 50% germination, plumule length, radicle length and seedling vigour

index.............................................................................................................................49

Table 4.1(a): Analysis of variance of data for shoot length, root length andshoot: root

ratio at vegetative stage under various As levels applied in soil..................................54

Table 4.2(a): Analysis of variance of data and mean±SE for fresh and dry weights (g)

and water contents of shoot at vegetative stage under various As levels applied in soil.

......................................................................................................................................56

Table 4.3(a): Analysis of variance of data for root fresh and dry weights (g) and

water contents of root at vegetative stage under various As levels applied in soil......57

Table 4.4(a): Analysis of variance of data for number of leaves, fresh and dry weights

(g) of leaf at vegetative stage under various As levels applied in soil.........................59

Table 4.5(a): Analysis of variance of data for leaf turgid weight, specific weight of

leaf, leaf area, leaf succulence and relative water contents of leaf at vegetative stage

under various As levels applied in soil........................................................................61

Table 4.6(a): ANOVA table for different agronomic and yield parameters of

sunflower cultivated in arsenic contaminated soil.......................................................63

Table 4.7(a): Analysis of variance for As accumulation in root, shoot, leaf, seed and

left over arsenic applied as various levels in soil.........................................................66

Table 4.8(a): ANOVA about bioaccumulation coefficient of arsenic in root, shoot,

leaf and seed of sunflower............................................................................................67

Table 4.9(a): Ratios of arsenic concentrations [As] among different sunflower plant

tissues...........................................................................................................................68

Table 4.10(a): ANOVA for phosphorus (P), calcium (Ca) and magnesium (Mg)

contents found in sunflower cultivars grown on arsenic contaminated soil................70

Table 4.11(a): ANOVA regarding potassium (K), boron (B) and copper (Cu) contents

in different organs of sunflower cultivated in arsenic contaminated soil....................73

Table 4.12(a): ANOVA for iron (Fe), manganese (Mn) and zinc (Zn) contents in

different organs of sunflower cultivated in arsenic contaminated soil........................76

Table 4.13(a): ANOVA for molybdenum (Mo), silver (Ag) and aluminum (Al)

contents in sunflower cultivars grown in arsenic contaminated soil............................80

Table 4.14(a): ANOVA for barium (Ba), bismuth (Bi) and cadmium (Cd) contents of

sunflower cultivars grown in arsenic contaminated soil..............................................83

Table 4.15(a): ANOVA for cobalt (Co), chromium (Cr) and lithium (Li) contents in

sunflower cultivars grown in arsenic contaminated soil..............................................87

Table 4.16(a): ANOVA for nickel (Ni), lead (Pb) and antimony contents in sunflower

cultivars grown in arsenic contaminated soil...............................................................90

Table 4.17(a): ANOVA for selenium (Se), strontium (Sr) and titanium (Ti) contents

in sunflower cultivars grown in arsenic contaminated soil..........................................94

Table 4.18(a): ANOVA for thallium (Tl) and vanadium (V) contents of sunflower


Table 4.1(b): Analysis of variance (ANOVA) of data for shoot length, root length

and shoot:root ratio at vegetative stage under various As levels applied through

irrigation water...........................................................................................................101

Table 4.2(b): Analysis of variance (ANOVA) of data about fresh weight, dry weight

and water contents of shoot and root at vegetative stage under various As levels

applied through irrigation water.................................................................................103

Table 4.3(b): Analysis of variance (ANOVA) of data for number of leaves, fresh,

dry, turgid and specific weight of sunflower leaf at vegetative stage under various As

levels applied through irrigation water......................................................................105

Table 4.4(b): Analysis of variance (ANOVA) of data for leaf succulence, leaf area

and relative water contents of sunflower leaf at vegetative stage under various As

levels applied through irrigation water......................................................................107

Table 4.5(b): ANOVA for stem length, root length, stem to root ratio, number of

leaves, capitulum diameter and hundred achene weight recorded in sunflower

cultivars irrigated through As contaminated water....................................................109

Table 4.6(b): ANOVA for arsenic (As) accumulated in sunflower, applied through


Table 4.7(b): ANOVA for As bioaccumulation coefficient (BC) of sunflower

irrigated through As contaminated water...................................................................113

Table 4.8(b): ANOVA for arsenic concentrations [As] ratios among different organs

of sunflower irrigated through As contaminated water..............................................115

Table 4.9(b): ANOVA for phosphorus (P), calcium (Ca) and magnesium (Mg)

contents in sunflower cultivars irrigated through arsenic contaminated water..........117

Table 4.10(b): ANOVA for potassium (K), boron (B) and copper (Cu) contents in

sunflower cultivars irrigated through arsenic contaminated water............................120

Table 4.11(b): ANOVA for iron (Fe), manganese (Mn) and zinc (Zn) contents in


Table 4.12(b): ANOVA for molybdenum (Mo), silver (Ag) and aluminum (Al)

contents of sunflower cultivars irrigated through arsenic contaminated water..........127

Table 4.13(b): ANOVA for barium (Ba), bismuth (Bi) and cadmium (Cd) contents in


Table 4.14(b): ANOVA for cobalt (Co), chromium (Cr) and lithium (Li) contents in

sunflower irrigated through arsenic contaminated water...........................................133

Table 4.15(b): ANOVA for nickel (Ni), lead (Pb) and antimony (Sb) contents in

sunflower irrigated through As contaminated water..................................................137

Table 4.16(b): ANOVA for selenium (Se) and strontium (Sr) contents of sunflower


Table 4.17(b): ANOVA for titanium (Ti), thallium (Tl) and vanadium (V) contents in


Table 4.1(c): Analysis of variance (ANOVA) of data for shoot length, root length and

shoot:root ratio at vegetative stage of two sunflower cultivars under various As levels

applied in soil and irrigation water.............................................................................147

Table 4.2 (c): ANOVA of data for fresh weight, dry weight and water contents of

shoot and root in two sunflower cultivars under different As levels in soil and


Table 4.3(c): Different interaction among varieties, salts and levels for fresh weight

and dry wt. of shoot and root in sunflower cultivated in arsenic contaminated soil and


Table 4.4 (c): ANOVA of data about number, fresh, dry, turgid and specific weight of

leaf and leaf area of two sunflower cultivars under different levels of As in soil and


Table 4.5(c): ANOVA of data for leaf succulence and RWC of leaf in two sunflower

cultivars under different As levels in soil and irrigation water..................................153

Table 4.6(c): Interaction (mean±SE) for leaf succulence and relative water contents

of leaf in two sunflower cultivars under different levels of arsenic in soil and


Table 4.7(c): ANOVA for stem length, root length, shoot to root ratio, number of

leaves, capitulum diameter and hundred achene weight of sunflower cultivars under

various As levels in soil and irrigation water.............................................................155

Table 4.8(c): ANOVA for different arsenic (As) contents found in root, shoot, leaf

and seed of sunflower cultivars and left over arsenic in soil.....................................157

Table 4.9(c): ANOVA for arsenic (As) bioaccumulative coefficient of sunflower

cultivars......................................................................................................................158

Table 4.10(c): ANOVA for relative ratios of arsenic concentrations in different

sunflower organs of two sunflower cultivars under different levels of arsenic in soil

and irrigation water....................................................................................................160

Table 4.11(c): ANOVA for phosphorus (P), calcium (Ca) and magnesium (Mg)

contents in sunflower cultivars grown in different levels of As in soil and irrigation

water...........................................................................................................................161

Table 4.12(c): ANOVA for potassium (K), boron (B) and copper (Cu) contents of

sunflower under As contaminated soil plus irrigation water.....................................165

Table 4.13(c): ANOVA for iron (Fe), manganese (Mn) and zinc (Zn) contents in

sunflower under different As conditions in soil and irrigation water........................169

Table 4.14(c): ANOVA for molybdenum (Mo), silver (Ag) and aluminum (Al)

contents in sunflower under different As conditions.................................................173

Table 4.15(c): ANOVA for barium (Ba) and bismuth (Bi) contents in sunflower

under different As conditions.....................................................................................176

Table 4.16(c): ANOVA for cadmium (Cd), cobalt (Co) and chromium (Cr) contents

in sunflower under different As conditions................................................................179

Table 4.17(c): ANOVA for lithium (Li), nickel (Ni) and lead (Pb) contents in

sunflower under different As conditions....................................................................183

Table 4.18(c): ANOVA for antimony (Sb), selenium (Se) and strontium (Sr) contents

in sunflower under different As conditions................................................................187

Table 4.19(c): ANOVA for titanium (Ti), thallium (Tl) and vanadium (V) contents in


List of Figures

Figure 4.1: Effect of arsenicals on germination percentage (G %age) of sunflower..50

Figure 4.2: Effect of arsenicals on mean germination time (MGT) of sunflower......50

Figure 4.3: Effect of arsenicals on time to 50% germination (T50) of sunflower......51

Figure 4.4: Effect of arsenicals on plumule length of sunflower................................51

Figure 4.5: Effect of arsenicals on radicle length of sunflower achenes....................52

Figure 4.6: Effect of arsenicals on seedling vigour index (SVI) of sunflower...........52

Figure 4.1(a): Shoot length, root length and shoot : root ratio of two sunflower

hybrids grown under different As levels in soil...........................................................54

Figure 4.2(a): Fresh and dry weight (g) and water contents of root in two sunflower

hybrids grown under different As levels in soil...........................................................58

Figure 4.3(a): No. of leaves, fresh and dry weight of leaf in two sunflower cultivars

grown under different As levels in soil........................................................................59

Figure 4.4(a): Leaf turgid weight, specific weight of leaf, leaf area, leaf succulence

and relative water contents of leaf at vegetative stage under various As levels in soil.

......................................................................................................................................61

Figure 4.5(a): Shoot length, root length, shoot to root ratio, number of leaves,

capitulum diameter and weight of hundred achenes under different arsenic

concentrations in soil....................................................................................................64

Figure 4.6(a): Left over arsenic and accumulation of arsenic in root, shoot, leaf and

seed of sunflower plants grown in arsenic contaminated soil......................................66

Figure 4.7(a): Bioaccumulation coefficient (BC) of root, shoot, leaves and seed of

sunflower cultivars grown under As contaminated soil...............................................68

Figure 4.8(a): Comparison of different ratios of arsenic concentrations determined in

different organs of sunflower grown in As contaminated soil.....................................69

Figure 4.9(a): Phosphorus (P), calcium (Ca) and magnesium (Mg) contents found in

root, shoot, leaf and seed of sunflower cultivars grown under arsenic contaminated

soil................................................................................................................................72

Figure 4.10(a): Potassium (K), boron (B) and copper (Cu) contents in sunflower


Figure 4.11(a): Iron (Fe), manganese (Mn) and zinc (Zn) contents in sunflower


Figure 4.12(a): Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in root,

shoot, leaves and seeds of sunflower cultivars grown in arsenic contaminated soil....81

Figure 4.13(a): Barium (Ba), bismuth (Bi) and cadmium (Cd) contents in root, shoot,

leaves and seeds of sunflower cultivars grown in arsenic contaminated soil..............85

Figure 4.14(a): Cobalt (Co), chromium (Cr) and lithium (Li) contents in root, shoot,

leaves and seeds of sunflower cultivars grown in arsenic contaminated soil..............88

Figure 4.15(a): Nickel (Ni), lead (Pb) and antimony (Sb) contents in root, shoot,

leaves and achnenes of sunflower cultivars grown in arsenic contaminated soil........92

Figure 4.16(a): Selenium (Se), strontium (Sr) and titanium (Ti) contents in root,

shoot, leaves and seeds of sunflower cultivars grown in arsenic contaminated soil....96

Figure 4.17(a): Thallium (Tl) and vanadium (V) contents of sunflower cultivars

grown in As contaminated soil.....................................................................................99

Figure 4.1(b): Shoot length, root length and shoot : root ratio of two sunflower

cultivars irrigated through different arsenic levels.....................................................102

Figure 4.2(b): Fresh weight, dry weight of shoot and shoot water contents of two

sunflower cultivars under different arsenic levels in irrigation water........................103

Figure 4.3(b): Number of leaves, fresh weight, turgid weight, dry weight and specific

weight of leaves in two sunflower cultivars under different levels of arsenic in


Figure 4.4(b): Graphs showing different parameters of sunflower grown under As contaminated irrigation water………………………………………………….109-111

Figure 4.5(b): Arsenic (As) accumulation in different plant parts of sunflower cultivars irrigated through As contaminated water…………………………………112

Figure 4.6(b): Arsenic bioaccumulation coefficient (BC) for sunflower cultivars

irrigated through As laden water................................................................................114

Figure 4.7(b): Ratios of arsenic concentrations [As] among different organs of


Figure 4.8(b): Phosphorus (P), calcium (Ca) and magnesium (Mg) contents in root,

shoot, leaves and seeds of sunflower cultivars irrigated through arsenic contaminated

water...........................................................................................................................118

Figure 4.9(b): Potassium (K), boron (B) and copper (Cu) contents in root, shoot,

leaves and seeds of sunflower cultivars irrigated through arsenic contaminated water.

....................................................................................................................................122

Figure 4.10(b): Iron (Fe), manganese (Mn) and zinc (Zn) contents in root, shoot,


....................................................................................................................................126

Figure 4.11(b): Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in root,

shoot, leaves and seeds of sunflower cultivars irrigated through arsenic contaminated

water...........................................................................................................................129

Figure 4.12(b): Barium (Ba), bismuth (Bi) and cadmium (Cd) contents in root, shoot,


....................................................................................................................................132

Figure 4.13(b): Cobalt (Co), chromium (Cr) and lithium (Li) contents in root, shoot,

leaves and seeds of sunflower irrigated through arsenic contaminated water...........135

Figure 4.14(b): Nickel (Ni), lead (Pb) and antimony (Sb) contents in root, shoot,

leaves and seeds of sunflower irrigated through As contaminated water..................139

Figure 4.15(b): Selenium (Se), and strontium (Sr) contents in root, shoot, leaves and

seeds of sunflower irrigated through As contaminated water....................................141

Figure 4.16(b): Titanium (Ti), thallium (Tl) and vanadium (V) contents in root,

shoot, leaves and seeds of sunflower irrigated through As contaminated water.......145

Figure 4.1(c): Shoot length and root length of two sunflower cultivars under different

levels of arsenic in soil and irrigation water..............................................................148

Figure 4.2(c): Water contents of shoot and root of sunflower cultivated in arsenic

contaminated soil and irrigated through As contaminated water...............................151

Figure 4.3(c): Number of leaves, fresh and specific weight of leaf and leaf area of

two sunflower cultivars under different levels of arsenic in soil and irrigation water.

....................................................................................................................................152

Figure 4.4(c): Shoot length, root length, number of leaves, capitulum diameter and

hundred achene weight of sunflower cultivated in As contaminated soil and irrigation

through As contaminated water.................................................................................155

Figure 4.5(c): Arsenic contents in root, shoot, leaf and seed along with left over

arsenic in soil of sunflower cultivars.........................................................................158

Figure 4.6(c): Arsenic (As) bio-accumulative coefficient of two sunflower cultivars

under different arsenic levels in soil and irrigation water..........................................159

Figure 4.7(c): Phosphorus (P), calcium (Ca) and magnesium (Mg) contents in

different organs of sunflower cultivars grown under As contaminated soil and


Figure 4.8(c): Potassium (K), boron (B) and copper (Cu) contents in sunflower

cultivated in As contaminated soil and irrigation water.............................................167

Figure 4.9(c): Iron (Fe), manganese (Mn) and zinc (Zn) contents in sunflower

cultivars under different As conditions......................................................................171

Figure 4.10(c): Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in


Figure 4.11(c): Barium (Ba) and bismuth (Bi) contents in sunflower under different

As conditions..............................................................................................................178

Figure 4.12(c): Cadmium (Cd), cobalt (Co) and chromium (Cr) contents in sunflower


Figure 4.13(c): Lithium (Li), nickel (Ni) and lead (Pb) contents in sunflower under

different As conditions...............................................................................................185

Figure 4.14(c): Antimony (Sb), selenium (Se) and strontium (Sr) contents in


Figure 4.15(c): Titanium (Ti), thallium (Tl) and vanadium (V) contents in sunflower


ABSTRACT

A series of four experiments was performed to evaluate effect of different

levels of two inorganic arsenicals on seed germination, vegetative and reproductive

growth or yield of sunflower cultivars. First experiment conducted in climatic room

using seeds of four sunflower cultivars revealed adverse effects of higher levels of

arsenic (6, 8 and 10 mg As/L) on seed germination. Reduction in germination

percentage, increased mean germination time, more days to 50 % germination and

stunted plumule and radicle growth with poor seedling vigour index depicted stressful

effects of arsenic for sunflower seedlings while lower levels of arsenic (2 and 4 mg

As/L) proved to be a little bit promoting for seeds to germinate. In further three pot

experiments arsenic was applied through soil, irrigation water as well as in

combination to two sunflower cultivars and different morphological, physiological

and plant water relation parameters were recorded. Arsenic bio-accumulative potential

as well as concentrations of 26 different micro, trace and heavy metal ions were also

determined in root, shoot, leaves and seeds of sunflower cultivars at crop maturity

using ICP-OES. As observed during the experimentation and concluded from physio-

chemical analysis of the plant organs, roots were found substantial sink for arsenic in

sunflower and least accumulation was recorded in seeds or achenes. Arsenic

application in soil proved more stressful than irrigation water alone and overall

arsenic application in combination was proved most deterrent for sunflower growth

and development. Plant accumulated arsenic according to its levels in rooting medium

and availability to plant because in aqueous rhizospheric environment it transforms

into various complex compounds and just a fraction is phytoextracted by sunflower

roots. Yield was also affected due to deleterious effects of higher concentrations of

arsenicals (80 and 100 mg As/kg soil) either as arsenate or arsenite with lower

capitulum diameter and reduced hundred achene weight. Both, cultivars or hybrids of

sunflower showed similar behavior towards its ability to cope with arsenic levels

more than 100 mg As/kg soil with very little phytoextraction potential and

accumulation in above ground biomass.

CHAPTER 1. INTRODUCTION

Eco-toxicity or environmental pollution being a major global concern

(Banejad and Olyaie, 2011) resulted from modern technology or industrial revolution

strengthened by “population explosion” along with mismanagement of resources.

Worldwide, metal contamination has increased in the biosphere as a result of rapid

urban and industrial growth (Lien and Wilkin, 2005). This situation is alarming in the

developing world where untreated waste water is extensively used for irrigation or is

disposed off in water resources (Kashif et al., 2009 and UNIDO, 2002).

All metals are naturally present in our environment while some of them are

essentially required as micro-nutrients (Taiz and Zeiger, 2006). Heavy metals are

defined as metals with a density higher than 5 g cm-3, therefore fifty three of the

naturally occurring elements are heavy metals (Ali, 2005). Being ubiquitous, arsenic

(As), is a trace metalloid which is carcinogenic (Matschullat, 2000 and Pigna et al.,

2009), occurring naturally in the Earth’s crust as the 20th most abundant element and a

component of more than 245 minerals (Mandal and Suzuki, 2002) virtually present in

all environmental media (Fitz and Wenzel, 2002).

Arsenic (As) contamination in ground water is a severe global environmental

problem (Yavuz et al., 2010). Many arsenic compounds present in the terrestrial and

marine environments have been detected (Francesconi et al., 2002 and Zhao et al.,

2009). Arsenate [As(V)] and arsenite [As(III)] are the primary inorganic As forms (Koch

et al., 2000). Arsenic toxicity depends on speciation; inorganic forms are more toxic

than organometallic species and inorganic As(III) is more toxic than As(V) (Kuehnelt et

al., 2000). Arsenic can enter the environment through weathering, volcanic activity

and biological activity (Meharg and Hartley-Whitaker, 2002).

Anthropogenic inputs from different agricultural and industrial practices, such

as the application of pesticides and chemical fertilizers, wastewater irrigation,

precipitation from heavy coal combustion, smelter wastes and residues from

metalliferous mining elevates the levels of As in soils along with contamination of

ground and surface water (Zhou and Huang, 2000; Zhang et al., 2002 and Sun et al.,

2009). The elevated levels of arsenic may set off a variety of problems, such as

ground water contamination, loss of soil flora as well as arsenic toxicity in plants,

1

animals and humans (Mahimairaja et al., 2005; Mukherjee and Bhattacharya et al.,

2001and World Health Organization, 2001).

Arsenic contamination is very unevenly distributed between the continents. In

terms of the exposed populations, by far the worst pollution is found in Asia,

especially in a band running from Pakistan, along the southern margins of the

Himalayan and Indo-Burman ranges, to Taiwan, which is referred to as the South and

South East Asian Arsenic Belt (SSAB) (Ravenscroft et al., 2009). Since the 1980s,

evidence has gradually unfolded that arsenic is naturally present in elevated levels in

part of tapped groundwater resources. At present, twelve countries in the region have

reported high levels of arsenic in part of their groundwater resources: Afghanistan,

Bangladesh, Cambodia, China, India, Lao PDR, Mongolia, Myanmar, Veit Nam,

Thailand, Nepal and Pakistan. (Heikens, 2006). The World Health Organization

(WHO) described the situation in Bangladesh (former Eastern Pakistan) as “the

largest poisoning of a population in history” (Smith et al., 2000). For the first time in

1980s the scientific explanation of the pollution in Bengal was presented (Jain and

Ali, 2000) showing that the cause was geological, and not anthropogenic, and acted as

a stimulus for testing in surrounding countries.

Following the arsenic crisis in Bangladesh and other neighboring countries,

Pakistan has recognized the need of assessing drinking water quality for arsenic

contamination. In this regard, the Government of Pakistan has undertaken many

initiatives with assistance from UNICEF since 1999. As a result of these initiatives,

the presence of arsenic contamination has been recognized and consequently an

arsenic mitigation programme, at national level has already been launched by the

Government of Pakistan with the assistance being provided by UNICEF. (Tameez,

2004). Alarming levels of ground water arsenic concentration has been observed

during the course of water quality surveys conducted by PCRWR during 2001, 2003

and 2004 (PCRWR, 2004). District Rahim Yar Khan has been declared as worst hit

arsenic contamination area (Islam-ul-Haq et al, 2007). Higher arsenic levels in

ground water and other food sources are also found in the area of Tehsil Pattoki,

District Ksur.

From contaminated water this poisonous metal enters in the food chain. Bio-

magnification makes it worse when it reaches up to the human level. A link between

arsenic contamination of ground water and increased risk of a number of vascular

2

diseases like blackfoot disease, cerebrovascular diseases, ischaemic heart disease has

been reported (Rahman, 2006). Recently the International Agency of Research on

Cancer classified arsenic in drinking water as a “Group 1” human carcinogen because

of evidences of elevated risk of bladder, skin and lung cancer (IARC, 2004). To

evaluate the possible health risk to humans consuming crops irrigated with arsenic

contaminated water, information is needed regarding the soil-to-plant transportation

of arsenic and to minimize the accumulation of arsenic in plants consumed directly by

humans, farm animals or wildlife (Meharg and Hartley-Whitaker, 2002). It is evident

that different types of plants, including both weeds and cultivated crops, can

accumulate substantial quantities of arsenic from the soil environment (Geng et al,

2006). Arsenic absorption by plants is influenced by many factors including plant

species (Williams et al., 2005), the concentration of arsenic in the soil

(Roychowdhury et al., 2005), soil properties such as pH and clay content (Dahal et

al., 2008), and the presence of other ions (Violante and Pigna, 2002).

There are different ways by which plants handle toxic heavy metals such as

phytoimmobilization, phytostabilization, rhizofilteration, phytovolatilization and

phytoextraction, the latter being most widely accepted for remediation of soils

contaminated with toxic heavy metals (Mahmood, 2010). Plants behave differently to

heavy metals; data about different crops like beans, members of Brassicaceae family,

mosses, ferns and certain medicinal plants are available about their sensitivity or

tolerance to arsenic. Plant species and even genotypes differ greatly in their ability to

take up, transport and accumulate arsenic within the plants. Plant mechanisms

affecting the root uptake and shoot transport of arsenic can also affect the expression

of arsenic toxicity in plants. Therefore, the selection of plant genotypes with high

ability to repress root uptake and shoot transport of arsenic is a reasonable approach to

alleviate adverse effects of arsenic toxicity in crop plants (Liu et al, 2012).

Sunflower (Helianthus annuus L.), of genus Helianthus, tribe Heliantheae and

family Asteraceae (Compositae) is considered as the world’s fourth largest oil seed

crop (Burke, 2003; Rodriguez et al., 2002). Being important for edible oil, it was

introduced in Pakistan in early sixties, in Pakistan various oilseed crops are being

grown including rapeseed-mustard, cottonseed, linseed, groundnut, sunflower,

sesame, soybean, canola, castor and safflower (Ahmad, 2009). Among these crops,

sunflower has considerable contribution towards the local production of edible oil in

Pakistan due to high yield potential as well as oil quality (Ali and Mirza, 2005). Data

3

regarding the area under cultivation and average yield of sunflower in Punjab

(Pakistan) since year 2000 are depicted in Table 1.1. The unique agro-climatic

conditions of Pakistan make it possible to cultivate sunflower during spring and

autumn seasons. Furthermore, its short duration, less water and fertilizer

requirements, high profitability due to its versatile role as a high proteinacious meal,

oil contents, medicinal use and preparation of textile dyes etc. attract the framers to

cultivate it over large areas in Pakistan, and therefore it is cultivated in almost all parts

of the country.

Table 1.1: Total estimates of sunflower crop in Punjab, Pakistan, from 2000-

2014.

Year Area (Acres) Production (Tones) Av. Yield (Mds/acre)

2000-01 45140 24920 14.79

2001-02 52979 28877 14.61

2002-03 92147 54885 15.96

2003-04 207839 122490 15.79

2004-05 145618 86356 15.89

2005-06 185465 122840 17.75

2006-07 223520 156258 18.73

2007-08 375827 324973 23.17

2008-09 160159 130510 21.83

2009-10 84294 65050 20.68

2010-11 80406 61253 20.41

2011-12 114258 94052 22.06

2012-13 127800 92200 19.34

2013-14 97300 71700 20.58

Source: Crop Reporting Services Punjab, Pakistan (2014).

Sunflower is adapted to a wide range of soil conditions, but grows best on well

drained, high water-holding capacity soils with a near neutral pH (6.5-7.5).

Production in high-stress soils such as those affected by heavy metals, drought and

salinity is not exceptional but compares favorably with other commonly grown

commercial crops (Iqbal, 2004). Seed and oil yield are severely reduced under

4

conditions of stress (Ahmad, 2009). Sunflower (Helianthus annuus L.) is cultivated in

Punjab (Pakistan) on 39375.913 hectares with average yield of 823.2 kg per acre and

average annual production of 71700 tones (GOP, 2014). There is also option of the

use of sunflower oil as biofuel (biodiesel) having similar composition and properties

to safflower and soy oils but it is economically not feasible (Darby and Halteman,

2011).

Arsenic contamination has emerged as a serious public health concern in

Pakistan (Islam-ul-Haq et al, 2007). In view of the increasing arsenic contamination

in agricultural soil and cultivation of sunflower over large area in Pakistan, it would

be of great interest to investigate the effect of arsenic on potential oilseed crop. The

major objectives of the proposed studies were, to evaluate phytoextraction potential of

selected sunflower cultivars/hybrids or extent of arsenic accumulation in various plant

parts such as root, stem, leaves and achenes (seeds) from soil having different level of

inorganic arsenicals (sodium arsenite and sodium arsenate), to determine the arsenic

bioaccumulation coefficient for sunflower crop which denotes the ratio of the

concentration of arsenic in the plant and in the growing medium, to understand that in

which condition arsenic is more phytoextracted by sunflower, either from

contaminated soil or contaminated irrigation water and to find out effect of different

arsenic levels on some morphological, physiochemical and yield parameters of

sunflower cultivars.

5

CHAPTER 2. REVIEW OF LITERATURE

2.1 Heavy metals

The term heavy metal or semimetal refers to a group of elements (metals and

semimetals or metalloids) associated with contamination or eco-toxicity (Banejad,

2011). Some define a heavy metal as an element having atomic mass greater than that

of sodium while others define it as a metal having density above 3.5-6.0 g cm -3,

including Arsenic (As), Copper (Cu), Cadmium (Cd), Chromium (Cr), Lead (Pb),

Mercury (Hg), Nickel (Ni), Silver (Ag) and Tin (Sn) etc. (Fayiga et al., 2007).

2.2 Arsenic (As)

Arsenic is the 33rd element of periodic table, discovered by Albertus Magnus

in 1250 (Rosen, 1999). The Greek word “arsenikon” meaning arsenic was primarily

used to mean male or potent (Rahman, 2006). According to IUPAC

recommendations, Arsenic belongs to group 15 also called VA of the periodic table of

the elements (below Phosphorus and above Antimony) having atomic number 33 and

74.92160 atomic mass. It is a metalloid ranking as the 20 th abundant element in the

earth crust, 14th in sea water and 12th in human body (Mandal and Suzuki, 2002).

2.3 Historical background

Sometimes known as the King of Poisons, arsenic has been known to

humankind for thousands of years, being used to harden bronze in the Middle East

around 3000 BC, and prized as a dye by the Egyptians, Greeks, and Romans. In the

first and second centuries AD, the Roman Emperors Nero and Mithridates and King of

Pontus, used arsenic to murder their enemies. It is super-toxic to biota and arsenical

dyes in wallpaper were responsible for the accidental poisoning of Napoleon

Bonaparte during his imprisonment on St Helena (Meharg, 2005), and known to be

the cause of the death of the American president Zachary Taylor (Feldmann, 2001).

Keeping in view the potency of arsenic, Environmental Protection Agency (US, EPA)

concluded that arsenic is a Group A carcinogen, known to trigger skin, bladder, and

lung cancers and thus has become a metaphor for poison (Bondada and Ma, 2003).

2.4 Characteristics of arsenic

It is colorless, tasteless and odorless. Elemental arsenic is a steel grey metal-

like material (Adriano, 2001). Arsenic has four oxidation states (3-, 0, 3+ and 5+),

6

arsenite (As3+) is dominant form in reducing while arsenate (As5+) is stable form in

oxygenated environments (Mattusch et al., 2000). Elemental arsenic is not soluble in

water while arsenic salts exhibit different solubilities depending upon pH and ionic

environment (WHO, 2001).

Four common oxidation states of arsenic are

As0 — as metalloid arsenic

As3- — as arsine gas

As3+ — as arsenites

As5+ — as arsenates

Mainly arsenic is found in two forms:

● Inorganic: combined with elements like oxygen (O), sulphur (S), and chlorine (Cl).

Arsenic acid (H3AsO4), Disodium arsenate (Na2HAsO4), and Sodium arsenite

(NaAsO2) are examples of inorganic arsenicals.

● Organic: combined with carbon (C) and hydrogen (H).

Dimethylarsinic acid or cacodylic acid (CH3)2AsO(OH), Disodium

methylarsenate (CH3AsO(ONa)2, and Carbasone (C2H9AsN2O4) are some examples of

organic arsenicals.

2.5 Arsenic minerals

Elemental arsenic is usually found in hydrothermal veins, but more commonly

it occurs either in primary arsenic-bearing minerals or adsorbed onto various mineral

phases such as iron and aluminium oxides, clays and iron sulphides, which are most

important stores of arsenic in nature (Lowers et al., 2007). There are more than 245

minerals having arsenic as a major constituent (Hug et al., 2001). The most common

primary and accessory arsenic minerals are Arsenic (As), Arsenolite (As2O3),

Orpiment (As2S3), Realgar (AsS), Arsenopyrite (FeAsS), Scorodite (FeAsO4•2H2O),

Enargite (Cu3AsS4), Cobaltite (CoAsS), Niccolite (NiAs) and Claudetite (As2O3)

(O’Day, 2006; Southam and Saunders, 2005). Over 200 different naturally occurring

arsenic minerals include approximately 60% arsenates, 20% sulfides and other

sulfosalts whereas remaining 20% are oxides, arsenides, arsenites, silicates and

elemental arsenic (Mandal and Suzuki, 2002). Some common arsenic minerals and

their occurrence are given in the following Table 2.1.

7

Table 2.1: Common arsenic minerals, their composition and occurrence.Arsenic Minerals Composition OccurrenceArsenic (Native)Orpiment

Realgar

NiccoliteRammelsbergiteSmaltiteProustiteSaffloriteSaligmanniteCobaltiteArsenopyrite

EnargiteTennantiteArsenolite and ClaudetiteScoroditeAnnabergiteHoernesiteHaematoliteConichalciteAdamiteDomeykiteLoellingitePharmacosiderite

AsAs2S3

AsS

NiAsNiAs2

CoAs2

Ag3AsS3

(Co, Fe)As2

PbCuAsS3

CoAsSFeAsS

Cu3AsS4

(Cu,Fe)12As4S13

As2O3

FeAsO4•2H2O(Ni,Co)3(AsO4)2•8H2O

Mg3(AsO4)2•8H2O(Mn,Mg)4Al(AsO4)(OH)8

CaCu(AsO4)(OH)Zn2(OH)(AsO4)

Cu3AsFeAs2

Fe3(AsO4)2(OH)3•5H2O

Hydrothermal veinsHydrothermal veins, hot springs, volcanic sublimation productVein deposits, associated with orpiment, clays and limestones Vein deposits and noritesMesothermal vein deposits-One of Ag mineralsMesothermal vein depositsHydrothermal veinsHigh temp. deposits, metamorphic rocksMost common arsenic mineral in mineral veinsHydrothermal veinsHydrothermal veinsSecondary mineral formed by oxidation of other arsenic mineralsSecondary mineralSecondary mineralSecondary mineral, smelter wastes-Secondary mineralSecondary mineralVeins and replacement depositsMesothermal vein depositsOxidation product of other As minerals

Source: Modified from Mandal and Suzuki, 2002.

2.6 Arsenic speciation

Arsenic toxicity depends on speciation; the molecular form, in which arsenic

occurs, commonly termed species (Table 2.2), affects both, its toxicity and mobility in

groundwater, as well as its removal during water treatment (Sharma and Sohn, 2009).

Inorganic forms of arsenic are more toxic than organometallic species and inorganic

arsenite (As(III)) is more toxic than arsenate (As(V)) (Matera and Hecho, 2001). In

water, arsenic occurs in one of two main forms, a reduced form, arsenite, with a

8

valence of 3+; and an oxidized form, arsenate, with a valence of 5+. These are often

referred to simply by their oxidation states as As(III) (arsenite) and As(V) (arsenate).

Arsenic also exists in the As(0) and As(3-) states, but these are of little importance in

natural waters (Azizur-Rahman, 2011). Arsenic can also exist in many organic forms,

of which the most common are monomethylated acids (MMA) and dimethylated acids

(DMA), both of which exist as As(III) and As(V) forms. These only ever occur as trace

components in natural waters, but they are important in plant and animal metabolism

(Akter et al., 2005).

Table 2.2: Arsenic species commonly found in environment and living organisms.

Name and abbreviations Chemical formula

Inorganic arsenicals

Arsenite (arsenious acid) AsIII

Arsenate (arsenic acid) AsV

Organic arsenicals

Monomethylarsonic acid MMAV

Monomethylarsonous acid MMAIII

Dimethylarsinic acid DMAV

Dimethylarsinous acid DMAIII

Dimethylarsinoyl ethanol DMAE

Trimethylarsine oxide TMAO

Tetramethylarsonium ion Me4As+

Arsenobetaine AB

Arsenobetaine 2 AB-2

Arsenocholine AC

Trimethylarsine TMAIII

Arsines AsH 3, MeAsH2,

Me2AsH

Ethylmethylarsines EtxAsMe3-x

Phenylarsonic acid PAA

Arylarsenicals used as animal feed additives

p-Arsanilic acid p-ASA

4-Nitrophenylarsonic acid 4-NPAA

4-Hydroxy-3-nitrophenlarsonic acid 3-

As(OH)3

AsO(OH)3

CH3AsO(OH)2

CH3As(OH)2

(CH3)2AsO(OH)

(CH3)2AsOH

(CH3)2AsOCH2CH2OH

(CH3)3AsO

(CH3)4As+

(CH3)3As+CH2COO-

(CH3)3As+CH2CH2COO-

(CH3)3As+CH2CH2OH

(CH3)3As

(CH3)xAsH3-x (x=0-3)

(CH3CH2)xAs(CH3)3-x (x=0-3)

C6H5AsO(OH)2

NH2C6H4AsO(OH)2

NO2C6H4AsO(OH)2

9

HNPAA

p-Ureidophenylarsonic acid p-UPAA

NO2(OH)C6H4AsO(OH)2

NH2CONHC6H4AsO(OH)2

Source: Modified from Cullen and Reimer, 1989.

2.7 Sources

Sources of metals into the ecosystem originate from both natural geological

processes and human activities. Natural sources include excessive weathering of

minerals and metal ions from rocks, displacement of certain contaminants from

ground-waters or subsurface soil, atmospheric deposition from volcanic activities,

transport of continental dusts, and forest fire (Ernst, 1998). In uncontaminated top-

soils, worldwide range for background As concentration is approximately 1-100

mg/kg, but often less than 5 mg/kg (Kabata-Pendias and Pendias, 2001).

2.7.1 Natural sources of Arsenic

The main natural sources of arsenic are weathering of As-containing rocks

which liberate inorganic arsenic compounds such as arsenic trioxide, arsenite and

arsenate, precipitation from the atmosphere, and volcanism (Tamaki and

Frankenberger, 1992).

Arsenic (As) is natural element widely distributed in the environment

originating either from arsenic in the soil parent material or from discharge of arsenic

onto land as a result of human activities. In some parts of the world, natural mineral

deposits and aquifer contain large quantities of arsenic which is the chief cause of

elevated levels of arsenic in soil and water. The main sources of arsenic

contamination are the non-ferric metallurgy and the application of arsenic-containing

herbicides (Stoeva et al., 2003).

Occurrence of arsenic in soil and natural water is dependent on the local

geology, hydrogeology and geochemical characteristics of the aquifer, and climate

changes as well as human activities. Natural sources of arsenic in water have been

attributed to several natural geochemical processes, including oxidation of arsenic-

bearing sulfides, de-sorption of arsenic from (hydro) oxides (e.g., iron, aluminum and

manganese oxides), reductive dissolution of arsenic bearing iron (hydro) oxides,

10

release of arsenic from geothermal water, and evaporative concentrations, as well as

leaching of arsenic from sulfides by carbonates (Kim, et al., 2000; Bennett and

Dudas, 2003). Arsenic pollutes the air through the smelting of sulfide ores. Airborne

arsenic may be inhaled, but also accumulates as fall out on soils, from where it may

be taken up by crops or enter in streams in runoff.

2.7.2 Unnatural or Anthropogenic pollution by arsenic

Since the beginning of the industrial revolution, the most common human

routes leading to the introduction of trace metals into the soil compartment include

● Disposal of industrial effluents

● Application of sewage sludge

● Deposition of airborne industrial particulates

● Military operations

● Mining and landfill operations

● Disposal of industrial solid and liquid wastes

● Use of agricultural chemicals

● Gas exhausts, and

● Production of heat and electricity from fuel combustion (Seward and

Richardson, 1990; Lasat, 2000). All these processes contribute significant amounts of

arsenic and other metals to the atmosphere.

There are a variety of anthropogenic sources responsible for emissions of As

but most dominating are smelting and refining of non-ferrous metals, iron and steel

production and coal burning (Chilvers and Peterson, 1987). In addition, chromated

copper arsenate compounds have been widely used as wood preservatives, although

this practice is now being discouraged (Hingston et al., 2001). Arsenic differs from

many of the common heavy metals in that the majority of the organo-As compounds

are less toxic than its inorganic compounds. Jarup (1992) reported that arsenic is

highly hazardous to human and animal health. A level of 0.1 g of arsenic trioxide

(As2O3) can prove potentially lethal and an ingested dose of 70-80 mg of arsenic

trioxide (As2O3) is deadly fatal to humans (Banejad and Olyaie, 2011).

2.8 Products and uses of arsenic

Humans have often polluted their environment with arsenic, usually in the

processing of geological materials such as coal and metaliferous ores (Han et al.,

11

2003). Other sources include wood burning, agricultural chemicals, glass and cement

manufacturing and waste incineration practices e-g arsenic trioxide is used in the

formation of glass and as a colorant, arsenic trioxide and pentoxide are used

industrially for the treatment of wood against insect attack and fungal decay, similarly

gallium arsenide is used to grow crystals in the semiconductors manufacturing

industry. Arsenic is also present as an impurity in coal and oil-based products such as

fuels like petrol, diesel and motor oil.

Agricultural application of arsenicals has introduced many arsenic compounds

to the soil environment. In the USA, although now largely abandoned, arsenical

pesticides were widely applied to orchards, cotton and rice fields resulting in serious

soil contamination (Peryea, 2002; Renshaw et al., 2006). For example, calcium

arsenate (Ca(AsO4)2 was used as an insecticide from the 1800s through the 1960s.

Currently, arsenic acid (H3AsO4), sodium arsenate (NaH2AsO4), sodium arsenite

(NaAsO2), and dimethyl arsenate or DMA ((CH3)2AsO2H) are being used as

defoliants, while disodium methyl arsenate or DSMA ((CH3AsO(ONa)2), mono

sodium methyl arsenate or MSMA (CH3AsOHNa), and MAA (CH3AsO2H2) are being

used as herbicides (Onken and Hossner, 1996). Arsenic is also mobilized by other

polluting activities such as landfill and oil spills (Burgess and Pinto, 2005).

Since the ancient times, inorganic arsenic compounds have been used in

medicine, Hippocrates (460 BC), the father of modern medicine and Galen (129 AD),

have used a paste for the treatment of ulcers, containing arsenic tetrasulphide (Squibb

and Fowler, 1983). Over the past one and a half century, arsenic is in use for treating

asthma, epilepsy, psoriasis, syphilis, amoebiasis, trypanosomiasis and dermatitis

herpetiformis (IARC, 1980; USRC, 2001 and WHO, 1981). Thomas Fowler in 1786

introduced Liquor Arsenicalis comprising of up-to 60 mg of arsenic in the form of

Potassium arsenite for various treatments (Meharg, 2005). In homeopathic and herbal

medicine arsenic is also in use (Kerr and Saryan, 1986; Dunbabin et al., 1992;

Mitchell-Heggs et al., 1990; Tay and Seach, 1975).

2.9 Arsenic pollution in Air, Water and Soil

Arsenic is ubiquitous in air, water, fuels as well as marine life. Arsenic

contamination may be greater in soils, by human activities through the use of various

arsenical pesticides, wood preservatives, industrial wastes and growth promoters for

plants and animals. In mining, pollution may occur as a result of improper disposal of

12

wastes from sulphide-rich ores (Abrahams and Thornton, 1987; Williams, 2001).

Arsenic concentrations vary in the environment as up-to 0.63 mg/m3 in urban air, up-

to 55 ppm in ground water, 0.03 to 0.25 ppm in soil and 0.023 to 0.25 ppm in plants

and one in every 60 people on the planet is living in an area where 50 ug/l or above of

arsenic is in ground water (T.J.W, 2000). It is concluded that one of the major sources

of arsenic in drinking water and high concentration in cereals, fruits and vegetables is

level of arsenic in contaminated soils (Welch et al., 2000). Arsenic pollutes the air

through the smelting of sulphide ores. Airborne arsenic may be inhaled but also

accumulates as fallout on soils from where it may be taken up by crops or enter

streams in runoff.

2.10 Arsenic and coal burning

Globally, the burning of coal has been the major anthropogenic input of

arsenic to the surface environment (Han et al., 2003). In coal arsenic exists largely as

arsenopyrite, it is emitted as arsenic trioxide from power plants. Some coals contain

high concentrations of arsenic, the worst case being in Guizhou province of China,

where power stations cause extensive air pollution, and even worse health effects

result from burning coal inside households (Ding et al., 2001). Another important

source of As emission into the atmosphere is coal-burning during electrical power

production and heating. Arsenic concentrations in coal from the USA, Australia and

the UK range from around 0.5 to 93 mg As kg-1 whereas fly ash particles can contain

up to 1700 mg As kg-1 (Peterson et al., 1981). Brown coal (from the Czechoslovakia)

was found to contain up to 1500 mg As kg-1 (Piver, 1983). Consequently, soil

contaminations in the surrounding of coal-fired power plants remains significant.

The earliest measurement of arsenic in natural water was done by the famous

German chemist Fresenius at Wiesbaden Spa in 1885 (Schwenzer et al., 2001). The

soil environment is an important sink for As compounds. Once As is deposited in soil,

it may build up very rapidly as it is slowly absorbed by plants, leaching loss,

methylation, etc. (Smith et al., 1998). Mukhopadhyay et al., (2000) reported that soils

subjected to irrigation water loaded with As are expected to elucidate better

deposition and concentration of As across and down a soil profile as having

concentrations ranging from 0.1 to 100 ppm or more depending on the types of rocks

under consideration (Cullen and Reimer, 1989). The worldwide median soil

concentration is 6.0 ppm, the concentration in uncontaminated soil rarely exceeds 40

13

ppm (Tamaki and Frankenberger, 1992). During the last few years, concern is

growing about the possible risks of irrigating with arsenic contaminated groundwater

resources (Heikens, 2006).

Recently, the introduction of arsenic into the food chain is a cause for great

concern. It is becoming apparent that ingestion of drinking water is not the only

elevated source of As to the diet. Long-term use of As contaminated water for

irrigation has resulted in elevated As levels in agricultural soils (Meharg and Rahman

2003; Roychowdhury et al., 2005; William et al., 2005; Dahal et al., 2008). Arsenic

very frequently can enter into human body through plants growing on As-

contaminated soils or contaminated groundwater used for irrigation purpose. Severe

As contamination in soils may cause a variety of problems such as loss of vegetation,

groundwater contamination, and arsenic toxicity in plants, animals and humans

(Fowler, 1983; Mukherjee and Bhattacharya et al., 2001).

2.11 Compatibility of arsenic with other elements

2.11.1 Arsenic (As) and iron (Fe):

In sedimentary rocks, arsenic might be precipitated with iron hydroxides and

sulfides as iron deposits, sedimentary iron ores and manganese nodules were also

found rich in arsenic (Mandal and Suzuki, 2002).

2.11.2 Arsenic and Phosphorus:

Arsenic and phosphorous (P) belong to group 15 (previously VA) of the

periodic table and they have similar electron configuration and chemical properties.

The presence of phosphates in the soil was reported to affect the mobility of arsenic,

with a high phosphate-containing soil possessing low arsenic retention (Sadiq, 1997).

Hence, the uptake of arsenic (as arsenate) and phosphate by plants is competitive. It

has been reported that phosphate suppresses plant uptake of arsenate (Woolson et al.,

1973; Khattak et al., 1991; Meharg and Macnair, 1991). On the other hand, enhanced

As uptake in the presence of phosphate has been reported by several authors. This

may be due to (a) solubility of phosphate and arsenate; (b) plant biomass; and (c) high

arsenic concentration in soils (Creger and Peryea, 1994; Jiang and Singh, 1994; Caille

et al., 2004). The study conducted by Tu and Ma (2003) indicated that interaction

between arsenate and phosphate influenced their availability in soils and their uptake

14

by plants and its growth. Quaghebeur and Rengel (2001) studied As-P interactions in

the rhizosphere and found that the presence of phosphate significantly increased

arsenic concentrations in shoots and roots of Holcus lanatus. As arsenic and

phosphate belong to the same chemical group, they have comparable dissociation

constants for their acids and solubility products for their salts, resulting in similar

geochemical behavior of arsenic and phosphate in soil (Adriano, 2001).

2.12 Arsenic pollution in Asia

In Asian countries before 2000, there were only five reported major incidents

of arsenic contamination in ground water, including Bangladesh, West Bengal, India

and China. But between 2000 and 2005 this problem has recognized in Nepal,

Afghanistan and Pakistan (Mukherjee et al., 2006). The environmental impacts of

arsenic are of great importance because of a crisis in Southeast Asia (Christen, 2001).

Millions of people have been exposed to elevated levels of arsenic from groundwater

which is the primary source of drinking water in the region. Berg et al., (2001)

reported that up to one million tube wells drilled into Ganges alluvial deposits may

have arsenic contamination up to 1000 μg/l in Bangladesh and West Bengal. While in

Vietnam, ground water contamination up to 3000 μg/l arsenic has been recorded. Vast

areas of Southeast Asian countries have to rely on arsenic contaminated ground water

for irrigation of staple crops (Abedin, et al., 2002). Consequently the problem is

magnified as along with contaminated drinking water people are also being exposed

to arsenic through ingestion of contaminated crops (because the crops are irrigated

with arsenic contaminated water), from livestock and their products because livestock

have been fed on arsenic contaminated vegetation (Berg et al., 2001).

Natural arsenic enrichment of groundwater around the world takes place by a

variety of geochemical processes (Smedley and Kinniburgh, 2002; Welch, et al.,

2000). In the last few decades, the demand and usage of groundwater has increased

due to pollution of major rivers throughout the world mainly by human, livestock and

industrial wastes especially in adjacent river floodplain deposits. This shift in water

usage has taken place particularly in developing countries (Nickson et al., 2000).

Saunders et al., (2008) reported that in early 1990’s it was inferred that higher arsenic

levels of ground water in young (Holocene) alluvial floodplain aquifers was an

important health problem especially in Southeast Asian countries. This higher arsenic

level of alluvial aquifer ground water was correlated with deposition of hydrous ferric

15

oxides (HFO) containing sorbed arsenic and organic matter naturally present in river

floodplain alluvium (Acharyya et al., 2000). The organic matter caused reductive

dissolution of hydrous ferric oxides releasing both the Fe(II) and arsenic in ground

water. Penny et al., (2003) included the metabolic effects of Fe-reducing bacteria and

Mn-reducing bacteria for releasing Fe, As and other trace elements like Co, Ni, Mn,

Ba and V etc in alluvial aquifer of the USA. Role of SO4-reducing bacteria in removal

of As, Fe, Co and Ni by co-precipitating them in biogenic pyrite was discussed by Lee

et al., (2007). Dowling et al., (2002) reported that Fe-reducing bacteria were

responsible for higher arsenic levels of Holocene alluvial aquifers in Bangladesh and

India. Islam et al., (2004) reported that his laboratory investigation on sediment cores

from Southeast Asia showed that Geobacter like Fe-reducing bacteria cause liberation

of arsenic from minerals. Saunders et al., (2005) confirmed the findings of Islam et

al., (2004) in his field investigation of Kansas city, MO, USA, by observing

abundance of Fe-reducing bacteria belonging to genus Geobacter in ground waters

with elevated arsenic levels whereas absence of these bacteria in arsenic free ground

waters. Sulfate-reducing bacteria especially Desolfovibrio desulfuricans were also

found in arsenic rich ground water. Keeping these findings in view, Saunders et al.,

(2005) summarized that such Fe and SO4-reducing bacteria were important in

geochemical cycling of arsenic in Southeast Asia. Lee et al., (2005) supported the

views of Saunders et al., (2005) about mobility of arsenic and stated that arsenic was

mobile under Fe-reducing conditions whereas immobile under sulfate-reducing

conditions, provided abundance of required electron donors and acceptors. Kirk et al.,

(2004) discussed arsenic bearing pyrite as the most important solid arsenic phase in

natural systems formed under sulfate reducing conditions. While O’Day et al., (2004)

reported pure As-S phase including realgar (AsS) and orpiment (As2S3) as solid

phases of arsenic formed under reducing conditions. Arsenic-bearing pyrite has also

been found in alluvial sediments in Bangladesh (Lowers, et al., 2007) and West

Bengal, India (Acharyya and Shah, 2007). Keimowitz et al., (2007) in his laboratory

experiment demonstrated that arsenic is released under iron-reducing conditions

whereas removed during biogenic sulfat-reduction. This is very useful finding for

bioremediation of arsenic, that SO4-reducing bacteria can remove arsenic under field

conditions. Here it is also proposed that arsenic adsorption on surfaces of sulfide

minerals (Fe-sulfides) is of chief importance for removal of arsenic by biogenic SO4-

reduction (Lee et al., 2008).

16

2.13 Arsenic contamination in Pakistan

In the big cities of Pakistan, untreated sewage water and industrial effluents

are directly discharged into canals and this polluted water is used for growing crops

especially vegetables and fodder crops in the hinterland of big cities (Khan et al.,

2003). It is too very perceptive that majority of the cities and industries in Pakistan

are lacking proper waste water treatment facilities and consequently large quantities

of such untreated municipal sewage and industrial effluents enter directly into surface

water causing rigorous pollution mainly due to heavy metals (Mukhtar et al., 2010).

City effluents, which are routinely used for growing crops in the pre-urban areas of

Pakistan, are one of the potential sources of metal pollution (Mussarat et al., 2007).

In Pakistan, Southern Punjab is facing a serious threat of water pollution. It

has been learnt through the findings of a field verification research, jointly conducted

by the team of Japanese researchers from Tokyo Institute of Technology and National

Institute of Advanced Industrial Science and Technology (AIST) that the water

arsenic level is >10 ppb, above WHO Guidelines. LDA, WASA has admitted before

the Lahore High Court that some of its tube-wells in the city are pumping out arsenic

contaminated ground water which is a serious health hazard. In view of arsenic poison

in water, Punjab and local government has been advised to modify its project of

water-Filtration plants installed in 150 union councils of Lahore into Arsenic

removing type plants to supply clean water to the citizens (Ahmad, 2010). Due to high

arsenic level found, there is an increase in cancer, still births, post-neonatal mortality

and other diseases. Increasing number of patients in Shaukat Khanum Memorial

Hospital and research center shows an alarming situation. The issue needs the urgent

attention of the government agencies responsible for planning and provision of basic

infrastructure and services including water and sanitation. According to the official

statistics of SKMH, by the year 2008 a total of 76,250 patients were registered,

197,730 patients were given chemotherapy treatment, 33,789 patients were surgically

operated, 717,562 got imaging studies and 12,777,05 pathology tests were conducted.

(Ahmad, 2010).

During a study on ground water samples obtained from Muzaffargarh district

of South Western Punjab, Central Pakistan, arsenic concentration up to 906 μg/l has

been recorded in shallow ground water of urban areas as well as >30 ppb in mango

17

fruit pulp collected from area of Rahim Yar Khan and similar high concentrations of

arsenic were also found in Pattoki area of District Kasur (Haque et al., 2007). These

elevated concentrations of As are either due to direct contamination with industrial or

agricultural chemicals or other anthropogenic factors (Nickson et al., 2005). Because

pesticides and phosphatic fertilizers are extensively used on cotton and sugarcane

crops, elevated As concentrations in ground water have been found due to application

of phosphatic fertilizers (Campos, 2002). In another study of ground water samples

from Quetta valley, Pakistan, arsenic concentrations up to 64.5μg/l has been recorded

by Shahab et al., 2009.

2.14 Toxicity of arsenicals

2.14.1 In Bacteria and Fungi:

Bacteria, fungi and yeast are able to form volatile methylated arsenic

compounds under aerobic and anaerobic conditions. Bacteria produce dimethylarsine

while fungi synthesize trimethylarsine. Soil bacteria are responsible for the

demethylation of both compounds (Tamaki and Frankenberger, 1992). In contrast to

heavy metals arsenic accumulation is observed in very few mushrooms such as wild

Agaricus spp., where concentrations up to 14 ppm have been described (Vetter, 1994).

The average arsenic concentration of edible mushroom does not exceed 0.5 ppm

usually (Ghosh, et al., 1999).

The reduced species (arsenite) is more toxic than the oxidized species

(arsenate) since it may react with sulfhydryl group of cysteine in structure and

enzyme proteins. Therefore oxidation of arsenite in micro(organisms) is an important

protective mechanism which is described for various species of genus Pseudomonas,

Xanthomonas and Achromobacter. However some bacteria are able to reduce arsenate

to the more toxic form arsenite. This process is described for some strains of

Pseudomonas fluorescens and Anabaena oscillaroides (Cullen and Reimer, 1989).

2.14.2 Toxicity of arsenic to plants:

Plants vary in their resistance or sensitivity to arsenicals. Arsenic uptake and

accumulation from soil by plants is influenced by certain factors as plant species, soil

arsenic concentration, soil properties, and pH (Matschullat, 2000; Adriano, 2001), the

presence of other ions such as acetate, carbonate, phosphate and iron (Fe) etc.

18

(Kabata-Pendias and Pendias, 2001), exposure time, the age of the plants and metal-

rhizosphere interactions (Fitz and Wenzel, 2002; Fitz et al., 2003). Koch, et al.,

(2000) reported that adverse effects or toxicity of arsenic depends upon the chemical

form or species it takes, for example arsenobetaine [(CH3)3As+CH2COO–] found in

mushrooms and marine animals is much less toxic than arsenous acid [As(OH)3].

Available information about the toxicity of arsenic to plants indicated that arsenic

is potentially toxic to some plant species. Fargasova, (1994), studied the toxic effects

of five metals including Cd2+, Cr6+, Pb2+, Hg2+ and As5+ on germination of mustard

seeds (Sinapis alba) and reported that after 72 hours the most toxic metal for seed

germination was As5+. Similarly Yu, (1999), performed a laboratory experiment in

which mung bean (Vigna radiate) seedlings were exposed to Cd2+, Cu2+, Hg2+, Zn2+

and As3+ and As5+ for 72 hours, and observed that arsenic proved most detrimental to

germination and seedling growth of mung bean than all other metals applied.

Reduction in biomass production and yields significantly at elevated arsenic

concentrations is reported in a variety of crops by Carbonell-Barrachina et al., 1998.

With soil application of only 50 mg As kg-1 reduction in yield of barley (Hordeum

vulgare. L), and rye grass (Lolium perenne. L) (Jiang and Singh, 1994), in wheat

(Triticum aestivum L.) (Zhang et al., 2009), rice (Oryza sativa L.) (Chaturvedi, 2006)

and maize (Zea mays L.) (Stoeva et al., 2003) was recorded.

Miteva (2002) in a laboratory experiment performed on tomatoes (Lycopersicon

esculentum), cultivated on soils (polluted with sublethal doses of arsenic including 15,

25, 50 and 100 mg/kg, in the form of As2O3) found an increase in root and stem

lengths and weights at lower concentrations (15, 25 mg/kg) of As. Whereas higher

concentrations of arsenic (50 and 100 mg/kg) caused reduction in growth of

vegetative and root system. An index of depression (ID), was also calculated on the

basis of morphological parameters and concluded that low concentration (15 mg/kg)

of arsenic in soil stimulated plant growth while higher levels of arsenic (50 & 100

mg/kg) caused depression of plant growth. No significant change of index of

depression was found in case of 25 mg/kg arsenic in soil. It was also concluded that

lower arsenic doses (15, 25 mg/kg) also stimulated the synthesis of photosynthetic

pigments which may be result of defense mechanism of plants to arsenic stress, while

higher doses (50 & 100 mg/kg) caused reduction in pigments representing poor

condition of those plants and lack of adaptive adjustment to higher arsenic levels in

soil. Miteva (2002) reported that 25 mg/kg arsenic in soil appeared to be threshold

19

value over which arsenic caused toxic changes in tomato plants and found the

concentration of arsenic higher in roots than leaves. With higher arsenic

concentrations in soil, the level of arsenic in root and leaves was also increased. First

leaf accumulated much more arsenic than the second leaf in tomato plants. It was

observed that primary leaves accumulate arsenic more intensively and tissue

concentrations of arsenic correlate with applied doses of element in soil. The young

plant tissues proved more sensitive and accumulated more arsenic than older ones,

and small quantities of arsenic seemed a bit beneficial for plant growth.

It is evident that different types of plants, including both weeds and cultivated

crops, can accumulate substantial quantities of arsenic from the soil environment

(Chakraborty and Das, 1997). Arsenic may be taken up by estuarine as well as

terrestrial plants. Usually arsenate is preferred compared to arsenite in view to plant

uptake (Aller et al., 1990).

To evaluate the possible health risk to humans consuming crops irrigated with

As contaminated water, information is needed regarding the soil-to-plant

transportation of As and to minimize the accumulation of As in plants consumed

directly by humans, farm animals or wildlife (Meharg and Hartley-Whitaker, 2002).

2.15 Adaptive mechanism in plants

Plants are not unique in having to protect themselves against the toxic effects

of metals. Thus, a variety of tolerance and resistance mechanisms have evolved,

including avoidance or exclusion, which minimizes the cellular accumulation of

metals, and tolerance, which allows plants to survive while accumulating high

concentrations of metals (Goldsbrough, 2000). The ability of plants accumulating

extraordinarily high concentrations of heavy metals was termed as hyperaccumulation

by Brooks et al., 1977, and in following Table 2.3 is given lower limits for

hyperaccumulation and normal range of elemental concentrations of some heavy

metals in plants.

Table 2.3: Normal ranges in plants and lower limits for hyperaccumulation of some heavy metals

Element Normal range

(μg/g)

Lower limit for hyperaccumulation

(μg/g)

Arsenic (As) 0.01 – 5 1000

Cadmium (Cd) 0.03 – 20 100

20

Cobalt (Co) 0.05 – 50 1000

Copper (Cu) 1 – 100 1000

Manganese (Mn) 5 – 2000 10,000

Nickle (Ni) 0.2 – 100 1000

Selenium (Se) 0.01 – 10 100

Thallium (Tl) 0 – 0.1 1000

Zinc (Zn) 5 – 2000 10,000

Modified from Reeves, et al., 1995; Leblanc, et al., 1997 and Ma et al., 2001.

Plants have developed three basic strategies for growing on contaminated and

metalliferous soils.

2.15.1 Metal excluders:

These plants effectively prevent metal from entering their aerial parts over a

broad range of metal concentrations in the soil; however, they can still contain large

amounts of metals in their roots.

2.15.2 Metal indicators:

These plants accumulate metals in their above-ground tissues and the metal

levels in the tissues of these plants generally reflect metal levels in the environment.

2.15.3 Accumulators:

These plant species (hyperaccumulators) can concentrate metals in their

above-ground tissues to levels far exceeding those present in the soil or in the non-

accumulating species growing nearby (Rahman, 2006 ; Mahmood, 2010).

Plants have internally developed certain mechanisms proposed for

hyperaccumulation and metal detoxification which involve sequestration of metal

away from sites of metabolism in cytoplasm either to cell wall or vacuole (also called

intracellular compartmentalization) (Baker et al., 2000 and US, EPA, 2000). The

definition of hyperaccumulation is based on various comparative surveys, indicating

that on metalliferous soils, most plants accumulate low concentrations of metals in

their shoots, while a few species, usually referred as metallophytes (plant species

endemic to metalliferous sites) accumulate distinctly high amounts of metal (Baker

and Brooks, 1989). Once the metals are taken up, they are concentrated in less

sensitive locations such as vacuoles, cell walls, epidermal cells and trichomes (Boyd

21

et al., 2000). For most plants, arsenic is predominantly concentrated in the roots, with

less accumulated in the shoots (Marin et al., 1993). Bhumbla and Keefer (1994) also

reported that As is not generally translocated readily to shoots and most of the As

taken up by crops tend to remain in the roots. Arsenic enters the leaf via the

transpiration stream (Gan et al., 2002).

Other adaptive characters include

i): Activation of alternative metabolic pathway which is less sensitive to metal ion

ii): Modification in enzyme structure

iii): Change in structure and composition of membranes altering permeability

and one of the major phenomenon is “Chelation” of metal ions with ligands (Salt et

al., 1998). Chelation reduces phytotoxicity by establishment of bonds between metal

ions and specific high affinity molecule called “ligands” thus concentration of free

metal ions in cell sap or cytoplasm is decreased (Dhankher, 2005). These intracellular

ligands have been categorized on the basis of specific electron donor centers (Baker et

al., 2000) such as

2.15.4 Oxygen donor ligands

There are numerous organic molecules having low molecular weight and are

involved in metal detoxification within plants (Brooks, 1998). There are plenty of

carboxylic acid anions in the cell of terrestrial plants which form complexes with

metal ions. Various concentrations of organic acids like oxalate, citrate, malate,

tartrate etc. found in leaves of metal tolerant plants strengthens the idea of these

molecules as chelators (Rauser, 1999). Carboxylates are considered to behave as

ligands for metal chelation in vacuole (Baker et al., 2000).

2.15.5 Sulfur donor ligands

There are certain molecules acting as organic ligands with sulfur donor centers

and form stable complexes with many metals. In plants two major classes of sulfur

containing metal chelating ligands have been identified, which play an important role

in metal tolerance, these are phytochelatins and metallothioneins.

Phytochelatins (PC’s) are composed of a family of peptides having general

structure (γ-GluCys)n-Gly, where, n = 2 to 11 (Goldsbrough, 2000 and Grill, 1987).

Studies indicate that phytochelatins are not synthesized by translation of mRNA

instead are product of an enzymatic reaction (Robinson, 1993 and Goldsbrough,

2000). The responsible enzyme was called phytochelatin synthase (Klapheck et al.,

22

1995 and Cobbett, 2000). Both metal sensitive and metal resistant plants produce

phytochelatins (Kramer et al., 1996) which have very important role in metal

detoxification and accumulation in higher plants (Maitani et al., 1996).

Metallothioneins are metal binding proteins which are cysteine rich and have

low molecular weight but are encoded by structural genes (Stillman et al., 1992 and

Stillman, 1995). Metallothioneins are important intracellular metal chelators of fungi

and mammals but in plants this task is best performed by phytochelatins (Robinson,

1990 and Van Hoof et al., 2001).

2.15.6 Nitrogen donor ligands

In plants nitrogen donor ligands generally comprise of amino acids and their

derivatives in which nitrogen donor centers become combined due to relatively high

affinity to some metals (Homer, et al., 1997). As reported by Baker et al., (2000) that

histidine is involved in Ni transport and translocation in hyperaccumulator plants.

2.16 Intracellular compartmentalization

Cell wall, cytosol and vacuoles are three main compartments in plants at

cellular level where excessive amounts of metals can be stored without any severe

harmful effects to plant (Verklaij and Schat, 1990). The central vacuole being suitable

as storage reservoir for excessively accumulated metals and similarly plant cell walls

are also a continuous matrix which provides a medium for exchange of metallic

cations by holding different concentrations of metals. Heavy metals are transported

acropetally via the xylem fluid in a complexed form, and the existence of free metal

ions in xylem sap appears unlikely, As(III) is normally complexed with thiols,

particularly phytochelatins, and the complex is stored within vacuoles (Meharg and

Hartley-Whitaker, 2002).

2.17 As contents of some edible crops and vegetables

The study or determination of arsenic in plants is of great importance in two

ways, firstly the arsenic uptake or accumulation by plant indicates that fraction of

metal in the soil which is bioavailable and secondly the accumulated or residual

arsenic in the plant is that amount which is then available to the next trophic level in

the food chain (Koch, et al., 2000). Das (2004) observed As-uptake and accumulation

in different crops including rice, wheat, maize, mustard, groundnut and potato etc. by

23

growing these crops in As-contaminated soils using irrigation water loaded with As.

Most of the crops grown in As-contaminated soils, which are subjected to irrigation

water loaded with As, exhibited an accumulation of As in crops, being varied with

different magnitude depending on the types of crops. However, the As accumulation

showed in the order of root > stem > leaf > grains > tubers. Similar observations were

made by Ghosh et al., (2006) who reported that As uptake by crops increased with the

degree of crop maturity. Marcus-Wayner and Rains (1982) also reported that As was

readily taken up by the roots of cotton plants but was not translocated to the shoots.

The brake fern (Pteris vittata L.) can accumulate 22630 mg As/kg (Wang et al.,

2002).

Table 2.4: Arsenic concentrations in some common edible crops

As conc. (mg/kg) in

Crops Root Shoot Leaves

Rice (Oryza sativa L.) 8.32 7.10 8.10

Wheat (Triticum aestivum L.) 13.12 6.90 5.69

Maize (Zea mays L.) 5.66 7.20 3.15

Mustard (Brassica juncea) 7.10 6.31 6.92

Groundnut (Arachis hypogaea L.) 3.0 2.23 2.10

Potato (Solanum tuberosum) 11.10 7.89 5.16

Plant Mean value of As in ug/kg

Spinach leaves (Spinacia oleraceae) 200-1500

Lettuce leaves (Lactuca sativa) 20-250

Onion bulbs (Allium cepa) 50-200

Potato tubers (Solanum tuberosum) 30-200

Reprinted from Aller et al. (1990).

2.18 Sunflower

Sunflower (Helianthus annuus L.), belongs to genus Helianthus, tribe

Heliantheae and family Asteraceae (Compositae). Genus name Helianthus is derived

24

from Greek word “helios” meaning sun and “anthos” meaning flower and the genus

has about 67 species (Ruffo et al., 2003).

It is an annual, broadleaf plant having an erect stem, strong taproot and a well-

developed spread of lateral surface roots. Usually stems are round early in the season

but later become angular and woody and oftenly remain unbranched. The leaves are

usually petiolate and three nerved, vary in shape from linear to ovate and are usually

entire or serrated. The color intensity of leaves also varies from light to dark green. It

is tolerant of both low and high temperatures, however, more tolerant to low

temperatures. The crop is particularly sensitive to high soil temperature during

emergence. Seeds are not affected by vernalisation (cold) in the early germination

stages. Sunflower seeds germinate ideally at 5°C, however, at least 14 to 21°C

temperatures are required for satisfactory germination. The optimum temperature for

growth is 23 to 28 °C, however, high temperatures up to 34 °C show little effect on

productivity. Extremely high temperatures have been shown to lower oil percentage

and also reduce seed fill and germination. In temperate regions, sunflower takes

approximately 11 days from planting to emergence, 33 days from emergence to head

visibility, 27 days from head visibility to first anther, 8 days from first to last anther,

and 30 days from last anther to maturity. Cultivar differences in maturity are usually

associated with changes in vegetative period before the head visibility, therefore, its

total growing period ranges from 125 to 130 days. Many hybrids of sunflower have

been developed to shorten the maturity period to less than 90 days (Berglund, 2007).

The inflorescence of sunflower (capitulum) or sunflower head is not a single

flower (as the name implies) but is made up of 1,000 to 2,000 individual flowers

joined at a mutual receptacle. The flowers around the circumference are ligulate ray

flowers without stamens or pistils; the remaining flowers are perfect flowers (with

stamens and pistils). Anthesis (pollen shedding) begins at the periphery and proceeds

to the center of the head. As many sunflower varieties have a degree of self-

incompatibility, pollen transfer among plants by means of insects is important,

therefore, bee colonies are helpful in causing an increased yields (Knodel, et al.,

2010). The achene or fruit of the sunflower comprises of a seed, oftenly called as

kernel, and an adhering pericarp, usually called the hull. In the absence of

fertilization, the achenes remain empty without any kernel inside. Size of achenes

may vary from 7 to 25 mm in length and 4 to 13 mm in width. They may be linear,

oval or almost round in structure. Average yield in dry land sunflower ranges

25

generally 1300 to 1500 pounds/acre, but in irrigated or highly rainfall conditions

yields over 2000 pounds/acre are not uncommon. Though oil content of the seed can

range from 35 to 50 percent, 40 to 42 percent is average. Oil yield extracted from the

sunflower seed can range from 35 up to 80 gallons per acre (Darby and Halteman,

2011).

2.19 Uses of sunflower

Two types of sunflower exist, oilseed and confectionary. The sunflower is

primarily cultivated for its seeds, which yield one of the world’s most important

sources of edible oil. Commercially available oilseed varieties of sunflower contain

from 39 to 49% oil in the seed (Putnam et al., 1990). The development of high quality

oilseed varieties is mainly based on high and better oil contents therefore seeds are

crushed to produce edible oil, leaving behind the sunflower meal as by-product. In

most of the developing world, fats through the edible oils are an important source of

calories for people at risk for malnutrition. Sunflower oil is an important natural

source of unsaturated fat (monounsaturated and polyunsaturated fatty acids) and could

be a better substitute of food sources containing relatively higher levels of potentially

harmful saturated fatty acids. Unsaturated fats have their role in moderating blood

cholesterol levels, reducing the risk of cardiovascular disease and may also be

beneficial for patients suffering from diabetes Type-2, and also a source of Vitamin E

more than any other vegetable oil (Dillivan, 2011). It is oftenly used in the form of

cooking oil, margarine, salad dressing oil and as snacks also. Industrially, it is also

used in certain paints, varnishes and plastics because of good semi-drying properties

without colour modification associated with oils high in linolenic acid. It can also be

used in manufacturing soaps and detergents. Sunflower oil is considered as a premium

oil because of its light colour, high level of unsaturated fatty acids, lack of linolenic

acid, lack of trans fat, bland flavour, high oxidative stability and high smoke points

(Burke, 2003). Other industrial uses include production of agrichemicals or pesticides,

surfactants, adhesives, fabric softeners, lubricants and coatings. A future high-

potential use will be as biofuel (on diesel engines) as the world is striving for a non-

polluted eco-friendly environment (Darby et al., 2009). Sunflower is used as silage

for animal feeds. Sunflower silage is richer in nutrients than corn but lower than

alfalfa hay. The non-dehulled or partly dehulled sunflower meal is also used for

26

ruminant animals and poultry feeds because of its high protein percentage (Dillivan,

2011).

27

CHAPTER 3. MATERIALS AND METHODS

Keeping in view the high concentrations of arsenic in soil, water and as a

result in food stuffs grown on such soils, a series of four experiments were conducted

in laboratory and wire house of Department of Botany, University of the Punjab,

Lahore to explore the arsenic uptake or accumulation potential of sunflower

(Helianthus annuus L.) cultivated on arsenic contaminated growth media (soil and

irrigation water). As it is concluded that one of the major sources of arsenic in

drinking water and high concentration in cereals, fruits and vegetables is level of

arsenic in contaminated soils (Welch et al., 2000). Following experimentation was

performed during the course of study.

Experiment 1: Toxicity of various levels of inorganic arsenicals on seed germination of four sunflower (Helianthus annuus L.) cultivars:

Germination experiment was performed in the climatic room of Department of

Botany, University of the Punjab, Lahore to evaluate the effects of different levels of

arsenic on germination of sunflower hybrids.3.1 Seed Material:

Seeds/achenes of four sunflower cultivars/hybrids were used during this lab

experiment, namely

1st cultivar = H1 = Hybrid 1 = FH-331

2nd cultivar = H2 = Hybrid 2 = FH-385

3rd cultivar = H3 = Hybrid 3 = FH-405

and 4th cultivar = H4 = Hybrid 4 = FH-415

Seeds were collected from the Oil Seed Department of Ayub Agriculture

Research Institute, Faisalabad. Achenes of sunflower cultivars were surface sterilized

using hydrogen peroxide (H2O2) solution for five minutes and rinsed thrice with

distilled water to prevent any kind of fungal contamination. Seeds/achenes were sown

in petriplates (9 cm diameter) lined with double filter paper Whattman No. 2.

28

3.2 Metal and Treatment plan:

Sodium salts of arsenic, Sodium arsenate (Na2HAsO4.7H2O) as source of AsV,

and Sodium arsenite (NaAsO2) as source of AsIII (Pigna, et al., 2009) of Sigma

Aldrich, Japan, were used for application of arsenic treatments composed in

Hoagland’s (Hoagland and Arnon, 1950) nutrient solution (Table 3.1). There were ten

treatments comprising of five different concentrations of arsenic (2, 4, 6, 8 and 10

mg/L) for both arsenic compounds, and the eleventh one, without any arsenic

contamination was kept as control.

T0 = Control (Hoagland’s nutrient solution, no arsenic compound)

T1 = 2 mg/L arsenic as Sodium arsenate (Na2HAsO4. 7H2O) source of AsV





T6 = 2 mg/L arsenic as Sodium arsenite (NaAsO2) source of AsIII


T8 = 6 mg/Larsenic as Sodium arsenite (NaAsO2) source of AsIII

T9 = 8 mg/Larsenic as Sodium arsenite (NaAsO2) source of AsIII


Table 3.1: Composition of Hoagland’s nutrient solution

Sr. No. Salts Stock solution (gL-

1)

ml of stock solution for 200 L (½ strength) Hoagland’s

solutionMacronutrients 1 L 250 ml 200 L 50 L 20 L 2 L

1 KH2PO4 136 g 34 g 100 ml 25 ml 10 ml 1 ml2 KNO3 101 g 25.2 g 500 ml 125 ml 50 ml 5 ml3 Ca (NO3)2. 4H2O 236 g 59 g 500 ml 125 ml 50 ml 5 ml4 MgSO4. 7H2O 246 g 61.4 g 200 ml 50 ml 20 ml 2 ml

Micronutrients1 H3BO3 2.86 g 0.72 g 100 ml 25 ml 10 ml 1 ml2 MnCl2. 4H2O 1.81 g 0.46 g 100 ml 25 ml 10 ml 1 ml3 ZnSO4. 7H2O 0.22 g 0.06 g 100 ml 25 ml 10 ml 1 ml4 CuSO4. 5H2O 0.08 g 0.02 g 100 ml 25 ml 10 ml 1 ml5 H2MoO4. H2O 0.02 g 0.006 g 100 ml 25 ml 10 ml 1 ml6 Fe-EDTA 37.33 g 9.34 g 100 ml 25 ml 10 ml 1 ml

(Prepared from Taiz and Zeiger, 2006).

29

20 seeds were placed in each petriplate. 10 ml of freshly prepared treatment

solution was applied daily after washing out the previous solution. The solutions were

changed every-day to ensure constant level of arsenic. The petri-dishes were placed in

climatic room under continuous white fluorescent light (PAR 300 μmol m-2 s-1) at 25

ºC ± 2 ºC. The experimental setup was in a completely randomized fashion with three

replicates. Data were collected daily for fourteen days. A seed was considered as

germinated when 5 mm of radicle had emerged out of seed coat. Arsenic tolerance

was assessed on the basis of germination percentage, time to 50% germination,

seedling vigour index, mean germination time, plumule length, radicle length and

seedling length were also calculated.

3.3 Germination percentage (GP):

Germinated seeds were counted daily according to the seedling evaluation

procedure in the Handbook of Association of Official Seed Analysts. The number of

germinated seeds was recorded every 24 hours (AOSA, 1990). Germination

percentage was calculated according to the formula given by Tanveer, et al., 2010.

Germination Percentage (GP) = Germinated Seeds × 100Total Seed

3.4 Mean Germination Time (MGT):

MGT was calculated in days according to the equation given by Ellis and

Roberts (1981) and Dezfuli, et al., 2008.

MGT= ΣDn / Σn

Where n is the number of seeds germinated on day D, and D is number of days

counted from the beginning of germination.

3.5 Seedling Vigour Index (SVI):

Vigour index for seedling was calculated using the formula given by Cokkizgin, 2010.

Vigour index (SVI) = [MPL + MRL] x GP

30

Where MPL = Mean Plumule Length

MRL = Mean Radicle Length

and GP = Germination Percentage

3.6 Days to 50% germination (T50):

The time to 50% germination (T50) was calculated according to the following

formula given by Coolbear, et al., (1984) modified by Farooq, et al., (2005) and

Farooq, et al., 2006.

T50 = ti + {(N/2) – ni}(tj – ti)

nj - ni

Where N is the final number of germinated seeds and ni, nj are cumulative number of

seeds germinated by adjacent counts at times ti and tj where ni < N/2 < nj.

Germinated seeds were collected after fourteen days and carefully detached

plumule and radicle were used for the estimation of plumule length and radicle length

with the help of a scale. Fresh weights of plumule and radicle were also calculated

using digital scientific balance, whereas dry weights were calculated after keeping

seedlings in electric oven for 72 hours at 600c.

After the completion of germination experiment in climatic room, next

experimentation was performed by planting the sunflower seeds of two selected

varieties or cultivars FH-385 as H1 and FH-415 as H2, in earthen pots having

capacity of 100kg soil and arsenic salts were applied in three different ways as

follows.

Experiment 2: Accumulation of arsenic in sunflower (Helianthus annuus L.)

cultivated on arsenic contaminated soil.

Experiment 3: Responses of sunflower to different levels of inorganic arsenicals

applied through irrigation water.

Experiment 4: Arsenic (As) accumulation in sunflower cultivated on arsenic

contaminated soil and irrigated through contaminated water.

31

All the three pot experiments were performed in the wire house of Botanical

Garden, University of the Punjab, Lahore to assess arsenic bio-accumulative potential

in two sunflower hybrids.

3.7 Pots

Earthen pots lined with polythene bags, having capacity of 100 kg soil were

used for this experiment. The pots were placed at proper distance from each other to

prevent any kind of contamination from neighboring treatment replica.

3.8 Soil

Pre-analyzed (of known qualities) soil (clay loam) locally called “GHESO”

(Generally called Highly Eroded Soil collected from the river Ravi basin) was

selected for the experiment. Soil was air dried ground and passed through 2 mm sieve

and mixed thoroughly. This soil was used for experiment and following parameters

(Table 3.2) were recorded prior to sowing of seeds.

3.9 Physiochemical Characteristics of experimental soil

Soil textural parameters were recorded according to the Bouyoucos

Hydrometer method as described by Bouyoucos, 1962 and Basu, 2011.

3.9.1 Determination of soil texture

Following materials were used for determination of soil texture

Hydrometer

Graduated cylinder (1000 ml)

Thermometer [digital with probe (Model SKU: 5457) from Alla instruments France]

Stop watch

Stirrer with cup

Beaker (varying capacities)

Perforated brass plunger and

Wash bottle

3.9.2 Reagents

Dispersing reagent (4%) was prepared by dissolving 40 g of sodium

hexametaphosphate and 10 g of sodium carbonate (Na2CO3) in distilled water, mixed

well and made the volume one liter

32

and

Amyl Alkohol.

3.9.3 Method

Weighed 40 g of air-dried soil (0.2 mm) into a 600 mL beaker and added 60 mL

dispersing solution. Covered the beaker with a watch-glass and left overnight.

Transferred contents of the beaker to a soil-stirring cup, and fill the cup to about

three-quarters with water. Stirred suspension at high speed for 3 minutes. Rinsed

stirring paddle into a cup, and allowed it to stand for 1 minute. Transferred suspension

into a 1L calibrated cylinder (hydrometer jar), and brought to volume with distilled

water.

3.9.4 Determination of Blank

Diluted 60 mL dispersing solution to 1L hydrometer jar with distilled water

and mixed well then inserted hydrometer, and took hydrometer reading referred as Rb

(reading of blank).

3.9.5 Determination of Silt and Clay

Mixed suspension carefully in the hydrometer jar using a special paddle, after

withdrawing the paddle, immediately inserted the hydrometer, a drop of amyl alcohol

was added to remove any froth and got hydrometer reading as Rsc (reading of silt and

clay).

Calculation of Percentage Silt plus Clay in soil:

% [Silt + Clay] (w/w) = (Rsc - Rb) × 100

Oven – dried soil (g)

Determination of Clay

Mixed the suspension in hydrometer jar with paddle then withdrew the paddle

carefully, leaving suspension undisturbed. After about 4 hours, inserted the

hydrometer, and got hydrometer reading Rc (reading for clay).

33

Percentage Clay in soil

% Clay (w/w) = (Rc - Rb) × 100

Oven – dried soil (g)

Percentage Silt in soil:

% Silt (w/w) = % [Silt + Clay (w/w)] – % [Clay (w/w)]

3.10 Determination of Sand

After taking readings for clay and silt, poured suspension through a 50 um

sieve and washed sieve until water passing the sieve was clear. Transferred the sand

from sieve to a 50 mL beaker of known weight. Allowed the sand to settle in the

beaker, and decanted excess water. Dried beaker with sand overnight at 105oC in an

oven and then cooled in a desiccator, and re-weighed beaker with sand.

Percentage Sand in soil:

% Sand (w/w) = Sand weight × 100

Oven-dried soil (g)

Weight of sand derived by using the formula

Sand weight (g) = [Beaker + Sand (g)] – [Beaker (g)]

After calculating the percentage of sand, silt, and clay, the soil was assigned a textural

class using the USDA textural triangle.

Table 3.2: Chemical composition of soil.Sr. No. Soil Properties Results

1 Soil Texture Clay loam2 Clay (%) 573 Sand (%) 294 Silt (%) 145 Organic matter (%) 0.746 Saturation percentage 527 Moisture percentage 188 Soil pH 7.89 Electrical conductivity (dSm-1) 2.3

10 Nitrogen (%) 4.711 Available Phosphorus (mg/kg dry soil) 13.5

34

12 Potassium (mg/kg dry soil) 78.113 Calcium (mg/kg dry soil) 10514 Arsenic (ug/kg dry soil) 0.3

3.11 Saturation percentage

The saturation percentage is defined as the ratio of the amount of water added

to saturate dry soil samples, to total mass of the fully dried soil. It is regarded as a

quantitative measure of soil texture, water-holding capacity, and cation exchange

capacity of soil and therefore, it is important for estimation of water use of plants

(Aali, et al., 2009). Initially dried soil samples were saturated with deionized water

and then oven dried at 105 C° for a period of 24 hrs.

3.12 Moisture contents (%)

Moisture percentage of soil was estimated by removing soil moisture (by

oven-drying a soil sample until the weight remained constant). The moisture

percentage (%) was calculated from the sample weight before and after drying with

the help of formula given by Standards Association of Australia, (1977) as

Moisture percentage or moisture contents (MC) = W2 – W3 × 100

W3 – W1

Where

W1 = Weight of container (g)

W2 = Weight of moist soil + container (g)

W3 = Weight of dried soil + container (g)

For the precision of results an appropriate Moisture factor was also calculated using

the formula

Moisture factor = 1 + MC 100

3.13 Soil pH

Soil pH is an indicative measurement of the chemical properties of a soil. A

strong relationship exists between soil pH and the solubility of various compounds, the

relative exchange capacity and the activity of micro-organisms (Haluschak, 2006). Soil

35

pH and EC was calculated using the multi-meter (HANNA), Model: HI9811-5, Hanna

Instruments, Woonsocket RI USA, made in Europe (Romania).

3.14 Application of arsenical treatments

Two sodium salts of arsenic, S1: Sodium arsenate (Na2HAsO4.7H2O) as source

of AsV, and S2: Sodium arsenite (NaAsO2) as source of AsIII (Pigna, et al., 2009) of

Sigma Aldrich, Japan, were used for arsenic application during all three experiments.

Both arsenicals were thoroughly mixed in soil (during experiment 2 & 4), at final

concentrations of 20, 40, 60, 80 and 100 mg (As) kg–1 dry soil according to scientific

publication of Liu et al., (2012). In experiment 3, soil was devoid of any arsenic

contamination prior to sowing but arsenic treatments were applied through irrigation

water (0, 2, 4, 6, 8 and 10 mg As/L water), whereas in experiment 4, double dose of

arsenic was used as soil was also pretreated with arsenic levels and crop was irrigated

through contaminated water too (five times during the course of study having same

concentrations used in experiment 3). Plants were watered using half strength

Hoagland’s (Hoagland and Arnon, 1950) nutrient solution throughout the course of

study. Overall there were eleven treatments comprising of ten different concentrations

of arsenic (five for each arsenic salt), and one without any arsenic contamination was

kept control in all three experiments.

Table 3.3: Treatments plan.Experiment. No. 2

(In the Soil)

Experiment No.3

(By irrigation water)

Experiment No.4

(In soil + water)

S1 S2 S1 S2 S1 S2

T0. Control T0. Control T0. Control

T1. 20mg As/kg soil

T2. 40mg As/kg soil

T3. 60mg As/kg soil

T4. 80mg As/kg soil

T5. 100mg As/kg soil

T1. 2mg As/L water

T2. 4mg As/L water

T3. 6mg As/L water

T4. 8mg As/L water

T5. 10mg As/L water

T1. 20mg As/kg soil + 2mg As/L water





S1: Sodium arsenate (Na2HAsO4. 7H2O) source of AsV

S2: Sodium arsenite (NaAsO2) source of AsIII

36

3.15 Meteorological Data

During the course of experimentation, data about maximum and minimum

temperatures, total rainfall and humidity of the surroundings of experimental site were

collected from the Meteorological Research Centre Lahore and were as following

Table 3.4: Meteorological data recorded during the course of experimentation.Year 2010 January February March April May June

Max. Temp. 17.3 22.8 30.9 38 39.8 39.2

Min. Temp. 7.8 11.9 19.2 24.3 26.8 27.9

Humidity % 91 75 69 41 38 46

Total Rainfall 0.3 mm 9.4 5.4 2.6 7.2 4.8

Year 2011 January February March April May June

Max. Temp. 16.6 21.2 28 32.9 39.9 37.2

Min. Temp. 7.4 10.4 15.2 20.6 25.1 27.4

Humidity % 86 75 62 40 32 32

Total Rainfall 0.3 28.5 9.4 16.5 6.4 47.8

3.16 Plant measurements and arsenic determination

The data were collected twice during the course of this study as shoot length

(cm), root length, number of leaves, fresh weight of shoot, dry weight of shoot as well

as fresh and dry weight of root were recorded at vegetative stage (at commencement

of flowering) whereas shoot length, root length, number of leaves and capitulum

diameter, weight of 100 achenes and arsenic contents in root, shoot, leaves and

achenes or seeds were also recorded at the time of final harvest or maturity. Plants

were uprooted carefully and separated into roots, shoots and leaves. Shoot and root

lengths were calculated using a wooden meter rod in centimeters. Fresh weights were

recorded using an electric topload balance and roots were washed completely with the

help of distilled water prior to weighing. Dry weights or biomass of different plant

parts were recorded after drying completely in an oven for 60 hours at 80oC as

described by Zhang et al., 2009).

3.17 Plant morphological observations

Plants were uprooted carefully, washed with tap water. The following

parameters were determined as follows.

37

a. Shoot length (cm):

The shoot length of the plants was measured from the soil surface in pots

to the tip of the plant with the help of meter rod.

b. Root length (cm):

Length of roots per plant was noted in centimeter scale.

c. Shoot Fresh Weight (g):

The shoot of each plant with fraction of petiole was separated from the

roots and their weight was noted in grams with the help of a scientific

topload balance.

d. Root Fresh Weight (g):

The roots were cut and thorough washed in distilled water with soft brush.

Fresh weight of root was noted in grams and the average of fresh weight of

root was determined.

e. Shoot Dry Weight (g):

The samples were kept in an oven at 70 oc for 48 hours. When they were

completely dry, the dry weight was registered in grams.

f. Root Dry Weight (g):

The roots wrapped in blotting paper were kept at a temperature of 70 oc in

an oven for drying. After drying, the dry weight was recorded in grams

and average of dry weights was determined.

g. Number of Leaves:

Number of leaves of each plant was counted.

h. Fresh Weight of Leaves (g):

The leaves were cut at the petiole junction at maturity and separately

wrapped in blotting paper. Their fresh weight was then noted in grams.

38

i. Dry Weight of Leaves (g):

Leaves were cut from petiole wrapped separately in blotting paper and

kept in an oven at 70 oc for 48 hours. After drying, the dry weight of

leaves was recorded separately and then average was also calculated.

3.18 Plant physiological or water relation parameters

Following different attributes were also recorded using top load balance and

computer software.

a. Fresh weight (FW) of leaf

b. Turgid weight (TW) of leaf

Turgid weight was taken by dipping the leaf completely in water for more than two

hours.

c. Dry weight (DW) of leaf

d. Leaf area (LA)

Leaf area was calculated with the help of computer software Image J (Rosband, W. S.

Image J, U. S. National Institute of Health, Bethesda, Maryland, USA,

http//rsb.info.nih.gov/ij/. 1997, 2008) using the pictures of leaves.

e. Succulence of leaf

Leaf succulence was calculated by using the formula given by Mantovani, (1997) as

Leaf succulence = [TW – DW] / LA

f. Relative water content of leaf

Water contents of leaf were calculated with the help of formula given by Pirzad et al.,

(2011) as

Relative water contents of leaf (%) = [(FW – DW) / (TW – DW)] × 100

g. Specific leaf weight

Specific leaf weight was calculated using the formula given by Steinbauer, (2000) as

Specific leaf weight = Leaf dry wt. / Leaf area

3.19 Yield components

Upon maturity of the crop, total seed yield per plant was recorded in the form of

following parameters

I. Hundred achene weight

II. Capitulum diameter

3.20 Preparation of plant samples for analysis:

39

3.20.1 Digestion of plant samples

Wet ashing of plant samples was performed according to (Richards ed. 1954).

3.20.2 Reagents:

Conc. HNO3 (A. R. grade)

Conc. HClO4 (A. R grade, 72%)

HCl (A.R grade)

Glass distilled water

Beaker 300 ml (Pyrex)

Volumetric flask 100 ml

Filter paper (Whatman No. 42)

3.20.3 Procedure

Transferred 1.0 g of oven dried, finely ground plant sample into 300 ml tall

beaker (Pyrex) and added 20 ml of conc. HNO3, wetting the entire sample. Covered

the beaker with watch glass and allowed it to stand for 2 hours for completion of

initial reaction.

Place the beaker on a hot plate inside the fume chamber. Continued heating

gently until solid particles disappeared. Removed the beaker from hot plate and

allowed it to cool.

Added 10 ml of 72% HClO4 and placed the beaker on hot plate again.

Continued heating gently first and then vigorously until white fumes given off during

heating subsided, and the contents became a colorless clear solution. Discontinued

heating when the solution was reduced to a volume about 2-3 ml.

3.20.4 Preparation of sample test solution

Left the contents of beaker to cool completely and then added 3 ml of distilled

(1:1) HCl and heated the contents on a hot plate until the solution was reduced to

about 2 ml. Cooled and added distilled water. Stirred the contents with a glass rod and

transferred the entire digest into a 100 ml volumetric flask. Made volume up to the

mark (100 ml) with glass distilled water. Mixed the contents (after stopper) by turning

the flask upside down 4-5 times and let it stand overnight. This diluted digest was

passed through a Whatman no. 42 filter paper. Discarded the initial 2-3 ml of filtrate

after rinsing the receiving flask with it. Collected the subsequent filtrate and preserved

40

it as sample test solution for the determination of elements. A blank test solution was

prepared in the same way, without taking a plant sample.

3.21 Macro and Micro-nutrient ions

Different macro and micro nutrient ions including total Calcium (Ca),

Potassium (K), Magnesium (Mg), Phosphorus (P), Boron (B), Copper (Cu), Iron (Fe),

Manganese (Mn), Molibdenum (Mo), and Zinc (Zn) in roots, stem and leaves of

harvested sunflower plants were determined for each treatment with the help of

Inductively Coupled Plasma, Optical Emission Spectrometer (ICP-OES).

3.22 Heavy metal and other ions detected in plants

Various heavy metal ions derived by plant from the soil such as Silver (Ag),

Aluminum (Al), Barium (Ba), Beryllium (Be), Bismith (Bi), Cadmium (Cd), Cobalt

(Co), Chromium (Cr), Lithium (Li), Nickel (Ni), Lead (Pb), Antimony (Sb), Selenium

(Se), Strontium (Sr), Titanium (Ti), Thalium (Tl), and Vanadium (V) were also

determined in roots, stem and leaves of sunflower plants treated with various

concentrations of arsenic in soil and irrigation water.

In everyday life, photographical industry, following electrochemistry and

medicine are the main sources of one of the most toxic heavy metal “silver(I)” ions

(Krizkova et al., 2008). Several mechanisms of silver ions have been suggested that

effect on vitally important processes and functions. One of the mostly cited is the

interactions between Ag+ and the vital molecule DNA (Song et al., 2005). It was

observed that silver ions interfere the replication process by binding rapidly on N7

guanine in dsDNA (Hossain and Huq, 2002).

The effect of excess concentrations of lead (Pb), cadmium (Cd), copper (Cu),

and zinc (Zn) on water relations in young sunflower (Helianthus annuus L.) plants

was studied by Kastori et al., (2008) in water culture under greenhouse conditions.

The accumulation of the heavy metals was found more intensive in the roots than in

the shoot. The rates of heavy metal accumulation in root were found in the following

decreasing order: Cu, Cd, Zn, and Pb. Their transport into the above‐ground parts

followed the order Zn, Cu, Pb, and Cd. Transpiration and relative water content were

significantly decreased by excess concentrations of the heavy metals. It was

concluded that excess concentrations of the heavy metals significantly affected plant

water status, causing water deficit and subsequent changes in the plants. The most

41

intensive effect on the plants was exerted by Cd, less intensive by Cu and Zn and the

least intensive by Pb. (Kastori et al., 2008).

Exposure of cabbage plants to excess (500 μM) of Co2+, Ni2+ and Cd2+ in sand

culture led to increased accumulation of the metals alongwith inhibition of growth and

induction of visible symptoms of metal toxicity. In addition to chlorosis, Co2+ treated

plants exhibited reddish purple coloration along leaf margins, Ni2+ treated plants

exhibited black spots near leaf margins, and Cd2+ treated plants developed purple

coloration along the leaf margins. At equimolar concentration, inhibition of growth

was most severe with excess Cd2+ and induction of visible symptoms was most severe

with excess Ni2+. Exposure to excess concentration of the heavy metals decreased the

uptake of Fe and its translocation to leaves. Each Co2+, Ni2+ and Cd2+ decreased water

potential and transpiration rate, associated with increase in diffusive resistance

showing development of water stress (Pandey and Sharma, 2002).

3.23 Arsenic accumulation in root, stem and leaves

The accumulation of arsenic was also determined in roots, stem and leaves.

Oven dried material was digested in HNO3/HClO4 and the concentrations of arsenic in

individual plant organs was determined with the help of Inductively Coupled Plasma,

Optical Emission Spectrometer (ICP-OES).

3.24 Arsenic bioaccumulation coefficient

The comparison of arsenic concentration in plant in relation to its

concentration in the environment (soil or water) is necessary to evaluate the efficiency

of plant in extracting metal. The bioaccumulation coefficient is defined as the ratio of

the concentration of arsenic in the plant and the concentration of arsenic in the

growing medium (Brooks and Robinson, 1998; Robinson et al., 2003).

The bioaccumulation coefficient was calculated according to the formula

discussed by Robinson, et al., (2005).

Bioaccumulation Coefficient (BC) = [As] in plant ÷ [As] in environment

Where [As] : Concentration of arsenic in mg/kg soil or in mg/L water

42

3.25 Arsenic contents of fruit or achenes

After calculating the yield parameters of sunflower fruit, achenes were oven

dried and digested to determine the arsenic concentrations or the bioaccumulation of

arsenic in sunflower fruit or achenes with the same instrument i-e ICP-OES.

3.26 Left over arsenic

After crop harvesting the soil was chemically analyzed to determine the left over

arsenic.

3.27 Statistical analysis:

Experiment was laid out in Completely Randomized Design (CRD) with three

replicates. The data regarding all morpho-chemical parameters was subjected to a

two-way analysis of variance (ANOVA) with the help of SPSS computer software,

version 16 (SPSS, software, 2008, Monterey, California) and to compare significance

of interaction means, least significance difference values (LSD) were also calculated

following the procedures adapted by Steel and Torrie (1984).

43

CHAPTER 4. RESULT AND DISCUSSION

4.1 EXPERIMENT 1

Toxicity of various levels of inorganic arsenicals on seed germination of four

sunflower (Helianthus annuus L.) cultivars

Seed germination parameters including germination percentage, mean

germination time, time to 50% germination, plumule and radicle length and seedling

vigour index revealed significant differences for all the four sunflower hybrids as

revealed by two way analysis of variance (ANOVA) (Table 4.1). Similarly significant

differences were found for different treatments and interaction of hybrids and

treatments for both sodium arsenate and sodium arsenite used for arsenic treatments

application.

4.1.1 Germination percentage (G %age)

Highly significant differences were found among different arsenic treatments

and sunflower hybrids (Table 4.1) for germination percentage. Considerable decrease

in germination percentage was recorded with increasing level of arsenic in all the four

sunflower cultivars used in this experiment as stated by Ahmad et al., (2009) that

higher concentrations of heavy metals suppresses the seed germination parameters.

Out of different treatments T5 and T10 (10 mg/L arsenic as sodium arsenate and

sodium arsenite respectively) affected most severely to all seed germination

parameters studied. Sodium arsenite posed more stress on germination percentage, as

inorganic As(III) is more toxic than As(V) (Winkel et al., 2008) and least value (55 %)

was calculated in treatment with 10 mg/L arsenic applied as sodium arsenite (Table

4.2, Figure 4.1), whereas with same concentration of sodium arsenate 73.33 %

germination percentage value was recorded. Maximum value (92.08 %) was noted in

control which was devoid of any arsenic application. Sunflower hybrid type H1 (FH-

331) showed best germination percentage with 90.56 % when arsenic was applied as

sodium arsenate while with sodium arsenite G %age was reduced to 75.83 %. The

minimum G %age 71.56 % was recorded in FH-385(H2). Achenes of H4 (FH-415)

responded in a same way (Table 4.3) with both kinds of arsenic salts in case of G

%age parameter.

44

4.1.2 Mean Germination Time (MGT)

Two way analysis of variance (ANOVA) revealed significant differences for

different hybrids in case of both salts of arsenic but there were non-significant

differences for various treatments (Table 4.1, Figure 4.2) whereas interaction of

hybrids into treatments revealed significant differences for sodium arsenate while

non-significant differences for sodium arsenite. Achenes or seeds belonging to

treatment T4 (8 mg/L arsenic) showed maximum value (5.28) for mean germination

time (MGT) as in table 2, while out of different cultivars H2 (FH-385) showed

minimum MGT value (3.46) and H1(FH-331) showed maximum MGT value (6.67).

Both of arsenic salts showed similar effects on mean germination time for sunflower

seeds (Table 4.2 & 4.3) as low doses caused the seeds to germinate early as compared

to control while high concentration of arsenic salts caused a little bit delay in

germination of sunflower seeds (Gulz et al., 2005) with higher MGT value.

4.1.3 Time to 50% germination (T50)

Significant differences were found for different sunflower hybrids/cultivars in case of

both sodium salts of arsenic whereas in case of treatments of arsenic, sodium arsenite

treatments showed non-significant differences. The interaction between treatments

and hybrids also showed significant differences (Table 4.1). Out of different

treatments T0 (control) showed same value (4.50 days) as time to 50% germination

for both of salts used. Treatment T2 (4 mg/L arsenic) of sodium arsenate gave the

least value for days to 50% germination (Table 4.2, Figure 4.3) which shows that low

concentration of arsenic boasts up the rate of seed germination (Cheng et al., 2006)

but high concentration of arsenic 10 mg/L arsenic in both forms of arsenic slows

down the rate of seed germination (Li et al., 2007) by giving maximum value (4.52

and 4.74 for sodium arsenate and arsenite respectively) for days to 50% germination.

4.1.4 Plumule Length (mm)

Different sunflower hybrids and various treatments of arsenic as well as

interaction of hybrid into treatments showed significant differences except the

interaction value in case of sodium arsenite application as inferred from the analysis

of variance (Table 4.1). Treatment means showed the maximum value (23.07 mm) in

45

case of T2 (4 mg/L arsenic) using sodium arsenite (Table 4.2, Figure 4.4) which is

130.9% of control (17.62 mm). This reveals that low concentrations of arsenic

increase the seedling growth up-to certain limit but after that more increase in

concentration inhibits the seedling growth (Liu and Zhang 2007; Geng et al., 2006) as

least value (14.56 mm) was recorded in highest concentration (10 mg/L) of sodium

arsenite. Sunflower cultivars behaved differently (Table 4.3) in case of plumule length

as out of different sunflower cultivars H3 (FH-405) gave maximum plumule length

(25.17 mm) whereas least vale (13.2 mm) was observed in H1 (FH-331).

4.1.5 Radicle Length (mm)

All the sunflower hybrids and different treatments of arsenic showed

significant differences except for the treatment into hybrid interaction values which

showed non-significant differences for sodium arsenite (Table 4.1). Highest mean

value (26.62 mm) of radicle length was recorded in case of T2 (4 mg/L arsenic) as

sodium arsenate while the least value (17.47 mm) was observed in case of T5 (10

mg/L arsenic) as sodium arsenite (Table 4.2, Figure 4.5), proving that in low

concentration arsenic promotes the seed germination but high concentrations of metal

inhibits the growth of seedling by suppressing root or radicle growth (Lux et al.,

2011). Out of different sunflower cultivars (Table 4.3) used in this experiment H3

(FH-405) gave maximum radicle length (28.07 mm) while H1 (FH-415) gave least

value (15.88 mm).

4.1.6 Seedling Vigour Index (SVI)

Highly significant differences were given by two way analysis of variance

(ANOVA) for sunflower cultivars and different arsenic treatments as well as

interaction of hybrids into treatments except for interaction value in case of sodium

arsenite which showed non-significant differences (Table 4.1). Maximum SVI value

(4296.96) for treatment means was recorded in case of T2 (4 mg/L arsenic) using

sodium arsenate while T10 (10 mg/L arsenic) using sodium arsenite gave least value

(1824.92) as shown in Table 4.2. Out of different sunflower cultivars, H2 (FH-385)

gave highest SVI value (4120.31) while H1 (FH-331) gave least value (2415.36) of

seedling vigour index (Table 4.3, Figure 4.6).

46

Table 4.1: Two way analysis of variance (ANOVA) for germination percentage (G %age), mean germination time (MGT), time to 50% germination (T50), plumule length (Pl L), radicle length (Rl L) and seedling vigour index (SVI) of sunflower cultivars.

Source D F

Mean square

Germ %age M G T T 50 Pl L Rl L S V I

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Hybrids (H)

Treatments (T)

H × T Interaction

Error

3

5

15

48

497.22**

554.72**

170.83**

53.12

536.11**

2064.72**

181.94**

68.75

33.93**

0.72ns

1.02**

0.41

26.39**

1.16ns

0.54 ns

0.49

10.04**

1.21**

1.04**

0.31

17.41**

1.13 ns

1.13*

0.53

307.90**

75.22**

13.97**

5.53

467.41**

137.91**

7.12 ns

6.48

441.56**

90.70**

15.93**

5.25

491.76**

139.52**

12.62 ns

9.88

7533128**

4639275.5**

770738.76**

264354.51

8406116.4**

9274712**

628847.1 ns

340110.31

CV (%)

Mean

8.631

84.44

10.85

76.39

12.825

5.023

14.96

4.68

13.338

4.158

16.92

4.29

12.207

19.274

12.68

20.07

9.909

23.13

13.39

23.47

14.402

3569.96

17.33

3365.39

Significance Level: 0.05** Highly significant ns Non significant

47

Table 4.2: Means of arsenical (sodium arsenate and sodium arsenite) treatments for germination %age, mean germination time, days to 50% germination, plumule length, radicle length and seedling vigour index.

Treatments

G %age M G T T 50 Pl L Rd L S V I

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

T0- Control

T1- 2 mg/L

T2- 4 mg/L

T3- 6 mg/L

T4- 8 mg/L

T5- 10 mg/L

LSD

92.08 a

90 a

87.08 ab

82.5 b

81.67 b

73.33 c

5.98

92.08 a

84.16 b

82.5 bc

75.83 c

68.75 d

55 e

6.81

5.02 ab

5.20 ab

4.76 ab

4.68 b

5.28 a

5.18 ab

0.52

5.02 a

4.63 ab

4.16 b

4.62 ab

4.65 ab

4.98 a

0.57

4.50 a

4.23 ab

3.73 c

4.01 bc

3.94 bc

4.52 a

0.37

4.50 ab

3.97 b

3.96 b

4.37 ab

4.19 ab

4.74 a

0.59

17.61 de

19.55 bc

22.94 a

21.1 ab

18.48 cd

15.94 e

1.93

17.62 c

22.97 a

23.07 a

21.96 ab

20.25 b

14.56 d

2.08

22.5 c

23.58 bc

26.62 a

25.11 ab

22.37 c

18.61 d

1.88

22.5 b

26.53 a

25.06 ab

25.11 ab

22.81 b

17.47 c

2.58

3670.5 bc

3908.67 ab

4296.96 a

3732.25 bc

3336.33 c

2475.08 d

422.04

3670.5 bc

4155.3 a

4098.62 ab

3533.96 c

2909.04 d

1824.92 e

478.70

Different letters indicate significant differences at 0.05 level.

48

Table 4.3: Means of sunflower cultivars for Germination %age, Mean Germination Time, days to 50% germination, plumule length, radicle length and seedling vigour index.

Sunflower Hybrids

G %age M G T T 50 Pl L Rd L S V I

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

Sodium arsenate

Sodium arsenite

H1: FH-331

H2: FH-385

H3: FH-405

H4: FH-415

LSD

90.56 a

83.06 b

78.06 c

86.11 ab

4.89

75.83 b

71.67 b

73.89 b

84.16 a

5.56

6.67 a

3.46 d

4.47 c

5.48 b

0.43

6.31 a

3.61 c

3.94 c

4.86 b

0.47

4.41 b

3.11 c

4.24 b

4.88 a

0.3

5.54 a

3.31 c

3.74 c

4.56 b

0.48

13.2 c

22.15 a

21.59 ab

20.15 b

1.58

13.65 d

22.95 b

25.17 a

18.51 c

1.71

15.88 c

26.94 a

25.37 b

24.35 b

1.54

17.01 c

27.35 a

28.07 a

21.45 b

2.11

2639.83 c

4120.31 a

3672.17 b

3847.56 ab

344.59

2415.36 c

3623.83 b

4021.44 a

3400.94 b

390.86

Different letters represent significant difference at 0.05 level.

49

Figure 4.1: Effect of arsenicals on germination percentage (G %age) of sunflower.

Figure 4.2: Effect of arsenicals on mean germination time (MGT) of sunflower.

50

Figure 4.3: Effect of arsenicals on time to 50% germination (T50) of sunflower.

Figure 4.4: Effect of arsenicals on plumule length of sunflower.

51

Figure 4.5: Effect of arsenicals on radicle length of sunflower achenes.

Figure 4.6: Effect of arsenicals on seedling vigour index (SVI) of sunflower.

52

4.2 RESULT & DISCUSSION EXPERIMENT NO. 2

Accumulation of arsenic in sunflower (Helianthus annuus L.) cultivated on arsenic contaminated soil

4.2.1 First harvest (at vegetative stage):

4.2.1.1 Agronomic attributes

Two way analysis of variance (ANOVA) of data regarding different

morphological or agronomic attributes recorded at vegetative (pre-anthesis) stage of

two sunflower varieties or cultivars, H1= FH-385 and H2= FH-415, cultivated in pre-

treated soil having six different levels (0, 20, 40, 60, 80 and 100 mg/kg soil) of two

salts containing inorganic arsenicals (S1= Sodium arsenate Na2HAsO4.7H2O as source

of As(v) and S2= Sodium arsenite NaAsO2 as source of As(III)) revealed that varieties

(V) showed significant (P<0.01) differences for shoot length, root length and

shoot:root ratio. Highest shoot length (40.50 ± 0.50) was recorded in control or T0 of

cultivar H2 (FH-415) plants whereas least value for shoot length (18.83 ± 0.87) in T5

(100 mg As/kg soil) of H1 (FH-385) plants (Figure 4.1(a)) revealing overall higher

values for shoot length in case of H2 (FH-415) as compared to H1 (FH-385).

Similarly highest root length (19.50 ± 0.50) was also noted in control plants of H2 as

compared to H1 which gave least value (9.00 ± 0.63) for root length (cm) when

maximum arsenic concentration (100 mg As/kg soil) was present. Shoot:root ratio

data revealed maximum value (2.48 ± 0.21) in case of T4 (80 mg As/kg soil) of H2

while minimum value (1.92 ± 0.04) in T1 (10 mg As/kg soil) in H1. These findings

proved that H2 showed better shoot and root growth than H1 under different levels of

arsenic in soil. Salts (S) of arsenic differed (P<0.05) significantly in case of shoot:root

ratio but non-significantly (P>0.05) for shoot and root length. Higher shoot length

value (33.53 ± 1.26) was found in case of S2 (sodium arsenite) with a little difference

(32.78 ± 2.05) in S1 (sodium arsenate) in plants belonging to H2 while H1 plants gave

less values (27.54 ± 1.65 and 27.28 ± 1.24) under S1 and S2 respectively depicting

that both salts of arsenic showed similar effects over shoot length, root length and

shoot:root ratio of sunflower plants. Different levels (L) of arsenic also showed

significant differences except for shoot:root ratio with a significant decrease in shoot

length and root length from T0-to-T5, by increasing arsenic concentration in soil with

least values for higher concentrations or levels of arsenic. Shao et al., (2011),

53

concluded similar findings in an experiment of wheat as they evaluated the effect of

arsenic on wheat and found that low concentration of arsenic caused an increase

whereas high concentration of arsenic caused reduction in shoot and root length as

well as biomass of plant. Interaction of varieties into salts and V × L differed

significantly for shoot:root ratio but non-significantly for shoot and root length

showing that levels of arsenic were equally effective on both varieties of sunflower,

whereas S × L was significant for shoot length but non-significant for root length and

shoot:root ratio revealing that shoot length and root length were both more effected by

levels of arsenic than kind of salts used in case of both cultivars used. Over all

interaction (V × S × L) was non-significant for all these three parameters.

Table 4.1(a): Analysis of variance of data for shoot length, root length andshoot: root ratio at vegetative stage under various As levels applied in soil.

Source D FMean square

Shoot Length (cm)

Root Length (cm)

Shoot : Root ratio

Varieties (V) 1 593.97** 22.22* 1.21**Salts (S) 1 1.02ns 5.56ns 0.24*

Levels (L) 5 492.52** 125.30** 0.03ns

InteractionsV × S 1 4.60ns 14.22ns 0.69**V × L 5 7.76ns 2.32ns 0.13*S × L 5 38.49** 4.98ns 0.06ns

V × S × L 5 7.08ns 2.32ns 0.07ns

Error 48 7.31 3.90 0.04

Figure 4.1(a): Shoot length, root length and shoot : root ratio of two sunflower hybrids grown under different As levels in soil.

Shoot length

54

Root length

Shoot to root ratio

Fresh and dry weights of shoot and shoot water contents data analysis also

revealed significant differences for varieties, salts and levels of arsenic applied. Both

cultivars of sunflower behaved similarly against various increasing levels of arsenic

giving higher values of shoot fresh and dry weights in H1 while relatively lower

values were recorded in case of H2. Maximum shoot fresh weight (14.47 ± 0.31) was

found in control plants of H1 whereas minimum fresh weight of shoot (6.03 ± 0.27)

was recorded in T5 (100 mg As/kg soil) of H2 showing much decrease in shoot fresh

weight in H2 as compared to H1 with increase in As concentrations in the soil. Salts

behaved similarly but average higher fresh weight of shoot (11.08 ± 0.38) was

recorded in H1 than in H2 in which less mean fresh weight (9.35 ± 0.46) was found.

Interaction of levels with salts and varieties also showed significant differences while

interaction of varieties into salts and overall interaction was non-significantly

(P>0.05) different (Table 4.2(a)).

Reduction in shoot water contents in FH-385 was small under 20, 40 and 60

mg As/kg soil and was evident in concentrations 80 and 100 mg As/kg soil whereas in

FH-415 this reduction in shoot water contents was extreme at each increased level of

arsenic than control plants. Results are in accordance with Pandey and Tripathy,

(2011), who evaluated the effect of different heavy metals including arsenic on

Albizia procera and found a positive correlation with shoot length, root length, leaf

area and biomass of the plant including fresh and dry weight of plant organs.

55

Table 4.2(a): Analysis of variance of data and mean±SE (standard error) for fresh and dry weights (g) and water contents of shoot at vegetative stage under various As levels applied in soil.


Fresh wt. shoot (g)

Dry wt. shoot (g)

Water contents shoot

Varieties (V) 1 53.74** 0.07* 49.93**Salts (S) 1 4.21* 0.098* 3.02*

Levels (L) 5 73.71** 4.49** 42.68**Interactions

V × S 1 2.35ns 0.03ns 2.88*V × L 5 2.36* 0.12** 3.19**S × L 5 2.45* 0.14** 1.82*

V × S × L 5 0.64ns 0.06** 0.59ns

Error 48 0.96 0.017 0.64

Shoot fresh weight (mean±SE) when arsenic in soilLevel Variety Mean

Hybrid 1 Hybrid 2T0 = Control 14.47 ± 0.31a 14.32 ± 0.27a 14.39 ± 0.20AT1 = 20 mg As/kg soil 11.90 ± 0.53b 10.48 ± 0.46cde 11.19 ± 0.40BT2 = 40 mg As/kg soil 11.17 ± 0.42bc 9.48 ± 0.31e 10.33 ± 0.36CT3 = 60 mg As/kg soil 10.82 ± 0.51bcd 8.23 ± 0.37f 9.53 ± 0.49CDT4 = 80 mg As/kg soil 9.93 ± 0.42de 7.55 ± 0.32f 8.74 ± 0.44DT5 = 100 mg As/kg soil 8.18 ± 0.78f 6.03 ± 0.21g 7.11 ± 0.50E

Dry weight of shoot Level Variety Mean

Hybrid 1 Hybrid 2T0 2.27 ± 0.06a 2.07 ± 0.10b 2.17 ± 0.06AT1 1.36 ± 0.17c 0.95 ± 0.04de 1.16 ± 0.10BT2 1.01 ± 0.08d 1.02 ± 0.08d 1.02 ± 0.06CT3 0.73 ± 0.02fg 0.80 ± 0.04ef 0.77 ± 0.02DT4 0.55 ± 0.04hi 0.63 ± 0.03gh 0.59 ± 0.03ET5 0.45 ± 0.04i 0.52 ± 0.03hi 0.48 ± 0.03E

Water contents of shoot Level Variety Mean

Hybrid 1 Hybrid 2T0 12.20 ± 0.37a 12.25 ± 0.34a 12.22 ± 0.24AT1 10.54 ± 0.34b 9.54 ± 0.46c 10.04 ± 0.31BT2 10.16 ± 0.39bc 8.46 ± 0.26de 9.31 ± 0.34CT3 10.09 ± 0.34bc 7.43 ± 0.34f 8.76 ± 0.46CDT4 9.39 ± 0.30cd 6.92 ± 0.32f 8.15 ± 0.43DT5 7.74 ± 0.60ef 5.51 ± 0.23g 6.62 ± 0.45E

Root fresh weight, dry weight and water contents of root also showed

significant differences for varieties and levels with highest fresh weight of root (3.12

± 0.10) was recorded in control plants of H1, while least value (0.45 ± 0.04) was also

noted in plants belonging to H1 under highest concentration (100 mg As/kg soil)

56

containing treatment (T5). Salts showed non-significant differences for root dry

weight (Table 4.3(a)), but differed significantly in case of fresh weight of root giving

higher mean value (1.45 ± 0.17) in H1 while H2 plants showed less mean value of

fresh weight of root (1.28 ± 0.15). Higher value of shoot fresh weight in case of S1

and least value in S2 revealed that role of S2 (Sodium arsenite) was relatively much

effective in reduction of root growth as compared to sodium arsenate (S1). Water

contents of root also decreased with increasing level of arsenic with no significant

effect of salt type applied in soil. Increase in arsenic concentration in soil caused

reduction in fresh and dry weights of root showing negative effect on growth and soil-

plant water relation parameters of root tissue because of interfering higher (60, 80 and

100 mg As/kg soil) levels of arsenic metal as compared to control plants. Interaction

of varieties with salts and levels gave non-significant differences but salts into levels

interaction showed significant differences for root fresh weight and water contents of

root depicting importance of levels of arsenic as compared to kind of salt and cultivar

of sunflower cultivated. Non-significant differences for overall interaction showed

that as a whole minute reduction was recorded in plants grown in arsenic

contaminated soil up to 100 mg As/kg soil in comparison with control plants grown in

normal soil devoid of any arsenic concentration.

Table 4.3(a): Analysis of variance of data for root fresh and dry weights (g) and water contents of root at vegetative stage under various As levels applied in soil.


Fresh wt. root (g)

Dry wt. root (g)

Water contents root

Varieties (V) 1 0.48* 0.015** 0.67**Salts (S) 1 1.22** 0.001ns 1.15**


V × S 1 0.12ns 0.09ns 0.11ns

V × L 5 0.07ns 0.07ns 0.07ns

S × L 5 0.27* 0.05ns 0.25**V × S × L 5 0.07ns 0.09ns 0.08ns

Error 48 0.08 0.04 0.04

57

Figure 4.2(a): Fresh and dry weight (g) and water contents of root in two sunflower hybrids grown under different As levels in soil.

Fresh weight of root when arsenic in soil

Dry weight of root

Water contents of root

4.2.1.2 Physiological and plant water relation parameters

In case of number of leaves and fresh and dry weights of leaf, only levels

showed significant differences whereas varieties differed significantly for fresh and

dry weight of leaf but non-significantly for number of leaves (Table 4.4(a)) revealing

that varieties of sunflower behaved similarly under both salts of arsenic applied but

only levels or concentrations of arsenic were effective. Salts showed significant

differences in case of leaf dry weight but non-significant (P>0.05) differences were

found in case of number of leaf and leaf fresh weight (g) showing impact of salts on

water relation attributes and physiology of sunflower leaves. Interactions V × S, V ×

L and S × L were significantly different for fresh and dry weight of leaf but non-

significantly for number of leaves whereas overall interaction V × S × L revealed

58

significant differences (P<0.05) for dry weight of leaf only but non-significant

differences for number of leaves and fresh weight of leaf revealing just a formal

impact of arsenic salts on number of leaves of both sunflower cultivars grown in soil

having different levels of arsenic.

Table 4.4(a): Analysis of variance of data for number of leaves, fresh and dry weights (g) of leaf at vegetative stage under various As levels applied in soil.


No. of leaves Fresh wt. leaf (g)

Dry wt. leaf (g)

Varieties (V) 1 0.00ns 1.31** 0.17**Salts (S) 1 0.22ns 0.22ns 0.02*


V × S 1 1.39ns 1.45** 0.13**V × L 5 0.67ns 0.47** 0.015**S × L 5 1.29ns 0.55** 0.02**

V × S × L 5 0.72ns 0.19ns 0.007*Error 48 1.18 0.11 0.003

Figure 4.3(a): No. of leaves, fresh and dry weight of leaf in two sunflower cultivars grown under different As levels in soil.

Number of leaves when arsenic in soil

Fresh weight of leaf

59

Dry weight of leaf

Varieties differed significantly (P<0.01) in case of leaf area, leaf succulence,

specific leaf weight and relative water contents of leaf but non-significant (P>0.05)

differences were found in case of leaf turgid weight (Table 4.5(a)) showing slightly

different behavior of both varieties under arsenic contamination with higher leaf area

value in H1 than H2 and higher leaf succulence value in H2 than in H1. Salts showed

significant differences for turgid and specific leaf weight and leaf area but non-

significant in case of leaf succulence and relative water contents of leaf showing

higher leaf area mean value (79.05 ± 4.47) using S1 while S2 gave less leaf area value

(64.56 ± 2.77) when S2 was used in case of H1 while H2 gave higher value of leaf

area when S2 was used than S1, similarly higher value for relative water contents of

leaf were observed in case of plants belonging to H1 under salt sodium arsenate

levels. Levels also gave significant differences for all parameters except for leaf

succulence, showing higher (70.46 ± 1.21) value for RWCLf (Relative Water

Contents of Leaf) in control plants of H1, and relatively lower for all treatments of H2

even in control plants also. V × S interaction gave significant differences for all

parameters but interaction between varieties and levels showed significant differences

in case of leaf area and turgid weight of leaf but non-significant for leaf succulence,

specific leaf weight and relative water contents of leaf as S1 gave higher value for leaf

succulence (6.14 ± 0.20) in plants belonging to H2, and lowest value (4.82 ± 0.19) in

H1 using same salt sodium arsenate whereas both cultivars behaved similarly in case

of S2. Salts into levels interaction was significantly different for all these parameters

except for leaf succulence and overall interaction V × S × L gave significant

differences for leaf area but non-significant for leaf turgid weight, specific leaf

weight, leaf succulence and relative water contents of leaf.

Gradual increase in leaf area was recorded in cultivar FH-385 under treatments 20, 40

and 60 mg As/kg but sudden drop was evident in 80 and 100 mg As/kg in soil under

sodium arsenate. In case of sodium arsenite first decrease and then increase than

60

control was recorded in leaf area of cultivar FH-385 whereas FH-415 showed similar

behavior without any remarkable difference from control plants. Leaves of cultivar

FH-415 gave higher values of leaf succulence than FH-385 and overall both varieties

and salts behaved similarly showing not any notable reduction than control in leaf

succulence or relative water contents of leaf (Figure 4.4(a)).

Table 4.5(a): Analysis of variance of data for leaf turgid weight, specific weight of leaf, leaf area, leaf succulence and relative water contents of leaf at vegetative stage under various As levels applied in soil.


Leaf turgid wt.

Leaf sp. wt.

Leaf area Leaf succulence

Relative water

contents of leaf

Varieties (V) 1 0.046ns 0.09** 2110.33** 9.44** 878.08**Salts (S) 1 0.76* 0.02** 713.79** 0.91ns 24.71ns

Levels (L) 5 0.67** 0.08** 569.25** 0.59ns 154.04**Interactions

V × S 1 1.29** 0.06** 1208.03** 6.41** 214.11**V × L 5 0.65** 0.01ns 769.14** 0.15ns 27.25ns

S × L 5 0.44* 0.05** 243.95** 0.26ns 133.07**V × S × L 5 0.19ns 0.01ns 372.51** 0.39ns 30.56ns

Error 48 0.15 0.07 13.17 0.64 27.55

Figure 4.4(a): Leaf turgid weight, specific weight of leaf, leaf area, leaf succulence and relative water contents of leaf at vegetative stage under various As levels in soil.

Turgid weight of leaf when arsenic in soil

Specific weight of leaf

61

Leaf area

Succulence of leaf

Relative water contents of leaf

4.2.2 Final harvest (at maturity):

Final harvest was taken at maturity of crop and the data obtained from this

study comprising on effect of various rhizospheric levels (Treatments) of inorganic

arsenicals or salts (S) applied through soil only, on growth and yield parameters and

phytoremediation potential of two sunflower cultivars (H1 and H2) are parameters

recorded were analyzed statistically.

4.2.2.1 Agronomic and yield parameters

Two way analysis of variance (ANOVA) revealed that varieties differed

significantly (P<0.01) only in case of number of leaves and capitulum diameter

whereas for all other parameters including shoot and root length and shoot : root ratio

and 100 achene weight, varieties and salts differed non-significantly (P>0.05),

although levels (L) of arsenic gave significantly different values except for shoot to

root ratio (Table 4.6(a)).

62

Table 4.6(a): ANOVA table for different agronomic and yield parameters of sunflower cultivated in arsenic contaminated soil.


Shoot length

Root length

Shoot : root

No. of leaves

Cap. Dia.

100 achene

wt.Varieties (V) 1 3.01ns 38.31ns 2.87ns 33.17** 12.66** 0.002ns

Salts (S) 1 84.84ns 10.59ns 1.41ns 15.96* 0.74ns 0.05ns

Levels (L) 5 7323.40** 95.83** 1.19ns 187.75** 64.64** 4.21**Interactions

V × S 1 3.25ns 0.28ns 0.002ns 0.33ns 0.61ns 0.31ns

V × L 5 90.04ns 3.11ns 0.39ns 4.15ns 3.98** 0.34ns

S × L 5 237.02ns 8.49ns 0.73ns 9.64* 1.64* 0.09ns

V × S × L 5 131.77ns 1.65ns 0.24ns 3.63ns 0.29ns 0.15ns

Error 48 176.69 11.35 0.86 3.87 0.64 0.16

A significant reduction in shoot and root length, shoot to root ratio, number of

leaves, capitulum diameter and 100 achene weight of sunflower plants was observed

with increasing level of both arsenic salts. Control plants gave maximum values of

shoot and root length (cm) while minimum value was recorded in T5 (100 mg As/kg

soil) of both arsenicals (Figure 4.5(a)), but overall plants coped with the stress if

applied by highest arsenic concentrations in soil medium. Both of varieties or

cultivars of sunflower behaved similarly in all these parameters recorded at crop

maturity. A reduction in shoot and root length was recorded under conditions of this

toxic metal like Madejon et al., (2003) who also reported considerable reduction in

shoot and root growth and biomass of sunflower plants under arsenic conditions

Yield attributes including diameter of capitulum (inflorescence) and weight of

hundred achenes showed significant reduction with increase in soil arsenic levels, and

both varieties behaved similarly as higher values in control plants and reduction in all

levels than control was evident in both cultivars of sunflower harvested finally at

maturity. A sudden drop is obvious in T2 (40 mg As/kg soil) when arsenic was in

arsenate form in the soil.

63

Figure 4.5(a): Shoot length, root length, shoot to root ratio, number of leaves, capitulum diameter and weight of hundred achenes under different arsenic concentrations in soil.

Shoot length (at maturity)

0

50

100

150

200

250

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

St L

2 (s

oil) Hybrid 1

Hybrid 2

Root length (at maturity)

0

5

10

15

20

25

30

35

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Rt L

2 (s

oil)

Hybrid 1Hybrid 2

Shoot to root ratio (at maturity)

0123456789

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Sht:R

t 2 (s

oil) Hybrid 1

Hybrid 2

Number of leaves (at maturity)

0

5

10

15

20

25

30

35

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

No.L

2 (s

oil)

Hybrid 1Hybrid 2

64

Capitulum diameter

0

5

10

15

20

25

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Cap.

Dia

(soi

l) Hybrid 1Hybrid 2

Hundred achene weight

00.5

11.5

22.5

33.5

44.5

5

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Wt.

100

(soi

l) Hybrid 1Hybrid 2

4.2.3 Arsenic accumulation in various plant tissues and left over arsenic

Arsenic accumulated in different tissues of sunflower plants was determined

with the help of ICP-OES (Inductively Coupled Plasma-Optical Emission

Spectrometry) after wet digestion of ground tissue of root, shoot, leaves and achenes

or seeds of experimental crop replicates. Two way analysis of variance of data

regarding arsenic accumulated in root, shoot, leaf and seed along-with left over

arsenic in remaining soil are depicted in Table 4.7(a)). Varieties of sunflower showed

significant differences (P<0.01) in case of As accumulated in roots and seeds but non-

significant differences (P>0.05) were found in case of As in shoot and leaves as well

as left over arsenic. Salts of arsenic showed significant differences in case of As in

root, shoot and leaf but non-significant differences were observed in As seed and left

over arsenic. Different levels (Treatments) of arsenic also showed significant

differences for all these parameters related to arsenic accumulation in different

sunflower plant organs. Highest assimilation or absorption of arsenic was recorded in

root tissue and least in seed or achenes whereas leaves were at second position in

arsenic accumulation. Although varieties behaved almost similarly but a gradual

increase in soil arsenic level caused increase in accumulation of arsenic and

resultantly higher contents in different organs.

65

Table 4.7(a): Analysis of variance for As accumulation in root, shoot, leaf, seed and left over arsenic applied as various levels in soil.


As root As shoot As leaf As seed Left over AsVarieties (V) 1 360.01** 3.72ns 14.42ns 2.88** 2.72ns

Salts (S) 1 263.35** 23.23** 96.98** 0.29ns 0.02ns

Levels (L) 5 2153.33** 127.96** 890.23** 14.54** 2417.65**Interactions

V × S 1 68.64* 6.54ns 606.22** 6.12** 56.18*V × L 5 39.26** 2.89ns 22.04** 1.29** 13.68ns

S × L 5 56.15** 7.67** 39.46** 0.18ns 12.50ns

V × S × L 5 23.56ns 0.91ns 53.44** 0.86* 37.81**Error 48 9.90 1.82 6.38 0.34 8.95

Left over arsenic was highest quantity due to chelation effect probably. Left

over arsenic in soil was recorded and maximum was retained in the form of arsenite

which is less assimilated as compared to sodium arsenate. Shoots accumulated less

arsenic but more than seeds or achenes (Figure 4.6(a)). The order of arsenic

accumulation remained root > leaves > shoot > seed while maximum concentration of

As was remained in the soil as left over arsenic.

Figure 4.6(a): Left over arsenic and accumulation of arsenic in root, shoot, leaf and seed of sunflower plants grown in arsenic contaminated soil.

4.2.4 Bioaccumulation coefficient (BC) of arsenic

Bioaccumulation coefficient of arsenic was determined and two way analysis

of variance showed significant (P<0.01) differences for varieties, salts and levels in

case of bioaccumulation coefficient values of root, shoot, leaves and seed of

sunflower cultivars (Table 4.8(a)). In case of BC root, varieties, salts and levels

caused significant differences and interaction among all these factors also differed

66

(P<0.05) significantly except for overall interaction among all three factors which

showed non-significant differences for BC root. Varieties, salts and levels also

showed significant differences and their interactions too in case of BC shoot and leaf

but BC seed was non-significantly differed under salts of arsenic used.

Table 4.8(a): ANOVA about bioaccumulation coefficient of arsenic in root, shoot, leaf and seed of sunflower.


BC root BC shoot BC leaf BC seedVarieties (V) 1 0.21** 0.01** 0.02** 0.003**

Salts (S) 1 0.07** 0.008** 0.008* 0.0002ns

Levels (L) 5 0.09** 0.02** 0.036** 0.004**Interactions

V × S 1 0.02* 0.002* 0.18** 0.002**V × L 5 0.01* 0.005** 0.02** 0.001**S × L 5 0.01** 0.004** 0.005* 0.0001ns

V × S × L 5 0.005ns 0.0003ns 0.014** 0.0002ns

Error 48 0.003 0.0005 0.002 0.00019

Highest values of bioaccumulation coefficient were observed in roots and

leaves while shoot was at third position with least in achenes or seeds of sunflower

(Figure 4.7(a)). H2 or FH-415 showed maximum bioaccumulation coefficient values

as compared to H1 (FH-385). A sudden rise in BC value for root and leaves was

evident in case of treatments having S2 (sodium arsenite) when 20 mg As/kg was

present in soil and then drop with next level (40 mg As/kg in soil) of arsenic as S2,

then again rise in roots of plants having 60 mg As/kg soil, same was situation in case

of Leaves BC. These ups and downs are more conspicuous in plants belonging to H2

or FH-415 whereas H1 (FH-385) behaved smoothly without sudden rises and falls of

BC values in root, shoot, leaves and seed of sunflower plants. This reveals low

phytoextraction potential of sunflower cultivars for arsenic which is in accordance

with the findings of Madejon et al., (2003) who cultivated sunflower in an abandoned

pyrite mine area.

67

Figure 4.7(a): Bioaccumulation coefficient (BC) of root, shoot, leaves and seed of sunflower cultivars grown under As contaminated soil

4.2.5 Comparisons of ratios among arsenic concentrations in root, shoot, leaves

and seed

Two way analysis of variance (ANOVA) for data regarding ratios of different

arsenic concentrations accumulated in sunflower tissues are depicted in Table 4.9(a)),

which reveals that significant (P<0.01) differences were observed for varieties in case

of ratio between [As] root and [As] seed along-with [As] shoot and [As] seed, while

non-significant (P>0.05) for [As] root and [As] shoot and [As] leaf to [As] seed.

Table 4.9(a): Ratios of arsenic concentrations [As] among different sunflower plant tissues


[As]root:[As]shoot [As]root:[As]leaf [As]root:[As]seedVarieties (V) 1 1.93ns 13.14* 984.35**

Salts (S) 1 0.67ns 11.50ns 2.56ns

Levels (L) 5 20.14** 4.38ns 143.96ns

InteractionsV × S 1 0.050ns 17.96* 416.55*V × L 5 20.95** 3.08ns 97.59ns

S × L 5 9.14** 4.21ns 16.66ns

V × S × L 5 0.099ns 3.98ns 61.53ns

Error 48 2.54 3.02 67.61


[As]shoot:[As]leaf [As]shoot:[As]seed [As]leaf:[As]seedVarieties (V) 1 0.88* 112.93** 69.66ns

Salts (S) 1 0.99** 1.86ns 91.04ns

Levels (L) 5 0.38* 7.15ns 118.50*Interactions

V × S 1 1.49** 37.72* 705.63**V × L 5 0.33* 31.70** 124.83**S × L 5 0.44** 11.17ns 20.66ns

V × S × L 5 0.25ns 7.14ns 50.82ns

68

Error 48 0.13 8.66 34.80

Highest accumulation of arsenic was occurred in root and minimum in seed

therefore ratio of arsenic concentration in root to arsenic concentration of seed was

found maximum than all other ratios (Figure 4.8(a)) and second peak values were

found in ratio of arsenic concentration of leaf to seed showing the difference between

arsenic accumulated in leaf as compared to seed and revealed that higher level of

arsenic was accumulated in leaves than seeds or achenes of sunflower plants.

Figure 4.8(a): Comparison of different ratios of arsenic concentrations determined in different organs of sunflower grown in As contaminated soil.



Phosphorus is important bio-element vital for growth and normal development

of plants. Arsenic and phosphorus having similar behavior in plants and because

arsenate mimics phosphate in its translocation in plant tissues. Analysis of variance

revealed that varieties showed significant differences (P<0.01) in case of [P] in root,

shoot and seed but non- significant (P>0.05) for leaf (Table 4.10(a)), levels differed

significantly in case of all tissue concentrations of P in sunflower. Both salts showed

mixed behavior as significant differences for shoot and seed [P] and while non-

significant for root and leaf as compared to arsenic concentrations in which salts

caused significant difference in case of root, shoot and leaf whereas only seed showed

non-significant differences. Out of different interactions varieties into levels

interaction showed significant differences for all phosphorus related parameters.

Calcium is important for proper growth of plants as is used in plant cell wall

formation and vital for many other developmental processes was also determined in

69

different tissues of sunflower cultivars. Analysis of variance revealed that varieties

differed significantly (P<0.01) when Ca was determined in root, leaf and seed while

non-significantly in case of shoot. Levels of arsenic also showed significant

differences except Ca in seed or achenes. Seed Ca contents showed that only varieties

differed significantly whereas salts, levels and all of interaction among these factors

showed non-significant differences as compared to root Ca contents in which all three

factors as well as all interactions differed significantly when arsenic was in soil.

Magnesium is central element in chlorophyll molecule so vital for all plants.

Significant differences were found in varieties, salts and levels in case of Mg

concentration in root, shoot, leaves and seeds of sunflower cultivars cultivated in

arsenic contaminated soil. Similarly significant differences were also evident in

interactions among varieties, salts and levels for root, shoot, leaves and seeds except

variety into salt interactions in case of Mg contents in root and seeds or achnes as

tabulated in Table 4.10(a).

Table 4.10(a): ANOVA for phosphorus (P), calcium (Ca) and magnesium (Mg) contents found in sunflower cultivars grown on arsenic contaminated soil.


P root P shoot P leaf P seedVarieties (V) 1 505.25** 381.62** 7.89ns 887.3**

Salts (S) 1 9.54ns 150.68** 2.25ns 1519.8**Levels (L) 5 158.33** 84.83** 239.96** 2787.7**

InteractionsV × S 1 53.51ns 43.80ns 17.76ns 244.1*V × L 5 90.89** 148.03** 110.08** 693.7**S × L 5 35.34ns 38.72* 130.05** 243.2**

V × S × L 5 72.60* 31.18ns 173.51** 554.4**Error 48 21.42 14.89 26.08 40.0


Ca root Ca shoot Ca leaf Ca SeedVarieties (V) 1 55428306** 73584ns 109110621** 502832**

Salts (S) 1 8280822** 3266ns 64031398** 29963ns

Levels (L) 5 27510229** 1359583** 117194912** 124903ns

InteractionsV × S 1 8234304** 880962** 579584ns 528ns

V × L 5 18674069** 463805** 105747447** 81809ns

S × L 5 23563998** 359700** 77154726** 76090ns

V × S × L 5 10543638** 1781542** 32506851** 50874ns

Error 48 508040 65303 5440308 56124


Mg root Mg shoot Mg leaf Mg seed

70

Varieties (V) 1 344385* 2293547** 6548798** 409232**Salts (S) 1 324903* 206913* 564549** 211532*

Levels (L) 5 534956** 1620719** 1950730** 107946*Interactions

V × S 1 837ns 1617751** 491430* 1044ns

V × L 5 223472** 1393020** 1692158** 136744*S × L 5 837461** 649748** 856568** 101122*

V × S × L 5 191722** 1215167** 702089** 133276*Error 48 50450 49690 74879 40208

Similar concentrations of P were found in root and shoot, but a little increase

was obvious in leaves whereas seeds showed maximum concentration especially in

plants which were grown in arsenate contaminated soil having 100 mg As/kg soil and

belonging to cultivar FH-415 (Figure 4.9(a)). A sharp rise was observed in

phosphorus concentration from T4 to T5 in case of both salts used showing that

arsenic is effective either present as arsenate or arsenite towards P concentrations in

plant tissues. It is evident that leaves and seeds or achenes are main sink for P in

sunflower plants.

Highest Ca contents were found in leaves of sunflower with a peak in case of

plants belonging to V2 under level T2 (40 mg As/kg soil) using arsenite than all other

plant organs either root, shoot and seeds revealing that leaf appeared to be strong sink

for Ca in sunflower plants grown in arsenic contaminated soil. In root tissue calcium

was in similar low concentration to shoot except a single peak in cultivar FH-415

under T4 (80 mg As/kg soil) having arsenate in soil, whereas seeds or achenes

showed least calcium contents in them in both cultivars and under both salts in all the

six levels of arsenic revealing that either arsenate or arsenite both as well as various

levels of these salts had negligible effects over Ca contents of sunflower achenes or

seeds obtained from plants of this experiment.

Leaves showed maximum Mg concentrations as compared to root, shoot and

seeds, whereas shoot was at the second level in accumulating magnesium ion while

some values were higher in roots too but seeds showed about similar values without

any prominent fluctuation (Figure 4.9(a)). V1 or 1st cultivar FH-385 showed

maximum conents of magnesium ion as compared to 2nd cultivar or V2 (FH-415).

Figure 4.9(a): Phosphorus (P), calcium (Ca) and magnesium (Mg) contents found in root, shoot, leaf and seed of sunflower cultivars grown under arsenic contaminated soil.

Phosphorus contents in root, shoot, leaf and seed when arsenic in soil

71

Calcium contents

Magnesium contents


and seeds of sunflower cultivars grown in arsenic contaminated soil

Potassium (K) another very important bio-element for plants having key role

in stomatal regulation and important in plant water relation parameters. ANOVA

regarding different concentrations of K in various tissues of sunflower cultivars

revealed significant differences in varieties, salts and levels with a few exceptions

(Table 4.11(a)). A lot of peaks and fluctuations in concentrations of K are evident that

sunflower showed mixed behavior towards accumulation of this bio-element in root,

shoot, leaves and seeds. Relatively higher values were found in root, shoot and leaves

72

by V2 (FH-415), while V1 (FH-385) gave higher K concentrations in sunflower

seeds.

Boron (B), as a micronutrient is important element for normal growth and

development of plants. Analysis of variance (ANOVA) revealed significant

differences (P<0.01) for varieties in case of boron concentration in leaf and seeds or

achenes of sunflower plants while non-significant (P>0.05) for root and shoot. Salts

showed significant differences in case of shoot and leaf boron levels while non-

significant in roots and seeds, whereas levels differed significantly for root, shoot and

leaves but non-significantly for seeds. All interactions among varieties, salts and

levels differed significantly for boron levels in root, shoot and leaves but non-

significant differences were found in case of boron in seeds of sunflower cultivars.

Copper (Cu) is important for proper functioning of many enzymes vital for a

few major metabolic pathways in plants. Two way analysis of variance revealed

significant differences in varieties for copper concentrations detected in shoot, leaves

and seed but non-significant for Cu in root (Table 4.11(a)). Salts showed significant

differences in case of shoot and leaves but non-significant for root and seeds, while

levels (L) caused significant differences (P<0.01) in copper concentrations of root,

shoot and leaves but in case of seeds levels did not effected significantly. Varieties

into levels interaction showed significant differences in all these four parameters

while in case of varieties into salts into levels interaction only shoot copper

concentrations showed non-significant differences, while all other organs root, leaves

and seeds showed significant (P<0.01) differences.

Table 4.11(a): ANOVA regarding potassium (K), boron (B) and copper (Cu) contents in different organs of sunflower cultivated in arsenic contaminated soil.


K root K shoot K leaf K seedVarieties (V) 1 2889788** 64809207** 45687575** 14914938**

Salts (S) 1 62334846** 44062656** 83879505** 1656916ns

Levels (L) 5 19043676** 40697847** 861944ns 2110836ns

InteractionsV × S 1 73443ns 129876ns 16148443** 362106ns

V × L 5 27850969** 46007098** 8416550** 3473879**S × L 5 13757063** 16337673** 20031126** 1283352ns

V × S × L 5 33192495** 19201873** 1652400ns 4642036**Error 48 82282 1072926 821008 973304


B root B shoot B leaf B seed

73

Varieties (V) 1 1.61ns 10.34ns 128.03** 139.92**Salts (S) 1 17.71ns 86.70** 185.12** 11.78ns

Levels (L) 5 43.88** 105.00** 966.81** 8.73ns

InteractionsV × S 1 199.43** 254.21** 960.61** 2.43ns

V × L 5 129.77** 103.15** 229.25** 4.38ns

S × L 5 107.37** 103.48** 448.14** 0.89ns

V × S × L 5 32.76** 167.30** 851.61** 2.01ns

Error 48 5.20 4.23 6.49 3.68


Cu root Cu shoot Cu leaf Cu seedVarieties (V) 1 0.009ns 31.48** 278.83** 83.21**

Salts (S) 1 15.65ns 37.89** 85.04** 1.10ns

Levels (L) 5 44.95** 13.97** 65.64** 9.45ns

InteractionsV × S 1 84.08** 9.53ns 6.90ns 61.23**V × L 5 33.51** 12.34** 105.00** 41.08**S × L 5 122.16** 3.24ns 109.95** 45.58**

V × S × L 5 21.68** 5.97ns 56.69** 35.23**Error 48 4.67 3.22 5.58 6.65

Out of different organs much fluctuation in K concentration was recorded in roots and shoots of sunflower cultivars with maximum values in V2 (FH-415), showing a sharp rise in plants belonging to T4 and T5 treatments or levels which contained maximum concentration of As in soil. Relatively stable values of K ion were found in seeds or achenes obtained from both cultivars but remained similar to root K ion concentration (Figure 4.10(a)).

Highest concentration of B was recorded in leaves with maximum fluctuations in both salts of arsenic. Level of boron in root and shoot tissues remained similar with seeds showing minimum fluctuation in boron values (Figure 4.10(a)). Both varieties behaved similarly but overall concentration of B remained almost similar in both cultivars with maximum in sunflower leaves.

Maximum levels of copper was recorded in seeds of sunflower cultivars, then 2ndly leaf and root showed accumulation of copper and minimum concentration was found in shoot. In seeds V2 (FH-415) gave values higher than FH-385 (V1) whereas in all other tissues higher values were found in V1 plants (Figure 4.10(a)). Comparatively higher values were found in plants having arsenate in their soil especially in root and leaves. Least concentration of Cu was found in shoot of plants grown in arsenite containing soil.

Figure 4.10(a): Potassium (K), boron (B) and copper (Cu) contents in sunflower cultivars grown in arsenic contaminated soil.

74

Potassium contents

Boron contents

Copper contents


and seeds of sunflower cultivars grown in arsenic contaminated soil

The data regarding iron (Fe) concentration found in root, shoot, leaves and

seed of sunflower was statistically processed and analysis of variance revealed

significant differences (P<0.01) caused by salts and levels of arsenic (Table 4.12(a)).

Varieties or cultivars also differed significantly except for leaf concentration of iron.

Interactions among varieties, salts and levels gave significantly different values for

root, shoot, leaves and seeds or achenes of sunflower. Variety into salt interaction

75

revealed non-significant differences (P>0.05) in case of seed and root iron levels.

Varieties showed non-significant differences in case of iron concentration in leaf too.

Two way analysis of variance revealed that varieties behaved differently for

Mn levels in root, shoot, and leaf while non-significant differences were found in seed

Mn concentration. Salts of arsenic caused significant differences in root and leaf Mn

level but behaved similarly in shoot and seed concentrations of Mn. Levels also

caused notable difference in root, shoot and leaf but non-significant differences were

observed in Mn contents in seed (Table 4.12(a)). All of the interactions among

varieties, levels and salts gave non-significant different results in case of Mn

concentrations of seed while in all other organs Mn contents were significantly

different.

Statistical analysis of the data regarding zinc concentration in different organs

of sunflower cultivars revealed that varieties, salts and levels of arsenic showed

significant (P<0.01) differences in all the four parameters comprising Zn in root,

shoot, leaves and seeds. Interactions among varieties, salts and levels also showed

reasonable differences except for variety into salt interaction in which non-significant

differences were observed in parameters Zn in shoot, leaf and seed (Table 4.12(a)).

Table 4.12(a): ANOVA for iron (Fe), manganese (Mn) and zinc (Zn) contents in different organs of sunflower cultivated in arsenic contaminated soil.


Fe root Fe shoot Fe leaf Fe seedVarieties (V) 1 27960504** 88239** 1955ns 63.38*

Salts (S) 1 29692387** 199468** 656139** 268.23**Levels (L) 5 12601815** 145295** 210796** 181.25**

InteractionsV × S 1 61121ns 62531** 115838** 0.26ns

V × L 5 7917222** 164864** 204347** 203.39**S × L 5 20549325** 141021** 418628** 22.71ns

V × S × L 5 7288897** 152105** 230041** 85.47**Error 48 92338 12.0 2106 11.99


Mn root Mn shoot Mn leaf Mn seedVarieties (V) 1 8382.5** 15.55** 545.16** 35.86ns

Salts (S) 1 2615.6** 5.69ns 208.76** 0.79ns

Levels (L) 5 3257.3** 5.72* 463.11** 16.34ns

InteractionsV × S 1 2072.8** 11.57* 229.27** 11.83ns

V × L 5 1350.6** 3.43ns 323.07** 22.20ns

S × L 5 7348.5** 5.11* 403.60** 1.08ns

76

V × S × L 5 929.6** 5.11* 142.98** 5.99ns

Error 48 11.0 1.73 8.78 10.76

SourceD F

Mean squareZn root Zn shoot Zn leaf Zn seed

Varieties (V) 1 550.0** 2641.43** 1966.4** 282.03**Salts (S) 1 1435.8** 316.01** 70.4* 369.92**


V × S 1 1874.8** 1.00ns 12.7ns 23.46ns

V × L 5 1780.8** 816.57** 598.6** 208.43**S × L 5 2934.7** 332.86** 1497.5** 400.68**

V × S × L 5 2005.0** 178.24** 522.6** 381.11**Error 48 8.9 9.45 10.5 11.15

Root gave maximum values of Fe especially in plants belonging to cultivar V2

(FH-415) grown in sodium arsenate contaminated soil, sodium arsenite showed less

impact on iron concentration in root of sunflower plants (Figure 4.11(a)). Much more

fluctuations in iron level were evident in both varieties but only in root organ while in

all other tissues very poor concentrations of iron were detected. Seeds showed least

values of Fe in them revealing the minimum accumulation of iron like shoots but in

shoot there was a single peak in T5 (100 mg As/kg soil) using arsenate in plants of V1

(FH-385). A similar peak of Fe contents was also find in leaves of plants belonging to

V2 under T3 (60 mg As/kg soil) when arsenate was in soil.

Highest concentration of Mn was recorded in roots but it was a sudden rise

and fall of just one value in cultivar FH-415 and in T4 (80 mg As/kg soil) using

sodium arsenate. Shoot showed least value for Mn (Figure 4.11(a)). After root, leaves

contained peak values of this element and especially in plants belonging to V2, in

case of both salts of arsenic used. Seeds or achenes also accumulated a little quantity

of Mn as compared to root and leaf.

Seeds showed maximum level of zinc in them as compared to all other plant

organs, although a highest peak was also found in root but average top values of zinc

concentration were found in seeds or achenes of sunflower cultivar V1 (FH-385)

grown in soil having either sodium arsenate or sodium arsenite. Occasional rises were

evident in FH-415 but followed by sudden fall as in root T4 (80 mg As/kg soil) gave

highest value but T5 (100 mg As/kg soil) gave similar reading with control. It seems

difficult to say that which organ showed minimum accumulation of Zn in it (Figure

4.11(a)), but maximum was in seeds. Both salts of arsenic behaved similarly towards

77

zinc accumulation in sunflower cultivars grown under arsenic contaminated

rhizospheric conditions especially in soil.

Figure 4.11(a): Iron (Fe), manganese (Mn) and zinc (Zn) contents in sunflower cultivars grown in arsenic contaminated soil.

Iron contents

Manganese contents

Zinc contents

4.2.9 Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in root, shoot,

leaves and seeds of sunflower cultivars grown in arsenic contaminated soil

78

Two way analysis of variance revealed significant differences for varieties,

salts and levels of arsenic in case of molybdenum concentration in root, shoot and

seeds but non-significant differences were observed in case of Mo concentration in

leaf (Table 4.13(a)). Out of different interactions among varieties, salts and levels,

shoot gave significantly different values of Mo while in root variety into salt showed

non-significant differences only whereas all interactions showed non-significant

differences when Mo contents were observed in leaf and seed of sunflower cultivars.

Two way analysis of variance revealed that varieties behaved similarly except

for silver contents in seed, whereas salts caused significant differences (P<0.05) in

leaf and seed silver contents as compared to root and shoot in which non-significant

(P>0.05) differences were found due to salts (Table 4.13(a)). Levels of arsenic also

behaved similarly except for root silver contents which were significantly different

(P<0.01). Most of interactions among varieties, salts and levels showed non-

significant differences, only variety into salt interaction in root and shoot and variety

into level and salt into level interaction in seed revealed significant differences.

Statistical analysis including analysis of variance (ANOVA) revealed

significant differences (P<0.01) in Al concentrations of root and leaf for varieties,

salts, levels and all their interactions (Table 4.13(a)). Aluminum contents of shoot

revealed non-significant differences in case of varieties and salts but significantly

different values were recorded in levels of arsenic applied, variety into salt interaction

also showed non-significantly (P>0.05) different values of shoot Al contents. In case

of seeds or achenes varieties and levels showed differences but salts behaved

similarly, whereas out of different interactions, only variety into salt interaction gave

similar values of Al concentration while all other interactions differed significantly.

79

Table 4.13(a): ANOVA for molybdenum (Mo), silver (Ag) and aluminum (Al) contents in sunflower cultivars grown in arsenic contaminated soil.


Mo root Mo shoot Mo leaf Mo seedVarieties (V) 1 7.52** 2.67** 1.08ns 2.52**

Salts (S) 1 0.89* 2.47** 0.34ns 0.92*Levels (L) 5 2.32** 4.41** 1.55ns 0.62*

InteractionsV × S 1 0.25ns 5.25** 6.47ns 0.11ns

V × L 5 3.54** 4.31** 6.21ns 0.34ns

S × L 5 0.67* 4.36** 0.54ns 0.21ns

V × S × L 5 0.54* 3.76** 1.28ns 0.05ns

Error 48 0.21 0.23 3.87 0.21


Ag root Ag shoot Ag leaf Ag seedVarieties (V) 1 2.77ns 0.84ns 1.40ns 740.03**

Salts (S) 1 0.001ns 0.071ns 2.88* 56.19*Levels (L) 5 11.17** 0.94ns 0.56ns 19.89ns

InteractionsV × S 1 10.67* 5.78** 0.94ns 36.96ns

V × L 5 4.39ns 0.97ns 1.39ns 20.91ns

S × L 5 2.79ns 0.84ns 0.78ns 32.04**V × S × L 5 1.63ns 0.66ns 0.84ns 53.03**

Error 48 2.41 0.58 0.63 9.28


Al root Al shoot Al leaf Al seedVarieties (V) 1 22008936** 2073ns 92391** 240.09**

Salts (S) 1 1415033** 2835ns 340663** 2.96ns

Levels (L) 5 4604128** 10231** 271110** 34.64**Interactions

V × S 1 636489** 3942ns 188413** 5.37ns

V × L 5 2729657** 8130** 236138** 40.47**S × L 5 17366493** 6823** 645197** 24.39*

V × S × L 5 632810** 3460* 281405** 83.95**Error 48 81126 1265 10026 9.36

Average higher Mo concentration was found in leaves with maximum

fluctuations in values whereas a single highest figure was evident in shoot of V1 (FH-

385) under arsenite salt. In root and leaf, FH-385 gave relatively higher values of Mo,

while in shoot and seeds, V2 (FH-415) gave higher Mo contents (Figure 4.12(a)). In

shoot organ a single rise was found in T3 (60 mg As/kg soil) followed by a sudden

drop in T4 and T5 using sodium arsenite. In seeds V1 gave relatively lower

concentration of molybdenum as compared to root where this cultivar gave higher

average Mo contents as compared to V2.

80

Concentration of silver was found maximum in seeds or achenes of V1 (FH-

385) especially in case of plants cultivated in arsenite containing soil than all other

organs of sunflower cultivars whereas minute rises were found in root organ of

sunflower plants. Overall V2 (FH-415) showed very little accumulation of silver ion

either in sunflower root, shoot or seeds (Figure 4.12(a)). In case of shoot a single

higher value was observed in T1 (20 mg As/kg soil) having arsenate in their soil.

Unevenly high level of silver was recorded in seeds of FH-385 grown in both arsenic

salts revealing higher potential of sunflower towards accumulation of silver in it. In

root a small peak of Ag contents was also observed in V1 under T2 (40 mg As/kg

soil) having arsenite in soil.

As shown in fig. , average higher Al contents were recorded in roots of

sunflower plants belonging to cultivar V2 (FH-415), especially in T4 (80 mg As/kg

soil) having sodium arsenate while arsenite also accumulated similarly in root organ.

Relatively lower levels of Al were accumulated in V1 (FH-385) with an exception in

root organ in which a single highest value of Al was found even higher than V2. A

slight increase in Al contents of leaf organ was also recorded but in shoot and seeds

very minute or negligible contents of aluminum were observed to be accumulated.

Figure 4.12(a): Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in root, shoot, leaves and seeds of sunflower cultivars grown in arsenic contaminated soil.

Molybdenum contents

81

Silver contents

Aluminum contents

4.2.10 Contents of barium (Ba), bismuth (Bi) and cadmium (Cd) found in root,

shoot, leaves and seeds of sunflower cultivars grown in arsenic

contaminated soil

Two way analysis of variance revealed significant (P<0.05) differences for Ba

concentration in root when observed for varieties while for salts, levels and all

interactions among varieties, levels and salts showed highly significant (P<0.01)

differences (Table 4.14(a)). Similarly shoot and leaf Ba contents also showed

significant differences in case of varieties, level and salts as well as all of the

interactions among them. In case of Ba contents of seed, varieties levels and their

interaction showed non-significant (P>0.05) differences. Salts into levels interaction

also showed non-significant differences only in case of Ba contents of sunflower

seeds.

Two way analysis of variance of data regarding Bi concentration in root

showed non-significant differences (P>0.05) for varieties but salts and levels showed

significant (P<0.01) differences, while in case of Bi in shoot varieties and salts

showed significant differences (Table 4.14(a)). In case of Bi in leaf, varieties, salts

and leaves showed significant differences whereas in case of bismuth contents of

82

seed, varieties and levels differed significantly but salts showed non-significant

differences. Variety into salts interaction showed significant differences in case of

bismuth contents in seed while non-significant differences for Bi in root, shoot and

leaves. Interaction of variety and leaves showed significant differences (P<0.05) in

case of Bi in root and significant (P<0.01) also for Bi in shoot, leaves and seeds. Salt

into level interaction gave significant differences in case of Bi in root and leaf but

non-significant differences were observed in case of Bi in shoot and seeds. Overall

interaction among varieties, salts and leaves gave non-significantly different values

for bismuth in root, shoot, leaf and seeds or behaved similarly.

Analysis of variance of data revealed that significant (P<0.01) differences

were found in varieties, salts and levels in case of Cd in root and interaction of variety

into salt and level also differed (P<0.05) significantly but salt into level and overall

interaction among variety into salt into level showed non-significant differences

(P>0.05). Cadmium in shoot showed significant differences for varieties, salts, levels

and all of their interactions (Table 4.14(a)). In leaf Cd contents showed non-

significant differences in varieties and salts and their interaction too, while variety

into level interaction differed significantly (P<0.05) but salts into level and variety

into salt into level interaction also showed non-significant differences. Cadmium

contents of seed also gave non-significant differences for all factors except for

interaction between variety into level in which significant (P<0.05) differences were

found.

Table 4.14(a): ANOVA for barium (Ba), bismuth (Bi) and cadmium (Cd) contents of sunflower cultivars grown in arsenic contaminated soil.

Source D F

Mean squareBa root Ba shoot Ba leaf Ba seed

Varieties (V) 1 72.3* 6830.08** 15817.1** 1.68ns

Salts (S) 1 2598.6** 509.02** 6385.6** 5.33ns


V × S 1 544.8** 257.42** 4095.7** 81.89**V × L 5 1451.7** 728.82** 2355.1** 3.06ns

S × L 5 2358.5** 260.01** 1710.7** 4.49ns

V × S × L 5 2387.1** 240.46** 2782.8** 69.18**Error 48 12.2 8.02 15.2 5.84


Bi root Bi shoot Bi leaf Bi seedVarieties (V) 1 0.74ns 39.34** 289.52** 142.46**

Salts (S) 1 95.75** 49.17** 124.29** 0.61ns

83

Levels (L) 5 51.43** 5.01ns 56.37** 32.86**Interactions

V × S 1 22.77ns 0.01ns 2.19ns 144.27**V × L 5 18.97* 11.53** 46.63** 52.56**S × L 5 95.37** 3.06ns 44.06** 10.49ns

V × S × L 5 1.89ns 2.28ns 9.69ns 19.59ns

Error 48 5.85 2.36 5.98 8.57


Cd root Cd shoot Cd leaf Cd SeedVarieties (V) 1 0.96** 0.76** 0.006ns 0.0012ns

Salts (S) 1 0.23** 0.75** 0.023ns 0.03ns

Levels (L) 5 0.16** 0.81** 0.09** 0.04ns

InteractionsV × S 1 0.17* 0.88** 0.024ns 0.01ns

V × L 5 0.07* 0.81** 0.026* 0.07*S × L 5 0.017ns 0.64** 0.0056ns 0.04ns

V × S × L 5 0.018ns 0.75** 0.0053ns 0.05ns

Error 48 0.026 0.59 0.0097 0.027

Average higher level of Ba was found in plant organs of cultivar V1 (FH-385),

with only two exceptions in case of root where V1 (FH-415) gave higher

accumulation of Ba in T4 (80 mg As/kg soil) using arsenate and T3 (60 mg As/kg

soil) under arsenite in soil. Overall highest concentration of Ba was found in leaf of

plants treated with T3 of V1 grown under arsenate conditions, showing a little rise in

Ba from T0, T1 and T2 (control, 20 and 40 mg As/kg soil respectively) followed by a

sudden drop in case of T3 when arsenate was in soil. Root, shoot and leaves showed

higher accumulation of Ba while minimum contents of Ba were found in seeds.

Maximum concentrations of Bi were detected in case of sunflower seeds or

achenes as compared to all other organs (Figure 4.13(a)). In case of leaf Bi was

detected less than seeds but higher than root and shoot. Out of both cultivars V1 (FH-

385) showed relatively higher contents of Bi in leaf, shoot and to some extent in root

as maximum Bi concentration in root was observed in T4 (80 mg As/kg soil) only. In

leaf higher levels of Bi were detected in V1 too showing a sudden rise in T3 (60 mg

As/kg soil) using arsenate while least concentration of Bi in leaf was found in V2

(FH-415). In case of leaf V1 showed rise in Bi contents with increasing level of As in

soil especially in the form of sodium arsenite while after T2 (40 mg As/kg soil) a

gradual decrease in Bi contents was observed in sunflower leaves. Random ups and

84

downs were found in Bi contents in seeds too with much higher values recorded in V2

(FH-415) under arsenate in soil.

Relatively low levels of Cd were recorded in all sunflower organs except an

extraordinary highest peak in shoot of V1 (FH-385) under T1 (20 mg As/kg soil)

having arsenate in soil (Figure 4.13(a)). In root, higher values of Cd concentrations

were found in V1 and in case of seeds higher values were found in V2 (FH-415) with

intermediate Cd contents in leaf either arsenate or arsenite was present in the soil. In

root least concentrations of Cd were found in V2 about in all treatments or levels of

arsenic used.

Figure 4.13(a): Barium (Ba), bismuth (Bi) and cadmium (Cd) contents in root, shoot, leaves and seeds of sunflower cultivars grown in arsenic contaminated soil.

Barium contents

Bismuth contents

Cadmium contents

85


leaves and seeds or achenes of sunflower cultivars grown in arsenic

contaminated soil

Data regarding cobalt concentrations in different organs of sunflower cultivars

was analyzed statistically and analysis of variance revealed that in case of cobalt

contents of root, varieties, salts, levels and their interactions showed significant

differences (P<0.01) except for variety into salts interactions which showed non-

significant (P>0.05) differences. In case of cobalt contents of shoot, only varieties

gave significant differences (P<0.05) while salts, levels and all interactions among

variety, salts and levels showed non-significant differences. Significant differences

(P<0.05) were also found for levels in case of cobalt in leaves while varieties, salts

and interactions among varieties, salts and levels differed non-significantly (Table

4.15(a)). Co contents of leaves were significantly different for varieties and levels and

interaction between variety into levels but non-significantly for salts and all remaining

interactions.

Two way analysis of variance regarding Cr concentration in various organs of

sunflower revealed that varieties, salts and levels gave significantly different (P<0.01)

Cr contents in root and shoot organs (Table 4.15(a)) while out of different interactions

variety into levels and salts into levels and overall interaction among varieties into

salts into levels showed significant differences while variety into salts showed non-

significant (P>0.05) differences in case of Cr contents in root. All three factors and all

interactions among V, S and L differed significantly in case of Cr contents of shoot.

In case of leaf contents of Cr, only salts showed significant differences (P<0.05) while

varieties, levels and all interactions showed non-significant differences. In case of

chromium in seeds non-significant differences were observed in all factors and

relevant interactions too.

Two way analysis of variance of the data regarding lithium in roots revealed

significant differences (P<0.01) in interactions between salts and levels while

varieties, salts, levels and all their other interactions showed non-significant (P>0.05)

differences. In case of Li contents of shoot all factors comprising of varieties of

sunflower, salts and levels of arsenic and all their interactions showed non-significant

differences (Table 4.15(a)). In leaf lithium contents were significantly different for

86

levels while non-significant differences were observed in salts and varieties, out of

different interactions only salt into levels differed significantly whereas other

interactions showed non-significant differences. Non-significant differences were

evident in case of Li contents in seed for varieties, salts and levels and all of their

interactions.

Table 4.15(a): ANOVA for cobalt (Co), chromium (Cr) and lithium (Li) contents in sunflower cultivars grown in arsenic contaminated soil.


Co root Co shoot Co leaf Co seedVarieties (V) 1 5.79** 0.24* 0.071ns 0.14*

Salts (S) 1 8.37** 0.21ns 0.07ns 0.01ns

Levels (L) 5 4.21** 0.01ns 0.16* 0.24**Interactions

V × S 1 0.09ns 0.08ns 0.04ns 0.02ns

V × L 5 1.01** 0.02ns 0.05ns 0.16**S × L 5 7.07** 0.05ns 0.061ns 0.005ns

V × S × L 5 0.65* 0.02ns 0.04ns 0.01ns

Error 48 0.23 0.03 0.05 0.03


Cr root Cr shoot Cr leaf Cr seedVarieties (V) 1 249.02** 34.61** 1.74ns 0.05ns

Salts (S) 1 142.69** 20.14** 22.75* 0.09ns

Levels (L) 5 65.50** 11.48** 1.89ns 0.44ns

InteractionsV × S 1 13.38ns 14.43* 0.97ns 0.46ns

V × L 5 119.19** 33.65** 3.53ns 0.21ns

S × L 5 140.71** 6.52* 6.37ns 0.19ns

V × S × L 5 83.35** 6.69* 3.65ns 0.26ns

Error 48 7.54 2.58 3.74 1.60


Li root Li shoot Li leaf Li seedVarieties (V) 1 27.90ns 3.17ns 0.73ns 0.032ns

Salts (S) 1 0.72ns 0.94ns 5.03ns 0.01ns

Levels (L) 5 7.82ns 1.71ns 10.33** 0.008ns

InteractionsV × S 1 8.23ns 0.03ns 1.46ns 0.01ns

V × L 5 18.13ns 5.12ns 2.25ns 0.004ns

S × L 5 51.12** 2.89ns 8.09** 0.014ns

V × S × L 5 9.64ns 2.12ns 4.94ns 0.014ns

Error 48 11.25 2.329 2.16 0.009

Cobalt contents were recorded maximum in sunflower roots especially in V2

(FH-415) in case of both arsenate and arsenite present in soil. An exceptionally

87

highest value was recorded in T4 (80 mg As/kg soil) in roots of both cultivars (Figure

4.14(a)). Shoot, leaves and seeds also accumulated Co but very less than roots and

especially in case of seeds in control plants higher cobalt level was found in V1 (FH-

385) under both salts but on average lowest Co contents were appeared in seeds

harvested from sunflower cultivars.

Maximum concentration of Cr was recorded in root organ belonging to V2

(FH-415) in T4 (80 mg As/kg soil) under arsenate and arsenite both salts with an

exceptionally highest value (Figure 4.14(a)). In root V2 showed highest contents of Cr

with relatively higher values. In shoot V1 (FH-385) gave high values in a few

treatments but then fallen as compared to V2. In leaf about similar contents of Cr

were found with seeds and were much less than root.

Roots of sunflower showed maximum contents of lithium either arsenate or

arsenite in soil with an extraordinary highest value in roots of V1 (FH-385) plants

under T4 (80 mg As/kg soil). Average higher values of Li in root were recorded in V2

(FH-415) with uneven rises and falls in values (Figure 4.14(a)). Lowest or negligible

Li contents were found in seeds and intermediate values for Li were found in shoot

and leaves of sunflower cultivars. Both of varieties and salts behaved in same way

and effected similarly against various levels of arsenate or arsenite.

Figure 4.14(a): Cobalt (Co), chromium (Cr) and lithium (Li) contents in root, shoot, leaves and seeds of sunflower cultivars grown in arsenic contaminated soil.

Cobalt contents

88

Chromium contents

Lithium contents

4.2.12 Nickel (Ni), lead (Pb) and antimony (Sb) contents found in root, shoot,

leaves and achenes of sunflower cultivars grown in arsenic contaminated

soil

Varieties, salts and levels of arsenic showed significant differences (P<0.01) in

case of Ni contents of root as revealed by analysis of variance (Table 4.16(a)). Out of

different interactions variety into level and salt into level showed significant

differences (P<0.05 and P<0.01 respectively) for Ni contents in root organ of

sunflower whereas variety into salt and variety into salt into level interaction gave

non-significant differences (P>0.05) in case of root Ni contents. Ni contents of shoot

and seed were non-significantly different in case of varieties, salts, levels and all of

their interactions. Varieties, levels and all their interactions gave significant

differences for Ni in leaf and out of all interactions only variety into salt showed non-

significant differences while all other factors along-with their interactions gave

significant different values.

Analysis of variance (ANOVA) of the data regarding Pb concentration

detected in sunflower grown in arsenic contaminated soil revealed that varieties and

89

levels showed significant differences (P<0.01) in case of all organs root, shoot, leaf

and seed (Table 4.16(a)). In case of salts except Pb in root which showed non-

significantly (P>0.05) different values all other organs shoot, leaf and seed gave

significantly different results. In seed and leaf all interactions except variety into salts

showed significant differences while in shoot all factors and their interactions showed

significant differences in values regarding Pb contents.

Analysis of variance of the data revealed that significant (P<0.01 and P<0.05)

differences were found in salts and levels when Sb in root organ were recorded,

varieties gave non-significant differences while all interactions among varieties, salts

and levels showed significant differences for Sb in root (Table 4.16(a)). Varieties and

levels gave significant differences while salts showed non-significant differences in

case of Sb in shoot and out of interactions, salt into level showed non-significant

differences whereas all other interactions showed significant differences. In leaf and

seed varieties, salts and levels showed significant differences while in leaf variety into

salt and in seed, variety into level showed non-significant differences and all other

interactions showed significant differences in Sb contents.

Table 4.16(a): ANOVA for nickel (Ni), lead (Pb) and antimony contents in sunflower cultivars grown in arsenic contaminated soil.


Ni root Ni shoot Ni leaf Ni seedVarieties (V) 1 36.68** 1.38ns 11.09** 0.55ns

Salts (S) 1 61.33** 0.03ns 3.27* 0.75ns

Levels (L) 5 25.66** 1.21ns 5.19** 0.31ns


V × L 5 14.43* 0.82ns 4.26** 0.18ns

S × L 5 38.83** 0.99ns 2.75** 0.35ns

V × S × L 5 4.68ns 0.80ns 7.74** 0.46ns

Error 48 4.34 0.56 0.58 0.345


Pb root Pb shoot Pb leaf Pb seedVarieties (V) 1 27.57** 42.16** 11.36** 17.98**

Salts (S) 1 1.45ns 9.31** 7.06* 5.36**Levels (L) 5 16.79** 16.07** 7.46** 3.82**


V × L 5 19.15** 10.14** 13.91** 1.21**S × L 5 65.55** 20.62** 8.43** 2.51**

V × S × L 5 14.63** 20.52** 8.24** 1.29**Error 48 2.83 0.88 1.46 0.34

90


Sb root Sb shoot Sb leaf Sb seedVarieties (V) 1 0.027ns 0.87** 0.94** 0.10**

Salts (S) 1 0.18* 0.007ns 1.23** 0.15**Levels (L) 5 0.36** 0.18** 1.71** 0.04**

InteractionsV × S 1 1.12** 0.39** 0.04ns 0.28**V × L 5 0.36** 0.17** 1.73** 0.01ns

S × L 5 0.258** 0.07ns 0.21* 0.03*V × S × L 5 0.32** 0.39** 0.54** 0.028*

Error 48 0.041 0.04 0.06 0.009

Roots of sunflower plants showed maximum Ni contents in them especially in

V2 (FH-415) whereas relatively lower values were found in shoot and in all other

organs V2 gave less values of Ni contents in them as compared to V1 (FH-385). Both

salts behaved similarly towards nickel accumulation in different plant organs. In case

of seeds both varieties behaved almost similarly and gave same values of Ni in seeds

for both salts of As used. In leaf too both salts effected similarly over Ni accumulation

showing a few uneven higher values as in T5 (100 mg As/kg soil) of arsenate salt

(Figure 4.15(a)). Shoot and seed showed minimum fluctuation and rises in Ni

concentrations.

In roots and leaves relatively higher concentration of Pb was found to be

accumulated irrespective of the salt of arsenic used. Both, cultivars or varieties of

sunflower also behaved in similar way against salts and levels of arsenic. Overall two

values of Pb contents were found highest in plants belonging to V1 (FH-385) one

from root and other from shoot and interestingly both peaks were observed in case of

arsenite (Figure 4.15(a)). In roots at first Pb concentrations was decreased with

increasing As in soil up-to T3 (60 mg As/kg soil) and then increased Pb contents were

observed, same was case in leaves where in case of V1 a gradual decrease in Pb

contents was recorded with increasing As level. Except a single peak in shoot,

average less accumulation of Pb was evident in shoot and seed organs of sunflower

cultivars grown in As contaminated soil.

Slightly uneven values were recorded about Sb in different organs of

sunflower plants showing two peaks in leaf of V1 (FH-385) one in case of arsenate

and other in case of arsenite. In root V2 (FH-415) showed maximum Sb accumulation

in T3 (60 mg As/kg soil) and then drop was observed in Sb contents. Seed showed

91

values with less fluctuations and relatively lower contents of Sb as compared to root,

shoot and leaf (Figure 4.15(a)). There was not any uniformity in the contents of Sb in

plant organs under As contaminated soil with an average high contents of Sb in case

of V1 plants while in seeds V2 gave higher values of Sb as compared to V1 under

arsenite, but in arsenate V2 showed less contents of Sb. In root and seed about same

contents of Sb were observed except a little variation in root of V1 when arsenate was

present in soil. Arsenite showed about similar effects in root, leaf and seeds.

Figure 4.15(a): Nickel (Ni), lead (Pb) and antimony (Sb) contents in root, shoot, leaves and achnenes of sunflower cultivars grown in arsenic contaminated soil.

Nickel contents

Lead contents

92

Antimony contents

4.2.13 Selenium (Se), strontium (Sr) and titanium (Ti) contents in root, shoot,

leaves and seeds of sunflower cultivars grown in arsenic contaminated soil

Significant differences (P<0.01) were observed in varieties and levels for the

contents of Se in root while salts gave non-significant differences (P>0.05), out of

different interactions significant differences were found except variety into salts

interaction for Se in root (Table 4.17(a)). In shoot Se contents were recorded and

analysis of variance revealed non-significant differences for varieties but significantly

different values for Se in root were recorded for salts, levels and all of their

interactions. In leaf, salts showed non-significant differences but varieties and levels

differed significantly and out of interactions variety into level and salt into level

showed non-significant differences while all other interactions differed significantly

for leaf Se contents, in case of seed Se contents, varieties, salts and levels all showed

non-significant differences but interactions differed significantly (P<0.01 and

P<0.05).

Varieties or cultivars showed significant (P<0.01) differences in Sr contents of

root and shoot but non-significant differences (P>0.05) in case of leaf and seed while

salts caused differences in Sr contents of leaf only and non-significant differences in

case of root, shoot and seed leaving behind. Levels of arsenic caused significant

differences in Sr contents of root and leaf only but shoot and seed were not affected

notably. Out of various interactions among varieties, salts and levels Sr contents of

leaf were differed significantly in all interactions (Table 4.17(a)). In case of Sr

contents of root all interactions showed significant differences except for variety into

93

salt whereas Sr level of seed and shoot were non-significantly differed for all

interactions among salts, varieties and levels.

Two way analysis of variance of the data revealed significant (P<0.01)

differences between varieties, salts and levels and all of their interactions in case of

titanium in root, shoot and leaves but only salts and interaction of variety into salt

showed non-significant differences (P>0.05) for titanium in seeds (Table 4.17(a)).

Table 4.17(a): ANOVA for selenium (Se), strontium (Sr) and titanium (Ti) contents in sunflower cultivars grown in arsenic contaminated soil.


Se root Se shoot Se leaf Se seedVarieties (V) 1 3.92** 0.001ns 5.82** 1.15ns

Salts (S) 1 0.12ns 10.05** 0.01ns 2.93ns

Levels (L) 5 0.31** 1.42** 2.38** 1.69ns

InteractionsV × S 1 0.12ns 0.91* 3.41** 7.93*V × L 5 0.29** 3.06** 0.77ns 4.29**S × L 5 0.24** 1.88** 0.61ns 3.46*

V × S × L 5 0.22** 0.51* 1.25* 3.41*Error 48 0.04 0.19 0.39 1.17


Sr root Sr shoot Sr leaf Sr seedVarieties (V) 1 1070.30** 765.77** 112.4ns 109.92ns

Salts (S) 1 16.57ns 193.42ns 12071** 10.58ns

Levels (L) 5 1724.99** 107.40ns 1504.2** 31.87ns

InteractionsV × S 1 49.00ns 146.18ns 7901.5** 2.63ns

V × L 5 796.08** 23.79ns 984** 25.72ns

S × L 5 919.76** 178.54ns 2702** 36.30ns

V × S × L 5 341.11** 141.08ns 1109.7** 28.57ns

Error 48 46.16 74.79 99.1 33.74


Ti root Ti shoot Ti leaf Ti seedVarieties (V) 1 19420.6** 370052** 355.73** 25.80*

Salts (S) 1 6887.3** 108486** 55.55* 5.36ns

Levels (L) 5 4868.9** 10435** 520.12** 80.93**Interactions

V × S 1 757.4** 104916** 1216.07** 2.17ns

V × L 5 2600.3** 13270** 864.05** 23.86**S × L 5 16863.6** 6358** 422.96** 78.66**

V × S × L 5 697.2** 6234** 198.15** 11.29*Error 48 14.3 13 10.61 3.905

There was a lot of variation in Se contents in different sunflower organs with

relatively lowest values in root and maximum in seeds and leaves too (Figure

94

4.16(a)). Some extraordinary high values of Se were found in seeds of V2 (FH-415)

with exception in arsenite in which V1 gave the maximum value of Se. Much

fluctuation was found in V2 plants while V1 gave smooth values in seed. Both salts of

arsenic behaved in same way in case of leaf and seed but in shoot arsenate treated

plants showed higher Se contents than arsenite. Contents of Se in both varieties

overlapped each other. In roots V2 showed least accumulation of Se as compared to

V1.

Strontium in leaf was recorded as maximum while in seeds was least

concentration of strontium found. In root and shoot about equal contents of Sr were

detected with a higher value by V2 (FH-415) in case of arsenate, while in shoot V1

(FH-385) gave higher Sr contents. In leaf highest Sr concentration was also by V2

when arsenate was in soil and lower most value was also given by V2 in case of

arsenite salt (Figure 4.16(a)). Seeds showed least values of Sr contents and both

varieties and salts behaved similarly even levels also not affected Sr contents in seed

or achene of sunflower.

Highest titanium (Ti) contents were found in shoot of sunflower belonging to

V1 (FH-385) under arsenate in soil (Figure 4.16(a)) whereas in all other plant organs

lower values of Ti were recorded as in roots, highest concentration of Ti was observed

in T4 (80 mg As/kg soil) under arsenate belonging to V2 (FH-415) but in case of

arsenite gradual decrease in Ti contents was recorded with increasing soil As level as

arsenite. In leaves and seeds very low contents of Ti were recorded in both salts either

arsenate or arsenite and by both of cultivars of sunflower. Hruby et al., (2002)

observed effects of different titanium concentrations on oat (Avena sativa L.) plants

and reported its beneficial as well as inhibitory effects on plant health status which are

in accordance with our findings during this experiment on sunflower.

95

Figure 4.16(a): Selenium (Se), strontium (Sr) and titanium (Ti) contents in root, shoot, leaves and seeds of sunflower cultivars grown in arsenic contaminated soil.

Selenium contents

Strontium contents

Titanium contents

4.2.14 Thallium (Tl) and vanadium (v) contents in root, shoot, leaves and seeds

of sunflower cultivars grown in arsenic contaminated soil

Statistical analysis of data revealed that varieties, salts and levels and all of

their interactions differed significantly in case of Tl contents of root and leaves

96

whereas only interaction among variety into salt into level showed non-significant

differences in case of thallium in shoot (Table 4.18(a)). Varieties differed

significantly (P<0.05) but salts showed non-significant differences in case of thallium

contents of seed. Levels of arsenic and interaction among variety into level, variety

into salt and overall interaction of variety into salt into level differed significantly in

case of thallium contents of seed.

Analysis of variance of the data revealed that varieties differed significantly in

case of vanadium contents in shoot only while contents of V in root, leaf and seeds

showed non-significant differences for varieties and salts also. Levels differed

significantly in case of vanadium contents of root and shoot (P<0.05) but non-

significant differences were found in case of V contents of leaf and seed (Table

4.18(a)). Out of different interactions, variety into salts interaction showed non-

significant differences for root, leaf and seed vanadium contents but significant

(P<0.01) differences were observed in vanadium contents of shoot, the remaining

interactions variety into level, salt into level and variety into salt into level interaction

showed significant differences only in case of vanadium contents of root but shoot,

leaf and seed showed non-significant differences.

Table 4.18(a): ANOVA for thallium (Tl) and vanadium (V) contents of sunflower cultivars grown in arsenic contaminated soil.


Tl root Tl shoot Tl leaf Tl seedVarieties (V) 1 18742.7** 96.18** 109.17** 21.342*

Salts (S) 1 3692.4** 125.35** 1320.98** 3.09ns


V × S 1 442.7** 151.43** 175.66** 1.65ns

V × L 5 2203.9** 94.63** 497.41** 33.21**S × L 5 15888.7** 19.54** 384.25** 85.75**

V × S × L 5 535.8** 16.39ns 228.79** 14.08**Error 48 18.6 7.38 8.91 3.84


V root V shoot V leaf V seedVarieties (V) 1 37.60ns 70.46** 0.01ns 3.77ns

Salts (S) 1 24.63ns 148.63** 0.28ns 1.82ns

Levels (L) 5 231.22** 25.43* 3.62ns 0.46ns


V × L 5 147.97** 94.56** 1.27ns 0.97ns

S × L 5 500.96** 17.64ns 5.08ns 0.91ns

V × S × L 5 326.67** 13.93ns 2.28ns 0.44ns

97

Error 48 11.33 7.43 2.68ns 1.13

Xiao et al., (2004) observed the accumulative potential of thallium

accompanied by arsenic and mercury and inferred that thallium is much more retained

in the soil but arsenic is more accumulated than thallium in plant organs which is in

accordance with our findings. Out of different plant organs, root showed maximum

accumulation of thallium especially in case of T4 (80 mg As/kg soil) belonging to

cultivar V2 (FH-415) whereas V1 (FH-385) showed a gradual increase in thallium

contents of root when arsenate was in soil (Figure 4.17(a)), similarly higher values of

thallium in root were also observed in V2 than V1. Shoot and seeds showed least

accumulation of Tl out of all organs of sunflower, leaves showed a bit higher Tl

accumulation than shoot and seeds especially in both inorganic arsenicals present in

soil.

Highest contents of vanadium were found in root when arsenate was in soil

and V1 (FH-385) gave the maximum vanadium contents in T5 (100 mg As/kg soil)

showing a gradual increase with increasing arsenate concentration in soil, while V2

(FH-415) showed maximum in T4 (80 mg As/kg soil), whereas in case of arsenite in

soil, V2 showed resembling values as compared to V1 in which T2 (40 mg As/kg soil)

gave highest value for vanadium contents in root (Figure 4.17(a)). In an experiment

on cucumber Tatar and coworkers found that vanadium (V), nickel (Ni) and lead (Pb)

along with different forms of iron (Fe) compounds effect the growth through

metabolites transport system and found the effectiveness of these elements

represented in a sequence as Ni > Pb > V (Tatar et al., 1999). In shoot less vanadium

was accumulated than root and higher values were recorded in V1, whereas in leaf

lower vanadium contents were observed and least in seeds either arsenate or arsenite

was in soil and both, cultivars or varieties behaved similarly giving very low

accumulation of vanadium in leaf and seed obtained from sunflower crop.

98

Figure 4.17(a): Thallium (Tl) and vanadium (V) contents of sunflower cultivars grown in As contaminated soil.

Thallium contents

Vanadium contents

4.2.15 Conclusion (Experiment 2)

Deleterious effects of higher arsenic concentrations in the form of both salts

arsenate as well as arsenite were evident on growth and yield parameters of both

sunflower cultivars especially in parameters collected at vegetative or pre-anthesis

stage. Root and shoot length and number of leaves as well as fresh and dry weight and

water contents of root and shoot were decreased with relative increase in arsenate and

arsenite concentrations or levels in soil. Parameters recorded at reproductive stage

revealed less conspicuous deterrent effects with a little reduction in yield. Although

maximum quantity of arsenic was retained in soil as left over arsenic but

comparatively highest accumulation of arsenic was recorded in roots of both cultivars

which proved effective sink as proved by bioaccumulative coefficient determination,

according to which order of arsenic accumulation was as left over arsenic in soil > in

99

root > in leaves > in shoot > in seeds or achenes. In contrast to arsenic highest

phosphorus (P) contents were found in seeds. Behavior of both cultivars of sunflower

remained similar towards accumulation of different metallic ions from soil as calcium

(Ca), magnesium (Mg) and boron (B) were found highest in leaves, copper (Cu) and

zinc (Zn) were also found highest in seeds but iron (Fe), nickel (Ni), aluminum (Al),

cobalt (Co), chromium (Cr), lithium (Li), lead (Pb), thallium (Tl) and vanadium (V)

were proved to be much accumulated in roots of sunflower cultivars. V1 (FH-331)

accumulated more silver (Ag) in seeds while strontium (Sr) was recorded highest in

leaves. Rations of arsenic concentrations [As] between different sunflower organs

were recorded and found highest in roots [As] and then in leaves [As] while least [As]

in shoot and seeds.

100

4.3 RESULTS AND DISCUSSION EXPERIMENT NO. 3

Responses of sunflower (Helianthus annuus L.) to different levels of inorganic arsenicals applied through irrigation water.


4.3.2 Agronomic parameters

Data were statistically analyzed considering three factors, two sunflower

varieties (V), two salts of arsenic (S) and six (0, 2, 4, 6, 8 and 10 mg/L water)

different levels of arsenic (L) applied through irrigation water used during this

greenhouse experiment. In contrast to arsenic contamination in soil, contaminated

water application showed non-significant (P>0.05) differences for varieties in case of

shoot, root length and shoot:root ratio, revealing that both varieties behaved similarly

and gave resembling values for shoot and root length against various arsenic

treatments, with a slight increase than control in lower arsenic levels but reduction

was recorded in both sunflower cultivars when higher level of arsenic was applied

through irrigation water. Salts caused significant differences (P<0.05) for shoot and

root length and (P<0.01) for shoot:root ratio, similarly levels also showed significant

differences except for shoot:root ratio as derived from analysis of variance (ANOVA)

of the data, showing that different levels of arsenic affected differently the plants and

showed reduction in shoot and root length of sunflower plants with an increase in

concentration of arsenic in irrigation water. Similarly reduction in root and shoot

growth was found by Yu et al., (2009), in an experiment on maize. Interaction of

varieties with salts (V × S) and levels (V × L) showed significant differences for shoot

length but non-significant for root length and shoot:root ratio (Table 4.1(b)). Salts into

level and overall interaction V × S × L showed non-significant (P>0.05) differences

for shoot and root length as well as shoot:root ratio depicting that up to 10 mg

arsenic/liter irrigation water has no significant deterrent effects on these growth

attributes of sunflower cultivars.

Table 4.1(b): Analysis of variance (ANOVA) of data for shoot length, root length and shoot:root ratio at vegetative stage under various As levels applied through irrigation water.


Shoot Length (cm)

Root Length (cm)

Shoot : Root ratio

101

Varieties (V) 1 6.42ns 1.13ns 0.03ns

Salts (S) 1 26.28* 15.12* 0.88**Levels (L) 5 330.66** 62.36** 0.036ns

InteractionsV × S 1 32.67* 6.12ns 0.064ns

V × L 5 22.15** 4.63ns 0.026ns

S × L 5 6.21ns 2.16ns 0.089ns

V × S × L 5 13.97ns 5.83ns 0.081ns

Error 48 6.31 3.09 0.052

Figure: 4.1(b). Shoot length, root length and shoot : root ratio of two sunflower cultivars irrigated through different arsenic levels.

Sunflower cultivars differed significantly in case of dry weight of shoot and

root but non-significant differences (P>0.05) were found in fresh weight and water

contents of shoot as well as root under different treatments of arsenic applied through

102

irrigation water. Salts also gave significant differences in case of fresh weight of shoot

and root and water contents of shoot and root but non-significantly for dry weight of

shoot, while levels differed significantly (P<0.01) for all parameters including fresh

and dry weights (g) as well as water contents of shoot and root (Table 4.2, 4.3(b)). In

case of fresh weight and water contents of shoot, all interactions among varieties, salts

and levels showed non-significant differences, while only varieties into salts

interaction showed significant differences when dry weight of shoot data was

statistically analyzed. V × L interaction data showed significant differences for fresh,

dry weight and water contents of root but non-significant in case of shoot. Salts into

levels interaction data differed significantly only in case of fresh weight and water

contents of root whereas overall interaction V × S × L showed non-significant

differences for all these six parameters.

Table 4.2(b): Analysis of variance (ANOVA) of data about fresh weight, dry weight and water contents of shoot and root at vegetative stage under various As levels applied through irrigation water.


Fresh wt. shoot (g)

Dry wt. shoot (g)

Water contents

shoot

Fresh wt. root

(g)

Dry wt. root (g)

Water contents

rootVarieties (V) 1 2.21ns 0.10* 1.36ns 0.015ns 0.013** 0.0009ns

Salts (S) 1 5.89* 0.03ns 5.98* 0.88** 0.051* 0.75**Levels (L) 5 57.16** 2.64** 36.45** 8.58** 0.077** 7.12**

InteractionsV × S 1 2.72ns 0.15** 4.17ns 0.01ns 0.001ns 0.018ns

V × L 5 0.57ns 0.01ns 0.74ns 0.09* 0.002* 0.069**S × L 5 0.96ns 0.004ns 0.92ns 0.12** 0.005ns 0.13**


Error 48 1.27 0.02 1.21 0.027 0.007 0.019

Figure 4.2(b): Fresh weight, dry weight of shoot and shoot water contents of two sunflower cultivars under different arsenic levels in irrigation water.

103

Table 4.3(b): Mean±SE for fresh, dry weight and water contents of root under various conditions of arsenic in irrigation water.

Fresh weight of root (Mean±SE) when arsenic in irrigation waterLevel Variety Mean

Hybrid 1 Hybrid 2T0 2.85 ± 0.05a 3.03 ± 0.11a 2.94 ± 0.06AT1 1.70 ± 0.09c 1.92 ± 0.07b 1.81 ± 0.06BT2 1.28 ± 0.14de 1.42 ± 0.12d 1.35 ± 0.09CT3 1.10 ± 0.14ef 0.93 ± 0.06fg 1.02 ± 0.08DT4 0.91 ± 0.07fg 0.74 ± 0.07gh 0.83 ± 0.06ET5 0.67 ± 0.03h 0.64 ± 0.06h 0.65 ± 0.03F

Dry weight of root (As in water)Level Variety Mean

Hybrid 1 Hybrid 2T0 0.257 ± 0.012a 0.282 ± 0.008a 0.269 ± 0.008AT1 0.188 ± 0.010b 0.255 ± 0.021a 0.222 ± 0.015BT2 0.150 ± 0.007c 0.208 ± 0.017b 0.179 ± 0.012CT3 0.107 ± 0.010de 0.113 ± 0.008d 0.110 ± 0.006DT4 0.080 ± 0.006ef 0.085 ± 0.008def 0.083 ± 0.005ET5 0.070 ± 0.006f 0.073 ± 0.013f 0.072 ± 0.007E

Water contents of root (As in water)Level Variety Mean

Hybrid 1 Hybrid 2T0 2.59 ± 0.06a 2.75 ± 0.06a 2.67 ± 0.05AT1 1.51 ± 0.08b 1.66 ± 0.05b 1.59 ± 0.05BT2 1.13 ± 0.14cd 1.21 ± 0.12c 1.17 ± 0.09CT3 0.99 ± 0.13de 0.82 ± 0.05fg 0.91 ± 0.07DT4 0.83 ± 0.07ef 0.66 ± 0.07gh 0.74 ± 0.05ET5 0.60 ± 0.03h 0.56 ± 0.05h 0.58 ± 0.03F

104

4.3.3 Physiological and water relation parameters

Varieties and salts showed non-significant differences while levels differed

significantly with similar higher number of leaves in control (T0) plants of both

varieties and resembling values for both salts while only higher concentrations of

arsenic caused prominent reduction in number of leaves in both sunflower cultivars,

whereas all interactions of varieties, salts and levels differed non-significantly. For

fresh weight of leaf, varieties, salts and levels showed significant (P<0.01) differences

giving higher fresh weight and dry weight in H1 plants and out of different

interactions only V × S showed non-significant differences in fresh weight and dry

weight of leaf while all other interactions variety into level and salts into level and

overall variety into salts into levels interaction gave significant differences (Table

4.3(b)). ANOVA regarding dry weight of leaf showed significant differences for

varieties and levels and their interaction too but non-significant (P>0.05) for salts. All

factors including varieties, salts and levels and their interactions showed significant

differences as a result of analysis of data regarding turgid weight and specific weight

of leaf except for S × L and overall interaction which showed non-significant

differences in case of specific weight of leaf. Different levels of arsenic caused

significant differences in all these parameters of both sunflower cultivars.

Table 4.3(b): Analysis of variance (ANOVA) of data for number of leaves, fresh, dry, turgid and specific weight of sunflower leaf at vegetative stage under various As levels applied through irrigation water.


No. of leaves

Fresh wt. leaf (g)

Dry wt. leaf (g)

Leaf turgid wt.

Sp. wt. leaf

Varieties (V) 1 0.50ns 4.03** 0.32** 5.69** 0.0017**Salts (S) 1 2.72ns 0.89** 0.01ns 3.56** 0.0010*

Levels (L) 5 12.95** 1.22** 0.09** 2.51** 0.0004*Interactions

V × S 1 0.22ns 0.15ns 0.002ns 0.80* 0.0012**V × L 5 0.16ns 2.38** 0.06** 3.72** 0.0005*S × L 5 0.58ns 0.65** 0.02* 1.33** 0.0002ns

V × S × L 5 0.63ns 0.87** 0.05** 1.15** 0.0002ns

Error 48 1.82 0.09 0.008 0.16 0.00016

105

Figure 4.3(b): Number of leaves, fresh weight, turgid weight, dry weight and specific weight of leaves in two sunflower cultivars under different levels of arsenic in irrigation water.

Number of leaves (As in water)

Interaction (mean±SE) for fresh wt. of leaf.Level Variety Mean

Hybrid 1 Hybrid 2T0 1.73 ± 0.11ef 2.31 ± 0.08cd 2.02 ± 0.11BT1 1.97 ± 0.25de 1.96 ± 0.08ef 1.96 ± 0.13BT2 2.87 ± 0.30b 1.95 ± 0.14ef 2.41 ± 0.21AT3 3.51 ± 0.12a 1.64 ± 0.17ef 2.57 ± 0.30AT4 2.45 ± 0.39c 1.68 ± 0.11ef 2.07 ± 0.23BT5 1.61 ± 0.11f 1.78 ± 0.11ef 1.69 ± 0.08C

Dry wt. of leaf (in water).

Interaction (mean±SE) for turgid wt. of leaf.S Level Variety Mean

Hybrid 1 Hybrid 2S1 T0 2.35 ± 0.18hi 3.26 ± 0.18def 2.81 ± 0.23D

T1 2.22 ± 0.20i 3.20 ± 0.31d-g 2.71 ± 0.27DT2 4.85 ± 0.41ab 2.79 ± 0.12e-i 3.82 ± 0.50ABCT3 4.49 ± 0.25ab 2.24 ± 0.10i 3.37 ± 0.52CT4 2.34 ± 0.15hi 2.37 ± 0.05hi 2.36 ± 0.07DT5 2.45 ± 0.13hi 2.74 ± 0.14f-i 2.59 ± 0.11D

S2 T0 2.35 ± 0.18hi 3.26 ± 0.15def 2.81 ± 0.23DT1 4.31 ± 0.23bc 3.40 ± 0.18de 3.86 ± 0.24ABT2 3.80 ± 0.15cd 2.99 ± 0.08e-h 3.39 ± 0.20BCT3 5.11 ± 0.48a 3.17 ± 0.40d-g 4.14 ± 0.51A

106

T4 4.42 ± 0.36bc 2.59 ± 0.23ghi 3.51 ± 0.45BCT5 2.66 ± 0.22f-i 2.58 ± 0.10ghi 2.62 ± 0.11D

Specific leaf weight (As in water).

Data about leaf area showed significant differences for sunflower varieties,

arsenic salts and levels and all their interactions. Overall higher leaf area value with

maximum (72.77 ± 6.10) was found in H1 when sodium arsenate was used while

minimum (56.70 ± 2.96) in case of H2 under same salt (Table 4.4(b)). Varieties and

levels and their interaction V × L showed significant differences in case of leaf

succulence and relative water contents of leaf as compared to salts and interaction

between salts into level which differed non-significantly for both these parameters

(Table 4.4(b)). Both salts of arsenic behaved similarly in sunflower cultivars while

levels or concentrations of arsenic caused significant differences in leaf succulence.

Overall interaction V × S × L differed significantly (P<0.01) in case of leaf

succulence and leaf area but non-significantly for relative water contents of leaf.

Table 4.4(b): Analysis of variance (ANOVA) of data for leaf succulence, leaf area and relative water contents of sunflower leaf at vegetative stage under various As levels applied through irrigation water.


Leaf succulence Leaf area Relative water contents of leaf

Varieties (V) 1 3.89** 1656.00** 394.15**Salts (S) 1 0.75ns 192.93** 76.84ns


V × S 1 0.26ns 754.14** 41.71ns

V × L 5 3.34** 1314.60** 116.04*S × L 5 0.69ns 182.65** 34.15ns

V × S × L 5 1.79** 631.09** 61.86ns

Error 48 0.30 13.32 47.78

Variety × Salt interaction (mean±SE) for leaf area under arsenic contaminated water.

S Variety Mean

107

Hybrid 1 Hybrid 2S1 72.77 ± 6.10a 56.70 ± 2.96d 64.74 ± 3.61AS2 63.02 ± 2.27b 59.90 ± 3.13c 61.46 ± 1.92BMean 67.89 ± 3.31A 58.30 ± 2.14B

Variety × Salt × Level interaction (mean±SE) for leaf succulence under As contaminated water.

S Level Variety MeanHybrid 1 Hybrid 2

S1 T0 3.94 ± 0.25l 6.55 ± 0.41a-d 5.25 ± 0.62T1 4.35 ± 0.21jkl 4.58 ± 0.43i-l 4.47 ± 0.22T2 4.23 ± 0.27kl 5.22 ± 0.27g-j 4.72 ± 0.28T3 6.10 ± 0.28c-g 5.45 ± 0.31e-i 5.78 ± 0.24T4 6.13 ± 0.39c-f 6.29 ± 0.25b-e 6.21 ± 0.21T5 6.57 ± 0.45abc 5.30 ± 0.44f-i 5.94 ± 0.40

S2 T0 3.96 ± 0.24l 6.57 ± 0.23abc 5.26 ± 0.60T1 5.07 ± 0.17h-k 4.63 ± 0.20i-l 4.85 ± 0.15T2 5.13 ± 0.33h-k 4.61 ± 0.29i-l 4.87 ± 0.23T3 5.07 ± 0.18h-k 5.65 ± 0.39d-h 5.36 ± 0.23T4 7.36 ± 0.55a 7.11 ± 0.21ab 7.24 ± 0.27T5 5.23 ± 0.32f-j 6.78 ± 0.22abc 6.00 ± 0.39

Variety × Level interaction (mean±SE) for RWC of leaf under As contaminated water.

Level Variety MeanHybrid 1 Hybrid 2

T0 68.97 ± 3.12a 65.33 ± 2.63ab 67.15 ± 2.02AT1 56.80 ± 3.21cd 51.92 ± 3.04d 54.36 ± 2.23DT2 60.32 ± 2.56bc 60.02 ± 3.63bc 60.17 ± 2.12BCT3 69.35 ± 2.60a 53.90 ± 2.45cd 61.63 ± 2.88ABCT4 68.37 ± 2.25a 61.91 ± 1.74abc 65.14 ± 1.67ABT5 56.28 ± 3.58cd 58.94 ± 2.61bcd 57.61 ± 2.15CD


The data about plants cultivated in normal or contamination free soil but were

irrigated (five times) through As contaminated water were analyzed statistically and

the results were inferred regarding all agronomic, physiological, yield and chemical

parameters observed at maturity or final harvesting stage of sunflower cultivars.

4.3.5 Morphological and yield parameters

Shoot or stem length differed significantly (P<0.01) in case of levels of arsenic

applied while varieties and salts showed non-significant differences (P>0.05). Root

length gave significant differences (P<0.05) in case of varieties and (P<0.01) in case

of levels of As but non-significantly differed for salts. No. of leaves, capitulum

108

diameter and weight of 100 achenes all differed significantly only in case of different

levels of As not for varieties and salts. Interactions among all these factors (varieties,

salts and levels) showed non-significant differences for about all parameters including

stem length, root length, stem:root ratio, number of leaves, capitulum diameter and

100 achene weight as tabulated under in Table 4.5 (b).

Table 4.5(b): ANOVA for stem length, root length, stem to root ratio, number of leaves, capitulum diameter and hundred achene weight recorded in sunflower cultivars irrigated through As contaminated water.


Stem length

Root length

Stem : root

No. of leaves

Cap. Dia.

100 achene

wt.Varieties (V) 1 235.04ns 38.43* 2.17ns 0.36 ns 0.54 ns 0.01ns

Salts (S) 1 165.95ns 22.86ns 3.54ns 1.87 ns 0.02ns 0.11ns


V × S 1 566.23ns 15.49ns 0.0005ns 10.22ns 0.46ns 0.70*V × L 5 199.48ns 5.28ns 0.99ns 2.71ns 5.23** 0.13ns

S × L 5 53.15ns 8.09ns 1.06ns 3.83 ns 0.16 ns 0.07ns


Error 48 300.08 8.88 1.31 2.84 1.08 0.126

In comparison with arsenic treatment in soil, irrigation water treatments

showed non-significant difference which is evident in graphs (Figure 4.4(b)). A slight

reduction from control was observed in shoot and root length, number of leaves, and

yield parameters. Mostly maximum values were found in control plants while with

increasing concentration of arsenate or arsenite in irrigation water only, a bit deterrent

effects were found with least values in higher concentrations (8 and 10 mg As/L

water). Both varieties or cultivars of sunflower behaved in similar way and both salts

of arsenic used also showed similar effects on sunflower plants, only levels of As

were effective in causing reduction or a little bit retarded growth.

Figure 4.4 (b): Graphs showing different parameters of sunflower grown under As contaminated irrigation water.

0

50

100

150

200

250

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

St L

2 (w

ater

) Hybrid 1Hybrid 2

109

0

5

10

15

20

25

30

35

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Rt L

2 (w

ater

) Hybrid 1Hybrid 2

0123456789

10

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Sht:R

t 2 (w

ater

) Hybrid 1Hybrid 2

0

5

10

15

20

25

30

35

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

No.L

2 (w

ater

) Hybrid 1Hybrid 2

0

5

10

15

20

25

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Cap.

Dia

(wat

er) Hybrid 1

Hybrid 2

110

00.5

11.5

22.5

33.5

44.5

5

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Wt.

100

(wat

er) Hybrid 1

Hybrid 2

4.3.6 Arsenic (As) contents accumulated in different sunflower organs and left

over arsenic, applied through irrigation water.

Significant differences (P<0.01) were found in varieties or cultivars of

sunflower for As contents of root, leaf and seeds but in case of As content of shoot,

varieties showed non-significant differences (Table 4.6 (b)). Salts also showed

significant (P<0.05) differences in root As contents, non-significant (P>0.05) in case

of shoot and highly significant (P<0.01) differences were found in leaf and seed As

contents. Levels of arsenic caused significant differences in case of all organs root,

shoot, leaf and seed of sunflower cultivars. Out of different interactions, variety into

salt and salt into level showed non -significant differences in root As contents but

variety into level and overall variety into salt into level interaction gave significant

differences in root As contents. Variety into level and salt into level interaction

showed significant differences in case of As in shoot but variety into salt and overall

interaction gave non-significant differences. As contents in leaf were different in case

of interaction between variety into level and salt into level but non-significant

differences were found for variety into salt and variety into salt into level. In seed, As

contents were different in case of interaction variety into level and salt into level but

non-significant differences were evident for variety into salt and variety into salt into

level interaction. Left over arsenic was significantly different in case of varieties and

levels but non-significant differences were found for salts and out of interactions

variety into salt and variety into level showed significant differences but salt into level

and variety into salt into level showed non-significant differences.

Table 4.6 (b): ANOVA for arsenic (As) accumulated in sunflower, applied through irrigation water.


As root As shoot As leaf As seed Left over AsVarieties (V) 1 6.43** 0.79ns 19.67** 12.73** 42.14**

Salts (S) 1 3.87* 0.43ns 25.92** 1.77** 9.03ns

111

Levels (L) 5 21.02** 0.42** 18.48** 3.90** 2638.70**Interactions

V × S 1 0.02ns 0.11ns 4.01ns 0.12ns 62.91**V × L 5 14.56** 2.97** 5.07* 1.59** 19.16**S × L 5 0.55ns 4.39** 11.29** 0.27* 10.98ns

V × S × L 5 3.36** 0.55ns 3.06ns 0.14ns 8.20ns

Error 48 0.65 0.24 1.80 0.11 4.72

In root and leaf a little higher concentration of As was found as compared to

shoot and seed with a gradual increase in levels of arsenic in irrigation water. Both

cultivars (varieties) behaved in a similar way towards different As salts and levels of

arsenic applied through irrigation water. Maximum contents of As were detected as

left over arsenic showing a gradual increase with increasing level of As in irrigation

water as a sharp elevation in contents of As is evident in Figure 4.5(b) for both

varieties of sunflower.

Figure 4.5(b): Arsenic (As) accumulation in sunflower cultivars irrigated through As contaminated water.

4.3.7 Arsenic bioaccumulation coefficient (BC) of sunflower cultivars

Varieties showed significant differences in case of BC of root (P<0.05) and

seed (P<0.01) but non-significant differences (P>0.05) were observed in

bioaccumulation coefficient of shoot and leaf (Table 4.7(b)) as revealed by analysis of

variance of the data. Salts revealed significant differences for BC of seed only while

bioaccumulation coefficient of root, shoot and leaf revealed non-significant

differences. Levels of arsenic showed significant differences for BC root, shoot and

seed but non-significantly for BC leaf. Variety into salt interaction showed non-

significant differences for BC root, shoot, leaf as well as seed, whereas variety into

level interaction showed significant differences for BC root, shoot, leaf (P<0.05) and

seed but salt into level interaction showed significant differences for BC shoot and

112

leaf while non-significant differences for BC root and seed. Overall interaction among

variety into salt into level showed non-significant differences for BC of all sunflower

organs including root, shoot, leaf and seed.

Table 4.7(b): ANOVA for As bioaccumulation coefficient (BC) of sunflower irrigated through As contaminated water.

Source D F

Mean squareBC root BC shoot BC leaf BC seed

Varieties (V) 1 0.021* 0.0005ns 0.0036ns 0.0086**Salts (S) 1 0.008ns 0.0002ns 0.0082ns 0.0017**

Levels (L) 5 0.093** 0.0034** 0.0041ns 0.0011**Interactions

V × S 1 0.006ns 0.0001ns 0.0017ns 0.0004ns

V × L 5 0.028** 0.0024** 0.0077* 0.0013**S × L 5 0.001ns 0.0025** 0.012** 0.0002ns

V × S × L 5 0.006ns 0.0004ns 0.0035ns 0.0002ns

Error 48 0.004 0.0003 0.0023 0.0001

Highest bioaccumulation coefficient value was observed in case of roots of

sunflower cultivars under various levels of both salts of arsenic in irrigation water. V2

showed overall higher values but occasionally V1 also showed higher

bioaccumulation coefficient value (Figure 4.6(b)). Seeds showed lowest value for

arsenic bioaccumulation coefficient with higher values for V1 (FH-385) in case of

both salts and V2 showed least values especially in case of arsenite in irrigation water.

In leaf arsenic bioaccumulation coefficient values were higher than shoot and seeds

but to some extent lower than leaf and root values with higher in case of V2 and lower

values for V1. Shoot showed a gradual increase in BC value with increase in arsenic

level but a sharp decrease was clear in case of highest As concentration or T5 (10

mg/L As in irrigation water) when As was as arsenate and in both varieties.

113

Figure 4.6(b): Arsenic bioaccumulation coefficient (BC) for sunflower cultivars irrigated through As laden water.

4.3.8 Arsenic concentration [As] ratios among various organs of sunflower

cultivars irrigated through As contaminated water.

The data regarding arsenic concentrations in different plant organs including

root , shoot leaves and seeds was analyzed statistically and two way analysis of

variance revealed that in case of [As] root to shoot, varieties and levels showed

significant (P,0.01) differences but salts showed non-significant differences (P>0.05).

Out of different interactions only variety into level interaction showed significant

differences but all other interactions including variety into salts, salts into level and

variety into salt into level showed non-significant differences (Table 4.8(b)). ANOVA

of the data regarding ratio between [As] root and leaf showed that only interaction

between salt into level was significantly (P<0.05) different but varieties, salts and

levels along with all other interactions showed non- significant differences. For [As]

root and seed ratio levels and interaction between varieties into level showed

significant differences while all other interactions, salt and level showed non-

significant differences. In case of [As] shoot : leaf ratio levels showed significant

differences and salt into level interaction also while other remaining factors and

interactions all differed non-significantly while for [As] shoot to seed ratio varieties

showed significant differences but levels and seeds non-significantly, variety into

levels and salt into level and variety into salt into level interaction differed

significantly but salt and levels and variety into salt interaction revealed non-

significant differences. [As] leaf and seed showed that variety and salt and levels gave

significant differences while out of interactions variety into level and salt into level

114

differed significantly (P<0.05) while non-significant differences were shown by

variety into salt and variety into salt into level interaction.

Table 4.8(b): ANOVA for arsenic concentrations [As] ratios among different organs of sunflower irrigated through As contaminated water.


[As]root:[As]shoot [As]root:[As]leaf [As]root:[As]seedVarieties (V) 1 708.51** 11.83ns 37.80ns

Salts (S) 1 88.05ns 13.47ns 46.87ns

Levels (L) 5 1555.76** 13.25ns 2917.14**Interactions

V × S 1 5.83ns 1.28ns 3.83ns

V × L 5 810.33** 14.52ns 1542.01**S × L 5 42.22ns 20.45* 57.86ns

V × S × L 5 19.57ns 14.56ns 54.66ns

Error 48 32.56 6.35 83.07


[As]shoot:[As]leaf [As]shoot:[As]seed [As]leaf:[As]seedVarieties (V) 1 0.005ns 322.37** 323.00**

Salts (S) 1 1.24ns 82.97ns 251.03**Levels (L) 5 2.71* 50.64ns 201.89**

InteractionsV × S 1 0.84ns 42.40ns 82.35ns

V × L 5 1.05ns 64.17* 60.88*S × L 5 5.19** 101.11** 74.56*

V × S × L 5 1.01ns 91.73* 51.15ns

Error 48 0.81 26.59 23.47

The ratio between [As] root and seed, and leaf and seed were highest when

comparison was drawn in graph (Figure 4.7(b)) although [As] root to shoot also

showed some higher values in V2 (FH-415) mostly when arsenate was in irrigation

water. A drop was evident with increase in arsenate level. Overall V2 gave higher

values for different concentration ratios of arsenic in plant organs of sunflower

cultivars which were periodically irrigated through arsenic contaminated water.

Minimum values were seen in ratio between [As] shoot and leaf in which both,

cultivars or varieties behaved similarly and overlapping values of arsenic

concentrations were found. Extraordinary higher values were found in ratio between

[As] root and seed and root and shoot given by V1 (FH-385).

115

Figure 4.7(b): Ratios of arsenic concentrations [As] among different organs of sunflower irrigated through As contaminated water.



contaminated water.

Two way analysis of variance of the data revealed significant (P<0.01)

differences in varieties and levels in case of P contents of shoot and root (Table

4.9(b)), while non-significant differences (P>0.05) were observed in salts for the

parameters P in root and shoot and the interaction between variety into salt and

variety into level for P in root while remaining interactions in case of P root gave

significantly different values. Similarly variety into salt interaction gave non-

significant differences for salts in case of P leaf and seed and also for varieties in P

leaf while out of different interactions variety into salt and salt into level in seed P and

variety into level and overall variety into salt into level showed non-significant

differences whereas variety into salt and salt into level in case of P leaf and variety

into level and variety into salt into level for P seed showed significant differences.

Significant differences (P<0.01) were shown by varieties, salts and levels

including all of their interactions in case of calcium contents in root, shoot and leaf of

sunflower cultivars (Table 4.9(b)). In case of calcium concentration in seed varieties,

salts and levels and interactions variety into salt and level, salt into level and variety

into salt into level showed non-significant differences (P>0.05).

116

Varieties, salts and levels and interaction among them all showed significant

differences in case of Mg contents in root and shoot (Table 4.9(b)). In leaf Mg

contents showed significant differences for varieties, salt and levels and only variety

into salt interaction showed non-significant differences while all other interactions

differed significantly (P<0.01). For magnesium in seed only salts showed significant

differences while varieties, levels and all interactions among varieties, salts and levels

showed non-significant differences (P>0.05).

Table 4.9(b): ANOVA for phosphorus (P), calcium (Ca) and magnesium (Mg) contents in sunflower cultivars irrigated through arsenic contaminated water.


P root P shoot P leaf P seedVarieties (V) 1 231.66** 172.76** 1.33ns 372.10*

Salts (S) 1 2.67ns 19.19ns 45.51ns 17.90ns


V × S 1 2.19ns 17.73ns 386.42** 180.25ns

V × L 5 9.50ns 130.57** 15.23ns 232.95**S × L 5 64.46** 46.54** 105.74** 31.58ns

V × S × L 5 43.50* 7.74ns 26.87ns 743.17**Error 48 12.86 12.09 12.34 52.72


Ca root Ca shoot Ca leaf Ca seedVarieties (V) 1 54467259** 92680414** 486492839** 6280ns

Salts (S) 1 12887829** 7076429** 141011353** 2035ns

Levels (L) 5 4780072** 3313367** 32965093** 15759ns

InteractionsV × S 1 5498470** 2390621** 160325281** 11012ns

V × L 5 6244492** 1524633** 229132491** 8413ns

S × L 5 1178423** 5409520** 89139603** 26271ns

V × S × L 5 2848326** 2338771** 75657728** 17899ns

Error 48 64044 93513 8939860 41739


Mg root Mg shoot Mg leaf Mg seedVarieties (V) 1 5415609** 10808491** 5659594** 2316ns

Salts (S) 1 740149** 2727218** 8646797** 399928**Levels (L) 5 1061101** 1607572** 2019890** 35756ns

InteractionsV × S 1 724148** 368105* 45170ns 112ns

V × L 5 1027799** 1655565** 8518584** 77258ns

S × L 5 486526** 1950939** 3689732** 29160ns

V × S × L 5 550975** 1039495** 501966** 25603ns

Error 48 54073 66697 43178 39627

117

Highest P contents were found in seeds of both cultivars and in case of both

inorganic arsenicals with less contents of P in leaf than seeds and root and shoot gave

resembling values for P contents (Figure 4.8(b)). In root and shoot V2 (FH-415)

showed higher values but overlapping with values obtained in case of V1 (FH-385)

under both salts of arsenic either arsenate or arsenite. No notable variations were

observed in case of shoot especially.

Leaves showed maximum calcium contents especially in case of arsenite and

cultivar V1 (FH-385) gave highest values (Figure 4.8(b)) of calcium ion

concentration. Root and shoot showed less accumulation or contents of Ca in them

and least was in seed. V1 showed overall higher calcium contents in all organs under

both inorganic salts of arsenic either arsenate or arsenite used in irrigation water. A

rise in calcium ion concentration was seen with increasing arsenite concentration but

up to T4 (8 mg As/L solution), after this level a sudden drop was seen in calcium

contents of V1 in case of sodium arsenite.

Highest level of magnesium was found in leaves under arsenite contaminated

water showing that firstly Mg contents were increased but at maximum As level (10

mg As/L solution) sharp drop was obvious in V1 (FH-385) but opposite situation was

recorded in V2 in which first Mg levels was decreased and then increased from T3 (6

mg As/L solution). Least variation in Mg contents was seen in case of seeds under

salts arsenate as well as arsenite and both cultivars behaved almost similarly.

Secondly higher values of Mg were recorded in shoot and occasionally in root also

where V1 showed higher Mg contents than V2 (FH-415) in case of arsenate and

arsenite.

Figure 4.8(b): Phosphorus (P), calcium (Ca) and magnesium (Mg) contents in root, shoot, leaves and seeds of sunflower cultivars irrigated through arsenic contaminated water.

118


and seeds of sunflower cultivars irrigated through arsenic contaminated

water.

In case of K contents of root and seed, varieties, salts, levels and all of their

interactions showed significant differences (P<0.01), while in case of potassium

contents in shoot, salts and interaction among variety into salt into level showed non-

significant differences (P>0.05) whereas variety, levels and their interactions differed

significantly. Varieties, salts and levels along-with their interactions differed

significantly except variety into salt and variety into salt into level interaction which

showed non-significant differences.

Varieties showed significant (P<0.05) differences while salts showed non-

significant differences but levels showed significant (P<0.01) differences in boron

contents of root when analysis of variance was calculated (Table 4.10(b)). Out of

different interactions only variety into salt differed non-significantly while all other

interactions differed significantly. Boron contents of shoot showed that significantly

different values were found in varieties, levels and salts and their interactions except

variety into salt interaction. In leaf boron contents only varieties showed non-

significant differences while salts, levels and all interactions among varieties, salts

and levels showed significant differences (P<0.01). ANOVA of the data revealed that

119

in case of B contents of seed only varieties behaved differently with significant

differences while salts and levels and all interactions among varieties, salts and levels


Analysis of variance of the data regarding copper (Cu) concentration in root

revealed significant differences in case of arsenic salts and levels and all of their

interactions but non-significant differences were found for varieties of sunflower

(Table 4.10(b)). Varieties, salts and levels differed also in case of copper contents of

shoot and out of various interactions variety into salts and variety into level and

variety into salt into level interaction showed non-significant differences while salt

into level showed significant differences. In case of Cu in leaf, significant differences

were found in varieties, salt and levels and all of their interactions whereas for copper

contents of seed, varieties and salts showed significant differences (P<0.05) and levels

showed non-significant differences and out of different interactions only variety into

level showed significant differences but all other interactions among varieties, salts

and levels showed non-significant differences.

Table 4.10(b): ANOVA for potassium (K), boron (B) and copper (Cu) contents in sunflower cultivars irrigated through arsenic contaminated water.


K root K shoot K leaf K seedVarieties (V) 1 84239075** 58353017** 79677681** 2974295**

Salts (S) 1 32801787** 197408ns 20025716** 2858727**Levels (L) 5 14818177** 27155754** 9555929** 360248**

InteractionsV × S 1 30002169** 3702075* 1715687ns 377314*V × L 5 4192030** 7749360** 5015567** 1529795**S × L 5 16878013** 42669548** 4013635** 410773**

V × S × L 5 43866421** 1146739ns 2652841ns 878310**Error 48 75406 519135 1131113 89999


B root B shoot B leaf B seedVarieties (V) 1 20.21* 152.51** 59.4ns 111.60**

Salts (S) 1 9.77ns 286.68** 473.3** 0.30ns

Levels (L) 5 30.47** 64.87** 1470.3** 7.59ns


V × L 5 100.65** 214.50** 2895.6** 1.44ns

S × L 5 56.03** 216.53** 2211.3** 2.95ns

V × S × L 5 13.65* 107.55** 321.9** 0.94ns

Error 48 4.43 8.12 16.3 4.81

Mean square

120

Source D F Cu root Cu shoot Cu leaf Cu seedVarieties (V) 1 11.63ns 30.51** 155.17** 58.59*

Salts (S) 1 56.32** 30.04** 118.42** 59.35*Levels (L) 5 29.93** 6.71* 65.65** 24.74ns

InteractionsV × S 1 47.79** 5.08ns 44.33* 21.18ns

V × L 5 60.03** 1.23ns 54.57** 29.00*S × L 5 22.43** 8.71** 69.79** 26.37ns

V × S × L 5 22.62** 1.42ns 81.99** 18.78ns

Error 48 5.34 2.16 6.64 12.02

Potassium contents in root showed enough variation in case of varieties as V1

(FH-385) showed increase in K contents with increasing As in irrigation water up to

T3 (6 mg As/L solution) then decrease with a sharp reduction from T4 to T5 (8 and 10

mg As/L respectively), while in V2 (FH-415) zigzag pattern with two peaks in T2 (4

mg As/L solution) and T5 were seen meaning higher value than V1 of potassium

contents in root under arsenate in irrigation water. In case of arsenite higher value was

noted in V1 while V2 showed continuous reduction in K contents with an increase in

As level in irrigation water while V1 showed a Z-scheme like graph (Figure 4.9(b))

with two peaks one in T1 (2 mg As/L solution) and highest value in T5 (10 mg As/L

solution). In shoot K contents in both varieties behaved same way with a little

difference that in case of arsenite after T4, K contents increased in V1 but decreased

in V2, and in case of arsenate gradual increase in K contents was seen in both

cultivars but V2 showed an increase at T1 as compared to V1 in which K contents

were decreased from control (T0) in T1 level. In leaves, a decrease was seen after the

rise in K contents in T1 in case of V1 but in V2 finally reduced K contents were seen

as compared to control plants irrigated through arsenate and arsenite. In seeds K

contents remained almost similar with resembling behavior of both cultivars and in

case of both arsenic salts.

Maximum contents of boron (B) were seen in leaf of sunflower under both

salts either arsenate or arsenite with highest value given by V2 (FH-415) plants

irrigated through arsenate. Boron contents in root were similarly lower like seeds but

in shoot a bit higher contents of boron were found with higher values given by control

(T0) and initial lowest levels (2 and 4 mg As/L solution) of arsenate and arsenite

(Figure 4.9(b)). Seeds showed minimum contents of boron with least variation in

values.

121

Seeds or achenes of sunflower plants showed maximum contents of copper

with a highest value in plants belonging to cultivar V2 (FH-415) irrigated through

arsenate containing water while in case of arsenite containing water irrigation

resembling values of copper contents in seeds were recorded (Figure 4.9(b)).

Secondly higher accumulation of Cu was found in leaves in which both salts and

varieties behaved differently as copper contents of leaf were remained similar in V1

(FH-385) in case of arsenate but in V2 first decrease and then increase was observed

in copper contents whereas in case of aresenite V2 showed gradual decrease in copper

contents with increase in arsenite concentration of irrigation water. Shoot showed

minimum accumulation of copper in both varieties while in root some variation was

observed in both varieties as first decrease was seen in V1 while V2 showed increase

in Cu contents from T0 (control) to T2 (4 mg As/L solution) and then decrease was

seen when arsenate was in irrigation water.

Figure 4.9(b): Potassium (K), boron (B) and copper (Cu) contents in root, shoot, leaves and seeds of sunflower cultivars irrigated through arsenic contaminated water.

122


and seeds of sunflower cultivars irrigated through arsenic contaminated

water.

Iron contents in root and leaf showed significant differences in case of

varieties, salts and levels and all of their interactions taken (Table 4.11(b)) whereas in

shoot only levels and interaction variety into seed showed significant differences

while varieties and salts and all remaining interactions among them all showed non-

significant differences (P>0.05). In case of Fe in seeds varieties, salts, levels and all of

their interactions showed non-significant differences when analysis of variance of the

data was calculated.

In case of Mn contents of root salts and levels and interactions among

varieties, salts and levels all differed significantly only varieties showed non-

significant differences (Table 4.11(b)) while significant differences were found in

levels but varieties and salts showed non-significant differences whereas interaction

between salt and level showed significant differences while all other interactions also

showed non-significant differences when Mn shoot contents were processed through

analysis of variance. In Mn contents of leaves varieties, salts and levels and all their

interactions showed significant differences (P<0.01) but in case of manganese

contents of seed varieties, levels, salts and all interactions except variety into salt

differed significantly.

Analysis of variance of the data about zinc contents of root revealed that

varieties, salts and levels showed non-significant differences and out of different

interactions only variety into level and variety into salt into level interaction showed

significant differences (P<0.01) while variety into salt and salt into level differed non-

significantly (Table 4.11(b)). In case of Zn contents of shoot varieties and levels and

interaction between variety into level and salt into level and variety into salt into level

123

showed significant differences but only salts and variety into salt interaction showed

non-significant differences, similarly in case of zinc contents of leaf salts and levels

and interactions variety into level and salt into level showed significant differences

but varieties and variety into salt interaction and overall variety into salt into level

interaction showed non-significant differences (P>0.05). In case of zinc contents of

seed all three factors and their interactions all showed non-significant differences.

Table 4.11(b): ANOVA for iron (Fe), manganese (Mn) and zinc (Zn) contents in sunflower cultivars irrigated through arsenic contaminated water.


Fe root Fe shoot Fe leaf Fe seedVarieties (V) 1 15764683** 3469ns 115907** 248.3ns

Salts (S) 1 15704603** 155ns 189048** 5.4ns

Levels (L) 5 6112408** 4684** 61605** 355.5ns

InteractionsV × S 1 862026* 8982** 185159** 156.4ns

V × L 5 3969368** 873ns 94155** 387.7ns

S × L 5 2790846** 1538ns 51190** 178.3ns

V × S × L 5 3200215** 1920ns 71110** 101.4ns

Error 48 171954 1060 9778 228.9


Mn root Mn shoot Mn leaf Mn seedVarieties (V) 1 445.9ns 0.109ns 973.36** 528.78**

Salts (S) 1 1268.2** 1.818ns 1953.65** 658.60**Levels (L) 5 3181.1** 7.324** 1066.59** 595.14**

InteractionsV × S 1 5322.0** 0.436ns 366.62** 52.50ns

V × L 5 4836.5** 1.467ns 1424.57** 279.15**S × L 5 2886.8** 22.410** 376.59** 355.40**

V × S × L 5 2630.4** 0.654ns 136.31** 146.59**Error 48 112.2 2.08 24.21 15.08


Zn root Zn shoot Zn leaf Zn seedVarieties (V) 1 153.24ns 3704.02** 233.7ns 244.1ns

Salts (S) 1 98.05ns 2.32ns 2762.8** 245.5ns

Levels (L) 5 101.95ns 631.19** 793.6** 193.1ns


V × L 5 287.09** 256.82** 371.3** 120.5ns

S × L 5 119.38ns 1236.87** 657.9** 53.0ns

V × S × L 5 240.94** 583.57** 138.4ns 23.1ns

Error 48 57.72 74.11 106.0 125.7

Iron contents were found highest in roots than all other organs with a different

behavior shown by both varieties under arsenate in irrigation water as seen in V1 first

124

decrease in iron contents up to T2 and then sudden rise in T3 and then again decrease

was seen in iron contents of root, in case of arsenite gradual decrease was seen in iron

contents with increasing arsenite in irrigation water (Figure 4.10(b)). Shoot and seeds

showed least contents of iron in them while in leaves a little higher contents of iron

were seen in both varieties showing almost similar behavior.

Roots and leaves showed maximum manganese (Mn) contents in both

varieties and especially in roots one higher value was observed in T3 (6 mg As/L

solution) of V1 (FH-385) when arsenate was used in irrigation water while T2 (4 mg

As/L solution) of V2 (FH-415) showed maximum Mn contents under arsenite in

irrigation water (Figure 4.10(b)). Shoot showed minimum Mn contents in case of

both salts of arsenic while leaves showed maximum contents of Mn with higher

variations in V2 showing highest manganese contents in T5 (10 mg As/L solution)

under arsenate and arsenite both salts, and V1 showed less variation in both salts. In

seed values of Mn contents overlapped in both varieties with a higher value in T2 of

V2 under arsenate and in arsenite too equal variation was observed in Mn contents of

seeds or achenes.

Zinc contents were found highest in seeds or achenes of sunflower with a little

variation in both cultivars or varieties and resembling values were recorded in case of

arsenate as well as arsenite while in case of root minimum contents of Zn were found

with a higher value in V1 (FH-385) under T3 (6 mg As/L solution) and in arsenite

irrigated water V2 (FH-415) showed the maximum contents of Zn under T1 and T2

(2, 4 mg As/L solution respectively (Figure 4.10(b)). In shoot Zn contents were found

higher than root and V1 showed an extraordinary higher value in T3 under arsenate

treatment but after T3 a fall in Zn contents is evident in case of T4 and T5 as

compared to arsenite application in which T5 gave highest zinc contents but plants

belonging to V2 showed similar behavior. In leaves Zn contents were in

correspondence with zinc contents of shoot but higher Zn contents were seen in case

of arsenite treatments in both cutivars.

125

Figure 4.10(b): Iron (Fe), manganese (Mn) and zinc (Zn) contents in root, shoot, leaves and seeds of sunflower cultivars irrigated through arsenic contaminated water.

4.3.12 Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in root, shoot,

leaves and seeds of sunflower cultivars irrigated through arsenic

contaminated water.

Varieties showed significant differences in case of Mo contents of root but

salts and levels showed non-significant differences while out of different interactions

only variety into level differed significantly (P<0.05) but all other interactions showed

non-significant differences (Table 4.12(b)). In case of Mo contents in shoot only

varieties and salts showed significant differences while levels and all of the

interactions showed non-significant differences similarly in case of molybdenum in

126

leaf varieties and salts and interaction variety into level showed significant differences

but levels and remaining interactions showed non-significant differences. Varieties

and salts also gave significant differences but levels and interactions among variety

into salt into level showed non-significant differences while remaining interactions

showed significant differences.

ANOVA regarding silver contents in root showed that varieties and levels and

all interactions among varieties, salts and levels differed significantly (P<0.01) only

salts showed non-significant differences (Table 4.12(b)). In case of silver contents of

shoot and leaf all the three factors varieties, salts and levels and all of their

interactions showed non-significant differences but in case of silver contents of seed

salt and level showed significant differences only varieties differed non-significantly

and out of different interactions salt into level and variety into salt into level showed

significant differences but variety into salt and variety into level showed non-

significant differences.

Analysis of variance of the data about aluminum contents in root organ

showed significant differences in varieties, salts, levels and all their interactions but in

case of aluminum in shoot only varieties and levels showed significant differences

while salts and all of the interactions among varieties, salt and levels showed non-

significant differences (Table 4.12(b)). Aluminum in leaf showed that significant

differences were found in varieties and salts and all possible interactions, only levels

showed non-significant differences. In seeds aluminum contents all the three factors

and their interactions showed non-significant differences.

Table 4.12(b): ANOVA for molybdenum (Mo), silver (Ag) and aluminum (Al) contents of sunflower cultivars irrigated through arsenic contaminated water.


Mo root Mo shoot Mo leaf Mo seedVarieties (V) 1 4.037** 1.029** 5.20* 3.65**

Salts (S) 1 0.012ns 0.181* 6.90* 1.61**Levels (L) 5 0.047ns 0.022ns 2.42ns 0.14ns

InteractionsV × S 1 0.39ns 0.027ns 4.37ns 0.47*V × L 5 0.31* 0.059ns 5.82** 0.37**S × L 5 0.14ns 0.020ns 2.42ns 0.36**

V × S × L 5 0.10ns 0.056ns 1.30ns 0.15ns

Error 48 0.11 0.027 1.13 0.09


Ag root Ag shoot Ag leaf Ag seed

127

Varieties (V) 1 6.07** 0.02ns 0.96ns 189.64ns

Salts (S) 1 0.001ns 0.30ns 0.43ns 317.98*Levels (L) 5 3.04** 0.76ns 0.13ns 248.87**

InteractionsV × S 1 9.02** 0.31ns 1.14ns 169.43ns

V × L 5 7.67** 0.83ns 0.21ns 109.15ns

S × L 5 7.06** 0.21ns 0.56ns 145.07*V × S × L 5 4.69** 0.21ns 0.19ns 144.02*

Error 48 0.25 0.34 0.33 56.19


Al root Al shoot Al leaf Al seedVarieties (V) 1 43018470** 61616** 1106241** 14.8ns

Salts (S) 1 24084993** 2136ns 400305** 76.1ns

Levels (L) 5 19326479** 12179* 57626ns 220.8ns

InteractionsV × S 1 25472358** 6416ns 291091** 243.4ns

V × L 5 23421222** 3466ns 230524** 181.2ns

S × L 5 11625426** 1938ns 121073** 234.4ns

V × S × L 5 12711469** 1401ns 123745** 83.9ns

Error 48 360399 4863 24816 109.6

Out of different organs leaves showed highest contents of Mo especially in V1

(FH-385) plants irrigated through arsenite contaminated water under treatments T2,

T3 and T4 (4, 6 and 8 mg As/L solution respectively) as compared to V2 which

showed decrease in Mo contents in same treatments (Figure 4.11(b)) and in case of

arsenate application T4 gave least value of Mo contents in both varieties. Least

variation in Mo content values was seen in shoot while in root V1 showed higher

contents than V2 in about all treatments as in shoot. In case of seeds or achenes V2

plants showed higher Mo contents in them as compared to V1 plants.

Seeds or achenes showed a bit higher accumulation of Ag irrespective of both

varieties and a highest value in T3 of V2 under arsenite was recorded while root

showed a little increase in silver contents in case of arsenate under T4 (8 mg As/L

solution) whereas silver in shoot and leaf was not detected notably.

Roots showed highest accumulation of aluminum than all other organs with

relatively higher values in arsenate under treatment T3 (6 mg As/L solution) of

cultivar V1 (FH-385). In shoot and seeds least detectable values of aluminum were

found but in leave a little higher values less than root were recorded in both salts and

in both varieties with a bit higher Al contents in V1 plants (Figure 4.11(b)).

128

Figure 4.11(b): Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in root, shoot, leaves and seeds of sunflower cultivars irrigated through arsenic contaminated water.

4.3.13 Barium (Ba), bismuth (Bi) and cadmium (Cd) contents in root, shoot,

leaves and seeds of sunflower cultivars irrigated through arsenic

contaminated water.

Data were processed and analysis of variance revealed that in case of barium

contents of root varieties and levels differed significantly but salts showed non-

significant differences similarly out of different interactions variety into salt showed

non-significant differences whereas all other interactions differed significantly (Table

4.13(b)). In shoot and leaf too varieties and levels showed significant differences in

129

barium contents while salts differed non-significantly while in case of interactions

only variety into salt into level in barium leaf showed non-significant differences

while all other interactions showed significant differences but in case of barium in

seed all the three factors and their interactions showed significantly different values.

Salts and levels showed significant differences while varieties showed non-

significant differences (Table 4.13(b)) in case of Bi in root, shoot and leaves, whereas

out of different interactions variety into level and variety into salt into level showed

significant differences but variety into salt and salt into level showed non-significant

differences in case of bismuth in root . In case of bismuth contents of shoot out of

interactions only salt into level showed significant differences but remaining all

interactions showed non-significant differences, similarly in case of bismuth contents

of leaf only variety into level showed significant differences but all remaining

interactions showed non-significant differences. In case of Bi contents of seed,

varieties, salts and levels showed non-significant differences and out of interactions

only variety into level differed significantly but variety into salt, salt into level and

variety into salt into level interactions showed non-significant differences among

them.

In case of cadmium contents of seed varieties of sunflower, salts and levels of

arsenic and interactions among these factors all showed significant (P<0.01)

differences (Table 4.13(b)), while in root only variety into salt interaction differed

(P<0.05) significantly but all the three factors and remaining interactions showed non-

significant differences. Similarly in case of Cd contents in shoot levels and

interactions variety into level, salt into level and variety into salt into level all showed

significant differences but in case of leaf cadmium contents only variety into salt

interaction showed significantly different values but non-significant differences were

shown by varieties, levels and salts and interaction among them.

Table 4.13(b): ANOVA for barium (Ba), bismuth (Bi) and cadmium (Cd) contents in sunflower cultivars irrigated through arsenic contaminated water.


Ba root Ba shoot Ba leaf Ba seedVarieties (V) 1 425.78** 2407.4** 1284.48** 287.44**

Salts (S) 1 82.80ns 176.2ns 15.69ns 270.67**Levels (L) 5 249.30** 1075.1** 419.70** 60.47**

InteractionsV × S 1 6.79ns 3015.1** 264.08* 249.61**V × L 5 557.11** 1726.8** 155.32* 37.05*S × L 5 211.13** 2523.9** 515.98** 48.78**

130

V × S × L 5 431.79** 1848.3** 68.92ns 48.01**Error 48 47.69 43.8 55.55 13.50


Bi root Bi shoot Bi leaf Bi seedVarieties (V) 1 4.43ns 0.33ns 3.54ns 42.97ns

Salts (S) 1 34.46** 13.02** 91.98** 25.56ns

Levels (L) 5 13.56* 3.72* 92.08** 26.13ns


V × L 5 30.21** 1.57ns 90.89** 32.77*S × L 5 9.61ns 7.51** 17.75ns 25.85ns

V × S × L 5 11.15* 1.34ns 15.13ns 17.39ns

Error 48 4.01 1.41 9.36 12.17


Cd root Cd shoot Cd leaf Cd seedVarieties (V) 1 0.010ns 0.130ns 0.165ns 4.05**

Salts (S) 1 0.101ns 0.173ns 0.035ns 4.05**Levels (L) 5 0.214ns 0.318** 0.045ns 4.39**

InteractionsV × S 1 0.448* 0.102ns 0.270* 4.34**V × L 5 0.242ns 0.399** 0.051ns 4.43**S × L 5 0.122ns 0.651** 0.044ns 4.80**

V × S × L 5 0.130ns 0.623** 0.052ns 4.36**Error 48 0.101 0.077 0.041 0.198

Least contents of barium were observed in seeds or achenes of sunflower

especially in case of arsenate irrigated water whereas in case of arsenite V1 (FH-385)

showed a bit higher contents of barium than V2 (Figure 4.12(b)) in seeds, whereas in

root, shoot and leaves similar behavior of both cultivars was recorded with same

resembling ups and downs in barium contents under both arsenicals arsenate as well

as arsenite and only a single value in V1 belonging to T5 (10 mg As/L solution) under

arsenite were recorded. Leaves showed higher values of barium contents in case of

arsenate application while in arsenite both varieties showed almost similar behavior.

Highest bismuth contents were found in seed or achenes of sunflower than all

other organs in both varieties with a bit higher value in T3 (6 mg As/L solution)

belonging to V2 under arsenate irrigated water (Figure 4.12(b)). Leaves showed

bismuth contents less than seeds but higher than root and shoot organs with average

higher values given by V2 (FH-415) in case of both salts either arsenate or arsenite.

Shoot showed the least contents of bismuth while roots showed a little higher contents

131

of bismuth than shoot with similar contents of Bi in both cultivars and under both

arsenicals.

There was not any notable variation in cadmium contents of root, shoot, leaf

and seed or achenes of sunflower recorded with only a few exceptional values like in

case of Cd contents in seed in V2 (FH-415) plants under T3 (6 mg As/L solution)

when arsenite was used in irrigation water (Figure 4.12(b)) and one value in root

under arsenite and in shoot both salts gave higher value, as a whole both varieties

showed similar behavior towards cadmium contents in sunflower irrigated through

arsenicals contaminated water of different levels.

Figure 4.12(b): Barium (Ba), bismuth (Bi) and cadmium (Cd) contents in root, shoot, leaves and seeds of sunflower cultivars irrigated through arsenic contaminated water.

132


leaves and seeds of sunflower irrigated through arsenic contaminated

water.

Statistical analysis of the data regarding cobalt contents in root and leaf

showed significant differences in varieties, salts and levels of arsenic as well as all

interactions taken among these factors (Table 4.14(b)) while in case of Co contents in

shoot and seed mostly non-significant differences were seen except salts and variety

into salt into level interaction which showed significant (P<0.05) differences in case

of cobalt contents of shoot.

Analysis of variance of the data regarding chromium contents in shoot showed

significant differences (P<0.01) for varieties, salts, levels and all their interactions,

similarly for Cr contents of leaf, except varieties, all factors and their interactions

showed significant differences in values (Table 4.14(b)). In case of Cr contents of root

varieties and variety into salt interaction showed non-significant differences while

salts and levels and remaining interactions among these factors showed significantly

different values. Non-significant differences were seen in varieties, salts levels and all

interactions for Cr in seed or achenes.

Analysis of variance (ANOVA) of the data revealed significant differences in

varieties, salts and levels and all their interactions in case of lithium contents in root

and seed while in leaf only levels showed non-significant differences but varieties and

salts and all interactions among varieties, salts and levels showed significantly

different values (Table 4.14(b)). Considering lithium contents of shoot, varieties, salts

and levels showed significant differences while out of interactions only variety into

level showed significant differences while all other interactions showed non-


Table 4.14(b): ANOVA for cobalt (Co), chromium (Cr) and lithium (Li) contents in sunflower irrigated through arsenic contaminated water.


Co root Co shoot Co leaf Co seedVarieties (V) 1 2.57* 0.004ns 0.88** 0.004ns

Salts (S) 1 1.86* 0.224* 0.88** 0.002ns

Levels (L) 5 3.48** 0.039ns 1.01** 0.010ns


V × L 5 4.91** 0.085ns 0.63** 0.015ns

S × L 5 2.74** 0.028ns 0.57** 0.009ns

V × S × L 5 2.93** 0.092* 0.81** 0.001ns

133

Error 48 0.42 0.035 0.078 0.012


Cr root Cr shoot Cr leaf Cr seedVarieties (V) 1 0.114ns 8.013** 2.588ns 0.588ns

Salts (S) 1 5.104* 4.401** 9.790** 0.418ns

Levels (L) 5 5.65** 3.417** 3.561* 0.130ns

InteractionsV × S 1 0.395ns 12.65** 7.443* 0.485ns

V × L 5 33.279** 9.135** 3.804** 0.259ns

S × L 5 8.642** 6.784** 2.884* 0.249ns

V × S × L 5 12.640** 8.026** 2.651* 0.255ns

Error 48 1.122 0.479 1.073 0.401


Li root Li shoot Li leaf Li seedVarieties (V) 1 144.81** 19.76** 9.78** 7.35**

Salts (S) 1 70.15** 8.20* 25.44** 6.97**Levels (L) 5 74.97** 2.93* 1.89ns 7.19**

InteractionsV × S 1 110.24** 1.41ns 12.11** 6.97**V × L 5 110.96** 3.16* 19.88** 7.19**S × L 5 48.37** 2.05ns 8.68** 6.82**

V × S × L 5 52.72** 2.58ns 3.02** 6.82**Error 48 7.14 1.15 0.82 0.44

Highest cobalt contents were found in roots with decrease in cobalt contents

except a single higher value in V1 (FH-385) under T3 (6 mg As/L solution) having

arsenate in irrigation water while in case of arsenite V2 (FH-415) showed highest

value in case of T2 (4 mg As/L solution) with relative decrease with increase in

arsenite concentration in irrigation water (Figure 4.13(b)). Cobalt contents in shoot

were very low in case of arsenate while in arsenite V2 showed a little increase in T3

and T4 (6 and 8 mg As/L respectively) and a value of V1 in T5 (10 mg As/L solution)

under arsenite. Leaf showed higher contents of Co than shoot and seed but less than

root with a stability in V1 under arsenate but two values one in T2 and other in T5 of

V2 and under arsenite a reduction was also seen in both varieties while seeds or

achenes showed least contents of cobalt.

Maximum chromium contents were observed in root (Figure 4.13(b))

especially under arsenate conditions and first increase and then decrease in Cr

contents was seen in both varieties with a highest value in V1 (FH-385) of T3 (6 mg

As/L solution), same trend was seen in case of arsenite conditions in which V2

showed first increase and then decrease but V1 showed an increase in chromium

134

under T5 (10 mg As/L solution). In shoot chromium contents were found intermediate

between root and leaf with a higher value in case of arsenate by V2 plants under T2 (4

mg As/L solution) while remaining values remained almost same, V1 showed

decrease in Cr contents from T0 to T2 and then a stability was seen, while in leaf Cr

contents a lot of variation was seen especially under arsenite conditions as V1 showed

an increase in chromium contents first up to T3(6 mg As/L solution) and then a

decrease was seen in T4 and T5 (8 and 10 mg As/L respectively), V2 showed opposite

behavior in case of arsenate as first decrease and then increase in Cr contents. In seed

Cr contents remain stable in control and all of the treatments of arsenate and arsenite

in both, cultivars or varieties of sunflower.

Roots showed highest lithium contents with a gradual decrease with increasing

arsenate concentration in irrigation water in V1 (FH-385) showing a highest value in

T3 (6 mg As/L solution), in case of arsenite decrease in Li contents was also seen in

V1 and V2 with a higher value in T2 (4 mg As/L solution) of plants belonging to V2

(Figure 4.13(b)). In shoot Li contents remain stable in both varieties and both salts

while in leaves higher contents of lithium were seen in arsenite treated plants showing

a rise in Li contents and then fall in V1 while V2 showed opposite behavior with first

decrease and then increase in leaf Li contents, but in seed except a single value

belonging to T4 (8 mg As/L solution) plants of V2 (FH-415) all treatment values were

undetectable for both varieties.

Figure 4.13(b): Cobalt (Co), chromium (Cr) and lithium (Li) contents in root, shoot, leaves and seeds of sunflower irrigated through arsenic contaminated water.

135

4.3.15 Nickel (Ni), lead (Pb) and antimony (Sb) contents in root, shoot, leaves

and seeds of sunflower irrigated through arsenic contaminated water.

Statistical analysis of the data comprising of analysis of variance revealed that

Ni contents in leaf showed significantly different values in varieties, salts, leaves and

all interactions similarly in case of shoot only varieties showed non-significant

differences while salts, levels and all interactions among these three factors showed

significant differences (Table 4.15(b)). In case of Ni contents of root, varieties and

levels and interaction between variety into level and salt into level and variety into

salt into level interaction showed significant differences in values but salt and variety

into salt interaction showed non-significant differences in values whereas in case of

Ni contents in seed or achenes all the three factors and their interactions showed non-


Varieties, salts and levels and all interactions among these factors showed

significant differences except for variety into level interaction in case of lead contents

of root and shoot (Table 4.15(b)) as shown by analysis of variance of the data, while

only levels showed significantly different values in case of Pb contents in leaf and all

interactions except salt into level interaction showed significantly different values

while in case of lead contents of seed variety and levels and salt into level and variety

into salt into level interaction showed significant differences whereas salts and variety

into salt and variety into level interaction showed non-significant differences.

136

Analysis of variance of the data regarding antimony contents of leaf revealed

that varieties, salts, levels and all interactions among these factors showed significant

differences (P<0.01), in shoot too the contents of Sb remained same as all the three

factors and their interactions except variety into level showed significant differences

(Table 4.15(b)) but in case of antimony contents of root only salt into level revealed

significant differences while all the three factors and their interactions showed non-

significant differences. Antimony contents of seed also showed that only levels and

interaction between variety into level and variety into salt into level interaction

showed significant differences while salts, levels and remaining interactions showed

non-significant differences in values.

Table 4.15(b): ANOVA for nickel (Ni), lead (Pb) and antimony (Sb) contents in sunflower irrigated through As contaminated water.


Ni root Ni shoot Ni leaf Ni seedVarieties (V) 1 6.55** 0.55ns 38.39** 0.075ns

Salts (S) 1 1.20ns 0.87* 8.71** 0.066ns

Levels (L) 5 15.94** 1.68** 8.88** 0.075ns

InteractionsV × S 1 0.06ns 5.27** 21.80** 0.075ns

V × L 5 11.58** 1.17** 7.30** 0.030ns

S × L 5 9.56** 1.32** 11.82** 0.021ns

V × S × L 5 7.77** 2.95** 15.94** 0.021ns

Error 48 0.79 0.21 0.65 0.286


Pb root Pb shoot Pb leaf Pb seedVarieties (V) 1 59.78** 4.93* 1.027ns 4.76*

Salts (S) 1 42.31* 6.61* 0.091ns 0.22ns

Levels (L) 5 19.47* 19.71** 7.78** 4.57**Interactions

V × S 1 45.77** 73.52** 35.56** 1.14ns

V × L 5 11.41ns 2.11ns 5.34* 0.90ns

S × L 5 40.59** 9.61** 4.67ns 3.59*V × S × L 5 20.86** 21.48** 10.00** 3.49*

Error 48 5.82 1.13 2.21 1.12


Sb root Sb shoot Sb leaf Sb seedVarieties (V) 1 0.0012ns 0.334* 5.46** 0.0107ns

Salts (S) 1 0.040ns 0.255* 0.72* 0.041ns

Levels (L) 5 0.071ns 0.256** 2.49** 0.212**Interactions

V × S 1 0.061ns 0.011* 1.49** 0.001ns

V × L 5 0.009ns 0.144ns 2.12** 0.312**

137

S × L 5 0.111* 0.532** 3.78** 0.05ns

V × S × L 5 0.018ns 0.206* 4.84** 0.102*Error 48 0.038 0.061 0.164 0.037

Nickel contents were found highest in root of V2 (FH-415) plants with a trend

showing decrease with an increase in arsenate concentrations and same effects were

recorded in both varieties from T3 (6 mg As/L solution) to T5 (10 mg As/L solution)

in case of arsenate application (Figure 4.14(b)), while in case of arsenite treated plants

higher values were found in T2 (4 mg As/L solution) of V2 but average higher

contents of Ni in root were shown by plants of V1(FH-385) belonging to T3 and T5.

In shoot well defined variation in values was seen in arsenate applied plants of V2

with decreasing trend and stability in case of arsenite treated plants while V1 plants

showed similar behavior in both salts. In leaf Ni contents showed increase in both

varieties with increasing arsenicals application with highest value recorded in T2 of

V2 plants whereas in seeds no variation was seen in both varieties as similar values

were recorded in all treatments including control plants.

In root lead contents were found maximum especially in case of arsenite

application in plants of V1 (FH-385) under lower concentration of arsenite but in case

of arsenate V2 (FH-415) showed less variation in values than V1 plants (Figure

4.14(b)), in arsenate highest concentration of lead was recorded in T5 (10 mg As/L

solution). In case of shoot V2 showed increase in lead contents with increase in

arsenate concentration and V1 showed less values variation while in arsenite

conditions V1 showed higher value in T2 (4 mg As/L solution). In leaves Pb contents

were found higher than shoot and seed with relatively stability in V1 plants but V1

showed variation under arsenate, while in case of arsenite V1 showed an increase in

lead contents with increasing arsenite but V2 showed gradual decrease in lead

contents. In seed lead contents remained almost stable with a little increase in V2

plants under arsenate condition but in arsenite similar values were recorded.

V2 (FH-415) showed a few higher values of antimony contents in leaf under

arsenate while in case of arsenite application V1 showed an elevation in Sb contents

with increase in arsenite concentration in irrigation water while V2 showed gradual

decrease in antimony contents in leaf (Figure 4.14(b)), while in root, shoot and seed

near zero values were mostly common in both varieties with a very little variation

among the contents of antimony in all these organs under various levels of arsenate

and arsenite.

138

Figure 4.14(b): Nickel (Ni), lead (Pb) and antimony (Sb) contents in root, shoot, leaves and seeds of sunflower irrigated through As contaminated water.

4.3.16 Selenium (Se) and strontium (Sr) contents in root, shoot, leaves and seeds

of sunflower irrigated through As contaminated water.

Varieties, salts and levels and all their interactions showed significant

differences when analysis of variance was calculated for selenium contents in root,

shoot and leaf of sunflower cultivars while in case of selenium contents of seed except

interaction between variety and salt and variety into salt into level interaction all

factors and interactions showed significant differences (Table 4.16(b)) whereas

139

varieties, salts and levels showed significant differences in case of selenium contents

of all four organs of sunflower plants.

ANOVA revealed that in case of Sr contents in root, shoot and leaf all the

three factors varieties or cultivars of sunflower, salts and levels of arsenicals showed

significant differences (P<0.01) among values whereas only in case of seed non-

significant differences were found in factors and interactions among factors all (Table

4.16(b)). Out of different interactions, variety into salt showed non-significant

differences for Sr contents in root and shoot and seed while remaining all interactions

among three factors showed significant differences of values as inferred from

statistical analysis of the data regarding these parameters.

Selenium (Se) contents were found unevenly distributed among different plant

organs as shoot and leaves showed higher contents with higher variation in values

(Figure 4.16(b)), root showed least contents of selenium especially in case of arsenate

application while in arsenite V1 showed a little higher values in T3 and T4 plants (6

and 8 mg As/L solution). In case of shoot V2 showed maximum contents of Se in all

treatments except control plants in case of both inorganic arsenicals applied. In case

of leaf also V2 showed a gradual decrease in Se contents with increasing level of

arsenate in irrigation water while in arsenite V1 showed highest values of selenium

contents in sunflower leaves but in seed a relative decrease in selenium contents was

seen in plants of V2 while V1 plants showed an increase in Se contents under arsenate

conditions but less variation under arsenite.

Table 4.16(b): ANOVA for selenium (Se) and strontium (Sr) contents of sunflower irrigated through As contaminated water.


Se root Se shoot Se leaf Se seedVarieties (V) 1 2.34** 9.65** 7.53** 5.04**

Salts (S) 1 4.30** 14.55** 13.93** 3.08*Levels (L) 5 1.21** 2.47** 6.56** 2.54*


V × L 5 1.18** 5.80** 20.91** 2.03*S × L 5 0.71** 3.15** 7.12** 2.67**

V × S × L 5 0.68** 3.04** 4.56** 1.20ns

Error 48 0.14 0.66 0.78 0.68


Sr root Sr shoot Sr leaf Sr seedVarieties (V) 1 293.99** 959.29** 5841.9** 4.96ns

Salts (S) 1 271.02** 895.84** 347.9** 3.67ns

140

Levels (L) 5 293.33** 456.47** 970.6** 3.02ns


V × L 5 424.55** 214.91** 1997.5** 4.04ns

S × L 5 221.27** 817.20** 859.8** 7.06ns

V × S × L 5 386.12** 79.89** 709.6** 2.51ns

Error 48 26.91 16.04 36.6 7.10

An increasing trend in strontium contents in root, shoot and leaf respectively

was noted in both sunflower cultivars overall (Figure 4.15(b)). In root V2 showed

increase in Sr contents with increase in arsenate concentration in irrigation water but

in case of arsenite application a gradual decrease was seen after T1 while V1 showed

decrease in case of arsenate and decrease in arsenite treatments. In case of shoot

decrease in Sr contents was shown by V2 under arsenate treatments after an increase

from control in T1 similarly in V1 first strontium contents were increased up to T3

and then decreased in T4 and T5 but in case of arsenite application an increasing trend

in Sr contents was seen in plants belonging to V2 and highest contents of strontium

were found in leaves with maximum variation noted in V1 plants from T3 to T5 under

arsenate whereas V2 showed decrease in Sr contents with increasing arsenate

concentration in irrigation water but in arsenite treated plants increasing trend of

strontium was shown by V1 plants in contrast to V2 which showed decrease after T2

in arsenite treated plants in Sr contents which remained same in seeds or achenes

found as yield and no variation in values was seen in control and arsenate or arsenite

treated plants.

Figure 4.15(b): Selenium (Se), and strontium (Sr) contents in root, shoot, leaves and seeds of sunflower irrigated through As contaminated water.

141

4.3.17 Titanium (Ti), thallium (Tl) and vanadium (V) contents in sunflower

cultivars irrigated through As contaminated water.

Statistical analysis comprising of analysis of variance of the data regarding

titanium contents in root and shoot showed that varieties, salts, levels and different

interactions drawn of these three factors showed significant differences (P<0.01)

while in case of Ti contents in leaf all the three factors and interactions except variety

into salt showed significant differences (Table 4.17(b)), but in case of titanium in

seeds obtained as yield out of three factors only levels showed significant differences

while varieties and salts showed non-significant differences among values whereas all

interactions among factors showed significant differences in values in case of titanium

contents of seeds.

ANOVA for thallium contents of root showed that varieties, salts, levels and

all their interactions showed significant differences among data (Table 4.17(b)). In

case of Tl contents of shoot varieties and levels and interactions except salt into level

all showed significantly different values and same was case in leaf thallium contents

except the interaction varieties into salt all interactions showed significantly (P<0.05)

different values of thallium contents in leaf, whereas in case of seed only levels

showed significant differences out of three factors while varieties and salts showed

non-significant differences but all interactions showed significant differences in data

obtained about thallium contents in sunflower plant organs.

Analysis of variance of data regarding vanadium contents in root showed that

varieties, salts, levels and all interactions among these three factors showed

significant differences among values (Table 4.17(b)). In case of shoot vanadium

contents, varieties and salts showed significant differences (P<0.01) while salts and

out of interactions only salt into level interaction showed non-significant differences

whereas all remaining interactions showed significant differences among data. In case

142

of vanadium contents of leaves only varieties and interaction variety into level

showed significant differences while salts, levels and remaining all interactions

showed non-significant differences, similarly in case of vanadium contents in seeds

levels and interaction variety into salt showed significant differences but varieties,

salts and all remaining interactions showed non-significant differences in values

obtained as a result of statistical analysis of the data collected.

Table 4.17(b): ANOVA for titanium (Ti), thallium (Tl) and vanadium (V) contents in sunflower irrigated through As contaminated water.


Ti root Ti shoot Ti leaf Ti seedVarieties (V) 1 12789.9** 413.90** 139.53** 7.82ns

Salts (S) 1 5953.2** 52.45** 314.88** 1.59ns


V × S 1 7519.1** 43.20** 35.04ns 25.59*V × L 5 11629.0** 155.84** 163.01** 69.17**S × L 5 3208.8** 163.23** 171.53** 34.04**

V × S × L 5 4601.6** 147.73** 28.86* 62.83**Error 48 8.3 4.97 9.64 4.43


Tl root Tl shoot Tl leaf Tl seedVarieties (V) 1 8414.3** 236.39** 337.22** 1.72ns

Salts (S) 1 1109.6** 3.18ns 2.53ns 1.64ns


V × S 1 2808.1** 114.86** 3.82ns 28.79*V × L 5 9212.0** 58.12** 66.69** 68.36**S × L 5 850.0** 12.69ns 50.04** 21.34**

V × S × L 5 1605.1** 49.55** 101.80** 69.56**Error 48 32.0 10.03 14.08 4.29


V root V shoot V leaf V seedVarieties (V) 1 128.48** 235.15** 102.74** 0.684ns

Salts (S) 1 143.59** 2.54ns 0.003ns 0.005ns

Levels (L) 5 85.33** 47.01** 0.891ns 1.267**Interactions

V × S 1 51.38* 119.30** 2.79ns 1.120*V × L 5 264.59** 57.58** 4.51* 0.459ns

S × L 5 136.61** 17.13ns 1.60ns 0.052ns

V × S × L 5 209.08** 38.81** 1.69ns 0.239ns

Error 48 12.20 8.63 1.72 0.259

143

Titanium contents in root remained higher than all other plant organs with

well-defined variation in values especially under arsenate conditions in which V1

(FH-385) showed in control and T3 (6 mg As/L solution) levels of arsenate (Figure

4.16(b)) similarly V2 (FH-415) showed gradual decrease in titanium contents with an

increase in arsenate concentrations similar behavior was also shown by both varieties

or cultivars under arsenite condition with gradual decrease in Ti contents except in

control and T2 (4 mg As/L solution) plants belonging to V1 and V2 respectively. Asli

and Neumann, (2009) in an experiment on maize seedling growth reported deleterious

effects of titanium on primary root growth of Zea mays L. Shoot and leaf of sunflower

showed similar values for titanium concentrations with a little higher value in leaf

than shoot but contents of titanium remained same with less variation in data. In seeds

least concentration of titanium was observed with very minute change in both

varieties and in all levels or treatments of arsenic salts.

Toxicity of Tl on plants has been reported by many researchers as Babula et

al,. (2008) reported its toxic impacts on growth and physiological parameters.

Thallium contents were found highest in roots with maximum value in case of V1

(FH-385) control plants and then a sudden drop at every level of arsenate or arsenite

application while V2 (FH-415) showed a higher value in T2 (4 mg As/L solution) and

then decrease in contents of thallium was seen in increased arsenite concentrations in

irrigation water (Figure 4.16(b)). Shoot thallium contents were found very low and

almost same in all treatments in V2 (FH-415) under arsenate and arsenite both but V1

showed a little higher contents of thallium in control, T1 and T2 (2, 4 mg As/L

respectively) but in higher concentrations of arsenate thallium contents remain same

in both varieties. LaCoste et al., (2001) performed an experiment on eleven edible

crops especially vegetables and reported accumulative potential of Tl and its toxic

effects on growth and metabolism of different plant organs. In our experiment on

sunflower crop leaf thallium contents remained a little higher than shoot and seed

with a little variation in values in both varieties and under both salts of arsenic and all

the five levels of arsenicals but values overlapped almost. In seed thallium contents

remained least but a little variation was seen in both varieties under different levels of

arsenate as well as arsenite.

Roots and shoots showed highest account of vanadium out of all sunflower

organs with a lot of variation in data (Figure 4.16(b)) as in root a gradual decrease in

V contents was seen in V1 (FH-385) plants except a higher value in T3 (6 mg As/L

144

solution) plants under arsenate treatment and in V2 (FH-415) first rise and then fall in

V contents was seen but T5 (10 mg As/L solution) showed maximum value in V2 as

compared to T5 in V1 which was least value of V under arsenate, in arsenite treated

plants higher variation among values was seen than arsnate in both varieties and

levels or treatments with rises and falls in both varieties. Some harmful ecological

impacts of vanadium are also mentioned by Yanguo et al., (2006). In shoot under

arsenate conditions V1 showed a decrease in V contents with increasing level of

arsenate but V2 showed same amount of vanadium with less variation similar was

situation in arsenite treated plants in which except control all plants showed almost

similar contents of vanadium in shoot in both varieties. In case of leaf vanadium

contents V1 showed stable values a little higher than V2 under arsenate as well as

arsenite and least contents of vanadium were found in seeds for both varieties and

under both arsenic salts used for arsenic application in irrigation water.

Figure 4.16(b): Titanium (Ti), thallium (Tl) and vanadium (V) contents in root, shoot, leaves and seeds of sunflower irrigated through As contaminated water.

145

4.3.18 Conclusion (Experiment 3):

Arsenic applied through irrigation water in uncontaminated soil also showed

deleterious effects on growth and development of both sunflower cultivars equally but

deleterious effects were conspicuous at vegetative or pre-anthesis stage than at

maturity or final harvest which showed very little effects of arsenic contaminated

irrigation. Reduction in shoot and root length, fresh and dry weight of shoot and root

were all decreased when calculated at vegetative stage under higher levels (6, 8 and

10 mg As/L solution) arsenic especially as arsenate. Less reduction in shoot, root

length, number of leaves, capitulum diameter and hundred achene weight. Very low

accumulation of arsenic was seen in all organs but a lot of arsenic was found as left

over arsenic in the soil. Roots showed highest bioaccumulation coefficient value.

Phosphorus was found highest in achenes while Ca, Mg and B was found higher in

leaves. Bi and Cu was much accumulated in seeds but Co, Cr, Fe, Mn, Al, Ti, Tl and

V were much accumulated in roots of both sunflower cultivars.

146

4.4 RESULTS AND DISCUSSION EXPERIMENT NO. 4

Arsenic (As) accumulation in sunflower (Helianthus annuus L.) cultivated on arsenic contaminated soil and irrigated through contaminated water.


4.4.2 Agronomic parameters:

Two way analysis of variance (ANOVA) were drawn regarding the data about

two sunflower cultivars (H1=FH-385 and H2=FH-415) grown in pretreated soil

having six levels (0, 20, 40, 60, 80 and 100 mg As/kg soil) of two arsenic salts

(S1=Sodium arsenate and S2=Sodium arsenite). These plants were irrigated (5 times)

through arsenic laden water having levels (0, 2, 4, 6, 8 and 10 mg As/liter water).

Plant morphological or agronomic parameters including shoot and root length & shoot

to root ratio showed significant differences (P<0.01) for both cultivars with relatively

higher values in plants belonging to H2. A gradual increase in shoot to root ratio was

observed in H2 while in contrast reduction was recorded in H1 with an increase in

arsenic concentration. Salts and levels of arsenic also showed significant differences

with maximum shoot length (39.33 ± 0.88) and root length (18.33 ± 0.88) in control

(T0) plants, H1 showed evident stress effects with least values of shoot (15.33 ± 1.45)

and root length (8.00 ± 1.00).Varieties and their interaction with salts and levels

showed significant differences for shoot:root ratio and shoot length but all interactions

among varieties, salts and levels differed non-significantly (P>0.05) in case of root

length (Table 4.1(c).

Table 4.1(c): Analysis of variance (ANOVA) of data for shoot length, root length and shoot:root ratio at vegetative stage of two sunflower cultivars under various As levels applied in soil and irrigation water.


Shoot Length (cm)

Root Length (cm)

Shoot : Root ratio

Varieties (V) 1 264.50** 12.50* 0.78**Salts (S) 1 46.72** 0.50ns 0.13*


V × S 1 133.38** 9.38ns 0.16*V × L 5 15.67* 4.56ns 0.41**S × L 5 13.42* 5.30ns 0.53ns

147

V × S × L 5 15.02* 1.25ns 0.47ns

Error 48 5.48 3.00 0.026

Highest shoot length value was recorded in control plants of both cultivars

with a gradual decrease as level of arsenate or arsenite increased with least value in

case of T5 (100 mg As/kg soil + 10 mg As/L solution) under arsenate conditions

while under arsenite in soil plus irrigation water, V1 (FH-385) showed decrease but

V2 (FH-415) values remained almost stable with less decrease in shoot length as

compared to V1 with increasing level or concentration of arsenite in soil and

irrigation water. A similar behavior was also find in case of root length with higher

values in control plants and decrease in root length with increasing level of arsenate or

arsenite (Figure 4.1(c)).

Figure 4.1(c): Shoot length and root length of two sunflower cultivars under different levels of arsenic in soil and irrigation water.

Interaction (mean±SE) for shoot : root ratio.Level Variety Mean

Hybrid 1 Hybrid 2T0 2.25 ± 0.05bcd 2.12 ± 0.03c-f 2.19 ± 0.03BCT1 2.26 ± 0.08bc 2.19 ± 0.06b-e 2.22 ± 0.05ABCT2 2.33 ± 0.10b 2.30 ± 0.08bc 2.32 ± 0.06ABT3 2.02 ± 0.07ef 2.68 ± 0.11a 2.35 ± 0.12AT4 2.06 ± 0.09def 2.18 ± 0.04b-e 2.12 ± 0.05C

148

T5 1.97 ± 0.06f 2.67 ± 0.10a 2.32 ± 0.12AB

Levels of arsenic and varieties of sunflower gave significantly different values

of fresh and dry weights of shoot and root and their water contents. Salts of arsenic

showed non-significant differences for shoot water contents and fresh weight of shoot.

Data analysis revealed that varieties differed significantly in all parameters except for

shoot fresh weight in which both varieties gave similar values but H1 showed higher

values (8.20 ± 0.73) under sodium arsenate and lower value (7.67 ± 0.59) of shoot

fresh weight under sodium arsenite in contrast to H2 in which inverse condition was

found with higher fresh weight of shoot under sodium arsenite and lower value under

sodium arsenate. Overall interaction of varieties, salts and levels (V×S×L) differed

non-significantly in case of fresh and dry weight of root and water contents of root.

Table 4.2 (c): ANOVA of data for fresh weight, dry weight and water contents of shoot and root in two sunflower cultivars under different As levels in soil and irrigation water.


Fresh wt. shoot

(g)

Dry wt. shoot

(g)

Water contents

shoot

Fresh wt. root

(g)

Dry wt. root (g)

Water contents

rootVarieties (V) 1 0.96ns 0.076* 1.57* 0.78** 0.017** 0.58**

Salts (S) 1 0.62ns 0.127** 0.18ns 0.28** 0.002* 0.23**Levels (L) 5 98.55** 5.09** 59.79** 10.28** 0.08** 8.53**

InteractionsV × S 1 9.32** 0.037ns 8.17** 0.08* 0.0004ns 0.06*V × L 5 0.74ns 0.038* 0.88* 0.22** 0.004** 0.17**S × L 5 5.93** 0.086** 4.95** 0.17** 0.0007ns 0.16**

V × S × L 5 0.69ns 0.094** 0.74* 0.013ns 0.00007ns 0.01ns

Error 48 0.43 0.015 0.29 0.017 0.0005 0.014

Fresh weight and dry weight of shoot and root were all decreased with

increasing concentration of arsenicals in the environment and both salts showed

similar deleterious effects (Table 4.3(c)) when higher concentration or level of

arsenical either arsenate or arsenite was present in the rhizospheric environment.

Highest fresh and dry weights were recorded in control plants devoid of any arsenical

treatments and reduction in fresh weight as well as dry weight was intensively

increased under higher concentrations of arsenate and arsenite i-e T3, T4 and T5 (60

mg As/kg soil + 6 mg As/L solution, 80 mg As/kg soil + 8 mg As/L solution and 100

mg As/kg soil + 10 mg As/L solution respectively).

149

Table 4.3(c): Different interaction among varieties, salts and levels for fresh weight and dry wt. of shoot and root in sunflower cultivated in arsenic contaminated soil and irrigated through As contaminated water.

Interaction (mean±SE) among varieties and salts for fresh wt. of shoot.S Variety Mean

Hybrid 1 Hybrid 2S1 8.20 ± 0.73a 7.25 ± 0.80b 7.73 ± 0.54AS2 7.67 ± 0.59b 8.16 ± 0.53a 7.91 ± 0.39AMean 7.93 ± 0.46A 7.70 ± 0.48A

Interaction (mean±SE) among varieties and levels for dry wt. of shoot.Level Variety Mean

Hybrid 1 Hybrid 2T0 2.000 ± 0.073b 2.150 ± 0.056a 2.075 ± 0.049AT1 1.198 ± 0.143d 1.433 ± 0.084c 1.316 ± 0.087BT2 0.912 ± 0.040e 0.982 ± 0.040e 0.947 ± 0.029CT3 0.635 ± 0.072fg 0.678 ± 0.045f 0.657 ± 0.041DT4 0.513 ± 0.063gh 0.462 ± 0.053hi 0.488 ± 0.040ET5 0.327 ± 0.069ij 0.272 ± 0.040j 0.299 ± 0.039F

Interaction (mean±SE) for fresh wt. of root.Level Variety Mean

Hybrid 1 Hybrid 2T0 2.84 ± 0.05b 3.10 ± 0.08a 2.97 ± 0.06AT1 1.34 ± 0.06e 2.02 ± 0.12c 1.68 ± 0.12BT2 1.25 ± 0.12e 1.57 ± 0.13d 1.41 ± 0.10CT3 0.84 ± 0.03fg 0.88 ± 0.05f 0.86 ± 0.03DT4 0.65 ± 0.04h 0.71 ± 0.07gh 0.68 ± 0.04ET5 0.47 ± 0.04i 0.38 ± 0.04i 0.42 ± 0.03F

Interaction (mean±SE) for dry wt. of root.Level Variety Mean

Hybrid 1 Hybrid 2T0 0.238 ± 0.010b 0.290 ± 0.010a 0.264 ± 0.010AT1 0.153 ± 0.013c 0.250 ± 0.015b 0.202 ± 0.017BT2 0.147 ± 0.010c 0.170 ± 0.014c 0.158 ± 0.009CT3 0.093 ± 0.006de 0.100 ± 0.006d 0.097 ± 0.004DT4 0.057 ± 0.003fg 0.073 ± 0.004ef 0.065 ± 0.004ET5 0.043 ± 0.004g 0.037 ± 0.005g 0.040 ± 0.003F

A gradual decrease in water contents of shoot and root was recorded (Figure

4.3(c)) in both cultivars or varieties of sunflower under both salts of arsenic and with

increase in concentration or level of arsenate or arsenite decrease in water contents of

shoot and root was observed.

150

Figure 4.3(c): Water contents of shoot and root of sunflower cultivated in arsenic contaminated soil and irrigated through As contaminated water.

4.4.3 Physiological and water relation parameters:

Salts and levels of arsenic gave significantly different values for number of

leaves, fresh, dry, turgid and specific weight of leaf as well as leaf area, while

varieties differed significantly in case of dry and turgid weight of leaf and leaf area

but non-significant differences were found in fresh weight, specific weight and

number of leaves, whereas interaction between variety and salt (V×S) revealed

significant differences (P<0.05) for fresh weight of leaf only, whereas salt into level

interaction showed significant differences (P<0.01) for all these water relation

parameters except for number of leaves. Similarly overall interaction also gave

significantly different values for all parameters other than leaf count (Table 4.4(c).

Table 4.4 (c): ANOVA of data about number, fresh, dry, turgid and specific weight of leaf and leaf area of two sunflower cultivars under different levels of As in soil and irrigation water.

Source D F

Mean squareNo. of leaves

Fresh wt. leaf

(g)

Dry wt. leaf (g)

Leaf turgid

wt.

Sp. Leaf wt.

Leaf area

Varieties (V)

1 1.68ns 0.0002ns 0.054** 1.91** 0.00011ns 1912.09**

Salts (S) 1 6.13* 2.78** 0.178** 7.01** 0.0032* 3676.82**Levels (L) 5 28.38** 2.29** 0.135** 6.11** 0.0021** 2814.42**

151

InteractionsV × S 1 0.35ns 0.23* 0.0004ns 0.039ns 0.00001ns 7.74ns

V × L 5 0.65ns 0.10* 0.011* 0.42ns 0.003** 91.76**S × L 5 1.29ns 0.57** 0.031** 2.32** 0.003** 435.71**

V × S × L 5 1.18ns 0.44** 0.017** 0.74* 0.002** 232.92**Error 48 1.11 0.036 0.003 0.25 0.0005 29.67

Interaction (mean±SE) for leaf dry wt.Level Variety Mean

Hybrid 1 Hybrid 2T0 0.597 ± 0.033a 0.547 ± 0.024a 0.572 ± 0.021AT1 0.415 ± 0.043bc 0.422 ± 0.029bc 0.418 ± 0.025BT2 0.393 ± 0.015bcd 0.368 ± 0.023cde 0.381 ± 0.014BT3 0.445 ± 0.042b 0.302 ± 0.042e 0.373 ± 0.035BCT4 0.327 ± 0.018de 0.325 ± 0.060e 0.326 ± 0.030CT5 0.313 ± 0.064e 0.197 ± 0.052f 0.255 ± 0.043D

Interaction (mean±SE) for turgid wt. of leaf.S Variety Mean

Hybrid 1 Hybrid 2S1 3.87 ± 0.18a 3.50 ± 0.17b 3.68 ± 0.13AS2 3.20 ± 0.24bc 2.92 ± 0.28c 3.06 ± 0.18BMean 3.53 ± 0.16A 3.21 ± 0.17B

Figure 4.4(c): Number of leaves, fresh and specific weight of leaf and leaf area of two sunflower cultivars under different levels of arsenic in soil and irrigation water.

152

Analysis of variance of the data regarding leaf succulence revealed that

varieties, salts, levels and all interactions among these factors showed significant

differences except variety into salt into level interaction (Table 4.5(c)) but in case of

relative water contents of leaf only varieties showed significant differences while out

of different interactions only variety into salt interaction showed significant

differences whereas all remaining interactions showed non-significant differences in

data.

Table 4.5(c): ANOVA of data for leaf succulence and RWC of leaf in two sunflower cultivars under different As levels in soil and irrigation water.


Leaf succulence Relative water contents of leaf

Varieties (V) 1 21.73** 1018.21**Salts (S) 1 5.19* 14.85ns

Levels (L) 5 2.43* 71.95ns

InteractionsV × S 1 8.13** 304.80*V × L 5 3.37** 73.23ns

S × L 5 2.98** 110.75ns

V × S × L 5 0.90ns 89.05ns

Error 48 0.78 46.44

Highest leaf succulence value was recorded in V2 in treatment T5 while least

value of leaf succulence (Table 4.6(c)) was found in T5 of V1 whereas relative water

153

contents were found highest in case of V2 under arsenite conditions but least water

contents were found in V1 under same salt.

Table 4.6(c): Interaction (mean±SE) for leaf succulence and relative water contents of leaf in two sunflower cultivars under different levels of arsenic in soil and irrigation water.Interaction (mean±SE) for leaf succulence.

Level Variety MeanHybrid 1 Hybrid 2

T0 4.38 ± 0.25d 4.58 ± 0.19cd 4.48 ± 0.15BT1 4.54 ± 0.22cd 4.80 ± 0.42cd 4.67 ± 0.23BT2 4.24 ± 0.25d 5.13 ± 0.44bcd 4.68 ± 0.28BT3 4.51 ± 0.23d 5.84 ± 0.49b 5.18 ± 0.33ABT4 4.73 ± 0.31cd 5.56 ± 0.46bc 5.14 ± 0.29ABT5 4.15 ± 0.27d 7.24 ± 1.05a 5.69 ± 0.69A

Interaction (mean±SE) for RWC of leaf.S Variety Mean

Hybrid 1 Hybrid 2S1 45.44 ± 1.75b 48.85 ± 1.86ab 47.14 ± 1.29AS2 40.42 ± 1.79c 52.05 ± 1.78a 46.23 ± 1.59AMean 42.93 ± 1.30B 50.45 ± 1.30A


4.4.4.1 Agronomic and yield parameters:

Statistical analysis of the data regarding different agronomic and yield

parameters was calculated and analysis of variance of data relevant to stem length

showed that varieties, salts and all interactions presented non-significant differences

but levels showed significant differences in data (Table 4.7(c)), similarly in case of

root length, varieties and levels showed significant differences but salts and all

interactions among factors showed non-significant differences in values. The data

regarding stem to root ratio showed completely non-significant differences in case of

varieties, salts, levels and all their interactions drawn similarly in case of number of

leaves only levels showed significant differences whereas varieties and salts and all

interactions among these factors showed non-significant differences in data. Out of

yield parameters analysis of variance of data regarding capitulum diameter revealed

that levels and interaction variety into level showed significantly different values

while all remaining factors and interactions showed non-significant differences. In

case of parameter hundred achene weight, levels of arsenic showed significant

154

differences in data whereas varieties, salts and all interactions among three factors

showed non-significant differences in data.

Table 4.7(c): ANOVA for stem length, root length, shoot to root ratio, number of leaves, capitulum diameter and hundred achene weight of sunflower cultivars under various As levels in soil and irrigation water.


Stem length

Root length

Stem : root

No. of leaves

Cap. Dia.

100 achene

wt.Varieties (V) 1 648.0ns 88.89** 4.47ns 18.00 ns 4.96 ns 0.028ns

Salts (S) 1 544.5ns 2.72ns 3.93ns 16.05 ns 0.03ns 0.109ns


V × S 1 346.7ns 2.00ns 0.001ns 1.38ns 0.15ns 0.51ns

V × L 5 306.8ns 12.52ns 1.30ns 3.93ns 10.11** 0.13ns

S × L 5 360.8ns 2.42ns 1.08ns 2.32 ns 0.74 ns 0.21ns


Error 48 241.0 6.86 1.37 6.71 1.63 0.13

Relative decrease in stem length, root length, number of leaves, capitulum

diameter and hundred achene weight was seen in both varieties with increase in

arsenate and arsenite level in soil plus irrigation water and both varieties behaved

similarly under both salts and different levels of arsenicals applied (Figure 4.7(c)).

Figure 4.7(c): Shoot length, root length, number of leaves, capitulum diameter and hundred achene weight of sunflower cultivated in As contaminated soil and irrigation through As contaminated water.

0

50

100

150

200

250

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

St L

2 (S

oil+

Wat

er)

Hybrid 1Hybrid 2

0

5

10

15

20

25

30

35

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Rt L

2 (S

oil+

Wat

er)

Hybrid 1Hybrid 2

155

0

5

10

15

20

25

30

35

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

No.L

2 (S

oil+

Wat

er)

Hybrid 1Hybrid 2

0

5

10

15

20

25

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Cap.

Dia

(Soi

l+W

ater

)

Hybrid 1Hybrid 2

00.5

11.5

22.5

33.5

44.5

T0 T1 T2 T3 T4 T5 T0 T1 T2 T3 T4 T5

S1 S2

Wt.

100

(Soi

l+W

ater

)

Hybrid 1Hybrid 2

4.4.4.2 Arsenic contents in different sunflower organs:

Analysis of variance of the data regarding As contents in different sunflower

organs revealed that in roots, salts and levels showed significant differences (P<0.01)

while varieties behaved similarly with non-significant differences in data and in case

of different interactions only variety into salt showed significant (P<0.05) differences

while remaining all interactions among varieties, salts and levels revealed non-

significant differences. In case of arsenic contents in shoot, all the three factors

including varieties, salts and levels and all interactions of these factors showed

significant differences in the data (Table 4.8(c)). Arsenic contents of leaf data analysis

revealed that only levels showed significant differences whereas varieties and salts

differed non-significantly but all interactions among data showed significant

differences among values recorded. Data analysis regarding arsenic contents of seeds

showed that out of factors varieties and levels showed significant differences but salts

showed non-significant differences and out of interactions all interactions except

156

variety into salt into level all showed significant differences in data. Left over arsenic

was also determined and statistical analysis of data revealed that varieties behaved

similarly but salts and levels of arsenic showed significant differences and out of

interactions except variety into level all interactions showed significant differences in

values.

Table 4.8(c): ANOVA for different arsenic (As) contents found in root, shoot, leaf and seed of sunflower cultivars and left over arsenic in soil.


As root As shoot As leaf As seed Left over AsVarieties (V) 1 9.8ns 307.48** 4.30ns 3.654** 6.81ns

Salts (S) 1 198.0** 116.05** 5.95ns 0.040ns 90.90*Levels (L) 5 5354.0** 1356.24** 3080.94** 23.483** 3337.79**

InteractionsV × S 1 60.9* 84.48** 86.02** 1.590** 147.63**V × L 5 24.5ns 87.35** 27.67* 1.029** 8.51ns

S × L 5 27.0ns 37.28** 24.05* 1.130** 32.48*V × S × L 5 24.8ns 78.21** 35.13** 0.369ns 62.47**

Error 48 13.0 5.40 9.03 0.217 12.70

Highest arsenic contents were recorded in roots and the order of arsenic

concentrations found in different sunflower organs was root > leaf > shoot > seed

(Figure 4.8(c)) and left over arsenic remained second highest after roots

accumulation. Both cultivars behaved similarly with gradual increase in arsenic

contents with increasing concentration of arsenicals in the treatment medium soil and

irrigation water as every rise in rhizospheric arsenic application caused increase in

contents of organs of sunflower. It was inferred from the observations that roots and

leaves proved good sink for arsenic in case of these sunflower cultivars as both

cultivars or varieties showed a little differences in values and showed overlapping

trend with least contents of arsenic in seeds or achenes which are most commonly

usable part of plant.

157

Figure 4.8(c): Arsenic contents in root, shoot, leaf and seed along with left over arsenic in soil of sunflower cultivars.

4.4.4.3 Arsenic bioaccumulative coefficient of sunflower cultivars:

Statistical analysis relevant to bioaccumulative coefficient (BC) of different

sunflower organs revealed that in case of BC root, salts and levels showed significant

differences while varieties and all interactions showed non-significant differences,

whereas in case of bioaccumulative coefficient of shoot, varieties, salts and levels all

showed significant differences and all interactions also showed significant differences

except variety into salt interaction (Table 4.9(c)). In case of BC leaf only levels and

interaction level into variety and level into salt showed significant differences while

salts and varieties and remaining interactions showed non-significant differences,

similarly in case of bioaccumulative coefficient of seed levels showed significant

differences whereas varieties, salts and all interactions among factors showed non-


Table 4.9(c): ANOVA for arsenic (As) bioaccumulative coefficient of sunflower cultivars.


BC root BC shoot BC leaf BC seedVarieties (V) 1 0.0042ns 0.0227** 0.0078ns 0.00013ns

Salts (S) 1 0.0741** 0.0056** 0.0033ns 0.00000ns


V × S 1 0.0105ns 0.0016ns 0.0039ns 0.00020ns

V × L 5 0.0103ns 0.0056** 0.0118** 0.00009ns

S × L 5 0.0168ns 0.0069** 0.0095* 0.00012ns

V × S × L 5 0.0050ns 0.0096** 0.0029ns 0.00005ns

Error 48 0.0089 0.0006 0.0032 0.00011

Bioaccumulative coefficient of root was recorded maximum with almost

similar behavior in both cultivars and under both arsenicals while secondly higher BC

158

value was in leaf with similar behavior of both cultivars under both arsenicals

arsenate as well as arsenite, whereas shoot showed intermediate value for

bioaccumulative coefficient and relative increase in values was recorded in contrast to

root and leaf BC with increasing concentration of arsenate as well as arsenite (Figure

4.9(c)). In seeds least bioaccumulative coefficient values were recorded and similar

behavior was seen in both cultivars.

Figure 4.9(c): Arsenic (As) bio-accumulative coefficient of two sunflower cultivars under different arsenic levels in soil and irrigation water.

Analysis of variance of the data regarding arsenic concentration ratios among

different organs of sunflower cultivars revealed that in case of root to shoot ratio and

root to leaf ratio of arsenic contents, levels and variety into level interaction showed

significant differences while salts and varieties and remaining interactions among

these three factors showed non-significant differences in values (Table 4.10(c)),

whereas in case of root to seed ratio of arsenic concentrations accumulated in organs

varieties and levels showed significant differences while out of different interactions

only salt into level interaction gave significantly different data while salts and other

interactions showed non-significant differences. The ratio between arsenic

concentration of shoot and arsenic concentration of leaf revealed that varieties, salts,

levels and all interactions among these three factors presented significantly different

(P<0.01) results, but in case of arsenic concentration ratio between shoot and seed

only levels differed significantly and out of different interactions variety into salt into

level interaction showed significant differences (P<0.05) while remaining interactions

remained similar with non-significant differences, similarly in case of arsenic

concentration of leaf to seed ratio analysis of variance revealed that levels caused

significant differences while varieties and salts showed non-significant differences but

out of interactions variety into level and salt into level showed significant differences

159

while variety into salt and variety into salt into level showed non-significant

differences in data.

Table 4.10(c): ANOVA for relative ratios of arsenic concentrations in different sunflower organs of two sunflower cultivars under different levels of arsenic in soil and irrigation water.


[As]root:[As]shoot [As]root:[As]leaf [As]root:[As]seedVarieties (V) 1 6.808ns 1.140ns 21368*

Salts (S) 1 14.779ns 0.448ns 24ns

Levels (L) 5 203.029** 3.367** 21290**Interactions

V × S 1 1.705ns 0.638ns 8189ns

V × L 5 19.168* 2.360** 8285ns

S × L 5 7.019ns 0.234ns 10179*V × S × L 5 15.169ns 0.055ns 8012ns

Error 48 6.591 0.467 3571


[As]shoot:[As]leaf [As]shoot:[As]seed [As]leaf:[As]seedVarieties (V) 1 0.621** 453.5ns 9623ns

Salts (S) 1 0.159** 1.2ns 3175ns

Levels (L) 5 0.362** 1785.0** 14339**Interactions

V × S 1 0.090* 712.5ns 8081ns

V × L 5 0.097** 150.3ns 7215*S × L 5 0.075** 204.4ns 6658*

V × S × L 5 0.190** 730.6* 5790ns

Error 48 0.021 270.5 2761

4.4.4.4 Phosphorus (P), calcium (Ca) and magnesium (Mg) contents in root,

shoot, leaf and seed of sunflower cultivars under different arsenic concentrations

in soil as well as irrigation water.

Varieties, salts and levels showed significant differences in case of phosphorus

contents of root while out of interactions salt into level interaction showed significant

(P<0.05) differences in values while in case of P contents of shoot varieties and levels

showed significant differences and variety into salt and variety into level interaction

showed significant differences among values (Table 4.11(c)) whereas in case of

phosphorus contents of leaf varieties and levels and all interactions except variety into

salt into level interaction showed significant differences among data similarly in case

of phosphorus in seed, salts and levels showed significant differences while out of

160

different interactions among the three factors variety into salt and salt into level

interaction showed significant differences in values recorded.

As revealed by ANOVA in case of calcium contents of root, arsenic salts,

levels and all interactions among varieties, salt and levels showed significant

differences only varieties showed non-significant differences, while in case of shoot

calcium contents all the three factors as well as interactions among three factors

showed significant differences (P<0.01) in data (Table 4.11(c)). Data regarding

calcium contents of leaf showed that varieties and levels caused significant

differences in values and all interactions except variety into salt showed significant

differences similarly in case of Ca contents in seed, varieties and all interactions

among varieties, salts and levels showed significant differences but salts and levels

showed non-significant differences among data.

Analysis of variance of the data regarding magnesium contents of different

sunflower organs revealed that varieties and levels and all interactions among

varieties, salts and levels showed significant differences among data only salts

showed non-significant differences in case of magnesium contents of root (Table

4.11(c)), while in case of shoot, varieties, salts and levels and all interactions except

variety into salt showed significant differences in data. Analysis regarding Mg

contents of leaf revealed that varieties, levels and all interactions showed significant

differences only salts showed non-significant differences among data. In case of

magnesium contents of seed, salts and levels showed significant differences while

varieties and all interactions among three factors showed non-significant differences

among data.

Table 4.11(c): ANOVA for phosphorus (P), calcium (Ca) and magnesium (Mg) contents in sunflower cultivars grown in different levels of As in soil and irrigation water.


P root P shoot P leaf P seedVarieties (V) 1 146.55* 183.20** 228.48** 48.30ns

Salts (S) 1 222.53** 4.00ns 79.93ns 849.34**Levels (L) 5 202.05** 151.48** 214.70** 852.31**

InteractionsV × S 1 27.28ns 158.87** 130.52* 423.55*V × L 5 34.81ns 52.66** 124.35** 85.53ns

S × L 5 66.77* 32.74ns 125.90** 447.81**V × S × L 5 36.74ns 30.44ns 44.93ns 97.01ns

Error 48 24.62 14.11 28.27 57.00

161


Ca root Ca shoot Ca leaf Ca seedVarieties (V) 1 16943ns 5003840** 103452629** 334859**

Salts (S) 1 254456* 1518243** 304051ns 11000ns

Levels (L) 5 1484105** 2280693** 131968546** 40844ns

InteractionsV × S 1 2492966** 4230680** 12092714ns 299303**V × L 5 677514** 2662796** 79484765** 115026*S × L 5 1174397** 1941889** 59788836** 187629**

V × S × L 5 806235** 632535** 31356138** 86985*Error 48 57479 66782 3585912 35468


Mg root Mg shoot Mg leaf Mg seedVarieties (V) 1 533341** 1263488** 906226** 71065ns

Salts (S) 1 135476ns 233857* 53447ns 176082*Levels (L) 5 295475** 457134** 2693604** 159826**

InteractionsV × S 1 641622** 1545ns 1330526** 60ns

V × L 5 185717** 1194949** 1955429** 28721ns

S × L 5 232025** 719540** 1165477** 47863ns

V × S × L 5 442261** 252852** 1080223** 68304ns

Error 48 41200 49862 44108 42393

Phosphorus contents in seeds were found maximum in case of both cultivars

with relatively higher values recorded in V2 (FH-415) under both salts or arsenic but

higher concentrations of arsenicals caused relative decrease in phosphorus contents

(Figure 4.10(c)). Leaves showed less P contents than seeds but higher than root and

shoot with overlapping values of phosphorus concentration in both varieties and under

both arsenicals. Root and shoot showed almost similar contents of phosphorus under

both arsenicals with similar behavior in both cultivars of sunflower as in case of

arsenite application V2 (FH-415) showed a little higher phosphorus contents in root

with considerably low effects of various levels of arsenate and arsenite in soil and

irrigation water.

Calcium contents of root remained similar in all different treatments or levels

of arsenic in case of both arsenicals applied similarly very little variation in data was

recorded in case of calcium contents of shoot with relatively higher values recorded in

plants belonging to V2 (FH-415) but overall same trend was seen in both cultivars

under various arsenic levels only Ca contents of leaf showed maximum variation with

highest contents and a decrease was seen in calcium contents in higher concentrations

of arsenic but overall both varieties showed a lot of variation in values (Figure

162

4.10(c)). Seed or achenes showed least contents of calcium in them with minimum

variation in data and almost similar behavior of both cultivars under both arsenicals.

Magnesium contents were found minimum in root with a decrease in V1 (FH-

385) from T0 (control) up to T4 (80 mg As/kg soil + 8 mg As/L solution) while

maximum value was in T5 (100 mg As/kg soil + 10 mg As/L solution) in V1 under

arsenate while V2 (FH-415) showed first increase from control in T1 (20 mg As/kg

soil + 2 mg As/L solution) then decrease in T2 (40 mg As/kg soil + 4 mg As/L

solution) and decrease was continuous up to T5, while in case of arsenite V1 showed

first increase and then decrease in magnesium contents while in V2 values remained

similar in all treatments or levels of arsnite (Figure 4.10(c)). Magnesium contents of

shoot were found a little higher than root as in case of arsenate V1 showed least

variation while V2 showed first decrease up to T2 and then increased Mg contents

were observed in T4 whereas under arsenite conditions V1 showed decrease in Mg

contents in higher arsenite conditions and in V2 level T1 showed least value of

magnesium contents but then rise in contents was observed after T1. Highest contents

of magnesium were found in leaves of control plants belonging to V1under arsenate

as well as arsenite and then decrease was seen in lower arsenate concentrations but

magnesium contents were increased under higher arsenate levels and similar trend

was seen in V2 under arsenate while in arsenite V1 showed decrease from control in

all levels of arsenite but V2 showed first increase and then decrease was seen. In case

of seed least variation was seen in Mg contents under both arsenicals and in both

varieties overlapping values were observed.

Figure 4.10(c): Phosphorus (P), calcium (Ca) and magnesium (Mg) contents in different organs of sunflower cultivars grown under As contaminated soil and irrigation water.

163

4.4.4.5 Potassium (K), boron (B) and copper (Cu) contents of sunflower under

different As levels in rooting medium.

Salts and levels caused significant differences in potassium contents of root

but varieties showed non-significant differences and all interactions among these three

factors showed significant differences among data as revealed by analysis of variance

(Table 4.12(c)). In case of K contents of shoot and leaves factors varieties, salts,

levels and all interactions except variety into salt showed significant differences in

data while in case of potassium contents of seed, salts and levels showed significant

differences in data while varieties showed non-significant differences and out of

interactions among factors all interactions except variety into salt showed significant

differences among analysis of variance data regarding potassium contents of seed or

achenes.

Analysis of variance of the data regarding boron contents in root revealed that

salts caused significant differences while varieties and levels showed non-significant

differences similarly all interactions among varieties of sunflower and salts and levels

of arsenic showed significant differences in data (Table 4.12(c)). In case of boron

contents of shoot, varieties, salts, levels and all interactions among these factors

except salt into level interaction showed significant (P<0.01) differences and in case

of leaf B contents, varieties and levels showed significant differences but salts showed

164

non-significant differences while all interactions except variety into salt showed

significant differences in data. Data analysis regarding boron contents in seed

revealed that varieties, salts and levels and all interactions among these factors

showed significant differences in values.

Data analysis regarding copper contents in root revealed that levels showed

significant differences while varieties and salts showed non-significant differences

and out of interactions only variety into salt and variety into salt into level interaction

showed significant differences but variety into level and salt into level interactions

showed non-significant differences as shown by analysis of variance of the data

(Table 4.12(c)). In case of Cu contents of shoot, varieties showed significant

differences but non-significant differences were seen in salts and levels similarly in

case of interactions only variety into level interaction showed significantly different

data while remaining interactions among factors showed non-significant differences.

Data analysis regarding copper contents of leaf revealed that varieties, salts and levels

showed significant differences while out of different interactions variety into level

and salt into level showed significant differences while variety into salt and variety

into salt into level interaction showed non-significant differences among data

similarly in case of Cu contents of seed, varieties, salts, levels and all interactions

except variety into salt showed significant differences in values recorded.

Table 4.12(c): ANOVA for potassium (K), boron (B) and copper (Cu) contents of sunflower under As contaminated soil plus irrigation water.


K root K shoot K leaf K seedVarieties (V) 1 6536ns 36072588** 8544930** 458804ns

Salts (S) 1 77966705** 1526718** 6094826** 4057465**Levels (L) 5 32595248** 26910843** 5954336** 7357110**

InteractionsV × S 1 2101250** 98050ns 92159ns 791179ns

V × L 5 23455778** 23682218** 3236210** 1627162**S × L 5 12256098** 5762113** 6673316** 3774535**

V × S × L 5 33897942** 8868831** 5411651** 3046249**Error 48 170937 177861 308866 454041


B root B shoot B leaf B seedVarieties (V) 1 10.035ns 106.313** 626.40** 7.088*

Salts (S) 1 158.361** 1780952** 102.70ns 25.884**Levels (L) 5 10.051ns 29.108* 405.76** 6.920**

InteractionsV × S 1 318.024** 314.545** 4.00ns 6.716*

165

V × L 5 75.481** 78.349** 548.07** 6.852**S × L 5 14.722* 9.477ns 937.75** 9.129**

V × S × L 5 20.525** 28.342* 1773.96** 6.218**Error 48 5.068 9.213 39.72 1.177


Cu root Cu shoot Cu leaf Cu seedVarieties (V) 1 0.044ns 16.801* 62.031* 654.44**

Salts (S) 1 0.062ns 5.963ns 113.578** 531.87**Levels (L) 5 22.474** 4.518ns 82.996** 119.25**


V × L 5 9.352ns 9.784* 61.794** 71.54**S × L 5 10.254ns 0.426ns 68.310** 142.70**

V × S × L 5 16.470* 4.100ns 22.439ns 74.80**Error 48 5.847 3.496 9.405 14.29

In case of potassium contents of root both varieties behaved a bit differently as

V1 showed first increase under low arsenate concentrations or levels and then relative

increase in K contents was recorded in maximum arsenate condition (Figure 4.11(c))

while V2 showed decrease in K contents under higher arsenate levels but in case of

arsenite conditions only T2 showed higher potassium level in root of both cultivars

and higher arsenite contamination caused reduction in K contents of root. Potassium

contents in shoot were also first increased in V2 under arsenate and then in maximum

arsenate a decrease was recorded in K contents of shoot of V2 plants while V1

showed less variation under different levels of arsenate as compared to V2 but in case

of arsenite conditions both cultivars showed first increase and then decrease in K

contents of shoot with increasing level of arsenite in rooting medium. Highest

contents of K in shoot were recorded in V2 plants under both arsenicals. K contents of

leaves were recorded first increased under low arsenate conditions but in T4 both

varieties showed opposite behavior as V1 showed highest while V2 showed least K

contents in shoot of sunflower plants but under arsenite gradual increase was recorded

in both cultivars with increasing arsenite concentrations. A lot of variation in K

contents of seed was also recorded in both sunflower cultivars with relatively higher

values in case of V2 plants under both arsenicals used while comparatively less

variation in seed K contents was recorded in plants belonging to V1 as compared to

V2 in which levels of arsenate and arsenite caused a remarkable variation in K

contents of seed. Overall K contents were recorded overlapping in all organs of

sunflower under both arsenicals used for contamination of rooting medium showing a

gradual rise from root to shoot to leaves to seeds.

166

Boron contents in leaves were found highest among all organs (Figure

4.11(c)). In root under arsenate conditions V2 showed the least values of boron

contents while in arsenite conditions this cultivar gave higher values than V1 plants

but values remained same with minimum variation under different levels of arsenate

and arsenite. Shoot showed resembling values of boron contents with root and both

cultivars showed less variation than leaves in which highest variation among B

contents at different arsenate and arsenite levels was recorded. Leaves of V2 plants

under T4 (80 mg As/kg soil + 8 mg As/L solution) presented highest value for boron

contents under arsenate conditions and under T1 in case of arsenite contamination

while V1 showed higher boron contents under T3 and T5 when arsenate was applied

and T3 and T4 under arsenite.

Similar copper contents were found in root and shoot with relatively

higher variation in values recorded in root than shoot under different arsenate and

arsenite levels as in case of root higher Cu contents were shown by plants of V2 under

arsenate while under arsenite V1 showed higher copper contents of root than V2

whereas in shoot both cultivars behaved similarly under both arsenicals and all

different levels of arsenicals used. A lot of variation among copper contents of leaf

was recorded in both cultivars under both arsenicals especially under arsenite the

reduction in Cu contents was recorded in both cultivars with increasing level or

concentration of arsenite. Copper contents in seed were recorded highest in both

cultivars under both arsenicals but under arsenate V1 showed maximum Cu contents

in level T3 whereas overall lower values were shown by plants belonging to V2 under

higher arsenite and arsenate levels.

Figure 4.11(c): Potassium (K), boron (B) and copper (Cu) contents in sunflower cultivated in As contaminated soil and irrigation water.

167

4.4.4.6 Iron (Fe), manganese (Mn) and zinc (Zn) contents in sunflower under

different arsenic salts and levels through soil plus irrigation water.

Analysis of variance of the data regarding iron contents in root revealed that

varieties and varieties into salt interaction showed significant (P<0.05) differences

whereas salts and levels of arsenic and remaining interactions among these three

factors showed non-signficant differences among values recorded (Table 4.13(c)). Fe

contents of shoot data analysis revealed that varieties of sunflower and levels of

arsenicals showed significant (P<0.01) differences while salts showed non-significant

differences similarly all interactions except variety into salt showed significant

differences in data while in case of iron contents of leaf, levels showed significant

differences but salts and varieties showed non-significant differences and all

interactions except salt into level showed significant differences in values whereas in

case of iron contents of seed all the three factors and interactions among these factors


Data analysis regarding manganese contents in root, shoot and leaf revealed

that varieties, salts and levels and all interactions among these factors showed

significant differences among values recorded (Table 4.13(c)) but in case of Mn

contents of seed, only variety into level interaction showed significant differences

168

while varieties, salts and levels and all other interactions showed non-significant

differences among data.

Analysis of data regarding zinc contents in sunflower root, shoot and leaves

revealed that varieties or cultivars of sunflower, salts and levels of arsenic showed

significant differences in values similarly all interactions among these three factors

also showed significant differences (P<0.01) except variety into alt interaction in case

of zinc contents of root only (Table 4.13(c)). In case of zinc contents of seed, levels

and all interactions among varieties, salts and levels showed significantly different

values while varieties and salts independently showed non-significant differences

among data recorded.

Table 4.13(c): ANOVA for iron (Fe), manganese (Mn) and zinc (Zn) contents in sunflower under different As conditions in soil and irrigation water.


Fe root Fe shoot Fe leaf Fe seedVarieties (V) 1 6394085* 50690** 17ns 336ns

Salts (S) 1 131767ns 216ns 746ns 0ns

Levels (L) 5 1374501ns 25214** 159079** 328ns

InteractionsV × S 1 7100840* 2353ns 44395* 93ns

V × L 5 2674647ns 32574** 74226** 1234ns

S × L 5 2212826ns 14689* 20525ns 443ns

V × S × L 5 1969606ns 18569** 95081** 185ns

Error 48 1328765 4667 9506 1504


Mn root Mn shoot Mn leaf Mn seedVarieties (V) 1 1169.67** 376.80** 44.35* 0.037ns

Salts (S) 1 794.28** 73.83** 51.60* 17.376ns

Levels (L) 5 407.66** 204.74** 640.10** 4.608ns

InteractionsV × S 1 49.67* 140.53** 192.37** 5.030ns

V × L 5 502.10** 336.72** 1114.55** 21.125*S × L 5 655.76** 85.46** 108.88** 13.363ns

V × S × L 5 116.16** 127.30** 281.89** 10.213ns

Error 48 8.06 7.04 10.15 7.425


Zn root Zn shoot Zn leaf Zn seedVarieties (V) 1 524.99** 165.89** 4660.3** 73.94ns

Salts (S) 1 73.53** 676.69** 4573.2** 65.53ns


V × S 1 31.50ns 139.19** 2906.5** 176.19**V × L 5 315.99** 1688.59** 1483.6** 99.65**

169

S × L 5 239.70** 204.95** 1861.3** 117.35**V × S × L 5 337.09** 294.48** 1026.8** 95.13**

Error 48 8.38 9.87 13.1 18.54

Roots showed highest iron contents in case of both arsenicals with variation in

both varieties under different levels of arsenate and arsenite (Figure 4.12(c)) as in case

of arsenate V2 plants gave maximum values under higher arsenate concentrations

while plants belonging to V1 showed decrease in iron contents with increase in

arsenate levels while under arsenite conditions T2 showed higher iron contents in root

in both cultivars or varieties of sunflower. Shoot showed lower iron contents than root

and leaves with less variation in different levels of arsenate as well as arsenite, while

leaves showed rise in iron contents with increasing arsenic concentrations in the form

of either arsenate or arsenite but seeds or achenes showed least iron contents with

uniform values under all levels of arsenate and arsenite.

In case of root a lot of variation was recorded in manganese contents under

different levels of arsenate as well as arsenite (Figure 4.12(c)) as both varieties

behaved almost similarly with a decreasing trend towards higher arsenic

concentrations with least values recorded in V1 plant roots but V2 plants showed

relatively higher Mn contents in roots except a maximum value in case of V1 plants

while under arsenite conditions V2 showed highest manganese contents than V1

plants especially under higher arsenite concentrations or levels. In shoot manganese

contents were recorded lowest than root, leaves and even seeds except a single highest

value in V2 plants under maximum arsenate level (T5) but under arsenite relatively

lower and smooth trend with little variation was shown by both cultivars with a little

increase in Mn contents under higher arsenite levels by V2 plants. Manganese

contents in leaves were higher than shoot and seed but in same range with root and

both cultivars showed much more variation in values under both arsenicals as under

arsenate both cultivars showed different behavior with increase in arsenate

concentrations but overall V2 showed a little higher manganese contents under both

arsenicals. In seeds both cultivars showed resembling values regarding Mn contents

with least variation in data and similar trend was seen in both, varieties or cultivars

under both arsenicals.

Zinc contents in root were found varied under different levels of arsenate as

well as arsenite but within a narrow range in arsenate than arsenite in both cultivars.

In root V1 plants showed maximum zinc contents under T5 using arsenate and T2

170

under arsenite while V1 showed similar behavior under both arsenicals (Figure

4.12(c)). In shoot Zn contents were found a little higher than root with similar trend

shown by V2 plants under both arsenic salts while V1 plants showed a little different

behavior under both arsenicals with less variation in values under different levels of

arsenate and a least value of Zn contents in shoot under T5 plants while in arsenite

treated plants T2 showed highest Zn contents and then in further levels of arsenite a

decrease in zinc contents of shoot was recorded. Zinc contents in leaves were found

same as shoot and both salts caused variation in both sunflower cultivars with overall

highest zinc contents in leaves of V2 plants under T1 and T2 using arsenite while V1

plants showed less zinc contents than control in all arsenate levels but in case of

arsenite V2 plants showed variation in values under different levels. In seeds average

highest Zn contents were found with same range of values in both cultivars under

arsenate as well as arsenite.

Figure 4.12(c): Iron (Fe), manganese (Mn) and zinc (Zn) contents in sunflower cultivars under different As conditions.

171

4.4.4.7 Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in sunflower

under different As conditions.

Analysis of variance of the data regarding Mo contents in root revealed that

levels of arsenicals showed significant differences while varieties and salts showed

non-significant differences and out of different interactions variety into salt and

variety into salt into level interaction showed significant differences but variety into

level and salt into level interaction showed non-significant differences (Table

4.14(c)). In case of molybdenum contents of shoot, varieties and levels showed

significant differences while salts behaved similarly while all interactions except

variety into salt showed significant (P<0.01) differences among data. Only levels

showed significant differences in case of Mo contents of leaf whereas varieties and

salts and all interactions among three factors showed non-significant differences,

similarly in case of molybdenum contents of seed, varieties and levels showed

significant differences and out of different interactions only variety into salt into level

interaction showed significant differences among all interactions of the factors

calculated.

Analysis of variance of the data regarding silver contents in root revealed that

varieties, salts and levels and all interaction among these factors showed significant

differences (P<0.01) among data (Table 4.14(c)) similarly in case of Ag contents of

shoot also varieties, salts and levels showed significant differences while all

interactions except variety into salt into level showed significant differences. In case

of silver contents of leaf too varieties and salts and all interactions showed significant

differences only levels showed non-significant differences (P>0.05) and in case of Ag

contents of seed all the three factors varieties, salts and levels and interactions among

these three factors showed significant differences among values recorded.

172

Data analysis regarding aluminum contents of root revealed that varieties and

levels and different interactions among varieties, salts and levels showed significant

differences while only salts showed non-significant differences (Table 4.14(c)). In

case of Al contents in shoot, varieties and levels showed significant (P<0.01)

differences while salts showed non-significant differences and all interactions among

varieties, salts and levels showed significant differences except variety into salt

interaction. Analysis of data regarding aluminum contents of leaves revealed that only

levels showed significant differences while varieties and salts showed non-significant

differences and out of different interactions variety into level and variety into salt into

level interaction showed significant differences, while in case of aluminum contents

of seed, salts and levels showed significant differences but varieties showed non-

significant differences and all interaction except variety into salt showed significant

differences among the values recorded.

Table 4.14(c): ANOVA for molybdenum (Mo), silver (Ag) and aluminum (Al) contents in sunflower under different As conditions.


Mo root Mo shoot Mo leaf Mo seedVarieties (V) 1 1.364ns 0.980** 1.789ns 1.467*

Salts (S) 1 0.710ns 0.002ns 2.160ns 0.024ns


V × S 1 3.786** 0.005ns 1.293ns 0.235ns

V × L 5 0.214ns 0.371** 2.203ns 0.524ns

S × L 5 0.072ns 0.172** 0.871ns 0.667ns

V × S × L 5 2.823** 0.084** 1.429ns 0.705*Error 48 0.404 0.013 1.091 0.291


Ag root Ag shoot Ag leaf Ag seedVarieties (V) 1 306.57** 254.93** 149.73** 673.9**

Salts (S) 1 415.63** 74.62** 58.95** 33.4*Levels (L) 5 53.19** 38.32** 2.76ns 925.2**

InteractionsV × S 1 252.26** 67.86** 49.08** 373.0**V × L 5 60.68** 19.50** 15.74** 1918.0**S × L 5 68.05** 5.49* 10.41** 600.6**

V × S × L 5 65.27** 11.53ns 7.12* 1113.8**Error 48 1.14 1.89 2.35 7.9


Al root Al shoot Al leaf Al seedVarieties (V) 1 2316632** 16551** 335ns 82.56ns

Salts (S) 1 40918ns 51ns 8573ns 242.29*

173

Levels (L) 5 261834* 25698** 77115** 385.53**Interactions

V × S 1 5805522** 928ns 27151ns 77.92ns

V × L 5 615625** 17312** 58945** 207.78**S × L 5 1020943** 8126** 29145ns 782.05**

V × S × L 5 486015** 10084** 83442** 293.79**Error 48 77654 923 14291 36.70

Molybdenum contents of root were found increasing in V2 plants under

arsenate while V1 plants under arsenite, while in shoot least values for Mo contents

were seen than root, leaves and seeds with relatively higher values recorded in plants

belonging to V2 cultivar in shoot (Figure 4.13(c)). Leaves showed highest Mo

contents in both cultivars with a lot of variation in data under different levels of

arsenate and arsenite but both cultivars showed an increasing trend towards higher

arsenicals concentrations. Seeds showed Mo contents in the same range with root but

less than leaves with increasing values towards higher arsenic treatments either as

arsenate or arsenite.

Highest variation among values was recorded in case of silver contents

of seed while root showed least variation under arsenate in both cultivars but under

arsenite V2 showed increasing silver contents with increasing arsenite concentrations

or levels whereas V1 showed lower silver contents except T4 plants (Figure 4.13(c)).

Silver contents in shoot and leaves were found same under different arsenate and

arsenite levels in case of V1 plants but plants belonging to V2 showed higher silver

contents under higher arsenate and arsenite levels in rooting medium. In case of seeds

silver contents were found very different under different levels of arsenate as well as

arsenite with decrease from control plants under different treatments of arsenate and

arsenite.

Highest aluminum contents were recorded in roots of both sunflower

cultivars with maximum concentrations shown by V2 plants under arsenate with an

increase in Al contents than V1 plants which showed a relative decrease in Al

contents with increasing arsenate concentrations while under arsenite both cultivars

showed overlapping values with highest in V1 plants under T2 of arsenite (Figure

4.13(c)). In shoot both cultivars showed resembling contents of aluminum with less

variation than root but in leaves both cultivars showed higher Al contents than shoot

but less than root with relative increase in Al contents in plants of V2 with an increase

in arsenite concentrations than in arsenate while V1 plants showed similar values

174

under different arsenite levels but a little variation was recorded in plants of V2 under

both arsenicals. In seeds least Al contents were found which can be said undetectable

in both sunflower cultivars.

Figure 4.13(c): Molybdenum (Mo), silver (Ag) and aluminum (Al) contents in sunflower under different As conditions.

4.4.4.8 Barium (Ba) and bismuth (Bi) contents in sunflower under different

arsenic conditions.

Statistical analysis of the data regarding barium contents in root revealed that

varieties, salts and levels showed significant (P<0.01) differences and all interactions

among these three factors except variety into salt interaction showed significant

differences among data recorded (Table 4.15(c)). Analysis of data about Ba contents

175

in shoot revealed that varieties and levels and all interactions among the factors

showed significantly different values whereas only salts showed non-significant

differences but in case of leaves, varieties, salts and levels and all their interactions

showed significant differences among data. In case of barium contents of seed also

varieties and levels showed significant differences while salts showed non-significant

differences whereas all interactions among the three factors showed significant

differences among data recorded.

Analysis of variance of the data regarding bismuth contents in root revealed

non-significant differences in varieties, salts and levels (Table 4.15(c)), similarly out

of different interactions only variety into salt interaction showed significant

differences (P<0.01) while remaining all interactions showed non-significant

differences among values recorded while in case of bismuth contents of shoot only

levels of arsenicals showed significant differences while varieties, salts and all

interactions among these three factors showed non-significant differences among data.

In case of Bi contents of leaves, salts and levels of arsenic showed significant

differences and out of different interactions variety into level and salt into level

interaction showed significant differences while remaining interactions showed non-

significant differences but in case of Bi contents in seed only levels of arsenic showed

significant differences and all interactions except salt into level showed significant

differences but factors varieties and salts showed non-significant differences among

different values of bismuth contents recorded.

Table 4.15(c): ANOVA for barium (Ba) and bismuth (Bi) contents in sunflower under different As conditions.


Ba root Ba shoot Ba leaf Ba seedVarieties (V) 1 852.50** 5124.8** 1228.84** 425.06**

Salts (S) 1 950.12** 19.1ns 435.08** 4.10ns


V × S 1 8.93ns 381.1** 276.32* 67.16*V × L 5 390.0** 2659.6** 234.25** 66.38**S × L 5 195.48** 242.2** 137.73** 44.57**

V × S × L 5 432.66** 244.4** 340.39** 73.33**Error 48 9.67 13.8 40.25 10.98


Bi root Bi shoot Bi leaf Bi seedVarieties (V) 1 0.754ns 0.001ns 7.586ns 43.32ns

Salts (S) 1 0.553ns 2.864ns 35.94* 26.10ns

176

Levels (L) 5 5.460ns 9.573** 89.97** 95.86**Interactions

V × S 1 71.26** 0.985ns 17.435ns 139.31**V × L 5 5.867ns 1.437ns 42.901** 48.39**S × L 5 6.625ns 0.622ns 49.968** 22.26ns

V × S × L 5 9.127ns 3.959ns 15.228ns 49.46**Error 48 7.158 2.621 8.620 11.90

A lot of variation in barium contents was observed in different organs of

sunflower cultivars under arsenate as well as arsenite (Figure 4.14(c)) as in roots V1

showed lower Ba contents than V2 plants under arsenate while in case of arsenite T3

of V2 plants showed maximum barium contents than all levels of arsenite out of both

cultivars. Similarly in shoot control plants of V2 showed highest Ba contents than all

levels of arsenate and arsenite with a relatively higher proportion of Ba contents in

plants belonging to V2 cultivar which showed relatively higher contents of barium

than V1 plants overall under both arsenicals except a single value in T4 plants using

arsenate but in arsenite treatments or levels overall V2 showed higher barium

contents. In case of barium contents of leaf also V2 plants showed relatively higher

Ba contents as compared to V1 plants under both arsenicals used but contents

remained in same range with root and shoot except a single higher value recorded in

plants of V2 cultivar under T5 using arsenite while in seeds average lowest barium

contents were recorded in both cultivars under arsenate and arsenite with relatively

higher Ba contents in plants of V2 under different levels of arsenate and arsenite.

Bismuth contents of root were recorded higher in V2 plants under arsenate as

compared to V1 while under arsenite conditions V1 showed a little higher Bi content

in levels T2 and T4 (Figure 4.14(c)) while in shoot both cultivars showed similar

values of Bi contents under both arsenicals and in both cultivars with a little variation

and in same range with bismuth contents of root. In leaves higher contents of Bi were

recorded in both cultivars with much variation in values as T1 of arsenate showed

minimum bismuth contents in both cultivars while under arsenite a decreasing trend

was seen in Bi contents with increasing level of arsenite in both cultivars but T1

plants of V1 cultivar showed a drop in bismuth contents than V2 plants. Seeds

showed relatively highest Bi contents than all organs of sunflower with handsome

variation under different levels or treatments of arsenate and arsenite with a highest

value in V1 plants under T3 level in arsenate conditions.

177

Figure 4.14(c): Barium (Ba) and bismuth (Bi) contents in sunflower under

different As conditions.

4.4.4.9 Cadmium (Cd), cobalt (Co) and chromium (Cr) contents in sunflower

under different arsenic conditions.

Data analysis regarding cadmium contents in root revealed that varieties, salts,

levels all interactions among these three factors showed significant differences

(P<0.01) among values recorded (Table 4.16(c)) while in case of Cd contents of shoot

only levels showed significant differences while varieties and salts showed non-

significant differences similarly out of different interactions among the three factors

only variety into level showed significant differences whereas remaining interactions

among varieties, salts and levels showed non-significant differences. In case of

cadmium contents of leaves, varieties and salts showed significant (P<0.05)

differences similarly levels and all interactions among varieties, salts and levels also

showed significant differences among data and in case of Cd contents of seed also

varieties, salts and levels and all interactions except variety into salt interactions also

showed significant differences among data recorded during the course of experiment.

Statistical analysis of the data regarding cobalt contents in root revealed that

levels of arsenicals showed significant differences while varieties of sunflower and

salts of arsenic used showed non-significant differences whereas all of the interactions

among these three factors also showed significant (P<0.01) differences among values

178

recorded (Table 4.16(c)). In case of Co contents of shoot salts and levels of arsenic

showed significant differences while varieties or cultivars of sunflower showed non-

significant differences but out of different interactions only salt into level interaction

showed significantly different values but remaining interactions showed non-

significant differences. Analysis of data regarding cobalt contents in leaves revealed

that levels of arsenicals showed significant differences while varieties and salts and all

interactions among the factors except variety into salt into level interaction showed

non-significant differences while in case of Co contents in seeds or achenes all the

three factors i-e varieties, salts and levels and their interactions showed non-

significant differences among different values recorded during the course of

experiment.

Two way analysis of variance of the data regarding chromium contents in root

revealed that varieties, salts and levels and all of the interactions among these three

factors showed significant differences among various values recorded (Table 4.16(c))

similarly in case of Cr contents of shoot all the three factors including varieties of

sunflower, salts and levels of arsenic and different interactions among these three

factors showed significant differences (P<0.01) among data. Data analysis regarding

chromium contents in leaves revealed that only levels of arsenicals showed significant

differences (P<0.05) while varieties of sunflower and salts of arsenic used showed

non-significant differences whereas out of different interactions only salt into level

interaction showed significant differences (P<0.05) while remaining interactions

showed non-significant differences (P>0.05) among the values recorded. In case of Cr

contents of seeds or achenes only levels of arsenic showed significant differences

while cultivars or varieties of sunflower and salts of arsenic used showed non-

significant differences and out of different interactions among these three factors only

variety into level interaction showed significant differences while remaining

interactions showed non-significant differences among the values recorded.

Table 4.16(c): ANOVA for cadmium (Cd), cobalt (Co) and chromium (Cr) contents in sunflower under different As conditions.


Cd root Cd shoot Cd leaf Cd seedVarieties (V) 1 0.744** 0.225ns 1.510* 1.274**

Salts (S) 1 1.366** 0.044ns 1.240* 0.220*Levels (L) 5 0.795** 0.537** 1.628** 0.317**


179

V × L 5 0.742** 0.288** 2.845** 0.308**S × L 5 0.739** 0.217ns 2.958** 0.335**

V × S × L 5 0.875** 0.006ns 2.116** 0.213**Error 48 0.064 0.081 2.262 0.043


Co root Co shoot Co leaf Co seedVarieties (V) 1 0.159ns 0.0006ns 0.0091ns 0.0435ns

Salts (S) 1 0.014ns 0.0800** 0.0023ns 0.0059ns

Levels (L) 5 0.334** 0.0320* 0.0900** 0.1917ns


V × L 5 0.338** 0.0150ns 0.0188ns 0.2845ns

S × L 5 0.240** 0.0470** 0.0142ns 0.0772ns

V × S × L 5 0.243** 0.0199ns 0.0334* 0.0454ns

Error 48 0.058 0.0099 0.0105 0.1960


Cr root Cr shoot Cr leaf Cr seedVarieties (V) 1 27.380* 31.787** 0.356ns 0.116ns

Salts (S) 1 83.033** 20.587** 1.674ns 0.041ns

Levels (L) 5 13.579* 12.701** 3.186* 1.301**Interactions

V × S 1 113.001** 5.249* 0.076ns 0.073ns

V × L 5 23.656** 17.190** 0.295ns 2.013**S × L 5 22.646** 19.246** 1.194* 0.015ns

V × S × L 5 13.600* 6.372** 0.639ns 0.271ns

Error 48 4.439 1.133 0.421 0.331

In root cadmium contents remained very low under all different levels of

arsenate in both cultivars while under arsenite V2 plants under level T4 showed

higher Cd contents while remaining levels showed similar lower contents in both

cultivars (Figure 4.15(c)). Cd contents in shoot were also found similar in both

cultivars except a little higher value in control plants of V1 and a little higher value

were recorded in V2 plants under arsenite levels or treatments. In leaves relatively

higher Cd contents were detected than root and shoot especially in plants belonging to

V1 under T3 and T4 levels of arsenate while under arsenite V2 plants showed higher

Cd contents especially in level T5 which showed highest value for Cd contents in

leaves than all organs of sunflower. In seeds or achenes also the plants of V2

exhibited higher values of cadmium contents than other plant organs under both

arsenicals used as compared to V1 plants.

Highest cobalt contents were recorded in roots with maximum variation

among data especially in V2 plants under arsenate in which T3 and T4 showed

180

maximum Co contents as compared to V1 plants in which a gradual reduction was

observed in different levels of arsenate but in case of arsenite V1 plants showed

higher cobalt contents than V2 plants especially in T2 level of arsenite (Figure

4.15(c)). Cobalt contents of shoot were recorded least under both arsenic salts with

much lower values recorded in V2 plants especially under higher levels of arsenate

while in leaves a gradual increase was observed in Co contents under arsenite levels

in plants belonging to V2 cultivar but in arsenate this cultivar showed least value in

T1 and V1 plants showed zero variation in first three levels but then rise was recorded

in T4 and T5. In case of seeds Co contents were found with much variation in both

cultivars as in arsenate T3 and T4 of V2 showed least value and in case of arsenite

this cultivar showed lower most values of Co contents under higher arsenite contents.

Chromium contents in root were found highest overall under both arsenicals or

salts of arsenic especially in plants belonging to V2 showed gradual increase in Cr

contents with increasing arsenate levels whereas V1 plants showed less chromium

contents under different levels of arsenate applied (Figure 4.15(c)) while in case of

arsenite highest Cr contents were recorded in plants of cultivar V1 under lower

arsenite concentrations than plants belonging to cultivar V2 but both cultivars showed

handsome variation in chromium contents of root. In shoot and leaves chromium

contents were found in similar range under both salts of arsenic as in shoot overall V2

plants showed a bit higher Cr contents than V1 plants under both arsenicals, same was

the situation in shoot in which both cultivars showed overlapping values of Cr

contents under different levels of both salts of arsenic, while chromium contents of

seeds or achenes were found also with minimum variation in values and relatively

lower Cr contents were recorded than root, shoot and leaves.

Figure 4.15(c): Cadmium (Cd), cobalt (Co) and chromium (Cr) contents in sunflower under different As conditions.

181

4.4.4.10 Lithium (Li), nickel (Ni) and lead (Pb) contents in sunflower under

different arsenic conditions in rooting medium.

Analysis of variance of the data regarding lithium contents in root revealed

that varieties, salts and levels and all interactions among these three factors showed

significant differences among data (Table 4.17(c)) while data analysis regarding Li

contents of shoot revealed that out of three factors only levels showed significant

differences but varieties or cultivars of sunflower and salts of arsenic showed non-

significant differences similarly all interactions among these three factors except salt

into level interaction showed significant differences (P<0.01) in values recorded. In

case of Li contents of leaf varieties and salts showed significant (P<0.05) differences

and levels also showed significant differences, similarly all interactions among these

three factors including varieties, salts and levels showed significant differences but in

case of lithium contents of seeds or achenes only varieties showed significant

differences while salts and levels of arsenic showed non-significant differences

whereas out of different interactions only variety into salt and salt into level

interaction showed significant differences but remaining interactions showed non-

significant differences among data recorded.

Statistical analysis of the data regarding nickel contents in root revealed that

salts and levels of arsenic showed significant differences (P<0.01) while varieties or

cultivars of sunflower showed non-significant differences whereas all interactions

182

among these three factors showed significant differences among data recorded (Table

4.17(c)). Data analysis regarding Ni contents of shoot revealed that varieties of

sunflower showed significant differences while salts and levels of arsenic showed

non-significant differences, similarly out of different interactions among these three

factors only salt into level interaction showed significant (P<0.05) differences but

remaining all interactions showed non-significant differences. In case of nickel

contents of leaves, levels of arsenicals showed significant differences while sunflower

varieties and salts of arsenic showed non-significant differences and only variety into

salt into level interaction showed significant (P<0.05) differences but remaining all

interactions showed non-significant differences in data. ANOVA regarding Ni

contents of seed or achenes revealed that varieties or cultivars of sunflower showed

significant differences while salts and levels of arsenic and all interactions among the

three factors showed non-significant differences among different values recorded.

ANOVA of the data regarding lead contents in root revealed that varieties

(P<0.05), salts and levels and all different interactions among these three factors

showed significant (P<0.01) differences among data recorded (Table 4.17(c))

similarly in case of Pb contents of shoot, varieties, salts and levels and all interactions

among these factors also showed significant differences among the data or values

recorded. Analysis of data regarding lead contents in leaves revealed that salts and

levels of arsenic showed significant differences but varieties showed non-significant

differences and all interactions among varieties, salts and levels showed significant

differences whereas in case of Pb contents of seed or achenes varieties of sunflower

and levels of arsenic showed significant differences but salts of arsenic showed non-

significant differences and all interactions among these three factors also showed

significant differences in values recorded and analyzed statistically.

Table 4.17(c): ANOVA for lithium (Li), nickel (Ni) and lead (Pb) contents in sunflower under different As conditions.


Li root Li shoot Li leaf Li seedVarieties (V) 1 2.108* 1.256ns 1.419* 0.2461**

Salts (S) 1 8.459** 0.039ns 2.139* 0.0550ns

Levels (L) 5 3.628** 4.417** 2.310** 0.023ns

InteractionsV × S 1 36.865** 4.248** 1.776* 0.141**V × L 5 8.585** 3.205** 1.312** 0.027ns

S × L 5 9.173** 0.381ns 1.279** 0.050*V × S × L 5 7.045** 4.001** 6.859** 0.043ns

183

Error 48 0.421 0.320 0.342 0.019


Ni root Ni shoot Ni leaf Ni seedVarieties (V) 1 1.397ns 2.391** 0.039ns 2.637**

Salts (S) 1 19.107** 2.793ns 1.298ns 1.125ns

Levels (L) 5 14.059** 0.474ns 1.811** 0.243ns


V × L 5 6.007** 0.118ns 0.278ns 0.331ns

S × L 5 5.336** 0.659* 0.485ns 0.481ns

V × S × L 5 14.714** 0.051ns 0.959* 0.261ns

Error 48 0.835 0.254 0.328 0.319


Pb root Pb shoot Pb leaf Pb seedVarieties (V) 1 33.37* 5.601** 13.18ns 8.694**

Salts (S) 1 484.33** 1.973* 103.78* 0.128ns


V × S 1 156.70** 7.144** 503.50** 7.334**V × L 5 32.35** 15.105** 116.90** 18.974**S × L 5 190.48** 5.269** 209.58** 4.375**

V × S × L 5 81.37** 13.877** 287.07** 2.315*Error 48 5.74 0.459 16.84 0.868

Roots of sunflower cultivars showed highest variation in lithium contents

especially higher values were recorded in cultivar V2 under arsenate levels while

under arsenite higher Li contents were found in V1 plants (Figure 4.16(c)) similarly in

shoot V2 plants showed higher contents of lithium under different levels of arsenate

whereas least value was recorded in plants belonging to the treatment with highest

arsenate treatment application or level and under different levels of arsenite plants

showed first increase and then decrease in Li contents in both cultivars or varieties. Li

contents in leaves were also in same range with root and shoot and higher Li contents

were found under higher arsenate and arsenite treatments especially in T4 and T5 in

both cultivars especially under arsenite V2 plants showed relatively higher Li contents

under T5 but plants belonging to V1 showed higher Li contents under T4 plants

whereas in seeds or achenes least lithium contents were found under all different

levels of arsenate and arsenite in both cultivars or varieties of sunflower.

Sunflower roots showed highest values of nickel contents especially in V2

plants under different levels of arsenate while under arsenite V1 plants belonging to

T4 showed maximum Ni contents (Figure 4.16(c)). In shoot, leaves and seeds or

184

achenes the contents of nickel remained similar with very less variation in values in

both cultivars under different levels of arsenate as well as arsenite as a little

fluctuation in Ni contents values was recorded in these organs of sunflower.

Lead contents in root were found higher under different levels of arsenate

especially in treatments or levels of higher arsenate (Figure 4.16(c)) while under

different levels of arsenite highest Pb contents were observed in V2 plants as

compared to plants belonging to cultivar V1. In shoot less lead contents were

observed than root under both salts of arsenic used as under arsenite levels V2 plants

showed higher values of lead contents but with least variation among values. In case

of leaves, Pb contents were found higher in V1 plants under higher arsenate levels but

in V2 plants a stability was observed in lead contents under different arsenate levels

whereas under arsenite conditions V2 plants showed higher lead contents especially a

highest value in T5 plants i-e under maximum concentration of arsenite. Seeds or

achenes showed minimum variation in values of lead contents under both arsenicals

and in both cultivars or varieties of sunflower.

Figure 4.16(c): Lithium (Li), nickel (Ni) and lead (Pb) contents in sunflower under different As conditions.

185

4.4.4.11 Antimony (Sb), selenium (Se) and strontium (Sr) contents in

sunflower under different As conditions.

Statistical analysis of the data regarding antimony contents in root revealed

that levels of arsenic showed significant differences (P<0.01) while varieties or

cultivars of sunflower and salts of arsenic used showed non-significant differences

(Table 4.18(c)) whereas out of different interactions among these three factors only

variety into salt interaction showed significant differences while remaining

interactions showed non-significant differences, similarly in case of antimony

contents of shoot also only levels showed significant (P<0.05) differences but

varieties and salts showed non-significant differences and out of all interactions only

variety into salt interaction showed significant differences while all other interactions

showed non-significant differences. In case of antimony contents in leaves, analysis

of variance of the data revealed that varieties, salts and levels all showed non-

significant differences while all of the different interactions showed significant

differences except variety into salt interaction but data analysis regarding antimony

contents in seed or achenes revealed that levels of arsenic showed significant

differences but varieties of sunflower and salts of arsenic showed non-significant

differences and out of different interactions variety into salt and variety into level

interaction showed significant (P<0.05) differences while remaining interactions


Two way analysis of variance of the data regarding selenium contents in root

revealed that salts of arsenic showed significant differences while varieties and levels

showed non-significant differences (Table 4.18(c)) whereas out of different

interactions variety into salt and variety into level showed significant differences but

remaining interactions showed non-significant differences while in case of Se

contents of shoot only levels of arsenic showed significant (P<0.05) differences but

varieties of sunflower and salts of arsenic showed non-significant differences and all

186

different interactions among these three factors showed significant differences except

variety into salt interaction. In case of selenium contents of leaves only levels of

arsenic showed significant differences (P<0.01) while varieties and salts showed non-

significant differences whereas all interactions among the three factors showed

significant differences among data recorded. Analysis of variance of the data related

to Se contents in seeds or achenes revealed that varieties or cultivars of sunflower and

levels of arsenic showed significant differences but salts of arsenic showed non-

significant differences and all of the interactions among these three factors showed

significant differences among different values recorded.

Data analysis regarding strontium contents in root revealed that varieties, salts

and levels showed significant differences (P<0.01) and all the interactions among

these three factors also showed significant differences among values recorded and

analyzed (Table 4.18(c)). Analysis of data about Sr contents in shoot revealed that

only levels showed significant differences while varieties and salts showed non-

significant differences and all interactions among these factors showed significant

differences whereas analysis about strontium contents of leaves revealed that varieties

of sunflower showed significant differences (P<0.05) and levels of arsenic also

showed significant differences but salts of arsenic showed non-significant differences

whereas all interactions among these factors showed significant differences among

values. In case of Sr contents of seeds or achenes varieties, salts and levels showed

non-significant differences and out of different interactions only variety into level

interaction showed significant differences while remaining all interactions showed

non-significant differences among various values recorded.

Table 4.18(c): ANOVA for antimony (Sb), selenium (Se) and strontium (Sr) contents in sunflower under different As conditions.


Sb root Sb shoot Sb leaf Sb seedVarieties (V) 1 0.002ns 0.0068ns 0.314ns 0.0012ns

Salts (S) 1 0.0005ns 0.0612ns 0.057ns 0.031ns

Levels (L) 5 0.268** 0.179* 0.120ns 0.088**Interactions

V × S 1 1.680** 0.451** 0.033ns 0.101*V × L 5 0.046ns 0.095ns 0.269* 0.058*S × L 5 0.145ns 0.010ns 0.332* 0.040ns

V × S × L 5 0.143ns 0.048ns 0.451** 0.014ns

Error 48 0.0769 0.057 0.109 0.017

Mean square

187

Source D F Se root Se shoot Se leaf Se seedVarieties (V) 1 0.124ns 1.034ns 3.075ns 13.081**

Salts (S) 1 3.046** 0.109ns 0.576ns 0.515ns

Levels (L) 5 0.428ns 1.009* 8.556** 2.348**Interactions

V × S 1 1.617* 0.000ns 6.078* 15.952**V × L 5 2.048** 3.576** 11.821** 0.801**S × L 5 0.537ns 1.626** 7.491** 2.311**

V × S × L 5 0.415ns 0.911* 11.290** 3.788**Error 48 0.253 0.312 1.249 0.202


Sr root Sr shoot Sr leaf Sr seedVarieties (V) 1 1177.98** 80.10ns 434.5* 33.27ns

Salts (S) 1 179.27** 50.77ns 277.9ns 0.88ns

Levels (L) 5 345.65** 608.38** 2823.5** 21.24ns


V × L 5 352.27** 589.20** 1840.3** 63.95**S × L 5 629.52** 325.06** 700.7** 17.56ns

V × S × L 5 224.24** 123.08** 331.5** 15.11ns

Error 48 10.43 22.63 83.0 13.78

An increasing trend was observed in shoot Sb contents with increasing level of

arsenite in plants belonging to V1 while V2 plants showed first increase under low

arsenite concentrations or levels and then decrease in antimony contents was recorded

under higher arsenite levels but in arsenate treated plants generally higher antimony

contents were recorded in plants belonging to V2 under different arsenate treatments

as compared to V1 plants (Figure 4.17(c)). In leaves different levels or concentrations

of arsenate caused a lot of variation in antimony contents of sunflower plants with a

highest value under T3 of arsenate in plants of V2 while V1 plants also showed two

peaks in T1 and T4 plants under arsenate whereas under arsenite first increase under

lower arsenite levels and then decrease under higher levels of arsenite was recorded in

both cultivars with a higher standard deviation values. Seeds or achenes of sunflower

cultivars showed antimony contents in same range with shoot and leaves as in

arsenate treated plants V2 cultivar showed average higher values of antimony than V1

plants while under arsenite treatments or levels plants belonging to V1 showed higher

antimony contents than V2 plants especially under higher arsenite concentrations.

Less variation was recorded in selenium contents of root especially under

arsenate conditions while under arsenite a lot of variation was seen and plants

belonging to V1 showed higher selenium contents in root under higher levels of

188

arsenite while V2 plants showed decrease in Se contents in treatments comprising of

higher arsenite concentrations (Figure 4.17(c)). In case of shoot selenium contents

were also found showing an increasing trend under arsenate in plants belonging to V1

while V2 plants showed a relative decrease towards higher arsenate concentrations

while in arsenite treated plants also V1 plants showed higher contents of selenium as

compared to V2 especially under higher concentrations of arsenite. in case of leaves

maximum variation in selenium contents was recorded in both cultivars as V1 plants

showed first decrease in selenium contents up to T3 and then increase was observed in

values of selenium contents in leaves under arsenate and on average higher Se

contents in leaves under arsenate were recorded in V2 plants while under arsenite

conditions a maximum value was recorded in V1 plants under level T4 while V2

plants showed a higher value in T1 plants of V2 cultivar and overall a decreasing

trend was observed with increasing arsenite concentrations. In seeds or achenes both

cultivars showed similar behavior under different levels or treatments of arsenate

showing first increase then decrease and then again increase under highest arsenate

concentrations while under different levels of arsenite V2 plants showed gradual

increase in selenium contents towards increasing arsenite concentrations or levels

while V1 plants showed relative decrease in Se contents under higher concentrations

of arsenite.

Strontium contents in root were found increasing gradually in plant s of V1

with increasing arsenate concentrations (Figure 4.17(c)) while V2 plants showed a

least value in level T1 of arsenate and then gradual rise in strontium contents up to T3

and then again fall in values was recorded while under arsenite conditions V1 plants

showed rise in Sr contents up to T2 and then fall in Sr contents was seen but in V2

plants very low variation was recorded in strontium contents under different levels or

treatments of arsenite. In shoot Sr contents were found in same range with root and

V2 plants showed relatively higher Sr contents than V1 plants under different arsenate

levels while under arsenite conditions V1 plants showed relatively higher Sr contents

in some treatments than V2 plants which showed less variation in Sr content values.

In leaves highest strontium contents were recorded in both cultivars of sunflower with

a lot of variation under different levels of arsenate as well as arsenite but in seeds or

achenes least contents of strontium were found with lowest variation under both

arsenicals in cultivar V1 and V2.

189

Figure 4.17(c): Antimony (Sb), selenium (Se) and strontium (Sr) contents in sunflower under different As conditions.

4.4.4.12 Titanium (Ti), thallium (Tl) and vanadium (V) contents in

sunflower under different conditions of arsenic.

Statistical analysis comprising of analysis of variance of the data regarding

titanium contents in root revealed that cultivars or varieties of sunflower, salts and

levels of arsenic and all interactions among these three factors showed significant

(P<0.01) differences in values (Table 4.19(c)) while in case of Ti contents of shoot,

varieties, salts and levels showed non-significant differences and out of different

interactions only variety into level interaction showed significant (P<0.05) differences

whereas remaining interactions showed non-significant differences but in case of

titanium contents of leaves, levels of arsenic showed significantly different values but

190

varieties and salts showed non-significant differences and variety into level and salt

into level interactions also showed significant differences but other remaining

interactions showed non-significant differences among different values recorded. The

data analysis about titanium contents of seeds or achenes revealed that varieties of

sunflower and levels of arsenic showed significant differences but salts showed non-

significant differences similarly all interactions except variety into level interaction

showed significant differences among values recorded.

Analysis of variance of the data regarding thallium contents of root revealed

that varieties, salts, levels and all different interactions among these three factors

showed significant differences while in case of Tl contents of shoot varieties, salts and

levels showed non-significant differences whereas out of all interactions only variety

into level interaction showed significant (P<0.01) differences and remaining

interactions showed non-significant differences (Table 4.19(c)) among data. In case of

thallium contents of leaves only levels of arsenic showed significant differences but

varieties and salts showed non-significant differences while all interactions among

varieties, salts and levels showed significant differences among values recorded. In

case of thallium contents of seeds or achenes varieties of sunflower and levels of

arsenic showed significant differences but salts of arsenic used showed non-

significant differences whereas all interactions except variety into level interaction

showed significantly different values.

Data analysis regarding vanadium contents in root revealed that varieties of

sunflower and levels of arsenic showed significant (P<0.01) differences but salts of

arsenic used showed non-significant differences while all interactions among these

three factors showed significant differences (Table 4.19(c)). Similarly in case of

vanadium contents of shoot varieties and levels showed significant differences but

salts showed non-significant differences and all interactions except variety into salt

showed significantly different values whereas in case of V contents of leaves as well

as seeds or achenes, all the three factors varieties, salts and levels and all interactions

among these factors showed non-significant differences among values recorded.

191

Table 4.19(c): ANOVA for titanium (Ti), thallium (Tl) and vanadium (V) contents in sunflower under different As conditions.


Ti root Ti shoot Ti leaf Ti seedVarieties (V) 1 1823.88** 31.26ns 18.46ns 121.368**

Salts (S) 1 2085.50** 1.45ns 0.64ns 1.717ns

Levels (L) 5 780.27** 19.58ns 551.58** 17.143*Interactions

V × S 1 395.84** 16.40ns 2.25ns 127.148**V × L 5 534.67** 31.58* 98.54** 12.892ns

S × L 5 1319.32** 2.08ns 69.33** 43.972**V × S × L 5 258.68** 10.41ns 15.46ns 26.846**

Error 48 25.51 11.29 15.13 6.675


Tl root Tl shoot Tl leaf Tl seedVarieties (V) 1 771.52** 14.851ns 3.277ns 169.004**

Salts (S) 1 237.00** 19.220ns 17.405ns 0.553ns

Levels (L) 5 50.40** 18.219ns 228.560** 28.813**Interactions

V × S 1 2013.06** 9.031ns 91.215** 175.188**V × L 5 443.14** 36.907** 35.574** 8.844ns

S × L 5 157.71** 6.938ns 34.673** 55.890**V × S × L 5 276.72** 8.487ns 91.632** 33.396**

Error 48 10.50 7.810 8.645 4.459


V root V shoot V leaf V seedVarieties (V) 1 9873.87** 15.401** 4.821ns 0.170ns

Salts (S) 1 38.08ns 0.106ns 0.456ns 0.034ns

Levels (L) 5 693.80** 69.390** 0.948ns 0.113ns


V × L 5 710.88** 103.950** 0.529ns 0.0578ns

S × L 5 187.39** 9.629** 0.516ns 0.0453ns

V × S × L 5 201.27** 18.230** 1.234ns 0.0969ns

Error 48 10.21 2.160 2.553 0.3962

Alcaraz-Lopez, et al., (2003) reported beneficial effects of titanium on growth

of plum tree (Prunus domestica) as inferred that foliar spray of titanium caused

increase in Ca, Fe, Cu and Zn contents in peel and flesh of plum and in our

experiment roots of sunflower showed highest contents of titanium especially under

different levels of arsenate in which V1 showed maximum Ti contents in T3 of both

cultivars but a drop was recorded in Ti contents under higher arsenate levels and same

trend was seen in V2 plants with relatively lower Ti contents under different arsenate

concentrations but under arsenite conditions less variation was recorded in Ti contents

192

with a single higher value in plants belonging to T2 in V1 plants (Figure 4.18(c)).

Shoots showed less contents of Ti with same range under all different levels of

arsenate as well as arsenite in both cultivars or varieties but in leaves a gradual

increase in Ti contents was recorded in both cultivars with increasing arsenate and

arsenite concentrations or levels. Similarly in seeds or achenes lower contents of Ti

were recorded in both cultivars under different levels of arsenate and in arsenite

treated plants V2 showed least contents of Ti than V1 plants.

Roots showed highest contents of thallium with maximum values recorded in

plants belonging to V2 while V1 plants showed decrease in Tl contents with increase

in arsenate levels but under different levels of arsenite V1 plants showed a higher

value of Tl contents in level T2 and then a decrease was observed in further higher

aresnite levels or treatments (Figure 4.18(c)). In shoot least variation was recorded Tl

contents in both cultivars under different levels of arsenate and arsenite but in leaves

thallium contents were recorded increased with increasing level of arsenate in plants

belonging to V1 cultivar while V2 plants showed less variation but under different

levels of arsenite a gradual increase was recorded in Tl contents under higher levels of

arsenite in both cultivars. Results are in accordance with Radic et al., (2009) who

reported that thallium is accumulated by many important crops as were observed its

toxic effects on leaves and roots and also reported that Tl accumulated 50-250 times

higher in roots than in shoots of broad bean plants. Thallium contents of seeds or

achenes were found similar in both sunflower cultivars with least variation in values

especially under arsenite conditions in V1 plants while plants belonging to V2 showed

least contents of thallium under different arsenite levels.

Highest vanadium contents were found in roots of V2 cultivar under both salts

of arsenic while V1 plants showed least variation in V contents under both arsenicals

(Figure 4.18(c)) similarly in shoot vanadium contents were found very low in both

cultivars with a few exceptions but in leaves and seeds or achenes vanadium contents

remained least than all organs even can be said undetectable. In cowpea plant

vanadium in the form of vanadate localized to the underground parts of the plant and

little was moved to the up-ground part of the plant as described by Furukawa, (2001),

and these findings have similarity with our situation in which vanadium remained

highest in roots only in case of V2 plants while V1 plants did not showed any specific

and mentionable concentrations of this heavy metal in the underground part or root of

the plant.

193

Figure 4.18(c): Titanium (Ti), thallium (Tl) and vanadium (V) contents in sunflower under different As conditions.

4.4.5 Conclusion (Experiment 4)

Eco-physiological responses of sunflower against various levels of arsenic

(and associated metals in soil) presents a great potential in present study. Higher

levels of arsenic (60, 80 and 100 mg As/kg soil) proved most stressful when applied

in combination as in soil along with irrigation water especially for vegetative or

growth parameters like shoot and root length as well as fresh and dry weight of shoot

and root and number of leaves. Yield parameters like capitulum diameter and hundred

achene weight were also affected and water contents of shoot and root were also

decreased with increasing levels of arsenic either as arsenate or arsenite. Reduction in

shoot and root length was conspicuous in V1 as compared to V2. Leaf area was

194

reduced much more under arsenite salt levels. Arsenic accumulation was found in

order as root > leaves > shoot > seeds but left over arsenic remained highest.

Bioaccumulation coefficient was found highest in root then in leaves and shoot while

least in achenes or seeds. Phosphorus accumulated highest in seeds while Ca and Mg

in leaves. K and Cu accumulated mostly in seeds while B in leaves. Fe, Al, Co, Cr and

Ni were much more accumulated in roots while Sr and Mn in leaves, Bi and Zn in

seeds but Ti, Tl and V all accumulated in roots as compared to all other sunflower

organs. Both sunflower cultivars behaved similarly in response to different ions

accumulation.

4.5 Future prospects

Being carcinogenic arsenic is toxic for metabolism of living organisms

including sunflower. The best way of coping with it is “source elimination” which is

applicable after awareness and precise evaluation of this element in environment.

Reduction in growth and yield represents its stressful effects over metabolic activities

controlled by enzymes. Concise and accurate structure of vital enzymes like

RUBISCO, ATP-synthase and NADP+H reductase in response to arsenate and

arsenite substitution with phosphate (P) would be assessed in vivo at different growth

stages which will be make through improvement in remote sensing technology along

with development in instrumentation.

195

4.6 Abbreviations

3-HNPAA 4-Hydroxy-3-nitrophenlarsonic acid4-NPAA 4-Nitrophenylarsonic acidAB ArsenobetaineAB-2 Arsenobetaine 2 AC ArsenocholineAD After DepartureAg SilverAs ArsenicAv. AverageBC Before ChristC CarbonCd Cadmium Cl Chlorinecm centimeterCr ChromiumCu CopperDMAE Dimethyl arsinoyl ethanolDMAIII Dimethyl arsinous acidDMAV Dimethyl arsinic acidEPA Environmental Protection Agencyet al and fellowsetc. EtceteraEtxAsMe3-x Ethyl methyl arsinesg gramH HydrogenHg MercuryIARC International Agency for Research on Cancer IUPAC International Union of Pure and Applied Chemistrykg kilogramMe4As+ Tetramethyl arsonium ionmg milligramMMAIII Monomethyl arsonous acidMMAV Monomethylarsonic acidNi NickelO Oxygen PAA Phenylarsonic acidp-ASA p-Arsanilic acidPb Leadppm Parts per millionp-UPAA p-Ureidophenylarsonic acidS SulphurSn TinTMAIII TrimethylarsineTMAO Trimethylarsine oxideUNICEF United Nations Children’s FundUS United States

196

USRC United States Revenue CutterVA Group Fifth A of periodic tableWHO World Health Organization

197

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